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] ~|||L'{I] [~ i[1[:]1 reviews properties of phenolic compounas Catherine A° R,ceoEvans, N,cho,as J° @,,,er and George Paganga

There is currently much interest in as bioactive components of . The roles of fruit, vegetables and red wine in disease prevention have been attrib- uted, in part, to the antioxidant properties of their constituent ( E and C, and the ). Recent studies have shown that many dietary polyphenolic constituents derived from plants are more effective than vitamins E or C, and thus might contribute significantly to the protective effects . It is now possible to establish the antioxidant activities of plant- derived in the aqueous and lipophilic phases, and to assess the extent to which the total antioxidant potentials of wine and tea can be accounted for by the activities of individual polyphenols.

tudies on the free -scavenging properties of properties, including: vitamins C and E; and flavonoids have permitted characterization of the ma- other micronutrients; carotenoids; ; S jor phenolic components of naturally occurring phyto- allium compounds; glucosinolates and ; dithiol- chemicals as antioxidants. Furthermore, the commercial thiones; isothiocyanates; protease inhibitors; fibre; and development of plants as sources of antioxidants that can folic acid. These compounds may act independently or in be used to enhance the properties of , for both nu- combination as anti- or cardioprotective agents by a tritional purposes and for preservation, is currently of variety of mechanisms. One such protective mechanism, major interest. Numerous epidemiological surveys have attributed to vitamins C and E and the carotenoids, is shown an inverse relationship between the intake of fruit, antioxidant (radical-scavenging) activity. vegetables and cereals and the incidence of coronary heart Recent work is also beginning to highlight the potential disease and certain . Many constituents of these role of other components, including the dietary components may contribute to their protective flavonoids, and phenolic acids, as im- portant contributing factors to the antioxidant activity of the (Ref. 1; see Table 1). The flavonoids are a Flavonol Flavone large class of compounds, ubiquitous in plants, and usually occurring as gly- cosides. They contain several phenolic hydroxyl functions attached to ring structures, designated A, B and C (Fig. 1). Structural variations within the rings subdivide the flavonoids into OH 0 OH 0 several families: • (e.g. and ), with the 3-hydroxy pyran-4-one C ring. • (e.g. luteolin, and -3-ol ), lacking the 3-hydroxyl group. • Flavanols (e.g. ), lacking the 2,3-double bond and the 4-one structure. • (e.g. ), in which OH 0 ~L~ the B ring is located in the 3 position on the C ring. OH These flavonoids often occur as gly- cosides, glycosylation rendering the Fig. 1. Structures of the flavonoids. The basic structure consists of the fused A and molecule less reactive towards free C rings, with the phenyl B ring attached through its 1' position to the 2-position radicals and more water-soluble, so of the C ring (numbered from the pyran ). Types shown include: flavonols permitting storage in the vacuole. (3-hydroxyflavones) [e.g. quercetin (3,5,7,3',4'-hydroxyl) and kaempferol (3,5,7,4'- Common glycosylation positions are: hydroxyl)]; flavones [e.g. luteolin (5,7,3',4'-hydroxyl), apigenin (5,7,4'-hydroxyl) and the 7-hydroxyl in flavones, isoflavones chrysin (5,7,-hydroxyl)]; flavan-3-ols [e.g. catechin (3,5,7,3',4'-hydroxyl)]; and and dihydroflavones; the 3- and 7- isoflavones [e.g. genistein (5,7,4'-hydroxyl)]. hydroxyl in flavonols and dihydro- flavonols; and the 3- and 5-hydroxyl in

