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Anti-obesity effects of chikusetsusaponins isolated from Panax japonicus rhizomes
© Han et al; licensee BioMed Central Ltd. 2005
Received: 08 January 2005
Accepted: 06 April 2005
Published: 06 April 2005
The rhizomes of Panax japonicus are used as a folk medicine for treatment of life-style related diseases such as arteriosclerosis, hyperlipidemia, hypertension and non-insulin-dependent diabetes mellitus as a substitute for ginseng roots in China and Japan. Obesity is closely associated with life-style-related diseases. This study was performed to clarify whether chikusetsusaponins prevent obesity induced in mice by a high-fat diet for 9 weeks.
We performed two in vivo experiments. In one, female ICR mice were fed a high-fat diet with or without 1 or 3% chikusetsusaponins isolated from P. japonicus rhizomes for 9 weeks. In the other, lipid emulsion with or without chikusetsusaponins was administered orally to male Wistar rats, and then the plasma triacylglycerol level was measured 0.5 to 5 h after the orally administered lipid emulsion. For in vitro experiments, the inhibitory effects of total chikusetsusaponins and various purified chikusetsusaponins on pancreatic lipase activity were determined by measuring the rate of release of oleic acid from triolein in an assay system using triolein emulsified with lecithin.
Total chikusetsusaponins prevented the increases in body weight and parametrial adipose tissue weight induced by a high-fat diet. Furthermore, consumption of a high-fat diet containing 1 or 3% total chikusetsusaponins significantly increased the fecal content and triacylglycerol level at day 3 compared with the high-fat diet groups. Total chikusetsusaponins inhibited the elevation of the plasma triacylglycerol level 2 h after the oral administration of the lipid emulsion. Total chikusetsusaponins, chikusetsusaponin III, 28-deglucosyl-chikusetsusaponin IV and 28-deglucosyl-chikusetsusaponin V inhibited the pancreatic lipase activity.
The anti-obesity effects of chikusetsusaponins isolated from P. japonicus rhizomes in mice fed a high-fat diet may be partly mediated through delaying the intestinal absorption of dietary fat by inhibiting pancreatic lipase activity. The present study clearly indicated that the saponin fractions of P. japonicus rhizomes had a significant anti-obesity action and supports the traditional usage as a substitute drug for ginseng roots.
The rhizomes of Panax japonicus C.A. Meyer (Japanese name; Chikusetsuninjin), have been used as a substitute for Ginseng roots (the roots of Panax ginseng C.A. Meyer). On the other hand, Ginseng roots are used as the remedy for life-style-related diseases such as arteriosclerosis hyperlipidemia, hypertension and non-insulin-dependent diabetes mellitus in China, Korea, Japan and Europe and there are a number of reports on the pharmacological studies of Ginseng roots [1–8]. It has been reported that the rhizomes of Panax japonicus have anti-ulcer action and fibrinolysis [9, 10]. However, the investigations of pharmacological effects of Panax japonicus rhizomes on life-style-related diseases such as obesity, arteriosclerosis hyperlipidemia, hypertension and non-insulin-dependent diabetes mellitus have been not thoroughly reported. Obesity is one of the fastest-growing major diseases in many areas of the world including Europe, the United States and Japan. Obesity results from an imbalance between energy intake and expenditure. Obesity is closely associated with life-style-related diseases such as hyperlipidemia, hypertension, arteriosclerosis and non-insulin-dependent diabetes mellitus and with increased risk of coronary heart disease . It has been reported that variations in total energy intake and diet composition are important in the regulation of metabolic processes [12, 13]. Furthermore, it has been suggested that dietary fat promotes body fat storage more effectively than dietary carbohydrate. Thus, inhibition of the digestion and absorption of dietary fat is a key to treating obesity. Dietary fat is not directly absorbed from the small intestine unless it has been subjected to the action of pancreatic lipase . In this study, we examined the effects of total chikusetsusaponins of P. japonicus rhizomes on obesity induced by long-term feeding of a high-fat diet.
Pancreatic lipase was purchased from Sigma Chemical Co. (St Louis, MO). Triglyceride E- and Total Cholesterol E-test kits were purchased from Wako Pure Chemical Co. (Osaka, Japan). 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). Orlistat (a lipase inhibitor) was purchased from Hong Kong Market. Other chemicals were of reagent grade.
The rhizomes of P. japonicus were obtained from Tochimoto Tennkaido Co. Ltd. (Osaka, Japan). Voucher specimens (No. PJ020817) are deposited in the Faculty of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto and the Second Department of Medical Biochemistry, School of Medicine, Ehime University.
Preparation of total saponins and five chikusetsusaponins from the rhizomes of P. japonicus
Composition of experimental high-fat diets
Total chikusetsusaponins (g/100 g diet)
High-fat diet (HF)
HF + 1% chikusetusaponins
HF + 3% chikusetsusaponins
HF + 0.012% orlistat
HF + 0.024% orlistat
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 Committees of Ehime University and Kumamoto Prefectural University.
