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Antioxidants in teaSheila A. Wiseman a , Douglas A. Balentine b & Balz Frei ca Unilever Research Laboratorium , Vlaardingen, The Netherlandsb Lipton , 800 Sylvan Avenue, Englewood Cliffs, NJ, 07632 Phone: 201 ‐894–7338 Fax: 201‐894–7338 E-mail:c Linus Pauling Institute , Oregon State University , Corvallis, OregonPublished online: 29 Sep 2009.

To cite this article: Sheila A. Wiseman , Douglas A. Balentine & Balz Frei (1997) Antioxidants in tea, Critical Reviews in FoodScience and Nutrition, 37:8, 705-718, DOI: 10.1080/10408399709527798

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Critical Reviews in Food Science and Nutrition, 37(8):705-718 (1997)

Antioxidants in Tea

Sheila A. Wiseman,1 Douglas A. Balentine,2 and Balz Frei3

1 Unilever Research Laboratorium Vlaardingen, The Netherlands; 2Lipton, Englewood Cliffs, New Jersey;3Linus Pauling Institute, Oregon State University, Corvallis, Oregon

* Address for conespondence: D. Balentine, Lipton, 800 Sylvan Avenue, Englewood Cliffs, NJ. 07632. Tel: 201 -894-7338; Fax: 201-894-7017; email: douglas.balentine@unilever.com

1. INTRODUCTION

Dietary factors play a major role in chronic dis-ease development, and health organizations active-ly endorse increasing consumption of fruits and vege-tables to improve public health.1"3 A number of thesechronic diseases, for example, cardiovascular dis-ease and cancer, have been linked to excess produc-tion of reactive oxygen species (ROS) and oxida-tive damage to biomolecules. Whereas fiber, vitamins,and minerals have been hypothesized as being impor-tant, the exact components of plants responsible forthe health benefits have not been identified, but anti-oxidants are likely candidates. Derived from plantmaterial and containing numerous biologically ac-tive components, tea beverage may also be proposedas a component of a healthy diet.4"8

Tea beverage is an infusion of the leaves of Ca-mellia sinensis. The majority of tea beverage is pre-

pared from two types of manufactured tea: black andgreen. Black tea, consumed typically in the U.S., Eu-rope, Africa, and India, is made by crushing and dryingfresh tea leaves to effect 'fermentation" prior to finalprocessing. During "fermentation", some of the cat-echins combine to form complex theaflavins andother undefined flavonoids, which provide distinc-tive flavor and color to black tea beverage (Figure 1).Oolong tea is a partially fermented tea product andhas a unique flavor and chemistry. Typically con-sumed in East Asian countries, green tea is preparedwhen the fresh leaves are processed rapidly to pre-vent "fermentation".

Black tea beverage typically contains approxi-mately 31% (wt/wt) flavonoids as 9% catechins, 4%theaflavins, 3% flavonols, and 15% undefined cat-echin condensation products. Green tea beveragecontains approximately 33% (wt/wt) flavonoids as3% flavonols and 30% catechins, the main form

OHOH

Theaflavins Catechin Condensation ProductsThemjblgens"

FIGURE 1. Chemical conversion of flavonoids during tea fermentation.

1040-8398/97/$.50©1997 by CRC Press LLC

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being epigallocatechin-3-O-gallate. A typical cupof tea contains approximately 600 mg of total sol-ids and 200 mg of flavonoids.

II. HEALTH BENEFITSOF ANTIOXIDANTS

Human cells are constantly exposed to ROS suchas superoxide, hydroxyl, and peroxyl radicals andhydrogen peroxide. These ROS are mostly pro-duced from endogenous sources, such as electrontransport chains, peroxisomes, and the cytochromeP-450 system. Other ROS and free radicals are gen-erated by the immune response, for example, mac-rophages or as a result of smoking, air pollution,or the influence of UV light.

Chronic exposure to ROS can damage DNA,membrane lipids, lipoproteins, and functional andstructural proteins.9"10 As constant oxidative stresshas been linked to the development of chronic diseasessuch as cancer, cardiovascular disease, cataracts,11"13

and dementia,14 its reduction is seen as beneficialto public health.

The human body has evolved antioxidant defensemechanisms to minimize the potential for radicaldamage.15 The extremely reactive hydroxyl radi-cal is particularly harmful, with a near diffusion-controlled rate constant of 109 tolO10 M-V1 enablingit to combine with virtually all molecules in livingsystems.16 Intracellular antioxidant enzymes func-tion as a first line of defense to neutralize ROS.The primary reaction is dismutation of superoxideradicals to hydrogen peroxide and oxygen by theenzyme superoxide dismutase, followed by remov-al of hydrogen peroxide that is catalyzed by cata-lase and glutathione peroxidase.17

The endogenous enzymatic defenses againstoxidative damage are not completely efficient,10-18

and a range of endogenous and exogenous free.radical scavenging antioxidants act as a secondline of defense. Vitamin E, vitamin C, and thecarotenoids are well-recognized antioxidant nu-trients,19"21 but fruits and vegetables also containflavonoids and other phytochemicals that are po-tent antioxidants22"25 and may contribute to de-fenses against oxidative damage.