1 52 April 1997,Vol. 2, No. 4 © 1997 ElsevierScience Ltd PII S1360-1385(97)01018-2 reviews

anthocyanidins2. The sugar most usually involved in the formation is glucose, although galactose, rhamnose, xylose and arab;nose also occur2, as well as disaccharides such as rutose. Antioxidant Sources ~tioxidant The variants are all activity ~ related by a common biosyn- (mM) thetic pathway (Fig. 2), incorpo- rating precursors from both the shikimate and the acetate-mal- E onate pathways 2. Further modi- fication occurs at various stages, resulting in alteration in the extent of hydroxylation, methy- Oenin Blac]~ grapes/red wine~ 1.8±0.02 lation, isoprenylation, dimeriza- Cyanidi~ Grapes, raspberries and strawberries. 4.4±0.12 tion and glycosylation (producing DeIphfnidin Aubergine skin: 4.4±0.11 O- or C-)3-6. F t~3.OIs Querce~in Onion, apple ski~, , black grapes, 4.7±0.10 Relationships between the tea and broccoli. structure and antioxidant Kaempferol EndiVe; leek, broccoli,grapefruit and tea 1.3±0.08 activity of The chemical activities of Flavones polyphenols in terms of their Rnti~ Onion, apple skin; berries, black grapes, 2.4±0.12 reducing properties as hydrogen- teaandbroccoli. or electron-donating agents pre- Lute61~ Lemon, oIive, and red pepper. 2.1 + 0.05 dicts their potential for action as Chrysin Fruit Skin: 1.4 + 0.07 Apigenin Celery and parSIey, free-radical scavengers (ant;oxi- 1.5 _+ 0.08 dants). The activity of an Flavan.3-ols ant;oxidant is determined by: (Epi)catecl)m:~ BI~ck grapes/red wine 2.4 _+ 0.02 • Its reactivity as a hydrogen- Epigall0ca~c}/i~ Teas~ 3.8 +- 0.06 or electron-donating agent Epigallbcateehin gattate Teas: &8 +_ 0.06 (which relates to its reduction 4.9 ± 0.02 potential). • The fate of the resulting ant;oxidant-derived radical, Citrus fi'uit. 1.9 ± 0.03 which is governed by its ability Nat;turin Ci~rus fruit. 0.8 u 0.5 (n~iringenim7-rutinoside} to stabilize and delocalize the Citrus fruit. li5 • 0;05 unpaired electron 7. Hesperidi~ . t.0~ 0.03 • Its reactivity with other ant;oxidants. L4 } 0:08 • The -chelating potential. Theaflavins Polyphenols possess ideal struc- ~eaflavin. Blacktea. 2.9 ± 0.08 tural chemistry for free 4.7 _+ 0.16 radical-scavenging activities, and 4.8 + 0.19 have been shown to be more effec- TheaflaVin digallate Black tea. 6.2 +_ 0.43 tive ant;oxidants in vitro than vit- Hydr0xye ares " amins E and C on a molar basis s'9. acid White grapes, olive, and asparagus 1.3 + 0.01 This is exemplified by studies Ch ic acid Apple, pear, cherry, tomato and peach. 1.3 _+ 0.02 using pulse radiolysis to investi- Ferulie acid Grains, tomato, cabbage and asparagus 1.9 + 0.02 gate the interactions of the p-Coumarie acid ~ite grapes, tomato, cabbage and 2.2 ± 0.06 ('OH), azide radi- asparagus. cal (N3'), anion (O2"-), peroxyl radical (LO0") and ~Measured as the TEAC (Trolox equivalent antioxidant activity) - the concentration of Trolox with model t-butyl alkoxyl radicals the equivalent anti0xidant activity of a 1 mM concentration of the experimental substance, (tBuO') with polyphenols, the rate constants of the reactions and the stability of the ant;oxidant radical 1°. In addition, the propen- the relative abilities of the compounds to scavenge free sity for metal , particularly and copper, supports radicals. This can be assessed 12'13 by applying the chromo- the role ofpolyphenols as preventative ant;oxidants in terms of genic indicator ABTS "+, the radical cation of 2,2'- inhibiting transition metal-catalysed free radical formationn. azinobis(3-ethylbenzothiazoline-6-sulphonic acid). The anti° There is a hierarchy of flavonoid and anti- oxidant activities (Table 1) relative to Trolox, the oxidant activities that is dependent on structure and defines water-soluble analogue, are entirely consistent

