¹2nd Department of Medical Biochemistry, School of Medicine, Ehime University, Ehime, Japan

²Central Research Laboratory, Ehime University, Ehime, Japan

³Koshiro Pharmaceutical Co. Ltd, Osaka, Japan

⁴The Pharmaceutical Institute, Dalian University, Dalian-shi Liaoning, People's Republic of China

Correspondence to: Y Kimura, 2nd Department of Medical Biochemistry, School of Medicine, Ehime University, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan

OBJECTIVE: Based on the inhibitory effects of teasaponin on pancreatic lipase activity in vitro, this study was performed to clarify whether teasaponin prevented obesity induced in mice by a high-fat diet for 11 weeks.

DESIGN: For in vitro experiments, assay for the inhibitory effects of teasaponin on pancreatic lipase activity was performed by measuring the rate of release of oleic acid from triolein in an assay system using triolein emulsified with lecithin, gum arabic, Triton X-100 or 4-methylumbelliferyloleate. For in vivo experiments, female ICR mice were fed a high-fat diet with or without 0.5% teasaponin for 11 weeks.

RESULTS: Teasaponin competitively inhibited the hydrolysis of triolein emulsified with lecithin, gum arabic, Triton X-100 or 4-methylumbelliferyloleate. Teasaponin inhibited the elevations of plasma triacylglycerol levels 3, 4 and 5 h after oral administration of lipid emulsion containing corn oil. Teasaponin suppressed the increases in body, parametrial adipose tissue weights and diameter in adipose cell size induced by a high-fat diet. Furthermore, feeding a high-fat diet plus teasaponin had no effect on stool frequency and content, but significantly increased triacylglycerol contents in feces as compared to feeding a high-fat diet.

CONCLUSIONS: The anti-obesity effects of teasaponin in high-fat diet-treated mice may be partly mediated through delaying the intestinal absorption of dietary fat by inhibiting pancreatic lipase activity.

International Journal of Obesity (2001) 25, 1459-1464

teasaponin; pancreatic lipase; high-fat diet

Introduction

Three kinds of tea, oolong, green and black, have been widely used as healthy drinks from ancient times all over the world, especially to prevent obesity and lipid metabolism. Among the three teas, oolong tea is traditionally reported to have anti-obesity and hypolipidemic actions. Kimura et al¹ reported that the three kinds of tea prevented the elevation of serum and hepatic total cholesterol and triacylglycerol, and liver injury and increase in serum transaminases in rats fed peroxidized oil for one week. Furthermore, we found that tea tannins, such as epicatechin gallate and epigallocatechin gallate, strongly inhibited lipid peroxidation in rat liver mitochondria and microsomes.² In the previous paper, we found that oolong tea prevented obesity induced by feeding a high-fat diet through inhibiting pancreatic lipase activity and accelerating catecholamine-induced fat mobilization.³ We reported that epicatechin gallate and epigallocatechin gallate had no effect on pancreatic lipase activity, but that saponin fraction inhibited pancreatic lipase activity.³ Furthermore, we found that caffein was isolated from oolong tea as an accelerating substance of norepinephrine-induced lipolysis.³ Therefore, we suggest that saponins of tea may have anti-obesity action through inhibiting pancreatic lipase activity in high-fat diet-treated obesity mice. In this experiment, we found that teasaponin (a mixture of theasaponins E₁ and E₂) isolated from oolong tea inhibited pancreatic lipase in vitro. Furthermore, an experiment was designed to clarify whether or not teasaponin prevented the obesity induced by long-term feeding of a high-fat diet containing 40% beef tallow.

Material and methods

Materials

Triolein, 4-methylumbelliferyloleate and pancreatic lipase were purchased from Sigma Chemical Co. (St Louis, MO). Gum arabic, the Triglyceride E- and total Cholesterol E-test kits were purchased from Wako Pure Chemical Co. (Osaka, Japan). Teasaponin are the mixtures of theasaponin E₁ and E₂. Laboratory pellet chow was purchased from CLEA Japan (Osaka, Japan). Beef tallow, casein, vitamin and mineral mixtures were purchased from Oriental Yeast Co. Ltd (Tokyo, Japan). Other chemicals were of reagent grade.

Isolation of teasaponin from the leaves of Thea sinensis

The leaves of Thea sinensis (5 kg) were extracted with methanol (3 l) under reflux three times. After removal of the solvent from the methanol solution under reduced pressures, the extract was subjected to ODS column chromatography (Chromatorex ODS DM 1020 T, Fuji Silysia Chemical Ltd, Japan) eluted with a mixture of water and methanol, to give the methanol eluted fraction (55 g). The methanol-eluted fraction was separated by silica gel column chromatography (Merck Co, Germany) eluted with a mixture of chloroform and methanol to furnish the saponins (31 g). The saponins were identified as a mixture of theasaponins E₁ and E₂ by the analysis of HPLC and the comparison of ¹³C-NMR data of theasaponins E₁ and E₂.⁴ The contents of theasaponins E₁ and E₂ in teasaponin were 88 and 12%, respectively, by the analysis of HPLC (YMC-pack, ODS-5, 20´250 mm i.d.) eluted with a 7:3 mixture of methanol and 1% acetic acid (v/v) as mobile phase.

