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Second Department of Medical Biochemistry, School of Medicine, Ehime University, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan and * The Pharmaceutical Institute, Dalian University, Dalian-shi, Liaoning 116622, Japan
1To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • Platycodi radix • inulin • pancreatic lipase • high fat diet • mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, the mechanism
of the antiobesity and antihyperlipidemic effects of Platycodi radix
remain to be clarified. We found that obesity was induced by feeding a
high fat diet containing 40% beef tallow for 10 wk to female mice
(Han et al. 1999b
). Furthermore, we found that chitosan
(Han et al. 1999a
) and oolong tea (Han et al. 1999c
) exerted antiobesity and/or antihyperlipidemic actions
that were mediated through delaying the intestinal absorption of
dietary fat by inhibiting pancreatic lipase activity. In preliminary
experiments, we found that an aqueous extract of Platycodi radix might
inhibit intestinal absorption of dietary fat by inhibiting hydrolysis
of the fat by pancreatic lipase. It is thus possible that similar
mechanisms are involved in the Platycodi radix actions. In preliminary
experiments, we examined the effects of the aqueous extract of
Platycodi radix on pancreatic lipase activity in vitro and on the
elevation of plasma triacylglycerol levels caused by the oral
administration of a lipid emulsion to rats. The principal study was
designed to clarify whether the aqueous extract of Platycodi radix
prevented the obesity induced by feeding mice a high fat diet for 8 wk. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Triolein and pancreatic lipase were purchased from Sigma Chemical (St. Louis, MO). Triglyceride E-Test and Total Cholesterol E-Test kits were purchased from Wako Pure Chemical (Osaka, Japan). Laboratory pellet diet was purchased from CLEA Japan (Osaka, Japan). Other chemicals were of reagent grade.
Animals.
Female ICR strain mice (3 wk old) and male Wistar King strain rats (6 wk old, 180 g body) were obtained from CLEA Japan and Charles River Japan (Yokohama, Japan), respectively, and housed for 1 wk with a 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 wk, the healthy animals were used.
Preparation of the aqueous extract of Platycodi radix.
The roots of Platycodon grandiflorum (Jacq.) A.DC. were obtained from Ji-Lin Sheng (Chang Chun, China). Aqueous extracts were prepared as follows: 1 kg of powdered dried plant material was refluxed with 10 L of water for 3 h, and the aqueous extract (300 g) was obtained.
Preparation of the crude saponin fractions and inulin from Platycodi radix.
The crude saponin fractions were prepared as follows: 10 kg of powdered
dried plant material was extracted with distilled water (100 L) for
3 h under reflux. After filtration and evaporation of the aqueous
solution, the aqueous extract (3.3 kg) was obtained. The aqueous
extract (3 kg) was extracted with 10 L of 95% ethanol at room
temperature for 8 h, and the ethanol solution was evaporated under
vacuum to obtain ethanol extracts (2.3 kg). The ethanol extract was
dissolved in water at a concentration of 100 g/L and then fractionated
on a macroporous adsorption resin D 101 column with water, 50 and 95%
ethanols as solvents. Crude saponins were obtained from the 50 and 95%
ethanol fractions. The yield was 
2.7%. Inulin was prepared from
aqueous extracts of Platycodi radix as follows: dried Platycodi radix
(100 g) was extracted with 1.4 L of boiling water for 2 h,
concentrated into 700 mL under reduced pressure, and the concentrated
solution was treated with activated charcoal powder. The
charcoal-treated solution was poured into 700 mL of ethanol and
kept at room temperature for 12 h. The precipitates were washed
three times with ethanol, dried and obtained as a white powder, which
was identified as inulin by comparison with an authentic sample. The
yield was 16 g.
Measurement of pancreatic lipase activity.
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 of 0.1
mol/L N-tris (hydroxymethyl)
methyl-2-aminoethanesulfonic acid buffer (pH 7.0) containing 0.1 mol/L
NaCl was sonicated for 5 min. This sonicated substrate suspension (0.1
mL) was incubated with 0.05 mL (10 U) of 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. The amount of oleic acid released
was determined by the method of Zapf et al. (1981)
with
a slight modification (Tsujita and Okuda 1983
). The
incubation mixture was added to 3.0-mL aliquots of a 1:1 (v/v) mixture
of chloroform and n-heptane containing 2% (v/v)
methanol and extracted by shaking the tubes horizontally for 10 min in
a shaker. The mixture was centrifuged at 2000 x g
for 10 min, and the upper aqueous phase was removed by suction. Copper
reagent (1.0 mL) was then added to the lower organic phase. The tube
was shaken for 10 min, the mixture was centrifuged at 2000 x g for 10 min and 0.5 mL of the upper organic phase,
which contained copper salts of the extracted free fatty acids, was
treated with 0.5 mL of 1 g/L bathocuproine in chloroform containing 0.5
g/L hydroxyanisole. The absorbance was then measured at 480 nm. Lipase
activity was expressed as moles of oleic acid released per liter of
reaction mixture per hour.