III. ANTIOXIDANT COMPONENTSOF TEA

All tea beverages are rich in flavonoids, par-ticularly catechins and flavonols, which scavengeROS and free radicals.26"28 Flavonoids effectivelystabilize free electrons through several proposedmechanisms, including delocalization of electrons,formation of intramolecular hydrogen bonds,29 andrearrangement of their molecular structure.30"31 Freecopper and iron, which may catalyze formation ofROS in vivo9 and are used to generate free radicalsin some test systems, are chelated by flavonoids.32-33

Recent interest in flavonoid compounds has beenheightened by epidemiological studies that haveidentified inverse associations between flavonoidconsumption and risk of chronic degenerative dis-eases such as cardiovascular disease and certaintypes of cancers.34-36 The antioxidant activity of fla-vonoids may be an important attribute of their pro-posed beneficial health properties. Tea beveragesare major contributors to the total daily flavonoidintake, and elucidation of their antioxidant poten-tial is therefore of interest.

To be regarded as effective in a biological sense,an antioxidant is a substance that when present atlow concentrations relative to an oxidizable sub-strate (e.g., lipid, protein, or DNA molecule), cansuppress, delay, or prevent oxidation of that sub-strate.37 Antioxidant potential is determined by chem-ical reactivity as an electron or hydrogen donor;the ability to delocalize and thus stabilize the un-paired electron; the reactivity with other antioxi-dants; and the reactivity with molecular oxygen.38

The chemical structures required for effectiveantioxidant activity of flavonoids and polyphenolshave been identified recently.2939"43 The most impor-tant features that have been identified in these stud-ies are an ort/io-dihydroxy catechol (3',4'-OH) ar-rangement on the B-ring (present in flavan-3-ols)that promotes formation of a stable phenoxyl radi-cal due to effective electron delocalization, and aC2-C3 double bond in the C-ring in combinationwith a C4 carbonyl group and the hydroxypyranonestructure (Figure 2). In addition to acting as effi-cient free radical scavengers based on these struc-tural features, flavonoids can also chelate free metalions32-33 via the catechol group on the B-ring, thus

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3',4' OH arrangement

H /

OH

Hydroxypyranonestructure

C2-C3 double bond

C-4 carbonyl

Flavan3-ols Flavonols

FIGURE 2. Structural features of flavonoids important to antioxidant chemistry.

reducing metal-catalyzed ROS production. The iron-chelating ability of catechins has been ranked: epi-gallocatechin (EGC)>epicatechin gallate (ECG) =epigallocatechin gallate (EGCG)>epicatechin(EC).44

IV. FREE RADICAL SCAVENGINGPROPERTIES OF TEA ANTIOXIDANTS

Chemical reactivity of tea extracts and individ-ual tea flavonoids has been characterized using invitro model systems based on scavenging of ROSor stable free radicals. Typical ROS-generating sys-tems employ UV light, radiation, pulse radiolysis,the hypoxanthine-xanthine oxidase system, or metalions (Fenton chemistry). The substrate for oxida-tion in these ROS-generating systems is frequentlylipids contained in tissue homogenates, liposomes,micelles, ghost cell membranes, or simple and com-plex lipid systems such as methyl linoleate or low-density lipoprotein (LDL). Typical markers for de-fining the extent of oxidation are conjugated dienesand oxygen consumption. Stable free radicals thathave been used to evaluate tea antioxidants includethe radical cation of the compound 2,2'-azinobis-(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS),the diphenyl-2-picrylhydrazyl radical (DPPH), andthe galvinoxyl radical.

A. Scavenging of Reactive OxygenSpecies

Tea and tea flavonoids consistently demonstratestrong in vitro scavenging ability against numer-ous physiologically significant ROS. Green and blackteas demonstrate strong antioxidant capacity againstboth peroxyl and hydroxyl radicals in the oxygenradical absorbing capacity (ORAC) assay. Thisassay determines the ability of an antioxidant toprevent oxidation of P-phycoerythrin by ROS rela-tive to Trolox, the water-soluble form of vitamin E.A cup of black or green tea was over three times moreeffective than a serving of most common vegeta-bles,45 and teas were over two times more effec-tive than a serving of most other common fruits.453

