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Division of Food Chemistry, National Institute of Health Sciences, Osaka Branch, 1-1-43, Hoenzaka, Chuo-ku, Osaka, 540-0006, Japan
2To 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: • gymnemic acids • fecal excretion • cholesterol • bile acids • gymnemagenin • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
This plant has been used in Ayurvedic medicine (Suttisri et al. 1995
) for the treatment of diabetes mellitus (Dixit and Pandey 1984
, Gupta 1961
, Jain and Sharma 1967
, Reddy et al. 1989
), eye diseases
(Dixit and Pandey 1984
), snake bite (Nagaraju and Rao 1990
, Shanmugasundaram et al. 1983
) and
mycosis of toes (Reddy et al. 1989
).
Gymnema sylvestre leaves or their extracts have been
widely used as health foods in tea bags, tablets, beverages and
confectioneries in recent years in Japan. The users of these health
foods often expect weight reduction or improvement of diabetes because
of their ability to suppress the taste of sweetness and inhibit glucose
absorption (Nakamura 1988
, Ueno 1997
).
The constituents which effectively work on diabetes in Gymnema
sylvestre leaves are gymnemic acids (Murakami et al. 1996
, Stöcklin 1969
) and conduritol A
(Fijimoto et al. 1991
, Miyatake et al. 1993
, Yamashita et al. 1991
). Gymnemic acids are
saponins with a triterpenoid structure (Fig. 1
).More than 10 kinds of gymnemic acid and related compounds were isolated
(Suttisri et al. 1995
). Murakami et al. (1996)
reported the contents of each gymnemic acid in leaves to
be 0.0050–0.012%. Because of the difficulty and tediousness of the
isolation of each gymnemic acid, the extract of Gymnema sylvestre
leaves was used to study the physiological effects of gymnemic acids.
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). They suppress taste sensitivity to sweetness
(Diamant et al. 1965
, Kurihara 1969
,
Warren and Pfaffmann 1959
), inhibit intestinal glucose
absorption (Hirata et al. 1992b
, Yoshioka 1986
) and lower plasma glucose (Abe 1993
,
Hirata 1992
, Hirata et al. 1992a
,
Kurata 1987
, Murakami et al. 1996
,
Yoshioka et al. 1985
) and insulin levels (Abe 1993
, Hirata 1992
, Hirata et al. 1992a
). Gymnemic acids change glucose utilization patterns and
enzyme activities in experimentally-induced diabetic animals
(Kato 1994
, Shanmugasundaram et al. 1983
), inhibit glucose-stimulated gastric inhibitory
peptide secretion in rats (Fushiki et al. 1992
).
Gymnemic acids also have an antieurodontic effect (Miyoshi et al. 1987
). There are few reports about the effect of gymnemic
acids on a lipid metabolism. Terasawa et al. (1994)
reported that the serum triglyceride and total cholesterol
concentration tended to be slightly lower in obese but not in lean rats
receiving long-term administration of a water extract of
Gymnema sylvestre. Ikemitsu (1990)
reported
that the intraperitoneal injection of gymnemic acids at a dose of 25
mg/kg had no effect on serum concentrations of total cholesterol, free
cholesterol, triglycerides or free fatty acids.
Saponins are defined as "any of numerous plant glycosides
characterized by foaming in water, and by producing hemolysis when
water solutions are injected into the bloodstream" (Price et al. 1987
). Generally, saponins have characteristic properties
such as hemolytic activity, cholesterol-binding properties and
bitterness (Price et al. 1987
).
The decreases in serum and hepatic cholesterol by ingestion of various
saponins were reported in animals fed a high-cholesterol diet.
Previous reports showed a cholesterol-lowering effect with an
unknown saponin in chickens (Griminger and Fisher 1958
)
and with quillaja saponin and soybean saponin in rats (Oakenfull et al. 1984
), and with Ginseng saponin (Yamamoto et al. 1983b
). Story et al. (1984)
indicated the
lowering of serum and hepatic cholesterol and triglycerides alfalfa
saponin in hyperlipidemic persons.
