Anti-obesity effects of lipase inhibitor CT-II, an extract from edible herbs, Nomame Herba, on rats fed a high-fat diet

Abstract

OBJECTIVE: To investigate the inhibitory effects of CT-II, extract of Nomame Herba, on lipase activity in vitro and on obesity in rats fed a high-fat diet in vivo.

DESIGN: The assay for the inhibitory effect of CT-II on lipase activity was performed by measuring released free fatty acids after the incubation of the medium with CT-II, porcine pancreatic lipase and triolein (experiment 1). In vivo experiments, lean rats or obese rats (570–718 g) were fed a high-fat diet containing 60% fat with or without CT-II for 8 weeks (experiment 2), for 14 days (experiment 3) or for 12 weeks (experiment 4), respectively.

MEASUREMENT: The time course of body weight, food intake, organ weight (parametrial fat, liver, heart and kidney) and plasma parameters (triglyceride, total cholesterol, glucose, AST, ALT and insulin), fecal output of total fat and total cholesterol were measured. Hepatic histological examinations were also performed.

RESULTS: CT-II inhibited the porcine lipase activity dose-dependently in vitro (experiment 1). Body and liver weight were reduced and hepatic histological examination showed an amelioration of fatty liver in CT II treated animals (experiment 2). CT-II significantly inhibited body weight gain and plasma triglyceride elevation in a dose-dependent manner, without affecting food intake in lean rats fed the high-fat diet. Elevated plasma AST and ALT were also decreased (experiment 3). When obese rats fed the high-fat diet were treated with CT-II for up to 6 months, body weight was initially reduced and thereafter weight gain was significantly suppressed. Total body fat was also significantly reduced and significant reduction of plasma AST and ALT was observed (experiment 4).

CONCLUSIONS: These results demonstrated that the lipase inhibitor CT-II is effective in preventing and ameliorating obesity, fatty liver and hypertriglyceridemia in rats fed a high-fat diet.

Introduction

Pancreatic lipase is the most important enzyme for the digestion of dietary triglycerides.1 The application of a lipase inhibitor was examined earlier as a treatment for obesity. Orlistat (Ro 18-0647), a hydrogenated derivative of lipstatin derived from Streptomyces toxitricini, is a potent inhibitor of gastric, pancreatic and carboxylester lipase,2 and has proved to be effective for the treatment of human obesity.3,4,5 The existence of lipase inhibitors in various foodstuffs has been researched, and the presence of these inhibitors in cereals, in wheat bran and wheat germ6 and in soybean7,8 has been reported. Lipase inhibitors have also been sought in natural products such as Chinese medicines and various herbs.9

A leguminous plant, Cassia mimosoides L. var. nomame Makino (Nomame Herba), is grown in Japan and China, and traditionally consumed as a tea in Japan. In the course of screening lipase inhibitors among natural products, an extract from Nomame Herba showed a remarkable inhibitory effect on lipase. In the present study, the inhibitory effect of this extract, CT-II, on lipase activity in vitro and the anti-obesity effects in rats fed a high-fat diet in vivo were examined.

Methods

Preparation of CT-II

CT-II was a fraction of aqueous ethanol extract from Nomame Herba, containing primarily proanthocyanidin (condensed tannin). Preparation of the fraction was as follows: 800 g of the aerial part of dried plant was extracted with 10 l of 50% ethanol (4 l, 3 l, 3 l, three extractions). The extract was subjected to a Diaion HP 20 column (Mitsubishi Chemical Corporation), washed with 30% ethanol and eluted with 80% ethanol. The eluted solution was evaporated and lyophilized to dryness, then, 24 g of CT-II powder was acquired. CT-II powder was utilized as the mixture with experimental diet in vivo.

