In recent years, many natural polysaccharides and
polysaccharide-protein complexes were isolated from fungi
and used as a source of therapeutic agents (Novak & Vetvicka,
2008). Among them, Cordyceps militaris, an entomopathogenic
fungus belonging to the class Ascomycetes, has been extensively
used as a crude drug and a folk tonic food in East Asia. C. militaris
is known as the Chinese rare caterpillar fungus and has similar
pharmacological activities to the well-known Chinese traditional
medicine Cordyceps sinensis (Gai, Jin, Wang, Li, & Li, 2004; Zheng &
Cai, 2004). The beneficial effects of Cordyceps on renal and hepatic
functions and immunomodulation-related antitumour activities
are most promising and deserve further attention (Paterson, 2008).
Various bioactive constituents from the Cordyceps species have
been reported, such as cordycepin, polysaccharides, antibacterial
and antitumour adenosine derivatives, ophicordin, an antifungal
agent, and L-tryptophan. Polysaccharides are considered one of
the major active components of Cordyceps. Purified polysaccha-rides from C. militaris have numerous biological activities, such as
antioxidant (Li, Li, Dong, & Tsim, 2001; Li et al., 2003), immunomod-ulatory
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Carbohydrate Polymers 89 (2012) 461– 466
Contents lists available at SciVerse ScienceDirect
Carbohydrate Polymers
jo u rn al hom epa ge: www.elsev ier .com
Cordyc ce t
activity
Mi Wang ang
Ying Lib,
a Institute of Tr tural U
b Department o cience
a r t i c l
Article history:
Received 18 Ja
Received in re
Accepted 8 Ma
Available onlin
Keywords:
Cordyceps mili
Cyclophospha
immunosuppr
Immunomodulation
Anti-oxidation activity in vivo
nd re
0 ma
s we
dy w
ters w
duced
leen
imm
perox
the malondialdehyde levels in vivo.
© 2012 Elsevier Ltd. All rights reserved.
1. Introdu
In rece
polysacchar
and used a
2008). Amo
fungus belo
used as a cr
is known a
pharmacolo
medicine Co
Cai, 2004). T
functions a
are most pr
Various
been report
and antitum
agent, and
the major a
rides from C
antioxidant
ulatory (Ch
∗ Correspon
∗∗ Correspon
E-mail add
0144-8617/$ –
doi:10.1016/j.ction
nt years, many natural polysaccharides and
ide-protein complexes were isolated from fungi
s a source of therapeutic agents (Novak & Vetvicka,
ng them, Cordyceps militaris, an entomopathogenic
nging to the class Ascomycetes, has been extensively
ude drug and a folk tonic food in East Asia. C. militaris
s the Chinese rare caterpillar fungus and has similar
gical activities to the well-known Chinese traditional
rdyceps sinensis (Gai, Jin, Wang, Li, & Li, 2004; Zheng &
he beneficial effects of Cordyceps on renal and hepatic
nd immunomodulation-related antitumour activities
omising and deserve further attention (Paterson, 2008).
bioactive constituents from the Cordyceps species have
ed, such as cordycepin, polysaccharides, antibacterial
our adenosine derivatives, ophicordin, an antifungal
L-tryptophan. Polysaccharides are considered one of
ctive components of Cordyceps. Purified polysaccha-
. militaris have numerous biological activities, such as
(Li, Li, Dong, & Tsim, 2001; Li et al., 2003), immunomod-
eung et al., 2009; Kim et al., 2008), antitumour (Park,
ding author. Tel.: +86 25 84395203; fax: +86 25 84398669.
ding author. Tel.: +86 21 34293460; fax: +86 21 34293396.
resses: ylhu@njau.edu.cn (Y.l. Hu), feiqun@gmail.com (F.Q. Xue).
Kim, Lee, Yoo, & Cho, 2009; Rao, Fang, Wu, & Tzeng, 2010), and
anti-inflammatory (Rao et al., 2010).
Previous studies on the immunomodulatory and antioxidant
effects of C. militaris polysaccharides (CMPs) in in vitro systems
have been conducted. CMPs can induce the functional activation of
macrophages through the upregulation of cytokine expression and
nitric oxide (NO) release (Lee et al., 2010), induce T-lymphocyte
proliferation and secretion of interleukin (IL)-2, IL-6, and IL-8 (Chen,
Zhang, Shen, & Wang, 2010), and stimulate the phagocytosis of
macrophages in vitro. These results confirm the important role of
CMPs in triggering immune responses. The CMPs fractions P70-
1 and CBP-1 were found to exhibit hydroxyl radical-scavenging
activity in vitro (Yu et al., 2007, 2009).
