The Pennsylvania State University. The Graduate School. Department of Food Science EFFECT OF THIOL-QUINONE REACTIONS ON

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1 The Pennsylvania State University The Graduate School Department of Food Science EFFECT OF THIOL-QUINONE REACTIONS ON POLYPHENOL AND LIPID INSTABILITY IN FOODS A Thesis in Food Science by Nausheel R. Unnadkat 2011 Nausheel R. Unnadkat Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science December 2011

2 The thesis of Nausheel R. Unnadkat was reviewed and approved* by the following: Ryan J. Elias Assistant Professor of Food Science Thesis Advisor Joshua D. Lambert Assistant Professor of Food Science John N. Coupland Professor of Food Science John D. Floros Professor of Food Science Head of the Department of Food Science *Signatures are on file in the Graduate School ii

3 ABSTRACT Polyphenols are attractive ingredients due to their purported health benefits, but their addition to foods is limited by their chemical instability, as they are rapidly oxidized under many conditions. This oxidation not only compromises the potential biological activity of the phenolic compound, but can also affect the chemical stability of the surrounding food matrix. Polyphenols bearing catechol or gallate groups, when oxidized to their benzoquinone forms, are strong electrophiles capable of reacting with nucleophilic thiols via 1,4-Michael addition reactions. These reactions are known to proceed in foods during processing and storage, yet little is known about the consequences of said reactions on polyphenol stability and lipid oxidation reactions. We hypothesize that thiols in foods accelerate catechol oxidation by capturing quinones via their sulfhydryl groups, thereby increasing hydrogen peroxide (H 2 O 2 ) production rates and, consequently, lipid oxidation rates. However, we also hypothesize that, browning reactions due to nonenzymatic polyphenol oxidation will be inhibited in the presence of thiols, as these compounds are expected to disrupt phenolic polymerization reactions. The stability of (-)-epigallocatechin gallate (EGCG) in the presence of several thiol-containing species [cysteine (Cys), glutathione (GSH), 3-mercaptohexan-1-ol (3SH), β-lactoglobulin (β-lg)] was followed in both aqueous and dispersed lipid systems. The test thiols were observed to inhibit browning, presumably by interfering with EGCG polymerization, although both cysteine and glutathione increased the rate of EGCG oxidation and H 2 O 2 production. EGCG oxidation and H 2 O 2 formation rates were lower in iii

4 the presence of β-lg compared to protein-free controls; however, EGCG was significantly more stable after β-lg s Cys residues were blocked with N-ethylmaleimide. Despite the observed increase in H 2 O 2 production rate in the presence of the thiols, a net antioxidant effect in was observed in emulsions. We attributed this antioxidant effect to the quenching of hydroxyl and lipid-derived radicals by EGCG and, possibly, the thiols sulfhydryl groups. These results suggest that thiols in foods accelerate polyphenol oxidation reactions and, in turn H 2 O 2 generation rates; however, this does not appear to compromise the oxidative stability of food lipids. iv

5 TABLE OF CONTENTS LIST OF FIGURES... ix LIST OF TABLES... xiv ACKNOWLEDGMENTS... xv Chapter 1: Literature Review Dietary Phenolics Structural Classification Sources of Polyphenols Lipid Oxidation Reactions in Foods Antioxidant Mechanisms of Flavonoids Radical Scavenging Activity of Polyphenols Metal Chelation Properties of Flavonoids Prooxidant Mechanisms of Flavonoids Stability of Polyphenols in Foods ph Effects on Polyphenol Stability Role of Transition Metals Reaction of Benzoquinones with Nucleophiles Reaction of Benzoquinones with Low Molecular Weight Thiols Reaction of Benzoquinones with Proteins Antioxidant Activity of Proteins in Presence of Benzoquinones Purpose and Significance Purpose v

6 1.4.1 Significance Hypothesis and Objectives Chapter 2: Materials and Methods Reactant Sources, Preparation and Analysis Reactant Sources Preparation of Cys-Blocked β-lactoglobulin Quantification of Sulfhydryl Groups Quantification of Residual NEM in Cys-Blocked β-lactoglobulin Kinetic Studies of Thiol-Quinone Reaction in Model Aqueous Solutions Sample Preparation and Storage Analysis of Samples Determination of Dissolved Oxygen Concentration Hydrogen Peroxide Analysis Residual EGCG Concentration Hydrogen Peroxide Analysis Residual Free Sulfhydryl Concentration Thiol-EGCG Adduct Formation Oxygen Radical Absorbance Capacity for Free LMW Thiols Effect of Polyphenol Oxidation on Lipid Oxidation in Model Food Emulsion Emulsion Preparation Sample Preparation and Emulsion Oxidation Analysis of Samples vi

7 Hydrogen Peroxide Analysis Residual EGCG Concentration Lipid Hydroperoxyl Radical Formation Lipid Hydroperoxide Analysis Thiobarbituric Acid Reactive Substances (TBARS) Statistical Analysis Chapter 3: Results and Discussion Quantifying Free Sulfhydryl Content Determination of Trace NEM in Cys-blocked β-lg Kinetic Studies of Thiol-Quinone Reaction in Model Aqueous Solutions Determination of Dissolved Oxygen Concentration Determination of Residual EGCG Concentration Hydrogen Peroxide Analysis Measurement of Non-Enzymatic Browning Residual Free Sulfhydryl Concentration Thiol-EGCG Adduct Formation Oxygen Radical Absorbance Capacity for Free LMW Thiols Thiol-Quinone Reactions in Model Food Emulsions and their Influence on Lipid Oxidation Reactions Residual EGCG Concentration Lipid Hydroperoxide Analysis Measurement of Thiobarbituric Acid Reactive Substances (TBARS) vii

8 Chapter 4: Conclusions and Future Studies Conclusions Future Studies Chapter 5: Appendix External Standard Curve for Low Molecular Weight Thiol Sources Hydrogen Peroxide Analysis in Emulsions Analysis of Lipid Radicals Bibliography viii

