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,2
*
Korea Institute of Bioscience and Biotechnology, KIST, Yusong, Taejon, 305-333, Korea, Departments of
Nutrition and Food Science and
**
Genetic Engineering, Kyungpook National University, Taegu, 702-701, and
Chungnam National University School of Pharmacy, Taejon, 305-764, Korea
2 To whom correspondence should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • hesperidin • naringin • HMG-CoA reductase • ACAT • tangerine peel extract • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). It is important to reduce excess
cholesterol to a level that is adequate for the maintenance of normal
body functions.
The regulation of plasma cholesterol levels involves factors that
influence both the extracellular and intracellular cholesterol
metabolism. The two key enzymes involved are
3-hydroxy-3-methyl-glutaryl-CoA
(HMG-CoA)3
reductase and acyl CoA:cholesterol O-acyltransferase (ACAT).
HMG-CoA reductase inhibitors are very effective in lowering plasma
cholesterol in most animal species including humans (Amin
1993
), and these inhibitors are now widely used in
hypocholesterolemic drugs (Lovastatin study groups I through IV
1993
). ACAT catalyzes the intracellular esterification of
cholesterol. ACAT is also involved in cholesterol absorption, hepatic
VLDL-cholesterol secretion, and cholesterol accumulation in the
vascular wall (Helgerud et al. 1981
, Suckling and Stange 1985
) by catalyzing cholesterol esterification. For
these reasons, ACAT inhibitors were used in test drugs as
cholesterol-lowering agents as well as antiatherosclerotic agents.
In recent years the use of HMG-CoA reductase inhibitors for
lowering plasma cholesterol levels has increased.
Some bioflavonoid compounds are associated with the prevention of
chronic diseases such as cancer and hyperlipidemia (Aboobaker et al. 1994
, Monforte et al. 1995
). Citrus fruits
contain various bioflavonoids. Among naturally occurring citrus
flavonoids, naringin and hesperidin were pharmacologically evaluated as
a potential anticancer agent (Aboobaker et al. 1994
) and
a potential anti-inflammatory agent (Emin et al. 1994
). We recently identified that naringin and hesperidin,
glycosylated citrus flavonoids, are two major bioflavonoids in
tangerine-peel extract. The present study was designed to examine
the effects of tangerine-peel extract and a mixture of these
bioflavonoids on the cholesterol metabolism of rats fed a
high-cholesterol diet.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Thirty male Sprague-Dawley rats weighing between 90 and 100 g
were purchased from Daehan Laboratory Animal Research Center Co.
(Chungbuk, Korea). The animals were individually housed in stainless
steel cages in a room with controlled temperature (20–23°C) and
lighting (alternating 12 h periods of light and dark) and fed a
pelletized commercial nonpurified diet for 6 d after arrival. They
were randomly divided into three groups (n = 10) and
fed a 1 g/100 g high cholesterol
diet4
with group 1 receiving a flavonoid mixture supplement (0.05 g naringin
+ 0.05 g hesperidin/100 g diet; Sigma Chemical Co. St. Louis,
MO), group 2 a tangerine-peel extract supplement (16.7 g/100 g
diet) prepared with ethanol, and group 3, no supplement. The rats were
given free access to water and food for 6 wk. The amount of
tangerine-peel extract mixed with the diet was based on an
equivalent of the total flavonoids in the hesperidin and naringin diet.
Dried tangerine peel weighing 6.7 kg was extracted with 80 L of 95%
ethanol for 24 h at 60°C. The extract was filtered and
concentrated using a high-capacity evaporator (EYELA Rotary vacuum
evaporator N-11; Tokyo Ridadidai Co., Ltd., Japan). The final weight of
the tangerine-peel concentrate was 1.7 kg with a density of 1.3
kg/L. Components of the tangerine-peel concentrate were: 100 g
of the tangerine-peel concentrate containing 39.1 g
H2O, 2.7 g crude protein, 1.8 g crude fat,
1.0 g crude ash, 20 g fructose, 16.5 g glucose, 8.6 g sucrose, 0.6 g hesperidin, 0.03 g naringin, and 9.67 g
other sugars. The hesperidin and naringin contents were analyzed by
HPLC (Rouseff 1988
).
The rats were given free access to food and distilled water. Every day for the last 5 d, feces were collected using metabolic cages and analyzed for fecal neutral sterols. The food consumption and weight-gain were measured every third day. At the end of the experimental period, the rats were anesthetized with Ketamine-HCl after withholding food for 12 h. Blood samples were taken from the inferior vena cava for the determination of plasma lipids. The livers were removed and rinsed with physiological saline. All samples were stored at -60°C until analyzed.
