Natural Antimicrobial Agents [1st edition 2018] 9783319670430, 9783319670454, 3319670433, 331967045X

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Sustainable Development and Biodiversity 19

Jean-Michel Mérillon Céline Rivière Editors

Natural Antimicrobial Agents

Sustainable Development and Biodiversity Volume 19

Series editor Kishan Gopal Ramawat Botany Department, M.L. Sukhadia University, Udaipur, Rajasthan, India

This book series provides complete, comprehensive and broad subject based reviews about existing biodiversity of different habitats and conservation strategies in the framework of different technologies, ecosystem diversity, and genetic diversity. The ways by which these resources are used with sustainable management and replenishment are also dealt with. The topics of interest include but are not restricted only to sustainable development of various ecosystems and conservation of hotspots, traditional methods and role of local people, threatened and endangered species, global climate change and effect on biodiversity, invasive species, impact of various activities on biodiversity, biodiversity conservation in sustaining livelihoods and reducing poverty, and technologies available and required. The books in this series will be useful to botanists, environmentalists, marine biologists, policy makers, conservationists, and NGOs working for environment protection.

More information about this series at http://www.springer.com/series/11920

Jean-Michel Mérillon Céline Rivière •

Editors

Natural Antimicrobial Agents

123

Editors Jean-Michel Mérillon Faculty of Pharmaceutical Sciences Institute of Vine and Wine Sciences University of Bordeaux Villenave d’Ornon France

Céline Rivière Faculty of Pharmaceutical Sciences Institute of Research in Agro-food and Biotechnology Charles Viollette University of Lille Lille France

ISSN 2352-474X ISSN 2352-4758 (electronic) Sustainable Development and Biodiversity ISBN 978-3-319-67043-0 ISBN 978-3-319-67045-4 (eBook) https://doi.org/10.1007/978-3-319-67045-4 Library of Congress Control Number: 2017962028 © Springer International Publishing AG 2018 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Printed on acid-free paper This Springer imprint is published by Springer Nature The registered company is Springer International Publishing AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

Plant Kingdom and Biodiversity: An Endless Source of Antimicrobials in Human and Plant Health Use of plants for various ailments is as old as human civilization. Continuous efforts are being made not only to improve the knowledge on the phytochemical composition and the biological activities of medicinal plants, but also to produce their bioactive secondary metabolites in high amounts through various high technologies. About 200,000 natural products of plant origin are known, and many more are being identified from higher plants and microorganisms. Some plant-based drugs are used since centuries and still remain at present time essential medicines. Morphine, obtained from poppy straw of Papaver somniferum, is on the WHO Model List of Essential Medicines and is widely used as antalgic, primarily to treat both acute and chronic severe pain. Drug discovery from medicinal plants or marine microorganisms continues to provide an important source of new drug leads. Research on new antibacterial and new antifungal agents represents a real and timely challenge of this century, in particular with the current contexts on the control of pathogenic agents in biomedicine, agriculture, and food industry. One of the main problems to fight human infections is the widespread of multidrug-resistant bacteria for which common antibiotics become less efficient. Among the most problematic Gram-positive bacteria are methicillin-resistant Streptococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), and multidrug-resistant Mycobacterium tuberculosis (XDR-TB) strains. Infections caused by resistant Gram-negative bacteria, in particular by extended-spectrum b-lactamase-producing Enterobacteriaceae, carbapenem-resistant Enterobacteriaceae, multidrug-resistant strains of Pseudomonas aeruginosa and Acinetobacter baumannii, also constitute a serious threat to public health worldwide. In agriculture, the management of crop diseases is submitted to two main constraints: (i) occurrence and widespread of fungicide resistance in most plant pathogenic fungi and (ii) societal and politic pressures aiming at reducing the use of conventional fungicides in crop protection because of their potential impacts on both the environment and human health. At last, in food

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industry, the use of conventional chemical preservatives to control food pathogens is also controversial. Thus, new strategies to control bacterial infections in human health are highly sought. A number of compounds found in plants are being cited as antimicrobials and resistance-modifying agents. The natural products can either have direct antibacterial action on resistant strains alone, or as synergists of potentiators of other antibacterial agents, or act as bacterial resistance-modifying agents (RMAs). In plant health, different types of biocontrol products are being developed since they have lower risks on the environment and human health than synthetic pesticides. The search for new antiparasitic and antiviral natural products is far from devoid of interest. According to the WHO report in 2013, malaria still represents some 207 million cases worldwide and more than 3 billion of people are still exposed to this risk. Similarly, about 350 million people are considered at risk of contracting leishmaniosis. The fight against some viruses also requires that the research on natural products continues. Hepatitis C virus (HCV) infection is a leading cause of chronic liver diseases. According to the World Health Organization, more than 150 million people are chronic carriers of HCV. The development of highly effective treatment regimens, including direct-acting antiviral drugs, has revolutionized the treatment of chronic hepatitis C infection. This new generation of antiviral drugs currently available on the market has a superior efficacy and a better safety profile than previous therapies. However, the cost of these treatments is very high and not easily accessible to an exposed population in developing countries. In addition, the appearance of treatment-resistant viral variants has been noted in some patients. Documenting the latest research in the field of different pathogenic organisms, this book compiles the recent information about natural sources of antimicrobials and their sustainable utilization in the following areas: (I) plants as a source of antibacterials (Human Health); (II) natural occurring antifungal natural products (Plant Health); (III) antiparasitic natural products (Human Health); (IV) antiviral natural products (Human Health). This book will be useful to researchers and students in microbiology, biotechnology, pharmacology, chemistry, and biology as well as medical professionals. Villenave d’Ornon, France Lille, France

Prof. (Dr.) Jean-Michel Mérillon Dr. Céline Rivière

Contents

Part I

Plants as a Source of Antibacterials (Human Health)

1

Antimicrobial Natural Products Against Campylobacter . . . . . . . . . Sonja Smole Možina, Anja Klančnik, Jasna Kovac, Barbara Jeršek and Franz Bucar

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2

An Overview of the Antimicrobial Properties of Hop . . . . . . . . . . . Laetitia Bocquet, Sevser Sahpaz and Céline Rivière

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3

How to Study Antimicrobial Activities of Plant Extracts: A Critical Point of View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Séverine Mahieux, Maria Susana Nieto-Bobadilla, Isabelle Houcke and Christel Neut

Part II

55

Natural Occurring Antifungal Natural Products (Plant Health)

4

Antifungal Activities of Essential Oils from Himalayan Plants . . . . Chandra Shekhar Mathela and Vinod Kumar

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5

Review Chapter: Fusarium Genus and Essential Oils . . . . . . . . . . . Martin Zabka and Roman Pavela

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6

Natural Agents Inducing Plant Resistance Against Pests and Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Ali Siah, Maryline Magnin-Robert, Béatrice Randoux, Caroline Choma, Céline Rivière, Patrice Halama and Philippe Reignault

Part III 7

Antiparasitic Natural Products (Human Health)

Antileishmanial and Antitrypanosomal Activities of Flavonoids . . . . . 163 Flore Nardella, Jean-Baptiste Gallé, Mélanie Bourjot, Bernard Weniger and Catherine Vonthron-Sénécheau

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Contents

8

Natural Products from Plants as Potential Leads as Novel Antileishmanials: A Preclinical Review . . . . . . . . . . . . . . . . . . . . . . 195 João Henrique G. Lago, Kaidu H. Barrosa, Samanta Etel T. Borborema and André G. Tempone

9

Insecticidal and Antimalarial Properties of Plants: A Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 Lucie Paloque, Asih Triastuti, Geneviève Bourdy and Mohamed Haddad

10 Antimalarial Terpenic Compounds Isolated from Plants Used in Traditional Medicine (2010–July 2016) . . . . . . . . . . . . . . . 247 Claire Beaufay, Joanne Bero and Joëlle Quetin-Leclercq Part IV

Antiviral Natural Products (Human Health)

11 Antimicrobial Capacities of the Medicinal Halophyte Plants . . . . . . 271 Faten Medini and Riadh Ksouri 12 Natural Products and Hepatitis C Virus . . . . . . . . . . . . . . . . . . . . . 289 Karin Séron, Marie-Emmanuelle Sahuc and Yves Rouillé Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329

Contributors

Kaidu H. Barrosa Center for Natural and Human Sciences, Federal University of ABC, Santo Andre, Brazil Claire Beaufay Pharmacognosy Research Group, Louvain Drug Research Institute, Catholic University of Louvain, Brussels, Belgium Joanne Bero Pharmacognosy Research Group, Louvain Drug Research Institute, Catholic University of Louvain, Brussels, Belgium Laetitia Bocquet Charles Viollette Research Institute, University of Lille (EA 7394), Lille, France Samanta Etel T. Borborema Centre of Parasitology, Adolfo Lutz Institute, São Paulo, Brazil Geneviève Bourdy UMR 152, IRD-UPS Pharma-DEV, University of Toulouse 3, Toulouse, France Mélanie Bourjot Laboratoire d’Innovation Thérapeutique UMR CNRS 7200, Faculty of Pharmacy, University of Strasbourg, Illkirch, France Franz Bucar Department of Pharmacognosy, Institute of Pharmaceutical Sciences, University of Graz, Graz, Austria Caroline Choma Charles Viollette Research Institute (EA 7394), ISA Lille, Lille, France Jean-Baptiste Gallé Laboratoire d’Innovation Thérapeutique UMR CNRS 7200, Faculty of Pharmacy, University of Strasbourg, Illkirch, France Mohamed Haddad UMR 152, IRD-UPS Pharma-DEV, University of Toulouse 3, Toulouse, France Patrice Halama Charles Viollette Research Institute (EA 7394), ISA Lille, Lille, France

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Contributors

Isabelle Houcke Laboratoire de Bactériologie, Faculté de Pharmacie, INSERM U995 LIRIC, University of Lille, Lille, France Barbara Jeršek Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia Anja Klančnik Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia Jasna Kovac Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia; Department of Food Science, The Pennsylvania State University, University Park, PA, USA Riadh Ksouri Laboratoire des Plantes Aromatiques et Médicinales, Center of Biotechnology of Borj Cedria, Hammam-Lif, Tunisia Vinod Kumar Department of Chemistry, Kumaun University, Nainital, Uttarakhand, India João Henrique G. Lago Center for Natural and Human Sciences, Federal University of ABC, Santo Andre, Brazil Maryline Magnin-Robert Unité de Chimie Environnementale et Interactions sur le Vivant (EA 4492), University of the Littoral Opal Coast, CS 80699, Calais, France Séverine Mahieux Laboratoire de Bactériologie, Faculté de Pharmacie, INSERM U995 LIRIC, University of Lille, Lille, France Chandra Shekhar Mathela Department of Chemistry, Kumaun University, Nainital, Uttarakhand, India Faten Medini Laboratoire des Plantes Aromatiques et Médicinales, Center of Biotechnology of Borj Cedria, Hammam-Lif, Tunisia Sonja Smole Možina Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia Flore Nardella Laboratoire d’Innovation Thérapeutique UMR CNRS 7200, Faculty of Pharmacy, University of Strasbourg, Illkirch Cedex, France Christel Neut Laboratoire de Bactériologie, Faculté de Pharmacie, INSERM U995 LIRIC, University of Lille, Lille, France Maria Susana Nieto-Bobadilla Laboratoire de Bactériologie, Faculté de Pharmacie, INSERM U995 LIRIC, University of Lille, Lille, France Lucie Paloque UPR8241 CNRS Laboratoire de Chimie de Coordination, University of Toulouse 3, Toulouse, France Roman Pavela Crop Research Institute, Ruzyne, Czech Republic

Contributors

xi

Joëlle Quetin-Leclercq Pharmacognosy Research Group, Louvain Drug Research Institute, Catholic University of Louvain, Brussels, Belgium Béatrice Randoux Unité de Chimie Environnementale et Interactions sur le Vivant (EA 4492), University of the Littoral Opal Coast, CS 80699, Calais, France Philippe Reignault Unité de Chimie Environnementale et Interactions sur le Vivant (EA 4492), University of the Littoral Opal Coast, CS 80699, Calais, France Céline Rivière Charles Viollette Research Institute (EA 7394), University of Lille, Lille, France Yves Rouillé Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL Center for Infection and Immunity of Lille, Lille, France Sevser Sahpaz Charles Viollette Research Institute (EA 7394), University of Lille, Lille, France Marie-Emmanuelle Sahuc Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL Center for Infection and Immunity of Lille, Lille, France Karin Séron Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL Center for Infection and Immunity of Lille, Lille, France Ali Siah Charles Viollette Research Institute (EA 7394), ISA Lille, Lille, France André G. Tempone Centre of Parasitology, Adolfo Lutz Institute, São Paulo, Brazil Asih Triastuti UMR 152, IRD-UPS Pharma-DEV, University of Toulouse 3, Toulouse, France Catherine Vonthron-Sénécheau Laboratoire d’Innovation Thérapeutique UMR CNRS 7200, Faculty of Pharmacy, University of Strasbourg, Illkirch, France Bernard Weniger Laboratoire d’Innovation Thérapeutique UMR CNRS 7200, Faculty of Pharmacy, University of Strasbourg, Illkirch, France Martin Zabka Crop Research Institute, Ruzyne, Czech Republic

Part I

Plants as a Source of Antibacterials (Human Health)

Chapter 1

Antimicrobial Natural Products Against Campylobacter Sonja Smole Možina, Anja Klančnik, Jasna Kovac, Barbara Jeršek and Franz Bucar

Abstract Campylobacteriosis is the world’s leading bacterial foodborne illness and the most frequently reported zoonosis in humans. The present review aims to present an overview of the alternative strategies to limit Campylobacter contamination and to prevent Campylobacter infections using natural products from various sources. Additionally, natural products may improve the sensory characteristics of foods and extend their shelf life. The most effective intervention is inhibiting Campylobacter growth and thus reducing their prevalence and levels in vitro and in vivo along the food supply chain and on food products. Further, development of innovative growth and virulence control strategies using natural products in subinhibitory concentrations that do not pose selective pressure, may be beneficial. At such low concentrations, natural products can act as resistance modulators (e.g., efflux pump inhibitors) and thus enhance anti-Campylobacter activity of antibiotics. Low doses of natural compounds that are not cytotoxic can prevent adhesion of Campylobacter to abiotic surfaces, hence preventing biofilm formation, or to biotic surfaces, hence preventing attachment to animal or human epithelial cells.







Keywords Natural products Antimicrobial Resistance mechanism Efflux pump inhibitors Anti-adhesion Campylobacter







S. S. Možina
A. Klančnik
J. Kovac
B. Jeršek Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia J. Kovac Department of Food Science, The Pennsylvania State University, University Park, PA 16802, USA F. Bucar (&) Department of Pharmacognosy, Institute of Pharmaceutical Sciences, University of Graz, Universitätsplatz 4, 8010 Graz, Austria e-mail: [email protected] © Springer International Publishing AG 2018 J.-M. Mérillon and C. Rivière (eds.), Natural Antimicrobial Agents, Sustainable Development and Biodiversity 19, https://doi.org/10.1007/978-3-319-67045-4_1

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1.1

S. S. Možina et al.

Introduction

Thermotolerant Campylobacter spp., mainly C. jejuni and C. coli, are the most common gastrointestinal bacterial foodborne pathogens in the European Union, and they can even surpass other infections due to Salmonella, Shigella and Escherichia coli (EFSA 2013). Campylobacter is generally regarded as being sensitive to environmental conditions outside of the animal and human host. However, it can survive the adverse conditions during food processing and storage (Humphrey et al. 2007; Klančnik et al. 2009, 2014), and can cause bacterial gastroenteritis in humans.

1.1.1

Campylobacter Contamination and Infection

Infections with Campylobacter are often contracted by eating and/or handling undercooked poultry meat or other cross-contaminated foods that are not thermally treated prior to ingestion (EFSA 2012). Outbreaks of Campylobacter are mostly associated with the consumption of contaminated water and poultry (Halberg Larsen et al. 2014). Broiler flocks are commonly colonized with Campylobacter through exposure to farm environmental factors. Once the pathogen is introduced to the farm, it can rapidly spread among birds in the flock via faecal-oral route (Sahin et al. 2015). Furthermore, when infected birds are slaughtered, the release of the intestinal content can lead to cross-contamination of the slaughterhouse environment and consequently of poultry meat from non-infected flocks (Reuter et al. 2010). Campylobacter can also be transmitted to humans through unpasteurized milk and dairy products from colonized dairy cattle, or by cross-contamination with manure. Additionally, people can be infected through direct contact with the faeces of infected farm or companion animals (Halberg Larsen et al. 2014). In addition to diarrhoeal disease, chronic complications associated with Campylobacter infection include reactive arthritis and Guillain–Barré syndrome. Severe autoimmune responses and axonal damage have been observed in Campylobacter-associated Guillain–Barré syndrome, which is mostly problematic in the poorer countries, where patients have limited access to health care and treatment (Nyati and Nyati 2013).

1.1.2

Campylobacter Control

The European Union recently banned the use of antibiotic growth promoters in animal feed. The awareness of increasing Campylobacter resistance has encouraged the search for new effective strategies to reduce the incidence of Campylobacter jejuni infection (Smole Možina et al. 2011). In the food industry, the problem arises

1 Antimicrobial Natural Products Against Campylobacter

5

due to modern food production facilities and the emergence and spread of resistance through intensive use of antimicrobial agents and the international trade in raw materials and food products. Campylobacter spp. has developed resistance to many of the available antibiotics, and as several infections are now not controllable with the available antibiotics, the regulatory bodies have restricted the use of antibiotics in the food supply chain (Cars et al. 2011). C. jejuni can also form mono- and multispecies biofilms which enhance survival in hostile environments, as their physiology and behaviour are significantly different from their planktonic counterparts (Reeser et al. 2007; Teh et al. 2014). It is known that C. jejuni can form biofilms on different abiotic surfaces including polystyrene or glass and on biotic surfaces, and adhere to animal and human intestinal cell lines, such as PSI, Caco2, H4 and HT29 cells (Šikić Pogačar et al. 2009, 2010, 2016; Kurinčič et al. 2016). The surfaces of equipment used for food handling, storage and processing thus represent major sources of Campylobacter cross-contamination (Dunne 2002; Simões et al. 2010; Nguyen et al. 2010). This can also lead to transmission of Campylobacter to food and to the next host. Due to the public health significance of C. jejuni infection, it is important to understand its survival in the environment and in the food supply chain (Joshua et al. 2006) and to control Campylobacter contamination, growth and transmission in the food supply chain. For example, in broiler meat production chain, the control is needed especially on preharvest, harvest and postharvest levels and is possible in several steps, such as (i) in primary production; (ii) during transportation and before slaughter; (iii) at slaughter, dressing and processing (EFSA 2011; Halberg Larsen et al. 2014). Conventional cleaning and sanitation regimens can contribute to inefficient biofilm control. In order to prevent Campylobacter persistence and cross-contamination at post-harvest level, effective cleaning and sanitation procedures must be in place in food processing environment. To improve the current situation, approaches for the prevention of Campylobacter contamination are focused on alternative antimicrobials of natural origin and on the early stage, such as safety of feed and drinking water for food animals. In addition, consumers are increasingly demanding fresh or minimally processed foods without added chemical additives with the longest possible shelf life. Natural antimicrobial substances are an alternative to replace synthetic chemical additives. The main purpose of many studies of natural antimicrobial substances is to obtain a substance with antimicrobial activity in the food and thus contribute to its stability and safety by growth inhibition or inactivation of spoilage and pathogenic microorganisms (Burt 2004; Lucera et al. 2012). Furthermore, new strategies using subinhibitory concentrations and targeting more specific mechanisms like quorum sensing, adhesion and biofilm formation and/or efflux pumps or other cell membrane functions could reduce Campylobacter growth and attenuate virulence. Such treatment is not bactericidal, so resistance is less likely to occur (Bensch et al. 2011; Kovač et al. 2015; Šikić Pogačar et al. 2016). However, public scientific evidence on resistance-modifying mechanisms, anti-adhesion and antiquorum-sensing activity of natural antimicrobials to reduce Campylobacter persistence is very limited.

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S. S. Možina et al.

Natural Products Inhibiting Growth of Campylobacter

Control of Campylobacter growth by natural products has been subject to numerous studies. Usually, cultivation of Campylobacter strains in vitro under microaerophilic conditions and application of extracts or pure compounds in increasing concentrations are applied. Natural antimicrobial products are isolated from different sources (Davidson et al. 2015): (i) Animal sources such as lysozyme (egg, milk), lactoperoxidase (milk), lactoferrin (milk), chitosan; (ii) Plant sources—species and their essential oils and extracts (i.e. cloves, cinnamon, oregano, thyme), berry fruits (i.e. cranberry, cloudberry, raspberry, strawberry), hop, olives, brassica, onion, garlic, grape; (iii) Microbial sources such as natamycin, nisin, other bacteriocins, fermentation metabolites, protective cultures, bacteriophages. For the application and potential use of natural products in controlling Campylobacter contamination and infection, it is important to study the target activity of natural products inside the Campylobacter cell. Only few studies have demonstrated mechanisms of antimicrobial activity. The activity of organic acids is known to be based on the ability of their undissociated form to penetrate through the cell membrane and to dissociate inside the cell, decreasing the intracellular pH value, thus disrupting homeostasis which is essential for the control of ATP synthesis, RNA and protein synthesis, DNA replication and cell growth. Besides the decrease in intracellular pH, the perturbation of membrane functions by organic acid molecules may be also responsible for the microbial inactivation. High concentration of anions (due to dissociation) inside the cells might result in an increased osmolarity and consequently the metabolic disruption (Hirshfield et al. 2003). To be able to react to potential development of resistance against natural products, it is essential to study the mechanisms of bacterial resistance to these products.

1.2.1

Methods for Identification of Antimicrobial resistance Activity

Several methods for measurement of minimal inhibitory concentration (MIC) values of natural products are available, but there is no standard procedure established. Since results from numerous reports of antimicrobial activity against Campylobacter need to be equivalent, it is important to evaluate different methods for antimicrobial activity assessment. The assay needs to be customized to suit individual bacterial species growth requirements by selecting the appropriate growth medium, incubation temperature, time and atmosphere and finally, the method for detection of bacterial growth inhibition. Thus, the comparison of

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reported activities of plant extracts and compounds should be made with caution, and the methods used for MIC determination (such as cultivation; bacterial growth evaluation; kinetics of inhibition via survival curves) have to be taken into account. An evaluation of different test methods like disc diffusion, agar dilution, broth macrodilution and microdilution assays revealed that broth microdilution assays are most suitable for fast screening of growth inhibitory potential of plant extracts and compounds (Klančnik et al. 2010). While for Gram-positive bacteria results from agar dilution and broth dilution were in good agreement, for Gram-negative bacteria like Campylobacter spp. lower MIC values were determined by broth dilution compared to agar dilution methods (Klančnik et al. 2010). A comparison of growth kinetics for 24 h by a macrodilution assay at certain MIC values could then be used to confirm the antibacterial effects. In case of Campylobacter spp., low MIC values seen in broth microdilution assays clearly correlate with the actual growth inhibitory effects of the test compounds (Klančnik et al. 2010). Critical point in the assessment of antibacterial activity in broth microdilution assays performed in 96-well microtiter plates is the way bacterial growth is examined. Since natural products, especially plant extracts, are rarely colourless, the determination of MIC by broth microdilution assays based upon measurement of bacterial optical density (OD) may be hindered. Therefore, growth indicators such as tetrazolium salts (Klančnik et al. 2010), resazurin (Kovač et al. 2014, 2015) or luminescent ATP detectors (Klančnik et al. 2009) associated with bacterial metabolic activity were found to be more appropriate to use for MIC determination of natural products. Viability determination of aerobic bacteria is possible by observing visible growth utilizing tetrazolium salts like XTT (2,3-Bis(2-methoxy-4-nitro-5sulphophenyl)-2H-tetrazolium-5-carboxanilide), INT (p-iodonitrotetrazolium violet) or resazurin (Klančnik et al. 2010). Tetrazolium salts are reduced by bacterial oxidative enzymes by acting as electron acceptors and are therefore not suitable for detection of metabolic activity in microaerophilic bacteria, such as Campylobacter (Klančnik et al. 2010). Thus, in case of Campylobacter growing under microaerophilic conditions with lower reduction kinetics, activity measurement of ATP or resazurin proved to be most suitable. ATP activity can be measured via bioluminescence after adding BacTiterGlo Reagent (Promega, USA) and incubation in the dark (Klančnik et al. 2009). Resazurin is converted by viable cells to the fluorescent resorufin product which can be detected by fluorescence signals measured with a microplate reader (excitation/emission wavelengths) after adding BacTiterBlue Reagent (Promega, USA) (Kovač et al. 2015). Apart from adjusting the growth detection methodology to suit the testing organism, also the potential interference of testing material with the growth detector needs to be ruled-out, in order to reliably determine the MICs. Natural products themselves, especially plant extracts rich in phenolic compounds, may namely reduce the growth detector resulting in false-positive growth results. From this aspect, the use of ATP detection systems for MIC determination of natural products seems to be the safest choice, although use of significantly more cost-effective resazurin is often appropriate, as well (Klančnik et al. 2012a, b; Kurinčič et al. 2012a; Kovač et al. 2014, 2015; Klančnik et al. 2012b).

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1.2.2

S. S. Možina et al.

Anti-Campylobacter Activity of Isolated Plant Compounds

Among substances of plant origin, the most active ones were found within isothiocyanates, diallysulphides and coumarins (see Table 1.1). In a study of Dufour et al. (2012), isothiocyanates (allyl-, benzyl-, ethyl-, 3-(methylthio)propyl-isothiocyanates) were tested against 24 C. jejuni isolates from chicken faeces, human infections and contaminated food together with two reference strains, NCTC 11168 and 81-176. They tested also the ggt mutant of 81-176 with deleted gene for c-glutamyl transpeptidase (GGT enzyme). Remarkable differences in anti-Campylobacter activity could be recorded with respect to the growth medium. Particularly, allyl isothiocyanate showed antibacterial effects with MIC of 50 to 200 mg/L measured by agar dilution assay and a higher effect with MIC of 5 to 10 mg/L measured by broth dilution assay. Also for benzyl isothiocyanate, a significant decline in MIC of 1.25 to 5 mg/L versus 0.625 to 1.25 mg/L measured by broth dilution assay could be observed. Both isothiocyanates were found to be bactericidal rather than bacteriostatic. In the preliminary screening (agar dilution assay and reference strain NCTC 11168), ethyl isothiocyanate and allyl isothiocyanate showed no or moderate activity (MIC  200 mg/L), whereas benzyl isothiocyanate and 3-(methylthio) propyl-isothiocyanate resulted in MIC values of 5 mg/L indicating the pronounced influence of the alkyl residue on the antibacterial activity of isothiocyanates and opening the window for exploring synthetic derivatives. Comparing wild-type 81-176 strain and its ggt mutant, let the authors conclude that the GGT enzyme might be involved in detoxification of isothiocyanates also in Campylobacter. However, resistance to isothiocyanate depended on additional, so far not unveiled other factors. Sulforaphane, an isothiocyanate originating from glucoraphanin which is abundant in brokkoli and other vegetables of the Brassicaceae family, revealed a similar potency as benzyl isothiocyanate with an MIC value of 15 mg/L measured by agar dilution but was not investigated further (Dufour et al. 2012). A series of diallyl sulphides abundant in essential oils of chives and garlic were tested against a number of food pathogens including C. jejuni ATCC 49349. A clear structure–activity relationship could be observed with increasing potency correlated to increasing number of sulphide groups, diallyl tetrasulphide being the most active (Rattanachaikunsopon and Phumkhachorn 2008). Alkaloids have been rarely investigated; however, the bisbenzylisoquinoline alkaloid cocsoline, isolated from the root bark of Epinetrum villosum (Menispermaceae), showed remarkably good activity on C. jejuni and C. coli (MIC of 15.62 to 31.25 mg/L; MBC 62.5 mg/L) (Otshudi et al. 2005). Among many phenolic compounds evaluated as growth inhibitors of Campylobacter spp., the prenylated 4-phenylcoumarin mammea A/AA isolated from the stem bark of Mammea africana (Calophyllaceae) showed remarkable high activity on C. jejuni ATCC 33291 in vitro with an MIC value of 0.25 mg/L measured by broth dilution assay. Other Gram-negative bacteria like Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Salmonella typhimurium were much less sensitive to this plant compound (MIC  16 mg/L).

1 Antimicrobial Natural Products Against Campylobacter

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Table 1.1 Anti-Campylobacter activity of selected compounds of plant origin Compound

Species

Assay; MIC (mg/L)

Viability

Reference

Allyl isothiocyanate (AITC)

C. jejuni

VIS

Dufour et al. (2012)

Benzyl isothiocyanate (BITC)

C. jejuni

VIS

Dufour et al. (2012)

a-Bisabolol

C. C. C. C. C. C. C. C. C. C.

AD; MIC 50– 200 BD; MIC 5–10 AD; MIC 1.25–5 BD; MIC 0.625–1.25 BMD; MIC 0.125–0.5

OD 600 nm; INT

Kurekci et al. (2013)

BMD; MIC 19.5–78

ATP activity

Klančnik et al. (2012b)

BMD; MIC 156–313

ATP activity

Klančnik et al. (2012b)

BMD; MIC 0.25

OD 600 nm INT

Kurekci et al. (2013)

BMD; MIC 15.62–31.25 MD; MBC 62.5 BMD; MIC 56; 12; 2; 1

n.i.

Otshudi et al. (2005)

n.i.

BMD; MIC 250 BD; MIC 600 BD; MBC 700 BMD; MIC 78– 313

n.i. OD

Rattanachaikunsupon and Phumkhachorn (2008) Rossi et al. (2007) Engels et al. (2011)

Carnosic acid Chlorogenic acid Cineole Cocsoline

jejuni coli jejuni coli jejuni coli jejuni coli jejuni coli

Diallyl mono-, di-, tri, tetrasulphide

C. jejuni

Elemicin Epigallocatechin gallate

C. jejuni C. jejuni

Ferulic acid Gallic acid

C. jejuni C. coli C. jejuni

Hepta-O-galloylglucose

C jejuni

Kaempferol-, myricetin- and quercetin glycosides Linalool

C. jejuni C. jejuni C. coli

Mammea A/AA

C. jejuni

Methyl gallate

C. jejuni

ATP activity

Klančnik et al. (2012b)

BMD; MIC 313

ATP activity

BD; MIC < 100 BD; MBC < 100 BMD; MIC 130–250 BMD; MIC 0.5–1 BMD; MBC 0.5–1 BMD; MIC 0.25

OD

Klančnik et al. (2012b) Engels et al. (2011)

BMD, MIC 60

INT VIS

VIS OD 590 nm INT

Madikizela et al. (2013) Duarte et al. (2016)

Canning et al. (2013) Madikizela et al. (2013)

(continued)

10

S. S. Možina et al.

Table 1.1 (continued) Compound

Species

Assay; MIC (mg/L)

Viability

Reference

Methyl isoeugenol Nerolidol

C. C. C. C. C.

BMD; MIC 150 BMD; MIC 0.5–1

n.i. OD 600 nm INT OD

Rossi et al. (2007) Kurekci et al. (2013)

Resazurin FL 550/ 959 nm OD

Kovač et al. (2015)

ATP activity

Klančnik et al. (2012b)

BMD; MIC 313

ATP activity

BMD; MIC 156–313

ATP activity

Klančnik et al. (2012b) Klančnik et al. (2012b)

BMD; MIC 0.125–0.25

OD 600 nm INT OD 600 nm INT

Pterostilbene (Pts), Pinosylvin (Ps); cyclodextrin inclusion complexes (Pts-CIC; Ps-CIC)

jejuni jejuni coli jejuni coli

a-Pinene

C. jejuni

Resveratrol (Rv); cyclodextrin inclusion complexes (Rv-CIC)

C. jejuni C. coli

Rosmarinic acid

C. jejuni C. coli C. jejuni

Sinapic acid Syringic acid a-Terpinene Terpinen-4-ol Thymol (Ty); thymol-b-D-glucopyranoside (t-gluc)

C. C. C. C. C. C. C. C.

jejuni coli jejuni coli jejuni coli jejuni coli

BMD; MIC Pts 50!400 Ps 25–50 Pts-CIC 128!1024 Ps-CIC 16–64 BMD; MIC 1000!2000 BMD; MIC Rv 50–100 Rv-CIC 64–256 BMD; MIC 78– 156

BMD; MIC 0.06

Silva et al. (2015)

Duarte et al. (2015)

Kurekci et al. (2013) Kurekci et al. (2013)

BMD Viable cell Epps et al. (2015) count Ty (1 mM): decrease in log CFU/ml for 1.88 to 4.80; t-gluc (1 mM): no effect Vanillic acid C. jejuni BMD; MIC 313 ATP activity Klančnik et al. 2012b ‘AD’ agar dilution; ‘BD’ broth dilution; ‘BMD’ broth microdilution method; ‘MIC’ minimal inhibitory concentration; ‘MBC’ minimal bactericidal concentration; ‘n.i.’ not indicated, ‘OD’ optical density, ‘VIS’ visually observation

However, when tested on normal and cancer cell lines, it became obvious that mammea A/AA displays a general cytotoxic effect which decreases its potential as anti-Campylobacter drug (Canning et al. 2013). Stilbenes like pinosylvin (25 to 50 mg/L) and resveratrol (50 to 100 mg/L) can be regarded as another promising group of plant phenolics with growth-inhibiting effects on C. jejuni and C. coli. Preparing cyclodextrin inclusion complexes might be an option to improve the

1 Antimicrobial Natural Products Against Campylobacter

11

solubility of this type of compounds (Silva et al. 2015; Duarte et al. 2015). Other compounds with phenolic structural features with antibacterial effects were flavonols like kaempferol, quercetin and myricetin glycosides (Madikizela et al. 2013), epigallocatechin gallate (Engels et al. 2011), hepta-O-galloyl glucose (Engels et al. 2011), methyl gallate (Madikizela et al. 2013), phenolic acids such as chlorogenic acid, rosmarinic acid, ferulic acid, gallic acid and syringic acid (Klančnik et al. 2012b), the phenolic terpenoids thymol (Epps et al. 2015) and carnosic acid (Klančnik et al. 2012b), or the phenylpropanoids elemicin and methyl isoeugenol (Rossi et al. 2007). Their anti-Campylobacter potency varied in a wide range between MIC values of 19.5 mg/L (C. jejuni/C. coli, carnosic acid) (Klančnik et al. 2012b) and 60 mg/L (C. jejuni, methyl gallate) (Madikizela et al. 2013) to 600 mg/ L (C. jejuni, epigallocatechin gallate) (Engels et al. 2011; Klančnik et al. 2012b). Mono- and sesquiterpenes isolated from essential oils generally can be regarded as moderately active, and promising structures within this class of compounds were terpinen-4-ol (C. jejuni, C. coli; MIC 0.6 ml/L) and a-bisabolol (C. jejuni; MIC 1.25 ml/L) (Kurecki et al. 2013). Campylobacters became resistant to the point where none of our available antibiotics work for some of the infections that confront patients and physicians in hospitals. However, the mechanisms of action of bioactive phytophenols must be investigated (Smole Možina et al. 2011). Resistance to phenolic compounds and plant extracts involves the CmeABC efflux system. This could be evidenced by comparing growth inhibitory effects on wild-type and efflux mutant strains in genes with crucial role in response and/or defence against antimicrobials (cmeB, cmeF, cmeR). In particular, gene knockout mutants of cmeB encoding the transport protein CmeB were highly susceptible to rosmarinic, chlorogenic or gallic acid compared to the wild-type strains with modulation factors of 64 to 128 mg/L, i.e. decreasing MIC values in mutant strains by 64 to 128-fold (Klančnik et al. 2012b). Similarly, increased susceptibilities could be shown for a number of herbal extracts like those of rosemary, sage, lemon balm, bearberry or grape leaves (Klančnik et al. 2012b). Finally, the influence of the gut microbiota on metabolisms of administered plant compounds and its influence on antimicrobial activity should not be neglected as shown by Epps et al. (2015). Thymol-O-b-D-glucoside exerted after coincubation with the gut bacterium Parabacteroides distasonis possessing b-glucosidase activity increased activity against C. coli and C. jejuni, and comparable results were observed after coincubation with porcine or bovine faecal microbes. These results shed light on the important role of metabolic capacities of gut microbiota to convert ingested natural products from inactive prodrugs to potentially antibiotic entities (Epps et al. 2015). This was also illustrated by urolithins, originating from ellagitannins by metabolic action of colon microbiota, which have been shown to inhibit quorum sensing of the enteropathogenic Yersinia enterocolitica (Gimenez-Bastida et al. 2012).

12

1.3

S. S. Možina et al.

Resistance Modulators and Campylobacter Efflux Pump Inhibitors from Natural Sources

Potentiation of antimicrobial activity can be achieved by a combination of different antimicrobials with synergistic antimicrobial effects or by antimicrobials together with resistance modulators. Resistance modulators are compounds or a mixture of compounds (e.g. plant extracts) that are able to enhance the activity of antibiotics without necessarily exhibiting direct antimicrobial activity themselves. By acting so, they can increase bacterial susceptibility to antibiotics and in some cases fully reverse bacterial resistance against antibiotics. Resistance-modifying effect can be achieved through interference of a modulator with specific or general mechanisms of antibiotic resistance existing in bacterial organisms. The modification of antibiotic target and inactivation of the antibiotic itself are here considered as specific mechanisms of resistance, in oppose to the insufficient influx of antimicrobial into a bacterium and efficient extrusion of antibiotic out of a bacterium through enhanced efflux, which are considered to be general, i.e. less specific, resistance mechanisms. The use of a b-lactam antibiotic amoxicillin together with a b-lactamase inhibitor clavulanic acid (Neu and Fu 1978) is a proof of concept of such resistance-modifying strategy in fight against antibiotic resistance. Its success demonstrates feasibility of applying narrow-spectrum modulators that target specific antibiotic-modifying enzymes, to protect the structural and functional integrity of an antibiotic. To date, the clavulanic acid, naturally produced by Streptomyces clavuligerus, which has the genes responsible for the biosynthesis of clavulanic acid and the b-lactam antibiotics clustered together in shared regulation network (Pradkar 2013), is a unique example of a drug licensed for use in conjunction with an antibiotic to enhance its activity, i.e. resistance modulator. It has been developed and patented for use in pharmaceutical formulation with antimicrobial agents in 1994, and the drug is still widely used in a variety of generic forms (FDA Orange Book 2015). Other clinically used drugs, such as synthetic verapamil and naturally occurring alkaloid reserpine, which have been licensed as calcium ion influx inhibitor and hypertensive, respectively (PubChem—Verapamil 2015a; PubChem—Reserpine 2015b), were demonstrated to have multidrug efflux-inhibitory activity in Gram-negative bacteria as well (Klančnik et al. 2012b; Kovač et al. 2014; 2015). Efforts have been placed into search for novel resistance modulators that could counteract accelerated evolution of antibiotic resistance observed under man-made selective pressure created since the beginning of antibiotic era in the past century (Baquero et al. 2009). We here reviewed natural products from plant sources possessing resistance-modifying or efflux-inhibitory activity in Campylobacter identified over the past years and the methodology used in this quest.

1 Antimicrobial Natural Products Against Campylobacter

1.3.1

13

Methods for Screening of Resistance Modulators and Efflux Inhibitors

Modulation of antibiotic resistance is determined using a modulation assay based on the principle of broth microdilution assay which is described in detail in Sect. 1.2.1. The modulation assay is a modified MIC assay, in which MICs of antibiotic alone and antibiotic in the presence of subinhibitory concentration of potential resistance modulator are determined and expressed as respective ratio called modulation factor (MF) (Lechner et al. 2008). Figure 1.1 presents the growth in modulation assay of C. jejuni detected using resazurin metabolic activity indicator. The modulation factor is calculated as in Eq. (1.1): MF ¼ MICAB =MICAB þ M

ð1:1Þ

where MICAB is the MIC of antibiotic and MICAB+M is the MIC of antibiotic in the presence of a resistance modulator. In this case, MICAB is 0.25 mg/L and MICAB+M is 0.031 mg/L resulting in MF 8. The modulation factors above two are considered to be good indicators of resistance-modifying activity, and compounds possessing such activity are often further investigated by exploring their efflux-inhibitory potential. Efflux-inhibitory activity of natural products can be evaluated using accumulation (Tegos et al. 2002; Lin et al. 2002; Garvey et al. 2011; Blanchard et al. 2014; Aparna et al. 2014; Kovač et al. 2014, 2015) and efflux assays (Lechner et al. 2008; Gröblacher et al. 2012a, b). There are two common approaches to quantifying the intracellular accumulation and efflux of efflux pump substrates by:

Fig. 1.1 Metabolic activity detection in modulation assay using resazurin. Growth of C. jejuni in the presence of antibiotic is represented by the blue curve with full squares and growth in the presence of antibiotic and resistance modulator by the red curve with full circles. The break point in the curve, at which the relative fluorescence signal (RFU) starts to increase, is defined as MIC

14

S. S. Možina et al.

(i) Real-time monitoring of intracellular substrate accumulation kinetics; (ii) Measurement of the accumulated endpoint concentration. The first approach can be performed by automated spectrofluorometric microplate reader kinetics measurement when using substrates such as ethidium bromide, Nile Red, DiBAC4-(3), berberine or Hoechst 33342 (Lin et al. 2002; Tegos et al. 2002; Garvey et al. 2011), fluorescence of which increases due to their binding with DNA after intracellular accumulation. Measuring the accumulation of antibiotics that do not have the same fluorescence properties becomes more time-consuming and laborious, since it requires manual sampling and processing of the sample, and finally measurement of antibiotic concentration employing chromatographic, spectrofluorometric or radiometric methods (Jeon et al. 2011; Shiu et al. 2013; Blanchard et al. 2014). Assays also differ among each other by the accumulation time, ranging from 10 min (Shiu et al. 2013) up to 60 min (Kovač et al. 2014). The advantage of measuring accumulation of an efflux pump substrate by determining the residual substrate concentration in the medium is that there are no limitations in terms of the substrate fluorescence properties. For example, HPLC and UPLC coupled with MS were successfully used to follow the accumulation of minocycline in Acinetobacter baumannii, as well as accumulation of linezolid in Escherichia coli and meropenem in Pseudomonas aeruginosa, respectively (Blanchard et al. 2014; Zhou et al. 2015). In addition to using a microplate reader, a microfluidic channel device can be used to evaluate the intracellular accumulation of efflux pump substrate using fluorescent microscope, as demonstrated in Escherichia coli using fluorescein-di-b-D-galactopyranoside (Matsumoto et al. 2011). To date, ethidium bromide and ciprofloxacin accumulation assays were performed in Campylobacter (Lin et al. 2002; Jeon et al. 2011; Kovač et al. 2014, 2015). Among reference efflux pump inhibitors, cyanide m-chlorophenyl hydrazone (CCCP) and reserpine resulted in best efflux inhibition in C. jejuni after 30 min, followed by verapamil (Fig. 1.2). Somewhat surprisingly, Phe-Arg-b-naphthylamide dihydrochloride (PAbN) and 1-(1-naphthylmethyl)-piperazine (NMP), which were demonstrated to produce substantial resistance modulation in combination with several antimicrobials in Campylobacter (Klančnik et al. 2012a, b; Kurinčič et al. 2012b), show poor efflux-inhibitory activity (unpublished data of JK; Fig. 1.2). This may be explained with the synergistic resistance-modifying effects of efflux inhibition and membrane permeabilization, as demonstrated in Pseudomonas aeruginosa (Lamers et al. 2013). The increased membrane permeability could negatively influence the intracellular accumulation of small molecules, such as ethidium bromide.

1 Antimicrobial Natural Products Against Campylobacter

15

Fig. 1.2 Ethidium bromide accumulation in Campylobacter jejuni NCTC 11168 in the presence of natural product (-)-a-pinene, reference inhibitors PAbN, NMP, verapamil, reserpine and CCCP

1.3.2

Natural Products as Campylobacter Resistance Modulators

Natural products have been demonstrated to be a valuable source of resistance modulators. The resistance-modifying agents from plant sources were comprehensively reviewed by Abreu et al. in 2012 and updated recently (Prasch and Bucar 2015). Among plant extracts, the ethanol extract of Alpinia katsumadai, a plant belonging to ginger family (Zingiberaceae), exhibited modulation of ciprofloxacin and erythromycin, ethidium bromide, bile salts and sodium deoxycholate in C. jejuni and C. coli at subinhibitory concentration 0.25 MIC (Klančnik et al. 2012a). The same study demonstrated similar effects for Rosmarinus officinalis L. extract from mint family (Lamiaceae). Beside crude plant extracts, also essential oils have been found to potentiate the antibiotic activity in Gram-negative bacteria. The antimicrobially inactive essential oil from Alpinia katsumadai seeds was demonstrated to modulate resistance against antibiotics ciprofloxacin and erythromycin, as well as the disinfectant triclosan, bile salts and ethidium bromide at half MIC concentration for over 256-fold (Kovač et al. 2014). Recently, two pure compounds responsible for resistance-modifying activity in Campylobacter were discovered. First one is catechin (-)-epigallocatechin gallate, a flavanol compound commonly found in green tea. It was confirmed as a modulator of resistance against a number of macrolide antibiotics in Campylobacter at concentrations 0.25 MIC (Kurinčič et al. 2012a). The second is (-)-a-pinene, a terpene

16

S. S. Možina et al.

compound present among others also in A. katsumadai seeds, the extracts of which were previously found to act resistance-modifying (Kovač et al. 2014, 2015).

1.3.3

Natural Products as Campylobacter Efflux Pump Inhibitors

Several synthetic (Blanchard et al. 2014; Vargiu et al. 2014) as well as natural (Aparna et al. 2014; Whalen et al. 2015) inhibitors of Gram-negative bacterial efflux pumps have been discovered in the last years; however, reports on inhibitors of Campylobacter efflux pumps are limited. Among plant natural products, the essential oil of Alpinia katsumadai (Zingiberaceae) has been identified as a potential inhibitor of Campylobacter efflux pumps (Kovač et al. 2014). The terpene compound (-)-a-pinene, abundant in A. katsumadai, pine trees, rosemary, lavender and turpentine, has been demonstrated to have inhibitory effects on major resistance– nodulation–division (RND) family efflux pump in Campylobacter, the CmeABC efflux pump. Furthermore, its activity extends to another newly discovered putative efflux protein Cj1687 (Kovač et al. 2015).

1.4

Inhibition of Adhesion and Biofilm Formation of Campylobacter by Natural Products

Several studies in recent years have provided evidence that this bacterial adhesion process is a multifactorial event, with the cooperative actions of several factors required mediating the adherence of C. jejuni to host cells (Ó Cróinín and Backert 2012; Backert and Hofreuter 2013). The precise molecular mechanisms involved in the attachment of C. jejuni remain largely unknown. Many factors (presented in Table 1.2) have been identified as critical to the adhesion and survival of Campylobacter in vivo and in nature, which include those related to survival under stress and to the basic biology of the stress response of the organism (Szymanski and Gaynor 2012; Sulaeman et al. 2010).

1.4.1

Methods for Identification of Adhesion

Given the tremendous clinical importance of adhesion and biofilm formation, it is somewhat surprising that there is no standard method for the investigation of cells during bacterial adhesion. Several methods that are based on different principles are available to detect the adhesion properties of pathogens. For bacteria, a common

1 Antimicrobial Natural Products Against Campylobacter

17

Table 1.2 Factors involved in the control of bacterial adhesion Aspect/factor

Reference

Material surface characteristics Surface chemistry; composition; topography; roughness; degree of hydrophobicity; hydrogen-bonding capacity Surface physicochemical factors Surface energy; surface charge; functional groups; hydration Bacterial properties Type; surface properties; morphology; cell membrane; host proteins/adhesins; bacterial motility; flagella; hydrophobicity; quorum-sensing inhibition; stress responses; extracellular polymeric matrix Environmental conditions pH; temperature; flow conditions; atmosphere and oxygen; nutrient composition

Garrett et al. (2008)

Lemos et al. (2014)

Reisner et al. (2006), Harvey et al. (2007), Rode et al. (2007), Dunne (2002), Sulaeman et al. (2010)

Reuter et al. (2010), Soni et al. (2008), Sulaeman et al. (2010)

method is to quantify the mass of the biofilm using crystal violet or safranin staining, followed by extraction of the bound dye with a solvent and measurement of its absorption (Reuter et al. 2010; Kurinčič et al. 2016). However, discussions remain, as the staining of live cells, dead cells and matrix with crystal violet can give misleading information. New methodologies might overcome the limitations of total biomass staining methods that appear when injured bacteria or viable but non-culturable bacteria are present. In some studies, the reduction of adhesion to polystyrene was measured in terms of cell culturability (according to CFU plate counting) and cell viability (according to a metabolic indicator) (Šikič Pogačar et al. 2015).

1.4.2

Natural Products as Campylobacter Anti-adhesives

Different concepts and approaches have been developed to achieve biomaterials that have such anti-infective properties. One of the new methodologies relies on the use of nanosized carriers to transport and controlling the release of an active antimicrobial (Liakos et al. 2014). Recently, there has been increasing interest in the use of phytochemicals for inhibition of adhesion and biofilm formation, particularly through the use of some natural compounds (Table 1.3). Anti-adhesion or antibiofilm formation activities have been shown for essential oils and for extracts from red wine, grape marc, pine bark and cranberry fruit, and also for specific natural compounds, such as ferulic acid, salicylic acid, phenyl isothiocyanate, trans-cinnamaldehyde, carvacrol, thymol, eugenol and others (Sandasi et al. 2010; Abreu et al. 2014; Lemos et al. 2014).

18

S. S. Možina et al.

Table 1.3 Treatments for the control of bacterial adhesion Treatment/compounds

Reference

Phytochemical addition Ferulic acid; salicylic acid; phenyl isothiocyanate; trans-cinnamaldehyde; carvacrol; thymol; eugenol; gallic acid; thyme extract; olive leaf extract; Quercus cerris; cinnamon essential oil; wine extracts; grape marc extracts; pine bark extracts; 7-hydroxycoumarin; indole-3-carbinol; saponin; okra fruit; Euodia ruticarpa; evocarpine and other quinoline fractions Biomaterials and biomaterial surfaces Cationic peptides; functionalized dendrimers; dendrimers; micro-/nanostructures; intrinsically antimicrobial materials; antimicrobial loading; antibiotic grafting; lysostaphin; phytoactive surfaces; dispersin B; cationic polymer grafting In vitro Anti-adhesion of Campylobacter Thyme extract; thyme postdistillation waste; olive waste; cayenne pepper; ginger; okra fruit extract; liquorice, Euodia ruticarpa

Simões et al. (2010), Abreu et al. (2014), Borges et al. (2013), Lemos et al. (2014), Soni et al. (2013), Bezek et al. (2016)

Campoccia et al. (2013)

Šikić Pogačar et al. (2016), Bensch et al. (2011), Bezek et al. (2016)

However, recently, we showed for the first time that a thyme extract is effective for inhibition of C. jejuni adhesion to a polystyrene surface and thus of biofilm formation, although this extract did not inhibit C. jejuni growth or kill C. jejuni cells at the effective concentration for the anti-adhesion activity (Šikić Pogačar et al. 2016). Additionally, the antimicrobial activity of this thyme extract was comparable to those of more well-known sources of plant phenolic compounds, like wine (Gañan et al. 2009) and grape skin, as have been tested against different foodborne pathogens (Katalinić et al. 2013). Adhered biomass is mostly evaluated using crystal violet staining, with measurement of the absorbance at 584 nm, which for the control, untreated C. jejuni K9/4 cells, is 0.13 ± 0.01 arbitrary units (a.u.) (Fig. 1.3). The adhered C. jejuni biomass to polystyrene can be significantly reduced by rosemary extract and (−)epigallocatechin gallate (EGCG), by more than 80% of the control adhesion (as shown in Fig. 1.3). The adhesion was significantly reduced also by rosmarinic acid with 50% reduction. Smaller reductions of 15% in the adhered C. jejuni biomass were noted for the tested ferulic acid. Interestingly, there was a similar reduction of adhered C. jejuni biomass at all tested concentrations which was similar to that seen at subinhibitory 0.25 MIC concentration (Klančnik et al. unpublished) (Fig. 1.3). Some research has been done for ferulic and gallic acids, which are known to act on the surface properties, particularly the occurrence of local rupture or pore

1 Antimicrobial Natural Products Against Campylobacter

19

Fig. 1.3 Absorbance determined with microtiter reader for biomass evaluation after dying with crystal violet for C. jejuni control cells and when natural compounds were added in MIC (minimal inhibitory concentration), 0.5 and 0.25 MIC concentrations

formation in the cell membranes, although again they can show different modes of action according to the concentrations used (Borges et al. 2013). Considering the mechanisms of anti-adhesion and the pronounced antimicrobial activities of thyme extract, it is known that more than one specific mechanism is involved, and thus, there are several targets in the cell activity (Šikić Pogačar et al. 2016). Additionally, a thyme postdistillation waste as a by-product of the agro-food industries and an olive (Olea europaea L.) leaf extract were tested as waste material. These residues have high economic burdens and can cause environmental problems; however, we showed that they are a source of bioactive phytochemicals that can inhibit C. jejuni adhesion at sub-bactericidal concentrations. Indeed, some studies have reported enhanced bacterial growth and induction of biofilm formation by essential oils or their components and also by antibiotics, such as aminoglycosides and b-lactams (Hoffman et al. 2005). The inability to inhibit biofilm growth at higher concentrations of olive leaf extracts confirms that C. jejuni cells in a biofilm respond adaptively and develop resistance (Šikić Pogačar et al. 2016). However, only a few studies have investigated the anti-adhesive activities of such natural substances against C. jejuni in different cell lines (Bensch et al. 2011). Low doses of thyme and olive extracts are not cytotoxic and can prevent specific cell adhesion of Campylobacter to PSI cells and to H4 human foetal small intestine cells (Šikić Pogačar et al. 2016). Again, these anti-adhesion activities were stable across a range of extract concentrations and occurred at lower concentrations in comparison with those for abiotic surfaces. Thus, these extracts can be used to modulate C. jejuni invasion and its intracellular survival, which represent the two most crucial mechanisms of colonization through which C. jejuni induces disease (Šikić Pogačar et al. 2016). These plant extracts can thus potentially be used as therapeutic agents, to replace certain bactericidal drugs (Rogers and Paton 2009).

20

1.5

S. S. Možina et al.

Antimicrobial Activity of Natural Products Against Campylobacter spp. in Food and Food Production

Campylobacteriosis is largely associated with the consumption of poultry meat, especially fresh broiler meat (Djenane et al. 2014; Humphrey et al. 2007). To reduce incidence of campylobacteriosis, different control measures in primary production of poultry meat production chain and in poultry meat manufacturing have been studied. These actions include also application of natural antimicrobial substances to reduce Campylobacter from poultry in farms and to reduce Campylobacter on poultry meats. Different strategies against Campylobacter in primary production include: (i) Feeding of chickens with components (probiotic, prebiotic and symbiotic antibacterial agents, natural plant extracts, organic acids, bacteriocins, combinations that act synergistically) that reduce colonization of poultry gut mucosa with Campylobacter; (ii) Vaccination of poultry against Campylobacter. Examples of application of natural antimicrobials against Campylobacter spp. in primary production are shown in Table 1.4. From examples, it is evident that in most cases adding of one antimicrobial component has no influence (Robyn et al. 2013), while the combination of more components is efficient. This is illustrated by the study of Baffoni et al. (2012) who showed that a microencapsulated combination of a prebiotic and a probiotic reduced C. jejuni counts by 1.1 log CFU/g in chicken faecal samples. Arsi et al. (2014) also studied the activity of a combination of thymol and carvacrol against C. jejuni in cecal contents. Although some combinations showed promising results, they were not replicated in at least two separate trials. The main limitation was rapid absorption of thymol and carvacrol in the upper intestine. Authors also stated other reasons for limited efficacy due to dietary interactions. Microencapsulation of antimicrobial compounds, for example propionic and sorbic acids combined with eugenol and thymol, showed significant reduction of C. jejuni in cecal contents (Grilli et al. 2013). Foods are complex systems, and use of natural antimicrobial substances is usually not possible without previous studies on food models and foods, since the results of in vitro studies of antimicrobial activities in microbiological culture media and laboratory conditions do not reflect the actual environment in the food. A higher concentration of natural antimicrobial substances is mostly needed to achieve the same effect in food in comparison with in vitro studies (Burt 2004; Table 1.5). Factors affecting the antimicrobial efficacy of natural antimicrobial substances in food are (Lucera et al. 2012): (i) Intrinsic factors such as pH, water activity, additives; (ii) Extrinsic factors such as temperature, relative humidity, modified or active packaging, vacuum, properties of naturally present microorganisms.

Propionic and sorbic acids combined with eugenol and thymol

Plant compounds

Thymol/carvacrol

Carvacrol

Thymol

Plant compounds

Galactooligosaccharide and microencapsulated Bifidobacterium longum subsp. longum (PCB133)

Prebiotic

Feed supplemented with microencapsula-ted organic acids and plant compounds

Natural plant compounds used as feed additive in chickens before slaughtering

Microencapsulated synbiotic mixture used as feed additive in broilers before slaughtering

Reduction of colonisation by competitive exclusion

Bacteria

Enterococcus faecalis MB 5259

Assay

Compound

In cecal

No log reduction 0 to 2.05 log reduction No log reduction

20 2.5 5 10 20 1.25/1.25 2.5/2.5

107 CFU/mL 107 CFU/mL 107 CFU/mL 107 CFU/mL 107 CFU/mL 107 CFU/mL 107 CFU/mL

2.1 log reduction 2.8 log reduction

1 for 14 days 3 for 14 days 5 for 14 days 10 for 14 days 1 for 21 days 3 for 21 days 5 for 21 days 10 for 21 days

105 CFU/mL 105 CFU/mL 105 CFU/mL 105 CFU/mL 105 CFU/mL 105 CFU/mL 105 CFU/mL 105 CFU/mL

5.2 log reduction

4.5 log reduction 3.3 log reduction

3.3 log reduction 3.4 log reduction

1.2 log reduction

0 to 2.03 log reduction In cecal

107 CFU/mL Feed

No log reduction No log reduction

0 to 01.95 log reduction No log reduction

No log reduction

10

107 CFU/mL

0 to 0.64 log reduction No log reduction

1.1 log reduction and 8.2 presence of Bifidobacterium 1.1 log reduction and 6.63 presence of Bifidobacterium In cecal

8.23 In faecal

8.23 7.41

2.5 5

21 days

14 days

21 days 21 days

Concentrations added (mg/L); Days

Log CFU/mL of C. jejuni

107 CFU/mL 107 CFU/mL

Feed additive Natural flora 8.48 log CFU/ mL 18.29 log CFU/ mL Feed additive

104 CFU/mL 108 CFU/mL

In vivo Conditions Inoculum

Table 1.4 Examples of studies of natural antimicrobials against Campylobacter jejuni in primary production

Grilli et al. (2013)

Arsi et al. (2014)

Baffoni et al. (2012)

Robyn et al. (2013)

Reference

1 Antimicrobial Natural Products Against Campylobacter 21

AD; MIC > 40

AD; MIC > 40

AD; MIC > 40

AD; MIC 0.6

BMD; MIC 0.08–0.16

BMD; MIC 1.03 BMD; MIC 0.63

Orange

Lemon

Citral

Linalool

Commercial Rosemary extract V40

Alpinia katsumadai seeds, ethanolic extract (AlpE) Epigallo-catechin-gallate (EGCG) AlpE/EGCG

AlpeE/EGCG at 37 °C 0

104 CFU/mL

No natural microflora

Minced meat

EGCG 0.63 at 37 °C

AlpeE/EGCG at 8 °C

104 CFU/mL

AlpeE 1.03 at 37 °C

EGCG 0.63 at 8 °C

104 CFU/mL

104 CFU/mL

AlpeE 1.03 at 8 °C

104 CFU/mL

AlpeE/EGCG at 37 °C

104 CFU/mL

0.08–0.16 at 37 °C

5.5x104 CFU/mL

104 CFU/mL

No natural MO

Chicken meat juice

EGCG 0.63 at 37 °C

0.31at 37 °C

103 CFU/mL

AlpeE 1.03 at 37 °C

0.31at 37 °C

105 CFU/mL

104 CFU/mL

0.31 at 37 °C

107 CFU/mL

104 CFU/mL

0.08–0.16 at 37 °C

103 CFU/mL

5 days; No natural microflora

0.08–0.16 at 37 °C

105 CFU/mL

Chicken meat juice sensory not tested

0.08–0.16 at 37 °C

107 CFU/mL

 72% growth inh.

54% growth inh.

 72% growth inh.

 81% growth inh.

60% growth inh.

 81% growth inh.

 80% growth inh.

69% growth inh.

 80% growth inh.

BD

> 2 log reduction

< 102 CFU/mL

< 102 CFU/mL

No growth inh.

No growth inh.

No growth inh.

No growth inh.

BD

5–6 log survival

No natural microflora

Chicken meat juice; sensory not tested;

Not tested

Not tested

Not tested

Not tested

Log CFU/mL differences

Assay; survival, reduction

Sensory acceptable; 109 cells/mL 0.6 at 37 °C

No natural microflora

Concentration added (mg/L); Temperature used (°C)

Conditions

Sensory not acceptable

Sensory not acceptable

Sensory not acceptable

Chicken skin; Sensory not acceptable

AD; MIC > 40

Sensory acceptance; Inoculum

Assay; MIC (mg/L)

Essential oils

In vivo

In vitro

Bergamot

Compound

Table 1.5 Examples of studies of natural antimicrobials against Campylobacter jejuni in food

(continued)

Klančnik et al. (2014)

Piskernik et al. (2011)

Fisher and Phillips (2006)

Reference

22 S. S. Možina et al.

Cinnamaldehyde

Apple-based edible films containing

Coriander

Essential oil

Compound

Table 1.5 (continued)

Not tested

1 at 32 °C 2.5 at 32 °C 5 at 32 °C

105 CFU/g 105 CFU/g

5 at 32 °C 1 at 4 °C 2.5 at 4 °C

105 CFU/g 105 CFU/g 105 CFU/g

No natural microflora 5 at 23 °C 15 at 23 °C 30 at 23 °C 5 at 4 °C 15 at 4 °C 30 at 4 °C

107 CFU/g 107 CFU/g 107 CFU/g 107 CFU/g 107 CFU/g 107 CFU/g

10 CFU/g Chicken breast Sensory not tested;

5 at 4 °C

2.5 at 32 °C

105 CFU/g

5

No natural microflora 1 at 32 °C

5 at 4 °C

105 CFU/g 105 CFU/g

2.5 at 4 °C

Lean beaf

1 at 4 °C

10 CFU/g 105 CFU/g

5

No natural microflora

AlpeE/EGCG at 8 °C

104 CFU/mL Chicken breast Sensory not tested;

EGCG 0.63 at 8 °C

104 CFU/mL

105 CFU/g

AlpeE 1.03 at 8 °C

104 CFU/mL

BMD; MIC 0.3–0.6

Concentration added (mg/L); Temperature used (°C)

Sensory acceptance; Inoculum

Assay; MIC (mg/L)

Conditions

In vivo

In vitro

2.3 log reduction

0.3 log reduction

0.2 log reduction

4.9 log reduction

4.9 log reduction

0.3 log reduction

BD

200 200

–

–

>500 >200

>500 –

2.5– 17.5

Helicobacter pylori

Candida albicans (yeast)

>500 –

>500 –

>200

Escherichia coli

–

–

12.5–50

10–500

12

11.6

25–300

2–16

–

Bacillus subtilis

Iso-a-acids or Iso-humulones

Humulone

a-acids in mixture

200

>200

>200

>200

–

–

>200

–

–

–

–

200

–

–

–

–

–

250

(Iso)humulic acid

200

>200

>200

>200

–

–

>200

–

–

–

–

>200

–

–

–

–

–

–

Hulupones

Table 2.3 Antimicrobial activity of acylphloroglucinol derivatives isolated from hops

200

>200

>200

>200

>200

–

–

–

–

–

–

>500

>500 –

>200

–

–

–

–

32

–

–

62

–

–

1–20

Lupulone

3.49  10−6−12.15  10−6

10

20

2–50

0.3

1–3.13

10

0.1

–

–

1

–

b-acids in mixture

(continued)

Mizobuchi and Sato (1985) Mizobuchi and Sato (1985)

Teuber (1970); Teuber and Schmalreck (1973); Schmalreck and Teuber (1975); Mizobuchi and Sato (1985); Simpson and Smith (1992); Bhattacharya et al. (2003); Yamaguchi et al. (2009); Čermák et al. (2015);

Source

2 An Overview of the Antimicrobial Properties of Hop 41

Strains

– –

–

–

Yellow fever virus

Plasmodium spp.

–

–

Human influenza virus –

–

–

Herpes simplex virus (HSV)

–

–

–

Human immunodeficiency virus (HIV)

–

–

–

Hepatitis B virus (HBV)

–

–

–

Cytomegalovirus (CMV)

Human rhinovirus

–

–

Bovine Viral Diarrhea Virus (BVDV)

Human respiratory syncytial virus (RSV)

Humulone

a-acids in mixture

–

NR

NR

NR

NR

NR

NR

9.2 (TC50 = 21 µg. mL−1; TI = 2.3)

9.5 (TC50 = 39 µg. mL−1; TI = 4.2)

4.7 (TC50 = 24 µg. mL−1; TI = 9.1)

Iso-a-acids or Iso-humulones

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

Hulupones

–

–

(Iso)humulic acid

–

NR

NR

NR

NR

NR

0.51 (TC50 = 0.21 µg.mL−1; TI < 1)

NR

0.13 (TC50 = 0.15 µg.mL−1; TI = 1.2)

NR

b-acids in mixture

2.5

–

–

–

–

–

–

–

–

–

Lupulone

Srinivasan et al. (2004)

Buckwold et al. (2004)

Source

Antibacterial and antifungal activities were expressed in MIC (in µg.mL−1): values or range of values if obtained by several data sources; antiviral and antiparasitic activities were expressed in IC50 (in µg. mL−1); NR = IC50 not reached; – = no data available in the literature Antiviral activities: IC50 = inhibitory concentration in µg.mL−1 required to reduce viral replication by 50%; TC50 = toxic concentration in µg.mL−1 required to reduce the number of viable cells by 50%. Extracts were tested at a maximum concentration of 100 µg.mL−1; Therapeutic index (TI): TC50/IC50

Antiparasitic (IC50 lg.mL−1)

Antiviral (IC50 lg.mL−1)

Antimicrobial activity

Table 2.3 (continued)

42 L. Bocquet et al.

Antiviral (IC50 lg.mL−1, except for HCV and for HIV)

Antifungal (MIC lg.mL−1)

Gram positive

Antibacterial (MIC lg.mL−1)

Gram negative

Strains

Antimicrobial activity

– >200 >200 >200 100 200 >200

– – – – – – – –

– – –

Staphylococcus epidermidis

Staphylococcus pyogenes

Streptococcus mutans

– –

–

– –

–

–

>200 >200 50 3.13 3.13 –

–

– –

–

–

Candida albicans (yeast)

Fusarium oxysporum

Mucor rouxianus

Trichophyton mentagrophytes

Trichophyton rubrum

Bovine Viral Diarrhea Virus (BVDV)a

Cytomegalovirus (CMV)a

Hepatitis B virus (HBV)

Human immunodeficiency virus (HIV)b

Herpes simplex virus (HSV-1)a

Herpes simplex virus (HSV-2)a

>200

100

–

6.25

Staphylococcus aureus

Escherichia coli

–

–

–

Propionibacterium acnes

–

–

–

–

–

–

–

–

–

17 (TC50 = 55 µg. mL−1;

NR

–

–

12 (TC50 = 25 µg. mL−1; TI = 2.2)

NR

>200

200

>200

>200

>200

>200

–

–

–

50

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

Kocuria rhizophila

–

–

–

Enterococcus faecalis

–

Chalcones IsoXN

DMX

Naringenin

8-PN

Flavanones 6-PN

Table 2.4 Antimicrobial activity of some prenylated flavonoids isolated from hops

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

DHX

1.5 (TC50 = 8.9 µg. mL−1;

2.7 (TC50 = 7.7 µg. mL−1; TI = 3.7)

0.82 (TC50 = 8.82 µg. mL−1; TI = 10.8)

–

2.5 (TC50 = 6.5 µg. mL−1; TI = 2.6)

1.7 (TC50 = 6.1 µg. mL−1; TI = 4.2)

3.13

3.13

50

>200

>200

>200

12.5

1

3

1–125

3

1

62

XN

(continued)

Buckwold et al. (2004); Wang et al. (2004); Gerhäuser, (2005); Lou et al. (2014)

Mizobuchi and Sato (1984); Gerhäuser (2005)

Mizobuchi and Sato (1984); Bhattacharya et al. (2003); Gerhäuser (2005); Yamaguchi et al. (2009); Rozalski et al. (2013);

Source

2 An Overview of the Antimicrobial Properties of Hop 43

Strains

–

–

Plasmodium spp.

–

–

–

Hepatitis C virus (HCV)c –

–

–

Yellow fever virus 18.8–50 and more

–

–

Human respiratory syncytial virus (RSV)

Plasmodium falciparum

–

–

–

–

–

–

–

–

–

–

–

–

6.6 (TC50 = 18 µg. mL−1; TI = 3.1)

–

14.4– 31.3

–

–

–

–

–

Human rhinovirusa

–

TI = 4.2) –

–

Human influenza virus

–

Chalcones IsoXN

DMX

Naringenin

8-PN

Flavanones 6-PN

–

4.4– 6.2

–

–

–

–

–

DHX

1.8

1–8.5

3.53 µM

–

–

NR

–

TI = 6.3)

XN

Herath et al. (2003); Srinivasan et al. (2004); Gerhäuser (2005); Frölich et al. 2005, 2009

Source

NR = IC50 not reached, – = no data available in the literature. Compounds are 6-prenylnaringenin (6-PN), 8-prenylnaringenin (8-PN), naringenin, isoxanthohumol (isoXN), desmethylxanthohumol (DMX), dihydro-xanthohumol (DHX), and xanthohumol (XN) a IC50 = inhibitory concentration in µg.mL−1 required to reduce viral replication by 50%; TC50 = toxic concentration in µg.mL−1 required to reduce the number of viable cells by 50%. Extracts were tested at a maximum concentration of 100 µg.mL−1; Therapeutic index (TI): TC50/IC50 b EC50 = inhibitory concentration in µg.mL−1 required to inhibit the cytopathic effects by 50%; CC50 = cytotoxic concentration in µg.mL−1 required to reduce the number of viable cells by 50% (C8166 cells). Therapeutic index (TI): CC50/EC50 c At 3.53 µM (1.25 µg.mL−1), xanthohumol decreased significantly the luciferase activity as compared to the negative control and induced a significant decrease of the HCV RNA level. No cytotoxic effect on the cell viability

Antiparasitic (IC50 lg.mL−1)

Antimicrobial activity

Table 2.4 (continued)

44 L. Bocquet et al.

2 An Overview of the Antimicrobial Properties of Hop

45

are more active against Gram-positive bacteria than against fungus. Mizobuchi and Sato (1984) also reported that MIC against Staphylococcus aureus is equal to 6.25 µg.mL−1 for xanthohumol and 6-prenylnaringenin (17.7 µM and 18.4 µM, respectively) (Gerhäuser 2005). Flavonoids, such as flavan-3-ols, flavonols, and tannins, isolated from hops, are also known to be good antibacterial agents. These compounds, very common in plants and not specific of hops, contribute to reinforce the antibacterial activity of hop’s female cones and probably contribute to the slight activity of its leaves (Karabín et al. 2016). The major monoterpenes and sesquiterpenes of hops’ essential oil showed moderate antibacterial effects against some Gram-negative bacteria (Proteus vulgaris, Escherichia coli, Pseudomonas aeruginosa) and some Gram-positive bacteria (Enterococcus faecalis and Streptococcus aureus) (Jirovetz et al. 2006) (Table 2.5). Few studies, however, described the activity of this plant and of its secondary metabolites against multidrug-resistant strains. One study conducted by Wendakoon et al. in 2012 has demonstrated the antibacterial activity of hops against strains resistant to antibiotics, specifically against Methicillin-resistant Staphylococcus aureus (MRSA). In addition, the mechanisms of the inhibitory activities observed are not expanded upon. Active molecules extracted from plants and combined with selected antibiotics could be a new strategy to treat certain infections, especially caused by multidrug-resistant bacteria (Gibbons 2008).

2.3.2

Antifungal Activity

The antifungal activity of hops boils down mainly to tests against some human pathogenic fungi. According to studies conducted by Langezaal et al. (1992), Gram-positive bacteria are more susceptible to hops’ extracts than fungi. The essential oil of hops is generally less active than an extract of cones. An exception exists with the fungus Fusarium mentagrophytes. In this case, the essential oil and the extract showed the same antifungal activity. The prenylated chalcone, xanthohumol, and the prenylated flavanone, 6-prenylnaringenin, have been identified as the most potent antifungal compounds of hops against Trichophyton mentagrophytes and T. rubrum, with a MIC equal to 3.13 µg.mL−1. They also showed a weak activity against Mucor rouxianus with a MIC of 50 µg.mL−1, corresponding to 141.2 µM for xanthohumol and 146.9 µM for 6-prenylnaringenin (Gerhäuser 2005). On the other side, these two compounds demonstrated a very low activity against pathogenic species of Fusarium as well as bitter acids (Langezaal et al. 1992; Mizobuchi and Sato 1984; Mizobuchi and Sato 1985). Antifungal activity against Penicillium and Aspergillus species was very low (Engelson et al. 1980; Mizobuchi and Sato 1985). Some authors also demonstrated that yeast species such as Saccharomyces cerevisiae and Saccharomyces pastorianus are not inhibited by normal concentrations of bitter acids (Schmalreck and Teuber 1975), allowing to

Gram positive

Antibacterial

Bacillus subtilis Enteroccus faecalis Staphylococcus aureus Staphylococcus pyogenes Gram Citrobacter negative fruendii Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Proteus vulgaris Antifungal Candida albicans (yeast) Inhibition diameters (in mm): values or range

Strains

Antimicrobial activity – 8

– 10 10 – – 8 – – 8 –

– – – – – 7 – – 8 –

–

–

–

8–19.5 8

10

–

25.3

26.9 –

Linalool

10

–

9

9 –

–

–

10

– –

–

8

8

10 –

–

–

11

– –

Sesquiterpenes a-Humulene b-Caryophyllene

of values if obtained by several data sources; – = no data available in the literature

25

12

– –

11

15 10

– 9 7

–

–

12

– 15

Geraniol

9

7

7

a-Terpineol

Monoterpenes b-Myrcene b-Pinene

Table 2.5 Antimicrobial activity of terpenes isolated from hops’ essential oil

Jirovetz et al. (2006)

Jirovetz et al. (2006); Kotan et al. (2007); Akdemir Evrendilek, (2015);

Source

46 L. Bocquet et al.

2 An Overview of the Antimicrobial Properties of Hop

47

safeguard cultured yeast strains vital to maintaining production of beer, while avoiding a bacterial contamination.

2.3.3

Antiviral Activity

The antiviral activity of extracts from hops and some of its metabolites was investigated against several DNA and RNA viruses. Xanthohumol showed a moderate antiviral activity against bovine viral diarrhea virus (BVDV), a surrogate model of hepatitis C virus, with an IC50 equal to 1.7 ± 1.4 µg.mL−1 and a therapeutic index TI (TI = cytotoxic concentration CC50/IC50) equal to 4.2 ± 0.99. This chalcone also showed a moderate activity against some herpes viruses including cytomegalovirus (CMV) (IC50 = 2.5 ± 0.56 µg.mL−1, therapeutic index = 2.6 ± 1.0), herpes simplex virus type 1 (IC50 = 2.7 ± 1.7 µg.mL−1, therapeutic index = 3.7 ± 1.8), and herpes simplex virus type 2 (IC50 = 1.5 ± 0.35 µg.mL−1, therapeutic index = 6.3 ± 3.3). Isoxanthohumol was less active. Iso-a-acids had also a moderate antiviral activity against BVDV (IC50 = 4.7 ± 5.1 µg.mL−1, therapeutic index = 9.1 ± 8.6) and against CMV (IC50 = 9.5 ± 0.5 µg.mL−1, therapeutic index = 4.2 ± 0.86) (Buckwold et al. 2004). Further studies showed that the antiviral effect of xanthohumol against BVDV-implicated several mechanisms including inhibition of BVDV-induced cytopathic effects, as well as inhibition of BVDV E2 expression and viral RNA levels (Zhang et al. 2009). Xanthohumol also could enhance the antiviral effect of interferon-a-2b against BVDV (Zhang et al. 2010). The study conducted by Lou et al. in 2014 confirmed the anti-hepatitis C virus activity of xanthohumol in a HCV cell culture (HCVcc) system and showed that this chalcone had an inhibitory effect on the replication of the virus. Xanthohumol could exert its antiviral activity at least in part by affecting diacylglycerol acyltransferase-1 (DGAT1) and/or microsomal triglyceride transfer protein (MTP) activity (Lou et al. 2014). An in vivo study also showed that xanthohumol may reduce hepatic inflammation, steatosis, and fibrosis induced by HCV mainly through inhibition of oxidative reaction, regulation of apoptosis, modulation of MTP activity, and inhibition of hepatic stellate cells (Yang et al. 2013). At last, xanthohumol was also effective against HIV-1 (human immunodeficiency virus). This phenolic compound inhibited HIV-1-induced cytopathic effects (EC50 = 0.82 µg/mL or 2 µM), the production of viral p24 antigen (EC50 = 1.28 µg/mL or 3.21 µM) and reverse transcriptase (EC50 = 0.50 µg/mL or 1.22 µM) in C8166 lymphocytes, with a therapeutic index (TI) equal to 10.8 (CC50 = 8.82 µg/mL or 21.51 µM) (Wang et al. 2004).

48

2.3.4

L. Bocquet et al.

Antiparasitic Activity

Chalcones are among the most effective class of natural compounds against Plasmodium. Frölich et al. (2005, 2009) have tested the antiparasitic activity of several chalcones from hops, including xanthohumol and seven derivatives. They showed that xanthohumol was the most potent inhibitor of the malarial protozoa Plasmodium falciparum with an inhibitory concentration corresponding to 50% for cell viability (IC50) of 8.2 and 24 µM depending on the strain. Herath et al. (2003) have tested the xanthohumol and several derivatives obtained by microbial transformation with the fungus Cunninghamella echinulata, against two strains of Plasmodium falciparum. They showed that xanthohumol is still one of the most active chalcone with IC50 between 1 and 3.3 µg.mL−1. The activity of xanthohumol and some bitter acids against Plasmodium sp. had also been highlighted by Srinivasan et al. (2004). The same authors showed that hop compounds are broadly active against protozoa, but more active against ciliates and flagellates than against amoebae. Beta acids and xanthohumol were more active than tetra-hydro-iso-alpha acids against all protozoa tested. The antimicrobial activities of hop’s extracts and of its main metabolites are summarized in the tables.

2.4

Safety and Bioavailability of Active Compounds

Most of the studies cited before have been assessed in vitro. Many parameters related to in vivo activities and clinical studies have to be considered for therapeutic applications, including bioavailability and safety in animals and in humans. Hops has been used in phytotherapy for many years. According to monograph established by the Committee on Herbal Medicinal Products (HMPC) in 2014, hops cones can be proposed for the management of moderate mental stress symptoms and to help sleep. It may be sold in the form of capsules or plant powder for preparation of herbal tea (European Medicines Agency 2014). In vivo studies as well as clinical studies have been carried out on these preparations, to check their safety as well as that of the active ingredients. A study carried out on mice during 4 weeks with a diet supplemented with 5  10−4 M of xanthohumol showed the safety of this metabolite which did not affect major organ functions. There was no difference in the metabolism of lipids, proteins, carbohydrates, and uric acid (Vanhoecke et al. 2005). Other in vivo studies have also confirmed the good safety profile of this main chalcone of hops in mice and rats (Hussong et al. 2005; Dorn et al. 2010). A more extensive pharmacokinetic study with xanthohumol has been assessed on 48 human subjects (24 men and 24 women). Each subject received 20, 60, or 180 mg of XN. Their blood was then analyzed at different times after ingestion, confirming the safety of the molecule for a short intake (Legette et al. 2014). Van Breemen et al. (2014) treated postmenopausal women with extracts of

2 An Overview of the Antimicrobial Properties of Hop

49

hops containing the same molecules (xanthohumol, isoxanthohumol, 6- and 8-prenylnaringenin, sex hormones, and prothrombin) over periods of 5 days 3 consecutive times at monthly interval. No effects on sex hormones and blood coagulation have been demonstrated during this clinical study (van Breemen et al. 2014). 8-prenylnaringenin taken as a single dose at 750 mg is known to be well tolerated by women, while demonstrating complete absorption and metabolic stability (Rad et al. 2006). Legette et al. (2012) have also studied the bioavailability of xanthohumol in rats. They confirmed that xanthohumol is absorbed by intestinal cells and transported in blood. The bioavailability depended on the dose taken and was 11, 13, and 33% for high, medium, and low doses, respectively, suggesting a saturation effect to a threshold dose. The part of xanthohumol that is not absorbed by the intestine can be transformed into isoxanthohumol by the intestinal microbiota (Legette et al. 2012). isoxanthohumol and more generally prenylflavonoids are not transformed during their passage through the stomach and small intestine (Żołnierczyk et al. 2015). The conversion of isoxanthohumol occurs in the colon because a third of the bacterial population is able to convert it into 8-prenylnaringenin (Possemiers et al. 2006). The latter could then be modified in the liver. Indeed, Nikolic et al. (2004) have shown that human hepatic microsomes are capable of modifying the molecule, thereby forming 12 different metabolites based on the prenylated chain or on the flavanone moiety. Information regarding bioavailability of other antimicrobial phenolic compounds isolated from hops, such as bitter acids, is yet limited (Karabin et al. 2015).

2.5

Industrial Applications

This fundamental research conducted on hops could lead to multiple applications, including cosmetics, food, and pharmaceutical sectors. In food industry, the use of iso-a-acids in beer is well known. If the antibacterial potential of hops in human health is very promising, hops could be also used as natural preservative. Bacterial multidrug resistance is a real public health issue, and the use of natural preservatives is a question more topical than ever. Indeed, the industry is in considerable need of replacements for preservatives in cosmetics and food. Because of many side effects observed with synthetic preservatives (Boberg et al. 2010; Lundov et al. 2011) such as the endocrine disrupting effects of parabens or contact allergy with methylisothiazolinone, research on natural preservatives, for which the safety is verified, is an urgent priority. Industry is confronted with a problematic health situation where a decrease in the concentration of preservatives is not always possible, rendering the antibacterial protection insufficient. In the cosmetic, pharmaceutical, and food industries, manufacturers are now encouraged to favor physical methods or to find alternative preservatives including natural preservatives (Varvaresou et al. 2009; Negi 2012). Various hop’s extracts have been already tested against foodborne pathogens to improve the preservation of meat, and promising results have been demonstrated (Kramer et al. 2015).

50

2.6

L. Bocquet et al.

Conclusion

Hop biosynthesizes original phenolic compounds responsible for many biological activities, which are mainly prenylated acylphloroglucinols and prenylflavonoids. Most of them are synthesize in glandular trichomes and are then found in the yellow resin of hop cones. These same parts are used in the brewing industry to bring bitterness and antiseptic properties. The antimicrobial potential of hop cones is known for several years; it is mainly directed against Gram-positive bacteria. The antimicrobial activity of leaves is weak in comparison with cones. Stems and rhizomes have not been studied for these properties; their composition is also unknown. Acylphloroglucinols (a- and b-acids) are responsible for a part of the activity. They have antibacterial and antiviral activities, but their antifungal potential is lower. The major prenylated chalcone, xanthohumol, is the most interesting compound because it has significant potential against fungus, parasites, virus, and bacteria including resistant strains. Some flavanones also show antimicrobial activities, including antifungal and antibacterial properties for 6-prenylnaringenin, and antiviral activity for isoxanthohumol. Whereas 8-prenylnaringenin does not show any antimicrobial activity, it is a potent phytoestrogen. Antimicrobial activities of these metabolites are of interest, but in vivo and clinical studies are still few and are required to prove their actual impact on human health. The pharmacokinetic and pharmacodynamics properties of these prenylated phenolic compounds must be further studied, in particular those of acylphloroglucinols.

