Honey: Current Research and Clinical Applications : Current Research and Clinical Applications [1 ed.] 9781611222838, 9781619426566

Citation preview

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012. ProQuest

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

FOOD AND BEVERAGE CONSUMPTION AND HEALTH

HONEY

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

CURRENT RESEARCH AND CLINICAL APPLICATIONS

No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

FOOD AND BEVERAGE CONSUMPTION AND HEALTH Additional books in this series can be found on Nova‘s website under the Series tab.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Additional e-books in this series can be found on Nova‘s website under the e-book tab.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

FOOD AND BEVERAGE CONSUMPTION AND HEALTH

HONEY CURRENT RESEARCH AND CLINICAL APPLICATIONS

JURAJ MAJTAN Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

EDITOR

Nova Science Publishers, Inc. New York Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‘ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book.

Library of Congress Cataloging-in-Publication Data Honey : current research and clinical applications / editor: Juraj Majtan. p. cm. Includes index. ISBN:  (eBook)

1. Honey. 2. Honey--Health aspects. I. Majtan, Juraj. TX560.H7.H66 2012 641.3'8--dc23 2011050984

Published by Nova Science Publishers, Inc. † New York Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Contents

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Preface

vii

Chapter I

Selected Topics on Honey Volatile Organic Compounds Research Igor Jerković

Chapter II

Honey Melanoidins: Emerging Novel Understanding on the Mechanism of Antioxidant and Antibacterial Action of Honey Katrina Brudzynski

1

17

Chapter III

Anticancer Activity of Honey and Its Phenolic Components Saravana Kumar Jaganathan and Mahitosh Mandal

39

Chapter IV

Honey and Microbes Laïd Boukraâ, Yuva Bellik and Fatiha Abdellah

61

Chapter V

Anti-Biofilm Activity of Natural Honey against Wound Bacteria Juraj Majtan , Jana Bohova, Miroslava Horniackova and Viktor Majtan

Chapter VI

Beyond the Direct Antibacterial Activity of Honey in Wound Healing: Immunomodulatory Effects Juraj Majtan

83

107

Chapter VII

Possible Anti-Diabetic Effects of Honey Karsten Münstedt and Philipp Teichfischer

121

Chapter VIII

Honey and Male Reproductive Health Mahaneem Mohamed

131

Chapter IX

Topical Application of Honey Biswa M. Biswal and Rajan Saini

143

Chapter X

Honey in Treatment of Burn Wounds Mutya Subrahmanyam

173

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

vi

Contents

Chapter XI

The Efficacy of Honey Dressing on Chronic Wounds and Ulcers Kamaruddin Mohd Yusoff , Zainabe Syed Akka, Anwar Suhaimi, Mohd Razif Mohd Ali and Mohd Yassim Mohd Yusoff

185

Chapter XII

Honey for Treating Eye Diseases Juraj Majtan, Martin Cernak, Nora Majtanova and Andrej Cernak

197

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Index

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

207

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Preface In last few years, with increasing frequency, modern medicine directs attention to natural products with biological and therapeutic properties and their use in clinical practice. The major arguments for implementing a natural product such as honey, are the low cost and absence of antimicrobial resistance risk. The antibiotic resistance is a serious problem worldwide and only two new antibiotics were approved in the last 4 years. We are now facing a global antibiotic resistance crisis that may negatively affect immunocompromised patients, infants and elder peoples. Historically, honey has been used in the treatment of a broad spectrum of wounds, in the treatment of cough and metabolic disorders and many other human diseases. It is cheap, readily available and shows no evidence of reduced efficacy over time. Due to a lack of strong scientific evidence to support its efficacy and its use in clinical practice, medical authorities tended to be skeptical. However, the current situation has positively changed and more and more clinical and research studies using honey have been published in peerreviewed journals. Isolation and characterization of new compounds from natural honey allow us to fully characterize the biological activities of honey. In the present book we have attempted to provide an updated overview of the current research and clinical application of honey. As a reading guide, chapters are organized around three main themes: the isolation and identification not-well studied compounds from honey; the biological activities; the clinical application in burn/wound management and in ophthalmology. Contributions gathered at the begging will lead the reader through the identification of volatile organic compounds from honey exhibited health and therapeutic qualities (Chapter I) and the role of Maillard reaction as an origin of bioactive compounds such as methylglyoxal and malanoidins in honey (Chapter II). Honey offers a broad spectrum of biological activities. Chapters III to VII will thus discuss antimicrobial, anti-biofilm, anti-inflammatory, anticancer, immunomodulatory and anti-diabetic effects of honey. Honey is traditionally consumed for enhancement of fertility and vitality among males in some populations. Therefore, the possible beneficial effect of honey on male reproductive health is described in Chapter VIII. Finally, the last chapters (Chapters IX, X, XI and XII) of the book disclose the direct medical and therapeutic potential of honey for treatment of burns and chronic wounds as well as ocular infections.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

viii

Juraj Majtan

This book has been written in order to stimulate and accelerate the clinical use of honey. In addition, clinicians and other health-care professionals as well as patients may now see honey in a positive light.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Dr. Juraj Majtan Bratislava, Slovak Republic November 2011

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

In: Honey: Current Research and Clinical Applications ISBN: 978-1-61942-656-6 Editor: Juraj Majtan © 2012 Nova Science Publishers, Inc.

Chapter I

Selected Topics on Honey Volatile Organic Compounds Research Igor Jerković Department of Organic Chemistry, Faculty of Chemistry and Technology, University of Split, Croatia

Abstract

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Five sources of honey volatile organic compounds (VOCs) have been proposed: 1) plant constituents (phytochemicals), 2) phytochemicals transformation by the bees, 3) direct compounds generation by the bees or compounds transfer from the combs environment, 4) their generation by honey thermal processing and 5) microbial or environmental contaminants. Honey VOCs are present at low concentrations as more or less complex mixtures of compounds with different organic functionalities. Ultrasonic solvent extraction (USE) has been used a suitable method to obtain reliable honey fingerprint of more and less volatile (low- and high-molecular) compounds in comparison to hydrodistillation (HD) or simultaneous distillation-extraction (SDE) that generate thermal artefacts. However, optimization of the extraction solvent polarity is necessary to obtain reliable VOCs profile. In addition, the use of headspace solid-phase microextraction (HS-SPME) for the same honey type often provides distinct headspace VOCs profiles in comparison to USE, and selection of HS-SPME fibre is needed prior to the extraction. The immediate contribution of the bees and combs to honey volatiles under controlled food-flow conditions (saccharose solution, no plant source) provided "saccharose honey" with VOCs constituted mainly of higher alcohols and saturated linear long-chain hydrocarbons. Identified chemical structures were related to the composition of combs and cuticular waxes, and less to the bee pheromones and all of them can be excluded as reliable botanical-origin biomarkers. On the other hand, as one of the most typical features of a particular honey, norisoprenoids, terpenes, benzene derivatives and others have been proposed as a quality marker for the authenticity of the floral origin. 

Department of Organic Chemistry, Faculty of Chemistry and Technology, University of Split, N. Tesle 10/V, 21000 Split, Croatia, E-mail: [email protected].

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

2

Igor Jerković Nowadays, more than 600 compounds have been identified as volatiles in honeys of different floral origins. Subsequently, some specific-marker volatile compounds have been suggested, e.g., methyl anthranilate for Citrus spp. honeys, 3- and 2aminoacetophenone for Castanea sativa honey, methyl syringate for Asphodelus microcarpus Salzm. et Viv. honey and others. Only a few compounds seem to be specific, and many of them can be found in variable concentrations in various honey types. In addition, isolated VOCs could exhibit health and therapeutic qualities. Therefore, selected USE extracts were further investigated in order to unlock their antiradical and antioxidant potential. The extracts showed remarkably higher antiradical capacity (DPPH assay) and antioxidant capacity (FRAP assay) in comparison to the honey. These findings reveals new potential of USE honey extracts for further more detail research on biological activity.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Introduction The chemical composition of honey is highly dependent on the plant origin and/or the nectar foraged by bees, but also on other factors such as bee species, geographic area, season, mode of storage, and even harvest technology and conditions. Determination, control and declaration of the honey botanical origin is still an open challenge since ubiquitous method of pollen analysis (melissopalynology) is not always adequate when pollen is underrepresented in the honey or for the honeydew characterization [1, 2]. In addition, melissopalynology is very time consuming and depends on the expert ability and judgment. Other traditional methods of honey characterization include organoleptic evaluation and determination of basic physico-chemical parameters [3]. However, the precise definition of a specific honey type is a very complex because the values of individual parameters vary greatly. The variation is due to the fact that bees do not collect nectar from one plant species only. In recent years, researchers have identified a number of phytochemicals in various honey types [4]. Phytochemicals are substances transferred form the plants to the honey and could be used for honey botanical characterization, but also can exhibit health-promoting activities (e.g. antioxidants or antimicrobial agents). It is known that accumulation of phytochemicals depends on climatic conditions (sunlight, moisture), soil characteristics, and other factors. Found phytochemicals in different honeys can be divided into the following groups: volatile compounds, phenolic compounds, carbohydrates, nitrogen containing compounds and other minor compounds.

Origin and Chemical Classification of Honey Volatile Organic Compounds Volatile organic compounds (VOCs) derived from the plant sources are likely to be responsible for the specific aroma of unifloral honeys and consequently were suggested as helpful for their classification. Research on honey volatiles began in the early 1960‘s and until now more than 600 compounds have been found [5]. Not all VOCs have impact on honey aroma. In general, the impact of a given compound depends on the extent to which the concentration exceeds its odor threshold (there is the possibility of synergetic and/or

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Selected Topics on Honey Volatile Organic Compounds Research

3

antagonistic interactions among different compounds). Taking into consideration that composition of some honey types has not yet been studied and that extraction and analyses methods are constantly improving, it is very likely that the number of identified compounds will further increase. Five sources of honey volatile organic compounds (VOCs) have been proposed [5, 6]:     

plant constituents (phytochemicals), phytochemicals transformation by the bees, direct compounds generation by the bees or compounds transfer from the combs environment, their generation by honey thermal processing or/and storage, microbial or environmental contaminants.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Honey VOCs are present at low concentrations as more or less complex mixtures. According to the organic functional groups the honey VOCs belong to 7 major groups: aldehydes, ketones, acids, alcohols, esters, hydrocarbons and cyclic compounds. The honey naturally VOCs belong mainly to the classes of terpenes, norisoprenoides, benzene derivatives and others. Terpenes are naturally occurring VOCs containing linked isoprene units (C5-units) derived from mevalonate or deoxyxylulose phosphate biosynthetic pathways via isopentenylpyrophosphate (IPP). They are typical compounds of many essential oils (e.g. lavender, sage, laurel and other aromatic plants) including semiterpenes (1 C5-unit), monoterpenes (2 C5-units) and sesquiterpenes (3 C5-units). However, chemical composition of the plant essential oil and corresponding honey VOCs is only in partial agreement and more often only a few essential oil components are found in the honey.

Figure 1. a) Monoterpenes generation from 2-IPP and 3-IPP, b) Structures of ubiquitous monoterpenes present in different honeys: cis- and trans-linalool oxides (1, 2), linalool (3), hotrienol (4), lilac aldehyde isomers (5, R = CHO), lilac alcohol isomers (5‟, R = CH2OH), (E) and/or (Z) 8hydroxylinalool (6), dill ether isomers (7), *- chiral carbon.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

4

Igor Jerković

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

The most abundant terpenes in all honeys are regular monoterpenes (composed of 2 linked C5-units "head to tail") derived from geranyl pyrophosphate (GPP) such as cis- and trans-linalool oxides, linalool, hotrienol, lilac aldehyde isomers, lilac alcohol isomers, (E)and/or (Z)-8-hydroxylinalool, dill ether isomers and others (Figure 1). Within the hive, warm (about 30 oC) and acidic pH (ripe honey) conditions exist. Moreover, it is known that nectar contains various amounts of enzymes, as it is also enriched with others by the bees. As a conclusion, various reactions are expected to occur in honey during ripening that can influence the phytochemicals originally transferred from the plants. For example, oxidation reactions are favored in citrus honey and linalool was found in very low proportions, compared to the amounts present in the nectar [7, 8]. Starting with linalool (3) an array of compounds can be formed [7, 8], as shown in Figure 2. Considering the acidic nature of honey, reduction reactions are not favored and it is plausible to assume that (E)-8-hydroxylinalool (8) is transformed by enzymatic ω-oxidation to lilac alcohols (5) that undergo oxidation to yield lilac aldehydes (5'). Dill ether (7) is produced from (E)-8-hydroxylinalool (8) via the allylic rearranged 8-hydroxygeraniol (10).

Figure 2. Linalool derived compounds (3) and formation of hotrienol (4).

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Selected Topics on Honey Volatile Organic Compounds Research

5

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Direct hydroxylation of linalool at the C8 position forms two isomers of 8hydroxylinalool which can give various products. Alternatively, epoxidation of linalool gives 6,7-epoxylinalool (11), which undergoes further oxidation generating furan linalool oxides (1, 2) in acidic conditions or heating and/or 2,6-dimethyl-3,7-octadiene-2,6-diol (12). Hotrienol is a known thermally generated product arising after the thermal degradation of either 2,6-dimethyl-3,7-octadiene-2,6-diol (12) or its glycoconjugate form (13) or the allylic rearranged 3,7-dimethyl-1,7-octadiene-3,6-diol (14). It is also possible to arise from (E)-8hydroxylinalool (15) and (Z)-8-hydroxylinalool (16). Nevertheless, some quantity seems to exist in non-thermally treated honey and has been considered as indicator of the honey freshness detected with much lower proportions in unripe than in ripe honey [9]. Norisoprenoides are carotenoid derived compounds with 3,5,5-trimethylcyclohex-2-enic structures (degraded-carotenoid-like structures) found in honeys of different botanical origins such as eucalyptus, thyme, heather, sulla and strawberry-tree [10, 11]. Norisoprenoids have been found as aroma contributors in a number of different matrices, such as tobacco, tea, flower scents, fruits, spices, grapes and wine. In addition to the most widespread C13-norisoprenoids, volatile C9-, C10-, and C11carotenoid metabolites are also frequently detected in nature [12]. The cleavage of the carotenoid chain is generally considered to proceed at different double bonds (Figure 3).

Figure 3. Relationships between the classes of degraded carotenoids (C9, C10, C13 and C15 norisoprenoids) with compounds identified in honeys: trans-β-damascenone (17), 3-hydroxy-trans-βdamascenone (18), 3-oxo-α-ionol (19), 3-hydroxy-7,8-dihydro-α-ionol (20), α-isophorone (21), 4hydroxy-α-isophorone (22), 4-ketoisophorone (23), 2-hydroxy-4-ketoisophorone (24), safranal (25), dehydrovomifoliol (26).

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

6

Igor Jerković

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Cleavage of C11=C12 bond produces C15-norisoprenoids via abscisic acid. Abscisic acid, a well-known growth hormone, is formed after cleavage of C40- carotenoids. Zeaxanthin is the first committed abscisic acid precursor [13]. A series of enzyme-catalyzed epoxidations and isomerizations, and final cleavage of the C40-carotenoid by a dioxygenation reaction yields the precursor xanthoxin, which is then further oxidized to abscisic acid [13]. trans,cisAbscisic acid along with trans,trans-abscisic acid were found in New Zealand Salix nectar honey [14]. Dehydrovomifoliol probably arises through degradation of abscisic acid. Cleavage of C9=C10 bond generates C13-norisoprenoids (trans-β-damascenone, 3-hydroxytrans-β-damascone, and 3-oxo-α-ionol). Safranal arises from the cleavage of C7=C8 bond. Degradation of carotenoids producing C9-norisoprenoids can yield α-isophorone, 4-hydroxyα-isophorone, 4-oxoisophorone, and 2-hydroxy-4-oxoisophorone. Benzene derivatives are derived from shikimate biosynthetic pathway and the most abundant ones in many honey types are phenylpropane derivatives and benzoic acid derivatives. The shikimate pathway begins with a coupling of phosphoenolpyruvate and Derythrose-4-phosphate. Shikimic acid itself is formed from 3-dehydroquinic acid via 3dehydroshikimic acid by dehydration and reduction steps. Ubiquitous phenylacetaldehyde can be generated in a honey from amino acid phenylalanine, either with the help of enzymes or by Strecker degradation (Figure 4). The content of phenylacetaldehyde in the honey depends on the phenylalanine content and on the storing conditions. Amino acid analyses have shown that the contents of phenylalanine in honeys differ largely and depend on the botanical origin of the honeys [15]. Consequently, different phenylacealdehyde contents can be expected in different honey types. Many researches have shown that phenylacetaldehyde is widespread and common in all honeys [16, 17] and it can not be consider as specific marker of the honey botanical origin.

Figure 4. Formation of phenylacetaldehyde.

Shikimate-pathway derivatives are often single para- and ortho-hydroxylation products of phenolic acids in distinction from meta-hydroxylation in the acetate pathway [18]. The relationship between major VOCs (originating from the shikimate pathway) identified in Salix spp. honeydew is presented in Figure 5.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Selected Topics on Honey Volatile Organic Compounds Research

7

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Figure 5. Relationship between selected honey VOCs from shikimate pathway [19].

Simple hydroxybenzoic acids such as found 4-hydroxybenzoic acid (p-salicylic acid) can be formed early in the shikimate pathway, but alternative routes exist in which cinnamic acid derivatives are cleaved at the double bond and lose two carbons from the side-chain. transCinnamic acid is produced after deamination (NH3 elimination) of L-phenylalanine by phenylalanine-ammonium lyase found exclusively in plants [20]. Salicylic acid (2-hydroxybenzoic acid) can arise in plants by hydroxylation of benzoic acid or by side-chain cleavage of 2-coumaric acid which is formed by hydroxylation of cinnamic acid (in microorganisms it is synthesized directly from isochorismic acid). Methyl salicylate can be derived by methylation of salicylic acid. Glucoside salicin, found in many willow species (chemotaxonomic marker of the genus Salix) is not derived from salicylic acid, but probably via glucosylation of salicylaldehyde and then reduction of carbonyl [18]. On the other hand degradation of Salix spp. glycosides releases different aglycones with structures related to saligenin (2-hydroxybenzyl alcohol) at acidic pH, probably by βglucosidase activity and others [21]. Another example of shikimate acid derivative found in the honeys is methyl syringate formed by deamination of tyrosine followed by sequence of hydroxylation, methylation, H2O addition, reverse aldol, oxidation and reverse Claisen reactions from 4-coumaric acid (Figure 6). Alternative route exists for methyl syringate formation by lignin breakdown using

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

8

Igor Jerković

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

ligninolytic enzymes as laccase [22]. It is interesting to note that methyl syringate was detected in honeys obtained from plants of different botanical families but only the asphodel honey reached the highest level [23]. Other volatiles found in the honey are derived from other biosynthetic pathways, heat treatment, storage, contaminants and/or others. Well known heat derived VOCs and few contaminants will be discussed further. Heating honey at temperatures as low as 50 oC leads to the formation of new volatile compounds, and their concentration vary significantly as a result of different heating conditions [24]. These heat derived compounds are known as artefacts (substances not present in original sample but produced during sample treatment).

Figure 6. Formation of methyl syringate.

It is well known that the Maillard or non-enzymatic browning reaction has a great impact on food flavors. The effect of different amino acids on the formation of Maillard related compounds (Figure 7) has been widely studied. As a result of the Maillard reaction, pyrazines, pyrroles, pyrones and, to a lesser extent, furanones increased if honey was submitted to the heating process, whereas alcohols, esters and acids remained unchanged [25]. The basic reaction is between free amino acids and reducing sugars present in the honey, generating Amadori compounds (from carbohydrates and amino groups) as Maillard reaction products. Dicarbonyl compounds are required for pyrazine formation from isoleucine, leucine, phenylalanine and methionine [26] (Figure 8). Also, 3-methyl-1-butanol in fresh honey arises from Strecker degradation or biodegradation of valine, leucine or isoleucine. These reactions produce, in particular, methylpyrazines. Varroa destructor Anderson and Trueman origin of the varroosis, is the most damaging parasite in honey bees (Apis mellifera L.) and represents a serious infestation of bee colonies. Components of the essential oil such as thymol, linalool, or camphor have been highly effective in controlling the varroosis. Different combinations of such components as well as different thymol application methods have been studied. Nevertheless, difficulties with

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Selected Topics on Honey Volatile Organic Compounds Research

9

natural treatments are the high level of residues (>0.8 mg/kg) found in honey after application [27]. This does not represent a risk for human health but may change the taste of the honey. Bogdanov et al. [28] found that the taste threshold of thymol in acacia and rape honey was ≈1.1-1.3 mg/kg honey.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Figure 7. Short scheme of carbonyl compounds produced from Maillard reaction.

Figure 8. Strecker degradation and formation of pyrazine.

Modern Methods of Honey Volatiles Extraction Naturally occurring VOCs are present in the honey at very low concentrations as more or less complex mixtures of volatile components of different functionality and relatively low molecular weight [3, 29]. Since artefacts can be generated due to the impact of heat (e.g. Maillard reactions), appropriate methods for volatiles isolation should be applied to obtain

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

10

Igor Jerković

representative honey volatile fingerprint. Most often it is necessary to combine the results of solvent extraction and headspace extraction to obtain representative and reliable profile of honey more and less volatile compounds. Solvent extraction methods - Solvent extraction (SE) at room temperature has been widely used for the isolation of honey VOCs (low- and high-molecular) with major advantage for obtaining thermolabile compounds. However, optimization of the extraction solvent polarity is necessary to obtain reliable honey VOCs profile. Ultrasound-assisted solvent extraction (USE) significantly reduces extraction times in comparison with traditional extraction and has been applied for honey VOCs isolation [30, 31]. The mechanical effect of ultrasound provides a greater penetration of solvent into matrix, via cavitation effects, and improves the extraction. Column extraction method is an alternative technique for isolating VOCs without the use of heat, and combines both solvent and a porous polymer. Honey is dissolved and passed through a column packed with porous polymer and the adsorbed constituents are then eluted with solvent [32]. Simultaneous steam distillation-extraction method (SDE) with the intention of avoiding sugar interference, proposed a two-step protocol which included preliminary extraction with acetone or dichloromethane followed by simultaneous Likens–Nickerson steam distillation and solvent extraction [33]. Since heat treatments can lead to artefacts, a modified version of the Likens–Nickerson method was adapted by using vacuum at room temperature. Headspace extraction methods - Static headspace (SHS) analysis has not been widely applied to analyze honey VOCs because of their low concentrations and low recoveries obtained for semi-volatile compounds [29]. Dynamic HS purge-and-trap techniques (DHS) have the advantage of identification and quantification of a wide range of volatiles, with sensitivity higher than that of static headspace [34]. Headspace solid-phase microextraction (HS-SPME) has recently been developed as a rapid, inexpensive and solvent-free technique [35]. This technique uses a fine fused silica fibre with a polymeric coating to extract organic compounds from the matrix and directly transfer them into the injector of a gas chromatograph for thermal desorption and analysis. In general, volatile extraction is best achieved when the polarity of the fibre matches the polarity of the analyte. Since gas chromatography-mass spectrometry (GC-MS) combines high separation efficiency and sensitivity and provides qualitative and quantitative data, it is usually the technique of choice for the analysis of the obtained extracts. Table 1. Different types of fibres (stationary phases and characteristics) Stationary phase/coating thickness PDMS: polydimethylsiloxane PA: polyacrilate CW/DBV: carbowax/divinylbenzene DVB/CAR/PDMS: divinylbenzene/carboxene/polydimethylsiloxane

Characteristics non-polar, for volatile compounds polar, for polar semivolatile compounds non-polar, for volatile compounds, amines and nitroaromatics compounds for odors

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Selected Topics on Honey Volatile Organic Compounds Research

11

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Organic Volatiles as Potential Chemical Markers of the Honey Botanical Origin One of the major concerns in chemical fingerprinting of the honey volatiles is to recognize those compounds that are generated by the bees or transferred from the combs environment. Namely, those compounds are ubiquitous in all honeys and can not be used for characterization of different honey types, although they can be present in significant percentages among analyzed VOCs. There are reports on the volatile emission of honeybees [36]. The colony odor on the cuticles of honeybees is a combination of cuticular hydrocarbons and compounds of the wax combs of the nest. The compounds from the comb wax include pheromones as well as floral scents brought to the nest via pollen and nectar. Compounds which are minor components of pheromonal secretions could become predominant in the vapor phase, if their volatility is high and vice versa. For example, little (E)-9-oxodec-2-enoic acid, the classical queen substance and predominant compound of head extracts, was found in the vapor phase [37]. In order to identify compounds that are generated by the bees or transferred from the combs environment research have been performed [38] on immediate contribution of the bees and combs to honey volatiles (blank-trial probe for chemical profiling of honey biodiversity). The bees were closed in a hive containing empty combs under controlled food-flow conditions (saccharose solution). The obtained saccharose honey probe samples were extracted and analyzed. A total of 66 compounds were identified. Higher alcohols made up ca. 50% of the total volatiles, mainly (Z)-octadec-9-en-1-ol, hexadecan-1-ol, and octadecan-1ol, with minor percentages of undecan-1-ol, dodecan-1-ol, tetradecan-1-ol, pentadecan-1-ol, and heptadecan-1-ol. Other abundant compounds were saturated long-chain linear hydrocarbons, C10–C25, C27, and C28, particularly C23, C25 and C27). Identified chemical structures were related to the composition of combs and cuticular waxes, and less to the bee pheromones. These findings can be considered as blank-trial probe (no plant source) for honey chemical profiling [38]. In order to distinguish different unifloral honeys it has been proposed to search for unique and characteristic VOCs in each honey type. It was reported that several honey types may be distinguished by characteristic compound. Methyl anthanilate (14) was reported as marker of citrus (Citrus spp.) honey [7], 3-aminoacetophenone (15) and 2-aminoacetophenone (16) for chestnut (Castanea sativa) honey [31], 2-hydroxy-5-methyl-3-hexanone (17) and 3-hydroxy5-methyl-2-hexanone (18) for eucalyptus honey [39] or methyl syringate (Figure 9) for Asphodelus microcarpus honey [23]. The use of individual marker compounds for the classification of unifloral honeys is probably only reasonable when they are quantitatively determined with defined specific concentration ranges for the unifloral honeys. However, only a few honey types contain specific compound, while the majority of honey samples are characterized by several compounds (e.g. benzoic acic, decanoic acid, dehydrovomifoliol for heather (Calluna vulgaris) honey [40], vomifoliol and methyl syringate for Mentha spp. honey [41]). Several groups of phytochemical VOC markers of floral origin may be differentiated belonging mainly to terpenes, noriozoprenoides and phenylpropane derivatives.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

12

Igor Jerković

Figure 9. Characteristic volatile organic compounds in honey.

The nectar from various flowers contributes to the production of every honey sample; therefore, the researchers should be very careful proposing the compounds found in low concentrations as floral markers. Moreover, this problem becomes even more remarkable when conclusions are made from a small number of samples. In addition, many compounds of biological and botanical origin found in honey are not stable (particularly volatile substances), and their structures may transform in the course of honey maturation and storage [5].

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Honey Organic Volatiles as Bioactive Compounds Health and therapeutic attributes of honey includes antimicrobial activity, promotion of wound healing, antioxidant activity along with energy source and prebiotic potential [4]. It is now recognized that many honey phytochemicals are bioactive compounds that can exhibit health-promoting activities such as antioxidant and/or antimicrobial activity. Bioactive capacity of different honeys varies by floral source as well by processing and storage conditions. Little information is available on the potential antioxidant activity of extracted honey VOCs, but several recent papers have been unlocking their potential. Honey is known to be rich in both enzymatic and non-enzymatic antioxidants. In vitro experiments on the inhibition of oxidation in different model systems (FRAP (ferric reducing antioxidant power) assay, DPPH (1,1-diphenyl-2-picrylhydrazyl) method, ORAC (oxygen radical absorbance capacity), TEAC (Trolox equivalent antioxidant activity) and others) using various honeys demonstrated a wide variation in the antioxidant capacity among floral sources [4]. In most cases it is necessary to use several tests to obtain good reliability. Components that were identified and/or quantified as honey antioxidants included phenolic compounds, ascorbic acid, the enzymes glucose oxidase, catalase, peroxidase and others. Additional research on single phenolic and other compounds in honey indicates that the antioxidant capacity is due to combination of a wide range of honey active compounds beyond phenolics [4]. In general, honey ultrasonic solvent extracts showed remarkably higher antiradical capacity (DPPH assay) and antioxidant capacity (FRAP assay) in comparison to the honey [42, 43]. These findings indicate the importance of USE extracts, not just for analytical purposes, but also reveals new potential for further antioxidant and biological activity research particularly in the scope of their chemical composition and isolation of pure

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Selected Topics on Honey Volatile Organic Compounds Research

13

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

compounds. The scavenging ability of the series of concentrations of the Amorpha fruticosa honey solvent extracts and the corresponding honey samples were tested by a DPPH assay [42] (Figure 10). Approximately 25 times lower concentration ranges (up to 2 g/L) of the extracts exhibited significantly higher free radical scavenging potential with respect to the honey samples. Antiradical activity (DPPH assay) of oak (Quercus frainetto Ten.) honeydew samples was 4.5 and 5.1 mmol TEAC/kg [43]. Corresponding ultrasonic solvent extracts showed several dozen times higher antiradical capacity in comparison to the honeydew. Antioxidant capacity (FRAP assay) of honeydew samples was 4.8 and 16.1 mmol Fe 2+/kg, while the extracts obtained by pentane and diethyl ether (1 : 2 v/v) showed antioxidant activity of 374.5 and 955.9 Fe2+/kg, respectively, and the dichloromethane extracts 127.3 and 101.5 mmol Fe2+/kg.

Figure 10. DPPH reduction percentage against increasing concentration of A. fruticosa honey extracts [42] obtained by ultrasonic solvent extraction with pentane : diethyl ether = 1 : 2 v/v.

A number of honey characteristics contribute to its antimicrobial activity [4]. However, well known honey peroxide-generating system does not account for all of the observed antibacterial activity. Support for the existence of non-peroxide antimicrobial factors comes from reports of continued activity after honey heat treatment, thereby inactivating the glucose oxidase, and after honey has been treated with catalase to remove the peroxide activity. Several VOCs with antibacterial activity are found in honey in small quantities that are too low to contribute significantly to antibacterial activity: terpenes, benzyl alcohol, 3,5dimethoxy-4-hydroxybenzoic acid (syringic acid), methyl-3,5-dimethoxy-4-hydroxybenzoate (methyl syringate), 3,4,5-trimethoxybenzoic acid, 2-hydroxy-3-phenylpropionic acid, 2hydroxybenzoic acid and 1,4-dihydroxybenzene [4].

Conclusion Volatile compound identification (particularly phytochemicals) with the purpose of assessing the botanical origin of honey has the potential to be useful strategy. However, only

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

14

Igor Jerković

a few honey types contain specific compound, while the majority of honey samples are characterized by several compounds (mainly terpenes, norisoprenoides and phenylpropane derivatives). In addition, honey VOCs can posses antioxidant and/or antimicrobial potential that has been unlocked recently. Honey solvent extracts containing VOCs showed remarkably higher antiradical and antioxidant capacities in comparison to the honey.

