Biotransformation and Bioavailability of Tea Polyphenols: Implications

Chapter 18

Biotransformation and Bioavailability of Tea Polyphenols: Implications for Cancer Prevention Research

Downloaded by MONASH UNIV on July 10, 2013 | http://pubs.acs.org Publication Date: July 21, 2005 | doi: 10.1021/bk-2005-0909.ch018

Joshua D. Lambert, Jungil Hong, Mao-Jung Lee, Shengmin Sang, Xiaofeng Meng, Hong Lu, and Chung S. Yang Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854

Consumption of tea (Camellia sinensis) has been suggested to prevent cancer, heart disease, and other diseases. Animal studies have shown that tea and tea constituents inhibit carcinogenesis at a number of organ sites including the skin, lung, oral cavity, esophagus, stomach, liver, intestine, colon, and prostate. A number of potential cancer prevention mechanisms for the tea polyphenols have been proposed based mainly on studies with human cancer cells. These include protection from oxidative stress, induction of oxidative stress, inhibition of enzymes (MAP kinases, cyclin-dependent kinases, telomerase, etc.), and inhibition of growth factor -related cell signaling (epidermal growth factor and others). Whereas some studies report effects of epigallocatechin-3 -gallate (EGCG) at submicromolar levels, most experiments require concentrations of greater than 10 or 20 μΜ to demonstrate the effect. In humans, mice, and rats, tea polyphenols undergo glucuronidation, sulfation, methylation, and ring fission. Recent reports also suggest that EGCG and other catechins may be substrates for active efflux. The peak

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© 2005 American Chemical Society In Phenolic Compounds in Foods and Natural Health Products; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

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plasma concentrations of EGCG, epigallocatechin (EGC), and epicatechin (EC) following oral administration of green tea are 0.04 -1 μΜ, 0.3-5 μΜ, and 0.1-2.5 μΜ, respectively. The plasma levels of theaflavins are much lower (~ 2 nM). The present chapter reviews the literature concerning the biotransformation and bioavailability of tea polyphenols. It is intended to serve as a guide for designing future bioavailability experiments and for interpreting mechanistic data regarding the actions of tea polyphenols in vitro and in vivo.

Tea [Camellia sinensis (Theaceae)] is second only to water in terms of worldwide popularity as a beverage. Consumption of tea has been suggested to have many health benefits, including the prevention of cancer and heart disease

ω. Green, black, and Oolong tea are the three major commercial types of tea and differ in how they are produced and in their chemical composition. Green tea is prepared by pan-frying or steaming fresh leaves to heat inactivate oxidative enzymes and then dried. By contrast, black tea is produced by crushing fresh tea leaves and allowing enzyme-mediated oxidation to occur in a process commonly known as fermentation. Green tea is chemically characterized by the presence of large amounts of polyphenolic compounds known as catechins (Figure 1). A typical cup of brewed green tea contains, by dry weight, 30-40% catechins including epicatechin (EC), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epigallocatechin-3-gallate (EGCG). Through fermentation, most of the catechins are converted to oligomeric theaflavins and polymeric thearubigins in black tea (Figure 1). The resulting brewed black tea contains 3-10% catechins, 2-6% theaflavins, and >20% thearubigins (2), Both green and black tea and their constituents have been extensively studied in vitro and in animal models of carcinogenesis (3). Whereas these compounds have been shown to inhibit tumor formation in a number of models of carcinogenesis, the epidemiological data of cancer prevention remains mixed. Likewise, the primary cancer preventive mechanisms of tea in animal models remain unclear. EGCG, the most widely studied catechin, has been shown to inhibit enzymes (topoisomerase, matrix metalloproteinases, and telomerase), growth factor signaling (epidermal growth factor, vascular endothealial growth factor) and transcription factors (AP-1, N F K B ) (3,4,5). None of these mechanisms have been firmly established in animals or humans.

In Phenolic Compounds in Foods and Natural Health Products; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

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Epicatechin: R, = R = H

Jheaflavin: R, = R = OH


2

2

Eoigallocatechin: R, = H; R = OH

Theaflavin-3-gallate: R, = Galloyl; R = OH

2

2

Epicatechin-3-gallate: Rj = Galloyl; R = H

Thcaflavin-3'-gallate: R, = OH; R = Galloyl

2

2

Epigallocatechin-3-gallate: R, = Galloyl; R = OH 2

Theaflavin^U'-digallate: R, = R = Galloyl 2

Figure 1. Structures of the tea polyphenols

The catechins have been shown to undergo considerable biotransformation and to have low bioavailability (Figure 2) (J). The theaflavins are even less bioavailable. This poor bioavailability confounds attempts to correlate in vitro findings with cancer prevention in animal models. Cell line studies typically require concentrations of compound in the 10-100 μΜ range. Such concentrations are typically not observed systemically. The low bioavailability of the tea polyphenols is likely due to their relatively high molecular weight and the large number of hydrogen-bond donating hydroxyl groups (6). These hydroxyl groups not only serve as functional handles for phase II enzymes but may also reduce the absorption of the compounds from the intestinal lumen. According to Lipinski's Rule of 5, compounds with a molecular weight greater than 500, greater than 5 hydrogen-bond donors, or 10 hydrogen-bond acceptors have poor bioavailability due to their large actual size (high molecular weight) or large apparent size (due to the formation of a large hydration shell) (6). In the present article, we discuss the current literature regarding the bioavailability of the tea polyphenols and the potential impact of these data on the study of tea polyphenols as cancer preventive agents in humans.