References Abram V, Čeh B, Vidmar M, Hercezi M, Lazíc N, Bucik V, Možina SS, Košir IJ, Kač M, Demšar L, Ulrih NP (2015) A comparison of antioxidant and antimicrobial activity between hop leaves and hop cones. Ind Crops Prod 64:124–134 Aghamiri V, Mirghafourvand M, Mohammad-Alizadeh-Charandabi S, Nazemiyeh H (2016) The effect of Hop (Humulus lupulus L.) on early menopausal symptoms and hot flashes: a randomized placebo-controlled trial. Complement Ther Clin Pract 23:130–135 Akazawa H, Kohno H, Tokuda H, Suzuki N, Yasukawa K, Kimura Y, Manosroi A, Manosroi J, Akihisa T (2012) Anti-Inflammatory and Anti-Tumor-Promoting Effects of 5-Deprenyllupulonol C and Other Compounds from Hop (Humulus lupulus L.). Chem Biodiv 9:1045–1053 Akdemir Evrendilek G (2015) Empirical prediction and validation of antibacterial inhibitory effects of various plant essential oils on common pathogenic bacteria. Int J Food Microbiol 202:35–41 Allsopp P, Possemiers S, Campbell D, Gill C, Rowland I (2013) A comparison of the anticancer properties of isoxanthohumol and 8-Prenylnaringenin using in Vitro models of colon cancer. BioFactors 39(4):441–447 Bhattacharya S, Virani S, Zavro M, Hass GJ (2003) Inhibition of Streptococcus mutans and other oral streptococci by hop (Humulus lupulus L.) constituents. Econ Bot 57(1):118–125

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Boberg J, Taxvig C, Christiansen S, Hass U (2010) Possible endocrine disrupting effects of parabens and their metabolites. Reprod Toxicol 30(2):301–312 Buckwold VE, Wilson RJH, Nalca A, Beer BB, Voss TG, Turpin JA, Buckheit RW III, Wei J, Wenzel-Mathers M, Walton EM, Smith RJ, Pallansch M, Ward P, Wells J, Chuvala L, Sloane S, Paulman R, Russell J, Hartman T, Ptak R (2004) Antiviral activity of hop constituents against a series of DNA and RNA viruses. Antivir Res 61(1):57–62 Cazarolli LH, Kappel VD, Zanatta AP, Suzuki DOH, Yunes RY, Nunes RJ, Pizzolatti MG, Silva FRMB (2013) Natural and synthetic chalcones: tools for the study of targets of action— insulin secretagogue or insulin mimetic. In: Attar U-Rahman (eds) Studies in natural products chemistry, vol 39. Elsevier Science BV, Amsterdam, pp 47–90 Čermák P, Palečková V, Houška M, Strohalm J, Novotná P, Mikyška A, Jurková M, Sikorová M (2015) Inhibitory effects of fresh hops on Helicobacter pylori strains. Czech J Food Sci 33 (4):302–307 Chadwick LR, Pauli GF, Farnsworth NR (2006) The pharmacognosy of Humulus lupulus L. (hops) with an emphasis on estrogenic properties. Phytomedicine 13(1–2):119–131 Clark SM, Vaitheeswaran V, Ambrose SJ, Purves RW, Page JE (2013) Transcriptome analysis of bitter acid biosynthesis and precursor pathways in hop (Humulus lupulus). BMC Plant Biol 13:1–12 De Keukeleire J, Ooms G, Heyerick A, Roldan-Ruiz I, Van Bockstaele E, De Keukeleire D (2003) Formation and accumulation of a-Acids, b-Acids, Desmethylxanthohumol, and Xanthohumol during flowering of hops (Humulus lupulus L.). J Agric Food Chemistry 51(15):4436–4441 De Keukeleire J, Janssens I, Heyerick A, Ghekiere G, Cambie J, Roldán-Ruiz I, Van Bockstaele E, De Keukeleire D (2007) Relevance of organic farming and effect of climatological conditions on the formation of a-Acids, b-Acids, Desmethylxanthohumol, and Xanthohumol in hop (Humulus lupulus L.). J Agric Food Chem 55(1):61–66 Dorn C, Bataille F, Gaebele E, Heilmann J, Hellerbrand C (2010) Xanthohumol feeding does not impair organ function and homoeostasis in mice. Food Chem Toxicol 48(7):1890–1897 Engelson M, Solberg M, Karmas E (1980) Anti-mycotic properties of hop extract in reduced water activity media. J Food Sci 45(5):1175–1178 European Commission (2014) Agriculture and rural development. Hop report for the harvest year 2014. https://ec.europa.eu/agriculture/sites/agriculture/files/hops/reports/pdf/report-2014_en. pdf Accessed 30 Nov 2016 European Medicines Agency (2014) Community herbal monograph on Humulus lupulus L., flos EMA/HMPC/682384/2013 (Committee on Herbal Medicinal Products-HMPC) Faivre C, Ghedira K, Goetz P, Lejeune R, Staub H (2007) Humulus lupulus L. Phytothérapie 5 (2):86–89 Frölich S, Schubert C, Bienzle U, Jenett-Siems K (2005) In Vitro Antiplasmodial activity of prenylated chalcone derivatives of hops (Humulus lupulus) and their interaction with Haemin. J Antimicrob Chemother 55(6):883–887 Frölich S, Schubert C, Jenett-Siems K (2009) Antimalarials from prenylated chalcone derivatives of hops. In: Preedy VR (ed) Beer in health and disease prevention. Academic Press, San Diego, pp 747–752 Gerhäuser C (2005) Broad spectrum anti-infective potential of xanthohumol from hop (Humulus lupulus L.) in comparison with activities of other hop constituents and xanthohumol metabolites. Mol Nutr Food Res 49(9):827–831 Gibbons S (2008) Phytochemicals for bacterial resistance—strengths, weaknesses and opportunities. Planta Med 74(6):594–602 Hall Amy J, Babish John G, Darland Gary K, Carroll Brian J, Konda Veera Reedy, Lerman Robert H, Bland Jeffery S, Tripp Matthew L, Hall AJ, Babish JG, Darland GK, Carroll BJ, Konda VR, Lerman RH, Bland JS, Tripp ML (2008) Safety, efficacy and anti-inflammatory activity of rho iso-alpha-acids from hops. Phytochemistry 69(7):1534–1547 Herath W, Ferreira W, Khan SI, Khan IA (2003) Identification and biological activity of microbial metabolites of xanthohumol. Chem Pharm Bull 51(11):1237–1240

52

L. Bocquet et al.

Hussong R, Frank N, Knauft J, Ittrich C, Owen R, Becker H, Gerhaüser (2005). A safety study of oral xanthohumol administration and its influence on fertility in Sprague Dawley rats. Mol Nutr Food Res 49(9):861–867 Jirovetz L, Bail S, Buchbauer G, Denkova Z, Slavchev A, Stoyanova A, Schmidt E, Geissler M (2006) Antimicrobial testings, gas chromatographic analysis and olfactory evaluation of an essential oil of hop cones (Humulus lupulus L.) from Bavaria and some of its main compounds. Sci Pharm 74:189–201 Karabin M, Hudcová T, Jelínek L, Dostálek P (2015) Biotransformations and biological activities of hop flavonoids. Biotechnol Adv 33:1063–1090 Karabín M, Hudcová T, Jelínek L, Dostálek P (2016) Biologically active compounds from hops and prospects for their use. Compr Rev Food Sci F 15(3):542–567 Kitamura K, Komiyama O, Tobe H (1998) Pharmaceutical composition for treating osteoporosis containing xanthohumol. US Patent 5,679,716 A Kotan R, Kordali S, Cakir A (2007) Screening of antibacterial activities of Twenty-One oxygenated monoterpenes. Z Naturforsch C 62(7–8):507–513 Kramer B, Thielmann J, Hickisch A, Muranyi P, Wunderlich J, Hauser C (2015) Antimicrobial activity of hop extracts against foodborne pathogens for meat applications. J Appl Microbiol 118(3):648–657 Krause E, Yuan Y, Hajirahimkhan A, Dong H, Dietz BM, Nikolic D, Pauli GF, Bolton JL, van Breemen RB (2014) Biological and chemical standardization of a hop (Humulus lupulus) botanical dietary supplement. Biomed Chromatogr 28(6):729–734 Langezaal CR, Chandra A, Scheffer JJC (1992) Antimicrobial screening of essential oils and extracts of some Humulus lupulus L. cultivars. Pharm Weekbl 14(6):353–356 Larson AE, Yu RR, Lee OA, Price S, Haas GJ, Johnson EA (1996) Antimicrobial activity of hop extracts against Listeria monocytogenes in media and in food. Int J Food Microbiol 33(2– 3):195–207 Legette L, Ma L, Reed RL, Miranda CL, Christensen JM, Rodriguez-Proteau R, Stevens JF (2012) Pharmacokinetics of xanthohumol and metabolites in rats after oral and intravenous administration. Mol Nutr Food Res 56(3):466–474 Legette L, Karnpracha C, Reed RL, Choi J, Bobe G, Christensen JM, Rodriguez-Proteau R, Purnell JQ, Stevens JF (2014) Human pharmacokinetics of xanthohumol, an anti-hyperglycemic flavonoid from hops. Mol Nutr Food Res 58(2):248–255 Lou S, Zheng YM, Liu SL, Qiu J, Han Q, Li N, Zhu Q, Zhang P, Yang C, Liu Z (2014) Inhibition of hepatitis C virus replication In Vitro by xanthohumol, a natural product present in hops. Planta Med 80(2–3):171–176 Lundov MD, Krongaard T, Menné TL, Johansen JD (2011) Methylisothiazolinone contact allergy: a review. Br J Dermatol 165(6):1178–1182 Mabberley DJ (2009) Mabberley’s Plant-Book. a portable dictionary of plants, their classification and uses. Third edition, completely revised. Cambridge University Press, Cambridge, UK, p 1021 Malizia RA, Molli JS, Cardell DA, Grau RJA (1999) Essential oil of hop cones (Humulus lupulus L.). J Essent Oil Res 11(1):13–15 Matousek J, Kocábek T, Patzak J, Stehlík J, Füssy Z, Krofta K, Heyerick A, Roldán-Ruiz I, Maloukh L, De Keukeleire D (2010) Cloning and molecular analysis of HlbZip1 and HlbZip2 transcription factors putatively involved in the regulation of the lupulin metabolome in hop (Humulus lupulus L.). J Agric Food Chem 58:902–912 Mizobuchi S, Sato Y (1984) A new flavanone with antifungal activity isolated from hops. Agric Biol Chem 48(11):2771–2775 Mizobuchi S, Sato Y (1985) Antifungal activities of hop bitter resins and related compounds. Agric Biol Chem 49(2):399–403 Mouratidis PXE, Colston KW, Tucknott ML, Tyrrell E, Pirianov G (2013) An investigation into the anticancer effects and mechanism of action of hop b-Acid Lupulone and its natural and synthetic derivatives in prostate cancer cells. Nutr Cancer 65(7):1086–1092

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53

Nagel J, Culley LK, Lu Y, Liu E, Matthews PD, Stevens JF, Page JE (2008) EST analysis of hop glandular trichomes identifies an O-methyltransferase that catalyzes the biosynthesis of xanthohumol. Plant Cell 20(1):186–200 Natarajan P, Katta S, Andrei I, Babu Rao Ambati V, Leonida M, Haas GJ (2008) Positive antibacterial co-action between hop (Humulus lupulus) constituents and selected antibiotics. Phytomedicine 15(3):194–201 Negi PS (2012) Plant extracts for the control of bacterial growth: efficacy, stability and safety issues for food application. Int J Food Microbiol 156(1):7–17 Nikolic D, Li Y, Chadwick LR, Grubjesic S, Schwab P, Metz P, van Breemen RB (2004) Metabolism of 8-prenylnaringenin, a potent phytoestrogen from hops (Humulus lupulus), by human liver microsomes. Drug Metab Dispos 32(2):272–279 Ohsugi M, Basnet P, Kadota S, Ishii E, Tamura T, Okumura Y, Namba T (1997) Antibacterial activity of traditional medicines and an active constituent lupulone from H. lupulus against Helicobacter pylori. J Trad Med 14:186–191 Paoletti T, Fallarini S, Gugliesi F, Minassi A, Appendino G, Lombardi G (2009) Anti-inflammatory and vascular protective properties of 8-prenylapigenin. Eur J Pharmacol 620(1–3):120–130 Possemiers S1, Bolca S, Grootaert C, Heyerick A, Decroos K, Dhooge W, De Keukeleire D, Rabot S, Verstraete W, Van de Wiele T (2006) The prenylflavonoid isoxanthohumol from hops (Humulus lupulus L.) is activated into the potent phytoestrogen 8-prenylnaringenin in vitro and in the human intestine. J Nutr 136(7):1862–1867 Rad M, Hümpel M, Schaefer O, Schoemaker RC, Schleuning WD, Cohen AF, Burggraaf J (2006) Pharmacokinetics and systemic endocrine effects of the phyto-oestrogen 8-prenylnaringenin after single oral doses to postmenopausal women. Br J Clin Pharmacol 62(3):288–296 Rozalski M, Micota B, Sadowska B, Stochmal A, Jedrejek D, Wieckowska-Szakiel M, Rozalska B (2013) Antiadherent and antibiofilm activity of Humulus lupulus L. derived products: new pharmacological properties. Biomed Res Int 2013:101089 Sakamoto K, Konings WN (2003) Beer spoilage bacteria and hop resistance. Int J Food Microbiol 89(2–3):105–124 Sansawat T, Lee HC, Zhang L, Ryser ET, Kang I (2016) Antilisterial effects of different hop acids in combination with potassium acetate and potassium diacetate at 7 and 37 °C. Food Control 59:256–261 Schmalreck AF, Teuber M (1975) Structural features determining the antibiotic potencies of natural and synthetic hop bitter resins, their precursors and derivatives. Can J Microbiol 21 (2):205–212 Simpson WJ, Smith AR (1992) Factors affecting antibacterial activity of hop compounds and their derivatives. J Appl Bacteriol 72(4):327–334 Simpson MG (2010) Plant systematics, 2nd edn. Academic Press (Elsevier), Burlington, USA. 740 p Small E (1978) A numerical and nomenclatural analysis of Morpho-Geographic taxa of Humulus. Syst Bot 3(1):37–76 Srinivasan V, Goldberg D, Haas GJ (2004) Contributions to the antimicrobial spectrum of hop constituents. Econ Bot 58(1):230–238 Stevens JF, Taylor AW, Deinzer ML (1999) Quantitative analysis of xanthohumol and related prenylflavonoids in hops and beer by liquid chromatography–tandem mass spectrometry. J Chrom A 832(1–2):97–107 Stevens JF, Page JE (2004) Xanthohumol and related prenylflavonoids from hops and beer: to your good health. Phytochemistry 65(10):1317–1330 Tan KW, Cooney J, Jensen D, Li Y, Paxton JW, Birch NP, Scheepens A (2014) Hop-Derived prenylflavonoids are substrates and inhibitors of the efflux transporter breast cancer resistance protein (BCRP/ABCG2). Mol Nutr Food Res 58(11):2099–2110 Teuber M (1970) Low antibiotic potency of isohumulone. Appl Microbiol 19(5):871

54

L. Bocquet et al.

Teuber M, Schmalreck AF (1973) Membrane leakage in Bacillus subtilis 168 induced by the hop constituents lupulone, humulone, isohumulone and humulinic acid. Arch Für Mikrobiol 94 (2):159–171 Tsurumaru Y, Sasaki K, Miyawaki T, Uto Y, Momma T, Umemoto N, Momose M, Yazaki K (2012) HIPT-1, a Membrane-Bound prenyltransferase responsible for the biosynthesis of bitter acids in hops. Biochem Biophys Res Commun 417(1):393–398 The Plant List (2013). http://www.theplantlist.org Accessed 30 Nov 2016 Van Breemen RB, Yuan Y, Banuvar S, Shulman LP, Qiu X, Alvarenga RF, Chen SN, Dietz BM, Bolton JL, Pauli GF, Krause E, Viana M, Nikolic D (2014) Pharmacokinetics of prenylated hop phenols in women following oral administration of a standardized extract of hops. Mol Nutr Food Res 58(10):1962–1969 Vanhoecke BW, Delporte F, Van Braeckel E, Heyerick A, Depypere HT, Nuytinck M, De Keukeleire D, Bracke ME (2005) A safety study of oral tangeretin and xanthohumol administration to laboratory mice. Vivo 19(1):103–107 Varvaresou A, Papageorgiou S, Tsirivas E, Protopapa E, Kintziou H, Kefala V, Demetzos C (2009) Self-preserving cosmetics. Int J Cosmet Sci 31(3):163–175 Villalobos-Delgado LH, Caro I, Blanco C, Bodas R, Andrés S, Giráldez FJ, Mateo J (2015) Effect of the addition of hop (infusion or powder) on the oxidative stability of lean lamb patties during storage. Small Rumin Res 125:73–80 Wang Q, Ding ZH, Liu JK, Zheng YT (2004) Xanthohumol, a novel anti-HIV-1 agent purified from hops Humulus lupulus. Antiviral Res 64(3):189–194 Wang G, Tian L, Aziz N, Broun P, Dai X, He J, King A, Zhao PX, Dixon RA (2008) Terpene biosynthesis in glandular trichomes of hop. Plant Physiol 148:1254–1266 Wendakoon C, Calderon P, Gagnon D (2012) Evaluation of selected medicinal plants extracted in different ethanol concentrations for antibacterial activity against human pathogens. J Med Act Plants 1(2):60–68 Yamaguchi N, Satoh-Yamaguchi K, Ono M (2009) In vitro evaluation of antibacterial, anticollagenase, and antioxidant activities of hop components (Humulus lupulus) addressing acne vulgaris. Phytomedicine 16(4):369–376 Yang M, Li N, Li F, Zhu Q, Liu X, Han Q, Wang Y, Chen Y, Zeng X, Lv Y, Zhang P, Yang C, Liu Z (2013) Xanthohumol, a main prenylated chalcone from hops, reduces liver damage and modulates oxidative reaction and apoptosis in hepatitis C virus infected Tupaia belangeri. Int Immunopharmacol 16:466–474 Zanoli P, Zavatti M (2008) Pharmacognostic and pharmacological profile of Humulus lupulus L. J Ethnopharmacol 116(3):383–396 Zhang N, Liu Z, Han Q, Chen J, Lou S, Qiu J, Zhang G (2009) Inhibition of bovine viral diarrhea virus in vitro by xanthohumol: comparisons with ribavirin and interferon-a and implications for the development of anti-hepatitis C virus agents. Eur J Pharm Sci 38:332–340 Zhang N, Liu Z, Han Q, Chen J, Lv Y (2010) Xanthohumol enhances antiviral effect of interferon a-2b against bovine viral diarrhea virus, a surrogate of hepatitis C virus. Phytomedicine 17:310–316 Zhao F, Watanabe Y, Nozawa H, Daikonnya A, Kondo K, Kitanaka S (2005) Prenylflavonoids and phloroglucinol derivatives from hops (Humulus lupulus). J Nat Prod 68:43–49 Żołnierczyk AK, Mączka WK, Grabarczyk M, Wińska K, Woźniak E, Anioł M (2015) Isoxanthohumol—Biologically active hop flavonoid. Fitoterapia 103:71–82

Chapter 3

How to Study Antimicrobial Activities of Plant Extracts: A Critical Point of View Séverine Mahieux, Maria Susana Nieto-Bobadilla, Isabelle Houcke and Christel Neut

Abstract Multiresistance to antibiotics is a global threat for our quality of life. New weapons are urgently needed, and natural compounds regain interest. Research on antimicrobial compounds should fulfill several claims—they should be only evaluated at concentrations that could be achieved on the target—the bacterial species tested should be in agreement with the chosen application—studies should not be limited to a single strain. Conventional antimicrobial research is conducted in vitro for substances affecting growth by determining the minimal inhibitory concentrations (MICs). Research can further be refined by determining the kill time, the growth rate at sub-MICs concentration, the post-antibiotic effect, and synergy with other compounds like antibiotics. But features not related to bacterial growth can also reduce the infectious risk like anti-biofilm, anti-adherence, and antitoxin strategies. They can be determined both in vitro and in vivo but only in vivo infection models show directly the effect on the expression of the pathogenicity. The search of the mode of action can give useful insights in toxicity and drug interactions. Scientific tools in genomics and chemistry are steadily increasing to improve this topic. Bio-guided isolation of active compounds enhances detection of active compounds in the yet underexploited nature.




Keywords Antibacterial natural compounds Minimal inhibitory concentration Growth curves Kill time Synergy with antibiotics







Abbreviations ATB ATCC CFU DSM FIC

Antibiotic American Type Culture Collection Colony-forming unit Deutsche Sammlung von Mikroorganismen Fractional inhibitory index

S. Mahieux
M. S. Nieto-Bobadilla
I. Houcke
C. Neut (&) Laboratoire de Bactériologie, Faculté de Pharmacie, INSERM U995 LIRIC, University of Lille, 3, Rue Du Professeur Laguesse BP 83, 59006 Lille, France e-mail: [email protected] © Springer International Publishing AG 2018 J.-M. Mérillon and C. Rivière (eds.), Natural Antimicrobial Agents, Sustainable Development and Biodiversity 19, https://doi.org/10.1007/978-3-319-67045-4_3

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MBC MIC MOA NCTC PAE TLC

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Minimal bactericidal concentration Minimal inhibitory concentration Mode of action National collection of type cultures Post-antibiotic effect Thin-layer chromatography

Introduction

Control of infection by antibiotics has transformed medicine. Invasive surgeries, organ transplantations, hip and knee prostheses improved quality of life for millions of people because the postsurgical infectious risk was under control. With increase of bacterial resistance, our quality of life is on risk (Brown and Wright 2016). Urgent work is necessary if we want to continue to be successful in fighting against bacteria (Roca et al. 2015). Both synthetic chemistry and discovery of new natural compounds with antibacterial activity might contribute to fulfill this gap. But high throughput studies on chemical libraries lead only to few hits. Natural compounds, already the first weapons, again regain interest. Plants evolved since a long time in the presence of bacteria and show remarkable resistance against bacterial diseases. Mainly secondary metabolites are reported more and more often as potent antimicrobial agents (Singh and Barrett 2006). They can be used in human medicine but also in food processing or animal facilities (O’Bryan et al. 2015; Nabavi et al. 2015). As the contact of bacteria with these natural products exists since a long time, the probability of rapid appearance of resistance seems low (Koehn and Carter 2005). The aim of this review is to point out relevant details in order to improve increasing research on natural products. Old studies all focus on the direct effect of substances on in vitro growing cells, and the effect should be at best direct death of the bacterial cell (bactericidal) or at least lowering of growth (bacteriostatic) (Brown and Wright 2016). These studies focus on minimal inhibitory concentration (MIC) determination. Giving the lack of new compounds and the evolving techniques available, newer studies focus on a narrower action targeting specific metabolic traits implicated for instance in the pathogenic strategies of a specific microorganism (Brown and Wright 2016; Vale et al. 2016).

3.2

In Vitro Studies

First studies on potential antimicrobial substances are usually in vitro studies on pure bacterial cultures; some simple points should be taken into consideration to allow as much conclusions as possible from these in vitro results.

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Studied Concentration

When extracting and purifying active substances from plants, it should be taken immediately in consideration in function of the possible application, which concentration can be obtained in/on human or animal tissues, on surfaces, etc. Too many works use concentrations higher than 5 or 10% for samples like essential oils (EOs). It is unlikely that these concentrations can be obtained in in vivo conditions. The definition of a validated threshold seems necessary to avoid publications of the presence of antimicrobial activities at non-physiological concentrations. We claim the limit of 1% or 10 g/L as the highest concentrations to be used in in vitro assays.

3.2.2

Solvents

One other critical point is the need of solvents for extraction of natural products. Secondary plant metabolites show often only poor solubility in water, and use of solvents is unavoidable. Solvents should be the less toxic as possible and an internal test of their influence on bacterial growth should be checked in each test. Differences in in vitro and in vivo studies can be occurring due to differences in solubility in different solvents and in biological fluids.

3.2.3

Choice of Microorganism

The choice of the tested bacterial strains should be adapted to the application. When food-spoiling bacteria are vised, bacteria naturally implicated need to be investigated, and when plant extracts are used for mouth rinsing, oral strains need to be checked. At a first time, aerobic strains can be tested as their development is rapid and the incubation in air easy. But when fastidious bacteria are implicated in the pathology, they should also be tested and all necessary methods can be adapted. The use of strains belonging to international collections available for all (like American Type Culture Collection (ATCC), DSM, NCTC) is important for reason of inter-laboratory comparison, but exclusive use of these strains needs to be avoided, as they are often isolated years ago and do no longer reflect what is today found in hospitals (in means of antibacterial resistance for instance) and they are maintained in synthetic culture medium during all these years (lowering the expression of virulence factors). A selection of several strains is necessary, some strains isolated recently from the clinical conditions investigated and one or two collection strains.

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3.2.4

Microbial Analysis

3.2.4.1

Culture Media

The medium most frequently used for antimicrobial tests is Mueller Hinton agar or broth. It can be cation-adjusted (Clinical and Laboratory Standards Institute 2012). But when studying bacteria with specific nutrient requirements, other media can be adapted; blood can be added for fastidious microorganisms. Media can be complemented with reducers (for anaerobes), vitamins or other growth enhancers. Incubation atmosphere and duration can be adapted. But controls should always been made using the same conditions.

3.2.4.2

Diffusion Tests

The first preliminary tests are usually still very simple disk diffusion tests like those used for antibiotic susceptibilities (Kirby-Bauer method) (EUCAST 2015). For these tests with antibiotics, known concentrations of the active antibacterial compound are used and this makes the tests reproducible. When using plant extracts, the amount of active substances is not known, the type of active substances (for instance, molecular weight) is not known, and so the diffusion rate is not known. For unknown substances, the diameter of diffusion zones is not relevant when comparing different extracts. So this first screening test is only useful as a rapid step to answer the question if there is any antibacterial activity.

3.2.4.3

MIC, MBC

A quantitative approach to measure the antibacterial activity is the determination of the minimal inhibitory concentration (MIC) defined as the smallest concentration with no visible growth (Clinical and Laboratory Standards Institute 2012). It can be determined both in liquid and in solid media. The method is always based on twofold dilutions of the compound. The range usually tested is comprised between 512 and 0.0675 mg/L (which are below the claimed limit in Chap. 1.1). It should be better expressed at a molar level, but pharmacological parameters are often expressed in mg/L which enables quick comparison with levels that can be achieved in biological tissues. When using solid medium, the diluted test substance is introduced in the molten agar medium before pouring in Petri dishes. One plate containing only the solvent is inoculated when beginning the test, and a second control plate is inoculated at the end of the assay to ensure the absence of contamination. Using a multipoint inoculator, up to 96 strains can be inoculated on the same plate (Fig. 3.1). This ensures a high reproducibility as all strains are inoculated on the same plate containing the

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same concentration of the compound. It makes it easy also to test immediately a large range of strains. When using broth MIC determination, the assay is independent for each strain. Twofold dilutions are made, and the test tubes (conventional tubes or microplates) are inoculated with the test strain. Negative (without bacteria, checking non-contamination) and positive controls (without the antibacterial compound, the solvent only is introduced) are added. After incubation, the presence of visible growth is checked. The test in broth allows also the determination of the minimal bactericidal concentration (MBC), in this case it is necessary to subculture aliquots of the last dilutions without visible growth to search survivors. When no survived bacteria are present, the compound is considered as bactericidal (as the initial bacterial concentration in the inoculum falls so low that no survivors are detected after incubation). A bactericidal action is preferred to a bacteriostatic action (when only bacterial multiplication is inhibited without death of the bacteria). MBC concentration cannot be established on solid media as bacteria are inoculated as spots on the agar medium. If a sufficient amount of strains have been tested, we can evaluate the MIC50 and the MIC90 which indicate the concentration inhibiting 50 and 90% of the tested strains. When the difference between the two values is low, strains have a low range of MICs, and greater differences in the two values indicate large variations between strains.

Fig. 3.1 MIC determination in solid medium using a Steers replicator for inoculation adapted both for aerobic and anaerobic bacteria

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Kill Time

For the next step in the description of the antibacterial activity, the time is taken into account. This test determines the time necessary to kill bacteria (Finberg et al. 2004). As we want to see the action of the compound on the number of viable bacteria present at the beginning of the assay, multiplication of bacteria should be avoided; the assay takes place in a non-nutrient diluent. A known number of bacteria are placed in the presence of a determined concentration of the test substance. Usually, a concentration of four times the MIC concentration is used in order to have an active concentration. At determined intervals, the survivors are outnumbered and the kill time curve is established (Fig. 3.2). In order to simplify the expression of results, the time necessary to lower the population by one log CFU/mL is indicated for each tested strain (Fig. 3.3). These results concern a synthetic blend of plant origin tested in our laboratory (Nieto-Bobadilla et al. 2015) with very short logarithmic reduction times for Gram-negative aerobic rods (a few minutes) when testing a concentration of 1% of the blend. A longer time is needed for Staphylococcus. At a concentration of four times the MIC, the effect is still fast.

3.2.4.5

Bacterial Growth at Sub-inhibitory Concentrations

As discussed above, MICs indicate only that no visible bacterial development takes places (troubling broth or producing colonies), but when more indications on bacterial development need to be established, it might be interesting to make

Fig. 3.2 Determination of the logarithmic reduction times (bactericidal kill time test)

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Fig. 3.3 Kill time results for CIN-102 at a concentration of 1% and 4xMIC concentration, a synthetic blend mimicking cinnamom essential oils, for 57 different strains

bacterial growth curves in the presence of sub-MICs concentrations. Indeed, if growth is still influenced by the tested product, in in vivo conditions, a positive effect can be observed at sub-inhibitory concentrations. Bacteria are inoculated in broth tubes containing either only the solvent (control) or the tested product at MIC, MIC/2 and MIC/4 concentration. Growth is allowed for determined intervals at 37 °C when aliquots are sampled, diluted, and plated (Fig. 3.4). The results for the same blend as above show here for an E. coli strain a bacteriostatic effect of the blend at the MIC concentration (Fig. 3.5). At half of this concentration, growth is nearly absent during 9 h when the control population increases by 3 log in count. At one-fourth of the MIC concentration after nine Fig. 3.4 Determination of the influence of tested substances at sub-inhibitory concentration on the growth of bacteria

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hours, the counts are still a 100-fold lower than in controls. The same effect on a Gram-positive coccus (Fig. 3.6) is less pronounced but shows still lower growth even at one-fourth of the MIC concentration. Only the MIC determination and the kill time will underestimate the effect on a bacterial population Fig. 3.5 Growth at sub-MICs for a strain of Escherichia coli

Fig. 3.6 Growth at sub-MIC concentration for a strain of Staphylococcus aureus

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Post-antibiotic Effect

The post-antibiotic effect (PAE) establishes the effect on growth of bacterial cells exposed to an antibacterial substance for a determined time at a concentration above the MIC, then the action of the substance is suppressed (either by dilution of the broth or by centrifugation and recovery of the bacteria in a new broth without the antibacterial compound) and bacterial growth is followed over time (Fig. 3.7) (Plachouras et al. 2007; Lorian 2005). Bacteria might be damaged by the presence of inhibitory compounds and need some time to recover. This time can be calculated in comparison to the control (Fig. 3.7). In clinical situations, the presence of a PAE is favorable, because in the absence of an active concentration of the antibacterial compound, the bacterial growth is still lowered. Here also, the PAE effect has been established for the same blend as above. The first example concerns a strain of Klebsiella pneumoniae (Fig. 3.8) where the PAE is of 3.3 h at the MIC concentration and of 4 h at 4 times the MIC concentration. The second example concerns Burkholderia cepacia, an emergent nosocomial pathogen very susceptible to our blend (Fig. 3.9). At 4 times the MIC concentration, the amount of bacteria decreases to the detection threshold and the few survivors grow up very late giving a PAE of 22 h. Bacteria damaged by the product during a contact of two hours need nearly one day to recover from this stress condition.

Fig. 3.7 Determination of the post-antibiotic effect (PAE)

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Fig. 3.8 Post-antibiotic effect on Klebsiella pneumoniae

Fig. 3.9 Post-antibiotic effect on Burkholderia cepacia

3.2.4.7

Synergy

When two active compounds do not target the same site of action, their simultaneous presence can have an effect greater than only the addition of the two actions. The simultaneous use of different targets in the same bacterium will also reduce the risk of development of resistance (Lorian 2005; Hemaiswarya et al. 2008). Synergies can be searched among two antibacterial agents but also on one antimicrobial compounds and one adjuvant (which needs not to have a proper antibacterial activity). It can for instance increase cell wall permeability allowing the administration of a lower dose of antibiotics (reducing the risk of resistance). It is useful to search

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synergy between new plant products with its proper antibacterial activity or adjuvant activity and antibiotics as clinicians will always hesitate to replace completely conventional treatments by new drugs but will accept easier to combine conventional and new treatments. The presence of synergy can be established by a checkerboard analysis when different concentrations of each product are present concomitantly (Fig. 3.10). The comparison of the MICs of the two products alone or in combination allows to establish the fractionary inhibitory concentration (FIC) index and to define the presence of synergy, addition, indifference, or antagonism (Lorian 2005). It can also be expressed by isobolograms (Fig. 3.11) (Zore et al. 2011). Eight bacterial strains belonging to eight different species are tested for their synergy with eight antibiotics of different families and the synthetic blend CIN-102 (Table 3.1). The first line for each strain indicates the concentration of first our blend and then the antibiotic’s concentration with the best synergy. The value below indicates the FIC index. When it is underlined and bold, a synergy is present. For each strain and each antibiotic, synergies exist showing the high interactions between the blend and antibiotics acting by different mechanisms. Antagonisms have never been observed. The different chapters above illustrate how a combination of different approaches on the in vitro action of antibacterial compounds on growing or non-growing bacteria. The criterion followed is always bacterial death or lack of development. The following chapters will treat another strategy aiming to inhibit virulence factors, like spatial organization of bacteria in form of biofilm or synthesis of specific virulence factors like toxins without inducing the death of bacteria (Roca et al. 2015; Vale et al. 2016; Fahra and Brown 2016).

Fig. 3.10 Interactions of products with antibiotics in order to calculate the fractionary inhibitory concentration (FIC) index indicating interaction between substances

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Fig. 3.11 Isobologram of synergy between various natural products (a: eugenol, linalol, citronellal, linalyl acetate, b: citral, and c: benzyl benzoate) and fuconazole on Candida albicans (ATCC 10231) (Zore et al. 2011)

3.3

Inhibition of Virulence Factors

This mode of action will avoid the emergence of resistance as the action of the compound does not affect the viability of the bacterial cell (Roca et al. 2015).

3.3.1

Anti-biofilm Action

The formation of biofilms is a spatial organization of bacteria strongly influencing the mode of action of antibacterial substances which underlines the importance of other specific mechanism. Bacteria can colonize and adhere to various supports. After this first step, they secrete extracellular substances enclosing the bacteria; pore formation allows circulation of nutrients but also intercellular signalization

Enterococcus faecium

Staphylococcus aureus

Acinetobacter baumannii

Pseudomonas aeruginosa

Enterobacter cloacae

Serratia marcescens

Klebsiella pneumoniae

Escherichia coli

4/37.5 0.38 150/4 0.50 150/16 0.50 37.5/4 0.38 600/8 0.98 5/1 0.52 5/0.125 0.52 600/0.03 1.00

5/16 0.27 150/1 0.27 150/1 0.38 5/0.25 0.02 600/1 0.50 150/0.03 0.50 5/0.25 0.27 150/0.25 0.25

MIC (mg/L) in combination (CIN-102/Antibiotic) ƩFIC index Imipenem Gentamicin 300/0.03 1.00 300/0.03 0.50 150/0.06 0.25 300/0.03 1.00 600/0.25 0.48 150/0.25 0.50 150/0.06 0.56 150/2 0.28

Vancomycin 150/4 0.56 300/0.03 0.50 300/4 0.56 150/16 0.75 600/0.5 0.49 150/0.25 0.56 75/0.03 0.25 150/4 0.31

Erythromycin

Table 3.1 Synergy between eight antibiotics and CIN-102 (Synergy: ƩFIC index  0.5)

Clindamycin 150/0.03 0.50 150/0.03 0.25 150/0.25 0.25 150/0.03 0.50 1250/0.03 1.00 150/0.25 0.50 75/8 0.50 600/0.03 1.00

Colistin 20/1 0.32 37.5/2 0.19 75/4 0.25 75/1 0.38 150/4 0.62 37.5/2 0.38 75/1 0.28 600/0.03 1.00

Amikacin 37.5/0.5 0.13 37.5/0.06 0.07 75/0.06 0.13 5/0.25 0.03 600/0.06 0.5 75/4 0.38 5/4 0.27 150/1 0.27

Tigecycline 5/0.5 0.52 300/0.03 0.52 300/0.03 0.52 300/0.03 1.02 5/16 0.50 300/0.03 1.01 5/0.25 0.52 5/0.5 0.51

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molecules. Available substrates for growth will lack soon, and the bacteria remain in a status without multiplication. The extracellular mass hinders the diffusion of antibacterial compounds. And antibacterial compounds acting on the division cycle of bacteria (as most of the antibiotics) will not be active as there is no multiplication. Biofilms are largely encountered in nature. We find them in the environment (in water supplies) but also in humans and animals in health (the mucus layer on the intestinal, oral, or vaginal mucosa is a biofilm composed both by host and bacterial materials) and in disease (pulmonary infections are often biofilm-depending but also infections on implants or in bones). About two-thirds of human infections are caused by bacteria present as biofilms (Kolenbrander et al. 2010; Bayramov and Neff 2016; Thakur et al. 2016). And the presence of biofilms is the main reason for failure of antibiotic (ATB) treatment. Substances even without antibacterial activity destroying the biofilm matrix like surfactants would be of great interest in different applications.

3.3.2

Anti-adhesion Formation

The first step in the interaction with host cells is adhesion. Only bacteria able to adhere on host cells can start a dialogue with the host. Anti-adhesion molecules can mimic the sugar moieties on the bacterial surface implicated in adhesion (Rane et al. 2014). One application concerns intestinal infection. Orally administered anti-adherence molecules cover the mucus layer and avoid bacterial because all adhesion sites on the mucus layer are already occupied by anti-adhesion molecules (Brufau et al. 2016). Other applications may be urinary or vaginal infections (Rane et al. 2014; Al-Ghazzewi and Tester 2016).

3.3.3

Anti-toxin Formation

Even more specific is the inhibition of toxin formation. But this step will always need species-specific strategies rather different from broad spectrum antibiotics. The targeted bacterium has to be identified before the application of this strategy. But in face of development of frequent antimicrobial resistance, we need to revisit empirical antibiotic treatment and come to personalized treatments starting by a clear diagnosis of the causative agents before the use of a specific treatment (Brown and Wright 2016; Vale et al. 2016). But this strategy will be without help in polymicrobial infection.

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Elucidating Mechanisms of Action

The knowledge of the mechanism of action (MOA) becomes more and more important, and the combination of different methods may be helpful. A good knowledge of the MOA can give us insight on toxicology (when the target is a metabolic pathway also present in mammalian tissues), leading to faster optimization of the new drug (Fahra and Brown 2016). One strategy used since a long time is the research of resistant mutants. Now with next-generation sequencing, it is possible to sequence the susceptible and the resistant strain to map drug-resistant mutation. This tool was useful to identify the MOA of pyridomycin on Mycobacterium tuberculosis (Hartkoorn et al. 2012). Affinity chromatography (when the investigated drug is immobilized on the support) allows identifying molecules linking to the drug (Zheng et al. 2015). The incorporation of radiolabeled precursors of metabolic pathways can help to elucidate the metabolic pathways influenced by the drug. For common species implicated in infections like Escherichia coli, Staphylococcus aureus, or Pseudomonas aeruginosa, a panel of mutants down-regulated for specific enzymes is now available. Penetration of the cell wall is a crucial step in the action of drugs. The damage of the cell wall can be first shown by electron microscopy resulting in spheroplast formation. Cell wall deficient bacteria can be compared to bacteria with their complete cell wall. The knowledge of efflux genes expression can help to evidence interactions with efflux pumps. Quorum-sensing enzymes are important regulators in biofilm formation; the influence of the drug on this signaling system is another task for MOA (Fahra and Brown 2016). The protein FtsZ participates in the formation of the cytoskeleton and is essential for cell division. A cell-free fluorescent polymerization assay helped to discover the first FtsZ inhibitor (Wang et al. 2003).

3.5 3.5.1

In Vivo Studies Experimental Infections in Animals

Here also, the chosen infection should be next to the disease focused in humans and be due to bacteria known to be targeted by the antimicrobial substances evidenced in vitro. Experimental infections in contrast to in vitro assays need not only growth but also the expression of the virulence mechanisms; they will be useful not only for classic antibiotics but also for new substances focusing on the inhibition of pathogenicity (Brown and Wright 2016).

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Bio-Guided Purification of Active Compounds: Bioautography

Bacterial methods can also been useful in helping to purify antimicrobial substances. Bioautography will help to localize these compounds on thin-layer chromatography (TLC). TLC plates are put in Petri dishes, and molten agar inoculated with the tested strains is poured on the plate. After incubation, growth is revealed by appropriate reagents. Zones without growth remain uncolored and correspond to zones with antibacterial activities. Substances migrating at this location can be further purified and analyzed (Potterat and Hamburger 2013).

3.7

Conclusion

In order to exploit the enormous natural reservoir of potential antimicrobial substances in plants, different strategies of bacteriological analyses are presented to help to improve the discovery of new molecules acting on the growth of bacteria but also on pathogenic features of specific microorganisms. In the era of evolving antimicrobial resistance against antibiotics, the urgent need of new agents might be fruitful when choosing as many different ways of research as possible.

References Al-Ghazzewi FH, Tester RF (2016) Biotherapeutic agents and vaginal health. J Appl Microbiol 121:18–27 Bayramov DF, Neff JA (2016) Beyond conventional antibiotics—new directions for combination products to combat biofilm. Adv Drug Deliv Rev. S0169-409X(16)30230-7 [Epub ahead of print] Brown ED, Wright GD (2016) Antibacterial drug discovery in the resistance era. Nature 529:336– 343 Brufau MT, Campo-Sabariz J, Bou R, Carné S, Brufau J, Vilà B, Marqués AM, Guardiola F, Ferrer R, Martín-Venegas R (2016) Salmosan, a b-galactomannan-rich product, protects epithelial barrier function in Caco-2 cells infected by Salmonella enterica Serovar Enteritidis. J Nutr 146:1492–1498 Clinical and Laboratory Standards Institute (2012) CLSI methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, M07-A9 EUCAST (2015) Breakpoint tables for interpretation of MICs zones and diameters. http://www. eucast.org Farha MA, Brown ED (2016) Strategies for target identification of antimicrobial natural products. Nat Prod Rep 33:668–680 Finberg RW, Moellering RC, Tally FP, Craig WA, Pankey GA, Dellinger EP, West MA, Joshi M, Linden PK, Rolston KV, Rotschafer JC, Rybak MJ (2004) The importance of bactericidal drugs: future directions in infectious disease. Clin Infect Dis 39:1314–1320

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Hartkoorn RC, Sala C, Neres J, Pojer F, Magnet S, Mukherjee R, Uplekar S, Boy-Röttger S, Altmann KH, Cole ST (2012) Towards a new tuberculosis drug: pyridomycin—nature’s isoniazid. EMBO Mol Med 4:1032–1042 Hemaiswarya S, Kruthiventi AK, Doble M (2008) Synergism between natural products and antibiotics against infectious diseases. Phytomedicine 15:639–652 Koehn FE, Carter GT (2005) The evolving role of natural products in drug discovery. Nat Rev Drug Discov 4:206–220 Kolenbrander PE, Palmer RJ Jr, Periasamy S, Jakubovics NS (2010) Oral multispecies biofilm development and the key role of cell-cell distance. Nat Rev Microbiol 8:471–480 Lorian V (2005) Antibiotics in laboratory medicine. Lippincott Williams & Wilkins, Baltimore Nabavi SF, Di Lorenzo A, Izadi M, Sobarzo-Sánchez E, Daglia M, Nabavi SM (2015) Antibacterial effects of cinnamon: from farm to food, cosmetic and pharmaceutical industries. Nutrients 7:7729–7748 Nieto-Bobadilla MS, Siepmann F, Djouina M, Dubuquoy L, Tesse N, Willart JF, Dubreuil L, Siepmann J, Neut C (2015) Controlled delivery of a new broad spectrum antibacterial agent against colitis: in vitro and in vivo performance. Eur J Pharm Biopharm 96:152–161 O’Bryan CA, Pendleton SJ, Crandall PG, Ricke SC (2015) Potential of plant essential oils and their components in animal agriculture—in vitro studies on antibacterial mode of action. Front Vet Sci 2:35 Plachouras D, Giamarellos-Bourboulis EJ, Kentepozidis N, Baziaka F, Karagianni V, Giamarellou H (2007) In vitro postantibiotic effect of colistin on multidrug-resistant Acinetobacter baumannii. Diagn Microbiol Infect Dis 57:419–422 Potterat O, Hamburger M (2013) Concepts and technologies for tracking bioactive compounds in natural product extracts: generation of libraries, and hyphenation of analytical processes with bioassays. Nat Prod Rep 30:546–564 Rane HS, Bernardo SM, Howell AB, Lee SA (2014) Cranberry-derived proanthocyanidins prevent formation of Candida albicans biofilms in artificial urine through biofilm—and adherence-specific mechanisms. J Antimicrob Chemother 69:428–436 Roca I, Akova M, Baquero F, Carlet J, Cavaleri M, Coenen S, Cohen J, Findlay D, Gyssens I, Heuer OE, Kahlmeter G, Kruse H, Laxminarayan R, Liébana E, López-Cerero L, MacGowan A, Martins M, Rodríguez-Baño J, Rolain JM, Segovia C, Sigauque B, Tacconelli E, Wellington E, Vila J (2015) The global threat of antimicrobial resistance: science for intervention. New Microbes New Infect 6:22–29 Singh SB, Barrett JF (2006) Empirical antibacterial drug discovery—foundation in natural products. Biochem Pharmacol 71:1006–1015 Thakur P, Chawla R, Tanwar A, Chakotiya AS, Narula A, Goel R, Arora R, Sharma RK (2016) Attenuation of adhesion, quorum sensing and biofilm mediated virulence of carbapenem resistant Escherichia coli by selected natural plant products. Microb Pathog 92:76–85 Vale PF, McNally L, Doeschl-Wilson A, King KC, Popat R, Domingo-Sananes MR, Allen JE, Soares MP, Kümmerli R (2016) Beyond killing: can we find new ways to manage infection? Evol Med Public Health 18:148–157 Wang J, Galgoci A, Kodali S, Herath KB, Jayasuriya H, Dorso K, Vicente F, González A, Cully D, Bramhill D, Singh S (2003) Discovery of a small molecule that inhibits cell division by blocking FtsZ, a novel therapeutic target of antibiotics. J Biol Chem 278:44424–44428 Zheng W, Li G, Li X (2015) Affinity purification in target identification: the specificity challenge. Arch Pharm Res 38:1661–1685 Zore GB, Thakre AD, Jadhav S, Karuppayil SM (2011) Terpenoids inhibit Candida albicans growth by affecting membrane integrity and arrest of cell cycle. Phytomedicine 18:1181–1190

Part II

Natural Occurring Antifungal Natural Products (Plant Health)

Chapter 4

Antifungal Activities of Essential Oils from Himalayan Plants Chandra Shekhar Mathela and Vinod Kumar

Abstract Himalayan temperate and subalpine flora with its diverse medicinal and aromatic species occupies an important position in the field of herbal pharmaceuticals. The spread of multidrug-resistant strains of fungi and relatively small number of antifungal drugs available made it necessary to look for new sources of antifungal molecules. This has led to the search for therapeutic alternatives, particularly among medicinal and aromatic plants and compounds isolated from them for their antifungal potential. Essential oils are naturally occurring phytochemicals with generally less deleterious side effects than corresponding synthetic drugs. Also, the resurgence of interest in natural control of human infectious fungal pathogens and increasing demand for effective, safe natural extracts and their constituents could lead to new antifungal agents. This could support the use of the plants in the treatment of various infective human diseases and protection of plant crops. This chapter gives an overview on the susceptibility of human and phytopathogenic fungi toward different essential oils and their chemical constituents, largely belonging to the tropical and subalpine Indian Himalayan region, viz. Nepeta, Erigeron, Aster, Cinnamomum, Thymus, Mentha, Senecio and their constituents such as new terpene iridoids, actinidine, nepetalactone, acetylenic esters, thymol, carvacrol and eugenol. Several of these have been found to possess high antifungal properties against various fungi.




Keywords Himalayan plants Essential oils Human and plant pathogenic fungi Iridoids





Antifungal activity
Actinidine
Nepetalactone

Abbreviations DMSO EOs GC-FT IR GC-MS

Dimethylsulfoxide Essential oils Gas chromatography-Fourier transform infrared Gas chromatography-mass spectrometry

C. S. Mathela (&)
V. Kumar Department of Chemistry, Kumaun University, Nainital 263 002, Uttarakhand, India e-mail: [email protected] © Springer International Publishing AG 2018 J.-M. Mérillon and C. Rivière (eds.), Natural Antimicrobial Agents, Sustainable Development and Biodiversity 19, https://doi.org/10.1007/978-3-319-67045-4_4

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g HCA IC50 LC-MS LC-NMR MAPs µg µL mg mL mm MIC PF PDA PCA NaCl Na2SO4

4.1

Gram Hierarchical cluster analysis 50% Inhibitory concentration Liquid chromatography-mass spectrometry Liquid chromatography-nuclear magnetic resonance Medicinal and aromatic plants Microgram Microliter Milligram Milliliter Millimeter Minimum inhibitory concentration Poison food Potato dextrose agar Principal component analysis Sodium chloride Sodium sulfate

Introduction

Medicinal and aromatic plants (MAPs) are nature’s gift to mankind as source of natural drugs since dawn of civilization. Plants are the part of traditional systems of medicine, modern medicines, nutraceuticals, food supplements, folk medicines, pharmaceutical intermediates, and chemical entities for synthetic drugs. Aromatic plants are a source of fragrances, flavors and health beverages. Himalaya is the best habitat of the herbal drug materials. Of nearly 3000 herbal drugs described in “Ayurvedic Materia Medica,” several occur in Himalayan region. Plants have a long history of herbal medicines as antiviral, antibacterial and antifungal agents (Bakkali et al. 2008). Considering the increasing demand for the herbal drugs in general and Himalayan medicinal plants in particular, it is important to initiate steps for large-scale production and developing high-yielding species. The majority of clinically used antifungals have various drawbacks in terms of toxicity and efficacy. Frequent use has led to the emergence of resistant strains. Additionally, in recent years public pressure to reduce the use of synthetic fungicides in agriculture has increased. Concerns have been raised about the environmental impact and the potential health risk related to the use of these compounds. Hence, there is a great need for novel antifungals belonging to a wide range of structural classes and biodegradable, selectively acting on new targets with fewer side effects. Natural alternatives that are user-friendly and possessing low toxicity to humans are desirable. Alternative methods of suppressing pathogenic and toxigenic

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fungi, based on the use of natural plant substances, often result in research focused on the development of highly effective essential oils (EOs). Plant EOs can provide alternative to synthetic antimicrobial drugs but research must be focused on plant-derived safer and biodegradable secondary plant metabolites. Our investigations on EOs and constituents of medicinal and aromatic plant (MAPs) species of important families, viz. Lamiaceae, Asteraceae, Apiaceae, Zingiberaceae, Lauraceae, have revealed potential antifungal EOs and their constituents against various human and plant pathogenic fungi (Bisht et al. 2010; Kumar et al. 2014a). Antifungal activity of EOs extracted from Aster peduncularis, A. thomsonii, Selinum tenuifolium and Cymbopogon jwarancusa were screened against Aspergillus tamarii, A. parasiticus, A. niger, A. terreus and A. ochraceus. Antifungal activity data showed that the oil from C. jwarancusa (>30 mm) was more active while that from A. thomsoni was found moderately active toward A. tamarii, A. parasiticus, A. terreus and A. ochraceus. A. terreus was sensitive to A. peduncularis and S. tenuifolium (Mathela and Sinha 1978). Antifungal properties of the EOs of Aster molliusculus, Thymus serpyllum and Cymbopogon citratus and their terpenoid constituents (hydrocarbons and oxygenated) were determined against 12 Aspergillus species, viz. A. terreus, A. flavus, A. niger, A. versicolor, A. ochraceus, A. parasiticus, A. quadrilineatus, A. rugulosus, A. fumigatus, A. flavipes, A. ustus and A. sejunctus; four Penicillium species, viz. P. nigricans P. melini, P. chrysogenum and P. brevicompactum; three Fusarium species, viz. F. monililorme, F. monililorme var. subglutinus and F. oxysporum (Agarwal et al. 1979, 1980). Results of antifungal examination showed that the Thymus serpyllum and Cymbopogon citratus EOs and their terpenoid constituents possess high degree of inhibitory activity (>35 mm growth inhibition) against test fungi. Among the terpene hydrocarbons, b-pinene, a-pinene, camphene and q-cymene showed moderate-to-low activity while the oxygenated terpenoids, viz. 1,8-cineol, a-terpineol and geraniol showed moderate-to-high activity (  15 mm). Carvacrol exhibited maximum activity against almost all tested fungi followed by its structural isomer, thymol. Among citral (geranial and neral mixture), citronellyl acetate, citronellol and geranyl acetate, citral was noticed to be very effective antifungal agent. The high antifungal activity of T. serpyllum oil was possibly largely because of phenolic constituents, carvacrol in particular. It showed maximum activity against A. versicolor, A. sejunctus and P. melini. C. citratus had remarkable growth inhibiting effect on A. terreus, P. nigricans, P. melini and P. chrysogenum. The activity of C. citratus is largely due to the presence of its major constituent, citral. The oil of A. molliusculus showed less activity in comparison with other oils. On the other hand, Fusarium species as a class are little affected by most of the oils and their constituents as compared to Penicillium and Aspergilli. Screening of 12 terpenoids against Aspergillus, Penicillium, and Fusarium species showed that the activity of thymol and carvacrol was comparable with the activity of standard antifungal antibiotics, viz. nystatin and talsutin (Mathela 1981).

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Phytochemistry of Essential Oils

The essential oils (EOs) may be defined as odiferous bodies of an oily nature and distributed in different plant parts, obtained from vegetable organs: flowers, leaves, barks, woods, roots, rhizomes, fruits and seeds. They are accumulated in secretary cells, cavities, channels and epidermic cells. They are mainly composed of lower isoprenoids, viz. mono- and sesquiterpenoids and are responsible for the characteristic odors. They are also associated with bioactivity of aromatic and medicinal plants. The major constituents of some of EOs screened for antifungal activity are given in Table 4.1.

4.2.1

Plant Collection

The plants belonging to Apiaceae, Asteraceae, Chenopodiaceae, Cupressaceae, Gentianaceae, Lamiaceae, Myrtaceae, Pinaceae, Piperaceae, Poaceae, Rutaceae, Valerianaceae, Verbenaceae, Zingiberaceae, etc., were collected from different locations of the Himalayan region of Uttarakhand (India) and identified by local taxonomist and confirmed by Botanical Survey of India, Dehradun. Voucher specimens have been deposited in the Phytochemistry Laboratory, Chemistry Department, Kumaun University, Nainital for reference. Table 4.1 Chemical markers of Himalayan Asteraceae and Lamiaceae species Plant species Erigeron species E. mucronatus E. karvinskianus E. annuus (L.) Pers Nepeta species N. leucophylla N. ciliaris N. elliptica N. clarkei Other species Calamintha umbrosa Aster indamellus Senecio nudicaulis Buch-Ham. ex D. Don.

Biochemical markers cis-Lachnophyllum ester, trans-2-cis-8-matricaria ester, caryophyllene oxide cis-Lachnophyllum ester, trans-2-cis-8-matricaria ester, caryophyllene oxide, germacrene D cis-Lachnophyllum ester, germacrene D d-Cadinene, caryophyllene oxide, 2,3-dihydrofarnesol, b-sesquiphellandrene, iridodial-b-monoenol acetate b-Caryophyllene, caryophyllene oxide, 2,3-dihydrofarnesol, a-trans bergamotene, iridodial-b-monoenol acetate (7R)-trans, trans-Nepetalactone Germacrene D, b-sesquiphellandrene, actinidine b-Pinene, linalool, b-caryophyllene, germacrene D, spathulenol, sabinene, germacren-D-4-ol, 1,8-cineole a-Muurolol, carvacrol acetate b-Caryophyllene, a-humulene, germacrene D, caryophyllene oxide

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Essential Oil Extraction

Essential oils are generally liquids and possess pleasant odor and essence. The extraction of the essential oils depends mainly on the rate of diffusion of the oil through the plant tissues to an exposed surface where the oil can be removed by a number of processes. There are different methods for the extraction of the oil from the plant materials such as steam distillation, hydrodistillation, liquid carbon dioxide or microwaves, low- or high-pressure distillation employing boiling water or hot steam, hydrodistillation being commonly in use. EOs were extracted by hydrodistillation, and the distillates were saturated with NaCl and extracted with n-hexane and dichloromethane. The organic phase was dried over anhydrous Na2SO4 and the solvent was distilled off in a rotary vacuum evaporator at 30 °C to get residual mass and preserved in a sealed vial at 4 ºC until further analysis. The extracted essential oils varied in yield, quality and quantity in the chemical composition depending upon the agro climate, plant organ, age, vegetative cycle stage and processing methods used for their handling and storage conditions.

4.2.3

Chemical Analysis

The complexity of the EOs was a real challenge for determining their reliable and accurate compositional data. But, advances in spectroscopic, chromatographic and hyphenated techniques, viz. GC-MS, GC-FTIR, LC-MS and LC-NMR have totally changed the scenario of compositional determination of essential oils. In the present study, the identification of the components was carried out by the comparison of both the GC Kovat indices and MS data (Adams 2007). New compounds were identified by spectral data (NMR, MS and IR) of isolates.

4.3

Essential Oils as Antifungal Agents

4.3.1

The Methods Used for Antifungal Activity

4.3.1.1

Poisoned Food Technique

Inhibitory activity of the EOs and compounds was determined by the poisoned food technique (Grover and Moore 1962) in PDA medium against the test fungi. The concentrations (25–500 µg/mL) of the oils and compounds were prepared by dissolving the requisite amounts in 10% DMSO and then added into 20 mL PDA to obtain the different final concentrations (Feng and Zheng 2007). Mycelial plugs (2 mm in diameter) from the edges of each culture were incubated in the center of each PDA plate (85 mm diameter). The control sets were prepared using 10%

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DMSO. The prepared plates were inoculated aseptically with assay disks of the test fungi and incubated at 26 ± 2 ºC for 3–7 days until the growth in the control plates reached the edge of the plates. Growth inhibition of each fungal strain was calculated as the percentage inhibition of radial growth relative to the control. The plates were used in triplicate for each treatment. IC50 values of constituents were obtained from linear regression equation. The relative growth inhibition of treatment compared to control was calculated by percentage, using the following formula: Inhibition (%) = [(C-T)/C]  100 where C and T are the radial mycelial growth (mm) of fungus in the control and treated samples, respectively.

4.3.1.2

Determination of Minimum Inhibitory Concentration (MIC)

The minimum inhibitory concentrations of the EOs and compounds were determined by agar dilution method (Mitscher et al. 1972). Samples were dissolved in 10% DMSO according to their respective known weights. A 10 µL spore suspension (106 per mL) of each test strain was inoculated in the test tubes in PDB medium and incubated for 5–7 days at less than 26 °C. The control tubes containing potato dextrose broth (PDB) medium were inoculated only with fungal suspension. The MIC was defined as the minimum concentration in µg/mL at which no visible growth was observed.

4.3.1.3

Spore Germination Assay

The fungal pathogens were cultured on potato dextrose agar (PDA) medium in sterilized petri dishes (85 mm diameter). Spores were harvested from the 10-day-old cultures of the respective pathogens in 10 mL sterile distilled water using an inoculation loop. Spore suspension was centrifuged (Megafuge 1.0, Heraeus Sepatech, Germany) at 2000 rpm for 5 min. to obtain the spores. The suspension was serially diluted up to 10−4 dilution to obtain optimum spores (about 250–500 spores) on center large square of hemocytometer (B.S. 748, I.S. 10269, Rohem, India). Finally, spores of each pathogen were counted with hemocytometer, and concentration (spore/mL) in each plate was calculated by using following formula. Spore per mL = n  25  104 where n is the average number of spore in medium square (0.04 mm2) of centered big square. Different concentrations of the essential oils were tested for spore germination assay using method given by Leelasuphakul et al. (2008) with slight modifications. Aliquots of 25 µL of the essential oil solutions at different concentrations (100–2000 µg/mL) were mixed with 25 µL of 5% dextrose solution and 50 µL of the mold spores suspension (approximately 106 spores/mL) in cavity slide which

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were incubated in a moist chamber at 26 ± 2 °C for 24 h. Each slide was then fixed in lacto phenol-cotton blue and observed under the microscope for spore germination. The spores that generated germ tubes were enumerated, and percentage of spore germination was calculated in comparison with the control assay. Each assay was performed in triplicate, and the results were expressed as the average of the three repetitions. The control (10% DMSO) was tested separately for spore germination of different fungi.

4.3.1.4

Agar Diffusion Method

The antifungal activity of the EOs was investigated by the disk diffusion method using 24–48 h grown strains reseeded on PDA. The cultures were adjusted to 106 CFU/mL with sterile water. 100 lL of the suspensions was spread over PDA plates to obtain uniform fungal growth. Filter paper disks (6.0 mm in diameter) were impregnated with 20 lL of the oils and then placed onto the agar plates which had previously been inoculated with the test microorganism. The plates were incubated at 30 °C for 24–48 h. The diameter of the inhibition zones (mean values) was measured in millimeter and considered as the zone of inhibition (ZOI). All experiments were performed in triplicate.

4.3.2

Activity Against Human Pathogens and Plant Pathogens

A survey of the literature revealed that various EOs have been used for treatment of infections and diseases caused by different pathogenic fungi (Cavaleiro et al. 2015, Jamalian et al. 2012, Policegoudra et al. 2012).

4.3.2.1

Essential Oils Against Human Pathogenic Fungi

Epidermophyton, Microsporum, and Trichophyton species are responsible for the fungal infections concerning the human skin, nails, and hair (Woodfolk 2005). The exposure to molds and their spores is assumed to be connected to asthmatic and allergic reactions. Many molds produce toxic molecules, mycotoxins, which represent a threat to human health since some of them act as carcinogens. Thus, biological efficacy of EOs was mostly targeted against the important allergenic, toxigenic and pathogenic fungi such as Aspergillus, Stachybotrys and Cladosporium (Lee 2003). These problematic species can cause numerous health problems due to their ability to produce extremely toxic and/or allergenic secondary metabolites on the surface of their spores (Amuzie et al. 2010). In most cases, Aspergilli can lead to invasive infections especially in patients with a weakened immune system. Around 20 species of the genus Aspergillus have been reported as

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the causative agents of opportunistic infections in human beings. Aspergillosis is an opportunistic infection which can attack the lungs, ears, eyes, digestive system, kidney and brain (Chakraborty et al. 2006). Yeasts are unicellular, eukaryotic organisms that belong to the kingdom of fungi. Two of these are Saccharomyces and Candida. Candida species are a natural part of the human flora in the gastrointestinal tract, genitourinary system, and on the skin, and they can cause infections in these body regions. The most common pathogens of Candidiasis are C. albicans followed by C. glabrata and C. tropicalis. In new borns, C. parapsilosis is a prevalent pathogen that can lead to candidiasis including candiduria (Achkar and Fries 2010). EOs extracted from root and leaf of three commercially important aromatic plants such as Acorus calamus, Origanum vulgare, and Cinnamomum tamala contain high quantity of (Z)-asarone (81.1–92.4%), thymol (53.2%), and (Z)-cinnamaldehyde (5.8–7.1%), respectively. The antifungal activity of the essential oils of A. calamus, O. vulgare and C. tamala was evaluated against Aspergillus flavus and A. niger. The O. vulgare essential oil exhibited the highest activity (Bisht et al. 2011). Cinnamomum tamala and C. cassia are well-known traditional Indian and Chinese medicinal plants, respectively. In India, the leaves of C. tamala are common Indian household spice. The oil is reported to be rich in eugenol, but the plant materials from different locations of Himalayan region were found to contain (E)-cinnamaldehyde (  80%) along with linalool (5.4%) and (E)-cinnamyl acetate (2.2–3.7%). Eugenol was not noticed in any of the samples (Joshi et al. 2009). Both the oil and pure cinnamaldehyde are effective in inhibiting the growth of Candida albicans, C. tropicalis, C. glabrata, C. krusei, three Aspergilli sp., one Fusarium sp., Microsporum gypseum, Trichophyton rubrum and T. mentagraphytes. The MICs of both oil and cinnamaldehyde for yeasts range from 100 to 450 lg/mL, for filamentous fungi from 75 to 150 lg/mL and for dermatophytes from 18.8 to 37.5 lg/mL (Linda et al. 2006).

4.3.2.2

Essential Oils Against Plant Pathogenic Fungi

Agriculture is the driving force in developing countries for broad-based economic growth. Major focus nowadays being in getting maximum yield of agricultural products in order to provide food for increasing population. The pre- and postharvest losses in crops due to fungi, insects, nematodes, bacteria and viruses amount to more than 12% (Montesinos 2003). Although highly effective, synthetic pesticides often have undesirable side effects such as toxicity to mammals and environmental pollution. Plant metabolites and plant-based pesticides appear to be one of the better alternatives as they are known to have minimal environmental impact and danger to consumers in contrast to the synthetic pesticides. Plant diseases caused by soil-borne plant pathogenics are considered as the major problem in agriculture production. Cultivated vegetables are prone to attack by several fungi at different stages of plant growth. Growth inhibition in many economically important crops caused by fungi is a major problem (Fletcher et al. 2006).

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Early blight disease (Alternaria solani), fusarium wilt (Fusarium oxysporum), damping-off, root rot, stem canker (Rhizoctonia solani), White Rust (Albugo candida), cottony rot, watery soft rot, root, stem or fruit rot (Sclerotonia sclerotiorum), leaf spots, blights and root rot (Curvularia lunata) are some of the important plant diseases (Agrios 2005, Steinkellneret al. 2008). Most of researches have focused on plant-derived safer and biodegradable secondary plant metabolites as fungicides as alternatives to synthetic materials (Chang et al. 2008, 2009, Lu et al. 2013).

4.3.3

Mode of Action of Essential Oils

It is believed that volatile oils, either inhaled or applied to the skin, act by means of their lipophilic fraction reacting with the lipid parts of the cell membranes and as a result, modify the activity of the calcium ion channels. At certain levels of dosage, the volatile oils saturate the membranes and show effects similar to those of local anesthetics. They can interact with the cell membranes by means of their physicochemical properties and molecular shapes and can influence their enzymes, carriers, ion channels and receptors. The studies report the effects of odors on memory and mood. The fragrance compounds are absorbed by inhalation and are able to cross the blood-brain barrier and interact with receptors in the central nervous system (Buchbauer and Jirovetz 1994). EOs have been extensively screened against a broad spectrum of fungi. Studies on the mode of action of EOs have been reported on fungal targets involved in cytoplasmatic and cell wall metabolism (Hammer et al. 2004).

4.3.4

Synergism Between the Compounds of Essential Oils

Since the EOs are complex mixture of numerous molecules, one might wonder if their biological effects are the results of a synergism of all the constituent molecules or the major constituents alone. Generally, the major components are found to reflect quite well the biophysical and biological features of the EOs from which they were isolated (Ipek et al. 2005), the quantum of their effect being dependent on their concentration when tested alone. But, in several instances, the synergistic function of different molecules present in an EO is also noticed. It is likely that the activity of the main components is modulated by other minor molecules (Hoet et al. 2006). It is possible that several components of the EOs play a role in defining the fragrance, the density, the texture, the color and above all, cell penetration, lipophilic or hydrophilic attraction and fixation on cell walls and membranes, and cellular distribution. This last feature is very important because the distribution of the oil in the cell determines the different types of radical reactions produced, depending on their compartmentation in the cell. In that sense, for biological

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purposes, it is more informative to study the entire oil rather than the major constituents alone (Bakkali et al. 2008).

4.3.5

Himalayan Essential Oils as Antifungal Agents

In the recent years, authors performed the in vitro activity screening of EOs and terpenoid constituents extracted from different species of Nepeta, Aster, Erigeron, Senecio, Calamintha and Cymbopogon, etc., against the common pathogenic fungi (Table 4.2, Figs. 4.1, 4.2, 4.3, 4.4 and 4.5). a. Himalayan Nepeta Species Over a dozen Nepeta species (Lamiaceae) occur in this region (Strachey 1974) and find application in folk medicines as antispasmodic, diuretic, and antiseptic. Feline attractant properties of Nepeta species are also known for the long time. Nepeta species possess biologically active secondary metabolites, viz. diastereomeric nepetalactones, iridomycin, terpene-alkaloid actinidine. They act as pheromones and catnip response factors. The EOs from six Himalayan Nepeta species, viz. N. leucophylla, N. discolor, N. govaniana, N. clarkei, N. elliptica and N. erecta have been screened for their in vitro antifungal activity in our laboratory. Among them, the essential oil from N. leucophylla showed the highest antifungal activity against Candida albicans and Trichophyton rubrum, followed by the essential oils from N. clarkei, N. govaniana and N. erecta (Bisht et al. 2010). The essential oil from N. leucophylla showed maximum antifungal activity against both C. albicans (20.0 mm, MIC = 0.78 lL/mL) and T. rubrum (19.2 mm, MIC = 0.19 lL/mL). The essential oils from N. elliptica, N. erecta and N. govaniana also showed significant activity against both the fungal strains. In search of novel iridodial derivatives, three new cyclopentano-monoterpene enol acetates have been isolated from N. leucophylla (Bottini et al. 1992). Iridodial is a very active gene repairing substance. Iridodial b-monoenol acetate, isolated from aerial parts of N. leucophylla and actinidine, a rare terpene alkaloid from N. clarkei (Fig. 4.2) were taken up for their in vitro antifungal activity against 11 fungi (Mathela and Joshi 2008). Iridodial b-monoenol acetate and actinidine were screened for antifungal activities against Aspergillus flavus, A. ochraceus, Penicillium citrinum and P. viridicatum, all known mycotoxin-producing taxa and Sclerotium rolfsii and Macrophomina phaseolina, potential soybean pathogens (Saxena and Mathela 1996). Iridodial b-monoenol acetate was most effective against S. rolfsii, while actinidine was highly active against M. phaseolina. The EOs possessing iridoid or lactone skeleton were noticed to possess higher antifungal activity as compared to those containing regular carbon skeleton. This might be due to their higher water solubility and diffusion coefficient through the medium and also due to their higher hydrogen bonding potential. This compound showed a better inhibiting capacity than did nystatin in the case of A. ochraceus, and comparable activities against most of the other test organisms were noticed.

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Table 4.2 Antifungal activity of essential oils and their constituents from Himalayan MAPs Essential oil/Constituents

Fungi

Reference

Aster peduncularis, A. thomsoni, Selinum tenuifolium, Cymbopogon jwarancusa Aster molliusculus, Thymus serpyllum, Cymbopogon citratus

Aspergillus species

Nepeta species: N. leucophylla, N. discolor, N. govaniana, N. clarkei, N. elliptica, N. erecta Iridodial b-monoenol acetate, actinidine

Candida albicans, Trichophyton rubrum

Mathela and Sinha (1978) Agarwal et al. (1979, 1980) Bisht et al. (2010)

b-Pinene, a-pinene, q-cymene, limonene, camphene,1,8-cineol, a-terpineol, citral, geraniol, linalool, thymol, carvacrol, citronellyl acetate, citronellol, geranyl acetate Erigeron mucronatus, E. annuus, E. karvinskianus

Nepeta elliptica, N. leucophylla, N. clarkei, N. ciliaris, Calamintha umbrosa

Iridodial b-monoenol acetate, actinidine

(7R)-trans, trans-Nepetalactone

Aspergillus, Penicillium

Aspergillus flavus, A. ochraceus, Penicillium citrinum, P. viridicatum Aspergillus, Penicillium

Fusarium oxysporum, Helminthosporium maydis, Alternaria solani, Rhizoctonia solani, Sclerotonia sclerotiorum, Curvularia lunata, Albugo candida Fusarium oxysporum, Helminthosporium maydis, Alternaria solani, Rhizoctonia solani, Sclerotonia sclerotiorum, Curvularia lunata, Albugo candida Sclerotium, Macrophomina, Fusarium species Fusarium oxysporum, Helminthosporium maydis, Alternaria solani, Rhizoctonia solani, Sclerotonia sclerotiorum, Curvularia lunata, Albugo candida

Saxena and Mathela (1996) Agarwal et al. (1979, 1980) Kumar et al. (2014a)

Kumar et al. (2014b, c)

Mathela and Joshi (2008) Kumar et al. (2014c)

Actinidine was found to be highly active against M. phaseolina and less active against S. rolfsii and A. ochraceus. The MICs for the mycotoxin-producing fungi were in the range of 0.004–0.011 g per flask with potential of being exploited to eliminate the pre- and postharvest losses of food crops. In continuation to this, the work was extended further to screen 8 mycotoxin-producing strains of Ascomycetes class Penicillium citrinum, P. purpuranum, Aspergillus flavus, A. fumigatus, A. parasiticus, A. ochraceus,

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Erigeron mucronatus DC.

Erigeron annuus (L.) Pers

Aster indamellus Grierson

Calamintha umbrosa Benth.

Nepeta ciliaris Benth.

Nepeta leucophylla Benth.

Nepeta elliptica Royle ex Benth.

Nepeta clarkei Hook. f.

Senecio nudicaulis Buch-Ham. ex D. Don.

Fig. 4.1 Some Himalayan aromatic plants

A. niger, A. nidulans, two yeasts, viz. Candida albicans and Rhodotorula muciloginosa and a plant pathogen Fusarium moniliforme (Mathela and Joshi 2008). The results of disk diffusion assay show the zones of inhibition due to actinidine were most significant against A. ochraceous (41.9 mm). The largest zone of inhibition for iridodial b-monoenol acetate among tested fungi was shown against A. fumigatus (46.6 mm) and P. citrinum (42.3 mm) and A. nidulans (34.3 mm). The activity of compounds was comparable to standard drug against A. ochraceous, A. niger and P. purpuranum after 48 h of incubation. Magnitude of inhibition effect shows iridodial b-monoenol acetate to be more effective as compared to actinidine against

4 Antifungal Activities of Essential Oils from Himalayan Plants O

87

H3CO

C OCH3

CHO

O

H H3C

H3C 1

OAc

2

3

O CHO

N

O

4

6

5 OH

CHO OH

8

7

9

Fig. 4.2 Chemical structures of terpenoid constituents showing antifungal activity. (1: cisLachnophyllum ester; 2: trans-2-cis-8-Matricaria ester; 3: Iridodial-b-monoenol acetate; 4: (7R)trans, trans-Nepetalactone; 5: Actinidine; 6: (E)-Cinnamaldehyde; 7: Carvacrol; 8: Thymol; 9: Citral)

Biplot (axes F1 and F2: 95.77 %) 10 8

S10

6 S3

F2 (5.81 %)

4

Group-II F2

0

S5 S6

F3

F4 S9

F5

F1

-2

S1

-4

S7

S8

Group-I

S4

-6 -8 -12

Group-III

S2

2

-10

-8

-6

-4

-2

0

2

4

6

8

10

12

F1 (89.96 %) Fig. 4.3 PCA of antifungal activity of essential oils and nepetalactone against five fungi. S1: Erigeron mucronatus, S2: E. karvinskianus, S3: E. annuus, S4: Calamintha umbrosa, S5: Nepeta elliptica, S6: N. leucophylla, S7: N. ciliaris, S8: N. clarkei, S9: Nepetalactone, S10: synthetic fungicide

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Dendrogram 700000 600000

Dissimilarity

500000 400000 300000 200000

F3

F1

F5

F4

0

F2

100000

Fig. 4.4 HCA based on the Euclidean distance between groups of the antifungal activity of essential oils. F1: Fusarium oxysporum, F2: Helminthosporium maydis, F3: Rhizoctonia solani, F4: Alternaria solani, F5: Sclerotonia sclerotiorum

k

l

k,l

100

j

H. maydis A. solani C. lunata

60

A. candida

i

0

trol

Con

E.

tus

ona

cr mu

a

E. k

nus

kia

s rvin

us

nnu

E. a

llus

me

da . in

A

a,b

a-g

a,b a-h f-h

a-c

a-h a-h

c-h

h

g-h a,b a-c

a-e a-f

a-h

a

a-d

a-g

a-h

20

e-h g-h b-h

40

d-h c-h

% Spore germination

80

F. oxysporum

ulis

ica

ud S. n

Fig. 4.5 Effect of essential oils on spore germination of tested fungi. Bars with different letters (a-l) are statistically different at the level of p < 0.05 according to the Duncan test

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fungi. Activity of both compounds was prominent up to 0.5–1 mg impregnation and comparable or even better than control clotrimazole (Figs 4.3 and 4.4). A recent study revealed that the EOs of some Nepeta species, viz. N. elliptica, N. leucophylla, N. clarkei and N. ciliaris, and (7R)-trans, trans-nepetalactone constituents were effective fungicides against soil-borne plant pathogenic fungi, viz. Fusarium oxysporum, Helminthosporium maydis, Alternaria solani, Rhizoctonia solani, Sclerotonia sclerotiorum, Curvularia lunata and Albugo candida (Kumar et al. 2014b, 2014c). The results of inhibitory effects of essential oils derived from N. leucophylla, N. ciliaris, and N. clarkei against tested phytopathogenic fungi revealed that more active being the oils containing oxygenated terpenoids. H. maydis was sensitive to all oils. The N. leucophylla oil possessing oxygenated iridoids was more effective against H. maydis, while N. ciliaris was more active against F. oxysporum. The results showed that oil of N. elliptica and (7R)-trans, trans-nepetalactone had potential as antifungal agent on fungal spore germination against tested plant pathogens. The in vitro antifungal activity of the oil observed in this study could be attributed to the presence of (7R)-trans, trans-nepetalactone. Slightly better activity of the oil could be because of synergism with the other constituents, viz. spathulenol and sesquiterpene hydrocarbons. Thus, these could lead to development of new antimicrobials to suppress common plant diseases, especially those caused by F. oxysporum, H. maydis and C. lunata. Nepetalactone might help in developing structurally related natural fungicides for many microbial phytopathogens which cause severe destruction to crop, vegetables, and ornamental plants. b. Himalayan Erigeron Species The genus Erigeron (Asteraceae) is represented by 8 species in this region and yields essential oils rich in biologically active polyacetylenic compounds besides terpenoids. The Erigeron species have a history of their use as folk medicines (Awen et al. 2010). Erigeron mucronatus, E. annuus, and E. karvinskianus growing in subalpine region possess isomeric polyacetylenic constituents, viz. isomeric matricaria and lachnophyllum esters accounting for 83.3, 69.3, and 30.1% of the essential oils from these species, respectively, in addition to mono- and sesquiterpenoids as minor constituents. The antifungal activity tested by poisoned food (PF) techniques against F. oxysporum, H. maydis, R. solani, A. solani and S. sclerotiorum demonstrated significant inhibition of the mycelial growth of all strains (p < 0.05) (Fig. 4.5). The oils (500 µg/mL) showed significant antifungal effect against tested fungi in the growth inhibition range of 37.6–85.5% with IC50 values ranging from 88.8 to 660.0 µg/mL as compared to standard fungicides (100% inhibition) with IC50 value in the range of 32.2–129.4 µg/mL. F. oxysporum, C. lunata and A. candida were highly susceptible to E. annuus oil with their IC50 values 120.7, 253.5, and 300.4 µg/mL, respectively (Kumar et al. 2014a). The essential oil of E. annuus showed strong inhibitory effect on the mycelial growth of phytopathogens such as F. oxysporum (85.5%), R. solani (82.0%) and S. sclerotiorum (65.1%) while the oil of E. mucronatus exhibited strong antifungal effect against H. maydis (83.9%), R. solani (78.0%) and F. oxysporum (77.3%), as

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compared to the oil of E. karvinskianus. Comparative study of IC50 and MIC values of Erigeron oils with fungicide (standard check) showed significant activity comparable to fungicide. The E. mucronatus and E. karvinskianus oils possessed significant activity against H. maydis with an IC50 values, 94.8 and 88.8 µg/mL, respectively, with MIC value 1000 µg/mL for each, while E. annuus oils showed an IC50 value 153.15 µg/mL with MIC value 1000 µg/mL against F. oxysporum. One of the fungal pathogen R. solani also displayed significant susceptibility to the oils from E. mucronatus, E. karvinskianus and E. annuus with IC50 values 232.9, 248.5, and 223.8 µg/mL, respectively (Kumar et al. 2014a). The significant effect of Erigeron oils on the spore germination of plant fungi (85.5%) could be attributed mainly to the presence of polyacetylenic chemical constituents, viz. cis-lachnophyllum ester and trans-2-cis-8-matricaria ester in the oils. Most of the studies on the mechanism of action for antifungal compounds have focused on their effects on cellular membrane. Actually, compounds not only attack cell walls and cell membranes, thereby affecting the permeability and release of intracellular constituents, but they also interfere with membrane function. According to Knobloch et al. (1989), the chemical components like limonene, a-pinene, b-pinene and lachnophyllum ester showed antimicrobial activity through disruption of cell membrane and mitochondria. Another important characteristic of these components is their hydrophobicity that enables them to penetrate the lipid component of the cell membrane and finally cause death of the cells. Thus, active polyacetylenic compounds might have several invasive targets which could lead to the inhibition of plant pathogenic fungi. c. Other Himalayan Species EOs isolated from aerial parts of Aster, Calamintha and Senecio species showed significant in vitro antifungal activity against phytopathogenic fungi responsible for plant diseases, viz. early blight disease, fusarium wilt, damping-off, root rot, white rust, stem or fruit rot. The EOs displayed potential as a mycelial growth inhibitor against all the test fungi. At the concentration of 500 µg/mL, F. oxysporum, H. maydis and R. solani were found to be the most inhibited (> 70%) fungal pathogens by the oils. The oil of C. umbrosa showed relatively lower inhibitory effect on the growth of F. oxysporum (67.4%), H. maydis (76.1%), R. solani (74.1%), A. solani (55.7%) and S. sclerotiorum (63.1%) with IC50 values 109.3 to 416.2 µg/mL (Kumar et al. 2014c). Aster indamellus oil also showed significant inhibition (50.9 to 72.9%) against F. oxysporum, H. maydis, R. solani, A. solani and S. sclerotiorum. H. maydis and R. solani were found most inhibited by the oil with IC50 values 186.1 µg/mL and 267.5 µg/mL, respectively. The Senecio nuducaulis oil exhibited fungal mycelial growth inhibition percentage ranging from 49.1 to 75.2%. F. oxysporum was most affected by the oil with IC50 values 262.4 µg/mL. There was a remarkable inhibition of germ tube formation as phytopathogenic spore germination by most of essential oils. The C. umbrosa oil was most effective in spore germination inhibition (92.8%) of C. lunata with IC50 value 557.4 µg/mL. A. indamellus and S. nudicaulis oils had significantly higher inhibitory effect on H. maydis and C. lunata inhibiting (> 90%) spore germination with IC50 values

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540.4 µg/mL and 589.7 µg/mL, respectively. Results showed lower inhibitory effect of oils on A. solani and A. candida spore germination as compared to the other fungal spore.

4.3.6

Statistical Analysis

The mean values and standard deviations were calculated for all tests. The data were analyzed using SPSS 16.0 statistical software. The results were calculated by one-way analysis of variance (ANOVA). The means were compared by Duncan Multiple Range tests at a level of significance of p < 0.05. The principal component analysis (PCA) and hierarchical cluster analysis (HCA) were performed using XLSTAT statistical computer software package, version 14, for evaluating correlation between antifungal activity and essential oils. The statistical analysis of the antifungal activity of the oils showed the significant difference among the oils and the fungal strains (p < 0.05). To evaluate the correlation between the antifungal activity and the essential oils, all the IC50 values of mycelia growth inhibition were subjected to the PCA and HCA analysis. The PCA horizontal axis explained 89.9% of the total variance, while the vertical axis a further 5.8% (Fig. 4.3). The HCA indicates three groups (I, II, and III) of fungi (Fig. 4.4). Group I represented by the F. oxysporum, R. solani and S. sclerotinia was the less resistant to the majority of Erigeron, Nepeta, Aster, Calamintha and Senecio EOs and nepetalactone compound. This group is divided into two subgroups Ia and Ib. Subgroup Ia was limited to S. sclerotinia which was characterized by the resistance to oils. Subgroup Ib represented by F. oxysporum and R. solani showed particularly less susceptibility. Group II is limited to the H. maydis. This strain showed more susceptibility for to all the EOs (IC50 43–177 µg/mL) and especially for Nepeta oils (IC50 < 90 µg/mL). Group III, consisting of A. solani which showed high resistance for all the EOs and nepetalactone as well.

4.4

Conclusion

Rich Himalayan biodiversity has been the source of safe and effective drugs in the indigenous system of medicine and has contributed in the development of a number of new therapeutic agents. Besides, these are sources of flavor chemicals and natural dyes. Large number of species belonging to Lamiaceae and Asteraceae are rich in volatile oils and are major source of bioactive natural products. Most studies were focused on their EO components such as terpenoids, iridoids, acetylenic and phenolic compounds, isomeric nepetalactones and furanoeremophilanes. EOs extracted from Himalayan plants and their constituents demonstrated antifungal activity against several human pathogenic fungi. Therefore, EOs have potential for development of

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natural agents in the prevention and control of fungal diseases and also in the preservation of food and food products from fungal spoilage and contamination. The biocontrol efficacy results of EOs from some species belonging to Lamiaceae and Asteraceae against soil-borne plant pathogens and other crop pests is a new dimension of the work carried out. Interesting results of studies in activity against soil pathogens and crop pests have been recorded with their potential as biochemical control in agricultural crops for replacement of synthetic insecticides/ pesticides by biomolecules of plant origin. Considering the in vitro reduction in mycelial growth and fungal spore germination, it can be concluded that the EOs have potential as possible biofungicides against microbial phytopathogens which cause severe destruction to crop, vegetables and ornamental plants. Acknowledgements The authors are grateful to Department of Chemistry, Kumaun University, Nainital, for providing laboratory facilities, Botanical Survey of India (BSI), Dehradun, for plant identification and Department of Plant Pathology, College of Agriculture, G.B. Pant University of Agriculture and Technology, Pantnagar, for their valuable help in carrying out some experiments.

