Astragalus polysaccharides lowers plasma cholesterol through mechanisms distinct from statins.

Abstract

To determine the efficacy and underlying mechanism of Astragalus polysaccharides (APS) on plasma lipids in hypercholesterolemia hamsters. The effect of APS (0.25 g/kg/d) on plasma and liver lipids, fecal bile acids and neutral sterol, cholesterol absorption and synthesis, HMG-CoA reductase activity, and gene and protein expressions in the liver and small intestine was investigated in twenty-four hypercholesterolemia hamsters. Treatment periods lasted for three months. APS significantly lowered plasma total cholesterol by 45.8%, triglycerides by 30%, and low-density lipoprotein-cholesterol by 47.4%, comparable to simvastatin. Further examinations revealed that APS reduced total cholesterol and triglycerides in the liver, increased fecal bile acid and neutral sterol excretion, inhibited cholesterol absorption, and by contrast, increased hepatic cholesterol synthesis and HMG-CoA reductase activity. Plasma total cholesterol or low-density lipoprotein-cholesterol levels were significantly correlated with cholesterol absorption rates. APS up-regulated cholesterol-7α-hydroxylase and LDL-receptor gene expressions. These new findings identify APS as a potential natural cholesterol lowering agent, working through mechanisms distinct from statins.

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Astragalus Polysaccharides Lowers Plasma Cholesterol
through Mechanisms Distinct from Statins
Yunjiu Cheng., Kai Tang., Suhua Wu*, Lijuan Liu, Cancan Qiang, Xiaoxiong Lin, Bingqing Liu
Division of Cardiology, the First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China
Abstract
To determine the efficacy and underlying mechanism of Astragalus polysaccharides (APS) on plasma lipids in
hypercholesterolemia hamsters. The effect of APS (0.25g/kg/d) on plasma and liver lipids, fecal bile acids and neutral
sterol, cholesterol absorption and synthesis, HMG-CoA reductase activity, and gene and protein expressions in the liver and
small intestine was investigated in twenty-four hypercholesterolemia hamsters. Treatment periods lasted for three months.
APS significantly lowered plasma total cholesterol by 45.8%, triglycerides by 30%, and low-density lipoprotein-cholesterol
by 47.4%, comparable to simvastatin. Further examinations revealed that APS reduced total cholesterol and triglycerides in
the liver, increased fecal bile acid and neutral sterol excretion, inhibited cholesterol absorption, and by contrast, increased
hepatic cholesterol synthesis and HMG-CoA reductase activity. Plasma total cholesterol or low-density lipoprotein-
cholesterol levels were significantly correlated with cholesterol absorption rates. APS up-regulated cholesterol-7a-
hydroxylase and LDL-receptor gene expressions. These new findings identify APS as a potential natural cholesterol lowering
agent, working through mechanisms distinct from statins.
Citation: Cheng Y, Tang K, Wu S, Liu L, Qiang C, et al. (2011) Astragalus Polysaccharides Lowers Plasma Cholesterol through Mechanisms Distinct from
Statins. PLoS ONE 6(11): e27437. doi:10.1371/journal.pone.0027437
Editor: Christian Schulz, Heart Center Munich, Germany
Received June 15, 2011; Accepted October 17, 2011; Published November 16, 2011
Copyright: ? 2011 Cheng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Guangdong Province Natural Science Foundation (No.06021338); Guangdong Province Science and Technology Program
(No. 2007B031508003); and National Ministry of Education Scholarly Exchanges Foundation (No. 200724). The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: wusuhua@hotmail.com
. These authors contributed equally to this work.
Introduction
Hypercholesterolemia is a contributing factor to atherosclerosis
and consequent cardiovascular and cerebrovascular disease.
Clinically, statins effectively lower plasma cholesterol by inhibiting
HMG-CoA reductase activity [1]. Nevertheless, some patients
under statin treatment can not tolerate statins well or do not reach
the low-density lipoprotein-cholesterol (LDL-C) goal recom-
mended by the US National Institutes of Health guidelines [2].
