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The chemistry of tea flavonoidsDouglas A. Balentine a , Sheila A. Wiseman b & Liesbeth C. M. Bouwens ba LIPTON , 800 Sylvan Avenue, Englewood Cliffs, NJ, 07632 Phone: 201–894–7338 Fax:201–894–7338 E-mail:b Unilever Research Laboratorium , Vlaardingen, The NetherlandsPublished online: 29 Sep 2009.

To cite this article: Douglas A. Balentine , Sheila A. Wiseman & Liesbeth C. M. Bouwens (1997) The chemistry of teaflavonoids, Critical Reviews in Food Science and Nutrition, 37:8, 693-704, DOI: 10.1080/10408399709527797

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Critical Reviews in Food Science and Nutrition, 37(8):693-704 (1997)

The Chemistry of Tea Flavonoids

Douglas A. Balentine,1* Sheila A. Wiseman,2 and Liesbeth C. M. Bouwens3

1Lipton, Englewood Cliffs, New Jersey; 2Unilever Research Laboratorium Vlaardingen, The Netherlands;3Unilever Research Laboratorium Vlaardingen, The Netherlands

* Address for correspondence: Douglas A. Balentine, Ph.D., LIPTON, 800 Sylvan Avenue, Englewood Cliffs, NJ 07632. Tel: 201-894-7338; Fax: 201-894-7017; E-mail: douglas.balentine@unilever.com

I. TEA

Tea is the fragrant brew prepared from the leavesof two varieties of the plant Camellia sinensis: assa-mica and sinensis. Originating in China and South-east Asia, tea has been cultivated and consumed formore than 2000 years.1 The Chinese value tea forits pleasant flavor and medicinal benefits, some ofwhich have scientific merit today (Table I).2-3

Tea was first introduced into continental Europeby the Dutch at the beginning of the sixteenth cen-tury and reached England and North America bythe mid-1600s. Tea has become an important com-mercial product throughout the world with annualproduction of 2,610,569 metric tons in 1996 (Fig-ure I).4 A comprehensive overview of the historyand agriculture of tea is available in "Tea: Cultiva-tion to Consumption".5

The distinctive color, flavor, and aroma of tearesult from chemical changes that occur during leafprocessing. Tea leaf contains flavonoids and methyl-xanthines (Table 2), unique bioactive compounds

that contribute to the organoleptic profile of thebeverage and are being studied to determine theirpotential role in prevention of chronic diseases suchas cancer and cardiovascular disease. The physio-logical activity of tea flavonoids is in part due toantioxidant function characterized by redox chem-istry and the ability to scavenge reactive oxygenspecies (ROS). The bioactivity of flavonoids alsoappears to be mediated by nonantioxidant mecha-nisms such as modulation of signal transductionpathways,6 proliferation at Gl phase of the cellcycle,7 and the immune response.8 However, themechanisms of tea bioactivity and their relevanceto human health remain to be established.

II. COMPOSITION OF FRESH TEA

AND BIOSYNTHESIS

OF TEA FLAVONOIDS

Flavonoids are 2-phenyl benzopyran (Figure 2)-based compounds that are subdivided into six class-

TABLE 1Traditional Health Claims for Tea

Traditional claims

Improved blood flowElimination of alcohol and toxinsClear urine and improve flowRelieves joint painImproved resistance to diseases

Adapted from Ref. 2.

Possible scientific basis

Vassodialation and decrease platelet activityIncreased activity of phase I and phase II enzymesDiuretic effectsAntiinflammatory activityPrevention of cancer and coronary heart disease

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

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<D4

Turkey " " " * ^ 1114,540

South America^56,500 " *^^

Kenya-*~~

257,162

BpS—

India779,996

/ /X /

Sri Lanka258,427

- \Bangladesh \55,129 •

Indonesia144,000

CIS(Former USSR)8,000

Japan\ 88,709

China\ 593,386

\\

Australia andPapua New Ginea8,100

FIGURE 1. World tea production 1996 (metric tons).Dow

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TABLE 2Composition of the Tea Leaf

Component

FlavanolsFlavonols and flavonol glycosidesPhenolic acids and depsidesOther polyphenolsCaffeineTheobromineAmino acidsOrganic acidsMonosaccharidesPolysaccharidesCelluloseProteinLigninLipidsChlorophyll and other pigmentsAshVolatiles

