Medicinal Foods as Potential Therapies for Type-2 Diabetes and Associated Diseases: The Chemical and Pharmacological Basis of their Action [1st ed.] 0081029225, 9780081029220, 9780081029237

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MEDICINAL FOODS AS POTENTIAL THERAPIES FOR TYPE-2 DIABETES AND ASSOCIATED DISEASES

MEDICINAL FOODS AS POTENTIAL THERAPIES FOR TYPE-2 DIABETES AND ASSOCIATED DISEASES The Chemical and Pharmacological Basis of their Action SOLOMON HABTEMARIAM Pharmacognosy Research Laboratories & Herbal Analysis Services UK, University of Greenwich, London, United Kingdom

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom © 2019 Elsevier Ltd. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN 978-0-08-102922-0 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Andre Gerhard Wolff Acquisition Editor: Erin Hill-Parks Editorial Project Manager: Megan Ashdown Production Project Manager: Stalin Viswanathan Cover Designer: Christian J. Bilbow Typeset by SPi Global, India

About the Author Dr Solomon Habtemariam is a Founder/ Owner of Herbal Analysis Services UK & Leader of the Pharmacognosy Research Laboratories at the University of Greenwich, Chatham-Maritime, UK. Dr Habtemariam received his BSc degree in Biology (minor: Chemistry) from the University of Addis Ababa and his Master’s degree (combinedstudies) in Pharmacology and Phytochemistry from the University of Strathclyde, Glasgow, UK. He stayed on at Strathclyde to study at doctoral level, studying on drug discovery researches and obtained his PhD in this area of research. After a number of years in teaching and post-doctoral research at the Strathclyde Institute for Drug Research and Strathclyde University, he joined the School of Science, University of Greenwich in September 1998.

Dr Habtemariam has been a leader of taught programmes and researches on bioassays & natural products-based drug development. The various researches that he has undertaken include the identification of novel compounds of natural-origin with potential antimicrobial, anticancer, anti-inflammatory, antidiabetic and antiobesity applications among others. He has published more than 171 scientific publications in peer reviewed journals and filed over three family of patents. He is also the author of a book entitled “The African and Arabian Moringa Species: Chemistry Bioactivity and Therapeutic Applications”. Details of his research activities and publications are available via his URL: http:// www.herbalanalysis.co.uk/.

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Preface Plants provide by far the most essential nutrients of the human diet such as carbohydrates, fats, proteins, vitamins and minerals. Collectively called plant foods, they are consumed to generate energy and maintain our normal body physiology or homeostasis. Beyond these vital nutrients, plants do also synthesize a variety of compounds that are called ‘secondary metabolites’ with functions often associated with defense against pathogens, herbivores or for allelochemical interactions. The exploitation of these valuable chemical compounds by mankind have been evident from prehistoric period of herbal medicines utilization to the significant proportion of today’s modern medicine which trace their origin back to natural products. The use of plants for flavoring and spices as well as exploitation for dyes, cosmetics, agrochemical and other industries also serve as good examples of plant secondary metabolites applications for human use. While advance in drug discovery and development in the last two centuries have made it possible to control/manage various diseases, or to improve the quality of life, there have also been continued challenges from emerging or the modern-era diseases that are not yet met by effective drug therapies. The classical example is the complex metabolic disease of type 2 diabetes (T2D). The epidemic proportion of recent increase in the incidence of this disease along with its major risk factor, obesity, brought significant burdens to our societies, governments and healthcare services. In the absence of drug of cure, a coordinated system of

intervention from disease prevention to management as well as public education is necessary. Measures of T2D management include life style changes such as dietary intervention, exercise, and weight loss where necessary, and drug interventions. At the forefront of dietary intervention measures are the balancing act of calorie intake and expenditure and promoting what are considered as good diets or healthy foods. Perhaps the most important therapeutic breakthrough in diabetes pharmacotherapy is associated with the discovery of insulin and its application that is dated back to the 1920s. Since then, numerous classes of compounds of biological/protein/peptide and small molecular weight compounds both from synthetic and natural product sources (or natural products-inspired synthesis) have been introduced. The requirement of longterm or life-time treatment coupled with the numerous side-effects of these drugs still push us, however, to desperately search for new therapies of T2D. In addition to insulin, the incretin analogues such as glucagon-like peptide mimics or exendin-4 from the saliva of the venomous Gila monster, which served as a synthesis framework for novel antidiabetic agents, underpins nature as a source of protein/peptide-based antidiabetic drugs. On the small-molecular weight side, our first line pharmacotherapy for T2D is still with metformin which is the biguanides class of compounds discovered form research based on the medicinal application of the plant Galega officinalis as antidiabetic agent. The rational for identifying novel drugs of natural-

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PREFACE

origin for T2D is thus as good as it has ever been and plants that have already been used by mankind for centuries and considered safe have even more added value if they are proven to be efficacious. At the junction between what constitute food and medicine are the medicinal foods. These include plant food ingredients which are known to contain a range of secondary metabolites that act through-drug like effects when they are consumed in sufficient amount. The various flavoring and spice plants such as ginger, garlic, turmeric, clove, fenugreek, etc. may be included into this category. While some plant foods are regularly consumed in some regions, they may be rare or considered medicinal in other regions of the world. For example, we have plants like Moringa which are eaten as staple food in East Africa and India while they are primarily used as herbal medicines in other parts of the world. While fruits like figs and olives are consumed as food, other plant parts of these plants such as their leaves are either routinely employed in herbal medicine applications or as herbal teas and infusions with some therapeutic implications. The vast arrays of herbal medicines are also recently repurposed as food supplements and are widely available in health shops as over-the-counter medicinal/nutraceutical products. Hence, the distinction between what we consider as food and medicine is even now becoming more and more blurred. This book is intended to provide a comprehensive resource on T2D and associated diseases from pathology to intervention by natural products that come under plant foods of medicinal importance. Section-A provides basic facts on T2D from significance as a global burden and prevalence (along with obesity) to its definitions, classifications and diagnostic criteria. In Section-B, the basic carbohydrate and lipid metabolism as well as pathological hallmarks of T2D are

extensively presented. In addition to the normal physiological regulations of carbohydrate/lipid metabolism from digestion in the gut to absorption, storage and utilization; pathological dysregulations and the major molecular/biochemical targets for drug therapy are outlined. One chapter is also dedicated in this section for pathological features of diabetes complication at macroand microvascular and/or cellular levels. In Section-C, an overview of the current therapeutic agents for T2D along with their mechanism of action are illustrated. The biosynthesis machinery of plants secondary metabolites and what classes of compounds are expected from natural sources are also discussed in this section. As such, the principles of drug action applicable to potential therapy of T2D by plant secondary metabolites are outlined. The choices of plants as medicinal foods was based on extensive review of the scientific literature and the profile of plant-based products in the market relevant to the food, nutraceutical and plant-based therapies. On these basis, three sections (Sections D–F) are dedicated to exhaustively appraise the chemistry and pharmacology of the chosen plants. With over 2270 chemical entries backed up with 4258 citations, this book is intended to provide readers with the most comprehensive and up-to-date information on the chemical and biological domains of these medicinal plants and/or foods. On the pharmacological side, all available resources from in vitro, the plethora of animal studies and human trials are systematically scrutinized. Mechanistic approach from insulin signaling and insulin resistance to general mechanisms of actions such as antioxidant and anti-inflammatory effects are extensively discussed. These include data from the chemical-, diet- and genetically-induced cellular and animal models of obesity and/or T2D. Along with

PREFACE

obesity, the T2D associated diseases such as hypertension, diabetes retinopathy, neuropathy, nephropathy and wound complications are all used to show pharmacological and clinical efficacies for the crude medicinal plant/food preparations as well as their active principles. Under each chapter, the historical perspectives of the plants as food and medicine, their botanical description and taxonomic significance are presented along with the relevant chemistry and pharmacology. Beyond the detailed pharmacological effects at in vitro, in vivo and clinical conditions, their pharmacokinetic and toxicological profiles are also discussed. Section-D exhibits fruits that have been shown to modulate T2D and the various components of metabolic syndrome. This include chapters on bilberries and blueberries, bitter melon (Momordica charantia L.), guava (Psidium guajava L.), okra (Abelmoschus esculentus (L.) Moench), papaya (Carica papaya L.), pomegranate (Punica grantum L.), prickly pear cactus (Opuntia species) and the various pumpkins (Cucurbita species). In addition to the fruits of these plants, discussions on other plant parts as emerging potential therapeutic agents are also included. Section-E is devoted to the flavour and spices groups that have shown to have significant health benefits in recent years and include chapters on cinnamon (Cinnamomum species), cloves (Syzygium aromaticum (L.) Merr. & L.M. Perry), fenugreek (Trigonella foenum-graecum L.), ginger (Zingiber officinale Roscoe), garlic (Allium sativum L.), and turmeric (Curcuma longa L.). Of the beverages included in Section-F are those known to modulate the diabetes pathology, but most importantly in reducing the diabetes prevalence/risk based on epidemiological and clinical data, are coffee, tea (Camellia sinensis (L.) Kuntze), rooibos (Aspalathus linearis (Burm. F.), and yerba mate (Ilex paraguariensis A. St.-Hil.). In the

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this section, one chapter is dedicated to include medicinal foods that are emerging to gain interest both scientifically and in the public domain as T2D modulating agents and include, cocoa (Theobroma cacao), coconut (Cocos nucifera), figs (Ficus carica), Moringa (Moringa oleifera and M. stenopetala), soybean (Glycine max), sweet potato (Ipomoea batatas), and walnuts (Juglans regia). The last chapter is dedicated to the large volume of information on herbal medicines that are now repurposed as food supplements. The merits of these products as dietary supplements with intention to modulate T2D and/or metabolic syndrome are scrutinized with the following key examples: Aloe vera (L.) Burm. F., Andrographis paniculata (Burm. F.), Wall. Ex Nees, banaba (Lagerstroemia speciose (L.) Pers), Cassia or Senna species (including Senna auriculata (L.) Roxband S. alata (L.) Roxb.), Ginkgo biloba L., ginseng (Panax. ginseng C.A. Mey, P. quinquefolius L. and P. notoginseng), gymnema (Gymnema sylvestre (Retz.) R.Br. ex Sm.), Jamun (Syzygium cumini (L.) Skeels), olive leaf extract (Olea europaea (L.)), scutellaria (Scutellaria baicalesis Georgi) and St John’s wort (Hypericum perforatum L.). Finally, this book is not intended to advocate the medicinal foods cited herein to serve as a replacement therapy to insulin, metformin or other antidiabetic pharmacotherapy in use today. It is a rather balanced appraisal of all the available data at hand with all negative and positive outcomes of pharmacological and clinical efficacies presented with the source of data variabilities, where appropriate, identified and scrutinized. In fact, the author, acknowledges that pharmacological data discrepancies for plant preparations, especially under clinical trial studies, reflects the challenges and frustrations of using medicinal foods or their products as medicine. Hence, the sources of controversies in efficacy and chemical composition variability for medicinal foods based on genetic and

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environmental factors that govern plant secondary metabolites; harvesting, processing, and manufacturing conditions; experimental models and designs including dosage, intervention time and assessment criteria, etc. are all taken into consideration. The author believes that the book would be an invaluable resource to scientist in drug discovery

researches, professionals in healthcare services, and those with interest in the herbal, food and pharmaceutical industries. Students and scientists in medical, pharmaceutical and food science disciplines are other beneficiaries of the book. Solomon Habtemariam

Acknowledgements I would like to thank a number of people who kindly gave me images of some plants used in this book, particularly Dr Helen Pickering of the Kew Botanical Garden, UK; Dr Jianhua Xie (State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, China); and Professor Susana Casal (REQUIMTE/Laboratory of Bromatology and Hydrology, Faculty of Pharmacy, Porto University, Rua Jorge Viterbo Ferreira, Porto, Portugal). Dr George Varghese of Kerala, my very good friend, who also supported me during my trip to

Kerala (India) has also been generous in introducing me to the medicinal foods of relevance in Asia. Images of a number of food ingredients and medicinal/nutraceutical products have been taken from health shops particularly Holland and Barret with the kind assistance of staff to whom I am grateful. I did extensively use the Kew Botanical Garden (London, UK) resources and this included acquiring images of the numerous medicinal plants in the garden and their excellent library services as well as online databases.

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C H A P T E R

1 Type-2 diabetes: Definition, diagnosis and significance O U T L I N E 1.1 Diabetes

3

1.2 Classification of diabetes

4

1.3 Diagnosis criteria: Diabetic and prediabetic conditions

5

1.4 Global prevalence of diabetes

7

1.5 The social and economic impact of diabetes

9

References

10

Further reading

10

1.1 Diabetes Diabetes or diabetes mellitus (DM) is a metabolic disorder characterized by either a total or partial loss of insulin secretion and/or resistance to insulin action leading to a chronic state of hyperglycaemia in the blood. DM can have multiple aetiology and almost inevitably involve some form of dysregulation in carbohydrate, fat, and/or protein metabolism. If not managed adequately, DM leads to complications including organ dysfunction and eventually death. The classical symptoms presented by DM patients include thirst, polyuria, blurred vision, unexplained weight loss, ketoacidosis, and hyperosmolar conditions. Unfortunately to DM sufferers, hyperglycaemia may not lead to the manifestation of these symptoms and complications and tissue damage often occur before diagnosis. The most common pathological hallmark of chronic/severe DM is retinopathy that could lead to blindness, nephropathy and/or renal failure, and neuropathy and autonomic dysfunctions that lead to limb ulcers and amputation. The link between diabetes and cardiovascular complications has also been well established and DM sufferers are under higher risk of peripheral, vascular, as well as cerebrovascular diseases. These pathological changes both at the molecular, cellular and systemic levels are discussed in Chapter 4.

Medicinal Foods as Potential Therapies for Type-2 Diabetes and Associated Diseases https://doi.org/10.1016/B978-0-08-102922-0.00001-8

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# 2019 Elsevier Ltd. All rights reserved.

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1. Type-2 diabetes: Definition, diagnosis and significance

1.2 Classification of diabetes The underlying causes of diabetes are either insufficient insulin production due to pancreatic β-cell destruction, insulin resistance, or a combination of both. For this, several pathogenic processes are involved leading to the development of abnormalities in carbohydrate, fat, and protein metabolism. Historically, DM was classified as either Insulin Dependent Diabetes Mellitus (IDDM) or Non-Insulin Dependent Diabetes Mellitus (NIDDM). The classification of DM is now, however, mainly based on the disease aetiology as follow: Type 1 diabetes (T1D): This includes either immune-mediated or idiopathic forms of β-cell destruction, which leads to absolute insulin deficiency. Insulin resistance is not common herein and therefore total dependency upon externally administered insulin is required for maintaining life. Type 2 diabetes (T2D): This involves a progressive loss of β-cell insulin secretion often coupled with insulin resistance. The disease has usually an adult onset that may begin with the body being insensitive to insulin and some form of insulin deficiency. The aetiology of T2D has a very strong component of genetic predisposition. Type 3 diabetes: This category includes a wide range of specific types of diabetes resulting from some form of genetic defects in insulin action or diseases. Examples include the monogenic diabetes syndromes such as the neonatal diabetes and maturity-onset diabetes of the young (MODY), diseases of the exocrine pancreas with cystic fibrosis as a well-established cause, and drug/chemical-induced diabetes including the prolonged usage of glucocorticoid following organ transplantation, management of AIDS, etc. Type 4 diabetes: Gestational diabetes mellitus (GDM) often diagnosed during the second or third trimester of pregnancy. Historical perspectives into the classification of diabetes starting from the two forms of insulin sensitive and insulin-insensitive types described by Himsworth in 1936 to the various aetiology-based definition of today are presented by Wild and Byrne (2013). With the aetiology of DM and its major risk factors including obesity are well established in recent years, the standardised classification and diagnostic criteria have been published by the World Health Organization (WHO). Such documents (e.g. that published in 1980) were also subjected to regular revisions. For example, the 1985 update described DM classification as IDDM and NIDDM, malnutrition-related diabetes mellitus (MRDM), gestational diabetes, and other types of diabetes. The 1999 WHO revision, on the other hand, replaces the IDDM and NIDDM, which reflect treatment rather than aetiology, by T1D and T2D, respectively. Under the present aetiology-based classification, the primary cause of T1D is pancreatic islet β-cell destruction where patients are prone to ketoacidosis. This includes the autoimmune-mediated destruction of β-cells and other forms called the idiopathic T1D where neither the pathogenesis nor the causes are known. When the specific cause of the β-cell destruction is identified, as in the cases of cystic fibrosis, mitochondrial defects, etc., they are not classified as T1D. In order to avoid ketoacidosis, coma and death, T1D patients need insulin administration. One common feature of T1D is the detection of antibodies as markers of the autoimmune β-cell destruction processes including anti-GAD (glutamic acid decarboxylase autoantibodies) or insulin antibodies. In the idiopathic T2D cases that is commonly expressed in non-Caucasians, however, markers of the autoimmune process may not be evident.

A. Type-2 diabetes: Prevalence and significance

1.3 Diagnosis criteria: Diabetic and prediabetic conditions

5

Unlike T1D, the insulin deficiency in T2D is relative rather than absolute and hence insulin resistance play major role. Considering that some degree of insulin production is maintained in T2D, these subjects do not need insulin treatment to survive at least in the first stage of the disease. For this very reason, the resulting hyperglycaemia in T2D may not be severe enough to cause noticeable symptoms or initiate early clinical detection. The common symptom of T1D, ketoacidosis, is not also common in T2D. As a result, T2D patients could be under high risk of developing macro- and microvascular complications that are outlined in Chapter 4. One of the common risk factor of T2D emerged in the last few decades is obesity and/or abdominal fat. The association of T2D with familial and/or genetic predisposition has been established and differences in the aetiology and severity of the diseases among different racial/ethnic groups have been reported. Readers must bear in mind, however, that a clear distinction between the various aetiology and even DM groupings may not be presented, i.e., a rather complex picture of mixed aetiological feature may be presented by patients. In T1D obese patients, for example, insulin resistance may also be common, while in T2D, patients may have even a higher level of insulin in their blood as compared to normal subjects but the excessive level of glucose would require even a further level of insulin production that cannot be met due to β-cell dysfunction. Hence, the increased level of insulin resistance demands for more insulin production that cannot be sustained by pancreatic β-cells. In some cases of T2D, normal insulin sensitivity coupled with impaired insulin secretion is also identified. In T2D, pharmacological treatment often in combination with lifestyle changes such as weight loss and dietary measures as well as physical activity are required for managing the diseases (see Chapter 5).

1.3 Diagnosis criteria: Diabetic and prediabetic conditions A further development by the WHO following DM classification was on diagnostic criteria of T2D. The major changes introduced in recent years and that adopted by various authorities including the American Diabetes Association (ADA, 2018) have been on lowering the cut point for fasting plasma/blood glucose (FBG) level from 7.8 mmol/L (140 mg/dl) to 7.0 mmol/L (126 mg/dL); the use of 2-h glucose tolerance test in addition to FBG level measurement and the introduction of glycated haemoglobin (HbA1c) level as a diagnostic measure. Hence, DM may be diagnosed as follow: • Fasting (no caloric intake for at least 8 h) plasma glucose (FBG)  126 mg/dL (7.0 mmol/L) or • 2-h plasma glucose  200 mg/dL (11.1 mmol/L) during an oral glucose tolerance test (OGTT). The WHO recommends glucose loading by taking 75 g of anhydrous glucose dissolved in water or • HbA1c  6.5% (48 mmol/mol) or • Classic diabetes symptoms are observed along with random plasma glucose  200 mg/dL (11.1 mmol/L) The common diagnostic tool for detection of T2D is FBG measurement. The 2-h plasma glucose after 75 g OGTT, and HbA1c are now used to confirm the disease but repeated measurements are often required to confirm positive diagnosis. T2D may also be identified

A. Type-2 diabetes: Prevalence and significance

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1. Type-2 diabetes: Definition, diagnosis and significance

through random screening, high risk group during routine check-up, and most commonly in patients presenting the classical symptoms. As discussed in the various chapters of this book, the same diagnostic criteria are used to monitor glycaemic control and efficacy of drugs in both experimental animals and human clinical studies. Recent approaches on diabetes intervention also gave considerable emphasis on managing prediabetic cases where an increased level of FBG is evident but is still below the threshold level of diabetes. This prediabetes state is often in the region of FBG of 100–125 mg/dL (5.6–6.9 mmol/L) and is also called impaired fasting glycaemia (IFG) or impaired glucose tolerance (IGT). Another marker of prediabetes is impaired glucose tolerance (IGT) of 140 and 199 mg/dL (7.9–11.0 mmol/L) plasma glucose in the 2-h OGTT or an HbA1c level of 5.7–6.4%. Some estimates suggest that unless a drastic measure is taken in the form of lifestyle changes and therapeutic interventions, about 70% of prediabetics could develop a full-blown diabetes in less than 10 years (Taba´k et al., 2012). In this context, the HbA1c level of >6% may be considered as high risk threshold for developing diabetes and is qualified for starting a therapeutic measure. The major risk factor of diabetes being obesity and age, screening for diabetes is also recommended for older and/or overweight people. Other risk factors for prediabetes/ diabetes are of course lack of physical activity, ethnicity/genetics, and clinical conditions such as cardiovascular diseases. Classification of diabetes, diagnosis, and treatment guidelines provided by the American Diabetes Association is a good reference material (ADA, 2018). The common diabetes symptoms that are often taken as indicators for initiating diagnosis are worth mentioning. These are related to the pathological changes and metabolic dysfunction associated with the disease and may give some clue about the class of diabetes as shown in Table 1.1.

TABLE 1.1 Common symptoms of diabetes System/organ affected

Symptom

Remark

CNS

Polyphagia

Excessive hunger or increased appetite

Polydipsia

Excessive thirst

a

Lack of energy and enthusiasm

Lethargy a

Near-unconsciousness or insensibility

Stupor Eye

Blurred vision

Respiratory system

Acetone smell in breatha Kussmaul breathinga

Systemic

Weight loss

Kidney/Urinary system

Polyurea

Hyperventilation due to diabetic ketoacidosis

Excessive urination

Glycoseurea/glucosuria Gastric a

Nausea, vomiting and abdominal pain

Presence of glucose in the urine a

Symptoms more common in T1D.

A. Type-2 diabetes: Prevalence and significance

1.4 Global prevalence of diabetes

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1.4 Global prevalence of diabetes The World Health Organisation (WHO) regularly publishes facts and figures for DM, and its 2016 global estimates (WHO, 2016) indicated 422 million adults aged over 18 years living with diabetes in 2014. The comparative assessment of diabetic cases over the years also indicated that the figure in 1980 was 108 million suggesting a drastic increase in diabetic cases. This corresponds to the global prevalence of diabetes growing from 4.7% in 1980 to 8.5% in 2014, during which time prevalence has increased or at the very best remained unchanged. For such assessment, the WHO divides the world countries into six zones/regions as shown in Fig. 1.1. The prevalence of diabetes for the different regions of the world by percentage and actual population size is also shown in Fig. 1.2. The highest rate of increase in T2D now appeared to be in the East Mediterranean region (EMR), while the largest number of diabetes sufferers are located in the Western pacific region followed by South-East Asia region (Fig. 1.3). Another significance of diabetes prevalence in recent years is the changing age boundary for the disease. From being a disease of the middle-aged and elderly people, T2D is now frequently seen in children and young people. The proportion of undiagnosed T2D has also been widely estimated to be between 24% and 62%. Another, and a rather disturbing figure by the WHO (2016), is on the prevalence of the major risk factor of DM, obesity, which is presented as follow:

FIG. 1.1 The WHO classification of the world regions for assessment of disease prevalence.

A. Type-2 diabetes: Prevalence and significance

8

1. Type-2 diabetes: Definition, diagnosis and significance

8

500

1980

Global Diabetes Cases (Millions)

Global Diabetes Prevalence (%)

10

2014

6 4 2

1980 2014

300 200 100 0

0 80

80

14

19

(A)

400

19

20

Year

(B)

14

20

Year

FIG. 1.2 Global prevalence of diabetes in percentage of population (A) and number (B). Source: WHO, 2016. Global report on diabetes http://apps.who.int/iris/bitstream/10665/204871/1/9789241565257_eng.pdf (Accessed 31.12.18). FIG. 1.3 Prevalence of diabetes across the world regions. Top panel (A) shows data in percentage, while lower panel (B) shows actual diabetes cases reported by the WHO (2016). Refer to Fig. 1.1 for the classification of world regions abbreviated in the X-axis.

A. Type-2 diabetes: Prevalence and significance

1.5 The social and economic impact of diabetes

9

Key fact: • Worldwide obesity has more than doubled since 1980. • In 2014, more than 1.9 billion adults, 18 years and older, were overweight. Of these, over 600 million were obese. • 39% of adults aged 18 years and over were overweight in 2014, and 13% were obese. • Most of the world’s population live in countries where overweight and obesity kills more people than underweight. • 41 million children under the age of 5 were overweight or obese in 2014. • The worldwide prevalence of obesity more than doubled between 1980 and 2014.

Hence, the diabetes epidemics appear to be going hand in hand with obesity. Moreover, the WHO figure in 2014 showing an estimated 41 million children under the age of 5 years in the world as overweight or obese is indeed alarming. In less economically developed countries such as Africa, the number of overweight or obese children also nearly doubled from 5.4 million in 1990 to 10.6 million in 2014 while nearly half of the children under 5 in Asia were classified as overweight or obese during the same time period.

1.5 The social and economic impact of diabetes As discussed in Chapter 4, diabetes is a leading cause of damage to the heart, blood vessels, eyes, kidneys, and nerves, among others. Inevitably, diabetes increases the risk of heart attacks and strokes. The endothelial dysfunction and reduced blood flow, together with neuropathy, increase the chance of foot ulcers which is susceptible to infection and eventually leads to limb amputation. Diabetes retinopathy due to extensive capillary formation or angiogenesis is a leading cause of blindness associated with the disease. Diabetes also remains to be the main cause of kidney failure making the disease as a priority area both for reducing the quality of life and cause of death. The direct medical costs and loss of work associated with diabetes is enormous and the disease along with obesity is considered a condition that might bankrupt the health care system of many countries in the very near future. The WHO indicates that in 2012, there were 1.5 million deaths worldwide directly caused by diabetes. The total burden of deaths from high blood glucose was even estimated to be higher and in 2012, it was 3.7 million, i.e., an additional 2.2 million deaths could be attributed to diabetes-associated diseases such as cardiovascular diseases, chronic kidney disease, and tuberculosis related to higher-than-optimal blood glucose. The WHO (2016) data further estimates that the largest number of deaths resulting from high blood glucose occurs in upper-middle income countries (1.5 million) and the lowest number in low-income countries (0.3 million). According to Diabetes UK (2018), the cost of diabetes to the National Health Service (NHS) was as follow: • Over £1.5 million an hour or 10% of the NHS budget for England and Wales, i.e., over £25,000 every minute • About £14 billion pounds a year for treating diabetes and its complications A. Type-2 diabetes: Prevalence and significance

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1. Type-2 diabetes: Definition, diagnosis and significance

• The cost of treating diabetes complications is higher than the cost of treating diabetes (blood glucose control) itself Data for the cost of diabetes in the United States have also been estimated by various government departments and organisations. The American Diabetes Association (2018) put the estimated cost of diagnosed diabetes in 2012 as $245 billion of which $176 billion was direct medical costs and $69 billion for the reduced productivity due to the diabetes burden. A 41% increase of the cost over a 5-year period was also reported. One could thus imagine the global cost of diabetes and associated diseases now and for the next few years.

References ADA (American Diabetes Association), 2018. The cost of diabetes. http://www.diabetes.org/advocacy/newsevents/cost-of-diabetes.html. (Accessed 31 December 2018). Diabetes UK, 2018. Cost of diabetes. Available at: https://www.diabetes.co.uk/cost-of-diabetes.html (Accessed 31 December 2018). Taba´k, A.G., Herder, C., Rathmann, W., Brunner, E.J., Kivim€aki, M., 2012. Prediabetes: a high-risk state for diabetes development. Lancet 379 (9833), 2279–2290. WHO, 2016. Global report on diabetes. http://apps.who.int/iris/bitstream/10665/204871/1/9789241565257_eng. pdf. (Accessed 31.12.18). Wild, S.H., Byrne, C.G., 2013. Commentary: sub-types of diabetes—what’s new and what’s not. Int. J. Epidemiol. 42, 1600–1602.

Further reading American Diabetes Association Standards of Medical Care in Diabetes, 2017. Classification and diagnosis of diabetes. Diabetes Care 40 (Suppl. 1), S11–S24.

A. Type-2 diabetes: Prevalence and significance

C H A P T E R

2 Glucose metabolism: Normal physiology, diabetic dysregulation, and therapeutic targets O U T L I N E 2.1 Introduction

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2.2 Carbohydrate digestion

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2.3 Mechanisms of glucose uptake and transport

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2.4 Glycogenesis

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2.5 Glycogenolysis

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2.6 Gluconeogenesis

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2.7 Hormonal regulation of glucose metabolism 2.7.1 Insulin 2.7.2 Glucagon and related peptides 2.7.3 Other hormones regulating glucose level

26 28 33 35

2.8 The AMP-activated protein kinase (AMPK) and cellular energy balance regulation

37

References

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2.1 Introduction The level of glucose in the blood is tightly regulated within a narrow window of concentration range through intricate balance of glucose generation and removal mechanisms. The primary source of glucose pool in the blood is derived from dietary carbohydrates from the gut, the release of glucose from storage, primarily in the liver through glycogenolysis, and synthesis of glucose (gluconeogenesis) in the body from other sources such as amino acids, lactic acids, and other reaction intermediates. Glucose is taken up from the blood virtually by all cells in the body for energy generation. Upon the complete oxidation of glucose through aerobic mechanism, 36 molecules of ATP are generated per molecule of glucose, while incomplete oxidation

Medicinal Foods as Potential Therapies for Type-2 Diabetes and Associated Diseases https://doi.org/10.1016/B978-0-08-102922-0.00002-X

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2. Glucose metabolism

through anaerobic or glycolytic route can generate two molecules of ATP per glucose molecule. Beyond energy generation, the metabolism of glucose in the energy generation process involving glycolysis and the ‘Krebs Cycle’, also known as the ‘Citric Acid Cycle’ or the ‘Tricarboxylic Acid Cycle (TCA)’ offer numerous essential ingredients for the synthesis of fatty acids, steroids, cholesterol, amino acids, the purines and pyrimidines and various other biological molecules. As excess amount of glucose is always excreted through the kidney, conservation of this vital metabolic source during postprandial excess in the blood is carried out in the liver and muscles by storing it in the form of glycogen. In the following sections, the major processes of glucose metabolism along with the key regulatory mechanisms and potential target areas for therapeutic interventions in type 2 diabetes (T2D) are discussed.

2.2 Carbohydrate digestion Large food particles are broken down into small pieces through mechanical (e.g. chewing in the mouth and the churning and grinding of food by the stomach) and chemical processes. While the mineral components of food along with others such as vitamins (micronutrients) are released from smaller food particles, major food nutrients such as carbohydrates, fats, and proteins are subjected to a number of enzymatic chemical processing throughout the gastrointestinal (GIT) system. The main carbohydrate sources of the human diet are complex polysaccharides such as starch, the disaccharide sucrose (our common sweet choice, table sugar), the lactose of milk origin, and the maltose mainly from the grain source (e.g. brewed products). The major carbohydrates of the human food from sources such as potatoes, wheat, corn, and rice are stored in the form of starch which is composed of the linear and helical amylose units and branched amylopectin. As explained in the later sections, the chemical composition of the polysaccharides with respect to the bonds between the sugar units are important features governing their digestibility. The water insoluble helical polymer of amylose is made of α-D-glucose monomers joined through α(1 ! 4) glyosidic bonds (Fig. 2.1). On the other hand, the water soluble amylopectin has the linear α(1 ! 4) glycosidic bonds that exhibit a repeating (every 24–30 glucose units) branching via α(1 ! 6) bonds (Fig. 2.1). For utilization by the body as an energy source, only the monomers of disaccharides and polysaccharides are taken up by the blood and hence complete breakdown of carbohydrates to their monomeric building blocks must be achieved in the gut: Starch ! glucose Sucrose ! glucose + fructose Lactose ! glucose + galactose Maltose ! glucose Enzymatic digestion of carbohydrate start in the mouth where the amylase with an optimum pH of 7 acts on starch to give di- and trisaccharides as well as smaller branched dextrin fragments. The enzyme acts on the α(1 ! 4) glycosidic bonds in the starch or amylose sugars. It does also cleave these bonds in the amylopectin although the branching points that contain α(1 ! 6) glycosidic bonds are unaffected. Until the enzyme is neutralized by the acidic environment in the stomach that denature it, salivary amlylase breaks down starch into smaller components in readiness to be acted by other enzymes. B. Normal physiological control of carbohydrate

2.2 Carbohydrate digestion

15

FIG. 2.1 Structures of amylose and amylopectin. The numbering of carbons of glucose molecules is also shown.

FIG. 2.2 The location of α-glucosidease in the intestine. The intestinal cells, enterocytes, have microvilli structural feature in the luminal or apical-end called brush border that contain a variety of carbohydrate digestive enzymes. These enzymes collectively called the α-glucosidases include maltase, sucrose, lactase, and α-dextrinase. The resulting glucose and other nutrients and minerals released from the food are absorbed to the cells to be transferred to the blood via the interstitial fluid at the junction between enterocytes and the blood vessels.

The major enzymatic digestion of carbohydrates occurs in the small intestine. As with the salivary amylase, pancreatic amylase with the optimum pH of 7 acts on polysaccharides to release small branches (dextrins—oligosaccharides containing at least one α (1 ! 6) glycosidic bond/branch), disaccharides (e.g. sucrose, maltose, and lactose), trisaccharides (matotriose), and glucose. The larger units (di-, tri-, and polysaccharides) are finally processed by the membrane-bound intestinal brush border enzymes (Fig. 2.2) as follow: • • • • •

Glucoamylase hydrolyses maltose and maltotriose Dextrinase hydrolyses dextrin by acting on the α(1 ! 6) glycosidic bonds Sucrase hydrolyses sucrose to generate glucose and fructose Lactase hydrolyses lactose to generate glucose and galactose Maltase converts maltose to glucose B. Normal physiological control of carbohydrate

16

2. Glucose metabolism

The resulting monomeric carbohydrates such as glucose, fructose, and galactose released by ectoenzymes of the brush border cells are then absorbed by the enterocytes (see Section 2.3 below for uptake mechanisms). Given that the α(1 ! 4) bond in carbohydrates of the human diet is the major significance, enzymes acting on this bond, collectively called α-glucosidases, are playing key role in making glucose available for uptake into the blood stream. Hence, inhibition of these enzymes constitutes a major mechanism of antidiabetic effect by pharmaceutical agents whereby the level of glucose in the blood is suppressed through inhibition of carbohydrate digestion. A number of complex carbohydrates including cellulose are not digested in the human body due to lack of the necessary enzymes. These indigestible polysaccharides are collectively called dietary fibres that may be soluble or insoluble and contribute to food/stool mobility within the digestive system (e.g. the colon).

Key facts: The major step in the digestion of carbohydrate and release of glucose into the blood stream involve the group of enzymes in the intestine collectively called α-glucosidases. Inhibition of these enzymes that cleaves the α(1 ! 4) bonds is one mechanism of antidiabetic effects by pharmaceutical agents and natural products such as medicinal foods. Indigestible carbohydrates or polysaccharides are dietary fibres that modify the normal function of the gut and also have applications in antidiabetic therapy.

2.3 Mechanisms of glucose uptake and transport Once carbohydrates are completely digested to release their monomers such as glucose, galactose, and fructose, they are absorbed by the intestinal brush-border cells via different transporters. The predominant means of glucose absorption across the cell membrane of these cells is through the Na+-dependent glucose transporter protein-1 (SGLT1). There is also another transporter, SGLT2 but its major role as glucose transporter is in the kidney for glucose reabsorption following glomerular filtration. These membrane proteins couple the transport of two molecules of Na+ ions with one molecule of glucose (Fig. 2.3). A number of studies (e.g. Kipnes, 2011; Musso et al., 2012) have established these two glucose transporters as follow: • The SGLT2 is a high-capacity and low-affinity protein, whereas SGLT1 which is lowcapacity and high-affinity protein transporter; • SGLT2 is predominantly expressed at the proximal renal tubules and accounts for reabsorption of up to 90% of glucose; • SGLT1 is predominantly expressed at the apical side of the intestinal brush border cells and late proximal renal tubules; • SGLT1 also transports galactose; • Both SGLT1 and SGLT2 can be targeted by antidiabetic agents to suppress the raised plasma glucose level in T2D.

B. Normal physiological control of carbohydrate

2.3 Mechanisms of glucose uptake and transport

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FIG. 2.3 Glucose absorption at the apical membrane of the enterocytes. The SGLT1 couples the transport of one molecule of glucose while co-transporting two molecules of Na+.

FIG. 2.4 Glucose exit from enterocytes via the basolateral membrane. The GLUT2 is primarily used to transport glucose down its concentration gradient without a need for ATP. The facilitated transport system through conformational changes of the protein is shown from A to D.

Once enterocytes are loaded with glucose, they release it to extracellular medium near the blood capillaries via the basolateral membrane through glucose transporter protein-2 (GLUT2). The transport of glucose by GLUT2 is a facilitated-diffusion as depicted in Fig. 2.4. GLUT2 has also shown to be involved in the entry of glucose into enterocytes through the apical cell membrane. The protein is translocated from the cytoplasmic vesicles into the apical membrane to enhance glucose transport. Readers should also bear in mind that the excess sodium ions gained in the enterocytes by the SGLT1 action could be cleared via the Na+/K+ energy dependent pump at the basolateral membrane. To date, several glucose transport proteins in the various tissues have been identified. All of them are mediating a passive facilitated diffusion system across cell membranes without requiring metabolic energy. The GLUT1 and GLUT3 are high affinity glucose transporters and play major role in the brain although they are also present in many cell types. They transport glucose efficiently throughout the normal range of plasma glucose concentration. The GLUT2 is a low affinity glucose transporter mainly located in the pancreas and liver. Unlike the high affinity GLUT1 and GLUT3, GLUT2 function during high plasma glucose levels as well as glucose absorption in the enterocytes following glucose excess after meal. Hence, GLUT2 can serve as a glucose sensor by which pancreatic β-cells and liver cells respond to

B. Normal physiological control of carbohydrate

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2. Glucose metabolism

high glucose concentration to respond by hormonal secretion (e.g. insulin) and glycogenesis, respectively. The GLUT4 is the major glucose transporter in muscles and adipose tissues and its function is highly regulated by hormones such as insulin. The protein is normally stored in intracellular vesicles and is translocated to the plasma membrane upon cellular stimulation by insulin. On the other hand, the glucocorticoids such as cortisol suppress the level of GLUT4 on plasma membrane. The GLUT5 is known to function in the ilium and the liver and known to be primarily transporting fructose. Obviously, more GLUT proteins will be characterized in the future and GLUT6 and GLUT7 are beginning to be understood; in the latter case as glucose-6-phosphate (G6P) transporter into the endoplasmic reticulum within the liver. The roles of GLUT4 in muscles and adipocytes, its functional regulation, and as a therapeutic target in diabetes are extensively discussed in the various sections of this book.

Key fact: Once glucose is released in the small intestine through the action of carbohydrate digesting enzymes, it is taken up to the blood steam via the enterocytes. The entry of glucose to this cells, primarily by SGLT1, and exit via GLUT2 can be targeted by drugs or natural products. Undigested carbohydrates or dietary fibres that physically hinder the absorption of glucose could also lower glucose availability to the blood.

2.4 Glycogenesis Glycogen is the major form of carbohydrate storage in animals or the starch equivalent in plants. When glucose concentration in the blood is elevated, primarily after meal, glycogenesis is initiated in the liver and muscle cells. While the storage of glucose in the liver is to maintain the level between meal, and hence for utilization by the body in general, glucose storage in the muscle is for utilization by the muscle itself. Chemically, glycogen is a highly branched polysaccharide or polymer of glucose. The cascade of events involved in the formation of glycogen from α-D-glucose is shown in Fig. 2.5. The process occurs within the cytosol and is energy dependent that is supplied by the ATP and uridine triphosphate (UTP). The reaction starts by the hexokinase (HK) or glucokinase (GK) enzyme-catalysed reaction that leads to the phosphorylation of glucose to form G6P which is further acted by phosphoglucomutase (PGM) to form glucose-1-phosphate (G1P). A further enzymatic action (glucose-1-phosphate uridylyltransferase) leads to the formation of UDP-glucose (see Fig. 2.5). The key enzyme in glycogen synthesis which is under regulation by various hormonal and metabolic mediators is glycogen synthase (GS). The synthesis of glycogen from UDP-glucose requires further enzyme-catalysed steps: The enzyme glycogen synthase generates a chain of glucose monomers with α(1 ! 4) glycosidic linkages while amylo-α(1,4 ! 1.6)-glucosyltransferase (also called

B. Normal physiological control of carbohydrate

2.4 Glycogenesis

19

FIG. 2.5 Glycogen synthesis. The conversion of glucose to glucose-1-phosphate (G1P) takes place via the G6P intermediate which is further acted by phosphoglucomutase (PGM). The enzyme glucose-1-phosphate uridylyltransferase, also known as UDP-glucose pyrophosphorylase, catalyses the formation of UDP-glucose from G1P and UTP. By using UDP-glucose as a source of glucose, the glycogen chain is elongated by adding one glucose molecule at the reducing end through the action of glycogen synthase (GS).

FIG. 2.6 The role of branching enzymes in the synthesis of glycogen.

amylo-(1,4 ! 1,6)-transglycosylase, 1,4-α-glucan branching enzyme, among others) is what is termed as the branching enzyme that transfers six or seven glucose residues from the growing linear chain (nonreducing end) to a branching α(1 ! 6) glycosidic chain of interior position. The glycogen synthesis requires fragment of glycogen that serves as a primer or nucleus on which more glucose units are added, and while total depletion of glycogen may not be the case, other primers such as protein sources (e.g. glycogenin in the liver and muscle cells) serve as primers. The intrinsic activity of glycogenin as glucosyl transferase is known to add glucose from UDP-glucose to its tyrosyl unit to initiate the reaction and making the glycogen primer. The overall α(1 ! 6) glycosidic chain branching formation is depicted in Figs. 2.6 and 2.7.

B. Normal physiological control of carbohydrate

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2. Glucose metabolism

FIG. 2.7

Schematic presentation of glycogen. The polysaccharides of glucose through α(1 ! 4) linkage and branching via α(1 ! 6) glycosidic bonds lead to highly branched layers of glycogen as an energy storage system. The branching system in glycogen not only increases water solubility but also the terminal residues that are readily acted upon by enzymes (glycogen phosphorylase, Section 2.5) to release glucose when required.

Key fact: As a key enzyme for the synthesis of glycogen, the glycogen synthase is highly regulated by various metabolic regulators. For example, it is activated by insulin released from pancreatic β-cells in response to postprandial increase in blood glucose or the G6P substrate precursor; while glucagon and sympathetic/adrenergic (epinephrine) stimuli inhibit it. It also serve as a therapeutic target for many drugs and natural products. Other therapeutic targets for natural products are the hexokinase/glucokinase enzymes which can be upregulated (expression or activity) to increase glycogen synthesis. Increased glycogen synthesis means reduced level of blood glucose.

2.5 Glycogenolysis The breakdown of glycogen to generate glucose is called glycogenolysis. It occurs in the cytosol of the cell and appear to be the reverse reaction of the glycogenesis: i.e. glycogenolysis occurs during fasting and/or between meals. The glycogen phosphorylase first brakes the α(1! 4) linkages on the outer branches of glycogen. As the enzyme is not active against the α(1 ! 6) bonds, it only degrade glycogen up to the branching points (called a limit

B. Normal physiological control of carbohydrate

2.5 Glycogenolysis

21

FIG. 2.8 The process of glycogenolysis. The breakdown of glycogen through the concerted action of two group of enzymes are shown. Note that the effect of glycogen phosphorylase that acts on α(1 ! 4) glycosidic bonds would be terminated at the α(1!6) branches until debranching enzymes remove this restriction and expose the next series of α(1 ! 4) glycosidic bonds.

dextrin). Hence, the main function of glycogen phosphorylase is to shorten glycogen by removing glucose from the outer core of the glycogen polymer as depicted in Fig. 2.8.

Key facts: Glycogen phosphorylase is activated by calcium ions, epinephrine and glucagon and inhibited by insulin and glucose. It does also serve as an important drug target for the control of glucose release from its storage.

After shortening of the glycogen chains by glycogen phosphorylase, branches are removed by the action of two enzymes. The oligo α(1! 6) to α(1 ! 4) glucan-transferase removes the outer three of the four glucose residues attached at a branch and transfer them to the nonreducing end of another chain, while the exposed α(1 ! 6) branch point is acted by the debranching enzyme, amylo-α-(1,6)-glucosidase. The removal of the branching further allows the phosphorylase enzyme to shorten the glycogen branches (Fig. 2.8). The released G1P through the glycolysis process is converted to G6P by the action of phosphoglucomutase in a reverse reaction of glycogenesis. Finally, removal of the phosphate group from G6P by the action of glucose-6-phosphatase (G6Pase) in the liver and kidney allows glucose to be free to diffuse to the blood stream. In muscles, the G6P goes through glycolysis for energy generation.

B. Normal physiological control of carbohydrate

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2. Glucose metabolism

Key facts: Since diabetes is associated with excess blood glucose level, inhibition of glycogenolysis can be seen as a therapeutic option. Hence, the last reaction step where glucose is released by the enzymatic action of G6Pase is one major target for natural products that is discussed in several sections of this book.

2.6 Gluconeogenesis Gluconeogenesis refers to the metabolic process that regenerates glucose form carbon substrates which are not normally classified as carbohydrates. In fact, the word derived from three words referring to: gluco; glucose, neo; new, and genesis; synthesis. The four most important sources of gluconeogenesis are the pyruvate, lactate, glycerol, and glucogenic amino acid (Fig. 2.9). One major route of glucose synthesis is from pyruvate through a reverse glycolysis pathway. Virtually all cells utilize glycolysis as means of energy generation and the biochemical intermediates therein and the associated pentose phosphate pathway are sources of many biochemical intermediates in the biosynthesis of various mico- and macromolecules. The cascade of reactions in glycolysis and the reverse order synthesis route of glucose production via gluconeogenesis are depicted in Fig. 2.10. The net reaction of glycolysis in generating pyruvate is the production of 2ATP and nicotinamide adenine dinucleotide (reduced form, NADH). Lactic acid is the glycolysis product in

FIG. 2.9 The four major sources of gluconeogenesis.

B. Normal physiological control of carbohydrate

2.6 Gluconeogenesis

FIG. 2.10

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The process of gluconeogenesis via glycolysis or pyruvate pathway. Glucose is utilized for energy generation through the glycolysis or cytoplasmic pathway leading to pyruvate formation. In the reverse pathway, glucose can be synthesized from pyruvate source when glucose concentration in the blood is critically low (e.g. starvation).

B. Normal physiological control of carbohydrate

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FIG. 2.11 Gluconeogenesis from lactic acid sources. Lactic acid as a metabolic end product could also be converted back to pyruvate to initiate glucose formation as depicted in Fig. 2.10.

muscle cells which must be removed to avoid muscle fatigue. Lactic acid is also a product of glycolysis from red blood cells. The chemical reaction involved in the production of lactic acid from pyruvate is shown in Fig. 2.11. By using the reducing agent NADH generated through the glycolysis process, lactate dehydrogenase reduces the ketone functional group of pyruvate at the middle carbon to a hydroxyl functional group. In the liver, lactic acid undergoes the reverse reaction to generate glucose along with ATP and NADH utilization (Fig. 2.11). The HK is an important enzyme that initiates glycolysis through phosphorylation of glucose to yield G6P. The liver and pancreas also have GK that function similarly, and in the liver where excess glucose is sequestered, the process is under the direct control of key endocrine hormones such as insulin and glucagon. As G6P does not pass cell membrane, its entrapment in the cell is a way of sequestering glucose and also a commitment to glycolysis unless it is diverted to other metabolic processes. Most importantly and relevant to our topic, the liver and kidney, where gluconeogenesis occur, G6Pase removes the phosphate group to release glucose. If glycolysis is primarily used to generate pyruvate for the gluconeogenesis, it does not seem economical as little energy is generated from the glycolysis pathway (unlike the TCA cycle) and even more energy is utilized for gluconeogenesis through the reverse reaction. Overall, the gluconeogenesis and glycolysis control is dependent by energy demand and supply. For example, under low ATP or high adenosine monophosphate (AMP) cellular environment (high energy demand) and high level of blood glucose or insulin signalling, the expression of genes for HK increases, i.e. increased glycolysis. On the other hand, when there is low blood glucose or glucagon signalling, the need for glucose production triggers the genes expression for G6Pase (gluconeogenesis). Glycerol is the principal product of fats hydrolysis by lipase and is released along with fatty acids. It should be noted that glycerol is not the principal route of glucose synthesis and utilized under long fasting condition where the glycogen storage is critically low. The key liver enzyme in this process is glycerol kinase that converts glycerol to glycerol-3phosphate (G3P) which is further acted upon by G3P dehydrogenase to give the glucose precursor dihydroxyacetone phosphate (DHAP, Fig. 2.12). As shown in Fig. 2.10, DHAP is a key glycolysis and gluconeogenesis intermediate. Many dietary amino acids can undergo chemical transformations to generate glucose and hence called glucogenic amino acids. The utilization of amino acids from structural proteins like those in muscle cells to generate glucose is extremely rare and could happen if all other alternative sources (glycogen, fats, etc.) are depleted. Most of the amino acids involved go through pathways that generate pyruvate while some go through oxaloacetate intermediate. Other amino acids may contribute to gluconeogenesis via entering the citric acid pathway (Fig. 2.13).

B. Normal physiological control of carbohydrate

2.6 Gluconeogenesis

25

FIG. 2.12 Gluconeogenesis from the glycerol source. Glycerol as a by-product of fat hydrolysis could be used for glucose formation during extreme starvation. DHAP is an important intermediate in the glycolysis and gluconeogenesis process from the pyruvate sources as depicted in Fig. 2.10.

FIG. 2.13 Gluconeogenesis from amino acids source. In rare cases, amino acids may be used in the synthesis of glucose via the various intermediates of the TCA cycle and pyruvate/glycolysis pathway. Amino acids including alanine, cysteine, glycine, serine, threonine, and tryptophan could be used to generate pyruvate while aspartate and asparagine could be used for the synthesis of oxaloacetate. The TCA intermediate, fumaric acid could be obtained from phenylalanine and tyrosine while succinyl-CoA could be obtained from isoleucine, methionine, threonine, and valine. Via the glutamic acid intermediate, α-ketoglutaric acid could be obtained from arginine, glutamine, histidine, and proline. The formation of glucose from pyruvate or oxaloacetate is shown in Fig. 2.10.

B. Normal physiological control of carbohydrate

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2. Glucose metabolism

Key facts: Gluconeogenesis is one major target for therapeutic agents and natural products. It occurs mainly in the liver and kidneys and the key enzymes such as G6Pase, PEPCK, and GAPDH as T2D targets are discussed in several chapters of this book.

2.7 Hormonal regulation of glucose metabolism Being the primary source of energy and the precursor of many biochemical reactions, glucose is a vital molecule that needs to be made available to virtually all cells in the body. As explained in Chapter 4, higher level of glucose concentration within the blood for a prolonged period has deleterious consequences. Hence, the regulation of glucose concentration within the window of the ‘normal range’ must be maintained through intricate balance of numerous hormonal and non-hormonal regulations. The highest amount of glucose in the blood coincide immediately after meal (postprandial) while the lowest point is between meals, and generally a plasma (not the whole blood) concentration of 4–6.7 mM or 70–120 mg/dL being considered as the normal range (see Chapter 1). As with the already explained mechanisms of glucose build-up in the blood (from the gut, storage, and synthesis) and its sequestration in tissues for storage, primarily in the liver and muscles (also adipose tissues, see Chapter 3), its removal for utilization is important. In the latter case, the most metabolically active organs such as the heart and the brain are worth mentioning. In parallel with oxygen consumption, the brain constitutes the site for the majority of glucose consummation accounting to 60–70% of the total glucose metabolism. The brain being poor in ATP storage and unable to utilize fatty acids for energy generation, as they are not even able to cross the blain brain barrier (BBB), only the ketone bodies such as the acetoacetate and hydroxybutyrate are being used as alternative energy source under severe glucose deprivation. The other vital organ that utilize large amount of glucose in the body is the heart. As explained in the previous section, the liver is the main site of glucose storage in the form of glycogen from which glucose is released to the circulation between meals. The liver is also a major site of gluconeogenesis. About 90% of glucose in the blood that does not derive directly from the diet therefore comes from the liver. As explained in the next chapter (Chapter 3), the liver is also the site of synthesis of lipoproteins and is involved in fatty acid and cholesterol metabolism that is critical both as energy source and metabolic regulations. The liver can also utilize alternative energy sources such as fatty acids and amino acids for its own energy generation while sparing glucose and ketone bodies for other most important organs such as the brain. Glucose can be excreted via the kidney and efficient recovery is vital to avoid wastage of this vital energy source. Even though the liver is the major organ for gluconeogenesis, the kidney also contributes to this process especially during prolonged starvation where glucose production is significantly enhanced in the kidney. The various organs involved in the supply and utilization of glucose are depicted in Fig. 2.14.

B. Normal physiological control of carbohydrate

2.7 Hormonal regulation of glucose metabolism

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FIG. 2.14 Major organs involved in the regulation of glucose level in the blood. Most of the glucose in the blood is derived from dietary source from intestinal absorption. Excess blood glucose is taken up for storage primarily by the liver while the skeletal muscles and adipose tissues do also take up and store significant amount. The brain and the heart are the major source of glucose utilization. The kidneys control glucose level via reabsorption mechanism following glomerular filtration. Glucose could also be synthesized in the liver and to some extent in the kidney through gluconeogenesis. The pancreas is a major organ for hormonal regulation of glucose.

As explained in the previous section, skeletal muscles are storing glucose in the form of glycogen, but unlike the liver, this reserve is kept for their own use. In the absence of exercise, skeletal muscles can maintain themselves with fatty acids as primary means of energy source and also can use ketone bodies (as with the brain) as an alternative source of energy in times of starvation. The large pool of protein stored in muscles may also be utilized in times of starvation for energy source. In this case, the released amino acids can be employed by the liver for gluconeogenesis but the muscle’s own storage of glucose remains a vital energy source under exercise condition. With respect to hormonal regulation of glucose metabolism, the pancreas plays major role as it is the site of synthesis of various digestive enzymes and also hormones such as insulin and glucagon. There actions relevant to glucose homeostasis are discussed in the following sections. The pancreas is a major exocrine gland with the vast majority of the cells involved in the production and secretion of digestive enzymes including the amylase along with trypsin, chymotrypsin and elastase. The acinar cells involved in the exocrine function constitute up to 98% of the pancreatic cellular mass. The endocrine function of the pancreas is carried out by the Islets of Langerhans; which is made up of the α, β, δ, and F cells that synthesize hormones unique to the cell types. The β-cells mainly located at the anterior lob but also found in other areas such as the posterior section are the factories of insulin. The α-cells specialise in glucagon production, the δ-cells in somatostatin while the F cells secrete pancreatic polypeptide. The two main endocrine hormones of insulin origin for glucose metabolism are the insulin and glucagon.

B. Normal physiological control of carbohydrate

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2. Glucose metabolism

2.7.1 Insulin 2.7.1.1 Production and release Insulin is produced in the form of a large precursor protein that needs to undergo a serious of processing to release a mature fully functional protein. Once packed into the secretary vesicles, the immature peptide proinsulin is processed by proteases to release insulin along with the inactive fragment C-peptide (Fig. 2.15). After release into the circulation and until degraded by the liver and the kidney, insulin has diverse function in the body. There is evidence to show that proinsulin is also released in small amount and has a weak insulin-like activity. On the other hand, the C-peptide has no activity but with longer half-life than insulin (about 5 min) and its release can be used as a measure of β-cell functional integrity. Structurally, the mature human insulin is composed of two polypeptide chains: Chain A with 21 amino acids and chain B of 30 amino acids (aa) length (Fig. 2.16). Chain A possess 4 cystins two of which at position 6 and 11 make a disulphide bridge within chain A, while FIG. 2.15 The molecular architecture of insulin precursor, proinsulin.

C Peptide

S

S A Chain

S

S

S

S B chain

FIG. 2.16

Structure of the human insulin. The amino acid sequence and the three disulphide bridge of the mature insulin are shown.

B. Normal physiological control of carbohydrate

2.7 Hormonal regulation of glucose metabolism

29

the other two at 7 and 20 positions are involved in inter-peptide disulphide bridges with 7 and 19 positions of B chain respectively. There is an incredible level of similarities between insulins from various animal sources with the position of the three disulphide bonds, the amino and carboxylic terminal amino acids of the A chain, and the carboxyl terminal of the B chain. This structural similarities account for the cross-species biological activity of insulin on the basis of which pigs insulin was employed to treat human diabetics. Insulin has also been shown to have the tendency to dimerise due to hydrogen bonding between the carboxyl-terminal of the B-chains which can also associate into hexamers when induced by metals such as zinc. The polymerization of insulin should also be dependent on its concentrations as lower concentrations in about a micromolar range generally favour the monomeric form. The clinical significance of these insulin multimers has been well understood. The monomers and dimers reach their target easily with the ease of their diffusion to the blood stream, while hexamers are delayed and possess slow onset of action. Hence, a number of recombinant insulins that differ with amino acid sequence at the carboxyl terminal-end of the B chain that allow different diffusion and onset of action have been developed for diabetes therapy. The major β-cell stimulant of insulin release is the elevated glucose level in the blood and the glucose-releasing hormone, glucagon. Once glucose is taken up into β-cells via GLUT2, it induces the release of insulin from packed vesicles. This quick onset of action also triggers the synthesis of fresh insulin through several steps from transcription to translation and post synthesis processing. The role of ATP-dependent potassium channels (KATP) in insulin release from pancreatic β-cells has also been well-established (Fig. 2.17). Following glucose entry into β-cells, glycolysis and the krebs cycle in the mitochondria generate ATP. The intracellular build-up of ATP concentration then triggers the shutting of the ATP-sensitive K+ channels. In the absence of outward flow of K+ ions, the membrane become depolarized which intern triggers the opening up of the voltage-dependent calcium channels. The influx of Ca2+ into the pancreatic β-cells leads to the intracellular calcium build-up (also called phase-1 increase) that further triggers the release of more Ca2+ from the intracellular stores (phase 2). It is the presence of Ca2+ in high concentration within the cytoplasm that triggers exocytosis—movement of secretary granules, they being part of the cell membrane and release their content, insulin and C-peptide. The overall regulatory mechanisms of insulin release from pancreatic β-cells are depicted in Fig. 2.17. Before the glucose concentration in the blood is raised (pre-postprandial concentrations) where typically below 90 mg/dL, potassium efflux is maintained via the KATP channels which keep the membrane polarized leaving the voltage-gated membrane Ca2+ channels closed. In the absence of calcium ions build-up in the cells, exocytosis is inhibited. When glucose is taken up by β-cells via the GLUT2, it undergoes oxidation by glucokinase that also acts as a glucose sensor. The rise in ATP means a closure of KATP channels, leading to membrane depolarization and the opening of voltage-gated Ca2+ channels. The regulation of insulin release also comes from neuronal and hormonal stimuli. This includes a receptor-mediated action via modulation of phospholipase C (PLC) or Adenylyl cyclase (AC) to induce positive influence by acetylcholine (Ach), cholecystokinin (CCK), glucagon, and GLP-1, or negative influence by epinephrine, norepinephrine, and somatostatin. The release of insulin from pancreatic β-cells takes place in two phases with the first rapid phase (5–10 min) constitute the release from the preformed and that already packed into the vesicles located close to the plasma membrane. The second phase is a slow release but can be

B. Normal physiological control of carbohydrate

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2. Glucose metabolism

FIG. 2.17

The process of insulin synthesis and release in the pancreas. The genes encoding insulin are first transcribed into mRNA in the DNA/nucleus (1). The mRNA then moves into the cytoplasm where the translation occurs in the ribosomes (2). The resulting preproinsulin formed is processed in the rough endoplasmic reticulum (RER) to proinsulin which is transported to the Golgi apparatus (3) to be cleaved and release insulin along with the C-peptide; both of which are loaded into the secretary granules (4). Under certain physiological conditions, secretary vesicles release their content out of the cell via exocytosis. AC, adenylyl cyclase; Ach, acetylcholine; CCK, cholecystokinin; GLP-1, glucagon-like peptide-1; PLC, phospholipase C.

sustained for hours and as long as the glucose concentration in the blood is maintained at higher level. Despite the biphasic nature of insulin release has been established over 50 years ago, its exact mechanism is still not known and several arguments including the presence of functionally distinct pools of insulin-containing granules as well as variability in locations of granules within the β-cells have been postulated (reviewed by Wang and Thurmond, 2009). The stimulatory effect of some amino acids such as leucine and arginine; intestinal hormones including the gastrin inhibitory peptide and CCK, and β-adrenergic agonists are also documented. Among the well-known negative modulators are cortisol, somatostatin, and catecholamines through actions on the α-adrenergic receptors that seems to override the opposite effect of β-adrenergic agonism. Hence, the stimulatory effect of β-adrenoceptor agonist may not be physiologically relevant for endogenous catecholamines.

Key facts: ATP-sensitive potassium channels which are unique to β-cells play key role in the glucosedependent insulin release from the pancreases. Interestingly, drugs such the sulfonylurea which has receptors (SUR1) associated with this channel mediate insulin release by modulating (switching of ) KATP channels.

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2.7.1.2 Mechanism of insulin action Insulin has diverse biological functions through multiple effects on tissues and organs. Its effect both under normal physiological and disease conditions need to be seen as that initiated through activation of its specific receptors located in almost all mammalian tissues. The density of insulin receptors (IR) in target organs varies and those organs such as the liver and adipose tissues appear to possess the highest density of IR. With over >300,000 receptors/ cell, insulin can induce action at concentrations less than a nanomolar or less concentrations. Upon binding with their agonist, IR can change their conformation to accelerate the rate of reaction and hence could be regarded as allosteric enzymes. First, the receptor is a large cylindrical tetrameric protein composed of two alpha and two β-polypeptide chains linked together by disulphide bonds. Second, the receptor has an intrinsic enzymatic activity with the β-subunit associated with tyrosine kinase activity that is regulated by the α-subunit upon binding with insulin. Hence, upon binding of the α-subunit with insulin, the kinase activity of the β-subunit is stimulated. Phosphorylation of the β-subunit intern recruits the insulin substrate (IRS) proteins that leads to a cascade of signal transductions. The insulin receptor substrate 1 (IRS-1) and ISR-2 are heavily involved in the metabolism of glucose and are discussed in the various chapters of this book along with the various modulators of insulin signalling such as natural products. Therapeutic targets including signal transduction events associated with IR activation also include several kinases, phosphatases, and transcription factors. A further note related to inulin signalling is that prolonged signalling of insulin receptors by high insulin concentration could lead to receptor desensitization and/or downregulation which are common features in T2D. As explained in the previous sections, the GLUT4 which is the main glucose transporter in skeletal muscles and adipose tissues is stored intracellularly and its translocation is upregulated by insulin (Fig. 2.18). The signal transduction pathway for insulin depends on the cell type and function. Among the best established regulators in its signal transduction pathway is the PI3K-Akt pathway that has diverse function in cellular metabolism, cell survival and growth. The key proteins involved in this process are the phosphatidylinositol 3-kinase (PI3K) and Akt or Protein kinase B. As a kinase, the PI3K phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) to yield PIP3 which further bind and activate 3-phosphoinositide-dependent protein kinase-1 (PDK1). PDK1 is known to phosphorylate and activate protein kinase C (PKC) and Akt that initiate GLUT4 translocation. The activation of Akt is also linked to the various known regulatory functions of insulin such as glycogenesis, gluconeogenesis, glycogenolysis, lipogenesis, protein synthesis, and cell survival. The mitogen-activated protein kinase (MAPK) system is also involved in insulin signalling related to cell growth and diverse other functions. These signal transduction pathways as targets for insulin and insulin like action by various drugs and natural products are discussed throughout the various chapters of this book. In relation to glycogen synthesis stimulation by insulin, activation of Akt leads to the phosphorylation and inactivation of glycogen synthase kinase 3 (GSK3). As discussed in the previous sections, one of the key enzymes in glycogen synthesis is GS which happen to be the substrate of phosphorylation and inactivation by GSK3. Hence, inactivation of GSK3 by insulin signalling via Akt is one mechanism of glycogen synthesis upregulation. The mammalian target of rapamycin (mTOR) is another key regulator of insulin signalling but mostly involved in the protein synthesis cascade that is not discussed in detail in this book.

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FIG. 2.18 Insulin signalling pathway. The binding of insulin with its cell surface receptors triggers a signal transduction pathway that leads to diverse biological function. In muscle cells and adipocytes, insulin triggers GLUT4 translocation from its internal storage to the cell membrane leading to glucose influx. The signal transduction pathway involved in insulin signalling is shown. Akt, protein kinase B; FOXO1, Forkhead box O1; GSK3, glycogen synthase kinase 3; IR, insulin receptor; IRS, insulin receptor substrate; mTOR, mammalian target of rapamycin; PDK1, 3-phosphoinositide-dependent protein kinase-1; PI3K, phosphatidylinositol 3-kinase; PIP2, phosphatidylinositol-4,5-diphosphate; PIP3, phosphatidylinositol (3,4,5)-triphosphate.

Gluconeogenesis mechanism being one major target of diabetes therapy, the Forkhead box O1 (FOXO1) which is also involved in lipogenesis by activating key genes is another target for many natural products and will be further discussed in this book. Other major targets of diabetes therapy related to insulin signalling are the negative regulators, of which, protein tyrosine phosphatase-1B (PTP1B) is by far the most emphasized in this book. By dephosphorylating activated IR and IRSs, PTP1B ameliorate insulin action and its dominant presence in diabetes makes it a key target by pharmacological agents. Further reading on insulin signalling are available from excellent reviews on the topic (e.g. Boucher et al., 2014; Huang et al., 2018; Petersen and Shulman, 2018).

Key facts: Downregulation of insulin signalling is a feature of diabetes and/or insulin resistance. Agents that mimic insulin action or upregulate the expression and/or activity of insulin signalling molecules such as IR, IRS, PI3K-Akt pathway have potential to treat diabetes and associated diseases. Similarly, removing the negative regulators of insulin signalling that are highly prominent in diabetes (e.g. PTP1B) confer antidiabetic effect.

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2.7.1.3 Summary of insulin action in major target organs or tissues Liver • • • • • • • •

Stimulates glycogen synthesis Inhibits glycogen breakdown Stimulates glycolysis Inhibits gluconeogenesis Stimulates lipid synthesis and release Stimulate protein synthesis Inhibits the breakdown lipids and proteins Increases fatty acid synthesis

Muscle • • • •

Stimulates amino acid uptake and protein synthesis Stimulates glucose uptake Stimulates glycogen synthesis Stimulates GLUT4 translocation

Adipose tissue • Stimulates glucose uptake • Stimulates the amount of lipoprotein lipase (release of free fatty acids from circulating lipoproteins) • Stimulates synthesis of glycerol-phosphate (required for triacylglycerol synthesis) • Stimulates hormone-sensitive lipase (crucial enzyme for triacylglycerol breakdown) • Promotes lipid synthesis • Inhibits lipid degradation Brain • Glucose uptake in the brain not dependent on insulin • May have other effects such as behavioural and hormonal control in the CNS

2.7.2 Glucagon and related peptides The glucagon is a peptide product of the α-cells of the pancreas which in many respects functionally oppose insulin (pancreatic β-cell hormone) in response to glucose. As discussed in the preceding section, insulin is an anabolic hormone that promotes the incorporation of glucose into vital organs such as the liver, muscle, and adipose tissue and promotes the synthesis of macromolecules (glycogen, fats, and proteins). In the opposite direction, glucagon promotes catabolic processes that increase glucose level in the blood stream via stimulation of glycogenolysis and gluconeogenesis among other effects. The delicate balance between insulin and glucagon levels could thus determine glucose concentration in the blood at a given time. A persistent excess glucagon and shortage of insulin, for example, could lead to hyperglycaemia conditions or diabetes. As with insulin synthesis discussed in the preceding section, the synthesis of glucagon starts with the production of preproglucagon protein which is processed to yield different protein

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2. Glucose metabolism

products depending on tissue types. In the pancreatic α-cells, the precursor protein for glucagon and related peptides is 160 amino acid (aa) long that is cleaved to give rise to glucagon (29 aa peptide) and others including the glucagon-like peptide-1 (GLP-1), GLP-2 and the glicentinrelated peptide. The release of the whole intact proglucagon (with aa sequence including GLP-1 and GLP-2) from pancreatic α cells is also known but the presence of other amino acids in this fragment appear to mask the biological activity of the peptide (GLP-1 and GLP-2). As discussed later, GLP-1 and GLP-2 are cleaved in the intestine from the proglucagon in their biologically active forms while glucagon released with masking amino acids is inactive. Hence, tissue specific post-translational processing of the same gene products leads to glucagon and related peptides with different tissue-specific hormonal products. The release of glucagon from the α-cells is stimulated by a variety of neuronal and hormonal stimuli (Fig. 2.19). Low plasma glucose level, catecholamines, and glucocorticoids are among the stimulators while glucose, insulin, and somatostatin are among the inhibitors. In what appears to be the opposite effect to insulin, glucagon acts on its receptors to elevate the intracellular cAMP levels. The function of glucagon is not as diverse as insulin and mainly manifested in the liver. It stimulates liver amino acid uptake, gluconeogenesis, and glucose release; and inhibits glycolysis and fatty acid synthesis. It is also worth noting that other hormones do play a role in the regulation of glucose metabolism. In addition to regulating glucagon and insulin release, epinephrine stimulates liver gluconeogenesis, muscle glycolysis, and glycogen breakdown. Similarly, glucocorticoids from adrenal cortex under stress conditions stimulate gluconeogenesis and glycogen synthesis in the liver, while reducing glucose uptake in muscles and adipose tissues.

FIG. 2.19

Regulation of glucagon release from pancreatic α-cells. Being highly dependent on glucose for an energy source, the brain regulates directly and indirectly the release of glucagon from α-cells. Stimulation of the adrenal medulla by the sympathetic neurons as a direct response of the brain to hypoglycemia results in the release of the hormone epinephrine. Likewise, the need for glucose during exercise stimulates epinephrine release from adrenal medulla. The pancreatic α-cells are also under the direct control of the sympathetic nervous system and stimulation of the neurons leads to norepinephrine release. These hormonal (epinephrine) and neuronal (norepinephrine) stimuli trigger glucagon release while at the same time suppressing insulin release.

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2.7.3 Other hormones regulating glucose level As discussed in the preceding section, the effects of catecholamines, epinephrine and norepinephrine, glucocorticoids, and growth hormone on glucose control mirror that of glucagon. Hence, they all act to raise the plasma glucose levels that generally oppose the action of insulin. The other two hormones worth mentioning are amylin and incretins (see the following section). 2.7.3.1 Amylin Also known as the islet amyloid polypeptide (IAPP; also islet/insulinoma amyloid polypeptide or diabetes-associated peptide), amylin is a 37 aa-residue that is secreted by pancreatic β-cells along with insulin in the ratio of approximately 100:1. As with insulin, amylin is synthesized as a precursor immature larger peptide: initially the 89-amino acid precursor undergoes two post-translational modifications. The 67 aa; proamylin after processing by proteases give the mature amylin whose amino acid sequence has been shown to be closely related to the calcitonin gene-related peptide (CGRP). Its co-secretion with insulin means that amylin is released during the spike in plasma glucose concentration right after meal. The glycaemic control of amylin is mediated through its action in slowing the gastric emptying and promoting satiety through central effects. Its main peripheral effect is in suppressing glucagon release. Hence, the postprandial spikes in blood glucose levels is suppressed by amylin. The effect of amylin is mediated through its receptors which belong to the large superfamily of cell surface G-protein-coupled receptors that have been under intense research during the last two decades. Readers may find a review articles by Hay et al. (2015) a useful reference material for amylin’s physiology, pharmacology, and clinical potential.

Key facts: One important feature to note here is that amylin is deficient in diabetes. Hence, amylin analogues such as pramlintide acetate (Symlin; AstraZeneca, Wilmington, DE) have clinical applications as antidiabetic drugs.

2.7.3.2 Incretins Incretins represent the group of hormones released to induce a decrease in blood glucose levels and hence supplement the action of insulin. They also have many other effects both centrally and peripherally. The two major incretins of interest to our topic are the intestinal peptides glucagon-like peptide-1 (GLP-1) and gastric inhibitory peptide (GIP; also called, glucose-dependent insulin tropic polypeptide or GIP). As discussed in Section 2.7.2, they are members of the glucagon peptide superfamily and are formed from the post-translational processing of the precursor preproglucagon protein. GLP-1 is a 42 aa long polypeptide produced by intestinal L-cells. The peptide is closely related to the glucagon of the pancreatic α-cells origin. In fact, it is a product of posttranslational modification of proglucagon, by a protease enzyme, prohormone convertase 1 (PC1) which is specific to GLP-1 synthesis in intestinal L-cells. Hence, the expression of the proglucagon gene is similar in both the pancreatic α-cells and in the intestinal L-cells and what leads to a different structurally and functionally

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different polypeptides is posttranslational processing in the two tissues. The whole length of the 42 aa in GLP-1 is still not required for biological activity and the cleavage of the first six amino acids from the N-terminus in the secretary vesicles liberate the functionally active compound. The two most predominant bioactive products released from the vesicles are the GLP-1 (7–36)-NH2 (the predominant form; about 80% of truncated GLP-1 forms) and GLP-1 (7–37) (about 20%). The release of GLP-1 is triggered by food intake and its function appear to suppress glucose level in the blood. Hence, the concentration of GLP-1 between meals is at its lowest. It is believed that the physical presence of food within the microvilli environment of the L-cells triggers GLP-1 secretion and may not be under direct control of neuronal signalling for insulin secretion. The general function of GLP-1 in glucose metabolism is depicted in Fig. 2.20 and is mediated via activation of its receptor (GLP-1R). Among the vital organs that have been shown to have GLP-1R are the pancreatic islet, brain, heart, intestine, kidney, liver, lung, and stomach. The effect of GLP-1 in fat and muscle tissues through indirect mechanisms (see Fig. 2.20) has also been suggested as many species have been shown to have no apparent GLP-1R expressed on the surface of these cells. Among the various functions are β-cell proliferation, increased heart rate, suppression of food intake, and many others listed in Fig. 2.20. The main drawback of using GLP-1 as a drug lies in its very short half-life (1.5 min) as it is rapidly degraded by the proteolytic action of dipeptidyl peptidase IV (DPP-IV, also named adenosine deaminase complex in G-protein or CD26 or cluster of differentiation 26) expressed on the surface of most cell types. The enzyme cleaves GLP-1 between alanine and glutamic acid at positions 8 leading to the formation of the inactive GLP-1 (9–36). Owing to the antidiabetic therapeutic potential of this peptide, a number of successful attempts in the

FIG. 2.20 General function of GLP-1. Once released from intestinal L cells in the GIT, the diverse function of GLP-1 is through hormonal effects by activating its receptor.

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search of novel DPP-IV inhibitors and stabilization of GLP-1 through substitution of some amino acid residues have been made. For example, It has been demonstrated that replacing alanine by valine at position 8 does not affect its biological effect but increased its half-life; though only to about 4–5 min). Good example of DPP-IV resistant long acting GLP-1 mimic developed through such approach is liraglutide (half-life of about 11–13 h). Exenatide is another example of DPP-IV resistant peptide (half-life of about 3–4 h) that has been originally isolated from a lizard. Although unwanted side effect including loss of activity after prolonged usage has been reported, these drugs are now effectively employed to treat T2D (see Chapter 5). Examples of other antidiabetic drugs that work via inhibiting DPP-IV include saxagliptin, sitagliptin, and vildagliptin. The diverse function of the enzyme in the various cell types however is still a major issue in suppressing its activity in long-term therapy. Pharmacological approach on GLP-1 based therapy of T2D has been reviewed by various groups (e.g. Cheang and Moyle, 2018) As with the GLP-1, the GIP is also a 42 aa long peptide but produced by the enteroendocrine K-cells predominantly located at the proximal region of the small intestine. It does also increase the glucose-induced secretion of insulin from pancreatic β-cells. The receptors for the GIP are also widely expressed including in the brain, heart and adipose tissues. With respect to T2D, it can also be seen that a longer half-life of GIP analogue could increase insulin secretion and hence offer antidiabetic potential. More importantly, the insulinotropic effect of GIP appears to be impaired in T2D. On the other hand, activation of the GIP receptors on adipocytes could lead to lipid accumulation and hence receptor antagonists may be used as weight loss strategy and improving insulin sensitivity under obesity conditions. The inactivation of the GIP is similar with GLP-1 and occur by cleavage with DPP-IV. More research on the role of GIP in diabetes and obesity are however needed to prove the potential of peptide mimics and receptor modulators as therapeutic agents.

Key facts: In addition to being a major site of carbohydrate digestion, the GIT is now regarded as a main source of endocrine hormones involved in carbohydrate and lipid metabolism. The GLP-1 and GIP are classical examples and pharmacological agents that mimic their action or prolong their half-life offer antidiabetic potential.

2.8 The AMP-activated protein kinase (AMPK) and cellular energy balance regulation One key metabolic regular of relevance to T2D is the AMP-activated protein kinase (AMPK). Numerous therapeutic agents and natural products discussed in this book act via augmenting the AMPK level and/or activity. Being sensitive to the metabolic energy level, the AMPK serves as a cellular metabolic sensor. As the level of ATP is depleted leading to the buildup of cellular AMP, AMPK is activated to induce diverse cellular function. Hence, its action as a metabolic sensor is based on its ability to detect the change in the AMP:ATP ratio. B. Normal physiological control of carbohydrate

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The AMPK is a trimeric protein complex composed of α, β, and γ subunits. With its binding site for AMP, the γ-subunit endows the ability of AMPK to sense the shift in AMP:ATP ratio. Upon binding with AMPK, the α-subunit undergoes conformational changes for activation to allow phosphorylation at threonine-172 position. The γ-subunit also binds with ATP through which threonine-172 dephosphorylation occur by allowing access to phosphatases. On the other hand, binding with AMP or ADP blocks access to phosphatases and hence lead to AMPK activation. The presence of different isoforms of the three AMPK subunits in various tissues or organs gives rise to different tissue-specific functions. By interacting with the α- and β-subunits, the calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) acts as an upstream kinase to phosphorylate the AMPK at the threonine-172 of the α-subunit. The major activator of CaMKK2 is calcium ions and could lead to AMPK activation irrespective of the status of AMP:ATP ratio. On the other hand, the allosteric activation of AMPK through AMP binding is via activation of the other upstream kinase, liver kinase B1 (LKB1, also known as Serine/Threonine Kinase 11—STK11). Hence, high concentration of AMPK leads to AMPK activation while high level of ATP inactivates it. As shown with classical example of bitter melon extracts and compounds (see Chapter 8), many natural products as well as antidiabetic agents (e.g. metformin and thiazolidinediones) exert their antidiabetic effect by activating AMPK. The AMPK is also activated by a variety of metabolic states such as oxidative stress and its primary function is to conserve energy by switching of anabolic processes that consume ATP, while augmenting catabolic pathways that produce ATP. The various functions of the AMPK activation discussed in this book include: • • • • • • • • • •

Stimulation of glucose uptake in skeletal muscle through GLUT4 upregulation; Stimulation of glycolysis in cells; Inhibition of glycogen synthesis and increased glygogenolysis; Inhibition of gluconeogenesis and lipid synthesis in the liver while increasing lipid oxidation; Inhibition of protein synthesis; Activation of autophagy; Stimulation of mitochondrial biogenesis and lipid oxidation in skeletal muscles; Activation of antioxidant defenses; Decrease in lipolysis and lipogenesis in adipose tissues; Decrease insulin secretion from pancreatic β-cells.

The role of AMPK in metabolic regulation under normal and pathological conditions as well as therapeutic targeting are reviewed in the various literature (e.g. Kjøbsted et al., 2018; Li et al., 2019; Long and Zierath, 2006; Umezawa et al., 2017).

References Boucher, J., Kleinridders, A., Kahn, C.R., 2014. Insulin receptor signaling in normal and insulin-resistant states. Cold Spring Harb. Perspect. Biol. 6(1), a009191. Cheang, J.Y., Moyle, P.M., 2018. Glucagon-like peptide-1 (GLP-1)-based therapeutics: current status and future opportunities beyond type 2 diabetes. Chem.Med.Chem. 13 (7), 662–671. Hay, D.L., Chen, S., Lutz, T.A., Parkes, D.G., Roth, J.D., 2015. Amylin: pharmacology, physiology, and clinical potential. Pharmacol. Rev. 67 (3), 564–600.

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References

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Huang, X., Liu, G., Guo, J., Su, Z., 2018. The PI3K/AKT pathway in obesity and type 2 diabetes. Int. J. Biol. Sci. 14 (11), 1483–1496. Kipnes, M.S., 2011. Sodium-glucose cotransporter 2 inhibitors in the treatment of type 2 diabetes: a review of phase II and III trials. Clin. Investig. 1, 145–156. Kjøbsted, R., Hingst, J.R., Fentz, J., Foretz, M., Sanz, M.N., Pehmøller, C., et al., 2018. AMPK in skeletal muscle function and metabolism. FASEB J. 32 (4), 1741–1777. Li, X., Liu, J., Lu, Q., Ren, D., Sun, X., Rousselle, T., et al., 2019. AMPK: a therapeutic target of heart failure-not only metabolism regulation. Biosci. Rep. 39 (1) 0 (pii: BSR20181767). Long, Y.C., Zierath, J.R., 2006. AMP-activated protein kinase signaling in metabolic regulation. J. Clin. Invest. 116 (7), 1776–1783. Musso, G., Gambino, R., Cassader, M., Pagano, G., 2012. A novel approach to control hyperglycemia in type 2 diabetes: sodium glucose co-transport (SGLT) inhibitors: systematic review and meta-analysis of randomized trials. Ann. Med. 44, 375–393. Petersen, M.C., Shulman, G.I., 2018. Mechanisms of insulin action and insulin resistance. Physiol. Rev. 98 (4), 2133–2223. Umezawa, S., Higurashi, T., Nakajima, A., 2017. AMPK: therapeutic target for diabetes and cancer prevention. Curr. Pharm. Des. 23 (25), 3629–3644. Wang, Z., Thurmond, D.C., 2009. Mechanisms of biphasic insulin-granule exocytosis—roles of the cytoskeleton, small GTPases and SNARE proteins. J. Cell Sci. 122 (7), 893–903.

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C H A P T E R

3 Lipid metabolism: Normal physiology, dysregulation under obesity and diabetes, and therapeutic targets O U T L I N E 3.1 Overview of lipids chemistry 3.1.1 Triacylglycerols or triglycerides (TGs) 3.1.2 Cholesterol and steroids

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3.2 Lipid digestion

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3.3 Lipid uptake from the intestine and transport system

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3.4 Utilization of lipids as an energy source

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3.5 Lipids storage

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3.6 De novo fatty acid synthesis

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3.8 Summary of energy homeostasis and hormonal control

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3.1 Overview of lipids chemistry Lipids represent structural groups of non-polar compounds that may serve living organism as energy storage (fats and oils); or by being an integral parts of cell membranes (phospholipids, glycolipids, and cholesterol), or as secondary metabolites like the terpenoids with diverse functions. A number of bioactive compounds involved in signal transduction pathways including in insulin signalling as well as cell-to-cell communications are of lipid origin.

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FIG. 3.1 Basic structures of lipids and triglycerides. The structures of glycerol and palmitic acid as an example of fatty acids representing the ‘R’ group of TG are shown. R1, R2, and R3 represent the three sites of FA esterification.

Lipids in the form of bile acids also play important role in food digestion and absorption in the gastro intestinal tract (GIT). Classical examples of lipids include: • • • • • • • •

Fatty acids (FAs) Triacylglycerols or triglycerides (TGs) Phospholipids Waxes Sphingolipids Glycosphingolipids Isoprenoids Steroids

One common physicochemical characteristic of all lipids is that they are hydrophobic or water insoluble due to the relatively large amount of hydrocarbon skeleton in their structure.

3.1.1 Triacylglycerols or triglycerides (TGs) The most abundant form of lipids, and that related to our topic in this book, come in the form of TGs which are chemically composed of esterified glycerol with FAs (Fig. 3.1). The composition of FAs in TG could vary as glycerol may be esterified with one, two or three different kinds of FAs. In a saturated fatty acid series, for example, the list could be numerous and the various types of fatty acids are listed in Table 3.1. As an energy source in human diet, however, very small number of FA esters of glycerol are utilized. The most common FAs both in plants and animals are of the C16 and C18 carbon length. Fatty acids are formed through the acetate metabolic pathway of biosynthesis starting from the simple 2-carbon building block, acetyl-CoA, precursor. Hence, many natural fatty acids do have an even number of carbon reflecting the number of acetate units that they came from. As shown by the palmitic acid structure in Fig. 3.1, FAs are amphipathic in nature with a long hydrocarbon chain giving them their hydrophobicity and a hydrophilic carboxylic acid end.

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3.1 Overview of lipids chemistry

TABLE 3.1

Some common fatty acids and their nomenclature

Common name

Systematic name

Structure

Carbon numbers

Propionic acid

Propanoic acid

CH3CH2COOH

C3

Butyric acid

Butanoic acid

CH3(CH2)2COOH

C4

Valeric acid

Pentanoic acid

CH3(CH2)3COOH

C5

Caproic acid

Hexanoic acid

CH3(CH2)4COOH

C6

Enanthic acid

Heptanoic acid

CH3(CH2)5COOH

C7

Caprylic acid

Octanoic acid

CH3(CH2)6COOH

C8

Pelargonic acid

Nonanoic acid

CH3(CH2)7COOH

C9

Capric acid

Decanoic acid

CH3(CH2)8COOH

C10

Undecylic acid

Undecanoic acid

CH3(CH2)9COOH

C11

Lauric acid

Dodecanoic acid

CH3(CH2)10COOH

C12

Tridecylic acid

Tridecanoic acid

CH3(CH2)11COOH

C13

Myristic acid

Tetradecanoic acid

CH3(CH2)12COOH

C14

Pentadecylic acid

Pentadecanoic acid

CH3(CH2)13COOH

C15

Palmitic acid

Hexadecanoic acid

CH3(CH2)14COOH

C16

Margaric acid

Heptadecanoic acid

CH3(CH2)15COOH

C17

Stearic acid

Octadecanoic acid

CH3(CH2)16COOH

C18

Nonadecylic acid

Nonadecanoic acid

CH3(CH2)17COOH

C19

Arachidic acid

Eicosanoic acid

CH3(CH2)18COOH

C20

Heneicosylic acid

Heneicosanoic acid

CH3(CH2)19COOH

C21

Behenic acid

Docosanoic acid

CH3(CH2)20COOH

C22

Tricosylic acid

Tricosanoic acid

CH3(CH2)21COOH

C23

Lignoceric acid

Tetracosanoic acid

CH3(CH2)22COOH

C24

Pentacosylic acid

Pentacosanoic acid

CH3(CH2)23COOH

C25

Cerotic acid

Hexacosanoic acid

CH3(CH2)24COOH

C26

Heptacosylic acid

Heptacosanoic acid

CH3(CH2)25COOH

C27

Montanic acid

Octacosanoic acid

CH3(CH2)26COOH

C28

Nonacosylic acid

Nonacosanoic acid

CH3(CH2)27COOH

C29

Melissic acid

Triacontanoic acid

CH3(CH2)28COOH

C30

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Variable length of FAs (short, medium, and large chain); fully saturated as exemplified by palmitic acid; mono or polyunsaturated chains with variable cis/trans geometric isomers, are all available. The chain length of FAs could determine their physical state with those smaller in size appearing as liquids, while higher molecular weight longer chains are solids. Many saturated fats do also appear as solid at room temperature, while the higher degree of unsaturation favours the liquid/oily state. Accordingly, vegetable fats appear as oils (low melting points) as they have higher percentage of unsaturated fatty acids, while animal fats appear as solid (higher melting points). The relationship between saturation and physical state (solid/ liquid forms, melting point, etc.) can be explained by the degree of packing of the various FA chains together. When double bonds are introduced in cis or trans configuration, the shape of the long chain hydrophobic backbone changes hindering the stacking of molecules, i.e. reduced van der Waals interactions, reduced melting point and increased fluidity. Hence, while stearic acid (C18) has a melting point of about 70 °C, oleic acid (C18 with one double bond) has 13 °C. Linoleic acid and γ-linolenic acid with two and three double bonds in the C18 carbon skeleton further have reduced melting point of up to
5 and
11 °C respectively. The kinked structure of the cis configuration making this changes in physical properties is shown in Fig. 3.2. The main subject of this chapter is to review FAs as a source of energy and also to outline what they are in structural terms, how they are processed, transported, and stored in mammalian cells. Hence, the common dietary fat as an energy source and excessive storage leading to obesity are of significance. The polyunsaturated FAs like those from fishes (omega-3 and omega-6) FAs are other forms that are often regarded as good FAs. The classic examples are shown in Fig. 3.3. The polyunsaturated FAs, such as linoleic acid and γ-linolenic acid, are not synthesized in human body and are called essential FAs that we must get from our dietary sources. On the other hand, arachidonic acid which is an integral part of cell membrane and that serve as a precursor for various signalling molecules (e.g. prostaglandins) is synthesized in our body from linoleic acid. Most of the unsaturated FAs do naturally occur in cis forms and hydrogenation reaction to saturate and make them in solid-form could convert some of them into trans state. Margarines and manufactured cooking oils by hydrogenation process are good examples. Foods of these kind are often classified as unhealthy option due to their effect on raising the level of low density lipoprotein (LDL) which is linked to pathologies, while the level of high density lipoprotein (HDL) is lowered. Cholesterol is transported mostly by LDL and raising the level of HDL while suppressing LDL is considered as a viable therapeutic option: i.e. reversing the effect of trans-fats.

3.1.2 Cholesterol and steroids The other important lipid biomolecules belong to the steroidal class that serve as hormones and various signalling functions in the body. In the context of our topic in this book, the chemistry and physiology of cholesterol is important and hence briefly described herein. Being an essential component of cell membranes and key precursor molecule for the synthesis of bile acids, vitamin D and various steroidal hormones, cholesterol has vital role in maintaining structural integrity and metabolic functions of mammalian cells. While the primary source of cholesterol in humans could be dietary sources, it can also be synthesized in the liver and adrenal gland from the simple primary metabolite precursor, acetyl-CoA. This is a multi-step reaction cascade which in many respect is similar with the terpenoids biosynthesis pathway in plants that leads to a 30-carbone precursor squalene (see Chapter 6). The pathway from squalene to cholesterol is B. Normal physiological control of carbohydrate

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FIG. 3.2 Structures of stearic, oleic and linoleic acids. Notice that they all have an 18 carbon skeleton. The cis double bond at C-9 of oleic and linoleic acid distorts their structures and a further cis double bond at C-11 of linoleic acid has even more adverse effect on its structure. The 2- (A) and 3-dimentional (B) structures are shown. The resulting difference in molecular stacking leads to deferent physical properties such as melting points or solid/oil states.

however unique to animals (does not happen in plants) and hence the main dietary source of cholesterol is non-plant sources (meat, meat, poultry, and dairy products). The various key biological targets of steroid synthesis will be addressed in the later sections of this chapter along with natural modulators. The biosynthesis of steroids starts with the B. Normal physiological control of carbohydrate

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FIG. 3.3 Structures of some important omega-3 and omega-6 fatty acids. Notice the carbon numbering starting from the last (omega) carbon in the FA chain.

action of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase that condenses acetyl-CoA with acetoacetyl-CoA. It is worth noting that there are two forms of the enzyme: the cytosolic isoform involved in cholesterol synthesis and the mitochondrial isoform involved in the ketone body synthesis. The conversion of the HMG-CoA to mevalonate by HMG-CoA reductase (HMGR) is the most important rate-limiting step in cholesterol synthesis that is often called the commitment step. This is because HMG-CoA can be utilized for either cholesterol biosynthesis via the HMGR effect or enter the ketogenesis pathway. The enzyme is regulated by negative feedback mechanism and hence steroids as well as glucagon suppress the enzyme through its phosphorylation, while insulin initiates the reverse (dephosphorylation) effect to activate the enzyme. Transcriptional regulation of the enzyme by these hormones is also vitally important in the regulation of cholesterol synthesis. Fundamentally, all terpenoids are constructed from 5-carbon skeletons originated from the basic isoprene units of isopentenyl pyrophosphate (IPP) and dimethylallylpyrophosphate (DMAPP). The polymerization of these isoprene units gives rise to the 10 (monoterpenoids), 15 (sesqueterenoids), 20 (diterpenoids), 30 (triterpenoids), etc. Steroids are derived from the triterpenoids skeleton (squalene), and in animals, cholesterol serve as an intermediate to undergo a serious of other reactions that lead to the synthesis of bile salts, hormones, etc. (see Figs. 3.4 and 3.5). In terms of therapeutic targets, HMGR is a key enzyme to modulate the biosynthesis of cholesterol and drugs such as statins act through inhibition of this enzyme. As outlined above,

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3.1 Overview of lipids chemistry

FIG. 3.4 Biosynthesis of cholesterol in animal cells. Cholesterol synthesis follows the terpenoids biosynthesis pathway in plants until squalene is obtained as the triterpenes precursor. Two units of farnesyl pyrophosphate are dimerised in head-tohead fashion to form squalene. Isopentenyl pyrophosphate (IPP) and dimethylallylpyrophosphate (DMAPP) are the basic 5-carbon building blocks or isoprene units from which all terpenoids are constructed from.

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FIG. 3.5 Synthesis of bioactive molecules in animals from cholesterol. Note the addition of glycine in structures 1 and 3 and taurine in structures 2 and 4.

phosphorylation of HMGR inhibits the enzyme function and is physiologically achieved by activating the 50 adenosine monophosphate-activated protein kinase (AMPK, Chapter 2). In a similar manner, the activation of the protein phosphatase inhibitor-1 (PPI-1) also inhibits HMGR: i.e. removing the action of dephosphorylating enzymes activates HMGR. Moreover, these physiological HMGR reductase inhibitors are activated when they are phosphorylated (e.g. PPI-1) leading to HMGR inhibition. The activated PPI-1 also inhibits phosphatases which inactivates AMPK via dephosphorylation. Hence, activation and inactivation of the HMGR is linked to the activities of AMPK and PPI-1 inhibitor that can also be modulated by drugs. The regulatory process of HMGR activity is depicted in Fig. 3.6. Waxes are esters of saturated FAs and long-chain alcohols. They are common in plants and honeycomb but play little role as energy source or in fat metabolism and/or obesity in humans and hence will not be discussed herein further.

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FIG. 3.6 Physiological regulation of HMGR activity. The reduction of HMG-CoA to form mevalonic acid is a key biosynthesis step in steroids and/or cholesterol synthesis and is regulated by various hormonal, neuronal and metabolic stimuli. Activation of the AMPK by increased AMP:ATP ratio and other stimuli (see Chapter 2) via its phosphorylation leads to inactivation of HMGR. The inhibition of HMGR activity via its phosphorylation is removed by the action of phosphatase enzymes which themselves are under the control of protein phosphatase inhibitor-1 (PPI-1). PPI-1 is activated by phosphorylation and its inactivation via dephosphorylation is promoted by insulin. On the other hand, agents that increase the level of cAMP such as glucagon and epinephrine promote PPI-1 phosphorylation (activation) via protein kinase A (PKA) activation. Activation of PPI-1 has antihypercholesterolaemic potential through inactivation of HMGR.

Key fact: Statins are competitive inhibitors of HMGR—the key rate-limiting enzyme in the biosynthesis pathway of cholesterol. As substrate for the enzyme, statins compete for the binding site of the HMGCR leading to the natural substrate, HMG-CoA, unable to access the enzyme. Inhibition of HMGR is associated with reduced level of blood cholesterol and hence reduced risk of cardiovascular diseases.

3.2 Lipid digestion Dietary fats in the form of TGs are the most energy-dense nutrients providing up to 9 kcal/g as compared to 4 kcal/g for carbohydrates or proteins. The enzymatic digestion of lipids starts

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FIG. 3.7 Enzymatic hydrolysis of triglycerides by the action of lipases.

in the stomach which contain gastric lipase (or acidic lipase). Within the acidic environment of the stomach, the enzyme is efficient in breaking down short to medium-range fatty acid-TGs. In comparison, its action on long FA containing TGs is slow while cholesteryl esters and phospholipids (phosphatidylcholine, PDC) are not hydrolysed by the enzyme. Gastric lipase, though not at optimal capacity, is also known to be active at pH of up to 7 and can still function while food is being moved through the duodenum. The enzymatic removal of the FA from C-3 position of glycerol gives a product slightly more polar that readily form emulsion with other food products. The emulsification process is further enhanced once the pancreatic juice and bile salts are mixed with the food in the duodenum. The most significant digestion of TGs come from the action of pancreatic lipases that liberate FAs from position C-1 and C-3 of the glycerol carbons. Hence, glycerol monoacylated at the C-2 position is the by-product of the pancreatic lipase enzymatic action. The emulsification process by the bile salts facilitates the interaction of the enzyme at the interface between the two phases: oil and aqueous media. The pancreas also produce a colipase which is liberated into the intestine in the form of precolipase form that is acted by the intestinal trypsin. Hence, efficient enzymatic activity of the pancreatic lipase is achieved in the presence of equal amount of colipase as well as bile salts in the intestine. The overall enzymatic hydrolysis of lipids by lipases is shown in Fig. 3.7.

Key fact: One way of limiting the level of lipids/fats in the body is through inhibition of pancreatic lipases activity in the intestine. This limits the availability of fats to be absorbed from the intestine. A number of natural products as well as drugs act through this mechanism.

As with TGs, phospholipids are also digested in the small intestine. It is worth noting that the bile products contain PDC and cholesterol along with bile salts. This PDCs together with the other food emulsions are forming the micelles that are subjected to hydrolysis by the action of phospholipase A2. Although the intestine has some phospholipase activity of its own, this enzyme is also released by the pancreas in its inactive form and is processed by the action of intestinal trypsin. By acting at the syn2 position (C-2 position of the glycerol skeleton) of PDC, the enzyme liberates FAs and lysophosphatidyl choline (Fig. 3.8).

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FIG. 3.8 Enzymatic digestion of phospholipids by phospholipase A2.

FIG. 3.9 Enzymatic action of cholesterol lipase.

Even though most dietary sterols are available in their free form, cholesteryl esters are subjected to hydrolysis by the action of the pancreatic enzyme, cholesterol esterase or carboxyl ester hydrolase. Beyond the cholestryl esters, this enzyme is known to hydrolyse TGs at all the three positions (C-1, C-2, and C-3) of the glycerol esterification sites and also phosphoglycerides as well as the monoactylated pancreatic lipase by-products of TGs (Fig. 3.9). One further important feature of this enzyme is its activation by the bile salts such as glycocholate that promotes conformational change to form aggregation. This process also appears to be resistant to proteolytic action by other intestinal enzymes.

3.3 Lipid uptake from the intestine and transport system As discussed above, the digestive process in the intestine breaks down TGs, phospholipids, and cholestryl esters to give major end-products as FAs, cholesterol, lysophosphatidylcholine, and monoacylated glycerol, among others. These highly non-polar compounds can readily pass cell membrane provided that they reach to the enterocytes by crossing the aqueous barrier. The formation of micelles through the action of bile salts still play key role in the absorption of lipids from the intestine. By incorporating themselves within the micelle, lipids can move through the aqueous phase in vesicular forms and freely cross the cell membrane of enterocytes. Lipid monomers and 2-monoacylglycerol can be absorbed by all regions

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FIG. 3.10 Resynthesis of triglycerides in intestinal cells. Once free FFs and monoacylglycerol enter the cells, they undergo a series of biosynthesis steps to form TGs. The common route of this synthesis reaction is via fatty acyl-CoA reactive intermediate that is added in a sequential manner through the catalytic acylation of glycerol acetyltransferases. Another route of TG formation is via the glycerol phosphate pathway. Phosphatidylcholine is also resynthesized in the enterocytes.

of the intestine although the jejunum appear to be the site for most of the absorption. The absorption process down a concentration gradient could be a passive process but the expression of several lipid-binding proteins in the small intestine that facilitate diffusion has also been reported (Buttet et al., 2014). Once in the cell, the lipid products are re-esterified to be exported into the blood stream in the form of chylomicrons. Within the cytoplasm, lipids mobility is facilitated by lipid-binding proteins which carries the absorbed FAs to the endoplasmic reticulum where they are acted by the acyl-CoA synthase (ACS). The resulting fatty acyl-CoAs are highly reactive and can go through the step-wise synthesis of TGs (Fig. 3.10). The action of mono- and diacylglycerol acyltransferase enzymes allows the synthesis of TGs (Fig. 3.10). The reaction can also go through a stepwise addition of FAs starting from glycerol-3-phosphate (G3P) through the action of glycerol phosphate acyltransferase and phosphatidate phosphatase as shown in Fig. 3.10. Detailed mechanisms of fat resynthesis in intestinal cells through the monoacylglycerol acyltransferase or G3P pathways have been published (Shi and Cheng, 2009; Yen et al., 2015). The monoacylglycerol acyltransferase synthesis route appears to be the predominant route accounting for up to 80% of the resynthesized TGs.

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FIG. 3.11 Pictoral representation of the chylomicron. The structure include imbedded apolipoproteins (such as ApoA, ApoB, ApoC, and ApoE) into the phospholipid and cholesterol outer surface. The highly nonpolar TGs and cholesterol esters are loaded at the core (shown in yellowcoloured core).

Once the resynthesis process is complete, lipids are either stored in the cytoplasm of the enterocytes as droplets or assembled in chylomicrons (CMs) for secretion to the blood stream (Fig. 3.11). The CMs contain TGs and cholesteryl esters at their core which is surrounded by monolayer of phospholipids that incorporate cholesterol and proteins. The assembly of the CMs starts from the endoplasmic reticulum (ER) where the synthesis starts followed by processing steps in the Golgi apparatus including packaging into vesicles for exocytosis. The mechanisms of assembly and the various protein and chylomicron components have been reviewed by various authors (e.g. Hussain, 2014). Exocytosis through the basolateralend of enterocytes disposes the lipids into the lymphatic system which moves them into the left subclavian vein. When they rich to their tissue destinations, the enzymatic action of lipoprotein lipase (LPL) acts on the core chylomicron TG to release FAs for cellular uptake. In this regard, the LPL located within the endothelial cell surfaces of capillaries plays vital role in clearing the TGs from CMs. The transport of lipids between the source-organs such as the intestine (dietary source) and the liver (synthesis) and their site of storage (e.g. adipose tissues) or utilization (e.g. muscles) is always a challenge as the nonpolar/hydrophobic compounds are travelling through aqueous media of body fluids (e.g. the blood). This challenge is largely overcome by using lipid transporting lipoproteins. The assembly of the lipids and proteins as a ball-like three dimensional structure (see Fig. 3.11) in this transport mechanism has been an interesting subject of research: • The lipids and protein components that are of hydrophilic nature are assembled to the aqueous-facing outer layer and this include phospholipids head, cholesterol and apolipoproteins (apo). • The nonpolar hydrophobic part of the assembly that include TGs and cholesterol esters are assembled to the inner part of the structure.

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To date, numerous lipoproteins that differ in density, which is a function of protein and lipid contents, are identified and the major five groups are as follow: • • • • •

HDL—high density lipoprotein LDL—low density lipoprotein IDL—intermediate density lipoprotein VLDL—very low density lipoprotein CMs

The density of lipoproteins is associated with the percentage of their protein content. For example, HDL has high protein content and is of high density but it is the smallest lipoprotein in size, i.e. more density is associated with the highest percentage of proteins. Often regarded as the good lipoprotein, the HDL transports lipids from tissues to the liver for excretion, while the LDL transports cholesterol from the liver to the tissues. Hence, the LDL is often associated with various cardiovascular pathologies such as atherosclerosis, while higher level of the HDL is considered to associate with less risks for cardiovascular diseases. The LDL also poses pathological challenges relating to atherosclerotic plaque formation through the distribution of cholesterol from the liver to tissues, especially the endothelium. Conversely, CMs and VLDL are involved in the transport of TGs. As already discussed, the CMs transport TGs of the dietary source released from the intestine to tissues for storage (adipose tissue) and utilization (muscle), and also to the liver where VLDL is formed and released to the circulation. The transport of lipids by the various career lipoproteins is a dynamic process where exchange of lipids and proteins takes place in a highly regulated manner. In the intestinal epithelial cells, the formation of the initial immature chylomicron (also called nascent chylomicron (nCM)), involves the assembly of TGs, phospholipids and cholesterol with apoproteins (apoB48, apoA-I, apoA-II, and apoA-IV). Released to the lymphatic vessels and then to the circulation via the subclavian vein, and hence bypassing the liver, the immature chylomicron receives apolipoproteins such as apoC-II and apoE from the HDL leading to the formation of the mature chylomicron (mCM). In target organs such as the muscles and adipose tissues, the endothelial LPL with its cofactor apoC-II releases FAs while glycerol is returned to the liver and kidneys. The apoA and apoC of mature chylomicron is then transferred to HDL while the remaining chylomicron with its cholesterol esters, apoE, and apoB48 is transferred to the liver for further processing including lysosomal degradation to release of FAs and glycerol. In the liver, TGs and cholesterol assembled with apoB100 to form VLDL and released to the circulation which further gains apoE and apoC-II from HDL as described for the chylomicron. Hence, VLDLs with its composition of lipids (TG, cholesterol, and cholesteryl esters) and lipoproteins (apoB-100, apoC-I, apoC-II, apoC-III, and apoE) is the major transporter of lipids to adipose tissues and muscles. Upon action by LPL and the release of FAs and glycerol, the apoCs are transferred back to the HDL. The remaining VLDL with its apoB-100 and apoE which could be converted or also called IDL is transported to the liver for processing with ApoE is transferred back to HDL. Therefore, the predominant apolipoprotein of LDL is apoB-100 while apoE in LDL is important for its absorption/take-up in the liver. The differential level/activity of LPL in adipose tissue and the skeletal muscle under feeding/starvation conditions is very important factor for determining fat storage or utilization. Postprandial excess is in favour of the pronounced enzymatic activity in adipose tissues, while starvation or exercise promotes more enzyme activity in the skeletal muscle. As B. Normal physiological control of carbohydrate

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55

expected, insulin as anabolic hormone upregulates the LPL expression leading to FAs storage in adipose tissue. It also stimulates FA and TG synthesis in the liver and adipose tissue and inhibits hormone-sensitive lipase (HSLPL) in adipose tissues.

