Structure and anti-tumor activity of a high-molecular-weight polysaccharide from cultured mycelium of Cordyceps gunnii

Article abstract of DOI:10.1016/j.carbpol.2012.01.068

Cordyceps gunnii (berk.) Berk (C. Gunnii) is well known as a Chinese rare caterpillar fungus and has similar pharmacological activity with C. sinensis. In this work, a high-molecular-weight polysaccharide (CPS) was isolated and purified from the mycelia of C. gunnii. The total sugar content of CPS was amounted to 92.84%. The result of HPLC indicated that CPS was a homogeneous polysaccharide. The estimated average molecular weight of CPS was 3.72 × 10⁶ Da. The specific rotation of CPS was recorded αD25=+134.2°. Its characteristic was determined by chemical analysis, gas chromatography, IR spectroscopy and NMR data. The results showed that CPS was mainly composed of glucose, and a small amount of rhamnose, arabinose, xylose, mannose and galactose with a molar ratio of Rha:Ara:Xyl:Man:Glu:Gal = 3.0:2.6:1.0:1.3:106.0:2.8. The main chain of CPS was majorly composed of α-(1 → 4) glucose. The tumor inhibition ratio on K562 cell by CPS was 56.65%.

Full text of DOI:10.1016/j.carbpol.2012.01.068

Carbohydrate Polymers 88 (2012) 1072–1076
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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Structure and anti-tumor activity of a high-molecular-weight polysaccharide
from cultured mycelium of Cordyceps gunnii
Zhen-yuan Zhua,∗, Nian Liua, Chuan-ling Sib,∗∗, Yang Liua, Li-na Dinga, Chen Jinga, An-jun Liua,
Yong-min Zhangc
a Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Science and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR
China
b Tianjin Key Laboratory of Pulp and Paper, College of Materials Science and Chemical Engineering, Tianjin University of Science and Technology, Tianjin 300457, PR China
c Université Pierre et Marie Curie-Paris 6, Institut Parisien de Chimie Moléculaire UMR CNRS 7201, 4 Place Jussieu, 75005, Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Cordyceps gunnii (berk.) Berk (C. Gunnii) is well known as a Chinese rare caterpillar fungus and has similar
pharmacological activity with C. sinensis. In this work, a high-molecular-weight polysaccharide (CPS)
was isolated and purified from the mycelia of C. gunnii. The total sugar content of CPS was amounted to
92.84%. The result of HPLC indicated that CPS was a homogeneous polysaccharide. The estimated average
molecular weight of CPS was 3.72 × 106 Da. The specific rotation of CPS was recorded [˛]2D5 = +134.2◦. Its
characteristic was determined by chemical analysis, gas chromatography, IR spectroscopy and NMR data.
The results showed that CPS was mainly composed of glucose, and a small amount of rhamnose, arabinose,
xylose, mannose and galactose with a molar ratio of Rha:Ara:Xyl:Man:Glu:Gal = 3.0:2.6:1.0:1.3:106.0:2.8.
The main chain of CPS was majorly composed of ␣-(1 → 4) glucose. The tumor inhibition ratio on K562
cell by CPS was 56.65%.
Received 12 December 2011
Received in revised form 15 January 2012
Accepted 20 January 2012
Available online 28 January 2012
Keywords:
Cordyceps gunnii
High-molecular-weight polysaccharide
Characteristic
Antitumor
© 2012 Elsevier Ltd. All rights reserved.
1. Introduction
entomogenous fungi such as C. sinensis (Leung, Zhao, Ho, & Wu,
2009; Liu, Zhong, Zhu, & Zhu, 2008; Liu, Leung, & Wu, 2008; Wu,
Cordyceps, one of the famous traditional Chinese medicines, has
been used as health food for a long time in China. Recently, owing to
its anti-tumor activity, anti-inflammatory activity, anti-aging effect
and improving immunity effect, Cordyceps has attracted much
attention (Wang, Yu, & Yuan, 2004). Cordyceps gunnii (berk.) Berk (C.
