The Chemistry and Biotransformation of Tea Constituents

Pharmacological Research 64 (2011) 87–99

Contents lists available at ScienceDirect

Pharmacological Research

jo urnal homepage: www.elsevier.com/locate/yphrs

Review

The chemistry and biotransformation of tea constituents

a,∗ b c d

Shengmin Sang , Joshua D. Lambert , Chi-Tang Ho , Chung S. Yang

Center for Excellence in Post-Harvest Technologies, North Carolina Agricultural and Technical State University,

North Carolina Research Campus, 500 Laureate Way, Kannapolis, NC 28081, USA

Department of Food Science, The Pennsylvania State University, 332 Food Science Building, University Park, PA 16802, USA

Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, NJ 08901, USA

Department of Chemical Biology, Rutgers University, 164 Frelinghuysen Road, Piscataway, NJ 08854, USA

a r t i c l e i n f o a b s t r a c t

Article history: Tea (Camellia sinensis, Theaceae) is one of the most widely consumed beverages in the world. The three

Received 30 December 2010

major types of tea, green tea, oolong tea, and black tea, differ in terms of the manufacture and chemical

Accepted 16 February 2011

composition. There are numerous studies in humans, animal models, and cell lines to suggest poten-

tial health beneﬁts from the consumption of tea, including prevention of cancer and heart diseases.

Keywords:

Many of the health beneﬁts have been attributed to the polyphenolic constituents in tea. Catechins

Tea

and their dimers (theaﬂavins) and polymers (thearubigins) have been identiﬁed as the major compo-

Chemistry

nents in tea. Methylation, glucuronidation, sulfation, and ring-ﬁssion metabolism represent the major

Stability

Biotransformation metabolic pathways for tea catechins. The present review summarizes the data concerning the chemistry

and biotransformation of tea constituents.

1. Introduction Different mechanisms of action have been proposed for the

observed beneﬁcial effects of tea polyphenols, based on studies in

Tea is one of the most widely consumed beverages in the world various cell line systems and animal models [1,4]. In the present

and is second only to water in popularity as a beverage. More than review, we will discuss the current knowledge on the chemistry

300 different kinds of tea are produced from the leaves of Camel- of tea, the analytical methods used to analyze tea constituents,

lia sinensis by different manufacturing processes. Generally they the stability and auto-oxidation of tea polyphenols, and the major

are divided into three types: green tea (non-fermented), oolong biotransformation pathways of tea constituents.

tea (semi-fermented), and black tea (fermented). About 78% of the

tea production worldwide is black tea, whereas green tea, mainly

2. The major components in tea

consumed in China and Japan, constitutes about 20%. Oolong tea is

partially fermented and constitutes about 2% of tea production. The

2.1. Catechins, ﬂavonols and ﬂavones

tea plant is considered native to south China and is now cultivated

in many other countries. The major tea-producing countries are

A typical tea beverage, with 2.5 g tea leaves in 250 mL hot

China, India, Japan, Sri Lanka, Indonesia and Central African coun-

water for a 3-min brew, usually contains 620–880 mg of water-

tries. Consumption of tea has been associated with many health

extractable solids [5,6]. Tea polyphenols, known as catechins,

beneﬁts including the prevention of cancer and heart disease [1–4].

usually account for 30–42% of the dry weight of the solids

These effects are attributed to the polyphenol compounds in tea.

in brewed green tea [5,6]. Catechins are members of a more

Catechins are the most abundant polyphenols in green tea. The

general class of ﬂavonoid, the ﬂavan-3-ols. They are character-

main pigments in black tea are theaﬂavins and thearubigins, which

ized by di- or tri-hydroxyl group substitution of the B ring and

are formed by the oxidation and polymerization of catechins dur-

the meta-5,7-dihydroxy substitution of the A ring. The struc-

ing the process known as fermentation. Although the thearubigins

−

tures of the four major catechins, ( )-epigallocatechin gallate

accounts up to 60% of the dry weight of black tea extract, the chem-

−

(EGCG), ( )-epigallocatechin (EGC), (−)-epicatechin gallate (ECG),

istry of thearubigins is still unclear.

and (−)-epicatechin (EC) are shown in Fig. 1. EGCG is the major

catechin in tea and may account for 50–80% of the total cat-

echins in tea. Gallocatechin gallate (GCG), gallocatechin (GC),

catechin gallate (CG), catechin (C), epigallocatechin digallates, epi-

∗

catechin digallate, 3-O-methyl EC, 3-O-methyl EGC, and afzelechin

Corresponding author. Tel.: +1 704 250 5710; fax: +1 704 250 5709.

E-mail addresses: [email protected], [email protected] (S. Sang). are present in smaller quantities [6]. 3 -O-methyl-EGCG and 4 -

88 S. Sang et al. / Pharmacological Research 64 (2011) 87–99

Fig. 1. Structures of the major catechins, ﬂavonols and ﬂavones in tea.

O-methyl-EGCG have been identiﬁed in different tea samples cream formation [6,9]. Their structures are well studied [6,9]. In

[7]. addition to the four major theaflavins (theaflavin; theaflavin 3-

Flavonols, including quercetin, kaempferol, myricitin, and their gallate; theaﬂavin 3 -gallate and theaﬂavin 3,3 -digallate) (Fig. 2),

glycosides (mono-, di-, and tri-) (Fig. 1), are also present in tea [6]. stereoisomers of theaﬂavins and a number of theaﬂavin derivatives,

They make up about 0.5–2.5% (w/w) extract as aglycone in tea infu- including theaﬂavic acids and theaﬂavates, have also been reported

sions. Apigenin, the only ﬂavone identiﬁed in tea, and its glycosides from black tea [12,6]. Our recent study showed that horseradish

have also been detected in tea but represent a very small fraction of POD can oxidize tea catechins to form theaﬂavin-type compounds

the tea polyphenols [6]. More recently, 19 O-glycosylated ﬂavonols, in the presence of H2O2 [12,13]. Eighteen benzotropolone deriva-

7 C-glycosylated flavones, 28 acylated glycosylated flavonols, and 3 tives, which include all the major theaflavins, theaflavates, and

flavonols were identified from green teas and fermented teas using theaflavic acids reported in black tea as well as several new

liquid chromatography with diode array and electrospray ioniza- benzotropolone derivatives, were synthesized by the reaction of

tion mass spectrometric detection (LC–DAD–ESI/MS) [8]. selected pairs of compounds, one with a vic-trihydroxyphenyl moi-

ety, and the other with an ortho-dihydroxyphenyl structure, using

2.2. Theaﬂavins, and thearubigins horseradish POD in the presence of H2O2 [12].

Thearubigins, which are red-brown or dark brown, are het-

The major polyphenols in black tea are theaﬂavins and thearu- erogeneous polymers of tea catechins [6,9]. Information about

bigins, which are formed by the oxidation and polymerization of their formation, structures and contribution to black tea quality

catechins during fermentation [6,9]. There are two major enzymes is still very limited. A partial structure of thearubigins from black

involved in the fermentation process in making black tea [6,9]. One tea was elucidated using chemical degradation, which indicated

is polyphenol oxidase (PPO), which plays a key role in the oxi- these compounds were heterogeneous polymers of ﬂavan-3-ols

dation of flavanols to black tea components, such as theaflavins and flavan-3-ol gallates having bonds at C-4, C-6, C-8, C-2 , C-

and thearubigins. It is mainly the action of this enzyme that pro- 5 , and C-6 in the ﬂavan-3-ol units [9]. In addition, the possible

duces theaﬂavins. Many studies have been carried out on the participation of theaﬂavins in the formation of thearubigins has

PPO-catalyzed formation of black tea oxidation products [6,9]. Per- been suggested [9]. It was shown that horseradish peroxidase

oxidase (POD), which can also catalyze the oxidation of o-diphenols would oxidize theaﬂavins into thearubigins-like compounds in the

to their quinones using peroxide, such as hydrogen peroxide, that presence of H2O2 [14]. We found that the galloyl ester group of

was formed by the action of PPO on certain ﬂavanols. In fresh tea theaﬂavin 3-gallate is as reactive as the B-ring (vic-trihydroxy) of

leaves the speciﬁc POD activity is more than ﬁve times higher than EGCG or EGC and the galloyl ester group of ECG, and can further

that of PPO and was found to increase during black tea manufac- react with EC to form the new theaﬂavin type tea catechin trimer,

turing [10]. During the ﬁring step of black tea processing PPO is theadibenzotropolone A, which was characterized from black tea

inhibited thermally, whereas POD remains active to a certain extent extract by LC/ESI–MS/MS [15]. Two additional peaks correspond-

[11]. Model oxidation systems have also been used to compare the ing to the same molecular weight as that of theadibenzotropolone

oxidation products obtained with tea PPO and with horseradish A were also found in black tea extract. To identify these unknown

POD [11]. However, the contribution of POD to the formation of peaks, two new dibenzotropolones, theadibenzotropolone B and C,

black tea pigment in tea fermentation is still not clear. together with one new tribenzotropolone, theatribenzotropolone

Theaﬂavins are orange or orange-red in color and possess a ben- A (Fig. 3), were synthesized by the reaction of theaﬂavins and

zotropolone skeleton that is formed from co-oxidation of selected tea catechins catalyzed by horseradish POD in the presence of

pairs of catechins, one with a vic-trihydroxyphenyl moiety, and H2O2 [13]. Theaﬂavin 3-gallate can react with not only EC to form

the other with an ortho-dihydroxyphenyl structure [12,6]. It is theadibenzotropolone A, but also catechin to form the isomer of

known that theaﬂavins make important contributions to the prop- theadibenzotropolone A, theadibenzotropolone B. Whereas the iso-

erties of black tea, such as color, ‘mouthfeel’, and extent of tea mer of theaﬂavin 3-gallate, neotheaﬂavin 3-gallate, could react