Key fact: The transport of TGs and cholesterol is aided with lipoproteins such as HDL, LDL, as well as several specific apolipoproteins of CMs, among others. Modulating the expression level of these proteins offer therapeutic options to manage obesity or lipid dysregulation.

3.4 Utilization of lipids as an energy source As explained in the previous section, LPL located at the luminal surface of capillary endothelial cells hydrolyses TGs located within the CMs and VLDL resulting in the release of free FA for tissue utilization. The FAs released from the circulation may be utilized as an energy source through mitochondrial oxidation, stored in adipose tissues, or processed by the liver (such as TG synthesis) and excreted as VLDL. As the amount of energy released from FAs is about twice higher than carbohydrates, fat accumulation in adipocytes is an effective system of energy storage in animals. Bearing in mind that the body already store carbohydrates in the liver and muscle cells in the form of glycogen, the storage of fat in adipocytes is indeed reserved for periods of carbohydrate starvation. The oxidation of FAs to generate energy occurs in the mitochondria. First, FAs in the cytosol has to be converted to fatty acyl-CoA as outlined in Fig. 3.12. The fatty acyl-CoA is however not able to pass the inner mitochondrial membrane and hence has to be converted into an ester of the amino alcohol, carnitine: fatty acyl carnitine. In the mitochondrion, fatty acyl carnitine reacts with coenzyme A (CoA) through utilization of ATP to convert it back to fatty acyl-CoA (Fig. 3.12). Promotion of FAs oxidation is a mechanism of action for lipid lowering agents discussed in the various sections of this book. For the β-oxidation process to occur in the mitochondrial matrix, the barrier of the inner mitochondrial membrane must be overcome by using the carnitine transport system. The carnitine shuttle uses carnitine palmitoyltransferases (CPT). These enzymes activity and/or expression level could be upregulated by natural products and the four isoforms of the enzyme are CPT1A, CPT1B, CPT1C, and CPT2. The conversion of fatty acyl-CoA to carnitine conjugates in readiness to pass the inner mitochondrial membrane is a function of CPT1. The distribution or expression of the various CPT1 isoforms is tissue-specific as follow: • CPT-1 or CPT1A occur in the brain, fibroblasts intestine, kidney, liver, lung, ovary pancreas, and the spleen; • CPT-1B predominantly expressed in the heart, skeletal muscle, and testis; • CPT1-C mostly occur in neurons; • Both CPT1 and CPT2 are primarily involved in the transport of long-chain FAs: i.e. transporting palmitoyl-CoA, oleoyl-CoA, and linoleoyl-CoA. On the other hand short or medium-chain FAs of up to 10 carbon length do not need this transport system. B. Normal physiological control of carbohydrate

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FIG. 3.12 The carnitine transport system of FAs entry into the mitochondrial matrix. FAs in the cytosol are subjected to acylation by the action of acyl-CoA synthases leading to their entrapment as fatty acyl-CoA thioesters do not pass cell membrane. By using the carnitine-based transport system, FAs enter into the mitochondrial matrix to undergo oxidation. CACT, carnitine acyl carnitine translocase; CPT, carnitine palmitoyl transferase; MM, mitochondrial membrane.

The transport process of fatty acids through cell membrane is generally through fatty acid transport proteins and the best characterized and that targeted by drugs is fatty acid translocase (CD36). Caveolins and plasma membrane fatty acid binding proteins are also involved, while carnitine acyl carnitine translocase (CACT) transports the carnitine conjugate of fatty acids across the mitochondrial membrane (Fig. 3.12).

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FIG. 3.13 The mitochondrial β-oxidation pathway.

The catabolic process of fatty acy-CoA oxidation in the mitochondria is called β-oxidation and is depicted in Fig. 3.13. As the name implies, the carbon at the β-position of the carbonyl undergoes oxidation to form a ketone. This is a multistep enzyme catalysed reaction, however, and starts by the formation of a double bond between the α- and β-carbons; followed by hydroxylation of the β-carbon; ketone formation at the β-carbon and finally the release of acetyl-CoA. The reaction continues until all the acetyl-CoA (two carbon) units are released from the fatty acid. The acetyl-CoA and NADH produced during β-oxidation are used in the citric acid cycle to generate ATP. Another important feature of fat utilization as an energy source during prolonged carbohydrate scarcity is through ketone bodies formation in the liver. The classic examples of ketone bodies are acetoacetate, β-hydroxybutyrate and acetone that are generated from B. Normal physiological control of carbohydrate

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acetyl-CoA of fatty acid sources. The ketone bodies level in the blood has to be regulated however and abnormally high level (ketosis) could lead to pathological state of ketoacidosis (low pH). This is a direct result of excess acetoacetate and β-hydroxybutyrate which are both acidic and lead to a significant drop in blood pH as commonly observed during prolonged starvation and diabetes. In such scenarios of a pH drop (e.g. to pH 7.1 during metabolic acidosis by ketone bodies), the oxygen carrying capacity of haemoglobin drops along with the depletion of bicarbonates that buffer the acids. An intriguing question is of course why acetylCoA generated from fatty acid oxidation is not going through the TCA cycle in the mitochondria of the liver itself just as with glucose oxidation to generate energy. Oxaloacetate that is required to generate citrate by using acetyl-CoA in the TCA cycle is, however, not available during starvation as it is rather used to synthesize glucose through a reverse glycolysis (gluconeogenesis) pathway. Hence, the need to generate energy through the ketone bodies which is paramount to the body is taking precedence. The sequence of reaction in ketogenesis leading to the production of the three ketone bodies is shown in Fig. 3.14. The production of acetone via the spontaneous non-enzymatic decarboxylation process is of limited significance and is excreted via the lung due to its volatile nature. The other two ketone bodies enter the

FIG. 3.14

Major route of ketone bodies formation.

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FIG. 3.15 Metabolism of ketone bodies in various organs. The production and clearance of ketone bodies in the various tissues are shown. Acetyl-CoA can be generated both from glucose (yellow arrows) and FAs (blue arrows) sources. Ketone bodies derived from FAs metabolism in the liver are used by non-hepatic tissues as an energy source.

circulation and reconverted to acetyl-CoA for energy generation by other tissues. In this case, the conversion of acetoacetate to acetoacetyl-CoA is catalysed by succinyl CoA-acetoacetate CoA transferase through a reaction that simultaneously converts succinyl-CoA to succinic acid. The liver appears to lack this enzyme and meets its energy requirement from glucose (dietary and gluconeogenesis source) while the ketone bodies it produced are utilized by other tissues to generate acetyl-CoA that goes through the TCA cycle. The preferred energy source especially in the brain is undoubtedly glucose but adaptation to this source of energy appears to be the case under prolonged starvation. The overall production and utilization of ketone bodies is depicted in Fig. 3.15.

Key fact: Ketone bodies serve as a source of energy during a period of prolonged glucose/carbohydrate starvation. One major adverse effect of ketone bodies is diabetic ketoacidosis which is most common in type 1 diabetes but also occur in type 2 diabetes (T2D).

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3.5 Lipids storage Lipids are stored in the form of TGs and in the human body, the white adipose tissues (WAT) appeared to be the major storage site. In contrast, the brown fat tissues (BAT) located at the supraclavicular region, deep neck, perineal region, and around the spinal cord of the intercostal regions are known to specialize in energy consumption instated of storage. Hence, a lot of attention has been given in recent years to brown fat tissues metabolism as potential targets for obesity. Through the non-shivering thermogenesis mechanism, BAT produces heat and hence are mainly involved in thermoregulation. One of the characteristic features of the BAT is also the higher mitochondrial content of their cells. They are also rich in iron, which gives them their brownish colouring, and a dense capillaries network as an adaptation to their higher demand for oxygen or dissemination of heat. The BAT also receives sympathetic innervation and their overall role in glucose metabolism and insulin sensitivity are currently under intense scrutiny. As an energy storage organ, the emphasis of this section lies on WAT and approaches in altering WAT to brown tissues by therapeutic agents (e.g. CarmoSilva and Cavadas, 2017; Hu and Christian, 2017; Silvester et al., 2018) and natural products are discussed in the various chapters of this book. WAT are composed of a variety of cells including adipocytes, preadipocytes, endothelial cells and immune cells. During the state of nutritional excess or positive energy balance, WAT store TGs as lipid droplets in adipocytes and the increased stimuli of nutritional excess further leads to an increase in the number of adipocyte (hyperplasia) or an enlargement in their size (hypertrophy). As the number of adipocytes is almost predetermined (probably during pre-adulthood stage), the increase in adipose tissue size or obesity in adulthood is mainly driven by hypertrophy though hyperplasia could still play a role. The localization of WAT has been an interesting area of intense debate with respect to targeting obesity and understanding the physiology of adipose tissues. Their major location appears to be the intraabdominal region around the omentum, the intestines and perirenal areas, and in the form of subcutaneous fat in the buttocks, thighs, and abdomen. They are also often classified as the visceral, muscle, epicardial, perivascular, kidney fat, etc. The accumulation of fat in the various regions has been linked to different pathological conditions: the upper, central, and lower body fat. In the latter case, metabolic complication is not very common. With respect to lipid accumulation in adipocytes, the released free FAs by the action of LPL in capillaries enter WAT via specific transporters (CD36, FATP, FABPpm) and used for TG synthesis. The accumulated TGs as an energy storage are then released by lipolysis when needed as fuel by tissues and organs. As shown later, the mobilization of lipids depends on the assembly of the TG, cholesterol, phospholipids, and other components in adipocytes in the form of lipid droplets. There are also proteins called perilipins associated with the surface of lipid droplets. As shown in the following texts, the regulatory mechanism of lipid mobilization in adipocytes involves the activity of perlilipins (see Fig. 3.17).

3.6 De novo fatty acid synthesis In addition to the direct relevance of dietary lipids in fat accumulation, obesity, and lipid dysregulation, FAs can also be synthesized from other sources primarily from excess B. Normal physiological control of carbohydrate

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FIG. 3.16 De novo synthesis of fatty acids in hepatic cells. Pyruvate and glycerol-3-phosphate (G3P) are products of glycolysis. Acetyl-CoA generated from the TCA cycle in the mitochondria supply citrate which then enter into the cytoplasm to regenerate acetyl-CoA for FAs synthesis. The enzymatic action of the key lipogenic enzyme, acetyl-CoA carboxylase (ACC), followed by fatty acid synthase (FAS) leads to the synthesis of palmitic acid. In addition, elongases and desaturases (e.g. stearoyl-CoA desaturase, SCD) give a variety of saturated and unsaturated (UFAs) long chain (LC-FAs) and very long chain (VLC-FAs) FAs.

carbohydrates. Thus, excess glucose in the liver that may not be stored as glycogen enters into the FA synthesis pathways in hepatic cells. Consequently, the primary driving force of lipogenesis via de novo FAs synthesis is excess carbohydrates. The synthesis of FAs from glucose is presented in Fig. 3.16. Acetyl-CoA is a product of the TCA cycle in the mitochondria and is also formed in the cytoplasm through various metabolic processes including during the conversion of citrate to oxaloacetate. Through the action of acetyl-CoA carboxylase (ACC), malonyl-CoA is formed from acetyl-CoA. A further action by fatty acid synthase (FAS) gives FAs such as palmitic acid with a 16-carbon saturated skeleton as a classical example. In addition, fatty acyl-CoA elongases produce long chain fatty acids, while unsaturated fatty acids could be formed through the action of destaturase enzymes as shown for stearoyl-CoA desaturase (SCD) in Fig. 3.16. All the saturated and unsaturated fatty acids along with G3P incorporation constitute TGs accumulated in fat storing cells. Throughout this book, the effects of natural products on lipogenesis as potential mechanism for antiobesity and/or lipid lowering effects are shown. This is primarily by modulating B. Normal physiological control of carbohydrate

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either the expression of the key lipogenic enzymes at varies levels or direct inhibition of the enzymatic activities. The key transcription factors involved and modulated by natural products include signal transducer and activator of transcription (STAT) family, sterol regulatory element-binding protein-1 (SREBP-1), the liver X receptors (LXRs), peroxisome proliferator activated receptors (PPARα, PPARβ/δ, and PPARγ), etc. Two of the major sites of lipogenesis discussed throughout the book are the liver and adipose tissues. The potential of transcriptional regulation of adipogenesis as therapeutic targets for obesity and associated disease have been extensively reviewed (e.g. Corrales et al., 2018; Guo et al., 2015; Gurzov et al., 2016; Kuri-Harcuch et al., 2019; Shimano and Sato, 2017; Song et al., 2018; Wang et al., 2015, 2017; Xu et al., 2018).

3.7 Hormonal control of lipid metabolism Since both fat storage and lipolysis are highly regulated, the amount of fat we store could be visualized as an imbalance between energy supply and demand. Adipocytes themselves are involved in the regulatory process as they are capable of producing signalling molecules such as leptin, adiponectin, tumour necrosis factor-α (TNF-α), and interleukin-1β (IL-1β). The secretary function of adipocytes is also closely linked to pathologies like diabetes and is discussed in Chapter 4. For example, chronic low grade inflammation in obese subjects can predispose people to T2D. The signalling molecules released by adipocytes are collectively called adipokinins and could be functionally grouped as follow: • Hormones involved in energy balance such as leptin and adiponectin; • Hormones such as adiponectin and resistin that are involved in glucose tolerance and insulin sensitivity; • Proinflammatory cytokines such as TNF-α, interleukin-6 (IL-6) and IL-1; • Proteins including LPL and retinol binding protein (RBP) with key role in lipid metabolism; • Regulators of vascular haemostasis including plasminogen activator inhibitor-1 and angiotensinogen; • Mediators of inflammation and stress responses including haptoglobin and metallothionein. The above list is by no means comprehensive as adipose tissues are known to release over 100 signalling molecules as hormones, cytokines, chemokines, and other mediators. Some of these adipokinenes play major role in insulin sensitivity and resistance as well as many other obesityrelated diseases. For example, leptin and adiponectin increase tissue sensitivity to insulin and stimulate β-oxidation of fatty acids. Under insulin resistance or diabetes, tissues develop resistance to these adipokines too and hence the increased level of leptin in obesity does not lead to improvement in insulin sensitivity, and the level of adiponectin in fact has been shown to decreases with increasing body fat content. For further details on this topic, readers are directed to good review articles (Francisco et al., 2018a, b; Pham and Lee, 2017; Xue et al., 2019). With respect to lipolysis and making FAs from WAT available for other tissues, the hormone sensitive lipolytic enzyme, hormone-sensitive lipase (HSL), in adipose tissue play

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major role. The enzyme is stimulated by adrenalin (epinephrine) and adrenocorticotrophic hormone. On the other hand, the anabolic hormone, insulin, inhibits lipolysis by suppressing HSL. The regulatory mechanism of the hormones is mediated by phosphorylation of the enzyme for activation and dephosphorylation to inactivate it. Under exercise condition and fasting state, catecholamines through the β-adenoceptor-mediated action (activation of adenylate cyclase) stimulate lipolysis. After postprandial nutritional excess, insulin suppress lipolysis by promoting the dephosphorylation of HSL and lowering the intracellular level of cAMP (via activation of phosphodiesterase that breakdown cAMP). The intricate balance of fat storage and lipolysis is depicted in Fig. 3.17. As explained in Chapter 4, the proinflammatory cytokines produced by adipocytes under obesity conditions are persistently higher and leads to insulin resistance. A number of other signalling molecules including catecholamines and even genetic factors play part in the regulation of the rate of supply and storage of FAs in adipose tissues. Another interesting variability in the rate of FAs storage/supply in adipocytes is governed by the anatomical location of the adipose tissues themselves: lower body fat, upper body subcutaneous fat, and visceral (intra-abdominal) fat. It is generally regarded as excess visceral fat is associated with greater risk of cardiovascular diseases than is accumulation of fat in the lower body. The major aspect

FIG. 3.17

Regulation of fat storage and lipolysis in adipocytes. The synthesis of TGs in adipocytes is essentially as explained for intestinal cells and follows the two routes of synthesis. After the reassembly of lipids in in the form of droplets within the cytosol, perilipins (lipid droplet-associated proteins), are also assembled on the surface and stored. The lipolysis process by HSL requires activation via hormonal regulations. This initiates the cAMP signalling cascade that leads to kinase-dependent phosphorylation and/or activation of HSL and lipid droplets via perilipins. The action of HSL liberates FAs and glycerol to the circulation.

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3. Lipid metabolism

of hyperlipidaemia and dyslipidaemia in obese subjects is the hypertriglyceridaemia. The increase in cardiovascular risk with increased level of plasma TGs and correlation between hypertriglyceridaemia and visceral fat (more than the subcutaneous fat) has been well established. As discussed in the preceding texts, the liver is transporting FAs by incorporating them in VLDL and excessive amount FAs undoubtedly lead to a scenario where LPL cannot coup with the release of FA to storage organs. This leaves the clearance of VLDL by macrophages and smooth muscle cells leading to atherosclerosis. It is evident that dyslipidaemia in obesity and the associated risk due to hyperlipidaemia are related to the high level of VLDL.

Key fact: HSL is activated by the fasting hormone (glucagon), exercise, and epinephrine to release FAs as a source of energy. Under fed state, insulin inhibits HSL. During insulin resistance and insufficient insulin (diabetes) cases, activated HSL is not suppressed by insulin resulting in the high blood FAs content. This increases β-oxidation, ketogenesis, and dyslipidaemia. This is in addition to the common problem of diabetes or insulin resistance, hyperglycaemia.

3.8 Summary of energy homeostasis and hormonal control • The hypothalamus in the brain acts as a centre to promote feeding. • The hypothalamus is activated by low glucose (starvation) and adeponectin released from adipose tissues as a result of low fat level. • The hypothalamus is also stimulated by the thyroid hormones T3/T4. • By releasing corticotrophin-releasing hormone (CRH), the hypothalamus activates the pituitary gland to release and adenocorticotrophin-releasing hormone (ACTH) that intern activate the adrenal cortex to release cortisol. Cortisol can also be released in response to starvation or stressful stimuli. As with other glucocorticoids, cortisol acts: • Promote gluconeogenesis in the liver; • Protein breakdown in muscles; • Increase TG breakdown in adipose tissues; • Reduce insulin-stimulated glucose uptake in skeletal muscles. • Via the sympathetic innervation, the hypothalamus also activate the adrenal medulla to release epinephrine. • In response to high fat level, adipocytes release leptin that suppress (negative feedback) the hypothalamic drive. • Glucose starvation stimulates pancreatic α-cells to release glucagon while glucose excess induces the release of insulin from β-cells and suppress the hypothalamic drive to feeding. Further reading material on this topics are review articles by Belfort-DeAguiar and Seo (2018); Greenhill (2017), Fr€ uhbeck et al. (2014), and Lee (2017).

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References

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References Belfort-DeAguiar, R., Seo, D., 2018. Food cues and obesity: overpowering hormones and energy balance regulation. Curr. Obes. Rep. 7 (2), 122–129. Buttet, M., Traynard, V., Tran, T.T., Besnard, P., Poirier, H., Niot, I., 2014. From fatty-acid sensing to chylomicron synthesis: role of intestinal lipid-binding proteins. Biochimie 96, 37–47. Carmo-Silva, S., Cavadas, C., 2017. Hypothalamic dysfunction in obesity and metabolic disorders. Adv. Neurobiol. 19, 73–116. Corrales, P., Vidal-Puig, A., Medina-Go´mez, G., 2018. PPARs and metabolic disorders associated with challenged adipose tissue plasticity. Int. J. Mol. Sci.. 19 (7) (pii: E2124). Francisco, V., Pino, J., Campos-Cabaleiro, V., Ruiz-Ferna´ndez, C., Mera, A., Gonzalez-Gay, M.A., et al., 2018a. Obesity, fat mass and immune system: role for leptin. Front. Physiol. 9, 640. Francisco, V., Pino, J., Gonzalez-Gay, M.A., Mera, A., Lago, F., Go´mez, R., et al., 2018b. Adipokines and inflammation: is it a question of weight? Br. J. Pharmacol. 175 (10), 1569–1579. Fr€ uhbeck, G., Mendez-Gimenez, L., Ferna´ndez-Formoso, J.-A., Ferna´ndez, S., Rodrı´guez, A., 2014. Regulation of adipocyte lipolysis. Nutr. Res. Rev. 27 (1), 63–93. Greenhill, C., 2017. Crosstalk between adipocytes and neurons. Nat. Rev. Endocrinol. 13, 438. Guo, L., Li, X., Tang, Q.Q., 2015. Transcriptional regulation of adipocyte differentiation: a central role for CCAAT/ enhancer-binding protein (C/EBP) β. J. Biol. Chem. 290 (2), 755–761. Gurzov, E.N., Stanley, W.J., Pappas, E.G., Thomas, H.E., Gough, D.J., 2016. The JAK/STAT pathway in obesity and diabetes. FEBS J. 283 (16), 3002–3015. Hu, J., Christian, M., 2017. Hormonal factors in the control of the browning of white adipose tissue. Horm. Mol. Biol. Clin. Investig. 31 (1) (pii: /j/hmbci.2017.31). Hussain, M.M., 2014. Intestinal lipid absorption and lipoprotein formation. Curr. Opin. Lipidol. 200–206. Kuri-Harcuch, W., Velez-delValle, C., Vazquez-Sandoval, A., Herna´ndez-Mosqueira, C., Fernandez-Sanchez, V., 2019. A cellular perspective of adipogenesis transcriptional regulation. J. Cell. Physiol. 234 (2), 1111–1129. Lee, M.J., 2017. Hormonal regulation of adipogenesis. Compr. Physiol. 7 (4), 1151–1195. Pham, T.X., Lee, J.Y., 2017. Epigenetic regulation of adipokines. Int. J. Mol. Sci.. 18 (8) (pii: E1740). Shi, Y., Cheng, D., 2009. Beyond triglyceride synthesis: the dynamic functional roles of MGAT and DGAT enzymes in energy metabolism. Am. J. Physiol. Endocrinol. Metab. 297, E10–E18. Shimano, H., Sato, R., 2017. SREBP-regulated lipid metabolism: convergent physiology—divergent pathophysiology. Nat. Rev. Endocrinol. 13 (12), 710–730. Silvester, A.J., Aseer, K.R., Yun, J.W., 2018. Dietary polyphenols and their roles in fat browning. J. Nutr. Biochem. 64, 1–12. Song, Z., Xiaoli, A.M., Yang, F., 2018. Regulation and metabolic significance of de novo lipogenesis in adipose tissues. Nutrients. 10 (10) pii: E1383. Wang, N., Kong, R., Luo, H., Xu, X., Lu, J., 2017. Peroxisome proliferator-activated receptors associated with nonalcoholic fatty liver disease. PPAR Res. 2017; 2017, 6561701. Wang, Y., Viscarra, J., Kim, S.J., Sul, H.S., 2015. Transcriptional regulation of hepatic lipogenesis. Nat. Rev. Mol. Cell. Biol. 16 (11), 678–689. Xu, P., Zhai, Y., Wang, J., 2018. The role of PPAR and its cross-talk with CAR and LXR in obesity and atherosclerosis. Int. J. Mol. Sci.. 19 (4) (pii: E1260). Xue, W., Fan, Z., Li, L., Lu, J., Zhai, Y., Zhao, J., 2019. The chemokine system and its role in obesity. J. Cell. Physiol. 234 (4), 3336–3346. Yen, C.L., Nelson, D.W., Yen, M.I., 2015. Intestinal triacylglycerol synthesis in fat absorption and systemic energy metabolism. J. Lipid Res. 56, 489–501.

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4.3 Macrovascular diseases

4.2 Chronic complications of T2D and pathological hallmarks 70 4.2.1 Microvascular or microangiopathic damage of the capillaries 70 4.2.2 Diabetes retinopathy 73 4.2.3 Diabetic neuropathy 73 4.2.4 Diabetic nephropathy 74 4.2.5 The diabetic foot ulcer and amputation 75

4.4 Molecular mechanisms of diabetes complications 4.4.1 T2D and advanced glycated end products (AGEs) 4.4.2 Oxidative stress in T2D pathology 4.4.3 Low-grade inflammation as a link between obesity and T2D References

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Before we describe the chronic pathological changes associated with type 2 diabetes (T2D), it is worth noting and revising the common symptoms that are outlined in Chapter 1. Diabetes complications could be both acute and chronic in nature. The acute diabetes complications are by and large related to the diabetes medical emergencies especially ketoacidosis and hypoglycaemia. The formation of ketone bodies and their role as energy source during diabetes are outlined in Chapter 3.

4.1 Acute emergencies The lack of insulin or its inability to induce its normal function means that peripheral cells cannot take up glucose from the blood. A state of starvation is thus set which intern initiates

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the mobilization of stored fats, carbohydrates and proteins to be used as a source of energy by converting them into glucose and ketone bodies. The dysregulation of glucose metabolism due to lack of insulin or its lack of action also means that ketone bodies level in the blood and their utilization as a main source of energy become significant, although the normal functioning of the body cannot be maintained in the long-term just by ketone bodies. This suggests that peripheral tissues are under starvation state despite persistent hyperglycaemia in T2D. The sorry state of the body with ketone bodies which are acidic in nature leading to acute ketoacidosis is one clinical feature of T2D though it is even more prevalent in type 1 diabetes (T1D). The physiological response to cellular starvation of glucose is to produce more glucose from the various sources (see Chapter 2) which further aggravate the persistent hyperglycaemia state of T2D. The excess glucose in the blood also means it cannot be fully reabsorbed by the kidney and hence the appearance of glucose in the urine—sweet urine. Water follows solutes and the excretion of more sugar by the kidney is coupled with excess water loss or urine and hence polyuria. As the physiological response to water loss is excessive thirst, polydipsia manifests as the initial symptoms of diabetes. In terms of medical emergencies, the most common case for T2D is perhaps the state of starvation or hypoglycaemia that could account to the clammy, pale and confused state of patients and could lead to full unconsciousness. The first manifestation of hypoglycaemia is the lack of glucose in the brain which could explain the confusion, unconsciousness and sometimes aggressive behavioural manifestation of the T2D patients.

4.2 Chronic complications of T2D and pathological hallmarks It is common knowledge that T2D is linked to a range of disease conditions such as cardiovascular problems and leads to blindness, kidney failure and limb amputation, etc. In this section we are looking at details of gross macro- and micro-changes that are common at biochemical, cellular, tissues and organ and system levels. A number of biochemical mechanisms including oxidative stress and inflammation play major role in the pathogenesis of macroand microvascular complications by hyperglycaemia associated with T2D. The pathogenic processes of the micro- and macrovascular changes are herein outlined first.

4.2.1 Microvascular or microangiopathic damage of the capillaries The microcirculatory system comprising of the arterioles, capillaries, and venules are the smallest functional unit of the circulatory system (Fig. 4.1). In this network of small blood vessels, often referred as the microvascular bed, oxygen and micronutrient coming from the arteriole side are delivered to tissues, while metabolic wastes are collected by the venule to be transported back to the heart. The ultimate exchange of nutrients/metabolites between tissues and the blood takes place in the capillaries and hence their anatomy and pathophysiological changes both in normal and diabetic states are given a lot more focus herein. Besides trafficking nutrients, gas and metabolites between tissues and the blood, the microcirculation also regulate blood flow to tissues/organs through myogenic responses that can dictate the volume of blood passing through the organ/tissue according to the local need. C. Pathophysiology of type 2 diabetes and therapeutic options

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FIG. 4.1 The microcirculatory system.

The permeability of the microvessels in the trafficking process is regulated by structural features of the endothelial cells (e.g. gap dimensions) and their immediate surroundings such the basement membrane which can considerably vary depending on organ or the microvascular beds. For example, the vascular bed in the kidney (glomeruli), the retina, the myocardium, the skin and the muscle have somehow specialist function requiring some physical or anatomical adaptation specific to their function. The biochemical changes that leads to microvascular damage will be discussed in the later sections and readers should know at this stage that hyperglycemia/diabetes induces not only cellular damage (e.g. endothelial cells) in the microvessels but also increase the development of diabetic microangiopathy. The cellular loss will affect a lot of haemostatic balance maintained by endothelial cells as well as the normal functioning of the vascular bed in nutrient/metabolite exchange between tissues and the blood. Such pathological changes ultimately attributes to hypoxia, wound healing delays, retinopathy and the various clinical features of T2D. The physiological response to hypoxia and diminished nutrient/metabolites exchange in the microvascular bed is neovascularization (e.g. diabetes retinopathy) while the platelet and vascular response to cellular damage could also lead to atherosclerosis. The cellular components of the capillaries are shown in Fig. 4.2 and constitute the endothelial cells monolayer that form the true cellular barrier between the blood and tissues. Endothelial cells are supported by a layer of protective matrix proteins forming the basement membrane. The specific function of the microvascular bed in the organ is governed by the nature of arrangement of endothelial cells in making the cellular barrier or gap junction between cells and the thickness of the basement membrane; both of which regulate materials to be exchanged between the blood and tissues. Small materials are exchanged by passing through the gap between endothelial cells which is very tight in C. Pathophysiology of type 2 diabetes and therapeutic options

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FIG. 4.2 The pericytes and other cellular components of the microvascular system.

the brain (blood–brain-barrier) and large in the kidneys (glomerular capillaries). Right outside the basement membranes are other cellular components of microvessels called the pericytes or mesangial cells forming the rather non-continuous cells which are now proven to play vital role in maintaining the normal function of the system. The key cellular alterations in diabetes are as follow: • The pericytes are group of cells closely associated with microvessels that regulate the physical maintenance of the vasculature. Through paracrine control, they are specifically involved in reparative process such as angiogenesis following endothelial damage. The increased apoptosis and degeneration of the pericytes have been shown to be associated in the common diabetic microangiopathy and/or microvascular damage in the retina, the kidney and the heart. Hence, diabetes hyperglycaemia initiate cellular damage including the pericytes; • Endothelial cells apoptosis is a common feature of diabetes in vital organs such as the eye. The first visible effect of such damage is the leakage of blood components to tissues. Moreover, diabetes is associated with impaired circulating endothelial progenitor cells and enhancing their function (by increasing their number or activity) has been suggested as one area of therapeutic options; • The basement membrane increase in thickness under diabetes condition. Increased production of extracellular matrix proteins, collagen and fibronectin, and of related enzymes (i.e. matrix metalloproteinases (MMPs)); • The above two (cellular loss and basement membrane thickness) restricts exchange of materials between the blood and tissues. Before the basement membrane gets too thickened, however, the barrier changes allow the leakage of content of capillaries into the tissues leading oedema that could alter the normal functioning of the organ. Good examples are the diabetes-associated retinal and macular oedema leading to visual problem and leakage of proteins from glomerular capillaries in the kidney; • Endothelial cell damage removes the paracrine and autocrine haemostasis control leading to cellular adhesion and platelet aggregation; • Microvessels eventually clogged up and blood circulation in the affected region ceased up—‘capillary drop out’;

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• Loss of capillaries leads to tissue ischaemia that promotes angiogenesis as commonly observed in diabetes retinopathy, maculopathy, and neuropathy. The common pathological feature of the above microcirculation abnormalities in diabetes results in the development of complications outlined in the following sections.

4.2.2 Diabetes retinopathy Diabetes retinopathy has been recognized as a major cause of blindness in adults in the developed world. After about 20 years life with diabetes, about 80% of T2D and nearly all T1D patients are expected to have developed some kind of retinopathy. Retinopathy in diabetes is primarily a widespread microvascular disease which includes vitreous haemorrhage, tractional retinal detachment, and endovascular glaucoma. The resulting hyperglycaemia in diabetes in known to be the cause for the micro-angiopathy and associated vascular leakage in the eye that results in diabetic macular oedema and capillary occlusion. The biochemical alterations that emanate from ischaemia and the release of vascular mediators such as the vascular endothelial growth factor (VEGF) triggers cellular proliferation and/or neovascularization. The development of these new blood vessels or neoangiogenesis has a tendency to bleed and vitreous haemorrhage is often accompanied by proliferation of other cell types such as fibroblasts, development of fibrotic membranes, retinal traction leading to retinal detachment. Hence, diabetic maculopathy and retinal ischaemia are two major factors that attribute to vision loss and/or disturbance in diabetes. With respect to the role of angiogenesis and the inflammation processes in diabetes retinopathy, several key cytokines have been proposed as therapeutic targets. Of these, the central role of VEGF has been established and anti-VEGF drugs including ranibizumab have already been approved (Boyer et al., 2009; Arevalo et al., 2010). The interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), and IL-6, are key proinflammatory cytokines that are significantly upregulated in diabetes and also linked to insulin resistance. Inevitably, the adhesion molecules such as intercellular cell adhesion molecule-1 are expressed following proinflammatory cytokines release and are upregulated in diabetic retinopathy (Demircan et al., 2006). As the levels of cytokines such as VEGF and IL-1β increases in diabetic retinas, inflammatory cascades and cellular apoptosis increases as evidenced from experiments that showed intravitreal injection of cytokines such as IL-1β into normal rats could increase retinal capillary cell apoptosis (Vincent and Mohr, 2007).

4.2.3 Diabetic neuropathy As explained in the previous sections, microvessels or capillaries dysfunction leading to tissue ischaemia could lead to acute or chronic peripheral nerve pathology collectively called diabetes neuropathy. A number of clinical symptoms of either focal or diffuse neuropathicorigin, or diabetic-induced demyelination leading to classical symptoms of hyperesthesia and allodynia, and burning, stabbing, shooting pains may be encountered. Other symptoms including numbness which could also attributes to the loss of sensation and eventually foot ulceration are other complications of diabetes. The common symptoms encountered in diabetic peripheral neuropathy are as follow:

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• Pain—Damage to small nerve fibers causes pain. For some, even wearing socks and shoes or the touch of sheets and blankets on their feet at night is excruciating; • Burning, stabbing or electric-shock sensations; • Numbness (loss of feeling)—Damage to large fibers; • Tingling—Damage to large fibers; • Muscle weakness—Damage to large fibers; • Poor coordination; • Muscle cramping and/or twitching; • Insensitivity to pain and/or temperature; • Extreme sensitivity to even the lightest touch; • Symptoms get worse at night.

4.2.4 Diabetic nephropathy Diabetic nephropathy is a classic example of microvascular complications and constitute as the main cause of the end-stage renal disease that govern the morbidity and mortality of T2D. Some estimate suggests that 30–47% end-stage renal diseases trace their cause as diabetes nephropathy. Some 25–40% diabetics are generally expected to develop diabetic nephropathy at some stage (Giullian et al., 2008). The disease is characterized by a relatively higher level of albumen excretion often called proteinuria or microalbuminuria that in a severe case will lead to proteinuria of over 500 mg excretion in 24 h. The clinical stages of diabetic nephropathy include normoalbuminuria, microalbuminuria, overt proteinuria, and finally end-stage renal disease. If untreated, patients with persistent microalbuminuria of 30–300 mg/day could go to what is regarded as an overt nephropathy of over 300 mg/day with a risk of cardiovascular disease development. As the disease progresses, glomerular filtration rate (GFR) declines. Guidelines on the disease classification and assessment are provided by the various government and relevant associations. The American Diabetes association (ADA, 2014), for example advocate screening result of estimated GFR (eGFR) 30 mg/g creatinine as indicators of diabetes kidney disease. Caution for the assessment methods are also given and even the eGFR has its limitations (ADA, 2014). Blood tests for blood urea nitrogen (BUN) and creatinine levels are routinely measured to determine kidney function. The BUN level of 7–20 mg/dL and creatinine of 0.8–1.4 mg/dL are considered normal; with females being considered to have a slightly lower creatinine level (0.6–1.2 mg/dL). Considering 100–150 mL/min GFR as a normal range, eGFR is also routinely employed as a method of assessment for kidney function. Creatinine clearance with 24 h urine collection in conjunction with blood sample analysis may also be used although the reliability of this methodology particularly at the advanced stage of the disease is often in question, i.e. creatinine clearance may not be exactly the same as GFR for various reasons. At the molecular level, diabetic nephropathy also involves cellular death or apoptosis and inflammation that are described in Section 4.4. These include cascades of events following hyperglycaemia such as activation of protein kinase C (PKC), increased reactive oxygen species (ROS) and advanced glycation end-products (AGEs) generation, increased expression of proinflammatory cytokines and transforming growth factor β (TGFβ), enhanced flux into the

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polyol and hexosamine pathways and dysregulation of the mitogen-activated protein kinase (MAPK) signalling pathway, etc. All these mechanisms are associated with microvascular complications including podocyte loss, thickening of the glomerular basement membrane, endothelial cell death, tubular atrophy, dropout, etc.

4.2.5 The diabetic foot ulcer and amputation Around 25% of people with diabetes will develop a diabetic foot ulcer (DFU) during their lifetime and the aetiology of the condition seems to be closely related to the neuropathic, ischaemic and/or neuroischaemic condition that are already described in the previous sections. Hence, diabetic foot ulcer is the combined effect of micro- and macrovascular dysfunction leading to impaired perfusion and ischaemia developed in the limbs. The relationship between peripheral neuropathy and DFU development could be explained as follow: • Loss of sensation means being unaware and/or unresponsive to some physical, chemical or thermal stimuli/injury; • Motor neuropathy like those of hammer toes and claw foot can cause foot deformities; • Autonomic neuropathy could lead to dry skin that is prone to fissures, cracking and callus. The type and prevalence of DFU are further summarized in Table 4.1. Many reports (e.g. NICE, 2016) indicate that more than 80% of limb amputations in diabetic patients is preceded by DFU. About 70% of diabetic people die within 5 years after having amputation, while the figure for those dying within 5 years of developing a DFU is about 50%. The loss of capillaries in the vascular bed of the skin makes it difficult for wound to heal quickly. Wound infection is TABLE 4.1

Typical features of DFUs according to aetiology.

Feature

Neuropathic

Ischaemic

Neuroischaemic

Sensation

Sensory loss

Painful

Degree of sensory

Callus/ necrosis

Callus present and often thick

Necrosis common

Minimal callus prone to necrosis

Wound bed

Pink and granulating, surrounded by callus

Pale and sloughy with poor granulation

Poor granulation

Foot temperature and pulses

Warm with bounding pulses

Cool with absent pulses

Cool with absent pulses

Other

Dry skin and fissuring

Delayed healing

High risk of infection

Typical location

Weight-bearing areas of the foot, such as metatarsal heads, the heel and over the dorsum of clawed toes

Tips of toes, nail edges and between the toes and lateral borders of the foot

Margins of the foot and toes

Prevalence

35%

15%

50%

Source: International Best Practice Guidelines (2013).

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also the major factor contributing to limb amputation in diabetes patients and diabetic foot infection in general is considered to take up to one-quarter of all diabetic hospital admissions in both Europe and the United States (Mendes and Neves, 2012).

4.3 Macrovascular diseases Considered as the primary cause of death in diabetes, the chance of macrovascular damages increased in diabetes by two- to fourfold. The most common macrovascular disorders in diabetes are atherosclerosis and arteriosclerosis. In the case of atherosclerosis, plaques comprising of lipids and fibrovascular tissue (atheroma) are developing in the vessel wall of large arteries. As the plaque increases in size, the lumen of the arteries narrows making perfusion to tissues reduced, and hence leading to ischaemia development. The classical example is the coronary artery disease or angina pectoris where myocardial ischaemia resulted from reduced blood supply to the heart. In the severe case where complete blockade like that by platelet aggregation occur, myocardial infarction or cell death (or heart attack) is the ultimate consequence. In the second example of macrovascular damage, arteriosclerosis, rigidity of the arteries wall or sclerosis means that the blood vessel loses its elasticity leading to an increased blood pressure. Diabetes is also associated with lipid dysregulation and often characterized by hyperlipidaemia and/or hypercholesterolaemia that aggravates the macrovascular dysfunction in diabetes.

4.4 Molecular mechanisms of diabetes complications 4.4.1 T2D and advanced glycated end products (AGEs) One of the best characterized mechanism of diabetic complication is through the nonenzymatic post-translational modification of proteins called glycation reactions that yield products collectively called AGEs. Structurally, the AGEs represent a heterogeneous group of modified biological molecules formed through glycation reaction followed by oxidation, dehydration and/or carbonylation processes. The best characterized route of AGE formation is the so called Maillard reaction whereby the carbonyl moiety of reducing sugars reacts with amino groups of proteins to form a Schiff base. The reaction is non enzymatic, slow and occur in few hours but as the Schiff base product is unstable, it undergoes intramolecular rearrangement to form an Amadori product (Fig. 4.3). Glycated haemoglobin (haemoglobin A1c (HbA1c)) is an example of an Amadori product (from glucose reaction with the N-terminal amino residue of valine on the haemoglobin β chain) that is widely used in clinical practice for diagnosis and regulation of diabetes. The Amadori products themselves undergo a slow oxidation reaction to yield reactive dicarbonyl compounds such as glyoxal, methylglyoxal (MG), 3-deoxyglucosone (3-DG), and deoxyglucosones in a period of a week to months (see Fig. 4.4). The final stage of the glycation reaction involves further oxidation, dehydration, and cyclization steps resulting

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FIG. 4.3 Reaction of glucose to form Amdori product and AGEs.

O

O H

H O

Glyoxal

O CH 3

H O

Methylglyoxal (MG)

OH OH

H O

OH

3-Deoxyglucosone (3-DG)

FIG. 4.4 Common reactive dicarbonyl compounds.

in the formation of the irreversible products that we call AGEs which are yellow-brown in colour, often fluorescent and insoluble adducts. The accumulation of AGEs over a long period leads to a variety of physiological and pathological changes as evidenced in T2D. The AGEs are divided into three main groups: • Fluorescent cross-linked AGEs such as crossline and pentosidine; • Non-fluorescent cross-linked AGEs including the imidazolium dilysine, alkyl formyl glycosyl pyrrole (AFGP) and arginine-lysine imidazole (ALI) cross-links; • Non-cross-linked AGEs such as pyrraline and N-carboxymethyllysine (Nε-(carboxymethyl)-lysine CML). The three best characterized AGEs derived from this glycoxidation process are the pentosidine, Nε-carboxymethyl-lysine (CML) and glucosepane (Fig. 4.5). In addition to the Maillard reaction pathway of the AGEs formation, lipid peroxidation and the glycolysis pathways are also known to be the route of formation of AGEs. Peroxidation of lipids by ROS could lead to the formation of reactive carbonyl compounds that ultimately form AGEs or advanced lipid end products. In the latter case, the marker of lipid peroxidation product, malondialdehyde (MDA), is a classic example. Similarly, the glycolysis pathway involves the oxidation of glucose to yield reactive carbonyl compounds such as methylglyoxal that undergoes a serious of reactions with proteins to yield the AGEs such as hydroimidazolone (MG-H1). The structures of the common AGEs are shown in Fig. 4.5. In summary, AGEs are formed endogenously through three distinct reactions pathways (Fig. 4.6). They can also be introduced into the body by a variety of ways including cigarette smoking which contain reactive glycation products and intake of high-AGE food

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FIG. 4.5 Structures of some common AGEs.

products including those acquired by food processing steps (oven frying or high temperature could induce AGE formation). Hence, significant amount of the AGEs (
10%) are introduced via the food and drinks. The AGEs are metabolized by the liver and excreted by the kidney. Numerous experimental and clinical evidences suggest that the levels of protein bound AGEs and AGE-free adducts are not only elevated in diabetes but also contribute to the micro- and macrovascular damages associated with the disease. The intracellular accumulation of AGEs is mediated via their specific cellular receptors (RAGEs). A number of ligands and receptors of AGEs have also been characterized in various cell types (e.g. smooth muscle cells, macrophages, endothelial cells and astrocytes) and among them are the lactoferrin, scavenger receptors types I and II, oligosaccharyl transferase-48 (OST-48), 80K-H phosphoprotein, galectin-3, and CD36. Moreover, interaction of RAGEs with a number of proteins including amyloid-β peptide (Aβ), β-sheet fibrils, S100/calgranulins, amphoterin and Mac-1

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FIG. 4.6 Summary of AGEs formation inside and outside the cell through the three common routes.

integrins have been observed. Hence, RAGEs are involved in diverse cellular functions via discrete signal transduction pathways. With respect to stimulation of RAGEs by AGEs, nuclear factor-κB (NF-κB) activation and the MAPK signalling pathway have been shown to be involved. This leads to gene transcription for cytokines and other signalling molecules including the endothelin-1, tissue factor and thrombomodulin and IL-1α, IL-6 and TNF-α. Undoubtedly, the expression of proinflammatory cytokines (TNF, IL-1, and IL-6) which are also known to be associated with insulin resistance leads to the expression of adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Hence, high levels of AGEs induce inflammation via activation of proinflammatory cytokines expression and adhesion molecules expression. The other common pathway linking diabetes and AGEs is through the generation of ROS that is implicated in diabetes pathology (see below). Moreover, the stable protein cross-linked products with extracellular matrix (ECM) proteins such as collagen alters the physical and functional characteristics of the proteins leading to the diabetes alterations in the basement membrane and/ or alterations of microvascular system that are discussed in the previous sections, i.e. excessive formation of AGEs can lead to a thickening of the microvessel, hypertension, endothelial dysfunction, loss of pericytes, decreased platelet survival and increased platelet aggregation. Such abnormalities undoubtedly promote procoagulation, tissue ischaemia and induction of growth factors (e.g. VEGF) with angiogenesis and neovascularization outcome. As a summary, Fig. 4.7 depicts the link between AGEs and diabetes.

4.4.2 Oxidative stress in T2D pathology Reactive oxygen species (ROS) is a terminology used in biology, chemistry and medicine to collectively describe oxygen derived radicals such as superoxide anion (O• 2 ), and hydroxyl radical (HO•) and other reactive non-radical species including hydrogen peroxide (H2O2) and

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FIG. 4.7 The link between diabetes and AGEs via oxidative stress and inflammation.

hypochlorous acid (HOCl). In a similar way, we also have reactive nitrogenous species (RNS) to describe the nitric oxide synthase (NOS) product, nitric oxide (NO), that possess multiple biological effects in the body; as well as other related compounds including peroxynitrite (OONO). Under normal physiological conditions, ROS and RNS are continuously produced to induce a variety of cellular functions mediated through a variety of direct effect on biological molecules or through signalling via activation/inhibition of enzymes and genes. In mammalian cell system, the best source of ROS is the cellular respiratory system of the mitochondrial electron transport system where leaked electrons may directly and prematurely reduce molecular oxygen to generate ROS via O• 2 . A number of enzymatic reactions such as the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine and cytochrome p450 systems, the arachidonic acid inflammatory pathway and many other biological processes do also generate ROS. A number of cytokines and death signalling molecules including apoptosis inducers (e.g. TNF-α) are also known to induce the generation of ROS. Both ROS and RNS are also generated through external factors such as drugs, toxins and other stimuli. Exposure to UV irradiation, for example, cleaves H2O2 to generate HO•. As a defence mechanism against microorganisms, white blood cells are employing ROS as a weapon and in this direction the enzyme myloperoxidase convert H2O2 to HOCl. Two of the most physiologically relevant ROS generating systems are further outlined below: 4.4.2.1 The NAD(P)H oxidase system in T2D The NAD(P)H oxidase is an efficient means of ROS generation in white blood cells but also found in vascular cells including podocytes and smooth muscle cells as well as in the kidneys. It sole purpose is to generate O• 2 and cytokines and AGEs which are implicated in diabetes are are known activate NAD(P)H oxidase (NOX) leading to ROS generation. Considering the primary function of this enzyme in neutrophils and macrophages is the ROS-mediated killing

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of microorganisms and therby serving as an inflammatory mediator, decades of research on this enzyme was related to oxidative burst phagocytosis. Several varities/isoforms of this enzyme has been discovered in recent years, however, with extensive variations in tissues distribution under normal and pathological conditions. Of the several isoforms known to date (NOX1, -2, -3, -4, and -5 and dual oxidase 1 and -2), Nox4 is predominantly found in the kidney where it mediate the generation of ROS under basal and pathological condition, particularly the diabetic nephropathy. Even though NOX2 (primarily of the leucocytes phagosomes) is also known to occur in the kidney, upregulation of expression of NOX4 appear to be the main renal oxidative stress and kidney injury that could be targeted by potential modulators. The overall reaction mechanism mediated by NOX is as follow: NADðPÞH + 2O2 $ NADP + + 2 O2  + H + Even though ROS genrated through the NAD(P)H system and others have profound effect in the body, the role played in the kidney with respect to diabetes is worth further scrutny. The kindeny is the second most important organ (next to the liver) for gluconeogenesis, an effect which is inhibited by insulin. In the proximal tubular cells, the regulation of glucose is also critical by reabsorbing filtered glucose into the blood. This glucose metabolism now understood to be regulated by NAD(P)H oxidase system and is dysregulated under T2D. Hence, considerable attention is now given to the modulation of the faulty glucose transport system in diabetic kidney tubules including the sodium/glucose cotransporters (SGLTs, see Chapter 2) across the brush border of the proximal tubules via NADPH oxidase. For extended review of the NAD(P)H oxidase role in kidneys under diabetic conditions, readers may refer review articles in the field (Rhee, 2016; Sedeek et al., 2013). The diverse role of ROS as cell signalling molecules to regulate numerous cellular functions such as cell growth, differentiation, survival mechanisms are also now extended to the NAD(P)H oxidase system. The role of NAD(P)H oxidase in cardiovascular system is also worth mentioning particularly angiotensin II being a known activator of the enzyme. Hence, it is relevant to hypertension and other common cardiovascular complications of T2D: further substantiating the role of ROS in diabetes pathology. 4.4.2.2 The mitochondrial respiratory system Mitochondrial respiratory chain employing complex chains I–III are the major sites of O• 2 generation (Fig. 4.8). The efficiency of mitochondria diminishes with age and pathological conditions such as diabetes suggesting the production of more ROS under disease state and/or old age. In the mitochondrial inner membrane where oxidative phosphorylation takes place, a gradient of proton (between the intermembrane space and inner mitochondrial region) is created as electrons are passed between careers (Fig. 4.8). While the electron being carried is eventually used to reduce molecular oxygen to form water at Complex IV, the proton gradient is used to couple the phosphorylation process to produce ATP. There is however a normal leakage of electron at complex I and III that directly form O• 2 from molecular oxygen. This makes the mitochondria as the major source of ROS that is even highly exaggerated under pathological conditions. The effects of various potential modulators of this ROS formation as antioxidants and/or antidiabetic mechanisms are discussed in the various chapters of this book.

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FIG. 4.8 The mitochondrial oxidative phosphorylation system and ROS generation. The mitochondrial electron transport system undertakes a sequence of oxidation-reduction reactions in four complexes (I-IV). Electrons are transferred from NAD(P)H through the system while the (H+) electrochemical gradient was created by pumping (complex I, II, and IV) it to the intermembrane space. While this electron transport is coupled with phosphorylation via the ATP synthase through the established (H+) gradient, O2 serve as a final electron acceptor at complex IV. On the other hand, a premature reduction of O2 at complexes I and III could lead to O• 2 formation.

4.4.2.3 Antioxidant defences Owing to the diverse pharmacology and toxicological effect of higher level of ROS, their production and elimination must be tightly regulated. As explained throughout this book, higher level of ROS are implicated in diabetes as well as a number of other pathologies. The regulation of ROS include a tight control of transition metals that promote ROS generation as a vast array of diseases including neurodegenerative disorders trace their origin thorough oxidative damage mediated by dysregulation of copper, iron and related transition metals. The best means of ROS regulation however remains through a range of antioxidant defenses as summarized below: Superoxide dismutase (SOD) enzymes primarily the CuZn and Mn isoforms acting on superoxide anion: SOD

2O2  + 2H + ƒ! H2 O2 + O2 Catalase (CAT) act on hydrogen peroxide to convert it to water CAT

2H2 O2 ƒ! 2H2 O + O2 The tripeptide glutathione (GSH) that removes ROS while itself oxidized to form GSSG (see Fig. 4.9). The two key oxidation-reduction enzymes are glutathione peroxidase (GPx) and reductase (GR). A number of dietary antioxidants and other nutritional factors that are discussed in this book also add to the antioxidant defense of the body. Hence, oxidative stress is a state when there is an imbalance between the formation of prooxidants (ROS/RNS) and antioxidant

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FIG. 4.9 Structures of reduced (GSH) and oxidized (GSSG) forms of glutathione.

defenses is in favour of the former, i.e. either the body antioxidant defense is weaker or exceptionally higher level of ROS/RNS are produced under pathological conditions. In T2D, generation as with other hyperglycaemia is directly associated with high level of O• 2 ROS. Hence, the above-mentioned diabetic nephropathy, microvascular injury and cellular death could be a direct result of oxidative stress. Throughout this book, emphasis is given to the role of natural products and other potential antidiabetic agents that modulate pharmacological effects via antioxidant mechanisms. This includes modulating the intricate balance of ROS and RNS generation and elimination as well as their diverse function in the body (Fig. 4.10). The various cellular and subcellular mechanisms including the role of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) are extensively discussed. Related to antioxidant defenses, transcriptional regulations of the cellular redox balance is critically important. As a valid therapeutic target for natural product, the transcription factor, erythroid 2-related factor 2 (Nrf2), is extensively discussed in the various chapters of this

FIG. 4.10 Summary of ROS/RNS generation and their inactivation.

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book. By binding to the antioxidant response element (ARE) of target genes, the Nrf2, regulates the expression of phase II detoxifying enzymes and antioxidants. These enzymes are collectively regarded as cytoprotective proteins and include the following: • Glutamate-cysteine ligase—critically involved in the synthesis of the antioxidant GSH; • GST enzymes at various cellular sites (mitochondrial and microsomal); • Multidrug resistance-associated proteins—membrane transporters for efflux and excretion of drugs and other compounds; • NAD(P)H quinone oxidoreductase 1 (Nqo1)—involved in the reduction and detoxification of highly reactive quinones. It ameliorate oxidative stress; • The UDP-glucuronosyltransferase (UGT)—enzyme for conjugation of drugs with glucuronic acid; • Heme oxygenase-1 (HO-1)—key enzyme in the production of the antioxidant and antiinflammatory biliverdin by breakdown haeme. The Nrf2/HO-1 axis as a regulator of both oxidative stress and inflammation will be highlighted in this book as it is a valid therapeutic target for diabetes and associated diseases. While breaking down haeme to release iron and carbon monoxide (CO), along with biliverdin, HO-1 boosts anti-inflammatory defenses by increasing the expression levels of IL-10 and IL-1 receptor antagonist. Extensive review articles describing the role of Nrf2/HO-1 in the various human pathologies have been published (e.g. Chen-Roetling and Regan, 2017; Furfaro et al., 2016; Landis et al., 2018; Loboda et al., 2016; Ndisang, 2017; Satoh et al., 2013; Wardyn et al., 2015).

4.4.3 Low-grade inflammation as a link between obesity and T2D Perhaps the best link between obesity and T2D is the common low grade but chronic inflammation associated with obesity that leads to insulin resistance. In a significant proportion of T2D sufferers, the level of inflammatory cytokines such as IL-6, and TNF-α as well as inflammation markers such as C-reactive protein (CRP) or high-sensitivity CRP (hs-CRP) and plasminogen activator inhibitor I are raised. The TNF-α gene expression is upregulated in adipose tissues during obesity, linking pro-inflammatory substances released from adipose tissues to insulin resistance in T2D. The role of proinflammatory cytokines in insulin resistance and T2D have been extensively studied and can be summarized as follow: • They inhibit insulin receptor signalling; • Promote hepatic fatty acid syntheses and induce the liver to produce more acute-phase proteins; • Recruit more inflammatory cells to adipose tissues; • Increase β-cell apoptosis and β-cell death; • Via the inflammatory mechanisms such as atherosclerosis, promote cardiovascular complications associated with diabetes; • Transcriptional activation such as NF-κB and activator protein-1 (AP-1) that are linked to diverse physiological and pathological conditions are activated by inflammatory mediators.

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In addition to the direct association between higher level of TNFα in obese subjects and under insulin resistance, the latter has been shown to be ameliorated by anti-TNF-α therapeutic approaches. IL-6 is the other best characterized cytokine that has been shown as a link between obesity and T2D. In recent years, the other potent proinflammatory cytokine, IL-1, is also emerging to play a role in insulin resistance. Virtually, all organs responding to insulin appear to be affected by the inflammatory mechanisms of diabetes which is outlined in the various sections of this book. The oxidative damage and inflammatory mechanisms should also be seen as a cooperative cascades of events that occur together in diabetes pathology. Pancreatic β-cell killing, for example, is associated with their extreme sensitivity to ROS (Wang and Wang, 2017) which cannot be seen separate from the inflammatory-mediated cell damage. The vascular injury in diabetes through oxidative stress, inflammation, and alteration of the haemodynamic balance (pro-coagulant cascades) have been shown to attribute to the micro- and macrovascular complications. The activation of NF-κB, for example, leads to the expression a range of adhesion molecules such as VCAM-1, ICAM-1 and E-selectin that mediate leucocyte infiltration and inflammation. The upregulation of expression of growth factors, such as VEGF, insulin-like growth factor-1 (IGF-1) and platelet-derived growth factor (PDGF) along with more inflammatory cytokines, such as IL-1 and TNF-α, and chemokines, such as monocyte chemoattractant protein-1 (MCP-1) further aggravate tissue injury in diabetes. The role of low density lipoprotein (LDL) in atherosclerosis and vascular injury is also known. For example, oxidized LDL is actively taken up by macrophage leading to their transformation into the foam cells in atherosclerosis development. The CRP concentration is also raised not only during inflammation but also under diabetes and cardiovascular damages. It is produced in the liver by stimulation of proinflammatory cytokines. Interestingly, adipocytes could also be stimulated by cytokines to release the CRP. The CRP intern stimulates various cells including endothelial cells to express adhesion molecules (e.g. ICAM-1) and hence facilitate leucocyte infiltration/inflammation. Hence, in the various antidiabetic assessments discussed in this book, the effect of potential modulators on CRP is discussed along with other obesity and cardiovascular disease markers such as low-density lipoprotein cholesterol (LDL-C) or high-density lipoprotein (HDL)-C. This kind of vascular injury through the combined action of oxidative stress and inflammation in diabetes has been reviewed (Domingueti et al., 2016). Overall, inflammation is now well accepted as a common link between obesity and insulin resistance and also attributes to the tissue damage that is observed under T2D pathology. Understanding the role of inflammation on T2D via the obesity pathway also requires the basic understanding of the adipose tissues cell population. Adipose tissues are largely composed of mature adipocytes, preadipocytes, fibroblasts, endothelial cells, histiocytes and macrophages. Preadipocytes can be induced to differentiate into macrophages-like cells but the adipocytes macrophage population are originated from monocytes of the blood and/or recruitment of blood monocytes bone-marrow progenitor cells of the haemopoisis process. As adipogenesis increases, the level of macrophage population and activation increases along with upregulated secretion of proinflammatory cytokines such as TNF-α, IL-1, and IL-6. This event accounts to the so called low grade inflammation associated with obesity that leads to increased insulin resistance. Furthermore, the production of adiponectin is suppressed as adipogenesis increased and low level of this hormone is known to be observed in obesity, T2D and during cardiovascular complications. This is associated with

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the known anti-inflammatory activity of adiponectin partly mediated by suppressing the levels of TNF-α and IL-6 output from macrophages and/or adipose tissues. On the other hand, leptin release from mature adipocytes increase with obesity and it is a proinflammatory mediator that promotes the release of TNF-α by macrophages. Readers should bear in mind that there are several other adipokines (e.g. omentin, visfatin, and vaspin) which are implicated in inflammation associated with obesity/T2D. The overall link between inflammation and obesity is depicted in Fig. 4.11. The increased levels of ROS, AGEs and angiotensin II are known to activate PKC that mediate the expression of VEGF to promote albuminurea and upregulation of TGF-β with implication of renal hypertrophy and sclerosis. This effect of TGF-β in diabetic nephropathy is also linked to increased collagen IV and fibronectin formation leading to enlargement of the basement membrane. PKC is also known to activate the NADPH oxidase thereby increasing ROS generation. These effects of PKC are also associated with other diabetes complications including diabetes retinopathy which is characterized by endothelial dysfunctions and capillary leakage. Various isoforms of PKC such as the α, β, and δ variants have also been characterized in recent years with distinct function in various tissues. The other pathway of diabetes pathology often cited in the literature is the polyol pathway (Fig. 4.12). The function of aldose dehydrogenase in removing aldehydes generated through the various mechanism of oxidative damage is also extended in the reduction of glucose to sorbitol. As sorbitol is oxidized by the enzymatic action of sorbitol dehydrogenase (SDH), the resulting fructose buildup initiates metabolic pathways leading to more AGEs and ROS generation. Furthermore, depletion of the antioxidant GSH and inactivation of glyceraldehyde-3-phosphate dehydrogenase (G3PD) leads to dysregulation of the pyruvate or glycolysis pathway (Fig. 4.12). Sorbitol is also linked to cellular damage through various

FIG. 4.11

The link between inflammation and T2D.

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FIG. 4.12

The polyol and aldose reductase pathway in diabetes pathology. Increased level of glucose in diabetes leads to the generation of toxic levels of aldehydes that initiate the activity of aldose reductase. The enzyme also convert glucose to sorbitol which is further acted by sorbitol dehydrogenase (SDH) to produce fructose. The NADP+ generated through the process is removed by the GSH and hence over activity leads to the depletion of the antioxidant store (GSH) while the increasing NADH/NAD+ balance that inhibits the activity of glyceraldehyde-3-phosphate dehydrogenase (G3PD) which is driven by NAD+.

means including osmotic changes. Excessive level of glucose has also been implicated to the formation of glucosamine that leads to glycosylation of proteins as observed in diabetes complications.

References ADA (American Diabetes Association), 2014. Diabetic kidney disease: a report from an ADA consensus conference. http://care.diabetesjournals.org/content/37/10/2864. Arevalo, J.F., Snchez, J.G., Wu, L., Berrocal, M.H., Alezzandrini, A.A., Restrepo, N., et al., 2010. Intravitreal bevacizumab for subfoveal choroidal neovascularization in age-related macular degeneration at twenty-four months: the Pan-American collaborative retina study. Ophthalmology 117 (10), 1974–1981. Boyer, D.S., Heier, J.S., Brown, D.M., Francom, S.F., Ianchulev, T., Rubio, R.G., 2009. A phase IIIb study to evaluate the safety of ranibizumab in subjects with neovascular age-related macular degeneration. Ophthalmology 116, 1731–1739.

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Chen-Roetling, J., Regan, R.F., 2017. Targeting the Nrf2-heme oxygenase-1 axis after intracerebral hemorrhage. Curr. Pharm. Des. 23 (15), 2226–2237. Demircan, N., Safran, B.G., Soylu, M., Ozcan, A.A., Sizmaz, S., 2006. Determination of vitreous interleukin-1 (IL-1) and tumour necrosis factor (TNF) levels in proliferative diabetic retinopathy. Eye 20, 1366–1369. Domingueti, C.P., Dusse, L.M., Carvalho, M.D., de Sousa, L.P., Gomes, K.B., Fernandes, A.P., 2016. Diabetes mellitus: the linkage between oxidative stress, inflammation, hypercoagulability and vascular complications. J. Diabetes Complications 30 (4), 738–745. Furfaro, A.L., Traverso, N., Domenicotti, C., Piras, S., Moretta, L., Marinari, U.M., Pronzato, M.A., Nitti, M., 2016. The Nrf2/HO-1 axis in cancer cell growth and chemoresistance. Oxid. Med. Cell. Longev. 20161958174. Giullian, J.A., Chuang, P., Lewis, J.B.L., 2008. Diabetic nephropathy. In: Brietzke, S.A. (Ed.), Endocrinology Board Review Manual, Endocrinology Vol. 7, Part 1, pp. 1–12. Available at: http://seminmedpract.com/pdf/brm_ Endo_V7P1.pdf. International Best Practice Guidelines, 2013. Wound Management in Diabetic Foot Ulcers. Wounds International, http://www.woundsinternational.com/media/best-practices/_/673/files/dfubestpracticeforweb.pdf. Landis, R.C., Quimby, K.R., Greenidge, A.R., 2018. M1/M2 Macrophages in Diabetic Nephropathy: Nrf2/HO-1 as Therapeutic Targets. Curr. Pharm. Des. 24 (20), 2241–2249. Loboda, A., Damulewicz, M., Pyza, E., Jozkowicz, A., Dulak, J., 2016. Role of Nrf2/HO-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism. Cell. Mol. Life Sci. 73 (17), 3221–3247. Mendes, J.J., Neves, J., 2012. Diabetic foot infections: current diagnosis and treatment. J. Diabetic Foot Complications 4 (2), 26–45. Ndisang, J.F., 2017. Synergistic interaction between heme oxygenase (HO) and nuclear-factor E2-related factor-2 (Nrf2) against oxidative stress in cardiovascular related diseases. Curr. Pharm. Des. 23 (10), 1465–1470. NICE (National Institute for health and Care Excellence), 2016. Diabetic foot problems: prevention and management. https://www.nice.org.uk/guidance/ng19/resources/diabetic-foot-problems-prevention-and-management1837279828933. (Accessed 8 January 2019). Rhee, E.P., 2016. NADPH oxidase 4 at the nexus of diabetes, reactive oxygen species, and renal metabolism. J. Am. Soc. Nephrol. 27 (2), 337–339. Satoh, T., McKercher, S.R., Lipton, S.A., 2013. Nrf2/ARE-mediated antioxidant actions of pro-electrophilic drugs. Free. Radic. Biol. Med. Dec. 65, 645–657. Sedeek, M., Nasrallah, R., Touyz, R.M., Hebert, R.L., 2013. NADPH oxidases, reactive oxygen species, and the kidney: friend and foe. JASN 24 (10), 1512–1518. Vincent, J.A., Mohr, S., 2007. Inhibition of caspase-1/interleukin-1beta signaling prevents degeneration of retinal capillaries in diabetes and galactosemia. Diabetes 56, 224–230. Wang, J., Wang, H., 2017. Oxidative stress in pancreatic beta cell regeneration. Oxid. Med. Cell. Longev.. 20171930261. Wardyn, J.D., Ponsford, A.H., Sanderson, C.M., 2015. Dissecting molecular cross-talk between Nrf2 and NF-κB response pathways. Biochem. Soc. Trans. 43 (4), 621–626.

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5.3 Common antidiabetic drugs under clinical use 5.3.1 Biguanides 5.3.2 Incretins and/or their mimetics 5.3.3 The sodium–glucose cotransporter-2 (SGL2) inhibitors 5.3.4 Sulfonylureas

5.3.5 Meglitinides—Rapid-acting secretagogues 5.3.6 Thiazolidinedione (TZD) or glitazones 5.3.7 Alpha-glucosidase inhibitors 5.3.8 Insulin 5.3.9 Amylin analogues 5.3.10 Other antidiabetic drugs of choice

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5.1 Type 2 diabetes diagnosis and management by lifestyle change The clinical diagnosis of diabetes is described in Chapter 1 and herein also briefly outlined to overview the goal of the pharmacotherapy. The two most widely used markers of type 2 diabetes (T2D) and that used to monitor therapeutic success are plasma glucose level and glycated haemoglobin (HbA1c) levels. The classical symptoms of diabetes including polyuria, polydipsia, unexplained weight loss or a combination of most of these symptoms may be presented to initiate the diagnosis. The diagnosis of T2D is commonly confirmed by the following criteria:

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• Fasting plasma glucose (FPG) often in the morning before meal (at least 8 h without meal) is consistently reaching to the level of 126 mg/dL or 7.0 mmol/L is regarded as a reliable diagnosis of T2D. • Oral administration of high glucose dose (75 g solution in water) showing a PG level of 200 mg/dL or 11.1 mmol/L over a period of 2 h. The test is called 2-h oral glucose tolerance test (OGTT). • The normal level of the HbA1C is below 5.7% and in chronic cases of diabetes, the level of HbA1c could be used as a disease marker. This criteria is however not taken precedence in the diagnosis of diabetes as many other pathological conditions (e.g. anaemia, haemolysis, like sickle cell disease, etc.) may raise the level of HbA1C. One of the topic of interest in diabetes intervention is lifestyle changes which is not addressed in this book. Readers should bear in mind that lifestyle change is the key to the prevention and control of various metabolic disorders including diabetes, obesity and cardiovascular diseases. Clinical studies on prediabetes for example has shown that lifestyle changes can prevent or delay the onset of diabetes: about 58% reduction in diabetes incidence in one particular study (Tuso, 2014). In whatever stage of diabetes that one can be in, exercise and dietary control should be the first lifestyle consideration to be included in the disease management. Other measures including a reduction in alcohol consumption should also be considered but the focus of our discussion in this book remains to be pharmacotherapy.

5.2 Pharmacological management approach for T2D As discussed in the various chapters, hyperglycaemia, is a characteristic feature of T2D which could be a result of various factors often in combination. Of these, the following mechanisms, sometimes called the ominous octet, are important as the current pharmacological modulators and/or clinically useful drugs act by modulating such mechanism(s): • • • • • • • •

Pancreatic β-cells dysfunction and/or deletion leading to a reduction in insulin secretion; Pancreatic α-cells producing high level of glucagon that antagonizes insulin; The liver producing increased level of glucose; Impaired glucose uptake in peripheral tissues such as skeletal muscle, liver, and adipose tissue; Diminished effect of incretin in the ileum; The brain developing insulin resistance and/or neuronal dysfunction; Increased level of lipolysis in adipose tissue leading to fatty acids (FAs) excess, i.e. insulin resistance and dyslipidaemia; Failure to excrete glucose via the kidney or higher level of renal glucose reabsorption.

Schematic presentation of these pathological sites which also serve as targets for the current drug therapeutic approaches are presented in Fig. 5.1. The application of pharmacological agents could vary depending on availability of drugs and the pathological state of the disease but drug therapy often start in combination with some measure of lifestyle changes. Other considerations are of course not just efficacy but also

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FIG. 5.1 Key pathological markers and targets of T2D.

cost, potential side effects, possible weight gain, hypoglycaemia risk, comorbidities, and preferences of the patients themselves. As indicated in the facts and figures section of this book (Chapter 1), pharmacological treatment of T2DM should be initiated when glycaemic control is not achieved or if HbA1c rises to 6.5%, say for example, after 2–3 months of lifestyle intervention. The guidelines of therapeutic intervention could vary from region to region or by country but the general guidelines for T2D management given by the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD) is centred in reducing the HbA1c in diabetes patients to less than 7.0% to reduce the incidence of microvascular diseases. The HbA1c level of 6.0–6.5% are considered appropriate for patients with recent disease onset, absence of cardiovascular disease and expected to have long life expectancy. The HbA1c level of 7.5–8.0%, may also be acceptable for “patients with a history of severe hypoglycaemia, limited life expectancy, advanced complications, extensive comorbid conditions, or trouble meeting glycaemic targets despite multipronged therapy” (Weiss, 2012). The general guidelines is also to employ lifestyle interventions by modifying dietary habit, increased physical exercise, and promoting weight loss in diabetes patients. For patients with an HbA1c less than 7.5%, pursuing lifestyle interventions for 3–6 months before starting medication is recommended. The various pharmacological treatment options are discussed in the following sections but the recommended general therapeutic guidelines are highlighted herein first. 1. The most widely used first-line cost-effective medication for T2DM is metformin. It is considered weight-neutral, does not increase the risk of hypoglycaemia, and may have cardiovascular benefits, although this is not entirely clear.

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2. For patients with HbA1c level of more than 9.0%, two pharmacological agents or insulin should be considered. Insulin should be strongly considered from the outset if the HbA1c level is 10.0–12.0%. 3. If monotherapy does not achieve or maintain the patient’s HbA1c target during 3 months of treatment, then a second oral agent, a glucagon-like peptide-1 (GLP-1) receptor agonist, or insulin should be considered. 4. If patient still makes no progress after the use of the second oral target, another agent with a different mechanism of action should be considered with insulin still the best option for patients with HbA1c remains over 8.5%. 5. Sulfonylurea insulin secretagogues are effective at controlling blood glucose levels, but are associated with some weight gain and a risk of hypoglycaemia. Shorter-acting secretagogues may be associated with reduced risk of hypoglycaemia but require more frequent dosing. 6. Thiazolidinediones do not increase the risk of hypoglycaemia and may be effective for longer period than sulfonylureas and metformin. One example, pioglitazone, showed a modest cardiovascular benefit in one trial, but has also been associated with a possible increase in risk of bladder cancer. 7. Injectable GLP-1 receptor agonists can cause moderate to significant weight loss, but can also cause nausea and vomiting. 8. Oral dipeptidyl peptidase-4 (DPP-4) inhibitors are weight-neutral. Neither they nor GLP-1 receptor agonists cause hypoglycaemia by themselves. 9. Insulin replacement therapy is frequently required in T2D, although not in as complex or intensive a fashion as in type 1 diabetes (T1D). The above guidelines are subject to regular updates and modification and hence readers are advised to consult up-to-date documents by professional bodies in their region. The ADA updated file in 2017, for example, is widely cited. They define clinically significant hypoglycaemia as glucose catechin (29) (2.4) > quercitrin (2.4, 52) > rutin (2.3, 53) > luteolin (2.2, 23) > epicatechin (1.6, 31). As part of the antidiabetic activity studies, peltatoside (54), hyperoside (46), isoquercetin (48) and guaijaverin (49) have also been isolated from the leaves (Eidenberger et al., 2013). Myricetin-3-O-β-D-glucoside (57) was also isolated from the leaves along with morin-3-O-α-L-arabopyranoside (63), quercetin (25), hyperin (46) and isoquercitrin (48) (Fu et al., 2009).

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TABLE 9.2 Entry

259

Some common flavonoid glycosiders of guava

Structural name

Trivial name

References

QUERCETIN (25)-BASED 46

3-O-β-D-galactopyranoside

Hyperin or hyperoside

Leaves (Eidenberger et al., 2013; Metwally et al., 2010; Shu et al., 2012; Wang et al., 2010; Zhu et al., 2013) Fruits (M€ uller et al., 2018)

47

3-O-β-D-xylopyranoside

Reynoutrin

Leaves (Shu et al., 2012; Zhu et al., 2013)

48

3-O-β-D-glucopyranoside

Isoquercitrin

Leaves (Eidenberger et al., 2013; Fu et al., 2009; Im et al., 2012; Metwally et al., 2010; Shu et al., 2012; Zhu et al., 2013) Fruits (M€ uller et al., 2018)

49

3-O-β-D-arabinopyranoside

Guaijaverin

Leaves (Eidenberger et al., 2013; Metwally et al., 2010; Shu et al., 2012; Wang et al., 2010; Zhu et al., 2013) Fruits (M€ uller et al., 2018)

50

3-O-α-L-arabinofuranoside

Avicularin

Leaves (Metwally et al., 2010; Shu et al., 2012; Wang et al., 2010) Fruits (M€ uller et al., 2018)

51

3-O-β-D-arabinopyranoside

52

3-O-α-L-rhamnopyranoside

Quercitrin

Leaves (Shu et al., 2012)

53

3-O-rutinoside

Rutin

Leaves (Wang et al., 2018) Fruits (dos Santos et al., 2017)

54

3-O-arabinoglucoside

Peltatoside

Leves (Eidenberger et al., 2013)

Leaves (Metwally et al., 2010)

MYRICERIN (27)-BASED 55

3-O-β-D-arabinopyranoside

Fruits (Flores et al., 2015)

56

3-O-β-D-xylopyranoside

Fruits (Flores et al., 2015)

57

3-O-β-D-glucoopyranoside

Fruits (Flores et al., 2015) Leaves (Fu et al., 2009)

ISOREHAMENTIN (26)-BASED 58

3-O-β-D-glucopyranoside

Fruits (Flores et al., 2015)

59

3-O-β-D-galactopyranoside

Fruits (Flores et al., 2015)

60

Phloretin-based

61

3-O-β-D-glucopyranoside

Phloridzin

Leaves and fruits M€ uller et al. (2018)

MORIN (28)-BASED 62

3-O-α-L-lyxopyranoside

Leaves (Arima and Danno, 2002)

63

3-O-α-L-arabopyranoside

Leaves (Arima and Danno, 2002; Fu et al., 2009) Continued

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TABLE 9.2 Some common flavonoid glycosiders of guava—Cont’d Entry

Structural name

Trivial name

References

Naringin

Leaves (Chen and Yen, 2007)

NARINGENIN (21)-BASED 64

7-O-α-L-rhamnopyranosyl-(1 !2)β-D-glucopyranoside or 7-Oneohesperidose

ANTHOCYANIDINS (33, 34)-BASED 65

Delphinidin-3-O-glucoside

Fruits (Flores et al., 2015)

66

Cyanidin-3-O-glucoside

Fruits (Flores et al., 2015)

FIG. 9.5 Diversity of sugars incorporated into the flavonoid aglycones in guava plant.

The flavonoid glycosides of the fruits are by and large similar with the leaves with quercetin glycosides appear to be very common. Anthocyanins being the colouring principle of many plant parts, their identification in the coloured guava fruits is not surprising. Delphinidin- (65) and cyanidin-3-glucosides (66) were isolated from the various extracts of the fruits (Flores et al., 2015). Two proanthocyanidins have also been identified that also appear to be formed from two gallocatechins or a galocatechin joined with catechin, or catechin/ epicatechin dimers (67–71). Proanthocyadins from guava fruits are exemplified by the

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261

identification of procyandin B1 (69) and B2 (70) (Fig. 9.6) (Flores et al., 2015). The identification of procyandin B1, B2 and B3 (71) from the leaves of guava has also been described by Okuda et al. (1984). The identification of polyphenols from the leaves of the guava plant in general can be traced back to the early days of phytochemistry as a subject. Seshadri and Vashishta (1965), for example, isolated quercetin, guaijaverin (49), leucocyanidin (33) and tannins in 1965. As part of spasmolytic effects study, these compounds were also isolated from the leaves (Lozoya et al., 1994). Earlier studies also showed the identification of (+)-gallocatechin (30) from the leaves as antimutagenic principle (Matsuo et al., 1994). Quantitative analysis of phenolic compounds in guava leaves by high performance liquid chromatography coupled with mass spectrometer (HPLC-MS) analysis has been carried out by numerous groups. A classic example is presented by the work of Dı´az-de-Cerio et al. (2015, 2016) where flavonols and flavan-3-ols were shown to be represent as the main phenolic compounds of guava leaves. Morin (28), quercitrin (52), guajaverin (49), procyanidin B (Fig. 9.6), and hyperin (46) constitute 41.2% of the sum of all phenolic compounds. As the compounds were not isolated and the method does not discriminate stereoisomers, several isomeric forms of compound have also entered in such listings. For example, hexahydroxydiphenoyl-D-glucose

FIG. 9.6 Procyanidins of guava.

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9. The chemical and pharmacological basis of guava

as a tannin derivative (see Section 9.3.4) appeared in three forms. Analysis of the various plant parts have also shown that the floral buds have the highest concentration of quercetin (25) (2036 mg/kg) followed by the mature leaf (1236 mg/kg) (Vargas et al., 2006). As part of an antimicrobial activity study, Arima and Danno (2002) have isolated four antibacterial compounds from the leaves of guava including morin-3-O-α-L-lyxopyranoside (62) and morin3-O-α-L-arabopyranoside (63) along with guaijavarin (49) and quercetin (25). The number of phenolic compounds that one can detect in the leaves and fruits is incredibly high. Dı´az-de-Cerio et al. (2016) identified 72 phenolic compounds from the aqueous ethanol extract of the leaves by means of HPLC coupled with electrospray ionization quadrupole timeof-flight tandem mass spectrometry (HPLC–ESI–QTOF–MS/MS) analysis. Their quantitative analysis also showed relative abundance of the compounds in two varieties: var. pomifera and pyrifera. They have also recorded some chemical composition difference in the fruits and leaves sourced from various regions such as Taiwan, China, Brazil, Thailand, USA, Mauritious, Spain, Mexico and Panama. The composition analysis study by Dı´az-de-Cerio et al. (2015) along with differences in the antioxidant effects also reported the order of concentration in aqueous extract of the leaves as follow: flavonols > flavan-3-ols > gallic and ellagic acid derivatives > benzophenones > flavanones. Through ESI-MS/MS study on the butanol fraction of the leaves, Im et al. (2012) also reported the identification of D-glucuronic acid, quercetin-3-glucuronide (110, Fig. 9.9), loganin (165, Fig. 9.13), and xanthyletin (96, Fig. 9.8) (Im et al., 2012). By using high-speed counter-current chromatography, Zhu et al. (2013) have isolated five flavonoid glycosides and one diphenylmethane glycoside from P. guajava leaves. The compounds were hyperin, isoquercetin, reynoutrin, guaijaverin and avicularin (Table 9.2), along with a benzophenone derivative (see Section 9.3.4). From the aqueous ethanol extract of the leaves, Shu et al. (2012) have also isolated kaempferol, quercetin, quercitrin and isoquercitrin as flavonoid aglycomnes (Fig. 9.4); and guaijaverin (49), avicularin (50), hyperoside (46) and reynoutrin (47) as glycosides (Table 9.2). Other compounds isolated along with these flavonoids were diphenylmethane (93) and benzophenone derivatives (see Section 9.3.4). The study by Chen and Yen (2007) also showed the presence in the leaves of epicatechin, (+)-catechin, quercetin, morin and naringin (Fig. 9.4) along with phenolic acids (ferulic acid, gallic acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, syringic acid, and 2-hydroxybenzoic acid (salicylic acid) Fig. 9.3). Following the demonstration of glucose transport inhibitory effect in vitro and glucose lowering effect in vivo, M€ uller et al. (2018) have done comparative analysis of the leaves and fruits of guava extracted with ethanol and supercritical CO2. They found striking differences based on the detection of compounds (by HPLC with diode array detection (DAD) and Orbitrap MS) from the concentrated and diluted extract as well as the extraction methods employed (Table 9.3). The aqueous and ethanol extraction appear to be optimized for obtaining phenolic compounds while the supercritical CO2 extraction is not suitable for such constituents in guava (Table 9.3). Several biological activates and phenolic compounds detection/isolation are accordingly done from polar extracts. Jang et al. (2014) directed their phytochemical analysis to enhance the anti-inflammatory activity of guava through phenolic composition analysis with respect to gallic acid (0.06 and 0.22 mg/g dry weight (d.w.)), gallocatechin (5 and 5.6 mg/g d. w.) and catechin. The highest amount they detected was glycolic acid followed gallocatechin. Other studies by Haida et al. (2011) also noted compositional differences between P. guajava L. var. pomifera and P. guajava L. var. pyrifera leaves. There are also several cultivars worth

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TABLE 9.3 Concentrations of compounds in the crude extracts of leaves and fruits of guava. Concentrated ethanol (cEtOH) and ethanol (EtOH) extract for the leaves and EtOH or supercritical (sCO2) extraction of the fruits (EF and SCF) are shown in mg/L concentrations.a Leaves (Mg/L)

Fruits (mg/L)

Compound

cEtOH

EtOH

EtOH

sCO2

Chlorogenic acid

354.0957

n.d.

47

14

Phloridzin

436.1376

35

n.d.

1.2

Phloretin

274.0847

45

n.d.

n.d.

Procyanidin B1

578.1430

n.d.

310

124

Procyanidin B2

n.d.

81

118

n.d.

(+)-Catechin

n.d.

236

78

n.d.

(
)-Epicatechin

n.d.

n.d.

46

n.d.

Gallocatechin

100

215

214

n.d.

Epicatechingallate

16

7.2

11

n.d.

Quercitrin

162

2.3

20

n.d.

Isoquercitrin/hyperoside

451

7.0

50

n.d.

Guaijaverin

728

n.d.

n.d.

n.d.

Avicularin

586

n.d.

n.d.

n.d.

Quercetin

698

n.d.

n.d.

n.d.

Ellagic acid

27

258

113

n.d.

Gallic acid

2.4

21

60.9

n.d.

DIHYDROCHALCONE DERIVATES

Flavan-3ols

FLAVONOLS

HYDROBENZOIC ACIDS

€ Adopted from Muller et al. (2018). n.d., Not determined.

a

mentioning and the study Chen and Yen (2007) is a good demonstration of compositional variabilities where differences for cultivars like cv. Hong Ba, Shi Ji Ba, Shui Jing Ba and Tu Ba were shown. All these studies highlight chemical composition differences in the same species of plants based on genotypic differences, environmental factors and extraction methods. Although not as prevalent as the flavonols, isoflanvonoids (72–88) are common constituents of the family Myrtaceae. The study by Lapik et al. (2005) employed HPLC–MS–selected ion monitoring (SIM) and immunoassay methods to detect compounds shown in Fig. 9.7. The malonate and acetate ester derivatives include, daidzin-600 -O-malonate (82), Daidzin-60 -O-acetate (83), glycitin-6-O-malonate (88), genistin-60 -O-malonate (85) and genistin-6-O-acetate (86).

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9. The chemical and pharmacological basis of guava

FIG. 9.7 Isoflavonoids of guava.

9.3.4 Unusual phenolics and tannins The leaves of guava have also been the source of unusual humulene-based meroterpenoids called ()-guajadial B (89) (Fig. 9.8). Although its biological activity is yet to be determined, this compound has also been obtained through a synthetic route (Gao et al., 2012). Shao et al. (2012a) have also identified three related unusual meroterpenoids from the leaves which they named guadial A (90), psiguadial C (91) and psiguadial D (92)—the compounds showed cytotoxicity against HepG2 and HepG2/ADM cells. Phenolic compounds incorporating terpenoids now appear to be common in guava. The further examples of these compounds are the biologically active meroterpenoids called psidals A-C (93–95). From the methanol (80% in water) extract of the leaves, Matsuzaki et al. (2010) isolated three compounds that they named guavinosides A-C (98–100) (Fig. 9.9). Guasinoside C (100) appears to be the glloyl derivative of quercetin, while the other compounds were 2,4,6trihydroxybenzophenone 4-O-(600 -O-galloyl)-β-d-glucopyranoside (98, guavinoside A), and 2,4,6-trihydroxy-3,5-dimethylbenzophenone 4-O-(600 -O-galloyl)-β-D-glucopyranoside (99, guavinoside B). As shown in Fig. 9.9, the α-L-arabinofuranoside derivative of quercetin or avicularin appears to be esterified with gallic acid to yield guavinoside C (100). From the leaves of guava, Shu et al. (2010) have also isolated five galloyl derivatives and identified them as 1-O-(1,2-propanediol)-6-O-galloyl-β-D-glucopyranoside (102), gallic acid, ellagic acid, ellagic acid-4-O-β-D-glucopyranoside (106) and quercetin-3-O-(600 -galloyl)

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265

FIG. 9.8 Other aromatic compounds including meroterpenoids.

β-D-galactopyranoside (101) (Fig. 9.9). Gallotannin derivatives are generally common in the plant and compounds such as 1-O-galloyl-β-D-glucose (103) (Fig. 9.9) have been isolated from the fruits (Fu et al., 2009). Seshadri and Vashishta, 1965, for example, isolated the hydrolyzable tannin, amritoside (107) in 1965 along with quercetin, guaijaverin (49) and leucocyanidin (33). Amritoside is composed of ellagic acid and the gentiobiose sugar component (107). Benzophenone, 2,6-dihydroxy-3-formaldehyde-5-methyl-4-O-(600 -O-galloyl-β-Dglucopyranosyl) diphenylmethane (108), and 2,6-dihydroxy-3,5-dimethyl-4-O-(600 -Ogalloyl-β-D-glucopyranosyl)-benzophenone (109) have been isolated from the leaves (Shu et al., 2012). The latter compound has also been isolated from the leaves by Zhu et al. (2013). Phenolic acid and ellagic acid glycosides have been isolated from the seeds of guava. The study by Salib and Michael (2004), for example, documented two phenolic glycosides and two new phenylethanoid glycosides which they identified as 1-O-3,4-dimethoxyphenylethyl-4-O3,4-dimethoxy cinnamoyl-6-O-cinnamoyl-β-D-glucopyranose (104) and 1-O-3,4-dimethoxyphenylethyl-4-O-3,4-dimethoxy cinnamoyl-β-D-glucopyranose (105) (Fig. 9.9). The seeds have also shown to be sources of a novel quercetin glucoside ester quercetin-3-O-β-D-(200 -O-galloyl glucoside)-40 -O-vinylpropionate (111) (Michael et al., 2002). It is not clear how the vinylpropionate (112) is attached to the flavonoid skeleton as an 40 -O-ester, however, and a structural revision of this compound is required.

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9. The chemical and pharmacological basis of guava

FIG. 9.9 Some unusual glycosides and simple tannin derivatives.

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267

Tannins are principal components of the guava plant and have been isolated from all parts including the leaves, fruits and stem-bark. From the stem-bark of guava, Tanaka et al. (1992) isolated six new complex tannins that they named guajavins A (113) and B (114), psidinins A (115), B (116) and C (117) and psiguavin (118) as well as many other derivatives (Fig. 9.10). Their structure contain favan-3-ols such as (+)-gallocatechin as well as hydrozable tannin moieties with C-glycosidic linkage. Since then, a number of similar tannin derivatives have also been isolated from the various parts. For example, Dı´az-de-Cerio et al. (2016) have detected compounds like stenophyllin A (121) and guavins (119,120) from the leaves (Fig. 9.11). Guavin B (122) was isolated along with procyandins from the leaves of guava (Okuda et al., 1984). Guavin B was also presented as the first example of ellagitannin with polyhydroxybenzophenone which also possess O-glucoside linkage: i.e. not an ester linkage (Fig. 9.11). The most comprehensive study on the identification of tannins from guava leaves were also carried out by Okuda et al. (1982) who reported the presence of the following compounds: pedunculagin (123), casuarictin (124), tellimargandin I (125), strictinin (126), isostrictinin (127), 2,3-hexahydroxydipenoylglucose (128), casuarinin (129), casuariin (130) and stachyurin (131).

9.3.5 Essential oils and other non-phenolic components Smith and Siwatibau (1975) have done one of the pioneering work on the hydro-distilled essential oil analysis of guava leaves. In their gas chromatography coupled with mass spectrometry (GC–MS) analysis and isolation studies, Fijian guava plants of the sweet, intermediate and sour fruits all have caryophyllene (132) as a major component but could be categorized as distinct chemotypes on the basis of β-bisabolene (142), β-selinene (139) and aromadendrene (134) (Fig. 9.12). Other compounds present in the oil in various amounts depending on the source were longicyclene (141), nerolidol (144), caryophyllene oxide (β-caryophyllene epoxide, 133) and sel-11-en-4α-ol (140) (Smith and Siwatibau, 1975). The study by Li et al. (1999) have documented caryophyllene (132) (18.81%), copaene (138) (11.80%), spathulenol/espatulenol (135) (10.27%) and eucalyptol (145) (7.36%) as the major components of essential oils of the leaves. In another study by Satyal et al. (2015), analysis of the hydro-distilled essential oil of the leaves indicated the major components as follow: (E)-nerolidol (144) (35.6%) and (E)-caryophyllene (132) (15.8%), with lower concentrations of (2Z,6E)-farnesol (143) (6.7%), and ledol (136) (5.5%). Hence, sesquterpenes appear to dominate the volatile components of the leaves (Fig. 9.12). Paniandy et al. (2000) studied the composition of essential oils obtained from the fresh fruits of guava by steam hydro-distillation and headspace solid-phase microextraction (SPME). The hydro-distillation method offer more compounds (61) than headspace SPME (21) with the overall number of compounds identified in the essential oils being 73. The major components were as follow: Headspace—hexanal (65.9%, 148), γ-butyrolactone (7.6%, 157), (E)-2-hexenal (7.4%, 149), (E,E)-2,4-hexadienal (2.2%, 150), (Z)-3-hexenal (2.0%, 151), (Z)-2-hexenal (1.0%, 152), (Z)-3hexenyl acetate (1.3%, 155) and phenol (1.6%, 147), hydro-distillation-β-caryophyllene (24.1%, 132), nerolidol (17.3%, 144), 3-phenylpropyl acetate (5.3%, 155) and caryophyllene

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FIG. 9.10

9. The chemical and pharmacological basis of guava

Tannins isolated from the stem-bark of guava.

D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

9.3 The chemistry of guava

FIG. 9.11 Other tannin components of guava.

D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

269

270

FIG. 9.12

9. The chemical and pharmacological basis of guava

Volatile components of guava.

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9.3 The chemistry of guava

271

oxide (5.1%, 133). It is astonishing to see such variability in composition for the two methods employed, but from the pharmacological point of view, all constituents detected by both methods should be considered. β-Caryophyllene (132) is still the major sesquiterpene component of the oil, and as with the leaves, in some reports concentrations as high as 95% of the hydrocarbon terpenoids fraction have been reported (Wilson and Shaw, 1978). Fresh ripe and aromatic Taiwanese guava (Psidium guajava L. cv. Chung-Shan-Yueh-Pa) fruits processed by steam distillation and solvent extraction have also been analyzed by GC–MS (Chen et al., 2006). Of the 65 compounds identified, the major constituents in guava fruits were: α-pinene (147), 1,8-cineole (eucalyptol, 145), β-caryophyllene (132), nerolidol (144), globulol (137), C6 aldehydes (unspecified), C6 alcohols (unspecified), ethyl hexanoate (162) and (Z)-3-hexenyl acetate (160). Jordan et al. (2003) also studied the volatile components of the fresh pink guava fruit puree and commercial guava essence by GC–MS analysis. While (E)-2-hexenal (149) is more relevant to the aroma of commercial essence than of the fresh fruit puree; they have detected variable contents of α-copaene (138), cinnamyl alcohol (156), (Z)-3-hexen-1-ol (160), limonene (146), 3-phenylpropanol (154), octanol (158), ethyl octanoate (159), along with other trivial compounds such as acetic acid, 3-hydroxy-2-butanone, 3-methyl-1-butanol, 2,3butanediol, 3-methylbutanoic acid, 6-methyl-5-hepten-2-one, and an unknown component. The study was however more directed to the aroma than potential medicinal uses.

9.3.6 Terpenoids 9.3.6.1 Mono- and sesquiterpenes As discussed in the preceding section, the presence of monoterpenes and sesquiterpenes in the essential oils of guava has been well documented (Fig. 9.12). These compounds are also shown to be incorporated into other structural groups to form unusual compounds as demonstrated by the structural diversity of meroterpenes (Fig. 9.8). Other sesquitepenes identified from the fruits were abscisic acid (163) and tupinionosides A (164) (Flores et al., 2015) (Fig. 9.13). The presence of loganin (165) in the leaves was also reported by Im et al. (2012). 9.3.6.2 Triterpenes Triterpenes appear to have a formidable occurrence in the various tissues of guava plant both in number and complexity. These are mainly based on oleonolic and ursolic acids

FIG. 9.13 Structures of further sesquiterpenes from guava.

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(Fig. 9.14) which may also be glycosylated (Fig. 9.15) or esterified either with p-coumaric or ferulic acids (Fig. 9.16). Other derivatives including sitosterol, lueol and betulinic acid derivatives (Fig. 9.15) also occur. Their structural features and isolation from the plant is described herein. Among the triterpenes widely known to occur in guava are pinfaensin (200), pedunculoside (201), guavenoic acid (170), madecassic acid (171), and asiatic acid (172) (Fig. 9.14). The fruits of guava are in fact proven to be a very rich source of triterpenes. Begum et al. (2002a) have isolated two new triterpenoids that they named guajavolide (2α,3β,6 beta,23-tetrahydroxyurs-12-en-28,20β-olide(194)) and guavenoic acid (2α,3β,6β,23tetrahydroxyurs-12,20(30)-dien-28-oic acid; 1170) along with oleanolic acid (166) from the fresh leaves. In a separate experiment, the authors (Begum et al., 2002b) have also isolated another two triterpenoids, 20β-acetoxy-2α,3β-dihydroxyurs-12-en-28-oic acid (guavanoic acid, 177), and 2α,3β-dihydroxy-24-p-Z-coumaroyloxyurs-12-en-28-oic acid (guavacoumaric acid, 213), along with six known compounds 2α-hydroxyursolic acid (176), jacoumaric acid (208), isoneriucoumaric acid (3β-hydroxy-2α-trans-p-coumaryloxy-urs-12-en-28-oic acid, 218), asiatic acid (172), ilelatifol D (185) and β-sitosterol-3-O-β-D-glucopyranoside (199) from the leaves. The isolation of ursolic acid (175) by Arthur and Hui (1954) and its 2-hydroxy derivative (2α-hydroxyursolic acid, 176) by Osman et al. (1974) was dated several decades ago. Oleanolic acid (166) (Arthur and Hui, 1954), maslinic acid (164) (Arthur and Hui, 1954; Osman et al., 1974) and arjunolic acid (168) (Sasaki et al., 1966) have also been recorded from the plant since the 1950s. In addition to oleanolic acid (167), and ursolic acid (175), Begum et al. (2004) isolated β-sitosterol (198) and uvaol (186), along with a novel compound that they named guajanoic acid (3β-p-E-coumaroyloxy-2α-methoxyurs-12-en-28-oic acid (219)) from the leaves. The identification of guajanoic acid along with β-sitosterol (198), uvaol (186), ursolic acid (175) and oleanolic acid (166) were also described from the leaves as part of bioactivity studies (Ghosh et al. (2010); but these compounds are already reported from the leaves previously (Begum et al., 2004). The further addition is also from the study by Begum et al. (2002c) that reported the isolation of a new triterpene 2α-hydroxy-3β-p-E-coumaroyloxyurs-12,18dien-28-oic acid (221) and two known terpenoids obtusinin (214) and goreishic acid I (190) from the leaves. Shao et al. (2012b) also isolated from the leaves four novel triterpenes that they named psiguanins A-D along with 13 known compounds. The structures of psiguanins A-D as shown in Fig. 9.14 were assigned as 2α,3β-dihydroxy-taraxer-20-en-28-oic acid (179), 2α,3β,12α,13β-tetrahydroxy-urs-28-oic acid (180), 2α,3β,12β,13β-tetrahydroxy-urs-28-oic acid (181), and 2α,3β,12β,13α-tetrahydroxy-urs-28-oic acid (182) respectively. Other compounds of interest were jacoumaric acid (208), 3β-O-trans-p-coumaroylmaslinic acid (215), 3β-Otrans-ferulyl-2α-hydroxy-urs-12-en-28-oic acid (212), eucalyptolic acid (216), 3β-O-ciscoumaroyl-2α-hydroxy-urs-12-en-28-oic acid (209), 3β-O-cis-p-coumaroylmaslinic acid (217), 3β-O-cis-ferulyl-2α-hydroxy-urs-12-en-28-oic acid (210), 6β-hydroxymaslinic acid (184), asiatic acid (172), urjinolic acid (168), 3β-acetylursolic acid (197), 3β,13βdihydroxyurs-11-en-28-oic acid (182), and 3β-hydroxyurs-11-en-28,13β-olide (183). The study by Fu et al. (2009) also identified nine tetriterpenes from the leaves including ursolic acid (175), 2α-hydroxyursolic acid (176) and 2α-hydroxyoleanolic acid (167) (Fig. 9.14), along with flavonoids and tannins.

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9.3 The chemistry of guava

FIG. 9.14 Triterpene components of guava.

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FIG. 9.15

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More triterpene components including glycosides.

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FIG. 9.16 Guava triterpenes acylated with p-coumaric and ferulic acids.

The study by Shu et al. (2009) also investigated the triterpene content of the fruits. They reported the isolation of nine triterpenoids, ursolic acid (175), 1β,3β-dihydroxyurs-12-en-28-oic acid (178), 2α,3β-dihydroxyurs-12-en-28-oic acid (176), 3β,19α-dihydroxyurs-12en-28-oic acid (188), 19α-hydroxylurs-12-en-28-oic acid-3-O-α-L-arabinopyranoside (196), 3β,23dihydroxyurs-12-en-28-oic acid (187), 3β,19α,23β-tri-hydroxylurs-12-en-28-oic acid (189), 2α,3β,19α, 23β-tetrahydroxyurs-12-en-28-oic acid (191) and 3α,19α,23,24tetrahydroxyurs-12-en-28-oic acid (193). The latter compound with 3α-hydroxyl configuration is interesting given the β-orientation at this site is the common stereochemistry for such compounds. Triterpenes have also been isolated from the roots. This include the study by Peng et al. (2017) that reported the identification of, 2α,3β,6β,23-tetrahydroxylurs-12,20(30)-dien-28-oic acid β-D-glucopyranoside (204), 2α,3β,6β,23-tetrahydroxylurs-12,18-dien-28-oic acid β-Dglucopyranoside (206), 2α,3β,23-trihydroxylurs-12,18-dien-28-oic acid β-D-glucopyranoside (205), nigaichigoside F1 (192), and 2α,3β,19α,23-tetrahydroxylurs-12-en-28-oic acid (191) along with asiaticoside (207) derivatives.

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Overall, guava appear to have a diverse group of triterpenes of oleanolic and ursolic acid derivatives. Both derivatives have diverse pharmacological activities and should be considered as active principles for treating diabetes and related diseases.

9.3.7 Carotenoids Mercadante et al. (1999) have isolated sixteen carotenoids from the flesh of Brazilian red guavas (P. guajava L.) of which thirteen were reported from the plant for the first time (Figs. 9.17 and 9.18). Beyond the detection of these carotenoids, quantitative analysis have shown the significance of guava fruits as sources of important carotenoids. The lycopene content of guava has especially been given a lot of attention in recent years due to the reported content in the fruits being higher than other common sources such as tomato, watermelon, mango, and papaya (Mangels et al., 1993; Oliveira et al., 2010; Vargas-Murga et al., 2016). Quantitative analysis of vitamin C and lycopene in various common foods (honey, red wine and fruits from guava and tomatoes) have also shown that guava being superior to other sources. The value presented for lycopene by Nwaichi et al. (2015) was 5 mg/100 g. The changes in carotene content and color with the maturity of the fruits have further been documented ( Jain et al., 2003).

9.4 Antidiabetic, antiobesity and lipid lowering effects of guava—Evidence from in vivo studies A number of animal models have been used to demonstrate the antidiabetic, lipid lowering and antiobesity effects of guava (Table 9.4). Both the fruits and the leaves have shown promising effects while numerous compounds isolated from the various parts of the plant could also be implicated as active principles. In this section, the gross pharmacological effects in animal models for the leaves and fruits are presented followed by detailed mechanism of action through further scrutiny of in vitro effects and studies on the active principles.

9.4.1 The pharmacology of guava fruits in diabetes and associated diseases In the streptozotocin (STZ)-induced diabetic mice, supplementation of diet with guava fruits powder was shown to improve glycaemic control and diabetes pathology markers of nephropathy (Li et al., 2015). Improvement of the triglyceride (TG) and inflammatory markers were other parameters that will be discussed under the mechanisms of action. The study by Lin and Yin (2012) was also in perfect agreement where supplementation of diet with the fruits extract could increase insulin level, and ameliorate hyperglycaemia along with various pathological markers in STZ-induced diabetic mice. In this model, the reduction of blood glucose by the fruits extract was also shown to be associated with pancreatic β-cell protection through antioxidant and anti-inflammatory mechanisms (Huang et al., 2011). The fruit peals extracted with water could also display hypoglycaemic effect in STZ rats when assessed both by fasting blood glucose (FBG) and postprandial glucose levels (Rai et al., 2007, 2009). The fruits and juices in STZ and alloxan-induced diabetic mice model have also been shown

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FIG. 9.17 Non-oxygenated carotenoids of guava.

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FIG. 9.18

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Oxygenated carotenoids of guava.

to display antidiabetic effects in various studies (Cheng and Yang, 1983; Yusof and Said, 2004). Improvement of glucose tolerance as a measure of reduction in postprandial glucose level was also demonstrated for guava fruits extract (400 mg/kg) (M€ uller et al., 2018). Lin et al. (2016) studied the effect of a rather complicated guava juice preparation with water and alcohol extraction followed by preparation in combination with trehalose. They have shown some improvement in oral glucose tolerance test (OGTT), plasma insulin and homeostatic model assessment of insulin resistance (HOMA-IR) in diabetic rats induced by a combination of high fructose, STZ and nicotinamide.

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TABLE 9.4

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In vivo antidiabetic, lipid lowering and antiobesity effect of guava Assay model and treatment protocol

Main outcome

Reference

Leaves and fruits extracts—Ethanol (80%) or Supercritical fluid extraction

OGTT in mice—400 mg kg

Decrease postprandial glucose

M€ uller et al., 2018

Leaves—Hot water extract

STZ-induced diabetic rats—100, 200 or 400 mg/kg, p.o. for 45 days

Dose dependently, raise insulin, glycogen, hexokinase, G6Pase dehydrogenase; suppress hepatic markers, gluconeogenic enzymes, and OGTT or FBG levels; attenuate the TG, TC, phospholipids, FFAs, and LDL levels and raised HDL levels; activate IRS-1, IRS-2, Akt, p-Akt, PI3K, GLUT2, AMPK, p-AMPK, and p-ACC.

Vinayagam et al., 2018

Leaves—Hot water extract

STZ-induced diabetic rats—100, 200 or 400 mg/kg, p.o. for 45 days

Reverse the following diabetes abnormalities: reduced level of insulin, accompanied by high blood glucose, LPO product (TBARS, LOOH) and, and inflammatory cytokines (IL-6, TNF-α; reduced levels of insulin and suppressed antioxidants (e.g., CAT, GR, SOD, GSH).

Jayachandran et al., 2018

P. guajava L. var. pyrifera leaf extract—Ethanol extract

HFD-induced diabetes/obesity in mice—5 mg/kg p.o. for 7 weeks

Reduce glucose and insulin resistance; improve the serum lipid profile (LDL-C and HDL-C ratio) in obese mice; no weight loss effect.