Estimation of body and parametrial adipose tissue weights, plasma triacylglycerol and total cholesterol in mice fed a high-fat diet for 9 weeks
Female ICR mice (3 weeks old) were divided into four groups that were matched for body weight, after 1 week of being fed laboratory pellet chow ad libitum. The control group continued to be fed laboratory pellet chow ad libitum. The mice consumed the high-fat diet or the high-fat diet containing 1 and 3% total chikusetsusaponins or 0.012 and 0.024% orlistat (positive control) for 9 weeks. The body weight of each mouse was measured once a week and the total amount food consumed was recorded 3 times per week. After the mice had been fed these diets for 9 weeks, blood was taken from each mouse by venous puncture under anesthesia with diethyl ether; the mice were then killed with an overdose of diethyl ether. Experiments were performed in a ventilated room. The plasma was prepared and frozen at -80°C until analysis. The liver and parametrial adipose tissue were dissected and weighed. Liver triacylglycerol and total cholesterol concentrations were measured using Wako Triglyceride E-Test and Total Cholesterol E-Test kits.
Fat excretion in feces of mice
Female ICR mice (3 weeks old) were housed for 1 week in a room maintained at 25 ± 1°C with 60% relative humidity and given free access to standard laboratory pellet chow and water. The mice consumed the high-fat diet or the high-fat diet containing 1% or 3% total chikusetsusaponins, or 0.025% orlistat for 3 days. Wet weight and triacylglycerol content in the feces obtained during the last 24 h were measured using the Wako Triglyceride E-Test kit.
Plasma triacylglycerol levels after oral administration of lipid emulsions to rats
Male Wistar rats were also housed for 1 week in the above same conditions. After rats had been deprived of food overnight, they were orally administered 1 mL of a lipid emulsion consisting of corn oil (3 mL), cholic acid (40 mg) and cholesteryl oleate (1 g) plus physiological saline (3 mL), the lipid emulsion (1 mL) plus total chikusetsusaponins (final concentration 1000 mg/kg body) or the lipid emulsion plus orlistat (final concentration 45 mg/kg body). Blood samples were taken from the tail vein 0, 0.5, 1, 2, 3, 4 and 5 h after administration of the lipid emulsion with or without total saponins or orlistat using a capillary tube (heparinized), and centrifuged at 5500 × g for 5 min in a Model KH-120 M (Kubota Co., Osaka, Japan) centrifuge to obtain the plasma. The plasma triacylglycerol concentration was determined using a Wako Triglyceride E-Test kit.
In vitro pancreatic lipase activity
Lipase activity in the porcine pancreas was assay as described previously . Enzyme activity was expressed as μmoles of oleic acid released per mL of reaction mixture per min.
All values are expressed as means ± s.e. Data were analyzed by one-way ANOVA, and then differences among means were analyzed using the Fisher's protected LSD test. Differences were considered significant at P < 0.05.
Fat excretion in feces of mice fed a high-fat diet with or without total chikusetsusaponins
Effects of total chikusetsusaponins and orlistat (a lipase inhibitor) on fat excretion on day 3 in feces of mice fed a high-fat diet
Feces weight (g)
Triacyglycrol in feces (μmol/g feces)
Laboratory chow pellet
1.04 ± 0.017*
5.05 ± 0.70*
High-fat diet (HF)
0.14 ± 0.035
36.66 ± 1.03
1% Total chikusetsusaponins
0.26 ± 0.012*
41.64 ± 3.48
3% Total chikusetsusaponins
0.31 ± 0.049
53.42 ± 4.86*
0.025 % orlistat (lipase inhibitor)
0.22 ± 0.040
394.38 ± 65.39*
Food consumption; body, parametrial adipose tissue and liver weights; and triacylglycerol content in the livers of mice fed high-fat diet with or without chikusetsusaponins for 9 weeks
Effects of total chikusetsusaponins and orlistat (a lipase inhibitor) on liver weight, hepatic triacylglycerol, and adipose tissue weight in mice fed a high-fat diet for 9 weeks.
