Hypocholesterolemic Effects of the Cauliflower Culinary-Medicinal Mushroom, Sparassis crispa (Higher Basidiomycetes), in Diet-Induced Hypercholesterolemic Rats

Abstract

The cauliflower culinary-medicinal mushroom, Sparassis crispa, possesses various biological activities that have been widely reported to have therapeutic applications. We examined the effects of S. crispa on serum cholesterol, hepatic enzymes related to cholesterol metabolism, and fecal sterol excretion in rats fed a cholesterol-rich diet for 4 weeks. Male Sprague-Dawley rats (8 weeks old) were randomly divided into 5 groups (n = 6 mice per group): normal diet (normal control [NC]), cholesterol-rich diet (cholesterol control [CC]), cholesterol-rich diet plus S. crispa fruiting body (SC), cholesterol-rich diet plus S. crispa extract (SCE), and cholesterol-rich diet plus S. crispa residue (SCR). SCE supplementation significantly enhanced hepatic cholesterol catabolism through the upregulation of cholesterol 7α-hydroxylase (CYP7A1) messenger RNA (mRNA) expression (2.55-fold compared with that in the NC group; P < 0.05) and the downregulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase mRNA expression (0.57-fold compared with that in the NC group; P < 0.05). Additionally, the SCE diet resulted in the highest fecal excretion of cholesterol and bile acid in hypercholesterolemic rats. In conclusion, mRNA expression of CYP7A1 and HMG-CoA reductase were significantly modulated by the absorption of SCE samples. Also, SCE samples had a significant effect on fecal bile acid and cholesterol excretion. These results suggest that SCE samples can induce hypocholesterolic effects through cholesterol metabolism and the reduction of circulating cholesterol levels.

Full text

International Journal of Medicinal Mushrooms, 17(10): 965–975 (2015)
965
1521-9437/15/$35.00 © 2015 Begell House, Inc. www.begellhouse.com
Hypocholesterolemic Effects of the Cauliower
Culinary-Medicinal Mushroom, Sparassis crispa
(Higher Basidiomycetes), in Diet-Induced
Hypercholesterolemic Rats
Ki Bae Hong,1 Sung-Yong Hong,1 Eun Young Joung,2 Byung Hee Kim,3 Song-Hwan Bae,4
Yooheon Park,1 & Hyung Joo Suh1,*
1
Department of Food and Nutrition, Korea University, Seoul, Republic of Korea; 2Department of Home Economic
Education, Jeonju University, Jeonju, Republic of Korea; 3Department of Food Science and Technology, Chung-Ang
University, Gyeonggi, Republic of Korea; 4Department of Food and Biotechnology, Hankyong National University,
Gyeonggi, Republic of Korea
*Address all correspondence to: Hyung Joo Suh, Department of Food and Nutrition, Korea University, Seoul 136-703, Republic of Korea; Tel.:
+82-2-940-2853; Fax: +82-2-940-2859; suh1960@korea.ac.kr.
ABSTRACT: The cauliower culinary-medicinal mushroom, Sparassis crispa, possesses various biological activi-
ties that have been widely reported to have therapeutic applications. We examined the eects of S. crispa on serum
cholesterol, hepatic enzymes related to cholesterol metabolism, and fecal sterol excretion in rats fed a cholesterol-
rich diet for 4 weeks. Male Sprague-Dawley rats (8 weeks old) were randomly divided into 5 groups (n = 6 mice per
group): normal diet (normal control [NC]), cholesterol-rich diet (cholesterol control [CC]), cholesterol-rich diet plus
S. crispa fruiting body (SC), cholesterol-rich diet plus S. crispa extract (SCE), and cholesterol-rich diet plus S. crispa
residue (SCR). SCE supplementation signicantly enhanced hepatic cholesterol catabolism through the upregulation
of cholesterol 7α-hydroxylase (CYP7A1) messenger RNA (mRNA) expression (2.55-fold compared with that in the
NC group; P < 0.05) and the downregulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase
mRNA expression (0.57-fold compared with that in the NC group; P < 0.05). Additionally, the SCE diet resulted in the
highest fecal excretion of cholesterol and bile acid in hypercholesterolemic rats. In conclusion, mRNA expression of
CYP7A1 and HMG-CoA reductase were signicantly modulated by the absorption of SCE samples. Also, SCE sam-
ples had a signicant eect on fecal bile acid and cholesterol excretion. These results suggest that SCE samples can
induce hypocholesterolic eects through cholesterol metabolism and the reduction of circulating cholesterol levels.
