Structural characterization of immunostimulating polysaccharide from cultured mycelia of Cordyceps militaris

Article abstract of DOI:10.1016/j.carbpol.2010.01.017

The water soluble crude polysaccharide obtained from cultured mycelia of Cordyceps militaris (CPM) by hot water extraction followed by ethanol precipitation was fractionated by DEAE cellulose and Sepharose CL-6B column chromatography. This fractionation process resulted in four polysaccharide fractions that were termed CPMN Fr I, CPMN Fr II, CPMN Fr III, and CPMN Fr IV. Of these fractions, CPMN Fr III was able to upregulate the functional events mediated by activated macrophages, such as production of nitric oxide (NO) and expression of cytokines (IL-1β and TNF-α). Its structural characteristics were investigated by a combination of chemical and instrumental analyses, including methylation, reductive cleavage, acetylation, Fourier transform infrared spectroscopy (FT-IR), and gas chromatography-mass spectrometry (GC-MS). Results indicate that CPMN Fr III was a high molecular mass polysaccharide with a random coil conformation of the β-1,4-branched-β-1,6-galactoglucomannan.

Full text of DOI:10.1016/j.carbpol.2010.01.017

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Carbohydrate Polymers 80 (2010) 1011–1017
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Structural characterization of immunostimulating polysaccharide
from cultured mycelia of Cordyceps militaris
Jong Seok Lee, Jeong Seok Kwon, Jong Seok Yun, Jung Woon Pahk, Won Cheol Shin, Shin Young Lee,
*
Eock Kee Hong
Department of Bioengineering and Technology, Kangwon National University, Chuncheon 200-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2009
Received in revised form 17 December 2009
Accepted 7 January 2010
Available online 15 January 2010
The water soluble crude polysaccharide obtained from cultured mycelia of Cordyceps militaris (CPM)
by hot water extraction followed by ethanol precipitation was fractionated by DEAE cellulose and
Sepharose CL-6B column chromatography. This fractionation process resulted in four polysaccharide
fractions that were termed CPMN Fr I, CPMN Fr II, CPMN Fr III, and CPMN Fr IV. Of these fractions,
CPMN Fr III was able to upregulate the functional events mediated by activated macrophages, such
as production of nitric oxide (NO) and expression of cytokines (IL-1b and TNF-a). Its structural char-
Keywords:
Cordyceps militaris
Immunostimulating polysaccharide
Macrophage activation
Random coil conformation
acteristics were investigated by a combination of chemical and instrumental analyses, including
methylation, reductive cleavage, acetylation, Fourier transform infrared spectroscopy (FT-IR), and
gas chromatography–mass spectrometry (GC–MS). Results indicate that CPMN Fr III was a high
molecular mass polysaccharide with
galactoglucomannan.
a
random coil conformation of the b-1,4-branched-b-1,6-
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
by liquid culture is shown as a promising alternative for fruiting
body (Ohta, 1990).
In recent years many natural polysaccharides and polysaccha-
ride–protein complexes, isolated from mushrooms, have been used
as therapeutic agents (Novak & Vetvicka, 2008). Among them,
Cordyceps militaris, an entomopathogenic fungus belonging to the
class Ascomycetes, has been reported to have beneficial biological
activities such as hypoglycemic (Kiho, Yamane, Hui, Usui, & Ukai,
1996), hypolipidemic (Yang et al., 2000), anti-inflammatory (Won
& Park, 2005), antitumor (Lin & Chiang, 2008; Park et al., 2005,
2009), anti-metastatic (Nakamura et al., 1999), immunomodula-
tory (Cheung et al., 2009; Kim et al., 2008), and antioxidant effect
(Yu et al., 2007, 2009). The fruiting bodies of wild C. militaris are
expensive because of host specificity and rarity in nature. There-
fore, the production of adequate quantities of the fruiting bodies
of wild C. militaris for wide spread use as a therapeutic agent is cur-
rently impractical. It takes a long time to complete the fruiting
body when solid culture is used. Liquid culture has the potential
to increase mycelial production in a compact space and shorter
time with less chance of contamination. The production of mycelia
Many studies have demonstrated that the polysaccharides from
basidiomycetes mushroom had highly beneficial therapeutic ef-
fects including (1) preventing oncogenesis after administering of
peroral medications prepared from these mushrooms or their ex-
tracts, (2) direct antitumor activity against various tumors, (3)
immunosynergism activity against tumors in combination with
chemotherapy, and (4) preventive effects on tumor metastasis
(Chihara, Maeda, Hamuro, Sasaki, & Fukuoka, 1969; Collins, Zhu,
Guo, Xiao, & Chen, 2006; Ng & Wang, 2005). It has been extensively
shown that the immunomodulating actions of polysaccharides are
dependent on their chemical composition, molecular weight, con-
formation, glycosidic linkage, degree of branching, etc. (Methacan-
on, Madla, Kirtikara, & Prasitsil, 2005; Yadomae & Ohno, 1996). As
a result of this phenomenon, several studies have been conducted
to determine accurately the structures of these different
polysaccharides.