Liver weight (g/100 g body weight)
Hepatic triacylglycerol (μmol/g)
Parametrial adipose tissue weight (g)
Laboratory chow pellet
5.20 ± 0.46*
22.3 ± 2.3*
0.71 ± 0.1*
High-fat diet (HF)
6.35 ± 0.76
141.8 ± 21.8
1.59 ± 0.3
1% Total saponins
6.13 ± 0.79
101.5 ± 6.8*
0.87 ± 0.1*
3% Total saponin
5.79 ± 0.47*
83.3 ± 6.7*
0.74 ± 0.1*
HF + 0.012% Orlistat
4.00 ± 0.11*
101.1 ± 4.8*
2.03 ± 0.12
Total chikusetsusaponins reduced the elevation of rat plasma triacylglycerol levels after oral administration of lipid emulsion to rats
Effects of total chikusetsusaponins and various purified chikusetsusaponins on pancreatic lipase activity in vitro
There are a number of reports showing that ginseng roots are clinically anti-allergic, anti-hypertensive, hypoglycemic, anti-thrombotic, and anti-arteriosclerotic [7, 8]. Recently, obesity is increasing in advanced countries including Europe, the United States and Japan. Obesity is closely associated with life-style-related diseases such as hyperlipidemia, hypertension, arteriosclerosis and non-insulin-dependent diabetes mellitus and with increased risk of coronary heart disease . Although the rhizomes of P. japonicus are used as a substitute drug for ginseng roots in Japan, the pharmacological effects of these rhizomes on life-style-related diseases have been not clarified. A methanol extract of these rhizomes and chikusetsusaponins such as chikusetsusaponins III, IV and V derived from P. japonicus rhizomes have been shown to promote the fibrinolysis . Yamahara et al.  reported that the saponin fractions and chikusetsusaponin III of P. japonicus exert anti-ulcer effects. However, there have hitherto been no reports on the inhibitory effects of chikusetsusaponins isolated from P. japonicus rhizomes on obesity induced by consumption of a high-fat diet. Dietary fat can increase body weight and adiposity in humans and animals more effectively than dietary carbohydrate [17, 20–22]. Dietary fat is not directly absorbed from the intestine unless it has been subjected to the action of pancreatic lipase . Therefore, the application of pancreatic lipase inhibitor was examined earlier as a treatment for high-fat diet-induced obesity in humans. It has been reported that a pancreatic lipase inhibitor, orlistat prevented obesity and hyperlipidemia through the enhancement of fat excretion in feces and the inhibition of pancreatic lipase . We found that feeding high-fat diet containing 40% fat content caused obesity [17, 19], but the high-fat diet containing 25% fat content slightly increased the body weight (data not shown). Anai et al. reported that feeding high-fat diet containing 60% fat for 2 weeks slightly increased the body weight . Furthermore, it has been reported that feeding high-fat diet containing 58% kcal fat for 16 weeks caused obesity . In the present study, to examine the effects of chikusetsusaponins on high-fat diet-induced obesity, we used the obesity model induced by feeding high-fat diet containing 40% fat for 9 weeks. We found that the administration of total chikusetsusaponins isolated from P. japonicus significantly suppressed the increase in body weight in mice fed a high-fat diet containing 40% beef tallow for 9 weeks. The treatment with total chikusetsusaponins also significantly reduced the final parametrial adipose tissue weight compared to that of the high-fat diet group. These inhibitions did not depend on decreased food or energy intake, because there was no significant difference between these in the groups fed the high-fat diet and the high-fat diet containing 1 or 3 % total chikusetsusaponin. Next, we examined the effects of total chikusetsusaponins on plasma triacylglycerol concentrations after oral administration of a lipid emulsion in rats, and found that the total chikusetsusaponins reduced the elevation of plasma triacylglycerol levels as measured by an oral lipid emulsion tolerance test. This finding suggests that total chikusetsusaponins may inhibit the uptake of dietary fat. In fact, total chikusetsusaponins strongly inhibited the pancreatic lipase activity, and therefore, we attempted to isolate substance(s) from total saponins that inhibit pancreatic lipase activity. Five chikusetsusaponins were isolated from total saponin fractions and identified as chikusetsusaponins III, IV and V and 28-deglucosyl-chikusetsusaponins IV and IV by the direct comparison with authentic samples. Among these five saponins, chikusetsusaponin III and 28-deglucosylchikusetsusaponins IV and V inhibited the pancreatic lipase activity, most strongly. The contents of total chikusetsusaponins in the MeOH extract are about 35%, and the contents of chikusetsusaponins III, IV and V in the total saponin fraction are about 10.4, 4.8 and 41.9%, respectively. On the other hand, the content of 28-deglucosylchikusetsusaponins IV and V in the total saponins are about 0.01 and 0.08%, respectively. Chikusetsusaponins IV and V are rapidly converted to 28-deglycosyl-forms by the treatment with alkaline solution. Therefore, it seems likely that the above 28-deglucosylchikusetsusaponin derive from chikusetsusaponins IV and V through the small intestine after orally administered chikusetsusaponins IV and V. Consequently, it is suggested that their metabolites (28-deglucosyl form) exhibit the inhibition of pancreatic lipase activity.
Total chikusetsusaponins isolated from P. japonicus may prevent high-fat-diet-induced increases in body weight and fat storage in adipose tissue by inhibiting intestinal absorption of dietary fat through the inhibition of pancreatic lipase activity, and the active components were identified here as chikusetsusaponins III and IV, 28-deglucosyl-chikusetsusaponins IV and V. The present study clearly indicated that the saponin fractions of P. japonicus rhizomes had a significant anti-obesity action and supports the traditional usage as a substitute drug for ginseng roots. Hence it might help in preventing obesity complications and serve as good adjuvant in the present armamentarium of anti-obesity drugs.
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