References Achkar JM, Fries BC (2010) Candida infections of the genitourinary tract. Clin Microbiol Rev 23 (2):253–273 Adams RP (2007) Identification of essential oil components by gas chromatography/mass spectrometry. Allured Publishing Corporation, Carol stream IL Agarwal I, Kharkwal HB, Mathela CS (1980) Chemical study and antimicrobial properties of essential oil of Cymbopogon citratus Linn. Bull Med Ethnobot Res 1:401–407 Agarwal I, Mathela CS, Sinha S (1979) Studies on the antifungal activity of terpenoids against Aspergilli. Indian Phytopathol 32(1):104–105 Agrios GN (2005) Plant pathology, 5th edn. Elsevier Academic Press, Burlington, MA, pp 446– 450 Amuzie CJ, Islam Z, Kim JK, Seo JH, Pestka JJ (2010) Kinetics of satratoxin G tissue distribution and excretion following intranasal exposure in the mouse. Toxicol Sci 116(2):433–440 Awen BZ, Unnithan CR, Ravi S, Lakshmanan AJ (2010) GC-MS analysis, antibacterial activity and genotoxic property of Erigeron mucronatus essential oil. Nat Prod Commun 5:621–624 Bakkali F, Averbeck S, Averbeck D, Idaomar M (2008) Biological effects of essential oils-a review. Food Chem Toxicol 46:446–475 Bisht DS, Padalia RC, Singh L, Pande V, Lal P, Mathela CS (2010) Constituents and antimicrobial activity of the essential oils of six Himalayan Nepeta species. J Serb Chem Soc 75(6):739–747 Bisht DS, Pal A, Chanotiya CS, Mishra D, Pandey KN (2011) Terpenoid composition and antifungal activity of three commercially important essential oils against Aspergillus flavus and Aspergillus niger. Nat Prod Res 25(20):1993–1998 Bottini AT, Dev V, Shah GC, Mathela CS, Melkani AB, Nerlo AT, Strum NS (1992) Cyclopentanomonoterpene enol acetates from Nepeta leucophylla. Phytochemistry 35:1653– 1657 Buchbauer G, Jirovetz L (1994) Aromatherapy-use of fragrances and essential oils as medicaments. Flav Fragr J 9:217–222 Cavaleiro C, Salgueiro L, Gonçalves MJ, Hrimpeng K, Pinto J, Pinto E (2015) Antifungal activity of the essential oil of Angelica major against Candida, Cryptococcus, Aspergillus and dermatophyte species. J Nat Med 69(2):241–248

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Chakraborty A, Marak RSK, Sing S, Gupta SO, Hurst SF, Padhye AA (2006) Brain abscess due to Aspergillus nidulans. J Med Mycol 16:100–104 Chang HT, Cheng YH, Wu CL, Chang ST, Chang TT, Su YC (2008) Antifungal activity of essential oil and its constituents from Calocedrus macrolepis var. formosana Florin leaf against plant pathogenic fungi. Bioresour Technol 99:6266–6270 Chutia M, Deka BP, Pathak MG, Sarma TC, Boruah P (2009) Antifungal activity and chemical composition of citrus reticulata Blanco essential oil against phytopathogens from North East India. LWT—Food Sci Technol 42:777–780 Feng W, Zheng X (2007) Essential oils to control Alternaria alternata in vitro and in vivo. Food Control 18:1126–1130 Fletcher J, Bender C, Budowle B, Cobb WT, Gold SE, Ishimaru CA et al (2006) Plant pathogen forensics: capabilities, needs, and recommendations. Microbiol Mol Biol Rev 70:450–471 Grover RK, Moore JD (1962) Toxicometric studies of fungicides against brown rot organisms Sclerotinia fructicola and S. laxa. Phytopathology 52:876–880 Hammer KA, Carson CF, Riley TV (2004) Antifungal effects of Melaleuca alternifolia (tea tree) oil and its components on Candida albicans, Candida glabrate and Saccharomyces cerevisiae. J Antimicrob Chemother 12:1–5 Hoet S, Stevigny C, Herent MF, Quetin-Leclercq J (2006) Antitrypanosomal compounds from leaf essential oil of Strychnosspinosa. Planta Med 72:480–482 Ipek E, Zeytinoglu H, Okay S, Tuylu BA, Kurkcuoglu M, Baser KH (2005) Genotoxicity and antigenotoxicity of Origanum oil and carvacrol evaluated by Ames salmonella microsomal test. Food Chem 93:551–556 Jamalian A, Shams-Ghahfarokhi M, Jaimand K, Pashootan N, Amani A, Razzaghi-Abyaneh M (2012) Chemical composition and antifungal activity of Matricaria recutita flower essential oil against medically important dermatophytes and soil-borne pathogens. J Med Mycol 22(4):308– 315 Joshi SC, Padalia RC, Bisht DS, Mathela CS (2009) Terpenoid diversity in the leaf essential oils of Himalayan Lauraceae species. Chem Biodivers 9(6):1364–1373 Knobloch K, Pauli A, Iberl B (1989) Antibacterial and antifungal properties of essential oils components. J Essent Oil Res 1:119–128 Kumar V, Mathela CS, Tewari G, Singh D, Tewari AK, Bisht KS (2014a) Chemical composition and antifungal activity of essential oils from three Himalayan Erigeron species. LWT- Food Sci Technol 56(2):278–283 Kumar V, Mathela CS, Tewari G, Singh D (2014b) Antifungal activity of Nepeta elliptica Royle ex Benth. oil and its major constituent (7R)-trans, trans-nepetalactone: a comparative study. Ind Crop Prod 55:70–74 Kumar V, Mathela CS, Tewari AK, Bisht KS (2014c) In vitro inhibition activity of essential oils from some Lamiaceae species against phytopathogenic fungi. Pestic Biochem Physiol 114:67– 71 Lee TG (2003) Health symptoms caused by molds in a courthouse. Arch Environ Health 58 (7):442–446 Leelasuphakul W, Hemmanee P, Chuenchitt S (2008) Growth inhibitory properties of Bacillus subtilis strains and their metabolites against the green mold pathogen (Penicillium digitatum Sacc.) of citrus fruit. Postharvest Biol Tec 48:113–121 Linda SMO, Li Yaolan, Sheung-Lau K, Hua W, Elaine YLW, Vincent ECO (2006) Antimicrobial activities of cinnamon oil and cinnamaldehyde from the Chinese medicinal herb Cinnamomum cassia Blume. Am J Chin Med 34(3):511–522 Lu M, Han Z, Xu Y, Yao L (2013) Effects of essential oils from Chinese indigenous aromatic plants on mycelial growth and morphogenesis of three phytopathogens. Flav Fragr J 28:84–92 Mathela CS (1981) In vitro antifungal examination of some terpenoids. Proc Natl Acad Sci India 51:513–516 Mathela CS, Joshi N (2008) Antimicrobial activity of Nepeta isolates. Nat Prod Commun 3 (6):945–950

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Mathela CS, Sinha GK (1978) Antibacterial and antifungal study of some indiginous essential oils. J Res Indian Med Yoga Homoeo 13(3):122–124) Mitscher LA, Leu RP, Bathala MS, Wu WN, Beal JL, White R (1972) Antimicrobial agents from higher plants, introduction, rationale and methodology. Lloydia 35:157–166 Montesinos E (2003) Development, registration and commercialization of microbial pesticides for plant protection. Int Microbiol 6:245–252 Policegoudra RS, Goswami S, Aradhya SM, Chatterjee S, Datta S, Sivaswamy R et al (2012) Bioactive constituents of Homalomena aromatica essential oil and its antifungal activity against dermatophytes and yeasts. J Med Mycol 22(1):83–87 Saxena J, Mathela CS (1996) Antifungal activity of new compounds from Nepeta leucophylla and Nepeta clarkei. Appl Environ Microbiol 62(2):702–704 Steinkellner S, Roswitha MR, Vierheilig H (2008) Germination of Fusarium oxysporum in root exudates from tomato plants challenged with different Fusarium oxysporum strains. Eur J Plant Pathol 122:395–401 Strachey R (1974) Catalogue of the plants of Kumaon and of the adjacent portions of Garhwal and Tibet. Periodical Experts, New Delhi, pp 142 Wood folk JA (2005) Allergy and dermatophytes. Clin Microbiol Rev 18(1):30–43

Chapter 5

Review Chapter: Fusarium Genus and Essential Oils Martin Zabka and Roman Pavela

Abstract The Fusarium genus belongs to the most significant fungi genera on earth. Enumeration of the large number of its importance aspects is beyond the means of one scientific discipline. Similarly, it comprises a huge group of many organisms, which are currently considered from many taxonomic viewpoints, in the light of developing modern methods and fungal taxonomy. The Fusarium genus includes a large number of species, which are pathogenic not only for plants, but also humans or livestock, where they cause local and systemic mycosis dangerous to life. In agriculture and plant protection, many species of the Fusarium genus constitute an enormous problem with huge economic impacts on plant and food production. One of the most dangerous properties is considered as the capability to produce chemically variegated and mostly extremely dangerous secondary toxic metabolites, which are known commonly as Fusarium mycotoxins. It is the Fusarium mycotoxins, which rank among the most dangerous mycotoxins. In conventional agricultural practice, but also in the treatment of human mycosis, elaborate strategies for fungicidal treatment exist that use synthetic fungicides. Thanks to the increasing number of cases of fungal resistance to synthetic fungicides and problems with residues in the ecosystem, the popularity of research in alternative and natural protection methods is increasing. Essential oils (EOs) are an excellent alternative based on natural mixtures and biodegradable substances of natural origin. The next chapter discusses the problems of fusaria from the above-mentioned aspects and includes the summary of research results published to date in the area of effect of EOs on the most common species of the Fusarium genus.










Keywords Essential oils Fusarium Fusariosis Pathogenic fungi Toxigenic fungi Antifungals Phenols Terpenes Mycosis Mycotoxins













M. Zabka (&)
R. Pavela Crop Research Institute, Drnovská 507, Praha 6, 161 06 Ruzyne, Czech Republic e-mail: [email protected] © Springer International Publishing AG 2018 J.-M. Mérillon and C. Rivière (eds.), Natural Antimicrobial Agents, Sustainable Development and Biodiversity 19, https://doi.org/10.1007/978-3-319-67045-4_5

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5.1

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The Genus Fusarium Introduction

Fungal species belonging to the genus Fusarium represent a really interesting group of filamentous fungi. Many different studies have been dedicated to this great topic of the Fusarium genus. The question of taxonomy of this genus remains unclear. More than 1000 species have been described so far, but there are many different modern concepts of taxonomical reduction. The difference depends on the preferred concept of speciation. Many of them have known their sexual state (teleomorphic form) often linked with the genera Gibberella (Watanabe et al. 2011; Summerell et al. 2010). The species of Fusarium occur mostly ubiquitously, with the capability of colonising many different substrates. These species can be isolated around the world, e.g. from plants, soil, water and air. From a biological viewpoint, the mentioned high adaptability to different conditions (Booth 1984) makes the Fusarium genus extremely successful throughout the long process of evolution. This feature of evolutionary susceptibility is probably one of the reasons why the Fusarium genus belongs to the most significant filamentous fungi, alongside the Aspergillus and Penicillium genera (Frisvad and Samson 1991). The significance of the Fusarium genus can be appropriately described from a practical point of view, where several of the most obvious aspects, especially those affecting different areas of human activity, are considered.

5.2

Fusarium Species as Significant Plant Pathogens

The Fusarium genus is considered extraordinarily significant primarily in the area of agriculture. There are plenty of known specialised Fusarium species which cause yield losses every year on a wide range of host crop plants. On the other hand, there are many species within the Fusarium genera having a broad spectrum of host plants. This review chapter only lists several basic Fusarium species which were used as target organisms for tests of essential oil antifungal activities, namely Fusarium oxysporum, F. graminearum, F. solani, F. verticillioides, F. tricinctum, F. poae. Fusarium oxysporum is a complex of many (over 100) specialised varieties of so-called formae speciales. F. oxysporum is commonly known as a cause of vascular wilt in many growing agricultural plants. On account of its many specialised forms, F. oxysporum affects a broad spectrum of hosts, causing severe losses in the case of many field crops, vegetables, flowers and plantation crops such as cotton, banana and oil palm (Agrios 1988; Smith et al. 1988; Gordon and Martyn 1997). F. graminearum (Gibberella zeae) is known mainly as a plant pathogen which produces symptoms of so-called Fusarium head blight, a serious disease on wheat and barley. This pathogen is responsible for immense economic losses every year (Goswami and Kistler 2004). F. solani (Haematonectria haematococca) is also a complex of formae speciales and is ubiquitous in soils all over the world.

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F. solani primarily affects the root system of many host plants, causing symptoms known as root rot in many agriculturally important crops, e.g. potatoes, tomatoes, legumes and members of the cucurbit family (Zhang et al. 2006). F. verticillioides (G. fujikuroi) is not considered a specialised pathogenic species and can be found as a pathogen on a wide range of crops. Although F. verticillioides shows no host specialisation, it mainly occurs on maize (Desjardins et al. 2000) and is generally associated with several diseases including stalk, kernel and ear rots known as Fusarium rots (White 1999). Both F. tricinctum and F. poae are species with an ability to affect many plants, but they are more often associated with severe head blight symptoms on winter wheat, barley and other grains worldwide (Kulik 2008; Stenglein 2009).

5.3

The Fusarium Genus and Dangerous Secondary Metabolites

Probably one of the most significant aspects of Fusarium infections in agricultural crops in the field, and in postharvest products, is the ability of many Fusarium species to produce really dangerous toxic secondary metabolites. These are commonly known as mycotoxins. There are three main chemical groups of fusarium mycotoxins—trichothecenes (type A and B), fumonisins and zearalenone. Frequently occurring fusarium trichothecenes include deoxynivalenol (DON), 3- and 15-acetyl-deoxynivalenol (3-,15-ADON), nivalenol (NIV), fusarenon-X (FUS-X), T-2 toxin (T-2) and HT-2 toxin (HT-2). Next, there are the fumonisins B1, B2, B3, and alpha and beta zearalenone (Desjardins 2006). Fusarium mycotoxin contaminations have been affecting mankind throughout history. The most known and discussed event is the tragedy which took place in the former Soviet Union in the 1930s and during WWII. In the Orenburg region of the Soviet Union, approximately one hundred thousand people died after suffering from alimentary toxic aleukia (ATA), a complex of symptoms due to fusarium mycotoxins in the consumed infected grains. Among the group of fusarium mycotoxins, T-2 toxin prevailed as the main suspect in this historical case (Yli-Mattila 2011). In the 1960s, high levels of T-2 toxin were also found in maize in the USA (Mirocha and Pathre 1973). Given the high toxicity and persistence of fusarium toxins, there was a controversial suspicion that they were being misused as a biological–chemical warfare substance known as “yellow rain” several times in military conflicts during the 1970s and 1980s in Afghanistan, Laos and Cambodia (Crocker 1988). Paradoxically, these bad historical experiences, e.g. with ATA, fortified the attempt to develop reliable and routine methods for mycotoxin detection in foodstuffs. Since that time, fusarium mycotoxins in foodstuffs have become one of the main subjects of risk assessment in the area of agricultural production and food safety (Turner et al. 2009; European Commission Regulation No 1881/2006).

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The Fusarium Genus as Human Pathogen

The severity of Fusarium species occurrence can be seen even in the area of human medicine. Many Fusarium species can infect and colonise human tissues, thereby causing a broad spectrum of less or more serious fungal infections. In those cases, many common species of Fusarium genus are causative agents of superficial, locally invasive or disseminated human mycoses. The clinical form of fusariosis depends mainly on the immune status of the host (Nucci and Anaissie 2007). Species of Fusarium represent a highly serious risk especially in the case of immunocompromised hosts (e.g. AIDS, post-transplant immunosuppression) or immunodeficiency due to other reasons, where fusariosis is typically found in its invasive and disseminated form (Kadri et al. 2015). But even in the case of an immunocompetent host, there are many known cases of serious fusarioses, including localised infections of the skin, cornea or nails. Keratomycoses and onychomycoses are mentioned most often, but cases of infections after surgical wounds, ulcers or otitis media have also been described. Fusarium species can also cause allergic reactions such as sinusitis in an immunocompetent host (Wickern 1993). Elimination of Fusarium through synthetic fungicides in the cases above, such as in the area of protecting agricultural crops in the field, and consequently preventing mycotoxin contamination of stored agricultural products, is sometimes problematic. For treatment of serious human mycoses such as fusarioses, utilising synthetic fungicides tends to be particularly problematic for many reasons. In this regard, the main problem is posed by the acute and chronic toxicity of synthetic fungicidal components, which have significant harmful side effects on human health, the environment and the long-term persistence of their residues (Zarn et al. 2003; Nakanishi 2007; Costa et al. 2008; Scordino et al. 2008; Gubbins and Heldenbrand 2010).

5.5

Essential Oils Definition and Common Composition

Essential oils (EOs) are defined as any volatile oils that have strong aromatic components and give a distinctive odour, flavour or scent to an aromatic plant. EOs are produced by more than 17,500 aromatic plant species commonly belonging to many angiospermic families, e.g. Lamiaceae, Rutaceae, Myrtaceae, Zingiberaceae and Asteraceae (Regnault-Roger et al. 2012). Bioactive components of EOs are the by-products of plant metabolism and are commonly referred to as volatile plant secondary metabolites. The compounds of EOs are synthesised and stored in complex secretory structures, viz. glandular trichomes, secretory cavities and resin ducts, and are present as droplets of fluid in the leaves, stem, flowers and fruits, bark, or roots of plants (Fahn 2000).

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EOs are very complex natural mixtures. They are often characterised by two or three major compounds at fairly high concentration (20–70%), compared to others components which are present in trace amounts. These major components determine the biological properties of the actual essential oil. It is possible to describe the components in many ways, but they simply represent two groups having different biosynthetic origins. The main group is composed of terpenes and terpenoids, and the other of aromatic and aliphatic constituents, all characterised by low molecular weight. EO components are synthesised in the cytoplasm and plastids of plant cells via malonic acid, mevalonic acid and methyl-D-erythritol-4-phosphate (MEP) pathways. Terpenes are basically hydrocarbons made up of several units of isoprene (C5). However, terpenoids are terpenes that have been biochemically modified via enzymes that add oxygen molecules and move or remove a methyl group (Burt 2004). Monoterpenes are formed from the coupling of two isoprene units (C10). They are the most representative molecules, constituting about 90% of EOs, and allow for a great variety of structures (acyclic, monocyclic, bicyclic) and several functions (carbures—e.g. myrcene, p-cimene, camphene; alcohols—e.g. linalool, menthol, borneol; aldehydes—e.g. geranial, citronellal; ketones—e.g. carvone, pulegone, camphor; esters—e.g. linalyl acetate, menthyl, citronellyl acetate; ethers—e.g. 1,8 cineole, menthofurane; peroxides—e.g. ascaridole; phenols—e.g. thymol, carcacrol). Examples of plants containing these compounds include bay, thyme, rosemary, parsley, hops, laurel, bay leaves, tea tree, mugwort, sweet basil, cannabis, ylang-ylang and wormwood, among others. Sesquiterpenes are formed from the assembly of three isoprene units (C15). The extension of the chain increases the number of cyclisations and thus leads to a great variety of structures. The functions of sesquiterpenoids are similar to those of monoterpenoids (carbures—e.g. azulene, b-caryophyllene, elemenes; alcohols— e.g. bisabol, cedrol, patchoulol; ketones—e.g. nootkatone, germacrone, turmerones). The principal plant sources for these compounds include mint, orange, angelica, rosemary, celery, sage and bergamot, among others (Banthorpe 1991). Derived from phenylpropane, aromatic compounds occur less frequently than terpenes. The biosynthetic pathways concentring terpenes and phenylpropanic derivatives are generally separated in plants but may coexist in some, with one major pathway taking over. The functions of aromatic compounds are similar to those of monoterpenoids and/or sesquiterpenoids and comprise aldehydes (cynnamaldehyde); alcohols (cinnamic alcohol); phenols (chavicol, eugenol); methoxy derivatives (anethole, estragole, methyleugenol) and methylene dioxy compounds (apiole, myristine, safrole). The principal plant sources for these compounds include anise, cinnamon, clove, fennel (Grayson 2000). EOs are defined as products obtained by the so-called hydrodistillation process, steam distillation, dry distillation or the mechanical cold pressing of plants. The classical method is based on the Clevenger steam distillation apparatus developed in 1928. Today, this method has been adapted for an industrial scale. Steam

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distillation requires large containers because of the low (generally 1000 mg/ l

MIC50 388 mg/l MIC50 353 mg/l

MIC50 295 mg/l MIC50 581 mg/l

MIC or MIC50

5 Review Chapter: Fusarium Genus and Essential Oils 109

Cinnamomum zeylanicum Blume

Cinnamonum spp.

Salvia officinalis L. Sample 4

Salvia officinalis L. Sample 3

Salvia officinalis L. Sample 2

Trans-cinnamaldehyde (74.0%)

Fusarium spp. (Christian and Goggi 2008) Fusarium tricinctum (Džamić et al. 2008)

MIC 800 ll/l

Fusarium verticillioides (Pinto et al. 2007) Fusarium verticillioides (Pinto et al. 2007) Fusarium verticillioides (Xing et al. 2014) Fusarium spp. (Christian and Goggi 2008) (continued)

MIC 5 ll/ml

MIC 2.5-5 ll/ml

MIC 60 ll/l

Lamiaceae

Lamiaceae

Lauraceae

Lauraceae

MIC 800 ll/l

Fusarium verticillioides (Pinto et al. 2007)

MIC 2.5 ll/ml

Lamiaceae

Lamiaceae

Fusarium verticillioides (Pinto et al. 2007)

MIC 15 ll/ml

Species (strain, if available)/references

MIC or MIC50

MIC 0.63 ll/ml

Lamiaceae

Linalyl acetate (52.83%), linalool (18.18%), a-terpineol (5%), a-pinene (4.57%), 1.8-cineole (2.29%), limonene (1.55%), b-caryophyllene (1.83%) and b-terpineol (1.19%) Camphor (20.5%), b-pinene (14.0%), cis-thujone (10.4%), camphene (7.3%), borneol (7.5%), a-humulene (6.7%), a-pinene (5.1%), 1.8-cineole (4.0%), bornyl acetate (3.2%), trans-thujone (2.1%), limonene (2.0%), myrcene (1.4%) Cis-thujone (32.9%), camphor (23.4%), 1.8-cineole (13.0%), camphene (3.8%), a-humulene (3.7%), trans-thujone (3.7%), b-pinene (2.7%), a-pinene (1.9%), borneol (1.8%), limonene (1.6%), myrcene (1.2%) b-pinene (12.8%), camphor (12.4%), a-humulene (11.5%), cis-thujone (8.8%), camphene (6.6%), borneol (5.7%), a-pinene (5.3%), 1.8-cineole (3.7%), bornyl acetate (2.6%), limonene (1.4%), trans-thujone (1.4%), myrcene (1.2%) Cis-thujone (37.1%), camphor (11.6%), 1.8-cineole (8.9%), trans-thujone (5.7%), camphene (4.2%), a-pinene (4.2%), a-humulene (3.5%), borneol (3.4%), b-pinene (2.7%), bornyl acetate (1.5%), limonene (1.4%) Trans-cinnamaldehyde (85%)

Salvia sclarea L.

Salvia officinalis L. Sample 1

Lamiaceae

Linalool (41.4%), lavandulol (10.2%), p-cymene (8.94%)

Thymus vulgaris L.

Family

Major detected compounds (  1%)

Plant (part, if available)

Table 5.1 (continued)

110 M. Zabka and R. Pavela

Trans-cinnamaldehyde (85.06%), o-methoxy-cinnamaldehyde (8.79%), cis-cinnamaldehyde (1.33%), phenylethyl alcohol (1.14%), benzaldehyde (1.04%) Cinnamyl aldehyde (45.13%), eugenol (7.47%), methyleugenol (5.23%), cinnamyl alcohol (5.13%), ethyl-cinnamate (3.86%), dihydro-eugenol (3.31%), geranial (1.79%), limonene (1.48%), nerol (1.06%), 1,8-cineol (1.01%) Citronnellal (66%), citronnellol (12%), citronnellyl acetate (4%), isopulegol (3%)

Cinnamomum cassia Blume

Myrtaceae

a-pinene (17.26%), a-terpineol (13.88%), b-pinene (11.28%)

Eugenol (83%)

Syzygium cumini (L.) Skeels.

Syzygium aromaticum (L.) Merrill & Perry

Myrtaceae

Myrtaceae

1,8-cineole (71.77%), a-pinene (11.47%), terpinen-4-ol (3.18%)

Melaleuca quinquenervia (Cav.) S. T. Blake Callistemon viminalis (Sol. ex Gaertn.) G. Don

Myrtaceae

Myrtaceae

Lauraceae

Lauraceae

Family

1,8-cineole (51%), a-terpineol (11%), viridiflorol (10%) spathulenol (3%)

Eucalyptus citriodora Hook

Cinnamomum zeylanicum Blume

Major detected compounds (  1%)

Plant (part, if available)

Table 5.1 (continued)

Fusarium solani (Ooi et al. 2006)

Fusarium oxysporum (El-Baroty et al. 2010)

Fusarium verticillioides (Fandohan et al. 2004) Fusarium verticillioides (Fandohan et al. 2004) Fusarium oxysporum Fusarium solani (Badawy and Abdelgaleil 2014) Fusarium oxysporum Fusarium solani (Badawy and Abdelgaleil 2014) Fusarium graminearum (Cardiet et al. 2012) (continued)

MIC 150 lg/ml

MIC 100 lg/ml

MIC 2.7 ll/ml

MIC 730 ll/l

MIC50 724 mg/l MIC50 970 mg/l

MIC50 932 mg/l MIC50 > 1000 mg/ l

MIC 4.0 ll/ml

Species (strain, if available)/references

MIC or MIC50

5 Review Chapter: Fusarium Genus and Essential Oils 111

Piperaceae

Eugenol (64.29%), methyleugenol (20.55%),b-carylophyllene (5.53%), myrcene (2.41%), 1.8-cineole (1.57%), alpha-humulene (1.45%)

a-humulene (16.4%), caryophyllene oxide (12.2%), viridiflorol (8.1%), globulol (7.4%), b-selinene (7.1%), spathulenol (6.2%), (e)-nerolidol (5.1%), linalool (4.5%), 3-pentanol (3.5%) Myrcene (28%), neral (citral b) (20%), geranial (citral a) (27%), geraniol (4%)

Pimenta dioica (L.) Merr.

Piper chaba Trel. & Yunck.

Citrus aurantifolia Swingle

Clausena anisata (Willd.) Hook. f. ex Benth. Ruta montana L.

Cymbopogon citratus (DC. ex Nees)

Myrtaceae

Eugenol (60.6%), (e) caryophyllene (26.9%), eugenol acetate (9.33%)

Syzygium aromaticum (L.) Merrill & Perry

Rutaceae

Rutaceae

Rutaceae

Estragol (93%)

2-butene (22.56%), 1-butene (38.33%), methylcyclopropane (15.47%), caryophyllene oxide (8.18%)

Limonene (40.19%), b-pinene (19.65%), a-citral (8.14%)

Poaceae

Myrtaceae

Myrtaceae

Eugenol (68.7%)

Syzygium aromaticum (L.) Merrill & Perry

Family

Major detected compounds (  1%)

Plant (part, if available)

Table 5.1 (continued)

MIC50 402 mg/l MIC50 852 mg/l

MIC 160 lg/ml MIC 140 lg/ml

MIC 1.3 ll/ml

MIC 1.3 ll/ml

MIC 250 lg/ml MIC 500 lg/ml

MIC 0.5 ll/ml MIC 0.6 ll/ml

Fusarium verticillioides (Fandohan et al. 2004) Fusarium verticillioides (Fandohan et al. 2004) Fusarium oxysporum Fusarium solani (Hammami et al. 2015) Fusarium oxysporum Fusarium solani (Badawy and Abdelgaleil 2014) (continued)

F. oxysporum F.verticillioides (Rana et al. 2011) Fusarium spp. (Christian and Goggi, 2008) Fusarium oxysporum Fusarium verticillioides (Zabka et al. 2009a) F. oxysporum F. solani (Rahman et al. 2011)

MIC 10 ll/ml MIC 12 ll/ml MIC 800 ll/l

Species (strain, if available)/references

MIC or MIC50

112 M. Zabka and R. Pavela

Limonene (89.23%), linalool (2.98%)

Linalool (43.2–65.97%), linalyl acetate (0.77–24.77%), a-terpineol (9.29–12.12%) (season’s variation impact on composition) Limonene (46.7%), geranial (19.0%), neral (14.5%), geranyl acetate (3.9%), geraniol (3.5%), a-caryophyllene (2.6%), nerol (2.3%), citronellal (1.3%), neryl acetate (1.1%) b-caryophyllene (32%), a-humulene (11%), c-cadinene (7%), 1,8-cineole (6%), spathulenol (6%), c-epi-eudesmol (5%), b-eudesmol (4%), a-phellandrene (2%) Myrcenone (36.5%), a-thujone (13.1%), lippifoli-1(6)ene-5-one (8.9%), limonene (6.9%), others (34.6%)

Citrus sinensis (L.) Osbeck

Citrus aurantium L. (Season’s variation)

Aloysia polystachya (Griseb.) Moldenke

Aloysia triphylla (L’Hér.) Britton

Lantana camara L.

Carvone (38.2%), a-thujone (30.3%), limonene (14.3%), others (17.2%)

Limonene (74.29%), linalool (4.61%), linalool oxide (4.18%)

Citrus paradisi Macfad.

Citrus reticulata Blanco

Rutaceae

Limonene (56.30%), b-pinene (8.81%), c-terpinene (6.42%)

Citrus limon (L.) Burm.f.

Verbenaceae

Verbenaceae

Verbenaceae

Rutaceae

Rutaceae

Rutaceae

Rutaceae

Family

Major detected compounds (  1%)

Plant (part, if available)

Table 5.1 (continued)

Fusarium verticillioides (Fandohan et al. 2004) Fusarium verticillioides (López et al. 2004) Fusarium verticillioides (López et al. 2004) (continued)

MIC 1.3 ll/ml

MIC 1000 ll/l

MIC 1500 ll/l

Fusarium oxysporum (Chutia et al. 2009)

Fusarium oxysporum Fusarium solani (Badawy and Abdelgaleil 2014) Fusarium oxysporum Fusarium solani (Badawy and Abdelgaleil 2014) Fusarium oxysporum Fusarium solani (Badawy and Abdelgaleil 2014) Fusarium culmorum (Ellouze et al. 2012)

Species (strain, if available)/references

MIC 0.2 ml/ 100 ml

MIC 6.9-9.6 mg/ ml

MIC50 382 mg/l MIC50 > 1000 mg/ l

MIC50 405 mg/l MIC50 983 mg/l

MIC50 366 mg/l MIC50 452 mg/l

MIC or MIC50

5 Review Chapter: Fusarium Genus and Essential Oils 113

Zingiber officinale Roscoe

Zingiberacae

Zingiberacae

Zingiberacae

a-zingiberene (23.85%), geranial (14.16%), (e,e)-a-farnesene (9.98%), camphene (8.43%), b-phellandrene (7.73%), neral (7.47%), b-sesquiphellandrene (7.04%), ar-curcumene (6.09%), 1,8-cineole (5.62%), a-phelladrene (1.43%), a-terpineol (1.10%) Zingiberene (40.7%), geranial (8.9%), elemol (5.9%), neral (4.5%), camphor (4.3%), limonene (3.7%), (e, e)-a-farnesene (3.6%), a-terpineol (3.0%), germacrene-D-4-ol (2.7%), eugenyl acetate (2.6%), eugenol (2.4%), geranyl acetate (2.2%), spathulenol (2.1%), a-cadinol (2.1%), (2e, 6e)a-farnesol (2.0%), (e)-b-ocimene (1.3%), a-phellandrene (1.2%), p-cymene (1%), sabinene (1%) b-sesquiphellandrene (25.16%), cis-caryophyllene (15.29%), zingiberene (13.97%), a-farnesene (10.52%), a-bisabolene (7.84%), ar-curcumene (6.62%), limonene (5.08%), b-bisabolene (3.34%)

Zingiber officinale Roscoe

Zingiber officinale Roscoe

Family

Major detected compounds (  1%)

Plant (part, if available)

Table 5.1 (continued)

Fusarium poae Fusarium verticillioides (Philippe et al. 2012)

Fusarium oxysporum (El-Baroty et al. 2010)

MIC 75 lg/ml

Fusarium verticillioides (Yamamoto-Ribeiro et al. 2013)

Species (strain, if available)/references

MIC > 1000 mg/L MIC > 1000 mg/L

MIC 2500 lg/mL

MIC or MIC50

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Moreover, within each group of EO chemical components, it is possible to discover similar compounds which exert a different level of antifungal activity. These differences are sometimes caused by slightly different molecular structures. The phenolic constituents of some EOs are considered to be the most potent antifungal compounds. But generally, there are also quite significant dissimilarities, which are frequently elucidated with the different structures of their molecules. According to a comparison of antifungal activity on the basis of the MIC values of individual phenolic compounds, thymol and its structural isomer, carvacrol, had the lowest estimated inhibitory values. Given the differences in molecular structures, these were the only compounds among those tested for which the isopropyl group is found on R2 and R5, and the methyl functional group on R5 and R2, respectively (Fig. 5.1). The presence of the hydroxyl group, along with a system of delocalised electrons, plays a very important role in the cell membrane activity of phenolic compounds (Ultee et al. 2002). Thymol and carvacrol are positional isomers, with their phenolic hydroxyl group at a different location. Their molecular structure and the relative position of the functional groups is responsible for their powerful ability to dissolve and accumulate in the cell membrane, resulting in cell membrane destabilisation. This can be attributed to more efficient proton transfer disruption (Rao et al. 2010; Ahmad et al. 2011). Eugenol and isoeugenol are less efficient than thymol and carvacrol. In the case of eugenol in particular, this could be explained by a shift in hydrophobicity arising from the different molecular structure and, above all, by the presence of the methoxy group, which results in a lower ability to release a proton from the hydroxyl group (Ben Arfa et al. 2006). On the other hand, essential oils with a high content of eugenol also provided an excellent antifungal effect (Zabka et al. 2009b). Other phenols exerted significantly lower efficacy and/ or there were larger differences, depending on the target fungal species. Other less effective compounds such as 2,6-dimethoxyphenol, vanillin and a majority of the

OH

H 3C

CH3

CH3

H 2C

H 2C CH3

CH3

O H 3C

HO

O

CH3 Thymol

Carvacrol H3C

OH

O

CH3 Eugenol H 3C

CH3

Methyleugenol

OH

CH3

CH3 OH

CH3

CH2 O

Cinnamaldehyde

H

CH3

Limonene

H 3C

CH3

Linalool

Fig. 5.1 Main components of EOs affecting antifungal activity

OH H 3C

CH3

Citronellol

H 3C

CH3

Geraniol

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phenolic acids showed little or no inhibitory effect, with efficacy only against some of the target fungal species. Thus, the effect of interspecies sensitivity to these compounds was more obvious there. The antifungal efficiency displayed by means of the MIC value varied in the case of phenolic compounds, from 76 lg/ml to values more than ten times higher (Zabka and Pavela 2013).

5.7

Conclusion

Besides antifungal activity against Fusarium and other fungal species (Zabka et al. 2014), dozens of plant EOs and extracts have tremendous biological activity against pathogenic bacteria, viruses, insects and oxidative deterioration (Bakkali et al. 2008; Prakash et al. 2010, 2015). These biological activities of EOs direct the attention of the scientific community towards the development of eco-friendly botanical pesticides. Being natural in origin, EOs and their components are considered environmentally favourable and user-friendly. Given the speed at which fungal resistance is currently developing, the supply of new, more effective synthetic fungicides is becoming insufficient. This results in excessive use and increased dosing of fungicides, leading to harmful loading of the natural ecosystem with non-biodegradable residues. Paradoxically, these omnipresent sublethal fungicide concentrations may be one of the causes of the accelerated occurrence of resistant forms; moreover, they may have a harmful effect on human health (Zarn et al. 2003; Jiang et al. 2005; Cus et al. 2010; Mullin et al. 2010). In the light of these problems, there is a growing need to research and develop new, environmentally safe substances which will undergo quick and natural degradation in the environment. Alternative methods of suppressing pathogenic and toxinogenic fungi, based on the use of natural plant substances, often result in research on the development of highly effective essential oils. Plant essential oils could be suggested as alternative sources for antifungal treatment (Singh et al. 2009; Kumar et al. 2010; Zabka et al. 2009b; Zabka et al. 2014). Acknowledgements This review was supported by the Ministry of Agriculture of the Czech Republic (project QJ1510160).

References Agrios GN (1988) Plant pathology, 3rd edn. Academic Press Inc, New York, p 803 Ahmad A, Khan A, Akhtar F, Yousuf S, Xess I, Khan LA, Manzoor N (2011) Fungicidal activity of thymol and carvacrol by disrupting ergosterol biosynthesis and membrane integrity against Candida. Eur J Clin Microbiol Infect Dis 30(1):41–50 Badawy ME, Abdelgaleil SA (2014) Composition and antimicrobial activity of essential oils isolated from Egyptian plants against plant pathogenic bacteria and fungi. Ind Crops Prod 52:776–782

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Bajpai VK, Rahman A, Kang SC (2007) Chemical composition and anti-fungal properties of the essential oil and crude extracts of Metasequoia glyptostroboides Miki ex Hu. Ind Crops Prod 26(1):28–35 Bajpai VK, Shukla S, Kang SC (2008) Chemical composition and antifungal activity of essential oil and various extract of Silene armeria L. Bioresour Technol 99(18):8903–8908 Bajpai VK, Lee TJ, Kang SC (2009) Chemical composition and in vitro control of agricultural plant pathogens by the essential oil and various extracts of Nandina domestica Thunb. J Sci Food Agric 89(1):109–116 Bakkali F, Averbeck S, Averbeck D, Idaomar M (2008) Biological effects of essential oils—a review. Food Chem Toxicol 46:446–475 Banthorpe DV (1991) Classification of terpenoids and general procedures for their characterization. In: Charlwood BV, Banthorpe DV (eds) Methods in plant biochemistry, vol 7. Terpenoids. Academic, London, pp 1–41 Bassole IH, Juliany R (2012) Essential oils in combination and her antimicrobial properties. Molecules 17:3989–4006 Ben Arfa A, Combes S, Preziosi-Belloy L, Gontard N, Chalier P (2006) Antimicrobial activity of carvacrol related to its chemical structure. Lett Appl Microbiol 43(2):149–154 Booth C (1984) The Fusarium problem: historical, economic and taxonomic aspects. In: Moss MO, Smith JE (eds) The applied mycology of Fusarium, pp 1–13 Burt S (2004) Essential oils: their antibacterial properties and potential applications in foods - a review. Int J Food Microbiol 94:223–253 Cardiet G, Fuzeau B, Barreau C, Fleurat-Lessard F (2012) Contact and fumigant toxicity of some essential oil constituents against a grain insect pest Sitophilus oryzae and two fungi, Aspergillus westerdijkiae and Fusarium graminearum. J Pest Sci 85(3):351–358 Christian EJ, Goggi AS (2008) Aromatic plant oils as fungicide for organic corn production. Crop Sci 48(5):1941–1951 Chutia M, Bhuyan PD, Pathak MG, Sarma TC, Boruah P (2009) Antifungal activity and chemical composition of Citrus reticulata Blanco essential oil against phytopathogens from North East India. LWT-Food Sci Technol 42(3):777–780 Costa LG, Giordano G, Guizzetti M, Vitalone A (2008) Neurotoxicity of pesticides: a brief review. Front Biosci 13:1240–1249 Crocker GB (1988) The “yellow rain” issue: evidence of chemical and toxin weapons use in Laos, Cambodia, and Afghanistan. Comments Toxicol 2(1):5–16 Cus F, Cesnik HB, Bolta SV, Gregorcic A (2010) Pesticide residues in grapes and during vinification process. Food Control 21(11):1512–1518 da Silva Bomfim N, Nakassugi LP, Oliveira JFP, Kohiyama CY, Mossini SAG, Grespan R et al (2015) Antifungal activity and inhibition of fumonisin production by Rosmarinus officinalis L. essential oil in Fusarium verticillioides (Sacc.) Nirenberg. Food Chem 166:330–336 Desjardins AE (2006) Fusarium mycotoxins, chemistry, genetics, and biology. The American Phytopathological Society, St. Paul. MN, USA, p 268 Desjardins AE, Plattner RD, Gordon TR (2000) Gibberella fujikuroi mating population A and Fusarium subglutinans from teosinte species and maize from Mexico and Central America. Mycol Res 104:865–872 Dubey NK (2011) Natural products in pest management. CAB International, London Džamić A, Soković M, Ristić M, Grujić-Jovanović S, Vukojević J, Marin PD (2008) Chemical composition and antifungal activity of Salvia sclarea (Lamiaceae) essential oil. Arch Biol Sci 60(2):233–237 El-Baroty GS, El-Baky HA, Farag RS, Saleh MA (2010) Characterization of antioxidant and antimicrobial compounds of cinnamon and ginger essential oils. Afr J Biochem Res 4(6):167– 174 El-Haci IA, Bekhechi C, Atik-Bekkara F, Mazari W, Gherib M, Bighelli A et al (2014) Antimicrobial activity of Ammodaucus leucotrichus fruit oil from Algerian Sahara. Nat Prod Commun 9(5):711–712

118

M. Zabka and R. Pavela

Ellouze I, Abderrabba M, Sabaou N, Mathieu F, Lebrihi A, Bouajila J (2012) Season’s variation impact on Citrus aurantium leaves essential oil: chemical composition and biological activities. J Food Sci 77(9):T173–T180 European Commission Regulation No 1881/2006 Off J Eur Union L364:5–24 Fahn A (2000) Structure and function of secretory cells. Adv Bot Res 31:37–75 Fandohan P, Gbenou JD, Gnonlonfin B, Hell K, Marasas WF, Wingfield MJ (2004) Effect of essential oils on the growth of Fusarium verticillioides and fumonisin contamination in corn. J Agric Food Chem 52(22):6824–6829 Farzaneh M, Ahmadzadeh M, Hadian J, Tehrani AS (2005) Chemical composition and antifungal activity of the essential oils of three species of Artemisia on some soil-borne phytopathogens. Commun Agric Appl Biol Sci 71(3 Pt B):1327–1333 Frisvad JC, Samson RA (1991) Filamentous fungi in foods and feeds: ecology, spoilage, and mycotoxin production. In: Arora DK, Mukerjii KG, Marth EH (eds) Foods and feeds. Handbook of applied mycology, vol 3. Marcel Dekker, New York, U.S.A, pp 31–68 Gherib M, Atik Bekkara F, Bekhechi C, Bighelli A, Casanova J, Tomi F (2014) Composition and antimicrobial activity of the essential oil from Algerian Warionia saharae Benth. & Hook. J Essent Oil Res 26(5):385–391 Gordon TR, Martyn RD (1997) The evolutionary biology of Fusarium oxysporum. Annu Rev Phytopathol 35:111–128 Goswami RS, Kistler HC (2004) Heading for disaster: Fusarium graminearum on cereal crops. Mol Plant Pathol 5(6):515–525 Grayson DH (2000) Monoterpenoids. Nat Prod Rep 17:385–419 Gubbins PO, Heldenbrand S (2010) Clinically relevant drug interactions of current antifungal agents. Mycoses 53(2):95–113 Hammami I, Smaoui S, Hsouna AB, Hamdi N, Triki MA (2015) Ruta montana L. leaf essential oil and extracts: characterization of bioactive compounds and suppression of crown gall disease. EXCLI J 14:83 Hossain MA, Ismail Z, Rahman A, Kang SC (2008) Chemical composition and anti-fungal properties of the essential oils and crude extracts of Orthosiphon stamineus Benth. Ind Crops Prod 27(3):328–334 Hussain AI, Anwar F, Shahid M, Ashraf M, Przybylski R (2010) Chemical composition, and antioxidant and antimicrobial activities of essential oil of spearmint (Mentha spicata L.) from Pakistan. J Essent Oil Res 22(1):78–84 Jiang QT, Lee TKM, Chen K, Wong HL, Zheng JS, Giesy JP, Lo KKW, Yamashita N, Lam PKS (2005) Human health risk assessment of organochlorines associated with fish consumption in a coastal city in China. Environ Pollut 136(1):155–165 Kadri SS, Remy KE, Strich JR, Gea-Banacloche J, Leitman SF (2015) Role of granulocyte transfusions in invasive fusariosis: systematic review and single-center experience. Transfusion 55(9):2076–2085 Kalemba D, Kunicka A (2003) Antibacterial and antifungal properties of essential oils. Curr Med Chem 10(10):813–829 Kulik T (2008) Detection of Fusarium tricinctum from cereal grain using PCR essay. J Appl Genet 49(3):305–311 Kumar A, Shukla R, Singh P, Anuradha DNK (2010) Efficacy of extract and essential oil of Lantana indica Roxb. against food contaminating moulds and aflatoxin B-1 production. Int J Food Sci Technol 45 (1):179–185 López AG, Theumer MG, Zygadlo JA, Rubinstein HR (2004) Aromatic plants essential oils activity on Fusarium verticillioides Fumonisin B1 production in corn grain. Mycopathologia 158(3):343–349 Mirocha CJ, Pathre S (1973) Identification of the toxic principle in a sample of poaefusarin. Appl Microbiol 26:719–724 Mullin CA, Frazier M, Frazier JL, Ashcraft S, Simonds R, vanEngelsdorp D, Pettis JS (2010) High levels of miticides and agrochemicals in North Američan apiaries: implications for honey bee health. PLoS ONE 5(3):e9754

5 Review Chapter: Fusarium Genus and Essential Oils

119

Nakanishi T (2007) Potential toxicity of organotin compounds via nuclear receptor signaling in mammals. J Health Sci 53(1):1–9 Nucci M, Anaissie E (2007) Fusarium infections in immunocompromised patients. Clin Microbiol Rev 20(4):695–704 Ooi LS, Li Y, Kam SL, Wang H, Wong EY, Ooi VE (2006) Antimicrobial activities of cinnamon oil and cinnamaldehyde from the Chinese medicinal herb Cinnamomum cassia Blume. Am J Chin Med 34(03):511–522 Philippe S, Souaïbou F, Jean-Pierre N, Brice F, Paulin A, Issaka Y, Dominique S (2012) Chemical composition and in vitro antifungal activity of Zingiber officinale essential oil against foodborne pathogens isolated from a traditional cheese wagashi produced in Benin. Int J Biosci (Ijb) 2(9):20–28 Pinto E, Salgueiro LR, Cavaleiro C, Palmeira A, Gonçalves MJ (2007) In vitro susceptibility of some species of yeasts and filamentous fungi to essential oils of Salvia officinalis. Ind Crops Prod 26(2):135–141 Prakash B, Shukla R, Singh P, Kumar A, Mishra PK, Dubey NK (2010) Efficacy of chemically characterized Piper betle L. essential oil against fungal and aflatoxin contamination of some edible commodities and its antioxidant activity. Int J Food Microbiol 142:114–119 Prakash B, Kedia A, Mishra PK, Dubey NK (2015) Plant essential oils as food preservatives to control moulds, mykotoxin contamination and oxidative deterioration of agri-food commodities—potentials and challenges. Food Control 47:381–391 Pujol I, Aguilar C, Fernandez-Ballart J et al (2000) Comparison of the minimum fungicidal concentration of amphotericin B determined in filamentous fungi by macrodilution and microdilution methods. Med Mycol 38(1):23–26 Rahman A, Hossain MA, Kang SC (2010) Control of phytopathogenic fungi by the essential oil and methanolic extracts of Erigeron ramosus (Walt.) BSP. Eur J Plant Pathol 128(2):211–219 Rahman A, Al-Reza SM, Kang SC (2011) Antifungal activity of essential oil and extracts of Piper chaba Hunter against phytopathogenic fungi. J Am Oil Chem Soc 88(4):573–579 Rana IS, Rana AS, Rajak RC (2011) Evaluation of antifungal activity in essential oil of the Syzygium aromaticum (L.) by extraction, purification and analysis of its main component eugenol. Braz J Microbiol 42(4):1269–1277 Randrianarivelo R, Sarter S, Odoux E, Brat P, Lebrun M, Romestand B et al (2009) Composition and antimicrobial activity of essential oils of Cinnamosma fragrans. Food Chem 114 (2):680–684 Rao A, Zhang YQ, Muend S, Rao R (2010) Mechanism of antifungal activity of terpenoid phenols resembles calcium stress and inhibition of the TOR pathway. Antimicrob Agents Chemother 54(12):5062–5069 Regnault-Roger C, Vincent C, Arnason JT (2012) Essential oils in insect control: low-risk products in a high-stakes world. Annu Rev Entomol 57:405–424 Reverchon E (1997) Supercritical fluid extraction and fractionation of essential oils and related products. J Supercrit Fluids 10:1–37 Ristic M, Sokovic M, Grubisic D et al (2004) Chemical analysis and antifungal activity of the essential oil of Achillea atrata L. J Essent Oil Res 16(1):75–78 Sampietro DA, Belizana MM, Baptista ZP, Vattuone MA, Catalán CA (2014) Essential oils from Schinus species of northwest Argentina: Composition and antifungal activity. Nat Prod Commun 9(7):1019–1022 Scordino M, Sabatino L, Traulo P, Gagliano G, Gargano M, Panto V, Gambino GL (2008) LC/ MS/MS detection of fungicide guazatine residues for duality assessment of commercial citrus fruit. Eur Food Res Technol 227(5):1339–1347 Shelef LA (1983) Antimicrobial effects of spices. J Food Saf 6:29–44 Singh P, Kumar A, Dubey NK, Gupta R (2009) Essential oil of aegle marmelos as a safe plant-based antimicrobial against postharvest microbial infestations and aflatoxin contamination of food commodities. J Food Sci 74(6):M302–M307 Smith IM, Dunez J, Phillips DH, Lelliott RA, Archer SA (eds) (1988) European handbook of plant diseases. Blackwell Scientific Publications, Oxford, p 598

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Stenglein SA (2009) Fusarium poae: a pathogen that needs more attention. J Plant Pathol 91 (1):25–36 Summerell BA, Laurence MH, Liew ECY et al (2010) Biogeography and phylogeography of Fusarium: a review. Fungal Divers 44(1):3–13 Turner NW, Subrahmanyam S, Piletsky SA (2009) Analytical methods for determination of mycotoxins: a review. Anal Chim Acta 632(2):168–180 Tyagi AK, Malik A (2011) Antimicrobial potential and chemical composition of Mentha piperita oil in liquid and vapour phase against food spoiling microorganisms. Food Control 22 (11):1707–1714 Ultee A, Bennik MHJ, Moezelaar R (2002) The phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen Bacillus cereus. Appl Environ Microbiol 68 (4):1561–1568 Villa-Ruano N, Pacheco-Hernández Y, Rubio-Rosas E, Lozoya-Gloria E, Mosso-González C, Ramón-Canul LG, Cruz-Durán R (2015a) Essential oil composition and biological/ pharmacological properties of Salmea scandens (L.) DC. Food Control 57:177–184 Villa-Ruano N, Pacheco-Hernández Y, Cruz-Durán R, Lozoya-Gloria E (2015b) Volatiles and seasonal variation of the essential oil composition from the leaves of Clinopodium macrostemum var. laevigatum and its biological activities. Ind Crops Prod 77:741–747 Voon CH, Bhat R, Rusul G (2012) Flower extracts and their essential oils as potential antimicrobial agents for food uses and pharmaceutical applications. Compr Rev Food Sci Food Saf 11:34–55 Watanabe M, Yonezawa T, Lee K, Kumagai S, Sugita-Konishi Y, Goto K, Hara-Kudo Y (2011) Molecular phylogeny of the higher and lower taxonomy of the Fusarium genus and differences in the evolutionary histories of multiple genes. BMC Evol Biol 11:322 White DG (1999) Compendium of corn diseases, 3rd edn. APS Press, p 78 Wickern GM (1993) Fusarium allergic fungal sinusitis. J Allergy Clin Immunol 92:624 Xing F, Hua H, Selvaraj JN, Zhao Y, Zhou L, Liu X, Liu Y (2014) Growth inhibition and morphological alterations of Fusarium verticillioides by cinnamon oil and cinnamaldehyde. Food Control 46:343–350 Yamamoto-Ribeiro MMG, Grespan R, Kohiyama CY, Ferreira FD, Mossini SAG, Silva E et al (2013) Effect of Zingiber officinale essential oil on Fusarium verticillioides and fumonisin production. Food Chem 141(3):3147–3152 Yli-Mattila T (2011) Detection of trichothecene-producing Fusarium species in cereals in Northern Europe and Asia. Agron Res 9:521–526 Yu CL, Kuang Y, Yang SX, Liu L, Liu CG (2014) Chemical composition, antifungal activity and toxicity of essential oils from leaves of Chimonanthus praecox and Chimonanthus zhejiangensis. Asian J Chem 26(1):254–256 Zabka M, Pavela R (2013) Antifungal efficacy of some natural phenolic compounds against significant pathogenic and toxinogenic filamentous fungi. Chemosphere 93(6):1051–1056 Zabka M, Pavela R, Slezakova L (2009a) Antifungal effect of Pimenta dioica essential oil against dangerous pathogenic and toxinogenic fungi. Ind Crops Prod 30(2):250–253 Zabka M, Pavela R, Slezakova L (2009b) Antifungal effect of Pimenta dioica essential oil against dangerous pathogenic and toxinogenic fungi. Ind Crop Prod 30(2):250–253 Zabka M, Pavela R, Prokinova E (2014) Antifungal activity and chemical composition of twenty essential oils against significant indoor and outdoor toxigenic and aeroallergenic fungi. Chemosphere 112:443–448 Zarn JA, Bruschweiler BJ, Schlatter JR (2003) Azole fungicides affect mammalian steroidogenesis by inhibiting sterol 14 alpha-demethylase and aromatase. Environ Health Perspect 111 (3):255–261 Zhang N, O’Donnell K, Sutton DA et al (2006) Members of the Fusarium solani species complex that cause infections in both humans and plantsare common in the environment. J Clin Microbiol 44(6):2186–2190

Chapter 6

Natural Agents Inducing Plant Resistance Against Pests and Diseases Ali Siah, Maryline Magnin-Robert, Béatrice Randoux, Caroline Choma, Céline Rivière, Patrice Halama and Philippe Reignault Abstract Plant resistance inducers, also referred to as elicitors, are agents that confer improved protection to pathogen or pest attacks by inducing host defense mechanisms. Such products are effective against a wide range of crop enemies, including viruses, bacteria, fungi, oomycetes, nematodes, and herbivores. The mode of action of these products differs from that of traditional pesticides because they do not target directly the bio-aggressor through antifungal activity, but they inhibit its development indirectly via the elicitation of plant defense reactions. In the current context of sustainable agriculture and growing demand for healthy food, plant resistance inducers are considered as an eco-friendly and promising alternative to conventional pesticides, and their implementation in integrated pest management strategy is strongly encouraged. Plant resistance inducers can be of synthetic or natural origin. This chapter will focus on resistance inducers of natural origin including living microorganisms, plant extracts, microbial cell-wall extracts, microbial metabolites, minerals, and ions. An overview on the market and recent advances on the regulation of these products as well as future challenges to promote their development and wide use in disease management programs will be described.










Keywords Resistance inducer Elicitor Plant protection Sustainable agriculture

A. Siah (&)
C. Choma
P. Halama Charles Viollette Research Institute (EA 7394), ISA Lille, 59000 Lille, France e-mail: [email protected] M. Magnin-Robert
B. Randoux
P. Reignault Unité de Chimie Environnementale et Interactions sur le Vivant (EA 4492), University of the Littoral Opal Coast, CS 80699, 62228 Calais, France C. Rivière Charles Viollette Research Institute (EA 7394), ISA Lille, University of Lille, 59000 Lille, France © Springer International Publishing AG 2018 J.-M. Mérillon and C. Rivière (eds.), Natural Antimicrobial Agents, Sustainable Development and Biodiversity 19, https://doi.org/10.1007/978-3-319-67045-4_6

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Introduction

Plants possess an array of intrinsic or natural defenses to protect themselves against pathogen attacks. When plants and pathogens interact, various molecules are released, that are of pathogen or plant origins. The recognition of some of these molecules by challenged plant cells is the prerequisite of the expression of plant immunity, a concept that emerged during the late 70s, and has become the theoretical basis for emerging disease control strategies. Such strategies aim at activating plant defenses by the use of agents or derived molecules mimicking natural attacks, products that are used today as an alternative to conventional chemical pesticides.

6.1.1

Historic of Induced Resistance

The description of plant defense mechanisms is linked to the first corresponding publications in the 70s which reported a discovery of microbial molecules able to induce in the plant the production of phytoalexins, secondary metabolites with antimicrobial activity. Such molecules are referred since then to as elicitors (Keen et al. 1972), and their subsequent use in practice by the early 80s led to the emergence of the concept of resistance inducers (Kuc 1982). Some of the earliest characterized elicitors were glucan sugars extracted from the cell wall of yeasts and oomycetes (Hahn and Albersheim 1978; Keen et al. 1983). These polysaccharides were early examples of molecules known as structural components of the cell wall having the ability to induce defense-related compounds in the plant. Further investigations allowed to highlight the great biochemical diversity and the wide distribution in different botanical families of phytoalexins (Kuc 1995). Plant immunity involves at the biochemical level the elicitation of many other defense mechanisms, including reactive oxygen species (ROS) metabolism, pathogenesisrelated (PR)-protein synthesis, and phenylpropanoid and octadecanoid pathways (Garcion et al. 2014). Plant immunity can occur at two distinct scales, either locally (local acquired resistance, LAR) or systemic (systemic acquired resistance, SAR). LAR is expressed by cells, tissues, or even organs that have been treated by an elicitor, whereas SAR occurs in parts of the plants spatially distant from the treated tissues or organs. SAR has been described for the first time at the end of the twentieth century (Görlach et al. 1996). LAR (such as HR; see below) is often a prerequisite to the expression of SAR, and SAR has been characterized as broader and more long-lasting than LAR. Other characteristic features of SAR are that it depends on the perception of salicylic acid (SA) and results on the expression of PR-protein encoding genes (Walters and Fountaine 2009). Beside SAR, the systemic expression of induced resistance can also consist of induced systemic resistance (ISR), triggered at the root level by certain beneficial microorganisms occurring in the

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rhizosphere (such as PGPR, PGPF, and AMF; see below). Although ISR also leads to a broad and long-lasting induced resistance, it relies on jasmonic acid (JA) perception and does not result in PR-protein biosynthesis (Pieterse et al. 1998). It turned out more recently that elicitor treatments do not always result directly in the expression of plant defense mechanisms, and that challenge inoculation may be necessary to occur before plant immunity is thereafter expressed. This phenomenon is referred to as “priming” and mainly consists of a potentiation of defenses after treatments and their actual expression after infection. Some resistance inducers such as BABA, an amino acid rarely found in nature (see below), have been shown to specifically exhibit such a mode of action, which in theory saves the plant from mobilizing its defense metabolism out of the infectious purpose (Conrath et al. 2002).

6.1.2

Categories of Elicitors

All plants, whether they are resistant or susceptible, respond to pathogen attack by the induction of a coordinated defense strategy. The innate ability of plants to detect pathogens is essential for their immunity (Altenbach and Robatzek 2007). Two distinct components of plant immunity have been established in the beginning of the twenty-first century (Jones and Dangl 2006). First, the perception of pathogen via elicitors (either endogenous, e.g., plant cell-wall fragments released consequently to the activity of cell-wall depolymerizing enzymes, molecules from flagella, pathogenicity determinants or effectors, or exogenous, e.g., microbial cell-wall fragments released by the cell-wall depolymerizing enzymes) is recognized by pattern recognition receptor (PRR) in the plant cytoplasmic membrane (Nürnberger et al. 2004). Such PRR can bind either to (i) molecular patterns associated to the pathogen (pathogen-associated molecular pattern, PAMP) or a non-pathogenic microorganism (microbe-associated molecular patterns, MAMPs), corresponding to exogenous elicitors or to (ii) patterns associated to cellular damage induced in the plant (damage-associated molecular pattern, DAMP), corresponding to endogenous elicitors. The recognition of such molecular patterns by PRR results in a cascade of cellular signaling leading to the expression of defense genes encoding for antimicrobial compounds such as phytoalexins and PR-proteins as well as phenolics and sugars (callose) involved in plant cell-wall reinforcement at the pathogen infection site. This first wave of defense is considered as efficient against a vast majority of pathogens and is referred to as PAMP-triggered immunity (PTI). Most of the terms used above are derived from those used in animal innate immunity, because of numerous common features between plant and animal immunities. Most virulent pathogens overcome PTI by injecting molecules into the plant cell, in order to escape its associated defense mechanisms. Such molecules are defined as “effectors,” and among them, there are virulence products originated from avr (avirulence) genes described in the gene-for-gene relationship determining race– cultivar specificity, which can potentially inhibit the expression of PTI. In resistant plants, effectors are recognized by membrane-associated or intracellular plant

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receptors corresponding to the products of R (resistance) genes in the gene-for-gene relationship and induce (i) a rapid, intense, and local resistance called hypersensitive response (HR) and involving the accumulation of SA and then (ii) SAR. Overall, this second wave of defense is referred to as effector-triggered immunity (ETI). Breeders who introduce major R genes in new varieties therefore capitalize on ETI since several decades. Except phytohormones such as SA or JA (see below), most of elicitors are considered as PAMP or DAMP and are therefore thought as PTI inducers. PTI is considered as less efficient than ETI, but more widespread among the plant kingdom (Boller and Felix 2009). Salicylic acid is the first phytohormone that exhibited resistance inducing activity and has shown its effectiveness in various plants against viruses, bacteria, fungi, and insects. In addition, as mentioned above, SA is a key determinant of the expression of SAR. However, although it can be used as resistance inducer, its application at high concentrations can result in plant phytotoxicity. Benzothiadiazole (BTH), a synthetic functional SA analog, also referred to as Acibenzolar-S-methyl (ASM), has been the first registered resistance inducer (Bion®, Syngenta) and by far the most extensively studied inducer against biotic stresses (Görlach et al. 1996). Beside SA, JA and ethylene are two other major phytohormones involved in plant defense pathway induction, i.e., the phenylpropanoid and octadecanoid pathways for SA and JA, respectively (Denancé et al. 2013). Their application leads to a direct and general defense induction and circumvents the molecular pattern recognition step.

6.2 6.2.1

Natural Resistance Inducers Living Microorganisms

Plants can be attacked by microbial pathogens but can also cooperate with beneficial microbes occurring in the rhizosphere and the phyllosphere. These microbes live as entophytes (inside plant tissue) or epiphytes (in the vicinity or on the surface of plant tissue, mainly roots) without causing any harmful effect on host plant. Beneficial microbes are referred to collectively as plant growth-promoting rhizobacteria (PGPR), plant growth-promoting fungi (PGPF), and arbuscular mycorrhizal fungi (AMF). These microbes can confer several positive effects to the plant such as promotion of plant growth (e.g., shoot length, biomass, leaf area, chlorophyll content, nitrogen content, and yield) and/or enhancement of resistance against abiotic and biotic stresses (Pérez-Montaño et al. 2014). Microbial species reported to induce resistance against biotic attacks belong to a wide range of genera, including Bacillus, Pseudomonas, Streptomyces, Enterobacter, and Burkholderia for PGPR, Trichoderma, Penicillium, Fusarium, and Heteroconium for PGPF and Glomus, Funneliformis and Rhizophagus for AMF. Plant inoculation with such microbes can confer resistance to a broad spectrum of pathogens or pests (Table 6.1). Resistance induction by beneficial

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Table 6.1 Examples of beneficial microbes reported to induce plant disease or pest resistance (from 2014 to present) Beneficial microorganism

Host plant

Plant growth-promoting rhizobacteria (PGPR) Bacillus subtilis Tomato

Bio-aggressor

Reference

Meloidogyne incognita

Adam et al. (2014) Fousia et al. (2015) Waewthongrak et al. (2015) Chandler et al. (2015) Wang et al. (2014)

Bacillus subtilis

Tomato

Bacillus subtilis

Citrus fruit

Pseudomonas syringae pv. tomato Penicillium digitatum

Bacillus subtilis

Rice

Rhizoctonia solani

Bacillus cereus

Loquat fruit

Bacillus cereus

Arabidopsis

Bacillus amyloliquefaciens

Tomato

Bacillus amyloliquefaciens

Lettuce

Colletotrichum acutatum Pseudomonas syringae pv. tomato Ralstonia solanacearum, Oidium neolycopersici Rhizoctonia solani

Bacillus oryzicola

Rice

Bacillus sp.

Soybean

Paenibacillus polymyxa

Tomato

Paenibacillus sp.

Tomato

Paenibacillus sp.

Cabbage

Pseudomonas fluorescens

Tomato

Pseudomonas fluorescens

Tomato

Pseudomonas fluorescens

Potato

Fusarium oxysporum f. sp. lycoersici Fusarium oxysporum f. sp. radicis-lycopersici Xanthomonas campestris pv. campestris Ralstonia solanacearum Alternaria alternata f. sp. lycopersici Streptomyces scabies

Pseudomonas fluorescens

Grapevine

Botrytis cinerea

Streptomyces rochei

Apple fruit

Streptomyces sp.

Oak

Streptomyces sp.

Eucalyptus

Botryosphaeria dothidea Microsphaera alphitoides Botrytis cinerea

Xanthomonas oryzae pv. oryzae, Burkholderia glumae Cercospora sojina

Niu et al. (2016) Yamamoto et al. (2015) Chowdhury et al. (2015) Chung et al. (2015) Tonelli and Fabra (2014) Mei et al. (2014) Sato et al. (2014) Ghazalibiglar et al. (2016) Murthy et al. (2014) Kumar et al. (2015) Arseneault et al. (2014) Gruau et al. (2015) Zhang et al. (2016a) Kurth et al. (2014) Salla et al. (2016) (continued)

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Table 6.1 (continued) Beneficial microorganism

Host plant

Bio-aggressor

Reference

Burkholderia phytofirmans Brevibacterium iodinum

Grapevine Pepper

Trdá et al. (2014) Son et al. (2014)

Enterobacter asburiae

Tomato

Carnobacterium sp.

Soybean

Botrytis cinerea Stemphylium lycopersici Tomato yellow leaf curl virus Fusarium oxysporum

Micromonospora sp.

Tomato

Botrytis cinerea

Streptomyces setonii, Bacillus cereus, Serratia marcescens Streptomyces setonii, Bacillus cereus Pseudomonas azotoformans, Paenibacillus elgii Pseudomonas fluorescens, Bacillus sp. Pseudomonas fluorescens, Streptomyces sp. Plant growth-promoting fungi Trichoderma harzianum Trichoderma harzianum

Tomato

Fusarium oxysporum f. sp. lycopersici

Tomato

Xanthomonas gardneri

Cucumber

Colletotrichum orbiculare Phoma sp.

(PGPF) Sunflower Grapevine

Rhizoctonia solani Plasmopara viticola

Trichoderma harzianum Trichoderma harzianum

Tomato Cucumber

Alternaria solani Podosphaera xanthii

Trichoderma harzianum

Oilseed rape

Erysiphe cruciferarum

Trichoderma harzianum

Pistachio

Verticillium dahliae

Trichoderma harzianum

Soybean

Trichoderma harzianum

Trichoderma viride

Cucumber, strawberry, bean, tomato Potato

Sclerotinia sclerotiorum Botrytis cinerea

Trichoderma viride

Black gram

Trichoderma asperellum

Onion

Adzuki bean Potato

Ralstonia solanacearum

Ralstonia solanacearum Fusarium oxysporum, Alternaria alternata Sclerotium rolfsii

Li et al. (2016) Jain and Choudhary (2014) Martínez-Hidalgo et al. (2015) Ferraz et al. (2014) Ferraz et al. (2015) Sang et al. (2014) Gupta et al. (2014a) Rosyidah et al. (2014) Singh et al. (2014) Banani et al. (2014) Selim (2015) Okon Levy et al. (2015) Alkooranee et al. (2015) Fotoohiyan et al. (20150 Zhang et al. (2016b) Okon Levy et al. (2015) Rosyidah et al. (2014) Surekha et al. (2014) Guzman-Valle et al. (2014) (continued)

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Table 6.1 (continued) Beneficial microorganism

Host plant

Bio-aggressor

Reference

Trichoderma asperelloides

Arabidopsis

Fusarium oxysporum

Rhodotorula aurantiaca

Melon

Acidovorax citrulli

Heteroconium chaetospira

Canola

Clonostachys rosea

Canola

Penicillium oxalicum

Pearl millet

Pythium oligandrum

Grapevine

Fusarium oxysporum

Pepper

Plasmodiophora brassicae Plasmodiophora brassicae Sclerospora graminicola Phaeomoniella chlamydospora Verticillium wilt

Penicillium citrinum, Aspergillus terreus Arbuscular mycorrhizal fungi Glomus fasciculatum Glomus fasciculatum Funneliformis mosseae Funneliformis mosseae, Rhizophagus irregularis

Sunflower

Alternaria alternata

Gupta et al. (2014b) Conceição et al. (2014) Lahlali et al. (2014) Lahlali and Peng (2014) Murali and Amruthesh (2015) Yacoub et al. 2016 Veloso et al. (2016) Waqas et al. (2015)

(AMF) Tomato Tomato Tomato Wheat

Fusarium oxysporum Alternaria alternata Alternaria solani Blumeria graminis f. sp. tritici

Nair et al. (2015a) Nair et al. (2015b) Song et al. (2015) Mustafa et al. (2016)

microbes was reported to be effective in both laboratory (Mustafa et al. 2016) and field conditions (Magnin-Robert et al. 2013). However, the protection efficacy levels conferred vary depending on the beneficial microbe used and the pathosystem considered. Resistance mechanisms by which beneficial microbes activate plant immunity involve mainly ISR (Zamioudis and Pieterse 2012). The first investigations which led to the discovery of this concept were undertaken by van Peer et al. (1991) who observed a trigger of plant-mediated resistance response to Fusarium wilt in aboveground plant parts after inoculation of carnation roots with a non-pathogenic Pseudomonas spp. strain. Since then, the ISR elicitation by microbes including AMF (Nair et al. 2015a) has been demonstrated in many plant species (e.g., Table 6.1). Interestingly, some beneficial microbes including PGPR did not induce ISR via the JA/ET pathway, but they activate this resistance mechanism via the SA pathway (e.g., Barriuso et al. 2008). Moreover, it has been reported that some PGPR activate both JA/ET and SA pathways during ISR (Walters and Bennett 2014). Another type of microbe-based resistance inducers is compost. Compost is the final product obtained after the aerobic degradation of different types of organic matter waste and can be used as substrates or substrate/soil amendments for plant

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cultivation because of its richness in nutrients and beneficial microorganisms such as Trichoderma hamatum and T. harzianum (Hoitink et al. 2006). Several studies reported that when applied to soils or container media, compost reduced the severity of both soil-borne and air-borne plant pathogens. For instance, an incorporation of composted paper mill sludge into a peat-based potting mix induced the formation of physical barriers at infection sites in tomato, thus limiting colonization by Fusarium oxysporum f. sp. radicis-lycopersici (Pharand et al. 2002). Also, an amendment of the substrate with olive marc compost enhanced resistance in Arabidopsis to Botrytis cinerea via a plant response that shares similarities with both SAR and ABA-dependent/independent abiotic stress responses (Segarra et al. 2013).

6.2.2

Plant Extracts

6.2.2.1

Spermatophyte Plant Extracts

The most studied plant extract for induced resistance is probably a commercial product, which consists in an aqueous formulation of a concentrated ethanolic extract from the giant knotweed Reynoutria sachalinensis (F. Schmidt) Nakai (Polygonaceae). The first formulation of the product has been commercialized in the 90s as Milsana® by Compo GmbH (Münster, Germany), and since 1998, by KHH BioSci Inc. and Biofa AG (Münsingen, Germany). Milsana® conferred protection of various plants against powdery mildew, such as cucumber against Sphaerotheca fuliginea (Daayf et al. 1995), roses against Sphaerotheca pannosa (Pasini et al. 1997), tomato against Leveillula taurica (Konstantinidou-Doltsinis et al. 2006), and wheat against Blumeria tritici f. sp. tritici (Randoux et al. 2006). Plant extracts from R. sachalinensis induced in plants’ local resistance more than systemic resistance (Schmitt 2002). In cucumber, an accumulation of fungitoxic phenolic compounds (considered as phytoalexins) was observed in leaves treated with Milsana® and infected with S. fuliginea (Daayf et al. 1995, 1997, 2000). Moreover, the induced resistance was correlated with increased chalcone synthase and chalcone isomerase activities, an accumulation of corresponding mRNA, as well as an accumulation of flavonoid compounds (Fofana et al. 2002). In wheat leaves, Milsana® induced lipoxygenase activity, hydrogen peroxide accumulation at the B. graminis f. sp. tritici penetration sites, and formation of abnormally long appressorial germ tubes on some fungal asexual spores (Randoux et al. 2006). However, Milsana® inhibited both B. graminis f. sp. tritici and L. taurica spore germination, thus suggesting an additional fungistatic direct effect of Milsana® against some pathogens. According to Biofa AG, the main active ingredient seems to be physcion, an anthraquinone derivative, which would be mainly found in the green plant material (www.abim.ch/fileadmin/abim/…/1_lehnhof_abim_2007.pdf). Since 2009, a new formulation of a R. sachalinensis extract is commercialized as

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Regalia® by Marrone Bio Innovations (Davis, CA, USA). Baysal-Gurel and Muiller (2015) confirmed its protective effect on greenhouse-produced tomato against powdery mildew. Various extracts from neem (Azadirachta indica A. Juss., Meliaceae) were tested as resistance inducers and showed a broad spectrum of activities. Neem leaf extracts appeared to induce defenses against Drechslera graminea in barley, leading to increased phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase activities and rapid accumulation of fungitoxic phenolic compounds (Paul and Sharma 2002). Fruit extracts of neem protect untreated and later emerging leaves of barley against D. graminea, where PAL and PO activities and SA concentration were higher, hence suggesting systemic acquired resistance induction by neem fruit extracts (Bhuvaneswari and Paul 2012). Azadirachtin A, the main active component of neem seed extract, is a natural limonoid known to be effective against many insects, while showing no apparent phytotoxicity (Ley et al. 1993). However, the toxicity profile of this molecule on human health is controverting due to some toxicological studies (Raizada et al 2001; Boeke et al. 2004). On tomato plants, foliar application of azadirachtin A elicited the JA pathway and enhanced the production of secondary metabolites such as polyphenols, flavonoids, phytoalexins, and auxin, thus suggesting that azadirachtin A induces ISR in tomato (Pretali et al. 2016). Many other plant extracts were tested for their ability to control plant diseases (Stangarlin et al. 2011). These extracts often displayed direct antimicrobial activities or a dual (antifungal and eliciting) effect. For instance, it was shown that a yucca extract inhibits directly the apple scab pathogen Venturia inaequalis and induces in apple seedlings an upregulation of two genes encoding for PR1 and PR8 proteins, suggesting that this yucca extract may also act as a resistance inducer (Bengtsson et al. 2009). Tillecur® (Biofa, Münsingen, Germany), based on the flour of white mustard, is commercialized as a plant strengthening agent that stimulates the resistance of germinating cereals to common bunt. In field experiments, a high control level (up to 94% efficacy) of bunt caused by Tilletia tritici was obtained with Tillecur® after seed treatment, and a mean of 78% efficacy against barley leaf stripe was observed under controlled conditions (Koch et al. 2006). Similarly, this product significantly decreased on bean the symptoms of anthracnose caused by Colletotrichum lindemuthianum (Tinivella et al. 2009). However, its mode of action is not yet described. Essential oils from various plants received also attention. Formerly, essential oils are known for their antimicrobial properties against common food-borne pathogens (Calo et al. 2015). Recently, an essential oil from Thymus capitatus L. (Hoffmanns & Link, Lamiaceae), a wild thyme species widespread in Tunisia, was shown to decrease gray mold and Fusarium wilt incidence on tomato plant (Ben-Jabeur et al. 2015). This protective effect was associated with an elicitation of defense responses, such as an increase in peroxidase activity. Likewise, an essential oil from Gaultheria procumbens L. (Ericaceae), mainly composed of methylsalicylate

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(MeSA), induced in Arabidopsis SA accumulation, SA-mediated defense responses, and resistance against the fungal pathogen Colletotrichum higginsianum (Vergnes et al. 2014).

6.2.2.2

Algae Extracts

Many marine algae extracts are already used in agriculture or horticulture and commercialized as biostimulants (Khan et al. 2009). Fucans are polysaccharides extracted from brown algae species; they are ingredients of most seaweed-based biostimulants and fertilizers. Klarzynski et al. (2003) described the eliciting effect of sulfated oligofucans in tobacco, leading not only to a local resistance to tobacco mosaic virus (TMV), which was associated with SA and phytoalexin accumulations and several PR-protein gene expressions, but also to a SA-dependent systemic resistance with SA and PR-1 accumulation. However, data about their eliciting and protective effects are scarce when compared to other algal polysaccharides. Similarly, carrageenans extracted from red algae are widely used as additives in food industries, but some of them were shown to elicit defense reactions in plants. They conferred in tobacco a protection against a broad range of pathogens including viruses, bacteria, and fungi and induced PAL activity and phenylpropanoid compounds accumulation (Vera et al. 2012). However, this eliciting effect depends on the carrageenan type, its sulfatation level, and the pathogen lifestyle (Stadnik and de Freitas 2014; Vera et al. 2012; Sangha et al. 2010). Only highly sulfated ʎ-carrageenan induced the resistance in Arabidopsis to the necrotrophic fungal pathogen Sclerotinia sclerotiorum (Sangha et al. 2010). The protection was correlated with an increased expression of JA-related genes such as AOS, PDF1.2, and PR3. Laminarins are polysaccharides extracted from brown marine algae, such as Laminaria digitata (Hudson) Lamouroux. Iodus40 and later Iodus2 and Vacciplant® are successive commercialized formulations of laminarins by Goëmar (Saint-Malo, France). Iodus40 was shown to induce protection in wheat against powdery mildew that was attributed to an increase in hydrogen peroxide accumulation at the fungal penetration site and a decrease in haustorium formation (Renard-Merlier et al. 2007). Defense reactions elicited by laminarin in grapevine-cell cultures include calcium influx, alkalinization of the extracellular medium, an oxidative burst, activation of two mitogen-activated protein kinases, expression of defense-related genes, increases in chitinase and b-1,3-glucanase activities, and the production of phytoalexins (Aziz et al. 2003). Surprisingly, the sulfated laminarin PS3 did not elicit early signaling events, except a strong plasma membrane depolarization (Gauthier et al. 2014). In grapevine leaves, laminarin elicited, 24 h after treatment, the expression of genes encoding for PR-proteins such as b-1,3-glucanase, serine-proteinase inhibitor and chitinase, and induced partial protection against Botrytis cinerea and Plasmopara viticola (Aziz et al. 2003). A transcriptome analysis of gene expression in grapevine leaves after laminarin or PS3 treatments showed that both molecules shared a common stress-responsive transcriptome, but PS3 specifically primed the SA-dependent defense pathway

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during P. viticola infection. A higher protection against this pathogen was obtained in PS3-treated plants (Gauthier et al. 2014). In tobacco, PS3 induced immunity against TMV infection, whereas laminarin induced only a weak resistance (Ménard et al. 2004). Ulvans isolated from green algae of the genus Ulva are also used as resistance inducers. These naturally sulfated heteropolysaccharides can reduce the disease severity in a broad range of host–pathogen interactions (Stadnik and de Freitas 2014). In Arabidopsis, ulvans induced resistance against Alternaria brassicicola and increased NADPH oxidase activity and hydrogen peroxide levels (de Freitas et al. 2015). In wheat cells, the production of hydrogen peroxide and subsequent oxidative burst elicited by chitin or chitosan were strongly primed by pretreatment with ulvan, which was associated with an enhanced resistance against powdery mildew in wheat and barley plants (Paulert et al. 2010). In Medicago trunculata, a nearly complete protection against Colletotrichum trifolii, the causal agent of anthracnose, was obtained after treatment with ulvan, which is an efficient elicitor of multiple defense responses in this plant species (Cluzet et al. 2004). Moreover, in apple fruit, a significant protection was obtained with ulvans and oligoulvans against B. cinerea and Penicillium expansum (Abouraïcha et al. 2015). Oligoulvans activated catalase, superoxide dismutase, peroxidase, polyphenoloxydase, and PAL activities and increased the levels of lignin and phenolic compounds, thus highlighting the role of phenylpropanoid pathway in oligoulvan-induced resistance. In tomato seedlings, oligoulvans reduced wilt development caused by Fusarium oxysporum f. sp. lycopersici and stimulated PAL activity, increased the content in phenolic compounds, and induced SA production in the leaves located above and below the elicitation site, hence suggesting SAR induction by oligoulvans (El Modafar et al. 2012). Most of the results obtained with algal polysaccharides suggest that they represent promising disease control compounds and a major source of plant resistance inducers (Stadnik and De Freitas 2014; Burketova et al. 2015).

6.2.2.3

Food Industry by-Product Extracts

Food industry by-products can provide extracts or molecules that can elicit defense reactions. For instance, extracts from grape marc, which is a winemaking by-product, induced local defense reaction in tobacco, Arabidopsis, and tomato (Benouaret et al. 2013; Goupil et al. 2012). Grape marc provides polyphenol-rich extracts that can induce local and systemic PR1 and PR2 gene upregulation in tobacco (Goupil et al. 2012). An upregulation of PR1, PR2, and PR3 genes was observed locally in tobacco leaves following grape marc extract infiltration (Benouaret et al. 2013). On suspension-cultured cells of tobacco, grape marc extract induced an extracellular alkalinization, an upregulation of defense-related genes such as PR3, but also PAL and CCOAOMT involved in phenylpropanoid and lignin synthesis, and elicited hypersensitive response (Benouaret et al. 2014).

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6.2.3

Microbial Cell-Wall Extracts

6.2.3.1

Chitosan

Chitin (or poly b-(1,4)-N-acetyl-D-glucosamine) is a structural component of the cell walls of many filamentous fungi (Younes and Rinaudo 2015) and of the exoskeletons of crustaceans (Kim and Rajapakse 2005). With a degree of acetylation of 90%, the chitin has limited applications due to its insolubility in both organic and water solvents, but it may be converted to chitosan (CHI) by deacetylation reactions to increase solubility (Kim and Rajapakse 2005). Since CHI is nontoxic and biodegradable biopolymer with antimicrobial and/or plant-immunity eliciting properties, it represents an interesting alternative to chemical pesticides in agriculture (Xing et al. 2015). CHI and derivatives have been shown to confer protection of various plants (monocotyledonous, dicotyledonous, herbaceous, and lignified plants) against a wide range of pests and diseases (Reignault and Walters 2007, El-Hadrami et al. 2010, Xing et al. 2015, Cooper and Horton 2015). CHI elicits a variety of plant defense responses such as PAL and peroxidase activities, callose apposition, lignin deposition, and phytoalexin and PR-protein accumulation (Agrawal et al. 2002; Aziz et al. 2006; Iriti and Faoro 2008; Faoro et al. 2008; Xing et al. 2015). For instance, wheat seed treatment with CHI induced an accumulation of phenolics and lignin in wheat seedlings and an inhibition of Fusarium graminearum transmission to the primary roots of germinating seedlings (Bhaskara Reddy et al. 1999). Moreover, CHI induced callose apposition at pathogen entry points during the initial hours of pathogen inoculation (Iriti et al. 2006), which exerts a determinant role in limiting microbial spread (Iriti and Faoro 2008). The perception by the plant of carbohydrate MAMPs, such as CHI, is mediated by PRR with lysin motifs: LysM-RLK and LysM-RLP (Monaghan and Zipfel 2012). After perception, a complex of signaling molecules is involved in CHI-mediated signal transduction. A fast accumulation of hydrogen peroxide was observed in Arabidopsis cells within one hour after CHI addition (Ndimba et al. 2003). In tomato cells, CHI induced a rapid production of nitric oxide and the release of phosphatidic acid after phospholipase D and C activation (Raho et al. 2011). A transient increase of free cytosolic Ca2+ concentration was detected in Arabidopsis guard cells exposed to CHI, in parallel to stomatal closure (Klüsener et al. 2002). CHI treatment appeared to modulate the content of phytohormones involved in the plant defense responses. For instance, the content of JA, and an important precursor of JA (12-oxo-phytodienoic), rapidly increased in leaves of rice seedlings treated with CHI (Rakwal et al. 2002). An upregulation of various genes involved in JA metabolism and in JA-mediated activation, as well as genes involved in ET perception and regulation, was observed in rape leaves treated with CHI, hence suggesting that CHI activates plant defense through JA/ET signaling pathway (Yin et al. 2006). CHI appeared also to work in a competitive manner with

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auxin (Coqueiro et al. 2015), while Iriti and colleagues (2009) observed an abscisic acid accumulation in bean leaves treated with CHI. Moreover, CHI oligosaccharides with a degree of polymerization from 2 to 10, and a degree of acetylation of 95%, induce resistance in Arabidopsis to TMV via the SA-mediated signaling pathway (Jia et al. 2016). These results suggest complicated interactions of CHI with various phytohormones. The protection of CHI by the plant appears to be dependent of the mode of application (foliar treatment or soil drenching), the product concentration, and the duration of resistance response (Yin et al. 2008; Falcón-Rodríguez et al. 2011; Jia et al. 2016). For instance, it has been shown that the optimal timing of CHI application for effective plant protection is one day for the tobacco–TMV pathosystem (Jia et al. 2016) and thee days for the rapeseed Sclerotinia sclerotiorum model (Yin et al. 2008). Moreover, the capacity of CHI and derivatives to induce plant defenses depends clearly on the physicochemical properties of these molecules, especially molecular weight, degree of polymerization, and degree of acetylation (Kim and Rajapakse 2005; Kauss et al. 1989; Vander et al. 1998; Lin et al. 2005; Aziz et al. 2006). Larger CHIs (10 kDa) have been reported to be less active in inducing phytoalexins accumulation in grapevine leaves than CHI molecules with low masses (1.5 and 3 kDa) (Aziz et al. 2006). Moreover, a clear influence of the degree of acetylation of CHI on the type of defense responses induced in tobacco leaves was observed; higher degree of acetylation favored the stimulation of PAL activity, whereas lower degree of acetylation induced higher peroxidase activity (Falcón-Rodríguez et al. 2011). In parallel, phytoalexin production increased with decreasing of the degree of acetylation in grapevine leaves pretreated with CHI (Aziz et al. 2006). Falcón-Rodríguez et al. (2011) also observed in tobacco a significant relationship between the reduction of the Phytophthora nicotianae infection and the increase in PAL activity, thus concluding that CHI physicochemical properties influence plant-induced defenses and the relevant plant resistance obtained.

6.2.3.2

Lipopolysaccharides

Lipolysaccharides (LPSs) are cell-surface compounds of gram-negative bacteria, localized in the outer membrane of the cell envelope (Dow et al. 2000). LPSs consist of a lipid (lipid A) linked to a core oligosaccharide (usually the sugar 3-deoxy-D-mannose-2-octulosonate) and an O-polysaccharide consisting of repeating units (O-antigen or O-side chain) (Dow et al. 2000). One of the first works revealing the ability of LPS to induce plant resistance was that of Leach et al. (1983), who showed that a purified LPS from Ralstonia solanacearum induces a localized induced resistance in tobacco that correlates with the synthesis of a 31.8 kDa polypeptides in leaf tissues (Leach et al. 1983). Furthermore, it has been reported that LPSs can induce resistance against various diseases caused by bacteria, fungi, or parasitic plants (Van Peer and Schippers 1992; Zeidler et al. 2010;

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Mabrouk et al. 2016). Moreover, the O-antigen part of the LPS molecule of non-pathogenic rhizobacteria is thought to be responsible for induced systemic resistance (ISR) in Arabidopsis (Newman et al. 2013). Purified LPSs from the PGPR Pseudomonas fluorescens induced resistance in various plants, such as carnation, tomato, and Arabidopsis (Van Peer and Schippers 1992; Duijff et al. 1997; Van Wees et al. 1997). LPSs have been reported to trigger various plant defense reactions, including Ca2+ influx, alkalization of the extracellular medium, ROS, nitric oxide accumulation, PR-protein encoding gene expression, callose and phenolic compound deposition, and phytoalexin synthesis (Barton-Willis et al. 1984; Erbs and Newmann 2003; Sun et al. 2012). LPS recognitions in plants remain little known (Newman et al. 2013). However, Desaki et al. (2012) indicated that the machinery recognizing LPSs is evolutionary conserved in monocots and eudicots.