gunnii), is also well known as the Chinese rare caterpillar fungus and
has similar pharmacological activity with C. sinensis. The anamorph
of Paecilomyces gunnii of C. gunnii has been isolated, verified and
identified (Liang, 1985). Many important secondary metabolic
products were found in C. gunnii mycelia including cordycepin,
cordycepic acid, polysaccharide and anti-ultraviolet radiation
constituents (Huang, Liang, & Liu, 1992). Polysaccharides have
been reported to account for the anti-tumor, anti-inflammatory,
antioxidant, steroidogenic, hypolipidemic and immunomodulatory
effects. Many polysaccharides and polysaccharide–protein com-
plexes, isolated from fungi, have attracted much attention recently
in the biochemical and medical areas due to their anti-cancer
effects. At present, although many studies on polysaccharides from
Sun, & Pan, 2006; Zhang, Li, Qiu, Chen, & Zheng, 2008) and C. militaris
(Hou et al., 2008; Kim et al., 2008; Wang, Wei, & Zhang, 2003; Wu,
Hu, Pan, Zhou, & Zhou, 2007; Yu et al., 2007) have been reported,
the anti-tumor activities of the polysaccharides from mycelium of
C. gunnii have been scarcely studied.
In our previous work, a low-molecular-weight polysaccharide
was isolated and purified from the mycelia of C. gunnii (Liu, Zhong,
et al., 2008; Liu, Leung, et al., 2008; Zhu, Si, et al., 2011). In this
paper, a novel high-molecular-weight polysaccharide (CPS) was
described. The structure of CPS was characterized and its anti-
tumor activity was confirmed by using human K562 cells.
2. Materials and methods
2.1. Materials
The C. gunnii mycelium and K562 cell were obtained from the
Key Laboratory of Food Nutrition and Safety, Ministry of Education,
College of Food Science and Biotechnology, Tianjin University of
Science and Technology, Tianjin, China.
The
standard
monosaccharides
(d-glucose,
d-xylose,
d-galactose, l-rhamnose, d-mannose, and d-arabinose), DEAE-
Sephadex A-25 and Sephadex G-100 were purchased from Sigma
Chemical Co. (St. Louis, MO, USA).
∗
Corresponding author. Tel.: +86 22 60601437; fax: +86 22 60601437.
Corresponding author. Tel.: +86 22 60602006; fax: +86 22 60602510.
E-mail addresses: zhyuanzhu@tust.edu.cn (Z.-y. Zhu), sichli@tust.edu.cn (C.-l. Si).
∗∗
0144-8617/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2012.01.068

Z.-y. Zhu et al. / Carbohydrate Polymers 88 (2012) 1072–1076
1073
2.2. Extraction and purification of polysaccharides
2.8. Methylation analysis
The C. gunnii mycelium was extracted three times by distilled
water at 80 ◦C for 2 h. The supernatant was mixed with 1 volume of
EtOH to obtain the crude polysaccharide (Liu, Zhong, et al., 2008;
Liu, Leung, et al., 2008; Zhu, Si, et al., 2011). The crude polysac-
charide was subjected to the Sevag method three times in order to
remove the protein. The obtained polysaccharide was then decol-
orized by AB-8 resin and purified by DEAE-Sephadex A-25 and
Sephadex G-100 (30 cm × 3 cm) with distilled water. Each fraction
showed only one main peak, and it was collected and freeze-
dried. Each fraction was determined by using a HPLC (Agilent-1200)
equipped with a TSKgel G4000 PWxl column (7.8 mm × 300 mm,
column temperature 30 ◦C) and Refractive Index Detector (RID,
detecting temperature 40 ◦C). The purified polysaccharide was
named CPS.
The methylation analysis was performed based on the Ciucanu
method (Ciucanu & Kerek, 1984). The sample was treated with 4 mL
of 90% formic acid for 6 h at 100 ◦C, then the residue was hydrolyzed
using 3 mL of 2 M TFA for 4 h at 110 ◦C. After removal of formic
acid, the hydrolysate was concentrated to dryness. The methylated
monosaccharides were converted into their corresponding alditol
acetates by reduction with NaBH4 at room temperature for 6 h. The
reduced polysaccharide was acetylated with acetic anhydride, and
dissolved in chloroform and ready for GC–MS analysis.
2.9. Cell lines
The K562 cell line was maintained in RPMI 1640 supplemented
with 10% fetal bovine serum, 100 mg/L streptomycin, and 100 mU/L
penicillin at 37 ◦C in a humidified atmosphere of 5% CO2.
