Isolation And Purification Of Endophytic Fungi Of Astragalus Membranaceus And The Study Of Its Secondary Metabolites Bioactivity

Aim:To isolate endophytic fungi from Roots and Leaves of Astragalus membranaceus evaluate the antibacterial,total reductive force and?-glucosidase inhibitory activities of their secondary metabolites.Methods:Preliminary study was conducted to assess the relationship between plants,rhizosphere microorganisms and soil physical and chemical factors.Soil and plant samples of Astragalus membranaceus were taken from four different sites in Gansu province with a rich history of cultivation of Chinese medicinal plants and high-throughput sequencing was performed to examine the rhizosphere bacterial communities of the four sites.Then,the diversity of the soil and plant endophytic bacteria was analyzed.For the study of bioactivity of secondary metabolites of the endophytic fungi of Astragalus membranaceus,the roots and leaves of Astragalus membranaceus were surface disinfected and sterile and then the endophytic fungi in the roots and leaves of Astragalus membranaceus were isolated and purified by tissue block culturing separation and sub-culturing.The purified colonies were identified by morphological methods and molecular biology methods.Extracts of their fermentation broth were tested for various bioactivities including antimicrobial,reducing power and–glucosidase inhibition.Filter paper disc method was used to screen for antibacterial activities against four indicators bacterial and the diameter of the zone of inhibition was measured.Also,Potassium ferricyanide method and pNPG method were used to measure the total reducing force and the rate of inhibition of?-glycosidase of secondary metabolites.Results:The preliminary studies showed that bacterial abundances in plant samples were higher than in the soil.In the endophytic fungi study,72 strains of endophytic fungi were isolated from the roots and leaves of Astragalus.The distribution was 56 endophytes from the root and 16 from the leaves.These endophytic fungi were identified by first studying their surface features and later by molecular biology and they were grouped accordingly.The diameter of the zone of inhibition showed that extracts of ethyl acetate had better antibacterial activities.Highest zone of inhibition was found in HQ_R-PDA13 which had high inhibition activity against all four strains.Four other extracts showed moderate inhibition against all the four strains.Also,16 extracts showed some level of antioxidant activity and among them,HQ-PDA-L₁ gave the strongest reducing power.10samples showed?-glycosidase inhibition rate?80%while there was no inhibition activities against?-glycosidase in the mycelia extract of HQ-PDA-R₂.Conclusion:Astragalus membranaceus has rich biodiversity of endophytic bacteria and fungi with varying degree of bioactivities.The high?-glycosidase inhibition rate of some of its extracts provide a potential source for obtaining?-glycosidase inhibitors for the treatment of type II diabetes.