Planta Med 2005; 71(4): 381-384
DOI: 10.1055/s-2005-864111
Letter
© Georg Thieme Verlag KG Stuttgart · New York

Structure Analysis and Antitumor Activity of (1→3)-β-D-Glucans (Cordyglucans) from the Mycelia of Cordyceps sinensis

Wu Yalin1 , Omar Ishurd1 , 2 , Sun Cuirong1 , Pan Yuanjiang1
  • 1Department of Chemistry, Zhejiang University, Hangzhou , P. R. China.
  • 2Bio-technology Research Center, Tripoli, Libya
Further Information

Prof. Dr. Pan Yuanjiang

Department of Chemistry

Zhejiang University

YuQuan Campus

Hangzhou 310027

People’s Republic of China

Phone: +86-571-8795-1264

Fax: +86-571-8795-1264

Email: panyuanjiang01@yahoo.com

Publication History

Received: June 9, 2004

Accepted: November 15, 2004

Publication Date:
27 April 2005 (online)

Table of Contents #

Abstract

The cell wall polysaccharides from Cordyceps sinensis were obtained from the fresh samples of the mycelia, the material was released by successive extractions with hot water and 0.05 M sodium hydroxide solutions. The extracts were fractionated by ion-exchange and gel-filtration chromatography, respectively. Analysis of the first fraction showed that cordyglucans were the unique component. Cordyglucans were found to exhibit potent antitumor activity, this activity could be correlated to their (1→3)-β-D-glucan linkages.

Cordyceps sinensis, a fungal parasite on the larvae of Lepidoptera, has been used as a tonic herb in Chinese traditional medicine for centuries. Many reports deal with its pharmacological activities [1]. Polysaccharides and nucleosides are often associated with these pharmacological activities of Cordyceps sinensis; however, to the best of our knowledge, the structures of the polysaccharides from Cordyceps sinensis have not yet been investigated. There is a strong tradition in Japan that Basidiomycetes belonging to the Polyporaceae family are effective against cancer. Ikekawa et al. have already reported that the hot-water extracts from the Polyporaceae family inhibit the growth of solid-tumor sarcoma 180 implanted subcutaneously in mice, and the activity is attributable to (1→3)-β-D-glucans [2]. The purpose of the present study is to contribute further information about the isolation and purification of β-D-glucans (cordyglucans) from Cordyceps sinensis [3].

The cell walls cordyglucans of Cordyceps sinensis were prepared as previously reported [4], and obtained in yield of 4 % based on the fresh weight of the mycelia. Analysis of the first fraction showed that D-glucans were unique compounds. Gel filtration of these glucans on Sepharose CL-4B indicated a range of molecular weight from 1 × 105 to 1 × 104 with a preponderance of material of high molecular weights. Ultracentrifugation analysis indicated the average molecular weight to be 12 860. The polysaccharide had [α]D 26: + 32° (c 0.1, water), which confirmed the D-configuration of cordyglucans.

The cordyglucans were twice methylated by the method of Hakomori [5]. After hydrolysis, reduction and acetylation, GLC of the alditol acetates from the fully methylated glucans showed three peaks (Table [1]). These results indicated a (1→3)-linked backbone with (1→6)-linked branches. After acetolysis, gel filtration on Sephadex G-25 gave only two fractions (A and B). The higher (A) molecular weight fraction was eluted with the void volume; the second (B) was composed of only glucose monomers, suggesting the presence of single D-glucosyl groups as side chains attached at O-6 of some of the main-chain units. The cordyglucans were submitted to periodate oxidation, borohydride reduction, and hydrolysis under mild conditions (Smith degradation) [6]. The anomeric proton signal at δ = 5.17 (d, J = 7.4 Hz) in the 1H-NMR spectrum confirmed that the sugar residues were linked β-glycosidically, which agrees with the presence of an IR band at 890 cm- 1 [7], [8]. The β-configuration of the D-glucosyl groups was clearly evidenced by the presence of two anomeric peaks in the 13C-NMR spectrum [9], [10]. The results suggested the basic structure 1 for the D-glucans of Cordyceps sinensis. [*]

