Life Sciences 74 (2004) 1621 – 1634

www.elsevier.com/locate/lifescie

Naringin alters the cholesterol biosynthesis and antioxidant

enzyme activities in LDL receptor-knockout mice under
cholesterol fed condition
Hye-Jin Kim a, Goo Taeg Oh b, Yong Bok Park c, Mi-Kyung Lee d,
Hyun-Ju Seo a, Myung-Sook Choi a,*
a
Department of Food Science and Nutrition Kyungpook National University, 1370 Sank-Yuk Dong Puk-Ku,
Daegu, 702-701, South Korea
b
Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yusong, Daejon,
305-600, South Korea
c
Genetic Engineering, Kyungpook National University, Daegu, 702-701, South Korea
d
Food and Bio-Industry Research Institute, Kyungpook National University, Daegu, 702-701, South Korea

Received 23 September 2002; accepted 21 August 2003

Abstract

The purpose of the current study was to evaluate the lipid lowering and antioxidant capacity of naringin
in LDL receptor knockout (LDLR-KO) mice fed a cholesterol (0.1 g/100 g) diet. As such, naringin or
lovastatin (0.02 g/100 g) was supplemented in a cholesterol diet for 6 weeks. The naringin and lovastatin
supplementation significantly lowered the plasma total cholesterol level compared to the control group. The
plasma and hepatic triglyceride level was only lowered by the lovastatin supplement, while the hepatic
cholesterol content was lowered by both the naringin and lovastatin supplements compared to the control
group. The hepatic HMG-CoA reductase activity was significantly lower in the naringin and lovastatin
supplemented groups than in the control group, whereas the ACAT activity was unaffected. The excretion of
total sterol was significantly higher in the naringin and lovastatin groups compared to the control group due
to significant changes in the acidic and neutral sterol, respectively. When comparing the hepatic antioxidant
enzyme activities, the superoxide dismutase, catalase, and glutathione reductase activities were all
significantly higher in the naringin-supplemented group than in the control group, while only the lovastatin
supplement increased the glutathione reductase activity. Accordingly, the current results confirmed that

* Corresponding author. Tel.: +82-53-950-6232; fax: +82-53-950-6229

E-mail address: mschoi@knu.ac.kr, (M.-S. Choi).

0024-3205/$ - see front matter D 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2003.08.026
1622 H.-J. Kim et al. / Life Sciences 74 (2004) 1621–1634

naringin lowers the plasma cholesterol level via the inhibition of hepatic HMG-CoA reductase activity and
increases the excretion of fecal sterol. Naringin was also found to improve the activities of hepatic
antioxidant enzymes against oxidative stress in a hypercholesterolemic animal model, i.e. cholesterol-fed
LDLR-KO mice.
D 2003 Elsevier Inc. All rights reserved.

Keywords: Naringin; Lovastatin; LDLR-KO; Cholesterol metabolism; Antioxidant enzyme activity