Dı´az-de-Cerio et al., 2017

Leaves—Ethyl acetate fraction or quercetin as active component

STZ-induced diabetic rats— 50 mg/kg, i.g. for 60 days

Increase insulin level; reduce the glycated haemoglobin and fructosamine levels; reduce the translocation of NF-κB from cytosol to nucleus in diabetic rats; downregulate the expression of TGF-β1, CTGF (Connective tissue growth factor) and BNP (brain natriuretic peptide). Protective effect of the extract was better than quercetin

Soman et al., 2016

Fruits excluding seeds— Concentrated water and ethanol extract from juice

High fructose plus STZ and nicotinamide-induced diabetic rats—(2, 4, 8, 20 20 mL/kg) of 40% guava juice p.o. for 4 weeks.

Improve OGTT, plasma insulin, HOMA-IR and HOMA-β BUT not HbA1c, level; decrease renal ROS, 4-hydroxynonenal, caspase-3/apoptosis, LC3-B/

Lin et al., 2016

Plant source

Continued

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TABLE 9.4 In vivo antidiabetic, lipid lowering and antiobesity effect of guava—Cont’d Assay model and treatment protocol

Plant source

Main outcome

Reference

Treatment in combination with trehalose.

autophagy and IL-1β/ pyroptosis. Content of quercetin in the guava juice sample was 0.633 μg/ml.

Fruits—red guava (redfleshed guava cultivar. Freeze-dried powder of edible portion)

STZ-induced diabetic mice—1%, 2%, and 5% in diet fed for 8 weeks

Improve blood glucose control, insulin resistance, creatinine, BUN, TG, non-esterified fatty acids, cholesterol, CRP, TNF-α, and IL-10; suppress iNOS and NF-κB expressions; activate PPAR-γ; increase GPx3 and ACO levels in the liver.

Li et al., 2015

Leaves—Aqueous extract

Fructose (15%) in water-induced diabetic rats—250 or 500 mg/kg, p.o. for 8 weeks

Reverse the high fructoseinduced raise in insulin, leptin and hepatic GLUT2 expression, insulin resistance, dyslipidemia, and hypertension; increase preference to pellet diet than fructose; reduce weight gain.

Mathur et al., 2015

Leaves

STZ-induced diabetic rats—leaf extract at a dosage of 300-mg/kg b.wt. for 30 days admin route not available

Reduce blood glucose, insulin, and HbA1c levels; restore activities of carbohydrate metabolizing enzymes.

Khan et al., 2013

Leaves—Ethyl acetate

STZ-induced diabetic rats—25 or 50 mg/kg, i.g. for 30 days

Decrease blood glucose, glycated hemoglobin (HbA1c) and fructosamine levels in a dose dependent manner; decrease cardiac isoform of liver alpha 2 macroglobulin; restore of serum transaminases (AST and ALT)

Soman et al., 2013

Leaves—Ethanol (70% v/v in water) extract

SHRSP.Z-Leprfa/Izm rats—2 g/ kg, p.o. for six weeks

No effect on water and food consumption or levels of FBG and insulin; reduce body weights; reduce plasma glucose at 60 and 120 min OGTT; reduce the values of AUC and QUICKI at 42 days post treatment; promote IRS-1, Akt, and PI3Kp85 expression and phosphorylation of IRS-1, AMPK, and Akt308, but not Akt473; increase the ratios of membrane to total GLUT4 expression and adiponectin receptor 1 transcription in the skeletal muscles.

Guo et al., 2013

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TABLE 9.4

281

In vivo antidiabetic, lipid lowering and antiobesity effect of guava—Cont’d

Plant source

Assay model and treatment protocol

Fruits—Aqueous and ethanol extracts

Main outcome

Reference

STZ-induced diabetic mice—1% or 2% in diet fed for 12 weeks

The 2% supplement can reduce glucose and BUN levels, increase insulin level in plasma of diabetic mice; reserve (dose-dependently) the suppressed GSH content and activity of CAT and GPx; decrease ROS, IL-6, TNF-α and IL-1β levels in the kidney; treatments at 2% suppress renal N (ε)-(carboxymethyl)lysine, pentosidine and fructose levels and renal activity of aldose reductase.

Lin and Yin, 2012

Leaves—70% (v/v) Ethanol extract

SHRSP.Z-Leprfa/IzmDmcr rats (SHRSP/ZF)—2 g/kg, p.o., for 6 weks

Decrease body weight gain, TG content of the liver and blood AST and ALT; increased genes expression for adiponectin receptors (AdipoR1) and AdipoR2; increase phosphorylation of AMPK and mRNA expression of PPAR-α; increase the hepatic mRNA expression of beta-oxidation enzymes (medium-chain acylCoA dehydrogenase and ACO) and mitochondrial fatty acid β-oxidation (CPT activity); increase phosphorylation of Akt at ser473 while suppressing mRNA level of PEPCK.

Yoshitomi et al., 2012a

Fruit—Freeze-dried powder

STZ-induced diabetic rats—125 or 250 mg/kg, p.o. for 4 weeks

Restore body weight loss; reduce blood glucose levels in a dosedependent manner; protect pancreatic β-cells against lipid peroxidation and DNA damage; inhibit NF-κB protein expression; restore the activities of antioxidant enzymes (SOD, CAT, and GPx).

Huang et al., 2011

Leaves—Ethyl acetate fraction

STZ-induced diabetic rats—25, 50 or100 mg/kg i.g. for 30 days.

Improve antioxidant status (decrease LPO and increase in the activity of CAT, SOD, GPx and GR); reduce glycated haemoglobin and fructosamine.

Soman et al., 2010

Continued

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TABLE 9.4 In vivo antidiabetic, lipid lowering and antiobesity effect of guava—Cont’d Assay model and treatment protocol

Plant source

Main outcome

Reference

Fruit peel—Aqueous extract

STZ-induced diabetic rats— 400 mg/kg p.o. for 3 weeks or after 8 h

Decrease FBG or post prandial glucose (OGTT) and urine sugar; increase haemoglobin level and body weight (body weight loss recovery).

Rai et al., 2007, 2009

Leaves—Aqueous and ethanol extracts

STZ and nicotinamide-induced diabetic rats—400 mg/kg p.o.

Acute and long-term administration reduce blood sugar; long-term administration increase insulin level and glucose utilization; increase activities of hepatic hexokinase, phosphofructokinase and G6PD.

Shen et al., 2008

Leaves extract—Butanol fraction obtained from methanol extract

Leprdb/Leprdb diabetic mice— 10 mg/kg i.p., 4 weeks

Reduce blood glucose and lipid droplets in the liver (histology); inhibit PTP1B activity in vitro.

Oh et al., 2005

Leaves—Aqueous extract

Alloxan-induced diabetic rats— 250 mg/kg p.o.

Screening in acute and sub-acute tests showed hypoglycemic effects.

Mukhtar et al., 2004

Fruits

STZ-induced diabetic mice
0.517 g/animal for 5 weeks

Insignificant reduction in FBG level

Yusof and Said, 2004

Fruits—Juice

Alloxan-treated diabetic mice— 1 g/kg., i.p.

hypoglycemic action in normal and diabetic animals.

Cheng and Yang, 1983

Abbreviations: ACC, acyl CoA carboxylase; ACO, acyl CoA oxidase; Akt, protein kinase B; AMPK, 50 adenosine monophosphate-activated protein kinase; ALT, alanine aminotransferase; AST, aspartate transaminase; BUN, blood urea nitrogen; CAT, catalase; CPT, carnitine palmitoyltransferase; CRP, C-reactive protein; FBG, fasting blood glucose; p.o., oral route of administration; G6Pase, glucose-6-phosphatase; G6PD, glucose-6-phosphate dehydrogenase; GLUT, glucose transporter; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, glutathione (reduced form); HDL, high density lipoprotein-cholesterol; HFD, high-fat diet; HOMA, homeostatic model assessment; HOMA-β, β cell function and insulin secretion; HOMA-IR, insulin resistance; i.g., intragastric route of administration; IL, interleukin; iNOS, inducible nitric oxide synthase; i.p., intraperitoneal route of administration; IRS, insulin receptor substrate; TG, triglyceride; TC, total cholesterol; FFAs, free fatty acids; LDL-C, low density lipoprotein-cholesterol; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; LOOH, lipid hydroperoxides; LPO, lipid peroxidation; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; OGTT, oral glucose tolerance tests; p-Akt, p-AMPK, and p-ACC are phosphorylated form of Akt, AMPK and ACC respectively; PPAR-γ, peroxisome proliferator-activated receptor-γ; PEPCK, phosphoenolpyruvate carboxykinase; PTP1B, protein tyrosine phosphatase 1B; QUICKI, quantitative insulin sensitivity check index; ROS, reactive oxygen species; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances; TNF-α, tumor necrosis factor-a.

9.4.2 The pharmacology of guava leaves in diabetes and associated diseases In a classical STZ-induced diabetic-rat model, antihyperglycaemic effect coupled with increased insulin levels have been shown for treatment with the leaves extract ( Jayachandran et al., 2018; Shen et al., 2008; Vinayagam et al., 2018). In the STZ-rat model, reduction of the glycated haemoglobin (HbA1c) and fructosamine levels (Soman et al., 2016) along with the diminished levels of liver enzyme markers in the serum (transaminases levels) (Soman et al., 2013) were also evident for the leaves extract. Improvement of glucose tolerance (OGTT) as a measure of reduction in postprandial glucose level was also demonstrated in

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either normal (M€ uller et al., 2018) or STZ-induced diabetes rats (Vinayagam et al., 2018). The lipid lowering effect of the leaves was further evident from studies on the leaves extract where the levels of TG, total cholesterol (TC), free fatty acids (FFAs) and low density lipoprotein cholesterol (LDL-C) were suppressed, while high density lipoprotein cholesterol (HDLdC) was increased in STZ-induced diabetic rats (Vinayagam et al., 2018). Other studies on the leaves include the alloxan-induced diabetic rat model where hypoglycaemic effects were shown (Mukhtar et al., 2004). Numerous other improvements in diabetes pathology have also been documented in the STZ model including restoration of body weight loss (Huang et al., 2011) and antioxidant status (Huang et al., 2011; Soman et al., 2010) that will be discussed under the mechanisms of action. In the classical high-fat diet (HFD)-induced obesity model in mice, Dı´az-de-Cerio et al. (2017) did not observe antiobesity effect but improvement of the serum lipid profile as a measure of LDL-C:HDL-C ratio was observed (Dı´az-de-Cerio et al., 2017). The lack of antiobesity effect should also be seen with caution as the dose tried was so small—only 5 mg/kg of the crude extract (p.o. for 7 weeks). In a fructose induced-diabetes model in rats, the augmented insulin and leptin levels along with insulin resistance and hepatic/lipid dysregulations could be reversed by the leaves extract (Mathur et al., 2015). By introducing fructose (15%) in water, Mathur et al. (2015) studied the effect of the aqueous leaves extract on diabetes and obesity markers. Beyond reversing insulin resistance (HOMA-IR value) and suppressing the raised level of glucose and insulin, antiobesity potential was demonstrated in a dose-dependent manner. After eight weeks of administration of the aqueous extract of the leaves (250 and 500 mg/kg), a preference for pellet diet than fructose leading to diminished total calories of 25.9% and 18.71%, respectively were recorded. This evidence also clearly indicated a loss of body weight gain in treated group even when compared to the nondiabetic control group. The suppression of fructose-induced fatty liver and hepatic expression of glucose transporter (GLUT2) is a further indication of potential antiobesity effect. A number of other experiments showing the antidiabetic effect of the leaves have also been presented including in the alloxan-induced diabetes rat model Okpashi et al. (2014). The experiment by Guo et al., 2013 on SHRSP.Z-Leprfa/Izm rats (SHRSP/ZF) as a model of spontaneous metabolic syndrome in animals was also worth mentioning. First, the dose used for the aqueous ethanolic extract was very high at 2 g/kg and no effect on FBG or insulin level were observed. Hence, even though the model was different from many other experiments such as the STZ- or alloxan-induced diabetic models, it appears to be in contradiction with other studies outlined above. On the other hand, their data was consistent with improvement of glucose tolerance and insulin sensitivity in the skeletal muscles of SHRSP/ZF rats as well as body weight lowering effects. In another study using this animal model (Yoshitomi et al., 2012a), the leaves extract at the same dose was also shown to suppress bodyweight gain and TG levels along with the serum level of liver enzymes such as aspartate transaminase (AST) and alanine aminotransferase (ALT). Another diabetes model employed was Leprdb/Leprdb diabetic mice (C57BLKS/J background) which was homozygous for the diabetes spontaneous mutation and hence exhibit diabetes pathology within their 4-weeks old age. By employing this model, Oh et al. (2005) have shown that the leaves extract display antidiabetic and lipid lowering effects. On the other hand, their in vitro study revealed protein tyrosine phosphatase 1B (PTP1B) inhibition that could in part explain the observed in vivo effects.

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Other plant parts of the plant may also be of therapeutic significance. For example, the stem-bark has been demonstrated to display antidiabetic effect in alloxan-induced model in rats (Mukhtar et al., 2006). A number of traditional herbal formula like the Chinese medicines containing guava leaves have also been shown to display antidiabetic effects (Wang and Chiang, 2012). Other plants related to guava (e.g., P. cattleianum) could also display antidiabetic effects in animal models (de Souza Cardoso et al., 2018).

9.5 Detailed mechanism of action from studies on guava extracts in vitro, in vivo and active principles 9.5.1 Modulation of insulin and other signaling molecules related to glycaemic and lipid control The experiment by Vinayagam et al. (2018) addressed the major mechanism of action of guava on STZ-induced diabetic rats. In their experiment using two doses of the water extracts of the leaves, they have demonstrated antidiabetic effect as evidenced from a reduced blood glucose level including in OGTT, increased insulin level, and recovery of the diabetesinduced weight loss. As a major glucose homeostasis target, their study on the liver shed some light on glucose metabolism both in storage (glycogen) and synthesis (gluconeogenesis). The hexokinase and glucose-6-phosphate dehydrogenase (G6PD) which are diminished in diabetes rats could be recovered by the extract treatment. On the other hand, the gluconeogenesis process with the key activity of glucose-6-phosphate (G6Pase) that converts glucose6-phosphate to glucose and phosphate is augmented in diabetes or insulin deficiency. This critical enzyme has also been shown to be suppressed by the leaves extract. These data together with the various biochemical markers that are reversed to normal level explain the antidiabetic mechanism of action for guava. Another similar study on the leaves was by Shen et al. (2008) that used the STZ and nicotinamide-induced diabetic model in rats. The reduction in blood glucose and increased insulin level after long-term administration of the aqueous and ethanol extracts (400 mg/kg, p.o.) of the leaves was coupled with increased glucose utilization and activities of hepatic hexokinase, phosphofructokinase and G6PD. As a major intracellular reducing agent, nicotinamide adenine dinucleotide phosphate (reduced state, NADPH) is required in numerous reactions including the mitochondrial energy metabolism, pentose phosphate pathway, and antioxidant pathways. The G6PD being the principal source of cellular NADPH, its inhibition by high level of glucose or diabetes both at the expression and activity levels triggers a host of metabolic abnormalities. One hallmark abnormality in diabetes is oxidative stress associated with G6PD loss leading to β-cells loss among others (Zhang et al., 2010). The increased level of expression of this enzyme by guava is thus expected to restore the redox balance and constitute another mechanism in addition to inhibition of gluconeogenesis (Fig. 9.19). Another well-defined mechanism of action for guava is modulation of insulin signaling molecules that are critical to both carbohydrate and lipid metabolism. The study by Vinayagam et al. (2018) on the leaves extract, for example, showed upregulation of the following in the liver of STZ-induced diabetic rats by guava treatment: IRS-1, IRS-2, Akt, p-Akt, PI3K, GLUT2, AMPK, p-AMPK, and p-ACC (Table 9.4). Hence, the insulin signaling pathway

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FIG. 9.19 Hexokinase, glucose-6-phosphatase and glucose-6-phosphate dehydrogenase as targets for guava leaves and fruits. Glucose utilization is promoted by activating hexokinase enzymes that are downregulated under diabetes conditions while the last step of gluconeogenesis is catalyzed by G6Pase which appears to be upregulated by diabetes. G6PD catalyzes a key process of the pentose phosphate pathway: it generates cellular reductant NADPH which is vital for the sequential reaction of antioxidant enzymes glutathione reductase (GR) and glutathione peroxidase (GPx) that remove reactive oxygen species (ROS) such as H2O2. Guava extracts upregulate hexokinases and G6PD while suppressing G6Pase.

from the insulin receptor substrates to PI3K/Akt pathway of the AMPK activation via its phosphorylation is involved. This suggest increased glucose uptake and utilization in insulin-sensitive vital organs. In the STZ-induced diabetic mice, the fruits of red guava given to animals as dietary supplements (1%, 2%, or 5% of diet) has also been shown to increase PPAR-γ, GPx3 and acyl CoA oxidase (ACO) in the liver (Li et al., 2015). While PPAR-γ is a well-established target both for diabetes and obesity, the effect on ACO is worth further scrutiny. Studies on SHRSP/ZF rats also suggest that the leaves extract display antiobesity effect not only by increasing genes expression for adiponectin receptors but also the hepatic mRNA levels for PPAR-α and β-oxidation enzymes (Yoshitomi et al., 2012a,b). In the latter case, the medium-chain acyl-CoA dehydrogenase and ACO and mitochondrial fatty acid β-oxidation (carnitine palmitoyltransferase (CPT activity) was augmented by the extract. The CPT is primarily responsible for the conversion of fatty acyl-CoAs into fatty acyl-carnitine molecules to facilitate fatty acids entry into the mitochondrial matrix for β-oxidation. Hence, both the leaves and fruit extracts of guava appear to work in a similar fashion to promote fatty acids entry into the mitochondria and their oxidation by key enzymes such as ACO. This explains induction of energy expenditure, antiobesity potential and increased insulin sensitivity by guava. One of the most comprehensive study on the antiobesity potential of guava leaves came from the study by Yoshitomi et al. (2012a,b) who used SHRSP.Z-Leprfa/IzmDmcr rats

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(SHRSP/ZF rats) as a model of metabolic syndrome. Beyond demonstrating the suppressive effect of the extract on weight gain and amelioration of liver damage marker enzymes such as ALT and AST, they have shown the involvement of adiponectin and related signaling pathways in fatty liver development that could be summarized as follow: • Even though obesity is known to decreases the plasma level of adiponectin, this was not demonstrated for the extract but the adiponectin receptor expression that was expected to diminish by hyperinsulinaemia appeared to be reversed. The expression of adiponectin receptor 1 and 2 was increased in diabetic rats; • The activity of AMPK and mRNA expression of PPAR-α, that are regulated by adiponectin receptors are also improved. By regulating the AMPK pathway, the effect of adiponectin to enhance glucose utilization as well as fatty acid oxidation appear to be established for the extract in this metabolic syndrome model. In the same way, the increased activity of adiponectin by the extract is expected to activate PPAR-α leading to initiation of fatty acid oxidation and ultimately lowering the tissue TG content; • Considering the critical role of PPAR-α in lipid catabolism, the increased genes expression level and activity of the medium-chain acyl-CoA dehydrogenase, ACO and CPT by the extract is in good agreement. As the rate-limiting enzyme of mitochondrial β-oxidation, the observed effect on CPT and various signaling cascades of adiponectin appears to lay down the foundation for the antiobesity, lipid lowering effect and subsequently the antidiabetic potential of the leaves extract. All these valuable data on the antiobesity potential demonstration for guava leaves using SHRSP.ZLeprfa/IzmDmcr rats could also be substantiated through in vitro studies. The study by Yoshitomi et al. (2012b) utilized the 3T3-L1 adipocyte differentiation as a model of pre-adipocytes transformation to mature adipocytes. In adipogenesis process, the expression of early adipocyte differentiation markers, CCAAT-enhancer-binding proteins such as C/EBP-β and C/EBP-δ, are followed by the induction of PPAR-γ and C/EBP-α. The inhibition of these markers (PPAR-γ and C/EBP-α) at mRNA level by the leaves extract along with various parameters of differentiation including cell mass was in line with the antiobesity potential of the plant. The downstream signaling markers of PPAR-γ, such as aP2, were also inhibited by the extract. In agreement with the in vivo experiment, activation of AMPK as demonstrated by the increased expression of p-AMPK is in support of body weight loss. Hence, the authors conclusion of the extract suppressing adipocyte differentiation by down-regulating the expression of PPAR-γ and C/EBP-α and downstream adipogenic genes as well as inhibition of clonal expansion by enhancing AMPK activity is well justified. As described in the various chapters of this book, PPAR-γ play critical role during the adipogenesis, adipocyte differentiation and/or lipid accumulation processes while PPARα stimulates the expression of genes primarily involved in the peroxisomal and mitochondrial fatty acids β-oxidation in vital organs such as the liver. Hence, the activation of the PPAR signaling in the liver by guava also explain the ameliorative effect of guava on weight gain/obesity or lipid dysregulation. The study by Guo et al. (2013) in the same animal model (SHRSP/ ZF rats) that showed enhancement of insulin sensitivity by the leaves extract was coupled with improvement of the above-mentioned signaling molecules in skeletal muscles: increased expression of IRS-1, Akt, and PI3Kp85 expression and IRS-1, AMKP, and Akt308. Inevitably, this signaling boost by the extract led to increased level of GLUT4 and adiponectin receptor-1

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on the cell surface of skeletal muscles (Guo et al., 2013). Increased glucose transports in muscle cells suggest more glucose uptake and insulin sensitivity. In line with the above-mentioned mechanism of action, evidence for the antidiabetic potential of aqueous extract of the leaves did also come from in vitro studies using high glucose-induced insulin-resistant mouse FL83B cells. The extract (200 and 400 μg/mL) was shown to increase glucose uptake in both normal and insulin-resistant cells leading to increased glycogen content (Liu et al., 2015). Mechanistically, enhancement of insulin receptor (IR) signaling including the phosphorylation of the IR, IRS PI3K, Akt (Ser), Glut2, and total glycogen synthase were shown (Liu et al., 2015). Cheng et al. (2009) have also studied the effect of the aqueous extracts of leaves on glucose uptake by rat clone 9 hepatocytes. The extract as well as quercetin (Fig. 9.4) as one of the main active components were shown to enhance glucose uptake by liver cells. The extract has also been shown to enhance the insulindependent and insulin-independent glucose transport in cultured 3T3-L1 adipocytes (Owen et al., 2008). Another direct evidence on the antidiabetic effect of guava fruits come from the study by Li et al. (2015). They used no processing or extraction steps of the plant material and rather used freeze-dried powder of the edible fruits in STZ-induced diabetes model. Their feeding experiment constitute three levels: low, medium and high as 1%, 2% and 5% diet respectively. For the readers’ clarity of this direct evidence, the following key observations are worth presenting: • Induction of GPx3 protein expression in the liver of 2% and 5% supplements; • Suppression of C-reactive protein (CRP) and TNF-α levels while the level of IL-10 was increased by all treatment (see Table 9.4); • Kidney function restored—all treatment suppressed the increased creatinine levels in diabetic mice; • Suppressed blood urea nitrogen (BUN) levels by all treatment—kidney function restored; • Blood lipid levels (TG and cholesterol) were significantly lower for all supplements; • ACO expression levels increased in mice livers by all supplements; • NF-κB and iNOS in the liver suppressed by all treatment (see anti-inflammatory mechanism in Section 9.5.3); • PPAR expression (protein and mRNA expression) levels in the epididymal fat increased for the 2% and 5% supplements; • The HOMA-IR index was reduced to normal level by the treatments. Even though the authors suggestion of quercetin, lycopene and ursolic acid as active principles needed further evidence, the antidiabetic effect of guava fruits appears to be substantiated in this classical animal studies. Another similar study by Lin and Yin (2012) was on 1% and 2% level of diet fortification by the aqueous and ethanolic extracts of the fruits for 12 weeks. Their data clearly showed anihyperglycaemic, antioxidant and anti-inflammatory mechanisms of the fruits (Table 9.4). The results also showed that even extraction by water and ethanol can lead to variability in the phenolic constituents although both preparations appear to be active as antidiabetic agents. Hence, the antidiabetic, lipid lowering and antiobesity effects of guava appear to be well established in animal models and the mechanism of action appeared to be diverse and include modulation of signaling molecules in carbohydrate and lipoid metabolism. By using preadipocyte differentiation and dexamethasone-induced

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insulin resistance in 3T3-L1 cells as a model, the study by Lin et al. (2013) have shown that ursolic acid as a constituent of guava leaves may act as an active principle. The compound (30 or 100 μM) could enhance basal and insulin-stimulated glucose uptake in insulin resistant adipocytes while decreasing FFAs production. Ursolic acid was also shown to increase adiponectin secretion in insulin resistant adipocytes while upregulating PPAR-γ protein (Lin et al., 2013).

9.5.2 Antioxidant mechanism The direct relevance of antioxidant potential of guava to its antidiabetic effect has been demonstrated through various experimental models (Table 9.4). This include inhibition of lipid peroxidation product while antioxidant defenses such as enzymes activities (CAT, GR, SOD, GR and/or GPx) and GSH levels were raised by guava leaves (Huang et al., 2011; Jayachandran et al., 2018) or by the fruits (Lin and Yin, 2012; Soman et al., 2010). While these enhancements of antioxidant defenses in diabetic animals could be a result of multiple mechanisms, a range of other related effects for the extracts and known active principles are outlined below. As polyphenolic-rich extracts, the leaves and fruits of guava preparations are expected to display antioxidant effects in a variety of assay models. For guava leaves, Irondi et al. (2016) reported the direct potent radical scavenging in 2,2-diphenyl-1-picrylhydrazyl (DPPH) (IC50, 13.4 μg/mL) and 2,20 -azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) (IC50, 3.2 μg/ mL) as well as xanthine oxidase (IC50, 13.2 μg/mL) and Fe2+-induced lipid peroxidation (IC50, 27.5 μg/mL) inhibitory effects. Considering the polyphenolic composition of the leaves listed in Section 9.3, the authors quantify the flavonoids level in the following order: quercetin > kaempferol > catechin > quercitrin > rutin > luteolin > epicatechin; while the concentrations of phenolic acids in the extract were in the order of caffeic acid > chlorogenic acid > gallic acids. In view of this reality, the antioxidant effect of the leaves is nothing but all expected. Guava fruits extracts from the various genotypes (Allahabad, Safeda, Fan Retief and Ruby Supreme) have also shown antioxidant effects when assessed by various in vitro (ABTS, DPPH, FRAP and ORAC) assays (Thaipong et al., 2006). As expected, variability depending on the plant sources have been reported and positive correlation of activity with the level of phenolic content in the extract was also observed. Variability in the antioxidant effects of some cultivars with differences in pulp color (white verses pink) have also been shown by Flores et al. (2015). In their study comprising seven cultivars of guava fruits, pink-pulp guavas (Barbie Pink, Homestead, Sardina 1, Sardina 2) had higher radical scavenging activity (DPPH and ABTS assays) than the white pulp cultivars (Yen 2 and Sayla), which intern was less than the red pulp guava cultivar (Thai Maroon). After demonstrating the pulp and peel of guava fruits as good sources of dietary fiber, Jimenez-Escrig et al. (2001) have further established antioxidant effects that appear to be correlated with their polyphenolic content. A number of other studies have also shown the antioxidant potential of guava. These include polyssacchrides from the fruits in DPPH radical scavenging (Zhang et al., 2016); leaves extract inhibiting nitric oxide (NO) production by macrophages (RAW264.7 cells) ( Jang et al., 2014b); and ABTS scavenging and inhibition of linoleic acid oxidation in vitro by the leaves (Chen and Yen, 2007).

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Closely related to the antioxidant mechanism of antidiabetic effect is also antiglycation pharmacology. As a measure of non-enzymatic glycation of proteins, glucose or other reducing sugars are incubated with bovine serum albumin (BSA) to induce the formation of Schiff base and subsequently fluorescent Amadori products. Fluorescence measurement or reduction of nitro blue tetrazolium (NBT) by Amadori products in alkaline solution to yield a colored monoformazan dye can then be measured spetrometrically. On this basis, Wu et al. (2009) have done extensive study on the antiglycation effect of both the leaves and fruits of guava extracted with hot water. The glycation process of BSA by glucose or in combination with Cu2+ as well as quantification of α-dicarbonyl compounds (glyoxal) as intermediates to advanced glycation end products (AGEs) formation have been shown to be suppressed by guava extracts. Moreover, the dried fruits were more potent than the leaves from few varieties (such as Shi Ji Ba and Tu Ba) as well as guava tea. The antiglycation effect was also demonstrated for the isolated compounds (gallic acid, ferulic acid, catechin and quercetin) with quercetin showing the strongest action. Hence, the phenolic antioxidant compounds are also the active principles for the antiglycation effect of guava extracts. Soman et al. (2010, 2013) showed antioxidant effects in vitro for the methanol extract of the leaves with the ethyl acetate (EtOAc) fraction displaying the maximum in vitro antioxidant (DPPH) and antiglycative potential as compared with others (hexane, chloroform and butanol fractions). Antiglycation effect and ferrous ion chelation have also been shown for the aqueous extracts of both the leaves and fruits of guava (Wu et al., 2009). In a model of α-dicarbonyl compounds-induced blood coagulation, the aqueous extract of guava leaves could reverse the decrease in thrombin clotting time induced by methylglyoxal. The extract and its active phenolic compounds including ferulic acid, gallic acid and quercetin also displayed protective effect against methylglyoxal-induced loss of activity of antithrombin III (Hsieh et al., 2007b); and antioxidant effect for the aqueous fruit extract in the DPPH scavenging, reducing (ferric) power, and copper-catalyzed human low-density lipoprotein (LDL) oxidation assays ( Jimenez-Escrig et al., 2001). The glyoxal and methylglyoxal-inducedinduced endothelial cells (human umbilical vein endothelial cell (HUVEC)) damage in vitro could also be ameliorated by guava leaves extract (Hsieh et al., 2007a). From all the data presented above, antioxidant effect should be considered as one mechanism for amelioration of pathologies by guava extracts and compounds in diabetesassociated diseases. This is by increasing the antioxidant capacity such as enzymes as demonstrated in animal models or suppression of ROS/glycation levels and activity by polyphenolic compounds.

9.5.3 Anti-inflammatory mechanisms Inflammation being one established link between obesity and diabetes, antiobesity and antidiabetic assays as shown in Table 9.4 often include measurements of inflammatory markers. In STZ-diabetic rats, the anti-inflammatory effect of guava leaves was associated with suppressed level of inflammatory cytokines such as IL-6 which TNF-α ( Jayachandran et al., 2018) which play significant role in insulin resistance. The inhibition of translocation of NF-κB from cytosol to nucleus in tissues of diabetic rats by the lives extract is a further example of anti-inflammatory effect in STZ-induced diabetic rats (Soman et al., 2016). For the

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fruits extracts, the study by Li et al. (2015) also showed similar improvement profile in inflammation associated with STZ-induced diabetes in mice. While the inflammatory marker CRP was suppressed along with TNF-α, the anti-inflammatory cyokine (IL-10) level in the liver was augmented. The level of expression of iNOS and NF-κB in the liver were also suppressed (Li et al., 2015). The suppressive effects of the fruits extract on the level of IL-6, TNF-α and IL-1β levels in STZ-induced diabetic mice kidneys were also shown (Lin and Yin, 2012). In vitro studies using the lipopolysaccharide (LPS)-induced cyclooxygenase-2 (COX-2) and iNOS expression in murine macrophage cell lines have shown that guava leaves extract can display anti-inflammatory effect by targeting the transcription factor, NF-κB (Choi et al., 2008). Inhibition of prostaglandin-E2 (PGE2) and NO production was also shown for the leaves extract in the LPS-activated RAW 264.7 cells ( Jang et al., 2014b). Im et al. (2012) have shown that the butanol fraction of guava leaves have inhibitory effect against matrix metalloproteinases (MMP)-9 and MMP-2 expression in lung cancer cells via downregulation of the ERK1/2 activation. Their ESI-MS/MS showed the major components of the fraction were D-glucuronic acid, quercetin 3-glucuronide, loganin, and xanthyletin. The antiangiogenic effect of the leaves extract has also been demonstrated by Peng et al. (2011) both in vitro and in vivo. In cancer cells, the expression of cytokines including vascular endothelial growth factor (VEGF), IL-6 and IL-8 were suppressed along with MMP-2 and MMP-9 that are critical for angiogenesis. On the other hand, the expression of tissue inhibitor of metalloproteinases 2 (TIMP-2) was induced and most critically, Chicken chorioallantoic membrane assay revealed a clear antiangiogenesis potential in vivo. The aqueous extract of guava leaves has been shown to display anti-inflammatory and analgesic effects in rats and mice. A dose-dependent effect (50–800 mg/kg, i.p.) against thermally- and chemically-induced nociceptive pain in mice and egg albumin-induced pedal (paw) edema in rats (Ojewole, 2006) were shown. While many active components including the terpenoids and flavonoids are likely to attribute to such effects, the essential oils obtained by steam distillation from the leaves have also been shown to display anti-inflammatory effects in carrageenan-induced paw edema and cotton pellets models in rats (Kavimani et al., 1997). With regard to the antinoceptive effect of the leaves, essential oils have also been implicated to play major role (Santos et al., 1998). The essential oil with β-caryophyllene (Fig. 9.12) as major component has also been shown to ameliorate the chemical (formalin and acetic acid) and thermal (hot-plate)-induced nociception in mice at doses of 200 and 400 mg/kg. Some of these effects may be through action at the CNS level, including potential depressant action (Meckes et al., 1996; Shaheen et al., 2000).

9.5.4 Effects on carbohydrate digestion or absorption Considering the polyphenol composition of the leaves and fruits, inhibitory effects on α-glucosidase and α-amylase activity, at least in vitro, by the extracts are also somehow expected. Direct evidence for this mechanism came from the study by Wang et al. (2010) on the leaves extract, fractions and purified compounds. The activity reported in higher mg/ml level was, however, very weak. For the seven compounds (quercetin, kaempferol, guaijaverin, avicularin, myricetin, hyperin and apigenin (Fig. 9.4 and Table 9.2)) isolated using the bioassay-guided fractionation, the IC50 values for the two enzymes were over

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3 mM. The potential additive and/or synergistic effects of flavonoids in enzyme inhibition including α-glucosidases have been outlined previously (Habtemariam, 2011) and herein, the α-glucosidase (sucrase and maltase) inhibitory effect of quercetin and myrecinin; hyperin and avicularin or kaempferol and quercetin combinations could be relevant. Indeed, the IC50 value obtained in the combination studies were lower than when all of the compounds were tested alone but the effect is still lower than a synergistic effect. The experiment by Wang et al. (2007) further added more relevance to the α-glucosidase inhibitory potential of guava leaves as they used α-glucosidase enzymes from small intestinal mucosa of diabetic mice induced by STZ. Their data once again were however in the potency range of 1 mg/ml. The water-soluble polysaccharides of guava fruits have also been investigated for their α-glucosidase inhibition. The study by Zhang et al. (2016) used hot water extraction and 90% ethanol precipitation to get the polysaccharides which were subjected to chromatographic isolation to obtain a novel polysaccharide (GP90-1B, Fig. 9.20). Interestingly, the crude polysaccharide preparation from which GP90-1B was isolated from showed antioxidant activity (DPPH radical scavenging) and α-glucosidase enzymes inhibition which was by order of magnitude better than acarbose. In fact, two of the polysaccharide fraction with EC50 of 2.27 μg/mL and 0.18 mg/mL were reported to be 1379 and 17-times better than acarbose (EC50–3.13 mg/ml). Even through more studies are required to confirm the predicted structure of the overall polysaccharide structure, previous studies on guava fruit polysaccharides also resulted in another predicted novel polysaccharide, GP70–2 (Fig. 9.21) as follow (Hua et al., 2014): FIG. 9.20

The structure of the polysaccharide GP90-1B. The sugar monomers of α-L-arabinose and α-D-glucose making the backbone structure with main linkages of (1 ! 5)-α-L-arabinose, (1 ! 2,3,5)-α-Larabinose and (1 ! 3)-α-L-arabinose. The branching linkages were composed of (1 ! 6)-α-D-glucose, (1!)-α-D-glucose and (1!)-α-L-arabinose.

FIG. 9.21 The predicted backbone structure of polysaccharide GP70.

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The leaves and fruits (ethanol (80%) or supercritical fluid extraction) extracts suppressing glucose uptake in Caco-2 cells by inhibiting SGLT1 was also suggested but this effect was observed at concentrations far higher than 25 mg/ml and even at these doses, GLUT2 was inhibited to a greater extent (M€ uller et al., 2018). It thus appear from the above observation that the polysaccharide fractions of guava could contribute to antidiabetic effects by suppressing glucose availability from the digestive system. The polyphenols from their known effect on α-amylase and α-glucosidase enzymes could also contribute to antidiabetic effect by inhibiting carbohydrate digestion.

9.5.5 Other mechanisms PTP1B being a negative regulator of insulin resistance, its inhibition is a valid target for diabetes therapy. This is not the well-defined mechanisms of action for guava, however, although the methanol extract of the leaves at the concentration of 30 μg/ml was shown to cause 87% inhibition in vitro (Oh et al., 2005). The observed activity is fairly good for the crude extract and may at least in part explain one mechanism of antidiabetic action for the leaves as demonstrated in the same study in vivo using the Lepr(db)/Lepr(db) mice model (Oh et al., 2005). While describing the identification of three novel sesquiterpene-based meroterpenoids, psidials A-C (Fig. 9.8), the enzyme inhibitory activity of psidial B and C against PTP1B were shown at the concentration of 10 μM (Fu et al., 2010). The ethanolic extract (80% in water) of guava leaves also showed a dose-dependent inhibition of dipeptidyl peptidase-4 (DPP4), with an IC50 of 380 μg/ml (Eidenberger et al., 2013). This activity level was not that potent but the authors further attempted to identify the active principles using the in vitro enzyme inhibition assay. For the isolated compounds, peltatoside, hyperoside, isoquercetin and guaijaverin (see Table 9.2 for structures), their IC50 were between 86 and 181 μM. Readers should bear in mind that this effect is rather moderate for an enzyme inhibition in vitro. Other compounds detected include hyperoside and other unconfirmed flavonoids glycosides of either quercetin or morin derivatives. With respect to the pharmacokinetic profiles of the indicated flavonoids in guava leaves, Eidenberger et al. (2013) have undertaken absorption studies in CaCo-2 epithelial cells a model of gastrointestinal tract absorption. They have shown that the uptake of flavonol glycosides (peltatoside, hyperoside, isoquercetin, guaijaverin and hyperoside, Table 9.2) into CaCo-2 cells from 380 μg/ml guava extract solution after 30 min incubation amounted to 2.2–5.3% of that theoretically available for absorption. This is in agreement with the general poor bioavailability of flavonoids described in the various parts of this book although they do have a plethora of pharmacological effects in vivo. Aldose reductase inhibitory effect of guava extracts has also been reported in vitro (Anand et al., 2016). In renoprotective study by extracts of guava fruit, a 2% dietary supplementation for 12 weeks was shown to suppress renal N (ε)-(carboxymethyl)lysine, pentosidine and fructose levels along with suppressed renal activity of aldose reductase (Lin and Yin, 2012). This mechanism also imply a retinoprotecive effect of guava under diabetes condition though data is yet to be provided in this area.

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9.6 Cardiovascular effects: Cardiac hypertrophy and hypertension The cardiac isoform of liver α2 macroglobulin is a major protein associated with early stages of cardiac hypertrophy and its downregulation could be seen as an indicator of potential benefit in tackling diabetes associated cardiovascular problems. In the STZ-model of diabetic rat, Soman et al. (2013) have shown that the EtOAc fraction of guava leaves could suppress the serum level a cardiac isoform of α-2 macroglobulin. The inotropic effect of the aqueous alcohol extract of the leaves has been demonstrated in guinea pig ileum preparations by Conde Garcia et al. (2003). In this model, the observed effect on the Bowditch phenomenon as an indicator of inward calcium current inhibition is worth mentioning. It was evident that concentrations in the range of 10–800 μg/ml could suppress myocardial force of contraction in a concentration-dependent manner; while the atrial relaxation time was increased and the positive staircase effect (Bowditch phenomenon) was abolished. It was suggested that such effect could be associated with inhibition of the common L-type calcium channels on cardiac tissues as well as blood vessels. The cholinergicdependent effect of the extract was also shown as atropine abolished the extracts’ effect in the isolated guinea pig left atrium preparation. They also reported that the acetic acid extract was 20-times more potent than the crude water-alcohol extract. From the preliminary mechanistic data provided by the authors, the predominant compound quercetin may contribute to such effect as it is a known inhibitor of L-type of voltage gated Ca2+ channel. Hence, quercetin has been shown to induce a range of cardiovascular effects including vasodilation (Hou et al., 2017) and cardioprotection (Guo et al., 2018) effects among others. In rat coronary artery, quercetin has been shown to induce vasospasmolytic effects by depressing the Ca2+ influx through L-type voltage-gated Ca2+ channels and augmentation of voltage-gated K+ channel activity in the myocytes (Hou et al., 2014). The dual effect and even activation of L-type Ca2+ channels in various organ system by quercetin is, however, also known including in rat tail artery smooth muscle cells (Saponara et al., 2002). Contrary to the above observation, the study by Olatunji-Bello et al. (2007) presented a different cardiactivity profile for the aqueous extracts of the leaves. A dose-dependent (0.25–2 mg/ml) effect on inducing contraction in rat aorta rings was unexpected. In fact, the authors reported that the sensitivity of the aortic rings to cumulative doses of the extract could be enhanced by phentolamine as if the mechanism was related to activation of α-adrenoceptor. They also reported that mechanism related to calcium channel could be implicated but perhaps to a lesser extent. Without trying to be not prejudice in these studies, even though the study by Garcia et al. (2003) being more comprehensive but also implicated in the atrium than aorta, more data is needed to establish the overall cardiovascular profile for guava extracts. In this direction, the experiment by Belemtougri et al. (2006) revealed that the leaves extract display antagonistic effects on the caffeine-induced calcium release from sarcoplasmic reticulum of cultured rat skeletal muscle cells in vitro. Hence, inhibiting the intracellular calcium release also appears to be one mechanism of action. The cardioprotective effect of guava leaves, fruits and active principles should also be seen hand in hand with their antioxidant effects. In a myocardial ischemia-reperfusion injury model using isolated rat hearts, the crude extracts as well as quercetin and gallic acid have been demonstrated to improve myocardial dysfunction as measured by reversal of the

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ischeamia-induced cardiac contracture (Yamashiro et al., 2003). Their antioxidant effect and suppression of malondialdehyde level in the heart preparation was evident. On the bases of antioxidant properties of the phenolic constituents of guava leaves and fruits, cardioprotective effects under diabetes or oxidative stress conditions is expected. The data on the inhibitory effects of the leaves extracts and its components on the α-dicarbonyl compounds-induced blood coagulation was also very promising. The study by Hsieh et al. (2007b) have also shown that the crude extract not only inhibit AGE formation but also reverse the methylglyoxal inhibition of antithrombin III activity. Moreover, the components of the extract notably ferulic acid, gallic acid and quercetin have similar effects in protection against methylglyoxal-induced loss of activity of antithrombin III. This data thus corresponds with the general antiglycation activity of the extract recorded both in vitro and in vivo as well as in the biological consequences expected for the glycation process. The antihypertensive effect of the leaves extract on STZ-model of diabetes in rats has been demonstrated by Ojewole (2005). In addition to the dose-dependent hypoglycaemic effect in normal (normoglycemic) and STZ-treated diabetic rats by oral administrations of the extract, intravenous administrations of the same doses could reduce the systemic arterial blood presure (BP) and heart rates of hypertensive animals. The inhibition of angiotensin converting enzyme (ACE), the enzyme that catalyzes the conversion of angiotensin I to angiotensin II in the renin– angiotensin–aldosterone system, has also been studied for guava extracts. Hence, the study by Irondi et al. (2016) that showed impressive potency (IC50, 21.06  2.04 μg/mL) for the polyphenolic-rich extract of the leaves is in line with potential antihypertensive effect of guava. The presence of a divalent cation Zn2+ at the active site of ACE makes it a primary target for phenolic acids and flavonoids that are abundant in the leaves/fruits extract. The orthodihydroxy functional groups of polyphenols as seen in caffeic/gallic acid and related compounds could be easily envisaged as the active structural moiety for such effects. In this direction, caffeic acid itself is known to have ACE inhibitory effects as well as antihypertensive property in hypertensive model animals (Bhullar et al., 2014; Li et al., 2005). The role of flavonoids in the same model is also worth mentioning as they have potent metal chelating effects, antioxidant properties as well as enzyme inhibitory effects. For example, the predominant flavonoid, quercetin is known to have ACE inhibitory effects (Al Shukor et al., 2013). In the study by Irondi et al. (2016), their quantification already noted the presence of phenolic compounds in the following order: flavonoids; quercetin > kaempferol > catechin > quercitrin > rutin > luteolin > epicatechin; and phenolic acids as caffeic acid > chlorogenic acid > gallic acids. The organoprotective effect of guava in diverse other systems is also worth mentioning given the vast array of compounds isolated from the plant are known to have such biological effects. For example, the hepatoprotective effects of the leaves extract have been demonstrated in vivo when liver damage was induced by paracetamol or carbon tetrachloride (Roy et al., 2006). Moreover, the doses for the aqueous extract used (250 and 500 mg/kg, p.o.) showed reversal of the elevated AST, ALT and alkaline phosphatase (ALP) as well as bilirubin like those demonstrated in antidiabetic studies. In acetaminophen-treated renal tubular endothelial cells, the fruits extract could also inhibit cytotoxicity (Wu et al., 2018). Hence, organo- and cytoprotective effects through antioxidant and anti-inflammatory mechanisms of guava extracts and/or their active components are all expected.

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9.7 Wound healing effects With respect to the claimed wound healing effect of guava leaves, the antimicrobial properties of guava has direct relevance. Staphylococcus aureus being a major bacterial agent implicated in would complication, the reported antimicrobial effect against this bacteria is worth mentioning. In fact, the leaves extract has been shown to inhibit the growth of antibioticsresistant clinical isolates of S. aureus (Anas et al., 2008; Nair and Chanda, 2007). Other studies also demonstrated the antimicrobial effect of the leaves extracts obtained by alcohol extraction (Dhiman et al., 2011), aqueous and acetone-water extracts (De Arau´jo et al., 2014), or spray dried extracts (Fernandes et al., 2014). Synergistic effect between the extract and standard antibiotics has also been demonstrated in both gram-negative and gram-positive bacteria (Betoni et al., 2006; Bezerra Morais-Braga et al., 2016). Abubakar (2009) has also performed antimicrobial susceptibility test for the leaves extract of guava in view of the potential use of the plant to tackle surgical wound, burns, skin and soft tissue infections. Even though the activity level (minimum inhibitory concentration (MIC)) that they reported were very high (6.25–50 mg/ml), effects against Proteus mirabilis, Streptococcus pyogenes, Escherichia coli, S. aureus and Pseudomonas aeruginosa have been reported. The antibacterial effect along with antioxidant and antitumor activity of ethanolic extract of the leaves have also been widely reported (Braga et al., 2014). Antimicrobial screening studies of the leaves extract obtained by methanol, acetone, N,Ndimethylformamide extraction have also be carried out by Nair and Chanda (2007). This study using 20 gram positive, 55 gram negative and 16 fungal strains made it the most comprehensive validation of antimicrobial effect of the leaves against clinically important strains. Arima and Danno (2002) have further attempted to isolate the antibacterial principles of the leaves. They identified two new flavonoid glycosides, morin-3-O-α-L-lyxopyranoside and morin-3-O-α-L-arabopyranoside (Table 9.2), together with two known flavonoids, guaijavarin and quercetin. As shown by the MIC studies, the activity level at 200 μg/ml or above for the two novel compounds against Salmonella enteritidis and Bacillus cereus suggest a rather weak activity. Considering the large number of compounds isolated from the leaves, many other compounds including tannins (Mailoa et al., 2014), triterpenes (Ghosh et al., 2010) and phenolic acids and flavonoids (De Arau´jo et al., 2014) have been implicated for a range of biological activities including antimicrobial, anti-inflammatory and analgesic effects. Similar experiments by other authors have also reported anti-inflammatory mechanisms through NF-κB modulation ( Jang et al., 2013, 2014). The different extraction methodologies with respect to optimizing the isolation of compounds that attributes to these biological effects have also been reported ( Jang et al., 2014b). Considering the role of the inflammatory cascade in the various stages of the wound healing process, the known anti-inflammatory properties of guava may also be relevant. The antidental plaque properties of the plant (Razak et al., 2006) and isolated compounds such as guaijaverin (Table 9.2) have also been reported (Prabu et al., 2006). Beyond the above-mentioned general antibacterial and already discussed antioxidant and antiinflammatory mechanisms, the wound healing effect of guava under diabetes condition is yet to be assessed.

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9.8 Overview of the guava antidiabetic principles The chemical composition of guava is primarily dominated by phenolic acids and flavonoids that are shown to have antidiabetic effects both in vitro and in vivo (e.g., Habtemariam and Varghese, 2014). In the various sections of this chapter, the various individual components of guava leaves and fruits as antioxidant, anti-inflammatory and various other mechanisms including cardiovascular effects are outlined. This section is therefore included to further add a little more information on these compounds without reiterating what is already mentioned. By using in vitro models, a number of studies have shown the antioxidant and antiglycative potential P. guajava leaves. After extracting the leaves with methanol and subsequently defatting with petroleum ether, re-extraction with the EtOAc has been shown to have a promise as an active fraction (Soman et al., 2010). Using the same strategy, Soman et al. (2016) have also tested the EtOAc fraction and its major component quercetin for cardioprotective effects in diabetic rats. Interestingly, the result obtained for the EtOAc fraction was better than the claimed active principle, quercetin. Hence, another active principle with greater activity or compounds acting in synergistic/additive fashion are expected. Following the demonstration of antidiabetic and antiobesity potential of the aqueous extract of guava leaves, Mathur et al. (2015) attempted to assess the chemical markers of the extract. By using LC-MS, they identified myrecetin, luteolin, kaempferol and guavanoic acid in the extract while the hydrolyzed extract contained quercetin as a predominant compound with the concentration of 9.9% w/w of the extract. Hence quercetin in the plant sample existing in its glycosylated form may also play major role for the observed activity. As evidenced by studies such as Zhu et al. (2013), quercetin glycosides are the dominant metabolites of the leaves. As outlined in the previous section, Irondi et al. (2016) put the relevance of flavonoids in the order of quercetin > kaempferol > catechin > quercitrin > rutin > luteolin > epicatechin; and phenolic acids caffeic acid > chlorogenic acid > gallic acids; by their concentrations. It is on this basis that this polyphenolic compounds as a whole are taken responsible for the observed effect of guava in diabetes and associated diseases. The effects of guava polyphenols are also extended to general organoprotective as well as specific effects on pancreatic β-cells. For example, quercetin can stimulate insulin release from Rat INS-1 β-Cells, by inhibiting transient KATP channels inhibition and increasing the level of intracellular Ca2+ concentrations (Kittl et al., 2016). This of course must be taken with a cautious note that higher concentrations of quercetin and related compounds could suppress pancreatic β-cells proliferation and rather induce apoptosis. Contrary to the effect of quercetin in suppressing L-type of Ca2+ channels in the cardiovascular system, it has been shown to activate this channels in pancreatic β-cells leading to insulin release (Bardy et al., 2013). The reported antispamodic effect of the plant as well as its active principles such as quercetin (Lutterodt, 1989) also account for the general gastrointestinal effect and/or uses of the plant in traditional medicine. Guava fruits polysaccharides (described as GP-1, GP-2, GP-3, and GP-4) were tested in HFD and STZ-induced diabetes in rats. When administered as the dose of 400 mg/kg (i.g.), a decreased levels of FBG, glucosylated serum protein, serum insulin, HOMA-IR, TC, TG and serum ALT, improved oral glucose tolerance, and increased insulin sensitivity were observed in diabetic rats ( Jiao et al., 2017). Histopathological observations also suggested that these polysaccharides (GP-1, GP-3, and GP-4) could alleviate injury in pancreatic islet cells and upregulate the genes expression of IR, IRS2, Akt, and GLUT4 ( Jiao et al., 2017). Hence, the polysaccharides as active principles could reproduce the antidiabetic effects observed for the crude D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

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297

plant extracts. As shown in Section 9.5.4, the structural identity of guava polysaccharides that act through modulation of carbohydrate digestion/absorption has been revealed. The experiment by Kuang et al. (2012) on the antidiabetic activity of guava leaves was directed towards the terpenoids class of secondary metabolites. Even though it is not clear how pure their preparation was to assert this distinction, their total terpenoids extract, in the STZinduced diabetic rats were shown to have a promise when administered (120, 240 mg/kg) for six weeks. The decrease in the levels of FPG was evident while the serum insulin and insulin sensitivity index were increased. As the level of BUN and creatinine were also decreased and the renal structural damages were improved, antidiabetic activity for the indicated extract could be confirmed in this model. Numerous triterpenes isolated from the plant including ursolic and oleonolic acids are known antidiabetic agents.

9.9 Clinical studies While the animal studies on potential therapeutic applications of guava for diabetes and associated diseases are fairly well-established, studies on human trials should be considered almost nonexistent. Literature search on ClinicalTrial.gov database for guava could only pick up one completed trial on the fruits for improvement of iron absorption. Hence, the level of enthusiasm on guava both on its marketing to the general public and experimental studies is yet to be paralleled with evidences coming from human studies. Singh et al. (1992) tested the hypothesis that guava fruits intake in humans could modify the serum total and HDL-C levels and on systemic blood pressure. In their 12 weeks randomized and single-blind study on 120 essential hypertension patients, they reported a significant net decrease in serum TC (9.9%), TG (7.7%) and BP (9.0/8.0 mmHg) with a significant net increase in HDL-C (8.0%) levels. With a caution of the methodology employed not being compressive; (e.g., adherence to guava consumption was assessed by questionnaires and weighing of guava intake by 24-h recall after 12 weeks of follow-up), the data was in agreement with the in vivo studies in animal models. In the authors follow-up study (Singh et al., 1993) using 145 hypertensives patients (72 patients test and 73 patients for placebo), the intake of soluble fiber (increased intake of soluble dietary fiber of 47.8  11.5 vs. 9.5  0.85 g/day) and a potassium-rich diet containing 0.5–1.0 kg of guava daily was associated with 7.5/8.5 mmHg net decrease in mean systolic and diastolic pressures. This BP reduction was also associated with a decrease in serum TC (7.9%), TG (7.0%) and an insignificant increase in HDL-cholesterol. In healthy human volunteers, the intake of 500 ml of freshly prepared guava fruit juice was shown to suppress the ex vivo collagen-induced platelet aggregation (Thaptimthong et al., 2016). In normal male subjects, the consumption of guava (400 g/day) was also shown to increase the total antioxidant status (Rahmat et al., 2004). Other clinical trial study on guava worth mentioning is a randomized trial on consumption of 200 g guava/day or 200 mg prior to and during the oral hygiene abstention period preventing the development of experimental gingivitis (Amaliya et al., 2018). Guava aqueous extracts prepared as 15 ml mouthwash twice a day applied in school children was also among herbal products that showed antibacterial efficacy against oral streptococci (Singla et al., 2018). In patients suffering from acute diarrhea, oral treatment every 8 h during 3 days with standardized guava leaves capsules (500 mg of QG-5) with quercetin as active ingredient were shown to suppress abdominal pain (Lozoya et al., 2002). D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

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While guava fruits are edible and its application as a human diet in juices and various other preparations are not of a concern as potentially toxic agent, other plant parts such as the leaves need more studies. The lack of data in humans means the toxicological and pharmacokinetics profiles of guava are also yet to be established.

9.10 Conclusions The guava plant has now been introduced from its indigenous American regions to many tropical Africa and Asian countries. The fruits are widely available to the general public as fresh produces in growing countries and juices and other preparations in food supermarkets of importing nations. All parts of the plants are known to be used in traditional medicine but the leaves and fruits appear to be those mostly exploited as healing agents. In line with their traditional applications, numerous animal models have been employed to show the potential of guava leaves and fruits as therapeutic agents for diabetes and associated diseases. Among those established so far are antihyperglycaemic, lipid lowering and antiobesity effects. There also appear to be well-established mechanisms of action for such effects as demonstrated in insulin signaling, adipogenesis process, anti-inflammatory and antioxidant mechanisms and various other lipid and glycaemic control mechanisms. Together with other general effects such organo-protective or amelioration of other diabetic pathologies such as nephropathies, hypertension and β-cell loss, the therapeutic potential of guava appear to be validated in animal experimental models and in vitro studies. The phytochemical study on guava is also comprehensive and the composition of the leaves and fruits comprise of polyphenols, terpenoids, carotenoids, essential oils, etc. Although most of the isolated compounds are already well-established as antidiabetic active principles, to what extent they contribute to the observed pharmacological effects of the crude extracts need further study. Clinical trials on human subjects are also lacking and the true potential of guava in diabetes and associated daises is yet to be demonstrated through well-designed clinical studies.

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Mathur, R., Dutta, S., Velpandian, T., Mathur, S.R., 2015. Psidium guajava Linn. Leaf extract affects hepatic glucose transporter-2 to attenuate early onset of insulin resistance consequent to high fructose intake: An experimental study. Pharm. Res. 7 (2), 166–175. Matsuo, T., Hanamure, N., Shimoi, K., Nakamura, Y., Tomita, I., 1994. Identification of (+)-gallocatechin as a bioantimutagenic compound in Psidium guava leaves. Phytochemistry 36, 1027–1029. Matsuzaki, K., Ishii, R., Kobiyama, K., Kitanaka, S., 2010. New benzophenone and quercetin galloyl glycosides from Psidium guajava L. J. Nat. Med. 64 (3), 252–256. Meckes, M., Calzada, F., Tortoriello, J., Gonza´lez, J.L., Martinez, M., 1996. Terpenoids isolated from Psidium guajava with depressant activity on central nervous system. Phytother. Res. 10, 600–603. Mercadante, A.Z., Steck, A., Pfander, H., 1999. Carotenoids from guava (Psidium guajava): isolation and structure elucidation. J. Agric. Food Chem. 47, 145–151. Metwally, A.M., Omar, A.A., Harraz, F.M., El Sohafy, S.M., 2010. Phytochemical investigation and antimicrobial activity of Psidium guajava L. leaves. Pharmacogn. Mag. 6 (23), 212–218. Michael, H.N., Salib, J.Y., Isaac, M.S., 2002. Acylated flavonol glycoside from Psidium gauijava L. seeds. Pharmazie 57, 859–860. Morais-Braga, M.F., Carneiro, J.N., Machado, A.J., Dos Santos, A.T., Sales, D.L., Lima, L.F., et al., 2016. Psidium guajava L. from ethnobiology to scientific evaluation: elucidating bioactivity against pathogenic microorganisms. J. Ethnopharmacol. 194, 1140–1152. Morton, J.F., 1987. Guava (Psidium guajava L.). In: Fruits of warm climates, Miami, FL, pp. 356–363. Also, available online: https://hort.purdue.edu/newcrop/morton/guava.html (accessed. 13 April 2019). Mukhtar, H.M., Ansari, S.H., Ali, M., Naved, T., Bhat, Z.A., 2004. Effect of water extract of Psidium guajava leaves on alloxan-induced diabetic rats. Pharmazie 59 (9), 734–735. Mukhtar, H.M., Ansari, S.H., Bhat, Z.A., Naved, T., Singh, P., 2006. Antidiabetic activity of an ethanol extract obtained from the stem bark of Psidium guajava (Myrtaceae). Pharmazie 61 (8), 725–727. M€ uller, U., St€ ubl, F., Schwarzinger, B., Sandner, G., Iken, M., Himmelsbach, M., et al., 2018. In vitro and in vivo inhibition of intestinal glucose transport by guava (Psidium guajava) extracts. Mol. Nutr. Food Res. 62 (11). e1701012. Nair, R., Chanda, S., 2007. In vitro antimicrobial activity of Psidium guajava L. leaf extracts against clinically important pathogenic microbial strains. Braz. J. Microbiol. 38, 452–458. Nwaichi, E.O., Chuku, L.C., Oyibo, N.J., 2015. Profile of ascorbic acid, β-carotene and lycopene in guava, tomatoes, honey and red wine. Int. J. Curr. Microbiol. App. Sci. 4 (2), 39–43. Oh, W.K., Lee, C.H., Lee, M.S., Bae, E.Y., Sohn, C.B., Oh, H., et al., 2005. Antidiabetic effects of extracts from Psidium guajava. J. Ethnopharmacol. 96 (3), 411–415. Ojewole, J.A., 2005. Hypoglycemic and hypotensive effects of Psidium guajava Linn. (Myrtaceae) leaf aqueous extract. Methods Find. Exp. Clin. Pharmacol. 27 (10), 689–695. Ojewole, J.A., 2006. Antiinflamatory and analgesic effects of Psidium guajava Linn (Myrtaceae) leaf aqueous extract in rats and mice. Methods Find. Exp. Clin. Pharmacol. 28, 441–446. Okpashi, V.E., Bayim, B.P., Obi-Abang, M., 2014. Comparative effects of some medicinal plants: Anacardium occidentale, Eucalyptus globulus, Psidium guajava, and Xylopia aethiopica. Extracts in alloxan-induced diabetic male Wistar albino rats. Biochem. Res. Int. 203051. Okuda, T., Yoshida, T., Hatano, T., Yazaki, K., Ashida, M., 1982. Ellagitannins of the Casuarinaceae, Stachyuraceae and Myrtaceae. Phytochemistry 21, 2871–2874. Okuda, T., Tsutomu, H., Kazufumi, Y., 1984. Guavin B, an ellagitannin of novel type. Chem. Pharm. Bull. 32, 3787–3788. Olatunji-Bello, I.I., Odusanya, A.J., Raji, I., Ladipo, C.O., 2007. Contractile effect of the aqueous extract of Psidium guajava leaves on aortic rings in rat. Fitoterapia 78, 241–743. Osman, A.M., Younes, M.E., Sheta, A.E., 1974. Triterpenoids of the leaves of Psidium guajava. Phytochemistry 13, 2015–2016. Oliveira, D.S., Lobato, A.L., Ribeiro, S.M., Santana, A.M., Chaves, J.B., 2010. Carotenoids and vitamin C during handling and distribution of guava (Psidium guajava L.), mango (Mangifera indica L.), and papaya (Carica papaya L.) at commercial restaurants. J. Agric. Food Chem. 58 (10), 6166–6172. Owen, P.L., Martineau, L.C., Caves, D., Haddad, P.S., Matainaho, T., Johns, T., 2008. Consumption of guava (Psidium guajava L) and noni (Morinda citrifolia L) may protect betel quid-chewing Papua new Guineans against diabetes. Asia Pac. J. Clin. Nutr. 17 (4), 635–643. Paniandy, J.C., Chane-Ming, J., Pieribattesti, J.C., 2000. Chemical composition of the essential oil and headspace solidphase microextraction of the guava fruit (Psidium guajava L.). J. Essent. Oil Res. 12, 153–158.

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Soman, S., Rauf, A.A., Indira, M., Rajamanickam, C., 2010. Antioxidant and antiglycative potential of ethyl acetate fraction of Psidium guajava leaf extract in streptozotocin-induced diabetic rats. Plant Foods Hum. Nutr. 65, 386–391. Soman, S., Rajamanickam, C., Rauf, A.A., Indira, M., 2013. Beneficial effects of Psidium guajava leaf extract on diabetic myocardium. Exp. Toxicol. Pathol. 65, 91–95. Soman, S., Rajamanickam, C., Rauf, A.A., Madambath, I., 2016. Molecular mechanisms of the antiglycative and cardioprotective activities of Psidium guajava leaves in the rat diabetic myocardium. Pharm. Biol. 54 (12), 3078–3085. Tanaka, T., Ishida, N., Ishimatsu, M., Nonaka, G., Nishioka, I., 1992. Tannins and related compounds. CXVI. Six new complex tannins, guajavins, psidinins and psiguavin from the bark of Psidium guajava L. Chem. Pharm. Bull. 40, 2092–2098. Thaipong, K., Boonprakob, U., Crosby, K., Cisneros-Zevallos, L., Hawkins, B.D., 2006. Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts. J. Food Compos. Anal. 19 (6–7), 669–675. Thaptimthong, T., Kasemsuk, T., Sibmooh, N., Unchern, S., 2016. Platelet inhibitory effects of juices from Pachyrhizus erosus L. root and Psidium guajava L. fruit: a randomized controlled trial in healthy volunteers. BMC complement. Altern. Med. 16, 269. USDA (United State Department of Agriculture) (2018). Psidium guajava L. https://plants.usda.gov/core/profile? symbol¼PSGU. (accessed 15.12.18). Vargas, A.D., Soto, H.M., Gonzalez, H.V.A., Engleman, E.M., Martinez, G.A., 2006. Kinetics of accumulation and distribution of flavonoids in guava (Psiduim guajava). Agrociencia 40, 109–115. Vargas-Murga, L., de Rosso, V.V., Mercadante, A.Z., Olmedilla-Alonso, B., 2016. Fruits and vegetables in the Brazilian household budget survey (2008–2009): Carotenoid content and assessment of individual carotenoid intake. J. Food Compos. Anal. 50, 88–96. Verma, A.K., Rajkumar, V., Banerjee, R., Biswas, S., Das, A.K., 2013. Guava (Psidium guajava L.) powder as an antioxidant dietary fibre in sheep meat nuggets. Asian-Australas. J. Anim. Sci. 26 (6), 886–895. Vinayagam, R., Jayachandran, M., Chung, S.S.M., Xu, B., 2018. Guava leaf inhibits hepatic gluconeogenesis and increases glycogen synthesis via AMPK/ACC signaling pathways in streptozotocin-induced diabetic rats. Biomed. Pharmacother. 103, 1012–1017. Wang, H.J., Chiang, B.H., 2012. Anti-diabetic effect of a traditional Chinese medicine formula. Food Funct. 3 (11), 1161–1169. Wang, B., Liu, H.C., Hong, J.R., Li, H.G., Huang, C.Y., 2007. Effect of Psidium guajava leaf extract on alpha-glucosidase activity in small intestine of diabetic mouse. Sichuan Da Xue Xue Bao Yi Xue Ban 38 (2), 298–301. Wang, H., Du, Y.-J., Song, H.-C., 2010. α-Glucosidase and α-amylase inhibitory activities of guava leaves. Food Chem. 123, 6–13. Wang, L., Luo, Y., Wu, Y., Liu, Y., Wu, Z., 2018. Fermentation and complex enzyme hydrolysis for improving the total soluble phenolic contents, flavonoid aglycones contents and bio-activities of guava leaves tea. Food Chem. 264, 189–198. Wilson, C.W., Shaw, P.E., 1978. Terpene hydrocarbons from Psidium guajava. Phytochemistry 17, 1435–1436. Wu, T.K., Liu, H.C., Lin, S.Y., Yu, Y.L., Wei, C.W., 2018. Extracts from guava fruit protect renal tubular endothelial cells against acetaminophen-induced cytotoxicity. Mol. Med. Rep. 17 (4), 5544–5551. Wu, J.W., Hsieh, C.L., Wang, H.Y., Chen, H.Y., 2009. Inhibitory effects of guava (Psidium guajava L.) leaf extracts and its active compounds on the glycation process of protein. Food Chem 113, 78–84. Yamashiro, S., Noguchi, K., Matsuzaki, T., Miyagi, K., Nakasone, J., Sakanashi, M., et al., 2003. Cardioprotective effects of extracts from Psidium guajava L and Limonium wrightii Okinawan medicinal plants, against ischemiareperfusion injury in perfused rat hearts. Pharmacology 67, 128–135. Yoshitomi, H., Guo, X., Liu, T., Gao, M., 2012a. Guava leaf extracts alleviate fatty liver via expression of adiponectin receptors in SHRSP.Z-Leprfa/Izm rats. Nutr Metab (Lond) 9, 13. Yoshitomi, H., Qin, L., Liu, T., Gao, M., 2012b. Guava leaf extracts inhibit 3T3-L1 adipocyte differentiation via activating AMPK. J. Nutr. Ther. 1, 107–113. Yusof, R.M., Said, M., 2004. Effect of high fibre fruit (guava-Psidium guajava L.) on the serum glucose level in induced diabetic mice. Asia Pac. J. Clin. Nutr. 13, S135. Zhang, Z., Liew, C., Handy, W., Zhang, D.E., Leopold, Y., Hu, J.A., Guo, J., Kulkarni, L., Loscalzo, R.N., J. Stanton R.C., 2010. High glucose inhibits glucose-6-phosphate dehydrogenase, leading to increased oxidative stress and β-cell apoptosis. FASEB J. 24 (5), 1497–1505. Zhang, Z., Kong, F., Ni, H., Mo, Z., Wan, J.B., Hua, D., Yan, C., 2016. Structural characterization, α-glucosidase inhibitory and DPPH scavenging activities of polysaccharides from guava. Carbohydr. Polym. 144, 106–114.

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Further reading

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Zhu, Y., Liu, Y., Zhan, Y., Liu, L., Xu, Y., Xu, T., Liu, T., 2013. Preparative isolation and purification of five flavonoid glycosides and one benzophenone galloyl glycoside from Psidium guajava by high-speed counter-current chromatography (HSCCC). Molecules 18 (12), 15648–15661.

Further reading Kenneth, S., Brekke, L., Johon, E., Donald, S., 1970. Volatile constituents in guava. J. Agric. Food Chem. 18, 598–599. Rai, P.K., Mehta, S., Watal, G., 2010. Hypolipidaemic and hepatoprotective effects of Psidium guajava raw fruit peel in experimental diabetes. Indian J. Med. Res. 131, 820–824. Wu, J.-W., Hsieh, C.-L., Wang, H.-Y., Chen, H.-Y., 2000. Inhibitory effects of guava (Psidium guajava L.) leaf extracts and its active compounds on the glycation process of protein. Food Chem. 113, 78–84.

D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

C H A P T E R

10 The chemical and pharmacological basis of okra (Abelmoschus esculentus (L.) Moench) as potential therapy for type 2 diabetes O U T L I N E 10.1 Introduction to Abelmoschus esculentus (L.) Moench

307

10.2 Botanical and taxonomic considerations

10.5 Effects of okra extracts and purified compounds on diabetes and associated diseases

308

10.6 Organoprotective and other effects 327

10.3 Economic significance

311

10.7 Toxicity

328

10.4 The chemistry of okra 10.4.1 Seed oil 10.4.2 Non-oil components 10.4.3 Polysaccharide components

313 313 315 318

10.8 Related species—Abelmoschus manihot (L.) Medik

329

10.9 Conclusion

329

References

329

319

10.1 Introduction to Abelmoschus esculentus (L.) Moench The family Malvaceae comprises of an accepted 246 genera of over 4000 species that occur as herbaceous, shrubs, and less often trees. Many species of the family are known to be cultivated as ornamental plants such as the mallow (the genus Malva of plants such as the musk, tree and marsh mallows) and hibiscus or for natural fibers such as cotton (the genus Gossypium) or jute (genus Corchorus). Another most important genus is cacao plant (Theobroma cacao),

Medicinal Foods as Potential Therapies for Type-2 Diabetes and Associated Diseases https://doi.org/10.1016/B978-0-08-102922-0.00010-9

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# 2019 Elsevier Ltd. All rights reserved.

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a source of cocoa beans or an ingredient of chocolate and its economic importance and pharmacology related to diabetes is discussed in Chapter 26. As represented by the small genus, Abelmoschus, some species of the Malvacea are also cultivated as vegetables. Commonly known in English as okra or lady finger, the immature fruits of commercial significance in this genus comes from the plant Abelmoschus escuentus. This accepted scientific name has been known by numerous synonyms in the scientific literature including the following: • • • • • • • • •

Abelmoschus bammia Webb Abelmoschus longifolius (Willd.) Kostel. Abelmoschus officinalis (DC.) Endl. Abelmoschus praecox Sickenb. Abelmoschus tuberculatus Pal & Singh Hibiscus esculentus L. Hibiscus hispidissimus A.Chev. nom. Illeg. Hibiscus longifolius Willd. Hibiscus praecox Forssk.

Of the above synonyms, Hibiscus esculentus L., has been extensively used in the older literature. The common name, okra, is also widely used in the scientific literature and hence all these names must be used while searching for information relevant to a particular field of study. In this chapter, the potential application of okra for diabetes and associated diseases is discussed from the botanical, chemical and pharmacological perspectives. While the plant has thousands of years of history of usage as food and traditional medicine applications, antidiabetic effect study for this plant is relatively new in the scientific literature. Within the last two decades, however, the interest in okra has significantly grown and the relevant chemistry and pharmacology of the immature edible fruits have been published fairly regularly. All these data are systematically presented herein with emphasis on the potential active principles and their mechanism of actions as antidiabetic, antiobesity and lipid lowering agents, among others.

10.2 Botanical and taxonomic considerations For a long time, the species belonging to the genus Abelmoschus of the family Malvaceae have been included as a section of the larger genera, Hibiscus. This was recognized in the Linnaeus book of Hortus Cliffortianus in 1773. The various synonyms of the plant as H. esculentus L., H. hispidissimus A. Chev. nom. Illeg., H. longifolius Willd and H. praecox Forssk all reflect this reality. The genus Abelmoschus comprising of a handful of species was recognized in 1787 by Medikus, but the number of accepted species even to date are only 12. The main distinguishing characteristics of the genus within the family have been described by many botanists with the work by Bates (1968) provided the most comprehensive and detailed classification. This included the caducous nature of the calyx and relationship of the calyx to the petal and staminal column aestivation. Among the widely cited features are the spathgaceous epicalyx and 5-toothed calyx that splits longitudinally along a suture. The further classification of the genera to the species level is also based on the number of dimensions and

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FIG. 10.1 Characteristic features of the genus Abelmoschus. The whole plant (A), flower (B) and exploding capsule (fruit) Abelmoschus manihot (L.) Medik. are shown. Pictures were courtesy of Dr. Helen Pickering (Kew Botanic Garden, London) and were taken from the grassland South-West region of Oman (Dhofar) at about 600 m altitude.

persistence of the epicalyx, the form and dimensions of the capsules such as the pedicels and the endumentum (Fig. 10.1). The native regions for the whole genus is the Eastern part of Africa from Eretria in the North to Tanzania in the South; and in Australasia from Pakistan in the West to China in the East and up to Austria to the South. The taxonomic relationship of the plant within the plant kingdom is as follow: Kingdom: Plantae Division: Magnoliophyta Class: Magnoliopsida Order: Malvales Family: Malvaceae Genus: Abelmoschus Species: esculentus As a cultivated plant (see next section), A. esculetus is grown across the globe in tropical and subtropical regions as well as in the drier Mediterranean (Middle East) regions. Okra appears to thrive in any frost-free hot and humid environment. The plant is annual and grows up to 4 m high, with alternate plalmate leaves of several (commonly five) lobes. The short-lived (around a day) hermaphrodite self-pollinated flowers of up to 8 cm in diameter carries five petals which vary in color from white to yellow often spotted at the base with red/purple color (Fig. 10.2). The flowers are similar with hibiscus and appear in solitary axillary forms with peduncle of up to 2.5 cm long. The immature fruits upon harvest have high mucilage

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FIG. 10.2 Abelmoschus escuentus (okra). Image of okra at flowering (A) and fruiting (B) stages. Courtesy: Wikipedia. https://upload.wikimedia.org/wikipedia/commons/thumb/4/4a/Okra%28Abelmoschus_esculentus%29.JPG/1280px-Okra% 28Abelmoschus_esculentus%29.JPG. Image of the fruits taken from the London markets (C) are also shown.

which biological effects are discussed in the following sections. The fruits become fibrous as it matures and if left uncollected, seed dispersal is via exploding pods. The okra fruit can grow up to 30 cm long with largely pointed tip. The narrow feature of the fruit size in diameter and long finger-like projection gave the fruits its common name: lady finger. Given the numerous cultivars developed over thousands of years of domestication, the botanical description of the plant cultivated in different regions could vary. The classical description from the Kew Science resource—Planet of the World online (POWO, 2018) is as follow: “Overview: An annual, erect herb up to 5 m (but typically about 2 m) tall. Stems succulent with scattered, stiff hairs. The whole plant has an aromatic smell resembling cloves. Leaves: Up to 50 cm wide and 35 cm long, deeply lobed, with toothed margins, hairy on both surfaces, especially on the nerves. Each leaf is borne on a petiole (leaf stalk) up to 50 cm long. Flowers: Showy, up to 8 cm in diameter, usually yellow with a dark red, purple or mauve centre, borne on a stout flower stalk (peduncle) up to 4 cm long. Stamens (male parts) united into a white, hairless column up to 2.5 cm long. Stigmas (female parts) dark purple. Calyx (whorl of sepals) and epicalyx (whorl of bracts) both present. Fruits: A capsule, 10–20 cm long, roughly circular in cross-section with a pointed end, usually 5-ribbed, borne at the leaf axils. Immature fruit can be purple-red, reddish-green, dark green, pale green or yellow. At maturity, fruits turn brown and split into segments.

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10.3 Economic significance

Seeds: Each fruit has up to 100 spherical or ovoid seeds bearing minute warts in concentric rows. Many cultivars are available, for example ‘Clemson Spineless’, ‘Indiana’, ‘Emerald’ (USA) and ‘Pusa Sawani’ (India).” The utilisation of okra by humans for thousands of years means that the plant is now largely a cultigen that passed through generations of selection processes. The use of okra by the Egyptians some 2000 BC has been known and its origin is often debated with some say the Upper Nile regions such as Ethiopia/Abyssinia while many other suggestions were equally made. According to the Kew Science—Plant of the World online resource (POWO, 2018), the native regions include India, Bangladish and Maynmar.

10.3 Economic significance An insight into the economic significance of okra in India, the largest okra producing country in the world, was presented by Kodandaram et al. (2017). Accounting to 72.9% of okra production in the world; taking cultivation area of 0.53 million hector; and annual production of 6.35 million tonnes, okra takes the share of 60% of the export of fresh vegetables. From the commercial production point of view, the main producer countries other than India are the ˜ ’te d’Ivoire, Egypt, Ghana, West African region including Benin, Burkina Faso, Cameroon, CA Mali and Nigeria. The major commercial output by these countries and others in Africa, Middle East, Asia and the American regions are shown in Table 10.1. TABLE 10.1

Global production of quantity (tonnes) of okra in 2016.a

Country

Quantity

Description

Albania

7666

Official data

Bahamas

482

IMb

Bahrain

822

IMb

Barbados

255

Official data

Belize

60

Official data

Benin

42,244

Official data

Brunei Darussalam

417

IMb

Burkina Faso

24,740

IMb

Cameroon

90,780

Official data

Congo

1523

IMb

˜ ’te d’Ivoire CA

112,966

IMb

Cyprus

1832

Official data

Djibouti

27

IMb

Egypt

57,721

IMb Continued

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TABLE 10.1 Global production of quantity (tonnes) of okra in 2016.—cont’d Country

Quantity

Description

Fiji

766

IMb

Ghana

66,360

Unofficial figure

Guatemala

6749

IMb

Guyana

10,731

Official data

India

5,507,000

Official data

Iraq

46,071

Official data

Jamaica

6922

Official data

Jordan

6299

Official data

Kenya

3941

Official data

Kuwait

7058

IMb

Lebanon

1045

IMb

Malaysia

55,856

Official data

Mali

241,033

Official data

Mauritius

1490

Official data

Mexico

29,602

Official data

Nigeria

1,978,286

IMb

Occupied Palestinian Territory

2449

IMb

Oman

6418

Official data

Pakistan

117,961

IMb

Philippines

30,529

Official data

Puerto Rico

133

IMb

Qatar

168

Official data

Saudi Arabia

46,478

IMb

Senegal

19,073

IMb

Sudan

287,300

Official data

Syrian Arab Republic

11,690

IMb

Trinidad and Tobago

1075

Official data

Turkey

29,529

Official data

United Arab Emirates

2007

IMb

United States of America

10,524

IMb

Yemen

24,356

IMb

a b

http://www.fao.org/faostat/en/#data/QC. FAO data based on imputation methodology.

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313

Okra is mainly cultivated for its fruits which are collected as immature pods with seeds. Many local and regional cuisines and recipes of okra are available for cooking in a variety of ways. The leaves are not common source of food but cooking in the same way as the fruits or salad-like uses to be eaten raw are not uncommon. In addition to the immature fruits and leaves, other plant parts even the petioles, stems, shoots, rhizomes, inflorescences, seeds in the various growing regions are known. The seeds of okra as a source of edible oil are also known. In addition to serving as a source of nutrition to humans, the leaves may be used as forage for cattle while the mucilage is known to have industrial applications. The utilization of the seeds for poultry feed; human consumption as a coffee substitute and medicinal as stomachic stimulant, antispasmodic and nervine have been reported in the various literature that report biological activities (see the following sections). The medicinal value of okra for treating ulcers, hemorrhoids, urinary disorders and diarrhea have also been cited in many literature. The most prominent uses advocated for okra in recent years are, however, for glycaemic control or diabetes and associated diseases which are elaborated further in the following sections.

10.4 The chemistry of okra The nutritional composition of okra is described in the various literatures and is not detailed in this chapter. As a source of carbohydrates, protein, mineral and even edible oils, various studies have listed the nutritional value of this fruit. The seeds as a meal with 21% protein, 14% lipids, and 5% ash composition has been reported by Savello et al. (1998). The authors also reported the mineral composition (per 100 g) of whole seeds as 135 mg of Ca and 335 mg of Mg with much lesser amounts ( palmitic (1) (19.3–21.84%) > α-linolenic acid (6) (14.60–20.98%) and then followed by oleic, stearic and behenic acid (3). Even though this composition could vary from plant to plant depending on various factors, the high nutritional value of okra seeds oil has been advocated based on the fatty acid composition, or the polyunsaturated fatty acids (PUFA) content and ratio of PUFA to saturated fatty acids, or favorable ratio of even n-6: n-3 fatty acids (Fig. 10.3) (Petropoulos et al., 2018).

10.4.2 Non-oil components On the basis of high performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) (HPLC–MS) analysis, Shui and Peng (2004) have identified four quercetin derivatives: quercetin-3-O-gentiobioside (quercetin-3-O-glucosyl-(1 ! 6)-glucopyranoside, 14), quercetin-3-O-glucopyraniside (isoquercitrin, 12), quercetin-3-O-(6-O-malonyl) glucopyranoside (17) and quercetin-3-O-xyloxyl-(1 ! 2)-glucopyranoside (18) along with ()-epigallocatechin (27) as major antioxidant components in fresh okra fruits (Fig. 10.4). The study by Arapitsas (2008) assessed the phenolic composition of okra fruits as two separate components: the skin (which should be regarded as the whole flesh) comprising 82.7% and the seeds comprising of 17.3%. Their HPLC coupled with ultraviolet (UV)/visible spectra and tandem mass spectrometry (MS/MS) allowed the detection of numerous polyphenolic compounds. Of these, the seeds were composed of oligomeric catechins (2.5 mg/g of seeds) and flavonol derivatives (3.4 mg/g of seeds) while phenolic compounds from the skins were represented by hydroxycinnamic acid and quercetin derivatives (0.2 and 0.3 mg/g of skins respectively) (Arapitsas, 2008). As the compounds were not isolated, the assignments of most of them was incomplete or at least tentative, but for the sake of clarity and valuable quantitative information, these compounds with their concentration level in the investigated plant parts are shown in Table 10.2. Some of the flavonoids reported by Arapitsas (2008) with structural assignment as tentative or not resolved altogether have been identified in other studies. Huang et al. (2017a,b), for example, identified quercetin 3-O-glucosyl-(1 ! 6)-glucoside (14), quercetin-3-O-xylosyl (1 ! 2) glucoside (18) and quercetin-3-O-glucoside (isoquercitrin, 12) from a highly polar

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10. The chemical and pharmacological basis of okra

FIG. 10.4 The structures of major flavonoids and ursolic acid identified from okra. * indicates the sugar configuration is yet to be confirmed: the B-type proanthociandin nature of oligomers have been established but the stereochemistry at C-2 and C-3 (catechin vs epicatechin) as well as linkage sites through C-4-C-6 or C4-C-8 positions have not yet been confirmed. The catechin group are, however, said to be predominant.

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TABLE 10.2

Phenolic composition of okra fruits parts. Concentrations (μg/g)

Compounds

Fruits

Skin/flesh

Seeds

p-Coumaroyl-hexoside

16.24

19.64

–

Sinapoyl-hexoside

38.04

46.00

–

Sinapoyl-hexoside

67.28

81.35

–

Sinapoyl-feruloyl

24.74

29.92

–

Sinapoyl-feruloyl

9.74

11.78

–

Sinapoyl derivative

14.84

17.95

–

Cinnamic derivative

3.87

4.68

–

Total Hydroxycinnamics

174.29

211.32

–

HYDROXYCINNAMIC DERIVATIVES

OLIGOMERIC CATECHINS (CATECHIN AND EPIGALLOCATECHIN DIMERS, TRIMERS AND TETRAMERS) Total

557.86

147.73

2518.44

Quercetin 3-O-diglucoside

363.41

93.50

1653.72

Myricetin 3-O-glucoside

3.96

–

21.36

Quercetin 3-O-glucosyl-xyloside

86.72

69.56

168.72

Quercetin 3-O-rutinoside

3.92

–

22.68

Quercetin 3-O-glucoside

320.95

117.44

1176.36

Isorhamnetin 3-O-glucosyl-pentoside

11.13

13.46

–

Quercetin 3-O-(malonyl)glucoside

72.47

26.18

293.76

Myricetin 3-O-rhmnoside

2.97

–

17.16

Kaempferol 3-O-glucoside

2.03

–

11.76

Myricetin

1.29

–

7.44

Kaempferol

Not detected

–

Not detected

Quercetin

0.69

–

3.96

Total

798.93

320.14

3387.12

FLAVONOLS

Adopted from Arapitsas, 2008.

water soluble fraction of the whole okra fruits extract. From this information, the highest amount of the flavonol detected in the seeds by Arapitsas (2008) appears to be quercetin3-O-gentiobioside (14). This was also confirmed by the study of Lu et al. (2016), along with the detection of quercetin-3-O-rutinoside (rutin, 15), isoquercitrin (12) and quercetin-3-O(malonyl) glucoside (17). From okra seeds, the (epi) gallocatechins and (epi) catechins okra

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oligomeric proanthocyanidins were also detected by using liquid chromatography coupled with matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) MS (Lu et al., 2016). They have detected B-type epi- or gallocatechin oligomers from dimer up to pentadecamer but dominated by galocatechin oligomers. The details of these proanthocyanidins is yet to be established and the two possible options of linkage for the epicatechin (22) and/or catechin (23) compounds are shown in Fig. 10.4 (compounds 24 and 25). Quercetin-3-O-sophoroside (16), rutin (15) and isoquercitrin (12) have been isolated as α-amylase inhibitory compounds of okra (Zeng et al., 2015). By using orthogonal high-speed counter-current chromatography separation coupled with UV and MS, the antioxidant (by measure of radical scavenging effects) principles of the dried okra fruits were also identified as quercetin-3-O-sophoroside (16), rutin (15), and isoquercitrin (12) (Wang et al., 2016). In their antidiabetic activity study, Fan et al. (2014a,b) also detected isoquercitrin (12), quercetin 3-O-gentiobioside (14) and rutin (15) along with quercetin (11) and ursolic acid (27) as minor components.

10.4.3 Polysaccharide components A number of studies (e.g., Sengkhamparn et al., 2009) have shown that okra polysaccharides belonging to the, pectins, xyloglucans, xylans, and celluloses are acidic and consists of galactose (Gal), rhamnose (Rha), and galacturonic acid (GalA). Of these, the backbone repeating units have been characterized as 4-α-GalpA-(1, 2)-α-L-Rhap-1- dimers and the presence of dimeric side chains of β-Galp-(1, 4)-β-Galp-1 (note—p represent the pyranose configuration of the sugar). The acetyl content was about 5.5% w/w. The study by Tomoda et al. (1980) also identified okra’s pectin polysaccharide with the rhamnogalacturonan I (RG-I) domain and with a 5.5% (w/w) degree of acetylation. The study by Liu et al. (2018) further led to the isolation of rhamnogalacturonan as a purified polysaccharide from okra pod. It was characterized as a pectin polysaccharide having ! 4)-D-GalpAMe-(1 ! 2)-L-Rhap-(1 ! as backbone and side chains including Galp-β-D-(1 ! 4)-Galp-β-D-(1 ! 4)-Galp-β-D-(1 ! 4)Galp-β-D-(1 ! 4)-Galp-β-D-(1 ! and Araf-α-L-(1 ! 6)-Galp-β-D-(1 ! 4)-Galp-β-D-(1 ! 4)-Galp-βD-(1 ! 4)-Galp-β-D-(1 ! (note—Me represent methyl derivative). Readers should note that although these backbones of the complex polysaccharides are established, plant pectin still have more complex compositions from the four main domains they are composed of. These are homogalacturonan (HG), rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II), and xylogalacturonan (XGA): • The HG domain—backbone consists of a linear polymer of 1, 4-linked α-D-galacturonic acid (GalpA) residues partially methyl esterified at the C-6 position and O-acetylated at the O-2 or O-3 positions. • RG-I—backbone of alternating α-L-rhamnose (Rhap) and α-D-GalpA repeating units [4)α-D-GalpA-(1, 2)-α-L-Rhap-(1]n, which is highly branched with α-L-arabinose (Araf )- and β-D-galactose (Galp)-rich side chains attached at the C-4 position of Rha residues (note— Ara represent arabinose). • RG-II—the most structurally diverse pectin, consisting of a 9 or 10 α-(1 ! 4)-D-GalpA unit HG backbone with four different oligosaccharide chains attached at C-2 or C-3 positions of the GalA residues.

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On the basis of the above, Zhang et al. (2018) isolated the pectin polysaccharide that they called WOP-2 and suggested to contain RG-I domain with backbone of ! 4)-α-D-GalpA(1 ! 2)-α-L-Rhap-(1 !; which was partly substituted at O-4 of Rhap by side chains, such as AG-II. Its molecular weight was estimated to be about 580 kDa. The monosaccharides composition was also determined and shown to be Rha (21.4%), GalA (34.9%), Gal (29.6%), GlcA (4.5%), Glc (5.9%), and Ara (3.7%) (note—GlA represent glucuronic acid). Hence, this data suggest that WOP-2 possess a rhamnogalacturonan I backbone with type-II arabinogalactan side-chains substituted partly at O-4 of Rhap. This identification was, however, not complete and the structure should be seen as a tentative proposal but the similarity with that reported by Liu et al. (2018) was evident.

10.5 Effects of okra extracts and purified compounds on diabetes and associated diseases One of the most popular medicinal claim for okra in recent years has been for diabetes and related diseases. Literature search on this subject in many databases such as PubMed could, however, yield beets > asparagus > eggplant ¼ turnips ¼ green beans ¼ carrots ¼ cauliflower. The viscous soluble dietary fibres of okra could also ameliorate glucose absorption through ultrafine membrane in vitro with implication of inhibiting glucose uptake from the gut (Khatun et al., 2010). In a rather rare hyperlipidaemic model of study in mice, Ngoc et al. (2008) injected tyloxapol (300 mg/kg, i.p.) to raise the lipid and cholesterol level. As tyloxapol inhibits plasma lipolytic activity, the TG level in the blood is raised and hence could serve as experimental hyperlipidaemia model in animal studies. When the high dose (Table 10.3) of the fruits extract were introduced orally immediately after tyloxapol, hypolipidaemic (TG and cholesterol) effect was demonstrated the following day. In high-fat-induced obese diabetic mice model, the antidiabetic effect of okra fruit extract was shown from a reduced blood glucose, insulin and TG and hepatic steatosis without lowering body weight or modulation of other lipids markers such as LDL-C, TC and HDL-C (Fan et al., 2014a,b). In this obesity model, okra extract appear to suppress the PPAR-γ mRNA levels and inhibited the expression of the PPAR-γ target gene, aP2. Readers should note that both agonists and antagonists of the PPAR system have been shown to offer some benefit in obesity and insulin resistance depending on the stage of adipogenesis. The PPARs are important in the adipogenesis process and hence their inhibition could offer fat accumulation while their expression or agonistic compounds could inhibit insulin resistance under full-fledged obesity conditions. Hence, the following notes are worth mentioning: • As explained in Chapter 5, T2D therapeutic agents include PPAR-γ agonists employed as insulin-sensitizing agents; • PPAR-γ antagonists have been extensively shown to suppress adipocyte differentiation through which they offer therapeutic option for insulin resistance and dyslipidaemia. This is mainly due to the role of PPAR-γ in mediating the differentiation process of adipocytes leading to hypertrophy and increased lipid accumulation in adipose tissues (obesity). Hence, and ironically, PPAR-γ has been proven through knockout studies to mediate the high-fat-mediated adipocyte hypertrophy and insulin resistance (Kubota et al., 1999; Jones et al., 2005). Through this mechanism, many natural products or other selective PPAR-γ antagonists (Rieusset et al., 2002) have been shown to reduce body weight, blood glucose and serum TG in HFD-induced obese mice (e.g., Goto et al., 2013). In an attempt to isolate the active principles, compounds detected as major components such as isoquercitrin (12) and quercetin-3-O-gentiobioside (14) were also tested for antidiabetic effects (Fan et al., 2014a,b). After two weeks of treatment (HFD containing 0.1% of isolated compounds in obese mice), both compounds could improve glucose tolerance (Table 10.3) but only isoquercitrin (12, not quercetin-3-O-gentiobioside, 14) reduce FBG. As with the crude extract, these compounds did not modify body weight or food intake. Both compounds also appear to lower TG as with the crude extract but they also lowered TC (but not LDL-C). Interestingly, these compounds could suppress body weight gain after longer treatment period (6 weeks) without altering food intake. The contradiction in longer treatment regime was that FBG was not suppressed although TG and TC by isoquercitrin (12) and TG by quercetin-3-O-gentiobioside (14) could be lowered. Not surprisingly, these known antioxidant compounds along with the crude okra extract have been shown to display radical scavenging effect in vitro (Fan et al., 2014a,b). Even though the experiments with these

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10. The chemical and pharmacological basis of okra

compounds appear to have a limitation such as just one dose being used, the observed effect is in line with potential antidiabetic effect associated with obesity. Another study by Fan et al. (2013) on what they called polysaccharides of okra were obtained by ethanolic extraction of the fruit but only contain 48.1% of polysaccharides while the rest were proteins and polyphenols including flavonoids. Hence, the data are presented in Table 10.3 as an aqueous extract instead of activity solely due to polysaccharides. The results clearly showed antihyperglycaemic, lipid lowering, improvement of glucose tolerance and insulin sensitivity in HFD-induced obese animals. The mechanism of action shown from the protein and gene expression studies in white adipose tissue (WAT) and liver tissues related to two groups of lipid and glucose regulatory nuclear receptor transcription factors were very impressive. The key findings were as follow: • Okra extract could suppress the liver X receptor (LXR) expression and their target genes (Table 10.3) that regulate cholesterol and fatty acid metabolism in liver tissues. Even though the TG level was not affected in this study, the diminished cholesterol level could attribute to the inhibitory effect of the extract on the major regulator of cholesterol and bile acid homeostasis, CYP7A1. Hence, the observed hypocholesterolaemic effect could be a result of inhibition of LXR or its target gene products. • The PPARs as major regulators of glucose and lipid metabolism as well as adipocyte differentiation are targeted by okra extract. This results are consistent with other studies discussed in the preceding texts where adipocyte differentiation by okra extract appears to be targeted. Screening studies on some plant seed extracts using 3T3-L1 adipocytes also identified okra among the active extracts that inhibit TNF-α production (Okada et al., 2010). This suggest potential anti-inflammatory effect. The α-glucosidase and α-amylase inhibitory effects of okra fruits or seeds as a potential antidiabetic mechanisms have also been extensively reported as with the common antioxidant effects. For the methanol extract of the fruits, Karim et al. (2014) reported a competitive-type inhibition against mammalian α-glucosidase and of mixed type against mammalian α-amylase. As with many other fruits and vegetables, cooking or increasing temperature could lower both the total polyphenol contents as well as antioxidant and/or inhibitory effect against carbohydrate digestive enzymes (α-glucosidase and α-amylase) (Karim et al., 2014). The aqueous extracts of both the fruit peels (flesh) and seeds have also been shown to display both α-glucosidase (IC50 ¼ 142.69 μg/mL and 150.47 μg/mL) and α-amylase (IC50 ¼ 132.63 μg/mL and 147.23 μg/mL respectively) inhibitory effects in vitro (Sabitha et al., 2012b). Lu et al. (2016) have further shown that the B-type proanthocyanidins predominantly gallocatechin oligomers from dimer up to pentadecamer isolated from okra seeds display potent carbohydrate digestive enzyme inhibition with IC50 values of 2.30  0.14 μg/mL (α-amylase) and 16.88  0.26 μg/mL (α-glucosidase). The identification of quercetin-3-O-sophoroside (16), rutin (15) and isoquercitrin (12) as the α-amylase inhibitory principles of okra has also been established using online two-dimensional high-speed countercurrent chromatography target-guided by ultrafiltration-HPLC system (Zeng et al., 2015). The identification of isoquercitrin (12) and to a less extent quercetin-gentiobioside (14) as α-glucosidase (intestinal maltase and sucrases or yeast α-glucosidases) inhibitors of okra have also been reported (Thanakosai and Phuwapraisirisan, 2013). Similarly, Hu et al. (2014) reported quercetin 3-O-gentiobioside (14) and isoquercitrin (12) as the two major components of the polyphenols of okra dried seed powder with significant biological activity. D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

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327

The hypoglycaemic and hypocholesterolaemic effects of okra polysaccharides have also been shown in rats (Lengsfeld et al., 2004a,b). Hence readers should bear in mind that the active principles of okra constitute both the phenolic and polysaccharides components as follow: • The presence of phenolic acids in okra fruits have been reported but their detailed structure is yet to be established. These hydroxycinnamic acid-type compounds are not predominant (Table 10.3) but could contribute to the overall antioxidant and antidiabetic effects reported for the flavonoids and polysaccharide components; • The flavonoids appear to play key role in the observed antidiabetic and antiobesity effects. Through antioxidant, anti-inflammatory and multiple effects in insulin signaling, their antidiabetic effects particularly for quercetin derivatives have been extensively reviewed in recent years (e.g. Habtemariam and Lentini, 2015; Habtemariam and Varghese, 2014). Quercetin and its glycosides such as, rutin, have also been shown to display antiinflammatory effect in the various model such as inflammatory bowel disease (Habtemariam and Belai, 2018) or neurodegenerative diseases (Habtemariam, 2016). The antioxidant effect such as radical scavenging properties and metal chelation effects for flavonoids such as quercetin are also highly optimized through their catecholic functional group of the B-ring and the 4-keto along with 5-OH and 3-OH positions (Habtemariam and Belai, 2018 and references there in). Hence, most of the antidiabetic properties of okra extract could be inferred from the high level of quercetin glycosides identified from the plant; • The polysaccharide components of okra are beginning to be clearly established both in their structural composition and pharmacological effects. Dietary fibers as one major structural groups of natural products that modify obesity, diabetes and a number of cardiovascular disorders have been extensively researched in recent years. The higher level of these components in okra combined with their demonstrated effect in experimental agents as discussed in this section imply their prominent role as active principles.

10.6 Organoprotective and other effects The ethanol extract (250 and 500 mg/kg) of the whole okra fruit powder has been shown to display hepatoprotective effect against carbon tetrachloride (CCl4)-induced liver toxicity as shown from the reduced level of elevation of serum AST, alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), cholesterol, TG and malondialdehyde (MDA), non-protein sulfhydryls and total protein levels in the liver tissues (Alqasoumi, 2012). In rat hepatocytes (BRL-3A cell line) exposed to the toxic effect of CCl4, the crude methanolic extract of okra dried seed powder and the major components quercetin 3-O-gentiobioside (14) and isoquercitirin (12) have been shown to alleviate cytotoxicity; reduce lipid peroxidation and liver damage markers (ALP and AST activities); while the levels of antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT) activities) were increased (Hu et al., 2014). As expected, the crude extracts and isolated compounds do also possess antioxidant effects as a measure of radical (2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide anions, and hydroxyl radical) scavenging (Hu et al., 2014). The gastroprotective effect of the methanolic extract of okra in ethanol-induced gastric ulcer in rats was also reported (Gurbuz et al., 2003). The neuroprotective effect of quercetin (11), rutin (15) D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

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and okra extract were tested on memory deficits induced by prolonged (21 days) treatment with dexamethasone. The reversal of cognitive deficits, including impaired of N-methyl-Daspartate (NMDA)-receptor positive cells of dentate gyrus cell proliferation, and protection against morphological changes in the CA3 region in dexamethasone-treated mice were reported (Tongjaroenbuangam et al., 2011). This data were in line with the known neneurprotective effect of quercetin (11) and its glycosides in neurodegenerative diseases (Habtemariam, 2016). There are also many other pharmacological effects of okra reported in recent years. The reported antiadhesive properties of the plant and polysaccharides fraction imply potential effect against infection of the gut by pathogenic bacteria (Deters et al., 2005; Lengsfeld et al., 2004a,b; Wittschier et al., 2007). Rhamnogalacturonans with a considerable amount of glucuronic acid as highly acid carbohydrate fractions of okra extract have also been isolated with antiadhesive effect against Helicobacter pylori in human gastric mucosa (Lengsfeld et al., 2004a,b). The immunomodulatory effect of crude and purified okra polysaccharide preparations have also been reported. The study by Chen et al. (2016) further led to the isolation of three polysaccharides with 600, 990, and 1300 kDa size. The first two were described to be mainly composed of galactose, rhamnose, galacturonic acid, and glucuronic acid, while that last one was mainly composed of galactose, rhamnose, galacturonic acid, glucuronic acid, and glucose. Beyond the rhamnogalacturonan I composition and general characteristic of the polysaccharides, their structures were not established in detail. They all appear however to enhance the proliferation of RAW264.7 macrophages and also increase the production of nitric oxide (NO) as well as expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-10 secretion. The 990 kDa polysaccharide also increased the spleen index, splenocyte proliferation, and cytokine secretion in normal and cyclophosphamide-induced immunosuppressed mice (Chen et al., 2016) suggesting the potential immunomodulatory effect of okra. This imply boosting the immune system under prevalent immunosuppressive pathological condition.

10.7 Toxicity Of course okra has been used as food for thousands of years and hence the primary concern is not on direct toxic effect when consuming okra products. No acute toxicity was also observed in rats at oral dose of 2000 mg/kg of peel and seed powders for two weeks (Sabitha et al., 2011) or many other studies when antidiabetic effects were reported in animal models. The potential drug interaction of okra with metformin was studied by using the filtered juice concentrate administered with metformin in alloxan-induced diabetic rats (Khatun et al., 2011). Even though the extract itself could inhibit the absorption of glucose from the gut after oral loading in normal animals, the antidiabetic effect of metformin in diabetic animals appear to be ameliorated by okra (Khatun et al., 2011). The dosage being crude and not determined in terms of bioactive component, the experiment should be seen as preliminary but has implication in drug interaction possibly inhibiting metformin absorption when co-administered with okra.

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References

329

10.8 Related species—Abelmoschus manihot (L.) Medik There is a growing interest to investigate the antidiabetic potential of the related species A. manihot that is also known in traditional medicine particularly in China. Antidiabetic effects have been shown for this plant with the emphasis appear to be on improvement in diabetic nephropathy under various experimental conditions (Ge et al., 2016; Liu et al., 2017; Mao et al., 2015; Zhou et al., 2012).

10.9 Conclusion The antidiabetic effect studies on okra is only getting momentum in recent years with many key issues still not addressed. The glucose lowering effects have been demonstrated at fairly low doses. There is a good set of data on lipid lowering and implication on weight loss but several experiments with contradictory results have been published: some showing weight loss, some with no effect; some showing TG lowering effects while others showed only one component (e.g. cholesterol without TG) modulated, etc. The dose regimen and in vitro experiments are very limited and hence more data are needed not only on showing antidiabetic properties but also for the whole set of diabetes-associated disorders from obesity to cardiovascular complication, retinopathy, neuropathy, etc. Most crucially, the clinical significance must be established in human subjects. Even though okra is a food product with no known toxicity, potential drug interaction with conventional antidiabetic agents is yet to be established. In the meantime, there is a clear indication that okra extract and the flavonoids and polysaccharide active components have potential application for diabetes and associated diseases that are worth further investigation.

References Adelakun, O.E., Oyelade, O.J., Ade-Omowaye, B.I.O., Adeyemi, I.A., Van de Venter, M., 2009. Chemical composition and the antioxidative properties of Nigerian okra seed (Abelmoschus esculentus Moench) flour. Food Chem. Toxicol. 47, 1123–1126. Alqasoumi, S.I., 2012. Okra Hibiscus esculentas L.: a study of its hepatoprotective activity. Saudi. Pharm. J. 20 (2), 135–141. Ames, J.M., Macleod, G., 1990. Volatile components of okra. Phytochemistry 29 (4), 1201–1207. Andras, C.D., Simandi, B., Orsi, F., Lambrou, C., Tatla, D.M., Panayiotou, C., et al., 2005. Supercritical carbon dioxide extraction of okra (Hibiscus esculentus L.) seeds. J. Sci. Food Agric. 85, 1415–1419. Arapitsas, P., 2008. Identification and quantification of polyphenolic compounds from okra seeds and skins. J. Food Chem. 110, 1041–1045. Bates, D.M., 1968. Notes on the cultivated Malvaceae 2, Abelmoschus. Baileya 16, 99–112. Ben-Chioma, A.E., Tamuno-Emine, D.G., Dan, D.B., 2015. The effect of Abelmoschus esculentus in alloxan-induced diabetic Wistar rat. IJSR 4 (11), 540–543. Camciuc, M., Bessie`re, J.M., Vilarem, G., Gaset, A., 1998. Volatile components in okra seed coat. Phytochemistry 48 (2), 311–315. Chen, H., Jiao, H., Cheng, Y., Xu, K., Jia, X., Shi, Q., 2016. In vitro and in vivo immunomodulatory activity of okra (Abelmoschus esculentus L.) polysaccharides. J. Med. Food 19 (3), 439. Deters, A.M., Lengsfeld, C., Hensel, A., 2005. Oligo- and polysaccharides exhibit a structure-dependent bioactivity on human keratinocytes in vitro. J. Ethnopharmacol. 102, 391–399.