Therefore, it is desirable to develop natural drugs that have
cholesterol-lowering effect comparable to statins, but could be
tolerated well by the patients.
Astragalus polysaccharides (APS), an extract of Radix Astrag-
ali, is one of the main efficacious principles. APS-I and APS-
II are well known to be the major structural components of
APS. APS-I (molecular weight=1,699,100 Da) consists of
arabinose and glucose in a molar ratio of 1:3.45, while APS-II
(molecular weight=1,197,600 Da) consists of rhamnose, arabi-
nose and glucose in a molar ratio of 1:6.25:17.86 [3]. In
China, APS has been extensively used to treat viral infection
[4,5], acute myocarditis [6], glomerulonephritis [7], diabetes
[8], tumor [9], and many other illnesses, with no toxic record in
clinic.
Previous reports indicated that some dietary soluble polysac-
charides lower plasma cholesterol via reduction of intestinal
cholesterol absorption [10,11,12] or interference with the
enterohepatic circulation of bile acids [13,14]. However, the
cholesterol-lowering effect of APS has not been well studied and
underlying mechanisms behind are still elusive. Hence, we are
here to determine how APS regulates plasma cholesterol and the
cholesterol metabolic pathways in hyperlipidemia hamsters.
Materials and Methods
Animals and diets
Twenty-four male Golden Syrian hamsters weighing 130–140 g
(Animal experimental center, Guangdong, China) were housed
one per cage with a 12 h light: dark cycle and humidity 50–60%.
The hamsters were fed a regular rodent cholesterol-free chow,
with free access to food and water. After two weeks of adaptation,
animals were weighed and randomly divided into 3 groups (n=8/
group): APS group, Simvastatin group and Control group. The
food of all the hamsters was switched to a high-fat diet for a period
of 3 months. The diet contained 10% lard, 10% yoke powder, 1%
cholesterol and 79% standard chow mix (Animal experimental
center, Guangdong, China). Diets were prepared once for the first
two weeks and on a weekly basis thereafter. For the next 3 months,
based on the high-fat diet, animals in APS group and Simvastatin
group were given orally APS at 0.25 g/kg/d and simvastatin at
10 mg/kg/d, respectively. Control animals received an equal
volume of vehicle (0.9% saline). The purity of APS was .95%
(Dabang animal Pharmaceutical Co, Ltd. Inner Mongolia, China).
Simvastatin tablets were obtained from Merck & Co., Inc. USA.
The study protocol was approved by the ethics committee of the
First Affiliated Hospital of Sun Yat-Sen University (Guangzhou,
China).
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Blood (0.5 mL) was collected from the tail vein into a
heparinized capillary tube at the end of month 0,1.5, 3, 4.5, and
6 after overnight fasting. Plasma was harvested for plasma lipids
and plasma liver enzyme measurements. At the end of month 0, 3,
6, fecal samples collected over 4 days for each animal were freeze-
dried, weighed, and ground to powder for bile acid and neutral
sterol analysis. A week prior to sacrifice, the hamsters were
gavaged with 2 mCi [3H]-b-sitosterol and 1 mCi [14C]-cholesterol,
and the feces were collected for three days for fractional
cholesterol absorption measurement. One hour before sacrifice,
40 mCi [3H] water was injected intraperitoneally for hepatic
cholesterol synthesis rate measurement. At the end of the
experiment, the hamsters were anesthetized with an i.p. injection
of sodium pentobarbital (40 mg/kg; Shanghai Chemical Reagent
Company, Beijing, China) and their livers and intestines obtained
were immediately frozen and stored at 280uC for histological
examination and measurement of lipids, HMG-CoA reductase
activity as well as gene and protein expressions.
Measurements of plasma and liver lipids and plasma liver
enzyme
Plasma concentrations of total cholesterol (TC), triglycerides
(TG), high-density lipoprotein-cholesterol (HDL-C) and low-
density lipoprotein-cholesterol (LDL-C) were measured in tripli-
cate using kit methods enzymatically following the instructions
provided (Xueyou Biotechnology Company, Guangzhou, China).