Introduction

Flavonoids are a group of naturally occurring compounds ubiquitous in the plant kingdom and
known to have a strong antioxidant effect. As such, a number of previous studies have investigated
the nature of the antioxidant capacities of bioflavonoids for treating various diseases. The flavonoid
glycoside naringin, a ‘‘bitter principal’’, is the major flavonoid present in grapefruits and
constitutes up to 10% of the dry weight (Ishii et al., 1996). Naringin has already been evaluated
as a potential anticancer (Aboobaker et al., 1994) and hypolipidemic (So et al., 1996) agent. It
exhibits antiexudative activity (Lambev et al., 1980), yet no mutagenic activity (Fisher, 1982). The
current authors also reported on the cholesterol-lowering effect of naringin via the inhibition of
HMG-CoA reductase in high-cholesterol fed (Shin et al., 1999) or high-fat and high-cholesterol fed
rats (So et al., 1996), and the plasma vitamin E, sparing effect of naringin in high-cholesterol
fed rabbits (Jeon et al., 2001). However, no studies have yet been performed using transgenic
animals.
Cholesterol appears to play a key role in regulating apo B lipoprotein secretion by the liver
(Thompson et al., 1996), and a strong correlation between the rate of cholesterol synthesis and apo B
lipoprotein production has been demonstrated in normal subjects (Watts et al., 1995). Homozygote
familial hypercholesterolemia subjects exhibiting increased cholesterol biosynthesis also exhibit
increased apo B lipoprotein production (Bilheimer et al., 1979). Hepatic HMG-CoA reductase is
the rate-limiting enzyme in the cholesterol biosynthetic pathway and its inhibitors are very effective
in lowering plasma cholesterol in most animal species, including humans (Amin et al., 1993), as
such, they are now widely used in hypocholesterolemic drugs (Lovastatin study groups I through IV,
1993).
LDLR-KO mice were chosen to confirm the previous findings on the effects of naringin associated
with the inhibition of HMG-CoA reductase in rats, as the cholesterol biosynthesis and plasma
cholesterol concentration are significantly increased in these animals due to the lack of an LDL
receptor. These mice developed through the targeted disruption of the LDL receptor gene (Ishibashi et
al., 1993) are thus a new animal model for studies on homozygous familial hypercholesterolemia. In
contrast to wild-type mice, LDLR-KO mice exhibit significantly elevated plasma cholesterol levels
when fed modest amounts of cholesterol.
Accordingly, the current study was carried out to confirm the effects of naringin on the plasma and
hepatic lipid levels, hepatic cholesterol-regulating enzymes, and sterol excretion using cholesterol-fed
LDLR-KO mice. The activities of the hepatic antioxidant enzymes were also determined since the
cardioprotective effects of phenolic compounds appear to be largely related to their action as
antioxidants.
 H.-J. Kim et al. / Life Sciences 74 (2004) 1621–1634 1623

Methods

Animal and diets

Twenty-one male homozygous mice deleted the LDL-R gene (LDLR-KO) were obtained from
Korea Research Institute of Bioscience and Biotechnology (Yusong, Korea). The animals were
individually housed in stainless steel cages in a room with controlled temperature (20–23 jC) and
lighting (alternating 12 h periods of light and dark) and fed a pelletized commercial nonpurified diet for
6 days after arrival. They were then randomly divided into 3 groups (n = 7) and fed a 0.1% (w/w)
cholesterol diet (control diet) or a diet supplemented with either 0.02% (w/w) naringin (Sigma
Chemical Co. St. Louis, MO) or 0.02% (w/w) lovastatin (Imported by Choongwae Pharma Corpora-
tion, Seoul, Korea) for 6 wks (Table 1). The composition of the mineral and vitamin mixture was
previously described (American Institute of Nutrition, 1977, 1980). Free access was given to food and
water. Blood was taken from tail vein for the determination of plasma total cholesterol concentration at
0 wk and after 2 and 4 wks. Every day for the last 4 days, the feces were collected and analyzed for
fecal sterols. The food consumption and body weight were measured daily and weekly, respectively. At
the end of the experimental period, the mice were anesthetized with Ketamin-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
70
jC until analyzed.

Plasma and hepatic lipids

The plasma cholesterol was determined using a commercial kit (Sigma) based on a modification of
the cholesterol oxidase method of Allain et al. (1974). The plasma triglyceride concentrations were

Table 1
Composition of diets (%)
Component Control Naringin Lovastatin
Casein 20.0 20.0 20.0
D,L-methionine 0.3 0.3 0.3
Corn starch 15.0 15.0 15.0
Sucrose 49.9 49.88 49.88
Cellulose powder 5.0 5.0 5.0
Corn oil 5.0 5.0 5.0
Cholinebitartrate 0.2 0.2 0.2
AIN-Minerala 3.5 3.5 3.5
AIN-Vitaminb 1.0 1.0 1.0
Cholesterol 0.1 0.1 0.1
Naringin 0.02
Lovastatin 0.02
Total 100 100 100
a
AIN-76 mineral mixture (Harlan Teklad Co. USA).
b
AIN-76A vitamin mixture (Harlan Teklad Co. USA).
1624 H.-J. Kim et al. / Life Sciences 74 (2004) 1621–1634

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. (1957). The dried lipid residues were dissolved in 1 mL of ethanol
for cholesterol and triglyceride assays. Triton X-100 and a sodium cholate solution (in distilled H2O)
were added to 200 AL 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 as used in the plasma analysis.