9 LIST OF FIGURES Figure 1-1. Structural classification of phenolics into 3-groups... 2 Figure 1-2: Structures of common flavanol and flavonols... 5 Figure 1-3. Proposed mechanism of polyphenol mediated lipid oxidation Figure 1-4. Proposed catalytic action of iron and copper in oxidation of catechols forming quinone and hydrogen peroxide Figure 1-5. Proposed overall mechanism of thiol-quinone covalent adduct formation 28 Figure 1-6. (a) Electrophilic sites of attack. Mechanism of (b) 1,2 Michael addition reaction and (c) 1,4 Michael addition reaction (with importance to thiol-quinone covalent adducts) Figure 1-7. Proposed Mechanism for Reduction of the Thiol-Quinone Adduct with Hydride Ions Resulting in Release of a Thiophenol from the Protein (Pr) Backbone Figure 3-1. Structures of the low molecular weight thiols used for study of thiol-quinone reaction Figure 3-2. Consumption of Dissolved oxygen in aqueous ph 4 buffered system for reaction between EGCG and low molecular weight thiols (Cys, GSH and 3SH) at 24⁰C. A control, with only EGCG, was used without any thiol reactant Figure 3-3. Consumption of Dissolved oxygen in aqueous ph 7 buffered system for reaction between EGCG and low molecular weight thiols (Cys, GSH, 3SH) at 24⁰C.. 82 Figure 3-4. Consumption of Dissolved oxygen in aqueous ph 7 buffered system for reaction between EGCG and proteins (β-lg and Cys-blocked β-lg or NEM-β-lg) at 24⁰C Figure 3-5. Structures of 4-methylcatechol and EGCG showing the o-dihydroxy positions Figure 3-6. Kinetics of consumption of EGCG in aqueous ph 4 buffered system for reaction between EGCG and low molecular weight thiols (Cys, GSH, 3SH) at 24⁰C.. 87 Figure 3-7. Kinetics of consumption of EGCG in aqueous ph 7 buffered system for reaction between EGCG and low molecular weight thiols (Cys, GSH, 3SH) at 24⁰C.. 89 ix

10 Figure 3-8. Consumption of EGCG in aqueous ph 7 buffered system for reaction between EGCG and protein (native control and NEM-β-lg) at 24⁰C Figure 3-9. Hydrogen peroxide generation kinetics in aqueous ph 4 buffered system for reaction between EGCG and low molecular weight thiols (Cys, GSH, 3SH) at 24⁰C.. 94 Figure Hydrogen peroxide generation kinetics in aqueous ph 7 buffered system for reaction between EGCG and low molecular weight thiols (Cys, GSH, 3SH) at 24⁰C.. 96 Figure Hydrogen peroxide generation kinetics in aqueous ph 7 buffered system for reaction between EGCG and proteins (native control and NEM-β-lg) Figure Color formation (measured by absorbance at 420 nm) in aqueous ph 4 buffered system containing EGCG and low molecular weight thiols (Cys, GSH, 3SH) Figure Superimposed wave-scans of absorbance of treatments containing only EGCG (broken line) and EGCG with Cys (solid line) Figure Proposed scheme showing quinone oligomerization and covalent adduct formation with a thiol Figure Color formation (measured by absorbance at 420 nm) in aqueous ph 7 buffered system containing EGCG and low molecular weight thiols (Cys, GSH, 3SH) Figure Color formation (measured by absorbance at 420 nm) in aqueous ph 7 buffered system containing EGCG and proteins (β-lg and NEM-β-lg) Figure Residual thiol concentration in aqueous ph 4 buffered system containing EGCG and low molecular weight thiols (Cys, GSH, 3SH) Figure Residual thiol concentration in aqueous ph 4 buffered system containing the controls- low molecular weight thiols (Cys, GSH, 3SH) Figure Residual thiol concentration in aqueous ph 7 buffered system containing EGCG and low molecular weight thiols (Cys, GSH, 3SH) Figure Residual thiol concentration in aqueous ph 7 buffered system containing controls- low molecular weight thiols (Cys, GSH, 3SH) Figure Sample SIR mass spectra showing corresponding peaks for adducts for the treatments containing EGCG and individual thiols x

11 Figure Thiol-EGCG adduct formation in aqueous ph 4 buffered system containing EGCG and low molecular weight thiols (Cys, GSH, 3SH) Figure Thiol-EGCG adduct formation in aqueous ph 7 buffered system containing EGCG and low molecular weight thiols (Cys, GSH, 3SH) Figure Proposed mechanism of peroxide quenching by 3SH Figure Oxygen radical absorbance capacity of thiols without any added EGCG. Area under the curve (AUC) of relative fluorescence values (F/F 0 ) vs. time Figure Oxygen radical absorbance capacity of thiols without any added EGCG. Changes in relative fluorescent intensity of fluorescein (Em, 485 nm; Ex, 520 nm) in the presence of AAPH and 100 µm thiol at 37 C. Fluorescence values (F) are given relative to the initial time values (F 0 ). The blank was prepared without any antioxidant, and the control was prepared without AAPH or antioxidant Figure EGCG depletion in 5% Tween-80 stabilized o/w emulsion in ph mm phosphate buffer containing EGCG and LMW thiols (Cys, GSH, 3SH), at 45⁰C Figure 3-28: Proposed mechanism of polyphenol mediated lipid oxidation, showing mechanisms for lipid hydroperoxyl, alkyl and alkoxyl radicals scavenging by the thiols and EGCG Figure EGCG depletion in 5% Tween-80 stabilized o/w emulsion in ph mm phosphate buffer containing EGCG and LMW thiols (Cys, GSH, 3SH), at 45⁰C Figure EGCG depletion in 5% Tween-80 stabilized o/w emulsion in ph mm phosphate buffer containing EGCG and proteins (β-lg or NEM-β-lg), at 45⁰C Figure Peroxide value in 5% Tween-80 stabilized o/w emulsion in ph mm phosphate buffer containing EGCG and Cys at 45⁰C Figure Peroxide value in 5% Tween-80 stabilized o/w emulsion in ph mm phosphate buffer containing EGCG and GSH at 45⁰C Figure Peroxide value in 5% Tween-80 stabilized o/w emulsion in ph mm phosphate buffer containing EGCG and 3SH at 45⁰C Figure Peroxide value in 5% Tween-80 stabilized o/w emulsion in ph mm phosphate buffer containing EGCG and Cys at 45⁰C Figure Peroxide value in 5% Tween-80 stabilized o/w emulsion in ph mm phosphate buffer containing EGCG and GSH at 45⁰C xi

