Handbook of Nutraceuticals and Functional Foods [3° ed.] 1498703720, 9781498703727

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Handbook of Nutraceuticals and Functional Foods Third Edition

Handbook of Nutraceuticals and Functional Foods Third Edition

Edited by

Robert E.C. Wildman Richard S. Bruno

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2020 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed on acid-free paper International Standard Book Number-13: 978-1-4987-0372-7 (Hardback) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http:// www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

To Amber, Gage, & Bryn for your daily support and love as well as Carol, Dave, & David for eternal inspiration! – Rob To Jenny, William, and Olivia – their support and love inspire me each and every day. – Rich Additional Support The editors would like to thank Brett Hanna for his contribution.

Contents Preface...............................................................................................................................................ix Editors................................................................................................................................................xi Contributors.................................................................................................................................... xiii

Unit I  Overview of Nutraceuticals and Functional Foods Chapter 1 Nutraceuticals and Functional Foods............................................................................3 Robert E.C. Wildman Chapter 2 Regulation of Nutraceuticals and Functional Foods................................................... 23 Rick Collins, Esq., Jay Manfre, Esq., and Robert E.C. Wildman

Unit II  Plant-Derived Nutraceuticals Chapter 3 Lycopene: Food Sources, Properties, and Effects on Human Health......................... 37 Jessica L. Cooperstone Chapter 4 Lutein in Neural Health and Disease.......................................................................... 55 Amy C. Long and Amy D. Mackey Chapter 5 Garlic: Chemistry, Function, and Implications for Health and Disease..................... 75 Sharon A. Ross and Craig S. Charron Chapter 6 The Role of Tocopherols in Health........................................................................... 105 Richard S. Bruno Chapter 7 Health Benefits of Green Tea.................................................................................... 127 Priyankar Dey, Geoffrey Y. Sasaki, and Richard S. Bruno Chapter 8 Scientific, Legal, and Regulatory Considerations for Cannabidiol........................... 147 Jay Manfre, Esq., Rick Collins, Esq., Marielle Kahn Weintraub, and Robert E.C. Wildman Chapter 9 Coffee as a Functional Beverage............................................................................... 159 Victoria Burgess, Lem Taylor, and Jose Antonio

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Chapter 10 Dietary Fiber and Coronary Heart Disease.............................................................. 173 Thunder Jalili, Eunice Mah, Denis M. Medeiros, and Robert E.C. Wildman Chapter 11 Anthocyanins and Their Health Benefits.................................................................. 189 Justin G. Martin, Gary D. Stoner, and Jairam K.P. Vanamala Chapter 12 Olive Oil and Health Benefits................................................................................... 211 Denis M. Medeiros and Meghan Hampton Chapter 13 Nutraceutical Herbs and Insulin Resistance............................................................. 223 Giuseppe Derosa and Pamela Maffioli

Unit III  Food Nutraceuticals from Animals Chapter 14 Protein as a Functional Food Ingredient for Optimizing Weight Loss and Body Composition.............................................................................................. 245 Paul J. Arciero, Michael J. Ormsbee, Robert E.C. Wildman, and Donald K. Layman Chapter 15 Nutraceutical Application of Creatine....................................................................... 267 Richard B. Kreider, Douglas S. Kalman, Jose Antonio, Tim N. Ziegenfuss, Robert E.C. Wildman, Darren G. Candow, and Chad M. Kerksick Chapter 16 Chicken Eggs and Human Health............................................................................. 295 Jonathan Merkle, Christopher Bailey, and Kevin Ruff Chapter 17 Dairy Milk: A Functional Beverage for Human Health...........................................307 Joshua D. McDonald and Richard S. Bruno Index............................................................................................................................................... 325

Preface The field of functional foods, and by association their bioactive food components, is a booming area of nutrition. This area was relatively unknown in the second half of the twentieth century, with only a few thousand total publications during this period, but then experienced exponential growth around the turn of the twenty-first century and now continues to flourish, with nearly 5000 scientific publications on an annual basis. It is difficult to imagine a more exciting field of nutrition research, education, and general health promotion.

Investigative opportunities are clearly endless. Large-scale epidemiological studies routinely identify health-promoting relationships with intakes of specific functional foods and the bioactive food components. These works have provided the foundational support for hypothesis-driven research at the cellular level, in animal models, and even controlled interventions in humans. Not only does the complement of these studies appear in the peer-reviewed literature, but knowledge is also disseminated rapidly across the globe through consumer publications and the entirety of the Internet. While the advent of many investigative techniques occurred in the latter half of the twentieth century to support the study of other critical areas of the nutrition field, the field of functional foods has not only prospered by these tools but also advances in experimental tools that permit detailed scientific inquiry. These advances have allowed scientists to objectively investigate some of the most ancient concepts in the application of foods as well as epidemiological relationships related to optimizing health and performance and the prevention and/or treatment of diseases. Throughout the twentieth century, nutrition recommendations often had a connotation of focusing on “what not to eat” and obtaining adequate dietary intakes of nutrients to prevent nutrition deficiency. For example, many recommendations focused on reducing intakes of sodium, saturated fat, cholesterol, and in some cases avoiding certain foods such as eggs. Today, there has been continued shift in focus to “what to eat” as the basis of many nutrition messages of public health importance. What remains clear over the past few decades is that those who eat a diet rich in more natural foods, such as fruits, vegetables, nuts, whole grains, and fish, tend to be protected from various chronic diseases. The incidences of certain cancers and heart disease are markedly lower than in populations that eat considerably lower amounts of these foods. For a while, many nutritionists believed that this observation was more of an association rather than cause and effect. This is to say that the higher incidence of disease was more the result of excess energy intake, fat and processed foods in conjunction with lower physical activity typically associated with the lower consumption of fruits, vegetables, and so on rather than the lack of these health-promoting foods. Thus, recommendations focused on limiting many of the “bad” food items by substituting them with foods that were not associated with the degenerative diseases, deemed “good” foods somewhat by ix

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Preface

default. With time scientists were able to better understand the composition and bioactivities of the “good” foods. Evidence quickly mounted as portrayed in the figure to support earlier beliefs that many natural foods are seemingly prophylactic and medicinal. Today we find ourselves at a critical crossroads to understand humanity’s relationship with nature. While the incorporation of various functional foods is health beneficial, the concept of nutraceuticals reminds us that other nonessential dietary constituents may also be critical for lowering the risk of chronic disorders and improving healthspan. For it is many functional foods that provide us with nutraceuticals that yield protection against the environment in which we exist and the potentially pathological events we internally create. Food was an environmental tool used in the sculpting of the human genome. It is only logical to think, then, that eating more natural foods such as fruits and vegetables would lead to a healthier existence. Advancement of scientific techniques, including various “omics” approaches, has not only allowed us to better understand the diet we are supposed to eat, but it has also opened the door to one of the most interesting events in commerce. Food companies are now able to market foods with approved health claims touting the nutraceutical or functional properties of the food. Food companies are also able to fortify existing foods with nutraceutical substances and/or create new foods designed to include one or more nutraceutical substances in their recipes. The opportunity afforded to food companies involved in functional foods appears without limitations at this time. Even though this book reviews numerous nutraceuticals and functional foods, the field is still relatively young and surely there is much more to be learned and applied to a healthier existence. It is hard to imagine that nutrition science would ever be more exciting than this. But perhaps some scientist wrote that very same thought less than a century ago during the vitamin and mineral boom. We truly hope you enjoy this book and welcome your comments and thoughts for future editions.

Editors Robert E.C. Wildman, PhD, is a native of Philadelphia, Pennsylvania, and attended the University of Pittsburgh (BS), Florida State University (MS), and Ohio State University (PhD). He is currently faculty at Texas Woman’s University in Denton, Texas. Dr. Wildman is author of The Nutritionist: Food, Nutrition, and Optimal Health and co-author of the textbooks Advanced Human Nutrition and Exercise and Sport Nutrition as well as creator of TheNutritionDr.com and founder of the International Protein Board. Dr. Wildman’s research focuses primarily on the impact of exercise and protein and amino acids on body weight, composition, and health and he presents around the world to educate and activate people to achieve better fitness and health. Richard S. Bruno, PhD, is a professor of human nutrition at The Ohio State University. He earned his doctorate in human nutrition from The Ohio State University (Columbus, Ohio), and both his MS and BS degrees in nutrition from the University of Delaware (Newark, Delaware). He also completed his postdoctoral training at the Linus Pauling Institute at Oregon State University (Corvallis, Oregon). Dr. Bruno is a bionutritionist and registered dietitian with expertise in the areas of phytochemical function and metabolism in relation to oxidative and inflammatory distress. His research program at The Ohio State University focuses on the bioavailability and metabolism of polyphenols and vitamin E, and their health-promoting functions to manage cardiometabolic disorders. Complementary areas of research also include the study of functional foods and their bioactive components to establish evidence-based dietary recommendations that enhance vascular endothelial function.

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Contributors Jose Antonio Department of Health and Human Performance Nova Southeastern University Davie, Florida Paul J. Arciero Department of Health and Human Physiological Sciences Skidmore College Saratoga Springs, New York Christopher Bailey Nutrition Sciences Stratum Nutrition Carthage, Missouri

Jessica L. Cooperstone Food Science and Technology The Ohio State University Columbus, Ohio Giuseppe Derosa Centre of Diabetes and Metabolic Diseases Fondazione IRCCS Policlinico San Matteo University of Pavia Pavia, Italy Priyankar Dey Department of Human Sciences The Ohio State University Columbus, Ohio

Meghan Hampton Naval Health Clinic Quantico Quantico, Virginia

Thunder Jalili Division of Nutrition University of Utah Salt Lake City, Utah

Richard S. Bruno Department of Human Sciences The Ohio State University Columbus, Ohio

Douglas S. Kalman Nutrition Research Unit Miami Research Associates Miami, Florida

Victoria Burgess Department of Health and Human Performance Nova Southeastern University Davie, Florida

Chad M. Kerksick School of Health Sciences Lindenwood University St. Charles, Missouri

Darren G. Candow Faculty of Kinesiology and Health Studies University of Regina Regina, Canada Craig S. Charron Food Components and Health Laboratory Agricultural Research Service United States Department of Agriculture Beltsville, Maryland Rick Collins, Esq. Collins Gann McCloskey & Barry PLLC Mineola, New York

Richard B. Kreider Department of Health & Kinesiology Texas A&M University College Station, Texas Donald K. Layman Department of Food Science & Human Nutrition University of Illinois Urbana, Illinois Amy C. Long Human Nutrition Abbott Nutrition Columbus, Ohio xiii

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Amy D. Mackey Human Nutrition Abbott Nutrition Columbus, Ohio Pamela Maffioli Centre of Diabetes and Metabolic Diseases Fondazione IRCCS Policlinico San Matteo University of Pavia Pavia, Italy Eunice Mah Biofortis Merieux NutriSciences Addison, Illinois Jay Manfre, Esq. Collins Gann McCloskey & Barry PLLC Mineola, New York Justin G. Martin Department of Chemical Engineering Pennsylvania State University University Park, Pennsylvania Joshua D. McDonald Department of Human Nutrition Winthrop University Rock Hill, South Carolina Denis M. Medeiros Department of Molecular Biology and Biochemistry University of Missouri-Kansas City Kansas City, Missouri Jonathan Merkle Nutrition Sciences Michael Foods Minneapolis, Minnesota Michael J. Ormsbee Institute of Sports Sciences & Medicine Nutrition, Food & Exercise Sciences Florida State University Tallahassee, Florida

Contributors

Sharon A. Ross Nutritional Science Research Group Division of Cancer Prevention National Cancer Institute National Institute Health Rockville, Maryland Kevin Ruff Human Nutrition Sciences Stratum Nutrition Carthage, Missouri Geoffrey Y. Sasaki Department of Human Sciences The Ohio State University Columbus, Ohio Gary D. Stoner Medical College of Wisconsin Wauwatosa, Wisconsin Lem Taylor School of Exercise & Sport Science University of Mary Hardin-Baylor Belton, Texas Jairam K.P. Vanamala Department of Food Science Pennsylvania State University University Park, Pennsylvania Marielle Kahn Weintraub President-Board of Directors US Hemp Authority Lexington, Kentucky Robert E.C. Wildman Department of Food & Nutrition Sciences Texas Woman’s University Denton, Texas Tim N. Ziegenfuss The Center for Applied Health Sciences Stow, Ohio

Unit I Overview of Nutraceuticals and Functional Foods

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Nutraceuticals and Functional Foods Robert E.C. Wildman

CONTENTS 1.1 Introduction...............................................................................................................................3 1.2 Defining Nutraceuticals and Functional Foods.........................................................................4 1.3 Classifying Nutraceutical Factors.............................................................................................. 5 1.4 Food and Nonfood Sources of Nutraceutical Factors................................................................ 6 1.5 Nutraceutical Factors in Specific Foods.................................................................................... 6 1.6 Mechanism of Action................................................................................................................8 1.7 Classifying Nutraceutical Factors Based on Chemical Nature............................................... 10 1.7.1 Isoprenoid Derivatives (Terpenoids)............................................................................ 11 1.7.2 Phenolic Compounds................................................................................................... 15 1.7.3 Carbohydrates and Derivatives.................................................................................... 17 1.7.4 Fatty Acids and Structural Lipids................................................................................20 1.7.5 Amino Acid–Based..................................................................................................... 21 1.7.6 Microbes (Probiotics).................................................................................................. 21 1.7.7 Minerals....................................................................................................................... 22 References......................................................................................................................................... 22

1.1 INTRODUCTION Interest in nutraceuticals and functional foods continues to grow, powered by progressive research efforts to identify properties and potential applications of nutraceutical substances, and coupled with public interest and consumer demand. Estimates vary; global market size for functional food ingredients is projected to exceed 250 billion by 2025. Among the principal reasons for the growth of the functional food market are current population and health trends. Across the globe, populations are aging. For instance, in 2015, the average projected life expectancy globally for those born that year was 71.4 years. Moreover, about 30 countries recorded average life expectancy at 80 years or above, including Japan, Singapore, and Switzerland, all above 83 years.1 Meanwhile, obesity is now recognized as a global epidemic as its incidence continues to climb in countries throughout the world. According to reports of the World Health Organization (WHO) in 2016, more than 1.9 billion adults aged 18 years and older were overweight, with over 650 million adults obese. In 2016, 39% of adults aged 18 years and over were overweight. Overall, about 13% of the world’s adult population (11% of men and 15% of women) were obese in 2016.2 In the United States of America in 2017, nearly 38% of adults were obese. Nearly 8% are extremely obese.3 Global trends show stability or favor an increased incidence of obesity versus a reduction. Meanwhile, heart disease continues to be a primary cause of death, responsible for one out of every four deaths in the U.S., and cancer, osteoporosis, and arthritis remain highly prevalent. Although genetics play a major role in the development of the diseases mentioned above, by and large most are considered preventable or could be minimized by a health-promoting diet and physical activity, weight management, and a healthier lifestyle, including environment. Additionally, 3

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people can optimize the health-promoting capabilities of their diet by way of supplementation and by consuming foods that have been formulated or fortified to include health-promoting factors. Another reason for the growing trend in functional foods is public education. People today are more nutrition savvy than ever before, the interest in health-related information being met by many informational resources. Every day people are exposed to media articles, blogs, and social media posts devoted to the relationship between diet and health. Numerous websites have been developed by government agencies such as the U.S. Department of Agriculture (USDA; www.nal.usda.gov) and organizations such as the American Heart Association (www.americanheart.org) and the American Cancer Society (www.cancer.org). Last, information-based entities abound on the Internet, including WebMD.com and TheNutritionDr.com.

1.2  DEFINING NUTRACEUTICALS AND FUNCTIONAL FOODS The term nutraceutical is a hybrid or contraction of nutrition and pharmaceutical. Reportedly, it was coined in 1989 by DeFelice and the Foundation for Innovation in Medicine.4 Restated and clarified in a press release in 1994, its definition was “any substance that may be considered a food or part of a food and provides medical or health benefits, including the prevention and treatment of disease. Such products may range from isolated nutrients, dietary supplements and diets to genetically engineered ‘designer’ foods, herbal products, and processed foods such as cereals, soups, and beverages.”5 At present there are no universally accepted definitions for nutraceuticals and functional foods, although commonality exists between the definitions offered by different health-oriented professional organizations. According to the International Food Information Council (IFIC), functional foods are foods or dietary components that may provide a health benefit beyond basic nutrition.6 The International Life Sciences Institute of North America (ILSI) has defined functional foods as “foods that by virtue of physiologically active food components provide health benefits beyond basic nutrition.”7 Meanwhile, Health Canada defines functional foods as “similar in appearance to a conventional food, consumed as part of the usual diet, with demonstrated physiological benefits, and/or to reduce the risk of chronic disease beyond basic nutritional functions.”8 The Nutrition Business Journal classified functional food as “food fortified with added or concentrated ingredients to functional levels, which improves health or performance. Functional foods include enriched cereals, breads, sport drinks, bars, fortified snack foods, baby foods, prepared meals, and more.”9 In the 2013 Academy of Nutrition and Dietetics (AND) position paper on nutraceuticals and functional foods, the authors state: “It is the position of the Academy of Nutrition and Dietetics to recognize that although all foods provide some level of physiological function, the term functional foods is defined as whole foods along with fortified, enriched, or enhanced foods that have a potentially beneficial effect on health when consumed as part of a varied diet on a regular basis at effective levels based on significant standards of evidence. The Academy supports Food and Drug Administration (FDA)-approved health claims on food labels when based on rigorous scientific substantiation.”10 Based on these statements, one can surmise that functional foods include everything from natural foods, such as fruits and vegetables endowed with antioxidants and fiber, to fortified and enriched foods, such as orange juice with added calcium or additional carotenoids, to formulated readyto-drink beverages containing protein, amino acids, vitamins, minerals, antioxidants, immunesupporting factors, etc. Regarding the term “nutraceutical,” the Nutrition Business Journal states that it uses the term nutraceutical for anything that is consumed primarily or particularly for health reasons. Based on that definition, a functional food would be a kind of nutraceutical.9 On the other hand, Health Canada states that nutraceuticals are a product that is “prepared from foods but sold in the form of pills or powders (potions), or in other medicinal forms not usually associated with foods. A nutraceutical is demonstrated to have a physiological benefit or provide protection against chronic disease.”8 Based on this definition and how functional foods are characterized, as noted previously, nutraceuticals would be distinct from functional foods.

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TABLE 1.1 Food Label Claim Guidelines Claim Nutrient content claim Qualified health claim

NLEA authorized health claims

Structure/function claim

Purpose

Example

Describe content of certain nutrients. Describe the relationship between food, food component, or dietary supplement and reduced risk of a disease or health related condition. This claim uses qualifying language because the evidence for this relationship is emerging and is not yet strong enough to meet the standard of significant scientific advancement set by the FDA. Characterize a relationship between a food, a food component, dietary ingredient, or dietary supplement and risk of a disease. Describes role of nutrient or ingredient intended to affect normal structure or function in humans. May characterize the means by which the nutrient or ingredient affects the structure or function. May describe a benefit related to a deficiency. Must be accompanied by a disclaimer stating that FDA has not reviewed the claim and that the product is not intended to “diagnose, treat, cure, or prevent any disease.”

“Fat-free,” “low sodium.” “Some scientific evidence suggests that consumption of antioxidant vitamins may reduce the risk of certain forms of cancer. However, FDA has determined that this evidence is limited and not conclusive.”

“Diets high in calcium may reduce the risk of osteoporosis.” “Calcium builds strong bones.”

Source: Adapted from International Life Sciences Institute of North America Web site, http://www.ilsi.org/, 2006.

The potential functions of nutraceutical/functional food ingredients are so often related to the maintenance or improvement of health that it is necessary to distinguish between a food ingredient that has function and a drug. The core definition of a drug is any article that is “intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease in man or other animals” (21  U.S.C.  321(g)(1)(B)). At the same time, certain health claims can be made for foods and ingredients that are associated with health conditions.11 In the U.S., such health claims are defined and regulated by the U.S. Food and Drug Administration (FDA). Health claims related to foods and ingredients include an implied or explicit statement about the relationship of a food substance to a disease or health-related condition (21 U.S.C.343(r)(1)(B) and 21 C.F.R.101.14(a)(1)). The major categories of health claims are listed in Table 1.1 with examples of each.

1.3  CLASSIFYING NUTRACEUTICAL FACTORS The number of purported nutraceutical substances is in the hundreds, and some of the more recognizable substances include isoflavones, tocotrienols, allyl sulfur compounds, fiber, and carotenoids. Considering a long and growing list of nutraceutical substances, organization systems are needed to allow for easier understanding and application. This is particularly true for academic instruction, as well as product formulation by food companies. Depending upon one’s interest and/or background, the appropriate organizational scheme for nutraceuticals can vary. For example, cardiologists may be most interested in those nutraceutical substances that are associated with reducing the risk factors of heart disease. Specifically, their interest may lie in substances purported to positively influence hypertension and hypercholesterolemia

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and to reduce free radical or platelet-dependent thrombotic activity. Accordingly, nutraceutical factors such as certain fibers, omega-3 fatty acids, phytosterols, quercetin, and grape flavonoids would be of interest. Meanwhile, oncologists may be more interested in those substances that target anticarcinogenic activities. These substances may be associated with augmentations of microsomal detoxification systems and antioxidant defenses, or they may slow the progression of existing cancer. Thus, their interest may lie in both chemoprevention or potential adjunctive therapy. On the other hand, the nutraceutical interest of food scientists working on the development of a functional food product will not only include physiological properties, but also stability and sensory properties, as well as issues of cost efficiency. To demonstrate this point, the anticarcinogenic triterpene limonin is lipid-soluble and intensely bitter, somewhat limiting its commercial use as a functional food ingredient.12 However, the glucoside derivative of limonin, which shares some of the potential anticarcinogenic activity of limonin, is water soluble and virtually tasteless, thereby enhancing its potential use as an ingredient.13 Whether it is for academic instruction, clinical trial design, functional food development, or dietary recommendations, nutraceutical factors can be organized in several ways. Cited below are a few ways of organizing nutraceuticals based upon food source, mechanism of action, and chemical nature.

1.4  FOOD AND NONFOOD SOURCES OF NUTRACEUTICAL FACTORS One of the broader models of organization for nutraceuticals is based upon their potential as a food source to humans. Here nutraceuticals may be separated into plant, animal, and microbial (i.e., bacteria and yeast) groups. Grouping nutraceutical factors in this manner has numerous merits and can be a valuable tool for diet planning, as well as classroom and seminar instruction. One interesting consideration with this organization system is that the food source may not necessarily be the point of origin for one or more substances. An obvious example is conjugated linoleic acid (CLA), which is part of the human diet, mostly as a component of beef and dairy foods. However, it is made by bacteria in the rumen of the cow. Therefore, issues involving the food chain or symbiotic relationships may have to be considered for some individuals working with this organization scheme. Because of conserved biochemical aspects across species, many nutraceutical substances are found in both plants and animals, and sometimes in microbes. For example, microbes, plants, and animals contain choline and phosphotidylcholine. This is also true for sphingolipids; however, plants and animals are better sources. Also, linolenic acid (18:3ω-3 fatty acid) can be found in a variety of food resources, including animal flesh, even though it is primarily synthesized in plants and other lower members of the food chain. Table 1.2 presents some of the more recognizable nutraceutical substances grouped according to food-source providers. Nonfood sources of nutraceutical factors have been sourced by the development of modern fermentation methods. For example, amino acids and their derivatives have been produced by bacteria grown in fermentation systems. The emergence of recombinant-genetic techniques has enabled new avenues for obtaining nutraceutical compounds. These techniques and their products are being evaluated in the arenas of the marketplace and regulatory concerns around the world. An example is the production of eicosapentaenoic acid (EPA) by bacteria, which is normally produced by some algae and bacteria. The EPA derived from salmon are produced by algae and are later incorporated in the salmon that consume the algae. Meanwhile, there is the potential to produce EPA by non–EPAproducing bacteria by importing the appropriate DNA through recombinant methods.14 The ability to transfer the production of nutraceutical molecules into organisms that allows for economically feasible production is cause for both optimism and discussion concerning regulatory and popular acceptance.

1.5  NUTRACEUTICAL FACTORS IN SPECIFIC FOODS In an organization model related to the one above, nutraceuticals can be grouped based upon relatively concentrated foods. This model is more appropriate when there is interest in a nutraceutical

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TABLE 1.2 Examples of Nutraceutical Substances Grouped by Food Source Plants β-Glucan Ascorbic acid γ-Tocotrienol Quercetin Luteolin Cellulose Lutein Gallic acid Perillyl alcohol Indole-3-carbonol Pectin Daidzein Glutathione Potassium Allicin

Animal

Microbial

Conjugated Linoleic Acid (CLA)

Saccharomyces boulardii (yeast)

Eicosapentaenoic acid (EPA) Docosahexenoic acid (DHA)

Bifidobacterium bifidum B. longum

Spingolipids Choline Lecithin Calcium Coenzyme Q10 Selenium Zinc Creatine Minerals

B. infantis Lactobacillus acidophilus (LC1) L. acidophilus (NCFB 1748) Streptococcus salvarius (subs. Thermophilus)

δ-Limonene Genestein Lycopene Hemicellulose Lignin Capsaicin Geraniol β-Ionone α-Tocopherol β-Carotene Nordihydrocapsaicin Selenium Zeaxanthin Minerals MUFA Note: The substances listed in this table include those that are either accepted or purported nutraceutical substances.

compound or related compounds, or when there is interest in a specific food for agricultural/ geographic reasons or functional food-development purposes. For example, the interest may be in the nutraceutical qualities of a local crop or a traditionally consumed food in a geographic region, such as pepper fruits in the southwestern United States, olive oil in Mediterranean regions, and red wine in western Europe and Northern California. There are several nutraceutical substances that are found in higher concentrations in specific foods or food families. These include capsaicinoids, which are found primarily in pepper fruit, and allyl sulfur (organosulfur) compounds, which are particularly concentrated in onions and garlic. Table 1.3 provides a listing of certain nutraceuticals that are considered unique to certain foods or food families. One consideration for this model is that for several substances, such as those just named, there is a relatively short list of foods that are concentrated sources. However, the list of food sources for other nutraceutical substances can be much longer and can include numerous seemingly unrelated foods. For instance, citrus fruit contain the isoflavone quercetin, as do onions, a plant food seemingly unrelated. Citrus fruit grow on trees, whereas the edible bulb of the onion plant (an herb)

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TABLE 1.3 Examples of Foods with Higher Content of Specific Nutraceutical Substances Nutraceutical Substance/Family Allyl sulfur compounds Isoflavones (e.g., genestein, daidzein) Quercetin Capsaicinoids EPA and DHA Lycopene Isothiocyanates β-Glucan CLA Resveratrol β-Carotene Carnosol Catechins Adenosine Indoles Curcumin Ellagic acid Anthocyanates 3-n-Butyl phthalide Cellulose Lutein, zeaxanthin Psyllium Monounsaturated fatty acids Inulin, Fructooligosaccharides (FOS) Lactobacilli, Bifidobacteria Catechins Lignans

Foods of Remarkably High Content Onions, garlic Soybeans and other legumes, apios Onion, red grapes, citrus fruit, broccoli, Italian yellow squash Pepper fruit Fish oils Tomatoes and tomato products Cruciferous vegetables Oat bran Beef and dairy Grapes (skin), red wine Citrus fruit, carrots, squash, pumpkin Rosemary Teas, berries Garlic, onion Cabbage, broccoli, cauliflower, kale, brussels sprouts Tumeric Grapes, strawberries, raspberries, walnuts Red wine Celery Most plants (component of cell walls) Kale, collards, spinach, corn, eggs, citrus Psyllium husk Tree nuts, olive oil Whole grains, onions, garlic Yogurt and other dairy Tea, cocoa, apples, grapes Flax, rye

Note: The substances listed in this table include those that are either accepted or purported nutraceutical substances.

develops at ground level. Other plant foods with higher quercetin content are red grapes—but not white grapes—broccoli (which is a cruciferous vegetable), and the Italian yellow squash. Again, these foods appear to bear very little resemblance to citrus fruit, or onions for that matter. On the other hand, there are no guarantees that closely related or seemingly similar foods contain the same nutraceutical compounds. For example, both the onion plant and the garlic plant are perennial herbs arising from a rooted bulb and are also cousins in the lily family. However, although onions are loaded with quercetin, with some varieties containing up to 10% of their dry weight of this flavonoid, garlic is quercetin void.

1.6  MECHANISM OF ACTION Another means of classifying nutraceuticals is by their mechanism of action. This system groups nutraceutical factors together, regardless of food source, based upon their proven or purported physiological properties. Among the classes would be antioxidant, antibacterial, antihypertensive, anti-hypercholesterolemic, anti-aggregate, anti-inflammatory, anti-carcinogenic, osteoprotective, and so on. Like the scheme just discussed, credible Internet resources may prove invaluable to this

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TABLE 1.4 Examples of Nutraceuticals Grouped by Mechanisms of Action Anticancer

Positive Influence on Blood Lipid Profile

Capsaicin

Antioxidant Activity

Antiinflammatory

Osteogenetic or Bone Protective

β-Glucan

CLA

Linolenic acid

CLA

Genestein

γ-Tocotrienol

Ascorbic acid

EPA

Soy protein

Daidzein

δ-Tocotrienol MUFA

β-Carotene Polyphenolics

DHA

Genestein Daidzein

Quercetin

Tocopherols

ω-3 PUFAs Resveratrol Tannins

Tocotrienols

GLA (gamma-linolenic acid)

Indole-3-carbonol Capsaicin Quercetin α-Tocopherol

FOS (fructooligosaccharides)

β-Sitosterol Saponins Guar Pectin

Ellagic acid

Inulin

α-Tocotrienol γ-Tocotrienol CLA Lactobacillus acidophilus Sphingolipids Limonene Diallyl sulfide Ajoene α-Tocopherol Enterolactone Glycyrrhizin Equol Curcumin Ellagic acid Lutein Carnosol L. bulgaricus

Curcumin

Calcium Casein phosphopeptides

Lycopene Lutein Glutathione Hydroxytyrosol Luteolin Oleuropein Catechins Gingerol Chlorogenic acid Tannins

Note: The substances listed in this table include those that are either accepted or purported nutraceutical substances.

approach. Examples are presented in Table 1.4. This model would also be helpful to an individual who is genetically predisposed to a medical condition or to scientists trying to develop powerful functional foods for just such a person. The information in this model would then be helpful in diet planning in conjunction with the organization scheme just discussed and presented in Table 1.3. It would also be helpful to a product developer trying to develop a new functional food, perhaps for heart health. This developer might consider the ingredients listed in several categories to develop a product that would reduce blood pressure, total and LDL-cholesterol levels, and inflammation. Some nutraceutical ingredients or mixtures are marketed on the basis that they have been used for many years in the practice of traditional or cultural medicine, that is, treatments for medical illness that have developed in cultural tradition because of trial and error. This rationale for use can be both compelling and a cause for concern. The plant and animal kingdoms contain many compounds that offer therapeutic benefit or danger; often the same compound offers both, with the difference being dependent upon the dose.15 While traditional medicines are assumed low risk for the most part, one 5-year study that followed over 1000 cases reported a possible or confirmed association between use and toxicity in nearly 61% of the cases.16 Thus, whereas a statement regarding traditional use seems to offer a sense of safety by virtue of use by many individuals over time, there always need to be systematic regulatory efforts to determine and document safety over time. What may be of interest is that there are several nutraceuticals that can be listed as having more than one mechanism of action. One of the seemingly most versatile nutraceutical families is the omega-3 polyunsaturated fatty acids (PUFAs). Their nutraceutical properties can be related to direct effects as well as to some indirect effects. For example, these fatty acids are used as precursors

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for eicosanoid substances that locally vasodilate, bronchodilate, and deter platelet aggregation and clot formation. These roles can be prophylactic for asthma and heart disease. Omega-3 PUFA may also reduce the activities of protein kinase C and tyrosine kinase, both of which are involved in a cell-growth-signaling mechanism. Here, the direct effects of these fatty acids may reduce cardiac hypertrophy and cancer-cell proliferation. Omega-3 PUFA also appears to inhibit the synthesis of fatty acid synthase (FAS), which is a principal enzyme complex involved in de novo fatty acid synthesis. Here the nutraceutical effect may be considered indirect, as chronic consumption of these PUFAs may theoretically lead to decreased quantities of body fat over time and slow the development of obesity. The obesity might then lead to the development of hyperinsulinemia and related physiological aberrations such as hypertension and hyperlipidemia.

1.7  CLASSIFYING NUTRACEUTICAL FACTORS BASED ON CHEMICAL NATURE Another method of grouping nutraceuticals is based upon their chemical nature. This approach allows nutraceuticals to be categorized under molecular/elemental groups. This preliminary model includes several large groups, which then provide a basis for subclassification or subgroups, and so on. One way to group nutraceuticals grossly is as follows: • • • • • • •

Isoprenoid derivatives Phenolic substances Fatty acids and structural lipids Carbohydrates and derivatives Amino acid–based substances Microbes Minerals

As scientific investigation continues, several hundred substances will probably be deemed nutraceuticals. As many of these nutraceutical compounds appear to be related in synthetic origin or molecular nature, there is the potential to broadly group many of the substances together (Figure 1.1). This scheme is by no means perfect, and it is offered “in pencil,” as opposed to being “etched in stone.” It is expected that scientists will ponder this organization system, find flaws, and suggest ways to evolve the scheme, or disregard it completely in favor of a better concept. Even at this point,

FIGURE 1.1  Organizational scheme for nutraceuticals.

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several “gray” areas are apparent. For instance, mixtures of different classes can exist, such as mixed isoprenoids, prenylated coumarins, and flavonoids. Also, phenolic compounds could arguably be grouped under a very large “amino acid and derivatives” category. Although most phenolic molecules arise from phenylalanine as part of the shikimic acid metabolic pathway, other phenolic compounds are formed via the malonic acid pathway, thereby circumventing phenylalanine as an intermediate. Thus, phenolics stand alone in their own group, whose most salient characteristic is chemical structure, not necessarily synthetic pathway.

1.7.1  Isoprenoid Derivatives (Terpenoids) Isoprenoids and terpenoids are terms used to refer to the same class of molecules. These substances are without question one of the largest groups of plant secondary metabolites. In accordance with this ranking, they are also the basis of many plant-derived nutraceuticals. Under this large umbrella are many popular nutraceutical families such as carotenoids, tocopherols, tocotrienols, and saponins. This group is also referred to as isoprenoid derivatives because the principal building block molecule is isoprene (Figure 1.2). Isoprene itself is synthesized from acetyl coenzyme A (CoA), in the wellresearched mevalonic acid pathway (Figure 1.3), and the glycolysis-associated molecules pyruvate and 3-phosphoglycerate in a less-understood metabolic pathway.17 In both pathways, the product is isopentenyl phosphate (IPP), and IPP is often regarded as the pivotal molecule in the formation of larger isoprenoid structures. Once IPP is formed, it can reversibly isomerize to dimethylallyl pyrophosphate (DMAPP) as presented in Figure 1.4. Both five-carbon structures are then used to form geranyl pyrophosphate (GPP), which can give rise to monoterpenes. Among the monoterpenes are limonene and perillyl alcohol. GPP can also react with IPP to form the 15-carbon structure farnesyl pyrophosphate (FPP), which then can give rise to the sesquiterpenes. FPP can react with IPP or another FPP to produce either the 20-carbon geranylgeranyl pyrophosphate (GGPP) or the 30-carbon squalene molecule, respectively. GGPP can give rise to diterpenes, while squalene can give rise to triterpenes and steroids. Lastly, GGPP and GPP can condense to form the 40-carbon phytoene structure, which then can give rise to tetraterpenes. Most plants contain so-called essential oils, which contain a mixture of volatile monterpenes and sesquiterpenes. Limonene is found in the essential oils of citrus peels, whereas menthol is the chief monoterpene in peppermint essential oil (Figure 1.5). Two potentially nutraceutical diterpenes in coffee beans are kahweol and cafestol.18 Both of these diterpenes contain a furan ring. As discussed by Miller and colleagues, the furan-ring component might be very important in yielding some of the potential antineoplastic activity of these compounds.19 Several triterpenes (examples in Figure 1.6) have been reported to have nutraceutical properties. These compounds include plant sterols; however, some of these structures may have been modified to contain fewer than 30 carbons. One of the most recognizable triterpene families is the limonoids. These triterpenes are found in citrus fruit and impart most of their bitter flavor. Limonin and nomilin are two triterpenoids that may have nutraceutical application, limonin more so than nomilin.20 Both of these molecules contain a furan component. In citrus fruit, limonoids can also be found with an attached glucose, forming a limonoid glycoside.21 As discussed above, the addition of the sugar group reduces the bitter taste tremendously and makes the molecule more water soluble. These properties

FIGURE 1.2  Isoprene.

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FIGURE 1.3  The mevalonic acid pathway.

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FIGURE 1.4  Formation of terpene structures. In addition: (1) FPP + FPP produces squalene (30 carbons) which yields triterpenes and steroids, and (2) GGPP + GGPP produces phytoene (40 carbons) which yields tetraterpenes.

may make it more attractive as a functional food ingredient. Saponins are also triterpene derivatives, and their nutraceutical potential is attracting interest.22,23 The carotenoids (carotenes and xanthrophils), whose name is derived from carrots (Daucus carota), are perhaps the most recognizable form of coloring pigment within the isoprenoid class. Carotenes and xanthrophils differ only slightly, in that true carotenes are purely hydrocarbon molecules (i.e., lycopene, α-carotene, β-carotene, γ-carotene); the xanthrophils (i.e., lutein, capsanthin, cryptoxanthin, zeaxanthin, astaxanthin) contain oxygen in the form of hydroxyl, methoxyl, carboxyl, keto, and epoxy groups. With the exception of crocetin and bixin, naturally occurring carotenoids are tetraterpenoids, and thus have a basic structure of 40 carbons with unique

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Handbook of Nutraceuticals and Functional Foods CH3

CH2

CH3

CH2 OH

H3C

CH2 Limonene

H3 C

CH3 Menthol

H3C

CH3 Myrcene

FIGURE 1.5  Structure of select monoterpenes.

FIGURE 1.6  Examples of triterpenes.

modifications. The carotenoids are pigments that generally produce colors of yellow, orange, and red. Carotenoids are also very important in photosynthesis and photoprotection. Different foods have different kinds and relative amounts of carotenoids. Also, the carotenoid content can vary seasonally and during the ripening process. For example, peaches contain violaxanthin, cryptoxanthin, β-carotene, persicaxanthin, neoxanthin, and as many as 25 other carotenoids; apricots

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contain mostly α-carotene, β-carotene, and lycopene; and carrots contain about 50–55 parts per million of carotene in total, mostly α-carotene, β-carotene, and γ-carotene, as well as lycopene. Many vegetable oils also contain carotenoids, with palm oil containing the most. For example, crude palm oil contains up to 0.2% carotenoids. Meanwhile, there are a few synthetic carotenoids, including β-apo-8′-carotenal (apocarotenal), and canthaxanthin. Beta-Apo-8′-carotenal (apocarotenal) imparts a light reddish-orange color, and canthaxanthin imparts an orange-red to red color.

1.7.2 Phenolic Compounds Like the terpenoids, phenolic compounds are also considered secondary metabolites. The base for this very diverse family of molecules is a phenol structure, which is a hydroxyl group on an aromatic ring. From this structure, larger and interesting molecules are formed such as anthocyanins, coumarins, phenylpropamides, flavonoids, tannins, and lignin. Phenolic compounds perform a variety of functions for plants, including defending against herbivores and pathogens, absorbing light, attracting pollinators, reducing the growth of competitive plants, and promoting symbiotic relationships with nitrogen-fixing bacteria. There are a couple of biosynthetic pathways that form phenolic compounds. The predominant pathways are the shikimic acid pathway and the malonic acid pathway. The shikimic pathway is more significant in higher plants, although the malonic acid pathway is also present.17 Actually, the malonic pathway is the predominant source of secondary metabolites in lower plants, fungi, and bacteria. The shikimic pathway is so named because an intermediate of the pathway is shikimic acid. Inhibition of this pathway is the purpose of a commercially available herbicide (Roundup). The malonic acid pathway begins with acetyl CoA. Meanwhile, in the shikimic pathway, simple carbohydrate intermediates of glycolysis and the pentose phosphate pathway (PPP) are used to form the aromatic amino acids phenylalanine and tyrosine. A third aromatic amino acid, tryptophan, is also a derivative of this pathway. As animals do not possess the shikimic acid pathway, these aromatic amino acids are diet essentials. Obviously, these amino acids are considered primary metabolites or products. Thus, it is the reactions beyond the formation of these amino acids that are of greater importance to the production of secondary metabolites. Once formed, phenylalanine can be used to generate flavonoids (Figure 1.7). The reaction that generates cinnamic acid from phenylalanine is catalyzed by one of the most-studied enzymes associated with secondary metabolites, phenylalanine ammonia lyase (PAL). The expression of PAL is increased during fungal infestation and other stimuli, which may be critical to the plant. From trans-cinnamic acid, several simple phenolic compounds can be made. These include the benzoic acid derivatives vanillin and salicylic acid (Figure 1.8). Also, trans-cinnamic acid can be converted to para-coumaric acid. Simple phenolic derivatives of para-coumaric acid include caffeic acid and ferulic acid. CoA can be attached to para-coumaric acid to form para-coumaryl CoA. Both para-coumaric acid and para-coumaryl CoA can also be used to form lignin-building blocks, paracoumaryl alcohol, coniferyl alcohol, and sinapyl alcohol. After cellulose, lignin is the most abundant organic molecule in plants. To continue the formation of other phenolic classes, para-coumaryl CoA can undergo further enzymatic modification, involving three malonyl CoA molecules, to create polyphenolic molecules such as chalcones and then flavonones. The basic flavonone structure is then the precursor for the flavones, isoflavones, and flavonols. Also, flavonones can be used to make anthocyanins and tannins via dihydroflavonols (Figures 1.7, 1.9, and 1.10). The flavonoids are one of the largest classes of phenolic compounds in plants. The basic carbon structure of flavonoids contains 15 carbons and is endowed with two aromatic rings linked by a 3-carbon bridge (Figure 1.11).17 The rings are labeled A and B. Whereas the simpler phenolic compounds and lignin-building blocks result from the shikimic pathway and are phenylalanine derivatives, formation of the flavonoids requires some assistance from both the shikimic pathway and the malonic acid pathway. Ring A is derived from acetic acid (acetyl CoA) and the malonic acid pathway (see the use of 3 malonyl CoA to form chalcones in Figure 1.7). Meanwhile, ring B and the

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FIGURE 1.7  Production of plant phenolic molecules via phenylalanine.

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FIGURE 1.8  Select coumarins (first row) and two benzoic-acid-derived phenolic molecules (second row).

3-carbon bridge are derived from the shikimic acid pathway.17 The flavonoids are subclassified based primarily on the degree of oxidation of the 3-carbon bridge. Also, hydroxyl groups are typically found at carbon positions 4, 5, and 7, as well as other locations. The majority of naturally occurring flavonoids are actually glycosides, meaning a sugar moiety is attached. The attachment of hydroxyl groups and sugars will increase the hydrophilic properties of the flavonoid molecule, while attachment of methyl esters or modified isopentyl units will increase the lipophilic character. Anthocyanins and anthocyanidins (Figure 1.9) are produced by plants and function largely as coloring pigments. Basically, anthocyanins are anthocyanidins with sugar moieties attached at position 3 of the 3-carbon bridge between rings A and B.17 These molecules help attract animals for pollination and seed dispersal. They are responsible for the red, pink, blue, and violet coloring of many fruits and vegetables, including blueberries, apples, red cabbage, cherries, grapes, oranges, peaches, plums, radishes, raspberries, and strawberries. Only about 16 anthocyanidins have been identified in plants and include pelargonidin, cyanidin, delphinidin, peonidin, malvidin, and petunidin. Although the flavonols and flavones are structurally like their close cousin anthocyanidins and the anthocyanidin-glycoside derivatives anthocynanins, they absorb light at shorter wavelengths and thus are not perceived as color to the human eye. However, they may be detected by insects and help direct them to areas of pollination. Because flavones and flavonols do absorb UV–B light energy (280–320 nm), they are believed to serve a protective role in plants. Also, certain flavonoids promote the formation of a symbiotic relationship between plant roots and nitrogen-fixing bacteria. The primary structural feature that separates the isoflavones from the other flavonoids is a shift in the position of the B ring. Perhaps the most ubiquitous flavonoid is quercetin. Hesperidin is also a common flavonoid, especially in citrus fruit.

1.7.3 Carbohydrates and Derivatives The glucose derivative ascorbic acid (vitamin C) is perhaps one of the most recognizable nutraceutical substances and is a very popular supplement. Ascorbic acid functions as a nutraceutical compound, primarily as an antioxidant. Meanwhile, plants produce some oligosaccharides that appear to function as prebiotic substances.

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FIGURE 1.9  Anthocyanidin (A) and molecular derivatives including anthocyanin (B).

Several plant polysaccharide families are not readily available energy sources for humans, as they are resistant to secreted digestive enzymes. These polysaccharides are grouped together along with the phenolic polymer compound lignin to form one of the most recognizable nutraceutical families—fibers. By and large the role of fibers is structural for plants. For example, cellulose and hemicellulose are major structural polysaccharides found within plant cell walls. Beyond providing structural characteristics to plant tissue, another interesting role of certain fibers is in tissue repair after trauma, somewhat analogous to scar tissue in animals. The non-starch polysaccharides can be divided into homogeneous and heterogeneous polysaccharides, as well as into soluble and insoluble substances. Cellulose is a homogeneous nonstarch polysaccharide, as it consists of repeating units of glucose monomers. The links between the glucose monomers are β1-4 in nature. These polysaccharides are found in plant cell walls as microfibril bundles. Hemicellulose is found in association with cellulose within plant-cell walls and is composed of a mixture of both straight-chain and highly branched polysaccharides containing pentoses, hexoses, and uronic acids. Pentoses such as xylans, mannans, galactans, and arabicans are found in relatively higher abundance. Hemicelluloses are somewhat different from cellulose in that they are not limited to glucose, and they are also vulnerable to hydrolysis by bacterial degradation. Another homopolysaccharide is pectin, where the repeating subunits are largely methylgalacturonic acid units. It is a jelly-like material that acts as a cellular cement in plants. The linkage between the subunits is also β1-4 bonds. The carboxyl groups become methylated in a seemingly random

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FIGURE 1.10  Basic tannin structure formed from phenolic units.

FIGURE 1.11  (A) Basic flavonoid carbon structure and (B) flavonoid structure production: Carbons 5 to 8 are derived from the malonate pathway and 2 to 4 and 1′ to 6′ are derived from the shikimic acid pathway via the amino acid phenylalanine. Carbons 2 to 4 comprise the 3-carbon bridge.

manner as fruit ripen. Chemically related to pectin is chitin. Chitin is not a plant polysaccharide but is found within the animal kingdom, although not necessarily in humans. It is a β1-4 homopolymer of N-acetyl-glucosamine found in shells or exoskeletons of insects and crustacea. Chitin has been positioned as a dietary ingredient for weight loss. Another family of polysaccharides that is worthy of discussion is glycosaminoglycans (GAGs). While these compounds are found in animal connective tissue, they are important to this discussion,

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as they are potential components of functional foods. At present, GAG and chondroitin sulfate are popular nutrition supplements being used by individuals recovering from joint injuries and suffering joint inflammatory disorders. Glycosaminoglycans are often referred to as mucopolysaccharides. They are characterized by their content of amino sugars and uronic acids, which occur in combination with proteins in secretions and structures. GAGs are responsible for the viscosity of body-mucus secretions and are components of extracellular amorphous ground substances surrounding collagen and elastin fibers, and cells of connective tissues and bone. Some examples of glycosaminoglycans are hyaluronic acid and chondroitin sulfate. Hyaluronic acid is a component of the ground substance found in most connective tissue, including the synovial fluid of joints. It is a jelly-like substance composed of repeating disaccharides of β-glucuronic acid and N-acetyl-d-glucosamine. Hyaluronic acid can contain several thousand disaccharide residues and is unique from the other glycosaminoglycans in that it will not interact with proteins to form proteoglycans. Chondroitin sulfate is composed of β-glucuronic acid and N-acetylgalactosamine sulfate. This molecule has a relatively high viscosity and ability to bind water. It is the major organic component of the ground substance of cartilage and bone. Both polysaccharides have β1-3 linkage between uronic acid and acetylated amino sugars but are linked by β1-4 covalent bonds to other polysaccharide units. Unlike hyaluronic acid, chondroitin sulfate will bind to proteins to form proteoglycans.

1.7.4  Fatty Acids and Structural Lipids At present, there are several fatty acids and/or their derivatives that have piqued the interests of researchers for their functional potential. These include the omega-3 PUFA found in higher concentrations in plants, fish, and other marine animals, and conjugated linoleic acid(CLA) produced by bacteria in the rumen of grazing animals such as cattle. The formation of CLA probably serves to help control the vitality of the released bacterial population in the rumen, whereas plants and fish use omega-3 fatty acids for their properties in membranes. Some plants also use omega-3 PUFA in a second messenger system to form jasmonic acid when plant tissue is under attack (i.e., by insect feeding). The CLA precursor, linoleic acid, and omega-3 PUFA are produced largely in plants. In processes very similar to those found in humans, plants construct fatty acids using two-carbon units derived from acetyl CoA. In humans and other animals, the reactions involved in fatty acid synthesis occur in the cytosol, whereas in plants they occur in the plastids. In both situations, FAS, acetyl CoA carboxylase enzymes, and acyl carrier protein (ACP) are major players. Plants primarily produce fatty acids to become components of triglycerides in energy stores (oils), as well as components of cell membrane glycerophospholipids and glyceroglycolipids, which serve roles similar to the phospholipids in humans. In fact, several of the plant glycerophospholipids are generally the same as phospholipids. Some of the major fatty acids produced include palmitic acid (16:0), oleic acid (18:1ω-9), linoleic acid (18:2ω-6), and linolenic acid (18:3ω-3). Grazing animals ingest linoleic acid, which is then metabolized to CLA by rumen bacteria. Herbivorous fish also ingest these fatty acids when they consume algae and other seaweeds and phytoplankton. Carnivorous fish and marine animals then acquire these PUFA and derivatives from the tissue of other fish and marine life. Fish will further metabolize the PUFA to produce longer and more unsaturated fatty acids such as DHA (docosahexaenoic acid, 22:6ω-3) and EPA (eicosapentaenoic acid, 20:5ω-3). The elongation and further unsaturation yield cell-membrane fatty acids more appropriately suited for colder temperatures and higher hydrostatic pressures, usually associated with deeper water environments. CLA is distinct from typical linoleic acid in that CLA is not necessarily a single structure. There seem to be as many as nine different isomers of CLA. However, the primary forms are mainly 9-cis, 11-trans, and 10-trans, 12-cis. From these positions, it is clear that the locations of the double bonds are unique. The double bonds are conjugated and not interrupted by methylene. Said another way, the double bonds are not separated by a saturated carbon but are adjacent. CLA is found mostly in the fat and milk of ruminant animals, which indicates that beef, dairy foods, and lamb are major dietary sources.

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Two other types of lipids in food products are structured lipids and diglycerides. Structured lipids are triglycerides that have undergone hydrolysis and re-esterification under conditions that resulted in triglycerides with new combinations of fatty acids. For example, a mixture of mediumchain triglycerides and fish oil taken through this process results in triglycerides that can contain medium-chain fatty acids and EPA, and DHA. The basic process results in the free fatty acids being randomly re-esterified to the glycerol backbones. However, the process can be manipulated to place specific fatty acids in preferred positions on the glycerol molecule. This option is quite expensive and thus has not been adopted by the food industry to any degree. However, the random re-esterification process has been used to produce structured triglycerides designed to facilitate the absorption of both medium-chain and long-chain omega-3 fatty acids. Diglycerides have been used as emulsifying agents in manufactured food products for many years. More recently, more specialized diglycerides, termed diacylglycerols (DAGs), have been produced by limited hydrolysis of triglycerides. This process results in a mixture of 1,2-diglycerides and 1,3-diglycerides. These diglycerides have absorption and metabolism characteristics like those of medium-chain triglycerides; that is, some of the fatty acids escape re-esterification within the cells of the small intestine and subsequent delivery to adipose tissue via the lymphatic system. Instead, they are delivered to the liver, where they are oxidized to produce energy and possibly to produce ketones. The result is an apparent caloric content that is somewhat less than the 9 kcal/g associated with most fats.

1.7.5 Amino Acid –Based This group has the potential to include intact protein (i.e., soy protein), polypeptides, amino acids, and nitrogenous and sulfur amino acid derivatives. Today, a few amino acids are also being investigated for their nutraceutical potential. Among these amino acids are arginine, ornithine, taurine, and aspartic acid. Arginine has been speculated to be cardioprotective in that it is a precursor molecule for the vasodilating substance nitric oxide (NO). Also, arginine may reduce atherogenesis. The non-proteogenic amino acid taurine may have blood pressure–lowering properties as well as antioxidant roles. However, the research in these areas is still inconclusive, and the effects of supplementation of these amino acids on other aspects of human physiology is unclear. Several plant molecules are formed via amino acids. A few of the most striking examples are isothiocyanates, indole3-carbinol, allyl sulfur compounds, and capsaicinoids. Another nutraceutical amino acid–derived molecule is folic acid, which is believed to be cardioprotective in its role of minimizing homocysteine levels. Other members of this group would include the tripeptide glutathione and choline. Several amino acid–derived molecules have nutraceutical potential as well. These include creatine, which is derived from three amino acids and found in meats. Creatine is an important short-term anaerobic energy reserve in muscle tissue, brain, kidneys, and other tissue. Higher intakes via supplementation can support gains and/or retention of strength and power and provide cellular protection during transient anoxia. In addition, carnitine might have application in supporting healthier blood glucose and lipid levels.

1.7.6 Microbes (Probiotics) Where the other groupings of nutraceuticals involve molecules or elements, probiotics involve intact microorganisms. This group largely includes bacteria, and its criteria are that a microbe must be resistant to: Acid conditions of the stomach, bile, and digestive enzymes normally found in the human gastrointestinal tract; able to colonize the human intestine; be safe for human consumption; and, lastly, have scientifically proven efficacy. Among the bacterial species recognized as having functional food potential are Lactobacillus acidophilus, L. plantarum, L. casei, Bifidobacterium bifidum, B. infantis, and Streptococcus salvarius subspecies thermophilus. Some yeasts have been noted as well, including Saccharomyces boulardii.

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1.7.7 Minerals Several minerals have been recognized for their nutraceutical potential and thus become candidates for functional food recipes. Among the most obvious is calcium with relation to bone health, colon cancer, and perhaps hypertension and cardiovascular disease. Potassium has also been purported to reduce hypertension and thus improve cardiovascular health. A couple of trace minerals have also been found to have nutraceutical potential. These include copper, selenium, manganese, and zinc. Their nutraceutical potential is usually discussed in relation to antioxidation. Copper, zinc, and manganese are components of superoxide dismutase (SOD) enzymes, whereas selenium is a component of glutathione peroxidase. Certainly, more investigation is required in trace elements considering their metabolic relationships to other nutrients and the potential for toxicity.

REFERENCES 1. World Health Organization Life Expectancy. http://www.who.int/gho/mortality_burden_disease/ life_tables/situation_trends/en/ 2. WHO | Obesity and overweight—World Health Organization—www.who.int/mediacentre/factsheets/ fs311/en/ 3. #stateofobesity http://stateofobesity.org/adult-obesity/ 4. Kalra, E.K. Nutraceutical—Definition and Introduction. AAPS PharmSci., 2003; 5: Article 25 (found at http://www.aapsj.org/). 5. DeFelice, S.L. What Is a True Nutraceutical? And What Is the Nature and Size of the U.S. Market? 1994, http://www.fimdefelice.org/archives/arc.whatisnut.html. 6. International Food Information Council Web site, http://ific.org/nutrition/functional/index.cfm, 2006. 7. International Life Sciences Institute of North America Web site, http://www.ilsi.org/, 2006. 8. Health Canada, http://www.hc-sc.gc.ca, 2006. 9. Sports Nutrition and Weight Management Report © 2018 Informa. https://www.nutritionbusinessjournal. com/reports/2018-sports-nutrition-and-weight-management-report/ 10. Academy of Nutrition & Dietetics. Position of the Academy of Nutrition and Dietetics: Functional Foods. J Acad. Nutr. Diet, 113: 1096–1103, 2013. 11. U.S. Food and Drug Administration. Center for Food Safety and Applied Nutrition, A Food Labeling Guide, http://www.cfsan.fda.gov. 12. Miller, E.G., Gonzales-Sanders, A.P., Couvillon, A.M., Binnie, W.H., Hasegawa, S., and Lam, L.K.T. Citrus liminoids as inhibitors of oral carcinogenesis. Food Technol., 48: 110–114, 1994. 13. Fong, C.H., Hasegawa, S., Herman, Z., and Ou, P. Liminoid glucosides in commercial citrus juices, J. Food Sci., 54: 1505–1506, 1990. 14. Yu, R., Yamada, A., Watanabe, K., Yazawa, K., Takeyama, H., Matsunaga, T., and Kurane, R., Production of eicosapentaenoic acid by a recombinant marine cyanobacterium, Synechococcus sp. Lipids, 35(10): 1061–4, 2000. 15. Duan L., Guo L., Wang L., Yin Q., Zhang C.M., Zheng Y.G., and Liu E.H. Application of metabolomics in toxicity evaluation of traditional Chinese medicines. Chin. Med., 13: 60, 2018 Dec 4. 16. Shaw, D., Leon, C., Kolev, S., and Murray, V., Traditional remedies and food supplements: A 5-year toxicological study (1991–1995), Drug Saf., 17: 342–56, 1997. 17. Taiz, L. and Zeiger, E., Plant defenses, in Plant Physiology, 2nd ed., Sinauer Associates, Sunderland, MA, 1998. 18. Wattenberg, L.W. and Lam, L.K.T., Protective effects of coffee constituents on carcinogenesis in experimental animals, Banbury Rep., 17: 137–145, 1984. 19. Miller, E.G., McWhorter, K., Rivera-Hidalgo, F., Wright, J.M., Hirsbrunner, P., and Sunahara, G.I., Kahweol and cafestol: Inhibitors of hamster buccal pouch carcinogenesis, Nutr. Cancer, 15: 41–46, 1991. 20. Miller, E.G., Gonzalez-Sanders, A.P., Couvillon, A.M., Binnie, W.H., Hasegawa, S., and Lam, L.K.T., Citrus limonoids as inhibitors of oral carcinogenesis, Food Technol., 110–114, 1994. 21. Hasegawa, S., Bennet, R.D., Herman, Z., Fong, C.H., and Ou, P., Limonoids glucosides in citrus, Phytochemistry, 28: 1717–1720, 1989. 22. Chang, M.S., Lee, S.G., and Rho, H.M., Transcriptional activation of Cu/Zn superoxide dismutase and catalase genes by panaxadiol ginsenosides extracted from Panax ginseng, Phytother. Res., 13(8): 641–644, 1999. 23. Lee, S.J., Sung, J.H., Lee, S.J., Moon, C.K., and Lee, B.H., Antitumor activity of a novel ginseng saponin metabolite in human pulmonary adenocarcinoma cells resistant to cisplatin, Cancer Lett., 144(1): 39–43, 1999.

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Regulation of Nutraceuticals and Functional Foods Rick Collins, Esq., Jay Manfre, Esq., and Robert E.C. Wildman

CONTENTS 2.1 Introduction............................................................................................................................. 23 2.2 Food & Drug Administration, Food, vs Dietary Supplements................................................24 2.2.1 History of the Food & Drug Administration and Dietary Supplements.....................24 2.2.2 Dietary Supplement Health and Education Act...........................................................24 2.3 Nutrition Product Labeling......................................................................................................26 2.3.1 Nutritional Labeling and Education Act......................................................................26 2.3.2 Marketing Claims........................................................................................................28 2.3.3 Structure Function and Benefit Claims....................................................................... 29 2.4 Manufacturing and Ingredients............................................................................................... 29 2.4.1 Good Manufacturing Practices.................................................................................... 29 2.4.2 New Dietary Ingredients............................................................................................. 29 2.4.3 Intellectual Property and Patents and Novel Ingredients............................................ 30 2.4.4 Product Testing Programs........................................................................................... 31 2.5 Product Safety.......................................................................................................................... 31 2.5.1 Adverse Event Reporting............................................................................................. 31 2.5.2 Adulterated Supplements............................................................................................. 32 2.5.3 A Safer Industry Ahead............................................................................................... 32 References......................................................................................................................................... 33

2.1 INTRODUCTION While the numerous sources of market data are not always in absolute agreement on market size, projections, and opportunities, they have been unified on the continued growth, interest, and general potential of the nutraceutical and functional food market. The notion that foods contain bioactive nutrients that can have immediate and long-term benefits is intriguing and aligns with the words of Hippocrates thousands of years ago—“Let food be thy medicine.” However, how information is communicated overlaps several industry components, including marketing, quality assurance/ control, regulatory, and legal. As outline in Chapter 1, targeting specific intact foods is the focus of building a nutrient-rich dietary platform, the list of nutraceutical endowed foods is overwhelming. Often, the concentration, isolation, and/or extraction of key foods or nutrients garners the most attention. Once the consumable nutritional offering delivery form transitions to pills, concentrated liquids, or powders packaged up and labeled, they become dietary supplements. While the nutritional information for conventional food is expressed on the Nutrition Facts panel, the nutrition offering in dietary supplements is captured in a Supplement Facts panel. Specific rules and regulations apply to dietary supplements and functional foods, as outlined by one or more federal entities like the Food & Drug Administration (FDA) in the United States. Most of the remaining chapter will provide an overview of some of the key regulatory aspects in the functional food and dietary supplement marketplace.

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2.2  FOOD & DRUG ADMINISTRATION, FOOD, VS DIETARY SUPPLEMENTS 2.2.1 History of the Food & Drug Administration and Dietary Supplements In the United States, the FDA oversees a vast number of activities and products, including foods (other than meat and poultry), human and animal drugs, and cosmetics.1 What began in 1862 as the Division of Chemistry (a single chemist in the U.S. Department of Agriculture) has evolved into the FDA as it exists today under the Department of Health and Human Services.1 The first regulation, the Pure Food and Drugs Act, was signed by President Theodore Roosevelt in 1906 and prohibited misbranded and adulterated food and drugs in interstate commerce.2 Although the Pure Food and Drugs Act was a significant step in the right direction, gaps existed in the commodities it covered, many hazardous consumer products remained on the market, and many products were not covered by the Act at all.2 President Franklin Roosevelt was elected in 1930 at a time when journalists and consumer protection organizations were pushing for Congress to replace the 1906 law.3 The catalyst for change came following the death of over 100 people, including many children, in 1937.3 Elixir Sulfanilamide was an untested new sulfa drug that was marketed by a Tennessee drug company.3 The solvent in Elixir Sulfanilamide was a chemical analogue of antifreeze, a highly toxic chemical used in motor vehicles, which can be fatal if consumed.3 On June 25, 1938, President Franklin Roosevelt signed the Food, Drug, and Cosmetic Act (FDCA), which created tighter controls over drugs and food, included new consumer protection provisions, and gave the government greater enforcement ability.4 The FDCA, as amended, continues to be in force to this day.4 Prior to 1994, dietary supplements could only be marketed and labeled the same way as “conventional foods.”5 This meant that the ingredients in dietary supplements had to be “generally recognized as safe” (GRAS) or specifically covered by a food additive regulation.5 During this time, dietary supplement claims were governed by the same standard as foods. As a result, claims made about a dietary supplement’s effect on the healthy structure or function of the body typically caused these supplements to be considered a misbranded or unapproved new drug by the FDA.5 The law was clearly not working from an industry perspective. Thus, the dietary supplement industry lobbied Congress to amend the FDCA as it pertained to dietary supplements.6 In 1994, Congress passed the Dietary Supplement Health and Education Act (DSHEA).4 Although still classifying dietary supplements as a “food” under the FDCA, DSHEA established a clearer and more practical regulatory framework for the regulation of dietary supplements. Among other things, DSHEA created a legal definition for “dietary supplements” and “dietary ingredients,” established specific labeling requirements, and authorized the FDA to establish current Good Manufacturing Practices (cGMP) regulations for supplements.7

2.2.2 Dietary Supplement Health and Education Act In October 1994, President Clinton signed DSHEA into law. The statute was enacted amid claims that the FDA was distorting the then-existing provisions of the FDCA to improperly deprive the public of safe and popular dietary supplement products. DSHEA defines a “dietary supplement” as a product that is intended to supplement the diet and contains one or more “dietary ingredients.” By definition, the “dietary ingredients” in these products may include vitamins; minerals; herbs or other botanicals; amino acids; dietary substances for use by man to supplement the diet by increasing the total dietary intake; and substances such as enzymes, organ tissues, and glandular extracts. Further, dietary ingredients may also include extracts, metabolites, or concentrates of those substances. Vitamins, minerals, and amino acids are defined by their ability to provide nutrients to the human body.8 For this reason, FDA has stated that synthetic versions of vitamins, minerals, and amino acids are considered “dietary ingredients” under DSHEA.8 On the other hand, herbs and botanicals are defined by their state of matter and not by their ability to provide nutrients to the human body. They include plants, algae, fungi, their exudates

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(secretions), and their physical parts.8 FDA has stated that synthetic copies of herbs and botanicals are not considered “dietary ingredients” because these substances have never been part of the herb or botanical in the first place.8 However, a recent decision by the United States Court of Appeals for the Eleventh Circuit holds contrary to FDA’s long-held position regarding synthetic botanicals. In United States of America v. Undetermined Quantities of All Articles of Finished and In-Process Foods, raw ingredients (bulk powders, bulk capsules), with any lot number, size, or type container, whether labeled, et al, the Court stated “If a product is indeed a dietary supplement because it contains a qualifying dietary ingredients – including, for example, an herb or other botanical – a manufacturer may take the dietary ingredient from nature or produce if artificially.”9 (p.14) This is a major decision, as the FDA has sent numerous warning letters to companies claiming that the ingredients contained in the company’s products render the product adulterated because the ingredient is a synthetic botanical. What will happen with this new determination remains to be seen. The most often confused aspect of the definition of a dietary ingredient is the phrase “a dietary substance for use by man to supplement the diet by increasing the total dietary intake.” Does this give the green light for anything to be a dietary ingredient because it can increase the dietary intake above zero? No; because the term “dietary substance” is not defined in the FDCA or other regulation, FDA interprets it according to its common, usual meaning.8 Based on the common, usual meaning of the terms contained in the phrase, FDA interprets this subsection to mean “food and food components that humans eat as part of their usual diet.”8 FDA makes clear that one cannot increase the “total dietary intake” of something that is not part of the human diet in the first place.8 Dietary supplements may be found in many forms, such as tablets, capsules, softgels, gelcaps, liquids, or powders, but may only be intended for oral ingestion. Dietary supplements cannot be marketed or promoted for sublingual, intranasal, transdermal, injectable, or any other route of administration except oral ingestion. For this reason, a dietary supplement that states on its label, “place drops under the tongue” (suggesting absorption through the mucosa) would be considered misbranded by the FDA. However, a dietary supplement that states on its label “place drops under the tongue and swallow” (suggesting absorption through the gastrointestinal tract) would likely be an acceptable method of delivery for a dietary supplement. A supplement can be found in other forms, including those that mirror “conventional foods” such as a bar or shake, as long as the information on its label does not represent the product as a conventional food or a sole item of a meal or diet. Whichever form the dietary supplement takes, the labeling and marketing must make clear that the product is not intended to be a meal by itself or used to replace a meal.10 One of the best examples of this is in the form of a bar. Protein bars can be found in almost every grocery store, supplement shop, and even convenience stores across America. What makes one bar a supplement while another is a food? Certainly, there are several factors that go into this determination, such as whether it is labeled with a Supplement Facts panel or a Nutrition Facts Panel and which ingredients are used. However, for the purposes of the definition of a dietary supplement under DSHEA, the determination of whether the bar is a food or a supplement comes down to the intended use of the product. If the bar is intended to be eaten in place of a meal or as a snack in between meals, then that bar is a conventional food and should be labeled accordingly. If the bar is intended to be consumed to provide an extra source of protein to the diet and is not marketed to be eaten in place of a meal, then the bar would be a supplement. Finally, DSHEA provides two sections about what a dietary supplement does, and does not, include.11 First, DSHEA states that a dietary supplement does include “an article that is approved as a new drug … certified as an antibiotic … or licensed as a biologic” if the article was marked as a dietary supplement or as a food prior to such approval, certification, or license.11 A perfect example of this is in the case of fish oil. Fish oil supplements have been marketed and sold in the United States for centuries, long before the passage of DSHEA. In the early 2000s, Reliant Pharmaceuticals developed a highly purified, chemically altered version of omega-3-acid ethyl esters that was put through the FDA drug approval process.12 The FDA-approved prescription fish oil Lovaza was sold by GlaxoSmithKline for the treatment of very high triglycerides.12 Because omega-3 fish oils were

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marketed and sold as dietary supplements prior to Lovaza’s approval as a drug, they are still able to be legally marketed and sold as dietary supplements notwithstanding the new drug approval. The same is true for other products that were marketed and sold as dietary supplements prior to their approval as drugs—vitamin D is available as a dietary supplement and as a prescription drug called Drisdol. Second, this section of DSHEA explains that a dietary supplement does not include “an article authorized for investigation as a new drug, antibiotic, or biological for which substantial clinical investigations have been instituted and for which the existence of such investigations has been made public” if the article was not marketed as a dietary supplement or food prior to such authorization.13 This means that if a pharmaceutical company conducts substantial clinical investigations for a substance, and the public is made aware of these investigations (typically through press releases from the company), then that substance cannot be a dietary supplement unless it was marketed as such prior to the investigations. This is one of the major obstacles being faced by companies that are selling cannabidiol (CBD) as a dietary supplement. The pharmaceutical company GW Pharmaceuticals began conducting substantial clinical investigations that were made public on its CBD drug Epidiolex in 2007. FDA has opined that CBD was not sold as a dietary supplement prior to GW Pharmaceuticals’ clinical investigations, and as a result is not a legal dietary supplement. Companies that market and sell CBD argue that CBD was sold as a dietary supplement prior to GW Pharmaceutical’s clinical investigations; however, at this point, FDA has expressed that there has not been evidence to validate the truth of that argument. Contrary to widespread mainstream media claims, DSHEA did not leave the industry unregulated. The dietary supplement industry is in fact regulated by the FDA as a direct result of DSHEA. The law ensures the authority of the FDA to provide legitimate protections for the public health. In addition to the FDA, the Federal Trade Commission (FTC) has jurisdiction over the marketing claims that dietary supplement manufacturers or companies make about their products. The FDA and FTC operate in a cooperative fashion to regulate the dietary supplement industry. In this respect, the extent to which information is shared and jurisdiction between these two entities overlaps with regard to the marketing and advertising of dietary supplements continues to increase.

2.3  NUTRITION PRODUCT LABELING 2.3.1 Nutritional Labeling and Education Act The Nutritional Labeling and Education Act (NLEA) of 1990 amended the FDCA to give the FDA authority to require nutrition labeling on most food packages, including dietary supplements.14 DSHEA further expanded on this in part by defining the term “dietary supplement” but also by requiring specific labeling requirements for dietary supplements.14 The labeling requirements for both foods and dietary supplements can be found in 21 C.F.R. 101. Labeling accuracy is important, and claims regarding nutrient levels must meet certain guidelines. In the U.S., the FDA classifies nutrients that are declared on Nutrient Facts and Supplement Facts as Class I or II as follows: • Class I Nutrients—Nutrients that are specifically added to food (e.g., fortified food) to increase its nutritional value or formulated as part of a dietary supplement. These nutrients include added vitamins, minerals, and fiber, as well as nutraceutical nutrients such as amino acids (e.g., taurine, citrulline, caffeine, plant extracts, etc.). Class I nutrients must be present at 100% or more of the value declared on the label all the way to the end of a product’s expiration date. Class I nutrients would include vitamins and minerals added to a breakfast cereal as well as nutrients formulated into a multivitamin/mineral supplement. In addition, if a specific nutrient is called out in the Statement of Identity on the front of the package (e.g., protein powder or protein bar), then the protein listed in the Nutrition Facts panel

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becomes a Class I nutrient. The same is true if a product makes a nutrient content claim somewhere on the package/label (e.g., “10 grams of protein”) in a food not commonly assumed to deliver that nutrient in significant amounts: then in this case protein could be classified as a Class I nutrient as well. • Class II Nutrients—Nutrients that are naturally occurring in an intact/near intact food (e.g., fruits, vegetables, oatmeal) or recipe foods (e.g., baked goods, peanut butter) can vary in concentration for reasons that cannot be controlled or predicted easily. Examples would be vitamin C in picked oranges or calcium in whole milk. Class II nutrients must be present at 80% or more of the value declared on the label. • Third Group Nutrients—In addition, specific nutrients of health-related interest are grouped together as a “third group,” which cannot exceed 120% of label claim. Third group nutrients include calories, sugars, total fat, saturated fat, cholesterol, and sodium. Five statements are required on the containers and packages of both dietary supplements and foods. These statements include: (1) the statement of identity; (2) the net quantity of contents; (3) the nutrition labeling; (4) the ingredient list; and (5) the name and place of business of the manufacturer, packer, or distributor.14 The statement of identity is the name of the dietary supplement or food product and must be placed on the principal display panel (the panel that faces the consumer). The statement of identity of a food, including dietary supplements, is the name specified by federal law or regulation, or the common or usual name of the food.15 For dietary supplements, the regulations specify that the statement of identity must include the term “dietary supplement”; however, the word “dietary” may be replaced with a description of the type of dietary ingredients in the product.15 For example, the statement of identity can be “protein supplement,” in the case of a protein powder that is marketed and sold as a dietary supplement. The net quantity of contents statement is located on the principal display panel and informs consumers of the amount of product that is in the container or package.16 The net quantity of contents statement can be expressed in weight, measure, numerical count, or a combination of numerical count and weight or measure.16 When expressed in weight or measure, the net quantity of contents statement must specify both metric and U.S. Customary System terms, with the U.S. terms listed first and the metric terms listed parenthetically.16 In the case of a dietary supplement marketed in the form of tablets, the net quantity of contents can be stated as the number of pills in the container (e.g., 60 tablets). In the case of a powdered dietary supplement, the net quantity of contents can be stated as the weight of the powder in the container (e.g., 3 LB [1.36 KG]). The nutrition label for a dietary supplement is called a “Supplement Facts” panel, while the nutrition label for conventional foods is called a “Nutrition Facts” panel.17 There are specific differences between a Supplement Facts panel and a Nutrition Facts panel. The key difference is that the Nutrition Facts panel for conventional foods cannot list nutrients for which the FDA has not determined a Recommended Daily Value (RDV), while the Supplement Facts panel for dietary supplements must list these nutrients. For example, while red meat contains creatine, the Nutrition Facts panel of a steak cannot list the amount of creatine it contains. A post-workout recovery powder that contains creatine as an active ingredient within the product would list the amount of creatine in the Supplement Facts panel. The ingredient list for dietary supplements and conventional foods is located directly underneath the Supplement or Nutrition Facts panel. The major difference between the ingredient labeling of dietary supplements as compared to conventional foods is that sources of dietary ingredients may be listed within the “Supplement Facts” panel itself.18 For example, in a dietary supplement containing calcium, calcium (as calcium carbonate) can be listed directly in the Supplement Facts panel. When listed in this manner, “calcium carbonate” would not be listed in the ingredient list. On the other hand, conventional foods are not permitted to list the source ingredients in the “Nutrition Facts” panel. Instead, conventional foods list all the ingredients contained in the product in the ingredient list underneath the Nutrition Facts panel.

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Finally, the name and place of business of the manufacturer, packer, or distributor must be placed on the principal display panel or the information panel (the panel to the right of the principal display panel).19 When the food or dietary supplement is not manufactured by the person, or company, whose name appears on the label, the name must be qualified by a phrase that reveals the connection between the person and the food or supplement.20 This could be stated as, “Manufactured for John’s Supplements,” “Distributed by John’s Supplements,” or another phrase that expresses the facts.20 The statement of the place of business includes the street address, city, state, and ZIP code, except that the street address can be omitted if it is shown in a current city directory or telephone directory.20 While the five statements above are mandatory for all foods, including dietary supplements, there are additional statements that may be required depending on certain circumstances. As explained below, for dietary supplements, the “FDA Disclaimer” is required if the product bears any “structure function claims” on its label.21 This disclaimer must be placed in a box either immediately adjacent to the claim with no “intervening material,” or elsewhere on the same panel or page that bears the statement. If the disclaimer is placed on another panel, it must be linked by a symbol (such as an asterisk) that refers to the disclaimer.21 For foods and dietary supplements that contain a “major food allergen,” the package or container must disclose the presence of the major food allergen. The Food Allergen Labeling and Consumer Protection Act of 2004 (FALCPA) requires that food labels must clearly identify the food source names of any ingredients that are one of the major food allergens. The eight major food allergens designated by FALCPA are milk, eggs, fish, crustacean shellfish, tree nuts, peanuts, wheat, and soybeans. Contrary to what is typically seen on food labels, the statute does not require a company to provide a warning about possible cross-contamination. This means that statements about a product being produced on “shared equipment” or “in a facility that also manufactures nuts” are not required.

2.3.2 Marketing Claims According to the 1990 Nutrition Labeling and Education Act, the FDA can review and approve health claims (claims describing the relationship between a food substance and a reduced risk of a disease or health-related condition) for dietary ingredients and foods. However, since the law was passed, the FDA has only approved a few health claims. The delay in reviewing health claims of dietary supplement ingredients resulted in a lawsuit, Pearson v. Shalala, filed in 1995. After years of litigation, in 1999, the U.S. Court of Appeals for the District of Columbia Circuit ruled that qualified health claims may be made about dietary supplements with approval by the FDA, as long as the statements are truthful and based on adequate science. Supplement or food companies wishing to make health claims or qualified health claims about supplements can submit research evidence to the FDA for review. The FTC also regulates the supplement industry, specifically in regard to truth in advertising. Unsubstantiated claims invite enforcement by the FTC (along with the FDA, state district attorney offices, groups like the Better Business Bureau, and plaintiff’s lawyers who file class action lawsuits). The FTC has typically applied a substantiation standard of “competent and reliable scientific evidence” to claims about the benefits and safety of dietary supplements. FTC case law defines “competent and reliable scientific evidence” as “tests, analyses, research, studies, or other evidence based on the expertise of professionals in the relevant area, that has been conducted and evaluated in an objective manner by persons qualified to do so, using procedures generally accepted in the profession to yield accurate and reliable results.” The FTC has claimed that this involves providing at least two clinical trials showing efficacy of the actual product, within a population of subjects relevant to the target market, supporting the structure/ function claims that are made. While the exact requirements are still evolving, the FTC has acted against several supplement companies for misleading advertisements and/or structure/ function claims.

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2.3.3 Structure Function and Benefit Claims In the United States, dietary supplements are classified as food products, not drugs, and there is generally no mandate to register products with the FDA or obtain FDA approval before producing or selling supplements to consumers. However, if a dietary supplement manufacturer makes claims about their product, the company must submit the claims to FDA within 30 days of marketing the product. Compare this, for example, with Canada, where under the Natural Health Product (NHP) Regulations enacted in 2004, supplements must be reviewed, approved, and registered with Health Canada. The rationale for the U.S. model is based on a presumed long history of safe use; hence, there is no need to require additional safety data. DSHEA also requires supplement marketers to include on any label displaying structure/ function claims (i.e., claims that the product affects the structure or function of the body) the mandatory FDA disclaimer “This statement has not been evaluated by the Food and Drug Administration. This product is not intended to diagnose, treat, cure, or prevent any disease.” Opponents of dietary supplements often cite this statement as evidence that the FDA does not review or approve dietary supplements. However, most dietary ingredients have been “grandfathered in” as DSHEA-compliant ingredients due to a long history of safe use, and those products containing new ingredients must generally be submitted by a notification to the FDA for a safety review prior to being brought to market.

2.4  MANUFACTURING AND INGREDIENTS 2.4.1 Good Manufacturing Practices When DSHEA was passed in 1994, it contained a provision requiring that the FDA establish and enforce current Good Manufacturing Practices (cGMPs) for dietary supplements. However, it was not until 2007 that the cGMPs were finally approved, and not until 2010 that the cGMPs applied across the industry, to large and small companies alike. The adherence to cGMPs has helped protect against contamination issues and should serve to improve consumer confidence in dietary supplements. The market improved as companies became compliant with cGMPs, as these regulations imposed more stringent requirements such as Vendor Certification, Document Control Procedures, and Identity Testing. These compliance criteria addressed the problems that had damaged the reputation of the industry with a focus on quality control, record keeping, and documentation. However, it does appear that some within the industry continue to struggle with compliance. In fiscal year 2017, it was reported that approximately 23.48% of the FDA’s 656 total cGMP inspections resulted in citations for failing to establish specifications for the identity, purity, strength, and composition of dietary supplements. Further, 18.47% of those inspected were cited for failing to establish and/or follow written procedures for quality control operations. Undoubtedly, relying on certificates of analysis from the raw material supplier without further testing, or failing to conduct identity testing of a finished product, can result in the creation of a product that fails to contain the proper amounts of ingredients it should, or contains something it should not contain, such as synthetic chemicals or even pharmaceutical drugs. All members of the industry need to ensure compliance with cGMPs.

2.4.2 New Dietary Ingredients Recognizing that new and untested dietary supplement products may pose unknown health issues, DSHEA distinguishes between products containing dietary ingredients that were already on the market and products containing new dietary ingredients (NDIs) that were not marketed prior to the enactment of the law. A “new dietary ingredient” is defined as a dietary ingredient that was not marketed in the United States before October 15, 1994. DSHEA grants the FDA greater control over

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supplements containing NDIs. A product containing an NDI is deemed adulterated and subject to FDA enforcement sanctions unless it meets one of two exemption criteria: Either (1) the supplement in question contains “only dietary ingredients which have been present in the food supply as an article used for food in a form in which the food has not been chemically altered”; or (2) there is a “history of use or other evidence of safety” provided by the manufacturer or distributor to the FDA at least 75 days before introducing the product into interstate commerce. The first criterion was silent in the law as to how and by whom presence in the food supply as food articles without chemical alteration is to be established. The second criterion—applicable only to new dietary ingredients that have not been present in the food supply—requires manufacturers and distributors of the product to take certain actions. Those actions include submitting, at least 75 days before the product is introduced into interstate commerce, information that is the basis on which a product containing the new dietary ingredient is “reasonably expected to be safe.” That information would include: (1) the name of the new dietary ingredient and, if it is an herb or botanical, the Latin binomial name; (2) a description of the dietary supplement that contains the new dietary ingredient, including (a) the level of the new dietary ingredient in the product, (b) conditions of use of the product stated in the labeling, or if no conditions of use are stated, the ordinary conditions of use, and (c) a history of use or other evidence of safety establishing that the dietary ingredient, when used under the conditions recommended or suggested in the labeling of the dietary supplement, is reasonably expected to be safe. In July 2011, the FDA released a Draft Guidance for Industry, entitled “Dietary Supplements: New Dietary Ingredient Notifications and Related Issues.” While a guidance does not carry the authority or the enforceability of a law or regulation, the FDA’s NDI draft guidance represented the agency’s current thinking on the topic. The guidance prompted great controversy, and FDA agreed to issue a revised draft guidance to address some of the issues raised by industry. In August 2016, FDA released a revised Draft Guidance that replaced the 2011 Draft Guidance. The purpose of the 2016 Draft Guidance was to help manufacturers and distributors decide whether to submit a premarket safety notification to FDA, help prepare NDI notifications in a manner that allows FDA to review and respond more efficiently and quickly, and improve the quality of NDI notifications. The 2016 Draft Guidance has been criticized by industry and trade associations for its lack of clarity and other problems. Some of these issues include the lack of clarity regarding pre-DSHEA (grandfathered) ingredients and FDA requiring an NDI notification even if another manufacturer has submitted a notification for the same NDI. The lack of clarity surrounding the “new” Draft Guidance has led to many NDI notifications being rejected by FDA for lack of safety data and other issues. Other companies have opted to utilize the “Self-Affirmed GRAS” route in order to “bypass” the NDI notification process. SelfAffirmed GRAS is a process in which a company engages a team of scientific experts to evaluate the safety of their ingredient. There is no requirement that the safety dossier be submitted to FDA, but it is retained by the company as an internal document that may be relied upon if the ingredient is challenged by the FDA. The FDA has expressed its concern with this practice and does not encourage dietary supplement manufacturers to use Self-Affirmed GRAS to avoid submitting NDI notifications. In any event, the likelihood of another revised Draft Guidance from the FDA becoming available in the future is high, and possibly more enforcement actions taken against companies that market an NDI without submitting a notification.

2.4.3  Intellectual Property and Patents and Novel Ingredients Companies can develop and pursue patents involving new processing and purification processes if the nutrient has not yet been extracted in a pure form or is not available in large quantities. Reputable raw material manufacturers conduct extensive tests to examine the purity of their raw ingredients. When working on a new ingredient, companies often conduct a series of toxicity studies on the new nutrient once a purified source has been identified. The company would then compile a safety dossier and communicate it to the FDA as a New Dietary Ingredient submission, with the hopes of it being allowed for lawful sale in the United States.

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When a powdered formulation is designed, the list of ingredients and raw materials is typically sent to a flavoring house and packaging company to identify the best way to flavor and package the supplement. In the nutrition industry, several main flavoring houses and packaging companies exist who provide these services for supplement companies. Most reputable dietary supplement manufacturers submit their production facilities to inspection from the FDA and adhere to cGMPs, which represent industry standards for good manufacturing of dietary supplements.

2.4.4 Product Testing Programs Some companies also submit their products for independent testing by third-party companies to certify that their products meet label claims. The certification services offered by these companies may include product testing, GMP inspections, ongoing monitoring, and use of branded markings indicating the products comply with inspection standards and screening for contaminants. More recently, companies have subjected their products for testing by third-party companies to inspect for banned or unwanted substances (e.g., Banned Substances Control Group, Informed Choice, NSF International, etc.). These types of tests help ensure dietary supplements made available to athletes do not contain substances banned by the International Olympic Committee, the World Anti-Doping Agency, or other athletic governing bodies (e.g., NFL, NCAA, MLB, NHL, etc.). While third-party testing does not guarantee that a supplement is void of banned substances, the likelihood is reduced. Moreover, consumers can request copies of the results of these tests, and each product that has gone through testing and earned certification can be researched online to help athletes, coaches, and support staff understand which products best meet their needs. In many situations, companies who are not willing to provide copies of test results or certificates of analysis should be viewed with caution, particularly for individuals whose eligibility to participate in athletics and employment might be compromised if a tainted product is consumed.

2.5  PRODUCT SAFETY 2.5.1 Adverse Event Reporting In response to growing criticism of the dietary supplement industry, the 109th Congress passed the first mandatory Adverse Event Reporting (AER) legislation for the dietary supplement industry. In December 2006, President Bush signed into law the Dietary Supplement and Nonprescription Drug Consumer Protection Act, which took effect on December 22, 2007. After much debate in Congress and input from the FDA, the American Medical Association (AMA), many of the major supplement trade associations, and a host of others all agreed that the legislation was necessary, and the final version was approved by all. In short, the Act requires that all “serious adverse events” regarding dietary supplements be reported to the Secretary of Health and Human Services. The law strengthened the regulatory structure for dietary supplements and built greater consumer confidence, as consumers have a right to expect that if they report a serious adverse event to a dietary supplement marketer, the FDA will be advised about it. An adverse event is any health-related event associated with the use of a dietary supplement that is adverse. A serious adverse event is an adverse event which (A) results in (i) death, (ii) a life-threatening experience, (iii) inpatient hospitalization, (iv) a persistent or significant disability or incapacity, or (v) a congenital anomaly or birth defect; or (B) requires, based on reasonable medical judgment, a medical or surgical intervention to prevent an outcome described under subparagraph (A). Once it is determined that a serious adverse event has occurred, the manufacturer, packer, or distributor (responsible person) of a dietary supplement whose name appears on the label of the supplement shall submit to the Secretary of Health and Human Services any report received of the serious adverse event accompanied by a copy of the label on or within the retail packaging of the dietary supplement. The responsible person has 15 business days to submit the report to the FDA after being notified of

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the serious adverse event. Following the initial report, the responsible person must submit follow-up reports of new medical information that they receive for 1 year. Although many dietary ingredients have been introduced into dietary supplements since October 1994 and have not been submitted to the FDA for a safety review, nutritional supplementation appears generally safe, especially when compared to prescription drugs. While there are over 50,000 dietary supplements registered with the Office of Dietary Supplement’s “Dietary Supplement Label Database,” a 2013 Annual Report (released in 2015) of the American Association of Poison Control Centers revealed zero fatalities occurred due to dietary supplements compared to 1692 deaths due to drugs. A 2015 report by the Centers for Disease Control alarmingly suggests that 2,287,273 emergency room visits were due to prescription drug-related events—dwarfing the 3266 emergency room visits due to dietary supplements (adjusted from 23,000 visits after excluding cases of older adults choking on pills, allergic reactions, unsupervised children consuming too many vitamins, and persons consuming ingredients not defined by DSHEA as a dietary supplement).22

2.5.2 Adulterated Supplements The FDA has various options to protect consumers from unsafe supplements. The Secretary of the Department of Health and Human Services (which falls under the FDA’s oversight) has the power to declare a dangerous supplement to be an “imminent hazard” to public health or safety and immediately suspend sales of the product. The FDA also has the authority to protect consumers from dietary supplements that do not present an imminent hazard to the public but do present certain risks of illness or injury to consumers. The law prohibits introducing adulterated products into interstate commerce. A supplement shall be deemed adulterated if it presents “a significant or unreasonable risk of illness or injury.” The standard does not require proof that consumers have actually been harmed or even that a product will harm anyone. It was under this provision that the FDA concluded that dietary supplements containing ephedra, androstenedione, and 1-3, Dimethylamylamine (DMAA) presented an unreasonable risk. Most recently, the FDA imposed an importation ban on the botanical Mitragyna speciosa, better known as Kratom. In 2016, the FDA issued Import Alert #54-15, which allows for detention without physical examination of dietary supplements and bulk dietary ingredients that are, or contain, Kratom. Criminal penalties are present for a conviction of introducing adulterated supplement products into interstate commerce. While the harms associated with dietary supplements may pale in comparison to those linked to prescription drugs, recent pronouncements from the U.S. Department of Justice confirm that the supplement industry is being watched vigilantly to protect the health and safety of the American public.

2.5.3 A Safer Industry Ahead As demonstrated, while some argue that the dietary supplement industry is “unregulated” and/or may have suggestions for additional regulation, manufacturers and distributors of dietary supplements must adhere to several federal regulations before a product can go to market. The safety of the dietary supplement industry has also been demonstrated in the relative infrequency in recalls. According to data obtained from the FDA and published in the American Herbal Products Association’s August publication, only 2% of more than 800 recalls initiated in 2019 involved dietary supplements.23 Further, before marketing products, manufacturers must have evidence that their supplements are generally safe to meet all the requirements of DSHEA and other FDA regulations. For this reason, over the last 20 years, many established supplement companies have employed research and development directors who help educate the public about nutrition and exercise, provide input on product development, conduct preliminary research on products and ingredients, and/or assist in coordinating research trials conducted by independent research teams (e.g., university-based researchers or clinical research sites). These companies also consult with marketing and legal teams with the responsibility of ensuring that structure/function claims do not misrepresent the results of research findings. This has increased job

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opportunities for sports nutrition specialists as well as enhancing external funding opportunities for research groups interested in exercise and nutrition research. Although some companies have falsely attributed research on different dietary ingredients or dietary supplements to their own products, suppressed negative research findings, and/or exaggerated results from research studies, the overall trend in the sports nutrition industry has been to develop scientifically sound supplements. This trend toward greater research support is the result of: (1) attempts to honestly and accurately inform the public about results, (2) efforts to obtain data to support the safety and efficacy of products for the FDA and the FTC, and/or (3) endeavors to provide scientific evidence to support advertising claims and increase sales. While the push for more research is due in part to greater scrutiny from the FDA and FTC, it is also in response to an increasingly competitive marketplace where established safety and efficacy attracts more consumer loyalty and helps ensure a longer lifespan for the product in commerce. Companies that adhere to these ethical standards tend to prosper, while those that do not will typically struggle to comply with the FDA and FTC guidelines, which results in a loss of consumer confidence and an early demise for the product.