Changes in fecal steroid excretion due to ingestion various saponins
were reported. Elevated fecal excretion of neutral steroids but not
bile acids was reported in rats administered soybean saponin
(Oakenfull et al. 1984
). Elevations in fecal excretion
of neutral steroids and bile acids were reported in rats administered
quillaja saponin (Oakenfull et al. 1984
), a saponin of
unknown origin (Oakenfull et al. 1979
) and Ginseng
saponin (Yamamoto et al. 1983a
), in pigs administered
soapwort saponin (Topping et al. 1970
), in healthy men
given a synthetic saponin tiqueside (Harris et al. 1997
), and in monkeys administered alfalfa saponins
(Malinow et al. 1981
). However, Calvert et al. (1980)
reported that soybean saponins do not affect fecal
excretion of neutral steroids and bile acids. Malinow et al. (1981)
reported that alfalfa saponins decreased the percentage
distributions of fecal deoxycholic acid (DCA) and lithocholic acid
(LCA). The increase in percentages of the fecal primary bile acids by a
saponin of unknown origin (Oakenfull et al. 1979
) and
those of primary bile acids and chenodeoxycholic acid (CDCA)-derived
bile acids by quillaja saponin and soybean saponin (Oakenfull et al. 1984
) were reported in rats.
Malinow et al. (1981)
found inhibition of cholesterol
absorption in monkeys by alfalfa saponins. Tiquesides inhibited
cholesterol absorption but not bile acid absorption in hamsters
(Harwood et al. 1993
). Tiquesides also inhibited
cholesterol absorption in hyperlipidemic patients (Harris et al. 1997
). Bile acids form large mixed micelles with soybean
saponins, quillaia saponins and soapwort saponins and aggregate in
aqueous solutions, which cause a decrease in cholate absorption in the
small intestine (Oakenfull 1986
, Sidhu and Oakenfull 1986
). Fenugreek saponins reduce bile acid absorption
in the rat everted sac (Stark and Madar 1993
). Thus, the
effects of saponins on the reduction in serum and hepatic lipids
concentrations and the increase in fecal neutral steroids and bile acid
excretion seem to be related to the interaction of saponins with
cholesterol and bile acids in the intestinal tract.
Gymnemic acids are saponins that have a bitter and astringent taste and
exert hemolytic activity when administered intravenously to rats
(Yoshioka et al. 1989
). In spite of the possibility that
gymnemic acids may affect cholesterol metabolism as described above,
there has been no information about their effect on fecal steroid
excretion to date. Therefore, we investigated the effect of gymnemic
acids on fecal steroid excretion in rats. Because very intricate and
tedious steps are required to isolate individual gymnemic acids, we
used the three kinds of extract from G. sylvestre leaves
with different gymnemic acids concentrations in this study.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cholesterol, 5
-cholestane, 5ß-cholanic acid, lithocholic acid
(LCA),3
deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), hyodeoxycholic
acid (HDCA) and cholic acid (CA) were purchased from GL Sciences Inc.
(Tokyo, Japan). Coprostanone was purchased from Sigma Chemical Co.
(St. Louis, MO). Coprostanol, nordeoxycholic acid (norDCA), 5-Cholenic
acid 3ß-ol, isolithocholic acid (ILCA), isodeoxycholic acid (IDCA),
murodeoxycholic acid (MDCA), 6-keto lithocholic acid (6KLCA),
12-keto lithocholic acid (12KLCA), -muricholic acid (MCA),
ß-muricholic acid (ßMCA), 
-muricholic acid (MCA), 7-keto
deoxycholic acid (7KDCA), and 12-keto chenodeoxycholic acid (12KCDCA)
were purchased from Steraloids, Inc. (Wilton, NH). Derivative reagents
for gas chromatography (GC) analysis, hexafluoroisopropanol and
trifluoroacetic acid anhydride, were purchased from GL Sciences Inc.
Ion-exchanged and redistilled water or distilled water for HPLC was
used throughout the experiments. Acetonitrile and ethanol used for
analysis were of HPLC grade. Other reagents were of the analytical
grade.