12 Figure Peroxide value in 5% Tween-80 stabilized o/w emulsion in ph mm phosphate buffer containing EGCG and 3SH at 45⁰C Figure Peroxide value in 5% Tween-80 stabilized o/w emulsion in ph mm phosphate buffer containing EGCG and proteins (β-lg and NEM-β-lg) at 45⁰C Figure TBARS values in 5% flax seed o/w Tween-80 stabilized emulsion in ph mm phosphate buffer containing EGCG and Cys at 45⁰C Figure TBARS values in 5% flax seed o/w Tween-80 stabilized emulsion in ph mm phosphate buffer containing EGCG and GSH at 45⁰C Figure TBARS values in 5% flax seed o/w Tween-80 stabilized emulsion in ph mm phosphate buffer containing EGCG and 3SH at 45⁰C Figure TBARS values in 5% flax seed o/w Tween-80 stabilized emulsion in ph mm phosphate buffer containing EGCG and Cys at 45⁰C Figure TBARS values in 5% flax seed o/w Tween-80 stabilized emulsion in ph mm phosphate buffer containing EGCG and GSH at 45⁰C Figure TBARS values in 5% flax seed o/w Tween-80 stabilized emulsion in ph mm phosphate buffer containing EGCG and 3SH at 45⁰C Figure TBARS values in 5% flax seed o/w Tween-80 stabilized emulsion in ph mm phosphate buffer containing EGCG and Proteins (β-lg and NEM-β-lg) at 45⁰C Figure 5-1. H 2 O 2 generation in 5% Tween-80 stabilized o/w emulsion in ph mm phosphate buffer containing EGCG and low molecular weight thiols (Cys, GSH, 3SH), at 45⁰C Figure 5-2. H 2 O 2 generation in 5% Tween-80 stabilized o/w emulsion in ph mm phosphate buffer containing EGCG and proteins (β-lg or NEM-β-lg), at 45⁰C Figure 5-3. EPR Spectrum Intensity for PBN spin adducts of lipid hydroperoxyl radicals in 5% flax seed o/w Tween-80 stabilized emulsion in ph mm phosphate buffer containing EGCG and Cys at 45⁰C xii

13 Figure 5-4. EPR Spectrum Intensity for PBN spin adducts of lipid hydroperoxyl radicals in 5% flax seed o/w Tween-80 stabilized emulsion in ph mm phosphate buffer containing EGCG and GSH at 45⁰C Figure 5-5. EPR Spectrum Intensity for PBN spin adducts of lipid hydroperoxyl radicals in 5% flax seed o/w Tween-80 stabilized emulsion in ph mm phosphate buffer containing EGCG and 3SH at 45⁰C Figure 5-6. EPR Spectrum Intensity for PBN spin adducts of lipid hydroperoxyl radicals in 5% flax seed o/w Tween-80 stabilized emulsion in ph mm phosphate buffer containing EGCG and Cys at 45⁰C Figure 5-7. EPR Spectrum Intensity for PBN spin adducts of lipid hydroperoxyl radicals in 5% flax seed o/w Tween-80 stabilized emulsion in ph mm phosphate buffer containing EGCG and GSH at 45⁰C Figure 5-8. EPR Spectrum Intensity for PBN spin adducts of lipid hydroperoxyl radicals in 5% flax seed o/w Tween-80 stabilized emulsion in ph mm phosphate buffer containing EGCG and 3SH at 45⁰C Figure 5-9. EPR Spectrum Intensity for PBN spin adducts of lipid hydroperoxyl radicals in 5% flax seed o/w Tween-80 stabilized emulsion in ph mm phosphate buffer containing EGCG and proteins (β-lg and NEM-β-lg) at 45⁰C xiii

14 LIST OF TABLES Table 1-1. Dietary sources of flavonoids (adapted from Rice-Evans et al.)... 7 Table 2-1. Certificate of Analysis for native β lactoglobulin Table 2-1. β-lactoglobulin amino acid profile, expressed in moles per mole pure protein Table 5-1. External standard curve for low molecular weight thiol sources xiv

15 ACKNOWLEDGMENTS I would like to sincerely thank my advisor, Dr. Ryan Elias for his constant guidance, advice, patience and unconditional support in my Masters studies at Penn State. I am lucky to have such a helpful and expert advisor in the field of work, who always encouraged me and honed my research analytical and data interpretation skills. I would also like to express my gratitude to my other committee members, Dr. Joshua Lambert and Dr. John Coupland, for their time, guidance and laboratory facility support. I am very grateful for the tuition support and assistantship from Dr. Ryan Elias and the Department of Food Science. My research would not have been possible without the help and timely motivation from my lab mates. Thanks and love to my family members for their moral support and selfless love. I express heartfelt gratitude for my friends at the yoga club, Penn State Vedic Society, for showing sheer enthusiasm in their services/ activities and events, and also keeping my engaged throughout my stay at Penn State in extracurricular activities. They were really full of fun and joy. I really loved the association of all such good people listed above. Apart from academic learning, I learnt (and have yet to learn) a lot of good lessons and qualities that inspired me, from each and every individual I encountered. I look forward to getting association of such nice and caring people in future too. xv

