Purification, Characterization, Immunomodulatory And Antioxidant Activities Of The Polysaccharides From The Cordyceps Kyushuensis

As is well known, Cordyceps sinensis, one of the famous traditional Chinese medicines, has been a kind of scarce resource due to excessive exploitation. So in recent years, people are actively seeking alternative Cordyceps to reduce the dependence on Cordyceps sinensis. Cordyceps kyushuensis is a kind of Cordyceps hosting of Bilineata larvae, and it had been artificial cultured. The polysaccharides, one of the main active ingredients, have been reported to account for the anti-tumor, anti-inflammatory, antioxidant, antiviral and immunomodulatory effects. At present, polysaccharide isolated from Cordyceps kyushuensis has been scarcely studied. Therefore, in this article polysaccharides isolated and purified from the fruit body of Cordyceps kyushuensis, their physical and chemical properties, the antioxidant and immunomodulatory activities were studied.First, crude polysaccharides were extracted from the fruiting bodies of Cordyceps kyushuensis by water-alcohol precipitation with the alcohol concentration of30%and50%. Two fractions were obtained, named CK-1and CK-2. Then the CK-2was fractionated by anion-exchange chromatography on a column DEAE-23cellulose eluted with different stepwise of NaCl (0.06M,0.1M,0.16M and0.32M). All four fractions were further purified on a column of Sephadex G-150, respectively, resulting in producing four purified polysaccharides named as CK-2A,CK-2B,CK-2C and CK-2D. The purity of the four purified polysaccharides was identified by HPLC and the results supported the conclusion that the four fractions were homogeneous polysaccharides.The physicochemical properties of CK-2A,CK-2B,CK-2C and CK-2D were analysised. The total sugar content were24.35%,17.33%,24.29%and18.22%, and the uronic acid content was lower than1.5%, and the protein content of CK-2D was the highest, at4.341%. The molecular weight of CK-2A,CK-2B,CK-2C and CK-2D were calculated as follow:7153,5945,5643and5642KDa, respectively. The results of a UV scan showed that the water solution of polysaccharides had characteristic absorption of polysaccharide at200nm and protein absorption at280, no absorption at260nm for nucleic acid. The IR spectra of CK-2A,CK-2B,CK-2C and CK-2D demonstrated the main absorption characteristics of the polysaccharide were exhibited between2800and3000cm-1, as well as the absorbance between950and1200cm-1indicating the presence of a pyranose ring. According to HPLC analysis of the four polysaccharides after TFA hydrolysis and PMP derivative, CK-2A was composed of Fru, Man, Glu and Gal with a content ratio of1:0.92:1.09:0.72. CK-2B and CK-2C were composed of Fru, Man and Gal in the proportion of1:0.63:0.61,1:1.65:1.4, as well as CK-2D was composed of Fru, Man and Gal with the proportion of1:2.06:1.97.Finally, the polysaccharides of CK-2A、CK-2B、CK-2C and CK-2D were determined for their antioxidant and immunomodulatory activities. We first determined the content of endotoxin in the polysaccharides and found that none existed in CK-2A, while there was a small amount of endotoxin in CK-2B, CK-2C and CK-2D. Further experiments showed that the equivalent of0.5μg/ml LPS of endotoxin had no obvious immunoregulating activity. In vitro experiments, splenic lymphocytes of normal mice were cultivated with different concentrations (125μg/ml、250μg/ml、500μg/ml、1000μg/ml) of polysaccharides and then detected by CCK-8Kit. Compared with blank control, in addition to the concentrations of500μg/ml and1000μg/ml of CK-2B, the rest could all significantly promote lymphocyte proliferation (P<0.01). In vivo detection of the effects of polysaccharide on lymphocyte proliferation, results revealed that four kinds of polysaccharides could not only significantly stimulate the proliferation of normal mice mixed lymphocyte, but also well promote the proliferation of ConA/LPS induced T/B lymphocyte (P<0.01), and could increase the levels of IL-2at the same time. The four polysaccharides could in different degrees enhance the abdominal macrophage phagocytosis in normal mice. However, in vitro polysaccharide couldn’t inhibit the proliferation of tumor cell B16. All the results showed that Cordyceps kyushuensis polysaccharides had a certain regulation of the immune system of mice and thus we speculated that polysaccharide played antitumor activity by immune regulating, rather than by directly killing tumor cells. Antioxidant experiment in vitro found that four kinds of polysaccharides all could in different degrees remove DPPH free radicals at the concentration of1-5mg/ml, of them CK-2D the highest clearance rate (35.6%) at lmg/ml and CK-2C the utmost (63.4%) at5mg/ml. Though the scavenging capacity of CK-2A and CK-2B to DPPH free radicals was relatively low, they had certain protective effect on the DNA damage induced by hydroxyl radicals, while the effect of CK-2C and CK-2D on DNA damage was not obvious and a possible reason was that polysaccharide could eliminate hydroxyl radicals, reducing the damage degree to DNA.