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of contributing hydroxyl groups by glycosylation Acetate-malonate ~O ~-....~/OH/ Shikimate pathway ~.....------~ _pat hway decreases the antioxidant activity. • For metal chelation, the two points of attachment of c JI C',nnh,s ' - Lignin transition metal ions to the flavonoid molecule are the TM H °H o-diphenolic groups in the 3',4'-dihydroxy positions in HO~..~ the B ring, and the ketol structures 4-keto, 3- hydroxy or 4-keto and 5- OH O / OH O hydroxy in the C ring of the OH O flavonols. However, it is Ohalcone likely that metals differ Dihydrochalcone with regard to chelation by polyphenols. Glycosylation HO~OH at all of these crucial hydroxyl positions influ- ences the antioxidant activ- ity of the flavonoids. This OH O is important when con- Flavone sidering the relationship between the antioxidant activity of the naturally H ~-~.~OH ~ HO. -.¢'..- .0. abundant glycosides and that of the aglycones. The half peak reduction potential (Ep/2) has also o- \ ,erocarpan been ascribed as a suitable (+)'Dihydr°flav°no°/ ~ ~ parameter for representing the scavenging activity of the flavonoids TM. This was rationalized on the basis .o.r o \ \ that both electrochemical oxidation and hydrogen- o. \ \ Ro,eno,d donating free radical-scav- enging involve the break- ing of the same phenolic / \ bond between oxygen and hydrogen, producing the phenoxyl radical and H', which consists of an elec- tron and an H ÷ ion. Thus a OH OH OH O flavonoid with a low value (-)-Epicatechin Flavonol for half peak reduction potential (i.e. <0.2 mV) is a Fig. 2. Inter-relationships between flavonoid classes=. Arrows indicate the principal biosynthetic good . The half routes. peak reduction potentials can be compared with the total antioxidant activity, with their structures. The structural arrangements im- measured as the Trolox Equivalent Antioxidant Capacity parting greatest antioxidant activity as determined from (TEAC), by applying the ABTS "÷ radical cation scavenging these studies are: assay. Table 2 reveals a broad agreement between the • The ortho 3',4'-dihydroxy moiety in the B ring (e.g. in TEAC value (at pH 7.4) and the Ep/2 value (at the same catechin, luteolin and quercetin). pH), in that flavonoids with efficient scavenging properties • The meta 5,7-dihydroxy arrangements in the A ring (e.g. have a TEAC value of >1.9 mM and Ep/2~<0.2 mV, in com- in kaempferol, apigenin and chrysin). parison with less efficient antioxidants with a TEAC of • The 2,3-double bond in combination with both the 4-keto <1.5 mM and Ep/2>0.3 mV (with the exception of group and the 3-hydroxyl group in the C ring, for electron kaempferol). It is interesting to note that the polyphenols delocalization (e.g. in quercetin), as long as the o-dihydroxy with the highest scavenging activities include those with structure in the B ring is also present. However, alterations the o-dihydroxy structure in the B ring. It has been in the arrangement of the hydroxyl groups and substitution reported that flavonoids with Ep/2 values of <0.06 mV

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undergo redox cycles under physiological conditions ~5''~ and are thus able to reduce transition metals ~7-~9.