16 Chapter 1: Literature Review 1.1 Dietary Phenolics Structural Classification Phenolic compounds represent the largest group of secondary metabolites in plant foods, with more than 6000 identified compounds. As a class, phenolics include a wide range of structures; however they all maintain at least one 6-member aromatic ring bearing one or more hydroxy substituents. With regard to their structure, the major groups of phenolic compounds are distinguished by a number of constitutive carbon atoms in conjunction with the basic phenolic skeleton. Those phenolics with most relevance to foods include the hydroxybenzoic acid derivatives (HBAs) with a general structure C 6 -C 1 (e.g. salicylic and gallic acids) (Figure 1-1). Variations in their basic structure include hydroxylations and methoxylations of the aromatic ring (1). The second group is that of those phenolic compounds having the general formula C 6 -C 3 (hydroxycinnamic acid derivatives), representing a series of trans-phenyl-3-propenoic acids differing in their ring substitution (1). The third and the largest group are represented by the flavonoids, with more than 4000 compounds characterized. They are also the most widespread and diverse, built upon a C 6 -C 3 -C 6 flavone skeleton, in which the three-carbon bridge between the phenyl groups is commonly cyclised with oxygen. Several sub-classes of flavonoid are differentiated on 1

17 the degree of unsaturation and degree of oxidation of the three-carbon segment (e.g. flavones, isoflavones, flavonols, flavanones, catechin, anthocyanins etc.). Within the various classes, further differentiation is possible, based on the number and nature of substituent groups attached to the rings (2, 3). Figure 1-1. Structural classification of phenolics into 3-groups Flavan-3-ols (often referred to as flavanols) are a class of flavonoids that possess the 2- phenyl-3,4-dihydro-2h-chromen-3-ol skeleton. These compounds include the catechins and the catechin gallates. Epigallocatechin and gallocatechin contain an additional phenolic hydroxyl group when compared to epicatechin and catechin (Figure 1-2), respectively, similar to the difference in pyrogallol compared to pyrocatechol. Catechin 2

18 gallates are gallic acid esters of the catechins; an example is epigallocatechin gallate (EGCG). Individual differences within each group result from the variation in number and arrangement of the hydroxyl groups as well as the alkylation by the gallate ring at the hydroxyl group of the C-ring. The most commonly occurring flavanols are those with dihydroxylation in the 4 and 5 positions of the B-ring, making them vicinal diol. (-)- Epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG) have an additional hydroxyl group at the 3 position making them vicinal triols. Flavonols represent yet another class, and possess hydroxyl groups at the 3 and/or 4 and/or 5 positions (Figure 1-2). Some common flavonols in foods include quercetin, myrecitin, rutin and kaempferol. The presence of hydroxyl groups on adjacent carbon atoms on the B-rings (and D-ring of digallates) is significant with respect to the redox chemistry of these flavonoids. The o-dior trihydroxy structures of these ring confers higher stability to the radical form and participates in electron delocalization (4). For example, the importance of the adjacency of the two hydroxyl groups in the ortho-diphenolic arrangement in the B ring of quercetin to its antioxidant activity (as hydrogen donating free radical scavengers) of 4.7 is revealed from a study of morin (3,5,7,2',4'-Pentahydroxyflavone) in which the dihydroxy groups are arranged meta to each other in the B ring, decreasing the value to [The assay that they used for the total antioxidant activity (TAA) or the Trolox equivalent antioxidant activity (TEAC) measured the concentration of Trolox solution with an equivalent antioxidant potential to a standard concentration of the compound under investigation. The TEAC reflects the ability of hydrogen-donating antioxidants to 3

19 scavenge the ABTS radical cation, absorbing in the near-ir region at 645, 734 and 815 nm compared with that of Trolox, the water-soluble vitamin E analog]. However, a related structure but with a lone 4'-OH group in the B ring, kaempferol, differing from quercetin in the absence of the 3 '-OH group from the B ring, has just 27% of the latter's antioxidant activity. When the B ring lacks the o-dihydroxy motif and contains only one hydroxy substituent, the monophenolic ring is significantly less effective hydrogen donor. In a study by Rice-Evans et al., the presence of a third OH group in the B ring did not enhance the effectiveness against aqueous phase radicals as in myricetin compared with quercetin (4). 4

20 Figure 1-2. Structures of common flavanol and flavonols 5

21 1.1.2 Sources of Polyphenols C 6 -C 1 group (hydroxybenzoic acid derivatives or HBAs) e.g. Generally, hydroxybenzoic acids (HBAs) are present as conjugates, although they can also be encountered as their free acids in some fruits (e.g. gallic acid in persimmons), after being released during fruit and vegetable maturation or food processing. A major source of gallic acid is green tea, where it exists as a part of the catechin structure such as ( )-epigallocatechin-3-gallate (EGCG) and ( )-epigallocatechin. C 6 -C 3 group (hydroxycinnamic acid derivatives or HCAs) Hydroxycinnamic acid derivatives (HCAs) are widely distributed as conjugates in plant material including many foods and beverages. The most common among them are caffeic (3,4-dihydroxycinnamic acid); ferulic, sinapic and p-coumaric acids. The best-known conjugate is 5-caffeoylquinic acid, commonly referred to as chlorogenic acid (CGA). Recent studies show that the human dietary intake of chlorogenic acids and other cinnamates ranges from 25 mg/day up to 1 g/ day depending upon the dietary constitution (5). The content of chlorogenic acid is relatively high in green coffee beans (6 10% dry basis). C 6 -C 3 -C 6 group (flavonoids) The main dietary sources of flavonoids include fruits, vegetables, and tea. A recent review indicates a daily intake of 6 60 mg/person/day (calculated based on the mean intake of flavonols of the German population calculated using data from the National German Food Consumption Survey) (6). The consumption of EGCG was estimated for 6