In vitro inhibitory effect of CT-II on lipase activity

The assay for lipase activity using triglyceride as the substrate was performed according to the method of Fukumoto et al.10 Briefly, the inhibitory effect of (pH 7.4) and 1 ml of 50% tetrahydrofuran (THF) solution of CT-II were mixed in an L-type test tube (φ18×120 mm), and 1 ml of the solution of porcine pancreatic lipase (Sigma type-II, 0.71 mg/ml as protein in McIlvaine buffer) was then added and incubated at 37°C for 15 min with shaking. The reaction was stopped by adding ethanol, and the released fatty acids were titrated by 0.05 M NaOH. The blank was measured using 50% THF without CT-II (experiment 1).

In vivo effects of CT-II on body weight, body fat and metabolites

Animals and diet.

Female Sprague–Dawley rats were used in the study. They were housed in individual stainless steel cages under a controlled temperature (24±2°C) and a 12 h light–dark cycle with free access to water and a high-fat powdered diet containing 60% fat, 7.5% carbohydrate, 24.5% protein (6.7 kcal/g) as in the previous study.11 Normal diet mainly containing defatted soybean meal and fish meal is referred to as extruded diet (CE-2, Clea Japan Inc.).

Procedures.

CT-II initially showed the inhibitory activity for lipase in the in vitro study. Since excessive intake of dietary fat contributes to the development of obesity, the preventive effects of the extract on the development of obesity in rats fed a high-fat diet were examined. After adaptation to the high-fat diet and laboratory conditions for 1 week, lean rats weighing 239–251 g were divided into two groups. The control group was given only the high-fat diet; the other group was given the high-fat diet with CT-II mixed in at a concentration of 2.5%. Body weight was measured every week. After 8 weeks of CT-II treatment, animals were killed, organ weights measured and the organs were histologically examined (experiment 2).

The effects of CT-II dose on body weight and plasma metabolites in rats were then examined. Lean rats weighing 265–298 g were divided into six groups. The control group was given only the high-fat diet as in experiment 2. The five other groups were given the high-fat diet with CT-II mixed in at concentrations of 0.5%, 1.0%, 1.5%, 2.5% and 3.5%, respectively. Body weight was measured at 4, 7, 11 and 14 days after the beginning of CT-II treatment, and daily food intake was measured during the 2 week treatment period. Thereafter, blood was taken from the jugular vein after 12 h fasting. The samples were centrifuged and plasma was separated and frozen until assay. Plasma triglyceride (TG), total cholesterol (TC), glucose, aspartate aminotransferase (AST, EC 2.6.1.1) and alanine aminotransferase (ALT, EC 2.6.1.2) were measured using the procedures described below in ‘Assays’ (experiment 3).

Finally, whether or not CT-II could reduce the obesity of rats fed the high-fat diet was evaluated. Rats in which obesity had been induced by the high-fat diet, weighing 570–718 g, were divided into two groups. The control group (mean body weight, 643 g) continued to be given only this diet. The second group (mean body weight, 641 g) was given the high-fat diet with CT-II mixed in at a concentration of 2.0%. Body weight was measured weekly. After 4 weeks of treatment, food intake, fecal output of total fat, and total cholesterol in feces were measured for 3 days. After 12 weeks of treatment, blood was taken from the heart after 12 h of fasting, and the plasma was separated and frozen until assay (TG, TC, glucose, AST, ALT and insulin; experiment 4).

Assays.

Glucose was assayed by the glucose oxidase method. Total cholesterol (TC), triglyceride (TG), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were assayed by a dry-chemical auto analyzer (AU-5200, OLYMPUS). Insulin was determined by immunological assay.12 Organ weights (parametrial fat, liver, heart and kidney) were measured and recorded as a percentage of final body weight together with the absolute values. Total carcass fat was determined by extraction from lyophilized homogenate of carcass without the skin. The carcass was skinned, and the subcutaneous fat attached to the skin was scraped off with a sharp knife or spatula. The skinless carcass and subcutaneous fat were combined and homogenized by twice running through a meat grinder (Meat Mincer β10, BONNY Inc.) with a 3.2 mm meshed plate, and then lyophilized. The extraction of lipids from the lyophilized homogenate of carcass without skin was done by Folch's method,13 and the extracts were evaporated in a vacuum and weighed. Total fat content was calculated as absolute value, and the percentage of final body weight.