References

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

[1]

Bianchi, F., Careri, M. and Musci, M. (2005). Volatile norisoprenoids as markers of botanical origin of Sardinian strawberry-tree (Arbutus unedo L.) honey: Characterisation of aroma compounds by dynamic headspace extraction and gas chromatography–mass spectrometry. Food Chemistry, 89, 527-532. [2] Castro-Vázquez, L., Díaz-Maroto, M. C. and Pérez-Coello, M. S. (2006). Volatile Composition and Contribution to the Aroma of Spanish Honeydew Honeys. Identification of a New Chemical Marker. Journal of Agricultural and Food Chemistry, 54, 4809-4813. [3] Anklam, E. (1998). A review of the analytical methods to determine the geographical and botanical origin of honey. Food Chemistry, 63, 549-562. [4] The National Honey Board. (2002). Honey - health and therapeutic qualities. Available from: http://www.honeystix.com/HoneyStix/ compendium.pdf. [5] Kaškoniené, V. and Venskutonis, P. R. (2010). Floral Markers in Honey of Various Botanical and Geographic Origins: A Review, Comprehensive Reviews in Food Science and Food Safety, 9, 620-634. [6] Cuevas-Glory, L. F., Pino, J. A., Santiago, L. S., Sauri-Duch, E. (2007). A review of volatile analytical methods for determining the botanical origin of honey. Food Chemistry, 103, 1032-1043. [7] Alissandrakis, E., Tarantilis, P. A., Harizanis, P. C. and Polissiou, M. (2007). Aroma investigation of unifloral Greek citrus honey using solid-phase microextraction coupled to gas chromatographic–mass spectrometric analysis, Food Chemistry, 100, 396–404. [8] Alissandrakis, E., Daferera, D., Tarantilis, P. A., Polissiou, M. and Harizanis, P. C. (2007). Ultrasound-assisted extraction of volatile compounds from citrus flowers and citrus honey, Food Chemistry, 100, 396–404. [9] Rowland, C. Y., Blackman, A. J., D_ Arcy, B., and Rintoul, G. B. (1995). Comparison of organic extractives found in leatherwood (Eucryphia lucida) honey and leatherwood flowers and leaves. Journal of Agricultural and Food Chemistry, 43, 753–763. [10] Bianchi, F., Careri, M. and Musci, M. (2005). Volatile norisoprenoids as markers of botanical origin of Sardinian strawberry-tree (Arbutus unedo L.) honey: Characterisation of aroma compounds by dynamic headspace extraction and gas chromatography–mass spectrometry, Food Chemistry, 89, 527–532. [11] Jerković, I., Tuberso, C. I. G., Gugić, M. and Dragan Bubalo, D. (2010). Composition of Sulla (Hedysarum coronarium L.) Honey Solvent Extractives Determined by GC/MS: Norisoprenoids and Other Volatile Organic Compounds, Molecules 2010, 15, 6375-6385.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Selected Topics on Honey Volatile Organic Compounds Research

15

[12] Winterhalter, P. and Russell, L. (2002). Carotenoid-derived aroma compounds; American Chemical Society, Washington DC, USA, pp. 1-17. [13] Nambara, E. and Marion-Poll, A. (2005). Abscisic acid biosynthesis and catabolism. Annual Review of Plant Biology, 56, 165–185. [14] Tan, S. - T., Wilkins, A. L., Holland, P. T., McGhie, T. K. (1989). Extractives from New Zealand unifloral honeys. 2. Degraded carotenoids and other substances from heather honey. Journal of Agricultural and Food Chemistry, 37, 1217-1221. [15] Cometto, P. M, Faye, P. F., Di Paola Naranjo, R. D., Rubio, M. A.and Aldo, M. A. J. (2003). Comparison of free amino acids profile in honey from three Argentinian regions. Journal of Agricultural and Food Chemistry, 51, 5079–5087. [16] Radovic, B. S., Careri, M., Mangia, A., Musci, M., Gerboles, M. and Anklam E. (2001). Contribution of dynamic headspace GC-MS analysis of aroma compounds to authenticity testing of honey. Food Chemistry, 72, 511-520. [17] Soria, A. C., Martínez-Castro and I., Sanz, J. (2003). Analysis of volatile composition of honey by solid phase microextraction and gas chromatography-mass spectrometry, Journal of Separation Science, 26, 793-801. [18] Dewick, P. M. (2002). The shikimate pathway: aromatic amino acids and phenylpropanoids. In: Medicinal natural products: a biosynthetic approach, 2nd edn. Wiley, New York, pp 121–164. [19] Jerković, I., Marijanović, Z., Tuberoso, C. I. G., Bubalo, D. and Kezić, N. (2010). Molecular diversity of volatile compounds in rare willow (Salix spp.) honeydew honey: identification of chemical biomarkers, Molecular Diversity, 14, 237–248. [20] Steeg, E. and Montag, A. (1988). Minorbestandteile des Honigs mit Aroma-Relevanz. I. Aromatische Carbonsäuren und Ester aromatischer Carbonsäuren, 84, 103–108. [21] Ruuhola, E., Julkunen-Tiitto, R., Vainiotalo, P. (2003). In vitro degradation of willow salicylates, Journal of Chemical Ecology, 29, 1083–1097. [22] Kulys, J., Krikstopaitis, K., Ziemys, A. and Schneider, P. (2002). Laccase catalyzed oxidation of syringates in presence of albumins. Journal of Molecular Catalyses B: Enzymatic, 18, 99–108. [23] Tuberoso, C. I. G., Bifulco, E., Jerković, I., Caboni, P., Cabras, P. and Floris, I. (2009). Methyl Syringate: A Chemical Marker of Asphodel (Asphodelus microcarpus Salzm. et Viv.) Monofloral Honey, Journal of Agricultural and Food Chemistry, 57, 3895–3900. [24] Visser, R., Allen, J. and Mand Shaw, J. G. (1988). The effect of heat on the volatile flavour fraction from a unifloral honey, Journal of Apiclutural Research, 27, 175–181. [25] Bonvehí, J. S. and Ventura Coll, F. V. (2003). Flavour index and aroma profiles of fresh and processed honeys, Journal of the Science of Food and Agriculture, 83, 275– 282. [26] Yaylayan, V. A. (2003). Recent Advances in the Chemistry of Strecker Degradation and Amadori Rearrangement: Implications to Aroma and Color Formation, Food Science and Technology Research, 9, 1–6. [27] Bogdanov, S. (2006). Contaminants of bee products, Apidologie, 37, 1–18. [28] Bogdanov, S., Kilchenmann, V., Imdorf, A. and Fluri P. (1998). Residues in honey after application of thymol against varroa using the Frakno Thymol Frame, American Bee Journal, 138, 610–611.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

16

Igor Jerković

[29] Cuevas-Glory, L. F., Pino, J. A., Santiago, L. S. and Sauri-Duch, E. (2007). A review of volatile analytical methods for determining the botanical origin of honey, Food Chemistry, 103, 1032–1043. [30] Alissandrakis, E., Daferera, D., Tarantalis, P. A., Polissiou, M., and Harizanis, P. C. (2003). Ultrasound-assisted extraction of volatile compounds from citrus flowers and citrus honey, Food Chemistry, 82, 575–582. [31] Jerković, I., Mastelić, J., Marijanović, Z., Klein, Ž. and Jelić, M. (2007). Comparison of hydrodistillation and ultrasonic solvent extraction for the isolation of volatile compounds from two unifloral honeys of Robinia pseudoacacia L. and Castanea sativa L., Ultrasonics Sonochemistry, 14, 750–756. [32] Shimoda, M., Wu, Y., and Osajima, Y. (1996). Aroma compounds from aqueous solution of haze (Rhus sucedánea) honey determined by absorptive column chromatography, Journal of Agricultural and Food Chemistry, 44, 3913–3918. [33] Bicchi, C., Belliardo, F., and Fratinni, C. (1983). Identification of the volatile components of some piedmontese honeys, Journal of Apicultural Research, 22, 130– 136. [34] Radovic, B. S., Careri, M., Mangia, A., Musci, M., Gerboles, M., Anklam, E., et al. (2001). Contribution of dynamic headspace GC–MS analysis of aroma compounds to authenticity testing of honey, Food Chemistry, 72, 511–520. [35] Pawliszyn, J. (1999). Application of solid phase microextraction, Ontario, Canada: Royal Society of Chemistry. [36] Gilley, D. C., DeGrandi-Hoffman, G. and Hooper, J. E. (2006). Volatile compounds produced by live European honey bee (Apis mellifera L.) queens, Journal of Insect Physiology, 52, 520-527. [37] Moritz, R. F. A. and Crewe, R. M. (1991). The volatile emission of honeybee queens (Apis mellifera L), Apidologie, 22, 205-212. [38] Jerković, i., Marijanović, Z., Ljubičić, I. and Gugić, M. (2010). Contribution of the Bees and Combs to Honey Volatiles: Blank-Trial Probe for Chemical Profiling of Honey Biodiversity, Chemistry and Biodiversity, 7, 1217-1229. [39] de la Fuente, E., Valencia-Barrera, R. M., Martínez-Castro, I. and Sanz, J. (2007). Occurrence of 2-hydroxy-5-methyl-3-hexanone and 3-hydroxy-5-methyl-2-hexanone as indicators of botanic origin in eucalyptus honeys, Food Chemistry, 103, 1176-1180. [40] Guyot, C., Scheirman, V. and Collin, S. (1999). Floral origin markers of heather honeys: Calluna vulgaris and Erica arborea, Food Chemistry, 64, 3-11. [41] Jerković, I., Hegić, G., Marijanović, Z. and Bubalo, D. (2010). Organic Extractives from Mentha spp. Honey and the Bee-Stomach: Methyl Syringate, Vomifoliol, Terpenediol I, Hotrienol and Other Compounds, Molecules, 15, 2911-2924. [42] Jerković, I., Marijanović, Z., Kezić, J. and Gugić, M. (2009). Headspace, Volatile and Semi-Volatile Organic Compounds Diversity and Radical Scavenging Activity of Ultrasonic Solvent Extracts from Amorpha fruticosa Honey Samples, Molecules, 14, 2717-2728. [43] Jerković, I. and Marijanović, Z. (2010). Oak (Quercus frainetto Ten.) Honeydew Honey—Approach to Screening of Volatile Organic Composition and Antioxidant Capacity (DPPH and FRAP Assay), Molecules, 15, 3744-3756.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

In: Honey: Current Research and Clinical Applications ISBN: 978-1-61942-656-6 Editor: Juraj Majtan © 2012 Nova Science Publishers, Inc.

Chapter II

Honey Melanoidins: Emerging Novel Understanding on the Mechanism of Antioxidant and Antibacterial Action of Honey Katrina Brudzynski1,2, 1

Department of Biological Sciences, Brock University, St. Catharines, Ontario, Canada 2 API-Medicals St. Catharines, Ontario, Canada

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Abstract Honey has well established antioxidant and antibacterial activities. However, the ever-important question concerning active agents and biochemical pathways underlying these activities remains elusive. In this review, the Maillard reaction is discussed as an origin of bioactive compounds in honey. Honey is presented as a ―reaction pot‖ where the main honey constituents – sugars, amino acids/proteins and polyphenols – are substrates and reactants in the Maillard reaction. During the process, several bioactive molecules are formed and sequestered to the brown polymeric melanoidins resulting in a gain-and-loss of antioxidant and antibacterial function in honey. The review is divided into three parts. The first part of the chapter briefly presents an overview of compounds involved in the antioxidant activity of honey to provide context for the argument that these compounds are structural and functional parts of melanoidins. The second part presents an overview of the Maillard reaction and formation of melanoidins. This forms the background for the postulated biological consequences of the Maillard reaction on honey function; namely, effects of protein modifications, formation of protein-polyphenol complexes and their sequestration to melanoidins. In this context, we discuss our research on honey melanoidins and their contribution to honey antioxidant and antibacterial activities. The third and final part of the chapter offers perspective on the functional consequences of 

Correspondence concerning this article should be addressed to: Dr. Katrina Brudzynski, Department of Biological Sciences, Brock University, 500 Glenridge Avenue, St. Catharines, Ontario, Canada, L2S 3A1, Tel. (905) 6885550, Fax: (905) 688-1855, Email: [email protected].

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

18

Katrina Brudzynski melanoidin formation. In conclusion, contrary to traditional views, honey functions result from a network of chemical interactions as opposed to the action of one molecule or one chemical reaction. Understanding this network is important in explaining honey health and therapeutic benefits.

Introduction

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

The underlying mechanism and compounds involved in antioxidant and antibacterial activities of honey has been a subject of considerable interest. The elucidation of the mechanism would significantly impact the recognition of honey as a health-promoting and therapeutic agent. With respect to compounds involved in honey‘s antioxidant activity, a range of constituents has been identified including enzymes such as glucose oxidase, catalase and non-enzymatic compounds such as flavonoids, phenolic acids, carotenoid derivatives, organic acids, Maillard reaction products, amino acids and proteins (Aljadi and Kamaruddin, 2004; Al-Mamary et. al. 2002; Gheldof and Engeseth, 2002; Gheldof et. al. 2002, Schramm et. al. 2003, Tan et. al. 1989). The challenge undertaken by many researchers was to determine which of these compounds and factors contributed most to honey‘s antioxidant activity. A frequent conclusion from these studies was that the antioxidant effect of a single or group of components was a fraction of the effect observed from honey as a whole (Gheldof et. al. 2002; Perez et al. 2007). The interactions between bioactive molecules – synergistic, inhibitory or antagonistic – were suspected and acknowledged in many studies however, the significance of these interactions within honey functions were not explained. The aim of this review is to provide literature evidence to support our hypothesis coming from preliminary observations that the Maillard reaction and formation of melanoidins in honey is a key mechanism underlying honey‘s antibacterial and antioxidant activities.

1. Overview of Compounds Involved in Antioxidant Activity of Honey 1.1. Contribution of Polyphenols to Antioxidant Activity of Honey The best defined factors linked with antioxidant activity of honeys are polyphenols, honey color, amino acids, and recently reported, the Maillard reaction products. The large number of studies described the existence of a highly significant correlation between antioxidant activity and polyphenolic content (Gheldof and Engeseth, 2002; Aljadi and Yusoff, 2004; Blasa et. al. 2006; Bertoncelj et. al. 2007; Estevinho et al., 2008). Subsequent research efforts aimed at establishing which phenolic class is mostly responsible for the antioxidant activity did not bring direct positive evidence (Gheldof et al, 2002). A predominance of certain individual polyphenols was, however, related to botanical origin of some honeys (Anklam, 1998; Yao et. al. 2003, Martos et. al. 2000; Ferreres et. al. 1998). Alternatively, the ratio of flavonoids to polyphenolic acid was employed to link groups of phenolics to antioxidant activity (Baltrušaitytė et. al. 2007). This was based on the known fact

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Honey Melanoidins

19

that the levels of radical scavenging activity of polyphenolic acids are generally lower than those of flavonoids (Rice-Evans et. al. 1996, Cai et. al. 2006). As a result, variation in the antioxidant activity between honeys of different color-hues (but derived from the same floral sources) could be explained by the differences in the abundance of flavonoids and phenolic acids (Baltrušaitytė et. al. 2007). However, when honeys were crudely fractionated using solid-phase extraction, isolated fractions rich in flavonoids were not the main contributors to the overall antioxidant activity of honey. In addition, the sum of antioxidant activities of fractionated phenolics was significantly lower than the antioxidant activity of honey as a whole (Gheldof et. al. 2002). The apparent lack of a direct relationship between specific groups of polyphenols and antioxidant activity on one hand, and the strong correlation between the total phenolic content and antioxidant activity on the other, implied that other phenolics, such as terpenoids and isoprenoids or polyphenolic-associated compounds such as the Maillard reaction products, may contribute to this activity.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

1.2. Common Structural Determinants for Antioxidant Activity and Color Honey color has long been recognized as a good predictor of its antioxidant activity. Higher antioxidant capacity was found for darker honeys (Chen et al., 2000; Frankel et. al.1998; Gheldof and Engeseth, 2002; Nagai et. al. 2001, Blasa et. al. 2006; Bertoncelj et. al. 2007), and usually the darker honeys had higher total polyphenol content (Gheldof and Engeseth, 2002; Al-Mamary et. al. 2002; Aljadi and Kamaruddin, 2004; Vela et. al. 2007). The basis for the close association between color and antioxidant activity lie in part in the presence of common structural determinants. As antioxidants, polyphenols may function as free radical scavengers (Salah et. al. 1995), hydrogen-donating agents (Rice-Evans et. al. 1996) and metal-chelators (Cao et. al. 1997). These functions are strongly influenced by the presence of the catechol moiety and a number of available hydroxyl groups. The enhanced radical scavenging activity of flavonoids is related to a better electron delocalization from their B-rings because of the presence of a 2, 3double bond in conjunction with the 4-oxo group in the C ring, and a presence of 3- and 5-OH groups in the C and A rings respectively (Bors et. al. 1990; Cai et. al. 2006; Rice-Evans et .al. 1996). The extended conjugated double bond system in phenolic structures has light absorbing properties and therefore is responsible for color. The conjugated system of more than 8 double bonds allows delocalization of π-electrons that absorb photons of light in the visible region. Flavonoids and long chain phenolics, terpenes, and carotenoids present in honey are both chromophores and antioxidants (D‘Arcy, 1997; Castro-Vazquez et. al. 2008, Paiva and Russell, 1999; Muller and Boehm, 2011). Thus, the strong correlation between honey color, antioxidant activity, and the total phenolic content arises, at least in part, from the common structural motif that imparts both color and antioxidant activity. The relationship between polyphenolic structure and antioxidant activity plays a significant role in melanoidin formation, as discussed in the next part of the chapter.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

20

Katrina Brudzynski

1.3. Amino Acids and Antioxidant Activity of Honey In contrast to the immense interest in the role of phenolics in the antioxidant activity of honey, relatively less attention has been given to the contribution of amino acids and proteins. Amino acids account for only 1% of honey, with proline being the most abundant amino acid amounting from 50 to 85% of the total amino acid count. Despite that, a significant correlation was found between the total amino acid content, proline and protein contents and antioxidant activity measured by DPPH method (Perez et. al. 2007). In another study, which used ORAC method, radical scavenging activity of the protein fraction contributed up to 16% of the total radical scavenging activity of the honey (Gheldof et al. 2002). Indeed, the correlation between the radical scavenging activity of amino acids and proline was higher than that of total phenolics (Perez et al .2007, Meda et al. 2005). These results point out to amino acids/proteins as a part of a mechanism that mediates antioxidant activity of honey.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

1.4. Maillard Reaction Products in Honey and Antioxidant Activity Recent studies have provided evidence that the Maillard reaction occurs in honey (Turkmen et al., 2006; Brudzynski and Miotto, 2011a, b). The Maillard reaction or nonenzymatic browning usually takes place in thermally processed foods such as coffee (Borelli et. al., 2002; Delgado-Andrade et. al., 2005; Nunes and Coimbra, 2007; Bekedam et. al. 2006; Gniechwitz et. al. 2008), cocoa beans (Summa et al., 2008) or bread crust (Borrelli et al., 2003). The final stage of the reaction is associated with the formation of brown melanoidins of high molecular weight, which exhibit antioxidant activity (for review, Manzocco et al., 2001; Wang et. al., 2011). The Maillard reaction products were long suspected ―perpetrators‖ of the browning of honeys observed during prolonged storage (Gonzales et. al. 1999), specifically for lightcolored honeys. Heat-treatment of honey, such as that used during honey harvesting and processing, increased brown pigment formation and its appearance coincided with the enhanced antioxidant activity (Turkmen et. al., 2006; Brudzynski and Miotto, 2011 a, b, Brudzynski and Kim, 2011). While the melanoidin formation in elevated temperatures is well documented, we have unexpectedly found significant amounts of high molecular weight brown polymers, which exhibited antioxidant activity in unheated honeys (Brudzynski and Miotto, 2011 a, b). By combining size-exclusion chromatography with activity guided fractionation, we have shown that unheated, raw honeys were composed of high molecular weight complexes ranging in size from 85-232 kDa (Table 1). The isolated complexes possessed several characteristic features of melanoidins: a strong radical scavenging activity (as measured by ORAC method), absorbance at A450 nm (which is a measure of the degree of browning), and a strong absorbance of UV light. Heat-treatment and long storage of honey accelerated the formation of these high molecular weight complexes (Brudzynski and Miotto, 2011 a, b). The findings that the Maillard reaction and melanoidin formation occurs in unheated honeys initiated our research into melanoidin composition and their role in antioxidant and antibacterial activities.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

21

Honey Melanoidins Table 1. Molecular size and antioxidant activity of unheated and heat-treated honey fractions

a

Honey 177 Fr. ORACa

177 Treated ORACa

148 ORACa

148 Treated ORACa

08 -5 ORACa

13 14 15 16

3730±155 3017±147 1073±132 102±8

1175 ±165 1479 ±48 1287 ±147 967 ±19

674±56 4710±645 2345±339 921±139

363 ±11 437 ±46 404 ±54 ---

1547±220 2979±202 2742±308 1125±67

08 -5 Treated ORACa 814±122 683 ± 34 409±102 ---

Molec. size (kDa) 180–232 140–180 109–140 85–109

Data shown as means (μmol TE/ L of 100% fraction) ± SD of at least three independent trials (n ≥ 9). RSD is < 15% for the values obtained.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Honey is a unique food product, in which high concentrations of catalytic sugars and a pool of free amino acids create conditions that can promote and facilitate the Maillard reaction. Furthermore, some characteristic Maillard reaction products such as hydroxymethylfurfural, glyoxal and methylglyoxal are observed in unheated honeys, although at low amounts (Tosi et. al. 2002; Adams et. al. 2008; Marvic et. al. 2008). Hydroxymethylfurfural and furosine (an early Maillard reaction product) are used as markers of honey quality and its freshness since their levels increase during heat processing and storage (Morales et. al. 2009; Villamiel et. al. 2001). The presented literature data and our findings provide a strong basis for the postulate that the Maillard reaction occurs in unheated honeys and that the reaction is further accelerated by heating and storage. Taking the development of antioxidant activity of honey as an example, we will present evidence that honey compounds exhibiting antioxidant activity are indeed the structural and functional parts of melanoidins – the brown pigments formed at the advanced stage of the Maillard reaction.

2. Honey Melanoidins: Composition and Antioxidant Activity 2.1. The Maillard Reaction and Melanoidin Formation The main substrates for the Maillard reaction are reducing sugars such as fructose and glucose and amino acids/proteins. The Maillard reaction is initiated by the condensation of a free amino group of amino acid or protein with a carbonyl group of a reducing sugar or a lipid breakdown product to form N-substituted glycosylamine (Schiff base) (Fayle and Gerrard, 2002, Hodge, 1953) (Figure 1). The resulting Schiff‘s base rearranges to form more stable Amadori products, which then undergo oxidative degradation and generate pools of deoxyglucosones, fission products and Strecker degradation products (Figure 1). Numerous active molecules that arise from each of these pools include: furfural and hydroxymethylfurfural; furanones, pyranones, diacetyl hydroxyaceton, pyruvaldehydes; furans; carbonyls and dicarbonyls; and finally Strecker aldehydes (Fayle and Gerrard, 2002, Hodge, 1953). Hydroxyfurfurals and dicarbonyls, methylglyoxal and glyoxal, are well known Maillard reactions products present in honeys. The Maillard reaction is further propagated by

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

22

Katrina Brudzynski

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

the interaction of these various Amadori degradation and fission products with amino acids or amino acid degradation products (Yaylayan, 1997). The reactions lead to formation of a number of N-containing heterocyclic compounds and the modified amino acid derivatives that include ε-fructosyl-lysine and its derivative furosine, glyoxal and methylglyoxal lysine dimers (GOLD and MOLD) and glyoxal and methylglyoxal arginine dimers (GODIC and MODIC) N-ε-carboxymethyllysine, pentosidine or pyrraline and others (Figure 1) (Nagaraj et. al. 1996, Biemel et. al. 2001, Henle et. al. 2005, Fu et. al. 1996). Ultimately, the multiple interactions between different pools of Maillard reaction products generate the advancedstage low molecular weight (LMW) end products (Yaylayan, 1997). The LMW products share some characteristic features: (a) they are chromophores of yellow colors, (b) they are involved in protein cross-linking and (c) they strongly absorb UV light and (d) possess radical scavenging activity (Hofman, 1998, Wang et. al. 201). Covalent binding between these low molecular weight color compounds to high molecular structures is proposed to be one of the mechanisms of melanoidin formation, and the process of acquiring antioxidant activity by melanoidins (Hofman, 1998, Delgado-Andrade and Morales, 2005). Similarly, sugar and Amadori degradation products, pentose and hexose-derived N-substituted pyrroles and furans derivatives have been found to be chromophores and had an exceptional propensity for polymerization (Tressl et. al. 1998).

Figure 1. Diagram representing a general scheme of the Maillard reaction and melanoidin formation. Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Honey Melanoidins

23

The final stage of the Maillard reaction is characterized by the formation of nitrogencontaining brown pigments-melanoidins (Hodge, 1953; Ames, 1992, Fayle and Gerrard, 2002) (Figure 1). The mechanism of melanoidin formation, their composition and structure have been a subject of several studies conducted on model systems as well as on melanoidins from foods. Based on the model systems, melanoidins were formed either by (a) polymerization of repeating units of furans or pyrroles (Tressl et. al. 1998), or (b) were arising either from sugar degradation products that form polymers through aldol-type condensation and/or from intact carbohydrates (Cämmerer and Kroh, 1995; Cämmerer et. al. 2002), or (c) resulted from protein cross-linking by low molecular weight colored compounds (Hofman, 1998). Melanoidin structures formed in foods proved to be much more complex due to a large pool of reactants, exceeds those of the model system. The best studied melanoidins are melanoidins of coffee brew. A characterization of their composition brought a novel observation of the presence of polyphenols (hydroxycinnamates) bound to proteinarabinogalactans complexes (Bekedam et. al. 2007, 2008a, 2008 b; Nunes 2010). The incorporation of polyphenols into melanoidins changed melanoidin functions, as discussed below.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

2.2. Honey Melanoidins: Honey Fractionation and Physicochemical Characterization of Fractions The size-exclusion chromatography (SEC) on Sepharose 4B in combination with an activity-guided fractionation of honeys allowed us to assign the radical scavenging activity to high molecular weight, brown colored fractions, which absorbed UV light and visible light at A420-450nm (the latter parameter is commonly used to assess degree of browning) (Figure 2, Table 1). Heat treatment of light-color honeys increased the size of aggregates and their radical scavenging activity (Figure 2 and 3). In contrast, heating of dark-color honeys caused formation of three types of complexes: insoluble high molecular weight brown polymers that precipitate out of honey solution and were removed before SEC, brown soluble complexes of very high molecular weight that eluted in void volume during honey fractionation (fr. 6 and 7, Figure 2) and soluble, light color complexes that eluted from the SEC column in the same position as the fractions from unheated honeys. The heat-induced formation of the first two types of HMW polymers caused re-distribution of the pool of available antioxidants between fractions (Figure 3). In general, however, heat-treatment increased radical scavenging (ORAC) activity of soluble honey melanoidins (Figure 3).

2.3. Presence of Polyphenols in Fractions and Antioxidant Activity The isolated melanoidin fractions from unheated and heated honeys were assessed for the phenolic content using the Folin-Ciocalteu method, after removal of reducing compounds through solid-phase extraction (Brudzynski and Miotto, 2011a, b). The concentration of polyphenols in fractions varied between honeys of different plant origin, with the highest levels being observed in the dark buckwheat honey (Figure 4). Heat-treatment of honeys resulted in the increased incorporation of polyphenols into melanoidins (Figure 4). This incorporation caused a shift in polyphenol distribution between fractions increasing their

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

24

Katrina Brudzynski

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

content in high molecular weight fraction (honey #177, fr. 12, 13, 14, Figure 4) by decreasing/depleting polyphenol content in fractions of lower molecular weight (honey # 177, fr. 15, 16, Figure 4). In contrast, heat-treatment of light- and medium- colored honeys resulted in the increased incorporation of polyphenols to all high molecular fractions (Figure 4).

Figure 2. Elution profiles of unheated and heat-treated honeys obtained from size exclusion chromatography on Sepharose 4B. Honeys (2 ml of 50% solution v/w in 0.15M NaCl) were eluted with water as a mobile phase and elution was monitored at 280 nm. The molecular size of eluted compounds was determined from the standar curve (protein gel filtration HMW calibration kit, GE Healthcare). The approximate size of of components was as follow: fr.13- 180 to 232 kDA, fr. 14- 140 to 180 kDa, fr. 15- 105 to 140 kDa and fr.16-85-109 kDa. Fractions 5 to 7 eluted in a void volume (in the same position as blue dextran). H 08-5 - light color dandelion honey, H 148 - medium color manuka honey and H 177 - dark color buckwheat honey. From Brudzynski and Miotto, 2011a.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Honey Melanoidins

25

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Figure 3. Antioxidant capacity of fractions obtained from size-exclusion chromatography. Antioxidant capacity was determined using oxygen radical absorbance capacity (ORAC) with 2,2‘-Azobis (2amidinopropane) dihydrochloride (AAPH) as a source of peroxyl radical. The concentration of antioxidant in honeys were calculated from the area-under-the curve (AUC) of the fluoresceine decay and compared to the AUC of the Trolox used as a standard. ORAC values of unheated honeys (crosshatched bars) and heat-treated honeys (black bars). From Brudzynski and Miotto, 2011a.

Figure 4. The total phenolic content in melanoidins fractions of unheated (crossed columns) and heattreated honeys obtained from SEC column. The total phenolic content was determined by using the Foli-Ciocalteu method. Gallic acid was used as a standard. From Brudzynski and Miotto, 2011b.

Thus, these results indicate that polyphenols are involved in honey melanoidin formation in a way which includes their attachment to the existing high molecular weight polymer. Their binding increases the size of the polymer. This process occurs in unheated honeys as Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

26

Katrina Brudzynski

evidenced by the presence of high molecular fractions containing polyphenols, but is greatly facilitated by high temperatures (Figure 2 and 4). The changes in the total phenolic content in unheated versus heated honeys were positively correlated with the changes in the antioxidant activity of melanoidin fractions (Figure 3 and 4). This correlation suggested that the presence of phenolics in melanoidins provided melanoidins with antioxidant activity brought by phenolics. In other words, melanoidin fractions acquired antioxidant activity because of the presence of polyphenols. It has been further observed, that honeys of different colors (light, medium or dark) differed in the amount and size of melanoidins produced (Figure 2, blue columns). Lightcolored honeys produced the lowest amounts of melanoidins and they were of shorter size. Their radical scavenging activity was also lower than that of melanoidins of darker honeys, so was their phenolics content (Figure 3, 4). These results suggested that the extent/size of melanoidin formation was related to honey color, and therefore to the types of polyphenols present in honeys and their radical scavenging capacity, as discussed in Part 1. It has to be emphasized at this point that polyphenols having catechol moiety and exhibiting high radical scavenging activity have been shown to possess increased ability to undergo auto-oxidation. In the presence of oxygen, or such oxidants as H2O2, they form semiquinone and quinone radicals which are further involved in oxidative polymerization and cross-linking with proteins (Cilliers and Singleton, 1991, Hotta et. al. 2001; Spencer, 1988). The readiness with which polyphenols auto-oxidize (pro-oxidant activity) and bind proteins determines the extent of polyphenol-protein complexation and in turn, the size of melanoidins to which the complexes are incorporated. Therefore, our results imply an existence of a link between the radical scavenging activity, color development and melnoidin sizes observed in honeys. Polyphenols are known constituents of coffee and wine melanoidins and have been shown to contribute to their color and antioxidant activity (Bekedam et al. 2008a; Gniechwitz et al. 2008, Rivero-Perez et. al. 2002). Analysis of the structure of coffee melanoidins revealed that phenolic compounds were covalently bound to the polysaccharides backbone (galactomannans and arabinogalactans) (Bekedam et. al.2007; Nunes and Coimbra, 2007; Delgado-Andrade, and Morales, 2005, Bekedam et. al. 2008), together with proteins and amino acid-derived compounds (Montavon et. al. 2003a; Nunes and Coimbra, 2010). Consistent with our findings, the decrease of polyphenols during thermal processing of foods adversely affected the content of antioxidants, such as the levels of ascorbic acid in tomato puree (Anese et. al. 1999), or chlorogenic acid in roasted green coffee beans (Del Castillo et. al. 2002). The decrease in antioxidant activity however has been compensated, at least in the case of coffee, by the incorporation of newly synthesized low-molecular weight phenolic compounds that possessed antioxidant activity in melanoidins (Nunes and Coimbra 2001; Nicoli et. al. 1999; Del Castillo et al. 2002). This observation emphasizes the fact that the melanoidin formation is a dynamic state determined by the difference between the rate of its degradation and de novo synthesis. Moreover, the higher degree of browning correlated to the increased presence of polyphenols and in most cases antioxidant activity (Bekedam et. al. 2008; Ortega-Heras et. al. 2009; Manzocco et. al. 2001). Overall, these studies give support for our findings that polyphenols are involved in melanoidin formation, and provided melanoidins with antioxidant activity. In a structure– depending way, polyphenols were capable of influencing the levels of antioxidant activity and the size of the melanoidin polymer in honey.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Honey Melanoidins

27

2.4. Proteins in Fractions The SDS-PAGE analysis of melanoidin fractions obtained from SEC revealed that they contained proteins. The number of protein bands differed in honeys of different botanical origin, however two protein bands of 82 kDa and 56-60 kDa were commonly observed in the melanoidin fractions for all honeys (Brudzynski and Miotto, 2011b). During chromatographic fractionation, the changes in the abundance of these proteins in melanoidin fractions followed the changes in the total phenolic levels. The characteristic feature of proteins on the SDS-gels was their diffuse, fuzzy appearance thereby suggesting their modification (Figure 5). This behavior of honey proteins on SDS-PAGE was noted before by several authors (White and Kushnir, 1967; Marshall and Williams, 1987; Baroni et. al. 2002). In an effort to characterize the compounds that modified proteins in honey, protein precipitates were extensively purified by several solvent-solvent extractions, followed by a LC-ESI-MS analysis of the removed ―contaminants‖. The LC-ESI-MS analysis demonstrated that the removed compounds contained phenolics, including flavonoids (manuscript in preparation). These results confirmed the presence of polyphenols in protein fraction and also indicated a relatively tight, potentially covalent binding between proteins and polyphenols.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

2.5. Polyphenol-Proteins Complexes and Mechanism of Their Formation After heat-treatment of honeys, the proteins of melanoidin fractions could be hardly visualized in SDS-PAGE; they seemed to disappear from the gel (Figure 5B). Our effort to increase the separation of proteins in SDS-PAGE was somewhat improved when the protein precipitate was first extracted with a mixture of three alcohols (ethanol, isopropanol and methanol) to remove polyphenols and then subjected to electrophoresis (Brudzynski and Miotto, 2011b). After the procedure, a clearly visible, high molecular weight protein polymer was observed on the border between the stacking and the resolving gels (Figure 5C). The disappearance of proteins from the electrophoretic profiles can be explained by the formation of large soluble protein-polyphenol complexes that precede their eventual precipitation but that were too large to enter the gel. The reduction in protein content and the disappearance of protein bands in the gels was also observed during the roasting of green coffee beans (Borrelli et al, 2002; Montavon et. al. 2003a, b). This phenomenon was thought to be a result of either the protein breakdown or protein polymerization and their association with melanoidins (Borrelli et al .2002; Montavon et al, 2003a). The roasting of green coffee beans caused an increase in polyphenol-protein complexations by the covalent attachment of chlorogenic acid to proteins (Montavon et al. 2003a, b; Rawel and Rohn, 2010). In view of these results, the general picture emerges that proteins in honeys are gradually bound to polyphenols and sequestered as protein-polyphenol complexes into melanoidins. Heating promotes the polyphenol-proteins complexation as evidenced by the formation of large soluble complexes (that are too big to enter SDS gels). As was mentioned before, the mechanism of protein complexation by polyphenols involves polyphenol auto-oxidation. The polyphenols structure/redox properties, and the degree of substitution of hydroxyl groups, determine their pro-oxidant activity. The instability of semiquinone and quinone radicals derived from ortho-diphenols (flavonoids for example) during auto-oxidation made them particularly reactive toward nucleophilic groups such as

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

28

Katrina Brudzynski

sulfhydryl, amine, amide groups of amino acids or proteins, leading to the formation of polyphenol-protein complexes (Methodieva et al. 1999, Cilliers and Singleton, 1991, Hotta et. al. 2001).