A Smith degradation to remove single (1→6)-β-D-glucopyranosyl branch units from the cordyglucans was performed according to [11]. The debranched cordyglucans had a low solubility in water and was recovered by centrifugation, washed with water, and freeze-dried. Results of bioassays of the cordyglucans and the debranched cordyglucans are given in Table [2]. Under ”complete regression” is given the number of mice in which the tumors completely disappeared after initial active growth, against the total number of mice in the group. It is evident that cordyglucan is effective against sarcoma 180 at a level of 0.8 mg/kg, where it shows almost 90 % inhibition with complete regression in 8 out of 10 mice tested. Our data are consistent with previous reports [12] concerning a possible correlation between antitumor activity and this type of (1→3)-β-D-glucans. Thus, there seemed to be a connection between antitumor activity and linearity of this type of compound. However, removal of branches of D-glucans gave an inactive glucan. This implies that linearity alone does not account for the activity.

Cordyceps sinensis produced cordyglucans of the same type as those of P. parasitica Dastur [13]. Cordyglucan is one of the most effective antitumor D-glucans reported [14], [15], and did not show toxic effects in the test animals, which remained in excellent physical condition throughout the testing period. Cordyglucan is readily available and may prove useful for evaluation of the antitumor action of glucans. The antitumor activity of these glucans is dose-dependent and tends to decrease with an increase in dose (Table [2]). A similar, dose-dependent, decrease of the antitumor activity has been noted in the case of lentinan [12]. It is probable that there are optimum doses for elicitation of a specific immunological response in the host. It is interesting that cordyglucans and certain other glucans, although having very similar structures, have different levels of activity. The difference in activity cannot be explained on the basis of present structural knowledge.

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Table 1 Molar ratio of the hydrolysis products of methylated native glucans and Smith-degraded glucans
O-Methyl-D-glucose Linkage indicated Molar ratios of glucans Smith degraded
Native
2,3,4,6-Tetra- Glc(→ 1.0 1.0
2,4,6-Tri- →3)-Glc(1→ 3.0 16.0
2,4-Di- →3)-Glc(1→
6
0.7 -
Table 2 Antitumor activity of cordyglucan and debranched derivatives from Cordyceps sinensis
Polysaccharide Daily dose (mg/kg) Tumor wt (g)a Inhibition (%) Complete regressionb
Cordyglucan 0.8 1.2 (0 - 8.8) 89.8 8/10
4.0 1.9 (0 - 7.1) 83.3 6/10
40.0 6.7 (0 - 16.9) 42.4 2/10
Debranched cordyglucan 0.8 6.3 (0 - 14.8) 46.2 2/10
4.0 9.6(7.5 - 19.9) 17.6 0/10
Control (negative) 0 11.7 (3.7 - 15.8) - 0/10
Control (CPA)* 0.8 8.3 (0 - 15.7) 29.1 1/10
40 4.5 (0 - 7.5) 61.5 7/10
a Average; in parentheses, minimum and maximum.
b Ratio of number of mice showing complete regression to number of mice tested.
Cyclophosphamide(CPA) was used as positive control in this test.
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Materials and Methods

Cordyceps sinensis mycelium (950 g) was used in this work. The mycelia obtained from Mr. Lijun Zhou, Biotechnological Co. Ltd. in Lishui (China). A voucher specimen (DFC 0423) is deposited at the Department of Chemistry, Zhejiang University, China.

Cordyglucans were isolated from mycelial walls, and extracted with boiling water (4 L, 100 °C) for 4 h. The supernatant solution was diluted with 95 % EtOH, washed with acetone, and dried. The insoluble residue was suspended in 0.5 M NaOH for 3 h at 80 °C and then centrifuged, neutralized with acetic acid and dialyzed. The non-dialyzed fraction was lyophilized and then fractionated by column chromatography on DEAE-cellulose (Pharmacia). Fractions were assayed for carbohydrate by the phenol-sulfuric acid method [16].

A solution of cordyglucans (3 mg) in distilled water (0.5 mL) was applied to a column (1.6 × 80 cm) of Sepharose CL-4B. The column was equilibrated and eluted with distilled water. The carbohydrate content of each fraction was determined with the anthrone reagent [17].

The cordyglucans (8 mg) were methylated by the method of Hakomori [5]. Methylated sugars were reduced with NaBH4, acetylated with acetic anhydride, and analyzed by GLC. The methylated sugars were analyzed by GLC-MS (methylated glucitol acetates) [18], [19].