1. Superoxide Radical

Extracts of green tea, pouchong tea, oolong tea,and black tea were efficient scavengers of super-oxide and hydrogen peroxide. Oolong tea (semi-fermented) was the best scavenger of both ROS.46

Epicatechin 3-0-gallate (ECG) and epigallocatechin3-0-gallate (EGCG), the main catechins in greenand black teas, were good scavengers of the super-oxide radical generated by the hypoxanthine-xan-thine oxidase system using electron spin resonance

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(ESR) spectrometry with 5,5-dimethyl-l-pyrroline-1-oxide (DMPO) as a spin-trapping agent.47 Thesedata were confirmed by another study showing thatEGCG is a highly efficient scavenger of the super-oxide radical generated by pulse radiolysis, react-ing with a rate constant of 7.2 x 105 M"1 s"1.28 Thereactivity of EGCG was found to be twice that ofeither epigallocatechin (EGC) or ECG, possibly dueto the presence of two antioxidant gallate moieties inthe EGCG molecule. However, EGC and EGCGwere found to have comparable superoxide scaveng-ing reactivities in a hypoxanthine-xanthine oxidasesystem.48 EGCG was the most potent radical-scaveng-ing catechin, and the catechins as a group were morepotent than vitamin C and vitamin E 49

Theaflavins were shown to be even better scav-engers of the superoxide radical than the gallocat-echins. In the case of the theaflavins, gallate substi-tution appeared to lower the rate of reactivity.50

2. Singlet Oxygen

Catechin (C) was the most efficient quencher ofsinglet oxygen when a range of flavonoids werecompared.51 The rate of quenching by catechin wasalmost 4 times greater than that of the flavonol quer-cetin, which generally displays a higher free radi-cal scavenging potency.52 The absence of the car-bonyl group in ring C of catechin (see Figure 2),when compared with quercetin, appeared to pro-mote singlet oxygen quenching ability.

3. Hydroxyl Radical

The catechins were ranked as follows in theirability to protect mitochondrial membranes fromoxidation induced by hydroxyl radicals: ECG>EGCG>EC>gallic acid (GA)>gallocatechin (GC)>EGC>C.53 Generation of the DMPO-OH spin ad-duct by photolysis of H2O2 was inhibited by catechinswith hydroxyl radical scavenging activity rankedas ECG>EC>EGCG»EGC.44 Green tea polyphe-nols scavenged hydroxyl radicals generated frompulse radiolysis with higher rate constants than quer-cetin, caffeic acid, or sinapic acid.54 The epicatechingallates were between two and ten times more ef-fective than Trolox in reducing hydroxyl radical-

induced oxygen consumption and malondialdehydeformation, two measures of ROS scavenging activ-ity. EGCG showed maximum inhibition of bothphoto-enhanced and Fe3+/ADP-induced lipid per-oxidation in epidermal microsomes compared withother green tea catechins.6 A good correlationwas found between the anticlastogenic, or chromo-somal mutation-inhibiting, effect and the in vitroantioxidant capacity of tea flavonoids when lino-leic acid micelles were oxidized by hydroxyl radi-cals produced by Fenton chemistry.55

4. Peroxyl Radical

Catechins and quercetin were 5 to 20 timesmore effective than a-tocopherol in protecting phos-phatidylcholine liposomes from oxidation by peroxylradicals induced in the aqueous phase by 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH).56 Thecatechins were consumed faster than a-tocopherol,possibly due to their localization near the surfaceof the phospholipid bilayer and their proximity toaqueous phase oxygen radicals.

Theaflavins were more effective than vitaminE in protecting erythrocyte ghost membrane lip-ids from oxidation (TBARS formation) induced byterf-butyl-hydroperoxide.57 The theaflavins inhibit-ed lipid peroxidation by up to 80%, while an equi-molar concentration of vitamin E (25 ]lM) inhib-ited formation of TBARS by only 30%. Theaflavindigallate exhibited the strongest antioxidative activ-ity in this system. In a similar system using tert-butyl-hydroperoxide-induced oxidation of rat liverhomogenates, both theaflavin and thearubigin frac-tions purified from black tea infusions inhibitedformation of TBARS more effectively than glu-tathione, ascorbic acid (vitamin C), a-tocopherol,and synthetic phenolic antioxidants,58 although ECG,EGC, and EGCG were more active. The activity oflyophilized black and green tea was similar.

Catechins are able to effectively inhibit in vitroperoxynitrite-mediated tyrosine nitration.59 Peroxy-nitrite is a reactive species generated in vivo (e.g.,during inflammatory responses) and has been showncapable of oxidizing LDL to more atherogenic forms.60

EGCG, ECG, and GA were the most efficient scav-engers of peroxynitrite.59 In a murine macrophage

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system mimicking inflammation, EGCG reducedlipopolysaccharide and interferon-y-induced nitriteproduction.61 EGCG also effectively scavenged hypo-chlorite, a product of the enzyme myeloperoxidasethat is formed in response to bacterial infection.62

Catechins and theaflavins are clearly effec-tive in vitro antioxidants based on their ability toscavenge a variety of physiologically importantROS.