REFERENCES 1. https://www.fda.gov/AboutFDA/WhatWeDo/History/FOrgsHistory/EvolvingPowers/ucm124403.htm 2. https://www.fda.gov/AboutFDA/Transparency/Basics/ucm214416.htm 3. https://www.fda.gov/AboutFDA/WhatWeDo/History/FOrgsHistory/EvolvingPowers/ucm054826.htm 4. https://www.fda.gov/AboutFDA/Transparency/Basics/ucm214416.htm 5. A Practical Guide to FDA’s Food and Drug Law and Regulation, Fifth Edition, Chapter 12—Page 352. 6. https://www.fda.gov/AboutFDA/WhatWeDo/History/FOrgsHistory/EvolvingPowers/ucm125632.htm 7. https://www.fda.gov/downloads/aboutfda/whatwedo/history/productregulation/ucm593507.pdf 8. https://www.fda.gov/downloads/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/ UCM515733.pdf 9. http://media.ca11.uscourts.gov/opinions/pub/files/201713376.pdf 10. https://www.fda.gov/Food/DietarySupplements/UsingDietarySupplements/ucm480069.htm 11. https://www.gpo.gov/fdsys/pkg/STATUTE-108/pdf/STATUTE-108-Pg4325.pdf 12. https://newdrugapprovals.org/tag/lovaza/ 13. https://www.gpo.gov/fdsys/pkg/STATUTE-108/pdf/STATUTE-108-Pg4325.pdf 14. https://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/ DietarySupplements/ucm2006823.htm 15. https://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/ DietarySupplements/ucm070594.htm 16. https://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/ DietarySupplements/ucm070596.htm 17. https://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/ DietarySupplements/ucm070597.htm 18. https://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/ DietarySupplements/ucm070611.htm 19. https://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/ DietarySupplements/ucm070519.htm 20. https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=101.5 21. https://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/ DietarySupplements/ucm070613.htm 22. Brown, A.C. An overview of herb and dietary supplement efficacy, safety and government regulations in the United States with suggested improvements. Part 1 of 5 series. Food. Chem. Toxicol. 2017, 107, 449–471, doi:10.1016/j.fct.2016.11.001. 23. http://www.ahpa.org/Portals/0/PDFs/AHPA_Report_Aug_2019--Recall%20Excerpt.pdf

Unit II Plant-Derived Nutraceuticals

3 Food Sources, Properties, and Lycopene

Effects on Human Health Jessica L. Cooperstone CONTENTS 3.1 Compound Category and Molecular Characteristics.............................................................. 37 3.2 Dietary Sources of Lycopene................................................................................................... 38 3.3 Effects of Food Processing on Lycopene Content and Profile................................................ 39 3.4 Bioavailability, Biological Distribution, and Metabolism of Lycopene..................................40 3.4.1 Bioavailability..............................................................................................................40 3.4.2 Factors Affecting Bioavailability of Lycopene............................................................ 41 3.4.3 Biological Distribution of Lycopene............................................................................ 42 3.4.4 Mammalian Lycopene Metabolism............................................................................. 42 3.5 Lycopene and Chronic Diseases.............................................................................................. 43 3.5.1 Cancer.......................................................................................................................... 43 3.5.2 Heart Disease...............................................................................................................44 3.5.3 Inflammation...............................................................................................................44 3.5.4 Skin and UV-Induced Sun Sensitivity......................................................................... 45 3.6 Conclusions.............................................................................................................................. 45 Acknowledgments............................................................................................................................. 45 References......................................................................................................................................... 45

3.1  COMPOUND CATEGORY AND MOLECULAR CHARACTERISTICS Dozens of studies have examined the relationship between fruit and vegetable intake and cancers, cardiovascular disease, and all-cause mortality.1 The Dietary Guidelines for Americans recommends that half of one’s plates should be fruits and vegetables, and to consume a variety of different types. There are numerous compounds in fruits and vegetables that could individually or synergistically contribute to improvements in human health, of which carotenoids are one. Carotenoids are a class of plant pigments that contribute to the coloring of many fruits and vegetables. For example, lycopene (Figure 3.1) imparts the red color found in tomatoes.2 Lycopene is one of nearly 700 carotenoids reported in nature, though only 40–50 carotenoids are commonly found in the human diet,3 with fewer detectable in human blood plasma.4 Carotenoids are derived from isoprene assembled head to tail, and often contain a series of conjugated double bonds, imparting yellow, orange, and red colors (though there are also some colorless carotenoids).5 Lycopene (C40H56) is a non-provitamin A lipophilic phytochemical. The deep red crystalline pigment was first isolated from Tamus communis (black bryony) in 1873, though at the time, the chemical identity of this purified compound was not known.6 Subsequently, in 1875, a crude mixture containing lycopene was obtained from tomatoes and this pigment was termed “solanorubine.”7 In 1903, the term lycopene was coined, differentiating it from “carotenes” from carrots because of its unique absorption spectrum.

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FIGURE 3.1  Structures of all-trans-lycopene and various cis-isomers.

In the Western diet, lycopene is the most consumed carotenoid as well as the most abundant human plasma.8 It is estimated that approximately 80% of dietary lycopene comes from tomatoes.9 As a result, it can be readily detected in a variety of biological tissues. Continued study of carotenoids,10 and more specifically lycopene,11 has contributed to our understanding of their potential role in human health. However, it is important to note that many studies (both observational and experimental) often conflate lycopene intake with tomato consumption (given that lycopene consumed overwhelmingly comes from tomato products).12 The health benefits associated with tomato consumption have been assumed to be due to the presence of lycopene, though this explicit link has not yet been conclusively demonstrated.

3.2  DIETARY SOURCES OF LYCOPENE In the U.S., it is estimated that lycopene contributes ∼28% of the total carotenoid intake,4 and a systematic review estimated lycopene intake to be ∼4.5 mg/d.8 Most lycopene consumed is from tomato products, though watermelon, pink grapefruit, guava, and papaya contain lycopene as well (Table 3.1).13 Interestingly, because a variety of food and host-dependent factors affect lycopene absorption, lycopene intake and plasma lycopene levels are often not well correlated,8 challenging the interpretation of both observational and interventional studies. Despite lycopene existing predominantly in plants and food in the all-trans configuration, cis configurations are equally prevalent or predominant in blood and tissues of animals and humans consuming lycopene9 (Figure 3.1). This has led to questions about the occurrence and effects of isomerization of lycopene in vivo.14

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TABLE 3.1 Lycopene Content of Select Foods, from the USDA Standard Reference 28 NDB Number 11546 11935 43217 06559 09139 09326 11529 09226 09112

Food Tomato paste Ketchup Tomato sauce Tomato soup Guava, raw Watermelon, raw Tomatoes, ripe, red, raw, year-round average Papaya, raw Grapefruit, raw, pink and red

Lycopene Content (mg/100 g) 28.8 12.1 13.9 5.35 5.20 4.53 2.57 1.83 1.42

Source: U.S. Department of Agriculture ARS, U.S. Department of Agriculture Agricultural Research Service NDL. USDA National Nutrient Database for Standard Reference, Release 2. 2016.

Changes in tomato color occur as chlorophylls degrade and carotenoid production increases, as chloroplasts transform into chromoplasts.15 As a result, lycopene content is highly dependent on fruit ripeness. Given the phenotypic and genetic diversity that exists within tomatoes, lycopene concentration can vary from nearly absent in yellow flesh mutants,16 to high pigment and dark green mutants, which can increase lycopene by more than threefold from traditional elite cultivars.17 Orange-colored fruits with the tangerine mutation accumulate less lycopene than red fruit, though this lycopene is more bioavailable.18 According to the USDA Standard Reference 28, raw, ripe tomatoes contain on average 2.57 mg/100 g fruit,13 though reports in the literature can range considerably, from 0.72–20 mg/100 g.19 This challenges estimations of lycopene intake given the larger variability of lycopene content among tomato fruits. Lycopene is also commonly available in purified supplement form or as tomato extracts, though the effects of consuming these supplements on disease risk are not well understood.

3.3  EFFECTS OF FOOD PROCESSING ON LYCOPENE CONTENT AND PROFILE Over 75% of tomatoes in the United States are consumed in the form of processed products,19 and, as a result, the effects of thermal processing on carotenoid profiles in red tomatoes have been extensively studied. Lycopene from tomato products is relatively stable to moderate heat processing, especially in the absence of fat.20,21 Extensive heat processing can cause degradation,19,22 though these conditions are not often used in traditional food processing. It has been shown that lycopene is more stable in a tomato matrix, compared to isolated, purified, or in solvents.50 Lycopene is stored in tomatoes as crystalline bodies in chromoplasts, relatively resistant to solubilization (a prerequisite for degradation) in the aqueous milieu of the fruit, with cell walls providing an additional barrier.15 When these protective factors are removed, lycopene is more prone to degradation and isomerization. This is observed in tangerine tomatoes where cis-lycopene is stored in lipid-dissolved droplets called plastoglobules and lycopene is labile to thermal degradation.24 The effect of fat addition to tomatoes on lycopene isomerization has yielded mixed results. The addition of 5% or 15% olive oil has been shown not to affect the lycopene isomer profile,20 while tomato juice heated in an oven at 180 °C with 10% safflower oil showed significant isomerization.25 Others have demonstrated that tomato dissolved in water underwent less isomerization compared to when dissolved in oil olive when subjected to 3 hours of heating at 75 °C.26

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Heating of tomato products can increase the bioavailability of lycopene.22,27 Lycopene from tomato paste has been demonstrated as more bioavailable than the same dose given from fresh tomatoes (though the isomeric profiles were not the same), with a mean area-under-the curve increase (representing bioavailability) of almost fourfold with the tomato paste treatment.27 The reason for this difference is proposed to be due to mechanical disruption of cells and the extraction of lycopene into a lipid-dissolved phase that is available for absorption.27 Additionally, lycopene from heat-processed tomato juice has also been shown to increase plasma lycopene more than that from unprocessed tomato juice with the same lycopene isomer distribution,28 suggesting increased bioavailability of heat-treated tomatoes independent of isomer profile. Lycopene is also more bioavailable from homogenized tomatoes,29 again suggesting that mechanical disruption plays a role in bioaccessibility. Lycopene can be isomerized by heat processing to a sauce enriched in cis-isomers, which also has increased bioavailability over a less processed tomato sauce.22

3.4 BIOAVAILABILITY, BIOLOGICAL DISTRIBUTION, AND METABOLISM OF LYCOPENE 3.4.1 Bioavailability In order for lycopene to potential impart health benefits, it is presumed that it must be first absorbed. Given that lycopene is a fat-soluble compound existing generally in crystals within food matrices, this requires destruction of cell walls and liberation from the chloroplast/chromoplast where it can be solubilized in mixed micelles and absorbed by the enterocyte, either passively or actively via transporters.30 A generalized schematic of lycopene absorption can be found in Figure 3.2.31

FIGURE 3.2  Generalized schematic of absorption of lycopene from foods. (Adapted from Yonekura L, Nagao A. Mol Nutr Food Res. 2007;51:107–15.)

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Typically, the bioavailability of lycopene from a single dose is determined by calculating the percentage of an administered dose that reaches circulation in the blood, often in the triglyceride-rich lipoprotein fraction, mainly composed of chylomicrons. This triglyceride-rich lipoprotein fraction is often used because it includes newly absorbed lycopene while ignoring lycopene circulating in the plasma as part of LDL or HDL,32 which can represent a significant fraction in blood given the relatively long half-life of lycopene and its cis isomers.14 Lycopene varies considerably between individuals in terms of its bioavailability given its dependence on a number of factors. Recent advances in this area have recently been well reviewed by Kopec and Failla,33 Harrison and Kopec,34 Moran and others,35 and Bohn and colleagues.36

3.4.2  Factors Affecting Bioavailability of Lycopene The acronym SLAMENGHI has been coined to note the factors that affect bioavailability of carotenoids:37 Species of carotenoid, molecular Linkage, Amount of carotenoid in the meal, food Matrix, Effectors of absorption/bioconversion, Nutrient status of the host, Genetic factors, Hostrelated factors, and Interactions. Molecular linkage (often, the presence of fatty acid esters) is not relevant to lycopene, as the lack of hydroxyl groups precludes ester formation. Extensive reviews exist on this topic;11,30,35,38–42 thus, only a summary will be covered here. A recent and comprehensive summary of the intrinsic and extrinsic factors known to affect lycopene/carotenoid metabolism has been published by Moran et al.35 Lycopene cis isomers have been shown to be more bioavailable than all-trans in a number of studies.18,22,23,27,28,43 This has been hypothesized to be because cis isomers are less likely to crystallize, and, as a result, are more bioaccessible (and thus more bioavailable) and easily incorporated into mixed micelles. Generally, lycopene (and carotenoid) bioavailability as a fraction of dose decreases as dose increases. When providing lycopene in the form of tomato beverages, only marginally more lycopene is absorbed in a 30-mg dose as compared to a 10-mg dose, and there is almost no significant increase in the amount absorbed from 30 to 120 mg.44,45 There is also considerable interindividual variation in lycopene absorption. In general, carotenoids found in foods are tightly bound within the food matrix, which may result in absorption difficulties and reduced bioavailability.46 Destruction of cellular structure (often via thermal processing or homogenization) has been demonstrated to increase the bioavailability of lycopene from tomatoes.28,29 Lycopene absorption is also affected by how lycopene is stored within the plant, with storage in lipid-dissolved droplets leading to higher bioaccessibility than when stored as crystals.18,47 The effects of carotenoid storage in plastids and the resulting impact on bioavailability have been thoroughly reviewed by Schweiggert and Carle.48 Because lycopene is lipophilic, its absorption is dependent upon the same processes that enable fat digestion and absorption. This includes solubilization by bile acids and digestive enzymes and incorporation into micelles. The simultaneous presence of dietary fat in the small intestine is recognized as an important factor for the absorption of lycopene.49 A number of studies have shown that fat is essential for the absorption of carotenoids.50–53 Lycopene circulates in blood primarily on LDL, unlike xanthophylls, which are roughly equally distributed on LDL and HDL.54 Dietary fiber has shown to be a negative effector of carotenoids. The effects of both dietary fat and fiber on lycopene (and carotenoid) absorption have been recently reviewed.33,55 Lycopene is handled by the body in a similar way as dietary lipid. Thus, any disorder, drug, or dietary compound that contributes to lipid malabsorption or that disrupts the micelle-mediated process could potentially reduce the bioavailability of lycopene as well as other carotenoids. Optimal carotenoid absorption occurs if these compounds can be effectively extracted from the food matrix and subsequently incorporated into the lipid phase of the chyme present in the gut. Consequently, patients with cholestasis, who are known to have difficulties with fat absorption, have lower plasma concentrations of lipophilic compounds, including lycopene, as compared to healthy control patients.56

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There are a number of genetic factors that have been associated with carotenoid and lycopene concentrations in blood.39 Single nucleotide polymorphisms (SNPs) in a number of genes involved (or hypothesized to be involved) in lycopene uptake and lipid metabolism have been thought to partially explain the large interindividual variation observed in lycopene bioavailability.39,57–61 A list of SNPs known or thought to affect carotenoid absorption or metabolism has recently been compiled,36 as well as a review on genetic factors involved in bioavailability of tomato carotenoids specifically.62 Host-related factors can also affect the absorption of lycopene, and have been recently reviewed.36 There has been no consistent association between plasma lycopene and age,63,64 though often, lower plasma lycopene has been associated with cigarette smoking.64,65 Many of these host-related factors are thought to contribute to the large interindividual variation in plasma lycopene levels noted.36 The interaction of all of these factors together is almost impossible to assess but cannot be ruled out as an effector of lycopene bioavailability.

3.4.3 Biological Distribution of Lycopene It is often thought that a prerequisite to the action of lycopene in target tissues is the presence of lycopene in those target tissues. It is common to measure lycopene in blood plasma/serum, and the analysis of lycopene in other human tissues is much less common, given the difficulty or impossibility of conducting biopsies. Weighted averages have been calculated by Moran et al.39 for concentrations of lycopene in serum/plasma9,66–74 and a variety of tissues, including testes,75,76 adrenals,75,76 liver,75–78 adipose,68,75,76,79 prostate,9,66,69,70,74,77 lung,77,78,80 kidney,75,76,78 colon,77,80 heart,75,76 skin,68,72,77,80,81 thyroid,75 ovary,75 breast adipose,67,71,80 spleen,75 and brain82 when concentrations of lycopene were available in plasma/serum and at least one other tissue. Some additional studies have reported concentrations of lycopene in plasma/serum as related to tissues, including prostate,83 since 2013. Data correlating carotenoids (including lycopene) in human milk, neonatal plasma, and material plasma has also been collected.84 By using 13C labeled lycopene, isolated from tomato callus culture grown with 13C-glucose, compartmental modeling has been used to estimate flow rates of lycopene to different tissues and fractional transfer coefficients.14

3.4.4 Mammalian Lycopene Metabolism Very little is known regarding the metabolism of lycopene in humans. Humans absorb a portion of lycopene intact; the extent of this absorption is dependent on a number of factors, as has been discussed. Given that lycopene is a non-provitamin A carotenoid, it cannot be cleaved by mammalian enzymes to yield vitamin A. However, it is known that lycopene predominates in foods as the alltrans isomer, while blood plasma and tissues are enriched in various cis-lycopene isomers.85 This has often been explained as an increase in bioavailability of cis isomers,18,27,43 though it has more recently been shown that in vivo isomerization from trans to cis also occurs.14 These cis isomers of lycopene could be considered in vivo lycopene metabolites. It has been hypothesized that isomerization is the first step toward degradation of lycopene in mammals. It has been hypothesized that lycopene could be cleaved oxidatively, in a way similar to vitamin A, to produce a series of products called lycopenoids.86 There are two mammalian enzymes that cleave carotenoids, β-carotene 15,15′-oxygenase 1 (BCO1) and β-carotene 9′,10′-oxygenase 2 (BCO2). BCO1 is known to cleave provitamin A carotenoids to produce retinal,87,88 though more recently human recombinant BCO1 expressed in Escherichia coli was shown to cleave lycopene with a similar catalytic efficiency to β-carotene.89 The products of lycopene cleavage by BCO1 (i.e., acycloretinal, acycloretinyl esters), however, have not been reported in mammalian systems. BCO2 has been demonstrated to eccentrically cleave β-carotene to produce apo-10-carotenal,90,91 while not acting on lycopene.91 Apolycopenoids, however, have been detected in tomato products,92 as well as in human plasma92 and mouse tissues,93 though it is unclear if these products are in fact bona fide metabolites or simply absorbed from the diet. Apolycopenals (aldehyde cleavage products smaller

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than lycopene) were not observed94 after the feeding of a 13C-labelled dose of lycopene.14 However, a 13C-labelled lycopene 1,2-epoxide was observed at about 2% of the levels of 13C-lycopene,94 though this epoxide was also present in the administered dose, so it is unclear whether it is an in vivo metabolite or absorbed from the dose. A recent study aimed to understand the relationship between plasma carotenoids and apocarotenoids after continued consumption of tomato juice.95 Apolycopenoids existed in tomato juices at 0.28% of the levels of lycopene, and despite feeding over 42 mg lycopene per day, lycopenoids were generally absent from blood plasma.95 This lends credibility to the idea that aldehyde cleavage products of lycopene are not primary carotenoid metabolites. Since many of the lycopenoids in tomato juice are not known to be produced from mammalian enzymes, it is hypothesized many of these lycopenoids are non-enzymatic, oxidative degradation products of lycopene. A ∼6-µg dose of 6,6′,7,7′,-14C-lycopene was fed to two healthy volunteers and radioactivity tracked over 42 days.96 It was found that radiolabeled lycopene reached maximum concentration in plasma at 6 hours and had a half-life of 5 days. Lycopene was isomerized from 92% trans at dosing to 50% trans at 24 hours, demonstrating extensive isomerization. An average of 18% of radioactivity was detected in urine with 3% in exhaled breath, suggesting that lycopene is broken down or metabolized to become sufficiently polar/small to be excreted via these pathways.96 Together, this leaves a chasm-sized gap in our understanding of what happens in vivo to lycopene after consumption that is still left to be filled.

3.5  LYCOPENE AND CHRONIC DISEASES There is widespread interest in lycopene and the role it may play in the health of humans. For this reason, a PubMed or Scopus search of lycopene yields thousands of articles, spanning associations with many different disease states or health measures. In this review, those studies that represent the consensus of the literature are presented, with a focus on meta-analyses of epidemiological studies and controlled intervention trials in humans. The intent is not to dismiss the importance of other pre-clinical data, which is invaluable to understanding the relationship between lycopene and health outcomes, but to summarize the consensus in the field as it relates to human health.

3.5.1 Cancer Epidemiological studies have correlated higher plasma lycopene levels with decreased risk of development of cancer at various sites. Given the multifactorial nature of cancer as a disease, as well as the long period in which the disease develops, it is challenging to demonstrate experimentally that consumption of a single compound (e.g., lycopene) can decrease risk of disease. However, below, meta-analyses of epidemiological studies, as well as human clinical trial interventions investigating the effect of lycopene (or, more often, tomato) on cancer outcomes are presented. Interest in the association between lycopene intake and prostate cancer increased with data published from the Health Professionals Follow-Up study suggesting an inverse relationship between tomato/lycopene consumption and prostate cancer risk.97,98 Since these initial reports, a number of meta-analyses and systematic reviews have been published investigating the relationship between tomato/lycopene and prostate cancer, and the strength of this relationship.99–104 These meta-analyses find a consistent association with increased tomato/lycopene intake and a decreased prevalence of prostate cancer, though the magnitude of this association ranges. There are a handful of intervention human clinical trials designed to glean additional information about the relationship between tomato/ lycopene and prostate cancer.83,105–109 Many studies report administering a lycopene supplement, when most often this lycopene supplement is a commercially available oleoresin of tomato called Lyc-o-Mato®. This should be kept in mind, as there are many additional phytochemicals in a tomato oleoresin aside from lycopene, which places tomato extracts somewhere between tomato foods and lycopene supplements. Some of these studies have found modulation of markers of growth and differentiation, or prostate-specific antigen, with tomato extracts,70,105–107,110–112 while others found

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no effect of treatment (though well tolerated).108,109,113–115 These results are still promising given the challenges of assessing what a nutritional intervention might do past middle age on a disease that takes years to progress. Investigating a preventative agent in a population where the seeds of disease are likely already planted challenges the interpretation of the current literature. The impact of lycopene in the context of other cancers is less studied. A few studies suggest that tomato/lycopene supplements can alter markers for colorectal cancer116,117 though recent metaanalyses of epidemiological studies have found a lack of an association.118 Relationships have been noted between increased lycopene and decreased risk for breast119,120 (with some reporting no effect)121,122 and gastric cancers,123 though a lack of intervention trials limits our understanding of the causal relationship between lycopene and cancers at these sites.

3.5.2 Heart Disease Cardiovascular disease (CVD) is a major cause of death in the Western world. Often, a diet rich in fruits and vegetables is recommended as a prevention measure for development of CVD.124–127 There are a number of published reviews on the topic of lycopene and heart health.128,129 Additionally, there are meta-analyses of both epidemiological and intervention trials.130–133 A comprehensive review on the effect of whole food (tomato) and supplement (lycopene) administration on risk factors associated with cardiovascular disease has been published recently, highlighting intervention trials that assess oxidation of LDLs, oxidative stress and damage, markers of inflammation, endothelial function, blood pressure, and blood lipids,130 which is summarized below. A recent systematic review of intervention trials investigating tomato products and lycopene on cardiovascular function evaluates studies that assess the effect of tomatoes/lycopene on blood lipids, blood pressure, and endothelial function.132 Oxidation of LDL is often measured in the context of nutritional interventions because of its suggestive indication of atherogenic activity. A number of intervention trials have investigated the effect of tomato extract, lycopene supplement (beadlets or purified), or tomato products on oxidation of LDLs, some finding a decrease in LDL oxidation,134–142 while others found no effect.143–151 There is little data about how tomato/lycopene supplementation affects a non-healthy population.130 Many studies where lycopene was provided as a supplement provide Lyc-o-Mato®, an oleoresin of tomato; thus, these studies do not represent true, single-compound supplementation with lycopene. Very few studies contained a true lycopene supplement arm.152

3.5.3  Inflammation Inflammation is an adaptive response triggered by noxious stimuli and conditions such as chemical or physical injury.153 In addition to activation of immune cells, inflammatory stimuli induce release of various chemical mediators, including cytokines and reactive oxygen species, that promote leukocyte recruitment to areas of injury to eliminate pathogens and/or repair damaged tissue.154 This inflammatory process is normally self limiting to prevent extensive damage to the host.155 However, in many cases, repeated exposure to harmful stimuli can trigger inappropriate regulation or failure to resolve inflammatory responses that leads to excess tissue damage and disease. Consumption of a tomato-rich diet has been suggested to decrease systemic inflammation.143 A recent study found that adding tomato juice to a usual diet for 2 months could increase adiponectin and decrease MCP-1 (a pro-inflammatory cytokine), while decreasing body weight, BMI, waist circumference, and percent body fat in healthy, normal-weight women.156 Lower plasma C-reactive protein (CRP) levels were observed in healthy males who consumed eight servings of carotenoid-rich fruits and vegetables per day for 4 weeks compared to men who consumed only two servings per day.157 In healthy subjects, consumption of 500 mL tomato juice/day for 2 weeks resulted in reduced CRP, but did not affect IL-1β or TNF-α.158 In a post-prandial feeding study of tomato paste, the rise in LDL oxidation and IL-6 as a result of a high-fat meal were attenuated in the tomato-containing meal compared to the control, but there was no effect on pro-inflammatory cytokines.140 Additionally, consumption of a beverage

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containing a tomato extract of 5.7 mg lycopene, 3.7 mg phytoene, and 2.7 mg phytofluene for 26 days resulted in 34% lower TNF-α compared to the placebo group, suggesting that even with low levels of inflammation, a dietary intervention with tomatoes can affect cytokine levels.160 A summary of studies investigating the effects of tomato/lycopene on markers of inflammation has been compiled by Burton-Freeman and Sesso.130 Often, studies measured multiple biomarkers of inflammation and observed effects on some but not all of these indices.139,140,143,145,156,158,160–164 Others found no effect of intervention.137,152,165–167

3.5.4 Skin and UV-Induced Sun Sensitivity Carotenoids function as accessory pigments in plants, helping quench free radicals168 and dissipate excess energy produced during the energy-intensive process of photosynthesis. Because of this function in planta, it has been postulated that carotenoids and lycopene may function via a similar mechanism in humans (although this has not been explicitly demonstrated). β-carotene was studied in the 1970s for its ability to reduce the photosensitivity response in patients that had erythropoetic protoporphyria (EPP). Those with this debilitating disease accumulate photosensitizing porphyrins in blood after exposure to the sun of as little to 10–20 minutes, causing burning and extreme redness and swelling.169 It has been shown that megadoses of β-carotene, up to 300 mg/day, can lessen this response.170–172 This ability of β-carotene to reduce this photosensitivity response made researchers question whether this idea could be exploited to reduce sunburn173 or prevent development of skin cancer174 (using animal models), and has been investigated using lycopene/tomato products, as lycopene accumulates in human skin.68,72,77,80,81,175 Feeding lycopene-rich foods prior to exposure to UV light has resulted in a dampened erythema response, suggesting that something about tomato consumption is altering the skin’s response to this damaging stimulus.176–181 This subject has been thoroughly reviewed elsewhere.10,159,176

3.6 CONCLUSIONS Lycopene, the predominant carotenoid in tomatoes; a Western diet; and blood plasma have been the subject of over a century of scientific research. Lycopene is absorbed and distributed among various tissues in humans, where it can exist for some time. There is a body of evidence suggesting that tomato consumption is beneficial for health. However, the exact function that lycopene plays is still an active area of research. Additional research is needed to further understand the specific role that lycopene may play in imparting health benefits to humans.

ACKNOWLEDGMENTS This work was supported by USDA-AFRI (2018-67017-27519), USDA Hatch Funds (OHO01470) and Foods for Health, a focus area of the Discovery Themes Initiative at The Ohio State University. This chapter was originally written by Richard S. Bruno, Robert Wildman Jr., and Steven J. Schwartz in editions 1 and 2 of this book.

REFERENCES

1. Aune D, Giovannucci E, Boffetta P, Fadnes LT, Keum NN, Norat T, Greenwood DC, Riboli E, Vatten LJ, Tonstad S. Fruit and vegetable intake and the risk of cardiovascular disease, total cancer and all-cause mortality—A systematic review and dose-response meta-analysis of prospective studies. Int J Epidemiol. 2017;46:1029–56. 2. Heber D. Vegetables, fruits and phytoestrogens in the prevention of diseases. J Postgrad Med. 2004;50:145–9. 3. Britton G, Liaaen-Jensen S, Pfander H, editors. Carotenoids Handbook. Basel, Switzerland: Birkhauser Verlag; 2004.

46

Handbook of Nutraceuticals and Functional Foods

4. Khachik F, Spangler C, Smith J, Canfield L, Steck A, Pfander H. Identification, quantification, and relative concentrations of carotenoids and their metabolites in human milk and serum. Anal Chem. 1997;69:1873–81. 5. Britton G. Structure and properties of carotenoids in relation to function. FASEB J. 1995;9:1551–8. 6. Hartsen F. Beiträge zur organischen chemie, ueber den einfluss der jahreszeiten auf die chemie der pflanzen. Chem Zentr. 1873;4:205. 7. Millardet A. Note sur une substance colorante nouvelle (solanorubine) decouverte dans la tomate. Bull Soc Sci Nancy. 1875;2:21–32. 8. Burrows TL, Williams R, Rollo M, Wood L, Garg ML, Jensen M, Collins CE. Plasma carotenoid levels as biomarkers of dietary carotenoid consumption: A systematic review of the validation studies. J Nutr Intermed Metab. 2015;2:15–64. 9. Clinton SK, Emenhiser C, Schwartz SJ, Bostwick DG, Williams AW, More BJ, Erdman Jr. JW. cis-trans lycopene isomers, carotenoids, and retinol in the human prostate. Cancer Epidemiol Biomarkers Prev. 1996;5:823–33. 10. Cooperstone JL, Schwartz SJ. Recent insights into health benefits of carotenoids. In: Carle R, Schweiggert R, editors. Handbook on Natural Pigments in Food and Beverages: Industrial Applications for Improving Food Color. Woodhead Publishing; 2016, pp. 473–97. 11. Story EN, Kopec RE, Schwartz SJ, Harris GK. An update on the health effects of tomato lycopene. Annu Rev Food Sci Technol. 2010;1:189–210. 12. Clinton SK. Lycopene: Chemistry, biology, and implications for human health and disease. Nutr Rev. 1998;56:35–51. 13. U.S. Department of Agriculture ARS, U.S. Department of Agriculture Agricultural Research Service NDL. USDA National Nutrient Database for Standard Reference, Release 2. 2016. 14. Moran NE, Cichon MJ, Riedl KM, Grainger EM, Schwartz SJ, Novotny JA, Erdman Jr. JW, Clinton SK. Compartmental and noncompartmental modeling of 13C-lycopene absorption, isomerization, and distribution kinetics in healthy adults. Am J Clin Nutr. 2015;102:1436–49. 15. Harris WM, Spurr AR. Chromoplasts of tomato fruits. II. The red tomato. Am J Bot. 1969;56:380–9. 16. Fray RG, Grierson D. Identification and genetic analysis of normal and mutant phytoene synthase genes of tomato by sequencing, complementation and co-suppression. Plant Mol Biol. 1993;22:589–602. 17. Levin I, de Vos C, Tadmor Y, Bovy A, Lieberman M, Oren-Shamir M, Segev O, Kolotilin I, Keller M, Ovadia R et al. High pigment tomato mutants—More than just lycopene (a review). Isr J Plant Sci. 2006;54:179–90. 18. Cooperstone JL, Ralston RA, Riedl KM, Haufe TC, Schweiggert RM, King SA, Timmers CD, Francis DM, Lesinski GB, Clinton SK, et al. Enhanced bioavailability of lycopene in humans when consumed as cis-isomers from tangerine tomatoes compared to red tomato juices, a randomized, cross-over clinical trial. Mol Nutr Food Res. 2015;59:658–69. 19. Shi J, Le Maguer M. Lycopene in tomatoes: Chemical and physical properties affected by food processing. Crit Rev Biotechnol. 2000;20:293–334. 20. Nguyen ML, Schwartz SJ. Lycopene stability during food processing. Proc Soc Exp Biol Med. 1998;218:101–5. 21. Nguyen ML, Francis DM, Schwartz SJ. Thermal isomerisation susceptibility of carotenoids in different tomato varieties. J Sci Food Agric. 2001;81:910–7. 22. Unlu NZ, Bohn T, Francis DM, Nagaraja HN, Clinton SK, Schwartz SJ. Lycopene from heat-induced cisisomer-rich tomato sauce is more bioavailable than from all-trans-rich tomato sauce in human subjects. Br J Nutr. 2007;98:140–6. 23. Simpson KL, Chichester CO. Metabolism and nutritional significance of carotenoids. Annu Rev Nutr. 1981;1:351–74. 24. Cooperstone JL, Francis DM, Schwartz SJ. Thermal processing differentially affects lycopene and other carotenoids in cis-lycopene containing, tangerine tomatoes. Food Chem. 2016;210:466–72. 25. Itsch ROB, Tto KOO, Seybold C, Fröhlich K, Bitsch R, Otto K, Böhm V, Fröhlich K, Bitsch R, Otto K et al. Changes in contents of carotenoids and vitamin E during tomato processing. J Agric Food Chem. 2004;52:7005–10. 26. Schierle J, Bretzel W, Bühler I, Faccin N, Hess D, Steiner K, Schüep W. Content and isomeric ratio of lycopene in food and human blood plasma. Food Chem. 1997;59:459–65. 27. Gartner C, Stahl W, Sies HH. Lycopene is more bioavailable from fresh tomatoes from tomato paste than. Am J Clin Nutr. 1997;66:116–22. 28. Stahl W, Sies H. Uptake of lycopene and its geometrical isomers is greater from heat-processed than from unprocessed tomato juice in humans. J Nutr. 1992;122:2161–6.

Lycopene

47

29. van het Hof KH, de Boer BC, Tijburg LBM, Lucius BRHM, Zijp I, West CE, Hautvast JGAJ, Weststrate JA, Hof KH Van, Boer BCJ De et al. Carotenoid bioavailability in humans from tomatoes processed in different ways determined from the carotenoid response in the triglyceride-rich lipoprotein fraction of plasma after a single consumption and in plasma after four days of consumption. J Nutr. 2000;130:1189–96. 30. During A, Harrison EH. Intestinal absorption and metabolism of carotenoids: Insights from cell culture. Arch Biochem Biophys. 2004;430:77–88. 31. Yonekura L, Nagao A. Intestinal absorption of dietary carotenoids. Mol Nutr Food Res. 2007;51:107–15. 32. van Vliet T, Schreurs WH, van den Berg H. Intestinal β-carotene absorption and cleavage in men: Response of β-carotene and retinyl esters in the triglyceride-rich lipoprotein fraction after a single oral dose of β-carotene. Am J Clin Nutr. 1995;62:110–6. 33. Kopec RE, Failla ML. Recent advances in the bioaccessibility and bioavailability of carotenoids and effects of other dietary lipophiles. J Food Compos Anal. 2017;68:16–30. 34. Harrison EH, Kopec RE. Digestion and intestinal absorption of dietary carotenoids and vitamin A. In: Physiology of the Gastrointestinal Tract. 6 edn. Elsevier Inc.; 2018, pp. 1133–51. 35. Moran NE, Mohn ES, Hason N, Erdman JW, Johnson EJ. Intrinsic and extrinsic factors impacting absorption, metabolism, and health effects of dietary carotenoids. Adv Nutr. 2018;9:465–92. 36. Bohn T, Desmarchelier C, Dragsted LO, Nielsen CS, Stahl W, Rühl R, Keijer J, Borel P. Host-related factors explaining interindividual variability of carotenoid bioavailability and tissue concentrations in humans. Mol Nutr Food Res. 2017;61:1600685. 37. Castenmiller JJ, West CE, Castenmiller. Bioavailability of carotenoids. Pure Appl Chem. 1997;69:2145. 38. Castenmiller JJM, West CE. Bioavailability and bioconversion of carotenoids. Annu Rev Nutr. 1998;18:19–38. 39. Moran NE, Erdman JW, Clinton SK. Complex interactions between dietary and genetic factors impact lycopene metabolism and distribution. In: Arch Biochem Biophys. Elsevier Inc.; 2013;539:171–80. 40. Harrison EH. Mechanisms involved in the intestinal absorption of dietary vitamin A and provitamin A carotenoids. Biochim Biophys Acta. 2012;1821:70–7. 41. Ross AC, Harrison EH. Vitamin A nutrition aspects of retinoids and carotenoids. In: Zempleni J, Suttie JW, Gregory III JF, Stover PJ, editors. Handbook of Vitamins. 5th edn. Boca Raton, FL: CRC Press; 2014, pp. 1–50. 42. van het Hof KH, West CE, Weststrate JA, Hautvast JG. Recent advances in nutritional sciences dietary factors that affect the bioavailability of carotenoids 1. J Nutr. 2000;503–6. 43. Unlu NZ, Bohn T, Francis D, Clinton SK, Schwartz SJ. Carotenoid absorption in humans consuming tomato sauces obtained from tangerine or high-beta-carotene varieties of tomatoes. J Agric Food Chem. 2007;55:1597–603. 44. Diwadkar-Navsariwala V, Novotny JA, Gustin DM, Sosman JA, Rodvold KA, Crowell JA, StacewiczSapuntzakis M, Bowen PE. A physiological pharmacokinetic model describing the disposition of lycopene in healthy men. J Lipid Res. 2003;44:1927–39. 45. Gustin DM, Rodvold KA, Sosman JA, Diwadkar-Navsariwala V, Stacewicz-Sapuntzakis M, Viana M, Crowell JA, Murray J, Tiller P, Bowen PE. Single-Dose pharmacokinetic study of lycopene delivered in a well-defined food-based lycopene delivery system (tomato paste-oil mixture) in healthy adult male subjects. Cancer Epidemiol Biomarkers Prev. 2004;13:850–60. 46. Zhou J, Gugger E, Erdman Jr. JW. The crystalline form of carotenes and the food matrix in carrot root decrease the relative bioavailability of beta- and alpha-carotene in the ferret model. J Am Coll Nutr. 1996;15:84–91. 47. Schweiggert RM, Kopec RE, Villalobos-Gutierrez MG, Högel J, Quesada S, Esquivel P, Schwartz SJ, Carle R. Carotenoids are more bioavailable from papaya than from tomato and carrot in humans: A randomised cross-over study. Br J Nutr. 2014;111:490–8. 48. Schweiggert RM, Carle R. Carotenoid deposition in plant and animal foods and its impact on bioavailability. Crit Rev Food Sci Nutr. 2017;57:1807–30. 49. Roodenburg AJC, Leenen R, Hof KH Van, Weststrate JA, Tijburg LBM. Amount of fat in the diet affects bioavailability of lutein esters but not alpha-carotene, beta-carotene and vitamin E in humans. Am J Clin Nutr. 2000;71:1187–93. 50. Kopec RE, Cooperstone JL, Schweiggert RM, Young GS, Harrison EH, Francis DM, Clinton SKS, Schwartz SJ. Avocado consumption enhances human postprandial provitamin A absorption and conversion from a novel high-β-carotene tomato sauce and from carrots. J Nutr. 2014;144:1158–66. 51. Unlu NZ, Bohn T, Clinton SK, Schwartz SJ. Carotenoid absorption from salad and salsa by humans is enhanced by the addition of avocado or avocado oil. J Nutr. 2005;135:431–6.

48

Handbook of Nutraceuticals and Functional Foods

52. Brown MJ, Ferruzzi MG, Nguyen ML, Cooper DA, Eldridge AL, Schwartz SJ, White WS. Carotenoid bioavailability is higher from salads ingested with full-fat than with fat-reduced salad dressings as measured with electrochemical detection. Am J Clin Nutr. 2004;80:396–403. 53. Goltz SR, Sapper TN, Failla ML, Campbell WW, Ferruzzi MG. Carotenoid bioavailability from raw vegetables and a moderate amount of oil in human subjects is greatest when the majority of daily vegetables are consumed at one meal. Nutr Res. 2013;33:358–66. 54. Goulinet S, Chapman M. Plasma LDL and HDL subspecies are heterogeneous in particle content of tocopherols and oxygenated and hydrocarbon carotenoids. Relevance to oxidative resistance and atherogenesis. Arterioscler Thromb Vasc Biol. 1997;17:786–96. 55. Goltz SR, Ferruzzi MG. Carotenoid bioavailability: Influence of dietary lipid and fiber. In: Tanumihardjo SA, editor. Carotenoids and Human Health. New York, NY: Springer Science+Business Media; 2013, pp. 111–28. 56. Floreani A, Baragiotta A, Martines D, Naccarato R, D’odorico A. Plasma antioxidant levels in chronic cholestatic liver diseases. Aliment Pharmacol Ther. 2000;14:353–8. 57. Borel P, Desmarchelier C, Nowicki M, Bott R. Lycopene bioavailability is associated with a combination of genetic variants. Free Radic Biol Med. 2015;83:238–44. 58. Ferrucci L, Perry JRBB, Matteini A, Perola M, Tanaka T, Silander K, Rice N, Melzer D, Murray A, Cluett C et al. Common variation in the β-carotene 15,15′-monooxygenase 1 gene affects circulating levels of carotenoids: A genome-wide association study. Am J Hum Genet. 2009;84:123–33. 59. Wang TT, Edwards AJ, Clevidence BA. Strong and weak plasma response to dietary carotenoids identified by cluster analysis and linked to beta-carotene 15,15’-monooxygenase 1 single nucleotide polymorphisms. J Nutr Biochem. 2013;24:1538–46. 60. Borel P. Genetic variations involved in interindividual variability in carotenoid status. Mol Nutr Food Res. 2012;56:228–40. 61. Zubair N, Kooperberg C, Liu J, Di C, Peters U, Neuhouser ML. Genetic variation predicts serum lycopene concentrations in a multiethnic population of postmenopausal women. J Nutr. 2015;145:187–92. 62. Desmarchelier C, Landrier J-F, Borel P. Genetic factors involved in the bioavailability of tomato carotenoids. Curr Opin Clin Nutr Metab Care. 2018;21:489–97. 63. Casso D, White E, Patterson R, Agurs-Collins T, Kooperberg C, Haines P. Correlates of serum lycopene in older women. Nutr Cancer. 2000;36:163–9. 64. Brady WE, Mares-Perlman JA, Bowen P, Stacewicz-Sapuntzakis M. Human serum carotenoid concentrations are related to physiologic and lifestyle factors. J Nutr. 1996;126:129–37. 65. Walmsley CM, Bates CJ, Prentice A, Cole TJ. Relationship between alcohol and nutrient intakes and blood status indices of older people living in the UK: Further analysis of data from the National Diet and Nutrition Survey of people aged 65 years and over, 1994/5. Public Health Nutr. 1998;1:157–67. 66. Van Breemen RB, Sharifi R, Viana M, Pajkovic N, Zhu D, Yuan L, Yang Y, Bowen PE, StacewiczSapuntzakis M. Antioxidant effects of lycopene in African American men with prostate cancer or benign prostate hyperplasia: A randomized, controlled trial. Cancer Prev Res. 2011;4:711–8. 67. Yeum K-J, Ahn S-H, de Paiva SAR, Lee-Kim YC, Krinsky NI, Russell RM. Correlation between carotenoid concentrations in serum and normal breast adipose tissue of women with benign breast tumor or breast cancer. J Nutr. 1998;128:1920–6. 68. Walfisch Y, Walfisch S, Agbaria R, Levy J, Sharoni Y. Lycopene in serum, skin and adipose tissues after tomato-oleoresin supplementation in patients undergoing haemorrhoidectomy or peri-anal fistulotomy. Br J Nutr. 2003;90:759–66. 69. Chen L, Stacewicz-Sapuntzakis M, Duncan C, Sharifi R, Ghosh L, van Breemen R, Ashton D, Bowen PE. Oxidative DNA damage in prostate cancer patients consuming tomato sauce-based entrees as a whole-food intervention. J Natl Cancer Inst. 2001;93:1872–9. 70. Schwarz S, Obermüller-Jevic UC, Hellmis E, Koch W, Jacobi G, Biesalski H-K. Lycopene inhibits disease progression in patients with benign prostate hyperplasia. J Nutr. 2008;138:49–53. 71. Shim E, Yeum K-J, Tang G, Ahn SH, Hwang J, Lee-Kim YC. Retinoids, carotenoids, and tocopherols in breast adipose tissue and serum of benign breast disease and breast cancer patients. Nutr Cancer. 2012;64:956–63. 72. Peng YM, Peng YS, Lin Y, Moon T, Baier M. Micronutrient concentrations in paired skin and plasma of patients with actinic keratoses: Effect of prolonged retinol supplementation. Cancer Epidemiol Biomarkers Prev. 1993;2:145–50. 73. Bowen P, Chen L, Stacewicz-Sapuntzakis M, Duncan C, Sharifi R, Ghosh L, Kim H-S, Christov-Tzelkov K, van  Breemen R. Tomato sauce supplementation and prostate cancer: Lycopene accumulation and modulation of biomarkers of carcinogenesis. Exp Biol Med. 2002;227:886–93.

Lycopene

49

74. Freeman VL, Meydani M, Yong S, Pyle J, Wan Y, Arvizu-Durazo R, Liao Y. Prostatic levels of tocopherols, carotenoids, and retinol in relation to plasma levels and self-reported usual dietary intake. Am J Epidemiol. 2000;151:109–18. 75. Kaplan LA, Lau JM, Stein EA. Carotenoid composition, concentrations, and relationships in various human organs. Clin Physiol Biochem. 1990;8:1–10. 76. Stahl W, Schwarz W, Sundquist AR, Sies H. cis-trans isomers of lycopene and beta-carotene in human serum and tissues. Arch Biochem Biophys. 1992;294:173–7. 77. Khachik F, Carvalho L, Bernstein PS, Muir GJ, Zhao D-Y, Katz NB. Chemistry, distribution, and metabolism of tomato carotenoids and their impact on human health. Exp Biol Med. 2002;227:845–51. 78. Schmitz HH, Poor CL, Wellman RB, Erdman Jr. JW. Concentrations of selected carotenoids and vitamin A in human liver, kidney and lung tissue. J Nutr. 1991;121:1613–21. 79. Parker RS. Carotenoid and tocopherol composition of human adipose tissue. Am J Clin Nutr. 1988;47:33–6. 80. Nierenberg DW, Stukel TA, Baron JA, Dain BJ, Greenberg ER, Group TSCPS. Determinants of increase in plasma concentration of ß-carotene after chronic oral supplementation. Am J Clin Nutr. 1991;53:1443–9. 81. Peng Y-M, Peng Y-S, Lin Y, Moon T, Roe DJ, Ritenbaugh C. Concentrations and plasma-tissue-diet relationships of carotenoids, retinoids, and tocopherols in humans. Nutr Cancer. 1995;23:233–46. 82. Craft N, Haitema T, Garnett K, Fitch K, Dorey C. Carotenoid, tocopherol, and retinol concentrations in elderly human brain. J Nutr Heal Aging. 2004;8:156–62. 83. Grainger EM, Hadley CW, Moran NE, Riedl KM, Gong MC, Pohar K, Schwartz SJ, Clinton SK. A comparison of plasma and prostate lycopene in response to typical servings of tomato soup, sauce or juice in men before prostatectomy. Br J Nutr. 2015;114:596–607. 84. Lipkie TE, Morrow AL, Jouni ZE, McMahon RJ, Ferruzzi MG. Longitudinal survey of carotenoids in human milk from urban cohorts in China, Mexico, and the USA. PLOS ONE. 2015;10:1–14. 85. Boileau TW-MM, Boileau AC, Erdman Jr JW. Bioavailability of all-trans and cis-isomers of lycopene. Exp Biol Med. 2002;227:914–9. 86. Lindshield BL, Canene-Adams K, Erdman Jr. JW. Lycopenoids: Are lycopene metabolites bioactive? Arch Biochem Biophys. 2007;458:136–40. 87. Wyss A, Wirtz G, Woggon W-D, Brugger R, Wyss M, Friedlein A, Bachmann H, Hunziker W. Cloning and expression of β,β-carotene 15,15′-dioxygenase. Biochem Biophys Res Commun. 2000;271:334–6. 88. von Lintig J, Vogt K. Filling the gap in vitamin A research. J Biol Chem. 2000;275:11915–20. 89. dela Seña C, Narayanasamy S, Riedl KM, Curley RW, Schwartz SJ, Harrison EH. Substrate specificity of purified recombinant human β-carotene 15,15′-oxygenase (BCO1). J Biol Chem. 2013;288:37094–103. 90. Kiefer C, Hessel S, Lampert JM, Vogt K, Lederer MO, Breithaupt DE, von Lintig J. Identification and characterization of a mammalian enzyme catalyzing the asymmetric oxidative cleavage of provitamin A. J Biol Chem. 2001;276:14110–6. 91. dela Seña C, Sun J, Narayanasamy S, Riedl KM, Yuan Y, Curley RW, Schwartz SJ, Harrison EH. Substrate specificity of purified recombinant chicken β-carotene 9′,10′-oxygenase (BCO2). J Biol Chem. 2016;291:14609–19. 92. Kopec RE, Riedl KM, Harrison EH, Curley RW, Hruszkewycz DP, Clinton SK, Schwartz SJ. Identification and quantification of apo-lycopenals in fruits, vegetables, and human plasma. J Agric Food Chem. 2010;58:3290–6. 93. Tan H-L, Moran NE, Cichon MJ, Riedl KM, Schwartz SJ, Erdman JW, Pearl DK, Thomas-Ahner JM, Clinton SK. β-carotene-9′,10′-oxygenase status modulates the impact of dietary tomato and lycopene on hepatic nuclear receptor-, stress-, and metabolism-related gene expression in mice. J Nutr. 2014;144:431–9. 94. Cichon MJ, Moran NE, Riedl KM, Schwartz SJ, Clinton SK. Identification of an epoxide metabolite of lycopene in human plasma using13C-labeling and QTOF-MS. Metabolites. 2018;8. 95. Cooperstone JL, Novotny JA, Riedl KM, Cichon MJ, Francis DM, Curley RW, Schwartz SJ, Harrison EH. Limited appearance of apocarotenoids is observed in plasma after consumption of tomato juices: A randomized human clinical trial. Am J Clin Nutr. 2018;108:1–8. 96. Ross AB, Vuong LT, Ruckle J, Synal HA, Schulze-ko T, Wertz K, Ru R, Liberman RG, Skipper PL, Tannenbaum SR et  al. Lycopene bioavailability and metabolism in humans: An accelerator mass spectrometry study. Am J Clin Nutr. 2011;93:1263–73. 97. Giovannucci E, Ascherio A, Rimm EB, Stampfer MJ, Colditz GA, Willett WC. Intake of carotenoids and retinol in relation to risk of prostate cancer. J Natl Cancer Inst. 1995;87:1767–76. 98. Gann PH, Ma J, Giovannucci E, Willett W, Sacks FM, Hennekens CH, Stampfer MJ. Lower prostate cancer risk in men with elevated plasma lycopene levels: Results of a prospective analysis. Cancer Res. 1999;59:1225–30. 99. Rowles JL, Katherine III, Catherine MR, Sookyoung CA, Ruopeng J, Jr. JWE. Processed and raw tomato consumption and risk of prostate cancer: A systematic review and dose–response meta-analysis. In: Prostate Cancer Prostatic Dis. US: Springer; 2017.

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100. Chen J, Song Y, Zhang L. Lycopene/tomato consumption and the risk of prostate cancer: A systematic review and meta-analysis of prospective studies. J Nutr Sci Vitaminol. 2013;59:213–23. 101. Xu X, Li J, Wang X, Wang S, Meng S, Zhu Y, Liang Z, Zheng X, Xie L. Tomato consumption and prostate cancer risk: A systematic review and meta-analysis. Sci Rep. 2016;6:1–8. 102. Etminan M, Takkouche B, Caamano-Isorna F. The role of tomato products and lycopene in the prevention of prostate cancer: A meta-analysis of observational studies. Cancer Epidemiol Biomarkers Prev. 2004;13:340–5. 103. Wang Y, Cui R, Xiao Y, Fang J, Xu Q. Effect of carotene and lycopene on the risk of prostate cancer: A systematic review and dose-response meta-analysis of observational studies. PLOS ONE. 2015;10:1–20. 104. Chen P, Zhang W, Wang X, Zhao K, Negi DS, Zhuo L, Qi M, Wang X, Zhang X. Lycopene and risk of prostate cancer: A systematic review and meta-analysis. Med. 2015;94:e1260. 105. Kucuk O, Sarkar FH, Sakr W, Khachik F, Djuric Z, Banerjee M, Pollak MN, Bertram JS, Wood Jr. DP. Lycopene in the treatment of prostate cancer. Pure Appl Chem. 2002;74:1443. 106. Kucuk O, Sarkar FH, Sakr W, Djuric Z, Pollak MN, Khachik F, Li Y-W, Banerjee M, Grignon D, Bertram JS et al. Phase II randomized clinical trial of lycopene supplementation before radical prostatectomy. Cancer Epidemiol Biomarkers Prev. 2001;10:861–8. 107. Kucuk O, Sarkar FH, Djuric Z, Sakr W, Pollak MN, Khachik F, Banerjee M, Bertram JS, Wood DP, Wood Jr DP. Effects of lycopene supplementation in patients with localized prostate cancer. Exp Biol Med. 2002;227:881–5. 108. Bunker CH, McDonald AC, Evans RW, de la Rosa N, Boumosleh JM, Patrick AL. A randomized trial of lycopene supplementation in Tobago men with high prostate cancer risk. Nutr Cancer. 2007;57:130–7. 109. Gann PH, Deaton RJ, Rueter EE, van Breemen RB, Nonn L, Macias V, Han M, Ananthanarayanan V. A phase II randomized trial of lycopene-rich tomato extract among men with high-grade prostatic intraepithelial neoplasia. Nutr Cancer. 2015;67:1104–12. 110. Ansari MS, Gupta NP. A comparison of lycopene and orchidectomy vs orchidectomy alone in the management of advanced prostate cancer. BJU Int. 2003;92:375–8. 111. Vaishampayan U, Hussain M, Banerjee M, Seren S, Sarkar FH, Fontana J, Forman JD, Cher ML, Powell I, Pontes JD, et  al. Lycopene and soy isoflavones in the treatment of prostate cancer. Nutr Cancer. 2007;59:1–7. 112. Barber NJ, Zhang X, Zhu G, Pramanik R, Barber JA, Martin FL, Morris JDH, Muir GH. Lycopene inhibits DNA synthesis in primary prostate epithelial cells in vitro and its administration is associated with a reduced prostate-specific antigen velocity in a phase II clinical study. Prostate Cancer Prostatic Dis. 2006;9:407–13. 113. Kumar NB, Besterman-Dahan K, Kang L, Pow-Sang J, Xu P, Allen K, Riccardi D, Krischer JP. Results of a randomized clinical trial of the action of several doses of lycopene in localized prostate cancer: Administration prior to radical prostatectomy. Clin Med Urol. 2008;1:1–14. 114. Jatoi A, Burch P, Hillman D, Vanyo JM, Dakhil S, Nikcevich D, Rowland K, Morton R, Flynn PJ, Young C et al. A tomato-based, lycopene-containing intervention for androgen-independent prostate cancer: Results of a phase II study from The North Central Cancer Treatment Group. Urology. 2007;69:289–94. 115. Clark PE, Hall MC, Borden LS, Miller AA, Hu JJ, Lee WR, Stindt D, D’Agostino R, Lovato J, Harmon M et al. Phase I-II prospective dose-escalating trial of lycopene in patients with biochemical relapse of prostate cancer after definitive local therapy. Urology. 2006;67:1257–61. 116. Walfisch S, Walfisch Y, Kirilov E, Linde N, Mnitentag H, Agbaria R, Sharoni Y, Levy J. Tomato lycopene extract supplementation decreases insulin-like growth factor-I levels in colon cancer patients. Eur J Cancer Prev. 2007;16:298–303. 117. Vrieling A, Voskuil DW, Bonfrer JM, Korse CM, Van Doorn J, Cats A, Depla AC, Timmer R, Witteman BJ, Van Leeuwen FE et al. Lycopene supplementation elevates circulating insulin-like growth factorbinding protein-1 and -2 concentrations in persons at greater risk of colorectal cancer. Am J Clin Nutr. 2007;86:1456–62. 118. Mannisto S, Yaun S-S, Hunter DJ, Spiegelman D, Adami H-O, Albanes D, Van Den Brandt PA, Buring JE, Cerhan JR, Colditz GA et al. Dietary carotenoids and risk of colorectal cancer in a pool analysis of 11 cohort studies. Am J Epidemiol. 2007;165:246–55. 119. Eliassen AH, Liao X, Rosner B, Tamimi RM, Tworoger SS, Hankinson SE. Plasma carotenoids and risk of breast cancer over 20 y of follow-up. Am J Clin Nutr. 2015;101:1197–205. 120. Eliassen AH, Hendrickson SJ, Brinton LA, Buring JE, Campos H, Dai Q, Dorgan JF, Franke AA, Gao Y, Goodman MT et al. Circulating carotenoids and risk of breast cancer: Pooled analysis of eight prospective studies. J Natl Cancer Inst. 2012;104:1905–16.

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121. Aune D, Chan DSM, Vieira AR, Rosenblatt DAN, Vieira R, Greenwood DC, Norat T. Dietary compared with blood concentrations of carotenoids and breast cancer risk: A systematic review and meta-analysis of prospective. Am J Clin Nutr. 2012;96:356–73. 122. Hu F, Wang Yi B, Zhang W, Liang J, Lin C, Li D, Wang F, Pang D, Zhao Y. Carotenoids and breast cancer risk: A meta-analysis and meta-regression. Breast Cancer Res Treat. 2012;131:239–53. 123. Yang T, Yang X, Wang X, Wang Y, Song Z. The role of tomato products and lycopene in the prevention of gastric cancer: A meta-analysis of epidemiologic studies. Med Hypotheses. 2013;80:383–8. 124. Dauchet L, Amouyel P, Hercberg S, Dallongeville J. Fruit and vegetable consumption and risk of coronary heart disease: A meta-analysis of cohort studies. J Nutr. 2006;136:2588–93. 125. He F, Nowson C, Lucas M, MacGregor G. Increased consumption of fruit and vegetables is related to a reduced risk of coronary heart disease: Meta-analysis of cohort studies. J Hum Hypertens. 2007;21:717–28. 126. Oyebode O, Gordon-Dseagu V, Walker A, Mindell JS. Fruit and vegetable consumption and all-cause, cancer and CVD mortality: Analysis of Health Survey for England data. J Epidemiol Community Health. 2014;68:856–62. 127. Sesso HD, Gaziano JM. Heart and vascular diseases. In: Krinsky NI, Mayne ST, Sies H, editors: Carotenoids in Health and Disease. New York, NY: Marcel Dekker; 2004, pp. 473–90. 128. Böhm V. Lycopene and heart health. Mol Nutr Food Res. 2012;56:296–303. 129. Mordente A, Guantario B, Meucci E, Silvestrini A, Lombardi E, Martorana GE, Giardina B, Bohm V, Böhm V. Lycopene and cardiovascular diseases: An update. Curr Med Chem. 2011;18:1146–63. 130. Burton-Freeman BM, Sesso HD. Whole food versus supplement : Comparing the clinical evidence of tomato intake and lycopene supplementation on cardiovascular risk factors. Adv Nutr. 2014;5:457–85. 131. Ried K, Fakler P. Protective effect of lycopene on serum cholesterol and blood pressure: Meta-analyses of intervention trials. Maturitas. 2011;68:299–310. 132. Cheng HM, Koutsidis G, Lodge JK, Ashor A, Siervo M, Lara J. Tomato and lycopene supplementation and cardiovascular risk factors: A systematic review and meta-analysis. Atherosclerosis. 2017;257:100–8. 133. Johnson EJ, Krinsky NI. Carotenoids and coronary heart disease. In: Britton G, Liaaen-Jensen S, Pfander H, editors. Carotenoids Volume 5: Nutrition and Health. Birkhauser Verkag Basek; 2009, pp. 288–300. 134. Agarwal S, Rao AV. Tomato lycopene and low density lipoprotein oxidation: A human dietary intervention study. Lipids. 1998;33:981–4. 135. Bub A, Watzl B, Abrahamse L, Delincée H, Adam S, Wever J, Müller H, Rechkemmer G. Moderate intervention with carotenoid-rich vegetable products reduces lipid peroxidation in men. J Nutr. 2000;130:2200–6. 136. Chopra M, O’Neill ME, Keogh N, Wortley G, Southon S, Thurnham DI. Influence of increased fruit and vegetable intake on plasma and lipoprotein carotenoids and LDL oxidation in smokers and nonsmokers. Clin Chem. 2000;46:1818–29. 137. Upritchard JE, Sutherland WH, Mann JI. Effect of supplementation with tomato juice, vitamin E, and vitamin C on LDL oxidation and products of inflammatory activity in type 2 diabetes. Diabetes Care. 2000;23:733–8. 138. Hadley CW, Clinton SK, Schwartz SJ. The consumption of processed tomato products enhances plasma lycopene concentrations in association with a reduced lipoprotein sensitivity to oxidative damage. J Nutr. 2003;133:727–32. 139. Briviba K, Kulling SE, Moseneder J, Watzl B, Rechkemmer G, Bub A. Effects of supplementing a lowcarotenoid diet with a tomato extract for 2 weeks on endogenous levels of DNA single strand breaks and immune functions in healthy non-smokers and smokers. Carcinogenesis. 2004;25:2373–8. 140. Burton-Freeman B, Talbot J, Park E, Krishnankutty S, Edirisinghe I. Protective activity of processed tomato products on postprandial oxidation and inflammation: A clinical trial in healthy weight men and women. Mol Nutr Food Res. 2012;56:622–31. 141. Visioli F, Riso P, Grande S, Galli C, Porrini M. Protective activity of tomato products on in vivo markers of lipid oxidation. Eur J Nutr. 2003;42:201–6. 142. Silaste ML, Alfthan G, Aro A, Kesäniemi YA, Hörkkö S. Tomato juice decreases LDL cholesterol levels and increases LDL resistance to oxidation. Br J Nutr. 2007;98:1251–8. 143. Ghavipour M, Saedisomeolia A, Djalali M, Sotoudeh G, Eshraghyan MR, Malekshahi Moghadam A, Wood LG, Moghadam AM, Wood LG. Tomato juice consumption reduces systemic inflammation in overweight and obese females. Br J Nutr. 2013;109:1–5. 144. McEneny J, Wade L, Young IS, Masson L, Duthie G, McGinty A, McMaster C, Thies F. Lycopene intervention reduces inflammation and improves HDL functionality in moderately overweight middleaged individuals. J Nutr Biochem. 2012;1:1–6.

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145. Gajendragadkar PR, Hubsch A, Mäki-Petäjä KM, Serg M, Wilkinson IB, Cheriyan J. Effects of oral lycopene supplementation on vascular function in patients with cardiovascular disease and healthy volunteers: A randomised controlled trial. PLOS ONE. 2014;9:e99070. 146. Steinberg FM, Chait A. Antioxidant vitamin supplementation and lipid peroxidation in smokers. Am J Clin Nutr. 1998;68:319–27. 147. Dugas TR, Morel DW, Harrison EH. Dietary supplementation with β-carotene, but not with lycopene, inhibits endothelial cell-mediated oxidation of low-density lipoprotein. Free Radic Biol Med. 1999;26:1238–44. 148. Sutherland WH, Walker RJ, De Jong SA, Upritchard JE. Supplementation with tomato juice increases plasma lycopene but does not alter susceptibility to oxidation of low-density lipoproteins from renal transplant recipients. Clin Nephrol. 1999;52:30–36. 149. Bub A, Barth SW, Watzl B, Briviba K, Rechkemmer G. Paraoxonase 1 Q192R (PON1-192) polymorphism is associated with reduced lipid peroxidation in healthy young men on a low-carotenoid diet supplemented with tomato juice. Br J Nutr. 2005;93:291. 150. Rao AV. Processed tomato products as a source of dietary lycopene: Bioavailability and antioxidant properties. Can J Diet Pract Res. 2004;65:161–5. 151. Shidfar F, Froghifar N, Vafa M, Rajab A, Hosseini S, Shidfar S, Gohari M. The effects of tomato consumption on serum glucose, apolipoprotein B, apolipoprotein A-I, homocysteine and blood pressure in type 2 diabetic patients. Int J Food Sci Nutr. 2011;62:289–94. 152. Thies F, Masson LF, Rudd A, Vaughan N, Tsang C, Brittenden J, Simpson WG, Duthie S, Horgan GW, Duthie G. Effect of a tomato-rich diet on markers of cardiovascular disease risk in moderately overweight, disease-free, middle-aged adults: A randomized controlled trial. Am J Clin Nutr. 2012;95:1013–22. 153. Wu X, Schauss AG. Mitigation of inflammation with foods. J Agric Food Chem. 2012;60:6703–17. 154. Arnson Y, Shoenfeld Y, Amital H. Effects of tobacco smoke on immunity, inflammation and autoimmunity. J Autoimmun. 2010;34:J258–65. 155. Aravindaram K, Yang NS. Anti-inflammatory plant natural products for cancer therapy. Planta Med. 2010;76:1103–17. 156. Li Y-F, Chang Y-Y, Huang H-C, Wu Y-C, Yang M-D, Chao P-M. Tomato juice supplementation in young women reduces inflammatory adipokine levels independently of body fat reduction. Nutrition. 2015;31:691–6. 157. Watzl B, Kulling SE, Moseneder J, Barth SW, Bub A. A 4-wk intervention with high intake of carotenoidrich vegetables and fruit reduces plasma C-reactive protein in healthy, nonsmoking men. Am J Clin Nutr. 2005;82:1052–8. 158. Jacob K, Periago MJ, Böhm V, Berruezo GR, Bohm V, Berruezo GR. Influence of lycopene and vitamin C from tomato juice on biomarkers of oxidative stress and inflammation. Br J Nutr. 2008;99:137–46. 159. Sies H, Stahl W. Nutritional protection against skin damage from sunlight. Annu Rev Nutr. 2004;24:173–200. 160. Riso P, Visioli F, Grande S, Guarnieri S, Gardana C, Simonetti P, Porrini M. Effect of a tomato-based drink on markers of inflammation, immunomodulation, and oxidative stress. J Agric Food Chem. 2006;54:2563–6. 161. Biddle MJ, Lennie TA, Bricker G V, Kopec RE, Schwartz SJ, Moser DK. Lycopene dietary intervention, a pilot study in patients with heart failure. J Cardiovasc Nurs. 2015;30:205–12. 162. Sánchez-Moreno C, Cano MP, de Ancos B, Plaza L, Olmedilla B, Granado F, Martín A. Consumption of high-pressurized vegetable soup increases plasma vitamin C and decreases oxidative stress and inflammatory biomarkers in healthy humans. J Nutr. 2004;134:3021–5. 163. Watzl B, Bub A, Briviba K, Rechkemmer G. Supplementation of a low-carotenoid diet with tomato or carrot juice modulates immune functions in healthy men. Ann Nutr Metab. 2003;47:255–61. 164. Kim H. Inhibitory mechanism of lycopene on cytokine expression in experimental pancreatitis. Ann N Y Acad Sci. 2011;1229:99–102. 165. Blum A, Monir M, Khazim K, Peleg A, Blum N. Tomato-rich (Mediterranean) diet does not modify inflammatory markers. Clin Invest Med. 2007;30:E70–4. 166. Denniss SG, Haffner TD, Kroetsch JT, Davidson SR, Rush JWE, Hughson RL. Effect of short-term lycopene supplementation and postprandial dyslipidemia on plasma antioxidants and biomarkers of endothelial health in young, healthy individuals. Vasc Health Risk Manag. 2008;4:213–22. 167. Markovits N, Amotz AB, Levy Y. The effect of tomato-derived lycopene on low carotenoids and enhanced systemic inflammation and oxidation in severe obesity. Isr Med Assoc J. 2009;11:598–601.

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168. Di Mascio P, Kaiser S, Sies H. Lycopene as the most efficient biological carotenoid singlet oxygen quencher. Arch Biochem Biophys. 1989;274:532–8. 169. Mathews-Roth MM, Pathak MA, Fitzpatrick TB, Harber LC, Kass EH. Beta-carotene as a photoprotective agents in erythropoetic protoporphyria. N Engl J Med. 1970;282:1231–1234. 170. Mathews-Roth MM. Carotenoids in erythropoetic protoporphyria and other photosensitivity diseases. Ann N Y Acad Sci. 1993;691:127–38. 171. Mathews-Roth MM, Pathak MA, Fitzpatrick TB, Harber LC, Kass EH. Beta-carotene as an oral photoprotective agent in erythropoetic protoporphyria. J Am Med Assoc. 1974;228:1004–8. 172. Mathews-Roth MM, Pathak MA, Fitzpatrick TB, Harber LH, Kass EH. Beta carotene therapy for erythropoietic protoporphyria and other photosensitivity diseases. Arch Dermatol. 1977;113:1229–32. 173. Epstein JH. Effects of beta-carotene on ultraviolet induced cancer formation in the hairless mouse skin. Photochem Photobiol. 1977;25:211–3. 174. Mathews-Roth MM, Krinsky NI. Carotenoids affect development of UV-B induced skin cancer. Photochem Photobiol. 1987;46:507–9. 175. Wingerath T, Sies H, Stahl W. Xanthophyll esters in human skin. Arch Biochem Biophys [Internet]. 1998;355:271–4. 176. Stahl W, Sies H. Beta-carotene and other carotenoids in protection from sunlight. Am J Clin Nutr. 2012;96:1179–84. 177. Aust O, Stahl W, Sies H, Tronnier H, Heinrich U. Supplementation with tomato-based products increases lycopene, phytofluene, and phytoene levels in human serum and protects against UV-light-induced erythema. Int J Vitam Nutr Res. 2005;75:54–60. 178. Stahl W, Heinrich U, Wiseman S, Eichler O, Sies H, Tronnier H. Dietary tomato paste protects against ultraviolet light-induced erythema in humans. J Nutr. 2001;1311449–51. 179. Rizwan M, Rodriguez-Blanco I, Harbottle A, Birch-Machin MA, Watson REB, Rhodes LE. Tomato paste rich in lycopene protects against cutaneous photodamage in humans in vivo: A randomized controlled trial. Br J Dermatol. 2011;164:154–62. 180. Heinrich U, Gärtner C, Wiebusch M, Eichler O, Sies H, Tronnier H, Stahl W, Gartner C, Wiebusch M, Eichler O et al. Supplementation with beta-carotene or a similar amount of mixed carotenoids protects humans from UV-induced erythema. J Nutr. 2003;133:98–101. 181. Césarini JP, Michel L, Maurette JM, Adhoute H, Béjot M. Immediate effects of UV radiation on the skin: Modification by an antioxidant complex containing carotenoids. Photodermatol Photoimmunol Photomed. 2003;19:182–9.

4

Lutein in Neural Health and Disease Amy C. Long and Amy D. Mackey

CONTENTS 4.1 Introduction............................................................................................................................. 55 4.2 Chemistry................................................................................................................................ 56 4.3 Dietary Sources and Bioavailability........................................................................................ 57 4.4 Safety of Lutein....................................................................................................................... 58 4.4.1 United States................................................................................................................ 58 4.4.2 Europe.......................................................................................................................... 58 4.4.3 Australia and New Zealand......................................................................................... 59 4.4.4 Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives..................................................................................... 59 4.5 Absorption and Digestion of Lutein........................................................................................ 59 4.6 Biology and Bioaccumulation.................................................................................................. 61 4.6.1 Lutein in Eye Health.................................................................................................... 61 4.6.2 Lutein and Eye Development....................................................................................... 61 4.6.3 Lutein and the Brain.................................................................................................... 62 4.6.4 Lutein in Neural Cell Membranes............................................................................... 62 4.6.5 Other Roles.................................................................................................................. 62 4.7 Lutein across the Lifespan in Relationship to Health and Disease......................................... 63 4.7.1 Lutein in Infancy and Childhood................................................................................ 63 4.7.2 Lutein and Visual or Cognitive Health of Infants and Children.................................64 4.7.3 Lutein in Adulthood..................................................................................................... 65 4.7.4 Lutein and Visual Performance................................................................................... 65 4.7.5 Lutein and Alzheimer’s Disease.................................................................................. 65 4.7.6 Age-Related Macular Degeneration............................................................................66 4.7.7 Cataract........................................................................................................................66 4.8 Summary.................................................................................................................................66 References......................................................................................................................................... 67

4.1 INTRODUCTION Carotenoids are a large class of naturally occurring pigments that are ubiquitous in nature. They are synthesized by plants and some microorganisms, as well as some non-photosynthetic bacteria and fungi. Carotenoids are an important and obligatory dietary constituent for many species.1 In higher plants, carotenoids are present in plastids, in the chloroplasts of photosynthetic tissues and the chromoplasts of fruits and flowers.1,2 The roles that carotenoids play in photosynthesis give important clues to their functions in humans. As accessory light-harvesting compounds, carotenoids transfer absorbed energy to chlorophylls to help fuel photosynthesis. This absorbed energy comes specifically from the 450–550 nm region of the light spectrum, the blue light region. Carotenoids also prevent damage from singlet oxygen, a free radical generated in plants as chlorophyll enters an energetic “triplet state” and reacts with molecular oxygen in the presence of light. The subsequent 55

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FIGURE 4.1  Chemical structures of lutein and closely related xanthophylls. Lutein differs from zeaxanthin and meso-zeaxanthin by the location of a single double bond. Zeaxanthin and meso-zeaxanthin are stereoisomers that differ in the orientation of a hydroxyl group.

formation of excited triplet states of carotenoids results in the dissipation of excess heat as carotenoids relax to a ground state.3 Photosynthetic organisms also benefit from the ability of carotenoids to stabilize pigment-protein complexes. Such complexes form the basic architecture of photosynthetic systems. These systems, although diverse among different organisms, follow a basic pattern of a light harvesting complex (LHC) coupled to a reaction center (RC). It appears that higher plants require lutein in order to establish stable LHCII complexes, alluding to the importance of lutein as a structural component in these assemblies.3 The carotenoids relative to humans are distinctly different in number from those that occur in nature: from a family of over 750 identified compounds, only about 40 carotenoids are present in the typical human diet, only about 20 are found in human blood, only 5 have been reported in the human infant brain, and only 3 (lutein, zeaxanthin, and mesozeaxanthin; Figure 4.1) accumulate specifically in the human retina.4,5 Bioselection of carotenoids therefore seems evident in the human body. Major roles of carotenoids in human health derive from their varying degrees of vitamin A and antioxidant activity. As one of the main carotenoids in human tissue and serum, lutein has many applications to human health. This chapter will focus on the chemical and biological properties of lutein in relation to human health.

4.2 CHEMISTRY Carotenoids are hydrocarbon molecules consisting of linked isoprene units, joined head to tail. The majority are derived from a C-40 backbone, containing 3 to 15 conjugated double bonds, whose structure determines the absorptive and antioxidant characteristics of the molecule. Modifications to the basic structure include chain elongation, isomerization, or degradation.1 Carotenoids are divided into two main classes: carotenes and xanthophylls, the latter containing at least one oxygen atom. The carotene family members are non-polar molecules, containing carbon and hydrogen only, and include α-carotene, β-carotene, and β-cryptoxanthin. Xanthophylls are oxygenated carotenoids, structurally characterized by the presence of hydroxyl groups attached to each of the two terminal β ionone rings in the molecule. Lutein and zeaxanthin are examples of xanthophylls. Zeaxanthin is a close structural isomer of lutein, and typically occurs in similar foods as lutein. The presence of

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hydroxyl groups increases the polarity and hydrophilicity of these compounds, facilitating reaction with singlet oxygen more readily than nonpolar carotenoids.6 An additional benefit to this polarity is the ability to modulate cell membrane dynamics. Polar carotenoids restrict the molecular motion of lipids, thereby increasing membrane rigidity.7

4.3  DIETARY SOURCES AND BIOAVAILABILITY Humans must obtain lutein from the diet, since it cannot be synthesized endogenously. Many commonly consumed fruits and vegetables contain lutein and zeaxanthin, with lutein content usually but not always exceeding zeaxanthin content.8,9 The highest concentrations of lutein are found in dark-green leafy vegetables such as kale, spinach, turnip greens, and collards.9 It has been reported that the bioavailability of lutein from a high-vegetable diet exceeds that of β-carotene, perhaps due to β-carotene conversion to vitamin A or the enhanced availability of a more polar carotenoid such as lutein in the aqueous environment of digestion.10,11 Lutein is highly bioavailable from egg yolks, owing to the lipid matrix of this food.12 Serum and tissue levels of lutein can be increased over time, indicating that lutein can be accumulated in the human body. Reported half-lives of lutein can vary from days, weeks, and months depending on the population, diet, dosing regimen, and design of the study. A relatively short half-life of 5.6 days has been reported,13 contrasting with other reports of much longer half-lives ranging from 33 to 76 days.14,15 Lutein occurs in a variety of tissues in the human body including lung, liver, and eye,16–18 although exactly how lutein is deposited and metabolized in various tissues is not fully understood. The concentration of lutein and zeaxanthin seems to be preferential to some tissues such as the human eye, where levels of these carotenoids can reach millimolar concentrations as they collectively form the macular pigment (MP).19 Evidence from a novel approach utilizing a plant biofactory suggests that sufficient quantities of pure lutein isotope can be produced, potentially providing an important avenue by which lutein’s distribution, metabolism, and function can be better understood.20 The current focus on healthy eating, along with multiple recommendations to consume a diet rich in fruits and vegetables, likely helps to bolster lutein intake in populations adhering to such recommendations. As such, however, studying the effects of lutein-depleted diets is difficult in humans adhering to these recommendations. Although some studies have reported success in administering low-carotenoid diets, compliance is an issue and in some populations, such as infants, consumption of a carotenoid-free diet may be viewed as unethical and oppositional to feeding breastmilk, which contains lutein. A well-studied primate animal model has provided data on the study of short- and long-term effects of lutein depletion across the lifespan. This model has been particularly important for the study of lutein in ocular disease, as nonhuman primates are the only animals with a macular structure closely resembling that in humans. The earliest use of this model described the basic model and study design, where monkeys were raised on normal or xanthophyll-free diets. In this early study, primates consuming the xanthophyll-free diet lacked MP and exhibited multiple abnormalities in the retina, including increased drusen-like bodies within the retinal pigment epithelium (RPE).21 Subsequent reports utilizing this model confirmed no detectable MP in subjects following a xanthophyll-free diet, but also showed that supplementing a portion of the initial subjects with pure lutein and or zeaxanthin (2.2 mg/kg per day) for 24 to 56 weeks resulted in a rapid increase in both serum lutein and zeaxanthin over the first 4 weeks, followed by a leveling off from 16 weeks onward. Peak MP optical density in these supplemented primates increased to a relatively steady level by 24 to 32 weeks in both lutein- and zeaxanthin-fed groups, suggesting that MP density may reach a plateau after a period of supplementation.22 A portion of the supplemented monkeys in this study received acute short-wavelength light exposures in the fovea and parafovea, with results showing that primates fed xanthophyll-free diets had a dip in the RPE cell density profile at the foveal center, rather than exhibiting a normal peak. Supplementation with xanthophylls reversed this abnormality to a more symmetric profile, indicating that RPEs are

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sensitive to depletion of xanthophylls.23 Utilizing a longer-term supplementation of xanthophylls from 24 to 101 weeks, an isomer of lutein, meso-zeaxanthin, was found to be deposited in the retinas of monkeys supplemented with lutein, although the diet did not contain meso-zeaxanthin. Thus, the discovery of lutein as the precursor of meso-zeaxanthin in the retina was an important finding.24 After long-term xanthophyll deficiency, lutein or zeaxanthin supplementation protected the fovea from blue light–induced damage.25 Lipofuscin accumulation, a marker of RPE cell damage, aging, and retinal disease, was higher in animals fed diets deficient in lutein and zeaxanthin, as well as omega-3 fatty acids. The increase corresponded to a mathematically calculated 12–20 year acceleration in lipofuscin accumulation compared to animals fed a standard diet.26

4.4  SAFETY OF LUTEIN Exposure to carotenoids primarily occurs through consumption of fruits and vegetables. Plasma levels of lutein/zeaxanthin increase following consumption of concentrated dietary sources of the carotenoids or supplements,27–29 and plasma levels of lutein/zeaxanthin are positively associated with fruit and vegetable consumption.30–33 Carotenoid intake from foods, even when ingested in large amounts (e.g., >30 mg carotenoid) are not known to be toxic.34,35 The Institute of Medicine evaluated the safety of lutein and zeaxanthin and concluded that no adverse effects, other than carotenodermia, have been reported from the consumption of carotenoids in foods, including lutein and zeaxanthin. No tolerable upper intake levels for lutein or zeaxanthin have been established.36 Lutein was recently cited as a case study for the re-examination of establishing dietary upper intake levels for bioactive nutrients.37 Although not determined to meet the classical definition of an essential nutrient in 2000,36 there is accumulating evidence that a Dietary Reference Intake should be established for lutein because of its role in eye health.38–40 In 2004, Alves-Rodrigues and Shao published a summary of the role of lutein in human health, reviewed lutein absorption and deposition, and presented information on safety of lutein intake.41 Based upon their review of the literature, the authors report that doses of 20 mg lutein per day for up to 6 months, or doses of 40 mg per day for over 2 months, were not associated with adverse effects in humans. The only side effect noted in human studies was carotenodermia, a reversible and harmless condition. The authors also review the oral animal toxicity studies conducted in rats and monkeys and the mutagenicity studies that were used to complete the GRAS determination for crystalline lutein. Similarly, Shao and Hatchcock found no adverse events in over 30 peer-reviewed studies involving lutein.42

4.4.1 United States Lutein is generally recognized as safe for use as an ingredient in a variety of food and beverage products, including baked goods and baking mixes, beverages and beverage bases, breakfast cereals, chewing gum, dairy product analogs, egg products, fats and oils, frozen dairy desserts and mixes, gravies and sauces, hard candy, infant and toddler foods (other than infant formula, at levels up to 1 mg/serving), milk products, processed fruit and vegetable products, soft candy, soups and soup mixes, medical foods intended as the sole item of the diet at levels up to 3 mg/serving,43,44 and as an ingredient in term infant formula at a maximum level of 250 µg/L.45

4.4.2 Europe In 2008, the European Food Safety Authority (EFSA)46 released a scientific opinion on the suitability of lutein in infant formula and follow-on formula; in the opinion, EFSA stated that the proposed use of 250 µg lutein/L in infant formula products raised no safety concerns. In 2010, EFSA re-evaluated lutein as a food additive and derived an acceptable daily intake (ADI) of 1 mg/kg body weight/day, based on a no-observed-adverse-effect-level (NOAEL) of 200 mg/kg body weight/day in a 90-day study in rats, with an additional 200-fold safety factor.47

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4.4.3 Australia and New Zealand In July 2008, the Food Standards Australia New Zealand (FSANZ) approved the addition of up to 250 µg lutein/L in infant formula products.48 In March 2009, FSANZ completed a First Review in which they recommended that use of up to 250 µg lutein/L be reduced to a level of up to 143 µg lutein/L (5 µg/100 kJ) in infant formula products. It was indicated that the evidence submitted to support the higher proposed levels of use in the original application did not demonstrate that lutein bioavailability from breast milk is higher than that from infant formula products. Given that there was no justification for the addition of greater amounts of lutein in infant formula, FSANZ adopted a conservative approach and reduced the permitted use levels of lutein in infant formula to reflect concentrations within the range present in breast milk. In 2009, FSANZ also recommended amending standards to permit the voluntary addition of lutein as a nutritive substance to formulated supplementary foods for young children up to a potential maximum concentration of 100 µg lutein/serving, or 500 µg lutein/L.49

4.4.4 Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives The Joint Food and Agriculture Organization (FAO)/WHO Expert Committee on Food Additives (JECFA) established a group Acceptable Daily Intake (ADI) for lutein from Tagetes erecta L. and synthetic zeaxanthin of up to 2 mg/kg body weight.50 In conjunction with dietary intake data from the U.S. Third National Health and Nutrition Examination Survey, 1988–1994 (NHANES III), the Institute of Medicine (IOM) used an expanded carotenoid database for foods reported in NHANES III to estimate the usual consumption of lutein and zeaxanthin by the total U.S. population greater than 2 months of age (29,015 individuals).36 The estimated mean and 90th percentile consumption of total lutein and zeaxanthin by the surveyed sample were 1.71 and 3.01 mg/person/day, respectively. Slightly higher mean intakes of 2.2 and 1.9 mg/person/day of total lutein and zeaxanthin, for men and women, respectively, were estimated from food frequency data obtained from 8341 adults in the 1992 National Health and Interview Survey using the USDA National Cancer Institute carotenoid food composition database.51 Kruger et al.52 estimated the intake of lutein and zeaxanthin using the dietary records for only those respondents to NHANES III who met their recommended daily intake of vegetables, as described in the Dietary Guidelines for Americans, and the carotenoid database employed by the IOM. The mean and 90th percentile for total lutein and zeaxanthin intakes for these individuals (5708 participants, approximately 25% of the total surveyed sample) were determined to be 3.83 and 7.29 mg/person/day, respectively. Therefore, estimated intakes of lutein and zeaxanthin based on recommended levels of vegetable consumption appear to be greater than twice the estimated actual intakes for the total U.S. population.52 Johnson et  al.53 estimated the individual intakes of lutein and zeaxanthin using lutein and zeaxanthin values in major dietary sources, applied to data from the NHANES 2003–2004 survey. Lutein intakes were reported to be significantly greater than zeaxanthin intakes among all age groups, both sexes, and all ethnicities.

4.5  ABSORPTION AND DIGESTION OF LUTEIN Lutein and other carotenoids are digested and absorbed similarly to dietary lipids. As a fat-soluble compound, dietary fat facilitates lutein absorption, although exact requirements for optimal absorption are varied. Relatively low levels of dietary fat in a meal are enough to enhance the carotenoid concentration in human plasma54,55 and increase the plasma response from carotenoids in raw salads.54–56 Carotenoids released from the food matrix are available for absorption by the intestinal epithelium via steps common to other lipid soluble compounds such as transfer to lipid droplets, incorporation into mixed bile salt micelles, uptake by enterocytes, and incorporation into

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chylomicrons for secretion into lymph. Release from the food matrix and pre-intestinal digestion are followed by transfer to mixed micelles during typical dietary lipid lipolysis.57 Once incorporated into mixed micelles in the intestinal lumen, the carotenoids are shuttled across the unstirred water layer and contact the brush border epithelium (enterocytes). Micelle dissociation occurs near the brush border, releasing carotenoids for uptake by the enterocyte,57 although the exact steps involved in this transfer are not completely understood.58 An important step for the absorption of carotenoids and other lipophilic compounds appears to be cleavage of phospholipids by phospholipase A2 (PLA2). The phospholipid content of micelles affects the cellular uptake of carotenoids. Uptake of micellar beta-carotene and lutein is suppressed by phosphatidylcholine in a dose-dependent manner, and the addition of PLA2 from porcine pancreas to the medium enhances the uptake of carotenoids from micelles containing phosphatidylcholine.59 Carotenoid uptake by enterocytes was previously thought to occur by simple diffusion; however, several studies support the existence of receptor-mediated transport of carotenoids in the apical membrane of enterocytes. Membrane proteins of intestinal cells, such as CD36 (cluster determinant 36), FAT (fatty acid translocase), NPC1L (Nieman Pick C1-like 1), and the ABCG5/G8 from the ABC transporters superfamily have been associated with the absorption of carotenoids.57 There is considerable interindividual variability in the absorption and tissue response to dietary carotenoids. It would follow that genetic variation in the expression of proteins involved in carotenoid metabolism could explain these biological effects in humans, and indeed such variants have been reported to affect carotenoid metabolism and carotenoid status.57,60,61 Once within enterocytes, the carotenoids are transported to the Golgi apparatus and assembled in nascent chylomicrons, which are secreted into the lymphatic system for their transport in the bloodstream.62 In the bloodstream, carotenoids are carried via lipoproteins. The degree to which each carotenoid is transported by specific lipoproteins can vary. Lutein and zeaxanthin are reported to be primarily associated with high-density lipoprotein HDL, consistent with their less hydrophobic nature relative to the carotenes.34 The specific components of HDL responsible for carotenoid binding remain to be identified. Low circulating levels of HDL as a result of a mutation in the ABCA1 transporter gene are characteristic of the Wisconsin hypoalpha mutant (WHAM) chicken. When these animals are fed a high-lutein diet, lutein levels increase in several organs, but not in retina, suggesting that HDL is critical for delivery of carotenoids to retinal tissue.63 Data suggests that the uptake of carotenoids is similar to that of cholesterol, and that HDL and the receptors of HDL such as SR-BI (scavenger receptor class B type 1) may be involved in this process. SR-BI, a member of the ATP-binding cassette (ABC) transporter super-family, mediates the selective uptake of cholesterol and cholesteryl esters by the liver and other steroidogenic tissues from HDL particles.64 Carotenoid transport in Caco-2 cells is shown to be decreased by ezetimibe, an inhibitor of cholesterol transport. This effect decreased with increasing polarity of the carotenoid molecule and required SR-BI.65 Zeaxanthin is exclusively dependent upon SR-BI for uptake in RPE cells, yet this mechanism only partly explains uptake of beta carotene.66 HDL-mediated transport of lutein is challenged by recent data showing that lutein is delivered in LDL despite its greater extent of association with HDL in serum. It is suggested that HDL-dependent uptake of zeaxanthin occurs via SR-B1, while LDL-dependent uptake of lutein occurs via the LDL receptor. By creating competition for cellular uptake by increasing the amounts of unenriched LDL, the authors showed that lutein uptake decreased by 27%.67 This finding is supported by other evidence that LDL complexes of lutein and HDL complexes of zeaxanthin and meso-zeaxanthin are taken up better by cells in culture.68 This group has also reported that all three scavenger receptor proteins (SRB1, SRB2, and CD36) are capable of binding and transporting macular carotenoids.68 Interesting data exists for how lutein might be transported to individual tissues, especially the human eye. Nature provides an example by which silkworms deliver lutein to the silk gland by both a specific cell-surface uptake protein and a specific binding protein.69 In the human macula, a similar process appears to involve the following binding proteins: (1) a lutein-binding protein, steroidogenic acute regulatory domain 3 (StARD3); (2) a zeaxanthin-binding protein, glutathione S-transferase P1; and (3) tubulin, which serves as a site for high-capacity, less specific binding protein of carotenoids in retina.70,71

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Polar lipid nutrients such as carotenoids may be less bioavailable than desired in supplements and are thus typically over-fortified in the product to ensure adequate delivery. Animal data using a lymph fistula model reported that the solubility of lutein can be significantly increased by mixing lutein in a specific combination of mono- and diglycerides (MDG) compared with standard triglyceride-based oils. This work was further corroborated in humans, where a single dose of MDG lutein resulted in a 129% increase in plasma lutein within the first 12 h compared with control.29

4.6  BIOLOGY AND BIOACCUMULATION 4.6.1 Lutein in Eye Health Lutein and zeaxanthin have suggested roles in modulating several diseases including cancer, heart disease, and stroke, as well as the eye-related disorders age-related macular degeneration (AMD) and cataract.72 Of all dietary carotenoids, only lutein and zeaxanthin concentrate within the lens17,73,74 and are predominant in the human retina. Lutein concentrates in the eye 1000 times greater than in the blood, and more specifically in the macula, a small, highly specialized area of the retina involved in relaying information to the visual cortex of the brain.75,76 In the primate macula, lutein, zeaxanthin, and meso-zeaxanthin compose the MP, often measured as macular pigment optical density (MPOD). MPOD has a multifunctional role in the retina, including improving visual performance and protecting against damaging light. As such, levels of MP in the eye are considered a “proxy” for overall health of the macula. There are several mechanisms by which lutein may protect against ocular diseases. Lutein has been shown to prevent oxidative damage to lipid membranes,77 and lutein molecules tend to orient themselves in membranes such that they maximize interactions to reduce oxidative stress and lend stability.78 The ability of supplemental lutein to accumulate in ocular tissues is a key aspect of its ability to prevent or deter ocular disease. Dietary or supplemental lutein intake in a variety of age groups is associated with an increase in both serum concentration and MP density.28,79,80 Population studies have shown positive relationships of varying strength among dietary lutein and zeaxanthin, their concentrations in blood, and MP density.81–83 As is true of many carotenoids, lutein absorbs light. The particular wavelengths of absorption correspond to approximately 450 nm, or the blue light region. Due to its high energy, blue light can penetrate tissues and cause cellular damage. Blue light is common in the environment and a constituent of the visible light spectrum: sources include sunlight and some forms of artificial lighting. Accordingly, lutein’s protective qualities on health of the eye fall into two major categories: (1) absorption or “filtering” of damaging light75 and (2) antioxidant ability.84 Lutein supplementation in healthy term newborns has been shown to increase antioxidant activity and decrease oxidative stress as compared to newborns who did not receive lutein supplementation.85 The selective accumulation of lutein in the retina may also be due to specific properties that afford membrane stability. Properties of lutein that may support membrane integrity include a high membrane solubility, transmembrane orientation, high chemical stability, and location in the most vulnerable regions of photoreceptors.4

4.6.2 Lutein and Eye Development The human eye is embryologically derived from surface ectoderm, neural ectoderm, and mesoderm by the end of 3 weeks gestation. The neural ectoderm is the same tissue from which the neural plate and other brain tissues are derived. All the basic structures of the eyes are present by the sixth week of gestation.86 Lutein is present in infant eyes during early gestation and predominates in the macula up to ∼2 years of life.87,88 The developing newborn eye may be vulnerable to damaging blue light and oxidative damage,89,90 partly due to the relatively transparent lens of an infant, which can allow more damaging blue light to reach the retina.91 The central portion of the retina, the fovea, changes dramatically after birth and evidence has shown that the infant retina “ages” rapidly due to increased

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oxidative stress. For example, the accumulation of lipofuscin is most rapid in the first years of life.92,93 The photoreceptors of the retina, rods and cones, are important for visual transmission and may be particularly vulnerable to damage due to their high concentrations of polyunsaturated fatty acids, particularly docosahexaenoic acid.