Total lipids were extracted from 0.5 g liver tissue in chloroform/
methanol (vol/vol =2/1). Liver TC and TG were measured using
the same method and reagents for plasma cholesterol [15]. Plasma
concentration of liver enzyme, alanine aminotransferase (ALT)
and aspartate aminotransferase (AST) were analyzed with kit
method (Xueyou Biotechnology Company, Guangzhou, China).
Fecal bile acid and neutral sterol excretion
The total bile acid content in 0.5 g of the ground feces was
determined using a 3a-hydroxysteroid dehydrogenase assay [16].
A second 0.5 g aliquot of feces were saponified with alkaline and
ethanol. Samples were then extracted in 15 ml of petroleum ether
to which 1.0 mg of 5-cholestene (Sigma) had been added as an
internal standard. Mass analysis of the extracted neutral sterols
was conducted by gas-liquid chromatography as described
previously [17].
Fractional cholesterol absorption
Fractional cholesterol absorption was measured by the fecal
isotope ratio method described by Turley et al [18]. At the end of
the experiment, the hamsters were gavaged with 2 mCi of [3H]-b-
sitosterol (American Radiolabeled Chemicals, St. Louis, MO) and
1 mCi of [14C]-cholesterol (Amersham Pharmacia, Piscataway, NJ)
dissolved in 100 ml of soybean oil. Each animal was individually
housed in a cage with a wire bottom and was allowed free access to
diet and water for 3 days. The feces were collected for three days,
1 g of feces from each animal was was extracted with chloroform/
methanol (vol/vol =2/1). The extracts were transferred to a
scintillation vial and dried at 65uC under N2. The [14C]
cholesterol and [3H] sitosterol counts were measured in a liquid
scintillation counter(Beckman, Palo Alto, CA). Fractional choles-
terol absorption was calculated using the following equation:
(14C/3H [dose]–14C/3H [fecal sample])/(14C/3H [dose])6100.
Hepatic cholesterol synthesis rate
The rate of in vivo cholesterol synthesis in liver was measured
as described [16]. At the end of the experiment, animals were
given an i.p. of 40 mCi of [3H] water (New England Nuclear
Corp.), and anesthetized, exsanguinated after 1 hour. Aliquots of
liver were saponified, and the radiolabeled digitonin-precipitable
sterols were extracted and measured. Cholesterol synthesis rates
were expressed as the nanomoles of [3H] water incorporated in
digitonin-precipitable sterols per hour per gram wet weight of
tissue.
Liver HMG-CoA reductase activity measurement
Frozen liver (400 mg) was homogenized and liver microsomal
protein was isolated by differential centrifugation, resuspended,
divided into several aliquots as previously described [19]. Liver
HMG-CoA reductase activity was determined according to the
method described by by van Heusden and Wirtz [20].
Measurement of gene expressions in the liver and small
intestine
Total RNA was extracted from liver (left lateral lobe) or small
intestine (jejunum) using TRIzol reagent (Invitrogen, USA). An
aliquot (1 mg) of RNA was transcribed to cDNA using Ominiscript
reverse transcriptase (Roche, USA) under the following condi-
tions:. 37uC for 1 h and 93uC for 5 min. The cDNA was diluted
1:10 using diethyl pyrocarbonate water, and the real-time PCR
reaction solution consisted of 5 ml cDNA, 12.5 ml QuantiTect
SYBR Green PCR kit (Roche, USA), 5.5 ml diethyl pyrocarbonate
water, and 1 ml forward and reverse primer (20 pmol) for a final
reaction volume of 25 ml. The primer sequences are presented in
Table 1. Quantitative real-time PCR analysis was performed on
an Applied Biosystems 7500 real-time PCR machine (Applied
Biosystems, USA) using the following conditions: 50uC for 2 min,
94uC for 10 min, and 40 cycles of 94uC for 10 s and 60uC for
1 min. The fluorescence measurement used to calculate threshold
cycle (Ct) was made at the 60uC point. A dissociation curve was
run at the end of the reaction to ensure a single amplification
product. One sample from the control was used as an external
calibrator. Gene expression was normalized to cyclophilin mRNA
concentration and presented as fold of the control by setting the
expression of the control group to 1.00.
