The Study On Pharmacological Activities Of Pigment And Exopolysaccharide Of Rhodotorula Glutinis Swjs-jm1

Objective:To look for antioxidant and immunomodulatory components,pigment and exopolysaccharide was extracted from the Rhodotorula glutinis SWJS-JM1,and the effect of pigment and exopolysaccharide on antioxidant and immunoregulatory activities were analyzed.Methods:(1)The pigment and exopolysaccharide components were extracted from the R.glutinis SWJS-JM1,and the content of the two kinds of substances were analyzed.(2)The effect of the two type chemical compounds on the cell growth were detected by MTT method,and the oxidative damage model of RAW264.7 cell was induced by H2O2.(3)The effect of the pigment from R.glutinis SWJS-JM1 on the production of ROS in the oxidative damaged RAW264.7 cells was determined by flow cytometry and observed by inverted fluorescence microscope.(4)The effect of the pigment from R.glutinis SWJS-JM1 on the HO-1 protein expression in the H2O2-induced RAW264.7 cells was investigated by western blot.(5)The effects of the pigment from R.glutinis SWJS-JM1 on the expression of Nrf-2 and the expression of genes related to antioxidative enzymes of oxidative damaged RAW264.7 cells were tested by Real-Time PCR.The effects of exopolysaccharide extract on the expression of related cytokines in RAW264.7 cells were also tested by Real-Time PCR.(6)The effect of exopolysaccharide extracts on the secretion of NO from RAW264.7 cells was examined by Griess’s reagent method(Griess method).Results:(1)The pigment extracts from R.glutinis SWJS-JM1 were analyzed by HPLC assay,the average content of beta carotene pigment was 8.62%,the average content of polysaccharide in the extract of exopolysaccharide from the R.glutinis SWJS-JM1 was 11.36%.(2)MTT results showed that exopolysaccharide extract between 25μg/mL and 400μg/mL,and the pigment between 20μg/mL and 80μg/mL had no obvious toxic effect on RAW264.7 cells.The RAW264.7 cells showed significant proliferation after incubation 48h with the concentration of exopolysaccharide between 25μg/mL and 400μg/mL.H2O2 stimulated RAW264.7 cells with different concentrations of 30min,IC50 value of 1171μmol/L.(3)The results of flow cytometry and the results of inverted fluorescence microscopy showed that pigment extract could significantly reduce the ROS level of H2O2-induced RAW264.7 cells.(4)Real-Time PCR results showed that the cells after incubation with the pigment extracts could significantly upregulated the expression of HO-1,SOD1 and SOD2 genes in H2O2-induced RAW264.7 cells,but the expression of Nrf2 gene was downregulated.At the same time,the western blot method was used to detect the expression of HO-1 protein in the cells and the results were in agreement with the results of Real-Time PCR.(5)Griess assay results showed that the amount of NO released from RAW264.7 cells significantly increased after treated with R.glutinis SWJS-JM1 exopolysaccharide,the results were consistent with the transcription level of nitric oxide synthase(iNOS)mRNA.IL-1β,IL-6,and TNF-α genes were up regulated after the incubation 24h.Conclusion:The main components of the extract of R.glutinis SWJS-JM1 were carotenoids,the average content of beta carotene was 8.62%.The low concentration of pigment extracts showed no significant toxicity to RAW264.7 cells,and it can significantly reduce the level of ROS in H2O2-induced RAW264.7 cells,Up regulation of the gene expression of antioxidative enzymes HO-1,SOD1 and SOD2,the pigment have good antioxidant activity.The average content of polysaccharide in the extract of exopolysaccharide from the R.glutinis SWJS-JM1 was 11.36%,it can promote the proliferation of RAW264.7 cells and production of NO,play a role of immune regulation by up regulation gene expression of iNOS,IL-1,IL-6,and TNF-a.Briefly,pigment extracts from R.glutinis SWJS-JM1 could play a role in antioxidant,R.glutinis SWJS-JM1 exopolysaccharide has capability of immunomodulatary by regulating the expression of inflammatory cytokine genes.This study could also lay the fundation for further exploitation and application of R.glutinis SWJS-JM1,especially for the two type chemical compounds as new potential resource of medicine.