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PI1 SOO24-3205(9!9)00607-4 CHINESE Life Sciences, Vol. 66, No. 5, pp. 41 I-423, zoo0 Copy-ight0 1999 Elsevk Sciexe Inc. F’rintcdin the USA. All t-i&bresaved 0024_3205/00/Esec tiunt mattu GREEN TEA LOWERSCHOLESTEROLLEVELTHROUGH AN INCREASEIN FECAL LIPID EXCRETION Teddy T.C. Yang and Marcel W.L. Koo Department of Pharmacology, Faculty of Medicine The University of Hong Kong, l/F, Li Shu Fan Building 5 Sassoon Road, Hong Kong (Received in final form September 9, 1999) Summary Lung Chen Tea, a Chinese green tea, has been found to lower serum and liver cholesterol. In this study, its dose response and mechanisms of action on cholesterol lowering in diet-induced hypercholesterolemic Sprague-Dawley rats were investigated. The activities of three major lipid metabolizing enzymes, 3hydroxy-3-methylglutaryl-coenzyme A (HMG-Co A) reductase, cholesterol 7ahydroxylase and fatty acid synthase (FAS), as well as fecal excretion of bile acids and cholesterol were examined. Lung Chen Tea administration for eight weeks significantly lowered the serum cholesterol in the 2% and 4% groups. The activities of the three enzymes were not affected by Lung Chen Tea, but the fecal bile acids and cholesterol excretions were significantly increased. These results demonstrated that Lung Chen Tea lowered plasma cholesterol by increasing fecal bile acids and cholesterol excretion. Further investigation is required to evaluate the exact mechanisms of action of Lung Chen Tea. Key Worak Lung Chen tea, green tea, cholesterol, HMG-CoA reductase, cholesterol 7a-hydroxylase, fatty acid synthase, bile acids Hypercholesterolemia is a major risk factor in coronary heart disease while maintaining the blood cholesterol level within normal range can reduce the risk of having coronary heart disease. Current drug treatments of hypercholesterolemia involve the use of statins, fibrates and bile acids binding resins. They lower cholesterol level by decreasing de nova synthesis of cholesterol through inhibition of HMG-CoA reductase, affecting lipoproteins metabolism and reducing absorption of cholesterol respectively. Corresponding The University address: Dr. M.W.L. Koo, Department of Pharmacology, Faculty of Medicine, of Hong Kong, l/F Li Shu Fan Building, 5 Sassoon Road, Hong Kong. 412 Tea Lowers Cholesterolby Lipid Excretion Vol. 66, No. 5, 2000 It has been shown that Japanese green tea and Lung Chen Tea, a Chinese green tea, lowered plasma cholesterol (1,2). Tea catechins, the most abundant components in green tea, have been implicated for its hypocholesterolemic effect. In this study, the effect of Lung Chen Tea on hepatic enzymes that regulate cholesterol synthesis, degradation, and lipogenesis were investigated. Diet-induced hypercholesterolemic rats were treated with different concentrations of Lung Chen Tea for eight weeks and their effects on HMG-CoA reductase, cholesterol 7ahydroxylase and FAS were examined. Materials and Methods Chemicals dithiothreitol (DTT), Acetyl CoA, ammonium formate, cholesterol oxidase, ethylenediaminetetraacetate (EDTA), ethylene glycol-bis (P-aminoethyl ether) N’N’NWtetraacetic acid (EGTA), (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCG), glucose 6-phosphate, glucose 6-phosphate dehydrogenase, 7P-hydroxycholesterol, hydroxysteriod dehydrogenase, leupeptin, malonyl CoA, l3-nicotinamide adenine dinucleotide (NAD+), P-nicotinamide adenine dinucleotide phosphate (NADP’), P-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), phenylmethylsulfonyl fluoride (PMSF), and sodium formate were purchased from Sigma Chemical Co. (St Louis, MO). 