HMG-CoA reductase and ACAT activities

The microsomes were prepared according to the method developed by Hulcher and Oleson (Hulcher
and Oleson, 1973) with a slight modification. The livers were homogenized in a 6-fold volume of an ice-
cold buffer (pH 7.0) containing 0.1 M of triethanolamine, 0.02 M of EDTA, and 2 mM of dithiothreitol,
pH 7.0. The homogenates were centrifuged for 10 min at 10,000  g and then at 12,000  g at 4 jC.
Next, the supernatants were ultracentrifuged twice at 100,000  g for 60 min at 4 jC. 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 (acyl CoA:cholesterol
acyltransferase) activities.
The HMG-CoA reductase activities were determined as described by Shapiro et al. (Shapiro et al.,
1974) with a slight modification using freshly prepared hepatic microsomes. An incubation mixture (120
AL) containing microsomes (50 f 100 Ag) and 500 nmol of NADPH (dissolved in a reaction buffer
containing 0.1 M of triethanolamine and 10 mM of EDTA) were preincubated at 37 jC for 5 min. Next,
50 nmol of [14C]-HMG-CoA (specific activity; 2.1420 GBq/mmol; NEMk Life Science Products, Inc.,
Boston, MA) was added and the incubation was continued for 15 min at 37 jC. The reaction was
terminated by the addition 30 AL of 6 M of HCl, then the resultant reaction mixture was incubated at 37
jC for a further 15 min to convert the mevalonate into mevalonolactone. The incubation mixture was
centrifuged at 10,000  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 benzene/acetone (1:1, v/v), and air-dried.
Finally, the Rf 0.3 f 0.6 region was removed by scraping using a clean razor blade, and the 14C
radioactivity was determined using a liquid scintillation counter (Packard Tricarb 1600TR; Packard
Instrument Company, Meriden, CT). The results were expressed as pmol mevalonate synthesized per
min per mg protein.
The ACAT activities were determined using freshly prepared hepatic microsomes as developed by
Erickson et al. (1980) and 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 Ag of cholesterol/mL. Next, reaction mixtures containing
20 AL of a cholesterol solution (6 Ag of cholesterol), 20 AL of a 1 M of potassium-phosphate buffer (pH
7.4), 5 AL of 0.6 mM bovine serum albumin, 50 f 100 Ag of the microsomal fraction, and distilled water
(up to 180 AL) were preincubated at 37 jC for 30 min. The reaction was then initiated by adding 5 nmol
of [14C]-Oleoyl CoA (specific activity; 2.0202 GBq/mmol; NEMk Life Science Products, Inc.) to a
final volume of 200 AL; the reaction time was 30 min at 37 jC. The reaction was stopped by the addition
500 AL of isopropanol/heptane (4:1, v/v), 300 AL of heptane, and 200 AL of 0.1 M potassium phosphate
(pH 7.4), then the reaction mixture was allowed to stand at room temperature for 2 min. Finally, an
 H.-J. Kim et al. / Life Sciences 74 (2004) 1621–1634 1625

aliquot (200 AL) of the supernatant was subjected to scintillation counting. The ACAT activity was
expressed as pmol cholesteryl oleate synthesized per min per mg protein.

Fecal sterols

The fecal neutral sterols were determined using a simplified micro-method developed by Czubayko et
al. (1992). Gas-liquid chromatography was carried out with a Shimazu gas chromatograph (Japan)
equipped with a hydrogen flame-ionization detector and Sack-5 capillary column (30 m  0.25 mm
i.d., 0.25 Am film; Supelco Inc., Bellefonate, PA, USA). Helium was used as the carrier gas. The
temperatures were set at 230 jC for the column and 300 jC for the injector/detector temperature. 5-a-
cholestane (Supelco Inc.) was used as the internal standard. The neutral sterol excretion was calculated
based on the sum of cholesterol, coprostanol, and coprostanone measured in each sample. The fecal bile
acid was extracted with t-butanol and quantified enzymatically with 3-a-hydroxysteroid dehydrogenase
(Michael and Ian, 1980).