The aim of this study was to better understand and characterize
the structural characteristics of the polysaccharide, CPMN Fr III,
which was isolated and purified from cultured mycelia of C. milita-
ris by gel filtration and ion exchange chromatography. To this end,
we investigated the release of NO and the production of cytokines
by macrophages that were activated by this polysaccharide as part
of the innate immune response. In addition, its chemical composi-
tion, molecular weight, conformation, degree of branching, and
glycosidic linkage were examined.
* Corresponding author. Address: College of Engineering, Department of Bioen-
gineering and Technology, Kangwon National University, 192-1, Hyoja-2-dong,
Chuncheon, Gangwon-do 200-701, Republic of Korea. Tel.: +82 33 250 6275; fax:
+82 33 243 6350.
E-mail address: ekhong@kangwon.ac.kr (E.K. Hong).
0144-8617/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2010.01.017

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2.5. Determination of NO production
2. Materials and methods
2.1. Materials
After pre-incubation RAW264.7 cells (1 ꢂ 106 cells/ml) for 18 h,
each polysaccharide (1000 lg/ml) or LPS (2.5 lg/ml) was added
and the mixture was incubated for an additional 24 h. Nitrite in
culture supernatants was measured by adding 100 l of Griess re-
agent (1% sulfanilamide and 0.1% N-[1-naphthyl]-ethylenediamine
dihydrochloride in 5% phosphoric acid) to 100 l samples. The ni-
trite concentration was determined at 540 nm using NaNO2 as a
standard.
The strain used in this study was C. militaris KCTC 6064, which
was cultivated for 11 days at 24 °C, 200 rpm, uncontrolled pH, and
a 2% (v/v) inoculum size in modified medium containing 80 g/l glu-
cose, 10 g/l yeast extract, 0.5 g/l MgSO4ꢀ7H2O, and 0.5 g KH2PO4.
After 11 days of cultivation, the culture broth was centrifuged at
5000 rpm for 20 min. Precipitated mycelia were washed three
times with distilled water, and then freeze-dried (Kwon, Lee, Shin,
Lee, & Hong, 2009). Dialysis tubing cellulose membranes, DEAE cel-
lulose, Sepharose CL-6B, standard dextrans, lipopolysaccharide
(LPS, Escherichia coli 0111:B4), laminarin, curdlan, and Congo red
were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fe-
tal bovine serum and RPMI1640 were obtained from GIBCO (Grand
Island, NY, USA). RAW264.7 macrophages were purchased from the
American Type Culture Collection (Manassas, VA, USA). All other
chemicals were of Sigma grade.
l
l
2.6. RT-PCR
To evaluate levels of LPS or CPMN Fr III-inducible mRNA expres-
sion, total RNA from CPMN Fr III-treated or untreated RAW264.7
cells was prepared by adding TRIzol reagent (Gibco-BRL) according
to the manufacturer’s protocol. The total RNA solution was stored
at ꢁ70 °C prior to subsequent use. Semiquantitative reverse tran-
scription-polymerase chain reaction (RT-PCR) was performed
using MuLV reverse transcriptase. Total RNA (1 lg) was incubated
with oligo-dT15 for 5 min at 70 °C, then mixed with a 5ꢂ first-
strand buffer, 10 mM dNTPs, and 0.1 M DTT. The reaction mixture
was further incubated for 5 min at 37 °C, then for 60 min after the
addition of 2 U of MuLV reverse transcriptase. Reactions were ter-
minated by heating for 10 min at 70 °C, and total RNA was depleted
by addition of RNase H. PCR was performed with the incubation
2.2. Extraction, fractionation and purification of water-soluble
polysaccharides
Lyophilized mycelia were extracted two times with three vol-
umes of distilled water at 121 °C for 2 h. Extracts were centrifuged
at 5000g for 20 min and filtered through 0.45 lm Whatman filter
paper to remove insoluble matter, then, freeze-dried. Polysaccha-
rides were precipitated from resuspended extracts using 95.0%
ethanol, collected by filtration through 0.45 lm Whatman filter pa-
per, resuspended and dialyzed against distilled water for 5 days to
remove low-molecular-weight compounds. The crude polysaccha-
rides, termed CPM, was dissolved in distilled water, centrifuged at
5000g for 20 min, and loaded onto a DEAE cellulose (Clꢁ) column
(2.5 ꢂ 50 cm) to separate neutral and acidic polysaccharides. The
resulting fractions were loaded onto a Sepharose CL-6B column
(2.3 ꢂ 80 cm) equilibrated with 0.5 N NaCl, then eluted with the
same solution to separate polysaccharides based on molecular
weight. Each polysaccharide fraction, derived from cultured myce-
lia of C. militaris, contained an endotoxin level that was below the
detection limit (0.0015 EU/ml) as assessed by an E-TOXATE kit
(Sigma, St. Louis, MO, USA).