S. Sang et al. / Pharmacological Research 64 (2011) 87–99 89

OH OH

OR2 OH OH OH

HO O OH HO O OH

O O

HO O HO O OH OH

OR1 OR

OH OH

Theaflavin: R1=R2=H NeoTheaflavin: R=H

Theaflavin 3-gallate: R1=Galloyl, R2=H NeoTheaflavin 3-gallate: R=Galloyl

Theaflavin 3'-gallate: R1=H, R2=Galloyl

Theaflavin 3,3'-digallate: R1=R2=Galloyl

OH OH OR OH OH OH

HO O OH HO O OH

O O

OH OH O OH O OH O OH O OH

HO O OH HO O

Theaflavate A: R=H

Theaflavate B: R=Galloyl NeoTheaflavate A

OH OH OH OR OH OH

HO O OH HO O OH

O O

OH OH

HOOC HOOC

Epitheaflavic acid: R=H Theaflavic acid

Epitheaflavic acid 3'-gallate: R=Galloyl

Fig. 2. Structures of the major theaﬂavins in black tea.

with EC to form theadibenzotropolone C. Interestingly, theatriben- erate benzotropolone-type polymers further indicating this type

zotropolone A, instead of the two isomers of theadibenzotropolone of oxidation is one of the major pathways for the formation of

expected, was obtained by the reaction between theaﬂavin 3,3 - thearubigins [16]. More recently, Kuhnert and co-workers reported

digallate and EC. We failed to obtain theaﬂavin 3 -gallate related that thearubigins are solely composed from low molecular weight

theadibenzotropolone derivatives. We further conﬁrmed the exis- compounds with a mass below 2100 g/mol based on results gener-

tence of theadibenzotropolone B in the black tea extract prepared ated from MALDI-TOF-MS [17]. Whether this conclusion is due to

from Yunnan Black tea using tandem mass spectrometry. In order the limitation of current analytical tools to detect larger molecular

to clearly understand the potential biological activities of black tea, weight components in thearubigins is a topic for further study in

it is important to determine the existence of these new di- or tri- order to completely understand the chemistry of thearubigins.

benzotropolone-type compounds in different black tea samples.

Our results clearly show that theaﬂavins can further react with 2.3. Other components

tea catechins to form di- or tri-benzotropolone-type compounds.

The observation that the galloyl ester group of theaﬂavins can Theasinensins, which are dimeric gallocatechins linked by C–C

be oxidized to form di- or tribenzotropolone skeletons strongly bonds, are present mainly in oolong tea. Theasinensin A (EGCG

implies that this type of oxidation is an important pathway to dimer), B (EGCG and EGC dimer), C (EGC dimer), and F (EGCG and

extend the molecular size of thearubigins. Theadibenzotropolone ECG dimer) are the most abundant ones (Fig. 4) [18]. Theasinensin

A and B are the first theaflavin type trimers of catechins in black A has been identified as the major oxidation product of EGCG under

tea. Using delayed pulsed ion extraction of ions generated via cell culture conditions as well [19].

the matrix-assisted laser desorption ionization (MALDI) technique, Gallic acid and its quinic acid ester, theogallin (Fig. 4), are the

on line with a Linear time-of-ﬂight (TOF) mass spectrometer, we major simple polyphenols found in tea [6]. The amount of gallic acid

found that theasinensins and/or procyanidins, which are dimers in black tea is signiﬁcantly increased due to the de-esteriﬁcation

or oligomers of catechins, could also react with catechins to gen- of the 3-galloyl substituted catechins either by native esterase or

90 S. Sang et al. / Pharmacological Research 64 (2011) 87–99

Fig. 3. Structures of theadibenzotropolone A, B, C and theatribenzotropolone A.

oxidative degallation during the fermentation. While the amount reported that there is no difference regarding the caffeine content

of theogallin will decrease during the fermentation process due between green and black teas indicating that caffeine is very stable

to the formation of a new theaﬂavin-type compound, theagallinin, during the fermentation process. Tea contains around 17% nitroge-

which is the condensation product between EC and theagallin [6]. nous materials as protein (∼6%) as well as amino and nucleic acids

∼

In addition, cinnamic acid derivatives of quinic acid, the coumaryl ( 8%). Among the 19 amino acids found in tea, theanine (␥-N-ethyl

and caffeoyl-quinic acids (Fig. 4) have also been identiﬁed from tea glutamine) (Fig. 4) is unique to tea. It is a signiﬁcant component of

[6]. both green tea and black tea, comprising about 3% (w/w) of extract

Tea water-extractable solids contain about 2–5% caffeine and solids [5,6]. Theanine has been associated with improved ﬂavor and

much smaller quantities of theobromine and theophylline (Fig. 4) antihypertensive effect. It can also be made synthetically on a com-

[5,6]. The amount of caffeine in tea beverage is determined by mercial scale with good yield. Potassium, calcium, magnesium, and

the leaf size, the brewing time, and the temperature. It has been aluminum are the predominant minerals found in the ash (10–15%)

S. Sang et al. / Pharmacological Research 64 (2011) 87–99 91

R O O 3 R2 HO R1 N N

HO O OH O N N R1

CH3

HO O OH Caffeine: R1=R2=Me

Theobromine: R1=H, R2=Me

OH Theophylline: R1=Me, R2=H OR2

OH NH2

Theasinensin A: R1=OH, R2=R3=Galloyl COOH

Theasinensin B: R1=OH, R2=Galloyl, R3=H

Theasinensin C: R1=OH, R2=R3=H

Theasinensin F: R1=H, R2=R3=Galloyl Theanine

COOR R

O OH

HO OH

HOOC Coumarylquinic acid: R=H

Gallic acid: R=H Chlorogenic acid: R=OH

Theogalline: R=Quinic acid HO OH

Fig. 4. Structures of the major other components in tea.

of the water-soluble solids of tea. Tea beverage is also a signiﬁcant of analysis time, resolving power, and solvent consumption. It was

source of ﬂuoride at ∼1 mg/serving. demonstrated that the separation of eight standard tea polyphenols

could be achieved in about 30 s while maintaining sufﬁcient resolu-

3. Analytical methods for tea constituents tion using UHPLC coupled to UV and mass spectrometry detectors

[27].

3.1. High performance liquid chromatography HPLC coupled to CoulArray electrochemical detector (ECD) is the

most commonly used analytical method to measure tea polyphe-

Numerous analytical methods have been developed to separate nols in biological ﬂuids. In general, the electrochemical detector

and quantify tea constituents. Reverse-phase high performance liq- has similar limits of detection for tea polyphenols as from the mass

uid chromatography (HPLC) followed by UV or electrochemical or detector. These are about 100 fold more sensitive than UV detector.

mass detection is the most widely used method for analysis of tea The high price of instrumentation and the tedious sample prepara-

polyphenols. The use of LC for the determination of tea constituents tion apparently limit the application of HPLC with mass detection

was ﬁrst reported in 1976 by Hoeﬂer and Coggon [20]. Five tea cate- (LC/MS) to the analysis of tea polyphenols in large sets of biologi-

chins (C, EC, ECG, EGC, and EGCG) were identiﬁed directly in a green cal ﬂuids with complex matrices. The clean cell activity using high

tea infusion in this study. Signiﬁcant improvements for the mea- electro potential in ECD cells after each run is another advantage of

surement of catechins by LC were reported by Goto and co-workers ECD compared to MS detection. Lee and co-workers reported the

in 1996. A C18 reverse-phase LC column with UV detection utilizing ﬁrst study of the analysis of plasma and urinary tea polyphenols

a water–acetonitrile–phosphoric acid mobile phase composition in human subjects employing LC with coulochem electrode array

was developed in this study to analyze eight tea catechins and detection [28]. The limits of detection for three tea catechins (EC,

caffeine [20]. The majorities of recently published analytical HPLC EGCC, and EGCG) were 1.0 ng/mL for this method.

methods have focused on optimization of the analysis time per LC with ﬂuorescence detection and chemiluminescence (CL)

sample, the conditions used to extract, detect, and characterize tea detection were also used to measure the levels of tea polyphe-

polyphenols found in infusions and biological matrices, as well as nols in biological ﬂuids. The concentration of (+)-catechin in

increasing sample throughput [21–25]. Fourteen tea constituents rabbit plasma following intravenous (i.v.) administration of the

(eight tea catechins, two methylated catechins, three purine alka- pure compound has been determined by ﬂuorescent detec-

loids, and gallic acid) were rapidly separated within 15 min by a tion with detection limit of 20 ng/mL [29]. Catechins can be

linear gradient elution of formic acid solution and methanol using determined by CL detection following reaction with hydrogen

a reverse-phase column [26]. Two new RP-HPLC-diode array detec- peroxide–acetaldehyde–horseradish peroxidase, which results in

tion (DAD) analyses of tea polyphenols were developed to facilitate a distinct chemiluminescent emission at 630 nm. A very sensitive

separation of catechins within 5 min and separation of catechins HPLC-CL method has been used to determine EGCG levels in human

(EC, ECG, EGC, and EGCG) and theaﬂavins (TF, TF-3G, TF-3 G, and TF- and rat plasma after a single oral supplementation of pure EGCG