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Dong, Z., Zhang, J.-G., Tian, K.-W., 2014. The fatty oil from okra seed: Supercritical carbon dioxide extraction, composition and antioxidant activity. Curr. Top. Nutraceutical Res. 12 (22), 75–84. Erfani-Majd, N., Tabandeh, M.R., Shahriari, A., Soleimani, Z., 2018. Okra (Abelmoscus esculentus) improved islets structure, and down-regulated PPARs gene expression in pancreas of high-fat diet and streptozotocin-induced diabetic rats. Cell J. 20 (1), 31–40. Fan, S., Zhang, Y., Sun, Q., Yu, L., Li, M., Zheng, B., et al., 2014a. Extract of okra lowers blood glucose and serum lipids in high-fat diet-induced obese C57BL/6 mice. J. Nutr. Biochem. 25 (7), 702–709. Fan, S., Guo, L., Zhang, Y., Sun, Q., Yang, B., Huang, C., 2013. Okra polysaccharide improves metabolic disorders in high-fat diet-induced obese C57BL/6 mice Mol. Nutr. Food Res. 57, 2075–2078. POWO (Plant of the World Online), 2018. Abelmoschus esculentus (L.) Moench. http://powo.science.kew.org/taxon/ urn:lsid:ipni.org:names:558006-1 (Accessed 10 January 2019). Fan, S., Zhang, Y., Sun, Q., Yu, L., Li, M., Zheng, B., et al., 2014b. Extract of okra lowers blood glucose and serum lipids in high-fat diet-induced obese C57BL/6 mice. J. Nutr. Biochem. 25 (7), 702–709. Ge, J., Miao, J.J., Sun, X.Y., Yu, J.Y., 2016. Huangkui capsule, an extract from Abelmoschus manihot (L.) medic, improves diabetic nephropathy via activating peroxisome proliferator-activated receptor (PPAR)-α/γ and attenuating endoplasmic reticulum stress in rats. J. Ethnopharmacol. 189, 238–249. Goto, T., Kim, Y.I., Takahashi, N., Kawada, T., 2013. Natural compounds regulate energy metabolism by the modulating the activity of lipid-sensing nuclear receptors. Mol. Nutr. Food Res. 57, 20–33. Gurbuz, I., Ustun, O., Yesilada, E., Sezik, E., Kutsal, O., 2003. Antiulcerogenic activity of some plants used as folk remedy in Turkey. J. Ethnopharmacol. 88, 93–97. Habtemariam, S., 2016. Rutin as a natural therapy for Alzheimer’s disease: insights into its mechanisms of action. Curr. Med. Chem. 23 (9), 860–873. Habtemariam, S., Belai, A., 2018. Natural Therapies of the inflammatory bowel disease: The case of rutin and its Aglycone, Quercetin. Mini Rev. Med. Chem. 18 (3), 234–243. Habtemariam, S., Lentini, G., 2015. The therapeutic potential of rutin for diabetes: an update. Mini Rev. Med. Chem. 15 (7), 524–528. Habtemariam, S., Varghese, G.K., 2014. The antidiabetic therapeutic potential of dietary polyphenols. Curr. Med. Chem. Biotechnology 15 (4), 391–400. Hu, L., Yu, W., Li, Y., Prasad, N., Tang, Z., 2014. Antioxidant activity of extract and its major constituents from okra seed on rat hepatocytes injured by carbon tetrachloride. Biomed. Res. Int. 2014, 341291. Huang, C.N., Wang, C.J., Lin, C.L., Lin, H.T., Peng, C.H., 2017a. The nutraceutical benefits of subfractions of Abelmoschus esculentus in treating type 2 diabetes mellitus. PLoS One. 12(12), e0189065. Huang, C.N., Wang, C.J., Lee, Y.J., Peng, C.H., 2017b. Active subfractions of Abelmoschus esculentus substantially prevent free fatty acid-induced β cell apoptosis via inhibiting dipeptidyl peptidase-4. PLoS One. 12(7), e0180285. Jarret, R.L., Wang, M.L., Levy, I.J., 2011. Seed oil and fatty acid content in okra (Abelmoschus esculentus) and related species. J. Agric. Food Chem. 59, 4019–4024. Kahlon, T.S., Chapman, M.H., Smith, G.E., 2007. In vitro binding of bile acids by okra, beets, asparagus, eggplant, turnips, green beans, carrot and cauliflower. Food Chem. 103, 6766–6780. Karim, M.R., Islam, M.S., Sarkar, S.M., Murugan, A.C., Makky, E.A., Rashid, S.S., et al., 2014. Anti-amylolytic activity of fresh and cooked okra (Hibiscus esculentus L.) pod extract. Biocat. Agric. Biotechnol. 3 (4), 373–377. Khatun, H., Rahman, A., Biswas, M., Islam, A.U., 2011. Water-soluble fraction of Abelmoschus esculentus L. interacts with glucose and metformin hydrochloride and alters their absorption kinetics after coadministration in rats. ISRN Pharm. 2011, 260537. Khatun, M.H., Rahman, M.A., Biswas, M., Islam, M.A., 2010. In vitro study of the effects of viscous soluble dietary fibers of Abelmoschus esculentus L. in lowering intestinal glucose absorption. BPJ 13 (2), 35–40. Kodandaram, M.H., Kumar, Y.B., Banerjee, K., Hingmire, S., Rai, A.B., 2017. Singh Field bioefficacy, phytotoxicity and residue dynamics of the insecticide flonicamid (50 WG) in okra (Abelmoschus esculenta (L) Moench). Crop Prot. 94, 13–19. Kubota, N., Terauchi, Y., Miki, H., Tamemoto, H., Yamauchi, T., Komeda, K., et al., 1999. PPAR gamma mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance. Mol. Cell 4, 597–609. Jamieson, G.S., Baughman, W.F., 1920. Okra seed oil. J. Am. Chem. Soc. 42 (1), 166–170. Jones, J.R., Barrick, C., Kim, K.A., Lindner, J., Blondeau, B., Fujimoto, Y., et al., 2005. Deletion of PPARgamma in adipose tissues of mice protects against high fat diet-induced obesity and insulin resistance. Proc. Natl. Acad. Sci. U.S.A 102, 6207–6212.

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Lengsfeld, C., Titgemeyer, F., Faller, G., Hensel, A., 2004a. Glycosylated compounds from okra inhibit adhesion of Helicobacter pylori to human gastric mucosa. J. Agric. Food Chem. 52, 1495–1503. Lengsfeld, C., Titgemeyer, F., Faller, G., Hensel, A., 2004b. Glycosylated compounds from okra inhibit adhesion of Helicobacter pyroli to human gastric mucosa. J. Agric. Food. Chem. 52, 1495–1503. Liu, J., Zhao, Y., Wu, Q., John, A., Jiang, Y., Yang, J., Liu, H., Yang, B., 2018. Structure characterization of polysaccharides in vegetable “okra” and evaluation of hypoglycemic activity. Food Chem. 242, 211–216. Liu, S., Ye, L., Tao, J., Ge, C., Huang, L., Yu, J., 2017. Total flavones of Abelmoschus manihot improve diabetic nephropathy by inhibiting the iRhom2/TACE signaling pathway activity in rats. Pharm. Biol. 56 (1), 1–11. Lu, Y., Demleitner, M.F., Song, L., Rychlik, M., Huang, D., 2016. Oligomeric proanthocyanidins are the active compounds in Abelmoschus esculentus moench for its α-amylase and α-glucosidase inhibition activity. J. Funct. Foods 20, 463–471. Mao, Z.M., Shen, S.M., Wan, Y.G., Sun, W., Chen, H.L., Huang, M.M., et al., 2015. Huangkui capsule attenuates renal fibrosis in diabetic nephropathy rats through regulating oxidative stress and p38MAPK/Akt pathways, compared to α-lipoic acid. J. Ethnopharmacol. 173, 256–265. Mishra, N., Kumar, D., Rizvi, S.I., 2016. Protective effect of Abelmoschus esculentus against alloxan-induced diabetes in Wistar strain rats. J. Diet. Suppl. 13 (6), 634–646. Ngoc, T.H., Ngo, Q.N., Van, A.T., Phung, N.V., 2008. Hypolipidemic effect of extracts from Abelmoschus esculentus L. (Malvaceae) on Tyloxapol-induced hyperlipidemia in mice. Warasan Phesatchasat. 35, 42–46. Okada, Y., Okada, M., Sagesaka, Y., 2010. Screening of dried plant seed extracts for adiponectin production activity and tumor necrosis factor-alpha inhibitory activity on 3 T3-L1 adipocytes. Plant Foods Hum. Nutr. 65, 225–232. Oyelade, O.J., Ade-Omowaye, B.I.O., Adeomi, V.F., 2003. Influence of variety on protein, fat contents and some physical characteristics of okra seeds. J. Food Eng. 57, 111–114. Peng, C.H., Chyau, C.C., Wang, C.J., Lin, H.T., Huang, C.N., Ker, Y.B., 2016. Abelmoschus esculentus fractions potently inhibited the pathogenic targets associated with diabetic renal epithelial to mesenchymal transition. Food Funct. 7 (2), 728–740. ˆ ., Barros, L., Ferreira, I.C.F.R., 2018. Chemical composition, nutritional value and antiPetropoulos, S., Fernandes, A oxidant properties of Mediterranean okra genotypes in relation to harvest stage. Food Chem. 242, 466–474. Ramachandran, S., Sandeep, V.S., Srinivas, N.K., Dhanaraju, M.D., 2010. Anti-diabetic activity of Abelmoschus esculentus Linn on alloxan-induced diabetic rats. Res. Rev. BioSci. 2010, 4. Rieusset, J., Touri, F., Michalik, L., Escher, P., Desvergne, B., Niesor, E., et al., 2002. A new selective peroxisome proliferator-activated receptor gamma antagonist with antiobesity and antidiabetic activity. Mol. Endocrinol. 16, 2628–2644. Sabitha, V., Ramachandran, S., Naveen, K.R., Panneerselvam, K., 2012a. Investigation of in vivo antioxidant property of Abelmoschus esculentus (L) moench. Fruit seed and peel powders in streptozotocin-induced diabetic rats. J. Ayurveda Integr. Med. 3 (4), 188–193. Sabitha, V., Panneerselvam, K., Ramachandran, S., 2012b. In vitro α-glucosidase and α-amylase enzyme inhibitory effects in aqueous extracts of Abelmoscus esculentus (L.) Moench. Asian Pac. J. Trop. Biomed. 2 (1), S162–S164. Sabitha, V., Ramachandran, S., Naveen, K.R., Panneersel-vam, K., 2011. Antidiabetic and antihyperlipidemic potential of Abelmoschus esculentus (L.) Moench. in streptozotocin-induced diabetic rats. J. Pharm. Bioall. Sci. 3, 397–402. Savello, P.A., Martins, F., Hull, W., 1998. Nutrition composition of okra seed meals. J. Agric. Food Chem. 28, 1163–1166. Savello, P.A., Martins, F., Hull, W., 1980. Nutrient composition of okra seed meals. J. Agric. Food Chem. 28, 1163–1166. Sengkhamparn, N., Bakx, E.J., Verhoef, R., Schols, H.A., Sajjaanantakul, T., Voragen, A.G.J., 2009. Okra pectin contains an unusual substitution of its rhamnosyl residues with acetyl and alpha-linked galactosyl groups. Carbohydr. Res. 344 (14), 1842–1851. Shui, G., Peng, L.L., 2004. An improved method for the analysis of major antioxidants of Hibiscus esculentus Linn. J. Chromatogr. A 1048, 17–24. Subrahmanyam, G.V., Sushma, M., Alekya, A., Neeraja, C.H., Harsha, H.S., Ravindra, J., 2011. Antidiabetic activity of Abelmoschus esculentus fruit extract. Int. J. Res. Pharm. Chem. 1, 17–20. Thanakosai, W., Phuwapraisirisan, P., 2013. First identification of α-glucosidase inhibitors from okra (Abelmoschus esculentus) seeds. Nat. Prod. Commun. 8, 1085–1088. Tian, Z.H., Miao, F.T., Zhang, X., Wang, Q.H., Lei, N., Guo, L.C., 2015. Therapeutic effect of okra extract on gestational diabetes mellitus rats induced by streptozotocin. Asian Pac. J. Trop. Med. 8 (12), 1038–1042.

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Tomoda, M., Shimada, K., Saito, Y., Sugi, M., 1980. Plant mucilages. XXVI. Isolation and structural features of a mucilage, ‘‘okra mucilage’’, from the immature fruit of Abelmoschus esculentus. Chem. Pharm. Bull. 28 (28), 2933–2940. Tomoda, M., Schimizu, N., Gonda, R., Kanari, M., Yamada, H., Hikino, H., 1989. Anticomplementary and hypoglycemic activity of okra and Hibiscus mucilages. Carbohydr. Res. 190 (2), 323–328. Tongjaroenbuangam, W., Ruksee, N., Chantiratikul, P., Pakdeenarong, N., Kongbuntad, W., Govitrapong, P., 2011. Neuroprotective effects of quercetin, rutin and okra (Abelmoschus esculentus Linn.) in dexamethasone-treated mice. Neurochem. Int. 59, 677–685. Uraku, A.J., Ajah, P.M., Okaka, A.N., Ibiam, U.A., Onu, P.N., 2010. Effects of crude extracts of Abelmoschus esculentus on albumin and total bilirubin of diabetic albino rats. Int. J. Sci. Nat. 1, 38–41. Wang, R., Liu, Q., Wu, Z., Wang, M., Chen, X., 2016. Target-guided isolation of polar antioxidants from Abelmoschus esculentus (L). Moench by high-speed counter-current chromatography method coupled with wavelength switching and extrusion elution mode. J. Sep. Sci. 39 (20), 3983–3989. Wang, H., Chen, G., Ren, D., Yang, S.T., 2014. Hypolipidemic activity of okra is mediated through inhibition of lipogenesis and upregulation of cholesterol degradation. Phytother. Res. 28 (2), 268–273. Wittschier, N., Lengsfeld, C., Vorthems, S., Stratmann, U., et al., 2007. Large molecules as anti-adhesive compounds against pathogens. J. Pharm. Pharmacol. 59, 777–786. Zeng, H., Liu, Q., Yu, J., Wang, M., Chen, M., Wang, R., et al., 2015. Separation of α-amylase inhibitors from Abelmoschus esculentus (L). Moench by on-line two-dimensional high-speed counter-current chromatography target-guided by ultrafiltration-HPLC. J. Sep. Sci. 38 (22), 3897–3904. Zhang, T., Xiang, J., Zheng, G., Yan, R., Min, X., 2018. Preliminary characterization and anti-hyperglycemic activity of a pectic polysaccharide from okra (Abelmoschus esculentus (L.) Moench). J. Funct. Foods 41, 19–24. Zhou, L., An, X.F., Teng, S.C., Liu, J.S., Shang, W.B., Zhang, A.H., Yuan, Y.G., Yu, J.Y., 2012. Pretreatment with the total flavone glycosides of Flos Abelmoschus manihot and hyperoside prevents glomerular podocyte apoptosis in streptozotocin-induced diabetic nephropathy. J. Med. Food. 15 (5), 461–468.

Further reading Anwar, F., Rashid, U., Ashraf, M., Nadeem, M., 2010. Okra (Hibiscus esculentus) seed oil for biodiesel production. Applied Energy 87 (3), 779–785. Linnaeus, C., 1773. Hortus Cliffortianus. Amsterdam. Ramachandran, K., Huang, H.H., Stehno-Bittel, L., 2015. A simple method to replace islet equivalents for volume quantification of human islets. Cell Transplant. 24 (7), 1183–1194. Sheu, S.-C., Lai, M.-H., 2012. Composition analysis and immuno-modulatory effect of okra (Abelmoschus esculentus L.) extract. Food Chem. 134 (4), 1906–1911. Uraku, A.J., Onuoha, S.C., Offor, C.E., Ogbanshi, M.E., Ndidi, U.S., 2011. The effects of Abelmoschus esculentus fruits on ALP, AST and ALT of diabetic albino rats. Int. J. Sci. Nat. 2, 582–586.

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C H A P T E R

11 The chemical and pharmacological basis of papaya (Carica papaya L.) as potential therapy for type-2 diabetes and associated diseases O U T L I N E 11.1 Botanical and taxonomic considerations 11.2 Papaya utilisation 11.2.1 Extent of papaya fruits production/utilisation 11.2.2 Uses as food and medicine 11.2.3 Enzyme production

334 337 337 340 341

11.3 The chemistry of papaya 341 11.3.1 Nutritional composition 341 11.3.2 Phenolic acids and flavonoids derivatives 342 11.3.3 Isothiocyanates and related compounds 344 11.3.4 Alkaloids 345 11.3.5 Enzymes and protein components 346

Medicinal Foods as Potential Therapies for Type-2 Diabetes and Associated Diseases https://doi.org/10.1016/B978-0-08-102922-0.00011-0

11.4 The pharmacology of papaya related to diabetes and associated diseases 11.4.1 In vitro studies 11.4.2 Antidiabetic and lipid lowering effects in animal models 11.4.3 Papaya and wound healing 11.4.4 Antihypertensive effect 11.4.5 Evidence of efficacy from human trials

347 347 349 352 355 356

11.5 Toxicological considerations

358

11.6 Conclusion

358

References

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Further reading

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# 2019 Elsevier Ltd. All rights reserved.

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11.1 Botanical and taxonomic considerations Papaya is one of the fastest growing tropical or subtropical fruit plants. With a potential to grow up to a height of 10 m, though quite commonly anywhere between 2 and 10 m, the plant can reach maturity within a year. As it grows very fast, its economic life is also short and is around 3–4 years. The papaya plant and fruits are known by numerous local names and among which ‘pawpaw’ is most common. As these local names are often used to describe other plants and fruits, however, the name papaya is only adopted in this chapter. Even though papaya has an appearance of a tree, the cylindrical single stem is hallow and the stem structure is composed of spongy-fibrous tissues. Hence, the papaya plant is considered as an herbaceous perennial tree. Branching is not common in papaya and occurs when the main shoot is damaged/cut or the plant reaches to maturity, thereby new branches start appearing from the lower-end of the stem. The large highly lobed or palmate leaves of papaya appear in alternate fashion at the apical end of the shoot leaving the stem in the middle to lower region free from leaves with the leaves scares very conspicuous (Fig. 11.1). One remarkable feature of the papaya tree is the polygamous nature of the plant: a behaviour that is governed not only by genetics but also the environment such as temperature, draught or a variety of other climatic conditions. Hence, a papaya tree could produce only a male (staminate), female (pistillate) or hermaphroditic (bisexual) flowers; or in the latter case even a tendency to either a female of male sex could manifests. Understanding this characteristics and selection of the best seeds to get the desired fruiting plant is an art that is being mastered by the papaya farmers. The description of papaya according to its floral/sexual characteristics is as follow (Yogiraj et al., 2014):

FIG. 11.1 The papaya plant. The leaves (A) of a young papaya plant grown in the UK (2 months old) and stem of a mature tree from Kew Garden UK showing the leaves scar (B) are shown.

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335

“Female and bisexual flowers are waxy, ivory white, and borne on short penducles in leaf axils, along the main stem. Flowers are solitary or small cymes of 3 individuals. Ovary position is superior. Prior to opening, bisexual flowers are tubular, while female flowers are pear shaped. Since, bisexual plants produce the most desirable fruit and are self–pollinating, they are preferred over female or male plants. A male papaya is distinguished by the smaller flowers borne on long stalks. Female flowers of papaya was pear shaped, when unopened whereas, bisexual flowers are cylindrical”.

Floral arrangement appearance and the above-described female and male flowers of papaya are exhibited in Fig. 11.2. Starting to flower within 5–8 months from planting, the papaya fruit yield comes in abundance (see Fig. 11.3). The papaya fruit is composed of the skin, pulp and seeds (Fig. 11.4) with variable composition depending on varieties or cultivars. The seeds could vary between 5% and 9%; skin 12–25% and pulp from 70% to 80%. The composition of the fruits could also vary depending on ripening state; with immature ones, for example, producing large amount of latex. Papaya belongs to Caricaceae, a small family of plants that comprise only six genera. The genus Carica L., intern comprises about 22 species that grow throughout the tropical and subtropical regions of the world. They are characterised by their poorly branched trees to shrubs as described for Carica papaya L. According to the USDA (2018) database, the taxonomic hierarchy of papaya is as follow:

FIG. 11.2 The papaya plant. Floral features (A) and male (B) and female (C) flowers are shown.

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336

FIG. 11.3

11. The chemical and pharmacological basis of papaya

The papaya plant. The fruits of papaya on mature plant at Kew Botanical Garden (London) is shown.

FIG. 11.4 The colour of papaya fruits based on maturity stages. The carotene content in papaya fruits vary as the fruits mature and change from the green (right) to the orange/yellow (left) colouration. The immature fruits have whitish-flesh colouration though colour variability also depends on the cultivar/variety/genotype of the plant.

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11.2 Papaya utilisation

337

Kingdom: Plantae—Plants Subkingdom: Tracheobionta—Vascular plants Superdivision: Spermatophyta—Seed plants Division: Magnoliophyta—Flowering plants Class: Magnoliopsida—Dicotyledons Subclass: Dilleniidae Order: Violales Family: Caricaceae—Papaya family Genus: Carica L.—papaya Species: Carica papaya L.—papaya With native range of tropical and subtropical America, the genus Carica L. comprises of four accepted species: Carica aprica V.M. Badillo, Carica augusti Harms, Carica chilensis (Planch. ex A.DC.) Solms and Carica papaya L. The native range of C. papaya stretches from Southern Mexico through Central America to Venezuela. C. papaya L. is also known by the following synonyms: • • • • • • • • • • • • • • • • • • • •

Carica citriformis J. Jacq. ex Spreng. Carica cubensis Solms Carica hermaphrodita Blanco Carica jamaicensis Urb. Carica jimenezii Bertoni Carica mamaya Vell. Carica papaya f. correae Solms Carica papaya var. jimenezii Bertoni Carica papaya f. mamaya (Vell.) Stellfeld Carica papaya F. portoricensis (Urb.) Solms Carica peltata Hook. & Arn. Carica pinnatifida Heilborn Carica portoricensis Urb. Carica posopora L. Carica pyriformis Willd. Carica rochefortii Solms Papaya pyriformis (Willd.) Baill. Vasconcellea peltata (Hook. & Arn.) A.DC. Papaya citriformis (J.Jacq. ex Spreng.) A.DC. Papaya peltata (Hook. & Arn.) Kuntze

11.2 Papaya utilisation 11.2.1 Extent of papaya fruits production/utilisation It is hard to imagine a tropical country where papaya is not cultivated. The global level of papaya production for the year 2016 is published by the Food and Agriculture Organisation of the United Nations (FAO) database, and is shown in Table 11.1. The highest producer was

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338

11. The chemical and pharmacological basis of papaya

TABLE 11.1 Global production quantity of papaya for the year 2016. Country

Tonnes

Flag

Ranking

Argentina

2129

Im

48

Australia

6261

Im

38

Bahamas

551

Im

54

Bangladesh

130,371

Of

15

Belize

10,791

Of

29

Bolivia (Plurinational State of )

18,216

Of

25

Brazil

1,424,650

Of

2

Cameroon

44

Im

63

Chile

2712

Im

47

China

156,646

Ag

14

Colombia

188,305

Im

9

Congo

3705

Im

43

Cook Islands

527

Im

56

Costa Rica

100,000

Of

17

˜ ’te d’Ivoire CA

15,951

Im

26

Cuba

212,579

Of

8

Democratic Republic of the Congo

215,263

Im

7

Dominica

83

Im

60

Dominican Republic

863,201

Of

5

Ecuador

50,901

Im

21

El Salvador

8151

Of

34

Ethiopia

50,396

Of

22

Fiji

4915

Im

41

French Guiana

742

Im

51

Ghana

5561

Un

40

Guadeloupe

43

Im

64

Guatemala

96,896

Of

18

Guinea-Bissau

3116

Im

45

Guyana

4207

Un

42

Haiti

99

Im

59

Honduras

7778

Im

35

India

5,699,000

Of

1

Indonesia

904,284

Of

4

Iran (Islamic Republic of )

189

Im

57

339

11.2 Papaya utilisation

TABLE 11.1

Global production quantity of papaya for the year 2016.—cont’d

Country

Tonnes

Flag

Ranking

Israel

61

Of

62

Jamaica

8582

Of

33

Kenya

128,131

Im

16

Malaysia

65,967

Of

19

Maldives

617

Im

53

Mali

54,664

Of

20

Mexico

951,922

Of

3

Morocco

172

Im

58

Mozambique

42,502

Im

23

Nepal

14,137

Of

27

Nigeria

836,702

Im

6

Oman

5772

Of

39

Pakistan

6642

Im

37

Panama

7750

Im

36

Paraguay

11,400

Im

28

Peru

169,437

Of

11

Philippines

162,481

Of

12

Puerto Rico

9705

Im

31

˜ #union RA

550

Im

55

Rwanda

2920

Of

46

Samoa

3471

Im

44

South Africa

9920

Of

30

Suriname

713

Of

52

Thailand

169,942

Im

10

Timor-Leste

1417

Im

49

Trinidad and Tobago

942

Of

50

Tunisia

72

Im

61

United States of America

8963

Of

32

Venezuela (Bolivarian Republic of )

160,955

Of

13

Yemen

25,929

Im

24

Zimbabwe

43

Im

64

a

http://www.fao.org/faostat/en/#data/QC. Abbreviations: Ag, Aggregate value that may include official, semiofficial, estimated or calculated data; In, FAO data based on imputation methodology; Un, unofficial figure; Data by the Food and Agriculture Organisation of the United Nations (FAO).a

340

11. The chemical and pharmacological basis of papaya

India followed by Cameroon and Mexico. The impressive list however indicated the widespread source of papaya as valuable fruits to be consumed in various forms but mainly presented to the general public as fresh whole fruits. Considerable amount of papaya fruits must also go to juice production as various preparations of papaya juices alone or in combination with others are widely available in food stores.

11.2.2 Uses as food and medicine It is needless to say that the primary purpose of papaya production is for its dietary fruits. Unripe or green fruits and leaves are also widely used in salads and cooking in Asian countries, though not common in the West. All parts of the plant including leaves, stem (and stembark), flowers, fruits and roots are known to have applications in traditional medicines. These practices are often different from region to region but mostly localised within the tropical areas where papaya grows. Papaya fruits, seeds, latex and extracts have been used traditionally to treat various ailments in humans across the world. The wound healing effect of papaya has been advocated for decades with proteoloytic mechanism of action has been suggested. This include the study by Sherry and Fletcher (1960) who reported the wound healing potential of the plant by scratching wounds and ulcers with papaya latex. The green fruits of C. papaya is one of the plants used in Trinidad and Tobago for treating diabetes and hypertension (Lans, 2006). The utilisation of the green fruits for hypertension, diabetes and cholesterol lowering effects is now widely advocated and routinely employed. The antifertility and abortifacient uses have also been well documented for the green fruits. The latex, mainly sourced from the green fruits are generally regarded as abortifacient but also employed to treat skin conditions such as dermatitis and psoriasis. In Malaysia, the decoction of papaya root as a means of birth control, preventing menstruation and for uterine contractions after birth are said to be employed (Ahmed and Ismail, 2003). In this connection, the bitter seeds are also employed as abortifacient, for pain management and menstrual problems. The medicinal claim for the leaves is also immense and range from cancer to antiinflammatory and pain management, fever, stomach pain, etc. Some entries also include the utilisation of the flowers for cough, bronchitis and respiratory problems. The root barks and others in traditional medicine associated with malaria infection have also been reported (Vigneron et al., 2005). In addition to papaya fruits presented to the public in varies format such as fresh or dietary supplements; fermented papaya preparations (FFP) are also widely available as commercialised functional food products in Asia, Europe and United States. In this sweet and granular product, the unripe fruits are fermented by yeast to make the product a combination of both drug and prebiotic preparation. The long-term fermentation papaya liquor by Aspergillus and yeast are generally known to be extracted to make FFP that are presented as tablets or powder. In one particular experiment, the FFP preparation was described as follow (You et al., 2017): “Immature papaya (half a month from maturity) were peeled and cut into 2 mm slices after removing the seeds. The prepared papaya was fermented for 3 months through Aspergillus oryzae (isolated from millet catsup and stored at our laboratory), and then fermented

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for 3 months using yeasts (Saccharomyces cerevisiae, provided by Desheng Biotechnology Co. Ltd., Zhejiang, China) with 5% glucose at 24–28°C. The FPEs were obtained by squeezing and filtering after fermentation. Total flavonoids, one of the important functional constituents in FPEs, were nearly 11.65 mg/ml detected by UV method.” The pharmacology of FFP related to the pathology of type 2 diabetes (T2D) and associated disease is presented in Section 11.4.

11.2.3 Enzyme production As with the other members of the Carica genera, papaya fruits both mature and immature forms are good sources of proteolytic enzymes. The immature fruits are the best source, however, and produce few enzymes with the best characterised and/or utilised being papain (see Section 11.3.5). This enzymes are extensively used in the various industries including as medicines, meat production as tenderising agent, in the textile and leather industry and beer brewery. Proteolytic enzymatic activity primarily by papain has also been employed from the plant including from industrial by-product sources (Thoma´s et al., 2009). The pharmacology of these enzymes as antimicrobial and wound healing agents is relevant to diabetic wound healing and discussed in Section 11.4.

11.3 The chemistry of papaya 11.3.1 Nutritional composition The level of ascorbic acid found in the freeze-dried pulp of papaya was estimated as 419 mg/100 g (Calvache et al., 2016). In the actual fruits, values for ascorbic acid between 45 and 77 mg/100 g fresh weight (FW) have been reported by various authors (e.g., Valente et al., 2011; Wall, 2006). Paes et al. (2015) have studied the lycopene content of raw or enzymatically hydrolysed papaya pulp and established various processing methodologies designed to retain the carotenoid. Several studies have also been directed in optimising the yield of dietary fibres from papaya by-products. Methodologies including microwaveassisted extraction have also been employed to maximise value-added products from papaya (Nieto Calvache et al., 2016). These dietary fibre concentrates do also have other soluble materials like polyphenols that further add to the antioxidant potential of the fibre preparation (Martı´nez et al., 2012). Carotenoids contribute to the colour of the edible parts of the papaya fruits which are generally yellow-orange in colour (Fig. 11.4). The red-fleshed cultivars often have a higher carotenods content than the yellow-fleshed, particularly in those carotenoids (e.g. lycopene) that are characteristically reddish in colour (Schweiggert et al., 2011). Papaya is generally considered to be a good source of lycopene, with average values ranging from 0.36 to 3.4 mg/ 100 g FW, and being ranked number 4 of overall foods in the USDA nutrient reference database, after red guava, water melon and tomatoes (Charoensiri et al., 2009; Gayosso-Garcı´a Sancho et al., 2011; Schweiggert et al., 2011, 2012, 2014). Carotenoids like β-cryptoxanthin and β-carotene are found in all types of papaya cultivars and are responsible for the yellow and orange hues in the flesh. On the other hand, lycopene, which confers red colours, is either

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not present or in very small amounts in yellow-fleshed fruit (Gayosso-Garcı´a Sancho et al., 2011; Marelli de Souza et al., 2008; Schweiggert et al., 2011; Wall, 2006). β-Cryptoxanthin, β-carotene and lycopene have been identified from papaya fruit flesh of C. papaya L. cv. Maradol (Gayosso-Garcı´a Sancho et al., 2011). The following carotenoids, along with vitamin C, have been reported to increase in the fruit flesh with ripening; lycopene (0.36 to 3.40 mg/100 g per dry weight (DW)), β-criptoxanthin (0.28 to 1.06 mg/100 g/ DW), β-carotene (0.23 to 0.50 mg/100 g/DW), and vitamin C (25.07 to 58.59 mg/100 g/ DW). The detection of lutein along with vitamin C is also reported by Franke et al. (2004). Lycopene, β-cryptoxanthin and β-carotene were identified in mesocarp as the major carotenoids in papaya fruits and tend to increase during ripening (Rivera-Pastrana et al., 2010). The seeds have also been reported as valuable sources of niacin, thiamine and riboflavin. Calvache et al. (2016) reported the total carotenoid content in the pulp and peel as 1.4 and 2.48 mg/100 g FW, respectively; while Gayosso-Garcı´a Sancho et al. (2011) reported 1–3 mg/ 100 g FW. As mentioned above, the carotenoids content is expected to increase in the fruit as ripening proceeds and experimental evidences have been provided to corroborate this observations (Herna´ndez et al., 2006). Calvache et al. (2016) have done a compositional analysis of papaya pulp dietary fibre concentrate obtained from the ethanol extract of the pulp and peel papayas. They found that the level of ascorbic acid and carotenoids were higher in the peels than pulp. While the total dietary fibre was slightly higher for the pulp, the peels had more insoluble dietary fibre; and the pulp is a very rich source of soluble dietary fibre by comparison. They found β-criptoxanthin and β-carotene in both peel and pulp though the higher amounts were in the peels; while lutein and zeaxanthin were detected only in the peel. Of the trivial compounds that is not given much attention in this section are the fatty acid compounds that have been identified from the various plant parts. The papaya seeds are the best source of such fatty acids, of which, the identification of oleic acid as antibacterial principle has been reported (Ghosh et al., 2017).

11.3.2 Phenolic acids and flavonoids derivatives A number of studies have been devoted in preliminary characterisation of papaya secondary metabolites in the various plant tissues such as total polyphenol contents expressed as gallic acid equivalents, the presence or absence of flavonoids, alkaloids, tannins, saponins, procyanidins, etc. This include comparative analysis of the various plant parts with respect to structural classes of compounds. For example, while saponins are reported to be found only in the leaves, tannins which are not detected in the pulp extract are found in both seeds and leaves (Ikpeme et al., 2011). Although variable concentrations are noted, the presence of alkaloids, glycosides, polyphenols and hydroxymethyl anthraquinones were reported in the seeds, leaves and pulp extracts. This kind of observation could be easily disputed, however, given variabilities in chemical composition of plants on the basis of various factors. Quantitative variations in such components for the various plant parts sourced from different regions have also been presented but this is of limited value in terms of identifying the active components. Hence, only literature with detailed study on phytochemical components are presented herein (see Figs. 11.5 and 11.6).

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R

OH

OH HO

HO

OH

O

HO

O

OH

HO

OH

OH

OH OH O 4 Myricetin

OH O 1 R=H Kaempferol 2 R=OH Quercetin 3 R=OMe Isorhamentin

OH O 5 Morin R

OH

OH

OH HO

O

HO

O OH

OH O 7 R=H Apigenin 8 R=OH Luteolin

O 6 Fisetin

R

R

OH HO

OH

O

HO

O

O OH O

O

HO

O

OH

OH O

O

O

HO

O

OH

O

OH

OH

OH

OH

HO

OH

O

OH

O OH

OH O

OH

O

OH

9 R=H Nicotiflorin 10 R=OH Rutin

11 R=H Clitorin 12 R=OH Manghaslin

FIG. 11.5 Flavonoids composition of papaya.

O

OH OMe

COOH HO

R R OH OH 13 R=H Protocatechuic acid 15 R=H p-Coumaric acid 16 R=OH=Caffeic acid 14 R=OH Gallic acid 17 R=OMe Ferulic acid HO COOH HO

O HO

OR OH

19 R=H Quinic acid 20 R=Caffeoyl chrlorogenic acidl

OH O

OR

21 R=H Malic acid 22 R=Caffeoyl 23 R=p-Coumaroyl 24 R=Feruloyl

FIG. 11.6 Phenolic acids and derivatives of papaya.

O

MeO

O

18 5,7-Dimethoxycoumarin

O

R

OH R=H p-Coumaroyl R=OH Caffeoyl R=OMe Feruloyl

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Lako et al. (2007) have studied the polyphenol composition of papaya fruits of Fijian origin and documented myricetin (4), quercetin (2), kaempferol (1) and morin (5) within 2–3 mg/ 100 g FW while fisetin (6) and isorhamentin (3) were below 1 mg/100 g FW. By using high-performance liquid chromatography-diode array detector-hyphenated with tandem mass spectrometry (HPLC-DAD-MS/MS-ESI), Gayosso-Garcı´a Sancho et al. (2011) have determined the level of phenolic acids in papaya fruits (Carica papaya L., cv. Maradol) of Mexican origin. Their analysis identified p-coumaric (15), ferulic (17) and caffeic (16) acids as principal components of the papaya skin. Phenolic compounds identified in the fruit skin tended to decrease with ripening. The compounds identified were ferulic acid (17, 277.49 to 186.63 mg/100 g DW), p-coumaric acid (15) (229.59 to 135.64 mg/100 g DW), and caffeic acid (16, 175.51 to 112.89 mg/100 g DW). The detection of myricetin (4), kaempferol (1), luteolin (8) and apigenin (7) but not quercetin in the fruits of Hawaii-origin have also reported by Franke et al. (2004). The study of Rivera-Pastrana et al. (2010) which reported ferulic acid (1.33–1.62 g/kg DW), caffeic acid (0.46–0.68 g/kg DW) and rutin (10) (0.10–0.16 g/kg DW) were found in ‘Maradol’ papaya fruit, which tended to decrease during ripening. As minor principles, a range of other phenolic acid derivatives and their glycosides have also been detected by HPLC. Brasil et al. (2014) analysed the methanolic extract of the leaves ESI
MS/MS analysis and identified ferulic acid, caffeic acid, gallic acid (14) and quercetin (2). Other compounds detected through further processing of the extract were flavonoids, quercetin (2), rutin (10), nicotiflorin (9), clitorin (11), and manghaslin (12). Canini et al. (2007) have studied the phytochemical composition of papaya leaves (of West Cameroon origin) by gas chromatography coupled with mass spectrometry (GC–MS) analysis. Of the flavonoids, kaempferol (1) and quercetin (2) were detected while the phenolic acid composition include protocatechuic (13), p-coumaric (15), caffeic (16) and chlorogenic (20) acids isolated along with 5,7-Dimethoxycoumarin (18). The quercetin (2) and kaempferol (1) composition of the leaves have also been confirmed from the study by Miean and Mohamed (2001). Ultra-high performance liquid chromatography coupled with triple-time of flight-mass spectrometry (UPLC-TripleTOF-ESI-MS) fingerprinting of C. papaya leaf from cultivar ‘Sekaki’ exhibited 17 peaks and with the limitation of the methodology not discriminating isomeric forms, the following compounds were unambiguously identified: quinic acid (19) and malic acid (21), and malate isomers; caffeoyl (22), p-coumaroyl malate (23) and feruloyl (24); quercetin-3-O-(200 ,600 -di-O-rhamnopyranosyl) glucopyranoside (manghaslin, 12), Kaempferol-3-O-(200 ,600 -di-O-rhamnopyranosyl) glucopyranoside (11, clitorin); quercetin-3O-rutinoside (10, rutin); kaempferol-3-O-rutinoside (nicotiflorin, 9) and the alkaloid carpaine (Afzan et al., 2012).

11.3.3 Isothiocyanates and related compounds Benzyl isothiocyanate (BITC, 26) is the chief or sole anthelmintic isothiocyanate compound in papaya seed extracts (Kermanshai et al., 2001). The BITC production occurs when the benzyl glucosinolate, present in the interior of the seeds, contacts the myrosinase enzyme, present in the seed’s surface. The enzyme catalyses the BITC production (Fig. 11.7). The extraction of this compound as a value-added product of the seeds from papaya production has been advocated. Barroso et al. (2016) have employed the supercritical CO2 extraction system to isolate D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

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11.3 The chemistry of papaya

OH HO

OH O

S

OH

N

Myrosinase

O O N S O O

C

S

26

25 OH HO HO N C

OH O O *

27 * (R) Prunasin 28 * (S) Sambunigrin

FIG. 11.7 Glucosinolate and isothiocyanate composition of papaya.

this compound in the oil obtained from crushed seeds. Rossetto et al. (2008) also established that the level of myrosinase activity was detected in the seeds with the highest benzylglucosinolate levels were detected in seeds, followed by the fruit peel and pulp. Myrosinase activity decreased in all tissues during fruit development. This suggest that all parts of the fruits are capable of producing BITC with the seeds playing major role. They also found that benzylglucosinolate (25) levels in the pulp did not decrease during ripening, suggesting glucosinolate and isothiocyanates are available as a dietary source from this fruits. The study by Bennett et al. (1997) also confirmed that BITC is exclusively formed with no other isothiocyanate compounds detected. The leaves, especially the younger ones, appear to have the highest amount as compared to the roots and stems. The seeds, however, remains to be the major source of this compound (Nakamura et al., 2007). The known anticancer effect of BITC as well as the fruits as glutathione S-transferase inducers (Nakamura et al., 2000) and cytotoxicity of the leaves in cancer cells (Otsuki et al., 2010) are in line with potential anticancer effect of the plant. Related to the isothiocyanates are the cyanogenic glucosides prunasin (27) and sambunigrin (28) that have been isolated from C. papaya leaves and stems (Seigler et al., 2002). These are compounds isolated in small amounts but have been detected in the leaves, stems and fruits (Spencer and Seigler, 1984).

11.3.4 Alkaloids Carpaine (29), an alkaloid with an intensely bitter taste and a strong depressant action on the heart, has been obtained mainly from the leaves, fruit and seeds (Hornick et al., 1978). Incredibly, the isolation of alkaloids from this plant is traced back to the 19th century. The publication by Govindachari et al. (1954), for example, cited the isolation of carpaine in 1890 for the first time from papaya leaves. Their own work also led to the isolation of D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

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H N

O O

H N

O O

O

N H

O

N H

O

30 Pseudocarpain

29 Carpaine

O

N O

O

N O

O O

N H

31 Dehydrocarpaine I FIG. 11.8

O

O O

N

32 Dehydrocarpaine II

Alkaloidal composition of papaya.

pseudocarpaine (30) as a novel compound. Numerous authors have reported these alkaloids from the various part and the study by Khuzhaev and Aripova (2000) reported their detection in the leaves, bark and heartwood of C. papaya. The common alkaloids from the various parts are now known to include carpaine, pseudocarpaine, dehydrocarpaine I (31) and II (32) (see Fig. 11.8). Dehydrocarpaine I and II were first isolated from the leaves of papaya by Tang, 1979. HPLC analysis have shown that carpaine is the major alkaloidal component of the leaves (Wang et al., 2015). The isolation of a trivial compound, choline, along with carpaine from the leaves was described by Ogan (1971).

11.3.5 Enzymes and protein components Papaya belong to a group of plants collectively called lactiferous for their ability to secrete white milk-like latex. Numerous plant families are known to specialise in such task and it is widely believed that this capacity is related to plant defences against infection. Some reports indicate that up to 35,000 plant species are known to produce latex. Not surprisingly, the release of latex from pants is induced by tissue damage such as that involving large-scale latex harvest from a rubber plant. The physiological adaptation to wounding by releasing latex is easy to understand. The milky latex released during tissue damage quickly set to coagulate and seal the damaged tissue and prevent potential pathogens entry into the plant tissue. The numerous pharmacological effects of the latex also fits to this conception. The ability to secrete latex by these plants is due to specialised cells called laticifers that are widely distributed throughout the plant tissues. In the case of papaya, the latex is a source of four well-characterised cysteine endopeptidases called papain, chymopapain, glycyl endopeptidase and caricain. Given the antimicrobial and wound healing properties of these endonucleases, papaya latex is a commercial product that gained lots of interest within the food (e.g., meat tenderising) and pharmaceutical industries. The best source for this products is the fully-grown but unripe papaya fruit. As ripening of the papaya fruit progresses, the latex amount diminishes. D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

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Fractionation and isolation of the enzymes from papaya latex have been studied by many authors. In the experiment by Azarkan et al. (2003), the isolation of chymopapain as a predominant enzyme, about 40% of enzymes present therein, has been described. The presence of a papaya endopeptidases has also contributed to the plant being a valuable latex for industrial applications. With respect to concentration in the latex, the well-known commercial product, papain (EC 3.4.22.2), is considered to be a minor component in the mixture of papaya latex endopeptidases. Caricain is another enzyme that is known to account to about 15% of proteinase A or papaya proteinase Ω (EC 3.4.22.30). Overall, the the latex from unripe papaya fruits is a mixture of highly valuable cysteine endopeptidases including papain, ficin, chymopapain A and B, papaya endopeptidase II, papaya endopeptidase IV and omega endopeptidase (Azarkan et al., 2003; Thoma´s et al., 2009). A number of other enzymes and proteins (pathogenesis-related proteins) such as chinitases, protease inhibitors, linamarase are also known to occur. As explained in the later sections, the significance of these proteins/enzymes is as wound healing agents in the plant preparations through diverse mechanisms including antibacterial effects. Papain both as antidefense protein (insect deterrence) and pharmaceutical/food industrial application as cysteine protease are of major significance and is discussed further in the following sections.

11.4 The pharmacology of papaya related to diabetes and associated diseases 11.4.1 In vitro studies Some of the key pharmacological activities related to potential diabetes therapy are summarised in Table 11.2. Considerable amount of in vitro experiments have been carried TABLE 11.2

In vitro effects of papaya preparations.

Preparation

Assay model

Key findings

References

Fermented papaya preparation (FPP)

PMA-stimulated respiratory burst activity of peripheral blood mononuclear cells (PBMC) from T2D patients

3 mg/ml—Improve the suppressed level Dickerson of respiratory burst (O%-); increased et al., 2012 phosphorylation of the p47phox subunit of NADPH oxidase; augment the protein and mRNA expression of Rac2; no effect on the AP-1 DNA binding activity but augment the Sp1 DNA binding activity.

Unripe Fruits— ethanol extract

Preliminary antibacterial screening— P. aeruginosa, Proteus murabilis, Enterobacter agglumerans, S aureus

Some activity—10 μl of the extract added Nayak et al., to agar. 2007

FPP

Antioxidant effect—radical (OH%) scavenging assay

50 mg/ml—Antioxidant

FPP

Platelets from patients with T2D

Raffaelli 50 μg/ml—Enhance Na+/K+-ATPase activity and membrane fluidity; increase et al., 2015 total antioxidant and SOD capacity; reduce conjugated diene levels

Imao et al., 1998

Continued

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TABLE 11.2 In vitro effects of papaya preparations.—cont’d Preparation

Assay model

Key findings

References

Fruits pulp and peel dietary fibre concentrate

Antioxidant effect (DPPH and ferric reducing power assays)

Peel more potent than pulp

Calvache et al., 2016

FPP

Aruoma 50 μg/ml—Reduce the genotoxic and Rat pheochromocytoma (PC12) and human cytotoxic effect of H2O2; reduce oxidative et al., 2006 damage. hepatoma (HepG2) cells treated with H2O2

FPP (commercial source—as gel, BioRex)

Antioxidant effect and antimicrobial (S. aureus) assay

1–5 mg/ml in vitro—suppress the generation of O.-2 (IC50 ¼ 5 mg/ml) and OH% radicals (IC50 ¼ 1.1 mg/ml) and total production of radicals in human blood cells (IC50 ¼ 2 mg/ml) by 50%; decrease bacterial catalase activity by 35%.

Mikhal’chik et al., 2004

Seeds— Methanol extract

Lymphocyte proliferation and complement-mediated haemolytic assays

Enhance phytohemagglutinin responsiveness; inhibit classical complement-mediated haemolytic pathway. i.e., immunostimulatory and anti-inflammatory effect.

MojicaHenshaw et al., 2003

Unripe fruits— Ethanol extract

Isolated rabbit arterial (aorta, renal and 10 μg/mL—Relax vascular muscle tone vertebral) strips which was attenuated by phentolamine

FPP

LPS/IFN-γ-activated RAW 264.7 macrophages

Unripe fruits

Antimicrobial (enteropathogens such as Bacteriostatic activity; scavenging action Bacillus subtilis, Enterobacter cloacae, on O.-2 and OH% radicals Escherichia coli, Salmonella typhi, Staphylococcus aureus, Proteus vulgaris, Pseudomonas aeruginosa, and Klebsiella pneumoniae) and antioxidant assay

Eno et al., 2000

Increase iNOS activity and nitrate Rimbach accumulation; stimulate TNF-α secretion. et al., 2000 Osato et al., 1993

Abbreviations: AP-1, Activator protein 1; iNOS, inducible nitric oxide synthase; NADPH, nicotinamide adenine dinucleotide phosphate (reduced form); PMA, phorbol 12-myristate 13-acetate; Rac2, Ras-related C3 botulinum toxin substrate 2; SOD, superoxide dismutase; Sp1, specificity protein 1 (a transcription factor); TNF-α, tumour necrosis factor α.

out to show the antioxidant, anti-inflammatory and antimicrobial effects of extracts from the various parts of the plant. The fruit pulp and peel as well as FPP display antioxidant effects when tested using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) or hydroxyl (OH%) radical scavenging assays as well as reducing power measurements using the ferric ions model (Calvache et al., 2016; Imao et al., 1998; Mikhal’chik et al., 2004). In a cellular system such as HepG2 hepatoma cells, the cytotoxicity induced by hydrogen peroxide (H2O2) could be ameliorated by FPP at fairly low concentrations (Aruoma et al., 2006). The leaves extract has been shown to suppress the H2O2-induced erythrocyte damage (Okoko and Ere, 2012); and the seeds extract protected cultured HepGe cells from H2O2 toxicity (Salla et al., 2016). The study by Amer et al. (2008) also showed that in vitro treatment of blood cells from β-thalassemic patients with FPP

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could enhance the glutathione (GSH) content of red blood cells, platelets and polymorphonuclear leukocytes, along with a reduction in the level of reactive oxygen species (ROS), membrane lipid peroxidation (LPO) and externalisation of phosphatidylserine. The antimicrobial effects recorded in vitro have been instrumental to assert that papaya extracts have potential therapeutic applications in wound healing process by suppressing secondary infections. A number of bacterial strains including those implicated in wound infection such as Staphylococcus aureus have been shown susceptibility to the extracts of the fruits (Mikhal’chik et al., 2004; Nayak et al., 2007; Osato et al., 1993). While general antioxidant activity is regarded as beneficial in diabetes complications, the wound healing process appear to follow a different perspective. As explained in the latter sections, the in vitro data supporting this mechanism is through enhancing leucocytes respiratory burst (Dickerson et al., 2012) and enhancement of leucocytes activation as demonstrated in the RAW264.7 macrophage model (Osato et al., 1993). The immunestimulatory effect of the seeds along with anti-inflammatory effect have also been reported (Mojica-Henshaw et al., 2003). Some of the doses employed in these studies are high but given the plant preparations are directly applied to wound areas as topical treatment, the relevance of the observation is not questionable. For example, the antioxidant and antimicrobial activity of FFP was in mg/ml range of concentrations but a 500-fold increase in the effectiveness of intracellular killing of Staphylococci was demonstrated for FFP as a commercial gel (Mikhal’chik et al., 2004). The proteolytic enzymes, chymopapain and papain are also known to display antimicrobial (Emeruwa, 1982) and antioxidant properties (Osato et al., 1993). Other in vitro data worth mentioning are enhancement of platelet function including their membrane integrity and fluidity as well as boosting their antioxidant defence under chronic diabetes condition (Raffaelli et al., 2015). As explained later under in vivo studies, some in vitro evidence also suggest vasorelaxant properties for the fruits (Eno et al., 2000).

11.4.2 Antidiabetic and lipid lowering effects in animal models As shown in Table 11.3, some studies relevant to this topic are presented, but the antidiabetic potential study on papaya plant is generally at its meagre stage. The study by Jua´rez-Rojop et al. (2012) is one of the few documented in animal studies to substantiate the potential antidiabetic property of the leaves. The doses used where not however measured as the extract was administered in drinking water instead of known amount given to animals. The glucose, cholesterol and total triglycerides (TG) lowering effects as well as reduction of liver toxicity markers and antioxidant parameters in the classical STZ-diabetic model is a good signature of antidiabetic potential. Reduction of blood glucose by the leaves extract were also demonstrated by other studies using spontaneously hypertensive rats (Brasil et al., 2014) or alloxan-induced diabetic rats (Sasidharan et al., 2011) at doses of 100–400 mg/kg. For the chloroform extract of the leaves, Ghosh et al. (2017) infact have shown glucose lowering effect and a decrease in TG level as well as liver toxicity markers at 31 or 64 mg/kg doses in STZ-diabetes model. Blood glucose level could also be lowered by the root bark extract in hypertensive model (Ravikant et al., 2012). Perhaps the most acclaimed and rather emerging trend of papaya as nutritional supplement came from the FPP which has also been demonstrated to lower blood glucose level

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TABLE 11.3 In vivo effects of papaya preparations. Preparation

Experimental model

Key findings

References

Roots— Aaqueous extract

Arsenate- induced toxicity in rats— 100 and 150 mg/kg. Administered together with vitamin C

Attenuate toxicity—reduce ASP, ALT, ALP and total bilirubin levels.

Ojo et al., 2017

Leaves— Chloroform extract

STZ-induced diabetic rats—31 or 62 mg/kg, p.o. for 20 days

Reduce FBG; little reduction in body weight; decrease serum TG but not cholesterol or HDL; reduce serum AST and ALT; increase insulin level in non-diabetic animals

Ghosh et al., 2017

Leaves— Methanolic extract

Spontaneously hypertensive rats— 100 mg/kg twice a day for 30 days

Reduce blood pressure and cardiac hypertrophy; angiotensin converting enzyme inhibitory activity.

Brasil et al., 2014

Leaves (fresh)— Aqueous extract

Mice receiving 2 g/mouse, p.o. for up to 21 days

Increased RBC and platelet count. Platelet count increase from Day 3 (3.4  0.18  105/μL), reaching almost a fourfold higher at Day 21.

Dharmarathna et al., 2013

Leaves— Aqueous extract

STZ-induced diabetic rats—0.75 g and 1.5 g/100 mL in drinking water for 4 weeks

Recover body loss; decrease FBG; cholesterol, TG and aminotransferases blood levels; no effect on insulin level in diabetic but increase in normal animals; improve pancreatic cell and hepatocytes recovery (histology); antioxidant effect; increase glycogen level in liver.

Jua´rez-Rojop et al., 2012

Leaves— Aqueous (hot) extract

Alloxan-induced diabetic rats— 400 mg/kg for 21 days (vehicle not mentioned)

Reduce glucose level and serum lipid profile levels (cholesterol, TG); no effect for 100 and 200 mg/kg dose;

Maniyar and Bhixavatimath, 2012

Ethanol extract of fruits

STZ-induced diabetic mice— 100 mg/kg

Reduce blood glucose; recover body weight; improve histology of pancreatic β cells and kidney.

Sasidharan et al., 2011

FPP

Obese Leprdb diabetic mice (db/ db)—200 mg/kg, p.o. 5 days/week for 8 weeks

Reduce blood glucose, TC and LDL; increase HDL; improve wound closure; increase ROS production and NO in macrophages and increase VEGF gene in wound tissues.

Collard and Roy, 2010

Root bark— Ethanol extract

Renal artery occluded hypertensive rats—25, 50 and 100 mg/kg, i.v.

Reduce the raised blood pressure via the renin-angiotensin system.

Ravikant et al., 2012

Dried papaya latex from unripe but mature fruits

Burns-induced wounds in mice— 1.0% and 2.5% topically

Increase hydroxyproline content (biochemical marker for collagen tissue); increase contraction; reduce epithelialization time.

Gurung and Skalko-Basnet, 2009

Green and ripe papaya epicarp—

Excised wound model in pregnant mice from day 10 and onwards after conception—1 g/kg, p.o.

Reduce wound healing time; contribute to embryonic resorption while green epicarp extract

Anuar et al., 2008

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TABLE 11.3 Preparation

351

In vivo effects of papaya preparations.—cont’d Experimental model

Aqueous extract

Key findings

References

contribute to premature delivery; differences between the two preparations in protein profile (SDSPAGE).

Leaves— Ethanol extract

Carrageenan- induced paw oedema, cotton pellet granuloma and formaldehyde- induced arthritis models—25-200 mg/Kg, p.o.

Positive anti-inflammatory effects in all models—slight mucosal irritation at high doses.

Owoyele et al., 2008

Unripe Fruits— Aqueous extract

STZ-induced diabetic rats—topical application of 100 mg/kg for 10 days

Excision and dead space wound model—Reduce wound area; fasten epithelization; increase hydroxyproline content, and wet and dry granulation tissue weight; also display antibacterial activity in five test species.

Nayak et al., 2007

FPP (commercial source—as gel, BioRex)

Burn trauma wound model in rats— topically up to 12 days

Reduce acute inflammatory response (day 12); wound area 2-fold lower than untreated; reduce ROS level in the whole blood cells; Reduce bacterial burden and survival in wounds; reduce bacterial catalase activity, while activities of other antioxidant enzymes (SOD) in the granulation tissue remained unchanged; reduce myeloperoxidase level.

Mikhal’chik et al., 2004

Unripe Fruits— Ethanol extract

Renal and DOCA-induced hypertension in rat—20 mg/kg, i.v

Reduce mean arterial blood pressure; effect mediated via alphaadrenoceptor activity (antagonised by propranolol but not by atropine and noradrenaline).

Eno et al., 2000

Abbrevations: ALP, alkaline phosphatase; ALT, Alanine transaminase; AST, aspartate transaminase, DOCA, deoxycorticosterone acetate; FBG, fasting blood glucose; FPP, fermented papaya preparation; i.v, intravenous, NO, nitric oxide; p.o., oral route of administration ROS, reactive oxygen species; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; SOD, superoxide dismutase; VEGF, vascular endothelial growth factor.

in the obese mice diabetes model along with total cholesterol (TC) lowering effect. It is thus reasonable to conclude that the current level of in vivo study is in line with glucose and lipid lowering effect of papaya extracts. There is no concrete evidence to show that papaya extracts have antiobesity effect, however. Improvement of the diabetes pathology obviously leads to recovery of weight loss (Sasidharan et al., 2011). While some studies showed an increase in high density lipoptotein-cholesterol (HDL-C) (Collard and Roy, 2010), some showed no effect even for cholesterol level (Ghosh et al., 2017). This could be differences in the doses employed in these experiments.

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The antioxidant and anti-inflammatory mechanisms demonstrated for papaya extracts are in line with potential benefit in treating diabetes pathology. This has been the case for the FPP (Mikhal’chik et al., 2004) and the leaves (Owoyele et al., 2008) in the various experimental models. Readers should bear in mind that the antioxidant effect of papaya even though well confirmed through a variety of assays is not as potent as that one can get in plants extracts. A comparative analysis of wheat sprouts, Morinda citrifolia, FFP and white tea, for example, have shown that FFP extract possesses the lowest antioxidant effects (Calzuola et al., 2006). The data by Zhang et al. (2006) have also shown that FFP have cytoprotective effects in SH-SY5Y neuronal cells from amyloid-β-induced ROS generation and cytotoxicity. Unlike guava, papaya was among the fruits that have been documented with total phenol content value lower than orange but the ascorbic acid content (as with guava) is far higher than orange. The antioxidant potential when assessed by radical scavenging (DPPPH) and chelating power (iron II) was not that potent (Lim et al., 2007). Anti-inflammatory effect for the leaves extract (50–200 mg/kg, i.p.) was shown in the egg albumin-induced paw oedema model in rats (Amazu et al., 2010).

11.4.3 Papaya and wound healing Before we discuss the therapeutic potential of papaya on diabetes wound, it is worth to briefly describe the process of wound healing. These processes and pharmacological intervention by natural products have been reviewed in the various literature (e.g. Tsala et al., 2013) and include at least four distinct phases: The haemostasis, inflammation, proliferation and maturation or remodelling stages. Haemostasis: This constitute as the immediate response to tissue injury and involve, vasoconstriction to reduce blood loss and clotting. At cellular level, platelets orchestrate the clotting cascade and together with leucocytes, a range of cytokines are released. These cytokines are involved in the later processes of coordinated inflammation and vascular activity and remodelling processes: Epidermal growth factor (EGF) ! Collagen synthesis and epithelialization; Fibroblast growth factors (FGF) ! Angiogenesis; Insulin-like growth factors (IGF1, IGF2) ! Collagen synthesis and fibroblast proliferation; Interleukin-1 (IL-1) and tumour necrosis factor α (TNF-α) ! Collagen synthesis angiogenesis; • Platelet-derived growth factor (PDGF) ! Collagen synthesis and macrophage migration; • Transforming growth factor β (TGF-β) ! Collagen synthesis, fibroblast proliferation, angiogenesis; • Vascular endothelial growth factor (VEGF) !Angiogenesis.

• • • •

The inflammatory phase—Constitute leucocyte immigration to the wound area. The first subgroup of leucocytes to accumulate are the neutrophils which deals with removal of dead cells and debris from injury site. They may contribute by releasing a range of growth factors and chemotaxis agents but their number will diminish as long as wound complication, say by bacterial infection, does not occur. The major inflammatory cells in the wound are macrophages. Monocytes from the blood transform themselves to efficient phagocytosis machines,

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macrophages, which also release a plethora of cytokines and growth factors. They stimulate cell proliferation (epidermal cells and fibroblasts) and extracellular matrix proteins synthesis and deposition, and also orchestrate angiogenesis. The fibrous tissue formation (fibroplasia) and collagen deposition are initiated at this stage. Although not as critical as macrophages, the involvement of lymphocytes, particularly, T-cells, are known in wound healing process. The proliferation phase—This constitute the major stage of the extracellular matrix (ECM) (e.g. proteoglycans and glycosaminoglycans) formation/deposition within the fibroplasia process. The process of new blood vessel formation (angiogenesis) from existing vessels and new capillary network formation are prevalent at this stage. Angiogenesis process classically distinguished as red granular appearance of the wound is a stage where vascualr endothelial growth factor (VEGF) and endothelial proliferation and tube-like formation predominates. The process of re-epithelialization also takes place in this stage where keratinocytes play major role and a number of matrix metalloproteinases (MMPs), tissue plasmin and adhesion molecules bringing similar cells together to form tissues play key roles. The maturation or remodelling phase—This is a stage following the active deposition and cellular proliferation stages and hence characterised by little metabolic processes. The stage is rather defined by reorganisation of tissues to form well defined and stronger collagen bundles, removal of less-defined capillaries and formation of stable scar tissues. Hence, this process lasts several months after the wound closure. Mikhal’chik et al. (2004) have done comprehensive study both in vitro and in vivo to show the potential wound healing effect of commercially available FPP (BioRex, 003117. I.392.07.2001). The preparation has antioxidant effect when tested in vitro using human peripheral blood neutrophils as inhibition of through hydroxyl and superoxide radicals (studied in H2O2-FeSO4 and xanthine-xanthine oxidase models). The production of reactive nitrogenous (RNS) and ROS by leukocytes in phorbol ester-stimulated preparations were also depressed while bactericidal effect against S. aureus associated with a reduction of catalase level was noted. In a thermal wound model in rats, topical treatment not only reduce the wound area but also reduced bacterial burden (S. aureus) through mechanism of lowering their catalase level. The ROS generation and myloperoxidase suppression was evident to suggest antioxidant and anti-inflammatory mechanisms. Experiments on the wound healing effect of papaya with respect to antimicrobial effects have also been demonstrated by various authors. This include a burn model of wound (Starley et al., 1999a, b; Mikhal’chik et al., 2004). An incredible level of information on the antidiabetic and wound healing potential of FPP came from the study by Collard and Roy (2010). They employed an obese diabetic (db/db) mice where they noted the following key observation after 0.2 g/kg oral supplementation for 8 weeks: • The reduction in blood glucose, total TG and TC level while enhancing the HDL was consistent with antidiabetic potential; • The lack of effect on weight gain appears to indicate lack of effect as antiobesity agent at the tested dose; • The improvement on wound closure from the full-thickness in skin and panniculus carnosum of dorsal wounds is in agreement with the claimed wound healing potential of the fermented fruit reported in the various literature;

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• Wound macrophages from the treated diabetic or non-diabetic animals group appear to produce more ROS and superoxide when stimulated; • Wound macrophages from the treated diabetic (as compared to placebo treated) animals produce more nitric oxide as well as inducible nitric oxide synthase (iNOS); • Treated diabetic mice group have enhanced level of CD68 and VEGF gene in wounds. While ROS generation is generally enhanced in diabetes and subsequently antioxidant and antiglycation therapy is advocated, wound healing processes have a different profile. With respect to tackling infection to avoid secondary infection, the leucocyte NADPH oxidase (see Chapter 4) that generate ROS activity play significant role. Not surprisingly, a high level of NADPH oxidase activity is linked to diabetes suggesting oxidative stress at the forefront of diabetes pathology. On the other hand, the reduced level ROS or NO is implicated in slow wound healing process in diabetes. In fact, macrophage dysfunction is a common feature of diabetic wounds which is characterised by increased apoptotic cell burden at the wound site and prolonged inflammatory phase (Khanna et al., 2010). Moreover, polymorphonuclear leukocytes (PMNs) from diabetes patients have also been known to display reduced level of phagocytosis and bacterial killing capacity (Bagdade et al., 1974). Hence, the effect of FPP in both systems (general diabetes pathology vs wound complication) appear to suggest a possible mechanism of action in wound repair and potential antimicrobial properties. The higher level of CD68 expression also suggest macrophage activation as this glycoprotein interact with LDL. Perhaps the most direct evidence on the potential wound healing effect of FPP came on enhanced expression level of VEGF suggesting augmented neovascurisiation and angiogenesis processes that is critical to wound healing process. Nayak et al. (2007) also employed the direct wound healing effect of topically applied papaya aqueous extract. As a marker of the wound healing process, they also demonstrated an increase in the collagen marker (hydroxyproline) as well as granulation tissue matrix following treatment. Interestingly, topical treatment of mush pulp of C. papaya containing papain and chymopapain for paediatric infected burns was effective for desloughing necrotic tissue, preventing infection and providing a granulating wound (Starley et al., 1999a, b). The effect of papain on infected wound healing by its own and in combination with other ingredients have also been evaluated by Telgenhoff et al. (2007). It was reported that an increase in dermal blood vessel formation, collagen I deposition, and mature collagen by papain treatment was consistent with its known wound healing effect. They have also shown qualitative measures to show a better healing process after treatment with papain as follow: • Greater number of keratinocytes observed in the epidermis; • The epithelial extensions that project into the underlying connective tissue in the skin which refereed as rete peg formation; • More vasculature formation; • Greater number of collagen birefringence to refer to organised orientation as oppose to outof-plane collagen orientation. Hence, the active components for the wound healing effect of papaya has been claimed to be associated with a rich proteolytic enzymes such as papain. The small molecular weight antioxidant compounds have also a role to play. Given the isolated compounds from the plant

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including phenolic acids and flavonoids are classical antioxidant compounds with antidiabetic effects in vitro and in vivo, their contribution to diabetes-related pathology should also be considered. Compounds such as caffeic acid, quercetin and rutin are good examples of papaya constituents that are reported to display antidiabetic effects both in vitro and in vivo through multiple mechanisms (Habtemariam, 2011, 2016; Habtemariam and Varghese, 2014). As a mechanism of wound healing effect, the antimicrobial effect and enzymatic property of the latex appear to be significant, however. Preliminary data suggest that the papaya-derived enzyme papain, when applied topically, may facilitate enzymatic wound debridement (Telgenhoff et al. (2007)). Hence, the application of exogenous enzymes like papain to promote a quick onset of debridement process have been advocated as therapeutic option in diabetes wound therapy. The enzyme’s ability to digest and breakdown necrotic tissue is expected to shorten the wound healing time.

11.4.4 Antihypertensive effect A number of studies have demonstrated the antihypertensive potential of papaya plant. By using the isolated mesenteric bed and aortic preparations, the methanol extract of the leaves has been shown to display vasorelaxant properties (Runnie et al. 2004). The antihypertensive activity of the methanolic extract of leaves of C. papaya (Ruby variety) has also been demonstrated by Brasil et al. (2014). By using a spontaneously hypertensive rat model, a reduction in blood pressure (BP) coupled with suppression of cardiac hypertrophy were noted. Furthermore, the possible mechanism of action via inhibition of angiotensin converting enzyme (ACE) activity was confirmed. Insight into the potential active principle has also been made given the methanolic extract with the known phenolic components were as follow: ferulic acid (17, 203.41  0.02 μg/g), caffeic acid (16, 172.60  0.02 μg/g), gallic acid (14, 145.70  0.02 μg/g), and quercetin (2, 47.11  0.03 μg/g). The flavonoids quercetin, rutin (10), nicotiflorin (9), clitorin (11), and manghaslin (12) were also present. Considering that flavonoids including the common flavonols (e.g., quercetin and kaempferol) detected in the plant have been shown to inhibit ACE (Guerrero et al., 2012), they may serve as the active principles for the observed antihypertensive effect. The correlation between hypertension and cardiac hypertrophy is evident and could be a critical problem in diabetes-associated cardiovascular complications. Moreover, angiotensin inhibition could also suppress cardiac hypertrophy. In fact, angiotensin II has been shown to induce hypertension and cardiac hypertrophy through its receptors in the kidney (Crowley et al., 2006). Improvement of baroreflex sensitivity in spontaneously hypertensive animals by quercetin is also in line with the observation of Brasil et al. (2014) for the leaves extract. The stems extract of papaya of Brazilian-origin has also been reported to display some inhibitory effect against ACE at the concentration of 100 μg/ml (Braga et al., 2007). The dichlomethane extract of dried fruits have also been demonstrated to display some ACE inhibitory effect in vitro (Loh and Hadira, 2011). The study by Eno et al. (2000) used both in vivo and in vitro models to study the potential antihypertensive effect of the fruits. The suppressive effect of the ethanolic extract on the deoxycorticosterone acetate-sodium chloride hypertensive rat model at fairly low dose (20 mg/kg, i.v.) confirmed antihypertensive effect. The mechanism of action could be

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complicated and is perhaps of multiple effects nature but in vitro studies using arterial strips clearly demonstrated the extract abolishing the contracture induced by phentolamine; perhaps suggesting involvement of α-adrenoceptors. Over all, animal studies have provided some evidence to substantiate the glucose and lipid lowering effects of papaya with implication of potential therapeutic application in diabetes and associated diseases. Perhaps the most concrete data so far for papaya is for treatment of diabetic wound primarily as a function of enzymatic activity of the extracts (especially from the green fruits) through antioxidant, antibacterial and enzymatic action of the wound healing process. The antihypertensive effect of the plant primarily through the action of small molecular weight compounds is gaining lots of interest and is likely to be employed for treating diabetes-related cardiovascular complications.

11.4.5 Evidence of efficacy from human trials According to the ClinicalTrials.gov database, there are four clinical trial entries for papaya related to diabetes (Table 11.4). These data merely shows the interest in the plant in the indicated area which are diabetes, hypertension and wound healing properties. TABLE 11.4 Human trials registered under ClinicalTrials.gov database.a Status

Study title

Conditions

Interventions

Completed (2014)—

Molecular and clinical effects of green tea and FPP on diabetes and cardiovascular diseases— 300 participants

• Assess the effect of green tea on diabetes; • Assess the effect of FPP on diabetes; • Effects of green tea and FPP on C-reactive proteins

• Dietary Supplement: Green tea

Active not recruiting

Nutritional regulation of wound inflammation: Part II

Diabetes

Dietary Supplement: FPP

Active not recruiting (From 2015–)

Nutritional regulation of wound inflammation: Part III

• Negative Pressure therapy; • Wound; • Open wound

Dietary Supplement: FPP

Completed (2009) 150 participants—A randomised, double-blind, placebo-controlled, doseranging, exploratory, 28-day study to examine the effects of AR9281 on BP and glucose tolerance in patients with mild to moderate hypertension and impaired glucose

Evaluation of soluble epoxide hydrolase (s-EH) inhibitor in patients with mild to moderate hypertension and impaired glucose tolerance

• Hypertension;

• Drug: AR9281

Impaired glucose tolerance

Drug: Placebo

a

Dietary Supplement: FPP

https://clinicaltrials.gov/

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The clinical trial by Danese et al. (2006) constitute two groups of 25 subject each representing diabetic and healthy adults group. The effect of FPP administered as 3 g daily, during lunch, for two months were assessed. Their study reported a reduction in glucose level of both diabetic and normal subjects receiving FPP. Oral administration of FPP (9 g/day, six weeks) to T2D patients followed by a twoo weeks washout period have been reported to enhance the induced “respiratory burst” in peripheral blood mononuclear cells while not influencing other blood parameters studied (Dickerson et al., 2015). THP-1 cells in vitro treated with this FPP (2.9 mg/ml) could also show enhanced cellular ATP and NADPH; higher mitochondrial membrane potential (Δψm) and oxygen consumption. The number of subjects employed in the study were not many, however, with only 22 recruited and 17 completed the study. Oral supplementation did not change blood glucose, glycated haemoglobin (HbA1c), or TC levels in T2D subjects. Hence, the results presented herein is in contradiction with that reported by Danese et al. (2006) for the glucose lowering effect of FPP at a far lower dose. The randomised, controlled clinical trial by Somanah et al. (2014) employed 127 prediabetic Mauritians with the treatment group receiving 6 g FPP dissolved in 200 ml (1 cup) water per day before mealtimes for a period of 14 weeks. After supplementation followed by a two weeks wash out period, a reduction in the rate of haemolysis and accumulation of protein carbonyls in the blood plasma of pre-diabetics were observed. The increase in the total antioxidant status in the FPP-supplemented pre-diabetics as well as FPP showing antioxidant effects in vitro led to the general conclusion that FPP has significance as antioxidant agent in humans. In another randomised controlled clinical trial involving 127 Mauritius diabetic subjects, the effect of 14 weeks supplementation with FFP (6 g FPP®/day) was studied (Somanah et al., 2012). For the 80% of participants (101 subjects) who completed the trial, a reduction in BP (systolic for men and both systolic and diastolic for women) after the washout period (14 weeks) was reported. No weight lowering effect were noted for the treatment. Although a reduction of fasting glucose level as well as cholesterol and LDL levels were observed, a significant change were not observed in this parameters as well as the HDL level. An improvement in total antioxidant status and a reduction in C-reactive protein (CRP) as a marker of inflammation and cardiovascular disease were of significant beneficial outcomes. On this basis, even though the observed effect was not spectacular as antidiabetic potential, the authors highlighted the potential efficacy of the FPP for the management of diabetes and for those at risk for cardiovascular disease, neurological disease and other conditions worsened by overt inflammation and oxidative stress. Some clinical trial studies on papaya were directed in other disease model could still have some implication to T2D therapy. The study by Marotta et al. (2004), for example, examined the commercially available FPP (Immun-Age 6 g/day nocte) in a six month therapy regimen in Helicobacter pylori-negative atrophic gastritis elderly patents. They have shown antioxidant effects as evidenced by the reversal of the increased malondialdehyde (MDA) and increased levels of mucosal MDA and xanthine oxidase. In a small (12 healthy elderly subjects) group study, Marotta et al. (2006a) have also shown that FPP administration (9 g/day for 4 weeks) could ameliorate red blood cell oxidative damage. A more convincing data by these authors (Marotta et al., 2006b) were also presented through a randomised, placebo-controlled, crossover study, involving 54 elderly patients. A three month supplementation followed by six weeks washout period have demonstrated enhanced antioxidant status as measured by redox status and DNA damage. When the inflammatory and antioxidant status in patents were D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

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assessed under cirrhosis condition, FPP (Immun-Age®, 9 g/day) supplementation at bedtime for six months could decrease oxidative status (as assessed by the levels of 8-OHdG (as DNA damage), reduced (GSH) and oxidised (GSSG) glutathione, glutathione peroxidase (GPx), and MDA levels) and cytokine balance (Marotta et al., 2007). The elevated serum level of TNF-α and of its soluble p75 receptor in cirrhosis was suppressed by FPP. All these and other related studies (e.g., Marotta et al., 2010) clearly indicate that the observed antioxidant effect in vitro and in vivo for FPP could be reproduced under clinical conditions. Fujita et al. (2017) investigated the effect of administration of FPP (9 g/day) to tube-fed patients for 30 days. They have shown that FPP can restore the capacity of peripheral blood mononuclear cell (PBMC) to kill pathogenic bacteria. They have also recorded a Firmicutes burden in the faecal microbiota with Clostridium scindens and Eggerthella lenta number reduced in most patients.

11.5 Toxicological considerations Acute toxicity tests have been carried out in a number of in vivo studies. After an oral dose of 2000 mg/kg, the leaves extract (methanol) for example had no lethality, macroscopic changes or haematological (total erythrocytes, leucocytes, haemoglobin, protein, and haematocrit, mean corpuscular volume, corpuscular haemoglobin, and corpuscular haemoglobin concentration) and biochemical (ALT, AST, urea, creatinine, and troponin I level) toxicity markers (Brasil et al., 2014). In a repeated dose regimen of 28-day toxicity study (Afzan et al., 2012), administration of up to 2 g/kg dairy p.o. did not cause mortality or histopathology of major organs including the liver. Body weight, food intake, water level, were not also altered but total protein, HDL-C, AST, ALT and ALP were elevated in a non-dose dependent manner (0.01, 0.14 and 2 g/kg). While the increase in HDL level may be beneficial, the increase in liver enzymes could be a sign of toxicity at this dose level. The LD50 of the unripe fruit was however low with one report showing LD50 value of 325.2 mg/kg, i.p., in mice (Eno et al., 2000). Nayak et al. (2007) reported that doses up to 4 g/kg of papaya fruit extract (aqueous) in animals did not show any sign of toxicity or mortality. Comparative assessment of the seeds, pulp and leaves extract on haematological parameters in rats receiving 100, 200 and 300 mg/ kg for one month was also performed by Ikpeme et al. (2011). The seeds and leaves extracts acted as leucocytes boosters at higher concentration of the extracts while the pulp extract reduced the leucocyte count, which seems to follow in a dose-dependent manner. In the study by Dharmarathna et al. (2013), oral administration of aqueous extract of fresh papaya leaves up to a dose of up 5 g/mouse per day was employed without causing any acute/subacute toxicity. This and a lower dose (2 g/mouse) however could increases platelet and RBC counts in the murine model (Table 11.3). Overall, while the leaves and unripe or immature/green fruits that are claimed to have abortifacient properties need to be taken with caution, the edible fruits appear to have little toxicity in general.

11.6 Conclusion The utilisation of papaya both as food and medicine has historical as well as practical significance. The glucose lowering effect of the plant, even though some evidence are presented D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

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in animal studies, need to be confirmed through comprehensive study, most importantly in human subjects. Papaya appear to have little effect as a weight lowering agent and the most prominent effect appear to be on improving the antioxidant status both in vitro, in vivo and under clinical conditions. The antihypertensive effect and most importantly potential treatment for diabetes wound are the most promising pharmacological properties of this plant. While small molecular weight compounds may play significant role for the antihypertensive effects, enzymatic activity appear to be the most relevant pharmacological principles for diabetic wound therapy. The most important development in recent years for papaya as a drug and nutraceutical agent is on FPP that appear to gain lots of attention. The phytochemistry study on the plant is at its meagre stage however and more research is required both on the identification of the active principles and providing concrete pharmacological evidence of efficacy.

References Afzan, A., Abdullah, N.R., Halim, S.Z., Rashid, B.A., Semail, R.H.R., Abdullah, N., et al., 2012. Repeated dose 28-days oral toxicity study of Carica papaya L. leaf extract in Sprague Dawley rats. Molecules 17, 4326–4342. Amer, J., Goldfarb, A., Rachmilewitz, E.A., Fibach, E., 2008. Fermented papaya preparation as redox regulator in blood cells of beta-thalassemic mice and patients. Phytother. Res. 22, 820–828. Ahmed, F.B, Ismail, G., 2003. Medicinal Plants Used by Kadazan Dusun Communities Around Crocker Range. ASEAN Review of Biodiversity and Environmental Conservation (ARBEC). Available at: https://www. academia.edu/4165060/MEDICINAL_PLANTS_USED_BY_KADAZANDUSUN_COMMUNITIES_AROUND_ CROCKER_RANGE (Accessed 10 January 2019). Amazu, L.U., Azikiwe, C.C.A., Njoku, C.J., Osuala, F.N., Nwosu, P.J.C., Ajugwo, A.O., et al., 2010. Antiinflammatory activity of the methanolic extract of the seeds of Carica papaya in experimental animals. Asian Pac. J. Trop. Med. 3 (11), 884–886. Anuar, N.S., Zahari, S.S., Taib, I.A., Rahman, M.T., 2008. Effect of green and ripe Carica papaya epicarp extracts on wound healing and during pregnancy. Food Chem. Toxicol. 46, 2384–2389. Aruoma, O.I., Colognato, R., Fontana, I., Gartlon, J., Migliore, L., Koike, K., et al., 2006. Molecular effects of fermented papaya preparation on oxidative damage, MAP kinase activation and modulation of the benzo[a]pyrene mediated genotoxicity. Biofactors 26, 147–159. Azarkan, M., El Moussaoui, A., van Wuytswinkel, D., Dehon, G., Looze, Y., 2003. Fractionation and purification of the enzymes stored in the latex of Carica papaya. J. Chromatogr. B 790, 229–238. Bagdade, J.D., Root, R.K., Bulger, R.J., 1974. Impaired leukocyte function in patients with poorly controlled diabetes. Diabetes 23, 9–15. Barroso, P.T.W., de Carvalho, P.P., Rocha, T.B., Pessoa, F.L.P., Azevedo, D.A., Mendes, M.F., 2016. Evaluation of the composition of Carica papaya L. seed oil extracted with supercritical CO2. Biotechnol. Rep. 11, 110–116. Bennett, R.N., Kiddle, G., Wallsgrove, R.M., 1997. Biosynthesis of benzylglucosinolate, cyanogenic glucosides and phenylpropanoids in Carica papaya. Phytochemistry 45 (1), 59–66. Braga, F.C., Serra, C.P., Viana Ju´nior, N.S., Oliveira, A.B., C^ ortes, S.F., Lombardi, J.A., 2007. Angiotensin-converting enzyme inhibition by Brazilian plants. Fitoterapia 78, 353–358. Brasil, G., Ronchi, S., do Nascimento, A., de Lima, E., Roma˜o, W., da Costa, H., et al., 2014. Antihypertensive effect of Carica papaya via a reduction in ACE activity and improved baroreflex. Planta Med. 80 (17), 1580–1587. Calvache, J.N., Cueto, M., Farroni, A., de Escalada Pla, M., Gerschenson, L.N., 2016. Antioxidant characterization of new dietary fiber concentrates from papaya pulp and peel (Carica papaya L.). J. Funct. Foods 27, 319–328. Calzuola, I., Gianfranceschi, G.L., Marsili, V., 2006. Comparative activity of antioxidants from wheat sprouts, Morinda citrifolia, fermented papaya and white tea. Int. J. Food Sci. Nutr. 57, 168–177. Canini, A., Alesiani, D., D’Arcangelo, G., Tagliatesta, P., 2007. Gas chromatography-mass spectrometry analysis of phenolic compounds from Carica papaya L. leaf. J. Food Compos. Anal. 20 (7), 584–590. Collard, E., Roy, S., 2010. Improved function of diabetic wound-site macrophages and accelerated wound closure in response to oral supplementation of a fermented papaya preparation. Antioxid. Redox Signal. 13 (5), 599–606.

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Charoensiri, R., Kongkachuichai, R., Suknicom, S., Sungpuag, P., 2009. Beta-carotene, lycopene, and alpha-tocopherol contents of selected Thai fruits. Food Chem. 113 (1), 202–207. Danese, C., Esposito, D., D’Alfonso, V., Cirene, M., Ambrosino, M., Colotto, M., 2006. Plasma glucose level decreases as collateral effect of fermented papaya preparation use. Clin. Ter. 157, 195–198. Crowley, S.D., Gurley, S.B., Herrera, M.J., Ruiz, P., Griffiths, R., Kumar, A.P., 2006. Angiotensin II causes hypertension and cardiac hypertrophy through its receptors in the kidney. Proc. Natl. Acad. Sci. U.S.A. 103, 17985–17990. Dharmarathna, S.L.C.A., Wickramasinghe, S., Waduge, R.N., Rajapakse, R.P.V.J., Kularatne, S.A.M., 2013. Does Carica papaya leaf-extract increase the platelet count? An experimental study in a murine model. Asian Pac. J. Trop. Biomed. 3 (9), 720–724. Dickerson, R., Banerjee, J., Rauckhorst, A., Pfeiffer, D.R., Gordillo, G.M., Khanna, S., et al., 2015. Does oral supplementation of a fermented papaya preparation correct respiratory burst function of innate immune cells in type 2 diabetes mellitus patients? Antioxid. Redox Signal. 22 (4), 339–345. Dickerson, R., Deshpande, B., Gnyawali, U., Lynch, D., Gordillo, G.M., Schuster, D., et al., 2012. Correction of aberrant NADPH oxidase activity in blood-derived mononuclear cells from type II diabetes mellitus patients by a naturally fermented papaya preparation. Antioxid. Redox Signal. 17 (3), 485–491. Emeruwa, A., 1982. Antibacterial substance from Carica papaya fruit extract. J. Nat. Prod. 45, 132–137. Eno, A.E., Owo, O.I., Itam, E.H., Konya, R.S., 2000. Blood pressure depression by the fruit juice of Carica papaya L. in renal and DOCA-induced hypertension in the rat. Phytother. Res. 14, 235–239. Franke, A.A., Custer, L.J., Arakaki, C., Murphy, S.P., 2004. Vitamin C and flavonoid levels of fruits and vegetables consumed in Hawaii. J. Food Compos. Anal. 17 (1), 1–35. Fujita, Y., Tsuno, H., Nakayama, J., 2017. Fermented papaya preparation restores age-related reductions in peripheral blood mononuclear cell cytolytic activity in tube-fed patients. PLoS One. 12(1). e016924. Gayosso-Garcı´a Sancho, L.E., Yahia, E.M., Gonza´lez-Aguilar, G.A., 2011. Identification and quantification of phenols, carotenoids, and vitamin C from papaya (Carica papaya L., cv. Maradol) fruit determined by HPLC-DAD-MS/MSESI. Food Res. Int. 44 (5), 1284–1291. Ghosh, S., Saha, M., Bandyopadhyay, P.K., Jana, M., 2017. Extraction, isolation and characterization of bioactive compounds from chloroform extract of Carica papaya seed and it’s in vivo antibacterial potentiality in Channa punctatus against Klebsiella PKBSG14. Microb. Pathog. 111, 508–518. Govindachari, L.T.R., Pai, B.R., Narasimhan, N.S., 1954. pseudoCarpaine, a new alkaloid from Carica papaya. J. Chem. Soc. 1847. Guerrero, L., Castillo, J., Quin˜ones, M., Garcia-Vallve, S., Arola, L., Pujadas, G., Muguerza, B., 2012. Inhibition of angiotensin-converting enzyme activity byflavonoids: structure-activity relationship studies. PLoS One 7, 1–11. Gurung, S., Skalko-Basnet, N., 2009. Wound healing properties of Carica papaya latex: in vivo evaluation in mice burn model. J. Ethnopharmacol. 121, 338–341. Habtemariam, S., 2011. α-Glucosidase inhibitory activity of kaempferol-3-O-rutinoside. Nat. Prod. Commun. 6 (2), 201–203. Habtemariam, S., Varghese, G.K., 2014. The antidiabetic therapeutic potential of dietary polyphenols. Curr. Pharm. Biotechnol. 15 (4), 391–400. Herna´ndez, Y., Lobo, M.G., Gonza´lez, M., 2006. Determination of vitamin C in tropical fruits: a comparative evaluation of methods. Food Chem. 96 (4), 654–664. Ikpeme, E.V., Ekaluo, U.B., Kooffreh, M.E., Udensi, O., 2011. Phytochemistry and haematological potential of ethanol seed leaf and pulp extracts of Carica papaya (Linn.). Pak. J. Biol. Sci., 14, 408–411. Hornick, C.A., Sanders, L.I., Lin, Y.C., 1978. Effect of carpaine, a papaya alkaloid, on the circulatory function in the rat. Res. Commun. Chem. Pathol. Pharmacol. 22 (2), 277–289. Imao, K., Wang, H., Komatsu, M., Hiramatsu, M., 1998. Free radical scavenging activity of fermented papaya preparation and its effect on lipid peroxide level and superoxide dismutase activity in iron-induced epileptic foci of rats. Biochem. Mol. Biol. Int. 45, 11–23. Jua´rez-Rojop, I.E., Dı´az-Zagoya, J.C., Ble-Castillo, J.L., Miranda-Osorio, P.H., Castell-Rodrı´guez, A.E., TovillaZa´rate, C.A., et al., 2012. Hypoglycemic effect of Carica papaya leaves in streptozotocin-induced diabetic rats. BMC Complement. Altern. Med. 12, 236. Kermanshai, R., McCarry, B.E., Rosenfeld, J., Summers, P.S., Weretilnyk, E.A., Sorger, G.J., 2001. Benzyl isothiocyanate is the chief or sole anthelmintic in papaya seed extracts. Phytochemistry 57, 427–435.

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Ojo, O.A., Ojo, A.B., Awoyinka, O., Ajiboye, B.O., Oyinloye, B.E., Osukoya, O.A., et al., 2017. Aqueous extract of Carica papaya Linn. roots potentially attenuates arsenic induced biochemical and genotoxic effects in Wistar rats. J. Tradit. Complement. Med. 8 (2), 324–334. Osato, J.A., Santiago, L.A., Remo, G.M., Cuadra, M.S., Mori, A., 1993. Antimicrobial and antioxidant activities of unripe papaya. Life Sci. 53, 1383–1389. Otsuki, N., Dang, N.H., Kumagai, E., Kondo, A., Iwata, S., Morimoto, C., 2010. Aqueous extract of Carica papaya leaves exhibits anti-tumor activity and immunomodulatory effects. J. Ethnopharmacol. 127 (3), 760–767. Owoyele, B.V., Adebukola, O.M., Funmilayo, A.A., Soladoye, A.O., 2008. Anti-inflammatory activities of ethanolic extract of Carica papaya leaves. Inflammopharmacology 16, 168–173. Paes, J., da Cunha, C.R., Viotto, L.A., 2015. Concentration of lycopene in the pulp of papaya (Carica papaya L.) by ultrafiltration on a pilot scale. Food Bioprod. Process. 96, 296–305. Raffaelli, F., Nanetti, L., Montecchiani, G., Borroni, F., Salvolini, E., Faloia, E., et al., 2015. In vitro effects of fermented papaya (Carica papaya, L.) on platelets obtained from patients with type 2 diabetes. Nutr. Metab. Cardiovasc. Dis. 25 (2), 224–229. Ravikant, T., Nishant, G., Shashipal, S., Samriti, T., Kumar, T.R., Vikas, V., et al., 2012. Antihypertensive effect of ethanolic extract of Indian Carica papaya L. root bark (Caricaceae) in renal artery occluded hypertensive rats. Int. J. Pharm. Clin. Res. 4, 20–23. Rimbach, G., Park, Y.C., Guo, Q., Moini, H., Qureshi, N., Saliou, C., et al., 2000. Nitric oxide synthesis and TNFalpha secretion in RAW 264.7 macrophages: mode of action of a fermented papaya preparation. Life Sci. 67, 679–694. Rivera-Pastrana, D.M., Yahia, E.M., Gonza´lez-Aguilar, G.A., 2010. Phenolic and carotenoid profiles of papaya fruit (Carica papaya L.) and their contents under low temperature storage. J. Sci. Food Agric. 90 (14), 2358–2365. Rossetto, M.R.M., Oliveira do Nascimento, J.R., Purgatto, E., Fabi, J.P., Lajolo, F.M., Cordenunsi, B.R., 2008. Benzylglucosinolate, benzylisothiocyanate, and myrosinase activity in papaya fruit during development and ripening. J. Agric. Food Chem. 56 (20), 9592–9599. Runnie, I., Salleh, M.N., Mohameda, S., Headb, R.J., Abeywardena, M.Y., 2004. Vasorelaxation induced by common edible tropical plant extracts in isolated rat aorta and mesenteric vascular bed. J. Ethnopharmacol. 92, 311–316. Salla, S., Sunkara, R., Ogutu, S., Walker, L.T., Verghese, M., 2016. Antioxidant activity of papaya seed extracts against H2O2 induced oxidative stress in HepG2 cells. LWT—Food Sci. Technol. 66, 293–297. Sasidharan, S., Sumathi, V., Jegathambigai, N.R., Latha, L.Y., 2011. Antihyperglycaemic effects of ethanol extracts of Carica papaya and Pandanus amaryfollius leaf in streptozotocin-induced diabetic mice. Nat. Prod. Res. 25 (20), 1982–1987. Schweiggert, R.M., Steingass, C.B., Heller, A., Esquivel, P., Carle, R., 2011. Characterization of chromoplasts and carotenoids of red- and yellow-fleshed papaya (Carica papaya L.). Planta 234, 1031–1044. Schweiggert, R.M., Steingass, C.B., Esquivel, P., Carle, R., 2012. Chemical and morphological characterization of Costa Rican papaya (Carica papaya L.) hybrids and lines with particular focus on their genuine carotenoid profiles. J. Agric. Food Chem. 60, 2577–2585. Schweiggert, R.M., Kopec, R.E., Villalobos-Gutierrez, M.G., H€ ogel, J., Quesada, S., Esquivel, P., Schwartz, S.J., Carle, R., 2014. Carotenoids are more bioavailable from papaya than from tomato and carrot in humans: a randomised cross-over study. Br. J. Nutr. 111 (3), 490–498. Seigler, D.S., Pauli, G.F., Nahrstedt, A., Leen, R., 2002. Cyanogenic allosides and glucosides from Passiflora edulis and Carica papaya. Phytochemistry 60, 873–882. Somanah, J., Aruoma, O.I., Gunness, T.K., Kowelssur, S., Dambala, V., Murad, F., et al., 2012. Effects of a short term supplementation of a fermented papaya preparation on biomarkers of diabetes mellitus in a randomized Mauritian population. Prev. Med. 54, S90–S97. Somanah, J., Bourdon, E., Rondeau, P., Bahorun, T., Aruoma, O.I., 2014. Relationship between fermented papaya preparation supplementation, erythrocyte integrity and antioxidant status in pre-diabetics. Food Chem. Toxicol. 65, 12–17. Sherry, S., Fletcher, A.P., 1960. Proteolytic enzymes: a therapeutic evaluation. Clin. Pharmacol. Ther. 1 (2), 202–226. Spencer, K.C., Seigler, D.S., 1984. Cyanogenic glycosides of Carica papaya and its phylogenetic position with respect to the Violales and Capparales. Am. J. Bot. 71, 1444–1447. Starley, I.F., Mohammed, P., Schneider, G., Bickler, S.W., 1999a. The treatment of prediatric burn using topical papaya. Burns 25 (7), 636–639.

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Starley, I.F., Mohammed, P., Schneider, G., Bickler, S.W., 1999b. The treatment of paediatric burns using topical papaya. Burns 25, 636–669. Tang, C.-S., 1979. New macrocyclic, Δ1-piperideine alkaloids from papaya leaves: dehydrocarpaine I and II. Phytochemistry 18 (4), 651–652. Telgenhoff, D., Lam, K., Ramsay, S., Vasquez, V., Villareal, K., Slusarewicz, P., et al., 2007. Influence of papain urea copper chlorophyllin on wound matrix remodeling. Wound Repair Regen. 15, 727–735. Thoma´s, G.E., Rodolfo, H.G., Juan, M.D., Georgina, S.F., Luis, C.G., Ingrid, R.B., Santiago, G.T., 2009. Proteolytic activity in enzymatic extracts from Carica papaya L. cv. Maradol harvest by-products. Process Biochem. 44, 77–82. Tsala, D.E., Amadou, D., Habtemariam, S., 2013. Natural wound healing and bioactive natural products. Phytopharmacology 4 (3), 532–560. USDA (United States Department of Agriculture, 2018). Classification for Kingdom Plantae Down to Species Carica papaya L. https://plants.usda.gov/java/ClassificationServlet?source¼display&classid¼CAPA23 (Accessed 10.1.19). Valente, A., Albuquerque, T.G., Sanches-Silva, A., Costa, H.S., 2011. Ascorbic acid content in exotic fruits: a contribution to produce quality data for food composition databases. Food Res. Int. 44 (7), 2237–2242. Vigneron, M., Deparis, X., Deharo, E., Bourdy, G., 2005. Antimalarial remedies in French Guiana: a knowledge attitudes and practices study. J. Ethnopharmacol. 98, 351–360. Wall, M.M., 2006. Ascorbic acid, vitamin A, and mineral composition of banana (Musa sp.) and papaya (Carica papaya) cultivars grown in Hawaii. J. Food Compos. Anal. 19 (5), 434–445. Yogiraj, V., Goyal, P.K., Chauhan, C.S., Goyal, A., Vyas, B., 2014. Carica papaya Linn: An Overview. Int. J. Herb. Med. 2 (5), 01–08. Wang, X., Hu, C., Ai, Q., Chen, Y., Wang, Z., Ou, S., 2015. Isolation and identification carpaine in Carica papaya L. leaf by HPLC-UV method. Int. J. Food Prop. 18 (7), 1505–1512. You, Z., Sun, J., Xie, F., Chen, Z., Zhang, S., Chen, H., et al., 2017. Modulatory effect of fermented papaya extracts on mammary gland hyperplasia induced by estrogen and progestin in female rats. Oxid. Med. Cell. Longev. 20178235069. Zhang, J., Mori, A., Chen, Q., Zhao, B., 2006. Fermented papaya preparation attenuates beta-amyloid precursor protein: beta-amyloid-mediated copper neurotoxicity in β-amyloid precursor protein and beta-amyloid precursor protein Swedish mutation overexpressing SH-SY5Y cells. Neuroscience 143, 63–72.

Further reading Azarkan, M., Wintjens, R., Looze, Y., Baeyens, D., 2004. Volant detection of three wound-induced proteins in papaya latex. Phytochemistry 65, 225–234. Cherian, T., 2000. Effect of papaya latex extract on gravid and non-graxvid rat uterine preparations in vitro. J. Ethnopharmacol. 70, 205–212. Dawkins, G., Hewitt, H., Wint, Y., Obiefuna, P.C., Wint, B., 2003. Antibacterial effects of Carica papaya fruit on common wound organisms. West Indian Med. J. 52, 290–292. Fibach, E., Tan, E.S., Jamuar, S., Ng, I., Amer, J., Rachmilewitz, E.A., 2010. Amelioration of oxidative stress in red blood cells from patients with beta-thalassemia major and intermedia and E-beta-thalassemia following administration of a fermented papaya preparation. Phytother. Res. 24, 1334–1338. Gopalakrishnan, M., Rajasekharasetty, M.R., 1978. Effect of papaya (Carica papaya Linn) on pregnancy and estrous cycle in albino rats of Wistar strain. Indian J. Physiol. Pharmacol. 22, 66–70. Habtemariam, S., Lentini, G., 2015. The therapeutic potential of rutin for diabetes: an update. Mini Rev. Med. Chem. 15 (7), 524–528. Oloyede, O.I., 2005. Chemical profile of unripe pulp of Carica papaya. Pak. J. Nutr. 4, 379–381. Schmidt, H., 1995. Effect of papain on different phases of prenatal ontogenesis in rats. Reprod. Toxicol. 9, 49–55.

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12 The chemical and pharmacological basis of pomegranate (Punica grantum L.) as potential therapy for type-2 diabetes and metabolic syndrome O U T L I N E 12.1 Introduction

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12.5 The chemistry of pomegranate 12.5.1 Polyphenol components of pomegranate 12.5.2 Triterpenes and sterols 12.5.3 Seed oil 12.5.4 Other compounds 12.6 Evidence of efficacy from in vitro and in vivo studies

12.6.1 General antioxidant effect 12.6.2 General anti-inflammatory effects 12.6.3 Effects of various plant parts

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12.1 Introduction Native to Persia and tightly associated with human civilisation in the Mediterranean region and beyond, pomegranate is a significant fruit crop of nutritional and medicinal significance. Its scientific name is Punica grantum L. and has been placed in the family Punicaceae for a long period until the recent taxonomic revision to group it under Lythraceae (see below). The Latin name of the fruit is known to be Malum granatum which means ‘grainy apple’ and appears to be the origin of the scientific name. The historical perspectives on the domestication and history of usage of pomegranate have been elegantly reviewed by various authors (e.g., Chandra et al., 2010). From its origin somewhere in Iran or Central Asia, it is believed to be first domesticated and cultivated during the Egyptian civilization from which it spreads to Europe. Ward (2010) employed a unique archaeobotanical study on the remains from the late fourteenth-century shipwreck near Turkey which had a cargo of numerous pomegranate seeds and pomegranate-shaped objects that were dated back to the Bronze Age of Eastern Mediterranean cultures. This implies that pomegranate was domesticated or being used by mankind around 3000 BCE. The plant is widely distributed to North Africa, North of Persia including Afghanistan and neighboring countries, India and as Far East as China. As a valuable crop, pomegranate is now cultivated all over the glob in regions of tropical or sub-tropical areas including in the USA and Mexico. Thousands of years of domestication and extensive cultivation under variable climatic and environmental conditions means that there are numerous accessions and genotypic variation, or at least in horticulture terms, varieties. While there are still few regions where natural verities still occur, such as in the Himalayan region (Rana et al., 2007), over 500 varieties of the species are known to be available. The plant is generally adapted to a hot and dry climate which are essential to maximize the yield and quality of the fruits under agricultural conditions. For extended review of pomegranate cultivation and horticulture, readers are directed to an article by Holland et al. (2009).

12.2 Botany and taxonomy The taxonomic hierarchy of the pomegranate (P. grantum) plant is shown below. The genus itself was first identified by Linnaeus and its description was included in his famous book entitled “The Species Plantarum” published in 1753. For a long time, the genus has been described as that belong to the family Punicaceae but recent phylogenetic relationship studies placed the genus under the family Lythraceae. The grouping of plants into several hierarchy, especially in the earlier times, were based on morphological, palynological, cytological, and anatomical relationship that collectively describe the phenotypes of the plants. There has been confusion about the genus Punica, however, and a more detailed study coming from genotypic relationship was needed. Conti et al. (1997), for example, have studied the interfamilial relationships in the order Myrtales which comprises the genus Punica and identified some molecular phylogeny and patterns of morphological evolution within the group. Although convincing data was not provided, ancestral relationship of the defined groups within the order that link few families have been presented. Some useful data on nuclear ribosomal DNA (nrDNA) also grouped other families such as Sonneratiaceae in the Lythraceae (Shi et al., 2000). The application of chloroplast rbcL gene, psaA-ycf3 spacer, and nrDNA internal transcribed spacer sequences as D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

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means of grouping the genus Punica among the family Lythraceae have also been described (Huang and Shi, 2002). Readers should bear in mind that many literature especially in the chemistry and pharmacology area of pomegranate still describe the plant as that belong to the family Punicaceae. Overall, the general consensus today is that the acceptable family for the genus Punica is under the family Lythraceae with the taxonomic hierarchy as follow: Kingdom: Plantae—Plants Subkingdom: Tracheobionta—Vascular plants Division: Magnoliophyta—Flowering plants Class: Magnoliopsida—Dicotyledons Subclass: Rosidae Order: Myrtales Family: Lythraceae—as Pomegranate family (not Punicaceae) Genus: Punica L.—pomegranate Species: Punica granatum L.—pomegranate The genus Punica represent two pomegranate species, Punica granatum L. and P. protopunica Balf. f., (syn. Socotria protopunica). Commonly known as pomegranate tree or Socotran pomegranate, P. protopunica Balf. f. has been known to be endemic to the Socotra Island of Yemen. While P. grantum has red flowers (Fig. 12.1), this species is distinguished by having pink trumpet-shaped flowers that would bear a smaller and less sweet fruits. The colour of the fruits are also yellowish-green or brownish-red upon maturity (ripening). Some earlier reports suggest that P. protopunica may have ancestral position within the genus. Pomegranate (from here on refereeing to P. grantum) is botanically described as a small tree of shrub that grows from 5 to 10 m high. The different varieties and domestication process over thousands of year’s means morphological variabilities is common including the dwarf and creeping bush varieties that grow not more than 2 m in height. P. granatum var. nana, for example, which has been described in some older literature as a separate species of its own, is a dwarf variety of pomegranate. According to Kewscience Plants of the World Online (KewPOTW, 2019) database, the brief description on the plant is as follow: Habit: Large shrub or small tree, the branches sometimes spiny; Leaves: Opposite, subopposite or fascicled, oblong to lanceolate or obovate, up to 7.5 cm. long, glabrous; Flowers: Orange-red or crimson, showy,
2.5 cm. wide; Calyx: Urceolate-funnel-shaped; Fruits: 3.5–12.5 cm. in diameter, but usually about the size of an orange; Seeds: Numerous, covered with pulp. The variability or existence of the numerous verities also means differing fruit types and yield that govern the marketing or horticultural value of the plants. The fruit size and colour which range from yellow to purple, but the usual colour being pink to red, are remarkable differences to note. The colour of the seed-coat could also vary from red to white, and genotypic variability also account to differences in juice content and taste including sweetness, acidity, astringency, etc. This extensive variability in the colour of the fruits (see Fig. 12.2) will also have implication in differences in chemical composition and hence biological activity diversity that will be discussed in the following sections. The plant is generally considered deciduous but evergreen verities and tendencies have been reported (e.g., in India). D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

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FIG. 12.1 The floral feature of pomegranate. The pomegranate blossom with intact petals (A) and conspicuous sepals and drying stamens after fertilization and petal fall (B) are shown. Courtesy: Wikipedia (https://en.wikipedia.org/wiki/Pomegranate).

In Europe, pomegranate is known by the following common name (Botanical-online, 2018): Catalan: Magraner Dutch: Granaatappel French: Grenade German: Granatapfel, Grenadine Italian: Melograno Portguese: Roma Spanish: Granado

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12.2 Botany and taxonomy

FIG. 12.2 Fruit variations of different pomegranate cultivars grown in Spain. Courtesy: Professor Susana Casal (REQUIMTE/Laboratory of Bromatology and Hydrology, Faculty of Pharmacy, Porto University, Rua Jorge Viterbo Ferreira, 228, 4050–313 Porto, Portugal).

