Effect of Green Tea Catechins on Plasma Cholesterol Level in Cholesterol-Fed Rats. Keiichiro MURAMATSU,1 Mayumi FUKUYO, and Yukihiko HARA 2

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1 J. Nutr. Sci. VitaminoL, 32, , 1986 Effect of Green Tea Catechins on Plasma Cholesterol Level in Cholesterol-Fed Rats Keiichiro MURAMATSU,1 Mayumi FUKUYO, and Yukihiko HARA 2 1 Laboratory of Food and Nutrition, Department of Agricultural Chemistry, Faculty of Agriculture, Shizuoka University, Ohya, Shizuoka 422, Japan 2 Food Research Laboratories, Mitsui Normn Co., Ltd., Fujieda 426, Japan (Received July 4, 1986) Summary Effects of tea catechins (tannins) on lipid metabolism were studied in male weanling rats fed a 25% casein diet containing 15% lard and 1% cholesterol for 28 days. Crude tea catechins prepared from green tea powder were supplemented at a 1% and 200 of the lard-cholesterol diet. The addition of 2% tea catechins slightly depressed growth but at the 1 level was without effect. Tea catechins decreased plasma total cholesterol, cholesterol ester, total cholesterol-hdl-cholesterol (VIDL-+LDL -cholesterol) and atherogenic index (VLDL-+ LDL-cholesterol/HDL -cholesterol). Hematocrit and plasma glucose were not altered by the addition of tea catechins. The liver weight, liver total lipids and cholesterol concentrations in rats fed the lard-cholesterol diet increased more than in the control rats, but the addition of tea catechins to the lard-cholesterol diet decreased those parameters. Tea catechin supplementation increased fecal excretion of total lipids and cholesterol. The results demonstrate that tea catechins exert a hypocholesterolemic effect in cholesterol-fed rats. Key Words tea catechins (tea tannins), epigallocatechin gallate, plasma cholesterol, atherogenic index Tea is a widely consumed traditional beverage. It is known that the astringent taste of green tea is due to polyphenol, the so-called "tea tannin," which makes up 10-15% (1) of the tea leaf. Tea tannin consists of four main catechin com ponents (1-4): (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epichatechin gallate (ECg) and (-)-epigallocatechin gallate (EGCg). EGCg is the predominating catechin, accounting for more than half of the total catechin content (5). Recent studies have shown that tea catechins have capillary resistance activity (6), and antioxidative (4, 7-9), antimutagenic (10-12) and antihepatotoxic (13) effects. Tea 613

2 614 K. MURAMATSU, M. FUKUYO, and Y. HARA catechins also alleviate the toxicity of heavy metal (14). It would be of interest to examine whether tea catechins have hypolipemic property in relation to the benefical effect of green tea, but information is limited (15, 16). The present study was designed to evaluate the effects of catechins prepared from green tea leaf on the lipid metabolism in rats fed diets containing cholesterol. MATERIALS AND METHODS Preparation of crude tea catechin mixture. A crude catechin mixture was prepared from spray-dried Indian green tea powder (Mitsui Normn Co., Shizuoka) by the procedure described in Fig. 1. Briefly, the green tea powder was dissolved in hot water and extracted three times with ethyl acetate, and the ethyl acetate was evaporated. The resulting concentrated solution was freeze dried to yield a crude catechin mixture. The purity of catechins was 72% (17). The contents of the main catechin components are listed in Table 1. Animals and diets. Male weanling Wistar strain rats (Shizuoka Agricultural Cooperative Association for Laboratory Animals, Hamamatsu) each weighing 55 to 60g were divided into groups of six rats. Animals were individually housed in stainless-steel cages with screen bottoms in a room maintained at Ž with about 50% relative humidity. The room was lighted from 06:00 to 18:00h. Fig. 1. Separation of crude catechin mixture from green tea leaves. Spray-dried Indian green tea powder (Mitsui Norin Co., Shizuoka) was used. Total catechin contents were determined by a colorimetric method using a ferrous tartrate reagent (17). J. Nutr. Sci. Vitaminol.

3 TEA CATECHINS AND PLASMA CHOLESTEROL 615 Table 1, Percentages of main catechins in crude catechin mixture prepared from Indian green tea. 1 1 Each catechin content was determined by high pressure liquid chromatography (4). This mixture also contained trace amounts of other catechins, (+)-gallocatechin, (+) -catechin and gallic acid. Small amounts of caffeine (2.0%), chlorophyll, flavanol glycosides, flavanone glycosides, flavone glycosides, antocyans, mono and polysac charides and organic acids were detected as concomitant impurities. Table 2. Composition of diets (%). The diets also contained retinyl acetate (2,OOOIU), ergocalciferol (200IU) and ƒ -tocopherol (0.01g) per 100g of diet. 1 25C (25% casein); 25CL(25C+15% lard); 25CLC (25CL+1% cholesterol); 25CLC+1% catechin (25CLC+1%tea catechins); 25CLC+2% catechin (25CLC+2% tea catechins). 2 Harper, A. E. (1959). J. Nutr., 68, Crude catechin mixture was prepared from green tea leaves (see Fig. 1). The composition of experimental diets is given in Table 2. The diets fed were the 25% casein (25C) diet, the 25C diet containing 15% lard (25CL), the 25CL diet containing 100 cholesterol (25CLC), and the 25CLC diets supplemented with 1 and 2% crude catechin mixture (25CLC+1% catechin and 25CLC+2% catechin). Caffeine (0.06%) in an amount equivalent to that contained in 2% crude catechins was added to the 25CLC diet, since it has been shown that caffeine has a Vol. 32, No. 6, 1986