3,3 -DG) within 10 min total analysis time [21]. The recent advance [30].

in liquid chromatography is the development of ultra-high pressure LC/MS especially with tandem mass spectrometry (MS/MS)

liquid chromatography (UHPLC) using columns packed with sub- detection is the most powerful technique for structure identiﬁca-

␮

2 m particles. This technology has shown clear beneﬁts in terms tion of trace quantities of tea constituents and the metabolites of

92 S. Sang et al. / Pharmacological Research 64 (2011) 87–99

◦

tea constituents in bioﬂuids [8,22,31,32]. Such structural informa- were autoclaved at 120 C for 20 min and speculated that the rela-

tion cannot be obtained when HPLC is used with DAD, ﬂuorescence, tively high amount of GCG found in some tea drinks was most likely

CL, or ECD. Electrospray ionization (ESI) and atmospheric pressure the epimerization product of EGCG during autoclaving.

chemical ionization (APCI) are the two most commonly used ion- The relationship between the chemical structure of tea cat-

ization methods to analyze tea constituents. Del Rio and co-workers echins and epimerization was further studied by Suzuki and

reported the application of HPLC-MS methods for the identiﬁca- co-workers [42]. They found that the epimeric isomers of (−)-

tion and analysis of more than 30 phenolics compounds and purine epicatechin, (−)-epicatechin-3-O-gallate, (−)-epigallocatechin,

−

alkaloids in green and black tea [22]. More recently, a standardized ( )-epigallocatechin-3-O-gallate, and (−)-epigallocatechin-

proﬁling method based on HPLC with diode array and electrospray 3-O-(3-O-methyl)-gallate in green tea extracts increased

◦

ionization mass spectrometric detection was applied to establish time-dependently at 90 C. The hydroxyl moiety on the B ring of

the chemical proﬁle of 41 green teas and 25 fermented teas [8]. catechins plays an important role in the epimerization in the order

More than 96 tea compounds were identiﬁed and over 30 phenolics 3 ,4 ,5 -triol type > 3 ,4 -diol type 3 ,5 -diol type. In addition,

are new for tea. This is the ﬁrst study to establish the chemi- they also found that the epimerization rates of authentic tea

cal proﬁle of tea and simultaneously detect C- and O-glycosylated catechins in distilled water are much lower than those in tea

ﬂavonoids, catechins, proanthocyanidins, pehnolic acid derivatives, infusion or in pH 6.0 buffer solution; the addition of tea infusion

and purine alkaloids. We have used tandem mass identiﬁed the to the authentic catechin solution accelerated the epimerization.

major metabolites of tea catechins in mice and human [31–33]. The addition of ethylenediaminetetraacetic acid, disodium salt

(Na2EDTA) decreased the epimerization in the pH 6.0 buffer solu-

tion indicating that metal ions in tea infusion may affect the rate

3.2. Capillary electrophoresis

of epimerization. Simultaneous degradation and epimerization of

EGCG were characterized over a wide temperature range [43,44].

Capillary electrophoresis (CE) is another common used

The degradation and epimerization are ﬁrst-order reactions and

method to separate and quantify tea constituents. Capillary zone

their rate constants followed Arrhenius equation. Two speciﬁc

electrophoresis (CZE), micellar electrokinetic capillary chromatog-

temperature points in the reaction kinetics were identiﬁed, at

raphy (MEKC), and non-aqueous capillary electrophoresis (NACE) ◦ ◦

44 and 98 C, respectively [43]. Below 44 C, the degradation was

with UV absorbance detection are the CE methods reported in ◦

more profound. Above 44 C, the epimerization from EGCG to

the literature for the determination of tea constituents [20,34–37].

GCG was faster than degradation. When temperature increased

In general, the MEKC method provides better separation, reso- ◦

to 98 C and above, the epimerization from EGCG to GCG became

lution and quantitation for a larger number of compounds than

prominent (Fig. 5).

does the CZE method. NACE is a method that can be used to

separate and analyze compounds that are difﬁcult to analyze in

4.2. Under laboratory conditions

aqueous systems. Horie and co-workers developed a CZE method

for simultaneous determination of ﬁve major tea catechins together

Our results indicated that in animal studies where EGCG or green

with caffeine, theanine, and ascorbic acid [34]. The separation was

tea extract (GTE) solutions were made fresh in deionized water

achieved using a fused-silica capillary column with a borax buffer

every two or three days, the catechins were rather stable at higher

at pH 8.0 and UV detection at 200 nm. Several MEKC methods

concentrations (0.32% EGCG or 0.6% GTE) [19]. At lower concentra-

with UV detection have been published to separate and deter-

tions (0.1% and 0.025% EGCG, or 0.2% and 0.05% GTE), up to a 20%

mine catechins in different tea samples [20,36,37]. Most of the

decrease in EGCG concentration could occur in two to three days. In

reported MEKC methods utilized sodium dodecyl sulfate micelles

general, the stability of EGC was about the same as EGCG, whereas

in the presence of a borate-based running buffer. A NACE method

ECG and EC were more stable, possibly due to the oxidation of the

has been reported to separate four major theaﬂavins in black tea

trihydroxyl structure on the B-ring of EGC and EGCG. We have also

within 10 min [35]. The optimized separation solution consisted

observed that EGCG and other tea polyphenols were less stable if

of acetonitrile–methanol–acetic acid (71:25:4, v/v/v) and 90 mM

the solution was made in tap water possibly due to the presence of

ammonium acetate.

metal ions.

Tea catechins have been used extensively in many cell cul-

4. Stability of tea constituents ture studies, but the stability under these experimental conditions

has been ignored by many investigators. We and other investiga-

4.1. In tea drinks tors have shown that tea catechins, especially EGCG, are unstable

under cell culture conditions and are subject to oxidation and poly-

Stability of tea catechins is an important issue to prepare com- merization, resulting in the formation of reactive oxygen species

mercial tea products and to understand the beneﬁcial health effects [19,45,46]. In McCoy’s 5A culture media, the half-life of EGCG was

of tea. The stability of tea catechins in tea drinks under either direct less than 30 min, and the half-life increased to 130 min in the pres-

brewing or industrial canning processes have been studied [38–41]. ence of HT-29 human colon adenocarcinoma cells [46]. It has been

Chen et al. reported that about 20% of tea catechins was lost when reported that in air-saturated Tris buffer (0.1 M, pH 9.0), EGCG

◦

they were heated in water for 7 h at 98 C [38]. In contrast, tea cat- undergoes auto-oxidation; oxygen is consumed and H2O2 is formed

◦ • −

echins in water remained unchanged for the same period at 37 C. [47]. The auto-oxidation of EGCG may generate O2 and quinones

The effect of the autoclave process used for sterilization in manu- [45–49]. Superoxide dismutase (SOD), which catalyzes the con-

• −

facture of tea drinks on the stability of tea catechins or pure EGCG version of O2 to H2O2 and O2, was shown by us and others

was also studied by Chen and co-workers. They found only 76% of to stabilize EGCG and decrease the overall EGCG-mediated H2O2

tea catechins remained in water after being autoclaved for 20 min generation [19,45–49]. We found that the stability of EGCG was

◦

at 120 C and the stability of tea catechins in solutions during auto- dramatically increased by the presence of SOD (5 U/mL) in the cell

claving was pH-dependent. Tea catechins were relatively stable at culture medium and the half-life of EGCG was increased to longer

pH 3 and 4, but it degraded readily at pH 5 and 6 during autoclaving. than 24 h [45].

About 80% tea catechins was lost in a solution of pH 6 buffer solu- Our further study on the stability of EGCG under cell culture

␮

tion during autoclaving for 20 min. In addition, they observed the conditions indicated that EGCG (20 M) underwent auto-oxidation

epimerization of EGCG to GCG after tea catechins and pure EGCG and EGCG dimers and other products were formed [19]. Several

S. Sang et al. / Pharmacological Research 64 (2011) 87–99 93

. O OH OH OH HO O HO O OH OH

HO O OH High tempera ture OH O O OH O O

O OH

OH OH O or anaerobic conditions HO OH HO OH OH H2O2 GCG OH OH OH EGCG 2H+

Cu+, Fe2+ - + EGCG + O2 •O2 + • EGCG + H O2

SOD

Dimer quinone H2O2 + O2

•O2 H+ HO HO EGCG quino ne OH OH O

OH OH O O O OH OH OH O O HO O OH OH OH O HO O HO O OH O O OH HO O OH OH HO O O OH OH O OH OH O OH OH EGCG

OH O

O OH HO EGCG quinon e HO OH P2 HO

Theasin ensin A

Fig. 5. The epimerization and auto-oxidation of EGCG.

factors, including pH, temperature, oxygen levels, antioxidant lev- idation of theasinensin A through the same mechanism as EGCG

els, metal ion, concentration of EGCG, and other ingredients in tea, oxidation. The dimer quinone can be further oxidized to form other

could affect the stability of EGCG. We have conducted a real-time dimers, such as P2.

mass data acquisition of EGCG solutions over a period of 24 h to EGCG quinone, the quinone of EGCG dimer, and reactive oxygen

further elucidate the mechanism of the stability of EGCG and the species (ROS) generated during EGCG auto-oxidation may trig-

kinetic proﬁles of products formation [33]. The starting EGCG con- ger a variety of biochemical reactions. For example, the radical

␮

centrations were 50 and 200 M. The time-dependent change of species may contribute to the inactivation of epidermal growth

EGCG concentration and product formation in Tris–HCl buffer (pH factor receptor (EGFR) and telomerase as reported in the literature