Percentage ofdry weight

25.03.05.03.03.00.24.00.54.0

13.07.0

15.06.03.00.55.00.1

(-)-epigallocatechin and (-)-epigallocatechin gal-late (Figure 3), colorless, water-soluble compoundsthat contribute bitterness and astringency to greentea. Flavonols such as quercetin, kaempferol, my-ricitin, and their glycosides, which are character-ized by a 4-oxo 3-hydroxy C ring, are found in tea(Figure 4). Flavonol glycosides make up 2 to 3%of the water-soluble extract solids of tea. The fla-vonol aglycones are not found in significant quan-tities in tea beverage due to their poor solubilityin water.9-10

OH

Adapted from Refs. 9, 10, 16.g

es: flavones, flavanones, isoflavones, flavonols, fla-vanols, and anthocyanins.

Flavanols and flavonols, the main classes foundin tea, are 30% of the dry weight of fresh leaf. Cat-echins (flavan-3-ols), the predominate form, are char-acterized by di- or tri-hydroxyl group substitutionof the B ring and the meta-5,7-dihydroxy substitu-tion of the A ring. Fresh tea leaf contains four majorcatechins: (-)-epicatechin, (-)-epicatechin gallate,

5'61

FIGURE 3. Major tea catechins.

Epicatechin EC H HEpicatechin gallate ECG Gallate HEpigallocatechin EGC H OHEpigallocatechin gallate EGCG Gallate OH

OGlycoside

OH O

FIGURE 4. Major tea flavonols.

5 4

FIGURE 2. Basic flavonoid structure (2-phenylbenzopyran).

Kaempferol glycosideQuercitin glycosideMyricitin glycoside

KaG HQuG OHMyG OH

HHOH

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A. Biosynthesis of Tea Flavonoids Proteins and Amino Acids

The pathways for the de novo biosynthesis offlavonoids in both soft and woody plants have beenelucidated and reviewed in detail.11 Flavonoids areformed from condensation reactions of cinnamicacids and acetic acid. Involved with the biosynthe-sis of tea flavonoids are the enzymes of the shiki-mate/arogenate pathway, especially 5-dehydroshUri-mate reductase, a key regulatory enzyme. Cinnamicacid biosynthesis is controlled by phenylalanineammonia lyase. The regulation of flavonoid bio-synthesis in tea has not been well characterized.

B. Methylxanthines

Tea leaf contains 2.5 to 4.0% caffeine (dry weightbasis) and much smaller quantities of the relatedmethylxanthine theobromine. Theophylline has beenreported as a tea constituent;12 however, it has notbeen detected in tea beverage using current analyt-ical techniques13 and is not formed by the biosyn-thetic pathway for methylxanthines in tea.14 A180 ml serving (6 ounces) of tea contains -60 mgof caffeine, compared with —100 mg of caffeine ina 180-ml serving of freshly brewed coffee. Black,green, and oolong tea beverages contain about thesame amount of caffeine when prepared using thesame amount of leaves. The amount of caffeine intea beverage is determined by the brewing condi-tions of time, temperature, leaf size, and amount oftea. Decaffeinated teas are available and containless than 5 mg of caffeine in a 180-ml serving.

C. Minor Phenolic Compoundsand Minerals

Tea beverage contains phenolic acids, gallicacid, and its quinic acid ester theogallin that areeasily detected by HPLC.15 Potassium, calcium, mag-nesium, and aluminum are the predominant miner-als found in the ash (10 to 15% w/w) portion of thewater-soluble extract solids of tea.16-17 Tea beverageis a significant source of fluoride at -1 mg/serving.

Tea beverage is —17% w/w nitrogenous ma-terials as protein (-6% w/w) and amino and nucleicacids (-1.0% w/w). Theanine (y-n-ethyl glutamine)(-3% w/w) is one of the 19 amino acids in greenand black tea16-18 and is unique to tea. Amino aciddegradation is involved with biogenesis of aroma.The distinctive flavor of Japanese green tea is inpart due to amino acids.

