Journal of Ethnopharmacology 151 (2014) 478484

Contents lists available at ScienceDirect

Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Effect of Cordyceps militaris extract and active constituents

on metabolic parameters of obesity induced by high-fat diet
in C58BL/6J mice
Seon Beom Kim a, Byeongwoo Ahn b, Myounghwan Kim b, Hyeong-Jin Ji b,
Sang-Kyung Shin b, In Pyo Hong c, Chul Young Kim d, Bang Yeon Hwang a, Mi Kyeong Lee a,n
a

College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 361763, Republic of Korea
Laboratory Animal Research Center, Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea
National Academy of Agricultural Science, Suwon, Gyunggi 441-707, Republic of Korea
d
College of Pharmacy, Hanyang University, Ansan, Kyeonggi 426-791, Republic of Korea
b
c

art ic l e i nf o

a b s t r a c t

Article history:
Received 23 September 2013
Received in revised form
28 October 2013
Accepted 28 October 2013
Available online 11 November 2013

Ethnopharmacological relevance: Cordyceps species which is well-known as winter worm summer grass
has long been used as tonics and stimulants to enhance energy, exhibiting a potential for energy
metabolism. Clinical trials have suggested their benecial effect on lipid metabolic disorders such as
hyperlipidemia.
Materials and methods: The effect of Cordyceps militaris on metabolic parameters was investigated using
C58BL/6J mice induced by high-fat diet (HFD). The effect was rst determined by assessing the body and
organ weight. For further investigation, sections of epididymal adipose tissue were stained with
hematoxylin and eosin and the size of epididymal adipocyte was measured by Image analysis system.
Fat accumulation in frozen liver sections was assessed by the Oil Red O staining and the plasma
biochemical parameters were also assessed. Active constituents were characterized using chromatographic and spectroscopic analysis.
Results: The administration of Cordyceps militaris extract (CE) at the dose of 100 mg/kg and 300 mg/kg
reduced body weight gain and food efciency ratio induced by HFD. The amount of epididymal fat and size
of adipocytes were also decreased by CE treatment. In addition, liver weight and fat deposition in liver
were dramatically reduced in CE-treated group. The treatment of CE also showed benecial effects on
plasma parameters related to lipid proles. Further study for the characterization of active constituents of
Cordyceps resulted in the isolation of two new compounds such as cordyrroles A (1) and B (7) together
with 12 known compounds including pyrrole alkaloids and nucleotide derivatives. Among the isolated
compounds, cordyrrole A signicantly inhibited adipocyte differentiation and pancreatic lipase activity,
whereas cordyrrole B was more effective at inhibiting pancreatic lipase. Cordycepin, a characteristic
compound of Cordyceps militaris, decreased the rate of adipocyte differentiation.
Conclusion: Treatment of CE inhibited HFD-induced metabolic disorders, mainly by improvement in
metabolic parameters. As active constituents, pyrrole alkaloids and nucleotide derivatives were characterized. These results suggested that Cordyceps militaris might be benecial for the treatment of metabolic
disorders obesity through the combined actions of diverse constituents.
& 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords:
Cordyceps militaris
Metabolic disorders
High-fat diet
Cordyrroles A and B

1. Introduction
Obesity stems from a prolonged imbalance between the levels
of energy intake and expenditure, with the surplus being stored as
body lipids. Metabolic disorders such as diabetes, atherosclerosis
n
Correspondence to: College of Pharmacy, Chungbuk National University, 410
Seongbong-ro, Heungduk-gu, Cheongju 361-763, Korea. Tel.: 82 43 261 2818;
fax: 82 43 268 2732.
E-mail address: mklee@chungbuk.ac.kr (M.K. Lee).

0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jep.2013.10.064

and liver diseases are closely related to obesity (Kopelman, 2000).

Fatty liver is the abnormal accumulation of triglycerides in the
cytoplasm of hepatocytes and further progresses to steatohepatitis
and advanced stages of liver diseases (Dai et al., 2013). Genetic and
environmental factors play a role in the development of obesity
and the diet is known to be one of the main environmental factors.
Especially, increased fat intake, in part due to Western diet, is
highly associated with body weight gain which can lead to obesity
and other related metabolic diseases. For the study of obesity
and the development of anti-obesity therapeutics, diverse animal