7.2) was recorded. Our results indicated that EGCG was unstable in a [45,53], and hydrogen peroxide may contribute to cell apoptosis

pH 7.2 buffer solution and several oxidation products were formed, [54,55]. We have demonstrated that, depending on the cell lines

including EGCG quinone, several dimers and dimer quinone, which and culture conditions, the EGCG-induced apoptosis can be com-

were also unstable. EGCG quinone has been proposed as the key pletely or partially blocked by the addition of catalase in the culture

intermediate for the formation of many EGCG oxidation products medium, suggesting that the apoptosis is mediated by H2O2 [54,55].

by us and others [50–52], but direct evidence for its existence has Vittal et al. found that catalase partially blocked EGCG-induced apo-

been lacking. We provided for the ﬁrst time the direct evidence ptosis and also abolished the effects on transforming growth factor

of the formation of EGCG quinone and dimer quinine [33]. Based (TGF) ␤ signaling in transformed human bronchial epithelial cells

on these observations, a mechanism of EGCG auto-oxidation is (21BES) [56]. We observed that the inhibition of EGFR phospho-

proposed (Fig. 5). Under neutral or slightly alkaline pH, EGCG is rylation caused by pre-incubation of the cell with EGCG could be

•

oxidized by molecular oxygen to form EGCG radical (EGCG ) and prevented by the presence of SOD, suggesting that the inhibition

•−

superoxide radical (O2 ) in a reaction probably catalyzed by trace of the EGFR signaling pathway is caused by the auto-oxidation of

+ 2+ •−

metal ions such as Cu or Fe . It is also possible that the O2 can be EGCG [45]. Interestingly, inclusion of SOD in the culture medium

•−

generated directly from molecular oxygen. The O2 can then react potentiated the activity of EGCG in inhibiting KYSE 150 cell growth,

•

with another EGCG molecule to form EGCG and hydrogen perox- indicating that these effects are independent of ROS production.

•

ide (H2O2). EGCG can react with molecular oxygen to form EGCG However, it is unclear whether the observed auto-oxidation of

•− •−

quinone and generate O2 . The O2 can then react with another EGCG occurs in vivo.

molecule of EGCG for the propagation of the chain reaction of EGCG

•−

auto-oxidation. The involvement of O in these reactions is sup-

2 4.3. Under in vivo conditions

ported by the observation that these reactions are inhibited by the

presence of superoxide dismutase (SOD) [45]. EGCG quinone can

Naasani et al. also reported that EGCG was unstable in human

react with EGCG to form EGCG dimer, such as theasinensin A. It can

and mouse plasma with theasinensin A and P2 as the two major

also react with another EGCG quinone to form the quinone of EGCG

oxidation products [53]. To determine if this phenomenon can be

dimer. This dimer quinone can also be generated from the autox-

observed in vivo, we attempted to detect those oxidation prod-

94 S. Sang et al. / Pharmacological Research 64 (2011) 87–99

ucts of EGCG in mouse plasma after EGCG administration. In this EGCG were methylated at much lower rates [67]. Cytosolic protein

study, we used intraperitoneal (i.p.) administration because it gives concentration, incubation time, concentration of S-adenosyl-l-

a higher plasma level of EGCG than oral administration. We chose methionine (SAM, the methyl donor), and incubation pH were the

1.5 h after the last treatment of EGCG because the Tmax of EGCG major factors affect the methylation of EC. It has been reported

for i.p. administration is approximately 30 min (Sang, unpublished that rat liver cytosol had higher COMT activity than that of humans

data). Therefore, 1.5 h plasma sample is compatible to the sample or mice. The small intestine had lower speciﬁc activity than the

obtained from incubating EGCG in plasma for 1 h. However, none of liver in the methylation of EGCG and EGC [63]. EGCG was methy-

the oxidative products could be detected from these plasma sam- lated by liver cytosolic COMT to 4 -O-methyl EGCG, and then 4 ,

ples using our sensitive tandem mass ion mapping method. Instead, 4 -di-O-methyl-EGCG. Glucuronidation on the B-ring or the D-ring

EGCG and its metabolites were detected as the major compounds in of EGCG greatly inhibited the methylation on the same ring, but

these samples. Our results indicate the observed auto-oxidation of glucuronidation on the A-ring of EGCG or EGC did not affect their

EGCG may not occur in vivo due to the higher antioxidative capacity methylation [63]. We have chemically synthesized 4 -O-methyl

(SOD, glutathione peroxidase, glutathione, and ascorbic acid) and EGC, 4 -O-methyl EGCG, and 4 , 4 -di-O-methyl-EGCG and con-

lower oxygen partial pressure in the internal organs. If oxidation ﬁrmed their structures in human plasma and urine samples using

of EGCG does occur in vivo, the oxidized EGCG could be reduced by LC/MS [66,68]. We found that 4 -O-methyl EGC and 4 , 4 -di-O-

reducing agents (e.g. ascorbic acid) to regenerate EGCG. This would methyl-EGCG were the major methylated metabolites of EGC and

be contrary to the anti-oxidative effects of tea catechins observed EGCG in human.

in vivo, but this topic need to be further studied in the future. The

difference between in vitro and in vivo systems should be consid- 5.2. Glucuronidation of tea catechins

ered in studies attempting to elucidate the mechanisms of action

of EGCG. UDP-glucuronosyltransferase (UGT)-catalyzed glucuronidation

is also a major pathway in Phase II metabolism. UGTs are a

multigenic family of membrane-bond enzymes that catalyze the

5. Biotransformation of tea constituents

binding of glucuronic acid from UDP-glucuronic acid on struc-

turally unrelated substances with a hydroxyl, carboxyl, amine,

Catechins have been demonstrated to undergo considerable

or thiol group. It has been reported that EC was efﬁciently

biotransformation [57,58]. Methylation, glucuronidation, sulfa-

glucuronidated with formation of two glucuronides, and was

tion, and ring-ﬁssion metabolism represent the major metabolic

not glucuronidated by the human liver, small intestinal and

pathways for tea catechins [58]. Fig. 6 shows the major biotransfor-

colon microsomes or by recombinant UGT1A7 [69]. Lu and co-

mative pathways for the tea catechins. Some catechins have been

workers have systematically characterized the glucuronidation

demonstrated to have low bioavailability, which is likely due to

of EGCG and EGC in human, mouse, and rat microsomes and

their relatively high molecular weight and the large number of

by nine different human UGT1A and 2B isozymes expressed in

hydrogen-bond donating hydroxyl groups [59]. According to Lip-

insect cells [70]. Four EGCG glucuronides (EGCG-7-O-glucuronide,

inski’s rule of 5, compounds which have ﬁve or more hydrogen

EGCG-4 -O-glucuronide, EGCG-3 -O-glucuronide, and EGCG-3 -O-

bond donors, or ten or more hydrogen bond acceptor, or molecu-

glucuronide) and two EGC glucuronides (EGC-3 -O-glucuronide

lar weight greater than 500 are usually poorly bioavailable due to

and EGC-7-O-glucuronide) were biosynthesized. EGCG-4 -O-

their large apparent size, due to the formation of a large hydration

glucuronide was identiﬁed as the major EGCG glucuronide in all

shell [59]. In addition, the efﬂux of these compounds by multidrug

incubations. Mouse small intestinal microsomes have the greatest

resistance-associated proteins (MRP) may play a vital role in lim-

catalytic efﬁciency (Vmax/Km) for the formation of EGCG-4 -O-

iting the bioavailability of tea catechins. MRP2 is located on the

glucuronide followed in decreasing order by mouse liver, human

apical surface of the intestine, kidney, and liver, where it transports

liver, rat liver, and rat small intestine [70]. The UGT-catalyzed glu-

compounds from the bloodstream into the lumen, urine, and bile,

curonidation of EGC was much lower than that of EGCG. Mouse

respectively. Hong et al. found that EGCG and its methyl metabo-

liver microsomes have the greatest catalytic efﬁciency (Vmax/Km)

lites were substrates for MRP1 and MRP2, but not for Pgp [60].

for the formation of EGC-3 -O-glucuronide followed in decreasing

Vaidyanathan and Walle have reported that treatment of Caco-2

order by human liver, rat liver, and rat and mouse small intestine

cells with MK-571 (an MRP2 inhibitor) enhances apical to basolat-

[70]. Using recombinant human UGT enzymes, it has been deter-

eral movement of EC and ECG, suggesting that both EC and ECG

mined that UGT1A1, 1A8, and 1A9 have highest activity toward

are the substrates of MRP2 [61,62]. The effects of MRP2 on the

EGCG, with the intestinal speciﬁc UGT1A8 having the highest cat-

bioavailability of the tea polyphenols remain to be determined

alytic efﬁciency. When the major glucuronidation site of EGCG is

in vivo.

occupied by a methyl group, the activities of UGT1A1, 1A3, and

1A8 are much lower. However, 4 -methylation can enhance glu-

5.1. Methylation of tea catechins curonidation at the same D-ring with UGT1A9. Overall, mice appear

to be more similar to humans than rats in the glucuronidation of

Catechol-O-methyltranserase (COMT), an enzyme ubiquitously EGCG and EGC. It has been reported that rat intestinal microsomes

present in high activity in humans and rodents, catalyzes the exhibited higher UGT activity on the galloyl group of EGCG and ECG

metabolic O-methylation of various catecholic compounds includ- compared to the ﬂavonoid ring, whereas rat hepatic UGT activity

ing the major tea catechins. COMT has been found in all mammalian was higher on the ﬂavonoid ring of EGCG and ECG compare to the

tissues investigated, with the highest activity in the liver, then galloyl groups [71].

the kidney and gastrointestinal tract [63]. The general function

of COMT is to eliminate the potentially active or toxic catechol 5.3. Sulfation of tea catechins

structures of endogenous and exogenous compounds. Methylated

catechins including 3 - and 4 -O-methyl-EC, 4 -O-methyl EGC, and Sulfotransferases (SULT) are transferase enzymes that catalyze

4 -O-methyl ECG and EGCG, and 4 , 4 -di-O-methyl-EGCG have the transfer of a sulfate group from a donor molecule to an acceptor

been observed in vivo and in incubations with rat liver homogenates alcohol or amine [72]. The most common sulfate donor is 3 -

[63–66]. Zhu and co-workers found that EC and EGC were good sub- phosphoadenosine-5 -phophosulfate (PAPS). It has been reported

strates for methylation by placental cytosolic COMT, but ECG and that the human liver cytosol efﬁciently sulfated EC mainly through

S. Sang et al. / Pharmacological Research 64 (2011) 87–99 95

Fig. 6. Major biotransformation pathways of the green tea catechins.