4.6.3 Lutein and the Brain The close anatomical and embryological relationship between the eye and the brain makes it logical that lutein’s proposed functions could extend to the human brain. Lutein is present in the adult brain and is the predominant carotenoid in the infant brain, including multiple anatomical areas important to memory and learning.5,94 In an infant primate model, feeding of a carotenoid-supplemented formula was found to enhance deposition of lutein in both serum and brain tissue of monkeys.95 Both MPOD and processing speed can be improved with lutein and zeaxanthin supplementation, demonstrating the beneficial effect of these dietary components on neural health.96 In older subjects with mild cognitive impairment, MPOD is shown to be significantly related to several cognitive attributes, including attention, language, and visual-spatial abilities. Fitting with its antioxidant role, lutein may ameliorate oxidative stress in neural tissues, as demonstrated by its ability to reduce malondialdehyde (MDA) levels in primary neuronal cell membranes.97

4.6.4 Lutein in Neural Cell Membranes The cell membrane is a dynamic array of molecules. Previous descriptions of the membrane as a “fluid mosaic” have been modified in recent years to incorporate data that suggests that membranes can segregate into regions. These regions are often characterized as “raft domains” and non-raft “bulk lipid” domains. Highly unsaturated lipids such as DHA seem to prefer an orientation close to non-raft proteins. Lutein, while an excellent antioxidant, may play a separate yet very important role in membrane physiology. The fact that lutein has two polar end groups that span the entire membrane means that it may afford structure and stability to the membrane, in addition to its important antioxidant role.7 In this way, each end of the lutein molecule “anchors” to the membrane bilayer, affording order and structure to that area of the membrane. The significance of lutein being found in similar areas of the membrane as DHA means that these nutrients can functionally interact in optimal ways to preserve DHA stability and, subsequently, membrane integrity and function. The membrane distribution of lutein in brain regions and its role as a neural antioxidant was recently evaluated in primates fed a stock diet containing ∼2 mg/day lutein or the stock diet plus a daily supplement of lutein (∼4.5 mg/day) and zeaxanthin (∼0.5 mg/day) for 6–12 months. Nuclear, myelin, mitochondrial, and neuronal plasma membranes were isolated from several brain regions, including prefrontal cortex, cerebellum, striatum, and hippocampus. Lutein was detected in all regions and membranes studied and lutein/zeaxanthin supplementation significantly increased total concentrations of lutein in serum, prefrontal cortex, and cerebellum. In the prefrontal cortex and striatum, mitochondrial lutein was inversely related to DHA oxidation products, supporting lutein’s potential role as an antioxidant in the brain.98

4.6.5 Other Roles Carotenoids, in addition to their antioxidant potential, have important non-antioxidant roles in biology. Apocarotenoids, molecules resulting from the oxidative cleavage of double bonds in the carotenoid molecule, serve as signaling molecules and assist plants in their interactions with the environment.99,100 Apocarotenoids are formed by chemical reactions in foods that contain carotenoids or by enzymatic cleavage of intact carotenoids. A member of the β-carotene oxygenase family, BCO2, has been found to catalyze the eccentric cleavage of xanthophylls, such as zeaxanthin and lutein and the acyclic carotenoid lycopene.101 Nature provides evidence that carotenoids may function

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in gene regulation. As plants are often exposed to far more light than they can use for photosynthesis, they have adapted by down-regulating their light harvesting systems in a mechanism termed nonphotochemical quenching (NPQ). Carotenoids participate in NPQ by regulating gene expression of proteins involved in light harvesting.3 Lastly, the stimulation of gap junction communication by carotenoids is not related to their antioxidant ability102 or their pro-vitamin A ability.103 In fact, there seems to be little correlation between the effects of carotenoids on gap junction communication and their ability to quench singlet oxygen.104 Metabolites of carotenoids often have similar or increased activity as the parent compound, as decomposition products of retinoic acid have been shown to enhance gap junction communication.105

4.7 LUTEIN ACROSS THE LIFESPAN IN RELATIONSHIP TO HEALTH AND DISEASE 4.7.1 Lutein in Infancy and Childhood Carotenoid status in the newborn depends on the nutritional status of the mother, but little is known about the transfer of carotenoids from the mother to the fetus. There is evidence that preterm birth is associated with almost absent MP.106 As lutein cannot be synthesized by humans, the nutritional status of the newborn will depend directly on its diet. Important dietary sources of this nutrient prior to food introduction are colostrum, human milk, and infant formula (Table 4.1). Lutein is ∼50% more concentrated in colostrum than in mature human milk.107 Levels of lutein in human milk are variable and generally reflective of maternal dietary intake. Mean concentrations of lutein/zeaxanthin in human milk from women in China and Japan (44 and 43 µg/L, respectively) are reported to be higher than those in the United States, perhaps due to different dietary habits.108 Lutein content in human milk decreases with lactation stage but appears to level off at approximately 2 months.109 Lipkie and colleagues report a similar decrease in lutein content with stage of lactation, and a median lutein content of samples from China, Mexico, and the U.S. across all stages as 114.4 nmol/L (∼65 µg/L).110 A selection of reported human milk levels are included in Table 4.1. Dietary carotenoid intake appears to have an impact on milk carotenoid levels, as consumption of carotenoid-rich vegetables or supplements by lactating women increased milk levels of these nutrients.111,112 Plasma concentrations of lutein are significantly correlated with intake and are similar to the breastfed infant for select supplemental concentrations studied in infant formula.113–115 A contemporary challenge facing many children today is the cumulative risk to ocular health from exposure to electronic devices with screens, such as smart phones or tablets that emit blue light. Data from a sample of individuals in developed countries born between the years of 1965 and 1996 show that an estimated 35% of this sample spends at least 9 hours a day on devices such as smartphones, tablets, and computers.116 An American Optometric Association (AOA) survey reports that 83% of children between the ages of 10 and 17 estimate that they use electronic devices 3 or more hours each day.117 A 2015 CHILDWISE Monitor survey conducted in the UK estimates that this number is actually much higher, at around 6 or more hours per day on screens and also highlights the concept of “multiscreening,” where children are viewing multiple screens at once without adequate breaks. Early behavioral and nutritional measures to ensure eye health will be important to combat later damage from such environmental stresses. Few studies have quantitatively examined lutein intake in young children. Johnson and colleagues report that intakes for all age groups from 1 to 18 years were significantly less than all older age groups. The average lutein intake reported was 279±21 µg/day, which is quite low.53 Given the unique ability of lutein to accumulate in the retina and brain, such a low early intake may hold later consequences for age-related cognitive and visual abnormalities. Various nutritional products for children now contain added lutein, such as kids’ growing-up milk beverages and various dietary supplements.

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TABLE 4.1 Selected Reports of Lutein Concentration in Human Milk Lutein (µg/L) 27.3 29.1 12.5 56.9a 62.6 100.7b,c 3.4 57.4 93 50.1 15.5 38.7 15 15 25 44 43 21.1 27.6 23.4 69 35 32 65b 40.1 35 32.6 25.2 145.2b,d

Country Mexico Japan UK USA Italy Northern Ireland Brazil Netherlands Germany Germany Cuba USA USA Australia Mexico China Japan USA USA USA USA

China, USA, Mexico USA South Korea USA Italy

Sampling Timeframe

Reference

1–12 months postpartum

146

27 days postpartum 30 days postpartum 1–41 days lactation 30–120 days Mean duration of lactation 4.2 months Day 4 postpartum Day 19, postpartum Early lactation 6–16 weeks 1–12 months postpartum

147 148 149 150 151 107 152 153 112

Week 12 28 days 56 days Weeks 1, 4, and 13 postpartum

109

Median value across all lactation stages

110

Milk collected from mothers with babies gestational age of 37 wks

154 155 156

Baseline After 6 weeks 3 and 13 weeks postpartum

114 113

157

Note: Data are mean values unless otherwise noted. Bolded values indicate lutein analyzed in combination with zeaxanthin. a Extrapolated from Figure 3. b Median value reported. c Converted from nmol/g milk fat using estimated 37 g fat/L from Jackson and Zimmer 2007. d Assumes 3.7 g/dL of milk fat.

4.7.2 Lutein and Visual or Cognitive Health of Infants and Children The benefit of lutein on visual and cognitive function in young people is difficult to detect due to the subject age and sensitivity of measurement. It is also somewhat difficult to detect improvement in such parameters in subjects already exhibiting normal or optimal visual and cognitive function, as is common in young people. However, several studies point to a benefit. MPOD measured in young children between the ages of 7 and 10 was positively and significantly associated with hippocampal-dependent memory performance.118 Using a modified test of inhibitory control paired with a neuroelectric measurement, Walk and colleagues report that MPOD in preadolescent children (mean age ~9 years) was associated with better performance on the behavioral task and neuroelectric indications of improved cognitive efficiency as exhibited by lower cognitive load.119

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4.7.3 Lutein in Adulthood The National Health and Nutrition Examination Survey (2003–2004) indicates that, on average, American adults consume only about 1.5 mg of lutein per day. For reference, a 0.5-cup serving of spinach contains approximately 11 mg of lutein.9 Recommended dark green vegetable intake in the Dietary Guidelines for Americans 2015 are 0.5 cups per week for a 1000 kcal level and up to 2 cups per week on a 2200 kcal diet. Over the course of a week, then, if one were aiming to achieve the recommended intake of dark green vegetables through spinach alone, the lutein intake per day (at a 0.5–2.0 cups per week recommendation) would range from 1.6 to 6.3 mg/day. Thus, an average intake of 1.5 mg/day demonstrates that the average American diet appears woefully lacking in lutein. Human adult brain carotenoid content reported by Craft and colleagues indicated that xanthophyll content exceeded carotene content, although β-cryptoxanthin was in the greatest concentration, regardless of region.94 Lutein tended to be higher in the frontal lobe compared with the occipital and was also higher in the gray matter than in the white matter. Additional and more recent evidence that lutein concentrates in brain tissue despite not being the major carotenoid in matched serum suggests preferential uptake of lutein in the brain.120 The content of lutein and zeaxanthin in the macula is significantly correlated with their levels in matched brain tissue, supporting that MPOD could be considered a biomarker of brain lutein concentrations, and related to the observation that MPOD is related to global cognitive function in adults.5,121,122 A larger study analyzing multiple regions determined that lutein was present in samples at higher concentrations than all other carotenoids, regardless of region.120 In adults, supplementation with both DHA and lutein significantly improved cognitive function.123 Older adults with higher levels of MPOD are reported to have significantly better global cognition, verbal learning and fluency, recall, processing speed, and perceptual speed than those with lower levels.124 Supplementation with lutein and zeaxanthin in young healthy adults increased MPOD significantly over the course of a year and resulted in significant improvements in spatial memory, reasoning ability and complex attention, above and beyond improvements due to practice effects.125 In a unique study using neuroimaging to measure the relationship of lutein and zeaxanthin to brain structure in community-dwelling older adults, serum lutein and zeaxanthin and MPOD were related to brain white matter integrity, particularly in regions vulnerable to age-related decline.126

4.7.4 Lutein and Visual Performance Visual perception and cognition are highly related.127 Both visual dysfunction and poor cognition can occur with increasing age, although the relationship between the two is not well defined. In a crosssectional analysis of two national data sets, NHANES (1999–2002) and the National Health and Aging Trends Study (NHATS, 2011–2015), vision dysfunction at distance and based on self-reports was associated with poor cognitive function.128 This underlies the importance of visual function in the maintenance of cognition. Indeed, MPOD is related to a number of visual performance parameters.129,130 Lutein supplementation in cataract patients was associated with improvements in visual acuity and reductions in glare sensitivity after a 2-year supplement.33 Stringham and Hammond measured changes in photostress recovery and glare disability after supplementing young healthy subjects with 12 mg of lutein and zeaxanthin per day for 6 months. Supplementation led to direct improvements in glare disability and photostress.130 A detailed analysis of how MP improves visibility reports a likely reduction in the veiling effects of blue haze, leading to the potential to see about 30% farther through the atmosphere compared to someone with little or no MP.131

4.7.5 Lutein and Alzheimer’s Disease The brain is particularly susceptible to oxidation due to its high metabolic activity, oxygen demand, and polyunsaturated fatty acid content.132 Indicators of both oxidative stress and low levels of antioxidants have been reported in Alzheimer’s disease patients,132,133 with demonstration that lutein can modulate

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interactions of amyloid beta with healthy cells.133 Lutein has also been shown to modulate the toxicity of amyloid beta by blocking nuclear factor kappa B (NF-κB) expression and upregulating the protective NF-E2-related factor 2 (NRF2) pathway in cerebrovascular endothelial cells.134 Lutein is an effective antioxidant and scavenger of nitric oxide, a compound with known relationship to cognitive disease.135 Due to its local concentration in areas of the eye and brain that can reach very high metabolic demand, lutein seems optimally positioned to exert a protective antioxidant effect in the brain. Min and colleagues have reported that high serum levels of lutein and zeaxanthin are associated with a lower risk of Alzheimer’s disease mortality in adults, suggesting that dietary intake of these xanthophylls may be an important and simple lifestyle habit for reducing mortality risk.136

4.7.6 Age-Related Macular Degeneration Age-related macular degeneration is the leading cause of blindness in the United States in the population over the age of 40;137 however, the incidence of AMD has declined in recent years.137,138 As reviewed by Mares,139 lutein and zeaxanthin intake are associated with a lower risk of developing AMD, presumably by reinforcing the concentrations of lutein and zeaxanthin in the macular pigment; however, there are other proposed mechanisms by which lutein and zeaxanthin modulate ocular diseases.140 Two large randomized controlled trial (RCT) intervention trials supported by the National Eye Institute, AREDS141 and AREDS2,142 investigated the effect of supplementation of select vitamins, minerals, and fatty acids on the development and progression of AMD. The original AREDS intervention used four different supplements containing (1) zinc, (2) antioxidants: Vitamins C, E, and beta-carotene, (3) zinc + antioxidants, or (4) placebo. The supplement containing the combined zinc and antioxidants yielded a significant OR of 0.72 for progression to advanced AMD compared to placebo.141 Following the relative success of the first AREDS trial, a second trial was undertaken to improve safety and efficacy of the zinc + antioxidants supplement. The AREDS2 trial included interventions that substituted lutein + zeaxanthin for beta-carotene and supplements including fish oil containing DHA and EPA. Although there was no statistical improvement in reducing further progression of AMD with any of the AREDS2 supplements compared to the original AREDS supplements,142 secondary analyses of the cohort revealed that subjects given lutein + zeaxanthin had a significant reduction in progression to advanced AMD (HR = 0.87, p = 0.04), demonstrating the AREDS2 supplement was an appropriate alternative to the original AREDS supplement in individuals with concerns about beta-carotene (smokers).143

4.7.7 Cataract Another leading cause of blindness globally is cataract. Cataract is a disease that clouds the lens and eventually blocks light from entering the eye. Cataract is successfully managed and treated with surgery; however, prevention of cataract development in both developed and developing countries would be far more cost effective. There is evidence to suggest a beneficial role of lutein and zeaxanthin in the protection against cataract development to help protect against oxidative damage to the lens.139,144 Recent evidence also suggests the effectiveness of lutein supplementation on cataract development was better in males compared to females.145

4.8 SUMMARY Lutein is an important nutrient for human health. It is a dihydroxy carotenoid with important functions in both plants and humans that center around its ability to absorb light and provide antioxidant protection. Novel roles for lutein include its ability to regulate gene expression and modulate cell membrane dynamics. Lutein specifically accumulates in the human macula and gives this structure its characteristic yellow pigmentation. It is this specific accumulation that makes lutein a promising nutrient for prevention and/or treatment of the age-related disease AMD. New

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evidence shows that lutein also accumulates in the brain, and more research is needed to determine the exact role it plays in this organ. Lutein is absorbed from the diet and is transferred to specific tissues by plasma lipoproteins and specific binding proteins. Many studies have established that the MP is related to dietary and plasma lutein levels, with higher intakes leading to higher pigment density. Lutein levels from a variety of regulatory bodies do not raise concern about the safety of lutein in various products. Studies in non-human primates have given us a unique understanding of the consequences of a diet lacking or devoid in xanthophylls. Such diets lead to lack of development of MP and disturbances in retinal development and function. While the relationship among eye function, MPOD, and plasma lutein is still a complex one, a case can be made that the average Westernized diet is low in lutein. The consequences of this modest intake may be more deleterious than previously thought, especially given the lifestyle changes in recent generations that include increased reliance on blue light–emitting devices. Although there is no requirement for lutein and no nutritional reference value or dietary recommendation has been established, the available data demonstrate that lutein may offer long-term benefits on neural function and development. Thus, its incorporation into a healthy diet is encouraged.

REFERENCES 1. Namitha KK, Negi PS. Chemistry and biotechnology of carotenoids. Crit Rev Food Sci Nutr. 2010; 50:728–60. 2. Nisar N, Li L, Lu S, Khin NC, Pogson BJ. Carotenoid metabolism in plants. Mol Plant. 2015; 8:68–82. 3. Hashimoto H, Uragami C, Cogdell RJ. Carotenoids and Photosynthesis. Subcell Biochem. 2016; 79:111–39. 4. Widomska J, Subczynski WK. Why has nature chosen lutein and zeaxanthin to protect the retina? J Clin Exp Ophthalmol. 2014; 5(1):326. 5. Vishwanathan R, Iannaccone A, Scott TM et al. Macular pigment optical density is related to cognitive function in older people. Age Ageing. 2014; 43:271–5. 6. Ojima F, Sakamoto H, Ishiguro Y, Terao J. Consumption of carotenoids in photosensitized oxidation of human plasma and plasma low density lipoprotein. Free Rad Biol Med. 1993; 15:377–84. 7. Gruszecki WI, Strzałka K. Carotenoids as modulators of lipid membrane physical properties. Biochim Biophys Acta. 2005; 1740:108–15. 8. Mangels A, Holden J, Beecher G, Forman M, Lanza E. Carotenoid content of fruits and vegetables: An evaluation of analytic data. J Am Diet Assoc. 1993; 93:284–96. 9. Perry A, Rasmussen H, Johnson EJ. Xanthophyll (lutein, zeaxanthin) content in fruits, vegetables and corn and egg products. J Food Comp Anal. 2009; 22:9–15. 10. van het Hof KH, Brouwer IA, West CE, Haddeman E, Steegers-Theunissen RP, Van Dusseldorp M, Weststrate JA, Eskes TK, Hautvast JG. Bioavailability of lutein from vegetables is five times higher than that of beta-carotene. Am J Clin Nutr. 1999; 70:261–8. 11. van Het Hof K, West C, Weststrate J, Hautvast J. Dietary factors that affect the bioavailability of carotenoids. J Nutr. 2000; 130:503–6. 12. Handelman G, Nightingale Z, Lichtenstein A, Schaefer E, Blumberg J. Lutein and zeaxanthin concentrations in plasma after dietary supplementation with egg yolk. Am J Clin Nutr. 1999; 70:247–51. 13. Thürmann PA, Schalch W, Aebischer JC, Tenter U, Cohn W. Plasma kinetics of lutein, zeaxanthin, and 3-dehydro-lutein after multiple oral doses of a lutein supplement. Am J Clin Nutr. 2005; 82(1):88–97. 14. Burri BJ, Neidlinger TR, Clifford AJ. Serum carotenoid depletion follows first-order kinetics in healthy adult women fed naturally low carotenoid diets. J Nutr. 2001; 131:2096–100. 15. Rock CL, Swendseid ME, Jacob RA, McKee RW. Plasma carotenoid levels in human subjects fed a low carotenoid diet. J Nutr. 1992; 122(1):96–100. 16. Khachik F, de Moura FF, Zhao DY, Aebischer CP, Bernstein PS. Transformations of selected carotenoids in plasma, liver, and ocular tissues of humans and in nonprimate animal models. Invest Ophthalmol Vis Sci. 2002; 43:3383–92. 17. Yeum K, Taylor A, Tang G, Russell R. Measurement of carotenoids, retinoids, and tocopherols in human lenses. Invest Ophthalmol Vis Sci. 1995; 36:2756–61. 18. Sommerburg O, Siems WG, van Kuijk FJ. Localization of carotenoids in different eye tissues. Biofactors. 2000; 11:3–6.

68

Handbook of Nutraceuticals and Functional Foods

19. Landrum JT, Bone RA, Moore LL, Gomez CM. Analysis of zeaxanthin distribution within individual human retinas. Methods Enzymol. 1999; 299:457–67. 20. Smith JW, Rogers RB, Jeon S, Rubakhin SS, Wang L, Sweedler JV, Neuringer M, Kuchan MJ, Erdman JW Jr. Carrot solution culture bioproduction of uniformly labeled (13)C-lutein and in vivo dosing in nonhuman primates. Exp Biol Med (Maywood). 2017; 242:305–15. 21. Malinow MR, Feeney-Burns L, Peterson LH, Klien ML, Neuringer M. Diet-related macular abnormalities in monkeys. IOVS. 1980; 19:857–63. 22. Neuringer M, Sandstrom M, Johnson E, Snodderly D. Nutritional manipulation of primate retinas, I: Effects of lutein or zeaxanthin supplements on serum and macular pigment in xanthophyll-free rhesus monkeys. Invest Ophthalmol Vis Sci. 2004; 45:3234–43. 23. Leung IY, Sandstrom MM, Zucker CL, Neuringer M, Snodderly DM. Nutritional manipulation of primate retinas, II: Effects of age, n-3 fatty acids, lutein, and zeaxanthin on retinal pigment epithelium. Invest Ophthalmol Vis Sci. 2004; 45:3244–56. 24. Johnson EJ, Neuringer M, Russell RM, Schalch W, Snodderly DM. Nutritional manipulation of primate retinas, III: Effects of lutein or zeaxanthin supplementation on adipose tissue and retina of xanthophyllfree monkeys. Invest Ophthalmol Vis Sci. 2005; 46:692–702. 25. Barker FM 2nd, Snodderly DM, Johnson EJ, Schalch W, Koepcke W, Gerss J, Neuringer M. Nutritional manipulation of primate retinas, V: Effects of lutein, zeaxanthin, and n-3 fatty acids on retinal sensitivity to blue-light-induced damage. Invest Ophthalmol Vis Sci. 2011; 52:3934–42. 26. McGill TJ, Renner LM, Neuringer M. Elevated fundus autofluorescence in monkeys deficient in lutein, zeaxanthin, and omega-3 fatty acids. Invest Ophthalmol Vis Sci. 2016; 57:1361–9. 27. Castenmiller JJM, West EE, Linssen JPH, van het Hof KH, Voragen AGJ. The food matrix of spinach is a limiting factor in determining the bioavailability of beta-carotene and to a lesser extent of lutein in humans. J Nutr. 1999; 129:349–55. 28. Bone RA, Landrum JT, Guerra LH, Ruiz CA. Lutein and zeaxanthin dietary supplements raise macular pigment density and serum concentrations of these carotenoids in humans. J Nutr. 2003; 133:992–8. 29. Marriage BJ, Williams JA, Chose YS, Maki KC, Vurma M, DeMichele S. Mono- and diglycerides improve lutein absorption in healthy adults: A randomized, double-blind, cross-over, single dose study. Br J Nutr. 2017; 118:813–21. 30. Campbell DR, Gross MD, Martini MC, Grandits GA, Slavin JL, Potter JD. Plasma carotenoids as biomarkers of vegetable and fruit intake. Cancer Epidemiol Biomarkers Prev. 1994; 3:493–500. 31. McEligot AJ, Rock CL, Flatt SW, Newman V, Faerber S, Pierce JP. Plasma carotenoids are biomarkers of long-term high vegetable intake in women with breast cancer. J Nutr. 1999; 129:2258–63. 32. Johnson EJ, Hammond BR, Yeum KJ, Qin J, Wang XD, Castaneda C, Snodderly DM, Russell RM. Relation among serum and tissue concentrations of lutein and zeaxanthin and macular pigment density. Am J Clin Nutr. 2000; 71:1555–62. 33. Olmedilla B, Granado F, Blanco I, Vaquero M. Lutein, but not alpha-tocopherol, supplementation improves visual function in patients with age-related cataracts: A 2-y double-blind, placebo-controlled pilot study. Nutrition. 2003; 19:21–4. 34. Olson JA. Absorption, transport, and metabolism of carotenoids in humans. Pure Appl Chem. 1994; 66:1011–6. 35. Omaye ST, Krinsky NI, Kagan VE, Mayne ST, Liebler DC, Bidlack WR. β-Carotene: Friend or foe. Fundam Appl Toxicol. 1997; 40:163–74. 36. Institute of Medicine (IOM). β-Carotene and other carotenoids. In: Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids. Panel on Dietary Antioxidants and Related Compounds, Subcommittees on Upper Reference Levels of Nutrients and Interpretation and Uses of DRIs, Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, Food and Nutrition Board, Institute of Medicine (IOM). Washington (DC): National Academy Press (NAP), 2000; pp. 325–82. 37. Yates AA, Erdman JW, Shao A, Dolan LC, Griffiths JC. Bioactive nutrients—Time for tolerable upper intake levels to address safety. Reg Toxicol Pharmacol. 2017; 84:94–101. 38. Biesalski HK, Erdman JW, Hathcock J et al. Nutrient reference values for bioactives: New approaches needed? A conference report. Eur J Nutr. 2013; 52:1–9. 39. Yetley EA, MacFarlane AJ, Greene-Finestone LS et al. Options for basing Dietary Reference Intakes (DRIs) on chronic disease endpoint: Report for a joint US-/Canadian-sponsored working group. Am J Clin Nutr. 2017; 105:249S–85S. 40. Ranard KM, Jeon S, Mohn ES, Griffiths JC, Johnson EJ, Erdman JW. Dietary guidance for lutein: Consideration for intake recommendations is scientifically supported. Eur J Nutr. 2017; S37–42. 41. Alves-Rodrigues A, Shao A. The science behind lutein. Toxicol Lett. 2004; 150(1):57–83.

Lutein in Neural Health and Disease

69

42. Shao A, Hathcock JN. Risk assessment for the carotenoids lutein and lycopene. Reg Toxicol Pharmacol. 2006; 45:289–98. 43. United States Food and Drug Administration. GRN No. 110. Lutein esters, 2002. 44. United States Food and Drug Administration. GRN No. 140. Crystalline lutein, 2004. 45. United States Food and Drug Administration. GRN No. 221. Suspended lutein, 2007. 46. European Food Safety Authority (EFSA). Scientific opinion of the Panel on Dietetic Products, Nutrition and Allergies [on a request from the European Commission on the] “Safety, bioavailability, and suitability of lutein for the particular nutritional use by infants and young children.” EFSA J. 2008; 823:1–24. 47. European Food Safety Authority (EFSA). Scientific opinion on the re-evaluation of lutein (E 161b) as a food additive. EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS). EFSA J. 2010; 8(7):1678. 48. Food Standards Australia New Zealand (FSANZ). First Review Report. Application A594: Addition of Lutein as a Nutritive Substance to Infant Formula Products. (4-09). 2009. 49. Food Standards Australia New Zealand (FSANZ). Final Assessment Report. Application A597: Addition of lutein to formulated supplementary foods for young children. (4-09). 2009. 50. Joint Expert Committee on Food Additives (JECFA). Lutein from Tagetes erecta L. In: Safety Evaluation of Certain Food Additives. Sixty-third Meeting of the Joint FAO/WHO Expert Committee on Food Additives, June 8–17, 2004, Geneva, Switz. (WHO Food Additives Series, no 54). Geneva, Switz.: World Health Organization (WHO), International Programme on Chemical Safety (IPCS), 2006; pp. 49–86, 637–638. 51. Nebeling LC, Forman MR, Graubard BI, Snyder RA. Changes in carotenoid intake in the United States: The 1987 and 1992 National Health Interview Surveys. J Am Diet Assoc 1997; 97:991–6. 52. Kruger C, Murphy M, DeFreitas Z, Pfannkuch F, Heimbach J. An innovative approach to the determination of safety for a dietary ingredient derived from a new source: Case study using a crystalline lutein product. Food Chem Toxicol 2002; 40:1535–49. 53. Johnson EJ, Maras JE, Rasmussen HM, Tucker KL. Intake of lutein and zeaxanthin differ with age, sex, and ethnicity. J Am Diet Assoc. 2010; 110:1357–62. 54. Brown MJ, Ferruzzi MG, Nguyen ML, Cooper DA, Eldridge AL, Schwartz SJ, White WS. Carotenoid bioavailability is higher from salads ingested with full-fat than with fat-reduced salad dressings as measured with electrochemical detection. Am J Clin Nutr. 2004; 80:396–403. 55. Unlu NZ, Bohn T, Clinton SK, Schwartz SJ. Carotenoid absorption from salad and salsa by humans is enhanced by the addition of avocado or avocado oil. J Nutr. 2005; 135(3):431–6. 56. Goltz SR, Campbell WW, Chitchumroonchokchai C, Failla ML, Ferruzzi MG. Meal triacylglycerol profile modulates postprandial absorption of carotenoids in humans. Mol Nutr Food Res. 2012; 56:866–77. 57. Reboul E. Absorption of vitamin A and carotenoids by the enterocyte: Focus on transport proteins. Nutrients. 2013 Sep 12; 5:3563–81. 58. Yonekura L, Nagao A. Intestinal absorption of dietary carotenoids. Mol Nutr Food Res. 2007; 51(1):107–15. 59. Sugawara T, Kushiro M, Zhang H, Nara E, Ono H, Nagao A. Lysophosphatidylcholine enhances carotenoid uptake from mixed micelles by Caco-2 human intestinal cells. J Nutr. 2001; 131:2921–7. 60. Borel P. Genetic variations involved in interindividual variability in carotenoid status. Mol Nutr Food Res. 2012; 56:228–40. 61. Herron KL, McGrane MM, Waters D, Lofgren IE, Clark RM, Ordovas JM, Fernandez ML. The ABCG5 polymorphism contributes to individual responses to dietary cholesterol and carotenoids in eggs. J Nutr. 2006; 136:1161–5. 62. Parker RS. Absorption, metabolism, and transport of carotenoids. FASEB J. 1996 Apr; 10(5):542–51. 63. Connor WE, Duell PB, Kean R, Wang Y. The prime role of HDL to transport lutein into the retina: Evidence from HDL-deficient WHAM chicks having a mutant ABCA1 transporter. Invest Ophthalmol Visual Sci. 2007; 48:4226–31. 64. Rigotti A, Miettinen HE, Krieger M. The role of the high-density lipoprotein receptor SR-BI in the lipid metabolism of endocrine and other tissues. Endocr Rev. 2003; 24(3):357–87. 65. During A, Dawson HD, Harrison EH. Carotenoid transport is decreased and expression of the lipid transporters SR-BI, NPC1L1, and ABCA1 is downregulated in Caco-2 cells treated with ezetimibe. J Nutr. 2005; 135:2305–12. 66. During A, Doraiswamy S, Harrison EH. Xanthophylls are preferentially taken up compared with betacarotene by retinal cells via a SRBI-dependent mechanism. J Lipid Res. 2008; 49:1715–24. 67. Thomas SE, Harrison EH. Mechanisms of selective delivery of xanthophylls to retinal pigment epithelial cells by human lipoproteins. J Lipid Res. 2016; 57:1865–78.

70

Handbook of Nutraceuticals and Functional Foods

68. Shyam R, Vachali P, Gorusupudi A, Nelson K, Bernstein PS. All three human scavenger receptor class B proteins can bind and transport all three macular xanthophyll carotenoids. Arch Biochem Biophys. 2017; 634:21–8. 69. Sakudoh T, Iizuka T, Narukawa J et al. A CD36-related transmembrane protein is coordinated with an intracellular lipid binding protein in selective carotenoid transport for cocoon coloration. J Biol Chem. 2010; 285:7739–51. 70. Bhosale P, Bernstein P. Vertebrate and invertebrate carotenoid binding proteins. Arch Biochem Biophys. 2007; 458:121–7. 71. Li B, Vachali P, Bernstein PS. Human ocular carotenoid-binding proteins. Photochem Photobiol Sci. 2010; 9:1418–25. 72. Ribaya-Mercado J, Blumberg J. Lutein and zeaxanthin and their potential roles in disease prevention. J Am Coll Nutr. 2004; 23:567S–87S. 73. Bates C, Chen S, Macdonald A, Holden R. Quantitation of vitamin E and a carotenoid pigment in cataractous human lenses, and the effect of a dietary supplement. Int J Vitam Nutr Res. 1996; 66:316–21. 74. Bernstein P, Khachik F, Carvalho L, Muir G, Zhao D, Katz N. Identification and quantitation of carotenoids and their metabolites in the tissues of the human eye. Exp Eye Res. 2001; 72:215–23. 75. Krinsky NI, Landrum JT, Bone RA. Biologic mechanisms of the protective role of lutein and zeaxanthin in the eye. Annu Rev Nutr. 2003; 23:171–201. 76. Landrum JT, Bone RA. Lutein, zeaxanthin, and the macular pigment. Arch Biochem Biophys. 2001; 385:28–40. 77. Woodall A, Britton G, Jackson M. Carotenoids and protection of phospholipids in solution or in liposomes against oxidation by peroxyl radicals: Relationship between carotenoid structure and protective ability. Biochim Biophys Acta. 1997; 1336:575–86. 78. Wisniewska A, Subczynski W. Effects of polar carotenoids on the shape of the hydrophobic barrier of phospholipid bilayers. Biochim Biophys Acta. 1998; 1368:235–46. 79. Nolan J, Stack J, O’connell E, Beatty S. The relationships between macular pigment optical density and its constituent carotenoids in diet and serum. Invest Ophthalmol Vis Sci. 2007; 48:571–82. 80. Bhosale P, Zhao da Y, Bernstein P. HPLC measurement of ocular carotenoid levels in human donor eyes in the lutein supplementation era. Invest Ophthalmol Vis Sci. 2007; 48:543–9. 81. Hammond BR, Curran-Celentano J, Judd S et al. Sex differences in macular pigment optical density: Relation to plasma carotenoid concentrations and dietary patterns. Vision Res. 1996; 36:2001–12. 82. Bone RA, Landrum JT, Dixon Z, Chen Y, Llerena CM. Lutein and zeaxanthin in the eyes, serum and diet of human subjects. Exp Eye Res. 2000; 71:239–45. 83. Curran-Celentano J, Hammond BR, Ciulla TA, Cooper DA, Pratt LM, Danis RB. Relation between dietary intake, serum concentrations, and retinal concentrations of lutein and zeaxanthin in adults in a Midwest population. Am J Clin Nutr. 2001; 74:796–802. 84. Chucair AJ, Rotstein NP, Sangiovanni JP, During A, Chew EY, Politi LE. Lutein and zeaxanthin protect photoreceptors from apoptosis induced by oxidative stress: Relation with docosahexaenoic acid. Invest Ophthalmol Vis Sci. 2007; 48:5168–77. 85. Perrone S, Longini M, Marzocchi B, Picardi A, Bellieni CV, Proietti F, Rodriguez A, Turrisi G, Buonocore G. Effects of lutein on oxidative stress in the term newborn: A pilot study. Neonatology. 2010; 97:36–40. 86. Mukherjee PK. Pediatric Ophthalmology. Delhi, India: New Age International Pvt Ltd., 2005. 87. Bone RA, Landrum JT, Fernandez L, Tarsis SL. Analysis of the macular pigment by HPLC: Retinal distribution and age study. Invest Ophthalmol Vis Sci. 1988; 29:843–9. 88. Handelman GJ, Dratz EA, Reay CC, van Kuijk JG. Carotenoids in the human macula and whole retina. Invest Ophthalmol Vis Sci. 1988; 29:850–5. 89. Hardy P, Dumont I, Bhattacharya M, Hou X, Lachapelle P, Varma DR, Chemtob S. Oxidants, nitric oxide and prostanoids in the developing ocular vasculature: A basis for ischemic retinopathy. Cardiovasc Res. 2000; 47:489–509. 90. Zimmer JP, Hammond BR Jr. Possible influences of lutein and zeaxanthin on the developing retina. Clin Ophthalmol. 2007; 1:25–35. 91. Dillon J, Zheng L, Merriam JC, Gaillard ER. Transmission of light to the aging human retina: Possible implications for age related macular degeneration. Exp Eye Res. 2004; 79:753–9. 92. Wing GL, Blanchard GC, Weiter JJ. The topography and age relationship of lipofuscin concentration in the retinal pigment epithelium. IOVS. 1978; 17:601–7. 93. Feeney-Burns L, Hilderbrand ES, Eldridge S. Aging human RPE: Morphometric analysis of macular, equatorial, and peripheral cells. IOVS. 1984; 25:195–200.

Lutein in Neural Health and Disease

71

94. Craft NE, Haitema TB, Garnett KM, Fitch KA, Dorey CK. Carotenoid, tocopherol, and retinol concentrations in elderly human brain. J Nutr Health Aging. 2004; 8:156–62. 95. Jeon S, Neuringer M, Johnson EE, Kuchan MJ, Pereira SL, Johnson EJ, Erdman JW. Effect of carotenoid supplemented formula on carotenoid bioaccumulation in tissues of infant rhesus macaques: A pilot study focused on lutein. Nutrients. 2017; 9. 96. Renzi LM, Dengler MJ, Puente A, Miller LS, Hammond BR Jr. Relationships between macular pigment optical density and cognitive function in unimpaired and mildly cognitively impaired older adults. Neurobiol Aging. 2014; 35:1695–9. 97. Vazhappilly R, Lai C-S, Kuchan M. Natural vitamin E and lutein protects DHA from peroxidation in neurons. Abstract presented at: American College of Nutrition 54th Annual Conference; November 13–16, 2013; San Diego, CA. 98. Mohn ES, Erdman JW Jr, Neuringer M, Kuchan MJ, Johnson EJ. Brain xanthophyll content and exploratory gene expression analysis: Subspecies differences in rhesus macaque. Genes Nutr. 2017; 12:9. 99. Walter MH, Floss DS, Strack D. Apocarotenoids: Hormones, mycorrhizal metabolites and aroma volatiles. Planta. 2010; 232:1–7. 100. Cazzonelli CI, Pogson BJ. Source to sink: Regulation of carotenoid biosynthesis in plants. Trends Plant Sci. 2010; 15:266–74. 101. Eroglu A, Harrison EH. Carotenoid metabolism in mammals, including man: Formation, occurrence, and function of apocarotenoids. J Lipid Res. 2013; 54:1719–30. 102. Stahl W, Ale-Agha N, Polidori CM. Non-antioxidant properties of carotenoids. Biol Chem. 2002; 383:553–8. 103. Zhang LX, Cooney RV, Bertram JS. Carotenoids up-regulate connexin-43 gene expression independent of their provitamin A or antioxidant properties. Cancer Res. 1992; 52:5707–12. 104. Stahl W, Sies H. Antioxidant activity of carotenoids. Mol Asp Med. 2003; 24:345–51. 105. Teicher V, Kucharski N, Martin H, van der Saag P, Sies H, Stahl W. Biological activities of apocanthaxanthinoic acids related to gap junctional communication. Arch Biochem Biophys. 1999; 365:150–5. 106. Bernstein PS, Sharifzadeh M, Liu A, Ermakov I, Nelson K, Sheng X, Panish C, Carlstrom B, Hoffman RO, Gellermann W. Blue-light reflectance imaging of macular pigment in infants and children. Invest Ophthalmol Vis Sci. 2013; 54:4034–40. 107. Schweigert FJ, Bathe K, Chen F, Büscher U, Dudenhausen JW. Effect of the stage of lactation in humans on carotenoid levels in milk, blood plasma and plasma lipoprotein fractions. Eur J Nutr. 2004; 43(1):39–44. 108. Canfield LM, Clandinin MT, Davies DP et al. Multinational study of major breast milk carotenoids of healthy mothers. Eur J Nutr. 2003; 42:133–41. 109. Song BJ, Jouni ZE, Ferruzzi MG. Assessment of phytochemical content in human milk during different stages of lactation. Nutrition. 2013; 29:195–202. 110. Lipkie TE, Morrow AL, Jouni ZE, McMahon RJ, Ferruzzi MG. Longitudinal survey of carotenoids in human milk from urban cohorts in China, Mexico, and the USA. PLOS ONE. 2015; 10:e0127729. 111. Allen LH, Haskell M. Estimating the potential for vitamin A toxicity in women and young children. J Nutr. 2002; 132:2907S–19S. 112. Canfield LM, Giuliano AR, Neilson EM, Yap HH, Graver EJ, Cui HA, Blashill BM. Beta-carotene in breast milk and serum is increased after a single beta-carotene dose. Am J Clin Nutr. 1997; 66:52–61. 113. Mackey AD, Albrecht D, Oliver J, Williams T, Long AC, Price PT. Plasma carotenoid concentrations of infants are increased by feeding a milk-based infant formula supplemented with carotenoids. J Sci Food Agric. 2013; 93:1945–52. 114. Bettler J, Zimmer JP, Neuringer M, DeRusso PA. Serum lutein concentrations in healthy term infants fed human milk or infant formula with lutein. Eur J Nutr. 2010; 49:45–51. 115. Price P, Mackey A, Oliver J, Albrecht D, Kuchan M. Infant plasma response to lutein in formula and human milk. Acta Biologica Cracoviensia 2011; 1.15 (abstract). 116. The Vision Council. Digital Eye Strain. Available at: https://www.thevisioncouncil.org/content/digitaleye-strain. Accessed 12 Feb 2018). 117. American Optometric Association. Survey Reveals Parents Drastically Underestimate the Time Kids Spend on Electronic Devices. Children’s Omnibus survey, 2014. Available at: http://www.aoa. org/newsroom/survey-reveals-parents-drastically-underestimate-the-time-kids-spend-on-electronicdevices?sso=y. Accessed 12 Feb 2018. 118. Hassevoort KM, Khazoum SE, Walker JA et  al. Macular carotenoids, aerobic fitness, and central adiposity are associated differentially with hippocampal-dependent relational memory in preadolescent children. J Pediatr. 2017; 183:108–14.e1.

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119. Walk AM, Khan NA, Barnett SM, Raine LB, Kramer AF, Cohen NJ, Moulton CJ, Renzi-Hammond LM, Hammond BR, Hillman CH. From neuro-pigments to neural efficiency: The relationship between retinal carotenoids and behavioral and neuroelectric indices of cognitive control in childhood. Int J Psycholphysiol. 2017; 118:1–8. 120. Johnson EJ, Vishwanathan R, Johnson MA et al. Relationship between serum and brain carotenoids, α-tocopherol, and retinol concentrations and cognitive performance in the oldest old from the Georgia Centenarian Study. J Aging Res. 2013; 2013:951786 121. Vishwanathan R, Kuchan MJ, Sen S, Johnson EJ. Lutein and preterm infants with decreased concentrations of brain carotenoids. J Pediatr Gastroenterol Nutr. 2014; 59:659–65. 122. Feeney J, Finucane C, Savva GM, Cronin H, Beatty S, Nolan JM, Kenny RA. Low macular pigment optical density is associated with lower cognitive performance in a large, population-based sample of older adults. Neurobiol Aging. 2013; 34:2449–56. 123. Johnson EJ, McDonald K, Caldarella SM, Chung HY, Troen AM, Snodderly DM. Cognitive findings of an exploratory trial of docosahexaenoic acid and lutein supplementation in older women. Nutr Neurosci. 2008; 11:75–83. 124. Lindbergh CA, Mewborn CM, Hammond BR, Renzi-Hammond LM, Curran-Celentano JM, Miller LS. Relationship of lutein and zeaxanthin levels to neurocognitive functioning: An fMRI study of older adults. J Int Neuropsychol Soc. 2017; 23:11–22. 125. Renzi-Hammond LM, Bovier ER, Fletcher LM, Miller LS, Mewborn CM, Lindbergh CA, Baxter JH, Hammond BR. Effects of a lutein and zeaxanthin intervention on cognitive function: A randomized, double-masked, placebo-controlled trial of younger healthy adults. Nutrients. 2017; 9:pii: E1246. 126. Mewborn CM, Terry DP, Renzi-Hammond LM, Hammond BR, Miller LS. Relation of retinal and serum lutein and zeaxanthin to white matter integrity in older adults: A diffusion tensor imaging study. Arch Clin Neuropsychol. 2017; 17:1–4. 127. Tacca MC. Commonalities between perception and cognition. Front Psychol. 2011; 302:358. 128. Chen SP, Bhattacharya J, Pershing S. Association of vision loss with cognition in older adults. JAMA Ophthalmol. 2017; 135:963–70. 129. Renzi LM, Hammond BR Jr. The relation between the macular carotenoids, lutein and zeaxanthin, and temporal vision. Ophthalmic Physiol Opt. 2010 Jul; 30(4):351–7. 130. Stringham JM, Hammond BR. Macular pigment and visual performance under glare conditions. Opt Vis Sci. 2008; 85:82–8. 131. Hammond BR Jr, Wooten BR, Engles M, Wong JC. The influence of filtering by the macular carotenoids on contrast sensitivity measured under simulated blue haze conditions. Vision Res. 2012; 15; 63:58–62. 132. Mecocci P, Polidori MC, Cherubini A, Ingegni T, Mattioli P, Catani M, Rinaldi P, Cecchetti R, Stahl W, Senin U, Beal MF. Lymphocyte oxidative DNA damage and plasma antioxidants in Alzheimer disease. Arch Neurol. 2002; 59(5):794–8. 133. Nakagawa K, Kiko T, Miyazawa T, Sookwong P, Tsuduki T, Satoh A, Miyazawa T. Amyloid β-induced erythrocytic damage and its attenuation by carotenoids. FEBS Lett. 2011; 585(8):1249–54. 134. Liu T, Liu WH, Zhao JS, Meng FZ, Wang H. Lutein protects against β-amyloid peptide-induced oxidative stress in cerebrovascular endothelial cells through modulation of Nrf-2 and NF-κb. Cell Biol Toxicol. 2017; 33:57–67. 135. Stringham JM, Garcia PV, Smith PA, Hiers PL, McLin LN, Kuyk TK, Foutch BK. Macular pigment and visual performance in low-light conditions. Invest Ophthalmol Vis Sci. 2015; 56:2459–68. 136. Min JY, Min KB. Serum lycopene, lutein and zeaxanthin, and the risk of Alzheimer’s disease mortality in older adults. Dement Geriatr Cogn Disord. 2014; 37:246–56. 137. Friedman DS, O’Colmain BJ, Muñoz B, Tomany SC, McCarty C, de Jong PT, Nemesure B, Mitchell P, Kempen J; Eye Diseases Prevalence Research Group. Prevalence of age-related macular degeneration in the United States. Arch Ophthalmol 2004; 122:564–72. 138. Huang GH, Klein R, Klein BE, Tomany SC. Birth cohort effect on prevalence of age-related maculopathy in the Beaver Dam Eye Study. Am J Epidemiol. 2003; 157(8):721–9. 139. Mares J. Lutein and zeaxanthin isomers in eye health and disease. Ann Rev Nutr. 2016; 36:571–602. 140. Bernestein PS, Li B, Vachali PP, Gorusupudi A, Shyam R, Henriksen BS, Nolan JM. Lutein, zeaxanthin, and meso-zeaxanthin: The basic and clinical science underlying carotenoid-based nutritional interventions against ocular disease. Prog Ret Eye Res. 2016; 50:24–66. 141. Age-Related Eye Disease Study Research Group. A randomized, placebo-controlled, clinical trial of high-dose supplementation with vitamin C and E, beta-carotene, and zinc for age-related macular degeneration and vision loss: AREDS report no. 8. Arch. Ophthalmol. 2001; 119:1417–36.

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142. Age-Related Eye Disease Study 2 Research Group. Lutein + zeaxanthin and omega-3 fatty acids for age-related macular degeneration: The Age-Related Eye Disease Study2 (AREDS2) randomized clinical trial. JAMA. 2013; 309:2005–15. 143. Age-Related Eye Disease Study 2 Research Group. Secondary analyses of the effects of lutein/zeaxanthin on age-related macular degeneration progression AREDS2 report no. 3. JAMA Ophthalmol. 2014; 132:142–9. 144. Mares J. Food antioxidants to prevent cataract. JAMA 2015; 313:1048–9. 145. Hayashi R, Hayashi S, Sakai M, Arai K, Chikuda M, Machida S. Gender difference in mRNA expression of aquaporin 8 and glutathione peroxidase in cataractous lens following intake of an antioxidant supplement. Exp Eye Res 2018; 168:28–32. 146. Jackson JG, Zimmer PJ. Lutein and zeaxanthin in human milk independently and significantly differ among women from Japan, Mexico, and the United Kingdom. Nutrition Research 2007; 27:449–53. 147. Gossage CP, Deyhim M, Yamini S, Douglass LW, Moser-Veillon PB. Carotenoid composition of human milk during the first month postpartum and the response to beta-carotene supplementation. Am J Clin Nutr. 2002 Jul; 76(1):193–7. 148. Cena H, Castellazzi AM, Pietri A, Roggi C, Turconi G. Lutein concentration in human milk during early lactation and its relationship with dietary lutein intake. Public Health Nutr. 2009 Oct; 12(10):1878–84. 149. Jewell VC, Mayes CB, Tubman TR, Northrop-Clewes CA, Thurnham DI. A comparison of lutein and zeaxanthin concentrations in formula and human milk samples from Northern Ireland mothers. Eur J Clin Nutr. 2004; 58:90–7. 150. Meneses F, Torres AG, Trugo NM. Influence of recent dietary intake on plasma and human milk levels of carotenoids and retinol in Brazilian nursing women. Adv Exp Med Biol. 2004; 554:351–4. 151. Tacken KJ, Vogelsang A, van Lingen RA, Slootstra J, Dikkeschei BD, van Zoeren-Grobben D. Loss of triglycerides and carotenoids in human milk after processing. Arch Dis Child Fetal Neonatal Ed. 2009; 94:F447–50. 152. Macias C, Schweigert FJ. Changes in the concentration of carotenoids, vitamin A, alpha-tocopherol and total lipids in human milk throughout early lactation. Ann Nutr Metab. 2001; 45(2):82–5. 153. Jackson JG, Lien EL, White SJ, Bruns NJ, Kuhlman CF. Major carotenoids in mature human milk: Longitudinal and diurnal patterns. Nutr Biochem. 1998; 9:2–7. 154. Hanson C, Lyden E, Furtado J, Van Ormer M, Anderson-Berry A. A comparison of nutritional antioxidant content in breast milk, donor milk, and infant formulas. Nutrients. 2016; 8:pii: E681. 155. Kim H, Yi H, Jung JA, Chang N. Association between lutein intake and lutein concentrations in human milk samples from lactating mothers in South Korea. Eur J Nutr. 2018; 57:417–421. 156. Sherry CL, Oliver JS, Renzi LM, Marriage BJ. Lutein supplementation increases breast milk and plasma lutein concentrations in lactating women and infant plasma concentrations but does not affect other carotenoids. J Nutr. 2014; 144:1256–63. 157. Costa S, Giannantonio C, Romagnoli C, Barone, G, Gervasoni J, Perri A, Zecca E. Lutein and zeaxanthin concentrations in formula and human milk samples from Italian mothers. Eur J Clin Nutr. 2015; 69, 531–2.

5 Chemistry, Function, and Garlic

Implications for Health and Disease Sharon A. Ross and Craig S. Charron CONTENTS 5.1 Introduction............................................................................................................................. 75 5.2 Garlic Composition and Chemistry......................................................................................... 76 5.3 Implications in Health............................................................................................................. 78 5.4 Antimicrobial Activity............................................................................................................. 78 5.5 Cancer...................................................................................................................................... 81 5.5.1 Nitrosamine and Heterocyclic Amine Formation....................................................... 82 5.5.2 Carcinogen Activity Modulation.................................................................................84 5.5.3 Cell Cycle Arrest/Apoptosis........................................................................................ 86 5.5.4 DNA Repair................................................................................................................. 86 5.5.5 Epigenetic Modulation................................................................................................. 87 5.5.6 Redox and Antioxidant Capacity................................................................................. 88 5.5.7 Immunocompetence/Immunonutrition........................................................................ 88 5.5.8 COX/LOX Pathways....................................................................................................90 5.5.9 Diet as a Modifier........................................................................................................90 5.6 Cardiovascular Disease........................................................................................................... 91 5.6.1 Cholesterol and Lipoproteins....................................................................................... 91 5.6.2 Blood Pressure.............................................................................................................92 5.6.3 Plaque and Platelet Aggregation..................................................................................92 5.7 Summary and Conclusions...................................................................................................... 93 Acknowledgments.............................................................................................................................94 References.........................................................................................................................................94

5.1 INTRODUCTION Garlic (Allium sativum) has been valued for its medicinal properties for centuries. In recent years, this interest has been reflected by numerous studies investigating the potential of garlic to reduce the risk of cardiovascular disease and cancer.1–4 The ability of garlic and related components to serve as antioxidants5 and influence immunocompetence6 and possibly cognitive function7 suggest that its health implications may be extremely widespread. A member of the Alliaceae family, garlic is one of the more economically important cultivated spices. Large amounts of garlic are produced annually in China and India. In 2015, 3.83 million cwt. of garlic were harvested from 23,600 acres in the United States.8 About 95% of this amount is produced in California. Although considerable consumption occurs as fresh garlic, it is also found as dehydrates, flakes, and salts in a variety of food preparations. Dozens of garlic supplements are also commercially available as essential oils, garlic-oil macerate, garlic powder, or garlic extract. Garlic has continued to be one of the top-selling herbs in the United States. 75

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Garlic is often referred to as a spice, herb, or vegetable. Along with onions, leeks, shallots, and chives, it is one of the major allium foods consumed by humans. The garlic bulb consists of several individual cloves, each weighing about 3 g. Actual garlic intakes are not known with certainty, especially as garlic is not typically considered in dietary assessment surveys. Intakes are thought to vary from region to region and from individual to individual. Annual per capita retail consumption of fresh garlic has ranged from 2.2 to 2.8 lbs. since 2000.9 Data used in a meta-analysis of colorectal and stomach cancer suggested that the mean intake (±SD) of raw and cooked garlic intake across all published reports was 18.3 ± 14.2 g per week, or about 6 cloves of garlic per week.10 Consumption ranged from none to 3.5 g per week (about 1 clove), whereas the highest intake exceeded 28.8 g per week (about 9 to 10 cloves). Although negative consequences are not always an outcome of high garlic intake, some individuals may be more susceptible to side effects than others. While their incidence is low, a spectrum of adverse allergic reactions can occur following contact with garlic.11,12 Even though garlic is recognized as a powerful irritant, a few reports of allergic contact dermatitis appear in the literature.13 Avoidance of direct contact seems to be the most logical approach for food handlers who are sensitive, but this may be more difficult than anticipated, as diallyl disulfide (DADS), an active irritant, penetrates most commercially available gloves.14 Excessive garlic intake has also been reported to lead to hemolytic anemia. The severity of the anemia correlates with a reduction in erythrocyte-reduced glutathione (GSH) and plasma ascorbic acid.15 Incubations of canine erythrocytes with sodium 2-propanyl thiosulfate from garlic were found to increase methemoglobin concentration and Heinz body occurrences, indicating that this compound may be the cause of oxidative damage in canine erythrocytes.16 Umar et  al.15 found that ascorbic acid or vitamin E supplements prevented the garlic-precipitated reduction in GSH and plasma ascorbic acid, thereby providing greater protection to the erythrocyte membrane. Other possible adverse effects of garlic include herb-drug interactions, particularly with regard to anticoagulant or antiretroviral therapy, and mild gastrointestinal discomfort, which is the most common adverse effect.17

5.2  GARLIC COMPOSITION AND CHEMISTRY The use of garlic typically centers on its unique flavor and odor characteristics. Unlike other foods, garlic is distinctive in that about 1% of its dry weight is sulfur.18 Garlic is of somewhat limited nutritional value because its total intake is typically low, although it is more nutritious than onions on a fresh-weight basis. A 3-g serving of garlic provides about 4.5 mg of potassium, 0.6 g of carbohydrate, and trace amounts of calcium, fiber, iron, and vitamin C. Table 5.1 provides some compositional information about garlic. Carbohydrates provide about 33% of garlic’s weight, whereas protein accounts for another 6.4%. Whereas many of garlic’s health benefits have been attributed to its sulfur components, other constituents, including arginine, selenium, oligosaccharides, and flavonoids, may also convey health benefits. The chemistry of sulfur compounds found in garlic is exceedingly complex and not completely understood.19 Regardless, it is known that the primary sulfur-containing constituents in garlic bulbs are γ-glutamyl-S-alk(en)yl-L-cysteines and S-alk(en)yl-L-cysteine sulfoxides. The content of S-alk(en)ylcysteine sulfoxide in garlic typically ranges between 0.53% and 1.3% of the fresh weight, with alliin (S-allylcysteine sulfoxide) the largest contributor.20 This variation likely reflects environmental factors, including climate or cropping conditions.21 Similarly, the processing method used can markedly influence the amounts and types of individual sulfur compounds. Alliin concentrations can increase during storage as a result of the transformation of γ-glutamylcysteines. In addition to alliin, garlic bulbs contain small amounts of (+)-S-methyl-Lcysteine sulfoxide (methiin) and (+)-S-(trans-1-propenyl)-L-cysteine sulfoxide, S-(2-carboxypropyl) glutathione, γ-glutamyl- S-allyl-L-cysteine, γ-glutamyl-S-(trans-1-propenyl)-L-cysteine, and γ-glutamyl-S-allylmercapto-L-cysteine.18,22

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TABLE 5.1 Content of Selected Components in Edible Garlic Component Water, g Energy, kcal Protein, g Total lipid (fat), g Carbohydrate, g Fiber, total dietary, g Calcium, mg Magnesium, mg Phosphorus, mg Potassium, mg Selenium, mcg Vitamin C, mg Folate, µg

Amount/100 g 58.6 149.0 6.4 0.5 33.1 2.1 181.0 25.0 153.0 401.0 14.2 31.2 3.1

Source: Data adapted from the USDA Nutrient Database for Standard Reference, Release 28 (May 2016).

The characteristic odor of garlic arises from allicin (thio-2-propene-1-sulfinic acid S-allyl ester) and oil-soluble sulfur compounds formed when the bulb is crushed or damaged. This membrane destruction yields organosulfur degradation products as a result of the release of the enzyme alliinase, which rapidly converts alliin to form the odiferous alkyl alkane-thiosulfinates, including allicin. Because allicin is unstable, it further decomposes to sulfides, ajoene, and dithiins.23,24 Tamaki and Sonoki25 reported that strong garlic flavor and scent were linked to a higher content of volatile sulfur. Not surprisingly, heating garlic reduced allyl mercaptan (AM), methyl mercaptan, and allyl methyl sulfide (AMS) concentrations and reduced its odor, possibly because of an inactivation of alliinase.25 The specific formulation of a garlic product has a clear impact on its sulfur compounds. Aged garlic extract (AGE) is sold in tablet, capsule, and liquid form, and is produced by extraction of chopped garlic in aqueous ethanol, followed by filtration and concentration. S-allylcysteine, S-trans-1-propenylcysteine, and S-allylmercaptocysteine arise as predominant compounds, whereas concentrations of γ-glutamylS-alkenylcysteines decrease due to the aging process.26 Steam-distilled garlic oil has high levels of mono- and polysulfides, particularly diallyl disulfide, diallyl trisulfide, allyl methyl disulfide, and allyl methyl trisulfide.27 This product is normally diluted 100–200-fold in vegetable oil. Another garlic formulation, oil-extracted garlic macerate, involves the extraction of macerated garlic in soybean or other vegetable oil, which is then sold as capsules. 2-vinyl-4-H-1,3-dithiin constitutes about half of the sulfur compounds on a weight basis, whereas 3-vinyl-4-H-1,2-dithiin constitutes less than a quarter.27 The composition of sulfur compounds in commercial preparations is highly variable. Nevertheless, the stability of some of them appears acceptable, according to Lawson and Gardner.28 They reported that the allyl thiosulfinates of blended fresh garlic were stable for at least 2 years when stored at –80°C. Likewise, they found the dissolution release of thiosulfinates from enteric-coated garlic tablets was near 95%, and the bioavailability, as determined by breath allyl methyl sulfide, was virtually complete and equivalent to that occurring with crushed fresh garlic. The S-allylcysteine (SAC) occurring in deodorized garlic preparations was found to be stable for 12 months when stored at ambient temperature. More compositional information should be provided about garlic preparations available in the marketplace, especially when claims are being made about a specific preparation.29 Greater attention to the types and

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amounts of active compounds in the various products will likely resolve some of the inconsistencies in the literature about the potential health benefits of garlic and commercially prepared extracts, solutions, or tablets. Standardization of the various garlic preparations with respect to one constituent is not a possibility, as the various preparations available in the marketplace have different active components. The development of reference assays that can evaluate the relative bioactivity/potency across preparations may be one of the only solutions for comparing the various preparations available. The in vivo pharmacokinetics of allyl sulfur compounds have been studied. Lachmann et al.30 reported the distribution of allicin and vinyldithiines in the form of an oil macerate of the 35S-labeled substance in rats. Overall, the absorption and the elimination of 35S-alliin was faster than for other garlic constituents, with maximum blood levels reached within the first 10 min after exposure. Alliin elimination from the blood was almost complete after 6 h. Maximum blood concentrations of 35S-allicin were not reached until 30 to 60 min after treatment, and for vinyldithiines, the maximum was not achieved until 120 min. Both allicin and vinyldithiines were present in blood at the end of their 72-h study. Urinary excretion suggested an absorption rate approximating 65% for allicin and 73% for vinyldithiines. Lawson and Wang suggested that allicin absorption in humans is about 95%, although precision was limited because of the rapid metabolism and absence in the blood after consumption.31 The presence of allicin in blood is transient. Allicin is rapidly transformed in the liver to DADS and allyl mercaptan and, even when incubated in heparinized blood, is converted within 5 min to DADS.32,33 DADS can also be further transformed into AM, allyl methyl sulfide, allyl methyl sulfoxide, and allyl methyl sulphone.34 Teyssier et al.35 provided evidence that DADS can be reconverted to diallyl thiosulfinate (allicin) in tissues principally by oxidation arising from cytochrome P450 monooxygenases, and to a limited extent by flavin-containing monooxygenases. Interestingly, their data suggest DADS is preferentially metabolized in human liver to allicin by cytochrome P450 2E1 (CYP2E1). As DADS can also cause autocatalytic CYP2E1 destruction, it is unclear how much allicin might be formed under physiological conditions. Flavin-containing monooxygenases in liver are probably responsible for the oxidization of S-allyl cysteine, among many other sulfur compounds.36 P450 monooxygenases do not appear to be involved in SAC metabolism. Rarely have comparisons of water- and oil-soluble compounds from garlic been examined in the same study. Preclinical studies suggest that garlic extracts of differing composition may inhibit cancer or cardiovascular disease, but it is difficult to compare studies due to the varying doses, times of exposure, animals, cell types, and other experimental procedures.37–41 Differences that occur in response to various preparations very likely relate to the content and effectiveness of individual sulfur constituents. The number of sulfur atoms present in the molecule seems to influence the response with diallyl trisulfide (DATS), generally found to be more effective than DADS, which is better than diallyl sulfide (DAS).42,43 Likewise, the presence of the allyl group generally enhances the response over that provided by the propyl moiety.44

5.3  IMPLICATIONS IN HEALTH Garlic and a host of its allyl sulfur compounds have been reported to possess a variety of health benefits. Notable among these are the antimicrobial, anticarcinogenic, and protective benefits against cardiovascular disease. Figure 5.1 illustrates some of the most common compounds associated with the health benefits of garlic, and their derivation from the processing of garlic. While there is a need for long-term intervention studies, a variety of preclinical and epidemiological studies suggest that key molecular targets involved in the risk of several diseases can be influenced by these organosulfur compounds arising from garlic.

5.4  ANTIMICROBIAL ACTIVITY Numerous plants are reported to act as antimicrobial agents. Those rich in tannins, terpenoids, alkaloids, flavonoids, and sulfur compounds have been found to be particularly effective. Historically,

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FIGURE 5.1  Organosulfur compounds from garlic and garlic preparations. (Reproduced from Charron, C.S. et al. in Encyclopedia of Food and Health, B. Caballero, P. Finglas, and F. Toldra, Editors. 2016, Academic Press: Oxford. p. 184–190.)

garlic extracts have been labeled as universal antibiotics.46 Considerable evidence indicates that garlic extracts can inhibit a range of Gram-negative and Gram-positive bacteria and serve as antifungal agents.47–49 Ruddock et al.50 examined the microbial activity of several garlic products found in the Canadian marketplace and observed a general trend toward increased in vitro antibacterial activity among those products containing higher amounts of allicin. Products with marginal antibacterial activity often contained lower concentrations of active constituents than their product labels indicated, which indicates the need to standardize garlic preparations used in research.

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Helicobacter pylori colonization of the gastric mucosa is responsible for most ulcers of the stomach and upper small intestine51 and increases risk of non-cardia gastric cancer.52 Studies by Cellini et al.53 provide rather convincing evidence that aqueous garlic extracts (2–5 mg/mL) inhibit H. pylori proliferation. Reduced effectiveness occurred when the garlic was heated prior to extraction.53 This depression in activity suggests the need for breakdown products from alliin to achieve a maximum response. As both DAS and DADS are recognized to elicit a dose-dependent depression in Helicobacter pylori proliferation in culture,54 a reduction in their formation may account for the loss of effectiveness caused by heating. Raw garlic extracts and three commercially available garlic tablets were found to vary in their efficacy, as indicated by a minimum inhibitory concentration in the range between 10 and 17.5 µg dry weight/mL.55 The ability of garlic to reduce H. pylori infection in humans is inconclusive. Although an epidemiological study suggested an association between increased garlic consumption and reduced H. pylori infection,56 three clinical studies testing different garlic preparations in H. pylori-infected subjects did not show efficacy.57–59 These interventions did not result in the elimination of the organism, change in the severity of gastritis, or a significant change in symptom scores. The studies were not randomized and had small sample sizes. A larger clinical study with 36 outpatients tested the effects of 4 g of garlic powder daily for 8 weeks, and concluded that the result was not different from the placebo.60 A study with 15 subjects reported that 3 g of garlic cloves consumed twice a day for 3 days suppressed H. pylori, but the small sample size and lack of placebo group limits drawing inferences from this result.61 Allium plants, including garlic, are effective in suppressing fungal growth.49 Allicin has been reported to be protective against Candida albicans and many other strains. These organisms were extremely sensitive to garlic extracts, some to a greater degree than to nystatin, a known effective antibiotic.62 Ajoene is also noted for its antimycotic activity both in vitro and in vivo. A fungal infection of the skin known commonly as ringworm and medically as tinea corporis can also be influenced by sulfur compounds found in garlic. Ledezma et al.63 found that treatment with ajoene (0.6% ajoene or 1% ajoene gel) was as effective as terbinafine (1% cream) in healing tinea corporis and tinea cruris in 70 soldiers with dermatophytosis. As ajoene can be prepared easily from garlic it may be particularly useful as a public health strategy, particularly in developing countries. The primary antimicrobial effect of garlic may reflect chemical reactions that take place with selected thiol groups of various enzymes and/or a change in the overall redox state of the organism. Specifically, the antimicrobial action of allicin and its breakdown products has been suggested to result from its rapid interaction with thiol or sulfide—containing molecules, including amino acids and cellular proteins within microbial organisms.49 An example of such an in vivo reaction is that between allicin and glutathione (GSH), which is thought to be the major intracellular mammalian thiol, and investigators have isolated the product of the reaction, established its structure, and examined its interaction with thiol-containing proteins.64 GSH reacted with allicin in the following fashion: 2GSH + CH2-CH-CH2(SO)-S-CH2-CH = CH2(allicin) → 2GS-S-CH2-CH = CH2(Sallylmercaptoglutathione) (GSSA) + H2O As proof of principle, in an in vitro setting, GSSA was found to react with the thiol-containing proteins papain and alcohol dehydrogenase from Thermoanaerobium brockii and inhibit their activity, whereas both proteins were reactivated using either reducing agent dithiothreitol or 2-mercaptoethanol. The concomitant release of allylmercaptan in these reactions indicated that the thioallyl moiety binds to inactivated proteins just as allicin has been shown to do. It is interesting to note that one enzyme that may be similarly affected by allicin breakdown products (i.e., DATS, SAC) is squalene monooxygenase. 65 Such activity may explain the antifungal properties of allicin, as squalene monooxygenase is an important enzyme for the formation of the fungal cell wall.66

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Despite promising in vitro results, there is relatively little clinical evidence for the efficacy of garlic as an antibiotic in humans. One research direction of particular importance may be that of determining the effects of garlic intake on the human gastrointestinal microbiome. Gastrointestinal microbiota metabolize dietary components and are modulated by dietary components, hence having a critical role in nutrient metabolism and health status.67 An in vitro study using the fecal inoculum of a single donor found that garlic powder had a temporal effect on gut commensal bacteria, but specific gut pathogens were not measured.68 The ample evidence that garlic compounds affect microorganisms in vitro suggests that garlic may have significant effects on the human gastrointestinal microbiome.

5.5 CANCER There is increasing awareness that several foods and certain dietary patterns may contribute to health, including a reduction in cancer risk.69–71 Although limitations exist in defining the precise role that garlic may have in the cancer process, garlic and garlic constituents have been actively studied using both epidemiological and laboratory investigations. There is epidemiological support for the association between increased intake of garlic and/or its active constituents with certain cancers, but the data are limited and show inconsistent results.72–77 More than 20 years ago, results from the Iowa Women’s Health Study, a prospective cohort study, found that the strongest association among fruits and vegetables for colon cancer risk reduction was for garlic consumption, with a reduced risk of approximately 50% in distal colon cancer associated with high garlic consumption.78 This association has since been examined in different populations. In the previous report of the World Cancer Research Fund/American Institute for Cancer Research,72 the evidence for the association between garlic and colorectal cancer was judged as “probably decreases risk,” whereas in their recent continuous update project on this relationship, it was judged as “limited-no conclusion.”77 The recent report included studies on garlic and garlic supplement intakes. Other cancer sites have also been studied. Results from a meta-analysis of 14 case control studies, 2 randomized controlled studies, and 1 cohort study with a total of 8621 cases and 14,889 controls found that high, low, and any garlic intake was associated with reduced risk of gastric cancer.73 However, recent results for two large prospective U.S. cohort studies—women in the Nurses’ Health Study (1984–2014) and men in the Health Professionals Follow-Up Study—found that garlic intake did not reduce gastric cancer risk or modify H. pylori infection.79 The authors suggested that further studies need to differentiate between cooked and uncooked garlic and consider different gastric cancer types. Interestingly, in a Chinese population, raw garlic intake compared to no garlic intake was associated with lower risk of development of lung cancer with a dose-response pattern.80 Garlic consumption has also been associated with decreased risk of prostate cancer. A meta-analysis of six case-control and three prospective cohort studies reported a significantly decreased risk of prostate cancer for intake of allium vegetables and that in subgroup analysis stratified by allium vegetable types, significant associations were observed for garlic.76 Epidemiologic approaches assist in determining associations with cancer risk in large populations, but they have limitations, including multiple testing and potential for false discovery. In addition, studying associations between garlic and cancer risk is also challenging due to the difficulty in assessment of intake levels. The common use of quantiles in epidemiologic studies makes it difficult to compare results across studies, as many cohorts have different ranges of garlic intake. Improved methods for assessment of garlic intake, including the amount, form, and preparation method, may help to further clarify the relationship between garlic and cancer risk. Few intervention studies have been performed to examine the efficacy of garlic in preventing or treating cancer. In a double-blind, randomized study of Japanese patients with colorectal adenomas, a higher-dose AGE was shown to reduce the risk of new colorectal adenomas compared to a lowerdose garlic extract.81 Due to observations of a case-control study of gastric cancer in Shandong, China, which indicated that persons in the highest quartile of intake of allium-containing vegetables (including garlic, garlic stalks, scallions, chives, and onions) had only 40% of the risk of those in the

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lowest quartile of intake,82 investigators included a garlic-supplementation arm (800 mg of garlic extract plus 4 mg steam-distilled garlic oil daily) in the Shandong Intervention Trial, a randomized multi-intervention trial to inhibit the progression of precancerous gastric lesion in this same region of China.83 The interventions included a 2-week course of amoxicillin and omeprazole for subjects who were seropositive for Helicobacter pylori and 7 years of oral supplementation with garlic or with a mixture of vitamin C, vitamin E, and selenium. The outcome of the trial showed that amoxicillin and omeprazole statistically significantly reduced the prevalence and average histological severity of precancerous gastric lesions, whereas the garlic and vitamin treatments did not.84 Another clinical trial in this same area of China examined the combination of synthetic DATS (200 mg/d of allitridum) plus selenium (100 mg every other day) for a 2-year period on the prevention of gastric cancer.85 Incidence of total or gastric cancer did not decrease significantly in the overall study population after 5 years, but in males only, the combined intervention resulted in a decreased relative risk for all tumors and gastric cancer. Preclinical models (Table 5.2) provide some of the most compelling evidence that garlic and its related sulfur components suppress cancer risk and alter the biological behavior of tumors. Overall, garlic and its associated sulfur components have been found to suppress the incidence of mammary, colon, skin, uterine, esophageal, lung, renal, forestomach, and liver cancers.86–94 Aberrant crypt foci (ACF) are a proposed early preneoplastic lesion of adenoma-carcinoma in humans and chemically induced colon cancer in rodents. In many preclinical studies, both water- and lipid-soluble allyl sulfur compounds administered to animals through their diet have been reported to inhibit ACF.95–97 Cancer protection may arise from several mechanisms, including blockage of carcinogen formation, suppressed bioactivation of carcinogens, enhanced DNA repair, reduced cell proliferation, and/or induction of apoptosis. It is possible, and quite probable, that several of these cellular events are modified simultaneously.

5.5.1 Nitrosamine and Heterocyclic Amine Formation Humans are exposed to an array of chemical substances through food sources that may be involved in cancer causation. Nitrosamines, heterocyclic amines (HCAs), and polycyclic aromatic hydrocarbons are potential dietary carcinogens that are not normally present in foods but may arise during preservation or cooking.98 Human exposure to these suspect carcinogens occurs through the ingestion or inhalation of preformed N-nitroso compounds (NOCs) or by the ingestion of precursors that are combined endogenously.99 Evidence points to the ability of garlic to suppress the formation of several NOCs.100,101 The ability of garlic to reduce NOCs may actually be secondary to an increase in the formation of nitrosothiols. Several sulfur compounds have been proposed to foster the formation of nitrosothiols, thereby reducing the quantity of nitrite available for NOC formation.100 Studies by Dion et al.102 revealed that not all allyl sulfur compounds are equally effective in stopping the formation of NOCs. The ability of SAC and its non-allyl analog S-propyl cysteine to retard NOC formation—but not DADS, dipropyl disulfide, and DAS—reveal the critical role that the cysteine residue has in this inhibition.102 As the content of allyl sulfur can vary among preparations, it is likely that not all garlic sources are equal in the protection they provide against NOC formation. Because of the potential for bacterial-mediated formation of nitrosamines, some of the protection against NOC exposure may also relate to antimicrobial properties associated with garlic.101 In a human study, providing 5 g garlic per day completely blocked the enhanced urinary excretion of nitrosoproline that occurred after ingesting supplemental nitrate and proline.103 The significance of this observation comes from the predictive value that nitrosoproline has for the synthesis of potential carcinogenic nitrosamines.104 Another human study suggested that 1 g of garlic per day may be sufficient to suppress nitrosoproline formation.105 The anticancer benefits attributed to garlic are also associated with the ability of its allyl sulfur compounds to suppress carcinogen bioactivation. Evidence from a variety of sources reveals that garlic is effective in blocking DNA alkylation, a primary step in nitrosamine carcinogenesis.91,106

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TABLE 5.2 Anticarcinogenic Effects of Garlic and/or Associated Allyl Sulfur Compounds Site/Carcinogen Bone marrow  Benzo[a]pyrene Buccal pouch  7,12-dimethylbenz[a]anthracene Colon  1,2-dimethylhydrazine  Azoxymethane Cervix  3-methylcholanthrene Esophagus  N-nitrosomethylbenzylamine Forestomach  7,12-dimethylbenz[a]anthracene  Benzo(a)pyrene  N-nitrosodiethylamine Gastric  Methylnitronitrosoguanidine Liver   Aflatoxin B1  N-nitrosodimethylamine Lung  Benzo(a)pyrene Mammary   7,12 Dimethylbenz(a)anthracene  N-methyl-N-nitrosourea   2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) Nasal  N-nitrosodiethylamine  N-nitrosodimethylamine  4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone Renal  N-diethylnitrosamine Skin   7,12 Dimethylbenz(a)anthracene  Benzo(a)pyrene   Vinyl carbamate

Garlic Compound

Host

Diallyl thioethers

Mouse239

Aqueous garlic extract

Hamster240

S-allylcysteine Diallyl sulfide Aqueous garlic extract

Rat241 Mouse88 Rat242

Aqueous garlic extract

Mouse89

Diallyl sulfide

Rat90

Diallyl sulfide Allyl group-containing derivatives Diallyl disulfide

Hamster93 Mouse243 Mouse244

Diallyl sulfide

Rat245

Fresh garlic, garlic oil Diallyl sulfide Garlic

Toad246 Rat247 Rat248

Diallyl sulfide, allyl methyl disulfide

Mouse243

Selenium-enriched garlic, garlic powder S-allylcysteine, diallyl disulfide, garlic powder Diallyl disulfide

Rat87,190 Rat116 Rat249

Diallyl sulfide Diallyl sulfide Diallyl sulfide Diallyl sulfide

Rat250 Rat250 Rat250 Mouse91

Diallyl disulfide

Rat92

Diallyl sulfide, diallyl disulfide, diallyl trisulfide Diallyl sulfide Diallyl sulfide

Mouse251,252 Mouse154 Mouse132

Note: The overall response to garlic and/or specific allyl sulfur components depends on the quantity provided and the amount of carcinogen administered. (Adapted from Ross, S.A. and Milner, J.A., in Handbook of Nutraceuticals and Functional Foods, Second Edition, R.E.C. Wildman, Editor. 2006, CRC Press: Boca Raton, FL. p. 73–99.)

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Consistent with this reduction in bioactivation, Dion et al.102 found that both water-soluble SAC and lipid-soluble DADS were effective in retarding the mutagenicity of N-nitrosomorpholine in Salmonella typhimurium TA100. A block in mutagenicity following aqueous garlic-extract exposure has also been noted following treatment with ionizing radiation, peroxides, adriamycin, and N-methyl-N-nitro-nitrosoguanidine.107 A block in nitrosamine bioactivation may reflect changes in several enzymes. However, substantial evidence points to the involvement of CYP2E1.108,109 An autocatalytic destruction of CYP2E1 may account for some of the chemoprotective effects of DAS, and possibly other allyl sulfur compounds.110 Variation in the concentration and overall activity of CYP2E1 may be an important variable in the degree of protection provided by garlic and associated allyl sulfur components. HCAs are produced by high-temperature cooking of protein-rich foods such as beef and chicken.98 Food preparation may be key, as addition of garlic powder to hamburger meat before cooking decreased the HCAs 2-amino-3,8-dimethylimidazo (4,5-f) quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) by 66.2% and 85.0%, respectively.111 In a study testing the ability of oil marinades containing various levels of onion, garlic, and lemon juice to inhibit HCA formation, increasing garlic and onion, but not lemon juice, in the marinades significantly decreased the formation of MeIQx.112 The concentrations of onion, garlic, and lemon that led to the maximal MeIQx reduction were 31.2%, 28.6%, and 14.6%, respectively. Once ingested, the in vivo bioactivation of heterocyclic amines to carcinogenic species is known to be initiated by N-oxidation. This reaction occurs primarily in the liver, and in humans is catalyzed by cytochrome P4501A2 (CYP1A2). Davenport and Wargovich113 reported the puzzling finding that in rats, the administration of a single bolus of 200 mg/kg DAS and AMS increased hepatic CYP1A2 protein (but not mRNA) by 282% and 70%, respectively. Acetylation or sulfation of the N-hydroxy-heterocyclic amine can also occur through the action of acetyltransferases (NAT) and sulfotransferases, which generate N-acetoxy and N-sulfonyloxy esters, electrophiles that are much more reactive with DNA. Several studies provide evidence that organosulfur compounds arising from garlic can effectively reduce NAT activity. Studies by Yu et al.114 demonstrated that a suppression in NAT mRNA expression accounts for the majority of the reduction in activity by garlic.

5.5.2 Carcinogen Activity Modulation Garlic and several of its allyl sulfur compounds can also effectively block the bioactivation and carcinogenicity of non-NOCs and HCAs (Table 5.2). This protection, which involves a diverse array of compounds and several target-tissue sites, suggests either multiple mechanisms of action or a widespread biological effect. Garlic extract and compounds have also been found to reduce the incidence of tumors resulting from treatment with methylnitrosurea (MNU), a known direct-acting carcinogen.115 Providing water-soluble SAC and lipid-soluble DADS at 57 µmol/kg in the diet has been reported to cause a comparable reduction in MNU-induced O6-methylguanine adducts bound to mammary cell DNA.116 However, not all evidence supports SAC for protection against MNU-induced mammary tumors.117 The reason for this discrepancy is unknown but may relate to the quantity of lipid in the diet or the quantity of carcinogen provided. If garlic compounds are effective blockers of these carcinogens, the mechanism(s) remain unresolved. Studies by Ludeke et al.118 revealed that DAS diminished the DNA hypermethylation of esophagus, liver, and nasal mucosa that arose from treatment with N-nitrosomethylbenzylamine. This finding suggests that the bioactivation of several carcinogens known to influence DNA methylation patterns119 may also be influenced by garlic and many of its sulfur constituents.101 Metabolic activation is required for many carcinogens used in studies aimed at examining the anticarcinogenic properties of garlic. Thus, possible cancer-preventive mechanisms include modulation

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of the activity of phase I enzymes, such as cytochrome P450s, which activate carcinogens, or phase II enzymes, such as glutathione S-transferases, that detoxify carcinogens. Recent observations show that the activity of several phase I enzymes, in addition to CYP1A2 and CYP2E1 discussed earlier, are modified following treatment with garlic or related sulfur compounds.113,120–122 The influence of organosulfur compounds (OSCs) on phase I metabolizing enzymes is reportedly quite diverse. For example, previous studies demonstrated that DAS competitively inhibited CYP2E1 activity, but robustly increased the transcriptional levels of CYP1A1, CYP2B1, and CYP3A1 in rat liver.121,123 A recent study evaluated organosulfur compounds of aged garlic extract—SAC, S-methyl-L-cysteine, and trans-S-1-propenyl-L-cysteine—for their effects on the activities of five major isoforms of human CYP enzymes: CYP1A2, 2C9, 2C19, 2D6, and 3A4.124 The authors found little CYP inhibition or activation after the human intake of these garlic compounds. Therefore, the role of garlic OSCs in carcinogenic biotransformation may be substrate specific. The significance of any slight induction of certain cytochrome P450 activities is not clear, but some reports suggest the induction of cytochrome P450 metabolic enzymes may increase the rate of clearance of toxic metabolites.125 Other enzymes and pathways are involved in the bioactivation or removal of carcinogenic metabolites in the observed protection from garlic constituents. Singh et al.126 provided evidence that the efficacy of various organosulfides to suppress benzo(a)pyrene tumorigenesis was correlated with their ability to induce NAD(P)H:quinone oxidoreductase (NQO), an enzyme involved with the removal of quinones associated with this carcinogen. Investigators have also found that this inductive effect of organosulfur compounds appears to be mediated by the resident antioxidant response element (ARE) enhancer sequence bound by the nuclear factor E2related factor 2 (Nrf2) in the NQO1 and the heme oxygenase 1(HO1) gene promoters.127 In fact, it was found that the organosulfur compounds DAS, DADS, or DATS differentially mediated the transcriptional levels of NQO1 and HO1. The third sulfur in the structure of OSCs appeared to have a major contribution to this bioactivity, and the allyl-containing OSCs were more potent than the propyl-containing OSCs. The data also suggested that the upregulation of detoxifying enzymes by garlic OSCs through Nrf2 protein accumulation and ARE activation might be partly due to the stress signals originating from the oxidative stress and/or calcium-dependent signaling pathways.127 More recently, findings suggest that DATS may directly interact with the Cys288 residue of Kelch-like ECH-associated protein-1 (Keap1), a cytosolic repressor of Nrf2, which partly accounts for DATs ability to induce Nrf2 activation and upregulate defensive gene expression.128 Changes in glutathione concentration and the activity of specific glutathione-S-transferases (GSTs), both factors involved in phase II detoxification, may be important in the protection provided by garlic. Both DADS and DATS have been shown to increase activity of GSTs in a variety of rat tissues.129 Moreover, gene expression of various GSTs (e.g., GSTp1, GSTa2, GSTm1) have been shown to be modulated by garlic constituents in several tissues.130 The preventive effects of garlic powders containing variable levels of sulfur compounds on the development of preneoplastic foci initiated by aflatoxin B1 (AFB1) in rats has been characterized.131 The ultimate metabolite of AFB1, aflatoxin B1-8,9-epoxide, is conjugated with glutathione by GST and more specifically by GSTA5; thus, GST was explored as a mechanism responsible for any chemoprotective properties of garlic against AFB1-induced carcinogenesis. Consumption of garlic was efficient in protecting against AFB1 carcinogenesis, and DADS treatment induced GST protein levels and activity, particularly GSTA5. Thus, not all GST isozymes may be influenced equally. Earlier evidence from Hu et al.44 provided support that the induction of glutathione S-transferase pi 1 (GSTP1) may be particularly important in the anticarcinogenic properties associated with garlic and allyl sulfur components. Modulation of both phase I and II enzymes by garlic oil was explored in nitrosodiethylamine (NDEA)-induced hepatocarcinogenesis.133 The authors found that changes of the activities, mRNA, and protein levels of phase I enzymes (including CYP2E1, CYP1A2, and CYP1A1) and phase II enzymes (including GSTs and UDP-glucuronosyltransferases) contributed to the protective effects of garlic oil against NDEA-induced hepatocarcinogenesis in rats.

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5.5.3 Cell Cycle Arrest/Apoptosis Evidence indicates that garlic constituents (i.e., DADS, DATS, S-allylmercaptocysteine [SAMC], ajoene) have the ability to suppress proliferation of several different cancer cells by blocking cellcycle progression and/or causing apoptosis (also known as programmed cell death).134–136 Several mechanisms have been cited for the effect of garlic constituents on cell cycle arrest, including reduced Cdk1/cyclin B kinase activity, activation of extracellular signal-regulated kinases (ERK1/2), or induction of phosphorylated checkpoint kinase-1.134,137,138 Knowles and Milner139 showed that the DADS-mediated suppression of Cdk1 kinase activity during cell-cycle arrest in G2/M was not due to direct interaction with the protein, but was associated with (a) a temporal and dose-dependent increase in cyclin B1 protein level, (b) a reduction in the level of Cdk1–cyclin B1 complex formation, (c) inactivating hyperphosphorylation of Cdk1, and (d) a decrease in Cdc25C protein level. The evidence suggests a complex and coordinated interaction of many factors for the observed DADSinduced cell-cycle arrest. Furthermore, gene expression analysis suggested that alterations in DNA repair and cellular adhesion factors may also be involved in the G2/M block following DADS exposure.140 Current knowledge of the mechanisms by which garlic compounds cause apoptosis indicates that they target various apoptosis-signaling molecules from initiation to execution, including mitogenactivated protein kinases (Jun N-terminal kinase (JNK), ERK1/2, and p38), P53, nuclear factor kappa-light-chain enhancer of activated B cells (NF–kB), B-cell lymphoma 2 (bcl-2) family, and caspases,135 but not all of the signaling molecules are affected by each of the garlic constituents. In many studies, however, the apoptotic effects of garlic constituents were triggered by increased intracellular production of reactive oxygen species (ROS), suggesting the importance of the intracellular redox environment for apoptosis induction.141 An example is shown by the ability of DADS to induce apoptosis, as well as cell-cycle arrest at the G2/M phase, in human A549 lung cancer cells in a time- and dose-dependent manner.136 In this study, DADS caused not only a dosedependent increase, but also a time-dependent change of ROS production, and an oxidative burst was found to be an early event, occurring less than 0.5 h after DADS treatment. These investigators hypothesized that the increased ROS may also act on the important signaling molecule in the observed DADS-induced cell cycle arrest. In a recent review of the activity of DATS in cancer prevention,142 several studies reported that DATS induces G2/M phase cell cycle arrest and that this may occur through the generation of ROS. Additionally, increased pro-apoptotic capacity as a result of regulating intrinsic and extrinsic apoptotic pathway components was widely reported following DATS treatment.142 The importance of the redox environment for apoptosis is also suggested by a study in which DATS increased hydrogen peroxide formation, lowered thiol levels, and induced caspase-3 activity in HepG2 cells.143

5.5.4 DNA Repair Exposing cells to mutagens, including intracellular by-products of cellular metabolism (ROS, endogenous alkylating agents) or extracellular influences (carcinogens, UV, or ionizing radiation), can cause DNA damage that is manifested as genomic instability, cellular senescence, and/or cell death. Initially the cell attempts to repair the damage, but if too extensive, a cascade of alternative cellular responses, including cell-cycle arrest or the induction of apoptosis, may occur. There are three major DNA repairing mechanisms: base excision, nucleotide excision, and mismatch repair. Very little information exists about garlic or its organosulfur constituents as a modifier of DNA repair, although evidence exists that pretreatment with garlic extracts has been reported to stimulate DNA repair in human fibroblasts following cadmium chloride, gamma-radiation, and 4-nitroquinoline-1-oxide treatment.144 Interestingly, investigators found that gene expression of DNA repair genes did not correlate with growth inhibition by DADS.145 Regardless, several studies have demonstrated that histone/chromatin modifications such as acetylation, methylation,

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and phosphorylation have a crucial role in DNA-repair processes, and some evidence suggests that garlic could influence one or more of these determinants of repair.

5.5.5 Epigenetic Modulation Cancer progression is probably also highly dependent on epigenetic changes. Several regulatory proteins, including DNA methyltransferases, methyl-cytosine guanine dinucleotide binding proteins, histone-modifying enzymes, chromatin-remodeling factors, and their multimolecular complexes, are involved in controlling the epigenetic process to influence the regulation of gene expression.146 Because epigenetic events can be influenced by several dietary components, they represent another plausible site for intervention with bioactive food components.146 Garlic and its constituents have been shown to influence gene expression both in vitro and in vivo,130 but few studies have examined whether epigenetic processes influence gene expression following garlic treatment. In one recent study, SAC treatment of human ovarian cancer A2780 cells was shown to decrease global DNA methylation levels, DNA methyltransferase (DNMT) activity, mRNA and protein levels of DNMT1. Additionally, SAC treatment resulted in re-expression of the mRNA and proteins of the silenced tumor suppressor gene CDKN1A accompanied by reduced cell proliferation and induced cell cycle arrest in the G1/S phase.147 Evidence suggests that some garlic constituents can influence histone homeostasis, which may influence gene expression and cellular phenotype. Lea et al.148 first reported that at least part of the ability of DADS to induce differentiation in DS19 mouse erythroleukemic cells might relate to its ability to increase histone acetylation. DADS caused a marked increase in the acetylation of H4 and H3 histones in DS19 and K562 human leukemic cells. Consistent with other studies, disulfide was found more effective than mono-sulfide. Moreover, these investigators found that the inhibition of cell proliferation by SAC and SAMC of DS19, Caco-2 human colon cancer, and T47D human breast cancer cells was associated with increased histone acetylation.149 Additionally, Druesne et al.150 reported DADS and allyl mercaptan, a metabolite of DADS, effectively increased histone H3 acetylation in cultured Caco-2 and HT-29 cells. The histone H4 hyperacetylation was found to occur preferentially at the lysine residues 12 and 16. The reason for this hyperacetylation was thought to be due to the observed reduction in histone deacetylase activity. This change in hyperacetylation was also accompanied by an increase in p21(waf1/cip1) expression, at mRNA and protein levels, again demonstrating that epigenetic events may influence subsequent gene expression patterns and lead to the accumulation of cells in the G2 phase of the cell cycle.150 Using an in vivo model, DADS was found to inhibit the growth of tumors in SCID mice bearing HL-60 peritoneal neoplasms, which was accompanied by increased expression of acetylated histone H3 and H4, as well as increased protein expression of p21WAF1.151 There is now ample evidence from preclinical studies that DADS, AM, and other garlic compounds increase histone acetylation.152 These observations are thought to be due to inhibition of histone deacetylase activity (HDAC).152 Nian et al.153 screened several garlic compounds for their ability to inhibit HDAC activity in vitro. AM was found to be the most potent HDAC inhibitor, acting as a competitive HDAC inhibitor in vitro, with a K i on the order of 24 µM for human HDAC8. Using HT29 cells, Nian et al.153 found that inhibition of HDAC activity by AM was associated with increased global histone acetylation, as well as localized hyperacetylation of histone H3 on the P21WAF1promoter. The garlic constituents DADS and AM, for which there is the most evidence for this activity, join the list of other food components with demonstrated weak histone deacetylase inhibitor activity.155 MicroRNAs are a class of small non-coding RNAs (approximately 23 nucleotides in length) that regulate post-transcriptional gene expression by binding to the 3′-untranslated region (3′-UTR) of target mRNAs, leading to mRNA cleavage or translational repression.156 MicroRNAs regulate the expression of a wide variety of target genes and are therefore involved in a broad range of biological processes, and their expression is dysregulated in cancer.157 Garlic compounds have been found to influence microRNA or miR expression in cancer cells. For example, DADS suppressed

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proliferation and invasion in human breast cancer cells by upregulating miR-34a to target repression of the cytoplasmic tyrosine kinase SRC mRNA, consequently reducing SRC/Ras/ERK oncogene signaling.158 These results add to the variety of gene regulatory mechanisms that may be modified by consumption of garlic and its constituents.