Preparation of hepatic antioxidant enzyme source

The preparation of the enzyme source fraction in the hepatic tissue was as follows. The hepatic
tissue was homogenized in a five-fold volume of a 0.25 M sucrose buffer, centrifuged at 600  g for
10 min to discard any cell debris, then the supernatant was centrifuged at 10,000  g for 20 min to
remove the mitochondria pellet. Finally, the supernatant was further ultracentrifuged at 105,000  g
for 60 min to obtain the cytosol supernatant. The amount of protein in the mitochondrial and cytosolic
fractions was measured according to the method of Bradford (1976) using bovine serum albumin as
the standard.

Antioxidant enzyme activities

The superoxide dismutase(SOD) activity was measured using Marklund and Marklund’s method
(Marklund and Marklund, 1974) with a slight modification. One hundred microliters of the cytosol
supernatant was mixed with 1.5 ml of a Tris-EDTA-HCl buffer (pH 8.5), then 100 Al of 7.2 mmol/
l pyrogallol was added and the reaction mixture incubated at 25 jC for 10 min. The reaction was
terminated by the addition of 50 Al of 1 mol/l HCl and measured at 420 nm. One unit was determined
as the amount of enzyme that determined as the amount of enzyme that inhibited the oxidation of
pyrogallol by 50%. The activity was expressed U/mg protein. Catalase (CAT) activity was measured
using Aebi’s (1974) method with a slight modification, in which the disapperance of hydrogen
peroxide was monitored spectrophotometrically at 240 nm for 5 min. A molar extinction coefficient of
0.041 mM
1 cm
1 was used to determine the CAT activity. The activity was defined as the amount of
enzyme which oxidized H2O2 Amol/min/mg protein. The glutathione peroxidase (GSH-Px) activity
was measured using Paglia and Valentine’s (1967) method with a slight modification. The reaction
mixture contained 2.6 ml of a 0.1 mol/l of Tris-HCl (pH 7.2) buffer, 100 Al of 30 mmol/l glutathione,
and 100 Al of 6 mmol/l NADPH. One hundred microliters of the cytosolic supernatant was added to
2.9 ml of the reaction mixture and incubated at 25 jC for 5 min. The reaction was initiated by the
addition of 100 Al of 7.5 mmol/l H2O2 and the absorbance was measured at 340 nm for 5 min. A
molar extinction coefficient of 6.22  103 (mmol/l)
1cm
1 was used to determine the activity. One
1626 H.-J. Kim et al. / Life Sciences 74 (2004) 1621–1634

Table 2
Effects of naringin and lovastatin supplementation for 6 wks on plasma lipids in cholesterol-fed LDLR-KO mice*
Control Naringin Lovastatin
y
Total cholesterol (mmol/L)
0 wk 5.64 F 0.51a 7.09 F 0.31ab 7.72 F 0.57b
2 wk 33.55 F 2.61NS 33.90 F 2.21 28.17 F 2.65
4 wk 34.76 F 4.39a 32.89 F 2.39a 22.73 F 2.62b
6 wk 40.70 F 5.11a 32.40 F 2.75b 19.49 F 1.92c

Triglyceride (mmol/L)
6 wk 2.55 F 0.32a 2.01 F 0.34ab 1.50 F 0.19b
NS
Not significantly different between groups at p < 0.05. abcMeans in the same row not sharing a common superscript are
significantly different between groups at p < 0.05.
*
Mean F SE.
y
Plasma cholesterol concentration was varied between groups prior to naringin and lovastatin supplementation. This
variation was normalized as shown in Fig. 1.

unit of GSH-Px was defined as the amount of enzyme which oxidized 1 Amol per min per mg protein.
Glutathione reductase (GR) activity was determined with the method of Pinto and Bartley (1969) by
monitoring the oxidation of NADPH at 340 nm. The reaction mixture contained 1 mM EDTA and 1
mM GSSG in 0.1 M potassium phosphate buffer (pH 7.4). The activity was expressed oxidized
NADPH nmol/min/mg protein. Glucose-6-phosphate dehydrogenase (G6PD) activity was determined
with the method of Pitkanen et al. (1997). The reaction mixture contained 55 mM Tris-HCl (pH 7.8),
3.3 mM MgCl2 buffer and 6 mM G-6-P. The activity was expressed reduced NADPH nmol/min/mg
protein.