E. Enzymes

The enzymes most important to the chemistryand manufacturing of tea are those responsible forthe biosynthesis of tea flavonoids and those in-volved in the conversion of fresh leaf into manu-factured commercial teas.

Polyphenol oxidase (PPO) (EC 1.14.18.1;monophenol monooxygenase [tyrosinase] or EC1.10.3.2; O-diphenol: O2 oxidoreductase) is oneof the more important enzymes involved with theformation of black tea polyphenols.16-19 The en-zyme is a metallo-protein thought to contain a bi-nuclear copper active site. Reviews of PPO are avail-able.19-21 Tea PPO reacts effectively with both 3'to 4 ' and 3' to 4' to 5-hydroxylated catechins withspecificity for the o-diphenol. PPO has good func-tionality in the pH range 4.6 to 5.6.9

Peroxidase (POD) (EC 1.11.1.7) is found infresh green leaf.22-23 It is a haemin-based enzymethat catalyzes the reductive decomposition of hy-drogen peroxide to water and organic peroxidespecies to the corresponding alcohol. PPO isthought to produce peroxide, which activates thePOD system. However, catalase is quite active intea and rapidly removes peroxides as they form,thereby limiting the role of POD in fermentationreactions.9-10

F. Analytical Methods

Methods are available to quantify many fla-vonoid phytochemicals in vegetables, fruit,24 andteas.25

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The total polyphenolic content of teas and otherpolyphenol-rich foods is best estimated using colori-metric techniques such as the Folin-Denis or Pruss-ian Blue assays that are based on redox reactionsmostly specific to phenolic groups.26 These nonspe-cific methods are useful for estimating the amountsof undefined polyphenols in tea beverage.

High-performance liquid chromatography (HPLQtechniques typically use reverse-phase C-18 meth-ods based on a gradient of acetonitrile and water toquantify the flavonoids in tea. These methods oftenpermit the simultaneous determination of flavonoidsand caffeine. Accurate HPLC methods for analy-sis of catechins,25-27 the flavonols—either as glyco-sides28 or hydrolyzed into free form,29 the theafla-vins25 and caffeine30 — are available. Gallic acid,theogallin, and the chlorogenic acids31 can also beanalyzed using HPLC methods. Methods are alsoavailable to estimate the level of catechins32-33 andtotal catechins34 in biological fluids.

Quantitative analysis of the undefined condensedflavonoids in black tea is not currently possible.One useful method of estimating the undefined poly-phenols in tea is to express these constituents as thedifference between known polyphenols determinedthrough quantitative methods such as HPLC and thetotal polyphenols estimated using the Folin-Denisor Prussian Blues assays. The latter assays can bemade more quantitative if defined polyphenolic mix-tures from tea are used as standards in lieu of gallicacid.

III. MANUFACTURING

Freshly harvested tea leaves require manufac-turing to be converted into green, oolong, and blackteas (Figure 5). Green tea is processed in a man-ner designed to prevent the enzymatic oxidationof catechins. Green tea consumption is increasingworldwide, but Japan, the People's Republic ofChina, North Africa, and the Middle East are tradi-tionally the sites of greatest consumption. Oolongtea is partially oxidized tea manufactured primarilyin the People's Republic of China and Taiwan. Blacktea is the dominantly manufactured tea product world-wide made through a polyphenol oxidase-catalyzedoxidation of fresh leaf catechins. Detailed reviewsof the manufacturing of tea are available.5-9

A. Chemistry of Tea Fermentation:Oxidation

Chemical reactions during the manufacture ofgreen, oolong, and black teas are responsible forthe development of their respective colors and fla-vors. During tea fermentation the colorless catechinsof green tea are converted to a range of products oforange-yellow to red-brown color through a series ofoxidative condensation reactions and numerous vola-tile flavor constituents are formed. These changes arereflected in the red-amber color, reduced astringency,and more complex flavor of black tea beverage.9-35

B. Flavanol Oxidation

The fermentation process is initiated by theoxidation of catechins to reactive quinones, a pro-cess catalyzed by the enzyme polyphenol oxidase.While the gallocatechins, epigallocatechin, and epi-gallocatechin gallate are preferred, polyphenol oxi-dase can use any catechin as a substrate to form thecomplex polyphenolic constituents found in blackand oolong teas.9-35