Western blot
Approximately 150 mg of liver tissue was homogenized
adequately prepared for Western blotting as described previously
[21]. An aliquot (50 mg) of sample proteins were denatured in
sample buffer containing SDS and b-mercaptoethanol, separated
on a 4–20% gradient SDS-PAGE gel, and electroblotted onto
nitrocellulose membranes. Nonspecific binding sites of the
membranes were blocked using defatted milk proteins followed
by the addition of antibodies against LDLR (Santa Cruz, CA,
USA). The relative amount of primary antibody was detected with
peroxidase-conjugated secondary antibody. Densitometry was
quantified using the BioRad Quantity one software. Similar
procedures were carried out with antibodies against cholesterol-
7a- hydroxylase (cyp7a-1) (Santa Cruz, CA, USA), ABCG5 (Santa
Cruz, CA, USA), ABCG8 (Santa Cruz, CA, USA), NPC1L1
(Santa Cruz, CA, USA) and b-actin (Santa Cruz, CA, USA). The
protein expression was expressed as a ratio of the target protein to
b-actin.
Histological examination
Liver tissues were rinsed with saline water (0.9%, w/w) and
fixed in methyl aldehyde solution (1:9, 0.01 M, pH 7.4,
NaH2PO3/Na2HPO 3), embedded in paraffin wax. Sections of
5 mm thickness were affixed to slides, deparaffinized, dehydrated
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and then stained with haematoxylin and eosin using routine
methods. Stained sections were observed under an optical
microscope and photographs were taken to record the histological
change.
Statistical analysis
Results were expressed as mean6standard deviation. One-way
ANOVA analysis of variance was used to analyze plasma and
liver lipids, plasma concentration of liver enzyme, cholesterol
absorption rates, cholesterol synthesis rates, and gene and
protein expressions using SPSS 17.0 (SPSS Inc., Chicago, IL,
USA). Relationships between plasma lipid concentrations and
intestinal cholesterol absorption rates were analyzed using
Pearson’s correlation coefficients. Significance level was set up
at p,0.05.
Results
All 24 hamsters survived the 6-month experiment. The mean
initial and final body weights for all 24 hamsters were 20565 g
and 25267 g,respectively. And no significant differences were
observed between groups.
Effect of APS on plasma lipoproteins
Before APS treatment, animals were fed a high fat diet for three
months, which significantly increased plasma TC, TG, LDL-C
and HDL-C and no intergroup differences were found (P.0.05).
Treatments of these hyperlipidemic animals with APS and
simvastatin for three months resulted in marked decreases in
plasma TC, TG, and LDL-C concentrations. Compared to the
control, APS and simvastatin treatments significantly (P,0.05)
decreased plasma TC by 45.8% and 53.8%, TG by 30%
and34.2%, LDL-C by 47.4% and 56.5%, respectively. Neverthe-
less, the difference failed to achieve statistical significance between
APS and Simvastatin group. Both APS and simvastatin reduced
(p,0.05) plasma HDL-C levels compared to the control group
(Figure 1).
APS increased fecal bile acid and neutral sterol excretion
To determine the effect of APS on whole-body cholesterol
turnover, the daily output of bile acids and neutral sterols in the
feces was measured. There were no significant differences
between groups for any fecal steroids prior to treatment.
However, after three months, the fecal bile acid and neutral
sterol excretion were significantly (P,0.05) higher in the APS
groups than in the Simvastatin and control group (Table 2). No
significant differences were found between Simvastatin and
control group.
APS inhibited intestinal fractional cholesterol absorption
To understand the mechanism by which the treatments affect
plasma cholesterol levels, we have measured fractional cholesterol
absorption rate in all animals of each group using the dual plasma
stable isotope ratio method. Compared with the control, APS
inhibited (p,0.05) fractional cholesterol absorption by 59.9%.