Fig. 1. Effects of naringin and lovastatin supplementation for 6 wks on weekly changes of the plasma cholesterol concentrations
of cholesterol-fed LDLR-KO mice. Means are normalized with 0 week value in each group. Mean F SE. abcMeans in the same
week not sharing a common superscript are significantly different between groups at p < 0.05.
 H.-J. Kim et al. / Life Sciences 74 (2004) 1621–1634 1627

Table 3
Effects of naringin and lovastatin supplementation for 6 wks on the hepatic lipids in cholesterol-fed LDLR-KO mice*
Control Naringin Lovastatin
a b
Total Cholesterol (mmol/g) 0.38 F 0.01 0.35 F 0.01 0.33 F 0.01b
Triglyceride (mmol/g) 0.28 F 0.03a 0.29 F 0.01a 0.19 F 0.02b
ab
Means in the same row not sharing a common superscript are significantly different between groups at p < 0.05.
* Mean F SE.

Statistical analysis

All data were presented as the mean F SE. Significant differences among the groups were
determined by one-way ANOVA using SPSS. Duncan’s multiple-range test was performed if differences
were identified between groups at p = 0.05.

Results

There were no significant differences in the food intake, weight gain or organ weights between the
groups (data not shown).

Plasma and hepatic lipids

The supplementation of naringin and lovastatin for 6 wks significantly lowered the plasma total
cholesterol levels compared to the control group (32.40 F 2.75 and 19.49 F 1.92 mmol/L vs 40.70 F
5.11 mmol/L, p < 0.05) (Table 2). Since the initial plasma cholesterol concentrations varied between the
groups prior to the naringin and lovastatin supplementation, these differences were normalized by
expressing the values based on the initial concentrations, as shown in Fig. 1. The plasma total cholesterol
levels of all three groups increased 3 to 6 fold at the end of the second week. Thereafter, the control
LDLR-KO mice that were only fed a cholesterol diet exhibited a more gradual increase for the remainder

Fig. 2. Effects of naringin and lovastatin supplementation for 6 wks on hepatic HMG-CoA reductase and ACAT activities in
cholesterol-fed LDLR-KO mice. Mean F SE. NSNot significantly different between groups at p < 0.05. abMeans not sharing a
common superscript are significantly different between groups at p < 0.05.
1628 H.-J. Kim et al. / Life Sciences 74 (2004) 1621–1634

Table 4
Effects of naringin and lovastatin supplementation for 6 wks on excretion of fecal acidic and neutral sterols in cholesterol-fed
LDLR-KO mice*
Control Naringin Lovastatin
NS
Feces (g/4 day) 0.50 F 0.05 0.50 F 0.03 0.49 F 0.04
Neutral Sterol (mg/g feces) 4.68 F 0.38a 5.48 F 0.28ab 6.59 F 0.74b
Acidic Sterol (mg/g feces) 0.26 F 0.01a 0.30 F 0.01b 0.26 F 0.01a
Total Sterol (mg/g feces) 4.94 F 0.38a 5.77 F 0.28b 6.85 F 0.74b
NS ab
Not significantly different between groups at p < 0.05. Means in the same row not sharing a common superscript are
significantly different between groups at p < 0.05.
* Mean F SE.

of the experimental period. However, the naringin and lovastatin supplementation attenuated the rise in
the plasma cholesterol concentration from the end of second wk, and resulted in significantly lower
concentration compared to the control group at the end of experiment. This effect was more dramatic
with the lovastatin supplement than the naringin supplement. The plasma and hepatic triglyceride levels
were only significantly lower in the lovastatin group compared to the control group, whereas the hepatic
total cholesterol content was lower in both the naringin and lovastatin groups (Tables 2 and 3). Naringin
also exhibited a tendency to lower the plasma triglyceride level compared to the control group (Table 2).