2.3. Determination of molecular weight by HPLC
2.10. In vitro anti-tumor activity against K562 cell
The molecular weights of CPS were determined by using a HPLC,
which was described above. A sample solution (20 ␮L) was injected
and run with purified water at 0.6 mL/min as mobile phase. The
standard curve was established using T-series Dextran as the stan-
dards (T-10, T-40, T-70, T-500 and T-2000) (Zhu, Liu, et al., 2011).
The anti tumor activity assays of K562 tumor cells was evaluated
in vitro using MTT assay. The K562 cells were seeded at a concen-
tration of 1 × 105 cell/mL in a volume of 0.1 mL in 96-well plates.
Cells were incubated with the CPS samples at concentrations of 25,
50, 100, 200 and 400 ␮g/mL. After 20 h, 44 h, 68 h, each well was
added 20 ␮L of 5 mg/mL of MTT and incubated for another 4 h. Then
the culture media were removed, 150 ␮L of DMSO was added to
each well. Absorbance at 490 nm was detected by microplate ELISA
reader. The inhibition ratio of K562 cell proliferation was calculated
as follows:
2.4. IR analysis
1 mg of CPS was mixed with 150 mg of dried KBr, and pressed
into disk for the analysis. The IR spectrum was recorded in the range
of 400–4000 cm−1 on a Fourier transformed IR spectrophotometer
(VECTOR-22).
(ODa − ODb)
˚ =
× 100%
ODa
where ODa is the absorbance value of negative control group, and
ODb is that of sample group (Liu, Song, Yang, Liu, & Zhang, 2007;
Liu, Lin, Gao, Ye, & Xi, 2007).
2.5. NMR spectroscopy
1H NMR and 13C NMR spectra were recorded on a Bruker
spectrometer (600 MHz) at a probe temperature of 298 K. Prior
to analysis, sample was exchanged twice with D2O upon freeze-
drying.
2.11. Statistical analysis
Data were expressed as means SD. Data in all the bioassays
were statistically evaluated by analysis of variance and P < 0.05 was
considered significant.
2.6. Monosaccharide analysis
Dried CPS sample (10.0 mg) was hydrolyzed for 6 h at 100 ◦C
with 4 mL of TFA (2 M). The soluble fraction was evaporated to dry-
ness under stream of nitrogen. The product was acetylated with
Ac2O-Pyridine (1:1, v/v) at 100 ◦C for 1 h. The sample was ready
for GC analysis. d-Glucose, d-xylose, d-galactose, l-rhamnose, d-
mannose, and d-arabinose were also derivatized as standard.
3. Results and discussion
3.1. Structural analysis
The yield of crude polysaccharide from C. gunnii mycelium was
12.96%. The crude polysaccharides were purified by AB-8 resin,
DEAE-A25 and Sephadex G-100, each showing a main peak (Fig. 1),
as detected by the phenol–sulfuric acid assay and HPLC (Fig. 2).
The total carbohydrate content was determined based on the
phenol–sulfuric acid method as d-glucose equivalents, and the total
sugar contents of CPS was 92.84%. The result of HPLC (Fig. 2D) indi-
cated that CPS was a homogeneous polysaccharide. The estimated
average molecular weight of CPS was 3.72 × 106 Da. The specific
rotation of CPS was recorded [˛]2D5 = +134.2◦.
2.7. Periodate oxidation and smith degradation analysis
10.0 mg of CPS sample was dissolved in 0.015 M sodium metape-
riodate (25 mL) and kept in the dark, with the absorption at 223 nm
monitored every 8 h. The reaction was completed after 56 h and
ethylene glycol (0.2 mL) was added to the solution to decompose
the excess of the reagent. Consumption of NaIO4 was measured by a
spectrophotometricmethod (Dixon & Lipkin, 1954) and the produc-
tion of formic acid was determined by titration with 0.01 M NaOH.
The reaction mixture was dialyzed against distilled water, and the
nondialysate was reduced with NaBH4 (25 mg) for 12 h. The pH was
adjusted to 5.0, the solution was dialyzed, and the nondialysate was
lyophilized, and then hydrolyzed with 2 M TFA at 110 ◦C for 4 h. The
hydrolysate was analyzed by GC.
The IR spectrum of CPS (Fig. 3) exhibited a strong band at
3385 cm−1, attributing to the hydroxyl stretching vibration. The
band at 2925 cm−1 was due to C H stretching vibration and the
band at 1417 cm−1 was assigned to C H bending vibration. The
three bands above are characteristic absorption peaks of polysac-
charide. The three bands at 1024–1154 cm−1 indicated pyranose.