B. Scavenging of Stable Free RadicalSpecies by Tea Antioxidants

In contrast to the rather indiscriminate reac-tions with short-lived, highly reactive radicals suchas the hydroxyl radical, reactions with more stablelong-lived free radicals require that antioxidantspossess a good hydrogen-donating ability. Tea fla-vonoids have been shown to possess such an abil-ity in both aqueous and lipophilic systems.

Tea flavonoids are effective scavengers in theaqueous phase of the stable free radical of ABTS.27-32-43

Black tea and green tea beverages both have a potentantioxidant capacity relative to Trolox.43 Antiox-idants from tea are significantly more potent radi-cal scavengers in this system than the well-recog-nized antioxidants vitamin E and vitamin C, thecarotenes and the xanthophylls63 (Table 1). The epi-catechins displayed 2.4 to 4.9 times27 and the thea-flavins 2.9 to 6.2 times32 the antioxidant activityof Trolox.

In the lipophilic phase, tea flavonoids scav-enge the DPPH radical more effectively than vita-min E48-53-64"66 (Table 2). EGCG is the most effec-tive scavenger of the DPPH radical among othertea flavonoids and unfermented (green) tea bever-age was more effective than black tea.

Radical scavenging by tea beverages and puri-fied tea catechins in aqueous and lipophilic (etha-nolic) phases has also been investigated using thestable Fremy's radical (potassium nitrosodisulfo-nate) and the galvinoxyl radical (2,6-di-terf-butyl-a-(3,5-di-f e/?butyl-4-oxo-2,5-cydohexadien-1 -ylidene)-/?-tolyloxy) in conjunction with ESR.67

Green tea (0.0025% lyophilized extract in water/ethanol) was a better radical scavenger in both theaqueous and ethanolic systems than black tea ofequivalent concentration. The catechins were effi-

TABLE1Relative Antioxidant Potentials of Vitamins,Tea Beverage, Flavonoids, Carotenes,and Xanthophylls

Antioxidant

VitaminsVitamin CVitamin E

Tea beverageGreen tea (1000 ppm)Black tea (1000 ppm)

Flavan 3-olsEpicatechinEpigallocatechinEpigallocatechin gallateEpicatechin gallate

TheaflavinsTheaflavinTheaflavin 3-monogaIlateTheaflavin 3'-monogallateTheaflavin 3-3'-digallate

FlavonolsQuercetinKaempferolRutin

FlavonesApigeninLuteoiin

CarotenesLycopenea-CaroteneP-Carotene

Xanthophyllsp-CryptoxanthinZeaxanthinLutein

Trolox equivalentantioxidant capacity

(TEAC, mM)a

1.0 + 0.021.010.03

3.8 + 0.033.5 + 0.05

2.5 + 0.023.8 ±0.064.8 ±0.064.9 ±0.02

2.9 ±0.084.7 ±0.164.8±0.196.2 ±0.43

4.7 ±0.101.3 ±0.082.4 ±0.06

1.5 ±0.082.1+0.05

2.9 ±0.151.3 ±0.041.9±0.10

2.0 ±0.021.4 ±0.041.5±0.10

a TEAC is the millimolar concentration of a Trolox Solu-tion having the antioxidant capacity equipment to a1.0 mMsolution of the substance under investigation.

cient scavengers in both aqueous and ethanolic phases,with EGCG displaying highest activity irrespectiveof the radical used. Ranking of the other tea flavo-noids appears to be system dependent (Table 2).

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TABLE 2Scavenging of Stable Free Radicals by Tea Antioxidants

Stable radical Polar/apolar Detection mode

Fremy's saltFrem/s saltGalvinoxylGalvinoxylABTS-ABTS-DPPH*DPPH-DPPH-

DPPH-

DPPH'

DPPH-

DPPH-

PolarPolarApolarApolarPolarPolarApolarApolarApolar

Apolar

Apolar

Apolar

Apolar

ESRESRESRESRabs 734 nmabs 734 nmESRabs 521 nmabs 517 nm

abs 517 nm

abs 520 nm

ESR

ESR

Relative scavenging capacity

EGCG>ECG>EC>EGC>C>GAGreen tea > black teaEGCG>ECG»GA>EOC>EGCGreen tea > black teaTF-dg>TF-mg>TFECG>EGCG(Q>EGC>GA>EC(CEGCG=ECG>EGC>EC(C»vit C>vit EEGCG»ECG(GA>EC=C=rutin>vit EPouchong tea>green tea>oolong tea

>black teaEGCG>ECG>GC>EC>GA>EGC>vit C

>TroloxECG>EGCG>EGC>GA>EC>C>vit C

=vit EECG(tet)>ECG(tri)>ECG(di) =EGCG

>EGC>EC>vit C>vit EECG>EGCG

Ref.