7a-Hydroxycholesterol was obtained from Steraloids, Inc. (Pauling, NV). QAE-Sephadex A25 (chloride form) was ordered from Pharmacia (Upsala, Sweden). [14C] HMG-CoA was from DuPont NEN (USA). Beckman ready gel liquid scintillation cocktail was from Beckman (Beckman Instruments Inc., USA). Animals and treatment Male Sprague-Dawley (SD) rats of 1 lo-130 g were used. They were housed in an airconditioned room at 20°C and 6570% relative humidity with a 12-h light-dark cycle. They were given free access to Purina rat chow (Ralston Purina, USA) and tap water for 1 week before the experiment. Afier 1 week of acclimatization, rats were randomly divided into groups and were assigned to different dietary treatments. Rats of the normal control group were fed with standard Purina rat chow and tap water ad libitum throughout the experimental period. Others were given cholesterol-enriched diet containing 1% cholesterol and 0.5% cholic acid (2) for 9 weeks after acclimatization. Rats of the disease model group were given free access to tap water for the 9 weeks period while the other three groups were given tap water for 1 week followed by 1%, 2% and 4% Lung Chen Tea for the following eight weeks. One percent, 2% and 4% tea solutions were prepared by soaking 5 g, 10 g and 20 g of tea leaves in 500 ml of boiled water for 30 minutes, and the tea solution was filtered before supplied to the rats (3). At the end of the experimental period, blood was collected from tail vein of the rats after 12-h of fasting. The animal was then killed by decapitation and the liver was perfused with cold normal saline before excision. It was blotted dry, and weighed before frozen in liquid nitrogen. The tissue samples were stored at -70°C until used. Vol. 66, No. 5,200O Tea Lowers Cholesterol by Lipid Excre.tion 413 Blood lipids determination Serum total cholesterol and triglyceride levels were assayed enzymatically (Cholesterol liquicolor and Triglycerides GPO liquicolor, Human Gesellschafi tir Biochemica und Diagnostica mbH, Germany) with the methods described by Roeschlau et al. (4) and Jacobs and VanDmark (5) respectively. Serum HDL-cholesterol level was determined after precipitation of apolipoprotein B containing lipoproteins by phosphotungstic acid and magnesium chloride (6). Measurement of tea catechins Lung Chen Tea was imported from West Lake of Hangzhou, China. It was prepared from the youngling buds of the tenderest leaves sprouting in the spring season. Catechins in tea solutions were analyzed with high performance liquid chromatography (HPLC) as described by Matsuda et al. (7) with minor modification. A HPLC solvent delivery system (ConstraMetric 3200, Waters) and Beckman Ultrasphere ODS column (5 urn, 4.6 x 250 mm) were used to separate the catechins in the tea solutions. Twenty microliters of tea sample were injected into the column and methanol:acetic acid (25%:0.1%) was used as the mobile phase. The flow rate was adjusted to 1 mYmin and the absorbance was set at 280 nm with a sensitivity of 0.01 absorbance units full scale (aufs). Determination of liver weight and lipids content Relative liver weight was represented as the ratio of liver to body weight. Liver cholesterol and triglyceride were extracted with chloroformmethanol (2:l) mixture (8). Cholesterol and triglyceride contents in the liver were measured with diagnostic kits and expressed as mg cholesterol or mg triglyceride/g liver. Determination of hepatic enzymes activities Hepatic FAS activity was determined with the method described by Nepokroeff et al. (9). Liver was homogenized in 1.5 volume of ice-cold phosphate-bicarbonate buffer (70 mM NaHC03, 85 mM KzHP04, 9 mM KHzPO~, 1 mM EDTA and 1 mM DTT at pH 8.0). The homogenate was centrifuged at 11,500 rpm (Beckman 52-21 Centrimge, USA) fot 10 minutes and ultracentrifuged at 40,000 rpm for 60 minutes at 4°C (Beckman L8-M ultracentrifuge, USA). One hundred microliters of the supematant and 1 ml of 500 FM potassium phosphate buffer at pH 7.0 containing 33 nmole acetyl CoA; 100 nmole malonyl CoA; 100 nmole NADPH and 1 umole EDTA were pre-incubated at 30°C for 10 minutes. One hundred micromoles NADPH in 10 pl of normal saline was added to initiate the reaction and the change in absorbance at 340 run was recorded for 5 minutes. Correction for auto-oxidation and other enzymatic oxidation of NADPH was made by measuring the absorbance in the reaction mixture with the omission of malonyl CoA. The activity of FAS was expressed as nmol NADPH oxidized/mg protein/minute. HMG-CoA reductase activity was measured by homogenizing the liver in 9 volumes of homogenizing buffer (50 