␤ d the SULT1A1 isoform. For the intestine, both SULT1A1 and SULT1A3 identiﬁed a new EGCG metabolite, 7-O- - -glucopyranosyl-EGCG-

contributed. Other SULT isoforms contributed very little. Sulfate 4 -O-␤-d-glucupyranoside (Fig. 5), in mouse urine sample collected

conjugation of EC also occurred in the rat liver. The apparent Km after administration of EGCG (50 mg/kg, i.p. or 200 mg/kg i.g.) using

value for this reaction was 8 times higher than in the human liver. LC/ESI–MS [32]. The structure of this metabolite was conﬁrmed by

n 2

Dr. Chung S. Yang’s laboratory has shown that EGCG is time- and the MS (n = 1–4) spectra as well as comparing the MS spectra of its

concentration-dependently sulfated by human, mouse, and rat liver daughter ions with those from EGCG standard and EGCG-4 -O-␤-d-

cytosol [73]. The rat cytosol has the greatest activity to sulfate EGCG, glucupyranoside standard. This is the ﬁrst report of glucosidation

followed by the mouse and the human liver cytosol. The sulfates of as a novel pathway in the metabolism of EGCG [32]. It is worth-

EC, EGC, and EGCG have been identiﬁed from rodent and human while to further study whether glucosidation of EGCG occurs in

samples mainly through LC/MS analysis. However, the chemical humans. It has been reported that rabbit liver microsome could

structures of those sulfate metabolites have not been characterized. efﬁciently catalyze the glucosidation of genistein to form genistein

The active sites for sulfation of tea catechins are still unknown. 7-glucoside [77]. Many other dietary ﬂavonoids, such as quercetin,

epicatechin, luteolin, and cyanidin, had the same A-ring structure as

5.4. Glucosidation of tea catechins that of EGCG and genistein. Further studies are needed to determine

whether glucosidation is similar to glucuronidation as a general

Glucosidation is a common metabolic pathway in plant, but biotransformation pathway to most of the dietary ﬂavonoids.

rarely in mammals. Glucosidation of exogenous and endogenous

compounds has been described previously [74–77]. Mammalian 5.5. Thiol conjugation of tea catechins

␤

UDP-glucosyltransferases utilizing phenols and UDP- -D-glucose

as substrates have been demonstrated [78–80]. It has been reported It has been reported that (+)-catechin can be metabolized

that EGCG can react with sucrose to form three EGCG glycosides by tyrosinase to form a cytotoxic o-quinone, which reacts with

catalyzed by glucansucrase produced by Leuconostoc mesenteroides glutathione to form mono-, bi-, and triglutathione conjugates of (+)-

B-1299CB [81]. The active sites of EGCG for glucosidation are posi- catechin and mono- and biglutathione conjugates of a (+)-catechin

tions 7 of the A-ring and 4 of the B-ring. More recently, we dimer [82]. When peroxidase and hydrogen peroxide were used,

96 S. Sang et al. / Pharmacological Research 64 (2011) 87–99

only monoglutathione conjugates of (+)-catechin were formed. In or sulfated form) is a major metabolite of EGC, reaching its peak

the presence of NADPH, rat liver microsomes also catalyzed oxi- level within the ﬁrst 2 h in human plasma at a concentration 4–6

dation of (+)-catechin leading to glutathione conjugate formation times higher than those of EGC [mainly as the glucuronidated form

[82]. (57–71%) or sulfated form (23–36%) and with only a small amount

In our study, we found that EGCG can be oxidized by perox- present as the free form (3–13%)] [28,57]. And the sulfated form

idase and hydrogen peroxide and then reacted with cysteine or of EC is more abundant (66%) than the glucuronidated form (33%)

glutathione to form conjugates [50]. The structures of the cysteine [28,69]. Our recent results from data-dependent MS analysis of

and glutathione conjugates of EGCG were identiﬁed using 2D NMR mouse urine samples after administration of EGCG by i.g. or i.p.

and MS. Two thiol conjugates of EGCG (2 -cysteinyl EGCG and 2 - show that phenolic groups of methylated EGCG (or glucuronidated

cysteinyl EGCG) were identiﬁed by LC/ESI–MS analysis from the or sulfated EGCG) can be further methylated, glucuronided and/or

urine samples of mice administered 200 or 400 mg/kg EGCG, i.p. sulfated to form multiple conjugated metabolites (Sang, unpub-

These conjugates were not found in urine samples of mice after lished data).

receiving EGCG at 50 mg/kg i.p. or in human urine following con- We have studied the human urinary metabolite proﬁle of tea

sumption of 3 g of decaffeinated green tea solids (containing 333 mg polyphenols using liquid chromatography/electrospray ionization

EGCG). At high doses, EGCG is believed to be oxidized to form EGCG tandem mass spectrometry with data-dependent acquisition. With

n 2 3

quinone, which can react with glutathione or cysteine to form the data-dependent MS analysis by collecting the MS and MS spec-

thiol conjugates. Our results suggest that detectable amounts of tra of the most intense ions in the sample, we identiﬁed more than

thiol conjugates of EGCG are formed only after rather high doses twenty metabolites of tea polyphenols from human urine sam-

of EGCG are given to the mice. Under normal physiological condi- ples collected at different time periods after consumption of green

tions, EGCG is metabolized through methylation, glucuronidation, tea. Consistent with previous observations [28,58,88,89], Phase II

and sulfation. At toxic doses of EGCG, these pathways may be satu- metabolites of EGCG and ECG were undetectable in this study.

rated, and the excessive amount of EGCG is oxidized to form EGCG Based on previous study on rats [90], they may excrete through

quinone, which can react with glutathione or cysteine to form bile. EGC glucuronide, methylated EGC glucuronide, methylated

thiol conjugates. It is not clear how EGCG is oxidized in this pro- EGC sulfate, EC glucuronide, EC sulfate, methylated EC sulfate, as

posed pathway. EGCG, with eight phenolic groups, appears to be too well as the glucuronide and sulfate metabolites of the ring ﬁs-

hydrophilic to be a good substrate for cytochrome P450 enzymes. It sion metabolites of tea catechins, M4, M6 and M6 , were the major

is possible that high concentrations of EGCG may be oxidized non- human urinary metabolites of tea polyphenols.

speciﬁcally by oxygenases/peroxidases. It is also possible that EGCG The metabolite proﬁle of tea catechins provided by this study

can induce ROS production in mitochondrial. The resulting quinone will help us to select the metabolites that can cover a 24 h time

then undergoes redox cycling to produce oxidative stress beyond period to reﬂect tea consumption. For example, we found that

the capacity of antioxidative enzymes (superoxide dismutase, glu- most of the urinary EC and EGC metabolites excreted within 9 h,

tathione peroxidase, and catalase). The thiol conjugate formation however, the ring-ﬁssion metabolites, M4, M6, and M6 , and their

may represent a mechanism to detoxify EGCG quinone. glucuronide and sulfate conjugates are the major metabolites in

the urine even at 12–24 h after tea ingestion. Since EC is also found

5.6. Microbial metabolism of tea catechins in cocoa and many fruits [91–93], metabolites of EC and its related

ring-fission product (M6) are not specific markers to reflect tea con-

The human colon contains 1000 bacterial species, amounting sumption, but they can be used as general markers to reﬂect fruits,

to over 100 trillion bacteria, which have enormous catalytic and chocolate, and tea consumption. Therefore, conjugated metabolites

hydrolytic potential of dietary components. Conolic microﬂora of EGC, methylated EGC, and its ring-ﬁssion products (M4 and M6 )

catalyzes the breakdown of polyphenols into simple compounds, can be used as the exposure markers to reﬂect tea consumption.

such as phenolic acids and their glycine conjugates, such as Using catechins and their metabolites to reﬂect tea consumption

derivatives of hippuric acid [58]. It has been reported that the will be a much more accurate method than the traditional way to

tea catechins are metabolized by intestinal ﬂora to form the recall tea consumption, which is based on how many cups of tea

ring ﬁssion products 5-(3 ,4 ,5 -trihydroxyphenyl)-␥-valerolactone consumed by the subjects per day, used in epidemiological studies.

