Studies On The Secondary Metabolites Of Endophytic Fungus From Pueraria Lobata Scavenging Free Radical Activity

The research took over 128 strains of endophytic fungus from the roots,stems,leaves and fruits of pueraria lobata,adopted DPPH-microplate assay and DPPH-TLC assay to check and inspect them.The results showed that many strains of endophytic fungus are featured by free radical activity of different degree.A strain 10y-4 collected from Xuan cheng had high free radical scavenging ability.In this study,take the free radical scavenging ratio as a guide factor,fermentation medium and conditions,extraction methods and purification processing of the bioactive components from the strain were optimized guided with the free radical scavenging-assay model were studied.The main research contents include:(1) A framework of collection,culture and separation the endophytic fungus,screening for active strains;(2) Studied the effects of nutritional and none-nutritional factor on active metabolites productions;(3) Bioactive constituent isolation and identification.The medicinal plant kudzu was collected from Huangshan,Xuancheng,Jingxian,and He fei.There were 128 strains separated from kudzu including 49 separated from leaves(account for 38.3%of the total separation strains),35 from stems(account for 27.3% of the total separation strains),40 from roots(account for 31.3%of the total separation strains),and 4 from fruits(account for 3.1%of the total separation strains).There were 128 strains screenrd preliminary by DPPH-microplate assay.The strains of HK-19(stem,Xuancheng),HK-53(leaf,Huangshan),HK-96(leaf,Hefei),4g-6(root, Huangshan),and 10y-4(leaf,Xuancheng) had high free radical scavenging ability,the value of them were over 70%.But the free radical activity of strain 10y-4 was 92.3%when the reaction time of 20 minute with consistency 2.5mg/ml.According to morphological characteristics of hyphae,conidiophores,conidiogenous cells,conidia etc.,guided with the free radical scavenging-assay model,the influence between different nutritional factors including carbon sources,nitrogen sources,mineral salt and different none-nutritional including pH,temperature,culture time on free radical activity were compared in this study.The results showed that the strain 10y-4 optimum carbon sources was the compounds of glucose and maltose,and optimum nitrogen sources was silkworm pupa powder.Combined with the mineral salt and growth factor,the optimal cultural medium of strain 10y-4 was:10g·L-1 glucose,10g·L^-1 maltose,20 g·L^-1 silkworm pupa powder,0.4 g·L^-1 MgSO₄·7H₂O,0.2 g·L^-1 Va.The results of the influence of non-nutrient factors such as pH,inocula amount,temperature and incubation time on the yield of anti-tumor bioactivity showed that the optimum pH was 6.5,the best inocula amount was 5%,the optimal temperature range is 25℃,and optimum incubation time was 10 days.The addition extracts from kudzu to the medium improved the production of endophytic fungus and increased endophytic fungus separation efficiency.Comparison of extraction solvents and conditions,physical and chemical characters tests of the activity components,an optimal extraction processing was obtained as follows: firstly,the liquid culture was condensed at 50℃to 1/5 volume of the broth.Secondly,3 times 95%ethanol was added to remove large molecule impurities such as polysaccharide. Thirdly,supernatant was condensed and extracted with 2 times of butyl alcohol for 3 times. The butyl alcohol extracts were condensed to dryness.The concentrated substance was crude bioactive product.HPLC-DAD-HRMS-Bioassay and bioactive guided isolation resulted in the active compound named G-1.High resolution MS and DAD analysis revealed that the compound molecular weight is 280,and the molecular formula is C₁₄H₁₆O₆.This compound physical description was yellow powder.