Gardner et al.67

Gardner et al.67

Gardner et al.67

Gardner et al.67

Miller et al.32

Salah et al.27

Nanjo et al.65

Fourneau et al.68

Yen and Chen46

Hong et al.53

Yoshida et al.64

Hatano et al.48

Uchida et al.47

Note: ABTS**, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) radical cation; DPPH*, 1,1-diphenyl-2-picrylhydrazyl radical; TF-dg, theaflavin digallate; TF-mg, theaflavin monogallate; TF, theaflavin; EGCG,epigallocatechin gallate; EGC, epigallocatechin; ECG, epicatechin gallate; EC; epicatechin; C, catechin; GA,gallic acid; Q, quercetin.

In conclusion, theaflavins and catechin gal-lates are more effective scavengers in vitro ofaqueous and lipophilic stable radicals than manyother flavonoids and the antioxidant vitamins.

C. Protection of Low-DensityLipoprotein (LDL) from OxidationIn Vitro and Ex Vivo

Mounting evidence links oxidation of LDLwith atherosclerosis.68 Unlike native LDL, oxidizedLDL is readily taken up by macrophages, induc-ing the formation of lipid-laden foam cells, whichare precursors of the atherosclerotic fatty streak.69

LDL particles that accumulate in macrophages arederived from the circulating plasma LDL pool. Itis feasible to suggest that factors influencing theoxidizability of plasma LDL may also be relevantto factors influencing oxidation of LDL in the blood

vessel wall. Such an approach was first advocatedand subsequently characterized by Esterbauer andcolleagues,70 and the role of antioxidants in pre-venting LDL damage in vitro or ex vivo by prooxi-dants subsequently has been studied in depth.

LDL oxidizes readily in vitro when incubatedwith excess copper, heme iron proteins, thermolabileazo compounds that induce peroxyl radical forma-tion, other oxidizing agents such as peroxynitrite,or a variety of cell types.71 Three consecutive phasesin the LDL oxidation process can be identified: alag time during which all endogenous antioxidantsare consumed, a propagation phase during which theoxidation reaction proceeds at a maximum rate,and a decomposition phase during which the initialproducts of lipid peroxidation undergo breakdownand modify the protein moiety of LDL (apolipopro-tein B).72 Products of LDL oxidation can be detect-ed as conjugated dienes, TBARS, apolipoproteinB fluorescence, and changes in relative electropho-retic mobility.

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Green tea and vitamin E extended the lag timeof copper-induced LDL oxidation more effective-ly than an equivalent (0.5 \\M) concentration ofvitamin C.73 On co-incubation, the tea flavonoidsECG, EGCG, and theaflavin were effective in pre-venting copper-induced oxidation of LDL74 (Table 3).Epigallocatechins, especially EGCG and EGC, inhib-ited copper-induced LDL oxidation in vitro at signif-icantly lower concentrations than vitamins C andE, P-carotene, synthetic phenolic antioxidants, andother plant flavonoids.75-76 Catechin-inhibited cop-per-mediated LDL oxidation in a dose-dependentmanner with complete inhibition at 69 jiM.77 Thelag phase of copper-induced LDL oxidation was in-creased by catechins, but a prooxidant effect wasobserved during the propagation phase.78 This obser-vation implies that similar to vitamin C, when fla-vonoids are present at an excessive concentrationtogether with transition metals, catechin phenoxylradicals abstract electrons from non-radical spe-cies and accelerate the chain reaction of lipid perox-idation. It is not known if such prooxidant reac-tions occur in vivo.

Theaflavins also inhibited copper-induced invitro LDL oxidation.32 Theaflavin mono- and digal-lates were considerably more active than theaflavin(Table 3). Theaflavins appear to be less active thanthe gallocatechins in their ability to inhibit LDL oxi-dation,27-32-79 which may be a consequence of theirgreater polarity and their reduced ability to partitioninto lipid environments. No data are currently avail-able on the inhibition of LDL oxidation by the unde-fined catechin condensation products of black tea.

Catechins ranked as antioxidants in the lipo-philic phase as follows when lipid peroxidation inLDL was catalyzed by metmyoglobin: ECG=EGCG=EC=C>EGC>GA.27 The same order ofeffectiveness was observed for the ability of thecatechins to spare a-tocopherol in LDL. The useof metmyoglobin in this system to induce LDLoxidation, rather than copper, eliminated any po-tential confounding effects of the tea catechins asmetal chelators and allowed assessment of theirlipid peroxyl radical scavenging properties.