S.B. Kim et al. / Journal of Ethnopharmacology 151 (2014) 478484

model of obesity has been developed using physiologically,

genetically and pharmacologically modication (Speakman et al.,
2008). Among them, high-fat diet (HFD) feeding in rodent can
induce obesity and metabolic disorders that resemble the human
metabolic syndrome (Buettner et al., 2007). Therefore, HFD
induced animal model is widely used for the development of
anti-obesity therapeutics.
Cordyceps species has its trivial name as winter worm summer
grass in Asian country, because of its different appearance in winter
and summer. Its characteristic feature, a pathogenic complex of the
fungus (Cordyceps militaris which belongs to Hypocreaceae Family)
and the caterpillar (Hepialus armoricanus which belongs to Hepialidae), enable to produce diverse skeleton of biologically active compounds. Cordyceps has long been used as tonics and stimulants to
enhance energy, which exhibited a potential for energy metabolism
(Panda and Swain, 2011). Many recent studies and clinical trials have
suggested their benecial effect on lipid metabolic disorders and its
related disorders such as hyperlipidemia and hyperglycemia (Paterson,
2008). Many commercial products are available in the market as
neutraceuticals.
Cordyceps is well known as a rich source of biologically active
components. Nucleosides and polysaccharides are reported as
major characteristic constituents of Cordyceps species. Cordyceps
have been used for treatment of several diseases such as cancer,
fatigue, hyposexualities (Koh et al., 2003; Lee et al., 2006; Kim
et al., 2010). Related to metabolic disorders, favorable roles of
Cordyceps militaris in the regulation of obesity and diabetes have
been reported (Yun et al., 2006; Shimada et al., 2008; Liu et al.,
2011). In the present study, the effect of Cordyceps militaris extract
(CE) was evaluated using high-fat diet (HFD)-induced obese mice.
Characterization of active constituents was also accomplished by
chromatographic and spectroscopic analysis.

2. Materials and methods

2.1. Materials
Dried Cordyceps militaris samples were identied and provided
by Rural Development Administration in November 2009. Cordyceps militaris samples were produced as previously reported
(Choi et al., 1999; Sung et al., 2002; Hong et al., 2010). Briey,
hyphal body suspension of Cordyceps militaris was cultured in rice
bran medium. After incubation, fruity bodies from rice bran
medium were harvested. Voucher specimen was deposited in the
herbarium of College of Pharmacy at Chungbuk National University (CBNU200911-CM). For the preparation of extract, Cordyceps
militaris was pulverized and extracted with 50% ethanol for 2 days.
The ltrate was evaporated in vacuo, which resulted in Cordyceps
militaris extract (CE) for further investigation.
2.2. In vivo anti-obesity effect of Cordyceps extract
2.2.1. Animal treatment
Male C57BL/6J mice (n 60, 4 weeks old, 15 710% g) were
purchased from Central Lab. Animal Inc., Seoul, Korea. All mice
were housed in a room with controlled temperature (2123 1C),
humidity (5560%), and lighting (12 h light/dark cycle), and given
water ad libitum. After acclimation for 1 week, mice were
randomly divided into four groups (n 15/group), normal diet
(ND), high-fat diet control (HFD), HFD CE100 (100 mg/kg of CE),
HFD CE300 (300 mg/kg of CE). Normal diet (ND) group was fed
with normal diet, and HFD, HFD CE100 and HFD CE300 groups
were fed with high fat diet (Table 1) ad libitum. CE sample was
diluted in distilled water (DW) and administered orally to mice to
HFD CE100 and HFD CE300 groups, and DW was administered

479

Table 1
Composition of experimental diets.
Unit: g%

ND

HFD

Formula
Protein
Carbohydrate
Fat

20
64
7

24
41
24

Ingredient
Casein
L-Cystein
Corn starch
Maltodextrin
Sucrose
Cellulose
Soybean oil
Lard
Cholesterol
Mineral mixture
Dicalcium phosphate
Calcium carbonate
Potassium citrate
Vitamin mixture
Choline bitartrate

20
0.3
39.7
13.2
10
5
7
0
0
3.5
0
0
0
1
0.3

23.3
0.3
8.5
11.7
20.1
5.8
2.9
20.7
0.5
1.2
1.5
0.6
1.9
1.2
0.2

orally to ND and HFD groups. At the end of experiment, mice were

anesthetized and blood, organs were collected. The protocol for
this study was approved by the Animal Care and Use Committee of
Chungbuk National University (Approved no. CBNUA-370-11-01).

2.2.2. Histopathology
Histological photograph of adipose tissue was analyzed based
on the parafn method using a light microscope. Epididymal
adipose tissue was xed with 10% neutral buffered formalin and
embedded in parafn block. Six m sections were cut and
mounted on glass slide. Parafn was removed with xylen and
alcohol. The sections were then stained with hematoxylin and
eosin (H&E). After dehydration by alcohol, the photograph was
taken with light microscope. The size of epididymal adipocyte was
calculated by Image analysis system (IPKR-1003, Saramsoft Co.,
Ltd., Korea). For the detection of lipid deposition in liver, liver
section were prepared from frozen liver and stained with Oil Red O
as previously reported (Fowler and Greenspan, 1985).