Hepatic HMG-CoA reductase and ACAT activities

The naringin and lovastatin supplements significantly lowered the HMG-CoA reductase activity by
30% and 46% compared to the control group (321.14 F 27.73 and 281.11 F 46.21 pmol/min/mg vs.
503.12 F 26.49 pmol/min/mg), respectively, as shown in Fig. 2. However, the hepatic ACAT activity
was not significantly different among groups.

Excretion of neutral and acidic sterol

The excretion of neutral sterol was significantly higher in the lovastatin group than in the control
group (6.59 F 0.74 mg/g vs. 4.68 F 0.38 mg/g, p < 0.05), whereas the excretion of acidic sterol, the
primary route of cholesterol output, was significantly higher in the naringin group than in the control or

Table 5
Effects of naringin and lovastatin supplementation for 6 wks on the hepatic antioxidant enzyme activities in cholesterol-fed
LDLR-KO mice*
Control Naringin Lovastatin
a b
SOD (unit/mg) 2.10 F 0.14 2.90 F 0.17 2.50 F 0.14a
CAT (nmol/mg/min) 3.50 F 0.51a 4.84 F 0.95b 3.53 F 0.30a
GSH-Px (nmol/mg/min) 21.66 F 2.91NS 21.54 F 1.45 23.88 F 3.16
GR (nmol/mg/min) 53.85 F 2.13a 67.96 F 4.05b 64.92 F 2.74b
G6PD (nmol/mg/min) 9.59 F 0.84NS 9.83 F 0.82 10.18 F 0.60
NS ab
Not significantly different between groups at p < 0.05. Means in the same row not sharing a common superscript are
significantly different between groups at p < 0.05.
* Mean F SE.
 H.-J. Kim et al. / Life Sciences 74 (2004) 1621–1634 1629

lovastatin group (0.30 F 0.01 mg/g vs. 0.26 F 0.01 and 0.26 F 0.01 mg/g, p < 0.05) (Table 4). When
these two sterols were added to determine the total fecal sterol, the naringin and lovastatin supplements
resulted in a significant increase in the fecal sterol compared to the control.

Hepatic antioxidant enzyme activities

The effects of the citrus bioflavonoid naringin on the hepatic activities of the antioxidant enzymes in
the LDLR-KO mice are shown in Table 5. The naringin supplementation significantly increased the
hepatic SOD, CAT, and GR activities compared to the control group, whereas the lovastatin
supplementation only increased the GR activity. However, the GSH-Px and G6PD activities were not
significantly affected by either the naringin or lovastatin supplementation.