KEY WORDS: medicinal mushrooms, Sparassis crispa, hypercholesterolemia, cholesterol, cholesterol 7α-hydroxylase,
3-hydroxy-3-methylglutaryl coenzyme A
ABBREVIATIONS: AI, atherogenic index; CC, cholesterol control group; CYP7A1, cholesterol 7α-hydroxylase;
FP, forward primer; HDL-C, high-density lipoprotein cholesterol; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme
A; LDL, low-density lipoprotein; mRNA, messenger RNA; NC, normal control group; PCR, polymerase chain reac-
tion; RP, reverse primer; SCE, hroup fed a cholesterol-rich diet + S. crispa extract; SCR, group fed a cholesterol-rich
diet + S. crispa residue; TC, total cholesterol.,
I. INTRODUCTION
Hypercholesterolemia is a disorder characterized by
a high level of blood cholesterol1 and is typically
a result of a combination of lifestyle, dietary, and
genetic factors. In addition, additive eects may be
caused by a single gene defect, resulting in familial
hypercholesterolemia.2 Hypercholesterolemia is a
dominant risk factor for the development and pro-
gression of atherosclerosis and related diseases,3
and is therefore a major health concern. A num-
ber of pharmacological and nonpharmacological
approaches have been used to regulate levels of blood
cholesterol. Although there has been considerable

International Journal of Medicinal Mushrooms
K.B. Hong et al.
966
progress in the management of hypercholesterol-
emia using synthetic drugs, the risk of undesirable
side eects posed by these agents has resulted in an
increasing demand for natural products with cho-
lesterol-reducing properties.4 Studies have shown
that the medicinal mushrooms Pleurotus ostreatus
and Volvariella volvacea lower blood cholesterol.5
In addition, Flammulina velutipes showed a hypo-
cholesterolemic eect by causing increased fecal
excretion of cholesterol and inducing the expression
of hepatic low-density lipoprotein (LDL).5,6
The culinary-medicinal cauliower mushroom,
Sparassis crispa (Wulfen) Fr. (Sparasidaceae,
Polyporales, higher Basidiomycetes), a species
with various medicinal properties, has been well
characterized, from its biological activity to its
genetic prole.7,8 S. crispa has a relatively high
β-glucan content (up to 43.6% of the dry weight
of the fruiting body),9 the primary structure of
which is a 6-branch 1,3-β-glucan, with a branch in
every third unit on the main chain. The β-glucan in
S. crispa is highly water-soluble and has an esti-
mated molecular weight of 510 kDa.10,11 Recent
studies have shown that β-glucan reduces blood
cholesterol and positively inuences lipid metabo-
lism12,13; hence S. crispa, which has high levels of
β-glucan, has signicant potential in the treatment
of hypercholesterolemia. Cholesterol homeostasis
in the liver is governed by several key regulatory
enzymes and receptors, including (1) cholesterol
7α-hydroxylase (CYP7A1), the enzyme involved in
the initial stages of the bile acid biosynthesis path-
way, and (2) 3-hydroxy-3-methylglutaryl-coenzyme
A (HMG-CoA) reductase, the enzyme that regulates
the rate-determining step in the cholesterol biosyn-
thesis pathway.16
Despite these ndings, the hypocholesterolemic
eects of S. crispa have not yet been evaluated.
In particular, there is a lack of comprehensive
understanding of the link between modulation of
the molecular mechanism and the consumption of
S. crispa. In this study we examined the eects of
S. crispa on serum cholesterol, hepatic expression of
cholesterol-related enzymes, and fecal sterol excre-
tion in rats fed a cholesterol-rich diet for 4 weeks.
The S. crispa used for this study was prepared using
a hot-water extraction method and divided into a
water-soluble extract and a water-insoluble residue.