2.2.3. Serum biochemical parameters

All mice were fasted for 12 h before sacrice. Blood was
collected and serum was obtained by the centrifugation at
3000 rpm for 10 min at 4 1C. The content of triglyceride (TG), total
cholesterol (TC), low-density lipoprotein cholesterol (LDL), highdensity lipoprotein cholesterol (HDL), alanine transaminase (ALT),
aspartate transaminase (AST) and alkaline phosphatase (ALP) were
determined using Hitachi7080 analyzer.
2.3. Characterization of active constituents
2.3.1. Extraction and isolation of compounds.
Cordyceps (1.5 kg) was extracted twice with 50% EtOH, which
yielded the ethanol extract (93.5 g). The ethanol extract was
suspended in H2O and partitioned successively with CH2Cl2,
EtOAc, and n-BuOH. The CH2Cl2 fraction (18.9 g) was subjected
to medium pressure liquid chromatography (MPLC) over silica gel
and eluted with hexane-EtOAcMeOH to give 22 subfractions
(CORM1CORM22). CORM16 was subjected to column chromatography over Sephadex LH-20 and eluted with CH2Cl2MeOH
to yield four subfractions (CORM16ACORM16D). Compounds 3
(1.9 mg) and 4 (4.0 mg) were obtained from CORE6 and CORM8,

480

S.B. Kim et al. / Journal of Ethnopharmacology 151 (2014) 478484

respectively, by semi-preparative high pressure liquid chromatography (HPLC) and eluted with MeCN-water and MeOH-water.
The n-BuOH fraction (67.9 g) was subjected to HP-20 column
chromatography with mixture of MeOH-water (0, 20, 40, 60, 80,
and 100% MeOH in water) to give six fractions (CORB1CORB6).
The CORB2 fraction was subjected to silica gel MPLC and eluted
with CH2Cl2MeOH to yield ten subfractions (CORB2ACORB2J).
Compound 5 (3.0 mg) was obtained from CORB2C3 by semipreparative HPLC and eluted with MeOH-water. Compounds 12
(1.5 mg), 13 (2.1 mg), and 14 (2.5 mg) were obtained from COR2G
by column chromatography over Sephadex LH-20 eluted with
MeOH, followed by semi-preparative HPLC eluted with MeOHwater. Compound 8 (2.4 mg) and 11 (3.4 mg) were obtained from
CORB2I by column chromatography over Sephadex LH-20 eluting
with MeOH. Compound 9 (1.4 mg) was obtained from CORB2J by
semi-preparative HPLC eluting with MeOH-water.
The CORB3 fraction was subjected to silica gel MPLC and eluted
with CH2Cl2MeOH to yield 12 fractions (CORB3ACORB3L). Compound 2 (1.7 mg) was obtained from CORB3A by column chromatography over Sephadex LH-20 eluted with MeOH, followed by
semi-preparative HPLC eluted with MeOH-water. Compound 1
(1.6 mg) was obtained from CORB3C by column chromatography
eluted with MeOH. Compound 10 (6.3 mg) was obtained from
CORB3D by column chromatography over Sephadex LH-20 eluted
with MeOH, followed by semi-preparative HPLC eluted with
MeOH-water.

2.3.2. Spectroscopic data of the new compounds

Cordyrrole A (1): Light brown amorphous gum; 25
D  177.01 (c
0.08, MeOH); UV (MeOH) max 294 nm; IRmax 3345, 1645 cm  1; 1H
NMR (CD3OD, 400 MHz) 9.34 (1H, s, H-1), 7.07 (1H, d, J 4.0 Hz,
H-3), 6.29 (1H, d, J 4.0 Hz, H-4), 5.06 (1H, dd, J 11.2, 6.8 Hz,
H-1), 4.66 (2H, d, J 2.0 Hz, H-6), 3.65 (1H, m, H-4a), 3.35 (1H, m,