Choloylglycine hydrolase (EC 3.5.1.24, 1500 unit) was purchased from Sigma Chemical Co. Commercial kits for the determination of serum lipids were purchased from Wako Pure Chemical Industries (Osaka, Japan). Sep-pakR C18 plus cartridge column was purchased from Waters Corporation (Milford, MA). Fused silica capillary columns DB-210 (0.25 mm i.d. x 30 m, film thickness 0.25 µm or 0.5 µm) were purchased from J&W Scientific (Folsom, CA). HPLC column Wakosil 5C18 HG (4.6 mm i.d. x 150 mm) was purchased from Wako Pure Chemical Industries.
Gymnema sylvestre extract (GSE, concentrations of gymnemic acids: 94 mg/g as gymnemic acids II or 58.87 mg/g as gymnemagenin), and G. sylvestre acid precipitates (GSA, concentrations of gymnemic acids: 258 mg/g as gymnemic acid II or 161.6 mg/g as gymnemagenin) were donated by Dai-Nippon Meiji Sugar Mfg. Co., Ltd. (Tokyo, Japan). Gymnemagenin (purity 90.4%) and G. sylvestre column fractionate (GSF, concentrations of gymnemic acids: 580 mg/g as gymnemic acids II or 363.3 mg/g as gymnemagenin) were donated by Maruzen Pharmaceuticals Co., Ltd. (Onomichi, Japan). Briefly, GSE is the ethanol/water extract of G. sylvestre leaves, GSA is the acid precipitate of GSE, and GSF is the column fractionate of GSA. Formulations of GSE, GSA and GSF were prepared before use. For each sample, 0.05–1.0 g was weighed, dissolved with hot water and adjusted to 5 mL. Each formulation was administered to rats at the dose of 5 mL/kg body weight.
Apparatus.
Shimadzu Model GC-14A gas chromatograph (Kyoto, Japan), equipped with a flame-ionization detector (FID), autosampler AOC-17A, and integrator C-R4A, was used for the determination of fecal steroids. Hewlett-Packard HP Series 1100 HPLC (Palo Alto, CA), equipped with a degasser G1322A, binary pump G1312A, thermostatted column compartment G1330A, autosampler G1329A, variable wavelength detector G1314A, and ChemStation, was used for the determination of gymnemagenin analysis. A Hitachi U-3210 spectrophotometer (Tokyo, Japan) was used for serum and hepatic lipid analysis.
Animals.
All the procedures involving animals were conducted in compliance with Japanese law (Bulletin of Prime Minister's Office No. 6, March 1980) and guidelines established by the National Institute of Health Sciences. Male rats of the Wistar-ST strain (4-wk-old) were purchased from Japan SLC, Inc. (Shizuoka, Japan) and kept in an air-conditioned room (23 ± 1°C, 50–60% humidity) lighted for 12 h/d (0700 to 1900). Rats were given free access to a commercial nonpurified diet (F-2, Funahashi Farm, Chiba, Japan) and water throughout the experiments. Composition of the diet was: moisture 7.67%, protein 20.22%, lipids 4.94%, fiber 3.66%, minerals 5.24%. The same lot of nonpurified diet was used in all experiments. The cholesterol concentration in the diet was 160.2 ± 15.8 µg/g (mean ± SEM, n = 6, determined by GLC), and no saponin was detected (n = 6, determined by HPLC). Rats weighing 134–154 g were used in Experiments 1–3, and those weighing 175–200 g were used in Experiment 4. Each group in Experiments 1–3 contained five rats, in Experiment 4, four rats.
In Experiment 1, GSE was administered orally at the doses of 0.1, 0.2, 0.5 and 1.0 g/kg for 22 d. In Experiment 2, GSA was administered orally at the doses of 0.05, 0.1, 0.5 and 1.0 g/kg for 22 d. In Experiment 3, GSF was administered orally at the doses of 0.05, 0.1, 0.5 and 1.0 g/kg for 22 d. Each sample was administered to individual rats between 0900 and 1100 h each day. Rats of the control group were administered water at 5 mL/kg body weight. Feces were collected on d 18–19 by housing each rat in a metabolic cage. Rats were food-deprived overnight on d 21. On d 22, rats were anesthetized with diethylether, and blood was collected by heart puncture. The liver was excised immediately after bleeding. To examine the effect of gymnemic acids on fecal steroid excretion more precisely, a single dose of GSF was administered to rats in Experiment 4. Four rats were administered GSF orally at a dose of 1.0 g/kg, and the fecal excretion patterns of steroids and gymnemic acids were investigated. Feces were collected on 0–1, 1–2 and 2–4 d from rats housed individually in metabolic cages.