II. MATERIALS AND METHODS
A. Mushroom Material and Its Preparation
Fruiting bodies of S. crispa obtained from Chung-
Ang University (Seoul, Korea) were freeze-dried
and pulverized. The resultant powder was treated
with hot water (1:10, w/v) at 100°C for 6 hours,
and the aqueous extract was ltered twice through
Whatman No. 2 lter paper under a vacuum on a
Buchner funnel. The ltrate and the residue were
subsequently freeze-dried and powdered. The
approximate composition of S. crispa fruiting body,
its aqueous extract, and the residue were determined
using the AOAC (Association of Ocial Analytical
Chemists) method; the results of the analysis are
shown in Table 1.
B. Animals and Diets
The experimental protocol for use of animals in this
study was approved by the Korea University Animal
Care Committee (Seoul, Korea). Male Sprague-
Dawley rats (8 weeks old) were obtained from
Daehan Biolink (Cheongju, Korea). The rats were
housed individually in stainless steel wire cages in
a room maintained at 24°C with 60% atmospheric
humidity and a 12-hour light/12-hour dark cycle.
After an adaptation period (1 week), the rats were
randomly divided into 5 groups (n = 6 mice per
group) that were assigned to the following diets:
normal diet (normal control [NC]), cholesterol-rich
diet (cholesterol control [CC]), cholesterol-rich diet
plus S. crispa fruiting body, cholesterol-rich diet
plus S. crispa extract (SCE), and cholesterol-rich
diet plus S. crispa residue (SCR). All cholesterol-
rich diets contained 1% supplemental cholesterol;
Table 2 provides the ingredient composition of the
diets used in this study. Fresh feces were collected
for 3 days during the 4th week. At the end of the
study, the rats were euthanized using carbon dioxide
asphyxiation, and the liver, spleen, and cecum were
isolated from the rats and weighed immediately.

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Hypocholesterolemic Eects of Sparassis crispa in Hypercholesterolemic Rats
967
C. Serum Cholesterol and Atherogenic
Index Assessment
Blood samples were collected from the common
carotid artery into nonheparinized centrifuge tubes
and centrifuged at 3,000 g at 4°C for 10 min to
obtain the serum. The concentrations of serum
total cholesterol (TC) and high-density lipoprotein
cholesterol (HDL-C) were measured using a DRI-
CHEM 3500 analyzer (Fuji Photo Film Co., Osaka,
Japan). The atherogenic index (AI) was calculated
based on measured TC and HDL-C values.
TABLE 1: Nutrient Composition of Sparassis crispa Samples
Composition (g/100 g) S. crispa Fruiting Body S. crispa Extract S. crispa Residue
Carbohydrate 24.10 ± 0.24 63.25 ± 1.14 1.85 ± 0.91
Dietary ber 64.36 ± 0.12 16.36 ± 1.17 90.40 ± 2.19
Soluble ber 2.56 ± 0.10 14.62 ± 1.68 1.03 ± 0.08
Insoluble ber 61.80 ± 0.23 1.74 ± 0.50 89.37 ± 2.11
Crude protein 3.92 ± 0.25 5.90 ± 0.09 3.52 ± 0.66
Crude fat 0.83 ± 0.00 0.89 ± 0.28 0.67 ± 0.07
Crude ash 3.06 ± 0.01 9.20 ± 0.81 0.85 ± 0.07
Moisture 3.73 ± 0.01 4.40 ± 0.88 2.71 ± 0.14
β-Glucan 24.00 ± 0.10 16.62 ± 0.14 7.38 ± 0.21
Values are mean ± standard error of the mean.