6.2.3.3

Peptidoglycans

Peptidoglycans (PGNs) are the only cell-wall components common to both gram-positive and gram-negative bacteria. They serve as a key structure of the bacterial cell by determining cell shape and providing resistance to internal turgor pressure (Dworking et al. 2014). PGNs are heteroglycan chains with polymeric alternating b-(1,4)-linked N-acetylglucosamine and N-acetylmuramic acid residues (Schleifer and Kandler 1972). PGN composition depends on the bacterial species and is considered as a taxonomic criterion (Schleifer and Kandler 1972). Application of a glutamate by-product produced by Corynebacterium glutamicum, which the main component was PGNs, led to a significant reduction of symptoms caused by Plasmopara viticola through an induction of plant defenses in grapevine (Chen et al. 2014). Pre-induction of tomato leaves with a purified PGN from Staphylococcus aureus inhibited the in planta growth of Pseudomonas syringae pv. tomato (Nguyen et al. 2010). PGNs act as elicitors in monocotyledonous or dicotyledonous plants and activate various typical early and late events of plant defense responses, such as extracellular alkalinization, Ca2+ influx, ROS accumulation, PR-protein encoding gene expression, chitinase activity, callose deposition, and phytoalexin synthesis (Felix and Boller 2003; Erbs et al. 2008; Liu et al. 2012; Chen et al. 2014). Erbs et al. (2008) reported that purified PGNs from the aggressive Xanthomonas campestris pv. campestris bacterium exhibit higher elicitor activity than PGNs from Agrobacterium tumefaciens, thus concluding that PGN defense-eliciting abilities appear to depend on subtle structural difference. Some LysM-RLP receptors, known to be able to bind to PGNs, were identified in the dicotyledonous Arabidopsis (LYM1 and LYM3) and in the monocotyledonous rice (LYP4 and LYP6) (Willmann et al. 2011; Liu et al. 2012).

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Flagellins

Flagella are important structures for gram-positive and gram-negative bacterial species since they possess several functions such as mobility, biofilm formation, protein export, and adhesion (Haiko and Westerlund-Wikström 2013). The major protein subunit, flagellin, belongs to molecules containing a pathogen-associated molecular pattern (PAMP) and plays a well-documented role in plant innate immunity (Newman et al. 2013). Flagellin from bacterial species differs in their central region, but has conserved N-terminal and C-terminal regions (Borrayo et al. 2013). Synthetic peptides comprising 15–22 amino acids of the highly conserved domain in the N-terminal of flagellin act as elicitors of plant defenses at sub-nanomolar concentrations (0.1–30 nM) in cells of dicotyledonous plants, such as Arabidopsis, tomato, potato, and tobacco (Felix et al. 1999). First described in Arabidopsis, the plasma membrane receptor Flagellin Sensing2 (FLS2) bounds flagellin to the extracellular domain of the receptor (Gomez-Gomez and Boller 2000). This event induces the heteromerization of FLS2 with Brassinosteroidinsensitive 1-associated kinase 1 (BAK1) and activate signaling cascades that finally culminate in the induction of defense responses (Heese et al. 2007). Zipfel and colleagues (2004) revealed that a 22-amino acid sequence (flg22) of the conserved N-terminal part of flagellin induced resistance in Arabidopsis through independent SA, JA, and ET signaling. Similarly, pretreatment with flg22 protects Arabidopsis plants against Botrytis cinerea infection via independent SA-, JA-, and ET-mediated signaling pathways (Ferrari et al. 2007).

6.2.3.5

Exopolysaccharides

Bacteria produce a wide range of exopolysaccharides, such as xanthan, sphingan, alginate, and cellulose via different biosynthesis pathways (Schmid et al. 2015). Exopolysaccharides can be homopolymeric or heteropolymeric and of diverse molecular weights, from 10 to 1000 kDa (Nwodo et al. 2012). Their functions include adherence of cells to surfaces, mobility, protection, and biofilm production (Nwodo et al. 2012). Purified exopolysaccharides from various PGPR (Bacillus cereus, B. amyloliquefaciens, Pseudomonas fluorescens, Burkholderia gladioli, and Bacillus polyxyma) were shown to induce systemic resistance in several plant species including Arabidopsis, cucumber, and wheat against fungal or bacterial pathogens (Kyungseok et al. 2008; Haggag et al. 2014; Jiang et al. 2016). In addition, exopolysaccharide applications elicited various defense responses, such as ROS accumulation, callose deposition, PR-protein encoding gene expression, and chitinase and peroxidase activities (Kyungseok et al. 2008; Haggag et al. 2014; Jiang et al. 2016). Purified exopolysaccharides from Pantoea agglomerans primed rice and wheat cells for better elicitation of plant defense during a subsequent elicitor treatment, while these exopolysaccharides directly elicited plant responses

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in tobacco and parsley cells, thus suggesting different recognitions and/or modes of action of P. agglomerans exopolysaccharides between monocotyledonous and dicotyledonous plants (Ortmann and Moerschbacher 2006; Ortmann et al. 2006).

6.2.4

Microbial Metabolites

6.2.4.1

Lipopeptides

Lipopeptides are amphiphilic molecules consisting of a short peptide chain linked to a lipid tail, produced by a variety of soil-borne or plant-associated bacterial genera such as Actinomyces, Streptomyces, Bacillus, and Pseudomonas (Raaijmakers et al. 2010). They are synthesized by a non-ribosomal enzyme complex called non-ribosomal peptide synthetase (NRPS), which confers considerable structural diversity to the molecules and results in the production of linear, branched, or cyclic low toxic compounds (Strieker et al. 2010; Deravel et al. 2014). Lipopeptides play a key role in the induction of plant immunity driven by beneficial microorganisms and have been reported to confer protection against a wide range of pathogens through antagonistic and/or induced resistance effects (Raaijmakers et al. 2010). For instance, Bacillus subtilis produces three main families of cyclic lipopeptides (surfactins, iturins, and fengycins). Purified mycosubtilin (iturin family) and surfactin from this bacterium activate distinct patterns of defense responses in grapevine leaves and result in a local long-lasting enhanced tolerance to the necrotrophic fungus Botrytis cinerea, although only mycosubtilin displayed direct antifungal activity (Farace et al. 2015). Mycosubtilin activated SA and JA signaling pathways, whereas surfactin mainly induced an SA-regulated response (Farace et al. 2015). Surfactin treatment did not influence the development of Bremia lactucae on lettuce plantlets, whereas treatment with mycosubtilin produced about seven times healthier plantlets than the control samples (Deravel et al. 2014). Treatment of cotton plants with purified iturins from Bacillus amyloliquefaciens suppressed the development of Verticillium dahliae by altering microsclerotial germination and by activating a set of defense reactions including ROS burst induction and Hog1 mitogen-activated protein kinase (MAPK) activation (Han et al. 2015). Treatment of tomato roots with the cyclic lipopeptide massetolide A from Pseudomonas fluorescens led to an increased leaf resistance to the oomycete Phytophthora infestans (Tran et al. 2007).

6.2.4.2

Harpins

Harpins are glycine-rich and heat-stable proteins produced by the type-III secretion system (T3SS) of gram-negative plant pathogenic bacteria. The first described harpin was HrpN, which is produced by the fire blight pathogen Erwinia

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amylovora. Furthermore, this harpin was identified in related pathogens such as Erwinia chrysanthemi (Yap et al. 2006). Messenger® and ProAct® are two commercially available harpin-based resistance inducers marketed as an alternative to fungicides for plant disease management. A number of reports have demonstrated the capacity of harpins to induce resistance and to confer plant protection against pathogen and pest attacks (Tayeh et al. 2014a). For instance, an application of Messenger® in field conditions allowed a reduction by 68% the incidence of Pierce’s disease caused by Xylella fastidiosa (Tubajika et al. 2007). Likewise, in cucumber, Messenger® conferred a decrease by 58.9 and 44.2% downy mildew (Pseudoperonospora cubensis) and gray mold (B. cinerea) extents, respectively (Yang and Ji 2009). Several other purified harpins were shown to induce plant resistance against diseases, such as Hpa1 from Xanthomonas oryzae pv. oryzicola that conferred resistance to Magnaporthe grisea (Che et al. 2011) and Alternaria alternata (Li et al. 2013).

6.2.4.3

Rhamnolipids

Rhamnolipids (RLs) are glycolipids synthetized by various bacteria species including some Pseudomonas (P. aeruginosa, P. chlororaphis, P. putida, P. fluorescens) and Burkholderia (B. pseudomallei, B. thailandensis) species (Vatsa et al. 2010; Toribio et al. 2010). The structure of RLs consists typically of 3hydroxyfatty acids linked through a beta-glycosidic bond to mono- or di-rhamnoses (Vatsa et al. 2010). Naturally produced RLs are always synthesized as mixtures of various RL congeners. The variability and distribution of the produced RLs may be attributed to diverse cultivation conditions and to strain-related variations (Abdel-Mawgoud et al. 2010). RLs have been described for their direct antimicrobial activity, virulence factors, and plant and animal immune modulation action (Abdel-Mawgoud et al. 2010; Vatsa et al. 2010). RLs were shown to induce plant resistance against bacteria, fungi, and oomycetes (Varnier et al. 2009, Sanchez et al. 2012). Sanchez et al. (2012) indicated that RL-mediated resistance involves different signaling pathways that depend on the pathogen lifestyle. Ethylene appears to be involved in RL-induced resistance to the biotrophic oomycete Hyaloperonospora arabidopsis and to the hemibiotrophic bacterium Pseudomonas syringae pv. tomato, whereas JA is essential for the resistance against the necrotrophic fungus B. cinerea (Sanchez et al. 2012). Moreover, SA have also a central role since SA-dependent defenses are potentiated by RLs following infection by B. cinerea or P. syringae pv. tomato (Sanchez et al. 2012). A variety of defense reactions are triggered by RLs, including Ca2+ influx, MAPK activation, ROS production, hypersensitive response-like induction, and the expression of various defense genes encoding for PR-proteins or involved in the phenylpropanoid and octadecanoid pathways (Varnier et al. 2009; Sanchez et al. 2012).

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Ergosterol

Ergosterol is the most predominant sterol in the fungal cell membranes and is of particular interest as a MAMP because of its potential to activate lipid-based signaling events (Klemptner et al. 2014). Granado et al. (1995) reported a stimulus– response perception system for ergosterol and showed that an ergosterol purified from Cladosporium fulvum spore extracts elicits some defense-related responses in tomato cells, including extracellular alkalinization. Furthermore, ergosterol was shown to enhance protection against B. cinerea and to trigger WRKY transcription factor, Vitis vinifera lipid transfer protein 1 (VvLTP1), and stilbene synthase gene expression in grape plantlets (Laquitaine et al. 2006).

6.2.4.5

Purified Microbial Proteins

Other purified proteins were reported to induce disease resistance in plants. A purified and characterized protein (PeBL1) secreted from the bacterium Brevibacillus laterosporus induced a typical hypersensitive response and systemic resistance in the tobacco Nicotiana benthamiana to both TMV and Pseudomonas syringae pv. tabaci (Wang et al. 2015). PeBL1 triggered defense responses in plants, including ROS production, extracellular medium alkalization, phenolic compound deposition, and defense-related gene expression. Similarly, MoHrip, a purified protein from Magnaporthe oryzae, was shown to induce resistance in rice against this pathogen (Chen et al. 2012). Other purified proteins of fungal origin were demonstrated to induce resistance against other pathogens such as Magnaporthe grisea (Yao et al. 2007) and Xanthomonas oryzae pv. oryzae (Peng et al. 2011) on rice, B. cinerea on tomato (Zhang et al. 2010), and TMV (Mao et al. 2010) and Pseudomonas syringae pv. tomato (Peng et al. 2011) on Arabidopsis.

6.2.5

Other Organic Molecules

6.2.5.1

b-Aminobutyric Acid

b-aminobutyric acid (BABA) is one of the most studied resistance inducers and has been shown to confer protection against a wide range of plant pathogens and pests. It is a non-protein amino acid rarely found in nature, known to possess great potential as a priming agent of defense reactions that are controlled by SA-dependent and -independent signaling pathways (Ton et al. 2005). However, a direct activation of plan defense responses by BABA, rather than a priming effect, was also reported in some pathosystems such as potato-Phytophthora infestans (Bengtsson et al. 2014) and rice-Meloidogyne graminicola (Ji et al. 2015). More recently, it was showed in strawberry fruits against Botrytis cinerea that the priming or direct activation of plant defenses depends on the concentration of BABA;

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concentrations higher than 100 mmol L−1 directly induce the defense response, whereas BABA at 10 mmol L−1 activates a priming response (Wang et al. 2016). BABA can confer a long-term priming effect (up to 28 days after treatment) and interestingly, even a transgenerational induced resistance as demonstrated in the wild potato Solanum physalifolium against Phytophthora infestans (Lankinen et al. 2016). Plant perception of BABA is mediated by an aspartyl-tRNA synthetase (Luna et al. 2014). BABA is effective as a foliar or fruit spray, soil drench, and seed treatment. For instance, tomato plants pretreated with BABA by soil drenching strongly reduced disease severity of bacterial wilt caused by Ralstonia solanacearum (leaf wilting index by 75.3% and vascular browning index by 69.9%), without any in vitro inhibitory activity on the pathogen (Hassan and Abo-Elyousr 2013). Foliar spray of Abyssinian mustard plants with BABA led to a significant reduction of lesions caused by Alternaria brassicae (Chavan and Kamble 2013). Postharvest treatment of strawberry fruits with BABA significantly reduced the symptoms of B. cinerea and induced different defense responses (Wang et al. 2016). Germinating seeds of tomato for one week in BABA-containing solutions significantly reduced lesion diameters of B. cinerea in 4-week-old plants (Luna et al. 2016). However, the efficacy of BABA could be conditioned by the mode of application. When applied on wheat seedlings as a soil drench, BABA significantly reduced weights of the insect Sitobion avenae, whereas foliar spray and seed treatment had no such effects (Cao et al. 2014). There are two isomers of BABA: a-aminobutyric acid (AABA) and c-aminobutyric acid (GABA). BABA is usually the more effective in the induction of plant resistance (Floryszak-Wieczorek et al. 2012), but GABA was also reported to induce resistance against some diseases. For example, a treatment of pear fruit with GABA induced strong resistance against the postharvest blue mold rot caused by Penicillium expansum (Yu et al. 2014).

6.2.5.2

Vitamins

Several vitamins were shown to induce disease resistance in plants. A treatment of rice plants with thiamine (vitamin B1) induced resistance against the root-knot nematode Meloidogyne graminicola by involving hydrogen peroxide and phenylpropanoid-mediated lignin production (Huang et al. 2016). An application of riboflavin (vitamin B2) on grapevine activated JA and callose biosynthesis pathways and provided a downy mildew disease reduction by 86%, without any direct fungicide effect (Boubakri et al. 2013a). Dipping pepper seedlings in a solution containing para-aminobenzoic acid (vitamin Bx) in field trials induced SAR and successfully protected pepper plants against Xanthomonas axonopodis pv. vesicatoria, without significant fitness allocation costs (Song et al. 2013). In addition to their beneficial effects on disease suppression, vitamins can also increase growth parameters as well as yields in treated plants (Song et al. 2013).

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Sugars

Sugars are recognized as signaling molecules in plants in addition to their typical roles as carbon and energy sources (Trouvelot et al. 2014). Trehalose, a non-reducing disaccharide commonly found in a wide variety of living organisms, is one of the most known sugars for its ability to induce disease resistance in plants. A foliar spray of wheat plantlets with trehalose strongly reduced powdery mildew symptoms caused by Blumeria graminis f. sp. tritici via the upregulation of chi4 precursor and lox gene expression as well as an induction of the LOX activity (Tayeh et al. 2014b). Trehalose was also reported to induce resistance in Arabidopsis against the green peach aphid Myzus persicae (Singh et al. 2011). On the other hand, a pretreatment of rice plants with sucrose drastically reduced rice blast symptoms caused by Magnaporthe oryzae (Gómez-Ariza et al. 2007). Other monosaccharides that rarely exist in nature, such as D-psicose and D-allose, have also been shown to elicit immunity and pathogen resistance in plants. For instance, a treatment of rice plants with D-allose or with D-psicose strongly reduced lesions of rice bacterial blight caused by Xanthomonas oryzae pv. oryzae (Kano et al. 2010, 2011). However, both sugars exhibited a significant inhibitory effect on root as well as shoot growth.

6.2.5.4

Amino Acids

A treatment of grapevine plants with methionine, a nutritionally essential sulfur-containing amino acid, activated ROS defense pathway and induced an upregulation of the expression of a battery of defense-related genes, resulting in significant protection level against Plasmopara viticola (Boubakri et al. 2013b). However, this amino acid exhibited a moderate antifungal activity against the oomycete. In pearl millet, an exogenous application of methionine induced defense-related genes and resistance against downy mildew caused by Sclerospora graminicola through a priming effect (Sarosh et al. 2005).

6.2.5.5

Biochar

Biochar is the solid coproduct of pyrolysis (thermal degradation) of biomass in the absence of oxygen. Several studies have demonstrated that soil-applied biochar can induce plant resistance against a wide range of pathogens. Soil amendment with biochar induced strawberry plant systemic resistance to three foliar fungal pathogens with different infection strategies: necrotrophic (B. cinerea), hemibiotrophic (Colletotrichum acutatum), and biotrophic (Podospharea aphanis) (Meller-Harel et al. 2012). Adding biochar to soil reduced by 50% the symptom extents of B. cinerea in different tomato genotypes, conferred by an induced systemic resistance involving a stronger and earlier hydrogen peroxide accumulation and JA pathway activation (Mehari et al. 2015). Biochar-amended potting medium at 1.2% reduced

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the susceptibility of rice to Meloidogyne graminicola, whereas direct toxic effects of biochar exudates on nematode viability, infectivity, or development were not observed (Huang et al. 2015). The increased plant resistance was associated with biochar-primed hydrogen peroxide accumulation as well as with the transcriptional enhancement of genes involved in the ethylene signaling pathway.

6.2.6

Minerals and Ions

6.2.6.1

Copper

Minerals and ions are not directly derived from living organisms, but they are considered as natural compounds, and some of them, such as copper (active substance of Bordeaux mixture), are widely used for plant protection in organic agriculture. Although copper is mainly considered as a product with direct biocide activity, it was shown to induce plant resistance against numerous pathogens, including oomycetes. When applied to grapevine leaves, copper sulfate (CuSO4) elicited substantial production of phytoalexins and provided significant suppression of both Botrytis cinerea and Plasmopara viticola (Aziz et al. 2006). Copper-based compounds were also reported to confer protection against olive leaf spot caused by Spilocaea oleagina (Obanor et al. 2013) and citrus black spot caused by Guignardia citricarpa (Hendricks et al. 2013).

6.2.6.2

Boron

Boron is a microelement that participates to plant growth and development, but it contributes also to plant protection. For instance, boron decreased significantly in both potato and tomato late blight severity caused by Phytophthora infestans without any direct biocide activity, thus indicating that boron may induce systemic acquired resistance against P. infestans (Frenkel et al. 2010). Boron also reduced symptoms of clubroot of canola caused by Plasmodiophora brassicae in laboratory and field conditions (Deora et al. 2011).

6.2.6.3

Silicon

Silicon (Si) is not considered as an essential element for most plants, but its absorption can induce protection against pathogen attack. When Si (potassium silicate, K2SiO3) was drenched weekly into the rhizosphere of zucchini plants, an increased level of Si was observed in the leaves, causing a suppression of powdery mildew caused by Podosphaera xanthii (Tesfagiorgis et al. 2014). In rice, Si confers protection against Cochliobolus miyabeanus though and induced resistance that functions independently of the classic immune hormones SA and JA, but via the

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abscisic acid and cytokinin pathways and probably by interfering with the production and/or action of fungal ethylene (Van Bockhaven et al. 2014). Likewise, when hydroponic soybean plants were supplied with Si, their resistance to rust caused by Phakopsora pachyrhizi was strongly increased (Arsenault-Labrecque et al. 2012). Si was also reported to be effective against other pathogens such as Acidovorax citrulli on yellow melon (Ferreira et al. 2015).

6.2.6.4

Calcium-Based Compounds

Calcium-based compounds have been shown to induce resistance in plants against pathogens since the calcium ion is a second messenger in numerous plant defense signaling pathways. Treatment of apple with calcium chloride (CaCl2) significantly reduced the decay lesion areas caused by B. cinerea or Penicillium expansum and induced an increase of the antioxidant activity and total phenolic compounds (Sharma et al. 2013). Calcium chloride also promoted an extended postharvest storage period of Guava infected by Collecttrichum simmonakii (Cruz et al. 2015). Similarly, calcium chloride protected pear fruit against B. cinerea, Penicillium expansum (Yu et al. 2012), and Alternaria alternata (Tian et al. 2006).

6.2.6.5

Phosphites

Phosphite-based compounds are mainly marketed as fertilizers, activators of natural defenses, or systemic fungicides because of their dual (biocide and eliciting) activity. Phosphite-induced resistance seems to be complex and relies on multiple processes (Lim et al. 2013). For instance, phosphite primes in Arabidopsis defense responses (callose papillae production and hydrogen peroxide accumulation) and enhances the expression of defense genes against Phytophthora cinnamomi (Eshraghi et al. 2011). Likewise, phosphite-mediated resistance in potato against Phytophthora infestans involves callose deposition as well as hydrogen peroxide and superoxide anion production (Machinandiarena et al. 2012), independently of the SA and JA hormones (Burra et al. 2014). By contrast, Lim et al. (2013) showed that treatment of potato with the phosphite-based product Confine™ induced resistance against P. infestans and triggered the SA-dependent defense responses, ROS and calcium-dependent pathways, as well as an induction of hypersensitive response and callose formation after pathogen attack. Moreover, Massoud et al. (2012) demonstrated the importance of the SA pathway in phosphite-induced resistance in Arabidopsis against Hyaloperonospora arabidopsidis.

6.2.6.6

Other Minerals and Ions

Nitric oxide (NO) is an important signal molecule involved in several plant responses to biotic and abiotic stresses. Treatment of peach fruit with a NO solution

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reduced disease incidence and lesion areas caused by Monilinia fructicola and induced chitinase and b-1,3-glucanase activities and the expression of PR-protein encoding genes, without any direct antifungal effect (Gu et al. 2014). Treatment of potato with Proalexin®, containing 85% potassium bicarbonate (KHCO3), significantly reduced the severity of late and tuber blight caused by P. infestans (Liljeroth et al. 2016). When applied on gerbera, Milstop®, containing also 85% potassium bicarbonate, conferred significant protection against the powdery mildew caused by Erysiphe cichoracearum (Moyer and Peres 2008). In field assays, potassium hydroxide (KOH) decreased the ascochyta blight caused by Ascochyta rabiei on chickpea (Ghazanfar et al. 2011). Aliette®, containing 80% fosetyl-aluminum, is known as a fungicide due to its direct effect on target pathogens, but it was also shown to induce resistance in several plants, including potato against P. infestans (Andreu et al. 2006).

6.3

Market and Regulation

Natural plant resistance inducers are a part of biocontrol products that include macroorganisms (insects, nematodes), microorganisms (fungi, bacteria, viruses), semiochemicals (pheromones, kairomones), and natural substances of plant, animal, or mineral origin (Ravensberg 2015). Biocontrol products are considered as an environmentally friendly alternative since they have lower risks on the environment and human health than synthetic pesticides; consequently, there is strong interest for their use in integrated pest management (IPM) strategy. In Europe, the use of biocontrol products is encouraged by the Directive 2009/128/CE aiming at reducing the use of conventional pesticides and promoting the deployment of agricultural inputs compatible with sustainable development. This directive requires member states to establish national action plans based on IMP to reduce their dependence on chemical control for plant protection (Villaverde et al. 2014). In this framework, several national initiatives were recently launched in Europe to encourage the reduction of chemical pesticides by using alternative approaches, such as Ecophyto plan in France, Green deal project in the Netherlands, Biopesticide track in Belgium, and Pesticide levy system in Denmark (Ravensberg 2015). Today, biocontrol products hold just 5% of the total crop protection market (approximately $3 billion in value worldwide), but this segment of the industry is growing and it is projected to increase by 8.84% annually, reaching more than 7% of the total crop protection market by 2025 (more than $4.5 billion in value worldwide) (Olson 2015). There are currently hundreds of biocontrol companies, most of them (over 230) are regrouped in the international biocontrol manufacturers association (IBMA) promoting their interests and activities. The regulation of biocontrol products differs strongly among countries and continents. In Europe, biopesticides including biocontrol products are evaluated according to the same regulations as their synthetic homologues, through the European pesticide regulation (EC) N° 1107/2009. Their registration also follows that designed for

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conventional pesticides (Villaverde et al. 2014). However, this situation is evolving and a simplified registration process for low-risk biocontrol products is currently under discussion with European authorities. The number of registered biocontrol products is strongly lower in Europe when compared to the USA (Dunham and Trimmer 2015), since regulation of biocontrol products is less constraint in this later country. In Europe, only two active substances of natural plant resistance inducers are registered as plant protection products: laminarin (Vacciplant®) from Goëmar, France, and COS-OGA, a complex of oligochitosans and oligopectates (PhytoSave®), from FytoFend, Belgium. Some political and societal obstacles still need to be overcome to allow a faster development of the market for biopesticides in Europe. At the political level, it seems important to bring more financial resources to research, especially applied research. Moreover, the Convention on Biological Diversity of 1992 and the Nagoya Protocol of 2010, although necessary for the protection of intellectual property rights, seem to slow down somewhat the development of biopesticides (e.g., restrictions on imports of some natural resources). At the societal scale, the understanding of biopesticides is not always clear: lack of understanding of terminology, negative and false public perception (risk for health of some biopesticides, higher cost for consumers), etc. (Ravensberg 2015). Another point to consider is the difficulty to establish unique risk assessment criteria because of the wide variety of biopesticides, such as natural products including living organisms (Villaverde et al. 2014).

6.4

Future Challenges

The protection efficacy of plant resistance inducers in laboratory and greenhouse conditions has been demonstrated for a wide range of pathosystems, as reviewed above. However, most of resistance inducers did not have their performances confirmed in field conditions, and a lack of stability in their field response was reported. This variability could be attributed to several factors, including climate conditions, plant cultivar, crop nutrition, and the extent to which plants are already induced (Walters et al. 2013). The nature of resistance inducer, the type of formulation, the dose, the timing (consideration of preventive activity) and frequency of application, as well as the application conditions such as the spraying technology used, can also strongly affect the level of product effectiveness. Although research in this area has increased over the last few years, the understanding of the impact of these influences on the expression of induced resistance is still poor. Further studies to understand how best to implement plant resistance inducers in practical crop protection and disease management programs are required. Nevertheless, it is already known that the efficacy of plant resistance inducers can be increased by their combination with conventional pesticides (Walters et al. 2010). The idea is that application of resistance inducers early in the season might reduce levels of pathogen inoculum in the crops, thereby allowing the use of less fungicides later in

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the season (Walters et al. 2013). The combined use of resistance inducers with distinct modes of action (i.e., inducing complementary defense reaction pathways) or various origins could also be a strategy to increase their efficacy. Several new approaches could be deployed to improve the efficacy of plant resistance inducers. The first challenge concerns breeding for cultivars with enhanced responsiveness to inducers. Indeed, the influence of host genotype on the level of induced resistance has been demonstrated in several species such as wheat against Zymoseptoria tritici (Ors et al. 2014). Moreover, it has been shown that both basal and induced resistance have been influenced by domestication, and that the level of basal resistance in common bean against P. syringae pv. Syringae was significantly higher in wild accessions than in modern cultivars (Córdova-Campos et al. 2012). Breeders should take into account the magnitude of plant responsiveness to resistance inducers in their genetic selection programs. Developing varieties more responsive to resistance inducers can motivate growers to use such alternative approaches in practice. Another promising strategy is the development of genetically modified (GM) plants containing SAR-related genes (Lyon et al. 2014). Several studies mainly on Arabidopsis highlighted genes induced by a wide range of resistance inducers. Some of the identified genes include potential signaling genes that are candidates for GM transformations. Transformation with transcription factors could be the most effective way forward ensuring an effective response to infection (Lyon et al. 2014). The idea here is the development of GM plants with inducer-responsive promoters ensuring resistance activation only after inducer application. Another way is the development of GM plants containing genes constrictively induced to provide continuous protection. However, this strategy is likely to be energy-demanding and therefore can lead to yield loss, since few reports showed that priming is likely to be less costly to the plant than direct triggering of resistance (e.g., Walters et al. 2009). The development of GM plants can be facilitated by using the new genome editing system—Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated protein 9 (CRISPR-Cas9)—a recently developed tool for the introduction of site-specific double-stranded DNA breaks (Bortesia and Fischer 2015). This system based on genome editing using artificial nucleases is considered a promising approach to modify genomes rapidly and in a precise and predictable manner.

6.5

Conclusion

Plant resistance inducers of natural origin have received during the last years much attention as potential main players for sustainable plant protection strategies. It is now established that such an approach represents and eco-friendly lever that can help farmers and growers to reduce the use of conventional inputs and to develop more sustainable cropping systems. However, with the exception of few success stories such as the extent use of potassium phosphite-based inducers to control late

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blight of potato and tomato (Alexandersson et al. 2016), their use in practice remains still limited. Few workers have assumed that resistance inducers can be used as a direct replacement for existing pesticides without the need to modify any other component of the agricultural system (Lyon et al. 2014). Evidence to date suggests that this is unlikely to be a very effective strategy and that resistance inducers may need to be considered as part of a new crop management approach, including the use of cultivars specifically chosen for appropriate inducers. Moreover, an effort should be performed by scientists and experimenters to improve the efficacy of resistance inducers in the field by better understanding the factors responsible for their response instability. The deployment of new emerging techniques such as untargeted “-omics” studies looking at genome-wide changes can form a good basis to better understand the plant response mechanisms in field conditions where multiple stresses occur. The industry can also contribute to the development of more performant products by optimizing formulation since formulation requirements for plant resistance inducers differ considerably from those of fungicides that target pathogen directly. Consideration of these constrains can contribute to the promotion of plant resistance inducers and their wide use in the framework of sustainable agriculture.

References Abdel-Mawgoud AM, Lépine F, Déziel E (2010) Rhamnolipids: Diversity of structures, microbial origins and roles. Appl Microbiol Biotechnol 86:1323–1336 Abouraïcha E, El Alaoui-Talibi Z, El Boutachfaiti R, Petit E, Courtois B et al (2015) Induction of natural defense and protection against Penicillium expansum and Botrytis cinerea in apple fruit in response to bioelicitors isolated from green algae. Sci Hortic 181:121–128 Adam M, Heuer H, Hallmann J (2014) Bacterial antagonists of fungal pathogens also control root-knot nematodes by induced systemic resistance of tomato plants. PLoS ONE 9(2):e90402 Agrawal GK, Rakwal R, Tamogami S, Yonekura M, Kubo A et al (2002) Chitosan activates defense/stress responses(s) in the leaves of Oryza staiva seedlings. Plant Physiol Biochem 40:1061–1069 Alexandersson E, Mulugeta T, Lankinen A, Liljeroth E, Andreasson E (2016) Plant resistance inducers against pathogens in solanaceae species - from molecular mechanisms to field application. Int J Mol Sci 17:1673 Alkooranee JT, Yin Y, Aledan TR, Jiang Y, Lu G et al (2015) Systemic resistance to powdery mildew in Brassica napus (AACC) and Raphanus alboglabra (RRCC) by Trichoderma harzianum TH12. PlosOne 10(11):e0142177 Altenbach D, Robatzek S (2007) Pattern recognition receptors: from the cell surface to intracellular dynamics. Mol Plant Microbe Interact 20:1031–1039 Andreu AB, Guevara MG, Wolski EA, Daleo GR, Caldiz DO (2006) Enhancement of natural disease resistance in potatoes by chemicals. Pest Manag Sci 62:162–170 Arsenault-Labrecque G, Menzies JG, Bélanger RR (2012) Effect of silicon absorption on soybean resistance to Phakopsora pachyrhizi in different cultivars. Plant Dis 96:37–42 Arseneault T, Pieterse CMJ, Gérin-Ouellet M, Goyer C, Filion M (2014) Long-term induction of defense gene expression in potato by Pseudomonas sp. LBUM223 and Streptomyces scabies. Phytopathology 104:926–932

6 Natural Agents Inducing Plant Resistance Against Pests …

147

Aziz A, Poinssot B, Daire X, Adrian M, Bezier A et al (2003) Laminarin elicits defense responses in grapevine and induces protection against Botrytis cinerea and Plasmopara viticola. Mol Plant Microbe Interact 16:1118–1128 Aziz A, Trotel-Aziz P, Dhuicq L, Jeandet P, Couderchet M et al (2006) Chitosan oligomers and copper sulfate induce grapevine defense reactions and resistance to gray mold and downy mildew. Phytopathology 96:1188–1194 Banani H, Roatti B, Ezzahi B, Giovannini O, Gessler G et al (2014) Characterization of resistance mechanisms activated by Trichoderma harzianum T39 and benzothiadiazole to downy mildew in different grapevine cultivars. Plant Pathol 63:334–343 Barriuso J, Ramos Solano B, Gutiérrez Mañero FJ (2008) Protection against pathogen and salt stress by four plant growth-promoting rhizobacteria isolated from Pinus sp. on Arabidopsis thaliana. Phytopathology 98:666–672 Barton-Willis PA, Wang MC, Holliday MR, Long MR, Keen NT (1984) Purification and composition of lipopolysaccharides from Pseudomonas syringae pathovar syringae. Physiol Plant Pathol 25:387–398 Baysal-Gurel F, Miller SA (2015) Management of powdery mildew in greenhouse tomato production with biorational products and fungicides. Sci Hortic 1069:179–184 Bengtsson M, Wulff E, Jørgensen HJL, Pham A, Lubeck M et al (2009) Comparative studies on the effects of a yucca extract and benzothiadiazole (BTH) on inhibition of Venturia inaequalis in apple leaves. Eur J Plant Pathol 124:187–198 Bengtsson T, Weighill D, Proux-Wéra E, Levander F, Resjö S et al (2014) Proteomics and transcriptomics of the BABA-induced resistance response in potato using a novel functional annotation approach. BMC Genom 15:315 Ben-Jabeur M, Ghabri E, Myriam M, Hamada W (2015) Thyme essential oil as a defense inducer of tomato against gray mold and Fusarium wilt. Plant Physiol Biochem 94:35–40 Benouaret R, Goujon E, Trivella A, Richard C, Ledoigt G et al (2013) Water extracts from winery by-products as tobacco defense inducers. Ecotoxicology 23:1574–1581 Benouaret R, Goujon E, Goupil P (2014) Grape marc extract causes early perception events, defence reactions and hypersensitive response in cultured tobacco cells. Plant Physiol Biochem 77:84–89 Bhaskara Reddy MV, Arul J, Angers P, Couture L (1999) Chitosan treatment of wheat seeds induces resistance to Fusarium graminearum and improves seed quality. J Agric Food Chem 47:1208–1216 Bhuvaneshwari V, Paul PK (2012) Transcriptional and translational regulation of defense enzymes induced by neem fruit extract in tomato. Arch Phytopathol Plant Protect 45:1–12 Boeke SJ, Boersma MG, Alink GM, van Loon JJ, van Huis A et al (2004) Safety evaluation of neem (Azadirachta indica) derived pesticides. J Ethnopharmacol 94:25–41 Boller T, Felix G (2009) A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annu Rev Plant Biol 60:379–406 Borrayo E, Martinez-Pena MD, Morales-Valencia JA, Cortes-Cruz M, Arteaga-Garibay R (2013) Flagellin complete sequence as an inter-specific molecular phylogenetic marker among bacteria. Afr J Microbiol Res 7:701–703 Bortesia L, Fischer R (2015) The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv 33:41–52 Boubakri H, Chong J, Poutaraud A, Schmitt C, Bertsch C et al (2013a) Riboflavin (Vitamin B2) induces defence responses and resistance to Plasmopara viticola in grapevine. Eur J Plant Pathol 136:837–855 Boubakri H, Waha MA, Chong J, Gertz C, Gandour S et al (2013b) Methionine elicits H2O2 generation and defense gene expression in grapevine and reduces Plasmopara viticola infection. J Plant Physiol 170:1561–1568 Burketova L, Trda L, Ott PG, Valentova O (2015) Bio-based resistance inducers for sustainable plant protection against pathogens. Biotechnol Adv 33:994–1004

148

A. Siah et al.

Burra D, Berkowitz O, Hedley PE, Morris J, Resjo S et al (2014) Phosphite-induced changes of the transcriptome and secretome in Solanum tuberosum leading to resistance against Phytophthora infestans. BMC Plant Biol 14:254 Calo JR, Crandall PG, O’Bryan CA, Ricke SC (2015) Essential oils as antimicrobials in food systems-a review. Food Control 54:111–119 Cao HH, Zhang M, Zhao H, Zhang Y, Wang XX et al (2014) Deciphering the mechanism of b-aminobutyric acid-induced resistance in wheat to the grain aphid. Sitobion avenae. PLoS ONE 9(3):e91768 Chandler S, Van Hese N, Coutte F, Jacques P, Höfte M et al (2015) Role of cyclic lipopeptides produced by Bacillus subtilis in mounting induced immunity in rice (Oryza sativa L.). Physiol Mol Plant Pathol 91:20–30 Chavan V, Kamble A (2013) b-Aminobutyric acid primed expression of WRKY and defence genes in Brassica carinata against alternaria blight. J Phytopathol 161:859–865 Che YZ, Li YR, Zou HS, Zou LF, Zhang B et al (2011) A novel antimicrobial protein for plant protection consisting of a Xanthomonas oryzae harpin and active domains of cecropin A and melittin. Microb Biotechnol 4:777–793 Chen M, Zeng H, Qiu D, Guo L, Yang X et al (2012) Purification and characterization of a novel hypersensitive response-inducing elicitor from Magnaporthe oryzae that triggers defense response in rice. PLoS ONE 7(5):e37654 Chen Y, Takeda T, Aoki Y, Fujita K, Suzuki S et al (2014) Peptidoglycan from fermentation by product triggers defense responses in grapevine. PLoS ONE 9(11):e113340 Chowdhury SP, Uhl J, Grosch R, Alquéres S, Pittroff S et al (2015) Cyclic lipopeptides of Bacillus amyloliquefaciens subsp. plantarum colonizing the lettuce rhizosphere enhance plant defence responses towards the bottom rot pathogen Rhizoctonia solani. Mol Plant Microbe Interact 28:984–995 Chung EJ, Hossain MT, Khan A, Kim KH, Jeon CO et al (2015) Bacillus oryzicola sp. nov., an endophytic bacterium isolated from the roots of rice with antimicrobial, plant growth promoting, and systemic resistance inducing activities in rice. Plant Pathol J 31:152–164 Cluzet S, Torregrosa C, Jacquet C, Lafitte C, Fournier J et al (2004) Gene expression profiling and protection of Medicago truncatula against a fungal infection in response to an elicitor from green algae Ulva spp. Plant, Cell Environ 27:917–928 Conceição CS, Felix KCS, Mariano RLR, Medeiros EV, Souza EB (2014) Combined effect of yeast and silicon on the control of bacterial fruit blotch in melon. Sci Hortic 174:164–170 Conrath U, Pieterse CM, Mauch-Mani B (2002) Priming in plant-pathogen interactions. Trends Plant Sci 7:210–216 Cooper WR, Horton DR (2015) Effects of elicitor of host plant defenses on pear psylla, Cacopsylla pyricola. Entomol Exp Appl 157:300–306 Coqueiro DSO, de Souza AA, Takita MA, Rodrigues CM, Kishi LT et al (2015) Transcriptional profile of sweet orange in response to chitosan and salicylic acid. BMC Genom 16:288 Córdova-Campos O, Adame-Álvarez RM, Acosta-Gallegos JA, Heil M (2012) Domestication affected the basal and induced disease resistance in common bean (Phaseolus vulgaris). Eur J Plant Pathol 134:367–379 Cruz AF, Medeiros NL, Benedet GL, Araújo MB, Uesugi CH et al (2015) Control of post-harvest anthracnose infection in guava (Psidium guajava) fruits with phosphites, calcium chloride, acetyl salicylic acid, hot water, and 1-MCP. Hortic Environ Biote 56:330–340 Daayf F, Schmitt A, Belanger RR (1995) The effects of plants extracts of Reynoutria sachalinensis on powdery mildew development and leaf physiology of long English cucumber. Plant Dis 79:577–580 Daayf F, Schmitt A, Belanger RR (1997) Evidence of phytoalexins in cucumber leaves infected with powdery mildew-following treatment with leaf extracts of Reynoutria sachalinensis. Plant Physiol 113:719–727 Daayf F, Ongena M, Boulanger R, El Hadrami I, Bélanger RR (2000) Induction of phenolic compounds in two cultivars of cucumber by treatment of healthy and powdery mildew-infected plants with extracts of Reynoutria sachalinensis. J Chem Ecol 26:1579–1593

6 Natural Agents Inducing Plant Resistance Against Pests …

149

de Freitas MB, Ferreira LG, Hawerroth C, Duarte MER, Noseda MD et al (2015) Ulvans induce resistance against plant pathogenic fungi independently of their sulfation degree. Carbohydr Polym 133:384–390 Denancé N, Sánchez-Vallet A, Goffner D, Molina A (2013) Disease resistance or growth: the role of plant hormones in balancing immune responses and fitness costs. Front Plant Sci 4:155 Deora A, Gossen BD, Walley F, McDonald MR (2011) Boron reduces development of clubroot in canola. Can J Plant Pathol 33:475–484 Deravel J, Lemière S, Coutte F, Krier F, VanHese N et al (2014) Mycosubtilin and surfactin are efficient, low ecotoxicity molecules for the biocontrol of lettuce downy mildew. Appl Microbiol Biotechnol 98:6255–6264 Desaki Y, Otomo I, Kobayashi D, Jikumaru Y, Kamiya Y et al (2012) Positive crosstalk of MAMP signaling pathways in rice cells. PLoS ONE 7:e51953 Dow M, Newman MA, von Roepenack E (2000) The induction and modulation of plant defense responses by bacterial lipolysaccharides. Annu Rev Phytopathol 38:241–261 Duijff BJ, Gianinazzi-Pearson V, Lemanceau P (1997) Involvement of the outer membrane lipopolysaccharides in the endophytic colonization of tomato roots by biocontrol Pseudomonas fluorescens strain WCS417r. New Phytol 135:325–334 Dunham WC, Trimmer D (2015) Evolution and future of biocontrol. The global biocontrol and biostimulants E-newsletter. http://www.2bmonthly.com/wp-content/uploads/2015/11/BillDunham-2BMonthly-Evolution-Future-of-Biocontrol-Industry-copy.pdf. Accessed 10 Nov 2016 Dworking J (2014) The medium is the message: interspecies and interkingdom signaling by peptidoglycan and related bacterial glycans. Ann Rev Microbiol 68:137–154 El Modafar C, Elgadda M, El Boutachfaiti R, Abouraicha E, Zehhar N et al (2012) Induction of natural defense accompanied by salicylic acid-dependant systemic acquired resistance in tomato seedlings in response to bioelicitors isolated from green algae. Sci Hortic 138:55–63 El-Hadrami A, Adam LR, El-Hadrami I, Daayf F (2010) Chitosan in plant protection. Mar Drugs 8:968–987 Erbs G, Newmann MA (2003) The role of lipopolysaccharides in induction of plant defence responses. Mol Plant Pathol 4:421–425 Erbs G, Silipo A, Aslam S, De Castro C, Liparoti V et al (2008) Peptidoglycan and muropeptides from pathogens Agrobacterium and Xanthomonas elicit plant innate immunity: structure and activity. Chem Biol 15:438–448 Eshraghi L, Anderson J, Aryamanesh N, Shearer B, McComb J et al (2011) Phosphite primed defence responses and enhanced expression of defence genes in Arabidopsis thaliana infected with Phytophthora cinnamomi. Plant Pathol 60:1086–1095 Falcón-Rodríguez AB, Costales D, Cabrera JC, Martínez-Téllez MA (2011) Chitosan physico-chemical properties modulate defense responses and resistance in tobacco plant against the oomycete Phytophtora nicotianae. Pest Biochem Physiol 100:221–228 Faoro F, Maffi D, Cantu D, Iriti M (2008) Chemical-induced resistance against powdery mildew in barley: the effects of chitosan and benzothiadazole. Biocontrol 53:384–401 Farace G, Fernandez O, Jacquens L, Coutte F, Krier F et al (2015) Cyclic lipopeptides from Bacillus subtilis activate distinct patterns of defence responses in grapevine. Physiol Mol Plant Pathol 16:177–187 Felix G, Boller T (2003) Molecular sensing of bacteria in plants. The highly conserved RNA-binding motif RNP-1 of bacterial cold shock proteins is recognized as an elicitor signal in tobacco. J Biol Chem 278:6201–6208 Felix G, Duran JD, Volko S, Boller T (1999) Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. Plant J 18:265–276 Ferrari S, Galletti R, Denoux C, De Lorenzo G, Ausubel FM et al (2007) Resistance to Botrytis cinerea induced in Arabidopsis by elicitors is independent of salicylic acid, ethylene, or jasmonate signaling but requires phytoalexin deficient3. Plant Physiol 144:367–379

150

A. Siah et al.

Ferraz HGM, Resende RS, Silveira PR, Andrade CCL, Milagres EA et al (2014) Rhizobacteria induces resistance against Fusarium wilt of tomato by increasing the activity of defense enzymes. Bragantia, Campinas 73:274–283 Ferraz HGM, Resende RS, Moreira PC, Silveira PR, Milagres EA et al (2015) Antagonistic rhizobacteria and jasmonic acid induce resistance against tomato bacterial spot. Bragantia, Campinas 74:417–427 Ferreira HA, do Nascimento CWA, Datnoff LE, de Sousa Nunes GH, Preston W, et al (2015) Effects of silicon on resistance to bacterial fruit blotch and growth of melon. Crop Prot 78:277-283 Floryszak-Wieczorek J, Arasimowicz-Jelonek M, Milczarek G, Janus L, Pawlak-Sprada S et al (2012) Nitric oxide-mediated stress imprint in potato as an effect of exposure to a priming agent. Mol Plant Microbe Interact 25:1469–1477 Fofana B, McNally DJ, Labbe C, Boulanger R, Benhamou N et al (2002) Milsana-induced resistance in powdery mildew-infected cucumber plants correlates with the induction of chalcone synthase and chalcone isomerase. Physiol Mol Plant Pathol 61:121–132 Fotoohiyan Z, Rezaee S, Bonjar GHS, Mohammadi AH, Moradi M (2015) Induction of systemic resistance by Trichoderma harzianum isolates in pistachio plants infected with Verticillium dahliae. J Nuts 6:95–111 Fousia S, Paplomatas EJ, Tjamos SE (2015) Bacillus subtilis QST 713 confers protection to tomato plants against Pseudomonas syringae pv tomato and induces plant defence-related genes. J Phytopathol 164:264–270 Frenkel O, Yermiyahu U, Forbes GA, Fry WE, Shtienberg D (2010) Restriction of potato and tomato late blight development by sub-phytotoxic concentrations of boron. Plant Pathol 59:626–633 Garcion C, Lamotte O, Cacas JL, Métraux JP (2014) Mechanisms of defence to pathogens: biochemistry and physiology. In: Walters DR, Newton AC, Lyon GD (eds) Induced resistance for plant defence: A sustainable approach to crop protection. Blackwell Publishing Ltd, Oxford, pp 106–136 Gauthier A, Trouvelot S, Kelloniemi J, Frettinger P, Wendehenne D et al (2014) The sulfated laminarin triggers a stress transcriptome before priming the SA- and ROS-dependent defenses during grapevine’s induced resistance against Plasmopara viticola. PLoS ONE 9(2):e88145 Ghazalibiglar H, Hampton JG, van Zijll de Jong E, Holyoake A (2016) Is induced systemic resistance the mechanism for control of black rot in Brassica oleracea by a Paenibacillus sp.? Biol Control 92:195-201 Ghazanfar MU, Wakil W, Sahi ST (2011) Induction of resistance in chickpea (Cicer arietinum L.) against Ascochyta rabiei by applying chemicals and plant extracts. Chil J Agric Res 71:52–62 Gómez-Ariza J, Campo S, Rufat M, Estopà M, Messeguer J et al (2007) Sucrose-mediated priming of plant defense responses and broad-spectrum disease resistance by overexpression of the maize pathogenesis-related PRms protein in rice plants. Mol Plant Microbe Interact 20:832– 842 Gomez-Gomez L, Boller T (2000) FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol Cell 5:1003–1011 Görlach J, Volrath S, Knauf-Beiter G, Hengy G, Beckhove U et al (1996) Benzothiadiazole, a novel class of inducers of systemic acquired resistance, activates gene expression and disease resistance in wheat. Plant Cell 8:629–643 Goupil P, Benouaret R, Charrier O, ter Halle A, Richard C et al (2012) Grape marc extract acts as elicitor of plant defence responses. Ecotoxicology 21:1541–1549 Granado J, Felix G, Boller T (1995) Perception of fungal sterols in plants. Plant Physiol 107:485– 490 Gruau C, Trotel-Aziz P, Villaume S, Rabenoelina F, Clément C et al (2015) Pseudomonas fluorescens PTA-CT2 triggers local and systemic immune response against Botrytis cinerea in grapevine. Mol Plant Microbe Interact 28:1117–1129 Gu R, Zhu S, Zhou J, Liu N, Shi J (2014) Inhibition on brown rot disease and induction of defence response in harvested peach fruit by nitric oxide solution. Eur J Plant Pathol 139:369–378

6 Natural Agents Inducing Plant Resistance Against Pests …

151

Gupta A, Khulbe D, Srinivas P, Ankita (2014a) Induction of systemic resistance in adzuki bean through seed bacterisation. Seed Sci Tech 42:332–343 Gupta KJ, Mur LAJ, Brotman Y (2014b) Trichoderma asperelloides suppresses nitric oxide generation elicited by Fusarium oxysporum in Arabidopsis roots. Mol Plant Microbe Interact 27:307–314 Guzman-Valle P, Bravo-Luna L, Montes-Belmont R, Guigon-Lopez C, Sepulveda-Jimenez G (2014) Induction of resistance to Sclerotium rolfsii in different varieties of onion by inoculation with Trichoderma asperellum. Eur J Plant Pathol 138:223–229 Haggag WWM, Hussein MM, Mehanna HM, El-Moneim D (2014) Bacteria polysaccharides elicit resistance of wheat against some biotic and abiotic stress. Int J Pharm Sci Rev Res 29:292–298 Hahn MG, Albersheim P (1978) Host-pathogen interactions XIV. Isolation and partial characterization of an elicitor from yeast extract. Plant Physiol 62:107–111 Haiko J, Westerlund-Wikström B (2013) The role of the bacterial flagellum in adhesion and virulence. Biology 2:1242–1267 Han Q, Wu F, Wang X, Qi H, Shi L et al (2015) The bacterial lipopeptide iturins induce Verticillium dahliae cell death by affecting fungal signalling pathways and mediate plant defence responses involved in pathogen-associated molecular pattern-triggered immunity. Environ Microbiol 17:1166–1188 Hassan MAE, Abo-Elyousr KAM (2013) Activation of tomato plant defence responses against bacterial wilt caused by Ralstonia solanacearum using DL-3-aminobutyric acid (BABA). Eur J Plant Pathol 136:145–157 Heese A, Hann DR, Gimenez-Ibanez S, Jones AM, He K et al (2007) The receptor-like kinase SERK3/BAK1 is a central regulator of innate immunity in plants. Proc Natl Acad Sci USA 104:12217–12222 Hendricks KEM, Donahoo RS, Roberts PD, Christman MC (2013) Effect of copper on growth characteristics and disease control of the recently introduced Guignardia citricarpa on citrus in Florida. Am J Plant Sci 4:282–290 Hoitink HA, Madden LV, Dorrance AE (2006) Systemic resistance induced by Trichoderma spp.: interactions between the host, the pathogen, the biocontrol agent, and soil organic matter quality. Phytopathology 96:186–189 Huang W, Ji H, Gheysen G, Debode J, Kyndt T (2015) Biochar-amended potting medium reduces the susceptibility of rice to root-knot nematode infections. BMC Plant Biol 15:267 Huang W, Ji H, Gheysen G, Kyndt T (2016) Thiamine-induced priming against root-knot nematode infection in rice involves lignification and H2O2 generation. Mol Plant Pathol 17:614–24 Iriti M, Faoro F (2008) Abscisic acid is involved in chitosan-induced resistance to tobacco necrosis virus (TNV). Plant Physiol Biochem 46:1106–1111 Iriti M, Sironi M, Gomarasca S, Casazza AP, Soave C et al (2006) Cell death mediated antiviral effect of chitosan in tobacco. Plant Physiol Biochem 44:893–900 Iriti M, Picchi V, Rossoni M, Gomarasca S, Ludwig N et al (2009) Chitosan antitranspirant activity is due to abscisic acid-dependent stomatal closure. Environ Exp Bot 66:493–500 Jain S, Choudhary DK (2014) Induced defense-related proteins in soybean (Glycine max L. Merrill) plants by Carnobacterium sp. SJ-5 upon challenge inoculation of Fusarium oxysporum. Planta 239:1027–1040 Ji H, Kyndt T, He W, Vanholme B, Gheysen G (2015) b-aminobutyric acid-induced resistance against root-knot nematodes in rice is based on increased basal defence. Mol Plant Microbe Interact 28:519–533 Jia X, Meng Q, Zeng H, Wang W, Yin H (2016) Chitosan oligosaccharides induces resistance to Tobacco mosaic virus in Arabidopsis via the salicylic acid-mediated signalling pathway. Nature https://doi.org/10.1038/srep26144 Jiang CH, Fan ZH, Xie P, Guo JH (2016) Bacillus cereus AR156 extracellular polysaccharides served as a novel micro-associated molecular pattern to induced systemic immunity to Pst DC3000 in Arabidopsis. Front Microbiol 9(7):664 Jones JDG, Dangl JL (2006) The plant immune system. Nature 444:323–32

152

A. Siah et al.

Kano A, Gomi K, Yamasaki-Kokudo Y, Satoh M, Fukumoto T et al (2010) A rare sugar, D-allose, confers resistance to rice bacterial blight with upregulation of defense-related genes in Oryza sativa. Phytopathology 100:85–90 Kano A, Hosotani K, Gomi K, Yamasaki-Kokudo Y, Shirakawa C et al (2011) D-Psicose induces upregulation of defense-related genes and resistance in rice against bacterial blight. J Plant Physiol 168:1852–1857 Kauss H, Jeblick W, Domard A (1989) The degree of polymerization and N-acetylation of chitosan determine its ability to elicit callose formation in suspension cells and protoplasts of Catharanthus roseus. Planta 178:385–392 Keen NT, Partidge J, Zaki A (1972) Pathogen-produced elicitor of a chemical defense mechanism in soybean monogenically resistant to Phytophthora megasperma var. sojae. Phytopathology 62:768 Keen NT, Yoshikawa M, Wang MC (1983) Phytoalexin elicitor activity of carbohydrates from Phytophthora megasperma f.sp. glycinea and other sources. Plant Physiol 71:466–471 Khan W, Rayirath UP, Subramanian S, Jithesk MN, Rayorath P et al (2009) Seaweed extracts as biostimulants of plant growth and development. J Plant Growth Regul 28:386–399 Kim SE, Rajapakse N (2005) Enzymatic production and biological activities of chitosan oligosaccharides (COS): a review. Carbohydr Polym 62:357–368 Klarzynski O, Descamps V, Plesse B, Yvin JC, Kloareg B et al (2003) Sulfated fucan oligosaccharides elicit defense responses in tobacco and local and systemic resistance against tobacco mosaic virus. Mol Plant Microbe Interact 16:115–122 Klemptner RL, Sherwood JS, Tugizimana F, Dubery IA, Piater LA (2014) Ergosterol, an orphan fungal microbe-associated molecular pattern (MAMP). Mol Plant Pathol 15:747–761 Klüsener B, Young JJ, Murata Y, Allen GJ, Mori IC et al (2002) Convergence of calcium signaling pathways of pathogenic elicitors and abscisic acid in Arabidopsis guard cells. Plant Physiol 130:2152–2163 Koch E, Weil B, Wächter R, Wohlleben S, Spiess H et al (2006) Evaluation of selected microbial strains and commercial alternative products as seed treatments for the control of Tilletia tritici, Fusarium culmorum, Drechslera graminea and D. teres. J Plant Dis Prot 113:150–158 Konstantinidou-Doltsinis S, Markellou E, Kasselaki A, Fanouraki MN, Koumaki CM et al (2006) Efficacy of Milsana, a formulated plant extract from Reynoutria sachalinensis, against powdery mildew of tomato (Leveillula taurica). Biocontrol 51:375–392 Kuc J (1982) Induced immunity to plant disease. Bioscience 32:854–860 Kuc J (1995) Phytoalexins, stress metabolism, and disease resistance in plants. Ann Rev Phytopathol 33:275–297 Kumar A, Gond SK, Mishra A, Sharma VK, Verma SK et al (2015) Salicylic acid and its role in systemic resistance induced by Pseudomonas fluorescens to early blight disease of tomato. Vegetos 28:12–19 Kurth F, Mailänder S, Bönn M, Feldhahn L, Herrmann S et al (2014) Streptomyces-induced resistance against oak powdery mildew involves host plant responses in defense, photosynthesis, and secondary metabolism pathways. Mol Plant Microbe Interact 27:891–900 Kyungseok P, Kloepper JW, Ryu CM (2008) Rhizobacterial exopolysaccharides elicit induced resistance on cucumber. J Microbiol Biotechnol 18:1095–1100 Lahlali R, Peng G (2014) Clonostachys rosea confers suppression of clubroot on canola via antibiosis and induced host resistance. Plant Pathol 63:447–455 Lahlali R, McGregor L, Song T, Gossen BD, Narisawa K et al (2014) Heteroconium chaetospira induces resistance to clubroot via upregulation of host genes involved in jasmonic acid, ethylene, and auxin biosynthesis. PLoS ONE 9(4):e94144 Lankinen A, Abreha KB, Alexandersson E, Andersson S, Andreasson E (2016) Nongenetic inheritance of induced resistance in a wild annual plant. Phytopathology 106:877–883 Laquitaine L, Gomès E, François J, Marchive C, Pascal S et al (2006) Molecular basis of ergosterol-induced protection of grape against Botrytis cinerea: induction of type I LTP promoter activity, WRKY, and stilbene synthase gene expression. Mol Plant Microbe Interact 19:1103–1112

6 Natural Agents Inducing Plant Resistance Against Pests …

153

Leach JE, Sherwood J, Fulton RW, Sequeira L (1983) Comparison of soluble proteins associated with disease resistance induced by bacterial lipopolysaccharide and viral necrosis. Physiol Plant Pathol 23:377–385 Ley SV, Denholm AA, Wood A (1993) The chemistry of azadirachtin. Nat Prod Rep 10:109–157 Li YR, Ma WX, Che YZ, Zou LF, Zakria M et al (2013) A highly-conserved single-stranded DNA-binding protein in Xanthomonas functions as a harpin-like protein to trigger plant immunity. PLoS ONE 8(2):e56240 Li H, Ding X, Wang C, Ke H, Wu Z et al (2016) Control of tomato yellow leaf curl virus disease by Enterobacter asburiae BQ9 as a result of priming plant resistance in tomatoes. Turk J Biol 40:150–159 Liljeroth E, Lankinen A, Wiik L, Burra DD, Alexandersson E et al (2016) Potassium phosphite combined with reduced doses of fungicides provides efficient protection against potato late blight in large-scale field trials. Crop Prot 86:42–55 Lim S, Borza T, Peters RD, Coffin RH, Al-Mughrabi KI et al (2013) Proteomics analysis suggests broad functional changes in potato leaves triggered by phosphites and a complex indirect mode of action against Phytophthora infestans. J Proteomic 93:207–223 Lin W, Hu X, Zhang W, Rogers WJ, Cai W (2005) Hydrogen peroxide mediates defence responses induced by chitosans of different molecular weights in rice. J Plant Physiol 162:937– 944 Liu B, Li JF, Ao Y, Qu J, Lo Z et al (2012) Lysin motif-containing proteins LYP4 and LYP6 play dual roles in peptidoglycan and chitin perception in rice innate immunity. Plant Cell 24:3406– 3419 Luna E, van Hulten M, Zhang Y, Berkowitz O, López A et al (2014) Plant perception of b-aminobutyric acid is mediated by an aspartyl-tRNA synthetase. Nat Chem Biol 10:450–456 Luna E, Beardon E, Ravnskov S, Scholes JD, Ton J (2016) Optimizing chemically induced resistance in tomato against Botrytis cinerea. Plant Dis 100:704–710 Lyon GD, Newton AC, Walters DR (2014) Induced resistance in crop protection: the future, drivers and barriers. In: Walters DR, Newton AC, Lyon GD (eds) Induced resistance for plant defence: a sustainable approach to crop protection. Blackwell Publishing Ltd, Oxford, pp 149– 170 Mabrouk Y, Mejri S, Belhadj O (2016) Biochemical mechanisms of induced resistance by rhizobial lipopolysaccharides in pea against crenate broomrape. Rev Bras Bot 39:107–114 Machinandiarena MF, Lobato MC, Feldman ML, Daleo GR, Andreu AB (2012) Potassium phosphite primes defense responses in potato against Phytophthora infestans. J Plant Physiol 169:1417–1424 Magnin-Robert M, Quantinet D, Couderchet M, Aziz A, Trotel-Aziz P (2013) Differential induction of grapevine resistance and defense reactions against Botrytis cinerea by bacterial mixtures in vineyards. Biocontrol 58:117–131 Mao J, Liu Q, Yang X, Long C, Zhao M et al (2010) Purification and expression of a protein elicitor from Alternaria tenuissima and elicitor-mediated defence responses in tobacco. Ann Appl Biol 156:411–420 Martínez-Hidalgo P, García JM, Pozo MJ (2015) Induced systemic resistance against Botrytis cinerea by Micromonospora strains isolated from root nodules. Front Microbiol 2(6):922 Massoud K, Barchietto T, Le Rudulier T, Pallandre L, Didierlaurent L et al (2012) Dissecting phosphite-induced priming in Arabidopsis infected with Hyaloperonospora arabidopsidis. Plant Physiol 159:286–298 Mehari ZH, Elad Y, Rav-David D, Graber ER, Meller-Harel Y (2015) Induced systemic resistance in tomato (Solanum lycopersicum) against Botrytis cinerea by biochar amendment involves jasmonic acid signaling. Plant Soil 395:31–44 Mei L, Liang Y, Zhang L, Wang Y, Guo Y (2014) Induced systemic resistance and growth promotion in tomato by an indole-3-acetic acid-producing strain of Paenibacillus polymyxa (double effect). Ann Appl Biol 165:270–279 Meller-Harel Y, Elad Y, Rav-David D, Borenstein M, Shulchani R et al (2012) Biochar mediates systemic response of strawberry to foliar fungal pathogens. Plant Soil 357:245–257

154

A. Siah et al.

Ménard R, Alban S, de Ruffray P, Jamois F, Franz G et al (2004) Beta-1,3 glucan sulfate, but not beta-1,3 glucan, induces the salicylic acid signaling pathway in tobacco and Arabidopsis. Plant Cell 16:3020–3032 Monaghan J, Zipfel C (2012) Plant pattern recognition receptor complexes at the plasma membrane. Curr Opin Plant Biol 15:349–357 Moyer C, Peres NA (2008) Evaluation of biofungicides for control of powdery mildew of gerbera daisy. Proc Fla State Hortic Soc 121:389–394 Murali M, Amruthesh KN (2015) Plant growth-promoting fungus Penicillium oxalicum enhances plant growth and induces resistance in pearl millet against downy mildew disease. J Phytopathol 163:743–754 Murthy KN, Uzma F, Srinivas CC (2014) Induction of systemic resistance in tomato against Ralstonia solanacearum by Pseudomonas fluorescens. Am J Plant Sci 5:1799–1811 Mustafa G, Randoux B, Tisserant B, Fontaine J, Magnin-Robert M et al (2016) Phosphorus supply, arbuscular mycorrhizal fungal species, and plant genotype impact on the protective efficacy of mycorrhizal inoculation against wheat powdery mildew. Mycorrhiza 26:685–697 Nair A, Kolet SP, Thulasiram HV, Bhargava S (2015a) Role of methyl jasmonate in the expression of mycorrhizal induced resistance against Fusarium oxysporum in tomato plants. Physiol Mol Plant Pathol 92:139–145 Nair A, Kolet SP, Thulasiram HV, Bhargava S (2015b) Systemic jasmonic acid modulation in mycorrhizal tomato plants and its role in induced resistance against Alternaria alternata. Plant Biol (Stuttg)17: 625-631 Ndimba BK, Chivasa S, Hamilton JM, Simon WJ, Slabas AR (2003) Proteomic analysis of changes in the extracellular matrix of Arabidopsis cell suspension cultures induced by fungal elicitors. Proteomics 3:1047–1059 Newman MA, Sundelin T, Nielsen JT, Erbs G (2013) MAMP (microbe-associated molecular pattern) triggered immunity in plants. Front Plant Sci 16(4):139 Nguyen HP, Chakravarthy S, Velasquez AC, McLane HL, Zeng L et al (2010) Methods to study PAMP-triggered immunity using tomato and Nicotiana benthamiana. Mol Plant Microbe Interact 23:991–999 Niu D, Wang X, Wang Y, Song X, Wang J et al (2016) Bacillus cereus AR156 activates PAMP-triggered immunity and induces a systemic acquired resistance through a NPR1-and SA-dependent signaling pathway. Biochem Biophys Res Commun 469(1):120–125 Nürnberger T, Brunner F, Kemmerling B, Piater L (2004) Innate immunity in plants and animals: striking similarities and obvious differences. Immunol Rev 198:249–266 Nwodo UU, Green E, Okoh AL (2012) Bacterial exopolysaccharides: Functionality and Prospects. Int J Mol Sci 13:14002–14015 Obanor FO, Walter M, Jones EE, Jaspers MV (2013) Efficacy of systemic acquired resistance inducers in olive leaf spot management. Australas Plant Pathol 42:163–168 Okon Levy N, Meller Harel Y, Haile ZM, Elad Y, Rav-David E et al (2015) Induced resistance to foliar diseases by soil solarization and Trichoderma harzianum. Plant Pathol 64:365–374 Olson S (2015) An analysis of the biopesticide market now and where it is going. Outlooks Pest Manage 26:5 Ors M, Couleaud G, Maumene C, Siah A, Randoux B, et al (2014) Cultivar- and dose-dependent efficacy of wheat resistance inducers against Septoria tritici blotch in laboratory and field conditions. XIII Meeting of the Working Group Biological control of fungal and bacterial plant pathogens, Uppsala, Sweden Ortmann I, Conrath U, Moerschbacher BM (2006) Exopolysaccharides of Pantoea agglomerans have different priming and eliciting activities in suspension-cultured cells of monocots and dicots. FEBS Lett 580:4491–4494 Ortmann I, Moerschbacher BM (2006) Spent growth medium of Pantoea agglomerans primes wheat suspension cells for augmented accumulation of hydrogen peroxide and enhanced peroxidase activity upon elicitation. Planta 224:963–970

6 Natural Agents Inducing Plant Resistance Against Pests …

155

Pasini C, Daquila F, Curir P, Gullino ML (1997) Effectiveness of antifungal compounds against rose powdery mildew (Sphaerotheca pannosa var. rosae) in glasshouses. Crop Prot 16:251– 256 Paul PK, Sharma PD (2002) Azadirachta indica leaf extract induces resistance in barley against leaf stripe disease. Physiol Mol Plant Pathol 61:3–13 Paulert R, Ebbinghaus D, Urlass C, Moerschbacher BM (2010) Priming of the oxidative burst in rice and wheat cell cultures by ulvan, a polysaccharide from green macroalgae, and enhanced resistance against powdery mildew in wheat and barley plants. Plant Pathol 59:634–642 Peng DH, Qiu DW, Ruan LF, Zhou CF, Sun M (2011) Protein elicitor PemG1 from Magnaporthe grisea induces systemic acquired resistance (SAR) in plants. Mol Plant Microbe Interact 24:1239–1246 Pérez-Montaño F, Alías-Villegas C, Bellogín RA, del Cerro P, Espuny MR et al (2014) Plant growth promotion in cereal and leguminous agricultural important plants: from microorganism capacities to crop production. Microbiol Res 169:325–336 Pharand B, Carisse O, Benhamou N (2002) Cytological aspects of compost-mediated induced resistance against fusarium crown and root rot in tomato. Phytopathology 92:424–438 Pieterse CM, van Wees SC, van Pelt JA, Knoester M, Laan R et al (1998) A novel signaling pathway controlling induced systemic resistance in Arabidopsis. Plant Cell 10:1571–1580 Pretali L, Bernard L, Butterfield TS, Trevisan M, Lucini L (2016) Botanical and biological pesticides elicit a similar Induced Systemic Response in tomato (Solanum lycopersicum) secondary metabolism. Phytochemistry 130:56–63 Raaijmakers JM, De Bruijn I, Nybroe O, Ongena M (2010) Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics. FEMS Microbiol Rev 34:1037–1062 Raho N, Ramirez L, Lanteri L, Gonorazky G, Lamattina L et al (2011) Phosphatidic acid production in chitosan-elicited tomato cells, via both phospholipase D and phospholipase C/ diacylglycerol kinase, requires nitric oxide. J Plant Physiol 168:534–538 Raizada RB, Srivastava MK, Kaushal RA, Singh RP (2001) Azadirachtin, a neem biopesticide: subchronic toxicity assessment in rats. Food Chem Toxicol 39:477–483 Rakwal R, Tamogami S, Agrawal GK, Iwahashi H (2002) Octadecanoid signaling component “burst” in rice (Oryza sativa L.) seedling leaves upon wounding by cut and treatment with fungal elicitor protein. Biochem Biophys Res Commun 295:1041–1045 Randoux B, Renard D, Nowak E, Sanssene J, Courtois J et al (2006) Inhibition of Blumeria graminis f. sp. tritici germination and partial enhancement of wheat defenses by Milsana. Phytopathology 96:1278–1286 Ravensberg W (2015) Crop protection in 2030: towards a natural, efficient, safe and sustainable approach. International Symposium Swansea University, 7–9 Sept 2015 Reignault P, Walters D (2007) Topical applications of inducers for disease control. In: Walters D, Lyon G (eds) Induced resistance for plant defence: a sustainable approach to crop protection. Blackwell Publishing Ltd, Oxford, pp 207–258 Renard-Merlier D, Randoux B, Nowak E, Farcy F, Durand R et al (2007) Iodus 40, salicylic acid, heptanoyl salicylic acid and trehalose exhibit different efficacies and defence targets during a wheat/powdery mildew interaction. Phytochemistry 68:1156–1164 Rosyidah A, Wardiyati T, Abadi AL, Maghfoer MD, Aini LQ (2014) Induced resistance of potato (Solanum tuberosum L.) toward Ralstonia solanacearum disease with combination of several bio-control microbes. J Biol Agric Healthc 4:90–98 Salla TD, Astarita LV, Santarém ER (2016) Defense responses in plants of Eucalyptus elicited by Streptomyces and challenged with Botrytis cinerea. Planta 243:1055–1070 Sanchez L, Courteaux B, Hubert J, Kauffmann S, Renault JH et al (2012) Rhamnolipids elicit defense responses and induce disease resistance against biotrophic, hemibiotrophic, and necotrophic pathogens that require different signaling pathways in Arabidopsis and highlight a central role for salicylic acid. Plant Physiol 160:1630–1641

156

A. Siah et al.

Sang MK, Kim EN, Han GD, Kwack MS, Jeun YC (2014) Priming-mediated systemic resistance in cucumber induced by Pseudomonas azotoformans GC-B19 and Paenibacillus elgii MM-B22 against Colletotrichum orbiculare. Phytopathology 104:834–842 Sangha JS, Ravichandran S, Prithiviraj K, Critchley AT, Prithiviraj B (2010) Sulfated macroalgal polysaccharides k-carrageenan and i-carrageenan differentially alter Arabidopsis thaliana resistance to Sclerotinia sclerotiorum. Physiol Mol Plant Pathol 75:38–45 Sarosh BR, Sivaramakrishnan S, Shetty HS (2005) Elicitation of defense related enzymes and resistance by L-methionine in pearl millet against downy mildew disease caused by Sclerospora graminicola. Plant Physiol Biochem 43:808–815 Sato I, Yoshida S, Iwamoto Y, Aino M, Hyakumachi M et al (2014) Suppressive potential of Paenibacillus strains isolated from the tomato phyllosphere against Fusarium crown and root rot of tomato. Microbes Environ 29:168–177 Schleifer KH, Kandler O (1972) Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36:407–477 Schmid J, Sieber V, Rehim B (2015) Bacterial exopolysaccahrides: biosynthesis pathways and engineering strategies. Front Microbiol 6:496 Schmitt A (2002) Induced responses by plant extracts from Reynoutria sachalinensis: a case study. In “induced resistance in plants against insects and diseases”. IOBC/wprs Bull 25:83–88 Segarra G, Santpere G, Elena G, Trillas I (2013) Enhanced Botrytis cinerea resistance of Arabidopsis plants grown in compost may be explained by increased expression of defense-related genes, as revealed by microarray analysis. PLoS ONE 8(2):e56075 Selim ME (2015) Effectiveness of Trichoderma biotic applications in regulating the related defense genes affecting tomato early blight disease. J Plant Pathol Microb 6:311 Sharma R, Singh D, Pal R (2013) Synergistic influence of pre-harvest calcium sprays and postharvest hot water treatment on fruit firmness, decay, bitter pit incidence and postharvest quality of royal delicious apples (Malus x domestica Borkh). Am J Plant Sci 4:153–159 Singh V, Louis J, Ayre BG, Reese JC, Pegadaraju V, Shah J (2011) Trehalose phosphate synthase11-dependent trehalose metabolism promotes Arabidopsis thaliana defense against the phloem-feeding insect Myzus persicae. Plant J 67:94–104 Singh BN, Singh A, Singh BR, Singh HB (2014) Trichoderma harzianum elicits induced resistance in sunflower challenged by Rhizoctonia solani. J Appl Microbiol 116:654–666 Son JS, Sumayo M, Hwang YJ, Kim BS, Ghim SY (2014) Screening of plant growth-promoting rhizobacteria as elicitor of systemic resistance against gray leaf spot disease in pepper. Appl Soil Ecol 73:1–8 Song GC, Choi HK, Ryu CM (2013) The folate precursor para-aminobenzoic acid elicits induced resistance against Cucumber mosaic virus and Xanthomonas axonopodis. Ann Bot 111:925–934 Song Y, Chen D, Lu K, Sun Z, Zeng R (2015) Enhanced tomato disease resistance primed by arbuscular mycorrhizal fungus. Front Plant Sci 28(6):786 Stadnik MJ, de Freitas MB (2014) Algal polysaccharides as source of plant resistance inducers. Trop Plant Pathol 39:111–118 Stangarlin JR, Kuhn OJ, Assi L, Schwan-Estrada KRF (2011) Control of plant diseases using extracts from medicinal plants and fungi. In: MendezVilas A (ed) Science against microbial pathogens: communicating current research and technological advances, pp 1033–1042 Strieker M, Tanovic A, Marahiel MA (2010) Nonribosomal peptide synthetases: structures and dynamics. Curr Opin Struct Biol 20:234–240 Sun A, Nie S, Xing D (2012) Nitric oxide-mediated maintenance of redox homeostasis contributes to NPR1-dependent plant innate immunity triggered by lipopolysaccharides. Plant Physiol 160:1081–1096 Surekha CH, Neelapu NRR, Siva Prasad B, Sankar Ganesh P (2014) Induction of defense enzymes and phenolic content by Trichoderma viride in Vigna mungo infested with Fusarium oxysporum and Alternaria alternata. Int J Agri Sci Res 4:31–40 Tayeh C, Siah A, Randoux B, Halama P, Walters DR et al (2014a) Topical application of inducers for disease control. In: Walters DR, Newton AC, Lyon GD (eds) Induced resistance for plant defence: A sustainable approach to crop protection. Blackwell Publishing Ltd, Oxford, pp 193–231

6 Natural Agents Inducing Plant Resistance Against Pests …

157

Tayeh C, Randoux B, Vincent D, Bourdon N, Reignault P (2014b) Exogenous trehalose induces defenses in wheat before and during a biotic stress caused by powdery mildew. Phytopathology 104:293–305 Tesfagiorgis HB, Laing MD, Annegarnet HJ (2014) Evaluation of biocontrol agents and potassium silicate for the management of powdery mildew of zucchini. Biol Control 73:8–15 Tian S, Wan Y, Qin G, Xu Y (2006) Induction of defense responses against Alternaria rot by different elicitors in harvested pear fruit. Appl Microbiol Biotechnol 70:729–734 Tinivella F, Hirata L, Celan M, Wrigh SAI, Amein T et al (2009) Control of seed-borne pathogens on legumes by microbial and other alternative seed treatments. Eur J Plant Pathol 123:139–151 Ton J, Jakab G, Toquin V, Flors V, Iavicoli A (2005) Dissecting the beta-aminobutyric acid-induced priming phenomenon in Arabidopsis. Plant Cell 17:987–999 Tonelli ML, Fabra A (2014) The biocontrol agent Bacillus sp. CHEP5 primes the defense response against Cercospora sojina. World J Microbiol Biotechnol 30:2503–2509 Toribio J, Escalante AE, Soberóron-Chavéz G (2010) Rhamnolipids: production in bacteria other than Pseudomonas aeruginosa. Eur J Lipids Sci Technol 112:1082–1087 Tran H, Ficke A, Asiimwe T, Höfte M, Raaijmakers JM (2007) Role of the cyclic lipopeptide massetolide A in biological control of Phytophthora infestans and in colonization of tomato plants by Pseudomonas fluorescens. New Phytol 175:731–742 Trdá L, Fernandez O, Boutrot F, Héloir MC, Kelloniemi J et al (2014) The grapevine flagellin receptor VvFLS2 differentially recognizes flagellin-derived epitopes from the endophytic growth-promoting bacterium Burkholderia phytofirmans and plant pathogenic bacteria. New Phytol 201:1371–1384 Trouvelot S, Héloir MC, Poinssot B, Gauthier A, Paris F et al (2014) Carbohydrates in plant immunity and plant protection: roles and potential application as foliar sprays. Front Plant Sci 5:592 Tubajika KM, Civerolo EL, Puterka GJ, Hashim JM, Luvisi DA (2007) The effects of kaolin, harpin, and imidacloprid on development of Pierce’s disease in grape. Crop Prot 26:92–99 Van Bockhaven J, Spıchal L, Novak O, Strnad M, Asano T et al (2014) Silicon induces resistance to the brown spot fungus Cochliobolus miyabeanus by preventing the pathogen from hijacking the rice ethylene pathway. New Phytol 206:761–773 Van Peer R, Niemann GJ, Schippers B (1991) Induced Resistance and phytoalexin accumulation in biological control of of fusarium wilt of carnation by Pseudomonas sp. strain WCS417r. Phytopathology 81:728–734 Van Peer R, Schippers B (1992) Lipopolysaccharides of plant growth-promoting Pseudomonas sp. strain WCS417r induce resistance in carnation to Fusarium wilt. Neth J Plant Pathol 96:119–132 Van Wees SCM, Pieterse CMJ, Trijssenaar A, Van’t Westende YAM, Hartog F, et al (1997) Differential induction of systemic resistance in Arabidopsis by biocontrol bacteria. Mol Plant Microbe Interact 10:716–724 Vander P, Varum KM, Domard A, El Gueddari NE, Moerschbacher BM (1998) Comparison of the ability of partially N-adetylated chitosans and chitooligosaccharides to elicit resistance reactions in wheat leaves. Plant Physiol 111:1353–1359 Varnier AL, Sanchez L, Vatsa P, Boudesocque L, Garcia-Brugger A et al (2009) Bacterial rhamnolipids are novel MAMPs conferring resistance to Botrytis cinerea in grapevine. Plant Cell Environ 32:178–193 Vatsa P, Sanchez L, Clément C, Baillieul F, Dorey S (2010) Rhamnolipid biosurfactants as new players in animal and plant defense against microbes. Int J Mol Sci 11:2095–5108 Veloso J, Alabouvette C, Olivain C, Flors V, Pastor V et al (2016) Modes of action of the protective strain Fo47 in controlling verticillium wilt of pepper. Plant Pathol 65:997–1007 Vera J, Castro J, Contreras RA, Gonzalez A, Moenne A (2012) Oligo-carrageenans induce a longterm and broad-range protection against pathogens in tobacco plants (var. Xanthi). Physiol Mol Plant Pathol 79:31–39

158

A. Siah et al.

Vergnes S, Ladouce N, Fournier S, Ferhout H, Attia F et al (2014) Foliar treatments with Gaultheria procumbens essential oil induce defense responses and resistance against a fungal pathogen in Arabidopsis. Front Plant Sci 5:477 Villaverde JJ, Sevilla-Moran B, Sandín-Espana P, Lopez-Goti C, Alonso-Prados JL (2014) Biopesticides in the framework of the European Pesticide Regulation (EC) No. 1107/2009. Pest Manag Sci 70:2–5 Waewthongrak W, Pisuchpen S, Leelasuphakul W (2015) Effect of Bacillus subtilis and chitosan applications on green mold (Penicilium digitatum Sacc.) decay in citrus fruit. Postharvest Biol Technol 99:44–49 Walters DR, Fountaine JM (2009) Practical application of induced resistance to plant diseases: an appraisal of effectiveness under field conditions. J Agric Sci 147:523–535 Walters DR, Bennett AE (2014) Microbial induction of resistance to pathogens. In: Walters DR, Newton AC, Lyon GD (eds) Induced resistance for plant defence: a sustainable approach to crop protection. Blackwell Publishing Ltd, Oxford, pp 149–170 Walters DR, Paterson L, Walsh DJ, Havis ND (2009) Priming for plant defense in barley provides benefits only under high disease pressure. Physiol Mol Plant Pathol 73:95–100 Walters DR, Paterson L, Havis ND (2010) Control of foliar diseases of spring barley using resistance elicitors. Proc Crop Prot North Britain 91–96 Walters DR, Ratsep J, Havis ND (2013) Controlling crop diseases using induced resistance: challenges for the future. J Exp Bot 64:1263–1280 Wang X, Wang L, Wang J, Jin P, Liu H et al (2014) Bacillus cereus AR156-induced resistance to Colletotrichum acutatum is associated with priming of defense responses in loquat fruit. PLoS ONE 9(11):e112494 Wang H, Yang X, Guo L, Zeng H, Qiu D (2015) PeBL1, a novel protein elicitor from Brevibacillus laterosporus strain A60, activates the defense responses and systemic resistance in Nicotiana benthamiana. Appl Environ Microbiol 81:2706–2716 Wang K, Liao Y, Xiong Q, Kan J, Cao S et al (2016) Induction of direct or priming resistance against Botrytis cinerea in strawberries by b-aminobutyric acid and their effects on sucrose metabolism. J Agric Food Chem 64:5855–5865 Waqas M, Khan AL, Hamayun M, Shahzad R, Kim YH et al (2015) Endophytic infection alleviates biotic stress in sunflower through regulation of defence hormones, antioxidants and functional amino acids. Eur J Plant Pathol 141:803–824 Willmann R, Lajunen HM, Erbs G, Newman MA, Kolb D et al (2011) Arabidopsis lysin-motif proteins LYM1 LYM3 CERK1 mediate bacterial peptidoglycan sensing and immunity to bacterial infection. Proc Natl Acad Sci USA 108:19824–19829 Xing K, Zhu X, Peng X, Qin Q (2015) Chitosan antimicrobial and eliciting properties for pest control in agriculture: a review. Agron Sustain Dev 35:569–588 Yacoub A, Gerbore J, Magnin N, Chambon P, Dufour MC et al (2016) Ability of Pythium oligandrum strains to protect Vitis vinifera L., by inducing plant resistance against Phaeomoniella chlamydospora, a pathogen involved in Esca, a grapevine trunk disease. Biol Control 92:7–16 Yamamoto S, Shiraishi S, Kawagoe Y, Mochizuki M, Suzuki S (2015) Impact of Bacillus amyloliquefaciens S13-3 on control of bacterial wilt and powdery mildew in tomato. Pest Manag Sci 71:722–727 Yang M, Ji X (2009) Studies on the induce-resistance effect of harpin on downy mildew and Botrytis cinerea of cucumber. Chin Agric Sci Bull 25:228–230 Yao Q, Yang XF, Liang Y, Xu F, Liu Z et al (2007) Expression of a Magnaporthe grisea elicitor and its biological function in activating resistance in rice. Rice Sci 14:149–156 Yap MN, Rojas CM, Yang CH, Charkowski AO (2006) Harpin mediates cell aggregation in Erwinia chrysanthemi 3937. J Bacteriol 188:2280-2284 Yin H, Li S, Zhao X, Du Y, Ma X (2006) cDNA microarray analysis of gene expression in Brassica napus treated with oligochitosan elicitor. Plant Physiol Biochem 44:910–916 Yin H, Zhao X, Du Y (2008) The primary study of oligochitosan inducing resistance to Sclerotinie scleraotiorum on B. napus. J Biotechnol 136:600–601

6 Natural Agents Inducing Plant Resistance Against Pests …

159

Younes I, Rinaudo M (2015) Chitin and chitosan preparation from marine sources. Structure, properties, applications. Mar Drugs 13:1173–1174 Yu T, Yu C, Lu H, Zunun M, Chen F et al (2012) Effect of Cryptococcus laurentii and calcium chloride on control of Penicillium expansum and Botrytis cinerea infections in pear fruit. Biol Control 61:169–175 Yu C, Zeng L, Sheng K, Chen F, Zhou T et al (2014) c-Aminobutyric acid induces resistance against Penicillium expansum by priming of defence responses in pear fruit. Food Chem 159:29–37 Zamioudis C, Pieterse CM (2012) Modulation of host immunity by beneficial microbes. Mol Plant Microbe Interact 25:139–150 Zeidler D, Dubery IA, Schmitt-Koplin P, Von Rad U, Durner J (2010) Lipopolysaccharides mobility in leaf tissues of Arabidopsis thaliana. Mol Plant Pathol 11:747–755 Zhang Y, Yang X, Liu Q, Qiu D, Zhang Y et al (2010) Purification of novel protein elicitor from Botrytis cinerea that induces disease resistance and drought tolerance in plants. Microbiol Res 165:142–151 Zhang Q, Yong D, Zhang Y, Shi X, Li B et al (2016a) Streptomyces rochei A-1 induces resistance and defense-related responses against Botryosphaeria dothidea in apple fruit during storage. Postharvest Biol Technol 115:30–37 Zhang F, Ge H, Zhang F, Guo N, Wang Y et al (2016b) Biocontrol potential of Trichoderma harzianum isolate T-aloe against Sclerotinia sclerotiorum in soybean. Plant Physiol Biochem 100:64–74 Zipfel C, Robatzek S, Navarro L, Oakeley EJ, Jones JDG et al (2004) Bacterial disease resistance in Arabidopsis through flagellin perception. Nature 428:764–767

Part III

Antiparasitic Natural Products (Human Health)

Chapter 7

Antileishmanial and Antitrypanosomal Activities of Flavonoids Flore Nardella, Jean-Baptiste Gallé, Mélanie Bourjot, Bernard Weniger and Catherine Vonthron-Sénécheau

Abstract This chapter focuses on the ability of flavonoid aglycones and glycosides to inhibit the growth of protozoan parasites of the genera Leishmania and Trypanosoma in different in vitro and in vivo models, namely pathogenic species for humans implicated in visceral (L. donovani, L. infantum) and cutaneous leishmaniasis (L. braziliensis, L. major, L. enriettii, L. mexicana), African sleeping sickness (T. brucei), and Chagas disease (T. cruzi). Several hundred naturally occurring flavonoids and their synthetic analogues were selected from a literature survey combining flavonoids exhibiting IC50 values smaller than 50 µM in one or more of the commonly used assays for antileishmanial or antitrypanosomal activity assessment. The trend of this survey indicates that the most active compounds against Leishmania spp. belong to chalcone, biflavone, and aurone classes, regardless of the performed assay. Few compounds of these classes exhibit submicromolar antileishmanial activity against axenic or intracellular amastigotes with no in vitro cytotoxicity. Flavones and flavonols are globally less active than chalcones, biflavones, and aurones. Flavones, flavan-3-ols, and isoflavones are less studied and less active. The most promising compounds against Trypanosoma brucei belong to synthetic chalcone, isoflavan, and isoflavanol classes, displaying submicromolar activities on trypomastigotes and selectivity indexes hundred times higher. On T. cruzi, flavan-3-ols are the most potent compounds, followed by flavones and flavonols. Globally, fewer compounds have been evaluated on T. cruzi than on T. brucei, and their activities and selectivity are often lower.