Statistical analysis.

Data were expressed as mean±s.e. and analyzed by Student's and Aspin-Welch t-test, or one-way ANOVA repeated measurements where appropriate; a P-value of <0.05 was considered significant.

Results

Figure 1 shows the effect of CT-II on lipase activity in vitro (experiment 1). CT-II inhibited the enzyme activity in a dose-dependent manner, and about 0.1 mg/ml of CT-II to 0.071 mg/ml of porcine pancreatic lipase resulted in 50% inhibition.

Figure 1
figure1

Effect of CT-II on lipase activity. Lipase activity was measured using porcine pancreatic lipase and triolein as substrate. Inhibiting effect was shown as the lowering of relative activity (%) against the lipase activity of control (0 mg/ml of CT-II).

Figure 2 shows the rate of body weight gain of lean rats fed the high-fat diet with or without CT-II (experiment 2). The gain of those fed the diet containing 2.5% of CT-II was significantly suppressed from 1 up to 8 weeks during the treatment (P<0.01). Table 1 shows the organ weight of rats. Parametrial fat and liver weight in animals with CT-II were significantly lower than in those without CT-II after 8 weeks of treatment (P<0.05). When organ sizes were expressed as a percentage of final body weight, parametrial fat was significantly lower (P<0.05) and the heart was significantly higher (P<0.05) in CT-II treated rats than in animals without CT-II after 8 weeks of treatment. Liver histology showed fewer fat droplets in lean rats fed the high-fat diet with CT-II than in the rats without CT-II (Figure 3). Food intakes after 4 weeks of treatment were 12.9 g/day without CT-II and 12.6 g/day with CT-II, respectively, showing no significant difference between the two groups (data not shown).

Figure 2
figure2

Effect of CT-II on body weight gain in lean rats fed the high-fat diet. Values are the mean±s.e. (%) (n=6). Changes in body weight gain at each treatment period are shown as a percentage of initial body weight. (•) control; () CT-II 2.5% in diet. ***Indicate significant differences by Student's and Aspin–Welch t-test comparing 2.5% of the CT-II group to the control group (0% of CT-II) (*P<0.05; **P<0.01).

Table 1 Organ weights of lean or obese rats fed the high-fat diet after each period of CT-II treatment
Figure 3
figure3

Comparison of liver histology in lean rats fed the high-fat diet after 8 weeks of CT-II treatment. Many fat droplets shown as large white spheres in untreated controls were eliminated or diminished in size.

The dose-dependent effects of body weight gain of rats fed the high-fat diet with and without CT-II are shown in Table 2. CT-II suppressed body weight gain in a dose-dependent manner. The rates of weight gain of the four groups fed the diet containing 1.0– 3.5% of CT-II were significantly suppressed (P<0.05 or P<0.01) compared to the rate of the control group (0% CT-II). There was no significant difference among groups in daily food intake during the two week experimental period. Plasma metabolites of rats fasted for 12 h after 2 weeks of treatment are shown in Table 3. There were no significant differences in plasma glucose or total cholesterol among the groups. Plasma triglycerides were lower in rats with CT-II treatment, with the concentrations measured for the groups given CT-II at 1.5, 2.5 and 3.5% being significantly lower than intrested rate (P<0.05). ASTs were lower in rats given CT-II of 1.0–3.5%, and ALTs were also lower in those given CT-II of 1.0, 1.5 and 3.5%.