Plasma and hepatic lipids.
Plasma cholesterol concentrations and HDL-cholesterol
concentrations were determined using a commercial kit (Sigma) based on
a modification of the cholesterol oxidase method of Allain et al. (1974)
. The HDL-fractions were separated using a Sigma
kit based on the heparin-manganese precipitation procedure
(Warnick and Albers, 1978
). The plasma triglyceride
concentrations were measured enzymatically using a kit from Sigma
Chemical Co., a modification of the lipase-glycerol phosphate
oxidase method (McGowan et al. 1983
). The hepatic lipids
were extracted using the procedure developed by Folch et al. (1956)
. The dried lipid residues were dissolved in 1 mL of
ethanol for cholesterol and triglycerides assays. Triton X-100 and
sodium cholate solutions (in distilled H2O) were added to
200 µL of the dissolved lipid solution to produce final
concentrations of 5 g/L and 3 mmol/L, respectively. The hepatic
cholesterol and triglycerides were analyzed with the same enzymatic kit
used in the plasma analysis.
Fecal neutral sterols.
The fecal neutral sterols were determined by a simplified
micro-method developed by Czubayko et al. (1991)
. A
gas–liquid chromatograph was carried out with a Hewlett-Packard
gas chromatograph (Model 5809; Palo Alto, CA) equipped with a hydrogen
flame-ionization detector and using a Sac-5 capillary column (30 m
x 0.25 mm i.d., 0.25 µm film; Supelco Inc., Bellefonte, PA).
Helium was used as a carrier. Temperatures were set at 230°C for the
column (isothermal) and 280°C for the injector/detector temperature.
The internal standard used was 5
-cholestane (Supelco Inc.). The
daily neutral sterol excretion was calculated from the amount of
cholesterol, coprostanol, and coprostanone in each fecal sample.
HMG-CoA reductase and ACAT activities.
Microsomes were prepared according to Hulcher and Oleson (1973)
with a slight modification. Two grams of the liver
tissues were homogenized in 4 mL of an ice-cold buffer (pH 7.0)
containing 0.1 mol/L of triethanolamine, 0.02 mmol/L of EDTA and 2
mmol/L of dithiothreitol, pH 7.0. The homogenates were centrifuged
twice at both 10,000 x g and 12,000 x g for 10 min at 4°C. Next the supernatants were
ultracentrifuged twice at 100,000 x g for 60 min at
4°C. The resulting microsomal pellets were then redissolved in 1 mL
of a homogenation buffer for protein determination (Bradford 1976
) and finally analyzed for HMG-CoA reductase and ACAT
activities.
The HMG-CoA reductase activities were determined as described by
Shapiro et al. (1974)
with a slight modification of
using freshly prepared hepatic microsomes. The incubation mixtures (120
µL) containing microsome (100–150 µg) and 500 nmol of NADPH
(dissolved in a reaction buffer containing 0.1 mol/L of triethanolamine
and 10 mmol/L of EDTA) were preincubated at 37°C for 5 min. Next, 50
nmol of [14C]-HMG-CoA (specific activity; 2.1420
GBq/mmol; NENTM Life Science Products, Inc., Boston, MA) were added,
and the incubation was continued for 15 min at 37°C. The reaction was
terminated by the addition of 30 µL of 6 mol/L of HCl, and the
resultant reaction mixture was incubated at 37°C for a further 15 min
to convert the mevalonate into mevalonolactone. The incubation mixture
was centrifuged at 10,000 x g for 5 min, and the
supernatant was spotted on a Silica Gel 60 F254 TLC plate
with a mevalonolactone standard. The plate was developed in
bezene–acetone (1:1, vol/vol) and air-dried. Finally, the region
Rf 0.3–0.6 was removed by scraping using a clean razor
blade and its 14C radioactivity was determined using a
liquid scintillation counter (Packard Tricarb 1600TR; Packard
Instrument Company, Meriden, CT). The results were expressed as
picomoles mevalonate synthesized · min-1 ·
mg microsomal protein-1.