Animals

Female ICR strain mice (3 weeks old) and male Wistar King strain rats (6 weeks old) were obtained from CLEA Japan (Osaka, Japan) and Charles River Japan (Yokohama, Japan), respectively, and housed for 1 week under a 12 h/12 h light/dark cycle in a temperature- and humidity-controlled room. The animals were given free access to food and water. After adaptation to the lighting conditions for 1 week, the healthy animals were used in these experiments. The experimental protocol was approved by the Animal Studies Committee of Ehime University.

Measurement of pancreatic lipase activity in vitro

Lipase activity was determined by measuring the rate of release of oleic acid from triolein. Briefly, a suspension of triolein (80 mg), phosphatidylcholine (10 mg) and taurocholic acid (5 mg) in 9 ml 0.1 M N-Tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) buffer (pH 7.0) containing 0.1 M NaCl was sonicated for 5 min. This sonicated substrate suspension (0.1 ml) was incubated with 0.05 ml (final concentration 5 units per tube) pancreatic lipase and 0.1 ml of various concentrations of sample solutions for 30 min at 37°C in a final volume of 0.25 ml. In addition, pancreatic lipase activity was determined using gum arabic or Triton X-100 as emulsifier; 45 mg gum arabic or 2.25 mg triton X-100, instead of lecithin, were used and the enzyme activity assayed as described above. Furthermore, we examined using 4-methylumbelliferyoleate as a synthetic substrate. Lipase activity was expressed as moles of oleic acid released per ml reaction mixture per min.

Measurement of plasma triacylglycerol levels after oral administration of lipid emulsion to rats

Lipid emulsions were prepared with 6 ml of corn oil, 80 mg of cholic acid, 2 g of chloestryloleate and 6 ml of saline in the absence or presence of teasaponin (final concentration 481 mg kg^-1 body weight). After male Wistar King strain rats had fasted overnight, they were orally administered 3 ml of lipid emulsion. Blood samples were taken from the tail vein at 0, 0.5, 1, 2, 3, 4 and 5 h after administration of the lipid emulsion with or without teasaponin using a heparinized-capillary tube, and centrifuged at 5500 g for 5 min in a Model KH-120M (Kubota Co., Japan) centrifuge to obtain the plasma. The plasma triacylglycerol was determined using a Triglyceride E-test kit.

Estimation of body and parametrial adipose tissue weights, plasma triacylglycerol and total cholesterol in mice fed a high-fat diet for 11 weeks

Female ICR mice (3 weeks old) were divided into three groups, with each group matched for body weight, after 1 week of being fed laboratory pellet chow ad libitum. The control group (n=10) continued to be fed laboratory pellet chow ad libitum. The high-fat diet group (n=14) was given the high-fat diet containing 40% beef tallow, 36% casein, 10% corn starch, 9% sugar, 4% mineral mixture and 1% vitamin mixture. The high-fat diet plus teasaponin group (n=12) was given the high-fat diet containing 40% beef tallow, 35.5% casein, 10% corn starch, 9% sugar, 4% mineral mixture and 1% vitamin mixture with teasaponin mixed in at a concentration of 0.5%. Body weight was measured weekly. The total amount of food intake by each mouse was recorded at least three times a week. To avoid auto-oxidation of the fat components, the feed was stored at-30°C and freshly prepared each day. After 11 weeks of consuming the indicated experimental diet, the blood of each mouse was taken by venous puncture under anesthesia with diethyl ether, and then the mice were killed with an overdose of diethyl ether. Experiments were performed in a ventilated room. The plasma was separated and frozen at-80°C until analysis. The parametrial adipose tissues were quickly removed and weighed. Celluarity of adipose tissues was determined by the methods of Hirsh and Gallian.⁵ Briefly, adipose tissue specimens, each 100 mg in weight, were immediately placed into plastic tubes containing 1.5 ml of 2% osmium tetraoxide in 0.05 M collidine-HCl buffer at pH 7.4, and tissues were fixed at 37°C for 72 h. After fixation, the contents of the plastic container were thoroughly washed through a nylon screen (250 µm) with distilled water. The filtrate contained most of the fixed free cells, but fibrous tissue and some intact shreds were gently rubbed by hand on the filter while washing. This procedure completely separated the tissue into free cells, and produced total recovery of cells in the filtrate. These cells were then collected and washed with distilled water using a finer screen (250 µm). The diameter of adipose cell suspensions was determined with osmium tetroxide-fixed cells by scanning electron micrography using a Hitachi H-500 scanning electron microscope (Hitachi Ltd, Hitachinaka, Japan).