In the present study, the fruiting body of C. militaris came
from Shanghai, which has been scarcely investigated. Successive
tests were conducted to evaluate the immune activation and reac-
tive oxygen species (ROS)-scavenging activity of CMP in vivo. The
details are reported in the current study.
2. Materials and methods
2.1. Material
Dry cultured C. militaris was obtained from Shanghai Dianzhi
Bioengineering Corp. (Shanghai, China). The material (No. 06-
01-0727) was identified by Associate Researcher X.H. Gao
of the Research Group of Dong Chong Xia Cao, Shanghai
see front matter © 2012 Elsevier Ltd. All rights reserved.
carbpol.2012.03.029eps militaris polysaccharides can enhan
in immunosuppressed mice
a,b, Xin Yu Mengb, Rui Le Yangb, Tao Qina, Xiao Y
Yuan liang Hua,∗, Fei Qun Xueb,∗∗
aditional Chinese Veterinary Medicine, College of Veterinary Medicine, Nanjing Agricul
f Pharmacy, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural S
e i n f o
nuary 2012
vised form 28 February 2012
rch 2012
e 19 March 2012
taris polysaccharides
mide-induced
ession
a b s t r a c t
To evaluate the immune activation a
taris polysaccharides (CMP) in vivo, 9
mice in the three experimental group
injection and 17.5, 35, or 70 mg/kg bo
cytic index, and biochemical parame
CMP was able to overcome the CY-in
thymus indices, and enhanced the sp
improve the antioxidation activity in
dismutase, catalase, and glutathione / locate /carbpol
he immunity and antioxidation
Wangb, Ke Yu Zhangb, Chen Zhong Feib,
niversity, Nanjing 210095, PR China
s, Shanghai 200241, PR China
active oxygen species scavenging activity of Cordyceps mili-
le BALB/c mice were randomly divided into six groups. The
re given cyclophosphamide at 80 mg/kg/d via intraperitoneal
eight CMP via gavage. The lymphocyte proliferation, phago-
ere measured. The results show that the administration of
immunosuppression, significantly increased the spleen and
lymphocyte activity and macrophage function. CMP can also
unosuppressed mice, significantly increase the superoxidase
idase levels and the total antioxidant capacity, and decrease
462 M. Wang et al. / Carbohydrate Polymers 89 (2012) 461– 466
Academy of Agricultural Sciences, Shanghai, China. RPMI 1640
was purchased from Gibco. The T-cell mitogen concanavalin
A (ConA)
(DMSO) w
Engineering
diphenyltet
Co. Assay
ondialdehy
(SOD), and
the Nanjing
Cyclophosp
Medicine C
albumin, Co
purchased f
membrane
All chemica
2.2. Polysac
Polysacc
described (
der of cultu
subsequent
2 h. The resu
and filtered
aqueous so
reduced pr
centrifugati
then suspen
(w/w) poly
anthrone w
2.3. Animal
Male BA
from Shan
Academy o
vided with
in a rodent
acclimatiza
were appro
of Agricultu
groups cons
used for ph
test, 3 were
the other ex
to their env
mice were
groups and
70 mg/kg bo
to 3, the ot
intraperiton
istered as fo
CMP groups
administere
istered via g
last drug ad
ficed via de
were excise
The thymu
formula, ind
2.4. Lymph
The mou
ficed mice
homogenised, and passed through a 40 m nylon cell strainer to
obtain single-cell suspensions in accordance with the method used
n, So
e we
esusp
m su
rp., C
lin. S
onta
or 3
ted f
ntrif
. DM
unt
red o
agoc
func
ce te
proc
ly in
speci
inal
.1% N
ible
nd th
ted a
OD1
t2
OD1
gocy
, B is
oche
orga
repa
salin
, and
, T-A
e MD
thod
chiy
idati
as m
Benz
omol
atisti
data
ndow
ana
’s te
nsid
ults
fect o
sple
ogno
dice
ose was purchased from Sigma. Dimethyl sulfoxide
as acquired from the Yixin Institute of Chemical
(Jiangsu, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-
razolium bromide (MTT) was purchased from Amresco
kits for the total antioxidant capacity (TAOC), mal-
de (MDA), catalase (CAT), superoxidase dismutase
glutathione peroxidase (GSH-Px) were purchased from
Jiancheng Bioengineering Institute (Nanjing, China).
