Structural Characterization And Immunomodulatory Effects Of A Polysaccharide Isolated From Cordyceps Cicadae

Cordyceps cicadae is a special form of medicinal mushroom which has its fungal fruit body formed on an insect larva. It is one of the species in the genus Cordyceps which has a record of about 750 identified species. Cordyceps cicadae display similar chemical constituents and pharmacological functions such as anti-fatigue, hyperglycemia, hyperlipidemia, reno-protective and immunomodulatory properties to those of Cordyceps sinensis. Polysaccharides are considered as one of the main active constituents found in Cordyceps, with active functions such as antioxidants, antitumor, antiviral, hypoglycemic, and immunomodulatory(improvement of macrophage functions, inducing T-lymphocyte proliferation and promotion of dendritic cell maturation) functions. In our study, we got the polysaccharide(CP50 and CP80) extracted from Cordyceps cicadae in hot water and isolated by precipitation in different concentrations of ethanol. CP50 showed higher reducing capacity and total antioxidant activity. CP50 was then further purified by DEAE–cellulose-52 and Sephadex G-100, then to analysis their structural characterizations and immunomodulatory effects.1.Study on the antioxidant activities of polysaccharides from Cordyceps cicadas. Two crude polysaccharide fractions(CP50 and CP80) were extracted from Cordyceps cicadae in hot water and isolated by precipitation in different concentrations of ethanol. The antioxidant activities were determined with DPPH radical scavenging assay, hydroxyl radical scavenging assay, total antioxidant assay, and reducing power assay. The results indicated that both CP50 and CP80 were effective in antioxidant activity. The activity of CP50 on radical-scavenging were relatively higher than that of CP80. CP50 also showed higher reducing capacity and total antioxidant activity. Therefore, CP50 was more superior to CP80 in antioxidant capacity, and more suitable to developed as antioxidants.2.Study on the purification and chemical properties of polysaccharides from Cordyceps cicadas. CP50 was then further purified by DEAE–cellulose-52 and Sephadex G-100, two purified fractions were obtained, namely CPA-1 and CPB-1. The UV and HPLC profiles appeared that both CPA-1 and CPB-1 were homogeneous, their average molecular weights were 2.82×104 Da and 1.19×104 Da, respectively. The results of chemical properties showed that both of the polysaccharides were white powder, they were soluble in water, but insoluble in ethanol, chloroform and other organic solvents. Both of them didn’t contain starch, protein, amino acids.3.Study on the structure characteristics of CPA-1 and CPB-1. The structural characterization was achieved using FT-IR, 1H NMR spectrum, HIO4 oxidation, Smith degradation and CD. The monosaccharide analysis was determined with the aid of GC. Our results indicated that CPA-1 was composed of mannose, glucose and galactose(molar ratio of 0.479:1.0:0.615), CPB-1 was also composed of mannose, glucose and galactose(molar ratio of 1:0.14:0.114). The results of sulfuric acid-phenol method indicated CPA-1 contained the monosaccharide about 82.3%, CPB-1 contained the monosaccharide about 79.2%. We use carbazol colorimetric method to check the content of glycuronic acid, the results addicated that CPA-1 contained about 0.956%, CPB-1contained about 1.87%. Both FTIR spectrums of CPA-1 and CPB-1 showed characteristic absorption peaks of polysaccharide. The signals of 1H NMR spectrum attributable to presence of α and β-type of glycosidic linkage in CPA-1 and CPB-1.Based on the results of HIO4 oxidation and Smith degradation, we speculate that the existence of α-(1→2) or(1→4) linkage in the main or branch chain in CPA-1, and CPB-1 showed the existence of 1→3 in glucose, 1→2、1→4 and little 1→6 linkage in mannose and galactose. In addition, the results showed that the CD spetrum of CPA-1 and CPB-1 changed significantly with external conditions(temperature, the pH value, ion intensity etc). It indicated that the conformation and asymmetry of the CPA-1 and CPB-1 were changed with the change of the external conditions.4. Immunomodulatory effects of a polysaccharide isolated from Cordyceps cicadae. Dendritic cells(DCs) are the most potent professional antigen-presenting cells. They act as a link between the adaptive and innate immune systems. DCs are responsible for the initiation of immune responses and they have been reported to interact with polysaccharides, as a means of connecting the innate and adaptive immunity. Our study revealed that CPA-1 and CPB-1 displayed a significant enhancement of DCs proliferation. They also changed the morphology of the pretreated DCs and significantly increased the surface antigen expressions of CD80/86, CD11 c and MHC class II. In a world, CPA-1 and CPB-1 could induce and activate DCs maturation as well as modulates DCs immune response.