mixture (2
Seoul, Korea], a 10ꢂ buffer [10 mM Tris–HCl, pH 8.3, 50 mM KCl,
0.1% Triton X-100], 250 M dNTPs, 25 mM MgCl2, and 1 U of Taq
ll of cDNA, 4 lM forward and reverse primers [Bioneer,
l
polymerase [Promega, USA]) under the following conditions: a
45 s denaturation step at 94 °C, a 45 s annealing step between 55
and 60 °C, a 60 s extension step at 72 °C, and a 7 min final exten-
sion step at 72 °C after 30 cycles. The primers used in this experi-
ment are indicated in Table 1. Ten microliters of PCR products were
electrophoresed on a 1.2% agarose gel and visualized by ethidium
bromide staining under ultraviolet light.
2.7. TNF-a production
The ability of CPMN Fr III to induce production of TNF-
a
in
RAW264.7 cells was determined by dissolving the polysaccharide
in the culture medium. Supernatants were harvested and the con-
centration of TNF-a was determined using an ELISA kit (Biosource
International, Camarillo, CA, USA), according to the manufacturer’s
2.3. Cell culture
instructions.
RAW264.7 cells were maintained in RPMI1640 that was supple-
mented with 100 U/ml penicillin, 100 lg/ml streptomycin, and 10%
2.8. Analysis of chemical properties
fetal bovine serum. Cells were grown at 37 °C in a humidified 5%
CO2 incubator.
The total sugar content of each polysaccharide was determined
using the phenol–sulfuric acid method (Chaplin & Kennedy, 1986),
2.4. Cell viability
Table 1
Primer sequences of genes investigated by RT-PCR analysis.
The effect of polysaccharides on the viability of RAW264.7 cells
was determined using the [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium] bromide (MTT) assay, which is based on
the reduction of a tetrazolium salt by mitochondrial dehydroge-
nase in viable cells. After pre-incubating RAW264.7 cells
Gene
Primer sequences
IL-1b
Fa
50-CAGATGAGGACATGAGCACC-30
50-CACCTCAAACTCAGACGTCTC-30
Rb
(1 ꢂ 106 cells/ml) for 18 h, polysaccharides (1000
lg/ml) or LPS
TNF-
Fa
a
50-TTGACCTCAGCGCTGAGTTG-30
50-CCTGTAGCCCACGTCGTAGC-30
(2.5 lg/ml) was added and the mixture was incubated for an addi-
tional 24 h. Fifty microliters of the MTT stock solution (2 mg/ml)
Rb
was then added to each well to attain a total reaction volume of
GAPDH
Fa
50-CACTCACGGCAAATTCAACGGCAC-30
50-GACTCCACGACATACTCAGCAC-30
200
for 5 min and the supernatants were aspirated. The formazan crys-
tals in each well were dissolved in 150 l dimethylsulfoxide and
the A540 was read on a scanning multiwell spectrophotometer.
ll. After incubation for 2 h, the plate was centrifuged at 800g
Rb
a
l
Forward.
Reverse.
b

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the total protein concentration was determined using the Bradford
method (Bradford, 1976), the hexosamine content was evaluated
using the Elson–Morgan method (Dische, 1962), and the uronic
acid content was assessed using the Blumenkrantz method (Blu-
menkrantz & Asboe-Hansen, 1973).