Table 2 Effect of different doses of CT-II on weight gain of lean rats fed the high-fat diet
Table 3 Effect of different doses of CT-II on plasma parameters after 2 weeks of treatment in lean rats fed the high-fat diet

Figure 4 shows body weight change of obese rats fed the high-fat diet with or without CT-II (experiment 4). The shape of the curve generated by the CT-II-treated rats differed from that of the group without the extract. Body weight initially decreased, then returned to the initial level by the 8th week, and thereafter rose in rats given CT-II; in this group it was significantly less than the control after 1 week of treatment (P<0.01). In contrast, body weight increased slowly but steadily in rats without CT-II. Total fat of the carcass excluding skin as a percentage of final body weight in rats fed the high-fat diet without CT-II, those fed the diet with 12 weeks treatment of CT-II and those fed a normal diet were 49%, 37% and 27%, respectively. The increase in body weight in rats fed the diet without CT-II can be accounted for by the increase in body fat (49% vs 27%), indicating that animals fed the high-fat diet became obese. Total fat excluding skin in obese rats given CT-II was significantly less (P<0.05) than that in obese rats without CT-II after 12 weeks of treatment (37% vs 49%). Plasma metabolites after 12 h fasting are shown in Table 4. There were no significant differences in plasma triglyceride, total cholesterol, glucose or insulin. AST and ALT were significantly lower in rats given CT-II (2%) (P<0.01), suggesting the amelioration of fatty liver. Liver weight was significantly decreased by the treatment (P<0.05), but change in liver size as a percentage of final body weight showed no statistical significance (Table 1). The weight of kidney increased significantly (P<0.01), however, no abnormal findings were observed histologically. No difference in parametrial fat pad weight was observed between the two groups.

Figure 4
figure4

Effect of CT-II on body weight gain in obese rats fed the high-fat diet. Values are the mean±s.e. (%) (n=6). Changes in body weight gain at each treatment period are shown as a percentage of initial body weight. (•) control; () CT-II 2.0% in diet. ***Indicate the significant differences by Student's and Aspin–Welch t-test comparing 2.0% of the CT-II group to the control group (0% of CT-II) (*P<0.05; **P<0.01).

Table 4 Effect of CT-II on plasma parameters after 12 weeks of treatment in obese rats fed the high-fat diet

After 4 weeks of treatment, food intake and fecal output of total fat, and total cholesterol in feces were measured over 3 days. Excretion of total lipid into feces in obese rats with CT-II was 4.30±0.24 (%) and was significantly larger (P<0.05) than the 3.55±0.32 (%) of that without CT-II. Cholesterol content in feces per day in the rats with CT-II was 20.56±2.27 (mg/day) and was also significantly larger (P<0.01) than the 11.06±1.49 (mg/day) of that without CT-II.

Discussion

With regard to lipase inhibitors, many studies of tetrahydrolipstatin (THL), which is catalytically hydrogenated from lipstatin isolated from Streptomyces toxytricini, have been reported. THL, a potent inhibitor of pancreatic lipase,2,14 selectively blocks fat absorption, and has been shown to have anti-obese and anti-hypercholesterolemic activity in several animal models,15,16 and in humans.3,4,5 It was reported that 50% inhibition of lipase activity was obtained by 0.63–0.85 to 0.1 μg/ml of partially purified porcine pancreatic lipase in a lipase assay system.2

CT-II inhibited porcine pancreatic lipase (crude lipase), and 50% inhibition of the lipase activity was observed with a concentration of less than 0.1 mg/ml in the present study. The ability to inhibit lipase is not comparable to that of THL because THL is a pure chemical agent while CT-II is a product of extraction and consists of multiple components. A small part of the active components of lipase inhibitors in CT-II were confirmed as 3′,4′,7-trihydroxyflavan-4α→8 catechin, and major active components were suggested to be 3′,4′,7-trihydroxyflavan oligomers.17 These are known as condensed tannins. It is well known that tannins can bind to various proteins and aggregate them. The mechanism of lipase inhibition by CT-II is not yet clear, however, we recognized that CT-II characteristically inhibits the activity of pancreatic lipase compared with other enzymes. For example, comparing the inhibitory effect on α-amylase of tea extract mainly including tannins (Sunphenon®) and CT-II using Bernfeld's method,18 IC50 of tea tannins (20 μg/ml) and that of CT-II (43 μg/ml) were on the same level; comparing the inhibitory effect on lipase, however, IC50 of CT-II was ca. 0.1 mg/ml as shown in Figure 1, while epigallocatechin gallate (EGCg), a main tannin in tea, showed only 39% inhibition at a concentration of 0.5 mg/ml.19 It may therefore be reasonable to consider that CT-II has high affinity to lipase and that the effect observed in this study resulted, in part, from its inhibition of lipase.