5.5.6 Redox and Antioxidant Capacity It is well documented that dysregulation of ROS is involved in the etiology of a variety of diseases, including cancer. As a result, attention has been given to the identification of antioxidants in human foods. How the classification of antioxidant activity in a food or food component translates to human health continues to be a topic of discussion. However, both water- and lipid-soluble organosulfur compounds from garlic have been studied for their antioxidant capacity. A variety of methods have been used to evaluate the total antioxidant activity of garlic preparations available in the marketplace. Any single method is insufficient, as the response depends not only on its ability to reduce oxidation radicals, but on its metal-chelating capabilities. Both alliin and allicin are have been shown to possess antioxidant properties in a Fenton oxygen-radical generating system.64,159 Additionally, the antioxidant actions of garlic and its constituents have been documented through their ability to scavenge ROS, inhibit lipid peroxide formation, retard low-density lipoprotein (LDL) oxidation, and enhance endogenous antioxidant systems.5,160 It should be noted that not all organosulfur compounds have been found to exhibit antioxidant properties. DADS, but not DAS, dipropyl sulfide, or dipropyl disulfide, has been found to inhibit liver microsomal-lipid peroxidation induced by NADPH, ascorbate, and doxorubicin.161 The presence of both the allyl and sulfur groups appears to magnify the antioxidant capabilities of the molecule. Both the number of sulfur atoms and the oxidation state of sulfur atoms can influence the overall antioxidant potential.162 Whereas allicin is effective in retarding methyl linoleate oxidation, it is less than that caused by α-tocopherol.163 Organosulfur compounds such as SAC are recognized to be powerful antioxidants and radical scavengers with the strong capacity to minimize oxidization.160 Antioxidant activity of garlic shows great variation depending on the genotype or species evaluated for activity.164,165 Moreover, processing of garlic may affect antioxidant efficacy; the heating of garlic can not only denature proteins, but also its antioxidant properties.166

5.5.7  Immunocompetence/Immunonutrition Diet is increasingly recognized to influence the development and functionality of immunocompetent cells. Several dietary components, including garlic extracts and allyl sulfur compounds, may have physiologically important immunomodulatory effects.6,167–170 Both an aqueous and an ethanolic extract of garlic powder significantly stimulated proliferation of rat spleen lymphocytes in culture, which was correlated with the upregulation of the Interleukin 2receptor alpha expression and an increase in interleukin (IL)-2 production.170 This data also suggested that the potentiating effect of the garlic extract on lymphocyte proliferation in vitro differed, depending on specific stimulators of cell proliferation, speculating that the in vivo response would depend on the type of responding cells. These investigators also demonstrated that aqueous and ethanolic extracts from two garlic powders significantly modulated proliferation of rat thymocytes and splenocytes in vitro to concanavalin A.171 Both garlic extracts significantly modulated lymphocyte proliferation, triggered by this potent T-cell mitogen, but the response was dependent on the type and dilutions of extracts, and concentrations of concanavalin A. Interestingly, at higher concentrations of the extracts, an inhibitory effect on T-cell proliferation was observed, whereas at lower concentrations, a significant increase in T-cell proliferation occurred. In an in vivo study, DAS treatment of BALB/c mice has been reported to block the suppression of the antibody response caused by N-nitrosodimethylamine to T-cell-dependent antigens, and the lymphoproliferative response to T-cell and the B-cell mitogens.172 These results support the concept that garlic may be a modulator of T cell-mediated immune functions in vivo.

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However, most studies have been carried out in vitro, and there is a need to design studies using well-defined systems and chemically pure active garlic compounds at defined concentrations, and to develop animal models to test the immunomodulatory effect of garlic in cancer prevention. It is important to note that very little research to determine the immunomodulatory effects of garlic on cancer or cancer prevention in humans has been performed. In one randomized double-blind clinical trial, the effects of AGE were evaluated on the quality of life (QOL) and immune functions of patients with advanced cancer of the digestive system.173 Although the authors reported no differences in QOL, they did find that the number of natural killer (NK)cells and NK cell activity increased significantly in the patients that consumed AGE. The immunomodulatory effects of garlic are not limited to sulfur compounds, as a protein fraction isolated from aged-garlic extract was also found to enhance cytotoxicity of human peripheral blood lymphocytes (PBL) against both natural killersensitive K562 and NK-resistant M14 cell lines.174 The mechanism(s) by which sulfur or non-sulfur components of garlic influence immunocompetence remains to be determined. Recent insights about their mechanism of action are suggested from the results of a human randomized crossover trial.175 In this study, a single meal containing raw, crushed garlic resulted in increased expression of immune function and cancer pathway–related genes in whole blood 3 h after consumption. The seven genes that were upregulated have a variety of functions, including roles in xenobiotic metabolism, inflammation, B cell and T cell development, apoptosis, and tumorigenesis, and five of these genes were also upregulated in the monocytic cell line Mono Mac 6 when treated with garlic extract. Garlic compounds may also be modulators of inflammatory molecules, including cytokines that exhibit a vast array of regulatory functions in both adaptive and innate immunity. DADS and AMS, in addition to DAS,176 demonstrated different effects on the production of cytokines in lipopolysaccharide (LPS)-activated macrophages. DAS inhibited both pro- and anti-inflammatory cytokines, including tumor necrosis factor (TNF)α, IL-β, IL-6, and IL-10, in stimulated macrophages. DADS enhanced pro-inflammatory cytokines IL-β and IL-6, but suppressed anti-inflammatory cytokine IL-10, indicating the effect of DADS may be more toward pro-inflammation. On the other hand, AMS, to a lesser extent, decreased production of nitric oxide (NO) and TNF-α in activated macrophages, but significantly enhanced IL-10 production, suggesting that AMS may be a potential anti-inflammation compound. Allicin and ajoene have been reported to cause a dose-dependent inhibition of the inducible nitric oxide synthase (iNOS) system in LPS-stimulated RAW 264.7 murine macrophages.177 Such inhibition has been correlated with a reduction in iNOS protein, as well as in its mRNA. Thus, changes in the amount or ratio of NO and peroxynitrite concentrations may be significant in the observed lowering of inflammation by garlic and associated sulfur components. DAS, DADS, and AMS have also been shown to display unique regulatory properties in suppressing NO in stimulated macrophages.178 DAS was found to decrease stimulated NO and prostaglandin E2 (PGE2) production by inhibiting inducible NO synthase and cyclooxygenase-2 expressions, and to indirectly enhance NO clearance. DADS inhibited activated NO production by decreasing inducible NO synthase expression and by directly clearing NO, whereas AMS suppressed NO mainly through its direct NO clearance activity. It has been hypothesized that consumption of garlic OSC may assist in shifting the balance from a pro-inflammatory to an anti-tumor response by dampening the pro-inflammatory response and/or strengthening the anti-tumor immunity toward tumor eradication.167 Using a colitis-induced colorectal cancer AOM/DSS mouse model, investigators recently examined whether DADS exerts its protective effects against colorectal tumors by suppressing inflammation.179 Supplementation with DADS resulted in a reduction in tumor incidence, tumor number, and tumor burden. Furthermore, the DADSsupplemented diet resolved the initial DSS-induced inflammation faster than those on the control diet, preventing prolonged inflammation and cellular transformation. Mechanistic studies using the human CRC SW480 cell line suggested that DADS interferes with nuclear translocation of pro-inflammatory transcription factor NF-κB and the expression of tumorigenic enzyme COX2 via the serine/threonine kinase GSK-3 (α/β) inactivation.179 These findings suggest that the antitumor effect of allyl sulfurs may be related to their anti-inflammatory as well as immune-stimulatory properties.

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5.5.8 COX/LOX Pathways Cyclooxygenase (COX), lipoxygenase (LOX), and pathway-associated enzymes have been found to activate carcinogens. Smith et al.180 reported that prostaglandin H synthase could metabolize the bay region diol of benzo(a)pyrene to electrophilic diol epoxides that were capable of binding to DNA. It has also been reported that both cyclooxygenase and lipoxygenase are involved with 7,12-dimethylbenz(a)anthracene (DMBA) bioactivation.181,182 Garlic may influence this bioactivation. For example, Song and Milner182 found that feeding DADS or SAC markedly reduced DMBA-induced DNA adducts in rat mammary tissue. Ali183 provided evidence that garlic could block cyclooxygenase activity. Moreover, garlic and associated sulfur components may also inhibit lipoxygenase activity.184 With regard to the influence of allyl sulfur compounds on the lipoxygenase and cyclooxygenase signaling pathways, DAS, DADS, and to a lesser extent AMS, were found to differentially regulate NO and PGE2 production in mouse RAW 264.7 macrophages stimulated by LPS.178 In another study, ajoene was found to act similarly to several non-steroidal anti-inflammatory drugs in that this garlic compound inhibited, in a dose-dependent fashion, the release of PGE2 from LPS-activated RAW 264.7 cells, which was associated with a dose-dependent inhibition of COX-2 enzyme activity.185 Collectively, these studies pose interesting questions about the role of both cyclooxygenase and lipoxygenase in not only forming prostaglandins, and therefore modulating tumor cell proliferation and immunocompetence, but also their involvement in the bioactivation of carcinogens. Clearly, additional attention is needed to clarify what role, if any, these enzymes have in determining the biological response to dietary garlic or its allyl sulfur components.

5.5.9 Diet as a Modifier Garlic’s influence on cancer processes cannot be considered in isolation, as certain dietary patterns and foods may influence the overall response. For example, the effects of combining tomato and garlic were examined using several carcinogenesis models.186–188 The combination suppressed the incidence and mean tumor burden of hamster buccal-pouch carcinomas more than either alone, and appeared to relate to a decrease in phase I enzymes and an increase in phase II enzyme activities. A variety of individual food components may also influence the response to garlic. Notable are the modifications made by the quantity of fat, selenium, methionine, and vitamin A in the diet.189–191 Amagase et al.190 and Ip et al.191 reported that selenium supplied either as a component of the diet or as a constituent of the garlic supplement enhanced the protection against DMBA mammary carcinogenesis beyond that provided by garlic alone. Suppression in carcinogen bioactivation, as indicated by a reduction in DNA adducts, may partially account for this combined benefit of garlic and selenium.189 Because both selenium and allyl sulfur compounds are recognized to suppress tumor cell proliferation and to induce apoptosis,192–194 the synergistic response to allyl sulfur and selenium may relate to changes in cancer-related processes other than those associated with carcinogen metabolism. Dietary fatty acid supply can influence the bioactivation of DMBA and ultimately the metabolites of this carcinogen, which binds to rat mammary cell DNA. A significant portion of the enhancement in mammary DNA adducts caused by increasing dietary corn oil consumption can be attributed to linoleic acid intake.195 Whereas exaggerated oleic acid consumption also increases DMBA-induced DNA adducts, it was found to be far less effective in promoting adduct formation than was linoleic acid. The influence of garlic in modifying the effect of corn oil on rat mammary cell DNA adducts resulting from DMBA treatment has been studied.190 Garlic supplementation was found to prevent the increase in DNA adducts caused by increasing dietary corn oil. The diversity of molecular targets that can be influenced by various food components demonstrates the complexity in dealing with nutrient–nutrient interactions. Although the effect of combining bioactive food components on garlic’s ability to influence cellular proliferation has not

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been adequately examined, there are potentially several combinations that would produce more dramatic effects. For example, and similar to information with chemical carcinogenesis mentioned above, there is evidence of a greater effect of allyl sulfur when combined with selenium than when provided alone.196 Likewise, a combination of garlic and onion oils was more effective in blocking the proliferation of HL60 cells in culture than when used singly.197 Furthermore, the combination of garlic and lemon aqueous extracts was more effective in inhibiting breast cancer in vitro and in vivo, using Balb/C mice inoculated with EMT6/P breast cancer cells, than either aqueous extract alone.198 The rationale for this study was that lemon extract may reduce the strong smell and flavor of garlic extract and thus enhance consumption. In addition, it was thought that the phytochemicals in lemon extract may act synergistically to enhance the anticancer activity of garlic constituents. Although the molecular basis for these enhanced effects needs to be investigated in more detail, they serve as proof-of-principle that interactions among food components must be considered when developing strategies for using diet for cancer prevention.

5.6  CARDIOVASCULAR DISEASE Garlic may have a role in the genesis and progression of cardiovascular disease. These effects may be mediated through a variety of biological responses, including modulation of serum lipids and fibrinogen concentrations, lowering arterial blood pressure, and/or an inhibition of platelet aggregation.

5.6.1 Cholesterol and Lipoproteins Several studies have attempted to clarify the effects that garlic has on serum total cholesterol, LDL, high-density lipoprotein (HDL), and triglycerides.1,199,200 While some studies have reported that garlic reduces LDL concentrations, others have not. Evaluating cardioprotective responses is made complicated by the use of various quantities of garlic, different preparations, variations in the duration of treatment, and qualities of study design. One randomized controlled clinical study compared the lipid-lowering effects of 4.0 g of raw garlic, an equivalent (based on allicin) dose of dried garlic, AGE, and a placebo in a moderately hypercholesterolemic population (LDL cholesterol, 130–190 mg/dL, mean 151 ± 15 mg/dL).201 Total cholesterol, LDL, HDL, total/HDL ratio, and triglycerides did not change in response to 6 months of any of the interventions (consumed 6 days/week).201 In contrast, in a study of 51 participants with mean baseline serum LDL = 186 ± 9 mg/dL, LDL decreased in men taking garlic powder tablets (350 mg/day) for 12 months compared to placebo, but not in women.202 This difference in response may have been associated with differences in the intervention period, the garlic dose, and/or the baseline LDL levels of the populations studied. A recent meta-analysis including 39 studies using various garlic preparations reported that total serum cholesterol and LDL cholesterol were reduced by 17 ± 6 mg/dL and 9 ± 6 mg/dL, respectively, in individuals with total cholesterol >200 mg/dL and who had taken the garlic preparation for more than 2 months.203 Triglycerides were not affected. Lipoprotein(a) [Lp(a)], which is related to LDL but is covalently bound to the protein apo(a), is an independent risk factor for cardiovascular disease. A recent metaanalysis did not indicate a significant effect of garlic supplementation on Lp(a) levels.204 As demonstrated for cancer models, lipid changes in response to intake of garlic preparations may depend on the formation of bioactive sulfur compounds. Jabbari et al.205 found that swallowing undamaged garlic had no lowering effect on serum lipids, but consuming crushed garlic reduced cholesterol, triglycerides, malondialdehyde, and blood pressure. Similarly, heating garlic modifies the ability of garlic to inhibit in vivo binding of mammary carcinogen [7,12-dimethylbenzene(a)anthracene, DMBA] metabolites to rat mammary epithelial cell DNA.206 Differential responses to garlic that is crushed or heated may be related to effects on the activity of alliinase in converting alliin to allicin. LDL oxidation has been investigated as a contributor to the initiation and progression of atherosclerosis.207 Some preclinical and clinical studies suggest that AGE may protect against

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oxidation.208 Munday et  al.209 found a modest reduced susceptibility of LDL particles to Cu+2mediated oxidation from subjects given 2.4 g of AGE daily for 7 d. Interestingly, a similar response was not observed when subjects were given 6 g of raw garlic as a daily supplement for 7 d. This result was repeated in subjects taking 1.2 g of AGE for 2 weeks.210 In contrast, Byrne et al.211 did not find that 900 mg of garlic powder for 6 months had an impact on LDL susceptibility to oxidation. It is unclear if the discrepancies in the literature about garlic and LDL oxidation relate to the subjects examined or the preparations used. DADS has been reported to protect human LDL, erythrocyte membranes, and platelets from oxidation and/or glycation.212 The protective effects of six organosulfur compounds (DAS, DADS, SAC, S-ethylcysteine, S-methylcysteine, and S-propylcysteine) were tested for their ability to reduce further oxidation and glycation in already partially oxidized and glycated samples from patients with non-insulin-dependent diabetes.213 These studies revealed that DAS and DADS were superior in delaying LDL oxidation compared to the four cysteine-containing compounds tested. However, the cysteine-containing agents were superior to DAS and DADS in delaying glycative deterioration in already partially glycated LDL. Both responses were highly concentration dependent. Thus, the content or potential for forming bioactive compounds likely explains much of the variability that has been observed in the published literature.

5.6.2 Blood Pressure Blood pressure is an important risk factor in cardiovascular disease. A large meta-analysis showed that lowering systolic blood pressure by 10 mm Hg or diastolic blood pressure by 5 mm Hg by any of the main classes of blood pressure–lowering drugs reduced cardiovascular disease events (fatal and non-fatal) by about 25% and stroke by about 33%.214 Diet, as well as age, sex, hormonal state, and genetic factors, probably influences blood pressure. Increasing evidence suggests garlic may be a dietary component with the ability to reduce blood pressure and cause relaxation in arterial walls. A recent meta-analysis which included 20 studies reported an average 5.1 mm Hg reduction in systolic blood pressure and an average 2.5 mm Hg reduction in diastolic blood pressure.215 The response to garlic was higher in hypertensive subjects, whose systolic blood pressure decreased by 8.7 mm Hg and diastolic blood pressure decreased by 6.1 mm Hg. Other studies suggest that garlic reduces blood pressure in hypertensive subjects but not in normotensive subjects.216 Formulations of AGE and garlic powder seemed to be similarly efficacious. Garlic treatment has been found to lead to a dose-dependent vasorelaxation in both endotheliumintact and mechanically endothelium-disrupted pulmonary arterial rings.217 This vasorelaxation was diminished by the administration of NG-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor. The inducible nitric oxide synthase is recognized to occur in human atherosclerotic lesions. Studies have demonstrated that garlic exerts its therapeutic effect by increasing NO production218 and by suppressing the reduction of cellular nitric oxide synthase by oxidized LDL.219 The relaxant effect on vascular smooth muscle appears to be mediated through a decrease in cGMP and the subsequent release in endothelium-derived relaxing factors, as well as a depression in prostaglandins via a suppression in cyclooxygenase activity.220,221 It is known that ROS counteract the vasodilating and antiproliferative actions of nitric oxide by rapidly degrading it to peroxynitrites. It is possible that part of the blood pressure changes caused by garlic may relate to its ability to reduce radical formation.78 Another mechanism by which blood pressure may be moderated by garlic involves hydrogen sulfide (H2S), which has been shown to be produced by metabolism of garlic polysulfides.222 H2S promotes sulfhydration of ATP-sensitive potassium channels, thereby opening voltage-sensitive channels and triggering subsequent relaxation of vascular smooth muscle cells.223

5.6.3 Plaque and Platelet Aggregation Acute coronary syndromes can occur when an unstable atherosclerotic plaque erodes or ruptures, thereby exposing the highly thrombogenic material inside the plaque to the circulating blood.224

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This exposure triggers a rapid formation of a thrombus that occludes the artery. Budoff et al.225 found in a pilot study that providing AGE for a year inhibited the rate of progression of coronary calcification compared to a placebo. A follow-up clinical trial with 65 patients reported that ingesting AGE (250 mg) supplemented with B vitamins, folic acid, and L-arginine daily for 1 year slowed the progression of subclinical atherosclerosis as assessed by coronary artery calcium scanning.226 In a study that measured low attenuation plaque (non-calcified plaque) in 55 subjects with metabolic syndrome, daily intake (2400 mg) of AGE for 1 year significantly decreased low attenuation plaque compared to placebo.227 Low attenuation plaque was an independent predictor of adverse cardiac events in a population with stable angina, and therefore the moderating effects of AGE on low attenuation plaque may be clinically important.228 Other garlic preparations have also been reported to inhibit plaque formation in humans. Providing 900 mg of garlic powder daily for 48 weeks in a randomized trial reduced arteriosclerotic plaque volumes in both the carotid and femoral arteries by 5%–18%.229 Another randomized trial determined that garlic powder (standardized to 2400 µg/day) ingested daily for 3 months was superior to placebo in retarding the progression of carotid intima-media thickness (CIMT), a measurement of the innermost two layers of the artery walls, and which is strongly associated with atherosclerosis.230 Time-released garlic powder tablets were effective in decreasing CIMT when taken daily for 1 year, whereas there was a moderate progression of CIMT in the placebo group.231 These investigators proposed that this result was a consequence of serum atherogenicity inhibition, a measurement of the ability of serum to induce cholesterol accumulation. Similarly, Zahid et al.232 suggested that garlic may exert its beneficial effect on plaque formation by reducing cholesterol as well as maintaining NO-mediated endothelial function, possibly secondary to an inhibition of LDL oxidation and an increase in HDL. Aggregates of activated platelets are also likely have a pivotal role in coronary syndromes. Garlic and some of its organosulfur components have been found to be potent inhibitors of platelet aggregation in vitro.220,233 Some of the platelet-inhibitory compounds arising from allium plants include ajoene, allicin, SAC, methylallyl trisulfide, and alk(en)nyl thiosulfates such as sodium 2-propenyl thiosulfate and sodium n-propyl thiosulfate. Heating garlic by boiling decreases its ability to inhibit platelet aggregation.233 Recently, Rahman et al.234 studied platelets from 14 subjects and found that AGE significantly decreased cGMP and cAMP, inhibited the binding of activated platelets to fibrinogen, and thereby prevented changes in platelet shape that favor aggregation. A few studies have documented that garlic can inhibit platelet aggregation in vivo. Steiner and Lin235 provided evidence that consumption of AGE reduced epinephrine and collagen-induced platelet aggregation, although it failed to influence adenosine diphosphate-induced aggregation. Their studies also provided evidence that platelet adhesion to fibrinogen could be suppressed by consumption of this garlic supplement. In a study with 23 subjects who had consumed 5 mL of AGE daily for 13 weeks, AGE did inhibit adenosine diphosphate-induced aggregation.236 Finally, DAS, a constituent of garlic oil, induced a reduction in adenosine-induced platelet aggregation in women with type 2 diabetes mellitus.237 Overall, the potential of garlic to reduce sterol synthesis, hyperlipidemia, hypertension, and thrombus formation make it a strong candidate for lowering the risk of heart disease and stroke. Nevertheless, the literature provides evidence for considerable variability in response. Additional studies are needed to help clarify who might benefit most from added garlic, and the most efficacious garlic formulations and dose levels.

5.7  SUMMARY AND CONCLUSIONS Garlic has significant physiological attributes that may promote health. Although it is possible that other allium foods possess similar health attributes, few comparative studies have been undertaken. As garlic causes relatively few side effects, except for possibly its lingering odor, there is little reason to avoid its use. Odor does not appear to be a necessary prerequisite for many of the benefits, as

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water-soluble SAC generally gives comparable benefits to those compounds associated with smell. Although garlic and its bioactive components may influence several key molecular events that are involved with health, to do so it must achieve an effective concentration within the target site, be in the correct metabolic form, and lead to changes in small molecular weight signals in the cellular milieu (metabolomic effects). Whereas most can savor the culinary experiences identified with garlic, some individuals, because of their gene profile and/or environmental exposure, may be particularly responsive to more exaggerated intakes.

ACKNOWLEDGMENTS We are indebted to the late Dr. John A. Milner—former director of the USDA Beltsville Human Nutrition Research Center (2012–2013) and former chief of the Nutritional Sciences Research Group, Division of Cancer Prevention, NCI, NIH (2000–2012)—for his passion and commitment toward understanding garlic chemistry and function in health and for his contribution to the second Handbook of Nutraceuticals in the chapter “Garlic: The Mystical Food in Health Promotion.”238

REFERENCES

1. Zeng, T., Zhang, C.L., Zhao, X.L., and Xie, K.Q., The roles of garlic on the lipid parameters: A systematic review of the literature, Crit Rev Food Sci Nutr, 53(3): 215–230, 2013. 2. Bahadoran, Z., Mirmiran, P., Momenan, A.A., and Azizi, F., Allium vegetable intakes and the incidence of cardiovascular disease, hypertension, chronic kidney disease, and type 2 diabetes in adults: A longitudinal follow-up study, J Hypertens, 35(9): 1909–1916, 2017. 3. Guercio, V., Galeone, C., Turati, F., and La Vecchia, C., Gastric cancer and allium vegetable intake: A critical review of the experimental and epidemiologic evidence, Nutr Cancer, 66(5): 757–773, 2014. 4. Ngo, S.N.T., Williams, D.B., Cobiac, L., and Head, R.J., Does garlic reduce risk of colorectal cancer? A systematic review, J. Nutr, 137: 2264–2269, 2007. 5. Banerjee, S.K., Mukherjee, P.K., and Maulik, S.K., Garlic as an antioxidant: The good, the bad and the ugly, Phytother Res, 17(2): 97–106, 2003. 6. Kyo, E., Uda, N., Kasuga, S., and Itakura, Y., Immunomodulatory effects of aged garlic extract, J Nutr, 131(3s): 1075S–1079S, 2001. 7. Mathew, B.C. and Biju, R.S., Neuroprotective effects of garlic: A review, Libyan J Med, 3(1): 23–33, 2008. 8. United States Department of Agriculture, N.A.S.S., Vegetables: 2015 Summary. 2016. 9. United States Department of Agriculture, E.R.S. Vegetable and Pulses Yearbook 2017, 2017, Last Updated: 4/6/2017, Date Accessed: 2/27/2018, Available from: https://www.ers.usda.gov/webdocs/ DataFiles/83086/Section%202_SandU%20Fresh.pdf?v=42831. 10. Fleischauer, A.T., Poole, C., and Arab, L., Garlic consumption and cancer prevention: Meta-analyses of colorectal and stomach cancers, Am J Clin Nutr, 72(4): 1047–1052, 2000. 11. Jappe, U., Bonnekoh, B., Hausen, B.M., and Gollnick, H., Garlic-related dermatoses: Case report and review of the literature, Am J Contact Dermat, 10(1): 37–39, 1999. 12. Lee, T.Y. and Lam, T.H., Contact dermatitis due to topical treatment with garlic in Hong Kong, Contact Dermatitis, 24(3): 193–196, 1991. 13. Burden, A.D., Wilkinson, S.M., Beck, M.H., and Chalmers, R.J., Garlic-induced systemic contact dermatitis, Contact Dermatitis, 30(5): 299–300, 1994. 14. Moyle, M., Frowen, K., and Nixon, R., Use of gloves in protection from diallyl disulphide allergy, Australas J Dermatol, 45(4): 223–225, 2004. 15. Umar, I.A., Arjinoma, Z.G., Gidado, A., and Hamza, H.H., Prevention of garlic (Allium sativum Linn)induced anaemia in rats by supplementation with ascorbic acid and vitamin E, Nig J Biochem Mol Biol, 13: 31–40, 1998. 16. Yamato, O., Sugiyama, Y., Matsuura, H., Lee, K.W., Goto, K., Hossain, M.A., Maede, Y., and Yoshihara, T., Isolation and identification of sodium 2-propenyl thiosulfate from boiled garlic (Allium sativum) that oxidizes canine erythrocytes, Biosci Biotechnol Biochem, 67(7): 1594–1596, 2003. 17. Borrelli, F., Capasso, R., and Izzo, A.A., Garlic (Allium sativum L.): Adverse effects and drug interactions in humans, Mol Nutr Food Res, 51(11): 1386–1397, 2007.

Garlic

95

18. Fenwick, G.R. and Hanley, A.B., The genus Allium. Part 2, Crit Rev Food Sci Nutr, 22(4): 273–377, 1985. 19. Block, E., Garlic and Other Alliums. 2010, Cambridge: The Royal Society of Chemistry. 20. Kubec, R., Svobodova, M., and Velisek, J., Gas chromatographic determination of S-alk(en)ylcysteine sulfoxides, J Chromatogr A, 862(1): 85–94, 1999. 21. Freeman, G.G. and Mossadeghi, N., Studies on the effect of sulphate nutrition on the flavour components of onion (Allium cepa), Biochem J, 118(2): 24P, 1970. 22. Sugii, M., Suzuki, T., Nagasawa, S., and Kawashima, K., Isolation of gamma-1-glutamyl-s-allylmercaptol-cysteine and s-allylmercapto-l-cysteine from garlic, Chem Pharm Bull (Tokyo), 12: 1114–1115, 1964. 23. Block, E., The chemistry of garlic and onions, Sci Am, 252(3): 114–119, 1985. 24. Lawson, L.D., Wang, Z.-Y.J., and Hughes, B.G., Identification and HPLC quantitation of the sulfides and dialk(en)yl thiosulfinates in commercial garlic products, Planta Med, 57(4): 363–370, 1991. 25. Tamaki, T. and Sonoki, S., Volatile sulfur compounds in human expiration after eating raw or heattreated garlic, J Nutr Sci Vitaminol, 45(2): 213–222, 1999. 26. Borek, C., Antioxidant health effects of aged garlic extract, J Nutr, 131(3 Suppl.): 1010S–1015S, 2001. 27. Lawson, L.D., The composition and chemistry of garlic cloves and processed garlic, in Garlic: The Science and Therapeutic Application of Allium Sativum L. and Related Species, H.P. Koch and L.D. Lawson, Editors. 1996, Williams & Waverly: Baltimore. p. 37–107. 28. Lawson, L.D. and Gardner, C.D., Composition, stability, and bioavailability of garlic products used in a clinical trial, J Agric Food Chem, 53(16): 6254–6261, 2005. 29. Staba, E.J., Lash, L., and Staba, J.E., A commentary on the effects of garlic extraction and formulation on product composition, J Nutr, 131(3): 1118S–1119, 2001. 30. Lachmann, G., Lorenz, D., Radeck, W., and Steiper, M., [The pharmacokinetics of the S35 labeled garlic constituents alliin, allicin and vinyldithiine], Arzneimittelforschung, 44(6): 734–743, 1994. 31. Lawson, L.D. and Wang, Z.J., Allicin and allicin-derived garlic compounds increase breath acetone through allyl methyl sulfide: Use in measuring allicin bioavailability, J Agric Food Chem, 53(6): 1974–1983, 2005. 32. Egen-Schwind, C., Eckard, R., and Kemper, F.H., Metabolism of garlic constituents in the isolated perfused rat liver, Planta Med, 58(4): 301–305, 1992. 33. Freeman, F. and Kodera, Y., Garlic chemistry: Stability of S-(2-propenyl) 2-propene-1-sulfinothioate (allicin) in blood, solvents, and simulated physiological fluids, J Agric Food Chem, 43: 2332–2338, 1995. 34. Germain, E., Auger, J., Ginies, C., Siess, M.H., and Teyssier, C., In vivo metabolism of diallyl disulphide in the rat: Identification of two new metabolites, Xenobiotica, 32(12): 1127–1138, 2002. 35. Teyssier, C., Guenot, L., Suschetet, M., and Siess, M.H., Metabolism of diallyl disulfide by human liver microsomal cytochromes P-450 and flavin-containing monooxygenases, Drug Metab Dispos, 27(7): 835–841, 1999. 36. Krause, R.J., Glocke, S.C., and Elfarra, A.A., Sulfoxides as urinary metabolites of S-allyl-L-cysteine in rats: Evidence for the involvement of flavin-containing monooxygenases, Drug Metab Dispos, 30(10): 1137–1142, 2002. 37. Modem, S., DiCarlo, S.E., and Reddy, T.R., Fresh garlic extract induces growth arrest and morphological differentiation of MCF7 breast cancer cells, Genes and Cancer, 3(2): 177–186, 2012. 38. Yan, J.Y., Tian, F.M., Hu, W.N., Zhang, J.H., Cai, H.F., and Li, N., Apoptosis of human gastric cancer cells line SGC 7901 induced by garlic-derived compound S-allylmercaptocysteine (SAMC), Eur Rev Med Pharmaco, 17(6): 745–751, 2013. 39. Kim, H.J., Han, M.H., Kim, G.Y., Choi, Y.W., and Choi, Y.H., Hexane extracts of garlic cloves induce apoptosis through the generation of reactive oxygen species in Hep3B human hepatocarcinoma cells, Oncology Reports, 28(5): 1757–1763, 2012. 40. Chiang, E.P.I., Chiu, S.C., Pai, M.H., Wang, Y.C., Wang, F.Y., Kuo, Y.H., and Tang, F.Y., Organosulfur garlic compounds induce neovasculogenesis in human endothelial progenitor cells through a modulation of microRNA 221 and the PI3-K/Akt signaling pathways, J Agric Food Chem, 61(20): 4839–4849, 2013. 41. Tang, F.-Y., Chiang, E.-P.I., Chung, J.-G., Lee, H.-Z., and Hsu, C.-Y., S-allylcysteine modulates the expression of E-cadherin and inhibits the malignant progression of human oral cancer, J Nutr Biochem, 20(12):1013–1020, 2009. 42. Sakamoto, K., Lawson, L.D., and Milner, J.A., Allyl sulfides from garlic suppress the in vitro proliferation of human A549 lung tumor cells, Nutr Cancer, 29(2): 152–156, 1997. 43. Sundaram, S.G. and Milner, J.A., Diallyl disulfide induces apoptosis of human colon tumor cells, Carcinogenesis, 17(4): 669–673, 1996. 44. Hu, X., Benson, P.J., Srivastava, S.K., Xia, H., Bleicher, R.J., Zaren, H.A., Awasthi, S., Awasthi, Y.C., and Singh, S.V., Induction of glutathione S-transferase π as a bioassay for the evaluation of potency of inhibitors of benzo(a)pyrene-induced cancer in a murine model, Int J Cancer, 73(6): 897–902, 1997.

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45. Charron, C.S., Milner, J.A., and Novotny, J.A., Garlic, in Encyclopedia of Food and Health, B. Caballero, P. Finglas, and F. Toldra, Editors. 2016, Academic Press: Oxford. p. 184–190. 46. Adetumbi, M.A. and Lau, B.H., Allium sativum (garlic)—A natural antibiotic, Med Hypotheses, 12(3): 227–237, 1983. 47. Verma, S., Kaur, S., Singh, J., and Garg, A., Anti-bacterial effect of garlic (Allium sativum L) extract on different pathogenic and non-pathogenic bacteria, Res J Pharm Biol Chem Sci, 6(3): 1103–1107, 2015. 48. Lai, P.K. and Roy, J., Antimicrobial and chemopreventive properties of herbs and spices, Curr Med Chem, 11(11): 1451–1460, 2004. 49. Davis, S.R., An overview of the antifungal properties of allicin and its breakdown products—The possibility of a safe and effective antifungal prophylactic, Mycoses, 48(2): 95–100, 2005. 50. Ruddock, P.S., Liao, M., Foster, B.C., Lawson, L., Arnason, J.T., and Dillon, J.A., Garlic natural health products exhibit variable constituent levels and antimicrobial activity against Neisseria gonorrhoeae, Staphylococcus aureus and Enterococcus faecalis, Phytother Res, 19(4): 327–334, 2005. 51. Siao, D. and Somsouk, M., Helicobacter pylori: Evidence-based review with a focus on immigrant populations, J Gen Intern Med, 29(3): 520–528, 2014. 52. Webb, P.M., Law, M., Varghese, C., and Forman, D., Gastric cancer and Helicobacter pylori: A combined analysis of 12 case control studies nested within prospective cohorts, Gut, 49(3): 347–353, 2001. 53. Cellini, L., Di Campli, E., Masulli, M., Di Bartolomeo, S., and Allocati, N., Inhibition of Helicobacter pylori by garlic extract (Allium sativum), FEMS Immunol Med Microbiol, 13(4): 273–277, 1996. 54. Chung, J.G., Chen, G.W., Wu, L.T., Chang, H.L., Lin, J.G., Yeh, C.C., and Wang, T.F., Effects of garlic compounds diallyl sulfide and diallyl disulfide on arylamine N-acetyltransferase activity in strains of Helicobacter pylori from peptic ulcer patients, Am J Chin Med, 26(3–4): 353–364, 1998. 55. Jonkers, D., van den Broek, E., van Dooren, I., Thijs, C., Dorant, E., Hageman, G., and Stobberingh, E., Antibacterial effect of garlic and omeprazole on Helicobacter pylori, J Antimicrob Chemother, 43(6): 837–839, 1999. 56. You, W.C. et al., Helicobacter pylori infection, garlic intake and precancerous lesions in a Chinese population at low risk of gastric cancer, Int J Epidemiol, 27(6): 941–944, 1998. 57. Aydin, A., Ersoz, G., Tekesin, O., Akcicek, E., and Tuncyurek, M., Garlic oil and Helicobacter pylori infection, Am J Gastroenterol, 95(2): 563–564, 2000. 58. McNulty, C.A.M., Wilson, M.P., Havinga, W., Johnston, B., O’Gara, E.A., and Maslin, D.J., A pilot study to determine the effectiveness of garlic oil capsules in the treatment of dyspeptic patients with Helicobacter pylori, Helicobacter, 6(3): 249–253, 2001. 59. Graham, D.Y., Anderson, S.Y., and Lang, T., Garlic or jalapeno peppers for treatment of Helicobacter pylori infection, Am J Gastroenterol, 94(5): 1200–1202, 1999. 60. Hekmatdoost, A., Ghobeh, M., Shaker-Hosseini, R., MirSattari, D., Rastmanesh, R., Rashidkhani, B., and Navai, L., The effect of garlic consumption on Helicobacter pylori treatment using urea breath test: A randomized clinical trial, J Nutr Sci Diet, 1(1): 21–27, 2015. 61. Zardast, M., Namakin, K., Esmaelian Kaho, J., and Hashemi, S.S., Assessment of antibacterial effect of garlic in patients infected with Helicobacter pylori using urease breath test, Avicenna J Phytomed, 6(5): 495–501, 2016. 62. Arora, D.S. and Kaur, J., Antimicrobial activity of spices, Int J Antimicrob Agents, 12(3): 257–262, 1999. 63. Ledezma, E., Marcano, K., Jorquera, A., De Sousa, L., Padilla, M., Pulgar, M., and Apitz-Castro, R., Efficacy of ajoene in the treatment of tinea pedis: A double-blind and comparative study with terbinafine, J Am Acad Dermatol, 43(5 Pt 1): 829–832, 2000. 64. Rabinkov, A., Miron, T., Mirelman, D., Wilchek, M., Glozman, S., Yavin, E., and Weiner, L., S-allylmercaptoglutathione: The reaction product of allicin with glutathione possesses SH-modifying and antioxidant properties, Biochimica et Biophysica Acta – Molecular Cell Research, 1499(1–2): 144– 153, 2000. 65. Gupta, N. and Porter, T.D., Garlic and garlic-derived compounds inhibit human squalene monooxygenase, J Nutr, 131(6): 1662–1667, 2001. 66. Balkis, M.M., Leidich, S.D., Mukherjee, P.K., and Ghannoum, M.A., Mechanisms of fungal resistance: An overview, Drugs, 62(7): 1025–1040, 2002. 67. Sonnenburg, J.L. and Bäckhed, F., Diet-microbiota interactions as moderators of human metabolism, Nature, 535(7610): 56–64, 2016. 68. Filocamo, A., Nueno-Palop, C., Bisignano, C., Mandalari, G., and Narbad, A., Effect of garlic powder on the growth of commensal bacteria from the gastrointestinal tract, Phytomedicine, 19(8–9): 707–711, 2012. 69. Milner, J.A., Molecular targets for bioactive food components, J Nutr, 134(9): 2492S–2498S, 2004.

Garlic

97

70. Sikand, G., Kris-Etherton, P., and Boulos, N.M., Impact of functional foods on prevention of cardiovascular disease and diabetes, Curr Cardiol Rep, 17(6): 39, 2015. 71. Mayne, S.T., Playdon, M.C., and Rock, C.L., Diet, nutrition, and cancer: Past, present and future, Nat Rev Clin Oncol, 13(8): 504–515, 2016. 72. American Institute for Cancer Research, Food, Nutrition, Physical Activity, and the Prevention of Cancer: A Global Perspective. 2007, World Cancer Research Fund/American Institute for Cancer Research: Washington, D.C. p. 1–16. 73. Kodali, R.T. and Eslick, G.D., Meta-analysis: Does garlic intake reduce risk of gastric cancer? Nutr Cancer, 67(1): 1–11, 2015. 74. Hu, J.Y., Hu, Y.W., Zhou, J.J., Zhang, M.W., Li, D., and Zheng, S., Consumption of garlic and risk of colorectal cancer: An updated meta-analysis of prospective studies, World J Gastroenterol, 20(41): 15413–15422, 2014. 75. Zhou, Y., Zhuang, W., Hu, W., Liu, G.J., Wu, T.X., and Wu, X.T., Consumption of large amounts of allium vegetables reduces risk for gastric cancer in a meta-analysis, Gastroenterology, 141(1): 80–89, 2011. 76. Zhou, X.F., Ding, Z.S., and Liu, N.B., Allium vegetables and risk of prostate cancer: Evidence from 132,192 subjects, Asian Pac J Cancer Prev, 14(7): 4131–4134, 2013. 77. World Cancer Research Fund/American Institute for Cancer Research, Diet, Nutrition, Physical Activity and Cancer: A Global Perspective. Continuous Update Project Expert Report 2018., 2018. 78. Steinmetz, K.A., Kushi, L.H., Bostick, R.M., Folsom, A.R., and Potter, J.D., Vegetables, fruit, and colon cancer in the Iowa women’s health study, Am J Epidemiol, 139: 1–15, 1994. 79. Kim, H., Keum, N., Giovannucci, E.L., Fuchs, C.S., and Bao, Y., Garlic intake and gastric cancer risk: Results from two large prospective US cohort studies, Int J Cancer, 143(5):1047–1053, 2018. 80. Myneni, A.A. et al., Raw garlic consumption and lung cancer in a Chinese population, Cancer Epidem Biomar Prev, 25(4): 624–633, 2016. 81. Tanaka, S. et al., Effects of aged garlic extract (AGE) on colorectal adenomas: A double-blinded study, Hiroshima J Med Sci, 53(3–4): 39–45, 2004. 82. You, W.C., Blot, W.J., Chang, Y.S., Ershow, A., Yang, Z.T., An, Q., Henderson, B.E., Fraumeni, J.F., Jr., and Wang, T.G., Allium vegetables and reduced risk of stomach cancer, J Natl Cancer Inst, 81(2): 162–164, 1989. 83. Gail, M.H. et al., Factorial trial of three interventions to reduce the progression of precancerous gastric lesions in Shandong, China: Design issues and initial data, Control Clin Trials, 19(4): 352–369, 1998. 84. Ma, J.L. et al., Fifteen-year effects of Helicobacter pylori, garlic, and vitamin treatments on gastric cancer incidence and mortality, J Natl Cancer Inst, 104(6): 488–492, 2012. 85. Li, H., Li, H.-Q., Wang, Y., Xu, H.-X., Fan, W.-T., Wang, M.-L., Sun, P.-H., and Xie, X.-Y., An intervention study to prevent gastric cancer by micro-selenium and large dose of allitridum, Chinese Med J Peking, 117(8): 1155–1160, 2004. 86. Singh, A. and Shukla, Y., Antitumour activity of diallyl sulfide on polycyclic aromatic hydrocarboninduced mouse skin carcinogenesis, Cancer Lett, 131(2): 209–214, 1998. 87. Ip, C., Lisk, D.J., and Stoewsand, G.S., Mammary cancer prevention by regular garlic and seleniumenriched garlic, Nutr Cancer, 17(3): 279–286, 1992. 88. Wargovich, M.J., Diallyl sulfide, a flavor component of garlic (Allium sativum), inhibits dimethylhydrazineinduced colon cancer, Carcinogenesis, 8(3): 487–489, 1987. 89. Hussain, S.P., Jannu, L.N., and Rao, A.R., Chemopreventive action of garlic on methylcholanthreneinduced carcinogenesis in the uterine cervix of mice, Cancer Lett, 49(2): 175–180, 1990. 90. Wargovich, M.J., Woods, C., Eng, V.W., Stephens, L.C., and Gray, K., Chemoprevention of N-nitrosomethylbenzylamine-induced esophageal cancer in rats by the naturally occurring thioether, diallyl sulfide, Cancer Res, 48(23): 6872–6875, 1988. 91. Hong, J.Y., Wang, Z.Y., Smith, T.J., Zhou, S., Shi, S., Pan, J., and Yang, C.S., Inhibitory effects of diallyl sulfide on the metabolism and tumorigenicity of the tobacco-specific carcinogen 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone (NNK) in A/J mouse lung, Carcinogenesis, 13(5): 901–904, 1992. 92. Takahashi, S., Hakoi, K., Yada, H., Hirose, M., Ito, N., and Fukushima, S., Enhancing effects of diallyl sulfide on hepatocarcinogenesis and inhibitory actions of the related diallyl disulfide on colon and renal carcinogenesis in rats, Carcinogenesis, 13(9): 1513–1518, 1992. 93. Nagabhushan, M., Line, D., Polverini, P.J., and Solt, D.B., Anticarcinogenic action of diallyl sulfide in hamster buccal pouch and forestomach, Cancer Lett, 66(3): 207–216, 1992. 94. Park, K.A., Kweon, S., and Choi, H., Anticarcinogenic effect and modification of cytochrome P450 2E1 by dietary garlic powder in diethylnitrosamine-initiated rat hepatocarcinogenesis, J Biochem Mol Biol, 35(6): 615–622, 2002.

98

Handbook of Nutraceuticals and Functional Foods

95. Ross, S.A., Finley, J.W., and Milner, J.A., Allyl sulfur compounds from garlic modulate aberrant crypt formation, J Nutr, 136(3): 852S–854, 2006. 96. Wargovich, M.J., Chen, C.D., Jimenez, A., Steele, V.E., Velasco, M., Stephens, L.C., Price, R., Gray, K., and Kelloff, G.J., Aberrant crypts as a biomarker for colon cancer: Evaluation of potential chemopreventive agents in the rat, Cancer Epidem Biomar Prev, 5(5): 355–360, 1996. 97. Wargovich, M.J., Jimenez, A., McKee, K., Steele, V.E., Velasco, M., Woods, J., Price, R., Gray, K., and Kelloff, G.J., Efficacy of potential chemopreventive agents on rat colon aberrant crypt formation and progression, Carcinogenesis, 21(6): 1149–1155, 2000. 98. Jakszyn, P., Agudo, A., Ibanez, R., Garcia-Closas, R., Pera, G., Amiano, P., and Gonzalez, C.A., Development of a food database of nitrosamines, heterocyclic amines, and polycyclic aromatic hydrocarbons, J Nutr, 134(8): 2011–2014, 2004. 99. Lijinsky, W., N-nitroso compounds in the diet, Mutat Res, 443(1–2): 129–138, 1999. 100. Shenoy, N.R. and Choughuley, A.S., Inhibitory effect of diet related sulphydryl compounds on the formation of carcinogenic nitrosamines, Cancer Lett, 65(3): 227–232, 1992. 101. Milner, J.A., Mechanisms by which garlic and allyl sulfur compounds suppress carcinogen bioactivation. Garlic and carcinogenesis, Adv Exp Med Biol, 492: 69–81, 2001. 102. Dion, M.E., Agler, M., and Milner, J.A., S-allyl cysteine inhibits nitrosomorpholine formation and bioactivation, Nutr Cancer, 28(1): 1–6, 1997. 103. Mei, X., Lin, X., Liu, J., Lin, X.Y., Song, P.J., Hu, J.F., and Liang, X.J., The blocking effect of garlic on the formation of N-nitrosoproline in humans, Acta Nutrimenta Sinica, 11: 141–145, 1989. 104. Ohshima, H. and Bartsch, H., Quantitative estimation of endogenous N-nitrosation in humans by monitoring N-nitrosoproline in urine, Methods Enzymol, 301: 40–49, 1999. 105. Cope, K., Seifried, H., Seifried, R., Milner, J., Kris-Etherton, P., and Harrison, E.H., A gas chromatography-mass spectrometry method for the quantitation of N-nitrosoproline and N-acetyl-Sallylcysteine in human urine: Application to a study of the effects of garlic consumption on nitrosation, Anal Biochem, 394(2): 243–248, 2009. 106. Haber-Mignard, D., Suschetet, M., Berges, R., Astorg, P., and Siess, M.H., Inhibition of aflatoxin B1- and N-nitrosodiethylamine-induced liver preneoplastic foci in rats fed naturally occurring allyl sulfides, Nutr Cancer, 25(1): 61–70, 1996. 107. Knasmuller, S., de Martin, R., Domjan, G., and Szakmary, A., Studies on the antimutagenic activities of garlic extract, Environ Mol Mutagen, 13(4): 357–365, 1989. 108. Wargovich, M.J., Diallylsulfide and allylmethylsulfide are uniquely effective among organosulfur compounds in inhibiting CYP2E1 protein in animal models, J Nutr, 136(3): 832S–834S, 2006. 109. Yang, C.S., Chhabra, S.K., Hong, J.Y., and Smith, T.J., Mechanisms of inhibition of chemical toxicity and carcinogenesis by diallyl sulfide (DAS) and related compounds from garlic, J Nutr, 131(3s): 1041s–1045s, 2001. 110. Jin, L. and Baillie, T.A., Metabolism of the chemoprotective agent diallyl sulfide to glutathione conjugates in rats, Chem Res Toxicol, 10(3): 318–327, 1997. 111. Rounds, L., Havens, C.M., Feinstein, Y., Friedman, M., and Ravishankar, S., Plant extracts, spices, and essential oils inactivate Escherichia coli O157:H7 and reduce formation of potentially carcinogenic heterocyclic amines in cooked beef patties, J Agric Food Chem, 60(14): 3792–3799, 2012. 112. Gibis, M., Effect of oil marinades with garlic, onion, and lemon juice on the formation of heterocyclic aromatic amines in fried beef patties, J Agric Food Chem, 55(25): 10240–10247, 2007. 113. Davenport, D.M. and Wargovich, M.J., Modulation of cytochrome P450 enzymes by organosulfur compounds from garlic, Food Chem Toxicol, 43(12): 1753–1762, 2005. 114. Yu, F.S., Yu, C.S., Lin, J.P., Chen, S.C., Lai, W.W., and Chung, J.G., Diallyl disulfide inhibits N-acetyltransferase activity and gene expression in human esophagus epidermoid carcinoma CE 81 T/ VGH cells, Food Chem Toxicol, 43(7): 1029–1036, 2005. 115. Lin, X.Y., Liu, J.Z., and Milner, J.A., Dietary garlic suppresses DNA adducts caused by N-nitroso compounds, Carcinogenesis, 15(2): 349–352, 1994. 116. Schaffer, E.M., Liu, J.Z., Green, J., Dangler, C.A., and Milner, J.A., Garlic and associated allyl sulfur components inhibit N-methyl-N-nitrosourea induced rat mammary carcinogenesis, Cancer Lett, 102(1– 2): 199–204, 1996. 117. Cohen, L.A., Zhao, Z., Pittman, B., and Lubet, R., S-allylcysteine, a garlic constituent, fails to inhibit N-methylnitrosourea-induced rat mammary tumorigenesis, Nutr Cancer, 35(1): 58–63, 1999. 118. Ludeke, B.I., Domine, F., Ohgaki, H., and Kleihues, P., Modulation of N-nitrosomethylbenzylamine bioactivation by diallyl sulfide in vivo, Carcinogenesis, 13(12): 2467–2470, 1992.

Garlic

99

119. Zhang, Y.J., Chen, Y., Ahsan, H., Lunn, R.M., Chen, S.Y., Lee, P.H., Chen, C.J., and Santella, R.M., Silencing of glutathione S-transferase P1 by promoter hypermethylation and its relationship to environmental chemical carcinogens in hepatocellular carcinoma, Cancer Lett, 221(2): 135–143, 2005. 120. Le Bon, A.M., Vernevaut, M.F., Guenot, L., Kahane, R., Auger, J., Arnault, I., Haffner, T., and Siess, M.H., Effects of garlic powders with varying alliin contents on hepatic drug metabolizing enzymes in rats, J Agric Food Chem, 51(26): 7617–7623, 2003. 121. Wu, C.-C., Sheen, L.-Y., Chen, H.-W., Kuo, W.-W., Tsai, S.-J., and Lii, C.-K., Differential effects of garlic oil and its three major organosulfur components on the hepatic detoxification system in rats, J Agric Food Chem, 50(2): 378–383, 2002. 122. Foster, B.C., Foster, M.S., Vandenhoek, S., Krantis, A., Budzinski, J.W., Arnason, J.T., Gallicano, K.D., and Choudri, S., An in vitro evaluation of human cytochrome P450 3A4 and P-glycoprotein inhibition by garlic, J Pharm Pharm Sci, 4(2): 176–184, 2001. 123. Brady, J.F., Ishizaki, H., Fukuto, J.M., Lin, M.C., Fadel, A., Gapac, J.M., and Yang, C.S., Inhibition of cytochrome P-450 2E1 by diallyl sulfide and its metabolites, Chem Res Toxicol, 4(6): 642–647, 1991. 124. Amano, H., Kazamori, D., and Itoh, K., Evaluation of the effects of S-allyl-L-cysteine, S-methyl-Lcysteine, trans-S-1-propenyl-L-cysteine, and their N-acetylated and S-oxidized metabolites on human CYP activities, Biol Pharm Bull, 39(10): 1701–1707, 2016. 125. Uno, S., Dalton, T.P., Derkenne, S., Curran, C.P., Miller, M.L., Shertzer, H.G., and Nebert, D.W., Oral exposure to benzo[a]pyrene in the mouse: Detoxication by inducible cytochrome P450 is more important than metabolic activation, Mol Pharmacol, 65(5): 1225–1237, 2004. 126. Singh, S.V., Pan, S.S., Srivastava, S.K., Xia, H., Hu, X., Zaren, H.A., and Orchard, J.L., Differential induction of NAD(P)H:quinone oxidoreductase by anti-carcinogenic organosulfides from garlic, Biochem Biophys Res Commun, 244(3): 917–920, 1998. 127. Chen, C., Pung, D., Leong, V., Hebbar, V., Shen, G., Nair, S., Li, W., and Kong, A.N., Induction of detoxifying enzymes by garlic organosulfur compounds through transcription factor Nrf2: Effect of chemical structure and stress signals, Free Radic Biol Med, 37(10): 1578–1590, 2004. 128. Kim, S., Lee, H.G., Park, S.A., Kundu, J.K., Keum, Y.S., Cha, Y.N., Na, H.K., and Surh, Y.J., Keap1 cysteine 288 as a potential target for diallyl trisulfide-induced Nrf2 activation, PLOS ONE, 9(1): e85984, 2014. 129. Munday, R. and Munday, C.M., Relative activities of organosulfur compounds derived from onions and garlic in increasing tissue activities of quinone reductase and glutathione transferase in rat tissues, Nutr Cancer, 40(2): 205–210, 2001. 130. Charron, C.S., Dawson, H.D., and Novotny, J.A., Garlic influences gene expression in vivo and in vitro, J Nutr, 146(2): 444s–449s, 2016. 131. Berges, R., Siess, M.H., Arnault, I., Auger, J., Kahane, R., Pinnert, M.F., Vernevaut, M.F., and le Bon, A.M., Comparison of the chemopreventive efficacies of garlic powders with different alliin contents against aflatoxin B1 carcinogenicity in rats, Carcinogenesis, 25(10): 1953–1959, 2004. 132. Surh, Y.J., Lee, R.C., Park, K.K., Mayne, S.T., Liem, A., and Miller, J.A., Chemoprotective effects of capsaicin and diallyl sulfide against mutagenesis or tumorigenesis by vinyl carbamate and N-nitrosodimethylamine, Carcinogenesis, 16(10): 2467–2471, 1995. 133. Zhang, C.L., Zeng, T., Zhao, X.L., and Xie, K.Q., Garlic oil attenuated nitrosodiethylamine-induced hepatocarcinogenesis by modulating the metabolic activation and detoxification enzymes, Int J Biol Sci, 9(3): 237–245, 2013. 134. Herman-Antosiewicz, A. and Singh, S.V., Signal transduction pathways leading to cell cycle arrest and apoptosis induction in cancer cells by allium vegetable-derived organosulfur compounds: A review, Mutat Res, 555(1–2): 121–131, 2004. 135. Wu, X., Kassie, F., and Mersch-Sundermann, V., Induction of apoptosis in tumor cells by naturally occurring sulfur-containing compounds, Mutat Res, 589(2): 81–102, 2005. 136. Wu, X.J., Kassie, F., and Mersch-Sundermann, V., The role of reactive oxygen species (ROS) production on diallyl disulfide (DADS) induced apoptosis and cell cycle arrest in human A549 lung carcinoma cells, Mutat Res, 579(1–2): 115–124, 2005. 137. Knowles, L.M. and Milner, J.A., Possible mechanism by which allyl sulfides suppress neoplastic cell proliferation, J Nutr, 131(3): 1061S–1066, 2001. 138. Ling, H., Lu, L.F., He, J., Xiao, G.H., Jiang, H., and Su, Q., Diallyl disulfide selectively causes checkpoint kinase-1 mediated G2/M arrest in human MGC803 gastric cancer cell line, Oncol Rep, 32(5): 2274–2282, 2014. 139. Knowles, L.M. and Milner, J.A., Diallyl disulfide inhibits p34(cdc2) kinase activity through changes in complex formation and phosphorylation, Carcinogenesis, 21(6): 1129–1134, 2000.

100

Handbook of Nutraceuticals and Functional Foods

140. Knowles, L.M. and Milner, J.A., Diallyl disulfide induces ERK phosphorylation and alters gene expression profiles in human colon tumor cells, J Nutr, 133(9): 2901–2906, 2003. 141. Filomeni, G., Rotilio, G., and Ciriolo, M.R., Molecular transduction mechanisms of the redox network underlying the antiproliferative effects of allyl compounds from garlic, J Nutr, 138(11): 2053–2057, 2008. 142. Puccinelli, M.T. and Stan, S.D., Dietary bioactive diallyl trisulfide in cancer prevention and treatment, Int J Mol Sci, 18(8), 2017. 143. Iciek, M., Kwiecien, I., Chwatko, G., Sokolowska-Jezewicz, M., Kowalczyk-Pachel, D., and Rokita, H., The effects of garlic-derived sulfur compounds on cell proliferation, caspase 3 activity, thiol levels and anaerobic sulfur metabolism in human hepatoblastoma HepG2 cells, Cell Biochem Funct, 30(3): 198–204, 2012. 144. L’Vova G, N. and Zasukhina, G.D., [Modification of repair DNA synthesis in mutagen-treated human fibroblasts during adaptive response and the antimutagenic effect of garlic extract], Genetika, 38(3): 306–309, 2002. 145. Konkimalla, V.S., Wang, G., Kaina, B., and Efferth, T., Microarray-based expression of DNA repair genes does not correlate with growth inhibition of cancer cells by natural products derived from traditional Chinese medicine, Cancer Genom Proteom, 5(2): 79–84, 2008. 146. Ross, S.A., Diet and DNA methylation interactions in cancer prevention, Ann NY Acad Sci, 983: 197–207, 2003. 147. Xu, Y., Su, D., Zhu, L., Zhang, S., Ma, S., Wu, K., Yuan, Q., and Lin, N., S-allylcysteine suppresses ovarian cancer cell proliferation by DNA methylation through DNMT1, J Ovarian Res, 11(1): 39, 2018. 148. Lea, M.A., Randolph, V.M., and Patel, M., Increased acetylation of histones induced by diallyl disulfide and structurally related molecules, Int J Oncol, 15(2): 347–352, 1999. 149. Lea, M.A., Rasheed, M., Randolph, V.M., Khan, F., Shareef, A., and desBordes, C., Induction of histone acetylation and inhibition of growth of mouse erythroleukemia cells by S-allylmercaptocysteine, Nutr Cancer, 43(1): 90–102, 2002. 150. Druesne, N., Pagniez, A., Mayeur, C., Thomas, M., Cherbuy, C., Duee, P.H., Martel, P., and Chaumontet, C., Diallyl disulfide (DADS) increases histone acetylation and p21(waf1/cip1) expression in human colon tumor cell lines, Carcinogenesis, 25(7): 1227–1236, 2004. 151. Zhao, J., Huang, W.G., He, J., Tan, H., Liao, Q.J., and Su, Q., Diallyl disulfide suppresses growth of HL-60 cell through increasing histone acetylation and p21WAF1 expression in vivo and in vitro, Acta Pharmacol Sin, 27(11): 1459–1466, 2006. 152. Druesne-Pecollo, N. and Latino-Martel, P., Modulation of histone acetylation by garlic sulfur compounds, Anti-Cancer Agents Med Chem, 11(3): 254–259, 2011. 153. Nian, H., Delage, B., Pinto, J.T., and Dashwood, R.H., Allyl mercaptan, a garlic-derived organosulfur compound, inhibits histone deacetylase and enhances Sp3 binding on the P21WAF1 promoter, Carcinogenesis, 29(9): 1816–1824, 2008. 154. Arora, A. and Shukla, Y., Induction of apoptosis by diallyl sulfide in DMBA-induced mouse skin tumors, Nutr Cancer, 44(1): 89–94, 2002. 155. Myzak, M.C., Ho, E., and Dashwood, R.H., Dietary agents as histone deacetylase inhibitors, Mol Carcinog, 45(6): 443–446, 2006. 156. Bartel, D.P., microRNAs: Target recognition and regulatory functions, Cell, 136(2): 215–233, 2009. 157. Zhang, B., Pan, X., Cobb, G.P., and Anderson, T.A., microRNAs as oncogenes and tumor suppressors, Dev Biol, 302(1): 1–12, 2007. 158. Xiao, X., Chen, B., Liu, X., Liu, P., Zheng, G., Ye, F., Tang, H., and Xie, X., Diallyl disulfide suppresses SRC/Ras/ERK signaling-mediated proliferation and metastasis in human breast cancer by up-regulating miR-34a, PLOS ONE, 9(11): e112720, 2014. 159. Rabinkov, A., Miron, T., Konstantinovski, L., Wilchek, M., Mirelman, D., and Weiner, L., The mode of action of allicin: Trapping of radicals and interaction with thiol containing proteins, Biochim Biophys Acta, 1379(2): 233–244, 1998. 160. Rahman, K., Garlic and aging: New insights into an old remedy, Ageing Res Rev, 2(1): 39–56, 2003. 161. Dwivedi, C., John, L.M., Schmidt, D.S., and Engineer, F.N., Effects of oil-soluble organosulfur compounds from garlic on doxorubicin-induced lipid peroxidation, Anticancer Drugs, 9(3): 291–294, 1998. 162. Atmaca, G., Antioxidant effects of sulfur-containing amino acids, Yonsei Med J, 45(5): 776–788, 2004. 163. Okada, Y., Tanaka, K., Fujita, I., Sato, E., and Okajima, H., Antioxidant activity of thiosulfinates derived from garlic, Redox Rep, 10(2): 96–102, 2005. 164. Petropoulos, S., Fernandes, A., Barros, L., Ciric, A., Sokovic, M., and Ferreira, I., Antimicrobial and antioxidant properties of various Greek garlic genotypes, Food Chem, 245: 7–12, 2018.

Garlic

101

165. Nencini, C., Menchiari, A., Franchi, G.G., and Micheli, L., In vitro antioxidant activity of aged extracts of some Italian allium species, Plant Foods Hum Nutr (Dordrecht, Netherlands), 66(1): 11–16, 2011. 166. Gorinstein, S. et al., Comparison of the bioactive compounds and antioxidant potentials of fresh and cooked Polish, Ukrainian, and Israeli garlic, J Agric Food Chem, 53(7): 2726–2732, 2005. 167. Schafer, G. and Kaschula, C.H., The immunomodulation and anti-inflammatory effects of garlic organosulfur compounds in cancer chemoprevention, Anti-Cancer Agents Med Chem, 14(2): 233–240, 2014. 168. Arreola, R., Quintero-Fabian, S., Lopez-Roa, R.I., Flores-Gutierrez, E.O., Reyes-Grajeda, J.P., CarreraQuintanar, L., and Ortuno-Sahagun, D., Immunomodulation and anti-inflammatory effects of garlic compounds, J Immunol Res, 2015: 401630, 2015. 169. Lamm, D.L. and Riggs, D.R., Enhanced immunocompetence by garlic: Role in bladder cancer and other malignancies, J Nutr, 131(3): 1067S–1070, 2001. 170. Colic, M. and Savic, M., Garlic extracts stimulate proliferation of rat lymphocytes in vitro by increasing IL-2 and IL-4 production, Immunopharm Immunot, 22(1): 163–181, 2000. 171. Colic, M., Vucevic, D., Kilibarda, V., Radicevic, N., and Savic, M., Modulatory effects of garlic extracts on proliferation of T-lymphocytes in vitro stimulated with concanavalin A, Phytomedicine, 9(2): 117–124, 2002. 172. Jeong, H.G. and Lee, Y.W., Protective effects of diallyl sulfide on N-nitrosodimethylamine-induced immunosuppression in mice, Cancer Lett, 134(1): 73–79, 1998. 173. Ishikawa, H., Saeki, T., Otani, T., Suzuki, T., Shimozuma, K., Nishino, H., Fukuda, S., and Morimoto, K., Aged garlic extract prevents a decline of NK cell number and activity in patients with advanced cancer, J Nutr, 136(3 Suppl): 816s–820s, 2006. 174. Morioka, N., Sze, L.L., Morton, D.L., and Irie, R.F., A protein fraction from aged garlic extract enhances cytotoxicity and proliferation of human lymphocytes mediated by interleukin-2 and concanavalin A, Cancer Immunol Immunother, 37(5): 316–322, 1993. 175. Charron, C.S., Dawson, H.D., Albaugh, G.P., Solverson, P.M., Vinyard, B.T., Solano-Aguilar, G.I., Molokin, A., and Novotny, J.A., A single meal containing raw, crushed garlic influences expression of immunity- and cancer-related genes in whole blood of humans, J Nutr, 145(11): 2448–2455, 2015. 176. Chang, H.P., Huang, S.Y., and Chen, Y.H., Modulation of cytokine secretion by garlic oil derivatives is associated with suppressed nitric oxide production in stimulated macrophages, J Agric Food Chem, 53(7): 2530–2534, 2005. 177. Dirsch, V.M., Kiemer, A.K., Wagner, H., and Vollmar, A.M., Effect of allicin and ajoene, two compounds of garlic, on inducible nitric oxide synthase, Atherosclerosis, 139(2): 333–339, 1998. 178. Chang, H.P. and Chen, Y.H., Differential effects of organosulfur compounds from garlic oil on nitric oxide and prostaglandin E2 in stimulated macrophages, Nutrition, 21(4): 530–536, 2005. 179. Saud, S.M., Li, W., Gray, Z., Matter, M.S., Colburn, N.H., Young, M.R., and Kim, Y.S., Diallyl disulfide (DADS), a constituent of garlic, inactivates NF-kappaB and prevents colitis-induced colorectal cancer by inhibiting GSK-3beta, Cancer Prev Res (Philadelphia, Pa.), 9(7): 607–615, 2016. 180. Smith, B.J., Curtis, J.F., and Eling, T.E., Bioactivation of xenobiotics by prostaglandin H synthase, Chem Biol Interact, 79(3): 245–264, 1991. 181. Li, N., Sood, S., Wang, S., Fang, M., Wang, P., Sun, Z., Yang, C.S., and Chen, X., Overexpression of 5-lipoxygenase and cyclooxygenase 2 in hamster and human oral cancer and chemopreventive effects of zileuton and celecoxib, Clin Cancer Res, 11(5): 2089–2096, 2005. 182. Song, K. and Milner, J.A., Heating garlic inhibits its ability to suppress 7, 12-dimethylbenz(a)anthraceneinduced DNA adduct formation in rat mammary tissue, J Nutr, 129(3): 657–661, 1999. 183. Ali, M., Mechanism by which garlic (Allium sativum) inhibits cyclooxygenase activity. Effect of raw versus boiled garlic extract on the synthesis of prostanoids, Prostaglandins Leukot Essent Fatty Acids, 53(6): 397–400, 1995. 184. Belman, S., Solomon, J., Segal, A., Block, E., and Barany, G., Inhibition of soybean lipoxygenase and mouse skin tumor promotion by onion and garlic components, J Biochem Toxicol, 4(3): 151–160, 1989. 185. Dirsch, V.M. and Vollmar, A.M., Ajoene, a natural product with non-steroidal anti-inflammatory drug (NSAID)-like properties? Biochem Pharmacol, 61(5): 587–593, 2001. 186. Velmurugan, B., Bhuvaneswari, V., Abraham, S.K., and Nagini, S., Protective effect of tomato against N-methyl-N’-nitro-N-nitrosoguanidine-induced in vivo clastogenicity and oxidative stress, Nutrition, 20(9): 812–816, 2004. 187. Bhuvaneswari, V., Abraham, S.K., and Nagini, S., Combinatorial antigenotoxic and anticarcinogenic effects of tomato and garlic through modulation of xenobiotic-metabolizing enzymes during hamster buccal pouch carcinogenesis, Nutrition, 21(6): 726–731, 2005.

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188. Kumaraguruparan, R., Chandra Mohan, K.V., Abraham, S.K., and Nagini, S., Attenuation of N-methylN’-nitro-N-nitrosoguanidine induced genotoxicity and oxidative stress by tomato and garlic combination, Life Sci, 76(19): 2247–2255, 2005. 189. Schaffer, E.M., Liu, J.Z., and Milner, J.A., Garlic powder and allyl sulfur compounds enhance the ability of dietary selenite to inhibit 7,12-dimethylbenz[a]anthracene-induced mammary DNA adducts, Nutr Cancer, 27(2): 162–168, 1997. 190. Amagase, H., Schaffer, E.M., and Milner, J.A., Dietary components modify the ability of garlic to suppress 7,12-dimethylbenz(a)anthracene-induced mammary DNA adducts, J Nutr, 126(4): 817–824, 1996. 191. Ip, C., Lisk, D.J., and Thompson, H.J., Selenium-enriched garlic inhibits the early stage but not the late stage of mammary carcinogenesis, Carcinogenesis, 17(9): 1979–1982, 1996. 192. Ganther, H.E., Selenium metabolism, selenoproteins and mechanisms of cancer prevention: Complexities with thioredoxin reductase, Carcinogenesis, 20(9): 1657–1666, 1999. 193. Sundaram, S.G. and Milner, J.A., Diallyl disulfide inhibits the proliferation of human tumor cells in culture, Biochim Biophys Acta, 1315(1): 15–20, 1996. 194. Knowles, L.M. and Milner, J.A., Depressed p34cdc2 kinase activity and G2/M phase arrest induced by diallyl disulfide in HCT-15 cells, Nutr Cancer, 30(3): 169–174, 1998. 195. Schaffer, E.M. and Milner, J.A., Impact of dietary fatty acids on 7,12-dimethylbenz[a]anthracene-induced mammary DNA adducts, Cancer Lett, 106(2): 177–183, 1996. 196. Tang, F., Zhou, J., and Gu, L., [In vivo and in vitro effects of selenium-enriched garlic on growth of human gastric carcinoma cells], Zhonghua Zhong Liu Za Zhi, 23(6): 461–464, 2001. 197. Seki, T., Tsuji, K., Hayato, Y., Moritomo, T., and Ariga, T., Garlic and onion oils inhibit proliferation and induce differentiation of HL-60 cells, Cancer Lett, 160(1): 29–35, 2000. 198. Talib, W.H., Consumption of garlic and lemon aqueous extracts combination reduces tumor burden by angiogenesis inhibition, apoptosis induction, and immune system modulation, Nutrition, 43–44: 89–97, 2017. 199. Reinhart, K.M., Talati, R., White, C.M., and Coleman, C.I., The impact of garlic on lipid parameters: A systematic review and meta-analysis, Nutr Res Rev, 22(1): 39–48, 2009. 200. Varshney, R. and Budoff, M.J., Garlic and heart disease, J Nutr, 146(2): 416S–421S, 2016. 201. Gardner, C.D., Lawson, L.D., Block, E., Chatterjee, L.M., Kiazand, A., Balise, R.R., and Kraemer, H.C., Effect of raw garlic vs commercial garlic supplements on plasma lipid concentrations in adults with moderate hypercholesterolemia: A randomized clinical trial, Arch Intern Med, 167: 346–353, 2007. 202. Sobenin, I.A., Pryanishnikov, V.V., Kunnova, L.M., Rabinovich, Y.A., Martirosyan, D.M., and Orekhov, A.N., The effects of time-released garlic powder tablets on multifunctional cardiovascular risk in patients with coronary artery disease, Lipids Health Dis, 9, 119, 2010. 203. Ried, K., Toben, C., and Fakler, P., Effect of garlic on serum lipids: An updated meta-analysis, Nutr Rev, 71(5): 282–299, 2013. 204. Sahebkar, A., Serban, C., Ursoniu, S., and Banach, M., Effect of garlic on plasma lipoprotein(a) concentrations: A systematic review and meta-analysis of randomized controlled clinical trials, Nutrition, 32(1): 33–40, 2016. 205. Jabbari, A., Argani, H., Ghorbanihaghjo, A., and Mahdavi, R., Comparison between swallowing and chewing of garlic on levels of serum lipids, cyclosporine, creatinine and lipid peroxidation in renal transplant recipients, Lipids Health Dis, 4, 11, 2005. 206. Song, K. and Milner, J.A., The influence of heating on the anticancer properties of garlic, J Nutr, 131(3): 1054S–1057, 2001. 207. Maiolino, G., Rossitto, G., Caielli, P., Bisogni, V., Rossi, G.P., and Calò, L.A., The role of oxidized lowdensity lipoproteins in atherosclerosis: The myths and the facts, Mediat Inflamm, 2013, 714653, 2013. 208. Lau, B.H.S., Suppression of LDL oxidation by garlic, J Nutr, 131(3 Suppl.): 985S–988S, 2001. 209. Munday, J.S., James, K.A., Fray, L.M., Kirkwood, S.W., and Thompson, K.G., Daily supplementation with aged garlic extract, but not raw garlic, protects low density lipoprotein against in vitro oxidation, Atherosclerosis, 143(2): 399–404, 1999. 210. Lau, B.H.S., Suppression of LDL oxidation by garlic compounds is a possible mechanism of cardiovascular health benefit, J Nutr, 136(3): 765S–768S, 2006. 211. Byrne, D.J., Neil, H.A., Vallance, D.T., and Winder, A.F., A pilot study of garlic consumption shows no significant effect on markers of oxidation or sub-fraction composition of low-density lipoprotein including lipoprotein(a) after allowance for non-compliance and the placebo effect, Clin Chim Acta, 285(1–2): 21–33, 1999.

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212. Ou, C.C., Tsao, S.M., Lin, M.C., and Yin, M.C., Protective action on human LDL against oxidation and glycation by four organosulfur compounds derived from garlic, Lipids, 38(3): 219–224, 2003. 213. Huang, C.N., Horng, J.S., and Yin, M.C., Antioxidative and antiglycative effects of six organosulfur compounds in low-density lipoprotein and plasma, J Agric Food Chem, 52(11): 3674–3678, 2004. 214. Law, M.R., Morris, J.K., and Wald, N.J., Use of blood pressure lowering drugs in the prevention of cardiovascular disease: Meta-analysis of 147 randomised trials in the context of expectations from prospective epidemiological studies, BMJ, 338(7705): 1245, 2009. 215. Ried, K., Garlic lowers blood pressure in hypertensive individuals, regulates serum cholesterol, and stimulates immunity: An updated meta-analysis and review, J Nutr, 146(2): 389S–396S, 2016. 216. Li, L., Sun, T., Tian, J., Yang, K., Yi, K., and Zhang, P., Garlic in clinical practice: An evidence-based overview, Crit Rev Food Sci Nutr, 53(7): 670–681, 2013. 217. Fallon, M.B., Abrams, G.A., Abdel-Razek, T.T., Dai, J., Chen, S.J., Chen, Y.F., Luo, B., Oparil, S., and Ku, D.D., Garlic prevents hypoxic pulmonary hypertension in rats, Am J Physiol, 275(2 Pt 1): L283–287, 1998. 218. Maslin, D.J., Brown, C.A., Das, I., and Zhang, X.H., Nitric oxide—a mediator of the effects of garlic? Biochem Soc Trans, 25(3): 408S, 1997. 219. Lei, Y.P., Liu, C.T., Sheen, L.Y., Chen, H.W., and Lii, C.K., Diallyl disulfide and diallyl trisulfide protect endothelial nitric oxide synthase against damage by oxidized lowdensity lipoprotein, Mol Nutr Food Res, 54(Suppl. 1): S42–S52, 2010. 220. Aqel, M.B., Gharaibah, M.N., and Salhab, A.S., Direct relaxant effects of garlic juice on smooth and cardiac muscles, J Ethnopharmacol, 33(1–2): 13–19, 1991. 221. Ashraf, M.Z., Hussain, M.E., and Fahim, M., Endothelium mediated vasorelaxant response of garlic in isolated rat aorta: Role of nitric oxide, J Ethnopharmacol, 90(1): 5–9, 2004. 222. Benavides, G.A., Squadrito, G.L., Mills, R.W., Patel, H.D., Isbell, T.S., Patel, R.P., Darley-Usmar, V.M., Doeller, J.E., and Kraus, D.W., Hydrogen sulfide mediates the vasoactivity of garlic, P Natl Acad Sci USA, 104(46): 17977–17982, 2007. 223. Jiang, B., Tang, G., Cao, K., Wu, L., and Wang, R., Molecular mechanism for H2S-induced activation of K ATP channels, Antioxid Redox Signal, 12(10): 1167–1178, 2010. 224. Patel, V.B. and Topol, E.J., The pathogenesis and spectrum of acute coronary syndromes: From plaque formation to thrombosis, Cleve Clin J Med, 66(9): 561–571, 1999. 225. Budoff, M.J., Takasu, J., Flores, F.R., Niihara, Y., Lu, B., Lau, B.H., Rosen, R.T., and Amagase, H., Inhibiting progression of coronary calcification using aged garlic extract in patients receiving statin therapy: A preliminary study, Prev Med, 39(5): 985–991, 2004. 226. Budoff, M.J., Ahmadi, N., Gul, K.M., Liu, S.T., Flores, F.R., Tiano, J., Takasu, J., Miller, E., and Tsimikas, S., Aged garlic extract supplemented with B vitamins, folic acid and l-arginine retards the progression of subclinical atherosclerosis: A randomized clinical trial, Prev Med, 49(2–3): 101–107, 2009. 227. Matsumoto, S. et al., Aged garlic extract reduces low attenuation plaque in coronary arteries of patients with metabolic syndrome in a prospective randomized double-blind study, J Nutr, 146(2): 427S–432S, 2016. 228. Versteylen, M.O. et al., Additive value of semiautomated quantification of coronary artery disease using cardiac computed tomographic angiography to predict future acute coronary syndrome, J Am Coll Cardiol, 61(22): 2296–2305, 2013. 229. Koscielny, J., Klussendorf, D., Latza, R., Schmitt, R., Radtke, H., Siegel, G., and Kiesewetter, H., The antiatherosclerotic effect of Allium sativum, Atherosclerosis, 144(1): 237–249, 1999. 230. Mahdavi-Roshan, M., Zahedmehr, A., Mohammad-Zadeh, A., Sanati, H.R., Shakerian, F., Firouzi, A., Kiani, R., and Nasrollahzadeh, J., Effect of garlic powder tablet on carotid intima-media thickness in patients with coronary artery disease: A preliminary randomized controlled trial, Nutr Health, 22(2): 143–155, 2013. 231. Sobenin, I.A., Korneev, N.V., Romanov, I.V., Shutikhina, I.V., Kuntsevich, G.I., Romanenko, E.B., Myasoedova, V.A., Revin, V.V., and Orekhov, A.N., The effects of garlic powder tablets in subclinical carotid atherosclerosis, Exp Clin Cardiol, 20(1): 629–638, 2014. 232. Zahid Ashraf, M., Hussain, M.E., and Fahim, M., Antiatherosclerotic effects of dietary supplementations of garlic and turmeric: Restoration of endothelial function in rats, Life Sci, 77(8): 837–857, 2005. 233. Ali, M., Bordia, T., and Mustafa, T., Effect of raw versus boiled aqueous extract of garlic and onion on platelet aggregation, Prostaglandins Leukot Essent Fatty Acids, 60(1): 43–47, 1999. 234. Rahman, K., Lowe, G.M., and Smith, S., Aged garlic extract inhibits human platelet aggregation by altering intracellular signaling and platelet shape change, J Nutr, 146(2): 410S–415S, 2016.

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235. Steiner, M. and Lin, R.S., Changes in platelet function and susceptibility of lipoproteins to oxidation associated with administration of aged garlic extract, J Cardiovasc Pharmacol, 31(6): 904–908, 1998. 236. Rahman, K. and Billington, D., Dietary supplementation with aged garlic extract inhibits ADP-induced platelet aggregation in humans, J Nutr, 130(11): 2662–2665, 2000. 237. Kumar, A., Nirmala, K., Prasad, M.P.R., Panpatil, V.V., Prasanna Krishna, T., Sesikeran, B., and Polasa, K., Reduction in platelet aggregation (in vitro) by diallyl sulphide in female participants with type 2 diabetes mellitus, J Pharmacol Toxicol, 6(4): 381–390, 2011. 238. Ross, S.A. and Milner, J.A., Garlic, the mystical food in health promotion, in Handbook of Nutraceuticals and Functional Foods, Second Edition, R.E.C. Wildman, Editor. 2006, CRC Press: Boca Raton, FL. p. 73–99. 239. Marks, H.S., Anderson, J.L., and Stoewsand, G.S., Inhibition of benzo[a]pyrene-induced bone marrow micronuclei formation by diallyl thioethers in mice, J Toxicol Environ Health, 37(1): 1–9, 1992. 240. Balasenthil, S., Arivazhagan, S., Ramachandran, C.R., and Nagini, S., Effects of garlic on 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis, Cancer Detect Prev, 23(6): 534–538, 1999. 241. Hatono, S., Jimenez, A., and Wargovich, M.J., Chemopreventive effect of S-allylcysteine and its relationship to the detoxification enzyme glutathione S-transferase, Carcinogenesis, 17(5): 1041–1044, 1996. 242. Sengupta, A., Ghosh, S., and Das, S., Tomato and garlic can modulate azoxymethane-induced colon carcinogenesis in rats, Eur J Cancer Prev, 12(3): 195–200, 2003. 243. Sparnins, V.L., Barany, G., and Wattenberg, L.W., Effects of organosulfur compounds from garlic and onions on benzo[a]pyrene-induced neoplasia and glutathione S-transferase activity in the mouse, Carcinogenesis, 9(1): 131–134, 1988. 244. Wattenberg, L.W., Sparnins, V.L., and Barany, G., Inhibition of N-nitrosodiethylamine carcinogenesis in mice by naturally occurring organosulfur compounds and monoterpenes, Cancer Res, 49(10): 2689– 2692, 1989. 245. Hu, P.J. and Wargovich, M.J., Effect of diallyl sulfide on MNNG-induced nuclear aberrations and ornithine decarboxylase activity in the glandular stomach mucosa of the Wistar rat, Cancer Lett, 47(1–2): 153–158, 1989. 246. el-Mofty, M.M., Sakr, S.A., Essawy, A., and Abdel Gawad, H.S., Preventive action of garlic on aflatoxin B1-induced carcinogenesis in the toad Bufo regularis, Nutr Cancer, 21(1): 95–100, 1994. 247. Guyonnet, D., Belloir, C., Suschetet, M., Siess, M.H., and Le Bon, A.M., Mechanisms of protection against aflatoxin B(1) genotoxicity in rats treated by organosulfur compounds from garlic, Carcinogenesis, 23(8): 1335–1341, 2002. 248. Samaranayake, M.D., Wickramasinghe, S.M., Angunawela, P., Jayasekera, S., Iwai, S., and Fukushima, S., Inhibition of chemically induced liver carcinogenesis in Wistar rats by garlic (Allium sativum), Phytother Res, 14(7): 564–567, 2000. 249. Suzui, N., Sugie, S., Rahman, K.M., Ohnishi, M., Yoshimi, N., Wakabayashi, K., and Mori, H., Inhibitory effects of diallyl disulfide or aspirin on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced mammary carcinogenesis in rats, Jpn J Cancer Res, 88(8): 705–711, 1997. 250. Hong, J.Y., Smith, T., Lee, M.J., Li, W.S., Ma, B.L., Ning, S.M., Brady, J.F., Thomas, P.E., and Yang, C.S., Metabolism of carcinogenic nitrosamines by rat nasal mucosa and the effect of diallyl sulfide, Cancer Res, 51(5): 1509–1514, 1991. 251. Dwivedi, C., Rohlfs, S., Jarvis, D., and Engineer, F.N., Chemoprevention of chemically induced skin tumor development by diallyl sulfide and diallyl disulfide, Pharm Res, 9(12): 1668–1670, 1992. 252. Shrotriya, S., Kundu, J.K., Na, H.K., and Surh, Y.J., Diallyl trisulfide inhibits phorbol ester-induced tumor promotion, activation of AP-1, and expression of COX-2 in mouse skin by blocking JNK and Akt signaling, Cancer Res, 70(5): 1932–1940, 2010.

6

The Role of Tocopherols in Health Richard S. Bruno

CONTENTS 6.1 Introduction........................................................................................................................... 105 6.2 History................................................................................................................................... 106 6.3 Functions................................................................................................................................ 107 6.3.1 Antioxidant................................................................................................................ 107 6.3.2 Non-Antioxidant........................................................................................................ 109 6.3.2.1 α-Tocopherol............................................................................................... 109 6.3.2.2 γ-Tocopherol............................................................................................... 110 6.4 Dietary Sources..................................................................................................................... 111 6.4.1 Food........................................................................................................................... 111 6.4.2 Dietary Supplements.................................................................................................. 111 6.5 Human Requirements and Dietary Intake............................................................................. 112 6.6 Bioavailability........................................................................................................................ 113 6.6.1 Digestion and Absorption.......................................................................................... 113 6.6.2 Hepatic Secretion....................................................................................................... 114 6.6.3 Hepatic Metabolism................................................................................................... 115 6.7 Deficiency.............................................................................................................................. 115 6.8 Toxicity.................................................................................................................................. 115 6.9 α- and γ-Tocopherol Interactions with Vitamin C................................................................ 116 6.10 Role in Chronic Disease Prevention...................................................................................... 116 6.10.1 Cardiovascular Disease............................................................................................. 116 6.10.2 Alzheimer’s Disease.................................................................................................. 117 6.10.3 Cancer........................................................................................................................ 118 6.10.3.1 Lung and Prostate Cancers......................................................................... 118 6.10.3.2 Colon Cancer.............................................................................................. 119 6.11 Conclusions............................................................................................................................ 119 Acknowledgments........................................................................................................................... 119 References....................................................................................................................................... 119

6.1 INTRODUCTION Vitamin E is the term that describes eight lipophilic, naturally occurring compounds including four tocopherols and four tocotrienols (Figure 6.1).1 Tocopherols have a saturated phytyl tail, whereas tocotrienols have an unsaturated tail. Within each class, four forms exist as α-, β-, γ-, and δ- that differ based on the number and position of methyl groups present on the chromanol head. When the chromanol head is fully methylated and the phytyl tail is saturated, this vitamer is identified as α-tocopherol. The most abundant forms of vitamin E found biologically and in the diet are α- and γ-tocopherol.2 Structurally, they are similar and differ only in that γ-tocopherol has an unsubstituted position on the chromanol head (Figure 6.1). Thus, due to the dietary and biological abundance of these vitamin E forms, a considerable body of knowledge has accumulated since their discovery. This 105

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FIGURE 6.1  Vitamin E structures: tocopherols and tocotrienols. Vitamin E consists of four tocopherols and four tocotrienols. α-tocopherol, either naturally occurring or from synthetic preparations, is widely consumed in supplement form. Synthetic preparations of α-tocopherol contain eight stereoisomers (2R forms: RRR, RSR, RRS, RSS; 2S forms: SRR, SSR, SRS, SSS) due to the three chiral centers (denoted by asterisks) present on the phytyl tail, whereas naturally occurring α-tocopherol exists solely in the RRR configuration.

chapter will therefore focus on these vitamin E forms, although greater emphasis will be placed on α-tocopherol because this is the only form of vitamin E that is essential for humans.

