Study On Antitumor Activities Of Polysaccarides From Submerged Fermentation Of Ganoderma Applanatum (Pers.) Pat

Ganoderma applanatum (Pers.ex Gray.)Pat belongs to Basidiomycota, Hymenomycetes, Aphyllophorales, Ganodermataceae, Ganoderma, traditional Chinese doctor recorded that it's feeble, taste thin and helpful to clear heat, remove stagnation, dissipate phlegm and relieve pain. Lots of studied exposed that polysaccharides from Ganoderma applanatum have many pharmaco-activities such as enhancing immunity, anti-viral, deducing blood glucose, antisepticise, eliminating inflammation, dephlogisticate, hepatoprotection and invigorating the stomach. However, some unique advantages of the polysaccharides'anti-cancer activity, such as high activity and low side effects provide basic theory to exploit Ganoderma applanatum's polysaccharides to be a new antitumor drug.In this study, we use Submerged Fermentation Fungal of Ganoderma applanatum (Pers.) Pat as the material, mono-factors experiment test the content of polysaccharides after water extraction with different temperature, dosage liquor, time and extraction times, the result shows that we can obtain the most yield in the condition:90℃,1:50 dosage liquor, extract 3h and 4 times. According to the result we design orthogonal test to decide the best project to extract GAFPⅠis:85℃,1:40 dosage liquor, extract 3h and 3 times. In addition, we use the same way to treat the insoluble residue from water extraction, make sure the best condition for GAFPⅢis 75℃,0.2M NaOH,1:40 dosage liquor, extract 3h and the best project for alkali extraction is: 75℃,0.2M NaOH,1:30 dosage liquor, extract 3h. Next, using sevag method to deproteinize GAFPⅠand GAFPⅢ, we get GAFPⅡand GAFPⅣ. The polysaccharides and protein of GAFPⅠ,GAFPⅡ, GAFPⅢ, GAFPⅣis tested by Phenol-Sulfuric acid and Folin-Phenol Method, the content are 86.14%.88.45%,13.57%,20.12% and 16%,3.2%,9.69%,0.46% respectively. The anti-tumor activity of 4 kinds of polysaccharides is explored in two aspects:in vitro and in vivo, which investigate the relevant mechanism furthermore.In vitro experiment, we use MTT assay to screen the best polysaccharides, which include SW1116,NCI-H446,SMMC-7721,Hela,MCF-7 cells, the results show that 4 kinds of GAFP could inhibit the growth of different tumor cells. However, GAFPⅠcould obviously affect the proliferation of SW1116 with dose-dependent relation, maximum inhibition ratio is 72.48%. fluorescence microscopy observe chromatin changes in early and late apoptosis, which prove that GAFPⅠcould cause apoptosis. Stain SW1116 cells with PI and FACS results show that GAFPⅠcould induce G2/M phase arrest in mitosis cycle, the antitumor activity is educed by inhibiting adlibitum cell division.Experiment in vivo revealed that GAFPⅠcould obviously inhibited the growth of S180 cells which transplant to mice, the tumor-inhibit rate have dose-dependent relation with GAFP I's concentration. Besides, thymus and spleen indices are changed after S180-bearing mice treat with GAFP. Lymphocyte conversion experiment show that GAFP I could increase the transformation efficiency and mitogen action of ConA to lymphocytic cells, and the ratio is also have dose-dependent relation with GAFP I's concentration. In addition, GAFP I could also promote the activity of SOD, CAT and decrease the content of lysozyme and MDA in blood serum of S180-bearing mice. Furthermore, Compared to the control bundle, we found that IL-2 and TNF-a in the treatment bundle appear striking rises. This reveal that the antitumor action of GAFP I in mice was mediated by enhancing antioxidation and regulating corpus immune system. At last, we use RT-PCR test the expression of tumor suppressor Rb gene, the findings indicated that GAFP I could obviously increase the expression of Rb gene in spleen, prove that GAFP I has antitumor activity, by means of regulating the expression of oncogene and anti-oncogene.