Analysis of serum and hepatic lipids.
Serum concentrations of total cholesterol and high density lipoprotein-cholesterol were determined using the commercial kits. A portion of liver was homogenized with chloroform/methanol solution (2:1, vol/vol) and filtered. Adequate amounts of the filtrate (lipid extract) were mixed with sodium cholate solution and evaporated completely under the nitrogen stream, and total cholesterol in residues was determined using the commercial kits.
Analysis of fecal steroids.
Collected feces were dried at 60°C overnight and ground into a
powder. Fecal neutral steroids and bile acids were analyzed by the
methods of Grundy et al. (1965)
and Setchell et al. (1983)
with some modifications.
To a portion of the ground dried feces, 5-cholestane, 5ß-cholanic
acid and norDCA were added as internal standards. Lipids in the feces
were extracted twice with ethanol and chloroform/methanol mixture (2:1,
vol/vol), respectively, by sonication and reflux. Fractions of
chloroform/methanol mixture and ethanol were collected and evaporated
completely. Neutral lipids were extracted with n-hexane after the
saponification with methanolic potassium hydroxide solution. Neutral
steroids were determined by GC–FID using 5-cholestane as an
internal standard. After the removal of methanol by evaporation, the
remaining fraction was neutralized with phosphoric acid, and applied to
a Sep-pakR C18 plus cartridge column
(Whitney and Thaler 1980
) that had been pre-washed
with methanol and water. The column was washed with water, and bile
acids were eluted with methanol. The eluate was evaporated completely,
redissolved with methanol and divided into two fractions. One portion
of the divided sample was acidified with concentrated HCl, and the free
bile acids were extracted with diethylether three times. For the other
portion of the fraction, methanol was evaporated, and deconjugation was
performed using choloylglycine hydrolase (EC 3.5.1.24) (Takikawa et al. 1982
). Total bile acids were extracted with diethylether
after the acidification with concentrated HCl. Free and total bile
acids were derivatized to hexafluoroisopropyl ester-trifluoroacetyl
(HFIP-TFA) derivatives (Imai and Tamura 1976
) and
determined by GC–FID using 5ß-cholanic acid and norDCA as an
internal standard.
GC conditions for steroid analysis were as follows: column, DB-210
(film thickness 0.25 µm for neutral steroids, 0.50 µm for bile
acids); carrier gas, He 1.5 mL/min; column temperature, 60°C (2 min)
10°C/min 180°C 5°C/min 230°C for neutral
steroids, 60°C (2 min) 10°C/min 235°C for bile acids;
injection port and detector temperature, 250°C; detector, FID;
injection method, splitless; injection volume, 2 µL.
Analysis of gymnemagenin.
Gymnemic acids precipitate in acidic solution (Suttisri et al. 1995
). Therefore, we analyzed gymnemic acids as gymnemagenin,
the aglycone of gymnemic acids, by its precipitation in the acidic
solution followed by alkaline and an acid hydrolysis (Nakamura et al. 1997
, Yokota et al. 1994
). Gymnemic acid
content can be calculated as gymnemic acid II by multiplying by a
coefficient of 1.5966 (809.00 molecular weight of gymnemic acid
II)/506.70 (molecular weight of gymnemagenin) to the value of
gymnemagenin (Yokota et al. 1994
).
For analysis of fecal gymnemagenin in aqueous fractions, a
portion of the ground dried feces was extracted with boiling water,
filtered by suction and cooled to ambient temperature. The filtrate was
acidified to pH 1 with concentrated HCl and centrifuged at 9,391
x g for 15 min at 4°C. The precipitate was dissolved in ethanol
and filtered. The filtrate was evaporated and redissolved with 50%
ethanol, and alkaline and acid hydrolyses were performed (Yokota et al. 1994
). The hydrolyzed matter was adjusted to pH 7.5–8.5
and one-tenth was applied to a Sep-pakR C18
plus cartridge column pre-washed with methanol, ethanol and water.