Keywords Flavonoid Leishmania Aurone Flavonol Isoflavonoid








Trypanosoma
Chalcone

F. Nardella Laboratoire d’Innovation Thérapeutique UMR CNRS 7200, Faculty of Pharmacy, University of Strasbourg, 74 route du Rhin CS 60024, 67401 Illkirch Cedex, France J.-B. Gallé
M. Bourjot
B. Weniger
C. Vonthron-Sénécheau (&) Laboratoire d’Innovation Thérapeutique UMR CNRS 7200, Faculty of Pharmacy, University of Strasbourg, 74 route du Rhin CS 60024, 67401 Illkirch, France e-mail: [email protected] © Springer International Publishing AG 2018 J.-M. Mérillon and C. Rivière (eds.), Natural Antimicrobial Agents, Sustainable Development and Biodiversity 19, https://doi.org/10.1007/978-3-319-67045-4_7

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Introduction Flavonoids

Flavonoids (chalcones, aurones, yellow flavonols) are almost universal plant pigments which contribute to the particularly attractive flowers’ color for entomophilic pollination. Flavonoids are found in leaves where some of them exert ultraviolet protective effects thanks to their absorption properties (approx. between 280 and 315 nm). Other flavonoids play a role in the relation between plants and phytophagous animals or as resistance agents to diseases (e.g., antifungal isoflavones) (Bruneton and Poupon 2016). Flavonoids share the same biosynthetic origin leading to the occurrence of a common structural feature: the 2-phenyl chromone moiety (or 3-phenyl chromone for isoflavonoids). Various classes of flavonoids can be defined according to the degree of oxidation of the central pyranic moiety, which can be opened or recyclized in a furanic moiety. Several thousand flavonoids are already described, and they are mostly water soluble. As heterosides, they are easily extractible from plant material using water or alcohol, even if a few of them, like rutinoside and hesperidoside, are poorly soluble in these solvents. Aglycones are generally soluble in apolar organic solvents. In this chapter, we will focus on flavonoids lato sensu: flavonols, flavones, flavanones, chalcones, dihydrochalcones, aurones, flavan-3-ols, isoflavonoids, and biflavonoids (Fig. 7.1). This class of compounds exhibits a broad spectrum of biological and pharmacological activities, generally associated with free radicals scavenging activity and antioxidant and anti-inflammatory activities, with therapeutic applications in cardiovascular diseases (veinotropic activity) and other diseases related with aging (skin aging, vascular aging, etc.) (Min-Hsiung et al. 2010; George et al. 2016). Nevertheless, recent experimental research revealed pharmacological activities of flavonoids in unexpected areas, like parasitic diseases (for review see Ntie-Kang et al. 2014; Nabavi et al. 2016).

7.1.2

Leishmaniasis and Trypanosomiasis

Leishmaniasis and trypanosomiasis are neglected parasitic diseases due to protozoan parasites, affecting millions of people in tropical regions. In these areas, people are accustomed to rely on plants for healing. Many reports based on traditional knowledge, dealing with isolation and identification of natural products (NPs) active against protozoan parasites, were published in the last decades. One of the last comprehensive reviews on NPs against neglected diseases was published by Schmidt and co-workers (Schmidt et al. 2012, Schmidt et al. 2012a; Llurba Montesmo et al. 2015).

7 Antileishmanial and Antitrypanosomal Activities of Flavonoids 3'

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If alkaloids, quinones, and terpenes show marked activity against trypanosomatidae, flavonoids are not outdone (Hoet et al. 2004; Schmidt et al. 2012a; Jones et al. 2013). The present chapter attempts to analyze quantitatively the ability of flavonoids and their synthetic analogues to inhibit the growth of trypanosomatidae parasites of the Leishmania and Trypanosoma genera with the aim to evaluate the potential of this kind of scaffold in the search of new bioactive compounds.

7.1.3

Methodology

To gather the activity of flavonoids against kinetoplastidae, we focused our bibliographic work on two human parasite genera Leishmania and Trypanosoma. SciFinder and Web of Science databases were searched with the keywords “class of flavonoid (i.e., flavones, flavonols, 2,3-dihydroflavonols, biflavonoids, aurones, chalcones, dihydrochalcones, flavan, flavan-3-ols, isoflavones, isoflavans, and isoflavanols) + Leishmania” or “class of flavonoid + Trypanosoma.” After discarding the publications with unsatisfying methodology, the data of 36 publications for Leishmania and 43 publications for Trypanosoma were treated. We retained the

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compounds displaying IC50 values lower than 50 µM on the in vitro-cultured parasites and recorded, if described, their activity versus mammalian cells. Concentration values expressed in mass concentration were converted into molar concentration to compare the activities of the different compounds. We selected compounds isolated from natural sources as well as synthetic analogues when they were investigated (mostly chalcones and aurones). We focused on compounds screened on phenotypic models describing the activity versus the whole parasites. Data obtained from enzymatic assays were discarded. In the case of compounds evaluated by several research groups, we retained the best IC50 values recorded for each form of the considered parasite. Regarding cytotoxicity, we also kept the lowest IC50 values on the different mammalian cell lines. The selectivity index (SI) was calculated as the ratio of cytotoxic activity to antiparasitic activity. We also recorded the in vivo data when they were available. We plotted the data regardless of the used test (based on cell redox metabolism, transformed parasites with a reporter gene, microscopy, etc.) but sensitive to the form of the parasite. For T. cruzi, for which only few data were available, the IC50 values against the different forms of the parasite were gathered on a single graph.

7.2 7.2.1

Major Diseases Caused by Trypanosomatidae Leishmaniasis

Leishmaniasis is a group of diseases caused by the bite of a female sandfly infected by one of the twenty pathogenic Leishmania species. Different clinical manifestations can occur depending on the parasite species and the immune system of the patient. They range from single cutaneous self-healing lesions to fatal visceral leishmaniasis (Sundar and Chakravarty 2014). Human leishmaniasis is distributed worldwide, but mainly in the tropics and subtropics. The World Health Organization estimates that about 12 million people are affected by leishmaniasis in 98 countries with more than 350 million people at risk. The annual incidence of new cases is about 2 million: 1.5 million for cutaneous leishmaniasis (CL) and 500,000 for visceral leishmaniasis (VL) (WHO 2010). Two morphological forms of the parasite are present during its life cycle (Kumar 2013; Center for Disease Control and Prevention 2015a) (Fig. 7.2). Leishmaniasis has three main clinical forms depending on the infecting species (WHO 2010): • Visceral leishmaniasis: the parasite invades the liver, the spleen, and the bone marrow causing a severe systemic disease, fatal if untreated. This manifestation is mainly due to the species L. donovani and L. infantum.

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Fig. 7.2 Life cycle of Leishmania spp. Promastigotes are injected into a vertebrate host during the insect blood meal and phagocytized by macrophages. They transform into amastigotes within the macrophage phagolysosome, where they survive and multiply. The amastigote form is responsible for the symptomatology of the disease. Adapted from (Center for Disease Control and Prevention 2015a)

• Mucocutaneous leishmaniasis: this manifestation is characterized by lesions in the mucosal tissue of the nose and the mouth and often progresses to massive tissue destruction and disfigurement. The principal species concerned are L. braziliensis and L. panamensis. • Cutaneous leishmaniasis leads to the development of self-healing but chronic skin ulcers at the site of sandfly bites. The most involved species are L. major, L. tropica, L. amazonensis, L. braziliensis, and L. peruviana. Until now, no vaccines are available for either human visceral or cutaneous leishmaniasis. The current treatments rely on chemotherapy, but available drugs are limited in number and suffer from various shortcomings: They mostly require long treatment courses with parenteral administration and have toxic side effects, and drug-resistant parasites are emerging (Sundar and Chakravarty 2014). Thus, there is an urgent need for new antileishmanial drugs. In this chapter, we focus on the activities of flavonoids against the various forms of the parasite. Several in vitro assays are available regarding those multiple forms (Gupta and Nishi 2011). The bioactivity against promastigotes is easy to evaluate, due to relatively simple culture conditions. However, this assay is the less relevant one because the promastigote form is exclusively present in the vector and proteomic studies showed significant modifications of the proteome between the promastigote and amastigote stages (Rosenzweig et al. 2008). The axenic amastigote form is closer to the one present in the vertebrate host but is obtained by artificial culture conditions and is therefore solely semi-predictive.

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Assays involving both forms do not take into account neither the crossing through the host cell membranes nor the peculiar environment inside the phagolysosome (Gupta and Nishi 2011). The most relevant in vitro assay for the evaluation of the antileishmanial compounds relies on the survival of intramacrophagic amastigotes. This model is much closer to the human physiopathology; nevertheless, it is more complex to perform due to the necessity of coculturing macrophages and parasites (Gupta and Nishi 2011). Various species have been used in the in vitro assays we considered, including species responsible for cutaneous (L. amazonensis, L. major, L. mexicana, L. peruviana, L. tropica), mucocutaneous (L. braziliensis), visceral (L. donovani, L. infantum), and nonpathogenic to human (L. enriettii) leishmaniasis.

7.2.2

Trypanosomiases

Trypanosomiases are vector-borne parasitic diseases caused by protozoan parasites belonging to the genus Trypanosoma. Two subgenera can infect humans: T. cruzi and T. brucei, the latter comprising two pathogenic species: T. brucei gambiense and T. brucei rhodesiense. T. brucei is responsible for human African trypanosomiasis (HAT), also known as sleeping sickness, and T. cruzi is responsible for Chagas disease, also known as American trypanosomiasis. The control of these diseases is complicated by the large reservoir of parasites in wild animals.

7.2.2.1

Sleeping Sickness

Sleeping sickness threatens 65 million of people living in sub-Saharan Africa where the vector is present (tsetse fly, Glossina sp.). People living in rural areas are the most exposed. In 2014, 3.796 new cases have been reported and the estimated number of actual cases is below 20,000. T. b. rhodesiense is responsible for less than 2% of cases, whereas T. b. gambiense infection represents more than 98% of the reported cases (WHO 2016a). The latter is endemic in 24 countries in West and Central Africa, whereas T. b. rhodesiense occurs mainly in 13 countries in eastern and southern Africa (Steinmann et al. 2015). The parasite is essentially transmitted during the blood meal of the tsetse fly, but other transmission routes exist like mother-to-child, sexual contact, or contaminated blood transmission. The parasite life cycle is described in Fig. 7.3 (Langousis and Hill 2014; Center for Disease Control and Prevention 2015b). The chronology of the disease will depend on the nature of the parasite. T. b. gambiense will induce a chronic infection, asymptomatic for months or even years, whereas T. b. rhodesiense pathology develops rapidly, within a few weeks or months after the infection. In both cases, the disease evolves in two stages. During the first stage of the infection, called haemo-lymphatic stage, trypanosomes multiply in subcutaneous tissues, blood, and lymph. Common symptoms are nonspecific like fever, headaches, itching, and joint pains. In the second stage, called meningo-encephalitic stage, the parasites reach the

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Fig. 7.3 Life cycle of Trypanosoma brucei sp. The life cycle of T. brucei involves two hosts: a mammalian definitive host and a vector insect from the genus Glossina. Body fluids trypomastigotes are responsible for the clinical form of sleeping sickness. Adapted from (Center for Disease Control and Prevention 2015b)

central nervous system. Symptoms are more specific and include behavioral changes, confusion, sensory perturbations, poor coordination, and sleep disturbance (which gave the disease its name). Without treatment, sleeping sickness is considered as fatal. The earlier the treatment is implemented, the higher the chances of recovery. The drugs used to cure the haemo-lymphatic stage are pentamidine and suramine. Three drugs are available to treat the meningo-encephalic stage: melarsoprol (highly toxic), eflornithine (only effective against T. b. gambiense), and nifurtimox associated with eflornithine (not studied for T. b. rhodesiense) (Bonnet et al. 2015; Sutherland et al. 2015; WHO 2016a). These treatments are poorly tolerated and need to be taken for a long period of time.

7.2.2.2

Chagas Disease

The World Health Organization (WHO) estimates that 7 million people are infected by T. cruzi worldwide, mainly in Latin America (WHO 2016b), and about 10,000 people die of complications from Chagas disease each year. This disease is responsible for an economic burden of 7.19 billion $US per year (Robertson et al. 2016). T. cruzi parasites are mainly transmitted by feces and urine of infected triatomine bugs (mostly from Triatoma, Panstrongylus, and Rhodnius genera) after a blood meal, when people scratch the bite area and introduce the parasite in damaged skin or in their mouth or eyes. Other transmission ways exist, like organ transplantation, blood transfusion, or foodborne transmission contaminated by

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feces/urine of the infected bug (Robertson et al. 2016). T. cruzi life cycle is described in Fig. 7.4. The disease presents two phases: an acute phase that lasts about 2 months and a chronic phase that develops years and decades after infection. During the acute phase, parasites are present at high levels in the blood, but this phase is almost asymptomatic. During the chronic phase, the parasite will infect predominantly cardiac and digestive muscles. Up to 30% of patients will develop cardiomyopathy and 10% will develop neurologic or digestive complications like megacolon or mega-esophagus (Morrot et al. 2016) that can lead ultimately to death. In Latin America, it is the major cause of death from cardiomyopathy (Chatelain 2015). Currently, no vaccine is available, but two drugs, benznidazole and nifurtimox, are available to treat Chagas disease. The treatment is 100% effective only if it is given soon after infection and its efficacy in the chronic phase has not been demonstrated. These drugs are not recommended for pregnant women and people with liver or kidney failure and severe heart diseases. A symptomatic treatment can be associated to treat cardiac or digestive manifestations. As it is the case for T. brucei, the used drugs induce a lot of side effects and should be taken for long duration to be effective. It is thus necessary to find new drugs that are more effective, safer, and cheaper for these two neglected diseases. In this chapter, we focus on the in vitro activities of flavonoids against trypomastigotes of T. brucei and trypomastigotes, amastigotes, and epimastigotes of T. cruzi. As reported earlier, T. b. gambiense is responsible for 98% of all sleeping sickness cases; this species should thus be preferred for in vitro screening.

Fig. 7.4 Life cycle of Trypanosoma cruzi. The life cycle of T. cruzi involves two hosts: a mammalian definitive host and a vector insect from the genera Triatoma, Panstrongylus, or Rhodnius. Bloodstream trypomastigotes and intracellular amastigotes are responsible for the clinical form of Chagas disease during the acute phase, whereas amastigotes are the main contributors during the chronic phase of the disease. Adapted from (Center for Disease Control and Prevention 2015c)

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Unfortunately, the limited propagation of this species in vitro and in vivo dramatically reduces its use. Therefore, T. b. rhodesiense and T. b. brucei are mostly used for screening campaigns. The bloodstream trypomastigote form being the most clinically relevant, it is the most common tested form (Sykes and Avery 2013). T. cruzi in vitro screening lacks well-established protocols. Screening campaigns are often realized on the amastigote form. To obtain this form, mammalian cells are infected with metacyclic trypomastigotes, form inoculated by the insect, that differentiate into intracellular amastigotes. Ultimately, infected cells will release bloodstream trypomastigotes, which are more clinically relevant than metacyclic trypomastigotes. It seems that during the acute phase, extracellular and intracellular forms are the main contributors to the disease, whereas during the chronic phase, amastigotes are more clinically relevant. Thus, in vitro models that assess the effect on both trypomastigotes and amastigotes may lead to more successful identification of lead compounds than models assessing the activity on these two forms separately (Sykes and Avery 2013). In the publications we analyzed, depending on the considered form, different techniques were used to assess the in vitro efficacy on parasites of the Trypanosoma genus. The activity against the epimastigote form is evaluated using tritiated thymidine uptake into the parasites’ DNA during its division, or microscopy count. The viability of trypomastigotes is evaluated using colorimetric or fluorimetric assays (MTT or resazurin assay), or microscopy count. The activity against the amastigotes is measured using the colorimetric assay of transformed parasites expressing the b-galactosidase reporter gene (C2C4 Tulahuen strain).

7.3

Antileishmanial Activity of Flavonoids

Nine classes of flavonoids, including 323 natural and synthetic compounds, exhibit antileishmanial activities below 50 µM. Chalcones from synthetic and natural sources, natural biflavones, and synthetic aurones are the most active compounds regardless the form of the parasite and the antileishmanial in vitro performed assay. Some flavones and flavonols also show submicromolar activities, but they are globally less active than the former ones. Flavones, flavan-3-ols, and isoflavones are less represented and mostly studied against Leishmania axenic amastigotes (Fig. 7.5).

7.3.1

Chalcones and Dihydrochalcones

Chalcone is the most representative class of natural or natural-derived flavonoids tested against Leishmania spp. due to the high number of synthetic analogues. The intensity of synthetic efforts underlines the putative importance of chalcones or dihydrochalcones as leads for antileishmanial drug development (Fig. 7.5).

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Flavan-3-ols Dihydrochalcones Chalcones Biflavones

IC 50 (µM)

0 0 0 1

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Fig. 7.5 Overview of the in vitro antileishmanial and cytotoxic activities of flavonoid classes. The most active compounds against Leishmania spp belong to chalcones (synthetic and natural), biflavones (exclusively natural), and aurones (exclusively synthetic), regardless the form of the parasite and the antileishmanial in vitro assay performed. Although flavones and flavonols include some compounds exhibiting submicromolar activity, they are globally less active than the formers. Flavones, flavan-3-ols, and isoflavones are less represented and were studied mostly against Leishmania axenic amastigotes. All the described compounds (323) were selected on the basis of their flavonoidic nature and their in vitro antileishmanial properties against promastigotes, axenic amastigotes, and/or intracellular amastigotes (IC50 values < 50 µM). Corresponding cytotoxicity against mammalian cells, if investigated, was also plotted. The vertical bar corresponds to the median of each dataset

The different combinations of substitutions essentially by hydroxylations, methoxylations of the A and B cycles, and prenylations offer a large diversity of molecules. Actually, the chalcone class is the most active class of flavonoids against Leishmania promastigotes (Fig. 7.6), with activities ranging from 0.22 to 49 µM (Chen et al. 1993; Nielsen et al. 1998; Torres-santos et al. 1999; Zhai et al. 1999; Kayser and Kiderlen 2001; Hermoso et al. 2003; Lunardi et al. 2003; Boeck et al. 2006; Borges-Argaez et al. 2007; Flores et al. 2007; Borges-Argaez et al. 2009; Bello et al. 2011; de Mello et al. 2014; Passalacqua et al. 2015). Eighteen compounds show strong submicromolar activity (Kayser and Kiderlen 2001; Hermoso et al. 2003; Boeck et al. 2006). The most active compound against promastigotes of L. donovani is the chalcone 1, isolated from Piper hispidum, with an IC50 value of 0.22 µM (Kayser and Kiderlen 2001) (Fig. 7.8). In contrast, few compounds are active against axenic amastigotes (one dozen), but some display good activities (Nielsen et al. 1998; Kayser and Kiderlen 2001; Salem and Werbovetz 2005, 2006; Ruiz et al. 2011). Interestingly, several dozens of chalcones (mainly of synthetic

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origin) are active in the micromolar or even the submicromolar range against intramacrophagic amastigotes (Zhai et al. 1999; Kayser and Kiderlen 2001; Boeck et al. 2006; Gupta et al. 2014; Passalacqua et al. 2015), but most of them show high cytotoxic activity (Fig. 7.7). The natural prenylated chalcone licochalcone A (2) bears submicromolar activity against intracellular amastigotes of L. donovani, and its synthetic analogues (3-5) demonstrate the same range of activity (0.42 < IC50 values < 0.81 µM). After intraperitoneal and oral administration, licochalcone A also exhibits significant in vivo antileishmanial effect in mice and hamsters infected respectively with L. major and L. donovani (Chen et al. 1994). The two synthetic chalcone derivatives 4 and 5 were further investigated on hamsters infected with L. donovani. After 6 days of intraperitoneal administration of 5 or 20 mg/kg body weight per day, the treated animals showed significant reduction of the parasite load in the liver (97 and 84%, respectively) and the spleen (88 and 70%) (Zhai et al. 1999). Two other synthetic chalcones, 6 and 7, were investigated in vivo by Gupta and colleagues (Gupta et al. 2014). These two compounds induced a significant reduction of the parasite load (48.54 ± 10.55% and 83.32 ± 12.37%, respectively) when hamsters were treated intraperitoneally for 5 and 10 days respectively at 50 mg/kg/day.

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Fig. 7.6 In vitro antileishmanial activity of flavonoid classes against the promastigote form. The described compounds (175) have been tested on promastigotes of the species L. amazonensis (Boeck et al. 2006; Mai et al. 2015; Passalacqua et al. 2015), L. braziliensis (Hermoso et al. 2003; Lunardi et al. 2003; Marin et al. 2009; Bello et al. 2011; Ramirez-Macias et al. 2012; de Mello et al. 2014), L. donovani (Nielsen et al. 1998; Kayser and Kiderlen 2001; del Rayo Camacho et al. 2004; Borges-Argaez et al. 2007; Flores et al. 2007; Rudrapaul et al. 2014; Akimanya et al. 2015), L. enriettii (Kayser et al. 1999; Kayser and Kiderlen 2001), L. infantum (Kayser et al. 1999; Hermoso et al. 2003; Ramirez-Macias et al. 2012; Passalacqua et al. 2015), L. major (Kayser et al. 1999; Zhai et al. 1999; Kayser and Kiderlen 2001), L. mexicana (Borges-Argaez et al. 2009), L. peruviana (Marin et al. 2009), and L. tropica (Hermoso et al. 2003). Corresponding cytotoxicity against mammalian cells, if investigated, was also plotted. The vertical bar corresponds to the median of each dataset

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Fig. 7.7 In vitro antileishmanial activity of flavonoid classes against the amastigote form. The described compounds have been tested on a axenic amastigotes (96 compounds) of L. amazonensis (Salvador et al. 2009; Ruiz et al. 2011), L. braziliensis (Ramirez-Macias et al. 2012), L. donovani (Kirmizibekmez et al. 2004; Weniger et al. 2004; Salem and Werbovetz 2005, 2006; Tasdemir et al. 2006; Kunert et al. 2008; Nour et al. 2010; Salem et al. 2011), L. infantum (Ramirez-Macias et al. 2012) and b intracellular amastigotes (78 compounds) of L. amazonensis (Boeck et al. 2006; Rizk et al. 2014; Passalacqua et al. 2015), L. braziliensis (Ramirez-Macias et al. 2012), L. donovani (Kayser et al. 1999; Zhai et al. 1999; Kayser and Kiderlen 2001; Gupta et al. 2014; Rudrapaul et al. 2014), and L. infantum (Ramirez-Macias et al. 2012; Roussaki et al. 2012; Passalacqua et al. 2015). Corresponding cytotoxicity against mammalian cells, if investigated, was also plotted. The vertical bar corresponds to the median of each dataset

7 Antileishmanial and Antitrypanosomal Activities of Flavonoids

7.3.2

175

Aurones

Few aurones were tested against Leishmania parasites, and the selected compounds for the current study are exclusively of synthetic origin (13 compounds). Nevertheless, Figs. 7.6 and 7.7b show that this class of flavonoids includes the most active compounds against promastigotes (0.15 < IC50 values < 1 µM) and intracellular amastigotes (0.1 µM < IC50 values < 16.8 µM), but also some cytotoxic compounds (Kayser et al. 1999; Roussaki et al. 2012). Compound 8 shows strong activity against L. enriettii promastigotes (IC50 value = 0.15 µM), an activity comparable to that of the most active chalcones, with a good selectivity (SI = 78), but its activity against intracellular amastigotes of L. donovani is less interesting with a mild IC50 value (6.6 µM) and a low selectivity (SI = 1.78) (Kayser et al. 1999) (Fig. 7.8). The benzoate derivative 9 exhibits a significant activity against intracellular amastigotes of L. donovani (IC50 value = 0.11 µM) together with a good selectivity (SI = 59) (Kayser et al. 1999). From a structural point of view, these compounds are closely related, differing only by the nature of the substitution in position 6, respectively a methoxy and a benzoyl group. Some (4,6)-dimethoxy aurones also show a notable activity in the low micromolar range (1.3 µM < IC50 values < 2.1 µM) against intracellular amastigotes of L. infantum (Roussaki et al. 2012). Finally, IC50 values for cytotoxic activities against monocytes and macrophages are rather high (6.5 µM < IC50 values < 100 µM), suggesting a real potential of this class of flavonoids against Leishmania parasites.

7.3.3

Flavonols

Synthetic analogues are somewhat less active against promastigotes than natural flavonols. Overall, antileishmanial activity of flavonols is comprised between 1 and 50 µM, but most of them exhibit IC50 values higher than 10 µM (Tasdemir et al. 2006; Marin et al. 2009; Salvador et al. 2009; Salem et al. 2011; Ramirez-Macias et al. 2012; Rudrapaul et al. 2014; Mai et al. 2015). The most active compound described so far is vitecetin (10), a naturally occurring flavonol isolated from Vitex peduncularis (Verbenaceae) by Rudrapaul and colleagues (Fig. 7.8). Vitecetin shows submicromolar activity against intracellular amastigotes of L. donovani (IC50 = 0.93 µM) with a good selectivity compared to THP-1-derived macrophages (SI = 133), as well as a quite good activity against promastigotes (IC50 = 2.4 µM, SI = 52) (Rudrapaul et al. 2014). Regarding antileishmanial activity against axenic amastigotes of L. donovani or L. amazonensis, polyhydroxylated aglycones like quercetin (Marin et al. 2009), galangin 3-methyl ether (Salvador et al. 2009), myricetin, apigenin, galangin, luteolin, and fisetin (Tasdemir et al. 2006) were the most active compounds (2 µM < IC50 < 6 µM). Only one glycosylated flavonol isolated from Lychnophora markgravii, tiliroside (11), showed a comparable

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activity with an IC50 value of 1.13 µM against axenic amastigotes of L. amazonensis (Salvador et al. 2009). Glycosylated flavonols like petioleroside, paenoside, astragalin (Ramirez-Macias et al. 2012), quercitrin, rutin, or hesperidin (Tasdemir et al. 2006) were less active (IC50 values > 10 µM). The flavonols, fisetin, quercetin, myricetin, and 3-hydroxyflavone which showed low micromolar activities against

CHALCONES R2 R1

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1 (Kayser and Kiderlen 2001; Ruiz et al. 2011) Piper hispidum (Piperaceae)

L. donovani promastigotes L. amazonensis axenic amastigotes L. donovani intracellular amastigotes Cytotoxic activity

2 Licochalcone R1 = OCH3, R2 = R5 = H, R3 = OH, R4 = C(CH3)2-CH=CH2 3 R1 = R2 = H, R2 = R3 = OCH3, R5 = CH2CH=CH2 4 R1 = R3 = H, R2 = R4 = OCH3, R5 = CH2CH=CH2 5 R1 = R3 = OCH3, R2 = R4 = H, R5 = CH2CH=CH2 (Zhai et al. 1999)

IC50 = 0.2 μM, SI = 7 IC50 = 0.8 μM, SI = 2 IC50 = 1.4 μM, SI = 1

L. major promastigotes L. donovani intracellular amastigotes

2

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4

7.2 μM

7.1 μM

6.0 μM 5.8 μM

0.9 μM

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O

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O

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OCH3 O

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N

O

6 (Gupta et al. 2014) Synthetic L. donovani intracellular amastigotes Cytotoxic activity

5

IC50 = 2.0 μM, SI = 163 IC50 = 325.4 μM (VERO cells)

7 (Gupta et al. 2014) Synthetic L. donovani intracellular amastigotes Cytotoxic activity

IC50 = 3.1 μM, SI = 47 IC50 = 146.5 μM (VERO cells)

AURONES H3CO

O

O OH O

8 (Kayser et al. 1999) Synthetic L. enriettii promastigotes L. donovani intracellular amastigotes Cytotoxic activity

L. enriettii promastigotes L. donovani intracellular IC50 = 6.6 μM, SI = 2 amastigotes IC50 = 11.7 μM (murine macrophages) Cytotoxic activity

IC50 = 0.2 μM, SI = 78

O

O

OH O

9 (Kayser et al. 1999) Synthetic IC50 = 0.3 μM, SI = 26

IC50 = 0.1 μM, SI = 59

IC50 = 6.5 μM (murine macrophages)

Fig. 7.8 Structure and biological activities of the most potent flavonoid compounds against Leishmania spp

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FLAVONOLS OCH3 OH H3CO

O

HO

O O

OH

HO

OH O

OCH3 OH

O

O O

10 Vitecetin (Rudrapaul et al. 2014) Vitex peduncularis (Verbenaceae) L. donovani promastigotes L. donovani intracellular amastigotes Cytotoxic activity

OH

O

OH OH

11 Tiliroside (Salvador et al. 2009) Lychnophora markgravii (Asteraceae)

IC50 = 2.4 μM, SI = 52 IC50 = 0.9 μM, SI = 133

L. amazonensis axenic amastigotes

IC50 = 1.1 μM

IC50 = 123.7 μM (human macrophages)

BIFLAVONES

FLAVONES OH

HO

O H3CO

O OH O

HO

O

OH OH O

OH O

12 Amentoflavone (Tasdemir et al. 2006; Rizk et al. 2014) Selaginella sellowii (Selaginellaceae) L. donovani IC50 = 11.1 μM, SI = 0.4 axenic amastigotes L. donovani intracellular IC50 = 0.2 μM, SI = 20 amastigotes Cytotoxic activity IC50 = 4.0 μM (murine fibroblasts)

13 Tectochrysin (Salvador et al. 2009) Lychnophora markgravii (Asteraceae)

L. amazonensis axenic amastigotes

IC50 = 0.6 μM

Fig. 7.8 (continued)

axenic amastigotes of L. donovani, were tested in vivo using the BALB/c mice model infected with the L. donovani HU3 strain. Only quercetin showed low insignificant in vivo activity after 5 days of intraperitoneal injection at a dose of 30 mg/kg (Tasdemir et al. 2006).

7.3.4

Biflavones

Biflavones are characterized by two flavone monomeric units covalently linked either with C-C or with C-O-C bonds. The monomeric units may be of the same structural type or not. Numerous reports are available in relation to their antiplasmodial activity (Ahmed et al. 2001; Suarez et al. 2003; Weniger et al. 2006;

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Ichino et al. 2006) and the correlation between interflavonyl linkage and activity (Weniger et al. 2006). Less data are available concerning their antileishmanial activity. Only 13 biflavones show IC50 values lower than 50 µM. The described activities cover axenic amastigotes and intracellular amastigotes with IC50 values ranging from 1.6 to 34 µM and 0.2 to 5.3 µM, respectively (Weniger et al. 2004; Tasdemir et al. 2006; Weniger et al. 2006; Kunert et al. 2008; Rizk et al. 2014). Amentoflavone (12) is the most active natural biflavonoid against intracellular amastigotes of L. amazonensis (IC50 value = 0.2 µM) with an acceptable selectivity (SI = 22). The range of the cytotoxic activities indicates a probable nonselective antileishmanial activity for most of the other compounds (Weniger et al. 2006). In contrast to antiplasmodial activity, the interflavonyl linkage type does not seem to have relevance regarding antileishmanial activity.

7.3.5

Miscellaneous

Only two natural flavan-3-ols showed antileishmanial activity with IC50 values below 50 µM. (-)-gallocatechin gallate and (-)-epigallocatechin gallate exhibited moderate activities against axenic amastigotes of L. donovani with IC50 values of 19.4 and 41.7 µM but without selectivity versus mammalian cells (SI of 1.7 and 0.77, respectively) (Tasdemir et al. 2006). Flavones were mainly evaluated against axenic amastigotes (Kirmizibekmez et al. 2004; Tasdemir et al. 2006; Salvador et al. 2009; Nour et al. 2010; Salem et al. 2011). They globally exhibited mild IC50 values ranging from 2.7 to 43.7 µM, except for tectochrysin (13), isolated from Lychnophora markgravii, which displayed an IC50 value of 0.56 µM on axenic amastigotes of L. amazonensis (Salvador et al. 2009) (Fig. 7.8). Luteolin and luteolin-7-O-glucoside, displaying respectively IC50 values of 2.8 and 2.5 µM on L. donovani axenic amastigotes, were evaluated in vivo but did not show any activity (Tasdemir et al. 2006). The antileishmanial activities of eight flavanones were evaluated on axenic amastigotes of L. donovani and L. amazonensis (Tasdemir et al. 2006; Salvador et al. 2009; Salem et al. 2011). Their IC50 values were comprised between 0.3 and 41.7 µM, and they showed low-to-moderate selectivity ranging from 3 to 18. Pinostrobin, isolated from Lychnophora markgravii, was the most active molecule of this subclass with an IC50 value of 0.31 µM on axenic amastigotes of L. amazonensis though the cytotoxicity of the molecule was not evaluated (Salvador et al. 2009). Among the seven isoflavones with IC50 values lower than 50 µM (Salem and Werbovetz 2006; Tasdemir et al. 2006), only one, biochanin A, exhibited an IC50 value lower than 10 µM with a mild selectivity (SI = 26.3). All the other compounds were only weakly active, without selectivity (IS < 3).

7 Antileishmanial and Antitrypanosomal Activities of Flavonoids

7.4

179

Anti-Trypanosoma brucei Activity of Flavonoids

Ten classes of flavonoids exhibit activities below 50 µM against the T. brucei trypomastigote form: flavones, flavonols, flavanones, dihydroflavonols, flavanols, chalcones, dihydrochalcones, isoflavones, isoflavans, and biflavonoids (Fig. 7.9). The most promising compounds are isoflavans, synthetic chalcones, and flavones. The other classes have mild activity and/or low selectivity. Some classes like dihydrochalcones and dihydroflavonols are poorly represented with only 3 described compounds. Two classes contain synthetic compounds: chalcones and dihydrochalcones; the other classes bear only natural molecules.

Isoflavones Isoflavans Flavonols Flavones Dihydroflavonols

Natural Synthetic Cytotoxicity

Flavanones Flavan-3-ols Dihydrochalcones Chalcones

0 0 1

IC 50 µM

0

0 0

0 1

1

.1 0

1

0

.0

1

Biflavonoids

Fig. 7.9 In vitro biological activity of natural and synthetic flavonoids described in the literature against T. brucei sp. and mammalian cells (cytotoxicity). The most active compounds against Trypanosoma brucei belong to isoflavans (exclusively natural), chalcones (synthetic and natural), and flavones (exclusively natural). Dihydroflavonols, flavan-3-ols, and dihydrochalcones were less studied. All the described compounds (217) were selected on the basis of their flavonoidic nature and their in vitro anti-Trypanosoma brucei properties against trypomastigotes (IC50 values < 50 µM). Corresponding cytotoxicity against mammalian cells, if investigated, was also plotted (Troeberg et al. 2000; del Rayo Camacho et al. 2002; del Rayo Camacho et al. 2004; Mbwambo et al. 2006; Salem and Werbovetz 2006; Tasdemir et al. 2006; van Baren et al. 2006; Weniger et al. 2006; Ganapaty et al. 2008; dos Santos et al. 2009; Bourjot et al. 2010; Nour et al. 2010; Kirmizibekmez et al. 2011; Mamadalieva et al. 2011; Salem et al. 2011; Qiao et al. 2012; Schmidt et al. 2012b; Hata et al. 2013; Roussaki et al. 2013; Almutairi et al. 2014; Hata et al. 2014; Mai et al. 2015; Nwodo et al. 2015; Omar et al. 2016). The vertical bar corresponds to the median of each dataset

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Isoflavans

Seven isoflavans (Hata et al. 2013, 2014; Omar et al. 2016) and five isoflavanols (Ganapaty et al. 2008; Hata et al. 2013, 2014) were described in the literature with IC50 values against T. brucei ranging from 0.01 to 30.5 µM. Hata et al. described in 2013 and 2014 a series of ten isoflavan quinones and hydroquinones extracted from Abrus precatorius (Fabaceae), some of them displaying strong trypanocidal activity. In an effort to obtain higher amounts of abruquinones I (14) and B (15) to proceed to in vivo experiments, they isolated abruquinones A (16), D (17), and L (18) with improved activity and selectivity (SI = 1725 and 480, respectively) (Hata et al. 2014). Despite the fact that they did not obtain enough material to evaluate these compounds in vivo, they isolated ten isoflavans, 6 of them displaying submicromolar activities on T. brucei. These results enable us to draw insights on the structure–activity relationship. Globally, isoflavans are more active than isoflavanols, and the presence of a hydroquinone or a quinone moiety in positions 2′ and 5′ seems to increase activity. The absolute configuration R in position 3 confers a better activity to the isoflavan than the S configuration if we consider the difference between abruquinones K (19) and L (18) or abruquinone I (14) and 7,8,3′,5′-tetramethoxyisoflavan-1′,4′-quinone (20). A substitution in position 8 seems to decrease the trypanocidal activity if we compare abruquinones B (15) and A (16), as well as abruquinones H (21), G (22), and K (19). Isoflavans having both a hydroxyl moiety in position 7 and a methoxy in position 6 are more toxic than isoflavanols or isoflavans harboring two methoxy in positions 6 and 7. In contrast, a hydroxyl moiety on position 8 seems to decrease the toxicity of abruquinone I (14) when compared to the methoxylated analog (20), and the stereochemistry in position 3 does not seem to interfere (Hata et al. 2013, 2014).

7.4.2

Chalcones and Dihydrochalcones

Seventy-nine chalcones were described to harbor an antitrypanosomal activity below 50 µM, including 74 synthetic compounds; only 3 dihydrochalcones are represented, two of them being synthetic (dos Santos et al. 2009; Qiao et al. 2012). In both cases, natural molecules (3.7 µM < IC50 values < 35.5 µM) (Salem and Werbovetz 2006; dos Santos et al. 2009) are less active than synthetic or hemisynthetic compounds (0.03 µM < IC50 values < 14.4 µM) (Troeberg et al. 2000; Qiao et al. 2012; Roussaki et al. 2013). Qiao et al. synthesized 54 chalcone– benzoxaborole hybrids displaying antitrypanosomal activities ranging from 0.03 to 14.4 µM. The authors were able to elucidate key positions to improve the activity: dihydrochalcones and compounds lacking the oxaborole moiety have a threefold to fivefold decrease in activity; compounds substituted in position 4 with a hydrogen-bounding capability are highly potent as well as 3-methoxy compounds. The combination of a 4-amino and a 3-methoxy substitution results in the most

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181

ISOFLAVANS AND ISOFLAVANOLS

Abruquinones I (14) R1 = H, R2 = OCH3, R3 = H, R4 = H, R5 = OCH3 (3R) B (15) R1 = OCH3, R2 = OCH3, R3 = OCH3, R4 = OCH3, R5 = H (3R) A (16) R1 = OCH3, R2 = OCH3, R3 = H, R4 = OCH3, R5 = H (3R) D (17) R1 = OCH3, R2 = OH, R3 = H, R4 = OCH3, R5 = H (3R) (20) R1 = H, R2 = OCH3, R3 = OCH3, R4 = H, R5 = OCH3 (3S) (Hata et al. 2013; Hata et al. 2014) Abrus precatorius (Fabaceae) 14

15

16

17

20

T. brucei trypomastigotes

0.3 μM

0.2 μM

0.02 μM

0.01 μM

0.9 μM

Cytotoxic activity (L6 cells)

22.1 μM

10.1 μM

34.5 μM

4.8 μM

3.9 μM

Abruquinones L (18) R1 = H, R2 = CH3, R3 = H, R4 = H (3R) K (19) R1 = OH, R2 = CH3, R3 = H, R4 = H (3S, 4R) H (21) R1 = OH, R2 = CH3, R3 = CH3, R4 = OH (3S, 4R) G (22) R1 = OH, R2 = CH3, R3 = CH3, R4 = OCH3 (3S, 4R) (Hata et al. 2013; Hata et al. 2014) Abrus precatorius (Fabaceae)

T. brucei trypomastigotes

Cytotoxic activity (L6 cells)

18

19

21

22

0.02 μM

0.11 μM

12.0 μM

17.0 μM

7.5 μM

57.3 μM

122 μM

51.8 μM

CHALCONES

Chalcone-benzoxaborole hybrid 23

Chalcone-benzoxaborole hybrid 24

(Qiao et al. 2012)

(Qiao et al. 2012)

Organic synthesis

Organic synthesis

T. brucei trypomastigotes

IC50 = 0.03 μM, SI = 157

IC50 = 0.07 μM, SI > 486

Cytotoxic activity

IC50 = 4.7 μM (L929 cells)

IC50 > 34 μM (L929 cells)

Fig. 7.10 Structures and biological activities of the most potent flavonoid compounds against T. brucei spp

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7,8-dihydroxyflavone

7,8,3',4'-tetrahydroxyflavone

25

26

(Tasdemir et al. 2006)

(Tasdemir et al. 2006)

Natural

Natural

T. brucei trypomastigotes

IC50 = 0.2 μM, SI = 116

IC50 = 1.7 μM, SI = 44

Cytotoxic activity

IC50 = 23.1 μM (L6 cells)

IC50 = 75.5 μM (L6 cells)

FLAVONOLS

Rhamnetin

3-hydroxyflavone

27

28

(Tasdemir et al., 2006)

(Tasdemir et al., 2006)

Natural

Natural

T. brucei promastigotes

IC50 = 1.6 μM, SI > 56

IC50 = 2.1 μM, SI = 29

Cytotoxic activity

IC50 > 90 μM (L6 cells)

IC50 = 60.4 μM (L6 cells)

Fig. 7.10 (continued)

potent compound, displaying an IC50 value of 0.03 µM (23). Although this compound has a cytotoxic activity of 4.7 µM, its selectivity index is over 100. Its deaminated analogue 24 displays an interesting antiparasitic activity as well (IC50 value = 0.07 µM), without cytotoxicity. Twelve compounds were assessed using a murine model of blood stage T. brucei infection. Mice treatment was initiated 24 h after infection by daily intraperitoneal injection of 50 mg/kg for 5 days; 23 and 24, the most potent compounds in vitro, permitted to obtain a 100% survival rate and a complete elimination of T. b. brucei parasites 30 days after infection. The other compounds were less active in vitro, reaching only to a 20% survival rate (Qiao et al. 2012).

7 Antileishmanial and Antitrypanosomal Activities of Flavonoids

7.4.3

183

Flavones

Forty-eight flavones are described in the literature with IC50 values ranging from 0.2 to 48.3 µM. Fourteen harbor activities lower than 10 µM (del Rayo Camacho et al. 2004; Salem and Werbovetz 2006; Tasdemir et al. 2006; dos Santos et al. 2009; Bourjot et al. 2010; Nour et al. 2010; Kirmizibekmez et al. 2011; Mamadalieva et al. 2011; Almutairi et al. 2014). The two most active molecules are hydroxylated flavones, substituted in positions 7 and 8: 7,8-dihydroxyflavone (25) and 7,8,3′,4′tetrahydroxyflavone (26) (Fig. 7.10) (Tasdemir et al. 2006). The additional 12 compounds that have IC50 values lower than 10 µM are hydroxylated flavones nonsubstituted in position 8, methoxylated, and/or prenylated flavones and are less active on Trypanosoma brucei (IC50 values between 4,3 and 9,5 µM) (Tasdemir et al. 2006; van Baren et al. 2006; dos Santos et al. 2009; Bourjot et al. 2010; Nour et al. 2010; Salem et al. 2011). Globally, aglycones are more potent than glycosides (Tasdemir et al. 2006). 7,8-dihydroxyflavone (25) was evaluated in vivo using infected mice with T. brucei brucei bloodstream forms and treated intraperitoneally

Isoflavonoids Flavonols Flavones Dihydroflavonols Natural Synthetic Cytotoxicity

Flavanones Flavan-3-ols Dihydrochalcones Chalcones

0 0 1

IC 50 µM

0

0 1

0

0 1

1

.1 0

-4

0 1

1

0

-6

Biflavonoids

Fig. 7.11 In vitro biological activity of natural and synthetic flavonoids described in the literature against the three forms of T. cruzi and mammalian cells (cytotoxicity). The most active compounds against Trypanosoma cruzi belong to flavan-3-ols (exclusively natural), chalcones (synthetic and natural), and flavones (exclusively natural). Isoflavonoids, flavanones, and biflavonoids were less studied. All the described compounds (73) were selected on the basis of their flavonoidic nature and their in vitro anti-Trypanosoma cruzi properties against trypomastigotes (Paveto et al. 2004; Sülsen et al. 2007; Ganapaty et al. 2008; Carvalho et al. 2012; da Rocha et al. 2014), amastigotes (Paveto et al. 2004; Mbwambo et al. 2006; Tasdemir et al. 2006; Weniger et al. 2006; Aponte et al. 2008; Carvalho et al. 2012; da Rocha et al. 2014; Sandjo et al. 2016), and epimastigotes (Lunardi et al. 2003; Sülsen et al. 2007; Borges-Argaez et al. 2009; Marín et al. 2011; Passalacqua et al. 2015; Beer et al. 2016) (IC50 values 2.109 IC50 = 0.1 μM, SI = 321 IC50 = 311.0 μM IC50 = 32.1 μM (L6 cells)

Gallocatechin gallate 30 (Paveto et al. 2004; Tasdemir et al. 2006) Camellia sinensis (Theaceae) IC50 = 0.12 pM, SI > 2.109 IC50 = 0.1 μM, SI = 323 nd IC50 = 32.3 μM (L6 cells)

CHALCONES N O

O

O

O

O

O

31 (Aponte et al. 2008) T. cruzi Trypomastigotes Amastigotes Epimastigotes Cytotoxic activity

O

N

O

32 (Aponte et al. 2008)

IC50 = 1.5 μM, SI = 2

IC50 = 1.9 μM, SI = 2

IC50 = 2.8 μM (VERO cells)

IC50 = 2.8 μM (VERO cells)

O O O

O

HO

O OH

O

33 (Aponte et al. 2008) T. cruzi Trypomastigotes Amastigotes Epimastigotes Cytotoxic activity

O

O

IC50 = 3.4 μM, SI = 12 IC50 = 40.9 μM (VERO cells)

Isocordoin 34 (Borges-Argaez et al. 2009)

IC50 = 7.0 μM, SI = 1 IC50 = 8.8 μM (MDCK)

Fig. 7.12 Structure and biological activities of the most potent flavonoid compounds against T. cruzi. nd = not determined, na = not active

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187

FLAVONES AND FLAVONOLS O O OH O O

O

O

O O

O

O O OH

O

O O

O

O

O

O

O O

O O

O

Eupatorin 35 (Beer et al. 2016) Stevia aristata & Stevia entreriensis (Asteraceae) T. cruzi Trypomastigotes Amastigotes Epimastigotes Cytotoxic activity

IC50 = 179.5 μM, SI > 8 na IC50 = 0.6 μM, SI > 2500 IC50 >1452 μM (VERO cells)

Astragalin-heptaacetate 36 (Marín et al. 2011) Hemisynthetic Delphinium staphisagria (Ranunculaceae)

IC50 = 0.8 μM, SI = 205 IC50 = 164 μM (VERO cells)

Fig. 7.12 (continued)

untreated control group. A dose of 0.16 mg/kg/day is not sufficient to obtain a significant effect (Güida et al. 2007). The authors attribute these results to the pharmacokinetics of EGCg (29). Actually, Lee et al. reported that an oral administration of 2 mg/kg of EGCg (29) to eight human volunteer leads to a peak plasma concentration around 80 ng/ml, one to two hours after administration. They obtained a 3.4-h elimination half-life and undetectable plasma levels after 24 h. The metabolites were eliminated in urine either as conjugated forms or flavan-3-ols in which the chromene ring have been opened (Lee et al. 2002). The future exploration of catechins should focus on the development of metabolically stable analogues with improved pharmacokinetic profiles.

7.5.2

Chalcones

Chalcone derivatives were evaluated on T. cruzi amastigotes, trypomastigotes, and epimastigotes. In this class, only one natural product is represented (Lunardi et al. 2003; Aponte et al. 2008; Borges-Argaez et al. 2009; Carvalho et al. 2012; Passalacqua et al. 2015; Sandjo et al. 2016). Their activity ranges between 1.5 and 44.5 µM (Borges-Argaez et al. 2009). The most active compounds of the class were 2′,4′-diallyloxy-6′-methoxychalcones, derivatives of the natural compound 2′,4′dihydroxy-4,6′-dimethoxydihydrochalcone, displaying IC50 values of 1.5 (31), 1.9 (32), and 3.4 µM (33), respectively, on T. cruzi amastigotes; 31 and 32 showed also cytotoxicity, with selectivity indexes lower than 2, whereas 33 had a better selectivity with an SI value of 12 (Aponte et al. 2008). Hemisynthetic derivatives of

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isocordoin (34) showed mild activities against T. cruzi epimastigotes with IC50 values of 1.5 µM for the methylated derivative and 1.8 µM for the acetylated derivative, and rather good selectivity indexes (110 and 24, respectively) when compared to the natural product (isocordoin, 7.0 µM against T. cruzi epimastigotes, SI 1.2). The substitution of the hydroxyls on cycle A seems to improve the selectivity toward T. cruzi (Borges-Argaez et al. 2009).

7.5.3

Flavones and Flavonols

Flavones and flavonols are two classes that display a better activity on T. cruzi epimastigotes (0.58 to 47.4 µM) (Sülsen et al. 2007; Fonseca-Silva et al. 2011; Beer et al. 2016) than on amastigotes (13.8 to 47.7 µM) (Tasdemir et al. 2006). The flavone eupatorin (35) is the most active compound on epimastigotes with an interesting selectivity index (SI > 2,500), but this compound has no activity on the trypomastigote and amastigote forms (IC50 values > 180 µM). Likewise, the flavone 5-desmethylsinensetin with an IC50 value of 1.12 µM against epimastigotes and a selectivity index above 1245 has no activity against the trypomastigote and amastigote forms (IC50 > 210 µM) (Beer et al. 2016). The hemisynthetic flavonol astragalin-heptaacetate (36) displays a better activity against the epimastigote forms than the natural product astragalin (IC50 values of 0.8 and 15.7 µM, respectively) and a similar cytotoxicity (164.0 and 174.3 µM, respectively). The acetylation of the hydroxyl moieties of astragalin, leading to an increase of the hydrophobicity of the compound, enables to improve about 20 times the activity. Astragalinheptaacetate (36) was evaluated in vivo using an acute model of T. cruzi infection. It allowed to reduce the blood infection of mice treated for five consecutive days (7 to 12 days postinfection, 1 mg/kg/day i.p.) by 59%, whereas benznidazole displayed only 16% inhibition when compared to the untreated control group (Marín et al. 2011).

7.5.4

Miscellaneous

The few described flavanones and isoflavonoids have low activities against T. cruzi trypomastigotes and amastigotes (IC50 values ranging from 10.1 to 45.6 µM) (Tasdemir et al. 2006; Ganapaty et al. 2008). Three biflavonoids have been evaluated against T. cruzi amastigotes (IC50 values ranging between 11.0 and 34.7 µM), but they have no selectivity (Mbwambo et al. 2006; Weniger et al. 2006) (Fig. 7.12).

7 Antileishmanial and Antitrypanosomal Activities of Flavonoids

7.6

189

Conclusion and Perspectives

The trend of this survey indicates that the most active flavonoids against Leishmania spp. belong to the chalcone, biflavone, and aurone classes, regardless of the performed assays. Few compounds of these classes exhibit submicromolar antileishmanial activity against axenic or intracellular amastigotes, without in vitro cytotoxicity. Flavones and flavonols are generally less active than the former. Flavones, flavan-3-ols, and isoflavones are less studied and less active (Fig. 7.5). Nevertheless, only few chalcone derivatives show significant activity in vivo in animal models. Isoflavans, synthetic chalcones, and flavones are currently the most promising classes of flavonoids, displaying submicromolar activities against T. brucei sp. and good selectivity. Two chalcone–benzoxaborole hybrids have a curative effect on mice treated with 50 mg/kg/day for 5 days using an acute model of sleeping sickness. Dihydrochalcones and dihydroflavonols are underrepresented, and an effort should be made to evaluate the activities of additional compounds belonging to these classes. Few compounds have been evaluated against T. cruzi, while the need for an improved management of Chagas disease is more pressing, especially since flavonoids are less active on T. cruzi than on T. brucei. Flavan-3-ols are highly potent compounds, but display discordant activities. Nevertheless, epigallocatechin gallate (29) induces a 60% survival rate in vivo, using an acute model of Chagas disease. Chalcone derivatives have low micromolar activities on T. cruzi amastigotes, whereas flavones and flavonols have an increased activity on epimastigotes and low activities on amastigotes and trypomastigotes. The low biodisponibility of flavonoids in general is currently an obstacle to the development of new drugs belonging to this family of compounds. However, the flavonoid moiety can provide an original scaffold in some therapeutic areas like parasitic diseases and enrich the therapeutic alternatives. Medicinal chemistry or vectorization efforts could address this issue in the future.

References Ahmed MS, Galal AM, Ross SA et al (2001) A weakly antimalarial biflavanone from Rhus retinorrhoea. Phytochemistry 58:599–602 Akimanya A, Midiwo JO, Matasyoh J et al (2015) Two polymethoxylated flavonoids with antioxidant activities and a rearranged clerodane diterpenoid from the leaf exudates of Microglossa pyrifolia. Phytochem Lett 11:183–187 Almutairi S, Edrada-Ebel R, Fearnley J et al (2014) Isolation of diterpenes and flavonoids from a new type of propolis from Saudi Arabia. Phytochem Lett 10:160–163 Aponte JC, Verástegui M, Málaga E et al (2008) Synthesis, cytotoxicity, and anti- Trypanosoma cruzi activity of new chalcones. J Med Chem 51:6230–6234 Beer MF, Frank FM, Germán Elso O, et al (2016) Trypanocidal and leishmanicidal activities of flavonoids isolated from Stevia satureiifolia var. satureiifolia. Pharm Biol:1–8

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F. Nardella et al.

Bello ML, Chiaradia LD, Dias LRS et al (2011) Trimethoxy-chalcone derivatives inhibit growth of Leishmania braziliensis: Synthesis, biological evaluation, molecular modeling and structure-activity relationship (SAR). Bioorg Med Chem 19:5046–5052 Boeck P, Falcao CAB, Leal PC et al (2006) Synthesis of chalcone analogues with increased antileishmanial activity. Bioorg Med Chem 14:1538–1545 Bonnet J, Boudot C, Courtioux B (2015) Overview of the diagnostic methods used in the field for human African Trypanosomiasis: what could change in the next years? Biomed Res Int 2015:1–10 Borges-Argaez R, Balnbury L, Flowers A et al (2007) Cytotoxic and antiprotozoal activity of flavonoids from Lonchocarpus spp. Phytomedicine 14:530–533 Borges-Argaez R, Vela-Catzin T, Yam-Puc A et al (2009) Antiprotozoal and cytotoxic studies on some isocordoin derivatives. Planta Med 75:1336–1338 Bourjot M, Apel C, Martin M-T et al (2010) Antiplasmodial, antitrypanosomal, and cytotoxic activities of prenylated flavonoids isolated from the stem bark of Artocarpus styracifolius. Planta Med 76:1600–1604 Bruneton J, Poupon E (2016) Pharmacognosie : Phytochimie, plantes médicinales, 5e edn Carvalho SA, Feitosa LO, Soares M et al (2012) Design and synthesis of new (E)-cinnamic N-acylhydrazones as potent antitrypanosomal agents. Eur J Med Chem 54:512–521 Center for Disease Control and Prevention (2015a) Parasites—Leishmaniasis. http://www.cdc.gov/ parasites/leishmaniasis/biology.html. Accessed 11 July 2016 Center for Disease Control and Prevention (2015b) Parasites—African Trypanosomiasis (also known as Sleeping Sickness). http://www.cdc.gov/parasites/sleepingsickness/biology.html. Accessed 12 July 2016 Center for Disease Control and Prevention (2015c) Parasites - American Trypanosomiasis (also known as Chagas Disease). http://www.cdc.gov/parasites/chagas/biology.html. Accessed 11 July 2016 Chatelain E (2015) Chagas disease drug discovery: toward a new era. J Biomol Screen 20:22–35 Chen M, Broegger Christensen S, Blom J et al (1993) Licochalcone A, a novel antiparasitic agent with potent activity against human pathogenic protozoan species of Leishmania. Antimicrob Agents Chemother 37:2550–2556 Chen M, Broegger Christensen S, Theander TG, Kharazmi A (1994) Antileishmanial activity of licochalcone A in mice infected with Leishmania major and in hamsters infected with Leishmania donovani. Antimicrob Agents Chemother 38:1339–1344 da Rocha CQ, Queiroz EF, Meira CS et al (2014) Dimeric flavonoids from Arrabidaea brachypoda and assessment of their anti-Trypanosoma cruzi activity. J Nat Prod 77:1345–1350 de Mello TFP, Bitencourt HR, Pedroso RB et al (2014) Leishmanicidal activity of synthetic chalcones in Leishmania (Viannia) braziliensis. Exp Parasitol 136:27–34 del Rayo Camacho M, Phillipson JD, Croft SL et al (2002) Assessment of the antiprotozoal activity of Galphimia glauca and the isolation of new nor-secofriedelanes and nor-friedelanes. J Nat Prod 65:1457–1461 del Rayo Camacho M, Phillipson JD, Croft SL et al (2004) in vitro antiprotozoal and cytotoxic activities of some alkaloids, quinones, flavonoids, and coumarins. Planta Med 70:70–72 dos Santos DAP, Braga PA de C, da Silva MF das GF et al (2009) Anti-African trypanocidal and antimalarial activity of natural flavonoids, dibenzoylmethanes and synthetic analogues. J Pharm Pharmacol 61:257–266 Flores N, Cabrera G, Jimenez IA et al (2007) Leishmanicidal constituents from the leaves of Piper rusbyi. Planta Med 73:206–211 Fonseca-Silva F, Inacio JDF, Canto-Cavalheiro MM, Almeida-Amaral EE (2011) Reactive oxygen species production and mitochondrial dysfunction contribute to quercetin induced death in Leishmania amazonensis. PLoS ONE 6:e14666 Ganapaty S, Pannakal ST, Srilakshmi GVK et al (2008) Pumilanol, an antiprotozoal isoflavanol from Tephrosia pumila. Phytochem Lett 1:175–178

7 Antileishmanial and Antitrypanosomal Activities of Flavonoids

191

George VC, Vijesh VV, Dehigaspege AI, Lakshmi CA, Anbarasu K, Kumar DR, Ethiraj R, Kumar RA, Rupasinghe HP et al (2016) Mechanism of action of flavonoids in prevention of inflammation-associated skin cancer. Curr Med Chem. https://doi.org/10.2174/ 0929867323666160627110342 Güida MC, Esteva MI, Camino A et al (2007) Trypanosoma cruzi: In vitro and in vivo antiproliferative effects of epigallocatechin gallate (EGCg). Exp Parasitol 117:188–194 Gupta S, Nishi (2011) Visceral leishmaniasis: experimental models for drug discovery. Indian J Med Res 133:27–39 Gupta S, Shivahare R, Korthikunta V et al (2014) Synthesis and biological evaluation of chalcones as potential antileishmanial agents. Eur J Med Chem 81:359–366. Hata Y, Ebrahimi SN, De Mieri M et al (2014) Antitrypanosomal isoflavan quinones from Abrus precatorius. Fitoterapia 93:81–87 Hata Y, Raith M, Ebrahimi SN et al (2013) Antiprotozoal isoflavan quinones from Abrus precatorius ssp. africanus. Planta Med 79:492–498 Hermoso A, Jiménez IA, Mamani ZA et al (2003) Antileishmanial activities of dihydrochalcones from piper elongatum and synthetic related compounds. Structural requirements for activity. Bioorganic Med Chem 11:3975–3980 Hoet S, Opperdoes F, Brun R, Quetin-Leclercq J et al (2004) Natural products active against African trypanosomes: a step towards new drugs. Nat Prod Rep 21:353–364 Ichino C, Kiyohara H, Soonthornchareonnon N et al (2006) Antimalarial activity of biflavonoids from Ochna integerrima. Planta Med 72:611–614 Jones AJ, Grkovic T, Sykes ML, Avery VM et al (2013) Trypanocidal activity of marine natural products. Mar Drugs 11:4058–4082 Kayser O, Kiderlen AF (2001) In vitro leishmanicidal activity of naturally occurring chalcones. Phyther Res 15:148–152 Kayser O, Kiderlen AF, Folkens U, Kolodziej H (1999) In vitro leishmanicidal activity of aurones. Planta Med 65:316–319 Kirmizibekmez H, Atay I, Kaiser M et al (2011) Antiprotozoal activity of Melampyrum arvense and its metabolites. Phytother Res 25:142–146 Kirmizibekmez H, Çalis I, Perozzo R et al (2004) Inhibiting activities of the secondary metabolites of Phlomis brunneogaleata against parasitic protozoa and plasmodial enoyl-ACP reductase, a crucial enzyme in fatty acid biosynthesis. Planta Med 70:711–717 Kumar A (2013) Leishmania and leishmaniasis. Springer, New York Kunert O, Swamy RC, Kaiser M et al (2008) Antiplasmodial and leishmanicidal activity of biflavonoids from Indian Selaginella bryopteris. Phytochem Lett 1:171–174 Langousis G, Hill KL (2014) Motility and more: the flagellum of Trypanosoma brucei. Nat Rev Micro 12:505–518. doi:10.1038/nrmicro3274, http://www.nature.com/nrmicro/journal/v12/n7/ abs/nrmicro3274.html#supplementary-information Lee M-J, Maliakal P, Chen L et al (2002) 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 A Publ Am Assoc Cancer Res Cosponsored by Am Soc Prev Oncol 11:1025–1032 Lunardi F, Guzela M, Rodrigues AT et al (2003) Trypanocidal and leishmanicidal properties of substitution-containing chalcones. Antimicrob Agents Chemother 47:1449–1451 Llurba Montesino N, Kaiser M, Brun R, Schmidt TJ et al (2015) Search for antiprotozoal activity in herbal medicinal preparations; new natural leads against neglected tropical diseases. Molecules 20:14118–14138 Mai LH, Chabot GG, Grellier P et al (2015) Antivascular and anti-parasite activities of natural and hemisynthetic flavonoids from New Caledonian Gardenia species (Rubiaceae). Eur J Med Chem 93:93–100 Mamadalieva NZ, Herrmann F, El-Readi MZ et al (2011) Flavonoids in Scutellaria immaculata and S. ramosissima (Lamiaceae) and their biological activity. J Pharm Pharmacol 63:1346– 1357

192

F. Nardella et al.

Marin C, Boutaleb-Charki S, Diaz JG et al (2009) Antileishmaniasis activity of flavonoids from consolida oliveriana. J Nat Prod 72:1069–1074 Marín C, Ramírez-Macías I, López-Céspedes A et al (2011) In vitro and in vivo trypanocidal activity of flavonoids from Delphinium staphisagria against Chagas disease. J Nat Prod 74:744–750 Mbwambo ZH, Kapingu MC, Moshi MJ et al (2006) Antiparasitic activity of some xanthones and biflavonoids from the root bark of Garcinia livingstonei. J Nat Prod 69:369–372 Min-Hsiung P, Ching-Shu L, Chi-Tang H et al (2010) Anti-inflammatory activity of natural dietary flavonoids. Food Funct 1:15–31 Morrot A, Villar SR, Gonzalez FB, Perez AR (2016) Evasion and immuno-endocrine regulation in parasite infection: two sides of the same coin in Chagas disease? Front Microbiol 7:704 Nabavi SF, Sureda A, Daglia M, Izadi M, Sobarzo-Sánchez E, Rastrelli L, Nabavi SM et al (2016) Flavonoids and Chagas’ disease: the story so far! Curr Top Med Chem Nielsen SF, Christensen SB, Cruciani G et al (1998) Antileishmanial Chalcones: Statistical Design, Synthesis, and Three-Dimensional Quantitative Structure-Activity Relationship Analysis. J Med Chem 41:4819–4832 Nour AMM, Khalid SA, Kaiser M et al (2010) The antiprotozoal activity of methylated flavonoids from Ageratum conyzoides L. J Ethnopharmacol 129:127–130 Ntie-Kang F, Amoa Onguéné P, Lifongo LL, Ndom JC, Sippl W, Meva’a Mbaze L et al (2014) The potential of anti-malarial compounds derived from African medicinal plants, part II: a pharmacological evaluation of non-alkaloids and non-terpenoids. Malaria J 13:81 Nwodo N, Okoye F, Lai D et al (2015) Evaluation of the in vitro trypanocidal activity of methylated flavonoid constituents of Vitex simplicifolia leaves. BMC Complement Altern Med 15:82 Omar RMK, Igoli J, Gray AI et al (2016) Chemical characterisation of Nigerian red propolis and its biological activity against Trypanosoma brucei. Phytochem Anal 27:107–115 Passalacqua TG, Dutra LA, de Almeida L et al (2015) Synthesis and evaluation of novel prenylated chalcone derivatives as anti-leishmanial and anti-trypanosomal compounds. Bioorg Med Chem Lett 25:3342–3345 Paveto C, Guida MC, Esteva MI et al (2004) Anti-Trypanosoma cruzi Activity of Green Tea (Camellia sinensis) Catechins. Antimicrob Agents Chemother 48:69–74 Qiao Z, Wang Q, Zhang F et al (2012) Chalcone–benzoxaborole hybrid molecules as potent antitrypanosomal agents. J Med Chem 55:3553–3557 Ramirez-Macias I, Marin C, Diaz JG et al (2012) Leishmanicidal activity of nine novel flavonoids from Delphinium staphisagria. Sci World J 2012:10 Rizk YS, de Arruda CCP, Fischer A et al (2014) In vitro activity of the hydroethanolic extract and biflavonoids isolated from Selaginella sellowii on Leishmania (Leishmania) amazonensis. Mem Inst Oswaldo Cruz 109:1050–1056 Robertson LJ, Devleesschauwer B, Alarcon de Noya B et al (2016) Trypanosoma cruzi: time for international recognition as a foodborne parasite. PLoS Negl Trop Dis 10:e0004656 Rosenzweig D, Smith D, Opperdoes F et al (2008) Retooling Leishmania metabolism: from sand fly gut to human macrophage. FASEB J 22:590–602 Roussaki M, Hall B, Lima SC et al (2013) Synthesis and anti-parasitic activity of a novel quinolinone-chalcone series. Bioorg Med Chem Lett 23:6436–6441 Roussaki M, Lima SC, Kypreou A-M et al (2012) Aurones, a promising heterocyclic scaffold for the development of potent antileishmanial agents. Int J Med Chem 2012:8 pp Rudrapaul P, Sarma IS, Das N et al (2014) New flavonol methyl ether from the leaves of Vitex peduncularis exhibits potential inhibitory activity against Leishmania donovani through activation of iNOS expression. Eur J Med Chem 87:328–335 Ruiz C, Haddad M, Alban J et al (2011) Activity-guided isolation of antileishmanial compounds from Piper hispidum. Phytochem Lett 4:363–366 Salem MM, Capers J, Rito S, Werbovetz K a (2011) Antiparasitic Activity of C-Geranyl Flavonoids from Mimulus bigelovii. Phyther Res 25:1246–1249

7 Antileishmanial and Antitrypanosomal Activities of Flavonoids

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Salem MM, Werbovetz KA (2006) Isoflavonoids and other compounds from Psorothamnus arborescens with antiprotozoal activities. J Nat Prod 69:43–49 Salem MM, Werbovetz KA (2005) Antiprotozoal compounds from Psorothamnus polydenius. J Nat Prod 68:108–111 Salvador MJ, Sartori FT, Sacilotto ACBC et al (2009) Bioactivity of flavonoids isolated from Lychnophora markgravii against Leishmania amazonensis amastigotes. Z Naturforsch C 64:509–512 Sandjo LP, de Moraes MH, Kuete V et al (2016) Individual and combined antiparasitic effect of six plant metabolites against Leishmania amazonensis and Trypanosoma cruzi. Bioorg Med Chem Lett 26:1772–1775 Schmidt TJ, Khalid SA, Romanha AJ, Alves TM, Biavatti MW, Brun R, Da Costa FB, de Castro SL, Ferreira VF, de Lacerda MV, Lago JH, Leon LL, Lopes NP, das Neves Amorim RC, Niehues M, Ogungbe IV, Pohlit AM, Scotti MT, Setzer WN, de N C Soeiro M, Steindel M, Tempone AG, et al (2012) The potential of secondary metabolites from plants as drugs or leads against protozoan neglected diseases—part I. Curr Med Chem 19:2128–2175 Schmidt TJ, Khalid SA, Romanha AJ, Alves TM, Biavatti MW, Brun R, Da Costa FB, de Castro SL, Ferreira VF, de Lacerda MV, Lago JH, Leon LL, Lopes NP, das Neves Amorim RC, Niehues M, Ogungbe IV, Pohlit AM, Scotti MT, Setzer WN, de N C Soeiro M, Steindel M, Tempone AG, et al (2012a) The potential of secondary metabolites from plants as drugs or leads against protozoan neglected diseases—part II. Curr Med Chem 19:2176–2228 Schmidt TJ, Rzeppa S, Kaiser M, Brun R (2012c) Larrea tridentata—Absolute configuration of its epoxylignans and investigations on its antiprotozoal activity. Phytochem Lett 5:632–638 Steinmann P, Stone CM, Sutherland CS et al (2015) Contemporary and emerging strategies for eliminating human African trypanosomiasis due to Trypanosoma brucei gambiense: review. Trop Med Int Heal 20:707–718 Suarez AI, Diaz MB, Delle Monache F, Compagnone RS (2003) Biflavonoids from Podocalyx loranthoides. Fitoterapia 74:473–475 Sülsen VP, Cazorla SI, Frank FM et al (2007) Trypanocidal and leishmanicidal activities of flavonoids from Argentine medicinal plants. Am J Trop Med Hyg 77:654–659 Sundar S, Chakravarty J (2014) An update on pharmacotherapy for leishmaniasis. Expert Opin Pharmacother 16:1–16 Sutherland CS, Yukich J, Goeree R, Tediosi F (2015) A literature review of economic evaluations for a neglected tropical disease: human African trypanosomiasis (“sleeping sickness”). PLoS Negl Trop Dis 9:e0003397 Sykes ML, Avery VM (2013) Approaches to protozoan drug discovery: phenotypic screening: Miniperspectives series on phenotypic screening for antiinfective targets. J Med Chem 56:7727–7740 Tasdemir D, Kaiser M, Brun R et al (2006) Antitrypanosomal and antileishmanial activities of flavonoids and their analogues: in vitro, in vivo, structure-activity relationship, and quantitative structure-activity relationship studies. Antimicrob Agents Chemother 50:1352–1364 Torres-santos EC, Moreira DL, Kaplan AC et al (1999) Methoxychalcone isolated from Piper aduncum on Leishmania amazonensis Selective Effect of 2J, 6J-Dihydroxy-4JMethoxychalcone isolated from Piper aduncum on Leishmania amazonensis. Antimicrob Agents Chemother 43:1234–1241 Troeberg L, Chen X, Flaherty TM et al (2000) Chalcone, acyl hydrazide, and related amides kill cultured Trypanosoma brucei brucei. Mol Med 6:660–669 van Baren C, Anao I, Leo Di Lira P et al (2006) Triterpenic acids and flavonoids from Satureja parvifolia. Evaluation of their antiprotozoal activity. Z Naturforsch C 61:189–192 Weniger B, Vonthron-Senecheau C, Arango GJ et al (2004) A bioactive biflavonoid from Campnosperma panamense. Fitoterapia 75:764–767 Weniger B, Vonthron-Senecheau C, Kaiser M et al (2006) Comparative antiplasmodial, leishmanicidal and antitrypanosomal activities of several biflavonoids. Phytomedicine 13:176–180 WHO (2010) Control of the leishmaniases. World Health Organ Tech Rep Ser 22–26

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F. Nardella et al.

WHO (2016a) Trypanosomiasis, human African (sleeping sickness). http://www.who.int/ mediacentre/factsheets/fs259/en/. Accessed 12 Jul 2016 WHO (2016b) Chagas disease (American trypanosomiasis). http://www.who.int/mediacentre/ factsheets/fs340/en/. Accessed 11 July 2016 Zhai L, Chen M, Blom J et al (1999) The antileishmanial activity of novel oxygenated chalcones and their mechanism of action. J Antimicrob Chemother 43:793–803

Chapter 8

Natural Products from Plants as Potential Leads as Novel Antileishmanials: A Preclinical Review João Henrique G. Lago, Kaidu H. Barrosa, Samanta Etel T. Borborema and André G. Tempone Abstract Different plant species have been used in the folk medicine to the treatment of several pathologies. In some poor regions of the world, the use of these extracts is the unique therapeutic source for the treatment of antiparasitic diseases, including leishmaniasis. The effects of these extracts are directly associated to the production and accumulation of specific active natural products/secondary metabolites—terpenoids, phenolic derivatives, alkaloids, lignoids. Several studies have been conducted for evaluation of in vitro antileishmanial activity of these compounds but there are only few reports that describe the preclinical evaluation. In this aspect, this chapter attempts to give an overview on the potential of such plant-derived natural products as antileishmanial leads, mainly those that displayed in vivo potential. Keywords Leishmaniasis Therapy Leishmania




8.1


Natural products
Plants
In vivo assays

Introduction

Leishmaniasis is a parasitic human disease caused by intracellular protozoan parasites of the genus Leishmania with several clinical manifestations (Ready 2014). Leishmaniasis represents a major global health problem and is endemic in India, Africa, America, and in subtropical Southwest Asia as well as the Mediterranean. Despite several natural products have been discovered as new prototypes to the treatment of Leishmaniasis, most of these studies report only in vitro assays.

J. H. G. Lago (&)
K. H. Barrosa Center for Natural and Human Sciences, Federal University of ABC, Santo Andre 09210580, Brazil e-mail: [email protected] S. E. T. Borborema
A. G. Tempone Centre of Parasitology, Adolfo Lutz Institute, São Paulo 01246902, Brazil © Springer International Publishing AG 2018 J.-M. Mérillon and C. Rivière (eds.), Natural Antimicrobial Agents, Sustainable Development and Biodiversity 19, https://doi.org/10.1007/978-3-319-67045-4_8

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Leishmaniaisis Epidemiology

Leishmaniasis is a vector-borne disease caused by intracellular protozoan parasites of the genus Leishmania belonging to the order Kinetoplastida and Trypanosomatidae family. Leishmania is transmitted to vertebrate hosts by the bite of blood-sucking female phlebotomine sand flies, belonging to the order Diptera and Psychodidae family (Ready 2013). Leishmaniasis represents a major global health problem and is one of the most important neglected tropical diseases that affect 350 million people in 98 countries, with a global incidence estimated at 0.2–0.4 million of cases of visceral leishmaniasis (VL) and 0.7–1.2 million of cases of cutaneous leishmaniasis (CL) occurring each year. It is estimated 20,000–40,000 deaths due to VL annually. More than 90% of global VL cases occur in only six countries: India, Bangladesh, Sudan, South Sudan, Ethiopia, and Brazil, and 90% of CL cases occur in Afghanistan, Algeria, Ethiopia, Sudan, Iran, Iraq, Saudi Arabia, Syria, Brazil, and Peru (Alvar et al. 2012). In 2004, among parasitic diseases, leishmaniasis was responsible for the second highest disease burden after malaria: 2,356,000 disability-adjusted life years (DALYs) (946,000 in women and 1,410,000 in men), a significant rank among communicable diseases (Desjeux 2004).

8.2.2

Pathogenesis

The Leishmania life cycle consists of two morphologically distinct stages: an extracellular flagellar form (promastigote) found in the gut of the invertebrate vector and an intracellular non-motile form (amastigote) that resides in the macrophages of mammalian host. Only female sand flies Phlebotomus spp. (Old Word) and Lutzomyia spp. (New World) transmit the disease to mammalians by inoculation of the promastigote form into the skin. In the vertebrate host organism, the parasites are internalized by dendritic cells and macrophages in the dermis. Promastigotes housed in cytoplasmic parasitophorous vacuoles transform into amastigotes by losing their flagella and replicate. They disseminate through the lymphatic and vascular systems, infect other monocytes and macrophages in the reticulo-endothelial system, causing dermal lesions or affecting internal organs with forms and tissue tropisms in humans that the outcome is dependent both the infecting species of Leishmania and the immune response of the host (Chappuis et al. 2007; McGwire and Satoskar 2014). Leishmania is delivered into skin together with molecules that modulate the bite site, including salivary proteins and parasite-derived promastigote secretory gel (Ready 2013). The two known subgenera—Leishmania Leishmania and Leishmania Viannia— were separated according to their ability to develop in the foregut (Suprapylaria)

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and hind-midgut (Peripylaria) of the sand fly, respectively. At least 21 species of Leishmania have been recorded as being infective to humans, which are transmitted by about 30 species of phlebotomine sand flies (Herwaldt 1999).

8.2.3

Clinical Presentation

Leishmaniasis consists of three main clinical syndromes: cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL). In CL, one or several ulcers can be observed in the skin. The ulcers can heal spontaneously, but cause disfiguring scars. Different species can infect the macrophages in the dermis and produce lesions, primarily L. (L.) major, L. (L.) tropica, L. (L.) aethiopica, L. (L.) infantum (Old world) and L. (L.) mexicana, L. (L.) amazonenesis, L. (V.) braziliensis, L. (V.) panamensis, L. (V.) peruviana, L. (V.) guyanensis and L. (L.) infantum (New world). In MCL, destructive ulcerations of the mucosa, extending from the nose and mouth to the pharynx and larynx occur, producing remarkable disfigurement. These lesions are not self-healing. The disease can be refractory to chemotherapy and patients could die from secondary super-infections and malnutrition. L. (V.) braziliensis and L. (V.) panamensis are responsible for cases of MCL. VL is the most severe form of the disease, progressive and is fatal if left untreated. The main symptoms are fever, hepatosplenomegaly, cachexia, and pancytopenia. This infection is caused by L. (L.) infantum (Mediterranean basin, Southwest and Central Asia, and Latin America) and L. (L.) donovani (Africa, Asia, and Indian subcontinent) species (Chappuis et al. 2007). Post-kala-azar dermal leishmaniasis (PKDL) is characterized by lesions rich in parasites, and these might favor anthroponotic transmission in Africa as well as in India (Desjeux et al. 2013). With the spread of HIV, VL has become increasingly prevalent. Individuals coinfected with HIV have a particular susceptibility to developing atypical presentations and increased severity. HIV-Leishmania coinfection is now reported in 35 endemic countries with high mortality and corresponds an important infectious reservoir (Singh 2014). The transmission characteristics of leishmaniasis can be differentiated by zoonotic transmission, and the parasite is transmitted from animal to vector to human and anthroponotic transmission, human to vector to human. Usually, zoonotic VL is found in areas of L. (L.) infantum transmission, whereas anthroponotic VL is found in areas of L. (L.) donovani transmission (Chappius et al. 2007). Furthermore, there is a continuing urbanization of zoonotic VL—L. (L.) infantum—and domestic transmission of zoonotic CL—L. (V.) braziliensis, L. (L.) mexicana—in Latin America. In addition, an epidemic of anthroponotic CL—L. (L.) tropica—in west Asia and VL—L. (L.) donovani—in east Africa, associated with mass movement of non-immune persons and increasing HIV-VL coinfection worldwide, except in southern Europe where highly active antiretroviral therapy (HAART) is available and cases of coinfection have declined (Murray et al. 2005). Furthermore, regional variations are commonly observed in both CL and VL even within, endemic

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regions, probably indicating an interaction between local parasite properties, vector biology, and host factors (Murray et al. 2005).

8.2.4

Control Strategies

Dogs are the main reservoir of L. (L.) infantum in zoonotic VL. In Brazil, the culling of seropositive dogs has long been recommended. However, the efficiency and acceptability of this control strategy have continuously discussed, as it has had limited impact on the incidence of human visceral leishmaniasis (Otranto and Dantas-Torres 2013). In Europe, dogs with clinical leishmaniasis are routinely treated with first-line drugs, including a combination of meglumine antimoniate and allopurinol. In Brazil, the treatment with drugs currently used for humans is forbidden, but veterinarians may adopt alternative protocols using drugs that are not used for humans (Dantas-Torres et al. 2012; Andrade et al. 2011; Manna et al. 2008). Vaccines are not available neither to prevent nor to treat human leishmaniasis, partly because of the complexity of the cellular immune response and the diversity of clinical manifestations and species. Otherwise, vaccines against canine leishmaniasis have effectively been tested in countries from South America and Europe, which could be an interesting approach aiming reduction of the transmission of Leishmania to people (Dantas-Torres 2009). Therefore, early diagnosis and treatment represent the best strategies to the control of leishmaniasis for both individual patients and for the community. In the absence of effective vector control measures and vaccines against leishmaniasis, effective chemotherapy remains the mainstay of treatment.

8.2.5

Current Drugs Used in the Treatment of Leishmaniasis

Although the number of treatment options for leishmaniasis has increased in the past decade, the existing therapies have serious drawbacks in terms of safety, resistance, stability, cost, and have low tolerability, long treatment duration, and difficult administration in field settings. Considering the current therapeutic options available for leishmaniasis, the choice for first-line and second-line treatment varies on the form of disease and is often guided by regional practice (Fig. 8.1). However, these drugs are toxic and have poor patient compliance because many of them require daily systemic (iv or im) administration for some weeks. Furthermore, the emergence of drug-resistant strains is rapidly increasing worldwide (McGwire and Satoskar 2014).

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Fig. 8.1 Structures of the current drugs used in antileishmanial treatment

Pentavalent antimonials, as sodium stibogluconate (Pentostam®) and meglumine antimoniate (Glucantime®), have been the first-line treatment for most forms of leishmaniasis for more than 70 years. In most cases, Antimonial therapy requires up to 28 days of parenteral administration; however, the drug toxicity, with frequent and intense side effects including cardiac arrhythmia and acute pancreatitis, and the emergence of significant resistance, limit the usefulness of the drug (Croft and Coombs 2003).

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Cutaneous disease heals by re-epithelialization with scarring. The treatment is used to accelerate cure, reduce scarring, and to prevent dissemination (e.g., mucosal disease) or relapse. Treatment is especially likely to be given for persistent lesions (>6 months) or lesions that are located over joints, multiple (5–10 or more), or large (4–5 cm or more) (Murray et al. 2005). Parenteral antimony presented satisfactory outcome for CL in all regions, and alternatively to systemic antimony, intralesional treatment is frequently used in a variety of regimens (Bumb et al. 2010). Conventional amphotericin B deoxycholate attaches to membrane ergosterol of parasites causing pores, which alter ion balance and result to cell death; it has replaced antimony in some areas where treatment failures occur. Although highly effective, adverse reactions as fever, chills, and rigor are common related, because of the needed of infusions and lengthy administration (20–30 days). Life-threatening adverse side effects such as hypokalemia (low potassium levels in the blood), nephrotoxicity, and first-dose anaphylaxis are common (Nagle et al. 2014). Lipid formulations of amphotericin B (AmBisome®) represent a macrophagetargeted treatment and induce side effects but to a lesser frequency than the free drug, being very effective in 5–10 day regimens. Although the cure rate with antimony is about 90%, in Europe, most patients now receive liposomal amphotericin B for treatment, which produces high-level efficacy in short-course regimens, reducing long hospital stays. However, the cost of this treatment restricts its use in most developing countries (Balasegaram et al. 2012). Paramomicyn, an aminoglycoside antibiotic similar to aminosidine, works by blocking protein synthesis by binding to 16S ribosomal RNA and has shown promising results in leishmaniasis treatment in India and Africa. The side effects include reversible high-tone otoxicity (damage to the inner ear), significant increase in hepatic transaminases and mild injection pain. The low cost for the 21-day course should provide an injectable alternative to amphotericin B in India and a potential substitute for antimony worldwide (Nagle et al. 2014). Pentamidine (Pentacrinat and Pentam®), is a multitarget drug, supposed to interfere with the biosynthesis of macromolecules such as DNA, RNA, phospholipids, and proteins. A wide array of adverse reactions have been reported with pentamidine use including hypoglycemia, diabetes, liver enzyme abnormalities, leukopenia, anemia, nephrotoxicity, cardiac arrhythmias, heart failure, and hypotension (McGwire and Satoskar 2014). Miltefosine (Impavido® and Miltex®) is an alkylphosphocholine initially developed as an anticancer drug. In 2002, it was approved in India as the first oral treatment of VL. In March 2014, the FDA approved oral miltefosine to treat visceral, cutaneous, and mucocutaneous leishmaniasis in patients aged  12 years. The most common adverse effect is associated with gastrointestinal disturbances and occasional hepato and nephrotoxicity. Due to teratogenic aspects associated to this drug, its use is forbidden in pregnant women and should be used with care in fertile women (Monge-Maillo and López-Vélez 2015). Although recent breakthroughs such as the development of miltefosine and paramomicyn as novel drugs and the reduction in the cost of liposomal amphotericin B, there is a definitive need for continued investment in leishmaniasis control

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(Chappuis et al. 2007). Furthermore, it is imperative to consider the access to drugs, which is determined by factors such as drug affordability, drug availability, proper forecasting, distribution and storage, drug quality, drug legislation and pharmacovigilance, and user-friendliness through appropriate packing and information leaflets (den Boer et al. 2011). The need to develop a single drug or drug formulation effective against all forms of leishmaniasis is a difficult purpose to achieve; not only the diversity of Leishmania, species differ intrinsically to drug sensitivity, but also the visceral and cutaneous sites of infection imposes differing pharmacokinetic requirements to be considered (Croft and Coombs 2003). Considering the intracellular localization of the parasite, antileishmanial drugs must be internalized by host cells. In order to search for new drugs, other important factors include rate of uptake, ability to resist intracellular degradation, intracellular trafficking, and possibility of host cytotoxicity should be evaluated. Therefore, when starting screening campaigns for new antileishmanials, one should consider the intra-macrophage amastigote assay, which is technically more difficult, but can identify selective compounds (Tempone et al. 2011). In this aspect, several secondary metabolites isolated from plant species acting directly in intracellular amastigotes were in vitro tested and displayed interesting results. Among these compounds, some natural products showed in vivo potential and should be considered as new hit compounds for Leishmaniasis.