mM Tris, 0.4 mM NaCl, 0.25 M sucrose, 1 mM EDTA, 10 mM DTT at pH 7.4) and centrifuged to obtain the microsomes, which was then re-suspended in the buffer (10). One hundred microliters of 100 mM sodium phosphate buffer containing 30 pM [14C] HMG-CoA (0.01 uCi), 10 mM imidazole, 5 mM DTT, 10 mM EDTA, 12 mM glucose 6- 414 Tea Lowers Cholesterol by Lipid Excretion Vol. 66, No. 5, 2000 phosphate, 0.5 unit glucose 6-phosphate dehydrogenase, 3 mM NADP+, at pH 7.4, was mixed with 25 ul of microsomal preparation in a 1.5 ml microcentrifuge tube. They were incubated at 30°C for 10 minutes and the reaction was terminated by the addition of 25 pl of 2 M HC1 and 10 mM mevalonolactone. The mixture was further incubated at 37’C for 30 minutes to allow lactonization of mevalonate, the enzymatic product of HMG-CoA reductase, to mevalonolactone. Five hundred microliters of 20 mM ammonium formate was added and then centrifuged at 10,000 rpm for 15 minutes at room temperature to pellet the denatured protein. Five hundred microliters of the supernatant was applied to the QAE-Sephadex A25 (formate form) column while the eluant was discarded. The QAE-Sephadex A25 (formate form) was prepared by soaking the QAE-Sephadex A25 (chloride form) overnight in 1 M sodium formate and then in 200 mM ammonium formate with several changes. A further 3 x 1 ml of 20 mM ammonium formate was loaded into the column and the eluant was collected into counting vials. Ten milliliters of scintillant (Beckman ready gel liquid scintillation cocktail, USA) was added to each counting vial and the radioactivity was determined with a scintillation counter (Beckman LS6500, USA). Blank samples were prepared in the same way except HCl was added before the addition of the microsomal preparation. The activity of HMG-CoA reductase was expressed as pm01 mevalonate/mg protein/minute. The activity of cholesterol 7a-hydroxylase was measured with reverse-phase HPLC (11). Liver was homogenized in 9 volumes of 100 mM potassium phosphate buffer at pH 7.2, consisting of 100 mM sucrose, 50 mM potassium chloride, 50 mM NaF, 5 mM EGTA, 3 mM DTT, 1 mM EDTA, 1 mM PMSF and 100 pM leupeptin. Microsomal preparation was obtained by centrifugation and it was homogenized in the original isolation buffer before the protein concentration was adjusted to 50 mg/ml. One milliliter of the reaction mixture contained 0.1 M potassium phosphate buffer, 50 mM NaF, 5 mM DTT, 1 mM EDTA, 20% glycerol, 0.015% Chaps, 1 mg microsomal preparation and 10 mM NADPH at pH 7.4. The mixture was preincubated at 37°C for 5 minutes and the reaction was initiated by the addition of 100 pl of 100 mM NADPH and incubated at 37°C for 20 minutes. The reaction was terminated by adding 30 pl of 20% sodium cholate and 1 pg 7P-hydroxycholesterol was added as an internal recovery standard. The conversion of 7a-hydroxycholesterol to 7a-hydroxy-4-cholesten-3-one was induced by the addition of 44 pl of 0.1% cholesterol oxidase in potassium phosphate buffer at pH 7.4, containing 1 mM DTT and 20% glycerol and incubated at 37°C for another 10 minutes. At the end of the incubation, the reaction was terminated by the addition of 2 ml of 95% ethanol. The products of the enzymatic reactions, 7a- and 7P-hydroxy-4-cholesten-3-one, were extracted three times with petroleum ether and re-suspended in 100 pl of acetonitrile:methano1(7:3). They were injected into a reverse-phase Beckman Ultrasphere-ODS column (5 pm, 4.6 x 250 mm) of a HPLC (Waters 600s Controller & 616 Pump, USA). The mobile phase used was 70% acetonitrile:30% methanol and the absorbance was monitored at 240 nm with a photodiode array detector (Waters 996, USA). The products were eluted at a flow rate of 0.8 ml/min for 18 minutes, thereafter, the rate was increased to 2.0 ml/min for a further 14 minutes to elute all cholesterol from the column. The activity of the cholesterol 7a-hydroxylase was expressed as pmol 7ct-hydroxycholesterolimg protein/minute. Determination ofprotein The protein concentration of the microsomal preparation for the assays of HMG-CoA reductase, cholesterol 7a-hydroxylase and FAS activities were measured