hamide (CY) was purchased from Jiangsu Hengrui
o., Ltd. (Lianyungang, Jiangsu, China). Bovine serum
omassie Brilliant Blue G-250, and cellulose sacks were
rom Sigma Chemical Co. (St. Louis, MO, USA). The filter
was purchased from Millipore Corp. (Billerica, MA, USA).
ls used in the experiments were of analytical grade.
charide extraction
harides from C. militaris were prepared as previously
Li, Yang, & Tsim, 2006; Yu et al., 2007). The dried pow-
red C. militaris was defatted with ethanol for 10 h and
ly extracted three times with hot water (100 ◦C) for
lting suspension was centrifuged (8000 × g for 20 min)
through a 0.45 m membrane (Millipore). The filtered
lution was concentrated to a specific volume under
essure. The dark brown precipitate was collected via
on and washed twice with ethanol. The precipitate was
ded in water and lyophilized to yield CMP with 41.2%
saccharide content, which was measured using vitriol-
ith anhydrous glucose as the standard control.
and experimental design
LB/c mice (8 weeks old, 18 h to 20 g) were purchased
ghai Slac Laboratory Animal Center of the Chinese
f Sciences (Shanghai, China). The animals were pro-
water and mouse chow ad libitum and were housed
facility at (22 ± 1) ◦C with a 12 h light-dark cycle for
tion. All procedures involving animals and their care
ved by the Ethics Committee of the Chinese Academy
ral Sciences. The mice were randomly divided into 6
isting of 15 mice each. Three mice from each group were
agocytic index determination in the carbon clearance
used for lymphocyte proliferation, and 9 were used for
periments. All animals were allowed one week to adapt
ironment before the treatment. Two groups of healthy
used as normal control (NS group) and positive control
treated once daily with physiological saline solution and
dy weight CMP, respectively, for 18 days. From days 1
her four groups of mice were given 80 mg/kg/d CY via
eal injection. From days 4 to 18, the mice were admin-
llows: model group, physiological saline solution; three
, 17.5, 35, or 70 mg/kg body weight CMP. CY (0.2 ml) was
d via intraperitoneal injection. The others were admin-
avage in 0.2 ml solutions. Twenty-four hours after the
ministration, the animals were weighed and then sacri-
capitation. The heart, liver, kidney, spleen, and thymus
d; the spleen and thymus were immediately weighed.
s and spleen indices were calculated according to the
ex (mg/g) = (weight of thymus or spleen)/body weight.
ocyte proliferation assay
se spleens were aseptically removed from the sacri-
using scissors and forceps in 0.1 M cold PBS, gently
by Yua
mixtur
were r
mediu
gen Co
penicil
plate c
tured f
incuba
was ce
carded
shaken
measu
2.5. Ph
The
clearan
to the
venous
Blood
the ret
2 ml 0
UV-vis
liver a
calcula
K = lg
where
Pha
weight
2.6. Bi
The
were p
logical
10 min
protein
and th
ric me
acid (U
autoox
level w
assay (
in nan
2.7. St
All
for Wi
tistical
Scheffe
was co
3. Res
3.1. Ef
The
and pr
mus in
with thng, Li, Li, and Dai (2006). The trythrocytes in the cell
re washed via hypo-osmostic haemolysis, and the cells
ended to a final density of 5 × 106 cells/ml in RPMI 1640
pplemented with 10% newborn bovine serum (Invitro-
arlsbad, CA, USA), 100 U/ml streptomycin, and 100 U/ml
pleen cells (100 l/well) were seeded into a 96-well
ining ConA (8 g/ml). The spleen cells were then cul-
days in 5% CO2 atmosphere at 37 ◦C, and then further
or 4.5 h with 10 l MTT (5 mg/ml) per well. The plate
uged at 200 × g for 15 min, and the supernate was dis-
SO (100 l) was added to each well, which was then
il all crystals dissolved. The absorbance at 570 nm was
n a microplate reader (Thermo Multiskan MK3, USA).
ytic index
tion of the macrophage cells was assessed via a carbon
st performed on three mice from each group according
edure of Wang et al. (2011). Each mouse was intra-
jected with diluted India ink at 100 l/10 g body weight.
mens were collected at 2 min (t1) and 10 min (t2) from
venous plexuses, and 20 l blood was then mixed with
a2CO3. The absorbance at 600 nm was measured on a
spectrophotometer with 0.1% Na2CO3 as the blank. The
e spleen were weighed, and the phagocytic index was
s follows:
− lg OD2
− t1
was for t1 and OD2 was for t2.