TABLE 2: Ingredient Composition of Experimental Diets
Composition
(g/100 g)
Study Groups
NC CC SC SCE SCR
Starch 61.80 60.55 60.55 60.55 60.55
Casein 18.00 18.00 18.00 18.00 18.00
Mineral mixture* 4.00 4.00 4.00 4.00 4.00
Vitamin mixture†1.00 1.00 1.00 1.00 1.00
Corn oil 5.00 5.00 5.00 5.00 5.00
Sucrose 5.00 5.00 5.00 5.00 5.00
Methionine 0.20 0.20 0.20 0.20 0.20
Sodium cholate — 0.25 0.25 0.25 0.25
Cholesterol — 1.00 1.00 1.00 1.00
Cellulose 5.00 5.00 — — —
S. crispa fruiting body — — 5.00 — —
S. crispa extract — — — 5.00 —
S. crispa residue — — — — 5.00
Total 100.00 100.00 100.00 100.00 100.00
* Mineral mixture (g/100 g mixture): CaPO4•2H2O, 14.6; KH2PO4, 25.7; NaH2PO4, 9.4; NaCl, 4.7; calcium lac-
tate, 35.1; ferric citrate, 3.2; MgSO4, 7.2; ZnCO3, 0.1; MnSO4•4H2O, 0.1; CuSO4•5H2O, 0.03; KI, 0.01.
†ICN Vitamin mixture, no. 904654, 1999.
CC, cholesterol control (cholesterol diet); NC, normal control (normal diet); SC, S. crispa fruiting body + cho-
lesterol diet; SCE, S. crispa extract + cholesterol diet; SCR, S. crispa residue + cholesterol diet.

International Journal of Medicinal Mushrooms
K.B. Hong et al.
968
D. Expression of Cholesterol-Related
Hepatic Enzymes at Levels of
Messenger RNA
Total RNA was extracted from the livers using
TRizol LS reagent (Invitrogen, Carlsbad, CA).
Quality-controlled RNA samples with a high opti-
cal density ratio (A260:A280 >1.8) were treated with
RQ1 RNase-free DNase I (Promega, Fitchburg, WI),
and 1 μg of total RNA was reverse transcribed using
SuperScript III Reverse Transcriptase (Invitrogen)
with oligo d(T) as the primer. Real-time polymerase
chain reaction (PCR) was performed on the comple-
mentary DNA generated using a Power SYBR Green
PCR Master Mix kit (Applied Biosystems, Foster
City, CA). Quantitative analyses were carried out
using StepOnePlus software version 2.0 (Applied
Biosystems), and the results were normalized to a
validated control gene, glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), using the ΔΔCt method.14
The following sequences of primers were used real-
time PCR: CYP7A1 (NM_012942.2): forward primer
(FP): 5′-CACCATTCCTGCAACCTTTT-3′, reverse
primer (RP): 5′-GTACCGGCAGGTCATTCAGT-3′;
HMG-CoA reductase (NM_013134.2): FP:
5′-TGCTGCTTTGGCTGTATGTC-3′, RP:
5′-TGAGCGTGAACAAGAACCAG-3′;
and GAPDH (NM_017008.4): FP:
5′-AGACAGCCGCATCTTCTTGT-3′, RP:
5′-CTTGCCGTGGGTAGAGTCAT-3′.
E. Fecal Cholesterol and Bile Acid
The levels of fecal cholesterol were measured using
a DRI-CHEM 3500 analyzer (Fuji Photo Film Co.).
Fecal bile acid was extracted using the method
described by Grundy et al.15 Briey, fecal solids
were homogenized and freeze-dried. A homog-
enized fecal sample (1 g) was extracted twice with
20 mL of absolute ethanol at 85°C for 1 hour, and
the extract was dried at 90°C. 2N sodium hydroxide
(4 mL) was added to the residue and autoclaved for
6 hours at 16–19 pounds of pressure (122–126°C).
Distilled water (4 mL) was added, and bile acid was
extracted 3 times with 20 mL of diethyl ether. A
supernatant containing neutral sterol was removed,
and the residue was acidied to pH 1–2 with 1.12 N
hydrogen chloride and extracted with diethyl ether.
The derived sterol was analyzed by gas chromatog-
raphy under following conditions: 25 m × 0.32 mm
inner diameter × 0.20 μm CP-SIL 19CB-1 capillary
column (Varian BV, Middelburg, the Netherlands),
helium (carrier gas) at a ow rate of 1.5 mL/min,
injector and detector temperatures of 295°C, and
an initial oven temperature of 265°C for 20 min.