by the Lowry method (12). Vol. 66. No. $2000 Determination Tea Lowers Cholesterolby Lipid Exation 415 offecal cholesterol and bile acids Feces of the last two days of the eight-week treatment period were collected. They were freezedried for 48 hours and stored at -7O’C until used. The feces were ground into fine powder and extracted with a chlorofotmmethanol (2:l) mixture in accordance to the method described by Folch et al. (8). The amount of fecal bile acids was determined enzymatically with the method of Turley and Dietschy (13). The reaction mixture that contained 750 pl Tris-HCl (0.133 M Tris, 0.666 mM EDTA, pH 9.5), 300 ~1 hydrazine sulphate (pH 9.5), 100 pl NAD+ (7 mM, pH 7.0), and 30 pl samples was pre-incubated at 30°C before the addition of 30 ul hydroxysteriod dehydrogenase preparation. It was incubated for another 60 minutes at 30°C in a shaking water bath and the absorbance of the mixture was recorded at 340 mn against reagent blank. Fecal cholesterol was determined using commercially available enzymatic kit (Cholesterol Liquicolor, Human Gesellschaft ftir Biochemica und Diagnostica mbH, Germany). Statistical analysis Data were expressed as the meansfSEM and the statistical significance was first analyzed with one-way ANOVA and the difference between groups were analzyed with post hoc test Tukey HSD. Difference of the catechins contents between different concentrations of green tea were analyzed with the Student’s unpaired two-tailed t-test. Results Body weight, food andfluid consumption during experimental period Table I shows the food and fluid consumption during the experimental period. There was no difference in food consumption among all rats in various treatment groups and fluid intake in the 1 % Lung Chen Tea group was similar to the tap water controls. Rats however consumed less 2% and 4% Lung Chen Tea @<O.OOl). No significant difference in the percentage increase in body weight was observed among all groups of rats (Table I). Quantities of catechins in the Lung Chen Tea solutions Figure 1 shows the concentration of catechins in l%, 2% and 4% Lung Chen Tea solutions. Four percent Lung Chen Tea contained the highest amount of all four major catechins @<O.OOl), while the catechin contents in 1% Lung Chen Tea were the lowest (p<O.OOl). Eflect on serum lipid levels There was no significant change in serum triglyceride level in all rats (Table II). Cholesterolenriched diet significantly increased the total cholesterol level (p<O.OOl). Rats treated with 2% and 4% Lung Chen Tea had lower serum total cholesterol than the 1% Lung Chen Tea group (~~0.05 and ~~0.02 respectively) and the Disease Model (~~0.05, and ~~0.02 respectively). Serum HDL-cholesterol level was increased significantly only in the 4% Lung Chen Tea treated group (pcO.05). 416 Tea Lowers Cholesterol by Lipid Excretion Vol. 66, No. 5, 2000 TABLE I Food, Fluid Consumptton Experimental Period Treatment Group n and Increase m Body 9 Cholesterol-enriched dret 10 Lung Chen Tea 1% 6 Lung Chen Tea 2% 9 Lung Chen Tea 4% 7 Disease Model during the lo-week Increase in Body Fluid Consumption Food Consumption (g/rat/day) Standard diet Normal Control Weight Weight (%) (g/rat/day) 27.9kO.7 35.9+0.7 233.7f10.4 30.4kO.7 32.751.6 30.3-fo.7 30.6k1.2 36.9kO.7 38.0&1.4 30.lf0.7* + 31.7*0.9* - 241.9k6.3 234.3k15.7 229.5+8.7 230.559.6 Values are expressed as means+SEM; * p<O.OOl when compared with the Disease Model; + p<O.OOl when compared with the 1% Lung Chen Tea group; n = no. of rats in each group. * 0.5 0.2 0 I I% 2% Lung Chen F1g. 4% Tea I Quantities of the four major tea catechins namely (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG), were determined in I%, 2% and 4% Lung Chen Tea solutions. Values are expressed as means+SEM of 6 determinants; * p<O.OOl when compared with the 1% and 2% Lung Chen Tea solutions; + p<O.OOl when compared with the 1% Lung Chen Tea solution. Effect on liver weight and liver lipid content Rats fed with cholesterol-enriched diet had a significant increase in relative liver weight Vol. 66, No. 5, 2000 Tea Lowers Cholesterol by Lipid Excretion 417 (p<O.OOl, Table III). Two