4 616 K. MURAMATSU, M. FUKUYO, and Y. HARA hypercholesterolemic effect (18). Crude catechin mixture, cholesterol and caffeine were incorporated at the expense of a-starch. Diets and water were provided ad libitum for 28 days. Body weight and food consumption were recorded daily. The feces of each rat were collected over 2 days before the end of the experimental period and frozen at -20 Ž. They were lyophilized, weighed and ground. Blood for the hematocrit test was drawn from the tail vein on the day prior to sacrifice. At the end of the experimental period, the rats were fasted overnight, then anesthetized with ether and blood was collected in heparinized syringes by heart puncture. Analytical methods. Hematocrit values were measured in heparinized mi, crotubes. Plasma glucose was determined using a glucose oxidase kit (Wako Pure Chemical Ind., Osaka). Total liver and feces lipids were extracted with chloroform methanol (3:1) by the method of Folch et al. (19) and determined gravimetrically. Total cholesterol in the plasma, liver and feces was measured by the method of Zak (20). Plasma high density lipoprotein (HDL) was separated by the heparine - Mn2+ precipitation method (21) followed by determination of HDL-cholesterol. Free cholesterol and HDL-cholesterol in the plasma were determined enzymatically using commercial kits (Wako Pure Chemical Ind., Osaka). Statistical analysis. Statistical evaluation of the experimental data was done by one-way analysis of variance (22). RESULTS Body weight gain, food intake and feed efficiency Lard (25CL)-and lard-cholesterol (25CLC)-fed rats had increased body weight gain and feed efficiency compared with the basal (25C) group. Body weight gain, food consumption and feed efficiency in rats fed the lard-cholesterol diet sup plemented with 1% crude tea catechins (25CLC+ 1% catechin) were not different from those of the control (25CLC) diet group but the addition of 2% tea catechins (25CLC+2% catechin) somewhat decreased growth and feed efficiency (Table 3). Table 3. Effect of tea catechins on growth, food intake and feed efficiency. 1 Mean }SE; n=6. Values without common superscripts are significantly different at p<0.05. J. Nutr. Sci. Vitaminol.

5 TEA CATECHINS AND PLASMA CHOLESTEROL 617 Organ weights Rats fed the 25CLC, 25CLC+1% catechin and 25CLC+2% catechin diets had larger liver size than the 25C and 25CL groups, but other relative organ weights did not significantly differ among the dietary groups (Table 4). Hematocrit, plasma glucose and cholesterol Hematocrit values and the plasma glucose level were not altered by the addition of tea catechins to the diets. The plasma total cholesterol level in rats fed the 25CL and 25CLC diets increased significantly compared to the 25C group. The addition of 1% and 2% tea catechins to the lard-cholesterol diet resulted in significantly lower levels of plasma total cholesterol, cholesterol ester and total cholesterol -HDL-cholesterol, i.e., very low density lipoprotein (VLDL)-+low density lipoprotein (LDL)-cholesterols. The free cholesterol level also decreased, but not to a statistically significant degree. In contrast, the HDL-cholesterol level showed a tendency to increase with the addition of 2% catechins. The atherogenic index (VLDL-+ LDL-cholesterols/HDL-cholesterol) in rats fed the 25CLC+1% catechin and 25CLC+2% catechin diets significantly decreased compared to that of rats fed the 25CLC diet (Table 5). Liver total lipids and cholesterol Liver total lipids in rats fed the 25CL diet increased more than in the 25C group. The 25CLC diet significantly increased liver total lipids and cholesterol levels Table 4. Effect of tea catechins on various organ weights. 1 Mean }SE; n=6. Values without common superscripts are significantly different at p<0.05. Vol. 32, No. 6, 1986

6 RA Table 5. Effect of tea catechms on hematocrit values and plasma glucose, plasma and liver lipid concentrations. 1 Mean }SE; superscripts n=6. Values without common are significantly different at p<0.05. Total 2 cholesterol-free cholesterol ( mg ~dl). 3 Total - HDL-chol/HDL-chol (ịe., -chol/ VLDL-+LDL HDL-chol).

