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1
Faculty of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto, Tsukide, Kumamoto 862-8502, Japan;
*
Department of Chinese Material Medicine, Chinese Material Medicine College of Jilin Agricultural University, Changchun-shi, Jilin 130118, China; and
Second Department of Medical Biochemistry, School of Medicine, Ehime University, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan
1To whom correspondence should be addressed. E-mail: yokim{at}m.ehime-u.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • Platycodi radix • crude saponins • platycodin D • pancreatic lipase • high fat diet • mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Obesity results from an imbalance between energy intake and expenditure. It is often associated with chronic diseases such as hyperlipidemia, hypertension and noninsulin-dependent diabetes mellitus and with increased risk of coronary heart diseases (2
). It has been reported that variations in total energy intake and diet composition are important in the regulation of metabolic processes. Furthermore, it has been suggested that dietary fat promotes body fat storage more effectively than dietary carbohydrate. Consistent with these suggestions, high fat diets can increase body weight and adiposity in humans and animals (3
–6
). Thus, inhibition of digestion and absorption of dietary fat is a key to treating obesity. Dietary fat is not absorbed from the intestine unless it has been acted upon by pancreatic lipase (7
). We reported that oolong tea (8
), chondroitin sulfate (9
) and Platycodi radix (10
) have antiobesity actions; they inhibit intestinal absorption of dietary fat by inhibiting pancreatic lipase activity. In China and Korea, pickled fresh roots of Platycodon gramdiflorum are consumed to prevent obesity. We reported previously that the aqueous extract (50 g/kg diet) of Platycodi radix prevented increases in body weight, adipose tissue weight and liver triacylglycerol in mice fed a high fat diet. Furthermore, we examined the effects of inulin isolated from Platycodi radix on lipid metabolism in mice with high fat diet–induced obesity and found that it had no effect. In in vitro experiments, crude saponin fractions prepared from aqueous extracts of Platycodi radix inhibited pancreatic lipase activity (10
). Therefore, we examined the effects of crude saponins of Platycodi radix on lipid metabolism and intestinal sucrase activity in mice with high fat diet–induced obesity to clarify whether the saponin fraction of Platycodi radix has antiobesity effects. We also examined the effect of the crude saponins on the elevation in plasma triacylglycerols caused by the oral administration of lipid emulsions in rats. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Triolein and pancreatic lipase (porcine pancreas) were purchased from Sigma (St. Louis, MO). Wako Triglyceride E-Test and Total Cholesterol E-Test Kits were purchased from Wako Pure Chemical (Osaka, Japan). The nonpurified diet, which contained (per 100 g) water, 8 g; crude carbohydrate, 51.3 g; crude protein, 24.6 g; crude lipid, 5.6 g; crude fiber, 3.1g; mineral mixture, 6.4 g; and vitamin mixture, 1 g, was purchased from CLEA Japan (Osaka, Japan). Beef tallow, cornstarch, casein, mineral mixture (AIN-76) (11
) and vitamin mixture (AIN-76) (11
) were purchased from Oriental Yeast (Tokyo, Japan). Fecal excretions were measured in mice housed in metabolic cages (CLEA Japan). The roots of Platycodon grandiflorum were obtained from Jilin Agricultural University (Changchun, China). Crude saponin fractions were isolated from the aqueous extract of Platycodi radix as previously described (10
). Platycodin D was the major saponin in Platycodi radix, with a concentration of 
550 g/kg in the crude saponins. The crude saponins of Platycodi radix and partially purified platycodin D isolated from Platycodi radix were used. Other chemicals were of reagent grade.
Diet compositions.
Previously, we reported that varying the casein concentration (220, 310, 340 and 360 g/kg diet) in the high fat diet containing 400 g/kg beef tallow did not affect body weight or parametrial adipose tissue weight (6
). Therefore, we added the crude saponins isolated from Platycodi radix to the high fat diet instead of casein. The compositions of the high fat diet and the high fat plus crude saponin diets are shown in Table 1
. To avoid autoxidation of the fat components, food was stored at -30°C. Although the high fat diet used in this study was deficient in linolenic acid, it did not affect the growth of mice (data not shown).
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Female ICR strain mice (3 wk old) and male Wistar King strain rats (6 wk old, 180 g) were obtained from CLEA Japan and Charles River Japan (Yokohama, Japan), respectively. The animals were housed for 1 wk in a room with a 12-h light:dark cycle and controlled for temperature and humidity. The animals had free access to food and water and were studied after 1 wk of adaptation to the lighting conditions. Mice and rats were treated according to the ethical guidelines of the Animal Center, School of Medicine, Ehime University and Prefectural University of Kumamoto. The Animal Studies Committee of Prefectural University of Kumamoto and Ehime University approved the experimental protocol.
In vitro pancreatic lipase activity.
Assay of lipase activity in the porcine pancreas was performed as described previously (6
). Enzyme activity was expressed as mmol oleic acid released/(L reaction mixture · h).