C. Theaflavins (TFs)

Theaflavins are a well-defined group of flavo-noid biopolymers produced through fermentation.Exhibiting a bright orange-red color in solution,they are important contributors of "brightness" andastringency, desirable attributes of tea beverage.36

Common to black tea are four main TFs thatvary in degree of gallation and several minor TFs,including the isotheaflavins and neotheaflavins (Fig-ure 6).10-35 The total TF content of black tea leavesdoes not usually exceed 2% and can be as low as0.3%. TF content can be readily determined by di-rect HPLC analysis of tea beverages.25 The quantityof TFs in black tea beverage is about one third thelevel of remaining catechins.

D. Theaflavic Acids and Theaflagallins

Gallic acid (GA) is not directly oxidized bypolyphenol oxidase but can be directly oxidized

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O)

00Fresh Leaf

\iijiii

iiiiiii iiiiiiiiiiiiiiiiiiiti

iiiiiiiiiiiiii

iiiiiii:::iiiiiii:::iiiiiiiii!

ii:::::::;

illIIIi•illiii

iiiiiii::::;:;iiiiiii•ilii:;pPr.:iiiiiiiiijiii:

::::iijl:

ii::::::;::

!:::..;:i:i::::::ii:tt:I::::::!:::::::::::::::I:-;:::-::-:!::::!:::::::::::;:::::;::::t:::;:

Green Tea Oolong Tea

:i:i:ii:iiii"•ii:i;;:S!

.;::::::..;:::::;::;

iiliiiiSHi

iiiiiii!:

Black Tea

FIGURE 5. Tea manufacturing process.

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OH

OR,

OH

O

OH

FIGURE 6. Theaflavins.

R2

TheaflavinTheaflavinTheaflavinTheaflavin

3-gallate3'-gallate3,3'-digallate

TFTF3GTF3'GTFDG

HGallateHGallate

HHGallateGallate

OH

HOOC

' '•OH OH

OH

to a gallic acid quinone.9'35 GA reacts with cat-echin quinones to form a group of compounds calledtheaflavic acids or with gallocatechin quinones toform theaflagallins such as epitheaflagallin (Fig-ure 7).37 The theaflavic acids are bright red, acidicsubstances present only in small quantities in black

35tea.

E. Bisflavanols (Theasinensins)

The paired condensation of two gallocatechinsform a group of colorless substances called bisfla-vanols (Figure 8).10-35 Reclassified as theasinesins38

and found in green and oolong teas,39 bisflavonolsare reactive compounds and are thought to rearrangeto form other undefined black tea flavonoids.

F. Condensed Tea Flavonoids

Catechins are reduced by ~85% during blacktea manufacturing, yet only ~10% can be account-ed for as theaflavins and theaflavic acids. The bal-ance of catechins form undefined, water-soluble

FIGURE 7. Epitheaflavlc acid and Epitheaflagallin.

polyphenolic substances thought to be the brownto black pigments of tea. These compounds havehistorically been termed "thearubigens", based onthe early work of Roberts.935 One subgroup of theseundefined flavonoids has been classified as proan-thocyandin polymers and form cyanidin and del-phinidin on acid hydrolysis.16 Recent attempts toseparate and purify the condensed flavonoids viachromatography and reverse phase HPLC have notled to further elucidation of these flavonoid conden-sation products.40-41 Black tea fractions defined astheafulvin40 and oolongtheanin39 have been isolated.

G. Consumption and Compositionof Tea Beverage

Hot black tea is most commonly consumedworldwide. Iced tea, however, accounts for 80% oftea consumption in the U.S. Tea is usually pre-pared from tea bags infused in hot water in a pro-portion of 1 g leaf to 100 ml water. The resulting

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TABLE 3Principal Components of Green and BlackTea Beverages (% wt/wt Solids)

OGallate] 0 H

OH OH

FIGURE 8. Bisflavonol A, or Theasinensin Aand Theassinensin F.

beverage typically has a solids concentration of~O.35% for a 3-min brew. A typical tea beveragecontains 2500 to 3500 ppm solids. Black and greentea beverages contain different polyphenols con-stituents due to the changes that occur during man-ufacturing (Table 3).

