Research On Anti-tumor And Immunity Function Of Extracellular Polysaccharides From Inonotus Obliquus

Inonotus obliquus (Fr.) Pilát is also known as "Chaga". It has been widely used as atraditional medicine in North America and Scandinavia. However, its artificial domesticationhas not yet become a mature technology and the increasing scarcity of resources results in therestricted phenomenon of wild Inonotus obliquus. For the further development and utilization ofInonotus obliquus, our study chose extracellular polysaccharide，intracellular polysaccharide andfermentation extract polysaccharide submerged fermentation as experimental material. Antumorexperiments were done on SKOV3. Extracellular polysaccharide was chosen to do the furtherexperiments Study the immune mechanisms of Inonotus obliquus via the experiments of normalmice and cyclophosphamide-induced immunization inhibition mice in vivo. And this paper alsostudied the effects of extracellular polysaccharide of Inonotus obliquus in mice on cough andphlegm.Meanwhile, immune-related contents of extracellular polysaccharide of Inonotusobliquus were detected, providing a theoretical basis for further study on functional foods andpharmaceuticals. The results were as follows:1Select two of the most commonly recognized polysaccharides having immune activity totest, which is glucan and mannan oligosaccharide. The results show that the glucan inextracellular polysaccharide of Inonotus obliquus contains0.1%. The mannan oligosaccharidehas not been detected. Intracellular polysaccharide contains82%glucan.Fermentation extractpolysaccharide contains20%glucan,2%.2Three kinds of polysaccharides from Inonotus obliquus in different concentration ofdrugs have inhibitory effect on SKOV3human ovarian cancer cells. The inhibition rates ofextracellular polysaccharide were25.1%、29.0%、23.5%、18.0%and16.7%, respectively. Theinhibition rates of intracellular polysaccharide were15.8%、28.6%、22.6%、17.8%and15.4%,respectively. The inhibition rates of fermentation extract polysaccharide were15.4%、16.7%、18.7%、23.7%and28.6%. The best concentration of three kinds of polysaccharides from Inonotusobliquus were50μg/mL、50μg/mL、400μg/mL.3To investigate the effects of extracellular polysaccharide of Inonotus obliquus oncyclophosphamide-induced immunosuppressant mice. The organ index, levels of TNFα and IL2were measured and lymphoproliferative experiment was done. After mice were injected withcyclophosphamide, thymus index and spleen of mice in model group were significantly lowerthan the control group, indicating that the immune function of mice with cyclophosphamide significantly inhibited and the formation of immune suppression model was completed. With themodel group comparison found positive control group and high-dose of extracellularpolysaccharide can significantly improve the immune suppression and thymus index of mice (P<0.01). Medium dose group and low dose group also significantly increased thymus index (P<0.05). Positive control group, high-dose and low dose of extracellular polysaccharide couldsignificantly improve immunosuppressant mice spleen index (P <0.05). It was found that themodel group compared with the control group, a significant reduction in serum IL-2and TNF-αlevels (P <0.01); serum levels of IL-2and TNF-α of extracellular polysaccharide groupincreased significantly compared with model group (P <0.01), and with increased serumconcentrations of IL-2and TNF-α levels are elevated in a dose-related nature. These resultsindicate that giving extracellular polysaccharide of Inonotus obliquus can inhibit and reduce thelevels of TNF-α and IL-2induced by cyclophosphamide in serum. Experimental results showthat compared with normal control group, group of high doses of Chaga normal mouse spleenCon A-induced lymphocyte proliferation has promoted (P <0.05). Positive control group ofnormal mice Con A-induced splenic lymphoid cell proliferation significantly promoted (P<0.01). Low dose and medium extracellular polysaccharide has promoting trend, but it has nostatistically significant (P>0.05). To test organ index, CD4+/CD8+, cytokines, mRNA expressionof lymphocyte in the intestinal cells and neutrophils, experiments using gavage to mice for14days. The results showed that in the conditon of the administration dosage were500mg/kg,250mg/kg,125kg/kg, high-dose and the medium dose significantly increased thymus index innormal mice (P <0.05). Low-dose group may also improve thymus index in normal mice, but itdo not have statistically significant (P <0.05). Positive control group and three dose groups ofextracellular polysaccharide could improve spleen index, but has no statistically significant (P>0.05). Studies have shown that compared with the negative control group the high dose ofextracellular polysaccharide can significantly increase the content of CD4+(P <0.01), whilesignificantly reducing the content of CD8+(P <0.05). It was found that in the group of high-dose of Chaga, serum levels of TNF-α were significantly lower (P <0.05) compared with thecontrol group. Compared with the control group, high-dose group significantly reduced thelevel of INFγ(P <0.05).High-dose of Chaga significantly increased serum levels of CRP (P<0.05). The level of IL6in mice serum was not significantly affected (P>0.05) These resultsindicate that giving extracellular polysaccharide of Chaga in a certain dose can decreased INFγand TNF-α levels in normal mice, and significantly improve CRP levels.Whereas, IL6is noimpact on the statistical significance (P>0.05). In the study of the intestinal sample, resultsshow that compared with the control group, high-dose of extracellular polysaccharide can significantly increased mRNA expression of IL-8R’s (P <0.05).High-dose of extracellularpolysaccharide significantly increased the the mRNA expression of CD62L (L-selectin)(P<0.05). Low-dose of extracellular polysaccharide could significantly improve IL6mRNAexpression levels. For blood sample, compared with the control group，high-dose of extracellularpolysaccharide can significantly improve the mRNA expression of CD62L (L-selectin)(P<0.01). Medium-dose of extracellular polysaccharide can significantly increased the mRNAexpression level of CD62L (L-selectin)(P <0.05). Select medium dose group of Inonotusobliquus (250mg/kg) conducted28days of ad libitum experiment. Results show that comparedwith the control group medium dose group of Inonotus obliquus significantly increased thymusindex (P <0.05). INFγ and TNF-α levels were increased significantly in normal mice (P>0.05),while for IL6and CRP, there were no statistically significant effects (P>0.05). Intestinalsamples showed that medium dose group of Inonotus obliquus significantly increased IL-8R andIL6mRNA expression of (P <0.01).4To study the antitussive and expectorant effects of Chaga, ammonia induced micecoughing model was used to evaluate the antitussive activity and the expectorant activity wasevaluated by volume of phenol red in mice’s tracheas. High and medium dose of Chaga couldsignificantly reduce the frequency of cough with ammonia and prolongen thedelitescence(P<0.01)compared with the blank control group, which show the antitussive effectsof Chaga High dose of Chaga also could increase the secreted volume of phenolsulfonphthaleinin mice’s tracheas(P<0.01) compared with the blank control group, which show the expectoranteffects of Chaga.