tic index ˛ = 3√K × A/(B + C), where A is the body
the liver weight, and C is the spleen weight.
mical assay
n homogenates (including the liver, heart, and kidney)
red in a 0.1 g/ml wet weight of ice-cold isotonic physio-
e. The samples were centrifuged at 2000 × g at 4 ◦C for
the supernates were used for the measurement of the
OC, MAD, CAT, SOD, and GSH-Px levels. The SOD activity
A and TAOC levels were measured via spectrophotomet-
s. The MDA level was detected using 2-thiobarbituric
ama & Mihara, 1978). The SOD activity was analysed via
on of pyrogallol (Marland & Marklund, 1974). The TAOC
easured using the ferric reducing/antioxidant power
ie & Strain, 1996). The enzyme activity was expressed
es per milligram of protein.
cal analysis
are presented as the mean ± SD, analysed using SPSS
s version 15.0 (SPSS Inc., Chicago, IL, USA). The sta-
lysis was evaluated via one-way ANOVA followed by
st to detect the intergroup differences. A P < 0.05 values
ered statistically significant.
f CMP on mouse spleen and thymus indices
en and thymus indices can reflect the immune function
sis of an organism. As shown in Fig. 1, the spleen and thy-
s of the model group remarkably decreased compared
of the normal group (P < 0.05). CMP increased the spleen
M. Wang et al. / Carbohydrate Polymers 89 (2012) 461– 466 463
Fig. 1. Effects mpar
group. Values
and thymu
manner at 1
the CY-indu
3.2. Effect o
Spleen l
the mechan
in Fig. 2, th
remarkably
(P < 0.05). C
liferation in
35, and 70 m
CMP is dire
3.3. Effect o
system
Carbon c
of CMP on
model grou
CMP effecti
dose-depen
phagocytic
4.51 to 4.74
function in
3.4. Antioxi
3.4.1. Effect
of the immu
Fig. 4 sh
(P < 0.01) in
trol group.
relative to t
3.4.2. Effect
of the immu
Fig. 5 sho
hearts, liver
groups. CM
activity com
3.4.3. Effect
organs of th
Fig. 6 s
(P < 0.01) in
control gro
activity com
Effect
mmu
7 s
1) in
l grou
OC a
Effect
mmu
8 sh
hear
oups
red t
cussi
is a c
ent i
uno
n, 2
ssion
tive
by C
n re
ine w
ongly
mmu
ation
e th
er p
ts th
e sy
acrop
ld, 20
iffere
romo
ing v
In prof CMP on the internal organ indices of the CY-induced mice. *P < 0.05, **P < 0.01 co
are means ± SD.
s indices in the CY-treated mice in a dose-dependent
7.5, 35, and 70 mg/kg, indicating that CMP can reverse
ced atrophy of immune organs.
f CMP on cellular immunity in mice
ymphocyte proliferation was examined to understand
ism of the immunoregulatory activity of CMP. As shown
e spleen lymphocyte proliferation of the model group
decreased compared with that of the normal group
MP significantly increased the spleen lymphocyte pro-
CY-treated mice in a dose-dependent manner at 17.5,
g/kg compared with the model group, suggesting that
ctly mitogenic for mouse splenocytes.
f CMP on the phagocytic activity of the macrophage
learance tests were performed to determine the effect
macrophage activation. The phagocytic index ˛ of the
p was lower compared with that of the NS group (Fig. 3).
vely increased the ˛ value of the CY-treated mice in a
dent manner. At the high CMP dose (70 mg/kg/d), the
activity was restored to above the normal level (from
), demonstrating that CMP can enhance the macrophage
CY-treated mice.
dant activity of CMP in vivo
of CMP on the activity of SOD in the different organs
nosuppressed mice
ows that CY significantly reduced the SOD activity
the hearts, livers and kidneys compared to the NS con-
All CMP doses significantly increased the SOD activity
he model group (P < 0.01).
of CMP on the activity of CAT in the different organs
3.4.4.
of the i
Fig.