percent and 4% Lung Chen Tea effectively reduced the elevation when compared with the 1% Lung Chen Tea group and the Disease Model groups (Table III). The liver cholesterol and triglyceride contents were increased by cholesterol feeding @<O.OOl, Table III). Two percent and 4% Lung Chen Tea suppressed the accumulation of liver cholesterol but the liver triglyceride level was not affected. TABLE II Serum Total Cholesterol, with Cholesterol-enriched HDL-Cholesterol and Triglyceride Levels after Treatment Diet and Lung Chen Tea for Eight Weeks Treatment Group n Serum Total Cholesterol Serum HDL (mg/dll Standard diet Normal Control 9 44.6k4.9 33.97U.9 60.5227.9 117.3+7.7** 121.5k8.7 97.8+5.2++ 90.3f2.9” ++ 21.74*2.7* 26.05t5.9 26.63f1.6 34.58+2.4+# 63.48f5.0 57.69f4.0 60.80f6.4 53.33k7.2 Cholesterol-enriched diet Disease Model IO Lung Chen Tea 1% 6 Lung Chen Tea 2% 9 Lung Chen Tea 4% 7 Serum Triglyceride (mg/dU Values are expressed as meansfSEM; * pcO.05, ** p<O.OOl when compared with the Normal Control; + p<O.OS, ++ ~~0.02 when compared with the Disease Model; ‘p<O.O5, “p<O.O2 when compared with the 1% Lung Chen Tea group, # pcO.05 when compared with the 2% Lung Chen Tea group; n = no. of rats in each group. EApeCton hepatic lipid metabolizing enzymes Figure 2 shows the activities of HMG-CoA reductase after 8 weeks treatment with Lung Chen Tea. Cholesterol-enriched diet significantly lowered the activity of HMG-CoA reductase when compared with the Normal Control @<O.OOl). There was no significantly difference in HMGCoA reductase activities in rats treated with Lung Chen Tea. A decreasing trend in FAS activity was observed in hypercholesterolemic rats but there was no statistical difference in enzyme activity when compared with the Normal Control (Figure 3). The activity of cholesterol 7a-hydroxylase is shown in Figure 4. Cholesterol 7ct-hydroxylase was not affected by the increase in cholesterol intake. However, there was a increasing trend of cholesterol 7a-hydroxylase activity in rats consuming 4% Lung Chen Tea (Figure 4). E#ect on fecal lipid excretion Fecal bile acids were significantly elevated in hypercholesterolemic rats (p<O.OOl, Figure 5). Four percent Lung Chen Tea significantly increased bile acids excretion when compared with the Disease Model (p~O.05, Figure 5). Cholesterol feeding significantly increased fecal cholesterol excretion when compared with rats Tea Lowers Cholesterolby Lipid Excretion 418 Vol. 66, No. 5, 2000 receiving standard diet (p<O.OO1, Figure 6). Four percent Lung Chen Tea increased excretion of cholesterol when compared with the Disease Model (~~0.05, Figure 6). FABLE 111 Relative Liver Weight, Liver Cholesterol with Lung Chen Tea for Eight Weeks Treatment Group n and Trlglycerlde Content after Treatment Liver Cholesterol (mg/g liver) Relative Liver Weight (g/lOOg body weight) Liver Triglyceride (mgig liver) Standard diet Normal Control 9 3.lkO.l 3.3kO.5 8.SkO.8 6.0f0.2* 6.3+_0.I 5.6kO.l’ +’ 5.4kO.2’ ++ 25.812.1* 25.0+1.7 18.5+1.7+‘+ 17.3fl.2” ++ 20.2?0.7* 20.5+3.3 21.1f0.8 18.8k1.3 Cholesterol-enriched dret Disease Model Lung Chen Tea 1% Lung Chen Tea 2% Lung Chen Tea 4% 10 6 9 7 Values are expressed as means+SEM; * p<O.OOl when compared with the Normal Control; * pco.05, ++ pcO.02 when compared with the Disease Model; ’ pcO.05, ‘+p<0.02 when compared with the 1% Lung Chen Tea group; n = no. of rats in each group. 7 FKIII Fed with standard diet K?ZZB Fed with cholesterol-enriched diet L Normal Control Disease I y. 2% Lung Chen Model 4% Tea Fig. 2 Effect of Lung Chen Tea on hepatic HMG-CoA reductase activity (nmol/min/mg protein). Values are expressed as means+SEM; T p~O.001 when compared with the Normal Control. Tea Lowers Choleskrol by Lipid Excretion Vol. 66, No. 5,200O 419 0 Fed with standard EZZ Fed with cholesterol-enriched diet diet ” Normal Control Disease Model 1% Lung 4% 2% Chen Tea Fig. 3 Effect of Lung Chen Tea on hepatic fatty acid synthase NADPWmin/mg protein). Values are expressed as meansGEM. significant difference between all groups. activity (nmol There was no x .