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Figure 5. AB: Protein profiles of melanoidin fractions eluted from SEC analysed by SDS-PAGE (10% polyacrylamide resolving gel and 5% stacking gel). A: Protein profiles in fractions of unheated buckwheat honey H177, B: Protein profiles of fraction F13 from unheated and heat-treated honeys. C Protein profiles of melanoidin fractions from SEC of unheated and heated (T) honeys. C: Protein profiles of fractions F13 of unheated buckwheat honey and F12, F13 and F14 of heated buckwheat honey after partial extraction of polyphenols. The high molecular material appears on the border between resolving and stacking gel (arrow). From Brudzynski and Miotto, 2011b

The degree of solubility of such complexes depends on the protein/polyphenol ratio. At low polyphenol concentrations, their attachment to proteins did not change protein hydrophilicity and such complexes remained soluble. With the increased concentration of polyphenols, the protein-polyphenol complexes became more hydrophobic and by crosslinking with other protein-polyphenols complexes they ultimately move from soluble to insoluble hydrophobic aggregates (Spencer et. al.1988). As shown in our experiments, the protein-polyphenol binding and growth in size can be significantly accelerated by the heattreatment of honey. The results discussed in this section indicated that both polyphenols and proteins participated in the melanoidin formation. The structural features of polyphenols seemed to be crucial in determining their antioxidant/pro-oxidant activity which in turn, influenced their binding to proteins, amount of protein-polyphenol complexes formed, the size of melanoidin polymers and their solubility.

3. Functional Consequence: Gain-and-Loss of Honey Function 3.1. Effects of the Maillard Reaction Products The Maillard reaction is a known source of bioactive compounds in various foods and for that reason it could also be of special interest within honey. The compounds generated from the Maillard reaction are endowed with novel properties that may have either beneficial or

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Honey Melanoidins

29

detrimental effects. For example, oxidative degradation of the Amadori products lead to the formation of dicarbonyl intermediates: methylglyoxal and glyoxal. Methylglyoxal has been recently shown to be a dominant component responsible for the antibacterial activity of manuka honey originating from Leptospermum spp. (Mavric et. al. 2008; Adams et al., 2008). Although the biochemical pathway leading to the antibacterial effect of methylglyoxal has not yet been explained, its cytotoxicity seemed to be linked to its pro-oxidant activity and the generation of free radicals (ROS) (Yim et. al. 2001; Kalapos, 2008). Methylglyoxal and glyoxal are the most potent glycating agents among the Amadori degradation products which, by reacting with the side chains of lysine, arginine, and cysteine residues can form cross-links between proteins (MOLD, GOLD, MODIC, GODIC, Figure 1) (Nagaraj et al. 1996, Biemel et. al. 2001; Henle; 2005; Yim et. al. 2001; Kalapos, 2008). In addition, free methylglyoxal, methylglyoxal modified proteins and ROS exert direct or indirect cytotoxic effects on bacterial cells. While this antibacterial effect could be considered beneficial from the view point of the therapeutic application of honey in wound healing, the same cytotoxic features of methylglyoxal toward human cells would pose a potential risk (Majtan, 2011). The accumulation of either free or protein-bound forms of methylglyoxal has been observed with aging (Frye et al. 1998), diabetes (Riboulet-Chavey et al. 2006), in non-diabetic uremic patients (Odani et al. 1998), and with atherosclerosis (Cantero et al. 2007). In this regard, one has to consider methylglyoxal as a double-edged sword where its cytotoxic action can be beneficial in one biological system and detrimental in the other. It appeared, however, that other redox-active compounds including polyphenols and melanoidins might also have dual functions (beneficial and potentially harmful), depending on the equilibrium (or the lack of it) between pro-oxidant and antioxidant activities.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

3.2. Effects of Polyphenol-Protein Complexes The major consequence of the advanced Maillard reaction in foods is the formation of polyphenol-protein complexes. Firstly, during protein cross-linking by polyphenols in the presence of O2 or H2O2, semiquinone radicals and hydroxyl radicals are formed; the latter from hydrogen peroxide via the metal-catalyzed Fenton reaction (Cao, Sofic and Prior, 1997; Fukumoto and Mazza, 2000; Sakihama et. al. 2002). These radicals have been shown to be cytotoxic, inducing strandbreaks in bacterial DNA (Sakihama et. al. 2002). Secondly, semiquinone radicals can further react with another polyphenols to form dimers or higher-order polymers. Polymerization of polyphenols increases the radical scavenging activity of the polymer (Hotta et al. 2001). This functionality could be transferred to melanoidins by incorporating polyphenol polymers (Delgado-Andrade et al. 2005, Tagliazucchi et al, 2010). Wen et al. (2005) observed a change in the type of antioxidant action of melanoidins from metal-chelating to pro-oxidant activity during the roasting of coffee beans. The loss of metal-chelating activity of melanoidin at higher temperatures can therefore result in gain- of- cytotoxic pro-oxidant function. Thus, the antioxidant status of melanoidins appeared to be important for its antibacterial potency. It is well known that metal ions Fe- and Mg- are crucial for bacterial growth and survival. Their chelation by melanoidins is one of the proposed mechanisms for antibacterial action (Rufián-Henares and de la Cueva, 2009).

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

30

Katrina Brudzynski

Thirdly, antibacterial action of melanoidins may also result from the inactivation of bacterial proteins due to their binding by semiquinones / quinones. Cross-linking of microbial cell wall peptides or membrane-bound enzymes might lead to a permanent destruction of cell membranes (Cowan, 1999). Further investigation is required to determine whether the ability to cross-link proteins by quinones is preserved or lost after their incorporation to melanoidins. However, melanoidins isolated from coffee and biscuits have been shown to cause irreversible cell membrane disruption in E. coli and bacterial death by chelating Mg-ions from the membranes (Rufián-Henares and de la Cueva, 2009, Rufián-Henares and Morales, 2008). The harmful effects of protein complexation by polyphenols and protein modification by Amadori products have been observed during seed and plant development. Protein crosslinking has been associated with the loss of enzymatic activities, the loss of membrane integrity and ultimately the loss of seed viability during seed storage (Murthy and Sun, 2000; Ravel and Rohn, 2010). Another aspect of the protein binding by polyphenols is that proteins have a relatively limited time when they are free and able to perform their original function. It is quite plausible then that the observed reduction of enzyme activities in honey during its storage – such as the decrease in diastase and invertase activities – could result from their gradual cross-linking and incorporation into melanoidins.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

3.3. Effect of Honey Melanoidins on Antibacterial and Antioxidant Activities In this context, we have recently shown that melanoidin formation during prolonged honey storage (one to two years) was associated with the decrease in antibacterial and antioxidative activity (Brudzynski and Kim, 2011). The decrease in antibacterial activity was rapid in the in the first three to six months of storage (Brudzynski and Kim, 2011) and was concomitant with significant reduction in protein levels as well as an increased rate of protein-polyphenol complex formation (manuscript in preparation). These changes in antibacterial activity were accompanied by physicochemical changes in honeys such as the darkening of color, increased browning, increased levels of UV absorption, and the appearance of melanoidins (Brudzynski and Kim, 2011). There is scarce and sometimes contradictory literature on the levels of antibacterial activity of honey during its storage. Some studies reported that the exposure of honey to heat (White and Subers, 1964) or its prolonged storage resulted in a loss of antibacterial activity (Radwan et. al. 1984), while other studies found no correlation between age and antibacterial activity (Allen et al. 1991; Rios et. al. 2001). It can be concluded that honey‘s antibacterial and antioxidant activities are influenced by the stages of the Maillard reaction. At the intermediate stage, the increased generation of dicarbonyl compounds (methylglyoxal) increases honey‘s antibacterial activity. Moreover, semiquinones and quinones derived from polyphenol auto-oxidation also contribute to the cytotoxicity against bacteria. In contrast to the intermediate stage, the advanced stage was associated with the decrease in antibacterial activity due to the increase of protein crosslinking, formation of polyphenol-protein complexes and their incorporation into melanoidins. The main picture emerging from the data presented here is that the Maillard reaction and melanoidin formation involves all compounds that are known to actively contribute to the antioxidant and antibacterial activity of honey: sugars, amino acids/proteins and polyphenols.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Honey Melanoidins

31

The ratio of antioxidants to pro-oxidants formed in the Maillard reaction is a key factor in the melanoidin formation which in the end, influences honey‘s functions. Therefore, the antioxidant activity of the Maillard reaction products and melanoidins are of significance for the antibacterial activity of honey.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Conclusion We have demonstrated that honey is composed of high molecular weight complexes comprised of polyphenols, proteins and sugars (Brudzynski and Miotto, 2011b). The following characteristics – the strong radical scavenging activity, the molecular size of these complexes, the degree of browning and the UV absorbance – allowed them to be identified as melanoidins. Heat treatment and long storage of honey increased binding and cross-linking of proteins by polyphenols, and accelerated incorporation of protein-polyphenol complexes into melanoidins. The stage of the Maillard reaction, intermediate versus the advanced stage in which melanoidins are synthesized, resulted in the gain-and-loss of antioxidant and antibacterial function in honey. At the intermediate stage, the Maillard reaction products such as methylglyoxal and semiquinones / quinones increased bacteriostatic and bactericidal properties of honeys. On the other hand, the protein modification by glycation, formation of protein-polyphenol complexes and their gradual sequestration into melanoidin polymers over time decreased their original antioxidant and antibacterial activities. The main picture emerging from the effects of the Maillard reaction on honey functions is that the antioxidant/pro-oxidant status of the Maillard reaction products and polyphenols are the cause of antibacterial activity of honey. Based on these data, we propose a new paradigm where the dynamic state of the Maillard reaction has an effect on honey‘s bioactivity. Within the context of a complex network of interactions, single molecules, such as oxygen and hydrogen peroxide can profoundly affect the chemical state of the Maillard reaction products and polyphenols and change their reactivity to other molecules. Red-ox properties of polyphenols are at the center of this dynamic system in honey since their pro-oxidant activity initiate protein-polyphenol binding, polyphenol polymerization and formation of melanoidins. Importantly, in a structure– depending way, polyphenols are capable of influencing the levels of antioxidant activity and the size of the melanoidin polymer in honey. Overall, the Maillard reaction and melanoidin formations are responsible for the gain-and-loss of antioxidant and antibacterial activities of honeys. A better understanding of these complex processes will help reveal the mechanisms of honey functions and predict honey‘s effect in different biological systems.

Acknowledgments The author wishes to thank Danielle Miotto, Linda Kim and Liset Maldonado-Alvarez for their research on this project and the Ontario Centres of Excellence for their financial support.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

32

Katrina Brudzynski

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

References Adams, A., Borrelli, C., Fogliano, V., and de Kimpe, N. (2005). Thermal degradation studies of food melanoidins. Journal of Agricultural and Food Chemistry 53, 4136- 4142. Aljadi, A. M., Kamaruddin, M. Y., Jamal, A. M., and Yassim, M. Y. (2000). Biochemical study on the efficacy of Malaysian honey on inflicted wound: an animal model. Medical Journal of Islamic Academy of Science 13, 125-132. Aljadi, A., and Yusoff, K. M. (2004). Evaluation of the phenolic contents and antioxidant capacities of two Malaysian floral honeys. Food Chemistry 85, 513–518. Allen, K.L., Molan, P. C and Reid, G. M. (1991). A survey of the antibacterial activity of some New Zealand honeys. Journal of Pharmacy and Pharmacology 43: 817–22. Al-Mamary., Al-Meeri, A. and Al-Habori, M. (2002). Antioxidant activities and total phenolics of different types of honey. Nutrition Research, 22, 1041-1047. Ames, J.M. (1992). The Maillard reaction. In B.J.F. Hudson, Biochemisrty of Food Proteins, (vol.4) (pp. 99-153). London, Elsevier Applied Science (Chapter 4). Anese, M., Manzocco, L., Nicoli, M. C., and Lerici, C. R. (1999). Antioxidant properties of tomato juice as affected by heating. Journal of the Science of Food and Agriculture 79, 750–754. Anklam, E. (1998). A review of the analytical methods to determine the geographical and botanical origin of honey. Food Chemistry, 63, 549-562. Baltrušaitytė, V., Venskutonis, P. R, and Čeksterytė V. (2007). Radical scavenging activity of different floral origin honey and beebread phenolic extracts. Food Chemistry, 101, 502514. Baroni, M.A.V., Chiabrando, G. A., Costa, C, and Wunderlin, D.A.(2002). Assessment of the Floral Origin of Honey by SDS-PageImmunoblot Techniques. Journal of Agricultural and Food Chemistry 50, 1362-1367. Bekedam, E. K., De Laat, M. P., Schols, H. A., Van Boekel, M.A and Smit, G. (2007). Arabinogalactan proteins are incorporated in negatively charged coffee brew melanoidins. Journal of Agricultural and Food Chemistry, 55, 761-768. Bekedam, E. K., Loots M.J., Schols, H.A., van Boekel, M, A. J. S, and Smit, G. (2008b). Roasting effect on formation mechanisms of coffee brew melanoidins. Journal of Agricultural and Food Chemistry 56, 7138- 7145. Bekedam, E.K., Schols, H. A., van Boekel, M. A. J. S. and Smit, G. (2008a). Incorporation of chlorogenic acids in coffee brew melanoidins. Journal of Agricultural and Food Chemistry 56, 2055-2063. Bekedam, E.K., Schols, H. A., van Boekel,M. A. J. S.,and Smit, G. (2006).High molecular weight melanoidins from coffee brew. Journal of Agricultural and Food Chemistry 54, 7658-7666. Bertoncelj, J., Doberšek, U., Jamnik, M., and Golob, T. (2007). Evaluation of the phenolic content, antioxidant activity and colour of Slovenian honey. Food Chemistry 105, 822828. Biemel, K. M., Buhler, H. P., Reihl, O., Lederer, M. O. (2001). Identification and quantitative evaluation of the lysine-arginine cross-links GODIC, MODIC, DODIC, and glucosepan in foods. Nahrung/Food, 45, 210- 214.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Honey Melanoidins

33

Blasa, M., Candiracci, M., Accorsi, A., Piacentini, M., Albertini, M., and Piatti, E. (2006). Raw Millefiori honey is packed full of antioxidants. Food Chemistry 97, 217–222. Borrelli , R.S., Visconti, A., Mennella, C., Anese, M.,and Fogliano, V. (2002). Chemical Characterization and Antioxidant Properties of Coffee Melanoidins. Journal of Agricultural and Food Chemistry 50, 6527-6533. Borrelli, R.C., Mennella, C., Barba, F., Russo, M., Russo, G. L., Krome, K., Erbersdobler, H. F., Faist, V., and Fogliano, V. (2003). Characterization of coloured compounds obtained by enzymatic extraction of bakery products. Food and Chemical Toxicology 41, 13671374. Bors, W., Heller, W., Michael, C and Saran, M. 1990. Radical chemistry of flavonoid antioxidants. Advances in Experimental Medicine and Biology, 264, 165-170. Brudzynski K, Kim L. (2011). Storage-induced chemical changes in active components of honey de-regulate its antibacterial activity. Food Chemistry 126, 1155 -1163. Brudzynski K, Miotto D. (2011b). Honey melanoidins. Analysis of a composition of the high molecular weight melanoidin fractions exhibiting radical scavenging capacity. Food Chemistry 127, 1023 -1030. Brudzynski K., Miotto: (2011a). The recognition of high molecular weight melanoidins as the main components responsible for radical-scavenging capacity of unheated and heattreated Canadian honeys. Food Chemistry 125, 570-575. Cai, Y-Z., Sun, M., Xing, J., Luo, Q., and Corke, H. (2006). Structure-radical scavenging activity relationship of phenolic compounds from traditional Chinese medicinal plants. Life Science, 78, 2872-2888. Cämmerer, B and Kroh, L. (1995). Investigation of the influence of reaction conditions on the elementary composition of food melanoidins. Food Chemistry 53, 55-59. Cämmerer, B., Jalyschko, W., and Kroh, L. (2002). Intact Carbohydrate Structures as Part of the Melanoidin Skeleton. Journal of Agricultural and Food Chemistry 50, 2083-2087. Cantero, A. V., Portero-Otin, M., Ayala, V., Augu, N., Sanson, M., Elbaz, M., Thiers, J-C., Pamplona, R., Salvayre, R and Negre-Salvayre, A. (2007). Methylgyoxal induces advanced glycation end product (AGEs) formation and dysfunction of PDGF receptor: beta: implications for diabetic atherosclerosis. The FASEB Journal, 21, 3096-3106. Cao, G, Sofic, E, and Prior, R, L. (1997). Antioxidant and pro-oxidant behaviour of flavonoids: Structure-activity relationships. Free Radical Biology and Medicine, 22,749760. Castro-Vazquez, L., Diaz-Maroto, M.C., and Perez-Coello, M. S. (2008). Influence of storage conditions on chemical composition and sensory properties of citrus honey. Journal of Agricultural and Food Chemistry, 56, 1999-2006. Chen, L., Mehta, A., Berenbaum, M., Zangeri,.A. R. and Engeseth, N.J. (2000). Honey from different floral sources as inhibitors of enzymatic browning of fruit and vegetable homogenates. Journal of Agricultural and Food Chemistry 48, 4997-5000. Cilliers, J.J.L. and Singleton, V.L.(1991). Characterization of the products of nonenzymic autoxidative phenolic reactions in a caffeic acid model system. Journal of Agricultural and Food Chemistry 39, 1298–1303. Cowan, M. M. (1999). Plant Products as Antimicrobial Agents. Clinical Microbiology Reviews, 12, 564-582. D'Arcy, B.R., Rintoul, G.B., Rowland, C.Y. and Blackman, A.J., (1997). Composition of Australian honey extractives. 1. Norisoprenoids, monoterpenes, and other natural

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

34

Katrina Brudzynski

volatiles from Blue Gum (Eucalyptus leucoxylon) and Yellow Box (Eucalyptus melliodora) honeys Journal of Agricultural and Food Chemistry, 45,1834-1843. Del Castillo, M. D., Ames, J. M and Gordon M.H. (2002). Effect of roasting on the antioxidant activity of coffee brews. Journal of Agricultural and Food Chemistry 50, 3698-3703. Del Castillo, M.D., Ferrigno, A., Acampa, I., Borrelli, R.C., Olano, A, Martinez-Rodriguez, A and Fogliano, V.(2007). In vitro release of angiotensin-converting enzyme inhibitors, peroxyl-radical scavengers and antibacterial compounds by enzymatic hydrolysis of glycated gluten. Journal of Cereal Science 45, 327-334. Delgado-Andrade, C., and Morales, F. J. (2005). Unraveling the Contribution of Melanoidins to the Antioxidant Activity of Coffee Brews. Journal of Agricultural and Food Chemistry 53, 1403–1407. Delgado-Andrade, C., Rufian-Henares , J., and Morales, F.J. (2005). Assessing the antioxidant activity of melanoidins from coffee brews by different antioxidant methods. Journal of Agricultural and Food Chemistry 53, 7832- 7836. Estevinho, L., Pereira, A., Moreira, L., Dias, L., and Pereira, E. (2008). Antioxidant and antimicrobial effects of phenolic compounds extracts of Northeast Portugal honey. Food Chemistry and Toxicology 46, 3774-3779. Fayle, S. E and Gerrard, J. A. (2002). The Maillard Reaction. Royal Society of Chemistry (Great Britain). Ferreres, F., Juan, T., Perez-Arquillue, C., Herrera-Marteache, A., Garcia-Viguera, C. and Tomas-Barberan, A. (1998). Evaluation of pollen as a source of kaempferol in rosemary honey. Journal of the Science of Food and Agriculture, 77, 506-510. Frankel, S., Robinson, G.E. and Berenbaum, M. R. (1998). Antioxidant content and correlated characteristics of 14 monofloral honeys. Journal of Apiculture Research, 37, 27-31. Frye, E. B., Degenhardt, T.P., Thorpe, R.S. and Baynes, J. W. (1998) Role of the Maillard reaction in aging of tissue proteins. Advanced glycation end product- dependent increase in imidazolium cross-links in human lens proteins. Journal of Biological Chemistry, 273, 18714- 18719. Fu, M-X., Requena, J. R., Jemkins, A. J., Lyons, T. J., Baynes, J. W and Thorpe, S. R. (1996). The advanced glycation Nε-(Carboxymethyl)lysine, is a product of both lipid peroxidation and glycoxidation reactions. The Journal of Biological Chemistry, 271, 9982-9986. Fukumoto, L. R and Mazza, G. (2000). Assessing antioxidant and pro-oxidant activities of phenolic compounds. Journal of Agricultural and Food Chemistry, 48, 3597-3604. Gheldof, N., and Engeseth N. J. (2002). Antioxidant capacity of honeys from various floral sources based on the determination of oxygen radical absorbance capacity and inhibition of in vitro lipoprotein oxidation in human serum samples. Journal of Agricultural and Food Chemistry 50, 3050–3055. Gheldof, N., Wang, X-H and Engeseth, N, J. (2002). Identification and Quantification of Antioxidant Components of Honeys from various Floral Sources. Journal of Agricultural and Food Chemistry 50, 5870 -5877. Gniechwitz, D., Reichardt, N., Ralph, J., Blaut, M., Steinhart, H and Bunzel, M. (2008). Isolation and characterization of a coffee melanoidin fraction. Journal of the Science of Food and Agriculture 88, 2153-2160.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Honey Melanoidins

35

Gonzales, P. A., Burin, L and del Pilar Buera, M. (1999). Color changes during storage of honeys in relation to their composition and initial color. Food Research International 32, 185- 191. Henle, T. (2005). Protein-bound advanced glycation endproducts (AGEs) as bioactive amino acid derivatives in foods. Amino Acids, 29, 313-322. Hodge, J. E. (1953).Chemistry of browning reaction in model systems. Journal of Agricultural and Food Chemistry 1, 928- 943. Hofmann, T. (1998). Studies on the relationship between molecular weight and the color potency of fractions obtained by thermal treatment of glucose/amino acid and glucose/protein solutions by using ultracentrifugation and color dilution techniques. Journal of Agricultural and Food Chemistry 46, 3891-3895. Hotta, H, Sakamoto, H, Nagano S, Osakai, T, Tsujino J. (2001). Unusually large number of electrons for the oxidation of polyphenolic antioxidants. Biochemica et Biophysica Acta, 1526, 159-167. Kalapos, M. P. (2008). The tandem of free radicals and methylglyoxal. Chemical and Biological Interactions, 171, 251-271. Majtan, J. (2011). Methylglyoxal- A potential risk factor of manuka honey in healing of diabeticulcers. Evidence-based Complementary and Alternative Medicine doi:10.1093/ecam/neq013. Manzocco, L., Calligaris, S., Mastrocola, D., Nicoli, M.C., and Lerici, C.R. (2001). Review of non-enzymatic browning and antioxidant capacity in processed foods. Trends in Food Science and Technology 11. 340-346. Marshall, T and Williams, K. M. (1987). Electrophoresis of Honey: Characterization of Trace Proteins from a Complex Biological Matrix by Silver Staining. Analytical Biochemistry 167, 301-303. Martos, I., Ferreres, F. and Tomas-Barberan, A. (2000). Identification of Flavonoid markers for the botanical origin if Eucalyptus honey. Journal of Agricultural and Food Chemistry, 48, 1498-1502. Mavric, E., Wittmann, S., Barth, G., and Henle, T. (2008). Identification and quantification of methylglyoxal as the dominant antibacterial constituent of Manuka (Leptospermum scoparium) honey from New Zealand. Molecular Nutrition and Food Research 52, 483489. Meda, A, Lamien, Ch. E., Romito, M., Millogo, J. and Nacoulma, O. G. (2005). Determination of the total phenolic, flavonoid and proline contents in Burkina Fasan honey, as well as their radical scavenging activity. Food Chemistry, 91, 571-577. Methodieva, D., Jaiswal, A.K., Cenas, N., Dickancaite, E and Segura-Aguilar., J. (1999). Quercetin may act as cytotoxic pro-oxidant after its metabolic activation to semiquinone and quinoidal product. Free Radical Biology and Medicine, 26, 107-116. Montavon, P., Duruz, E., Rumo, G., and Pratz, G. (2003a). Evolution of green coffee protein profiles with maturation and relationship to coffee cup quality. Journal of Agricultural and Food Chemistry 51, 2328-2334. Montavon, P., Mauron A-F., and Duruz, E. (2003b). Changes in green coffee protein profiles during roasting. Journal of Agricultural and Food Chemistry 51,2335-2343. Morales, V, Sanz, L. M, Martin-Alvarez, P.J. and Corzo, N. (2009). Combine use of HMF and furosine to assess fresh honey quality. Journal of the Science of Food and Agriculture, 89, 1332-1338.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

36

Katrina Brudzynski

Muller, L and Boehm, V. (2011). Antioxidant activity of β- carotene compounds in different in vitro assays. Molecules 16, 1055-1069. Murthy, U.M.N. and Sun, W. Q. (2000). Protein modification by Amadori and Maillard reaction during seed storage: roles of sugar hydrolysis and lipid peroxidation. Journal of Experimental Botany, 51, 1221-1228. Nagai, T., Sakai, M., Inoue, R., Inoue, H. and Suzuki, N. (2001). Antioxidative activities of some commercially honeys, royal jelly, and propolis. Food Chemistry, 75: 237-240. Nagaraj, R., Shipanova, I. N and Faust, F. (1996). Protein cross-linking by the Maillard reaction. Isolation, characterization and in vivo detection of a lysine cross-link derived from methylglyoxal. Journal of Biological Chemistry, 32, 19338-19345. Nicoli, M.C, Anese, M, Parpinel, M. (1999). Influence of processing on the antioxidant properties of food and vegetables. Trend in Food Science and Technology, 10, 94- 100. Nunes, F. M and Coimbra, M. A. (2010). Role of hydroxycinnamates in coffee melanoidin formation. Phytochemical Reviews 9, 171–185. Nunes, F. M., and Coimbra, M. A. (2001). Chemical characterization of the high molecular material extracted with hot water from green and roasted Arabica coffee. Journal of Agricultural Food Chemistry, 49, 1773-1782. Nunes, F. M., and Coimbra, M. A. (2007). Melanoidins from Coffee Infusions. Fractionation, Chemical Characterization, and Effect of the Degree of Roast. Journal of Agricultural Food Chemistry, 55, 3967-3977. Odani, H., Shinzato, T., Usami, J., Yoshihiro Matsumoto, Y., Brinkmann Frye, E., John W. Baynes, J. W and and Kenji Maeda, K. (1998). Imidazolium cross-links derived from reaction of lysine with glyoxal and methylglyoxal are increased in serum proteins of uremic patients: evidence for increased oxidative stress in uremia. FEBS Letters, 427, 381-385. Ortega-Heras, M and Gonzales-Sanjose, M. L. (2009). Binding capacity of brown pigments present in special Spanish sweet wines. LWT-Food Science and Technology, 42, 17291737. Paiva, S. A, Russell, R. M. (1999). Beta-carotene and other carotenoids as antioxidants. Journal of American Collage of Nutrition, 18, 426–433. Perez, R. A., Iglesias, M. T., Pueyo, E., Gonzalez, M and de Lorenzo, C. (2007). Amino acid composition and antioxidant capacity of Spanish honeys. Journal of Agricultural Food Chemistry, 55, 360-365. Radwan, S.S, El-Essawy, A. A and Sarham, M. M. (1984). Experimental evidence for the occurrence in honey of specific substances active against microorganisms. Zentralblatt für Mikrobiologie 139, 249–255. Rawel, H. and Rohn, S. (2010). Nature of hydrocinnamate-protein interaction. Phytochemical Reviews 9, 93-109. Riboulet-Chavey, A., Pierron, A., Durand, I., Murdaca, J., Giudicelli, J and Van Obberghen, E. (2006). Methylglyoxal Impairs the Insulin Signaling Pathways Independently of the Formation of Intracellular Reactive Oxygen Species. Diabetes 55, 1289-1299. Rice-Evans, C. A., Miller, J. N. and Paganga, C. (1996). Structure-antioxidant activity relationship of flavonoids and phenolic acids. Free Radical Biology and Medicine, 20, 933-956.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Honey Melanoidins

37

Rios, A. M, Novoa, M. L and Vit, P. (2001). Effects of extraction, storage conditions and heating treatment on antibacterial activity of Zanthoxylum fagara honey from Cojedes, Venezuela. Revista Cientifica 11, 397-402. Rivero-Perez, M. D, Perez-Magarino, S, Gonzales-San Jos, M, L. (2002). Role of melanoidins in sweet wines. Analytica Chimica Acta, 458, 169-175. Rufian-Henares, J. A and Morales, J. A. (2007). Effect of in vitro enzymatic digestion on antioxidant activity of coffee melanoidins and fractions. Journal of Agricultural Food Chemistry 55, 10016-10021. Rufián-Henares, J. A., and de la Cueva S. P. (2009). Antimicrobial activity of coffee melanoidins -A study on their metal-chelating properties. Journal of Agricultural Food Chemistry 57, 432-438. Rurián-Henares, J.A, and Morales, F.J. (2008). Antimicrobial activity of melanoidins against Escherichia coli is mediated by a membrane-damage mechanism. Journal of Agricultural Food Chemistry, 56, 2357-62. Sakihama, Y., Cohen, M. C., Grace, S. C. and Yamasaki, H. (2002). Plant phenolic antioxidant and pro-oxidant activities: phenolics-induced oxidative damage mediated by metals in plants. Toxicology, 177, 67–80). Salah, N., Miller, N. J., Paganga, G., Tijburg, L., Bolwell, G. P., and Rice-Evans, C. (1995). Polyphenolic flavonols as scavengers of aqueous phase radicals and as chain-breaking antioxidants. Archives of Biochemistry and Biophysics, 332, 339-346. Schramm, D., Karim, M. and Schrader, H. R. (2003) Honey with high levels of antioxidants can provide protection to healthy human subjects. Journal of Agricultural Food Chemistry, 51, 1732-35. Spencer, C., Cai, Y., Martin, R., Gaffney, S. H., Goulding, P. N., Magnolato, D., Lilley, T. ZH and Haslam, E. (1988). Polyphenol complexation-some thoughts and observations. Phytochemistry, 27, 2397-2409. Summa, C., McCourt, J., Cammerer, B., Fiala, A., Probst, M., Kun, Sz., Anklam, E and Wagner, K-H. (2008). Radical scavenging activity, anti-bacterial and mutagenic effects of Cocoa bean Maillard Reaction products with degree of roasting. Molecular Nutrition and Food Research 52, 342- 361. Tagliazucchi, D., Verzelloni, E. and Conte, A. (2010). Contribution of melanoidins to the antioxidant activity of traditional balsamic vinegar during aging. Journal of Food Biochemistry, 34, 1061-1078. Tan, S. T., Wilkins, A. L., Holland, P. T., McGhie, T. K. 1989. Extractives from New Zealand unifloral honeys. 2. Degraded carotenoids and other substances from heather honey. J. Agric. Food Chem, 37, 1217-1221. Tosi E., Ciappini, M, Re E, and Lucero, H. (2002). Honey thermal treatment effects on hydroxymethylfurfural content. Food Chemistry. 77, 71-74. Tressl, R., Wondrak, T.G., and Garbe, L-A. (1998). Pentoses and hexoses as source of new melanoidin-like Maillard polymers. Journal of Agricultural Food Chemistry 46, 17651776. Turkmen, N., Sari, F., Poyrazoglu, E. S., and Velioglu, Y. S. (2006). Effects of prolonged heating on antioxidant activity and color of honey. Food Chemistry 95, 653–657. Vela, L., de Lorenzo, C. and Perez, R.A. (2007). Antioxidant capacity of Spanish honeys and its correlation with polyphenol content and other physicochemical properties. Journal of the Science of Food and Agriculture, 87,1069-1075.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

38

Katrina Brudzynski

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Villamiel, M, Del Castillo, M.D, Corzo, N and Olano, A. (2001). Presence of furosine in honey. Journal of the Science of Food and Agriculture, 81, 790- 793. Wang, H-Y., Qian, H. and Yao, W-R. (2011). Melanoidins produced by the Maillard reaction: Structure and biological activity. Food Chemistry, 128, 573-584. Wen et al. (2005). Effects of roasting on properties of the Zinc-chelating substance in coffee brews. Journal of Agricultural and Food Chemistry, 53, 2684-2689). White, J.W and Kushnir, I. (1967). Composition of honey. VII. Proteins. Journal of Apicultural Research 6,163–78. White, J.W and Subers, M, H. (1964). Studies on honey inhibine.4. Destruction of the peroxide accumulation system by light. Journal of Food Science 29, 819-828. Yao L, Nivedita Datta N,Tomás-Barberán FA, Ferreres F, Martos I, Singanusong R. 2003. Flavonoids, phenolic acids and abscisic acid in Australian and New Zealand Leptospermum honeys. Food Chemistry, 81, 159- 168. Yaylayan, V. A. (1997). Classification of the Maillard reaction: A conceptual approach. Trends in Food Sciemce and Technology, 8, 13-18. Yim, M. B., Yim, H. S., Lee, C., Kang, S. O. and Chock, P. B. (2001). Protein glycation: creation of catalytic sites for free radicals generation. Annals of New York Academy of Science, 928, 48-53.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

In: Honey: Current Research and Clinical Applications ISBN: 978-1-61942-656-6 Editor: Juraj Majtan © 2012 Nova Science Publishers, Inc.

