Effect of Substrate Dependent Mushroom Polysaccharide Extracts on IL-7 Up Regulation and Cancer Immunotherapy and, their Probable Mode Of Action

Abstract

Ethnopharmacological relevance: Fungi have been used in traditional systems both as food and medicine. In China the mushrooms are widely used in Traditional Chinese Medicine (TCM). On one hand residue resulting from medicinal plants are treated as waste, yet the en- vironment in which medicinal plants including fungi grow greatly influences the potency and quantities of bioactive secondary metabolites thereof. Since Mushrooms are widely consumed for immunomodulatory purposes especially for people suffering from tumors, this study sought to find out whether there is enhanced effect of mushroom polysaccharides extracts of Trametes robiniophila murr a Chinese fungus, grown on media supplemented with Chinese herbal drug residues both in vitro and in vivo as an immunotherapeutic remedy for cancer. Methodology: We used Intestinal Epithelial Cells (IEC) known to produce IL-7 cytokine which works on the immune system and on the postulation that polysaccharide extracts work by up regulating IL-7. The polysaccharide extracts investigated were obtained from mushrooms grown on media supplemented with Chinese herbal residues. The Cell Counting Kit-8 (CCK-8) proliferation assay procedure was used to asses' cell proliferation. Extract concentrations ranging from 0.00001 - 100µg/ml was used in the cell proliferation assay. IEC-6 cells grown in media supplemented with 100µg/ml mushroom polysaccharides extracts was used in vitro and RT-PCR technique employed to evaluate up-regulation of IL-7 in a time dependent manner ranging from 3 to 72 hours. The extract with best IL-7 expression was selected for further evaluation in vivo at 30mg/mice/day in mice infected with CT-26 colon cancer tumor cells. Cyclophosphamide at 20mg/mice/day was taken as the positive control drug. Flow cytometry was employed to evaluate the peripheral blood CD4 + and CD8 + lymphocytes. The extract was further tested in vitro on CT-26 cells (100 & 200 µg/ml), to explore direct inhibition. Results: 1µg/ml was found to give optimum IEC-6 cell growth however; 100µg/ml was determined as the ED 50 and used as the minimum evaluation concentration for IL-7 expression. The polysaccharide extracts obtained from mushrooms supplemented with the Chinese herb Gancao (Glycyrrhiza glabra L. var. glandulifera) residues were found to up-regulate IL-7 best. 6 hours exposure of cells to extract gave optimal IL7 expression of over 1.2 times at 100µg/ml compared to polysaccharide extracts from mushrooms grown on normal media. Extracts obtained at temperatures ranging between 70-90 o C were found to be most ideal. The extract was found to increase the CD4 + and CD8 +