(M4), 5-(3 ,4 -dihydroxyphenyl)-␥-valerolactone (M6) and 5-(3 ,5 -

␥

dihydroxyphenyl)- -valerolactone (M6 ) (Fig. 6) [83,84]. We have 5.8. Biotransformation and bioavailability of theaﬂavins and

found that these ring ﬁssion products are present in human urine thearubigins

(4–8 ␮M) and plasma (0.1–0.2 ␮M) approximately 13 h after oral

ingestion of 20 mg/kg decaffeinated green tea [83]. Peak urine con- The biotransformation and bioavailability of theaﬂavins are less

centrations of 8, 4, and 8 ␮M have been demonstrated for M4, well characterized due to the very low systematic bioavailability

M6, and M6 , respectively, following ingestion of 200 mg EGCG. of theaflavins. Theaflavin, theaflavin 3-gallate, theaflavin 3 -gallate,

M6 was previously shown to form during anaerobic incubation of methylated theaﬂavin 3,3 -digallate, and gallic acid were identiﬁed

ECG and EC with human intestinal bacteria [85]. Further degrada- as the major metabolite of theaﬂavin 3,3 -digallate in mouse fecal

tion of these compounds by intestinal ﬂora results in the formation sample collected after administration of theaﬂavin 3,3 -digallate

of lower molecular weight phenolic acids [85]. 4-Hydroxybenzoic 200 mg/kg, i.g. using liquid chromatography/electrospray ioniza-

acid, 3,4-dihydroxybenzoic acid, 3-methoxy-4-hydroxy-hippuric tion tandem mass spectrometry (Sang, unpublished data). Both

acid, and 3-methoxy-4-hydroxybenzoic acid (vanillic acid) have glucuronidated and sulfated metabolites were not detectable using

been identiﬁed as the major tea metabolites from the human urine our established LC/MS method. Our results indicate that theaﬂavin

samples collected at 6–48 h after drinking tea (about 400 mg tea 3,3 -digallate is a substrate of COMT and can be degraded by gut

catechins) [86]. micro flora to form gallic acid, theaflavin and theaflavin mono-

gallate. Mulder et al. reported that the maximum concentration

5.7. Metabolic proﬁle of tea catechins and potential exposure of theaﬂavin in human plasma and urine was only 1 ng/mL and

markers of tea consumption 4.2 ng/mL, respectively, following consumption of 700 mg of pure

theaﬂavins mixture, equivalent to about 30 cups of black tea [94].

Following tea ingestion, EGCG is present mainly in the free form Neither theaﬂavin mono- and di-gallates were detectable in this

in the plasma [87]. 4 -O-methyl EGC (mostly in the glucuronidated study. However, our recent results have shown that none of the

S. Sang et al. / Pharmacological Research 64 (2011) 87–99 97

theaﬂavins are detectable by LC/MS (limits of detection <1 ng/mL) [10] Mahanta PK, Boruah SK, Boruah HK, Kalita JN. Changes of polyphenol oxidase

and peroxidase activities and pigment composition of some manufactured

in human plasma and urine following consumption of 1200 mg

black teas (Camellia sinensis L.). J Agric Food Chem 1993;41:272–6.

of theaﬂavins mixture by 8 volunteers (4 men and 4 women)

[11] Finger A. In vitro studies on the effect of polyphenol oxidase and peroxidase

(Sang, unpublished data). A recent study found that theaﬂavin is in the formation of black tea constituents. J Sci Food Agric 1994;66:293–305.

[12] Sang S, Lambert JD, Tian S, Hong J, Hou Z, Ryu JH, et al. Enzymatic synthe-

bioavailable in mouse prostate [95]. The mouse prostate tissue

sis of tea theaﬂavin derivatives and their anti-inﬂammatory and cytotoxic

concentration of theaﬂavin was 1.5 nmol/g after giving 171.6 mg

activities. Bioorg Med Chem 2004;12:459–67.

theaﬂavin/kg body weight in the diet for two weeks [95]. How- [13] Sang S, Tian S, Stark RE, Yang CS, Ho CT. New dibenzotropolone deriva-

tives characterized from black tea using lc/ms/ms. Bioorg Med Chem

ever, the author did not report the plasma level of theaﬂavin in

2004;12:3009–17.

this study. The bioavailability and biotransformation of theaﬂavins

[14] Subramanian N, Venkatesh P, Ganguli S, Sinkar VP. Role of polyphenol oxidase

need to be further studied. Because of their large molecular weight, and peroxidase in the generation of black tea theaﬂavins. J Agric Food Chem

thearubigins are probably not absorbed in the small intestine. Stud- 1999;47:2571–8.

[15] Sang S, Tian SY, Meng XF, Star RE, Rosen RT, Yang CS, et al. Thead-

ies have shown that higher molecular weight polyphenols are

ibenzotropolone a, a new type pigment from enzymatic oxidation of

metabolized by the microbiota and their metabolites may play an −

( )-epicatechin and (−)-epigallocatechin gallate and characterized from

important role in black tea chemopreventive action [96–101]. How- black tea using lc/ms/ms. Tetrahedron Lett 2002;43:7129–33.

[16] Menet MC, Sang S, Yang CS, Ho CT, Rosen RT. Analysis of theaﬂavins and

ever, the bacterial metabolites of thearubigins are still unknown.

thearubigins from black tea extract by maldi-tof mass spectrometry. J Agric

Our recent results found that lower molecular weight phenolic

Food Chem 2004;52:2455–61.

acids are the major microbial metabolites of thearubigin-like com- [17] 1Kuhnert N, Drynan JW, Obuchowicz J, Clifford MN, Witt M. Mass spec-

trometric characterization of black tea thearubigins leading to an oxidative

pounds in black tea (Sang, unpublished results). This topic needs to

cascade hypothesis for thearubigin formation. Rapid Commun Mass Spectrom

be further studied. 2010;24:3387–404.

[18] Hashimoto FN, Nishioka GI. Tannins and related compounds Lxix. Isolation

and structure elucidation of b,b -linked bisﬂavanoids, theasinensins d-g and

6. Concluding remarks oolongtheanin from oolong tea. Chem Pharm Bull 1988;36:1676–84.

[19] Sang S, Lee MJ, Hou Z, Ho CT, Yang CS. Stability of tea polyphenol (−)-

epigallocatechin-3-gallate and formation of dimers and epimers under

As discussed in other chapters of this volume, inconsisten-

common experimental conditions. J Agric Food Chem 2005;53:9478–84.

cies concerning the health effects of tea consumption still exist. [20] Dalluge JJ, Nelson BC. Determination of tea catechins. J Chromatogr A

Although many animals and epidemiological studies have demon- 2000;881:411–24.

[21] Neilson AP, Green RJ, Wood KV, Ferruzzi MG. High-throughput analysis of

strated the efﬁcacy of tea constituents in the prevention of chronic

catechins and theaﬂavins by high performance liquid chromatography with

diseases, other studies have failed to demonstrate such beneﬁcial

diode array detection. J Chromatogr A 2006;1132:132–40.

effect. A clearer understanding of the chemistry, stability, bioavail- [22] Del Rio D, Stewart AJ, Mullen W, Burns J, Lean ME, Brighenti F, et al. Hplc-msn

analysis of phenolic compounds and purine alkaloids in green and black tea.

ability and biotransformation of tea polyphenols will provide the

J Agric Food Chem 2004;52:2807–15.

biochemical basis for understanding many of the existing results

[23] Khokhar S, Magnusdottir SG. Total phenol, catechin, and caffeine contents

and planning new studies. Among the tea constituents, catechins, of teas commonly consumed in the united kingdom. J Agric Food Chem

2002;50:565–70.

especially EGCG, and caffeine have been well studied. However,

[24] Lee MJ, Prabhu S, Meng X, Li C, Yang CS. An improved method for the

information on black tea polyphenols is still lacking. The bio-

determination of green and black tea polyphenols in biomatrices by high-

chemistry and biological activity of thearubigins are almost totally performance liquid chromatography with coulometric array detection. Anal

Biochem 2000;279:164–9.

unknown. These large molecular weight black tea polyphenols,

[25] Unno T, Sagesaka YM, Kakuda T. Analysis of tea catechins in human plasma

even though not absorbed, may exert effects in the oral-digestive

by high-performance liquid chromatography with solid-phase extraction. J

tract through direct contact or through their microbial metabolites. Agric Food Chem 2005;53:9885–9.

[26] Hu B, Wang L, Zhou B, Zhang X, Sun Y, Ye H, et al. Efﬁcient procedure for

Further study on the microbial metabolites of thearubigins could

isolating methylated catechins from green tea and effective simultaneous

signiﬁcantly contribute to our understanding of the biological activ-

analysis of ten catechins, three purine alkaloids, and gallic acid in tea by

ities of black tea. This is important because black tea is the major high-performance liquid chromatography with diode array detection. J Chro-

form of tea consumed world-wide. It would be a major advance- matogr A 2009;1216:3223–31.

[27] Guillarme D, Casetta C, Bicchi C, Veuthey JL. High throughput qualitative

ment in the ﬁeld of tea research if most of the key metabolites

analysis of polyphenols in tea samples by ultra-high pressure liquid chro-

can be measured to serve as exposure markers for epidemiological

matography coupled to uv and mass spectrometry detectors. J Chromatogr A

studies, and the effects of tea consumption on metabolism can be 2010;1217:6882–90.

[28] Lee MJ, Wang ZY, Li H, Chen L, Sun Y, Gobbo S, et al. Analysis of plasma and uri-

analyzed as possible effect markers. Some of the new metabolomic

nary tea polyphenols in human subjects. Cancer Epidemiol Biomarkers Prev

approaches may help to advance this ﬁeld of research. 1995;4:393–9.