Flavonoids in Citrus and grapes The flavonols and their glycosides, the flavanols and the , are the predominant flavonoid classes found Ftavonoid ~tioxidant Half ~ak reduction in fruit. The genus Citrus is characterized by a substantial activity. (n~) pete (mV) accumulation of glycosides, which are not found in many other fruit, at the expense of the accumulation of Quercetin 4.7 0.03 flavanols and anthocyanins. Some of these flavanone and Rutir~ 2~4 0.18 Catechin 2.4 0,16 flavanolol compounds are intermediates in the biosynthetic LUteolin 2A 0.18 pathways that lead from hydroxycinnamic acids and mal- Taxifolin- .... 1,9 0.15 onyl coenzyme A to anthocyanins, and flavonols 1.5 >1 (Fig. 2). Narihgen~ 1.:5 0.6 Each plant species is characterized by a particular fla- ~ 1.4 0.4 vanone glycoside pattern 2°. The flavanone aglycones are the .... 1.3 0.12 5,7-dihydroxy structures naringenin and hesperetin, and their 4'-methoxylated derivatives, and hes- ~Measured as the TEAC (Trolox equivalent antioxidm~t activity) - peretin; the major glycosides are and , the ConcentrationofTr01oX with the equivalent antioxidant activity the 7-rutinosides of naringenin and hesperetin. Lemon peel contains two flavanone glycosides, hesperedin and , the 7-rutinosides of hesperetin and eriodic- scavenger~4~~ tyol, respectively. In grapefruit, (the 7-neohesperi- doside of naringenin) is predominant and is accompanied by narirutin (naringenin 7-rutinoside). Naringenin constitutes 88% (v/v) of flavanones in the juice. Naringin also predomi- Citrus and in the skins of certain red cultivars of Vitis nates in the juice of sour orange (56-71%, v/v), along with vinifera. Ferulic acid usually forms only a small percentage other neohesperidosides - particularly the 7-neohesperido- of the total hydroxycinnamic acid in fruit, but, again, can side of (). Only the 7-~-rutinosides reach 50% (v/v) of the total hydroxycinnamates in certain are present in sweet orange, hesperidin being the dominant Citrus. flavanone glycoside. In juice of sweet orange, hesperidin is There is great interest as to whether the polyphenolic accompanied by narirutin. Citrus fruits also contain several constituents of red wine are an important factor contribut~ flavones, some of which are polymethoxylated flavones, such ing to protection from coronary heart disease. Grapes and as nobiletin and sinensetin in orange peel. wine contain large amounts of polyphenols at concen- Another family of phenolics found in Citrus are the trations in the range 1.0-1.8 ~g ml 1. The major polyphenols phenylpropanoids, and in particular the hydroxycinna- of V. vinifera are: caftaric acid, the ester of car- mates. The most widely distributed phenolic components in feic acid; the blue-red pigment malvidin-3-glucoside, as the plant tissues are the hydroxycinnamic acids, p- coumaric, caffeic and ferulic COOH COOH adds, which are synthe- sized via the shikimate pathway (Fig. 3). These are present in many diets at higher concentrations than the flavonoids and antho- cyanins. Their occurrence is Phenylalanine Cinnamic acid usually in various conju- gated forms resulting from enzymatic hydroxylation, O-glycosylation, O-methyla- COOH COOH COOH COOH COOH tion or esterification of p- coumaric acid. is frequently the most abundant phenyl- propanoid in fruit and is the major representative of hydroxycinnamates in Cit- OH OH OH OH OH rus fruit. Although p- Tyrosine p -Coumaric acid Caffeic acid Feruiic acid Sinapic acid coumaric acid is less abun- dant, in general, than Fig. 3. Intermediates in biosynthesis. Arrows indicate the principal biosyn- thetic routes. caffeic acid in most fruit, it predominates in certain