Green tea is most commonly used in Asia, espe-cially in Japan and China. The numerous reports ofhealth benefits associated with green tea drinkinghave increased the awareness of green tea in the U.S.

Sugar, lemon, herbs, fruit flavors, and spicesare commonly added to tea beverages as flavoringagents.

IV. REDOX PROPERTIES OF TEAANTIOXIDANTS

Catechins and flavonols scavenge ROS andfree radicals42-43 through several proposed media-

Flavonoid

CatechinsTheaflavinsSimple polyphenolsFlavonolsOther polyphenolsTheanineAmino acidsPeptides/proteinOrganic acidsSugarsOther carbohydratesCaffeinePotassiumOther minerals/ash

Green tea

30-42

226336274

3-65

5-8

Black t

3-102-6

31

23336274

3-65

5-8

Adapted from Refs. 9, 10, 16.

nisms, including delocalization of electrons, for-mation of intramolecular hydrogen bonds,44 and re-arrangement of their molecular structure.45-46 Thesetea polyphenols may also prevent oxidative reac-tions by chelating free copper and iron, which maycatalyze formation of ROS in vivo.47'49 The redoxchemistry of tea flavonoids provides a chemicalbasis for describing chemical reactivitiy as an elec-tron donor and, thus, antioxidant functionality.

The structural features thought to be responsiblefor flavonoid antioxidant activity are an o-dihydroxycatechol (3',4'-OH) arrangement on the B ring, ei-ther di- or tri-hydroxy-substituted catechins or fla-vonols, and the C2-C3 double bond in the C ringconjugated with a C4 carbonyl group found in fla-vonols.44-50-51 Antioxidant function is also thoughtto be promoted by C5, C7 dihydroxylation on theA ring.52 The antioxidant activity of catechins' isdetermined by the B ring catechol structure and isfurther enhanced in gallocatechins by the 5' hy-droxy group on the B ring.

The reduction potential of a compound pro-vides an estimate of the energy required to donatean electron; the lower the reduction potential, theless energy required to donate an electron and thehigher the expected antioxidant activity. The reduc-tion potentials of catechins, gallocatechins, querce-tin, and rutin for donation of a single electron were

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determined using differential pulse voltammetry withreference to a saturated calomel electrode (SCE).The measurements were made at 20°C and pH 6.15using 1 xaM aqueous solutions of the catechins andgallocatechins and 0.5 mM solutions of quercetinand rutin in 50% methanol (Table 4). Gallocatechinshad lower redox potentials than the simple catechins.The 5'-OH group in the B ring of the gallocatechinsis thought to allow for easier electron donation andthus better antioxidant activity.

Comparison of the redox potentials of catechinsand catechin gallates demonstrates that introductionof a gallate ester into the B ring of the catechinmolecule increases the energy required for electrondonation. The redox potential of EGC was lowerthan that of EGCG (0.09 V vs. 0.14 V). Differenc-es in redox potential between the catechin and epi-catechins were also observed with all epi formshaving significantly lower values. The differencewas most evident between EGC and GC, which haveredox values of 0.09 V and 0.13 V, respectively.The redox potentials of the simple catechins, EC,C, and EGC were similar. The redox potential ofquercetin was similar to the non-gallated epicat-echins, while the quercetin glycoside rutin has aredox potential higher than the catechin gallates.The redox potentials do not fully predict the rank-ing of these flavonoids as antioxidants in biologi-cally based systems.42-53154 Other mechanisms im-portant to determination of overall antioxidantfunctionality are delocalization of electrons, forma-tion of intramolecular hydrogen bonds,44 rearrange-

ment of molecular structure,4546 reactivity withother antioxdants, and chain termination events.