Statistical analysis

Data are expressed as means±s.e. Statistical analyses were performed by the Fisher's protected LSD test to determine significance (P<0.05) using super ANOVA software (Abacus Concepts, Berkeley, CA).

Results

Effects of teasaponin fractions prepared from three teas and isolated teasaponin on pancreatic lipase activity in vitro

As shown in Table 1, saponin fraction prepared from oolong, green and black teas inhibited the lipase activity using triolein emulsified with lecithin. Among the three teas, the saponin fractions of oolong tea showed the greatest inhibition. The discrepancy of inhibition of three teas on pancreatic lipase activity may be due to the difference of saponin contents in three teas. As shown in Figure 1a, isolated teasaponin inhibited the pancreatic lipase activity dose-dependently in the following assay systems, using triolein emulsified with lecithin, gum arabic and Triton X-100. Furthermore, isolated teasaponin inhibited the pancreatic lipase activity using 4-methylumbelliferyloleate as a substrate.

We performed the analysis by Lineweaver-Burk plot for characterization of the mechanism of teasaponin on pancreatic lipase activity. Teasaponin was a competitive inhibitor, and the K_m and V_max values of the lipase activity for lecithin-emulsified triolein were 1.42 mg ml^-1 and 476.2 nkat l^-1, respectively. The K_i value of teasaponin was 0.25 mg ml^-1.

Effects of teasaponin on the plasma triacylglycerol levels after oral administration of lipid emulsion to rats

Figure 2 shows the serial changes in plasma triacylglycerol concentration when lipid emulsion with or without teasaponin was administered orally to rats. At 3, 4 and 5 h after administration of teasaponin, the plasma triacylglycerol concentrations were significantly lower than those in the control group.

Effects of teasaponin on food consumption, triacylglycerol contents in feces, body and parametrial adipose tissue weights in mice fed a high-fat diet for 11 weeks

The mean food consumption per week per mouse was significantly different between the control group and high-fat diet groups, being 430±12 kJ in the control group and 487±17 kJ in the high-fat diet group, but not significantly different between the high-fat and high-fat plus 0.5% teasaponin diet groups, being 487±17 kJ (high-fat diet) and 582±59 kJ (high-fat plus 0.5% teasaponin). Feeding a high-fat diet plus teasaponin had no effect on stool frequency and content, but significantly increased triacylglycerol contents in feces as compared to feeding a high-fat diet (triacylglycerol contents in feces: control group, 4.3±0.2; high-fat diet group, 37.2±6.7; high-fat plus 0.5% teasaponin group, 81.9±26.4 mmol/g). Figure 3 shows the changes in body weights of the groups during the experiments. Feeding a high-fat diet for 11 weeks caused significant increases in body weights at 5 to 11 weeks compared to the control group (laboratory pellet chow). Feeding a high-fat diet with 0.5% teasaponin significantly suppressed the increase in body weight compared to the high-fat diet during the treatment period. The final parametrial adipose tissue weights of the groups are shown in Table 2. The final parametrial adipose tissue weight was significantly increased by feeding a high-fat diet compared to the control group. That in animals with a high-fat diet containing 0.5% teasaponin was significantly reduced as compared to the high-fat diet group.

In addition, oral administration of teasaponin did not caused hemolysis of erythrocyte in mice fed a high-fat diet and lipid emulsion-treated rat (data not shown).

Effects of tea saponin on the diameter of fat cells in mice fed a high-fat diet for 11 weeks

As shown in Table 2, the diameter of fat cells was significantly greater in the high-fat diet group than in the control group, and teasaponin completely prevented the high-fat diet-induced increase in cell diameter.

Discussion

In recent years, some studies have reported the anti-allergic,⁶ antihypertensive,⁷ antimicrobial and anti-inflammatory⁸ effects of teasaponin, but yet the inhibitory effects of teasaponin on pancreatic lipase activity have been not reported. Pancreatic lipase is the most important enzyme for the digestion of dietary triacylglycerols. It is well known that dietary fat is not directly absorbed from the intestine unless it has been subjected to the action of pancreatic lipase.⁹ The application of pancreatic lipase inhibitor was examined earlier as a treatment for diet-induced obesity in humans. It has been clinically reported that a pancreatic lipase inhibitor orlistat (Ro 18-0647) prevented obesity and hyperlipidemia through the increment of fat excretion into feces and the inhibition of pancreatic lipase.^{10,11,12,13,14} In preliminary experiments, we found that saponin fractions prepared from oolong, green and black teas inhibited pancreatic lipase, and the inhibition rations were 100, 75 and 55% at a concentration of 2 mg ml^-1, respectively. The yields of teasaponin of oolong, green and black tea are 0.06, 0.065 and 0.029%, respectively. The difference in teasaponin content among the three teas may result from the hydrolysis of saponin during the processing from fresh leaf to dried leaf. Therefore, the discrepancy of the inhibition of lipase activity by saponin fractions obtained from three teas may be due to the difference of teasaponin contents. Next, we isolated teasaponin from oolong tea, and the teasaponin was found to be a mixture of theasaponins E₁ and E₂. Teasaponin inhibited the pancreatic lipase activity in the following assay systems, using triolein emulsified with lecithin, gum arabic or Triton X-100 as a substrates, or 4-methylumbelliferyloleate as a synthetic substrate.