[29] Ho Y, Lee YL, Hsu KY. Determination of (+)-catechin in plasma by

high-performance liquid chromatography using ﬂuorescence detection. J

References Chromatogr B Biomed Appl 1995;665:383–9.

[30] Nakagawa K, Miyazawa T. Chemiluminescence-high-performance liquid

chromatographic determination of tea catechin (−)-epigallocatechin 3-

[1] Grove KA, Lambert JD. Laboratory, epidemiological, and human intervention

gallate, at picomole levels in rat and human plasma. Anal Biochem

studies show that tea (Camellia sinensis) may be useful in the prevention of

1997;248:41–9.

obesity. J Nutr 2010;140:446–53.

[31] Sang S, Lee MJ, Yang I, Buckley B, Yang CS. Human urinary metabolite proﬁle of

[2] Higdon JV, Frei B. Tea catechins and polyphenols: health effects, metabolism,

tea polyphenols analyzed by liquid chromatography/electrospray ionization

and antioxidant functions. Crit Rev Food Sci Nutr 2003;43:89–143.

tandem mass spectrometry with data-dependent acquisition. Rapid Commun

[3] Naito Y, Yoshikawa T. Green tea and heart health. J Cardiovasc Pharmacol

2009;54:385–90. Mass Spectrom 2008;22:1567–78.

[32] Sang S, Yang CS. Structural identiﬁcation of novel glucoside and glucuronide

[4] Yang CS, Wang X, Lu G, Picinich SC. Cancer prevention by tea: animal

metabolites of (−)-epigallocatechin-3-gallate in mouse urine using liquid

studies, molecular mechanisms and human relevance. Nat Rev Cancer

2009;9:429–39. chromatography/electrospray ionization tandem mass spectrometry. Rapid

Commun Mass Spectrom 2008;22:3693–9.

[5] Balentine DA, Wiseman SA, Bouwens LC. The chemistry of tea ﬂavonoids. Crit

[33] Sang S, Yang I, Buckley B, Ho CT, Yang CS. Autoxidative quinone for-

Rev Food Sci Nutr 1997;37:693–704.

mation in vitro and metabolite formation in vivo from tea polyphenol

[6] Harbowy ME, Balentine DA. Tea chemistry. Crit Rev Plant Sci 1997;16:415–80.

(−)-epigallocatechin-3-gallate: studied by real-time mass spectrometry com-

[7] Chiu FL, Lin JK. Hplc analysis of naturally occurring methylated cate-

bined with tandem mass ion mapping. Free Radic Biol Med 2007;43:

chins, 3 - and 4 -methyl-epigallocatechin gallate, in various fresh tea

362–71.

leaves and commercial teas and their potent inhibitory effects on inducible

[34] Horie H, Mukai T, Kohata K. Simultaneous determination of qualitatively

nitric oxide synthase in macrophages. J Agric Food Chem 2005;53:

7035–42. important components in green tea infusions using capillary electrophoresis.

J Chromatogr A 1997;758:332–5.

[8] Lin LZ, Chen P, Harnly JM. New phenolic components and chromatographic

[35] Wright LP, Aucamp JP, Apostolides Z. Analysis of black tea theaﬂavins by non-

proﬁles of green and fermented teas. J Agric Food Chem 2008;56:8130–40.

aqueous capillary electrophoresis. J Chromatogr A 2001;919:205–13.

[9] Haslam E. Thoughts on thearubigins. Phytochemistry 2003;64:61–73.

98 S. Sang et al. / Pharmacological Research 64 (2011) 87–99

[36] Weiss DJ, Austria EJ, Anderton CR, Hompesch R, Jander A. Analysis of green [65] Okushio K, Suzuki M, Matsumoto N, Nanjo F, Hara Y. Identiﬁcation of (−)-

tea extract dietary supplements by micellar electrokinetic chromatography. epicatechin metabolites and their metabolic fate in the rat. Drug Metab Dispos

J Chromatogr A 2006;1117:103–8. 1999;27:309–16.

[37] Weiss DJ, Anderton CR. Determination of catechins in matcha green tea by [66] Meng X, Sang S, Zhu N, Lu H, Sheng S, Lee MJ, et al. Identiﬁcation and

micellar electrokinetic chromatography. J Chromatogr A 2003;1011:173–80. characterization of methylated and ring-ﬁssion metabolites of tea cat-

[38] Chen Z, Zhu QY, Tsang D, Huang Y. Degradation of green tea catechins in tea echins formed in humans, mice, and rats. Chem Res Toxicol 2002;15:

drinks. J Agric Food Chem 2001;49:477–82. 1042–50.

[39] Wang HHK. Epimerisation of catechins in green tea infusions. Food Chem [67] Zhu BT, Patel UK, Cai MX, Conney AH. O-methylation of tea polyphenols

2000;70:337–44. catalyzed by human placental cytosolic catechol-o-methyltransferase. Drug

[40] Zhu QYZA, Tsang D, Huang Y, Chen ZY. Stability of green tea catechins. J Agric Metab Dispos 2000;28:1024–30.

Food Chem 1997;45:4624–8. [68] Meng X, Lee MJ, Li C, Sheng S, Zhu N, Sang S, et al. Formation and identi-

−

[41] Komatsu YS, Hisanobu S, Saigo Y, Matsuda H, Hara RK. Studies on preservation ﬁcation of 4 -o-methyl-( )-epigallocatechin in humans. Drug Metab Dispos

of constituents in canned drinks. Part ii. Effects of ph and temperature on 2001;29:789–93.

reaction kinetics of catechins in green tea infusion. Biosci Biotechnol Biochem [69] Vaidyanathan JB, Walle T. Glucuronidation and sulfation of the tea ﬂavonoid

1993;57:907–10. (−)-epicatechin by the human and rat enzymes. Drug Metab Dispos

[42] Suzuki M, Sano M, Yoshida R, Degawa M, Miyase T, Maeda-Yamamoto 2002;30:897–903.

M. Epimerization of tea catechins and o-methylated derivatives of [70] Lu H, Meng X, Li C, Sang S, Patten C, Sheng S, et al. Glucuronides of tea

(−)-epigallocatechin-3-o-gallate: relationship between epimerization and catechins: enzymology of biosynthesis and biological activities. Drug Metab

chemical structure. J Agric Food Chem 2003;51:510–4. Dispos 2003;31:452–61.

[43] Wang R, Zhou W, Jiang X. Reaction kinetics of degradation and epimerization [71] Crespy V, Nancoz N, Oliveira M, Hau J, Courtet-Compondu MC, Williamson

of epigallocatechin gallate (egcg) in aqueous system over a wide temperature G. Glucuronidation of the green tea catechins (−)-epigallocatechin-3-gallate

range. J Agric Food Chem 2008;56:2694–701. and (−)-epicatechin-3-gallate, by rat hepatic and intestinal microsomes. Free

[44] Wang R, Zhou W, Wen RA. Kinetic study of the thermal stability of tea cat- Radic Res 2004;38:1025–31.

echins in aqueous systems using a microwave reactor. J Agric Food Chem [72] Negishi M, Pedersen LG, Petrotchenko E, Shevtsov S, Gorokhov A, Kakuta

2006;54:5924–32. Y, et al. Structure and function of sulfotransferases. Arch Biochem Biophys

[45] Hou Z, Sang S, You H, Lee MJ, Hong J, Chin KV, et al. Mechanism of action 2001;390:149–57.

of (−)-epigallocatechin-3-gallate: auto-oxidation-dependent inactivation of [73] Lu H. Mechanistic studies on the phase ii metabolism and absorption of tea

epidermal growth factor receptor and direct effects on growth inhibition in catechins. Toxicology. Ph.D., New Brunswick, Rutgers, The State University of

human esophageal cancer kyse 150 cells. Cancer Res 2005;65:8049–56. New Jersey; 2002.

[46] Hong J, Lu H, Meng X, Ryu JH, Hara Y, Yang CS. Stability, cellular uptake, bio- [74] Matern H, Matern S, Gerok W. Formation of bile acid glucosides by a

transformation, and efﬂux of tea polyphenol (−)-epigallocatechin-3-gallate sugar nucleotide-independent glucosyltransferase isolated from human liver

in ht-29 human colon adenocarcinoma cells. Cancer Res 2002;62:7241–6. microsomes. Proc Natl Acad Sci USA 1984;81:7036–40.

[47] Mochizuki M, Yamazaki S, Kano K, Ikeda T. Kinetic analysis and mechanistic [75] Boberg M, Angerbauer R, Kanhai WK, Karl W, Kern A, Radtke M, et al. Biotrans-

aspects of autoxidation of catechins. Biochim Biophys Acta 2002;1569:35–44. formation of cerivastatin in mice, rats, and dogs in vivo. Drug Metab Dispos

[48] Nakayama T, Enoki Y, Hashimoto K. Hydrogen peroxide formation during cat- 1998;26:640–52.

echin oxidation is inhibited by superoxide dismutase. Food Sci Technol Int [76] Tang C, Hochman JH, Ma B, Subramanian R, Vyas KP. Acyl glucuronidation and

1995;1:65–9. glucosidation of a new and selective endothelin et(a) receptor antagonist in

[49] Miura YH, Tomita I, Watanabe T, Hirayama T, Fukui S. Active oxygens gener- human liver microsomes. Drug Metab Dispos 2003;31:37–45.

ation by ﬂavonoids. Biol Pharm Bull 1998;21:93–6. [77] Labow RS, Layne DS. The formation of glucosides of isoﬂavones and

[50] Sang S, Lambert JD, Hong J, Tian S, Lee MJ, Stark RE, et al. Synthesis and struc- of some other phenols by rabbit liver microsomal fractions. Biochem J

ture identiﬁcation of thiol conjugates of (−)-epigallocatechin gallate and their 1972;128:491–7.

urinary levels in mice. Chem Res Toxicol 2005;18:1762–9. [78] Radominska A, Little J, Pyrek JS, Drake RR, Igari Y, Fournel-Gigleux S,

[51] Hernandez I, Alegre L, Munne-Bosch S. Enhanced oxidation of ﬂavan-3-ols and et al. A novel udp-glc-speciﬁc glucosyltransferase catalyzing the biosynthe-

proanthocyanidin accumulation in water-stressed tea plants. Phytochemistry sis of 6-o-glucosides of bile acids in human liver microsomes. J Biol Chem

2006;67:1120–6. 1993;268:15127–35.