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of cardiovascular diseases and cancers, and as markers of lipid . Green tea consumption has been associ- ated with lowered cardiovascular risk through decreased serum and triacylglyceride, increased high den- Concentration in Concentration in red wine (rag 1-1) (mg 1"1) sity lipoprotein and a decrease in indicators of atherogen- esis 28. Animal studies in vivo demonstrated that green tea Catechin 191 35 stimulates hepatic UDP-glucuronyl transferase activity24 Epicatechin 82 21 and provides protection against kidney , Gallic acid 95 7 whereas black tea showed enhanced protection against 3 0 peroxidation of liver 25. Theaflavins are formed during the Malvidin-3 - 24 1 manufacture of black and oolong teas from the enzymatic glucoside oxidation of the flavanols, catechin and gallocatechin, by Rutin 9 0 oxidase. The reaction involves the oxidation of Quercetin 8 0 the B rings to quinones, followed by a 'Michael' addition of 9 0 the gallocatechin quinone to the catechin quinone, prior to 1.5 0 carbonyl addition across the ring and subsequent decar- ~Data from Ref. 21. boxylation26. Theaflavins also possess in vitro antioxidative properties against lipid peroxidation, in erythrocyte mem- branes and microsomes27, and suppress mutagenic effects induced by (the gallic acid moiety of the major ; and the flavan-3-ol catechin 2°. The theaflavins is essential for this activity27). Epigallocatechin mean amounts of phenolic constituents found in a range of gallate, one of the major polyphenolic constituents of green wines are shown in Table 3 (Ref. 21). Unlike other classes of tea, suppresses the production of superoxide radicals and flavonoids, monomeric flavan-3-ols are generally found in hydrogen peroxide by tumour promoter-activated human free rather than glycosylated or esterified form in fruit. neutrophils ~s. Grape is a fruit of high flavan-3-ol content and the major The flavanols catechin and epicatechin, their gallo forms monomers are (+)-catechin and (-)-epicatechin. Epicatechin with three hydroxyl groups adjacently placed on the B ring is reported as being the most abundant fiavonoid in skin and their gallate esters are major constituents of green tea. extracts of several white cultivars. Polyphenols constitute about 42% of the dry weight compo- sition of green tea extract, of which 26.7% are catechin-gal- Black grape skin and the antioxidant activity of wines late components: (11.16%); epicat- The total antioxidant activities of a range of red wines 8 echin gallate (2.25%); epigallocatechin (10.32%); epicatechin vary from 12-14 mM for Californian Pinot Noir, Rioja and (2.45%); and catechin (0.53%) (Ref. 29). As seen in the par- Bouzy Rouge, through to about 16 mM for Australian Shi- ent catechin structure (Fig. 1), there is no delocalization of raz, and 23 mM for red Bordeaux and Chianti. Even though electrons across the structures between the A and B rings the antioxidant activities of the wines vary over a factor of because of saturation of the heterocyclic pyran ring. 2, the ratios of activities to the total phenol content are Studies on the antioxidant activities of the individual approximately the same (about a factor of 10), indicating constituent catechin and catechin-gallate esters of green the direct relationship between the two. This is further tea have shown that in vitro they are more effective anti- underlined by the reduced antioxidant activity (total anti- oxidants on a molar basis than (Ref. 30). This oxidant activity = 1.5 mM) of a white wine from the same ranking of reactivity follows the order: epicatechin gallate grape as Bouzy Rouge, but without the black grape skin. ~epigallocatechin gallate>epigallocatechin>epicatechin-- Based on the data 21'22 for the phenolic composition of catechin. This is consistent with the number and arrange- wine, and data for the TEAC values of polyphenols 8'9, a ment of phenolic hydroxyl groups s. These findings are in figure can be calculated for the contribution that these agreement with the hierarchies of antioxidant activities constituents make to the antioxidant activity of wine. Cal- against the 1,1-diphenyl-2-picrylhydrazyl radical 31 and culating the mean total antioxidant activity from the meas- superoxide radical reduction 32. ured antioxidant activities of the individual constituents, The enhanced values for the catechin gallate esters in only 25% of the measured value can be accounted for. relation to the reflect the additional contribution Although Frankers compositional figures are low compared from the trihydroxybenzoate, gallic acid. Epigallocatechin with the data of others 2°'21, and in view of the constancy is structurally similar to catechin and epicatechin, with an of the total antioxidant activity:polyphenol ratios, the additional hydroxyl group adjacent to the o-diphenolic remainder of the activity is presumably derived from structure in the B ring enhancing the antioxidant activity. unidentified antioxidants such as vitamin C, other poly- The linkage of gallic acid to the epicatechin or epigallo- phenols and phenolic acids, but especially formed catechin structure via esterification at the 3 position again from the polyphenols. increases the antioxidant potential, as in epicatechin gal- late or epigallocatechin gallate. The total antioxidant activ- Flavonoids as contributors to the antioxidant activity of ity of catechin gallate-rich green tea extract at 1000 ppm tea (mg 1-1) gives a value of 3.78 _+ 0.03 mM (n = 9). The antioxi- The importance of catechins, gallocatechins, catechin- dant capacities of the polyphenolic constituents in green gallate esters and related phytochemicals as dietary anti- tea in relation to their concentrations are used to calculate oxidants has been investigated. Both in vivo and in vitro their predicted contributions to the antioxidant potential, studies on the effects of green and black teas, and their and the result of 2.95 n~ is reasonably consistent with the polyphenolic constituents, have been undertaken in models measured value of the proportionately combined catechin