‘1: > .: sm 80 2, rn, A.j 70 ; i; r0 5‘ z 4J z _c 0 I Fed @ZZZ?A Fed with with standard diet cholesterol-enriched diet 60 50 .r E 0 E 2 40 30 20 2 2 10 t z 0 Normal Control Disease M ode1 1% 4% 2% Lung Chen Tea Fig. 4 Effect of Lung Chen Tea on hepatic cholesterol protein). Values are expressed as means*SEM. 7a-hydroxylase activity (pmol/min/mg 420 Tea Lowers Cholesterol by Lipid Excretion CIIIIEX EZZZ Fed Fed with with Normal Control Vol. 66, No. 5, 2000 standard diet cholesterol-enriched diet 1y. Disease M ode1 4% 2% Lung Chen Tea Effect of Lung Chen Tea on fecal bile acids excretion. Feces were collected on the last two days of experimental period. Values are expressed as means?SEM; ’ p<O.OOl when compared with the Normal Control; * ~~0.05 when compared with the Disease Model. I BZZI Fed Fed with with standard diet cholesterol-enriched diet * T + z P) =; b) 0 _sS 0 m h: Lr, 12 IO 8 6 4 2 Normal Control Disease model I% Lung 2% Chen 4% Tea Fig. 6 Effect of Lung Chen Tea on fecal cholesterol excretion. Feces were collected on the last two days of experimental period. Values are expressed as means&EM; ’ p<O.OOl when compared with the Normal Control; * pcO.05 when compared with the Disease Model. Vol. 66, No. 5, 2000 Tea Lowers Cholesterolby Lipid Excretion 421 Discussion Green tea and tea catechins have been shown to be hypolipidemic (1,2), however, the effect of green tea on the major lipid metabolizing enzymes has not been investigated. In this study, hypercholesterolemic rats were treated with Lung Chen Tea and its effects on HMG-CoA reductase, cholesterol 7ol-hydroxylase, fatty acid synthase (FAS), bile acids and cholesterol excretion were examined. Due to the bitter taste of Lung Chen Tea, rats drank significantly less 2% and 4% Lung Chen Tea. However, it did not affect the apparent growth of the rats, since the percentage increase in body weight was similar over the experimental period (Table I). Analysis of the tea catechins content showed that the amount of catechins increased with the concentration of Lung Chen Tea (Figure 1). Epigallocatechin gallate (EGCG) which accounted for almost 70% (w/w) of the total catechins (Figure 1) was most abundant in Lung Chen Tea. Even though the consumption of 2% and 4% Lung Chen Tea was lower than that of I%, the intake of catechins was still higher than that of 1% Lung Chen Tea. Lung Chen Tea dose dependently lowered serum total cholesterol level (Table II). However, 1% Lung Chen Tea that contained lower content of catechins did not prevent the elevation (Table II). Lung Chen Tea (4%) also reduced the HDL lowering effect of excessive cholesterol intake @<0.05, Table II). Since HDL facilitates translocation of cholesterol from peripheral tissue; like arterial walls to liver for catabolism, thus a relative increase in HDL may slow down the atherogenic process (14). Cholesterol-enriched diet has no effect on serum triglyceride level and rat consuming Lung Chen Tea has similar serum triglyceride level when compared with control animal. As plasma triglyceride level is determined by hcpatic triglyceride synthesis, release from the liver, and the activity of lipoprotein lipase, it thus showed that Lung Chen Tea has little effect on these processes (15). The relative liver weight in cholesterol fed rats was significantly increased due to excessive accumulation of cholesterol and triglyceride (Table III). As described by Fungwe et al. (16), cholesterol has an stimulatory effect on hepatic fatty acid biosynthesis and the incorporation of newly synthesized fatty acid into hepatic triglyceride. Thus, liver triglyceride content was significantly increased in hypercholesterolemic rats. Lung Chen Tea lowered the liver cholesterol content and thus reduced the relative liver weight (Table III), however, it has no effect on liver triglyceride level. It has been reported that tea catechins inhibited intestinal absorption of cholesterol (1) and this may be one of the mechanisms by which Lung Chen Tea lowered plasma cholesterol levels. A reduction in intestinal cholesterol absorption prevents the accumulation of cholesterol in the liver. Since the expression of LDL receptor is controlled by