7 TEA CATECHINS AND PLASMA CHOLESTEROL 619 Table 6. Effect of tea catechins on fecal excretion of total lipids and cholesterol. 1 Mean }SE; n=6. Values without common superscripts are significantly different at p<0.05. compared to the 25C and 25CL groups but the addition of 1% and 2% tea catechins to the lard-cholesterol diet decreased those parameters significantly (Table 5). Fecal total lipids and cholesterol Rats fed the 25CLC diet showed marked increases in fecal total lipids and cholesterol as compared to the 25C group (data not shown), which were further increased by the addition of 1% and 2% tea catechins (Table 6). DISCUSSION There is a great deal of evidence that dietary fat, cholesterol, sugar, protein and fiber can influence plasma cholesterol levels and atherogenesis in experimental animals and in humans. Relatively little attention has been paid to polyphenols and tannins in foodstuffs. Wursch reported a reduction in plasma cholesterol in rats fed polymerized tannins (23). Okuda et al. (15) observed that tea tannins inhibit the elevation of serum cholesterol levels when administered to rats fed a peroxidized corn oil diet. Fukuo et al. (16) reported that the blood total cholesterol level in human subjects who habitually of drinking a mixture of egg yolks and green tea was within the normal range, and suggested that tannins in green tea may be involved in the maintenance of normal blood cholesterol levels. The results of the present study indicated that the plasma total cholesterol levels and atherogenic index in rats fed the lard-cholesterol diet were raised, but the supplementation of 1% and 2% tea catechins to the diet reduced both parameters. The purity of crude tea catechins used in this study was 72%, and thus the influence of impurities in the preparation cannot be disregarded. However, in an another experiment using pure EGCg, the main component of tea catechins, we proved that EGCg decreases plasma cholesterol levels as well as crude tea catechins (24). The importance of plasma cholesterol and lipoprotein concentrations in Vol. 32, No. 6, 1986

8 620 K. MURAMATSU, M. FUKUYO, and Y. HARA atherosclerosis has been indicated by several workers. Increased levels of plasma cholesterol, LDL- and VLDL-cholesterols are a risk factor contributing to the development of coronary heart diseases (25, 26). Our results suggest that catechins (tannins) in green tea exert a hypocholesterolemic effect, and therefore have a protective effect against the atherosclerotic process. Lowered plasma cholesterol was also observed when 0.5% of crude catechins or EGCg (24) supplemented the lard-cholesterol diet. Massive oral doses of plant phenolic compounds (tannins, tannic acid and gallotannic acid) given to rats have been reported to cause growth depression and toxicity (27-30). The present experiment showed that slight growth depression occurred with 2% tea chatechins added to the diet. Growth depression would probably occur if a higher amount of tea catechins were supplemented to the diet. We did observe growth retardation in rats by adding the 3% tea catechins to their diet (unpublished data). The mechanism(s) by which dietary tea catechins result in lowered plasma cholesterol levels is not clear. It may be assumed that the enhanced excretion of cholesterol-the lower absorption of cholesterol by tea catechins-is primarily responsible for this phenomenon. The other possible mechanism of tea catechin action may be increased excretion of bile acids and alteration of endogenous cholesterol metabolism. Experiments are in progress to elucidate the mechanism of action. REFERENCES 1) Maeda, S., and Nakagawa, M. (1977): General chemical and physical analyses on various kinds of green tea. Chagyo Kenkyu Hokoku (in Japanese), No. 45, ) Hirose, S., and Tamada, S. (1979): A quantitative determination of flavanols in tea by high pressure liquid chromatography. Chagyo Kenkyu Hokoku (in Japanese), No. 50, ) Saijo, R. (1981): Changes of catechin contents in tea leaves during development. Chagyo Gijutsu Kenkyu (in Japanese), No. 61, ) Matsuzaki, T., and Hara, Y. (1985): Antioxidative activity of tea leaf catechins. Nippon Nogeikagaku Kaishi (J. Agric. Chem. Soc. Jpn.), 59, ) Roberts, E. A. H. (1962): The Chemistry of Flavonoid Compounds, ed. by Geissman, T. A., Pergamon Press, New York, pp ) Das, D. N. (1963): Effect of tea and its tannins upon capillary resistance of guinea-pigs. Ann. Biochem. Exp. Med., 23, ) Okuda, T., Kimura, Y., Yoshida, T., Hatano, T., Okuda, H., and Arichi, S. (1983): Studies on the activities of tannins and related compounds from medicinal plants and drugs. I. Inhibitory effects on lipid peroxidation on mitochondoria and microsomes of liver. Chem. Pharm. Bull., 31, ) Kimura, Y., Okuda, H., Mori, K., Okuda, T., and Arichi, S. (1984): Effect of extracts of various kinds of tea on lipid metabolic injury in rats-fed peroxidized oil. Nippon Eiyo Shokuyro Gakkaishi (J. Jpn. Soc. Nutr. Food Sci.), 37, ) Hatano, T., Yoshida, T., Fujita, Y., Okuda, T., Kimura, Y., Okuda, H., and Arichi, S. (1984): Effect of tannins of wakan-yaku on lipid peroxidation and fat cells. Wakan- J. Nutr. Sci. Vitaminol.

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10 622 K. MURAMATSU, M. FUKUYO, and Y. HARA 29) Glick, Z., and Joslyn, M. A. (1970): Effect of tannic acid and related compounds on the absorption and utilization of proteins in the rat. J. Nutr., 100, ) Joslyn, M. A., and Glick, Z. (1969): Comparative effects of gallotannic acid and related phenolics on the growth of rats. J. Nutr., 98, J. Nutr. Sci. Vitaminol.