6.2 HISTORY Vitamin E was discovered in 1922 as a compound necessary to sustain reproductive ability in rodents.3 Evans and Bishop determined that rodents fed diets containing rancid fat (i.e., vitamin E deficient) produced offspring that were mostly sterile in the first generation and completely sterile in the second generation, and fetal resorption occurred despite the presence of normal ovarian structure and function. Around this time period, the same conclusion was formed by Barnett Sure, who was performing similar dietary experiments, but he coined the term “vitamin E” because vitamins A, B, C, and D were already identified.4 Further work led to the isolation of α-tocopherol from wheat germ, which exhibited biologic activity of vitamin E.5 In the subsequent year, β- and γ-tocopherols were isolated from vegetable oils, but these vitamin E homologs were demonstrated to have lower biological activity than α-tocopherol.6 While these non-α-tocopherol forms of vitamin E reportedly have vitamin E biological activity, compound purity and the sensitivity of analytical methods utilized have been questioned. For example, commercially available γ-tocopherol is typically ∼97% pure, with much of the “contamination” attributed to α-tocopherol. Thus, some early research regarding vitamin E biological activity may need to be reconsidered. Historically, the fetal resorption assay has been used to define vitamin E biological activity despite this assay being laborious.7 However, the assay provides useful information since it quantifies the amount of vitamin E necessary to maintain the maximal number of live fetuses. While vitamin E deficiency can be induced in laboratory animals, it is quite difficult to do so in humans. Horwitt and others8–10 studied the effects of chronically low vitamin E intakes among hospitalized patients. After nearly 2 years of the 6-year-long investigation, circulating vitamin E decreased into the

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deficient range, but overt signs of clinical deficiency did not develop despite increased sensitivity of erythrocytes to hydrogen peroxide–induced hemolysis. Despite the efforts of chronic dietary vitamin E restriction in humans, symptoms of vitamin E deficiency (i.e., peripheral neuropathy, spinocerebellar ataxia, skeletal myopathy, pigmented retinopathy) have not been observed in the laboratory. Further, humans usually become vitamin E deficient only secondary to other pathologies, including fat maldigestion disorders,11 dysfunctional lipid metabolism,12 and severe protein-energy malnutrition.13 However, the discovery of the α-tocopherol transfer protein and its rarely occurring mutation have led to the identification of vitamin E deficiency independent of other pathologies in humans,14 which has been confirmed in α-tocopherol transfer protein knock-out mice.15

6.3 FUNCTIONS 6.3.1 Antioxidant The most well-known function of vitamin E is its chain-breaking antioxidant activity that terminates the cyclic propagation of lipid peroxidation (Figure 6.2).16 Indeed, vitamin E is a peroxyl radical scavenger that protects polyunsaturated fatty acids from lipid peroxidation.17 Vitamin E “outcompetes” the propagation reactions such that a single vitamin E molecule should be able to protect ∼1000 lipid molecules from the chain reaction propagation step.18 This phenomenon is attributed to the higher rate constant between vitamin E and peroxyl radicals compared with the reaction rate between PUFAs and peroxyl radicals. When vitamin E scavenges peroxyl radicals, it loses an electron while terminating the propagation reaction, and becomes oxidized to form a tocopheroxyl radical (Figure 6.2). Tocopheroxyl radicals can then: (1) undergo additional oxidation to generate a tocopherol quinone,19 (2) react with another radical to yield a non-reactive product, (3) be recycled to a native tocopherol by other antioxidants (e.g., vitamin C),2 or (4) theoretically reinitiate lipid peroxidation through a process referred to as tocopherol-mediated peroxidation.20 Of all the tocopherols, α-tocopherol has the strongest antioxidant activity based on a technique that assesses the inhibited autooxidation of styrene (i.e., peroxyl radical generation).21 Antioxidant activities of tocopherols are as follows: α > γ > β > δ, with respective rate coefficients of 320, 140,

FIGURE 6.2  Chain breaking antioxidant activity of α- and γ-tocopherol. Oxidative stress results in the formation of carbon-centered radicals that react with oxygen to generate peroxyl radicals. (A) In the absence of α- or γ-tocopherol, lipid peroxidation is propagated in a cyclic manner following the regeneration of a carboncentered radical. (B) In the presence of α- or γ-tocopherol, peroxyl radicals are scavenged to terminate the cyclic progression of lipid peroxidation. Abbreviations: R•, carbon-centered radical; ROO •, peroxyl radical; R-OO-H, lipid hydroperoxide; RH, polyunsaturated fatty acid; α- or γ-T, α- or γ-tocopherol; α- or γ-T•, α- or γ-tocopheroxyl radical. (Adapted from Burton GW, Traber MG. Annu Rev Nutr. 1990;10:357–82.165)

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130, and 44 × 104 (M−1 s−1). α-Tocopherol likely exhibits superior antioxidant activity because it contains three methyl groups (Figure 6.1) on the chromanol head that function to stabilize phenoxyl radicals. Other tocopherols are lacking one or more methyl groups and hence have lower antioxidant activity. In agreement, β- and γ-tocopherol each have two methyl groups but at differing positions on the chromanol head, and each has approximately the same antioxidant activity. Despite in vitro antioxidant activity of each vitamin E form, whether each form exhibits in vivo antioxidant function is questionable because of considerable differences in bioavailability. Although there are eight forms of vitamin E, only α- and γ-tocopherols are generally detected in tissues and plasma of non-users of dietary supplements (discussed further under the “Bioavailability” section). In fact, plasma α-tocopherol concentrations (∼20–40 µmol/L) are significantly higher than those of γ-tocopherol (∼1–5 µmol/L), whereas the other six vitamin E forms are generally low (90% of American men and women do not consume diets that meet the Estimated Average Requirement (EAR; 12 mg/d) for α-tocopherol.55 Data from the National Health and Nutrition Examination Survey also indicate that median α-tocopherol intakes from food alone for men and women (19–30 years) were only 9.4 and 6.4 mg, respectively.1 However, these values might be underestimated due to several measurement errors. These include underreporting of total energy64 and fat intake,65 the amounts of fats and oils used in food preparation, the uncertainty of the specific oils consumed, and inaccuracies in the food composition databases.1 Nonetheless, Americans likely do not meet dietary α-tocopherol recommendations, and the available data concerning intakes are likely inaccurate. Because diet assessment is limited for evaluating α-tocopherol adequacy, circulating α-tocopherol is an important biomarker of status that must be considered. An observation made in humans undergoing chronic depletion of α-tocopherol was that overt clinical deficiency did not occur

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but that the susceptibility of hydrogen peroxide–induced hemolysis was greater when circulating α-tocopherol decreased to 30 µmol/L is associated with a lower risk of mortality. While this makes for a compelling case to achieve higher circulating α-tocopherol, caution is needed in interpreting α-tocopherol as a biomarker of adequacy. This is because hyperlipidemia likely “traps” α-tocopherol in the circulation,29,67 resulting in physiological deficiency at target tissues. For this reason, it has been indicated that novel biomarkers of α-tocopherol adequacy are needed.1

6.6 BIOAVAILABILITY 6.6.1 Digestion and Absorption Since vitamin E is lipophilic, its absorption from the intestinal lumen is dependent on the same processes that enable fat digestion and uptake into the enterocytes (Figure 6.5). In addition to pancreatic esterases that are required to cleave fatty acids from triglycerides, bile acid secretion is equally important to promote the formation of mixed-micelles that enable vitamin E absorption.68 In fact, the absence of either results in poor vitamin E absorption, which is why vitamin E deficiency occurs in patients secondary to biliary obstruction, cholestatic liver disease, pancreatitis, or cystic fibrosis.69 Following enterocyte uptake, vitamin E absorption into the lymphatic system is dependent on chylomicron synthesis and secretion (Figure 6.5). In the enterocytes, chylomicrons containing

FIGURE 6.5  Vitamin E bioavailability. Dietary vitamin E, including all tocopherols and tocotrienols, are similarly incorporated into micelles, absorbed at the small intestine, and packaged into chylomicrons prior to lymphatic transport. Chylomicrons containing newly absorbed vitamin E can be catabolized by lipoprotein lipase to facilitate the transfer of vitamin E to peripheral tissues or lipoproteins. The majority of vitamin E, as part of a chylomicron remnant, is taken up at the liver in a receptor-mediated process. The liver, through the actions of α-tocopherol transfer protein (α-TTP), then preferentially packages and secretes VLDL containing mostly α-tocopherol (α-T) into the circulation. Other vitamin E forms (E) are secreted from the liver to a much lesser extent and are otherwise directed for elimination as vitamin E catabolites (i.e., CEHCs; carboxyethyl-hydroxychromanols).

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triglycerides, cholesterol, phospholipids, and apolipoprotein are synthesized.70 During that process, vitamin E is incorporated into chylomicrons and secreted into the lymph (Figure 6.5). In healthy individuals, the absorption of vitamin E was estimated to range between 15% and 45% when using radioactive α-tocopherol.71 However, in thoracic-duct-cannulated rats, the absorption of vitamin E became less efficient with increasing amounts of α-tocopherol ingested.72 The bioavailability of α-tocopherol was examined in healthy humans who consumed test meals of varying lipid amounts (0–11 g fat) along with deuterium-labeled RRR-α-tocopherol (22 mg) that was impregnated into apple pieces.73 Data indicated that dietary fat dose-dependently increased α-tocopherol bioavailability, with up to a threefold increase when 11 g of fat was co-ingested with the deuterium-labeled α-tocopherol. Further, at least 33% of the dose was absorbed during the 11-g fat trial, and because there was a linear relationship between dietary fat level and absorption, it was estimated that 0.33 mg of α-tocopherol was absorbed for each gram of fat consumed. However, despite demonstrating the importance of total fat on α-tocopherol bioavailability, the optimal quantity or type of fat was not determined in these studies.

6.6.2 Hepatic Secretion Differences in plasma concentrations between various vitamin E forms were initially attributed to differences in intestinal absorption.68 However, the application of deuterated tocopherols provides an understanding that differences in circulating vitamin E forms are not attributable to their enterocyte uptake or their secretion as part of chylomicrons.59,74 The liver is actually responsible for the preferential secretion of α-tocopherol into the plasma,75 with VLDL containing newly absorbed vitamin E (primarily as α-tocopherol).76 Hepatic discrimination of the various vitamin E forms is attributed to the function of the α-tocopherol transfer protein.1 This protein was first identified by Catignani and Bieri,77 purified and characterized from liver,78,79 and later crystallized.80 The α-tocopherol transfer protein is expressed mainly in the liver81 and functions to preferentially transfer/secrete α-tocopherol from the liver (Figure 6.5) to the plasma82 by a mechanism that is incompletely understood. In comparison to α-tocopherol, other vitamin E forms bind with significantly less affinity to this protein (Table 6.2),83 thus explaining the hepatic discrimination of various vitamin E forms. In the absence of functional α-tocopherol transfer protein, such as in α-tocopherol transfer protein knock-out mice84 or humans with a genetic defect,85 the result is vitamin E deficiency. TABLE 6.2 Binding Affinity of the α-Tocopherol Transfer Protein with Vitamin E Vitamin E Form RRR-α-Tocopherol

α-Tocopherol Transfer Protein Binding Affinity (% of RRR-α-Tocopherol) 100

β-Tocopherol

38

γ-Tocopherol

9

δ-Tocopherol

2

α-Tocopherol acetate

2

α-Tocopherol quinone

2

SRR-α-Tocopherol

11

α-Tocotrienol

12

Source: Data adapted from Hosomi et al. FEBS Lett. 1997;409:105–8. Note: The α-tocopherol transfer protein has the highest binding affinity for naturally occurring stereoisomer of α-tocopherol (RRR-α-tocopherol).

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6.6.3 Hepatic Metabolism Hepatic excretion of α- and γ-tocopherol occurs via two predominant pathways. They can either be excreted intact or in their oxidized forms into bile. Alternatively, tocopherols can be catabolized in a cytochrome P450-dependent manner to yield a final excretory product, CEHC (α- or γ-carboxyethylhydroxychroman; Figure 6.3) that is eliminated in either the urine or bile. Tocopherol metabolism occurs predominately in the liver. The specific P450 enzyme responsible for initiating the metabolism of tocopherols has been widely investigated,86–90 with evidence pointing to cytochrome P450 4F2.91,92 Tocopherol metabolism is initiated by ω-hydroxylation of the phytyl tail, followed by several cycles of β-oxidation that each remove two carbon units from the phytyl tail until the final product, CEHC, is produced. CEHC is then often glucuronidated or sulfated prior to biliary or urinary excretion. α- and γ-tocopherols are catabolized at different rates. This was exemplified in healthy humans who ingested equal amounts of deuterium-labeled α- and γ-tocopherol.93 Data show that plasma γ-tocopherol disappeared three times faster than α-tocopherol. Further, plasma deuterium-labeled α-CEHC was undetectable, whereas plasma deuterium-labeled γ-CEHC was readily detectable. The rates of disappearance of γ-CEHC and γ-tocopherol were also similar, suggesting that γ-tocopherol disappearance is largely attributed to its P450-mediated metabolism. Although the diet contains mostly γ-tocopherol, human plasma contains significantly more α-tocopherol compared with γ-tocopherol. This is explained by the higher binding affinity of the α-tocopherol transfer protein to α-tocopherol compared with γ-tocopherol (Table 6.2). Since γ-tocopherol is not well recognized by the α-tocopherol transfer protein, it is more actively metabolized to γ-CEHC than α-tocopherol is catabolized to α-CEHC.93 In fact, plasma γ-CEHC concentrations are generally in the range of 50–150 nmol/L, whereas α-CEHC concentrations are either low (635,000 deaths annually.119 Oxidative stress is well implicated in atherosclerosis.120 A key atherogenic initiator is the oxidation of LDL. This occurs in the subendothelial space of the vascular wall following the

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translocation of LDL from the circulation across the single-cell endothelial layer. This prompts macrophage-mediated uptake of oxidized LDL and the formation of lipid-laden foam cells, and drives pro-inflammatory responses that provoke endothelial dysfunction and injury that provoke atherogenesis. Epidemiological data suggest that circulating α-tocopherol is inversely related to cardiovascular risk.121,122 Individuals with coronary risk factors such as hypertriglyceridemia and low HDL also have low circulating α-tocopherol.123 The cardioprotective role of α-tocopherol is supported by studies showing that the susceptibility of LDL to oxidation is alleviated by a supplementation regimen that enriches α-tocopherol in LDL.124,125 α-tocopherol also inhibits smooth muscle cell proliferation, platelet adhesion and aggregation, and endothelial monocyte adhesion.126 Despite the promise of epidemiological evidence, randomized controlled trials examining α-tocopherol on cardiovascular risk have yielded inconsistent outcomes. Many clinical trials have reported neutral benefits of α-tocopherol, whereas others indicate that α-tocopherol supplementation lowers cardiovascular risk. These include the CHAOS trial,127 the SPACE trial,128 the Transplant Associated Arteriosclerosis Study,129 and the ASAP study.115 The CHAOS and SPACE trials evaluated the effects of α-tocopherol alone, whereas the other two investigated co-treatment of α-tocopherol and ascorbic acid. The CHAOS study showed a 47% reduction in coronary artery disease–associated death and nonfatal myocardial infarction by α-tocopherol supplementation. Although it occurred without statistical significance, the SPACE trial reported a 39% decrease in coronary artery disease–related mortality while observing a significant 70% decrease in myocardial infarction rate. In the Transplant Associated Arteriosclerosis Study, α-tocopherol and ascorbic acid co-treatment significantly reduced evidence of atherosclerosis. Similarly, hypercholesterolemic patients from the ASAP study had slower rates of intima media thickening with co-supplementation but not by either antioxidant alone. The benefits of α-tocopherol and ascorbic acid were most pronounced in men who smoked compared with nonsmokers. The Women’s Health Study, a placebo-controlled trial examining α-tocopherol supplementation for 10 years, indicated no cardioprotective benefit of α-tocopherol.130 However, among older women (>65 years of age) who represented only 10% of the cohort but accounted for 31% of all study endpoints, there was a significant reduction in major cardiovascular events in association with a 49% lower risk of cardiovascular-related death.

6.10.2 Alzheimer’s Disease Because lipid peroxidation–related oxidative stress is implicated in Alzheimer’s disease, it has been hypothesized that α-tocopherol would prevent or slow its etiologic progression. This is consistent with brain tissue containing large amounts of PUFAs that are susceptible to lipid peroxidation in agreement with evidence that patients with Alzheimer’s disease have increased concentrations of the lipid peroxidation biomarker malondialdehyde.131 Studies in vitro indicate that α-tocopherol attenuates hydrogen peroxide–mediated cytotoxicity132 and amyloid β-protein-induced cell death.133 Similar benefits also occur in association with limiting NFκB inflammation.134 Studies in rodents support that supplementation increases brain α-tocopherol concentrations135,136 and improves cognitive performance in aged rats.137 Supplementation of α-tocopherol in rodents also alleviates neurotoxin-induced oxidative stress that is otherwise associated with impaired water maze performance.138 Others also report that α-tocopherol in rodents protects against brain lipofuscin accumulation139 and hippocampal ischemic neural damage.140 A prospective observational study in older adults suggested that vitamin E supplement users have a lower risk of Alzheimer’s disease.141 In a separate prospective study, vitamin E from food, but not supplements, and no other antioxidants were associated with a lower risk of Alzheimer’s disease.142 Data from participants of the Chico Health and Aging Project also suggested that higher intakes of α-tocopherol equivalents (i.e., vitamin E derived from tocopherols and tocotrienols) were associated with a reduced incidence of Alzheimer’s disease.143 α- and γ-Tocopherol were also reported to have independent associations with lower Alzheimer’s disease risk, and intakes of either tocopherol were associated with slower rates of cognitive decline.

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Based on these findings, it is not surprising that several randomized controlled trials have investigated vitamin E in the treatment or prevention of Alzheimer’s disease. The PREADViSE trial was concluded as a cohort study, but was initiated as a double-blind randomized clinical trial investigating α-tocopherol, selenium, or their combined use.144 Neither α-tocopherol alone nor selenium prevented dementia among 3786 persons >60 years of age who continued into the cohort study from an initial population of 7540 men who were enrolled into the intervention. In a separate double-blind, placebo-controlled trial in patients with mild to moderate Alzheimer’s disease, α-tocopherol (2000 IU/d) compared with placebo resulted in slower cognitive decline.145 This differed from the outcome of another report indicating that α-tocopherol supplementation (2000 IU/d; 3 years) failed to protect against the progression from mild cognitive impairment to Alzheimer’s disease in patients having an amnestic subtype of mild cognitive impairment at the time of enrollment.146 Consistent with these disparities, it has been suggested that the benefits of α-tocopherol in Alzheimer’s disease patients may be dependent upon whether an antioxidant effect can be observed.147 A separate line of evidence, which has been investigated limitedly, also suggests that γ-tocopherol may have a role to protect against Alzheimer’s disease. Specifically, γ-tocopherol scavenges peroxynitrite to form nitro-γ-tocopherol (Figure 6.3). In the brain of postmortem Alzheimer disease patients, levels of 5-nitro-γ-tocopherol were reported to be increased by 2–3-fold.52

6.10.3 Cancer Dietary antioxidants have received attention based on evidence that intakes of fruits and vegetables are inversely related to cancer incidence.148 Antioxidants such as vitamin E may have an anticancer benefit consistent with oxidative stress disrupting apoptotic processes and provoking DNA damage.149 Most studies examining vitamin E on cancer risk have focused on α-tocopherol, although γ-tocopherol has received some attention.150 Regardless of the vitamin E form, there is a lack of evidence to support an anti-cancer benefit of α-tocopherol in humans, although studies in vitro are generally supportive. 6.10.3.1  Lung and Prostate Cancers The ATBC trial tested whether α-tocopherol and β-carotene supplementation, alone and in combination, would protect against lung cancer development in a cohort of >29,000 male smokers.151 One of the largest nutrition interventions in history, this trial was terminated prematurely because β-carotene increased lung cancer incidence; α-tocopherol had no effect on lung cancer incidence. In a secondary analysis of the ATBC trial, α-tocopherol was suggested to lower prostate cancer incidence among those receiving supplements.152 This preventative effect of α-tocopherol was lost, however, when data were examined from the post-intervention follow-up period, suggesting that the benefits ceased when supplementation was terminated. In the CARET trial, which provided β-carotene and retinol for a potential benefit on lung cancer risk, it was observed that low serum α-tocopherol was associated with a higher risk of prostate cancer.153 These findings and others led to the planning of SELECT, which examined prostate cancer incidence in >35,000 men who were randomized in a double-blind, placebo-controlled manner to receive selenium and α-tocopherol (alone and in combination) for 7–12 years.154 After a median follow-up period of 5.5 years, neither selenium nor vitamin E, alone or in combination, affected prostate cancer risk. Because interim data analysis indicated that supplementation was unlikely to yield a favorable outcome, SELECT was terminated, and participants were instructed to cease using supplements. However, data collection continued during the post-intervention follow-up phase. Compared with placebo, the prior use of α-tocopherol supplements alone was associated with a 17% higher risk of developing prostate cancer.155 These outcomes were disappointing because evidence in vitro indicated favorable effects of α-tocopherol on prostate cancer risk.156–158 Separate evidence also supports that γ-tocopherol inhibits prostate tumor cell growth to a greater extent than α-tocopherol,159 and that γ-tocopherol induces

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apoptosis in androgen-responsive prostate cancer cells while dysregulating sphinolipid metabolism in association with increased death of prostate cancer cells.160 6.10.3.2  Colon Cancer The gastrointestinal tract is thought to be a major site on which antioxidants could exert protective benefits.161 Epidemiological studies examining vitamin E relative to colon cancer risk have been inconsistent, although patients with colon cancer often have lower circulating vitamin E concentrations.162,163 Secondary analysis of the ATBC trial suggested a lower incidence of colorectal cancer, but a greater frequency of cancers of the stomach.151 Findings of the Women’s Health Study similarly indicated that long-term α-tocopherol supplementation had no effect on the incidence of total cancer, including colon cancer as well as cancer-related mortality.130 These works in relation to other studies have suggested that perhaps γ-tocopherol has greater potential compared with α-tocopherol for colon cancer prevention,164 but this has not been rigorously examined through controlled trials.

6.11 CONCLUSIONS Considerable effort has been invested to establish vitamin E requirements, define its trafficking and metabolism along the gut-liver axis, and understand its potential role to alleviate chronic disease risk. Clear evidence supports its antioxidant function, especially in relation to terminating the cyclic progression of lipid peroxidation, and its interaction with the antioxidant vitamin C. Despite these mechanistic insights, the available evidence is less clear regarding its role to manage chronic disease risk. This suggests that dietary α-tocopherol supplements may have limited benefit for most individuals and that focus is needed to target those persons with compromised vitamin E status. Depending on the index of adequacy used, up to ∼80% of Americans may have suboptimal circulating α-tocopherol even though overt vitamin E deficiency is rarely observed. Because a substantial proportion of Americans fail to achieve recommended intakes of dietary α-tocopherol, and often obtain it from foods that are low in α-tocopherol, dietary modification is encouraged to obtain α-tocopherol from α-tocopherol-rich foods (Figure 6.4). Further study is also needed to establish the health benefits of γ-tocopherol. Despite γ-tocopherol having antioxidant activity in humans, it is not actively maintained in the circulation relative to α-tocopherol but rather preferentially catabolized to γ-CEHC. Future studies need to examine the independent and additive benefits of γ-tocopherol and γ-CEHC, and whether γ-tocopherol has a clear health-promoting role that can be demonstrated through carefully controlled clinical trials.

ACKNOWLEDGMENTS This work was supported by grants from the United States Department of Agriculture, the National Dairy Council, and the Ohio Agricultural Research and Development Center and Center for Applied Plant Sciences at The Ohio State University.

REFERENCES

1. Institute of Medicine (U.S.). Panel on Dietary Antioxidants and Related Compounds. Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids: A Report of the Panel on Dietary Antioxidants and Related Compounds, Subcommittees on Upper Reference Levels of Nutrients and of Interpretation and Use of Dietary Reference Intakes, and the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, Food and Nutrition Board, Institute of Medicine. Washington, D.C.: National Academy Press; 2000. 2. Bruno RS, Traber MG. Cigarette smoke alters human vitamin E requirements. J Nutr. 2005;135:671–4. 3. Evans HM, Bishop KS. On the existence of a hitherto unrecognized dietary factor essential for reproduction. Science. 1922;56:650–1.

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4. Sure B. Dietary requirements for reproduction. II. The existence of a specific vitamin for reproduction. J Biol Chem. 1924;58:693–709. 5. Evans HM, Emerson OH, Emerson GA. The isolation from wheat germ oil of an alcohol, α-tocopherol, having the properties of vitamin. J Biol Chem. 1936;113:319–32. 6. Emerson OH, Emerson GA, Mohammad A, Evans HM. The chemistry of vitamin E. Tocopherols from various sources. J Biol Chem. 1937;122:99–107. 7. Machlin LJ. Handbook of Vitamins. 2nd, rev. and expand ed. New York: M. Dekker; 1991. 8. Horwitt MK. Erythrocyte survival time and reticulocyte levels after tocopherol depletion in man. Am J Clin Nutr. 1963;12:99–106. 9. Horwitt MK, Harvey CC, Duncan GD, Wilson WC. Effects of limited tocopherol intake in man with relationships to erythrocyte hemolysis and lipid oxidations. Am J Clin Nutr. 1956;4:408–19. 10. Horwitt MK. Vitamin E and lipid metabolism in man. Am J Clin Nutr. 1960;8:451–61. 11. Sokol RJ, Heubi JE, Iannaccone ST, Bove KE, Balistreri WF. Vitamin E deficiency with normal serum vitamin E concentrations in children with chronic cholestasis. N Engl J Med. 1984;310:1209–12. 12. Rader DJ, Brewer HB, Jr. Abetalipoproteinemia. New insights into lipoprotein assembly and vitamin E metabolism from a rare genetic disease. JAMA. 1993;270:865–9. 13. Kalra V, Grover J, Ahuja GK, Rathi S, Khurana DS. Vitamin E deficiency and associated neurological deficits in children with protein-energy malnutrition. J Trop Pediatr. 1998;44:291–5. 14. Cavalier L, Ouahchi K, Kayden HJ, Di Donato S, Reutenauer L, Mandel JL, Koenig M. Ataxia with isolated vitamin E deficiency: Heterogeneity of mutations and phenotypic variability in a large number of families. Am J Hum Genet. 1998;62:301–10. 15. Terasawa Y, Ladha Z, Leonard SW, Morrow JD, Newland D, Sanan D, Packer L, Traber MG, Farese RV, Jr. Increased atherosclerosis in hyperlipidemic mice deficient in alpha -tocopherol transfer protein and vitamin E. Proc Natl Acad Sci U S A. 2000;97:13830–4. 16. Burton GW, Ingold KU. Vitamin E as an in vitro and in vivo antioxidant. Ann N Y Acad Sci. 1989;570:7–22. 17. Burton GW, Cheeseman KH, Doba T, Ingold KU, Slater TF. Vitamin E as an antioxidant in vitro and in vivo. Ciba Found Symp. 1983;101:4–18. 18. Buettner GR. The pecking order of free radicals and antioxidants: Lipid peroxidation, alpha-tocopherol, and ascorbate. Arch Biochem Biophys. 1993;300:535–43. 19. Terentis AC, Thomas SR, Burr JA, Liebler DC, Stocker R. Vitamin E oxidation in human atherosclerotic lesions. Circ Res. 2002;90:333–9. 20. Upston JM, Terentis AC, Stocker R. Tocopherol-mediated peroxidation of lipoproteins: Implications for vitamin E as a potential antiatherogenic supplement. Faseb J. 1999;13:977–94. 21. Burton GW, Doba T, Gabe EJ, Hughes L, Lee FL, Prasad L, Ingold KU. Autoxidation of biological molecules. 4. Maximizing the antioxidant activity of phenols. J Am Chem Soc. 1985;107:7053–65. 22. Handelman GJ, Packer L, Cross CE. Destruction of tocopherols, carotenoids, and retinol in human plasma by cigarette smoke. Am J Clin Nutr. 1996;63:559–65. 23. Leonard SW, Bruno RS, Paterson E, Schock BC, Atkinson J, Bray TM, Cross CE, Traber MG. 5-nitrogamma-tocopherol increases in human plasma exposed to cigarette smoke in vitro and in vivo. Free Radic Biol Med. 2003;35:1560–7. 24. Frei B, England L, Ames BN. Ascorbate is an outstanding antioxidant in human blood plasma. Proc Natl Acad Sci U S A. 1989;86:6377–81. 25. Botti H, Batthyany C, Trostchansky A, Radi R, Freeman BA, Rubbo H. Peroxynitrite-mediated alpha-tocopherol oxidation in low-density lipoprotein: A mechanistic approach. Free Radic Biol Med. 2004;36:152–62. 26. Sacheck JM, Blumberg JB. Role of vitamin E and oxidative stress in exercise. Nutrition. 2001;17:809–14. 27. Traber MG, Buettner GR, Bruno RS. The relationship between vitamin C status, the gut-liver axis, and metabolic syndrome. Redox Biol. 2018;21:101091. 28. Mastaloudis A, Leonard SW, Traber MG. Oxidative stress in athletes during extreme endurance exercise. Free Radic Biol Med. 2001;31:911–22. 29. Mah E, Sapper TN, Chitchumroonchokchai C, Failla ML, Schill KE, Clinton SK, Bobe G, Traber MG, Bruno RS. alpha-tocopherol bioavailability is lower in adults with metabolic syndrome regardless of dairy fat co-ingestion: A randomized, double-blind, crossover trial. Am J Clin Nutr. 2015;102:1070–80. 30. Bruno RS, Ramakrishnan R, Montine TJ, Bray TM, Traber MG. {alpha}-tocopherol disappearance is faster in cigarette smokers and is inversely related to their ascorbic acid status. Am J Clin Nutr. 2005;81:95–103. 31. Basu S, Helmersson J. Factors regulating isoprostane formation in vivo. Antioxid Redox Signal. 2005;7:221–35.

The Role of Tocopherols in Health

121

32. Bruno RS, Leonard SW, Atkinson J, Montine TJ, Ramakrishnan R, Bray TM, Traber MG. Faster plasma vitamin E disappearance in smokers is normalized by vitamin C supplementation. Free Radic Biol Med. 2006;40:689–97. 33. Roberts LJ, 2nd, Oates JA, Linton MF, Fazio S, Meador BP, Gross MD, Shyr Y, Morrow JD. The relationship between dose of vitamin E and suppression of oxidative stress in humans. Free Radic Biol Med. 2007;43:1388–93. 34. Boscoboinik D, Szewczyk A, Azzi A. Alpha-tocopherol (vitamin E) regulates vascular smooth muscle cell proliferation and protein kinase C activity. Arch Biochem Biophys. 1991;286:264–9. 35. Chatelain E, Boscoboinik DO, Bartoli GM, Kagan VE, Gey FK, Packer L, Azzi A. Inhibition of smooth muscle cell proliferation and protein kinase C activity by tocopherols and tocotrienols. Biochim Biophys Acta. 1993;1176:83–9. 36. Devaraj S, Li D, Jialal I. The effects of alpha tocopherol supplementation on monocyte function. Decreased lipid oxidation, interleukin 1 beta secretion, and monocyte adhesion to endothelium. J Clin Invest. 1996;98:756–63. 37. Freedman JE, Farhat JH, Loscalzo J, Keaney JFJ. Alpha-tocopherol inhibits aggregation of human platelets by a protein kinase C-dependent mechanism. Circulation. 1996;94:2434–40. 38. Cominacini L, Garbin U, Pasini AF, Davoli A, Campagnola M, Contessi GB, Pastorino AM, Lo Cascio V. Antioxidants inhibit the expression of intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 induced by oxidized LDL on human umbilical vein endothelial cells. Free Radic Biol Med. 1997;22:117–27. 39. Chan AC, Wagner M, Kennedy C, Mroske C, Proulx P, Laneuville O, Tran K, Choy PC. Vitamin E up-regulates phospholipase A2, arachidonic acid release and cyclooxygenase in endothelial cells. Akt Ernahr-Med. 1998;23:1–8. 40. Chan AC, Wagner M, Kennedy C, Chen E, Lanuville O, Mezl VA, Tran K, Choy PC. Vitamin E up-regulates arachidonic acid release and phospholipase A2 in megakaryocytes. Mol Cell Biochem. 1998;189:153–9. 41. Wu D, Liu L, Meydani M, Meydani SN. Vitamin E increases production of vasodilator prostanoids in human aortic endothelial cells through opposing effects on cyclooxygenase-2 and phospholipase A2. J Nutr. 2005;135:1847–53. 42. Devaraj S, Jialal I. Alpha tocopherol supplementation decreases serum C-reactive protein and monocyte interleukin-6 levels in normal volunteers and type 2 diabetic patients. Free Rad Biol Med. 2000;29:790–2. 43. Devaraj S, Jialal I. Alpha-tocopherol decreases tumor necrosis factor-alpha mRNA and protein from activated human monocytes by inhibition of 5-lipoxygenase. Free Radic Biol Med. 2005;38:1212–20. 44. Suzuki YJ, Packer L. Inhibition of NF-kappa B DNA binding activity by alpha-tocopheryl succinate. Biochem Mol Biol Int. 1993;31:693–700. 45. Calfee-Mason KG, Spear BT, Glauert HP. Vitamin E inhibits hepatic NF-kappaB activation in rats administered the hepatic tumor promoter, phenobarbital. J Nutr. 2002;132:3178–85. 46. Jiang Q, Elson-Schwab I, Courtemanche C, Ames BN. Gamma-tocopherol and its major metabolite, in contrast to alpha-tocopherol, inhibit cyclooxygenase activity in macrophages and epithelial cells. Proc Natl Acad Sci U S A. 2000;97:11494–9. 47. Jiang Q, Ames BN. Gamma-tocopherol, but not alpha-tocopherol, decreases proinflammatory eicosanoids and inflammation damage in rats. Faseb J. 2003;17:816–22. 48. Christen S, Jiang Q, Shigenaga MK, Ames BN. Analysis of plasma tocopherols alpha, gamma, and 5-nitro-gamma in rats with inflammation by HPLC coulometric detection. J Lipid Res. 2002;43:1978–85. 49. Christen S, Woodall AA, Shigenaga MK, Southwell-Keely PT, Duncan MW, Ames BN. Gammatocopherol traps mutagenic electrophiles such as NO(X) and complements alpha-tocopherol: Physiological implications. Proc Natl Acad Sci U S A. 1997;94:3217–22. 50. Hoglen NC, Waller SC, Sipes IG, Liebler DC. Reactions of peroxynitrite with gamma-tocopherol. Chem Res Toxicol. 1997;10:401–7. 51. Morton LW, Ward NC, Croft KD, Puddey IB. Evidence for the nitration of gamma-tocopherol in vivo: 5-nitro-gamma-tocopherol is elevated in the plasma of subjects with coronary heart disease. Biochem J. 2002;364:625–8. 52. Williamson KS, Gabbita SP, Mou S, West M, Pye QN, Markesbery WR, Cooney RV, Grammas P, Reimann-Philipp U et al. The nitration product 5-nitro-gamma-tocopherol is increased in the Alzheimer brain. Nitric Oxide. 2002;6:221–7. 53. Church DF, Pryor WA. Free-radical chemistry of cigarette smoke and its toxicological implications. Environ Health Perspect. 1985;64:111–26.

122

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54. Dial S, Eitenmiller RR. Tocopherols and tocotrienols in key foods in the U.S. diet. In: Ong ASH, Niki E, Packer L, editors. Nutrition, Lipids, Health, and Disease. Champaign, IL: AOCS Press; 1995. pp. 327–42. 55. Maras JE, Bermudez OI, Qiao N, Bakun PJ, Boody-Alter EL, Tucker KL. Intake of alpha-tocopherol is limited among US adults. J Am Diet Assoc. 2004;104:567–75. 56. McLaughlin PJ, Weihrauch JL. Vitamin E content of foods. J Am Diet Assoc. 1979;75:647–65. 57. Bieri JG, Evarts RP. Gamma tocopherol: Metabolism, biological activity and significance in human vitamin E nutrition. Am J Clin Nutr. 1974;27:980–6. 58. Muller PY, Netscher T, Frank J, Stoecklin E, Rimbach G, Barella L. Comparative quantification of pharmacodynamic parameters of chiral compounds (RRR- vs. all-rac-alpha tocopherol) by global gene expression profiling. J Plant Physiol. 2005;162:811–7. 59. Traber MG, Burton GW, Ingold KU, Kayden HJ. RRR- and SRR-alpha-tocopherols are secreted without discrimination in human chylomicrons, but RRR-alpha-tocopherol is preferentially secreted in very low density lipoproteins. J Lipid Res. 1990;31:675–85. 60. Burton GW, Traber MG, Acuff RV, Walters DN, Kayden H, Hughes L, Ingold KU. Human plasma and tissue alpha-tocopherol concentrations in response to supplementation with deuterated natural and synthetic vitamin E. Am J Clin Nutr. 1998;67:669–84. 61. Traber MG, Rader D, Acuff RV, Brewer HB, Jr., Kayden HJ. Discrimination between RRR- and allracemic-alpha-tocopherols labeled with deuterium by patients with abetalipoproteinemia. Atherosclerosis. 1994;108:27–37. 62. Bruno RS, Leonard SW, Li J, Bray TM, Traber MG. Lower plasma alpha-carboxyethyl-hydroxychroman after deuterium-labeled alpha-tocopherol supplementation suggests decreased vitamin E metabolism in smokers. Am J Clin Nutr. 2005;81:1052–9. 63. Valk EE, Hornstra G. Relationship between vitamin E requirement and polyunsaturated fatty acid intake in man: A review. Int J Vitam Nutr Res. 2000;70:31–42. 64. Mertz W, Tsui JC, Judd JT, Reiser S, Hallfrisch J, Morris ER, Steele PD, Lashley E. What are people really eating? The relation between energy intake derived from estimated diet records and intake determined to maintain body weight. Am J Clin Nutr. 1991;54:291–5. 65. Briefel RR, Sempos CT, McDowell MA, Chien S, Alaimo K. Dietary methods research in the third National Health and Nutrition Examination Survey: Underreporting of energy intake. Am J Clin Nutr. 1997;65:1203S–9S. 66. McBurney MI, Yu EA, Ciappio ED, Bird JK, Eggersdorfer M, Mehta S. Suboptimal serum alphatocopherol concentrations observed among younger adults and those depending exclusively upon food sources, NHANES 2003–2006. PLOS ONE. 2015;10:e0135510. 67. Michels AJ, Leonard SW, Uesugi SL, Bobe G, Frei B, Traber MG. Daily consumption of Oregon hazelnuts affects alpha tocopherol status in healthy older adults: A pre-post intervention study. J Nutr. 2018;148:1924–30. 68. Traber MG. Vitamin E. In: Shils ME, Olson JA, Shike M, Ross AC, editors. Modern Nutrition in Health and Disease. Baltimore, MD: Williams & Wilkins; 1999. pp. 347–62. 69. Sokol RJ. Vitamin E deficiency and neurological disorders. In: Packer L, Fuchs J, editors. Vitamin E in Health and Disease. New York, NY: Marcel Dekker, Inc.; 1993. pp. 815–49. 70. Cohn JS, McNamara JR, Cohn SD, Ordovas JM, Schaefer EJ. Plasma apolipoprotein changes in the triglyceride-rich lipoprotein fraction of human subjects fed a fat-rich meal. J Lipid Res. 1988;29:925–36. 71. Blomstrand R, Forsgren L. Labelled tocopherols in man. Intestinal absorption and thoracic-duct lymph transport of dl-alpha-tocopheryl-3,4-14C2 acetate dl-alpha-tocopheramine-3,4-14C2 dl-alpha-tocopherol(5-methyl-3H) and N-(methyl-3H)-dl-gamma-tocopheramine. Int Z Vitaminforsch. 1968;38:328–44. 72. Traber MG, Kayden HJ, Green JB, Green MH. Absorption of water-miscible forms of vitamin E in a patient with cholestasis and in thoracic duct-cannulated rats. Am J Clin Nutr. 1986;44:914–23. 73. Bruno RS, Leonard SW, Park S, Zhao YY, Traber MG. Human vitamin E requirements assessed with the use of apples fortified with deuterium-labeled alpha-tocopheryl acetate. Am J Clin Nutr. 2006;83:299–304. 74. Traber MG, Burton GW, Hughes L, Ingold KU, Hidaka H, Malloy M, Kane J, Hyams J, Kayden HJ. Discrimination between forms of vitamin E by humans with and without genetic abnormalities of lipoprotein metabolism. J Lipid Res. 1992;33:1171–82. 75. Traber MG, Rudel LL, Burton GW, Hughes L, Ingold KU, Kayden HJ. Nascent VLDL from liver perfusions of cynomolgus monkeys are preferentially enriched in RRR- compared with SRR-alphatocopherol: Studies using deuterated tocopherols. J Lipid Res. 1990;31:687–94. 76. Kayden HJ, Traber MG. Absorption, lipoprotein transport, and regulation of plasma concentrations of vitamin E in humans. J Lipid Res. 1993;34:343–58.

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77. Catignani GL, Bieri JG. Rat liver alpha-tocopherol binding protein. Biochim Biophys Acta. 1977;497:349–57. 78. Sato Y, Hagiwara K, Arai H, Inoue K. Purification and characterization of the alpha-tocopherol transfer protein from rat liver. FEBS Lett. 1991;288:41–5. 79. Yoshida H, Yusin M, Ren I, Kuhlenkamp J, Hirano T, Stolz A, Kaplowitz N. Identification, purification, and immunochemical characterization of a tocopherol-binding protein in rat liver cytosol. J Lipid Res. 1992;33:343–50. 80. Min KC, Kovall RA, Hendrickson WA. Crystal structure of human alpha-tocopherol transfer protein bound to its ligand: Implications for ataxia with vitamin E deficiency. Proc Natl Acad Sci U S A. 2003;100:14713–8. 81. Arita M, Sato Y, Miyata A, Tanabe T, Takahashi E, Kayden HJ, Arai H, Inoue K. Human alphatocopherol transfer protein: cDNA cloning, expression and chromosomal localization. Biochem J. 1995;306(Pt 2):437–43. 82. Traber MG. Vitamin E, nuclear receptors and xenobiotic metabolism. Arch Biochem Biophys. 2004;423:6–11. 83. Hosomi A, Arita M, Sato Y, Kiyose C, Ueda T, Igarashi O, Arai H, Inoue K. Affinity for alphatocopherol transfer protein as a determinant of the biological activities of vitamin E analogs. FEBS Lett. 1997;409:105–8. 84. Schock BC, Van der Vliet A, Corbacho AM, Leonard SW, Finkelstein E, Valacchi G, Obermueller-Jevic U, Cross CE, Traber MG. Enhanced inflammatory responses in alpha-tocopherol transfer protein null mice. Arch Biochem Biophys. 2004;423:162–9. 85. Cellini E, Piacentini S, Nacmias B, Forleo P, Tedde A, Bagnoli S, Ciantelli M, Sorbi S. A family with spinocerebellar ataxia type 8 expansion and vitamin E deficiency ataxia. Archiv Neurol. 2002;59:1952–3. 86. Birringer M, Drogan D, Brigelius-Flohe R. Tocopherols are metabolized in HepG2 cells by side chain omega-oxidation and consecutive beta-oxidation. Free Radic Biol Med. 2001;31:226–32. 87. Parker RS, Sontag TJ, Swanson JE. Cytochrome P4503A-dependent metabolism of tocopherols and inhibition by sesamin. Biochem Biophys Res Commun. 2000;277:531–4. 88. Sontag TJ, Parker RS. Cytochrome P450 omega-hydroxylase pathway of tocopherol catabolism. Novel mechanism of regulation of vitamin E status. J Biol Chem. 2002;277:25290–6. 89. Kluth D, Landes N, Pfluger P, Muller-Schmehl K, Weiss K, Bumke-Vogt C, Ristow M, Brigelius-Flohe R. Modulation of Cyp3a11 mRNA expression by alpha-tocopherol but not gamma-tocotrienol in mice. Free Radic Biol Med. 2005;38:507–14. 90. Traber MG, Siddens LK, Leonard SW, Schock B, Gohil K, Krueger SK, Cross CE, Williams DE. alpha-Tocopherol modulates Cyp3a expression, increases gamma-CEHC production, and limits tissue gamma-tocopherol accumulation in mice fed high gamma-tocopherol diets. Free Radic Biol Med. 2005;38:773–85. 91. Farley SM, Leonard SW, Taylor AW, Birringer M, Edson KZ, Rettie AE, Traber MG. Omegahydroxylation of phylloquinone by CYP4F2 is not increased by alpha-tocopherol. Mol Nutr Food Res. 2013;57:1785–93. 92. Bardowell SA, Stec DE, Parker RS. Common variants of cytochrome P450 4F2 exhibit altered vitamin E-{omega}-hydroxylase specific activity. J Nutr. 2010;140:1901–6. 93. Leonard SW, Paterson E, Atkinson JK, Ramakrishnan R, Cross CE, Traber MG. Studies in humans using deuterium-labeled alpha- and gamma-tocopherols demonstrate faster plasma gamma-tocopherol disappearance and greater gamma-metabolite production. Free Radic Biol Med. 2005;38:857–66. 94. Schultz M, Leist M, Petrzika M, Gassmann B, Brigelius-Flohe R. Novel urinary metabolite of alphatocopherol, 2,5,7,8-tetramethyl-2(2’-carboxyethyl)-6-hydroxychroman, as an indicator of an adequate vitamin E supply? Am J Clin Nutr. 1995;62:1527S–34S. 95. Lebold KM, Ang A, Traber MG, Arab L. Urinary alpha-carboxyethyl hydroxychroman can be used as a predictor of alpha-tocopherol adequacy, as demonstrated in the Energetics Study. Am J Clin Nutr. 2012;96:801–9. 96. Betancor-Fernandez A, Sies H, Stahl W, Polidori MC. In vitro antioxidant activity of 2,5,7,8-tetramethyl2-(2’-carboxyethyl)-6-hydroxychroman (alpha-CEHC), a vitamin E metabolite. Free Radic Res. 2002;36:915–21. 97. Ouahchi K, Arita M, Kayden H, Hentati F, Ben Hamida M, Sokol R, Arai H, Inoue K, Mandel JL, Koenig M. Ataxia with isolated vitamin E deficiency is caused by mutations in the alpha-tocopherol transfer protein. Nat Genet. 1995;9:141–5. 98. Ban R, Takitani K, Kim HS, Murata T, Morinobu T, Ogihara T, Tamai H. Alpha-tocopherol transfer protein expression in rat liver exposed to hyperoxia. Free Radic Res. 2002;36:933–8.

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99. Kim ES, Noh SK, Koo SI. Marginal zinc deficiency lowers the lymphatic absorption of alpha-tocopherol in rats. J Nutr. 1998;128:265–70. 100. Shaw HM, Huang C. Liver alpha-tocopherol transfer protein and its mRNA are differentially altered by dietary vitamin E deficiency and protein insufficiency in rats. J Nutr. 1998;128:2348–54. 101. Hathcock JN, Azzi A, Blumberg J, Bray T, Dickinson A, Frei B, Jialal I, Johnston CS, Kelly FJ et al. Vitamins E and C are safe across a broad range of intakes. Am J Clin Nutr. 2005;81:736–45. 102. Kappus H, Diplock AT. Tolerance and safety of vitamin E: A toxicological position report. Free Radic Biol Med. 1992;13:55–74. 103. Miller ER, 3rd, Pastor-Barriuso R, Dalal D, Riemersma RA, Appel LJ, Guallar E. Meta-analysis: Highdosage vitamin E supplementation may increase all-cause mortality. Ann Intern Med. 2005;142:37–46. 104. Gerss J, Kopcke W. The questionable association of vitamin E supplementation and mortality—Inconsistent results of different meta-analytic approaches. Cell Mol Biol (Noisy-le-grand). 2009;55(Suppl):OL1111–20. 105. Abner EL, Schmitt FA, Mendiondo MS, Marcum JL, Kryscio RJ. Vitamin E and all-cause mortality: A meta-analysis. Curr Aging Sci. 2011;4:158–70. 106. Eidelman RS, Hollar D, Hebert PR, Lamas GA, Hennekens CH. Randomized trials of vitamin E in the treatment and prevention of cardiovascular disease. Arch Intern Med. 2004;164:1552–6. 107. Shekelle PG, Morton SC, Jungvig LK, Udani J, Spar M, Tu W, M JS, Coulter I, Newberry SJ, Hardy M. Effect of supplemental vitamin E for the prevention and treatment of cardiovascular disease. J Gen Intern Med. 2004;19:380–9. 108. Vivekananthan DP, Penn MS, Sapp SK, Hsu A, Topol EJ. Use of antioxidant vitamins for the prevention of cardiovascular disease: Meta-analysis of randomised trials. Lancet. 2003;361:2017–23. 109. Ashor AW, Siervo M, Lara J, Oggioni C, Afshar S, Mathers JC. Effect of vitamin C and vitamin E supplementation on endothelial function: A systematic review and meta-analysis of randomised controlled trials. Br J Nutr. 2015;113:1182–94. 110. Afri M, Ehrenberg B, Talmon Y, Schmidt J, Cohen Y, Frimer AA. Active oxygen chemistry within the liposomal bilayer. Part III: Locating vitamin E, ubiquinol and ubiquinone and their derivatives in the lipid bilayer. Chem Phys Lipids. 2004;131:107–21. 111. Huang J, May JM. Ascorbic acid spares alpha-tocopherol and prevents lipid peroxidation in cultured H4IIE liver cells. Mol Cell Biochem. 2003;247:171–6. 112. May JM, Qu ZC, Mendiratta S. Protection and recycling of alpha-tocopherol in human erythrocytes by intracellular ascorbic acid. Arch Biochem Biophys. 1998;349:281–9. 113. Bisby RH, Parker AW. Reaction of ascorbate with the alpha-tocopheroxyl radical in micellar and bilayer membrane systems. Arch Biochem Biophys. 1995;317:170–8. 114. Burton GW, Wronska U, Stone L, Foster DO, Ingold KU. Biokinetics of dietary RRR-alpha-tocopherol in the male guinea pig at three dietary levels of vitamin C and two levels of vitamin E. Evidence that vitamin C does not “spare” vitamin E in vivo. Lipids. 1990;25:199–210. 115. Salonen JT, Nyyssonen K, Salonen R, Lakka HM, Kaikkonen J, Porkkala-Sarataho E, Voutilainen S, Lakka TA, Rissanen T et al. Antioxidant Supplementation in Atherosclerosis Prevention (ASAP) study: A randomized trial of the effect of vitamins E and C on 3-year progression of carotid atherosclerosis. J Intern Med. 2000;248:377–86. 116. Salonen RM, Nyyssonen K, Kaikkonen J, Porkkala-Sarataho E, Voutilainen S, Rissanen TH, Tuomainen TP, Valkonen VP, Ristonmaa U et al. Six-year effect of combined vitamin C and E supplementation on atherosclerotic progression: The Antioxidant Supplementation in Atherosclerosis Prevention (ASAP) Study. Circulation. 2003;107:947–53. 117. Buettner GR. Commentary on “Faster plasma vitamin E disappearance in smokers is normalized by vitamin C supplementation.” Free Radic Biol Med. 2006;40:555. 118. Betteridge DJ. What is oxidative stress? Metabolism. 2000;49:3–8. 119. Heron M. Deaths: Leading causes for 2016. Natl Vital Stat Rep. 2018;67:1–76. 120. Stocker R, Keaney JF, Jr. Role of oxidative modifications in atherosclerosis. Physiol Rev. 2004;84:1381–478. 121. Harris A, Devaraj S, Jialal I. Oxidative stress, alpha-tocopherol therapy, and atherosclerosis. Curr Atheroscler Rep. 2002;4:373–80. 122. Jialal I, Devaraj S. Scientific evidence to support a vitamin E and heart disease health claim: Research needs. J Nutr. 2005;135:348–53. 123. Miwa K, Okinaga S, Fujita M. Low serum alpha-tocopherol concentrations in subjects with various coronary risk factors. Circ J. 2004;68:542–6. 124. Fuller CJ, Chandalia M, Garg A, Grundy SM, Jialal I. RRR-alpha-tocopheryl acetate supplementation at pharmacologic doses decreases low-density-lipoprotein oxidative susceptibility but not protein glycation in patients with diabetes mellitus. Am J Clin Nutr. 1996;63:753–9.

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125. Devaraj S, Adams-Huet B, Fuller CJ, Jialal I. Dose-response comparison of RRR-alpha-tocopherol and all-racemic alpha-tocopherol on LDL oxidation. Arterioscler Thromb Vasc Biol. 1997;17:2273–9. 126. Devaraj S, Jialal I. The effects of alpha-tocopherol on critical cells in atherogenesis. Curr Opin Lipidol. 1998;9:11–5. 127. Stephens NG, Parsons A, Schofield PM, Kelly F, Cheeseman K, Mitchinson MJ. Randomised controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study (CHAOS). Lancet. 1996;347:781–6. 128. Boaz M, Smetana S, Weinstein T, Matas Z, Gafter U, Iaina A, Knecht A, Weissgarten Y, Brunner D et al. Secondary prevention with antioxidants of cardiovascular disease in endstage renal disease (SPACE): Randomised placebo-controlled trial. Lancet. 2000;356:1213–8. 129. Fang JC, Kinlay S, Beltrame J, Hikiti H, Wainstein M, Behrendt D, Suh J, Frei B, Mudge GH et al. Effect of vitamins C and E on progression of transplant-associated arteriosclerosis: A randomised trial. Lancet. 2002;359:1108–13. 130. Lee IM, Cook NR, Gaziano JM, Gordon D, Ridker PM, Manson JE, Hennekens CH, Buring JE. Vitamin E in the primary prevention of cardiovascular disease and cancer: The Women’s Health Study: A randomized controlled trial. JAMA. 2005;294:56–65. 131. Palmer AM, Burns MA. Selective increase in lipid peroxidation in the inferior temporal cortex in Alzheimer’s disease. Brain Res. 1994;645:338–42. 132. Behl C, Davis JB, Lesley R, Schubert D. Hydrogen peroxide mediates amyloid beta protein toxicity. Cell. 1994;77:817–27. 133. Behl C, Davis J, Cole GM, Schubert D. Vitamin E protects nerve cells from amyloid beta protein toxicity. Biochem Biophys Res Commun. 1992;186:944–50. 134. Behl C. Vitamin E protects neurons against oxidative cell death in vitro more effectively than 17-beta estradiol and induces the activity of the transcription factor NF-kappaB. J Neural Transm. 2000;107:393–407. 135. Leonard SW, Terasawa Y, Farese RV, Jr., Traber MG. Incorporation of deuterated RRR- or all-racalpha-tocopherol in plasma and tissues of alpha-tocopherol transfer protein—Null mice. Am J Clin Nutr. 2002;75:555–60. 136. Pillai SR, Traber MG, Steiss JE, Kayden HJ, Cox NR. Alpha-tocopherol concentrations of the nervous system and selected tissues of adult dogs fed three levels of vitamin E. Lipids. 1993;28:1101–5. 137. Socci DJ, Crandall BM, Arendash GW. Chronic antioxidant treatment improves the cognitive performance of aged rats. Brain Res. 1995;693:88–94. 138. Wortwein G, Stackman RW, Walsh TJ. Vitamin E prevents the place learning deficit and the cholinergic hypofunction induced by AF64A. Exp Neurol. 1994;125:15–21. 139. Monji A, Morimoto N, Okuyama I, Yamashita N, Tashiro N. Effect of dietary vitamin E on lipofuscin accumulation with age in the rat brain. Brain Res. 1994;634:62–8. 140. Hara H, Kato H, Kogure K. Protective effect of alpha-tocopherol on ischemic neuronal damage in the gerbil hippocampus. Brain Res. 1990;510:335–8. 141. Morris MC, Beckett LA, Scherr PA, Hebert LE, Bennett DA, Field TS, Evans DA. Vitamin E and vitamin C supplement use and risk of incident Alzheimer disease. Alzheimer Dis Assoc Disord. 1998;12:121–6. 142. Morris MC, Evans DA, Bienias JL, Tangney CC, Bennett DA, Aggarwal N, Wilson RS, Scherr PA. Dietary intake of antioxidant nutrients and the risk of incident Alzheimer disease in a biracial community study. JAMA. 2002;287:3230–7. 143. Morris MC, Evans DA, Tangney CC, Bienias JL, Wilson RS, Aggarwal NT, Scherr PA. Relation of the tocopherol forms to incident Alzheimer disease and to cognitive change. Am J Clin Nutr. 2005;81:508–14. 144. Kryscio RJ, Abner EL, Caban-Holt A, Lovell M, Goodman P, Darke AK, Yee M, Crowley J, Schmitt FA. Association of antioxidant supplement use and dementia in the Prevention of Alzheimer’s Disease by Vitamin E and Selenium Trial (PREADViSE). JAMA neurology. 2017;74:567–73. 145. Dysken MW, Sano M, Asthana S, Vertrees JE, Pallaki M, Llorente M, Love S, Schellenberg GD, McCarten JR et al. Effect of vitamin E and memantine on functional decline in Alzheimer disease: The TEAM-AD VA cooperative randomized trial. JAMA. 2014;311:33–44. 146. Petersen RC, Thomas RG, Grundman M, Bennett D, Doody R, Ferris S, Galasko D, Jin S, Kaye J et al. Vitamin E and donepezil for the treatment of mild cognitive impairment. N Engl J Med. 2005;352:2379–88. 147. Lloret A, Badia MC, Mora NJ, Pallardo FV, Alonso MD, Vina J. Vitamin E paradox in Alzheimer’s disease: It does not prevent loss of cognition and may even be detrimental. J Alzheimers Dis. 2009;17:143–9. 148. Block G, Patterson B, Subar A. Fruit, vegetables, and cancer prevention: A review of the epidemiological evidence. Nutr Cancer. 1992;18:1–29. 149. Bjelakovic G, Nikolova D, Simonetti RG, Gluud C. Antioxidant supplements for preventing gastrointestinal cancers. Cochrane Database Syst Rev. 2004:CD004183.

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150. Wagner KH, Kamal-Eldin A, Elmadfa I. Gamma-tocopherol—An underestimated vitamin? Ann Nutr Metab. 2004;48:169–88. 151. Albanes D, Heinonen OP, Huttunen JK, Taylor PR, Virtamo J, Edwards BK, Haapakoski J, Rautalahti M, Hartman AM et al. Effects of alpha-tocopherol and beta-carotene supplements on cancer incidence in the Alpha-Tocopherol Beta-Carotene Cancer Prevention Study. Am J Clin Nutr. 1995;62:1427S–30S. 152. Virtamo J, Pietinen P, Huttunen JK, Korhonen P, Malila N, Virtanen MJ, Albanes D, Taylor PR, Albert P. Incidence of cancer and mortality following alpha-tocopherol and beta-carotene supplementation: A postintervention follow-up. JAMA. 2003;290:476–85. 153. Goodman GE, Schaffer S, Omenn GS, Chen C, King I. The association between lung and prostate cancer risk, and serum micronutrients: Results and lessons learned from beta-carotene and retinol efficacy trial. Cancer Epidemiol Biomarkers Prev. 2003;12:518–26. 154. Lippman SM, Klein EA, Goodman PJ, Lucia MS, Thompson IM, Ford LG, Parnes HL, Minasian LM, Gaziano JM et al. Effect of selenium and vitamin E on risk of prostate cancer and other cancers: The Selenium and Vitamin E Cancer Prevention Trial (SELECT). JAMA. 2009;301:39–51. 155. Klein EA, Thompson IM, Jr., Tangen CM, Crowley JJ, Lucia MS, Goodman PJ, Minasian LM, Ford LG, Parnes HL et al. Vitamin E and the risk of prostate cancer: The Selenium and Vitamin E Cancer Prevention Trial (SELECT). JAMA. 2011;306:1549–56. 156. Ni J, Chen M, Zhang Y, Li R, Huang J, Yeh S. Vitamin E succinate inhibits human prostate cancer cell growth via modulating cell cycle regulatory machinery. Biochem Biophys Res Commun. 2003;300:357–63. 157. Sigounas G, Anagnostou A, Steiner M. dl-alpha-tocopherol induces apoptosis in erythroleukemia, prostate, and breast cancer cells. Nutr Cancer. 1997;28:30–5. 158. Hartman TJ, Dorgan JF, Virtamo J, Tangrea JA, Taylor PR, Albanes D. Association between serum alpha-tocopherol and serum androgens and estrogens in older men. Nutr Cancer. 1999;35:10–5. 159. Moyad MA, Brumfield SK, Pienta KJ. Vitamin E, alpha- and gamma-tocopherol, and prostate cancer. Semin Urol Oncol. 1999;17:85–90. 160. Jiang Q, Wong J, Ames BN. Gamma-tocopherol induces apoptosis in androgen-responsive LNCaP prostate cancer cells via caspase-dependent and independent mechanisms. Ann N Y Acad Sci. 2004;1031:399–400. 161. Bjelakovic G, Nikolova D, Simonetti RG, Gluud C. Antioxidant supplements for prevention of gastrointestinal cancers: A systematic review and meta-analysis. Lancet. 2004;364:1219–28. 162. Campbell S, Stone W, Whaley S, Krishnan K. Development of gamma (gamma)-tocopherol as a colorectal cancer chemopreventive agent. Crit Rev Oncol Hematol. 2003;47:249–59. 163. Stone WL, Krishnan K, Campbell SE, Qui M, Whaley SG, Yang H. Tocopherols and the treatment of colon cancer. Ann N Y Acad Sci. 2004;1031:223–33. 164. Yang CS, Suh N. Cancer prevention by different forms of tocopherols. Top Curr Chem. 2013;329:21–33. 165. Burton GW, Traber MG. Vitamin E: Antioxidant activity, biokinetics, and bioavailability. Annu Rev Nutr. 1990;10:357–82.

7

Health Benefits of Green Tea Priyankar Dey, Geoffrey Y. Sasaki, and Richard S. Bruno

CONTENTS 7.1 Introduction........................................................................................................................... 127 7.2 History of Green Tea............................................................................................................. 128 7.3 Processing and Composition................................................................................................. 128 7.3.1 Green Tea Processing................................................................................................ 128 7.3.2 Catechin Structure and Composition......................................................................... 129 7.3.3 Flavonoids, Caffeine, and Nutrients.......................................................................... 129 7.4 Green Tea Catechin Bioavailability....................................................................................... 130 7.4.1 Catechin Absorption.................................................................................................. 130 7.4.2 Catechin Metabolism................................................................................................. 130 7.4.3 Microbial Metabolism............................................................................................... 131 7.5 Safety and Toxicity................................................................................................................ 132 7.6 Bioactivity of Catechins........................................................................................................ 132 7.7 Benefits of Green Tea for Chronic Disease Prevention......................................................... 133 7.7.1 Obesity....................................................................................................................... 133 7.7.2 Diabetes..................................................................................................................... 134 7.7.3 Nonalcoholic Fatty Liver Disease.............................................................................. 134 7.7.4 Cardiovascular Disease............................................................................................. 136 7.7.5 Cancer........................................................................................................................ 137 7.7.5.1 Prostate Cancer........................................................................................... 137 7.7.5.2 Breast Cancer.............................................................................................. 137 7.7.5.3 Hepatocellular Carcinoma.......................................................................... 137 7.7.5.4 Other Cancers............................................................................................. 138 7.8 Conclusion............................................................................................................................. 138 References....................................................................................................................................... 138

7.1 INTRODUCTION Tea (Camellia sinensis) is the most commonly consumed prepared beverage in the world, and ranks only second to that of water for all beverages consumed.1,2 Green tea is rich in polyphenolic catechins, especially epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin (EC; Figure 7.1). Of these, EGCG has received the most extensive study because it is thought to be primarily responsible for the health benefits of green tea. Although green tea is consumed less frequently than black tea, its popularity has increased due to growing knowledge of its health benefits. For example, epidemiological evidence supports that green tea consumption decreases the risk of cardiometabolic disorders and some forms of cancer.3–15 These findings have laid the foundation to define the bioactivities of green tea, with evidence indicating that catechins improve health status through antioxidant, anti-inflammatory, and metabolic benefits.

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FIGURE 7.1  Major green tea catechins and their metabolites of microbial origin. Parental catechins include those that are gallated (epigallocatechin gallate, EGCG; epicatechin gallate, ECG) and non-gallated (epicatechin, EC; catechin, CAT; epigallocatechin, EGC). Microbial metabolism by gut microbiota bacteria result in the formation of tri-hydroxy (3′,4′,5′-trihydroxyphenyl-γ-valerolactone), di-hydroxy (3′,4′- or 3′5′-dihydroxyphenylγ-valerolactone), and mono-hydroxy (3′-hydroxyphenyl-γ-valerolactone) metabolites that are further degraded to derivatives of benzoic acid, proprionic acid, and hippuric acid. (Adapted from Feng WY. Curr Drug Metab. 2006;7(7):755–809; Sang S et al. Pharmacol Res. 2011;64(2):87–99.)

7.2  HISTORY OF GREEN TEA The tea plant (Camellia sinensis) originated in China prior to spreading throughout Asia and Europe before reaching the New World by the seventeenth century.16 The discovery of tea is connected to the cultural history of China, with its first mention dating back nearly 5000 years ago.2 Although Emperor Shen Nung is credited with its discovery in 2737 bc, the first written record appeared around 400 bc in the Erh Ya, a Chinese dictionary.17,18 In 780 AD, Lu Yu authored The Classic of Tea to popularize tea while also providing among the earliest records of green tea processing.18 Green tea was developed initially by the Chinese, who exposed the tea leaves to hot steam or heat prior to drying.17 Throughout Chinese history, green tea leaf preparations varied from steaming and compressing tea leaves into bricks or as powdered tea formation.18 Green tea became prevalent in Japan by the sixth century, coinciding with the preparation of matcha, a powdered green tea that continues to be popular.18 Tea production exceeds three billion kilograms annually.2,19 Of this, green tea represents ∼20%–22% of global tea production, with most being consumed in Asia.2 However, the production of green tea is on a trajectory to outpace that of black tea20 due to consumer demand for healthy beverages and dietary supplements.21,22

7.3  PROCESSING AND COMPOSITION 7.3.1 Green Tea Processing Green, black, and oolong teas are all derived from the leaves and buds of Camellia sinensis. Although each is prepared from the same plant, they differ in post-harvest processing. The leaves intended for green tea are unfermented, whereas those for black and oolong tea are fully and partially fermented, respectively. Tea leaves used for the manufacture of green tea are processed rapidly upon harvesting including high temperature exposure by steaming or pan frying prior to being rolled

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TABLE 7.1 Catechin Content of Freshly Brewed Green Tea, Black Tea, and Oolong Tea Epicatechin (EC) Epicatechin gallate (ECG) Epigallocatechin (EGC) Epigallocatechin gallate (EGCG) Total Catechins

Green Tea

Black Tea

Oolong Tea

20.0 25.2 26.8 105.0 177.0

15.2 12.5 6.8 14.6 49.1

8.3 14.7 21.8 66.2 111.0

Source: Data adapted from Neilson AP et al. J Chromatogr A. 2006;1132(1–2):132–40. Note: Green tea is non-fermented, thereby preserving catechin content (mg/250 mL), whereas catechins are degraded with fermentation. Partially fermented oolong tea has less catechin content than green tea, but retains a greater proportion of catechins compared with fully fermented black tea.

and dried.2,18 This inactivates polyphenol oxidase and peroxidases that otherwise cause oxidation (i.e., fermentation) of the catechins (Table 7.1).23,24

7.3.2 Catechin Structure and Composition The phytochemical composition of green tea is influenced by its growing conditions, including geographical location, climate, season, and leaf maturity.2,25,26 Catechins (flavan-3-ols; Figure 7.1) are the major polyphenols in green tea and account for ∼30%–40% of the dry weight of tea leaves.18,25,27 Catechins have a 2-phenyl benzopyran structure with aromatic A- and B-rings that are connected to a 3-carbon oxygenated heterocylic C-ring.27,28 Catechins are characterized by a meta-5,7-dihydroxy substitution on the A-ring and either a di-hydroxy (EC, ECG) or tri-hydroxy (EGC, EGCG) substitution on the B-ring.27,29 EGCG and ECG also contain a gallic acid moiety that is esterified to the 3-carbon of the C-ring.30 EGCG is the most abundant catechin, and can account for 50%–80% of the total catechin content of green tea.29 Green tea also contains low levels of catechin isomers (e.g., gallocatechin gallate), dimers (e.g., EGC-di-gallate), and methylated metabolites (e.g., 3-methyl-EC).27,29,31 Although the catechin content varies by commercial source and brewing technique, freshly brewed green tea has a greater amount of total catechins than both black and oolong teas, with EGCG being most abundant, followed by EGC, ECG, and EC (Table 7.1).32

7.3.3  Flavonoids, Caffeine, and Nutrients Catechins represent ∼70% of the total flavonoids in green tea and the remaining flavonoid content is other flavanols and polymeric flavonoids.23 Non-catechin flavanols include quercetin, myricetin, kaempferol, and their glycosides.27,33 Modifications of flavanols (e.g., glucosylation, rhamnosylation, rutinosylation) have also been identified in green tea.34 Phenolic acids (e.g., gallic and quinic acids) also provide a small proportion of the total polyphenol content of green tea.25,27,29 Further, small amounts of proteins, carbohydrates, lipids, vitamins, and minerals are present in green tea, with each representing 3.5 times,75 suggesting that adverse effects may be alleviated if catechins are co-ingested with food. Concurrent ingestion of green tea may also interfere with prescription drug metabolism to induce a hepatotoxicity.76,77 There are few or no adverse effects when 800 mg EGCG (∼10 times the amount in a serving of freshly brewed green tea) is ingested on a single occasion or chronically.38,75,78 Further, no significant adverse effects were reported when EGCG (800 mg/day) was ingested for up to 1 year in overweight men,79 post-menopausal women,80 or persons with type 2 diabetes.81 The available evidence therefore supports that green tea is safe when consumed as recommended.

7.6  BIOACTIVITY OF CATECHINS Extensive study has focused on the antioxidant function of catechins because of their multiple hydroxyl groups that are capable of redox reactions. Catechins are able to directly scavenge reactive oxygen/nitrogen species (e.g., superoxide radical, peroxyl radicals) and prevent downstream oxidative damage in vitro.82,83 They are also effective chelators that prevent iron- and copper-induced free radical generation.84 Catechins also exhibit indirect antioxidant activities by inducing host antioxidant enzymes, such as glutathione peroxidase and superoxide dismutase.84,85 Notably, though, green tea catechins likely protect against oxidative damage independent of Nrf2 signaling that upregulates cytoprotective antioxidant defenses.86 On the other hand, pro-oxidant activities of catechins, at least

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under in vitro conditions, modulate cellular signaling that may help to prevent cancer.82,87 Green tea catechins also exhibit anti-inflammatory activities. Studies in preclinical models show that EGCG prevents the nuclear translocation of nuclear factor κB, a pro-inflammatory transcription factor, by limiting IκB phosphorylation84,88 and DNA binding activity.89 Green tea catechins also decrease pro-inflammatory responses under the transcription control of activator protein-1 by inhibiting phosphorylation of upstream kinases (i.e., extracellular signal-related protein kinases and c-jun kinases).90 However, the benefits of green tea or its catechins on inflammation have been less clear, with outcomes of some studies showing a favorable benefit and others showing a neutral effect.91–94

7.7  BENEFITS OF GREEN TEA FOR CHRONIC DISEASE PREVENTION The putative antioxidant and anti-inflammatory activities of green tea catechins have been investigated for their role to enhance metabolic function, especially in relation to obesity, cardiometabolic risk, and cancer. Several meta-analyses have examined the beneficial effects of green tea consumption on diabetes, liver diseases, and cancers (Table 7.2), whereas studies in preclinical models typically use purified catechins or green tea extract (GTE) that is enriched in catechins. In the below sections, a summary of preclinical and clinical studies is presented to provide foundational knowledge that continues to support research into the human health benefits of green tea and its catechins.

7.7.1 Obesity Obesity is already at epidemic proportions and is expected to worsen consistent with estimates indicating that >50% of the population worldwide will be obese by 2030.95 Evidence suggests that green tea or its catechins may help to alleviate obesity through gut-level mechanisms that limit dietary lipid absorption; peripheral effects that upregulate energy expenditure, inhibit adipogenesis, and stimulate lipid oxidation; or through satiating benefits that limit energy intake.96 In obese adults participating in a 12-wk exercise program, the daily consumption of green tea (625 mg catechins) decreased abdominal adipose mass by 7.7% and subcutaneous adipose mass by 8.7% while also decreasing circulating triglyceride concentrations compared with those not receiving green tea.97 Similarly, GTE supplementation in exercising mice fed a high-fat diet TABLE 7.2 Select Meta-Analyses Examining Green Tea Consumption in Relation to Chronic Disease Risk Major Study Outcomes Green tea consumption is associated with lower fasting glucose, HbA1c levels, and fasting insulin Green tea consumption is associated with a reduced risk of liver diseases (hepatocellular carcinoma, steatosis, hepatitis, cirrhosis) Green tea consumption is associated with lower plasma total cholesterol and LDL-C in obese subjects Green tea consumption is associated with a reduced the risk for cardiovascular disease, intracerebral hemorrhage, and cerebral infarction Green tea consumption is associated with lower systolic and diastolic blood pressure Green tea consumption is associated with a lower risk of coronary artery disease Green tea consumption is associated with a reduced risk of liver cancer, especially among women Green tea consumption is associated with a lower risk of lung cancer  Green tea consumption is associated with a lower risk of prostate cancer

Studies

Pooled Subjects

Reference

17

1133

108

15

826,149

161

21

1704

138

9

259,267

125

13

1367

162

5

53,586

163

9

468,968

163

12 10

107,537 96,511

10 164

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significantly reduced body mass and adiposity compared with exercise or GTE supplementation alone.98 In overweight and obese persons, 12-wk supplementation with GTE containing EGCG (90 mg) and caffeine (50 mg) increased energy expenditure while also decreasing body mass and waist circumference.99 This is potentially attributed to GTE stimulating thermogenesis by increasing the release of noradrenaline that mediates non-shivering energy expenditure.100 Others suggest that GTE increases thermogenesis due to catechins that inhibit the activity of catecholO-methyltransferase that otherwise degrades norepinephrine.101 In support, an intervention in obese Thai adults demonstrated that GTE supplementation decreased body mass and increased resting energy expenditure and the respiratory quotient.102 Potentially, the benefits of GTE to reduce obesity risk are attributed to effects on adipokines. For example, a double-blind, placebo-controlled trial in Taiwanese women demonstrated that GTE supplementation improved dyslipidemia while increasing circulating adiponectin concentrations; the latter were inversely related to body mass. An emergent investigative area is the study of chronic “low-grade” inflammation in relation to obesity risk. Chronic supplementation of GTE to mice fed a high-fat lowered adipose expression of the inflammatory parameters TNF-α and IL-10 and decreased body mass in association with increased circulating adiponectin concentrations and an upregulation of the lipolytic pathway.103

7.7.2 Diabetes Numerous studies support that GTE inhibits the progression of diabetes and related metabolic complications (e.g., insulin resistance, hyperglycemia, hepatic nephropathy, glycemic hepatotoxicity). Indeed, epidemiological studies have suggested that chronic green tea consumption is strongly correlated with a lower risk of diabetes,104,105 and controlled studies in obese mice indicate benefits of green tea to reduce circulating glucose and improve insulin sensitivity.106,107 In a meta-analysis of 17 clinical trials (n = 1133 subjects), the consumption of green tea for 2–6 months was associated with lower blood glucose, lower insulin, and lower levels of HbA1c.108 In normoglycemic humans, acute supplementation of GTE limited glycemic excursions otherwise induced by a 75 g oral glucose tolerance test.109 Studies in rodents have suggested that the glucoselowering activity of GTE may be attributed gallated catechins (i.e., EGCG and ECG) that limit intestinal absorption of glucose by interacting with glucose transporters.110 Others have suggested that GTE may reduce glycemic excursions otherwise induced by starch ingestion by inhibiting the activities of α-amylase and α-glucosidase.111 Further study in vitro has provided evidence that catechins inhibit pancreatic α-amylase in a non-competitive manner to prevent starch digestion112 and that EGCG most strongly inhibits α-glucosidase activity compared with other catechins.113 In Taiwanese persons with type 2 diabetes mellitus, decaffeinated green tea three times daily for 18 weeks lowered circulating triglyceride concentrations along with insulin resistance.81 Further, circulating glucagon-like peptide-1 was significantly increased, which is important for regulating circulating glucose. Green tea has also been shown to manage the risk of diabetic nephropathy.114 Consumption of green tea polyphenols for 12 weeks lowered albuminuria, which was suggested to be improved by reducing podocyte apoptosis by activating the WNT pathway. Similar benefits also occurred in rats in which EGCG supplementation prevented nephropathy as evidenced by reduced renal fibrosis, mesangial cell hyperplasia, and improved morphometry of Bowman’s capsules within the renal system.115

7.7.3 Nonalcoholic Fatty Liver Disease Nonalcoholic steatohepatitis (NASH) is an early stage of nonalcoholic fatty liver disease (NAFLD) that is regarded as the hepatic manifestation of metabolic syndrome and significantly increases the risk for more progressive disorders including fibrosis, cirrhosis, and potentially hepatocellular carcinoma (HCC). Of concern is that NASH afflicts >70 million Americans, and its multifacted etiology that is characterized by inflammation, oxidative distress, and dysregulated lipid and glucose metabolism makes it difficult to manage.30,116

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Epidemiological evidence from Japan suggested that the daily consumption of >10 cups of green tea is associated with reduced levels of liver injury (i.e., alanine and aspartate aminotransferase; ALT and AST), circulating ferritin, triglyceride, total cholesterol, LDL-C, and increased HDL-C.117 Although there are no non-invasive diagnostic measures of NASH, elevations in ALT and AST are commonly observed in these persons. No large-scale clinical trials have examined green tea to manage NASH. However, findings of a double-blind, placebo-controlled trial (n = 80 patients) indicated that GTE supplementation for 3 months lowered serum ALT, AST, and alkaline phosphatase (ALP), but ALT and ALP and not AST were similarly lowered among those allocated to placebo.118 In a separate trial (n = 17 NAFLD patients), a higher dose of catechins was reported to decrease serum ALT and urinary isoprostanes to a greater extent than a lower dose of catechins or placebo.119 These observations require further confirmation, but their promise has been well established by studies in rodent models of NASH. In genetically obese, leptin-deficient mice, dietary GTE supplementation inhibited histological evidence of liver steatosis and reduced hepatic triglyceride levels without affecting hepatic antioxidants or circulating adiponectin.120 These benefits were likely attributed to GTE limiting adipose lipogenesis and the flux of free fatty acids to the liver, where they get esterified and stored as triglyceride.85 Separate studies in leptin-adequate rodents also indicate that GTE prevents or treats diet-induced NASH (Figure 7.4).121,122 Although early findings suggested that GTE protects against liver injury by upregulating Nrf2-dependent antioxidant defenses,85 studies in Nrf2-deficient mice

FIGURE 7.4  Benefits of GTE on nonalcoholic fatty liver disease in experimental rodents. Chronic feeding of a high-fat (HF) diet in male C57BL6 mice induces liver steatosis and hepatocellular ballooning compared with those fed a low-fat (LF) diet. Dietary supplementation of GTE in LF mice does not alter histological evidence of NAFLD (LF + GTE), whereas it reduces liver steatosis and hepatocellular ballooning in mice fed a HF diet (HF + GTE). (Data are representative of those reported in a study published by an author of this chapter. Li J et al. J Nutr Biochem. 2017;41:34–41.)

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clearly showed that GTE provides extensive Nrf2-independent hepatoprotection during NASH.86 Follow-up studies in rodents fed a high-fat diet suggested that GTE alleviates NASH by inhibiting NFκB activation by downregulating signaling from the pro-inflammatory receptors tumor necrosis factor receptor-1 (TNFR1) and Toll-like receptor-4 (TLR4).122 When studies were conducted in mice lacking intact TLR4 signaling, GTE was shown to protect against NASH in wild-type mice to the extent observed in TLR4 mutant mice that were fully protected from NASH.123 This suggested that GTE-mediated inhibition of TLR4/NFκB limits the induction of TNFα that is otherwise needed to induce TNFR1 signaling. The importance of limiting TLR4 signaling was further supported by studies in mice with NASH. Indeed, GTE decreased serum endotoxin (i.e., the ligand for TLR4) in association with improving intestinal integrity and mRNA expression of tight junctions.122–124 Thus, at least in rodents, GTE protects against NASH by limiting gut-derived endotoxin translocation to the liver, where it would activate TLR4/NFκB inflammation that induces hepatocellular injury. Future, well-controlled studies are needed to translate these pre-clinical findings to improve the health of humans with or at-risk of developing NASH.

7.7.4 Cardiovascular Disease Cardioprotective benefits of green tea were supported by findings of a large meta-analysis (n = 259,267 individuals) indicating that daily consumption lowers the risk of cardiovascular disease, intracerebral hemorrhage, and cerebral infarction.125 Separate findings from an observational study (n = 8552 adults) also suggested a lower risk of cardiovascular-related morbidity among those consuming high levels of green tea.126 The benefits of green tea on cardiovascular risk may be attributed to its vasoprotective activity that was observed in coronary artery disease patients who received dietary EGCG supplementation.127 Similar benefits on vascular function were also observed among healthy persons who were supplemented with EC.128 Smokers who were instructed to consume green tea daily also had lower levels of C-reactive protein and oxidized LDL in association with reduced concentrations of the platelet activation marker P-selectin.129 These findings suggest that the anti-inflammatory activities of green tea or its catechins help to improve vascular endothelial function. Invasive studies in rodents have also revealed that EGCG increases the activation of endothelial nitric oxide synthase,130 which is critical for nitric oxide–dependent vascular function. Anti-inflammatory activities of catechins that decrease the expression of cellular adhesion molecules131 and the recruitment of monocytes132 are also likely to improve vascular health. Related activities, including inhibiting matrix metalloproteinase133 and increasing endothelial prostacyclin production,134 may also contribute to the benefits of green tea to maintain vascular function. It has also been proposed that the antioxidant activities of catechins that reduce the accumulation of reactive oxygen species and reactive nitrogen species (RNS) may also lower cardiovascular risk. For instance, intraperitoneal administration of EGCG in hypercholesterolemic mice upregulated the expression of cytoprotective genes that detoxify reactive species while also attenuating atherosclerotic plaque accumulation.135 Consistent with LDL oxidation being an early mediator of atherosclerosis, green catechins were reported to prevent copper-induced oxidation of LDL in vitro with the following potencies: EGCG > ECG > EC > EGC.136 These effects are potentially due to the metal chelating function of catechins.137 Whether this chelator function helps to lower cardiovascular risk in humans remains unclear, however, because catechins have poor bioavailability and concentrations studied under in vitro conditions are often at levels difficult to achieve in humans through normal consumption. Lipid-lowering activities of green tea catechins are also likely to manage the risk of cardiovascular disease. A meta-analysis of 21 studies involving 1704 overweight and obese subjects established that green tea consumption lowered total cholesterol by 3.38 mg/dL and LDL-C by 5.29 mg/dL without affecting circulating triglyceride or HDL-C.138 Although the authors indicate that more high-quality and large-scale trials are needed to confirm this effect, the findings are of potential

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importance because a lowering of circulating cholesterol by 1% reduces cardiovascular disease risk by 2%.139 The mechanism by which green tea lowers cholesterol is not fully defined, but may involve catechins interfering with the emulsification, digestion, and micellar solubilization of dietary fat and cholesterol140 or the downregulation of genes involved in lipogenesis and cholesterol biosynthesis.141

7.7.5 Cancer Lifestyle modification, especially dietary approaches, continues to be a leading recommendation for cancer chemoprevention. Although green tea has been examined for its potential benefits in reducing cancer risk, controlled trials in humans are lacking, thereby placing reliance on findings from rodent and cellular model systems. In the below sections, evidence concerning green tea or its catechins on the risk of various cancers will be summarized. 7.7.5.1  Prostate Cancer The incidence of prostate cancer in Asian countries where green tea is widely consumed is lower than that in the United States.142 A study from China in patients with histologically confirmed prostate adenocarcinoma and healthy persons suggested that prostate cancer risk was inversely related to the frequency of green tea consumption and especially lower among those drinking >3 cups per day.143 In a cohort from Japan, findings from 49,920 middle-aged men suggested that the regular consumption of >5 cups of green tea was also associated with lower prostate cancer risk.11 Studies in transgenic mice that spontaneously develop metastatic prostate cancer have demonstrated that dietary supplementation with green tea polyphenols increases survival and dramatically inhibits prostate cancer incidence.144 The anti-tumor benefit may be due to green tea polyphenols lowering insulin-like growth factor-I and cellular proliferation.144 Studies in androgen-sensitive and -insensitive prostate cancer cells also support that EGCG upregulates apoptosis.145 Others have also reported in a human prostate cancer cell line that ECG has greater activity than other catechins to induce apoptosis by upregulating mitochondrial depolarization and increasing intracellular ROS.146 7.7.5.2  Breast Cancer The association between green tea consumption and the prevalence, recurrence, and risk of breast cancer has received study. A meta-analysis supported that the consumption of green tea consumption was inversely associated with both the recurrence and overall risk of breast cancer.147 These benefits may be explained by findings supporting that the regular consumption of green tea is associated with lower serum estrogen levels.148 This association also persistent regardless of catechol-O-methyltransferase genotype. In the MCF-7 breast cancer cell line, EGCG decreased matrix metalloproteinase by interfering with the transmembrane receptor integrin, and also reduced vascular endothelial growth factor and NFκB activation.149 EGCG also inhibits the aggressive growth and invasion of tamoxifen-resistant MCF-7 breast carcinoma cells by limiting epidermal growth factor receptor signaling.150 7.7.5.3  Hepatocellular Carcinoma The incidence of hepatocellular carcinoma has been largely attributed to viral infections and alcohol abuse. However, concern now exists for NAFLD because of its mediating effects on liver cancer risk that are expected to outpace those due to other historical risk factors. Individuals consuming green tea for >30 years had a lower risk of developing HCC.151 Liver cancer risk was also inversely related to green tea consumption among 41,761 Japanese persons who were followed for 9 years.13 It is thought that the antioxidant and anti-inflammatory activities of green tea catechins are responsible for these beneficial effects at the liver – both through metabolic effects and altering signal transduction pathways.152 For example, EGCG inhibits insulin-like growth factors and induces apoptosis in HCC cells153 and inhibits hepatocarcinoma cell proliferation by attenuating vascular endothelial growth factor.154 EGCG also attenuates platelet-derived growth factor-induced cellular proliferation and collagen expression in

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hepatic stellate cells.155 This is of importance because liver fibrosis provokes the risk toward HCC. In genetically obese mice exposed to a liver carcinogen, EGCG limited tumorigenesis in association with attenuating several pro-oncogenic pathways (e.g., signal transducer and activator of transcription-3; extracellular signal-regulated kinase; c-Jun NH(2)-terminal kinase). Further, these were also associated with lower levels of pro-inflammatory mediators (TNFα, IL-1B, IL-6, IL-18) and liver steatosis. This suggests that the early management of NASH may help to alleviate HCC risk, but controlled trials in humans are needed before clear recommendations can be established. 7.7.5.4  Other Cancers Cancers at other sites have also received study in relation to green tea consumption patterns, but to a lesser extent and with somewhat equivocal outcomes. Findings of a meta-analysis, which was composed of 13 observational studies, suggested an inverse association between green tea consumption and stomach cancer in case-control studies but not in cohort studies.156 The risk of esophageal cancer was also lower among Chinese women but not men who consumed higher levels of green tea.157 Interestingly, a benefit was observed in both men and women when data were stratified to consider only nonsmokers and non-users of alcohol. Other prospective observational studies (n = 69,710 Chinese women) also suggest a lower risk of colorectal cancers among those who are regular green tea consumers.158 Paradoxically, one cohort study (n 102,137; 11 y) from Japan observed no significant association between green tea consumption and pancreatic cancer, whereas another population-based case-control study from China (n = 931 colon, 884 rectal, and 451 pancreatic cancer cases vs. 1552 healthy controls) observed an inverse relation with increased green tea use.159,160

7.8 CONCLUSION Originally consumed for its recreational pleasures, green tea has gained significant interest for its health benefits that are mediated through its polyphenolic catechins. Indeed, green tea is one of few foods rich these compounds, which is evident by the considerably lower levels present in black and oolong teas that are derived from the same tea plant. Greater study of the bioavailability and metabolism of catechins, especially those processes mediated by the gut microbiota, are expected to help define the mechanism of action of these health-promoting compounds. In addition, large-scale randomized controlled trials are needed to confirm the cardiometabolic and anti-cancer benefits observed in preclinical models and observational studies. Likewise, although green tea appears to be safe when consumed at reasonable levels, more study is needed to fully establish its safety and upper limits of recommended consumption, and to identify why certain persons may be susceptible to the rarely observed mild adverse effects.

REFERENCES 1. McKay DL, Blumberg JB. The role of tea in human health: An update. J Am Coll Nutr. 2002;21(1):1–13. 2. Hayat K, Iqbal H, Malik U, Bilal U, Mushtaq S. Tea and its consumption: Benefits and risks. Crit Rev Food Sci Nutr. 2015;55(7):939–54. 3. Zhang C, Qin YY, Wei X, Yu FF, Zhou YH, He J. Tea consumption and risk of cardiovascular outcomes and total mortality: A systematic review and meta-analysis of prospective observational studies. Eur J Epidemiol. 2015;30(2):103–13. 4. Kuriyama S. The relation between green tea consumption and cardiovascular disease as evidenced by epidemiological studies. J Nutr. 2008;138(8):1548S–53S. 5. Saito E, Inoue M, Sawada N, Shimazu T, Yamaji T, Iwasaki M, Sasazuki S et al. Association of green tea consumption with mortality due to all causes and major causes of death in a Japanese population: The Japan Public Health Center-based Prospective Study (JPHC Study). Ann Epidemiol. 2015;25(7):512–8 e3. 6. Kokubo Y, Iso H, Saito I, Yamagishi K, Yatsuya H, Ishihara J, Inoue M, Tsugane S. The impact of green tea and coffee consumption on the reduced risk of stroke incidence in Japanese population: The Japan public health center-based study cohort. Stroke. 2013;44(5):1369–74.

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7. Kuriyama S, Shimazu T, Ohmori K, Kikuchi N, Nakaya N, Nishino Y, Tsubono Y, Tsuji I. Green tea consumption and mortality due to cardiovascular disease, cancer, and all causes in Japan: The Ohsaki study. JAMA. 2006;296(10):1255–65. 8. Tang J, Zheng JS, Fang L, Jin Y, Cai W, Li D. Tea consumption and mortality of all cancers, CVD and all causes: A meta-analysis of eighteen prospective cohort studies. Br J Nutr. 2015;114(5):673–83. 9. Yuan JM. Cancer prevention by green tea: Evidence from epidemiologic studies. Am J Clin Nutr. 2013;98(6 Suppl):1676S–81S. 10. Tang N, Wu Y, Zhou B, Wang B, Yu R. Green tea, black tea consumption and risk of lung cancer: A meta-analysis. Lung Cancer. 2009;65(3):274–83. 11. Kurahashi N, Sasazuki S, Iwasaki M, Inoue M, Tsugane S. Green tea consumption and prostate cancer risk in Japanese men: A prospective study. Am J Epidemiol. 2008;167(1):71–7. 12. Yang G, Zheng W, Xiang YB, Gao J, Li HL, Zhang X, Gao YT, Shu XO. Green tea consumption and colorectal cancer risk: A report from the Shanghai Men’s Health Study. Carcinogenesis. 2011;32(11):1684–8. 13. Ui A, Kuriyama S, Kakizaki M, Sone T, Nakaya N, Ohmori-Matsuda K, Hozawa A, Nishino Y, Tsuji I. Green tea consumption and the risk of liver cancer in Japan: The Ohsaki Cohort study. Cancer Causes Control. 2009;20(10):1939–45. 14. Marventano S, Salomone F, Godos J, Pluchinotta F, Del Rio D, Mistretta A, Grosso G. Coffee and tea consumption in relation with non-alcoholic fatty liver and metabolic syndrome: A systematic review and meta-analysis of observational studies. Clin Nutr. 2016;35(6):1269–81. 15. Vernarelli JA, Lambert JD. Tea consumption is inversely associated with weight status and other markers for metabolic syndrome in US adults. Eur J Nutr. 2013;52(3):1039–48. 16. Sharangi AB. Medicinal and therapeutic potentialities of tea (Camellia sinensis L.)—A review. Food Res Int. 2009;42(5):529–35. 17. Weisburger JH. Tea and health: A historical perspective. Cancer Lett. 1997;114(1–2):315–7. 18. Harbowy ME, Balentine DA. Tea chemistry. Crit Rev Plant Sci. 1997;16(5):415–80. 19. Yang CS, Wang X, Lu G, Picinich SC. Cancer prevention by tea: Animal studies, molecular mechanisms and human relevance. Nat Rev Cancer. 2009;9(6):429–39. 20. Chang K. World tea production and trade: Current and future development: Food and Agriculture Organization of the United Nations; 2015. 21. Cabrera C, Artacho R, Gimenez R. Beneficial effects of green tea—A review. J Am Coll Nutr. 2006;25(2):79–99. 22. Sarma DN, Barrett ML, Chavez ML, Gardiner P, Ko R, Mahady GB, Marles RJ, Pellicore LS, Giancaspro GI, Low Dog T. Safety of green tea extracts: A systematic review by the US Pharmacopeia. Drug Saf. 2008;31(6):469–84. 23. Feng WY. Metabolism of green tea catechins: An overview. Curr Drug Metab. 2006;7(7):755–809. 24. Wang Y, Ho CT. Polyphenolic chemistry of tea and coffee: A century of progress. J Agric Food Chem. 2009;57(18):8109–14. 25. Graham HN. Green tea composition, consumption, and polyphenol chemistry. Prev Med. 1992;21(3):334–50. 26. Dai W, Qi D, Yang T, Lv H, Guo L, Zhang Y, Zhu Y et al. Nontargeted analysis using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry uncovers the effects of harvest season on the metabolites and taste quality of tea (Camellia sinensis L.). J Agric Food Chem. 2015;63(44):9869–78. 27. Balentine DA, Wiseman SA, Bouwens LC. The chemistry of tea flavonoids. Crit Rev Food Sci Nutr. 1997;37(8):693–704. 28. Manach C, Scalbert A, Morand C, Remesy C, Jimenez L. Polyphenols: Food sources and bioavailability. Am J Clin Nutr. 2004;79(5):727–47. 29. Sang S, Lambert JD, Ho CT, Yang CS. The chemistry and biotransformation of tea constituents. Pharmacol Res. 2011;64(2):87–99. 30. Masterjohn C, Bruno RS. Therapeutic potential of green tea in nonalcoholic fatty liver disease. Nutr Rev. 2012;70(1):41–56. 31. Khan N, Mukhtar H. Tea polyphenols for health promotion. Life Sci. 2007;81(7):519–33. 32. Neilson AP, Green RJ, Wood KV, Ferruzzi MG. High-throughput analysis of catechins and theaflavins by high performance liquid chromatography with diode array detection. J Chromatogr A. 2006;1132(1–2):132–40. 33. Lin LZ, Chen P, Harnly JM. New phenolic components and chromatographic profiles of green and fermented teas. J Agric Food Chem. 2008;56(17):8130–40.