Purified green tea polyphenols prevented oxi-dative LDL modification by macrophages.80 LDLoxidation induced by various cells (mouse J774 mac-rophages, human monocyte-derived macrophages,and vascular endothelial cells) was inhibited by cat-

echin, which also inhibited macrophage uptake anddegradation of cell-modified LDL.77

Information about the association between teaflavonoids and lipid-rich LDL particles can be ob-tained from oxidation of LDL, which has been isolat-ed from plasma preincubated with tea flavonoids.Incubation of plasma with 50 mg/1 green tea ex-tract (equivalent to 160 \iM catechins based on anapproximate molecular weight for green tea cat-echins of 400 kDa) significantly extended the lagphase of copper-mediated LDL oxidation.81 Blacktea was less effective on a weight basis. A concen-tration of 100 mg/1 (equivalent to approximately320 |lM catechins) was required to enhance resis-tance of LDL to oxidation.

The effects of the individual catechins havebeen assessed in a similar manner.82 LDL isolatedfrom plasma containing 200 [iM EGCG or ECGhad a significantly longer lag time than LDL isolat-ed from catechin-free plasma. Preincubation of plas-ma with EGCG prolonged the lag time to more thanthree times that of the control, whereas only a two-fold increase in lag time was seen with an equiva-lent concentration of vitamin E. Higher concentra-tions of EC and EGC (400 \xM in plasma) wererequired to achieve significant effects. In the samestudy, the theaflavins were more effective than thecatechins in preventing LDL oxidation. The thea-flavins were ranked as follows in prolonging lagtime: theaflavin digallate >theaflavin monogallate>theaflavin. However, the theaflavins were foundto be less effective than the catechins in co-incuba-tion studies.32 The ranking of theaflavin antioxi-dant activity in preincubation of plasma and co-incubation with LDL test systems was found to besimilar (Table 3).

The ability of tea antioxidants to inhibit LDLoxidation in vivo is also determined by absorptionand distribution of these compounds into LDL andeanonly be assessed through intervention trials. Con-sumption of green or black tea (six cups per dayfor 4 weeks) by volunteers had no effect on exvivo, copper-mediated LDL oxidation.81 In thisstudy, 642 mg/d catechins were obtained fromgreen tea, while 215 mg/d catechins and 15 mg/dtheaflavins were consumed as a result of black teaconsumption. In a separate study, a slight butsignificant increase in resistance of LDL to exvivo oxidation was found following consumption

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10TABLE 3Prevention of In Vitro LDL Oxidation by Tea Antioxidants

Prooxidant

Cu2+

Cu2+

Cu2*

Cu2+

Cu2*Cu2+

Cu2*

Cu2+

Cu2+

Cu2+

Cu2+

Cu2+

Cu2+

Cu2+

Cu2+

PeroxynitriteMetmyoglobin

MacrophagesMacrophages

MacrophagesJ774 macrophages, vascular

endothelial cells, humanmonocyte-macrophages

Detected parameter

Conjugated dienes (lag phase)

Conjugated dienes (lag phase)Conjugated dienes (lag phase)

TBARS

Conjugated dienes (lag phase)

TBARSConjugated dienes (lag phase)

Conjugated dienes (lag phase)apo B fragmentationConjugated dienes (lag phase)TBARSapo B fragmentationTBARSTBARSTBARSREMTBARS/REM

TBARSLDL uptake by scavenger

receptorapo B fluorescence/REM

TBARS

Relative scavenging capacity

TF-dg>EGCG>TF-mg>ECG>TF>EGC>ECa

EGCG>vit Ea

GT (50 mg/ml) > BT (50 mg/ml)fl

EGC and EC inhibitory in initiation phase, accelerative inpropagation phase

GT(vit E>vit CTF-dg>TF3-mg>TFEGCG>EC>quercetin>chlorogenic acid >resveratrol>rutin>vit E

>hesperetin>genistein

sesaminol>Q>EGCG>TF»myricetin>BHT >a-tocopherolEGCG>BHT>a-tocopherolEGCG>ECG>EC>C>EGCECG>EGCG>EC>C>EGC>BHTEGCG>C

Flavanols most effective flavonoidsEGCG>EGC>ECG>C>BHT>vit C>vit E >(i-carotene(+)-C complete inhibition at 20 ng/mlECG>GA>EC=EGC=EGCGEGCG=ECG=EC=C>EGC>GAGT polyphenols inhibited TBARs formationGT polyphenols inhibited LDL uptake by scavenger receptors

GT polyphenols inhibited apo B fragmentationInhibition by (+)-C at 50 ug/ml

Ref.