H-4b), 2.27 (2H, m, H-2), 2.01 (2H, m, H-3) ppm; 13C NMR
(CD3OD, 100 MHz) 178.8 (C-1), 170.5 (C-6), 143.9 (C-2), 132.0
(C-5), 125.8 (C-3), 109.8 (C-4), 56.5 (C-1), 55.4 (C-6), 41.5 (C-4),
28.8 (C-2), 22.0 (C-3) ppm; ESIMS (negative mode) m/z: 221
[M H]  ; HRESIMS (positive mode) m/z: 245.0911 (calc for
C11H14N2O3 245.1004).
Cordyrrole B (7): White amorphous powder; UV (MeOH) max
265 nm; IRmax 3316, 2927, 1730, 1617 cm  1; 1H NMR (CD3OD,
400 MHz) 8.28 (1H, s, H-7), 8.26 (1H, s, H-2), 5.98 (1H, d,
J 7.6 Hz, H-1), 4.76 (1H, dd, J 5.2, 6.4 Hz, H-2), 4.35 (1H, m,
H-3), 4.32 (2H, t, J 5.6 Hz, H-2), 4.19 (1H, dd, J 2.5, 5.2 Hz, H-4
a), 3.91 (2H, m, H-1), 3.90 (1H, dd, J 2.4, 12.8 Hz, H-5), 3.76 (1H,
dd, J 2.4, 12.8 Hz, H-4b), 2.05 (3H, s, COCH3) ppm; 13C NMR
(CD3OD, 100 MHz) 152.0 (C-2), 148.2 (C-4), 115.6 (C-5), 154.9
(C-6), 140.2 (C-7), 39.1 (C-1), 62.8 (C-2), 171.4 (COCH3), 19.3 (COCH3),
89.8 (C-1), 74.0 (C-2), 71.2 (C-3), 86.8 (C-4), 62.1 (C-5) ppm;
ESIMS (positive mode) m/z: 354 [MH]; HRESI-TOF-MS (positive
mode) m/z: 354.1408 (calc for C14H19N5O6 354.1414).

2.4. In vitro activity of compounds

2.4.1. Pancreatic lipase activity
Pancreatic lipase inhibitory activity was evaluated using a
method reported previously (Kim et al., 2012). The enzyme
solution was prepared by reconstituting porcine pancreatic lipase,
and pancreatic lipase activity was determined by measuring the
hydrolysis of p-NPB to p-nitrophenol at 405 nm using a microplate
reader. Relative pancreatic lipase activity (%) was calculated as
(activity of compound with substrate negative control of
compound without substrate)/(activity of without compound
and with substrate negative control of without compound and
substrate)  100.

Fig. 1. Effect of CE100 and CE300 on body weight (A), FER (C) and organ weight (C) in HFD-induced obese mice. FER was calculated as total weight gain/total food intake.
Results are expressed as the mean 7 SE (n 1215). np o0.05 compared with ND group, #p o0.05 compared with HFD group.

S.B. Kim et al. / Journal of Ethnopharmacology 151 (2014) 478484

2.4.2. Adipocyte differentiation

3T3-L1 mouse embryo broblasts (ATCC CL-173) were obtained
from the American Type Culture Collection (Manassas, VA). The cells
were stimulated to differentiate with differentiation medium containing DMEM with 10% FBS, 0.5 mM 3-isobutyl-1-methyl-xanthine,
1 M insulin and 1 M dexamethasone for 2 days (day 2). Cells were
then maintained in DMEM supplemented with 10% fetal bovine
serum (FBS) and 1 M insulin for another 2 days (day 4), followed by
culturing with DMEM with 10% FBS for an additional 4 days (day 8).
The cultures were treated with 100 M isolated compounds for the
entire culture period (days 08). Lipid droplets in cells were stained
with Oil Red O. The Oil Red O stain was dissolved in isopropyl alcohol
and optical density was measured at 520 nm using ELISA plate reader
for the quantitative analysis (Choi et al., 2011).
2.5. Statistical analysis
The evaluation of statistical signicance was determined by
Levene's test followed by one-way ANOVA or Turkey HSD-test
with a value of p o0.05 considered to be statistically signicant.

3. Results and discussion

3.1. In vivo effect of the CE on HFD-induced obesity
The effect of CE on obesity was investigated using HFD-induced
male C58BL/6J mice. Diet-induced obesity in rodents has been
used as an animal model to investigate environmental effect. HFDfed rodents become obese and show distinctive symptoms such as
increase of adipose tissue, disturbance of lipid metabolism, hyperinsulinemia and fatty liver, which are typically associated with
human obesity (Sclafani and Springer, 1976). In our study, the body

