Tea Catechin (EGCG) and Its Metabolites as Bioantioxidants - ACS

Chapter 21

Tea Catechin (EGCG) and Its Metabolites as Bioantioxidants I. Tomita, M. Sano, K. Sasaki, and T. Miyase

Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: September 24, 1998 | doi: 10.1021/bk-1998-0701.ch021

School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422, Japan

After a single administration of 100mg (-)-epigallocatechin gallate (EGCG)/kg body weight orally to SD rats, its concentration changes in the plasma and bile were investigated using HPLC with electrochemical detector (ECD). The highest concentration in plasma was observed one hour after administration. Not only free, but also conjugated forms of E G C G (e.g. glucuronide and sulfate) were detected in both biological fluids. When E G C G was incubated with rat plasma or bile at 37 degrees C, most of it disappeared after 30 min, while about 20% of the antioxidative activities remained in both fluids. Two new dimerized products of EGCG, P-1 and P-2, were isolated from the incubation mixture with bile. They were identified as theasinensin A (P-1), and a new product (P-2). Though the yield of these 2 products from E G C G was small, their antioxidative activities comparable to that of EGCG, should be worth consideration.

It is known that (-)-epigallocatechin gallate(EGCG) is the major tea catechin in green tea leaves, and it exerts powerful antioxidative activities in vitro(I,2), as well as in vivo experimental systems(3-5). We have shown that the oral administration of E G C G (50mg/kg) to rats once per day, for 7 days, inhibited /-butyl hydroperoxide (BHP) induced peroxidation in the liver and kidney slices up to 32% and 25%, respectively^). We have also reported that feeding rats with 1% polyphenon 100 (catechin mixture of (+)-gallocatechin 1.4%, (-)-epicatechin 5.8%, (-)-epicatechin gallate 12.5%, (-)-epigallocatechin 17.6% and E G C G 53.9% supplied by Mitsui Norin Co.Ltd.) for 18 months, significantly suppressed the increase of TBARS level in plasma(d). It also suppressed the increase of triglyceride, total cholesterol and phospholipid in the plasma. These results indicate

©1998 American Chemical Society

In Functional Foods for Disease Prevention I; Shibamoto, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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that catechins exert their activity in vivo after they were absorbed and distributed in rat tissue and organs. In relation to the activity of EGCG in vivo, its absorption, distribution and metabolic fate are of recent concern among investigators. Unno et al. (7,8\ and Okushio et al. (9), proved that EGCG was absorbed from rat or human intestinal tracts and it was present as such in plasma using GCMS and LC-MS analysis. The highest concentration of E G C G in rat or human plasma was detected 1 or 2 hours after the administration and decreased quickly thereafter. Lee et al.(/0), reported that catechins were present in their conjugated forms in the plasma and urine of humans who ingested 1.2g of decaffeinated green tea, containing 235mg catechins in total. The analysis was carried out by HPLC with the coulochem electrode array detection system. In vivo antioxidant effects of tea in men who ingested 300 ml of tea extract, prepared from 6g green tea leaves was recently reported by Serafini et al.(5), Maximum plasma radical trapping activity for peroxyl radicals was attained 30 min after the dosage of green tea. In the present study, we have investigated the time course of the levels of free E G C G and the conjugated forms (glucuronide and sulfate) in the plasma and bile, after the oral administration of EGCG to free-moving rats with catheters. We also tried to identify the chemical structures of two compounds produced during the incubation of E G C G with bile. Materials and Methods Chemicals: E G C G was prepared from green tea leaves, which was processed at Shizuoka Tea Experimental Station. Its purity was confirmed to be ca. 98% by H NMR. 3,7-Dimethyl amino 10-N-methyl carbamoyl phenothiazin (MCDP) for antioxidative assay was obtained from Kyowa Medex Co. Ltd.(Tokyo, Japan). Beta-Glucuronidase (G-7896) and sulfatase (S-9754) were purchased from Sigma Chemical Co. (St.Louis. M O , USA). A l l other chemicals used were of reagent grade quality. ,

Animals: Male Sprague-Dawley rats (8 weeks old) were obtained from Japan SLC Inc. (Hamamatsu, Japan). Rats underwent both venous, and biliary catheterization to allow free moving, and enteral feeding under unanesthetized conditions. Blood and bile samples from the above rats were collected time-dependently for a period of 24 hours. The plasma was obtained by centrifuging the blood at 1500xg for 10 min. These samples were mixed with 1/5 volume of ascorbic acid-EDTA solution(/0) and stored at -80 degrees C until analysis. Blood and bile used for the in vitro metabolic study of E G C G were collected from rats under E G C G anesthesia. Extraction of E G C G and its dimerization products from plasma and bile: A n aliquot of the sample was mixed with 1/4 volume of 0.1 H HC1. The mixture was extracted 2 times with the same volume of ethyl acetate . The combined extract was evaporated and dried under N2 gas. The residue was dissolved in 25% acetonitrile aqueous solution and used for the analysis of polyphenol and for the determination of antioxidant activities.