Discussion

Vascular disease is a prevalent disorder leading to coronary heart disease and strokes attributed to
atherosclerosis, a complex disease process often initiated by hypercholesterolemia. A number of
previous epidemiological studies have implied a role for flavonoids in reducing the risk of coronary
heart disease based on the antioxidant activity of these compounds (Hertog et al., 1993, 1995; Knekt et
al., 1996). The investigation of naturally occurring compounds as regulators of the cholesterol
metabolism has particular therapeutic importance, as evidenced by the discovery of the HMG-CoA
reductase inhibitor derived from fungal fermentation products. The current authors have already
speculated on the cholesterol-lowering action of naringin that would seem to be mediated by the
inhibition of HMG-CoA reductase in rats (Shin et al., 1999). Mice, in which specific enzymes,
transporters, and apoproteins can be either deleted or overexpressed, offer a powerful model for
examining these processes and further elucidating changes in the quantitative physiology of lipoproteins,
in general, and of LDL-C, in particular. When the LDL receptor activity is reduced, for example, by
either genetic or environmental manipulation, LDL-C production invariably increases (Bilheimer et al.,
1979; Ishibashi et al., 1994; Spady et al., 1987). Accordingly, the current study was designed to confirm
that naringin lowers the plasma cholesterol concentration in LDL-receptor deficient mice in association
with the inhibition of cholesterol biosynthesis under cholesterol-fed conditions by comparing its effect to
the HMG-CoA reductase inhibitor lovastatin.
The current study demonstrated that cholesterol (0.1%, w/w) supplementation for 6 wks increased the
plasma total cholesterol level 7.5 fold (>40 mmol/L) in the absence of a functional LDLR. However,
naringin and lovastatin supplementation both significantly lowered the plasma total cholesterol level
compared to the control group in LDLR-KO mice, although the plasma cholesterol-lowering effect of
lovastatin was more potent than that of naringin. Both supplements also tended to lower the hepatic
cholesterol content compared to the control group. In a related study, tangerine-peel extract and a
mixture of naringin were found to be potent agents for the inhibition of HMG-CoA reductase and
beneficial in lowering plasma cholesterol levels in rats resistant to developing atherosclerosis (Bok et al.,
1999). In the current study, naringin and lovastatin supplementation significantly inhibited the hepatic
HMG-CoA reductase activity in cholesterol diet fed LDLR-KO mice. As such, the cholesterol
biosynthesis, supposedly highly elevated in LDLR-KO mice due to a lack of LDLR (Bisgaier et al.,
1997), was concomitantly lowered by naringin, as indicated by the lower hepatic HMG-CoA reductase
1630 H.-J. Kim et al. / Life Sciences 74 (2004) 1621–1634

activity compared to the control group. In a previous study, although LDLR-deficient mice failed to
develop atherosclerosis with a chow diet, when challenged with a high-fat and high-cholesterol diet,
their plasma cholesterols were >1500 mg/dL and they developed atherosclerosis throughout the aorta
(Endo, 1992).
Currently, HMG-CoA reductase inhibitors are widely used to lower the plasma total and LDL-
cholesterol levels in the treatment of hypercholesterolemia due to their efficacy as lipid-lowering drugs
(Bilheimer et al., 1984). However, lovastatin supplementation was not found to contribute to lowering
the plasma cholesterol concentration in high-cholesterol fed rats (Lee et al., 2001). This may be due to
differences in the lipoprotein metabolism among animal species (Tall, 1993). Well-known HMG-CoA
reductase inhibitor drugs do not have a hypocholesterolemic effect in rats (Endo et al., 1979), whereas
they do in hamsters, rabbits (Ma et al., 1986), and humans (Lovastatin study groups I through IV).
However, in LDLR-KO mice fed a cholesterol diet, lovastatin supplementation was found to
significantly inhibit the HMG-CoA reductase activity compared to the control group.
HMG-CoA reductase inhibitors have been reported to regulate the cholesterol metabolism via the up-
regulation of LDL receptors, thereby increasing the removal rate of both LDL and LDL precursors from
the blood (Brown and Goldstein, 1986). However, the effectiveness of HMG-CoA reductase inhibitors
on the up-regulation of LDLR was not anticipated in LDLR-deficient subjects (Brown and Goldstein,
1986; Bilheimer et al., 1983; Reihnér et al., 1990). Yet, in the current study, naringin and lovastatin
supplementation seemed to lower the plasma and hepatic cholesterol levels without affecting the LDL
receptors as the animals lacked LDL receptors. Thus, in spite of the LDLR deficiency, naringin and
lovastatin supplementation still resulted in lowering the HMG-CoA reductase activity in LDLR-KO
mice, resulting in less cholesterol available for VLDL or LDL production. As reported by Auerbach et
al. (Auerbach et al., 1995), atorvastatin, an HMG-CoA reductase inhibitor, lowered the plasma LDL-
cholesterol in dietary induced hypercholesterolemia in normal rabbits through the direct inhibition of
LDL production rather than enhanced clearance. In addition, Arad et al. (Arad et al., 1990) suggested
that lovastatin acts, in part, by limiting LDL-production in specific human hyperlipidemia. On the other
hand, hepatic ACAT activity which catalyzes esterification of hepatic free cholesterol was not affected by
naringin and lovastatin supplement that is different result from rats (Bok et al., 2000). ACAT offers the
newly synthesized cholesteryl esters during assembly of VLDL of liver. These results suggest that the
inhibition of ACAT by naringin supplement is limited to these LDLR-KO mice, although the reason is
unknown. In current study, hypocholesterolemic effect of naringin was mediated by the inhibition of
cholesterol synthesis rather than of its esterification.
Interestingly, the fecal sterol excretion was significantly changed in the naringin and lovastatin groups
compared to the control group. The neutral sterol excretion was increased by the lovastatin supplement,
while the acidic sterol excretion was increased by the naringin supplement. However, as the fecal sterol
level was relatively higher in the neutral sterol than in acidic sterol, lovastatin supplement seemed to be
more effective in excretion of fecal sterol compared to the control group. Accordingly, supplementation
of lovastatin could lower the plasma cholesterol concentration to a much greater extent than naringin via
high excretion of neutral sterol although its exact mechanism can not be elucidated. This suggests that
lovastatin and naringin may lower the reabsorption of cholesterol and bile acid via enterohepatic
circulation, although the specific mechanism is still unclear. The intestine and liver would seem to be the
primary site for polyphenol metabolism. These compounds can undergo methylation, hydroxylation,
reduction of the carbonyl group in the pyrane ring, and a conjugation reaction in the liver (Bourne and
Rice-Evans, 1997). As such, polyphenol metabolites may influence the reabsorption of neutral or acidic
 H.-J. Kim et al. / Life Sciences 74 (2004) 1621–1634 1631

