The Extraction Process And Anticancer Activity Of Polysaccharide From Inonotus Obliquus

Tumor is still a clinical disease difficult to cure,its main clinical treatment method include surgery,chemotherapy,radiotherapy,which may damage the immune function and make patients recover slowly while play a therapeutic role,and bring both hope of survival and the larger trauma to the patients.Therefore,It's an urgent mission to looking for a natural medicine to cure malignant tumors which with great curative effect and low toxicity.Inonotus obliquus(Fr)Pilát,also known as oblique fibrobacteria,Betula albus,tea(????)and Qianggan,etc.is commonly known as chaga in Russia,belonging to the genus Hymenochaetales of Hymenochaetaceae,mainly distributed in the latitude 45~50° area.Its main component polysaccharide has variety physiological functions such as anti-tumor,immune regulation,anti-oxidation and hypoglycemic.However,current research mainly focused on the separation,purification and structural features of single components from Inonotus obliquus,few studies on the comprehensive coverage extract of Inonotus obliquus,in spite of the most significant feature of traditional natural medicine is its overall efficacy.This project based on the Inonotus obliquus from Daxinganling in China,studied on the extraction process,anticancer activity in vitro/vivo and toxicology of polysaccharides extracts.The results were as follows:1.The optimum extraction process of Inonotus obliquus polysaccharide(IOPS)wasdeterminedThe extraction of water-soluble polysaccharides from Inonotus obliquus was carried out by cellulose-assisted water extraction and alcohol precipitation method,The response surface methodology(RSM)was used to optimize the extraction process on the basis of single factor experiments.The yield of polysaccharides was chosen as the response variable and measured by phenol-sulfuric acid method.An extraction condition with solid-liquid ratio 1:30,extraction temperature 90 ?,extraction time 5 h and enzymatic hydrolysis 65 min was optimal.The extraction yield under the optimized conditions was(3.575±0.24)%,agreeing with the predicted value 3.598%.The purity of IOPS acquired after protein removal by sevage method up to(66.80 ± 1.46)%.2.Assure the anti-cancer activity of polysaccharides from Inonotus obliquus(IOPS)and its serum in vitroCCK-8 method was used to detect the growth inhibitory activity of IOPS and its serum in HepG2 and MCF-1 cells,and the cell apoptosis was detected by AO / EB staining.The results showed that IOPS had no obvious inhibitory effect on MCF-7 and HepG2 cells,while the inhibition rate exceed 50% after treated by the IOPS serum with high/medium dosage at 48 h/72 h.Combined with AO/EB staining results,we can conclude that the serum of IOPS can inhibit the activity of tumor cells efficiently,and correlated with the dose positively.3.Evaluation the anti-cancer activity of Inonotus obliquus polysaccharide(IOPS)on tumor-bearing animals in vivo successfulConstruct athymic mice with hepatocellular carcinoma by subcutaneous inoculation,intragastric administration of cyclophosphamide and IOPS high,medium and low dose,respectively.The results showed that the antitumor effect of IOPS showed dose-dependent,the tumor inhibition rate(TIR)of high,medium and low dose of IOPS to female nude mice is 56.63%,38.00% and 36.64%,respectively.The tumor inhibition rate(TIR)of high,medium and low dose of IOPS to male nude mice is 64.37%,38.49% and 40.13%,respectively.The group of CTX and(CTX + IOPS)showed TIR to female mice were 84.53% and 70.01%,TIR to male mice were 84.27% and 73.01%,respectively.The tumor weight in female mice of all the drug groups showed significant difference from those of model group(P <0.001).The tumor weight in male mice of group IOPS high dose,CTX and(CTX + IOPS)showed significant difference to model group(P <0.001).The results of ELISA showed that the dose increase of IOPS could significantly increase the expression levels of IL-2 ?IFN-? and TNF-?,HE staining showed that IOPS could protect liver by reduce deformation and necrosis of hepatocytes.4.Certain the toxicological properties of Inonotus obliquus polysaccharide(IOPS)MTT assay was used to detect the cytotoxicity of IOPS to normal liver cells,acute toxicity and genotoxicity of IOPS were evaluated by acute toxicity test and sperm abnormality test in mice.The results showed that IOPS had no significant inhibitory effect on LO2 cells,and the mice did not show any toxic reaction in a daily dose of 5000 mg/kg.In the mouse sperm abnormality experiment,the positive control group showed significant difference to the normal group(P <0.05),and no significant difference between the IOPS group and the normal group(P ?0.05).All the evidence indicate that IOPS is not toxic.