However, there is no difference between Simvastatin and Control
group (Table 3). Fractional cholesterol absorption rates were
strongly associated with plasma TC (r=0.723, p,0.0001;
Figure 2A) and LDL-C (r=0.783, p,0.0001; Figure 2B).
APS up-regulated cholesterol synthesis rates
As reported previously [22], cholesterol synthesis changed
reciprocally with the internal cholesterol absorption. To examine
the effect of APS on cholesterol synthesis in the liver, we measured
in vivo cholesterol synthesis rate in all animals using tritiated
water. Expectedly, compared to Simvastatin and Control group,
the rate of hepatic cholesterol synthesis in APS group was
increased by 33.4% (p,0.05) and 66.8% (p,0.05), respectively
(Table 3).
Effect of APS on cholesterol metabolism associated gene
and protein expressions in the liver and small intestine
In addition to cholesterol absorption and synthesis, quantitative
real-time PCR and western blot were used to measure the
expression of key genes of cholesterol metabolism in the livers
(Figure 3). LDLR is involved in liver clearance of LDL-C from
circulation. The present study showed that mRNA and protein
abundance of LDLR was higher in APS and simvastatin treated
hamsters than the control. The difference did not reach statistical
significance between APS group and Simvastatin group. The
current study shows both APS and simvastatin elevated hepatic
cyp7a-1 gene expression significantly compared to the control
(P,0.001), whereas APS demonstrated superior ability in this
effect (P,0.05).
Table 1. Primer sequences used for real-time PCR analysis.
GenePrimer sequence NCBI Accession Number
cyp7a-159-ACCTGCCGGTACTAGACAGCA-39
NM_ 012942
59-TGCGGATATTCAAGGATGCA-39
LDL-R59-ACCTGCCGGTACTAGACAGCA-39
NM_175762
59-GAACTTGGGTGAGTGGGCAC-39
NPC1L159-CCTGACCTTTATAGAACTCACCACAGA-39
DQ897680
59-GGGCCAAAATGCTCGTCAT-39
ABCG5 59-TGATTGGCAGCTATAATTTTGGG-39
AF312713
59-GTTGGGCTGCGATGGAAA-39
ABCG859-TGCTGGCCATCATAGGGAG-39
AF324495
5-TCCTGATTTCATCTTGCCACC-39
Cyclophilin59-CAAATGCTGGACCAAACACA-39
X17105
59-CAGTCTTGGCGGTGCAGAT-39
doi:10.1371/journal.pone.0027437.t001
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Figure 1. Changes of plasma lipids in response to APS and simvastatin treatments. APS and simvastatin significantly lowered total
cholesterol (TC), triglycerides (TG), high-density lipoprotein –cholesterol (HDL-C) and low-density lipoprotein-cholesterol (LDL-C) concentrations
compared to the control. # P,0.05 compared with control group.
doi:10.1371/journal.pone.0027437.g001
Table 2. Effect of APS and simvastatin on fecal excrection of bile acids and neutral sterol.
Fecal lipidFeeding periodControl (n=8) APS (n=8)Simvastatin (n=8)
bile acidsMonth 012.2462.8913.3563.1712.4662.29
(mmol/d/100 g BW) Month 323.9864.11 24.8764.7622.0164.19
Month 637.8567.14 51.9968.17#*
29.4365.16
neutral sterolsMonth 018.7862.95 20.1463.4319.5163.19
(mmol/d/100 g BW)Month 3 48.6767.7049.0766.7647.0266.36
Month 6 75.0768.4192.2368.10#*
61.7067.03
The daily total bile acids and neutral sterols were measured and expressed per 100 g body weight (BW). Values are means6SDs of 8 animals per group.
#P,0.05 compared with Control group,
*P,0.05 compared with Simvastatin group.
doi:10.1371/journal.pone.0027437.t002
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In order to understand how APS affect cholesterol absorption,
we measured their gene and protein expressions in the small
intestine. We found that APS did not show a significant effect on
the mRNA and protein levels of ABCG5, ABCG8 or NPC1L1 in
the small intestine. Similarly, simvastatin did not affect cholesterol
transport associated gene and protein expressions compared to the
control (Figure 3).