211 Analysis and assay for antioxidant potency. The analysis of E G C G and the dimerization products was performed by HPLC with ECD(77). Chemical structures of the dimerization products were elucidated on the basis of spectroscopic evidence such as U V , N M R , F A B - M S , SI-MS and CD. Incubation of the plasma and bile with beta-glucuronidase or sulfatase was carried out in the presence of ascorbic acid-EDTA by the method of Lee et al(/0). Antioxidant activities (AOA) were evaluated using M C D P and radical inducers (/-butyl hydroperoxide, H2O2), or a thiobarbituric acid (TBA) assay with rat brain homogenates by the method of Sano, et al.(/2) and Stocks, et al.(75), respectively.


Results and Discussion Time course of the contents of E G C G and its conjugated forms in plasma and bile. After EGCG(100mg/kg-body weight) was administered orally to SD rats, portal blood and bile were periodically collected through free moving cannulation system. As shown in Figure 1, the maximum E G C G level was found 60 and 90 min after administration in plasma and bile, respectively. The level of E G C G in plasma decreased sharply after 1 hour, which is in accord with the results of Unno et al (7). On the contrary , the level of E G C G in bile decreased slowly with another peak in 5 hours, which might be a reflection of reabsorption through the intestinal tract. When the plasma or bile was treated with beta-glucronidase (500 units), or sulfatase (40 units), at 37 degrees C for 45 min in the presence of vitamin C and EDTA, their level changed as shown in Figure 2. The portion of glucronide to that of sulfate, and free form in plasma, increased after 120 min, while it was almost unchanged in bile. Their ratio was found to be 1:1:1, for glucronide, sulfate and free form. Time course of antioxidative activity vs. metabolic change of E G C G in plasma and bile. When E G C G (100 ug/ml) was incubated with rat plasma or bile at 37 degrees C for 60 min, its content decreased sharply. Almost no E G C G was found after 45 min., as shown in Figure 3. The antioxidative activities (AOA) with BHP or H2O2, however, decreased rather moderately, and the activities remained in both plasma and bile after 45 min (see Figure 3). It was also found that antioxidative activities evaluated as TBARS with rat brain homogenate-autoxidation system(75) remained after 45 min (see also Figure 3). We, then, examined whether E G C G might be converted into other compounds with antioxidative activities in these fluids. Isolation and identification of 2 dimeric products from E G C G . Plasma or bile, to which E G C G (100 ug/ml) was added, and incubated at 37 degrees C for 30 min, was analyzed by HPLC. It was found that 2 new peaks (P-l and P-2) appeared, different from that of E G C G ( Figure 4). The elucidation of the chemical structure of P-l and P-2 were conducted using the products isolated from the reaction mixture of E G C G with bile. P - l isolated as an amorphous powder has a molecular formula of 24

C44H34O22

and [alpha] D was -187.5D. The chemical structure was identical to that of theasinensin A(14) as shown in Figure 5. P-2 was a new compound, and afforded tridecaacetate by treatment with as amorphous powder. The molecular formula was determined as C43H32O21 on the basis of secondary ion mass spectroscopy (SI-MS)


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500

0

2

— :

4 6 Time (h) Plasma, — ο - :

24

Bile

Figure 1. EGCG levels in rat plasma and bile following the oral administration of EGCG. Values are average of duplicate determinations from two rats.

Figure 2. Free and conjugated EGCG in rat plasma and bile.


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Antioxidative activities

EGCG


AOA

BHP

AOA

TBARS

Η2Ο2

brain

- o - Plasma, -»~Blle Figure 3. Fluctuation of antioxidative activities of EGCG in rat plasma and bile. Values are mean±SE (plasma:n=4, bile:n=5). Significantly different from antioxidative activity of EGCG (*P

Tea Catechin (EGCG) and Its Metabolites as Bioantioxidants - ACS

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