8.3

IN VIVO Antileishmanial Potential of Natural Products from Plants

Several secondary metabolites with potential antileishmanial activity have been isolated from different plant species. These compounds, distributed in alkaloids, terpenoids, phenolic derivatives, pyrones, naphtoquinones, and iridoids (Table 8.1 and Fig. 8.2), represent potential structures for the study of new drug candidates for Leishmaniasis. The flavonoid (-)-epigallocatechin 3-O-gallate (1), an abundant phenolic derivative from the green tea Camellia sinensis (Theaceae), has been evaluated against L. amazonensis-infected BALB/c mice (MHOM/BR/75/LTB0016 strain) with interesting results. The oral treatment at 30 mg/kg/day significantly reduces the lesion size and parasite burden. In spite of the toxicity aspects for kidney and liver tissues were observed in treated mice, additional analysis showed lack of relevant alterations in creatinine, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) serum levels (Inacio et al. 2013). Piper rusbyi (Piperaceae) produces pyrones with activity against cutaneous leishmaniasis (Leishmania amazonensis) in BALB/c model. Bioactivity-guided fractionation of crude extract provided two bioactive compounds: (+)-(7R,8S)epoxy-5,6-didehydrokavain (2) and flavokain B (3). Compound 3 showed in vivo

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Table 8.1 Natural products (1–41) isolated from plant species with in vivo antileishmanial activity Compound

Plant species

Tested against Leishmania sp.

Reference

(-)-epigallocatechin-3-O-gallate (1)

L. amazonensis

(+)-(7R,8S)epoxy-5,6-didehydrokavain (2) flavokain B (3)

Camellia sinensis Piper rusbyi

L. amazonensis

Piper rusbyi

L. amazonensis

16a-hydroxycleroda-3,13,14Zdien-15,16-olide (4) plumbagin (5)

Polyalthia longifolia Pera benensis

L. donovani

3,3’-biplumbagin (6)

Pera benensis

8,8’-biplumbagin (7)

Pera benensis

4-hydroxy-1-tetralone (8)

Ampelocera edentula Zanthoxylum chiloperone Zanthoxylum chiloperone Chenopodium ambrosioides Chenopodium ambrosioides Chenopodium ambrosioides Pluchea carolinensis Pluchea carolinensis Pluchea carolinensis Pluchea carolinensis Myrica rubra

Inacio et al. (2013) Flores et al. (2007) Flores et al. (2007) Misra et al. (2010) Fournet and Barrios (1992) Fournet and Barrios (1992) Fournet and Barrios (1992 Fournet et al. (1994) Ferreira et al. (2002) Ferreira et al. (2002) Monzote et al. (2014) Monzote et al. (2014) Monzote et al. (2014) Montrieux et al. (2014) Montrieux et al. (2014) Montrieux et al. (2014) Montrieux et al. (2014); Tasdemir et al. (2006) Mittra et al. (2000) Mittra et al. (2000) Muzitano et al. (2009) (continued)

ascanthin-6-one (9) 5-methoxycanthin-6-one (10) ascaridole (11) carvacrol (12) caryophyllene oxide (13) caffeic acid (14) ferulic acid (15) rosmarinic acid (16) quercetin (17)

L. amazonensis/L. venezuelensis L. amazonensis/L. venezuelensis L. amazonensis/L. venezuelensis L. amazonensis/L. venezuelensis L. amazonensis L. amazonensis L. amazonensis L. amazonensis L. amazonensis L. amazonensis L. amazonensis L. amazonensis L. donovani L. donovani

Fagopyrum esculentum Vitex negundo

L. donovani

Kalanchoe pinnata

L. amazonensis

L. donovani

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Table 8.1 (continued) Compound

Plant species

Tested against Leishmania sp.

Reference

flavone A (18)

Vitex negundo

L. donovani

hesperidin (19)

Citrus sinensis

L. donovani

diosmin (20)

Citrus sinensis

L. donovani

rutin (21)

Fagopyrum esculentum Glycyrrhiza ralensis Citrus sinensis

L. donovani

Sen et al. (2005) Sen et al. (2005) Sen et al. (2005) Sen et al. (2005) Chen et al. (1994) Arruda et al. (2009) Tasdemir et al. (2006) Mittra et al. (2000) Mittra et al. (2000) Tasdemir et al. (2006) Tasdemir et al. (2006) Tasdemir et al. (2006) Saha et al. (2013) Germonprez et al. (2005) Kyriazis et al. (2013) Khaliq et al. (2009) Muzitano et al. (2009)

licochalcone A (22) limonene (23) luteolin (24)

L. major/L. donovani L. amazonensis

Terminalia chebula Fagopyrum esculentum Vitex negundo

L. donovani

L. donovani L. donovani

myricetin (27)

Gonocytisus angulatus Senegalia berlandieri Myrica rubra

lyoniside (28)

Saraca indica

L. donovani

maesabalides I–VI (29–34)

Maesa balansae Olea europaea

L. infantum

Peganum harmala Kalanchoe pinnata

L. donovani

Kalanchoe pinnata Helietta apiculata Nyctanthes arbor-tristis Matricaria chamomilla

L. amazonensis

luteolin-7-O-glucoside (25) fisetin (26)

oleuropein (35) peganine hydrochloride (36) quercetin-3-Oa-L-arabinopyranosyl-(1 ! 2)a-L-rhamnopyranoside (37) quercetin-3-Oa-L-rhamnopyranoside (38) c-fagarine (39) calceolarioside A (40) (-)-a-bisabolol (41)

L. donovani L. donovani

L. donovani

L. donovani

L. amazonensis

L. amazonensis L. donovani L. infantum

Muzitano et al. (2009) Ferreira et al. (2010) Poddar et al. (2008) Corpas-López et al. (2015)

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Fig. 8.2 Structures of natural products (1–41) with in vivo antileishmanial activity

activity at 5 mg/kg/day dosage after eight weeks of treatment significantly reducing the lesion sizes (Flores et al. 2007). The terpenoid 16a-hydroxycleroda-3,13,14Z-dien-15,16-olide (4), isolated from Polyalthia longifolia (Annonaceae), showed no cytotoxicity against J774A.1 macrophages and was orally administered to male Golden hamsters (Mesocricetus auratus) infected with L. donovani (MHOM/80/Dd8) at four dose schedules: 20, 50, 100, and 250 mg/kg for five days. At higher doses, the compound was effective to reduce the parasite burden in spleen by 91% (±2%). Regarding the mechanism of action, in silico molecular docking indicated DNA topoisomerase I interaction

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Fig. 8.2 (continued)

through hydrophobic forces and five strong hydrogen bonds, which may have inhibited the enzyme and ultimately lead to cell apoptosis (Misra et al. 2010). Popularly used to the treatment of cutaneous leishmaniasis caused by L. braziliensis, crude extracts from Pera benensis (Euphorbiaceae) afforded the naphtoquinones plumbagin (5), 3,3’-biplumbagin (6), and 8,8’-biplumbagin (7). BALB/c mice infected with L. amazonensis (IFLA/BR/67/PH 8) or L. venezuelensis

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Fig. 8.2 (continued)

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Fig. 8.2 (continued)

(VE/74/PM-H3) were treated by subcutaneous route and intralesional injection with 5–7. Compound 5 displayed higher efficacy after eight weeks of treatment at 2.5 and 5 mg/kg/day. The obtained results indicated that the lesion size (4 mm) of treated animals was equivalent to the standard drug (Glucantime®) in comparison to lesion size of the untreated controls (6.4 mm). Additionally, this compound showed lack of toxic effects (Fournet and Barrios 1992).

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Used by indigenous population in Bolivia for treatment of cutaneous leishmaniasis caused by L. braziliensis, the extracts from stem bark of Ampelocera edentula Kuhlm. (Ulmaceae) were subjected to a bioactivity guided fractionation to afford the bioactive 4-hydroxy-1-tetralone (8). This compound was tested in L. amazonensis (PH8) and L. venezuelensis infected BALB/c mice. After 14 days of treatment, the treatment using one single dose of 8 at 50 mg/kg and Glucantime at 112 mg/kg indicated that the plant metabolite presented a superior efficacy (Fournet et al. 1994). From extract of stem bark from Zanthoxylum chiloperone var. angustifolium Engl. (Rutaceae), it was isolated two bioactive alkaloids, identified as ascanthin-6-one (9) and 5-methoxycanthin-6-one (10). Female BALB/c mice infected with L. amazonensis (MHOM/IFLA/BR/67/PH8) were treated with 10 mg/kg daily for 14 days (oral route) and 4 days (intralesional route). Despite the parasite load reduction after treatment with compound 9, the obtained results displayed no significant difference to the untreated group. Furthermore, it demonstrated no superior efficacy to the standard drug (Glucantime), which was administered by subcutaneous route (100 mg/kg for 10 days) and reduced by 91% the parasite burden in lesions (Ferreira et al. 2002). Leaves of Chenopodium ambrosioides (Chenopodiaceae) produce an essential oil which has been used by native people against parasitic diseases, including leishmaniasis. Crude essential oil and purified terpenoids ascaridole (11), carvacrol (12), and caryophyllene oxide (13) were tested in BALB/c mice at 30 mg/kg (intralesional route) on a 4 days interval during 14 days. Only the crude essential oil prevented lesion development caused by L. amazonensis (MHOM/77BR/LTB0016) strain, statistically superior to the control treatment (Glucantime). Interestingly, the purified compounds 11, 12, and 13 were individually tested but showed no potential effect, leading to animal death after three days of treatment (Monzote et al. 2014). Phenolic derivatives caffeic (14), ferulic (15), and rosmarinic (16) acids as well as quercetin (17), isolated from Pluchea carolinensis (Asteraceae), were tested against MHOM/77BR/LTB0016 strain of L. amazonensis in BALB/c mice parasite burden and lesion decrease models. After 15 days of infection, animals were treated with a solution of purified compounds in DMSO (30 mg/kg) at five doses, which were administered in an intralesional route each 4 days for 45 days. Compounds 14–17 displayed in vitro selectivity indexes (SI) of 11, 17, 20, and 10, respectively, but only phenolic acids (caffeic, ferulic, and rosmarinic) managed to control lesion size and parasite load in treatments groups. Additionally, the obtained results indicated that compound 15 displayed superior efficacy (highest and fastest) in lesion size reduction and no trace of parasite was found after six weeks of treatment. In conclusion, compounds 14–16 displayed superior activity when compared to Glucantime (30 mg/kg) after six weeks of treatment (Montrieux et al. 2014). As flavonoids are phenolic derivatives with antioxidant properties, some derivatives were used to mitigate anemia and parasitaemia associated with visceral leishmaniasis. Flavone A (18), isolated from Vitex negundo (Lamiaceae) leaves, hesperidin (19) and diosmin (20), obtained from Citrus sinensis (Rutaceae), rutin (21) isolated from Fagopyrum esculentum (Polygonaceae) and quercetin (17), from different plant species, were orally administered at 5, 10, 20, 30, and 40 mg/kg in a

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biweekly schedule for 30 days. Dosages ranging from 10 to 40 mg/kg proved to be effective in restoring the Syrian golden hamsters hemoglobin (Hb) decreased levels caused by L. donovani (MHOM/IN/1983/AG83) infection, indicating the efficacy rate as 17 > 18 > 19 > 20 > 21. Quercetin (17) presented the greatest performance for the subsequently in vivo tests, achieving the highest effect in preventing the lipid peroxidation (oxidative damage to membranes cell), suppression of protein carbonyl increase, rise of erythrocytes antioxidant capacity, correction of anemia levels during visceral leishmaniasis and spleen parasitaemia reduction. Further studies evaluated the efficacy of compound 17 combined with the reference drug sodium stibogluconate (SAG). The obtained results showed superior prevention of reactive oxygen species (ROS) formation (57.9% mixture; 23.7% SAG; 47.4% quercetin), higher proteolytic degradation reduction, prolonged red cells life span, and decreased spleen load of parasites for the associated treatment (Sen et al. 2005). Licochalcone A (22), isolated from Glycyrrhiza ralensis (Fabaceae), was examined against L. major (MHOM/IVL67/LRC-L137) and L. donovani (MHOM/ KE/85/NLB 439) with promising results. At doses of 2.5 and 5.0 mg/kg (daily for 39 days) using intraperitoneally administration, it managed to completely prevent lesion development in L. donovani-infected BALB/c mice. In male Golden hamsters, the intraperitoneally route at doses of 20 mg/kg daily for 6 days induced a superior rate than 96% of parasite load reduction in liver and spleen. The daily oral treatment for 6 days managed to reduce 65% and 85% of L. donovani loads in liver and spleen, respectively, at doses of 5 to 150 mg/kg. Spleen loads of parasites assessed through [3H]-thymidine uptake from infected hamsters displayed 1% of the observed parasites from untreated control group (Chen et al. 1994). Limonene (23), a commonly hydrocarbon monoterpene isolated from Citrus sinensis (Rutaceae) as well as several other plant species, was tested in female C57BL/6 mice against L. amazonensis (MHOM/BR/1973/M2269) infection. This monoterpene was topically delivered in ointments vehicles (10% wt/wt) for 19 weeks and intrarectally at 100 mg/kg/day doses for 2 weeks. Average lesion size was significantly reduced in 80% of the intrarectally treated animals. After 16 weeks of treatment completion, the animal condition remained, whilst untreated control group developed growing manifestation of the disease. Through limiting dilution assay, the parasite load displayed a 99.9% reduction in 80% of the animals. The topical treatment managed to completely heal 67–86% of animals and 13 weeks after treatment interruption very scarce or no trace of parasites were found with no side effects detected in such case (Arruda et al. 2009). Tasdemir et al. (2006) evaluated the in vivo antileishmanial activity (BALB/c models) of six flavonoid derivatives isolated from different plant species: luteolin (24) from Terminalia chebula (Combretaceae), luteolin-7-O-glucoside (25) from Gonocytisus angulatus (Fabaceae), fisetin (26) from Senegalia berlandieri (Fabaceae) as well as quercetin (17) and myricetin (27) from Myrica rubra (Myricaceae). Mice infected with L. donovani (HU3) were intraperitoneally treated with 30 mg/kg for 5 days but only quercetin (17) proved mildly active inhibiting 15.3% of infection.

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Flavonoids quercetin (17) and luteolin (24) were also obtained from Fagopyrum esculentum (Polygonaceae) and from Vitex negundo (Lamiaceae). Concerning their in vivo potential, male Golden hamsters infected with L. donovani (AG83) were orally treated with 3.5 mg/kg of 24 and 14 mg/kg of 17 for 4 weeks and the spleen parasitaemia was assessed. In spite of the inferior concentration flavonoid 24 was able to reduce 80% of parasite load, while compound 17 displayed 90% of reduction at four times the higher dose (Mittra et al. 2000). The lignan lyoniside (28) was isolated from Saraca indica (Fabaceae) stem barks and was evaluated against visceral leishmaniasis. This compound was tested in BALB/c infected with L. donovani (AG83) models at 2.5 and 5 mg/kg via intraperitoneally route on a 2 times a week regimen during three weeks of treatment. It was determined that through the nitric oxide and ROS lignans induced production liver and spleen parasite burden was almost fully eliminated. Poor cytotoxic effect in murine peritoneal macrophages was detected at concentration up to 100 lM (Saha et al. 2013). Several antiprotozoal saponins were isolated from the leaves of the Vietnamese medicinal plant Maesa balansae (Myrsinaceae). Maesabalides I–VI (29–34) were evaluated against L. infantum (MHOM/MA-(BE)/67) in BALB/c mouse model. Infection and treatment commenced simultaneously via subcutaneous injections and seven days after the single dose of 0.2 mg/kg of 31 displayed more than 90% of liver burden reduction (Germonprez et al. 2005). Oleuropein (35), an iridoid derivative isolated from Olea europaea (Oleaceae), was in vivo tested in a visceral leishmaniasis model. BALB/c mice infected with L. donovani (zymodeme MON-2, strain MHOM/IN/1996/THAK35) were intraperitoneally treated every day with 45, 15, and 5 mg/kg for 3 days with pure 35. All dosages proved to reduce spleen parasite burden significantly, reaching up to 79.5%, 86.8%, and 57.2% (45, 15, and 5 mg/kg, respectively). Liver parasite load displayed 58.1%, 62.0%, and 65.7% of reduction rate after 28 days of treatment, respectively. Six weeks after treatment completion, spleen parasite load diminished by 99.7%, 99.8%, and 96.9%, respectively (Kyriazis et al. 2013). Peganum harmala (Zygophyllaceae) is medicinal plant commonly used in India to the treatment of parasitic diseases. Fractionation of bioactive extract from seeds afforded a bioactive alkaloid identified as peganine hydrochloride (36). This compound was orally delivered in three dosages of 50, 100, and 200 mg/kg in L. donovani (MHOM/80/Dd8)-infected hamsters for 5 days. The highest dose managed to inhibit 87.5% of parasite splenic burden, the 100 mg/kg inhibited slightly less of 79.6% whilst the minimum dosage proved inactive. Control drug miltefosine at 40 mg/kg inhibited 95.5% of parasites (Khaliq et al. 2009). Flavonoid glycosides derived from aqueous extract of Kalanchoe pinnata (Crassulaceae) were investigated as antileishmanial drugs. Quercetin (17), quercetin-3-O-a-L-arabinopyranosyl-(1 ! 2)-a-L-rhamnopyranoside (37), and quercetin-3-O-a-L-rhamnopyranoside (38) were orally tested in L. amazonensis

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(MHOM/BR/75/Josefa strains) BALB/c models at 16mg/kg. Compounds 17, 37, and 38 reduced parasite load by 76%, 65%, and 57%, respectively, demonstrating a potential oral therapeutic treatment with these compounds (Muzitano et al. 2009). The furoquinoline alkaloid c-fagarine (39) was isolated from Helietta apiculata (Rutaceae), a shrub found in South America. Mice (BALB/c model) infected with L. amazonensis (MHOM/IFLA/BR/67/PH8) were treated for 15 days with 10 mg/ kg with via oral administration. Compared to the reference drug Glucantime, compound 39 displayed superior efficacy. In spite of the superior concentration of 100 mg/kg and subcutaneous delivery route, the drug control reduced lesion weight by 66.9% whilst the alkaloid 39 displayed 90.5% of effectiveness. The reference drug and 39 reduced parasite burden by 95.2% and 97.4%, respectively, with satisfactory toleration by the animals, indicating a potential lead for cutaneous therapy (Ferreira et al. 2010). The bioactivity guided fractioning of Nyctanthes arbor-tristis (Oleaceae) leaves yielded the bioactive phenylpropanoid calceolarioside A (40). In golden hamsters infected with L. donovani (Ag83), compound 40 was able to reduce spleen (84%) and liver (79%) parasite load at 20 mg/kg doses. The treatment was performed on a daily bases for three consecutive weeks with increasing dosages (Poddar et al. 2008). Derived from chamomile, Matricaria chamomilla (Asteraceae) is essentially composed by the monocyclic sesquiterpene (-)-a-bisabolol (41). BALB/c mice infected with L. infantum promastigotes were treated with 200 mg/kg oral doses of 41, using meglumine antimoniate (104 mg SbV/kg) as a standard drug. In addition to the lack of toxicity, this sesquiterpene was more effective than the standard drug in decreasing the parasite burden in spleen (71.6%) and as effective as the standard drug in liver (89.2%) (Corpas-López et al. 2015).

8.4

Conclusion

This chapter presented an overview concerning the secondary metabolites isolated from different plant species with in vivo potential against Leishmania spp. Several compounds such as mono, sesqui, di, and triterpenoids as well as steroids, iridoids, saponins, naphtoquinones, flavonoids, phenylpropanoids, pyrones, alkaloids, and lignoids displayed activity. Among these compounds, an important metabolite is quercetin, a non-glycosylated flavonol widely distributed in several plant species. The data presented in this chapter shows the immense wealth of interesting natural compounds with in vivo activity against Leishmania spp. and the high potential of natural products for the discovery of new leads for the treatment of a neglected parasitic disease as leishmaniasis.

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References Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, den Boer M (2012) Leishmaniasis worldwide and global estimates of its incidence. PLoS ONE 7:e35671 Andrade HM, Toledo VPCP, Pinheiro MB, Guimarães TMPD, Oliveira NC, Castro JA, Silva RN, Amorim AC, Brandão RMSS, Yoko M, Silva AS, Dumont K, Ribeiro ML, Bartchewsky W, Monte SJH (2011) Evaluation of miltefosine for the treatment of dogs naturally infected with L. infantum (=L. chagasi) in Brazil. Vet Parasitol 181:83–90 Arruda DC, Miguel DC, Yokoyama-Yasunaka JKU, Katzin AM, Uliana SRB (2009) Inhibitory activity of limonene against Leishmania parasites in vitro and in vivo. Biomed Pharmacother 63:643–649 Balasegaram M, Ritmeijer K, Lima MA, Burza S, Ortiz Genovese G, Milani B, Gaspani S, Potet J, Chappuis F (2012) Liposomal amphotericin B as a treatment for human leishmaniasis. Expert Opin Emerg Drugs 17:493–510 Bumb RA, Mehta RD, Ghiya BC, Jakhar R, Prasad N, Soni P, Lezama-Davila C, Satoskar AR (2010) Efficacy of short-duration (twice weekly) intralesional sodium stibogluconate in treatment of cutaneous leishmaniasis in India. Br J Dermatol 163:854–858 Chappuis F, Sundar S, Hailu A, Ghalib H, Rijal S, Peeling RW, Alvar J, Boelaert M (2007) Visceral leishmaniasis: what are the needs for diagnosis, treatment and control? Nat Rev Microbiol 5:873–882 Chen M, Christensen SB, Theander TG (1994) Antileishmanial activity of Licochalcone A in mice infected with Leishmania major and in hamsters infected with Leishmania donovani. Antimicrob Agents Chemother 38:1339–1344 Corpas-López V, Morillas-Márquez F, Navarro-Moll MC, Merino-Espinosa G, Díaz-Sáez V, Martín-Sánchez J (2015) (−)-a-Bisabolol, a promising oral compound for the treatment of visceral leishmaniasis. J Nat Prod 78:1202–1207 Croft SL, Coombs GH (2003) Leishmaniasis—Current chemotherapy and recent advances in the search for novel drugs. Trends Parasitol 19:502–508 Dantas-Torres F (2009) Canine leishmaniosis in South America. Parasit Vectors 2:S1 Dantas-Torres F, Solano-Gallego L, Baneth G, Ribeiro VM, de Paiva-Cavalcanti M, Otranto D (2012) Canine leishmaniosis in the old and new worlds: unveiled similarities and differences. Trends Parasitol 28:531–538 Den Boer M, Argaw D, Jannin J, Alvar J (2011) Leishmaniasis impact and treatment access. Clin Microbiol Infect 17:1471–1477 Desjeux P (2004) Leishmaniasis: current situation and new perspectives. Comp Immunol Microbiol Infect Dis 27:305–318 Desjeux P, Ghosh R, Dhalaria P, Strub-Wourgaft N, Zijlstra EE (2013) Report of the Post Kala-Azar Dermal Leishmaniasis (PKDL) consortium meeting, New Delhi, India, 27–29 June 2012. Parasit. Vectors 6:196 Ferreira ME, de Arias AR, Yaluff G, de Bilbao NV, Nakayama H, Torres S, Schinini A, Guy I, Heinzen H, Fournet A (2010) Antileishmanial activity of furoquinolines and coumarins from Helietta apiculata. Phytomedicine 17:375–378 Ferreira ME, Rojas de Arias A, Torres de Ortiz S, Inchausti A, Nakayama H, Thouvenel C, Hocquemiller R, Fournet A (2002) Leishmanicidal activity of two canthin-6-one alkaloids, two major constituents of Zanthoxylum chiloperone var. angustifolium. J Ethnopharmacol 80:199– 202 Flores N, Cabrera G, Jiménez IA, Piñero J, Giménez A, Bourdy G, Cortés-Selva F, Bazzocchi IL (2007) Leishmanicidal constituents from the leaves of Piper rusbyi. Planta Med 73:206–211 Fournet A, Barrios A (1992) Effect of natural naphthoquinones in BALB/c mice infected with Leishmania amazonensis and L. venezuelensis. Trop Med Parasitol 43:219–222 Fournet A, Barrios AA, Muñoz V, Hocquemiller R, Roblot F, Cavé A (1994) Antileishmanial activity of a tetralone isolated from Ampelocera edentula, a Bolivian plant used as a treatment for cutaneous leishmaniasis. Planta Med 60:8–12

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Germonprez N, Maes L, Van Puyvelde L, Van Tri M, Tuan DA, De Kimpe N (2005) In Vitro and in vivo anti-leishmanial activity of triterpenoid saponins isolated from Maesa balansae and some chemical derivatives. J Med Chem 48:32–37 Herwaldt BL (1999) Leishmaniasis. Lancet 354:1191–1199 Inacio JDF, Canto-Cavalheiro MM, Almeida-Amaral EE (2013) In vitro and in vivo effects of (−)epigallocatechin 3-O-gallate on Leishmania amazonensis. J Nat Prod 76:1993–1996 Khaliq T, Misra P, Gupta S, Reddy KP, Kant R, Maulik PR, Dube A, Narender T (2009) Peganine hydrochloride dihydrate an orally active antileishmanial agent. Bioorg Med Chem Lett 19:2585–2586 Kyriazis JD, Aligiannis N, Polychronopoulos P, Skaltsounis A-L, Dotsika E (2013) Leishmanicidal activity assessment of olive tree extracts. Phytomedicine 20:275–281 Manna L, Reale S, Picillo E, Vitale F, Gravino AE (2008) Interferon-gamma (INF-c), IL4 expression levels and Leishmania DNA load as prognostic markers for monitoring response to treatment of leishmaniotic dogs with miltefosine and allopurinol. Cytokine 44:288–292 McGwire BS, Satoskar AR (2014) Leishmaniasis: clinical syndromes and treatment. QJM 107:7– 14 Misra P, Sashidhara KV, Singh SP, Kumar A, Gupta R, Chaudhaery SS, Gupta S Sen, Majumder HK, Saxena AK, Dube A (2010) 16alpha-Hydroxycleroda-3,13(14) Z-dien-15,16-olide from Polyalthia longifolia: a safe and orally active antileishmanial agent. Br J Pharmacol 159:1143–1150 Mittra B, Saha A, Chowdhury AR, Pal C, Mandal S, Mukhopadhyay S, Bandyopadhyay S, Majumder HK (2000) Luteolin, an abundant dietary component is a potent anti-leishmanial agent that acts by inducing topoisomerase II-mediated kinetoplast DNA cleavage leading to apoptosis. Mol Med 6:527–541 Monge-Maillo B, López-Vélez R (2015) Miltefosine for visceral and cutaneous leishmaniasis: drug characteristics and evidence-based treatment recommendations. Clin Infect Dis 60:1398– 1404 Montrieux E, Perera WH, García M, Maes L, Cos P, Monzote L (2014) In vitro and in vivo activity of major constituents from Pluchea carolinensis against Leishmania amazonensis. Parasitol Res 113:2925–2932 Monzote L, Pastor J, Scull R, Gille L (2014) Antileishmanial activity of essential oil from Chenopodium ambrosioides and its main components against experimental cutaneous leishmaniasis in BALB/c mice. Phytomedicine 21:1048–1052 Murray HW, Berman JD, Davies CR, Saravia NG (2005) Advances in leishmaniasis. Lancet 366:1561–1577 Muzitano MF, Falcão CAB, Cruz EA, Bergonzi MC, Bilia AR, Vincieri FF, Rossi-Bergmann B, Costa SS (2009) Oral metabolism and efficacy of Kalanchoe pinnata flavonoids in a murine model of cutaneous leishmaniasis. Planta Med 75:307–311 Nagle AS, Khare S, Kumar AB, Supek F, Buchynskyy A, Mathison CJN, Chennamaneni NK, Pendem N, Buckner FS, Gelb MH, Molteni V (2014) Recent developments in drug discovery for leishmaniasis and human african Trypanosomiasis. Chem Rev 114:11305–11347 Otranto D, Dantas-Torres F (2013) The prevention of canine leishmaniasis and its impact on public health. Trends Parasitol 29:339–345 Poddar A, Banerjee A, Ghanta S, Chattopadhyay S (2008) In vivo efficacy of calceolarioside A against experimental visceral leishmaniasis. Planta Med 74:503–508 Ready P (2014) Epidemiology of visceral leishmaniasis. Clin, Epidemiol, p 147 Ready PD (2013) Biology of phlebotomine sand flies as vectors of disease agents. Annu Rev Entomol 58:227–250 Saha S, Mukherjee T, Chowdhury S, Mishra A, Chowdhury SR, Jaisankar P, Mukhopadhyay S, Majumder HK (2013) The lignan glycosides lyoniside and saracoside poison the unusual type IB topoisomerase of Leishmania donovani and kill the parasite both in vitro and in vivo. Biochem Pharmacol 86:1673–1687

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Sen G, Mandal S, Saha Roy S, Mukhopadhyay S, Biswas T (2005) Therapeutic use of quercetin in the control of infection and anemia associated with visceral leishmaniasis. Free Radic Biol Med 38:1257–1264 Singh S (2014) Changing trends in the epidemiology, clinical presentation, and diagnosis of Leishmania–HIV co-infection in India. Int J Infect Dis 29:103–112 Tasdemir D, Kaiser M, Brun R, Yardley V, Schmidt TJ, Tosun F, Rüedi P (2006) Antitrypanosomal and antileishmanial activities of flavonoids and their analogues: in vitro, in vivo, structure-activity relationship, and quantitative structure-activity relationship studies. Antimicrob Agents Chemother 50:1352–1364 Tempone AG, Martins de Oliveira C, Berlinck RGS (2011) Current approaches to discover marine antileishmanial natural products. Planta Med 77:572–585

Chapter 9

Insecticidal and Antimalarial Properties of Plants: A Review Lucie Paloque, Asih Triastuti, Geneviève Bourdy and Mohamed Haddad

Abstract Parasitic diseases remain a major burden on global human and veterinary health. They affect more than two billion people worldwide causing considerable morbidity and mortality and are a major constraint on livestock production, especially in the world’s poorest communities. The immense suffering caused by these illnesses and the consequential loss of productivity is a major drain on the limited resources of the populations in which they occur. Most modern and effective drugs for parasitic diseases present no financial viability for the pharmaceutical industry since affected people have limited financial resources. Although financial return on investment is insufficient for drug discovery process and development, there is a constant desperate need for new chemical entities presenting new mechanisms of action. Higher plants, marine organisms, and microorganisms provide immense opportunities for the discovery of new drugs and drug leads. The screening of these natural sources thus remains one of the most attractive routes to discovering and developing new drugs. This article reviews the importance of natural products as a source of antiparasitic drugs and discusses some of the research challenges. Keywords Natural products Drug discovery

9.1


Antimicrobial
Human/animal parasitic disease

Introduction

Parasitic diseases are a major burden on global human and veterinary health, especially in the world’s poorest areas where their impact significantly drains already limited resources. Most modern and effective drugs for parasitic diseases L. Paloque UPR8241 CNRS Laboratoire de Chimie de Coordination, University of Toulouse 3, 31062 Toulouse, France A. Triastuti
G. Bourdy
M. Haddad (&) UMR 152, IRD-UPS Pharma-DEV, University of Toulouse 3, 31062 Toulouse, France e-mail: [email protected] © Springer International Publishing AG 2018 J.-M. Mérillon and C. Rivière (eds.), Natural Antimicrobial Agents, Sustainable Development and Biodiversity 19, https://doi.org/10.1007/978-3-319-67045-4_9

215

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L. Paloque et al.

present no financial viability for the pharmaceutical industry since affected people have limited financial resources. Although the financial return on investment is insufficient for drug discovery process and development, new reasonably priced antiparasitic drugs presenting new mechanisms of action remain a constant and urgent need (Hertweck 2015; Pink et al. 2005; Ndjonka et al. 2013). Over the past few years, the pharmaceutical industry has diverted much of their focus from approaches based on high-throughput screening (HTP) of combinatorial libraries and genomics to those investigating natural products, mainly with bacteria and fungi, especially endophytes, as important sources of biologically active compounds. Marine organisms have also proven an interesting source of antiparasitic compounds and have stimulated the search in other natural sources, which today remains a major strategy and one of the most attractive routes to discovering and developing new drugs (McKerrow 2015; Butler et al. 2014; Fernańdez-Álvaro et al. 2016; Colley 2000). Higher plants produce a large diversity of bioactive compounds with a huge amount of structurally differing secondary metabolites having evolved during plant development as a means for plants to defend themselves against herbivores and microorganisms. These compounds exert various biological activities, including antimicrobial or antiparasitic (Kayser et al. 2003; Newman and Cragg 2012, 2016; Ngo et al. 2013; Blunt et al. 2016). Within parasites, they interfere with vital systems, such as the gene-expression machinery, neuronal signal transduction, and membrane integrity, as well as with cytoskeletal proteins and enzymes (Müller and Hemphill 2013; Ziegler et al. 2013; Horn and Duraisingh 2014; Wink 2010, 2015; Zhou et al. 2004; Hussain et al. 2014; Saifi et al. 2013).

9.2 9.2.1

Overview of Parasitic Infections Main Parasitic Diseases

Upon an exhaustive review of the literature and public health database, Woolhouse and Gowtage-Sequeria listed 1,407 human pathogen species as being defined by “species infectious to and capable of causing disease in humans under natural transmission conditions”. Among these, 344 (25%) are parasitic with, respectively, 57 and 287 species of protozoa and helminths (Woolhouse and Gowtage-Sequeria 2005). These parasitic pathogens are responsible for a wide variety of symptoms from benign to severe or lethal. The main pathogenic protozoan and helminth parasites of human concern, according to the World Health Organization (WHO) and the Center for Disease Control and Prevention (CDC) databases (WHO 2016; CDC 2016), are listed in Table 9.1. Other parasitic diseases associated with moderately severe symptoms and/or middle incidence occurring in humans are free-living amebic infections; cryptosporidiosis, cyclosporiasis, cystoisosporiasis and babesiosis due to Apicomplexa; giardiasis and trichomoniasis due to protozoan flagellates; strongyloidiasis,

Trypanosomatidae

Trypanosoma cruzi Trypanosoma spp

Trypanosoma brucei

Theileria spp Toxoplasma gondii Leishmania spp

Plasmodium falciparum Plasmodium vivax Plasmodium malariae Plasmodium ovale Plasmodium knowlesi Plasmodium spp

Babesia spp Cryptosporidium spp Eimeria spp

Protozoa

Apicomplexa

Parasite name

Classification

Main concern Worldwide Worldwide Worldwide

Babesiosisa Cryptosporidiosisa Coccidiosis (Eimeriosis)

Human African Trypanosomiasisb (Sleeping sickness) American Trypanosomiasisb (Chagas Disease) Bovine trypanosomiasis

Africa

South America

Africa

Tropical and subtropical areas

Leishmaniasisa,

Worldwide Worldwide Worldwide

Avian malaria Theileriosis Toxoplasmosisa

Tropical and subtropical areas

Malaria

b

Geographical distribution

Disease name

Table 9.1 Main protozoan and helminth parasites affecting humans and livestock

(continued)

9 Insecticidal and Antimalarial Properties of Plants: A Review 217

Wuchereria bancrofti

Dictyocaulus viviparus Haemonchus contortus Necator americanus Onchocerca volvulus Ostertagia ostertagi Teladorsagia circumcincta Trichostrongylus spp Trichuris trichiura

Ascaris lumbricoides Brugia malayi

Ancylostoma spp

Helminths

Nematoda

Parasite name

Classification

Table 9.1 (continued) Main concern

Worldwide

Gastrointestinal nematode infections Ancylostomiasisb, c

c

Lymphatic filariasisb

Trichuriasisb,

Onchocerciasisb (River blindness) Gastrointestinal nematode infections

Worldwide

Lung infection

Tropical area

Worldwide

Worldwide

Africa, Latin America, Middle East

Worldwide

Tropical areas

Worldwide

Lymphatic filariasisb

Ascariasis

Worldwide

b, c

Ancylostomiasisb,

c

Geographical distribution

Disease name

(continued)

218 L. Paloque et al.

Taeniasis ¤

Taenia spp

z

Cysticercosis

a, b

Echinococcosisa,

Schistosomiasisb b

e

b, e

Paragonimiasisb,

Opisthorchiasis

Taenia solium

Echinoccocus spp

Paragonimus westermani Schistosoma spp

Opisthorchis spp

d

Worldwide

Worldwide

Africa, Latin America, Middle East, Southeast Asia, China, Corsica (France) Worldwide

Americas, Africa, Southeast Asia

Asia, Europe

Worldwide

Fascioliasisa,

Fasciola hepaticad

b, e

Asia

Clonorchiasisb,

Clonorchis sinensisd

e

Geographical distribution

Main concern

Disease name

Parasite name

Zoonotic Disease b Neglected Tropical Disease c Soil-Transmitted Helminth Infections d Liver fluke e Foodborne Trematodiases

a

Cestoda

Trematoda

Classification

Table 9.1 (continued)

9 Insecticidal and Antimalarial Properties of Plants: A Review 219

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L. Paloque et al.

enterobiasis, toxocariasis, loaiasis, trichinellosis and trichostrongylosis due to Nematoda; echinostomiasis and heterophiasis due to Trematoda; hymenolepiasis and diphyllobothriasis due to Cestoda (CDC 2016).

9.2.2

Burden of Parasitic Diseases

In 2016, WHO estimated 50% of the world population to be at risk of contracting one of the main parasitic diseases listed in Table 9.1. While malaria mortality has seen a historical decrease over the last decade resulting in 438 000 deaths in 2015 (WHO 2015), the death toll relating to foodborne trematodiases, leishmaniasis, or schistosomiasis remains unacceptable. The impact of a disease on human population health is expressed in Disability-Adjusted Life Years (DALYs), which measures the years of life lost due to premature mortality, disability, or ill health. It depends on the burden of the disease on the life expectancy (Torgerson 2013). The main human parasitic pathogens are responsible for the loss of 140 000 to 55 million DALYs depending on the disease (Table 9.2), totaling over 75 million DALYs (Hotez et al. 2014; WHO GBD 2016). The DALY metrics consider neither the economic impact of diseases, which—especially neglected tropical diseases— may trap people in a cycle of poverty and illness, nor the direct cost of surveillance, prevention, and treatment measures (Hotez et al. 2014). The global socioeconomic burden of parasitic pathogens is thus mainly due to their direct impact on human health but also concerns their impact on livestock as sources of food/financial asset or as tools in agricultural production, especially in low-income countries where they represent wealth and valuable cultural benefit (Torgerson 2013). In 2014, the worldwide livestock population was 4 708.7 million (FAOSTAT 2016) and is expected to grow further in order to feed an estimated 9 billion people in 2050 (United Nations Population Fund 2012). Antiparasitic drugs

Table 9.2 Estimated DALYs of the main human parasitic diseases (Hotez et al. 2014; WHO GBD 2016)

Disease name

DALYs

Malaria Sleeping sickness Chagas Disease Leishmaniasis Soil-Transmitted Helminth Infections Lymphatic filariasis River blindness Schistosomiasis Foodborne trematodiases Cysticercosis Echinococcosis Total

55 111 000 1 264 000 528 000 3 374 000 5 266 000 2 839 000 598 000 4 026 000 1 880 000 500 000 140 000 75 526 000

9 Insecticidal and Antimalarial Properties of Plants: A Review

221

comprise the principal focus of the animal pharmaceutical industry (Lanusse et al. 2014), with parasitic infections representing a major constraint on efficient livestock production and in many developing countries exerting a profound impact on local poverty. For example, coccidiosis remains a major and recurring problem to affect the poultry industry (one of the most important food suppliers in the world), leading to a global economic impact in excess of $3 billion USD per annum (Blake and Tomley 2014; Chapman 2014). Most grazing ruminants are infected by a variety of helminths (mixed infections are common) which exert a major impact on farming efficiency (Charlier et al. 2014; Roeber et al. 2013). Indeed, gastrointestinal nematode infections cause non-quantifiable economic losses as they affect all farms, even those in developed countries (Geurden et al. 2015). In addition, fasciolosis is responsible for worldwide economic annual losses of around $2 000 million USD in the livestock industry (Khan et al. 2013), and the prevalence of echinococcosis among livestock in some European countries reached 65% in the last decade (Cardona and Carmena 2013). Improving animal health not only helps to reduce the socioeconomic burden of parasitic diseases but it also reduces their incidence in humans in the case of zoonotic diseases. The main pathogenic protozoan parasites and helminths to affect livestock are listed in Table 9.1. Numerous human and/or animal parasitic pathogens share common features, such as host genus and/or species preference, mode of transmission, intra-host location, and clinical effects. An example of this is Babesia bovis, which belongs to the same phylum Apicomplexa as Plasmodium falciparum, and is responsible for intravascular hemolysis, anemia, and the accumulation of parasitized red blood cells in microvasculature associated with cerebral symptoms, respiratory distress, and multivisceral failure in cattle (Gohil et al. 2013).

9.3 9.3.1

Antiparasitic Drugs Importance of Natural Products in Drug Discovery

Natural products and their derivatives have historically provided major sources of new and innovative therapeutic agents. This remains true today despite a definite de-emphasis on natural product discovery and research by many pharmaceutical companies, in particular the large ones, in favor of approaches based on HTP screening of combinatorial libraries and genomics. Nature offers exceptional complexity and molecular diversity for exploitation as a source of drugs, many of which are almost impossible to produce commercially by synthetic means. Drugs of natural origin can be classified into three groups: (i) original natural products; (ii) products derived or chemically synthesized from natural products; or (iii) synthetic products based on natural product structures. Close to half of the most important pharmaceuticals are either natural products or their derivatives, illustrating the importance of natural products in the discovery of leads in drug

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Table 9.3 All anti-infective (anti-bacterial, -fungal, -parasitic, and -viral) drugs (n = 326) for human use. Adapted from Newmann and Cragg (2016) Indication

Total

B

Anti-bacterial 141 1 Antifungal 32 1 Antiparasitic 15 – Anti-viral 138 14 Total 326 16 Percentage 100 4.9 B: Biological macromolecule; N: mixture); ND: Natural product pharmacophore); V: Vaccine; NM:

N

ND

S

S/NM

S*

S*/NM

V

11 71 29 – – 1 28 – 3 25 3 – – – 2 5 5 – 2 – 1 – 4 14 5 24 17 60 13 83 73 8 26 18 89 4.0 25.5 22.4 2.4 8.0 5.5 27.3 Unaltered natural product; NB: Botanical drug (defined derivative; S: Synthetic drug; S*: Synthetic drug (NP Mimic of natural product

development for the treatment of human diseases (Cragg et al. 1997). In their recent review, Newman and Cragg (2016) analyzed the number of natural products providing a source of new drugs for human use worldwide, accounting first for all global diseases combined across 34 years from January 1st, 1981, to December 31st, 2014, and secondly for all approved antitumor drugs from 1950 (earliest so-far identified) to December 2014 (Table 9.3). The authors demonstrated that, overall, the major disease areas to have been investigated (in terms of numbers of drugs approved) in the pharmaceutical industry are infectious diseases (microbial, parasitic, and viral), cancer, hypertension, diabetes, and inflammation, all with over 50 approved drug therapies. However, a detailed look at these major disease areas reveals that among 326 anti-infective drugs (against bacterial, fungal, parasitic, and viral infection) approved and on the pharmaceutical market between 1981 and 2014, 15 compounds are antiparasitics including two natural products and five natural product derivatives.

9.3.2

Approved Natural Products as Antiparasitic Drugs

Tables 9.4 and 9.5 report the 78 molecules currently used against the main pathogenic parasites of human and/or livestock concern. Among these 78 compounds, 23 are natural products or natural product derivatives belonging to highly diverse chemical classes; their structures are presented in Fig. 9.1. Despite their small number, these natural products are of great importance in the fight against parasitic diseases worldwide. Artemisinin and its derivatives, recommended in combination with other antimalarial drugs (ACT) by the WHO as first-line treatment for Plasmodium falciparum infections since 2001, largely participated toward the strong reduction (60% in 15 years) in global malaria death (WHO 2015). In the UK only, ionophore compounds (monensin, lasalocid, narasin, and salinomycin) account for more than 70% of anticoccidial drugs used, with drugs against Eimeria representing 40% of all antimicrobials sold for use in farm and domestic animals

Primaquine

Paromomycin (6)

Miltefosine

Proguanil

Nicarbazin

Diminazene

Alkylphosphocholine

Biguanide derivative

Bisnitrophenylurea

Diamidine (triazene)

S

S

S

N Streptomyces krestomuceticus S

S

Chloroquine

Aminoside

S

Amodiaquine, Piperaquine

8-aminoquinoline

S

Quinoline N Cinchona succiruba S

Lumefantrine, Halofantrine

Mefloquine

S

Amprolium

Aminopyrimidine/ Pyridinium Amino-alcohol

Quinine (5)

N Streptomyces lasaliensis (cpd 1) Streptomyces cinnamonensis (cpd 2) Streptomyces albus (cpd 3) Streptomyces aureofaciens (cpd 4) S

Lasalocid (1), Monensin (2), Salinomycin (3), Narasin (4)

Ionophore compounds

Origin, source organism

Compound

Chemical class

Table 9.4 Approved antiprotozoan compounds

Babesia

Eimeria

Plasmodium

Leishmania

Leishmania

Plasmodium

Plasmodium

Babesia, Plasmodium Plasmodium

Plasmodium

Plasmodium

Eimeria

Eimeria

Parasite

Usage

Quiroz-Castaneda et al. (2015), Chapman (2014) Mosqueda et al. (2012) (continued)

CDC (2016), WHO (2016), Braga et al. (2011) CDC (2016), WHO (2016)

CDC (2016), WHO (2016)

Quiroz-Castaneda et al. (2015), Chapman (2014)

Ref

9 Insecticidal and Antimalarial Properties of Plants: A Review 223

S S

Pyrimethamine

Trimethoprime

2,4-diaminopyridine

S ND Semisynthetic derivative of erythromycin produced by Saccharopolyspora erythraea N Streptomyces ambofaciens

Atovaquone

Buparvaquone Azithromycin (8)

Hydroxynaphtoquinone

Macrolide

Spiramycin (9)

ND Semisynthetic derivative of lincomycin produced by Streptomyces lincolnensis S

Clindamycin (7)

Lincosamide

S

Ketoconazole

Imidazole derivative

S

Pentamidine

Diamidine

Origin, source organism

Compound

Chemical class

Table 9.4 (continued)

Toxoplasma

Babesia, Plasmodium, Toxoplasma Theileria Babesia

Babesia, Toxoplasma

Leishmania

Plasmodium

Toxoplasma, Plasmodium Toxoplasma

Trypanosoma brucei Leishmania

Trypanosoma

Parasite

Usage

(continued)

Morrison (2015) CDC (2016), WHO (2016)

CDC (2016), WHO (2016)

Braga et al. (2011)

CDC (2016), WHO (2016)

Geerts et al. (2001)

Ref

224 L. Paloque et al.

Allopurinol

Halofuginone (11)

Purine analogue

Quinazolinone

Polyene

Phenanthridinium

N-methyl glucamine, Meglumine antimoniate, Sodium stibogluconate Isometamidium, Homidium (ethidium bromide) Amphotericin B (10)

Pentavalent antimoniae derivatives

S

Nitazoxanide

Eflornithine

S

Nifurtimox

Ornithine derivative

S

Benznidazole

Nitroheterocycle (2-nitroimidazole) Nitroheterocycle (5-nitrofurane) Nitroheterocycle (5-nitrothiazole)

ND Synthetic halogenated derivative of febrifugine, isolated from a Chinese herb, Dichroa febrifuga

N Streptomyces nodosus S

S

S

S

S

Suramine

Naphthalenepolysulfonic acid derivative

Origin, source organism

Compound

Chemical class

Table 9.4 (continued)

Cryptosporidium

Leishmania

Leishmania

Trypanosoma

Trypanosoma brucei Leishmania

Trypanosoma cruzii Trypanosoma cruzii Cryptosporidium

Trypanosoma brucei

Parasite

Usage

(continued)

Shahiduzzaman and Daugschies (2012)

Geerts et al. (2001) CDC (2016), WHO (2016)

CDC (2016), Checkley et al. (2015) CDC (2016), WHO (2016)

Ref

9 Insecticidal and Antimalarial Properties of Plants: A Review 225

S

Sulphaquinoxaline

Fluconazole, Itraconazole

Triazole derivative

ND Semisynthetic derivatives of oxytetracycline produced by Sreptomyces rimosus S

Trivalent arsenic Melarsoprol S derivative Ureide/Imidazoline Imidocarb S derivative N (natural product), ND (derived from a natural product), S (synthetic)

Doxycycline (16)

Tetracycline

Sulfanilamide S

Origin, source organism

Artemisinin (12), Artemether (13), Artesunate (14), Dihydroartemisinin (15) Sulfadiazine, Sulfamethoxazole Sulfadoxine

Sesquiterpen lactone

N/ND Artemisia annua (cpd 12) Semisynthetic derivatives of artemisinin (cpds 13, 14, 15) S

Compound

Chemical class

Table 9.4 (continued) Parasite

Trypanosoma brucei Babesia

Leishmania

Plasmodium

Plasmodium, Toxoplasma Plasmodium

Toxoplasma

Plasmodium

Usage

Mosqueda et al. (2012)

CDC (2016), WHO (2016)

Braga et al. (2011)

CDC (2016), WHO (2016)

Ref

226 L. Paloque et al.

S S S S

S

S

Clorsulon Bithionol Monepantel Albendazole

Mebendazole

Triclabendazole

Origin, source organism

Compound

Chemical class

Aniline-bis-sulfonamide Bis-phenolthioether Benzamide derivative Benzimidazole

Table 9.5 Approved anthelmintics compounds

Paragonimus

Soil-transmitted helminths, Echinococcus Fasciola

Helminths Helminths Helminths Soil-transmitted helminths, Lymphatic filariae, Clonorchis, Opisthorchis, Echinococcus Fasciola

Parasite

Usage

(continued)

CDC (2016), Khan et al. (2013), Knubben-Schweizer and Torgerson (2015) CDC (2016)

Khan et al. (2013), Knubben-Schweizer and Torgerson (2015) CDC (2016)

CDC (2016), WHO (2016)

Olliaro et al. (2011), Epe and Kaminsky (2013)

Ref

9 Insecticidal and Antimalarial Properties of Plants: A Review 227

Compound

Meniclofolan

Febantel Avermectins (abamectin) (17) Ivermectin (18)

Levamisole Emodepside (19)

Milbemycin (20), Moxidectin (21)

Nitroxynil Nitroscanate Piperazine Closantel, Rafoxanide Niclosamide

Chemical class

Bisnitrophenol

Diphenylthioether Heteroside with macrocyclic lactone aglycone

Imidazothiazole derivative Lactonic macrocyclic peptide

Macrocyclic lactone

Nitrophenol Nitrothiocyanodiphenylether Piperazine Salycilanilide

Table 9.5 (continued)

Taenia

S

Helminths

Helminths Helminths Helminths Helminths

Helminths Ascaris, Onchocerca, Trichuris, Lymphatic filariae Helminths Helminths

Helminths Helminths

Fasciola

Parasite

S ND Semisynthetic derivative of PF1022A, isolated from Camellia japonica N Streptomyces hygroscopicus (Cpd 20) Streptomyces cyanogriseus (Cpd 21) S S S S

S N Streptomyces avermitilis ND Semisynthetic derivative, by chemical reduction, of avermectins

S

Origin, source organism Usage

CDC (2016), WHO (2016) (continued)

Olliaro et al. (2011), Epe and Kaminsky (2013)

CDC (2016), WHO (2016)

Khan et al. (2013), Knubben-Schweizer and Torgerson (2015) Olliaro et al. (2011), Epe and Kaminsky (2013)

Ref

228 L. Paloque et al.

Derquantel (22)

Praziquantel

Diethylcarbamazine

Pyrantel

Tetrahydroisoquinoline/ Piperazinone derivative

Ureide

Vinylthiophene derivative/ Tetrahydropyrimidine derivative

Origin, source organism

S

S

ND Semisynthetic derivative, by chemical reduction, of paraherquamide isolated from Penicillium simplicissimum S

N (natural product), ND (derived from a natural product), S (synthetic)

Compound

Chemical class

Spiroindole derivative

Table 9.5 (continued)

Necator, Ancylostoma

Helminths Helminths

Helminths Taenia, Schistosoma, Paragonimus, Opisthorchis, Clonorchis Lymphatic filariae

Helminths

Parasite

Usage

Olliaro et al. (2011), Epe and Kaminsky (2013) CDC (2016), WHO (2016)

CDC (2016), WHO (2016)

Olliaro et al. (2011), Epe and Kaminsky (2013)

Ref

9 Insecticidal and Antimalarial Properties of Plants: A Review 229

Fig. 9.1 Structures of the 22 natural products or natural product derivatives approved as antiparasitic drugs

230 L. Paloque et al.

231

Fig. 9.1 (continued)

9 Insecticidal and Antimalarial Properties of Plants: A Review

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L. Paloque et al.

(Blake and Tomley 2014). More than 1.5 billion treatments with ivermectin (Mectizan®) have been provided (202 million in 2015) to fight onchocerchiasis and lymphatic filariasis since the beginning of the Mectizan Donation Program initiated by Merck & Co in 1987 (Goldman Sachs 2015). Moreover, the discovery of artemisinin and ivermectin as antiparasitic treatments was awarded in 2015 by the Nobel Prize in Physiology or Medicine (Nobel Prize 2015).

9.3.3

The Rationale Behind Use of Antiparasitic Drugs

Because human and animal parasites share biological features common to the genus or phylum to which they belong, some antiparasitic compounds (7/23) are used in both human and veterinary medicine and/or on related parasites. Indeed, chloroquine exerts activity on both human and avian Plasmodium spp (Braga et al. 2011). Atovaquone is used in the treatment of human malaria as well as human babesiosis and human toxoplasmosis (CDC 2016) caused by two related parasites, Babesia and Toxoplasma, respectively, belonging to the same phylum Apicomplexa. Albendazole, ivermectin, pyrantel pamoate, and praziquantel are all used indifferently on numerous species of helminth affecting humans and/or animals (Olliaro et al. 2011; Epe and Kaminsky 2013; CDC 2016) (Table 9.3). In the same way, research into new antiparasitic compounds explores the biological and biochemical similarities between human/animal affecting, or related parasite species. Screening of the existing library of Medicines for Malaria Venture (MMV) Open Access Malaria Box (400 compounds exerting antiplasmodial activity) led to the identification of 19 compounds with significant activity against Cryptosporidium parvum (Bessoff et al. 2014). In vitro and in vivo studies have also been conducted to improve the efficacy of artemisinin derivatives on animal babesiosis (Kumar et al. 2003; Goo et al. 2010; Mazuz et al. 2013; Iguchi et al. 2015). On the contrary, while all anthelmintics were initially developed for veterinary use (Geary et al. 2010), some are today included in the Drug for Neglected Diseases (DNDi) portfolio. Indeed, two derivatives of a veterinary antibiotic are in preclinical study for the treatment of onchocerciasis and lymphatic filariasis, and emodepside, an anthelmintic compound used to treat cats and dogs, is now in phase-I clinical trials for the treatment of onchocerciasis (DNDi 2016).

9.3.4

Need for New Antiparasitic Compounds

Control over the major human and animal parasitic diseases depends largely on drugs threatened by issues of drug resistance. As regards the development of antiparasitic drug resistance, the question is not “if” but “when.” Indeed, all parasites, whatever the phylum to which they belong, have already developed or are able to develop resistance against all therapeutic compounds. Resistance to the

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anthelmintic drugs was established an average of 10 years after its introduction in the field (Waller 2006) and resistance of Plasmodium to atovaquone occurred in only a few months (Kessl et al. 2007). Artemisinin, the spearhead of malaria control, is now threatened by the emergence and spread of artemisinin resistance in Asia, associated with a novel multidrug tolerance phenotype (Paloque et al. 2016). Such parasitic drug resistance alongside a lack of efficient vaccines against most parasites, as well as the continuing high burden of human and animal parasitic diseases worldwide, altogether equate to a desperate need for new chemical entities presenting new mechanisms of action.

9.4

New Antiparasitic Drug Discovery

Drug discovery for parasitic diseases follows different basic approaches, as reviewed by Pink and colleagues (Pink et al. 2005), which include the repurposing of existing drugs and the de novo discovery of novel active compounds unrelated to known drugs.

9.4.1

Repurposing of Existing Drugs

The repurposing of existing drugs is an increasingly important cost- and time-saving strategy used by the pharmaceutical industry. Indeed, the cost and duration of drug development, from drug discovery to Food and Drug Administration (FDA) approval, are estimated at $1.5 billion USD and 14 years compared to $300 million USD and 6 years for drug repositioning (Nosengo 2016). The repurposing of existing drugs can be based on their use on a related organism or on their mechanism of action (MOA) being useful in the treatment of another disease. An example of this is the repurposing of the antimalarial drug library for anti-Babesia or anti-Cryptosporidium activities, as previously detailed (see Sect. 9.3.3). The repurposing of veterinary medicines for use in humans could allow a considerable reduction in the time required to develop new drugs, as available data are essentially equivalent to those required for human medicine licensing (Olliaro et al. 2011). Advantages of veterinary over human drug development processes include the possibility to assess new drugs directly in the target species at the beginning of the process, allowing for a higher proportion of drugs selected for development successfully passing safety assessments (Lathers 2003). However, criteria are more stringent for veterinary medicines dedicated to animals for human consumption (Olliaro et al. 2011) than they are for medicines destined for humans, mainly to enhance consumer safety with regard to chemical residues in meat and dairy products. In 2009, the global market for veterinary drugs comprised livestock (58%) and domestic animals (42%) and in these two groups, antiparasitic

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drugs represented 28% of veterinary therapeutics making them the most important market segment for the veterinary pharmaceutical industry (Crosia 2011).

9.4.2

De Novo Discovery of Antiparasitic Drugs

The de novo discovery of novel antiparasitic drugs is based on high-throughput screening of diverse compound libraries against whole parasites, or against molecular targets subsequent to target-based drug design. All available antiparasitic drugs have been identified through the whole parasites strategy, in which investigations into potential targets are carried out after the compound efficacy has been confirmed (Müller and Hemphill 2016). The target-based drug design strategy explores the molecular and biochemical differences between parasites and their mammalian hosts. The ideal target is strongly specific to the parasite or has a low degree of similarity with host homologues and is essential for parasite survival. The main drug targets that have been identified in protozoan parasites and helminths, have been recently reviewed by Pink et al. (2005), Rana and Misra-Bhattacharya (2013), and Müller and Hemphill (2013 and 2016) and are presented in Table 9.6. Most of these targets are enzymes belonging to highly conserved cellular process; structural differences between the parasitic target and its mammalian homologue form the basis for their selectivity. Such is the case for plasmodial dihydro-orotate dehydrogenase, which displays sufficient structural difference from the human homologue to allow the design of specific inhibitors (Müller and Hemphill 2016). Some other targets, such as the fatty acid biosynthesis type-II pathway, are not found in mammalian cells and are highly specific to parasites (Shears et al. 2015). The unique enzymatic system trypanothione/trypanothione reductase found within Trypanosomatidae acts, like its homologue systems glutathione/glutathione reductase and thioredoxin/thioredoxin reductase present in most eukaryotic cells, in thiol redox homeostasis (Krauth-Siegel and Comini 2008). Another specific target of Trypanosomatidae and of fungus is ergosterol and ergosterol biosynthesis, with ergosterol replacing almost all cholesterol in the membrane bilayer of these organisms (De Souza and Rodrigues 2009). Anthelmintics mainly act by disturbing two basic processes vital to worm survival: worm motility via damage to the nervous system and integrity via cuticule and cytoskeleton degradation. Knowledge of each drug’s MOA alongside the identified parasite therapeutic targets should help improve opportunities to discover new antiparasitic compounds. The different methodological approaches existing to identify the MOA employed by antiparasitic compounds, including genetic crosses, phenotypic alterations, and genomic, transcriptomic and proteomic strategies, have been recently reviewed by Skinner-Adams et al. (2016). The advantages and disadvantages of the whole parasite strategy versus the target-based drug design strategy for de novo discovery of antiparasitic drugs have been discussed recently by Müller and Hemphill (2016), focusing on protozoan parasites. Any identification

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Table 9.6 Main antiprotozoan and anthelmintic therapeutic targets Parasites

Drug target

Apicomplexa Pink et al. (2005), Müller and Hemphill (2013, 2016)

Enzymes

Proteins

Trypanosomatidae Pink et al. (2005), Müller and Hemphill (2013, 2016)

Cellular process Plasmepsins I, II, IV, Cysteine protease falcipain

Hemoglobin degradation

Type-II enoyl-acyl carrier protein reductase (FabI)

Fatty acid biosynthesis type-II

1-Deoxy-D-xylulose 5-phosphate reductoisomerase, MECP synthase

Isoprenoid biosynthesis

Purine nucleoside phosphorylase, Dihydro-orotate dehydrogenase, Inosine monophosphate dehydrogenase,

Nitrogen bases metabolism

Protein phosphatase, Threonine peptidase, peptide deformylase, N-myristoyltransferase, Cysteine proteases, protein farnesyltransferase

Protein metabolism

Lactate dehydrogenase, Glucose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase

Energy metabolism

Helicases

Nucleic acid metabolism

Cyclic nucleotide phosphodiesterases

Intracellular signaling

Cyclin-dependent protein kinases

Cell cycle regulation

7,8-Dihydro-6-hydroxymethylpterin pyrophosphokinase, Dihydrofolate reductase Dihydropteroate synthase

Folate biosynthesis

Cytochrome b

Respiratory pathway

Myosin interacting protein

Cellular motility

Transporters

Hexose transporter

Glucose uptake

Enzymes

Trypanothione reductase

Defense against chemical/oxidant stress

Topoisomerase 1B

Nucleic acid metabolism

Ornithine decarboxylase

Polyamine biosynthesis

Type-II enoyl-acyl carrier protein reductase (FabI)

Fatty acid biosynthesis type-II

Protein farnesyltransferase, Serine protease, Oligopeptidase B

Protein metabolism

Cyclic nucleotide phosphodiesterases

Intracellular signaling

(continued)

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Table 9.6 (continued) Parasites

Drug target

Sterol

Helminths Rana and Misra-Battacharya (2013)

Enzymes

Structural protein Receptors

Cellular process Cysteine protease cruzain

Hemoglobin degradation

Sterol C-14 a-demethylase

Ergosterol biosynthesis

Ergosterol

Cellular membrane integrity

Polo-like kinase (eukaryotic protein kinase)

Cellular division

Cathepsin B1, asparagine tRNA synthetase

Protein metabolism

Glutathione-S-transferase

Glutathione metabolism

Mitochondrial complex I & II

Respiratory pathway

Topoisomerase II

Nucleic acid metabolism

Chitinase

Chitin metabolism

Lactate dehydrogenase

Energy metabolism

Beta-tubulin

Cytoskeleton integrity

G-protein-coupled receptor

Cell signaling

Latrophilin receptors, Nicotinic acetylcholine receptor

Nervous system cell signaling

Transporter

Serotonin transporter

Ion channels

Ligand-gated Ca2+ and K+ ion channels, calcium-activated potassium channel, cys-loop ligand-gated ion channels, glutamate-gated chloride channels

Ion homeostasis, Neurotransmission

of a new active compound by screening against molecular targets needs confirmation by parasite viability assays.

9.4.3

First Step in Evaluating Antiparasitic Activity

In vitro assays on whole parasites have offered a less expensive and ethically unlimited alternative to in vivo testing, thus replacing it as the first step in drug discovery. They allow the simultaneous evaluation of large numbers of compounds as well as their potential synergism or antagonism in combination. Drug effectiveness is usually quantified by determining the inhibitory concentration 50% (IC50), defined as the concentration of drug required to inhibit by 50% the parasite viability compared to the appropriate control. Current standard methods of assessing viability in protozoan parasites mainly involve incorporation of specific radiolabeled or fluorescent probes, enzymatic activities, and use of reporter genes.

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In helminthes, however, such methods mainly examine motility, essential for the worms to resist expulsion by bowel peristalsis. The three methods that are generally used are fecal egg count reduction test (FECRT), egg hatch test (EHT), and larval development test (LDT) (Königová et al. 2003; Vernerova et al. 2009). Nonetheless, screening on whole parasites is limited to those parasite species and stages that can be routinely maintained in vitro (Skinner-Adams et al. 2016). Screening against certain clinically relevant stages of Plasmodium and Leishmania infection, namely hypnozoites and intracellular amastigotes, respectively, is now possible but remains challenging. Constraints surrounding the culturing of helminths render the throughput of screening assays against these whole parasites at least ten times less than for protozoa (Pink et al. 2005). Compounds likely to be toxic, mutagenic, highly reactive, unstable, or intractable to chemical modifications should be cast aside to ensure “drug likeness” (Pink et al. 2005), and the selectivity of compounds to target parasites should always be assessed by determining the therapeutic/selectivity index in selected mammalian cell lines.

9.4.4

Alternative Use of Natural Products

Although the consumption of certain food products for their medicinal value has long been in existence, the term nutraceutical was first defined only in 1989 as “a food (or part of a food) that provides medical or health benefits, including the prevention and/or treatment of a disease” (Kalra 2003; DeFelice 2002). Since then, nutraceutical practice has become increasingly important in livestock to control parasitic diseases. In a context of increasing monitoring of chemical residues in meat and dairy products, the use of nutraceutical products is emerging as an alternative and attractive strategy that has been easily embraced by eco- and health-conscious consumers (Muthamilselvan et al. 2016). While scientific backing is required to support the rational use of nutraceutical products, many are already commercially available, in particular for the prevention and treatment of coccidiosis in the poultry industry (Muthamilselvan et al. 2016; Quiroz-Castaneda et al. 2015; Abbas et al. 2012). For example, curcumin extract from the Zingiberaceae Curcuma longa has demonstrated broad range pharmacological activity, exerting antiparasitic, antimicrobial, anti-inflammatory, and powerful antioxidant activities. Dietary supplementation with Curcuma longa enhanced coccidiosis resistance in chickens, as demonstrated by an increase in their systemic humoral and cellular immunity. Treated animals showed increased body weight, reduced fecal oocyst shedding, and decreased gut lesions (Kim et al. 2013). In another report, the combination of curcumin and luteolin preserved meat quality after coccidiosis infection in broiler chickens (Rajput et al. 2014). Use of nutraceutical products to control helminthiasis in the livestock industry has also been studied, and many bioactive ingredients and phytochemicals of plant origin have been identified in livestock feed (Hoste et al. 2015). Tannins possess biological activity against gastrointestinal helminths such as Ascaris suum in pig, Haemonchus contortus in goats and sheep (Singh et al. 2015),

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or Cryptosporidium in calves (Hoste et al. 2015) and were also reported to inhibit the life cycle of Eimeria by penetrating the wall of the oocyst and damaging the cytoplasm (Molan et al. 2009). In helminths, tannins inhibited newly hatched larvae migration, and reduced worm motility and survival, leading to a shortened life cycle (Williams et al. 2014a). The principal MOA of tannins probably involves their binding to proline-rich proteins on the cuticle or in the digestive system of the larvae, forming complexes with nutrients thus inhibiting their availability for larval growth (Williams et al. 2014b). Such natural substances could be used as nutraceutical products either directly by their incorporation into the animal feed or in a concentrated form after extraction from the bioactive plant.

9.5 9.5.1

Reality of Using Natural Products in Drug Discovery Natural Product Chemistry for Drug Discovery

Regarding the methodology of natural products research, the use of ethnopharmacological knowledge is one of the most attractive ways to reduce empiricism and enhance the probability of success in new drug-finding processes (Patwardhan 2005; Cordell and Colvard 2012). The use of herbal remedies is often justified by their long-standing empirical-based use in various cultures, despite no mechanistic knowledge on their pharmacological activities or active constituents (Atanasov et al. 2015). The term “herbal drugs” denotes plants or plant parts transformed into phytopharmaceuticals by means of simple processes involving harvesting, drying, and storage (EMEA 1998). By virtue of their hugely diverse metabolite content, whole plant rather than one single purified compound may offer greater therapeutic benefit. However, some plants used in herbal medicines can also be highly toxic and case reports exist of serious adverse events after administration of herbal remedies. The raw herb contains complicated mixtures of organic chemicals, which may include metabolites such as flavonoids, glycosides, sterols, alkaloids, saponins, and terpenes as well as other small compounds. It is thus difficult to determine which components are bioactive. Besides the limited accessibility of plant material, several other problems not applicable to synthetic drugs often influence the quality of herbal drugs. Firstly, in most cases, the active compound(s) is/are unknown. Secondly, the plant origin is inconstant, with variability caused by differences in soil composition, altitude, current climate, processing, and storage conditions. Chemo-varieties and chemo cultivars also exist. In addition, extraction, as well as isolation processes (Bucar et al. 2013; Jones and Kinghorn 2012) may affect the molecular structures of these products (transformation and degradation) and thus their biological activities. Consequently, a well-defined and constant composition is crucial for the production of a quality drug. Standardization of herbal medicines aimed at creating a uniform product for clinical trials (constant parameters, efficacy, safety, and reproducibility) would be a step in the right direction. However, the lack

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of assurance of a renewable supply of natural products and the problem of sustainable supply of herbal material cannot meet the challenges posed by the demands for increasing compound amounts due to issues re-isolating from the plant sources (Atanasov et al. 2015). The molecular complexity of most bioactive natural products (number of asymmetric centers, functional groups, etc.) makes a daunting task of their total or semi-synthesis (David et al. 2015). Researchers thus need to broaden their horizons with regard to their natural product sourcing. To overcome the supply issue, endophytes (fungi or bacteria) could be better exploited as a readily renewable and inexhaustible source of novel structures bearing pharmaceutical potential. Endophytic fungi are ubiquitously present in nature, occurring in all plants on earth. Particularly tropical leaves appear to represent endophyte biodiversity hotspots. Endophytes seem an obviously rich and reliable source of genetic diversity and bioactive natural products. The majority of natural products occurring in these microorganisms have proven antimicrobial activity, implicated in plant host protection. Endophytes have also been demonstrated to produce some important pharmaceuticals produced by their host plants (e.g., the world’s first billion-dollar anticancer drug paclitaxel or camptothecin), highlighting the enormous potential in the horizontal gene transfer between host and endophyte.

9.5.2

Ethical and Legal Constraints in Natural Drug Discovery

Ecological and legal considerations have an important influence on the accessibility of plants as a source of drug discovery, especially laws dealing with plant access and sharing of benefits (ABS, as well as patentability issues with local governments in the countries of origin. As shown above, biodiversity in nature represents a source of a surprisingly high number of antiparasitic natural substances, some of which have had a beneficial impact on the epidemiologic status of certain populations in the world. Much of this vast source undoubtedly remains unexplored, giving hope to the discovery of much needed new treatments such as antimalarials or antitrypanocidals in a still unexploited living organism. In 2014, David (David et al. 2015) estimated that 95% of the world’s biodiversity remained to be evaluated (known biodiversity being estimated as 2 million species of plant, fungus, and microorganisms). The challenge now is finding ways of efficiently accessing and evaluating this natural chemical diversity. Besides all the technical aspects of such a search involving a multi-disciplinary team, and well-equipped laboratories, one important consideration is the now stricter measures of control over research on biodiversity (encompassing traditional knowledge) outlined in the Nagoya protocol ratified by 78 countries during the 2016 Convention on Biological Diversity (CDB 2016). The CDB sets out three objectives: the conservation of biological diversity, the sustainable use of its components, and the fair and equitable sharing of the benefits

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arising out of the utilization of genetic resources. The Nagoya protocol concerns the third objective of the CDB and was adopted at the Earth Summit in Rio de Janeiro in 1992. Despite its initial setup as an international convention aimed at ensuring the preservation of the environment while apprehending the diversity of its constituent elements and their interactions, the CBD has, for the most part, become an instrument toward crystallizing dreams of planetary equity and hopes of economic prosperity. Indeed, founded on the use of “green gold,” new biotech industries are envisaged to develop the medicines of tomorrow (Aubertin and Filoche 2011). The “Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization” is a strategic action plan and a financial mechanism for implementation of the Convention. Reciprocity and equity are the key words, in the sense of a contract engaging one country providing biodiversity and another aiming to use it for R&D purposes, and the sharing of any benefits with the provider. For developing countries, benefit-sharing means putting an end to biopiracy, with the belief that there is a market for genetic resources which would generate instant income. While ABS contracts imply that countries having ownership of their genetic resources are in a position to control their access, numerous questions remain and issues unresolved regarding conservation. From a practical point of view, the Nagoya protocol stipulates that all public and private researchers planning to carry out R&D on biodiversity in the search for bioactive natural products are required to seek a Prior Informant Consent (PIC) and negotiate Mutually Agreed Terms (MAT) and a Material Transfer Agreement (MTA) with a representative of the source country. However, the situation remains far from unified, with signatory countries having to implement these contracts in their own laws to ensure that the objective of the regimen be fulfilled. The result is that legal steps vary according to geographic location: in Europe, some countries (Austria, Sweden, and Denmark) even chose to make their resources freely available, while France and Spain wrote a specific legislation for access to their biodiversity. Peru has a long experience with these contracts, while Laos is only just implementing new laws. All researchers, private and public companies or research institutes, are thus responsible for informing themselves on the laws of the country in which they wish to work ensuring that all legal obligations are satisfied.

9.6

Conclusion

Continued disturbance to the natural ecosystems of parasites and/or vectors by human activities (mainly changes in land use or agricultural practice), human population migration and climate change which modify parasitic disease distribution could also lead to the re-emergence or increasing prevalence of certain illnesses in a given geographical area (Patz et al. 2000; Woolhouse and Gowtage-Sequeria 2005). The current constant progression of endemic dengue and chikungunya cases across Europe is an example of such a phenomenon. Furthermore, the development

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and spread of antiparasitic drug resistance even against the best available drugs, imposes an urgent need for new antiparasitic drugs. For any new identified target, its therapeutic relevance needs demonstrating in all disease-relevant parasitic stages, as does the transferability of in vitro activity to in vivo conditions. By virtue of their hugely diverse metabolite content, whole plants or microorganisms are believed to provide more therapeutic benefit compared to their individual purified constituents. Most drugs based on natural products are derived from natural sources, such as plants or microorganisms, which have been chemically modified to improve their bioactivity against parasites. After a number years of diminished interest in natural products, with pharmaceutical industry favoring approaches based on HTP screening of combinatorial libraries and genomics, the past few years has seen renewed interest, especially in bacteria and fungi, particularly endophytes, as important sources of biologically active compounds. Marine organisms have also received recognition as an interesting source of antiparasitic compounds. Over the past few years, a new model of R&D, classified under the broad term of public-private partnerships for product development (PPPs), aims to associate the competences and research capacity of academia, pharmaceutical industry, and contract researchers to create a focused research association addressing all aspects of drug discovery, especially for neglected diseases. Natural products will maintain their position in this R&D landscape with some already included in the MMV and DNDi portfolio, the two major PPPs dedicated to parasitic diseases. However, a major challenge over the next years will be, among others, finding unexplored ways to efficiently exploit non-plant sources of natural products, like endophytes or marine microorganisms, toward maintaining the greatest possible diversity of chemical entities within the development pipeline of drugs for antiparasitic diseases. The recent development and implementation of new technologies offer unique opportunities in the screening of natural products and will re-establish them as a major source for drug discovery.

References Abbas R, Colwell D, Gilleard J (2012) Botanicals: an alternative approach for the control of avian coccidiosis. Worlds Poult Sci J 68(2):203–215 Atanasov AG, Waltenberger B, Pferschy-Wenzig EM, Linder T, Wawrosch C, Uhrin P, Temml V, Wang L, Schwaiger S, Heiss EH, Rollinger JM, Schuster D, Breuss JM, Bochkov V, Mihovilovic MD, Kopp B, Bauer R, Dirsch VM, Stuppner H (2015) Discovery and resupply of pharmacologically active plant-derived natural products: A review. Biotechnol Adv 33 (8):1582–1614 Aubertin C, Filoche G (2011) The Nagoya protocol on the use of genetic resources: one embodient of an endless discussion. Sustenabilidade em Debate 2(1):51–64 Bessoff K, Spangenberg T, Foderaro JE, Jumani RS, Ward GE, Hustona CD (2014) Identification of Cryptosporidium parvum active chemical series by Repurposing the open access malaria box. Antimicrob Agents Chemother 58(5):2731–2739

242

L. Paloque et al.

Blake DP, Tomley FM (2014) Securing poultry production from the ever-present Eimeria challenge. Trends Parasitol 30(1):12–19 Blunt JW, Copp BR, Keyzers RA, Munro MH, Prinsep MR (2016) Marine natural products. Nat Prod Rep 33(3):382–431 Braga ÉM, Silveira P, Belo NO, Valkiũnas G (2011) Recent advances in the study of avian malaria: an overview with an emphasis on the distribution of Plasmodium spp in Brazil. Mem Inst Oswaldo Cruz 106:3–11 Bucar F, Wube A, Schmid M (2013) Natural product isolation - how to get from biological material to pure compounds. Nat Prod Rep 30:525–545 Butler MS, Robertson AAB, Cooper MA (2014) Natural product and natural product derived drugs in clinical trials. Nat Prod Rep 31:1612–1661 Cardona GA, Carmena D (2013) A review of the global prevalence, molecular epidemiology and economics of cystic echinococcosis in production animals. Vet Parasitol 192(1–3):10–32 CDB (2016) https://www.cbd.int/abs/nagoya-protocol/signatories/default.shtml CDC (2016) http://www.cdc.gov/dpdx/ Chapman HD (2014) Milestones in avian coccidiosis research: a review. Poult Sci 93:501–511 Charlier J, van der Voort M, Kenyon F, Skuce P, Vercruysse J (2014) Chasing helminths and their economic impact on farmed ruminants. Trends Parasitol 30(7):361–367 Checkley W, White AC Jr, Jaganath D, Arrowood MJ, Chalmers RM, Chen XM, Fayer R, Griffiths JK, Guerrant RL, Hedstrom L, Huston CD, Kotloff KL, Kang G, Mead JR, Miller M, Petri WA Jr, Priest JW, Roos DS, Striepen B, Thompson RC, Ward HD, Van Voorhis WA, Xiao L, Zhu G, Houpt ER (2015) A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for cryptosporidium. Lancet Infect Dis 15(1):85–94 Colley DG (2000) Parasitic diseases: opportunities and challenges in the 21st century. Mem Inst Oswaldo Cruz 95:79–87 Cordell GA, Colvard MD (2012) Natural products and traditional medicine: turning on a paradigm. J Nat Prod 75(3):514–525 Cragg GM, Newman DJ, Snader KM (1997) Natural products in drug discovery and development. J Nat Prod 60(1):52–60 Crosia PJ (2011) Worldwide market of veterinary drugs: trend analysis for the last ten years. Bull Acad Vet France 164(1):21–25 David B, Wolfender JL, Dias DA (2015) The pharmaceutical industry and natural products: historical status and new trends. Phytochem Rev 14(2):299–315 DeFelice SL (2002) FIM rationale and proposed guidelines for the nutraceutical research & education Act - NREA, November 10, 2002. Foundation for Innovation in Medicine. http:// www.fimdefelice.org/archives/arc.researchact.html De Souza W, Rodrigues JCF (2009) Sterol biosynthesis pathway as target for anti-trypanosomatid drugs. Interdiscip Perspect Infect Dis. https://doi.org/10.1155/2009/642502 DNDi (2016) http://www.dndi.org/diseases-projects/portfolio/ EMEA (1998) Quality of herbal medicinal products. Guidelines, European Agency for the Evaluation of Medicinal Products (EMEA), London Epe C, Kaminsky R (2013) New advancement in anthelmintic drugs in veterinary medicine. Trends Parasitol 29(3):129–134 FAOSTAT (2016) http://faostat3.fao.org/home/E Fernańdez-Álvaro E, Hong WD, Nixon GL, O’Neill PM, Calderoń F (2016) Antimalarial chemotherapy: natural product inspired development of preclinical and clinical candidates with diverse mechanisms of action. J Med Chem 59:5587–5603 Geary TG, Woo K, McCarthy JS, Mackenzie CD, Horton J, Prichard RK, de Silva NR, Olliaro PL, Lazdins-Helds JK, Engels DA, Bundy DA (2010) Unresolved issues in anthelmintic pharmacology for helminthiases of humans. Int J Parasitol 40(1):1–13 Geerts S, Holmes PH, Diall O, Eisler MC (2001) African bovine trypanosomiasis: the problem of drug resistance. Trends Parasitol 17(1):25–28

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Geurden T, Chartier C, Fanke J, Frangipane di Regalbone A, Travaersa D, von Samason-Himmelstjerna G, Demeler J, Vanimisetti HB, Bartam DJ, Denwood MJ (2015) Anthelmintic resistance to ivermectin and moxidectin in gastrointestinal nematodes of cattle in Europe. Int J Parasitol Drugs Drug Resist 5(3):163–171 Gohil S, Herrmann S, Günther S, Cooke BM (2013) Bovine babesiosis in the 21st century: advances in biology and functional genomics. Int J Parasitol 43(2):125–132 Goldman Sachs (2015) Mectizan donation program, Annual highlights. http://www.goldmansachs. com/media-relations/press-releases/current/pdfs/2013-q4-results.pdf Goo YK, Terkawi MA, Jia H, Aboge GO, Ooka H, Nelson B, Kim S, Sunaga F, Namikawa K, Igarashi I, Nishikawa Y, Xuan X (2010) Artesunate, a potential drug for treatment of Babesia infection. Parasitol Int 59(3):481–486 Hertweck C (2015) Natural products as source of therapeutics against parasitic diseases. Angew Chem Int Ed Engl 54(49):14622–14624 Horn D, Duraisingh MT (2014) Antiparasitic chemotherapy: from genomes to mechanisms. Annu Rev Pharmacol Toxicol 54:71–94 Hoste H, Torres-Acosta JFJ, Sandoval-Castro CA, Mueller-Harvey I, Sotiraki S, Louvandini H, Thamsborg SM, Terrill TH (2015) Tannin containing legumes as a model for nutraceuticals against digestive parasites in livestock. Vet Parasitol 15;212(1–2):5–17 Hotez PJ, Alvarado M, Basanez MG, Bolliger I, Bourne R, Boussinesq M, Brooker SJ, Brown AS, Buckle G, Budke CM, Carabin H, Coffeng LE, Fèvre EM, Fürst T, Halasa YA, Jasrasaria R, Johns NE, Keiser J, King CH, Lozano R, Murdoch ME, O’Hanlon S, Pion SD, Pullan RL, Ramaiah KD, Roberts T, Shepard DS, Smith JL, Stolk WA, Undurraga EA, Utzinger J, Wang M, Murray CJ, Naghavi M (2014) The global burden of disease study 2010: interpretation and implications for the neglected tropical diseases. PLoS Negl Trop Dis 8(7): e2865 Hussain H, Al-Harrasi A, Al-Rawahi A, Green IR, Gibbons S (2014) Fruitful decade for antileishmanial compounds from 2002 to late 2011. Chem Rev 114(20):10369–10428 Iguchi A, Matsuu A, Matsuyama K, Hikasa Y (2015) The efficacy of artemisinin, artemether, and lumefantrine against Babesia gibsoni in vitro. Parasitol Int 64(2):190–193 Jones WP, Kinghorn AD (2012) Extraction of plant secondary metabolites. Methods Mol Biol 864:341–366 Kalra EK (2003) Nutraceutical: definition and introduction. AAPS pharmSci 5(3):E25 Kayser O, Kiderlen AF, Croft SL (2003) Natural products as antiparasitic drugs. Parasitol Res 90: S55–S62 Kessl JJ, Meshnick SR, Trumpower BL (2007) Modeling the molecular basis of atovaquone resistance in parasites and pathogenic fungi. Trends Parasitol 23(10):494–501 Khan MK, Sajid MS, Riaz H, Ahmad NE, He L, Shahzad M, Hussain A, Khan MN, Iqbal Z, Zhao J (2013) The global burden of fasciolosis in domestic animals with an outlook on the contribution of new approaches for diagnosis and control. Parasitol Res 112(7):2421–2430 Kim DK, Lillehoj HS, Lee SH, Jang SI, Lillehoj EP, Bravo D (2013) Dietary Curcuma longa enhances resistance against Eimeria maxima and Eimeria tenella infections in chickens. Poult Sci 92(10):2635–2643 Knubben-Schweizer G, Torgerson PR (2015) Bovine fasciolosis: control strategies based on the location of Galba truncatula habitats on farms. Vet Parasitol 208(1–2):77–83 Königová A, Várady M, Čorba J (2003) Comparison of in vitro methods and faecal egg count reduction test for the detection of benzimidazole resistance in small strongyles of horses. Vet Res Commun 27(4):281–288 Krauth-Siegel RL, Comini MA (2008) Redox control in trypanosomatids, parasitic protozoa with trypanothione-based thiol metabolism. Biochim Biophys Acta 1780(11):1236–1248 Kumar S, Gupta AK, Pal Y, Dwivedi SK (2003) In-vivo therapeutic efficacy trial with artemisinin derivative, buparvaquone and imidocarb dipropionate against Babesia equi infection in donkeys. J Vet Med Sci 65(11):1171–1177 Lanusse C, Alvarez L, Lifschitz A (2014) Pharmacological knowledge and sustainable anthelmintic therapy in ruminants. Vet Parasitol 204(1–2):18–33

244

L. Paloque et al.

Lathers CM (2003) Challenges and opportunities in animal drug development: a regulatory perspective. Nat Rev Drug Discov 2(11):915–918 Mazuz ML, Golenser J, Fish L, Haynes RK, Wollkomirsky R, Leibovich B, Shkap V (2013) Artemisone inhibits in vitro and in vivo propagation of Babesia bovis and B. bigemina parasites. Exp Parasitol 135(4):690–694 McKerrow JH (2015) Recognition of the role of Natural Products as drugs to treat neglected tropical diseases by the 2015 Nobel prize in physiology or medicine. Nat Prod Rep 32:1610– 1611 Molan AL, Liu Z, De S (2009) Effect of pine bark (Pinus radiata) extracts on sporulation of coccidian oocysts. Folia Parasitol (Praha). 56(1):1–5 Morrison WI (2015) The aetiology, pathogenesis and control of theileriosis in domestic animals. Rev Sci Tech 34(2):599–611 Mosqueda J, Olvera-Ramirez A, Aguilar-Tipacamu G, Canto GJ (2012) Current advances in detection and treatment of babesiosis. Curr Med Chem 19(10):1504–1518 Müller J, Hemphill A (2013) New approaches for the identification of drug targets in protozoan parasites. Int Rev Cell Mol Biol 301:359–401 Müller J, Hemphill A (2016) Drug target identification in protozoan parasites. Expert Opin Drug Discov 11(8):815–824 Muthamilselvan T, Kuo TF, Wu YC, Yang WC (2016) Herbal remedies for coccidiosis control: a review of plants, compounds, and anticoccidial actions. Evid Based Complement Alternat Med:1–19 Ndjonka D, Rapado LN, Silber AM, Liebau E, Wrenger C (2013) Natural products as a source for treating neglected parasitic diseases. Int J Mol Sci 14(2):3395–3439 Newman DJ, Cragg GM (2012) Natural products as sources of new drugs over the 30 years from 1981 to 2010. J Nat Prod 75(3):311–335 Newman DJ, Cragg GM (2016) Natural products as sources of new drugs from 1981 to 2014. J Nat Prod 79(3):629–661 Ngo LT, Okogun JI, Folk WR (2013) 21st century natural product research and drug development and traditional medicines. Nat Prod Rep 30(4):584–592 Nobel Prize (2015) http://www.nobelprize.org/nobel_prizes/medicine/laureates/2015/ Nosengo N (2016) Can you teach old drugs new tricks? Nature 14;534(7607):314–316 Olliaro P, Seiler J, Kuesel A, Horton J, Clark JN, Don R, Keiser J (2011) Potential drug development candidates for human soil-transmitted helminthiases. PLoS Negl Trop Dis 5(6): e1138 Paloque L, Ramadani AP, Mercereau–Puijalon O, Augereau JM, Benoit–Vical F (2016) Plasmodium falciparum: multifaceted resistance to artemisinins. Malar J 15:149 Patz JA, Graczyk TK, Geller N, Vittor AY (2000) Effects of environmental change on emerging parasitic diseases. Int J Parasitol 30(12–13):1395–1405 Patwardhan B (2005) Ethnopharmacology and drug discovery. J Ethnopharmacol 100(1–2):50–52 Pink R, Hudson A, Mouriès MA, Bendig M (2005) Opportunities and challenges in antiparasitic drug discovery. Nat Rev Drug Discov 4(9):727–740 Quiroz-Castaneda RE, Dantan-Gonzalez E (2015) Control of avian coccidiosis: future and present natural alternatives. Biomed Res Int. https://doi.org/10.1155/2015/430610 Rajput N, Ali S, Naeem M, Khan MA, Wang T (2014) The effect of dietary supplementation with the natural carotenoids curcumin and lutein on pigmentation, oxidative stability and quality of meat from broiler chickens affected by coccidiosis challenge. Br Poult Sci 55(4):501–509 Rana AK, Misra-Bhattacharya S (2013) Current drug targets for helminthic diseases. Parasitol Res 112:1819–1831 Roeber F, Jex AR, Gasser RB (2013) Impact of gastrointestinal parasitic nematodes of sheep, and the role of advanced molecular tools for exploring epidemiology and drug resistance—an Australian perspective. Parasit Vectors 6:153

9 Insecticidal and Antimalarial Properties of Plants: A Review

245

Saifi MA, Beg T, Harrath AH, Altayalan FSH, Quraishy SA (2013) Antimalarial drugs: mode of action and status of resistance. Afr J Pharm Pharmacol 7(5):148–156 Shahiduzzaman M, Daugschies A (2012) Therapy and prevention of cryptosporidiosis in animals. Vet Parasitol 188(3–4):203–214 Shears MJ, Botté CY, McFadden GI (2015) Fatty acid metabolism in the Plasmodium apicoplast: drugs, doubts and knockouts. Mol Biochem Parasitol 199(1–2):34–50 Singh S, Pathak AK, Sharma RK, Khan M (2015) Effect of tanniferous leaf meal based multi-nutrient blocks on feed intake, hematological profile, immune response, and body weight changes in Haemonchus contortus infected goats. Vet World 8(5):572–579 Skinner-Adams TS, Sumanadasa SD, Fisher GM, Davis RA, Doolan DL, Andrews KT (2016) Defining the target of antiparasitic compounds. Drug Discov Today 21(5):725–739 Torgerson PR (2013) One world health: socioeconomic burden and parasitic disease control priorities. Vet Parasitol 195(3–4):223–232 United Nations Population Fund (2012) Population dynamics in the least developed countries: challenges and opportunities for development and poverty reduction. http://www.unfpa.org/ sites/default/files/pub-pdf/CP51265.pdf Vernerova E, Vondrova R, Kisova H, Svobodova V, Hera A (2009) Detection of benzimidazole resistance in gastrointestinal nematode parasites of sheep in the Czech Republic. Veterinari Medicina 54(10):467–472 Waller PJ (2006) From discovery to development: current industry perspectives for the development of novel methods of helminth control in livestock. Vet Parasitol 139(1–3):1–14 WHO (2015) World Malaria Report. http://www.who.int/malaria/media/world-malaria-report2015/en/ WHO (2016) http://www.who.int/en/ WHO GBD (2016) http://www.who.int/healthinfo/global_burden_disease/estimates/en/index1. html Wink (2010) Occurrence and function of natural products in plants, in Phytochemistry and Pharmacognosy, [Eds.John M.Pezzuto, Massuo Jorge Kato], in Encyclopedia of Life Support Systems(EOLSS), Developed under the Auspices of the UNESCO, Eolss Publishers, Oxford, UK Wink M (2015) Modes of action of herbal medicines and plant secondary metabolites. Medicines 2 (3):251–286 Williams AR, Fryganas C, Ramsay A, Mueller-Harvey I, Thamsborg SM (2014a) Direct anthelmintic effects of condensed tannins from diverse plant sources against Ascaris suum. PLoS ONE 9(6):e97053 Williams AR, Ropiak HM, Fryganas C, Desrues O, Mueller-Harvey I, Thamsborg SM (2014b) Assessment of the anthelmintic activity of medicinal plant extracts and purified condensed tannins against free-living and parasitic stages of Oesophagostomum dentatum. Parasit Vectors 7:518 Woolhouse MEJ, Gowtage-Sequeria S (2005) Host range and emerging and reemerging pathogens. Emerg Infect Dis 11(12):1842–1847 Zhou S, Koh HL, Gao Y, Gong ZY, Lee EJ (2004) Herbal bioactivation: the good, the bad and the ugly. Life Sci 74(8):935–968 Ziegler S, Pries V, Hedberg C, Waldmann H (2013) Target identification for small bioactive molecules: finding the needle in the haystack. Angew Chem Int Ed Engl 52(10):2744–2792

Chapter 10

Antimalarial Terpenic Compounds Isolated from Plants Used in Traditional Medicine (2010–July 2016) Claire Beaufay, Joanne Bero and Joëlle Quetin-Leclercq

Abstract Malaria is an infectious tropical disease mainly affecting children and sub-Saharan Africa, being one of the major death causes with almost half of the world population at risk. Therefore and to face increasing parasite resistance to available treatments, there is an urgent need to discover new antimalarial drugs. Nature and mostly plants are a resourceful supplier of potential active metabolites, as shown by the successful example of the sesquiterpene lactone: artemisinin. Terpenoids are occurring widely in nature and display a lot of biological activities among which antiplasmodial effects. This review is a compilation of terpenic compounds isolated from plants with moderate (2 µM < IC50  11 µM) to strong (IC50  2 µM) activity against different strains of Plasmodium published from 2010 to July 2016. Forty-seven references are identified.