It seems possible that teasaponin may prevent high-fat diet-induced obesity through inhibiting of pancreatic lipase activity. To test this possibility, we first examined the effects of teasaponin on plasma triacylglycerol concentrations after oral administration of a lipid emulsion containing corn oil in rats, and found that teasaponin significantly reduced the elevation in plasma triacylglycerol levels after oral administration of a lipid emulsion. Based on these findings, we designed in vivo experiments to examine the effects of teasaponin on obesity in mice induced by feeding a high-fat diet. Previously we reported that the variation of casein content in the high-fat diet from 22 to 36% did not affect both body weight or parametrial adipose tissue weight. That is, 22-36% casein content of the high-fat diet caused similar degrees of obesity.¹⁵ In this study, we found that the administration of teasaponin significantly suppressed the increase in body weight in mice fed a high-fat diet containing 40% beef tallow for 11 weeks. The treatment with teasaponin also significantly reduced the final parametrial adipose tissue weight as compared to that of high-fat diet group. These inhibitions did not depend on decreased food or energy intake because there was no significant difference between the high-fat diet and the high-fat diet containing 0.5% teasaponin groups. It was reported that the weights of the epididymal and retroperitoneal adipose tissue deposits were significantly higher in the high-fat fed rats than in the low-fat fed rats.¹⁶ Furthermore, there are reports that both adipocyte size and number are increased in animals with obesity caused by a high-fat diet.^17,18 The measurement of the diameter of adipose cells after 11 weeks of feeding with a high-fat diet containing 0.5% teasaponin showed that tea saponin completely suppressed increases in the diameter of adipose cells compared to that of high-fat diet group. This finding shows that the teasaponin suppressed fat accumulation in the parametrial adipose cell induced by a high-fat diet.

It has been reported that the plant saponins cause hemolysis.¹⁹ Among these saponins, various saikosaponins isolated from Bupleum falcatum L. caused hemolysis.^20,21 On the other hand, ginseng saponin isolated from Panax ginseng C. A. Meyer was reported to inhibit the hyperosmotic hemolysis of erythrocyte.²² Furthermore, Nguyen et al reported that the oral administration of total saponins from Panax vietnamensis protected carbon tetrachloride-induced hepatotoxicity in mice.²³ In this study, we found that the oral administration of teasaponin did not cause hemolysis of erythrocytes in vivo.

In conclusion, teasaponin may prevent the high-fat diet-induced increases in both body and parametrial adipose tissue weights by inhibiting intestinal absorption of dietary fat via inhibition of pancreatic lipase activity.

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Figure 1 Effects of teasaponin on pancreatic lipase activity. (a) Results are expressed as means±s.e. of four experiments. (open circle) Triolein emulsified with lecithin; (solid circle) triolein emulsified with gum arabic; (solid square) Triolein emulsified with triton X-100; (solid triangle) 4-methylumbelliferyloleate. (b) Lineweaver-Burk plots of the released oleic acid with lecithin-emulsified triolein as substrate in the presence of various concentrations of teasaponin. Tea saponin concentrations used: (open circle) 0 mg/ml; (solid circle) 0.5 mg/ml.

Figure 2 Effects of teasaponin on rat plasma triacyglycerol levels after oral administration of a lipid emulsion. The procedure is described in Materials and methods. (circle) lipid emulsion; (square) lipid emulsion plus teasaponin. Each point represents the means±s.e. of four rats. *P<0.05, significantly different from lipid emulsion only treated group.

Figure 3 Effects of teasaponin on body weight in mice fed a high-fat diet for 11 weeks. (open circle) Control; (solid circle) high-fat diet; (solid square) high-fat diet+0.5% teasaponin. Results are expressed as means±s.e. of 10-14 mice. *P<0.05, significantly different from high-fat diet group.

Table 1 Effects of saponin fraction isolated from oolong, green and black tea on pancreatic lipase activity

Table 2 Effects of teasaponin on parametrial adipose weight and the diameter of adipose cells of mice fed a high-fat diet for 11 weeks