2.14. Determination of glycosidic linkage
Permethylated CPMN Fr III was extracted in dichloromethane
and reductive cleavage was performed using a combination of tri-
methylsilyl methanesulfonate and trifluoride etherate as the cata-
lyst as previously described (Rolf & Gray, 1982). The reaction was
allowed to proceed for 8–12 h at room temperature, then was
quenched by addition of sodium bicarbonate. The organic layer
was separated with a syringe and products were isolated and acet-
ylated. Glycosidic linkage was analyzed by GC–MS on a Micromass
apparatus (Waters Corp., Milford, MA, USA) equipped with an HP-
5MS column and a temperature program of 120–180 °C at 5 °C/min
and 180–250 °C at 2 °C/min). Mass spectra were obtained at an ion
2.9. Analysis of monosaccharide composition
Monosaccharide composition and ratios were determined by
first hydrolyzing the polysaccharide with 2 M trifluoroacetic acid
(TFA) in a sealed tube at 100 °C for 4 h. Acid was removed by re-
peated evaporation using a vacuum distillation device. The hydro-
lysate was then dissolved in 1.0 ml of distilled water and filtered
energy of 70 eV, a current intensity of 500 lA and temperature of
250 °C.
through a 0.2 lm PTFE membrane. The aqueous hydrolysate was
analyzed by reverse-phase HPLC using an ED50 electrochemical
detector (Dionex, Sunnyvale, CA, USA) under the following condi-
tions: column: CarboPac PA10 Analytical Column (4 ꢂ 240 mm);
solvent: A, deionized water, B, 200 mM NaOH; program: 0–
20 min (B conc. 8%), 20–40 min (B conc. 25%), 40–70 min (B conc.
8%); flow rate: 0.9 ml/min; temp.: 30 °C. Glucose, galactose, man-
nose, and fucose were used as monosaccharide standards.
2.15. Statistical analysis
A Student’s t-test and a one-way ANOVA were used to deter-
mine the statistical significance of the differences between the val-
ues determined for the various experimental and control groups.
Data are expressed as means standard errors (SEM) and the re-
sults are taken from at least three independent experiments per-
formed in triplicate. P values of 0.05 or less were considered to
be statistically significant.
2.10. Determination of molecular weight
The molecular weight of the polysaccharide fractions was deter-
mined by gel filtration using a Sepharose CL-6B packed column. A
standard curve was prepared based on the elution volume and the
molecular weight. Standard dextrans (MW: 670, 410, 150, and
25 kDa) were used for calibration.
3. Results
3.1. Purification and fractionation
In the first stage of purification and fractionation, ion exchange
chromatography through a DEAE-cellulose column was used to
separate neutral polysaccharides from acidic fractions. The yield
of the neutral fraction (CPMN) and the acidic fraction (CPMA) ob-
tained from the crude polysaccharide extract CPM was 0.668 g/g
and 0.052 g/g, respectively (Fig. 1A). The molecular distribution
of CPMN was investigated using gel filtration chromatography
with a Sepharose CL-6B column, resulting in four polysaccharide
fractions, namely CPMN Fr I (0.018 g/g), CPMN Fr II (0.125 g/g),
CPMN Fr III (0.408 g/g), and CPMN Fr IV (0.049 g/g) (Fig. 1B).
2.11. Analysis of helix-coil transition
The conformational structure of the polysaccharides in solution
was determined by characterizing Congo red–polysaccharide com-
plexes. The transition from a triple-helical arrangement to the sin-
gle-stranded conformation was examined by measuring the kmax of
Congo red–polysaccharide solutions at NaOH concentrations rang-
ing from 0.01 to 0.5 N. Polysaccharide aqueous solutions (1 mg/ml)
containing 100 ll of 0.5 mg/ml Congo red were treated with differ-
ent concentrations of NaOH. Visible absorption spectra were
recorded with a UV/vis spectrophotometer at each alkali concen-
tration (Ogawa & Hatano, 1978; Ogawa, Tsurugi, & Watanabe,
1973).
3.2. Macrophage activation by polysaccharides
To examine whether polysaccharides purified from cultured
mycelia of C. militaris were able to stimulate the functional activa-
tion of macrophages, macrophage-like RAW264.7 cells were incu-
2.12. Identification of anomeric configuration
bated with 1000 lg/ml of each polysaccharide and NO production
To ascertain the presence or absence of the
a or b configuration
was measured and compared to the amount produced by the un-
treated control group. Polysaccharide-treated cells produced larger
amounts of NO than untreated cells (Fig. 2A). To address whether
CPMN Fr III elicits innate immune responses in macrophages, RT-
PCR and ELISA assays were used to examine induction of transcrip-
tional gene upregulation and increased expression of proinflamma-
tory cytokines. These experiments showed that CPMN Fr III
strongly triggers the expression of proinflammatory cytokines
in each polysaccharide, b-linked polysaccharides were detected
using a Fungi-Fluor Kit (Polysciences, Warrington, PA, USA). Each
sample was dissolved in distilled water and the solution was
placed on a slide and dried in an oven. Following the addition of
methanol, each sample dried for an additional 20 min. Fungi-Fluor
Solution A (cellufluor, water, and potassium hydroxide) was used
as a dye. A few drops were added to each sample and the mixtures
were incubated for 3 min. After washing with distilled water, the
fluorescence level was determined using a UV Illuminator (Vilber
Lourmat Inc., France).