Keywords Terpene Plant compounds Malaria Plasmodium falciparum





Antiplasmodial activity

C. Beaufay and J. Bero—contributed equally to this work. C. Beaufay (&)
J. Bero
J. Quetin-Leclercq Pharmacognosy Research Group, Louvain Drug Research Institute, Catholic University of Louvain, Avenue E. Mounier, B1.72.03, 1200 Brussels, Belgium e-mail: [email protected] J. Bero e-mail: [email protected] J. Quetin-Leclercq e-mail: [email protected] © Springer International Publishing AG 2018 J.-M. Mérillon and C. Rivière (eds.), Natural Antimicrobial Agents, Sustainable Development and Biodiversity 19, https://doi.org/10.1007/978-3-319-67045-4_10

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Introduction

Malaria is a tropical disease caused by a protozoan of the genus Plasmodium transmitted to mammals by an infected female mosquito of the genus Anopheles. There are several species of Plasmodium responsible for human malaria, P. falciparum causing the most severe cases. In the last World Malaria Report of 2015, about 214 million cases were reported, and especially in Africa. Even if the number of reported cases is decreasing (18% less in 15 years) (World Health Organization (WHO) 2015), there is an urgent need to discover new active drugs to fight this global health problem. Indeed, there is an overall spread of resistance to available treatments, such as chloroquine but also the current one being artemisinin-based combination therapies (ACTs) (Ashley et al. 2014; Sibley 2015). In 2015, a new vaccine has been approved for the first time by a medicines regulatory authority to be used outside European Union (European Medicines Agency (EMA) 2015). This vaccine provides modest protection decreasing after one year for children aged from 5 to 17 months. It is an important step in the fight against malaria but much work has still to be done in its optimization to become a significant tool toward malaria control (Clemens and Moorthy 2016; Hoffman et al. 2015; White et al. 2015; RTS 2015). In this area, plants used in traditional medicine are a rich source of active original compounds, structurally innovative and with new mechanisms of action. Moreover, the innate affinity for biological receptors of natural compounds is a complementary advantage to consider (Ginsburg and Deharo 2011). The perfect example remains the actual used drug, artemisinin, a sesquiterpene lactone isolated from Artemisia annua, used for thousands years in Chinese traditional medicine. This discovery was awarded recently by the Medicine Nobel Prize in 2015 (Zhai et al. 2016). Moreover, according to WHO, about 80% of developing countries are relying on traditional remedies in their usual health care. This shows that plants and natural products can actively participate in the fight against malaria. This review is an update of the literature supplementing the previous papers on antimalarial compounds isolated from plants used in traditional medicine published from 2005 up to 2009 (Bero et al. 2009; Bero and Quetin-Leclercq 2011). It only gathers the most active natural terpenic metabolites (IC50  11 µM) that may have some interests for further development published from 2010 to July 2016. It focuses on terpenoids, as most active molecules isolated in our laboratory (Bero et al. 2013) belong to this chemical class. Moreover, according to previous results, a higher number of moderately to strongly active natural compounds belong to the terpenoid chemical class, along with alkaloids. Reasonable activity is considered as IC50 between 2 and 11 µM and strong one as lower than 2 µM. Activities were assessed on different strains of P. falciparum species, among which are chloroquine-sensitive (NF54, 3D7, D6, F32, D10, HB3), chloroquine-resistant (FcB1, W2, Dd2, MRC-pf-303, FCR3), and/or multidrug-resistant (K1) strains, to find effective compounds against resistant malaria. In some papers, cytotoxicity is also evaluated on different cell lines to calculate the selectivity index (SI) and estimate the

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therapeutic window: L6, rat skeletal myoblast; CHO, Chinese hamster ovary cell; A2780, human ovarian cancer cell; WI38, human fibroblast; MRC5, human fetal lung fibroblast; Vero, monkey kidney fibroblast; LLC/MK2, monkey kidney epithelial cell; MCF-7, human breast cancer cell; KB, nasopharyngeal epidermoid carcinoma cell. Some natural terpenoids were also cited in other reviews, as in Amoa Onguéné et al. and more recently in Silva et al. (Silva et al. 2015; Amoa et al. 2013).

10.2

Monoterpenes

The CH2Cl2-MeOH (1:1) extract of the aerial parts of Sphaeranthus bullatus Mattf. (Asteraceae) contains three known moderately active carvotacetone derivatives (1–3), named, respectively, 3-acetoxy-7-hydroxy-5-tigloyloxycarvotacetone, 3,7-dihydroxy-5-tigloyloxycarvotacetone, 3-acetoxy-5,7-dihydroxycarvotacetone with IC50 values of 4.3, 2.8, 2.5 µM on D6 and 6.2, 3.2, 2.8 µM on W2, respectively, (Machumi et al. 2012) (Fig. 10.1).

10.3

Sesquiterpenes

The separation of the n-hexane extracts of Ferula pseudalliacea Rech. F. (Apiaceae) roots gave new and known sesquiterpene coumarins among which sanandajin (4) and methyl galbanate (5) showing some activity on K1 strain (IC50 = 2.6 and 7.1 µM, respectively), but also cytotoxicity on L6 cells with IC50 of 9.4 and 4.3 µM (SI = 3.6 and 0.6) (Dastan et al. 2012). The main active constituent of Dicoma anomala subsp. Gerrardii (Asteraceae) was dehydrobrachylaenolide (6) which showed antiplasmodial activity in vitro with IC50 of 1.9 and 4.1 µM, respectively, on D10 and K1 with a selectivity index of 9.2 and 4.2 compared to CHO cells (Becker et al. 2011). Athroisma proteiforme (Humbert) Mattf. (Asteraceae) contains athrolides C (7) and D (8) which showed antiplasmodial activities with IC50 values of 6.6 and OH O

O O

O 1

OH O

O HO

O 2

OH O

O

O O

OH 3

Fig. 10.1 Monoterpenes with moderate in vitro activity against D6 and W2 strains of Plasmodium falciparum

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7.2 lM against HB3 strain and 5.5 and 4.2 lM against Dd2 one, respectively. Cytotoxicities were evaluated against A2780 cells, with IC50 values of 0.6 and 0.4 lM, respectively (Pan et al. 2011). Purifications from the dichloromethane extract of Inula montbretiana DC. (Asteraceae) resulted in the isolation of four sesquiterpenic lactones: 9b-(3′,4′epoxy-3′-methylpentanoyloxy)-parthenolide (9a + 9b) and 9b-(3′-oxo-2′-methylbutanoyloxy)-parthenolide (10a + 10b), representing two diastereomeric mixtures and exhibiting in vitro activities against K1 with IC50 of 9.2 µM with SI < 1 and 4.1 µM with SI = 34.7, respectively, compared to L6 cells (Gokbulut et al. 2012). Two known sesquiterpenic lactones (11 and 12) were isolated from the aerial parts of Acanthospermum hispidum D.C. (Asteraceae): (15-acetoxy-8b[(2-methylbutyryloxy)]-14-oxo-4, 5-cis-acanthospermolide) and (9a-acetoxy-15-hydroxy-8b-(2-methylbutyryloxy) -14-oxo-4, 5-trans-acanthospermolide). They showed in vitro antiplasmodial activity with IC50 of 2.9 and 2.2 lM, respectively, against 3D7. Their selectivity indices were, respectively, of 4.9 and 18.5 compared to WI38 cells (Ganfon et al. 2012). Psilostachyin (13) and peruvin (14), were isolated from an organic extract of Ambrosia tenuifolia Sprengel (Asteraceae) and were tested on F32 and W2 strains for their antiplasmodial activities and on lymphoid cells for cytotoxicity (IC50 = 2.1 and 1.1 µM, respectively, on F32 with SI of 11.6 and 34.4, 6.4, and 18.9 µM, respectively, on W2 with SI of 3.8 and 2.0) (Sulsen et al. 2011). The dichloromethane extract of the aerial parts of Eupatorium perfoliatum L. (Asteraceae) led to the isolation of a guaianolide (15) which exhibits antiplasmodial activity in vitro against K1 with IC50 of 2.0 µM and was less cytotoxic against L6 cells (IC50 of 16.2 µM; SI around 8) (Maas et al. 2011). The ethanol extract of leaves and twigs of Piptocoma antillana Urb. (Asteraceae) afforded two new goyazensolide-type sesquiterpene lactones named 5-O-methyl-5-epiisogoyazensolide (16) and 15-O-methylgoyazensolide (17), together with the known compounds 1-oxo-3,10-epoxy-8-(2-methylacryloxy)15-acetoxygermacra-2,4,11(13)-trien-6(12)-olide (18) and 5-epiisogoyazensolide (19). They displayed moderate antimalarial activity against Plasmodium falciparum Dd2, with IC50 values of 6.2, 2.2, 8.0 and 9.0 lM, respectively (Liu et al. 2014b). The bio-guided fractionation of whole plant dichloromethane extracts from Dicoma tomentosa Cass. (Asteraceae) led to the isolation of urospermal A-15-Oacetate (20), with moderate antiplasmodial activity (IC50 = 2.9 and 2.4 µM against 3D7 and W2 strains, respectively). Nevertheless, this compound displayed only a restrained selectivity (SI = 3.3 with IC50 = 9.5 µM on WI38) (Jansen et al. 2012). The dichloromethane extract of aerial parts of Centaurea salmantica L. (Asteraceae) contains a guaianolide, cynaropicrin (21) (Ha 2003) reasonably active on Plasmodium falciparum K1 (IC50 = 2.99 µM) (Zimmermann et al. 2012). The leaf and tuber of Vernonia guineensis Benth. (Asteraceae) contain the known sesquiterpene lactones vernopicrin (22) and vernomelitensin (23) highly active with IC50 of 1.8 and 1.4 µM on HB3 and 2.3 and 1.6 µM on Dd2 strains, respectively (Toyang et al. 2013a, b).

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9-epideoxymuzigadial, 9-deoxymuzigadial, muzigadial, and 3-a-acetoxypolygodial (24–27) were isolated from the leaves of Canella winterana L. Gaertin (Canellaceae) and showed antiplasmodial activity with IC50 of 4.3, 9.4, 1.2, and 9.5 µM on D10 with SI of 1.8, 15.5, 3.8, and 20.7 compared to CHO cells, respectively (Grace et al. 2010). Muzigadiolide (28), 11a-hydroxymuzigadiolide (29), mukaadial (30), and ugandensidial (31) were isolated from the stem barks of Warburgia ugandensis Sprague (Canellaceae). The active compounds showed activity with IC50 of 7.2, 6.4, 6.4, and >97.4 µM and 7.3, >114.0, 7.9, and 10.6, respectively, against 3D7 and K1. Cytotoxicity tests were only made on the extract from which compounds were isolated (IC50 = 5.6 µg/ml on KB cells) (Wube et al. 2010). Four sesquiterpenoid derivatives were identified in the CH2Cl2/CH3OH (1:1) extract of seeds of Salacia longipes (Oliver) N. Hallé var. camerunensis (Loes.) N. Hallé (Celastraceae): salaterpene A, B, C, and D (32–35) together with a known compound, 2b-acetoxy-1a, 6b, 9b-tribenzoyloxy-4b-hydroxy-dihydrob-agarofuran (36) exhibiting a moderate to good potency on W2 strain with IC50 of 2.0, 1.8, 2.6, 2.4, and 1.7 µM, respectively (Mba’ning et al. 2013). A known sesquiterpenic acid, cyperenoic acid (37), was isolated from the rhizomes of Jatropha isabellei Müll. Arg. (Euphorbaceae) and exhibited a reasonable activity on 3D7 and K1 strains (8.1 > IC50 > 12.5 µM) (Hadi et al. 2013). Lipiferolide (38) was isolated from Liriodendron tulipifera L. (Magnoliaceae) and exhibited antiplasmodial activity with IC50 of 5.9 µM on D10 and 7.5 µM on Dd2 with SI of 0.8, and 0.6, respectively, indicating a higher cytotoxicity on CHO cells (Graziose et al. 2011). Known drimane sesquiterpenes, 1-b-(p-methoxycinnamoyl)-polygodial, and 1-b-(p-cumaroyloxyl)-polygodial (39–40), isolated from the chloroform stem bark extract of Drimys brasiliensis Miers (Winteraceae) showed moderate to high activity on FcR3 strain with IC50 of 4.9 and 1.0 µM, respectively (Claudino et al. 2013). Figures 10.2 and 10.3 show the structures of sesquiterpenes with moderate or promising activity in vitro against various strains of Plasmodium falciparum.

10.4

Diterpenes

Andrographolide, a diterpenic lactone (41), was purified from the methanolic extract of Andrographis paniculata Nees (Acanthaceae) and has antiplasmodial activity in vitro against MRC-pf-303 with IC50 of 9.1 lM. No cytotoxicity assay was realized on the pure compound (Mishra et al. 2011). Serratol, a cembrane-type diterpene (42), was isolated from the gum resin of Boswellia serrata Roxb. (Burseraceae) and showed antiplasmodial activity with IC50 of 2.5 µM on K1 with selectivity index of 15.6 compared to L6 cells (Schmidt et al. 2011).

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Fig. 10.2 Sesquiterpenes with moderate or promising activity in vitro against various strains of Plasmodium falciparum

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Fig. 10.3 Sesquiterpenes with moderate or promising activity in vitro against various strains of Plasmodium falciparum (continued)

Chemical investigation of the ethyl acetate extract of Neoboutonia macrocalyx Pax. (Euphorbiaceae) bark resulted in the identification of diterpenoids among which the known montanin (43) showing reasonable antiplasmodial activity on D6 and W2 strains with IC50 of 22.7 and 10.2 µM, respectively, but this compound is known for its higher cytotoxicity (IC50 of 5.7 µM on MRC5 cell line) (Namukobe et al. 2015). The rhizomes of Jatropha isabellei Müll. Arg. (Euphorbaceae) contain reasonably active diterpenes, jatrophone (44), and jatropholone A–B (45a–b) with an IC50 range assessed between 8.1 and 12.5 µM on 3D7 and K1, except one IC50 lower than 4.1 µM for jatrophone on K1 strain (Hadi et al. 2013). Two new diterpene alkaloids, caesalminines A and B (46–47), possessing a tetracyclic cassane-type furanoditerpenoid skeleton with a c-lactam ring, were isolated from a methanolic extract of the seeds of Caesalpinia minax Hance (Fabaceae) and presented IC50 values on K1 strain of 0.4 and 0.8 lM, respectively (Ma et al. 2014). The dichloromethane extract of Salvia miltiorrhiza Bunge (Lamiaceae) roots led to the identification of tanshinone-type diterpenoids, miltirone (48), tanshinone IIa

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(49), 1,2 dihydrotanshinquinone (50), methylenetanshinquinone (51), tanshinone I (52), and methyltanshinonate (53). The antiplasmodial activities were determined on K1 and the selectivity on L6 cells (IC50 = 5.0, 4.0, 5.3, 6.3, 7.2, and 4.1 µM and SI = 0.3, 1.9, 0.8, 1.9, 1.7, and 1.7, respectively) (Slusarczyk et al. 2011). The n-hexane extract of the roots of Salvia sahendica Boiss. and Buhse (Lamiaceae) contains known abietane-type diterpenoids and a new one, sahandol (54). Some of them exhibited middle (12-deoxy-salvipisone (55) (IC50 = 8.8 µM), sahandinone (56) (IC50 = 5.1 µM), and sahandol (IC50 = 4.7 µM)) to strong (D9-ferruginol (57) (IC50 = 0.9 µM), and 7a-acetoxyroyleanone (58) (IC50 = 1.3 µM)) activity on K1 strain. Although, only two of these compounds showed a selectivity index higher than three compared to cytotoxicity on L6-cells (SI = 0.06, 0.08, 3.2, 17.2, and 0.15, respectively) (Ebrahimi et al. 2013). Aphadilactones A–D (59–62), four diterpenoid dimers, were isolated from Aphanamixis grandifolia Blume (Meliaceae) and showed significant antimalarial activities on Dd2 with IC50 values of 0.2, 1,4, 0.2, and 0.1 µM, respectively (Liu et al. 2014a). Activity-guided isolation of the n-hexane and dichloromethane extracts from the bark of Cupania cinerea Poepp. and Endl. (Sapindaceae) afforded two diterpenic glycosides (63 and 64) named cupacinoside and 6’-de-O-acetylcupacinoside, together with active triterpenes (122–125, Fig. 10.7). These diterpenes showed in vitro activities against K1 with IC50 of 1.3 and 2.1 lM and selectivity indices of 8.9 and 4.1 compared to L-6 cells cytotoxicity (Gachet et al. 2011). Three abietane diterpenoids (65–67) were isolated from Clerodendrum eriophyllum Gürke (Verbenaceae): taxodione, 6-hydroxysalvinolone, and 6,11,12,16-tetrahydroxy-5,8,11,13-abietatetra-en-7-one. They showed antiplasmodial activity with IC50 of 3.8, 5.5, and 8.7 µM, respectively, against D6 and 3.8, 7.6, and 13.9 µM, respectively, against W2. The IC50 for cytotoxicity were >15.1, 13.6 and >13.7 µM on Vero cells (Machumi et al. 2010). Four new other metabolites, derived from beilschmiedic acid, cryptobeilic acids A–D, were isolated from the MeOH-CH2Cl2 bark extract of Beilschmiedia cryptocaryoides Kosterm. (Lauraceae), together with a known compound, tsangibeilin B. Cryptobeilic acids B (68), D (69), and tsangibeilin B (70) were moderately active on NF54 strain with IC50 of 5.35, 10.8, and 8.2 µM, respectively, and weakly cytotoxic on L6 cells with IC50 of 20.4, 61, and 21.5 µM, respectively (Talontsi et al. 2013). Structures of diterpenes with moderate or promising activity in vitro against various strains of P. falciparum are given in Fig. 10.4.

10.5

Triterpenes

Two protostane triterpenoids, alisol B 23-monoacetate (71), and alisol G (72) were isolated from the ethyl acetate extract of roots of Alisma plantago-aquatica L. (Alismataceae). They have IC50 of 5.4 and 7.0 µM against Plasmodium falciparum

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Fig. 10.4 Diterpenes with moderate or promising activity in vitro against various strains of Plasmodium falciparum

K1 strain. Cytotoxicities were evaluated on L6 cells with IC50 of 11.8 and 14.0 µM (Adams et al. 2011). Phytochemical investigation of the leaves of Centella asiatica (L.) Urb. (Apiaceae) resulted in the isolation of two antiplasmodial triterpenic saponins: 23O-acetylmadecassoside (73) and madecassoside (74) active on D6 and W2 with IC50 of 0.6 and 0.7 and 1.2 and 0.9 lM, respectively, without any cytotoxicity

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observed against Vero cells at the highest concentration tested (Rumalla et al. 2010). One reasonably active ursane triterpene, brein (75), was isolated from 70% ethanolic extract of aerial parts of Kleinia odora (Forssk) DC (Asteraceae) with IC50 on K1 strain of 9.7 µM. This antiplasmodial activity was selective according to cytotoxicity test on MRC-5 SV2 fibroblasts (IC50 > 64 µM) (Al Musayeib et al. 2013). 2b, 3b, 19a-trihydroxy-urs-12-en-28-oic acid (76) was isolated from the stem bark of Kigelia africana (Lam.) Benth (Bignoniaceae) and tested in vitro against W2 and two field isolates, CAM10 and SHF4 (IC50 = 1.6, 2.2, and 8.0 µM, respectively). The cytotoxicity was evaluated on LLC/MK2 cells, and the selectivity indices were 12.0, 8.8, and 2.4, respectively (Zofou et al. 2011). Pomolic acid (77) and 3-acetylpomolic acid (78) were isolated from Markhamia tomentosa K. Schum. ex Engl. (Bignoniaceae) and exhibited antimalarial activity with IC50 of 7.3 and 3.1 µM against K1 with SI of 1.2 and 7.8, respectively compared to L6 cells (Tantangmo et al. 2010). Balsaminoside A (79) and karavilagenin E (80) were isolated from the methanol extract of aerial parts of Momordica balsamina L. (Cucurbitaceae) and exhibited antiplasmodial activity against two Plasmodium strains (IC50 values for balsaminoside A = 4.6 and 4.0 µM, and for karavilagenin E = 7.4 and 8.2 µM, respectively, on 3D7 and Dd2) with selectivity indices close to four compared to MCF-7 cells (Ramalhete et al. 2011). Another bioassay-guided fractionation of this extract led to the isolation of a new cucurbitane-type triterpenoid, balsaminoside B (81), along with the known glycosylated cucurbitacins, cucurbita-5,24-diene-3b,23 (R)-diol-7-O-b-D-glucopyranoside (82), and kuguaglycoside A (83). These compounds were evaluated for their antiplasmodial activity and have IC50 of 2.9, 3.4, and 3.9 µM against 3D7 and 6.3, 7.2, and 4.7 µM against Dd2, respectively. Cytotoxicities were evaluated on MCF-7 cell line, and selectivity indices were 4.8, 7.0, and 3.5, respectively, for 3D7 and 2.3, 3.3, and 2.9, respectively, for Dd2 (Ramalhete et al. 2010). Hexane extract of aerial parts of Datisca glomerata (C. Presl) Baill (Datiscaceae) contains seven cucurbitacin glycosides, datiscosides I–O, along with two known compounds, datiscoside and datiscoside B. Two of them, datiscosides K (84) and N (85), showed some activity on D10 strain with IC50 of 7.7 and 8.8 µM, respectively, with no cytotoxicity on CHO but a high one on L6 with IC50 of 0.1 and 11.3 µM, respectively (Graziose et al. 2013). Leaves ethyl acetate extract of Neoboutonia macrocalyx Pax. (Euphorbiaceae) led to the isolation of new cycloartane triterpenes: neomacrolactone (86), 22a-acetoxyneomacrolactone (87), 6-hydroxy neomacrolactone (88), 22a-acetoxy6-hydroxyneomacrolactone (89), 6,7-epoxyneomacrolactone (90), 4-methylenneomacrolactone (91), neomacroin (92), in addition to the known cycloartane triterpenoid, 22-de-O-acetyl-26-deoxyneoboutomellerone (93). These isolated

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compounds were evaluated for antiplasmodial activity against the chloroquine-resistant FcB1/Colombia strain of Plasmodium falciparum and for cytotoxicity against the KB and MRC-5 cells. The two first compounds were less interesting while others showed moderate to high antimalarial activity (IC50 of 2.1, 2.5, 1.5, 2.7, 9.4, 1.9, 3.2, and 2.1 µM, respectively) but with significant cytotoxicity except 22a-acetoxyneomacrolactone and neomacroin (24.0 and 25.0% inhibition at 8.8 µM and 10.0 and 24.0% inhibition at 9.5 µM, on KB and MRC5 cells, respectively) (Namukobe et al. 2014). The rhizomes of Jatropha isabellei Müll. Arg. (Euphorbiaceae) hold a reasonably active triterpene, acetyl-aleuritolic acid (94) with IC50 range between 8.1 and 12.5 µM on 3D7 and K1 strains (Hadi et al. 2013). 18b-glycyrrhetinic acid (95) identified in methanol extract from roots of Glycyrrhiza glabra L. (Fabaceae) showed moderate activity on NF54 strain with IC50 of 3.6 µM (Kalani et al. 2013). Salvadione C (96), perovskone B (97), two new triterpenoids, and hydrangenone (98), a new heptacyclic isoprenoid, were isolated from an antiplasmodial n-hexane extract of aerial parts from Salvia hydrangea DC. ex Benth. (Lamiaceae). Compounds showed in vitro antiplasmodial activity against K1, with IC50 values of 1.4, 0.2, and 1.4 lM and selectivity indices of 86.2, 69.6, and 6, respectively compared to L6 cells (Farimani et al. 2012). A limonoid was isolated from abundant residual pressed seed material of Carapa guianensis Aubl. (Meliaceae): 6a-acetoxygedunin (99) tested against Plasmodium falciparum K1 strain and showing moderate activity with IC50 of 7 µM without cytotoxicity (IC50 > 185 µM) on MRC-5 cells (Pereira et al. 2014). New tirucallane-type triterpenoids were isolated from the bark of Entandrophragma congoense A. Chev. (Meliaceae). Prototiamin A, B, C, E, F, G (100–105) showed good activity on Plasmodium falciparum NF54 strain, with IC50 of 0.7, 1.3, 0.4, 0.9, 1.4, 1.3 µM, respectively. Prototiamin D (106) along with congoensin A/B (107–108) and agladoral A (109) displayed a moderate activity with IC50 of 2.0, 5.5, 6.1, and 2.4 µM, respectively. Cytotoxicities, evaluated on L6 cell line, were moderate to low with IC50 of 104.7, 25.2, 12.1, 12.7, 6.8, 9.0, 21.0, 10.6, 57.4, and 61.6 µM, respectively (Happi et al. 2015a, b). Three new limonoids, rubescins B, C (110–111) and E (112) and a known havanensin type limonoid TS3 (113) isolated from the roots and stem bark of Trichilia rubescens Oliv. (Meliaceae), along with other new limonoids, exhibited antiplasmodial in vitro activity on strain 3D7 with IC50 values of 6.7, 6.2, 1.1, and 0.8 lM, respectively (Armelle et al. 2016; Tsamo et al. 2013). Eight triterpenic esters this last update, cis and trans isomers of 27-O-coumaroyloxy- and 27-O-feruloyloxy-ursolic and -oleanolic acids (114–121), were isolated from the a dichloromethane extract of Keetia leucantha (K. Krause) Bridson (Rubiaceae) twigs. Their mixtures showed an antiplasmodial IC50 of about 2.6 µM on 3D7 strain with selectivity index of 34.6 on WI38. The same range of activity was observed for all isolated isomers pairs (Bero et al. 2013).

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Activity-guided isolation of the n-hexane and dichloromethane extracts from the bark of Cupania cinerea Poepp. and Endl. (Sapindaceae) afforded a lactonized triterpene: cupacinoxepin (122), together with the known lupenone (123), betulone (124), and taraxerol (125) but also two diterpenic glycosides (63 and 64, Fig. 10.4). These triterpenes showed in vitro activities against K1 with IC50 of 8.7, 4.7, 3.0, and 8.5 lM. Cytotoxicities were evaluated against L6 cells and showed IC50 of >90, >90, 22.2 and >90 µM (Gachet et al. 2011). The antimalarial activities of physalins B, D, F, and G (126–129), isolated from Physalis angulata L. (Solanaceae), were evaluated for their antimalarial activity and showed IC50 of 2.8, 5.5, 2.2, and 6.7 µM on W2, respectively, with selectivity indices of 12.3, 10.4, 5.9, and 5.6 on BALB/c mice splenocytes (Sa et al. 2011). Figures 10.5, 10.6, and 10.7 show the structures of triterpenes cited in the text.

10.6

Discussion

Comparing abundances of active compounds in the different terpenic subclasses, we observed that active sesquiterpenes are most often lactones as it was observed in 2005–2009 (Bero et al. 2009; Bero and Quetin-Leclercq 2011) and that a very small amount of active monoterpenes were identified between 2010 and 2016, compared to no one before. The diterpene class was pointed out to be very interesting in 2005–2008 with a high number of active compounds but not in 2009 and the number of new active isolated compounds is decreasing. For triterpenes, active metabolites isolation remains stable, with less moderately and more highly active ones. It also has to be noted that selectivity against mammalian cells is not always determined, but it is a crucial step in assessing a drug potential for further research. Compared to the previous results from 2005 to 2009, we observed a same trend in the discovery of active triterpenic compounds with the highest number of isolated promising molecules which could be considered as lead compounds for further investigations (Fig. 10.8). In this last update, the best activities were observed for aphadilactones derivatives, diterpenes isolated from Aphanamixis grandifolia Blume (Meliaceae), mainly aphadilactones A, C, and D (59, 61, 62) with IC50 on Dd2 of 0.2, 0.2, and 0.1, µM respectively. Perovskone B (97), a triterpene isolated from Salvia hydrangea DC. ex Benth. (Lamiaceae) also exhibited a strong activity with an IC50 of 0.2 µM on K1. Moreover, this compound showed a good selectivity compared to L6 cells (SI = 69.6), one of the highest indices from this review along with prototiamin A (100). Indeed, this tirucallane-type triterpenoid isolated from the bark of Entandrophragma congoense A. Chev. (Meliaceae) displayed a very low cytotoxicity on L6 cells (IC50 = 104.7 µM) with a promising antiplasmodial activity on NF54 strain (IC50 = 0.7 µM) leading to a remarkable SI of 156.3.

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Fig. 10.5 Triterpenes with moderate or promising activity in vitro against various strains of Plasmodium falciparum

Concerning differences between families, the highest number of antiplasmodial metabolites was extracted from Asteraceae (mainly sesquiterpenes and some monoterpenes). However, the most active natural compounds gathered from 2010 to July 2016 were isolated from Meliaceae, because of the eleven triterpenes isolated from the bark of Entandrophragma congoense A. Chev. while Caesalpiniaceae was pointed out in the previous review (Fig. 10.9).

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Fig. 10.6 Triterpenes with moderate or promising activity in vitro against various strains of Plasmodium falciparum (continued)

Terpenoids are the most extended class of vegetal secondary metabolites and can be an alternative source of new promising drugs, as shown by this review and by the successful example of artemisinin. As they are mainly lipophilic compounds, they usually do not follow the Lipinski’s rules for good oral bioavailability (Leeson 2012). In order to increase oral bioavailability, loading compounds in nanoemulsions or lipid microparticles can reduce poor aqueous solubility and low stability leading to an enhanced activity (Chinaeke et al. 2015; Dwivedi et al. 2015; Thakkar and S B 2016). Furthermore, Lipinski’s rules have exceptions as shown by some

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Fig. 10.7 Triterpenes with moderate or promising activity in vitro against various strains of Plasmodium falciparum (continued)

ether artemisinin derivatives exhibiting a higher lipophilicity than b-arteether (log P > 6) but also a better oral antimalarial activity profile (Singh et al. 2006). It is also interesting to highlight that in some cases, plant crude extracts exhibit a higher antiplasmodial efficiency than active isolated pure compounds. Indeed, the complexity and diversity of extracted interacting compounds can lead to additive or synergic pharmacologic effects. The best example remains artemisinin which activity on Plasmodium falciparum is potentiated in vitro by flavonoids present in Artemisia annua tea. The same is observed for quinine whose in vitro activity against resistant strain is improved thanks to synergism between different Cinchona alkaloids (Ginsburg and Deharo 2011; Deharo and Ginsburg 2011; Rasoanaivo et al. 2011). Moreover, component or herbal mixtures fit with WHO guidelines about combination therapies in the fight against parasite resistance occurrence and development and can influence bioavailability (Elfawal et al. 2012).

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Fig. 10.8 Number of compounds with good (IC50  2 µM) or moderate (2 < IC50  11 µM) activity in vitro against various strains of P. falciparum, classified according to their chemical classes

Fig. 10.9 Number of compounds with good (IC50  2 µM) or moderate (2 < IC50  11 µM) activity in vitro against various strains of P. falciparum, classified according to their family from 2010 to 2016

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Finally, some predictive tools based on chemo-informatics profiling and structure–activity relationships are developed to predict, guide, and facilitate antiplasmodial drug development from natural extracts or for pure compounds (Egieyeh et al. 2016; Zhang et al. 2013).

10.7

In Vivo Activities

To conclude, we will discuss some antiplasmodial compounds isolated from plants which were evaluated for their in vivo antimalarial activity, but the list is very short. According to other reviews, we can note in some cases a low in vitro activity associated with a good antimalarial activity in vivo as for oleanolic acid (Bero et al. 2009). Thereby, it is essential to evaluate in vivo efficacy of isolated compounds in a next step. Hinokitiol (130), an antimalarial component of essential oil from Thujopsis dolabrata Siebold and Zucc. (Cupressaceae), showed in vivo activity by percutaneous administration on mice infected by Plasmodium berghei NK-5. Faneous oil with 10% of hinokitiol at a dose of 385 mg/kg thrice a day for three days reduced the parasitemia at 5.6% versus 28.7% for the negative control at day nine (Fujisaki et al. 2012). 18b-glycyrrhetinic acid (93) identified in methanol extract from roots of Glycyrrhiza glabra L. (Fabaceae) showed a dose dependent activity (6.7, 1.5 and 0% parasitaemia at 62.5, 125 and 250 mg/kg respectively) in mice infected with P. berghei K173 treated per os (6 days test (Boniface and Pal 2013)) compared to 20.6% parasitaemia in infected non-treated animals. Toxicity risk parameters (mutagenicity, tumorigenicity, irritation, effect on reproduction) calculated in silico were estimated as low and the treatment as safe (Kalani et al. 2013). A known labdane diterpenoid called otostegindiol (131) isolated from the 80% methanol leaf extract Otostegia integrifolia Benth (Lamiaceae) displayed a significant antimalarial activity against Plasmodium berghei ANKA strain at doses of 25, 50, and 100 mg/kg per os with parasitaemia inhibition values of 50.2, 65.6, and 73.2%, respectively, in the 4-day suppressive assay of Peters (Endale et al. 2013). A limonoid isolated from abundant residual pressed seed material of Carapa guianensis Aubl. (Meliaceae): 6a-acetoxygedunin (97) suppressed parasitaemia versus untreated control by 40 and 66% at oral doses of 50 and 100 mg/kg/day, respectively, in BALB/c mice infected with chloroquine-sensitive Plasmodium berghei NK65 strain in the 4-day suppressive test of Peters (Pereira et al. 2014). a-Santalol (132) isolated from essential oil of Santalum album L. (Santalaceae), was administered subcutaneously to mice infected by NK-5 strain at a dose of 100 µl thrice a day for 3 days. Parasitaemia was significantly reduced compared to negative control at day 7 post-infection (29.4 versus 48.5% respectively) (Fujisaki et al. 2012). Treatment of P. berghei NK65-infected mice with physalin D (127), isolated from Physalis angulata L. (Solanaceae), significantly decreased parasitaemia by

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Fig. 10.10 Terpenes with in vivo activity against various strains of P. berghei

about 65% at the eighth-day post-infection at 100 mg/kg/day for four consecutive days by the intraperitoneal route (Sa et al. 2011). Figure 10.10 shows the structures of terpenes with in vivo activity not already mentioned above.

10.8

Conclusion

Because of the rapid emergence of resistance to most of the antiplasmodial drugs, there is an urgent need to find new leads with high activity, biodisponibility, and/or original mode of action. As shown in this review gathering 129 in vitro and 6 in vivo active compounds on Plasmodium, natural products can provide interesting structures but further studies are necessary to determine the most interesting compounds and their modes of action. If a selection had to be done to analyze in more details the activity of some of these molecules, those fulfilling as much as possible the Lipinski’s rules should be first selected as well as those having shown in vivo effects and a high selectivity.

References Adams M, Gschwind S, Zimmermann S, Kaiser M, Hamburger M (2011) Renaissance remedies: Antiplasmodial protostane triterpenoids from Alisma plantago-aquatica L. (Alismataceae). J Ethnopharmacol 135(1):43–47 Al Musayeib NM, Mothana RA, Gamal AA, Al-Massarani SM, Maes L (2013) In vitro antiprotozoal activity of triterpenoid constituents of Kleinia odora growing in Saudi Arabia. Molecules 18(8):9207–9218 Amoa OP, Ntie-Kang F, Lifongo LL, Ndom JC, Sippl W, Mbaze LM (2013) The potential of anti-malarial compounds derived from African medicinal plants, part I: a pharmacological evaluation of alkaloids and terpenoids. Malar J 12:449 Armelle TT, Pamela NK, Pierre M, Muller IB, Marat K, Sass G, Ephrem NA (2016) Antiplasmodial Limonoids from Trichilia rubescens (Meliaceae). Med Chem Ashley EA, Dhorda M, Fairhurst RM, Amaratunga C, Lim P, Suon S, Sreng S, Anderson JM, Mao S, Sam B, Sopha C, Chuor CM, Nguon C, Sovannaroth S, Pukrittayakamee S, Jittamala P, Chotivanich K, Chutasmit K, Suchatsoonthorn C, Runcharoen R, Hien TT, Thuy-Nhien NT, Thanh NV, Phu NH, Htut Y, Han KT, Aye KH, Mokuolu OA, Olaosebikan RR, Folaranmi OO,

10

Antimalarial Terpenic Compounds Isolated from Plants …

265

Mayxay M, Khanthavong M, Hongvanthong B, Newton PN, Onyamboko MA, Fanello CI, Tshefu AK, Mishra N, Valecha N, Phyo AP, Nosten F, Yi P, Tripura R, Borrmann S, Bashraheil M, Peshu J, Faiz MA, Ghose A, Hossain MA, Samad R, Rahman MR, Hasan MM, Islam A, Miotto O, Amato R, MacInnis B, Stalker J, Kwiatkowski DP, Bozdech Z, Jeeyapant A, Cheah PY, Sakulthaew T, Chalk J, Intharabut B, Silamut K, Lee SJ, Vihokhern B, Kunasol C, Imwong M, Tarning J, Taylor WJ, Yeung S, Woodrow CJ, Flegg JA, Das D, Smith J, Venkatesan M, Plowe CV, Stepniewska K, Guerin PJ, Dondorp AM, Day NP, White NJ (2014) Spread of artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med 371(5):411–423 Becker JV, van der Merwe MM, van Brummelen AC, Pillay P, Crampton BG, Mmutlane EM, Parkinson C, van Heerden FR, Crouch NR, Smith PJ, Mancama DT, Maharaj VJ (2011). In vitro anti-plasmodial activity of Dicoma anomala subsp. gerrardii (Asteraceae): identification of its main active constituent, structure-activity relationship studies and gene expression profiling. Malar J 10:295 Bero J, Frederich M, Quetin-Leclercq J (2009) Antimalarial compounds isolated from plants used in traditional medicine. J Pharm Pharmacol 61(11):1401–1433 Bero J, Herent MF, Schmeda-Hirschmann G, Frederich M, Quetin-Leclercq J (2013) In vivo antimalarial activity of Keetia leucantha twigs extracts and in vitro antiplasmodial effect of their constituents. J Ethnopharmacol 149(1):176–183 Bero J, Quetin-Leclercq J (2011) Natural products published in 2009 from plants traditionally used to treat malaria. Planta Med 77(6):631–640 Boniface PK, Pal A (2013) Substantiation of the ethnopharmacological use of Conyza sumatrensis (Retz.) E.H.Walker in the treatment of malaria through in-vivo evaluation in Plasmodium berghei infected mice. J Ethnopharmacol 145(1):373–377 Chinaeke EE, Chime SA, Onyishi VI, Attama AA, Okore VC (2015) Formulation development and evaluation of the anti-malaria properties of sustained release artesunate-loaded solid lipid microparticles based on phytolipids. Drug Deliv 22(5):652–665 Claudino VD, da Silva KC, Cechinel FV, Yunes RA, Delle MF, Gimenez A, Salamanca E, Gutierrez-Yapu D, Malheiros A (2013) Drimanes from Drimys brasiliensis with leishmanicidal and antimalarial activity. Mem Inst Oswaldo Cruz 108(2):140–144 Clemens J, Moorthy V (2016) Implementation of RTS, S/AS01 malaria vaccine-the need for further evidence. N Engl J Med 374(26):2596–2597 Dastan D, Salehi P, Reza GA, Zimmermann S, Kaiser M, Hamburger M, Reza KH, Ebrahimi SN (2012) Disesquiterpene and sesquiterpene coumarins from Ferula pseudalliacea, and determination of their absolute configurations. Phytochemistry 78:170–178 Deharo E, Ginsburg H (2011) Analysis of additivity and synergism in the anti-plasmodial effect of purified compounds from plant extracts. Malar J 10(Suppl 1):S5 Dwivedi P, Khatik R, Chaturvedi P, Khandelwal K, Taneja I, Raju KS, Dwivedi H, Singh SK, Gupta PK, Shukla P, Tripathi P, Singh S, Tripathi R, Wahajuddin PS, Dwivedi AK, Mishra PR (2015) Arteether nanoemulsion for enhanced efficacy against Plasmodium yoelii nigeriensis malaria: an approach by enhanced bioavailability. Colloids Surf B Biointerfaces 126:467–475 Ebrahimi SN, Zimmermann S, Zaugg J, Smiesko M, Brun R, Hamburger M (2013) Abietane diterpenoids from Salvia sahendica–antiprotozoal activity and determination of their absolute configurations. Planta Med 79(2):150–156 Egieyeh SA, Syce J, Malan SF, Christoffels A (2016) Prioritization of anti-malarial hits from nature: chemo-informatic profiling of natural products with in vitro antiplasmodial activities and currently registered anti-malarial drugs. Malar J 15:50 Elfawal MA, Towler MJ, Reich NG, Golenbock D, Weathers PJ, Rich SM (2012) Dried whole plant Artemisia annua as an antimalarial therapy. PLoS ONE 7(12):e52746 Endale A, Bisrat D, Animut A, Bucar F, Asres K (2013) In vivo antimalarial activity of a labdane diterpenoid from the leaves of Otostegia integrifolia Benth. Phytother Res 27(12):1805–1809 European Medicines Agency (EMA) (2015) First malaria vaccine receives positive scientific opinion from EMA. http://www.ema.europa.eu/docs/en_GB/document_library/Press_release/ 2015/07/WC500190447.pdf

266

C. Beaufay et al.

Farimani MM, Taheri S, Ebrahimi SN, Bahadori MB, Khavasi HR, Zimmermann S, Brun R, Hamburger M (2012) Hydrangenone, a new isoprenoid with an unprecedented skeleton from Salvia hydrangea. Org Lett 14(1):166–169 Fujisaki R, Kamei K, Yamamura M, Nishiya H, Inouye S, Takahashi M, Abe S (2012) In vitro and in vivo anti-plasmodial activity of essential oils, including hinokitiol. Southeast Asian J Trop Med Public Health 43(2):270–279 Gachet MS, Kunert O, Kaiser M, Brun R, Zehl M, Keller W, Munoz RA, Bauer R, Schuehly W (2011) Antiparasitic compounds from Cupania cinerea with activities against Plasmodium falciparum and Trypanosoma brucei rhodesiense. J Nat Prod 74(4):559–566 Ganfon H, Bero J, Tchinda AT, Gbaguidi F, Gbenou J, Moudachirou M, Frederich M, Quetin-Leclercq J (2012) Antiparasitic activities of two sesquiterpenic lactones isolated from Acanthospermum hispidum D.C. J Ethnopharmacol accepted Ginsburg H, Deharo E (2011) A call for using natural compounds in the development of new antimalarial treatments-an introduction. Malar J 10(Suppl 1):S1 Gokbulut A, Kaiser M, Brun R, Sarer E, Schmidt TJ (2012) 9beta-hydroxyparthenolide esters from Inula montbretiana and their antiprotozoal activity. Planta Med 78(3):225–229 Grace MH, Lategan C, Mbeunkui F, Graziose R, Smith PJ, Raskin I, Lila MA (2010) Antiplasmodial and cytotoxic activities of drimane sesquiterpenes from Canella winterana. Nat Prod Commun 5(12):1869–1872 Graziose R, Grace MH, Rathinasabapathy T, Rojas-Silva P, Dekock C, Poulev A, Lila MA, Smith P, Raskin I (2013) Antiplasmodial activity of cucurbitacin glycosides from Datisca glomerata (C. Presl) Baill. Phytochemistry 87:78–85 Graziose R, Rathinasabapathy T, Lategan C, Poulev A, Smith PJ, Grace M, Lila MA, Raskin I (2011) Antiplasmodial activity of aporphine alkaloids and sesquiterpene lactones from Liriodendron tulipifera L. J Ethnopharmacol 133(1):26–30 Hadi V, Hotard M, Ling T, Salinas YG, Palacios G, Connelly M, Rivas F (2013) Evaluation of Jatropha isabelli natural products and their synthetic analogs as potential antimalarial therapeutic agents. Eur J Med Chem 65:376–380 Happi GM, Kouam SF, Talontsi FM, Lamshoft M, Zuhlke S, Bauer JO, Strohmann C, Spiteller M (2015a) Antiplasmodial and Cytotoxic Triterpenoids from the Bark of the Cameroonian Medicinal Plant Entandrophragma congoense. J Nat Prod 78(4):604–614 Happi GM, Kouam SF, Talontsi FM, Zuhlke S, Lamshoft M, Spiteller M (2015b) Minor secondary metabolites from the bark of Entandrophragma congoense (Meliaceae). Fitoterapia 102:35–40 Hoffman SL, Vekemans J, Richie TL, Duffy PE (2015) The march toward malaria vaccines. Vaccine 33(Suppl 4):D13–D23 Jansen O, Tits M, Angenot L, Nicolas JP, De MP, Nikiema JB, Frederich M (2012) Anti-plasmodial activity of Dicoma tomentosa (Asteraceae) and identification of urospermal A-15-O-acetate as the main active compound. Malar J 11:289 Kalani K, Agarwal J, Alam S, Khan F, Pal A, Srivastava SK (2013) In silico and in vivo anti-malarial studies of 18beta glycyrrhetinic acid from Glycyrrhiza glabra. PLoS ONE 8(9): e74761 Leeson P (2012) Drug discovery: chemical beauty contest. Nature 481(7382):455–456 Liu J, He XF, Wang GH, Merino EF, Yang SP, Zhu RX, Gan LS, Zhang H, Cassera MB, Wang HY, Kingston DG, Yue JM (2014a) Aphadilactones A-D, four diterpenoid dimers with DGAT inhibitory and antimalarial activities from a Meliaceae plant. J Org Chem 79(2):599–607 Liu Y, Rakotondraibea LH, Brodie PJ, Wiley JD, Cassera MB, Goetzc M, Kingstona DG (2014b) Antiproliferative and antimalarial sesquiterpene lactones from Piptocoma antillana from Puerto Rico. Nat Prod Commun 9(10):1403–1406 Ma G, Sun Z, Sun Z, Yuan J, Wei H, Yang J, Wu H, Xu X (2014) Antimalarial diterpene alkaloids from the seeds of Caesalpinia minax. Fitoterapia 95:234–239 Maas M, Hensel A, Costa FB, Brun R, Kaiser M, Schmidt TJ (2011) An unusual dimeric guaianolide with antiprotozoal activity and further sesquiterpene lactones from Eupatorium perfoliatum. Phytochemistry 72(7):635–644

10

Antimalarial Terpenic Compounds Isolated from Plants …

267

Machumi F, Samoylenko V, Yenesew A, Derese S, Midiwo JO, Wiggers FT, Jacob MR, Tekwani BL, Khan SI, Walker LA, Muhammad I (2010) Antimicrobial and antiparasitic abietane diterpenoids from the roots of Clerodendrum eriophyllum. Nat Prod Commun 5 (6):853–858 Machumi F, Yenesew A, Midiwo JO, Heydenreich M, Kleinpeter E, Tekwani BL, Khan SI, Walker LA, Muhammad I (2012) Antiparasitic and anticancer carvotacetone derivatives of Sphaeranthus bullatus. Nat Prod Commun 7(9):1123–1126 Mba’ning BM, Lenta BN, Noungoue DT, Antheaume C, Fongang YF, Ngouela SA, Boyom FF, Rosenthal PJ, Tsamo E, Sewald N, Laatsch H (2013) Antiplasmodial sesquiterpenes from the seeds of Salacia longipes var. camerunensis. Phytochemistry 96:347–352 Mishra K, Dash AP, Dey N (2011) Andrographolide: a novel antimalarial diterpene lactone compound from Andrographis paniculata and its interaction with curcumin and artesunate. J Trop Med 2011:579518 Namukobe J, Kiremire BT, Byamukama R, Kasenene JM, Akala HM, Kamau E, Dumontet V (2015) Antiplasmodial compounds from the stem bark of Neoboutonia macrocalyx pax. J Ethnopharmacol 162:317–322 Namukobe J, Kiremire BT, Byamukama R, Kasenene JM, Dumontet V, Gueritte F, Krief S, Florent I, Kabasa JD (2014) Cycloartane triterpenes from the leaves of Neoboutonia macrocalyx L. Phytochemistry 102:189–196 Pan E, Gorka AP, Alumasa JN, Slebodnick C, Harinantenaina L, Brodie PJ, Roepe PD, Randrianaivo R, Birkinshaw C, Kingston DG (2011) Antiplasmodial and antiproliferative pseudoguaianolides of Athroisma proteiforme from the Madagascar dry forest. J Nat Prod 74 (10):2174–2180 Pereira TB, Rocha e Silva LF, Amorim RC, Melo MR, Zacardi de Souza RC, Eberlin MN, Lima ES, Vasconcellos MC, Pohlit AM (2014) In vitro and in vivo anti-malarial activity of limonoids isolated from the residual seed biomass from Carapa guianensis (andiroba) oil production. Malar J 13:317 Ramalhete C, da Cruz FP, Lopes D, Mulhovo S, Rosario VE, Prudencio M, Ferreira MJ (2011) Triterpenoids as inhibitors of erythrocytic and liver stages of Plasmodium infections. Bioorg Med Chem 19(24):7474–7481 Ramalhete C, Lopes D, Mulhovo S, Molnar J, Rosario VE, Ferreira MJ (2010) New antimalarials with a triterpenic scaffold from Momordica balsamina. Bioorg Med Chem 18(14):5254–5260 Rasoanaivo P, Wright CW, Willcox ML, Gilbert B (2011) Whole plant extracts versus single compounds for the treatment of malaria: synergy and positive interactions. Malar J 10(Suppl 1):S4 Rts SCTP (2015) Efficacy and safety of RTS, S/AS01 malaria vaccine with or without a booster dose in infants and children in Africa: final results of a phase 3, individually randomised, controlled trial. Lancet 386(9988):31–45 Rumalla CS, Ali Z, Weerasooriya AD, Smillie TJ, Khan IA (2010) Two new triterpene glycosides from Centella asiatica. Planta Med 76(10):1018–1021 Sa MS, de Menezes MN, Krettli AU, Ribeiro IM, Tomassini TC, dos Ribeiro SR, de Azevedo WFJ, Soares MB (2011) Antimalarial activity of physalins B, D, F, and G. J Nat Prod 74(10):2269–2272 Schmidt TJ, Kaiser M, Brun R (2011) Complete structural assignment of serratol, a cembrane-type diterpene from Boswellia serrata, and evaluation of its antiprotozoal activity. Planta Med 77 (8):849–850 Sibley CH (2015) Infectious diseases. Understanding artemisinin resistance. Science 347 (6220):373–374 Silva GN, Rezende LC, Emery FS, Gosmann G, Gnoatto SC (2015) natural and semi synthetic antimalarial compounds: emphasis on the terpene class. Mini Rev Med Chem 15(10):809–836 Singh C, Chaudhary S, Puri SK (2006) New orally active derivatives of artemisinin with high efficacy against multidrug-resistant malaria in mice. J Med Chem 49(24):7227–7233 Slusarczyk S, Zimmermann S, Kaiser M, Matkowski A, Hamburger M, Adams M (2011) Antiplasmodial and antitrypanosomal activity of tanshinone-type diterpenoids from Salvia miltiorrhiza. Planta Med 77(14):1594–1596

268

C. Beaufay et al.

Sulsen V, Gutierrez YD, Laurella L, Anesini C, Gimenez TA, Martino V, Muschietti L (2011) In vitro antiplasmodial activity of sesquiterpene lactones from Ambrosia tenuifolia. Evid Based Complement Alternat Med 2011:352938 Talontsi FM, Lamshoft M, Bauer JO, Razakarivony AA, Andriamihaja B, Strohmann C, Spiteller M (2013) Antibacterial and antiplasmodial constituents of Beilschmiedia cryptocaryoides. J Nat Prod 76(1):97–102 Tantangmo F, Lenta BN, Boyom FF, Ngouela S, Kaiser M, Tsamo E, Weniger B, Rosenthal PJ, Vonthron-Senecheau C (2010) Antiprotozoal activities of some constituents of Markhamia tomentosa (Bignoniaceae). Ann Trop Med Parasitol 104(5):391–398 Thakkar M, S B (2016) Combating malaria with nanotechnology-based targeted and combinatorial drug delivery strategies. Drug Deliv Transl Res 6(4):414–425 Toyang NJ, Krause MA, Fairhurst RM, Tane P, Bryant J, Verpoorte R (2013a) Antiplasmodial activity of sesquiterpene lactones and a sucrose ester from Vernonia guineensis Benth. (Asteraceae). J Ethnopharmacol 147(3):618–621 Toyang NJ, Wabo HK, Ateh EN, Davis H, Tane P, Sondengam LB, Bryant J, Verpoorte R (2013b) Cytotoxic sesquiterpene lactones from the leaves of Vernonia guineensis Benth. (Asteraceae). J Ethnopharmacol 146(2):552–556 Tsamo TA, Mkounga P, Njayou FN, Manautou J, Kirk M, Hultin PG, Nkengfack AE (2013) Rubescins A, B and C: new havanensin type limonoids from root bark of Trichilia rubescens (Meliaceae). Chem Pharm Bull (Tokyo) 61(11):1178–1183 White MT, Verity R, Griffin JT, Asante KP, Owusu-Agyei S, Greenwood B, Drakeley C, Gesase S, Lusingu J, Ansong D, Adjei S, Agbenyega T, Ogutu B, Otieno L, Otieno W, Agnandji ST, Lell B, Kremsner P, Hoffman I, Martinson F, Kamthunzu P, Tinto H, Valea I, Sorgho H, Oneko M, Otieno K, Hamel MJ, Salim N, Mtoro A, Abdulla S, Aide P, Sacarlal J, Aponte JJ, Njuguna P, Marsh K, Bejon P, Riley EM, Ghani AC (2015) Immunogenicity of the RTS, S/AS01 malaria vaccine and implications for duration of vaccine efficacy: secondary analysis of data from a phase 3 randomised controlled trial. Lancet Infect Dis 15(12):1450– 1458 World Health Organisation (WHO) (2015) World Malaria Report. http://www.who.int/malaria/ publications/world-malaria-report-2015/report/en/ Wube AA, Bucar F, Gibbons S, Asres K, Rattray L, Croft SL (2010) Antiprotozoal activity of drimane and coloratane sesquiterpenes towards Trypanosoma brucei rhodesiense and Plasmodium falciparum in vitro. Phytother Res 24(10):1468–1472 Zhai X, Wang Q, Li M (2016) Tu Youyou’s Nobel Prize and the academic evaluation system in China. Lancet 387(10029):1722 Zhang L, Fourches D, Sedykh A, Zhu H, Golbraikh A, Ekins S, Clark J, Connelly MC, Sigal M, Hodges D, Guiguemde A, Guy RK, Tropsha A (2013) Discovery of novel antimalarial compounds enabled by QSAR-based virtual screening. J Chem Inf Model 53(2):475–492 Zimmermann S, Kaiser M, Brun R, Hamburger M, Adams M (2012) Cynaropicrin: the first plant natural product with in vivo activity against Trypanosoma brucei. Planta Med 78(6):553–556 Zofou D, Kengne AB, Tene M, Ngemenya MN, Tane P, Titanji VP (2011) In vitro antiplasmodial activity and cytotoxicity of crude extracts and compounds from the stem bark of Kigelia africana (Lam.) Benth (Bignoniaceae). Parasitol Res 108(6):1383–1390

Part IV

Antiviral Natural Products (Human Health)

Chapter 11

Antimicrobial Capacities of the Medicinal Halophyte Plants Faten Medini and Riadh Ksouri

Abstract Halophytes grow in many regions of the world where they are exposed and survive to harsh environmental conditions. As a consequence, these species developed adaptive responses including the synthesis of several bioactive molecules that made them plants of significant economic potential as well as a potential source of newly discovered medicine. Moreover, several salt marsh plants have traditionally been used for medical, nutritional, and even artisanal purposes. Currently, an increasing interest is granted to these species because of their high content in bioactive compounds (primary and secondary metabolites) such as polyunsaturated fatty acids, carotenoids, vitamins, sterols, essential oils (terpenes), polysaccharides, glycosides, and phenolic compounds. This chapter reviews available literature about the status of a wide variety of halophytic species traditionally used as medicinal virtue. It aims to highlight the importance of halophytes as a potential source of antimicrobial and antiviral agents and indicate their prospective utilization at industrial scale. Keywords Halophytes Antifungal ability

11.1


Antiviral activity
Antibacterial capacity

Introduction

Salty areas, which accounts for 9.5 billion hectares of the world’s soil, are convenient habitats for a number of shrubby plants and trees (Jones et al. 2012). Halophytic vegetation includes all these classes and grows in different saline biotopes. They are found on sand dunes or rocky coasts, in saline depressions (sabkhas), in saline inland deserts, and in salt marshes. There are more than 2500 halophyte species known worldwide to possess some salinity tolerance, and among F. Medini
R. Ksouri (&) Laboratoire des Plantes Aromatiques et Médicinales, Center of Biotechnology of Borj Cedria, BP 901, 2050 Hammam-Lif, Tunisia e-mail: [email protected] © Springer International Publishing AG 2018 J.-M. Mérillon and C. Rivière (eds.), Natural Antimicrobial Agents, Sustainable Development and Biodiversity 19, https://doi.org/10.1007/978-3-319-67045-4_11

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them, several could be suitable candidates to be used as cash crops (edible plants, fodder, fuel, medicine, chemicals, and ornamentals) (Abdelly et al. 2006). Since the halophyte plants grow in environmental constraint conditions, they regulate a number of novel biosynthesis pathways to synthesize different secondary metabolites that help them sustain themselves in the fragile ecosystem. In plants, unfavorable environmental conditions such as salt constraint increase production and accumulation of reactive oxygen species (ROS), leading to cellular damage, metabolic disorders, and senescence processes (Menezes-Benavente et al. 2004). Halophytes are known for their ability to withstand and quench these toxic ROS, since they are equipped with a powerful antioxidant system (Jaleel et al. 2009; Ksouri et al. 2008, 2009). Interestingly, the natural antioxidants contained in halophytes exhibit a strong biological activity, sometimes exceeding many natural antioxidants from medicinal glycophytes or synthetic antioxidants that are being restricted due to their potential carcinogenicity (Suhaj 2006). Many natural antioxidants have already been isolated from different kinds of plants; among them, phenolic compounds are in the forefront as they are largely distributed in the plant kingdom. Actually, these compounds have been suggested to be associated with several health-promoting activities such as anticancer, antibacterial, antifungal, antiviral activities (Ksouri et al. 2009). A review of the halophyte literature reveals a broad spectrum of research activity where traditional medicinal practices are used to treat various diseases, microbial infections and aging processes, particularly in the rural areas, where folk medicine remains a major source to cure minor ailments (Ksouri et al. 2012). In this later review, authors detail the folkloric medicinal uses and they report recent insights into the biological activities of extracts, and nutraceuticals identified from Tunisian halophytes. Natural products, such as plant extracts, standardized extracts or pure compounds, provide numerous opportunities for new drug discoveries because of the unmatched availability of chemical diversity (Bhuwan et al. 2011). According to the World Health Organization (WHO), more than 80% of the world’s population rely on traditional medicine for their primary healthcare needs (Hassan et al. 2009). For these reasons, phytochemicals are gaining importance as a potential source for antiviral agents. In fact, prolonged therapy with the available antiviral drugs has resulted in some undesirable effects and has induced the emergence of drug-resistant virus strains (Roizman et al. 2007). Therefore, the development of new antiviral agents and complementary therapy with currently available drugs are still needed. When one considers that a single plant may contain up to thousands of constituents, the possibilities of making new discoveries become evident. The crucial factor for the ultimate success of an investigation into bioactive plant constituents is thus the selection of plant material and the proper extraction and purification process of the active compounds (Hostettmann et al. 1995). Since the plants are considered as the most potent source of novel compounds, halophyte plants are thus studied to discover some novel antimicrobial molecules of

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pharmaceutical importance. That is, in this chapter, we are reporting some previous and recent studies that highlighted the efficiency of many halophyte species as antiviral and antimicrobial agents.

11.2

Experimental Approach

The search for antimicrobial agents from plants has been a growing interest in the last few decades. Many papers on the antimicrobial efficacy of plant essential oils and extracts have been dedicated to research methods (Eloff 1998; Lahlou 2004; Rios and Recio 2005; Camargo et al. 2008; Rechter et al. 2006). However, there still remains a number of disparities in the techniques, where there is a need to explore standardization. As microbiological methods incorporate viable test micro-organisms. Antimicrobial test systems should ideally be simple, rapid, reproducible, inexpensive, and maximize sample throughput in order to cope with a varied number of extracts and fractions. There are several in vitro and in vivo methods reported in the current literature to study the antiviral activities of plant/herbal extracts or plant-derived molecules. Most commonly, researchers are using the cytopathic effect (CPE) on virus-infected for preliminary studies and/or screening of large numbers of molecules/extracts. This activity was characterized by evaluating their effect on the pretreatment, the virucidal activity and the effect on the adsorption or post-adsorption period of the viral cycle (Camargo et al. 2008; Rechter et al. 2006). For the antibacterial activity, the CLSI (NCCLS 2003) has standardized the agar dilution method for quantitative determination of antibiotics. Broth dilution methods for inhibitory determination are also recommended, using different principles to assess microbial growth or its inhibition. The two most common methods used to investigate the antimicrobial activity of the halophyte plants are disc diffusion and minimum inhibitory concentration (MIC) assays. Other antimicrobial methods have been used to assess activity; these include broth-based turbidometric assay.

11.3

Antimicrobial Activities of Halophyte Species (Table 11.1)

11.3.1 Antiviral Activity Unlike microbial cells, which are free-living entities, viruses are obligate intracellular parasites, which contain little more than bundles of gene strands of either RNA or DNA, and may be surrounded by a lipid-containing envelope. Every strain of virus has its own unique arrangement of surface molecules, which helps in its attachment to host cells. Following attachment, viruses utilize the host cell they

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invade to propagate new viruses. Hence, their spread in nature have been attributed to differences in their genetic compositions, means of transmission, efficient replication within host cells, and their ability to persist in the host and thus cause various diseases in humans, animals, and plants alike (Bagla et al. 2012). The low efficiency of the existing treatment coupled with the development of resistant mutants as well as the potential toxic effect of presently available therapeutics arouse the need for finding new antiviral drugs. Natural products remain an important source of biologically active substances, especially for the treatment of infectious diseases. Approximately 44% of the antiviral drugs approved between 1981 and 2006 were natural products, semi-synthetic natural product analogues or synthetic compounds based on natural-product pharmacophores (Newman and Cragg 2007). According to the literature, a great number of halophytes are traditionally used to heal viral infections particularly in the rustic areas, where the major source to treat diseases remains the traditional medicine (Ksouri et al. 2012). For instance, Plantago major L. is a perennial plant that belongs to the Plantagiaceae family. This is a commonly used medicinal herb in Taiwan, which has been utilized in the treatment of a number of diseases related to the skin, respiratory organs, digestive organs, reproduction, circulation, cancer prevention, and against infections (McCutcheon et al. 1995). It has also been used as a remedy for colds and viral hepatitis (Ksouri et al. 2012). This plant has shown to contain five classes of biologically active compounds, namely benzoic compounds (vanillic acid), flavonoids (baicalein, baicalin, luteolin), iridoid glycoside (aucubin), phenolic compounds (caffeic, chlorogenic, ferulic, and p-coumaric acids), and triterpenes (oleanolic and ursolic acids) (Samuelsen 2000). Its ferulic and caffeic acids have been demonstrated to possess in vitro activity against herpes simplex virus 2 (HSV-2) (Bourne et al. 1999). This traditional herbal remedy has been used for treating colds, conjunctivitis, and hepatitis for hundreds of years in Taiwan. The aqueous extract of P. major was found to possess a slight activity against herpes virus (HSV-2). In contrast, certain pure compounds belonging to the five different classes of chemicals found in extracts of this plant exhibited potent antiviral activity. Among them, caffeic acid exhibited the strongest activity against HSV-1 (EC50 = 15.3 µg/mL), HSV-2 (EC50 = 87.3 µg/mL), and ADV-3 (EC50 = 14.2 µg/mL), whereas chlorogenic acid possessed the strongest anti-ADV-11 with EC50 of 13.3 µg/mL (Chiang et al. 2002). The low antiviral activity of the aqueous extract of P. major may be explained by the low concentrations of these phenolic compounds present in the extract. An extensive long-term study of the species of Limonium has showed that their derivatives demonstrate wide therapeutic effects (Zhusupova et al. 2002). There are over than 300 species of Limonium Mill. Phytopreparations obtained on the basis of Limonium Mill possess antiviral, hepatoprotector, anti-inflammatory, antiburn properties (Zhusupova et al. 2002). For example, Limonium sinense has been used to treat hemorrhage and improve blood circulation (Chiu and Chang 1992). The phytochemical profile of the ethanolic extract of L. sinense consists of a mixture of flavonols, flavonol glycosides, flavonol glycoside gallates, flavones, flavanones, and flavan-3-ols (Lin and Chou 2000; Lin et al. 2000). Bioactivity analyses indicate that epigallocatechin 3-

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O-gallate and samarangenin B isolated from the plant’s roots can inhibit herpes simplex virus type-1 infection (Lin et al. 2000). In addition, the study of Hsu et al. (2015) indicated that the water extract from the underground part of this specie exhibited potent inhibitory activity against HCV at non-cytotoxic concentrations. It targeted early HCV infection without affecting viral replication, translation, and cell-to-cell transmission and blocked viral attachment and post-attachment entry/ fusion steps. Bioactivity analysis of major constituents from the extract through viral infectivity/entry assays revealed that gallic acid also inhibits HCV entry. Study conducted by Medini et al. (2014a, b) on L. densiflorum extracts reported a good antiviral activity of ethanol and methanol extracts. They were most potent in HSV-1 inhibition than H1N1 influenza virus. The most potent inhibition was observed with ethanol extract which exhibited high levels of virucidal activity against HSV-1 with an IC50 of 6 µg/mL. Also, it inhibits the replication of the virus by 75% when added after penetration of the virus, and by 100% when added during the viral attachment. This extract showed a good protection of MDCK cells against influenza. Methanol extract showed a moderate antiviral capacity against both viruses. While dichloromethane has excellent antiherpes potential, results were inappropriate because it was toxic to Vero cells. An in vitro bio-guided fractionation of this extract was undertaken by preparative chromatographic techniques. On the basis of nuclear magnetic resonance techniques, the structures of the isolated compounds were determined as gallic acid, epigallocatechin gallate, quercitrin, dihydrokaempferol, pinoresinol, N-trans-ferulolyl tyramine, and myricetin 3-O-a-rhamnopyranoside and myricetin 3-O-L-arabinofuranoside (Medini et al. 2016). The isolated molecules were evaluated for their virucidal against HSV-1. Results showed that gallic acid and epigallocatechin gallate have a strong effect, while pinoresinol and N-trans-ferulolyl tyramine have a moderate activity. However, the other molecules were inactive (Medini et al. 2016). Premanathan et al. (1999) studied the antiviral properties of Mangrove halophytes against RNA viruses (Newcastle disease virus, encephalomyocarditis virus, Semliki forest virus, and human immunodeficiency virus) and DNA viruses (vaccine virus and hepatitis B virus) and concluded that plants belonging to the family Rhizophoraceae, in particular Bruguiera cylindrica and Rhizophora mucronata, presented the highest potential against all the tested viruses and, therefore, represented a source of antiviral substances. In addition, the study of Banerjee et al. (2012) was carried out to find out the efficiency of mangrove leaf extracts against the Hepatitis B virus and results suggested the powerful activity of Suaeda maritima which might be due to the presence of chemical constituents such as phenolic acids, flavonoids, and tannins. Indeed, leaves from S. maritima are traditionally known for their use as a medicine for hepatitis (Bandaranayake 2002) and its antiviral and hepatoprotective properties were scientifically reported (Ravikumar et al. 2011; Padmakumar and Ayyakkannu 1997) with the presence of triterpenoids, sterols (Subramanyam et al. 1992). The inhibitory effect of the S. maritima leaf extract can be directly used as a blocking agent of the viral surface antigen or inhibitory agent of viral polymerase enzymes involved in the DNA replication (Mohan et al. 2011). Viruses can also cause several infections in animals. In this context, Bagla et al. (2012) described the antiviral

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activity of halophytes against animal-infecting viruses such as feline herpes virus (FHV-1), canine distemper virus (CDV), and lumpy skin disease virus (LSDV). Authors depicted a strong inhibition of the CDV and LSDV with acetone and methanol extracts of the halophyte Podocarpus henkelii (Podocarpaceae) which inhibited the replication of the viruses by 75% at 3 mg/ml. Moreover, excellent capacity was also found with the hexane extracts of Plumbago zeylanica (Plumbaginaceae) and Carissa edulis (Apocynaceae) against CDV. In fact, these extracts reduced viral-induced structural changes in the host cells by 50 and 75%, respectively. Bagla et al. (2012) showed a traditional use of the species Podocarpus latifolius and Podocarpus falcatus in the treatment of canine distemper infection in dogs and thus confirm the high antiviral activity of Podocarpus ssp suggesting the presence of antiviral compounds in plant of this genus and their potential source for new antiviral molecules. Usually, viral infections are accompanied with profound changes in cell/tissue metabolism, which leads to generation of reactive oxygen species. The latter may enhance the pathogenesis of the infection. For example, it was found that the main cause of mortality from influenza virus-induced pneumonia is cytotoxicity, which is determined by a considerably increased level of superoxide before viral replication in the bronchial epithelial cells occurs (Akaike et al. 1996). Several studies showed the powerful antioxidant capacity of halophytes. In the light of this result, we can conclude that these plants not only have an inhibitory effect against viruses, but also can be of great value in preventing the inception or the progression of virus infection. Falleh et al. (2011) reported the first study on the identification of the most active phenolics in the halophyte Mesembryanthemum edule by LC/ESI-MS/ MS after fractionation. The results obtained in this study demonstrate that this edible halophyte is a valuable source of active polyphenolics, mainly procyanidins and propelargonidins which were very potent for in vitro DPPH and ABTS free radical scavenging and b-carotene bleaching inhibition. These polyphenol-type compounds with several-OH groups show antibacterial and antiviral activities, cardioprotective, and anticancer ability (Okuda 2005).

11.3.2 Antibacterial and Antifungal Capacities The diversity of plants offers many possibility of finding novel structures with antibacterial and antifungal properties. A great number of halophytic medicinal plants are, according to ethno-medical uses, able to treat microbial infections (Kouri et al. 2012), and many species were proven to have an interesting antimicrobial activity in vitro. The majority of studies dedicated to the antimicrobial activity of the halophyte plants focus essentially on plant extracts. Screening publications are initially the first choice of investigation with the intention to identify plants with potential antimicrobial activity for further study. A summary of several screening studies from halophyte plants is presented in Table 11.1. The antimicrobial studies have been undertaken on specific species such as Limonium densiflorum (Medini

Limonium delicatilum

Shoot

Antifungal

Antibacterial

Antiviral

Antifungal

Antibacterial

E. coli S. aureus S. thyphi P.aureginosa L. monocytogenes C. krusei C. parapsilosis C. glabatra C. albicans HSV-1 Influenza E. coli S. aureus S. thyphi P.aureginosa L. monocytogenes C. krusei C. parapsilosis C. glabatra C. albicans

Methanilc Acetonic Ethanolc

50 mg/mL

100 µg/mL

50 mg/mL

50 mg/mL

(continued)

Medini et al. (2014a, b)

Medini et al. (2011)

Référence

Limonium densiflorum

Range of crude extract

Plant part

Species

Extract

Table 11.1 Antibacterial, antifungal, and antiviral activities of some halophyte plants Microbes

Antimicrobial Capacities of the Medicinal Halophyte Plants

Activity

11 277

Shoot

Shoot

Crithmum maritimum

Crithmum maritimum

S. epidermidis M. luteus E. coli P. aeruginosa B. cereus B. thuringiensis L. monocytogenes B. subtilis E. faecalis Klebsiella sp S. arizonae C. albicans C. glabrata C. kefyr C. holmii C. sake S. aureus E. coli P. aeruginosa B. cereus S. aureus E. coli P. aeruginosa

Flowers

Antibacterial

Antibacterial

Antifungal

S. aureus

Leaves

Microbes

Tamarix gallica

Activity

Plant part

Species

Table 11.1 (continued)

Chloroform Water

Acetonic

Methanolic

Methanolic

Extract

100 mg/mL

(continued)

Meot-Duros et al. (2009)

Jalleli et al. (2014)

Boulaaba et al. (2015)

100–500 µg/mL

100 mg/mL

Référence

Range of crude extract

278 F. Medini and R. Ksouri

Shoot

Shoot

Inula crithmoïdes

Retama raetam

Cakile maritima

Eryngium maritimum

Antibacterial

Antifungal Antibacterial

S. epidermidis L. monocytogenes E. Faecalis M. luteus E. coli S. aureus S. typhimurium V. vulnificus V. alginolyticus V. cholerae V.parahaemolyticus

B. cereus L. monocytogenes M. Luteus S. arizonae S. montevideo E. carotovora P. marginalis P. fluorescens C. albicans S. aureus E. coli P. aeruginosa B. cereus B. cereus A. hydrophila Hexane 70% MeOH/ H2O Ethyl-acetate Water

Acetonic

Extract

100 mg/mL

100 mg/mL

Range of crude extract

Saada et al. (2014)

Jalleli et al. (2014)

Référence

(continued)

Species

Microbes

Table 11.1 (continued) Activity

Antimicrobial Capacities of the Medicinal Halophyte Plants

Plant part

11 279

Plant part

Leaves

Shoot

Species

Limoniastrum monopetalum

Artemisia campestris

Table 11.1 (continued)

Antifungal

Antibacterial

Antifungal

Activity

S. epidermidis P. aeruginosa M. luteus C. albicans C. glabrata C. kefyr C. holmii C. sake B. thuringiensis S. aureus L. monocytogenes E. coli S. typhimurium A. hydrophila V. vulnificus V. alginolyticus V. cholerae V.parahaemolyticus C. albicans C. glabrata C. kefyr C. holmii C. sake

S. aureus

Microbes

Methanolic

70% MeOH/ H2O

Extract

50–300 mg/mL

1 mg/mL

Range of crude extract

(continued)

Megdiche et al. (2015)

Trabelsi et al. (2009)

Référence

280 F. Medini and R. Ksouri

Plant

Plant

Leaves

Leaf Stem Root Seed

Salicornia herbacea

Acanthus ilicifolius

Hippophaë rhamnoides

Antibacterial

Antibacterial

Antibacterial

Antifungal

Ethyl-acetate Methanolic

B. cereus E. coli P. aeruginosa C. albicans C. neoformans S. aureus streptococcus S. enteritidis P. aeruginosa E. coli K. pneumoniae S. epidermidis P. aeruginosa S. pyogenes L. plantarum C. albicans T. rubrum P. aeruginosa E. coli B. cereus E. durans S. aureus C. albicans Ethanol

Chloroform Acetone Ethanol Methanol Water

60% MeOH/ H2O

Hexane

S. aureus

Michel et al. (2012)

100 µg/mL

(continued)

Govindasamy and Arulpriya (2013)

Essaidi et al. (2013)

Saad et al. (2011)

100 mg/mL

20–100 mg/mL

100 mg/mL

Référence

Lumnitzera littorea

Range of crude extract

Species

Extract

Plant part

Table 11.1 (continued) Microbes

Antimicrobial Capacities of the Medicinal Halophyte Plants

Activity

11 281

Plant part

Fractions of stem leaf root

Fractions of areal parts

Shoot

Shoot Shoot Leaf Shoot

Shoot Shoot

Species

Mesembryanthemum edule

Limoniastrum guyonianum

Diplotaxis harra

Plantago major Limonium sinense Suaeda maritima Podocarpus henkelii

Plumbago zeylanica Carissa edulis

Table 11.1 (continued)

Antiviral Antiviral

Antiviral Antiviral Antiviral Antiviral

Antibacterial

Antibacterial

Antibacterial

Activity P. aeruginosa B. cereus S. aureus E. coli M. luteus S. typhimurium E. faecum S. aureus E. coli E. feacalis P. aeruginosa P. aeruginosa E. coli M. luteus HSV-2 HCV HCV CDV LSDV CDV CDV

Microbes

Water Water Water Methanolic Acetonic Hexane Hexane

Methanol

70% MeOH/ H2O

60% MeOH/ H2O

Extract

mg/mL mg/mL mg/mL mg/mL 3 mg/mL 3 mg/mL

1 1 1 3

1 mg/mL

1 mg/mL

1 mg/mL

Range of crude extract

Balga et al. (2012) Balga et al. (2012)

Chiang et al. (2002) Hsu et al. (2015) Ravikumar et al. (2011) Balga et al. (2012)

Falleh et al. (2013a, b)

Trabelsi et al. (2013)

Falleh et al. (2013a, b)

Référence

282 F. Medini and R. Ksouri

11

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283

et al. 2011), Limonium delicatilum (Medina et al. 2014a, b), Tamarix gallica (Boulaaba et al. 2014), root, leaves, and stems of Mesembryanthimum edule (Falleh et al. 2013a, b), Hippophaë rhamnoides (Michel et al. 2012), Crithmum maritimum (Jalleli et al. 2014; Meot-Duros et al. 2009), and Salicornia herbacea (Essaidi et al. 2013) where the majority of activities ranged from 1 to 500 mg/mL. Seasonal variation studies on halophyte extracts demonstrated little antimicrobial variability between the periods. In this context, Medini et al. (2011) and Trablesi et al. (2009) showed that plants collected in summer (flowering stage) were more potent that those collected in winter (vegetative stage). In addition, extracts having activities where MIC values are below 8 mg/ml (Fabry et al. 1998) are considered to possess some antimicrobial activity, and natural products with MIC values below 1 mg/ml are considered noteworthy (Gibbons 2004; Rios and Recio 2005). The majority of publications (Table 11.1), reporting antimicrobial activities on halophyte plant extracts below 1 mg/ml include (Boulaaba et al. 2015; Falleh et al. 2013a, b; Trabelsi et al. 2013, Michel et al. 2012) showing that halophyte plants can be a good source for antimicrobial agents. In the study of Meot-Duros et al. (2008), apolar fractions of Eryngium maritimum were more active than polar ones. Eryngium maritimum presented a strong antibacterial activity against two of the three Pseudomonas species tested (Pseudomonas aeruginosa and Pseudomonas fluorescens). Falleh et al. (2008) showed that various C. cardunculus organs (leaves and seeds) exhibited an interesting power against several human pathogenic bacteria, possibly due to their specific phenolic composition (principally syringic and transcinnamic acids, as major phenolic acids, in addition to epicatechin and quercetrin as major flavonoids). Furthermore, Tamarix extracts showed appreciable antibacterial properties against human pathogen strains (Ksouri et al. 2009). The mean inhibition zone of T. gallica was from 0 to 6.5 mm when the concentration increased from 2 to 100 mg/mL. The highest activity was recorded against M. luteus, and the lowest one was observed against E. coli. Concerning antifungal tests, organ extracts showed a moderate activity against the tested Candida only at the highest extract concentration (100 mg/mL). In addition, L. sinense and L. tetragonum show antiviral activity (Yuh-Chi et al. 2002), whereas L. axillare and L. californicum show antibacterial activities (Kandil et al. 2000; Sakagami et al. 2001). A comparative study of the antibacterial action of volatile oils of Tamarix boveana flowers showed strong antibacterial activity against Staphylococcus epidermidis and Escherichia coli (500 mg/ml), attributed to the presence of a high concentration of aldehydes (22.21%) and eugenol (Burt 2004; Saenz et al. 2004; Saïdana et al. 2008). Eugenol has been found to inhibit production of amylase and proteases by the sensitive bacteria and to deteriorate cell wall and cause a high degree of cell lysis (Thoroski et al. 1989). The hydroxyl group on eugenol is thought to bind to proteins, preventing enzyme action in the bacteria (Wendakoon and Sakaguchi 1995). In other hand, camphene, present in flowers, of T. boveana seemed to ameliorate antibacterial activity. The antibacterial nature of the T. boveana essential oil studied was apparently related again to the presence of b-caryophyllene (Saïdana et al. 2008). This compound showed in vitro activity against E. coli, P. aeruginosa, and S. aureus. The essential oil of the obligate

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halophyte Sesuvium portulacastrum exhibited antibacterial activity against Acetobacter calcoacetica, Bacillus subtilis, Clostridium sporogenes, Clostridium perfringens, E. coli, Salmonella typhii, Staphylococcus aureus, and Yersinia enterocolitica. This activity against almost all the sensitive bacteria may be due to camphene found in its essential oil (Magwa et al. 2006). In addition, the oil exhibited antifungal activity against C. albicans, Aspergillus niger, A. flavus, and Penicillium notatum. Leaf antibacterial activity of Thespesia populnea (Malvaceae) is due to the known triterpene lupeol, and gossypol was the active ingredient in the flowers, which accounted for its antifertility activity (Goyal and Rani 1989).