Chapter III

Anticancer Activity of Honey and Its Phenolic Components Saravana Kumar Jaganathan1 and Mahitosh Mandal2, 1

Department of Biomedical Engineering, PSNA college of Engineering and Technology, Dindugal, Tamilnadu, India 2 School of Medical Science and Technology, Indian Institute of Technology, Kharagpur, West Bengal, India

Abstract Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Cancer is a class of diseases in which a cell or a group of cells display uncontrolled growth, invasion, and sometimes metastasis. Most of the drugs used in cancer are apoptotic inducers, hence apoptotic nature of any natural/artificial compound is considered vital. Honey has a long history of human consumption and is used for both medical and domestic needs. Phenolic profile, antioxidant ability as well as anticancer property of various honey types were studied recently. A number of researchers have investigated the effect of crude honey on cancer. Research conducted on the effect of honey against colon cancer cells provided valuable clues of honey induced apoptosis. Honey transduced the apoptotic signal via initial depletion of intracellular non protein thiols, consequently reducing the mitochondrial membrane potential (Ψ) and increasing the reactive oxygen species (ROS) generation. Time kinetic analysis of honey treated cells using flow cytometry showed the increasing accumulation of hypodiploid nuclei in the sub-G1 phase of cell cycle indicating apoptosis Honey induced apoptosis by upregulating the p53 and modulating the expression of pro and anti-apoptotic proteins. Honey exhibited moderate antitumor and significant antimetastatic effects in five different strains of rat and mouse tumors. Further the antitumor activity of certain chemotherapeutic drugs such as 5-fluorouracil and cyclophosphamide were facilitated by honey. In another research, honey was showed to induce apoptosis in T24, RT4, 253J and MBT-2 bladder cancer cell lines. They showed significant inhibition of the proliferation 

Correspondence concerning this article should be addressed to: Dr Mahitosh Mandal, School of Medical Science and Technology, Indian Institute of Technology, Kharagpur, Tel: +91-3222-283578, Fax: 91-3222-255303, Email address: [email protected].

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

40

Saravana Kumar Jaganathan and Mahitosh Mandal of T24 and MBT-2 cell lines by 1-25% honey and of RT4 and 253J cell lines by 6-25% honey. Further in the in vivo studies, intralesional injection of 6 and 12% honey significantly inhibited bladder tumor cell growth. Moreover, some of the phenolic honey components of honey were found to induce apoptosis. We concentrated on the some major polyphenols available in the honey which exhibited antiproliferative effect on various cancer cell lines. The list of compounds reviewed for their anticancerous activity is caffeic acid (CA), caffeic acid phenyl ester (CAPEs), chrysin (CR), galangin (GA), quercetin (QU), acacetin (AC), kaempferol (KF), pinocembrin (PC), pinobanksin (PB), and apigenin (AP). In succinct, this chapter aimed in summarizing the anticancer and molecular mechanism of honey and above-mentioned polyphenols against various cancer cell lines.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

1. Introduction Prevention is better than cure and this is very true in case of cancer. Chemoprevention was defined as the administration of agents to prevent induction, to inhibit or to delay the progression of cancer [1], or as the inhibition or reversal of carcinogenesis at a premalignant stage [2]. Chemoprevention utilizes appropriate pharmacological agents [3, 4] or of dietary agents, consumed in diverse forms like macronutrients, micronutrients, or non-nutritive phytochemicals [5-7]. Consumption of antioxidants have been related to the several preventative effects against different diseases such as cancer, coronary diseases, inflammatory disorders, neurological degeneration, aging, etc., [8, 9] led to search for natural foods rich in antioxidants. Although honey has been used since long time, only recently its antioxidant property came to limelight [10]. Honey has some minor constituents compared to its major sugar level, which is believed to have antioxidant properties [11, 12]. Some to mention were flavonoids and phenolic acids [13, 14], certain enzymes (glucose oxidase, catalase), ascorbic acid [15], carotenoid-like substances [16], organic acids [13], amino acids and proteins [17]. Phytochemicals are one wide class of nutraceuticals found in plants which are extensively researched by scientists for their health-promoting potential. Honey has a wide range of phytochemicals including polyphenols which act as antioxidants. Polyphenols and phenolic acids found in the honey vary according to the geographical and climatic conditions. Some of them were reported as a specific marker for the botanical origin of the honey. Considerable differences in both composition and content of phenolic compounds have been found in different unifloral honeys [18]. Terpenes, benzyl alcohol, 3, 5-dimethoxy-4-hydroxybenzoic acid (syringic acid), methyl 3, 5-dimethoxy-4-hydroxybenzoate (methyl syringate), 3, 4, 5trimethoxybenzoic acid, 2-hydroxy-3-phenylpropionic acid, 2-hydroxybenzoic acid and 1, 4dihydroxybenzene are some of the phytochemicals ascribed for the antimicrobial activity of honey [19]. Among these phytochemicals, polyphenols were reported to have antiproliferative potential. In this review, we summarized the compositional chemistry and antiproliferative potential of crude honey and some of its important polyphenols in various cancer cells.

2. Source and Compositional Chemistry of Honey Honey bees collect the nectar from various floral sources and store it as honey which serves as food for bees during winter. Honey bees make a journey of nearly 55,000 miles to

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Anticancer Activity of Honey and Its Phenolic Components

41

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

gather nectar from approximately 2 million flowers for accumulating one pound of honey. In the bee-hive, we can find thee types of bees namely the queen, drone and worker bees. Among them, only worker bees collect and regurgitate the nectar number of times, in order to partially digest the nectar, before storing in the honey comb. During the collection of nectar, pollen can be included into the honey though variety of ways. As the honeybee visits the flower in hunt of nectar, some of the flower's pollen falls into the nectar collected by the bee and stored in the stomach which will be regurgitated along with nectar. Moreover some pollen grains often attach themselves to the various parts of the honey bee body like legs, antenna, hairs, and also in the eyes of visiting bees which will get entangled in the hive and thereby paving entry into the honey. Airborne pollen is also another route of entry for pollen into the honey which got transferred though wind currents. Honey bees use its wings to fan the honey comb, to evaporate most of the water from nectar thereby avoiding the fermentation of honey.

Figure 1. Color variation of honey samples from dark amber (top-left dish) to whitish yellow (bottomright dish). Flavor of the honey depends upon the color, generally darker the honey stronger the flavor and quality.

Figure 2. Pie-chart of honey composition indicating the percentage share of various sugars, water and other minor constituents.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

42

Saravana Kumar Jaganathan and Mahitosh Mandal

The color of the honey collected by the bees varies according to the floral source and its mineral content, which usually ranges from water white to dark amber. Flavor of the honey depends upon the color, generally darker the honey stronger the flavor and quality (Figure 1). It has been reported more than 300 unique varieties of honey depending upon the floral sources from United States alone. Honey mainly composed of sugars and water which accounts roughly 79.6 % and 17.2 % respectively (Figure 2). Major sugars of honey are levulose and dextrose which constitutes 38.19 % and 31.28 % correspondingly, remaining are the sucrose 1.3 % and maltose 7.3 %. Honey minor constituents include acids (.57 %), protein (.266 %), nitrogen (0.043 %), amino acids (0.1 %), a little amount of minerals (.17 %), and a number of other minute quantities of components like pigments, flavor and aroma substances, phenolics compounds, colloids, sugar alcohols and vitamins which all together accounts for the 2.1 % of whole honey composition [20-21].

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

3. Anti-Anticancerous Property of Crude Honey Few researchers studied the effect of crude honey in cancer. In a recent research conducted by Jaganathan et al. illustrated the apoptosis inducing ability of the honey. They showed honey induced apoptosis in human colon cancer cells by arresting the cells at subG1 phase. Honey possessing higher phenolic and tryptophan content was more potent in inhibiting the colon cancer cell proliferation. Honey-induced apoptosis was associated with Caspase-3 activation and PARP cleavage. DNA fragmentation assay in HT 29 cells displayed typical ladder pattern confirming apoptosis [22]. Honey transduced the apoptotic signal via initial depletion of intracellular non protein thiols, consequently reducing the mitochondrial membrane potential (MMP) and increasing the reactive oxygen species (ROS) generation. An increasing earlier lipid layer break was observed in the treated cells compared to the control. Honey induced apoptosis was accompanied by up-regulating the p53 and modulating the expression of pro and anti-apoptotic proteins [23]. In further studies, they studied antitumor activity of two selected honey samples and eugenol (one of the phenolic constituents of honey) against murine Ehrlich ascites and solid carcinoma models. Honey containing higher phenolic content was found to significantly inhibit the growth of Ehrlich ascites carcinoma as compared to other samples. When honey containing higher phenolic content was given at 25% (volume/volume) intraperitoneally (i/p), the maximum tumor growth inhibition was found to be 39.98%. However, honey was found to be less potent in inhibiting the growth of Ehrlich solid carcinoma [24]. In another research they proposed honey with multitude of phenolic and flavonoid compounds may be considered as a plausible candidate for reversing the multidrug resistance by inhibiting the P-gp proteins [25]. Research led by Orsolic et al. showed that water soluble derivative of propolis and its associated phenolic compounds have anti-metastatic effect on the tumor mice models before and after the injection of tumor cells. Further they showed honey could exert anti-metastatic effect when given before tumor cell injection [26]. In the study conducted by Tarek et al. honey was proven to be a very effective agent for repressing the growth of bladder cancer cell lines (T24, RT4, 253J and MBT-2) in vitro. Further honey was found to be effective when administered intralesionally or orally in the MBT-2 bladder cancer implantation models. There was also a significant difference between the final tumor volume (P< 0.05) in the Intra lesion (IL) honey-treated groups (IL

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Anticancer Activity of Honey and Its Phenolic Components

43

6% honey) compared to the IL saline group. The difference between the final tumor volume or weight in the IL saline group and the control group was not significant [27]. Research conducted by Gribel and Pashinskii indicated that honey exhibited moderate antitumor and significant anti-metastatic effects in five different strains of rat and mouse tumors. Moreover, the antitumor activity of certain chemotherapeutic drugs such as 5-fluorouracil and cyclophosphamide were also facilitated by the honey [28]. It has been elucidated that polyphenols are anticarcinogenic, anti-inflammatory, anti-atherogenic, antithombotic, immune modulating and also act as antioxidants [29–34]. Hence antitumor properties of honey could be attributed to the polyphenols found in the honey. Moreover, with the evolution of extraction procedure for various polyphenols, which had been attributed with anti-cancerous property of honey, researchers concentrated on the polyphenolic compounds extracted from the honey rather than crude honey itself. Phenolic compounds or polyphenols are the important groups of compounds occurring in plants, where they are widely distributed, comprising at least 8000 different known structures [35]. It is also produced by plants as a secondary metabolite. Some of these phenolic compounds were also available in the honey. In general, phenolic compounds can be divided into at least 10 types depending upon their basic structure: simple phenols, phenolic acids, coumarins and isocoumarins, naphthoquinones, xanthones, stilbenes, anthaquinones, flavonoids and lignins. Flavonoids constitute the most important polyphenolic class, with more than 5000 compounds already described. Flavonoids are the natural antioxidants exhibiting a wide range of biological effects including antibacterial, anti-inflammatory, antiallergic, antithombotic and vasodilatory actions [29]. Various polyphenols were reported in honey. Polyphenols found in the honey was used a marker for particular type of honey. For example, flavanol kaempferol as an indicator for rosemary honey [36, 37] and quercetin for sunflower honey [38]. The hydroxy-cinnamates like caffeic acid, ferulic acid and p-coumaric acid have been found in the chest-nut honey [39]. Characteristic flavonoids of propolis like pinocembrin, pinobanksin and chrysin were also found in the most European honey samples [38]. In this review, we concentrated on the some major polyphenols available in the honey which exhibited antiproliferative effect on various cancer cell lines. The list of compounds reviewed for their anti-cancerous activity are Caffeic acid (CA), Caffeic acid phenyl ester (CAPE), Chrysin (CR), Galangin (GA), Quercetin (QU), Acacetin (AC), Kaempferol (KF), Pinocembrin (PC), Pinobanksin (PB), and Apigenin (AP)

3.1. Role of Individual Polyphenols in Cancer 3.1.1. Effect of Caffeic Acid and Its Esters in Animal Model and Cancer Cell Lines Caffeic acid is a naturally occurring phenolic compound present in the honey. Research conducted by Hirose et al. studied the carcinogenicity of low dietary levels of the antioxidants like butylated hydroxyanisole (BHA), caffeic acid, sesamol, 4-methoxyphenol (4-MP) and catechol. These antioxidants were eminent target of the fore-stomach or glandular stomach and these were examined for their predominant effects in alone or in combinations, for a twoyear period experiment. Carcinogenicity study was undertaken in groups of 30–31 male F344 rats, by treating with 0.4% BHA, 0.4% caffeic acid, 0.4% sesamol, 0.4% 4-MP and 0.16%

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

44

Saravana Kumar Jaganathan and Mahitosh Mandal

catechol either alone or in combination for up to 104 weeks and then killed. The ultimate average body weights of rats having basal diets were higher than those treated with antioxidants alone, and were lowest in the combinational groups. Moreover the relative liver and/or kidney weights were greater than before in the BHA, sesamol, catechol and combination groups. It led to the conclusion, that the occurrences and frequencies of forestomach histopathological lesion were increased by exposing to antioxidants except in the case of BHA. The incidences and/or multiplicities of fore-stomach papillary or nodular (PN) hyperplasia were appreciably increased in the groups treated with 4-methoxyphenol, caffeic acid and the antioxidants in combination, as compared with the basal diet group. Studies on medium-term multiorgan carcinogenesis model, suggested an increase in the occurrence of fore-stomach papillomas in each high dose group and no synergistic effect were observed in combinations. In the low dose case, the incidence of fore-stomach papillomas was significantly increased only in the combination group. The effect on the other organs particularly colon tumors, was significantly decreased only in the high dose combination group. Hence it can be inferred that at low dose levels, the phenolic compounds can exhibit additive/synergistic effect on carcinogenesis [40]. From these early experiments, caffeic acid is still listed under older Hazard Data sheets as a potential carcinogen. Rao et al. performed a detailed study by synthesizing thee caffeic acid esters namely methyl caffeate (MC), phenylethyl caffeate (PEC) and phenylethyl dimethylcaffeate (PEDMC) and examined them against the 3, 2'-dimethyl-4-aminobiphenyl (DMAB, a colon and mammary carcinogen) induced mutagenecity in Salmonella typhimurium strains TA 98 and TA 100. Both the strains of Salmonella subsisted (survival rate >98%) concentration of about 2,500 μM CA, 150 μM MC, 70 μM PEC and 80 μM PEDMC/plate. Moreover 150μM MC, 40-80μM of PEDMC, 40-60μM of PEC significantly inhibited the DMAB induced mutagenecity in both strains. The outcome of these experiments placed MC at a concentration greater than 225 μM and PEC and PEDMC at a level greater than 60 μM were toxic. CA exhibited significant toxicity only at above 2500 μM concentration. In colon cancer cell line (HT-29), cytotoxicity effect of CA, PEC, PEDMC and MC were evaluated. The growth inhibitory effect of these compounds were measured after exposing cells for a period of 48 h. CA was found to be the least effective in inhibiting the growth of HT-29 cells when compared to its ester analogs. To further corroborate the growth inhibitory effects, synthesis of polynucleotide and protein synthesis after incubating the HT-29 cells with these agents for 48 h were investigated. It has been observed that at the concentration of 175 μM of MC, 40 μM of PEC and 60 μM of PEDMC blocked the DNA, RNA and the protein synthesis. Moreover ornithine decarboxylase (ODC) activity was inhibited at concentrations of 150 μM MC, 40 μM PEC and 20 μM PEDMC. Tyrosine protein kinase (TPK) activity was also inhibited at concentrations of 100μM of MC, 30μM of PEC and 20 μM of PEDMC [41]. In their followup studies made by them, reported the inhibitory effects of methyl caffeate (MC) and phenylethyl caffeate (PEC) on azoxymethane (AOM)-induced ornithine decarboxylase (ODC), tyrosine protein kinase (TPK) and arachidonic acid metabolism in liver and colonic mucosa of male F344 rats. They depicted the inhibitory effects of caffeic acid, MC, PEC, phenylethyl-3-methylcaffeate (PEMC), and phenylethyl dimethyl caffeate (PEDMC) on in vitro arachidonic acid metabolism in liver and colonic mucosa. Finally they investigated the effects of PEC, PEMC, and PEDMC on AOM-induced aberrant crypt foci (ACF) formation in the colon of F344 rats. For a period of five weeks, groups of F334 rats were fed with diets containing 600 ppm of MC or PEC for biochemical studies and 500 ppm of PEC, PEMC or

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Anticancer Activity of Honey and Its Phenolic Components

45

PEDMC for ACF studies. After two weeks, subcutaneous injection of AOM was given once in a week for two consecutive weeks, for all animals except the vehicle-treated groups. Biochemical studies were performed by sacrificing the animal after 5 days. In case of ACF study, F334 rats were sacrificed after 9 weeks latter for analyzing ACF in colon. The colonic mucosa and liver of the rats were analyzed for the orinithine decarboxylase activity, tyrosine protein kinase activity (TPK), lipoxygenase and cyclooxygenase metabolites. PEC diet significantly inhibited AOM-induced ODC and TPK activities in liver and colon. It had been observed that PEC diet significantly repressed the AOM-induced lipoxygenase metabolites 8(S) - and 12(S)-hydroxyeicosatetraenoic acid (HETE). The animals fed the MC diet exhibited a moderate inhibitory effect on ODC and 5(S)-, 8(S)-, 12(S)-, and 15(S)-HETEs and a significant effect on colonic TPK activity. However, both the MC and PEC diets showed no significant inhibitory effects on cyclooxygenase metabolism. ACF were significantly inhibited in the animals fed with PEC (55%), PEMC (82%), or PEDMC (81%). The results of the study indicated that PEC, PEMC and PEDMC present in the honey, inhibited AOMinduced colonic preneoplastic lesions, ODC, TPK, and lipoxygenase activity, which were relevant to the colon carcinogenesis [42]. Huang et al. showed the strong repressive effect of CAPE application on 12-0tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion and production of 5hydroxymethyl-2‘-deoxyuridine (HMdU) in the deoxyribonucleic acid (DNA) of the mouse skin. They established the inhibitory effect of CAPE on TPA-induced tumor promotion by topical application of CAPE in CD-I mice previously treated with 7, 12dimethylbenz[a]anthracene (DMBA). They applied CAPE in concentration ranging from 1, 10, 100 or 3000 nmol together with 5 nmol of TPA twice a week for 20 weeks. At the above concentrations, CAPE inhibited the number of skin papillomas by 24, 30, 45 and 70 % and tumor size per mouse was decreased by 42, 66, 53 and 74 % respectively. Moreover topical application of 5 nmol of TPA twice weekly for 20 weeks to mice produced an average of 12.6 HMdU residues per 104 normal bases in epidermal DNA. Topical application of 1, 10, 100 or 3000 nmol of CAPE along with 5 nmol of TPA twice weekly for 20 weeks to DMBAinitiated mice decreased the levels of HMdU in epidermal DNA by 40-93 %. CAPE at 1.25, 2.5, 5, 10 or 20 μM inhibited the incorporation of [ 3H]-thymidine into DNA in cultured HeLa cells by 32, 44, 66, 79 and 95 % respectively. Similarly incorporation of [ 3H]-uridine into RNA was inhibited by 39, 43, 58, 64 and 75 % whereas incorporation of [ 3H]-leucine into protein was inhibited by 29, 30, 37, 32 or 47 % respectively. These results indicated that CAPE is a potent inhibitor of DNA synthesis but it is somewhat less effective in inhibiting RNA synthesis and it is least effective in inhibiting the protein synthesis [43]. The molecular basis of CAPE action was elucidated by Natrajan et al. Since NF-кB has a role in these activities, they examined the effect of CAPE on this transcription factor in an exhaustive manner. They pre-incubated the U-937 cells with CAPE with various concentrations for 2 h before treating with TNF (0.1 nM) for 15 min. CAPE inhibited the TNF-dependent activation of NF-кB in a dose-dependent manner with maximum effect occurring at 25 μg/ml. NF-кB activation induced by the phorbol ester, phorbol-12-myristate 13-acetate (PMA), ceramide, okadaic acid and hydrogen peroxide were also inhibited by CAPE. It prevented the translocation of p-65 subunit of NF-кB to the nucleus without affecting the TNF-induced IкBα degradation. It does not showed any inhibitory effect on the other transcription factors like AP-1, TFIID and oct-1. To study further precisely about the role of CAPE in inhibiting NF-кB various structural analogues of CAPE were examined. It

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

46

Saravana Kumar Jaganathan and Mahitosh Mandal

has been configured that a bicyclic, rotationally constrained, 5, 6-dihydroxy form showed supremacy, whereas 6, 7-dihydroxy variant was least active in inhibiting the NF-кB. With these findings they concluded that CAPE is a potent and a specific inhibitor of NF-кB activation and this may provide the molecular basis for its multiple immunomodulatory and anti-inflammatory activities of CAPE [44]. In another study initiated by Lee et al. investigated the cytotoxicity potential of CAPE and the molecular mechanism of its action in C6 glioma cells. The results of the experiments indicated C6 glioma cells underwent internucleosomal DNA fragmentation after 24 h treatment with CAPE (50 μM). FACS analysis of CAPE-treated C6 glioma cells showed increasing accumulation of hypodiploid nuclei (24% at 36 hour) in time-dependent fashion. Further results showed that CAPE induced the release of cytochome-c from mitochondria into the cytosol after 3 h of treatment resulting in the activation of caspase-3 (CPP32) from the beginning of 3 h. Moreover the cleavage of PARP (substrate of CPP32) started within 12 h after CAPE treatment. CAPE enhanced the serine phosphoryaltion of p53 after 0.5 h and the protein level of p53 was increased after 3 h. CAPE treatment also enhanced the expression of Bax and Bak and resulted in the reduced level of B-cell lymphoma/leukemia-2 gene (Bcl2) protein (after 36 h). Moreover they reported that CAPE application activates the extracellular signal-regulated kinase (ERKs) and p38 mitogen-activated protein kinase (p38 MAPK) in the C6 glioma cells. Further they showed that expression of p53, phospho-serine 15 of p53, Bax and inactivate form of CPP32 were suppressed by a pre-treatment of a specific p38 MAPK inhibitor, SB203580. Hence they concluded p53 dependent apoptosis in C-6 glioma cells were mediated by p38 MAPK [45]. Chung et al. showed both CA and CAPE selectively inhibited Matrix Metalloproteinases2 (MMP-2) and MMP-9. CAPE inhibited strongly with IC50 of 2-5 μM whereas CA requires 10-20 μM. But MMP-1, 3, 7 and Cathepsin–K were not completely inhibited by both of them. CA and CAPE had a dose-dependent inhibitory effect on the proliferation of HEPG2 cells. In HepG2 cells, CA at the concentration of 200 μg/mL reduced the cell viability to 61% compared to the control, and the treatment with CAPE (at low concentration of 20 μg/mL) reduced the viability to 72% compared of the control. CAPE and CA suppressed the MMP-9 expression, exposed to phorbol 12-myristate 13-acetate (PMA), by blocking the NF-кB activity in HEPG2 cells. They also confirmed that CA (20 mg/kg) and CAPE (5 mg/kg) repressed the growth of HepG2 tumor xenografts in nude mice as well as liver metastasis when administered subcutaneous or orally. Finally they concluded their observation as follows that CA and its derivative CAPE: 1) inhibited the enzymatic activity of MMP-9 that plays an important role in cancer invasion and metastasis, 2) blocked the invasive potential though the suppression of MMP-9 gene transcription by inhibiting NF-кB function in PMAstimulated HepG2 cells and 3) suppressed the growth of HepG2 cell xenografts in nude mice. Therefore, these two drugs were reported as strong candidates for treatment of cancer and metastasis via dual mechanisms (dual inhibition of metastasis-specific enzyme activity and gene transcription) [46]. Further in a recent study initiated by H.J. Hwang et al. investigated the effect of CAPE on tumor invasion and metastasis in HT 1080 fibrosarcoma cells by determining the regulation of matrix metalloproteinase‘s (MMPs). HT 1080 cells were treated with increasing concentration of CAPE and the m-RNA transcripts of MMP-2 and MMP-9 were analyzed using semi-quantitative RT-PCR. Both MMP-2 and 9 proteins level were significantly suppressed at dose dependent manner. Gelatin zymography also indicated constitutively

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Anticancer Activity of Honey and Its Phenolic Components

47

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

expressed MMP-2 and 9 proteins in HT 1080 cells which gradually reduced after treating with CAPE. To further corroborate the down-regulation of MMP-2, activation studies of proMMP2 were performed using organomercuric compound, 4-aminophenylmercuric acetate (APMA), and the result indicated the down regulation of MMP-2 by CAPE. It has been shown that m-RNA levels of Tissue inhibitor of matrix metalloproteinase‘s (TIMPs) and Membrane type- Matrix Metalloproteinase‘s (MT-1 MMPs) were also reduced significantly. CAPE also inhibited the cell invasion, cell migration and colony formation of tumor cells. Thus CAPE acts as a vital anti-metastatic agent, by inhibiting the metastatic and invasive potential of malignant cells [47]. Moreover some researchers investigated the possible UVC (280-100nm) protective properties of caffeic acid in human diploid fibroblast and A-431 epidermoid cancer cell lines. The UVC safeguarding effect of CA in two different concentrations (55.5 μM and 166.5 μM) were clearly illustrated both in transformed and normal cells. A marked difference in the proliferation of normal and transformed cells when irradiated to UVC radiation was observed when cell was grown in DMEM media containing CA. CA‘s protective effect was distinct in the transformed cells compared to normal cells [48]. In a sequential study by Vanisree et al, explained protective effect of CA against UVB (280- 320 nm) radiation induced IL-10 expression and the activation of the Mitogen-activated Protein Kinase (MAPK‘S) in mouse skin. CA inhibited the IL-10 promoter transcription, measured using in vivo transgenic IL-10 promoter-luciferase reporter gene base assay. IL-10 mRNA expression and protein production in the mouse skin was significantly repressed by CA. It has also been shown the upstream regulators like extracellular regulated protein kinase (ERK), c-Jun N-Terminal protein, p-38 mitogen activated protein kinase (p38 MAPK) and the downstream transcription factors like activator protein (AP-1) and nuclear factor kappa B (NF-кB) were also inhibited by CA in mouse skin. From these experiments it was inferred that CA could be used as a topical agent against harmful UVB irradiation [49]. 3.1.2. Effect of Chrysin and Its Derivatives in Cancer Cell Lines Chrysin (5, 7-dihydroxyflavone) is a natural and biologically active compound extracted from honey, plants and propolis. It possesses potent anti-inflammatory, antioxidant properties and promotes cell death by perturbing cell cycle progression. In a recent study conducted by Weng et al. illustrated the molecular mechanism of action of chrysin against C6-glioma cells. In an anti-proliferation assay performed on C6 glioma cells, chrysin inhibited the cell proliferation after 24, 48 and 72 h. After 72 h of incubation with 50 μM of chrysin, 90 % of cell proliferation was inhibited. Flow cytometry analysis reported that by 30 and 50 μM treatments after 24 h, chrysin increased the proportion of cells in the G1 phase of the cell cycle from 69 to 79 % and 83 % and decreased the proportion of S phase cells from 11.4 to 6.1 % and 2.8 % respectively. The proportion of G2/M phase cells changed from 17.9 to 12.2 % and 9.2 %, after 30 and 50 μM treatments. It has been found that levels of phosphorylation of Retinoblastoma (Rb) protein in C6 glioma cells decreased after treating with 30μM of chrysin. Moreover in chrysin-treated cells, it has been demonstrated that cyclin dependent kinase inhibitor (p21Waf1/Cip1) levels to be increased significantly without the change in p53 protein level. To depict the role of p38 in chrysin-mediated p21Waf1/Cip1induction, they used p38 specific inhibitor which resulted in the lowering of p21 Waf1/Cip1 level. Furthermore they showed that proteosome activity, cyclin dependent kinase 2 (CDK2) and 4 (CDK4) were also inhibited by chrysin. These results suggested that chrysin exerts its growth-inhibitory effects

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

48

Saravana Kumar Jaganathan and Mahitosh Mandal

either though activating p38-MAPK leading to the accumulation of p21Waf1/Cip1 protein or mediating the inhibition of proteosome activity [50]. In another study by Woo et al. reported the chrysin-mediated apoptosis in U-937 cancer cell lines. DNA fragmentation assay of chrysin treated cells after 12 h showed typical internucleosomal fragmentation of DNA. FACS analysis of treated cells showed marked increase of accumulation of subG1 cells after 12 h. Decreased proenzyme level of caspase-3 after chrysin treatment indicated the importance of activated caspase-3 in apoptosis. Further the activation of phospho-lipase C- γ (PLC-γ), a down stream target of caspase-3 in chrysin treated cells confirmed the role of caspase-3 in chrysin treated U937 cells. Western blotting analysis of chrysin treated cells indicated the reduction in the level of XIAP (a member of Inhibitor of Apoptosis Proteins) and cytochrome c induction in dose dependent manner. Mitogen activated protein kinase (MAPK) does not have any role in the signaling pathway as shown by western blot analysis, whereas Akt- signaling played significant role in chrysin mediated apoptosis of U937 cells. It has been shown that inhibition of Akt phosphorylation in U937 cells by the specific PI3K inhibitor, LY294002, significantly enhanced the apoptosis. Overexpression of a constitutively active Akt (myr-Akt) in U937 cells inhibited the induction of apoptosis, activation of caspase 3 and PLC-γ1 cleavage by chrysin [51]. Further Zheng et al. synthesized 13 derivatives of chrysin and tested it for anti-cancer effect against human gastric adenocarcinoma cell line (SGC-7901) and colorectal adenocarcinoma (HT-29) cells. These derivatives were formed mainly by alkylation, halogenation, nitration, methylation, acetylation and trifluoromethylation. MTT assay revealed that 5, 7-dimethoxy-8-iodochrysin and 8-bromo-5-hydroxy-7-methoxychrysin have the strongest activities against SGC-7901 and HT-29 cells respectively. The compound 5, 7Dihydroxy-8-nitrochrysin was found to have strong activities against both SGC-7901 and HT-29 cells [52]. Zhang et al. tried to improve the biological properties of chrysin by synthesizing diethyl chrysin-7-yl phosphate (CPE: C19H19O7P) and tetraethyl bisphosphoric ester of chrysin (CP: C23H28O10P2) though a simplified Atheron–Todd reaction. In Mass spectroscopy analysis, CPE formed complexes with lysozyme and hence phosphate esters of chrysin enhanced the interaction with proteins compared to unmodified chrysin. Cultured human (HeLa) cell lines were treated by CR, CP and CPE with 10 μM for 24, 48, and 72 h. The Cell viability markedly declined in time-dependent fashion. Moreover methyl green-pyronin staining, PCNA immunohistochemistry and TUNEL techniques were also employed to study the effect of CR, CPE and CP in the cultured HeLa cell lines. It favored their hypothesis that all CR, CP and CPE could inhibit proliferation and induce apoptosis in the following order of inhibition potency CP> CPE> CR. Hence they suggested CP and CPE as a new potential candidate for human cervical cancer [53]. 3.1.3. Effect of Galangin in Leukemia Cancer Cell Line Charles et al. described the antiproliferative effect of galangin on human leukemia (HL60) cell line. Trypan blue exclusion method indicated the remarkable decrease in the cell viability after treating with 100 μM for 24 h. Galangin of 1-10 μM exerted antiproliferative effect which is evident after 48 h of incubation. Early and late apoptosis were detected using annexin-V-FITC and PI staining using 100μM galangin and these results correlated with the results of trypan-blue method reported already. Active caspase-3, a hallmark of apoptosis process, was detected after 24 h and 72 h of incubation with 50 and 10 μM of galangin respectively. Cell cycle analysis indicated the increase in the subG1 phase of galangin (>10

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Anticancer Activity of Honey and Its Phenolic Components