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34. Dai W, Tan J, Lu M, Xie D, Li P, Lv H, Zhu Y et al. Nontargeted modification-specific metabolomics investigation of glycosylated secondary metabolites in tea (Camellia sinensis L.) based on liquid chromatography-high-resolution mass spectrometry. J Agric Food Chem. 2016;64(35):6783–90. 35. Yang CS, Chen L, Lee MJ, Balentine D, Kuo MC, Schantz SP. Blood and urine levels of tea catechins after ingestion of different amounts of green tea by human volunteers. Cancer Epidemiol Biomarkers Prev. 1998;7(4):351–4. 36. Lee MJ, Maliakal P, Chen L, Meng X, Bondoc FY, Prabhu S, Lambert G, Mohr S, Yang CS. Pharmacokinetics of tea catechins after ingestion of green tea and (-)-epigallocatechin-3-gallate by humans: Formation of different metabolites and individual variability. Cancer Epidemiol Biomarkers Prev. 2002;11(10 Pt 1):1025–32. 37. Lee MJ, Wang ZY, Li H, Chen L, Sun Y, Gobbo S, Balentine DA, Yang CS. Analysis of plasma and urinary tea polyphenols in human subjects. Cancer Epidemiol Biomarkers Prev. 1995;4(4):393–9. 38. Chow HH, Cai Y, Alberts DS, Hakim I, Dorr R, Shahi F, Crowell JA, Yang CS, Hara Y. Phase I pharmacokinetic study of tea polyphenols following single-dose administration of epigallocatechin gallate and polyphenon E. Cancer Epidemiol Biomarkers Prev. 2001;10(1):53–8. 39. Renouf M, Marmet C, Guy PA, Beaumont M, Lepage M, Williamson G, Dionisi F. Dose-response plasma appearance of green tea catechins in adults. Mol Nutr Food Res. 2013;57(5):833–9. 40. Fung ST, Ho CK, Choi SW, Chung WY, Benzie IF. Comparison of catechin profiles in human plasma and urine after single dosing and regular intake of green tea (Camellia sinensis). Br J Nutr. 2013;109(12):2199–207. 41. Clifford MN, van der Hooft JJ, Crozier A. Human studies on the absorption, distribution, metabolism, and excretion of tea polyphenols. Am J Clin Nutr. 2013;98(6 Suppl):1619S–30S. 42. Chen L, Lee MJ, Li H, Yang CS. Absorption, distribution, elimination of tea polyphenols in rats. Drug Metab Dispos. 1997;25(9):1045–50. 43. Zhu M, Chen Y, Li RC. Pharmacokinetics and system linearity of tea catechins in rat. Xenobiotica. 2001;31(1):51–60. 44. Kim S, Lee MJ, Hong J, Li C, Smith TJ, Yang GY, Seril DN, Yang CS. Plasma and tissue levels of tea catechins in rats and mice during chronic consumption of green tea polyphenols. Nutr Cancer. 2000;37(1):41–8. 45. Neilson AP, Ferruzzi MG. Influence of formulation and processing on absorption and metabolism of flavan-3-ols from tea and cocoa. Annu Rev Food Sci Technol. 2011;2:125–51. 46. Zhang L, Zheng Y, Chow MS, Zuo Z. Investigation of intestinal absorption and disposition of green tea catechins by Caco-2 monolayer model. Int J Pharm. 2004;287(1–2):1–12. 47. Nakagawa K, Okuda S, Miyazawa T. Dose-dependent incorporation of tea catechins, (-)-epigallocatechin3-gallate and (-)-epigallocatechin, into human plasma. Biosci Biotechnol Biochem. 1997;61(12):1981–5. 48. Neilson AP, Hopf AS, Cooper BR, Pereira MA, Bomser JA, Ferruzzi MG. Catechin degradation with concurrent formation of homo- and heterocatechin dimers during in vitro digestion. J Agric Food Chem. 2007;55(22):8941–9. 49. Lambert JD, Sang S, Yang CS. Biotransformation of green tea polyphenols and the biological activities of those metabolites. Mol Pharm. 2007;4(6):819–25. 50. Yang CS, Sang S, Lambert JD, Lee MJ. Bioavailability issues in studying the health effects of plant polyphenolic compounds. Mol Nutr Food Res. 2008;52(Suppl 1):S139–51. 51. Del Rio D, Calani L, Cordero C, Salvatore S, Pellegrini N, Brighenti F. Bioavailability and catabolism of green tea flavan-3-ols in humans. Nutrition. 2010;26(11–12):1110–6. 52. Kohri T, Suzuki M, Nanjo F. Identification of metabolites of (-)-epicatechin gallate and their metabolic fate in the rat. J Agric Food Chem. 2003;51(18):5561–6. 53. Sang S, Lee MJ, Yang I, Buckley B, Yang CS. Human urinary metabolite profile of tea polyphenols analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry with datadependent acquisition. Rapid Commun Mass Spectrom. 2008;22(10):1567–78. 54. Li C, Meng X, Winnik B, Lee MJ, Lu H, Sheng S, Buckley B, Yang CS. Analysis of urinary metabolites of tea catechins by liquid chromatography/electrospray ionization mass spectrometry. Chem Res Toxicol. 2001;14(6):702–7. 55. Monagas M, Urpi-Sarda M, Sánchez-Patán F, Llorach R, Garrido I, Gómez-Cordovés C, Andres-Lacueva C, Bartolomé B. Insights into the metabolism and microbial biotransformation of dietary flavan-3-ols and the bioactivity of their metabolites. Food Funct. 2010;1(3):233–53. 56. Lu H, Meng X, Li C, Sang S, Patten C, Sheng S, Hong J et al. Glucuronides of tea catechins: Enzymology of biosynthesis and biological activities. Drug Metab Dispos. 2003;31(4):452–61. 57. Takano M, Yumoto R, Murakami T. Expression and function of efflux drug transporters in the intestine. Pharmacol Ther. 2006;109(1–2):137–61.

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58. Vaidyanathan JB, Walle T. Glucuronidation and sulfation of the tea flavonoid (-)-epicatechin by the human and rat enzymes. Drug Metab Dispos. 2002;30(8):897–903. 59. Lu H, Meng X, Yang CS. Enzymology of methylation of tea catechins and inhibition of catechol-Omethyltransferase by (-)-epigallocatechin gallate. Drug Metab Dispos. 2003;31(5):572–9. 60. Sang S, Yang CS. Structural identification of novel glucoside and glucuronide metabolites of (-)-epigallocatechin-3-gallate in mouse urine using liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom. 2008;22(22):3693–9. 61. Sang S, Lambert JD, Hong J, Tian S, Lee MJ, Stark RE, Ho CT, Yang CS. Synthesis and structure identification of thiol conjugates of (-)-epigallocatechin gallate and their urinary levels in mice. Chem Res Toxicol. 2005;18(11):1762–9. 62. Schantz M, Erk T, Richling E. Metabolism of green tea catechins by the human small intestine. Biotechnol J. 2010;5(10):1050–9. 63. Auger C, Mullen W, Hara Y, Crozier A. Bioavailability of polyphenon E flavan-3-ols in humans with an ileostomy. J Nutr. 2008;138(8):1535S–42S. 64. Stalmach A, Mullen W, Steiling H, Williamson G, Lean ME, Crozier A. Absorption, metabolism, and excretion of green tea flavan-3-ols in humans with an ileostomy. Mol Nutr Food Res. 2010;54(3):323–34. 65. Meng X, Sang S, Zhu N, Lu H, Sheng S, Lee MJ, Ho CT, Yang CS. Identification and characterization of methylated and ring-fission metabolites of tea catechins formed in humans, mice, and rats. Chem Res Toxicol. 2002;15(8):1042–50. 66. Roowi S, Stalmach A, Mullen W, Lean ME, Edwards CA, Crozier A. Green tea flavan-3-ols: Colonic degradation and urinary excretion of catabolites by humans. J Agric Food Chem. 2010;58(2):1296–304. 67. Li C, Lee MJ, Sheng S, Meng X, Prabhu S, Winnik B, Huang B, Chung JY, Yan S, Ho CT, Yang CS. Structural identification of two metabolites of catechins and their kinetics in human urine and blood after tea ingestion. Chem Res Toxicol. 2000;13(3):177–84. 68. Calani L, Del Rio D, Luisa Callegari M, Morelli L, Brighenti F. Updated bioavailability and 48 h excretion profile of flavan-3-ols from green tea in humans. Int J Food Sci Nutr. 2012;63(5):513–21. 69. Mazzanti G, Di Sotto A, Vitalone A. Hepatotoxicity of green tea: An update. Arch Toxicol. 2015;89(8):1175–91. 70. Mazzanti G, Menniti-Ippolito F, Moro PA, Cassetti F, Raschetti R, Santuccio C, Mastrangelo S. Hepatotoxicity from green tea: A review of the literature and two unpublished cases. Eur J Clin Pharmacol. 2009;65(4):331–41. 71. Isomura T, Suzuki S, Origasa H, Hosono A, Suzuki M, Sawada T, Terao S, Muto Y, Koga T. Liver-related safety assessment of green tea extracts in humans: A systematic review of randomized controlled trials. Eur J Clin Nutr. 2016;70(11):1340. 72. Lambert JD, Kennett MJ, Sang S, Reuhl KR, Ju J, Yang CS. Hepatotoxicity of high oral dose (-)-epigallocatechin-3-gallate in mice. Food Chem Toxicol. 2010;48(1):409–16. 73. Wang D, Wang Y, Wan X, Yang CS, Zhang J. Green tea polyphenol (-)-epigallocatechin-3-gallate triggered hepatotoxicity in mice: Responses of major antioxidant enzymes and the Nrf2 rescue pathway. Toxicol Appl Pharmacol. 2015;283(1):65–74. 74. James KD, Kennett MJ, Lambert JD. Potential role of the mitochondria as a target for the hepatotoxic effects of (-)-epigallocatechin-3-gallate in mice. Food Chem Toxicol. 2017;111:302–9. 75. Chow HH, Hakim IA, Vining DR, Crowell JA, Ranger-Moore J, Chew WM, Celaya CA, Rodney SR, Hara Y, Alberts DS. Effects of dosing condition on the oral bioavailability of green tea catechins after single-dose administration of Polyphenon E in healthy individuals. Clin Cancer Res. 2005;11(12):4627–33. 76. Yang CS, Pan E. The effects of green tea polyphenols on drug metabolism. Expert Opin Drug Metab Toxicol. 2012;8(6):677–89. 77. Salminen WF, Yang X, Shi Q, Greenhaw J, Davis K, Ali AA. Green tea extract can potentiate acetaminophen-induced hepatotoxicity in mice. Food Chem Toxicol. 2012;50(5):1439–46. 78. Chow HH, Cai Y, Hakim IA, Crowell JA, Shahi F, Brooks CA, Dorr RT, Hara Y, Alberts DS. Pharmacokinetics and safety of green tea polyphenols after multiple-dose administration of epigallocatechin gallate and polyphenon E in healthy individuals. Clin Cancer Res. 2003;9(9):3312–9. 79. Frank J, George TW, Lodge JK, Rodriguez-Mateos AM, Spencer JP, Minihane AM, Rimbach G. Daily consumption of an aqueous green tea extract supplement does not impair liver function or alter cardiovascular disease risk biomarkers in healthy men. J Nutr. 2009;139(1):58–62. 80. Dostal AM, Samavat H, Bedell S, Torkelson C, Wang R, Swenson K, Le C et al. The safety of green tea extract supplementation in postmenopausal women at risk for breast cancer: Results of the Minnesota Green Tea Trial. Food Chem Toxicol. 2015;83:26–35.

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81. Liu CY, Huang CJ, Huang LH, Chen IJ, Chiu JP, Hsu CH. Effects of green tea extract on insulin resistance and glucagon-like peptide 1 in patients with type 2 diabetes and lipid abnormalities: A randomized, double-blinded, and placebo-controlled trial. PLOS ONE. 2014;9(3):e91163. 82. Sang S, Hou Z, Lambert JD, Yang CS. Redox properties of tea polyphenols and related biological activities. Antioxid Redox Signal. 2005;7(11–12):1704–14. 83. Valcic S, Burr JA, Timmermann BN, Liebler DC. Antioxidant chemistry of green tea catechins. New oxidation products of (-)-epigallocatechin gallate and (-)-epigallocatechin from their reactions with peroxyl radicals. Chem Res Toxicol. 2000;13(9):801–10. 84. Higdon JV, Frei B. Tea catechins and polyphenols: Health effects, metabolism, and antioxidant functions. Crit Rev Food Sci Nutr. 2003;43(1):89–143. 85. Park HJ, DiNatale DA, Chung M-Y, Park Y-K, Lee J-Y, Koo SI, O’Connor M, Manautou JE, Bruno RS. Green tea extract attenuates hepatic steatosis by decreasing adipose lipogenesis and enhancing hepatic antioxidant defenses in ob/ob mice. J Nutr Biochem. 2011;22(4):393–400. 86. Li J, Sapper TN, Mah E, Rudraiah S, Schill KE, Chitchumroonchokchai C, Moller MV, McDonald JD, Rohrer PR, Manautou JE. Green tea extract provides extensive Nrf2-independent protection against lipid accumulation and NFκB pro-inflammatory responses during nonalcoholic steatohepatitis in mice fed a high-fat diet. Mol Nutr Food Res. 2016;60(4):858–70. 87. Lambert JD, Elias RJ. The antioxidant and pro-oxidant activities of green tea polyphenols: A role in cancer prevention. Arch Biochem Biophys. 2010;501(1):65–72. 88. Nomura M, Ma W, Chen N, Bode AM, Dong Z. Inhibition of 12-O-tetradecanoylphorbol-13-acetateinduced NF-kappaB activation by tea polyphenols, (-)-epigallocatechin gallate and theaflavins. Carcinogenesis. 2000;21(10):1885–90. 89. Yang F, de Villiers WJ, McClain CJ, Varilek GW. Green tea polyphenols block endotoxin-induced tumor necrosis factor-production and lethality in a murine model. J Nutr. 1998;128(12):2334–40. 90. Chung JY, Huang C, Meng X, Dong Z, Yang CS. Inhibition of activator protein 1 activity and cell growth by purified green tea and black tea polyphenols in H-ras-transformed cells: Structure-activity relationship and mechanisms involved. Cancer Res. 1999;59(18):4610–7. 91. Basu A, Du M, Sanchez K, Leyva MJ, Betts NM, Blevins S, Wu M, Aston CE, Lyons TJ. Green tea minimally affects biomarkers of inflammation in obese subjects with metabolic syndrome. Nutrition. 2011;27(2):206–13. 92. de Maat MP, Pijl H, Kluft C, Princen HM. Consumption of black and green tea had no effect on inflammation, haemostasis and endothelial markers in smoking healthy individuals. Eur J Clin Nutr. 2000;54(10):757–63. 93. Ryu OH, Lee J, Lee KW, Kim HY, Seo JA, Kim SG, Kim NH, Baik SH, Choi DS, Choi KM. Effects of green tea consumption on inflammation, insulin resistance and pulse wave velocity in type 2 diabetes patients. Diabetes Res Clin Pract. 2006;71(3):356–8. 94. Nantz MP, Rowe CA, Bukowski JF, Percival SS. Standardized capsule of Camellia sinensis lowers cardiovascular risk factors in a randomized, double-blind, placebo-controlled study. Nutrition. 2009;25(2):147–54. 95. Finkelstein EA, Khavjou OA, Thompson H, Trogdon JG, Pan L, Sherry B, Dietz W. Obesity and severe obesity forecasts through 2030. Amer J Prev Med. 2012;42(6):563–70. 96. Huang J, Wang Y, Xie Z, Zhou Y, Zhang Y, Wan X. The anti-obesity effects of green tea in human intervention and basic molecular studies. Eur J Clin Nutr. 2014;68(10):1075–87. 97. Maki KC, Reeves MS, Farmer M, Yasunaga K, Matsuo N, Katsuragi Y, Komikado M et al. Green tea catechin consumption enhances exercise-induced abdominal fat loss in overweight and obese adults. J Nutr. 2009;139(2):264–70. 98. Shimotoyodome A, Haramizu S, Inaba M, Murase T, Tokimitsu I. Exercise and green tea extract stimulate fat oxidation and prevent obesity in mice. Med Sci Sports Exerc. 2005;37(11):1884–92. 99. Dulloo AG, Duret C, Rohrer D, Girardier L, Mensi N, Fathi M, Chantre P, Vandermander J. Efficacy of a green tea extract rich in catechin polyphenols and caffeine in increasing 24-h energy expenditure and fat oxidation in humans. Am J Clin Nutr. 1999;70(6):1040–5. 100. Dulloo A, Seydoux J, Girardier L, Chantre P, Vandermander J. Green tea and thermogenesis: Interactions between catechin-polyphenols, caffeine and sympathetic activity. Int J Obes. 2000;24(2):252. 101. Shixian Q, VanCrey B, Shi J, Kakuda Y, Jiang Y. Green tea extract thermogenesis-induced weight loss by epigallocatechin gallate inhibition of catechol-O-methyltransferase. J Med Food. 2006;9(4):451–8. 102. Auvichayapat P, Prapochanung M, Tunkamnerdthai O, Sripanidkulchai BO, Auvichayapat N, Thinkhamrop B, Kunhasura S, Wongpratoom S, Sinawat S, Hongprapas P. Effectiveness of green tea on weight reduction in obese Thais: A randomized, controlled trial. Physiol Behav. 2008;93(3):486–91.

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103. Cunha CA, Lira FS, Rosa Neto JC, Pimentel GD, Souza GI, da Silva CMG, de Souza CT, Ribeiro EB, Sawaya ACHF, Oller do Nascimento CM. Green tea extract supplementation induces the lipolytic pathway, attenuates obesity, and reduces low-grade inflammation in mice fed a high-fat diet. Mediators Inflamm. 2013;2013. 104. Oba S, Nagata C, Nakamura K, Fujii K, Kawachi T, Takatsuka N, Shimizu H. Consumption of coffee, green tea, oolong tea, black tea, chocolate snacks and the caffeine content in relation to risk of diabetes in Japanese men and women. Br J Nutr. 2010;103(3):453–9. 105. Toolsee NA, Aruoma OI, Gunness TK, Kowlessur S, Dambala V, Murad F, Googoolye K et  al. Effectiveness of green tea in a randomized human cohort: Relevance to diabetes and its complications. Biomed Res Int. 2013;2013:412379. 106. Ortsater H, Grankvist N, Wolfram S, Kuehn N, Sjoholm A. Diet supplementation with green tea extract epigallocatechin gallate prevents progression to glucose intolerance in db/db mice. Nutr Metab (Lond). 2012;9:11. 107. Tsuneki H, Murata S, Anzawa Y, Soeda Y, Tokai E, Wada T, Kimura I, Yanagisawa M, Sakurai T, Sasaoka T. Age-related insulin resistance in hypothalamus and peripheral tissues of orexin knockout mice. Diabetologia. 2008;51(4):657–67. 108. Liu K, Zhou R, Wang B, Chen K, Shi LY, Zhu JD, Mi MT. Effect of green tea on glucose control and insulin sensitivity: A meta-analysis of 17 randomized controlled trials. Am J Clin Nutr. 2013;98(2):340–8. 109. Park JH, Jin JY, Baek WK, Park SH, Sung HY, Kim YK, Lee J, Song DK. Ambivalent role of gallated catechins in glucose tolerance in humans: A novel insight into non-absorbable gallated catechin-derived inhibitors of glucose absorption. J Physiol Pharmacol. 2009;60(4):101–9. 110. Kobayashi Y, Suzuki M, Satsu H, Arai S, Hara Y, Suzuki K, Miyamoto Y, Shimizu M. Green tea polyphenols inhibit the sodium-dependent glucose transporter of intestinal epithelial cells by a competitive mechanism. J Agric Food Chem. 2000;48(11):5618–23. 111. Lochocka K, Bajerska J, Glapa A, Fidler-Witon E, Nowak JK, Szczapa T, Grebowiec P, Lisowska A, Walkowiak J. Green tea extract decreases starch digestion and absorption from a test meal in humans: A randomized, placebo-controlled crossover study. Sci Rep. 2015;5:12015. 112. Miao M, Jiang B, Jiang H, Zhang T, Li X. Interaction mechanism between green tea extract and human alpha-amylase for reducing starch digestion. Food Chem. 2015;186:20–5. 113. Yilmazer-Musa M, Griffith AM, Michels AJ, Schneider E, Frei B. Grape seed and tea extracts and catechin 3-gallates are potent inhibitors of alpha-amylase and alpha-glucosidase activity. J Agric Food Chem. 2012;60(36):8924–9. 114. Borges CM, Papadimitriou A, Duarte DA, Lopes de Faria JM, Lopes de Faria JB. The use of green tea polyphenols for treating residual albuminuria in diabetic nephropathy: A double-blind randomised clinical trial. Sci Rep. 2016;6:28282. 115. Mohan T, Velusamy P, Chakrapani LN, Srinivasan AK, Singh A, Johnson T, Periandavan K. Impact of EGCG supplementation on the progression of diabetic nephropathy in rats: An insight into fibrosis and apoptosis. J Agric Food Chem. 2017;65(36):8028–36. 116. Neuschwander-Tetri BA. Non-alcoholic fatty liver disease. BMC Med. 2017;15(1):45. 117. Imai K, Nakachi K. Cross sectional study of effects of drinking green tea on cardiovascular and liver diseases. BMJ. 1995;310(6981):693–6. 118. Pezeshki A, Safi S, Feizi A, Askari G, Karami F. The effect of green tea extract supplementation on liver enzymes in patients with nonalcoholic fatty liver disease. Int J Prev Med. 2016;7. 119. Sakata R, Nakamura T, Torimura T, Ueno T, Sata M. Green tea with high-density catechins improves liver function and fat infiltration in non-alcoholic fatty liver disease (NAFLD) patients: A double-blind placebo-controlled study. Int J Mol Med. 2013;32(5):989–94. 120. Bruno RS, Dugan CE, Smyth JA, DiNatale DA, Koo SI. Green tea extract protects leptin-deficient, spontaneously obese mice from hepatic steatosis and injury. J Nutr. 2008;138(2):323–31. 121. Park HJ, Lee JY, Chung MY, Park YK, Bower AM, Koo SI, Giardina C, Bruno RS. Green tea extract suppresses NFkappaB activation and inflammatory responses in diet-induced obese rats with nonalcoholic steatohepatitis. J Nutr. 2012;142(1):57–63. 122. Li J, Sapper TN, Mah E, Moller MV, Kim JB, Chitchumroonchokchai C, McDonald JD, Bruno RS. Green tea extract treatment reduces NFκB activation in mice with diet-induced nonalcoholic steatohepatitis by lowering TNFR1 and TLR4 expression and ligand availability. J Nutr Biochem. 2017;41:34–41. 123. Li J, Sasaki GY, Dey P, Chitchumroonchokchai C, Labyk AN, McDonald JD, Kim JB, Bruno RS. Green tea extract protects against hepatic NFkappaB activation along the gut-liver axis in diet-induced obese mice with nonalcoholic steatohepatitis by reducing endotoxin and TLR4/MyD88 signaling. J Nutr Biochem. 2017;53:58–65.

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124. Dey P, Sasaki GY, Wei P, Li J, Wang L, Zhu J, McTigue D, Yu Z, Bruno RS. Green tea extract prevents obesity in male mice by alleviating gut dysbiosis in association with improved intestinal barrier function that limits endotoxin translocation and adipose inflammation. J Nutr Biochem. 2019;67:78–89. 125. Pang J, Zhang Z, Zheng TZ, Bassig BA, Mao C, Liu X, Zhu Y et al. Green tea consumption and risk of cardiovascular and ischemic related diseases: A meta-analysis. Int J Cardiol. 2016;202:967–74. 126. Nakachi K, Matsuyama S, Miyake S, Suganuma M, Imai K. Preventive effects of drinking green tea on cancer and cardiovascular disease: Epidemiological evidence for multiple targeting prevention. Biofactors. 2000;13(1–4):49–54. 127. Widlansky ME, Hamburg NM, Anter E, Holbrook M, Kahn DF, Elliott JG, Keaney JF, Jr., Vita JA. Acute EGCG supplementation reverses endothelial dysfunction in patients with coronary artery disease. J Am Coll Nutr. 2007;26(2):95–102. 128. Schroeter H, Heiss C, Balzer J, Kleinbongard P, Keen CL, Hollenberg NK, Sies H, Kwik-Uribe C, Schmitz HH, Kelm M. (-)-Epicatechin mediates beneficial effects of flavanol-rich cocoa on vascular function in humans. Proc Natl Acad Sci U S A. 2006;103(4):1024–9. 129. Lee W, Min WK, Chun S, Lee YW, Park H, Lee DH, Lee YK, Son JE. Long-term effects of green tea ingestion on atherosclerotic biological markers in smokers. Clin Biochem. 2005;38(1):84–7. 130. Lorenz M, Wessler S, Follmann E, Michaelis W, Dusterhoft T, Baumann G, Stangl K, Stangl V. A constituent of green tea, epigallocatechin-3-gallate, activates endothelial nitric oxide synthase by a phosphatidylinositol-3-OH-kinase-, cAMP-dependent protein kinase-, and Akt-dependent pathway and leads to endothelial-dependent vasorelaxation. J Biol Chem. 2004;279(7):6190–5. 131. Ludwig A, Lorenz M, Grimbo N, Steinle F, Meiners S, Bartsch C, Stangl K, Baumann G, Stangl V. The tea flavonoid epigallocatechin-3-gallate reduces cytokine-induced VCAM-1 expression and monocyte adhesion to endothelial cells. Biochem Biophys Res Commun. 2004;316(3):659–65. 132. Hong MH, Kim MH, Chang HJ, Kim NH, Shin BA, Ahn BW, Jung YD. (-)-Epigallocatechin-3-gallate inhibits monocyte chemotactic protein-1 expression in endothelial cells via blocking NF-kappaB signaling. Life Sci. 2007;80(21):1957–65. 133. El Bedoui J, Oak MH, Anglard P, Schini-Kerth VB. Catechins prevent vascular smooth muscle cell invasion by inhibiting MT1-MMP activity and MMP-2 expression. Cardiovasc Res. 2005;67(2):317–25. 134. Mizugaki M, Ishizawa F, Yamazaki T, Hishinuma T. Epigallocatechin gallate increase the prostacyclin production of bovine aortic endothelial cells. Prostaglandins Other Lipid Mediat. 2000;62(2):157–64. 135. Chyu KY, Babbidge SM, Zhao X, Dandillaya R, Rietveld AG, Yano J, Dimayuga P, Cercek B, Shah PK. Differential effects of green tea-derived catechin on developing versus established atherosclerosis in apolipoprotein E-null mice. Circulation. 2004;109(20):2448–53. 136. Miura S, Watanabe J, Tomita T, Sano M, Tomita I. The inhibitory effects of tea polyphenols (flavan3-ol derivatives) on Cu2+ mediated oxidative modification of low density lipoprotein. Biol Pharm Bull. 1994;17(12):1567–72. 137. Forester SC, Lambert JD. Antioxidant effects of green tea. Mol Nutr Food Res. 2011;55(6):844–54. 138. Yuan F, Dong H, Fang K, Gong J, Lu F. Effects of green tea on lipid metabolism in overweight or obese people: A meta-analysis of randomized controlled trials. Mol Nutr Food Res. 2018;62(1). 139. Velayutham P, Babu A, Liu D. Green tea catechins and cardiovascular health: An update. Curr Med Chem. 2008;15(18):1840–50. 140. Koo SI, Noh SK. Green tea as inhibitor of the intestinal absorption of lipids: Potential mechanism for its lipid-lowering effect. J Nutr Biochem. 2007;18(3):179–83. 141. Shrestha S, Ehlers SJ, Lee JY, Fernandez ML, Koo SI. Dietary green tea extract lowers plasma and hepatic triglycerides and decreases the expression of sterol regulatory element-binding protein-1c mRNA and its responsive genes in fructose-fed, ovariectomized rats. J Nutr. 2009;139(4):640–5. 142. Johnson J, Bailey H, Mukhtar H. Green tea polyphenols for prostate cancer chemoprevention: A translational perspective. Phytomedicine. 2010;17(1):3–13. 143. Jian L, Xie LP, Lee AH, Binns CW. Protective effect of green tea against prostate cancer: A case-control study in southeast China. Int J Cancer. 2004;108(1):130–5. 144. Gupta S, Hastak K, Ahmad N, Lewin JS, Mukhtar H. Inhibition of prostate carcinogenesis in TRAMP mice by oral infusion of green tea polyphenols. Proc Natl Acad Sci U S A. 2001;98(18):10350–5. 145. Gupta S, Ahmad N, Nieminen AL, Mukhtar H. Growth inhibition, cell-cycle dysregulation, and induction of apoptosis by green tea constituent (-)-epigallocatechin-3-gallate in androgen-sensitive and androgeninsensitive human prostate carcinoma cells. Toxicol Appl Pharmacol. 2000;164(1):82–90. 146. Chung LY, Cheung TC, Kong SK, Fung KP, Choy YM, Chan ZY, Kwok TT. Induction of apoptosis by green tea catechins in human prostate cancer DU145 cells. Life Sci. 2001;68(10):1207–14.

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147. Ogunleye AA, Xue F, Michels KB. Green tea consumption and breast cancer risk or recurrence: A metaanalysis. Breast Cancer Res Treat. 2010;119(2):477–84. 148. Wu AH, Arakawa K, Stanczyk FZ, Van Den Berg D, Koh WP, Yu MC. Tea and circulating estrogen levels in postmenopausal Chinese women in Singapore. Carcinogenesis. 2005;26(5):976–80. 149. Sen T, Moulik S, Dutta A, Choudhury PR, Banerji A, Das S, Roy M, Chatterjee A. Multifunctional effect of epigallocatechin-3-gallate (EGCG) in downregulation of gelatinase-A (MMP-2) in human breast cancer cell line MCF-7. Life Sci. 2009;84(7–8):194–204. 150. Farabegoli F, Papi A, Orlandi M. (-)-Epigallocatechin-3-gallate down-regulates EGFR, MMP-2, MMP-9 and EMMPRIN and inhibits the invasion of MCF-7 tamoxifen-resistant cells. Biosci Rep. 2011;31(2):99–108. 151. Li Y, Chang S-C, Goldstein BY, Scheider WL, Cai L, You N-CY, Tarleton HP, Ding B, Zhao J, Wu M. Green tea consumption, inflammation and the risk of primary hepatocellular carcinoma in a Chinese population. Cancer Epidemiol. 2011;35(4):362–8. 152. Shimizu M, Shirakami Y, Sakai H, Kubota M, Kochi T, Ideta T, Miyazaki T, Moriwaki H. Chemopreventive potential of green tea catechins in hepatocellular carcinoma. Int J Mol Sci. 2015;16(3):6124–39. 153. Shimizu M, Shirakami Y, Sakai H, Tatebe H, Nakagawa T, Hara Y, Weinstein IB, Moriwaki H. EGCG inhibits activation of the insulin-like growth factor (IGF)/IGF-1 receptor axis in human hepatocellular carcinoma cells. Cancer Lett. 2008;262(1):10–8. 154. Shirakami Y, Shimizu M, Adachi S, Sakai H, Nakagawa T, Yasuda Y, Tsurumi H, Hara Y, Moriwaki H. (-)-Epigallocatechin gallate suppresses the growth of human hepatocellular carcinoma cells by inhibiting activation of the vascular endothelial growth factor-vascular endothelial growth factor receptor axis. Cancer Sci. 2009;100(10):1957–62. 155. Sakata R, Ueno T, Nakamura T, Sakamoto M, Torimura T, Sata M. Green tea polyphenol epigallocatechin3-gallate inhibits platelet-derived growth factor-induced proliferation of human hepatic stellate cell line LI90. J Hepatol. 2004;40(1):52–9. 156. Myung SK, Bae WK, Oh SM, Kim Y, Ju W, Sung J, Lee YJ, Ko JA, Song JI, Choi HJ. Green tea consumption and risk of stomach cancer: A meta-analysis of epidemiologic studies. Int J Cancer. 2009;124(3):670–7. 157. Gao YT, McLaughlin JK, Blot WJ, Ji BT, Dai Q, Fraumeni JF, Jr. Reduced risk of esophageal cancer associated with green tea consumption. J Natl Cancer Inst. 1994;86(11):855–8. 158. Yang G, Shu XO, Li H, Chow WH, Ji BT, Zhang X, Gao YT, Zheng W. Prospective cohort study of green tea consumption and colorectal cancer risk in women. Cancer Epidemiol Biomarkers Prev. 2007;16(6):1219–23. 159. Ji BT, Chow WH, Hsing AW, McLaughlin JK, Dai Q, Gao YT, Blot WJ, Fraumeni JF, Jr. Green tea consumption and the risk of pancreatic and colorectal cancers. Int J Cancer. 1997;70(3):255–8. 160. Luo J, Inoue M, Iwasaki M, Sasazuki S, Otani T, Ye W, Tsugane S. Green tea and coffee intake and risk of pancreatic cancer in a large-scale, population-based cohort study in Japan (JPHC study). Eur J Cancer Prev. 2007;16(6):542–8. 161. Yin X, Yang J, Li T, Song L, Han T, Yang M, Liao H, He J, Zhong X. The effect of green tea intake on risk of liver disease: A meta analysis. Int J Clin Exp Med. 2015;8(6):8339–46. 162. Peng X, Zhou R, Wang B, Yu X, Yang X, Liu K, Mi M. Effect of green tea consumption on blood pressure: A meta-analysis of 13 randomized controlled trials. Sci Rep. 2014;4:6251. 163. Huang YQ, Lu X, Min H, Wu QQ, Shi XT, Bian KQ, Zou XP. Green tea and liver cancer risk: A metaanalysis of prospective cohort studies in Asian populations. Nutrition. 2016;32(1):3–8. 164. Guo Y, Zhi F, Chen P, Zhao K, Xiang H, Mao Q, Wang X, Zhang X. Green tea and the risk of prostate cancer: A systematic review and meta-analysis. Medicine. 2017;96(13).

8

Scientific, Legal, and Regulatory Considerations for Cannabidiol Jay Manfre, Esq., Rick Collins, Esq., Marielle Kahn Weintraub, and Robert E.C. Wildman

CONTENTS 8.1 Introduction........................................................................................................................... 147 8.2 Potential for Nutraceutical Benefit of Cannabidiol................................................................ 147 8.3 Analytical Methods for Hemp and Cannabidiol.................................................................... 149 8.4 The Farm Bill........................................................................................................................ 150 8.5 The Food and Drug Administration and the Food, Drug, and Cosmetic Act....................... 152 8.6 Federal Legislation on the Horizon....................................................................................... 155 8.7 The Future of Cannabidiol..................................................................................................... 155 References....................................................................................................................................... 155

8.1 INTRODUCTION Both marijuana and hemp are produced from the same species of plant, Cannabis sativa, though different varieties are cultivated for specific characteristics . Cannabidiol (CBD) is one of over 100 naturally occurring cannabinoids found in both marijuana and hemp. Although both marijuana and hemp come from Cannabis sativa, hemp typically has a much lower concentration of delta-9 tetrahydrocannabinol (THC), the psychoactive chemical found in marijuana that produces a “high” when consumed.1 The World Health Organization’s Expert Committee on Drug Dependence discussed CBD during its 39th meeting, which took place in November 2017 in Geneva, Switzerland. The World Health Organization reported that when consumed by humans, pure CBD does not exhibit the effects indicative of abuse, dependence potential, or any public health-related problems.2 Although pure CBD does not produce a “high” or cause dependence in users, the legal status of CBD in the United States has been mired in an intricate web of regulatory and legal considerations that are worthy of examination.

8.2  POTENTIAL FOR NUTRACEUTICAL BENEFIT OF CANNABIDIOL Groundbreaking work by Dr. Ralph Mechoulam and his colleagues in 1963 discovered and defined the chemical structure of CBD (Figure 8.1), followed by the chemical structure of THC a year later.3 Alynn Howlett further defined the activation of delta-9-tetrahydrocannabinol on a specific cannabinoid receptor, CB1, found in areas of the brain involved in movement, stress, and cognitive function.4 These findings, in addition to identification of specific G-protein-coupled receptors for THC,5 and the discovery of two endogenous cannabinoids, arachidonoyl-ethanolamide (AEA), referred to as anandamide and arachidonoyl glyceride (2-AG),4–6 laid the substantial foundation for the discovery of the endocannabinoid system (ECS).7,8 The ECS was named after the Cannabis sativa plant and psychoactive ingredient THC that led to its discovery. This biological system is involved 147

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FIGURE 8.1  Cannabidiol (CBD).

in homeostatic and physiologic functions.9 The ECS has also been shown to play an important role in CNS development, neuroplasticity, and neuroprotective properties.10–12 THC has a higher binding affinity for CB1 receptors, which are found in more abundance in the CNS in, comparison to CB2 receptors, found throughout the peripheral nervous system (PNS).13,14 CB2 receptors were originally found in spleen cells15 but were later identified throughout the PNS. Although there are some similarities in composition to CB1 receptors, CB2 receptors are thought to be more involved in the modulation of immune function and inflammation.16 Interestingly, CBD has been discovered to indirectly impact both CB1 and CB2 receptors, by modulating the affinity of other cannabinoids, including THC, and compounds binding to these receptor types 10. Understanding this mechanism of action may help us better comprehend the positive effects CBD appears to have on various physiological processes, discussed further in this chapter. Traditional synaptic signaling involves neurotransmitters being released from the presynaptic terminal, then diffusing to the postsynaptic terminal, where they bind and activate receptors. However, the CB1 receptor is theorized to use a less common form of signaling, called retrograde signaling.12 Retrograde signaling occurs when a diffusible messenger is released from the postsynaptic terminal and travels “backward” across the synaptic cleft, where it activates receptors on the presynaptic cell (Figure 8.2). Several studies of both the endocannabinoid system and endogenous cannabinoids have revealed their involvement in numerous physiological processes, including appetite,17 pain sensation, control of chronic pain,18–20 and regulation of immune cell functions.21 Endocannabinoid compounds, such as cannabidiol, have been shown to modulate various disease pathology and movement disorders.22–24

FIGURE 8.2  Anandamide mediates retrograde synaptic signaling via cannabinoid receptors

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There is little research available on the direct effects of CBD supplementation in conjunction with exercise in human subjects; however, it has been suggested that the endocannabinoid system partakes in adaptive responses to exercise. One theory of this adaptation is evident by the activation of the endogenous cannabinoid, anandamide, during exercise.25 Anandamide acts as a vasodilator, leading to hypertension and facilitating blood flow. Additionally, studies have demonstrated that both endocannabinoids and exogenous cannabinoids can act as bronchodilators,26 affecting the respiratory system and therefore possibly facilitating breathing during exercise. The degree to which endocannabinoids and phytocannabinoidsncrease regenerative properties, such as “healthy bone, tendon, ligament, muscular and connective tissue integrity27 is still being researched However, Dr. Hector Lopez theorizes how hemp-derived CBD products may help balance and optimize ECS physiology27. The addition of a CBD extract product may reduce anxiety levels, increase quality sleep, and contribute to an optimized diet. Numerous studies have demonstrated both anti-inflammatory and analgesic properties of CBD. CBD has also been shown to have antioxidant properties and neuroprotective qualities. A study published by the American Medical Association (2017) found that 99% of deceased NFL players were found to have chronic traumatic encephalopathy (CTE)29. CTE is a neurodegenerative disorder characterized by a buildup of abnormal tau protein manifesting in memory loss, aggression, depression, and impaired judgment. Research suggests that the neuroprotective effects of CBD may offer a preventative measure from concussions resulting from high-impact sports and exercise. Although the possible neuroprotective effects from CBD are intriguing, the anti-inflammatory effects may be another catalyst for the increase in popularity among athletes. Reduced inflammation in muscle and tissue following intense training and exercise, as well as reduced anxiety, may contribute to faster healing times and reduced performance anxiety during competitions. Although further studies are needed to better understand the impact CBD can have on both pre- and post-workout routines, there has been clear shift in the attitude toward CBD in sports. The World Anti-Doping Agency (WADA) officially removed CBD from its 2018 list of prohibited substances, although THC remains a prohibited substance.

8.3  ANALYTICAL METHODS FOR HEMP AND CANNABIDIOL Through the US Hemp Authority, the need to have relevant and defined testing regulations was voiced by the industry and made a priority. Numerous members of the industry have come together through these and other trade organizations, such as the Hemp Industries Association, National Hemp Association, and American Herbal Products Association, in an effort to assist in the writing of these self-regulation standards, until clear direction is developed at both the state and federal levels. The US Hemp Round Table’s mission to “Join citizens from across government, the agricultural industry, U.S. manufacturers, the small business community, and beyond, to support legislation (Senate Bill 2667 and House Bill 5485) that would establish hemp as an agricultural commodity, removing it completely and permanently from the purview of the Controlled Substances Act.” In addition, the US Hemp Authority, which developed “stringent self-regulatory standards and comprehensive guidance for hemp growers and processors,” is a part of the hemp industry’s objective to provide high standards, best practices, and self-regulation, giving confidence to consumers and law enforcement that hemp products are safe and legal. CBD regulations continue to evolve at the state and federal level; meanwhile, third-party analytical testing laboratories are attempting to comply with these changes. This is accomplished by monitoring the state regulations passed and by closely setting internal testing standards to mimic dietary supplement testing and regulations that are more clearly defined by the FDA. For example, in July 2018, the State of Indiana passed a regulation, Engrossed Senate Bill No. 52 (ESB52), that all industrial hemp-derived CBD products must have a certificate of analysis from an ISO 17035:2005 accredited third-party analytical laboratory for the compound THC. This regulation was written to ensure compliance with the federal definition of hemp. Following this regulation, laboratories that

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wished to be in compliance for their customers selling CBD products had to get their cannabinoid potency methods ISO accredited by an approved accreditor, suggested in ESB52. In addition to state testing regulations, there are very explicit labeling regulations that affect all products sold in CA listed under the Safe Drinking Water and Toxic Enforcement Act, better known as Prop 65, that CBD product retailers and manufacturers must also strictly follow. Although the legal status of CBD as a dietary ingredient is still not on solid ground, new hemp companies and numerous “mainstream” food and dietary supplement companies are formulating new products with hemp extracts and launching these products on the US market. In response to these new products, globally recognized standard-developing organizations, such as AOAC International and ASTM, have launched programs to ensure the hemp and cannabis industries will have access to developed and published technical standards for test methods, materials, processes, and other consensus standards. The following excerpt written by Johnathan S Miller and Nolan M Jackson, of Frost Brown Todd LLC, discusses the confusing landscape of industrial hemp-derived products. They explain how federal statements regarding CBD products have increased uncertainty of the legal status of those products. “While the Farm Bill and Omnibus Law provide protections for the sales of hemp-derived CBD—especially when the hemp is grown as part of a state-authorized pilot program—some federal agencies objected, and began to issue non-legally-binding statements that suggested that CBD was not permitted for sale under federal law. Most concerning to the hemp industry, the FDA concluded that CBD could not be marketed as a dietary supplement, and that the interstate sale of food products containing CBD was not legal. However, the FDA has left the question open to further input from the industry and did not signal that any enforcement actions were imminent.”28

8.4  THE FARM BILL When presented with the question, “Is CBD legal?” most supporters cite the Agricultural Improvement Act of 2018 (the 2018 Farm Bill) as the federal act that gives CBD the “green light” to be sold in the United States so long as the CBD comes from hemp. Although this argument “sounds good,” we must examine the text of the 2018 Farm Bill to determine what, in fact, it authorizes and what it does not. “Farm Bills” are typically passed about every 5 years to create or reauthorize certain federal programs. The Agriculture Act of 2014 (the 2014 Farm Bill) was signed into law by President Obama on February 7, 2014, and included Section 7606, entitled “Legitimacy of Industrial Hemp Research.”30 This was the first step along the journey of legalizing the growing and cultivating of hemp within the United States. Section 7606 of the 2014 Farm Bill allowed for an institution of higher education or a state department of agriculture to grow or cultivate industrial hemp if: “1. the industrial hemp [was] grown or cultivated for purposes of research conducted under an agricultural pilot program or other agricultural or academic research; and 2. the growing or cultivating of industrial hemp [was] allowed under the laws of the state in which such institution of higher education or state department of agriculture [was] located and such research occur[ed].”30 The 2014 Farm Bill defined “industrial hemp” as “the plant Cannabis sativa L. and any part of such plant, whether growing or not,” with a THC concentration of “not more than 0.3% on a dry weight basis.”30 Under the 2014 Farm Bill, if the legal requirements of Section 7606 were met, then growing and cultivating industrial hemp was permitted. As can be seen from the plain language of the Act, industrial hemp was only permitted to be grown by an institution of higher education or state department of agriculture for purposes of research. While the 2014 Farm Bill was a historical piece of legislature, the Act did not allow simply anyone who wanted to grow or cultivate industrial hemp to do so. Due to the increased media attention and general confusion surrounding the sale of CBD, specifically the product sold by CW Hemp called Charlotte’s Web, the DEA released a statement to The Cannabist on CBD, hemp, and the 2014 Farm Bill. The DEA stated, “It is important to correct a misconception that some have about the effect of the Agricultural Act of 2014 (which some refer to as the ‘farm bill’) on the legal status of ‘Charlotte’s Web’/CBD oil. Section 7606 of the Agricultural

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Act of 2014 authorizes institutions of higher education (e.g., universities) and state Departments of Agriculture to grow and cultivate ‘industrial hemp’ (defined under the Act as marijuana with a THC content of 0.3% or less) for agricultural research purposes where permitted under state law. However, the Agricultural Act of 2014 does not permit such entities, or anyone else, to produce nonFDA-approved drug products made from cannabis. Thus, the CSA and FDCA restrictions sic remain in effect with respect to the production of ‘Charlotte’s Web’/CBD oil for human consumption.”31 Contrary to widespread belief among those in the marijuana and hemp industries, the 2014 Farm Bill did not give the “green light” to those who sought to sell CBD derived from hemp for commercial purposes. On December 20, 2019, President Trump signed the Agriculture Improvement Act of 2018 (the 2018 Farm Bill).32 Senate Majority Leader Mitch McConnell of Kentucky introduced the Hemp Farming Act, a standalone bill that sought to legalize hemp in April of 2018. The provisions of that bill were later included in the Farm Bill of 2018.33 There are several differences between the 2014 Farm Bill and the 2018 Farm Bill. The first major difference is the way that “hemp” is defined. As explained above, the 2014 Farm Bill allowed for the growing and cultivation, subject to limitations, of “industrial hemp,” which it defined as “the plant Cannabis sativa L. and any part of such plant, whether growing or not, with a delta-9 tetrahydrocannabinol concentration of not more than 0.3% on a dry weight basis.”30 The 2018 Farm Bill, on the other hand, more broadly defines the term “hemp” as “the plant Cannabis sativa L. and any part of that plant, including the seeds thereof and all derivatives, extracts, cannabinoids, isomers, acids, salts, and salts of isomers, whether growing or not, with a delta-9 tetrahydrocannabinol concentration of not more than 0.3% on a dry weight basis.”32 Notably, the 2014 Farm Bill used the term “industrial hemp,” while the 2018 Farm Bill simply states “hemp.” Additionally, the 2018 Farm Bill includes within its definition of hemp the specific chemical constituents of the plant, including CBD (Figure 8.3).32 The second, and arguably most impactful, distinction between the 2014 Farm Bill and the 2018 Farm Bill is that the 2018 version amends the CSA.32 The very first line of the 2014 Farm Bill stated, “Notwithstanding the Controlled Substances Act …”30 As a result, under the 2014 Farm Bill, the only parts of the hemp plant that were not considered a controlled substance were those parts that were specifically excluded as a controlled substance from the CSA—“the mature stalks of such plant, fiber produced from such stalks, oil or cake made from the seeds of such plant, any other compound, manufacture, salt, derivative, mixture, or preparation of such mature stalks (except the resin extracted therefrom), fiber, oil, or cake, or the sterilized seed of such plant which is incapable of germination.”34 This meant that all chemical constituents and extracts of hemp, including CBD, were Schedule 1 controlled substances. The 2018 Farm Bill specifically amends the CSA to exempt “hemp” from the definition of “marihuana” and, as a result, “hemp” is no longer a controlled substance.32 That amendment removes hemp, including CBD derived from hemp, as defined in the 2018 Farm Bill, from the oversight authority of the Drug Enforcement Administration (DEA). Furthermore, the 2018 Farm Bill removes the THC contained in hemp (not more than 0.3%

FIGURE 8.3  Farm Bill–driven separation between marijuana and hemp.

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on a dry weight basis) from the CSA.32 However, at this time, marijuana is still federally illegal; thus, CBD and THC that are derived from the marijuana plant are still Schedule I controlled substances. The third most significant change is how the two versions differ with respect to who is permitted to grow and cultivate hemp. The 2014 Farm Bill specifically stated that “an institution of higher education or a State department of agriculture may grow or cultivate industrial hemp if—(1) the industrial hemp [was] grown or cultivated for purposes of research conducted under an agricultural pilot program or other agricultural or academic research; and (2) the growing or cultivating of industrial hemp [was] allowed under the laws of the State in which such institution of high education or State department of agriculture [was] located and such research occurs.”30 The 2018 Farm Bill removes the restriction on growing and cultivating “for purposes of research” and removes the ability of the individual states to restrict the growing and cultivation of hemp. The 2018 Farm Bill provides that if a state or Native American tribe wants to have primary regulatory authority over the production of hemp in their State or Tribal territory, they must submit a plan to the Secretary of Agriculture, under which the State or tribe will monitor and regulate the production.32 The Secretary has 60 days following the receipt of a State or Tribal plan to either approve or disapprove its plan.32 If a State or Tribal government does not submit a plan, or their plan is rejected by the Secretary, the production of hemp in that State or tribal territory will be subject to a plan established by the Secretary.32 Accordingly, the 2018 Farm Bill makes the growing and cultivation of hemp legal in all 50 US states and Native American territories.

8.5 THE FOOD AND DRUG ADMINISTRATION AND THE FOOD, DRUG, AND COSMETIC ACT If a product is composed of or contains CBD that was extracted from hemp grown pursuant to the 2018 Farm Bill, can that product be lawfully marketed and sold as a dietary supplement? In order to answer that question, we must analyze the regulatory framework of the Food and Drug Administration. The FDA regulates foods, drugs, and cosmetics, among other consumer goods, under the Food Drug and Cosmetic Act (FDCA). Currently, President Donald Trump is seeking to consolidate federal food safety under a single agency under the US Department of Agriculture. President Barack Obama also sought to consolidate food safety; however, Congress would not extend President Obama the power to do so. If the Trump Administration’s plan ultimately comes to fruition, food safety would be removed from the FDA’s jurisdiction and the FDA would be renamed the “Federal Drug Administration.” Whether or not this change occurs, dietary supplements will continue to be regulated by the FDA. Congress passed the Dietary Supplement Health and Education Act of 1994, which was signed by President Bill Clinton in October of 1994.35 Although still classifying dietary supplements as “food” under the FDCA, DSHEA established a clearer and more practical framework for the regulation of dietary supplements. Among other things, DSHEA created a legal definition for a “dietary supplement” as a product (other than tobacco) intended to supplement the diet that contains one or more “dietary ingredients.”35 By definition, “dietary ingredients” in these products may include vitamins, minerals, herbs or other botanicals, amino acids, and dietary substances for use by man to supplement the diet by increasing the total dietary intake.35 Dietary ingredients can also include extracts, metabolites, or concentrates of the preceding substances.35 As explained above, both hemp and marijuana come from the plant Cannabis sativa L. and therefore fit under the DSHEA definition of botanicals. Because dietary ingredients also include “extracts, metabolites, or concentrates” of botanicals, products containing CBD, which has been extracted from the cannabis plant, would also fall within the general definition of a dietary supplement. Although CBD appears to qualify as a dietary supplement based on the general definitions set forth above, other provisions of the FDCA and DSHEA must be examined to determine the regulatory status of CBD as a dietary supplement. In addition to the categories of dietary

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ingredients, DSHEA also distinguishes between those ingredients marketed and sold prior to the passage of DSHEA and those marketed and sold after DSHEA. A “new dietary ingredient”(NDI) is defined as a dietary ingredient that was not marketed in the United States before October 15, 1994.35 A product containing an NDI is deemed adulterated and subject to FDA enforcement sanctions unless it meets one of two exemption criteria: either (1) the supplement in question contains “only dietary ingredients which have been present in the food supply as an article used for food in a form in which the food has not been chemically altered”, or (2) there is a “history of use or other evidence of safety” provided by the manufacturer or distributor to the FDA at least 75 days before introducing the product into interstate commerce.35 CBD that has been isolated from hemp, or hemp oil containing amounts of CBD in excess of those naturally contained in the plant, would not meet the first exemption under DSHEA because the “form has been chemically altered.” Certain companies have chosen to try to limit their exposure to risk by selling products containing “FullSpectrum Hemp Extract.” The reasoning behind this is that “Full-Spectrum Hemp Extract” is purportedly extracted from hemp in a form that has not been chemically altered. On December 20, 2018 (the same date the 2018 Farm Bill was signed by President Trump), the FDA completed its evaluation of three Generally Recognized as Safe notices for food ingredients derived from hemp seed.36 The FDA reviewed the GRAS submissions and had no questions regarding the company’s conclusion that the ingredients are GRAS under their intended conditions of use.36 While this is a great step forward, it is important to note that the hemp ingredients contain only trace amounts of THC and CBD, which the seeds might pick up through contact with other parts of the plant during the harvesting and processing.36 At the time of this writing, no GRAS notice has been accepted by the FDA for hemp ingredients that have been derived from parts of the hemp plant that have more than a trace level of CBD. As a result, in order to market and sell CBD as a dietary supplement, the manufacturer or distributor would need to submit an NDI notification to FDA demonstrating the safety of CBD.35 That information would include: (1) the name of the new dietary ingredient and the Latin binomial name, and (2) a description of the dietary supplement that contains the new dietary ingredient, including (a) the level of the new dietary ingredient in the product, (b) conditions of use of the product stated in the labeling, or if no conditions of use are stated, the ordinary conditions of use, and (c) a history of use or other evidence of safety establishing that the dietary ingredient, when used under the conditions recommended or suggested in the labeling of the dietary supplement, is reasonably expected to be safe.35 Assuming that the manufacturer or distributor of a CBD supplement submitted an NDI notification to the FDA and sufficiently demonstrated the safety of the product, would that NDI notification ultimately be successful? At the current time, the answer is most likely no. In addition to the distinction between “old” and “new” dietary ingredients, DSHEA also defines what a dietary supplement does not include. DSHEA states that a dietary supplement does not include: an article that is approved as a new drug under section 505, certified as an antibiotic under section 507, or licensed as a biologic under section 351 of the Public Health Service Act (42 U.S.C. 262), or an article authorized for investigation as a new drug, an article authorized for investigation as a new drug, antibiotic, or biological for which substantial clinical investigations have been instituted and for which the existence of such investigations has been made public, which was not before such approval, certification, licensing, or authorization marketed as a dietary supplement or as a food unless the Secretary, in the Secretary’s discretion, has issued a regulation, after notice and comment, finding that the article would be lawful under this Act.35

In 2007, GW Pharmaceuticals began conducting clinical investigations on its CBD drug Epidiolex. On June 25, 2018, the FDA approved Epidiolex for the treatment of seizures associated with LennoxGastaut syndrome and Dravet syndrome for patients 2 years of age and older.37 This means that,

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under DSHEA, in order for CBD to potentially be considered a dietary supplement, CBD would have had to have been legally marketed as a dietary supplement or food prior to GW Pharmaceuticals’ clinical investigations in 2007. On the FDA website, there is a page entitled, “FDA and Marijuana: Questions and Answers.”38 Question 12 on this website asks, “Can products that contain THC or cannabidiol (CBD) be sold as dietary supplements?” In its answer, the FDA concludes that products containing CBD cannot be sold as dietary supplements because there is no evidence that CBD was lawfully marketed as a dietary supplement or food before the new drug investigation for CBD was authorized. The FDA does state that interested parties can present the agency with evidence that they believe has bearing on the issue, but “[FDA’s] continuing review of information that has been submitted thus far has not called [FDA’s] conclusions into question.”38 Based on the FDA’s position and subsequent approval of CBD as a pharmaceutical drug, it appears that unless the industry can come forth with evidence to support that CBD was lawfully marketed as a dietary supplement or food prior to 2007, CBD sold as a dietary supplement will continue to be an adulterated or misbranded drug and subject to enforcement. The Hemp Industries Association has publicly stated that it provided FDA with evidence that hemp extract and CBD was marketed prior to 2007, but the FDA has not offered its opinion on such. Aside from the NDI notification and GRAS determinations explained above, there is another potential way for CBD to be a permissible dietary ingredient and supplement. The FDA is a regulatory agency within the US Department of Health and Human Services (HHS). The Secretary of HHS has the authority under the FDCA to issue a regulation, following notice and comment, finding CBD lawful in dietary supplements.35 The FDA released a statement on December 20, 2018 (the date the 2018 Farm Bill was signed), offering some potential hope for the future of CBD as a dietary supplement.39 In the press release, the current FDA Commissioner, Scott Gottlieb, M.D., stated, “pathways remain available for the FDA to consider whether there are circumstances in which certain cannabis-derived compounds might be permitted in a food or dietary supplement. Although such products are generally prohibited to be introduced in interstate commerce, the FDA has authority to issue a regulation allowing the use of a pharmaceutical ingredient in a food or dietary supplement. We are taking new steps to evaluate whether we should pursue such a process.”39 Prior to this statement, the FDA’s position on CBD as a dietary supplement seemed impervious to change. While there has been no change to the FDA’s formal position on the legality of CBD as a dietary supplement, this statement offers a glimmer of hope for the future of CBD as a supplement. Putting aside for the moment the various hurdles facing CBD as a dietary ingredient, it is important to also understand that dietary supplements can never be marketed to diagnose, treat, cure, or prevent any disease state. Multiple companies that sell products that are, or contain, CBD have received warning letters from the FDA for making “disease claims” related to their CBD supplements. The FDA defines a disease as, “damage to an organ, part, structure or system of the body such that it does not function properly, or a state of health leading to such dysfunctioning.”35 Many companies selling CBD as dietary supplements tout the benefits of CBD on conditions like cancer, anxiety, dementia, arthritis, epilepsy, and inflammation. Based on the FDA’s definition of a disease, these types of claims about a dietary supplement cause that product to be deemed a misbranded or adulterated drug. These types of claims are “low-hanging fruit” for the FDA because rather than arguing over the regulatory status of CBD as a dietary supplement, the claims alone cause these CBD products to be considered adulterated and misbranded drugs under the FDA definition, subjecting the marketers to enforcement actions. Further, when companies make these disease claims, it only serves to increase the level of scrutiny surrounding the CBD market. In addition to the FDA, the Federal Trade Commission regulates the marketing and advertising of dietary supplements. Companies that make claims about their CBD products that are not substantiated risk enforcement actions from the FTC. In addition to the warning letters sent to companies making disease claims, the FDA has also issued warning letters because certain products containing CBD also contained high levels of THC.

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8.6  FEDERAL LEGISLATION ON THE HORIZON On June 27, 2018, Senate Democratic Leader Chuck Schumer introduced the Marijuana Freedom and Opportunity Act cosponsored by Senators Bernie Sanders, Tim Kaine, and Tammy Duckworth.40 The Marijuana Freedom and Opportunity Act would remove marijuana from the list of controlled substances under the CSA and allow states to decide how to treat marijuana possession. Although not directly mentioning CBD, by removing marijuana from the list of controlled substances, it would effectively remove marijuana extract from the CSA as well. This is due to the fact that the scheduling of marijuana extract is dependent upon marijuana itself being a Schedule I controlled substance. If ultimately signed into law, this Act would remove CBD derived from marijuana from the CSA and DEA jurisdiction—a significant win for CBD and the marijuana industry as a whole.

8.7  THE FUTURE OF CANNABIDIOL Based on the laws and regulations as they currently exist in the United States, the FDA considers CBD to bean adulterated and misbranded drug if labeled and sold as a dietary supplement. Despite this, the CBD market continues to grow and expand, with individual states legalizing marijuana for medical and recreational purposes and creating state-specific CBD laws. Further, with the current “opioid epidemic” in the United States, it appears that the FDA and DEA have “bigger fish to fry” when determining the proper allocation of resources. While companies selling CBD are currently choosing to accept the risk of enforcement, the Farm Bill of 2018 removed hemp from the CSA, allowing farmers to grow and cultivate hemp for commercial purposes and taking enforcement away from the DEA. Additionally, if signed into law, the Marijuana Freedom and Opportunity Act would legalize marijuana and remove marijuana-derived CBD from the list of controlled substances where it is currently a Schedule I drug as marijuana extract. While all of these developments would benefit the marijuana and hemp industry, the regulatory status of CBD as a dietary supplement ultimately comes down to the FDA. While the FDA’s position on CBD remains that it is not a legal dietary supplement, the comments by former Commissioner Scott Gottlieb and the FDA’s actions related to the three GRAS notifications led to speculation suggesting a future reconsideration of the FDA’s position. On May 31, 2019, the FDA held a public hearing on CBD with over 100 speakers and 10 hours of testimony. The purpose of the hearing was to obtain scientific data and information about the safety, manufacturing, marketing, labeling, quality, and sale of products containing cannabis or cannabis-derived compounds. The FDA began the hearing by expressing safety concerns regarding side effects, drug interactions, dosing, and adolescent use. While those concerns may not have been fully addressed at that time, the hearing was a step forward in assessing how the FDA might establish a pathway to regulate CBD products.

REFERENCES 1. National Conference of State Legislatures (Last updated 2019, Nov. 1) State Industrial Hemp Statutes. http://www.ncsl.org/research/agriculture-and-rural-development/state-industrial-hemp-statutes.aspx 2. WHO Expert Committee on Drug Dependence: Thirty-ninth report. Geneva: World Health Organization; 2017 (WHO Pre-Review Report; Agenda Item 5.2). http://www.who.int/medicines/access/controlledsubstances/5.2_CBD.pdf 3. Gaoni, Y. and Mechoulam, R. 1964. Isolation, structure, and partial synthesis of an active constituent of hashish. J Am Chem Soc, 86, 1646–1647. 4. Mechoulam, R. and Shvo, Y. 1963. Hashish-I: The structure of cannabidiol. Tetrahedron, 19, 2073–2078. 5. Devane, W.A., Dysarz, F.A., Johnson, M.R., Melvin, L.S., and Howlett, A.C. 1988. Determination and characterization of a cannabinoid receptor in rat brain. Mol Pharmacol, 34, 605–613. 6. Matsuda, L.A., Lolait, S.J., Brownstein, M.J., Young, A.C., and Bonner, T.I. 1990. Structure of a cannabinoid receptor and functional expression of the clones cDNA. Nature, 346, 561–564. 7. Devane, W.A., Hanus, L., Breuer, A. et al. 1992. Isolation and structure of a brain constituent that bind to the cannabinoid receptor. Science, 258, 1946–1949.

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8. Mechoulam, R., Ben-Shabat, S., Hanus, L. et al. 1995. Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem Pharmacol, 50, 83–90. 9. Sugiura, T., Kondo, S., Sukagawa, A. et al. 1995. 2-Arachidonoylglycerol: A possible endogenous cannabinoid receptor ligand in brain. Biochem Biophys Res Commun, 215, 89–97. 10. Fine, P.G. and Rosenfeld, M.J. 2013. The endocannabinoid system, cannabinoids, and pain. Rambam Maimonides Med J, 4, e0022. 11. Alvarez, F.J., Lafuente, H., Rey-Santano, M.C. et al. 2008. Neuroprotective effects of the nonpsychoactive cannabinoid cannabidiol in hypoxic-ischemic newborn piglets. Pediatr Res, 64, 653–658. 12. Lu, H. and Mackie, K. 2016. An introduction to the endogenous cannabinoid system. Biol Psychiatry, 79, 516–525. 13. Alger, B.E. 2002. Retrograde signaling in the regulation of synaptic transmission: Focus on endocannabinoids. Prog Neurobiol, 68, 247–286. 14. Hanuš, L., Breuer, A., Tchilibon, S. et  al. 1999. HU-308: A specific agonist for CB2, a peripheral cannabinoid receptor. Proc Natl Acad Sci, 96, 14228–14233. 15. Pertwee, R.G. 1997. Pharmacology of cannabinoid CB1 and CBD2 receptors. Pharmacol Ther, 74, 129–180. 16. Munro, S., Thomas, K.L., and Abu-Shaar, M. 1993. Molecular characterization of a peripheral receptor for cannabinoids. Nature, 365, 61–65. 17. Hulsebosch, C.E. 2012. Special issue on microglia and chronic pain. Exp Neurol, 234, 253–254. 18. Dietrich, A. and McDaniel, W.F. 2004. Endocannabinoids and exercise. Br J Sports Med, 38, 536–541. 19. Donvito, G., Nass, S.R., Wilkerson, J.L. et al. 2018. The endogenous cannabinoid system: A budding source of targets for treating inflammation and neuropathic pain. Neuropsychopharmacology, 43, 52–79. 20. Woodhams, S.G., Chapman, V., Finn, D.P., Hohmann, A.G., and Neugebauer, V. 2017. The cannabinoid system and pain. Neuropharmacology, 124, 105–120. 21. Basu, S. and Dittel, B. 2011. Unraveling the complexities of cannabinoid receptor 2 (CB2) immune regulation in health and disease. Immunol Res, 51, 26–38. 22. Johnson, J.R., Burnell-Nugent, M., Lossignol, D., Ganae-Motan, E.D., Potts, R., and Fallon, M.T. 2010. Multicenter, double-blind, randomized, placebo-controlled, parallel-group study of the efficacy, safety, and tolerability of THC:CBD extract and THC extract in patients with intractable cancer-related pain. J Pain Symptom Manage, 39, 167–179. 23. Richardson, D., Pearson, R.G., Kurian, N. et al. 2008 Characterization of the cannabinoid receptor system in synovial tissue and fluid in patients with osteoarthritis and rheumatoid arthritis. Arthritis Res Ther, 10, R43. 24. Valastro, C., Campanile, D., Marinaro, M. et al. 2017. Characterization of endocannabinoids and related acylethanolamides in the synovial fluid of dogs with osteoarthritis: A pilot study. BMC Vet Res, 6, 309. 25. Dietrich, A. and McDaniel, W.F. 2004. Endocannabinoids and exercise. Br J Sports Med, 28, 526–541. 26. Calignano, I., Katona, F., Desarnaud, A. et al. 2000. Bidirectional control of airway responsiveness by endogenous cannabinoids. Nature, 408, 96–101. 27. Lopez, H. 2018, September 5. Potential Role of Hemp-Derived Full-Spectrum CBD Oil in Rehabilitation and Physical Therapy. http://rehab-insider.advanceweb.com/potential-role-of-hemp-derivedfull-spectrum-cbd-oil-in-rehabilitation-and-physical-therapy/ 28. Miller, J.S. and Jackson, N.M. 2017. The evolving law and regulation of industrial hemp in the United States. Louis D. Brandeis School of Law University of Louisville, 8, 12–37. 29. Mez, J., Daneshvar, DH., Kiernan, PT. et al. 2017. Clinicopathological Evaluation of Chronic Traumatic Encephalopathy in Players of American Football. JAMA, 318(4):360–370. doi:10.1001/jama.2017.8334 30. Public Law 113–79: Agricultural Act of 2014. https://docs.wixstatic.com/ugd/89d621_884253959e9541 6aa5973114084f10b0.pdf 31. Wallace, A. 2017, July 5. In the DEA’s words: Agency Stance on CBD, Hemp Products and the Farm Bill. The Cannabist. https://www.thecannabist.co/2017/07/05/dea-statement-cbd-hemp-farmbill-controlled-substances-act/83100/ 32. Public Law 115-334: Agriculture Improvement Act of 2018. https://www.agriculture.senate.gov/imo/ media/doc/Agriculture%20Improvement%20Act%20of%202018.pdf 33. Angell, T. 2018, June 28. U.S. Senate Votes to Legalize Hemp after Decades-Long Ban Under Marijuana Prohibition. Forbes. https://www.forbes.com/sites/tomangell/2018/06/28/u-s-senate-votes-to-legalizehemp-after-decades-long-ban-under-marijuana-prohibition/#1ec754b5418a 34. Controlled Substances Act, 21 U.S.C. 812(c). 35. Dietary Supplement Health and Education Act, 21 U.S.C.A. Ch. 9.

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36. U.S. Food & Drug Administration. 2018, Dec. 20. FDA Responds to Three GRAS Notices for Hemp Seed-Derived Ingredients for Use in Human Food. https://www.fda.gov/Food/NewsEvents/ ConstituentUpdates/ucm628910.htm 37. U.S. Food & Drug Administration. 2018, June 25. FDA Approves First Drug Comprised of an Active Ingredient Derived from Marijuana to Treat Rare, Severe Forms of Epilepsy. https://www.fda.gov/ NewsEvents/Newsroom/PressAnnouncements/ucm611046.htm 38. U.S. Food & Drug Administration (Last updated 2018, Dec. 20) FDA and Marijuana: Questions and Answers. https://www.fda.gov/NewsEvents/PublicHealthFocus/ucm421168.htm#dietary_supplements 39. U.S. Food & Drug Administration. 2018, Dec. 20. Statement from FDA Commissioner Scott Gottlieb, M.D., on Signing of the Agriculture Improvement Act and the Agency’s Regulation of Products Containing Cannabis and Cannabis-Derived Compounds. https://www.fda.gov/NewsEvents/Newsroom/ PressAnnouncements/ucm628988.htm 40. Senate Democrats. 2018, June 27. Schumer Introduces Marijuana Freedom and Opportunity Act— New Legislation Would Decriminalize Marijuana at Federal Level. https://www.democrats.senate. gov/newsroom/press-releases/schumer-introduces-marijuana-freedom-and-opportunity-act_-newlegislation-would-decriminalize-marijuana-at-federal-level

9

Coffee as a Functional Beverage Victoria Burgess, Lem Taylor, and Jose Antonio

CONTENTS 9.1 Introduction........................................................................................................................... 159 9.2 Introduction to Coffee and Caffeine...................................................................................... 159 9.3 Doses of Caffeine.................................................................................................................. 160 9.4 Coffee and Caffeine in Weight Loss and Energy Expenditure............................................. 160 9.5 Effects of Coffee (Caffeine) in the Brain and Body.............................................................. 161 9.6 Exercise Performance with Coffee and Caffeine Consumption............................................ 162 9.7 Caffeine Consumption Timing for Performance................................................................... 164 9.8 Health-Related Issues in Coffee Consumption...................................................................... 164 9.8.1 Blood Pressure........................................................................................................... 164 9.8.2 Cardiovascular Disease............................................................................................. 165 9.8.3 Diabetes..................................................................................................................... 165 9.8.4 Cancer........................................................................................................................ 166 9.9 Conclusion and Closing Remarks.......................................................................................... 166 References....................................................................................................................................... 168

9.1 INTRODUCTION Caffeine is the most commonly consumed drug in the world and has been used for centuries for a variety of reasons. Caffeine is a common substance in the diets of a variety of individuals ranging from athletes to elderly. Today, caffeine can be found in numerous products such as sports gels, energy drinks, and alcoholic beverages.1 Coffee is likely to be the primary delivery system for caffeine today. For most people, caffeine and coffee are synonymous, but these two should not be thought of as the same. There are other ingredients in coffee besides caffeine that exert a biological effect. Caffeine can have many effects in the body, but typically caffeine is thought of as a way to boost a person’s energy level on both a psychomotor level (i.e., awareness) as well as a physiological level (i.e., energy), which is clearly a role that caffeine can play.2 These two factors alone are probably the sole reason that many people consume coffee as part of their daily ritual, and this aspect of coffee consumption is very important. The following chapter will discuss the background of coffee and its role as a functional food. This discussion will include types of coffee, the ingredients in coffee, the effects of coffee on energy metabolism, and its role as a drink that can enhance various aspects of health and possibly prevent or reduce the risk of some diseases. The effects of caffeine on various diseases and health conditions need to be discussed due to the fact that caffeine is the active ingredient in coffee in most preparations.

9.2  INTRODUCTION TO COFFEE AND CAFFEINE The popularity of coffee has increased dramatically over the last decade. Drinking coffee is a ritual that suits a variety of situations, from jump-starting your day to an aspect of social engagement. Traditionally, caffeine is typically associated with coffee consumption, and this is probably the most popular form of caffeine in the U.S. today. There are many different types of coffee, and they usually differ on factors 159

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TABLE 9.1 Typical Caffeine Content of Several Coffee Products Double espresso (2 oz) Drip-brewed coffee (8 oz) Instant coffee (8 oz) Decaf coffee (8 oz) Tea, black (8 oz) Tea, green (8 oz) Tea, white (8 oz) Coffee energy drinks (8.3 oz) Ben and Jerry’s coffee fudge frozen yogurt (8 oz)

55–100 mg 80–130 mg 70–95 mg 1–5 mg 45 mg 20–30 mg 15 mg 50–160 mg 85 mg

such as taste or flavor, type of preparation, and the caffeine content of various types. Obviously, the brand of the product and the flavor usually have something to do with the content of caffeine, but most caffeine in food products typically contains chocolate or coffee flavoring. Caffeine content can range from a couple of milligrams in an ounce of milk chocolate to ∼115 mg in 12 oz of a Red Bull energy drink. The most popular form of caffeine ingestion is via coffee, and the content typically depends on the method and duration of brewing; caffeine concentrations can range from 65 mg in 7 oz of instant coffee to 175 mg in 7 oz of drip-brewed coffee. Table 9.1 gives a comprehensive list of the caffeine contents of various consumer products.

9.3  DOSES OF CAFFEINE The caffeine content of coffee is one of its important aspects for many people when they drink coffee. Caffeine is a derivative of methylxanthine and is found in numerous consumer products in the U.S.3 The following section will address the topic of caffeine doses, particularly the doses used in research settings and/or the doses that are allowed to be used by athletes before “caffeine doping” is reached. All of these factors are important in considering how much caffeine one should ingest for both optimal performance and safety. Whether you are drinking coffee, tea, or taking caffeine pills, the amount of caffeine that an individual consumes is important to consider. Research has focused on varying levels of caffeine ingestion to determine optimal doses for different situations. In research trials, the most commonly used dose of caffeine is approximately 6 mg/kg of body weight, and this dose has been shown to give improved endurance exercise capacity and performance.3–6 Other doses have been used as well in research trials, with increases in performance still evident. Doses ranging from 1.5–2.9 mg/kg of body weight have been reported to increase performance.6 Alertness, mood, and cognitive processes have been found to improve after a low dose of caffeine (∼200 mg), and these findings support other evidence that caffeine can have an ergogenic effect at intakes as low as 1–3 mg/kg of body weight.7–11 On the other hand, doses as high as 13 mg/kg of body weight have been used in research, and have been found to reduce RPE during submaximal exercise and increase exercise performance.4, 5 Caffeine is not banned or restricted by the IOC.

9.4  COFFEE AND CAFFEINE IN WEIGHT LOSS AND ENERGY EXPENDITURE Like other stimulants, caffeine has been advertised and sold as a way to stimulate energy expenditure and weight loss. This potential effect on weight management is important to coffee’s role as a functional food. The fact that coffee is consumed by so many people and can be a potent dose of caffeine could indicate that daily coffee consumption can be important in augmenting energy expenditure and, as a corollary, weight loss. As discussed with caffeine’s role as an ergogenic aid in endurance exercise, caffeine can stimulate both lipolysis and energy expenditure.12 Many studies have been performed on the results of caffeine

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ingestion, with some examining caffeine alone, whereas others have examined caffeine combined with various herbal and vitamin products like ephedra, green tea extracts, calcium, tyrosine, chromium picolinate, capsaicin, garcinia cambogia, and so on. Caffeine has even been examined when combined with another popular stimulant in the U.S., cigarette smoking, in which caffeine was suggested to increase energy expenditure in an additive manner from the smoking stimulant.13 This has been stated to be due to the thermogenic effect of both nicotine and caffeine, and when combined, produces additive effects.14, 15 The method of administration has varied from coffee and/ or tea ingestion to administering caffeine-containing pills. The following section will discuss the pertinent research involving caffeine and possibly coffee’s effect on energy expenditure as well as possible weight loss that could result from this decreased energy state. Some of the original work on caffeine and energy expenditure came out of the American Journal of Clinical Nutrition in the late 1980s. Initial findings suggested that a single dose of 100 mg of caffeine had a significant effect on the resting metabolic rate (an increase of 3%–4% over 150 min) in a variety of populations. These findings led the authors to suggest that caffeine can have a significant effect on energy balance at a commonly consumed dose and possibly have positive effects in the treatment of obesity.13 Subsequent studies confirmed these findings, with one study reporting that caffeine ingestion increased energy expenditure by 7%, while also lowering plasma insulin and norepinephrine levels and increasing the appearance of free fatty acids in the blood.16 Koot and Deurenberg reported similar findings of a 7% increase in energy expenditure for 3 h following ingestion of 200 mg of caffeine, which was administered as coffee.17 Clearly, older studies have shown caffeine to have an effect on the metabolic rate of humans, and recent research has continued to back this notion. More recent research on the effects of caffeine continues to support its role in increasing energy expenditure. As mentioned earlier, caffeine is now being combined with a variety of products to promote a thermogenic effect. One example in the literature used the combination of capsaicin, catechins, caffeine, tyrosine, and calcium. This study reported an increase in energy expenditure of 2% over a 7-d period when these products were ingested as bioactive food products.18 Another recent study that looked at caffeine alone found an increase in energy expenditure of 13%, with the doubling of lipid turnover. These researchers concluded that the effects of caffeine alter energy expenditure and are mediated via the sympathetic nervous system. Furthermore, they explain the lipid mobilization action of caffeine in two ways: increased mobilization alone is insufficient to drive oxidation, or large increments in lipid turnover can result in an increase in lipid oxidation.13 This lipid turnover rate, however, largely depends on the individual’s body composition profile. The leaner individual has a higher oxidation after coffee consumption than one who is overweight.15 In recent years, it has become quite common and popular to consume green tea for weight loss and energy expenditure due to its two main components in the ingredients, caffeine and catechin. Clearly, caffeine does play a role in metabolism and energy expenditure. One can debate how much caffeine is necessary and the optimal time to consume it. One solution to this is to incorporate it into products that consumers use daily or at least regularly. Even coffee, which is consumed many times a day by millions of people, has now been modified by adding some of these products, plus additional caffeine. These products do have some credibility, and early research has found some functional coffee beverages to have significant effects on energy expenditure, body weight, and fat loss when compared to regular caffeinated coffee (Experimental Biology Meeting, 2006; Ron Mendel, Ph.D., personal communication). It has yet to be determined whether caffeine and the many products that contain significant amounts of it have long-term effects on energy balance. Despite this, the role of coffee as a functional food is intriguing because of the popularity of consumption on a broad scale.

9.5  EFFECTS OF COFFEE (CAFFEINE) IN THE BRAIN AND BODY Caffeine and caffeinated coffee can have a stimulatory effect on mental performance. This effect of consuming caffeine has been well documented. One study, in particular, suggested that consuming caffeinated beverages can maintain both cognitive and psychomotor performance throughout the day.19 Because coffee is a caffeinated beverage, these beneficial effects could be associated with

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daily coffee consumption. In fact, additional research has focused specifically on the effects of caffeinated vs. decaffeinated coffee on various cognitive function variables. The results of this study suggest that lifetime and current consumption of caffeinated coffee may be associated with better cognitive performance among women, especially in elderly populations.20 Further, caffeinated coffee consumption has been shown to decrease the risk of Alzheimer’s disease.21

9.6  EXERCISE PERFORMANCE WITH COFFEE AND CAFFEINE CONSUMPTION As we have discussed, caffeine is a popular drug all over the world as well as a frequently used ergogenic aid among athletes. There is substantial research that supports the fact that caffeine consumption can have beneficial effects on exercise performance. The following section will discuss the evidence to support caffeine’s role in exercise performance. Caffeine has been shown to improve performance and increase endurance during prolonged exercise, and in smaller amounts in shorter-term endurance performance.4, 22 This enhanced performance in endurance exercise is typically not associated with elevations in VO2 max and/or any parameters related to it, but it could allow an individual to compete at a higher power output or give the ability to train longer.23 Other reported benefits include a reduction in perceived leg pain induced by exercise24 and improved psychomotor performance (reaction time) during exercise.2 Improved concentration, improved cognitive performance after exercise,25 a reduction or delay in fatigue,26 and enhancement in alertness22 have also been reported. The benefits of caffeine consumption are clear. The evidence supporting a functional role for coffee consumption on exercise performance is discussed next. The research on coffee and caffeine intake on exercise performance began in the 1970s and is still being conducted today. One classic study was performed by Costill and colleagues to determine the effects of caffeine ingestion on performance during prolonged exercise.27 This study utilized a cycle ergometer at 80% of VO2 max until exhaustion following the consumption of either decaffeinated or regular coffee (330 mg of caffeine) to determine the physiological effects of caffeine. The results found that the caffeine group exercised longer (90.2 min) than the decaffeinated group (75.5 min), and the caffeine group also showed an enhanced fat-burning effect. In addition, the caffeine group also reported a lower rating of perceived exertion during the exhaustive exercise bout.27 Other studies have shown similar results when coffee was used as the means of caffeine administration. A more recent study determined that various forms of caffeine ingestion all resulted in significant increases in time-to-exhaustion exercise when compared to placebo groups. Furthermore, this study demonstrated that prior coffee consumption did not decrease the ergogenic effect of anhydrous caffeine ingestion on exercise performance.28 While in the past, studies were sparse as far as coffee as a means of caffeine administration, recent studies have examined this method of consumption. It has been noted that caffeine consumed in the form of coffee improves performance in the same way other forms of caffeine are consumed. When ingesting coffee during low-intensity exercise, fat oxidation significantly increased. In addition to increased performance after the ingestion of coffee, it has also been found to decrease perceived exertion during performance.29 In addition to these ergogenic effects, caffeine has not been associated with any negative effects on exercise performance including rehydration status, ion imbalance, or any other negative effects on exercise performance.1, 30 Caffeine consumption stimulates a mild diuresis similar to water, but there is no evidence that the fluid–electrolyte imbalance is negatively affected on exercise performance. In fact, caffeine consumption doses ranging from 100–680 mg of caffeine have rarely affected the differences in urine output when compared to placebo. The effect on fluid–electrolyte imbalance is also affected by caffeine tolerance, and the chance of affecting it is reduced in individuals that regularly consume caffeine. Overall, whether it is coffee or another caffeine-containing product, individuals who consume caffeine in moderation and maintain a typical diet will not incur any detrimental fluid–electrolyte imbalances.31 Further, sweat rate and heat dissipation are not altered by the addition of caffeine alone.32

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Despite all of these reported benefits, the mechanism of action for the respective effects is still unclear. Traditionally, the benefits of endurance exercise were associated with increased free fatty acid oxidation27, 33 and subsequent sparing of muscle glycogen.34, 35 These effects of caffeine ingestion are most likely due to the competitive antagonism of adenosine receptors at physiological concentrations, especially at low doses.36 Despite the findings of these studies, many other studies disagree with the mechanism of action in which caffeine is exhibiting these effects.7, 37 The primary argument in these studies is that performance enhancement has been shown to occur without changes in catecholamine or FFA/glycerol concentrations during exercise.7 Together, these findings suggest that caffeine has an effect at the level of the skeletal muscle, which could be the result of the ergogenic effect.7, 37 Recent studies are in agreement with the notion that the effect of caffeine is mediated at the skeletal muscle level. One study found that anaerobic exercise performance was increased following caffeine ingestion resulting from stimulation of the skeletal muscle by caffeine.38 This has been found to promote greater force output due to excitation contraction coupling.39 Other studies have suggested that caffeine has an effect on calcium release via the ryanodine receptor,40 and this release was not a result of adenosine antagonism.41 In addition, studies have found that caffeine ingestion can potentiate submaximal skeletal muscle contractile force,3, 42 thus eliciting an ergogenic effect. This is most pronounced in slow twitch muscles.43, 44 The most recent study exhibiting these findings found that caffeine ingestion of 6 mg/kg of body weight potentiated contraction force during low frequency stimulation.3 These authors suggested that, in view of the known effects of caffeine on the ryanodine receptor, these data are consistent in demonstrating that caffeine can potentiate calcium release from the SR and further suggest that caffeine’s ergogenic effects are at least partly mediated by direct effects at the skeletal muscle level.3, 7 In addition, the researchers suggest that since caffeine ingestion has no effect on MVC, high-frequency stimulation is consistent with the fact that caffeine has lesser to no effects on maximal strength and high-intensity exercises,3 as has traditionally been thought to be the case. Additionally, caffeine does not impair protein synthesis, mTOR signaling, or muscle hypertrophy, as some studies have suggested.45 Another possible mechanism that may partially explain caffeine’s ergogenic effects involves its relationship to RPE and perceived pain. Research has suggested that caffeine ingestion increased high-intensity cycling performance; the authors reported that the reduction in RPE, as well as an elevation in blood lactate concentration, could be the reason for the ergogenic effect.46 A metaanalysis on caffeine ingestion and RPE levels also suggests that caffeine reduces RPE levels during exercise, thus eliciting an important ergogenic effect.11 These studies agree with a previously cited report that caffeine ingestion significantly reduced leg muscle pain ratings during moderate-intensity cycling exercise. The researchers suggested that caffeine’s hypoalgesic properties could play a role in improving exercise performance.24 Although they are not the same, RPE and perceived leg pain could be associated with one another, thus suggesting that the decreased RPE and/or perceived pain resulting from caffeine ingestion could be one factor in the ergogenic effects of endurance exercise performance.21, 37, 38 In addition to leg muscle pain ratings and caffeine, RPE breathing has also been found to be significantly lower following the ingestion of caffeine during cycling trials.48 Caffeine was, however, found to increase feelings of nervousness and restlessness post-exercise after consuming caffeine This should be taken into consideration when supplementing with caffeine for performance. In conclusion, despite the fact that the mechanism of action is somewhat still debated, caffeine consumption can result in improved exercise performance on a variety of levels. Caffeine is the most commonly consumed drug in the world, and athletes frequently use it as an ergogenic aid. Caffeine consumption improves performance and endurance during prolonged, exhaustive exercise and, to a lesser degree, caffeine enhances short-term, high-intensity athletic performance. In addition, caffeine improves concentration, reduces fatigue, and adds to alertness; all of these factors can improve performance in different events. Habitual intake does not diminish caffeine’s ergogenic properties. Caffeine is safe and does not cause significant dehydration or electrolyte imbalance during exercise. The role of coffee ingestion has also been shown to be an effective way of administrating caffeine as an ergogenic aid, thus substantiating coffee’s role as a functional food.

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9.7  CAFFEINE CONSUMPTION TIMING FOR PERFORMANCE Caffeine is absorbed rapidly into the blood stream and peaks within 30–60 minutes; thus, timing for performance is important.49 When taking within 60 minutes, enhanced performance has been found. Different sources of caffeine have been found to peak at different times after ingestion; however, all were within 60 minutes of intake. For example, in a random, double blind, placebo-controlled study, coffee, cola, and caffeine capsules were ingested at different times. Saliva caffeine levels peaked at 42 minutes for coffee, 39 minutes for cola, and 67 minutes for the capsule. 50 Additionally, consuming caffeine during performance has been found to enhance performance. In a study that examined a 2-hour time trial, researchers found that taking 1 mg/kg of caffeine every 20 minutes enhanced performance significantly.49

9.8  HEALTH-RELATED ISSUES IN COFFEE CONSUMPTION Based on the fact that billions of individuals worldwide drink coffee, it could be assumed that if there were negative side effects to drinking coffee, the problems would be manifest in large populations of coffee consumers; however, there is no evidence that such harm occurs. In fact, there are data to suggest that coffee consumption may indeed confer numerous health benefits.

9.8.1 Blood Pressure One very important marker of health that affects millions of people across the world is blood pressure. The role of coffee consumption and its effects on blood pressure have been studied, and these studies have shown consistent results. One large-scale study examined over 3000 Japanese males who were 48–56 years old and undergoing preretirement health screenings. These individuals completed self-administered questionnaires to determine average coffee intake over the past year. The significant findings of this study revealed that regular coffee drinkers had lower blood pressure than individuals who did not consume coffee. In addition, this effect was demonstrated at all levels of alcohol consumption, cigarette smoking, obesity, and glucose intolerance. Thus, the major conclusions of this study suggest that habitual coffee consumption does not have adverse effects on blood pressure, and drinking coffee does have significant beneficial effects on the blood pressure levels in this population.51 A recent meta-analysis found that while caffeine intake produces a slight increase in blood pressure for ≥3 hours, in long-term coffee use, there is no association with coffee consumption and increased blood pressure or risk of cardiovascular disease, even in hypertensive patients.52 Other studies examining the relationship between coffee consumption and blood pressure have found similar results. One study examined over a thousand adults during health checkups and revealed that coffee consumption had no significant effects on blood pressure in these individuals or on total or HDL blood cholesterol levels. In addition, these findings revealed a negative correlation between coffee consumption and serum triglycerides in these individuals. These findings further support the beneficial effects of coffee consumption in these populations and show that drinking coffee does not adversely affect these cardiovascular risk factors in adults.53 Even when consuming a higher intake of coffee (around five cups per day), it was found to only have a small increase in blood pressure.54 Despite these positive findings, it is important to note that individuals who currently have high blood pressure should be more cautious with coffee drinking and should probably consult a physician before drinking coffee on a regular basis. This suggestion is supported by research that indicates that reducing or restricting coffee intake may have a beneficial effect on controlling high blood pressure in some populations.55, 56 Overall, habitual coffee consumption does not seem to lead to negative effects on blood pressure, even with those who are predisposed to high blood pressure. In addition, moderate coffee consumption may have a beneficial effect on blood pressure levels.

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9.8.2  Cardiovascular Disease One of the most significant health issues over the last 30 years has been the prevalence of cardiovascular disease. Over the last decade or so, a false impression has risen around the relationship between coffee consumption and an increased risk of heart problems. However, coffee consumption not only does not increase the risk, but, as we will discuss later, it also has beneficial effects on some of the contributing factors that result in cardiovascular disease, like type 2 diabetes and hypertension. In fact, a recent meta-analysis found that habitual consumption of three to five cups of coffee per day is associated with a 15% reduction in the risk of CVD.57 Other studies have examined the relationship between coffee consumption and various aspects of cardiovascular disease, some of which will be discussed next. One of these studies was performed on over 85,000 middle-aged registered nurses in the U.S. It examined the 10-year incidence of coronary heart disease (CHD) and found no association with caffeine intake from all sources and CHD. In addition, there was no association between CHD and decaffeinated coffee consumption in this population.58 A more recent study conducted in hospitalized patients who had confirmed acute myocardial infarction (heart attack) found that coffee consumption was not associated with the overall rate of death in these individuals.59 Additional research has supported the idea that coffee consumption does not increase one’s risk of cardiovascular disease. These studies report consistent findings, such as no significant effect of coffee consumption on general mortality and/or cardiovascular disease-associated mortality in men. A lower rate of general mortality was associated with coffee consumption in women.60 The risk of occurrence for a nonfatal heart attack is not associated with coffee consumption in men, and the all-cause mortality rate was decreased by increasing coffee consumption in women.61 In fact, in a large U.S. cohort study, men who drank six or more cups of coffee per day had a 10% lower risk of death, and women had a 15% lower risk.62 Thus, the evidence seems to support the understanding that moderate coffee consumption does not increase an individual’s risk for developing cardiovascular disease. In addition, there is some evidence to suggest that moderate consumption may have some beneficial effects as well, thus providing evidence to support the role of coffee as a functional food.

9.8.3 Diabetes There is a plethora of fairly recent research to support the inverse relationship between coffee consumption and type 2 diabetes.62–70, 80 The following section will discuss some of the more relevant examples of the research examining the association with drinking coffee and type 2 diabetes. One general consensus reached in examining the relationship between coffee and type 2 diabetes is that coffee drinking is associated with higher insulin sensitivity and a lower risk of type 2 diabetes.64, 68, 70, 79, 81 This is important due to the fact that type 2 diabetes is a disease that is characterized by a severe reduction in insulin sensitivity, thus leading to adverse metabolic effects on the body. One study demonstrating the evidence to support this was conducted in about 8,000 healthy individuals aged 35–56 years, who were administered questionnaires to obtain information regarding coffee consumption as well as other general factors. The overall findings of this study demonstrated that high coffee consumption (five cups per day) was inversely associated with insulin resistance, thus promoting a positive effect on insulin metabolism.62 In a recent meta-analysis, it was found that those who drank four to six cups and more than six to seven cups of coffee per day had a lower risk of type 2 diabetes than those who drank less than two cups per day.63 Further support from the Nurses’ Health Study and Health Professionals’ Follow-up Study that examined approximately 42,000 men and 84,000 women found another inverse association between coffee intake and type 2 diabetes, following the adjustment for age, body mass index, and other risk factors. Additional findings from this study found that total caffeine intake from all sources was associated with a significantly lower risk for diabetes in men and women.70 When looking at the progression of type 2 diabetes on prediabetic individuals, it was found that drinking three or more cups of coffee per day had the greatest preventive effect on diabetes onset.71

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Thus, the evidence is clear, and in some cases overwhelming, that drinking moderate to high amounts (four to six cups per day) of coffee has a protective effect on the development of type 2 diabetes in men and women. The implication of reducing the risk of diabetes affects not only the individual, but also clearly our society and economy, due to the substantial costs related to treating this disease. Diabetes is the seventh leading cause of death by disease in the United States.72 Its incidence will probably continue to rise in the future, with one study projecting that one out of three U.S. adults could have diabetes by 2050.73 Knowing what we now know about the protective effects coffee can have on this disease, it is clear that this fact alone should justify a role for coffee as a functional food.

9.8.4 Cancer In a recent meta-analysis, coffee was found to have inverse associations on cancer of many types such as oral, pharynx, liver, colon, prostate, endometrial, and melanoma cancers.74 A recent study on breast cancer in relation to coffee and tea intake found that women who consume a higher amount of coffee (three to four cups/day) when compared to one to two cups/day had a decrease in breast cancer risk. Tea, on the other hand, was found to increase breast cancer risk.75 Breast cancer is not the only form that has been studied in regards to coffee consumption and risk of disease. Other research has suggested that drinking regular coffee (i.e., not decaf) may decrease the risk of developing other types of cancer in men and women. It was noted in one study that the risk for oral/pharyngeal and esophageal cancer was found to decrease with higher coffee consumption.76 Another study that examined the risk of colon and rectal cancer found a decreased risk of cancer by 26% with those who consume coffee compared to non-coffee drinkers.77 Caffeinated beverages have no effect on the risk of thyroid cancer, and coffee intake has been shown to have no association with the risk of pancreatic cancer.47 As you can see, the evidence is pretty clear that frequent coffee consumption does not increase the risk for developing cancer and in some cases, coffee intake is associated with having a preventive effect (see Table 9.2). To summarize, it is clear that coffee and caffeine consumption has been studied in various aspects of health and disease. Some of the more prevalent diseases in our country were discussed in the text. Coffee has been studied in other aspects of health and disease as well (Table 9.2). Despite traditional beliefs, it is now becoming apparent that both occasional and habitual coffee drinking, which is accompanied by caffeine consumption, does not have a negative affect on health, even in those who suffer from high blood pressure, cardiovascular disease, cardiac arrhythmias, heart failure, or diabetes.78 Furthermore, drinking coffee does seem to have beneficial effects on one’s health, and not all of these are contributed by caffeine. Taken together, these data provide further evidence to support the role of coffee as a functional food.