The column was washed with water, and gymnemagenin was eluted with
ethanol. Gymnemagenin content was determined by HPLC (Yokota et al. 1994
). Urinary and the serum gymnemagenin was determined by
the above method after acidification to pH 1 with concentrated HCl.
For the determination of gymnemagenin in the lipid-soluble fraction, a portion of the ground dried feces was extracted with chloroform/methanol mixture (2:1, vol/vol), and neutral lipids were removed with n-hexane by the method described in the fecal steroid analysis section. The remained aqueous layer was centrifuged at 9,391 x g for 15 min at 4°C. Content of the gymnemic acids was determined as gymnemagenin by the method described above.
HPLC conditions for gymnemagenin analysis were as follows:
column, Wakosil 5C18 HG; mobile phases, (A)
acetonitrile/water/phosphoric acid 800:200:1 (vol/vol/vol), (B)
water/phosphoric acid 1000:1 (vol/vol); a gradient program, (A) 25% (0
min) 75% (20 min) 100% (21–25 min) 25% (26 min); column
oven temperature, 40°C; detector, UV 
213 nm; injection volume, 20
µL.
Statistics.
Data are expressed as means ± SEM. Statistical analyses were performed by one-way ANOVA with subsequent Dunnett's multiple comparison test using the "Statlight" program (Yukms, Tokyo, Japan). Dose-response was examined by linear regression analysis after one-way ANOVA. Probability values lower than 0.05 were accepted as significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One rat administered 1.0 g/kg of GSA and one administered 1.0 g/kg of
GSF died on d 1–2. Hypertrophy of the stomach wall was observed in two
other rats administered 1.0 g/kg of GSF, and those had smaller body
weight gains. Body weight gain and food intakes decreased as the doses
of GSA and GSF increased (Table 1,
P < 0.01), whereas GSE had no effect. A significantly
lower than control body weight gain was observed in rats administered
1.0 g/kg of GSA and 0.5 or 1.0 g/kg of GSF. Significantly lower food
intakes were observed in rats administered 1.0 g/kg of GSA
(P < 0.05) and more than 0.1 g/kg of GSF compared to
controls. No significant differences in relative liver weights or dry
fecal weights were due to the administration of GSE, GSA or GSF. GSE,
GSA and GSF did not affect serum total and HDL cholesterol (data not
shown). Hepatic cholesterol was significantly lower than control only
in the rats administered 0.5 or 1.0 g/kg of GSF.
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Because GSF contains the highest amounts of gymnemic acids, it was used
to investigate in detail the effects of gymnemic acids on fecal steroid
excretion. Excretions of cholesterol, coprostanone and total neutral
steroids (total of cholesterol, coprostanol and coprostanone, which are
the intestinal metabolites of cholesterol) increased in
dose-dependent manners (Table 2,
P < 0.01). Significantly greater excretions of
cholesterol and total neutral steroids and a significantly lower ratio
of coprostanol/cholesterol were observed in the rats administered
at least 0.1 g/kg of GSF (P < 0.05). Excretion of
gymnemagenin in lipid-soluble and aqueous fractions increased in a
dose-dependent manner (P < 0.001). Most of the
fecal gymnemagenin was present in the lipid-soluble fraction rather
than in the water-soluble fraction. No gymnemagenin was detected in
the urine and serum even in rats administered 1.0 g/kg.
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P < 0.05). Excretion of total bile acids, DCA, 6KLCA
and the ratio of CA-derived bile acids/CDCA-derived bile acids
increased (P < 0.05) but that of 5-cholenic acid
3ß-ol decreased (P < 0.01) in a dose-dependent
manner.