Ishikawa et al.82

Ishikawa et a!.62

van net Hof et al.»1

Yamanaka et al.78

Luo et al.73

Miller et al.32

Vinson et al.75

Miura et al.79

Miura et al.79

Miura et al.74

Miura et al.74

Miura et al.74

Vinson et al.75

Vinson et al.76

Mangiapane et al.77

Pannala et al.59

Salah et al.27

Zhenhua et al.80

Zhenhua et al.80

Zhenhua et al.80

Mangiapane et al.77

Note: TF-dg, theaflavin digallate; TF-mg, theaflavin monogallate; TF, theaflavin; EGCG, epigallocatechin gallate; EGC, epigallocatechin; ECG, epicatechin- gallate; EC; epicatechin; C, catechin; GA, gallic acid; GT, green tea; BT, black tea; Q, quercetin; BHT, butylated hydroxytoluene; TBARS, thiobarbituric

acid reactive substances; REM, relative electrophoretic mobility.

a Antioxidants preincubated with plasma prior to LDL isolation.

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of 750 ml black tea/d (containing 372 mg total cat-echins and 4 mg theaflavin digallate) for 4 weeks.82

The mean lag time for LDL oxidation was signifi-cantly increased from 54 to 62 min following blacktea consumption, whereas no change in lag timewas observed in the group consuming water. Totalcatechin consumption was higher in the latter82 study(372 vs. 215 mg/d), although theaflavin consump-tion was lower (4 vs. 15 mg/d). These differencesin tea antioxidant consumption may account forthe demonstration of a significant effect on ex vivoLDL oxidation in the study of Ishikawa et al.82

That the effect is due to some other dietary vari-able cannot be ruled out, as statistical compari-sons were made only within treatment groups andnot with the control group. The findings from thisstudy are, however, consistent with an increasedin vivo antioxidant activity from tea drinking, andthis aspect will be clarified as more studies arereported.

In conclusion, results from in vitro studies, usingeither copper, metmyoglobin, or cells as prooxidants,show that tea flavonoids (catechins and theaflavins)effectively scavenge aqueous and lipophilic radi-cals and protect LDL from oxidation more effectivelythan established antioxidants. The gallocatechinswere consistently the most protective flavonoids.Gallate substitution of theaflavin-enhanced LDLprotective activity. The inhibition mechanism of thetea flavonoids did not appear to be dependent onmetal ion chelation properties. LDL high in vitroplasma concentrations of tea are necessary to re-cover small amounts of bound tea flavonoids fromisolated LDL. The levels of tea flavonoids boundto LDL in vivo remain to be established.

V. EFFECT OF TEA FLAVONOIDSON IN VIVO ANTIOXIDANT CAPACITYAND INHIBITION OF OXIDATIVEDAMAGE

A good direct method for determining the anti-oxidant activity of a compound in vivo in either ani-mals or man is not currently available. Biomarkersof oxidative damage such as oxidized deoxynucle-osides (8-hydroxydeoxyguanosine (8-OHdG)), plas-ma and urinary thiobarbituric reactive substances(TBARS) or malondialdehyde (MDA) values, micro-nuclei formation, sister chromatid exchange, F2-iso-

prostanes, and LDL oxidation ex vivo (see above)are often employed as surrogates for in vivo antiox-idant activity. In biomarker studies, oxidative stressis often induced by radiation, chemical oxidants,tobacco smoke, or diet.

A. Animal Models

Tea flavonoids consistently reduced oxidativedamage in animals exposed to radiation, chemicaloxidants, or dietary stress. Mice fed several dif-ferent flavonoids were gamma-irradiated to cre-ate hydroxyl radical-induced DNA damage in vivo.Treatment with hydroxylated flavan-3-ols and fla-vonols significantly reduced formation of micro-nucleated reticulocyctes, a marker of DNA dam-age.83 EGCG provided to mice in drinking waterfor 1 month significantly blocked radiation-inducedoxidative damage to hepatic lipids and improvedthe 30-d survival time after irradiation by 33%.84

The carcinogens 2-nitropropane and 4-(methyl-nitrosamino)-l-(3-pyridyl)-l-butanone (NNK) in-duce oxidative damage to DNA. Rats provided greentea infusion or a tea polyphenol isolate (standard-ized to the same level of EGCG as the green tea) for2 weeks prior to exposure to 2-nitropropane had sig-nificantly lower levels of hepatic 8-OHdG adductsand lipid peroxides.85 In another study, NNK-in-duced 8-OHdG adducts and lung tumors were sig-nificantly reduced in mice drinking green tea orEGCG.86 Oral administration of tea flavonoids torodents apparently protects hepatic and pulmonarytissues from oxidative damage induced by chemi-cal carcinogens.