Plasma triacylglycerol levels after oral administration of lipid emulsions to rats.
After rats had been deprived of food overnight, they were orally administered 2 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) or the lipid emulsion (2 mL) plus the crude saponins (final concentration 375 mg/kg body). Blood samples were taken from the tail vein 0, 0.5, 1, 2, 3 and 4 h after administration of the lipid emulsion with or without the crude saponin fraction using a capillary tube (heparinized), and centrifuged at 5500 x g for 5 min in a Model KH-120 M (Kubota, Japan) centrifuge to obtain the plasma. The plasma triacylglycerol concentration was determined using a Wako Triglyceride E-Test kit. In addition, in a similar experiment, we examined the effect of platycodin D on plasma triacylglycerol after oral administration of the lipid emulsion to rats. After rats had been deprived of food overnight, 2 mL of lipid emulsion or the lipid emulsion (2 mL) plus platycodin D (final concentration 244 mg/kg body) were administered orally.
Fat excretion in feces of mice.
The mice consumed the high fat diet or the high fat diet containing 10 or 30 g/kg crude saponins for 3 d. Samples of feces were obtained from each mouse at intervals of 24 h and the triacylglycerol was measured using the Wako Triglyceride E-Test kits as previously described (6
).
Body, liver and parametrial adipose tissue weights, and liver triacylglycerol and total cholesterol concentrations.
The body weight of each mouse was measured once each week and the total amount of food consumed was recorded 3 times per week. After 9 wk of feeding the diets, blood was taken from each mouse by venous puncture while 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 the Wako Triglyceride E-Test and Total Cholesterol E-Test kits as previously described (10
). The small intestine of each mouse was removed and washed with physiological saline and stored at -20°C until intestinal sucrase and maltase activities were determined.
Intestinal enzyme and protein analysis in high fat diet–treated mice.
The small intestinal segments were homogenized in 1.5 mL of ice-cold 80 mmol/L sodium phosphate buffer (pH 7.0) using a Kinematica homogenizer (Brinkmann Instruments, Westbury, NY). Sucrase (EC 3.2.1.48) and maltase (EC 3.2.1.20) assays were based on a modification of the method of Dahlqvist (12
) using 50 mmol/L sucrose or maltose as substrates. The protein content of the homogenates was determined using a kit (Bio-Rad, Hercules, CA). Enzyme activity was expressed as µmol substrate hydrolyzed/(h · mg protein).
Statistical analysis.
All values are expressed as means ± SEM. Data were analyzed by one-way ANOVA, and then differences among means were analyzed using the Bonferroni/Dunn test or Dunnett’s test. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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At 2, 3 and 4 h after oral administration of the corn oil emulsion with or without crude saponins (375 mg/kg body), the plasma triacylglycerol concentration was significantly lower in the saponin-treated rats than in the controls (Fig. 1A
). Platycodin D (244 mg/kg body) prevented the increase in rat plasma triacylglycerol after oral administration of the lipid emulsion at 1, 2 and 3 h after administration (Fig. 1
B).
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Feeding the high fat diet for 3 d reduced the stool content (0.22 ± 0.05 g, n = 4) at d 3 compared with mice fed a nonpurified diet (2.33 ± 0.17 g, n = 4). Mice fed the high fat plus 30 g/kg crude saponins for 3 d had greater fecal content (1.11 ± 0.05 g) and triacylglycerol (51.9 ± 7.79 µmol/g feces) at d 3 compared with the high fat diet group (feces, 0.22 ± 0.05 g and triacylglycerol, 25.8 ± 4.12 µmol/g feces) (P < 0.01).
Energy intake, body, and tissue weights, and hepatic lipids in mice fed a high fat diet with or without crude saponins.
Energy intakes did not differ among mice fed the high fat diet or the diet with 3.5 or 30 g/kg crude saponins [values expressed in kJ/(wk · mouse) (mean ± SEM): high fat diet group (533.9 ± 17.4); high fat diet plus 3.5 g/kg crude saponins (610.4 ± 32.1); and high fat diet plus 30 g/kg crude saponins (591.9 ± 37.8)]. However, mice fed the high fat diet plus 10 g/kg crude saponins consumed more energy [657.3 ± 24.1 kJ/(wk · mouse)] than those fed the high fat diet [533.9 ± 17.4 kJ/(wk · mouse)]. Body weight (Fig. 2
) was lower at wk 1 in mice fed the high fat diet containing 10 or 30 g/kg crude saponins compared with those fed the high fat diet alone. Similarly, final parametrial adipose tissue weight was reduced in mice fed the high fat diet containing 10 or 30 g/kg crude saponins (0.78 ± 0.06 g and 0.59 ± 0.07 g, respectively) compared with those fed the high fat diet alone (1.45 ± 0.13 g). Mice fed the high fat diet containing 3.5 g/kg crude saponins did not differ from controls in the body or final parametrial adipose tissue weights.