Measurement of plasma triacylglycerol level after oral administration of lipid emulsion in rats.
After male Wistar king strain rats had been deprived of food overnight, 3 mL of lipid emulsion (6 mL corn oil, 80 mg cholic acid, 2 g chloestryloleate plus 6 mL saline) or the lipid emulsion (3 mL) plus the aqueous extract of Platycodi radix (final concentration, 570 mg/kg body) were administered orally to the rats. 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 aqueous extract of Platycodi radix using a capillary tube (heparinized), and centrifuged at 5500 x g for 5 min in a Model KH-120M (Kubota, Japan) centrifuge to obtain the plasma. The plasma triacylgylcerol concentration was determined using a Triglyceride E-Test Wako kit.
Body, liver and parametrial adipose tissue weights, and liver triacylglycerol and total cholesterol concentrations.
Female ICR mice (3 wk old) were divided into five groups, with each
group matched for mean body weight, after 1 wk of feeding. The basic
composition of the experimental diet was as follows (g/100 g food):
beef tallow, 40; cornstarch, 10; sugar, 9; vitamin mixture, 1; mineral
mixture, 4; and casein, 36. The composition of the diet for each
experimental group is shown in Table 1
. Previously, we reported that the variation in casein concentration in
the high fat diet (22–36%) did not affect either body weight or
parametrial adipose tissue weight. That is, 22- 36% casein in the high
fat diet caused similar degrees of obesity (Han et al. 1999a
). On the basis of these facts, we added the aqueous
extract of Platycodi radix and inulin to the high fat diet instead of
casein. To avoid autooxidation of its fat components, food was stored
at -30°C and freshly prepared each day. Each mouse was weighed once
a week and the weight was recorded. The total amount of food consumed
by each mouse was recorded at least three times per week.
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Statistical analysis.
The results are expressed as means ± SEM. The data were analyzed by ANOVA. Fisher’s Protected Least Significant Difference tests were used to determine the significance of differences among the groups. P-values < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The aqueous extract of Platycodi radix inhibited the pancreatic lipase
activity in a dose-dependent manner in the assay system using
triolein emulsified with phosphatidylcholine. Inulin isolated from the
aqueous extract of Platycodi radix did not inhibit pancreatic lipase
activity in vitro (Fig. 1
).
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At 2, 3 and 4 h after administration of the aqueous extract of
Platycodi radix, the plasma triacylglycerol concentration was
significantly lower in treated rats than in the control group
(Fig. 2
).
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Energy consumption at 4–8 wk was not significantly different among the high fat diet and the high fat diet plus aqueous extract of Platycodi radix (2 or 5%) or inulin (0.5 or 1%) groups as follows: 753.0 ± 33.0 kJ/(wk · mouse) in the high fat diet group; 712.7 ± 35.2 kJ/(wk · mouse) in the high fat diet plus 2% aqueous extract of Platycodi radix group; 682.8 ± 45.8 kJ/(wk · mouse) in the high fat diet plus 5% aqueous extract of Platycodi radix group; 655.1 ± 42.7 kJ/(wk·mouse) in the high fat diet plus 0.5% inulin group; and 662.4 ± 20.7 kJ/(wk·mouse) in the high fat diet plus 1% inulin group.
Consumption of the high fat diet for 8 wk caused significant increases
in body, parametrial adipose tissue and liver weights. Body weight at 3
to 8 wk (Fig. 3
) and final parametrial adipose tissue (Fig. 4
) and liver weights (Table 2
) were significantly reduced by consumption of the high fat diet
containing 5% aqueous extract of Platycodi radix compared with feeding
the high fat diet alone. Feeding the high fat diet containing 2%
aqueous extract of Platycodi radix or 0.5 or 1% inulin did not reduce
the body or parametrial adipose tissue weights. On the other hand,
feeding the high fat diet containing 2% aqueous extract of Platycodi
radix or 0.5 or 1% inulin significantly reduced liver weight.
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Feeding the high fat diet caused fatty liver with accumulation of
triacylglycerol and total cholesterol (Table 2)
. The accumulation of
hepatic triacylglycerol caused by the high fat diet was significantly
decreased by feeding the high fat diets containing a 5% aqueous
extract of Platycodi radix compared with feeding the high fat diet
alone. However, consumption of the high fat diet plus the aqueous
extract of Platycodi radix did not affect hepatic total cholesterol
concentration. Furthermore, the aqueous extract of Platycodi radix had
no effects on plasma lipid levels after mice were fed the high fat diet
(data not shown). Consumption of the 0.5 and 1% inulin diets did not
affect liver triacylglycerol or total cholesterol concentrations.
Effect of crude saponin fractions on pancreatic lipase activity (in vitro).