49

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

μM) treated cells. This was illustrated further in DNA fragmentation assay, in which they could observe typical ladder pattern after 24 h of 100 μM galangin exposure. Forward and side scatter changes were predominantly observed after 24 h and 72 h incubation with 100 μM galangin. Galangin treated cells displayed reduced forward scatter indicative of decreased relative size, and enhanced side scatter indicative of increased internal complexity. Rhodamine median florescence intensity measured as an indicator of ROS levels, showed no evidence for intracellular oxidative stress as a key-player of cytotoxicity and significant phagocyte-like differentiation was not detected [54]. 3.1.4. Effect of Quercetin in Cancer Cell Lines Kang et al. investigated the role of quercetin as an anti-cancer agent in HL-60 cells. From their experiments they inferred the concentration dependent inhibition of HL-60 cell proliferation between the ranges of 10 to 80 μM. They showed cells incubated with 10 μM displayed inhibition on the growth of HL-60 cells. It was 17.1, 27.3, 40.1, and 52.7% after 24, 48, 72, and 96 h of treatment. Cell cycle analysis indicated that quercetin (20, 40, and 60 μM) increased the number of cells in the G2/M phase from 7.6% to 12.4%, 19.1%, and 23.5% correspondingly, and decreased the population of G0/G1, cells from 46.2% to 40.2%, 32.1%, and 34.5% respectively, without significant changes in the S-phase cell population after 24 h treatment. Quercetin showed remarkable inhibitory effect on the activities of cytosolic Protein Kinase C (PKC) and membrane TPK of HL-60 cells in vitro, with IC50 values of about 30.9 and 20.1 μM respectively, but did not have the effect on membrane PKC or cytosolic TPK activity. It has also repressed the complete activity of phosphoinositides like phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and Phosphatidylinositol 4, 5-bisphosphate (PIP2) at the concentration of 80 μM. Hence they concluded the inhibitory effect of quercetin on the growth of HL-60 cells may be related to its inhibitory effects on PKC and/or TPK in vitro and/or on the production of phosphoinositides [55]. Csokay et al. studied the effect of quercetin in K562 human leukemia cells. Treatment with quercetin (5.5 µM) activated both apoptosis and differentiation programs. After 1 h exposure to the drug it resulted in apoptosis of the leukemia cells. Differentiation of K562 cells was observed at least after 12 h of exposure. They attributed these effects to the early downregulation of c-myc and Ki-ras oncogenes and rapid reduction of Inositol-1, 4, 5-triphosphate (IPs) concentrations [56]. Robaszkiewicz et al. illustrated the effect of quercetin in A-549 cells. They found quercetin exerted both antioxidant and pro-oxidant properties depending upon the concentration used. Quercetin in low concentration (1-20 μM) promoted the cell proliferation whereas higher concentration (50-200 μM) showed the concentration dependent cytotoxicity. The lower concentration (10 μM) of quercetin produced increased number of live cells, repressing the number of cells in the apoptotic and necrotic portions. On the other hand if the concentration was above 50 μM, it reduced the number of live cells by increasing the apoptotic/necrotic fractions. Quercetin decreased production of reactive oxygen species in the cells producing peroxides in the medium. They also found incubating with low concentrations of quercetin led to a small increase in Total Antioxidant Capacity (TAC) of cell extracts but higher concentrations of the quercetin led to a progressive decrease in the TAC of cell extracts. Total thiol content of the cells followed a pattern similar to TAC. Hence they suggested that cellular effects of quercetin are complex and include both antioxidant effects and induction of oxidative stress due to formation of reactive oxygen species in the

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

50

Saravana Kumar Jaganathan and Mahitosh Mandal

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

extracellular medium [57]. In another study made by Elizandra et al. substantiated that quercetin may act differently on cancer and normal neuronal tissue. Quercetin decreased the cell viability in glioma cell cultures resulting in necrotic and apoptotic cell death. It also arrested the glioma cells in the G2 checkpoint of the cell cycle, and decreased the mitotic index. Furthermore, they demonstrated quercetin was able to protect the hippocampal organotypic cultures from ischemic damage. These results showed that although it induced growth inhibition and cell death in the U138MG human glioma cell line, still it has a cytoprotective effect in normal cell cultures [58]. Indap et al. examined the antiproliferative effect of quercetin both in vitro and in vivo. They showed quercetin could exert antiproliferative effect against MCF-7 cell line in a dose and time dependent manner with IC50 value of 10 µg/ml. Further quercetin was found to arrest the MCF-7 cell growth in G2/M phase of cell cycle. Moreover it was shown that quercetin inhibited the tumor growth by more than 58 % in mice grafted with mammary carcinoma and it extended the survival ability of sarcoma 180 bearing mice by 2.3 times. Further quercetin enhanced the inhibitory effect of mitomycin C in mammary adenocarcinoma. Finally they concluded these effects were mediated in part by the often poorly vascularised and hypoxic regions of tumors [59]. In a recent study initiated by Choi et al. studied the anti-cancer effect of quercetin against breast cancer cell (MDA-MB-435). MTT assay revealed that quercetin showed inhibitory effect on MDA-MB-435 cell growth in a time and dose dependent manner. Further cell cycle analysis of quercetin treated cells showed significant increase in the accumulation of cells at subG1 phase. Further quercetin treatment increased Bax expression but decreased the Bcl2 levels. Cleaved caspase-3 and PARP expression were also increased by quercetin [60]. 3.1.5. Effect of Acacetin in Liver and Lung Cancer Cell Lines Hsu et al. investigated the antiproliferative effect of acacetin in human liver cancer cell line (HepG2). The maximum inhibitory effect (nearly 72 %) was observed at a concentration 20 μg/mL after 48 h. The IC50 value was observed to be 10.44μg/ml for the HepG2 cells. Flow cytometry results indicated an increase in the G1 phase of cells from 31.1 to 61.6 and 76.5 % at a concentration of 10 and 20 μg/mL respectively. DNA fragmentation assay of cells treated with acacetin indicated the number of cells undergoing apoptosis increased to about 4fold at 10 μg/mL and 8-fold at 20 μg/mL after 48 h. Further it has been demonstrated that acacetin increased the induction of p53 and its downstream target, p21/WAF1 as assayed by Enzyme linked Immuno-sorbent assay (ELISA). Fas ligand assay indicated that FasL, mFasL and sFasL increased in a dose-dependent manner. Pro-apoptotic Bax protein level also increased due to acacetin treatment at 24 and 48 h. In their continuity study, they examined the role of acacetin in human non-small cell lung cancer A549 cells. They reported the antiproliferative effect was significant in dose-dependent manner and the IC50 value was found to be 9.46 μM. Cell cycle analysis of A-549 cells treated with 5 and 10 μM of acacetin indicated an increase in G1 phase from 34.7 to 42.6 and 61.2% respectively. Similarly DNA fragmentation assay indicated the number of cells undergoing apoptosis increased from about 3.2 fold to 8.1 fold at 5 and10 μM of acacetin, respectively after 48 h. Similar to the observation in HepG2 cells, acacetin increased the induction of p53 and its downstream target, p21/WAF1 as assayed by Enzyme linked Immuno-sorbent assay (ELISA). Fas ligand assay indicated that FasL, mFasL, and sFasL increased in a dose-dependent manner. They

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Anticancer Activity of Honey and Its Phenolic Components

51

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

concluded that p53 and Fas/FasL apoptotic system may participate in the antiproliferative activity of acacetin in HepG2 and A549 cells [61, 62]. 3.1.6. Effect of Kaempferol in Lung and Leukemia Cells Henry et al. explored the significance of Kaempferol induced apoptosis in human lung non-small carcinoma cells (H460).Trypan blue exclusion assay, demonstrated the varying concentration of kaempferol reduced the cell viability in the dose-dependent manner with an IC50 value of 50μM. Lactate dehydrogenase (LDH) assay indicated cell death is due to apoptosis since there is no release of LDH enzyme with the cells treated with kaempferol. ROS production is not the cause for the cytotoxicity observed, since the oxidant-sensitive fluorescent probe, CM-H2DCFDA signal doesn‘t showed any change after kaempferol treatment. Mitochondrial membrane potential measured using fluorescent probe 3, 3‘-dihexyloxacarbocyanine (DiOC6), a mitochondrion-specific and voltage-dependent dye, indicated no change after treating with kaempferol at varying concentrations for 16 h. Kaempferol (50μM) induced Apoptosis inducing factor (AIF) from mitochondria to nucleus and elicited DNA fragmentation and condensation in H460 cells. The levels of pro-caspase 3 were decreasing after exposing to kaempferol for 8 h. Moreover protein levels of Mn SOD and Cu/Zn SOD increased during treatment with 50 μM kaempferol for 24 h [63]. Bestwick et al. reported the kaempferol antiproliferative effect in the pro-myelocytic leukemia cells (HL60). Dose-dependent inhibition of HL-60 was observed over 72 h exposure to kaempferol. FACS analysis reported treatment of cells with Kaempferol (10 μM) decreased the cell growth. After 5 h of treatment the proportion of cells in S-phase increased compared to decrease in the G1 phase. 100 μM of kaempferol induced an initial accumulation in Sphase and then G2/M as the time course progressed from 48 to 96 h. Phosphodityl serine exposure without membrane damage as indicated by annexin V-FITC binding, a feature of the early pre-necrotic phase of apoptosis, was only observed for a minor proportion of cells treated with ≥20 μM kaempferol following either 24 h or 72 h treatments. After exposing the cells with kaempferol for 24 and 72 h, a decrease in the mitochondrial potential followed by enhanced expression of active caspase-3 were observed. Retinoic acid treatment results nearly 5 % differentiation of the cell population indicated by phorbol 12-myristate 13-acetate stimulated nitro blue tetrazolium NBT reduction over 72 h of treatment with 100 μM of kaempferol. Multi-parametric flow cytometric analysis revealed distinct sub populations of cells with decreased size, typical of apoptosis and necrosis, possessing heightened caspase-3 activity followed by decreased anti-apoptotic Bcl2 expression and changes in the membrane asymmetry and integrity. The remaining population had elevated active caspase-3 but no change or a moderate increase in Bcl2 expression and no plasma membrane alterations. Hence kaempferol growth inhibitory effect on HL-60 leukemia cells is due to heterogeneous response mainly dominated by cell cycle alternation although some degree of cytotoxicity results from apoptotic as well as non-apoptotic process [64]. 3.1.7. Role of Pinocembrin and Pinobanksin in Cancer Cell Lines Suresh Kumar et al. showed cytotoxicity of pinocembrin against a variety of cancer cells including normal lung fibroblasts with relative non toxicity to human umbilical cord endothelial cells. Pinocembrin induced loss of mitochondrial membrane potential (MMP) with subsequent release of cytochome c and processing of caspase-9 and -3 in colon cancer cell line HCT 116. The initial trigger for mitochondrial apoptosis appears to be by the

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

52

Saravana Kumar Jaganathan and Mahitosh Mandal

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

translocation of cytosolic Bax protein to mitochondria [65]. Pinobanksin has been reported to exert antioxidant activity by lowering the Fe (II) induced lipid peroxidation as well as inhibiting the mitochondria membrane permeability transmission (MMPT) [66]. 3.1.8. Effect of Apigenin in Cancer Cells Apigenin belongs to the flavonoid family and it is widely reported for its antitumor effects in various cell lines. It had exerted antiproliferative effect against colon, breast, cervical, neuroblastoma and liver cancer cell lines. Wang et al. studied the effect of apigenin on the cell growth and cell cycle in the colon carcinoma cell lines like SW480, HT 29 and Caco-2. Cell count and protein content of the apigenin treated cells showed reduction compared to the control. Apigenin inhibited the cell growth with the IC 50 values of 40, 50, and 70 µM for the SW480, HT-29, and Caco-2 cells respectively. Flow cytometric analysis of apigenin (80 µM) treated cells resulted in G2/M arrest of 64 %, 42 % and 26 % in SW480, HT-29, and Caco-2 cells respectively. They had also reported the inhibition of p34cdc2 kinase and cyclin B1 proteins in the apigenin treated cells [67]. Way et al. demonstrated the antiproliferative nature of apigenin against breast cancer cells. They reported apigenin was found to be more potent in inhibiting the HER2/neu- over expressing cells (MDA-MB-453 cells) compared to basal level HER2/neu-expressing cells (MCF-7). For instance, 40 µM of apigenin resulted in 48 % inhibitory effect in MDA-MB-435 whereas in MCF-7 it caused only 31 % growth inhibition. They examined the role of HER2/HER3-PI3K/Akt pathway in the apigenin induced apoptosis and showed that it inhibited directly the PI3K activity first, consequently inhibiting the Akt kinase activity. Moreover they demonstrated the inhibition of HER2/neu autophosphorylation and transphosphorylation resulting from depleting HER2/neu protein in vivo [68]. In another study by Zheng et al. elucidated the apoptosis induced by apigenin in human cervical cancer cell HeLa. It was found that apigenin could decrease the cell viability with an IC50 of 35.89 µM. DNA fragmentation assay and flow cytometric analysis of apigenin treated cells confirmed the apoptosis induction. They had observed increased expression of p21/WAF1 and p53. Further Fas/APO-1 and caspase-3 increase and Bcl2 reduction in the apigenin treated HeLa cells confirmed the apoptosis induction [69]. Torkin et al. reported that apigenin could induce apoptosis in neuroblastoma cells like NUB-7 and LAN-5. Apigenin repressed the cell viability in a dose-dependent manner in human neuroblastoma cell lines with an EC50 = 35 µmol/L in NUB-7, and EC50 = 22 µmol/L in LAN-5 after 24 h. Moreover it was found to inhibit the colony forming ability and NUB-7 xenograft tumor growth in nonobese diabetic mouse model. They had shown apigenin induced apoptosis was mediated though p53 as it enhanced the expression of p53 and p53 induced gene products like p21WAF1/CIP1 and Bax [70]. Recent research by Chiang et al. suggested the antiproliferative effect of apigenin in HepG2, Hep3B and PLC/PRF/5 cells. It was found that apigenin could inhibit the cell growth of the above reported liver cancer cells but not the normal murine liver BNL.CL2 cells. IC50 was observed to be 8.02 µg/ml for HepG2, 2.16 µg/ml for Hep3B and 22.73 µg/ml for PLC/PRF/5. In addition, DNA ladder and flow cytometric analysis indicated apoptosis in the HepG2 cells. Apigenin treated cells were arrested at G2/M phase of the cell cycle. Further they observed increasing accumulation of p53 and p21/WAF1 in the treated cells [71].

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Anticancer Activity of Honey and Its Phenolic Components

53

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Conclusion Chemoprevention utilizes appropriate pharmacological agents [3, 4] or of dietary agents, consumed in diverse forms like macronutrients, micronutrients, or non-nutritive phytochemicals. Crude honey rich in phenolic compounds have been reported for its antiproliferative effect. It has been shown honey can be considered as a plausible candidate for induction of apoptosis through ROS and mitochondria-dependent mechanism. Various polyphenols reported in honey are found to be anti-cancerous. Some of the polyphenols of honey like Caffeic acid (CA), Caffeic acid phenyl ester (CAPE), Chrysin (CR), Galangin (GA), Quercetin (QU), Acacetin (AC), Kaempferol (KF), Pinocembrin (PC), Pinobanksin (PB) and Apigenin (AP) have evolved as promising pharmacological agents in treatment of cancer. The summaries of individual polyphenols were tabulated under Table 1. Caffeic acid has been reported as a carcinogen in initial studies, but the same caffeic acid along with combination of other antioxidant has been shown to suppress colon tumors in rats. Chung et al. showed that oral administration of Caffeic acid and Caffeic acid phenyl esters (CAPE) reduced liver metastasis, mediated by the dual inhibition of NF-KB and MMP-9 enzyme activity [46]. Natarajan et al. demonstrated that CAPE is known to have antimitogenic, anticarcinogenic, anti-inflammatory and immunomodulatory properties [44]. CAPE‘s anti-inflammatory and anti-cancer property has also been shown to protect skin cells when exposed to ultra-violet radiation and UVB radiation [49]. Weng et al. showed the growth inhibitory effect of chrysin in C6 glioma cells was either though activating p38-MAPK which leads to the accumulation of p21Waf1/Cip1 protein or mediating the inhibition of proteasome activity [50]. In another study by Woo et al. it has been elucidated that chrysin induces apoptosis in association with the activation of caspase-3 and Akt signal pathway, that plays a crucial role in chrysin-induced apoptosis in U937 cells [51]. Galangin expressed antiproliferative effect on HL-60 cells on dose dependent manner and also induced DNA fragmentation without loss of membrane integrity [54]. Quercetin also inhibited the HL-60 cell proliferation in association with the inhibition of cytosolic Protein Kinase C (PKC) and membrane Tyrosine Protein Kinase (TPK) in-vitro [55]. It has been reported that quercetin in low concentration promoted cell proliferation of A-549 cells, whereas in higher concentration it inhibited cell proliferation and survival [57]. Further quercetin exerted antiproliferative effect against glioma and breast cancer cells [58-60]. Acacetin, another important flavanoid inhibited the proliferation of A549 cells, induced apoptosis and blocked the cell cycle progression at G1 phase. It also improved the expression of p53 and Fas ligands [61]. In another study, it has been shown to inhibit HepG2 cell proliferation and provoke apoptosis by enhancing the p53 and Fas ligands as in the case of A549 cells [62]. Kaempferol induced apoptosis in H460 cells which was accompanied by significant DNA condensation and increasing ATP levels. It also changed the expression of Caspase 3 and Apoptosis Inducing Factor (AIF) levels [63]. Bestwick et al. reported recently that kaempferol growth inhibitory effect on HL-60 leukemia cells due to heterogeneous response mainly dominated by cell cycle alternation although some degree of cytotoxicity results from apoptotic as well as non-apoptotic process [64]. Pinocembrin induced loss of mitochondrial membrane potential (MMP) with subsequent release of cytochrome c and processing of caspase-9 and -3 in colon cancer cell line HCT 116 [65]. Apigenin exerted

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

54

Saravana Kumar Jaganathan and Mahitosh Mandal

antiproliferative effect against colon, breast, cervical, neuroblastoma and liver cancer cell lines [67-71]. Table 1. Summary of in vitro studies of honey polyphenols Compound CA,MC, PEDMC, PEC

CAPE

CAPE

CAPE

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

CA, CAPE

CAPE Chrysin

Chrysin Galangin

Quercetin

Quercetin

Cell Line tested Observation/ Result HT -29 Toxicity: CA > 2500 μM PEC > 60 μM PEDMC > 60 μM MC > 225 μM Inhibition of DNA/RNA: 150 μM MC, 40 μM PEC and 20 μM PEDMC TPK activity down-regulation: 100μM of MC, 30μM of PEC and 20 μM of PEDMC ODC activity down-regulation: 150 μM MC, 40 μM PEC and 20 μM PEDMC HeLa Subst nce Inhibition Concentration Percentage DNA 95 % 20 μM RNA 75 % 20 μM protein 47 % 20 μM U-937 a) Maximum inhibition of NFкB at 25 μg/ml after TNF treatment. b) No inhibitory effect on AP-1, TFIID, and Oct-1. c) Structural analogue 5, 6 dihydroxy strongly inhibited the NF-кB. C6 glioma a ) DNA fragmentation at 50 μM after 24 h b ) p-p53 ↑, active Caspase 3↑, Bak and Bax ↑, Bcl2 ↓ HepG2 a) CA and CAPE inhibited MMP-2 and9 with IC50 of 10-20 μM and 2-5 μM. b) CA at the concentration of 200 μg/mL reduced the cell viability to 61% viability compared to the controls, and the treatment with CAPE (20 μg/mL) in HepG2 cells reduced the viability to 72% of the controls HT 1080 a) m-RNA levels of MMP-2 and MMP-9 were inhibited ↓ b) m-RNA levels of TIMP-1 and MT-1 MMP level decreased ↓ C-6 glioma a) 72 h of incubation with 50μM of chrysin, inhibited ↓ 90% of cellproliferation b) p21Waf1/Cip1 levels increased ↑, CDK2 and CDK4 were inhibited↓ U-937 a ) PLC-γ and active Caspase-3 level increased ↑ b ) XIAP level decreased ↓ whereas cytochome-C level ↑ HL-60 a) Galangin of 1-10 μM also promoted antiproliferative effect which is evident after 48 h of incubation. b) Active Caspase 3 ↑, a hallmark of apoptosis process, was detected after 24 h and 72 h of incubation with 50 and 10 μM of galangin respectively. c) Cell cycle analysis indicated the increase in the subG1 phase ↑of galangin (>10 μM) treated cells. HL-60 a )Quercetin had a remarkable inhibitory effect ↓ on the activities of cytosolic PKC and membrane TPK from HL-60 cells in vitro, with IC50 values of about 30.9 and 20.1 μM, respectively, b) Quercetin repressed ↓ the complete activity of phosphoinositides like PI, PIP, and PIP2 at the concentration of 80 μM. A-549 a) Quercetin in low concentration (1-20 μM) promoted the cell proliferation ↑ whereas higher concentration (50-200 μM) showed the concentration dependent cyotoxicity ↓. b) Increase in TAC ↑of cell extracts but higher concentrations of the quercetin led to a progressive decrease in the TAC ↓

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Reference no 41

43

44

45 46

47 50

51 54

55

57

55

Anticancer Activity of Honey and Its Phenolic Components Compound Quercetin Quercetin Quercetin

Acacetin Acacetin Kaempferol

Pinocembrin

Cell Line tested Observation/ Result K562 (a) reduction of c-myc and Ki-ras oncogenes ↓ (b) fall in Inositol-1,4,5-triphosphate (IPs) concentrations ↓ Glioma cell (a) arrested the glioma cells in the G2 checkpoint of the cell cycle (b) decreased the mitotic index MCF-7 (a) IC50 value of 10 µg/ml. (b) cell cycle arrest at G2/M phase (c) inhibited the tumor growth by more than 58 % in mice grafted with mammary carcinoma HEPG2 a) IC50 value = 10.44μg/ml b) p53 ↑, p21Waf1↑, FasL ↑, mFasL ↑, sFasL ↑ and Bax ↑ A-549 a) IC50 value = 9.46μM b) p53 ↑, p21Waf1↑, FasL ↑, mFasL ↑, sFasL ↑ and Bax ↑ HL-60 a) Mitochondrial potential decreased ↓ caspase-3 level increased ↑ b) Kaempferol growth inhibitory effect on HL-60 leukemia cells due to heterogeneous response mainly dominated by cell cycle alternation although some degree of cytotoxicity results from apoptotic as well as non-apoptotic process

HCT116

a) Mitochondrial potential decreased ↓ b) BAX translocates in to mitochondria c) Cyt-C release c) Caspase-3 and Caspase 9 level increased (↑)

Reference no 56 58 59

62 61 64

65

(a) Inhibits the mitochondria membrane permeability transition ↓ (b) Lowers the lipid peroxidation ↓

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Pinobanksin

Apigenin Apigenin Apigenin

Apigenin

Apigenin

(a) inhibition of p34cdc2 kinase (b) cyclin B1 ↓ (c) ) IC50 values: HT 29 = 50 μM SW480 = 40 μM Colon cancer Cac0-2 = 70 μM Breast Cancer (a) inhibiting the HER2/neu- over expressing cells (MDA-MB-453 cells) compared to basal level HER2/neu-expressing cells (MCF-7) Hela (a) IC50 = 35.89 µM (b) p21/WAF1 ↑ (c) p53 and caspase-3 increased ↑ (d) Bcl2 decreased Neuroblastoma (a) EC50 = 35 µmol/L in NUB-7 (b) EC50 = 22 µmol/L in LAN-5 (c) p53 and p21WAF1/CIP1 ↑ (d) Bax ↑ Liver cancer (a) IC50 was observed to be 8.02 µg/ml for HepG2, 2.16 µg/ml for Hep3B and 22.73 µg/ml for PLC/PRF/5 (b) G2/M cell cycle arrest (c) Increase of p53 and p21/WAF1 ↑ Rat liver Mitochondria

66

67 68 69

70 71

This chapter has clearly demonstrated certain honey polyphenols tested in laboratorial setups showed to be a promising pharmacological agent for inhibiting cancer cell proliferation. After generating more in-depth and exhaustive information of these compounds jointly in in-vitro and in-vivo studies, clinical trials have to be initiated to further validate these compounds in medical applications.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

56

Saravana Kumar Jaganathan and Mahitosh Mandal

References [1] [2] [3]

[4] [5] [6] [7] [8]

[9] [10]

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

[11]

[12]

[13] [14]

[15]

[16]

[17] [18]

Sporn, M. B. and Newton, D. L. (1979). Chemoprevention of cancer with retinoids. Fed. Proc., 38, 2528–2534. Kelloff, G. J. (2000). Perspectives on cancer chemoprevention research and drug development. Adv. Cancer Res., 78, 199–334. Kelloff, G. J. and Boone, C. W. (1994). Cancer chemopreventive agents. Drug development status and future prospects I, J. Cell Biochem. (Suppl. 20). New York, NY: Wiley-Liss. Kelloff, G. J., Hawk, E. T. and Sigman, C. C. (2004). Cancer Chemoprevention Volume 1: Promising Cancer Chemopreventive Agent, Totowa, NJ: Humana Press. Ferguson, L. R. (1994). Antimutagens as cancer chemopreventive agents in the diet. Mutat. Res., 307, 395–410. Ferguson, L. R., Philpott, M. and Karunasinghe, N. (2004). Dietary cancer and prevention using antimutagens. Toxicol.,198,147–159. Surh, Y. J. (2003). Cancer chemoprevention with dietary phytochemicals. Nature Rev. Cancer, 3, 768–780. Wollgast, J. and Anklam, E. (2000). Review on polyphenols in Theobroma cacao: changes in composition during the manufacture of chocolate and methodology for identification and quantification. Food Res. Int., 33, 423–447. Madhavi, D. V., Despande, S. S., Salunkhe, D. K., Madhavi, D. L., Deshpande, S. S. and Salunkhe, D. K. (1996). Food Antioxidants. New York, NY: Marcel Dekker. Food and Agriculture Organization. (1996). Agricultural Services Bulletin Food and Agriculture Organization. Rome. Italy. Antony, S. M., Han, I. Y., Rieck, J. R. and Dawson, P. L. (2000). Antioxidative effect of maillard reaction products formed from honey at different reaction times. J. Agric. Food Chem., 48, 3985–3989. Vit, P., Soler, C. and Tom´as-Barber´an, F. A. (1997). Profiles of phenolic compounds of Apis mellifera and Melipona spp. honeys from Venezuela. Unters Forsch. Lebensm. Z., 204, 43–47. Cherchi, L., Spanedda, C., Tuberoso, C. and Cabras, P. (1994). Solid-phase extraction and HPLC determination of organic acid in honey. J. Chromat. A, 669, 59–64. Davies, A. M. C. and Harris, R. G. (1982). Free amino acid analysis of honeys from England and Wales: application to determination of the geographical origin of honeys. J. Apic. Res., 21, 168–173. White, J. W. (1975). Composition of honey. In Crane, E. (Ed.), Honey, a comprehensive survey. (pp. 157-206). London (UK): Bee Research Association and Chalfont St Peter. Tan, S. T, Wilkins, A. L., Holland, P. T. and McGhie, T. K. (1989). Geographical discrimination of honeys though the employment of sugar patterns and common chemical quality parameters. J. Agri. Food Chem., 37, 1217–1221. White, J. W. (1978). The protein content of honey. J. Apic. Res., 17, 234–238. Amiot, M. J., Aubert, S., Gonnet, M. and Tacchini, M. (1989). Les composés phénoliques des miels: étude préliminaire surl identify cation et la quantifi cation par familles. Apidologie, 20, 115-125.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Anticancer Activity of Honey and Its Phenolic Components

57

[19] World Wide Wounds. Honey as a topical antibacterial agent for treatment of infected wounds. Last Modified. February 15, 2002. http://www.worldwidewounds.com /2001 /november/Molan/honey-as-topical-agent.html. [20] National Honey Board. Honey and Bees. Last accessed April 14, 2007. http://www.honey.com/consumers. [21] Todd, F. E. and Vansell, G. H. (1942). Pollen grains in nectar and honey. J. Econ. Entomol., 35, 728-731. [22] Jaganathan, S. K. and Mandal, M. (2009). Honey constituents and its apoptotic effect in colon cancer cells. J. Api. Prodcut. Api. Med. Sci.,1, 29-36. [23] Jaganathan, S. K. and Mandal, M. (2010). Involvement of non-protein thiols, mitochondrial dysfunction, reactive oxygen species and p53 in honey-induced apoptosis. Invest. New Drugs, 5, 624-633. [24] Jaganathan, S. K., Mondhe, D., Wani, Z. A., Pal, H. C. and Mandal, M. (2010). Effect of honey and eugenol on Ehrlich ascites and solid carcinoma. J. Biomed. Biotechnol., No. 989163. [25] Jaganathan, S. K. (2011). Can flavonoids from honey alter multidrug resistance? Med. Hypotheses, 76, 535-537. [26] Orsolic, N., Knezevi, A., Sver, L., Terzi, S., Hackenberger, B. K. and Basi, I. (2004). Influence of honey bee products on transplantable tumours. J. Vet. Compar. Oncology, 4, 216-226. [27] Tarek, S., Naoto, M., Mizuki, O., Kazunori, H., Koji, K., Toru, S. and Hideyuki, A. (2003). Antineoplastic activity of honey in an experimental bladder cancer implantation model: In vivo and in vitro studies. Int. J. Urol., 10, 213-219. [28] Gribel, N. V. and Pashinskiĭ, V. G. (1990). The antitumor properties of honey Voprosi Onkologii J., 36, 704-709. [29] Cook, N. C. and Sammon, S. (1996). Flavonoids - Chemistry, metabolism, cardioprotective effects and dietary sources. Nutr. Biochem., 7, 66–76. [30] Catapano, A. L. (1997). Antioxidant effect of flavonoids. Angiology, 48, 39–44. [31] Loku, K., Tsushida, T., Takei, Y., Nakatani, N. and Terao, J. (1995). Antioxidant activity of quercitin monoglucosides in solution and phospholipid bilayers. Biochem. Biophys. Acta, 1234, 99–104. [32] Salah, N., Millar, N. J., Paganda, G., Tijburg, L., Bolwell, G. P. and Rice-Evans, C. (1995). Polyphenolic flavanols as scavengers of aqueous phase radicals and as chainbreaking antioxidants. Arch. Biochem. Biophys., 322, 339–346. [33] Serafın, M., Ghiselli, A. and Ferro-Luzzi, A. (1996). In vivo antioxidant effect of green and black tea in man. Eur. J. Clin. Nutr., 50, 28–32. [34] Vinson, J. A., Hao, Y., Su, X. and Zubik, L. (1998). Phenol antioxidant quantity and quality in foods: vegetables. J. Agric. Food Chem., 46, 3630–3634. [35] Bravo, L. (1998). Polyphenols: Chemistry, dietary sources, metabolism, and nutritional significance. Nutr. Rev., 56, 317–333. [36] Ferreres, F., Blazquez, M. A., Gil, M. I. and Tomas-Barberan, F. A. (1994). Separation of honey flavonoids by micellar electrokinetic capillary chromatography. J. Chromat., 669, 268–274. [37] Ferreres, F., Juan, T., Perez-Arquillue, C., Herrera-Marteache, A., Garcia-Viguera, C. and Tomas-Barberan, F. (1998). Evaluation of pollen as a source of kaempferol in rosemary honey. J. Sci. Food Agric., 77, 506–510.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

58

Saravana Kumar Jaganathan and Mahitosh Mandal

[38] Tomas-Barberan, F. A., Martos, I., Ferreres, F., Radovic, B. S. and Anklam, E. (2001). HPLC flavonoid profiles as markers for the botanical origin of European unifloral honeys. J. Sci. Food Agric., 81, 485–496. [39] Cherchi, A., Spanedda, L., Tuberoso, C. and Cabras, P. (1994). Solid-phase extraction and HPLC determination of organic acid in honey J. Chromat., 669, 59–64. [40] Hirose, M., Takesada, Y., Tanaka, H., Tamano, S., Kato, T. and Shirai, T. (1998). Carcinogenicity of antioxidants BHA, caffeic acid, sesamol, 4- methoxyphenol and catechol at low doses, either alone or in combination, and modulation of their effects in a rat medium-term multi- organ carcinogenesis model. Carcinogenesis, 19, 207-212. [41] Rao, C. V., Desai, D., Simi, B., Kulkarni, N., Amin, S. and Reddy, B. S. (1993). Inhibitory effect of caffeic acid esters on azoxymethane-induced biochemical changes and aberrant crypt foci formation in rat colon. Cancer Res., 3, 4182-4188. [42] Rao, C. V., Desai, D., Kaul, B., Amin, S. and Reddy, B. S. (1992). Effect of caffeic acid esterson carcinogen-induced mutagenecity and human colon adenocarcinoma cell growth. Chem. Biol. Interact., 84, 277-290. [43] Huang, M. T., Ma. W., Yen, P., Xie, J. G., Han, J., Frenkel, K., Grunberger, D. and Conney, A. H. (1996). Inhibitory effects of caffeic acid phenethyl ester (CAPE) on 12O-tetradecanoylphorbol-13-acetate-induced tumor promotion in mouse skin and the synthesis of DNA, RNA and protein in HeLa cells. Carcinogenesis, 17, 761-765. [44] Natarajan, K., Singh, S., Burke, T. R., Grunberger, D. and Aggarwal, B. B. (1996). Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-kappa B. PNAS, 93, 9090-9095. [45] Lee, Y. J., Kuo, H. C., Chu, C. Y., Wang, C. J., Lin, W. C. and Tseng, T. H. (2003). Involvement of tumor suppressor protein p53 and p38 MAPK in caffeic acid phenethyl ester-induced apoptosis of C6 glioma cells. Biochem. Pharmacol., 66, 2281–2289. [46] Chung, T. W., Moon, S. K., Chang, Y. C. and Ko, J. H. (2004). Novel and therapeutic effect of caffeic acid and caffeic acid phenyl ester on hepatocarcinoma cells: complete regression of hepatoma growth and metastasis by dual mechanism. FASEB J., 80, 16701681. [47] Hwang, H. J., Park, H. J., Chung, H. J., Min, H. Y., Park, E. J., Hong, J. Y. and Lee, S. K. (2006). Inhibitory effects of caffeic acid phenethyl ester on cancer cell metastasis mediated by the down-regulation of matrix metalloproteinase expression in human HT1080 fibrosarcoma cells. J. Nutr. Biochem., 17, 356– 362. [48] Nereadil, J; Veselska, R. and Slanina J. (2003). UVC protective effect of Caffeic acid on Normal and transformed skin cells in vitro. Folia Biol., 49, 197-202. [49] Staniforth, V., Chiu, L. Y. and Yang, N. S. (2006). Caffeic acid suppresses UVB radiation-induced expression of interleukin-10 and activation of mitogen-activated protein kinases in mouse. Carcinogenesis, 27, 1803–1811. [50] Weng, M. S., Ho, Y. S. and Lin, J. K. (2005). Chrysin induces G1 phase cell cycle arrest in C6 glioma cells though inducing p21Waf1/Cip1 expression: Involvement of p38 mitogen-activated protein kinase. Biochem. Pharmacol., 69, 1815–1827. [51] Woo, K. J., Jeong, Y. J., Park, J. W. and Kwona, T. K. (2004). Chrysin-induced apoptosis is mediated though caspase activation and Akt inactivation in U937 leukemia cells. Biophys. Biochem. Res. Comm., 325, 1215–1222. [52] Zheng, X., Meng, W. D., Xu, Y. Y., Cao, J. G. and Qing, F. L. (2003). Synthesis and Anticancer Effect of Chrysin Derivatives. Bioorg. Med. Chem. Lett., 13, 881–884.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Anticancer Activity of Honey and Its Phenolic Components