11.4

Conclusion

Plants remain an important source to combat serious diseases in the world. The traditional medicinal methods, especially the use of medicinal plants, still play a vital role to cover the basic health needs in the developing countries. The medicinal value of halophytes plants lies in the chemical active substances they produce. In addition to their capacity to tolerate salt, halophytes represent a potent source of bioactive compounds with multiple and powerful biological activities that can be useful for industrial purposes. This fact highlights the promising medicinal and economical features of halophytes as compared to other non-halophytic species. In the foreseeable future, special focus will be on those secondary metabolites that exhibit these species with high biological activities. Safety, edibility, and in vivo efficacy studies on these plant bioactive substances must also be conducted.

References Abdelly C, Barhoumi Z, Ghnaya T, Debbez A, Ben Hamed K, Ksouri R, Talbi O, Zribi F, Ouerghi Z, Smaoui A, Huchzernmeyer B, Grignon C (2006) Potential utilisation of halophytes for the rehabilitation and valorisation of salt-affected areas in Tunisia. In: Ozturk M, Waisel Y, Khan MA, Gork G (eds) Biosaline agriculture and salinity tolerance in Plants, Tome I, pp 161–170 Akaike T, Noguchi Y, Ijiri S, Setoguch K, Suga M, Zeng Y, Dietzschold B, Maeda H (1996) Pathogenesis of influenza virus-induced pneumonia: involvement of both nitric oxide and oxygen radicals. Proc Natl Acad Sci USA 93:2448–2453 Bagla VP, McGaw LJ, Eloff JN (2012) The antiviral activity of six South African plants traditionally used against infections in ethnoveterinary medicine. Vet Microbiol 155:198–206 Bandaranayake WM (2002) Bioactivities, bioactive compounds and chemical of mangrove plants. Wet Ecol Man 10:421–422 Banerjee MB, Ravikumar S, Gnanadesigan M, Rajakumar B, Anand M (2012) Antiviral, antioxidant and toxicological evaluation of mangrove associate from South East coast of India. Asian Pac J Trop Biomed:S1775–S1779

11

Antimicrobial Capacities of the Medicinal Halophyte Plants

285

Bhuwan B, Mishra-Vinod K, Tiwari J (2011) Natural products: an evolving role in future drug discovery. Eur J Med Chem 46:4769–4807 Boulaaba M, Snoussi M, Saada M, Mkadmini K, Smaoui A, Abdelly C, Ksouri R (2014) Antimicrobial activities and phytochemical analysis of Tamarix gallica extracts. Ind Crops Prod 76:1114–1122 Boulaabaa M, Snoussi M, Saadaa M, Mkadmini KH, Ab S, Abdelly CH, Ksouri R (2015) Antimicrobial activities and phytochemical analysis of Tamarix gallica extracts. Ind Crops Prod 76:1114–1122 Bourne KZ, Bourne N, Reising SF, Stanberry LR (1999) Plant products as topical microbicide candidates: assessment of in vitro and in vivo activity against herpes simplex virus type 2. Antivir Res 42:219–226 Burt S (2004) Essential oils: their antibacterial properties and potential applications in foods—a review. Int J Food Microbiol 94:223–253 Camargo F, Cortezb DAG, Ueda-Nakamurac T, Nakamurac CV, Dias Filho BP (2008) Antiviral activity and mode of action of a peptide isolated from Sorghum bicolor. Phytomedicine 15 (3):202–208 Chiang LC, Chiang W, Chang MY, Ng LT, Lin CC (2002) Antiviral activity of Plantago major extracts and related compounds in vitro. Antivir Res 55:53–62 Chiu NY, Chang KH (1992) The illustrated medicinal plants of Taiwan. SMC Publishing Inc., Taipei, Taiwan CLSI (NCCLS) (2003) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically: approved standard 23. National Committee for Clinical Laboratory Standards, Wayne, PA, USA Eloff JN (1998) A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Med 64:711–713 Essaidi I, Brahmi Z, Snoussi A, Ben Haj Koubaier A, Casabianca H, Abe N, El Omri A, Moncef Chaabouni M, Bouzouita N (2013) Phytochemical investigation of Tunisian Salicornia herbacea L., antioxidant, antimicrobial and cytochrome P450 (CYPs) inhibitory activities of its methanol extract. Food Control 32:125–133 Fabry W, Okemo PO, Ansorg R (1998) Antibacterial activity of East African medicinal plants. J Ethnopharmacol 60:79–84 Falleh H, Ksouri R, Chaieb K, Karray-Bouraoui N, Trabelsi N, Boulaaba M, Abdelly C (2008) Phenolic composition of Cynara cardunculus L. organs, and their biological activities. C R Biol 331:372–379 Falleh H, Msilini N, Oueslati S, Ksouri R, Magne CH, Lachaal M (2013a) Diplotaxis harra and Diplotaxis simplex organs: assessment of phenolics and biological activities before and after fractionation. Ind Crops Prod 45:141–147 Falleh H, Oueslati S, Guyot S, Ben Dali A, Magné C, Abdelly C, Ksouri R (2011) LC/ESI-MS/MS characterisation of procyanidins and propelargonidins responsible for the strong antioxidant activity of the edible halophyte Mesembryanthemum edule L. Food Chem 127:1732–1738 Falleh H, Trabelsi N, Bonenfant-Magné G, Le Flochc G, Abdelly CH, Magnéb CH, Ksouri R (2013b) Polyphenol content and biological activities of Mesembryanthemum edule organs after fractionation. Ind Crops Prod 42:145–152 Gibbons S (2004) Anti-staphylococcal plant natural products. Natural Prod Rep 21:263–277 Govindasamy CH, Arulpriya M (2013) Antimicrobial activity of Acanthus ilicifolius: Skin infection pathogens. Asian Pac J Trop Dis 3(3):180–183 Goyal MM, Rani KK (1989) Antibacterial activity of the natural products from the leaves of Thespesia populnea. Acta Cienc Indica Chem 15:117–124 Hassan A, Rahman S, Deeba F, Mahmud S (2009) Antimicrobial activity of some plant extracts having hepatoprotective effects. J Med Plants Res 3(1):020–023 Hostettmann K, Marston A, Wolfender JL (1995) In phytochemistry of plants used in traditional medicine. Clarendon Press, Oxford, pp 17–45

286

F. Medini and R. Ksouri

Hsu WS, Chang SP, Lin LS, Li CHL, Richardson CH, Lin CH, Lin LT (2015) Limonium sinense and gallic acid suppress hepatitis C virus infection by blocking early viral entry. Antivir Res 118(2015):139–147 Jaleel CA, Ksouri R, Gopi P, Manivannan J, Ines H, Al-Juburi Z, Chang- Xing S, Hong-Bo PR (2009) Antioxidant defense responses: physiological plasticity in higher plants under abiotic constraints. Acta Physiol Plant 31:427–436 Jallali I, Zaouali Y, Missaoui I, Smeoui A, Abdelly CH, Ksouri R (2014) Variability of antioxidant and antibacterial effects of essential oils and acetonic extracts of two edible halophytes: Crithmumn maritimum L. and Inula crithmoïdes L. Food Chem 145(2014):1031–1038 Jones A, Panagos P, Barcelo S, Bouraoui F, Bosco C, Dewitte O, Gardi C, Erhard M, Hervas J, Heiderer R, Jef-fery S, Lukewille A, Marmo L, Montanarella L, Olazabal C, Petersen JE, Penizek V, Strassburger T, Toth G, Van Den Eeck-aut M, Van Liedekerke M, Verheigen F, Viestova E, Yigini Y (2012) The state of soil in Europe, Copenhagen, European Environment Agency. http://ec.europa.eu/dgs/jrc/downloads/jrcreference report 2012 02 soil.pdf Kandil FE, Ahmed KM, Hussieny HA, Soliman AM (2000) A new flavonoid from Limonium axillare. Archiv der pharmazie. J Pharm Med Chem 333:275–277 Ksouri R, Falleh H, Megdiche W, Trabelsi N, Baya M, Chaieb K, Bakrouf A, Magné M, Abdelly CH (2009) Antioxidant and antimicrobial activities of the edible medicinal halophyte Tamarix gallica L. and related polyphenolic constituents. Food Chem Toxicol 47:2083–2091 Ksouri R, Ksouri WM, Jallali I, Debez A, Magné CH, Hiroko I, Abdelly CH (2012) Medicinal halophytes: potent source of health promoting biomolecules with medical, nutraceutical and food applications. Crit Rev Biotechnol:1–38 Ksouri R, Megdiche W, Falleh H, Trabelsi N, Boulaaba M, Smaoui A, Abdelly C (2008) Influence of biological, environmental and technical factors on phenolic content and antioxidant activities of Tunisian halophytes. Compte Rendu Biol 331:865–873 Lahlou M (2004) Methods to study the phytochemistry and bioactivity of essential oils. Phytother Res 18:435–448 Lin LC, Chou CJ (2000) Flavonoids and phenolics from Limonium sinense. Planta Med 66:382–383 Lin LC, Kuo YC, Chou CJ (2000) Anti-herpes simplex virus type-1 flavonoids and a new flavanone from the root of Limonium sinense. Planta Med 66:333–336 Magwa ML, Gundidza M, Gweru N, Humphrey G (2006) Chemical composition and biological activities of essential oil from the leaves of Sesuvium portulacastrum. J Ethnopharmacol 103:85–89 McCutcheon AR, Roberts TE, Gibbons E, Ellis SM, Babiuk LA, Hancock REW, Tower GHN (1995) Antiviral screening of British Colombian medicinal plants. J Ethnopharmacol 49:101–110 Medini F, Fellah H, Ksouri R, Abdelly CH (2014a) Total phenolic, flavonoid and tannin contents and antioxidant and antimicrobial activities of organic extracts of shoots of the plant Limonium delicatulum. J Taibah Univ Sci 8:216–224 Medini F, Ksouri R, Falleh H, Megdiche W, Trabelsi N, Abdelly CH (2011) Effects of physiological stage and solvent on polyphenol composition, antioxidant and antimicrobial activities of Limonium densiflorum. J Med Plants Res 5(31):6719–6730 Medini F, Megdiche W, Mshvildadze V, Pichette A, Legault J, St-Gelais A, Ksouri R (2016) Antiviral-guided fractionation and isolation of phenolic compounds from Limonium densiflorum hydroalcoholic extract. Compte Rendu Chimie Medini M, Legault J, Pichette A, Abdelly CH, Ksouri R (2014b) Antiviral efficacy of Limonium densiflorum against HSV-1 and influenza viruses. S Afr J Bot 92:65–72 Megdiche-Ksouri W, Trabelsia N, Mkadmini KH, Bourgoua S, Noumi A, Snoussi M, Barbria R, Tebourbi O, Ksouri R (2015) Artemisia campestris phenolic compounds have antioxidant andantimicrobial activity. Ind Crops Prod 63:104–113 Menezes-Benavente L, Karam Teixeira F, Alvim Kamei CL, Margis- Pinheiro M (2004) Salt stress induces altered expression of genes encoding antioxidant enzymes in seedlings of a Brazilian indica rice (Oryza sativa L.). Plant Sci 166:323–331 Meot-Duros L, Le Floch G, Magn´e CH (2008) Radical scavenging, antioxidant and antimicrobial activities of halophytic species. J Ethnopharmacol 116:258–262

11

Antimicrobial Capacities of the Medicinal Halophyte Plants

287

Meot-Duros L, Cérantola S, Talarmin H, Le Meur C, Le Floch G, Magné C (2009) New antibacterial and cytotoxic activities of falcarindiol isolated in Crithmum maritimum L. leaf extract. Food Chem Toxicol 48(2):553–557 Michel T, Destandau E, Le Floch G, Lucchesi ME, Elfakir C (2012) Antimicrobial, antioxidant and phytochemical investigations of sea buckthorn (Hippophaë rhamnoides L.) leaf, stem, root and seed. Food Chem 131:754–760 Mohan K, Paramasivam R, Chandran P, Veerasami V, Gnanasekaran A, Anushka S, Murugesan M (2011) Inhibition of hepatitis B virus DNA polymerase and modulation of TH1 & TH2 cytokine secretion by three Indian medicinal plants and its correlation with antiviral properties. J Pharmacol Res 4(4):1044–1046 Newman DJ, Cragg GM (2007) Natural products as sources of new drugs over the last 25 years. J Nat Prod 70(3):461–477 Okuda T (2005) Systematics and health effects of chemically distinct tannins in medicinal plants. Phytochemistry 66:2012–2031 Padmakumar K, Ayyakkannu K (1997) Antiviral activity of marine plants. Indian J Virol 13:33–36 Premanathan M, Kathiresan K, Nakashima H (1999) Mangrove halophytes: a source of antiviral substances. S Pac Study 19:1–2 Ravikumar S, Inbanesan SJ, Suganthi P, Gnanadesigan M (2011) In vitro antiplasmodial activity of ethanolic extracts of mangrove pants from South East coast of India against chloroquine-sensitive Plasmodium falciparum. Parasitol Res 108:873–878 Rechter S, Konig T, Auerochs S, Thulke S, Walter H, Dornenburg H, Walter C, Marschall M (2006) Antiviral activity of Arthrospira-derived spirulan-like substances. Antivir Res 72:197e206 Rios JL, Recio MC (2005) Medicinal plants and antimicrobial activity. J Ethnopharmacol 100:80–84 Roizman B, Knipe DM, Whitley RJ (2007) Herpes simplex viruses. In: Fields virology, 5th edn. pp 2501–2601 Saad M, Taher M, Susanti D, Qaralleh H, Binti NA, Rahim A (2011) Antimicrobial activity of mangrove plant (Lumnitzera littorea). Asian Pac J Trop Med:523–525 Saada M, Falleh H, Jalleli I, Snoussi M, Ksouri R (2014) Phenolic profile, biological activities and fraction analysis of the medicinal halophyte Retama raetam. S Afr J Bot 94:114–121 Saenz MT, Tornos MP, Alvarez A, Fernandez MA, Garcia MD (2004) Antibacterial activity of essential oils of Pimenta racemosa var. terebinthina and Pimenta racemosa var. grisea. Short Report. Fitoterapia 75:599–602 Saïdana D, Mahjoub MA, Boussaada O, Chriaa J, Chéraif I, Daamid M, Mighrib Z, Helal AN (2008). Chemical composition and antimicrobial activity of volatile compounds of Tamarix boveana (Tamaricaceae). Microbiol Res 163:445–455 Sakagami Y, Murata H, Nakanishi T, Inatomi Y, Watabe K, Iinuma M, Tanaka T, Murata J, Lang FA (2001) Inhibitory effect of plant extracts on production of verotoxin by enterohemorrhagic Escherichia coli O157:H7. J Health Sci 47(5):473–477 Samuelsen AB (2000) The traditional uses, chemical constituents and biological activities of Plantago major L. A review. J Ethnopharmacol 71:1–21 Subramanyam C, Rao KB, Rao CV, Rao BV (1992) Chemical examination of Suaeda monoica and Suaeda maritima. Acta Cienca Indica (C) 18:7–8 Suhaj M (2006) Spice antioxidants isolation and their antiradical activity: a review. J Food Compos Anal 19:531–537 Thoroski J, Blank G, Biliaderis C (1989) Eugenol induced inhibition of extracellular enzyme production by Bacillus cereus. J Food Prot 52(6):399–403 Trabelsi N, Megdiche W, Ksouri R, Falleh H, Oueslati S, Soumaya B, Hajlaoui H, Abdelly CH (2009) Solvent effects on phenolic contents and biological activities of the halophyte Limoniastrum monopetalum leaves. LWT-Food Sci Technol 43:632–639 Trabelsia T, Oueslati S, Henry-Vitrac C, Waffo-Téguob P, Medini F, Mérillon J, Abdelly CH, Ksouri R (2013) Phenolic contents and biological activities of Limoniastrum guyonianum fractions obtained by centrifugal partition. Ind Crops Prod 49:740–746

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Wendakoon CN, Sakaguchi M (1995) Inhibition of amino acid decaboxylase activity of Enterobacter aerogenes by active components of spices. J Food Prot 58:280–283 Yuh-Chi K, Lie-Chwen L, Wei-Jern T, Cheng-Jen C, Szu-Hao K, Yen-Hui H (2002) Samarangenin B from Limonium sinense suppresses herpes simplex virus type 1 replication in vero cells by regulation of viral macromolecular synthesis. Antimicrob Agents CH 46 (9):2854–2864 Zhusupova G, Rachimov K, Shalakhmetova T, Abilov ZH (2002) Biodiversity, biomolecular aspects of biodiversity and innovative utilization. Edited by sener kluwer academic/Plennium publisher

Chapter 12

Natural Products and Hepatitis C Virus Karin Séron, Marie-Emmanuelle Sahuc and Yves Rouillé

Abstract Hepatitis C is a major health problem worldwide. For a long time, it has not been possible to grow in cell culture the virus responsible for this disease. This impeded the development of efficient antiviral treatments. In the past decade, the establishment of a hepatitis C virus (HCV) cell culture model led to numerous studies aiming at finding chemical compounds with anti-HCV activity. A number of natural compounds were isolated and identified from plants, which inhibit various steps of the HCV life cycle. A review of the current knowledge on plant-derived natural compounds with anti-HCV activities is presented in this chapter.










Keywords Hepatitis C virus Antiviral agent Plant extract Traditional medicine Bioactive molecule

12.1

Introduction

Hepatitis C is a major cause of chronic hepatitis and often leads to serious complications, such as cirrhosis and hepatocellular carcinoma (Levrero 2006; Messina et al. 2015). Between 130 and 170 million people are infected with hepatitis C virus (HCV) worldwide. The prevalence of HCV infection varies considerably between countries and can be greater than 10% in some countries like Egypt (Lavanchy 2011). HCV is an enveloped, single-stranded RNA virus (Lindenbach et al. 2007). HCV isolates are divided into seven major genotypes, based on sequence homology. In patients, many different and closely related HCV sequences of the same strain, named quasi-species, are usually found. This molecular diversity results from the lack of proofreading ability of HCV RNA polymerase and provides an

K. Séron (&)
M.-E. Sahuc
Y. Rouillé Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL Center for Infection and Immunity of Lille, 59000 Lille, France e-mail: [email protected] © Springer International Publishing AG 2018 J.-M. Mérillon and C. Rivière (eds.), Natural Antimicrobial Agents, Sustainable Development and Biodiversity 19, https://doi.org/10.1007/978-3-319-67045-4_12

289

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Fig. 12.1 Schematic representation of HCV infectious cycle. HCV enters cells via attachment to non-specific factors present at the cell surface, followed by binding to specific entry factors (receptors). The virus is endocytosed, and the viral envelope fuses with the endosomal membrane. The viral RNA is released in the cytoplasm and translated by ribosomes in a polyprotein, which is cleaved by viral and cellular proteases to produce mature viral proteins. The RNA is replicated. Virion assembly takes place at the vicinity of the lipid droplets (LD). The virions are transported and released out of the cell by secretion. Alternatively, they can be transmitted to neighbouring cells via cell-to-cell transfer. ER: endoplasmic reticulum

advantage to the virus for adapting to new environments, such as an antiviral treatment or the passage to a new patient. Good reviews on the biology of HCV are available (Bartenschlager et al. 2011; Lindenbach and Rice 2013; Moradpour and Penin 2013; Paul and Bartenschlager 2015; Popescu et al. 2014). Therefore, the HCV life cycle will not be described in detail here. HCV is a spherical viral particle with an average diameter of *60 nm. A capsid and an envelope protect the viral genome. The envelope is made of a lipid bilayer in which are embedded envelope glycoproteins E1 and E2 and cellular apolipoproteins. Its mode of transmission is parenteral. Before the development of HCV diagnostic tests, blood transfusions contributed to most transmission cases. Nowadays, HCV is spread by contaminated medical equipment, as well as intra-venous drug use, piercing practices and acupuncture using contaminated materials. The HCV life cycle can be divided into three major steps: entry,

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Fig. 12.2 Schematic representation of HCV genomic organization. Capsid (C) in blue, envelope protein E1 and E2 in green, P7 and NS2 non-structural proteins in pink, and NS3 to NS5B non-structural proteins necessary for replication in purple

replication and assembly/release (Fig. 12.1). At each step, different sets of viral proteins and host factors are involved. HCV entry is initiated by particles binding to non-specific entry factors, low-density lipoprotein receptor and glycosaminoglycans, at the cell surface (Dubuisson et al. 2008). Then, HCV interacts with specific entry factors: scavenger receptor type B member 1 (Scarselli et al. 2002), the tetraspanin CD81 (Pileri et al. 1998), and tight junction proteins claudin-1 (Evans et al. 2007) and occludin (Ploss et al. 2009). The viral particles are internalized by via clathrin-dependent endocytosis and are transported into endosomes, where the acidic pH triggers the fusion of the viral envelope and the endosomal membrane catalysed by glycoproteins E1 and E2 (Blanchard et al. 2006). The fusion of endosomal membrane and viral envelope results in the release of the viral RNA genome into cell cytoplasm. The released viral RNA contains a single open reading frame, which is translated into a polyprotein precursor of about 3000 amino acid residues (Fig. 12.2). The structural proteins are found at the amino-terminal part of the polyprotein, while in the carboxy-terminal part are present the non-structural proteins. Structural proteins are components of infectious viral particles, whereas non-structural proteins are involved in the replication of the viral genome and the production of new infectious particles in infected cells, but are not incorporated into infectious particles. The mature forms of structural and non-structural proteins are generated by proteolytic cleavages of the polyprotein precursor, and all these proteins are initially associated with the endoplasmic reticulum (ER) of the host cell. Structural proteins are core, the capsid protein and envelope glycoproteins E1 and E2. Core is sequentially cleaved from the polyprotein by two cellular proteases: a signal peptidase, which releases core from the polyprotein, and a signal peptide peptidase, which generates the mature form of the protein (McLauchlan et al. 2002). E1 and E2 are anchored at the surface of the viral particles by a trans-membrane domain. E1 and E2 are N-glycosylated, and their folding is assisted by chaperone proteins of the ER, like

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calnexin (Dubuisson and Rice 1996). They are generated by signal peptidase-mediated cleavages of the polyprotein and initially associated as non-covalent heterodimers in the membrane of the ER. Then, during virion morphogenesis, E1E2 heterodimers trimerize (Falson et al. 2015), and these trimers of E1E2 heterodimers are stabilized by disulphide bonds at the surface of viral particles (Vieyres et al. 2010). The non-structural proteins are p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B. p7 belongs to the viroporin family of viral proteins that form ion channels. It plays a role in virus assembly and release by a mechanism that is not well understood. NS2 has a cysteine protease activity in its C-terminal domain, which cleaves the polyprotein between NS2 and NS3. NS2 also has a role in viral particle assembly, and this function is borne by its N-terminal membrane domain. NS3 is a serine protease that cleaves the polyprotein at the C terminus of NS3, NS4A, NS4B and NS5A, and releases these non-structural proteins as well as NS5B from the polyprotein. In addition to its protease activity, NS3 also has a helicase activity, which is involved in the replication of the RNA genome. NS4A forms a non-covalent complex with NS3 and is a co-factor of its protease activity. NS4B is a polytopic membrane protein involved in the formation of membrane rearrangements that are associated with the replication of the viral genome. NS5A is a phosphoprotein with no enzymatic activity, which is involved both in the replication of the viral genome and in the assembly of new infectious viral particles. NS5B is the RNA-dependent RNA polymerase (RdRp). It is involved in the formation of a negative RNA strand, which is used as a template for the synthesis of new viral RNA genomes (Moradpour and Penin 2013). The replication of the viral genome occurs in rearranged membranes originating from the ER. These membrane reorganizations are named ‘membranous web’ (Egger et al. 2002). They are made of single-membrane and double-membrane vesicles (Ferraris et al. 2010; Romero-Brey et al. 2012). Their formation is induced by NS4B and NS5A, respectively. NS3/4A, NS4B, NS5A and NS5B are the viral components of replication complexes. Together with p7, NS2 and structural proteins, they also participate in the assembly of new viral particles. The assembly of new viral infectious particles occurs in association with lipid droplets (Miyanari et al. 2007). New virions bud into the ER and follow the secretory pathway to get out of the cell. During or after the budding event, viral particles associate with apolipoproteins and probably form hybrid structures named lipoviroparticles (Bartenschlager et al. 2011). Lipoviroparticles are formed via the very low-density lipoprotein (VLDL) pathway of hepatocytes (Huang et al. 2007). Particles secreted into the extracellular medium can infect new hepatocytes and initiate a new round of infection. The infection can also be spread to new cells by cell-to-cell transmission (Timpe et al. 2008). To date no vaccine is available against HCV. This is mainly due to the wide variety of viral isolates that makes difficult the development of a vaccine (Tarr et al. 2015). In recent decades, various treatments have been established. Before 2011, a standard therapy combining pegylated interferon alpha and ribavirin (a guanosine analogue) was administered to patients (Liang and Ghany 2013).

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The first direct-acting antiviral agents (DAA), telaprevir and boceprevir, which are inhibitors of the NS3/4A protease activity, were released in 2011. A triple therapy associating pegylated interferon, ribavirin and a NS3/4A inhibitor was effective only against genotype 1 HCV-infected patients and induced significant side effects. In 2013, sofosbuvir, a polymerase inhibitor, has been marketed. This molecule has an anti-HCV efficacy of up to 90%, independently of the viral genotype. Other DAA have now been released. For example, daclatasvir is a strong inhibitor of NS5A. When this molecule is combined with sofosbuvir, sustained virological response can reach up to 100%, with very few side effects (Pawlotsky 2014). However, these treatments are very expensive and are not accessible to all people infected (Graham and Swan 2015). Moreover, there is a risk of selecting viral variants resistant to DAA, which will require new treatments (Pawlotsky 2016). Plants have been used for thousands of years in traditional medicine worldwide, and 80% of the world population is dependent of these therapies. Furthermore, it is estimated that over a quarter of the marketed medicines are from plant origin (Balunas and Kinghorn 2005; Cragg and Newman 2005). In Africa, 28 million people are infected with HCV (Lavanchy 2011). Plants used in traditional medicine against liver diseases constitute a reservoir of bioactive molecules and can be a valuable alternative for discovering new anti-HCV drugs. This review will focus on natural products isolated from plants, from crude plant extracts to purified molecules, which were described to interfere with the HCV infectious cycle. We will not report on natural products with indirect impact on HCV, such as hepatoprotective or immunomodulatory activities. Antiviral agents have many different modes of action and can inhibit different steps of the virus life cycle. Entry inhibitors can be separated into different classes, inhibitors targeting the early steps of entry, i.e. the attachment of the virus to the cell surface and the interaction with the specific HCV entry factors such as CD81 or Claudin-1, and inhibitors targeting the late steps of entry, i.e. endocytosis and envelope fusion. Furthermore, inhibitors could directly target the viral particle or a cellular component. Natural compounds inhibiting HCV entry belong to all these different classes of inhibitors even if, for many of them, their mechanism of action is not well understood. Furthermore, some compounds are described to interfere with different steps of the HCV life cycle, meaning that the same molecule may have different mechanisms of action. Compounds inhibiting the replication might inhibit one of the different proteins involved in this step. Inhibitors of NS3/4A protease activity, of NS5B polymerase activity or of NS5A have been described. Different tools are available to study HCV in vitro and have evolved in the past decades. The entire HCV life cycle can be achieved in cell culture using HCV strain JFH1 and human hepatoma-derived cells Huh-7 (Wakita et al. 2005), or Huh-7-derived subclones which yield higher viral titres (Blight et al. 2002). This cell culture system has been named HCVcc. In addition to HCVcc, specific steps of the HCV life cycle can be studied with other tools. The entry step can be monitored using HCVpp, which are retroviral particles pseudotyped with HCV envelope glycoproteins (Bartosch et al. 2003). Their entry, up to the fusion of the viral envelope and a cellular membrane, is carried out by HCV E1E2 glycoproteins.

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Then, post-fusion entry events depend on the retroviral nucleocapsid. Using this system allows to study host cell attachment, receptors engagement, virion endocytosis and envelope fusion. The replication step can be studied using replicons (Blight et al. 2000; Lohmann et al. 1999). Replicons are minimal replication units. For HCV, replicons contain both untranslated regions of the viral genome and non-structural proteins NS3 to NS5B. In addition, they also contain a selection system and/or an enzymatic or fluorescent marker. The replicon RNA is translated in vitro and introduced into Huh-7 cells using electroporation. Cells harbouring a functional replicon can be selected for using the selection system. Alternatively, the replication can be quantified over time after electroporation using the enzymatic marker (usually a luciferase) without selection. There is no animal model for in vivo study of HCV. Surrogate models to in vivo studies exist, which allow studying HCV infection in its natural host cell, the human hepatocyte. These models include the use of primary human hepatocytes (PHH) in cell culture (Podevin et al. 2010) or in chimeric mice with a humanized liver (Meuleman and Leroux-Roels 2008).

12.2

Plant Natural Products with Anti-HCV Activity

Phytochemicals can be divided into different groups on the basis of their chemical structure. Plant-derived molecules inhibiting HCV infection belong to different chemical classes, phenolic compounds, terpenoids and alkaloids. Indeed, a correlation between the chemical group and the antiviral activity is not easy to establish. Furthermore, antiviral capacity of crude plant extracts without identification of the active substance is also reported.

12.2.1 Phenolic Compounds 12.2.1.1

Lignans and Flavanolignans

Silymarin, a standardized extract of Milk thistle (Silybum marianum, Asteraceae), contains a mixture of flavonolignans, among them a mixture of two diastereoisomers, silibinin A and B (also known as silybin A and B). These two flavonolignans are the most widely studied compounds among the natural products with anti-HCV capacities. Both silymarin and silibinin showed a direct antiviral activity against HCV in vitro but also a hepatoprotective activity in vivo in animal model (Polyak et al. 2013; Ramasamy and Agarwal 2008). However, only the succinate derivative of silibinin A and B, Legalon SIL, has a beneficial effect on HCV-infected patients, probably due to the lack of solubility and bioavailability of silymarin and silibinin (Polyak et al. 2013). Silibinin has been shown to reduce viral load in HCV-infected patients (Ferenci et al. 2008) and to prevent graft reinfection after liver transplantation (Beinhardt et al. 2010; Eurich et al. 2011; Neuman et al. 2010).

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Regarding their direct activity on the HCV life cycle, silymarin and silibinin have been shown to inhibit HCV infection in vitro at multiple steps of the virus life cycle, including entry (Blaising et al. 2013; Wagoner et al. 2011, 2010). Silymarin appears to interfere with a late phase of HCV entry, the fusion step, but has no impact on the attachment step. Furthermore, by studying the activity of silibinin, Blaising et al. (2013) showed that both molecules might also interfere with clathrin-mediated endocytosis. Cell-to-cell transmission is an important way of HCV dissemination. Silymarin is able to interfere with HCV cell-to-cell spread (Wagoner et al. 2010). Furthermore, inhibition of HCV replication via the inhibition of HCV NS5B RNA-dependent RNA polymerase activity was demonstrated for silibinin (Ahmed-Belkacem et al. 2010; Wagoner et al. 2011) and silymarin (Wagoner et al. 2010) in vitro with some differences on efficacy depending on the genotypes. However, the activity of silymarin against RNA replication in vivo was not demonstrated. By transiently expressing HCV core gene in Huh-7 cells, Ashfaq et al. (2011a) showed that silymarin induces a decrease in HCV core expression. An inhibition of infectious virion production was also observed in HCV-infected cells treated with silymarin (Wagoner et al. 2010), as well as an inhibition of microsomal triglyceride transfer protein (MTP) activity and apolipoprotein B secretion. A viral kinetic model shows that silibinin might also affect HCV virion release (Guedj et al. 2012). Silibinin was tested against HCV replicon (Esser-Nobis et al. 2013). Activity against HCV replication was demonstrated for genotype 1a and 1b but not 2a. By analysing silibinin HCV-resistant virus obtained in cell culture, mutations in NS4B were identified. Interestingly, mutations in NS4B were also identified in hepatitis C patients treated with silibinin after liver transplantation suggesting a new mechanism of action for silibinin. Honokiol, a lignan extracted from the bark of Magnolia officinalis (Magnoliaceae), a plant commonly used in Asian traditional medicine, was demonstrated to have multiple effects on the HCV life cycle, inhibiting both entry and replication (Lan et al. 2012). Furthermore, an inhibition of IRES-mediated translation of HCV RNA was observed for this compound at high concentration (>30 µM). This was demonstrated using luciferase reporter gene expression assay and confirmed by measuring the expression of HCV non-structural proteins. By studying the anti-HCV capacity of Brazilian plant extracts, Jardim et al. (2015) identified four compounds able to inhibit HCV replication using replicon and HCVcc systems. These compounds are APS, a natural alkaloid isolated from Maytenus ilicifolia (Celastraceae), two tetrahydrofuran lignans 3*43 and 3*20 and secolignan 5*362 from Peperomia blanda (Piperaceae). Very interestingly, the authors show that these compounds are active against daclatasvir NS5A-resistant mutant Y93H in a replicon system. It would be interesting to identify the target of these compounds. Another lignan, the 3-hydroxy caruilignan molecule isolated from Swietenia macrophylla (Meliaceae) stem extract, was shown to inhibit HCV replication as demonstrated with a HCV replicon system (Wu et al. 2012). The antiviral effect appears to correlate with the induction of the IFN response.

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Tannins

Flavan-3-ols and Condensed Tannins Epigallocatechin-3-gallate (EGCG), the major flavonoid present in green tea (Camellia sinensis, Theaceae) extract, has been identified by us and other to inhibit HCV entry (Calland et al. 2015, 2012; C. Chen et al. 2012; Ciesek et al. 2011; Fukazawa et al. 2012). This molecule, tested in HCVcc and HCVpp systems and PHH, acts directly on the viral particle and impairs the attachment of the virus to the cell surface. Interestingly, a pan-genotypic activity of this compound against HCV was observed. A proposed mechanism of action is the direct action of EGCG on E1E2 envelope glycoprotein leading to an alteration of the structure of the viral envelope, without its disruption, and blockade of the binding of HCV to the cells (Calland et al. 2015; Colpitts and Schang 2014). An inhibition of HCV envelope binding to cell surface heparan sulphate by EGCG was suggested (Colpitts and Schang 2014). EGCG was also shown to inhibit cell-to-cell transmission (Calland et al. 2012; Ciesek et al. 2011). By structure activity relation analysis, we identified a compound related to EGCG, delphinidin, an anthocyanidin which is a plant pigment of flowers and berries, very abundant in blueberries (Vaccinium corymbosum, Ericaceae) or bilberries (Vaccinium myrtillus), that inhibits HCV entry with the same mechanism of action (Calland et al. 2015). Although EGCG has been well characterized as an entry inhibitor, C. Chen et al. (2012) showed that this molecule could also inhibit HCV replication. Rhodiola kirilowii (Crassulaceae) is a plant used in Chinese Tibetan traditional medicine. R. kirilowii rhizome extract was shown to be active against HCV NS3 protease expressed in Cos-7 cells (Zuo et al. 2007). Different compounds were purified from the extract and identified. Among them, EGCG and (-)-epicatechin-3O-gallate (ECG) and their dimers rhodisin and 3,3′-digalloylprocyanidin B2 were found to actively inhibit NS3 protease. Interestingly, the same compounds (-)epicatechin and ECG were inactive or exhibited weak antiviral activity, respectively, on HCVcc entry (Calland et al. 2012). Other compounds isolated from green tea, like epicatechin isomers, were shown to inhibit HCV replication in both replicon and HCVcc systems (Lin et al. 2013). An additive inhibitory effect was observed when the compounds were used in combination with interferon alpha or protease inhibitors. An indirect effect, i.e. the downregulation of Cox2, was also observed. Furthermore, an anti-inflammatory effect was demonstrated with the inhibition of tumour necrosis factor (TNF)-a, interleukin (IL)-1b and inducible nitrite oxide synthase gene expression. The major constituents of grape (Vitis vinifera L., Vitaceae) seed extract epicatechin, catechin and gallic acid (Sharaf et al. 2012) were tested for their anti-HCV activity. An inhibition of early step of HCV infection, entry, was observed for crude extract, epicatechin and catechin but not for gallic acid. A moderate inhibition was also observed for the post-entry step, the replication step. Procyanidin B1, a dimer of (-)- epicatechin and (+)- catechin, extracted from Cinnamomi cortex (Cinnamomum verum, Lauraceae), has been shown to inhibit HCVpp entry in a dose-dependent manner (Li et al. 2010). However, the predominant

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effect was observed on replication. The authors showed that procyanidin B1 is able to inhibit HCV RNA synthesis. San-Huang-Xie-Xin-Tang (SHXT) is a formula used in Chinese traditional medicine and composed of different plants, Rhei rhizoma (Rheum officinale, Polygonaceae), Scutellaria radix (Scutellaria baicalensis, Lamiaceae) and Coptidis rhizome (Coptis chinensis, Ranunculaceae). Fractionation of SHXT extract identified the (+)catechin containing fraction as the active fraction against HCV replication using a replicon cell line (Lee et al. 2011). An attenuation of Cox2 and NFjB activation seems to explain the antiviral effect. An effect of catechin and naringenin (see below) on intracellular infectious virion assembly was also described by Khachatoorian et al. (2012). Taken together, these reports highlight the anti-HCV potential of catechins as anti-HCV agents. Procyanidin is also present in Pycnogenol®, an extract of outer bark from Pinus pinaster ssp. atlantica (Pinaceae, French maritime pine extract). With taxifolin, another polyphenol present in this extract, they were shown to inhibit HCV replication in cell culture using a replicon and in humanized mice (Ezzikouri et al. 2015). Synergy with interferon and ribavirin was observed, and Pycnogenol® was able to inhibit telaprevir-resistant mutant. Pycnogenol® is known to have antioxidant capacity. Reactive oxygen species (ROS) production was significantly decreased by Pycnogenol® in replicon cell line. Interestingly, flavonoids present in another Pinus species, Pinus massoniana Lamb bark extract, were shown to inhibit HCV replication using a replicon cell line and HCVcc (Wang et al. 2015). The authors demonstrated an inhibition of the activity of NS3 protease using a recombinant protein assay. Proanthocyanidin, purified from crude extract of blueberry (Vaccinium virgatum Aiton, Ericaceae) leaves (Takeshita et al. 2009), was shown to inhibit replication of HCV subgenomic RNA. This inhibitory effect seems to be due to the capacity of proanthocyanidin to bind to heterogenous nuclear ribonucleoproteins (hnRNP) or eukaryotic translation elongation factor 3 (eIF3). Co-infection of patients with HIV and HCV often occurs in different populations. A pan-African natural product library was screened against HIV (Tietjen et al. 2015). The authors identified ixoratannin A-2, and an alkaloid, boldine, as new inhibitors of HIV. They tested these two compounds against HCV using HCVcc system and showed that ixoratannin A-2 inhibits HCV replication. Such compound could be potentially helpful for treating patients suffering of hepatitis C and AIDS. However, a better understanding of their mechanism of action is needed. Gallic Acid and Hydrolysable Tannins Gallic acid (a phenolic acid), identified in grape seed extract (Sharaf et al. 2012) as an anti-HCV agent, was also isolated from Limonium sinense (Plumbaginaceae), a plant used in traditional medicine. Root water extract from L. sinense was shown to inhibit HCV infection at the entry step, more precisely during attachment and fusion/endocytosis (Hsu et al. 2015). Among the compounds isolated from the plant extract, gallic acid was shown to be the most active one, with an inhibitory activity on viral entry. Furthermore, antiviral activity of the plant extract and gallic acid was confirmed in PHH. However, the activity of gallic acid did not completely correlate

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to that of the crude extract, showing that other active compounds might not have been identified yet or that the antiviral activity requires a combination of different molecules. It is worth noting that gallic acid was identified as HCV entry inhibitor in two independent studies. Gallic acid and quercetin were also identified to be the active compounds present in an Indonesian plant extract of Kalanchoe pinnata (Crassulaceae) (Aoki et al. 2014). Gallic acid inhibited both entry and replication, and quercetin only replication. Pomegranate (Punica granatum, Lythraceae) fruit peel and juice extracts were tested for their activity against HCV NS3 protease (Reddy et al. 2014). Both were active, the peel extract being the most active. Purified compounds were then isolated from the peel extract and the major constituents, punicalin, punicalagin and ellagic acid (phenols), tested against NS3 protease. All of them displayed anti-protease activity. The efficacy of punicalin and punicalagin against HCV replication was confirmed using HCVcc. Interestingly, both compounds were shown to strongly inhibit HCV entry, but not (or in addition to) replication with this assay. Among 400 medicinal plant extracts tested, a methanol extract of flower buds of Rosa rugosa (Rosaceae) was shown to inhibit VSV pseudotyped with E1E2 envelope glycoproteins of HCV (Tamura et al. 2010). Three active ellagitannin compounds, tellimagrandin I, eugeniin and casuarictin, were purified that could potentially inhibit HCV entry. As polyphenols are widely present across the plant kingdom, they are often identified as active compounds in plant extracts containing anti-HCV activity. The chemical structures of some of them, identified as potential anti-HCV agents, are not completely elucidated. Many different studies analysed the activity of plant extracts or isolated compounds using an in vitro protease assay using recombinant NS3. In the mangrove plant Excoecaria agallocha (Euphorbiaceae), used in traditional Chinese medicine, more than 20 polyphenols were isolated and tested for their anti-NS3 protease activity using a recombinant protease (Li et al. 2012). One new polyphenol, excoecariphenol D, and three already known polyphenols, corilagin, geraniin and chebulagic acid, were identified as active substances against the HCV protease. This activity was confirmed for excoecariphenol D and corilagin using a replicon-containing cell line. Similarly, three polyphenols were isolated from Galla Chinese (Rhus chinensis, Anacardiaceae) (Duan et al. 2004), and all of them displayed an anti-NS3 protease activity in vitro. Compounds isolated from Saxifraga melanocentra (Saxifragaceae), another plant used in Chinese traditional medicine (Zuo et al. 2005), were also tested against purified NS3 protease. Polyphenols purified from the ethyl acetate fraction were shown to inhibit HCV NS3 protease with penta-O-galloyl-b-D-glucoside being the most active. Finally, two oligophenols extracted from Stylogyne cauliflora (Primulaceae) plant extract were shown to be the most active among others against recombinant NS3/4A protease (Hegde et al. 2003). Taken together, these data suggest that compounds with polyphenol structure are potent antiviral inhibitors of the NS3/4A HCV protease.

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Flavonoïds

Flavonols Quercetin is a flavonol often identified to be the active substance present in different plant extracts with anti-HCV capacities like in K. pinnata (Aoki et al. 2014). It was first shown to inhibit HCV IRES-mediated translation via the inhibition of HSP70 (Gonzalez et al. 2009; Khachatoorian et al. 2012). Recently, a clinical phase I study was conducted to test the safety of quercetin in HCV-infected patients. The results are promising, because the authors demonstrated the safety of the compound even at high dose and a tendency to a decrease of HCV viral load in patients (Lu et al. 2015). Thirty plants from Ayurvedic medicine (India Siddha medicine) were screened for their capacity to inhibit HCV protease activity (Berdichevsky et al. 2003). Four plant extracts were identified as potential NS3 inhibitors. Among them, Embelia ribes (Primulaceae) seed extract was more thoroughly studied (Bachmetov et al. 2012). Different compounds were isolated from the crude extract, and quercetin was shown to inhibit HCV NS3 protease activity. This activity was confirmed in a replicon cell line, and a reduction of particle production was demonstrated in Huh-7 cells using HCVcc. Another Embelia species, E. schimperi, was identified among others as a potential source of anti-HCV compounds in a screening of seventy plant extracts from Sudanese traditional medicine for their anti-HCV NS3 protease activity (Hussein et al. 2000). Seven of them, Acacia nilotica (Fabaceae), Boswellia carterii (Burseraceae), E. schimperi (Primulaceae), Quercus infectoria (Fagaceae), Trachyspermum ammi (Apiaceae), Piper cubeba (Piperaceae), and Syzygium aromaticum (Myrtaceae), showed some anti-HCV activity. Further experiments were conducted on E. schimperi and compounds isolated from this plant extract. Two benzoquinones, embelin and 5-O-methylembelin, appear to be the active compounds and display anti-NS3 protease activity. In this case, quercetin was not identified to be involved in the anti-protease activity. Finally, crude methanol extract from aerial parts of Solanum rantonnetii (Solanaceae) was shown to inhibit HCVcc infection at the entry step (Rashed et al. 2014). Different compounds were isolated, and among them, quercetin 3-methyl ether was the most active. It is worth noting that quercetin was often identified in computational docking studies as potential inhibitor of different HCV non-structural protein confirming its potential as an anti-HCV agent. As different flavonoids have been described to inhibit NS5B RdRp, Ahmed-Belkacem et al. (2014) investigated the inhibitory capacity of different plant-derived compounds and unravelled their mechanism of action. They identified quercetagetin as the most potent inhibitor of RdRp activity and showed that this molecule interferes with the RNA binding activity of NS5B. Quercetagetin is the first non-nucleoside inhibitor of NS5B that binds to the fingers domain of the protein. Flavones Ladanein is a flavone isolated from Marrubium peregrinum (Lamiaceae) that inhibits HCV entry in vitro, at a post-attachment step (Haid et al. 2012). To confirm the antiviral activity of this natural compound, the authors chemically synthesized

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the molecule that was shown to exhibit similar anti-HCV capacities. Its effect is similar to anti-CD81 antibodies and, to a lesser extent to ITX5061, an inhibitor of SRB1, meaning that ladanein could interfere with the entry factors CD81 or SRB1 (Haid et al. 2012). This compound is active against all HCV genotypes, and the inhibitory effect was also demonstrated in PHH. The bioavailability of ladanein was assessed in mice, demonstrating its potential use in antiviral therapy. Apigenin is a flavonoid present in different plants like parsley (Petroselinum crispum, Apiaceae) or flowers of chamomile (Asteraceae) which was shown to inhibit maturation of microRNA (miRNA) (Ohno et al. 2013). Among these miRNA, miR122 is the most abundant in liver cells and is essential for the replication of HCV RNA (Gentzsch et al. 2011; Randall et al. 2007). Shibata et al. (2014) demonstrated that apigenin inhibits miR122 maturation and HCV replication. The inhibition of maturation appears to result from an inhibition of the phosphorylation of TRBP, a protein essential for the DICER machinery. These results confirmed data previously obtained demonstrating that apigenin and luteolin (another flavonoid) can inhibit HCV infection and HCV replication in cells stably expressing HCV replicon (Liu et al. 2012). These two compounds were first identified by a pharmacophore and a structure-based study approach using NS5B as a target. The anti-NS5B polymerase activity was confirmed in vitro for luteolin (Liu et al. 2012). Very interestingly, apigenin and luteolin extracted from Eclipta alba (Asteraceae), a plant used in Ayurvedic Indian traditional medicine, were also shown to inhibit HCV replication (Manvar et al. 2012). The authors demonstrated that the plant extract is able to inhibit NS5B RNA-dependent RNA polymerase in vitro. This activity was confirmed in cells expressing a subgenomic replicon. Citrus unshiu (Rutaceae) peel fractions and nobiletin, a flavonoid purified from one of these fractions, inhibit HCV infection in lymphoblastic leukaemia cell line, MOLT-4 cells (Suzuki et al. 2005). However, the antiviral capacity of this molecule should be analysed in a more relevant system, hepatoma cell lines, to confirm that nobiletin is an anti-HCV agent. Flavanones Naringenin, a flavanone present in grapefruit (Citrus x paradisi, Rutaceae), was shown to decrease HCV titre, core secretion, HCV RNA level and apolipoprotein B secretion via the inhibition of MTP activity as well as HMGR and ACAT2 enzymes (Nahmias et al. 2008). This effect of naringenin on HCV was confirmed by Goldwasser et al. (2011) who showed that naringenin affects virion assembly via an inhibition of MTP activity and an activation of PPAR alpha. Furthermore, the inhibitory activity of naringenin was confirmed in PHH. Finally, Khachatoorian et al. (2012) reported an effect of naringenin on intracellular infectious virion assembly. Chalcones Lucidone, a chalcone extracted from Lindera erythrocarpa (Lauraceae), was demonstrated to be active against HCV replication using HCV replicon and HCVcc (Chen et al. 2013). It dose-dependently decreases HCV RNA levels in both systems.

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The authors further analysed the mechanism of action of the compounds by looking at cellular protein expression levels. They showed that lucidone upregulates HO-1 leading to an increase of IFN response and an inhibition of NS3/4A protease. A synergistic effect of lucidone combined with other DAA or interferon alpha was also observed. Two other chalcones isoliquiritigenin, and licochalcone A isolated from G. uralensis and G. glabra will be mentioned in the paragraph Saponins as anti-HCV compounds (Adianti et al. 2014; Sekine-Osajima et al. 2009).

12.2.1.4

Stilbenes and Phenols

Vitisin B, a resveratrol tetramer, was recently identified as a new inhibitor of HCV replication that targets NS3 helicase (Lee et al. 2016). This compound extracted from grape (Vitis vinifera, Vitaceae) root is very highly active with an IC50 within the nanomolar range (6 nM). A direct binding of vitisin B to NS3 protein was observed. Furthermore, a synergistic effect is observed when vitisin B was combined with sofosbuvir (NS5B inhibitor). A study of 173 extracts from medicinal plants, identified Mori cortex radices, the root epidermis of Morus alba (Moraceae) methanol extract for its anti-HCV activity (Lee et al. 2007). An inhibition of the replication was observed in a replicon-containing cell line. Five compounds were isolated from the ethyl-acetate soluble fraction. Moracin P and Moracin O (two benzofurans) were the most potent ones for inhibiting HCV replication. The authors confirmed the inhibition of NS3 helicase activity by both compounds.

12.2.1.5

Coumarins and Coumestans

Coumarins are compounds present in numerous plants used in traditional medicine, and some of them have been identified as anti-HCV agents like the activity of wedelolactone, a coumestan derivative, chalepin (Wahyuni et al. 2014), glycycoumarin (Adianti et al. 2014; Sekine-Osajima et al. 2009), glycerol and glycyrin (Adianti et al. 2014). The activity of wedelolactone against NS5B polymerase activity was reported by Kaushik-Basu et al. (2008). It was confirmed with wedelolactone isolated from Eclipta alba (Asteraceae) (Manvar et al. 2012).

12.2.1.6

Diarylheptanoids

Curcumin, a linear diarylheptanoid present in rhizomes of the spice turmeric of the ginger family (Curcuma longa, Zingiberaceae), and responsible of the yellow colour

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of the turmeric, has also been shown to inhibit HCV entry in a pan-genotypic manner (Anggakusuma et al. 2014). Curcumin inhibits HCV binding and fusion acting both at early and late steps of entry by altering the virion envelope fluidity. Curcumin was shown to be active in PHH. This compound acts directly on the virus without disrupting the viral envelope. Curcumin is also able to inhibit HCV cell-to-cell transmission. Similarities in the mechanism of action of curcumin, EGCG and delphinidin are observed. In addition to its action on HCV entry, curcumin was described to inhibit HCV replication via an indirect mechanism of action, the induction of the heme oxygenase-1 and the suppression of the PI3 K-Akt and Akt-SREBP-1 pathways (Chen et al. 2012; Kim et al. 2010). Combined with interferon alpha, this molecule strongly inhibits HCV replication in a replicon cell line.

12.2.1.7

Phenolic Acids and Derivatives

The inflorescences of Scabiosa comosa and S. tschilliensis (Caprifoliaceae) and their purified compounds were tested for their antioxidant capacity and their anti-HCV activity (Ma et al. 2015). Both plants are commonly used in traditional medicine for the treatment of liver diseases. Two compounds 3,5-dicaffeoylquinic acid (DCQA, phenolic acid) and chlorogenic acid (phenylpropanoid) showed anti-HCV activity and antioxidant activity. Caffeic acid phenethyl ester (phenylpropanoids) from propolis prepared from honeybee hives (Shen et al. 2013) and its derivate caffeic acid n-octyl ester actively inhibit HCV replication both in replicon and HCVcc systems. Moreover, these compounds are active in combination with other HCV inhibitors like interferon alpha 2b, daclatasvir and VX-222 with a synergistic effect.

12.2.1.8

Quinones

As mentioned ahead, two benzoquinones, embelin and 5-O-methylembelin, were described to have anti-NS3 protease activity (Hussein et al. 2000).

12.2.2 Terpenoids 12.2.2.1

Monoterpenes

Loliolide, a monoterpenoid lactone extracted from Phyllanthus urinaria, another plant belonging to the Phyllanthaceae family, was identified as a new inhibitor of HCV entry (Chung et al. 2016). This molecule inhibits HCV attachment and is active against different genotypes in the HCVpp system.

12

Natural Products and Hepatitis C Virus

12.2.2.2

303

Iridoids

Lamiridosin A/B, two iridoid aglycone isomers extracted from the flowering top of Lamium album L. (Lamiaceae), have been shown to inhibit HCV entry (Zhang et al. 2009). The authors also showed that the inhibition does not result from a blockade of the interaction of E1E2 envelope glycoproteins with CD81. New iridoid glycoside compounds were isolated from Anarrhinum orientale (Plantaginaceae) aerial part extract, tested for their anti-NS3 protease activity and found to have moderate or weak anti-protease activity (Salah El Dine et al. 2011).

12.2.2.3

Sesquiterpene Lactones

Artemisinin, a sesquiterpene lactone isolated from Artemisia annua (Asteraceae), is a molecule used in Chinese traditional medicine for more than 2000 years. This compound and its derivatives are used for the treatment of Plasmodium falciparum, and clinical studies are underway to test their efficacy against helminth parasites and cancer. Paeshuyse et al. (2006) demonstrated the anti-HCV effect of artemisinin combined with hemin in a replicon system. These results need to be confirmed in the HCVcc system to validate the anti-HCV activity of this broadly used anti-malarial drug. High-throughput screening of natural compounds isolated from American and African plants revealed that new pseudoguaianolides isolated from Parthenium hispidum (Asteraceae) extract inhibit HCV replication using a replicon-based assay (Hu et al. 2007). The antiviral activity of these compounds on replication was not confirmed in the HCVcc system so far. Recently, two compounds isolated from water extracts of the leaves of the wild Egyptian artichoke (Cynara cardunculus, Asteraceae) have been identified as new inhibitors of HCV entry (Elsebai et al. 2016). They are two sesquiterpene lactones, grosheimol and cynaropicrin. Both are active against different HCV genotypes and are able to inhibit cell-to-cell transmission.

12.2.2.4

Diterpenes

Different compounds extracted from roots of Flueggea virosa. Phyllanthaceae were tested against HCV using the HCVcc system (Chao et al. 2014). Dinorditerpenes extracted from this plant, which are rare in nature, exhibit anti-HCV activity with an unknown mechanism of action.

12.2.2.5

Triterpenoids

Cynomorium songaricum Rupr. (Cynomoriaceae) is a plant used in Chinese traditional medicine. Triterpenes were isolated from C. songaricum stem extract and

304

K. Séron et al.

tested against NS3 protease (Ma et al. 2009). Malonyl ursolic acid hemiester was the most potent in inhibiting HCV NS3 protease activity. Interestingly, this compound is also an inhibitor of HIV protease (Ma et al. 1999). Furthermore, the resistance to heat of malonyl ursolic acid hemiester was tested. The results showed that this compound is quite unstable upon heating. Drying process of herbs used in traditional medicine in ovens may alter the composition of these herbs and the bioactivity of the compounds. Five plants used in Chinese traditional medicine were tested for their anti-NS5B polymerase activity, and Fructus Ligustri Lucidi, the dried ripen fruit of Ligustrum lucidum (Oleaceae) extract, was shown to significantly inhibit NS5B polymerase activity (Kong et al. 2007) in a cell expression system. Active compounds were characterized later by the same group to be oleanolic and ursolic acids (pentacyclic triterpenoids) (Kong et al. 2013). The antiviral effect was confirmed using HCVcc and in a replicon-containing cell line. An inhibition of NS5B polymerase was confirmed for these two compounds. Recently, a compound extracted from Schisandra sphenanthera (Schisandraceae), schizandronic acid, was identified as an inhibitor of HCV entry at the fusion step. This compound has a pan-genotypic activity and is active in primary human hepatocytes (Qian et al. 2016).

12.2.2.6

Saponins

Glycyrrhizin (a triterpenoid saponin) is one of the natural compounds with multiple effects on HCV infection. It is extracted from Glycyrrhiza glabra (liquorice, Fabaceae) and has been used for the treatment of chronic hepatitis in Japan, resulting in an alanine transaminase-lowering effect. A role of this molecule against HCV infection was reported by Matsumoto et al. (2013) who observed a reduction of virion release in HCV-infected cells upon glycyrrhizin treatment and by Ashfaq et al. (2011b) who also showed that glycyrrhizin induces a decrease of HCV titre in the culture medium of HCV-infected cells. Two phenolic compounds were also isolated from Glycyrrhizae radix (Glycyrrhiza uralensis, Chinese liquorice, Fabaceae), a plant of the same family. These two products, isoliquiritigenin (a chalcone) and glycycoumarin (coumarin), were shown to inhibit HCV replication in a replicon system (Sekine-Osajima et al. 2009). Their mechanism of action is not known but does not depend on IRES-mediated translation. Because they are often used in Chinese traditional and modern medicines, other compounds were isolated from Glycyrrhiza family plants and tested against HCV. Glycycoumarin, glycerol, glycyrin (coumarins), liquiritigenin (flavonoid) isolated from G. uralensis and isoliquiritigenin, licochalcone A (two chalcones) and glabridin (isoflavan) isolated from G. glabra inhibit HCV infection (Adianti et al. 2014). All these compounds inhibit a post-entry step of the HCV life cycle, i.e. the replication step. Kim TW et al. (2012) first described the potential anti-HCV capacity of Platycodon grandiflorus (Campanulaceae) hot water extract using a replicon

12

Natural Products and Hepatitis C Virus

305

system. The anti-HCV activity was confirmed by the same group who isolated triterpenoid saponins from root crude extract of P. grandiflorum and demonstrated their antiviral activity using both replicon and HCVcc systems (Kim et al. 2013). Furthermore, they identified the RdRp NS5B as the target of the molecules. Few natural compounds so far described are direct inhibitors of NS5B polymerase. Saikosaponin b2, a glycoside bearing a triterpene structure, isolated from B. kaoi roots (Bupleurum chinensis, Apiaceae), a plant used in Eastern Asia for the treatment of liver disease, was shown to inhibit HCV entry at various stages, attachment, post-attachment and endocytosis/fusion (Lin et al. 2015). The crude plant extract is also active against HCV. A direct effect on the virion was demonstrated. Interestingly, saikosaponin b2 is active in PHH and against different HCV genotypes.

12.2.3 Alkaloids An alkaloid mentioned ahead, APS from Brazilian plant extracts, was described as a HCV replication inhibitor (Jardim et al. 2015). Few other alkaloids were identified as active anti-HCV agents in the same study. Different alkaloids, myriberine A (Huang et al. 2013) and quinolizine alkaloids (Cao et al. 2014), were isolated from Myrioneuron faberi (Rubiaceae), a plant from China. All the compounds were reported to inhibit HCV replication. Six compounds were extracted from Ruta angustifolia leaves (Rutaceae), a plant used in Indonesian traditional medicine for the treatment of liver disease and jaundice (Wahyuni et al. 2014). Among the compounds identified, an alkaloid, the pseudane IX and chalepin, a furocoumarin, displayed the strongest anti-HCV activity tested with the HCVcc system. Both molecules inhibit HCV replication. Moreover, c-fagarine, arborinine and kokusaginine, also isolated from R. angustifolia, exert a moderate anti-HCV activity. Among the isolated compounds, only scopoletin, a coumarin, did not show any anti-HCV activity, even at high concentration.

12.2.4 Porphyrins Morinda citrifolia (Rubiaceae) fruits and leaves are used in traditional medicine. Methanol leave extract and fractions of M. citrifolia exhibit anti-HCV activity in cell culture (Ratnoglik et al. 2014). Two purified compounds isolated from these fractions, pheophorbide (a breakdown product of chlorophyll) and its catabolite pyropheophorbide a, possess anti-HCV capacities both at entry and post-entry steps.

306

K. Séron et al.

12.2.5 Crude Plant Extracts Several plant extracts from medicinal plants of different areas have been described to inhibit HCV infection. Bark, leaf, stem, root, fruit peel, fruit juice are used to produce the crude extract. Even if the active compounds are not identified, the anti-HCV activity is worth mentioning. This concerns different plants from Indonesia, Toona sureni (Meliaceae), Melicope latifolia (Rutaceae), Melanolepis multiglandulosa (Euphorbiaceae) and Ficus fistulosa (Moraceae) (Wahyuni et al. 2013), and plants from Cameroon, Entada africana (Fabaceae), Trichilia dregeana (Meliaceae), Detarium microcarpum (Fabaceae), Phragmanthera capitata (Loranthaceae) and Khaya grandifoliola (Meliaceae) (Galani et al. 2016, 2015, 2014). T. sureni and M. latifolia leave extracts were shown to inhibit both entry and replication, whereas M. multiglandulosa stem and F. fistulosa leave extracts inhibit the entry step (Wahyuni et al. 2013). However, HCV genotype 1a, 2b and 7a were more resistant to M. multiglandulosa and F. fistulosa extracts. E. africana is used in traditional medicine in Cameroon (Galani et al. 2014). Plant extract and fractions of this plant were tested using a HCV replicon system. The methylene chloride-methanol fraction was shown to inhibit HCV replication. Furthermore, these compounds also induce the expression of two interferon-stimulated genes, HO-1 and 2′-5′ oligoadenylate synthetase-3. In contrast, T. dregeana, D. microcarpum and P. capitata extracts were shown to inhibit HCV entry (Galani et al. 2015). More recently, a study was conducted to study the anti-HCV capacity of bark extract of K. grandifoliola (Galani et al. 2016), showing that one of the fractions from the crude extract was able to inhibit both HCV replication in a replicon system, at high concentration, and HCV entry demonstrated using the HCVpp system, at lower concentration. Compounds present in this fraction from K. grandifoliola extract probably inactivate the virus before entry, impairing its attachment to the cell surface. This plant that potentially contains agents inhibiting both HCV entry and replication might be further studied to characterize the active compounds. Different articles have reported the antiviral activity of plant extracts from Pakistanese traditional medicine. Twelve extracts were tested for their anti-HCV activity in a replicon cell line (Rehman et al. 2011). Methanolic extract of A. nilotica (Fabaceae) and its acetone fraction were shown to inhibit HCV replication. This confirms results obtained by Hussein et al. (2000), showing that A. nilotica extract is able to inhibit HCV NS3 serine protease activity. Moreover, N-butanol-methanol extract of A. confusa (Fabaceae), a plant from Taiwan used in traditional medicine, inhibits HCV replication in a replicon-containing cell line (Lee et al. 2011). This extract was combined with anti-protease inhibitors, and a synergistic inhibitory effect was observed. Anti-HCV activity could be due to inhibition of Cox-2 expression. Ten more different Pakistanese plant extracts were tested against HCV from the serum of an infected patient and against recombinant NS3 protease (Javed et al. 2011). Among them, Solanum nigrum (Solanaceae) seed extract was shown to

12

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307

be active. As mentioned before, another Solanaceae, S. rantonnetii, was also described to have anti-HCV capacities (Rashed et al. 2014). Another similar study reports the anti-HCV activity of Portulaca oleracea (Portulacaceae) extract which is active against HCV present in serum of infected patients and recombinant NS3 protease (Noreen et al. 2015). Phyllanthus amarus (Phyllanthaceae) whole plant extract was tested against NS3 protease and NS5B polymerase of HCV (Ravikumar et al. 2011). Both enzymes were inhibited by the extract. The inhibition of replication was confirmed using a replicon system. The inhibitory activity from root and leaf of P. amarus was then tested separately, and, surprisingly, the root extract inhibited NS3 protease activity, whereas the leaf extract was a strong inhibitor of NS5B polymerase. Taken together, these results showed that this plant extract potentially contains two different inhibitors of HCV replication targeting different viral enzymes.

12.3

Chemically Synthesized Derivatives of Natural Compounds

Natural compounds may serve as scaffolds for the synthesis of antiviral agents with enhanced inhibitory capacity compared to the native molecules. Derivatives from coumarin, artemisinin (Blazquez et al. 2013; Obeid et al. 2013) and EGCG (Bhat et al. 2014; Rivero-Buceto et al. 2015) have been described. Based on their structure similarities with coumestan, a structure-activity relation study was performed with different coumarins to test their potential anti-NS5B activity (Nichols et al. 2013). Some synthesized molecules have, indeed, an anti-polymerase activity.

12.4

In Silico Studies

In silico docking analyses might also reveal the potential antiviral capacity of some natural compounds present in plant extracts. This was done for NS3/4A protease with the identification of quercetin, catechins, and, with lower docking affinity, of resveratrol and luteolin (Fatima et al. 2014), A. nilotica phytochemicals (Khan et al. 2013b), and quercetin derivatives from Amelanchier alnifolia (Rosaceae) (Khan et al. 2013a). 3-Deacetyl-3-cinnamoyl-azadirachtin, extracted from Azadirachta indica (Meliaceae) leaves, is another example of potential NS3 protease inhibitor (Ashfaq et al. 2015). According to computational docking study, HCV p7 ion channel could also be inhibited by quercetin (Mathew et al. 2015). Naringenin and quercetin were identified as potential NS2 protease inhibitors using a similar approach (Lulu et al. 2015).

Peperomia blanda (Jacq.) Kunth

Peperomia blanda (Jacq.) Kunth

Peperomia blanda (Jacq.) Kunth

Lignan 3*(20

Secolignan 5*362

Swietenia macrophylla King Magnolia officinalis Rehder & E.H. Wilson

Plant name

Lignan 3*43

Honokiol

1-Phenolic compounds Lignans and flavonolignans 3-Hydroxy caruilignan

Bioactive molecule

Table 12.1 Molecules from plant origin with anti-HCV activity

Piperaceae

Piperaceae

Piperaceae

Magnoliaceae

Meliaceae

Plant family

Entry Replication IRES-mediated translation Non-structural protein synthesis Replication Inhibition of daclatasvir NS5A-resistant mutant Y93H Replication Inhibition of daclatasvir NS5A-resistant mutant Y93H Replication Inhibition of daclatasvir NS5A-resistant mutant Y93H

Replication

Viral step or protein target

38.9

8.2

4.0

1.2

10.5

IC50 (µM)

Jardim et al. (2015)

Jardim et al. (2015)

Jardim et al. (2015)

Lan et al. (2012)

Wu et al. (2012)

Reference

(continued)

308 K. Séron et al.

Chlorogenic acid

Chebulagic acid

Scabiosa comosa Fisch. ex Roem. & Schult. S. tschilliensis Grüning

Rosa rugosa Thunb. Rheum officinale Baill Scutellaria baicalensis Georgi Coptis chinensis Franch (San-Huang-Xie-Xin-Tang) Excoecaria agallocha L.

Silybum marianum (L.) Gaertn

Silymarin/Silibinin

Tannins Casuarictin (+)Catechin

Plant name

Bioactive molecule

Table 12.1 (continued)

Dipsacaceae

Euphorbiaceae

Rosaceae Polygonaceae Lamiaceae Ranunculaceae

Asteraceae

Plant family

Replication NS3

Entry Replication Virion assembly

Entry (endocytosis/ fusion) Cell-to-cell spread Virion release Core NS4B NS5B Decrease viral load in patients Prevention of liver graft reinfection

Viral step or protein target

22.2

42.3

40.0– 100.0

IC50 (µM)

Ma et al. (2015)

Liu et al. (2012)

(continued)

Tamura et al. (2010) Khachatoorian et al. (2012), Lee et al. (2011)

Ahmed-Belkacem et al. (2010), Ashfaq et al. (2011a), Beinhardt et al. (2010), Blaising et al. (2013), Esser-Nobis et al. (2013), Eurich et al. (2011), Ferenci et al. (2008), Guedj et al. (2012), Neuman et al. (2010), Wagoner et al. (2011, 2010)

Reference

12 Natural Products and Hepatitis C Virus 309

Excoecaria agallocha L.

Vaccinium corymbosum L.

Rhodiola kirilowii (Regel) Maxim Punica granatum L.

Rhodiola kirilowii (Regel) Maxim Camellia sinensis (L.) Kuntze Rhodiola kirilowii (Regel) Maxim Vitis vinifera L. Camellia sinensis (L.) Kuntze Rhodiola kirilowii (Regel) Maxim

Corilagin

Delphinidin

Digalloylprocyanidin B2

(-)-Epicatechin3-O-gallate (ECG) Epicatechin isomers

Epigallocatechin-3-gallate (EGCG)

Ellagic acid

Plant name

Bioactive molecule

Table 12.1 (continued)

Entry (early step, binding) Cell-to-cell spread Replication NS3 Direct effect on virion

Theaceae Crassulaceae Vitaceae

Theaceae Crassulaceae

Entry Replication NS3

Crassulaceae

Replication NS3 Entry (early step, binding) Cell-to-cell spread Direct effect on virion NS3

Viral step or protein target

Replication NS3/4A NS3

Lythraceae

Crassulaceae

Ericaceae

Euphorbiaceae

Plant family

5–21

50.0– 75.0

8.55

60.0

0.91

3.7

13.5

IC50 (µM)

(continued)

Calland et al. (2012), C. Chen et al. (2012), Ciesek et al. (2011), Colpitts and Schang (2014), Fukazawa et al. (2012), Zuo et al. (2007)

Lin et al. (2013) Sharaf et al. (2012) Zuo et al. (2007)

Zuo et al. (2007)

Reddy et al. (2014)

Zuo et al. (2007)

Calland et al. (2015)

Liu et al. (2012)

Reference

310 K. Séron et al.

Rosa rugosa Thunb. Excoecaria agallocha L.

Kalanchoe pinnata (Lam.) Pers. Limonium sinense (Girard) Kuntze Vitis vinifera L. Excoecaria agallocha L.

Ixora L. Saxifraga melanocentra Franch Vaccinium virgatum Aiton

Cinnamomum verum J. Presl Pinus pinaster ssp. atlantica Villar Punica granatum L.

Punica granatum L

Eugeniin Excoecariphenol D

Gallic acid

Ixoratannin A2 Penta-O-galloyl-b-D-glucoside

Procyanidin B1

Punicalin

Punicalagin

Proanthocyanidin

Geraniin

Plant name

Bioactive molecule

Table 12.1 (continued)

Lythraceae

Lythraceae

Lauraceae Pinaceae

Ericaceae

Rubiaceae Saxifragaceae

Euphorbiaceae

Crassulaceae Plumbaginaceae Vitaceae

Rosaceae Euphorbiaceae

Plant family

Replication Entry NS3/4A protease Entry Replication NS3/4A protease

Replication Binding to hnRNP Entry Replication

Replication NS3 Replication NS3 protease

Entry Replication NS3 Entry Replication

Viral step or protein target

100.0

>150.0

Reddy et al. (2014)

Reddy et al. (2014)

(continued)

Ezzikouri et al. (2015), Li et al. (2010)

Takeshita et al. (2009)

* 0.15 15.0 25.0– 103.7

Tietjen et al. (2015) Zuo et al. (2005)

Liu et al. (2012)

Aoki et al. (2014) Hsu et al. (2015) Sharaf et al. (2012)

Tamura et al. (2010) Liu et al. (2012)

Reference

23.0 0.68

33.1

36.4

12.6

IC50 (µM)

12 Natural Products and Hepatitis C Virus 311

Luteolin

Licochalcone A Lucidone

Ladanein BJ486K Liquiritigenin

Glabridin Isoliquiritigenin

Tellimagrandin I Flavonoïds Apigenin

Glycyrrhiza uralensis Fisch. ex DC. Glycyrrhiza glabra L. Lindera erythrocarpa Makino Eclipta alba (L.) Hassk.

Glycyrrhiza glabra L. Glycyrrhiza glabra L. Glycyrrhiza uralensis Fisch. ex DC. Marrubium peregrinum L.

Petroselinum crispum (Mill.) Fuss Chamomile flowers Eclipta alba (L.) Hassk.

Rhodiola kirilowii (Regel) Maxim Pinus pinaster ssp. Atlantica Villar Rosa rugosa Thunb.

Rhodisin

Taxifolin

Plant name

Bioactive molecule

Table 12.1 (continued)

Asteraceae

Fabaceae Lauraceae

Fabaceae

Lamiaceae

Fabaceae Fabaceae

Apiaceae Asteraceae Asteraceae

Rosaceae

Pinaceae

Crassulaceae

Plant family

Replication NS5B

Replication Replication

Entry (post-attachment) Replication

Replication Inhibition of miR122 maturation NS5B Replication Replication

Entry

Replication

NS3

Viral step or protein target

1.1– 11.3

7.3 1.1

2.5– 10.0 64.0

19.1 14.4– 24.1

4.3– 7.9

49.3– 197.1 1.7

0.77

IC50 (µM)

Liu et al. (2012) Manvar et al. (2012) (continued)

Adianti et al. (2014) Chen et al. (2013)

Adianti et al. (2014)

Haid et al. (2012)

Adianti et al. (2014) Adianti et al. (2014), Sekine-Osajima et al. (2009)

Liu et al. (2012), Manvar et al. (2012), Shibata et al. (2014)

Tamura et al. (2010)

Ezzikouri et al. (2015)

Zuo et al. (2007)

Reference

312 K. Séron et al.

Ruta angustifolia Pers. Glycyrrhiza uralensis Fisch. ex DC. G. inflata Batalin

Vitis vinifera L.

Vitisin (resveratrol tetramer)

Coumarins Chalepin Glycycoumarin

Morus alba L.

Kalanchoe pinnata (Lam.) Pers Embelia ribes Burm. f. Solanum rantonnetii Carrière

Quercetin

Moracin P

Citrus unshiu Marcov. Eriocaulon L.

Nobiletin Quercetagetin

Morus alba L.

Citrus x paradisi Macfad.

Naringenin

Stilbenes and benzofurans Moracin O

Plant name

Bioactive molecule

Table 12.1 (continued)

Rutaceae Fabaceae Fabaceae

Vitaceae

Moraceae

Moraceae

Crassulaceae Primulaceae Solanaceae

Rutaceae Eriocaulaceae

Rutaceae

Plant family

Replication Replication

Replication NS3 helicase Replication NS3 helicase Replication NS3 helicase

HCV infection NS5B RNA binding Entry Replication IRES-mediated translation NS3 Virion release Decrease viral load in patients

HCV production Assembly

Viral step or protein target

5.4 23.8– 42.0

0.006

35.6

80.8

2.8– 6.1 4.9

109.0

IC50 (µM)

Natural Products and Hepatitis C Virus (continued)

Wahyuni et al. (2014) Adianti et al. (2014), Sekine-Osajima et al. (2009)

Lee et al. (2016)

Lee et al. (2007)

Lee et al. (2007)

Aoki et al. (2014), Bachmetov et al. (2012), Gonzalez et al. (2009), Khachatoorian et al. (2012), Lu et al. (2015)

Goldwasser et al. (2011) Khachatoorian et al. (2012), Nahmias et al. (2008) Suzuki et al. (2005) Ahmed-Belkacem et al. (2014)

Reference

12 313

Caffeic acid n-octyl ester Quinones 5-O-methylembelin Embelin 2-Terpenoids Monoterpenes Loliolide

Phenolic acids and derivatives 3,5-dicaffeoylquinic acid

Diaryheptanoids Curcumin

Wedelolactone

Phyllanthaceae

Phyllanthus urinaria L.

Entry (attachment) Cell-to-cell spread

NS3 NS3

Primulaceae Primulaceae

E. schimperi Vatke E. schimperi Vatke

Entry (binding and fusion) Alteration of envelope fluidity Cell-to-cell spread Replication

NS5B

Replication

Replication

Viral step or protein target

Replication

Dipsacaceae

Zingiberaceae Vitaceae

Asteraceae

Fabaceae

Fabaceae

Plant family

Scabiosa comosa Fisch. ex Roem. & Schult. S. tschilliensis Grüning Propolis

Curcuma longa L. Vitis vinifera L.

Glycyrrhiza uralensis Fisch. ex DC. Glycyrrhiza uralensis Fisch. ex DC. Eclipta alba (L.) Hassk.

Glycyrin

Glycerol

Plant name

Bioactive molecule

Table 12.1 (continued)

2.4

46 21

1.5

8.46– 18.3

7.7– 36.1

12.5

18.8

IC50 (µM)

Chung et al. (2016)

Hussein et al. (2000) Hussein et al. (2000)

Shen et al. (2013)

Ma et al. (2015)

(continued)

Anggakusuma et al. (2014) M.-H. Chen et al. (2012), Kim et al. (2010)

Kaushik-Basu et al. (2008) Manvar et al. (2012)

Adianti et al. (2014)

Adianti et al. (2014)

Reference

314 K. Séron et al.

Phyllanthaceae

Parthenium hispidum Raf.

Flueggea virosa Roxb. Ex Willd.

Pseudoguaianolides Diterpenes Dinorditerpenes

Ursolic acid

Schizandronic acid

Oleanolic acid

Schisandra sphenanthera Rehd. Et Wils Ligustrum lucidum Ait

Cynomorium songaricum Rupr. Ligustrum lucidum Ait

Asteraceae

Cynara cardunculus L.

Grosheimol

Triterpenoids Malonyl ursolic acid hemiester

Asteraceae Asteraceae

Artemisia annua L. Cynara cardunculus L.

Oleaceae

Schisandraceae

Oleaceae

Cynomoriaceae

Asteraceae

Lamiaceae

Plant family

Lamium album L.

Plant name

Iridoids Lamiridosin A/B Sesquiterpene lactones Artemisinin Cynaropicrin

Bioactive molecule

Table 12.1 (continued)

Replication NS5B

Replication NS5B Entry (fusion)

NS3

Infection

Replication Entry Cell-to-cell spread Entry Cell-to-cell spread Replication

Entry

Viral step or protein target

2.9 10.6

2.9 10.6 11.4

5.5

5.0– 7.5

1.0

1.3

2.3

IC50 (µM)

(continued)

Kong et al. (2013, 2007)

Qian et al. (2016)

Kong et al. (2013, 2007)

Ma et al. (2009)

Chao et al. (2014)

Hu et al., (2007)

Elsebai et al. (2016)

Paeshuyse et al. (2006) Elsebai et al. (2016)

Zhang et al. (2009)

Reference

12 Natural Products and Hepatitis C Virus 315

Bupleurum chinensis D.C.

Platycodon grandiflorus (Jacq.) A. DC.

Saikosaponin b2

Triterpenoid saponins

Rutaceae Rubiaceae

Ruta angustifolia Pers.

Morinda citrifolia L.

Morinda citrifolia L.

Pseudane IX 4-Porphyrins Pyropheophorbide a

Pheophorbide

Rubiaceae

Rubiaceae

Myrioneuron faberi Hemsl.

Quinolizine alkaloids

Rubiaceae

Myrioneuron faberi Hemsl.

Celastraceae

Campanulaceae

Apiaceae

Fabaceae

Plant family

Myriberine A

Maytenus ilicifolia (Schrad.) Planch.

Glycyrrhiza glabra L.

Saponins Glycyrrhizin

3- Alkaloids APS

Plant name

Bioactive molecule

Table 12.1 (continued)

Entry Post-entry Entry Post-entry

Replication

Replication

Replication Inhibition of daclatasvir NS5A-resistant mutant Y93H Replication

Decrease core expression Virion release Entry (attachment, post-attachment, endocytosis/ fusion) Direct effect on virion Replication

Viral step or protein target

0.5

0.37

0.9– 4.7 5.1

29.6

Ratnoglik et al. (2014)

Ratnoglik et al. (2014)

Cao et al. (2014), Huang et al. (2013) Cao et al. (2014), Huang et al. (2013) Wahyuni et al. (2014)

Jardim et al. (2015)

Kim et al. (2013, 2012)

*4– 40 2.3

Lin et al. (2015)

Ashfaq et al. (2011b), Matsumoto et al. (2013)

Reference

16.1

17.0

IC50 (µM)

316 K. Séron et al.

12

Natural Products and Hepatitis C Virus

317

Table 12.2 Crude plant extracts with anti-HCV activity Plant name

Plant family

Viral step or protein target

IC50 (µg/ml)

Reference

Acacia confusa Merr. Acacia nilotica Willd. ex Delile

Fabaceae Fabaceae

Replication Replication NS3

5.0 40.5

Boswellia carteri Birdw. Cynara cardunculus L.

Burseraceae Asteraceae

23.0 299.0

Detarium microcarpum Guill. & Perr. Eclipta alba L. Hassk.

Fabaceae

NS3 Entry Cell-to-cell spread Entry

Lee et al. (2011) Hussein et al. (2000), Rehman et al. (2011) Hussein et al. (2000) Elsebai et al. (2016)

1.4

Galani et al. (2015)

Asteraceae

11.3

Manvar et al. (2012)

Embelia schimperi Vatke Entada africana Guill. & Perr.

Primulaceae Fabaceae

38.0

Hussein et al. (2000) Galani et al. (2014)

Ficus fistulosa Reinw. ex Blume Flueggea virosa Roxb. ex Willd. Glycyrrhiza uralensis Fisch. ex DC. Kalanchoe pinnata (Lam.) Pers. Khaya grandifoliola A. Juss.

Moraceae

Replication NS5B NS3 Entry Replication Entry

5.7–15.0

Phyllanthaceae

Infection

17.2

Wahyuni et al. (2013) Chao et al. (2014)

Fabaceae

Replication

8.0-(20.0

Adianti et al. (2014)

Crassulaceae

Infection

17.2

Aoki et al. (2014)

Meliaceae

2.3 27.1 10.0

Galani et al. (2016)

Ligustrum lucidum Ait

Oleaceae

Entry Replication NS5B

Limonium sinense (Girard) Kuntze Melicope latifolia (DC.) T.G. Hartley Melanolepis multiglandulosa (Reinw. ex Blume) Rchb. f. & Zoll. Morinda citrifolia L.

Plumbaginaceae

Entry

9.7

Rutaceae

Entry Replication Entry

3.5 2.1 6.2–17.1

Wahyuni et al. (2013) Wahyuni et al. (2013)

Entry Post-entry Entry

6.1-(20.6

Ratnoglik et al. (2014) Galani et al. (2015)

Replication NS3/NS5B Entry (attachment) Cell-to-cell Replication

5.0-(20.0

Euphorbiaceae

Rubiaceae

Phragmanthera capitata (Spreng.) Balle Phyllanthus amarus Schumach. & Thonn. Phyllanthus urinaria L.

Loranthaceae

Pinus pinaster ssp. atlantica Villar

Pinaceae

Phyllanthaceae Phyllanthaceae

13.1

10.5

5.7

Kong et al. (2013, 2007) Hsu et al. (2015)

Ravikumar et al. (2011) Chung et al. (2016)

Ezzikouri et al. (2015)

(continued)

318

K. Séron et al.

Table 12.2 (continued) Plant name

Plant family

Viral step or protein target

IC50 (µg/ml)

Reference

Piper cubela L.f. Platycodon grandiflorus (Jacq.) A. DC. Portulaca oleracea L. Punica granatum L. Quercus infectoria Olivier Rhus chinensis, Galla Chinese Ruta angustifolia Pers.

Piperaceae Campanulaceae

NS3 Replication

18 2.83–35

Portulacaceae Lythraceae Fagaceae Anacardiaceae Rutaceae

NS3 NS3 NS3 NS3 Replication

Solanum nigrum L. Solanum rantonnetii Carrière Stylogyne cauliflora (Mart. & Miq.) Mez Syzygium aromaticum (L.) Merr. & L.M. Perry Toona sureni (Blume) Merr.

Solanaceae Solanaceae Primulaceae

Infection Entry NS3

6.9

Hussein et al. (2000) Kim et al. (2013, 2012) Noreen et al. (2015) Reddy et al. (2014) Hussein et al. (2000) Duan et al. (2004) Wahyuni et al. (2013) Javed et al. (2011) Rashed et al. (2014) Hegde et al. (2003)

Myrtaceae

NS3

33.0

Hussein et al. (2000)

Meliaceae

Trachyspermum ammi (L.) Sprague Trichilia dregeana Sond. Vitis vinifera L.

Apiaceae

Entry Replication NS3

13.9 2.0 56.0

Wahyuni et al. (2013) Hussein et al. (2000)

Meliaceae Vitaceae

Entry Entry

16.1

Galani et al. (2015) Sharaf et al. (2012)