A sample (13 mg) was oxidized with 0.05 M NaIO4 (13 mL) at 4 °C in the dark for one week. Oxidation was stopped by addition of 1,2-ethanediol. The material was dialyzed and reduced with NaBH4, then hydrolyzed in 0.5 M trifluoroacetic acid and fractionated by chromatography on Sephadex G-15 (1.6 × 80 cm).

Acetolysis of glucans (11 mg) was performed according to Dubourdieu et al. [4]. Acetylated sugars were extracted with chloroform washed with concentrated aqueous sodium hydrogen carbonate, dried (CaCl2) and concentrated. Each residue was dissolved in acetone (4 mL), and 0.2 M sodium hydroxide (4 mL) was added. After 30 min at 4 °C, the reaction was stopped by adding Dowex 50-X8 (H+) resin to pH 5. The resin was removed and the filtrate was concentrated at 40 °C under reduced pressure. The residue was eluted from a column of Sephadex G-15 with distilled water [20].

Exo-(1→3)-β-D-glucanase was prepared from a culture of Basidiomycetes sp. QM 806 according to the procedure of Peterson and Kirkwood [21]. To a solution of the polysaccharide (10 mg) in Sörensen phosphate buffer (pH 6.5, 2 mL) was added 45 μL of β-D-glucanase suspension (5 mg/mL). The mixture was stirred and dialyzed at 37 °C overnight against the same buffer. The dialyzed solution was heated to ∼100 °C for 5 min to inactivate the enzyme and then centrifuged, and the supernatant solution was dialysed against distilled water and freeze-dried. The dialysate was deionized using ion-exchange resin and concentrated to a syrup. Analytical results from TLC and PC revealed only glucose.

Female ICR/JCL mice (Academy of Medical Sciences in Zhejiang Province, Hangzhou, China), at least 6-week-old, 10 per group, were used. During the experimental period, they were housed in standard environmental conditions (bred at the Central Animal House of College of Animal Science, Hangzhou, China) and supplied with tap water and standard diet ad libitum. Food and water consumption as well as body weight gain were monitored daily over the treatment period. The care and treatment of the mice were in accordance with the guidelines established by the publication of Health Service Policy on Humane Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee.

Antitumor activity was tested by the method of Chihara et al. [22]. ICR/JCL mice weighing about 20 g were used for the antitumor assay. Seven-day-old ascites of sarcoma 180 were transplanted subcutaneously into the right groin of mice. The test samples, dissolved in saline, were intraperitoneally injected daily for 10 days, starting 24 h after tumor implantation. The growth of tumors was charted weekly for 5 weeks. At the end of the 5th week, the mice were killed, and the tumors were extirpated and weighed. The inhibition ratio was calculated.

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Acknowledgments

The authors are grateful to NSFC of China (20 365 037) and the Forestry Department (Zhejiang University, China) for the identification of the plant. This work was supported by a grant from Biotechnological Co. Ltd ( Lishui, China) .