9.9  CONCLUSION AND CLOSING REMARKS This chapter has discussed the various aspects of coffee consumption in both acute and chronic instances. Coffee is one of the most popular beverages in the world and is consumed by millions of people every day. Coffee’s most intriguing and studied ingredient is caffeine. Both coffee and caffeine have been studied in a variety of situations, from psychomotor effects, to performance enhancement effects in exercise, to drinking coffee to prevent a number of diseases. As this chapter has demonstrated, coffee consumption is not dangerous by any means and in most cases can have a multitude of beneficial effects. Traditionally, these beneficial effects have been attributed to the caffeine content of coffee, but we now know that this is not the case in every situation, and the additional ingredients of coffee may also provide beneficial effects. In most cases, a functional food has a special effect on a particular population, but it is clear that the benefits of drinking coffee cover a wide spectrum of the population, and the benefits are not defined in isolated situations. The role that coffee consumption has in preventing some of the most devastating and prevalent diseases should justify the classification of coffee as a functional beverage.

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TABLE 9.2 Coffee and Caffeine Consumption and Health Author

Type of Study

Wadhawan and Anand82 Rodriguez et al.57

Cohort

Mesas et al.52

Review

Habitual coffee drinkers

Poole et al.83

Review

Adult population

Lee et al.84

Cross-sectional

Korean population

Schmit et al.77

Case-control

Drinkers and non drinkers

Antwi et al.85

Case-control

Drinkers and non-drinkers

Je et al.86

Meta-analysis

Cases of death

Wijarnpreecha et al.87

Review

Coffee drinkers and non-drinkers

Miranda et al.88

Cross-sectional

557 Brazilian men and women

Rhee et al.89

Prospective study

Lukic et al.90

Experimental

112,935 postmenopausal women 104,080 Norwegian women

Larsson et al.91

Cohort study

Individuals with gallbladder disease

Qi and Li92

Meta-analysis

Case and controls of patients with Parkinson’s disease

Suliga et al.93

Cross-sectional

Obese, overweight, and normal-BMI individuals

Review

Population Individuals with liver disease Habitual coffee drinkers

Observations of Study Coffee and caffeine consumption improves liver enzymes and decreases mortality. Habitual consumption does not increase risk of CVD. In fact, the consumption of coffee decreased the risk of CVD by 15%. Blood pressure temporarily increases after the consumption of coffee; however, had no long-term increase on blood pressure. Coffee was found to reduce the risk of CVD, cancer, neurological, metabolic, and liver conditions. Higher coffee consumption was associated with 38% lower odds ratio for stroke in women. A decrease in odds of developing colorectal cancer was found in drinkers over non-drinkers. Also, the consumption of decaffeinated coffee was also found to have an inverse association. Consumption of caffeinated coffee is associated with a reduced risk of renal cell carcinoma. Coffee consumption reduced risk of total mortality. Even high intake of decaffeinated coffee has been found to have a lower risk of mortality. Nonalcoholic fatty liver disease was found to decrease in those who drank coffee, compared to those who don’t. Increased coffee consumption was associated with lower risk of cardiovascular risk factors. Drinking coffee, caffeinated or decaffeinated coffee, is not a risk factor in hypertension in post-menopausal women. Low, moderate, and high coffee consumption was found to reduce the risk of malignant melanoma. Two or more cups of coffee per day was associated with a reduced risk of gallbladder cancer. Coffee and tea consumption was found to decrease Parkinson’s disease risk. This reached a maximum at around three cups/­day Lower coffee consumption was related to abdominal obesity, high cholesterol.

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REFERENCES

1. Graham, T.E. Caffeine and exercise: Metabolism, endurance and performance. Sports Med. 2001; 31, 785–807. 2. Kruk, B., Chmura, J., Krzeminski, K. et al. Influence of caffeine, cold and exercise on multiple choice reaction time. Psychopharmacology (Berl). 2001; 157, 197–201. 3. Tarnopolsky, M., Cupido, C. Caffeine potentiates low frequency skeletal muscle force in habitual and nonhabitual caffeine consumers. J Appl Physiol. 2000; 89, 1719–1724. 4. Bruce, C.R., Anderson, M.E., Fraser, S.F. et al. Enhancement of 2000 meter rowing performance after caffeine ingestion. Med Sci Sports Exerc. 2000; 32, 1958–1963. 5. Anderson, M.E., Bruce, C.R., Fraser, S.F. et al. Improved 2000 meter rowing performance in competitive oarswomen after caffeine ingestion. Int J Sport Nutr Exerc Metab. 2000; 10, 464–475. 6. Talanian, J.L., Spriet, L.L. Low and moderate doses of caffeine late in exercise improve performance in trained cyclists. Appl Physiol Nutr Metab. 2016; 41(8), 850–855. 7. Graham, T.E., Spriet, L.L. Metabolic, catecholamine, and exercise performance responses to various doses of caffeine. J Appl Physiol. 1995; 78, 867–874. 8. Kovacs, E.M., Stegen, J.H.C.H., Brouns, F. Effect of caffeinated drinks on substrate metabolism, caffeine excretion, and performance. J Appl Physiol. 1998; 85, 709–715. 9. Pasman, W.J., van Baak, M.A., Jeukendrup, A.E., de Haan, A. The effect of different dosages of caffeine on endurance performance time. Int J Sports Med. 1995; 16, 225–230. 10. Spriet, L.L. Exercise and sport performance with low doses of caffeine. Sports Med, 2014; 44(Suppl 2):S175–184. doi, 10.1007/s40279-014-0257-8 11. Doherty, M., Smith, P.M. Effects of caffeine ingestion on rating of perceived exertion during and after exercise: A meta-analysis. Scand J Med Sci Sports. 2005; 15(2), 69–78. 12. Graham, T.E., Spriet, L.L. Performance and metabolic responses to a high caffeine dose during prolonged exercise. J Appl Physiol. 1991; 71, 2292–2298. 13. Acheson, K.J., Gremaud, G., Meirim, I. et al. Metabolic effects of caffeine in humans: Lipid oxidation or futile cycling? Am J Clin Nutr. 2004; 79, 40–46. 14. Perkins, K.A., Sexton, J.E., Epstein, L.H., DiMarco, A., Fonte, C., Stiller, R.L., Jacob, R.G. Acute thermogenic effects of nicotine combined with caffeine during light physical activity in male and female smokers. Am J Clin Nutr. 1994; 60(3), 312–319. doi, 10.1093/ajcn/60.3.312 15. Bracco, D., Ferrarra, J.M., Arnaud, M.J., Jequier, E., Schutz, Y. Effects of caffeine on energy metabolism, heart rate, and methylxanthine metabolism in lean and obese women. Am J Physiol. 1995; 269(4 Pt 1):E671–678. 16. Collins, L.C., Cornelius, M.F., Vogel, R.L., Walker, J.F., Stamford, B.A. Effect of caffeine and/or cigarette smoking on resting energy expenditure. Int J Obes Relat Metab Disord. 1994; 18, 551–556. 17. Dulloo, A.G., Geissler, C.A., Horton, T., Collins, A., Miller, D.S. Normal caffeine consumption: Influence on thermogenesis and daily energy expenditure in lean and post obese human volunteers. Am J Clin Nutr. 1989; 49, 44–50. 18. Poehlman, E.T., LaChance, P., Tremblay, A. et al. The effect of prior exercise and caffeine ingestion on metabolic rate and hormones in young adult males. Can J Physiol Pharmacol. 1989; 67, 10–16. 19. Koot, P., Deurenberg, P. Comparison of changes in energy expenditure and body temperatures after caffeine consumption. Ann Nutr Metab. 1995; 39, 135–142. 20. Belza, A., Jessen, A.B. Bioactive food stimulants of sympathetic activity: Effect on 24-h energy expenditure and fat oxidation. Eur J Clin Nutr. 2005; 59, 733–741. 21. de Mendonca, A., Cunha, R.A. Therapeutic opportunities for caffeine in Alzheimer’s disease and other neurodegenerative disorders. J Alzheimers Dis. 2010; 20(Suppl 1):S1–S2. 22. Paluska, S.A. Caffeine and exercise. Curr Sports Med Rep. 2003; 2, 213–219. 23. Graham, T.E. Caffeine, coffee and ephedrine: Impact on exercise performance and metabolism. Can J Appl Physiol. 2001; 26(Suppl.):S103–S119. 24. Motl, R.W., O’Connor, P.J., Dishman, R.K. Effect of caffeine on perceptions of leg muscle pain during moderate intensity cycling exercise. J Pain. 2003; 4, 316–321. 25. Hogervorst, E., Riedel, W.J., Kovacs, E., Brouns, F., Jolles, J. Caffeine improves cognitive performance after strenuous physical exercise. Int J Sports Med. 1999; 20, 354–361. 26. Applegate, E. Effective nutritional ergogenic aids. Int J Sport Nutr. 1999; 9, 229–239. 27. Costill, D.L., Dalsky, G.P., Fink, W.J. Effects of caffeine ingestion on metabolism and exercise performance. Med Sci Sports. 1978; 10, 155–158. 28. McLellan, T.M., Bell, D.G. The impact of prior coffee consumption on the subsequent ergogenic effect of anhydrous caffeine. Int J Sport Nutr Exerc Metab. 2004; 14, 698–708.

Coffee as a Functional Beverage

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29. Higgins, S., Straight, C.R., Lewis, R.D. The effects of preexercise caffeinated coffee ingestion on endurance performance: An evidence-based review. Int J Sport Nutr Exerc Metab. 2016; 26(3), 221–239. doi, 10.1123/ijsnem.2015-0147 30. Fiala, K.A., Casa, D.J., Roti, M.W. Rehydration with a caffeinated beverage during the nonexercise periods of three consecutive days of twice-a-day practices. Int J Sport Nutr Exerc Metab. 2004; 14, 419–429. 31. Armstrong, L.E. Caffeine, body fluid -electrolyte balance, and exercise performance. Int J Sport Nutr Exerc Metab. 2002; 12, 189–206. 32. Goldstein, E.R., Ziegenfuss, T., Kalman, D., Kreider, R., Campbell, B., Wilborn, C., Antonio, J. International society of sports nutrition position stand: Caffeine and performance. J Int Soc Sports Nutr. 2010; 7(1), 5. 33. Ryu, S., Choi, S.K., Joung, S.S. et  al. Caffeine as a lipolytic food component increases endurance performance in rats and athletes. J Nutr Sci Vitaminol (Tokyo). 2001; 47, 139–146. 34. Erickson, M.A., Schwarzkopf, R.J., McKenzie, R.D. Effects of caffeine, fructose, and glucose ingestion on muscle glycogen utilization during exercise. Med Sci Sports Exerc. 1987; 19, 579–583. 35. Spriet, L.L., MacLean, D.A., Dyck, D.J., Hultman, E., Cederblad, G., Graham, T.E. Caffeine ingestion and muscle metabolism during prolonged exercise in humans. Am J Physiol. 1992; 262:E891–E898. 36. Holtzman, S.G., Mante, S., Minneman, K.P. Role of adenosine receptors in caffeine tolerance. J Pharmacol Exp Ther. 1991; 256, 62–68. 37. Mohr, T., Van Soeren, M., Graham, T.E., Kjaer, M. Caffeine ingestion and metabolic responses of tetraplegic humans during electrical cycling. J Appl Physiol. 1998; 85, 979–985. 38. Bell, D.G., Jacobs, I., Ellerington, K. Effect of caffeine and ephedrine ingestion on anaerobic exercise performance. Med Sci Sports Exerc. 2001; 33, 1399–1403. 39. Davis, J.K., Green, J.M. Caffeine and anaerobic performance: Ergogenic value and mechanisms of action. Sports Med. 2009; 39(10), 813–832. 40. Penner, R., Neher, E., Takeshima, H., Nishimura, S., Numa, S. Functional expression of the calcium release channel from skeletal muscle ryanodine receptor cDNA. FEBS Lett. 1989; 259, 217–221. 41. Fryer, M.W., Neering, I.R. Actions of caffeine on fast- and slow-twitch muscles of the rat. J Physiol. 1989; 416, 435–454. 42. Lopes, J.M., Aubier, M., Jardim, J., Aranda, J.V., Macklem, P.T. Effect of caffeine on skeletal muscle function before and after fatigue. J Appl Physiol. 1983; 54, 1303–1305. 43. Weber, A., Herz, R. The relationship between caffeine contracture of intact muscle and the effect of caffeine on reticulum. J Gen Physiol. 1968; 52(5), 750–759. 44. Tallis, J., James, R.S., Cox, V.M., Duncan, M.J. The effect of physiological concentrations of caffeine on the power output of maximally and submaximally stimulated mouse EDL (fast) and soleus (slow) muscle. J Appl Physiol. 2012; 112(1), 64–71. 45. Moore, T.M., Mortensen, X.M., Ashby, C.K., Harris, A.M., Kump, K.J., Laird, D.W., Thomson, D.M. The effect of caffeine on skeletal muscle anabolic signaling and hypertrophy. Appl Physiol Nutr Metab. 2017; 42(6), 621–629. 46. Doherty, M., Smith, P., Hughes, M., Davison, R. Caffeine lowers perceptual response and increases power output during high-intensity cycling. J Sports Sci. 2004; 22, 637–643. 47. Mack, W.J., Preston-Martin, S., Dal Maso, L. et al. A pooled analysis of case-control studies of thyroid cancer: Cigarette smoking and consumption of alcohol, coffee, and tea. Cancer Causes Control. 2003; 14, 773–785. 48. Killen, L.G., Green, J.M., O’Neal, E.K., McIntosh, J.R., Hornsby, J., Coates, T.E. Effects of caffeine on session ratings of perceived exertion. Eur J Appl Physiol. 2013; 113(3), 721–727. 49. Cox, G.R., Desbrow, B., Montgomery, P.G., Anderson, M.E., Bruce, C.R., Macrides, T.A., Burke, L.M. Effect of different protocols of caffeine intake on metabolism and endurance performance. J Appl Physiol. 2002; 93(3), 990–999. 50. Liguori, A., Hughes, J.R., Grass, J.A. Absorption and subjective effects of caffeine from coffee, cola and capsules. Pharmacol Biochem Behav. 1997; 58(3), 721–726. 51. Wakabayashi, K., Kono, S., Shinchi, K. et al. Habitual coffee consumption and blood pressure: A study of self-defense officials in Japan. Eur J Epidemiol. 1998; 14, 669–673. 52. Mesas, A.E., Leon-Munoz, L.M., Rodriguez-Artalejo, F., Lopez-Garcia, E. The effect of coffee on blood pressure and cardiovascular disease in hypertensive individuals: A systematic review and meta-analysis. Am J Clin Nutr. 2011; 94(4), 1113–1126. doi, 10.3945/ajcn.111.016667 53. Lancaster, T., Muir, J., Silagy, C. The effects of coffee on serum lipids and blood pressure in a U.K. population. J R Soc Med. 1994; 87, 506–507. 54. Geleijnse, J.M. Habitual coffee consumption and blood pressure: An epidemiological perspective. Vasc Health Risk Manag. 2008; 4(5), 963–970.

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55. Rakic, V., Burke, V., Beilin, L.J. Effects of coffee on ambulatory blood pressure in older men and women: A randomized controlled trial. Hypertension. 1999; 33, 869–873. 56. Hakim, A.A., Ross, G.W., Curb, J.D. et al. Coffee consumption in hypertensive men in older middle- age and the risk of stroke: The Honolulu heart program. J Clin Epidemiol. 1998; 51, 487–494. 57. Rodriguez-Artalejo, F., Lopez-Garcia, E. Coffee consumption and cardiovascular disease: A condensed review of epidemiological evidence and mechanisms. J Agric Food Chem. 2018; 66(21), 5257–5263. 58. Willett, W.C., Stampfer, M.J., Manson, J.E. et al. Coffee consumption and coronary heart disease in women: A ten-year followup. JAMA. 1996; 275, 458–462. 59. Mukamal, K.J., Maclure, M., Muller, J.E., Sherwood, J.B., Mittleman, M.A. Caffeinated coffee consumption and mortality after acute myocardial infarction. Am Heart J. 2004; 147, 999–1004. 60. Jazbec, A., Simic, D., Corovic, N., Durakovic, Z., Pavlovic, M. Impact of coffee and other selected factors on general mortality and mortality due to cardiovascular disease in Croatia. J Health Popul Nutr. 2003; 21, 332–340. 61. Kleemola, P., Jousilahti, P., Pietinen, P., Vartiainen, E., Tuomilehto, J. Coffee consumption and the risk of coronary heart disease and death. Arch Intern Med. 2000; 160, 3393–3400. 62. Park, S.Y., Freedman, N.D., Haiman, C.A., Le Marchand, L., Wilkens, L.R., Setiawan, V.W. Association of coffee consumption with total and cause-specific mortality among nonwhite populations. Ann Intern Med. 2017; 167(4), 228–235. 63. Muley, A., Muley, P., Shah, M. Coffee to reduce risk of type 2 diabetes?: A systematic review. Curr Diabetes Rev. 2012; 8(3), 162–168. 64. Carlsson, S., Hammar, N., Grill, V., Kaprio, J. Coffee consumption and risk of type 2 diabetes in Finnish twins. Int J Epidemiol. 2004; 33, 616–617. 65. Gerber, D.A. Coffee consumption and type 2 diabetes mellitus. Ann Intern Med. 2004; 141, 323; author reply 323–4. 66. Glaser, J.H., Glaser, S.K. Coffee consumption and type 2 diabetes mellitus. Ann Intern Med. 2004; 141, 323; author reply 323–4. 67. Louria, D.B. Coffee consumption and type 2 diabetes mellitus. Ann Intern Med. 2004; 141, 321; author reply 323–4. 68. Rosengren, A., Dotevall, A., Wilhelmsen, L., Thelle, D., Johansson, S. Coffee and incidence of diabetes in Swedish women: A prospective 18-year follow-up study. J Intern Med. 2004; 255, 89–95. 69. Ranheim, T., Halvorsen, B. Coffee consumption and human health—Beneficial or detrimental? Mechanisms for effects of coffee consumption on different risk factors for cardiovascular disease and type 2 diabetes mellitus. Mol Nutr Food Res. 2005; 49, 274–284. 70. Salazar-Martinez, E., Willett, W.C., Ascherio, A. et al. Coffee consumption and risk for type 2 diabetes mellitus. Ann Intern Med. 2004; 140, 1–8. 71. Lee, J.H., Oh, M.K., Lim, J.T., Kim, H.G., Lee, W.J. Effect of coffee consumption on the progression of type 2 diabetes mellitus among prediabetic individuals. Korean J Fam Med. 2016; 37(1), 7–13. 72. American Diabetes Association. Statistics about diabetes. 2018. Available from http://www.diabetes.org/ diabetes-basics/statistics/ 73. Boyle, J.P., Thompson, T.J., Gregg, E.W., Barker, L.E., Williamson, D.F. Projection of the year 2050 burden of diabetes in the US adult population: Dynamic modeling of incidence, mortality, and prediabetes prevalence. Popul Health Metr. 2010; 8, 29. 74. Wang, A., Wang, S., Zhu, C., Huang, H., Wu, L., Wan, X., Zhao, H. Coffee and cancer risk: A metaanalysis of prospective observational studies. Sci Rep. 2016; 6, 33711. doi, 10.1038/srep33711 75. Oh, J.K., Sandin, S., Strom, P., Lof, M., Adami, H.O., Weiderpass, E. Prospective study of breast cancer in relation to coffee, tea and caffeine in Sweden. Int J Cancer. 2015; 137(8), 1979–1989. 76. Zhang, Y., Wang, X., Cui, D. Association between coffee consumption and the risk of oral cancer: A meta-analysis of observational studies. Int J Clin Exp Med. 2015; 8(7), 11657–11665. 77. Schmit, S.L., Rennert, H.S., Rennert, G., Gruber, S.B. Coffee consumption and the risk of colorectal cancer. Cancer Epidemiol Biomarkers Prev. 2016; 25(4), 634–639. 78. Chrysant, S.G. The impact of coffee consumption on blood pressure, cardiovascular disease and diabetes mellitus. Expert Rev Cardiovasc Ther. 2017; 15(3), 151–156. 79. Soriguer, F., Rojo-Martinez, G., de Antonio, I.E. Coffee consumption and type 2 diabetes mellitus. Ann Intern Med. 2004; 141, 321–323; author reply 323–4 80. Tuomilehto, J., Hu, G., Bidel, S., Lindstrom, J., Jousilahti, P. Coffee consumption and risk of type 2 diabetes mellitus among middle-aged Finnish men and women. JAMA. 2004; 291, 1213–1219. 81. Van Dam, R.M., Pasman, W.J., Verhoef, P. Effects of coffee consumption on fasting blood glucose and insulin concentrations: Randomized controlled trials in healthy volunteers. Diabetes Care. 2004; 27, 2990–2992.

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82. Wadhawan, M., Anand, A.C. Coffee and Liver Disease. J Clin Exp Hepatol. 2016; 6(1), 40–46. doi:10.1016/j.jceh.2016.02.003 83. Poole, R., Kennedy, O.J., Roderick, P., Fallowfield, J. A., Hayes, P.C., Parkes, J. Coffee consumption and health: Umbrella review of meta-analyses of multiple health outcomes. BMJ. 2017; 359, j5024. doi:10.1136/bmj.j5024 84. Lee, J., Lee, J. E., Kim, Y. Relationship between coffee consumption and stroke risk in Korean population: The Health Examinees (HEXA) Study. Nutr J. 2017; 16(1), 7. doi:10.1186/s12937-017-0232-y 85. Antwi, S.O., Eckel-Passow, J.E., Diehl, N.D., Serie, D.J., Custer, K.M., Arnold, M.L., Parker, A.S. Coffee consumption and risk of renal cell carcinoma. Cancer Causes Control. 2017; 28(8), 857–866. doi:10.1007/ s10552-017-0913-z 86. Je, Y., Giovannucci, E. Coffee consumption and total mortality: A meta-analysis of twenty prospective cohort studies. Br J Nutr. 2014; 111(7), 1162–1173. doi:10.1017/S0007114513003814 87. Wijarnpreecha, K., Thongprayoon, C., Ungprasert, P. Coffee consumption and risk of nonalcoholic fatty liver disease: A systematic review and meta-analysis. Eur J Gastroenterol Hepatol. 2017; 29(2), e8–e12. doi:10.1097/MEG.0000000000000776 88. Miranda, A.M., Steluti, J., Fisberg, R.M., Marchioni, D.M. Association between Coffee Consumption and Its Polyphenols with Cardiovascular Risk Factors: A Population-Based Study. Nutrients. 2017; 9(3). doi:10.3390/nu9030276 89. Rhee, J.J., Qin, F., Hedlin, H.K., Chang, T.I., Bird, C.E., Zaslavsky, O., Winkelmayer, W.C. Coffee and caffeine consumption and the risk of hypertension in postmenopausal women. Am J Clin Nutr. 2016; 103(1), 210–217. doi:10.3945/ajcn.115.120147 90. Lukic, M., Licaj, I., Lund, E., Skeie, G., Weiderpass, E., Braaten, T. Coffee consumption and the risk of cancer in the Norwegian Women and Cancer (NOWAC) Study. Eur J Epidemiol. 2016; 31(9), 905–916. doi:10.1007/s10654-016-0142-x 91. Larsson, S.C., Giovannucci, E.L., Wolk, A. Coffee Consumption and Risk of Gallbladder Cancer in a Prospective Study. J Natl Cancer Inst. 2017; 109(3), 1–3. doi:10.1093/jnci/djw237 92. Qi, H., Li, S. Dose-response meta-analysis on coffee, tea and caffeine consumption with risk of Parkinson’s disease. Geriatr Gerontol Int. 2014; 14(2), 430–439. doi:10.1111/ggi.12123 93. Suliga, E., Koziel, D., Ciesla, E., Rebak, D., Gluszek, S. Coffee consumption and the occurrence and intensity of metabolic syndrome: A cross-sectional study. Int J Food Sci Nutr. 080/09637486.2016.1256381

10

Dietary Fiber and Coronary Heart Disease Thunder Jalili, Eunice Mah, Denis M. Medeiros, and Robert E.C. Wildman

CONTENTS 10.1 Dietary Fiber Definition and Classification........................................................................... 173 10.1.1 Fiber Consumption and Recommendation................................................................ 174 10.1.2 Description of Common Dietary Fibers.................................................................... 175 10.2 Physical and Physiological Properties of Fiber..................................................................... 178 10.3 Relationship between Cholesterol Levels and Coronary Heart Disease............................... 179 10.3.1 Role of Fiber in Reducing Serum Cholesterol........................................................... 180 10.3.2 Mechanisms for Lowering of Serum Cholesterol by Fiber....................................... 182 10.3.3 Other Relevant Considerations for Fiber and Coronary Heart Disease Risk............ 183 10.3.4 Fiber as Adjunct Therapy to Statin Medication......................................................... 184 10.4 Health Claims Associated With Fiber and Coronary Heart Disease.................................... 185 References....................................................................................................................................... 186

10.1  DIETARY FIBER DEFINITION AND CLASSIFICATION Dietary fiber is generally described as plant material that is resistant to human digestive enzymes. Most of these plant materials fall into the category of non-starch polysaccharides, with the exception of plant lignins, which are actually polyphenolic in nature. Dietary fiber also may include resistant starches inherent in foods or created during processing of foods. Several definitions of dietary fiber exist in the United States, based on analytical methods used to isolate and quantify fiber and physiological effects. The following are definitions proposed by the Panel on the Definition of Dietary Fiber, assembled by the Food and Nutrition Board, under the oversight of the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes1: (1) Dietary Fiber consists of non-digestible carbohydrates and lignin that are intrinsic and intact in plants, (2) Functional Fiber consists of isolated, non-digestible, and carbohydrates that have beneficial physiological effects in humans, and (3) Total Fiber is the sum of Dietary Fiber and Functional Fiber The Food and Drug Administration (FDA) defines dietary fiber as “non-digestible soluble and insoluble carbohydrates (with 3 or more monomeric units), and lignin that are intrinsic and intact in plants” or if not intact and intrinsic, must be “isolated or synthetic non-digestible carbohydrates (with 3 or more monomeric units) determined by FDA to have physiological effects that are beneficial to human health.”2 The FDA has a definitive list of non-digestible carbohydrates that meet their dietary fiber definition (Table 10.1). These consist of non-digestible carbohydrates that can be declared as dietary fiber and also those that currently under consideration as dietary fiber. The National Academy of Medicine (formerly the Institute of Medicine) recommended phasing out the terms soluble fiber and insoluble fiber, although food labels may still include soluble and insoluble fiber data.3 Soluble (water) fibers include pectin (pectic substances), gums, and mucilages, whereas the insoluble fibers include cellulose, hemicellulose, lignin, and modified cellulose. 173

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TABLE 10.1 Non-Digestible Carbohydrates That Meet the FDA Dietary Fiber Definition and Recognized Beneficial Physiological Effects Non-Digestible Carbohydrate Beta-glucan Psyllium husk Cellulose Guar gum Pectin Locust bean gum Hydroxypropylmethylcellulose Mixed plant cell wall fibersa Arabinoxylana Alginatea Inulin and inulin-type fructansa High amylase starch (resistant starch 2)a Galactooligosaccharidea Polydextrosea Resistant maltodextrin/dextrina

a

b

Beneficial Physiological Effect Reduction of coronary heart disease risk Reduction of coronary heart disease risk Improved laxation Attenuation of blood cholesterol levels Attenuation of blood cholesterol levels Attenuation of blood cholesterol levels Attenuation of blood cholesterol levels Variesb Attenuation of blood glucose and insulin levels Attenuation of postprandial glucose levels Improvements in bone mineral density and calcium absorption Attenuation of postprandial insulin levels Improvements in calcium absorption Reduction in energy intake at a subsequent meal Improvements in calcium absorption and body retention, as well as bone formation

Mixed plant cell wall fibers are ingredients that contain two or more of the following plant cell wall fibers in varying proportions: cellulose, pectin, lignin, beta-glucan, and arabinoxylan. The associated beneficial physiological effect is dependent on the composition of the fibers. These non-digestible carbohydrates are not confirmed as dietary fiber and are under consideration as of June 2018.

The concept of soluble and insoluble fibers were introduced as an attempt to assign physiologic effects to chemical types of fiber; soluble fibers (from oat, barley, and psyllium) have health claims for their ability to lower blood lipid levels, while wheat bran and other more insoluble fibers are typically linked to laxation. Some of the better food sources of soluble fibers are fruit, legumes, oats, and some vegetables. Meanwhile, those foods noted to be richer sources of insoluble fibers include cereals, grains, legumes, and vegetables. A third category of fiber, resistant starches, are now an accepted member of the fiber family, and are found in foods such as oats, rice, and legumes. Some of these foods are also good sources of soluble and insoluble fibers. The term “resistant starch” was first used to describe the fraction of starch that resisted hydrolysis by α-amylase and pullulanase in vitro.4 Resistant starch (RS) is any starch not digested in the small intestine. RS is a broad and diverse range of materials and a number of different types exist, categorized as RS type 1–5. Food sources of RS include a variety of plant sources including oats, rice, grains, legumes, potatoes and potato starch, and green bananas.

10.1.1  Fiber Consumption and Recommendation The amount of fiber present within the human diet can vary geographically. In more industrially developed countries, such as the United States, fiber consumption is relatively lower than in other societies as a direct result of Western dietary patterns. For example, the average intake of fiber in the United States is only about 12 to 15 g daily. Table 10.2 provides the percentage of the total weight of select foods that is attributable to fiber. This consumption falls well below current recommendations of the World Health Organization of 25 to 40 g of fiber daily. The American diet tends to derive less than one-half of its dietary carbohydrate intake from fruit, vegetables, and whole grains. In contrast, the people of some African societies are known to eat as much as 50 g of fiber daily.

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TABLE 10.2 Fiber Content of Select Foods Food Almonds Apples Lima beans String beans Broccoli Carrots Flour, whole wheat Flour, white wheat Oat flakes Pears Pecans Popcorn Strawberries Walnuts Wheat germ

Fiber (% Weight) 3 1 2 1 1 1 2 4 weeks) of RS supplementation can generate more obvious effects on TC and LDL-C levels, and higher dose (>20 g/d) of RS also had a lowering effect on TG level. One study on wheat-based RS4 demonstrated reductions in total cholesterol, LDL-cholesterol, and HDL-cholesterol following consumption of a blend of 70% wheat flour/30% wheat-based RS4 for 26 weeks.36

10.3.2 Mechanisms for Lowering of Serum Cholesterol by Fiber There are several possible mechanisms in which soluble fiber is thought to reduce serum cholesterol levels; many are related to the ability of soluble fibers to form viscous gels in the intestinal tract. Among these potential mechanisms are reduced cholesterol absorption in the presence of soluble fiber, increased excretion of bile acids, an alteration of bile acid type present in the gut, and possible influences of short-chain fatty acid production by intestinal flora upon cholesterol synthesis. It has been proposed that soluble fiber reduces plasma cholesterol through its ability to bind bile acids in the gastrointestinal tract. As soluble fibers bind bile acids in the intestinal tract, micelle formation is altered and reabsorption of bile acids is subsequently impaired, resulting in the excretion of the fiber–bile complex through the feces. There are two classes of bile acids, primary and secondary. Primary bile acids (cholic and chenodeoxycholic acid) are those synthesized directly from the liver, whereas secondary bile acids (deoxycholic and lithocholic acid) are produced after modification of primary bile acids by bacterial action in the colon. It has been demonstrated that consumption of oat bran increases the loss of bile acids by twofold and specifically increases the loss of deoxycholic acid (secondary bile acid) by 240% in human subjects.37 It was also concluded that the pool of bile acids was not decreased, even though bile acid excretion is increased.37 Another human study done with soluble fiber from psyllium found increased bile acid turnover of both primary bile acids as well.32 These studies point to the fact that bile excretion is increased when high amounts of soluble fiber are eaten. Usually bile is reabsorbed in the large intestine and reused in emulsification of fats; however, since a constant pool is required, the excreted bile must be replaced to keep bile levels adequate for digestive

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needs. Theoretically, this would indicate that bile acid synthesis would be increased under these conditions and, indeed, an increase in bile acid synthesis has also been observed in individuals consuming high amounts of soluble fiber.32 Specifically, the synthesis of deoxycholic acid has been found to increase with consumption of a high-fiber diet. This may have further beneficial effects, as deoxycholic acid has been shown to decrease absorption of dietary cholesterol.37 Replacement of bile can be achieved two ways: (1) more hepatic cholesterol can be dedicated for bile synthesis instead of being exported in the circulation as very low-density lipoprotein, and (2) increased hepatic cholesterol demand will upregulate synthesis and activity of LDL receptors, allowing for greater amounts of VLDL remnants and LDL to be removed from circulation. The overall effect of these alterations is a reduction in LDL and total cholesterol levels. With regard to the first point, data from animal studies demonstrate an increased rate of cholesterol synthesis in the livers of psyllium-fed hamsters.30 Specifically, the enzymatic activity of HMG CoA reductase, the ratelimiting enzyme for hepatic cholesterol synthesis, is observed to be increased three- to fourfold in hamsters fed soluble fiber.38 This effect is thought to be transcriptionally mediated, as mRNA levels have been found to be similarly increased in the same model.38 Alterations of LDL receptor activity are also possible under the influence of psyllium fiber; however, this has been found to occur in experimental animals fed high-fat and high-cholesterol diets. Usually consumption of a high-fat diet tends to depress LDL receptor activity, but hamsters fed high-fat and high-cholesterol diets in conjunction with high dietary soluble fiber demonstrate a restoration of LDL receptor expression to normal levels.38 Examination of the effects of oat bran consumption reveals a divergence in the mechanism of action between soluble fiber from oats vs. that of psyllium. Both have the ability to bind to bile acids and facilitate their excretion; however, they differ in their secondary influence on hepatic cholesterol synthesis. As mentioned above, psyllium fiber fed to animals has been found to increase hepatic cholesterol synthesis. Paradoxically, soluble fiber from oat bran has been found to depress hepatic cholesterol synthesis.39 Bacterial fermentation of soluble fiber from oats results in the production of short-chain fatty acids, specifically propionate, that are absorbed in the colon and travel to the liver via the portal vein. Data from in vitro studies demonstrate an inhibition of hepatic cholesterol and fatty acid synthesis under the influence of propionate.39 The apparent paradox of psyllium fiber increasing cholesterol synthesis and oat fiber decreasing cholesterol synthesis may be explained by the fact that psyllium is very poorly fermented by bacteria in the colon; hence, little propionate is produce to decrease hepatic cholesterol synthesis. In the final analysis, it seems that oat bran may be able to reduce cholesterol levels in a twopart fashion: increasing bile loss and decreasing endogenous hepatic cholesterol synthesis, thus resulting in a shift of serum cholesterol for bile synthesis. Psyllium may reduce serum cholesterol levels through only one relevant mechanism, the loss of bile acids. Furthermore, in spite of the increase in HMG CoA reductase activity and cholesterol synthesis under the influence of psyllium, hepatic cholesterol content continues to be markedly reduced in animals fed a high-psyllium diet.38 Therefore, it seems that this upregulation is barely enough to meet the demands of bile acid synthesis, and obviously not enough to contribute significantly to VLDL exportation and hence LDL cholesterol levels. As one can conclude after careful consideration of the cited studies in this section, even though the net effect of soluble fiber consumption is well established, the specific biochemical events that occur in cholesterol metabolism are still incompletely understood and require more thorough testing.

10.3.3 Other Relevant Considerations for Fiber and Coronary Heart Disease Risk Fiber has also been implicated in reducing risk for CHD through mechanisms other than plasma cholesterol modification. One such example is through modification of blood clotting ability. An enhanced clotting ability coupled with atherosclerosis increases the risk of developing an occlusion in the coronary arteries and subsequent myocardial infarction. The ability of the blood to clot

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is dependent upon fibrinogen levels and quality of the resulting fibrin network. Pectin has been found to influence the concentration and quality of fibrin networks in the blood and reduce the tensile strength of these networks. Pectin supplements have been shown to decrease the strength and quality of fibrin networks; these types of networks are thought to be less atherogenic than fibrin networks under normal conditions and thus may represent another vehicle for reducing risk for CHD.40,41 It has been demonstrated that individuals consuming 18.5 g or more of dietary fiber had a 42% risk for elevated plasma C-reactive protein compared to those consuming 8.5 g or less. Similar findings were reported after analysis of survey data from the National Health and Nutrition Examination data as well. Using this data, a 41% lower risk of elevated C-reactive protein was found in individuals consuming high-fiber diets, after adjusting for smoking, BMI, physical activity, total energy, and fat intake.36 Finally, a meta-analysis also reported that six out of seven clinical trials examined reported significant reductions in plasma C-reactive protein resulting from high-fiber dietary interventions.42 Given the role of C-reactive protein as a plasma marker of inflammation, and as a marker of atherosclerosis, it is noteworthy that dietary fiber may act in ways beyond its cholesterol lowering ability. Since the 1980s, it has been proposed that LDL particle size could play an important role in increasing risk for coronary heart disease. However, the most recent reviews that have examined all the evidence to date state that LDL particle size has not been independently associated with CHD risk.43 Therefore, clinical practice does not yet include LDL particle size as a risk factor that should be screened. Nonetheless, the impact of dietary fiber on LDL particle size has been studied, and there are reports that indicate fiber and food sources of fiber can increase, decrease, or not affect LDL particle size. For example, soluble fiber has been shown to significantly reduce the levels of small dense LDL particles. In a study that gave 14 g fiber per day from oat cereal to overweight middle aged men, overall LDL levels were reduced by 5%, but more importantly, levels of small LDL particles were reduced by 17%.44 In contrast, a dietary portfolio containing fiber, nuts, phytosterols, and vegetable protein did not demonstrate a greater reduction in small LDL particles compared to overall LDL levels.45 Finally, a recent study that added almonds (a source of dietary fiber) to an existing statin regimen found that LDL particle size actually increased.46 Given the limited number of studies published thus far, more research is needed to define the role of fiber effects on small LDL particle content, and whether any changes in particle size are clinically relevant to disease risk. Whole grains have also been shown to be protective against CHD, as demonstrated by an inverse relationship between whole grain consumption CHD.47–49 However, it still remains unclear whether this association is due to the fiber content of whole grains or other components of whole grains such as phytochemicals, antioxidants, folate, vitamin B6, monounsaturated fatty acids, or n-3 polyunsaturated fatty acids that may act to reduce CHD risk. In spite of a certain degree of confusion regarding the individual contribution of whole grain–derived fiber in reducing CHD risk, the overall beneficial effect of whole grains in general should not be overlooked.

10.3.4  Fiber as Adjunct Therapy to Statin Medication Current medical practice is to use statin drugs to reduce elevated plasma cholesterol levels. There are many types of statin drugs used today, but they all share the common feature of inhibiting the hepatic enzyme HMG CoA reductase. Since dietary fiber is thought to reduce cholesterol levels through other mechanisms in addition to HMG CoA inhibition, it has been proposed that combining fiber therapy with medication may be an effective approach to reduce cholesterol. A recent study examined the precise role of dietary fiber as adjunct therapy to statin medication and found that hypercholesterolemic patients taking 10 g of psyllium per day along with a 10 mg dose of simvastatin had the same degree of cholesterol reduction as those taking 20 mg of simvastatin alone.43 Other

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studies have reported that fiber (carboxymethyl cellulose) in combination with statin drugs can further lower LDL.5 However, guar gum supplementation combined with lovastatin increased the rate of endogenous cholesterol production, while overall LDL levels remained unchanged.50 Finally, pectin and oat bran consumption combined with lovastatin actually increased LDL compared to just lovastatin alone.51 These data demonstrate that dietary fiber may have differential effects based on the type of fiber consumed and the type of statin taken. Therefore, it may be prudent to be cautious with fiber supplementation, beyond what is present in a normal healthy diet. Furthermore, given the ambiguity on how various types of statins respond to adjunct fiber supplementation therapy, it is also recommended for individuals to check cholesterol levels after a period of fiber supplementation to verify that cholesterol levels are improving.

10.4 HEALTH CLAIMS ASSOCIATED WITH FIBER AND CORONARY HEART DISEASE The U.S. Food and Drug Administration allows food manufacturers to use certain health claims related to the link between dietary fiber and a reduced risk of heart disease. For example, upon review of the research literature, the FDA recognizes the relationship between fruit, vegetables, and grain products that contain fiber, particularly soluble fiber, and a reduced risk of CHD. Foods that apply for related health claims would include fruit, vegetables, and whole-grain breads and cereals. To qualify, foods must meet criteria for low saturated fat, low fat, and low cholesterol. The foods must contain, without fortification, at least 0.6 g of soluble fiber per reference amount, and the soluble fiber content must be listed on the label. The health claim must use the terms fiber, dietary fiber, some types of dietary fiber, some dietary fibers, or some fibers and coronary heart disease or heart disease in discussing the nutrient–disease link. The term soluble fiber may be added. A sample health claim may read: Diets low in saturated fat and cholesterol and rich in fruits, vegetables, and grain products that contain some types of dietary fiber, particularly soluble fiber, may reduce the risk of heart disease, a disease associated with many factors.

More specific to soluble fiber, the FDA has to date reviewed and authorized three sources of soluble fiber (oat, barley, and psyllium) to be eligible for use of a health claim with regard to a reduction in the risk of CHD (Table 10.5). In doing so, the FDA acknowledges that in conjunction with a low-saturated-fat and low-cholesterol diet, certain soluble fiber foods may favorably influence total cholesterol and LDL levels and thus lower the risk of heart disease. Foods and supplements meeting this criteria may contain oat bran, whole oat flour, oatrim, whole grain barley, dry milled barley, barley betafiber, and psyllium seed husk. Again, in order for a food manufacturer to use such a health claim on a food label, the food must meet criteria for low saturated fat, low cholesterol, and low fat. The food must provide oat- and/or barley-based eligible ingredients in at least 0.75 g of soluble fiber per serving. Foods that contain psyllium seed husk must contain at least 1.7 g of soluble fiber per serving. In addition, a claim must indicate the daily dietary intake of the soluble fiber source necessary to reduce the risk of heart disease. Also, the claim must indicate the contribution that one serving of the product will make toward that intake level. Further still, the soluble fiber content must be stated in the nutrition label. In the health claim, the food manufacturer must state “soluble fiber” qualified by the name of the eligible source of soluble fiber and “heart disease” or “coronary heart disease” in describing the nutrient–disease association. A sample claim may read as follows: Diets low in saturated fat and cholesterol that include 3 grams or more of beta-glucan soluble fiber from either oats or barley may reduce the risk of heart disease. One serving of dried oats provides 2 grams of this soluble fiber.

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TABLE 10.5 Eligible Soluble Fiber Sources for the Health Claim on the Relationship between Soluble Fiber and Risk of Coronary Heart Disease Food Ingredient

Ingredient Requirement

Oat bran

Oat bran fraction is not more than 50% of the original starting material and provides at least 5.5% (dwb) β-glucan soluble fiber and a total dietary fiber content of 16% (dwb), and such that at least one-third of the total dietary fiber is soluble fiber

Rolled oat

Provides at least 4% (dwb) of β-glucan soluble fiber and a total dietary fiber content of at least 10%

Whole oat flour

Provides at least 4% (dwb) of β-glucan soluble fiber and a total dietary fiber content of at least 10% (dwb)

Oatrim

Has β-glucan soluble fiber content up to 10% (dwb) and not less than that of the starting material (dwb)

Whole grain barley

Has β-glucan soluble fiber content of at least 4% (dwb) and a total dietary fiber content of at least 10% (dwb)

Dry milled barley

Contain at least 4% (dwb) of β-glucan soluble fiber and at least 8% (dwb) of total dietary fiber, except barley bran and sieved barley meal, for which the minimum β-glucan soluble fiber content is 5.5% (dwb) and minimum total dietary fiber content is 15% (dwb)

Barley betafiber

Has β-glucan soluble fiber content of at least 70% on a dry weight basis Has a purity of no less than 95%, such that it contains 3% or less protein, 4.5% or less of light extraneous matter, and 0.5% or less of heavy extraneous matter, but in no case may the combined extraneous matter exceed 4.9%

Psyllium husk/psyllium seed husk

Abbreviation:  dwb, dry weight basis.

REFERENCES 1. Dietary Reference Intakes: Energy, Carbohydrates, Fiber, Fat, Fatty Acids, Cholesterol, Protein and Amino Acids. Washington D.C.: Institute of Medicine: Food and Nutrition Board. National Academies Press; 2005. 2. Food and Drug Administration. The Declaration of Certain Isolated or Synthetic Non-Digestible Carbohydrates as Dietary Fiber on Nutrition and Supplement Facts Labels: Guidance for Industry. 2018. 3. Dietary Reference Intakes: Proposed Definition of Dietary Fiber. Washington, D.C.: Institute of Medicine, Food and Nutrition Board. National Academies Press; 2001:1–64. 4. Englyst HN, Wiggins HS, Cummings JH. Determination of the non-starch polysaccharides in plant foods by gas-liquid chromatography of constituent sugars as alditol acetates. Analyst. 1982;107:307–318. 5. Makki K, Deehan EC, Walter J and Backhed F. The impact of dietary fiber on gut microbiota in host health and disease. Cell Host Microbe. 2018;23:705–715. 6. Benjamin EJ, Blaha MJ, Chiuve SE, Cushman M, Das SR, Deo R, de Ferranti SD et al. American Heart Association Statistics C and Stroke Statistics S. Heart disease and stroke statistics—2017 update: A report from the American Heart Association. Circulation. 2017;135:e146–e603. 7. Leading Causes of Death in Females, 2015 current listing. Centers for Disease Control and Prevention. 2018. 8. Kannel WB, Gordon T and Castelli WP. Role of lipids and lipoprotein fractions in atherogenesis: The Framingham study. Prog Lipid Res. 1981;20:339–348. 9. Martin MJ, Hulley SB, Browner WS, Kuller LH and Wentworth D. Serum cholesterol, blood pressure, and mortality: implications from a cohort of 361,662 men. Lancet. 1986;2:933–936. 10. Simons LA. Interrelations of lipids and lipoproteins with coronary artery disease mortality in 19 countries. Am J Cardiol. 1986;57:5G–10G. 11. Stamler J, Wentworth D and Neaton JD. Is relationship between serum cholesterol and risk of premature death from coronary heart disease continuous and graded? Findings in 356,222 primary screenees of the Multiple Risk Factor Intervention Trial (MRFIT). JAMA. 1986;256:2823–2828.

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12. Anderson JW and Hanna TJ. Impact of nondigestible carbohydrates on serum lipoproteins and risk for cardiovascular disease. J Nutr. 1999;129:1457S–1466S. 13. Todd S, Woodward M, Tunstall-Pedoe H and Bolton-Smith C. Dietary antioxidant vitamins and fiber in the etiology of cardiovascular disease and all-causes mortality: results from the Scottish Heart Health Study [In Process Citation]. Am J Epidemiol. 1999;150:1073–1080. 14. Wolk A, Manson JE, Stampfer MJ, Colditz GA, Hu FB, Speizer FE, Hennekens CH and Willett WC. Long-term intake of dietary fiber and decreased risk of coronary heart disease among women. JAMA. 1999;281:1998–2004. 15. Brown L, Rosner B, Willett WW and Sacks FM. Cholesterol-lowering effects of dietary fiber: A metaanalysis. Am J Clin Nutr. 1999;69:30–42. 16. Anderson JW, Zettwoch N, Feldman T, Tietyen-Clark J, Oeltgen P and Bishop CW. Cholesterollowering effects of psyllium hydrophilic mucilloid for hypercholesterolemic men. Arch Intern Med. 1988;148:292–296. 17. Behall KM, Scholfield DJ and Hallfrisch J. Effect of beta-glucan level in oat fiber extracts on blood lipids in men and women. J Am Coll Nutr. 1997;16:46–51. 18. Gerhardt AL and Gallo NB. Full-fat rice bran and oat bran similarly reduce hypercholesterolemia in humans. J Nutr. 1998;128:865–869. 19. Olson BH, Anderson SM, Becker MP, Anderson JW, Hunninghake DB, Jenkins DJ, LaRosa JC et al. Psyllium-enriched cereals lower blood total cholesterol and LDL cholesterol, but not HDL cholesterol, in hypercholesterolemic adults: Results of a meta-analysis. J Nutr. 1997;127:1973–1980. 20. Romero AL, Romero JE, Galaviz S and Fernandez ML. Cookies enriched with psyllium or oat bran lower plasma LDL cholesterol in normal and hypercholesterolemic men from Northern Mexico. J Am Coll Nutr. 1998;17:601–608. 21. Whyte JL, McArthur R, Topping D and Nestel P. Oat bran lowers plasma cholesterol levels in mildly hypercholesterolemic men. J Am Diet Assoc. 1992;92:446–449. 22. DeGroot AP, Luyken R and Pikaar NA. Cholesterol lowering effects of rolled oats. Lancet. 1963;2:303–304. 23. Anderson JW, Story LS, Sieling B, Chen WJL, Pertro MS and Story J. Hypocholesterolemic effects of oat bran or beans intake for hypercholesterolemic men. Am J Clin Nutr. 1984;40:1145–1155. 24. Ripsin CM, Keenan JM, Jacobs DR, Jr., Elmer PJ, Welch RR, Van Horn L, Liu K et al. Oat products and lipid lowering. A meta-analysis. JAMA. 1992;267:3317–3325. 25. Thies F, Masson LF, Boffetta P and Kris-Etherton P. Oats and CVD risk markers: A systematic literature review. Br J Nutr. 2014;112(Suppl 2):S19–S30. 26. Johnston TP, Korolenko TA, Pirro M and Sahebkar A. Preventing cardiovascular heart disease: Promising nutraceutical and non-nutraceutical treatments for cholesterol management. Pharmacol Res. 2017;120:219–225. 27. Fischer MH, Yu N, Gray GR, Ralph J, Anderson L and Marlett JA. The gel-forming polysaccharide of psyllium husk (Plantago ovata Forsk). Carbohydr Res. 2004;339:2009–2017. 28. Anderson JW, Jones AE and Riddell-Mason S. Ten different dietary fibers have significantly different effects on serum and liver lipids of cholesterol-fed rats. J Nutr. 1994;124:78–83. 29. Kritchevsky D, Tepper SA and Klurfeld DM. Influence of psyllium preparations on plasma and liver lipids of cholesterol-fed rats. Artery. 1995;21:303–311. 30. Turley SD, Daggy BP and Dietschy JM. Cholesterol-lowering action of psyllium mucilloid in the hamster: Sites and possible mechanisms of action. Metabolism. 1991;40:1063–1073. 31. Coats AJ. The potential role of soluble fibre in the treatment of hypercholesterolaemia. Postgrad Med J. 1998;74:391–394. 32. Everson GT, Daggy BP, McKinley C and Story JA. Effects of psyllium hydrophilic mucilloid on LDLcholesterol and bile acid synthesis in hypercholesterolemic men. J Lipid Res. 1992;33:1183–1192. 33. Anderson JW, Allgood LD, Turner J, Oeltgen PR and Daggy BP. Effects of psyllium on glucose and serum lipid responses in men with type 2 diabetes and hypercholesterolemia. Am J Clin Nutr. 1999;70:466–473. 34. Pal S and Radavelli-Bagatini S. Effects of psyllium on metabolic syndrome risk factors. Obes Rev. 2012;13:1034–1047. 35. Yuan HC, Meng Y, Bai H, Shen DQ, Wan BC and Chen LY. Meta-analysis indicates that resistant starch lowers serum total cholesterol and low-density cholesterol. Nutr Res. 2018;54:1–11. 36. Nichenametla SN, Weidauer LA, Wey HE, Beare TM, Specker BL and Dey M. Resistant starch type 4-enriched diet lowered blood cholesterols and improved body composition in a double blind controlled cross-over intervention. Mol Nutr Food Res. 2014;58:1365–1369. 37. Leiss O, von Bergmann K, Streicher U and Strotkoetter H. Effect of three different dihydroxy bile acids on intestinal cholesterol absorption in normal volunteers. Gastroenterology. 1984;87:144–149.

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38. Horton JD, Cuthbert JA and Spady DK. Regulation of hepatic 7 alpha-hydroxylase expression by dietary psyllium in the hamster. J Clin Invest. 1994;93:2084–2092. 39. Wright RS, Anderson JW and Bridges SR. Propionate inhibits hepatocyte lipid synthesis. Proc Soc Exp Biol Med. 1990;195:26–29. 40. Veldman FJ, Nair CH, Vorster HH, Vermaak WJ, Jerling JC, Oosthuizen W and Venter CS. Dietary pectin influences fibrin network structure in hypercholesterolaemic subjects. Thromb Res. 1997;86:183–196. 41. Veldman FJ, Nair CH, Vorster HH, Vermaak WJ, Jerling JC, Oosthuizen W and Venter CS. Possible mechanisms through which dietary pectin influences fibrin network architecture in hypercholesterolaemic subjects. Thromb Res. 1999;93:253–264. 42. North CJ, Venter CS and Jerling JC. The effects of dietary fibre on C-reactive protein, an inflammation marker predicting cardiovascular disease. Eur J Clin Nutr. 2009;63:921–933. 43. Allaire J, Vors C, Couture P and Lamarche B. LDL particle number and size and cardiovascular risk: Anything new under the sun? Curr Opin Lipidol. 2017;28:261–266. 44. Davy BM, Davy KP, Ho RC, Beske SD, Davrath LR and Melby CL. High-fiber oat cereal compared with wheat cereal consumption favorably alters LDL-cholesterol subclass and particle numbers in middleaged and older men. Am J Clin Nutr. 2002;76:351–358. 45. Lamarche B, Desroches S, Jenkins DJ, Kendall CW, Marchie A, Faulkner D, Vidgen E et al. Combined effects of a dietary portfolio of plant sterols, vegetable protein, viscous fibre and almonds on LDL particle size. Br J Nutr. 2004;92:657–663. 46. Ruisinger JF, Gibson CA, Backes JM, Smith BK, Sullivan DK, Moriarty PM and Kris-Etherton P. Statins and almonds to lower lipoproteins (the STALL Study). J Clin Lipidol. 2015;9:58–64. 47. Jacobs DR, Jr., Meyer KA, Kushi LH and Folsom AR. Whole-grain intake may reduce the risk of ischemic heart disease death in postmenopausal women: The Iowa Women’s Health Study [see comments]. Am J Clin Nutr. 1998;68:248–257. 48. Kushi LH, Meyer KA and Jacobs DR, Jr. Cereals, legumes, and chronic disease risk reduction: Evidence from epidemiologic studies. Am J Clin Nutr. 1999;70:451S–458S. 49. Moreyra AE, Wilson AC and Koraym A. Effect of combining psyllium fiber with simvastatin in lowering cholesterol. Arch Intern Med. 2005;165:1161–1166. 50. Uusitupa MI, Miettinen TA, Happonen P, Ebeling T, Turtola H, Voutilainen E and Pyorala K. Lathosterol and other noncholesterol sterols during treatment of hypercholesterolemia with lovastatin alone and with cholestyramine or guar gum. Arterioscler Thromb. 1992;12:807–813. 51. Vaquero MP, Sanchez Muniz FJ, Jimenez Redondo S, Prats Olivan P, Higueras FJ and Bastida S. Major diet-drug interactions affecting the kinetic characteristics and hypolipidaemic properties of statins. Nutr Hosp. 2010;25:193–206.

11

Anthocyanins and Their Health Benefits Justin G. Martin, Gary D. Stoner, and Jairam K.P. Vanamala

CONTENTS 11.1 Introduction........................................................................................................................... 189 11.2 Classification.......................................................................................................................... 190 11.2.1 Nutrient Category...................................................................................................... 190 11.2.2 Molecular Characteristics.......................................................................................... 190 11.3 Structure-Activity Relationships of Anthocyanins................................................................ 191 11.3.1 Degree and Pattern of B-Ring Hydroxylation........................................................... 192 11.3.2 Degree of Glycosylation or Acylation....................................................................... 192 11.4 Sources of Anthocyanins....................................................................................................... 193 11.5 Biological Aspects................................................................................................................. 196 11.5.1 Digestion/Absorption................................................................................................. 196 11.5.2 Metabolism, Distribution, and Storage...................................................................... 198 11.5.3 Excretion....................................................................................................................200 11.5.4 Toxicity Potential.......................................................................................................200 11.6 Functional Applications.........................................................................................................200 11.6.1 Health Promotion.......................................................................................................200 11.6.2 Disease Prevention and Application.......................................................................... 201 11.6.2.1 Obesity........................................................................................................ 201 11.6.2.2 Cancer.........................................................................................................202 11.6.2.3 Cardiovascular Disease...............................................................................204 11.6.3 Physical Performance Aspects...................................................................................205 11.7 Frontiers in Research.............................................................................................................205 11.7.1 Anthocyanins–Gut Microbiota Interactions..............................................................205 11.7.2 Colon Cancer Stem Cell Apoptosis...........................................................................205 References.......................................................................................................................................206

11.1 INTRODUCTION Anthocyanins (ACNs) are a class of flavonoids that give many fruits and vegetables their red, blue, or purple coloration. The color of ACNs, as well as their stability, is dependent on the pH of the environment. ACNs are one of the most common plant bioactive compounds, with more than 600 variations. A bioactive compound is a compound that is not essential to maintain bodily functions but has pronounced effects on living tissue. ACNs are examples of bioactive food components that exhibit anti-proliferative and anti-inflammatory activity and can counter carcinogenesis both in vitro and in vivo. This chapter will provide a discussion of the characteristics, synthesis, and bioavailability of ACNs, as well as their digestion and excretion pathways. It also discusses their anti-inflammatory and proapoptotic properties that influence several diseases such as heart disease and cancer. Finally, we discuss the new frontiers in ACN research.1

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11.2 CLASSIFICATION 11.2.1 Nutrient Category ACNs are responsible for the red, blue, and purple coloration of plants and are mostly found in their flowers and fruits and in lower quantities in the roots and stems of plants. The higher density of ACNs in flowers attracts pollinators, thus increasing the chance for the species to reproduce. Stress from wounds, high exposure to sunlight, or drought increases ACN production in plants, thus indicating that ACNs play a crucial role in a plant’s defense system. The amount of ACNs in fruit is highly dependent on the food source. For example, in one study, the black raspberry had 589 mg cyanidin-3 glucoside equivalents/100 g, while the thornberry had only 150 mg cyanidin-3 glucoside equivalents/100 g.2 Within the same type of fruit, the season of harvest and the conditions for storage influenced the amount of ACNs in the fruit. Because of the abundance of ACNs in fruit, they are the most highly consumed flavonoid. A flavonoid is any plant pigment that has the same basic structure as flavone (Figure 11.1), a compound responsible for the white/yellow pigmentation in plants. ACNs are a class of flavonoids that humans ingest when eating fruit, vegetables, and grains with red, blue, or purple coloration. Grapes and blueberries account for the bulk of the ACNs consumed due to their high levels of ACNs and the amount of these fruits consumed. Processed foods like jams and wine are also sources of dietary ACNs. Currently, ACNs are being explored as new food dyes since there is a growing demand for natural food colorants.3

11.2.2 Molecular Characteristics The basic structures of ACN aglycones are depicted in Figure 11.2. Subtle differences in the different functional groups (OH, H, OCH3) of these molecules account for the 25 different aglycone forms. However, six aglycone forms make up nearly 95% of all anthocyanins. These six aglycones and their abbreviations are delphinidin (DEL), cyanidin (CYA), petunidin (PET), peonidin (PEO), malvidin C

A

B

FIGURE 11.1  The basic structure of a flavone with rings named, from left to right, A, C, and B.

FIGURE 11.2  Common ACN species with the corresponding R-groups.

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FIGURE 11.3  pH-induced structural transformations of cyanidin. (Adapted from Welch CR et al. Curr. Anal. Chem. 2008;4:75–101.)

(MAL), and pelargonidin (PEL). The attached groupings change the polarity of the aglycone. Aglycones with an alcohol (OH) group are the most polar, followed by ether (OCH3), and those with an alkane (H) group are the least polar. The addition of sugar molecules to the C3, C5, or C7 positions of the aglycone, a process called glycosylation, leads to the formation of ACNs. Many ACNs are acylated with aromatic or aliphatic acids, which are hydrophobic. Glycosylation increases water solubility, while acylation decreases solubility. The solubility of the molecule is increased once digested, as ACNs undergo a process known as glucuronidation or sulfation, which takes place in the liver.4 Oxygen, enzymes, temperature, light, and especially pH can break down or alter ACNs. At different pH values, ACNs assume four different forms in aqueous solutions. As illustrated in Figure 11.3, the main species of ACN present at pH values below 2 is red flavylium; for pH values between 3 and 6, the main species is the carbinol pseudo base; and at a pH of slightly less than or equal to 7, then the main species is the quinoidal ACN. The jump from the red flavylium to the carbinol pseudo base occurs through hydration of the C2 position on the flavylium cation.5,6 High-performance liquid chromatography (HPLC) paired with photodiode array detectors is used to detect the presence of the anthocyanin isomers. This analytical process works well when the ACNs are within plants, but once consumed by mammals, most ACNs are bio-transformed into metabolites, which may not be detectable with visible light. However, HPLC with mass spectroscopy or nuclear magnetic resonance spectroscopy can be used to detect ACN metabolites in bodily fluids.7

11.3  STRUCTURE-ACTIVITY RELATIONSHIPS OF ANTHOCYANINS Understanding the structure-activity relationship of ACNs enables us to understand the biological activity of the different species of ACNs. It has been found that the bioactivity of ACNs depends on both the degree and pattern of hydroxylation of the B-ring and the degree of glycosylation or acylation.8 Figure 11.4 shows the structure of the B-ring.

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FIGURE 11.4  The ring designations are shown in the figure along with carbon numbering.

11.3.1 Degree and Pattern of B-Ring Hydroxylation The greater the number of hydroxyl groups on the B-ring of the aglycone structure of the ACNs, the greater their antioxidant activity. Specifically, ACNs with more hydroxylation of the B-ring reduced the rate of iron-induced lipid peroxidation, the process in which free radicals capture electrons from the phospholipids that are integral to cell membranes, thus damaging the cell.9 This conclusion was drawn from a study that found DEL, an aglycone with three hydroxyl groups, to more effectively inhibit lipid peroxidation (70%) compared with CYA, which has two hydroxyl groups (60%), and PEL with one hydroxyl group (40%).10,11 The explanation for this phenomenon is the o-di-hydroxyphenyl structure on the B-ring because it is a determinant for the antioxidant radicals by electron delocalization. If the hydroxyl groups at the C-3 position of the B-ring are substituted with methoxy groups, then antioxidant activity decreases. A possible explanation is that the added methoxy groups affect the ability of the aglycone to find damaging oxyradicals by altering the redox potential, which reduces lipid peroxidation reactions. The evidence in support of this is that the MAL and PEO aglycones with 3′,5′-dimethoxyl and 3′-methoxyl substituents, respectively, showed much lower antioxidant capabilities than 3′,4′,5′-trihydroxylated DEL.11 Another structure-activity relationship study found that greater B-ring hydroxylation leads to greater proapoptotic activity in human leukemia cells by ACNs.12,13 Therefore, it is not surprising that the aglycones with two or more hydroxyl groups on the B-ring (CYA, DEL, PET) are more potent in inducing apoptosis than MAL, PEO, or PEL, which have only one B-ring hydroxyl group. Indeed, DEL was found to exhibit the highest potency to induce apoptosis in the leukemic cells relative to the other ACNs. Another study demonstrated that DEL and CYA suppressed LoVo and LoVo/ADR (colorectal carcinoma) cell growth, whereas MAL and PEL produced much lower levels of cytotoxicity.14 Overall, the degree of B-ring hydroxylation plays a significant role in the biological activity of aglycones.

11.3.2 Degree of Glycosylation or Acylation The degree of glycosylation and acylation of aglycones also impact the antioxidant capabilities and bioactivity of ACNs. The antioxidant properties and biological activities of ACNs increase with a decreasing degree of glycosylation. Given that aglycones are devoid of glycosylation, they have greater levels of oxidation than their parent ACNs as measured by their ability to inhibit the fluorescence intensity decay of the extrinsic probe, 3-[p-(6-phenyl)-1,3,5-hexatrienyl]-phenylpropionic acid, due to free radicals generated during metal ion-induced peroxidation. It was also found that the anticancer properties of ACNs are affected by their glycosylation and acylation patterns.10 For example, when the number of sugar moieties on CYA glycosides was decreased, both the antioxidant and

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proinflammatory cyclooxygenase inhibitory activity increased.1,15–17 In studies in vitro, acylation of potato ACNs with different cinnamic acid derivatives led to different antioxidant activities. When potato ACNs were acylated with p-coumaric acid, they exhibited higher antioxidant activity when compared to ACNs acylated with ferulic acid and caffeic acid moieties,18 further suggesting that acylation patterns can affect the antioxidant properties of ACNs.

11.4  SOURCES OF ANTHOCYANINS The highest concentrations of ACNs are found in fruits and vegetables that are purple, blue, and red. Table 11.1 lists some common sources of ACNs, along with the notable species of ACNs found within each source and includes a measure of the ACN content in terms of milligrams of cyanidin (3-glucoside) equivalents per 100 grams of food source. The putative health benefits of ACNs are becoming more apparent with recent preclinical and clinical data. Thus, there is a growing interest in the fortification of foods with ACNs and the development of ACN supplements to counter a growing epidemic of chronic-inflammation–promoted diseases. Examples of these fortified foods and supplements are listed in Tables 11.2 and 11.3. TABLE 11.1 Natural Sources of ACNs

Food Sources Acai Acerola

Black Currant

Black Raspberry

Boysenberry

Cherry

Chokeberry Cranberry

Eggplant

Identified ACNs cyanidin (3-glucoside) cyanidin (3-glucoside) cyanidin (3-rahmnoside) pelargonidin (3-rahmnoside) delphinidin (3-glucoside) delphinidin (3-o-rutinoside) cyanidin (3-glucoside) cyanidin (3-o-rutinoside) cyanidin (3-o-sophoroside) cyanidin (3-o-2G-glucosylrutinoside) cyanidin (3-o-glucoside) cyanidin (3-o-2G-xylosylrutinoside) cyanidin (3-o-rutinoside) cyanidin (3-o-sophoroside) cyanidin (3-o-2G-glucosylrutinoside) cyanidin (3-o-glucoside) cyanidin (3-o-rutinoside) cyanidin (3-2G-glucosylrutinoside) cyanidin (3-rutinoside) pelargonidin (3-rutinoside) cyanidin (3-glucoside) cyanidin (3-glucoside) delphinidin (3-glucoside) malvidin (3-glucoside) pelargonidin (3-glucoside) delphinidin (3-rutinol-5-glucoside)

ACN Content (mg cyanidin (3-glucoside) Equivalents/100 g)

Reference

320 144

19,20 21,22

190–270

23

589

24,25

1609

1,25

350–400

1,26

1480 60–200

20,23,27 1,20

750

1,28 (Continued)

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TABLE 11.1 (Continued) Natural Sources of ACNs

Food Sources

Elderberry

Lingonberries

Mexican Blackberry Mulberry

Onion

Identified ACNs delphinidin (3-(4″-p-coumaroyl-rham (1-6) glucoside)-5-glucoside) pelargonidin (3-sophoroside-5-glucoside) cyanidin (3-glucoside) cyanidin (3-sambubioside) cyanidin (3-sambubioside-5-glucoside) cyanidin (3-galactoside) cyanidin (3-arabinoside) cyanidin (3-glucoside) cyanidin (3-o-glucoside) cyanidin (3-glucoside) cyanidin (3-rutinoside) pelargonidin (3-glucoside) cyanidin (3-o-glucoside) cyanidin (3-laminaribioside)

ACN Content (mg cyanidin (3-glucoside) Equivalents/100 g)

Reference

100

21,29

1

21,30

361–495

31

191

26,32

25

1,33

1

1,34

1642

1,35

20

36,37

20–120

22,38

cyanidin (3-(3″-malonyl)glucoside) delphinidin (3,5-digalactoside) cyanidin (3-(3″-acetoyl)glucoside) delphinidin (3,5-diglucoside) cyanidin (3-(6″-malonyl glucoside))

Pigmented Orange Purple Corn

Purple-Sweet Potato Purple-Fleshed Potato

cyanidin (3-(6″-malonoyl-laminaribioside)) delphinidin (3-glucoside) cyanidin (3-(malonyl)(acetoyl)glucoside) cyanidin (3-o-glucoside) cyanidin (3-o-(6″-malonyl-glucoside)) cyanidin (3-glucoside) pelargonidin (3-glucoside) peonidin (3-glucoside) cyanidin (3-(6-malonyl-glucoside)) pelargonidin (3-(6-malonyl-glucoside)) cyanidin (3-(dimalonyl-glucoside)) peonidin (3-(6-malonyl-glucoside)) cyanidin (3-(6-acetate-glucoside)) cyanidin (3-caffeylferulysophoroside-5-glucoside) peonidin (3-caffeylferulysophoroside-5-glucoside) petunidin (3-rutinoside-5-glucoside) malvidin (3-rutinoside-5-glucoside) cyanidin (3-o(6-o-malonyl-β-d-glucoside) peonidin (3-(p-coum)-isophoro-5-glucoside) peonidin (3-rutinoside-5-glucoside) petunidin (3-(p-coum)-rutinoside-5-glucoside) peonidin (3-caffeyl-rutinoside-5-glucoside) pelargonidin (3-(p-coum)-rutinoside-5-glucoside)

(Continued)

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TABLE 11.1 (Continued) Natural Sources of ACNs

Food Sources

Red Grape

Strawberry

Wild Blueberry

Radish Red Cabbage

Red Wine

Identified ACNs pelargonidin (3-(4-ferul-rutinoside)-5-glucoside) peonidin (3-(p-coum)-rutinoside-5-glucoside) malvidin (3-(p-coum)-rutinoside-5-glucoside) delphinidin (3-glucoside) cyanidin (3-glucoside) petunidin (3-glucoside) peonidin (3-glucoside) malvidin (3-glucoside) peonidin (3-acetylglucoside) malvidin (3-acetylglucoside) malvidin (3-caffeoylglucoside) petunidin (3-p-coumaroylglucoside) malvidin (3-p-coumaroylglucoside) pelargonidin (3-glucoside) cyanidin (3-glucoside) pelargonidin (3-rutinol) cyanidin (3-galactoside) cyanidin (3-arabinoside) delphinidin (3-galactoside) delphinidin (3-glucoside) delphinidin (3-(6″-p-coumaroyl-rham (2-1) glucoside)-5-(6″-malonyl)-glucoside) cyanidin (3-diglucoside-5-glucoside) cyanidin (3-(sinapoyl)-diglucoside-5-glucoside) cyanidin (3-glucosyl)(sinapoyl)(p-coumaroyl) sophoroside-5-glucoside) cyanidin (3-glucosyl)(sinapoyl)(feruloyl) sophoroside-5-glucoside) cyanidin (3-diferuloylsophoroside-5-glucoside) cyanidin (3-(coumaroyl)sophoroside-5-glucoside) cyanidin (3-(feruloyl)sophoroside-5-glucoside) cyanidin (3-diferuloylsophoroside-5-glucoside) cyanidin (3-(sinapoyl)(feruloyl)sophoroside-5-glucoside) cyanidin (3-(sinapoyl)(sinapoyl)sophoroside-5-glucoside) delphinidin (3-o-glucoside) cyanidin (3-o-glucoside) petunidin (3-o-glucoside) malvidin (3,5-o-diglucoside) malvidin (3-o-glucoside trimer) peonidin (3-o-glucoside) malvidin (3-o-glucoside) pyranopeonidin (3-o-glucoside) carboxypyranomalvidin (3-o-glucoside) malvidin (3-o-caftaric acid)

ACN Content (mg cyanidin (3-glucoside) Equivalents/100 g)

Reference

888

39,40

15–35

1,21

558

1,41

11–60

1,28

25

1,42

24–35

43,44

(Continued)

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TABLE 11.1 (Continued) Natural Sources of ACNs

Food Sources

Thornberry

Identified ACNs carboxypyranopetunidin (3-o-glucoside) pyranomalvidin (3-o-glucoside) malvidin (3-o-coumaroylglucoside-5-o-glucoside) malvidin (3-o-glucoside-(8,8)-methylmethyne-catechin) carboxypyranomalvidin (3-o-acetylglucoside) methylpyranomalvidin (3-o-caftaric acid) petunidin (3-o-acetylglucoside) delphinidin (3-o-caffeoylglucoside) methylpyranomalvidin (3-o-glucoside) malvidin (3-o-glucoside-(8,8)-methylmethyne-epicatechin) carboxypyranopetunidin (3-o-coumaroylglucoside) monocoumaroyl malvidin (3-o-glucoside trimer) carboxypyranomalvidin (3-o-caffeoylglucoside) malvidin (3-o-acetylglucoside) pyranomalvidin (3-o-caffeoylglucoside) delphinidin (3-o-coumaroylglucoside) pyranopetunidin (3-o-coumaroylglucoside) peonidin (3-o-caffeoylglucoside) malvidin (3-o-caffeoylglucoside) carboxypyranomalvidin (3-o-coumaroylglucoside) cyanidin (3-rutinoside) cyanidin (3-glucoside) peonidin (3-rutinoside) peonidin (3-glucoside)

ACN Content (mg cyanidin (3-glucoside) Equivalents/100 g)

150

Reference

21,45

11.5  BIOLOGICAL ASPECTS An obstacle in linking health benefits to ACNs is that ACN levels measured in plasma and urine samples are very low compared to those used in in vitro studies. For a human that ingests between 68 and 1300 mg of ACNs, the maximum plasma ACN concentrations occur within 0.5 to 4 hours, with a maximum concentration between 1.4 and 592 nM.50 This low concentration of ACNs can be attributed to the fact that most of the ACNs are converted to metabolites and separating the metabolites from the parent ACNs is technically challenging. However, it may be worth the effort to identify and quantitate the metabolites since they may exhibit more biological activity than the parent ACNs. Emerging evidence suggests that the high bioactivity of ACNs is due to their conversion to circulating phenolic metabolites.50 Interestingly, individual ACNs are not as bioactive as mixtures of ACNs that originate from different food sources, iterating the importance of diversity in our food choices.

11.5.1 Digestion/Absorption When ACNs reach the acidic stomach, they become stabilized due to their pH-dependent nature. When the absorption of ACNs was studied in rats, it was found that they are rapidly absorbed

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TABLE 11.2 Food Products Enriched with ACNs for Putative Health Benefits

Fortified Food Product Tomato Puree

Dark Chocolate

Bread

Identified ACNs cyanidin (3-xylosylglucosyl-galactoside) cyanidin (3-xylosyl-galactoside) sinapoyl p-coumaroyl cyanidin (3-sambubioside) delphinidin (3-glycoside) cyanidin (3-glycoside) petunidin (3-glycoside) peonidin (3-glycoside) malvidin (3-glycoside) cyanidin (3-sambubioside) cyanidin (3-xylosyl-rutinoside) cyanidin (3-glucoside) cyanidin (3-rutinoside) pelargonidin (3-glucose) cyanidin (3-glucoside)

ACN Content (mg cyanidin (3-glucoside) Equivalents/100 g)

Reference

5

46

25 at 40°C, pH 4.5

47

up to 4% content of bread flour

48

TABLE 11.3 Supplemental Sources of ACNs Supplementation Sources Chokeberry Mother Juice

Elderberry Concentrate

Processed Acai Powder

Nonorganic Acai Powder

ACN Source

ACN Content

cyanidin (3-galactoside) cyanidin (3-glucoside) cyanidin (3-arabinoside) cyanidin (3-xyloside) cyanidin (3-diglucoside) cyanidin (3-sambubioside-glucoside) cyanidin (3-glucoside) cyanidin (3-sambubioside) cyanidin (3-glucoside) cyanidin (3-sambubioside) cyanidin (3-rutinoside) peonidin (3-rutinoside) cyanidin (3-glucoside) cyanidin (3-sambubioside) cyanidin (3-rutinoside) peonidin (3-rutinoside)

100 mg ACNs/Daily Dose

Reference 49

98 mg ACNs/Daily Dose

49

1 mg/100 g

45

27 mg/100 g

45

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in the stomach and excreted into bile as parent molecules and their metabolites. The absorption process may be carried out by bilitranslocase, an organic anion membrane carrier in the gastric mucous membrane. An in situ gastric perfusion study confirmed the absorption of ACNs in the stomach of rats by observing high concentrations of ACNs in the portal veins and in the systemic circulation.51 Any unabsorbed ACNs move to the duodenum, where the highly acidic stomach acids are neutralized with sodium bicarbonate. The rising pH destabilizes the ACNs, promoting their breakdown into metabolites in the small intestine. The small intestine has a high capacity to absorb nutrients, including flavonoids, so it is reasonable to suspect that ACNs get absorbed through similar means because of similarity in structure.51 A study using a Ussing chamber lined with sections of rat intestines showed the highest absorption of ACNs occurs in the jejunum (55.3%), followed by the duodenum (10.4%). There was no evidence of ACN absorption in the ileum or colon.51 Flavanone glycosides pass from the duodenum to the ileum, where microbiota convert them into their respective aglycones. These aglycones are then partially absorbed and metabolized into gut-derived phenolic compounds.52 The currently accepted mechanisms for the absorption of ACNs in the small intestines are divided into three categories:34 • Sodium-glucose co-transportation actively transports ACNs. • Passive diffusion of aglycones after extracellular hydrolysis of the glucoside via lactate phlorizin hydrolase at the brush order (microvilli-covered surfaces in the small intestine). • Metabolism of the ACNs into small molecules, followed by their deglycosylation into aglycones, which are then actively transported across the intestine or subjected to further metabolism. In one study, the absorption of ACNs was investigated by administering 27 ± 6.7 nmol of cyanidin 3-glucoside to male rats via stomach tube. The rats were euthanized at 30, 60, 120, and 180 minutes after receiving the ACNs to examine the contents of the bladder, stomach, and small intestines. It was found that ACNs decreased linearly throughout the gastric lumen, peaking at 120 minutes. The small intestines had a 7.5% uptake of the initial dose, and throughout the GI tract, the levels of the cyanidin 3-glucosides were relatively stable, containing 75%–79% of the initial dose of ACNs.53 Most ACNs reach the colon and are catabolized by microbiota into phenolic compounds, which are absorbed through the intestinal lining and also metabolized into phase II conjugates.52 Figure 11.5 shows the pathway of digestion, absorption, and excretion of ACNs and their metabolites in the body.51

11.5.2 Metabolism, Distribution, and Storage Metabolism starts in the mouth, as ACNs can undergo metabolism by saliva. 55 Then, phase I and II ACN metabolites from the liver enter the colon after they have been excreted back into the intestines via the enterohepatic cycle. The enterohepatic circulation increases the time that metabolites spend in the plasma, thus expanding the time the body is exposed to the metabolites. Bile returns hepatic metabolites to the lumen of the intestine. ACNs that make it past the stomach and small intestines unabsorbed travel to the colon, where the gut bacteria aid in their metabolism. Microbial enzymes, including α-rhamnosidase, β-glucosidase, and β-glucuronidase, catalyze the deconjugation of the ACNs, producing aglycones, which are broken down into smaller compounds. The major metabolites of the ACNs are protocatechuic acid (PCA, 3,4-dihydroxybenzoic acid), syringic acid (3,4-dimethoxy benzoic acid), vanillic acid (3-methoxy-4-hydroxybenzoic acid), and 4-hydroxybenzoic acid from CYA, MAL, PEO, and PEL, respectively. The colonic epithelia absorb the resulting aglycones and phenolic acids. Microbial metabolism uniquely produces the phenolic acids and other non-phenolic metabolites. Important enzymes for conjugation that are found in the liver, kidneys, and intestines are methyltransferases for methylation; uridine diphosphoglucose glucuronosyl transferase and uridine diphosphoglucose dehydrogenase for glucuronidation; and

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FIGURE 11.5  Digestion, absorption, and metabolism of ACNs. Berries and certain color-fleshed vegetables are known to contain greater levels of ACNs [mg of cyanidin (3-glucoside)/100 g fresh weight]. Given that anthocyanin-containing foods are known to have putative health benefits, there is a growing interest in ACN metabolism. This figure depicts the pathway that ACNs take through the first pass of human digestion and illustrates how they are absorbed into the body and ultimately excreted (adapted from Reference 34). When ACNs are orally consumed, they come in contact with saliva, which begins the first steps of ACN metabolism; then in the stomach, many ACNs are absorbed through the stomach lining and reach the liver, and ACNs that are not absorbed in the stomach continue to the small intestine. In the small intestines, the ACNs become unstable due to basic conditions produced from bile, leading to the formation of metabolites. Also, in the colon, metabolites are formed from the remaining parent ACNs by the gut microbiota. Any unabsorbed parent ACNs or metabolites now leave the body in feces. The ACNs and the metabolites that were absorbed go to the liver, where some are delivered back to the small intestines in the bile or enter systemic circulation. ACNs and metabolites in the bloodstream are expelled in urine after passage through the kidney. A recent study found that in blood samples 6 hours after consumption, 44% of the parent ACNs were found to be metabolized into protocatechuic acid.54 This study found that protocatechuic acid, a potential gut bacteria metabolite, is the major metabolite formed in humans from cyanidin glucosides.

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sulfotransferases for sulfation.4,6,7,56 When all of the metabolites are considered, ACNs are much more bioavailable than previously considered.52 Distribution studies of ACNs in rats have shown that they are found in higher concentrations in the jejunum, stomach, kidney, and brain. A recent study in rats fed tart cherries indicated that most of the ACNs accumulated in the kidney and bladder. In lower concentrations, ACNs were found in the liver, heart, and brain. The polarity of the parent ACN plays a crucial role as to how metabolism will take place and ultimately where the metabolite or ACN will be distributed in the body. For example, cyanidin 3-rutinoside was not found in the brains of rats, but the more polar and complex version, cyanidin 3-rutinoside-5-glucoside, was found in the brain.57 Pigs supplemented with a diet that contained up to 4% blueberries for 4 weeks resulted in the accumulation of ACNs in the liver, eyes, cortex, and cerebellum, which implies that ACNs may provide health benefits to the eyes and brain once distributed to these regions. The storage of ACNs does not occur, due to their high-water solubility. Instead, they accumulate and are then expelled in urine and feces within 4 hours after consumption.4,58 However, their metabolites can remain in the circulation for at least 48 hours.59

11.5.3 Excretion In both humans and animals, most of the unabsorbed ACNs are excreted in feces, while the absorbed ACNs are excreted through urine. The parent ACNs and their metabolites that enter the systemic circulation are available to tissues, but once in the systemic circulation, they eventually are excreted in urine. In humans, less than 3% of the initial dosage of ACNs are excreted in urine as the parent ACN. A maximum rate of parent ACN excretion through urine happens between 1 and 4 hours after consumption.4 A pharmacokinetic study in 11 human volunteers given oral administration of 45 grams of freeze-dried black raspberries containing the specific ACNs cyanidin-3-glucoside, cyanidin-3-sambubioside, cyanidin-3-rutinoside, and cyanidin-3-xylosyl-rutinoside daily for 7 days revealed peak levels of anthocyanins in blood plasma at 1–2 hours and in the urine at 4 hours postconsumption. Less than 1% of the administered ACNs were expelled in urine, indicating that the ACNs were highly metabolized by the host and/or gut bacteria.60,61 Two factors determine how ACNs leave the body: The site of conjugate production and the type of conjugate. In the case of glucuronide conjugates, when formed in the intestines, they enter the systemic circulation and are excreted through urine, while those formed in the liver are excreted in feces. Some ACNs re-enter the jejunum and are met with bile and either absorbed by the colon and re-enter the enterohepatic circulation or are excreted in feces.17 Interestingly, in the case of quercetin, more than 50% is exhaled as CO2 rather than excretion through the urine and feces.62 These results lend support to the concept that the human body and our gut bacteria extensively metabolize ACNs and other plant flavonoids, and metabolites are highly bioavailable to tissue before excretion.

11.5.4 Toxicity Potential ACNs exhibit little to no toxicity in animals or in humans because they are water soluble and tend not to accumulate in the body.63 Thus, dietary ACNs with putative anti-inflammatory activity can be used to counter chronic inflammation.63

11.6  FUNCTIONAL APPLICATIONS 11.6.1 Health Promotion Recent research on plant polyphenols has resulted in an increased interest by the public in the health benefits of these compounds. Particularly, consumers are interested in ACNs, which show great potential for exerting health benefits after consumption. For example, their ability to suppress

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chronic inflammation could have major benefits against the numerous inflammation-promoted chronic diseases that plague societies across the globe.64

11.6.2 Disease Prevention and Application 11.6.2.1 Obesity ACNs may have the ability to prevent and reduce obesity. Obesity was originally thought to be caused solely by the storing of excess energy due to energy imbalance. However, more recent studies indicate that chronic low-grade inflammation in the adipose tissue plays a crucial role in obesity. ACNs appear to be very effective in suppressing this inflammation. A study revealed that the mixture of ACNs found within red cabbage microgreen (the younger and smaller version of red cabbage), blueberry, black currant, mulberry, cherry, black elderberry, black soybean, chokeberry, and jaboticaba peel had greater potency than single ACNs.65–70 For example, mice fed a high-fat diet with red cabbage microgreen had decreased weight gain and lower low-density lipoprotein, triacylglycerol, and cholesterol levels than control mice given the high-fat diet. Plasma levels of the pro-inflammatory cytokines CRP and TNF-α were also reduced in these mice. When blueberries were added to the high-fat diets of these mice, there was a reduction in body weight and in blood levels of glucose, TNF-α, and IL-6 as well as an improved insulin resistance.71 Black currant, mulberry, cherry, black elderberry, black soybean, and jaboticaba peel all decreased weight gain and lowered the triacylglycerol and cholesterol levels in high-fat diet mice. Chokeberry extract was added to the drinking water of mice fed a high-fat diet, which decreased inflammation.71 Another study showed that Zucker fatty rats fed a diet containing 2% (wt/wt) blueberry powder had reduced intraperitoneal fat and elevated peroxisome proliferator-activated receptor gamma levels in white adipose tissue and skeletal muscle than rats on control diet.52 Also, Zucker rats fed a diet containing 8% (wt/wt) blueberry powder for 8 weeks had increased blood adiponectin levels and reduced levels of inflammatory markers in white adipose tissue and improved dyslipidemia relative to those on control diet.52 Therefore, dietary ACNs can change the expression levels of adipocytokines. A study utilizing ACN-containing black chokeberry juice in male C57BL/6J mice found up to a 30% decrease in epidydimal fat.52 Further, there was a positive change in adiponectin levels. Thus, this study revealed that the black chokeberry juice aided in preventing weight gain. Rats fed black chokeberry fruit for 4 weeks had reduced visceral fat and hyperglycemia via inhibition of pancreatic lipase, thus reducing lipid absorption in the intestines.52 Goka, the fruit of Acanthopanax senticosus, when fed to mice produced results similar to those in mice fed either sweet orange or cornelian cherries: improved glucose and insulin tolerance/sensitivity, reduced insulin in blood plasma, and reduced hepatic lipid concentrations.52 In one human trial, subjects with a BMI over 23 or a waist circumference over 90 cm were administered either a placebo or an ACN-containing black soybean extract. This treatment resulted in reductions in abdominal fat, triacylglycerol, and low-density lipoprotein levels.72 However, another trial in which 16 healthy subjects aged 18–65 were administered purple carrots containing an average of 118 mg ACNs/day resulted in no loss in body weight or change in inflammation markers.73 Another study examined the effects of oral consumption of 45 g/day of freeze-dried black raspberries on plasma levels of interleukin-6 (IL-6), C-reactive protein (CRP), and tumor necrosis factor-alpha (TNF-α) in older obese males. During the 14-day trial, one group consumed 45 g/day of black raspberries for 4 days, while the other group did not. Then on the sixth day, a high fat/ high-calorie breakfast was consumed. Blood samples were taken before the breakfast and 1, 2, 4, 8, and 12 hours after consumption. Then before the crossover, there was a 2-day period where no freeze-dried raspberries were consumed. IL-6 was significantly lowered (P = 0.03, n = 10) by the black raspberries (34.3 ±  7.6  pg · mL−1 · h−1) compared to the high fat/high-calorie breakfast alone (42.4 ±  17.9  pg · mL−1 · h−1). No significant differences were found in plasma CRP and TNF-α levels. These results suggest that freeze-dried black raspberries may ease inflammation after consuming

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high fat/high-calorie meals in older obese males.74 However, additional human trials are needed to verify if ACNs have an effect on obesity in humans. 11.6.2.2 Cancer 1. In vitro: ACNs may be able to act as a “natural chemopreventive intervention” for many cancers and specifically colon cancer because of the high concentration of ACNs in the colon.75 ACNs in vitro elicit several anti-carcinogenic effects, including antioxidant activity, activation of phase II metabolizing enzymes, induction of apoptosis, inhibition of cell proliferation, cell cycle arrest, and stimulation of cell differentiation, as well as other anticancer mechanisms.76–83 Along with their anti-proliferative effects, the pro-apoptotic effects of ACNs in vitro have been investigated extensively. Most research points to biomarkers of the effects of ACNs on both the intrinsic and extrinsic pathways for apoptosis in cancer cells. The intrinsic pathways disrupt mitochondrial function; promote apoptosome formation; and activate caspase-9 and effector caspases 3, 6, and 7 to induce apoptosis.84 Extrinsic pathways involve the interplay of Fas/Fas ligand coupled to the caspase-8 signaling pathway.84   The intrinsic pathway of apoptosis involves the Bcl-2 proteins that control mitochondrial permeability. Bcl-2 is an anti-apoptotic protein found in the mitochondrial wall that inhibits the release of cytochrome C. Bax, a pro-apoptotic member of the same family, is found in the cytosol and can move to the mitochondria when given the appropriate signal; it promotes the release of cytochrome C.1,85,86 When cytochrome C is released from mitochondria, it binds to Apaf-1 and forms an activation complex with caspase-9, which then activates caspases-3, 6, and 7. These caspases, in turn, induce cellular apoptosis. An example of the ability of ACNs to induce apoptosis through the intrinsic pathway is when sweet potato greens were shown to increase caspase-3 in prostate cancer PC-3 cells but not caspase-8, which is part of the extrinsic pathway. ACNs can also induce caspase-mediated mitochondrial apoptosis through activation of the p38-MAPK signaling pathway and suppression of protein kinase B (Akt) in HCT- 116 cells. This activation ultimately releases cytochrome C, which complexes with caspase-9, leading to activation of caspase-3 and ultimately cellular apoptosis. ACNs can also induce apoptosis intrinsically through pro-oxidant activities selective to cancer cells. HL-60 cells treated with delphinidin showed elevated levels of ROS, which signal to activate JNK and JNK activates caspase-3, thus inducing apoptosis. ACNs selectively target cancer cells because cancer cells cannot survive the elevated ROS levels, as antioxidant enzymes are low in cancer cells compared to normal cells. Normal cells can cope with the additional ROS due to greater endogenous antioxidant enzyme levels.1,87–90   In the extrinsic pathway, apoptosis is induced through the activation of death receptors Fas, TNFR1, DR3, and DR4/DR5. These receptors bind to extrinsic ligands (FasL, TNF-α, Apo3L, and Apo2L) and transduce intracellular signals that lead to cell death. In the case of the Fas/FasL, signaling pathway trimerization is induced. Fas trimerization activates caspase-8 through the adapter protein FADD. Caspase-8 stimulates apoptosis through two cascades: (1) it can directly cleave and activate caspase-3, or (2) it can act through Bid and Bc I-2 family proteins to cause the mitochondrial membrane to become depolarized and activate apoptosis via caspase-3.1,84,91 Some ACNs can induce apoptosis through this extrinsic pathway. For example, ACNs from Vitis coignetiae Pulliat induced apoptosis in human leukemia cells by activating caspase-8 and down-regulating Bcl-2 pathways.88   In addition to apoptosis, ACNs have been shown to inhibit cancer cell proliferation. Grape, bilberry, and chokeberry ACN rich extracts were tested for their ability to inhibit, delay, or reverse colon carcinogenesis. Colon-cancer-derived HT-29 and nontumorigenic colonic NCM460 cells were exposed to CAN-rich extracts containing 10–75 µg of monomeric anthocyanin/mL for up to 72 hours. All of the ACN-rich extracts inhibited HT-29 cell growth, with the chokeberry extract being the most effective. The chokeberry extract at 25 µg/mL

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inhibited HT-29 cell growth by about 50% after 48 hours of treatment. None of the extracts inhibited the growth of NCM460 cells at this concentration. Therefore, this study concluded that the ACNs are more potent inhibitors of the growth of tumorigenic colon cells than nontumorigenic colon cells.92 Another recent study investigated the effects of ACNs and their sugar-free counterparts, the anthocyanidins, on the proliferation of lung cancer cells. Individual anthocyanidins (aglycones) were found to be significantly more effective than their respective anthocyanins (glycones) in inhibiting the growth of non-small-cell lung cancer (NSCLC) cells. It was also found that mixtures of anthocyanidins in equimolar concentrations were more potent in antiproliferative activity than the individual anthocyanidins. In conclusion, it appears that in vitro, the ACNs act as effective anti-cancer agents by inducing apoptosis and by inhibiting cell growth. However, it should be noted that the concentration of ACNs used in vitro are much higher than the levels achieved by absorption in vivo. 2. Animal model studies: Studies of the effects of anthocyanins to influence the development of cancer in animal model systems have focused primarily on esophageal cancer in rats. Wang et al. (2009) examined the anti-cancer activity of ACNs using a rat model of N-nitrosomethylbenzylamine (NMBA)–induced esophageal tumorigenesis.91 All rats were exposed to NMBA and then different groups of rats were provided with the following diets: groups 1–3 each contained ∼3.8 µmol ACNs/g diet (group 1—5% whole black raspberry powder, group 2—an anthocyanin-rich fraction, group 3—an organic solvent-soluble extract), group 4—an organic-insoluble (residue) fraction containing only 0.02 µmol anthocyanins/g diet, group 5—a hexane extract, and group 6—a sugar fraction, all derived from black raspberries). Groups 5 and 6 had low concentrations of ACNs, and the control group had no ACNs. After 30 weeks, it was found that groups 1–3 equally suppressed tumor growth and reduced tumor count, suggesting that the anthocyanin fraction of black raspberries is nearly equally effective in inhibiting esophageal tumor development as whole black raspberry powder. Group 4 was also effective, suggesting that the residue or fiber fraction of black raspberries also exhibits anti-cancer properties. As might be expected, the hexane extract (group 5) and the sugar fraction (group 6) had no anti-cancer activity.91 Once it was determined that the ACNs in black raspberries have anticancer properties against NMBA-induced esophageal cancer, a major metabolite of the black raspberry ACNs, protocatechuic acid, was studied to see if it had similar effects. In this study, rats were first injected with NMBA three times a week for 5 weeks. They were then fed diets containing either: (1) 6% black raspberry powder, (2) an ACN-enriched fraction of black raspberries containing the same amount of ACNs (3.8 µmol/g) as in the 6% black raspberry powder diet, or (3) protocatechuic acid at a concentration (500 ppm) that might be expected to be produced by enteric metabolism of the anthocyanins present in diets 1 or 2. The results showed that all diets effectively reduced tumorigenesis, but diet 1 was somewhat more potent than diets 2 and 3. Nevertheless, this study demonstrated that at least one prominent ACN metabolite, protocatechuic acid, exhibits significant anticancer activity.92 Using the same protocol in a separate study, these investigators examined the effects of diets 1, 2, and 3 on inflammatory biomarkers in rat plasma and esophagus. All three diets compared to the control diet suppressed the expression of cytokine IL1β (pro-inflammatory) and increased expression of cytokine IL10 (anti-inflammatory). Also, all three diets increased the expression of IL12, a cytokine that activates natural cytolytic killer and CD8+ T cells, and decreased infiltration of macrophages and neutrophils into the esophagus. This study concluded that it is possible that one mechanism by which the ACNs in black raspberries and protocatechuic acid inhibit NMBA-induced rat esophageal tumorigenesis is by altering cytokine expression and innate immune cell trafficking into tumor tissues.74 3. Human studies: DNA methylation is an important mechanism in gene regulation, and the deregulation of the methylation of DNA is often a crucial step toward carcinogenesis.