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Figure 2. Biotransformation of the tea catechins.

Biotransformation The catechins are subject to extensive biotransformation including methylation, glucuronidation, sulfation, and ring-fission metabolism (Figure 2). Recent studies on the enzymology of EGC and EGCG methylation have shown that EGC is methylated to form 4'-0-methyl-(-)-EGC and EGCG is methylated by catechol-O-methyltransferase (COMT) to form 4"-0-methyl-(-)-EGCG and 4 ,4"-0-dimethyl-(-)-EGCG (7). At low concentrations of EGCG, the dimethylated compound is the major product. Rat liver cytosol shows higher COMT activity toward EGCG and EGC than did human or mouse liver cytosol. Additionally, the K and V values are higher for EGC than for EGCG (e.g. in human liver cytosol, K is 4 and 0.16 μΜ for EGC and EGCG, respectively). ,

m

m a x

m


216 Studies of EGCG and EGC glueuronidation reveal that EGCG-4"-0glucuronide is the major metabolite formed by human, mouse, and rat microsomes (8). Mouse small intestinal microsomes have the greatest catalytic efficiency ( V / K ) for glueuronidation followed, in decreasing order, by mouse liver, human liver, rat liver, and rat small intestine. Human UGT1A1, 1A8, and 1A9 have that highest activity toward EGCG, with the intestinalspecific UGT1A8 having the highest catalytic efficiency. EGC-3-O-glucuronide is the major product formed by microsomes from all species with the liver microsomes having a higher efficiency than intestinal microsomes. Based on these studies, it appears that mice are more similar to humans than are rats in terms of enzymatic ability to glucuronidate tea catechins. While these similarities must still be confirmed in vivo, this information will aid in choosing the most appropriate animal model to study the potential health benefits of tea constituents. Vaidyanathan et al have shown that EC undergoes sulfation catalyzed by human and rat intestinal and liver cytosol, with the human liver being the most efficient (9). Further studies have revealed that sulfotransferase (SULT)lAl is largely responsible for this activity in the liver, whereas both SULT1A1 and SULT1A3 are active in the human intestine. The catalytic efficiency for SULT1A1 and SULTlA3-mediated sulfation of EC are 5834 and 55 \ihlmmlm%. EGCG is also time- and concentration-dependently sulfated by human, mouse, rat liver cytosol (10). The rat has the greatest activity followed by the mouse and the human. Anaerobic fermentation of EGC, EC, and ECG with human fecal microflora has been shown to result in the production of the ring fission products 5-(3',4 ,5'trihydroxyphenyl)-y-valerolactone (M4), 5-(3',4 -dihydroxyphenyl)-y-valerolactone (M6), and S-^^'-dihydroxyphenyO-y-valerolactone (M6 ) (Fig 2) (//). We have found these ring fission products are present in human urine and plasma approximately 3 h after oral ingestion of 20 mg/kg decaffeinated green tea (12). The compounds have a T of 7.5 - 13.5 h and reach peak plasma concentrations of 100-200 nM. Peak urine concentrations of 8, 4, and 8 μΜ have been demonstrated for M4, M6, and M6 , respectively following ingestion of 200 mg EGCG. M4, M6, and M6 retain the polyphenolic character of the parent compound; have the addition of a potentially, biologically-active valerolactone structure; and may therefore have biological activities. In animals, the phase II metabolism reactions likely compete with one another. The relative concentration of each enzyme and their activities for the tea polyphenols determine the metabolic profile in vivo. Since EGCG has a lower K for COMT than UGTs, methylation may be favored at physiological (usually low) concentrations. Indeed, EGCG is first methylated to form 4"-0-methylEGCG and then further methylated to form 4',4"-di-0-methyl-EGCG in vivo (13). At high doses, glueuronidation becomes more prominent, leading the


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max

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217 formation of EGCG-4"-glucuronide in the mouse (8). This compound can be further methylated on the B-ring to produce different methylated metabolites. This is consistent with the observation that four mono-methylated and two dimethylated compounds are found in mouse urine after hydrolysis with βglucuronidase and sulfatase following administration of high doses of EGCG to the mouse (13). The methylated compounds were found to have similar peaks heights: if conjugation had not preceeded methylation, 4"-0-methyl-EGCG peak would have been the predominant mono-methylated metabolite.


Active Efflux Active efflux has been shown to limit the bioavailability and cellular accumulation of many compounds. The multidrug resistance-associated proteins (MRP) are ATP-dependent efflux transporters that are expressed in many tissues and are overexpressed in many human tumor types. MRP1 is located on the basolateral side of cells and is present in nearly all tissues. The physiological function of this protein is to transport compounds from the interior of the cells into the interstitial space (14). In contrast, MRP2 is located on the apical surface of the intestine, kidney, and liver, where it transports compounds from the bloodstream into the lumen, urine, and bile, respectively (14). Recent studies on EGCG uptake in our laboratory have shown that indomethacin (MRP inhibitor) increases the intracellular accumulation of EGCG, EGCG 4"-0-methyl-EGCG, or 4',4"-di-0-methyl-EGCG by 10-, 11-, or 3-fold in Madin-Darby canine kidney (MDCKII) cells overexpressing MRP-1 (15). Similarly, treatment of MRP-2 overexpressing MDCKII cells with MK-571 (MRP-2 inhibitor) results in 10-, 15-, or 12-fold increase in the intracellular levels of EGCG, 4"-

Biotransformation and Bioavailability of Tea Polyphenols: Implications

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