481

weight of HFD group was signicantly increased compared to

that of ND after 2 weeks diet. However, the body weight gains of
HFD CE100 and HFD CE300 groups were signicantly reduced
compared to HFD groups, after 21 days and 10 days treatment,
respectively (Fig. 1A). On day 42, at the end of experiment, the
body weight gains of CE100 [4.29 g] and CE300-treated groups
[4.01 g] were reduced dramatically compared to HFD group
[8.39 g]. The relative ratio of epididymal fat was also signicantly
increased in HFD group compared to ND group (Fig. 2A). Treatment of CE100 and CE300 signicantly reduced the relative ratio
of epididymal fat in dose related manner. The liver weight in HFD
group was signicantly increased compared to ND group in our
study. However, the increase of liver weight induced by high
fat diet was signicantly attenuated in CE100 and CE300 groups
compared to HFD group. The treatment of CE100 and CE300,
however, did not affect the relative weight spleen and heart
whereas relative weight of kidney was slightly reduced (Fig. 1C).
Food intake was higher in the ND group than in HFD group, but did
not differ signicantly among the HFD, HFD CE100 and
HFD CE300 groups. However, food efciency ratio (FER) of
HFD CE100 and HFD CE300 groups were signicantly reduced
compared to HFD group (Fig. 1B).
High ratio of fat consumption accompanies excessive growth
of adipose tissue in both cell number and cell size, and consequently induces fat accumulation. As shown in Fig. 2A, the size of
adipocyte in epididymal tissue was greatly increased in HFD
group, as analyzed by image analyzer after H&E staining. However,
the size of adipocyte in CE100 and CE300-treated groups was
dramatically decreased compared to HFD (Fig. 2B). HFD diet also
induced fat accumulation in liver, which was easily observed
by Oil Red O staining (Fig. 2C). However, treatment of CE100
and CE300 greatly reduced fat accumulation in liver. Especially, fat
accumulation in HFD CE300 group was almost completely

Fig. 2. Effect of CE100 and CE300 on lipid parameters in HFD-induced obese mice. (A) Sections of epididymal adipose tissue stained with H&E (magnication, 200  ).
(B) Quantitation of the size of epididymal adipocyte by Image analysis system. (C) Fat accumulation in frozen liver sections stained with Oil Red O. (D) Plasma biochemical
parameters. Data are expressed as mean 7 SD (n 1215 per group). ALP, alkaline phosphatase (IU/l); AST, aspartate transaminase (IU/l); ALT, alanine transaminase (IU/l);
HDL, high-density lipoprotein (mg/dl); LDL, low-density lipoprotein (mg/dl); TC, total cholesterol (mg/dl); TG, triglyceride (mg/dl); np o 0.05 compared with ND group,
#
p o 0.05 compared with HFD group.

482

S.B. Kim et al. / Journal of Ethnopharmacology 151 (2014) 478484

reduced comparable to ND group. These result shows that CE100

and CE300 efciently inhibit fat accumulation in liver and epididymal adipocyte tissues.
Persistent high fat and cholesterol diets cause damage to liver,
which results in change of enzymes and lipid proles. HFD caused
elevation of plasma level of ALP, AST and ALT, which was recovered
in HFD CE100 and HFD CE300 groups. In addition, HFD-induced
increases of TC, LDL and TG were signicantly decreased in CE100
and CE300-treated groups. HDL, however, was decreased in HFD
group, which was signicantly recovered in CE100 and CE300treated group (Fig. 2D).

3.2. Structural elucidation

3.2.1. Structural determination of new compounds
Compound 1 was obtained as a pale brown amorphous powder
with a molecular formula of C11H14N2O3, determined by HRESI-MS
(m/z 245.0910 [M Na] ; calc 222.1004). The IR spectrum showed
the presence of an amine group (3345 cm  1) and carbonyl group
(1645 cm  1). The maximum UV absorption was observed at
293 nm, which is characteristic of a pyrrole alkaloid (Chin et al.,
2003). The presence of pyrrole ring was also supported by the
characteristic J value of the pyrrole signals at H 7.08 (1H, d,
J 4.0 Hz), 6.29 (1H, d, J 4.0 Hz) together with four carbon signals
at C 143.9, 132.0, 125.8, and 109.8 in the 1H- and 13C NMR spectra.
The presences of aldehydes and hydroxymethyl moieties were
easily deduced from the signals at [H 9.34 (1H, s) and C 178.8]
and [H 4.66 (2H, d, J 2.0) and C 55.4], respectively, in the HSQC
spectrum. The HSQC Spectrum of 1 also showed a methine signal
at [H 5.06 (1H, dd, J 11.2 6.8) and C 56.5] and three methylene
signals at [H 2.27 (2H, m) and C 28.8], [H 2.01 (2H, m) and C
22.0] and [H 3.65 (1H, m), 3.35 (1H, m) and C 41.5]. An additional
carbonyl signal was observed at C 170.5 in the 13C NMR spectrum,. The HMBC correlations were observed between H 3.65