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12.3 Uses as food and medicine The primary use of pomegranate is for its delicious fruits that are eaten fresh as fruit or in salad dressing or processed in various forms such as juices for global market. The concentrated juices are also stored and sold while the delicious fleshy cover of the seeds or arils are similarly presented as food. Numerous processed food products of pomegranate are available and include as teas and food colouring (dye) and pharmaceutical products. Food products of pomegranate origin also include jellies and jam, while the fermented beverage products include wine. Food products decorated with pomegranate toppings, sauces and other forms such as dipping are very common. The intense colour of the fruits have also been the subject of edible dye extraction that are widely employed in food, beverage and pharmaceutical industries. Considerable amount of the pomegranate fruit (over 20%) also constitute the seeds or arils (Fig. 12.3) that contribute to the various uses of the fruits from colour to food and medicine. In recognition of a superfood status that pomegranate gained in recent years, FIG. 12.3 The pomegranate seeds. The pomegranate seeds are enclosed in a soft jelly seed pods called arils which are eaten raw or processed to make other products.

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utilization as functional food and nutraceutical source have been reviewed in the various literature (e.g., Johanningsmeier and Harris, 2011). Readers should bear in mind that there is a great deal of exaggerated claims for medicinal foods or superfoods in recent years and the approach taken in this book is not to glorify the claim but to present the scientific evidences in the most unbiased manor from the scientific perspectives. The long history of pomegranate usage as a dye has also been extended to utilization in the textile industry in earlier civilizations. The historical perspective of dye and ink production in India using pomegranates and other plants has been reviewed ( Jha, 2001). As explained in the following pharmacology sections (under Section 12.6), pomegranate seeds oil is also a valuable product and constitute an established industry of its own. The rich polyphenolic composition of pomegranate has also attracted lots of interest for cosmetic product development owing to the potential protective effect of these chemicals against ultraviolet (UV) radiation. The high tannins content of the plants including the bark and roots also attributed to the traditional use of the plant in the tanning industry. The tannin content of the various part of the plant can range from 10% to close to 30% making the plant one of the favored source of leather tanning natural source in ancient civilizations. The healing power of pomegranate has also been long known and various literature cite practices dating back to the time of Hippocrates. The superfood status and medicinal uses of the plant is now widely reported and include for heart conditions, improving sexual desire and obesity, among others. In fact, almost all parts of the plant are known to be used in traditional medicine to treat various disease conditions. The historical perspective of pomegranates healing power and mythology has been reviewed by Langley (2000) and asserted that pomegranate has been given scared place by all major religions (Christianity, Buddhism, Islam, Judaism and Zoroastrianism) even before its medical properties were recorded. It was also presented as a chosen logo of medical profession to show the human body, and a heartbeat. Exemplary presentation of pomegranate in this connection is the coats of arms of the British Medical Association, the Royal College of Midwives (from the time of Catherine of Aragon’s coat of arms), the Royal College of Physicians of London ( middle of the sixteenth century) and The Royal College of Obsericians and Gynecologists (Langley, 2000). In the latter case, pomegranate has been largely seen as a symbol of “life, fertility, and regeneration”. The myth of pomegranate as a symbol of fertility, abundance, and good luck has also been mentioned in the various literature with citations from the Old Testament of the Bible. Usage for expelling parasites as anthelmintic agent by the fruits preparations, the leaves and roots as wound healing agents and the flowers as reputed medicine for managing diabetes are among those mentioned in the various literature. The most prevalent usage in earlier literature, however, appears to be for gastrointestinal problem primarily for expelling tapeworm and related parasites. The rind and root-bark also appeared to be the most favored for such purposes during the, Egyptian and Roman time and beyond. Considering the claim for obesity and type 2 diabetes (T2D) therapy by this plant has now been gaining lots of momentum, the primary focus of this chapter is to present the chemical and pharmacological basis of these potential benefits. Readers should bear in mind that the list of medicinal claim in traditional medicine is rather long and uses including treating diarrhea and dysentery and microbial infections; hemorrhage and cardiovascular indications, ulcer, antipyretic and implication of inflammatory conditions; respiratory illness, etc. are all mentioned.

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12.4 Plant preparations for antidiabetic use The utilization of pomegranate as food and medicine is based on the fruits and flowers (which are both edible) of the plant prepared as extracts, juices, fresh fruits consumption and other forms. As detailed in the following sections, a large body of evidences are presented to show the antidiabetic effects and related pharmacology of the fruit juice and flowers. The best characterized and that utilized part is the juices which are prepared from the crushed fruits excluding the seeds. The juice in this case could be obtained by pressure extraction. By removing the water of the juice concentrate, the dried juice often presented as an extract is ready for animal test or for medicine preparations. The juice concentrates are also diluted and made available to customers in various forms. The peels which are rich sources of polyphenols are also becoming very popular for the extract production and antidiabetic effect study. As industrial by-products particularly from the juice production, considerable amount of pomegranate material is gone to either waste or value-added products. This include uses for animal fodder and others but they are also being studied for their medicinal importance. The dried arils are also widely available in the market for food and medicine. Approaches for standardization of such products have been presented by Thakur et al. (2010) but this is limited to physical appearance such as colour, texture, taste and aroma instead of chemistry. The dried pomegranate as raisins are also common products sold in the market. The seeds extract and seed oil have also becoming very popular both in food/medicinal usage and pharmacological studies. Their antidiabetic potential have also been extensively studied and hence will be included in the chemical and pharmacology scrutiny herein. In the case of the leaves, solvent extraction primarily by alcohol or aqueous alcohol is used as with the flowers, given the active components are considered polar phenolic compounds. Several oil extraction methodologies are available for the seeds to release mostly the fatty acid components unique to the plant, while solvent extraction methodologies are also used to isolate the polar components. The numerous pharmacological effects on the seed or seed oil however focus on one polyunsaturated fatty acid, punicic acid, as a major active principle (see the following chemistry section).

12.5 The chemistry of pomegranate As outlined in the various sections of this book, listing the nutritional composition of medicinal foods is not part of the antidiabetic potential scrutiny approaches in this book. The energy and macro- and microcomponents of these products are widely available in the literature as well as the food products themselves when presented in the market. The general description of a pomegranate fruit compositional analysis, for example, can be summarized as follow (El-Nemr et al., 1990): “The edible parts of pomegranate fruit represented 52% of total fruit weight, comprising 78% juice and 22% seeds. The fresh juice contained 85.4% moisture, 10.6% total sugars, 1.4% pectin, 0.1 g/100 ml total acidity (as citric acid), 0.7 mg/100 ml ascorbic acid, 19.6 mg/100 ml free amino nitrogen and 0.05 g/100 ml ash. Meanwhile, the seeds are a rich source of total lipids, protein, crude fibers and ash representing 27.2%, 13.2%, 35.3% and 2.0%, respectively,

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and also contained 6.0% pectin and 4.7% total sugars. The iron, cupper, sodium, magnesium and zinc contents of the juice were lower than those of seeds, except potassium which was 49.2 ppm in the juice.” Beyond the oil composition, the protein contents of the seeds can rich as high as 17% by dry weight (Elfalleh et al., 2010). While these components could contribute to our general wellbeing by supplying valuable nutrients and additional antioxidant properties, we are focusing on the plant secondary metabolites (see Chapter 6) or drug-like molecules which are extensively discussed in the following sections.

12.5.1 Polyphenol components of pomegranate Being the major components of pomegranate fruits, the numerous pharmacological effects of the plant are often linked to polyphenolic compounds which are regarded as the active principles. The vast majority of literature associated with research on this plant are also based on estimation of the polyphenol contents and some form of antioxidant activity tests. The total phenolic content reported as gallic acid equivalent and often in combination with some form of structural class determination as flavonoids or tannin contents are common literature reports on pomegranate. The common active principles as gallic acid derivatives, flavonoids and tannins are outlined below. The composition of these components in the various cultivars of pomegranate could considerably vary and understanding their chemistry and available extraction methodologies would be important to maximize biological activity or consistency of industrially processed products. The colour of pomegranates for example vary, and with it, the phenolic composition such as anthocyanins that govern the fruit peel colour. 12.5.1.1 Phenolic acids and related compounds Phenolic acids include the shikimic acid products of C6-aromatic and either C-1 or C-3 side chain attachments. The classical examples of C6-C1 class compounds are gallic acid (4), p-hydroxy-benzoic acid (1), vanillic acid (3) and synapic acid (5) which were among the various phenolic derivatives (1–38) isolated from pomegranate (Figs. 12.4 and 12.5). As examples of phenylpropanoids as (C6-C3 compounds), p-coumaric (10), caffeic (11) and ferulic (12) acids are also common constituents of pomegranate. The quinic acid ester of caffeic acid (chlorogenic acid, 16) has also been isolated. Ellagic acid (23) appear to be the principal component of pomegranate and exist in its free and various other forms (e.g., tannins, see the following section). The fruits of pomegranate including the peels have been known to contain predominantly ellagic acid and gallic acids. Up to 50% of the polyphenols of the peels, for example, have been reported to be ellagic acid (23). The concentration of these individual components in the fruits of pomegranate could considerably vary depending of geographical locations and cultivars. The extraction methodologies could also play major role given that these compounds could not be equally extracted by solvents of variable polarity ranges. Alcoholic extracts often in aqueous mixture are the common extraction medium employed but others including supercritical CO2 and enzyme-assisted and pressurized water extraction methodologies have been reported. Considering the large volume of literature describing the extraction of phenolic compounds from pomegranate fruits, it is not possible to put an exhaustive list of literature but some key references are listed herein (e.g., C ¸ am and Hışıl,

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O

COOH

COOH

R R OH R OH OH OH 6 Catechol 1 R=H p-Hydroxy benzoic acid 4 R=OH Gallic acid 5 R=OMe Sinapic acid 2 R=OH Protocatechuic acid 3 R=OMe Vanillic acid OH O O COOH COOH COOH COOH

H

MeO

OMe OH 7 Syringaldehyde

COOH

R OH

R

8 cis-p-Coumaric acid

HO

O

9 Coutaric acid

10 R=H p-Coumaric acid 11 R=OH Caffeic acid 12 R=OMe Ferulic acid

HO

O OH

O 15 Coumestrol

OH

HO COOH O OH

HO

OH

HO

16 Chlorogenic acid

R2 O

OH OH

17 Neochlorogenic acid O

R A=

HO

OH HO HO OH

O OH

O

R1

B = HO

OH 18 R1=H, R2=A 19 R1=OMe, R2=A

O

O O

FIG. 12.4

13 R=H Cinnamic aicd 14 R=OH O-Coumaric acid

HO COOH O

O

MeO

R

OH

OH

20 R=A Icariside D1 21 R=B Phenethyl rutinoside

O HO

HO HO OH Rutinose

O OH

Simple phenolic acid derivatives of pomegranate.

2010; Dikmen et al., 2011; Elfalleh et al., 2011; Mansour et al., 2013; Mena et al., 2013; Mushtaq et al., 2015; Qu et al., 2012). Given the high polar nature of these phenolics, aqueous extract especially in hot extraction methodologies have been shown to yield a higher level of phenolic acids than organic solvents, while methanol as expected can extract a lot more than acetone or ethyl acetate. The stability of polyphenols in the extracted fruits could also vary depending on the method of industrial processing and storage time (see a review article by Ka˚rlund et al., 2014). Fingerprint analysis of product to ascertain qualitative and quantitative assessment of the known active ingredients are thus necessary. The simple phenyl propanoids such as p-coumaric (10), caffeic (11), ferulic (12) and chlorogenic (16) acids should be expected in the fruits and fruit juices (e.g., Artik et al., 1998). Gallic acid (4) itself is ubiquitously present in the fruit juice, peel and flowers while its derivative, methyl gallate, is rare and was isolated from the heartwood (El-Toumy et al., 2001). While the trans form p-coumaric acid (10) has been isolated from fruit peals, juice, D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

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12.5 The chemistry of pomegranate

OR2

OH HO

HO

O

R1O

O

OH

O

OH OH

R1 23 H 24 H 25 H 26 H 27 Me

22 Hexahydroxydiphenol

OH

OH O HO

HO

OH

R2 H Me Me Me H

O

R3 H H Me H Me O

O OH

O

O

O

O

OR3 OR4

28 Gallagic acid O

OH O

O

HO

29 3¢-O-Methyl-3,4-methylenedioxy-ellagic acid OH OH HO OH O O O O

OH

O HO O

OH OH

O

HO

OH 30 Valoneic acid dilactone OH HO

O OH

HO

O

O

32 Brevifolin

O

O

O O OH KO S O OH HOOC

OH OH

O

OH OH 31 Gallagyldilacton (Terminalin) + –

OH

ROOC

O

O

OH

OH

OH

O

OR3 OR R4 4 H Ellagic acid M3 3,3¢-Di- O-methyl-ellagic acid Me 3,3¢,4¢-Tri- O-methyl-ellagic acid H 3- O-Methyl-ellagic acid H 4,4¢-Di- O-methyl-ellagic acid

O

O OH

HO

HO O

O

O

O O O 33 R=H Brevifolin carboxylic acid 34 R= CH3 CH2 Ethyl brevifolin carboxylate

O

O

O

35 O

O O

O

HO

O O O

O

HO O

36 Pomegranatate

OH HO

9

8 7 6a

O

4a

COOH

HO 2

O

HO

3

6

O

O

OH

10 10a 1

OH

OH 37 Urolithin M-5

O 38 Phyllanthusiin E

FIG. 12.5 Gallic acid and protocatechuic acid derivatives of pomegranate.

seeds and leaves by various authors, the cis form (cis-p-coumaric acid, 8) along with p-coumaric acid (10), coutaric acid (9), protocatechuic acid (2), neochlorogenic acid (5-Ocaffeoylquinic acid, 17) have been isolated from the fruit peel and juice (Ambigaipalan et al., 2016). Fischer et al. (2011) also reported the detection of chlorogenic (16), p-coumaric

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(10), gallic (4), neochlorogenic (17) and protocatechuic (2) acids from the fruit peel and juice. Caffeic acid (11) and ferulic (12) acids are considered ubiquitous antioxidant and biologically active compounds that are common in the fruit peel, juice, seed, and leaves of pomegranate (Artik et al., 1998; Calani et al., 2013). Other simple phenolics detected in the fruit peel and seeds include catechol (6) (Artik et al., 1998) and syringaldehyde (7) (Mena et al., 2013). The seeds of the plant have also shown other small molecular weight compounds of phenolic acid derivatives. For example, Wang et al. (2004) isolated coniferyl 9-O-[β-Dapiofuranosyl(1 ! 6)]-O-β-D-glucopyranoside (18) and sinapyl 9-O-[β-D-apiofuranosyl (1 ! 6)]-O-β-D-glucopyranoside (19), along with five known compounds, 3,30 -di-Omethylellagic acid (3), 3,30 ,40 -tri-O-methylellagic acid (4), phenethyl rutinoside (21), and icariside D1 (20). Representing the coumestans group of compounds, coumestrol (15) has been reported from the seeds (Moneam et al., 1988). While ellagic acid (23) is expected in all pomegranate tissues (leaves, fruit peel and flowers; see Nawwar et al., 1994a,b; Wang et al., 2006) at variable concentrations, the methyl derivatives of ellagic acid have also been obtained from pomegranate (Wang et al., 2004). These include 3,30 -di-O-methylellagic acid (24) and 3,3,40 -tri-O-methylellagic acid (25) from the seeds (Wang et al., 2004), and 3-O-methylellagic acid (26), 4,40 -Di-O-methylellagic acid (27), and 30 O-methyl-3,4-methylenedioxy-ellagic acid (29) isolated from the heartwood (El-Toumy et al., 2001; El-Toumy and Rauwald, 2003). Hexahydroxydiphenol (22), gallagic acid (4) and gallagyldilacton (terminalin, 31) are components of tannins that may also be available in their free form. For example, they have been isolated from the stem-bark of pomegranate (Tanaka et al., 1986a). Gallagic acid (28) is particularly important as it is one of the major component of pomegranate tannins and its free form is also identified in the peels (Glazer et al., 2012). Derivatives like brevifolin (32) and brevifolin carboxylic acid (33) or its 10-monopotassium sulphate (35) have been isolated form the leaves, flowers or the heartwood (El-Toumy and Rauwald, 2003; Hussein et al., 1997; Yuan et al., 2013). The ethyl derivative of brevifolin carboxylic acid, ethyl brevifolin carboxylate (34), has also been identified in the flowers (Wang et al., 2006). The other derivative of hexahydroxydiphenol is 3,4,8,9,10-pentahydroxydibenzo[b,d]pyran-6-one (37) that has been isolated from the leaves (Nawwar et al., 1994b). This compound also called urolithin M-5 (37) is one of the metabolic product of ellagic acid-derived tannins after bacterial transformation in the gut (see Section 12.8). Other ellagic acid derivatives are pomegranatate (36) and phyllanthusin E (38) that have been isolated from the flowers (Wang et al., 2006). Valoneic acid dilactone (30) is a further ellagic acid derivative with one more gallate added through oxygen bridge between the ellagic acid and C-2 position of gallic acid (30) and has been detected in the fruits (juice, mesocarp and peel) by high-performance liquid chromatography (HPLC) coupled to electrospray ionization mass spectrometric (ESI-MS) and diode array (DAD) detection (HPLC-DAD ESI/MS) (Fischer et al., 2011). 12.5.1.2 Flavonoids The other phenolic compounds of pomegranate fruits are flavonoids that are reported in numerous literature. The various class of flavonoids in plants are discussed in Chapter 6 of which pomegranate appear to contain flavonols, flavones, flavanones, flavans, flavan-3-ols, anthocyanins and isoflavonoids (Figs. 12.6–12.11). The common flavans identified from the

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12.5 The chemistry of pomegranate

OH

OH O

HO

O

R2O

O

HO

OH

OH

OH

OH OR

OR1

OR OH O 39 R=H Kaempferol 41 R= Glc Astragalin 42 R=Rut Nictoflorin

OH O OH O 43 R1=H, R2=H Quercetin 50 Myricetin 44 R1=Rut, R2=H Rutin 45 R1=Gal , R2=H Hyperoside 46 R1=Rha , R2=H 47 R1=Ara , R2=H Guaiaverin 48 R1=Glc, R2=H Isoquercetin 49 R1=H, R2=Glc Quercetin 7-O-glucoside

OH OH

OH Glc

O

OH

O

O

Glc

O

OH

HO

O OR

OH

O

O

OH O

OH O HO 51 Amurensin

OMe 52 Phellatin

CH2OH O

CH2OH OH O OH

OH

OH

OH OH

OMe

O

HO

OR

OH

OH O

OH O 53 OH O CH3 OH OH

54a b-D-Glucopyranoside (Glc)

54b b-D-Glactopyranoside Gal) 54c a-L-Rhamnopyranoside (Rha) O OH O O O O HO OH OH CH3 OH OH OH OH OH OH OH OH 56 Rutinose (Rut) 55a b-D-Arabinopyranoside (Ara) 55b b-D-Xylopyranoside (Xyl)

FIG. 12.6 Flavonols composition of pomegranate.

fruits (e.g. peels) are catechin (83) and epicatechin (84) while epigallocatechin (86) has also been detected. The most common flavonols are quercetin (43) and its rutinoside, rutin (44). Myricetin (50) and kaempferol (39) have also been detected in the fruits while the flavones are represented by luteolin (59) and apigenin (57). Other flavonoids in polymeric forms from procyanidins to tannins (see below) have also been reported. Flavonoid glycosides of the flavonol and favone skeletons have also been detected in the fruits of pomegranate. For example, the study by Abdulla et al. (2017) listed hyperoside (45), quercetin-3-O-arabinoide (47), kampferol-3-O-rutionoside (42), astragalin (41), luteolin-4-O-glucoside (62) and luteolin-30 O-glucoside (60), quercetin-7-O– (64) and quercetin-30 -O– glucoside (60), luteonin-30 -Oarabinoside (61) along with luteolin (59). Nawwar et al. (1994a) have also isolated apigenin-40 -O-β-glucopyranoside (58), luteolin-30 -O-β-glucopyranoside (60) and luteolin-30 O-β-xylopyranoside (63) from the leaves along with an alkaloid. A more detailed qualitative

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FIG. 12.7

12. The chemical and pharmacological basis of pomegranate

Flavones composition of pomegranate.

OH OH +

O

HO

OH +

O

HO

OR1 OR2 66 R1=R2=H Pelargonidin 67 R1=R2=Glc Pelargonidin 3,5-diglucoside 68 R1=Glc, R2=H Pelargonidin 3-diglucoside

OR1 OR2 69 R1=R2=H Cyanidin 70 R1=Glc, R2=H Cyanidin 3-glucoside 71 R1=R2=Glc Cyanidin 3,5-diglucoside 72 R1=Rut , R2=H Cyanidin 3-rutinoside OMe

OH

OH

OH O+

HO

OH

O+

HO

OR

OR1 OR2

OH

73 R1=R2=H Delphinidin 76 R=Herxose Peonidin hexoside 74 R1=R2=Glc Delphinidin 3,5-diglucoside 75 R1=Glc, R2=H Delphinidin 3-glucoside (Myrtillin)

FIG. 12.8

Anthocyanidins and anthocyanins of pomegranate.

analysis of these flavonoid constituents of the various pomegranate plant parts is presented in the following sections. The flavonol group of compounds in their free and glycosylated forms are shown in Fig. 12.6. They are represented by the kaepferol (39), quercetin (43) and myricetin (50) that possess one, two and three hydroxyl groups in the B-ring respectively. The common glycosides encountered in pomegranate flavonoids are the six-carbon pyranosides, β-Dglucopyranoside (Glc, 54a), β-galacopyranoside, (Gal, 54b) α-L-rhamnopyranoside (Rha, 54c); five-carbon pyranosides, β-D-arabinopyranoside (Ara, 55a) and β-D-xylopyranoside

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12.5 The chemistry of pomegranate

OH OH HO

O

RO

OH

OH

O

RO

O OH

HO OH O

OH O 78 R=Rut Naringin

77 Pinocembrin

HO

O

Rut = Rutinosyl Xyl = Xylosyl

OH

OH HO

OH O 79 R=Xyl Granatumflavanyl xyloside

OMe OH

R1

O HO

OH

OH O 80 Punicaflavanol

R2

O OH OH

O

O

O

OH O OH 81 R1=R2=OH 82 R1=H, R2=OMe

FIG. 12.9 Flavanones composition of pomegranate.

(Xyl, 55b); and rutinoside (α-L-rhamnopyranosyl-(1! 6)-β-D-glucopyranose, Rut, 56) (Fig. 12.6) derivatives. Their abbreviation as shown in Fig. 12.6 is used throughout this chapter. Kaempferol (39) (van Elswijk et al., 2004) and myricetin (50) (Zhao et al., 2014) have been detected in the fruit peel but quercetin (43) was the predominant flavonol in its free form and occur in the fruit peel, juice, seeds and leaves (Pande and Akoh, 2009; van Elswijk et al., 2004). Quercetin-3,40 -dimethyl ether 7-O-α-L-arabinofuranosyl(1 ! 6)-β-D-glucoside (53) is a rare flavonol detected from the stem-bark (Chauhan and Chauhan, 2001). Astragalin (kaempferol-3-O-glucoside, 41) and 7-O-glycosylated kaepferol with isoprene unit attachment at C-8 (amurensin or noricaritin 7-β-D-glucopyranoside, 51) or C-6 (phellatin, 52) positions occur in the juice (Ambigaipalan et al., 2016). While kaempferol-3-O-rhamnoglucoside (Nictoflorin, 42) is detected in the juice (Mena et al., 2012), quercetin-3-O-glucoside (48), quercimeritrin (quercetin-7-O-glucoside, 49) and quercetin-3-O-rhamnoside (46) are components of the fruit peel (Ambigaipalan et al., 2016). Rutin (44) is another predominant favonol detected in the juice (Artik et al., 1998). Except for tricetin (65, which was isolated from the fruit peals and flowers (Lal et al., 2011; Xie, et al., 2008), the flavones of pomegranate (Fig. 12.7) are dominated by apigenin (57) which is detected in the peels in its free form (Zhao, et al., 2014) and luteolin (59) occur in the fruit peel and flowers (Lal et al., 2011; Xie, et al., 2008). Apigenin-4-O-β-glucopyranoside (58), luteolin-30 -O-β-glucopyranoside (60), luteolin-30 -O-β-xylopyranoside (63) are constituents of the leaves (Nawwar et al., 1994a); while cynaroside (luteolin-7-O-glucoside, 64) is known to be present in the fruit peels (van Elswijk et al., 2004). The intense colour of pomegranate fruits attributes to the anthocyanin content in the peels. Hence, the degree of coluoration at different stages of fruits development and cultivar variation means variable anthocyanins content. Around 30% of all the anthocyanins in the fruits could be accounted in the peels, and as expected, the degree of fruit colour variation has

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12. The chemical and pharmacological basis of pomegranate

OH OH OH

OH HO

HO

OH

OH HO

O

O

R

O

O OH O OH

OH

OH

O

OH O

OH O

OH OH

83 Catechin

85 R=H Epicatechin gallate 86 R=Gallate Epigallocatechin gallate

84 Epicatechin

OH

OH

OH

OH HO

O OH OH HO

HO

R1 OH

OH OH

O

O

HO

OH OH O

R2 OH

HO

OH

OH

87 R1=OH, R2=H 88 R1=R2=OH 89 R1=H, R2=OH

90 Procyanidin B1

OH HO

HO

OH OH HO

OH OH

OH

OH

O

HO HO

92 Procyanidin B3

91 Procyanidin B2

O

OH

O

OH

OH

O

OH

OH

HO

FIG. 12.10

HO

O

OH O

OH

OH

OH O

OH

OH

OH HO

OH

OH

HO

93 Procyanidin A2

Flavon-3-ols composition of pomegranate.

OH HO

O OH OR

O

94 R=Glc Hovetrichoside C

FIG. 12.11

HO

OH OR O 95 R=H Phloretin 96 R=Glc Phlorizin

OH

HO

O R

O

OH

97 R=H Daidzein 98 R=OH Genistein

Other flavonoids classes of pomegranate.

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381

implications on anthocyanins both in qualitative and quantitative terms. The three common aglycone components of the anthocyanins are pelargonidin (66), cyanidin (69), and delphinidin (73) which differ from each other by their degree of oxygenation pattern in the B-ring (Fig. 12.8). These compounds along with their glycosides have been isolated from the fruits by various authors (Akhavan et al.; 2015; El-Nemr et al., 1990; (Borochov-Neori et al., 2011, Gil et al., 1995, Hernandez et al., 1999). Hence, the common oxygenation pattern of ringA and C of pomegranate anthocyanins is at C-3, C-5 and C-7 position. Glycosylation is most common at C-3 position, primarily with glucose and rarely rutinose (Fig. 12.8). The C-3 and C-5 di-glycosylation pattern is also common in pomegranate anthocyanins (Fig. 12.8). Other less defined anthocyanins are cyanidin-pentoside and cyanidin-hexocyde which are reported in small amounts. This kind of reports however come from liquid chromatography–mass spectrometry (LC-MS) analysis where the compounds are not isolated but rather characterized on the basis of their mass spectrometry profile. As with the phenolic acids, extensive literature reports on the flavonoids components analysis of the fruits are available and only key representative references are cited herein (e.g., Abid et al., 2017; Khalil et al., 2017; Li et al., 2006; Masci et al., 2016; Mousavinejad et al., 2009). The composition of processed fruits could also show considerable variation depending on storage time and packaging methodologies (e.g., Mphahlele et al., 2017) leading to differential biological activity. Other environmental, climate and growing condition that vary the flavonoid contents of plants must also be considered. For example, Borochov-Neori et al. (2011) have reported anthocyanin accumulation changes inversely with season’s temperatures. While cyanidin is generally more abundant, the delphinidin accumulation appear to be enhanced in cooler seasons. On the other hand, monoglucosylated anthocyanins are more common in cooler temperatures and their concentration decline during seasonal warming where the diglucosides concentration increases. Mena el al. (2012) also showed that sustained water deficiency (or irrigation) of the pomegranate plant could affect the colour and phytochemical characteristics of the juice. The arils of ripe fruits later in the season have also been shown to contain more soluble phenolics that account to differences in antioxidant effects (Borochov-Neori, et al., 2009). Hence, both seasonal and climatic variations as well as growing conditions would govern the chemical composition and ultimately biological activities of the pomegranate plants. With respect to the known therapeutic potential of anthocyanins, readers should bear in mind that there are numerous varieties with less intense colouring of the fruit and the general consensus is that the tannin-like compounds and their phenolic skeleton are a lot more therapeutically relevant components than the anthocyanins. Nevertheless, the juice preparation of the fruits have been shown to contain generous amounts of the anthocyanins shown in Fig. 12.8 (e.g., Go´mez-Caravaca et al., 2013; Noda et al., 2002). At least six compounds representing the flavanone skeleton have been identified from pomegranate (Fig. 12.9). Eriodictyol-7-O-α-L-arabinofuranosyl (1 !6)-β-D-glucoside and (1 ! 6)-β-D-glucoside (81) and naringenin-4’methyl ether 7-O-α-L-arabinofuranosyl (1 ! 6)-β-D-glucoside (82) have been obtained from the stem-bark (Srivastava et al., 2001); granatumflavanyl xyloside (79) from the flowers (Bagri et al., 2010), naringin (naringenin7-O-rhamnoglucoside, 78) from the fruit peel (Lansky et al., 2007), pinocembrin (77) from the juice (Mena et al., 2012) and punicaflavanol (80) from the flowers (Bagri et al., 2010). Of the flavan 3-ol compounds (Fig. 12.10), catechin (83) was detected in the fruit peel, juice and leaves and epicatechin (84) which was also detected in the seeds (Ambigaipalan et al.,

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2016; Mena et al., 2012; Pande et al., 2009); while epicatechin gallate (85) and apigallocatechin3-O-gallate (86) were detected in the fruits (Ambigaipalan et al., 2016, de Pascual-Teresa et al., 2000). The flavan 3-ol dimers have been exclusively detected in the fruit peel and include gallocatechin-(4 ! 8)-catechin (87), gallocatechin-(4 ! 8)-gallocatechin (88), catechin(4 ! 8)-gallocatechin (89), procyanidin B1 (90), procyanidin B2 (91) and procyanidin B3 (92) and procyanidin A2 (93) (Ambigaipalan et al., 2016, Plumb et al., 2002). Other flavonoid classes (Fig. 12.11) are represented by hovetrichoside C (94) which was in the flowers (Yuan et al., 2013) and the dihydrochalcone, phloretin (95) from the juice (Mena et al., 2012) and its glycoside, phlorizin (96), from the flower (Yuan et al., 2013). Isoflavonoids are not as common as the other group of flavonoids but genistein (97) and daidzein (98) have been reported from the seeds (Moneam et al., 1998). 12.5.1.3 Tannins Undoubtedly, the major components of pomegranate fruits including those with pharmacological significance belong to tannins (Figs. 12.12–12.16). Ellagic acid (23) and gallic acid (4) being the major phenolic components which are also the principal components of tannins. As oppose to the condensed tannins which are not common in pomegranate (rarely procyanidin of undescribed nature reported), the gallotannins and ellagitannins are predominant components. These are also what we call hydrolysable tannins. For the sake of clarity, tannins are broadly classified into four classes: the hydrolysable (mainly gallo- and ellagitannins), the proanthocyanidins oligomers and polymers of flavonoids mainly catechin (83) and epicatechin (84) making the condensed tannins, the so called complex tannins that are composed of catechins and gallic/ellagic acids, and the oligomers of phloroglucinol making phlorotannins. The structures of tannins from pomegranate tissues are shown in Figs. 12.12–12.16. The best characterized tannin of pomegranate as active principle is punicalalagin (99, 100). Its structure often presented as hexahydroxydiphenoyl (HHDP)gallagyl-hexoside is drawn as a pyranoside glucose unit as shown in Fig. 12.12. Quantitative analysis using LC-MS techniques have listed numerous other tannins some which are not fully characterized and tentatively presented as isomers or hexoside, pentosides derivatives. Numerous tannins with distinct structural identification are, however, presented with impressive level of complexity in the oxidation patterns of the gallate/ellagate moieties. The study by Satomi et al. (1993), for example, listed punicalin (101, 102), punicalagin (99 and 100), granatin B (108), gallagyldilactone (31), casuarinin (109), pedunculagin (135) and tellimagrandin I (113) as classical examples of tannins isolated from the pericarp of pomegranate fruits. The stereochemistry at the C-1 position of the pyranose glucose offer structural variability as in punicalagin and punicalain derivatives (99–102). The CdC bridge formation between neighboring gallates as in pedunculagin (141/142) vs tellimagrandin I (113), or granatin A (107) vs granatin B (108) along with the opening of the sugar unit to occur in acyclic form (109–111, 126–133) give enormous structural diversity to this compounds. Ellagitannins are generally seen as classical examples of the active components of pomegranate and occur in the various parts of the fruits such as the epicarp, pulp and the seeds. As tannins are highly polar water soluble compounds, extraction methodologies employing aqueous medium are preferred for their extraction. Literature report on tannin composition of pomegranate are extensive and selected few are shown herein (Fischer et al., 2011; Khalil et al., 2017; Orak et al., 2012; Nawwar et al., 1994a; Qu et al., 2012; Saad et al., 2012; Satomi et al., 1993) but readers

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12.5 The chemistry of pomegranate

OH

OH O

HO

OH

O

HO OH

HO O

HO O

O O

OH

O HO

O

OH

O

OH

O O OH OH

O

OH

HO O HO OH

O O

OH

O

O

O

HO

OH HO

OH

OH OH

O

HO

OH

O

O HO

99 1a-OH Punicalagin A 100 1b-OH Punicalagin B

O 101 1a-OH Punicalin A 102 1b-OH Punicalin B

HO OH

OH O

HO HO O

O

O OH

O HO

OH

OH

HO

R1O

OH

O O

O

O

OH O CH3

O HO

HO O

HO

OH

OH OH

HO

O

O

OH

HO

OH

O

O

O O

O O

O

O

O

OH HO OH

OH

O O

HO

OH

HO

O

O

O

OH

O O

O

O

HO

OH

OH

O

104 R1=R2=H Eschweilenol C 105 R1=Me, R2=H 106 R1=R2=Me

OH 103 2-O-galloylpunicalin (2-O-galloyl-4,6-(S,S)-gallagyl-D-glucose) HO

OH O

OH OH

OH OH

HO

OR2

O

O

OH

OH 107 Granatin A

HO OH

O

O O HO HO OH

108 Granatin B

FIG. 12.12 Tannins of pomegranate-1.

should bear in mind punicalagin being the predominant compound of significance as a major active component. The contribution by Tanaka et al. (1986a, b, 1990) have been particularly significant in establishing the structures of granatin A (107) and B (108), punicalin (101 and 102), punicalagin (99 and 100), punigluconin (130) and punicacortein A-D (126–129); while the contribution by Satomi et al. (1993) was in the assignment of corilagin (137), gallagyldilacton

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12. The chemical and pharmacological basis of pomegranate

D

D D D D D D D D

FIG. 12.13

Tannins of pomegranate-2.

(31), pedunculagins (140, 141) and tellimagrandin I (113). As mentioned above, Satomi et al. (1993) have also identified several of these and related tannins from pomegranate. The collective abundance of such compounds govern the biological activities of pomegranate as that dictated by polyphenolic compounds.

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12.5 The chemistry of pomegranate

OH

OH HO

HO

OH

OH

OH

OH OH

OH O OH O H OH O O OH

H H O H H H2C

OH

H H O H H H2C

O

OH OH

126 Punicacortein A OH

O OH O H OH O OH O

127 Punicacortein B

OH O

OH OH OH OH

OH HO

OH

HO

OH

OH

OH OH

OH HO HO

OH

O

O

HO HO

O

H H O O H OH H HC OH 2

O

OH O H O OH

OH

OH HO

O

HO O O

O

HO

HO

HO OH 128 Punicacortein C OH HO OH

HO OH

COOH H O O H O H O H OHO H2C O

HO

COOH H OH HO H O H O H O O H2C O

OH 131 Lagerstannin C

O

HO

129 Punicacortein D

OH

HO

OH OH OH

OH OH OH OH OH

HO

O

O

O OH

130 Punigluconin

O

OH O

O HO

HO

O

OH O H OH O

OH

OO HO

O

H H O H H O OH H2C

OH OH OH OH

HO HO

COOH H O O H O O H H O O CH2

O

HO HO HO

O O

OH

O

OH OH OH OH

OH 132 Lagerstannin B

OH OH

FIG. 12.14 Tannins of pomegranate-3.

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12. The chemical and pharmacological basis of pomegranate

OH

OH

HO

HO

O

HO

O

O

O

OH

HO

OH

HO

O O

OH O

HO

O O

OH O

OH OMe

H

O

O

O

O OH O

O

O OH O

HO OMe

133 Punicatannin A HO

OH

O HO

OH OMe

H

O O O

HO

O HO

OH O

O

O O

HO

HO O

OMe

134 Punicatannin B

OH OH

HO

OH

HO HO

O

O

O

OH HO

OH

O O

HO

HO

O

O

O

O

HO O

HO

O

O HO

OH OH HO OH OH 135 R1=R2= Gallate Punicafolin HO

OH

O

O

O

OH

O

O

O

OH OH OH 136 α1 Galloyl isoorilagin 137 β1 Galloyl corilagin

RO O

HO

O

HO

OH

OH

HO

O

HO

OH

O HO

OH

HO

OH HO O

HO HO

O

O

O

O O

HO O

O O

OH 138 R=H 2,3-(S)-hexahydroxydiphenoyl-D-glucose 139 R=Galooyl 6-O-galloyl-2,3-(S)hexahydroxydiphenoyl-D-glucose

OH

O

140 Tercatain HO

OH OH

FIG. 12.15

Tannins of pomegranate-5.

D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

OH

387

12.5 The chemistry of pomegranate

OH

OH

HO

HO O

O

HO

HO

O

O O

O O

HO

O

HO

O

OH

O

HO

O

O

O

O O

OH

O

OH

OH

HO

OHHO

OH

OH

HO

OHHO

OH

OH

142 Pedunculagin II

OH

OH HO O HO

HO O

OH OH

O O

O O

HO

HO

O

OH

HO

OH O

O O O HO

O OH

O

HO

OH

OH

OH

HO

O

O

OH

HO

OH OH OH

O

O O

O

O

OH

O

HO

O

HO

OH

OH

OH

O

OH

OH

141 Pedunculagin I

O O

O

O

HO

HO OH OH

O O

HO

O

HO O

HO O

O

O

OH

O O

O

O

O O

OH

O OH

HO

OH

OH

HO HO

OH OH

n

OH

HO

OH

147 Oenothein B

143 n = 1 Eucalbanin B 144 n = 2 Eucarpanin T1 145 n = 3 Pomegraniin A 146 n = 4 Pomegraniin B

FIG. 12.16 Tannins of pomegranate-6.

It is interesting to note again that the fruits, flowers, leaves and seeds of pomegranate which are used as antidiabetic medication are all known to contain ellagitannins. Tanaka et al. (1985, 1986a,b) and others have also isolated ellagitanins from the bark and leaves of the plant. The relationship between the various isolated tannins appears to be rather simple despite the structural complexity of these higher molecular weight compounds. Two gallic acid molecules oxidized to make the hexahydroxy-diphenyldicarboxylic acid is the basic skeleton that provide the dilactone ellagic acid (23). Even though the structural complexity via methylation, esterification and

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12. The chemical and pharmacological basis of pomegranate

glycosylation of the various hydroxyl groups are possible, a further addition of gallate unit as well as CdC bridging appear to be the main routes of structural diversity. The leaves are not extensively studied for pharmacological effects but have also been shown to be the source of diverse tannins. It predominantly contain, however, the gallotannin-type of hydrolysable tannins. The study by Nawwar et al. (1994b) identified brevifolin carboxylic acid (33), brevifolin (32), corilagin (137), 1,2,3-tri-O-galloyl-β-4C1-glucopyranose (118), 1,2,6-tri-O-galloyl-β-4C1glucopyranose (120), 1,4,6-tri-O-galloyl-β-4C1-glucopyranose (122), ellagic acid (23), 3,4,8,9,10pentahydroxydibenzo[b,d]pyran-6-one (37), granatin-B (108) and punicafolin (135) from the leaves. Punicafolin has been used as classical example of the tannins from the leaves and its derivatives with less galloyl content (e.g. 3,6-(R)-hexahydroxydiphenoyl-(α/β)-1C4-glucopyranose (150), see Fig. 12.17) are also known to occur (Nawwar et al. 1994b). The leaves have also been shown to contain 1,2,4-tri-O-galloyl-β-D-glucopyranose (119), 1,3,4-tri-O-galloyl-β-Dglucopyranose (121) along with various unidentified tannins (Hussein et al., 1997). Studies on accumulation and movement of ellagic acid and tannins in the leaves tissues along with their biological activities have also been assessed (Lan et al., 2009). An interesting insight into the stereochemistry of these tannins is the glucose designation as 1C4 form in some reports. Glucose, and other monosaccharides in their pyranose form as six-membered ring display isomeric chair confirmation designated as 1C4 (148) or 4C1 (149) forms (Fig. 12.17). As with other molecules, the conformation with the list energy level and most stable predominates, and the 1C4 confirmation of β-D-glucopyranose (148) with the 1,3–diaxially positioned ring substituents leading to repulsive van der Waals is of higher energy or unflavoured confirmation. On the other hand the 4C1 conformation of β-D-glucose (149) is the more favored. As mentioned above some of the pomegranate tannins are specifically mentioned as that of the least favored conformation 1C4 that should be noted when reviewing the structures (Fig. 12.12–12.16). From the qualitative analysis point of view, the fruits and/or peels contain casuarinin (109) (Satomi et al., 1993), corilagin (137) (Satomi et al., 1993), granatin A (107) and B (108) (Steinmetz, 2010), lagerstannin B (132) (Fischer et al., 2011), pedunculagin I (141) and II (142) FIG. 12.17

Isomeric chair confirmations of glucopyranose. The predominant glucopyranose isomer is the 4C1-glucopy ranose but the minor 1C4-glucopyranose in the tannin components of pomegranate was also known. A good example is 3,6-(R)hexahydroxydiphenoyl-(α/β)-1C4glucopyranose (150).

D

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D

12.5 The chemistry of pomegranate

389

(Calani et al., 2013, Glazer et al., 2012; Tanaka et al., 1986a), punicalin (101, 102) (Glazer et al., 2012; Fischer et al., 2011) and tellimagrandin I (113) (Satomi et al., 1993). The arils have also shown to contain eucalbanin B (143), eucarpanin T1 (144), pomegraniin A (145) and B (146), oenothein B (147), punicalagin A (99) and B (100) and punicalin (101, 102) (Ito et al., 2014). In pomegranate juice, the following tannins have been identified: 6-O-galloyl-2,3-(S)hexahydroxydiphenoyl-D-glucose (139) (Calani et al., 2013), 2,3-(S)-hexahydroxydiphenoyl-Dglucose (138), lagerstannin C (131) (Calani et al., 2013), punicalagin I (141) and II (142) (Calani et al., 2013; Fischer et al., 2011), and punicalin (101, 102) (Fischer et al., 2011; Tanaka et al., 1986a). The tannins components of the flowers are isocorilagin (136) (Yuan et al., 2012); hippomanin A (115) and gemin D (116) (Yuan et al., 2013), 1,2-di-O-galloyl-4,6-O-(S) hexahydroxydiphenoyl β-D-glucopyranoside (117) (Xie et al., 2008); gallic acid 3-O-β-D-(60 -Ogalloyl)-glucopyranoside (112) (Yuan et al., 2013); punicatannin A (133) and B (134) (Yuan et al., 2012); 1,2,6-tri-O-galloyl-β-D-glucopyranose (120) (Xie et al., 2008) and 3,4,6-tri-Ogalloyl-β-D-glucopyranose (123) (Yuan et al., 2013). Tanaka et al. (1985) isolated several tannins from the leaves including corilagin (137), granatin A (107) and B (108), 1,2,3,4,6-penta-Ogalloyl-β-D-glucose (126), 1,2,4,6-tetra-O-galloyl-β-D-glucose (124), punicafolin (135) and strictinin or 1-O-galloyl-4,6-(S)-hexahydroxydiphenoyl-D-glucose (114) (Tanaka et al., 1985). Other tannins from the leaves are 1,2,3-tri-O-galloyl-β-glucopyranose (118), 1,2,6tri-O-galloyl-β-glucopyranose (120), and 1,4,6-tri-O-galloyl-β-glucopyranose (122) (Nawwar et al., 1994a,b), 1,2,4-tri-O-galloyl-β-glucopyranose (119) and 1,3,4-tri-O-galloyl-β-glucopyranose (121) (Hussein et al., 1997), tercatain (140) or 1,4-di-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-βglucopyranose (140) (Hussein et al., 1997). The stem bark has been extensively studied for its tannin contents and the identified compounds include castalagin (110), casuariin (111), casuarinin (109), 6-O-galloyl-2,3-(S)hexahydroxydiphenoyl-D-glucose (139) and 6-O-galloyl-2,3-(S)-hexahydroxydiphenoyl-Dglucose 2,3-(S)-hexahydroxydiphenoyl-D-glucose (139), pedunculagin I (141), 2-Ogalloylpunicalin (2-O-galloyl-4,6-(S,S)-gallagyl-D-glucose, 103), punicacortein A-D (126– 129), punicalagin A (99) and B (100), punicalin (101, 102) and punigluconin (130) (e.g., Tanaka et al., 1986a, 1986b). Many tannins have also been isolated from the heartwood including eschweilenol C (ellagic acid 4-O-α-L-rhamnopyranoside, 104), 2-O-galloylpunicalin (2-Ogalloyl-4,6-(S,S)-gallagyl-D-glucose, 103), 3-O-methylellagic acid 4-O-α-L-rhamnopyranoside (105) and 3,40 -O-dimethylellagic acid 4-O-α-L-rhamnopyranoside (106) (El-Toumy and Rauwald, 2003), and punicalin (101, 102) (El-Toumy et al., 2001; El-Toumy and Rauwald, 2002). Numerous articles on quantitative analysis study are available for pomegranate components and the study by Fischer et al. (2011), for example, described the composition of the peel, mesocarp and aril juices by HPLC-DAD–ESI/MS analysis. Of the 48 compounds detected, 9 were anthocyanins, 2 gallotannins, 22 ellagitannins, 2 gallagyl esters, 4 hydroxybenzoic acids, 7 hydroxycinnamic acids and one dihydroflavonol. Their study have clearly shown that ellagitannins were found to be the predominant phenolics in all samples investigated, among them the punicalagin concentration ranged from 11 to 20 g/kg dry matter of the mesocarp and peel as well as 4–565 mg/L in the juices. Not surprisingly, the antioxidant effect of such extracts primarily attributed by the tannins has been demonstrated (e.g., Abid et al., 2017; Akhavan et al., 2015). It is also important to stress that the predominant compounds reported from pomegranate being hydrolyzable tannins (Fischer et al., 2011, Seeram et al., 2005), their monomers

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12. The chemical and pharmacological basis of pomegranate

could be released after hydrolysis in the small intestine. Where the fermented products are employed as food or medicine, a somehow different polyphenolic profile should be expected. For example, Sepu´lveda et al. (2018) reported ellagic acid as the main components of the fermented pomegranate as compared to intact ellagitannins being abundant in the fresh preparations. The study by Abdulla et al. (2017) using HPLC-QTOF-MS also listed 50 polyphenols detected of which 35 were hydrolysable tannins while 15 were flavonoids. The presence of flavan-3-ol monomers, and procyanidin dimers and trimers are known to occur in pomegranate beverages but they are less defined and characterized than the hydrolysable tannins (Borges and Crozier, 2012). Proanthocyanidins are rare in the fruits and when they were detected, they are dominated by procyanidin dimers (Ambigaipalan et al., 2016). 12.5.1.4 Lignans Lignans (Fig. 12.18) are another class of phenolic compounds that are formed from two C6-C3 units or phenyl propanoids such as caffeic and ferulic acids. Although they are by no means major components, lignans have been isolated from the various parts of pomegranate. Medioresinol H

MeO HO

O

MeO

OH OH

O

HO

H OMe

H

MeO

OH OH

HO

H OMe

OMe

OH

OH

OH

151 Isolariciresinol

153 Secoisolariciresinol

152 Matairesinol

OMe

OMe

OH

OH O

O H

H

H MeO

MeO

R1

OMe H O

O R2O

HO OMe

155 R1 =R2 =H Pinoresinol 156 R1 =H, R2 =Me Phylligenin 157 R1 =OMe, R2 =H Medioresinol

154 Syringaresinol

OMe

OMe O

OH O

O O OH

HO

OH

OH

OH

158 Pomegralignan

FIG. 12.18

O

O HO

O

OH HO OH

OMe OH

HO

OMe O OH

O O

OH

159 Punicatannin C

Lignans composition of pomegranate. D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

12.5 The chemistry of pomegranate

391

(157) and syringaresinol (154) appear to be found in the juice, seeds and wood (Bonzanini et al., 2009), while isolariciresinol (141), phylligenin (156), pinoresinol (155) and secoisolariciresinol (150) have been detected either in the juice or fruit peels (Fischer et al., 2012, Mena et al., 2013). Matairesinol (142) was detected in the wood (Bonzanini et al., 2009). The lignin glycosides are also represented by pomegralignan (158) which was isolated from the arils and peels (Ito et al., 2014), while punicatannin C (159) occur in the flowers (Yuan, et al., 2013).

12.5.2 Triterpenes and sterols The triterpenes and phytosterols group of compounds are encountered in pomegranate (Fig. 12.19) but they are not considered as the major active components. As most of them are extremely nonpolar, they can be obtained by solvent extraction and in the expressed oils of the seeds. Their identification is largely based on MS-based detection often in combination with some kind of chromatography. The fruit peels contain punicanolic acid (177), and fatty acid (myristate (164) or laurate (163) conjugates of β-sitosterol (Lal et al., 2011), while the flowers contain asiatic acid (176), maslinic acid (175), oleanolic acid (174), punicanolic acid (177), ursolic acid (173), and β-sitosterol (161) (Xie et al., 2008) as well as daucosterol (162) (Wang et al., 2006). The seeds contain stigmasterol (169) (Kaufman and Wiesman, 2007), β-sitosterol (161) (Kaufman and Wiesman, 2007), daucosterol (162) (Wang et al., 2004) and campesterol (160) (Kaufman and Wiesman, 2007). Betulinic acid (172) has also been detected in the leaves (Brieskorn and Keskin, 1955). The isolation of these compounds from the seeds and flowers have also been reported in other studies (e.g., Ahmed et al. 1995; Fu et al., 2014). Other triterpenes (see Fig. 12.20) are also detected as part of the seed oil composition.

12.5.3 Seed oil The seeds of pomegranate are rich sources of oil but they, though variable depending various factors, have been reported to constitute below 20% by weight. The seeds however constitute a significant portion of the pomegranate fruits with variability from 9% to over 50% by weight, with the general estimate being about 20%. The extraction and composition analysis of pomegranate seed oils obtained from the various cultivars have been published and cold pressing, solvent extraction (non-polar solvents such as hexane) and supercritical CO2 are routinely employed, while others such as microwave-assisted extraction are evolved in recent years. The most important note to make on the seed oil is that approximately 80% by weight is accounted by punicic acid (9Z,11E,13Z-octadeca-9,11,13-trienoic acid, 178, Fig. 12.20) (Vito Verardo et al., 2014). This unusual omega-5 polyunsaturated fatty acid of 18-carbon skeleton bear three conjugated double bonds. It has been shown to display numerous pharmacological effects including anti-cancer, anti-diabetes, anti-obesity, antioxidant, and anti-inflammatory properties (Holic et al., 2018). Hence, the seeds oil could be regarded as a rich source of potentially useful polyunsaturated fatty acids dominated by just one fatty acid, punicic acid. Linoleic acid (cis, cis-9,12-octadecadienoic acid or (9Z,12Z)-octadeca-9,12-dienoic acid, 179) and oleic acid (cis-9-octadecenoic acid or (9Z)-Octadec-9-enoic acid, 180) are the other components worth mentioning but their content are far less than 10%. Hence, the pharmacological effects attributed to the oil has been primarily reported as that accounted for by punicic acid. In terms of pomegranate seeds are by-products from juice and other food production from the D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

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12. The chemical and pharmacological basis of pomegranate

H

H

H

H

H H H HO 160 Campesterol

H RO

H H

H

161 R= H β-Sitosterol 162 R=Glc Daucosterol 163 R= Laurate 164 R=Myristate

HO

H

165 Stigmastanol

H H

H

H

H

H

H

H

HO

HO

HO 167 Brassicasterol

166 5-en-Avenasterol

H 168 Citrostadienol

H

H

H

H H

H

HO

HO

COOH

H

R

H

COOH

171 R= CH2OH Betulinol 172 R=COOH Betulinic acid

HO

H HO

174 R=H Oleanolic acid 175 R=OH Maslinic acid

173 Ursolic acid

H

HO

H

HO

R

H

HO

170 24-Methylene-cycloartanol

169 Stigmasterol

H

H

H

H H

H H

COOH

H OH

176 Asiatic acid

OH H H

COOH

H HO 177 Punicanolic acid

FIG. 12.19

Triterpenes and sterols composition of pomegranate.

fruits, the seeds oil is regarded as a value adding benefit to the pomegranate industry. Variability in fatty acid composition based on different varieties and extraction methods should be expected. The study by Fadavi et al. (2006), for example, presented linolenic acid as the predominant component (31.8–86.6%) of Iranian pomegranate seed oils followed by linoleic acid (0.7–24.4%), oleic acid (0.4–17.4%), stearic acid (2.8–16.7%), and palmitic acid (0.3–9.9%). It begs a question whether this was due to an error or misidentification of punicic acid as linolenic acid. D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

393

12.5 The chemistry of pomegranate

O

O HO

HO O HO

179 Linoleic acid

178 Punicic acid

180 Oleic acid

181 Squalene

HO

R1

R2 182 R1 = R2 δ-Tocopherol 183 R1 = H, R2 = Me γ-Tocopherol 184 R1 = Me R2 = H β-Tocopherol 185 R1 = R2 = Me α-Tocopherol

FIG. 12.20 Seed oils composition of pomegranate. Phytosterols present in the seed oils as minor components are shown in Fig. 12.19.

The tocopherol compositions of the seeds oil have also been reported in the various literature. All α-, β-, γ- and δ-tocopherols (182–185) have been reported in good amounts. The study by Fernandes et al. (2015) showed that γ-tocopherol appears to be the most abundant, followed by α-tocopherol and δ-tocopherol in the various cultivars studied. Considering the composition variability resulting from differences in cultivars, growing conditions and extraction methodologies, there are always discrepancies on the reported quantitative data. The order of abundance for the tocopherols for example reported by Elfalleh et al. (2011) was as follow: δ-tocopherol > α-tocopherol > γ-tocopherol. As with many seed oils, triterpene aglycones of sterols nature have been detected in pomegranate seeds oils. These include campesterol (160), β-sitosterol (161), stigmastanol (165) and Δ5-avenasterol (166). Many other related compounds such as brassicasterol (167), citrostadienol (168) and stigmasterol (169) in smaller amounts have also been detected while other triterpenes known to occur in the oil are squalene (181), 24-methylene-cycloartenol (170) and betulinol (171) (see Fig. 12.19). These group of compounds have relatively minor significance for the antidiabetic effect of pomegranate and are not discussed in detail. Readers can refer to the classical composition analysis reports of this kind (Fernandes et al., 2015; Verardo et al., 2014 and references there in). The carotenoids content of pomegranate seeds oils is generally considered low. Glycolipids such as cerebroside are also known to occur in the seeds. Tsuyuki et al. (1981) isolated N-palmitoyl cerebroside, while glycerides including those incorporating punicic acid (178) have also been isolated (Fatope et al., 2002; Lal, et al., 2011; Yusuph and Mann, 1997).

12.5.4 Other compounds Although not as major components, alkaloids have also been isolated from the leaves, bark and roots of pomegranate. As medicinal significance, perhaps those detected in the fruit extract are worth mentioning and include tryptamine (186), serotonin (187) and melatonin (188) (Badria, 2002). Punigratane (197) was isolated from the fruits (Rafiq et al., 2016) while N-(20 ,50 dihydroxyphenyl)pyridinium chloride (196) was isolated and identified as a novel compound from the leaves (Nawwar et al., 1994a,b). The barks of the plant have been shown D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

394

12. The chemical and pharmacological basis of pomegranate

NH2 R

NH MeO

O

N H 186 R=H Tryptasmine 187 R=OH Serotonin

189 R=H Norhygrine 190 R=Me Hygrine

OH N H

191 R=H Peelletierine 192 R=Me N-Methylpelletierene

N R

188 Melatonin

O N R

O

N H

OH N

193 Sedridine

194

N 195

R N N+

Cl– OH

N O

HO 196

FIG. 12.21

197 Punigratane

198 R=Me Pseudopelletierene 199 R=H Norpseudopelletierene

Alkaloids composition of pomegranate.

to be the source of pelletierine-type alkaloids (Neuhofer et al., 1993) including peelletierine (191), N-methylpelletierene (192), norpseudopelletierene (199) and pseudopelletierene (198); while piperidine-type alkaloids including hygrine (190), norhygrine (189) and related compounds, sedridine (193), 2-(20 -Hydroxypropyl)Δ1-piperideine (194) and 2-(20 Propenyll)Δ1-piperideine (195), have also been isolated (Fig. 12.21, Neuhofer et al., 1993). The identification of N-(20 ,50 -dihydroxyphenyl)pyridinium chloride (196) from the leaves has also been described (Nawwar et al., 1994a,b). Esterogenic compounds including sex hormones such as estrone,17-α-estradiol, testosterone, estriol as well as coumestrol based on a flavonoid skeleton have been isolated from the seeds (Abd et al., 1998; Moneam et al., 1988). Volatile components of the terpenoids nature are also known to occur (Va´zquez-Arau´jo et al., 2011) though their pharmacological relevance has not been demonstrated.

12.6 Evidence of efficacy from in vitro and in vivo studies In this section, comprehensive evidences coming from enzyme-based, cell-free and cellbased assays, and various animal models are presented. Key data corroborating the antidiabetic and other mechanisms related to potential benefit for metabolic syndrome are outlined in Table 12.1 (in vitro) and Table 12.2 (in vivo).

D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

TABLE 12.1 Summary of key findings related to the antidiabetic and related activities of pomegranate in vitro. Bioassay

Key findings

References

Fruit juice and compounds

Lipase, α-glucosidase and dipeptidyl peptidase-4 (DPP4) enzyme assay; adipocytes derived from 3T3-L1 cells

Juice, ellagic acid, punicalagin and urolithin A inhibit lipase, α-glucosidase and dipeptidyl peptidase-4; metabolite urolithin A displayed anti-adipogenic properties in a dose-dependent manner—reduce TG accumulation and gene expression related to adipocyte formation (adiponectin, PPAR-γ, GLUT4, and FABP4)

Les et al., 2018

Flowers—70% Ethanol extract

Chang liver cells

25–200 μg/ml—Reduce MDA and increase SOD and GPx levels; decrease TNF-α and IL-8 levels.

Yan et al., 2017

Flowers—Methanol extract

HepG-2 cells

10–100 μg/ml—Increase PPAR-α and ACO.

Xu et al., 2017

Fruits peel— Methanol extract

Antioxidant and enzyme inhibition assay

Radical scavenging effect; α-glucosidase inhibition (IC50–0.0453 μg/mL; LDL oxidation (IC50–16.2 μg/ mL; ACE inhibition properties (IC50–0.77 μg/mL).

Arun et al., 2017

Flowers—Methanol extract

Enzyme inhibition assay on seven Tunisian verities

α-Amylase inhibitory effect- only one variety (Zaghwani) was weakly active—(IC50 of 185.92
2.00 μg/mL); α-glucosidase inhibitory effect range from IC50 of 29.77 to 48.02 μg/ml.

Bekir et al., 2016

Leaves -Methanol: water (70:30) extract—ethyl acetate fraction

Enzyme inhibition assay

α-amylase: C50–187.82 μg/ml; α-glucosidase inhibition assay IC50–94.25 μg/ml

Patel et al., 2014

Fruits juice of commercial sourcedried and ellagic acid

3T3-L1 adipocytes

100 μg/ml for crude extract and 20, 40, and 70 μM for ellagic acid—Reduce the intracellular protein (not mRNA) levels of resistin in differentiated cells.

Makino-Wakagi et al., 2012

Fruit juice and active components

Human hepatoma HuH7cell line

Juice, punicalagin, gallic acid, and less so ellagic acid, dose-dependently increased cell-associated and hepatocyte-secreted PON1 arylesterase activity; secreted PON1 protect LDL and HDL oxidation; upregulation of PON1 expression involve PPARγPKA-cAMP signaling.

Khateeb et al., 2010

12.6 Evidence of efficacy from in vitro and in vivo studies

D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

Preparation

395

continued

396

TABLE 12.1 Summary of key findings related to the antidiabetic and related activities of pomegranate in vitro.—cont’d Bioassay

Key findings

References

Flowers—Methanol extract

Human THP-1-differentiated macrophage cells

10, 50, and 100 μg/ml—enhance PPAR-γ mRNA and protein expression; Increase PPAR-γ-dependent mRNA expression and activity of lipoprotein lipase.

Huang et al., 2005a

Flowers—Methanol extract

Cultured human embryonic kidney 293 cells

100 and 300 μM—activates PPAR-α

Huang et al., 2005b

Flowers—Methanol extract

α-glucosidase activity

0.25–32 μg/ml—Dose-dependent inhibition.

Li et al., 2005

Fruit—Juice

Human coronary artery endothelial cells

7 or 14 μL—Ameliorate the shear stress-induced eNOS suppression; increase ELK-1, and the p-JUN protein levels; increase the level of NOS downregulated by oxidized low-density lipoprotein

de Nigris et al., 2005, 2006, 2007b

Fruits—Juice

J774A.1 macrophages

Increase expression (mRNA, protein) and activity of PON2; effect dependent on PPAR-γ and AP-1 activation; reduce macrophage oxidative status.

Shiner et al., 2007

Fruits—Juice

J774.A1 macrophages

Suppress oxidized-LDL degradation and cholesterol biosynthesis in macrophages.

Fuhrman et al., 2005

Flowers—Methanol extract

Human embryonic kidney 293 cells

100 and 300 μM -activates PPAR-α.

Huang et al., 2005b

Abbreviations: ACE, Angiotensin-converting enzyme; ACO, acyl-CoA oxidase; cAMP, Cyclic adenosine monophosphate; AP-1, Activator protein 1; ELK-1, ETS transcription factor  1; eNOS endothelial nitric oxide (NO) synthase; FABP4, Fatty acid binding protein 4; GLUT4, Glucose transporter type 4; GPx, glutathione peroxidase; HDL, High-density lipoproteins; LDL, low-density lipoproteins; IL-8, interleukin-8; MDA, malondialdehyde; NOS, nitric oxide synthase; PKA, protein kinase A; PON, paraoxonase; p-JUN, phosphorylated JUN; PPAR-γ, Peroxisome proliferator-activated receptor-γ; SOD, superoxide dismutase; TG, triglyceride; TNF-α, tumor necrosis factor-α.

12. The chemical and pharmacological basis of pomegranate

D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

Preparation

TABLE 12.2 Summary of key findings showing antidiabetic and related pharmacology for pomegranate preparations in vivo. Main outcome

References

Fruit—Juice (seeds excluded)

Alloxan-induced diabetic rats—3 ml/ animal, p.o. for 4 weeks

Reduce blood glucose; restore electrocardiographic parameters (diabetes-induced increase in heart rate and ECG parameters), serum glucose, biomarkers and lipid profiles (reverse the increased AST, ALT, ALP, Creatinine kinase, LDH, creatinine and albumin levels).

Chakraborty et al., 2017

Seeds oil

STZ-induced diabetic rats—0.36 and 0.72 mg/kg, p.o. for 3 weeks

Increase antioxidant enzymes activity (SOD, GST and PON-1) and decrease oxidative stress in kidney and heart homogenates (mitochondrial fraction).

Mollazadeh et al., 2017

Flowers—70% Ethanol extract

Apo E/ mice 140 mg/kg, i.g. for up to 16 weeks

Reduce body weight, visceral fat mass, serum levels of TC and LDL-C, alleviates (after 16 weeks) liver steatosis (histology), lower liver weight and TG content; reduce serum ALT and AST levels.

Yan et al., 2017

Fresh juice or seed powder

STZ-nicotinamide-induced diabetic rats—1 ml juice or 100 mg powder, p.o. for 21 days

Non-significant reduction of plasma glucose and no effect on insulin level; Reduce TC and TG, and inflammatory markers (IL-6, TNF-α, NF-κB) and restore islets of Langerhans (histology).

Taheri Rouhi et al., 2017

Seed oil

STZ-induced diabetic rats—0.4 and 0.8 mL/kg, p.o. for three weeks

Decrease in tissue MDA content, serum glucose, creatinine and urea levels as well as urine markers (volume and protein level); reverse thiol depletion and improve histology of the heart.

Mollazadeh et al., 2016

Fruits peel and leaves—95% Ethanol extract

STZ-induced diabetic rats—100 and 200 fruits and 200 mg/kg leaves, p.o. for 4 weeks

Reduce blood glucose, TC and TG levels; increase HDL-C.

Salwe et al., 2015

Fruits peel extract containing 40% total polyphenols

STZ-induced diabetic mice—400 mg/kg, p.o. for 4 weeks

Decrease blood glucose; increases insulin secretion and the pancreas weight index and histology; increase antioxidative capacity (GSH and the T-AOC level)

Wang et al., 2015

Flavonoid-rich fraction of leaves

STZ-induced diabetic rats—200 mg/kg, p.o. for 28 days

Decrease fasting glucose level and in OGTT test, TC, TG, LDL-C, and VLDL-C, increase HDLdC; decreased the alteration in GFR by decreasing serum creatinine, BUN, and increasing creatinine clearance; normaliz the levels of GSH, SOD, MDA, and CAT in the kidney; improve kidney histology; recover body weight loss.

Ankita et al., 2015

397

Experimental model

12.6 Evidence of efficacy from in vitro and in vivo studies

D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

Preparation

continued

Experimental model

Main outcome

References

Fruits—Extract (commercial source)

Hypercholesterolemic diet in pigs— 625 mg for 10 days

Prevented diet-induced impairment of endothelial relaxation; activate the vascular Akt/eNOS pathway

Vilahur et al., 2015

Seed oil

STZ-Nicotinamide-induced diabetic rats—600 mg/kg, p.o. for 28 days

Increase serum insulin level and GPx activity; no effect on blood glucose level.

Nekooeian et al., 2014

Fruits or seed-juice or seed powder (100 mg/ml)

STZ-nicotinamide (STZ-NA)-induced diabetic rats—1 ml juice or 100 mg seed powder per animal, i.g. 21 days

Increase plasma levels of insulin, antioxidant enzymes (SOD and CAT) and total antioxidant status; reduce plasma MDA and GGT; improve liver histology; Higher effect for the seed juice).

Aboonabi et al., 2014

Leaves—Methanol: water (70:30) extract—ethyl acetate fraction

STZ-induced diabetic rats—50, 100, 200 mg/kg, p.o. 28 days

Reduce glucose level in OGTT; decrease fasting blood glucose and hemoglobin; reduce intestinal glucose absorption; increase glycogen content (e.g., skeletal muscle); increase serum total protein; no effect on plasma insulin level; decrease TC, TG, LDL-C and VLDL-C while increasing HDLdC; increase the level of liver SOD, CAT and GSH and reduce MDA; improve body weight; improve histology of pancreas, liver and kidney.

Patel et al., 2014

Fruits extract (commercial juice dried)

Ovariectomized mice—30 mg/kg for 12 weeks

Reduce the resistin level in serum and white adipose tissues.

Makino-Wakagi et al., 2012

Flowers—Methanol extract

Normal mice—0.25 or 0.5% supplement in food for 55 weeks

Ameliorate aging-induced abnormal increases in the HOMA-IR, glucose concentrations during oral glucose tolerance test, and adipose insulin resistance index.

Wang et al., 2012

Leaves—90% Ethanol extract

Alloxan-induced diabetic rats—500 mg/ kg, p.o. for 1 week

Decrease blood glucose; increase glycogen content in the liver, cardiac, and skeletal muscles; reduce intestinal glucose absorption; decrease serum TC, TG, LDL; increase serum HDL level.

Das and Barman, 2012

Fruits—Juice

– ovariectomized ddY mice- 30 mg/kg body weight/day) for 12 weeks

Reduce serum level of resistin with no effect on adiponectin.

Makino-Wakagi et al., 2012

12. The chemical and pharmacological basis of pomegranate

D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

Preparation

398

TABLE 12.2 Summary of key findings showing antidiabetic and related pharmacology for pomegranate preparations in vivo.—cont’d

High-fat diet-induced obesity and insulin resistance model in C57Bl/J6 mice- 1% seed oil as food additive for 122 weeks

Reduce body weight and fat mass; no effect on food intake or energy expenditure; no effect on glucose or insulin levels.

Vroegrijk et al., 2011

Fruits—Juice

STZ-induced diabetic rats—100 and 300 mg/kg; p.o. for 4 weeks

Reduce mean arterial BP and vascular reactivity changes to catecholamines and Ang II; decrease TBARS while increasing the activity of CAT, GR, SOD in pancreas and kidney; Also inhibit ACE activity in vitro.

Mohan et al. (2010)

Fruits peels— Methanol extract

STZ-induced diabetic rats—10 and 20 mg/kg, p.o. for 4 weeks

No effect on glucose level; enhance antioxidant enzymes (CAT in the liver, SOD in the liver and kidney, GPx in liver, kidney and RBC, and GR in the liver); reduce lipid peroxidation (MDA levels) in liver, kidney and RBC; enhance serum antioxidant status.

Althunibat et al., 2010

Flowers—Aqueous extract

STZ-induced diabetic rats—250 and 500 mg/kg, p.o. for 21 days

Reduce fasting blood glucose, TC, TG, LDL-C, VLDL-C and tissue LPO; elevate HDLdC, GSH content and antioxidant enzymes (GPx, GR, GST, SOD and CAT) in the pancreas.

Bagri et al., 2009

Flowers—Methanol extract

Zucker diabetic fatty rats—500 mg/kg, p. o. for 6 weeks

Reduce ratio of liver weight to tibia length, hepatic TG contents and lipid droplets; enhance hepatic gene expression (PPAR-α, CPT-1 and ACO, and reduced SCD-1); minimal effects on expression of genes responsible for synthesis, hydrolysis or uptake of FA and TG.

Xu et al., 2009

Flowers—Aqueous extract

STZ-induced diabetic rats—250 and 500 mg/kg, p.o. for 21 days

Reduce FBG, TC, TG, LDL-C, VLDL-C and tissue LPO levels; increase HDL-C, GSH content and antioxidant enzymes (CAT, SOD and GR).

Bagri et al., 2009

Fruits extract, fruits juice and seed oil

Obese Zucker rats—atherogenic diet with supplements.

Extract and juice -decrease the expression of vascular inflammation markers, TSP, and cytokine TGFβ1; seed oil only affect TSP-1 expression; plasma

de Nigris et al. 2007a

12.6 Evidence of efficacy from in vitro and in vivo studies

D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

Seed oil

continued

399

400

TABLE 12.2

Summary of key findings showing antidiabetic and related pharmacology for pomegranate preparations in vivo.—cont’d Experimental model

Main outcome

References

nitrate and nitrite levels increased (fruit juice and extract); Extract increase eNOS expression. Flowers—Methanol extract

Zucker diabetic fatty rats—500 mg/kg, p. o. for 6 weeks

No effect on plasma glucose level in lean or obese rats model but reduced blood glucose in OGTT in obese animals; enhance mRNA of PPAR-γ and GLUT4 expression in cardiac tissues.

Huang et al., 2005a

Flowers—Methanol extract

Zucker diabetic fatty rats—500 mg/kg, p. o. for 6 weeks

Lowered plasma FA levels; inhibits cardiac overexpression of mRNAs encoding for FA transport protein, PPAR-α, carnitine palmitoyltransferase-1, acyl-CoA oxidase and 50 AMP-activated protein kinase α2; restore downregulated cardiac acetyl-CoA carboxylase mRNA expression.

Huang et al., 2005b

Flowers—Methanol extract

Zucker diabetic fatty rats—500 mg/kg p. o. for 2 weeks

Reduce collagen deposition in left ventricular coronary artery tissues; suppress fibronectin and collagen I and III mRNAs expressions; increase cardiac endothelin (ET)-1 mRNA expression; restore the down-regulated mRNA expression of IκB-α.

Huang et al., 2005c

Flowers—Methanol extract

Zucker diabetic fatty rats—500 mg/kg, p. o. for 6 weeks

Lower blood glucose in diabetic (not normal) animals; reduce glucose level in sucrose-, but not glucose-loaded mice.

Li et al., 2005

Fruit—Juice

Hypercholesterolemic mice— 31 μl, p. o., for 4 weeks

Reduce atherosclerotic lesions in low- and highprone areas by perturbed shear stress; reduce level of lipid-laden macrophage foam cells; increase eNOS activity in low- and high-prone areas; increase in p-JUN and ELK-1 protein levels

de Nigris et al., 2005, 2007b

12. The chemical and pharmacological basis of pomegranate

D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

Preparation

STZ-induced diabetic rats—300 and 600 mg/kg, p.o.

Hypoglycemic effect.

Das et al., 2001

Fruits juice and tannin fraction

Apolipoprotein E-deficient (E(0)) mice given juice or fraction via drinking water for 2–4 months

Reduce serum PON activity; macrophage lipid peroxide, oxidized-LDL uptake and cholesterol esterification; increase cholesterol efflux from macrophages, suppress atherosclerotic lesion size, plasma lipid peroxidation stress and macrophage oxidative stress

Kaplan et al., 2001

Flowers—Aqueousethanolic (50%) extract

Alloxan diabetic rats—300, 400 and 500 mg/kg, p.o. Acute test on OGTT

Hypoglycaemic effect at 30 and 90 min after OGTT in normal animals 2 h; hypoglycaemic in alloxan model after 1 and 2 h

Jafri et al., 2000

Abbreviations: ACO, acyl-CoA oxidase; Akt, Protein kinase B; Ang II, angiotensin II; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate transaminase; BP, blood pressure; BUN, blood urea nitrogen; CAT, catalase; CPT-1, carnitine palmitoyltransferase-1; ECG, electrocardiogram; eNOS, endotrhelial nitric oxide synthase; FAS, fatty acid synthase; FBG, fasting blood glucose; GGT, gamma-glutamyltransferase; GFR, glomerular filtration rate; GPx, glutathione peroxidase; GR, glutathione reductase; peroxidase; GST, glutathione-Stransferase; GSH, glutathione; HDLdC, high-density lipoprotein (HDL)-cholesterol; HOMA-IR, homeostatic model of assessment for insulin resistance; i.g., intragastric; IκB-α, inhibitor-κBα; IL-6, interleukin 6; LDH, lactate dehydrogenase; LDL-C, low-density lipoprotein (LDL) cholesterol; LPO, lipid peroxidation; MDA, malondialdehyde; OGTT, oral glucose tolerance test; p. o., oral administration; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; p-JUN, phosphorylated JUN; PPAR, peroxisome proliferator-activated receptor; PON, paraoxonase; RBC, red blood cell; SCD-1, stearoyl-CoA desaturase-1; SOD, superoxide dismutase; STZ, Streptozotocin; TC, total cholsterol; TG, triglyceride; TGFβ1; transforming growth factor β-1; T-AOC, total antioxidant capacity; TBARS, thiobarbituric acid reactive substances; TSP, thrombospondin; VLDL-C, very-low-density lipoprotein (VLDL) cholesterol.

12.6 Evidence of efficacy from in vitro and in vivo studies

D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

Seeds—Methanol extract

401

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12.6.1 General antioxidant effect Considering the predominant phytochemical contents of pomegranate are polyphenolic compounds including phenolic acids, flavonoids and tannins, the in vitro antioxidant effects of the plant is by far the most studied pharmacology. The numerous studies on the estimation of the polyphenolic content of the fruits and other plant parts are also associated with some kind of antioxidant effect assessments (e.g., Gil et al., 2000). It is not therefore worthwhile to list the large volume of literature and studies on the direct radical scavenging or inhibition of reactive oxygen species (ROS) generation in vitro by the crude extracts or the known active phenolic compounds. Comparative antioxidant activity with respect to crude polyphenolic level estimation of the various plant tissues and the fruit juice preparations from different genotypic or accessions have also been routinely studied (e.g., Tzulker et al., 2007). The pomegranate peels extracts have been generally reported to display a higher level of antioxidant capacity than the pulp which is also in good correlation with total phenolics, flavonoids and tannins contents in these plant tissues (Li et al., 2006). Diabetes is characterized by persistent oxidative stress resulting from high level of ROS generation coupled with suppressed level and activity of antioxidant enzymes. Hyperglycaemia which leads to glycation end product formation is also linked to excessive production of ROS under diabetic conditions (see Chapter 4). The well-known antioxidant effect of pomegranate preparations in vitro (e.g., Gil et al., 2000) and the reported boosting of antioxidant defenses discussed in the following sections are therefore classical examples of antidiabetic mechanisms of action for the plant. In an in vitro antiglycation assays, the fruits extract as well as punicalin (101/102), punicalagin (99/100), ellagic acid (23), and gallic acid (4) have been shown to suppress the formation of advanced glycation end products (AGEs) from bovine serum albumin and sugars; an effect which can be reproduced for the fruit extract in high-fat diet (HFD) and high-sucrose fed KK-A(y) mice (Kumagai et al., 2015). A reduced glycation end products such as glycoalbumin, hemoglobin A1c (HbA1c), and serum AGEs have been noted. Liu et al. (2014) have also shown that pomegranate fruit extract, its phenolic constituents (punicalagin (99/100), ellagic acid (23) and gallic acid (4)), as well as their metabolites (urolithin A and urolithin B (Fig. 12.22) display anti-glycation activities as well as carbonyl scavenging effects. As expected for potent antioxidant agents, pomegranate juice and extracts have been shown to protect cells from UVB-mediated damage (Afaq et al., 2009). The general antioxidant effect and polyphenolic content (e.g., tannins) of pomegranate have also been shown to account for their anticancer effects in vitro (Dikmen et al., 2011; Kashiwada et al., 1992; Les et al., 2015). Not surprisingly, ellagitannin-rich pomegranate extract inhibits angiogenesis in prostate cancer in vitro and in vivo (Sartippour et al., 2008).

12.6.2 General anti-inflammatory effects Considering the role of inflammation in diabetes (see Chapter 4), it is worth reviewing the known anti-inflammatory effect of pomegranate compounds. Numerous studies have shown that one of the well-established link between obesity and diabetes is via the state of low-grade but chronic inflammation under obesity conditions. The continuous release of proinflammatory cytokines and chemokines from adipose tissues and immune cells leads to the accumulation and/or activation macrophages and other leucocytes population. Of

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OH O

HO

OH OH

HO O

O

O HO

O OH O

O O

OH

OH OH OH Glycosylation; oxidation

O HO

Intestinal breakdown

O HO

Punicalagin (99/100)

O

O

HO

OH

O

OH

OH OH OH COOH HO

O

OH

HO COOH

+ HO HO

OH OH 4 Gallic acid

COOH

OH

COOH 4

OH

O

OH HO HO

OH

O

22 Hexahydroxydiphenol

OH O 23 Ellagic acid

Interstinal bacteria transformation OH HO

R HO

O

HO

O 200 R=H Urolithin A 201 R=OH Urolithin C

O

O

OH O

HO

O 203 R=H Urolithin D 204 R=OH Urolithin M-5

202 Urolithin B O

OH

O

O 205 8-O-Methylurolitin A

FIG. 12.22

OH

OH

O MeO

OH

R

HO R 206 R=H Isourolithin B 207 R=OH Isourolithin A

O

HO O 208

Metabolism of pomegranate tannins. Metabolites identified following an intake of punicalagin

are shown.

the inflammatory cytokines, tumor necrosis factor-α (TNF)-α and interleukin (IL)-6 (IL-6) are by far the best characterized inducers of insulin resistance (Asghar and Sheikh, 2017; Cusi et al., 2000; Singh et al., 2009). Through activation of the ubiquitous transcription factor, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), TNF-α and IL-6, inflammation suppress insulin signaling that is outlined in Chapter 2. This include the inhibition of the insulin-mediated activation of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and

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protein kinase B (PKB or Akt) pathways of glucose uptake and inhibition of gluconeogenesis. The mitogen-activated protein kinase (MAPK) pathway is also closely associated with insulin signaling (Chapter 2). Accordingly, pomegranate juice, total pomegranate ellagitannins, and punicalagin (99/100) have all been shown to suppress inflammatory cell signaling by inhibiting the TNF-α-induced Akt activation which is needed for NF-κB activity as well as NF-κB mobilization (Adams et al., 2006). The calcium inophore-induced expression of inflammatory cytokines (IL-6 and IL-8) in basophilic cell line (KU812) has also been shown to be inhibited by the polyphenol-rich pomegranate fruit extract via suppressing the MAPK pathway (c-jun N-terminal kinase (JNK)- and extracellular-regulated kinase (ERK) dependent) and NF-κB activation (Rasheed et al., 2009). The ERK and JNK activation is induced by a variety of cellular and environmental stimuli that trigger the expression of proinflammatory cytokines such as TNF-α, IL-6 and IL-1. Hence, one known mechanism of inflammation-induced insulin resistance is inhibited by pomegranate and its active constituents. These cytokines expression and action also requires NF-κB that serve as a therapeutic target for numerous disease conditions including cancer, inflammation and diabetes. Evidence on the general anti-inflammatory effect of pomegranate extract also came from animal studies where inflammation and joint damage in rheumatoid arthritis could be inhibited (Shukla et al., 2008a). In this model of collagen-induced arthritis in mice, dietary supplements containing pomegranate fruits extract (13.6 and 34 mg/kg) could ameliorate the arthritis score and the level of inflammatory cytokines (IL-1β, IL-6 and TNF-α) at the arthritis joint. In mouth macrophages stimulated by LPS, the pomegranate fruits extract (20 μg/ml) could inhibit the phosphorylation of JNK without affecting the level of JNK—i.e., inhibition of its activation. The activation of NF-κB by lipopolysaccharide (LPS) in macrophages is regulated via the phosphorylation of the inhibitory protein IκB (or inhibitor-κBα) by the IκB kinase (IKK) complex. The subsequent degradation of IκB to allow NF-κB mobilization to the nucleus is the major event that also serve as a ubiquitous target by many natural products. Although the precise mechanism was not established, NF-κB inhibition was demonstrated for pomegranate fruit extract in macrophages and leads to the inhibition of LPS-induced cytokines and nitric oxide (NO) release (Shukla et al., 2008a). The inhibition of IL-1β-induced expression of matrix metalloproteinases (MMP-1, 3, and  13 protein) in human chondrocytes in vitro by the fruits extracts of pomegranate (6.25–25 μg/mL) has also been shown to be mediated through inhibition of activation of MAPK and NF-κB (Ahmed et al., 2005). Detailed analysis also revealed inhibitory effect on p38-MAPK and the IL-1β-induced phosphorylation of IκBα and the DNA binding activity of the transcription factor NF-κB (Ahmed et al., 2005). In another study by the same authors, inhibition of cyclooxygenase-2 (COX2) activity ex vivo and IL-1β-induced prostaglandin-E2 (PGE2) production in human chondrocytes were demonstrated for the fruits extract in vitro (Shukla et al., 2008b). In both animal model of lung inflammation and human alveolar cells, pomegranate juice has been shown to reduce emphysematous changes and injury secondary to cigarette smoking (Husari et al., 2016). In another anti-inflammatory effect study using rat colitis model of colon inflammation, pomegranate extract and its metabolite, urolithin-A, have been shown to display promising effect (Larrosa et al., 2010). Collagenase (MMP-1), gelatinase (MMP-2, MMP-9), stromelysin (MMP-3), marilysin (MMP-7), elastase (MMP-12), and tropoelastin inhibitory effect of pomegranate juice and extracts have also been demonstrated (Afaq et al., 2009) suggesting antiinflammatory effects. A review of literature on anti-inflammatory effect of pomegranate

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preparations is also available (Danesi and Ferguson, 2017). All these known antiinflammatory effect of pomegranate are likely to account to the antidiabetic properties discussed in the following sections.

12.6.3 Effects of various plant parts 12.6.3.1 Fruits As shown in Tables 12.1 and 12.2, the pulp, juice and seeds of pomegranate have been assessed in various diabetes and obesity models both in vitro and in vivo. Considerable attention have also been given to study the peels as separate component given large volume of these products could be left behind as industrial by-products of the juice production. This include the alloxan-induced diabetes model through which the antidiabetic effect of the peels and whole fruits (excluding seeds) extracts have been demonstrated (Chakraborty et al., 2017; Parmar and Kar, 2007). These data clearly showed a reduced level of blood glucose along with favorable hematological biomarkers of liver toxicity (aspartate transaminase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) enzymes), improved kidney functions (creatinine clearances) and antioxidant status. The antidiabetic effect on streptozotocin (STZ) and often in combination with nicotinamide-induced diabetes models have also been established in various studies (Aboonabi et al., 2014; de Nigris et al., 2007a; Mohan et al. (2010); Salwe et al., 2015; Taheri Rouhi et al., 2017; Wang et al., 2015). The antioxidant effect of the fruit juices and seeds in STZ-nicotinamide-induced diabetic rats have also been demonstrated Aboonabi et al. (2014). These studies have at least few common beneficial effects reported but the discrepancies in dosage, juice preparation (whole or peels) and outcome is worth mentioning. For example, Taheri Rouhi et al. (2017) did not find a significant effect on the level of glucose and insulin, while a reduced level of glucose was reported by others (Salwe et al., 2015; Wang et al., 2015 for the peel extract). The study by Althunibat et al. (2010) also revealed the potential health benefit of the peels extract when assessed using the STZ-induced diabetes model. Despite the comprehensive antioxidant effect shown, however, glucose lowering effect was not demonstrated by their studies. One common source of discrepancy is of course the doses used which in this case was only 10 and 20 mg/kg (p.o.) (Althunibat et al. (2010) instead of over 100 mg/kg used by other authors. Increase in plasma level of insulin was reported by Aboonabi et al. (2014); while increase in antioxidant enzyme activity was the most prevalent outcome in many studies and hence catalase (CAT), glutathione (GSH) reductase (GR), superoxide dismutase (SOD) in pancreas and kidney were augmented (Mohan et al., 2010). The anti-inflammatory effect in the STZ-induced diabetic model has also been reported and the suppressed level of proinflammatory cytokines such as IL-6, TNF-α as well as NF-κB was evident in the pomegranate juice treated animals (Taheri Rouhi et al., 2017). Les et al. (2018) studied the lipase, α-glucosidase and dipeptidyl peptidase-4 (DPP4) inhibitory effect of pomegranate juice and active components, ellagic acid (23) and punicalagin (99/100) as well as the metabolite of the latter, urolithin A (Fig. 12.22). The pomegranate juice as well as the purified compounds were shown to display potent inhibition with IC50 value of 0.0055, 0.015, 0.025, 0.38 and 1.01 mg/ml for punicalagin, urolithin-A, ellagic acid, acarbose and the juice respectively. For lipase inhibition, an IC50 values were 0.00074, 0.032, 0.092, 0.16 and 2.50 mg/ml for orlistat, urolithin-A, ellagic acid, punicalagin and pomegranate juice

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respectively; while DPP-4 inhibition was with an IC50 value of 0.025, 0.059 and 0.095 mg/ml for ellagic acid, punicalagin and urolithin-A, respectively. The α-glucosidase activity of the active principles have also been studied by many authors and ellagitannins-enriched fraction and punicalagin (99/100), punicalin (101/102), and ellagic acid (23) were identified as α-glucosidase inhibitors (IC50 of 140.2, 191.4, and 380.9 μmol/L, respectively) (Bellesia et al., 2015). The compounds also suppressed 3T3-L1 adipocytes differentiation, could reduce adipogenesis, and decrease triglycerides (TG) accumulation. Punicalagin and urolithin-A (200), have been shown to decrease the TG content by half in the adipocytes, with IC50 of 0.027 and 0.002 mg/ml, respectively. The mRNA levels of key adipogenic genes as markers of adipocyte differentiation were also studied and adiponectin, peroxisome proliferatoractivated receptor (PPAR)-γ (PPAR-γ), Glucose transporter type 4 (GLUT4) and Fatty acid binding protein-4 (FABP4) were suppressed particularly by urolithin A. Hence, both the juice and active ingredients are capable of displaying antidiabetic and antiobesity effect in vitro. Readers should bear in mind that tannin-like compounds do interact non-specifically with proteins and their enzyme inhibitory activity is somehow expected. This is particularly common for punicalagin (99/100) which showed inhibition of enzymes including protein disulfide-isomerase A3 (PDIA3) reductase activity (Giamogante et al., 2018). Some of the reported activity in inhibition of lipogenesis and lipolysis by the fruit juice was however at rather high doses and the experiment by Les et al. (2017) is good example where 1 and 10 mg/ml juice were shown to have effect in vitro. The data available so far appear to corroborate the antiatherogenic potential of pomegranate fruits and juices. A reduction in atherogenic potential (assessed as oxidative stress, TG and cholesterol metabolism) of macrophages are also demonstrated in pomegranate-treated rats under various conditions (Rosenblat et al., 2015). A number of in vitro studies have demonstrated that the fruit extracts and active components including, punicalin (101/102), punicalagin (99/100), ellagic acid (23), and gallic acid (4) could suppress the formation of AGEs from bovine serum albumin and sugars. The study by Kumagai et al. (2015) further revealed that the fruit extract with its high polyphenols composition could display antiglycation effect in vivo in a HFD and high-sucrose diet in KK-A(y) mice. They have shown that the fruit juice can suppress glycation products, glycoalbumin, HbA1c, and serum AGEs. The sugar fractions of the fruits have also shown potential benefit as suppressive effect on cellular peroxide levels in J774A.1 macrophage cell-line and mouse peritoneal macrophages were demonstrated, including that obtained from STZ-induced diabetic mice (Rozenberg et al., 2006). More importantly, consumption of the sugar fraction in diabetic mice for 10 days (in drinking water) have been shown to reduce the total peroxide levels and increment in cellular GSH content in peritoneal macrophages. The sugars treatment also suppressed paraoxonase 2 (PON2) activity (see details below to show this as a contradiction to other studies). The study by de Nigris et al. (2007a) also showed the antiatherogenic effect of the fruits extract in obese-animal model where the expression of vascular inflammation markers, thrombospondin and cytokine transforming growth factor β-1 (TGFβ1) have been inhibited. On the other hand, the expression level of endothelial nitrix oxide synthase (NOS) which is known to be suppressed under diabetic condition is enhanced. Under oxidative stress condition, the activity of PON2 is enhanced in cells like macrophages and induce inhibitory effect on cell-mediated oxidative modification of low density lipoprotein (Ng et al., 2001). Suppression of this enzyme under diabetic condition was claimed to

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offer favorable outcome but this should be considered with caution as PON2 is a ubiquitous intracellular protein that can protect cells against oxidative damage. In fact, Shiner et al. (2007) have shown the upregulation of PON2 both at expression and activity level in macrophages by pomegranate juice in vitro. They have shown potential antioxidant effect of the enzyme expressed by the juice via PPAR-γ and AP-1 activation (Shiner et al. (2007). The role of paraoxonase gene family in atherogenesis has also been well established with PON-1 displaying antioxidative and antiatherogenic properties (Ng et al., 2001, 2005). As discussed later, a number of studies have shown that pomegranate juice can augment the serum level of PON-1 both in atherogenic mice and in human trials (e.g. Aviram et al., 2004; Kaplan et al., 2001). From all the available literature, it can be concluded that both PON-1 and PON-2 have antiatherogenic effect but while PON-2 is expressed in tissues and macrophages, PON-1 is predominantly expressed in the liver and is released into the circulation. Thus, PON-2 deficiency aggravates atherosclerosis in mice (Ng et al., 2006). The PON-2 activation-induced protection of macrophages from oxidative stress and generation of excess ROS (Shiner et al., 2007) is thus in line with many other studies. Moreover, the effect of the pomegranate juice in enhancing PON-2 in macrophages is shown to be mediated via activation of transcription factors PAPR-γ and AP-1. More evidence on the effect of pomegranate juice on PON-2 came from studies on peritoneal macrophages collected from normal or PON1KO mice or PON2KO mice (Rosenblat et al., 2010). It has been shown that pomegranate juice consumption (200 μg of gallic acid equivalents/mouse/day, for 1 month period) can increase the serum level of PON-1 catalytic activities and upregulate PON2 expression in macrophages. It was also observed that pomegranate juice consumption could reduce the macrophage cellular cholesterol content and cholesterol biosynthesis rate as well as HDL-mediated cholesterol efflux. From the knockout studies, however, the pomegranate-induced stimulation of macrophage PON2 confer antioxidative properties in macrophages but this effect was not related to the action of the extract on macrophage cholesterol and TG metabolism. A more convincing effect of the pomegranate fruit juice and active components on PON came from the study by Khateeb et al. (2010) that revealed unequivocal evidence on PON-1 upregulation. Their study on cultured hepatocytes showed that the juice as well as punicalagin (99/100), gallic acid (4), and to a lesser extent, ellagic acid (23), all increase the expression and secretion of PON-1 leading to protection of LDL and HDL from oxidation. Activation of PPAR-γ and its signaling pathway was reported as a mechanism for this beneficial effect as revealed from specific antagonist studies. The upstream signaling cascade of PPAR-γ including protein kinase A (PKA) and cyclic AMP (cAMP) could also lead to stimulation of hepatocyte for PON-1 upon activation. Considering PON-1 as an HDL-associated lipo-lactonase that protect both LDL and HDL from oxidation, it could also protect macrophages from oxidative stress (though primarily done by PON-2), but more importantly stimulate cholesterol efflux from macrophages (see above). Hence, it has critical role in reducing atherosclerosis development and lesion. Accordingly, the anti-atherosclerotic properties of pomegranate juice and the tannin fraction has been demonstrated in long-term experiment in apolipoprotein E-deficient (E(0)) mice after administration via drinking water for up to four months (Kaplan et al., 2001). A range of atherosclerosis markers (e.g. lesion size) have been shown to be suppressed, coupled with enhancement of PON-1 activity in the serum, suppression of macrophage oxidative stress, oxidized-LDL uptake and cholesterol esterification; while cholesterol efflux in macrophages was augmented.

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The possible therapeutic potential of pomegranate fruit polyphenols in the cardiovascular system under sheer stress conditions have also been studied using hypercholesterolaemic LDL receptor-deficient (LDLR/) mice and cultured human Human coronary artery endothelial cells (de Nigris et al., 2005, 2007b). It has been shown that the reduced level of NO under sheer stress condition both in vitro and in vivo could be recovered by using the juice (Tables 12.1 and 12.2). The level of atherosclerosis in arterial samples examined by immunoassay was reduced while redox-sensitive genes that are downregulated by sheer stress (in vitro or in vivo) (ELK-1 and p-JUN) appear to be augmented. The doses used did not appear to alter plasma cholesterol levels but the level of lipid-laden macrophage foam cells were reduced. The downregulation of endothelial NOS (eNOS) activity induced by oxidized lowdensity lipoprotein could also be ameliorated by the fruit juice (de Nigris et al., 2006). The authors also assessed the effect of the fruit juice on NO and arterial function in obese Zucker rats (de Nigris et al. 2007a). The reduction in vascular inflammation markers, thrombospondin (TSP), and cytokine TGFβ1 coupled with induction of NOS expression was reported. Hence, the downregulated NOS and/or NO release in obesity and metabolic syndrome could be ameliorated by pomegranate supplementation along with improvement in lipid profile as shown from amelioration of the rise in LDL, arterial pressure and heart rate (de Nigris et al. 2007a). In a comparative analysis study, the fruit juices and extracts have shown to be superior to the seed oils in inducing these effects, perhaps owing to the polyphenolic active principles in them. In pigs fed with a 10-day hypercholesterolaemic diet, supplementation with 625 mg/day of a pomegranate extract prevented the impairment of endothelial relaxation via vascular Akt/endothelial NO-synthase activation and lower monocyte chemoattractant protein-1 expression. Hence, in addition to improving the antioxidant status, the hyperlipidaemiainduced coronary endothelial dysfunction could be recovered by the extract via activation of the Akt/endothelial NOS pathway (Vilahur et al., 2015). Beyond the diabetes model, the cardioprotective effect of pomegranate juice in various models have also been demonstrated. Jadeja et al. (2010), for example, have shown that juice supplementation could attenuate the isoproterenol-induced cardiac necrosis in rats. The cigarette smoking-induced cardiac hypertrophy, vascular inflammation and injury could also be ameliorated by supplementation of pomegranate juice in rats (Al Hariri et al., 2016). The raised oxidative stress associated with increased levels of IL-1β, TNF-α, fibronectin, and leptin receptor (ObR) in rat’s aorta was shown to be ameliorated (Al Hariri et al., 2016). The fruit and juice of pomegranate have also shown to suppress platelet aggregation, calcium mobilization, thromboxane A2 production, and hydrogen peroxide (H2O2) formation, induced by collagen and arachidonic acid (Mattiello et al., 2009). The angiotensin II-induced hypertension in diabetic rats could also be ameliorated by pomegranate juice (Mohan et al., 2010). The coronary angiotensinconverting enzyme (ACE) activity in hypertensive female rats could also be suppressed by the peels extract along with oxidative stress markers (Dos Santos et al., 2016). ACE inhibition has also been validated through in vitro studies by Arun et al. (2017) who also showed inhibitory effect on LDL oxidation. The data by Delgado et al. (2016) further corroborated the cardioprotective effect of the fruits as the peels extract (by alcohol) has been shown to enhance the endothelium-dependent coronary artery relaxation in the isolated perfused heart from the spontaneously hypertensive ovariectomized rats.