The ACAT activities were determined using freshly prepared hepatic
microsomes according to the method developed by Erickson et al. (1980)
as modified by Gillies et al. (1986)
. To
prepare the cholesterol substrate, 6 mg of cholesterol and 600 mg of
Tyloxapol (Triton WR-1339; Sigma) were each dissolved in 6 mL of
acetone, mixed well and completely dried in N2 gas. The
dried substrate was then redissolved in 20 mL of distilled water to a
final concentration of 300 µg of cholesterol/mL. Next, reaction
mixtures containing 20 µL of a cholesterol solution (6 µg
cholesterol), 20 µL of a 1 mol/L of potassium-phosphate buffer
(pH 7.4), 5 µL of 0.6 mmol/L bovine serum albumin, 50–100 µg of
microsomal fraction, and distilled water (up to 180 µL) were
preincubated at 37°C for 30 min. The reaction was then initiated by
adding 5 nmol of [14C]-Oleoyl CoA (specific activity;
2.0202 GBq/mmol, NENTM Life Science Products, Inc.) to a final volume
of 200 µL; the reaction time was 30 min at 37°C. The reaction was
stopped by adding 500 µL of an isopropanol–heptane mixture (4:1,
vol/vol), 300 µL of heptane and 200 µL of 0.1 mol/L potassium
phophate (pH 7.4), and the reaction mixture was allowed to stand at
room temperature for 2 min. Finally, an aliquot (200 µL) of the
supernatant was subjected to scintillation counting. The ACAT activity
was expressed as pmoles of cholesteryl oleate synthesized ·
min-1 · mg microsomal protein-1.
The inhibitory effects of hesperidin and naringin on HMG-CoA reductase and ACAT were also examined in vitro using rat microsomes as enzyme sources. Hesperidin and naringin, prepared in methanol, were separately added to the aforementioned mixtures in varying concentrations (25–2500 µmol/L) and then incubated at 37°C. The inhibitory activities of both HMG-CoA reductase and ACAT were measured to identify a one-half maximal inhibitory concentration of the inhibitor (IC50).
Statistical analysis.
All data were presented as the mean ± SE. Significant differences
among the groups were determined by one-way ANOVA using SSPS.
Duncan's multiple-range test was performed if differences were
identified between groups at = 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasma and hepatic lipids.
The supplementations of the naringin + hesperidin mixture and
tangerine-peel extract both significantly lowered the levels of
plasma and hepatic cholesterol and hepatic triglycerides (Table 1
).However, the plasma HDL-cholesterol and triglyceride concentrations
were not significantly different among the groups. The ratios of HDL to
total cholesterol were significantly higher in the two experimental
groups than in the control, whereas the opposite was true for the
atherogenic index.
|
The HMG-CoA reductase and ACAT activities in the mixed bioflavonoid
group and the tangerine-peel extract group were significantly lower
than those of the control group (Fig. 1
),The mixed bioflavonoids apparently inhibited liver cholesterol
biosynthesis and the esterification of hepatic cholesterol by 28.3 and
23.7%, respectively, while the tangerine-peel extract inhibited by
37.0 and 32.0%, respectively. However, when tested in vitro, neither
the bioflavonoid mixture nor tangerine-peel extract inhibited the
activities of HMG-CoA reductase and ACAT (IC50 < 1
mmol/L, not shown in figure).
|
The control group excreted about 250% as much neutral sterol as the
two experimental groups (P < 0.01, Fig. 2
).The mixture of flavonoids and tangerine-peel extract may have
increased the efficiency of the utilization of exogenous cholesterol,
since the cholesterol intake did not differ among groups. Since all
rats consumed a high-cholesterol diet, cholesterol constituted the
greatest proportions of the neutral sterol in feces, followed
consecutively by coprostanone and coprostanol. Cholesterol and
coprostanone excretions were both significantly higher in the control
than in the other two groups (P < 0.05, Fig. 2
).
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
yet do in hamsters, rabbits (Ma et al. 1986
) and humans
(Lovastatin study groups I through IV 1993
). This might
be due to differences in lipoprotein metabolism among animal species
(Tall 1993
). Rats lack the cholesteryl ester transfer
protein and are resistant to developing atherosclerosis due to
high-cholesterol feeding, while hamsters and rabbits are like
humans and are susceptible to atherosclerosis. Despite these
differences, rats can be used as an animal model to study cholesterol
metabolism if experimental conditions are appropriately controlled.
Dosages of HMG-CoA reductase inhibitors have to be very high to
exert a hypocholesterolemic response in rats (Amin et al.
1993
, Shand and West 1995
).