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gallate constituents of 2.78 + 0.03 raM. Thus 78% of the no initiating radical species need be added - as long as antioxidant activity of green tea extracts can be accounted redox-cycling agents, such as copper ions or heine , for by the catechins and catechin gallate esters. Taking into are applied to propagate the decomposition of the minimal account the antioxidant activity of the polyphenolic con- levels of endogenous pre-existing 3s. stituents of green tea and their relative abundances, the Assessing the relative efficacies of the polyphenolic con- order of contribution to the antioxidant effectiveness within stituents of green tea to protect LDL from oxidation demon- green tea is: epigallocatechin~-epigallocatechin gallate>epi- strates the low concentration (<1 IxM) required for 50% catechin gallate=epicatechin>catechin. inhibition of LDL oxidation 3°. Specifcally, whereas 0.75 FM The effectiveness of theaflavin as an antioxidant is epigallocatechin and 1.2 IxM gallic acid were required, for increased by esterification with gallate, and is further epicatechin, catechin, epicatechin gallate and epigallocat- enhanced as the digallate ester 33. This can be predicted from echin gallate concentrations between 0.25 and 0.38 ~,oM the previous studies on the catechins and catechin-gallate were effective. The three- to fivefold differences in concen- esters showing that, in the case of the flavanols, increasing tration required to inhibit LDL oxidation by 50% for epigal- numbers of hydroxyl groups as ortho diphenolics or tri- locatechin or gallic acid and the rest presumably relate to phenolics, as with incorporation of gallate esters or in the the relative partitioning abilities of the constituents within gallocatechins, progressively augments the antioxidant the LDL particle and their accessibility to the lipid peroxyl activities of these polyphenols against radicals generated radicals: epigallocatechin and gallic acid are more in the aqueous phase. Thus the hierarchy of antioxidant hydrophilic and therefore have less access to the lipid per- activities of these phytochemicals can be rationalized by oxyl radicals. Miura et al. 39 also found that epigallocatechin consideration of their structural chemistry. was the least effective catechin in protecting LDL from copper-mediated oxidation and epigallocatechin gallate the Antioxidant activity of polyphenols in the lipophilic phase most effective, with a threefold decrease in the concen- One of the key mechanisms relating the antioxidant sta- tration required for 50% inhibition of oxidation. The cate- tus of the blood and decreased risk of coronary heart disease chin/gallate family of tea components was also studied for relates to the postulate that oxidation of the low density its ability to conserve the a- located within the lipoproteins (LDL) is an early event in . Bio- LDL particles and protect it from oxidation. Monitoring the chemical and clinical studies have suggested that oxidized consumption of ~-tocopherol within the LDL, when chal- LDL has an array of atherogenic properties through the for- lenged with a pro-oxidant in the presence of catechin mation of lipid hydroperoxides and the products derived polyphenols, confirms that epigallocatechin is indeed the from these. Oxidatively modified LDL can be taken up read- least effective in protecting vitamin E. This reflects its ily by macrophages via the scavenger receptor to form foam lesser contribution to increasing the resistance of LDL to cells, an early marker of the development of atherosclerotic