(H-4a) and C 22.0 (C-3), between 3.35 (H-4b) and C 28.8 (C-2),
170.5 (C-6), and between H 5.06 (H-1) and C 28.8 (C-2), 170.5
(C-6), suggesting the presence of a 2-piperidone ring (Ghosh et al.,
2009). The position of the 2-peperidone ring was determined to be
N-1 by the correlation between at H 5.06 (H-1) and C 132.0
(C-5), 143.9 (C-2) in the HMBC spectrum. The positions of the
aldehyde and hydroxymethyl moiety were C-2 and C-5, respectively, by the HMBC correlation between H 9.34 (H-1) and C
143.9 (C-2), and between H 4.66 (H-6) and C 132.0 (C-5). Based
on these data, the structure of 1 was determined as shown in Fig. 3
and named cordyrrole A.
Compound 7 was obtained as a white amorphous powder with
a molecular formula of C14H19N5O6, determined by HRESIMS (m/z
354.1408 [M H] ; calcd 354.1414). The 1H- and 13C NMR spectra
of 7 showed characteristic signals for adenosine, a major constituent of Cordyceps (Furuya et al., 1983). The 1H and 13C NMR of 7
also showed two methylene signals at [H 3.91 (2H, m) and 4.32
(2H, t, J 5.6 Hz)] and [c 39.1 and 62.8], which was very similar to
those of N6-(2-hydroxyethyl)adenosine.18However, additional signals for an acetyl moiety were observed at [H 2.05 (3H, s), C 19.3
and 171.4] in the HMBC spectrum, which suggested 7 as an
acetylated form of N6-(2-hydroxyethyl)adenosine. The position of
an acetyl moiety was determined from the HMBC correlation
between C 171.4 (COCH3) and H 4.32 (H-2). Taken together,
the structure of 7 was determined as shown and was named
cordyrrole B.

3.2.2. Identication of known compounds

Twelve known compounds were identied, by the spectroscopic
data analysis and comparison with literature values including 5(hydroxymethyl)-1-(2-oxopiperidin-3-yl)-1H-pyrrole-2-carbaldehyde
(2), dihydrouracil (3), uracil (4), nicotinamide (5), N6-(2-hydroxyethyl)
adenosine (6), cordycepin (8), adenosine (9), 2-O-methyladenosine
(10), xanthosine (11), 2-deoxyuridine (12), uridine (13), thymine (14)

Fig. 3. (A) Structures of compounds 114 from Cordyceps militaris and (B) key HMBC correlations of new compounds, cordyrroles A (1) and B (7).

S.B. Kim et al. / Journal of Ethnopharmacology 151 (2014) 478484

483

in the adipocyte, which might have disturbed fat accumulation in

the liver induced by HFD. Collectively, inhibition of fat absorption
and fat accumulation by the constituents of CE are responsible for
the reduction of fat accumulation in liver and adipocyte, which
leads to recover liver function and lipid metabolism.

4. Conclusion

Fig. 4. Effects of isolated compounds on adipocyte differentiation and pancreatic

lipase. Activity was measured at 100 M and expressed as mean 7 SD of three
independent experiments each performed in triplicate. The optical density of
controls in adipocyte differentiation and pancreatic lipase were 0.934 7 0.059
and 0.4777 0.024, respectively. npo 0.05 compared with control.

(Furuya et al., 1983; Jiang, Gerwick, 1991; Beigelman et al., 2000;