Tangerine-peel extract and the mixture of naringin and hesperidin
also inhibited the ACAT in this study. Inhibiting ACAT may also be a
possible therapeutic approach for decreasing plasma cholesterol
concentrations. For instance, the treatment of different species with
selective ACAT activity inhibitors has resulted in variable decreases
in plasma cholesterol (Marezetta 1994
, Williams et al. 1989
). However, the extent of the role of intestinal or
hepatic ACAT in the cholesterol metabolism of humans is still unclear
(Harris et al. 1990
).
This study has identified decreased plasma cholesterol and hepatic cholesterol concentrations and decreased fecal neutral sterols in rats supplemented with tangerine-peel extract and tangerine-peel flavonoids. These results suggest that, in high-cholesterol fed rats, intestinal ACAT activity and cholesterol absorption likely are not decreased by these supplements. Cholesterol biosynthesis was concomitantly reduced by these citrus bioflavonoids as indicated by the decreased hepatic HMG-CoA reductase activities. Since the cholesterol intake was about the same for all groups, the supplementation of these compounds seemed to promote an efficient utilization of dietary cholesterol, i.e., a possible increase of cholesterol uptake by tissues. The absorption of intestinal cholesterol was not decreased by these supplements as indicated by the lower amount of fecal neutral sterols compared to controls.
The combination of generally recognized concepts of hepatic cholesterol
homeostasis and our present findings would suggest that several
mechanisms underlie the metabolic effects of these bioflavonoids. Our
results suggest that citrus bioflavonoids reduce cholesterol
biosynthesis through the inhibition of hepatic HMG-CoA reductase
and ACAT, resulting in lower hepatic cholesterol level. Accordingly,
the plasma cholesterol concentration falls, resulting in an increased
absorption of dietary cholesterol and contributing to a simultaneous
decrease in fecal cholesterol excretion in citrus
bioflavonoid-supplemented rats. HMG-CoA reductase activity is
normally decreased by a high-cholesterol diet, whereas the
activities of hepatic ACAT and cholesterol-7--hydroxylase are
increased (Björkhem 1985
, Field et al.
1982
). However, the presence of citrus bioflavonoids in a
high-cholesterol diet significantly decreased the activities of
both hepatic HMG-CoA reductase and ACAT. Reduced ACAT activity may
lead to less cholesteryl ester being available for VLDL packing,
thereby resulting in a reduction of VLDL secretion from the liver, as
suggested by Carr et al. (1992)
. Nevertheless, the
plasma triglyceride levels were not significantly different among the
groups in this study, suggesting that the hepatic ACAT inhibition did
not affect VLDL secretion. Another hypothesis is that the potential
increase of hepatic cholesterol uptake by LDL receptors could
accelerate cholesterol catabolism in citrus
bioflavonoid-supplemented rats. Interestingly, neither naringin nor
hesperidin inhibited HMG-CoA reductase or ACAT in vitro, suggesting
that both may undergo some structural changes to become active either
in the intestine or another organ. A further conjecture is that the
gene expression of these enzymes could be inhibited by naringin or
hesperidin in high-cholesterol-fed rats. More studies are needed, with
various animal models, to explain the inhibitory activities of citrus
bioflavonoids on HMG-CoA reductase and ACAT in vivo as well as
their hypocholesterolemic effects.
|
FOOTNOTES |
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3 Abbreviations used: ACAT, acyl CoA:cholesterol
O-acyltransferase (EC 2.3.1.26); HMG-CoA,
3-hydroxy-3-methyl-glutaryl-CoA; IC50, one-half maximal
inhibitory concentration of inhibitor.
4 Composition of diets: Each kilogram of diet
contained 10 g cholesterol, 200 g casein, 3 g
DL-methionine, 150 g corn starch, 50 g cellulose,
50 g corn oil, 2 g choline bitartrate, 35 g AIN-76
mineral mix (American Institute of Nutrition 1977
), and
10 g of vitamin mixture, which furnished per kilogram of diet:
thiamin HCl, 0.6; riboflavin, 0.6; pyridoxine HCl, 0.7; nicotinic acid,
0.003; D-calcium pantothenate, 0.0016; folate, 0.2;
D-biotin, 0.02; cyanocobalamin, 0.001; retinyl palmitate
premix, 0.8; DL- tocopheryl acetate, premix, 20;
cholecalciferol, 0.0025; menaquinone, 0.05; antioxidant, 0.01; sucrose,
finely powdered, 972.8, 0.5 g naringin and hesperidin or 167 g tangerine-peel extract.
Manuscript received September 5, 1998. Initial review completed October 19, 1998. Revision accepted February 23, 1999.
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