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Considering the active principles of the fruits, the polyphenolic compounds as principal constituents have been implicated both from their abundance in the fruits and other parts as well as their known pharmacology. They are potent antioxidant compounds reported in the various literature and also display antiglycation effect both in vitro and in vivo (Kumagai et al., 2015). Ellagic acid (23) is a known antioxidant and anti-inflammatory compound both in vitro and in vivo (Lee et al., 2010). As with the fruit juice preparations, it can protect endothelial cells and others from oxidized LDL-induced cell damage and apoptosis (Ou et al., 2010). The endothelium-dependent and independent vasorelaxation induced by ellagic acid has been reported in rat thoracic aortic rings preparations (Yilmaz and Ustam, 2013). In line with the anti-α-glucosidase effect of pomegranate preparations (Table 12.1), pomegranate ellagitannins inhibit the enzyme in vitro and reduce starch digestibility under simulated gastro-intestinal conditions (Bellesia et al., 2015). High level of circulating resistin is common in subjects with obesity and T2D. As a product of adipocytes (but also produced by other tissues), the increased level of this adipocytokine is an established link between obesity and T2D. Hence, the low grade but sustained inflammation in obese subjects that is known to be correlated with increased insulin resistance is also correlated with increased resistin level. The experiment by Makino-Wakagi et al. (2012) in cultured adipocytes and animal model using ovariectomized ddY investigated whether effect on resistin level could serve as possible mechanism of action for the pomegranate polyphenols such as ellagic acid (23). They have shown in vivo that pomegranate juice administration could suppresses the raised resitin level in the blood induced by removal of the ovary, while in vitro experiment showed suppression of its release from 3T3-L1 adipocytes. A mechanism, of action where acceleration of the protein degradation in the adipocytes without affecting its synthesis was postulated. Interestingly, gallic acid (4) could reproduce this effect while the seed oil active component, punicic acid (178) didn’t. In line with the reported inhibitory effect of the fruit extract on resistin secretion from differentiated murine 3T3-L1 adipocytes, ellagic acid (23) supplementation (0.1% of food) has been shown to reduce the serum resistin and improves hepatic steatosis and serum lipid profile in KK-A(y) mice fed with HFD (Yoshimura et al. (2013). In this obese T2D model, ellagic acid (23) improved the serum lipid profile and hepatic steatosis, and reduced serum resistin levels without altering mRNA expression levels in adipose tissue. They have also shown that supplementation with ellagic acid could upregulate the mRNA expression level of several genes in the liver including PPAR-α (Makino-Wakagi et al., 2012). 12.6.3.2 Flowers The in vivo and in vitro antidiabetic and antiobesity potential studies (Tables 12.1 and 12.2) on pomegranate preparations have also been dominated by extracts from the flowers. Huang et al. (2005a) examined the therapeutic potential of the flowers of pomegranate in fat-induced diabetes model (Zucker diabetic fatty rats—a genetic model of obesity and T2D). Their data showed that the flowers extract does not alter glucose level in both lean and fat-induced diabetes model but possess a selective glucose lowering effect during glucose loading in obese rats. Their in vitro study on THP-1-derived macrophage cell line also showed that the extract could enhance the mRNA and protein level expression of PPAR-γ. The PPAR-γ-mediated transcription of both the PPAR-γ and lipoprotein lipase genes were also enhanced by the extract. With the proven hypothesis that diabetes leads to the downregulation of GLUT4 in

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cardiac muscles and PPAR-γ activation could upregulate the GLUT4 level, the effect of the extract in enhancing both GLUT4 and PPAR-γ levels is an interesting observation. On the basis of their preliminary study, the authors assert that the polyphenol constituents particularly, gallic acid, is responsible for the observed activity. This is based on the abundance of gallic acid (4) in the active fraction and its effect on lipoprotein lipase mRNA expression and the enzyme activity; but a rather high concentration (300 μM) used by the authors is not a convincing conclusion. The direct antidiabetic effect of the flowers extract has also been shown in the Zucker diabetic fatty rats where glucose lowering effect (in contradiction to the report by Huang et al., 2005a) where shown after oral administration of the flowers extract (500 mg/ kg) for two weeks (Li et al., 2005). The possible mechanism of action could also be shown to be attributed through α-glucosidase inhibition not only from in vitro experiment showing inhibition at low concentration range of the extract, but also the effect on sugar loading test in animal models. Gallic acid (4) and ellagic acid (23) as components of the methanolic flowers extract have also been shown to display a selective inhibition against α-glucosidase (vs α-amylase) (Kam et al., 2013). While the increased glucose in the blood from sucrose loading was inhibited by the flowers extract, no effect on glucose loading was observed suggesting an effect on the intestinal sucrase enzymes. Using the same model and protocol, the authors (Huang et al., 2005b) in another study have also demonstrated that long-term oral administration of the flowers extract (500 mg/kg) could reduce cardiac and plasma TG and cholesterol contents. The selective inhibitory effect on fat-induced diabetic (not lean animals) of the extract on cardiac overexpression of mRNAs encoding for fatty acid transport protein, PPAR-α, carnitine palmitoyltransferase-1, acyl-CoA oxidase and 50 -AMP-activated protein kinase α2; while restoring the downregulated cardiac acetyl-CoA carboxylase has also been demonstrated. The flowers extract increasing the uptake of fatty acids in cardiac and fatty acids oxidation in diabetic animals coupled with direct activation of PPAR-α in vitro (human embryonic kidney 293 cells) also appear to suggest the possible mechanism of antidiabetic action (Huang et al., 2005b). Another contribution in this field by Huang et al. (2005c) also came from a study on Zucker diabetic fatty rats that revealed the potential of the flowers extract (500 mg/kg, p.o.) to ameliorate cardiac fibrosis. In addition to suppression of fibronectin and collagen I and III levels in the left ventricular and coronary artery, inhibition of the NF-κB inhibition both in vitro and in vivo have been shown. Interestingly, the in vivo effect was mediated through reversing the downregulated mRNA expression of inhibitor-κBα in Zucker diabetic fatty rats. Further evidence on the antidiabetic mechanism came from in vitro studies (Bekir et al., 2016) on potential α-glucosidase and α-amylase inhibitory properties of the flowers extract. In a screening study based on seven Tunisian pomegranate verities (Chetoui, Espagnoule, Gabsi, Garsi, Rafrafi, Zaghwani and Zehri), only one variety (Zaghwani) with rather very weak α-amylase inhibitory effect (IC50 of 185.92
2.00 μg/mL) were observed. Their α-glucosidase inhibitory effects were however good and ranged from IC50 value of 29.77 (Rafrafi) to 48.02 (Zehri) μg/mL. Other enzyme inhibition including acetyl- and butyrylcholinesterases have also been noted (Bekir et al., 2016). The potential effect of the methanol extract of pomegranate flowers on non-alcoholic fatty liver disease have been investigated by Xu et al. (2009). In their Zucker diabetic fatty rat model, the extract did not change body weight, but it reduced liver weight and the ratio of liver:body weight in ZDF rats; reduced hepatic TG contents and fatty droplets without

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altering hepatic total cholesterol (TC) contents. Treatment with the flowers extract also enhance the PPAR-α, carnitine palmitoyltransferase (CPT-1), acyl-CoA oxidase (ACO) and stearoyl-CoA desaturase (SCD-1) mRNAs in both lean and fat rats; while the hepatic PPAR-γ, fatty acid synthase (FAS), scetyl-CoA carboxylase (ACC), sterol regulatory element-binding transcription factor 1 (SREBP-1), fatty acid transport protein and lipoprotein lipase mRNA expression were not affected by the treatment. The in vitro experiment on HepG2 hepatocytes cells in the same study also showed enhancement of PPAR-α and ACO expression. Considering that PPAR-α activation mediates the expression of genes regulating lipid oxidation, and insulin resistance could be ameliorated by PPAR-α agonists, the observed effect of the extract on these system could validate the therapeutic potential of the flowers for targeting obesity and insulin resistance. Most importantly: • CPT-1 as a critical rate-determining regulator of β-oxidation in hepatocytes is augmented by the extract; • SREBP-1 which activates most genes required for de novo synthesis of fatty acid and TG synthesis not altered; • PPAR-γ stimulate hepatic gene expression involved in fatty acid uptake, storage and synthesis (Loviscach et al., 2000). However, expression of PPAR-γ in liver is low and not altered by the extract. In the same year, another publication by Bagri et al. (2009) also investigated the antidiabetic effect of the flowers extracted with hot water in STZ-induced diabetes rat model. They have shown that the extract (250 mg/kg/day, p.o. for 3 weeks) can display antidiabetic effects that are be summarized as follow: • Reduction of fasting blood glucose (FBG) which was raised by diabetes; • Improvement in lipid profile as suppression of TC, TG, cholesterols of low density lipoprotein (LDL-C) and very low density lipoprotein (VLDL-C) and tissue lipid peroxidation (LPO) levels; and increase in the levels of high density lipoprotein cholesterol (HDL-C) and GSH content were observed; • The content of antioxidant enzymes GSH peroxidase (GPx), GR, GSH-S-tranferase (GST), SOD and CAT in the pancreas that is normally diminished under diabetes condition was raised by the extract. The effect of the flowers extract in the alloxan-induced diabetes model has also been reported by Jafri et al. (2000). When the hot water extracts were administered (300, 400 and 500 mg/kg, p.o.), glucose lowering effect was demonstrated in oral glucose tolerance test (OGTT) in both normal and diabetic animals. Hypoglycaemic effect was also noted in diabetic animals in this alloxan model. Even though the duration of treatment is not mentioned and it looks an acute administration regimen, a similar result for the positive control tolbutamide (500 mg/kg, p.o.) was observed. Hence, the observed dose-dependent effects were in good agreement with potential antidiabetic effects. The experiment by Yan et al. (2017) further corroborated that pomegranate flower polyphenols inhibit the development of non-alcoholic steatohepatitis fatty liver. Their experiment was on Apo E/ mice which are highly susceptible to atherosclerosis and other cardiovascular disorders. They have shown that supplementation with the polyphenols-rich extract (for 16 weeks) could suppress the HFD-induced gains in body weight, hepatic lipid accumulation,

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and the size of lipid droplets in the epididymal fat pads. The levels of total cholesterol, TG, and free cholesterol, thiobarbituric acid reactive substances (TBARS) were reduced; while HDL-C levels did not change. Altered expression profiles of several lipid metabolism-related genes, including ACC, AMPK, CPT-1α, FAS, LDLR, Leptin, LXR, PON1, PPAR, SirT3, and SREBP could also be corrected. The results thus clearly shows the anti-inflammatory activities of the extract and potential protection of the liver from obesity-induced damage. Finally, the experiment by Wang et al. (2012) assessed the long-term effect of the flowers extract (0.25% or 0.5% of diet) in normal mice and recorded amelioration of aging-induced insulin resistance after 55 weeks of feeding. They have shown that aging mice exhibit an increased level of homeostatic model assessment of insulin resistance (HOMA-IR) index coupled with a decline in glucose clearance in the OGTT assessments that is reversed by pomegranate flowers extract. From the mechanistic point of view, a number of experiments undertaken in vitro also supported the possible anti-inflammatory mechanism of action of the flowers extract. The study by Danesi and Ferguson (2017) employed the LPS-induced RAW264.7 cells where concentrations (10, 25, 50, 100 μg/ml) of pomegranate flowers extract (ethanol) have shown dosedependent inhibition on production of NO (IC50 value ¼ 31.8 μg/mL), PGE2 (IC50 value ¼ 54.5 μg/mL), IL-6 (IC50 value ¼ 48.7 μg/mL), IL-1β (IC50 value ¼ 71.3 μg/mL) and TNF-α (IC50 value ¼ 62.5 μg/mL) (Xu et al., 2017). It has also been shown that the phosphorylation of ERK1/2, p38, JNK and translocation of the NF-κB p65 subunit were inhibited suggesting the possible mechanism of action of the flower extract as anti-inflammatory agent. Readers should note that suppression of the NOS in macrophages, which is triggered by the NF-κB-mediated activation of the inflammatory cascade, is of benefit in diabetes, while the endothelial NOS that is downregulated under diabetic condition need to be augmented as demonstrated for pomegranate preparations. Anti-inflammatory effect in Chang liver cells has also been shown as evidenced from reduction in TNF-α and IL-8 expression while boosting antioxidant defense (Yan et al., 2017). Furthermore, the antiobesity potential of the flowers extract was evident in various cellular models including 3T3-L1 adipocytes where the level of resistin is suppressed (an effect which is also reported for ellagic acid) (MakinoWakagi et al., 2012); increase in PPAR-α and ACO in liver (Xu et al., 2009) and kidney (Huang et al., 2005b) cells. In view of the high level of tannins and other phenolic compounds in the flowers extract, the α-glucosidase activity (Li et al., 2005) is somehow expected and could contribute to the potential antidiabetic effects. Beyond the above-mentioned antidiabetic, antiobesity and antihyperlipidaemic effects, the flowers extracts have also been shown to possess numerous other pharmacological effects. For example, an improvement in memory that is shown to be deteriorated in experimental diabetes has been reported (Cambay et al., 2011). The wound healing effect of pomegranate has not been investigated but preliminary experiment on the flowers extract (200 mg/kg) have shown some promise in alloxan-induced diabetic rats (Pirbalouti et al., 2010). The reported weaker effect could be due to the extraction solvent being diethyl ether which is not optimum to extract the known highly polar polyphenolic compounds of the plant. 12.6.3.3 Seed oil and punicic acid Hontecillas et al. (2009) have shown that the predominant component of pomegranate seed oil, punicic acid (178), could induce a dose-dependent increase PPAR-α and PPAR-γ reporter activity in 3T3-L1 cells and can bind (although weakly) to the ligand-binding domain of

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human PPAR-γ. In a genetically obese db/db mice and a model of diet-induced obesity, the compound (178) has also been shown to improve glucose homeostasis and suppress inflammation after oral administration (Hontecillas et al., 2009). These include a decrease in fasting plasma glucose concentrations, improvement in glucose-metabolism, suppression of NF-κB activation, TNF-α expression and upregulation of PPAR-α- and PPAR-γ-responsive genes in skeletal muscle and adipose tissues. Another convincing data on the potential benefit of punicic acid (178) to ameliorate obesity and insulin resistance came from the study on the seed oil using HFD-induced obesity model in mice (Vroegrijk et al., 2011). It has been shown that consumption of pomegranate seed oil (1%) along with the HFD for 12 weeks could lower body weight with no effect on food intake or energy expenditure. The reduction in body fat mass coupled with enhancement of insulin sensitivity in peripheral tissues (e.g., muscles) but not in the liver was reported. As the seed oil did not affect the level of insulin or glucose concentrations in the blood (in this study), the potential mechanism appears to be linked to antiobesity effect and/or amelioration of insulin resistance. Another similar data on antiobesity and antidiabetic effect of the pomegranate seed oil came from the study by McFarlin et al. (2009) who used almost identical HFD-induced obesity model in CD-1 mice. Thy found that energy intake was not altered by pomegranate seed oil feeding but suppressive effect on leptin and enhancement of adiponectin levels were reported. Insulin sensitivity could also be improved by the seed oil, but in contradiction with the study by Vroegrijk et al. (2011), a reduced level of insulin was noted in CD-1 mice treated in this experiment. In a model of TNF-α-induced insulin resistance in 3T3-L1 adipocytes, punicic acid has been shown to improve glucose uptake, ROS accumulation, mitochondrial biogenesis and energetics in TNF-α treated cells. It also ameliorated the TNF-α-induced alterations in proteins associated with mitochondrial dynamics like mitochondrial fission 1 protein (FIS1) and mitochondrial dynamin Like GTPase (OPA1) (Anusree et al., 2015). TNF-α being one of the best characterized cytokine in mediating the obesity- or low-grade inflammation-induced insulin resistance, the observed effect in vitro is one possible mechanism of the health benefit by the seeds oil. Readers may thus generalize that the pomegranate seed oil and punicic acid (178) may remove insulin resistance by ameliorating the obesity or inflammation-induced loss of insulin sensitivity in peripheral tissues. Moreover, this effect is similar with the thiazolidinedione group of antidiabetic agents discussed in Chapter 5, but such drugs are associated with numerous undesirable side effects. As always, natural products especially those known to be employed as food and exhibit little or no side effect have tremendous potential to be develop further as a novel and safe therapeutic option. An investigation by Bassaganya-Riera et al. (2011) also corroborated the notion of anti-inflammatory mechanism of ameliorating insulin resistance by punicic acid (178) from their study in mice where intestinal inflammation was suppressed via activation of PPAR-γ. A further study by Anusree et al. (2014) showed that punicic acid (30 μM) can upregulate adiponectin secretion and GLUT4 expression and translocation in 3T3-L1 adipocytes in vitro. This effect was also coupled with suppression of ROS generation and increased glucose uptake. Their molecular modeling study also showed high binding affinity of punicic acid (178) to the PPAR-γ ligand binding domain which was supported by in vitro study to conclude punicic acid as a PPAR-γ agonist. Other studies showing the antidiabetic potential of the seeds oil in vivo include that by Mollazadeh et al. (2017) in STZ-induced diabetic rats and in vitro studies using high glucose-treated H9c2 cells. Their key finding was to show boosted antioxidant defenses at

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very low doses (0.36 and 0.72 mg/kg, p.o.). They have also shown hypoglycaemic effect and improved kidney function along with histological improvement of major organs such as the heart (Mollazadeh et al., 2016). On the other hand, an increase in the serum level of insulin and antioxidant status without alteration in blood glucose level has been reported by Nekooeian et al. (2014) for a fairly high dose in STZ-nicotinamide model. The increase in insulin level along with antioxidant defense (enzyme and antioxidant status) by the seed juice has also been reported by other authors in STZ-nicotinamide model (Aboonabi et al., 2014). The hypoglycaemic effect of the methanol extract of the seed (300 and 600 mg/kg, p.o.) has also been shown in STZ diabetic rats (Das et al., 2001). The seed oil did also show some characteristic features of the fruit extract and juices, though to a lesser extent, in boosting the expression of NOS (and nitrate accumulation) and improvement of arterial functions (relaxation and inflammation) in obese Zucker rats (de Nigris et al., 2007a). As with the fermented juice, the cold pressed seed oil of pomegranate could ameliorate the activities of COX and lipoxygenase suggesting possible anti-inflammatory activity (Schubert et al., 1999). In experimentallyinduced colon inflammation (2,4,6 trinitrobenzenesulfonic acid (TNBS) in rats, inhibitory effect on TNF-α-induced neutrophil ROS overproduction has also been demonstrated (Boussetta et al., 2009). The methanolic extracts of the seeds have also been shown to display hypoglycaemic effect in STZ-induced diabetes model in rats (Das et al., 2001). Readers should note that in STZ and other diabetic models where pancreatic β-cells are depleted, increasing the insulin level is a valid therapeutic approach but this depend on the extent of the surviving β-cells. On the other hand, a HFD-induced model raises the insulin level and a reduction in insulin level by therapeutic agent is expected as glycaemic control is established. Hence, some of the discrepancies in the effect of pomegranate preparations on insulin level may arise from the variability in the experimental models employed. The overall in vitro and in vivo data suggest that there is a clear therapeutic potential for pomegranate seed oil as antidiabetic agent and/or metabolic syndrome conditions. 12.6.3.4 Leaves Patel et al. (2014) presented by far the most compressive data on the antidiabetic potential of the pomegranate leaves. They have first extracted the plant material with methanol:water (70:30) from which the ethyl acetate fraction was taken for analysis. Their dose-dependent effect (50, 100 and 200 mg/kg) after 28 days of oral treatment showed hypoglycaemic effect in the OGTT and FBG level measurements. For the highest dose employed, a reduction in haemoglobin lipid peroxidation, improvement in lipid profile and antioxidant status (Table 12.2) have been shown. Intestinal glucose absorption and increase in glycogen content coupled with structural improvement of major organs have also been observed. The only parameter that was not altered was the level of insulin in the blood. Their in vitro enzyme inhibition assay also showed a pronounced α-glucosidase and some α-amylase inhibitory effect for the extract/fraction. The antidiabetic effect of the leaves has also been similarly studied in STZ-induced diabetic animal models at the same dose (200 mg/kg), where improvement in lipid profile was reported (Salwe et al., 2015). In a comparative analysis, the fruit peels extract appear to be more potent than the leaves extract (Salwe et al., 2015). In alloxan-induced diabetes model, the ethanolic extract of pomegranate leaves (500 mg/ kg, p.o.) administered for one week has been shown to lower blood glucose coupled with increase in glycogen content in the liver, cardiac, and skeletal muscle (Das and Barman, 2012).

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A reduced intestinal glucose absorption was also reported suggesting possible inhibition of carbohydrate digestive enzymes persistent to the known phenolic compositions that attributes to such mechanism. The reduction in the serum TC, TG, LDL and increase in serum HDL were also persistent with the antihyperlipidaemic activity as with its antidiabetic effects. Doses of 200 and 400 mg/kg of the aqueous and methanolic extracts of the leaves have also shown hypoglycaemic effect (FBC and OGTT) in alloxan-induced diabetes model; with the aqueous extract being more potent (Patil et al., 2013). The antilipase activity of the leaves extract has also been demonstrated both via in vitro enzyme assays and in normal mice receiving acute dose of 200–800 mg/kg (Yu et al., 2017).

12.7 Evidence of efficacy from clinical studies Pomegranate is one of the medicinal foods that benefited from the good number of clinical trials. Kerimi et al. (2017) have done two randomized, crossover, controlled trials in healthy subjects and compared the acute effect of pomegranate juice and a polyphenol-rich extract supplement on the bread-derived postprandial blood glucose concentration. Their double-blinded study for the supplements included each of 16 healthy volunteers. They found that pomegranate juice could display hypoglycaemic effect when compared to a control solution containing the equivalent amount of sugars. In contrast, the pomegranate supplement, or a solution containing the malic and citric acid components of the juice, was ineffective. The pomegranate polyphenol punicalagin (99/100) was also shown to be a very effective inhibitor of human α-amylase in vitro too, comparable to the drug acarbose. Neither the pomegranate extract nor the individual component polyphenols inhibited 14 C-D-glucose transport across differentiated Caco-2/TC7 cell monolayers, but they inhibited uptake of 14C-glucose into Xenopus oocytes expressing the human glucose transporter type 2 (GLUT2). Furthermore, some of the predicted pomegranate gut microbiota metabolites modulated 14C-D-glucose and 14C-deoxy-D-glucose uptake into hepatic HepG2 cells. They concluded that pomegranate polyphenols, when present in a beverage but not in a supplement, can reduce the postprandial glycaemic response of bread, whereas microbial metabolites from pomegranate polyphenols exhibit the potential to further modulate sugar metabolism much later in the postprandial period. The data by Huang et al. (2017) also supported this observation where lack of efficacy of pomegranate supplementation for glucose management, insulin levels and sensitivity were reported from a systematic review and meta-analysis. Their study was based on sixteen eligible trials with 538 subjects but this could be equally argued from other meta-analysis studies and primary data as discussed below. In contrast to this assessment, in an acute study using 40 male and 45 female diabetic patients where pomegranate juice (1.5 ml/kg juice) was administered orally, a reduced FBC, increased β-cell function, and decreased insulin resistance among T2D participants were observed three hours after juice administration (Banihani et al., 2014). In a randomized double-blind clinical trial study by Faghihimani et al. (2016) involving 80 patients (28 men) with T2D, capsules containing 1000 mg twice daily (2000 mg PSO) were given for eight weeks. Of the favorable responses recorded were FBG, insulin, HbA1c, ALT, and HOMA.

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A randomized clinical trial performed on 60, 40–65 years old diabetic patients by Sohrab et al. (2017) on the other hand was on 200 ml of pomegranate juice after daily intake for six weeks which showed an outcome of reduced oxidized LDL and anti-oxidized LDL antibodies; while the total serum antioxidant capacity and arylesterase activity of PON were increased. In a quasi-experiment trial by Shishehbor et al. (2016) where 40 T2D diabetic patients were asked to consume 50 g of the juice daily for four weeks, an increase in both TC and HDLC was noted without significant change in serum TG, LDL-C, FBC, and BP. Even though IL-6 was suppressed, the level of TNF-α and C-reactive protein (CRP) level were not altered. Overall, the antioxidant index was the most visible improvement observed. Sohrab et al. (2015) have also done a 12-week intervention study based on a randomized, double-blind, placebo-controlled trial using 44 T2D patients (age range 56
6.8 years) receiving 250 ml/ day of pomegranate juice (n ¼ 22) and placebo (n ¼ 22). The total antioxidant capacity and malondialdehyde (MDA) level could be decreased by pomegranate juice but the AGE markers including carboxy methyl lysine (CML) and pentosidine were not affected. Furthermore, Gonzalez-Ortiz et al. (2011) tried to assess the beneficial effects of pomegranate juice on insulin secretion and sensitivity in patients with obesity. They used a randomized, doubleblind, placebo-controlled clinical trial but only on 20 obese subjects. The patient group (10) who received 120 ml of pomegranate juice for one month did not show change in insulin secretion or insulin sensitivity but the increased weight and adiposity were suppressed. Many clinical trials on the polyphenol-rich juices like grapes and pomegranate have shown to have beneficial effect in improving endothelial dysfunction under clinical conditions (Kelishadi et al., 2011). In this regard, more specific outcomes from pomegranate preparations as antihypertensive and cardioprotective effects have also been done and are outlined in this section. After administration of two capsules of pomegranate polyphenols (POMx, 1 capsule ¼ 753 mg polyphenols) daily for four weeks, Basu et al. (2013) reported no significant effect on body weight and blood pressure (BP), glucose and lipids both in healthy controls and in T2D patients. Lipid oxidation and inflammation, oxidized LDL and serum CRP did not differ at week-4 but MDA level was reduced. The number of subjects and randomization is however not clear. Mathew et al. (2012) also conducted a small randomized, controlled crossover trial with nineteen young, healthy men, consuming drinks containing pomegranate extract. They have shown that the consumption of a single drink did not decrease postprandial plasma TG concentrations, but suppressed the postprandial increase in systolic BP following the high-fat meal. The study by Tsang et al. (2012) on pomegranate juice included only twelve males and sixteen females in a randomized, placebo-controlled cross-over study (BMI: 26.77 and mean age of 50.4 years). They have shown that consumption of 500 ml of pomegranate juice (vs 500 ml of a placebo drink) for four weeks could reduce cortisol/cortisone ratios in urine and saliva, systolic BP, fasting plasma insulin and HOMA-IR index. This data should be seen however as effect on normal non-diabetic subjects. Parsaeyan et al. (2012) assessed the potential effect of pomegranate juice on the activity of the HDL-associated enzyme, PON-1, that prevents lipoprotein oxidation. In fifty patients with T2D who consumed 200 ml of pomegranate daily for a period of six weeks, the levels of FBG, TC, LDL-C and MDA were shown to be decreased. This was associated with increased PON and arylesterase activity of PON-1 but no change in HDL-C level was reported. Readers should bear in mind that the antioxidant effect of HDL-associated PON-1 has been shown to suppress diabetes development and stimulate insulin release from β-cells

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(Koren-Gluzer et al., 2011). Another study linked to PON-1 activity of pomegranate juice (prepared from whole fruit) was conducted by Fuhrman et al. (2010) where oxidative stress under diabetic condition can impair the binding of this enzyme to HDL while the juice polyphenols increased this binding. A further clinical-related study on this enzyme was by Rock et al. (2008) who correlate pomegranate juice consumption (50 ml/day for four weeks) could increase the activities of HDL-associated PON1 arylesterase, paraoxonase, and lactonases. The binding of PON-1 protein to HDL was shown to be enhanced by the juice. Other studies on the polyphenol-rich extracts including those from pomegranate have shown a reduction in LPO in patients with T2D after chronic treatment (Fenercioglu et al., 2010). The study by Esmaillzadeh et al. (2006) also assessed the effects of concentrated pomegranate juice on the lipid profiles in diabetic patients with hyperlipidaemia. Their pilot study involved 22 diabetic Iranian diabetic patients who received concentrated pomegranate juice for eight weeks. They reported a reductions in TC, LDL-C, LDL-C:HDL-C, and TC:HDL-C ratio. This effect was however without a change in the serum level of TG and HDL-C concentrations. Even though one may question the methodology employed, some beneficial effect consistent with other studies have been reported. Their previous study (2004) on concentrated pomegranate juice (40 g/day for eight weeks) in 22 otherwise healthy diabetic patients also reported similar results. The small trial by Rosenblat et al. (2006) also included the participation of ten healthy subjects (controls) and 10 T2D patients who consumed the juice (50 ml per day for three months). They noted that the supplementation could not suppress serum glucose, cholesterol and TG levels. The positive effect was however on serum lipid peroxides and TBARS levels which was reduced by the treatment. The serum thiol and PON-1 activity was also shown to be increased. A reduced cellular peroxides (by 71%), and increased GSH levels (by 141%) in macrophages of patients with T2D was recorded for the juice. The cellular uptake of oxidized-LDL was suppressed by the treatment. With respect to anti-inflammatory effect, Sohrab et al. (2014) studied the relationship between pomegranate juice consumption and inflammatory markers in patients with T2D. Their randomized, double-blind clinical trial study on 44 patients with T2D (40–65 years old) showed that subjects receiving 250 ml/day juice for 12 weeks have decreased levels of high sensitive CRP (hsCRP) and IL-6. Aviram and Dornfeld (2001) have conducted a number of clinical studies over several years. Their study in 2001 was on seven hypertensive males and three females with mean BP levels of 155
7/83
7 mmHg. Only two were diabetic and two were hyperlipidaemic. In their assessment of serum angiotensin II converting enzyme activity after two weeks of pomegranate juice consumption (50 ml contained 1.5 mmol of total polyphenols per day), they noted a reduced enzyme activity in seven out of the ten hypertensive patients. Their study in 2004 (Aviram et al., 2004) with 19 patients who were randomized to either pomegranate (50 ml of juice per day which contain 1.5 mmoles of total polyphenols for a period of one year, and five out of them agreed to continue for up to 3 years) or placebo group. The key outcome was that systolic BP was reduced after one year of the juice consumption coupled with intima-media thickness reduction by up to 30%, increased serum PON-1 activity by 83%, whereas serum LDL basal oxidative state and LDL susceptibility to copper ion-induced oxidation were both reduced, by 90% and 59%, respectively. The study by Lynn et al. (2012) also assessed the potential effect of the pomegranate juice on BP in healthy young and middle-aged men and women. The 51 healthy adults (30–50 years) in this study consumed

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330 ml/day of pomegranate juice or control drink for four weeks. A fall in BP (systolic, diastolic or mean arterial) was recorded but the BP was not correlated with changes in the serum level of ACE suggesting a different mechanism of action. The clinical trial by Asgary et al. (2013) was on small group of thirteen hypertensive men aged 39–68 years receiving pomegranate juice (150 ml/day) following a 12 h fast. They reported a decrease both in systolic and diastolic BP without any effect on circulating levels of CRP, intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM1), E-selectin, and IL-6. The study being a single arm and rather acute study, only effects after 4–6 h or administration of the juice was measured. Their following study (Asgary et al., 2014) on twenty-one hypertensive patients (aged 30–67 years) receiving pomegranate juice (150 ml/ day in a single occasion between lunch and dinner; n ¼ 11) or the same amount of water (n ¼ 10) was however extended for a period of two weeks. In this case, in addition to the systolic and diastolic BP reduction, the serum levels of VCAM-1 was also suppressed. The contradiction was however with the elevated level of E-selectin observed while ICAM-1, hsCRP, lipid profile parameters, apolipoproteins and IL-6 were not altered. The clinical trial by Sumner et al. (2005) also examined the effect of pomegranate on the myocardial perfusion in patients with coronary heart disease. In a randomized trial using 45 patients with stable coronary heart disease, consuming 240 ml/day of pomegranate juice (POM Wonderful, Los Angeles, California) for three months has shown a reduction in stress-induced ischemia without a change in blood sugar, HbA1c, weight, or BP in either group. The same dose of 240 ml/day of pomegranate juice were also employed by Davidson et al. (2009) to study the effect of pomegranate juice on carotid intima-media thickness in men and women at moderate risk for coronary heart disease. The number of men (45 to 74 years old) and women (55–74 years old) study subjects were however higher with 146 in the treatment and 143 in the placebo beverage. They did not observe difference in anterior and posterior carotid intima-media thickness progression rate but suggested potential benefit in subjects with increased oxidative stress and disturbances in the TG-rich lipoprotein/HDL axis. On the other hand, Haghighian et al. (2016) employed thirty eight obese women (BMI 30–35) with dyslipidaemia grouped those receiving 500 mg pomegranate peel extract (n ¼ 19) or placebo (n ¼ 19) daily for eitht weeks. They reported a decrease systolic but not diastolic BP. While the serum level of TC was reduced, other parameters such as HDLdC, and BMI were not affected. Sahebkar et al. (2016a, 2016b and 2017) have done three meta-analysis study on pomegranate juice. They concluded that pomegranate juice consumption could benefits in BP management; there was no indication of benefit from assessment of the CRP levels, and the available randomized clinical trial data is not in support of pomegranate consumption improving lipid profile in human. The suppression of systolic BP appears to be consistent while diastolic pressure could be suppressed at very high doses. In a systematic review of randomized clinical trials, the conclusion made by Gbinigie et al. (2017) was that the evidence for pomegranate supplementation to improve BP management is weak. The striking contrast in some studies showing a reduction in BP while some showed no effect and variability in methodology and quality of data often associated with small subject group size have been highlighted. While this comment is generally true to the vast array of clinical trials on medicinal foods and other natural products, meta-analysis of randomized controlled trials on the effect of pomegranate juice on BP has proven the consistent benefits of pomegranate juice consumption on BP

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(Sahebkar et al., 2017). One must therefore conclude that the antidiabetic and antiobesity potential of pomegranate in humans certainly deserve more clinical trial studies. A standardized methodology and plant material with known ingredients of defined quantity would lower the discrepancies between studies. While there is undoubtedly huge benefit of taking pomegranate as food, the question of it being used as a replacement to the currently useful drugs is not answered. The large number of animal studies showing the antiobesity, lipid lowering and antioxidant effect even under clinical trial cases are promising reports in favor of continued research on the plant.

12.8 Pharmacokinetics and toxicological perspectives Several toxicological studies have been undertaken as part of the in vivo animal studies. For example, repeated oral administration of high doses of the pomegranate ellagitannin (23) and punicalagin (99/100) in rats for 37 days were reported to be not toxic (Cerda et al., 2003). The authors have detected five punicalagin-related metabolites in liver and kidney, that is, two ellagic acid derivatives, gallagic acid (4), 3,8-dihydroxy-6H-dibenzo[b,d]pyran6-one glucuronide (which is a glucuronide of urolithin A, 200), and 3,8,10-trihydroxy-6H-dibenzo[b,d]pyran-6-one (207). Meerts et al. (2009) have also undertaken in vitro and in vivo experiments addressing the acute and chronic (28-day toxicity) administration of pomegranate seed oil. They have even included doses of up to 14,214 mg/kg and did not observe any effect up to 4.3 g/kg but excessively high doses that are not used in the biological assay could increase hepatic enzyme activities in plasma (AST, ALP and ALT) or liver-tobody weight ratios. For the leaves extract, Patel et al. (2014) did also use a massive dose of 5000 mg/kg and observed no lethality or any toxic reaction in animals at a single large dose within the two weeks observation period. Up to 2000 mg/kg body weight of the leaves and fruit peel of the plant have also shown to be devoid of toxicity in acute toxicity study (Salwe et al., 2015). Overall, considering that pomegranate is a common food product consumed by mankind for thousands of years, acute toxicity is not a general concern at least for the edible parts (fruits). Seeram et al. (2006, 2004) have studied the metabolites of ellagitannin following administration of pomegranate juice in human subjects. They have reported that ellagic acid (23) could be detected in the plasma of all subjects and the elimination half-life was 0.71 +/ 0.08 h. The free and conjugated metabolites of ellagic acid detected in plasma and urine were dimethylellagic acid glucuronide (DMEAG) and hydroxy-6H-benzopyran-6-one derivatives (urolithins, Fig. 12.22). The detection of DMEAG was regarded as a biomarker pomegranate juice intake as it is detected early. The glucoronide of urolithin A (200) and urolithin B (202) were also detected in the urine but the health benefit of the juice was claimed to be due to urolithins that are formed by intestinal bacteria. More evidence on the potential role of uroithins also came from another human studies by Cerda´ et al. (2004) where the in vitro transformation of ellagitannins to hydroxy-6H-dibenzopyran-6-one derivatives by colonic microflora of healthy humans has been shown. The metabolite though not identified has been reported to reach up to 18.6 μM in plasma, suggesting the possible generation of bioactive metabolite by intestinal bacteria. Various other studies (Mertens-Talcott et al., 2006;

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Toma´s-Barbera´n et al., 2014) including in humans have identified the urolithins that might have biological significance as active principles of pomegranate elagitannins. The antioxidant effect of these compounds as potential active principle following transformation of pomegranate ellagitannins by intestinal bacterial have also been shown (Bialonska et al., 2009). As the compounds lose their polyphenolic nature while being transformed to a smaller urolithin skeleton, however, they display lower level of in vitro antioxidant effects (Cerda´ et al., 2004). Urolithin A glucuronide and its aglycone urolithin A (200) have also been shown to ameliorate the TNF-α-induced inflammation and associated molecular markers (e.g., IL-8 expression and endothelial cell migration) in human aortic endothelial cells (GimenezBastida et al., 2012). The effect of urolithin A (200) in attenuating the oxidized-LDL-induced endothelial dysfunction via mechanisms including modulation of microRNA-27 and ERK/ PPAR-γ pathway further put the link between polyphenols and their active metabolites as pharmacologically active principles of pomegranate (Han et al., 2016). The evidence from the study by Tang et al. (2017) also documented urolithin A (200) alleviating the myocardial ischaemia/reperfusion injury both in vivo and in vivo via PI3K/Akt pathway as shown for the pomegranate preparations. As a cardioprotective pathway induced by exercise and other stimuli, the PI3K/Akt signaling pathway also appear to be stimulated by pomegranate metabolites. The scheme of metabolites formation following tannins intake is depicted in Fig. 12.22. Patients taking antidiabetic drugs should also take caution about the potential drug interaction with medicinal plants and/or certain food ingredients. For example, pomegranate has been shown to reduce the metformin maximum plasma concentration (Awad et al., 2016). The complete inhibition of human CYP2C9 by pomegranate juice and increased tolbutamide bioavailability in rats through action on intestinal metabolism has also been reported (Nagata et al., 2007).

12.9 General summary and conclusion The thousands of years of historical perspective in the medicinal and nutritional value of pomegranate appear to be substantiated by numerous in vitro, in vivo and clinical evidences. With respect to the potential antidiabetic and benefit for metabolic syndrome, the following conclusion could be made: • The in vitro, in vivo and clinical evidences suggest that pomegranate fruits have antioxidant effect. Irrespective of the pathology, this general mechanism is of benefit to ameliorate a range of disease conditions but primarily diabetes and/or metabolic syndrome through the various mechanisms highlighted in this book. The polyphenolic components primarily the gallate- and elligitannin-based compounds appear to account to this pharmacology. • The anti-inflammatory mechanism in in vitro and animal models is conclusive but this needs further evidence in clinical conditions given the data is patchy and in some cases even contradictory. • The antihypertensive effect of pomegranate has been extensively researched and several mechanism including inhibition of ACE and restoring amelioration of the diabetesinduced endothelial dysfunction were postulated. The clinical evidence is however D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

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variable with many studies showing benefit while others show no effect and hence more clinical data is required. • The antiobesity potential in animal models and particularly in adipocytes in culture were demonstrated but once again the clinical evidence is not clear with variable effects, some showing weight lowering while others not. This once again could be due to discrepancies on the plant materials used with respect to variability in active components and doses. • Lipid lowering effect has been demonstrated in the various model and subject to further clinical trial, the evidence for continued use of pomegranate is justifiable. • One unique mechanism that has been specifically highlighted for pomegranate in all models (in vitro, in vivo and clinical trials) is the universal effect on upregulating PON and hence deserves a little more analysis as shown below. The paraoxonases are group of enzymes represented by PON-1, PON-2 and PON-3. In addition to the organophosphatase activity, the hydrolytic activity of PONs include actonase activity (e.g., against homocysteine, thiolactone) and arylesterase activity (e.g., against phenyl acetate), organophosphatase activity (e.g., against paraoxon). PON-1 is known to display all these enzymatic activities while PON-2 and PON-3 display only lactonase activity although PON3 is likely to have some arylesterase effects. PON-1 is predominantly expressed and secreted to the blood stream by the liver but other organs including the lungs, heart, brain, small intestine, and kidney are known to produce it. On the other hand PON2 is not released to the blood but localized within the intracellular domain but most probably the endoplasmic reticulum and nuclear membranes. Hence, unlike PON-1 (and PON-3), PON-2 is not associated with HDL or LDL. PON-2 is produced by almost all human tissues but its role in macrophage and pathological role in atherosclerosis and lipid metabolism have been extensively studded. PON3 which is expressed primarily in the liver, and at lower level in other tissues such as kidney and the gastrointestinal tract has also antioxidant and antiatherosclerotic activities. PON1 activity in the blood is inversely proportional to the risk of atherosclerosis and related diseases. In diabetes and associated cardiovascular complications, PON-1 is dissociated from HDL in blood and became inactive. Its antiatherogenic effect of summarized as follow: • PON-1 in the antioxidant system is to protect LDL and HDL against oxidative processes. Hence, it prevents the formation of atherogenic oxidized-LDL which is atherogenic. • PON-1 hydrolyze the oxidized phospholipids and hydroxides of cholesteryl linoleate contained in oxidized-LDL molecules. The phagocytosis of oxidized LDL by macrophages transform them into fat-laden foam cells and initiation of atherosclerosis is thus tackled by PON-1. • PON-1 stimulates cholesterol efflux from macrophages through HDL. • PON-1 also suppresses the differentiation of monocytes into macrophages As with PON-1, PON-2 reduces lipid peroxides in macrophages and inhibits LDL oxidation. This has protective effect in macrophages as antioxidant mechanism and hence its concentration increases under oxidative stress. For detailed pharmacology of PON-1 and PON-2, readers are directed to review articles in the field (Kowalska et al., 2015; Shih and Lusis, 2009). The antioxidant effect of pomegranate and perhaps its beneficial effect in lipid metabolism as in metabolic syndrome cases could be associated to its upregulation of PON-1 and PON-3 activity.

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Most of the work on pomegranate pharmacology have focussed on the gallate and ellagitannin-based compounds such as tannins. The flavonoids including the major colour component of the pericarp, anthocyanins, are not at the forefront of these studies. Readers should bear in mind that these compounds are potent antidiabetic agents and review article showing comprehensive data on anthocyanins is available (Belwal et al., 2017). Pomegranate anthocyanins are also known to be transformed in the gut mainly due to pH changes (PerezVicente et al., 2002), and their real contribution in the pharmacology of the fruit preparations needs more assessment. The pomegranate plant overall has tremendous potential as antidiabetic agent that needs further research especially in the clinical trial area which has been almost exclusively done on the fruit juice preparations. The available data is clearly in support of polypharmacology principle of general effects by polyphenols through multi drug! multi target ! complex disease(s) principle (Habtemariam, 2017). Overall, the current available data is not in support of pomegranate to be used as a replacement to metformin or other antidiabetic agent but its superfood status as value adding nutritional agent with multiple benefits in diabetic and/or metabolic syndrome conditions has been demonstrated.

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The roles of PON1 and PON2 in cardiovascular disease and innate immunity. Curr. Opin. Lipidol. 20 (4), 288–292. Shiner, M., Fuhrman, B., Aviram, M., 2007. Macrophage paraoxonase 2 (PON2) expression is up-regulated by pomegranate juice phenolic anti-oxidants via PPAR gamma and AP-1 pathway activation. Atherosclerosis 195, 313–321. Shishehbor, F., Mohammad, S.M., Zarei, M., Saki, A., Zakerkish, M., Shirani, F., Zare, M., 2016. Effects of concentrated pomegranate juice on subclinical inflammation and cardiometabolic risk factors for Type 2 diabetes: a quasiexperimental study. Int. J. Endocrinol. Metab.. 14(1)e33835. Shukla, M., Gupta, K., Rasheed, Z., Khan, K.A., Haqqi, T.M., 2008a. Consumption of hydrolyzable tannins-rich pomegranate extract suppresses inflammation and joint damage in rheumatoid arthritis. Nutrition 24, 733–743. Shukla, M., Gupta, K., Rasheed, Z., Khan, K.A., Haqqi, T.M., 2008b. Bioavailable constituents/metabolites of pomegranate (Punica granatum L) preferentially inhibit COX2 activity ex vivo and IL-1beta-induced PGE2 production in human chondrocytes in vitro. J. Inflamm. (Lond) 13, 5–9. Singh, B., Arora, S., Goswami, B., Mallika, V., 2009. Metabolic syndrome: a review of emerging markers and management. Diabetes Met. Syndrome: Clin. Res. Rev. 3 (4), 240–254.

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Verardo, V., Garcia-Salas, P., Baldi, E., Segura-Carretero, A., Caboni, M.F., 2014. Pomegranate seeds as a source of nutraceutical oil naturally rich in bioactive lipids Research article. Food Res. Int. 65 (Part C), 445–452. Vilahur, G., Padro´, T., Casanı´, L., Mendieta, G., Lo´pez, J.A., Streitenberger, S., Badimon, L., 2015. Polyphenol-enriched diet prevents coronary endothelial dysfunction by activating the Akt/eNOS pathway. Rev. Esp. Cardiol. (Engl. Ed.) 68 (3), 216–225. Vroegrijk, I.O., van Diepen, J.A., van den Berg, S., Westbroek, I., Keizer, H., Gambelli, L., et al., 2011. Pomegranate seed oil, a rich source of punicic acid, prevents diet-induced obesity and insulin resistance in mice. Food Chem. Toxicol. 49, 1426–1430. Wang, R.F., Xie, W.D., Zhang, Z., Xing, D.M., Ding, Y., Wang, W., et al., 2004. Bioactive compounds from the seeds of Punica granatum (Pomegranate). J. Nat. Prod. 67, 2096–2098. Wang, R., Wei, W., Wang, L., Liu, R., Yi, D., Du, L., 2006. Constituents of the flowers of Punica granatum. Fitoterapia 77, 534–537. Wang, J., Rong, X., Um, I.S., Yamahara, J., Li, Y., 2012. 55-Week treatment of mice with the unani and ayurvedic medicine pomegranate flower ameliorates ageing-associated insulin resistance and skin abnormalities. Evid. Based Complement. Alternat. Med. 2012, 350125. Wang, J.Y., Zhu, C., Qian, T.W., Guo, H., Wang, D.D., Zhang, F., Yin, X., 2015. Extracts of black bean peel and pomegranate peel ameliorate oxidative stress-induced hyperglycemia in mice. Exp. Ther. Med. 9 (1), 43–48. Ward, C., 2010. Pomegranates in eastern Mediterranean contexts during the Late Bronze Age. World Archaeol. 34, 529–541. Xie, Y., Morikawa, T., Ninomiya, K., Imura, K., Muraoka, O., Yuan, D., et al., 2008. Medicinal flowers. XXIII. New taraxastane-type triterpene, punicanolic acid, with tumor necrosis factor-a inhibitory activity from the flowers of Punica granatum. Chem. Pharm. Bull. 56, 1628–1631. Xu, K.Z., Zhu, C., Kim, M.S., Yamahara, J., Li, Y., 2009. Pomegranate flower ameliorates fatty liver in an animal model of type 2 diabetes and obesity. J. Ethnopharmacol. 123, 280–287. Xu, J., Zhao, Y., Aisa, H.A., 2017. Anti-inflammatory effect of pomegranate flower in lipopolysaccharide (LPS)stimulated RAW264.7 macrophages. Pharm. Biol. 55 (1), 2095–2101. Yan, D., Wei, Y.Y., Li, X.M., Sun, X.C., Wang, Z., Aisa, H.A., 2017. PFP alleviates nonalcoholic steatohepatitis fatty liver in both Apo E/ mice and Changliver cell[S]. Am. J. Transl. Res. 9 (6), 3073–3083. Yilmaz, B., Ustam, C., 2013. Ellagic acid-induced endothelium-dependent and endothelium-independent vasorelaxation in rat thoracic aortic rings and the underlying mechanism. Phytother. Res. 27, 285–289. Yoshimura, Y., Nishii, S., Zaima, N., Moriyama, T., Kawamura, Y., 2013. Ellagic acid improves hepatic steatosis and serum lipid composition through reduction of serum resistin levels and transcriptional activation of hepatic para in obese, diabetic KK-A(y) mice. Biochem. Biophys. Res. Commun. 434 (3), 486–491. Yu, X., Wang, X.-P., Lei, F., Jiang, J.-F., Du, L.-J., 2017. Pomegranate leaf attenuates lipid absorption in the small intestine in hyperlipidemic mice by inhibiting lipase activity. Chin. J. Nat. Med. 15 (10), 732–739. Yuan, T., Ding, Y., Wan, C., Li, L., Xu, J., Liu, K., et al., 2012. Antidiabetic ellagitannins from pomegranate flowers: inhibition of α-glucosidase and lipogenic geneexpression. Org. Lett. 14, 5358–5361. Yuan, T., Wan, C., Ma, H., Seeram, N.P., 2013. New phenolics from the flowers of Punica granatum and their in vitro α-glucosidase inhibitory activities. Planta Med. 79, 1674–1679. Yusuph, M., Mann, J., 1997. A triglyceride from Punica granatum. Phytochemistry 44, 1391–1392. Zhao, X., Yuan, Z., Fang, Y., Yin, Y., Feng, L., 2014. Flavonols and flavones 70 changes in pomegranate (Punica granatum L.) fruit peel during fruit development. J. Agric. Sci. Technol. 16, 1649–1659.

Further reading Aviram, M., Dornfeld, L., Rosenblat, M., Volkova, N., Kaplan, M., Coleman, R., et al., 2000. Pomegranate juice consumption reduces oxidative stress, atherogenic modifications to LDL, and platelet aggregation, studies in humans and in atherosclerotic apolipoprotein E-deficient mice. Am. J. Clin. Nutr. 71, 1062–1076. Esmaillzadeh, A., Tahbaz, F., Gaieni, I., Alavi-Majd, H., Azadbakht, L., 2004. Concentrated pomegranate juice improves lipid profiles in diabetic patients with hyperlipidemia. J. Med. Food 7 (3), 305–308. He, L., Xu, H.G., Liu, X., He, W.H., Yuan, F., Hou, Z.Q., et al., 2011. Identification of phenolic compounds from pomegranate (Punica granatum L.) seed residues and investigation into their antioxidant capacities by HPLC-ABTS(+) assay. Food Res. Int. 44, 1161–1167.

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Lansky, E.P., Newman, R.A., 2007. Punica granatum (pomegranate) and its potential for prevention and treatment of inflammation and cancer. J. Ethnopharmacol. 109, 177–206. Linnaeus, C., 1753. Species plantarum. vol. I. Salvius, Stockholm, Sweden, p. 472. Seeram, N.P., Lee, R., Heber, D., 2004. Bioavailability of ellagic acid in human plasma after consumption of ellagitannins from pomegranate (Punica granatum L.) juice. Clin. Chim. Acta 348 (1–2), 63–68. Seeram, N.P., Zhang, Y., McKeever, R., Henning, S.M., Lee, R.P., Suchard, M.A., et al., 2008. Pomegranate juice and extracts provide similar levels of plasma and urinary ellagitannin metabolites in human subjects. J. Med. Food 11, 390–394.

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C H A P T E R

13 The chemical and pharmacological basis of prickly pear cactus (Opuntia species) as potential therapy for type 2 diabetes and obesity O U T L I N E 13.1 Introduction

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13.2 Taxonomic and botanical perspectives

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13.3 Common uses of prickly pear 13.3.1 Food production 13.3.2 Traditional medicine 13.3.3 Food supplements 13.3.4 Other uses

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13.4 The chemistry of cactus pears 13.4.1 Betalains 13.4.2 Polyphenols 13.4.3 Terpenoids composition 13.4.4 Seeds oil 13.4.5 Polysaccharides components

443 444 445 449 449 450

13.5 Antidiabetic, antiobesity, and lipid lowering effects of prickly pears

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Medicinal Foods as Potential Therapies for Type-2 Diabetes and Associated Diseases https://doi.org/10.1016/B978-0-08-102922-0.00013-4

13.5.1 Antidiabetic effect demonstrated through animal studies 451 13.5.2 Antiobesity and lipid lowering potential 453 13.5.3 Some common mechanisms of antidiabetic, lipid lowering, and antiobesity effects 455 13.6 Active principles of prickly pear 13.6.1 Polysaccharides 13.6.2 Cactus pear pigments 13.6.3 Other compounds

458 458 459 462

13.7 Diuretic effects

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13.8 Human studies on prickly pears

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13.9 General summary and conclusions 465 References

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Further reading

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# 2019 Elsevier Ltd. All rights reserved.

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13.1 Introduction Cactus pear or prickly pear refers to the genus Opuntia of the Cactaceae family. The genus is known to be represented by over 200 species. Cactus has a wide distribution throughout the drier regions of the world including the Americas, the Mediterranean, Europe, Asia, Africa, and Australia regions. Mexico is known for its diverse flora of this genus with over 126 species reported (Reyes-Ag€ uero and Aguirre Rivera, 2011) of which about 78 species are native. Many species are domesticated for their uses with Opuntia ficus-indica being the most common both in Mexico and many other countries. In fact the ‘prickly pear’ in many literature is used to refer to this species (O. ficus-indica) which is also known by its other common name, the Indian fig-opuntia. In the Mediterranean region such as Spain, distribution along the coast lines, Andalucia, Murcia, the Balearic and Canary Islands and many areas from the sunny slope to roadsides and abandoned fields have been reported (Andreu et al., 2018). The distribution of the genus is also outlined by DeFelice (2004). Commercial production in the native and other countries is mainly based on domesticated O. ficus-indica and some insight about the domestication process of this plant has been illustrated by Griffith (2004). The plant is regarded as extremely efficient in converting water into biomass making it ideal commercial plant to grow in arid environments. The success of this plant in its world-wide distribution is also based on its efficient vegetative propagative ability that makes it as a noxious weed in some places. According to Griffith (2004) and references cited there in, the use of Opuntia as human food goes back by at least some 9000 to 12,000 years.

13.2 Taxonomic and botanical perspectives Native to the New World of South and North Americas, the genus Opuntia Mill. belongs to the Cactaceae family. According to the Kew Science Plants of the World database (KewPOTW, 2018a), the natural distribution is said to be throughout the Americas from British Columbia to the Strait of Magellan while the long list of countries where it has been introduced include as follow: Albania, Algeria, Andaman Is., Angola, Ascension, Austria, Bahamas, Baleares, Bangladesh, Benin, Bermuda, Bulgaria, Canary Is., Cape Provinces, Cape Verde, Cayman Is., China South-Central, China Southeast, Corse, Czechoslovakia, East Aegean Is., Eritrea, Ethiopia, Fiji, France, Free State, Gambia, Great Britain, Greece, Guinea, Gulf of Guinea Is., Hainan, India, Italy, Kenya, Korea, Kriti, KwaZulu-Natal, Lesotho, Libya, Madeira, Mauritius, Morocco, Namibia, Netherlands, New Caledonia, Nicobar Is., Northern Provinces, Portugal, Rodrigues, Rwanda, Reunion, Sardegna, Senegal, Sicilia, Somalia, South China Sea, Spain, St. Helena, Sudan, Swaziland, Switzerland, Tanzania, Tunisia, TurksCaicos Is., West Himalaya, Western Sahara, Yugoslavia, and Zaı¨re. There are at least 123 accepted species of the genus Opuntia. The most common Opuntia being O. ficus-indica (L.) Mill., its botanical description, according to the Flora of Tropical east Africa, is as follow (Kew-PTOW, 2018b): Habit—Shrubby or arborescent, up to 4(
5) m tall, often with a cylindrical trunk. Spines—Joints elliptic to narrowly obovate, flattened, often 30–40 cm long, 15–20 cm broad, 1–1.5 cm thick, greyish-green; glochids yellow, deciduous; spines usually none, sometimes 1 or more, up to 1.5 cm long, bristle-like. D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

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Leaves—Leaves subulate, 3–4 mm long, early deciduous. Flowers—Flowers 5–8 cm in diameter; perianth spreading, yellow or orange, longer than the style and stamens. Ovary—Ovary cylindrical, 3.5–5 cm long, with many areoles, the upper bearing bristles up to 1.5 cm long. Fruits—Fruit ellipsoidal or obovoid, 5–9 cm long, 3–6 cm in diameter, variable in colour, with edible pulp. Habitat—Roadsides and by rivers; grasslands on pumice dust. Distribution—Presumably native to America but not known wild there widely cultivated and naturalized around the Mediterranean Sea, the Red Sea, in Africa and Australia. Cultivated in Kenya. The naming of the most popular cactus pear as Indian fig is intriguing. Fig belongs to the genus Ficus and the Ficus benghalensis L. (India?) does not resemble cactus in any form. As the plant was originally called Ficus indica, the name O. ficus-indica must have been a revision by botanists such as the Linnaeus as this taxonomic naming was known to be recognized by the beginning of the 16th century (Donkin, 1977). Images of some common Opuntia species are exhibited in Figs 13.1–13.9, highlighting the edible leaves and fruits. The cactus exhibit the best exemplary adaptation features of plants occupying the desert or semidesert environment. These succulent plants minimize water loss by having thick, waxy FIG. 13.1

Opuntia dillenii (Ker Gawl.) Haw. Native to native range is Mexico and S. Jamaica. Image of the vegetative part (A) and fruiting bodies (B) were taken from a plant growing in Somaliland. Courtesy: Dr Helen Pickering, Kew Botanical Garden, United Kingdom.

D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

FIG. 13.2 Opuntia ficus-indica (L.) Mill. Vegetative parts of the plant (A) and the edible young cladodes (also called ‘nopal’) (B) are shown. Image taken from Kew Botanical Garden, United Kingdom.

FIG. 13.3 Opuntia ficus-indica (L.) Mill. (A) Floral and (B) fruiting bodies. Courtesy of Wikipedia (https://en.wikipedia. org/wiki/Opuntia_ficus-indica).

13.2 Taxonomic and botanical perspectives

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FIG. 13.4 Opuntia lasiacantha Pfeiff. Native to Mexico is a source of edible prickly pear. Image taken from Kew Botanical Garden, United Kingdom.

FIG. 13.5 Opuntia macrocentra Engelm. Native to the United States and Mexico and introduced to the Canary Islands. Image taken from Kew Botanical Garden, United Kingdom.

cuticle and they also have a shallow root system to maximize water uptake during the short or sporadic rainy season. This level of efficiency in water absorption and storage has been well studied and over 80% of the cladodes is actually water or serving as a water reservoir (Snyman, 2007). The mucilage of the stem/leaves which constitute ~ 14% of the dry weight is also known to hold about 30% of the total water of the reserve parenchymatous tissues. Biochemically, these plants are adapted to minimize water lose during photosynthesis by taking up CO2 at night and to this end master the Crassulacean acid metabolism or an alternative photosynthetic pathway.

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FIG. 13.6 Opuntia microdasys (Lehm.) Pfeiff. Native to Mexico Gulf, Mexico Northeast, Mexico Northwest, Mexico Southwest but introduced in Africa, United States, and Europe. Picture taken from Hargeisa, Somalia (Courtesy: Dr Helen Pickering, Kew Botanical Garden, United Kingdom).

FIG. 13.7 Opuntia monacantha (Willd.) Haw. Native to South America (Brazil Northeast, Brazil South, Brazil Southeast, Paraguay, Uruguay) and introduced to the United States, Europe, Africa, and Asia. Image taken from Kew Botanical Garden, United Kingdom.

O. ficus-indica is also the common source of prickly pear in Mexico with great commercial significance. The name ‘prickly pears’ or ‘cactus pears’ is in fact commonly used for the highly coloured fruits (red, orange, purple, and lime green fruits) but given the medicinal and nutritional values of other plant parts, the name should not be associated only to the fruits. Readers, once again should also be reminded that products with prickly pear label could include many other Opuntia species other than the most popular variety, O. ficusindica. The variability in fruit colouring for prickly pear is amazing making both the products in the market (Fig. 13.10) as well as Opuntia species as valuable ornamental plant (see Figs 13.1–13.10). D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

13.3 Common uses of prickly pear

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FIG. 13.8 Opuntia robusta H.L.Wendl. ex Pfeiff. Native to Mexico and introduced to South America (Argentina) and Africa (Algeria and South America). Images taken from Kew Botanical Garden (United Kingdom) showing the earlier maturity stage (A) and fully matured fruits (B).

FIG. 13.9 Opuntia stricta (Haw.) Haw. Native to Mexico but introduced to the United States, the Caribbean, Africa, Europe and the Far East regions. Picture taken from Somalia (Courtesy: Dr Helen Pickering, Kew Botanical Garden, United Kingdom).

13.3 Common uses of prickly pear 13.3.1 Food production The most important parts of the plant of food and medicinal significance are cladodes, flowers, fruits, and seeds. Edible stems known as pads, vegetable, cladodes, nopales, or pencas are all consumed as vegetable in salads and other preparations. In many scientific literatures, these young stem segments, referred to as cladodes or nopales, are eaten. The utilization of domesticated O. ficus-indica for commercial production has been largely for the fresh sweet fruits (often called ‘tunas’). Mexico and to a less extent Italy are known for D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

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FIG. 13.10

Variations in fruit colour of prickly pear. The mature fruits on the cladodes (A) and pealed and ready to eat fruits (B) are shown. Courtesy of Wikipedia (https:// upload.wikimedia.org/wikipedia/commons/9/9e/ Prickly_pears.jpg).

the highest production and consumption levels while world-wide interest in consumption and commercial exploitation have grown over the years. The vegetable parts such as the cladodes are known for being good source of dietary fibres. In addition to being ingredients of fresh foods, the use of the cladodes and fruits in preparing jams, alcoholic beverages, natural liquid sweeteners and animal feed have been widely known. Both for human consumption and as animal feed, the large-scale utilization of these plants in drought-prone countries, such Ethiopia has been advocated in recent years. As a food source of food for human consumption, the cactus pear fruits are perhaps the most important parts to be exploited by mankind for centuries. Once the thick peel is removed, a highly flavoured coloured pulp containing numerous seeds is available for fresh consumption. Numerous preparations of dried or preserved cactus pear fruits in the form of juices (Gurrieri et al., 2000), jams (Sawaya et al., 1983), syrups and other processed preparations are available in the market. Flavoured drinks as well as alcoholic beverages such as wine are also commonly available (Bustos, 1981). On the other hand, the fruits as natural food colourant mainly due to the presence of the betalain pigments (see Section 13.4) are very common.

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Beyond the food value and various pharmacological activities of the cactus pear, other dimensions of the use include as natural colourants. The highly coloured components of the fruits with characteristic purple, yellow, green, or red appearance have been employed in the food industry (Castellar et al., 2006; Dı´az-Sanchez et al., 2006). A number of studies in improving the industrial processes of extraction of these colourants to improve their physicochemical properties and reducing microbial degradation (due to high sugar level of the fruits) have also been reported (Castro-Mun˜oz et al., 2018; Leon-Martı´nez et al., 2010). Nopal is extensively used in traditional Mexican cuisines as well as animal fodder.

13.3.2 Traditional medicine Feugang et al. (2006) were among the various authors who outlined the medicinal and nutritional importance of the genus cactus pear (Opuntia spp.). The medicinal properties of the cladodes/nopal and fruits include for treating arteriosclerosis, diabetes, gastritis, and hyperglycemia. In Mexico, ‘nopalitos’, ‘cladodes’, or ‘pencas’ are names associated with the spineless flat stems and have been known to be used for centuries for treating burns and diabetes. Other claims include for treating inflammation, oedema, and indigestion.

13.3.3 Food supplements At the borderline between what are regarded as foods and medicines are the functional foods and other formulations of cactus pears that are sold in the various local shops and supermarkets. For example, Cacti-Nea® is a dehydrated water extract obtained from the fruits of O. ficus-indica. The extract is standardized with the two principal betalain components that give the powdered product its distinctive colour (amber-to-red). The chemistry of the betalains (betanin and indicaxanthin) is described in Section 13.4.

13.3.4 Other uses Opuntia ficus-indica (along with other Opuntia species) has been grown from pre-Columbian times as a host plant for cochineal insects (Dactylopius coccus) for the production of valuable, vivid red and purple dyes (Donkin, 1977; Nobel, 1994). Considering the plant can yield very high biomass of the leaves/stems, utilization in the form of ethanol production has also been advocated (Retamal et al., 1987).

13.4 The chemistry of cactus pears The composition of the cladodes includes micro- and macro-nutrients and/or minerals that are not discussed in this book as bioactive compounds. Comparative analysis of such composition in the various parts of the plant parts are published (Andreu et al., 2018). The fibre and polyphenolic components such as flavonoids and other phenolics are of interest as potential drug molecules (Ginestra et al., 2009; Medina-Torres et al., 2011). The fibre of

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the plant parts such as the nopal include the soluble parts of mucilage and pectin as well as and insoluble fibre components of cellulose, hemicellulose and lignin. The fruits consist of the peal, pulp, and seeds of which the latter two are important as food and medicine preparations (Jimenez-Aguilar et al., 2014). As a food source, the pulp provides glucose, fructose, and pectin in good amounts (Zenteno-Ramirez et al., 2015). As with the cladodes, the fruits are rich sources of flavonoids and other polyphenols but also contain betalains (Jimenez-Aguilar et al., 2014; Stintzing et al., 2005). The presence of β-carotenes, polyphenols, and vitamin C which contributes to the antioxidant activity has also been reported (e.g. Stintzing et al., 2005).

13.4.1 Betalains The pigments of cactus pear fruits attributes to the water-soluble pigments that are known to be stored within the vacuole and collectively called the betalains. The two most predominant betalains of cactus fruits are betacyanins representing the (red-violet) and betaxanthins for yellow-orange pigments. The betanin (1) and indicaxanthin (2) are the best characterized of the betacyanins and betaxanthins respectively (Fig. 13.11). The presence of these two pigments in different proportion accounts to the various colouration of the cactus pear fruits ranging from purple to orange (Castellanos-Santiago and Yahia, 2008; Cejudo-Bastante et al., 2014; Forni et al., 1994; Ferna´ndez-Lo´pez and Almela, 2001; Stintzing et al., 2001; Castellar et al., 2003, 2006). As described in the following sections, these two pigments also display an array of biological activities including antioxidant effects (Abdel-Hameed et al., 2014; Butera et al., 2002; Castellar et al., 2012; Kanner et al., 2001; Pedreno and Escribano, 2001; Butera et al., 2002; Stintzing et al., 2005; Yeddes et al., 2013). Other effects also include potential anticancer effects as evidenced from in vitro apoptosis inducing effects in leukaemia cell line, among others (Sreekanth et al., 2007). The colour diversity of these two compounds in a mixture could give rise to their potential use as food colourant, particularly in the pH rage of 4–7 where they are reported to be stable (Albano et al., 2015). Hence, research on the various cultivars yielding from the orange to purple fruits has been the subject of intense research along with their potential benefits primarily as antioxidants. As natural colourant, the stability of betalains extracted from cactus pear has also been studied under different extraction methodologies and storage conditions. For HO

OH

HO O

HO O HO

H

H HOOC

N H

1 Betanin

FIG. 13.11

H

N+ COOH

COOH

+

N COOH

H HOOC

N H

COOH

2 Indicaxanthin

Structures of common betalains.

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13.4 The chemistry of cactus pears

example, when betanin from O. lasiacantha Pfeiffer (red prickly pear, Fig. 13.4) was spray dried using maltodextrin as coating material and kept in storage for 24 weeks, pigment retention (86.2%  3.9%) was found to be similar to the values found in the red beet pigment (Dı´azSanchez et al., 2006). Further stabilization of the yellow cactus pear colour using additives such as ascorbic acid in juice products has also been documented (El Gharras et al., 2008).

13.4.2 Polyphenols The phenolic acids and flavonoids derivatives of prickly pear are shown in Figs 13.12 and 13.13 respectively. The study by Guzma´n-Maldonado et al. (2010) has shown that gallic acid (6), catechin (15), vanillic acid (5), 4-hydroxybenzoic acid (3), epicatechin (16), and vanillin (8) could be detected in the fruit pulp. They were not however of major component with the highest amount detected being catechin (15) at 4 mg/100 g from O. humifusa Raf. (Chungbuk, Korea). Quercitrin (quercetin-3-O-rhamnoside, 27), taxifolin (18), quercetin (24), isorhamnetin (29) have also been identified from the plants (Kim et al., 2015). The aqueous ethanol extract of O. ficus-indica var. saboten has also been shown to yield seven major flavonols: isorhamnetin-3-O-glucoside (30), isorhamnetin-3-O-rutinoside (31), kaempferol (19), kaempferol-3-O-glucoside (20), kaempferol-7-O-neohesperidoside (23), quercetin (24) and quercetin-3-methylether (25) (Son et al., 2014). Isorhamnetin-3-O-rutinoside (31) was the most abundant of them all. These compounds were isolated as part of the antioxidant principles including oxidative stress amelioration in neuronal (PC12) cells. Other flavonols including dihydrokaempferol (17), kaempferol (19), narcissin (isorhamnetin-3-O-rutinoside, 31), quercetin (24) and quercetin-3-methylether (25) were identified in O. ficus-indica (Dok-Go et al., 2003; Lee et al., 2003). 4-Hydroxybenzoic acid (3), ferulic acid (13) and salicylic acid (7) have been reported from the cladodes. In the high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (HPLC–MS–MS) study by Guevara-Figueroa et al. (2010), gallic (6), coumaric (11, 140.8 μg/g), 3,4-dihydroxybenzoic (protocatechuic acid, 4), 4-hydroxybenzoic (3), ferulic (13, 347.7 mg/g), and salicylic (7) acids were detected from COOH

COOH

O

COOH OH

H

O

MeO R HO OH OH OH OH 8 Vanillin 6 Gallic acid 3 R=H p-Hydroxybenzoic acid 7 Salicylic acid 4 R=OH Protocatechuic acid 5 R=OMe Vanillic acid HO COOH COOH R COOH O COOH HO

O

HO OH

OH 10 Eucomic acid

11 R=H p-Coumaric acid 12 R=OH Caffeic acid 13 R=OMe Ferulic acid

O

9 Coumarin

OH OH

14 Chlorogenic acid

FIG. 13.12 Phenolic acids and derivatives of prickly pear. D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

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13. The chemical and pharmacological basis of prickly pear cactus

HO

O

OH

OH

OH HO

R

OH

OH

HO

O

OH

OH

OH

OH O

OH

OH

17 R=H Dihydrokaempferol 18 R=OH Taxifolin

16 Epicatechin

15 Catechin

OH OH R2O

O OR OH O

24 25 25 27 28

R=H Quercetin R=Me Quercetin-3-methylether R=Glc Quercetin-3-O-glucoside R=Rha Quercetin-3-O-rhamnoside R=Rut Rutin

OH O

OH O

OH OH OH OH Neo = Neohesperosyl OH

O O

O

OH

OH

OMe OH

OH Glc O OR

O

O

OMe O

OH

OH HO

O

OR1 OH O 19 R1=R2=H Kaempferol 20 R1=Glc, R2=H 21 R1=H, R2=Glc 22 R1=Rut, R2=H Nicotiflorin 23 R1=H, R2=Neo

HO

O

O

HO

OH OH Rut = Rutinosyl

OR OH O

29 R=H Isorhamnetin 30 R=Glc Isorhamnetin-3-O-glucoside 31 R=Rut Isorhamnetin 3-O-rutinoside (Narcissin) 32 R=Pentosyl Isorhamnetin-3-O-pentoside 33 R= Neo

34 R=Pentosylglucosyl

O

OH

OH OH OH Glc = Glucosyl O

OH

OH OH Rha = Rhamnosyl

FIG. 13.13

Some common flavonoids composition of prickly pear.

the cladodes of O. species. Other HPLC–MS studies on the cladodes of O. ficus-indica cv. Milpa Alta, of Mexican origin have also confirmed the detection of quercetin (24), kaempferol (19) and isorhamnetin (29) (Avila-Nava et al., 2014). The presence of p-hydroxybenzoic acid (3), vanillic acid (5) and ferulic acid (13) were also reported from O. fragilis (Abramovitch et al., 1968). The presence of flavonoids in the various tissues of O. species has been well documented. In fact, the detection of isorhamnetin (29) in the flowers of O. ficus-indica was reported as far back as in 1961 (Arcoleo et al., 1961). The flowers were also known to contain a range of other flavonoids including quercetin 3-O-glucoside (25), quercetin-3-O-rutinoside (rutin, 28) and kaempferol-3-O-glucoside (20) (Clark and Parfitt, 1980; Clark et al., 1980). From the young cladodes of O. ficus-indica of cultivars Copena V1, Tojo vigour and AtlixcoMexico, eucomic acid (10); chlorogenic acid (14); quercetin 3-O-rhamnosyl-(1–2)-

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[rhamnosyl-(1–6)]-glucoside (38); isorhamnetin-O-glycosides (uncharacterized); isorhamnetin-3-O-(pentosylglucoside)-7-O-glucoside (uncharacterized, 34); isorhamnetin-3-O-rutinoside (31); isorhamnetin-3-O-pentose (32) were detected by HPLC– MS–MS (Astello-Garcı´a et al., 2015). From the O. ficus-indica. Gafsa, of Tunisian origin, Ben-Saad et al. (2017) detected phenolic acids as gallic acid (6), catechin (15), caffeic acid (12), epicatechin, vanillic acid (5), and coumarin (9). Flavonoids as rutininoside of isorhamnetin (narcissin, 31), quercetin (24) and kaempferol (19) were also detected by HPLC (Fig. 13.13). Similarly, a study by Msaddak et al. (2017) on spineless cladodes from O. ficusindica f. inermis, Gabes, of Tunisian origin identified quercetin (24), quercetin-3-O-glucoside (25), kaempferol (19), kaempferol-3-O-glucoside (20), kaempferol-3-O-rutinoside (22), isorhamnetin (29), isorhamnetin-3-O-glucoside (30), isorhamnetin-3-O-neohesperidoside (33), quercetin (24), phenolics p-coumaric acid (3) and zataroside-A (39, Fig. 13.15) by HPLC–MS. In the latter case, the phenolic compound is terpenoids origin (Fig. 13.15). The presence of kaempferol (19) and isorhamnetin glycosides (glucoside and rhamnoside) in the Opuntia ficus-indica cladodes have also been reported by Ginestra et al. (2009).

FIG. 13.14 Complex flavonoid glycosides of prickly pear.

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FIG. 13.15

13. The chemical and pharmacological basis of prickly pear cactus

Some terpenoids components of prickly pear.

A complex flavonoid glycoside esterified with caffeic acid as exemplified by the isorhamnetin derivative (isorhamnetin-3-O-(600 -O-E-feruloyl)neohesperidoside) (36) has also been identified (see Fig. 13.14) as a radical scavenging principle (IC50 ¼ 45.58 μg/mL) of cladodes of O. ficus-indica var. saboten of Korean origin (Saleem et al., 2006). Other known compounds isolated in the study were quercetin (24), kaempferol (19) (Lee et al., 2003), quercetin-3-O-methyl ether (25) (Lee et al., 2003), 2,3-dihydrokaempferol (17) (Lee et al., 2003), isorhamnetin-3-O-glucoside (25), 2,3-dihydroquercetin (18), coumaric acid (11), kaempferol 7-O-glucoside (21), ferulic acid (13), isorhamnetin-3-O-neohesperidoside (33), isorhamnetin-3-O-rutinosyl-40 -O-β-D-glucoside (35), isorhamnetin-3-O-(2,6-dirhamnosyl)glucoside (37), zataroside-A (39), n-butyl-β-D-fructopyranoside and 4-O-glucosyl-mapic acid. Some studies have also focused on the polyphenolic composition variabilities within the wild and commercial varieties of nopal tablets. Clear difference in the phenolic acids (gallic, coumaric, 3,4-dihydroxybenzoic, 4-hydroxybenzoic, ferulic, and salicylic acid) contents were noted while five flavonoids (isoquercitrin (25), isorhamnetin-3-O-glucoside (30), nicotiflorin (kaempferol-3-O-rutinoside, 22), rutin (28), and narcissin (isorhamnetin-3-rutinoside, 31)) were found in all varieties (Guevara-Figueroa et al., 2010). Kuti (2004) has done an impressive comparative study on the flavonoids content of some cactus pear species and varieties. It was reported that quercetin (24) was predominant in the fruits of all cactus pear varieties, while kaempferol (19) was found in fruits of the green-skinned (O. ficus-indica), the purple-skinned (O. lindheimeri), and red-skinned (O. streptacantha) cactus pears. Isorhamnetin (29) was found in fruits of the green-skinned and the purple-skinned cactus pears. Kaempferol (19) was not also detectable in the fruits of yellow-skinned cactus pear (O. stricta var. stricta) and isorhamnetin (29) was not detectable in red-skinned or yellow-skinned cactus pear fruits. For the fruits composition, quercetin (24) derivatives were shown to account for 62.2% (43.2 μg/g fresh weight (FW)), kaempferol (19) derivatives for 3.2% and isorhamnetin (29) accounted for 34.7% of total flavonoids in green-skinned cactus pear fruits. In purple-skinned fruit variety, the total flavonoids consisted of quercetin (24) as 96.8% (90.5 μg/g FW), while it was 93.1% quercetin (51.0 μg/g FW) in the red-skinned, and 100% quercetin (9.8 μg/g FW) in yellow-skinned cactus pear fruits. In these cactus pear fruit varieties examined, the total flavonoid contents ranged from as low as  9.8  3.0 μg/g fresh weight in yellow-skinned cactus pear fruits to as high as  93.5  12.4 μg/g FW in purple-skinned cactus pear fruits.

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13.4 The chemistry of cactus pears

13.4.3 Terpenoids composition As mentioned in the preceding section, zataroside-A (39) was isolated from the spineless cladodes of O. ficus-indica f. inermis, Gabes, of Tunisian origin (Msaddak et al., 2017). The study by Saleem et al. (2006) on the leaves of O. ficus-indica var. saboten of Korean origin revealed the presence of other ionol derivatives of terpenoids origin: opuntiside A ((6R)9,10-dihydroxy-4,7-megastigmadien-3-one-9-O-β-D-glucopyranoside, 40) and B ((6S)-9,10dihydroxy-4,7-megastigmadien-3-one-9-O-β-D-glucopyranoside 41) (Fig. 13.15).

13.4.4 Seeds oil Ciriminna et al. (2017) have studied the seed oil composition of O. ficus-indica. The relevance of this study was based on a number of report describing the seeds coming in significant volume from cactus pears fruits harvest. Various estimates, for example, suggest that the seeds constitute 0.24 g per gram pulp. The seeds are generally known to contain minerals, amino acids and proteins among others but the major components are cellulose and lipids that accounts to 45.1% and 23% by weight respectively. Although minor components, the proanthocyanidins which are largely oligomers of flavonoids (e.g. catechin and epicatechin) and gallic acid esters are important particularly in antioxidant properties. The amount of oils that can be extracted from the seeds obviously depend on the species, genotypic makeup and environmental conditions. For O. ficus-indica seeds, variabilities between 7% and 15% (w/w) have been reported (Chougui et al., 2013; Ciriminna et al., 2017; Coskuner and Tekin, 2003; El Finti et al., 2013). The oil composition of the seeds (Fig. 13.16) appear to be dominated by linoleic acid (42) that could be as high as 63% under O

O OH

OH

42 Linoleic acid

43 Oleic acid

H H

H

H

H H HO

HO 44 β-Sitosterol

H

H

45 Campesterol

HO

H

H

46 Stigmasterol

O HO

47 γ-Tocopherol

FIG. 13.16 Major components of prickly pear seeds oil. Linoleic acid is the predominant fatty acid followed by oleic acid while the phytosterol composition of the oil is predominated by β-sitosterol. γ-Tocopheol was also identified as a major tocopherol component of the oil.

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different extraction methodologies. Other components reported include palmitic acid, as the second most dominant (12%) and to a less extent stearic and oleic acids. The sterols composition of the extractable oils is significant and can be as high as 28% of which β-sitosterol (44) is predominanat (22%) and campesterol (45, 4%) and stigmasterol (46, 2%) also detected (Ghazi et al., 2013). Ramadan and Morsel (2003a,b) also suggested that the β-sitosterol (44) content could be up to 6 g/kg of the seed oil. About 1% of the oil is vitamin E (γ-tocopherol, 47). Readers have to bear in mind that these compositions could vary depending on the oil source and specific data on the various genotypes and verities are available (e.g. Ciriminna et al., 2017). The presence of undecylic, lauric, myristic, pentadecylic, palmitic, and stearic acids in O. fragilis extracts have also been noted even in earlier studies (Abramovitch et al., 1968). Other composition studies on the oil of O. ficus-indica seeds made similar conclusions (Coskuner and Tekin, 2003; Ramadan and Morsel, 2003b; Salvo et al., 2002; Sawaya and Khan, 1982). Various extraction methodologies can be employed to obtain the seed oil. Extraction by nonpolar solvents like hexane in cold or hot (such as Soxhlet extraction) is common (Ennouri et al., 2006). In this method, the ratio of saturated fatty acids (12.43%) to monounsaturated (18.19%) and polyunsaturated fatty (70.29%) was reported to be 1:1.5:5.6. Specifically, the composition of linoleic acid was (70.29%) while others were, oleic (16.77%), palmitic (9.32%), stearic (3.11%), and palmitoleic acid (1.42%). All these findings suggest that the oil of cactus pear seed oil is rich in an essential fatty acid, linoleic acid, which has been shown to have a plethora of health benefits including for cardiovascular problems.

13.4.5 Polysaccharides components Dietary fibres are polysaccharides of two forms. The integral parts of plant structures such as cell walls include cellulose, hemicellulose and lignin which are insoluble in water and hence called the insoluble fibre. On the other hand, non-structural polysaccharides such as gum, mucilage and pectins constitute the soluble fibres. Both the soluble and insoluble components of dietary fibres are known for their health benefits including digestibility, water retention and food mobility within the gut and can also possess some antimicrobial effects. The prickly pear cladodes are generally known for their high dietary fibres and in this direction, the major polysaccharide components of O. ficus-indica have been fairly characterized. The fibrous components as mucilage and pectin are composed of complex carbohydrates/polysaccharides with sugar monomers of arabinose, galactose, rhamnose, and galacturonic acid. The study by Combo et al. (2011) has shown pectins as a heteropolysaccharides with a high content of galacturonic acid monomers linked by α-(1 ! 4) bonds. The presence of some acetylated sugars or esterified with methyl groups have also noted. Biological effects reported for these compounds include inhibition of the formation of advanced glycation end products (AGEs) which is relevant to diabetes pharmacology (Zhao et al., 2007). The method of extraction of these pectins under various temperature, pH media and ultrafiltration techniques have also been described (de Wit et al., 2016). A number of climatic and genetic variations have been shown to attribute to variabilities in fibre content of cactus. POLOF and POLOS were two polysaccharides isolated from the cladodes of O. ficus-indica and O. streptacantha with components of monosaccharides shown to comprise arabinose, furanose, galactose,

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glucose, rhamnose, arabinopyranose, and galactopyranose (Alarcon-Aguilar et al., 2003). Interest on such polysaccharides have grown in recent years as they have been shown to display hypoglycaemic effects at an oral (p.o.) dose of 500 mg/kg (Alarcon-Aguilar et al., 2003).

13.5 Antidiabetic, antiobesity, and lipid lowering effects of prickly pears 13.5.1 Antidiabetic effect demonstrated through animal studies Ibanez-Camacho et al. (1983) have studied the hypoglycaemic effect of O. streptacantha sap in various species and experimental conditions. They found that the hypoglycaemic effect of the sap was observed under moderate increase of blood sugar but not either in normoglycaemic or pancreatectomized animals. By using the in vivo hypoglycaemic bioassay in rabbits, the effect of saps collected from O. streptacantha throughout the year was also studied to see if there was seasonal variability in biological activities. It was reported that the sap has shown consistent activity suggesting the potential use of the plant by harvesting the sap at any season of the year (Meckes-Lozoya and Ibanez-Camacho, 1989). A preparation of fresh blend of cladodes by cutting them of into pieces and taking the whole liquefied shake before breakfast as a possible therapy for diabetes has been tested (Andrade-Cetto and Wiedenfeld, 2011). In the streptozotocin (STZ)-induced diabetes model in rats, this total extract and O. streptacantha juice (filtrate) failed to display hypoglycaemic effect after administration of the extracts (whole extract 135 mg/kg and juice 27 mg/kg). At the same doses, however, both extracts were shown to display effect in oral glucose tolerance test (OGTT). Similar result was observed when these extract preparations were tested in (100 mg/kg for the whole extract and 4 mL/kg for the juice) after a load of 3 g/kg of maltose, hence showing anti-hyperglycaemic effect (Becerra-Jimenez and Andrade-Cetto, 2012). Such a discrepancy could be accounted for by the selected doses in view of the cladodes extract act at relatively high doses (e.g. see below). Leem et al. (2016) have administered the aqueous extracts of dried cladodes of O. ficusindica var. saboten (1 g/kg and 2 g/kg) in db/db (C57BL/6J db/db) diabetic mice. The study was based on the plant cultivated in the southern part of Korea. Their result demonstrated a dose-dependent amelioration of hyperglycaemia (62% decrease), hyperinsulinaemia, and glucose tolerance when tested by OGTT. In this model, insulin level in diabetic mice was twofold higher than non-diabetic (heterozygous littermates (db/
)) mice suggesting insulin resistance appears to be ameliorated by the extract treatment. Their analysis of insulin sensitivity index by QUICKI (quantitative insulin sensitivity check index) and homeostatic model assessment of insulin resistance (HOMA-IR) index also showed a dose-dependent improvement in insulin sensitivity in diabetic mice. Histological examination also revealed an increase in β-cell mass in diabetic mice. One of the hallmark of diabetes in β-cell loss (necrosis and atrophy) following prolonged exposure to high glucose level is thus improved by treatment with O. ficus-indica var. saboten. The most studied cactus pear plant for antidiabetic effect remains to be O. ficus-indica. This include the seed oil (2 mL/kg) of O. ficus-indica in alloxan-induced diabetic mice displaying antihyperglycaemic effect along with protection of islets of langerhans against tissue alterations (morphological assessment) (Berraaouan et al., 2015). In STZ-induced diabetic rats,

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O. ficus-indica seeds oil (1 or 2 mL/kg, p.o. single dose) could also decrease postprandial hyperglycaemia (60 min after glucose loading) (Berraaouan et al., 2014). This effect was also observed in healthy animals where a decrease in intestinal glucose absorption by about 25.42% was observed (Berraaouan et al., 2014). Another study on the seed oils was in rats which were fed lipid-enriched diet in rats where treatment (25 g/kg for 9 weeks) with the oil was shown to decrease the plasma glucose, total cholesterol (TC) and low density lipoproteincholesterol (LDL-C), very low density lipoprotein-cholesterol (VLDL-C) without effect on the level of high density lipoprotein-cholesterol (HDL-C) (Ennouri et al., 2006). The levels of glycogen contents in the liver and skeletal muscle were increased by treatment with O. ficus-indica seeds oil (Ennouri et al., 2006). In STZ-induced diabetic rats, nopal extract (50 mg/kg) from O. ficus-indica could also reduce postprandial blood glucose level (Nun˜ez-Lo´pez et al., 2013). A more comprehensive study on nopal came from studies on obese Zucker (fa/fa) rats fed with diet containing 4% nopal for 7 weeks. In addition to lipid lowering and antiobesity effects (see Section 13.5.2), improvement in postprandial glycaemic/insulin control was observed along with reduction in liver marker enzymes such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (Moran-Ramos, et al., 2012). The potential antidiabetic effects of the stem and the fruits when combined at the ratio of 75:25 (combination studies) were also studied in normal rats receiving 6–176 mg/kg (p.o.). The most significant observation from this study was the reduction in blood glucose and/ or during glucose tolerance test, and increase in the basal plasma insulin levels that was considered a direct action on pancreatic β-cells (Butterweck et al., 2011). This effect was also similar with the aqueous cladode extract when administered at a dose of as low as 6 mg/kg (Butterweck et al., 2011). Other effects related to diabetes for the various parts of O. ficus-indica and proprietary preparations will be discussed under mechanisms of actions and elsewhere in the following sections. A number of other cactus pear plants other than O. ficus-indica have also been demonstrated to display antidiabetic effects in vivo. In the STZ-induced diabetes model in rats, combination treatment of insulin and O. fuliginosa Griffiths stems extract (claimed to be purified with undescribed methodology) for 7 weeks (p.o.) followed by extract alone has been studied (Trejo-Gonza´lez et al., 1996). In such drug combination, blood glucose was not only shown to be normalized but also a small amount of O. fuliginosa extract was required (1 mg/kg per day). On this basis, the authors suggested possible active components other than dietary fibres which are known to act at high doses. The dried stems of O. humifusa were also tested in the STZ-induced diabetes model at the dose of 250 or 500 mg/kg (p.o. for 7 weeks) (Hahm et al., 2011). It was reported that the stem powder can reduce blood glucose as well as triglycerides (TG), cholesterol and LDL-C; while the HDL-C level was increased. Other markers of diabetes such as the elevated ALT and AST were shown to be suppressed by the treatment along with improvement in relative β-cell volume of pancreas for the treatment group with 500 mg/kg of the stem. By using STZ-treated pigs, the antidiabetic potential of O. lindheimeri Englem extract (250 or 500 mg/kg) were also assessed by Laurenz et al. (2003). In an acute effect study, a decrease in blood glucose in a dose- and time-dependent manor was observed. It was reported that the hypoglycaemic effect could be observed within 1 h of the plant administration, with maximal effects occurring at 4 h after administration. The aqueous cladodes extract of O. megacantha was also shown to display hypoglycaemic effect in STZ-rats by

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administering 200 mg/kg for 5 weeks (Bwititi et al., 2001). The extract has been shown to suppress the plasma glucose level both in the STZ-diabetic and non-diabetic rats. It has also been demonstrated that the extract can increase urinary Na+ output in diabetic and non-diabetic rats leading to low plasma sodium level. Even though administration of the leaves extract was shown to increase glomerular filtration rate (GFR) in diabetic rats, it did also increase the plasma creatinine and urea concentrations in both diabetic and non-diabetic rats suggesting some possible toxic side effects. The data which was consistent with the authors’ previous study (Bwititi et al., 2001) thus indicate that the use of this plant cladodes must be taken with caution until this potential toxic effect to the kidneys is clarified. Chahdoura et al. (2017) have recently assessed the antidiabetic potential of aqueous flowers extract of O. microdasys in the fructose and alloxan-induced diabetes rat model. Oral administration of the extract at the dose of 200 mg/kg for 28 days could lower the levels of glucose and hepatic marker enzymes and products including alkaline phosphatase (ALP), AST and ALT, lactate dehydrogenase (LDH), γ-glutamyltransferase (γ-GT), total bilirubin, TC, LDL-C, and TG; while HDL-C was increased. The suppressive effect of the extract on the diabetes-induced oxidative stress by improving the level of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) peroxidase (GPx) activity and the levels of protein carbonyl and malondialdehyde (MDA) in the liver and restoration of the histological architecture of the liver. O. monacantha cladode extracted with water was also shown to decrease blood glucose level and enhance the HDL level in STZ-induced diabetic rats when administered (100, 200, and 300 mg/kg) for 4 weeks (Yang et al., 2008). In the STZ-induced diabetic pigs, the antidiabetic effect of O. lindheimeri Engelm extract (250 or 500 mg/kg body weight, p.o.) was demonstrated in a dose- and time-dependent manner. The hypoglycaemic effect could be seen within 1 h of administration with maximal effects reached at 4 h. As described in the previous section, α-glucosidase and α-amylase inhibitory effect of the extract at concentrations higher that 0.1 mg/mL have also been shown (Chahdoura et al., 2017). The hypoglycaemic effect of O. robusta Wendl. was also established in STZ-induced diabetic rats along with O. ficus-indica Mill. and O. lindheimeri Engelm. (Enigbokan et al., 1996).

13.5.2 Antiobesity and lipid lowering potential In the preceding section where the antidiabetic effects of the various species of Opuntia were described, lipid lowering effects were also demonstrated. In some cases, TG, cholesterol and LDL-C were reduced while the HDL-C level was increased as demonstrated for O. humifusa (Hahm et al., 2011) or O. microdasys (Chahdoura et al., 2017). HDL-C was enhanced by O. monacantha cladode (Yang et al., 2008) while O. ficus-indica seed oil given as a supplement (25 g/kg seed oil to the diet) failed to alter HDL-C level although it suppressed the plasma level of TC, LDL-C and VLDL-C (Ennouri et al., 2006). It is thus reasonable to assume that at least some components of the lipid profile could be modulated by Opuntia spp. Cardenas-Medellin et al. (1998) investigated the antiobesity potential of O. ficus-indica cladodes in rats. They have shown that rats fed with 12% nopal had lower weight gains when compared with those fed with 6% nopal or the control diet. On the other hand, nopal supplementation did not affect the level of glucose, TC and HDL-C levels but there was a 34% reduction in LDL-C levels for the 12% nopal feeding. These results suggest that O. ficusindica stems/cladodes could have antiobesity and lipid lowering effects. In experiments

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employing guinea pigs feeding with high cholesterol diet, supplementation with crude pectins of cactus pear (2.5 g/100 g diet) has shown a decrease in plasma LDL and hepatic cholesterol concentrations (Fernandez et al., 1994). While the hepatic acyl-coenzyme A (CoA):cholesterol acyltransferases (ACAT) activity was reduced by pectin supplementation, the level of hepatic 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase activity was not altered. The mechanism of HMG-CoA inhibition thereby limiting fat synthesis is the primary target for the statins that does not seem to be shared by the cactus pears pectins. On the other hand, ACAT is membrane-bound protein that synthesizes cholesteryl esters from long-chain fatty acyl-CoA and cholesterol. Moreover, the hepatic apolipoprotein B/E receptor expression (Bmax) in guinea pigs fed with cactus pears pectins supplementation under similar experimental condition was found to be around 60% higher (Ferna´ndez et al., 1992). While the receptor-mediated LDL fractional catabolic rates were 190% higher in treated animals, apolipoprotein LDL flux rates were not affected. Hence, apolipoprotein B/E receptor expression could also account to the plasma LDL lowering effect by prickly pear pectins. Moran-Ramos et al. (2012) also employed the Obese Zucker (fa/fa) rats model to study the potential antiobesity and lipid lowering effect of nopal from O. ficus-indica. By feeding rats with 4% nopal for 7 weeks, they have achieved about 50% reduction in hepatic TG coupled with improvement of other biochemical and histological markers all suggesting the amelioration of hepatic steatosis by increasing fatty acid oxidation and VLDL synthesis; decreasing oxidative stress; and improving liver insulin signalling in obese Zucker (fa/fa) rats. The key observations for the treatment were: • Lowered hepatic lipid droplet and TG (50% reduction) content (histology) and attenuated hepatic steatosis; • Liver weight of the nopal groups was 20% lower than control group; • Reduced LDL particles and serum transaminases; • Increase the serum level of adiponectin; • Upregulated genes involved in hepatic fatty acid oxidation such as transcription factor peroxisome proliferator-activated receptor (PPAR)-α (PPAR-α), and its target genes Cpt1 and Aox; • Increased genes involved in lipid export and production of carnitine palmitoyltransferase-1 and microsomal TG transfer proteins in the liver; • Suppressed the liver reactive oxygen species (ROS) and MDA levels, while SOD level (mRNA) was increased; • Lowered insulin concentration; • Increased insulin receptor substrate-1 (IRS1) and protein kinase B (Akt) phosphorylation in the liver. Even when glucose lowering effect was not demonstrated for O. ficus-indica supplementation (12% supplementation of diet for 1 month) in rats, reduction in body weight gain and blood LDL level was achieved (Cardenas-Medellin et al., 1998). With a caution of more research needed in this area, including in mechanistic studies, prickly pear appear to show antiobesity effect in animals.

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13.5.3 Some common mechanisms of antidiabetic, lipid lowering, and antiobesity effects 13.5.3.1 Glycaemic control Leem et al. (2016) studied the effect of dried stem powder aqueous extracts of O. ficus-indica var. saboten on α-glucosidase activity and intestinal glucose absorption by assessing the Na+-dependent glucose uptake in brush border membrane vesicles. First, the enzyme inhibitory activity was demonstrated in a concentration-dependent manner with 10 mg/mL of the extract inhibiting α-glucosidase activity by 64.9%. Such level of concentration, however, is highly unlikely to be therapeutically significant unless combined with other mechanisms of action. The lack of effect of aqueous extracts of dried O. streptacantha Lem. cladodes in α-glucosidase inhibition has also been reported (Becerra-Jimenez and Andrade-Cetto, 2012). The aqueous extract of O. ficus-indica var. saboten, however, dose-dependently increased glucose uptake in L6 muscle cells (Leem et al., 2016). Interestingly, the 50 adenosine monophosphate-activated protein kinase (AMPK) and p38 mitogen activated protein kinase (MAPK) phosphorylations were shown to be stimulated by the extract, while inhibitors of both the AMPK (compound C) and p38 MAPK (SB203580) could ameliorate the effect of the extract. Unlike the α-glucosidase enzyme inhibition effect, the effect of the extract in glucose uptake was mediated in micromolar concentration ranging from 1 to 100 μM. As expected, the extract could also increase the translocation of the glucose transporter 4 (GLUT4) to the plasma membrane. The AMPK signalling pathway has been well studied in recent years and established as a key pharmacological target for treating both T2D and obesity. The p38 MAPK as a component of the downstream AMPK signalling system is also another target that is involved in glucose uptake including that stimulated by insulin in target cells (e.g. muscle cells). One of the ultimate effector mechanism of the AMPK/p38 MAPK activation is the recruitment of GLUT4 to the plasma membrane leading to upregulation of glucose uptake. One possible mechanism of action of prickly pear extract is therefore by improving insulin sensitivity or tackling insulin resistance by enhancing glucose uptake in muscle cells through the AMPK/p38 MAPK signalling pathway. In human chondrocyte culture stimulated with proinflammatory cytokine interleukin (IL)1β (IL-1β), the lyophilized extracts obtained from O. ficus-indica (L.) cladodes could also protect cartilage alteration (Panico et al., 2007). The inhibitory effects of aqueous flowers extract of O. microdasys on some carbohydrate metabolizing enzymes, pancreatic α-amylase (IC50 ¼ 2.55  0.41 mg/mL) and intestinal α-glucosidase (IC50 ¼ 0.17  0.012 mg/mL) activities in vitro has also been reported (Chahdoura et al., 2017). This level of activity is slightly better than the stems/cladodes extract in the various test systems and perhaps could play more role in the antidiabetic effect of the flowers. 13.5.3.2 Antioxidant effects In the animal models of antidiabetic and antiobesity assessments, a reduction in the level of ROS and lipid peroxidation (LPO) products as well as increased antioxidant defences including GSH, SOD, CAT, GPx have been observed (Chahdoura et al., 2017; Moran-Ramos et al., 2012). In mice subjected to zearalenone (fusarial mycotoxin) intoxication and oxidative stress, the cladodes extract of O. ficus-indica (25, 50, and 100 mg/kg) could ameliorate oxidative damage as shown by the reduced levels of kidney and liver MDA and protein carbonyls (Zourgui

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et al., 2008). Modulation of CAT activity and the expression of the heat shock proteins were also shown for all tested doses. In carbon tetrachloride (CCl4)-induced hepatotoxicity in rats, administration of O. ficus-indica fruits juice (3 mL/rat) for 9 consecutive days could display hepatoprotective effects: restored normal hepatic parenchyma within 72 h (histology) and reduced the increased plasma level of liver enzymes (Galati et al., 2005). Hence, the classic example of oxidative damage model in the liver could be suppressed by the fruits juice of prickly pear. Most of the demonstrated biological activities for Opuntia species in vitro constitute studies on antioxidant effects. In a study by Kuti (2004), the ascorbic acid concentration (μg/g FW) in fruit extracts were in the following order O. streptacantha (815) >O. ficus-indica (458) > O. stricta var. stricta (437) > O. lindheimeri (121). In view of carotenoids play a role in antioxidant effects, their concentration is also quantified and was found in the following order: O. stricta var. stricta (23.7) >O. streptacantha (14.6) >O. lindheimeri (6.7) >O. ficus-indica (2.9). Similarly, the cladodes of prickly pear are also known to contain ascorbic acid and carotenoids. The fresh stems of Mexican nopal, for example containing 7–22 mg ascorbic acid and 11.3–53.5 μg total carotenoids (Stintzing et al., 2001). The most important antioxidant effects, however, came from the flavonoid composition that has been reported in the previous section. Hence, the numerous reports (e.g. Kuti, 2004) tried to correlate in vitro antioxidant effects with the level of flavonoids contents. Comparative radical scavenging and ferric-reducing ability of O. ficus-indica cladode and fruits were assessed with the higher effect recorded for fruit peel than in cladodes (Andreu et al., 2018). O. ficus-indica fruit extract has also shown to display radical scavenging (ABTS.+, DPPH, and FRAP assays) and inhibits H2O2-induced DNA damage in lymphocytes (Siriwardhana et al., 2006). The observed effect in the range of 0.0625–0.5 mg/mL was also promising and was consistent with other studies where the fruits have potent antioxidant activity. Even though the concentration range showing activity was not that impressive (0.5, 1, 1.5, and 2 mg/mL), antiglycation, radical scavenging and LPO inhibition were also shown for the O. ficus-indica cladodes (Chaouch et al., 2015). The study by Kim et al. (2014) also assessed O. ficus-indica stems for radical scavenging and cytotoxicity in cancer cells (SW480 colon and MCF7 breast cancer cells). In their experiment, the plant material were extracted with hexane, ethyl acetate (EtOAc), acetone, methanol (MeOH), or MeOH:water (80:20). It was reported that the MeOH extract had the highest amount of polyphenolic compounds and the acetone extract exhibited the most potent radical (DPPH and ABTS.+) scavenging effect; inhibition of cyclooxygenase-2 (COX-2) and increased the Bax/ Bcl2. The inhibition of COX-2 is indicative of anti-inflammatory effect which is also relevant to diabetes therapy. The DPPH radical scavenging effect for the seed oil of O. ficus-indica has also been reported (Berraaouan et al., 2015). In a concentration range (0.1, 0.5, and 1.0 mg/mL), inhibition of AGEs formation has also been reported for the aqueous extract O. monacantha, cladodes (Zhao et al., 2007). The cladodes of O. humifusa Raf. were also investigated for antioxidant effect using DPPH and xanthine oxidase assays along with lipopolysaccharide (LPS)-activated RAW264.7 cells (Cho et al., 2006). Beyond antioxidant effect, inhibition of the expression of inducible nitric oxide synthase (iNOS) and IL-6 were reported while the EtOAc active fraction gave quercetin (24) as the most probable active principle (Cho et al., 2006).

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In addition to direct radical (DPPH) effects, the juice of the whole fruits obtained from the Sicilian cultivars of prickly pear (O. ficus-indica (L.) Mill.) could ameliorate the ulcerogenic activity of ethanol in rats (Galati et al., 2003). As shown by light microscopy, an increase in mucus production and the restoration of the normal mucosal architecture was observed by the juice administration. In their attempt to standardize the juice product with the known active principles of the fruits, the authors identified ferulic acid (13) as the principal hydroxycinnamic acid component while the mean concentration of total phenolic compounds in the juice was reported as was 746 μg/mL. Rutin and isorhamnetin were also identified as principal components of the flavonoid fraction. Hence, the known antioxidant polyphenols could be implicated to the observed antioxidant and antiulcerative effect of the fruit juice. With respect to O. ficus-indica extract, Nakahara et al. (2015) have shown that its antioxidant effect in human keratinocytes cells was mediated through activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) and the downstream antioxidant enzyme; NAD(P)H: quinone oxidoreductase 1 (NQO1). Upon activation, the NQO1 can inhibit the generation of ROS in these cells when stimulated with tumour necrosis factor-α (TNF-α) or benzo[α]pyrene. The further evidence of the role of Nrf2 in the antioxidant effect of O. ficus-indica extract came from the lack of effect in the Nrf2 knockdown keratinocytes. The effect of extract in Nrf2-NQO1 upregulation was also shown to be associated through activation of the aryl hydrocarbon receptor (AHR). The AHR activation by the extract intern upregulate the expression of epidermal barrier proteins: filaggrin and loricrin. Hence, the extract could prevent the Th2 cytokine-mediated downregulation of filaggrin and loricrin expression in an AHRdependent manner. It was thus concluded that the antioxidant effect of the extract is also associated with potent activation of the AHR- Nrf2-NQO1 signalling cascades that is of interest in other disease conditions. The organoprotective effects of O. ficus-indica, have been demonstrated in the various animal models. By using the lithium-induced liver injury in rats, Ben-Saad et al. (2017) have shown that treatment of animals with 100 mg/kg cladodes aqueous extract (intraperitoneal (i.p.) for 60 days) showed significant hepatoprotective effects as assessed by haematological parameters. The Li2CO3-treated rats showed increased blood level of glucose, cholesterol, TG and of AST, ALT, ALP, and LDH activities which were reversed by the extract; while in hepatic tissues, the raised LPO level and decreased activities of SOD, CAT, and GPx were all ameliorated by the extract treatment. Hence, this assay model could be seen both as oxidative-induced injury and hyperglycaemia that seems to be reversed by the extract. The authors also demonstrated antioxidant effects in vitro by assessing direct radical scavenging, reducing power and Metal (Fe2+) chelating activity. Readers should note however that the direct antioxidant effect observed was in IC50 values greater or equal to 0.36 mg/mL, making the extract a weak antioxidant agent. Since HPLC profiling of the extract showed the presence of rutin, isorhamnetin, quercetin, and kaempferol (Fig. 13.13) (Ben-Saad et al., 2017), which are all known for their antioxidant effects, the observed activity is in line with the phenolic composition of the plant as active principles. 13.5.3.3 Anti-inflammatory mechanisms Opuntia ficus-indica cladodes significantly inhibited the carrageenan-induced oedema probably by affecting prostaglandins synthesis (Galati et al., 2000). In fact, COX-2 inhibition

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by the methanol extract of O. ficus-indica stems extract has been demonstrated (Kim et al., 2014). Beyond antioxidant effects, Matias et al. (2014) have explored the anti-inflammatory effect of O. ficus-indica juice in intestinal inflammation by using the human Caco-2 cells. The reduction of IL-8, nitric oxide (NO) and TNF-α expression coupled with blockade of degradation of IκBα were reported for the flavonoid-rich concentrate. They also isolated the main flavonoids (namely isorhamnetin and its derivatives such as isorhamnetin 3-Orhamnose-rutinoside and isorhamnetin 3-O-rutinoside) and phenolic acids (such as ferulic and eucomic acids) as potentially active components (Figs 13.12 and 13.13). Park et al. (2001) have studied the anti-inflammatory effect of ethanol extract of O. ficus-indica dried stems using the adjuvant-induced pouch granuloma model in mice. After detecting activity for the crude extract, their bioassay-guided isolation study revealed the identification of β-sitosterol (44) as the active principle. In other models of inflammation such as the acetic acid writhing (analgesic model) and carrageenan-induced rat paw oedema, the ethanol extracts of the fruits and stems of O. ficus-indica have been shown to display activity (Park et al., 1998). Oral administrations of these extracts could suppress paw oedema and the leukocyte migration of carboxymethyl cellulose-pouch model in rats: they also suppress the release of β-glucuronidase (a lysosomal enzyme) from rat neutrophils and display protective effect on gastric mucosal layers (Park et al., 1998). Although the above results are good indicators of the anti-inflammatory mechanism of action for prickly pear preparations as antidiabetic agents, more direct evidence in animal and cellular models need to be generated.

13.6 Active principles of prickly pear 13.6.1 Polysaccharides There is no doubt that dietary fibres play a role in the hypoglycaemic effects of cactus pear stems via inhibition of intestinal glucose absorption. In the hypoglycaemic effect of the stems extract in diabetic db/db mice (Leem et al., 2016), the good efficacy demonstrated at a rather high dose of 1 and 2 g/kg of O. ficus-indica var. saboten is worth a scrutiny. The dietary fibre component of the extract was 56% (dry weight (DF) basis). The high fibre contents of the cactus pear cladodes have also been reported by various other authors and can reach to over 60% (Bensado´n et al., 2010). In Chapter 17, the antidiabetic potential of soluble dietary fibre from Trigonella foenum-graecum (e.g. Hannan et al., 2007) is discussed and mechanisms including inhibition of carbohydrate digestion and absorption, and even effect on peripheral insulin resistance are all expected for dietary fibres. The potential cholesterol lowering effects of prickly pear pectins in guinea pigs have been studied (Ferna´ndez et al., 1992, 1994). In a diet containing cholesterol, the addition of 2.5 g prickly pear pectin/100 g diet has been shown to reduce plasma and hepatic cholesterol concentrations (LDL); reduce hepatic acyl CoA:cholesterol acyltransferase activity; and increase hepatic apolipoprotein B/E receptor expression (Ferna´ndez et al., 1992, 1994). Polysaccharides isolated from O. dillenii cladodes (labelled as, ODP-Ia, ODP-Ib, and ODPII0 ) have been tested for antidiabetic effects in STZ-induced diabetes mice model (Zhao et al.,

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2011). The polysaccharides were extracted by using an ultrasonic extraction method followed by diethylaminoethyl (DEAE)-Sepharose fast-flow column chromatography system. The ODP-Ia was active and its administration (50, 100, and 200 mg/kg, p.o. for 3 weeks) could reverse body weight loss and increase the hepatic levels of glycogen, HDL-C, and activities of SOD and GPx in diabetic mice. The polysaccharide (ODP-Ia) has also been reported to reduce the intake of food and water (even though body weight increases); the fasting levels of blood glucose, TC, TG, blood urea nitrogen (BUN), and MDA; and the activity of glucose-6phosphatase (G6Pase). However, ODP-Ia did not significantly increase insulin levels in the mice with STZ-induced diabetes. The structure of this active polysaccharide as well as ODP-II0 which also showed some activity have not been determined. The authors’ preliminary study indicated that “ODP-Ia was comprised mainly of rhamnose, arabinose, galactose and glucose, with 15.13% (w/w) of arabinuronic acid. ODP-Ib was composed of rhamnose, arabinose, and galactose, with 52.66% (w/w) of arabinuronic acid while ODP-II0 was composed of rhamnose, glucose, with 26.38% (w/w) of arabinuronic acid.” The study by Gao et al. (2015) on O. dillenii Haw. fruits also shed some light into the contribution of polysaccharides in the reported effects on STZ-induced diabetic rats. They have isolated a novel water-soluble polysaccharide fraction with molecular weight of 6479.1 kDa which was consisted of rhamnose, xylose, mannose, and glucose in the molar ratio of 14.99:1.14:1.00:6.47. The isolation was based on the combination of anion exchange (DEAE52 cellulose column) and size-exclusion (Sephacryl S-400 column) chromatography systems. Oral administration (50, 100, or 200 mg/kg for 28 days) of this polysaccharide could decrease food and water intake, urine production, organ weights and blood glucose level, and increased body weight in diabetic animals. An increased level of activities of SOD, GPx, and CAT, and decreased MDA level in serum, liver, kidney, and pancreas in STZ-induced diabetic rats have also been achieved by the polysaccharide. An improvement in the structure integrity of pancreatic islet tissue also suggests the antidiabetic potential of the polysaccharide (Gao et al., 2015). The increased body weight in this model should not be seen as obesity induction but a rather recovery of bodyweight loss induced by STZ in this diabetic animals. Polysaccharides isolated from O. ficus-indica and O. streptacantha by dialysis have also been reported to display some antidiabetic effects in alloxan model in mice when administered at the dose of 500 mg/kg (intragastric (i.g.)) (Alarcon-Aguilar et al., 2003).

13.6.2 Cactus pear pigments 13.6.2.1 Antioxidant effects of indicaxanthin and betanin Most of the in vitro and animal studies on the biological activities of cactus fruits are related to the nutritional and antioxidant components of various species and cultivars (Ferna´ndez-Lo´pez et al., 2010; Galati et al., 2003; Madrigal-Santilla´n et al., 2013; Piga et al., 2003; Yahia and Mondrago´n-Jacobo, 2011). Human trials showing the decrease in oxidative stress following the intake of cactus pear (O. ficus-indica) fruit supplements have also been shown (Tesoriere et al., 2004a,b). Of all the antioxidant effect studies, the polyphenol content analysis coupled with in vitro antioxidant effects primarily, radical scavenging, predominate. Accordingly, Ferna´ndez-

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Lo´pez et al. (2010) assessed antioxidant constituents and in vitro antioxidant capacity in the extracts of three species of Spanish red-skinned cactus pear fruits (O. ficus-indica, O. undulata, and O. stricta). They found that O. ficus-indica fruits extract showed the strongest antioxidant potential and taurine content. While O. stricta fruits were reported to be the richest in ascorbic acid and total phenolics, O. undulata fruits had the highest carotenoid content. In line with the chemical studies reported in the previous section, the flavonoids quercetin (24) and isorhamnetin (29) were detected (Ferna´ndez-Lo´pez et al., 2010) possibly implying their role as the major antioxidant principles. The two principal colour components of the fruits, indicaxanthin (2) and betanin (1), could also play major role given their abundance in the cactus pear fruits. Both compounds have been shown to have direct antioxidant effects in vitro when assessed by radical scavenging effects. Tesoriere et al. (2007) have conducted the kinetics of the lipoperoxyl radical-scavenging activity of indicaxanthin (2) both in solution and unilamellar liposomes. The result showed that the compound acts as a chain-terminating lipoperoxyl radical scavenger in solution whereas its incorporation in liposomes could prevent lipid oxidation by inducing a lag period as well as decreasing the propagation rate of the reaction. In cellular system, protective effect against damage and cytotoxicity induced by a variety of agents has also been observed. Good example is endothelial cells protection (Gentile et al., 2004). When oxidative haemolysis was induced by cumene hydroperoxide, indicaxanthin (2) can offer protective effect in concentration range of 1.0–10 μM (Tesoriere et al., 2006). Moreover, the compound could also prevent lipid and haemoglobin oxidation as well as reducing vitamin E and GSH depletion (Tesoriere et al., 2006). These effects could also be relevant in oxidative stress condition of β-thalassemia as ex vivo data also suggest protection of RBC from thalassemic patients (Tesoriere et al., 2006). The further extension of these studies were in humans were the distribution of betanin (1) and indicaxanthin (2) in red blood cells (RBCs) isolated from healthy volunteers taking dietary betalains (cactus pear fruit meal) were investigated (Tesoriere et al., 2005). The authors reported that the peak concentration of indicaxanthin (1.03  0.2 μM) was observed in RBCs isolated at 3 h after fruit feeding, whereas the concentration at 5 h was about half, and even smaller amounts were measured at 8 h. Indicaxanthin (2) was not detected at 1 h. In contrast, betanin (1, 30.0  5.2 nM) was found only in RBCs isolated at 3 h from fruit feeding. The addition of betalains to the RBC isolated from healthy human volunteers also made the cells resistant to cumene hydroperoxide-induced haemolysis (Tesoriere et al., 2005). Betanin and indicaxanthin from sicilian prickly pear (O. ficus-indica) fruit extracts have been shown to inhibit organic hydroperoxide-stimulated RBC membrane lipid oxidation, as well as the metal-dependent and -independent LDL oxidation: they were also good Trolox scavengers (Butera et al., 2002). In addition to the free radical scavenging and antioxidant properties of betalains from cactus pear (O. ficus-indica), they protect endothelium from cytokine-induced redox state alteration, through inhibition of expression of intercellular adhesion moplecule-1 (ICAM-1) (Gentile et al., 2004). Hence endothelial protective effect via both antioxidant and anti-inflammatory mechanism is evident for these natural pigments. The contribution of indicaxanthin (2) and betanin (1) in the biological activities of O. ficusindica fruit have been studied by many authors. This includes haemolysis studies where the compounds prevent toxicity induced by cumene hydroperoxide (Tesoriere et al., 2006).