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Small intestine weight and sucrase and maltase activities in mice fed a high fat diet with or without crude saponins.
The weights of small intestine (0.71 ± 0.03 g, n = 16) in mice fed the high fat diet for 9 wk were lower (P < 0.04) than in those fed the nonpurified diet (0.93 ± 0.06 g, n = 11). Those fed the high fat diet plus all levels of crude saponins had greater small intestine weights than those fed the high fat diet alone (Table 2
). Sucrase [0.70 ± 0.10 µmol/(h · mg protein)] and maltase [1.92 ± 0.18 µmol/(h · mg protein)] activities of small intestine in mice fed the high fat diet were lower (P < 0.01) than in those fed the nonpurified diet [sucrase, 1.90 ± 0.22 and maltase, 2.17 ± 0.22 µmol/(h · mg protein)]. Mice fed the high fat diet plus 3.5 g/kg crude saponins, but not the two higher levels, had greater sucrase activity than those fed the high fat diet alone (Table 2)
. Maltase activity did not differ among the diet groups (data not shown).
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The major component of the crude saponins, Platycodin D, inhibited pancreatic lipase activity 80% at 1 g/L (Table 3
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Crude saponins (375 mg/kg) reduced the elevation of plasma triacylglycerol after oral administration of a lipid emulsion to rats (Fig. 1
A). Furthermore, mice fed the high fat diet plus 30 g/kg crude saponins had greater fat excretion into feces compared with those fed the high fat diet for 3 d. These results suggest that crude saponins reduce the small intestinal absorption of dietary fat by inhibiting pancreatic lipase activity. Mice fed to the high fat diet for 9 wk had greater body weight and adipose tissue mass than those fed the nonpurified diet (6
,8
,9
). By feeding the high fat diet for a longer period (9 wk), we could examine the effects of various doses of crude saponins on diet-induced obesity with fat storage. Mice fed the high fat diet plus 10 or 30 g/kg crude saponins had lower body and final parametrial adipose tissue weights than those fed the high fat diet alone (Fig. 2)
. Furthermore, feeding a high fat diet containing 3.5, 10 or 30 g/kg crude saponins significantly decreased the hepatic triacylglycerol and total cholesterol concentrations compared with feeding a high fat diet alone, without decreasing energy intake. The inhibitory effects of the crude saponins on lipid metabolism in mice fed a high fat diet were stronger than those of the aqueous extract of Platycodi radix reported previously (10
). It has been reported that various saponins isolated from foodstuffs have antiobesity actions (13
,14
). Therefore, we suggest that the antiobesity actions of the aqueous extract might be due to the crude saponins of Platycodi radix. Among the aqueous extracts, the crude saponins and purified saponin platycodin D, platycodin D inhibited the elevations of plasma triacylglycerol the most. Furthermore, pure platycodin D inhibited pancreatic lipase in vitro the most. These results suggest that the inhibitory effect of the aqueous extract or crude saponins of Platycodi radix on plasma triacylglycerol might be due to a saponin, platycodin D. Experiments are now in progress to isolate other saponins from the crude mixture. In future experiments, we will test the effects of various purified saponins such as platycodins D, E and F on high fat diet–induced obesity.
Recently, Black et al. (15
) reported that a high fat diet reduced intestinal sucrase and maltase activities by stimulating pancreatic protease secretion. In addition, Goda and Takase (16
) reported that rats fed high fat diets had shortened microvilli with slightly greater diameters, and reduced surface area of microvilli per enterocyte. The reduction in microvillar surface area was accompanied by a decrease in total proteins of the brush border membranes as well as a decrease in the activities of microvillar-stalked disaccharidases, i.e., sucrase-isomaltase and lactase. In this study, sucrase activity was significantly greater in mice fed the high fat diet plus the lowest level of crude saponins (3.5 g/kg diet). We also found that the high fat diet plus 10 or 30 g/kg crude saponins significantly increased the small intestine weight compared with mice fed the high fat diet alone (Table 2)
. The high fat diet caused obesity with fat storage and a reduction in small intestinal sucrase activity without elevating plasma insulin (data not shown). Thus, it seems likely that high fat diet–induced obesity did not involve plasma insulin. Rather, feeding a high fat diet to mice increases the secretion of pancreatic lipase, and consequently, sucrase activity of the small intestine may be reduced. Therefore, the antiobesity action by crude saponins of Platycodi radix may be due to the inhibition of pancreatic lipase activity; consequently, crude saponins may prevent the reduction in small intestinal sucrase activity. Further work is required to clarify the alteration of small intestinal enzymes in mice fed a high fat diet for a longer term. In conclusion, the antiobesity effect of crude saponins of Platycodi radix in mice fed a high fat diet may be due to the inhibition of intestinal absorption of dietary fat, and one of the active substances was identified as platycodin D.
Manuscript received 12 December 2001. Initial review completed 9 January 2002. Revision accepted 16 May 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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