22 Japan to be as high as 1 g/ per day (7). The largest number of flavonoids is represented by the sub-group of flavonols and flavanols, usually found in plants bound to sugars as O- glycosides. The most important representatives of flavonols are kaempferol, quericitin and myrecitin and those of flavanols are (-)-epicatechin, (-)-epigallocatechin and their gallate esters. Sugars are dominantly bound to flavonoids via a β-glycosidic bond. Sugar molecules can bind to various positions in the parent flavonoid, although there is a preference for the C3-position (Figure-1-1). Aglycones of flavonols do not occur in fresh plants but may occur as a result of food processing. Table 1-1. Dietary sources of flavonoids (adapted from Rice-Evans et al.) (4) Phenolic Group Common Examples Food Sources Hydroxybenzoic acids (HBAs) (C 6 -C 1 ) Benzoic acid Gallic acid Vanillic acid Gum benzoin Great burnet, green and black teas Pennell Protocatechuic acid Cinnamon Phenyl propanoids or hydroxy cinnamic acids (HCAs) (C 6 -C 3 ) Flavonols (C 6 -C 3 -C 6 ) Ferulic acid Caffeic acid Chlorogenic acid Epicatechin, catechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate Wheat, corn, rice, tomatoes, spinach, cabbage White grapes, coffee, spinach, olives, cabbage Apples, pears, cherries, plums, peaches, anis Green and black teas Red wine Flavonols (C 6 -C 3 -C 6 ) Kaempferol Broccoli, radish, grapefruit, black tea, endive 7

23 Quercitin Myrecitin Lettuce, broccoli, apple skin, berries, olive Cranberry, grapes, red wine Flavanones (C 6 -C 3 -C 6 ) Naringin Peel of citrus fruits Flavones (C 6 -C 3 -C 6 ) Anthocyanidins (C 6 -C 3 -C 6 ) Chrysin Apigenin Malvidin Cyanidin Apigenidin Fruit skin Celery, parsley Red grapes, red wine Cheery, raspberry, strawberry, grapes Colored fruit and peels 8

24 1.2 Lipid Oxidation Reactions in Foods Lipid peroxyl radicals can be preformed during oil processing or can be formed in presence of a catalyst, reactive singlet oxygen which is formed by activation from transition metal ions. Activation of molecular oxygen to form singlet oxygen: Fe 2+ + O 2 Fe 3+ + O 2-1 O 2 Singlet oxygen can exist in five different configurations with the most common in foods being the 1 Δ state (where electrons exist in the same orbital). Because singlet oxygen is more electrophilic than triplet oxygen (triplet oxygen, 3 O 2, is a biradical), it can react directly with high electron density double bonds. Since the electrons in singlet oxygen match the spin direction of the electron in double bonds, it can react with an unsaturated fatty acid to directly form lipid hydroperoxides 1500 times faster than triplet oxygen (8). If I represent an unsaturated (mono or polyunsaturated) lipid molecule as LH, then the lipid autoxidation can be summarized as follows (8) LH + In* L (lipid alkyl radical) + H +...Initiation Step (Hydrogen Abstraction) Step 2: Isomerization Step (because of double bonds) L + 1 O 2 LOO + LH LOOH + Fe 2+ LOO (lipid hydroperoxyl radical)... Propagation Step LOOH (lipid hydroperoxide) + L.Propagation Step LO (alkoxyl radical) + Fe 3+ + OH LO Break down products (aldehydes, semialdehydes, acids, epoxides, alcohols) L + L L-L L + LOO LOOL Termination Steps 9

25 1.2.1 Antioxidant Mechanisms of Flavonoids Primary antioxidants donate hydrogen directly to lipid hydroperoxyl radicals and the resulting antioxidant radical does not react with substrate as they often have bulky groups and are relatively stable. The major (primary) antioxidants currently used in foods are monohydroxy or polyhydroxy phenol compounds with various ring substitutions. R + AH R + AH ROO + AH RH + A (inhibits at the reaction initiation step) RA ROOH + A (inhibits at the reaction propagation step) ROO + A ROOA RO + AH ROH + A (inhibits at the reaction termination step) RO + A ROA A A Antioxidant + O 2 Oxidized Antioxidant Secondary antioxidants work by non-radical mechanisms. These are usually of two subtypes, depending on their mechanism of action: metal chelators (e.g., ethylene diamine tetraacetic acid (EDTA), phosphoric acid, and polyphenols) and peroxide destroyers (reduce peroxides to stable alcohols) (e.g., methionines and Cys residues, phosphines). They are frequently referred to as hydroperoxide decomposers as they decompose hydroperoxides into non-radical, non-reactive, and thermally stable products. They are often used in combination with primary antioxidants to yield synergistic stabilization effects. 10

26 The protection of lipids against oxidative damage can be ascribed to (i) scavenging of hydroxyl, peroxyl, or synthetic radicals, (ii) termination of chain reactions in the lipid phase, involving peroxyl radicals and hydroperoxides, (iii) chelation of divalent cation metals used to initiate oxidative events, and/or (iv) interactions with other initiators, such as ascorbate, which may reduce and recycle the flavonoid radical. If a compound inhibits the formation of free alkyl radicals in the initiation step, or if the chemical compound interrupts the propagation of the free radical chain, the compound can delay the start or slow the lipid oxidation rates. The initiation of free radical formation can be delayed by the use of metal chelating agents, singlet oxygen inhibitors, and peroxide stabilizers. For a free radical scavenger to be an effective antioxidant in foods it must be more oxidatively labile than the unsaturated fatty acids and the resulting radical (on the protein chain) must not be powerful enough to promote lipid oxidation (2, 9). The activity of different types of antioxidants can vary significantly depending on whether the lipids are triacylglycerols, methyl esters, or free fatty acids. The location of antioxidants in aqueous, bulk lipid or in heterophasic systems has an important effect on their activity. Antioxidants that can promote metal reduction can alter the formation of lipid oxidation markers such that lipid hydroperoxides do not increase (due to their decomposition) while increasing the formation of secondary lipid oxidation products (e.g., propanal). In order for an antioxidant to be effective, it must be able to inhibit the formation of volatile secondary lipid oxidation products that are perceived as rancidity (10). 11