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References

  • 1 Zhu J S, Halpern G M, Jones K. The scientific rediscovery of an ancient Chinese herbal medicine: Cordyceps sinensis: part I.  J Altern Complement Med.. 1998;  4 289-303
  • 2 Ikekawa T, Uehara N, Maeda Y, Nakanishi M, Fukuoka F. Antitumor activity of aqueous extracts of edible mushrooms.  Cancer Res. 1969;  29 734-5
  • 3 Stone B A, Clarke A E. Chemistry and biology of (1→3)-β-glucan. Victoria, Australia; La Trobe University Press 1992
  • 4 Dubourdieu D, Ribereau P, Fournet B. Structure of the extracellular β-D-glucan from Botrytis cinerea .  Carbohydr Res. 1981;  93 294-9
  • 5 Hakomori S. A rapid permethylation of glycolipid, and polysaccharide catalyzed by methylsulfinyl carbanion in dimethyl sulfoxide.  J Biochem. 1964;  55 205-8
  • 6 Dixon J S, Lipkin D. Spectrophotometric determination of vicinal glycols (Application to the determination of ribofuranosides).  Anal Chem. 1954;  26 1092-3
  • 7 Ishurd O, Zahid M, Ahmad V U, Pan Y. Isolation and structure analysis of a glucomannan from the seeds.  J Agric Food Chem. 2001;  49 3772-4
  • 8 Ishurd O, Sun C, Xiao P, Ashour A. A neutral β-D-glucan from dates of the date palm, Phoenix dactylifera L.  Carbohydr Res. 2002;  337 1325-8
  • 9 Ishurd O, Yousef A, Wanxing W, Ashour A. An alkali-soluble heteroxylan from seeds of Phoenix dactylifera L.  Carbohydr Res. 2003;  338 1609-12
  • 10 Saito H, Okhi T, Takasuka N, Sasaki T. A 13C-N.M.R.-spectral study of a gel-forming, branched(1 - 3)-β-D-glucan, (lentinan) from Lentinus edodes, and its acid-degraded fractions. Structure, and dependence of conformation on the molecular weight.  Carbohydr Res. 1977;  58 293-305
  • 11 Goldstein I J, Hay G W, Lewis B A, Smith F. Controlled degradation of polysaccharides by periodate oxidation, reduction, and hydrolysis.  Methods Carbohyd Chem. 1965;  5 361-70
  • 12 Chihara G, Maeda Y, Hamuro G, Sasaki T, Fukuoka F. Inhibition of mouse sarcoma 180 by polysaccharides from Lentinus edodes (Berk.) Sing.  Nature. 1969;  222 687-8
  • 13 Bartnicki S. Cell wall chemistry morphogenesis, and taxonomy of fungi.  Annu Rec Microbiol. 1968;  22 87-108
  • 14 Sasaki T, Takasuka N. Further study of the structure of lentinan, an anti-tumor polysaccharide from Lentinus edodes .  Carbohydr Res. 1976;  47 99-104
  • 15 Misaki A, Nasu M, Sone Y, Kishida E, Kinoshita C. Comparison of structure and antitumor activity of polysaccharides isolated from Fukurotake, the fruiting body of Volvariella volvacea .  Agric Biol Chem. 1986;  50 2171-83
  • 16 Dubois M, Gilles K A, Hamilton J K, Rebers P A, Smith F. Colorimetric method for determination of sugars and related substances.  Anal Chem. 1956;  28 350-6
  • 17 Shields R, Burneu W. Determination of protein-bound carbohydrate in serum by a modified anthrone method.  Anal Chem. 1960;  32 885-6
  • 18 Bjorndal H, Lindberg B, Svenndon S. Mass spectrometry of partially methylated alditol acetates.  Carbohydr Res. 1967;  5 433-41
  • 19 Perret J, Bruneteau M, Michel G, Marais M F, Joseleau J P, Ricci P. Effect of growth conditions on the structure of β-d-glucans from Phytophthora parasitica Dastur, a phytopathogenic fungus.  Carbohydr Res. 1992;  17 231-6
  • 20 Bayard B, Montreuil J. Études sur les glycoprotéines. XLVIII. Description d’un procédé de fractionnement des acétolysats.  Carbohydr Res. 1972;  24 427-43
  • 21 Peterson D R, Kirkwood S. Studies on the structure and mechanism of an exo-(1→3)-β-D-glucanase from Basidiomycete QM806.  Carbohydr Res. 1975;  41 273-83
  • 22 Chihara G, Hamuro J, Maeda Y, Arai Y, Fukuoka F. Fractionation and purification of the polysaccharides with marked antitumor activity, especially lentinan, from Lentinus edodes (Berk.) Sing. (an edible mushroom).  Cancer Res. 1970;  30 2776-81