F. Statistical Analysis
All statistical analyses were performed using the
SPSS software version 12.0 (IBM/SPSS, Chicago,
IL). Statistical dierences between groups were
evaluated using 1-way analysis of variance and
Tukey multiple tests. All data were 2-sided with a
5% signicance level and are reported as the mean
± standard error of the mean.
III. RESULTS
A. Food Intake, Body Weight Gain, and
Relative Organ Weight
Figure 1 illustrates the food intake among animals
over the 4-week study period. At the 4th week, food
consumption among rats in the CC group (28.92 g/
day) was signicantly higher than those fed a nor-
mal diet (25.42; P < 0.05). By contrast, rats fed a
diet with S. crispa supplementation (the cholesterol-
rich diet plus S. crispa fruiting body, SCE, and SCR
groups) had slightly less daily food intake than those
in the CC group. Initial body weights of rats were
not signicantly dierent because of the random-
ization of animals. The lower food intake by rats in
the S. crispa supplementation groups was reected
by a lower nal body weight for these animals, but
the dierence was not signicant. The average rela-
tive organ weights are presented in Table 3. The
relative liver weight in rats fed a cholesterol-rich
diet showed a noteworthy increase resulting from
the accumulation of excess dietary cholesterol
(NC: 3.39 vs. 4.83 g/100 g BW in the NC and
CC groups, respectively; P < 0.05). By contrast,
rats receiving S. crispa supplementation did not

Volume 17, Number 10, 2015
Hypocholesterolemic Eects of Sparassis crispa in Hypercholesterolemic Rats
969
FIG. 1: Eect of Sparassis crispa on food intake in rats fed a high-cholesterol diet for 4 weeks. Values are the mean
± standard error of the mean of 6 rats/group. Means with dierent superscript letters are signicantly dierent at
P < 0.05 (Tukey multiple range test). CC, cholesterol control (cholesterol diet); NC, normal control (normal diet);
SC, S. crispa fruiting body + cholesterol diet; SCE, S. crispa extract + cholesterol diet; SCR, S. crispa residue +
cholesterol diet.
TABLE 3: Eect of Sparassis crispa on Relative Organ Weights in Rats Fed a High-Cholesterol Diet for 4
Weeks
Organ Weight
(g/100 g BW)
Study Groups
NC CC SC SCE SCR
Liver 3.39 ± 0.20b4.83 ± 0.11a4.71 ± 0.21a4.95 ± 0.34a4.52 ± 0.27a
Spleen 0.25 ± 0.01 0.27 ± 0.01 0.25 ± 0.02 0.31 ± 0.03 0.28 ± 0.02
Values are the mean ± standard error of the mean of 6 rats/group. Means with dierent superscript letters are
signicantly dierent at P < 0.05 (Tukey multiple range test).
BW, body weight; CC, cholesterol control (cholesterol diet); NC, normal control (normal diet); SC, S. crispa
fruiting body + cholesterol diet; SCE, S. crispa extract + cholesterol diet; SCR, S. crispa residue + cholesterol
diet.
have any alteration in their relative organ weights
compared with the groups eating a cholesterol-rich
diet. Furthermore, gross examination of the internal
organs of the rats treated with S. crispa revealed no
detectable abnormalities (data not shown).
B. Serum Cholesterol
The eects of S. crispa on serum cholesterol in rats
fed a cholesterol-rich diet for 4 weeks are shown
in Fig. 2. Hypercholesterolemia was accompanied
by changes in serum lipid prole. Rats receiving
dietary cholesterol had a signicant increase in TC
levels and a decrease in HDL-C levels (P < 0.05),
thus resulting in a higher AI in the CC groups
(3.00) compared with the NC (0.39; P < 0.05). This
cholesterol-induced increase in AI was somewhat
ameliorated in rats treated with S. crispa, however,
but the dierences were not statistically signicant.

International Journal of Medicinal Mushrooms
K.B. Hong et al.
970
(mRNA) levels of CYP7A1 and decreased levels
of HMG-CoA reductase. Animals in the SCE group
displayed remarkable upregulation of CYP7A1
(2.55-fold compared with the NC group; P < 0.05),
and this could probably result in enhanced hepatic
cholesterol catabolism. Additionally, supplementa-
tion with the S. crispa extract and residue further
enhanced the cholesterol-rich diet–induced sup-
pression of the mRNA levels of hepatic HMG-CoA
reductase; the transcript levels of HMG-CoA reduc-
tase in the SCE and SCR groups were 0.57-fold and
0.54-fold lower, respectively, compared with the NC
group (P < 0.05).