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A recent study reported that multiple genes associated with the Wnt signaling pathway were reduced in expression in human colon cancer cell lines HCT116, Caco-2, and SW480 via DNA methylation. Treatment of these cell lines with black raspberry ACNs for 3 days at 0.5, 5, and 25 µg/mL restored the normal expression of these genes by suppressing protein levels of the DNA methyltransferases, DNMT1 and DNMT3B. Ultimately, this led to a reduced mRNA expression of two principal genes associated with cellular proliferation, β-catenin and c-Myc, thus providing a mechanism by which ACNs inhibit cell proliferation in colon cancer cells.93 In one human clinical trial, 16 patients with solid tumors were administered 40 mg/kg doses of Recancostat supplements with an active ingredients ratio of 5:1:1.25 for glutathione, cysteine, and ACNs for 1–30 weeks.93 This treatment did not result in increased inhibition of tumor growth compared to chemotherapy alone. However, in another study, treatment of 25 colorectal cancer patients with 0.5–2.0 g of ACNs/day for 7 days before surgery demonstrated the ability of the ACNs to stimulate apoptosis of colon cancer cells. For all patients combined, the apoptotic index in the colorectal tumors increased from 3.6% to 5.3% of epithelial cells.94 A trial of black raspberry powder was conducted in patients with familial adenomatous polyposis (FAP) who had undergone subtotal colectomy with ileorectal anastomosis.95 In this study, seven patients received 20 g of black raspberry powder mixed with water orally three times per day (60 g/day total), plus two rectal suppositories, each containing 700 mg of black raspberry powder, 1 hour before bed. Another seven subjects did not receive oral black raspberry powder but were treated with the rectal suppositories. The treatment period was 9 months. Patients receiving both oral black raspberry powder and the rectal suppositories had a 53% decrease in polyps, and there was a 25% decrease in patients receiving only the suppositories. This study demonstrated that black raspberry powder containing appreciable levels of ACNs regresses polyps in FAP patients. Another trial was conducted in 20 colorectal cancer patients who received a total of 60 g/day of black raspberry powder orally for 1–9 weeks.96 This treatment resulted in suppressed proliferation and angiogenesis as well as elevated apoptosis (the apoptotic effect was not significant) in the tumors, indicating that ACN containing black raspberry powder exhibits potential anti-cancer activity against human colorectal tumors.96 Thus, in vitro and in vivo studies of ACNs in humans have revealed some of the mechanisms for their anti-proliferative and proapoptotic activities against colorectal cancer. They are likely to exhibit similar effects on other types of human cancers as well. Additional studies are required to evaluate the potential chemopreventive activity of ACNs in humans, and these should include an emphasis on determining the role of the local microbiome and its potential influence on the anti-cancer activity of the ACNs. 11.6.2.3  Cardiovascular Disease Another bioactivity of ACNs is to protect against cardiovascular disease. Consuming 1–2 portions of strawberries, raspberries, or blueberries a day (equivalent to 44 mg of ACNs) provides enough ACNs to reduce the risk of CVD (cardiovascular disease) significantly.59 Eating fruit containing at least 12 mg/day of ACNs cuts the risk of CHD (coronary heart disease) by 12%–32%. For older men and women, the risk was cut by 12%–21%, while for young to middle-aged women, the risk was cut by 32%. Indeed, for every 15 mg increase in ACN consumption, the risk for MI decreased by 17%.59 Studies examining a possible correlation between ACN intake and CVD mortality had mixed results. One study showed no correlation,2 while two other studies found a 9%–14% reduction in risk comparing low to high intake of ACNs.59,97 Though the mechanisms through which ACNs reduce the risk of CVD are not fully elucidated, emerging evidence suggests that they can reduce the stiffening of arteries as well as blood pressure.98 A higher intake of ACNs led to a 4-mmHg decrease in systolic blood pressure, which is similar to what occurs in cessation of smoking. In several short-term studies (less than 2 months), it was found that daily blueberry consumption (350 g fresh weight) lowered systolic and diastolic blood pressure by 5%–6%.59 A recent 3-month study administered 78 and 155 mg/day of ACNs from

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strawberries and observed that high intake resulted in beneficial changes to LDL-cholesterol levels and there were improvements in cholesterol efflux capacity.59 ACNs prevent atherosclerosis from developing and alter cell signaling pathways that are involved with heart inflammation and reduce infarct size after coronary occlusion and perfusion. A potential factor as to why there is individual variation in positive response to ACN intake could be that the ACN metabolites involved in preventing CVD are produced to differing extents by gut microbiota, whose concentration varies from one person to another. Higher concentrations of certain microbes will allow one person to create more ACN metabolites compared to others. Therefore, CV health could be dependent on the gut microbial flora and their metabolites.58,59

11.6.3 Physical Performance Aspects Possible physical performance-enhancing aspects of ACNs were tested with a double-blind clinical test where 54 female and male subjects who were physically active were split into two groups that were administered either a 100 mg ACN pill or a placebo pill for 6 weeks, and there was an untreated control group. The subjects were asked to continue following their physical activity routine. The results showed that total body water, soft lean mass, and percent body fat did not change in any significant amounts. However, the observed max VO2, which is the maximum amount of oxygen an individual can use during intense exercise, increased in the ACN group (48.65 ± 4.73 vs. 52.62 ± 5.04); (P ≤ 0.0001), and there was a significant difference between the two groups (52.62 ± 5.04 for intervention group vs. 49.61 ± 5.33 for placebo); (P = 0.003).99

11.7  FRONTIERS IN RESEARCH 11.7.1 Anthocyanins–Gut Microbiota Interactions There is growing interest in harnessing the health benefits of dietary anthocyanins. However, only a few studies have considered the reciprocal interaction of ACNs and gut microbiota in understanding the biological activities of ACNs. Emerging evidence suggests that putative health benefits of ACNs may depend on: (1) their ability to modulate the gut microbiome and (2) biotransformation of ACNs into their metabolites. For example, ACNs can elevate beneficial bacteria such as Bifidobacteria spp.100 Further, ACNs reduced the ratio between Firmicutes to Bacteroidetes as well as increasing Akkermansia muciniphila and concomitantly suppressing insulin resistance and low-grade inflammation in mice consuming a high-fat diet.80 Interestingly, Bifidobacterium spp. is known to break down ACNs into metabolites. Both ACNs and their gut bacterial metabolites are known to exert anti-inflammatory activity in vivo.76 However, recent studies suggest that gut bacterial metabolites of ACNs may be largely responsible for their biological activity, as they tend to remain in the circulation for 48 hours, unlike ACNs that remain for only 4 hours.58,76 To better capture the anti-inflammatory and anti-cancer activity of ACNs, there is a critical need for studies focused on deciphering the reciprocal interaction of the ACNs and the gut microbiota, as bioavailability of ACNs differs significantly among individuals.

11.7.2 Colon Cancer Stem Cell Apoptosis Another interesting area of research is potential to target cancer stem cells using ACNs, as a variety of cancers are known to originate from cancer stem cells. A recent report suggests that ACNs found in purple-fleshed potatoes suppress colon cancer stem cell proliferation as well as elevate apoptosis.68 This in vitro study utilized colon cancer stem cells with functioning p53 and shRNA-attenuated p53 and exposed these cells to ACNs-containing purple-fleshed potato extract (5.0 µg/mL). Interestingly, this extract suppressed cell growth in a p53-independent manner. Another study showed that the feeding of ACN containing baked purple-fleshed potatoes (20% wt/wt) to rats reduced the occurrence of azoxymethaneinduced colon tumors.68 In addition, the potato ACNs reduced the number of stem cells in colonic crypts

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that stained positively for nuclear β-catenin and increased the number of cells stained for apoptosis. This study concluded that purple-fleshed potatoes can reduce colon cancer stem cells in vitro and suppress tumorigenesis in vivo by suppressing Wnt/β-catenin signaling and increasing mitochondrial-mediated apoptosis. Further research is needed to determine if ACNs from other vegetables and fruits have the same effect and if ACNs are effective in eliminating colon cancer stem cells in humans.68 Indeed one in vitro study lends support to the contention that ACNs can target human colon cancer stem cells. An ACN-containing extract of Eugenia jambolana, more commonly known as the java plum, was found to be effective in eliminating colon cancer stem cells at 30 µg/mL medium via suppression of proliferation and induction of apoptosis. Thus, there is a critical need to further evaluate ACNs for their anti-cancer activity against cancer stem cells in animal models and eventually in humans.

REFERENCES 1. Vanamala J, Radhakrishnan S, Reddivari L and Massey A. Anthocyanins as Apoptotic Regulators. In Novel Apoptotic Regulators in Carcinogenesis (eds. Chen GG and Lai PBS). Springer, Netherlands, 2012, 93–122. 2. Mursu J et al. Flavonoid Intake and the Risk of Ischaemic Stroke and CVD Mortality in Middle-Aged Finnish Men: The Kuopio Ischaemic Heart Disease Risk Factor Study. Br. J. Nutr. 2008;100:890–895. 3. Fukumoto LR and Mazza G. Assessing Antioxidant and Prooxidant Activities of Phenolic Compounds. J. Agric. Food Chem. 2000;48:3597–3604. 4. Kay CD. Aspects of Anthocyanin Absorption, Metabolism and Pharmacokinetics in Humans. Nutr. Res. Rev. 2006;19:137. 5. Tsao R. Chemistry and Biochemistry of Dietary Polyphenols. Nutrients 2010;2:1231–1246. 6. He J and Giusti MM. Anthocyanins: Natural Colorants with Health-Promoting Properties. Annu. Rev. Food Sci. Technol. 2010;1:163–187. 7. Wang Y and Ho C-T. Metabolism of Flavonoids. In: Food Factors for Health Promotion (ed. Yoshikawa, T. (Kyoto)). Karger: Basel, 2009, 61, 64–74. 8. Welch CR, Wu Q and Simon JE. Recent Advances in Anthocyanin Analysis and Characterization. Curr. Anal. Chem. 2008;4:75–101. 9. Singleton B. Astaxanthin Health Benefits|LIVESTRONG.COM. Available at: https://www.livestrong. com/article/160675-astaxanthin-health-benefits/. (Accessed: 1st March 2018). 10. Seeram NP and Nair MG. Inhibition of Lipid Peroxidation and Structure–Activity-Related Studies of the Dietary Constituents Anthocyanins, Anthocyanidins, and Catechins. 2002. 11. Tsuda T, Shiga K, Ohshima K, Kawakishi S and Osawa T. Inhibition of Lipid Peroxidation and the Active Oxygen Radical Scavenging Effect of Anthocyanin Pigments Isolated from Phaseolus vulgaris L. Biochem. Pharmacol. 1996;52:1033–1039. 12. Lazze MC et al. Anthocyanins Induce Cell Cycle Perturbations and Apoptosis in Different Human Cell Lines. Carcinogenesis 2004;25:1427–1433. 13. Hou DX et al. International Journal of Oncology. University of Crete, Faculty of Medicine, Laboratory of Clinical Virology, 2003, 23. 14. Cvorovic J et al. Oxidative Stress-Based Cytotoxicity of Delphinidin and Cyanidin in Colon Cancer Cells. Arch. Biochem. Biophys. 2010;501:151–157. 15. Nurmi T et al. Metabolism of Berry Anthocyanins to Phenolic Acids in Humans. J. Agric. Food Chem. 2009;57:2274–2281. 16. McGhie TK and Walton MC. The Bioavailability and Absorption of Anthocyanins: Towards a Better Understanding. Mol. Nutr. Food Res. 2007;51:702–713. 17. Ichiyanagi T, Shida Y, Rahman MM, Hatano Y and Konishi T. Bioavailability and Tissue Distribution of Anthocyanins in Bilberry (Vaccinium myrtillus L.) Extract in Rats. J. Agric. Food Chem. 2006;54:6578–6587. 18. Ling ZC, Chun GH, Yuan DZ and Zhao Q. Pharmacological and Nutritional Activities of Potato Anthocyanins. African J. Pharm. Pharmacol. 2009;2:463–468. 19. Schauss AG et al. Antioxidant Capacity and Other Bioactivities of the Freeze-Dried Amazonian Palm Berry, Euterpe oleraceae Mart. (Acai). J. Agric. Food Chem. 2006;54:8604–8610. 20. Cretu GC and Morlock GE. Analysis of Anthocyanins in Powdered Berry Extracts by Planar Chromatography Linked with Bioassay and Mass Spectrometry. Food Chem. 2014;146:104–112.

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21. Fang J. Classification of Fruits Based on Anthocyanin Types and Relevance to their Health Effects. Nutrition 2017;31:1301–1306. 22. Ribeiro da Silva LM, Teixeira de Figueiredo EA, Silva Ricardo NM, Pinto Vieira IG, Wilane de Figueiredo R, Brasil IM, Gomes CL. Quantification of Bioactive Compounds in Pulps and By-Products of Tropical Fruits from Brazil. Food Chem. 2014;143:398–404. 23. Koeppen BH and Herrmann K. Flavonoid Glycosides and Hydroxycinnamic Acid Esters of Blackcurrants (Ribes nigrum). Zeitschrift fϋr Leb. und -forsch. 1977;164:263–268. 24. Wada L and Ou B. Antioxidant Activity and Phenolic Content of Oregon Caneberries. J. Agric. Food Chem. 2002;50:3495–3500. 25. McGhie TK, Ainge GD, Barnett LE, Cooney JM and Jensen DJ. Anthocyanin Glycosides from Berry Fruit Are Absorbed and Excreted Unmetabolized by Both Humans and Rats. J. Agric. Food Chem. 2003;51:4539–4548. 26. Wu T, Yin J, Zhang G, Long H and Zheng X. Mulberry and Cherry Anthocyanin Consumption Prevents Oxidative Stress and Inflammation in Diet-Induced Obese Mice. Mol. Nutr. Food Res. 2016;60:687–694. 27. Qin B and Anderson RA. An Extract of Chokeberry Attenuates Weight Gain and Modulates Insulin, Adipogenic and Inflammatory Signalling Pathways in Epididymal Adipose Tissue of Rats Fed a FructoseRich Diet. Br. J. Nutr. 2012;108:581–587. 28. Jing P, Zhao S, Ruan S, Sui Z, Chen L, Jiang L and Qian B. Quantitative Studies on Structure– ORAC Relationships of Anthocyanins from Eggplant and Radish Using 3D-QSAR. Food Chem. 2014;145:365–371. 29. Duymus HG, Göger F and Baser KH. In Vitro Antioxidant Properties and Anthocyanin Compositions of Elderberry Extracts. Food Chem. 2014;155:112–119. 30. Zheng W and Wang SY. Oxygen Radical Absorbing Capacity of Phenolics in Blueberries, Cranberries, Chokeberries, and Lingonberries. J. Agric. Food Chem. 2003;51:502–509. 31. Cuevas-Rodríguez EO et  al. Characterization of Anthocyanins and Proanthocyanidins in Wild and Domesticated Mexican Blackberries (Rubus spp.). J. Agric. Food Chem. 2010;58:7458–7464. 32. Xiao T, Guo Z, Sun B and Zhao Y. Identification of Anthocyanins from Four Kinds of Berries and Their Inhibition Activity to α-Glycosidase and Protein Tyrosine Phosphatase 1B by HPLC–FT-ICR MS/MS. J. Agric. Food Chem. 2017;65:6211–6221. 33. Petersson EV, Liu J, Sjöberg PJ, Danielsson R and Turner C. Pressurized Hot Water Extraction of Anthocyanins from Red Onion: A Study on Extraction and Degradation Rates. Anal. Chim. Acta 2010;663:27–32. 34. Grosso G et al. Red Orange: Experimental Models and Epidemiological Evidence of its Benefits on Human Health. Oxid. Med. Cell. Longev. 2013;2013. 35. Li J, Lim SS, Lee JY, Kim JK, Kang SW, Kim JL and Kang YH. Purple Corn Anthocyanins Dampened High-Glucose-Induced Mesangial Fibrosis and Inflammation: Possible Renoprotective Role in Diabetic Nephropathy. J. Nutr. Biochem. 2012;23:320–331. 36. Ju J-H et al. Anti-Obesity and Antioxidative Effects of Purple Sweet Potato Extract in 3T3-L1 Adipocytes In Vitro. J. Med. Food 2011;14:1097–1106. 37. Ahmed M, Akter MSTS and Eun J-B. Optimization Conditions for Anthocyanin and Phenolic Content Extraction From Purple Sweet Potato Using Response Surface Methodology. Int. J. Food Sci. Nutr. 2011;62:91–96. 38. Madiwale GP, Reddivari L, Holm DG and Vanamala J. Storage Elevates Phenolic Content and Antioxidant Activity but Suppresses Antiproliferative and Pro-Apoptotic Properties of Colored-Flesh Potatoes against Human Colon Cancer Cell Lines. J. Agric. Food Chem. 2011;59:8155–8166. 39. Muñoz-Espada AC, Wood KV, Bordelon B and Watkins BA. Anthocyanin Quantification and Radical Scavenging Capacity of Concord, Norton, and Marechal Foch Grapes and Wines. J. Agric. Food Chem. 2004;52:6779–6786. 40. Liazid A, Barbero GF, Azaroual L, Palma M and Barroso CG. Stability of Anthocyanins from Red Grape Skins under Pressurized Liquid Extraction and Ultrasound-Assisted Extraction Conditions. Molecules 2014;19:21034–21043. 41. DeFuria J et al. Dietary Blueberry Attenuates Whole-Body Insulin Resistance in High Fat-Fed Mice by Reducing Adipocyte Death and Its Inflammatory Sequelae. J. Nutr. 2009;139:1510–1516. 42. Huang H et  al. Red Cabbage Microgreens Lower Circulating Low-Density Lipoprotein (LDL), Liver Cholesterol, and Inflammatory Cytokines in Mice Fed a High-Fat Diet. J. Agric. Food Chem. 2016;64:9161–9171.

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43. Frankel EN, Waterhouse AL and Teissedre PL. Principal Phenolic Phytochemicals in Selected California Wines and Their Antioxidant Activity in Inhibiting Oxidation of Human Low-Density Lipoproteins. J. Agric. Food Chem. 1995;43:890–894. 44. Oliveira J, da Silva M, Teixeira N, De Freitas V and Salas E. Screening of Anthocyanins and AnthocyaninDerived Pigments in Red Wine Grape Pomace Using LC-DAD/MS and MALDI-TOF Techniques. J. Agric. Food Chem. 2015;63:7636–7644. 45. Mikulic-Petkovsek M, Stampar F, Veberic R and Sircelj H. Wild Prunus Fruit Species as a Rich Source of Bioactive Compounds. J. Food Sci. 2016;81:C1928–C1937. 46. Gerardi C, Albano C, Calabriso N, Carluccio MA, Durante M, Mita G, Renna M, Serio F and Blando F. Techno-Functional Properties of Tomato Puree Fortified with Anthocyanin Pigments. Food Chem. 2018;240:1184–1192. 47. Gültekin-Özgüven M, Karadağ A, Duman S, Özkal B and Özcelik B. Fortification of Dark Chocolate with Spray Dried Black Mulberry (Morus nigra) Waste Extract Encapsulated in Chitosan-Coated Liposomes and Bioaccessability Studies. Food Chem. 2016;201:205–212. 48. Sui X, Zhang Y and Zhou W. Bread Fortified with Anthocyanin-Rich Extract from Black Rice as Nutraceutical Sources: Its Quality Attributes and In Vitro Digestibility. Food Chem. 2016;196:910–916. 49. Vlachojannis C, Zimmermann BF and Chrubasik-Hausmann S. Quantification of Anthocyanins in Elderberry and Chokeberry Dietary Supplements. Phyther. Res. 2015;29:561–565. 50. Kay CD, Pereira-Caro G, Ludwig IA, Clifford MN and Crozier A. Anthocyanins and Flavanones Are More Bioavailable Than Previously Perceived: A Review of Recent Evidence. Annu. Rev. Food Sci. Technol. 2017;8:155–180. 51. Fang J. Bioavailability of Anthocyanins. Drug Metab. Rev. 2014;46:508–520. 52. Azzini E, Giacometti J and Russo GL. Antiobesity Effects of Anthocyanins in Preclinical and Clinical Studies. Oxid. Med. Cell. Longev. 2017;2017. 53. He J, Wallace TC, Keatley KE, Failla ML and Giusti MM. Stability of Black Raspberry Anthocyanins in the Digestive Tract Lumen and Transport Efficiency into Gastric and Small Intestinal Tissues in the Rat. J. Agric. Food Chem. 2009;57:3141–3148. 54. Vitaglione P et al. Protocatechuic Acid Is the Major Human Metabolite of Cyanidin-Glucosides. J. Nutr. 2007;137:2043–2048. 55. Mallery SR et al. Effects of Human Oral Mucosal Tissue, Saliva, and Oral Microflora on Intraoral Metabolism and Bioactivation of Black Raspberry Anthocyanins. Cancer Prev. Res. (Phila). 2011;4:1209–1221. 56. van Duynhoven J et al. Metabolic Fate of Polyphenols in the Human Superorganism. Proc. Natl. Acad. Sci. U. S. A. 2011;108(Suppl 1):4531–4538. 57. Kirakosyan A, Seymour EM, Wolforth J, McNish R, Kaufman PB and Bolling SF. Tissue Bioavailability of Anthocyanins from Whole Tart Cherry in Healthy Rats. Food Chem. 2015;171:26–31. 58. Mueller D et al. Human Intervention Study to Investigate the Intestinal Accessibility and Bioavailability of Anthocyanins from Bilberries. Food Chem. 2017;231:275–286. 59. Cassidy A. Berry Anthocyanin Intake and Cardiovascular Health. Mol. Aspects Med. 2017. 60. Stoner GD et al. Pharmacokinetics of Anthocyanins and Ellagic Acid in Healthy Volunteers Fed FreezeDried Black Raspberries Daily for 7 days. J. Clin. Pharmacol. 2005;45:1153–1164. 61. Cassidy A and Minihane AM. The Role of Metabolism (and the Microbiome) in Defining the Clinical Efficacy of Dietary Flavonoids. Am. J. Clin. Nutr. 2017;105:10–22. 62. Walle T, Walle UK and Halushka PV. Carbon Dioxide is the Major Metabolite of Quercetin in Humans. J. Nutr. 2001;131:2648–2652. 63. Kuspradini H. Antioxidant and Toxicity Properties of Anthocyanin Extracted from Red Flower of Four Tropical Shrubs. Nusant. Biosci. 2016;8:135–140. 64. Lee YM et al. Dietary Anthocyanins Against Obesity and Inflammation. Nutrients 2017;9:1–15. 65. Dragano NRV et al. Freeze-Dried Jaboticaba Peel Powder Improves Insulin Sensitivity in High-Fat-Fed Mice. Br. J. Nutr. 2013;110:447–455. 66. Kim SY et al. Inhibitory Effect of Anthocyanin-Rich Black Soybean Testa (Glycine max (L.) Merr.) on the Inflammation-Induced Adipogenesis in a DIO Mouse Model. J. Funct. Foods 2015;14:623–633. 67. Farrell NJ et al. Black Elderberry Extract Attenuates Inflammation and Metabolic Dysfunction in DietInduced Obese Mice. Br. J. Nutr. 2015;114:1123–1131. 68. Charepalli V et al. Anthocyanin-Containing Purple-Fleshed Potatoes Suppress Colon Tumorigenesis via Elimination of Colon Cancer Stem Cells. J. Nutr. Biochem. 2015;26:1641–1649. 69. Benn T et al. Polyphenol-Rich Blackcurrant Extract Prevents Inflammation in Diet-Induced Obese Mice. J. Nutr. Biochem. 2014;25:1019–1025.

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70. Wu T et al. Blueberry and Mulberry Juice Prevent Obesity Development in C57BL/6 Mice. PLoS One 2013;8:e77585. 71. Qin B and Anderson RA. An Extract of Chokeberry Attenuates Weight Gain and Modulates Insulin, Adipogenic and Inflammatory Signalling Pathways in Epididymal Adipose Tissue of Rats Fed a FructoseRich Diet. Br. J. Nutr. 2012;108:581–587. 72. Lee M, Sorn SR, Park Y and Park H-K. Anthocyanin Rich-Black Soybean Testa Improved Visceral Fat and Plasma Lipid Profiles in Overweight/Obese Korean Adults: A Randomized Controlled Trial. J. Med. Food 2016;19:995–1003. 73. Wright ORL, Netzel GA and Sakzewski AR. A Randomized, Double-Blind, Placebo-Controlled Trial of the Effect of Dried Purple Carrot on Body Mass, Lipids, Blood Pressure, Body Composition, and Inflammatory Markers in Overweight and Obese Adults: The QUENCH Trial. Can. J. Physiol. Pharmacol. 2013;91:480–488. 74. Peiffer DS et al. Dietary Consumption of Black Raspberries or Their Anthocyanin Constituents Alters Innate Immune Cell Trafficking in Esophageal Cancer. Cancer Immunol. Res. 2016;4:72–82. 75. de Sousa Moraes LF, Sun X, do Carmo Gouveia Peluzio M and Zhu M-J. Anthocyanins/Anthocyanidins and Colorectal Cancer: What Is Behind the Scenes? Crit. Rev. Food Sci. Nutr. 2017;0:1–13. 76. Larrosa M et al. Polyphenol Metabolites from Colonic Microbiota Exert Anti-Inflammatory Activity on Different Inflammation Models. Mol. Nutr. Food Res. 2009;53:1044–1054. 77. Charepalli V et al. Eugenia jambolana (Java Plum) Fruit Extract Exhibits Anti-Cancer Activity against Early Stage Human HCT-116 Colon Cancer Cells and Colon Cancer Stem Cells. Cancers (Basel). 2016;8:29. 78. Reddivari L, Vanamala J, Chintharlapalli S, Safe SH and Miller JC. Anthocyanin Fraction from Potato Extracts is Cytotoxic to Prostate Cancer Cells through Activation of Caspase-Dependent and CaspaseIndependent Pathways. Carcinogenesis 2007;28:2227–2235. 79. Madiwale GP, Reddivari L, Stone M, Holm DG and Vanamala J. Combined Effects of Storage and Processing on the Bioactive Compounds and Pro-Apoptotic Properties of Color-Fleshed Potatoes in Human Colon Cancer Cells. J. Agric. Food Chem. 2012;60:11088–11096. 80. Roopchand DE et  al. Dietary Polyphenols Promote Growth of the Gut Bacterium Akkermansia muciniphila and Attenuate High-Fat Diet-Induced Metabolic Syndrome. Diabetes 2015;64:2847–2858. 81. Zikri NN et al. Black Raspberry Components Inhibit Proliferation, Induce Apoptosis, and Modulate Gene Expression in Rat Esophageal Epithelial Cells. Nutr. Cancer 2009;61:816–826. 82. Afgar A et al. MiR-339 and Especially miR-766 Reactivate the Expression of Tumor Suppressor Genes in Colorectal Cancer Cell Lines through DNA Methyltransferase 3B Gene Inhibition. Cancer Biol. Ther. 2016;17:1126–1138. 83. Wang L-S et al. Black Raspberry-Derived Anthocyanins Demethylate Tumor Suppressor Genes through the Inhibition of DNMT1 and DNMT3B in Colon Cancer Cells. Nutr. Cancer 2013;65:118–125. 84. Elmore S. Apoptosis: A Review of Programmed Cell Death. Toxicol. Pathol. 2007;35:495–516. 85. Brunelle JK and Letai A. Control of Mitochondrial Apoptosis by the Bcl-2 Family. J. Cell Sci. 2009;122:437–441. 86. Harris MH and Thompson CB. The Role of the Bcl-2 Family in the Regulation of Outer Mitochondrial Membrane Permeability. Cell Death Differ. 2000;7:1182–1191. 87. Shin DY et al. Induction of Apoptosis in Human Colon Cancer HCT-116 Cells by Anthocyanins through Suppression of Akt and Activation of p38-MAPK. Int. J. Oncol. 2009;35:1499–1504. 88. Lee SH et al. Induction of Apoptosis in Human Leukemia U937 Cells by Anthocyanins through DownRegulation of Bcl-2 and Activation of Caspases. Int. J. Oncol. 2009;34:1077–1083. 89. Lo C-W, Huang H-P, Lin H-M, Chien C-T and Wang C-J. Effect of Hibiscus Anthocyanins-Rich Extract Induces Apoptosis of Proliferating Smooth Muscle Cell via Activation of P38 MAPK and p53 Pathway. Mol. Nutr. Food Res. 2007;51:1452–1460. 90. Van Laethem A et al. Activation of p38 MAPK Is Required for Bax Translocation to Mitochondria, Cytochrome C Release and Apoptosis Induced by UVB Irradiation in Human Keratinocytes. FASEB J. 2004;18:1946–1948. 91. Fulda S and Debatin K-M. Extrinsic versus Intrinsic Apoptosis Pathways in Anticancer Chemotherapy. Oncogene 2006;25:4798–4811. 92. Zhao C, Giusti MM, Malik M, Moyer MP and Magnuson BA. Effects of Commercial AnthocyaninRich Extracts on Colonic Cancer and Nontumorigenic Colonic Cell Growth. J. Agric. Food Chem. 2004;52:6122–6128. 93. Bode U, Hasan C, Hülsmann B and Fleischhack G. Recancostat Compositum Therapy Does Not Prevent Tumor Progression in Young Cancer Patients. Klin. Pädiatrie 1999;211:353–355.

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94. Thomasset S et  al. Pilot Study of Oral Anthocyanins for Colorectal Cancer Chemoprevention. Cancer Prev. Res. (Phila). 2009;2:625–633. 95. Wang L-S et al. A Phase Ib Study of the Effects of Black Raspberries on Rectal Polyps in Patients with Familial Adenomatous Polyposis. Cancer Prev. Res. (Phila). 2014;7:666–674. 96. Wang L-S et al. Modulation of Genetic and Epigenetic Biomarkers of Colorectal Cancer in Humans by Black Raspberries: A Phase I Pilot Study. Clin. Cancer Res. 2011;17:598–610. 97. McCullough ML et al. Flavonoid Intake and Cardiovascular Disease Mortality in a Prospective Cohort of US Adults. Am. J. Clin. Nutr. 2012;95:454–464. 98. Johnson SA et al. Daily Blueberry Consumption Improves Blood Pressure and Arterial Stiffness in Postmenopausal Women with Pre- and Stage 1-Hypertension: A Randomized, Double-Blind, PlaceboControlled Clinical Trial. J. Acad. Nutr. Diet. 2015;115:369–377. 99. Yarahmadi M et  al. The Effect of Anthocyanin Supplementation on Body Composition, Exercise Performance and Muscle Damage Indices in Athletes. Int. J. Prev. Med. 2014;5:1594–1600. 100. Morais CA, de Rosso VV, Estadella D and Pisani LP. Anthocyanins as Inflammatory Modulators and the Role of the Gut Microbiota. J. Nutr. Biochem. 2016;33:1–7.

12

Olive Oil and Health Benefits Denis M. Medeiros and Meghan Hampton

CONTENTS 12.1 Introduction........................................................................................................................... 211 12.2 Nutritional Components of Olives......................................................................................... 211 12.3 Olive Oil................................................................................................................................ 212 12.4 Coronary Heart Disease........................................................................................................ 213 12.4.1 Fatty Acids in the Mediterranean Diet...................................................................... 213 12.4.2 Other Olive Constituents and Their Effects.............................................................. 214 12.4.3 Olives as Sources of Antioxidants............................................................................. 214 12.4.4 Olive Oil and Inflammation....................................................................................... 216 12.4.5 Hypertension and Olive Oil Consumption................................................................ 216 12.5 Cancer.................................................................................................................................... 217 12.5.1 Breast Cancer and Olive Oil...................................................................................... 217 12.5.2 Prostate Cancer and Olive Oil................................................................................... 218 12.5.3 Other Cancers and Olive Oil..................................................................................... 218 12.5.4 Summary and Future Need for Cancer Research and Olive Oil............................... 218 12.6 Other Disease Conditions and Olive Oil............................................................................... 218 12.7 Reviews and Consensus Reports........................................................................................... 219 12.8 Summary............................................................................................................................... 219 References....................................................................................................................................... 219

12.1 INTRODUCTION The olive is a common name for a plant family and its representative genus, and for the fruit of the olive tree. There are approximately 900 species of olives in 24 genera. Most of us are familiar with the olive that is cultivated for its fruit, which are sometimes referred to as drupes. Olives for eating are harvested or picked when they are either unripe or ripe. The unripe olives are green and remain so during pickling. Ripe olives are dark bluish when fresh and turn blackish during pickling. Olives have been associated with Mediterranean cultures for some time. The cultivated olive is originally native to the eastern Mediterranean region but is cultivated throughout that area and in other parts of the world that have climates like the Mediterranean area. The genus and species of the cultivated olive is Olea europea, which is grown between the 30th and 45th parallels. Spain, Italy, and Greece are the major producers of olives, with Spain being the biggest producer, followed by Italy and then Greece. Other producers in the area include Portugal, Turkey, Morocco, Tunisia, and France. More countries and regions of the world (United States, Canada, Japan, Chile, Argentina, New Zealand, and Australia) are cultivating olives because of interest in the health benefits of the Mediterranean diet. In the United States, most of the production is in California due to its more Mediterranean-like climate. Olive trees normally thrive in regions where there are mild winters and hot summers. The trees cannot normally tolerate temperatures below 10°C, but they can withstand hot temperatures and are drought resistant.

12.2  NUTRITIONAL COMPONENTS OF OLIVES Harvesting of olives may influence their nutrient composition. A point worth noting is to not let them over-ripen, as the acidity level will increase too much. If the harvest is too early, there is limited oil 211

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TABLE 12.1 Selected Nutrient Compositions of Olives Nutrient

1 Large Olive (4.4 g)

Macronutrients Water Energy Protein Total lipid Carbohydrate Total dietary fiber Ash (minerals)

3.52 g 5.05 Kcal 0.037 g 0.47 g 0.28 g 0.14 g 0.10 g

Lipids Palmitic acid (16:0) Stearic acid (18:0) Oleic acid (18:1) Linoleic acid (18:2) Linolenic acid (18:3)

0.05 g 0.01 g 0.34 g 0.04 g 0.003 g

Source: From USDA Nutrient Database for Standard Reference, Release 12. U.S. Department of Agriculture, Agriculture Research Service, Nutrient Data Laboratory Home Page, http:// www.nal.usda.gov/fnic/foodcomp, 1998.

in the olive. When the olives turn green, it is a good time to pick them. The acidity and oil content will continue to increase as they turn purple and black. For the most part, the nutrient composition of olives shown in Table 12.1 is representative. One large olive will supply 5.1 Kcal. Most of the caloric value is supplied by fat, followed by carbohydrate and protein. Olive oil is derived from the fresh, ripe fruit and makes up about 20% of the olive by weight. One of the most studied aspects of olives is the fatty acid content, with the oil being a good source of the monounsaturated fatty acid oleate. Oleate may range from 56% to 84% of the fatty acid content.1 Olive oil also contains the saturated fatty acids palmitoleate and stearate in small amounts, the polyunsaturated fatty acids linoleate, and to a small degree linolenate.2 Linoleate may make up 3%–21% of the fatty acid content.1

12.3  OLIVE OIL The best-quality olive oil is termed virgin oil or extra virgin olive oil (EVO). This is the oil that is first expressed under light pressure during processing and not further refined. This process is a very significant part of olive oil production. The fact that it has fewer polyunsaturated fatty acids than other oils gives it a better shelf life. Furthermore, it has a mixture of tocopherols, including vitamin E, which can give a protective effect.4 Figure 12.1 illustrates the method commonly used in production of extra virgin olive oil. The olives can be hand-picked from the olive tree, or the trees may be beaten with poles to loosen the olives. Some machines collect the olives into nets as a tractor shakes the branches of the olive trees. Most olive oil on the market is expressed under heavy pressure and undergoes further refinement. Olives should be processed within 24 h of picking, especially if the weather is hot. They should be processed regardless within 72 h of picking. Typically, olive oil may oxidize easily and produce a strong flavor. Thus, protection from light and heat will increase its shelf life considerably.3 It is important that during processing no heat or chemicals be used to produce extra

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FIGURE 12.1  Scheme of the production of extra virgin olive oil. After olives are harvested and cleaned, within 72 h they must be crushed and made into a pulp. After that point, the pulp is cold-pressed in the absence of light and oxygen and the oil expelled and bottled in opaque glass bottles.

virgin olive oil. After the olives are harvested, they are washed and then ground up with the pits into a pulp in a mill made from stainless steel. Before industrialization, granite rocks were used to grind the olives. All the oil is pressed from the pulp and then collected. If the process occurs above 86°C, it is no longer considered “cold press,” which is one of the characteristics of extra virgin olive oil. Using this method, 90% of the oil is extracted from the olives. To obtain the remaining 10% requires heat and/or a chemical process, producing an oil that would not be considered extra virgin olive oil. The three basic grades of olive oils that consumers have access to are: (1) extra virgin, (2) virgin, and (3) olive oils. The interest in the health benefits of olive oil is due to the low incidence of coronary heart disease and even cancer, particularly breast cancer, in cultures that consume a “Mediterranean diet.” This diet is rather high in fruits, vegetables, grains, and legumes, but low in meat. Much of the evidence that links the Mediterranean diet to a lower incidence of coronary heart disease has centered around the relatively high oleate, but low saturated, fat content. In fact, this diet is associated with a lower incidence of several chronic diseases.5,6 Diets in the Mediterranean area are characterized by a high content of oleic acid compared to diets in other Northern European cultures and North America. It is well known that monounsaturated fatty acids may lower blood cholesterol levels and may increase HDL cholesterol levels, which could be a link between olive consumption and the lower incidence of coronary heart disease. With respect to olive oil intake and cancer rate, the mechanisms for such observations are less clear. This review will focus on two health conditions as affected by olive consumption: Coronary heart disease and cancer—but will also consider generalized health aspects. The components of olives, in addition to fatty acids, will be evaluated, as will other compounds such as polyphenols.

12.4  CORONARY HEART DISEASE 12.4.1  Fatty Acids in the Mediterranean Diet Prescription of a Mediterranean diet to patients who have had a myocardial infarct decreases the risk of a second cardiovascular accident,7 which may be due to several factors. It is commonly accepted that saturated fatty acids are twice as effective at raising blood cholesterol as are polyunsaturated

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and monounsaturated fatty acids at lowering blood cholesterol. The consensus appears to be that mono- and polyunsaturated fatty acids are similar in terms of their cholesterol-lowering abilities. Several studies have shown that monounsaturated fatty acids decrease total blood cholesterol, LDL cholesterol, apolipoprotein B, and triglycerides, with no changes in HDL cholesterol and Apo-I plasma levels.8 Elder and Kirchgessner9 reported that rats fed linseed oil, as opposed to olive oil, had lower concentrations of cholesterol, triglycerides, and phospholipids in plasma and lipoproteins, but a higher susceptibility of LDL to lipid peroxidation. This latter factor, the susceptibility of lowdensity lipoprotein cholesterol to oxidation, yields a more potent atherogenic compound and may be more significant. Also, although polyunsaturated fatty acids may lower blood lipids,10,11 they elevate the oxidative susceptibility of LDL, in contrast to fats that contain elevated saturated and monounsaturated fatty acids.12,13

12.4.2 Other Olive Constituents and Their Effects It is not always clear if the resistance to oxidation resulting from the Mediterranean diet is due only to oleic acid or to other non-triglyceride components present in oleic acid-rich oils. The minor constituents of virgin olive oil are nonglycerides such as hydrocarbons, monoglyceride esters, tocopherols, alkanols, flavonoids, anthocyanins, hydroxy and dihydroxyterpenic acids, sterols, polyphenols, and phosopholipids.1,14,15 The Mediterranean diet is high in polyphenolic compounds, and olives have a high amount of these substances. The level of these compounds is variable, with 50–800 mg/kg olive oil reported, and is dependent upon several agronomic factors, including soil, degree of olive ripeness, and cultivar or olive variety.1 There are a number of phenolic compounds in extra virgin oil (Table 12.2). The simple phenolic compounds are hydroxytyrosol (3,4-dihydroxyphenylethanol), tyrosol, and phenolic acids such as vanillic and caffeic acids. The complex phenolic compounds are tyrosol, hydroxytyrosol esters, oleuropein, and its aglycone. Oleuropein is the phenol that contributes primarily to the bitter taste of olives,1,15 but other phenolic compounds may contribute some bitterness as well. In addition to the phenolic compounds described, newer information has revealed the presence of the lignan class of phenolics such as (+)-1-acetoxypinoresinol, (+)-pinoresinol, and (+)-1-hydroxypinoriesinol.4 For extra virgin olive oil, the levels of these lignans can be as high as 100 mg/kg in the oils, but variation does exist.4

12.4.3 Olives as Sources of Antioxidants Phenols are very good antioxidants. The greater the phenol content in virgin olive oil, the better the oxidative stability. Hydroxytyrosol can donate a hydrogen to free radicals, thereby neutralizing their potential harmful effects, as demonstrated in Figure 12.2. Another factor is that hydroxytyrosol can chelate metal ions, which are themselves prooxidant agents. However, it is important that metal TABLE 12.2 Phenolic Compounds in Extra Virgin Olive Oil Hydroxytyrosol Tyrosol Oleuropein Vanillic acid Caffeic acid Lignans: (+)-1-acetoxypinoresinol (+)-pinoresinol (+)-1-hydroxypinoriesinol

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FIGURE 12.2  Hydroxytyrosol’s antioxidant mechanism. Hydrogen is donated from the hydroxyl groups of the phenol ring structure to free radicals to generate stable compounds. Two hydrogen atoms per compound can react, resulting in a carbonyl structure on the phenol ring of the hydroxytyrosol.

ions be removed during processing, as their presence can lead to partial degradation of the phenolic compounds in the oil.16 With respect to the ability of the various phenolic compounds to protect against LDL cholesterol oxidation, both hydroxytyrosol and oleuropein inhibit CuSO4-induced oxidation of LDL, and the effect appears to be dose dependent. Luteolin and lutean aglycon are both effective in protecting against LDL oxidation.17,18 Visioli and Galli1 reported that oleuropein and hydroxytyrosol are equally or more effective than other antioxidants such as butylated hydroxytoluene (BHT), vitamin C, and vitamin E. Incubation of LDL with olive oil phenolics (oleuropein or hydroxytyrosol) reduced the fall in vitamin E levels. Normally, virtually all of the vitamin E would have disappeared in 30 min, but 80% remained in the presence of phenols. A lower number of compounds such as isoprostanes, malonaldehyde, and lipid peroxides were present. The presence of these substances is relatively indicative of free-radical activity. Also, both phenolic compounds prevented the oxidation of linoleic and docosahexaenoic compounds in the LDL phosopholipids. Phenols can also inhibit platelet aggregation. Reduced TXB2 and LTB4 production by activated leukocytes is a known effect of olive phenolics.1,15 In one study, Nicolaiew et al.14 used 10 normolipidemic subjects in a crossover design in which they received virgin olive oil or sunflower oil for 3 weeks each. Plasma levels of LDL cholesterol did not change in both diets in either the fasting or postprandial states. LDL oxidation, as measured by the formation of conjugated dienes, decreased after the olive oil diet. The results were mixed, in that there was a decrease in the level of conjugated dienes at the beginning and at the end of the oxidation reaction, but the total diene production (maximal-diene at time zero) in the presence of CuSO4 did not differ.

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Many studies on olive oil are linked to studies on the Mediterranean diet where other dietary factors could play a role in the findings. However, recent studies have demonstrated that antioxidant capacity is enhanced by adherence to the Mediterranean diet.19 The Attica area of Greece studied 3042 male and female adult subjects without evidence of cardiovascular disease. Total antioxidant capacity of the serum samples were obtained. This approach involves determining the extent to which the addition of exogenous hydrogen peroxide reacts with antioxidants already in the serum. Total antioxidant capacity was positively correlated with fruits, vegetables, and olive oil intake as found in a Mediterranean diet, but inversely correlated with consumption of red meat. Furthermore, low oxidized LDL cholesterol levels were reported for those with greater total antioxidant capacity.

12.4.4 Olive Oil and Inflammation Another theory has emerged suggesting olive oil and its phenolic compounds are mediators of inflammation. Miles et al.20 conducted a systematic study of the different components of olive oil such as vanillic, p-coumaric, syringic, homovanillic and caffeic acids, kaempferol, oleuropein glycoside, and tyrosol to determine the degree to which they were able to inhibit the proinflammatory effects of lipopolysaccharide using diluted human blood cultures. They studied several cytokines, and the results suggested that the phenolic compounds all had differing degrees of inhibiting cytokines at different concentrations. For instance, oleuropein glycoside and caffeic acid decreased the concentration of interleukin-1β. However, oleuropein at a concentration of 10− 4 M inhibited interleukin-1β production by 80%, but caffeic acid only reduced it by 40% at the same concentration. Kaemferol decreased prostaglandin E2 by 95% at a concentration of 10− 4 M. These phenolics did not appear to affect interleukin-6 or tumor necrosis in this in vitro study. Moreover, a study on human subjects who consumed the Mediterranean diet reported that serum levels of tumor-necrosis factor-α and vascular cell adhesion molecule (VCAM)-1 were markedly decreased.21 Further, the authors were able to separate out the effects of olive oil vs. other components of the diet and found that both gave similar results. Ruano et al.22 studied endothelial function in hypercholesterolemic men as a result of an acute response to a meal high in virgin olive oil. Five men and 16 women from Cordoba, Spain, with cholesterol between 200 and 350 mg/100 mL were also studied. In a crossover design, subjects received two fat meals consisting of 60 g of white bread and 40 mL of virgin olive oil with either low or high phenolic acid content. Venous blood was sampled for periods of time after ingestion up to 240 min after consumption. Ischemic reactive hyperemia was measured with a laser-Doppler probe. Subjects on the high phenolic acid olive oil diet had significantly greater increases in ischemic reactive hyperemia starting 120 min after meal ingestion than those on the low phenolic acid diet. This suggested improved endothelial function among these subjects. Further analysis revealed a reduction in oxidative stress and an increase in nitric oxide metabolites. Hydroxytyrosol, present in olives, is a diphenolic compound common in extra virgin olive oil and may be a potent antioxidant. The superoxide radical (O2• −) and nitric oxide (NO−) react rapidly to form peroxynitrite (ONOO−), which is a chemical that is very reactive and can cause tissue damage. Nitric oxide may contribute to inflammatory diseases and cardiovascular disease. Hydroxytyrosol has been shown to be highly protective against the peroxynitrite-dependent nitration of tyrosine and DNA damage by peroxynitrite in vitro.23 On the other hand, oleuropein can increase nitric oxide release by cultured macrophages after endotoxin challenge by increasing nitric oxide-synthase expression. This may be beneficial in the sense that NO may guard against infectious agents and parasites.24

12.4.5 Hypertension and Olive Oil Consumption There is some evidence that olive oil may lower blood pressure. One study reported that a diet enriched with olive oil reduced the mean blood pressure in adult men and women.25 For those with hypertension, a crossover study in women revealed that olive oil, as opposed to high oleicacid sunflower oil, significantly reduced both systolic and diastolic blood pressure.26 This suggests

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that constituents of olive oil other than fatty acids may be contributing to these findings. A recent study by Alonso and Martinez-Gonzalez27 in Spain of 6863 adults, the Seguimiento Universidad de Navarra (SUN) study, revealed lower blood pressure among men who consumed more olive oil in their diets, but no such relationship was observed among women. Furthermore, Fito et al.28 reported that extra virgin olive oil, as compared to refined olive oil, lowered systolic blood pressure in hypertensive patients. However, diastolic blood pressure, blood glucose, lipids, and even oxidized LDL cholesterol did not differ between the refined olive oil group and the extra virgin olive oil group.

12.5 CANCER 12.5.1 Breast Cancer and Olive Oil In modern cultures, a switch from a low-fat diet that contains a high proportion of monounsaturated fatty acids to a high-fat diet containing a high proportion of saturated fatty acids may be contributing to the increased incidence of cancer, including breast cancer. There is geographic variation in the incidence of breast cancer, and this variation is coincident with the consumption of a high oleic acid intake derived from olive oil, typical of the Mediterranean diet.29 Case control studies have yielded evidence of a protective association between oleic acid or olive oil consumption and breast cancer. Animal experiments indicate that oleic acid may be protective when ingested in a vehicle both very high in oleic acid and very low in linoleic acid, which is typical of olive oil. Consumption of olive oil has been shown to reduce mammary tumor incidence even when compared with safflower oil, which contains similar amounts of oleic acid but higher levels of linoleic acid.30,31 Moreover, experiments with feeding rats a 15% olive oil diet significantly reduced tumor incidence caused by the carcinogenic compound, 9,10-dimethyl-1,2-benzanthracene.32 Simsonsen et al.33 hypothesized that an olive oil diet could reduce susceptibility of tissue structures to damage by free radicals, and thus the incidence of breast cancer. This research group used gluteal fat aspirates and measured the fatty-acid profiles of subjects from various European cultures. The study included 291 postmenopausal incident breast cancer patients and 351 control subjects. Oleic acid showed a strong inverse relationship with breast cancer in Spanish cultures, but not among subjects from Berlin, Northern Ireland, the Netherlands, and Switzerland, or non-Spanish residents. One reason for the failure of this study to show any relationship of oleic acid levels to breast cancer in the non-Spanish population could be because olive oil contains other compounds such as phenols and flavonoids, which are good antioxidants. Moreover, the Spanish residents obtained their oleic acid from olive oil, whereas the other residents obtained theirs from other sources, which possibly explains these results. Epidemiological studies have yielded consistent results on the association of monounsaturated fatty acids or olive oil consumption and the incidence of breast cancer. Omega-6 fatty acids enhance carcinogenesis promotion,34,35 but omega-3 fatty acids from fish inhibit this phase.36,37 The impact of omega-6 fatty acid-rich diets is thought to be related to eicosanoid products, such as prostaglandins E2 and F2α, and thromboxane B2, which are elevated in N-nitrosomethylurea-induced rat mammary cancer.38 On the other hand, there have been epidemiology studies reporting a higher risk from increased polyunsaturated fat consumption for breast cancer. Landa et al.39 studied 100 breast cancer subjects and 100 controls using a food-frequency instrument. Those with breast cancer reported lower intakes of fish, fruits, and vegetables compared to controls. Those with breast cancer also had lower intakes of vitamin C and monounsaturated fatty acids. Martin-Moreno et al.40 used a case-control study in Spain and examined specific nutrient intakes using a food-frequency questionnaire in 762 newly diagnosed breast cancer women and compared this to 988 randomly selected control females. Both total fat and type of fat intake were not associated with breast cancer in either pre- or postmenopausal women, after adjustment for energy intake. However, a lower risk of breast cancer was reported in those who consumed higher amounts of olive oil. Trichopoulou et al.41 used a semi-quantitative food-frequency instrument administered to 820 women with breast cancer and 1548 control women from Greece to estimate the intakes of olive oil, margarine, and other food items. After adjustment for some other

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potential confounding factors, increased olive oil consumption was associated with a significantly reduced risk for breast cancer. Margarine consumption was associated with a greater risk of breast cancer. They also reported that fruit and vegetable consumption was inversely related to breast cancer in the same study. In a much larger study in Italy, 2564 women hospitalized with breast cancer were compared to 2588 women admitted to the same hospital for other health conditions not related to breast cancer, hormone problems, or gastric disorders.42 Using a food-frequency questionnaire, this study demonstrated an inverse relationship with olive oil and other vegetable oil consumption and the incidence of breast cancer. No relationship for butter or margarine were reported.

12.5.2 Prostate Cancer and Olive Oil Olive oil may protect against prostate cancer. Southern European populations of Greece, Italy, Portugal, and Spain have lower rates of prostate cancer, and perhaps the Mediterranean diet that is high in olive oil may be a factor.43,44 Studies have suggested that diets high in olive oil may afford protection against prostate cancer. Hodge et al.45 reported in a case-controlled study of 858 men below 70 years of age with prostate cancer, compared to 905 age-frequency-matched men in Australia, that diets with high levels of olive oil, tomatoes, and allium-containing vegetables reduce the risk of prostate cancer. However, the association with olive oil in that study was weak. It was also unclear whether the fatty acids or the antioxidants in olive oil were the responsible factor. Many studies on prostate cancer have reported inconsistent results for the effect of fatty acid intake on prostate cancer.46,47 However, margarine consumption was related to an increased risk of prostate cancer. A New Zealand study revealed that diet patterns high in monounsaturated fatty acid-rich vegetable oils reduced the risk of prostate cancer in 317 prostate cancer cases compared to 480 controls.48 However, the association was with the foods high in monounsaturated fatty acids and not the fatty acids per se. This suggested that other components in these foods (e.g., phenolic compounds) could be contributing factors.

12.5.3 Other Cancers and Olive Oil In addition to the role of olive oil in lowering the incidence of breast tumors, later studies have suggested that other cancer types may benefit from a diet high in olive oil. Franceschi et  al.49 examined cases of 512 men and 86 women from Northeastern Italy who had cancer of the oral cavity and pharynx and compared them to 1008 men and 483 women controls who had been admitted to area hospitals for ailments other than neoplastic conditions. Subjects were administered a dietary questionnaire to evaluate fat intake and other lifestyle aspects. Risk for these cancers was reduced by at least 50% in subjects with the highest intakes of several food items, including poultry, fish, raw and cooked vegetables, citrus fruits, and olive oil.

12.5.4 Summary and Future Need for Cancer Research and Olive Oil While much of the work on olive oil intake and cancer has focused upon the monounsaturated fatty acid content, the antioxidant compounds present may play an important role in its benefits as it apparently does for heart disease, as reviewed in earlier text. Furthermore, studies examining the antioxidant effects of olive oil on various cancers are surprisingly limited and thus afford more opportunity for investigation.

12.6  OTHER DISEASE CONDITIONS AND OLIVE OIL Heart disease and cancer are the two diseases that show a reduction in risk with increased olive oil intake as found in the Mediterranean diet. Recently it has been suggested that metabolic syndrome can be prevented by adherence to the Mediterranean diet.50 Metabolic syndrome consists of a combination of conditions, including hypertension, abdominal obesity, increased fibrinogen, insulin resistance, increased blood viscosity, and uric acid levels.51 These conditions predispose the individuals to be

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at high risk for cardiovascular disease. Esposito et al.52 conducted a randomized clinical trial among 90 men and women with metabolic syndrome. The intervention group followed a Mediterranean diet and the control group followed the prudent diet where carbohydrates provided 50%–60%, protein 15%–20%, and total fat less than 30%. Two years later, serum C-reactive protein levels, whose elevation indicates metabolic syndrome, were significantly reduced. Furthermore, insulin resistance, interleukin-6, and improved endothelial function were found in those on the Mediterranean diet. Interestingly, some recent studies have suggested that a combination of fish oil with olive oil may prove beneficial in treatments of inflammation-related conditions. The omega-3 fatty acids present in fish oils have a well-known impact upon attenuating the inflammatory response. Berbert et al.53 reported that treatment of rheumatoid arthritis patients with fish oil plus olive oil resulted in superior relief of clinical arthritic symptoms as opposed to those supplemented with fish oil only. The control group received soy oil. Subjects consuming both supplements of fish and olive oils were better able to withstand pain on handgrip tests, had reduced duration of morning stiffness, and increased time before fatigue became apparent. Camuesco et al.54 reported that fish oil and olive oil were superior in reducing inflammation in the colons of rats that were induced to develop colitis with dextran sodium sulfate (DSS). Tumor necrosis factor-α and LTB4 levels were reduced in rats treated with both fish and olive oils.

12.7  REVIEWS AND CONSENSUS REPORTS Several reviews and international conference consensus reports exist for the health benefits of virgin olive oil.55–59 In addition to the influence of extra virgin olive oil upon cardiovascular disease and cancer, the report goes further to suggest that EVO may be protective against age-related cognitive decline and Alzheimer’s disease. They further recommended that olive oil intake is especially important during the first decades of life and particularly that EVO intake should begin before puberty and continue throughout life. The most relevant molecular effects of EVOO involved in the prevention or resolution of liver damage are: (1) activation of the nuclear transcription factor erythroid-derived 2-like 2 (Nfr2), inducing the cellular antioxidant response; (2) inactivation of the nuclear transcription factor-κB (NF-κB), preventing the cellular inflammatory response; and (3) inhibition of the PERK pathway, preventing endoplasmic reticulum stress, autophagy, and lipogenic response.

12.8 SUMMARY Clearly, the monounsaturated content of the Mediterranean diet, with respect to the intake of olive oil, plays a significant role in the lower incidence of both coronary heart disease and cancer, particularly breast cancer. The antioxidant compounds present in extra virgin olive oil allow for protection against LDL cholesterol oxidation and thus spare other antioxidant nutrients. The role of these same antioxidants in protecting against various cancers should be pursued. Therefore, further examination of olive oil intake and other cancer types may also yield beneficial information. The health benefits of olive oil due to its active compounds should focus more on those agronomic factors that optimize their content. Additionally, further knowledge on the genetic regulation of the production of antioxidant phenolic compounds would be worthwhile. Increasing the content of these valuable nutrients to protect against both coronary heart disease and cancers is a good example of functional food for health. Furthermore, extraction of these compounds from olives and concentrating them for clinical trials, both animal and human, may provide better insights into their utility as nutraceuticals for the future.

REFERENCES 1. Visioli, F. and Galli, C. The effect of minor constituents of olive oil on cardiovascular disease: New findings. Nutr. Rev. 56: 142–147, 1998. 2. USDA Nutrient Database for Standard Reference, Release 12. U.S. Department of Agriculture, Agriculture Research Service, Nutrient Data Laboratory Home Page, http://www.nal.usda.gov/fnic/ foodcomp, 1998.

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3. Freeland-Graves, J.M. and Peckham, G.C. Foundations of Food Preparation. 5th ed. MacMillan, New York, 1987. 4. Owen, R.W., Mier, W., Giacosa, A., Hull, W.E., Spiegelhalder, B., and Bartsch, H. Identification of lignans as major compounds in the phenolic fraction of olive oil. Clin. Chem. 46: 976–988, 2000. 5. World Health Organization Study Group. Diet, Nutrition, and the Prevention of Chronic Diseases. World Health Organization Tech. Rep. Ser. 16, 2003. 6. Ganji, V. and Kafai, M.R. Demographic, health, lifestyle and blood vitamin determinants of serum total homocysteine concentrations in the third National Health and Nutrition Examination Survey, 1988–1994. Am. J. Clin. Nutr. 77: 826–833, 2003. 7. Renaud, S., DeLorgeril, M., Delaye, J., Guidollet, J., Jacquard, F., Mamelle, N., Martin, J.L., Monjaud, I., Salen, P., and Toubol, P. Cretan Mediterranean diet for prevention of coronary heart disease. Am. J. Clin. Nutr. 61: 1360–1367, 1995. 8. Baggio, G., Pagnan, A., Muraca, M., Martini, S., Opportuno, A., Bonanome, A., Ambrosio, G.B., Ferrari,  S., and Crepaldi, G. Olive oil-enriched diet: Effect on serum lipoprotein levels and biliary cholesterol saturation. Am. J. Clin. Nutr. 47: 960–964, 1998. 9. Elder, K. and Kirchgessner, M. Concentrations of lipids in plasma and lipoproteins and oxidative susceptibility of low-density lipoproteins in zinc-deficient rats fed linseed oil or olive oil. J. Nutr. Biochem. 8: 461–468, 1997. 10. Stangl, G.I., Kirchgessner, M., Reichlmayr-Lais, A.M., and Eder, K. Serum lipids and lipoproteins from rats fed different dietary oils. J. Anim. Physiol. Anim. Nutr. 71: 87–97, 1994. 11. Balasubramaniam, S., Simons, L.A., Chang, S., and Hickie, J.B. Reduction in plasma cholesterol and increase in biliary cholesterol by a diet rich in n-3 fatty acids in the rat. J. Lipid Res. 26: 684–689, 1985. 12. Scaccini, C., Nardini, M., D’Aquino, M., Gentili, V., Di Felice, M., and Tomassi, G. Effect of dietary oils on lipid peroxidation and on antioxidant parameters of rat plasma and lipoprotein fractions. J. Lipid Res. 33: 627–633, 1992. 13. Parthasarthy, S., Khoo, J.C., Miller, E., Barnett, J., Witztum, J.L., and Steinberg, D. Low density lipoprotein rich in oleic acid is protective against oxidative modification: Implication for dietary prevention of atherosclerosis. Proc. Natl. Acad. Sci. USA 87: 3894–3898, 1990. 14. Nicolaiew, N., Lemort, N., Adorni, L., Berra, B., Montorfano, G., Rapelli, S., Cortesi, N., and Jacotot, B. Comparison between extra virgin olive oil and oleic acid rich sunflower oil: Effect on postprandial lipemia and LDL susceptibility to oxidation. Ann. Nutr. Metab. 42: 251–260, 1998. 15. Visioli, F. and Galli, C. Olive oil phenols and their potential effects on human health. J. Agric. Food Chem. 46: 4292–4296, 1998. 16. Angerosa, F. and DiGiacinto, L. Oxidation des huiles d’olive vierges par le métaux manganese et nickel, Note 1 (Metal-induced oxidation of virgin olive oils: Manganese and nickel. Note 1), Rev. Fr. Corps Gras. 40: 41–44, 1993. 17. Visioli, F., Bellomo, G., Montedoro, G.F., and Galli, C. Low-density lipoprotein oxidation is inhibited in vitro by olive oil constituents. Atherosclerosis 117: 25–42, 1995. 18. Visioli, F. and Galli, C. Oleuropein protects low-density lipoprotein from oxidation. Life Sci. 55: 1965–1971, 1994. 19. Pitsavos, C., Panagiotakos, B.B., Tzima, N., Chrysohoou, C., Economou, M., Zampelas, A., and Stefanadis, C. Adherence to the Mediterranean diet is associated with total anti-oxidant capacity in healthy adults: The ATTICA study. Am. J. Clin. Nutr. 82: 694–699, 2005. 20. Miles, E.A., Zoubouli, P., and Calder, P.C. Differential anti-inflammatory effects of phenolic compounds from extra virgin olive oil identified in human whole blood cultures. Nutrition 21: 389–394, 2005. 21. Serrano-Martinez, M., Palacios, M., Martinez-Losa, E., Lezaun, R., Maravi, C., Prado, M., Martinez, J.A., and Martinez-Gonzalez, M.A. A Mediterranean dietary style influences TNF-alpha and VCAM- 1 coronary blood levels in unstable angina patients. Eur. J. Nutr. 44: 349–354, 2005. 22. Ruano, J., Lopez-Miranda, J., Fuentes, F., Morena, J.A., Bellido, C., Perez-Martinez, P., Lozano, A., Jiménez, Y., and Jiménez, F.P. Phenolic content of virgin olive oil improves ischemic reactive hyperemia in hypercholesterolemic patients. J. Am. Coll. Cardiol. 46: 1864–1868, 2005. 23. Deiana, M., Aruoma, O.I., Bianchi, M.L.P., Spencer, J.P.E., Kaur, H., Halliwell, B., Aeschbach, R., Banni, S., Dessi, M.A. and Corongiu, F.P. Inhibition of peroxynitrite dependent DNA base modification and tyrosine nitration by the extra virgin olive oil-derived antioxidant hydroxytyrosol. Free Radic. Biol. Med. 26: 762–769, 1999. 24. Visioli, F., Bellosta, S., and Galli, C. Oleuropein, the bitter principles in olives, enhances nitric oxide production by murine macrophages. Life Sci. 62: 541–546, 1998.

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25. Lahoz, C., Alonso, R., Porres, A., and Mata, P. Las Dietas Enriquecidas en Ácidos Gracos Monoin— saturadoes y Ácidos Gracos Poliinsaturado Omega 3 Disminuyen la Presion Artersial, sin Modificar la Concentración de Insulina Plasmática en Sujetos Sanos. Med. Clin. (Barc). 112: 133–137, 1999. 26. Ruiz-Gutierres, V., Muriana, F.J., Guerrero, A., Cert, A.M., and Villar, J. Plasma lipids, erythrocyte membrane lipids and blood pressure of hypertensive women after ingestion of dietary oleic acid from two different sources. J. Hypertens. 14: 1483–1490, 1996. 27. Alonso, A. and Martinez-González, M.Á. Olive oil consumption and reduced incidence of hypertension: The SUN study. Lipids. 39: 1233–1238, 2004. 28. Fito, M., Cladellas, M., de la Torre, R., Marti, J., Alcántara, M., Pujadas-Bastardes, M., Marrugat, J., Bruguera, J., López-Sabater, M.C., Vila, J. et al. Antioxidant effect of virgin olive oil in patients with stable coronary heart disease: A randomized, crossover, controlled, clinical trial. Atheroscleorsis 181: 149–158, 2005. 29. Berrino, F. and Muti, P. Mediterranean diet and cancer. Eur. J. Clin. Nutr. 43: 49–55, 1989. 30. Cohen, L.A., Thompson, D.O., Maeura, Y., Choi, K., Blank, M.E., and Rose, D.P. Dietary fat and mammary cancer. I. Promoting effects of different dietary fats on N-nitrosomethylurea-induced rat mammary tumorigenesis. J. Natl. Cancer Inst. 77: 33–42, 1986. 31. Lasekan, J.B., Clayton, M.K., Gendron-Fitzpatrick, A., and Ney, D.M. Dietary olive and safflower oil in promotion of DMBA-induced mammary tumorigenesis in rats. Nutr. Cancer 13: 153–163, 1990. 32. Zusman, I. Comparative anticancer effects of vaccination and dietary factors on experimentally-induced cancers. In vivo 12: 675–689, 1998. 33. Simsonsen, N.R., Navajas, J.F.C., Martin-Moreno, J.M., Strain, J.J., Huttnen, J.K., Martin, B.C., Thamm,  M. et  al. Tissue stores of individual monounsaturated fatty acids and breast cancer: The EURAMIC study. Am. J. Clin. Nutr. 68: 134–141, 1998. 34. Carrol, K.K. Experimental evidence of dietary factors and hormone-dependent cancers. Cancer Res. 35: 3374–3383, 1975. 35. Cohen, L.A., Thompson, D.O., Maeura, Y., Choi, K., Blank, M.E., and Rose, D.P. Dietary fat and mammary cancer. I. Promoting effects of different dietary fats on N-nitrosomethylurea-induced rat mammary tumorigenesis. J. Natl. Cancer Inst. 77: 33–42, 1986. 36. Jurkowski, J.J. and Cave, W.T., Jr. Dietary effects of menhaden oil on the growth and membrane lipid composition of rat mammary tumors. J. Natl. Cancer. Inst. 74: 1145–1150, 1985. 37. Cohen, L.A., Chen-Backlund, J.Y., Sepkovic, D.W., and Sugie, S. Effect of varying proportions of dietary menhaden corn oil on experimental rat tumor promotion. Lipids 28: 449–456, 1993. 38. Karmali, R.A., Thaler, H.T., and Cohen, L.A. Prostaglandin concentrations and prostaglandin synthase activity in N-nitrosomethylurea-induced rat mammary adenocarcinoma. Eur. J. Clin. Oncol. 19: 817–823, 1983. 39. Landa, M.C., Frago, N., and Tres, A. Diet and the risk of breast cancer in Spain. Eur. J. Cancer Prev. 3: 313–320, 1994. 40. Martin-Moreno, J.M., Willett, W.C., Gorgojo, L., Banegas, J.R., Rodriguez-Artalejo F., FernandezRodrigues J.C., Maisonneuve, P., and Boyle, P. Dietary fat, olive oil intake and breast cancer risk. Int. J. Cancer. 58: 774–780, 1994. 41. Trichopoulou, A., Katsouyanni, K., Stuver, S., Tzala, L., Gnardellis, C., Rimm, E., and Trichopoulos, D. Consumption of olive oil and specific food groups in relation to breast cancer in Greece. J. Natl. Cancer. Inst. 87: 110–116, 1995. 42. La Vecchia, C., Negri, E., Franceschi, S., Decarli, A., Giacosa, A., and Lipworth, L. Olive oil, other dietary fats, and the risk of breast cancer (Italy). Cancer Causes Control. 6: 545–550, 1995. 43. Helsing, E. Traditional diets and disease patterns of the Mediterranean, circa 1960. Am. J. Clin. Nutr. 61 (Suppl.): 1329S–1337S, 1995. 44. Mezzanotte, G., Cislagni, C., Decarli, A., and LaVecchia, C. Cancer mortality in broad Italian geographic areas, 1975–1977. Tumori 72: 145–152, 1986. 45. Hodge, A.M., English, D.R., McCredie, M.R.E., Severi, G., Boyle, P., Hopper, J.L., and Giles, G.G. Foods, nutrients and prostate cancer. Cancer Causes Control. 15: 11–20, 2004. 46. Clinton, S.K. and Giovannucci, E. Diet, nutrition, and prostate cancer. Annu. Rev. Nutr. 18: 413–440, 1998. 47. Kolonel, L.N. Fat, meat and prostate cancer. Epidemiol. Rev. 23: 72–81, 2001. 48. Norrish, A.E., Jackson, R.T., Sharpe, S.J., and Skeaff, C.M. Men who consume vegetable oils rich in monounsaturated fat: Their dietary patterns and risk of prostate cancer (New Zealand). Cancer Causes Control 11: 609–615, 2000.

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49. Franceschi, S., Favero, A., Conti, E., Talamini, R., Volpe, R., Negri, E., Barzan, L., and La Vecchia, C. Food groups, oils and butter, and cancer of the oral cavity and pharynx. Br. J. Cancer. 80: 614–620, 1999. 50. Panagiotakos, D., and Polychronopoulos, E. The role of Mediterranean diet in the epidemiology of metabolic syndrome: Converting epidemiology to clinical practice. Lipids Health Dis. 4: 7, 2005 http:// www/lipiworld.com/content/4/1/7 51. Hansen, B.C. The metabolic syndrome X. Ann. N. Y. Acad Sci. 892: 1–24, 1999. 52. Esposito, K., Marfella, R., Ciotola, M., DiPalo, C., Giugliano, F., Giuliano, G., D’Armiento, M., D’Andrea, F., and Gingliano, D. Effect of a Mediterranean-style diet on endothelial dysfunction and markers of vascular inflammation in the metabolic syndrome: A randomized trial. JAMA 292: 1440–1446, 2004. 53. Berbert, A.A., Kondo, C.R.M., Almendra, C.L., Matsuo, T., and Dichi, I. Supplementation of fish oil and olive oil in patients with rheumatoid arthritis. Nutrition 21: 131–136, 2005. 54. Camuesco, D., Galvez, J., Nieto, A., Comalada, M., Rodrigues-Cabezas, M.E., Concha, A., Xaus, J., and Zarzuelo, A. Dietary olive oil supplemented with fish oil, rich in EPA and DHA (n-3) polyunsaturated fatty acids, attenuates colonic inflammation in rats with DDS-induced colitis. J. Nutr. 135: 687–694, 2005. 55. Perez-Jimenez, F. International conference on the health effect of virgin olive oil. Eur. J. Clin. Invest. 35: 421–424, 2005. 56. Nocella, C., Cammisotto, V., Fianchini, L., D’Amico, A., Novo, M., Castellani, V., Stefanini, L., Violi, F., and Carnevale, R. Extra virgin olive oil and cardiovascular diseases: Benefits for human health. Endocr. Metab. Immune Disord. Drug Targets 18:(1): 4–13, 2018. 57. Gambino, C.M., Accardi, G., Aiello, A., Candore, G., Dara-Guccione, G., Mirisola, M., Procopio, A., Taormina, G., and Caruso, C. Effect of extra virgin olive oil and table olives on the immune inflammatory responses: Potential clinical applications. Endocr. Metab. Immune Disord. Drug Targets 18(1): 14–22, 2018. 58. Santangelo, C., Vari, R., Scazzocchio, B., De Sanctis, P., Giovannini, C., D’Archivio, M., and Masella, R. Anti-inflammatory activity of extra virgin olive oil polyphenols: Which role in the prevention and treatment of immune-mediated inflammatory diseases? Endocr. Metab. Immune Disord. Drug Targets 18(1): 36–50, 2018. 59. Soto-Alarcon, S.A., Valenzuela, R., Valenzuela, A., and Videla, L.A. Liver protective effects of extra virgin olive oil: Interaction between its chemical composition and the cell-signaling pathways involved in protection. Endocr. Metab. Immune Disord. Drug Targets 18(1): 75–84, 2018.

13

Nutraceutical Herbs and Insulin Resistance Giuseppe Derosa and Pamela Maffioli

CONTENTS 13.1 Introduction........................................................................................................................... 223 13.2 Medical Plants.......................................................................................................................224 13.2.1 Agaricus blazei........................................................................................................224 13.2.2 Amorphophallus konjac..........................................................................................224 13.2.3 Ascophyllum nodosum and Fucus vesiculosus....................................................... 230 13.2.4 Avena sativa............................................................................................................ 231 13.2.5 Berberis aristata..................................................................................................... 231 13.2.6 Cinnamomum aromaticum..................................................................................... 232 13.2.7 Cyamopsis tetragonoloba....................................................................................... 232 13.2.8 Cynara scolymus..................................................................................................... 233 13.2.9 Curcuma longa........................................................................................................ 233 13.2.10 Gymnema sylvestre................................................................................................. 233 13.2.11 Glycine max............................................................................................................ 234 13.2.12 Ilex paraguariensis................................................................................................. 234 13.2.13 Lagerstroemia speciosa.......................................................................................... 234 13.2.14 Momordica charantia............................................................................................. 234 13.2.15 Morus alba.............................................................................................................. 235 13.2.16 Opuntia ficus-indica............................................................................................... 235 13.2.17 Panax quinquefolius............................................................................................... 235 13.2.18 Phaseolus vulgaris.................................................................................................. 235 13.2.19 Plantago ovata........................................................................................................ 235 13.2.20 Stevia rebaudiana................................................................................................... 236 13.2.21 Trigonella foenum-graecum.................................................................................... 236 13.2.22 Syzygium cumini...................................................................................................... 236 13.3 Phytoconstituents................................................................................................................... 237 13.3.1 α-Lipoic Acid.......................................................................................................... 237 13.3.2 Essential Fatty Acids (n-3 PUFAs).......................................................................... 237 13.4 Conclusions............................................................................................................................ 238 References....................................................................................................................................... 238

13.1 INTRODUCTION Insulin is a hormone produced by the endocrine pancreas, in particular by pancreatic β-cells, in response to hyperglycemia. Under normal conditions, muscle cells and fat cells are equipped with insulin receptors. After a meal, pancreatic β-cells increase insulin secretion, insulin binds to its receptors in muscle and adipose tissue with migration of glucose transport glucose 4 (GLUT4) channels, quiescent in the cellular cytoplasm, to the surface of the membrane cell, allowing glucose entrance in the cell. Also, the liver is equipped with insulin receptors: after the meal, insulin binds to hepatic receptors and with a cascade of signals inhibits glycogenolysis in favor of glycogenesis. Moreover, insulin links to its receptors in liver 223

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cause opening of glucose transport glucose 2 channels (GLUT2), allowing glucose to enter the hepatic cell. Insulin resistance is a condition characterized by a reduced capacity of muscular, adipose, and liver cells to respond to insulin; these cells become less sensitive to the action of insulin, making more insulin needed to perform the same actions. In the initial stages, the pancreas produces more insulin to maintain normal blood glucose levels with greater work by the β-cells and hyperinsulinemia. Over time, the pancreas begins to fail to compensate for the greater need of insulin due to insulin resistance; when this happens, glycemia begins to rise, initially after meals, and then even fasting. This constant increase in fasting blood sugar can, over the years, lead to type 2 diabetes mellitus. However, insulin resistance does not only cause diabetes, because hyperinsulinemia is also considered a predictor of future cardiovascular events.1,2 Insulin resistance, in fact, can increase fibrinogen in the blood and is closely associated with hypertension in the obese and not obese.3 For this reason, prevention is certainly important; since insulin resistance is almost always linked to excessive weight, the first intervention should be an appropriate lifestyle. Lifestyle changes require the patient to be educated to follow a proper diet, in order to obtain a correction of excess body weight. An appropriate nutritional plan is based on the model of the Mediterranean diet and provides a total fat intake of less than 30%; a supply of saturated fatty acids below 10%; a supply of fibers, half soluble, exceeding 15 g/1000 kcal; a carbohydrate intake of 45%–60%; and proteins equal to 15%–20%. Patients should limit saturated fat intake to less than 7% of daily caloric needs; monounsaturated fatty acids, such as olive oil and other vegetable oils, are recommended.4 Patient should also be encouraged to increase physical activity and, in particular, to practice aerobic activity for at least 30–40 minutes, 3–4 times a week. If the patient is a smoker, he or she should be encouraged to quit. In addition to this, it is also important to address all cardiovascular risk factors (dyslipidemia, hyperglycemia, hypertension, smoking). Following an adequate healthy lifestyle, however, is not always easy, and nutraceuticals can be helpful to patients for this reason. The science of nutraceuticals is constantly evolving; different substances have shown a favorable effect in controlling lipid profile, glycemia, hypertension, insulin resistance, and metabolic parameters.5,6 In the next pages, we will describe the main nutraceuticals with evidence of a certain action on glycemia and insulin resistance. For the description of how various nutraceuticals act, please see Table 13.1 and Figures 13.1–13.7. For a description of their chemical formula, see Table 13.2.

13.2  MEDICAL PLANTS 13.2.1  Agaricus blazei Many edible mushrooms and traditional plants have been widely screened for use as remedies for natural products for the control of diabetes. Agaricus blazei is a common mushroom in South America and Asia, and has been widely used in traditional medicine as a remedy for certain types of cancers and diabetes.7–9 In Asia, including the Republic of Korea, fruiting bodies of Agaricus blazei have a considerable reputation as a potent remedy for diabetes mellitus. A preliminary study in our laboratory showed that dried culture broth from submerged cultures of Agaricus blazei inhibits α-glucosidase activity in vitro and exhibits hypoglycemic action in streptozotocin-induced diabetic Sprague-Dawley rats. However, the molecule responsible for the hypoglycemic response, free of β-glucans and glycoproteins, is not known. Dietary supplementation with an extract of Agaricus blazei Murill has a protective effect against obesity induced by a high-fat diet. This is not due to decreased food intake but to increases in both energy expenditure and locomotor activity (especially during the dark period, which is when rodents are active), as well as decreases in pancreatic lipase activity within jejunum. As a result of the decreased body weight gain and fat mass, both plasma insulin and leptin concentrations are back to normal values.10

13.2.2  Amorphophallus konjac Konjac extract (KE) was refined from Amorphophallus konjac K.Koch, a kind of Chinese herb. KE is a kind of white crystal grain obtained from its tuber. Its main component is Konjac glucomannan,11 which is a kind of excellent edible fiber. It was reported12,13 that this polysaccharide could decrease

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TABLE 13.1 Chemical Formula Name

Chemical Formula

Fucus vesiculosus

Fucoidan

Avena sativa

Avena sativa

Berberis aristata

Berberine

Curcuma longa

Curcumin

Gymnema sylvestre

Antrachinones

(Continued)

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TABLE 13.1 (Continued) Chemical Formula Name

Chemical Formula

Glycine max

Glycine max

Lagerstroemia speciosa

Corosolic acid

Panax quinquefolius

Ginsenoides

n-3 PUFAs

α-lipoic acid

α-lipoic acid

Nutraceutical Herbs and Insulin Resistance

FIGURE 13.1  Possible mechanism of action: Stomach and bowel.

FIGURE 13.2  Possible mechanism of action: Pancreas.

FIGURE 13.3  Possible mechanism of action: Adipose tissue and muscle.

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FIGURE 13.4  Possible mechanism of action: Liver.

FIGURE 13.5  Possible mechanism of action: Gut hormone and enzyme.

FIGURE 13.6  Possible mechanism of action: Nucleus.

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FIGURE 13.7  Possible mechanism of action: Insulin receptor.

TABLE 13.2 Mechanisms of Action Molecule Agaricus blazei Amorphophallus konjac Ascophyllum nodosum and Fucus vesiculosus Avena sativa

Berberis aristata

Cinnamomum aromaticum

Cyamopsis tetragonoloba

Cynara scolymus

Mechanism of Action It increases adiponectin increase and has a metformin-like action and an α-glucosidase inhibition. It delays stomach emptying and increases adiponectin by adjusting the rate of absorption of nutrients by small bowel, thereby increasing insulin sensitivity. They modulate the activity of α-glucosidase. β-glucan is reported to increase the viscosity of food bolus, delay gastric emptying, and lengthen intestinal transit time; slow the absorption of nutrients especially the carbohydrates; and enhance satiety. It was also reported that β-glucan could slow the appearance of glucose in plasma, resulting in longer-lasting insulin secretion, which exerts a prolonged inhibition of endogenous glucose production. It improves insulin resistance partly through activation of adenosine monophosphateactivated protein kinase (AMPK) and increased phosphorylation of insulin receptor; inhibition of α-glucosidase activity. It activates the insulin receptor by multiple mechanisms that included increased autophosphorylation of the insulin receptor, increased glucose transporter 4 receptor (GLUT4) synthesis and activation, inhibition of pancreatic and intestinal amylase and glucosidase, and increases glycogen synthesis in the liver, thus improving insulin sensitivity and glycemic control. Its facilitates glucose uptake into peripheral tissues. It acts as a real insulin sensitizer. It restores the activity of glucose-6-phosphatase and fructose-1,6-bisphosphatase. It decreases mass transfer and modulates intestinal absorption of glucose. It is a powerful inhibitor of glucose 6-phosphate translocase, an essential component of the hepatic glucose 6-phosphatase system that regulates the homeostasis of blood glucose. In addition, dicaffeoylchinic acid derivatives can also play a hypoglycemic role in modulating the activity of α-glucosidase and consequently the catabolism of dietary carbohydrates. (Continued)

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TABLE 13.2 (Continued) Mechanisms of Action Molecule Curcuma longa

Gymnema sylvestre Ilex paraguariensis Lagerstroemia speciosa

Momordica charantia

Morus alba

Opuntia ficus-indica Panax quinquefolius

Phaseolus vulgaris

Plantago ovata Stevia rebaudiana Trigonella foenum-graecum Eugenia jambolana

Mechanism of Action It improves glycolipid metabolism through upregulation of the expressions of adipose triglyceride lipase and hormonesensitive lipase, peroxisome proliferator–activated receptor γ/α (PPARγ/α), and CCAAT/enhancer binding protein α (C/EBPα) in adipose tissue. It increases the release of glucose insulinotropic peptide, a potent stimulator of insulin secretion by K cells of the small intestine. It interferes with glucose absorption in the gut by decreasing SGLT1 expression. It upregulates GLUT4 expression or increases its translocation. Some of these mechanisms are mediated by PPAR encoded by different genes. Ligand-regulated transcription factors control gene expression (such as GLUT4) by binding to specific response elements within promoters. It upregulates GLUT4 expression or increases its translocation. Some of these mechanisms are mediated by PPAR encoded by different genes. Ligand-regulated transcription factors; they control gene expression (such as GLUT4) by binding to specific response elements within promoters. It modulates the activity of α-glucosidase and significantly upregulates the expressions of phosphorylated AMPK, and it might induce the conversion of white preadipocytes to beige adipocytes by activating AMPK. It affects insulin secretion by increasing the release of glucose insulinotropic peptide, a potent stimulator of insulin secretion by K cells of the small intestine. It upregulates GLUT4 expression or increases its translocation. Some of these mechanisms are mediated by PPAR encoded by different genes. Ligand-regulated transcription factors control gene expression (such as GLUT4) by binding to specific response elements within promoters. It contains soluble fiber, which contributes to an increase in self-reported satiety and a reduced rate of both gastric emptying and nutrient access by alimentary digestive enzymes. Legume-derived resistant starch and slowly digestible starch are also associated with improved glycemic response and lower post-prandial glucose concentrations. It decreases abdominal white adipose tissue ratio and white/brown adipocyte size and upregulates mRNA expressions of peroxisome proliferator activated receptors (PPARs). It enhances the first phase insulin response and concomitantly suppresses the glucagon levels. It inhibits intestinal glucosidase and has a positive effect on glycolytic and gluconeogenic enzymes to restore glucose homeostasis; it induces a significant increase in GLUT translocation. It inhibits DPP-4.

total cholesterol (TC) and blood glucose, fat, and excretion. Recent studies indicated that KE could obviously improve glucose tolerance in diabetic patients and animals.14 This was confirmed by Mao et al, who showed that KE might not only improve insulin resistance and increase insulin sensitivity, but also lower fasting plasma glucose (FPG) and glycogen in liver and skeletal muscle, but it had no effect on the release of insulin. The experimental results revealed that KE might improve insulin sensitivity by increasing glucose usage of non-oxidation approach, not depending on the release of insulin.15

13.2.3  Ascophyllum nodosum and Fucus vesiculosus Ascophyllum nodosum and Fucus vesiculosus are brown seaweed species harvested off the coast of Sea Nord that are commercially available as a nutritional supplement and feed additive. The polyphenolic composition is represented by phlorotannins, able to inhibit α-amilase and α-glucosidase with an

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important hypoglycemic action in vivo16,17 and in particular post-prandial glucose (PPG). Phlorotannins slow carbohydrate absorption with a noncompetitive (not focalized on catalitic site in competition with the substrate) and reversible mechanism of inhibition of the enzymes involved in carbohydrate degradation.16 The inhibiting action toward the activity of these enzymes results, in animal models (rats), in a reduction of glycemia and insulinemia after administration of amids and glucose. Derosa et  al. conducted a study about the effects of a hypoglycemic nutraceutical containing Ascophyllum nodosum and Fucus vesiculosus in a ratio of 95/5 and chromium picolinate.18 Also, Ascophyllum nodosum and Fucus vesiculosus act in an acarbose-like mechanism, and also, in this case, we recorded a similar reduction of glycated hemoglobin (HbA1c), FPG, PPG, and HOMA-IR compared to placebo, suggesting that reducing glucose absorption can be a valid option to prevent diabetes.

13.2.4  Avena sativa Oats, which are considered unique among the cereals, belong to the Poaceae family and are known as “Jai” or “Javi” in the Indian subcontinent. In the mid-1980s, oats were recognized as a healthy food, helping prevent heart disease, and then became more popular in human nutrition. The common oat (Avena sativa) is the most important crop among the cultivated oats. Oats are suitable for human consumption as oatmeal, rolled oats, and other oat-enriched products. Recent studies in food and nutrition have revealed the importance of the various components of oats, such as dietary fiber, especially β-glucan, minerals, and other nutrients.19 Oats and oat-enriched products have been proven to control blood glucose concentrations and to be helpful in the treatment of diabetes. Several studies have suggested that oats and oat-enriched diets can significantly decrease insulin responses, FPG, and PPG in overweight and type 2 diabetic subjects,20–25 which is mainly attributed to the markedly functional properties and enormous importance of β-glucan in human nutrition. β-glucan is a kind of high-molecular-weight polysaccharide exhibiting high viscosity at relatively low concentrations, which can reduce mixing of the food with digestive enzymes and delay gastric emptying. Increased viscosity also retards the absorption of glucose.

13.2.5  Berberis aristata Berberine is a standardized extract of Berberis aristata, an Indian medicinal plant of the Berberidaceae family, an isoquinolinic alkaloid that has been shown to decrease glucose levels and improve insulin resistance through activating of MAP-kinase and increasing the phosphorylation of insulin receptors. In addition to a positive effect on glucose metabolism, berberine also regulates the expression of the receptor for low-density lipoproteins, leading to a 30% decrease in total cholesterol and 25% in LDL cholesterol. The extract of Berberis aristata is present in various combinations and also available with the extract of Silybum marianum, a potential inhibitor of enterocyte-associated glycoprotein and known hepatoprotector. Berberis aristata, in fact, is easily absorbed at the intestinal level, but is characterized by poor oral bioavailability, as it is the substrate of several ABC transporters, such as P-glycoprotein, which causes enterocitary re-extrusion. The extract of Silybum marianum, being a P-glycoprotein inhibitor, is able to allow berberine to bypass P-glycoprotein and be absorbed at a higher concentration. The patented combination of Berberis aristata/Silybum marianum based on 588 mg of Berberis extract aristata titrated to 85% in berberine (equivalent to 500 mg of berberine) and 105 mg of extract of Silybum marianum titrated to 65% in flavanolignanes has shown good efficacy in reducing insulin resistance in several studies. A first study was conducted by Di Pierro et al.;26 it was a single-blind, randomized, controlled, 4-month trial, conducted on 69 normocholesterolemic patients with type 2 diabetes mellitus and non-optimal glycemic control (HbA1c between 7.0% and 9.0%). Patients were randomized to Berberis aristata at a dose of two tablets/day, corresponding to 500 mg of berberine, or to Berberis aristata/Silybum marianum at a dose of two tablets/day for 120 days. The therapy taken before enrollment was not changed. Both Berberis aristata and Berberis aristata/Silybum marianum reduced FPG (−19.05%,

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and −18.13%, respectively) and HbA1c (−7.18% and −12.35%, respectively), with a better effect of Berberis aristata/Silybum marianum on HbA1c. The results are not surprising since it has been said above that Silymarin serves to increase the bioavailability of berberine, so a combination of these is more effective. Although the data of Di Pierro et al. suggest that there is an improvement in insulin resistance due to the improvement of glyco-metabolic control, the first study to directly evaluate the improvement of insulin resistance with Berberis aristata/Silybum marianum was conducted by Derosa et  al.27 This randomized, placebo-controlled, 14-month, double-blind, randomized study was conducted on 105 overweight, normotensive Caucasian patients aged ≥18 years, euglycemic and hypercholesterolemic, with cholesterol values between 200 and 240 mg/dL and triglycerides (Tg)