Effect of APS on liver HMG-CoA reductase activity
After 3-month treatment, HMG-CoA reductase activity in APS
group, Simvastatin group and control group were 36.2465.62
pmole/min/mg protein, 12.6062.03 pmole/min/mg protein and
22.8666.45 pmole/min/mg protein, respectively. Statistical
analysis found significant difference among the three groups.
Relative to the control and Simvastatin group, APS significantly
increased the HMG-CoA reductase activity by 58.5% (P,0.05)
and 1.87 fold (P,0.05), respectively.
Effect of APS on liver lipids, enzymes and histopathology
In comparison to the control, APS and simvastatin significantly
(P,0.001) reduced liver TC by 45.6% and 41.9%, and TG by
41.8% and 40.9%, respectively (Table 4). There is no significant
difference found between APS and Simvastatin group. To
determine whether APS have hepatotoxic effect on hamsters,
we have measured the plasma concentration of ALT and AST
and performed histological examination of the livers. Our study
shows APS and simvastatin significantly decreased the concen-
trations of plasma ALT and AST (P,0.05), and markedly
ameliorated liver fatty degeneration compared to the control
(Figure 4).
Discussion
Inhibiting HMG-CoA reductase activity and increasing hepatic
LDLR expression are primary mechanisms of statins therapy for
hyperlipidemia. Here, we identify a new cholesterol-lowering
drug, APS, that effectively reduces plasma cholesterol to levels
comparable to that of statins, but may work through different
mechanisms. In our study, a 45.8% reduction of TC, 30%
reduction of TG, and 47.4% reduction of LDL-C were achieved in
hyperlipidemic hamsters after 3-month treatment of APS, similar
to statins that lowered LDL-C by 56.5%. Previous study by Wu
CZ et al. showed Ogi-Keishi-Gomotsu-To-Ka-Kojin (OKGK), a
Chinese medicine composite that contains a small amount of APS
(10.07%) may also exert a hypolipidemic effect in hamsters [23].
Yet they did not make clear which ingredient really works, as
Panax ginseng, a crude drug of OKGK was also reported to show
antihypercholesterolemic action in human, chicken and rat
[24,25].
The small intestines are implicated in regulating cholesterol
homeostasis through affecting cholesterol absorption. An inhibi-
tion of intestinal absorption results in lower levels of circulating
cholesterol. As ezetimibe and sitosterol, which act directly at the
level of intestine, lower plasma cholesterol by inhibiting intestinal
fractional cholesterol absorption [26,27]. Our results revealed that
APS led to a profound reduction in intestinal cholesterol
absorption and a markedly accelerated rate of fecal neutral sterol
excretion. Correlation between cholesterol absorption rates and
plasma TC or LDL-C concentrations were also observed,
suggesting a hitherto unknown mechanism that APS reduces
plasma cholesterol by interfering with the intestinal cholesterol
absorption.
Previous researchers have identified Niemann-Pick C1 like 1
(NPC1L1) as a cholesterol uptake transporter [28] and the ATP-
binding cassette (ABC) proteins ABCG5 and ABCG8 as
cholesterol efflux transporter [29]. However, our studies indicated
that the sterol transporters, ABCG5, ABCG8, and NPC1L1 in the
Figure 2. Relationships between plasma lipids and intestinal
cholesterol absorption. Intestinal cholesterol absorption rates are
significantly correlated with the plasma LDL-C (A) and TC (B) levels,
respectively.
doi:10.1371/journal.pone.0027437.g002
Table 3. Effect of APS on fractional cholesterol absorption
and cholesterol synthesis rates in hamsters.
GroupAbsorption (%) Synthesis (nmol/h/g tissue)
Control61.2367.67278645
APS24.5368.78#*
371653#*
Simvastatin 56.44611.12 220639
Values are means6SD of 8 animals per group.
#P,0.05 compared with Control group,
*P,0.05 compared with Simvastatin group.
doi:10.1371/journal.pone.0027437.t003
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