C. Transcriptional Regulation of Hepatic
Enzymes Related to Cholesterol
Metabolism
To investigate whether the cholesterol-lowering
mechanism of S. crispa involved transcriptional
processes, the levels of messenger RNA of choles-
terol-regulating hepatic enzymes such as CYP7A1
and HMG-CoA reductase were determined (Fig. 3).
Dietary cholesterol seemed to inuence transcrip-
tional levels of CYP7A1 and HMG-CoA reductase
since animals receiving a cholesterol-rich diet for
4 weeks tended to have increased messenger RNA
NC CC SC SCESCR
0
50
100
150
200
b
a
ab
a
a
Serum levels of TC (mg/dL)
NC CC SC SCESCR
0
20
40
60
80
a
bb
ab
b
Serum levels of HDL-C (mg/dL)
NC CC SC SCESCR
0.0
1.5
3.0
4.5
6.0
b
a
aa
a
Athe rogenic inde x (AI)
FIG. 2: Eect of Sparassis crispa on serum cholesterol in rats fed a high-cholesterol diet for 4 weeks: total cho-
lesterol (TC) (A), high-density lipoprotein cholesterol (HDL-C) (B), and atherogenic index ([TC − HDL-C]/HDL-C)
(C). Values are the mean ± standard error of the mean of 6 rats/group. Means with dierent superscript letters are
signicantly dierent at P < 0.05 (Tukey multiple range test). CC, cholesterol control (cholesterol diet); NC, normal
control (normal diet); SC, S. crispa fruiting body + cholesterol diet; SCE, S. crispa extract + cholesterol diet; SCR,
S. crispa residue + cholesterol diet.
(A) (B)
(C)

Volume 17, Number 10, 2015
Hypocholesterolemic Eects of Sparassis crispa in Hypercholesterolemic Rats
971
D. Fecal Excretion of Cholesterol and Bile
Acid
The fecal levels of cholesterol and bile acid are pre-
sented in Fig. 4 and Table 4, respectively. Cholesterol
absorption had no eect on fecal cholesterol, but
among the groups receiving the cholesterol-rich
diet, levels of cholesterol in feces were highest in
the SCE group (CC group: 416.10 μmol/g feces,
SCE group: 468.03 μmol/g feces; P < 0.05), lead-
ing to the inference that supplementation with S.
crispa extract signicantly enhances the excretion
of dietary cholesterol through feces. Furthermore,
supplementation with the S. crispa extract also
resulted in the highest fecal excretion of bile acid in
hypercholesterolemic rats (CC group: 3.84 μmol/g
feces, SCE group: 5.34 μmol/g feces; p < 0.05).
Animals in the SCE group had an exceptionally
higher excretive ratio of secondary bile acid (0.90,
resulting from bacterial action in the colon) in com-
parison with animals in CC group (0.87; P < 0.05).
IV. DISCUSSION
Culinary-medicinal mushrooms are a potential
source of dietary ber: fungal cell walls contain chi-
tin, other hemicelluloses, mannans, and one of the
most interesting functional components, β- glucans.
These compounds, homo- and heteroglucans with
β(1→3), β(1→4), and β(1→6) glucosidic linkages,
are known to play a key role in the benecial prop-
erties of mushrooms, including reduction of blood
cholesterol and blood glucose concentrations.5
S. crispa is a culinary-medicinal mushroom
comprising more than 40% β-glucan.17 The β-glucan
of S. crispa comprises a β-(1→3)-D-glucan back-
bone with single β-(1→6) or β-(1→2)-D-glucosyl
side branching units occurring every 3 or 4 resi-
dues.9,18 In this study 3 dierent samples (a fruiting
body, extract, and residue) were found to contain
24.00, 16.62, and 7.38 g/100 g β-glucan, respec-
tively (Table 1). The body contains signicantly
more soluble β-glucan than the extract or residue.