feedback inhibition of intracellular cholesterol, reduction in hepatic cholesterol accumulation in turn stimulates the production of more high affinity LDL receptors (16). This results in an increase in clearance of cholesterol from the circulation by LDL receptor and thus lowers blood cholesterol (17). In the present study, the three major lipid metabolizing enzymes in the liver were investigated. HMG-CoA reductase activities in cholesterol-enriched diet groups were lower than that of the standard diet group (Figure 2). HMG-CoA reductase is the rate-determining enzyme for cholesterol synthesis and its activity is regulated by the feedback inhibition of cholesterol and sterols (18). Increase in cholesterol intake resulted in an accumulation of cholesterol and triglyceride in the liver (Table III) and thus suppressed the transcription of HMG-CoA reductase gene, In this study no effect of Lung Chen Tea on HMG-CoA reductase activity was observed 422 Tea Lowers Cholesterolby Lipid Excretion Vol. 66, No. 5, 2000 (Figure 2), therefore the hypocholesterolemic effect of Lung Chen Tea may not be explained by the suppression of cholesterol synthesis. This result supported the findings of Chisaka et al. (19) that (-)-epigallocatechin gallate (EGCG) isolated from Japanese Green Tea, did not affect the in vitro incorporation of j4C-acetate into cholesterol in liver slices obtained from normal or hypercholesterolemic rats. Hepatic FAS is the key enzyme in fatty acid synthesis and its level is regulated by hormones and nutrition intake of lipids and carbohydrates (20). Fatty acids synthesized by FAS will be incorporated with glycerol to form triglyceride. From the present experiment, an increase in cholesterol intake has led to a slight but insignificant decrease in FAS activity (Figure 3). Rats consuming Lung Chen Tea has no inhibitory effect on the activity FAS. These are in line with the absence of effect of Lung Chen Tea on serum and liver triglyceride levels (Table II). Conversion of cholesterol to bile acids is the major pathway of cholesterol elimination and it accounts for about 50% of daily cholesterol excretion (21). Cholesterol 7a_hydroxylase, the rate determining enzyme in the conversion of cholesterol to bile acids, is mainly regulated by the feedback inhibition of bile acids re-absorbed from the intestine (22). It was found that cholesterol-enriched diet did not affect the activity of cholesterol ‘la-hydroxylase. Rats consuming 4% Lung Chen Tea showed a rise in cholesterol 7a-hydroxylase activity but not reached significant level (Figure 4). Fecal excretion of bile acids and cholesterol were found to be increased in cholesterol-fed rats, while 4% Lung Chen Tea further enhanced their excretion (Figures 5 and 6). The increase in the excretion of bile acids and cholesterol seems to activate cholesterol 7a-hydroxylase. The increase in cholesterol 7a-hydroxylase can enhance the conversion of liver cholesterol to bile acids for excretion. This leads to a decrease in hepatic cholesterol content which in turn stimulated LDL receptor expression and lowered blood cholesterol level (23). Two percent Lung Chen Tea reduced serum and liver cholesterol levels without significantly increased the fecal cholesterol and bile acids excretion, thus implying that other mechanisms may contribute to the hypocholesterolemic effect of Lung Chen Tea. The present study demonstrated that Lung Chen Tea has hypocholesterolemic effect. It lowered serum total cholesterol and elevated HDL-cholesterol level. It has no inhibitory effect on the de novo synthesis of cholesterol and lipogenesis. One of its mechanisms of hypocholesterolemic action may be due to the promotion of cholesterol and bile acids excretion. Acknowledgments The authors would like to thank Mr. H.C. Leung for his excellent technical assistance. The present study was partly supported by the Hsin Chong K.N. Godfrey Yeh Education Fund of the University of Hong Kong. Vol. 66, No. $2000 Tea LowersCholesterolby Lipid Excretion 423 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. I. 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