Prof. Dr. Pan Yuanjiang

Department of Chemistry

Zhejiang University

YuQuan Campus

Hangzhou 310027

People’s Republic of China

Phone: +86-571-8795-1264

Fax: +86-571-8795-1264

Email: panyuanjiang01@yahoo.com

#

References

  • 1 Zhu J S, Halpern G M, Jones K. The scientific rediscovery of an ancient Chinese herbal medicine: Cordyceps sinensis: part I.  J Altern Complement Med.. 1998;  4 289-303
  • 2 Ikekawa T, Uehara N, Maeda Y, Nakanishi M, Fukuoka F. Antitumor activity of aqueous extracts of edible mushrooms.  Cancer Res. 1969;  29 734-5
  • 3 Stone B A, Clarke A E. Chemistry and biology of (1→3)-β-glucan. Victoria, Australia; La Trobe University Press 1992
  • 4 Dubourdieu D, Ribereau P, Fournet B. Structure of the extracellular β-D-glucan from Botrytis cinerea .  Carbohydr Res. 1981;  93 294-9
  • 5 Hakomori S. A rapid permethylation of glycolipid, and polysaccharide catalyzed by methylsulfinyl carbanion in dimethyl sulfoxide.  J Biochem. 1964;  55 205-8
  • 6 Dixon J S, Lipkin D. Spectrophotometric determination of vicinal glycols (Application to the determination of ribofuranosides).  Anal Chem. 1954;  26 1092-3
  • 7 Ishurd O, Zahid M, Ahmad V U, Pan Y. Isolation and structure analysis of a glucomannan from the seeds.  J Agric Food Chem. 2001;  49 3772-4
  • 8 Ishurd O, Sun C, Xiao P, Ashour A. A neutral β-D-glucan from dates of the date palm, Phoenix dactylifera L.  Carbohydr Res. 2002;  337 1325-8
  • 9 Ishurd O, Yousef A, Wanxing W, Ashour A. An alkali-soluble heteroxylan from seeds of Phoenix dactylifera L.  Carbohydr Res. 2003;  338 1609-12
  • 10 Saito H, Okhi T, Takasuka N, Sasaki T. A 13C-N.M.R.-spectral study of a gel-forming, branched(1 - 3)-β-D-glucan, (lentinan) from Lentinus edodes, and its acid-degraded fractions. Structure, and dependence of conformation on the molecular weight.  Carbohydr Res. 1977;  58 293-305
  • 11 Goldstein I J, Hay G W, Lewis B A, Smith F. Controlled degradation of polysaccharides by periodate oxidation, reduction, and hydrolysis.  Methods Carbohyd Chem. 1965;  5 361-70
  • 12 Chihara G, Maeda Y, Hamuro G, Sasaki T, Fukuoka F. Inhibition of mouse sarcoma 180 by polysaccharides from Lentinus edodes (Berk.) Sing.  Nature. 1969;  222 687-8
  • 13 Bartnicki S. Cell wall chemistry morphogenesis, and taxonomy of fungi.  Annu Rec Microbiol. 1968;  22 87-108
  • 14 Sasaki T, Takasuka N. Further study of the structure of lentinan, an anti-tumor polysaccharide from Lentinus edodes .  Carbohydr Res. 1976;  47 99-104
  • 15 Misaki A, Nasu M, Sone Y, Kishida E, Kinoshita C. Comparison of structure and antitumor activity of polysaccharides isolated from Fukurotake, the fruiting body of Volvariella volvacea .  Agric Biol Chem. 1986;  50 2171-83
  • 16 Dubois M, Gilles K A, Hamilton J K, Rebers P A, Smith F. Colorimetric method for determination of sugars and related substances.  Anal Chem. 1956;  28 350-6
  • 17 Shields R, Burneu W. Determination of protein-bound carbohydrate in serum by a modified anthrone method.  Anal Chem. 1960;  32 885-6
  • 18 Bjorndal H, Lindberg B, Svenndon S. Mass spectrometry of partially methylated alditol acetates.  Carbohydr Res. 1967;  5 433-41
  • 19 Perret J, Bruneteau M, Michel G, Marais M F, Joseleau J P, Ricci P. Effect of growth conditions on the structure of β-d-glucans from Phytophthora parasitica Dastur, a phytopathogenic fungus.  Carbohydr Res. 1992;  17 231-6
  • 20 Bayard B, Montreuil J. Études sur les glycoprotéines. XLVIII. Description d’un procédé de fractionnement des acétolysats.  Carbohydr Res. 1972;  24 427-43
  • 21 Peterson D R, Kirkwood S. Studies on the structure and mechanism of an exo-(1→3)-β-D-glucanase from Basidiomycete QM806.  Carbohydr Res. 1975;  41 273-83
  • 22 Chihara G, Hamuro J, Maeda Y, Arai Y, Fukuoka F. Fractionation and purification of the polysaccharides with marked antitumor activity, especially lentinan, from Lentinus edodes (Berk.) Sing. (an edible mushroom).  Cancer Res. 1970;  30 2776-81

Prof. Dr. Pan Yuanjiang

Department of Chemistry

Zhejiang University

YuQuan Campus

Hangzhou 310027

People’s Republic of China

Phone: +86-571-8795-1264

Fax: +86-571-8795-1264

Email: panyuanjiang01@yahoo.com

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