sterol, since certain flavonoid metabolites can be recycled via the enterohepatic biliary route (Bourne and
Rice-Evans, 1997).
The plasma and hepatic triglyceride level was only significantly lowered in the lovastatin-supple-
mented group compared to the control group in the current study. The exact mechanism responsible for
the triglyceride-lowering action of statin drugs is still unknown, yet most likely reflects a decreased
production of endogenous triglycerides (Witztum, 1996).
Oxidative stress is one of the causative factors that link hypercholesterolemia with the pathogenesis of
atherosclerosis. This stress results from an imbalance between the production of free radicals and the
effectiveness of the antioxidant defense system (Halliwell, 1996). Dietary flavonoids appear to have
physiological antioxidant properties, which quench reactive oxygen and nitrogen species, thereby
potentially contributing against the pathogenesis of cardiovascular disease. Following administration
of eriocitrin, a flavonoid glycoside present in lemon, plasma exhibited an elevated resistance effect to
lipid peroxidation (Miyake et al., 2000). The hepatic antioxidant enzyme activities of the control group
might be decreased due to the dramatic increase of cholesterol. In the present study, the hepatic SOD and
CAT activities were only elevated by naringin supplementation. The increased SOD and CAT activities
in the liver may have been due to the activation of the enzymes by naringin, thereby resulting in a lower
superoxide anion level. This is because SOD generates H2O2 as a metabolite, which is more toxic than
oxygen radicals in cells and need to be scavenged by CAT or GSH-Px (Pigeolot et al., 1990). In contrast,
the hepatic GSH-Px activity was unaffected by naringin or lovastatin supplementation. The higher CAT
and/or SOD activity may have led to a reduced reactive oxygen species level in the liver in the naringin
supplemented group, as Ng et al. (2000) recently reported that naringin inhibited lipid peroxidation in
normal rats.
In conclusion, it is very clear that naringin is capable of reducing the plasma cholesterol concentration
via the inhibition of hepatic HMG-CoA reductase even under LDL receptor-deficient conditions. As
such, naringin supplementation seemed to be beneficial for lowering the plasma cholesterol in
cholesterol-fed LDLR-KO mice and alters the antioxidant status. However, more studies are needed
to elucidate its mechanism on hypocholesterolemic and antioxidative properties in LDLR-KO mice.

Acknowledgements

This study was supported by a grant of the Korea Health 21 R and D project, Ministry of Health and
Welfare, Republic of Korea. (HMP-00-B-22000-0044).

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