(P < 0.0
contro
the TA
3.4.5.
of the i
Fig.
in the
trol gr
compa
4. Dis
CY
tant ag
to imm
& Bara
suppre
protec
caused
CMP ca
In l
are str
their i
stimul
improv
in canc
sugges
immun
and m
Boqwa
from d
of mac
activat
2006).nosuppressed mice
ws the marked reductions CAT activity (P < 0.01) in the
s and kidneys of mice in the CY-treated and NS control
P (17.5, 35, and 70 mg/kg) significantly increased CAT
pared to the model group (P < 0.01).
of CMP on the activity of GSH-Px in the different
e immunosuppressed mice
hows the significant reductions in GSH-Px activity
the hearts, livers and kidneys of the CY-treated and NS
ups. All CMP doses significantly increased the GSH-Px
pared to the model group (P < 0.01).
to induce t
upregulatio
and IL-1)
as the prod
ner. They p
nitric oxide
secretion o
enzymatic
the current
the spleen
index ˛ in
can also en
function in ed with the NS group; #P < 0.05, ##P < 0.01 compared with the model
of CMP on the activity of TAOC in the different organs
nosuppressed mice
hows the remarkable reductions in TAOC activity
the hearts, livers and kidneys of the CY-treated and NS
ps. CMP (17.5, 35, and 70 mg/kg) significantly increased
ctivity compared to the model group (P < 0.01).
of CMP on the activity of MDA in the different organs
nosuppressed mice
ows the significant increases in MDA levels (P < 0.01)
ts, livers and kidneys of the CY-treated and NS con-
. All CMP doses significantly decreased the MDA levels
o the model group (P < 0.01).
ons
ytotoxic chemotherapeutic drug that acts as an impor-
n tumour treatment. However, its administration leads
suppression, which may be life-threatening (Hong, Yan,
004). Traditional Chinese medications for immuno-
treatment are available. In the present study, the
effects of CMP in reversing the immunosuppression
Y treatment were investigated. The results indicate that
verse the CY-induced atrophy of immune organs.
ith the usage of Cordyceps in China, Chinese medicines
recommended for the ageing population to enhance
ne system and prevent possible infection. Immuno-
itself is regarded as one of the important strategies to
e body
′
s defense mechanism in elderly people as well as
atients. A significant amount of experimental evidence
at polysaccharides from mushrooms enhance the host
stem by stimulating natural killer cells, T-cells, B-cells,
hage-dependent immune system responses (Dalmo &
08; Dennert & Tucker, 1973). Polysaccharides obtained
nt natural sources represent a structurally diverse class
lecules, which exert their antitumour action mostly by
arious immune system responses (Schepetkin & Quinn,
evious studies, Cordyceps polysaccharides were found
he functional activation of macrophages through the
n of cytokine expression (tumour necrosis factor alpha
and nitric oxide (NO) release (Lee et al., 2010), as well
uction of IL-6 and IL-10 in a dose-dependent man-
romote the mRNA and protein expressions of inducible
synthase, induce T-lymphocyte proliferation and the
f IL-2, IL-6, and IL-8, and increase the phagocytic and
activities of the acid phosphatase of macrophages. In
study, the administration of CMP significantly enhanced
lymphocyte proliferation and increased the phagocytic
a dose-dependent manner, thereby implying that CMP
hance the spleen lymphocyte activity and macrophage
CY-treated mice.
464 M. Wang et al. / Carbohydrate Polymers 89 (2012) 461– 466
Fig. 2. Effect of CMP on the spleen lymphocyte proliferation in CY-treated mice. *P < 0.05, **P < 0.01 compared with the NS group; #P < 0.05, ##P < 0.01 compared with the
model group. Values are means ± SD.
Fig. 3. Effect of CMP on the phagocytic index in the CY-treated mice. *P < 0.05,**P < 0.01 compared with the NS group; #P < 0.05, ##P < 0.01 compared with the model group.
Values are means ± SD.
Fig. 4. Effect of CMP on the SOD activity in the hearts, livers and kidneys of the immunosuppressed mice. *P < 0.05, **P < 0.01 compared with the NS group; #P < 0.05, ##P < 0.01
compared with the model group. Values are means ± SD.
Fig. 5. Effect of CMP on the CAT activity in the hearts, livers and kidneys of the immunosuppressed mice. *P < 0.05, **P < 0.01 compared with the NS group; #P < 0.05, ##P < 0.01
compared with the model group. Values are means ± SD.
M. Wang et al. / Carbohydrate Polymers 89 (2012) 461– 466 465
Fig. 6. Effect of CMP on the GSH-Px activity in the hearts, livers and kidneys of the immunosuppressed mice. *P < 0.05, **P < 0.01 compared with the NS group; #P < 0.05,
##P < 0.01 compared with the model group. Values are means ± SD.
Fig. 7. Effect o presse
##P < 0.01 com
Free-rad
with a num
such as sup
(collectively
tissue dama
(Simic, Berg
ing amount
rides have p
damage in l
& Chang, 1
2003). Cord
the anti