The crude saponin fraction strongly inhibited pancreatic lipase
activity in the assay system using triolein emulsified with
phosphatidylcholine (Figure 5
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In this
investigation, we found that feeding the aqueous extract of Platycodi
radix for 8 wk prevented obesity and the associated increases in
parametrial adipose tissue weight and hepatic triacylglycerols caused
by consumption of a high fat diet containing 40% beef tallow.
Verger (1984)
reported that dietary fat was hydrolyzed
during digestion by pancreatic lipase. The two main products formed by
the hydrolysis of pancreatic lipase are fatty acids and
2-monoacylglycerols (Hernell et al. 1990
). These
lipolytic products are mixed with bile salts, dispersed as micelles and
carried in this form to the site of fat absorption. Lipid absorption
takes place in the apical part of the plasma membrane of epithelial
cells or enterocytes lining the gut. Previously, we found that the
glucosamine polymer chitosan, which has a positive charge, inhibited
the hydrolysis of triolein emulsified with phosphatidylcholine in a
pancreatic lipase activity assay. On the basis of these results, we
designed in vivo experiments and clarified that chitosan prevented the
increase in body and parametrial adipose tissue weights induced by a
high fat diet through inhibition of the intestinal absorption of
dietary fat (Han et al. 1999a
). In this study, we first
attempted to examine the effects of the aqueous extract of Platycodi
radix on pancreatic lipase activity. The aqueous extract of Platycodi
radix inhibited the hydrolysis of triolein emulsified with
phosphatidylcholine in the pancreatic lipase activity assay. On the
basis of this result, we performed experiments that examined the
reducing effect of the aqueous extract of Platycodi radix on plasma
triacylglycerols elevated in rats after oral administration of a lipid
emulsion containing corn oil and clarified its effect. The results
suggested that the aqueous extract of Platycodi radix might exert
antiobesity and antihyperlipidemic actions, which were mediated through
delaying the intestinal absorption of dietary fat by inhibiting
pancreatic lipase activity. On the other hand, in the long-term
experiments, hyperlipidemia was not caused by feeding the high fat diet
or by food deprivation after consumption of a high fat diet (data not
shown). Therefore, long-term feeding of a high fat diet did not
elevate plasma lipids; rather, it caused fatty liver and an increase in
the parametrial adipose tissue weight. In the long-term (8 wk)
experiment, we found that 50 g/kg food aqueous extract of Platycodi
radix significantly reduced both body and parametrial adipose tissue
weights, although no significant differences in energy consumption were
found. However, there is a discrepancy concerning the action of the
aqueous extract of Platycodi radix between the results on plasma
triacylglycerol level after oral administration of the lipid emulsion
to rats and those on parametrial adipose tissue weight and liver
triacylglycerol concentration in mice fed the high fat diet. The effect
of the extract was faster acting in rat plasma triacylglycerol levels
than in the mouse parametrial adipose tissue weight and liver
triacylglycerol concentration (Figs. 2
, 4
and Table 2
). It is likely
that this discrepancy is explained by the different time frames of the
two studies. As shown in Figure 2
, the plasma triacylglycerol level was
traced for 4 h after oral administration of the lipid emulsion. On
the other hand, the parametrial adipose tissue weight and liver
triacylglycerol concentration were estimated after 8 wk of consuming
the high fat diet. During the longer experimental period, accumulation
of triacylglycerol in parametrial adipose tissue and liver may be
heavily affected by metabolism of plasma chylomicrons in other tissues
such as muscle, in which the high level of plasma chylomicrons is
metabolized preferentially compared with a lower level, by lipoprotein
lipase. It seems likely that such a preferential utilization of a high
level of chylomicrons may minimize the effect of the aqueous extract of
Platycodi radix.
It has been reported in clinical studies that a pancreatic lipase
inhibitor, orlistat (Ro 18–0647), prevented obesity and hyperlipidemia
after treatment for 12 wk through inhibition of fat absorption
(Drent et al. 1995a and 1995b
, Drent and Van der veen 1995
, Hauptman et al. 1992
). To clarify the
active substances of Platycodi radix, we examined the effects of the
major components of Platycodi radix, the inulin and saponin fractions,
on pancreatic lipase activity. Inulin had no effect on pancreatic
lipase activity, and high fat diet–induced obesity in mice was not
prevented by the administration of inulin.
It has been reported that various saponins isolated from foodstuffs
have antiobesity (Kawano-Takahashi et al. 1986
) or
hypolipidemic (Kimura et al. 1983
, Oakenfull et al. 1979
, Sirtori et al. 1977
, Yamamoto et al. 1975
) actions. We found that the crude saponin fractions
isolated from Platycodi radix strongly inhibited pancreatic lipase
activity. Therefore, it seems likely that the antiobesity and
hypolipidemic actions of the aqueous extract of Platycodi radix may be
attributed in part to its crude saponin fractions.
Experiments are now in progress to clarify the antiobesity action of pure saponin isolated from Platycodi radix in mice fed a high fat diet.
Manuscript received February 7, 2000. Initial review completed April 28, 2000. Revision accepted July 24, 2000.
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