27 Radical Scavenging Activity of Polyphenols The antioxidant activity of flavonoids depends upon the arrangement of functional groups about the core structure. The specific arrangement of substituents (ortho/ meta/ para) is perhaps a greater determinant of antioxidant activity than the flavan backbone alone. Consistent with most polyphenolic antioxidants, both the configuration and total number of hydroxyl groups substantially influence several mechanisms of antioxidant activity. Free radical scavenging capacity is primarily attributed to the high reactivities of hydroxyl substituents that participate in the following reaction: A-OH + ROO A-O + ROOH where, A-OH represents polyphenol; A-O is the resulting antioxidant or polyphenolic free radical In order to function as an antioxidant, the phenolic compound must be capable of forming a resonance stabilized phenoxyl radical which, in turn, can react with other available radicals (e.g. lipid peroxyl radical) through a series of reactions, consequently deactivating them. The B-ring 3,4 -dihydroxyl configuration in flavanols is the most significant determinant of scavenging of reactive oxygen species and prevention of lipid peroxidation (11). This arrangement is a salient feature of the most potent scavengers of peroxyl and superoxide radicals. Hydroxyl groups on the B-ring have low activation energy to donate hydrogens to hydroxyl, peroxyl, lipid hydroperoxyl radicals, thus stabilizing them and giving rise to a relatively stable flavonoid radical. Thus they act as chain-breaking antioxidants. The antioxidant activity of polyphenols also stems from 12

28 their ability to donate electrons to the free radicals, thereby preventing them from propagating lipid peroxidation reactions. However, the reactivity of the phenoxyl radicals should remain relatively low, such that it should not promote the radical formation (e.g. by H-subtraction), from unsaturated fatty acids. Delocalization of unpaired electrons on the flavan skeleton (especially the B-ring) results in resonance stabilization of antioxidant radical. Among structurally homologous flavones and flavanones, their peroxyl and hydroxyl scavenging activity increases linearly and curvilinearly, respectively, according to the total number of OH groups (11). For example, the peroxyl radical scavenging ability of luteolin (which possesses two hydroxyl substituents at the 3,4 positions in the B-ring) substantially exceeds kaempferol (which possesses only one hydroxyl substituent at 3 position) (12). Oxidation of a flavonoid occurs on the B-ring when the catechol is present, yielding a fairly stable ortho-semiquinone radical (as will be discussed in details in section 1.3.1) through facilitating electron delocalization. Flavones lacking catechol or o-trihydroxyl (pyrogallol) systems form relatively unstable radicals and are weak radical scavengers. The significance of other hydroxyl configurations (e.g. the -OH groups on A- and C-rings) is less clear, but beyond increasing total number of hydroxyl groups, A-ring substitution correlates little with antioxidant activity (13). For flavonoid type polyphenols (e.g. EGCG), the gallate ester group has an important role in the antioxidant activity of the catechins. As shown in Figure 1-2, both the D-ring (3-OH) position of the gallate group as well as the B-ring (5`-OH) position of gallyl group can be potentially contribute to antioxidant activity. Catechins (including epicatechins) with three hydroxyl groups in 13

29 the B ring are gallocatechins and those esterified to gallic acid at the 3-OH group in the C ring are catechin gallates. There is no electron delocalization between the A and B rings due to the saturation of the heterocyclic ring; the antioxidant activity responds broadly to the tenet that the structures with the most hydroxyl groups exert the greatest antioxidant activity. The catechin-gallate esters reflect the contribution from gallic acid (3,4,5- trihydroxybenzoic acid). This structural advantage confers an enhancement of the TEAC value to 4.7 ± 0.10 mm. The catechin structure with a TEAC value of 2.4 ± 0.02 mm can be modified to enhance its antioxidant potential to 4.7 as in EGCG (4.75 ± 0.06 mm) by ester linkage via the 3-OH group to gallic acid and incorporation of an additional 5'-OH group in the B ring (Figure 1-2). The insertion of a third adjacent hydroxyl group in the B ring as in epigallocatechin (EGC) enhanced the antioxidant activity to 3.8 ± 0.06 mm (14). The antioxidant potentials of the tea catechins, on a molar basis, against oxygen radicals (e.g. superoxide or hydroperoxyl radicals) generated in the aqueous phase are, in order of decreasing effectiveness, epicatechin gallate ~ epigallocatechin gallate > epigallocatechin > gallic acid > epicatechin catechin. Free radical scavenging by flavonoids is also highly dependent on the presence of a free OH group at the 3-position on the B-ring. The flavonoid heterocycle contributes to antioxidant activity by (i) the presence of a free 3-OH, and (ii) permitting conjugation between the aromatic rings. The closed C-ring itself may not be critical to the activity of flavonoids (15). The torsion angle of the B-ring with respect to the rest of the molecule strongly influences free radical scavenging ability. Flavonols and flavanols with a 3-OH are planar (e.g. quercetin), while the flavones and flavanones, which lack this feature, are 14

30 slightly twisted (Figure 1-2). Planarity permits conjugation, electron dislocation, and a corresponding increase in flavonoid phenoxyl radical stability. It is postulated that B-ring hydroxyl groups form hydrogen bonds with the 3-OH, aligning the B-ring with the heterocycle and the A-ring. Eliminating this hydrogen bond causes a minor twist of the B-ring, compromising its electron delocalization capacity. Due to this intramolecular hydrogen bonding, the influence of a 3-OH is potentiated by the presence of a 3,4 - catechol, explaining the potent antioxidant activity of flavan-3-ols and flavon-3-ols that possess the latter feature (12). The physical environment can change the partitioning of the phenolic compounds into the emulsifier (interface) of an emulsion. Antioxidant activity is strongly affected by the physical composition of the test system, partly due to partitioning of the antioxidants between the phases being important, and the relative activity of antioxidants of different polarity varies significantly in different multiphase systems (16). Polyphenol hydrophobicity and partitioning is another large factor involved in determining the polyphenol s reactivity in inhibiting lipid oxidation (17, 18). Polyphenols partitioned into the lipid may not readily undergo oxidation by metals in the aqueous phase and may better act as antioxidants for the lipid, but antioxidant activity resulting from metal chelation may be lost when polyphenols are within the lipid droplets (19). 15