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Protective effect in combination with vitamin E has also been studied where the two compounds shown favourable outcomes (Tesoriere et al., 2003, 2007). In human studies by Tesoriere et al. (2004a,b), both compounds have been reported to be bioavailable although indicaxanthin (2) appears to be absorbed a lot more (20 times better) than betanin (1). In this study, a randomized, crossover, double-blind study, was employed on 18 healthy volunteers receiving either 250 g fresh fruit pulp or 75 mg vitamin C twice daily for 2 weeks, with 6 weeks washout period between the treatments. After supplementation with cactus pear fruit, 8-epi-prostaglandin F2α (8-epi-PGF2α) and MDA decreased by approximately 30% and 75%, respectively; GSH:GSSG shifted towards a higher value; and LDL hydroperoxides were reduced by almost one-half. On the other hand, supplementation with vitamin C did not significantly affect any marker of oxidative stress. Interestingly, the level of biological activity with respect to LDL levels appears to be correlated with the plasma level of betanin and indicaxanthin. Other experiments by the same authors also suggested that these two pigments in the fruits O. ficus-indica serve as the antioxidant principles of the plant (Tesoriere et al., 2005). LDL in ex vivo from plasma enriched with betanin or indicaxanthin has also shown to be resistant to copper-induced oxidation (Tesoriere et al., 2003). Various studies have also reported the high vitamin C content of cactus pear fruits and an estimation of 20–40 mg/100 g has been suggested (El Kossori et al., 1998; Ramadan and Morsel, 2003a,b). The variability in ascorbic acid content of the fruits depending on fruit colours and/or genotypes has further been studied. For example, the red and purple prickly pear varieties are known to contain higher levels as compared to the green, even though the latter appear to be consumed in larger amount due to favourable pH/acidity level (Cansino et al., 2013; Zafra-Rojas et al., 2013). Methods of extraction to maximize the yield of ascorbic acid during industrial processing is also available (Cruz-Cansino et al., 2016). 13.6.2.2 Antidiabetic effects of cactus pear pigments Sutariya and Saraf (2017) studied the antidiabetic effect of betanin (1) from Opuntia elatior Mill by using the STZ-induced diabetes model in rats. Administration of 25, 50, and 100 mg/ kg p.o. for 8 weeks have been shown, not only to reduce blood glucose, but also attenuate the diabetic kidney injury as shown by the reduced level of proteinuria, serum creatinine, and BUN levels. Restoration of the antioxidant enzyme activities in kidney tissues and histological improvements of glomerular surface area for betanin treatment was recorded. Furthermore, betanin was able to modulate the mRNA and protein expression of transforming growth factor-β (TGFβ), type IV collagen, α-smooth muscle actin (α-SMA) and E-cadherin in the kidney (Sutariya and Saraf, 2017). In both Type 1 diabetes (T1D) and T2D, an increased level of expression of TGFβ in glomerular and tubular system of the kidney is well known. The crosstalk between ROS and TGFβ dysregulation in diabetes is also known as ROS can increase the protein level expression of TGFβ. Type IV collagen expression in the kidney is also augmented by STZ treatment along with α-SMA (marker of myofibroblasts). On the other hand, E-cadherin (common cell–cell adhesion molecule) is expected to decrease in diabetic kidney as cellular and tissue degradation means a loss of junctional proteins and/or adhesion molecules. The observed result for betanin (1) where LPO was suppressed and antioxidant status in renal tissue (reduction in thiobarbituric acid reactive substances (TBARS) level and

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increased the level of SOD and CAT); prevented morphological changes in diabetic kidney and normalization of protein expression dysregulation (Sutariya and Saraf, 2017) were thus in agreement with amelioration of diabetic nephropathy by betanin.

13.6.3 Other compounds The impressive list of polyphenolic compounds including phenolic acids, flavonoids and their derivatives are displayed in Section 13.4.2. These compounds are primarily responsible for the in vitro antioxidant effect of prickly pear (see Section 13.5.3.2). For example, the presence of rutin, isorhamnetin, quercetin, and kaempferol (Fig. 13.13) in the active fraction to account for the antioxidant effect of prickly pear has been reported (Ben-Saad et al., 2017). Rutin and isorhamnetin were also identified as principal components of the flavonoid fraction (Galati et al., 2003). Although most of the emphasis on the antidiabetic effect of prickly pear so far focused mainly on the polysaccharides and betalains, there is no doubt that the polyphenol composition could also contribute to these effects. The antidiabetic effect of these dietary polyphenols has been reviewed (Habtemariam and Varghese, 2014) and compounds such as quercetin and its glycosides possess antidiabetic effect both in vitro and in vivo (Habtemariam, 2011, Habtemariam and Lentini, 2015). As an active principle for prickly pear (O. ficus-indica), one of the best studied compound was isorhamnetin glycosides and elaborated with a little more detail herein. In high-fat diet (HFD)-induced obese mice, the dehydrated pads/stems extract (80% methanol, v/v) added to diet as 0.3% and 0.6% supplement for 12 weeks led to less weight gain, and reduced TC and LDL-C levels. A reduction in glucose and insulin level coupled with improvement of glucose tolerance (and HOMA-IR) was also in line with antidiabetic and antiobesity potential of O. ficus-indicus cladodes. Moreover, in animal models and in isolated pancreatic islets, higher insulin concentration for the extract treated group were observed; while stimulation of insulin secretion in vitro was shown to be associated with increased glucose transporter 2 (GLUT2) and peroxisome PPAR-γ mRNA content. All these data together with the increased energy expenditure by the extract in treated obese animals suggest potential application of prickly pear in the obesity-diabetes axis. While the size of adipocytes was shown to be reduced, hepatic IRS1 phosphorylation (at the yr-608 and S6 K thr-389 sites) was increased as hepatic steatosis was ameliorated, i.e. O. ficus-indica extract can increase insulin signalling in the liver in vivo. The authors did not isolate the individual active principles but the active fraction composed of isorhamnetin glycosides with the following composition by HPLC: • • • • •

Isorhamnetin-3-O-glucosyl-rhamnosyl-rhamnoside (32.6%) Isorhamnetin-3-O-glucosyl-rhamnosyl-pentoside (29.5%) Isorhamnetin-3-O-hexose-methylpentose-pentoside (6.1%) Isorhamnetin-3-O-glucosyl-pentoside (12.0%) Isorhamnetin-3-O-glucosyl-rhamnoside (19.8%)

These active principles are not fully characterized but as an active fraction, they collectively suppressed hepatic lipid content associated with reduced mRNA expression of the endoplasmic reticulum stress markers (e.g. ATF6, XBP1, and CHOP) along with reduced mRNA expression level of key lipogenic factors such as the transcription factor sterol regulatory

D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

13.7 Diuretic effects

463

FIG. 13.17 Mechanisms of antiobesity, lipid lowering, and antihyperglycaemic effects of O. ficus-indica and isorhamnetin glycosides as active principles (Rodrı´guez-Rodrı´guez et al., 2015). The triple action of the extract and active fractions are shown.

element-binding protein 1 (SREBP-1), fatty acid synthase (FAS) and stearoyl CoA desaturase 1 (SCD-1). In line with increased fatty acid oxidation induced by isorhamnetin glycosides, the levels of carnitine palmitoyltransferase-1 (CPT-1) and alternative oxidase (AOX) were increased. Hence, the active principles together as well as the crude extract of prickly pear cladodes could increase energy expenditure and suppress adipogenesis while boosting insulin release (Fig. 13.17). In this study (Rodrı´guez-Rodrı´guez et al., 2015), the HDL-C level was suppressed along with LDL-C and TG but the improvement of lipid profile, antiobesity and antidiabetic potential appears to be validated by this study (Fig. 13.7).

13.7 Diuretic effects The diuretic effect of the fruits, flowers and cladode as dehydrated water extract Cacti-Nea® have been demonstrated by various authors (Bisson et al., 2010; Galati et al., 2002). When administered at the dose of 240 mg/kg (p.o. for 7 days), Cacti-Nea® was shown to increase the urine volume as well as reduce body weight gain in rats (Bisson et al., 2010). On the basis that the diuretic effect of Cacti-Nea® having no impact on the natriuresis and kaliuresis, an effect on the proximal tubules as possible mechanism of action was suggested. Even though the authors did not exclude organic compounds as active constituents, they speculated the high potassium level in the fruits as a potential diuretic principles. In this direction, the high potassium level of the pulp that reaches 900 and 2170 mg/kg (Piga, 2004) with potential osmotic effect on the proximal tubule have been suggested. The chronic diuretic effects of Cacti-Nea® was generally reported to be comparable with that of the standard drug hydrochlorothiazide and its chronic

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13. The chemical and pharmacological basis of prickly pear cactus

oral administration increase the blood globular levels of GPx (Bisson et al., 2010). The 15% infusion of cladodes, flowers and non-commercial fruits (as industrial waste) could also induce diuresis: effect more marked for the fruit (Galati et al., 2002). More research in this field, particularly effect on hypertension associated with diabetes, is need.

13.8 Human studies on prickly pears The study by Frati et al. (1990) gave some insight into the acute hypoglycaemic effect of cactus pear cladode. They administered unprocessed grilled O. streptacantha stems in T2D and healthy fasting subjects to see the short-term effect on blood glucose and insulin levels. While they observed a reduction of blood glucose within 60 min in diabetic patients even below the control healthy level, they also noted similar reduction for blood insulin. These glucose and insulin lowering effect was not observed in healthy subjects. In a similar experimental protocol using eight T2D diabetic subjects and six healthy individuals, oral administration of 500 g of broiled stems of O. streptacantha has also been shown to lower serum glucose but not C-reactive peptide (CRP) in diabetic patients while no effect was observed in healthy subjects (Frati et al., 1991). They also reported that a second dose of O. streptacantha, 2 h after the first one, did not improve the acute hypoglycaemic effect. Various experiments on the acute antidiabetic effect of O. streptacantha and other cactus pear preparations on T2D patients have also been demonstrated by various authors (Frati et al., 1990; Frati-Munari et al., 1983, 1987, 1988a,b, 1989, 1990). Another acute experiment was also performed on nopal extract (capsules containing 10.1 g extract) from O. ficus-indica where the extract failed to lower blood glucose in T2D patients but was effective in lowering postprandial hyperglycaemia after dextrose load (Frati-Munari et al., 1989). Similarly, Lopez-Romero et al. (2014) conducted a small pilot study on 14 patients with T2D patients consuming fresh nopal in high carbohydrate breakfast with or without 300 g steamed fresh nopal. Even though the study was rather preliminary and the control group population was small (n ¼ 7) with a different age and BMI profile than the patients, they reported a reduction in postprandial blood glucose and serum insulin as well as increased antioxidant activity in healthy people and patients with T2D. In a trial by Linare`s et al. (2007), a patented dehydrated leaves of O. ficus-indica, NeOpuntia, was studied at one centre. Their randomized placebo controlled 6 weeks study was doubleblind in nature with 68 women (59 subjects completed) of ages 20–55, with metabolic syndrome and a BMI 25–40. They have reported that the 42 females above 45 years of age, had a significant increase in HDL-C levels with NeOpuntia and a tendency towards decreased TG levels after intake of 1.6 g per meal of NeOpuntia for up to 42 days. At the same time, there was a decrease in LDL-C levels (Linare`s et al., 2007). The study by Godard et al. (2010) assessed the effect of OpunDia supplementation in prediabetic males and females. Their double-blind placebo controlled study was of 16-week period and included participants (age range of 20–50 years) taking either 200 mg OpunDia (n ¼ 15), or placebo (n ¼ 14). The study was an acute phase study with OGTT with a 400 mg bolus of OpunDia given 30 min before orally ingesting a 75 g glucose drink. The study showed the acute blood glucose lowering effects and the long-term safety of the proprietary product OpunDia, thus supporting the traditional use of Opuntia ficus-indica for blood glucose management. D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

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Other small trials include that by Wolfram et al. (2003) that involve daily consumption of 250 g in eight healthy volunteers and eight patients with mild familial heterozygous hypercholesterolemia on various parameters of platelet function. Daily prickly pear consumption, beside its action on lipids and lipoproteins, reduced the platelet proteins (platelet factor 4 and beta-thromboglobulin), ADP-induced platelet aggregation and improved platelet sensitivity (against PGI2 and PGE1) in volunteers as well as in patients. The same group also conducted another pilot study on prickly pear (O. robusta) on glucose- and lipid-metabolism in nondiabetics with hyperlipidaemia (Wolfram et al., 2002). It was reported that administration of prickly pear edible pulp (250 g/day) for 8 weeks could decrease TC (12%), LDL-C (15%), apolipoprotein B (9%), TG (12%), fibrinogen (11%), blood glucose (11%), insulin (11%) and uric acid (10%), while body weight, high-density lipoprotein-cholesterol, apolipoprotein A-I, and lipoprotein(a) remained unchanged. In a randomized double-blind cross-over study involving 11 subjects, oral intake of O. ficus-indica cladode and fruit skin (OpunDia™, 1000 mg) in combination with leucine was shown to enhance the carbohydrate-induced insulin release (Deldicque et al., 2013). Another trial was a crossover protocol on heart rate (HR) variability in high-level athletes where O. ficus-indica given as a diet supplement was shown to decreases HR (Schmitt et al., 2008). Hence, while noting the human studies on prickly pear is far from complete, some favourable effects on glycaemic and lipid control appears to be demonstrated. Trials under chronic diabetic condition and treatment regimen with well-designed large-scale study is still awaiting.

13.9 General summary and conclusions Mexico being the hotspot of cactus pear or Opuntia species origin and diversity, it is also a country where large-scale commercial production occur. Represented by the most utilized species, O. ficus-indica, numerous species are introduced throughout the world occupying mainly drier regions but also in temperate regions including Europe. The stems or nopales or cladodes are used as food and source of deity fibres both for human consumption and as animal fodders. The fruits with an array of fascinating colours due to the betalains composition are source of food and natural dyes among other uses. The medicinal uses of the various plant parts including for diabetes have seen these natural resources appearing as dietary supplements with various proprietary brands developed and are already in use. Opuntia species as cactus exhibit morphological and biochemical adaptations to thrive in drier environments. The efficiency of propagation with a small piece of the stem/cladode giving rise to the whole plant often make the species notorious weeds growing in roadside and unused lands. This very nature of efficient biomass production with little care and in land least suited for other food crops have given even more motivation in recent years to exploit cactus pear for its diverse potential applications. This chapter displayed the vast array of compounds isolated from cactus pear. The flavonoids composition is dominated by isorhamnetin glycosides though others including quercetin and kaempferol both as aglycones and glycosides are characterized. Phenolic acids such as hydroxycinnamic acids and gallic acid derivatives are also known to occur. These compounds are primarily responsible for the direct radical scavenging properties as well as inhibition of ROS generation or action.

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The betalains primarily betanin and indicaxanthin are responsible for the diverse colouration of the fruits which also attracted lots of interest as natural colouring agents. In addition to displaying antioxidant effects, they have been shown to induce antidiabetic effects in the various animal models. The polysaccharides are the other class of compounds that are shown to account for the antidiabetic effects both the fruits and cladodes/stems of the cactus pear plants. Inhibition of carbohydrate digestion through action on digestive enzymes or glucose absorption, modulation of insulin signalling and genes expression appear to be involved for the antidiabetic, antiobesity and lipid lowering effects. Although evidence in animal models and some studies in human subjects show antidiabetic effects, more clinical evidence of efficacy is required to confirm the true potential of these plants as antidiabetic agents. Most of the studies appear to substantiate the acute glucose lowering effects in OGTT both in animals and humans. Chronic treatment and well-designed large-scale clinical trial is still needed. The data available so far suggest antiobesity and lipid lowering effect but the diversity of the plants in species and variety means data on a more standardized products showing unequivocal bioactivity is needed. We are not still in a position to see cactus pear as a replacement to metformin but modulation of diabetes is well expected by these plants either by their own or perhaps as an adjuvant therapy with existing antidiabetic drugs.

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An anti-inflammatory principle from cactus. Fitoterapia 72, 288–290. Piga, A., 2004. Cactus pear: a fruit of nutraceutical and functional importance. J. Prof. Assoc. Cactus 6, 9–22. Piga, A., del Caro, A., Pinna, I., Agabbio, M., 2003. Changes in ascorbic acid, polyphenol content and antioxidant activity in minimally processed cactus pear fruits. LWT: Food Sci. Technol. 36, 257–262. Ramadan, M.F., Morsel, J.-T., 2003a. Recovered lipids from prickly pear (Opuntia ficus-indica (L.) Mill) peel: a good source of polyunsaturated fatty acids, natural antioxidant vitamins and sterols. Food Chem. 83, 447–456. Ramadan, M.F., Morsel, J.T., 2003b. Oil cactus pear (Opuntia ficus-indica). Food Chem. 82, 339–345.

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Wolfram, R., Budinsky, A., Efthimiou, Y., Stomatopoulos, J., Oguogho, A., Sinzinger, H., 2003. Daily prickly pear consumption improves platelet function. Prostaglandins Leukot. Essent. Fatty Acids 69 (1), 61–66. Yahia, E.M., Mondrago´n-Jacobo, C., 2011. Nutritional components and anti-oxidant capacity of ten cultivars and lines of cactus pear fruit (Opuntia spp.). Food Res. Int. 44, 2311–2318. Yang, N., Zhao, M., Zhu, B., Yang, B., Chen, C., Cui, C., et al., 2008. Anti-diabetic effects of polysaccharides from Opuntia monacantha cladode in normal and streptozotocin-induced diabetic rats. Innov. Food Sci. Emerg. Technol. 9, 570–574. Yeddes, N., Cherif, J.K., Guyot, S., Sotin, H., Ayadi, M.T., 2013. Comparative study of antioxidant power, polyphenols, flavonoids and betacyanins of the peel and pulp of three Tunisian Opuntia forms. Antioxidants 2, 37–51. Zafra-Rojas, Q.Y., Cruz-Cansino, N., Ramı´rez-Moreno, E., Delgado-Olivares, L., Villanueva-Sa´nchez, J., Alanı´sGarcı´a, E., 2013. Effects of ultrasound treatment in purple cactus pear (Opuntia ficus-indica) juice. Ultrason. Sonochem. 20, 1283–1288. Zenteno-Ramirez, G., Juarez-Flores, B.I., Aguirre-Rivera, J.R., Ortiz-Perez, M.D., Zamora-Pedraza, C., RendonHuerta, J.A., 2015. Evaluation of sugars and soluble fiber in the juice of prickly pear varieties (Opuntia spp.). Agrociencia 48, 141–152. Zhao, M., Yang, N., Yang, B., Jiang, Y., Zhang, G., 2007. Structural characterization of water-soluble polysaccharides from Opuntia monacantha cladodes in relation to their anti-glycated activities. Food Chem. 105, 1480–1486. Zhao, L.Y., Lan, Q.J., Huang, Z.C., Ouyang, L.J., Zeng, F.H., 2011. Antidiabetic effect of a newly identified component of Opuntia dillenii polysaccharides. Phytomedicine 18 (8–9), 661–668. Zourgui, L., Golli, E.E., Bouaziz, C., Bacha, H., Hassen, W., 2008. Cactus (Opuntia ficus-indica) cladodes prevent oxidative damage induced by the mycotoxin zearalenone in Balb/C mice. Food Chem. Toxicol. 46, 1817–1824.

Further reading Abdel-Nabey, A.A., 2001. Chemical and technological studies on prickly pear (Opuntia ficus indica) fruits. Alex. J. Agric. Res. 46, 61–70. Brahmi, D., Bouaziz, C., Ayed, Y., Ben Mansour, H., Zourgui, L., Bacha, H., 2011a. Chemopreventive effect of cactus Opuntia ficus indica on oxidative stress and genotoxicity of aflatoxin B1. Nutr. Metab. 8, 73. Brahmi, D., Ayed, Y., Bouaziz, C., Zourgui, L., Hassen, W., Bacha, H., 2011b. Hepatoprotective effect of cactus extract against carcinogenicity of benzo(a)pyrene on liver of Balb/C mice. J. Med. Plant Res. 5, 4627–4639. Brahmi, D., Ayed, Y., Hfaiedh, M., Bouaziz, C., Mansour, H.B., Zourgui, L., Bacha, H., 2012. Protective effect of cactus cladode extract against cisplatin induced oxidative stress, genotoxicity and apoptosis in balb/c mice: combination with phytochemical composition. BMC Complement. Altern. Med. 12, 111. Bwititi, P., Musabayane, C.T., Nhachi, C.F., 2000. Effects of Opuntia megacantha on blood glucose and kidney function in streptozotocin diabetic rats. J. Ethnopharmacol. 69, 247–252. Chavez-Santoscoy, R.A., Gutierrez-Uribe, J.A., Serna-Saldı´var, S.O., 2009. Phenolic composition, antioxidant capacity and in vitro cancer cell cytotoxicity of nine prickly pear (Opuntia spp.) juices. Plant Foods Hum. Nutr. 64, 146–152. Ibanez-Camacho, R., Meckes-Lozoya, M., 1983. Effect of a semipurified product obtained from Opuntia streptacantha L. (a cactus) on glycemia and triglyceridemia of rabbit. Arch. Invest. Med. 14, 437–443. Majdoub, H., Roudesli, S., Picton, L., Le Cerf, D., Muller, G., Grisel, M., 2001. Prickly pear nopals pectin from Opuntia ficus-indica physico-chemical study in dilute and semi-dilute solutions. Carbohydr. Polym. 46, 69–79. Meckes-Lozyoa, M., Roman-Ramos, R., 1986. Opuntia streptacantha: a coadjutor in the treatment of diabetes mellitus. Am. J. Chin. Med. 14, 116–118. Zorgui, L., Ayed-Boussema, I., Ayed, Y., Bacha, H., Hassen, W., 2009. The antigenotoxic activities of cactus (Opuntia ficus-indica) cladodes against the mycotoxin zearalenone in Balb/c mice: prevention of micronuclei, chromosome aberrations and DNA fragmentation. Food Chem. Toxicol. 47, 662–667. Zou, D.M., Brewer, M., Garcia, F., Feugang, J.M., Wang, J., Zang, R., et al., 2005. Cactus pear—a natural product in cancer chemopretection. Nutr. J. 4, 25.

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C H A P T E R

14 The chemical and pharmacological basis of pumpkins (Cucurbita species) as potential therapy for type-2 diabetes O U T L I N E 14.1 Introduction

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14.3 Ornamental, food, and medicinal values

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14.4 Antidiabetic effects of C. pepo 479 14.4.1 Antidiabetic effects in animals models 479 14.4.2 Chemical components of C. pepo 480

14.5 Antidiabetic effect of C. moschata 487 14.6 Antidiabetic effects of C. ficifolia

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14.7 Antidiabetic effect of C. maxima

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14.8 Antidiabetic effect of C. mixta

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14.9 Conclusions

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References

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Further reading

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14.1 Introduction Pumpkins represent a diverse group of plants collectively known as either pumpkins or squash in the genus Cucurbita of the family Cucurbitaceae. The family comprise valuable edible plants including cucumbers, melons, watermelon and loofahs (Luffa species), pumpkins, squashes, and gourds. In the latter case, the family is often called the gourd family. Their characteristic feature is being mostly herbaceous climbing vines and the male and female flowers are separate. The family is represented with well-over 118 genera and 845 species which grow throughout the tropics and warm temperate regions. Cucurbita is, however, a small genus with around 27 species, of which, the most cultivated and economically important species

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are C. pepo, C. moschata, C. maxima, C. mixta, and C. ficifolia. While the name squash is widely applied to the various fruit cultivars of C. pepo, C. mixta, or C. maxima that are mostly used in their immature form, the pumpkins are normally associated to the mature edible fruits of any Cucurbita fruits that may be eaten in any form—vegetable, juice, cooked as pie, etc. Numerous species and varieties of the domesticated edible fruit named as pumpkins occur all over the world. The diversity of their fruit size is astonishing and range from few grams to hundreds of kilograms. The most common pumpkin is of course C. pepo L. which has a history of thousands of years of cultivation. Others such C. maxima, C. moschata, and C. argyrosperma are also called pumpkins or squashes. The three most economically important member of the genus that represent pumpkins are, however, C. pepo L. (sumer squash), C. maxima Duchesne (winter squash), and C. moschata Duchesne. The pumpkins as well as various squashes are consumed in various forms and their nutrition value include source of carbohydrate as well as fatty acids, fibre, carotenoids, vitamins, and minerals. They are also equally important as animal fodder. The common morphological features of pumpkins are shown in Fig. 14.1. As an ornamental plant, the broad leaves, creeping stems that grows horizontally and spread in just few weeks of the summer growth, and beautiful

FIG. 14.1 Common morphological features of the pumpkin plant. The male flower (A), female flower (B), floral detail (C), and fruit development (D) in pumpkin are shown.

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FIG. 14.2

Variations of pumpkin fruits. The pumpkin fruits could vary in size, colour, and shape based on species, verities, and many other factors. Some common forms from those growing in our experimental garden are shown.

male/female separate flowers are some common features of pumpkin. The fruits of pumpkins also show extensive diversity in size, shape, and colour and some from our experimental gardens are shown in Fig. 14.2.

14.2 Botanical and taxonomic perspectives Pumpkins are among the Cucurbitaceous plants which are believed to be originated in the tropical America and introduced to Europe around 500 years ago (Whitaker, 1947). The most common domesticated pumpkins belong to the C. argyrosperma, C. ficifolia, C. maxima, C. mostacha and C. pepo along with other wild species. Their taxonomic relationship with the plant kingdom is as follow: Kingdom: Plantae—Plants Subkingdom: Tracheobionta—Vascular plants Superdivision: Spermatophyta—Seed plants

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Division: Magnoliophyta—Flowering plants Class: Magnoliopsida—Dicotyledons Subclass: Dilleniidae Order: Violales Family: Cucurbitaceae—Cucumber family Genus: Cucurbita L. The botanical description or identification key for the five most important Cucurbita species is as follow (Whitaker and Davis, 1962): • Plant perennial, seeds black, or tan: C. ficifolia; • Plants annual; seeds white, buff or tawey: four species, C. moschata, C. pepo, C. maxima, and C. mixta; • Penducle hard, smoothly grooved, flared at fruit attachment foliage non-stipulate: C. moschata; • Stems hard, angular; peduncle angular, grooved; peduncle hard, sharply angular, grooved, foliage stipulate: C. pepo; • Plants annual; seed white, buff, or towey; stem soft, round; peduncle soft, terete, enlarged by soft cork; C. maxima; • Peduncle hard, greatly enlarged in diameter by hard cork, not flared at fruit attachment; foliage non-stipulate: C. mixta. The variation in sizes and colours of pumpkins are exhibited in a rather simplistic presentation by Boisset (1997) in a book entitled Pumpkins and Squashes. The three most edible species are described to be distinguished by their fruit stems or peduncles with “…C. maxima have large, corkey or spongy stems which are round and larger at the base (nearest the fruit). The stems of C. moschata are angled and fleshy, rather than rounded and ropy, opening out toward the fruit, becoming distinctively star-shaped and knobby. The stems of C. pepo are rigid all around, widening slightly at the base.” Cucurbita pepo subspecies pepo is the most common and highly variable pumpkin owing to the various varieties resulting from the extended domestication period. C. pepo is known to be native to the America probably in Mexico. Archeological sites in Mexico showing the domestication of C. pepo around 7000 BC or some 10,000 years ago have been documented (Smith, 1997). This length of domestication and selection process appear to be responsible for the incredible diversity in this group that culminated in sub species, and varieties and cultivars. It is believed that the plant was introduced to Europe during the 16th century from which it was distributed to all over the world (Paris, 2001). C. pepo is largely a cultivated pumpkin representing the commercial production all under extensive cultivars, varieties and even sub-species. The sub-species of C. pepo include the following: 1. 2. 3. 4. 5.

C. pepo ssp. pepo, C. pepo ssp. ovifera var. ovifera, C. pepo ssp. ovifera var. texana, C. pepo ssp. ovifera var. ozarkana, C. pepo ssp. Fraternal

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While the first two of the above-mentioned subspecies are highly used as crops, the rest are known to occur as wild varieties (Kates et al., 2017). The presence of several cultivars also means differences in the chemical composition of the various pumpkin products (Nawirska-Olsza nska et al., 2013).

14.3 Ornamental, food, and medicinal values In addition to the C. pepo fruits being the major source of food, the flowers and young leaves are also known to be eaten as vegetables. The seeds which are extensively used for oil production are edible with several health claims including a rich source of zink. The ornamental value of the plant should also be acknowledged with several varieties of the fruits in shape and colour occur. Paris (2001) described the variations as follow: “acorn, cocozelle, crookneck, scallop, straightneck, vegetable marrow, and zucchini squash; the orange, grooved, Halloween and pie pumpkins of the United States and Canada; black-green and green-striped, sometimes naked seeded pumpkins of Europe and Asia Minor; ribbed, grey-green, and green-striped pumpkins of Mexico; as well as small-fruited, often striped, ornamental and wild gourds, and assorted other forms.” Cucurbita maxima Duchesne. is also called winter squash, giant pumpkin, hubbard squash, kabocha squash or pumpkin. The giant pumpkin is a good example of this group to represent variations in fruit size but other variations also include in colour (e.g. red, orange, pink, or blue green) and shape of fruits in a range of buttercup, hubbard, banana, and some other kinds of squash, and the turban gourds As with C. pepo, the species exhibit extensive diversity with respect to fruit shape and size as well as other morphological variations. From the horticultural point of view, the various groups of C. maxima include (1) Australian Blue, (2) Banana, (3) Buttercup, (4) Hubbard, (5) Mammoth Pumpkin (or Show Pumpkin), (6) Turban, and (7) Zapallito; and many others (Kaz´mi nska et al., 2017). Cucurbita moschata Duchesne—represent Butternut squash, Seminole pumpkin, canned pumpkin, crookneck squash. This is known to grow best in humid tropics. It is believed to be originated in the North Western part of South America. It is now well distributed throughout tropics of Asia, America, and Africa, The economic importance of C. ficifolia is not as high as other pumpkins particularly C. pepo but its medicinal value as antidiabetic agent is emerging to give the plant lots of significance. The wound healing properties, uses for haemorrhoids and fever have also been reported. The antidiabetic effect in the Americas has been extensively reported (AlarconAguilar et al., 2002). In addition to their nutritional value, the pumpkin fruits are extensively used for Halloweens parties and related tradition in the Western countries (Fig. 14.3). While the fruits are eaten in various cooked preparations, the seeds are also used as source of oil and as nutritional supplements. For these purposes, both the unprocessed seeds and seeds with shells removed and ready to eat are all available in food supermarkets and health shops (see Fig. 14.4.). Various species of pumpkins as well as the fruit juice of C. maxima are known to be used for their hypoglycaemic effects in Mexico (Andrade-Cetto and Heinrich, 2005). The medicinal

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FIG. 14.3 Pumpkin in the Western culture. Beyond the nutritional value of pumpkin as human food, its production in Western countries is to meet the large demand for the Halloweens’ day: a celebration observed in several countries on 31 October. Halloween parties and children in Halloween costumes going around the neighbourhood for trick-or-treating and household leaving carved pumpkins into jack-o’-lanterns are common traditions.

FIG. 14.4

Pumpkin seeds available to the public. The various food stores and health shops present pumpkin seeds as whole roasted seeds (A) or as hulled or shelled pumpkin seeds (B).

value especially in food supplement applications may not explicitly indicate the source species of pumpkin, however. Pumpkin seeds, pumpkin oil, or related products are thus sold as a generic product that could greatly vary in their chemical compositions. Considering the growing reports on pumpkins for type 2 diabetes (T2D) therapy and their popularity as dietary supplements, this chapter is designed to scrutinize the chemical and pharmacological basis of such therapeutic applications.

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14.4 Antidiabetic effects of C. pepo 14.4.1 Antidiabetic effects in animals models Sedigheh et al. (2011) directly administered (1 or 2 g/kg, for 4 weeks) the dried fruit powder of C. pepo L. orally (p.o.) in alloxan-induced diabetic rats. The antidiabetic and lipid lowering effects of the preparation was evident as a decrease in glucose, triglycerides (TG), low density lipoprotein-cholesterol (LDL-C), and C-reactive protein (CRP) were demonstrated for the lower dose; while the higher dose further reduced the level of total cholesterol (TC). Along with suppression of the inflammatory marker, CRP, improvement in the structural integrity of the Langerhans islets was also shown as revealed by histology. Other experiments by the same authors (Asgary et al., 2010) revealed the antidiabetic effect of the fruits in alloxaninduced diabetic rats model as evidenced from amelioration of the diabetes-induced liver damage and restoration of the diabetes-induced abnormalities in fasting blood glucose (FBG), cholesterol, TG, LDL-C, and high density lipoprotein-cholesterol (HDL-C) levels. Sedigheh et al. (2011) also showed that administration of the fruits powder (1 or 2 g/kg, p.o. for 4 weeks) of C. pepo in alloxan-induced diabetic rats have similar effects: decrease glucose, TG, LDL, CRP, and cholesterol, while β-cells histology was improved. Liu et al. (2018) have shown that pumpkin (C. pepo, lady godiva) polysaccharide (1000 mg/ kg, intragastric (i.g.) for 4 weeks) administered in the high-fat diet (HFD) and streptozotocin (STZ)-induced diabetic rats could improve insulin tolerance; decrease serum glucose, TC, and LDL-C; while the level of HDL-C was increased. Furthermore, change in the composition of the gut microbiota where selective enrichment of the Bacteroidetes, Prevotella, Deltaproteobacteria, Oscillospira, Veillonellaceae, Phascolarctobacterium, Sutterella, and Bilophila were observed for the treatment group with pumpkin polysaccaharise (Liu et al., 2018). There is evidence to suggest that antidiabetic therapy by the current drugs including metformin could change the gut microbiota (Liu et al., 2018; Shin et al., 2014) with implication of improved glucose homeostasis. The effect of probiotics as weight lowering agents has also been associated with the gut microbiota as evidenced from the study on Lactobacillus supplementation (Park et al., 2013). The data provided by Lie et al. (2018) is therefore a good starting point to further explore the microbiota-based mechanism of action of pumpkin polysaccharides as antidiabetic agents. The authors also asserted that short chain fatty acids released from the selected microbiota may contribute to the antidiabetic effect that needs further investigation. In a diet-induced obese rats, the hydro-alcoholic extract of pumpkin (100 and 200 and 400 mg/kg, 6 weeks) was also shown to decrease TG, LDL-C, and liver enzymes while the levels of HDL-C and glutathione (GSH) increased (Ghahremanloo et al., 2017). Abou Seif (2014) investigated the effect of pumpkin (C. pepo L) seeds oil (50 mg/kg, 3 times per week for 3 weeks) in alcohol-induced hepatotoxicity and oxidative stress in rats. It improved liver oxidative stress markers and antioxidant defense system in normal and alcohol-administered rats. The increase in GSH level as well as glutathione-S-transferase (GST) and catalase (CAT) activities and amelioration of the elevated activities of alanine transaminase (ALT) and alkaline phosphatase (ALP), total bilirubin (not aspartate transaminase (AST), and γ-Gamma-glutamyl transferase (γ-GT) activities); along with improved histology of the liver, all suggested the beneficial health promoting effect of the seeds oil.

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The experiment by Gossell-Williams et al. (2008) also suggested the potential health benefit of pumpkin oil (undescribed origin) in ovariectomized rats supplemented with the oil (40 mg/kg, p.o. for 5 days per week for 12 weeks). They reported that the higher level of TC, LDL-C, TG, and lower HDL-C in ovariectomized animals could be reversed by the oil. The blood pressure (BP) lowering effect (in both normal and ovariectomized) was also demonstrated from the suppression of both systolic (SBP) and diastolic (DBP) BP in these animals. The antihypertensive and cardioprotective effect of pumpkin seeds oil (40 or 100 mg/kg) have also been studied in nitric oxide (NO) synthase (NOS) inhibitor (N(ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME))-induced hypertension in rats (El-Mosallamy et al., 2012). After oral administration of the oil for 6 weeks, the following beneficial effects were recorded: • Reduction of the increased BP induced by L-NAME; • Amelioration of L-NAME-induced electrocardiogram, (ECG) changes including prolongation of the RR interval, increased P wave duration, and ST elevation; • Reduction of oxidative stress induced by L-NAME—i.e. reduced malondialdehyde (MDA) level; • Increased the level of NO as revealed by its metabolites levels; • Protected against L-NAME-induced pathological alterations in the heart and aorta. The C. pepo seeds extract obtained by extraction of with nonpolar solvent, petroleum ether, and polar hydroalcohol have also been investigated in the STZ-induced diabetes rats (Kaur et al., 2017). After 45 days of administration (100, 200, and 400 mg/kg), both extracts have been shown to improve body weight loss, reduce blood glucose level, and abolish the kidney hypertrophy index. In the latter case, a reduction in the levels of creatinine, blood urea nitrogen, TC, TG, advanced glycation end products (AGEs), and albumin in urine were recorded. Hence, antidiabetic effect via amelioration of oxidative stress and nephroprotective effect are evident for the seeds. With the hope of identifying the active principles responsible for the various health benefit claims of pumpkin, numerous chemical analysis studies were undertaken. For example, Quanhong et al. (2005) isolated protein-bound polysaccharide that consist mainly of polysaccharide (approximately 41.21%) and protein (approximately 10.13%) and showed hypoglycaemic activity in alloxan-induced diabetic rats. While increasing the levels of serum insulin, reduction in blood glucose including improvement in glucose tolerance was noted for the polysaccharide at the dose of 500 or 1000 mg/kg. Many other studies have also revealed the antidiabetic effect of pumpkin polysaccharides in animal models. This also includes alloxan-induced diabetes in rats (Xiong and Cao, 2001). In the following section, the various classes of compounds isolated from C. pepo and their possible role as antidiabetic agents are outlined.

14.4.2 Chemical components of C. pepo The nutritional composition and some common antioxidant effect of pumpkin (C. pepo) has been widely reported (e.g. Oloyede et al., 2012). In this section, compounds that belong to the various classes of plant secondary metabolites with potential effect on diabetes pathology are discussed. D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

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14.4 Antidiabetic effects of C. pepo

14.4.2.1 Polyphenols By using liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) analysis, Nawirska-Olsza nska et al. (2013) identified the following compounds in pumpkin seeds: caffeic acid tri-hexoside (unknown), p-hydroxybenzoic acid (1), vanillic acid (3), caffeic acid (5) derivative (unknown), p-coumaric acid (4), sinapic acid (6), caffeic acid derivative (unknown), and dihydroxybenzoic acid (2). Phenolic glycosides have also been isolated from the seeds of C. pepo. In a classical experiment, Li et al. (2005) isolated eight new phenolic glycosides that they names as cucurbitosides F-M (Fig. 14.5): 4-(2-hydroxyethyl)phenyl 5-O-(2-S-2methylbutyryl)-β-D-apiofuranosyl(l!2)-β-D-glucopyranoside (7), 4-(2-hydroxyethyl)phenyl 5-O-(3-methylbutyryl)-β-D-apiofuranosyl(l!2)-β-D-glucopyranoside (8), 4-(2-hydroxyethyl) COOH

OH

COOH

OH

Glc = O R

R2

OH 1 R=H p-Hydroxybenzoic acid 2 R=OH Protocatechuic acid 3 R-OMe Vanillic acid

R1

OH

4 R1=R2=H p-Coumaric acid 5 R1=OH, R2=H Caffeic acid 6 R1=R2=OMe Sinapic acid

A O B

OH HO HO R2O

OH

HO

O

O

RO

OH OH

O

OH O

H O

R1O

MeO

OH HO H

HO F

MeO

OH OH

HO H OH

MeO OH

HO

19 R1=R2=H 20 R1=Glc, R2=H 21 R1=H, R2=Glc

O

H2N

O

OMe

OR2

N

E

14 R=A Cucurbitoside K 15 R=C Cucurbitoside L 16 R=D Cucurbitoside M 17 R=E 18 R=F

OMe

C D

O

OH OH

7 R1=H, R2=A Cucurbitoside F 8 R1=H, R2=B Cucurbitoside G 9 R1=H, R2=C Cucurbitoside H 10 R1=H, R2=D Cucurbitoside I 11 R1-OMe, R2=A Cucurbitoside J 12 R1=H, R2=A 13 R1=H, R2=E

MeO

O

HO

R1

O

HO H

O

OH

O

OH

HO

22

23

FIG. 14.5 Phenolic compounds of C. pepo.

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14. The chemical and pharmacological basis of pumpkins

phenyl 5-O-nicotinyl-β-D-apiofuranosyl(l!2)-β-D-glucopyranoside (9), 4-(2-hydroxyethyl)phenyl 5-O-(4-aminobenzoyl)-β-D-apiofuranosyl(l!2)-β-D-glucopyranoside (10), 4-(2hydroxyethyl)-2-methoxyphenyl 5-O-(2-S-2-methylbutyryl)-β-D-apiofuranosyl(l!2)-βD-glucopyranoside (11), 4-(hydroxymethyl)phenyl 5-O-(2-S-2-methylbutyryl)-β-D-apiofuranosyl (l! 2)-β-D-glucopyranoside (14), 4-(hydroxymethyl)phenyl 5-O-nicotinyl-β-D-apiofuranosyl (l! 2)-β-D-glucopyranoside (15), and 4-(hydroxymethyl)phenyl 5-O-(4-aminobenzoyl)-βD-apiofuranosyl(l! 2)-β-D-glucopyranoside (16). The antidiabetic effects of polyphenolic compounds are well understood and some caffeic acid derivatives, as revealed for other pumpkin species, are shown to display antidiabetic effects (see Section 14.5). The other polyphenolic compounds of significance are lignans 19–23 which were isolated from the seeds along with phenolic glycosides (12, 13, 17, 18) (Kikuchi et al. (2015). Their pharmacological effects related to diabetes are also discussed in the following section. 14.4.2.2 Terpenoids One of the diverse group of secondary metabolites isolated from pumpkins appear to be the triterpenes. These are particularly important for the seeds as numerous novel compounds have been isolated from them in the past few decades. From the seeds of C. pepo (zucchini variety), Appendino et al. (1999) have isolated two interesting multiflorane triterpenoid esters that bear the para-aminobenzoic acid moiety (Fig. 14.6). The compounds were 3-O-p-Aminobenzoyl-29-Obenzoylmultiflora-8-ene-3α,7β,29-triol (25) and 3-O-p-Aminobenzoyl-29-Obenzoylmultiflora-7,9(11)-diene-3α,29-diol (27). A further work by Appendino et al. (2000) has added two more novel multiflorane p-aminobenzoates, identified as 7-epi zucchini factor A (26) and debenzoyl zucchini factor B (28). Even though multiflorane p-aminobenzoates were not detected, the presence of bryonolic acid (24) has been reported in zucchini sprouts. The study by Tanaka et al. (2013) on the seeds of C. pepo (Zuchini variety) also led to the isolation of three novel multiflorane-type triterpenoids, 3α-p-nitrobenzoylmultiflora-7:9(11)-diene-29-benzoate (29), 3α-acetoxymultiflora-7:9(11)-diene-29-benzoate (30), and 3α-acetoxymultiflora-5(6):7:9 (11)-triene-29-benzoate (31), along with two known related compounds 3α-p-aminobenzoylmultiflora-7:9(11)-dien-29-benzoate (27) and 5α,8α-peroxymultiflora-6:9(11)-diene-3α,29dibenzoate (32). The antidiabetic effects of these compounds is yet to be determined but their cytotoxic activities in cancer cell lines as well as their inhibitory activities on melanogenesis have been reported (Tanaka et al., 2013). In the study by Kikuchi et al. (2015), an impressive list of compounds isolated from C. pepo (zucchini) seeds has been presented. This includes triterpenes, diterpenes, lignans, and phenolic glycosides. The structures of the three triterpene were novel compound (24S)-stigmasta-7,22E,25-trien-3-one (33) and known compounds 34 and 35. Two new ent-kaurene-type diterpenes were presented in Fig. 14.7 and were assigned to 12α-(β-D-glucopyranosyloxy)-7β-hydroxykaurenolide (40) and 7β-(β-D-glucopyranosyloxy)12α-hydroxykaurenolide (41). Of the the14 compounds isolated altogether were also the glucosides (12, 13, 17, 18) and lignans (19–23) (Fig. 14.6). When all the isolated compounds were tested for lipopolysaccharide (LPS)-stimulated NO production inhibitory effects in RAW264.7 mouse macrophages, only compound 35 showed a promise with IC50 value of 15.5 μM. This is interesting considering all other compounds, even structurally related (notice the side chain of the terpenoids that features a ketone group in 35 vs 33 and 34), have IC50 values over 30 μM.

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COOH

OR1

H

H

H

H

7

H H

HO

R2

24 Bryonolic acid

25 R1=A, R2=B 7--βOH 26 R1=A, R2=B 7-αOH

OH

R2O R1=A, R2=B R1=H, R2=B R1 A, R2=C R1=A, R2=Ac

27 28 29 30 OR

OR H

H

Ac

OR1

H

O

RO

O

O

O

31 R=A

H

H 33

32 R=A

O

O

H HO

H

H

HO

H

H

OH

O

H

34

36

35 A O

Glc O

O H

OH OH

B

O

OH

37

C

O

O

O OH RO

O H

OH

NH2

O

O

OH

OH

O H

Glc O

38 R=Glc 39 R=Rutinose

O HO HO

O HO OH

NO2 O OH OH

Rutinosyl Glc = Glucopyranosyl

FIG. 14.6 Triterpenes of P. pepo.

From the fruits of C. pepo cv dayangua, Wang et al. (2007) have isolated cucurbitacin L 2-Oβ-D-glucopyranoside (36), cucurbitacin K 2-O-β-D-glucopyranoside (37), and two hexanorcucurbitane glycosides: 2,16-dihydroxy-22,23,24,25,26,27-hexanorcucurbit-5-en-11,20-dione 2-O-β-D-glucopyranoside (38) and 16-hydroxy-22,23,24,25,26,27-hexanorcucurbit-5-en-11,

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HO HO O

HO

OH

OH O

O OH O

FIG. 14.7

O 40

O

O

O OH

HO HO

OH

41

Diterpenes of C. pepo.

20-dione 3-O-α-L-rhamnopyranosyl-(1 !2)-β-D-glucopyranoside (39). Considering the antidiabetic effects of triterpenes highlighted in the various chapters of this book, it would be interesting to examine the potential health benefits of triterpenes from pumpkin seeds. 14.4.2.3 Tocopherols The vitamins and tocopherols content of pumpkins have been routinely assessed and reported. Tocopherols in pumpkin seeds, for example, range between 16.3 mg/100 g and 46.7 mg/100 g with C. pepo varieties higher than C. maxima. Variabilities with respect to α-, β-, γ-tocopherols has also been reported for the various cultivars (Nawirska-Olsza nska et al. (2013). Ten of the related compounds known to occur in C. pepo along with the tocopherols have been reported as follow (Fig. 14.8): α-, β-, δ-, and γ-tocopherols (48–51); β-carotene (43), lutein (44), cucurbitine (47), β-sitosterol (56), campesterol (57), and squalene (42) (Bharti et al., 2013). As with many pumpkin preparations, C. pepo fruits and seeds extracts, including those sourced from industrial by-products, have been demonstrated to display antioxidant effects (Saavedra et al., 2015). Carotenoids and tocopherols being known antioxidants and occur in abundance within C. pepo fruits, their antidiabetic effects have also been investigated. The study by Bharti et al. (2013) employed poloxamer-407 (PX-407)-induced diabetic rats model where 2 and 5 g/kg administration of the tocopherol fraction of pumpkin seeds for 3 weeks suppressed the levels of glucose and hemoglobin A1c (HbA1c). In this model, the raised insulin level in diabetic animals was also suppressed by tocopherol treatment. The homeostatic model assessment of insulin resistance (HOMA-IR) index was also reduced suggesting amelioration of insulin resistance by the tocopherol fraction. Furthermore, the TC and TG levels could be suppressed by the lower, but not the higher dose, suggesting potential toxicity or antagonistic effects of compounds at higher dose. An increased level of GSH, total thiols, GST, superoxide dismutase (SOD), CAT, pancreatic weight, and histological improvement of pancreatic damage were also observed. The ameliorating effect on lipid peroxidation (LPO) as measured from the reduction in thiobarbituric acid reactive substances (TBARS) was a further example of the antioxidant mechanisms of antidiabetic effects for the tocopherol fraction. In addition, the treatment also scored another potential antidiabetic beneficial outcome via increasing the glucagon like peptide (GLP) level of caecum in treated animals. The docking study by Bharti et al. (2013) on the tocopherol components of pumpkin is not conclusive but provided some insight into the potential interaction of these compounds with diabetic target molecules such as protein-tyrosine phosphatase 1B (PTP1B), dipeptidyl

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O ON+

42 Squalene

45 Trigonelline O OH 43 β-Carotene

N

HO

46 Nicotinic acid O OH NH2 HN

OH

44 Lutein

47 Cucurbitine

HO

HO O

O 49 β-Tocopherol

48 α-Tocopherol HO

HO

O

O

51 δ-Tocopherol

50 γ -Tocopherol O OH 52 Palmitic acid

OH 53 Stearic acid

H H

O H

H

HO 56 β-Sitosterol

O OH

54 Oleic acid

O OH

55 Linoleic acid

H H H

H

HO 57 Campesterol

FIG. 14.8 Other classes of compounds of C. pepo including tocopherols, carotenoids, sterols, and alkaloids.

peptidase IV (DPP-IV), and proliferator-activated receptor-γ (PPAR-γ). They have also shown interaction as a ligand to DPP-IV for α-, β-, δ-, and γ-tocopherol. These compounds also showed affinity for binding with the active sites of PPAR-γ but no interaction was observed with PTP1-B. More research in this area is needed to prove if the observed effect has therapeutic relevance in cellular/animal models.

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14.4.2.4 Seeds oil About 11–31% of the seed content of C. pepo by weight is accounted by the oil (Bharti et al., 2013). For a variety of usage, pumpkin seeds oil has been exploited for centuries at commercial level. Solvent extraction (mainly hexane), expeller pressing, supercritical CO2 extraction, aqueous enzymatic process, and enzyme-catalyzed reaction technologies, microwave, ultrasound, high pressure, extrusion, electrical discharge, and cavitation systems have been adopted for the extraction of pumpkin seeds oil. The scrutiny of these extraction methods is beyond the topic of this book (see Li et al., 2016 and references there in) but a brief outline of the oil composition is described. Fatty acid composition analysis of the seed oil generally show the following composition (Fig. 14.8): palmitic acid (52, 11.53–12.48%) and stearic acid (53, 3.08–5.09%) as principal components of saturated fatty acids; oleic acid (54, 37.35–37.54%) as the predominant ingredient of monounsaturated fatty acids; and linoleic acid (55, 44.51–47.67%) as the main compound of polyunsaturated fatty acids (Li et al., 2016; Nawirska-Olsza nska et al., 2013). Higher content of fatty acids were reported from C. maxima than cultivars of C. pepo. On the other hand, C. pepo varieties displayed better antioxidant properties regardless of the extraction solvent used than C. maxa (Nawirska-Olsza ska et al., 2013). The pressed seed oil of C. pepo is considered to be a rich source of n caroteinoids including lutein and β-carotene (43, 10.1%). In addition to the above-mentioned pigments, the presence of violaxanthin, luteoxanthin, auroxanthin epimers, lutein epoxide, flavoxanthin, chrysanthemaxanthin, 9(90 )-cis-lutein, 13(130 )-cis-lutein, 15-cis-lutein (centralcis)-lutein, α-cryptoxanthin, β-cryptoxanthin, and α-carotene have been reported in the oil in small quantities (Matus et al., 1993). These minor components are prevalent in other species, however, and will be discussed in the following sections. The α-amylase inhibitory activity (IC50 45.46  1.29 μg/mL) of this pumpkin seed oil was reported while the α-glucosidase inhibition activity was relatively weak (IC50 > 1000 μg/mL) (Li et al., 2016). 14.4.2.5 Polysaccharides The extraction of polysaccharides from pumpkin has been outlined by various authors. Liu et al. (2018) extracted polysaccharides from the fruits (excluding the seeds) using water, and then removed the proteins using Sevag reagent followed by precipitation through repeated alcohol treatment. In this case, they could obtain the polysaccharides with molecular weight of the main component as 8720 Da and composition of 1,6-αglucosyl, 1,2,6-α-mannosyl, 1,3,6-linked-mannosyl, 1,2,6-α-galactosyl, 1,2,6-α-galactosyl, terminal fucosyl, and terminal glucose. In a similar experiment, Song et al. (2015) processed pumpkin (C. pepo, lady godiva variety) to obtain this polysaccharide as the main component while their previous pioneering work showed 1,2,6-trisubstituedgalactopyranosyl, 1,6-disubstitued-galactopyranosyl, 1,4,6-trisubstitued-glucopyranosyl, 1,3-disubstituedglucopyranosyl, terminal-glucopyranosyl, and terminal-fucopyranosyl, with a molar proportion of 1:4:2:2:2:1 (Song et al., 2011). Readers should note that this polysaccharide was obtained from the fleshy part of pumpkin fruit with the exclusion of peels and seeds. The antidiabetic effect of the tocopherols (also present in the seed oils) extracted from the seeds are already discussed in the previous section. The seeds oil of commercial sources may not even indicate the source species of the pumpkin and various effects including antidiabetic properties are outlined in the section for C. moschata that appears to be investigated in greater detail. D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

14.5 Antidiabetic effect of C. moschata

487

In view of the known components such as trigonelline (45) and nicotinic acid (46) in pumpkin, attempt to link the antidiabetic pharmacology with these compounds (Fig. 14.8) have also been made. For example, Yoshinari et al. (2009) demonstrated that trigonelline and nicotinic acid from the methanol extract of pumpkin seeds could improve glucose tolerance and in non-obese T2D Goto-Kakizaki rats. This effect was also in line with their observation on pumpkin paste concentrate and the purified compounds (45, 46)-fed diabetic rats were lower activity of liver fatty acid synthase (FAS), and higher activity of liver carnitine palmitoyl transferase (CPT) and glucokinase were observed. Hence, inhibition of fatty acid synthesis by the alkaloids may serve as a mechanism for antiobesity effect. More research is required, however, to show the role of these compounds as the active principles, i.e. whether the concentration of these compounds in pumpkin accounts to the observed antidiabetic effects remains to be established.

14.5 Antidiabetic effect of C. moschata Beyond utilization for food, the fruits of C. moschata have been employed in many Asian countries as medicine including for blood glucose management ( Jiang and Du, 2011). In an attempt to isolate the active antidiabetic principles of this plant, Jiang and Du (2011) have isolated new tetrasaccharide glyceroglycolipids (Fig. 14.9) from the fruits that they named QGMG-3 (1-O-(9Z,12Z,15Z-octadecatrienoyl)-3-O-[β-D-galactopyranosyl-(1 !6)-O-β-Dgalactopyranosyl-(1 !6)-O-β-D-galactopyranosyl-(1! 6)-O-β-D-galactopyranosyl] glycerol, 59) and QGMG-2 (1-O-(9Z,12Z-octadecadienoyl)-3-O-[β-D-galactopyranosyl-(1 ! 6)-O-β-Dgalactopyranosyl-(1 !6)-O-β-D-galactopyranosyl-(1! 6)-O-β-D-galactopyranosyl] glycerol, 58). Structurally, both compounds contain a lipid or glyceryl fatty acid ester with different saturation label (omega-3 vs omega-6). The sugar components were, however, composed of repeating units of just one sugar unit, galactose. In STZ-and high fat-induced diabetic mice receiving 50 mg/kg (i.p for 4 weeks), their glucose lowering effect was evident with QGMG-3 (59) being more active. The compounds however did not modify the body weight of the animals. It is worth noting that galactolipids such as 1,2-di-O-α-linolenoyl-3-O-β-galactopyranosyl-sn-glycerol have been isolated from various plants and shown to display a range of biological activities including anti-inflammatory and anticancer effects (Hou et al., 2007). Other parts of the plant have also been shown to display antiobesity effects in animal models. For example, the antidiabetic effect (STZ-induced mice model) of the stems was studied by Chang et al. (2014) who showed antihyperglycaemic effect for the methanol extract at fairly low doses (50 and 100 mg/kg, p.o. for 6 days). Interestingly, the insulin-stimulated glucose uptake in FL83B hepatocyte cells in vitro, which was suppressed by TNF-α, could be ameliorated by the extract at the concentration of 100 μg/ml. On the basis of these information, attempt to isolate the active principles led to the identification of nine compounds that were characterized as an apocarotenoid, loliolide (70) phenolic acids such as salicylic acid (71) p-hydroxycinnamic acid (4) and ferulic acid (72); four lignans [(+)-(1R,2S,5R,6S)-2,6-di(40 hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane] (60), pinoresinol (61), 4-ketopinoresinol (63), and syringaresinol (62); and 2 steroids [(22E,24R)-24-methyl-6β-methoxy-5α-cholesta-7,22diene-3β,5-diol] (73), and 3β-hydroxy-(22E,24R)-ergosta-5,8,22-trien-7-one (74) (Fig. 14.9).

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OH R

O

O O

R

OH HO

HO

R = HO

OH O

HO

58 QGMG-2

HO

O

O

O

HO

OH HO O

OH HO

O

O

HO O

OH HO

HO

O

O

O O

59 QGMG-3

R1

HO

OH

OMe HO

O

R2

H

H O

O H

R1

RO

64 Dehydrodiconiferyl alcohol

OH OH

O

HO

RO

OH OH 65 R=A Cucurbitosides A 66 R=B Cucurbitosides B

O

OH

O

HO

O O

O

MeO OMe

63 4-Ketopinoresinol

OH

HO

OH O

O

HO

O

RO

OH OH

O

70 Loliolide

HO

O

O O

R A R=H B R=OH

OH OH

67 R=A Cucurbitosides C 68 R=B Cucurbitosides D

69 R=A Cucurbitosides E

R

O

COOH

HO 4 R=H 72 R=OMe ferulic acid

71 Salicylic acid

HO

HO

OH

O

HO

COOH OH HO

OH

HO

OH

R2 60 R1=R2=H 61 R1=R2=OMe, R2=H Pinoresinol 62 R1=R2=OMe Syringaresinol

HO

O H

O

OH

O

OMe 73

FIG. 14.9

OH

74

Some common constituents of C. moschata.

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14.5 Antidiabetic effect of C. moschata

489

The biological activity study in vitro using glucose uptake study showed the following important observation: • Screening at 20 μM (or 40 μM) identified 73 and 74, as promoters of glucose uptake in normal hepatocytes in the same way as insulin; • Compounds 73 and 74 did not act via insulin receptor substrate (IRS) and protein kinase B (Akt) that insulin utilize but activate the 50 adenosine monophosphate-activated protein kinase (AMPK) (2–30 μM); • In insulin-resistant cells (induced by TNF-α), 72 (ferulic acid), 62 (syringaresinol), and 9 were identified as promotors of glucose uptake within 2–50 μM. They enhance the phosphorylation of AMPK and AMPK substrate ACC-1 (acetyl-CoA carboxylase-1). Their AMPK-dependent insulin-like effect was confirmed by inhibitor studies; • Phosphorylation of Akt and its substrate (AS160) induced by insulin (not in the absence of insulin) were enhanced by ferulic acid (72) and syringaresinol (62) whereas 73 activated Akt only in the presence of insulin (insulin sensitizer) and promote phosphorylation of AS160 in both the absence and presence of insulin. Compound 73 was therefore regarded as insulin sensitizer and insulin substitute. Antidiabetic drugs including metformin which are known to increase glycogen synthesis and inhibit gluconeogenesis (partly by suppressing gene expression for rate-limiting enzymes) are known to activate the AMPK. Activation of the AMPK by metformin could also enhance glucose uptake by upregulating the glucose transporter-4 (GLUT4). The diverse pharmacological effect of natural products discussed in this book also highlight that phosphorylation of AMPK leads to its activation resulting in the inactivation of ACC or the lipogenesis pathway; while fatty acid oxidation is increased. For example, the effect of AMPK in suppressing the expression of the transcription factor, sterol regulatory element-binding protein-1 (SREBP-1) and lipogenesis genes (e.g. FAS) has been reiterated. Feruic acid (72) is a known antioxidant compound that processes numerous pharmacological effects including antidiabetic action in both alloxan-, STZ-, and obesity-induced diabetic models (Prabhakar et al., 2013; Ramar et al., 2012; Song et al., 2014) and its potential antiobesity effects has also been demonstrated. If it is indeed found in the plant in sufficient amount to explain the pharmacological effect of C. moschata, its therapeutic potential is understandable. It is interesting to note that syringaresinol (62) which is structurally similar with the other lignans is active while others that do not happen to have tri-oxygenation pattern in their aromatic rings (60–63) failed to be active. In the study by Choi et al. (2007), the water-soluble extract from stems of C. moschata showed antiobesity effects by controlling lipid metabolism in a HFD-induced obesity mouse model. At a dose of 500 mg/kg (p.o. for 8 weeks), antiobesity effect as a function of suppressing body weight gain, fat storage, and inhibition of fatty-liver tissue development were observed. At molecular level, suppression of the mRNA of lipogenic genes (SREBP1c and stearoyl-CoA desaturase-1 (SCD-1)) while promoting lipolytic genes (peroxisome proliferator-activated receptor (PPAR)-α, acetyl-CoA-1 (ACO-1), carnitine palmitoyltransferase 1 (CPT-1), and uncoupling protein (UCP)-2) have been demonstrated. Subsequent study on the identification of the active anti-lipogenic principles from the stems of C. moschata have been carried out in vitro using 3T3-L1 adipocytes. The inhibition of fatty liver development and increased hepatic β-oxidation activity, suppression of TG, cholesterol, and leptin;

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and increased adiponectin levers were in line with antiobesity and lipid lowering effects. The isolated compound characterized as dehydrodiconiferyl alcohol (64, Fig. 14.9) could reduce adipocyte differentiation and the accumulation of intracellular TG in a dose-dependent manner (0.7–70 μM). They have also shown that the compound abolished the expression of adipogenic transcription factors in the same way as the crude plant extract. Thus, PPAR-γ and CCAAT/enhancer binding protein (C/EBP)-α, as well as a lipogenic transcription factor, SREBP-1c were suppressed. The mitotic clonal expansion of preadipocytes was also suppressed as evidenced from inhibition of DNA replication and cell cycle markers (cyclinA and Cdk2). Another relevant data presented from the study was the inhibitory effect of dehydrodiconiferyl alcohol (64) on the phosphorylation, DNA binding, and centromeric localization of C/EBPβ. The expression of SREBP-1c and SCD-1 (but not PPAR-γ and C/EBPα) was also inhibited. Other phytochemical components that attribute to the general health promoting effects of C. moshata must also be considered. The seeds, for example, which are not primarily used as food are known to contain about 40% of carotenoids and provitamin A (or β-carotene, 43). Methodologies for extracting valuable carotenoids such as lutein, β-carotene, α-carotene, and zeaxanthin from various parts including seeds and peels have also been described (e.g. Song et al., 2018). Quantitative analysis of the various parts of the plant have also been carried out and this include the listing of several carotenoids in addition to β-carotene (43) and lutein (44) that are shown as components of C. pepo (Fig. 14.8). They include high content of vitamin A (78), α-carotene (79), ζ-carotene (75), zeaxanthin (81), violaxanthin (83), β-carotene 5,6-epoxide (84), β-cryptoxanthin (80), taraxanthin (85), luteoxanthin (86), auroxanthin (87), phytofluene (76), neurosporene (77), and neoxanthin (82) (Fig. 14.10). The relative abundance of the components could vary depending on cultivar, geographical, environmental conditions, and plant parts even with the fruit tissues. Several references in this context are available (Gonza´lez et al., 2001; Murkovic et al., 2002; Noseworthy & Loy, 2008; Rodriguez-Amaya et al., 2008; Toshiro et al., 1986). Jun et al. (2006) obtained the alcohol insoluble polysaccharide from pumpkin (C. moschata Duch) peels which led to the isolation of water soluble pectic polysaccharide fractions. Even through the structures of these large molecular weight compounds were not characterized, they have shown to enhance the growth of Lactobacillus brevis, Bifidobacterium bifidum, and Bifidobacterium longum, while suppressing Escherichia coli and Clostridium perfringens growth in vitro. Numerous studies have also focused on the dietary fibre composition and potential application of pumpkin fruits powder within the food industry. Methodologies for maximizing the yield of fibre extraction from (C. moschata Duchesne ex Poiret), for example, have been outlined by various authors (de Escalada Pla et al., 2007; Prakash Maran et al., 2013). Some of the acidic polysaccharides display antioxidant effects in vitro when tested on radical scavenging assay (e.g. 2,2-diphenyl-1-picrylhydrazyl (DPPH)) and hydrogen peroxide (H2O2)induced antioxidant GSH and SOD depletion, and cytotoxicity in cultured mouse peritoneal macrophages (Yang et al., 2007). The characterization of these polysaccharides was based on their monomeric composition analysis which was as follow: galactose (11.5%), arabinose (9.8%), xylose (4.4%), and rhamnose (2.8%). In the study by Jin et al. (2013), they have demonstrated first the hypoglycaemic efficacy in the alloxan-induced acute diabetes model by administration of the aqueous extract of the fruits. The observed glucose lowering effect at the dose of 200 mg/kg (i.p.) was effective both in normal and diabetic animals in acute (72 h)

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75 ς-Carotene

76 Phytofluene

77 Neurosporene

78 Vitamin A

OH

79 α-Carotene

80 β-Cryptoxanthin

HO

HO HO

OH

81 Zeaxanthin

C

O

82 Neoxanthin

O HO

OH

O

OH

83 Violaxanthin

O 84 β-Carotene 5,6-cpoxide

HO

O

OH

85 Taraxanthin

O O

86 Luteoxanthin

HO

O O

OH OH

87 Auroxanthin

HO

FIG. 14.10 Carotenoids of C. moschata other than β-carotene and lutein.

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14. The chemical and pharmacological basis of pumpkins

study. The polysaccharides isolated also showed antidiabetic effect but they were not further characterized except for compositional analysis revealing the four monosaccharide (glucose, galactose, arabinose, and rhamnose) components. A more elaborative work on the characterization of polysaccharides from C. moschata came from the study by Wang et al. (2017). From the dried fruits pulp using solvent extraction (water and alcohol), alcohol fractionations followed by purification with Sephadex G-200 gel chromatography, a purified polysaccharide was obtained. Their heteropolysaccharide was shown to be composed of rhamnose, arabinose, glucose, and galactose as its main compositions while inositol was reported to be present as a minor component. In alloxan-induced diabetes mice model, administration of the active polysaccharide 150 mg/kg (i.p.) showed hypoglycaemic effect in acute study (7 h) while increasing the insulin and glycogen level. Enhancement of β-cell (NIT-1 cells) proliferation was also reported for these polysaccharides. Similar study by Wang et al. (2018) on the polysaccharides of C. moschata seeds identified the mannose, glucose, and galactose composition in a molar ratio of 1.00:4.26:5.78 and a molecular weight of 3728 g/mol. The proposed backbone of the structure was! 6)-β-D-Galp-(1 !, ! 6)α-D-Glcp-(1 !, and ! 3,6)-β-D-Manp-(1 ! with branching at O-3 and O-6 of ! 3,6)-β-D-Manp(1 !. Branch linkages were composed of α-D-Glcp-(1 ! and !4)-α-D-Galp-(1 ! (Fig. 14.11). The protein-bound polysaccharides isolated from the fruits with approximate 41.21% polysaccharide and 10.13% protein have also shown to display antidiabetic effect in alloxaninduced diabetic rat model (Quanhong et al., 2005). In addition to reduction of blood glucose, and increase the levels of serum insulin and improvement in glucose tolerance have been noted for 500 and 1000 mg/kg doses administered p.o. for 10 days. Other evidences on the antidiabetic effect of C. moschata polysaccharides came from studies on alloxan-induced diabetic rabbits (Zhang et al., 2013). Administration (75 mg/kg for 21 days i.v.) of polysaccharides from the dried seeds (peals removed) pulp was shown to recover body weight loss; reduced blood glucose, serum lipid (TC, TG), and glycosylated hemoglobin (HbA1c) levels. The regeneration of damaged pancreatic islets by stimulating β-cell proliferation was also reported for the polysaccharides. Although ion exchange and gel chromatography led to the isolation of the heteropolysaccharide consisting of glucose, galactose, arabinose, rhamnose, and little amount of hexuronic acid, with a molecular weight of 1.15  105 Da; details of the structure was not established. Many studies on pumpkin components may also be published without detailing the species from which the fruit was sourced. In the polysaccharide characterization study by Zhu et al. (2015), for example, the polysaccharides of the pumpkin fruit sourced from local market in China was described. Although this is likely to be of C. moschata, it could be any other species discussed in this chapter. They described the isolation of a polysaccharide with a molecular weight of 23 kDa and composition of D-arabinose, D-mannose, D-glucose, and D-galactose with a molar ratio of 1:7.79:70.32:7.05. In vitro cell culture studies using freshly α-D-Glcp-(1→4)-α-D-Glcp-(1→6)

α-D-Glcp-(1→4)-α-D-Glcp-(1→3)

→6)-β-D-Galp-(1→6)-α-D-Glcp-(1→3)-β-D-Manp-(1→6)-β-D-Galp-(1→6)-α-D-Glcp-(1→6)-β-D-Manp-(1→6)-β-D-Galp-(1→

88

FIG. 14.11 Proposed structural backbone of C. moschata polysaccharide. The pyranosides of mannose (Manp), glucose (Glcp), and galactose (Galp) are suggested to form the backbone of the polysaccharide.

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14.6 Antidiabetic effects of C. ficifolia

493

isolated pancreatic cells from rats showed protection from STZ-induced cytotoxicity via increasing the antioxidant SOD level and reduction of and MDA and NO production. The concentration-dependent effect shown were within 0.5–1 mg/ml range which is perhaps not highly potent but still significant considering the very high molecular weight of the polysaccharide. Some polysaccharides isolated from pumpkin pulp (C. moschata) have also shown to display prebiotic activities as evidenced by in vitro experiments that showed selective enhancement of Lactobacilli growth (Du et al., 2011). The protein components of the seeds have also attracted some interest as antidiabetic agent. The study by Teugwa et al. (2013), for example, assessed the globulin proteins stored in the seeds of Cucurbitaceae seeds including C. moschata. In their oral glucose tolerance test (OGTT) in normal rats, administration of the crude proteins (50 mg/kg, p.o.) displayed hypoglycaemic effects. An interesting array of phenolic glycosides has been isolated from the seeds of C. moschata. In the first series, five novel compounds labelled as cucurbitosides A-E (65–69) were identified by Koike et al. (2005) as follow: 2-(4-hydroxy)phenylethanol 4-O-(5-O-benzoyl)-βD-apiofuranosyl(1 ! 2)-β-D-glucopyranoside (65), 2-(4-hydroxyphenyl)ethanol 4-O-[5-O-(4hydroxy)benzoyl]-β-D-apiofuranosyl(1 !2)-β-D-glucopyranoside (66), 4-hydroxybenzyl alcohol 4-O-(5-O-benzoyl)-β-D-apiofuranosyl(1! 2)-β-D-glucopyranoside (67), 4-hydroxybenzyl alcohol 4-O-[5-O-(4-hydroxy)benzoyl]-β-D-apiofuranosyl(1! 2)-β-D-glucopyranoside (68), and 4-hydroxyphenyl 5-O-benzoyl-β-D-apiofuranosyl(1! 2)-β-D-glucopyranoside (69). Their structures are shown in Fig. 14.9. The pharmacological relevance of these compounds is yet to be established. Perhaps not of pharmacological interest but a new fatty acid, 13-hydroxy-9,11,15octadecatrienoic acid, has also been isolated from the leaves (Bang et al., 2002). Other effects reported in the scientific literature for C. moschata fruit include hepatoprotective effects. In the study by Shayesteh et al. (2017), the aqueous extract (50 μg/mL) was shown to ameliorate hepatocyte cytotoxicity induced by cumene hydroperoxide (oxidative stress model) or glyoxal (carbonylation model). In addition to inhibition of protein carbonylation in glyoxal-induced carbonyl stress, inhibition of hepatocyte lysis, ROS production, LPO, glutathione depletion, mitochondrial membrane potential collapse, lysosomal damage, and cellular proteolysis were all recorded for the extract.

14.6 Antidiabetic effects of C. ficifolia The fruits juice preparation of C. ficifolia has been shown to display antihyperglycaemic effects in a preliminary screening study by Roman-Ramos et al. (1995). A number of similar experiments by the same authors have also shown the antihyperglycaemic effect of the plant extract (Roman-Ramos et al., 1991, 1992) in alloxan-induced diabetes model. The data by Alarcon-Aguilar et al. (2002) in the same model was also of similar significance which once again demonstrated potential antidiabetic effect in vivo. In this case, the freeze-dried juice (500 mg/kg, i.p. or p.o. for 14 days) in alloxan-diabetic mice showed an acute hypoglycaemic effect. By using the STZ-induced diabetes model, Xia and Wang (2006a, 2007, 2009) published three articles showing that oral administration of the fruit extracts (300 and 600 mg/kg for 30 days) could reduce hyperglycaemia and glycosylated haemoglobin levels, while increasing plasma insulin level and pancreatic β-cell number and total haemoglobin. The LPO of D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

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14. The chemical and pharmacological basis of pumpkins

pancreatic tissues could also be suppressed by oral feeding of the fruit extracts (Xia and Wang, 2009). A reduction in the TG and lactate dehydrogenase (LDH) levels while increasing the HDL level coupled with improvement in oral glucose tolerance test have also been recorded. Miranda-Perez et al. (2016) have studied the effect of the aqueous extract of C. ficifolia (9, 18, 36, and 72 μg/ml) along with the suspected active component (D-chiro-inositol, (50, 100, 200, and 400 μM) on RINm5F pancreatic β-cells. The extract but not D-chiro-inositol was found to increase [Ca2+]i and insulin secretion. The extract was not found to block K+ATP channel but it did exert an effect by increasing [Ca2+]i from the endoplasm reticulum. Xia and Wang (2006b) previously, however, showed that D-chiro-inositol (89) found in C. ficifolia has antidiabetic effect in the STZ-induced diabetes rat model. Their experiment was based on the extract of C. ficifolia equivalent to 10 or 20 mg/kg D-chiro-inositol displaying antidiabetic effect after oral administration for 30 days. Just like the crude extract, the pure compound (89) also lowered blood glucose, and increased the levels of hepatic glycogen, total haemoglobin and plasma insulin. An improvement in glycaemic control in OGTT was observed in the same way as 20 mg/kg of synthetic D-chiro-inositol under the same experimental condition. Considering the significant level of D-chiro-inositol in the fruit extract of C. ficifolia, many authors tried to associate antidiabetic effects with this compound as an active principle. The study by RomanRamos et al. (2012), for example, quantified 3.31 mg/g of the active fraction that displayed antidiabetic effect (at 200 mg/kg, p.o. 15 days) in the STZ-induced diabetes model. They have shown that the fraction increase GSH and decrease MDA levels in the liver; reduce the inflammatory cytokine TNF-α; but raised the levels of IL-6 and interferon-γ (IFN-γ) as well as the antiinflammatory cytokine, IL-10 in the serum. Other pharmacologically active principles should also be considered in this direction where the study by Jessica et al. (2017) is relevant. In their alloxan-induced diabetes model in mice, they have shown that the aqueous extract of the fruits augment the level of glycogen in the liver along with glycogen synthase enzyme. The level of glycogen hydrolyzing enzyme, glycogen phosphorylase, on the other hand, was suppressed. The liver-protective (hepatoprotective) effect could also be confirmed from the histology study while the extract appeared to have no effect on glucose uptake transporter, GLUT2, in the liver nor on glucagon receptor. From the active fractions, they have isolated the following compounds: stigmast-7,22-dien-3-ol (also called chondrillasterol (91), and stigmast-7-en-3-ol (92), p-coumaric acid (4), p-hydroxybenzoic acid (1), and salicin (Fig. 14.12). Although the authors assert that the dose employed contained 11.6 μg/kg of p-coumaric acid (4) that could account to the observed antidiabetic effect, conclusive data in support of the hypothesis was not given. Xia and Wang (2006a) also studied the antidiabetic effects of the aqueous fruits extract of C. ficifolia in STZ-induced diabetes rat model. They have shown that oral administration of the extract (300 and 600 mg/kg) for 30 days could reduce blood glucose, glycosylated haemoglobin, and an increase in plasma insulin and total haemoglobin. As expected, the crude fruit extract in the same model (STZ-induced diabetic rat) and 30 day treatment regimen, not only reduce blood glucose and improve glycaemic control under OGTT, but also improve the lipid profile (Xia and Wang, 2009), i.e. a reduction in TG, LDL and increase in HDL level were recorded. The antidiabetic effect in the STZ-model in rats has also been shown to be associated with an increase in plasma insulin and β-cell mass (Xia and Wang, 2007). Dı´az-Flores et al. (2012) experimented on the STZ-induced diabetes mice model to understand the potential effect of C. ficifolia Bouche (aqueous extract of fruits) on the glutathione

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14.7 Antidiabetic effect of C. maxima

COOH OH HO

COOH

OH

HO

OH

O

OH

OH

89 D- chiro-Inositol

4 p-Coumaric acid

H

OH HO

OH

OH

OH OH

1 p-Hydroxybenzoic acid

H

H

HO

O

90 Salicin

H

HO 92

91

FIG. 14.12 Phytochemical components of C. ficifolia.

redox cycle. Their conclusive data have shown that the fruits extract display hypoglycaemic effect (200 mg/kg, p.o. for 30 days) with concomitant improvement on GSH redox state, increasing the glutathione pool, GSH and GSH:GSSG ratio in liver, pancreas, kidney, and heart tissues. Other favourable effects included inhibition of polydipsia, hyperphagia and plasma LPO. Acosta-Patin˜o et al. (2001) undertook a short term clinical study on T2D patients receiving a raw extract of C. ficifolia (fresh immature fruits) or potable water in a single dose of 4 mL/kg in two different sessions at least separated by 1 week. Blood measurement 5 h later showed decrease in blood glucose levels. The patient population (n ¼ 10) and diversity was however not clarified. In a combination treatment strategy with probiotic yogurt, a small clinical study by Bayat et al. (2016) using 80 participants have shown beneficial effect in suppressing the level of blood glucose and inflammatory marker. Administration of 100 g C. ficifolia along with yogurt (150 g) for 8 weeks could also reduce HbA1c, hyper sensitive CRP, and LDL-C (though not significant) and BP (both SBP and DBP). With respect to the active principles responsible for the antidiabetic effect of the plant, little is really known apart from the general information at hand on the chemistry of other related pumpkins. The contribution of D-chiro-inositol and polysaccharides discussed in the preceding texts are good starting point but further studies in this field are necessary.

14.7 Antidiabetic effect of C. maxima The α-Amylase and α-glucosidase inhibitory activity of C. maxima seeds was assessed in vitro by Kushawaha et al. (2016). They have reported IC50 value for α-amylase and α-glucosidase inhibitory effects as 7.00  0.29 and 11  0.36 mg/mL, respectively. The

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14. The chemical and pharmacological basis of pumpkins

α-glucosidase inhibitory activity of the methanolic extract of the flowers with IC50 value of 610.52  60.33 μg/mL has also been reported (Acharya and Bratati, 2015). As with many other pumpkin preparations, the aerial parts have also been shown to display antioxidant effects in vitro when tested in DPPH, NO, superoxide, H2O2, and lipid peroxide radical models (Saha et al., 2011). The reported activity profile was however by no means impressive as a pharmacologically relevant therapeutic agent. The antioxidant potential of the water soluble polysaccharides has also been demonstrated at relatively higher concentrations (Nara et al., 2009). In the STZ-induced diabetic rat model, however, the seeds extracts could reduce the FBG level both in normal and diabetic rats, with more pronounced effect in the latter case. The report by Kushawaha et al. (2018) corroborated the antidiabetic effect of the seeds extract as administration at a dose of 200 mg/kg (p.o. for 28 days) could offer antihyperlipidaemic effect in STZ-induced diabetes rat model. A reduction in TC, TG, LDL-C, and very low density lipoprotein (VLDL-C); and other favourable parameters including weight recovery and glucose/protein level in the urea and increase in HDL were achieved. They also reported the lack of toxicity even when 10-times more extract than the effective dose was used. A reduction of FBG by the aqueous extract (200 and 250 mg/kg, p.o., for 28 days) of C. maxima seeds in STZ-induced diabetics rat along with hypoglycaemic effect in both diabetic and normal animals (but more pronounced in diabetics) in OGTT test were also established (Kushawaha et al., 2016). Additional evidence of the STZ-induced diabetes rat model was provided by Lal et al. (2011) who reported that C. maxima fruit juice and aqueous extract (excluding seeds) could suppress blood glucose in a dose (100 and 200 mg/kg)-dependent manner after oral administration for 28 days. From the seeds of C. maxima, Kikuchi et al. (2014) isolated three new multiflorane-type triterpene esters, 7α-hydroxymultiflor-8-ene-3α,29-diol 3-acetate-29-benzoate (99), 7αmethoxymultiflor-8-ene-3α,29-diol 3,29-dibenzoate(93), and 7βmethoxymultiflor-8-ene-3α,29-diol 3,29-dibenzoate (94). The known compounds isolated from the seeds at the same time included multiflora-7,9(11)-diene-3α,29-diol 3,29-dibenzoate (96). Although some melogenesis inhibitory activity (compound 99) and moderate cytotoxicity in cancer cells were reported, their antidiabetic effects have not yet been investigated. From the seeds of C. maxima, Kikuchi et al. (2013) have also isolated multiflorane-type triterpenes; 7α-methoxymultiflor-8-ene-3α,29-diol 3-acetate-29-benzoate (99), 7-oxomultiflor-8ene-3α,29-diol 3-acetate-29-benzoate (100), and multiflora-7,9(11)-diene-3α,29-diol 3-p-hydroxybenzoate-29-benzoate (95), along with three known compounds, multiflora-7,9 (11)-diene-3α,29-diol 3,29-dibenzoate (96), multiflora-7,9(11)-diene-3α-29-diol 3-benzoate (97), and multiflora-5,7,9(11)-triene-3α,29-diol 3,29-dibenzoate (98). The isolation and identification of cucurbita-5,24-dienol (101) (Akihisa et al., 1986) and α- (102) and β-amyrin (103) (Cattel et al., 1979) from the seeds of C. maxima have also been reported (see Fig. 14.13). The isolation of polysaccharides from C. maxima is also emerging to gain interest (Zhou et al., 2014) and more research in this direction would prove the potential exploitation of these class of antidiabetic compounds as with the other Cucurbita species.

D. Potential modulators of type-2 diabetes and associated diseases: Super fruits

497

14.9 Conclusions

OR1

OR1

H

H

H

H

R2 O

R2

R2O O

95 R1=A, R2=B 96 R1=R2=A 97 R1=H, R2=A

93 R1=A, R2=α -OMe 94 R1=A, R2= β-OMe OR1

OR1

H

R A R=H B R=OH

H Ac

R2 O

H O

R2 99 R1=A R2= α OMe, βH 100 R=O

98 R1=R2=A

H

H

H

H

HO

H

HO

101 Cucurbita-5,24-dienol

H H

H

HO

102 α -Amyrin

103 β -Amyrin

FIG. 14.13 Chemical components of C. maxima.

14.8 Antidiabetic effect of C. mixta The antidiabetic effect of C. mixta is yet to be demonstrated.

14.9 Conclusions Pumpkin is a diverse group Cucurbita species that represent numerous species, subspecies and cultivars. The pumpkin fruits, seeds, or other plant parts are therefore expected to differ from each other not only in their phenotypic appearance but also in chemical composition.

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As dietary supplement and nutritional value, the seeds are emerging to gain more and more significance in recent years within the food industry. This chapter exhibited that both the fruits and the seeds are proven to show antidiabetic, antiobesity, and lipid lowering effect in the various animal models. Phytochemical studies also showed a range of compounds that potentially attribute to the observed pharmacological effects but most of the isolated compounds are yet to be evaluated for their antidiabetic potential. Of those shown to contribute as active components range from the carotenoids, tocopherol, and lipid composition to phenolic compounds such as ferulic acid. Other small molecular weight compounds such as D-chiro-inositol and trigonelline are among the active principles but the polysaccharides of pumpkin have emerged as the dominant antidiabetic principles. The triterpenes are other major compounds of pumpkin but their pharmacology is hardly studied. As antidiabetic agents, the best studied pumpkin species to data are C. pepo, C. moschata, C. ficifolia, and C. maxima. The leaves, flowers, and stems or simply the aerial parts also need to be thoroughly investigated for antidiabetic action. From all the available data discussed in this chapter, it is fair to conclude that these species have potential as antidiabetic agents but evidence of efficacy from human studies is yet to be provided.

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Further reading Adams, G.G., Imran, S., Wang, S., Mohammad, A., Kok, M.S., Gray, D.A., et al., 2014. The hypoglycaemic effect of pumpkin seeds, trigonelline (TRG), nicotinic acid (NA), and D-chiro-inositol (DCI) in controlling glycaemic levels in diabetes mellitus. J. Crit. Rev. Food Sci. Nutr. 54 (10), 1322–1329. Aristatile, B., Alshammari, G.M., 2017. In vitrobiocompatibility and proliferative effects of polar and non-polar extracts of cucurbita ficifolia on human mesenchymal stem cells. Biomed. Pharmacother. 89, 215–220. Lee, J., Kim, D., Choi, J., Choi, H., Ryu, J.H., Jeong, J., et al., 2012. Dehydrodiconiferyl alcohol isolated from Cucurbita moschata shows anti-adipogenic and anti-lipogenic effects in 3T3-L1 cells and primary mouse embryonic fibroblasts. J. Biol. Chem. 287, 8839–8851. Procida, G., Stancher, B., Catenia, F., Zacchignaa, M., 2013. Chemical composition and functional characterisation of commercial pumpkin seed oil. J. Sci. Food Agric. 93, 1035–1041.

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C H A P T E R

15 The chemical and pharmacological basis of cinnamon (Cinnamomum species) as potential therapy for type-2 diabetes and associated diseases O U T L I N E 15.1 Introduction

506

15.2 Taxonomic and botanical descriptions

507

15.3 Medicinal uses

510

15.6.3 The active principles of cinnamon: Evidence of efficacy from studies in vitro and animal models 528 15.6.4 Evidence of efficacy from human trials 534 15.6.5 Other pharmacological effect of cinnamon relevant to T2D 537

15.4 The chemistry of the cinnamon bark 511 15.4.1 Volatile components 512 15.4.2 Non-volatile components 514 15.5 General pharmacology

519

15.6 Antidiabetic, antiobesity, and antihyperlipidaemic effects 521 15.6.1 Evidence of efficacy from studies on animal models: Cinnamon powder and crude extracts 521 15.6.2 General mechanism of action—Crude extracts 523

Medicinal Foods as Potential Therapies for Type-2 Diabetes and Associated Diseases https://doi.org/10.1016/B978-0-08-102922-0.00015-8

15.7 Toxicological profile

538

15.8 Overview of pharmacokinetics profile

539

15.9 General summary and conclusions 540 References

542

Further reading

550

505

# 2019 Elsevier Ltd. All rights reserved.

506

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15.1 Introduction As one of the precious holly herb mentioned in the Old Testament of the Bible, the use of cinnamon by humans goes back to antiquity. During the Egyptians civilizations around 3000 BC, cinnamon was known to be used not only for its fragrance and flavouring but also for mummifying dead bodies. The culture of using cinnamon for its fragrances and employing it to dead bodies was also known to be extended to the Roman times. Whether to remove the bad odour and/or preservation of dead bodies, the practice of making someone holly in Christianity as experienced by the Moses, or being the common household spice ingredient in every kitchen that goes from aroma to medicine today; cinnamon has a long history of use by humans. From the industrial exploitation point of view, the massive trade of this spice in Europe from the 16th century owe it to its discovery in Sri Lanka by the Portuguese. As with the other spices such as clove (see Chapter 16), the competition by the European nations for this valuable resource has its own long history. In this case too, the Portuguese had absolute monopoly on cinnamon trade from its Native Sri Lanka until they lose it to the Dutch by the middle of the 17th century; which then went to the British when the Island (by then called Ceylon and hence the name Ceylon Cinnamon) was ceased by the very late 18th century. Some of the historical perspective of the cinnamon industry in the earlier days is reviewed (Wijesekera, 1978). The cinnamon tree belongs to the genus Cinnamomum (Lauraceae family) which comprise about 257 species. Only a handful of species are, however, exploited as the spice, cinnamon (Table 15.1). The name cinnamon is believed to be adopted from a Greek word that refers to sweet wood. While the principal cinnamon utilized for fragrances and flavouring is the bark, all the other plant parts are known to be employed in traditional medicine and culinary uses. TABLE 15.1 Cinnamomum species exploited as sources of the spice cinnamon. Common name

Botanical name

Indigenous region

True cinnamon or Ceylon cinnamon or Mexican cinnamon

Cinnamomum zeylanicum Blume or Cinnamomum verum J. Presl

Sri Lanka

Cassia cinnamon or Chinese cinnamon

Cinnamomum aromaticum Nees or Cinnamomum cassia (L) J. Presl

China

Indonesian cassia

Cinnamomum burmannii (Nees & T. Nees) Blume

Indonesian islands of Sumatra and Java

Vietnamese cinnamon or Saigon

Cinnamomum loureiroi Ness

Vietnam

Indian cassia

Cinnamomum tamala (Buch.-Ham.) T. Nees & Eberm

North-eastern region of India and Myanmar

Indigenous cinnamon

Cinnamomum osmophloeum Kaneh.,

Taiwan’s natural hardwood forests

Various local names

Cinnamomum bejolghota (Buch.-Ham.)

Central and Eastern Himalayas and Andaman Islands

The four main sources are Ceylon cinnamon, Chinese cinnamon, Indonesian cassia, and Vietnamese cinnamon while other local cinnamon species with increasing application also include Indian cassia and others.

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Throughout this chapter, cinnamon refers to the bark of the indicated Cinnamomum species, unless specified. The central part of the bark collected from young trees are dried and sold either as cinnamon sticks, powdered and packed in various forms for the food industry or processed as an extract or formulated in either tablets/capsules for use as medicinal and/or nutraceutical purposes. Cinnamon tea often in combination with other herbs is also commonly available to the public (Fig 15.1). The degree of utilization of these cinnamon species depend on the part of the world where these plants grow and/or marketed. For example, cassia cinnamon is cheaper and generally more popular in some part of Europe (e.g. Italy and Czech) when compared to the true cinnamon (Blahova and Svobodova, 2012; Lungarini et al., 2008). According to the Food and Agriculture Organization of the United Nations (FAO), Indonesia is a major producer of cinnamon, followed by China and Sri Lanka (Table 15.2). The designation of cinnamon by the United States Food and Drug Administration (FDA) as GRASS and its consumption as spice or for whatever purpose in small amount is a proof of cinnamon as safe food/nutraceutical ingredient. In fact, the production of cinnamon is mostly as spice for food flavouring but utilization for essential oil and medicinal uses are also common.

15.2 Taxonomic and botanical descriptions According to the Kew Science Plant of the WORLD Online (Kew-POTW, 2019), there are 257 accepted species of the genus Cinnamomum Schaeff with native distribution almost exclusively in the Australasia: From India in the West to Japan in the East and Australia in the South. More specifically, Andaman Island., Assam, Bangladesh, Bismarck Archipelago, Borneo, Cambodia, Caroline Is., China North-Central, China South-Central, China Southeast, Christmas I., East Himalaya, Fiji, Guatemala, Hainan, India, Japan, Jawa, Korea, Laos, Lesser Sunda Island, Malaya, Maluku, Myanmar, Nansei-shoto, Nepal, New Guinea, New South Wales, Nicobar Island, Ogasawara-shoto, Philippines, Queensland, Santa Cruz Is., Solomon Island, Sri Lanka, Sulawesi, Sumatera, Taiwan, Thailand, Tibet, Vietnam, and West Himalaya. Except for a limited region in Central America, other regions of Cinnamomum occurrence are through late introduction and included West Africa, Northern, Central and Southern America and the Caribbean regions: Alabama, Argentina Northeast, Brazil Southeast, Cook Island, Cuba, El Salvador, Florida, Georgia, Guinea, Gulf of Guinea Island, Honduras, Leeward Island, Louisiana, Mauritius, Mississippi, Norfolk Island, North Carolina, R
eunion, Samoa, Seychelles, Society Island, South Carolina, Texas, Trinidad-Tobago, and Windward Island. The most distinct characteristics of the genus are their fragrant leaves and other plant tissues that, according to the description by Wuu-Kuang (2011), display the following common features: Shrubs or trees to 50 m tall, with or without buttresses. Bark, root and crushed leaves often with a characteristic smell of cinnamon (cinnamic aldehyde), cloves (eugenol), sassafras (safrole), camphor (camphor) or a combination of these odours. Twig terete or angular, usually apically angular or subangular, 1–5 mm diam, hairy or glabrous. Terminal buds not perulate or rarely perulate, glabrous or hairy. Leaves opposite to subopposite or rarely alternate, rarely at twig-end the leaves are arranged closely in spiral; triplinerved,

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FIG. 15.1 Cinnamon preparations available to the general public. Cinnamon barks are available in food markets either in large quantities as bundles (especially in source countries) or packed in plastic bags or bottles in smaller quantities. The powdered cinnamon packed in loose plastic bags or bottled are also widely available in food stores and in health shops. Cinnamon teas often in combination with other herbs are also available in food supermarkets while cinnamon oils (leaves and bark) and tablets/capsules from bark extracts are available in health shops.

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15.2 Taxonomic and botanical descriptions

TABLE 15.2

World production of cinnamon in 2016.a

Country

Tonnes

China

77,055

Comoros

15

IMb

Dominica

52

IMb

Grenada

73

IMb

Indonesia

91,300

IMb

Madagascar

2460

IMb

Sao Tome and Principe

59

IMb

Seychelles

3

Official data

Sri Lanka

16,931

IMb

Timor-Leste

111

IMb

Viet Nam

35,516

IMb

a b

Flag description

http://www.fao.org/faostat/en/#data/QC. FAO data based on imputation methodology.

trinerved or rarely penninerved, if trinerved or triplinerved, the lateral veins ascend toward the leaf tip or between 1/2–2/3 of the lamina length; mature blades glabrous above, glabrous or hairy below, frequently glaucous below, margin entire; major intercostal veins scalariform, subscalariform or rarely reticulate; minor intercostal veins reticulate or scalariform. Inflorescences axillary or subterminal; paniculate-cymose with 1–3 order branching, flowers of the ultimate branch arranged in cyme, rarely racemiform; rachis angular; bracts caducous or persistent. Flowers bisexual, trimerous, appressed hairy; receptacle tube shallow, 0.5–3 mm deep; perianth lobes 6 in 2 whorls, equal; fertile stamens 9, in 3 whorls, filaments 1/4–3/4 the length of stamen; anthers 2- or 4-locular, if 4-locular the locules of the upper pair smaller than that of the lower pair, anther of the first and second whorls of stamens introrse, those of the third whorl extrorse-latrorse; third whorl stamens with 2 stipitate reniform glands attached on each side of the filaments; the gland stalks free or fused with the filaments; staminodes 3, in the fourth whorl, stipitate, hairy, apex sagittate or hastate; ovary superior, stigma subpeltate, peltate, discoid or trilobed. Fruits ellipsoid, obovoid, ovoid to globose seated on cupule, drupaceous, epicarp waxy, glabrous, pericarp thin or thick, often fragrant; cupule small to well-developed, subtending the lower part of the fruit; perianth lobes persistent, partly persistent or caducous. Seeds 1 per fruit, smooth, glabrous; endosperm absent; germination hypogeal.

Wuu-Kuang (2011) also provided the key to the classification of the various Cinnamomum species. The general taxonomic hierarchy to the common commercially relevant cinnamon is shown below: Kingdom: Plantae—plantes, planta, vegetal, plants Subkingdom: Viridiplantae—green plants Infrakingdom: Streptophyta—land plants Superdivision: Embryophyta Division: Tracheophyta—vascular plants, tracheophytes Subdivision: Spermatophytina—spermatophytes, seed plants, phan
erogam Class: Magnoliopsida

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FIG. 15.2 Image of C. zeylanicum growing in Kerala, India.

Superorder: Magnolianae Order: Laurales Family: Lauraceae—laurels Genus: Cinnamomum Schaeff.—cinnamon The common appearance of the cinnamon plant as exemplified by C. zeylanicum is shown in Fig. 15.2. As shown for C. burmannii (Fig. 15.3), one other common feature for most of the species is their leaves appearing reddish when they are young and turning to dark green as they age.

15.3 Medicinal uses The culinary use of cinnamon including for food flavouring, seasoning, and colouring and as preservatives for food and drinks are all common to the readers. For this purpose, the essential oil components of the plant are of interest and the major constituent, cinnamaldehyde, in fresh and cooked foods is of great significance. The emphasis of this chapter is, however, to look at the benefit of cinnamon beyond its sweet and spicy flavouring potential of our food/ drinks and herein are listed some general medicinal claims: • Treating chronic bronchitis; • A range of inflammatory disorders from, vaginitis, neuralgia, rheumatism, to heal wounds; • Pharmaceutical preparations of cinnamaldehyde for its flavour and other effects; • Mouth problems from freshness of breath to tooth ache; • Nutraceutical or supplement for a plethora of heath claims including type 2 diabetes (T2D). Cinnamon is one of the popular medicinal plants in the Indian or Ayurvedic systems of medicine where it is routinely used for a range of respiratory, digestive and gynecological ailment (Ranasinghe et al., 2013). The oils obtained from the barks of cinnamon tree have been employed in traditional systems of medicine such as the Indian Ayurvedic and Unani E. Potential modulators of type-2 diabetes and associated diseases: Spices

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511

FIG. 15.3 Appearance of cinnamon leaves. Notice colour differences in young and older leaves. Courtesy: Wikipedia (https://upload.wikimedia.org/wikipedia/ commons/f/f1/Starr_090213-2452_Cinnamomum_ burmannii.jpg).

systems as well as various other Eastern medicine. In the Western medicine, the use of cinnamon (e.g. C. verum and C. aromaticum) for a variety of ailments including loss of appetite, dyspeptic complaints, bloating, and flatulence has been common. In the Western markets including food retailers, cinnamon is available to the general public in the form of dried sticks of bark (or quills), powdered bark powder or extracts. Depending on the source plant, plant parts and geographical origin, one should expect variability in the active constituents of these preparations leading to variable health outcomes. Moreover, these available spices may be a mixture of cinnamon obtained from different regions and species and hence prediction of their pharmacological effects may be quite difficult. The use of cinnamon in the Chinese traditional medicine has been well documented too but the inclusion of this spice in this book is based on the claimed use of the plant in modifying metabolic syndrome or T2D. The chemistry and pharmacology of the cinnamon plants that attribute to these medicinal applications are discussed in the following sections.

15.4 The chemistry of the cinnamon bark The nutritional composition of cinnamon with respect to water, ash, protein, carbohydrate, fibres, minerals, or energy levels is available in nutritional databases and product levels. The focus of this section is to scrutinize, the chemistry of the plant that attributes to E. Potential modulators of type-2 diabetes and associated diseases: Spices

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pharmacological effects through drug-like effects. In this regard, cinnamon has two major types of bioactive components: the volatile and non-volatile secondary metabolites. The most important volatile components that predominantly occur in the plant and also of antidiabetic effect is cinnamaldehyde. While the non-volatile components comprise type A and B procyanidins and also have pharmacological significance. It is important to note from the outset that cinnamon represent a number of plant species grown in different countries. Chemical diversity is therefore inevitably common in cinnamon products even far greater than the diversity discussed for a given one species in other chapters based on genetic varieties, growing conditions including soil, climate and other environmental factors, as well as harvesting time and stage of the plant growth (maturity levels and seasons). Only the major classes of compounds representing the cinnamon product as potentially active principles are therefore presented in this section.

15.4.1 Volatile components The essential oil content of the barks ranging from 1% to 4% by weight could be obtained but in all cases the most abundant component is cinnamaldehyde that can be isolated in the yield of 60–80% but up to 90% yield can also be obtained. This cinnamaldehyde that will be discussed in this chapter is the trans-form (1) while the cis-cinnamaldehyde (2) occurs in minute concentration. Readers should note that cinnamaldehyde in this chapter therefore refers to the trans isomeric form (1) (Fig. 15.4). Comparative assessment of yield from different bark sources, for example, showed a yield variability between 0.72% H

O

O

H

O H

OMe O

MeO 1 trans-Cinnamaldehyde

3

OH 4 Eugenol

9 α-Phellandrene

10 β-Phellandrene

2 cis-Cinnamaldehyde

O 5

OH

6 Linalool

7 Camphene

8 Limonene

H

H H

H

12 α-Muurolene

FIG. 15.4

11 p-Cymene

13 α -Calacorene

14 Copaene

H

H

15 β-Caryophyllene

16 α -Guaiene

Essential oil components of cinnamon.

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513

and 3.08% with C. loureirii (3.08%), and C. verum 1.14% oils (Li et al., 2013a). In addition to composition differences based on species, variations of the essential oil components in cinnamon could emanate from differences in habitats, plant parts, harvest seasons and even from the method of extraction and/or analysis. Hence, the volatile oils of cinnamon can greatly vary depending on a host of genetic and environmental conditions. The second most important components of the essential oils that may be obtained with up to 10% yield is eugenol (4). Other components such as α-guaiene (16) can be obtained in good yield but the several dozens of components listed in the essential oil of cinnamons are trace amounts that may not contribute much to the biological effects far beyond modifying the aroma or the fragrances. Gas chromatography coupled with mass spectrometry (GC–MS) analysis of cinnamon (unknown) bark oil reported by Kim et al. (2015) listed 17 compounds of which cinnamaldehyde (1, 64.49%), eugenol (4, 16.57%), linalool (6, 4.87%) and limonene (8, 2.53%), while the rest were less than 2%. As a component of C. verum essential oil, cinnamaldehyde (1, 56.3), β-phellandrene (10, 6.3%), p-cymene (11, 4.1%), linalool (6, 4.1%), β-caryophyllene (15, 3.2%), camphene (7, 2.7%), α-phellandrene (9, 2.1%) and eugenol (4, 2%) were dominant components among the 27 compounds listed (Choi et al., 2016). The study by Li et al. (2013a) showed that the amount of trans-cinnamaldehyde (1) in the C. loureirii barks was 81.97% and that in the C. verum was 74.49%. In contrast, the bark oils contained small amounts of cis-cinnamaldehyde (2, 0.72–2.29%), α-guaiene (16, 1.51–7.62%), copaene (13, 0.58–3.86%), 2-propenal, 3-(2-methoxyphenyl) (3, 0.98–2.59%), α-muurolene (12, 0.11–1.83%) and α-calacorene (13, 0.57–1.28%). The cinnamaldehyde content of 80–95% in the indigenous C. loureirii was also described (Weiss, 2002). Hence, compounds listed in Fig. 15.4 are of major relevance to the pharmacology of the plant related to cinnamon essential oils. The predominant components should be regards as aromatic phenyl propanoids dominated by cinnamaldehyde and to a small extent eugenol; while monoterpenoids and sesquiterpenes do also occur. The essential oils of the leaves are also extensively investigated. In a classical comparative assessment study of the hydro-distilled oils, the highest yield obtained was 1.5% and was for C. cassia followed by C. zeylanicum, C. pauciflorum, C. burmannii, and C. tamala (Wang et al., 2009). While cinnamaldehyde is present in all species and appear as the major component of C. cassia and C. burmannii leaves, the trivial compound, 3-ethoxy-1,2-propanediol, was also a major component in C. cassia; while eugenol was dominant in C. zeylanicum, C. pauciflorum, and C. burmannii essential oils of the leaves. 5-(2-Propenyl)-1,3-benzodioxide (or 3,4(methylenedioxy) allylbenzene, 6) as a major component of C. tamila leaves oil was also suggested. Hence, the genetic diversity of essential oils is also demonstrated for the leaves. Other studies on the leaves as source of essential oil with comparative assessment of the various developmental stages and interspecies variations are also available (bin Jantan et al., 2008; Li et al., 2016). The high cinnamaldehyde content dictates the sweet flavouring properties of cinnamon bark oil while it also contribute to the known antibacterial effect of cinnamon in addition to the polar proanthocyanidins (Shan et al., 2007). One should also bear in mind variabilities of oil content based on fresh, dried or freeze-dried; and extraction methods including solvent extractions, steam distillation, Soxhlet and hydro-distillation, supercritical fluids, pressurized liquid extraction, ultrasound and microwave extractions.