The band at 850 cm−1 showed ␣-polysaccharides (Barker, Bourne,
Stacey, & Whiffen, 1954).

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Z.-y. Zhu et al. / Carbohydrate Polymers 88 (2012) 1072–1076
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
A
0
0
5
10
15
20
25
Tube
2.5
B
2
1.5
1
0.5
0
0
5
10
15
Tube
1.6
C
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
5
10
Tube
Fig. 1. Elution profiles of CPS on AB-8 (A), CPS on DEAE-Sephadex A-25 (B), and CPS
on Sephadex G-100 (C).
The 1H NMR spectrum (Fig. 4a) of CPS showed one anomeric
H at ı 5.29 which indicated that the CPS was mainly composed of
one type of sugar. In addition, the 13C NMR spectrum (Fig. 4b) also
showed one anomeric C at ı 99.76, which confirmed that the CPS
was mainly composed of one type of sugar.
Fig. 2. HPLC profiles of crude polysaccharide (A), purified by AB-8 (B), purified by
DEAE-A25 (C), CPS (D). The results supported the conclusion that CPS was homoge-
neous polysaccharides.
The Monosaccharide analysis showed that the CPS
mainly contained glucose (Fig. 5a), Table
1
gave the
monosaccharides composition and the molar ratio of CPS,
Rha:Ara:Xyl:Man:Glu:Gal = 3.0:2.6:1.0:1.3:106.0:2.8.
The results of periodate oxidation demonstrated that the con-
sumption of NaIO4 was 0.153 mmol and no formic acid for CPS.
The GC analysis of the Smith degradation of the periodate-oxidized
CPS showed that it mainly contained erythritol and little glycerol
and arabinose (Fig. 5b). The molar ratio of glycerol, erythritol and
arabinose was 2.29:15.66:1.00. The periodate oxidation and smith
degradation analysis indicated that the linear chain of CPS was
mainly composed of (1 → 4) glucose, small amount of (1 → 2) linked
monosaccharides, (1 → 3) arabinose and no (1 → 6) linked saccha-
rides were detected.
Table 1
Monosaccharides composition and molar ratio of CPS.
Name
Rha
3.0
Ara
2.6
Xyl
1.0
Man
1.3
Glu
Gal
2.8
The fully methylated CPS was hydrolyzed with acid, converted
into alditol acetates, and analyzed by GC–MS (Table 2). GC–MS was
Molar ratio
106.0

Z.-y. Zhu et al. / Carbohydrate Polymers 88 (2012) 1072–1076
1075
Fig. 3. IR spectrum of CPS.
Fig. 5. GC profiles of monosaccharides of CPS (a) and GC analysis of the Smith
degradation of the periodate-oxidized CPS (b).
3.2. Anti-tumor activity of CPS
In this work, K562 cells were cultured with different concen-
trations of CPS for different time. Fig. 6 demonstrated anti-tumor
activities of CPS which were determined by inhibition ratio, when
K562 cells were subjected to coculture with different concen-
trations of CPS. At 24 h coculture on K562, the CPS exhibited a
relatively lower inhibition ratio, about <10% at the concentrations
from 50 to 400 ␮g/mL. At 48 h, the activity had significant dif-
ference; the inhibition ratio reached 43.67% at 50 ␮g/mL of CPS.
Between CPS concentration at 50 and 200 ␮g/mL, the inhibition
ratios on K562 cells ranged from 43.67% to 32.97%. In addition,
when CPS concentration increased from 200 to 400 ␮g/mL, the inhi-
bition on K562 cells significantly enhanced from 32.97% to 56.65%.
At 72 h, the inhibition ratio on K562 cell was reduced compared
with the inhibition ratio at 48 h.
Fig. 4. 1H NMR spectra (a) and 13C NMR spectra (b) of CPS.
performed to indicate the presence of five components, namely
2,3,6-tri-o-methyl-glucose, 6-o-methy-glucose, 2,4-di-o-methyl-
arabinose, 3,4-di-o-methyl-ribose and 1,4,6-tri-o-methy-mannose.
Table 2
Results of the methylation analysis of CPS.