In experiment 2, the administration of CT-II inhibited body weight gain in lean rats fed the high-fat diet dose-dependently. This inhibition did not depend on decreased food intake because there was no difference in the amount of food consumed between CT-II-treated and non-CT-II-treated rats. Plasma TG was also lowered dose-dependently. AST and ALT were reduced after 2 weeks of CT-II treatment, and liver histology after 2 months of treatment showed an amelioration of fatty liver. The results also demonstrated that CT-II treatment prevented fatty liver.

When obese rats fed the high-fat diet were treated with CT-II, the body weight was initially reduced, and thereafter body weight gain was suppressed. The obese rats fed the high-fat diet for 9 months showed an apparent high composition of body fat, and CT-II treatment reduced both body weight and body fat. Body composition analysis revealed that the reduction of body weight was due to a decrease in body fat. The decrease in plasma TG observed in lean rats fed the high-fat diet 2 weeks after CT-II treatment in experiment 3 was not observed in obese rats fed this diet in experiment 4. The reason for these different results has not yet been determined. One possible explanation may be that plasma TG in obese rats in this experiment was within normal range. The reduction of AST and ALT was also noted in obese rats, confirming the amelioration of fatty liver. The improvement of liver functions might be a consequence of the improvement of obesity by the inhibitory effect of CT-II on lipase. On the other hand, it is known that tannins (polyphenols) have anti-oxidative activity, and antioxidants, such as β-carotene, vitamin E, selenium and astaxanthin, might cause plasma transaminase levels to decrease as a consequence of prevention against oxidative damage.20,21 Thus, there is also a possibility that the anti-oxidative ability of CT-II contributed to the decrease in elevated plasma transaminase levels.

The balance test between administered fat and fat excreted in feces after 4 weeks of treatment showed an increase in the latter, suggesting the inhibition of intragastrointestinal lipase by CT-II, which would have resulted from the suppression of hydrolysis and absorption of triglyceride as shown in vitro in the present study. Thus, inhibition of pancreatic lipase activity was also demonstrated in vivo. The excretion of cholesterol in feces also increased. THL reportedly inhibits cholesterolesterase as well as lipase activity.15 There is therefore a possibility that absorption of cholesterolester was suppressed by the CT-II inhibition of cholesterolesterase. However, CT-II did not reduce plasma cholesterol, although it increased cholesterol excretion in the feces, so that more research is necessary to understand this observation.

An adverse effect of lipase inhibitor THL is reportedly the highly frequent occurrence of diarrhea in human.3,5 However, rats treated with CT-II in the present study showed no diarrhea and no abnormal blood chemistry, suggesting the extract has fewer adverse effect than THL.

In summary, lipase inhibitor CT-II was effective in preventing and ameliorating obesity, fatty liver and hypertriglyceridemia in rats fed a high-fat diet. The results suggest that CT-II may be a useful substance for preventing and treating obesity and hypertriglyceridemia in humans.

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Yamamoto, M., Shimura, S., Itoh, Y. et al. Anti-obesity effects of lipase inhibitor CT-II, an extract from edible herbs, Nomame Herba, on rats fed a high-fat diet. Int J Obes 24, 758–764 (2000). https://doi.org/10.1038/sj.ijo.0801222

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Keywords

  • Cassia mimosoides
  • lipase inhibitor
  • obesity
  • fatty liver
  • hypertriglyceridemia

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