Jurczyk et al., 2000; Zhu et al., 2011; Zhang, Xu, 2011). (Fig. 3)
3.3. In vitro activity of isolated compounds
The effects of the isolated compounds on adipocyte differentiation
were investigated using 3T3-L1 preadipocytes as an assay system.
Among the isolated compounds, compounds 1 and 8 signicantly
reduced fat accumulation in differentiated 3T3-L1 cells (Fig. 4). The
inhibitory effects of isolated compounds on pancreatic lipase were
investigated employing porcine pancreatic lipase and p-nitrophenyl
butyrate as the substrate. Among the isolated compounds, two new
compounds 1 and 7 showed the most signicant inhibitory effects
(Fig. 4). Other compounds also signicantly inhibited pancreatic lipase
activity.
3.4. Effect of Cordyceps and its compounds on lipid metabolism
In our present study, increase of fat accumulation and abnormal
lipid metabolism were observed in HFD group, which was improved in
HFDCE100 and HFD CE300 groups. Especially, the liver weight and
fat accumulation in liver were greatly increased in HFD groups, which
was reduced in HFD CE100 and HFDCE300 groups comparable to
ND group. Therefore, treatment of CE100 and CE300 inhibited fat
accumulation into liver, which resulted in decrease of liver weight.
HFD-induced increase of epididymal fat weight and adipocyte size
were also improved in HFDCE100 and HFD CE300 groups. In
addition, biochemical parameters related to lipid metabolism, such
as TC, HDL, LDL and TG were also recovered in HFDCE100 and
HFDCE300 groups. Taken together, treatment of CE100 and CE300
mainly act on inhibition of fat accumulation, which further resulted in
decrease in liver weight and lipid proles in blood. Therefore, CE
might be effective in liver dysfunction induced by HFD, which was
supported by the improvement of ALP, AST and ALT parameters in
HFDCE100 and HFDCE300 groups.
Taken together, oral administration of CE at 100 and 300 mg/kg
dramatically improved many parameters of HFD-induced obesity.
However, food intake was not affected by the treatment of CE100
and CE300 groups, which resulted in the decrease of FERs in the
HFD CE100 and HFD CE300 groups (Fig. 2B). Therefore, the
decrease in body weight in the HFD CE100 and HFD CE300
groups was achieved partially by a decrease in FER not by a loss of
appetite. Compounds isolated from Cordyceps, including two new
compounds, inhibited pancreatic lipase activity (Fig. 2). Therefore,
treatment of CE reduced fat absorption by the inhibition of
pancreatic lipase. These compounds also reduced fat accumulation

Conclusively, two new compounds, cordyrroles A and B, were

isolated from Cordyceps together with twelve known compounds.
They were effectively inhibited pancreatic lipase activity and
adipocyte differentiation. Cordyceps extract was also improved
many metabolic parameters in high-fat diet animal model. These
results suggested that CE might be benecial for the prevention
and/or treatment of obesity and related metabolic disorders.

Acknowledgements
This study was supported by a grant from the Korean Rural
Development Administration (Agenda Program, PJ0067792010),
and by Basic Science Research Program (2012-0008023) and
Medical Research Center Program (2010-0029480) through the
National Research Foundation of Korea.

Appendix A. Supplementary material

Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.jep.2013.10.064.
References
Beigelman, L., Haeberli, P., Sweedler, D., Karpeisky, A., 2000. Improved synthetic
approaches toward 2mpO-methyl-adenosine and guanosine and their N-acyl
derivatives. Tetrahedron 56, 10471056.
Buettner, R., Schlmerich, J., Bolheimer, C., 2007. High-fat diets: modeling the
metabolic disorders of human obesity in rodents. Obesity 15, 798808.
Chin, Y.W., Lim, S.W., Kim, S.H., Shin, D.Y., Suh, Y.G., Kim, Y.B., Kim, Y.C., Kim, J., 2003.
Hepatoprotective pyrrole derivatives of Lycium chinensis fruits. Bioorg. Med.
Chem. Lett. 13, 7981.
Choi, I.Y., Choi, J.S., Lee, W.H., Yu, Y.J., Joung, G.T., Ju, I.O., Choi, Y.K., 1999. The
condition of production of articial fruiting body of Cordyceps militaris. Korean
J. Mycol. 27, 243248.
Choi, K.M., Shin, E., Liu, Q., Yoo, H.S., Kim, Y.C., Sung, S.H., Hwang, B.Y., Lee, M.K.,
2011. Hydroxyframoside B, a secoiridoid of Fraxinus rhynchophylla, inhibits
adipocyte differentiation in 3T3-L1 cells. Planta Med. 77, 10201023.
Dai, F.J., Hsu, W.H., Huang, J.J., Wu, S.C., 2013. Effect of pigeon pea (Cajanus cajan L.)
on high-fat diet-induced hypercholesterolemia in hamsters. Food Chem.
Toxicol. 53, 384391.
Fowler, S.E., Greenspan, P., 1985. Application of Nile red, a uorescent hydrophobic
probe, for the detection of neutral lipid deposits in tissue section: comparison
with Oil Red O. J. Histochem. Cytochem. 33, 833836.
Furuya, T., Hirotani, M., Matsuzawa, M., 1983. N6-(2-hydroxyethyl)adenosine, a
biologically active compound from cultured mycelia of Cordyceps and Isaria
species. Phytochemistry 22, 25092512.
Ghosh, S.C., Muthaiah, S., Zhang, Y., Xu, X., Hong, S.H., 2009. Direct amide synthesis
from alcohols and amines by phosphine-free ruthenium catalyst systems. Adv.
Synth. Catal. 351, 26432649.
Hong, I.P., Kang, P.D., Kim, K.Y., Nam, S.H., Lee, M.Y., Choi, Y.S., Kim, N.S., Kim, H.K.,
Lee, K.G., Humber, R.A., 2010. Fruit body formation on silkworm by Cordyceps
militaris. Mycobiology 38, 128132.
Jiang, Z.D., Gerwick, W.H., 1991. Novel pyrroles from the oregon red alga
gracilariopsis lemaneiformis. J. Nat. Prod. 54, 403407.
Jurczyk, S.C., Horlacher, J., Devined, K.G., Benner, S.A., Battersby, T.R., 2000.
Synthesis and characterization of oligonucleotides containing 2-deoxyxanthosine using phosphoramidite chemistry. Helv. Chim. Acta 83, 15171524.
Kim, E.S., Lee, K.J., Oh, K.H., Ahn, J.H., Kim, S.B., Liu, Q., Hwang, B.Y., Lee, M.K., 2012.
Inhibitory effects of marine algae extract on adipocyte differentiation and
pancreatic lipase activity. Nat. Prod. Sci. 18, 153157.
Kim, H.S., Kim, J.Y., Kang, J.S., Kim, H.M., Kim, Y.O., Hong, I.P., Lee, M.K., Hong, J.T.,
Kim, Y., Han, S.-B., 2010. Cordlan polysaccharide isolated from mushroom
Cordyceps militaris induces dendritic cell maturation through toll-like receptor
4 signaling. Food Chem. Toxicol. 48, 19261933.