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A significant increase in the excretion of coprostanol, cholesterol and
total neutral steroids (Table 4,
P < 0.01) and a significant decrease in the ratio of
coprostanol/cholesterol (P < 0.05) were observed only
from d 0–1 after administration of GSF. Within 3 d, 12.6% of the
administered gymnemic acids were found as gymnemagenin. More than 84%
of all gymnemagenin was found within 1 d, and more than 84.7%
existed in the lipid-soluble fraction throughout the experimental
period. Gymnemagenin was not detected in urine during the 4 d
after administration.
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).Excretion of total bile acids (P < 0.01) and 12KLCA
(P < 0.01), the ratio of CA-derived bile
acids/CDCA-derived bile acids (P < 0.01) and primary
bile acids (P < 0.05) increased, but LCA excretion
decreased (P < 0.05) at d 0–1 compared to baseline
(before administration). Ratios of free bile acids were 88.4–96.3%,
and there was no significant change during the experimental period.
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Positive correlations were detected between fecal gymemagenin and fecal
cholesterol (Table 6,
P < 0.01), total neutral steroids (P
< 0.01), total bile acids (P < 0.05) and CA/CDCA
ratios (P < 0.05) in rats that underwent both repeated
(Experiment 3) and single administration (Experiment 4) of GSF.
Positive correlations were detected between fecal gymnemagenin and
coprostanone (P < 0.001) or DCA (P < 0.01) in rats administered repeatedly (Experiment 3), while there were
positive correlations between fecal gymnemagenin and coprostanol
(P < 0.01) or 12KLCA (P < 0.001) in
rats administered a single dose of GSF (Experiment 4). No significant
correlations were detected for other factors.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Gupta 1961
, Jain and Sharma 1967
, Reddy et al. 1989
, Suttisri et al. 1995
). In spite of the cholesterol-binding properties of
saponins, the effects of gymnemic acids on cholesterol metabolism were
not elucidated. The present study demonstrated for the first time that
a high dose of gymnemic acids increases fecal excretion of cholesterol
and CA-derived bile acids in rats.
Body weight gain and food intakes decreased in a dose-dependent
manner (P < 0.01) in the rats administered GSA and GSF
(Table 1)
. The decrease in body weight gain may have been due to the
decrease in food intake. Marquet et al. (1997)
considered that the amphiphilic characteristics of the molecules were
responsible for their deleterious effects on the digestive tract. So,
the hypertrophy of stomach wall in the rats administered 1.0 g/kg of
GSF suggests that gymnemic acids may irritate the epithelium of the
gastrointestinal tract. On the contrary, relative weight of the liver
and fecal dry weight were not affected (Table 1)
.
There were no effects on the levels of serum total and HDL cholesterol
due to gymnemic acids although hepatic total cholesterol was
significantly lower in the rats administered 0.5 or 1.0 g/kg of GSF
compared to the control group (data are not shown). These results may
be caused by a normal diet (cholesterol concentration was 0.02%). In
animals fed a high cholesterol diet, ingestion of various saponins
decreases serum and hepatic cholesterol (Griminger and Fisher 1958
, Oakenfull et al. 1984
, Yamamoto et al. 1983b
).
A high dose of GSF significantly changed fecal steroid excretions in
rats that underwent repeated or single administrations (Tables 2
3
4
5)
.
Dose-dependency was observed in the increase of fecal excretion of
cholesterol, total neutral steroids, DCA, 6KLCA, total bile acids and
ratio of CA-derived bile acids/CDCA-derived bile acids
(P < 0.05) and in the decrease of fecal
coprostanol/cholesterol ratio and fecal 5-cholenic acid 3ß-ol
excretion (P < 0.01) on d 18–19 in rats administered
daily for 22 d (Tables 2
3)
. Changes in fecal steroid excretion
occurred within 1 d after GSF ingestion. There were significant
increases in fecal excretion of coprostanol, cholesterol, total neutral
steroids, 12KLCA, total bile acids, fecal CA-derived bile
acids/CDCA-derived bile acids ratio and primary bile acids and a
significant decrease in the fecal coprostanol/cholesterol ratio in rats
from d 0–1 after administration of GSF 1.0 g/kg compared to baseline
(Tables 4
and 5)
. Coprostanol is a major intestinal metabolite of
cholesterol whereas DCA and 12KLCA are major secondary bile acids
derived from CA in rats. The increase in fecal total bile acids
excretion seems to be due to the increase in CA-derived bile acids
excretion. These results indicate that a high dose of GSF increases
fecal excretion of cholesterol and CA-derived bile acids.