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15.4.2 Non-volatile components The non-volatile components of cinnamon include various polyphenolic groups such as flavonoids of the catechins and proanthocyanidin types, and tannins. Other compounds of the cinnamic acid derivatives, coumarins and many other secondary metabolites are all common that may also differ in composition depending on a variety of conditions. For example, caffeic (28), ferulic (29), p-coumaric (27), protocatechuic (18), and vanillic (20) acids were identified from the water extract bark of C. zeylanicum (Khuwijitjaru et al., 2012). By using LC–MS/ MS analysis, Klejdus and Kova´cik (2016) performed qualitative and quantitative analysis of cassia cinnamon and the most predominant phenolic compounds in their free form were cinnamic acid (26) followed by protocatechuic acid (18) but the list was bigger for the trace amount of detections such as gallic acid (19), p-hydroxy benzoic acid (17), p-hydroxybenzaldehyde (22), 3,4-dihydroxybenzldehyde (23), salicylic acid (25), syringic acid (21), vanillic acid (20), vanillin (24), caffeic acid (28), chlorogenic acid (31), ferulic acid (29), p-coumaric acid (27) and sinapic acid (30) (Fig. 15.5). Isolated from the various cinnamon species, proanthocyanidins are now emerging as the predominant phenolic active constituents. Structurally, proanthocyanidins are dimers, trimers or polymers of the flavonoids class of secondary metabolites which are ubiquitously found in plants. The basic biosynthetic pathway of flavonoids synthesis including flavanols is described in Chapter 6. Polymerization of flavonoids particularly of the flavan-3-ols and flavan-3,4-diols lead to the formation of proanthocyanidins or what are usually termed as the condensed tannins. In some literature, leucoanthocyanidins refers to polymers comprising monomeric flavan-3,4-diols while proanthocyanidins are represented as the oligomers of

O

COOH

R2 OH 17 R1=R2=H p-hydroxybenzoic acid 18 R1=OH, R2=H Protocatechuic acid 19 R1=R2=OH Gallic acid 20 R1=OMe, R2=H Vanillic acid 21 R1=R2=OMe Syringic acid R1

H

COOH

R1

R2 R1 R2 OH OH 25 Salicylic acid 22 R1=R2=H p-Hydroxybenzaldehyde 23 R1=R2=OH 3,4-dihydroxybenxaldehyde 24 R1=OMe, R2=H Vanillin

COOH COOH HO COOH O

R1 R2 26 R1=R2=H Cinnamic acid 27 R1=H, R2=OH p-Coumaric acid 28 R1=OH Caffeic acid 29 R1=OMe Ferulic acid

FIG. 15.5

MeO

OMe OH

30 Sinapinic acid

HO

O OH 31 Chlorogenic acid

Some common phenolic acid derivatives of cinnamon.

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OH OH

15.4 The chemistry of the cinnamon bark

515

monomeric flavan-3-ols. Procyanidin referring to both the leucoanthocyanidins and proanthocyanidins group is the better terminology to adopt as both groups are often found in plants as complex polymers containing various combinations of monomers. The common monomers of the proanthocyanidins found in cinnamon are shown in Fig. 15.6. In the inner bark of Ceylon cinnamon (C. zeylanicum L.), the composition of complex proanthocyanidins has been shown to constitute (epi)gallocatechin and (epi)catechingallate units. The combinations of (epi)catechin, (epi)catechingallate, (epi)gallocatechin, and (epi) afzelechin, to polymerize in a highly heterogeneous mixture of procyanidins, prodelphinidins, and propelargonidins has been proposed (Mateos-Martin et al., 2012). In quality control terms, these are real nightmares where the heterogeneity does not allow accurate standardization of the various herbal preparations of cinnamon. The major chemical structures of the flavan-3-ol units in proanthocyanidins and the major cinnamon procyanidin, type A procyanidin, ( Jiao et al., 2013) is shown in Fig. 15.7. For the sake of clarity, perhaps it is worth describing the structural distinction between procyanidins type A and type B which are both present in cinnamon. Since the proanthocyanidins oligomers in cinnamon are composed of mainly flavan-3-ols where epicatechin (33) or catechin (37) serves as monomers, they are called procyanidins. In highly polymerized form of these compounds, condensed tannins are formed which also occur in cinnamon. The most common procyanidin types in nature are the linkage catechol/epicatechin with another unit through C4 ! C8 or C4 ! C6 bridge to form the B-type of procyanidin. In some plants as exemplified by cinnamon, the A-type procyanidins are formed through the 4! 8 linkage as in the B-type but also 2β catechin/epicatechin linked with the O-7 group of the other flavonoid to make a double bridge (Fig 15.7). The diversity of the dimeric product alone is astonishingly large as exemplified in the following examples through 4 !8 or 4! 6 linkage:

FIG. 15.6 Common flavonoid aglycones that exist in their free form but also serve as monomers of protoanthocyanidins or procyanidins.

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15. Antidiabetic effects of cinnamon

OH

OH

OH

OH O

HO

O

HO

O

O

OH

OH

OH

OH

OH

OH HO

O

O HO

HO

OH

OH HO

HO

OH O

42

HO

OH 43

HO OH

OH O

HO

O

OH

O HO

OH

HO

O HO HO

44

45

O

O

OH HO

O OH

HO

46 α-OH Cinnamtannin B1 47 β -OH Cinnamtannin D1

OH OH

O OH

OH OH HO

O HO

OH

OH O

HO

PA-1 HO

OH

HO 48 Parameritannin A1

FIG. 15.7

OH

HO

HO OH

OH O

HO OH

HO OH

OH

HO OH

OH

OH

OH HO

OH HO

HO

HO HO

O OH

O

O

HO

O

OH

OH

O

HO

OH OH HO

OH

O

HO

OH

OH OH

OH

Examples of common A-type procyanidins isolated from cinnamon.

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15.4 The chemistry of the cinnamon bark

• • • • • • •

517

Procyanidin B1: epicatechin-(4β! 8)-catechin Procyanidin B2: epicatechin-(4β! 8)-epicatechin Procyanidin B3: catechin-(4α! 8)-catechin Procyanidin B4: catechin-(4α! 8)-epicatechin Procyanidin B5: epicatechin-(4β! 6)-epicatechin Procyanidin B6: catechin-(4α! 6)-catechin Procyanidin B8: catechin-(4α! 6)-epicatechin

As shown in Fig. 15.7, the structure of polyphenolic compounds isolated from cinnamon (e.g. C. burmannii) are dimers or doubly linked procyanidin type-A polymers (Anderson et al., 2004; Jiao et al., 2013). The polymerization increases from dimeric to higher order both in type A and type B forms. Eerier studies have shown the identification of polymeric catechin/ epicatechin units from cinnamon bark (C. loureirii Nees) but not the monomeric leucoanthocyanidins or polyphenolic derivatives of benzoic/cinnamic acid (Buchalter, 1971). Anderson et al. (2004) have also isolated polyphenol type-A polymers (e.g. 44) from the water extract of cinnamon that display insulin-like biological activity. The polymers were composed of monomeric units with a molecular mass of 288. Two trimers with a molecular mass of 864 and a tetramer with a mass of 1152 were isolated. Their protonated molecular masses indicated that they are A-type doubly linked procyanidin oligomers of the catechins and/or epicatechins. It is likely that more complex tannins or proanthocyanidins will be isolated from cinnamon. From cinnamon (C. zylanicum) bark was extracted with hydro alcohol (90%), pentameric (not fully characterized) or trimeric (45) procyanidin A were isolated (Connell et al., 2016). While studying the immunosuppressive effects of C. tamala or C. cassia bark, Chen et al. (2014) isolated five procyanidin oligomers compounds: epicatechin-(4β! 8, 2-O-7)epicatechin-(4β ! 8)-epicatechin (cinnamtannin B1, CTB-1, 46), epicatechin-(4β! 8, 2-O-7)epicatechin-(4β ! 8)-catechin (cinnamtannin D1, CTD-1, 47); epicatechin-(4β ! 8, 2-O-7)[epicatechin-(4β !6)]-epicatechin-(4β! 8)-epicatechin (parameritannin A1, PA-1 48); along with the B-type procyanidins epicatechin-(4β ! 8)-epicatechin (49) and epicatechin(4β! 8)-epicatechin(4β !8)-epicatechin (50) (Figs 15.7 and 15.8). The structural assignment

OH

OH

OH OH HO

OH

OH

OH

OH OH

O

OH

O

HO

O

HO

FIG. 15.8 Examples of procyanidin B2 compounds isolated from cinnamon.

OH

OH

OH HO

OH

O OH

OH HO

OH OH

O OH

49

OH

50

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of cinnamon procyanidins must be considered incomplete as they still need further isolation and structural determinations. One of the most comprehensive chemical analysis of cinnamon (C. cassia Barks) for phenolic constituents was carried out by Luo et al. (2013) who isolated three new phenolic glycosides called cinnacassosides A–C (51–53) as 70 -hydroxy-90 -β-glucopyranosyloxyl secoisolariciresinol (cinnacassoside A, 51); dihydrodehydrodiconiferyl alcohol-9-β-D-apiofuranosyl (1!6)-β-Dglucopyranoside (cinnacassoside B, 52); and 3,4-dimethoxy-5-hydroxy-phenol β-D-apiofuranosyl (1! 6)-β-D-glucopyranoside (cinnacassoside B, 53) (Fig. 15.9). Of the 15 known compounds isolated together with these novel compounds, some (56–59, 61, 163–68) were identified from the species for the first time. The list of compounds included (see Fig. 15.9) cinnacasside C (54), rosavin (55), ()-(70 S,8S,80 R)-4,40 -dihydroxy-3,30 ,5,50 -tetramethoxy-70 ,9-epoxylignan-90 -ol-7-one (56), lino-cinnamarin (57), leonuriside (58), 3,4-dimethoxyphenol β-D-apiofuranosyl (1!6)-β-D-glucopyranoside (59) [26], 3,4,5-trimethoxyphenol (1! 6)-β-D-glucopyranoside (60), 3-trimethoxy-4-hydroxyphenol β-D-apiofuranosyl β-D-apiofuranosyl (1!6)-β-D-glucopyranoside (61), rosarin (62), ()-lyoniresinol 3α-O-β-Dglucopyranoside (63), spicatolignan B (64), methyl 3-methoxy-4-(β-D-allopyranosyloxy) benzoate (65), 6-hydroxy-2-(4-hydroxy- 3,5-dimethoxyphenyl)-3,7-dioxabicyclo-[3.3.0]-octane (66), evofolin B (67), and 2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-1,3-propanediol (68). It is worth mentioning that compounds 54 58 were shown to inhibit overproduction of fibronectin, collagen IV, and interleukin (IL)-6 (IL-6) under high-glucose in mesangial cells at 10 μM, while all the other compounds were not active when tested at this fixed concentration. Several other secondary metabolites have been isolated from cinnamon although the polyphenols of the water extract, primarily procyanidins, are generally regarded as the active components for the antidiabetic effects of cinnamon barks. Of the other compounds isolated (Fig. 15.10) include cinncassiol D4 (69) and cinncassiol D4 glucoside (70) as two diterpene constituents of the bark of C. cassia (Nohara et al., 1982). The hexacyclic diterpene, cinncasiol E (71), was also isolated from C. cassia (Nohara et al., 1985). As part of the tyrosinase inhibitory activity study on the methanol extract of the twigs of C. cassia, several compounds have been isolated with cassiferaldehyde (72), cinnacassinol (74), icariside DC (75), and dihydrocinnacasside (73) identified with weak activity (IC50 values ranging from 0.24 to 0.94 mM) (Ngoc et al., 2009). Numerous compounds have been isolated from C. tenuifolium but the novel entries worth showing are tenuifolide A (76), isotenuifolide A (77), and tenuifolide B (78), a new secobutanolide, secotenuifolide A (79), and one new sesquiterpenoid, tenuifolin (80) (Lin et al., 2009). From the hot water extract of barks of C. cassia, cassioside (81), the derivatives of compound 53 as trimethoxyl as 3,4,5-trimthoxypherol-β-D-apiofurrrosyl-(1 !6)-β-glucopyranoside (82), and cinnoside (83) were isolated as antiulcerogenic active principles (Shiraga et al., 1988). From the bark of C. cassia, Miyamura et al. (1983) also isolated seven compounds: lyoniresinol 3α-O-β-Dglucopyranoside (63), 66, 3,4,5-trimethoxyphenol 1-O-β-D-apiofuranosyl-(1 ! 6)-β-Dglucopyranoside (82), epicatechin derivatives (84, 85) and trans-cinnamaldehyde derivatives with cyclic glycerol 1,3-acetal structures (86, 87). Finally, one trivial compound but with toxicological interest for cinnamon application is coumarin (88) and its derivatives and will be discussed in Section 15.7.

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15.5 General pharmacology

O OH

HO

O OH

O

HO O

HO HO

O

HO HO

HO HO

O OH

O

OH

O HO

HO HO

OH

HO Glc = Glucose

O

C

HO HO D

HO

HO

B

O

O

O

HO O

52

OH O Glc

OH

O B

O O

O

OH O 53

Glc

O O

O

O

OH

53 O

O C

O

O

OH 51

55

O OH

Glc

O HO

R1

O

R2

HO O

O 57

OH O O D

B

O

O

H

O

O

O

67

O

OH OH

O O

O

66

O

HO

OH H

HO

Glc O 65

64 OH

O

OH

O

O OH 63

O HO

Glc

O

O O

HO

O

HO

62

59 R1=OMe, R2=H 60 R1=OMe, R2=OMe 61 R1=OH, R2=H

56

O

O Glc 58

O

O

O

O OH

HO

B

OH

HO

HO

OH

O OH

O

68

FIG. 15.9 Lignans and other aromatic compounds from the bark of C. cassia.

15.5 General pharmacology One common pharmacological effects of essential oil is antioxidant, antimicrobial and antiinflammatory effects which have been demonstrated for cinnamon oil (Singh et al., 2007; Tung et al., 2008). The antiulcer (Amr and Maysa, 2010) and other pharmacological effects

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RO

HO OH

H H O HO HO

H

OH O OH

OHO

H

OH

O OH

HO

HO 72 Cassiferaldehyde 73 Dihydrocinnacasside

71 Cinncassiol E 69 R=H Cinncassiol D4 70 R=Glc Cinncassiol D4 2-O-glucoside

HO

OH

HO

Glc O O

O

OH

O

O HO

O O

O

O

O

HO OH

O OH 75 Icariside DC

HO

OH HO

O

OH

OH O

C25H51

OH

O

HO

OH

O

HO

O

H O O 76 Tenuifolide A

OH

74 Cinnacassinol

H

HO

C25H51 O

O

O

77 Isotenuifolide A

C18H37

HO

O OMe

O

O

O

O

79 Secotenuifolide A

B

O

OH

O O

B

O

O

O

C Glc

OH

H

78 Tenuifolide B

O

MeO

C23H47

MeO

80 Tenuifolin

O

HO

H

O

OH OH

B=

O

OH

O

OH R

82

83 Cinnoside

O O

O

HO Glc =

O MeO

FIG. 15.10

84 R=H, Me 85 R= -CH2-

HO

R2

86 R1=H, R2=OH 87 R1=OH, R1=H

OH OH

O

R1

OH OMe

OH

HO

81 Cassioside O

OH

O

O

88

Other miscellaneous compounds of cinnamon.

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have also been recorded but those specifically relevant to antidiabetic effect of cinnamon are included in the following sections.

15.6 Antidiabetic, antiobesity, and antihyperlipidaemic effects 15.6.1 Evidence of efficacy from studies on animal models: Cinnamon powder and crude extracts In a screening study for potential antidiabetic effect, cinnamon was among spices (apple pie spice, cloves, bay leaves, and turmeric) which was reported in 1990 to potentiate insulin activity by more than threefold (Khan et al., 1990). A number of animal studies since then have shown not only the glucose lowering effect of cinnamon but also the various pathologies associated with chronic diabetes. In addition to the antidiabetic effect, improvement of glycaemic control by cinnamon as assessed by oral glucose tolerance test (OGTT) was among the best studied pharmacology for cinnamon in various models. Ranasinghe et al. (2012a), for example, showed that C. zeylanicum (Ceylon cinnamon) (600 mg/kg, equivalent to 6 g in humans) caused faster decline in blood glucose in OGTT, as well as inducing suppressive effect on food intake and improvement of lipid parameters in streptozotocin (STZ)-induced diabetic rats. Improvement of glucose tolerance has also been established for cinnamon (C. cassia and C. zeylanicum) in normal animals (Verspohl et al., 2005). By the measure of glucose utilization and insulin signalling, Qin et al. (2004) have investigated the role of cinnamon supplementation in drinking water (300 mg/kg for 3 weeks) in rats fed with a high-fructose diet (HFRD). When 3 mU/kg/min insulin infusions was employed on in HFRD-fed rats, the decreased glucose infusion rate was improved by cinnamon administration to the controls normal diet-fed animals. In insulin resistance induced by a high fat/high fructose diet, cinnamon given as a 20 g/kg of diet supplement has also been shown to improve insulin sensitivity along with reversal of the change in pancreas weight and mesenteric white fat accumulation (Couturier et al., 2010). A high fructose diet in rats receiving high doses of cinnamon bark (C. zeylanicum) extract in a 60-day experimental protocol could also reduce the levels of glucose, insulin, protein-bound sugars and hyperlipidaemia to near normal level (Kannappan et al., 2006). The doses was, however not well quantified and stated to be 2 mL/rat from 10 g/100 mL extract and 10-times dilution. In HFD- and alloxan-induced obese diabetic rats, the aqueous extract of cinnamon (C. zeylanicum L.) extract (at doses of 200 and 400 mg/kg, p.o. for 6 weeks) reduced body weight and body fat mass; normalized serum levels of liver enzymes (alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT)); improved lipid profile (reduce total cholesterol (TC), triglyceride (TG), low density lipoprotein-cholesterol (LDL-C) and increased high density lipoprotein-cholesterol (HDL-C)), antioxidant defences (superoxide dismutase (SOD), glutathione (GSH) peroxidase (GPx) and catalase (CAT) enzymes in renal tissues); decreased blood glucose and leptin; and increased insulin serum levels (Shalaby and Saifan, 2014). In hypercholesterolaemic rats fed with 1% cholesterolenriched diet for 15 days, the significant increase in the TG, TC, LDL-C, liver enzyme markers (AST, ALT, and ALP), homocysteine (Hcy) and hepatic malondialdehyde (MDA) were suppressed by cinnamon extract (Amin and Abd El-Twab, 2009). Moreover, the suppressed

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level of antioxidant enzyme activities (SOD and CAT) and nitric oxide (NO) levels were shown to be improved by cinnamon extract (Amin and Abd El-Twab, 2009). Another diabetic model employed for cinnamon was the genetically obese ob/ob mice where cinnamon extract (0.8 g/kg, p.o. for 6 weeks) was not only shown to suppress the fasting blood glucose (FBG) levels and glucose tolerance but also improve insulin sensitivity, brain function and the insulin-stimulated locomotor activity (Sartorius et al., 2014). In this T2D diabetic db/db mice model, administration of C. cassiae extract (50, 100, 150, and 200 mg/kg, p.o.) for 6 weeks has also been shown to suppress glucose levels in a dosedependent manner with the highest dosage (200 mg/kg) shown to induce comparable level with the non-diabetic control groups (Kim et al., 2006). A significant increase in the serum levels of insulin and HDL-C coupled with reduction in the levels of TG and TC were also among the favourable outcomes of cinnamon treatment. In addition to the effect of cinnamaldehyde (20 mg/kg) discussed in Section 15.6.3.1, the oil (100 mg/kg and 200 mg/kg, 28 days) of C. tamala, with the cinnamaldehyde content of only 44.898%, showed comparable effect with standard drug glibenclamide (0.6 mg/kg) in inducing body weight recovery and reduction of blood glucose, glycosylated hemoglobin and TC in STZ-induced diabetic animals (Kumar et al., 2012). In the T2D animal model of KK-A(y) mice which exhibits obesity coupled with hyperglycaemia and increased HbA1c level, cinnamon oil (hydro-distillation from commercial cinnamon bark in Chinese market) (100 mg/kg, intragastric (i.g.) for 35 days) was shown to reduce blood glucose, plasma C-reactive peptide (CRP), serum TG, TC, and blood urea nitrogen levels, while the serum HDL-C level was significantly increased (Ping et al., 2010). Furthermore, glucose tolerance and pancreatic islets β-cells functions were improved. This oil with the composition of cinnamaldehyde as 78.513% could attribute to the main antidiabetic effect of this major compound that will be discussed in Section 15.6.3.1. The polyphenolic water soluble components that will be discussed in Section 15.6.3.2 are also considered as active principles of cinnamon and display a range of antidiabetic effects. For example, the polyphenolic oligomer-rich extract of C. parthenoxylon bark have shown to display antidiabetic effect in the STZ-induced diabetic rats’ model when administered at doses of 100, 200, and 300 mg/kg (p.o. for 14 days). The extracts decreased glucose level in diabetic rats by 11.1%, 22.5%, and 38.7%, respectively ( Jia et al., 2007). Significant hypoglycaemic effect in OGTT induced by the extract also suggests the antidiabetic potential of the plant. As outlined above, there is now overwhelming evidence to suggest that cinnamon could induce antidiabetic effect in various animal models. In addition to reduction of glucose and glycated haemoglobin levels, the antihyperlipidaemic effects of cinnamon are evident in the various diabetes models as lipid dysregulation were shown to be ameliorated. In view of these known effects of cinnamon, Nayak et al. (2017) also studied the anti-atherosclerotic potential of the aqueous extract of C. zeylanicum bark extract in insulin resistance associated with the dexamethasone-induced atherosclerosis, increased atherogenic Index and dyslipidaemia. They reported that the extract (500 mg/kg and 250 mg/kg) significantly prevented dyslipidaemia and maintained atherogenic index to control level. Treatment with the extract also protected the aorta from atherosclerosis (by 40.3% and 30.2% respectively). Further insight into the mechanisms of action as well as active principles of cinnamon are presented in the following sections.

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15.6.2 General mechanism of action—Crude extracts 15.6.2.1 Carbohydrate digestion The inhibition of intestinal α-glucosidase and pancreatic α-amylase by various cinnamon species were investigated by Adisakwattana et al. (2011). For the intestinal maltase activity, the Thai cinnamon extract was found to be the most potent inhibitor (IC50 values of 0.58  0.01 mg/mL) while Ceylon cinnamon was the most potent in the intestinal sucrase and pancreatic α-amylase inhibition (IC50 values of 0.42  0.02 and 1.23  0.02 mg/mL, respectively). Interestingly, these extracts showed additive effect in intestinal α-glucosidase and pancreatic α-amylase inhibition when combined with the well-known clinically useful antidiabetic agent, acarbose that act through the same mechanism. By using the T2D animal model (db/db) in mice, Kim et al. (2006) have studied the effect of cinnamon extract (C. cassia) on intestinal glycosidases (sucrase, maltase and lactase) in the small intestine. They found that the extract (200 mg/kg, p.o.) could decrease sucrase in proximal and middle segments; maltase activity in the proximal and distal segments, while lactase activity decreased only in the proximal segment. As explained in the previous texts, the dose of cinnamon extract employed in this study showed total reversal of glucose level in diabetes animals. While conducting clinical trial on cinnamon, Beejmohun et al. (2014) also studied the effect of Ceylon cinnamon extracts (water and hydro-alcoholic extract) on carbohydrate digestion both in vitro and in vivo. The more potent extract was the hydro-alcoholic extract which showed pancreatic α-amylase activity in vitro with IC50 of 25 μg/mL and in vivo study using starch tolerance tests in rats at the dose of 12.5 mg/kg. Arachchige et al. (2017) investigated the effects of ethanol (95%) and dichloromethane: methanol (1:1, v/v) bark and leaves extracts of Ceylon cinnamon for antiamylase and antiglucosidase activities. The bark extract showed significantly higher activities for antiamylase (IC50: 214  2–215  10 μg/mL) when compared to the leaves extract. The methanol extract of the cinnamon bark of C. zeylanicum has also been shown to display a dosedependent inhibitory activity against the yeast α-glucosidase (IC50 value of 5.83 μg/mL) and mammalian (rat-intestinal) α-glucosidase (IC50 value of 670 μg/mL). Furthermore, the postprandial hyperglycaemia after the oral intake of the cinnamon extract (300 mg/kg) was suppressed by 78.2% and 52.0% in maltose and sucrose loaded STZ-induced diabetic rats respectively (Mohamed Sham Shihabudeen et al., 2011). 15.6.2.2 Glucose production The crude extract of cinnamon and its polyphenol-enriched fraction have been shown to lower FBG levels in diet-induced obese hyperglycaemic mice when tested at 300 and 600 mg/kg respectively (Cheng et al., 2012). These two preparations did also display a dose-dependent inhibition of hepatic glucose production in vitro (rat hepatoma cells) with significant levels of inhibition obtained at 25 μg/mL as well as suppression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase): two major regulators of hepatic gluconeogenesis. As shown below, mechanisms related to insulin signalling and genes expression modulation attribute to the inhibition of glucose production in vital organs such as the liver.

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15.6.2.3 Gene and protein expression/activation By measuring the insulin potentiation activity of cinnamon through glucose oxidation assay in rat epididymal fat cells, Imparl-Radosevich et al. (1998) have shown that inhibition of protein tyrosine phosphatase-1 (PTP-1) should be considered as a potential mechanism of antidiabetic effect by cinnamon. As an inhibitor (Wortmannin) of phosphatidylinositol-4,5bisphosphate 3-kinase (PI3K) suppressed the response to both insulin and that induced by cinnamon, the effect of cinnamon through action at some stage of upstream of PI3K activity was proposed. Moreover, cinnamon has been show to stimulate the autophosphorylation of a truncated form of the insulin receptor in addition to inhibition of PTP-1 that inactivates the insulin receptor. Hence, effect through protein phosphorylation-dephosphorylation reactions in intact adipocyte that mirrors insulin’s effect was proposed, i.e. protein tyrosine phosphatase is a target for cinnamon. The water extract of cinnamon (C. burmannii) was also tested for its effect on the expression of genes encoding adipokines, glucose transporter (GLUT) family, and insulin-signalling components in mouse 3T3-L1 adipocytes (Cao et al., 2010). GLUT4 and protein tristetraprolin (TTP) levels in adipocytes did also increase by the extract when tested at the concentration of 100 μg/mL. Readers should note that TTP (also called the zinc finger protein 36 homolog) is one of the negative feedback loop of inflammation that interferes with the production of TNFα by destabilizing its mRNA, i.e. it is an anti-inflammatory gene (ZFP36) or a protein. The extract also increased GLUT1 mRNA levels by several fold at 2-, 4-, and 16-hour treatment periods, while the expression of genes encoding insulin-signalling pathway proteins (glycogen synthase kinase 3β (GSK3B), IGF1R, insulin-like growth factor (IGF)-2 receptor, and PI3K receptor-1) were suppressed. Previous studies by Cao et al. (2007) have also shown that the water extract of cinnamon (100 μg/mL of C. burmannii) enhance the mRNA levels of TTP by about sixfold in mouse 3T3-L1 adipocytes while its effect at higher concentrations decreased insulin receptor-β (IRβ) protein and insulin receptor (IR) mRNA levels, and the biphasic pattern of effect on GLUT4 mRNA levels was reported. Experimental evidences to demonstrate the improvement of insulin action via increasing glucose uptake in vivo through enhancement of the insulin-signalling pathway in skeletal muscles have also been presented (Qin et al., 2003). This include a higher level of insulinstimulated tyrosine phosphorylation levels of IRβ and IR substrate-1 (IRS-1) in cinnamon extract-treated (300 mg/kg, p.o.) rats. Furthermore, the IRS-1/PI3K association significantly increased by cinnamon leading to improved activation of PI3K activity or glucose uptake. In the study by Qin et al. (2012), freshly isolated intestinal enterocytes were used to investigate the effect of cinnamon polyphenols ex vivo for insulin signalling and intestinal lipoprotein expression at genes and protein levels. Their data revealed that cinnamon significantly decreased the amount of apolipoprotein-B48 secretion, inhibited the mRNA expression of genes of inflammatory cytokines (IL-1β, IL-6, and tumour necrosis factor-α (TNF-α)) and induced the expression of the anti-inflammatory gene, Zfp36 (or TTP). Cinnamon extract also increased the mRNA expression of genes leading to increased insulin sensitivity, including IR, IRS-1, IRS-2, PI3K, and protein kinase B (e.g. Akt1), and decreased phosphatase and tensin homologue deleted on chromosome 10 (PTEN) expression. The cinnamon extract also inhibited genes associated with increased cholesterol, TG, and apolipoprotein-B48 levels (e.g. Abcg5, Npc1l1, Cd36, Mttp, and Srebp1c) and facilitated ATP-binding cassette

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transporter (ABCA1) expression. On the other hand, the phospho-p38 mitogen-activated protein kinase (p-p38 MAPK), c-Jun N-terminal kinase (JNK), and extracellular-signal-regulated kinase expressions (ERK) were stimulated by the extract though no changes in protein levels were observed. Hence, cinnamon could be regarded as an agent that regulates genes associated with insulin sensitivity, inflammation, and cholesterol/lipogenesis metabolism as well as the activity of the MAPK signalling pathway in intestinal lipoprotein metabolism. The effects of cinnamon on central expression of genes related to immune system, hypothalamicpituitary-adrenocortical axis function, and cerebral plasticity in rats have also been outlined (Marissal-Arvy et al., 2014). The water extract (30 μg/mL) of cinnamon (C. zeylanicum) has also been shown to induce glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes in vitro by stimulating the phosphorylation of 50 adenosine monophosphate-activated protein kinase (AMPK) and acetylCoA carboxylase (ACC) (Shen et al., 2014). Interestingly, the AMPK inhibitor and liver kinase B1 (LKB1) siRNA blocked the cinnamon-induced glucose uptake; while glucose tolerance tests in T2D model rats (Otsuka Long-Evans Tokushima Fatty (OLETF) rat—a model of type 2 diabetes) could be improved by the extract (100 mg/kg for 15 weeks). Absalan et al. (2012) also demonstrated that the hydro-alcoholic cinnamon extract (100 or 1000 μg/mL) in C2C12 myoblastic cells can induce GLUT4 translocation from intracellular to cell surface. In the various sections of this book, the AMPK is presented as a ubiquitous target for natural products to increase insulin sensitivity and upregulate glucose uptake via GLUT4 expression in muscle and improvement of lipid profile in adipose tissues. Qin et al. (2004) have shown the antidiabetic effect of cinnamon in HFRD-fed rats: an effect which was found to be blocked by about 50% by the inhibitor of nitric oxide synthase (NOS), N-monomethyl-L-arginine. The suppressed levels of muscular insulin-stimulated IRβ and IRS-1 tyrosine phosphorylation and IRS-1 associated with PI3K in HFRD-fed rats were shown to be significantly improved by cinnamon treatment. It could thus be concluded that administration of cinnamon could ameliorate in HFRD-induced development of insulin resistance by enhancing insulin signalling and possibly via the NO pathway in skeletal muscles. In order to investigate the mechanisms of insulin resistance and lipid metabolism improvement by cinnamon extract, Sheng et al. (2008) employed HFD-induced obesity and db/db diabetic mice model in vivo as well as 3T3-L1 adipocyte in vitro. They have reported that cinnamon (200 or 600 μg/mL) can increase the expression of peroxisome proliferator-activated receptors (PPPARs), PPAR-γ/α, and their target genes (such as lipoprotein lipase (LPL), CD36, GLUT4, and ACC); increase the trans-activities of both full length and ligand-binding domain of PPAR-γ and PPAR-α. These data suggest that the antidiabetic and antihyperlipidaemic effects of water extract of cinnamon could be via dual activation of PPAR-γ and -α. This mechanism was also consistent with the demonstration of cinnamon extract (400 mg/kg for 3 weeks) improving hyperglycaemia and intraperitoneal glucose tolerance test (IPGTT) or hyperlipidaemia and liver function in animal models. In db/db mice, the antihyperglycaemic and antihyperlipidaemic action of C. cassiae bark extract (200 mg/kg, p.o. for 12 weeks) was associated with increased PPAR-α mRNA (liver) and PPAR-γ mRNA (adipose tissue) expression levels. This is in line with the reduction of FBG and postprandial (2 h) blood glucose levels as well as serum lipids and hepatic lipids improvement in diabetic animals (Kim and Choung, 2010). Hence, induction of PPARs expression by cinnamon could

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be seen as a mechanism of action similar with the thiazolidinediones (TZDs) class of antidiabetic drugs that act through PPAR-γ agonistic effects. Shen et al. (2010) used STZ-induced diabetic rats and 3T3-L1 adipocytes to investigate the antidiabetic mechanism of action of aqueous extract of cinnamon (C. zeylanicum) bark. After treatment with a relatively small dose (at 30 or 100 mg/kg, p.o.) for 22 days, the reduction in hyperglycaemia, hyperlipidaemia (improvement in TC, TG, and HDL) and nephropathy (histology and plasma creatinine level) score was shown to be associated with upregulation of uncoupling protein-1 (UCP-1) and GLUT4 in brown adipose tissues and muscles. Furthermore, upregulation of GLUT4 translocation and increase in the glucose uptake was evident in cultured adipocytes in vitro when treated by cinnamon extract (30 μg/mL). In the mitochondria of brown adipose tissues, the UCP-1 use the mitochondrial membrane potential to transduce heat production instead of ATP and hence allowing energy expenditure in the absence of exercise. Increasing the level of UCP-1 or its activity; or browning of white adipose tissue to express more UCP proteins are regarded as mechanisms of antiobesity or lipid lowering effects. Based on data from experiments on extracts (C. cassia) added at the various stage of 3T3-L1 adipocyte differentiation, cinnamon may boost lipid storage in white adipocytes and increase fatty acid oxidation capacity throughout the initiation stage of differentiation. Genes related to adipogenesis and lipogenesis were also enhanced when cinnamon extract was administered during the initiation stage of differentiation but not when administered during the preadipocyte and post differentiation stages (Lee et al., 2016). While variable response may be obtained for the different cinnamon species and disease models, genes and proteins expression and/or activation appear to be the main target for cinnamon to induce antidiabetic, antiobesity, and/or antihyperlipidaemic effects. 15.6.2.4 Insulin release Numerous reports on the antidiabetic effect of cinnamon outlined its insolinotropic effect. In insulin secreting cell line (INS-1 cells), for example, the induction of insulin release by cinnamon (C. cassia and C. zeylanicum) was observed (Verspohl et al., 2005). This is due to the β-cell protective effect as well as stimulation of insulin production by the active components that will be discussed in Section 15.6.3. 15.6.2.5 Antioxidant and antiglycation effects Besides the general antioxidant effect of the essential oils, the water soluble fractions of cinnamon have been shown to demonstrate antioxidant effects both in vivo and in vitro or even more specifically on diabetes models. The increased in antioxidant defences such as SOD, CAT, GPx and reduction of lipid peroxidation (LPO) products such as MDA are classical examples of antioxidant mechanism exhibited as part of the antidiabetic effects in animal models (Section 15.6.1). In a high fat/high fructose diet-induced insulin resistance model in rats, cinnamon administration has been shown to alleviate oxidative stress in the brain (Couturier et al., 2016). Since reactive oxygen species (ROS) are involved in this deleterious process, the antioxidant mechanism of cinnamon is suggested to contribute to the antidiabetic properties of cinnamon products. As shown for the active principles in the following sections, direct radical scavenging and other mechanisms are also involved for amelioration of oxidative stress by cinnamon.

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By altering the stress-activated protein kinases pathway, primarily P38-MAPKs, cinnamon can also induce antioxidant and anticancer activities (Schoene et al., 2009). The negative effects of a high fat/high fructose diet on behaviour, brain insulin signalling and Alzheimer’s disease-associated changes have also been ameliorated by cinnamon (Anderson et al., 2013). Some evidence coming from clinical trial whereby cinnamon administration increase the plasma thiol (SH) groups while decreasing the plasma MDA levels in diabetic subjects (Roussel et al., 2009) is also in line with the antioxidant effect of cinnamon. In a glial cell (C6 glial cells) model of antioxidant study (Panickar et al., 2009), cinnamon polyphenols have been shown to attenuate cell swelling and mitochondrial dysfunction following oxygen-glucose deprivation. This effect is partly due to reversal of the decline in depolarization of the inner mitochondrial membrane potential (ΔΨm) induced by oxidative stress. While procyanidins such as the type A polymers are regarded as the main antioxidant principles of cinnamon (Panickar et al., 2012), other lipophilic compounds such as eugenol and cinnamaldehyde have also been shown to display general antioxidant effects as well as modulation of the mitochondrial membrane potential and respiratory chain complexes (Usta et al., 2002). Water-soluble polyphenolic polymers from cinnamon (polyphenol type-A polymers) that account to the glucose regulatory and antioxidant effects have further been isolated by Anderson et al. (2004). Two trimers with a molecular mass of 864 and a tetramer with a mass of 1152 were also isolated. The A type doubly linked procyanidin oligomers of the catechins and/or epicatechins was suggested but not fully characterized. Arachchige et al. (2017) investigated the effects of ethanol (95%) and dichloromethane: methanol (DCM:M; 1:1, v/v) bark and leaves extracts of Ceylon cinnamon for antiglycation and glycation reversing potential in bovine serum albumin- (BSA-) glucose and BSA– methylglyoxal models in vitro. In this model, the glycation reversing effect for the bark in BSA–glucose model (EC50: 94.33  1.81–107.16  3.95 μg/mL) was better than leaves extract. In contrast, glycation reversing in BSA–methylglyoxal (EC50: ethanol: 122.15  6.01 μg/mL) and antiglycation in both BSA–glucose (IC50: ethanol: 15.22  0.47 μg/mL) and BSA– methylglyoxal models (IC50: DCM:M; 278.29  8.55 μg/mL) were significantly higher in the leaves extract. Jin and Cho (2011) screened the water extracts of commercially available (South Korea) spices for a range of pharmacological effects. They included cinnamon (unknown variety) which contained cinnamaldehyde as high as 82.6%. Their findings include: • Cinnamon extract may protect apoA-I from non-enzymatic glycation by fructose as less formation of multimeric bands, and modification of the apoA-I tertiary structure, i.e. antiglycation effect was demonstrated; • Antioxidant activity was observed from the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity; • Antiobesity and hypolipidaemic activity in hypercholesterolaemic zebrafish model was observed though the effect was not as good as clove. In HepG2 cells damage induced by 30 mM D-ribose damage, the ethanol extract of C. osmophloeum could ameliorate oxidative stress while increasing the expression of ghrelin gene as well as mRNAs of ghrelin processing enzyme, furin, and mboat4. The level of ghrelin hormones in the culture media increasing by 4–9 times was also suggested as the mechanism of cytoprotective from oxidative damage (Liu et al., 2017).

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15.6.2.6 Anti-inflammatory mechanisms A number of studies have shown the anti-inflammatory and analgesic effect of cinnamon. Studies on the aqueous extract of C. cassia on experimental allergic reaction (Nagai et al., 1982a) suggest an inhibitory effect on the complement-dependent allergic reaction. At high concentration, the extract also inhibited the immunological haemolysis, chemotactic migration of neutrophils in response to complement activated serum, and the generation of chemotactic factors; while the type IV reaction, contact dermatitis, was not affected by the extract. On the other hand, the extract only slightly inhibited the production of haemolytic plaque forming cells. Other studies have similarly demonstrated the anti-inflammatory effect of aqueous extract of cinnamon on experimental glomerulonephritis (Nagai et al., 1982b). More specifically to diabetes, the anti-inflammatory effect of cinnamon has been demonstrated from the inhibitory effects on genes and proteins of proinflammatory cytokines such as IL-1β, IL-6, and TNF-α and anti-inflammatory genes/proteins induction such as TTP. This will be further outlined for the active principles. 15.6.2.7 Cardiovascular effects The favourable effect of various cinnamon preparations (e.g. C. cassia and C. burmannii) on blood pressure (BP) and cardiovascular system have been demonstrated by various studies (Chen, 1981; Preuss et al., 2006). More specifically to diabetes, the addition of cinnamon (8%, w/w in diet) was shown to ameliorate the sucrose-induced BP elevations in spontaneously hypertensive rats (Preuss et al., 2006).

15.6.3 The active principles of cinnamon: Evidence of efficacy from studies in vitro and animal models 15.6.3.1 Cinnamaldehyde Babu et al. (2007) have studied the antidiabetic effect of cinnamaldehyde isolated from C. zeylanicum. When the compound was administered (5, 10, and 20 mg/kg, p.o. for 45 days) in STZ-induced diabetic rats, a dose-dependent decrease in plasma glucose level (reaching up to 63.29% of control) was achieved. The compound (20 mg/kg) significantly decreased glycosylated hemoglobin (HbA1C), the serum levels of TC and TG while at the same time markedly increased plasma insulin, hepatic glycogen and HDL-C levels. It did also reverse the diabetes-mediated alteration of plasma enzyme (AST, ALT, ALP, lactate dehydrogenase, and acid phosphatase) levels to near normal level. Even though the dose used was higher than the reference antidiabetic glibenclamide, (0.6 mg/kg), the high LD50 value of cinnamaldehyde (1850  37 mg/kg) and known use of the compound in various in vivo models is encouraging. In fact, this dose is far lower than the common antidiabetic agent metformin in the various animal models described in this book. In the last decade, numerous other studies using the STZ-induced diabetic rat model have also been used to demonstrate the efficacy of cinnamaldehyde as an antidiabetic agent when administered orally or via intragastric gavage. This include doses of 40 mg/kg for 3 weeks (Lee et al., 2013); 20 mg/kg for 28 days (Kumar et al., 2012); 60 days (Anand et al., 2010); or 6 weeks (El-Bassossy et al., 2011). Similarly, the alloxan-induced diabetic rat model was used to show

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the antidiabetic effect of cinnamaldehyde at doses of 5, 10, and 20 mg/kg when administered by intraperitoneal (i.p.) route for 4 weeks (Mishra et al., 2010). Another animal model was the HFD-induced diabetic mice where cinnamaldehyde was shown to display antidiabetic effect at doses as small as 5 and 10 mg/kg (p.o.) after 14 weeks of daily administration (Khare et al., 2016) or when it was used as 0.2% food supplement in 36 days intervention (Camacho et al., 2015). Similarly, doses of 0.1%, 0.5%, or 1% food supplement with cinnamaldehyde in high fat and high fructose meal could induce antidiabetic effect as shown in a 30 days intervention study (Tamura et al., 2012). In a combination of HFD and STZ in rats, 40 mg/kg dose (i.g.) displayed antidiabetic effect in 4 weeks study (Li et al., 2009; Zhang et al., 2008). Finally, intervention (25–100 mg/kg, i.g. for 35 days) study in the KK-Ay mice model has shown the effectiveness of cinnamaldehyde as an antidiabetic agent. One communality in all the above-mentioned animal studies is that cinnamaldehyde was effective when administered for a period of few weeks; perhaps explaining why acute or one day intervention by 10 mg/kg (Hafizur et al., 2015) in diabetic animals was not effective (see below). The severity of diabetes in the animal models could vary. For example, STZ may be administered via the intravenous (i.v.) or i.p. routes or its dosage could vary between 30–40 mg/kg in combination studies (e.g. with HFD) to 50–90 mg/kg by its own. The duration of study could also vary making the STZ model either T1D or T2D depending on the severity of β-cell loss. In all diet-, chemical- and/or obesity induced diabetes models, however, the therapeutic potential of cinnamaldehyde through oral route has been demonstrated. Considering that the volatile oil of cinnamon was shown to contain cinnamaldehyde up to 98%, the whole hydro-distillate oil from the dried inner bark of C. zeylanicum Blume has been used as cinnamaldehyde (Mishra et al., 2010). Further effects and biochemical mechanisms that attributes to the antidiabetic effects of cinnamaldehyde are outlined below. 15.6.3.1.1 Cinnamaldehyde and glycaemic control

Bernardo et al. (2015) have shown that cinnamon (C. burmannii) tea administration (6 g into 100 mL water) could slightly reduce the plasma glucose level after OGTT. This is consistent with the human studies where improvement of postprandial glycaemic control was demonstrated for cinnamon powder (see Section 15.6.4). The improvement of insulin sensitivity and glycaemic control by cinnamaldehyde is a result of effects at various levels including in fat/ adipose tissues and muscles. The study by Anand et al. (2010) showed that cinnamaldehyde in STZ-induced diabetic rats (20 mg/kg, p.o. for 60 days) could effectively increase the muscle and hepatic glycogen content; while in isolated pancreatic islets, it enhanced insulin release. Furthermore, the compound reduced blood glucose, HBA1c and glucose level in GTT by its own and as an active principle of C. zeylanicum. Hence, cinnamaldehyde: • Reversed the reduced pyruvate kinase activity in diabetic kidney and liver; • Reduced the increased phosphoenol pyruvate carboxykinase activity in the diabetic liver and kidney; • Increased the GLUT4 level in skeletal muscles that is suppressed under diabetic condition. The above data for cinnamaldehyde was consistent with inhibition of gluconeogenesis enzymes and increased glucose uptake shown as mechanism of action for cinnamon extracts. Other studies (e.g. Kumar et al., 2012) further showed that cinnamaldehyde increase glycogen content in the liver and other organs. In C2C12 skeletal muscle cells, the expression of GLUT4

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was shown to increase in a dose-dependent manner (10, 20, and 50 μm) with about threefold increase observed for the highest dose used (Nikzamir et al., 2014). Further insight into this mechanism of action for cinnamaldehyde treatment (40 μm) also came from studies by Gannon et al. (2015) in C2C12 myocytes with the following key observations noted: • It increased myocyte enhancer factor 2 (MEF2) along with glucose GLUT4 content; • It increased the expression AMPK, PPAR-γ coactivator 1a (PGC-1a), cytochrome c (CytC), as well as PPAR-α and PPAR-β/δ, i.e. it increase mitochondrial biogenesis; • It increased glucose-mediated lipid biosynthesis without elevating the level of PPAR-γ and sterol receptor element binding protein 1c (SREBP-1c) expression. It also upregulate fatty acid synthase (FAS). The increased level of FAS by cinnamaldehyde may explain why in some experiments antiobesity effect was not observed or sometimes increased adipogenesis are the outcomes of cinnamon treatment. This could be due to the differential effects of active principles, in this case cinnamaldehyde. As antiobesity and/or lipid lowering agent are rather expected to downregulate FAS, more research in this area seems to be needed to clarify the effect of cinnamaldehyde on this enzyme level. The enhancement of insulin sensitivity could also be a result of direct effect on insulin receptors as demonstrated by Li et al. (2009) where the antidiabetic effect of cinnamaldehyde in STZ-induced diabetic rats was associated with increased expression IRS-1, while the level of p85 (a negative regulator of skeletal muscle insulin signalling) was lowered in the gastrocnemius. With regard to the association of insulin resistance in adipocytes with suppressed level of GLUT4 expression, the role of cinnamaldehyde in the secretion of retinol binding protein 4 (RBP4) from adipocyte has been investigated. In STZ plus HFD-induced diabetic model of rats, the hypolipidaemic, hypoglycaemic, insulin sensitizing and suppressing LDL-C and homeostasis model assessment of insulin resistance (HOMA-IR) index were associated with reduction of RBP4 levels and upregulated GLUT4 protein expression in tissues (Zhang et al., 2008). In view of the positive correlation of plasma level of RBP4 and T2D or insulin resistance, the effect of cinnamaldehyde in this system could be considered as a valid mechanism of antidiabetic action. The glycaemic control induced by cinnamaldehyde could also be related to inhibition of carbohydrate digestive enzymes. In this regard, cinnamaldehyde was identified as the active principle for the α-amylase inhibitory properties of cinnamon (C. verum Presl.) at submicrogram concentration levels (Okutan et al., 2014). 15.6.3.1.2 Antiobesity and lipid lowering effects

There is significant overlap between the increased insulin sensitivity and glycaemic control by cinnamaldehyde and its effect on adipose tissues. In high fat and high sucrose-fed obese mice, cinnamaldehyde as 0.5% or 1.0% supplement could suppress the weight of the mesenteric adipose tissue coupled with increased UCP-1 protein levels in the interscapular brown adipose tissue (Tamura et al., 2012). Transient receptor potential-ankyrin receptor 1 (TRPA1) agonistic effect was also demonstrated. In a ghrelin secreting cell line (MGN3-1 cells), the addition of cinnamon could up-regulate the expression of TRPA1 and IR genes. Treatment of mice with cinnamaldehyde also reduce cumulative food intake and gastric emptying rates leading to antiobesity outcome through multiple effects including modulation of TRPA1

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and ghrelin secretion in mouse stomach epithelial cells (Camacho et al., 2015). As explained in the following sections, TRAPA1 as a mechanism for cinnamon action also gained interest in clinical trials. TRAPA1 being a nonselective cation channel including high activity for Ca2+ and Na+, its activation could also increase insulin secretion from rat islet β-cell-derived RINm5F cells (Numazawa et al., 2012) or GLP-1 secretion from enteroendocrine L cells (Emery et al., 2015). Hence, the above-mentioned effect of cinnamaldehyde reported by Camacho et al. (2015) and that for the crude extract by Sartorius et al. (2014) are in line with potential antidiabetic and antiobesity mechanism of action via TRAPA1 activation. Data coming from HFD-induced mice and 3T3-L1 preadipocyte cells also suggest the following effect for cinnamaldehyde (Khare et al., 2016): • It (40 μm) inhibits lipid accumulation in vitro; • It reduce weight gain, decrease fasting-induced hyperphagia, as well as circulating leptin and leptin/ghrelin ratio; • It increases anorectic gene expression in hypothalamus and lipolytic gene expression in visceral white adipose tissue; • It decreases serum IL-1β and inflammatory gene expression in visceral white adipose tissue. The inhibition of adipogenesis by cinnamaldehyde was further studied by using the transformation of 3T3-L1 preadipocytes when differentiated into adipocytes in vitro as commonly used for many natural products. The study by Huang et al. (2011) outlined the following observations for cinnamaldehyde when tested at doses of 10–40 μM: • It reduced lipid accumulation; • It downregulated the expression of PPAR-γ, C/EBPα, and SREBP1 in concentrationdependent manners; • It upregulated AMPK and ACC. Furthermore, the authors (Huang et al., 2011) also demonstrated that cinnamaldehyde (40 mg/kg) in HFD-fed animals could ameliorate weight gains, insulin resistance index, plasma TG, nonesterified fatty acid (NEFA), and cholesterol levels. In obese mice fed for 5 weeks with cinnamaldehyde containing diet, body weight gain and improvement of glucose tolerance even without detectable modification of insulin secretion were also observed (Camacho et al., 2015). Enhancement of insulin sensitivity as well as increased fatty acid β-oxidation and energy uncoupling in skeletal muscle and adipose tissue could be achieved by cinnamaldehyde via the expression of PPAR-δ and PPAR-γ target genes, such as aP2 and CD36 (Li et al., 2015). Hence, while adipogenesis is inhibited by suppressing the level of key transcription factors such as PPAR-γ, insulin sensitivity in mature adipose tissues could be enhanced by upregulating these transcription factors. When fatty-sucrosed diet along with STZ was introduced at the 7th day of gestation in rats, pre-mating treatment of cinnamaldehyde could ameliorate gestational diabetes as evidenced from reduced hyperphagia and glucose intolerance (Hosni et al., 2017). It also reduced the levels of fructosamine, TC, TG, leptin, TNF-α, MDA, and NO, while it significantly increased HDL-C, adiponectin, liver glycogen, GSH, and CAT activity in pregnant rats. In addition, cinnamaldehyde administration upregulated the mRNA expression level of PPAR-γ and also

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ameliorated the number of viable fetuses, implantation loss sites, fetal glucose and insulin levels (Hosni et al., 2017). 15.6.3.1.3 Organoprotective effects

Stimulation of insulin release by cinnamaldehyde is one mechanism of its antidiabetic effect as shown in primary β-cell culture from STZ-treated rats (Anand et al., 2010). Through antioxidant mechanism, the pancreatic β-cell protective effect in STZ-rats (e.g. Subash-Babu et al., 2014) has been demonstrated in many experiments. Inhibition of the high glucoseinduced hypertrophy in renal interstitial fibroblasts by cinnamaldehyde was also shown (Chao et al., 2010). The antihypertensive potential of cinnamaldehyde was also noted and its administration (20 mg/kg for 6 weeks) in STZ or fructose-induced diabetic rats prevented the hyperglycaemia associated impaired vascular reactivity and increased BP. More specifically, the increased response to phenylephrine (PE) and KCl or Ca2+ influx and decreased response to acetyl choline (ACh) associated with insulin resistance could be ameliorated by cinnamaldehyde treatment (El-Bassossy et al., 2011). The neuroprotective effects of cinnamaldehyde have been well established and neurochemical deficits including ACh under diabetes and the associated memory dysfunction could be normalized by cinnamaldehyde treatment ( Jawale et al., 2016). This is an addition to the anti-inflammatory effects as evidenced from inhibition of proinflammatory cytokines (TNF-α and IL-6) expression in the hippocampus and cortex. The antipyretic effect of cinnamyl compounds from C. cassia was reported by Kurokawa et al. (1998). When enriched with cinnamaldehyde, the alcoholic (ethanol or methanol) extract of cinnamon (C. verum) (1, 2, or 4 mg/kg, p.o. for 2 weeks) could also ameliorate the collagen-induced arthritis in mice (Qadir et al., 2018). In mouse aortic rings and human umbilical vein endothelial cells (HUVECs), cinnamaldehyde (10 μM) could abolish endothelial dysfunction induced by high glucose via activation of the nuclear factor (erythroid-derived 2)-like (Nrf2). While ROS generation was inhibited and NO level is preserved in endothelial cells, Nrf2 expression and its translocation to the nucleus leading to increased heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), catalase and GPx1 expression were observed (Wang et al., 2015). The antioxidant effect of cinnamaldehyde and or cinnamon could thus be in part orchestrated by induction of the transcription factor Nrf2 that activates genes expression for key antioxidant as well as anti-inflammatory enzymes/proteins. 15.6.3.2 Polar and phenolic components as active principles Li et al. (2013b) obtained the cinnamon polyphenol-rich extract by extracting the bark first with 70% (v/v) ethanol followed by sequential fractionation with chloroform and n-butanol in water. In STZ-HFD-induced diabetic mice, the butanol fraction (regarded as polyphenol fraction—0.3, 0.6, 1.2 g/kg) was shown to downregulate the serum glucose and insulin levels, while the levels of oxidative stress makers were reduced (increased SOD, GPx in pancreatic tissues while MDA level was suppressed) along with improvement in histological markers of the pancreas. The lipid profile (TG, TC, LDL-C lowered, and HDL-C increased) was also improved while the inducible NOS (iNOS) and nuclear factor-κB (NF-κB) levels in pancreatic tissues were suppressed. Im et al. (2014) studied the effect of water soluble extracts of C. zeylanicum containing 45% and 75% gallic acid equivalents (GAE) that they prepared and in comparison with standard

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aqueous extract, contained 15% GAE. The better antioxidant potential, hypoglycaemic and hypolipidaemic effects, and significant decrease in other biochemical disease markers for the higher polyphenolic extract (200 mg/kg 30 days) in STZ-induced diabetic rats were noted. The efficacy of polyphenol extracts in lowering blood glucose levels and ameliorating oxidative stress was further demonstrated in humans by administrating ‘procynZ-45’ containing 45% polyphenols at a relatively low dosage (2 doses of 125 mg per day for 30 days) taken by 15 volunteers who had elevated FBG levels; but not involved in any medication. As explained in the previous section, the water extract of cinnamon that was shown to regulate genes associated with insulin sensitivity, inflammation, and cholesterol/lipogenesis metabolism (Qin et al., 2012), contained 0.74% of a type-A tetramer with a molecular weight of 1152 and four type-A trimeric isomers, each with a molecular weight of 864; and the overall type-A polymers constituted 4.10% in the extract. Lu et al. (2011) also tested the antidiabetic therapeutic potential of procyanidin oligomers in polyphenolic oligomer-rich extracts of cinnamon samples (barks of C. cassia and C. japonica Sieb). When administered at doses of 200 and 300 mg/kg in HFD-fed and low-dose STZ-induced diabetic mice for 14 days, a significant reduction in blood glucose level was observed. The active fractions were demonstrated to contain the A- and B-type procyanidin oligomers but their exact structures were not determined. The extracts also enhance glucose uptake in vitro when tested on insulin-resistant and normal HepG2 cells. The effect of an isolated polyphenolic compound as a trimer (compound 43, Fig. 15.7) with the same molecular mass as (864 Da) from C. burmannii has also been demonstrated in mouse 3T3-L1 adipocytes (Cao et al., 2007). As with the crude water extract, the isolated compound increased IRβ, GLUT4 and TTP levels in the adipocytes. Other compounds as mixtures of trimers and tetramers are identified but the exact structures and distinction between catechin and epicatechin monomers are not established. As with the above-mentioned outcomes for some cinnamon preparations, similar results were obtained for the crude extract and/or polyphenolic oligomer-rich fraction of barks of C. parthenoxylon (Jack) Nees when tested in STZ-induced diabetic rats ( Jia et al., 2009). In this case, oral administration of the extract (100, 200, and 300 mg/kg) over 14 days decreased the blood glucose levels both in diabetic and normal rats. In the STZ-induced diabetic rats, feeding of animals with 3% cinnamon or 0.002% procyanidin-B2 enriched extract of C. zeylanicum for 12 weeks in vivo could suppress glucose or HbA1c levels (Muthenna et al., 2014). Cinnamon as well as the procyanidin B2-fraction could also effectively ameliorate diabetic nephropathy as evidenced from reduced urinary albumin and creatinine. As a mechanism of action, the inhibition of N-carboxy methyl lysine (CML) build-up in the kidney, the AGE-induced loss of expression of glomerular podocyte proteins such as nephrin and podocin could be reversed by the intervention (Muthenna et al., 2014). The authors’ previous study also established that the procyanidin-B2 fraction of cinnamon could inhibit the formation of glycosylated haemoglobin in human blood under ex vivo conditions; while in diabetic rats, cataract development could be delayed through inhibition of AGEs (Muthenna et al., 2013). In view of the presence of several phenolic compounds including the monomeric catechin, epicatechin, and procyanidin A2 and B2, the antidiabetic activity of the aqueous extract has been linked to antiglycation and antioxidant activities. Their ability to trap reactive carbonyl species such as MGO and particularly the formation of MGO-procyanidin B2 can explain their antiglycation and/or antioxidant properties (Peng et al., 2008).

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The pathological damage in mouse pancreatic β-cells induced by high-sugar and HFD was shown to be ameliorated by treatment with the polyphenol fractions of cinnamon (Li et al., 2013b). At the same time, a reduction in iNOS and NF-κB expressions suggest the antioxidant and anti-inflammatory mechanisms of action for these cinnamon products. The glucose lowering effect of 18% polyphenol enriched cinnamon (C. cassia) extract in STZ-induced diabetes model have also been demonstrated when added at 200 mg/kg dose in a 30 day treatment regimen. The structure of HPLC-purified cinnamon (C. burmannii) polyphenol fraction is a doubly linked procyanidin type-A polymer (Quin et al., 2007). On the basis of these structures offering antidiabetic potential, supplement such as Cinnulin PF® representing the water soluble fraction containing relatively high levels of the double-linked procyanidin type-A polymers of flavonoids (4.5% type-A trimeric and tetrameric polymers) have been developed (Rafehi et al., 2012). However, both the A- and B-type of procyanidins must be taken for consideration as active principles. For example, the extract of C. cassia rich in B-type and A-type procyanidin oligomers (200 and 300 mg/kg body wt. 14 days) in HFD and low-dose STZ-induced diabetic mice could reduce blood glucose while increasing glucose uptake in vitro in insulin-resistant HepG2 cells (Lu et al., 2011). Analysis of procyanidin oligomer-rich extracts showed that C. cassia extract (CC-E) and C. tamala extract were rich in B- and A-type procyanidin oligomers, respectively. Antidiabetic assay using the diabetic (db/db) mice treated with either of these extracts (both 200 mg/kg per day, for 4 weeks) showed positive outcomes for both, but variability was observed between the two extracts. While C. cassia promoted lipid accumulation in the adipose tissue and liver, C. tamala mainly improved the insulin concentration in the blood and pancreas (Chen et al., 2012). This differential effect may suggest variabilities in the different outcomes of cinnamon treatment in humans (see Section 15.6.4). Considering the known antioxidant effects of catecholic compounds such as catechin and epicatechin and their polymers, the direct radical scavenging and ROS removal by these compounds is not surprising. Jayaprakasha et al. (2006) isolated five compounds from the water extract of cinnamon (C. zeylanicum) fruit powder including protocatechuic acid, cinnamtannin B-1 (46), lignans and flavonoids that showed antioxidant and radical scavenging activities. Other compounds including flavonoids and those shown in Section 15.5 may also contribute to the antidiabetic property of cinnamon. For example, a hydroxychalcone with insulin mimetic effect in 3T3-L1 adipocytes has been reported. Its effect include increasing glucose uptake and glycogen synthesis via activation of PI3K as specific inhibitors (wortmannin and LY294002), abolished its effects. PI3K activity was enhanced, glycogen synthase kinase-3β inhibited; IR was phosphorylated and synergistic effect with insulin was observed ( Jarvill-Taylor et al., 2001). From all available data so far and their concentrations in cinnamon preparation, cinnamaldehyde and the polyphenolic rich water soluble fractions dominated by procyanidins are regarded as the main antidiabetic principles.

15.6.4 Evidence of efficacy from human trials Numerous randomized clinical trials (RCTs) have been conducted on cinnamon to test its potential for reducing the serum level of blood glucose and/or HbA1c or improvement of lipid profile. The daily doses of cinnamon powder used in these studies could vary from

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1 g (Blevins et al., 2007; Crawford, 2009; Hasanzade et al., 2013; Mirfeizi et al., 2016; Zare et al., 2018); 1.2 g (Wainstein et al., 2011); 1.5 g (Vanschoonbeek et al., 2006), 2 g (Akilen et al., 2010), 3 g (Azimi et al., 2014; Talaei et al., 2017; Vafa et al., 2012) to 1–6 g (Khan et al., 2003). The cinnamon powder may not be specified in these trials but those described are mostly sourced from C. cassia while entries for C. zeylanicum or C. verum are also available. Some studies use cinnamon extract (e.g. Mang et al., 2006; Lu et al., 2012) instead of powder which allowed lowering the dosage to around 300 mg per day. Mang et al. (2006), for example, stated that 336 mg of the aqueous extract of C. cassia, was equivalent to 3 g of cinnamon powder. The patient groups used for cinnamon treatment are mostly between 25 and 100 subjects with few exceptions; e.g. 140 in the trial by Zare et al. (2018). While cinnamon administration in these studies was universally through the oral route, the duration of treatment varied from 6 to 16 weeks. For daily doses of 1 g or higher, many studies appear to show a significant reduction in the FBG (e.g. Akilen et al., 2010; Azimi et al., 2014; Khan et al., 2003; Lu et al., 2012; Mang et al., 2006; Mirfeizi et al., 2016; Vafa et al., 2012; Zare et al., 2018). Some studies included HbA1c measurement and found reduction by cinnamon treatment (Akilen et al., 2010; Crawford, 2009; Lu et al., 2012; Mirfeizi et al., 2016; Vafa et al., 2012; Zare et al., 2018). A rather small cross-sectional study using a total of 93 participants (47 men and 46 women) with untreated prediabetes also reported the association between cinnamon usage and a better working memory on the basis of working mini-mental state examination (MMSE) (Wahlqvist et al., 2016). Other studies not showing favourable effect either for FBG or HbA1c need to also be mentioned. For example, Talaei et al. (2017) reported that cinnamon supplementation had no significant effects on glycaemic and inflammatory indicators in patients with T2D after intervention with 1 g/day cinnamon supplement for 8 weeks. Similarly, the trial by Hasanzade et al. (2013) in T2D patients receiving cinnamon (1 g or 2 capsules of 500 mg per day) for 60 days did not change the FBG or glycosylated haemoglobin level in diabetic patients. The cinnamon cassia powder 1.5 g/day in 12-week intervention study too did not have any significant effect in reducing FBG, HbA1c and serum lipid profile in T2D patients (Suppapitiporn et al., 2006). Even a higher dose of 3 g/day cinnamon supplement for 8 weeks in a double-blind, RCT study using 44 T2D patients study failed to show significant changes in the level of FBG, insulin, HbA1c, HOMA-IR, carboxymethyl lysine (AGE), total antioxidant capacity, and MDA levels (Talaei et al., 2017). A trial on type 1 diabetes (T1D) patients with administration of 1 g/day cinnamon powder for 13 weeks, similarly did not show favourable glycaemic control as assessed by HbA1c level or requirement of insulin intake (Altschuler et al., 2007). It is now obvious that this dose is insufficient to induce antidiabetic effects in humans even though some evidence could even contradict this argument. In a 12-week trial with 18 T2D patients (9 women and 9 men), for example, 1 g of C. cassia decreased their blood sugar levels by about 30 mg/dL, which was comparable to oral medications available for diabetes (Hoehn and Stockert, 2012). In poorly controlled T2D Iraqi patients, intake of 1 g of cinnamon for 12 weeks was shown to reduce FBG and glycosylated haemoglobin; increase the level of serum GSH and SOD, while reducing the serum level of MDA (Sahib, 2016). The data discrepancy for the lipid profile is even more prevalent. There are few reports were no improvement in lipid profile was reported, while some showed a reduction in TG, TC and LDL-C (Khan et al., 2003); TC and LDL-C (Zare et al., 2018); only TG

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(Azimi et al., 2014; Vafa et al., 2012) or increase in HDL-C (Zare et al., 2018); but the vast majority of studies do not show favourable effect on lipid profile. In order to understand the impact of cinnamon intervention in diabetes and/or associated diseases, some examples of the clinical trials on either prediabetics, T2D or obese subjects are worth mentioning. From the earlier studies, the trial by Roussel et al. (2009) on overweight or obese subjects using the dried aqueous extract of C. cassia (250 mg/day for 12 weeks) showed a small but significant reduction in FBG. The study by Ziegenfuss et al. (2006) on the aqueous extract of cinnamon powder used a dose twice higher (500 mg of water soluble extract that was equivalent to 10 g powder) in prediabetic and metabolic syndrome patients and showed a reduction in FBG without improvement in the lipid profile. Another trial on prediabetics was that by Anderson et al. (2016) who showed a reduction in FBG for the water extract (500 mg/day for 9 weeks) of cinnamon powder along with lipid profile improvement for all components (reduction in TG, TC, LDL-C and increase in HDL-C). The combination of cinnamon extract (bark of C. cassia which was rich in polyphenol type-A polymers or oligomeric proanthocyanidins-A) with chromium was also tried out in the RCT on overweight or obese prediabetic patients. Four month treatment with this extract (228 mg/day) as a dietary supplement was shown to suppress the FBG level and also increased fat-free mass (Liu et al., 2015). The trial by Gupta Jain et al. (2017) on metabolic syndrome patients was also recorded antidiabetic efficacy for cinnamon (3 g powder per day for 16 weeks) as a reduction in both FBG and HbA1c levels were observed along with improvement in lipid profile (reduction in TG, TC, LDL-C and increase in HDL-C). In healthy human subjects, the intake of cinnamon (3 g/day) for 2 weeks improve glycaemic control (OGTT) and insulin sensitivity but the effect was quickly lost upon cessation of cinnamon feeding (Solomon and Blannin, 2009). In a 30 min of OGTT, such effect may be regarded as small or marginal though significant reduction even in humans taking cinnamon powder (5 g of cinnamon) taken 12 h before OGTT were effective in glycaemic control and improvement of insulin sensitivity (Solomon and Blannin, 2007). Hlebowicz et al. (2007) also showed that 6 g cinnamon with rice pudding reduces postprandial blood glucose and delays gastric emptying without affecting satiety. Magistrelli and Chezem (2012) further showed that the addition of cinnamon (6 g powder) to the cereal meal significantly reduced 120-min glucose in normal weight and obese adults. In these studies, the hypoglycaemic effect of cinnamon in postprandial glucose overshoot depends on the timing of the assessment and variability in data depending on 30 or 120 min have been noted (Hlebowicz et al., 2007; Solomon and Blannin, 2007). In the study by Hlebowicz et al. (2009), administration of 3 g cinnamon not only reduced postprandial serum insulin but also increased GLP-1 concentrations without significantly affecting blood glucose, gastric inhibitory polypeptide (GIP), the ghrelin concentration, satiety, or the gastric emptying rate in healthy subjects. This is in contrast to the effect of 6 g cinnamon reducing postprandial blood glucose reported by the same authors (Hlebowicz et al., 2007) but the reported effect on delayed gastric emptying without affecting satiety is interesting. Another interesting study by Markey et al. (2011) also included assessment on gastric emptying effect for cinnamon along with arterial stiffness, postprandial lipidaemia, glycaemia, and appetite responses to high-fat breakfast. There single-blind randomized crossover study was based only on nine healthy subjects who consumed 3 g cinnamon that did not alter their postprandial response to a high-fat test meal. In view of some contradictory data in the postprandial effect of cinnamon, more data in this area,

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particularly on T2D subjects, are needed. An OGTT study in small number (n ¼ 10) of young, sedentary, and obese women showed that administration of 5 g dose of Cassia cinnamon could reduce the peak blood glucose response and improve glucose tolerance but insulin sensitivity (or insulin resistance) was not altered (Gutierrez et al., 2016). A RCT on women with polycystic ovarian syndrome (n ¼ 66) with cinnamon powder capsules intake of 1.5 g/day (in 3 doses) for 12 weeks, however, could reduce fasting insulin and insulin resistance (Hajimonfarednejad et al., 2018). The implication of polyphenols as the active principle of cinnamon was investigated by Im et al. (2014) who used water soluble extracts of Cinnamomum zeylanicum that contained 45% and 75% GAE. After showing antidiabetic effect in STZ-induced diabetic rats, their preparation of 45% GAE that they called ‘procynZ-45’ was shown to be effective (125 mg twice daily for 30 days) when tested in small number of human volunteers (n ¼ 15) who had elevated FBG. In nonalcoholic fatty liver disease patients, Askari et al. (2014) recorded daily supplementation with 1.5 g/day (two capsules of 750 mg each) of cinnamon powder for 12 weeks could reduce the HOMA index, FBG, TC, TG, ALT, AST, γ-glutamine transpeptidase (GGT), and high-sensitivity CRP (no effect on HDL-C level). Finally, the trial by Borzoei et al. (2018) was among the best positive outcome for demonstrating the hypolipidaemic potential of cinnamon where administration (1.5 g per day for 8 weeks) in overweight or obese metabolic syndrome patents could reduce TC, TG, LDL-C while increasing HDL-C. Overall, the contradictory human trial data does not allow to make firm conclusion on the antiobesity potential of cinnamon on human subjects. The lack of effect for C. cassia powder on body weight change in T2D patients for 1 g (Blevins et al., 2007), 1.2 g (Wainstein et al., 2011) or 2 g (Akilen et al., 2010) per day supplement for 12 weeks were reported. The lack of effect on body weight for the 3 g supplement of C. verum in T2D for 8 weeks, or in prediabetics treated with 500 mg/day extract for 9 weeks (Anderson et al., 2016) are among the reports that dampen the enthusiasm for as a cinnamon therapeutic agent for obesity. On the other hand, high doses such as 3 g (C. zeylanicum (Vafa et al., 2012) or unknown source (Gupta Jain et al., 2017)) in T2D, or prediabetic and metabolic syndrome by 10 g equivalent cinnamon powder (unspecified source; Ziegenfuss et al., 2006) or smaller doses such as 1.5 g/day for 12 weeks in T2D (Zare et al., 2018), 1 g for 13 weeks (Mirfeizi et al., 2016) were reported for favourable outcomes in T2D and/or metabolic syndrome. Readers should note that, in full blown diabetes associated with significant weight loss, the lack of effect on weight reduction may not be seen as a negative outcome. Hence, both the weight lowering effect of cinnamon in obesity as well as body weight recovery in T2D not associated with obesity could be equally seen as favourable outcomes. Considering the contradictory data in human trials as well as some reports in animal experiments for the crude extract, depending on the plant /species source and the active principle, cinnamaldehyde (that may negatively affect obesity/lipid profile), more data is needed in this field.

15.6.5 Other pharmacological effect of cinnamon relevant to T2D There are also a number of other disease areas for cinnamon implicated as a therapeutic agent including cancer, neurodegenerative diseases and inflammatory disorders. In the

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latter case, the double-blind RCT involving 36 women with rheumatoid arthritis have shown that cinnamon intake of 4 capsules of 500 mg cinnamon powder daily for 8 weeks could reduce the serum levels of CRP and TNF-α along with various other disease scores (Shishehbor et al., 2018). Although no significant changes were observed for FBG, lipid profile, liver enzymes, etc., the potential anti-inflammatory effect suggests that cinnamon could be at least used as adjuvant therapy for inflammatory disorders. Consumption of cinnamon (short term) is known to be associated with a notable reduction in SBP and DBP. In a randomized treatment with 1.2 g/day cinnamon in T2D patients, however, a fall in SBP was not achieved (Wainstein et al., 2011); perhaps owing the relatively small dosage used in the study. Mesripour et al. (2016) showed that the water extract C. zeylanicum bark could improve memory performance in alloxan-induced diabetic mice. In alloxan-induced diabetic rats, C. cassia at the dose of 400 mg/kg (3 months) that did not alter blood glucose, HbA1C, and lipid profile, could normalize the changes in nervous ischiadicus (neuronal and myelin sheath integrity) suggesting potential benefit in diabetic neuropathy (Bahceci et al., 2009). C. cassia Blume improving cognitive dysfunction and energy and glucose dysregulation by reducing neuroinflammation and hippocampal insulin resistance in β-amyloid-infused rats have also been reported (Park et al., 2017). The association between the antidiabetic effect (also antiobesity and antisteatosis) and changes in gut microbiota and markers of gut barrier induced by cinnamon treatment has been suggested (Van Hul et al., 2018). In HFD-fed mice treated with cinnamon bark for 8 weeks, the changes in microbiota (e.g. Peptococcus, Desulfovibrio, Lactococcus, Allobaculum, and Roseburia) were altered by treatment with the extract. More data in this field is however required to make firm conclusions.

15.7 Toxicological profile As a starting point, cinnamon is a nontoxic food ingredient that can be used in small amount as spice without any restriction. For application as drug with doses far higher than normal usage, however, a comprehensive clinical data need to be presented. The numerous clinical trial on humans with doses of cinnamon up to 10 g/day effectively used without any complication, however, still suggest that cinnamon is generally nontoxic in humans. Some precautions to be taken on the basis of some adverse reactions reported in the literature are addressed below. In one case report, a patient taking cinnamon bark powder (C. cassia, 1 g daily) leading to a leg swelling was reported (Crawford and Crawford, 2018). Even though this is just a one case report, the authors assert the known effect of cinnamon and its active component cinnamaldehyde in upregulating the mRNA expression level of PPAR-γ that implies similar pharmacological antidiabetic mechanism and common side effects as thiazolidinediones. Hence, diurnal proximal sodium retention in diabetic and hypertensive cases demonstrated for pioglitazone (Zanchi et al., 2010) and the general trend of increased water retention known for thiazolidinediones (e.g. Alema´n-Gonza´lez-Duhart et al., 2016; Berlie et al., 2007) was speculated to apply for cinnamon.

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When doses of 20-time higher (0.4 g/kg) than that effectively used as antidiabetic agent were administered in rats, no toxicity or behavioural changes were observed for cinnamaldehyde (Kumar et al., 2012). As part of toxicological and dermatological assessment study, cinnamaldehyde was also among the compounds found to have low order of toxicity by the oral and dermal route of exposure; no significant potential to produce genotoxic effects in vivo; or no toxic or persistent metabolites formed from it are reported (Bickers et al., 2005). Skin irritation may be encountered with uses of cinnamaldehyde but the plethora of formulation approaches such as novel intravenous submicrometer emulsion systems (Zhao et al., 2015) are also available to limit these potential adverse effects. The potential toxic components of cinnamon such as the coumarins that are not tolerated at high doses and could cause liver toxicity, and generally seen as non-genotoxic carcinogens in rodents among other effects (e.g. Abraham et al., 2010) should also be considered. While the safety record of cinnamon in animals and humans is generally good, high concentrations of coumarin intake from cinnamon added to children oatmeal porridge in Norway has been reported (Fotland et al., 2012). Some cinnamon species (e.g. C. zeylanicum) are known to contain trace amount of coumarin while others such as C. cassia have been shown to contain incredibly high amount (up to 1 g/kg) (e.g. Woehrlin et al., 2010). Wide range of coumarin content in the bark sticks and powdered cinnamon has been reported by various groups (Blahova and Svobodova, 2012; He et al., 2005; Lungarini et al., 2008; Woehrlin et al., 2010). Several European Union regulations scrutinized the safe amount of coumarins taken in herbs and spices and the (EC) No 1334/2008 (European Parliament and Council, 2008) directive for cinnamon containing foods is a good example. Many cases of products such as cinnamon cookies and other products in Europe exceeding the current EU regulation have been reported (Ballin and Sørensen, 2014). Hence, cinnamon should not be taken as a generic name that represents one standardized product. It is rather a heterogeneous product that varies on the level of potentially toxic components and hence a quality control measure for each source or species must be available to guarantee high level of safety as a clinical product.

15.8 Overview of pharmacokinetics profile Han and Cui (2012) investigated the antidiabetic effect of cinnamon oil formulated in normal saline containing 3% Tween 80 or when carried by liquid-loadable tablets or the self-emulsifying formulation carrier. After oral administration (100 mg/kg), the plasma concentration–time profile and pharmacokinetic parameters (Cmax), AUC and F values) were suggested to be significantly improved for the liquid-loadable tablet career. In alloxaninduced diabetes rats, the improvement of blood glucose and the HbA1c in the hyperglycaemic rats as well as pancreatic β-cells recovery in the 45 days study period was also far better for the liquid-loadable tablets as a carrier. Pharmacokinetics study for the active principle, cinnamaldehyde, is also available. In a gas chromatography–mass spectrometry (GC–MS) detection method development by Zhao et al. (2014), cinnamaldehyde after absorption in rats rapidly oxidize (60%) to cinnamic acid but its long half-life (6.7  1.5 h) was also noted. 14C-labelled cinnamaldehyde study in rats using a single (5 and 50 mg/kg) or multiple dose levels also showed the degradation of the compound to benzoic acid through

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β-oxidation (Sapienza et al., 1993). The excretion of the compound in the urine was mainly as hippuric acid, though far smaller concentrations than benzoic and cinnamic acids, were also detected. In these rats subjected to multiply high dosage (500 mg/kg), benzoic acid appears to be the main urinary products with the recoveries of the radiolabel after 24 h in the urine were 80.4%, 80.6%, and 81.9% for the dose for the 5, 50, and 500 mg/kg, respectively. In contrast, faecal excretion of radiolabel after multiple dosing at 24 h was low: 6.3%, 6.9%, and 4.5% of the administered radioactivity at the 5, 50, and 500 mg/kg dose levels, respectively. Good tissue distribution from observation in the fat, liver and gastro-intestinal tract was also reported (Sapienza et al., 1993). Proanthocyanidin-rich cinnamon extract (C. zeylanicum) incorporated into solid lipid microparticles (SLMs) by spray chilling technique using vegetable fat as carrier was available (Tulini et al., 2016). There is a trend now to make formulations of cinnamon and its oil to make them more effective and palatable. This will undoubtedly improve the efficacy of cinnamon products if they were to be used as pharmaceutical/nutraceutical products.

15.9 General summary and conclusions This chapter has shown the fascinating story of cinnamon from being a common spice of flavoring our food to source of pharmaceutically active compounds such as cinnamaldehyde. It is shown that cinnamon represent a range of plant species belonging to the genus Cinnamomum with four species mainly traded at international level. While the generic class of components of the oil and highly water soluble extracts are similar, there are large differences in the chemical composition between the various species. This is in addition to the universal challenge of standardizing herbal products due to differences in growing conditions, genotypic/variety differences, seasonal changes, plant developmental stage, etc. These differences also appear to be well represented in the cinnamon pharmacology. The animal model and in vitro studies on cinnamon powder, extract and active principles primarily as cinnamaldehyde and procyanidins are in good agreement with potential applications of cinnamon for treating T2D and the associated hyperlipidaemia. Diverse mechanisms of action related to antioxidant pharmacology where ROS could be readily removed by cinnamon polyphenols, AGEs formation is inhibited, and antioxidant defenses are augmented in diabetes models suggest antidiabetic mechanisms as well as amelioration of the diabetes-associated organ damage (pancreases, liver, kidney, etc.). Some of the specific effects also include activation of the Nrf2-HO-1 pathway leading to the expression of diverse antioxidant enzymes and proteins. The key inflammatory pathways such as cytokines (TNF-α and IL-6) that are upregulated under obesity condition to induce insulin resistance also appear to be targeted by cinnamon and its compounds. In addition, anti-inflammatory genes/ protein expression such as Zfp36 (or TTP) further laid down the anti-inflammatory mechanisms of antidiabetic effect by cinnamon products. This could be partly due to some common mechanisms such as inhibition of iNOS and NF-κB expressions. The plethora of experimental models where antidiabetic effects demonstrated for cinnamon products is incredibly large and range from chemical to fat/diet and genetically-induced diabetes and obesity models. While hyperglycaemia and HbA1c levels were suppressed in

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these diabetic models, glycogen level was raised and glucose production suppressed by downregulating key gluconeogenesis enzymes such as pyruvate kinase activity and phosphoenol pyruvate carboxykinase. Cinnamon and its active principles also modulate IR, IRS-1, IRS-2, PI3K, Akt1, PTEN, and various other insulin signaling molecules/pathways. This leads to GLUT4 upregulation and glucose uptake in muscles and adipose tissues. Both for the glycaemic and lipid regulation, the effect of cinnamon and its active principles in modulating the genes and protein levels of PPAR-γ, C/EBPα, and SREBP1, AMPK, ACC, PGC-1a and SREBP-1c account to the reported favourable effects. Other interesting data as a mechanism of action presented for cinnamon include digestive enzymes inhibition, TRAPA1 activation that have implication to gastric activity and GIT hormones (e.g. GLP-1) release. Effects on UCP-1 protein and RBP4 are other examples of mechanism of action for cinnamon. While all the animal studies and in vitro data are convincingly shown antidiabetic and antihyperlipidaemic effects with questionable antiobesity property, the human trials are diverse and often with contradictory outcomes. One has to admire the number of human trials published in the scientific literature for cinnamon that included antidiabetic and/or antihyperlipidaemic effects. The range of regional trials are also diverse and include, for example, Pakistan (Khan et al., 2003, 2010), Iraq (Sahib, 2016), Thailand (Suppapitiporn et al., 2006); Germany (Mang et al., 2006), United Kingdom (Akilen et al., 2010), France (Beejmohun et al., 2014), United States (Blevins et al., 2007), China (Lu et al., 2012), India (Sharma et al., 2012), and Iran (Mirfeizi et al., 2016; Vafa et al., 2012). In these experiments, trials higher than 1 g/day for cinnamon powder appear to be the cases and FBG were reduced in many cases, some components of lipid profiles like TG and LDL-C appear to be modified more than HDL-C. The aqueous extracts are also tried out routinely to reduce FBG in prediabetics (Ziegenfuss et al., 2006) but the doses appear to correspond to over 1 g of the powder (e.g. 3 g/day Mang et al., 2006). A number of meta-analysis studies on the existing clinical data also showed inconclusive evidence with some in favour while others in support of using cinnamon as a valid therapeutic agent (Akilen et al., 2012; Allen et al., 2013; Davis and Yokoyama, 2011; Leach and Kumar, 2012; Ranasinghe et al., 2012b). Davis and Yokoyama (2011) with limited data input suggesting a reduction in FBG by cinnamon powder/extract; Allen et al. (2013) from 10 clinical trials suggesting a reduction in FBG and TC and LDL-C while even increasing HDL-C were reported. The greatest discrepancy in these clinical data undoubtedly arose from the heterogeneity in the preparation of cinnamon both in source and dosage form; variability in the subjects involved both on the clinical stage of diabetes and their status; as well as experimental protocols including blinding. The cinnamon used in the studies was C. cassia, C. zeylanium (Ceylon), or C. aromaticum while in some studies they were not specified. The composition of these cinnamon preparations with respect to active components are not known while their preparation could be either home-made in the lab, or obtained from either commercial or other sources. In many cases, the dose-related effect has not been demonstrated and just activity or lack of it have been reported at one fixed dosage. The dietary and physical states (including exercise) of the patients while receiving the drugs were not elaborated. Most importantly, most of the clinical studies were conducted with small number of subjects and in short duration. A number of critical commentaries on these clinical trials are available (e.g. Costello et al., 2016) and on the basis of the clinical data available so far, a modest effect on FBG and HbA1c reduction is

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possible but a significant therapeutically relevant clean data on the antidiabetic potential of cinnamon is yet to be demonstrated. There is no doubt that some diabetes patients may opt to use cinnamon as an alternative therapy for treating diabetes but from the available data so far, it cannot be recommended as a substitute to the existing antidiabetic agents. Looking ahead into resolving the unanswered questions related to the efficacy of cinnamon lies on the development of standardized aqueous extracts of C. species that well-designed clinical trials can utilize. For doses far higher than 1 g/day, cinnamon is likely to display antidiabetic effect and has potential as an adjuvant therapy at the very minimum.

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Sheng, X., Zhang, Y., Gong, Z., Huang, C., Zang, Y.Q., 2008. Improved insulin resistance and lipid metabolism by cinnamon extract through activation of peroxisome proliferator-activated receptors. PPAR Res. 2008, 581348. Shiraga, Y., Okano, K., Akira, T., Fukaya, C., Yokoyama, K., Tanaka, S., et al., 1988. Structures of potent antiulcerogenic compounds from Cinnamomum cassia. Tetrahedron 44, 4703–4711. Shishehbor, F., Rezaeyan Safar, M., Rajaei, E., Haghighizadeh, M.H., 2018. Cinnamon consumption improves clinical symptoms and inflammatory markers in women with rheumatoid arthritis. J. Am. Coll. Nutr. 3, 1–6. Singh, G., Maurya, S., DeLampasona, M.P., Catalan, C.A., 2007. A comparison of chemical, antioxidant and antimicrobial studies of cinnamon leaf and bark volatile oils, oleoresins and their constituents. Food Chem. Toxicol. 45, 1650–1661. Solomon, T.P.J., Blannin, A.K., 2007. Effects of short-term cinnamon ingestion on in vivo glucose tolerance. Diabetes Obes. Metab. 9, 895–901. Solomon, T.P.J., Blannin, A.K., 2009. Changes in glucose tolerance and insulin sensitivity following 2 weeks of daily cinnamon ingestion in healthy humans. Eur. J. Appl. Physiol. 105, 969–976. Subash-Babu, P., Alshatwi, A.A., Ignacimuthu, S., 2014. Beneficial antioxidative and antiperoxidative effect of cinnamaldehyde protect streptozotocin-induced pancreatic beta-cells damage in wistar rats. Biomol. Ther. 22, 47–54. Suppapitiporn, S., Kanpaksi, N., Suppapitiporn, S., 2006. The effect of cinnamon cassia powder in type 2 diabetes mellitus. J. Med. Assoc. Thail. 89 (suppl 3), S200–S205. Talaei, B., Amouzegar, A., Sahranavard, S., Hedayati, M., Mirmiran, P., Azizi, F., 2017. Effects of cinnamon consumption on glycemic indicators, advanced glycation end products, and antioxidantstatus in type 2 diabetic patients. Nutrients 9, 991. Tamura, Y., Iwasaki, Y., Narukawa, M., Watanabe, T., 2012. Ingestion of cinnamaldehyde, a trpa1 agonist, reduces visceral fats in mice fed a high-fat and high-sucrose diet. J. Nutr. Sci. Vitaminol. 58, 9–13. Tulini, F.L., Souza, V.B., Echalar-Barrientos, M.A., Thomazini, M., Pallone, E.M.J.A., Favaro-Trindade, C.S., 2016. Development of solid lipid microparticles loaded with a proanthocyanidin-rich cinnamon extract (Cinnamomum zeylanicum): potential for increasing antioxidant content in functional foods for diabetic population. Food Res. Int. 85, 10–18. Tung, Y.T., Chua, M.T., Wang, S.Y., Chang, S.T., 2008. Anti-inflammation activities of essential oil and its constituents from indigenous cinnamon (Cinnamomum osmophloeum) twigs. Bioresour. Technol. 99, 3908–3913. Usta, J., Kreydiyyeh, S., Bajakian, K., Nakkash-Chmaisse, H., 2002. In vitro effect of eugenol and cinnamaldehyde on membrane potential and respiratory chain complexes in isolated rat liver mitochondria. Food Chem. Toxicol. 40, 935–940. Vafa, M., Mohammadi, F., Shidfar, F., Sormaghi, M.S., Heidari, I., Golestan, B., et al., 2012. Effects of cinnamon consumption on glycemic status, lipid profile and body composition in type 2 diabetic patients. Int. J. Prev. Med. 3, 531–536. Van Hul, M., Geurts, L., Plovier, H., Druart, C., Everard, A., Sta˚hlman, M., et al., 2018. Reduced obesity, diabetes, and steatosis upon cinnamon and grape pomace are associated with changes in gut microbiota and markers of gut barrier. Am. J. Physiol. Endocrinol. Metab. 314 (4), E334–E352. Vanschoonbeek, K., Thomassen, B.J., Senden, J.M., Wodzig, W.K., van Loon, L.J., 2006. Cinnamon supplementation does not improve glycemic control in postmenopausal type 2 diabetes patients. J. Nutr. 136, 977–980. Verspohl, E.J., Bauer, K., Neddermann, E., 2005. Antidiabetic effect of Cinnamomum cassia and Cinnamomum zeylanicum in vivo and in vitro. Phytother. Res. 19 (3), 203–206. Wahlqvist, M.L., Lee, M.S., Lee, J.T., Hsu, C.C., Chou, Y.C., Fang, W.H., et al., 2016. Cinnamon users with prediabetes have a better fasting working memory: a cross-sectional function study. Nutr. Res. 36 (4), 305–310. Wainstein, J., Stern, N., Heller, S., Boaz, M., 2011. Dietary cinnamon supplementation and changes in systolic blood pressure in subjects with type 2 diabetes. J. Med. Food 14, 1505–1510. Wang, R., Wang, R., Yang, B., 2009. Extraction of essential oils from five cinnamon leaves and identification volatile compounds compositions. Innov. Food Sci. Emerg. Technol. 10, 289–292. Wang, F., Pu, C., Zhou, P., Wang, P., Liang, D., Wang, Q., et al., 2015. Cinnamaldehyde prevents endothelial dysfunction induced by high glucose by activating nrf2. Cell. Physiol. Biochem. 36, 315–324. Weiss, E.A., 2002. Spice Crops. CABI Publishing, London, pp. 55–56. Wijesekera, R., 1978. Historical overview of the cinnamon industry. Crit. Rev. Food Sci. Nutr. 10 (1), 1–30.

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Woehrlin, F., Fry, H., Abraham, K., Preiss-Weigert, A., 2010. Quantification of flavoring constituents in cinnamon: high variation of coumarin in cassia bark from the German retail market and in authentic samples from Indonesia. J. Agric. Food Chem. 58, 10568–10575. Wuu-Kuang, S., 2011. Taxonomic revision of Cinnamomum (Lauraceae) in Borneo. Blumea 56, 241–264. Zanchi, A., Maillard, M., Jornayvaz, F.R., Vinciguerra, M., Deleaval, P., Nussberger, J., et al., 2010. Effects of the peroxisome proliferator-activated receptor (PPAR)-gamma agonist pioglitazone on renal and hormonal responses to salt in diabetic and hypertensive individuals. Diabetologia 53, 1568–1575. Zare, R., Nadjarzadeh, A., Zarshenas, M.M., Shams, M., Heydari, M., 2018. Efficacy of cinnamon in patients with type II diabetes mellitus: a randomized controlled clinical trial. Clin. Nutr. pii: S0261-5614(18)30114-6. Zhang, W., Xu, Y.C., Guo, F.J., Meng, Y., Li, M.L., 2008. Anti-diabetic effects of cinnamaldehyde and berberine and their impacts on retinol-binding protein 4 expression in rats with type 2 diabetes mellitus. Chin. Med. J. 121, 2124–2128. Zhao, H., Xie, Y., Yang, Q., Cao, Y., Tu, H., Cao, W., et al., 2014. Pharmacokinetic study of cinnamaldehyde in rats by gc-ms after oral and intravenous administration. J. Pharm. Biomed. Anal. 89, 150–157. Zhao, H., Yuan, J., Yang, Q., Xie, Y., Cao, W., Wang, S., 2015. Cinnamaldehyde in a novel intravenous submicrometer emulsion: pharmacokinetics, tissue distribution, antitumor efficacy, and toxicity. J. Agric. Food Chem. 63, 6386–6392. Ziegenfuss, T.N., Hofheins, J.E., Mendel, R.W., Landis, J., Anderson, R.A., 2006. Effects of a water-soluble cinnamon extract on body composition and features of the metabolic syndrome in pre-diabetic men and women. J. Int. Soc. Sports Nutr. 3, 45–53.

Further reading Akilen, R., 2013. Effects of cinnamon consumption on glycemic status, lipid profile and body composition in type 2 diabetic patients. Int. J. Prev. Med. 4, 379–380. Baker, W.L., Gutierrez-Williams, G., White, C.M., Kluger, J., Coleman, C.I., 2008. Effect of cinnamon on glucose control and lipid parameters. Diabetes Care 31, 41–43.

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16 The chemical and pharmacological basis of cloves (Syzygium aromaticum (L.) Merr. & L.M.Perry) as potential therapy for type 2 diabetes and associated diseases O U T L I N E 16.1 Introduction

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16.2 Botanical and taxonomic perspectives

16.5 Antidiabetic effects—Crude extract and preparations

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16.3 Culinary and medicinal uses of clove

16.6 Antidiabetic effects—Active principles of cloves

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16.4 The chemistry of cloves 16.4.1 Essential oils 16.4.2 Flavonoids 16.4.3 Phenolic acids and derivatives 16.4.4 Tannins 16.4.5 Terpenoids

556 556 558 559 560 561

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16.8 General summary and conclusion 572 References

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16. The chemical and pharmacological basis of cloves

16.1 Introduction Clove is a spice name representing a small reddish brown dried flower buds of Syzygium aromaticum (Synonym Eugenia caryophyllata) of the family Myrtaceae. Known to be native to the Maluku Islands (or Moluccas or often called the Spice Islands) of Indonesia, clove is among the first group of spices known to be traded by humans. Its world-wide cultivation however had to wait until the 17th century when the European nations compete for its access along with other spices. The Portuguese lost the clove trade monopoly when they were forced out of the spice islands of Indonesia by the Dutch. In order to maintain high prece through limited ownership and supply, the Dutch colonizers were known to eradicate cloves from all the indigenous islands except for Amboina and Ternate. It was only by the late 18th century when the French was successful in smuggling the plant and cultivate it elsewhere (e.g. Mauritius in 1770) that led to the end of the Dutch monopoly and the beginning of spread of clove’s cultivation. It took another century for the new-found clove cultivation to shape the world market, however, and by then an essential oil extraction from the clove flower buds has opened up a new market. By the 1920s, clove was planted in Madagascar. According to the data by the Food and Agriculture Organization (FAO) of the United Nations (http://www.fao.org/faostat/en/#data/QC), the world biggest producer (in tonnes) in the year 2016 was Indonesia (139,522) with yield far higher than the combined total of other producers including Madagascar (20,821), Tanzania (8915), Sri Lanka (4991), Comoros (2699), Kenya (2077), China (1243), Malaysia (217), and Grenada (37). Today, the clove plant (S. aromaticum (L.) Merril & Perry) is commercially exploited for the dried flower buds (cloves), clove oil and oleoresin. The new addition is of course medicinal value including type 2 diabetes (T2D) which is the main focus of this chapter.

16.2 Botanical and taxonomic perspectives The botanical description of the clove plant (S. aromaticum) is as follow (Teuscher, 2006): Up to 20 m tall, evergreen tree with pyramid-shaped top. The petiolated leaves are leathery, elliptic to lanceolate, 9 to 12 cm long, 3.5 cm to 4.5 cm wide. The terminal flowers are arranged in tripartite paniculate corymbs. Flowers 10 to 14 mm long, with 2 scale-like prophylls, tubular calyx 1 to 1.5 cm long, 4 thick sepals, 4 petals, whitish pink to carmine-red, numerous stamina, inferior ovary, which is partially enclosed by and fused with a tubular hypanthium (cup-like structure of the flower axis). The fruit is a dark red berry, 2.5 cm to 3 cm long, 1.3 to 1.5 cm wide, crowned by 4 curved sepals and containing 1, rarely 2 seeds.

The evergreen clove tree is adapted to tropical climate around the see with altitude not exceeding 300 m. In many literatures, the clove tree is described to have an average height of 8–12 m although it has a potential to grow up to 20 m high. The most precious part of the plant, the flowers (Fig. 16.1), occur after a long period of growth of around 8–10 years (some reports say after 6 years) with full flowering appear at about 20 years. The economic return of the clove crop thus comes after two decades of waiting but sustained for several decades as the plant is known to provide flowers on annual bases for over 80 years. The unopened flower buds in clusters or inflorescences bearing the cloves are handpicked, and

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16.2 Botanical and taxonomic perspectives

FIG. 16.1 The clove plant during flowering period. Courtesy: Wikipedia (https://en. wikipedia.org/wiki/Clove).

FIG. 16.2

Image of cloves—dried flow

buds.

sun-dried (Fig. 16.2). The appearance of a clove—nail-like—that gave its name is known to derive from the French word ‘clou’ or English word ‘clout’ referring to the nail. The flower buds transform from pale to green and then pink to bright reddish colour when they are ready for collection before the flower opens. The cloves collected with care not to damage the young branches are less than 2 cm long and the overall yield from the tree vary with age but can reach up to 7 kg per tree annually. Yield could vary, however, from far smaller in younger to some report even say up to 9 kg from mature trees. It is interesting to note the floral feature of the plant from which clove derives. The long nail feature comes from the calyx ending with four sepals and the unopened four petals or rather pieces of the chalice forming the central ball-like (nail head) feature at the centre. Upon sundrying which normally takes 3–4 days, the darker-brown clove appears. The hard-dry feature together with its preservation with its chemical components like eugenol, clove can be kept for a loner period without losing its aroma.

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From taxonomic point of view, the genus and the family of clove have been one of the most interesting topics for the last few centuries. The genus Syzygium Gaertn comprised of around 1200 species that occur in the Old World tropical and subtropical regions of Africa, Asia, Australia, New Zealand and the Southwest Pacific regions. Some estimates even suggest that up to 1800 species may be represented by the genus making it as the largest genera of woody flowering plants in the world. The name Syzygium was suggested to be introduced in 1756 and as a genus replacing Caryophyllus, which now represents a different group of plants. There has been a great deal of discussion that even continued to date with respect to the confusion surrounding the genus Eugenia and Syzygium. Several revisions on the basis of the classical morphological characteristics were made but the lack of discrete variations in flowering, fruiting and other features or simply lack of demarcation line between the related genera and species has been a divisive subject for botanists. In fact, the similarity of the floral features of Syzygium species is astonishing. The one from Syzygium paniculatum (magenta cherry or Bush cherry) maintained at the Kew Botanical Garden (United Kingdom) is shown in Fig. 16.3. For the benefit of readers interested in this area, a useful review article is available (Ahmad et al., 2016). Some characteristics such as vascular features and evolutionary and phylogenetic data from DNA sequence have also been employed and the recent consensus has been to put Eugenia and Syzygium as not so close relatives. There are still no concrete data separating the two genera, however, and the unified genera for Eugenia including Syzygium and classification based on placing the Old World species as Eugenia and the New World as Syzygium are among the several suggestions made in recent years. The most economically important species of the genus is S. aromaticum, which is discussed in this chapter, but many species are also aromatic and being used in traditional medicines including for treatment of diabetes. Those top in the list with traditional medicine significance include S. cordatum Hochst. ex Krauss, S. jambos (L.) Alston., S. suborbiculare (Benth.) T.G. Hartley & L.M.Perry, S. corynocarpum (A.Gray) M€ ull.Berol., and S. cumini (L.) Skeels. In the

FIG. 16.3 Syzygium paniculatum (magenta cherry or Bush cherry) maintained at the Kew Botanical Garden (United Kingdom). Image of floral features (A) and fruits (B) that are incredibly similar with other species of the genus make the taxonomy a difficult task.

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latter case (S. cumini), the plant is well-known for treating diabetes and discussion on its old traditional medicinal value now being repurposed for use as food supplements is included in Chapter 26. The taxonomic hierarch of clove is shown below: Kingdom: Plantae—Plants Subkingdom: Tracheobionta—Vascular plants Superdivision: Spermatophyta—Seed plants Division: Magnoliophyta—Flowering plants Class: Magnoliopsida—Dicotyledons Subclass: Rosidae Order: Myrtales Family: Myrtaceae—Myrtle family Genus: Syzygium P. Br. ex Gaertn.—Syzygium Species: Syzygium aromaticum (L.) Merr. & L.M.Perry—Clove

16.3 Culinary and medicinal uses of clove The primary use of clove is for flavoring and seasoning or other culinary uses. The essential oils of primarily eugenol composition have been traditionally used for treating many diseases. The preservative nature of the oils associated with antimicrobial activity has also been a significant feature in food preservation and traditional medicine. Treating bad breath and dental care with clove has been reported throughout the history of traditional uses of clove by mankind. In fact, the essential oil has antibacterial effect against those strains responsible for dental caries and periodontal diseases (Cai and Wu, 1996). The antimicrobial activities of the essential oil against a range of gram-positive and gram-negative species have also been routinely reported (e.g. Gon˜i et al., 2009; Heredia-Guerrero et al., 2018). With this implication, control of foodborne pathogens and spoilage microorganisms (Beuchatn, 2001); control of Listeria (Cressy et al., 2003), and fungi (Chami et al., 2005; Kalemba and Kunicka, 2003) using clove essential oil is common. Preparations of clove oil to make it applicable in preservation of food products such as antifungal effects in meat products by microcapsule preparation (Wang et al., 2018); clove oil nanoemulsions and selfmicroemulsifying drug delivery systems (Kheawfu et al., 2018) or liposomes (Sebaaly et al., 2015) to maximize aqueous miscibility for internal use have been outlined. The development of clove buds powder as antibacterial structures and films for food packaging applications have also been extensively researched (e.g. Abdali and Ajji, 2015). The composition of essential oils from the leaves and stem could differ although eugenol is still the major component. Due to toxicity, even for eugenol, care has to be taken and mostly the essential oil from the clove flower buds is used in traditional medicine. Recent interest on the use of clove as antidiabetic agent is gaining momentum. This is based on some pharmacological data and traditional uses that led to the inclusion of clove to the long list of natural food ingredients being developed as dietary supplements. This chapter assesses the chemical and pharmacological basis of its potential to treat diabetes and associated diseases.

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16.4 The chemistry of cloves Clove buds are packed-full of oils, terpenoids and phenolic compounds ranging from simple aromatic acids to complex tannins. These compounds can be extracted by a variety of methods and presented in the following sections based on the structural class that they belong to.

16.4.1 Essential oils The chemistry of clove is dominated by its essential oil which can also be extracted from the leaves and other plant parts. The clove flower buds oil is dominated by eugenol (1) that constitute from 70% to 85%, while the other two main components are eugenyl acetate (2, up to 15%) and β-caryophyllene (3, 5–12%) (Fig. 16.4). Hence, around 99% of the oil is composed of these three compounds. The minor components of the oil that may contribute to the odour of the oil include methylsalicylate and methylamylketone. The essential oil extracted from the clove buds could account to 15–20% by weight and hence is significant, while the leaves could yield less than 5% of essential oil. Even the stem and fruits also contain significant amount of oil but for eugenol extraction, the clove buds, stems and leaves appear to be mostly exploited. Classical examples of oil yield and variability based on sources are described by Srivastava et al. (2005). Different methods of extraction including microwave assisted extraction with

(A)

(B) FIG. 16.4 Oil components of clove. The essential oil composition of clove flower buds (Panel A) predominantly contain eugenol and to a less extent eugenol acetate followed by β-caryophyllene and caryophyllene oxide. The fixed oil (Panel B) is dominated by oleic and linoleic acids while others in smaller amount include palmitic, stearic, decanoic, and γ-linolenic acids.

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16.4 The chemistry of cloves

intention of maximizing eugenol content (Kapadiya et al., 2018), environmentally friendly method of extraction with microwave methods (Kapadiya et al., 2018), and supercritical CO2 (de Oliveira et al., 2016) have been employed. In the latter case, eugenol content not exceeding 63% and (E)-caryophyllene (up to 15%) and eugenol acetate (up to 20%) was reported (de Oliveira et al., 2016). As an exemplary composition analysis, the essential oil clove flower buds obtained by the classical hydro-distillation method and that showed antibacterial activity against Staphylococcus aureus is shown in Table 16.1 (Xu et al., 2016). The oil obtained with the yield of 12.8% has been shown to contain 22 compounds though most of them are in trace amount with 11 compounds (half of all detected) less than 0.1% each. TABLE 16.1 Chemical composition of essential oil from clove bud extracted by hydrodistillation.a Compound

(%)

Eugenol

76.23

β-Caryophyllene

11.54

Caryophyllene oxide

4.29

Eugenyl acetate

1.76

α-Caryophyllene

0.64

β-Selinene

0.25

α-Selinene

0.16

Eucalyptol

0.14

4-Allylanisole

0.13

(
)-β-Cadinene

0.12

Anethol

0.11

2-Pinene