31 Metal Chelation Properties of Flavonoids Free ferrous iron is quite sensitive to oxygen and easily oxidizes to its ferric state. This oxidation is coupled to the reduction of dioxygen to superoxide. Further reduction of superoxide, or its protonated form the hydroperoxyl radical, to hydrogen peroxide is facilitated by abstraction of a hydrogen from a flavonoid. The reaction of ferrous iron with hydrogen peroxide generates the hydroxyl radical (i.e., the Fenton reaction), which is capable of promoting the oxidation of surrounding lipids. Hydroxyl radicals are the most reactive oxygen radical species known, and are thought to react with organic material as diffusion-controlled rates. In food systems containing high concentrations of transition metals (i.e., iron, copper), Fenton chemistry is predicted to be an important generator of ROS in foods; however, few studies have directly investigated this. Fenton-induced oxidation has been shown to be strongly inhibited by flavonoids with 3,4 -catechol, 4-oxo, and 5-OH arrangements (20). Chelating complexes with divalent cations may form between the 5-OH and 4-oxo group, or between the 3, 4 and/ or 5 -hydroxyl groups. Chelation of a divalent cation does not necessarily render the flavonoid inactive, as the complex retains ROS scavenging activity. By virtue of both metal-chelating properties and radical scavenging ability, polyhydroxylated flavonoids may offer considerable benefit as inhibitors of the Fenton reaction in foods such as wine. EGCG is known to preferentially bind transition metal ions (e.g., ferric ions; Fe 3+ ) with a 1:2 metal:ligand ratio (21), or a 2:1 metal:ligand ratio when examined with a molar excess (pseudo first order conditions) of Fe 3+, with 16

32 complexation occurring at both B- and D-rings (22). That these polyphenols are often more effective inhibitors of metal-induced oxidation compared to non-metal induced oxidation, lends support to the role of metal chelation in flavonoid inhibition of free radical damage. 17

33 1.2.2 Prooxidant Mechanisms of Flavonoids Whether tea catechins act as antioxidants or prooxidants appears to be dependent on the method to evaluate oxidation and the lipid used in the test system. EPR has shown that all green tea polyphenols can undergo auto-oxidation at alkaline (ph 13) conditions, which leads to oxidation of the B ring (23). Similar oxidative reactions have also been shown to occur at physiological ph (7.4) (24). When EGCG and EGC react with H 2 O 2, the A-ring of both compounds undergo oxidation followed by decarboxylation to form two oxidation products of EGCG and one oxidation product of EGC (25). This is a prooxidant effect as such reactions produce the hydroxyl radical in the presence of iron or copper (Fenton). Prooxidant activity is directly proportional to the total number of hydroxyl groups. In a report by Hanasaki and colleagues, a series of mono- and dihydroxy flavonoids demonstrated no detectable prooxidant activity, while multiple hydroxyl groups, especially in the B-ring, significantly increased production of hydroxyl radicals in a Fenton system. The latter compounds included myricetin and baicelein, both of which have a pyrogallol structure in the A-ring, which has also been reported to promote hydrogen peroxide production from which Fenton reaction may generate highly reactive hydroxyl radicals (26). There is also evidence that the unsaturated 2,3-bond and 4-oxo arrangement of flavones may promote the formation of ROS induced by divalent copper in the presence of oxygen (11). Collectively, this suggests that some of the same structural attributes that optimize antioxidant capacity may also exacerbate damage to unsaturated lipid molecules. However, considering the role of flavonoid radical stability 18

34 in prooxidant behavior postulated by Bors and colleagues, structural advantages to radical stability that increase antioxidant activity, such as a 3,4 -catechol, 3-OH, and conjugation between the A- and B-rings, may modulate the adverse pro-oxidative effects of flavonoids (27). Divalent iron or copper cations increase the production of free radicals in a concentration-dependent manner. While the presence of iron may accelerate prooxidant effects, chelation of iron by the flavonoid may theoretically modify this process. In addition, high ascorbate concentrations attenuate generation of ROS by flavonoids and it is postulated that vitamin C status modulates the prooxidant tendency of these compounds (28). Figure 1-3. Proposed mechanism of polyphenol mediated lipid oxidation a (29) a For simplicity, oxidation of catechol is depicted 19

35 1.3 Stability of Polyphenols in Foods Most dietary polyphenols, including EGCG, are readily oxidized in foods, especially at neutral and alkaline ph values. With respect to the chemical reactivity of phenolic compounds with two ortho hydroxyl groups, two reaction steps are involved. The first reaction step consists of the oxidation of the o-diphenols (B- or D galloyl ring of EGCG) into o-quinones via a semiquinone intermediate. The phenolic compounds are prone to both enzymatic and non-enzymatic (metal-catalyzed) oxidation in the presence of oxygen (10). Enzymatic oxidation reaction consumes oxygen and is catalyzed by the enzymes o- diphenolase or polyphenol oxidase (PPO) (catecholase activity). Similarly the laccases (E.C ) oxidize o-diphenols as well as p-diphenols forming their corresponding quinones. Flavonoids with a catechols structural motif undergo oxidation in their B ring. Catechins with both catechol and gallate moieties, can undergo oxidation both in their B and D rings. Non enzymatic or metal-catalyzed oxidation takes place in slightly alkaline conditions whereby polyphenols are easily oxidized in presence of oxygen, as shown in (10). Phenolic compounds are highly reactive towards oxygen and more precisely with reactive species of oxygen (ROS), which are the activated oxygen species formed during O 2 reduction in the presence of metals. The formation of those species requires the addition of four electrons to O 2 as shown in the schematic below: 20

36 O 2 +e, H + HO 2 HO 2 +e, H + H 2 O 2 H 2 O 2 +e, H + OH (+H 2 O) OH (+H 2 O) + e, H + (2) H 2 O It is proposed that oxygen and the catechol do not interact directly, but do so via redox active transition metals (e.g. Fe, Cu), where these metals perform an essential catalytic function (30). These reactions (Figure 1-4 and 1-5), occur concomitantly with the reduction of oxygen to ROS, namely superoxide, or its protonated version (hydroperoxyl radicals) and, eventually, H 2 O 2 (31-33). The process is initiated by the coordination of the catechol with Fe(III) (Figure 1-5). The complex is unstable and electron transfer within the complex results in the formation of Fe(II) and the semiquinone radical, which dissociates. The semiquinone may then be oxidized by Fe(III) or disproportionate to produce the benzoquinone, while the Fe(II) is reoxidized to the ferric state by oxygen to continue its catalytic function. As a result of the latter interaction, oxygen is reduced first to the hydroperoxyl radical and then to hydrogen peroxide, probably by the catechol (32). This latter reaction is classically referred to as the Fenton reaction, and results in the production of highly oxidizing hydroxyl radicals ( OH) in foods due to the ubiquity of trace levels of iron and copper. For the case of EGCG, oxidation can occur at the B- or the D-ring while concomitantly producing ROS such as superoxide (O 2 ) and hydrogen peroxide (H 2 O 2 ). 21