59

[53] Zhang, T., Chen, X., Qu, L., Wu, J., Cui, R. and Zhao, Y. (2004). Chrysin and its phosphate ester inhibit cell proliferation and induce apoptosis in Hela cells. Bioorg. Med. Chem., 12, 6097–6105. [54] Bestwick, S., and Milne, L. (2005). Influence of galangin on HL-60 cell proliferation and survival. Cancer. Lett, 243, 80–89. [55] Kang, T. B. and Liang, M. C. (1997). Studies on the inhibitory effects of quercetin on the growth of HL460 leukemia cells. Biochem. Pharmacol., 54, 1013-1018. [56] Csokay, B., Prajda, N., Weber, G. and Olah, E. (1997). Molecular mechanisms in the antiproliferative action of quercetin. Life Sci., 60, 2157-2163. [57] Robaszkiewicz, A., Balcerczyk, A. and Bartosz, G. (2006). Antioxidative and prooxidative effects of quercetin on A549 cells. Cell Biol. Int., 31, 1245-1250. [58] Braganhol, E., Zamin; L. L., Canedo, A. D., Horn, F., Tamajusuku, A. S., Wink, M. R., Salbego, C. and Battastini, A. M. (2006). Antiproliferative effect of quercetin in the human U138MG glioma cell line. Anti Cancer Drugs, 17, 663-671. [59] Indap, M. A., Radhika, S., Motiwale, L. and Rao, K. V. K. (2006). Antitumor activity and pharmacological Manipulations for increased therapeutic gains. Ind. J. Pharmaceut. Sci., 68, 465-469. [60] Choi, E. J., Bae, S. M. and Ahn, W. S. (2008). Antiproliferative effects of quercetin though cell cycle arrest and apoptosis in human breast cancer MDA-MB-453 cells. Arch. Pharmcol. Res., 31, 1281-1285. [61] Hsu, Y. L., Kuo, P. L., Liu, C. F. and Lin, C. C. (2004). Acacetin-induced cell cycle arrest and apoptosis in human non-small cell lung cancer A549 cells. Cancer Lett., 212, 53-60. [62] Hsu, Y. L., Kuo, P. L., and Lin, C. C. (2003). Acacetin inhibits the proliferation of Hep G2 by blocking cell cycle progression and inducing apoptosis. Biochem. Pharmacol., 67, 823-829. [63] Leung, H. W., Lin, C. J., Hour, M. J.., Yang, W. H., Wang, M. Y. and Lee, H. Z. (2007). Kaempferol induces apoptosis in human lung non-small carcinoma cells accompanied by an induction of antioxidant enzymes. Food Chem. Toxicol., 45, 20052013. [64] Bestwick, C. S., Milne, L. and Duthie, S. J. (2002). Kaempferol induced inhibition of HL-60 cell growth results from a heterogeneous response, dominated by cell cycle alterations. Chem. Biol. Interact., 170, 76-85. [65] Kumar, M. A., Nair, M., Hema, P. S., Mohan, J. and Santhoshkumar, T. R. (2006). Pinocembrin triggers Bax-dependent mitochondrial apoptosis in colon cancer cells. Mol. Carcinog,, 46, 231-241. [66] Santos, A. C., Uyemura S. A., Lopes, J. L., Bazon, J. N., Mingatto, E. F. and Curti, C. (1998). Effect of naturally occurring flavonoids on lipid peroxidation and membrane permeability transition in mitochondria. Free Radic. Biol. Med., 24, 1455-1461. [67] Wang, W., Heideman, L., Chung, C. S., Pelling, J. C, Koehler, K. J. and Birt, D. F. (2000). Cell-cycle arrest at G2/M and growth inhibition by apigenin in human colon carcinoma cell lines. Mol. Carcinog., 28, 102-110. [68] Way, T. D., Kao, M. C. and Lin, J. K. (2004). Apigenin induces apoptosis though proteasomal degradation of HER2/neu in HER2/neu-overexpressing breast cancer cells via the phosphatidylinositol-3'- kinase/Akt-dependent pathway. J. Biol. Chem., 279, 4479-4489.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

60

Saravana Kumar Jaganathan and Mahitosh Mandal

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

[69] Zheng, P. W., Chiang, L. C., Lin, C. C. (2005). Apigenin induced apoptosis though p53-dependent pathway in human cervical carcinoma cells. Life Sci., 76, 1367–1379. [70] Torkin, R., Lavoie, J. F., Kaplan, D. R. and Yeger, H. (2005). Induction of caspasedependent, p53-mediated apoptosis by apigenin in human neuroblastoma. Mol. Cancer Ther., 4, 1-11. [71] Chiang, L. C., Ng, L. T., Lin, I. C., Kuo, P. L. and Lin, C. C. (2006). Antiproliferative effect of apigenin and its apoptotic induction in human Hep G2 cells. Cancer Lett., 237, 207–214.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

In: Honey: Current Research and Clinical Applications ISBN: 978-1-61942-656-6 Editor: Juraj Majtan © 2012 Nova Science Publishers, Inc.

Chapter IV

Honey and Microbes Laïd Boukraâ,1, Yuva Bellik2 and Fatiha Abdellah1 1

Veterinary Institute Ibn-Khaldun University of Tiaret,Tiaret, Algeria Faculty of Life and Natural Sciences Ibn-Khaldun University of Tiaret, Tiaret, Algeria

2

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Abstract Honey and other bee products were subjected to laboratory and clinical investigations during the past few decades and the most remarkable discovery was their antibacterial activity. Honey has been used since ancient times for the treatment of some diseases and for the healing of wounds but its use as an anti-infective agent was superseded by modern dressings and antibiotic therapy. However, the emergence of antibiotic resistant strains of bacteria has confounded the current use of antibiotic therapy leading to the re-examination of former remedies. Honey has a strong antibacterial activity. Even antibiotic-resistant strains such as epidemic strains of methicillin resistant Staphylococcus aureus (MRSA) and vancomycine resistant Enterococcus (VRE) have been found to be as sensitive to honey as the antibiotic-sensitive strains of the same species. Sensitivity of bacteria to bee products varies considerably within the product and the varieties of the same product. Botanical origin plays a major role in its antibacterial activity. Honey has been found to be able to treat many infectious disorders such as wound infection, gastroenteritis, sinusitis and ophthalmic disorders.

Keywords: Honey, antibacterial, antiviral, antifungal, prebiotic

Introduction Natural products have been used for thousands of years in folk medicine for several purposes. Among them, honey has attracted increased interest in recent years due to its 

Correspondence concerning this article should be addressed to: Laid Boukraâ. Veterinary Institute, Ibn Khaldun University of Tiaret 14000 Tiaret, Algeria. Email: [email protected]; Tel: + 213 7 95306930; Fax: +213 46 45 1444.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

62

Laïd Boukraâ, Yuva Bellik and Fatiha Abdellah

antimicrobial activity against a wide range of pathogenic microorganisms. Various studies attribute antibacterial, antifungal, anti-inflammatory, antiproliferative and anticancer potentiating properties to honey [1]. Approximately 70% of bacteria that cause infections in hospitals are resistant to at least one of the antibiotics most commonly used to treat infections. This antibiotic resistance is driving up health care costs, increasing the severity of disease, and the fatality of certain infections. Sepsis is another serious medical condition resulting from severe inflammatory response to systemic bacterial infections [2]. More desirably, honey has capacity to bind bacterial endotoxin and neutralize bacterium induced inflammatory response. Because of the dual capability to kill bacteria and neutralize endotoxins, this antimicrobial natural product holds great promise as a new class of antimicrobial and anti-sepsis agents [3]. The major antimicrobial properties are correlated to the hydrogen peroxide level which is determined by relative levels of glucose oxidase and catalase [4] whereas the non-peroxide factors that contribute to honey antibacterial and antioxidant activity are lysozyme, phenolic acids and flavonoids [5]. Apart from antibacterial properties, honey also plays a therapeutic role in wound healing and the treatment of eye and gastric ailments. This is partly due to its antioxidant activity [6] since some of these diseases have been recognized as being a consequence of free radical damage [7].

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Microbiological Quality of Honey Honey, is a very concentrated sugar solution with a high osmotic pressure, making impossible the growth of any microorganisms. It contains fewer microorganisms than other natural food; especially there are no dangerous Bacillus species. The microbes of concern in honey are primarily yeasts, molds and spore-forming bacteria. These microorganisms may be involved in activities such as spoilage of provisions, production of enzymes, antibiotics, mycotoxins and growth factors (vitamins, amino acids), metabolic conversion of provisions, and inhibition of competing microorganisms [8]. Honey contains Bacillus bacteria, causing the dangerous bee pests, but these are not toxic for humans. That is why, in order to prevent bee pests, honey should not be disposed in open places, where it can easily be accessed by bees. However, a number of bacteria are present in honey, most of them being harmless to man. Recent extensive reviews covered the main aspects of honey microbiology and the possible risks [5, 9, 10]. The presence of Clostridium botulinum spores in honey was reported for the first time in 1976 [11]. Since then there were many studies in honey all over the world. In some of them no C. botulinum was found, in others, few honeys were found to contain the spores [5, 9, 12, 13]. Honey does not contain the Botulinus toxin, but the spores can theoretically build the toxin after digestion. Very few cases of infant botulism after ingestion of honey have been reported lately and this has been attributed to C. botulinum spores present in honey. These findings have led the health authorities of some countries (US, UK) to label honeys that is not given to infants until one year of age. There are many countries which find that such notice is unnecessary. Indeed, honey is not the only source of C. botulinum spores as it can be found in any natural food. Honey contains naturally different osmotolerant yeast, which can cause undesirable fermentation. Osmotolerant yeasts can particularly develop in honeys with high moisture content. It has been found that some honey types, e.g. rape,

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Honey and Microbes

63

sunflower and also honeys from tropical countries has a higher content of osmotolerant yeast [14] and are less stable than other honeys with normal yeast counts.

The Antimicrobial Properties of Honey The antibacterial effectiveness of honey is attributed to its physico-chemicals characteristics and phytochemical compounds.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Physico-Chemical Properties It has been demonstrated in many studies that honey has antibacterial effects, attributed to its high osmolarity, low pH, hydrogen peroxide content, and content of other, uncharacterized compounds [15]. The low water activity of honey is inhibitory to the growth of the majority of bacteria, but this is not the only explanation for its antimicrobial activity. Molan [16] has studied sugar syrups of the same water activity as honey and found them to be less effective than honey at inhibiting microbial growth in vitro. Honey is mildly acidic, with a pH between 3.2 and 4.5. The low pH alone is inhibitory to many pathogenic bacteria and, in topical applications at least, could be sufficient to exert an inhibitory effect. When consumed orally, the honey would be so diluted by body fluids that any effect of low pH is likely to be lost.2 Hydrogen peroxide was identified as the major source of antibacterial activity in honey [17]. It is produced by the action of glucose oxidase on glucose, producing gluconic acid. This is inhibited by excessive heat and low water activity [17]. The fact that the antibacterial properties of honey are increased when diluted was clearly observed and reported in 1919 [18]. The explanation for this apparent paradox came from the finding that honey contains an enzyme that produces hydrogen peroxide when diluted [19]. This agent was referred to as ‗inhibine‘ prior to its identification as hydrogen peroxide [20]; hydrogen peroxide is a wellknown antimicrobial agent, initially hailed for its antibacterial and cleansing properties when it was first introduced into clinical practice [19]. In more recent times, it has lost favour because of inflammation and damage to tissue [21–23]. However, the hydrogen peroxide concentration produced in honey activated by dilution is typically about 1,000 times less than in the 3% solution commonly used as an antiseptic [16]. Although the level of hydrogen peroxide in honey is very low, it is still effective as an antimicrobial agent. It has been reported that hydrogen peroxide is more effective when supplied by continuous generation with glucose oxidase than when added in isolation [24]. Additional non-peroxide antibacterial factors have been identified [25]. Moreover, the antibacterial components of medical-grade honey have been completely characterized by Kwakman and colleagues [26]. Besides the presence of hydrogen peroxide, some minerals particularly copper and iron present in honey may lead to the generation of highly reactive hydroxyl radicals as part of the antibacterial system [16, 27]. Therefore, there must be mechanisms involved in honey to control the formation and removal of these reactive oxygen species. Contributions of free radicals and quenching properties of honeys in wound healing have been demonstrated by Henriques and colleagues [28].

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

64

Laïd Boukraâ, Yuva Bellik and Fatiha Abdellah

Phytochemical Properties of Honey

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Recently, methylglyoxyal has been successfully identified as the dominant bioactive component in manuka honey (Leptospermum scoparium) as well as in honeys of similar floral source (Leptospermum polygalifolium: Beringa honey) and its concentration was correlated to the non-peroxide activity of the honey [29, 30]. Phenolic compounds are amongst the most important groups of compounds occurring in plants, which are found to exhibit anticarcinogenic, anti-inflammatory, antiatherogenic, antithrombotic, immunemodulating and analgesic activities and which may exert these functions as antioxidants [31]. They are also present in honey and have been reported to have some chemoprotective effects in humans [32]. The phenolic acids are generally divided into two subclasses: the substituted benzoic acids and cinnamic acids, whereas the flavonoids present in honey are categorised into three classes with similar structure: flavonols, flavones and flavanones. These contribute significantly to honey colour, taste and flavour and have beneficial effects on health [33]. The composition of honey, including its phenolic compounds, is variable depending mainly on the floral source and also other external factors including seasonal and environmental factors as well as processing [32]. Thus, with different compositions of active compounds in honey collected from different locations, differences in honey properties are to be expected (Fig 1). Davidson et al. [34] have shown that individual phenolic compounds have growth inhibition on a wide range of Gram-positive and Gram-negative bacteria. Other honey therapeutic action studies carried out had been mainly on screening the raw honey samples on antimicrobial activity [35; 36] and on antioxidant capacity [37, 38]. As not all honeys are created equal in terms of antimicrobial activity because of differences in levels of nonperoxide factors, which vary by floral source and processing (Table 1), a comparative study has been conducted to set out the antibacterial potency of Sahara honey and those of northern Algerian origin. Results have shown that Sahara honey is more potent against bacteria compared to other Algerian honeys. This is most likely due to its phenolic and flavonoid components [39].

Figure 1. Non-peroxide activity of different honeys against Staphylococcus aureus [130]. Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

65

Honey and Microbes

Table 1. Minimum concentration of honey (%, v/v) in the growth medium needed to completely inhibit the growth of various species of wound-infecting bacteria [85] Species Manuka Honey + Catalase Other Honey Escherichia coli 3.7 7.1 Proteus mirabilis 7.3 3.3 Pseudomonas aeruginosa 10.8 6.6 Salmonella typhimurium 6.0 4.1 Serratia marcescens 6.3 4.7 Staphylococcus aureus 1.8 4.9 Streptococcus pyogenes 3.6 2.6 The Manuka honey had catalase added to remove hydrogen peroxide, so that only the unique Leptospermum antibacterial was being tested.

Honey Fractions Michalkiewicz et al. [40] have investigated the solid-phase extraction (SPE) methods that affect the recovery of phenolic acids (such as gallic acid, p-HBA, p-coumaric, vanillic, caffeic and syringic acid) and some flavonols (rutin, quercetin and kaempferol) in honey. Several other studies involving SPE procedures to remove matrix components from honey using polystyrene non-ionic sorbents Amberlite XAD type [4, 41-43], a strongly acidic ionexchange resin such as Dowex 50WX8 [44] or Bond Elut C-18 cartridges have also been performed [45, 46].

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Table 2. Relative distribution of antibacterial activity in different honey fractions [130] Honey

% antibacterial activity in different fractions * acidic basic non-polar St. Mic. St. Mic. St. Mic Manuka NZ 100 75 0 10 0 5 Sunflower It 58 46 13 15 16 25 Rape CH 25 40 7 33 63 22 Lavender Fr 25 27 34 30 23 29 Mountain CH 24 25 60 25 8 25 Blossom S. America 62 73 13 20 9 7 Honeydew CH 45 46 26 15 26 15 Honeydew CH 32 31 37 31 19 31 Honeydew CH 43 26 22 26 19 26 Honeydew Europe 43 32 25 31 26 37 average 46 42 24 24 21 22 standard deviation 23 18 17 8 17 10 minimum 24 25 0 10 0 5 maximum 100 75 60 33 63 37 * - values of individual honeys. St - Staphylococcus aureus. Mic- Micrococcus luteus.

volatile St. Mic. 0 10 13 15 5 5 18 14 8 24 16 0 2 24 12 6 15 23 6 0 10 12 6 9 0 0 18 24

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

66

Laïd Boukraâ, Yuva Bellik and Fatiha Abdellah

In a recent study, it has been reported that SPE fractions act differently against the tested strains [47]. At present, solid-phase extraction (SPE) represents a suitable way to extract, clean and preconcentrate target analytes from environmental or food samples. However, one problem associated with ordinary SPE is its low selectivity, due to many unwanted interfering substances of similar hydrophobicity/hydrophilicity [47]. It is concluded that the majority of the antibacterial properties of honey lie in the acidic portion while the polar portion also contributes to some activity (Table 2).

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Variation in the Potency of the Antibacterial Activity In almost all studies in which more than one type of honey has been used, differences in the antibacterial activity of the honeys have been observed. The degree of difference observed has in some cases been very large, and in many others where it has been smaller this possibly is the result of a more limited range of testing rather than of less variance in the activity of the honeys. In many studies the antibacterial activity of different honeys has been compared by way of the "inhibine number" determined by the method devised by Dold and Witzenhausen for such comparisons who coined the term "inhibine number" in 1955 to describe the degree of dilution to which a honey will retain its antibacterial activity [48]. This is a term that has been widely used since as a measure of the antibacterial activity of honey. The "inhibine number" involves a scale of 1 to 5 representing sequential dilutions of honey in steps of 5% from 25% to 5%. There have since been various minor modifications to this method so that the actual concentration corresponding to the "inhibine number" reported may vary. One modification has been to estimate fractional "inhibine numbers" by visual assessment of partial inhibition on the agar plate with the concentration of honey that just allows growth [49-51]. In other studies activity was found to range over a four-fold difference in concentration in the dilution series [52, 53]. With some honeys not active at the highest concentration tested in some of the studies, and others still active at the greatest dilutions, it is possible that if greater and lesser degrees of dilution had been included in the testing then a wider range of activities would have been detected. One study using a wider range of dilutions (honey from 50 to 0.25%) found the minimum inhibitory concentrations of the honeys tested to range from 25 to 0.25% [54]. Another, testing from 50 to 0.4% found the minimum inhibitory concentrations to range from >50 (i.e. not active at 50%) to 1.5% [55]. Other studies with wide ranges tested also found some honeys without activity at the highest concentration tested, and other honey with activity at the lowest concentration tested: the ranges were 20-0.6% [56] and 50-1.5% [57, 58]. When the data are examined, activities are seen to be fairly well spread over these ranges. Duisberg and Warnecke [50] plotted the distribution of the activity of 131 samples of honey tested, and found that it deviated from a normal Gaussian distribution because of the large number of samples with low activity. (In 7% of the samples the activity was below the level of detection). They attributed this to destruction of activity by exposure to heat and light, and estimated that 50% of the samples had lost more than half of their original activity, and 22% had lost more than three quarters. The Antiviral Properties of Honey A large amount of research has established the potent antibacterial activity of honey, but its activity against viral species has been the subject of only a small number of studies. These Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Honey and Microbes

67

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

were with viruses which cause localised infections in which honey could be used topically. Recent studies demonstrating the safety of intrapulmonary administration of honey in sheep and humans raised the possibility of using honey to treat respiratory infections. Attempts at isolating the antiviral component in honey demonstrated that the sugar was not responsible for the inhibition of respiratory syncytial virus (RSV), but that methylglyoxal may play a part in the greater potency of Manuka honeys against RSV. Addition of honey to moderately infected cells was observed to halt the progression of infection, thus showing the potential benefit of using honey as a treatment in individuals already experiencing symptoms of infection. It was shown that sugar, in similar levels found in 2% natural honey, is not an effective antiviral agent. In contrast, further investigations determined that methylglyoxal had potent antiviral activity even at very low concentrations, and may therefore be the component responsible for the greater inhibition seen in the high-NPA honeys (NPA=Non Peroxide Activity). It is concluded from the findings in this study that honey may possibly be an effective antiviral treatment for the therapy of respiratory viral infections, and provides justification for future in vitro studies and clinical trials [59]. Previous studies suggest other roles that honey may play if used as a treatment for viral infections as well as having antibacterial activity which would protect patients from secondary infections, honey has also been found to have some immunostimulatory effects [60, 61], which would also augment the direct antiviral action and therefore contribute to the clearing of virus and healing of infection by the body‘s own defenses. The Antifungal Properties of Honey Although an earlier brief review [62] of the biological effects of honey expressed the opinion that honey had no effect on fungi beyond its osmotic action, many recent studies show that some honeys, at least, must have antifungal factors present, as some fungi are inhibited under conditions where the sugar content of the honey is clearly not responsible. Boukraâ and colleagues have reported the potency of different varieties of honey against fungi [63, 64].

Honey and Infection Honey has been found to be effective against a large number of bacteria causing infections. Table 3 summarizes the susceptibility of the most common pathologic agents of infectious diseases.

Honey to Prevent Wound Infections An ample variety of micro-organisms may colonise the burn wound, proliferate on and within the eschar, progress in depth and initiate a systemic infection which remains a major cause of death among people with burns [65, 66] and any means of preventing will lead to higher survival rates in burned patients.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

68

Laïd Boukraâ, Yuva Bellik and Fatiha Abdellah

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Table 3. Infection caused by bacterial pathogens that are sensitive to the antibacterial activity of honey [16] Infection Anthrax Diphteria Diarrhea, septicemia, urinary infections wound infections Ear infections, meningitis, respiratory infections, sinusitis Pneumonia Meningitis Tuberculosis Infected animal bites Septicemia, urinary infections, wound infections Urinary infections, wound infections Diarrhea Septicemia Typhoid Wound infections Septicemia, wound infections Dysentery Abscesses, boils, carbuncles, impetigo wound infections Urinary infections Dental caries Ear infections, meningitis, pneumonia Sinusitis Ear infections, impetigo, puerperal fever rheumatic fever, scarlet fever, sore throat, wound infections Cholera

Pathogen Bacillus anthracis Corynebacterium dithteriae Escherichia coli Haemophilus influenzae Klebsiella pneumoniae Listeria monocytogenes Mycobacterium tuberculosis Pasteurella multocida Proteus species Pseudomonas aeruginosa Salmonella species Salmonella cholerae-suis Salmonella typhi Salmonella typhimurium Serratia marcescens Shigella species Staphylococcus aureus Streptococcus faecalis Streptococcus mutans Streptococcus pneumoniae Streptococcus pyogenes

Vibrio cholerae

Prevention and treatment of burn wound infection includes proper wound dressing [67], surgical debridement, and systemic and topical antimicrobial therapy [68, 69]. Third-degree burn wound eschar is avascular and frequently several millimeters distant from the patient‘s microvasculature. Therefore, systemically administered antimicrobial agents may not achieve therapeutic levels by diffusion to the wound, where microbial numbers are usually very high [68]. In addition, systemic antibiotics can lead to the development of drug resistant respiratory and urinary tract infections [70]. Silver sulfadiazine cream (SSD), while being effective, is not without systemic complications which include neutropenia, erythema sultiforme, crystalluria, and methaemoglobinaemia [71]. The emergence of antibiotic resistant bacteria, particularly methicillin-resistant Staphylococcus aureus (MRSA), has posed problems in the management of chronic wound infections. MRSA is now the major cause of nosocomial infections [72, 73] and is increasing in prevalence on burns units. Mortality and

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Honey and Microbes

69

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

morbidity from infection are greater when caused by antimicrobial-resistant bacteria [74]. Pseudomonas aeruginosa is one of the main organisms responsible for drug resistant nosocomial infections, particularly among people with burns [75, 76]. What is even more worrying is that increased resistance to vancomycin in S. aureus has recently been reported [77]. Many studies [77-82] have shown that honey has antibacterial activity in vitro, and clinical case studies have shown that application of honey to severely infected cutaneous wounds is capable of clearing infection from the wound and improving healing. Honey has an osmolarity sufficient to inhibit microbial growth [81], but when used as a wound contact layer, dilution by wound exudate may reduce the osmolarity to a level that ceases to control infection [83]. The antimicrobial activity is measured as the minimum inhibitory concentration (MIC), i.e. the minimum concentration necessary for complete inhibition of growth. While there is insufficient data to clearly identify the antibacterial activity of all honeys, there is some evidence of high levels in honeydew honey from the conifer forests of the mountainous regions of central Europe [84] and manuka honey (Leptospermum scoparium) from New Zealand [16]. Willix et al. [85], studying the effectiveness of 26 types of honey against bacterial strains isolated from wounds, reported that manuka honey had the highest levels of non-peroxide activity. Methylglyoxal has been recently isolated and identified as the dominant antibacterial fraction of manuka honey [29, 30, 86]. Antibioticresistant strains have also been studied and found to be as sensitive to honey as the antibioticsensitive strains of the same species [77, 79-82]. Wounds infected with MRSA have been cleared of infection and healed by application of honey including a leg ulcer [83], cavity wounds resulting from haematomas [87], and surgical wounds [88]. Also vancomycinresistant enterococci (VRE) have been reported to be sensitive to honey [89]. The most frequently isolated bacteria from burns and wounds, namely S. aureus and P. aeruginosa, have been found to be sensitive to honey action [90, 91]. Topical application of honey to burn wounds and other wounds has been found to be effective in controlling infection and producing a clean granulating bed [92-95].

Honey to Treat Gastrointestinal Disorders Oral administration of honey to treat and protect against gastrointestinal infections such gastritis, duodenitis and gastric ulcerations and rotavirus has been reported. The findings have demonstrated that the prevention of bacterial adherence caused by honey was through effect on bacteria rather than epithelial cells [96].

Gastroenteritis Infections of the intestinal tract are common throughout the world, affecting people of all ages. The infectious diarrhoea exacerbates nutritional deficiencies in various ways, but as in any infection, the calorific demand is increased. Pure honey has bactericidal activity against many enteropathogenic organisms, including those of the Salmonella and Shigella species, and enteropathogenic E. coli [97]. In vitro studies of Helicobacter pylori isolates which cause gastritis have been shown to be inhibited by a 20% solution of honey. Even isolates that exhibited a resistance to other antimicrobial agents were susceptible [98]. In a clinical study,

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

70

Laïd Boukraâ, Yuva Bellik and Fatiha Abdellah

the administration of a bland diet and 30 mL of honey three times a day was found to be an effective remedy in 66% of patients and offered relief to a further 17%, while anaemia was corrected in more than 50% of the patients [99]. A clinical study of honey treatment in infantile gastroenteritis was reported by Haffejee and Moosa [100] They found that by replacing the glucose (111 mmol/l) in the standard electrolyte-containing oral rehydration solution recommended by the World Health Organisation/UNICEF [101], as well as the solution of electrolyte composition 48 mmol/ l sodium, 28 mmol/l potassium, 76 mmol/l chloride ions, with 50 ml/l honey [102], the mean recovery times of patients (aged 8 days to 11 years) were significantly reduced. Honey was found to shorten the duration of diarrhoea in patients with bacterial gastroenteritis caused by organisms such as Salmonella, Shigella and E. coli. They recommended that honey was a safe substitute for glucose as long as it provided 111 mmol/l each of glucose and fructose. The high sugar content of honey means that it could be used to promote sodium and water absorption from the bowel.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Gastric Ulcers Clinical and animal studies have shown that honey reduces the secretion of gastric acid. Additionally, gastric ulcers have been successfully treated by the use of honey as a dietary supplement [103]. An 80% recovery rate of 600 gastric ulcer patients treated with oral adminstration of honey has been reported [103]. Radiological examination showed that ulcers disappeared in 59% of patients receiving honey. Animal experiments have shown that the administration of a honey solution via a tube in the stomach of rabbits prior to them being administered with 0.5 g ethanol per kg body weight accelerated alcoholic oxidation. A more recent animal study [104] showed that honey administered subcutaneously or orally before oral administration of ethanol affords protection against gastric damage and reverses changes in pH induced by ethanol. A controlled clinical trial demonstrated the use of fructose in the treatment of acute alcoholic intoxication. A small but significant increase occurred in the rate of fall of blood-ethanol levels and it was concluded that fructose may be beneficial in shortening the duration of alcoholic intoxication [105].

Honey for Respiratory Infections Honey has been found to be effective in killing bacterial biofilms formed by Pseudomonas aeruginosa and Staphylococcus aureus which play a major role in the pathophysiology of chronic rhino-sinusitis [106]. In a study involving 105 children aged 2-18 years with upper respiratory tract infections of 7 days or less and night-time coughing, a single night-time dose of buckwheat honey was an effective alternative treatment for symptomatic relief of nocturnal cough and sleep difficulty, compared to a single dose of dextromethorphan (DM). Researchers from the Penn State College of Medicine asked parents to give either honey, honey-flavored dextromethorphan (DM), or no treatment to the children. The first night, the children did not receive any treatment. The following night they received a single dose of buckwheat honey, honey-flavored DM, or no treatment 30 minutes before bedtime. The trial was partially blind as parents could not distinguish between the honey and the medication, although those administering no medication were obviously aware of the fact.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Honey and Microbes

71

Parents were asked to report on cough frequency and severity, how bothersome the cough was, and how well both adult and child slept, both 24 hours before and during the night of the dosage. Significant symptom improvements were seen in the honey-supplemented children, compared with the no treatment group and DM-treated group, with honey consistently scoring the best and no-treatment scoring the worst. Based on parental "symptom points," children treated with honey improved an average of 10.71 points compared with 8.39 points for DMtreated children and 6.41 points for those who were not treated [107]. Sinusitis, a sinus infection, is an inflammation of the paranasal sinuses. This condition is mainly caused by a bacterial, fungal, viral, infection or allergic or autoimmune issues. The pressure and headache caused by a sinus infection are due to the fact that bacteria form layers of living material (biofilms) that coat the surface of the sinus cavities. A study from the University of Ottawa shows manuka honey to be more effective (in many cases) at destroying bacteria that cause sinus infections than antibiotics. The research was restricted to laboratory tests (in vitro). Manuka honey destroyed bacteria free-floating in liquid. It didn't completely rid the bacteria in a biofilm, but was still able to kill 63 to 91 per cent of different bacteria types [108].

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Honey for Eye Infections Honey is hyperosmotic, hygroscopic, and has a pH of 3.9 [109]. The acidic hyperosmotic milieu with the presence of inhibin (glucose oxidase) confers unique antimicrobial properties to honey [110]. Its topical use improves visual acuity in eyes with epithelial corneal oedema, decreases pain from bullous keratopathy, and allows anterior and posterior segment visualization facilitating anterior and posterior segment laser therapy. Compared to saline or dextran [111] honey is inexpensive, universally available, contains no preservatives, and does not induce allergy. However, initial applications result in stinging and its effect is short lasting. The therapeutic potential of uncontaminated pure honey is grossly underutilized [112].

Honey as a Prebiotic The oligosaccharides are of potential nutritional importance among the minor constituents [113]. This group of low molecular weight polysaccharides is of interest because, while they are neither hydrolyzed nor absorbed in the upper part of the gastrointestinal tract [114], they may beneficially affect the health of the consumer by selectively stimulating the growth and/or activity of desirable bacteria in the colon [115, 116]. Thus, these essential bacteria, of which Bifidobacterium spp. are the most widely studied, dominate the walls of the colon through their unique ability to digest the mucin secreted by cells of the epithelium. In this position, they protect the host by competing for available nutrients and space against bacterial or fungal pathogens, an antagonism that is reinforced by the secretion of organic. It has been established that different honeys contain specific oligosaccharides, e.g. isomaltose and melezitose in New Zealand honey [117] and raffinose in Italian honey [118] and it is likely that one or more of these compounds would prove stimulatory to Bifidobacterium spp. [119]. Equally relevant is the fact that the lactobacilli in the small intestine, e.g. Lactobacillus

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

72

Laïd Boukraâ, Yuva Bellik and Fatiha Abdellah

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

acidophilus, could well be affected advantageously by the sugars and/or other components in honey [120, 121]. It has been found that the activity of certain species of Bifidobacterium in the colon can be stimulated by fructo-oligosaccharides [122] and the oligosaccharides from soya beans [123]. The importance of these oligosaccharides lies in the fact that the galactosidic linkages present cannot be digested by humans [124] but, on entering the colon, they can be metabolised by Bifidobacteria. Although the stimulatory role of oligosaccharides on the gut flora(s) has received most attention, it has been speculated that the same components in honey could inhibit the development of pathogens like Helicobacter pylori or Staphylococcus aureus in the body. More specifically, it has been proposed that the oligosaccharides could become attached to the cell walls of the bacteria and prevents adhesion to human tissues [125]. Bifidobacteria are considered important in the health of the gastrointestinal tract. Clinical studies have associated other beneficial effects such as immune enhancement and anti-carcinogencity with the presence of gastrointestinal Bifidobacteria. To increase numbers of these beneficial bacteria, prebiotics may be incorporated into the diet. Currently the most common prebiotics are non-digestible oligosaccharides such as fructo-oligosaccharides (FOS), galactooligosaccharides (GOS) and inulin. Honey contains a variety of oligosaccharides varying in degree of polymerisation. A study by Kajiwara et al. [126] of the department of Food Science and Nutrition, Michigan State University, showed that honey enhanced the growth, activity and viability of commercial strains of Bifidobacteria typically used in the manufacture of fermented dairy products. It was shown that the effect of honey on intestinal Bifidobacterium species was similar to that of commercial oligosaccharides (FOS, GOS and inulin), and that it was due to the synergistic effect of the carbohydrate components of honey, rather than just a single component.