This is probably because of their dierent molecular
structures or, more simply, the simultaneous pres-
ence of β-glucan molecules with dierent degrees
of polymerization and, consequently, with dier-
ent molecular weights. Soluble β-glucans seem to
have stronger physiological activities than insoluble
β-glucans.19
The ß-glucan in S. crispa has been shown to
cause substantial reduction in blood cholesterol
in animals and humans.20–22 In addition, β-glucan
decreases levels of free fatty acids in blood and
causes a marginal increase in HDL-C levels.23 A pos-
sible mechanism of action of β-glucan in cholesterol
NC CC SC SCESCR
0.0
0.4
0.8
1.2
1.6
a
a
ab
bb
HMG-CoA reductase
Relative gene expression
NC CC SC SCESCR
0
1
2
3
4
aab
ab
ab
b
CYP7A1
Relative gene expression
FIG. 3: Eect of Sparassis crispa on gene expression of hepatic cholesterol 7α-hydroxylase (CYP7A1) (left) and
3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (right) in rats fed a high-cholesterol diet for 4 weeks.
Values are the mean ± standard error of the mean of 6 rats/group. Means with dierent superscript letters are signi-
cantly dierent at P < 0.05 (Tukey multiple range test). CC, cholesterol control (cholesterol diet); NC, normal control
(normal diet); SC, S. crispa fruiting body + cholesterol diet; SCE, S. crispa extract + cholesterol diet; SCR, S. crispa
residue + cholesterol diet.

International Journal of Medicinal Mushrooms
K.B. Hong et al.
972
regulation could be ber binding or an increase
in viscosity in the intestine, leading to decreased
bile acid reabsorption.24 Intestinal viscosity may
depend on the solubility and molecular weight of
the β-glucan present in situ. It has been hypothesized
that, upon ingestion of β-glucan, its low molecular
weight and a propensity to form viscid solutions
result in increased intestinal viscosity, leading to
reduced absorption of bile acid and cholesterol, and
ultimately lowering blood cholesterol and altering
digestive enzyme activities.25,26 Since we used water
to isolate β-glucan from S. crispa fruiting bodies, the
β-glucan concentration in the aqueous extract could
be more than that in the fruiting bodies or residue.
The water solubility and molecular weight of
β-glucan may also inuence its hypocholesterol-
emic eect. Indeed, it has been postulated that
the viscosity of β-glucan in the intestinal tract,
FIG. 4: Eect of Sparassis crispa on fecal cholesterol in rats fed a high-cholesterol diet for 4 weeks. Values are the
mean ± standard error of the mean of 6 rats/group. Means with dierent superscript letters are signicantly dierent
at P < 0.05 (Tukey multiple range test). CC, cholesterol control (cholesterol diet); NC, normal control (normal diet);
SC, S. crispa fruiting body + cholesterol diet; SCE, S. crispa extract + cholesterol diet; SCR, S. crispa residue +
cholesterol diet.
TABLE 4: Eect of Sparassis crispa on Fecal Bile Acid in Rats Fed a High-Cholesterol Diet for 4 Weeks
Bile Acid
(μmol/g Feces)
Study Groups
NC CC SC SCE SCR
CA 1.22 ± 0.21c1.23 ± 0.09c1.33 ± 0.04a1.21 ± 0.32c1.28 ± 0.07b
CDCA 0.84 ± 0.02c0.85 ± 0.03c0.98 ± 0.12a0.83 ± 0.09c0.91 ± 0.08b
DCA 10.24 ± 0.98b10.22 ± 1.00b12.69 ± 1.12a12.34 ± 0.89a11.89 ± 0.76a
LCA 4.08 ± 0.32b3.89 ± 0.45b3.99 ± 0.03b5.34 ± 0.29a4.43 ± 0.78b
Total 16.38 ± 1.32c16.19 ± 1.02c18.99 ± 1.24ab 19.72 ± 1.04a18.51 ± 0.98b
P/B 0.87 ± 0.01b0.87 ± 0.02b0.88 ± 0.04b0.90 ± 0.04a0.88 ± 0.03b
Values are the mean ± standard error of the mean of 6 rats/group. Means with dierent superscript letters are
signicantly dierent at P < 0.05 (Tukey multiple range test).