37 Figure 1-4. Proposed catalytic action of iron and copper in oxidation of catechols forming quinone and hydrogen peroxide (34) 22

38 1.3.1 ph Effects on Polyphenol Stability The Fenton reaction leads to the formation of free superoxide (O 2 ) and peroxide (O 2 2 ) radicals before eventually producing hydroxyl radicals. These are better oxidants than O 2 and may be directly reduced by phenolic molecules (Figure 1-5; reactions 1 3) (35). At low ph, the hydroperoxyl radical dominates, which is more reactive than its deprotonated counterpart (i.e., superoxide, which dominates at higher ph). The pka of hydroperoxyl radical is 4.88 so that at neutral ph 7 the vast majority of superoxide is in the anionic form, O 2 (36). Hence, the oxidation of phenolic compounds in the presence of trace amounts of transition metals, initiates a cascade of chemical transformations (Figure 1-5; reactions 4 5). Semiquinone radicals and quinones, both catechol derivatives, are formed, on the one hand, while oxygen is reduced to hydrogen peroxide in the presence of transition metal ions, such as Fe 2+ (Figure 1-5; reaction 1-5) (35) and, on the other hand, by direct oxidation by Fe 3+ under acidic conditions (Figure 1-5; reaction 5). The acidic conditions are important in coordinating preferentially to ferric ions to reduce the formal reduction potential of the Fe 3+ / Fe 2+ couple (35). This will enable the redox cycling of Fe 3+ to Fe 2+ and hence polyphenol oxidation can proceed. At neutral ph (ph 7), H 2 O 2, by itself, is not a potent oxidant (E 0 = 320 mv at ph 7.0 for the H 2 O 2, H + / H 2 O, OH couple), however it is easily reduced to yield highly reactive hydroxyl radicals (E 0 = 2310 mv at ph 7.0 for the HO, H + / H 2 O couple) by transition metal catalysts. Formation of hydroxyl radicals drives the oxidation of Fe 2+ to Fe 3+, which eventually accelerates catechol oxidation to form semiquinone. 23

39 A change in ph alters the charge of the polyphenolic antioxidants and might also alter the charge of the emulsifier depending on its character (anionic, cationic, or nonionic). These changes may influence the location of the antioxidants because of repulsive or attractive forces between antioxidant and emulsifier and thereby also influence the efficacy of the antioxidants in an emulsion. Hagerman et al. studied the radical chemistry of the plant polyphenolics, epigallocatechin gallate (EGCG) and epigallocatechin (EGC) using electron paramagnetic resonance spectroscopy and observed that radical species are formed spontaneously in aqueous solutions at low ph without external oxidant. The spectra of the (added) Zn(II) stabilized EGCG radicals were mainly assigned to the gallyl radical and the anion gallyl radical, with only 10% of the signal assigned to a radical from the galloyl ester. Using spectral simulations, they established a pka of 4.8 for the EGCG radical. The electrochemical redox potentials of EGCG varied from 1000mV at ph 3 to 400mV at ph 8. Moreover, the polyphenolics did not produce hydroxyl radicals unless reduced metal ions such as Fe(II) were added to the system (37). A study with caffeic acid showed that the rate of oxidation increased with ph (range 4 to 8) such that a good correlation was found between ph and phenolate ion concentration. Therefore, it was proposed that the reaction was initiated by single electron transfer from phenolate ions directly to oxygen, producing the semiquinone and superoxide, which then reacted further to yield a quinone and hydrogen peroxide (38). It was previously shown that the rate of polyphenol oxidation accelerates with increasing ph (39). However, the reduction potentials of the O 2 / H 2 O 2 and quinone/ polyphenol redox couples have the same ph dependency, each being reduced by 59 mv per unit increase in ph. Danilewicz et al. suggested that the increase in the rate of oxidation is due to the much reduced stability of 24

40 the quinone, which by decomposing with the increasing rapidity as ph increases, draws the reaction forward by displacing the redox process. For example: at ph 4, Cys allows EGCG to react with O 2 at ph 3.6 by adding to quinone; however at ph 7, its effect on reaction rate is much reduced and it was proposed that the rate of quinone decomposition is comparable to the rate of thiol addition (40). 25

41 1.3.2 Role of Transition Metals In a previous study, the oxidation of ethanol by hydrogen peroxide was found to be dependent on metals, such as iron and copper, to generate hydroxyl radicals by way of the Fenton reaction (as shown in reaction 3 in Figure 1-5). The initial oxidation of the polyphenols, which was previously conducted under forcing conditions (pure oxygen at 55 C over 35 days) to achieve an adequate reaction rate at ph 3.5, was understood to also have possibly been mediated by trace quantities of these metals (35). Recent studies in model and real wine have showed that the rate of oxidation of (+)- catechin increases with iron concentration (41). However, a decrease in (+)-catechin concentration is also observed without added iron, and since some products differed from those observed with added iron, an iron-independent degradation pathway was proposed. One of these products had spectral properties similar to those of the (+)-catechin oxidation product, dehydrodicatechin A, which indicated that the iron-free reaction involved oxidation with formation of the quinone. However, as noted above, trace metal impurities may still mediate this proposed iron-free process. Some studies have demonstrated the existence of a metal-independent pathway, but it remains to be seen whether or not this can contribute significantly to polyphenol oxidation under wine conditions (42). Furthermore, Danilewicz et al. provides strong evidence that iron is an essential catalyst in oxidative processes and that its action is greatly influenced by the presence of other transition metals, such as copper. 26