Honey and Threatening Bacteria: How Safe Is Honey? Although it is known that honey inhibits the growth of the major part of pathogenic bacteria, spore-forming micro-organisms such as Clostridium botulinum are still able to survive in honey and there may be a risk of wound botulism when it is applied on the wound. Thus, gamma irradiation that is used to sterile heat-sensitive medical items such as surgical gloves and polymeric dressing was suggested as a possible alternative to sterilise honey for medical use [127, 128]. Moreover, although Clostridium botulinum spores are inactive in the honey itself, once inside a digestive tract they can multiply and cause a potentially fatal disease of the nervous system called infant botulism. By the time of a child‘s first birthday, there are usually enough beneficial bacteria in the digestive tract to make it an inhospitable environment for Clostridium botulinum, meaning that honey can be eaten safely. In 2002 an expert study of the Health and Consumer Directorate of the European Commission carried out on ―Honey and microbiological hazards‖ [129]. It was concluded that: ―The level and frequency of contamination of honey with spores of C.botulinum appear generally to be low, although limited microbiological testing of honey has been performed. The routes by which spores of C.botulinum contaminate honey have not been

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Honey and Microbes

73

precisely identified. Although some geographical regions of the world can be associated with a particular type of C. botulinum in the soil, it is not possible to identify countries as the origin of honey with a greater risk of containing C. botulinum. C. botulinum can survive as spores in honey but cannot multiply or produce toxins due to the inhibitory properties of honey. At present there is no process that could be applied to remove or kill spores of C. botulinum in honey without impairing product quality. Microbiological testing would not be an effective control option against infant botulism, due to the sporadic occurrence and low levels of C. botulinum in honey‖.

Conclusion

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

In the developing world a reluctance to stay with simple and still effective methods, treatments, and equipment, in order to ‗catch up‘ with the developed world, comes at a cost. It is suggested that honey could be used instead of high-tech products such as the new recombinant growth factors. This may receive low priority in modern protocols, but should rate highly in resource-based health delivery. In this respect, honey appears superior to the expensive modern hydrocolloid wound dressings which are favoured in the art as a moist dressing. Although it is known that honey inhibits the growth of the major part of pathogenic bacteria, spore-forming micro-organisms such as Clostridium botulinum are still able to survive in honey and there may be a risk of botulism when it is applied on the wound or ingested. Thus, gamma irradiation that is used to sterile heat-sensitive medical items such as surgical gloves and polymeric dressing was suggested as a possible alternative to sterilise honey for medical use. Using honey as a medicine means that it should be free of any additives and residues. A number of medical grade honeys are now available on the international market. These have guaranteed levels of antibacterial activity and are free of residues and microbial contaminants.

References [1] [2]

[3] [4]

[5] [6]

Skiadas, PK; Lascaratos, JG. Dietetics in ancient Greek philosophy: Plato‘s concepts of healthy diet. European Journal of Clinical Nutrition, 2001, 55, 532-537. Martin, GS; Mannin, DM; Eaton, S; Moss, M. The epidemiology of sepsis in the United States from 1979 through 2000. New England Journal of Medicine, 2003, 348, 15461554. Finaly, BB; Hancock, RE. Can innate immunity be enhanced to treat microbial infections? Nature Reviews Microbiology, 2004, 2, 497-504. Weston, RJ; Broncklebank, LK; Lu, Y. Identification and quantitative levels of antibacterial components of some New Zealand honeys. Food Chemistry, 2000, 70, 427-435. Snowdon, JA; Cliver, DO. Microorganisms in honey. International Journal of Food Microbiology, 1996, 31, 1–26. Gheldof, N; Wang, XH; Engeseth, NJ. Identification and quantification of antioxidant components of honeys from various floral sources. Journal of Agricultural and Food Chemistry, 2002, 50, 5870–5877.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

74 [7] [8]

[9] [10] [11] [12] [13] [14]

[15] [16] [17] [18]

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

[19]

[20]

[21]

[22]

[23] [24]

[25]

Laïd Boukraâ, Yuva Bellik and Fatiha Abdellah Aljadi, AM; Kamaruddin, MY. Evaluation of the phenolic contents and antioxidant capacities of two Malaysian floral honeys. Food Chemistry, 2004, 85, 513–518. Goerzen, DW. Microflora associated with the alfalfa leaf cutting bee, egachile rotundata (Fab) (Hymenoptera: Megachilidae) in Saskatchewan, Canada. Apidologie, 1991, 22, 553-561. Cliver, DO. Honey, human pathogens, and HACCP. Dairy, Food Environmental Sanitation 2000; 20, 261-263. Zucchi, P; Bassignani, V; Carpana, E. Honey microbiology. Industrie Alimentari, 2001, 40, 1346-1350. Huhtanen, CN; Knox, D; Shimanuki, H. Incidence and origin of Clostridium botulinum spores in honey. Journal of Food Protection, 1981, 44, 812-814. Debodt, G ; Vlayen, P. Miel et botulisme. Les Carnets du CARI Abeilles et Cie 1994, 46, 14-16. Tanzi, MG; Gabay, MP. Association between honey consumption and infant botulism. Pharmacotherapy 2002, 22, 1479-1483. Timmroth, R; Speer, K; Beckh, G; Lulmann C. Comparison of European honeys to tropical honeys - effects of yeast cell numbers on the concentration of especially selected components. Apimondia abstracts Ireland 2005, Apimondia International Apicultural Congress Dublin, Ireland; Dublin, Ireland; pp 110. Molan, PC. The antibacterial properties of honey. Chem. NZ 1995; 59(4):10–14. Molan, PC. The antibacterial activity of honey. 1. The nature of the antibacterial activity. Bee World, 1992, 73, 5–28. White, JW; Subers, MH. Studies on honey inhibine. Effect of heat. Journal of Apicultural Research, 1964, 3, 45–50. Sackett, WG. Honey as a carrier of intestinal diseases. Bull Colorado State University Agricultural Experiment Station, 1919, 252, 1–18. White, JW; Subers, MH; Schepartz, AI. The identification of inhibine, the antibacterial factor in honey, as hydrogen peroxide and its origin in a honey glucose-oxidase system. Biochimica Biophysica Acta, 1963, 73, 57–70. Dold, H; Du, DH; Dziao, ST. [Detection of the antibacterial heat and light-sensitive substance in natural honey]. Zeitschrift fur Hygiene Infektionskrankheiten, 1937, 120, 155–167. Saissy, JM; Guignard, B; Pats, B; Guiavarch, M; Rouvier, B. Pulmonary oedema after hydrogen peroxide irrigation of a war wound. Intensive Care Medicine, 1995, 21, 287– 288. Salahudeen, AK; Clark, EC; Nath, KA. Hydrogen peroxide-induced renal injury. A protective role for pyruvate in vitro and in vivo. Journal of Clinical Investigation, 1991, 88, 1886–1893. Halliwell, B; Cross, CE. Oxygen-derived species: their relation to human disease and environmental stress. Environmental Health Perspectives, 1994, 102, 5–12. Pruitt, KM; Reiter, B. Biochemistry of peroxidase system: antimicrobial effects; in Pruitt KM, Tenovuo JO (eds). The Lactoperoxidase System: Chemistry and Biological Significance. New York, Marcel Dekker, 1985, pp 144–78. Allen, KL; Molan, PC; Reid, GM. A survey of the antibacterial activity of some New Zealand honeys. Journal of Pharmacy and Pharmacology, 1991, 43, 817–822.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Honey and Microbes

75

[26] Kwakman, PH ; te Velde, AA ; de Boer, L ; Speijer, D ; Vandenbroucke-Grauls, CM ; Zaat, SA. How honey kills bacteria. FASEB Journal, 2010, 24, 2576-2582. [27] McCarthy, J. The antibacterial effects of honey: Medical fact or fiction? American Bee Journal, 1995, 135, 341-342. [28] Henriques, A; Jackson, S; Cooper, R; Burton, N. Free radical production and quenching in honeys with wound healing potential. Journal of Antimicrobial Chemotherapy, 2006, 58, 773-777. [29] Adams, CJ; Boult, CH; Deadman, BJ; Farr, JM; Grainger, MN; Manley-Harris, M; Snow, MJ. Isolation by HPLC and characterisation of the bioactive fraction of New Zealand manuka (Leptospermum scoparium) honey. Carbohydrate Research, 2008, 343, 651-659. [30] Mavric, E; Wittman, S; Barth, G; Henle, T. Identification and quantification methylglyoxal as the dominant antibacterial constituent of Manuka (Leptospermum scoparium) honeys from New Zealand. Molecular Nutrition and Food Research, 2008, 52, 483-489. [31] Vinson, JA; Hao, Y; Su, X; Zubik, L. Phenol antioxidant quantity and quality in foods: vegetables. Journal of Agricultural and Food Chemistry, 1998, 46, 3630–3634. [32] Arreaz-Roman, D; Gomez-Caravaca, AM; Gomez-Romero, M; Segura-Carretero, A; Fernandez-Gutierrez, A. Identification of phenolic compounds in rosemary honey using solid-phase extraction by capillary electrophoresis–electrospray ionization-mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis, 2006, 41, 1648– 1656. [33] Estevinho, L; Pereira, AP; Moreira, L; Dias, LG; Pereira, E. Antioxidant and antimicrobial effects of phenolic compounds extracts of Northeast Portugal honey. Food and Chemical Toxicology, 2008, 46, 3774-3779. [34] Davidson, PM; Sofos, JN; Brenem, AL. Antimicrobials in Foods (third ed.), Marcel Dekker Inc., New York. 2005, 291–306. [35] Taormina, PJ; Niemira, BA; Bauchat, LR. Inhibitory activity of honey against foodborne pathogens as influenced by the presence of hydrogen peroxide and level of antioxidant power. International Journal of Food Microbiology, 2001, 69, 217–225. [36] Basualdo, C; Sgroy, V; Finola, MS; Marioli, JM. Comparison of the antibacterial activity of honey from different provenance against bacteria usually isolated from skin wounds, Veterinary Microbiology, 2007, 124, 375–381. [37] Rauha, JP; Remes, S; Heinonen, M; Hopia, A; Kahkonen, M; Kujala, T; Pihlaja, K; Vuorela, H; Vuorela, P. Antimicrobial effects of Finnish plant extracts containing flavonoids and other phenolic compounds. International Journal of Food Microbiology, 2000, 56, 3–12. [38] Frankel, S; Robinson, GE; Berenbaum, MR. Antioxidant capacity and correlated characteristics of fourteen unifloral honeys. Journal of Apicultural Research, 1998, 37, 27–31. [39] Boukraa, L; Niar, A. Sahara Honey Shows Higher Potency Against Pseudomonas aeruginosa Compared to North Algerian Types of Honey. Journal of Medicinal Food, 2007 10, 712-714. [40] Michlkiewicz, A; Biesaga, M; Pyrzynska, K. Solid phase extraction procedure for determination of phenolic acids and some flavonols in honey. Journal of Chromatography A, 2008, 1187, 18–24.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

76

Laïd Boukraâ, Yuva Bellik and Fatiha Abdellah

[41] Tomas-Barberan, FA; Bilazquez, MA; Garcia-Viguera, C; Ferreres, F; Tomas-Lorenete, FA. Comparative study of different Amberlite XAD resins in flavonoid analysis. Phytochemical Analysis, 1992, 3, 178 -181. [42] Martos, I; Ferreres, F; Tomas-Barberan, FA. Identification of flavonoid markers for the botanical origin of Eucalyptus honey. Journal of Agricultural and Food Chemistry, 2000, 48, 1498-1502. [43] Yao, L; Jiang, Y; Singanusong, R; Datta, N; Raymont, K. Phenolic acids and abscisic acid in Australian Eucalyptus honeys and their potential for floral authentication. Food Chemistry, 2004, 86, 169-177. [44] Paramas, AMG; Barez, JAG; Marcos, CC; Garciavillanova, RJ; Sanchez, JS. HPLCfluorimetric method for analysis of amino acids in products of the hive(honey and beepollen). Food Chemistry, 2006, 95, 148-156. [45] Dimitrova, B; Gevrenova, R; Anklam, E. Analysis of phenolic acids in honeys of different floral origin by solid-phase extraction and high-performance liquid chromatography. Phytochemicals Analaysis, 2007, 18, 24. [46] Korta, E; Bakkali, A; Berrueta, LA; Gallo, B; Vincente, F. Study of semi-automated solid-phase extraction for the determination of acaricide residues in honey by liquid chromatography. Journal of Chromatography A, 2001, 930, 21-29. [47] Kirnpal-Kaur, BS; Hern-Tze T; Boukraa L; Siew-Hua G. Different solid phase extraction fractions of tualang (Koompassia excelsa) honey demonstrated diverse antibacterial properties against wound and enteric bacteria. Journal of ApiProduct and ApiMedical Science, 2011, 3, 59-65. [48] Dold, H; Witzenhausen, R. Ein verfahren zur beurteilung der örtlichen inhibitorischen (keimvermehrungshemmenden) wirkung von honigsorten verschiedener herkunft. Zeitschrift für Hygiene und Infektionskrankheiten, 1955, 141, 333-337. [49] Adcock, D. The effect of catalase on the inhibine and peroxide values of various honeys. Journal of Apicultural Research, 1962, 1, 38-40. [50] Duisberg, H; Warnecke, B. Erhitzungs- und lichteinfluß auf fermente und inhibine des honigs. Zeitschrift für Lebensmittel-Untersuchung und Forschung, 1959, 111, 111-119. [51] Stomfay-Stitz, J; Kominos, SD. Über bakteriostatische wirkung des honigs. Zeitschrift für Lebensmittel-Untersuchung und Forschung, 1960, 113, 304-309. [52] Chwastek, M. Jakosc miodówpszczelich handlowych na podstawie oznaczania ich skladników niecukrowcowych. czesc II. Zawartosc inhibiny w miodach krajowych. Rocziniki Panstwowego Zakladu Higieny, 1966, 17, 41-48. [53] Molan, PC; Smith, IM; Reid, GM. A comparison of the antibacterial activity of some New Zealand honeys. Journal of Apicultural Research, 1988, 27, 252-256. [54] Agostino Barbaro, AD; Rosa, CLA; Zanelli, C. Atttività antibatterica di mieli Siciliani. Quaderni della Nutrizione, 1961, 21, 30-44. [55] Dustmann, JH. Antibacterial effect of honey. Apiacta, 1979, 14, 7-11. [56] Buchner, R. Vergleichende untersuchungen über die antibakteriellen wirkung von blüten und honigtauhonigen. Südwestdeutscher Imker, 1966, 18, 240-241. [57] Christov, G ; Mladenov, S. Propriétés antimicrobiennes du miel. Comptes rendus de l'Académie bulgare des Sciences, 1961, 14, 303-306. [58] Khristov, G; Mladenov, S. Honey in surgical practice: the antibacterial properties of honey. Khirurgiya (Moscow), 1961, 14, 937-946 (original in Bulgarian).

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Honey and Microbes

77

[59] Zareie, PP. Honey as an antiviral agent against respiratory syncytial virus Master of Science in Biological Sciences at The University of Waikato. 2008, Waikato, New Zealand. [60] Tonks, AJ; Cooper, RA; Jones, KP; Blair, S; Parton, J; Tonks A. Honey stimulates inflammatory cytokine production from monocytes. Cytokine, 2003, 21, 242–247. [61] Tonks, AJ; Dudley, E; Porter, NG; Parton, J; Brazier, J; Smith, EL; et al. A 5.8-kDa component of manuka honey stimulates immune cells via TLR4. Journal of Leukocyte Biology, 2007, 82, 1147-1155. [62] Haydak, MH; Crane, E; Duisberg, H; Gochnauer, TA; Morse, RA; White, JW; Wix, P. Biological properties of honey. In Crane, E (ed) Honey: a comprehensive survey. Heinemann; London, UK, 1975; pp 258-266. [63] Boukraâ, L; Benbarek, H; Ahmed, M. Synergistic action of starch and honey against Aspergillus niger in correlation with diastase number. Mycoses, 2008, 51, 520–522. [64] Boukraâ, L; Bouchougrane, S. Additive action of honey and starch against Candida albicans and Aspergillus niger. Revista Iberoamericana de Micologia, 2007, 24, 309313. [65] Bauer, GJ; Yurt, RW. Burns; in Mandel GL, Bennett JE, Dolin R (eds): Principles and Practice of Infectious Diseases, ed 6. Philadelphia, PA, Churchill-Livingstone, 2005. [66] Soares de Macedo, JL; Santos, JB. Bacterial and fungal colonization of burn wounds. Memorias do Instituto Oswaldo Cruz, 2005, 100, 535–539. [67] Edwards, JV; Bopp, AF; Batistie, SL; Goynes, WR. Human neutrophil elastase inhibition with a novel cotton-alginate wound dressing formulation. Journal of Biomedical Materials Research, 2003, 66, 433–440. [68] Monafo, WW; West, MA. Current treatment recommendation for topical burn therapy. Drugs, 1990, 40, 364–373. [69] Monafo, WW; Bessey, PQ. Wound care; in Herendon DN (ed): Topical Burn Care. London, Saunders, 2002, pp. 109–119. [70] Dacso, CC; Luterman, A; Curreri, PW. Systemic antibiotic treatment in burned patients. Surgical Clinics of North America, 1987, 67, 57–68. [71] Keswani, MH; Vartak, A; Patil, A; Davies, JWL. Histological and bacteriological studies of burn wounds treated with boiled potato peel dressings. Burns, 1990, 16, 137– 243. [72] Blaser, G; Santos, K; Bode, U; Vetter, H; Simon, A. Effect of medical honey on wounds colonised or infected with MRSA. Journal of Wound Care, 2007, 16, 325–328. [73] Yasunori, M; Loughreya, A; Earle, JAP; Millar, BC; Raod, JR; Kearnse, A. Antibacterial activity of honey against community-associated methicillin resistant Staphylococcus aureus (CA-MRSA). Complementary Therapies in Clinical Practice, 2008, 14, 77–82. [74] Hsueh, P; Chen, WH; Luh, K. Relationships between antimicrobial use and antimicrobial resistance in Gram-negative bacteria causing nosocomial infections from 1991–2003 at a university hospital in Taiwan. International Journal of Antimicrobial Agents, 2005, 26, 463–472. [75] Estahbanati, HK; Kashani, PP; Ghanaatpisheh, F. Frequency of Pseudomonas aeruginosa serotypes in burn wound infections and their resistance to antibiotics. Burns, 2002, 28, 340–348.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

78

Laïd Boukraâ, Yuva Bellik and Fatiha Abdellah

[76] Altopark, U; Erol, S; Akcay, M; Celebi, F; Kadanali, A. The time related changes of antimicrobial resistance patterns and predominant bacterial profiles of burn wounds and body flora of burned patients. Burns, 2004, 30, 660–664. [77] Cooper, RA; Molan, PC; Harding, KG. The sensitivity to honey of Gram-positive cocci of clinical significance isolated from wounds. Journal of Applied Microbiology, 2002, 93, 857–863. [78] French, VM; Cooper, RA; Molan, PC. The antibacterial activity of honey against coagulase-negative staphylococci. Journal of Antimicrobial Chemotherapy, 2005, 56, 228–231. [79] Lusby, PE; Coombes, A; Wilkinson, JM. Honey: a potent agent for wound healing? Journal of Wound Ostomy and Continence Nursing, 2002, 29, 295–300. [80] Natarajan, S; Williamson, D; Grey, J; Harding, KG; Cooper, RA. Healing of an MRSAcolonized, hydroxyurea- induced leg ulcer with honey. Journal of Dermatological Treatment, 2001, 2, 33–36. [81] Simon, A; Traynor, K; Santos, K; Blaser, G; Bode, U; Molan, P. Medical honey for wound care – still the ‗latest resort‘? eCAM, 2009, 6, 165–173. [82] Henriques, AF; Jenkins, RE; Burton, NF; Cooper, RA. The intracellular effects of manuka honey on Staphylococcus aureus. European Jounral for Clinical Microbiology and Infectious Diseases, 2009, 29, 45-50. [83] Chirife, J; Herszage, L; Joseph, A; Kohn, ES. In vitro study of bacterial growth inhibition in concentrated sugar solutions: microbiological basis for the use of sugar in treating infected wounds. Antimicrobial Agents and Chemotherapy, 1983, 23, 766–773. [84] Molan, PC. The antibacterial activity of honey. 2. Variation in the potency of the antibacterial activity. Bee World, 1992, 73, 59–76. [85] Willix, DJ; Molan, PC; Harfoot, CG. A comparison of the sensitivity of woundinfecting species to the antibacterial activity of manuka honey and other honey. Journal of Applied Bacteriology, 1992, 73, 388–394. [86] Adams, CJ; Manley-Harris, M; Molan, PC. The origin of methylglyoxal in New Zealand manuka (Leptospermum scoparium) honey. Carbohydrate Research, 2009, 344, 1050–1053. [87] Wadi, M; Al-Amin, H; Farouq, A; Kashef, H; Khaled, SA. Sudanese bee honey in the treatment of suppurating wounds. Arab Medico, 1987, 3, 16–18. [88] Dunford, C; Cooper, R; White, RJ; Molan, P. The use of honey in wound management. Nursing Standard, 2000, 15, 63–68. [89] Dunford, C; Cooper, R; Molan, P. Using honey as a dressing for infected skin lesions. Nursing Times, 2000, 96, 7–9. [90] Boukraa, L; Benbarek, H; Aissat, S. Synergistic action of starch and honey against Pseudomonas aeruginosa in correlation with diastase number. Journal of Alternative and Complementary Medicine, 2008, 14, 181–184. [91] Boukraa, L; Amara, K. Synergistic action of starch on the antibacterial activity of honey. Journal of Medicinal Food, 2008, 11, 195–198. [92] Subrahmanyam, M. Topical application of honey treatment of burns. British Journal of Surgery, 1991, 78, 497-498. [93] Bulman, MW. Honey as a surgical dressing. The Middlesex Hospital Journal, 1955, 15, 18-19.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Honey and Microbes

79

[94] Subrahmanyam, M. Honey dressing for burns: an appraisal. Annals of Burns and Fire Disasters, 1996, 9, 33-35. [95] Subrahmanyam, M. Honey dressing versus boiled potato peel in the treatment of burns: A perspective randomised study. Burns, 1996, 22, 491-493. [96] Al-Jabri, A; Mahrouqui, ZA; Basil, N; Herbert, N. Inhibition effect of honey on the adherence of Salmonella to intestinal epithelial cells in vitro. International Journal of Food Microbiology, 2005, 103, 347-351. [97] Jeddar, A; Kharsany, A; Ramsaroop, UG; Bhamjee, A; Haffejee, IE; Moosa, A. The antibacterial action of honey: an in vitro study. South Africa Medical Journal, 1985, 67, 257-258. [98] Ali, AT; Chowdhury, MN; Al-Humayyd, MS. Inhibitory effect of natural honey on Helicobacter pylori. Tropical Gastroenterology, 1991, 12, 139-143. [99] Salem, SN. Treatment of gastroenteritis by the use of honey. Islamic Medicine, 1981, 1, 358-362. [100] Haffejee, IE; Moosa, A. Honey in the treatment of infantile gastroenteritis. British Medical Journal, 1985, 290, 1866-1867. [101] World Health Organisation. Treatment and prevention of dehydration in diarrhoeal diseases. A guide for use at the primary level. Geneva: WHO, 1976, 1-13. [102] Chatterjee, A; Mahalanabis, D; Jalan, KN. Oral rehydration in infantile diarrhoea. Controlled trial of a low sodium glucose electrolyte solution. Archives of Disease in Childhood, 1978, 53, 284-289. [103] Kandil, A; El-Banby, M; Abdel-Wahed, GK; Abdel- Gawwad, M; Fayez, M. Curative properties of true floral and false non-floral honeys on induced gastric ulcers. Journal of Drug Research, 1987, 17, 103-106. [104] Ali, AT. Prevention of ethanol-induced gastric lesions in rats by natural honey and its possible mechanism of action. Scandinavian Journal of Gastroenterology, 1991, 26, 281-288. [105] Brown, SS; Forrest, JA; Roscoe, P. A controlled trial of fructose in the treatment of acute alcoholic intoxication. Lancet, 1972, 2, 898-900. [106] Alandejani, T; Marsan, J; Ferris, W; Slinger, R; Chan, F. Effectiveness of honey on Staphylococcus aureus and Pseudomonas aeruginosa biofilms. Otolaryngology – Head and Neck Surgery, 2009, 141, 114-118. [107] Paul, IM; Beiler, J; McMonagle, A; Shaffer, ML; Duda, L; Berlin, CM. Effect of honey, dextromethorphan, and no treatment on nocturnal cough and sleep quality for coughing children and their parents. Archives of Pediatrics and Adolescent Medicine, 2007, 161, 1140-1146. [108] http://www.squidoo.com/manuka-honey-medical-use accessed on September 24, 2011. [109] Burgett, DM. Antibiotic systems in honey, nectar and pollen. In: Morse RA, Nowogrodzki R (eds). Honey, Bee Pests, Predators, and Diseases. Ithaca: Cornell University, 1990, 330–340. [110] McInerney, RJF. Honey: a remedy rediscovered. Journal of the Royal Society of Medicine, 1990, 83, 127. [111] Quiroz-Mercado, H; Salinas-van Orman, E; Morales-Canton, V; de Regil-Romero, M; Cuevas-Cancino, D. The use of dextran after keratoplasty for visualizing the vitreous cavity. Ophthalmic Surgery and Lasers, 1998, 29, 980–984.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

80

Laïd Boukraâ, Yuva Bellik and Fatiha Abdellah

[112] Mansour, AM; Traboulsi, E. Honey as a substitute for Healon in experimental anterior segment surgery in animals. Pakistan Journal of Ophthalmology, 1985, 1, 136. [113] Al-Qassemi, RAS; Robinson, RK. Some special nutritional properties of honey-a review. Nutrition and Food Science, 2003, 33, 254-260. [114] Cummings, JH; Macfarlane, GT; Englyst, HN. Prebiotic digestion and fermentation. American Journal of Clinical Nutrition, 2001, 73, 415-420. [115] Chow, J. Probiotics and prebiotics: A brief overview. Journal of Renal Nutrition, 2002, 12, 76-86. [116] Haddadin, MSY; Awaisheh, SS; Robinson, RK. Production of yoghurt with probiotic bacteria isolated from infants in Jordan. Pakistan Journal of Nutrition, 2004, 3, 290293. [117] Weston, RJ; Brocklebank, LK. The oligosaccharide composition of some New Zealand honeys. Food Chemistry, 1999, 64, 33-37. [118] Oddo, LP; Piazza, MG; Sabatini, AG; Accorti, M. Characterisation of unifloral honeys. Apidologie, 1995, 26, 453-465. [119] Itsaranuwat, PK; Al-Haddad, HS; Robinson, RK. The potential therapeutic benefits of consuming health-promoting fermented dairy products. International Journal of Dairy Technology, 2003, 56, 203-210. [120] Qiao, H, Duffy, LC; Griffiths, E; Dryja, D; Leavens, A; Rossman, J; Rich, G; Riepenhoff-Talty, M; Locniskar, M. Immune responses in rhesus Rotavirus-challenged balb/c mice treated with Bifidobacteria and prebiotic supplements. Pediatric Research, 2002, 51, 750-755. [121] Rossi, M; Corradini, C; Amaretti, A; Nicolini, M; Pompei, A; Zanoni, S; Matteuzzi, D. Fermentation of fructooligosaccharides and inulin by Bifidobacteria: a comparative study of pure and fecal cultures. Applied Environmental Microbiology, 2005, 71, 61506158. [122] Shin, HS; Lee, JH; Pestka, JJ; Ustunol, Z. Growth and viability of commercial Bifidobacterium spp. in skim-milk containing oligosaccharides and inulin. Journal of Food Science, 2000, 65, 884-887. [123] Liu, K. Soybeans: Chemistry, Technology and Utilisation, Chapman and Hall, 1997, London. [124] Gopal, PK; Sullivan, PA; Smart, JB. Utilisation of galacto-oligosaccharides as selective substrates for growth by lactic acid bacteria including Bifidobacterium lactis and Lactobacillus rhamnosus. International Dairy Journal, 2001, 1, 19-25. [125] Somal, NA; Coley, KE; Molan, PC; Hancock, BM. Susceptibility of Helicobacter pylori to the antibacterial activity of manuka honey, Journal of the Royal Society of Medicine, 1994, 87, 9-12. [126] Kajiwara, S; Gandhi, H; Ustunol, Z. Effect of honey on the growth of and acid production by human intestinal Bifidobacterium spp.: An in vitro comparison with commercial oligosaccharides and inulin. Journal of Food Protection, 2002, 65, 214218. [127] Molan, PC; Allen, KL. The effect of gamma-irradiation on the antibacterial activity of honey. Journal of Pharmacy and Pharmacology, 1996, 48, 1206–1209. [128] Postmes, T; van den Bogaard, AE; Hazen, M. The sterilization of honey with cobalt 60 gamma radiation: a study of honey spiked with spores of Clostridium botulinum and Bacillus subtilis. Experientia, 1995, 51, 986–989.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Honey and Microbes

81

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

[129] European Commission. Honey and microbiological hazards. Report European Commission of Health and Consumer Protection, Directorate-General, 2002, 1-40. [130] Bogdanov, S. Antibacterial substances in honey. Bee Product Science, 2008, 1-10.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

In: Honey: Current Research and Clinical Applications ISBN: 978-1-61942-656-6 Editor: Juraj Majtan © 2012 Nova Science Publishers, Inc.

Chapter V

Anti-Biofilm Activity of Natural Honey against Wound Bacteria Juraj Majtan1,2 , Jana Bohova1, Miroslava Horniackova2 and Viktor Majtan2 1

Institute of Zoology, Slovak Academy of Sciences, Bratislava, Slovakia 2 Department of Microbiology, Faculty of Medicine, Slovak Medical University, Bratislava, Slovakia

Abstract Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Chronic, non-healing wounds are major health care problems worldwide. It is now accepted that the tissue of all chronic wounds is colonised by polymicrobial flora. Bacterial populations within chronic wounds are typically arranged into highly organised biofilms that protect bacteria from antimicrobial therapy and the patient‘s immune system. It has recently been suggested that biofilm is one of the major contributing factors toward impaired healing. There is an urgent need for introducing novel or reemerging effective strategies that target bacterial biofilms. Honey has been successfully used for treating non-healing wounds associated with bacterial biofilms, including diabetic and pressure ulcers. One of the major benefits of honey is that there is no risk of developing antimicrobial resistance. However, the mechanisms that are involved in honey-induced wound healing are only partially known. This chapter discusses the current knowledge about potential anti-biofilm activity of honey and, most importantly, shows that selected Slovak honeydew honey is able to both inhibit biofilm formation and disrupt already pre-formed biofilms of wound clinical isolates in vitro. This observation suggests that honey contains compounds with antibiofilm activity likely to be different from antibacterial compounds, which can block the attachment of bacteria to host tissue. In already colonised chronic wounds, honey substances may penetrate the biofilm structures and disrupt biofilms.



Correspondence concerning this article should be addressed to: Dr. Juraj Majtan, Institute of Zoology, Slovak Academy of Sciences, Dubravska cesta 9, 845 06 Bratislava, Slovakia, Tel.: +421-2-59302647, Fax.: +421-259302646, E-mail address: [email protected].

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

84

Juraj Majtan, Jana Bohova, Miroslava Horniackova et al. However, further studies are needed to characterise whether anti-biofilm compounds are present in every natural honey as well as define their bee-derived or phytochemical origin.