oxidation, whereas the consumption of ~-tocopherol was lesions (see Ref. 34). delayed and prolonged by epigallocatechin gallate and epi- The antioxidant status of LDL and plasma are important catechin gallate. Their abilities to enhance the resistance of determinants of the susceptibility of-the LDL to peroxi- low density lipoprotein to oxidation and to prolong the lifeo dation. The major antioxidant localized in LDL is the time of ~-tocopherol within LDL is in the sequence: epi- dietary phenolic a-tocopherol, the level of which is an impor- gallocatechin gallate=epicatechin gallate=epicatechin=cate- rant contributor to the resistance of LDL to oxidation. Of the chin>epigallocatechin>gallic acid. This is consistent with other dietary antioxidants, the extent of incorporation of the their partition coefficients, gallic acid and epigallocatechin flavonoids and polyphenols is unclear, although interesting being preferentially localized in the aqueous phase and studies 35-37 have suggested a relationship between cardiopro- thus having less access to the lipid peroxyl radicals. tection and the increased consumption of flavonoids from The ability of the hydroxycinnamates to enhance the dietary sources such as onions, apples, tea and red wine. resistance of LDL to oxidation shows that chlorogenic and The constituents of red wine are a source of particular inter- caffeic acids have a higher peroxyl radical scavenging abil- est because of the French paradox 37, that the Southern ity than monophenolics such as p-coumaric acid 4°'41. French show low mortality from coronary heart disease, Methoxylation of one hydroxyl group of caffeic acid to form despite their high intake and tendencies. ferulic acid decreases the efficiency of the scavenging reac- Because oxidation of LDL is implicated in the - tion with peroxyl radicals (i.e. caffeic is substantially more esis of atherosclerosis, the enhancement of the resistance of effective in inhibiting LDL oxidation). Ferulic acid is much LDL to oxidation is one of the models used by many more effective than p-coumaric acid. The electron-donating researchers for investigating the efficacy of dietary phyto- methoxy group allows increased stabilization of the result- chemicals as antioxidants against radicals generated in the ing aryloxyl radical through electron delocalization after lipophilic phase. The LDL particle contains a-tocopherol, hydrogen donation by the hydroxyl group. These data are the major lipophilic antioxidant in plasma, in its outer again consistent with the partitioning characteristics of the monolayer, and carotenoids in the inner core. Free radical- hydroxycinnamates, and with results from studies under- mediated peroxidation of polyunsaturated fatty acids leads taken in lipophilic systems to establish the structural cri- to the formation of lipid hydroperoxides through peroxi- teria for the activity of polyhydroxyflavonoids in enhancing dation. Oxidative and reductive decomposition of hydroper- the stability of dispersions (especially methyl oxides amplifies the peroxidation process. The presence of linoleate), , oils and low density lipoproteins 7'11'3°'3~'42. chain-breaking phenolic antioxidants provides a means of intercepting the peroxidation process by reducing the Bioavailability alkoxyl or peroxyl radicals to alkoxides or hydroperoxides, There is a paucity of information on the absorption, respectively. In order to study the antioxidant activity of pharmacokinetics, metabolism and excretion of dietary polyphenols as scavengers of propagating peroxyl radicals, polyphenols in humans and their handling by the