Sauvaire et al. (1991)
indicated that the fenugreek
saponins were in part (about 57%) hydrolyzed to sapogenin in the
digestive tract of diabetic dogs. It is likely that the fecal
gymnemagenin of rats in this study reflects the unchanged gymnemic
acids because the hydrolysis or degradation of gymnemic acids may also
occur in the digestive tract.
Changes in fecal steroid excretion seem to be compatible with the
increase of fecal gymnemagenin. Significant correlations were detected
between fecal gymnemagenin and fecal excretion of cholesterol,
coprostanone or coprostanol (intestinal metabolites of cholesterol),
total neutral steroids, DCA or 12KLCA (CA-derived bile acids), total
bile acids, and ratios of fecal coprostanol/cholesterol and CA/CDCA
(P < 0.05) in rats underwent repeated or single
administration of GSF (Table 6)
. These correlations strongly suggest
than interactions of gymnemic acids with steroids, especially with
cholesterol and CA-derived bile acids, occur in the intestinal
tract of rats.
The contribution of gymnemic acids to the change in fecal steroid
excretion seems to be the same in lipid-soluble and aqueous
fractions (Table 6)
. The fact that most of the gymnemagenin existed in
the lipid-soluble fraction (Tables 2
and 4)
indicates that gymnemic
acids might have an affinity to bind cholesterol and CA-derived
bile acids rather than CDCA-derived acids and form the
water-insoluble complexes. Changes in steroid excretion by GSF
administration are acute (Tables 4
and 5)
. Therefore, the increase in
fecal excretion of cholesterol and CA-derived bile acids due to GSF
administration might be due to an interruption with the formation of
micelles that contain cholesterol and bile acids in the gut and the
following interference with absorption of cholesterol or reabsorption
of bile acids.
We previously investigated the contents of gymnemagenin in health foods
in Japan (Nakamura et al. 1997
). Gymnemagenin intake
from health foods was estimated as less than 1.23 mg/kg based on the
assumption that the body weight of human is 50 kg (Nakamura et al. 1997
). A significant increase in fecal neutral steroids
excretion was observed in the rats administered more than 0.1 g/kg of
GSF (Table 3)
, i.e., more than 36.33 mg/kg as gymnemagenin. Because of
the bitter and astringent taste and sweetness-suppressing effect of
gymnemic acids or G. sylvestre leaves' extract, it might be
impossible to take a dose of gymnemic acids sufficiently high to
increase fecal excretion of neutral steroids (more than 36.33 mg/kg as
gymnemagenin).
We examined the effect of gymnemic acids on fecal steroid excretion in rats fed a normal diet. Different results may be obtained in hypercholesterolemic rats. Further studies including examination of the toxic effects of gymnemic acids are needed to clarify the effect of gymnemic acids on cholesterol metabolism.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: CA, cholic acid; CDCA,
chenodeoxycholic acid; DCA, deoxycholic acid; FID, flame-ionization
detector; GC, gas chromatography; GSA, Gymnema sylvestre acid
precipitate; GSC, Gymnema sylvestre column fractionate; GSE, Gymnema
sylvestre extract; HDCA, hyodeoxycholic acid; HFIP-TFA,
hexafluoroisopropyl ester-trifluoroacetyl; IDCA, isodeoxycholic acid;
ILCA, isolithocholic acid; 6KLCA, 6-ketolithocholic acid; 12KLCA,
12-ketolithocholic acid; 7KDCA, 7-ketodeoxycholic acid; 12KCDCA,
12-ketochenodeoxycholic acid; LCA, lithocholic acid; MDCA,
murodeoxycholic acid; MCA, -muriocholic acid; ßMCA,
ß-muricholic acid; MCA, -muricholic acid; norDCA,
nordeoxycholic acid.
Manuscript received July 20, 1998. Initial review completed September 23, 1998. Revision accepted February 23, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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