[52] Tanaka T, Matsuo Y, Kouno I. A novel black tea pigment and two new oxidation [79] Gessner T, Jacknowitz A, Vollmer CA. Studies of mammalian glucoside conju-

products of epigallocatechin-3-o-gallate. J Agric Food Chem 2005;53:7571– gation. Biochem J 1973;132:249–58.

8. [80] Magdalou J, Antoine B, Ratanasavanh D, Siest G. Phenobarbital induction of

[53] Naasani I, Oh-Hashi F, Oh-Hara T, Feng WY, Johnston J, Chan K, et al. Blocking cytochrome p-450 and udp-glucuronosyltransferase in rabbit liver plasma

telomerase by dietary polyphenols is a major mechanism for limiting the membranes. Enzyme 1982;28:41–7.

growth of human cancer cells in vitro and in vivo. Cancer Res 2003;63:824– [81] Moon YH, Lee JH, Ahn JS, Nam SH, Oh DK, Park DH, et al. Synthesis, structure

30. analyses, and characterization of novel epigallocatechin gallate (egcg) glyco-

[54] Yang GY, Liao J, Li C, Chung J, Yurkow EJ, Ho CT, et al. Effect of black and sides using the glucansucrase from leuconostoc mesenteroides b-1299cb. J

green tea polyphenols on c-jun phosphorylation and h(2)o(2) production Agric Food Chem 2006;54:1230–7.

in transformed and non-transformed human bronchial cell lines: possible [82] Moridani MY, Scobie H, Salehi P, O’Brien PJ. Catechin metabolism: glutathione

mechanisms of cell growth inhibition and apoptosis induction. Carcinogene- conjugate formation catalyzed by tyrosinase, peroxidase, and cytochrome

sis 2000;21:2035–9. p450. Chem Res Toxicol 2001;14:841–8.

[55] Yang GY, Liao J, Kim K, Yurkow EJ, Yang CS. Inhibition of growth and induction [83] Li C, Lee MJ, Sheng S, Meng X, Prabhu S, Winnik B, et al. Structural identiﬁcation

of apoptosis in human cancer cell lines by tea polyphenols. Carcinogenesis of two metabolites of catechins and their kinetics in human urine and blood

1998;19:611–6. after tea ingestion. Chem Res Toxicol 2000;13:177–84.

[56] Vittal R, Selvanayagam ZE, Sun Y, Hong J, Liu F, Chin KV, et al. Gene expres- [84] Li C, Meng X, Winnik B, Lee MJ, Lu H, Sheng S, et al. Analysis of urinary metabo-

sion changes induced by green tea polyphenol (−)-epigallocatechin-3-gallate lites of tea catechins by liquid chromatography/electrospray ionization mass

in human bronchial epithelial 21bes cells analyzed by DNA microarray. Mol spectrometry. Chem Res Toxicol 2001;14:702–7.

−

Cancer Ther 2004;3:1091–9. [85] Meselhy MR, Nakamura N, Hattori M. Biotransformation of ( )-epicatechin

[57] Yang CS, Maliakal P, Meng X. Inhibition of carcinogenesis by tea. Annu Rev 3-o-gallate by human intestinal bacteria. Chem Pharm Bull (Tokyo)

Pharmacol Toxicol 2002;42:25–54. 1997;45:888–93.

[58] Feng WY. Metabolism of green tea catechins: an overview. Curr Drug Metab [86] Pietta PG, Simonetti P, Gardana C, Brusamolino A, Morazzoni P, Bom-

2006;7:755–809. bardelli E. Catechin metabolites after intake of green tea infusions. Biofactors

[59] Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computa- 1998;8:111–8.

tional approaches to estimate solubility and permeability in drug discovery [87] Chow HH, Cai Y, Alberts DS, Hakim I, Dorr R, Shahi F, et al. Phase i phar-

and development settings. Adv Drug Deliv Rev 2001;46:3–26. macokinetic study of tea polyphenols following single-dose administration

[60] Hong J, Lambert JD, Lee SH, Sinko PJ, Yang CS. Involvement of mul- of epigallocatechin gallate and polyphenone. Cancer Epidemiol Biomarkers

tidrug resistance-associated proteins in regulating cellular levels of Prev 2001;10:53–8.

−

( )-epigallocatechin-3-gallate and its methyl metabolites. Biochem Biophys [88] Lee MJ, Maliakal P, Chen L, Meng X, Bondoc FY, Prabhu S, et al.

Res Commun 2003;310:222–7. Pharmacokinetics of tea catechins after ingestion of green tea and (−)-

[61] Vaidyanathan JB, Walle T. Cellular uptake and efﬂux of the tea ﬂavonoid epigallocatechin-3-gallate by humans: formation of different metabolites

−

( )epicatechin-3-gallate in the human intestinal cell line caco-2. J Pharmacol and individual variability. Cancer Epidemiol Biomarkers Prev 2002;11:1025–

Exp Ther 2003;307:745–52. 32.

[62] Vaidyanathan JB, Walle T. Transport and metabolism of the tea ﬂavonoid [89] Yang CS, Chen L, Lee MJ, Balentine D, Kuo MC, Schantz SP. Blood and urine

(−)-epicatechin by the human intestinal cell line caco-2. Pharm Res levels of tea catechins after ingestion of different amounts of green tea by

2001;18:1420–5. human volunteers. Cancer Epidemiol Biomarkers Prev 1998;7:351–4.

[63] Lu H, Meng X, Yang CS. Enzymology of methylation of tea catechins and inhi- [90] Chen L, Lee MJ, Li H, Yang CS. Absorption, distribution, elimination of tea

bition of catechol-o-methyltransferase by (−)-epigallocatechin gallate. Drug polyphenols in rats. Drug Metab Dispos 1997;25:1045–50.

Metab Dispos 2003;31:572–9. [91] Soleas GJ, Diamandis EP, Goldberg DM. Wine as a biological ﬂuid: history,

[64] Okushio K, Suzuki M, Matsumoto N, Nanjo F, Hara Y. Methylation of tea cat- production, and role in disease prevention. J Clin Lab Anal 1997;11:287–

echins by rat liver homogenates. Biosci Biotechnol Biochem 1999;63:430–2. 313.

S. Sang et al. / Pharmacological Research 64 (2011) 87–99 99

[92] Terao J. Dietary ﬂavonoids as antioxidants in vivo: conjugated metabolites of [97] Deprez S, Brezillon C, Rabot S, Philippe C, Mila I, Lapierre C, et al. Polymeric

(−)-epicatechin and quercetin participate in antioxidative defense in blood proanthocyanidins are catabolized by human colonic microﬂora into low-

plasma. J Med Invest 1999;46:159–68. molecular-weight phenolic acids. J Nutr 2000;130:2733–8.

[93] Weisburger JH. Chemopreventive effects of cocoa polyphenols on chronic [98] Forester SC, Waterhouse AL. Metabolites are key to understanding health

diseases. Exp Biol Med (Maywood) 2001;226:891–7. effects of wine polyphenolics. J Nutr 2009;139:1824S–31S.

[94] Mulder TP, van Platerink CJ, Wijnand Schuyl PJ, van Amelsvoort JM. Analysis [99] Larrosa M, Luceri C, Vivoli E, Pagliuca C, Lodovici M, Moneti G, et al. Polyphe-

of theaflavins in biological fluids using liquid chromatography-electrospray nol metabolites from colonic microbiota exert anti-inflammatory activity on

mass spectrometry. J Chromatogr B Biomed Sci Appl 2001;760:271– different inﬂammation models. Mol Nutr Food Res 2009;53:1044–54.

9. [100] Monagas M, Khan N, Andres-Lacueva C, Urpi-Sarda M, Vazquez-Agell M,

[95] Henning SM, Aronson W, Niu Y, Conde F, Lee NH, Seeram NP, et al. Lamuela-Raventos RM, et al. Dihydroxylated phenolic acids derived from

Tea polyphenols and theaﬂavins are present in prostate tissue of humans microbial metabolism reduce lipopolysaccharide-stimulated cytokine secre-

and mice after green and black tea consumption. J Nutr 2006;136:1839– tion by human peripheral blood mononuclear cells. Br J Nutr 2009;102:201–

43. 6.

[96] Clifford MN, Copeland EL, Bloxsidge JP, Mitchell LA. Hippuric acid as a [101] Rios LY, Gonthier MP, Remesy C, Mila I, Lapierre C, Lazarus SA, et al. Chocolate

major excretion product associated with black tea consumption. Xenobiotica intake increases urinary excretion of polyphenol-derived phenolic acids in

2000;30:317–26. healthy human subjects. Am J Clin Nutr 2003;77:912–8.