The redox potential of catechins and gallocat-echins was also determined using pulse radiolysisrelative to the hydrogen electrode (NHE) at pH7.43 All values relative to the NHE electrode havebeen corrected to the equivalent SCE value by sub-tracting a factor of 0.19 V to allow for comparisonof results. The same ranking of catechin redox poten-tial was found using both methods, but the valuesfound relative to the NHE electrode were clearlyhigher (possibly due to differences in pH): EGC0.23 V, EGCG 0.24 V, C 0.38 V, and EC 0.38 V.Determinations using the NHE show that EGC andEGCG have lower redox potentials than the redoxstandards Trolox (0.29 vs. SCE),55 rutin (0.41 vs.SCE),45 and 4-methoxyphenol (0.55 v.s SCE).56

Due to the lower redox potential of the EGC andEGCG radicals compared with the vitamin E radi-cal (~D 0.06 V), it is feasible that electron transferfrom the gallocatechins to the vitamin E radical (i.e.,regeneration of vitamin E) would occur in biolog-ical systems. The gallocatechins are potentially rel-evant biological antioxidants based on their redoxproperties.

The redox potential of theaflavins have beendetermined using both pulse voltammetry, with ref-erence to a SCE, and pulse radiolysis, with refer-ence to the NHE.57 At pH 6.15 relative to the SCE,the first redox potentials of the theaflavins are the-aflavin (TF) 0.16 V, theaflavin di-gallate (TFdG)0.19 V, theaflavin-3'-gallate (TF3'-G) 0.19 V, and

TABLE 4Redox Potentials of Catechins, Gallocatechins, Quercetin,and Rutin

Component 1st redox potential vs. SCE (V)

Epigallocatechin (EGC)Quercetin (Q)Gallocatechin (GC)Epigallocatechin gallate (EGCG)Gallocatechin gallate (GCG)Epicatechin (EC)Epicatechin gallate (ECG)Catechin (C)Rutin (R)Gallic acid (GA)

0.090.110.130.140.150.190.200.200.230.25

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theaflavin-3-gallate (TF3-G) 0.20 V; these valuesare similar to those of simple catechins and higherthan those of the gallocatechins. The reduction poten-tials of theaflavin radicals induced by pulse radioly-sis in neutral media (E, vs. SCE) are: TF = 0.32Vand TFdG = 0.35V;57 these values are higher thanthose of the gallocatechins and Trolox. The redoxpotentials of the theaflavins were similar, althoughgallate esterification tended to increase the redoxvalue. TF had the lowest first reduction potential;however, the TF gallates have higher antioxidantactivity than TF in the lipid phase,48 demonstrat-ing that redox potential is not a perfect indicatorof antioxidant activity.

Reduction potentials of the tea flavonoid radi-cals are also lower than those of the alkylperoxyl(E? = 0.86 V vs. SCE)58 and superoxide (E, = 0.75vs. SCE)59 radicals and therefore have the potentialto neutralize these reactive species based on redoxproperties. A good quantitative correlation was notobserved between the redox potential and abilityto inhibit lipid peroxidation,44 but a good qualita-tive relationship was found. Flavonoid compoundswith reduction potentials up to 0.2 V (vs. SCE)possess good antioxidant activity and flavonoidswith higher values are moderate antioxidants. Com-pounds that could not be oxidized at 1 V are veryweak or even inactive antioxidants. Flavonoids withredox potentials less than 0.06 V are able to redoxcycle under physiological conditions60 and are po-tentially prooxidant. None of the tea catechins dem-onstrated such a low reduction potential, althoughHodnick et al. determined the redox potential ofquercetin to be 0.06 V.

A. Protein Binding

Flavonoids readily bind to proteins through hy-drophobic interactions and hydrogen bonding.61 Athigh in vitro concentrations, tea flavonoids bind toproline-rich proteins and can readily inhibit enzy-matic activity accounting for many of the report-ed effects of tea on enzyme inhibition. However,at low concentrations binding of flavonoids to pro-teins appears to be more specific and may involvereceptor sites, and chemical structures suggest thattea flavonoids can function specifically modulatereceptors, enzymes, or regulatory proteins in vivo.

Tea flavonoids are competitive inhibitors of micro-somal glucuronidase,62 slow cell proliferation throughmodulation of API activation,6 and decrease growthof tumors cell lines at the Gl phase of the cell cycle.7

These properties of tea flavonoids suggest that thephysiological effects of tea on processes involvedwith chronic diseases such as cancer and coronaryheart disease involve more than simple antioxidantmechanisms. A better understanding of the physi-ological effects of tea and potential mechanismsrequires much more research.

ACKNOWLEDGMENTS

The authors thank Michael Albano for his edi-torial and technical assistance in the preparation ofthis manuscript.

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