CA, cholic acid; CC, cholesterol control (cholesterol diet); CDCA, chenodeoxycholic acid; DCA, deoxycho-
lic acid; LCA, lithocholic acid; NC, normal control (normal diet); P/B: secondary bile acid per total bile acid;
SC, S. crispa fruiting body + cholesterol diet; SCE, S. crispa extract + cholesterol diet; SCR, S. crispa residue
+ cholesterol diet.

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Hypocholesterolemic Eects of Sparassis crispa in Hypercholesterolemic Rats
973
which is positively related to its solubility in water
and its molecular weight, is an important deter-
minant of its LDL-cholesterol-lowering eects.
Highly water-soluble β-glucan with a moderate to
high molecular weight may reduce serum LDL-
cholesterol levels more than that with low water
solubility and a low molecular weight. This dier-
ence in eect is explained by the assumption that a
higher intestinal viscosity lowers the reabsorption
of bile acids, leading to increased excretion of bile
acids. Increased bile acid excretion promotes bile
acid synthesis from cholesterol, which increases
LDL-cholesterol uptake in the liver.27 This could
provide an explanation for the attenuation of a
diet-induced increase in blood cholesterol among
animals in the SCE group.
We investigated the effects of S. crispa on
cholesterol metabolism in rats, focusing on the
expression of CYP7A1 and HMG-CoA reductase
at mRNA levels; these are hepatic enzymes cen-
tral to the metabolism of cholesterol and bile acid.
CYP7A1 is the rate-limiting enzyme in the major
bile acid biosynthesis pathway,28 and inhibition of
the intestinal absorption of bile acids and increases
in hepatic CYP7A1 activity play an important role
in reducing levels of cholesterol in vivo.29 In our
study, animals in the SCE group showed enhanced
hepatic CYP7A1 expression (Fig. 3). This overex-
pression of CYP7A1 probably resulted in marked
activation of the bile acid biosynthesis pathway and
decreased HMG-CoA reductase activity.16,29 Thus,
upregulation of CYP7A1 expression could be one
way to eectively lower blood cholesterol. The nd-
ings in our study strongly support the hypothesis
that S. crispa extract–mediated enhancement of
CYP7A1 activity could potentially result in lower-
ing blood cholesterol.
The conversion of cholesterol to bile acid is
the major pathway for cholesterol elimination and
accounts for approximately 50% of daily cholesterol
excretion into feces.30 One possible explanation for
the eect may be cholesterol metabolism–related
enzymes. The increase in excretion of cholesterol
and bile acid seems to be the result of activation
of CYP7A1. Bile acid is synthesized from choles-
terol, and the rate of bile acid synthesis is mainly
controlled by expression of hepatic CYP7A1.31 The
increase in CYP7A1 may enhance the conversion
of cholesterol to bile acid for excretion, thereby
leading to decreased hepatic and blood cholesterol
levels.3 We found that S. crispa extract supplemen-
tation may be associated with an increase in fecal
excretion of cholesterol and bile acid in hyper-
cholesterolemic rats (Fig. 3, Table 4). Thus, the
possible S. crispa extract–mediated enhancement
of CYP7A1 also facilitates cholesterol excretion
from the gastrointestinal system, hence reducing
its levels in blood.
S. crispa extract supplementation in diets
enhanced hepatic cholesterol catabolism through
the upregulation of CYP7A, which led to a decrease
in the expression of hepatic HMG-CoA reductase
at the mRNA level and an increase in the fecal
excretion of cholesterol and bile acid, especially
secondary bile acid.
V. CONCLUSION
S. crispa extract seems to have the potential to
reduce levels of cholesterol in the blood and to
possess benecial properties as a useful functional
food. While current studies have demonstrated the
pharmacological eects of S. crispa, more studies
are needed to support its role in the medical manage-
ment of hypercholesterolemia.
ACKNOWLEDGMENTS
This work was carried out with the support of the
Cooperative Research Program for Agriculture
Science & Technology Development (project no.
PJ9070212011), Rural Development Administration,
Republic of Korea.
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