TNF-a and interleukin-1b (IL-1b) (Fig. 2B and C).
3.3. Chemical properties and monosaccharide composition
2.13. Methylation of CPMN Fr III
The total sugar content of CPMN Fr III was 92.34%. Its major su-
gar constituents are mannose (72.22%), galactose (18.61%) and glu-
cose (9.17%) (Table 2 and Fig. S1). The contents of proteins,
hexosamine and uronic acid of this polysaccharide are 0.21%,
0.12% and 0.33%, respectively (Table 2).
CPMN Fr III was methylated according to the method developed
by Ciucanu and Kerek, using powdered NaOH in Me2SO–MeI (Ciu-
canu & Kerek, 1984). Methylation was confirmed by measuring the
FT-IR spectrum.

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6B packed column (Fig. 3A). The molecular weight of this fraction
was then determined by gel filtration chromatography to be
210 kDa using dextrans as standards (Fig. 3B).
3.5. Identification of helix-coil transition
A shift in the visible absorption maximum of Congo red is in-
duced by the presence of polysaccharides and can thus be used
to provide conformational information. The absorption maximum
of dextran, which has a random coil conformation, was around
450 nm (Fig. 4). Curdlan exhibits a triple-helical conformation,
which was demonstrated by the shift in the absorption maximum
at 0.24 M NaOH. However, the absorption maximum of laminarin,
which has a different triple-helical conformation, was around
550 nm. Based on this analysis, CPMN Fr III was found to exhibit
a random coil conformation similar to that of dextran.
3.6. Identification of anomeric configuration
To ascertain the presence or absence of the
a or b configuration
in CPMN Fr III, the Fungi-Fluor Kit was used. The Fungi-Fluor stain-
ing solution, cellufluor, binds nonspecifically to b-linked polysac-
charides, thus enabling their rapid detection. While dextran,
which is an a-glucan, did not exhibit fluorescence in the presence
of cellufluor, a signal was clearly observed for curdlan, which is a b-
glucan. CPMN Fr III displayed a fluorescence signal very similar to
that of curdlan, indicating that it is a b-linked polysaccharide
(Fig. 5).
Fig. 1. Isolation and purification of polysaccharides extracted from cultured
mycelia of Cordyceps militaris. (A) Ion exchange chromatogram of the crude
polysaccharides, CPM, on a DEAE-cellulose column. (B) Gel filtration chromatogram
3.7. Glycosidic linkage of the polysaccharide
of the neutral polysaccharide fraction, CPMN, on
a Sepharose CL-6B column
(fraction number of ion exchange chromatography: 14–18).
CPMN Fr III exhibited an IR absorption spectrum characteristic
of a polysaccharide, with bands at 1080 cmꢁ1 (C@O), 2800–
2900 cmꢁ1 (CAH), and 3400 cmꢁ1 (OAH). Glycosidic linkage anal-
ysis of permethylated CPMN Fr III was performed by the reductive
cleavage method. The polysaccharide was shown to be fully meth-
ylated, as indicated by the disappearance of the band at 3400 cmꢁ1
3.4. Homogeneity and molecular weight
The homogeneity of CPMN Fr III was confirmed by refractiona-
tion through gel filtration chromatography using a Sepharose CL-
Fig. 2. Immunostimulating effects of polysaccharide, CPMN Fr III, purified by DEAE cellulose and Sepharose CL-6B chromatography. (A) Effect of purified polysaccharides on
NO synthesis in murine macrophage-like cells. RAW264.7 cells (1 ꢂ 106 cells/ml) were stimulated by each polysaccharide fraction (1000
lg/ml) for 24 h. Supernatants were
collected and NO concentration was determined using the Griess reagent, as described in Section 2. (B) The effect of CPMN Fr III on the expression of cytokines. RAW264.7
cells (1 ꢂ 107 cells/ml) were incubated with CPMN Fr III (1000
lg/ml) or LPS (2.5 lg/ml) for 6 h. Cytokine mRNA levels were determined by semiquantitative RT-PCR. The
results shown are from one of three experiments performed. (C) The effect of CPMN Fr III on TNF-
a
production. RAW264.7 cells (1 ꢂ 106 cells/ml) were stimulated by CPMN Fr
III (1000
lg/ml) for 6 h. Supernatants were collected and TNF-a concentration was determined by ELISA, as described in Section 2. Data (A and C) represent means SEM of
three independent experiments performed in triplicate.