Keywords: Biofilm, wound bacteria, honeydew honey, resistance

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Introduction Most microbiological studies have involved the use of single-species microorganisms cultured in liquid media. The microorganisms cultured in this manner are described as being in a planktonic state. Planktonic bacterial cells are more susceptible to the effects of antibiotics and to environmental and host factors. The antibiotics currently in use are chosen because they are able to kill planktonic cells. However, microorganisms exist within natural and clinical environments in an entirely different form from these artificially grown laboratory strains. Microorganisms have adapted to survive within hostile environments by existing as adherent populations (sessile microorganisms). A large collection of groups of microorganisms adhering to a surface is referred to as a biofilm [1]. According to the definition by Donlan and Costerton [1] , biofilm is a microbially-derived sessile community characterised by cells that are irreversibly attached to a substratum, interface or to each other, embedded in a matrix of extracellular polymeric substances that they have produced, and exhibit an altered phenotype with respect to growth rate and gene transcription. Biofilms are ubiquitous and their presence in many areas associated with health care in particular is generally regarded as unfavourable. It has been known for decades that some chronic bacterial infections are caused by the ability of bacteria to form biofilms [2-8]. The classic example of biofilm involvement in chronic infections is non-healing dermal wounds. Biofilm growth and its persistence within wounds have recently been suggested as being a contributing factor toward impaired healing [4,9,10]. Chronic wounds offer ideal conditions for biofilm production because proteins (collagen and fibronectin) and damaged tissues are present, thus allowing attachment. Chronic wounds such as diabetic foot ulcers, pressure ulcers and venous leg ulcers are a major worldwide health care problem and are associated with decreasing quality of life and significant patient morbidity. Recent estimates indicate that 1 to 2% of the population in developing countries will experience chronic skin wounds during their lifetime [11]. In the United States, annual chronic wound treatment costs in excess of US$25 billion [12]. Nearly 2% of European health budgets are spent on the impaired healing of chronic wounds [13]. Global wound care expenditures amount to $13 to $15 billion annually [14]. There are also substantial indirect costs through loss of income, depression, deconditioning and impact on friends and family. Using cultural and molecular techniques, it has been shown that chronic wound pathogenic biofilm is generally polymicrobial, with certain species such as Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) often predominant. Recently, several studies have demonstrated that a wide variety of bacteria with different physiological and phenotypic preferences are common as part of the pathogenic biofilm communities in chronic wounds [15-18]. In addition, facultative and strict anaerobic Gram-

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Anti-Biofilm Activity of Natural Honey against Wound Bacteria

85

positive cocci are the most prevalent bacterial populations within diabetic and pressure ulcers, as determined by pyrosequencing [15]. The formation of a biofilm is dynamic and the early stages of formation can occur within hours of inoculation [19,9]. Following irreversible adhesion of pioneering bacteria to a surface, adherent bacteria begin to grow and extracellular polymers are produced, resulting in bacteria being eventually embedded in a complex three dimensional matrix of hydrated polymeric substances. Bacterial cell-cell communication is a critical function for ensuring survival in harsh environments and the mechanisms underlying this communication include cell-cell signalling and quorum sensing [20]. During quorum sensing, signal molecules, or autoinducers, are produced and secreted by the bacterial cells. When the critical bacterial population density is reached, extracellular signalling occurs and allows the population to change its phenotype to become a biofilm [21]. In fact, many bacterial physiological functions, such as luminescence, virulence, motility, sporulation and biofilm formation, are regulated by quorum sensing systems [22,23]. Although regulation by quorum sensing is highly conserved in bacteria, its molecular mechanisms, as well as the chemical nature of the autoinducers, differ significantly between Gram-positive and Gram-negative bacteria [24]. In Gram-negative bacteria, autoinducers belong to the acyl-homoserine lactones (AHLs) [25] while in Gram-positive bacteria, autoinducers are short peptides (5 to 50 amino acids) [24,26]. Unlike planktonic bacteria, bacterial biofilms consist of about 80% extracellular polymeric substances (EPS), composed of polysaccharides, proteins and nucleic acids, and about 20% bacterial cells [27]. The exopolysaccharides represent the major component of the macromolecules, accounting for 40 to 95% of the microbial EPS [28]. However, the composition of EPS is known to fluctuate and the variations have been shown to be dependent on the adherent bacterial community, availability of nutrition and environmental conditions. EPS confers many benefits to a biofilm; it not only provides the biofilm‘s cohesive forces, but also absorbs organic and inorganic materials that act as nutrients for the proliferating bacteria. Furthermore, EPS can influence physicochemical processes such as diffusion and fluid frictional resistance. Bacterial EPS primarily possess backbone structures containing 1,3- or 1,4-β-linked hexose residues that are rigid and generally poorly soluble or insoluble, whereas some EPS molecules are more readily soluble in water. Production of EPS by bacteria in culture or in aggregates depends on a number of factors, such as microbial species, growth phases, nutritional status and environmental conditions. With the recent confirmation of the presence of biofilms in wounds and their role in delaying wound healing, we investigated the anti-biofilm activity of honey against wound bacteria. In this chapter, we will determine whether (a) honey is able to prevent biofilm formation of wound bacteria at sub-inhibitory concentrations, and (b) honey can disrupt already pre-formed biofilm on a plastic surface.

Biofilm Susceptibility to Antimicrobial Agents Bacteria that reside within mature biofilms are highly resistant to many traditional therapies. Bacterial cells can change their proteome to exist in a sessile state with low metabolic levels and down-regulated cellular activities [29]. As a result of their low metabolic activity, microorganisms in biofilms are far more difficult than planktonic microorganisms to

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

86

Juraj Majtan, Jana Bohova, Miroslava Horniackova et al.

eradicate using conventional antimicrobial agents [9,30]. It has been reported that some biofilms can persist at concentrations of antibiotics and antimicrobial agents 100 to 1,000 times higher than those concentrations that can inhibit planktonic cells [31,32,1,33]. However, even the long-term use of high doses of antimicrobial agents does not ensure the eradication of microorganisms within the biofilm [34,35]. The resistance that bacteria exhibit when they grow in biofilms is not due to ‗classic‘ genetic mechanisms (i.e., genetic mutation and genetic exchange) but is instead determined by some peculiarity of biofilm growth. Previous studies have shown that several bacteria respond to antibiotic treatment by increasing polysaccharide synthesis and biofilm formation [36]. O‘Toole and Stewart [37] supposed that this response was an evolutionary adaptation of bacteria against various microbially produced antibiotics. In the case of biofilms, there are several factors and mechanisms responsible for protecting microorganisms: (a) reduction of antimicrobial penetration and diffusion into the biofilm, (b) microenvironment within the biofilm, (c) biofilm growth rate and (d) development of persister cells. Antimicrobial molecules must diffuse through the biofilm matrix in order to inactivate the encased cells. Reduced or incomplete penetration of antimicrobial agents into biofilms has already been demonstrated [38,35]. Suci et al. [39] demonstrated a delayed penetration of ciprofloxacin into P. aeruginosa biofilms; what normally required 40 seconds for a sterile surface required 21 minutes for a biofilm-containing surface. The EPS matrix of a biofilm generally has a net negative charge. Gordon et al. [40] and Costerton et al. [41] proposed that this charge could serve to sequester positively charged compounds, thereby preventing exposure of the cells embedded within the biofilm. Furthermore, the EPS charge is also able to inhibit diffusion of negatively charged molecules (e.g., aminoglycosides) into the biofilms by electrostatic repulsion [42,43]. Another hypothesis for enhanced biofilm tolerance to antimicrobial molecules relates to the altered chemical environment of the biofilm. The increased bacterial density within biofilms causes waste accumulation and an altered environment (e.g., low pH, low pO2 and high pCO2), which might lead to the cleavage of the nucleus of β-lactam class of antibiotics. Tresse et al. [44] found that Escherichia coli (E. coli) cells were more resistant to an aminoglycoside as oxygen tensions decreased. Reduced biofilm susceptibility to antimicrobial agents is not necessarily related to the thickness of the biofilm [45]. Within the biofilm, a small subpopulation of microorganisms exhibiting a biofilm-specific phenotype might develop [46-48]. These extremely resistant cells, called persisters, typically represent 0.1% or less of the bacterial population and may express a temporary multidrug tolerance to antibiotics [49]. Persister cells do not grow at high concentrations of antibiotic and are thus not resistant to antimicrobials per se [38]. Rather, persisters are considered highly tolerant to antimicrobial agents because they do not die, and further, may represent a recalcitrant subpopulation that can seed a new culture with normal susceptibility [50].

Anti-Biofilm Strategies Treatment of bacterial biofilm in the wound is complicated by the mechanisms underlying biofilm growth. Furthermore, mixed species biofilms have complementary metabolic strategies for obtaining nutrients and degrading host immune molecules [51,52]. As

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Anti-Biofilm Activity of Natural Honey against Wound Bacteria

87

mentioned above, current antibiotics may have little long-term effect on preventing or treating established biofilms as most of these antibiotics are designed to target metabolically active planktonic bacterial cells, while bacterial cells embedded in EPS matrix are unresponsive [27]. Biofilm control strategies in wound care include physical (manual and ultrasound debridement), chemical (ionic silver, iodine, gallium and ethylendiaminetetraacetic acid ) and biological (xylitol, dispersin B, quorum sensing inhibitors, lactoferrin and honey) approaches [53]. Available antimicrobial and anti-biofilm agents function by different mechanisms. Each may be effective in suppressing biofilm on the surface of a wound, but in routine clinical use, they appear to apply only to selective pressure and biofilms may become less responsive to inhibitors within a short period of time. Despite extensive efforts in developing new antibiofilm agents, no antimicrobial drug has yet been found that completely eradicates adherent microbial populations.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Honey With increasing frequency, modern medicine is focusing on natural products with antimicrobial activity and their use in clinical practice. The major arguments for implementing natural products, such as honey, are low cost and the absence of antimicrobial resistance risk. Honey can inhibit the growth of a wide range of bacteria, fungi, protozoa and viruses [54,55]. Manuka honey derived from the floral source of Leptospermum scoparium in New Zealand has been claimed to have particularly high antimicrobial activity against various bacterial species [56-59]. It is well documented that honey can act as an antimicrobial and antioxidative agent in addition to being an immunomodulator with both pro-inflammatory and anti-inflammatory effects [60-64]. As well as the two well-characterised major antibacterial factors in honey, hydrogen peroxide and high osmolarity, methylglyoxal [65,66] and bee defensin-1 [67] also act as antibacterial substances. In addition, phenolic compounds found in dark honeys are partially responsible for antibacterial activity [68-70].

Clinical Evidence for the Use of Honey in Management of Chronic Wounds All the above mentioned antibacterial factors are crucial for eradicating bacterial pathogens in chronic wounds. Moreover, the findings from animal studies and from several randomised clinical trials involving more than 2,000 participants have provided compelling evidence that honey can accelerate wound healing [58]. On the other hand, the quality of the reported trials is variable and the evidence to date supports honey only as a treatment for mild to moderate superficial and partial thickness burns [71]. Authors of a recent systematic review assert that there is insufficient evidence to guide clinical practice for other wound types [71]. Jull and co-workers established that honey-impregnated dressings did not significantly improve venous ulcer healing at 12 weeks compared with usual care [72]. Moreover, these dressings did not significantly improve the time for healing, change in ulcer area, incidence of infection or quality of life. On the other hand, two very recent clinical trials have suggested

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

.

88

Juraj Majtan, Jana Bohova, Miroslava Horniackova et al.

that healing times and incidence of infection after treatment with honey are reduced compared to conventional treatment, and the results are of clinical significance [73,74]. The main limitation of the above two trials is that insufficient numbers of recruited patients were included to reach statistical significance. Manuka honey is the type of honey most often studied in randomised controlled studies. Six trials (n=701) [75,72-74,76,77] recruited participants with chronic wounds. Non-manuka honey was recently compared to povidone iodine in treating Wagner type II diabetic foot ulcers [78]. Ulcer healing was not significantly different between the two groups and the authors deduced that honey dressing represents a safe alternative dressing for treating diabetic foot ulcers. In addition, Eddy and Gideonsen [79] reported a case where ordinary honey was applied to a patient‘s heel and forefoot diabetic ulcers in order to avoid leg amputation. Granulation tissue appeared within two weeks and the ulcers healed in 6 to 12 months.

Honey as an Anti-Biofilm Agent

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Honey is one of the promising biological agents with anti-biofilm activity. Natural honey represents an ideal agent that meets all criteria to be therapeutically useful in treating biofilmassociated infections (Figure 1). Honey is practical, nontoxic, has few side effects and is inexpensive. In addition, an important advantage of honey is the fact that there is no risk of developing antimicrobial resistance.

Figure 1. Anti-biofilm activity of honey. Honey exerts anti-biofilm effects through partially known mechanisms and molecules.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Anti-Biofilm Activity of Natural Honey against Wound Bacteria

89

Until now, there have only been very few studies investigating the effect of honey on biofilms [80-82]. Authors of these studies found that honey, in particular manuka honey, a pronounced medical grade honey, is effective in killing P. aeruginosa and methicillinresistant Staphylococcus aureus bacterial biofilms. They supposed that substances in honey are able to diffuse through the established biofilm matrix. However, Okhira et al. [83] demonstrated that P. aeruginosa biofilms were not significantly inhibited by 20% solution of manuka honey while cultures of planktonic cells of P. aeruginosa were susceptible to manuka honey at concentrations less than 10%. The maximal reduction of biofilm in the presence of 40% honey solution was reported at 11 hours of incubation. Interestingly, in all cultures of P. aeruginosa exposed to honey, there appeared to be an increase in biofilm biomass after 24 hours. This observation provides important evidence that the level of active honey within a dressing is critical if biofilm is inhibited rather than stimulated in a wound. In a very recent study, manuka honey at a concentration of 10% was able to affect the formation of biofilms in oral pathogenic Streptococcus mutans and also inhibited streptococcal adherence to a glass surface at sub-minimal inhibition concentrations (subMICs) [84]. These findings suggest that manuka honey might be able to reduce oral pathogens within dental plaque.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Inhibitory Activity of Honey in Lectin-Mediated Biofilm Formation in Pathogens The sugar content of honey may be important beyond the osmotic pressure it exerts. Honey is rich in carbohydrates (approximately 79%, including around 38% fructose, 31% glucose, 1% sucrose and 9% other sugars), but its protein content is only around 0.7% [8587]. Lerrer et al. [88] have shown that fructose in honey prevents the establishment of P. aeruginosa infection by blocking the bacterial sugar binding protein, or ‗lectin‘, whose natural target is the fucose molecules that are expressed on target tissue cells. The matrix of biofilm is itself a polysaccharide and it has become apparent that sugar molecules are also used as chemical messages between bacterial species [89]. Thus, honey can block lectinmediated bacterial biofilm formation and adhesion to tissue cells. Examination of honeys from different sources using lectins such as Concanavalin A (lectin from the plant Canavalia ensiformis), PA-IIL (lectin from P. aeruginosa) and UEA-I (lectin from the plant Ulex europaeus) demonstrated an apparent variability of lectin specificities, but significantly high uniformity among the diverse honeys themselves [88]. The fact that most of the honey-inhibiting activities toward Concanavalin A and PA-IIL were removed by dialysis indicates that they are mainly associated with low molecular weight components [88]. These results are in accordance with both the reported honey composition [86] and the Concanavalin A and PA-IIL shared affinity to fructose [90,91], which constitutes almost 40% of the honey mass. Furthermore, it has been demonstrated that glycoproteins found in honey strongly inhibit Concanavalin A and partially inhibit bacterial PA-IIL [88]. No inhibitory activity of honey was detected for plant lectin UEA-1. The protein content of honey, primarily the 55 kDa glycoprotein, major royal jelly protein 1 (MRJP1, which is the dominant protein of honey [87]), might be responsible for honey‘s inhibitory effect on lectins. In addition, it has been reported that some other biological effects can also be attributed to MRJP1 [92-94,61]. It enhances proliferation of hepatocytes [92], stimulates tumor necrosis

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

90

Juraj Majtan, Jana Bohova, Miroslava Horniackova et al.

factor α secretion from murine macrophages [61], and peptides derived from MRJP1 possess potent antihypertensive [93] and antimicrobial activity [94]. On the other hand, MRJP1 is not able to activate human epidermal keratinocytes, whose activation is necessary for proper wound healing [64].

Honey Polyphenols Disrupt Bacterial Biofilm Honey has a wide range of phytochemicals, including polyphenols, a class of natural products possessing a diverse range of pharmacological properties. Polyphenols, including flavonoids and phenolic acids, are found in honey as residual secondary metabolites and have been studied for their potential use as a botanical and geographical marker and also to explain the antibacterial properties of honey. Several studies have demonstrated that honey phenolic compounds are partially responsible for honey antibacterial and antioxidant activity [68,95-97]. Moreover, some polyphenols have been shown to inhibit bacterial biofilm formation [98,99]. According to the study of Blanco et al. (2005) [98], green tea polyphenols interfere with the polysaccharides that form the glycocalyx, disrupting their interactions and thus inhibiting biofilm formation by ocular staphylococcal isolates. The naringenin has recently exhibited prominent antagonistic activity against E. coli and Vibrio harveyi biofilm formation and may serve as the lead compound for antipathogenic drug discovery [99]. Thus, honey polyphenols are promising compounds contributing to the disruption of bacterial biofilm.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Quorum Sensing Inhibitory Activity of Honey As mentioned above, honey contains various polyphenolic compounds. It has been shown that phenolic compound extracts obtained from dark honey samples have stronger antioxidant activity then clear honey samples [68]. This observation, also attributed to the differences in phenolic compound profiles that are dependent on the honey‘s geographical origin (flora predominance), are in accordance with other investigations that state that dark honey samples have phenolic compounds with higher microbiological inhibitory properties. One of the interesting properties of some polyphenolic compounds is the ability to inhibit cell to cell communication or quorum sensing (QS). Little information has been published regarding anti-quorum sensing activity of honey [81,95,100]. The authors of a recent study [95] tested 29 honey samples of different origins for their capacity to inhibit production of AHLs in Chromobacterium violaceum. Compared to control, all unifloral honey samples showed a significant drop in AHL production, even at low concentrations of honey (0.1g/ml), although significant differences were observed according to floral origin. Similar to this study, it has been shown that chestnut honey and its extracts inhibit or degrade AHLs in Erwinia carotovora, Yersinia enterocolitica and Aeromonas hydrophila [81]. The obtained results suggest that most active compounds, which inhibit or degrade AHLs, are in the aqueous extract of honey. Thus, the QS inhibitory activity seems to be

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Anti-Biofilm Activity of Natural Honey against Wound Bacteria

91

related to water soluble compounds rather than to the phenolic compounds mainly present in the methanolic extract. Furthermore, biofilm formation of all tested strains was significantly reduced by chestnut honey and its aqueous extract. Only slight reductions were observed when the methanolic extract was added to the culture media of all strains.

Materials and Methods Bacterial Isolates Bacterial clinical isolates (n=40) from non-healing wounds were collected from the Departments of Clinical Microbiology and Wound Care Centres across Slovakia and transported to the Department of Medical Microbiology, School of Medicine, Slovak Medical University (Bratislava, Slovakia). These clinical isolates were collected during the period from March to September 2010.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Antibiotic Susceptibility Test Antibiotic susceptibility was determined by the disc diffusion method with 18 antibiotic discs according to the criteria of the Clinical and Laboratory Standards Institute (CLSI) [101] on Mueller-Hinton agar (Oxoid, Hampshire, UK). The following antimicrobial agents (disk content indicated in parentheses) were tested: amoxicillin/clavulanic acid (20/10 μg), ampicillin (10 μg), aztreonam (30 μg), carbenicillin (100 μg), cefepime (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg), cefuroxime (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), gentamicin (10 μg), meropenem (10 μg), ofloxacin (5 μg), oxacillin (1 μg), streptomycin (10 μg), piperacillin/tazobactam (40 µg), tetracycline (30 μg) and trimethoprim (5 μg). E. coli ATCC 25922 was used as the control.

Honey Samples The antibacterial activity of two honeydew honey samples against wound clinical isolates was determined by comparison to the commercially available active manuka honey imported from New Zealand (Nature‘s Nectar, UMF 15+). Honeydew honeys used in this study are monofloral honeys derived from the floral source of Abies spp. localised in Cergov (Bardejov, Slovakia) and Volovske Vrchy (Stara Voda, Slovakia), respectively. To distinguish the effect of the antibacterial components of honey from any osmotic effect, artificial honey, a control solution with sugar content similar to that of natural honey, was also used for comparison. The artificial honey was prepared by dissolving 39 g Dfructose, 31 g D-glucose, 8 g maltose, 3 g sucrose and 19 g distilled water. All honey samples were stored in darkness at 2 to 5°C when not in use. Fifty percent (w/v) stock solution of each type of honey was prepared by weighing 20g of honey and bringing the volume up to 40 ml of Tryptone Soya Broth (TSB).

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

92

Juraj Majtan, Jana Bohova, Miroslava Horniackova et al.

Further dilutions of stock solution of natural honeys were made to obtain honey concentrations of 25%, 22.5%, 20%, 17.5%, 15%, 12.5%, 11.25%, 10%, 8.75%, 7.5%, 6.25%, 5% and 3.75%. Dilution of 50%, 25% and 12.5% of artificial honey was used. Artificial honey was tested at three concentrations of 12.5%, 25% and 50%. For characterisation of honey-induced biofilm dispersion, 50% (w/v) stock solution of honey was prepared in distilled water instead of TSB and rendered by filtration through 0.22 µm microbial filter.

Minimum Inhibitory Concentration (MIC) Assay Determination of MIC was conducted according to the recommendation of the Clinical and Laboratory Standards Institute (CLSI) [102] with minor modification of using TSB instead of Mueller Hinton broth. Briefly, one bacterial colony was suspended in phosphate buffered saline (PBS) buffer, pH 7.2, and the turbidity of suspension was adjusted to 10 8 colony forming unit (CFU)/mL and diluted with medium to final concentration of 107 CFU/mL. Ten µl aliquots of suspension were inoculated in each well of sterile 96-well polystyrene plates. The final volume in each well was 100 µL, consisting of 90 µL of sterile medium or diluted honey and 10 µL of bacterial suspension. After 18 h of incubation at 37°C, bacterial growth inhibition was determined by monitoring the optical density at 490 nm. The MIC was defined as the lowest concentration of antibiotic inhibiting bacterial growth. All tests were performed in triplicate and were repeated three times.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Test Tube Biofilm Assay A loopful of cells from blood agar plate was transferred to a polystyrene culture tube containing 4 mL of sterile PBS solution, vortexed for 30 s and the turbidity of cell suspension was adjusted to 108 CFU/mL. Ten µL aliquots of each bacterial suspension were inoculated in polystyrene tube containing 1 ml TSB. After 24 h incubation of bacterial cultures at 35 °C, the remaining attached bacteria were washed with PBS and fixed with methanol. The tubes were stained with 2% (w/v) crystal violet, then rinsed with water and dried. The amount of biofilm biomass was quantified by destaining the biofilms with 1 ml of 33% acetic acid and then measuring the absorbance in an automated microtiter plate reader set at 570 nm.

Biofilm Inhibition Assay The most effective honey against wound planktonic bacteria was selected for further characterisation of bacterial biofilm inhibition. Biofilms were cultured in polystyrene tubes as described above, except that TSB broth was enriched with sub-inhibitory concentrations of honey (5, 10 or 15% of honey solution). The remaining attached bacteria were quantified as described in the previous section.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Anti-Biofilm Activity of Natural Honey against Wound Bacteria

93

Biofilm Killing Assay After incubation of wound bacteria in TSB medium for 24 h, biofilms were rinsed with sterile PBS and then treated with 1 ml of 40 and 50% honey solution for another 24 h. These concentrations of honey were chosen because they completely kill planktonic bacteria. The remaining attached bacteria were quantified as described above.

Statistics Analysis Results are presented as the mean with standard error (SEM). All data were statistically analysed from five independent experiments using one-way ANOVA to determine whether there were differences between treatments and control. P values smaller than 0.05 were considered to be significant. Analyses were performed using GraphPad Prism (GraphPad Software Inc., USA).

Results

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Antibiotic Susceptibility Testing Among all isolates, 32 clinical isolates (80%) were multi-drug resistant against 3 to 10 antibiotics. Meropenem and tetracycline resistance was found only in one and two isolates, respectively. Two isolates, namely Klebsiella oxytoca 2392/10 and P. aeruginosa 964/10, were sensitive to all antibiotics. It was observed that the prevalence of cephalosporin resistance, including cefepime, a fourth-generation cephalosporin, was high (42.5%) in the collected wound isolates. Antibiotic resistance profiles of all isolates are summarised in Table 1. Monitoring of the resistance to antibiotics of the bacteria in wound infections is important and valuable as it provides practical guidance of rational selection of antimicrobial agents for clinicians. Treatment of serious skin infections associated with deep chronic wound infection should include broad-spectrum coverage of aerobes and anaerobes. The use of topical application of antibiotics is not justified for the routine treatment of biofilm-associated infections. Topical antibiotics can provoke delayed hypersensitivity reactions [103], superinfections [104] and, most importantly, select for resistance. It has been suggested that resistance of bacteria in wound biofilms is not acquired via mutations or mobile genetic elements [105]. However, the polymicrobial nature of chronic wounds is likely to provide an appropriate environment for genetic exchange between bacteria. Indeed, the first two cases of vancomycin-resistant S. aureus in the USA were both isolated from chronic wound patients [106,107]. The use of topical antibiotics in chronic wound infections is not recommended and systemic antibiotics should only be used in the treatment of invasive tissue infections (e.g., osteomyelitis, cellulitis and abscess formation) due to the risk of developing antibiotic resistance.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

94

Juraj Majtan, Jana Bohova, Miroslava Horniackova et al.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Table 1. Incidence of antibiotic resistance in 40 wound clinical isolates Wound isolate Acinetobacter baumannii Acinetobacter calcoaceticus Citrobacter koseri Corynebacterium spp. Enterobacter cloacae Enterococcus faecalis Enterococcus spp. Enterococcus spp. Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Klebsiella oxytoca Klebsiella pneumoniae Klebsiella pneumoniae Morganella morganii Morganella morganii MRSA MRSA Proteus mirabilis Proteus mirabilis Proteus vulgaris Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas mendocina Streptococcus agalactiae Stenotrophomonas maltophilia Staphylococcus aureus Staphylococcus aureus coagul. neg. Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus

No. 2414/10 945/10 2392/10 2384/10 2383/10 2392/10 904/10 1007/10 2394/10 692/10 725/10 729/10 893/10 904/10 921/10 1041/10 2392/10 2384/10 2405/10 2383/10 2444/10 1758/10 1637/10 2460/10 719/10 2392/10 2460/10 2383/10 694/10 964/10 788/10 2384/10 2383/10 2421/10 2449/10 706/10 1004/10 1026/10 955/10 1034/10

Antibiogram AMC, ATM, C, CAZ, CIP, CRO, CXM, FEP, G, OFX AMC, ATM, C, CAZ, CIP, CRO, CXM, FEP, OFX AMC, CAZ, CXM, G CIP, OFX AMC, CXM C, CIP, G, OFX, OX, S, T AMC, AMP, CAR, CIP, OFL, OX, S, TMP C, G OFX, OX, S, T, TMP AMC, CIP, OFX AMC, ATM, CAZ, CIP, CRO, CXM, FEP, G, OFX AMC, CIP, CRO, CXM, FEP, G, OFX ATM, CAZ, CIP, CRO, CXM, OFX AMC, CAZ, CIP, CRO, CXM, FEP, OFX AMC, ATM, CIP, CRO, CXM, FEP, OFX, TZB AMC, ATM, CAZ, CIP, CRO, CXM, FEP, G, OFX AMC, ATM, CAZ, CIP, CRO, CXM, FEP, G, OFX nr AMC, ATM, C, CAZ, CIP, CRO, CXM, FEP, G, OFX C, CXM AMC, ATM, C, CAZ, CRO, CXM, G, OFX AMC, ATM, CXM AMC, AMP, CAR, CIP, OFX, OX, S AMP, CAR, S nr AMC, ATM, CAZ, CIP, CRO, CXM, FEP, G, OFX CXM AMC, C, CRO, CXM, OFX AMC, C, CXM AMC, C, CIP, CRO, CXM, OFX nr AMC, C, CIP, CRO, CXM, G, OFX AMC, CIP, OFX, S, TMP AMC, ATM, CAZ, CRO, CXM, MEM AMP, C, CAR, S AMC, AMP, CAR, OX AMP, CAR, S CIP, S AMC, AMP, C, CAR, S AMC, AMP, C, CAR, G, S S

MRSA – Methicillin-resistant Staphylococcus aureus; nr – non-resistant. AMC, amoxicillin/clavulanic acid; ATM, aztreonam; AMP, ampicillin; C, chloramphenicol; CAR, carbenicillin; CAZ, ceftazidime; CIP, ciprofloxacin; CRO, ceftriaxone; CXM, cefuroxime; FEP, cefepime; G, gentamicin; MEM, meropenem; OX, oxacillin; OFX, ofloxacin; S, streptomycin; T, tetracycline; TMP, trimethoprim; TZB, tazobactam.

Determination of MICs of Honeys The MIC values of the three natural honeys and artificial honey are shown in Table 2. The MICs for honeydew honey from Bardejov and Stara Voda ranged from 10 to 20% and 15 to above 25%, respectively, while those for active manuka honey ranged from 12.5% to

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Anti-Biofilm Activity of Natural Honey against Wound Bacteria

95

above 25%. Honeydew honey (Bardejov) had lower MIC values than manuka and honeydew honey (Stara Voda) against 24 of the tested wound isolates, where the MIC values of honeydew honey were below 12.5% in 11 isolates. In three isolates, manuka honey had lower MIC values compared to both honeydew honeys. Artificial honey inhibited the growth of all isolates at a concentration of 50% and, in some cases, at concentrations higher than 50%.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Table 2. Minimum inhibitory concentration (%) of different honeys toward wound clinical isolates

Wound isolate (n=40) Acinetobacter baumannii Acinetobacter calcoaceticus Citrobacter koseri Corynebacterium spp. Enterobacter cloacae Enterococcus faecalis Enterococcus spp. Enterococcus spp. Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Klebsiella oxytoca Klebsiella pneumoniae Klebsiella pneumoniae Morganella morganii Morganella morganii MRSA MRSA Proteus mirabilis Proteus mirabilis Proteus vulgaris Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas mendocina Streptococcus agalactiae Stenotrophomonas maltophilia Staphylococcus aureus Staphylococcus aureus coagul. neg. Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus

No. 2414/10 945/10 2392/10 2384/10 2383/10 2392/10 904/10 1007/10 2394/10 692/10 725/10 729/10 893/10 904/10 921/10 1041/10 2392/10 2384/10 2405/10 2383/10 2444/10 1758/10 1637/10 2460/10 719/10 2392/10 2460/10 2383/10 694/10 964/10 788/10 2384/10 2383/10 2421/10 2449/10 706/10 1004/10 1026/10 955/10 1034/10

Type of honey Honeydew (Bardejov) 20 20 20 15 25 20 20 20 15 20 15 15 12.5 12.5 25 20 15 25 20 12.5 12.5 10 10 20 12.5 20 20 15 15 20 12.5 20 20 20 20 12.5 10 10 20 15

Honeydew (Stara Voda) > 25 25 > 25 20 > 25 > 25 25 25 25 25 25 20 20 20 > 25 25 25 > 25 > 25 20 20 15 15 > 25 20 20 25 25 20 25 20 25 25 15 25 20 25 20 25 20

Manuka (UMF 15+) 20 20 25 15 25 20 20 25 25 25 25 20 20 20 20 20 20 25 25 20 20 20 20 > 25 25 25 20 25 25 20 20 20 12.5 > 25 20 15 20 20 15 20

MRSA – Methicillin-resistant Staphylococcus aureus.

Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

96

Juraj Majtan, Jana Bohova, Miroslava Horniackova et al.

These results are in accordance with our previous research [108], where honeydew honey (Bardejov) was more efficient than manuka honey (UMF 15+) against multi-drug resistant Stenotrophomonas maltophilia clinical isolates. In a recent study by Tan et al. [109], the antibacterial activity of Malaysian tualang honey and manuka honey (UMF 10+) was tested against wound clinical isolates. The MIC values ranged between 10% and 25% for both honeys. However, manuka honey exhibited better activity against wound isolates. Newly identified honeys have advantages over or similarities with manuka honey due to enhanced antimicrobial activity, local production and/or greater selectivity against medically important pathogens [57]. In addition to manuka honey, honeydew honey from Slovakia [108], tualang honey from Malaysia [109-111] and ulmo honey from Chile [112] could be used as a possible alternative therapy for wound healing. However, it would be very important to compare effectiveness of these types of honey in randomised controlled clinical trials recruiting participants with chronic wounds or burns.

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

Biofilm Formation by Wound Isolates All collected wound clinical strains were tested for their ability to form biofilms in polystyrene culture tubes. The capacity of each isolate to develop biofilm was not uniform (Figure 2). Interestingly, isolates of S. aureus, including MRSA strains, as well as P. aeruginosa isolates were only weak biofilm producers. Several other groups have also examined the ability of clinical isolates of MRSA to form biofilm [113,114]. O‘Neill et al. [114] studied biofilm formation in 114 clinical isolates of MRSA and found that only 9% had the ability to form fully established biofilm. In another study [113], it has been demonstrated that only 20.5% of all 763 MRSA isolates produced fully established biofilm on a polystyrene platform. These results showed that not every bacterial species is able to form biofilm, suggesting that clinical biofilm can consist of single bacterial species even though the infection is polymicrobial.

Figure 2. Biofilm formation by wound clinical isolates. Data represent mean ± SEM from five independent experiments. Honey: Current Research and Clinical Applications : Current Research and Clinical Applications, Nova Science Publishers, Incorporated, 2012.

Anti-Biofilm Activity of Natural Honey against Wound Bacteria

97

In this chapter, we found that clinical isolates of Proteus spp., Enterobacter cloacae, Stenotrophomonas maltophilia and E. coli were the most effective in forming biofilms on a plastic surface. Among all strong biofilm producers, Proteus mirabilis 719/10, Enterobacter cloacae 2383/10 and E. coli 893/10 were selected for further study.

Honey and Inhibition of Biofilm Formation by Wound Isolates

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

The most logical approach to prevent bacterial biofilm formation is by inhibiting the initial binding of the bacterium to the tissue or biomaterial. In this chapter, honeydew honey (Bardejov) was found to be the most effective honey against wound clinical isolates and was selected for characterisation of possible anti-biofilm activity. The best three biofilm producers Proteus mirabilis 719/10, Enterobacter cloacae 2383/10 and E. coli 893/10 were cultured with 5, 10 and 15% of sub-MICs of honeydew honey. It was found that honeydew honey at 10% of its sub-MIC value was able to inhibit the binding of Proteus mirabilis bacterial cells to the plastic surface (Figure 3). A statistically significant inhibition of biofilm formation was even more notable in the case of E. coli, where honeydew honey at 5 and 10% of sub-MICs prevented biofilm formation (P