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Flavonoid Dose Observation

Catechin44 5.8 g (based on 83 mg kg-1 for a 70 kg man). 26% of administered dose excreted within 24 h. Major m-hydroxyphenyl propionic acid, detected in plasma after 6 h.

3.O-methyl-catechin45 2 g. Plasma levels, 11-18 ~g m1-1within 2 h; urine, 38% excreted within 120 h as glucuronides and sulphates.

Quercetin ~.47 64 mg from fried onions. 89-100 mg Plasma concentration about 1 }1~, 2 h later. equivalent from supplements. Quercetin4s 4 g supplement. Not detected in urine or plasma after oral dosage. Decaffeinated green tea 29 88 mg epigallocatechin gaUate, Plasma levels, 46-268 ng m1-1, 82-206 ng mt-1, 82 mg epigallocatechin, 33mg epicatechin undetectable and 40-80 ng m1-1, respectively. gallate, and 32 mg epicatechin.

"Basal fiavonoid plasma levels in nonsupplemented humans49: rutin 0.72 ~M; combined other glycosides of flavonols 0.6-3.8 ~M; and phloridzin 0.6-1.6 ~M.

. Much early evidence43 indicated that compound48. Green tea consumption led to the detection of absorption of the hydroxyaromatic acids, benzoates, all the major constituents, with the exception of epicate- phenylacetates and hydroxycinnamates might occur, but chin gallate, in plasma 29. More recently, studies 49 have there was a requirement for splitting of the polyphenols in shown the presence of several dietary flavonoids in human the gastrointestinal tract to lower molecular mass forms plasma from nonsupplemented subjects, including phlo- prior to absorption and conversion to hydroxyaromatic ridzin, quercetin glucosides and quercetin rutinosides. acids 43. Hydrolysis of flavonoid glycosides, ring fission and It should also be emphasized, however, that certain the reductive metabolism of phenyl acyl fragments are car- flavonoids in chemical systems autoxidize readily, es- ried out by intestinal microorganisms. The catabolism of pecially quercetin, myricetin, quercetagetin and delphini- quercetin, for example, is proposed to form hydroxyaro- din 1~,~°. Furthermore, there are some reports of the pro- matic acids with two-carbon side chains on the aromatic oxidant activity of some polyphenols in the presence of ring, caused by the presence of the 3-hydroxyl group on the metal ions in which excessive concentrations (25-100 ~M) C ring of quercetin. However, in the absence of a 3- accelerate hydroxyl radical formation and DNA damage in hydroxyl group, as in hesperidin, hydroxyaromatic acids vitro ~1. with three-carbon side chains are formed, which then undergo p-oxidation to benzoic acid derivatives. The meta- Conclusions bolic transformation of caffeic acid, for example, has been The studies described in this review demonstrate the proposed to proceed through the methylation of the pheno- antioxidant properties of polyphenolic constituents of lic hydroxyl groups, dehydroxylation in the para position, plants, and will help in the identification of the active con- hydrogenation of the side chain, p-oxidation and formation stituents in beverages, vegetables and fruit that may help of conjugates, as in the metabolism of flavonoids. These sustain antioxidant status and protect against free radical studies suggest that the conjugation reaction with glu- damage. This should be useful information for identifying curonide or sulphate is perhaps the most common final foods that are rich in these protective components, for the step in the with intact flavonoids. development of safe food products and additives with Table 4 summarizes the current (limited) available appropriate antioxidant properties. information concerning the absorption and uptake of a range of polyphenols in humans. Evidence exists that cat- Acknowledgements echin is absorbed by the human gut 44, and other investi- C.A.R-E. acknowledges the Biotechnology and Biological gations involving oral adminstration of 3-O-methyl-[U- Sciences Research Council, the Ministry of Agriculture, 14C]-catechin in three volunteers showed that the Fisheries and Food and Unilever for financial support. supplement occurred in plasma 45. Peak levels were observed within 2 h of administration. The time course of plasma quercetin was studied in two subjects after inges- References 1 Rice-Evans, C. and Miller, N.J. (1996) Antioxidant activities of tion of fried onions containing quercetin glycosides to the flavonoids as bioactive components of food, Biochem. Soc. Trans. 24, level of quercetin aglycone equivalent to 64 mg, and on 790-795 supplementation with pure quercetin glycosides46'47. 2 Markham, K.R. (1982) Techniques in Flavonoid Identification, Quercetin was detected in plasma after hydrolysis, Academic Press whereas, in one early study, supplementation with 3 Harborne, J.B. and Mabry, T.J., eds (1982) Advances in Flavonoid quercetin suggested that there was no absorption of this Research, Chapman & Hall

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