Excess intake of some dietary nutrients can over-whelm normal antioxidant defense systems lead-ing to an oxidative stress condition. Rats fed a highprotein stress diet (18% (w/w) casein and 0.75%(w/w) adenine) supplemented with either a catechinmixture or EGCG excreted less urinary methylgua-nidine, an indirect renal marker for hydroxyl rad-ical damage.87 Supplementation of a diet high inpolyunsaturated fat (30% perilla oil) with catechins(1 % of diet) reduced the level of oxidative damage(TBARS) in rat plasma and erythrocytes and hada sparing effect on plasma a-tocopherol.88 Ex vivolipid peroxidation determined in rat liver was reducedfollowing feeding of diets containing 3% (w/w)

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green or black tea for 50 d.89 Long-termsupplementation (19 months) of green tea polyphe-nols to rat diets significantly reduced the level ofplasma TBARS.90

Although the evidence for an in vivo role for teaflavonoids is still limited, tea flavonoids demonstratein vivo protective activity against oxidative stressinduced by radiation, chemicals, or diet in animalstudies.

B. Human Studies

1. Effect of Tea Flavonoids on PlasmaAntioxidant Activity

One study has demonstrated a significant in-crease in plasma total antioxidant status in humanvolunteers between 30 and 60 min after consump-tion of 300 ml of either green or black tea preparedby infusion of 2 g of tea leaves with 100 ml water.91

However, in a separate study, an enhancement ofthe serum total antioxidant activity after consump-tion of 500 ml black tea (1.0 g tea leaf infused with100 ml water) was not found.92 A single drink of teamay not be sufficient to induce a consistently mea-surable change in plasma antioxidant status.

In a 4-week intervention trial in non-smokers,consumption of green or black tea (six cups perday) had no effect on plasma levels of malondial-dehyde, LDL lipid hydroperoxide levels, or plasmalevels of antioxidant vitamins (ascorbic acid, cc-tocopherol, or carotenoids).81 However, green teaconsumption did result in a small but significantincrease in the total antioxidant activity of plasma.

2. Effects of Tea Flavonoids onBiomarkers of Oxidative DNA Damage

Tobacco smoke contains many ROS that cancause oxidative tissue damage in smokers.93 Tea drink-ing has been shown to reduce oxidative biomarkersin chronic smokers, such as increased sister chroma-tid exchange (SCE) and micronucleation in lym-phocytes.94-95 In a case-controlled population study,tea drinking among smokers was associated with asignificantly lower level of smoking-induced micro-nuclei in peripheral-blood lymphocytes.94 Also, smok-ers who consumed 2 to 3 cups of green tea per day

for 6 months had significantly lower levels of SCEin mitogen-stimulated white blood cells than smok-ers who consumed 2 to 3 cups of coffee or otherbeverages per day.95 Although evidence is still lim-ited, tea drinking appears to protect against smok-ing-induced oxidative damage, suggesting an invivo antioxidant effect.

VI. CONCLUSION

Tea is an abundant source of flavonoid anti-oxidants, mostnotably gallatedcatechins andtheafla-vins, which are effective in vitro scavengers of ROSand free radicals in both the aqueous and lipophil-ic phases. Proposed mechanisms of tea flavonoidantioxidant activity include hydrogen donating abil-ity, delocalization of electrons, and metal ion che-lation. Tea beverage has greater in vitro antioxi-dant capacity than most fruits and vegetables perserving and is more potent than vitamins C, E,and the carotenoids. Tea beverage and its flavonoidsprotect LDL from oxidation following co-incuba-tion in vitro, and there is some evidence to sug-gest that tea drinking also protects LDL ex vivo.Tea flavonoids demonstrate in vivo protection insmokers and in rodents exposed to oxidative stressinduced by radiation, chemicals, or diet, and in-creased plasma antioxidant status has been foundin human volunteers after tea consumption. Morein vivo data are required to establish the efficacy oftea flavonoids as biological antioxidants, in par-ticular in relation to established antioxidants. Effi-cacy in vivo will be dependent on factors in addi-tion to those that can be demonstrated in vitro, suchas free radical scavenging activity and metal ionchelation. These factors include bioavailability andlocalization to the required site of oxidative chal-lenge at effective levels and the potential for pro-oxidant effects. Available evidence from the limit-ed number of in vivo studies suggests that tea beverageis potentially a good dietary source of non-nutrientantioxidants, but additional studies are required.

ACKNOWLEDGMENTS

The authors thank Michael Albano for hiseditorial and technical assistance in preparation

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of this manuscript and Professor A. Bast and Dr.G.R.M.M. Haenen for their critical review.

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