Methylation positions
Linkages
Major mass fragments
(m/z)
2,3,6-tri-o-methyl-glucose
1,4-Linked Glc
43, 45, 87, 99, 101, 113,
117, 233
6-o-methy-glucose
2,4-di-o-methyl-arabinose
1,2,3,4-Linked Glc
1,3-Linked Ara
43, 101, 117
43, 89, 101, 117, 131, 159,
173, 233
3,4-di-o-methyl-ribose
1,4,6-tri-o-methy-mannose
1,2-Linked Rib
1,2,3-Linked Man
43, 89, 101, 117, 189
43, 87, 101, 117
Fig. 6. The anti tumor activity of CPS.

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Z.-y. Zhu et al. / Carbohydrate Polymers 88 (2012) 1072–1076
4. Conclusions
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production. Journal of Ethnopharmacology, 120(3), 366–371.
The results of our study showed that the average molecu-
lar weight of the polysaccharide from C. gunnii mycelium was
3.72 × 107 Da. The CPS was d-glucan containing ␣-(1 → 4)-linked
backbone. Preliminary biological tests suggested that CPS signifi-
cantly inhibit the growth of K562 cell in vitro.
Leung, P. H., Zhao, H., Ho, K. P., & Wu, J. Y. (2009). Chemical properties and a ntiox-
idant activity of exopolysaccharides from mycelial culture of Cordyceps sinensis
fungus Cs-HK1. Food Chemistry, 114(4), 1251–1256.
Liang, Z. Q. (1985). Isolation and identification of the conidial stage of Cordyceps
gunnii. Acta Mycologica Sinica, 4, 162–166.
Liu, A. J., Song, W., Yang, N., Liu, Y. J., & Zhang, G. R. (2007). Cartilage polysaccharide
induces apoptosis in human leukemia K562 cells. Cell Biology and Toxicology, 23,
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Liu, C. H., Lin, Q. X., Gao, Y., Ye, L., & Xi, T. (2007). Characterization and antitumor
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Acknowledgments
Liu, A. J., Zhong, Y. R., Zhu, C. M., & Zhu, Z. Y. (2008). Extraction isolation and analysis
of the polysaccharides from Cordyceps gunnii (Berk.) Berk. Modern Food Science
and Technology, 24, 28–31.
Liu, Y. S., Leung, P. H., & Wu, J. Y. (2008). Exopolysaccharide production in batch and
semi-continuous fermentation of Cordyceps sinensis. Journal of Biotechnology,
136(S1), S301–S302.
Wang, B. J., Wei, M., & Zhang, L. P. (2003). Studies on structure and properties of
water soluble polysaccharide from fruiting body of Cordyceps militaris (L.) Link.
Chemical Research in Chinese Universities, 19(1), 34–37.
Wang, Z. S., Yu, Y. X., & Yuan, Q. S. (2004). Bioactive components of Cordyceps (Fr.)
Link fungi. Chinese Traditional and Herbal Drugs, 10, 8–11.
This work was financially supported by Program for Changjiang
Scholars and Innovative Research Team in University (IRT1166),
National Agricultural Innovation Project (no. 2011GB2A100009),
Natural Science Foundation of Tianjin City (nos. 09JCZDJC21800,
09JCYBJC15800), the Foundation of Tianjin Educational Commit-
tee (no. 20090604), National Natural Science Foundation of China
(NSFC, 31170541), Program for New Century Excellent Talents in
University (NCET-10-0951).
Wu, Y. L., Sun, C. R., & Pan, Y. J. (2006). Studies on isolation and structural features
of a polysaccharide from the mycelium of a Chinese edible fungus (Cordyceps
sinensis). Carbohydrate Polymers, 63, 251–256.
Wu, Y. L., Hu, N., Pan, Y. J., Zhou, L. J., & Zhou, X. X. (2007). Isolation and characteriza-
tion of a mannoglucan from edible Cordyceps sinensis mycelium. Carbohydrate
Research, 342, 870–875.
Yu, R. M., Yang, W., Song, L. Y., Yan, C. Y., Zhang, Z., & Zhao, Y. (2007). Structural
characterization and antioxidant activity of a polysaccharide from the fruiting
bodies of cultured Cordyceps militaris. Carbohydrate Polymers, 70, 430–436.
Zhang, W. Y., Li, J., Qiu, S. Q., Chen, J. P., & Zheng, Y. (2008). Effects of the exopolysac-
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