484

S.B. Kim et al. / Journal of Ethnopharmacology 151 (2014) 478484

Koh, J.H., Kim, K.M., Kim, J.M., Song, J.C., Suh, H.J., 2003. Antifatigue and antistress
effect of the hot-water fraction from mycelia of Cordyceps sinensis. Biol. Pharm.
Bull. 26, 691694.
Kopelman, P.G., 2000. Obesity as a medical problem. Nature 404, 635643.
Lee, H., Kim, Y.J., Kim, H.W., Lee, D.H., Sung, M.K., Park, T., 2006. Induction of
apoptosis by Cordyceps militaris through activation of caspase-3 in leukemia
HL-60 cells. Biol. Pharm. Bull. 29, 670674.
Liu, Q., Hong, I.P., Ahn, M.-J., Yoo, H.-S., Han, S.-B., Hwang, B.Y., Lee, M.K., 2011. Antiadipogenic activity of Cordyceps militaris in 3T3-L1 cells. Nat. Prod. Commun. 6,
18391841.
Panda, A.K., Swain, K.C., 2011. Traditional uses and medicinal potential of Cordyceps
sinensis of Sikkim. J. Ayurveda Integr. Med. 2, 913.
Paterson, R.R.M., 2008. CordycepsA traditional Chinese medicine and another
fungal therapeutic biofactory? Phytochemisty 69, 14691495.
Sclafani, A., Springer, D., 1976. Dietary obesity in adult rats: similarities to
hypothalamic and human obesity syndromes. Physiol. Behav. 17, 461471.
Shimada, T., Hiramatsu, N., Kasai, A., Mukai, M., Okamura, M., Yao, J., Huang, T.,
Tamai, M., Takahashi, S., Nakamura, T., Kitamura, M., 2008. Suppression of

adipocyte differentiation by Cordyceps militaris through activation of the aryl

hydrocarbon receptor. Am. J. Physiol. Endocrinol. Metab., Endocrinology and
Metabolism 295, E859E867.
Speakman, J., Hambly, C., Mitchell, S., Krl, E., 2008. The contribution of animal
models to the study of obesity. Lab. Anim. 42, 413432.
Sung, J.M., Choi, Y.S., Shrestha, B., Park, Y.J., 2002. Investigation on articial fruiting
of Cordyceps militaris. Korean J. Mycol. 30, 610.
Yun, Y., Han, S., Lee, S., Ko, S., Lee, C., Ha, N., Kim, K., 2006. Anti-diabetic effects of
CCCA, cmESS, and cordycepin from Cordyceps militaris and the immune
responses in streptozotocin-induced diabetic mice. Nat. Prod. Sci. 9, 291298.
Zhang, X., Xu, Y.Z., 2011. NMR and UV Studies of 4-thio-2treptozotocin-inducits
derivatives. Molecules 16, 56555664.
Zhu, L., Liang, Y., Lao, D., Zhang, T., Itoc, Y., 2011. Preparative separation of highpurity cordycepin from Cordyceps militaris (L.) link by high-speed countercurrent chromatography. J. Liq. Chromatogr. Related Technol. 37, 491499.