Liquid Culture Medium Optimization And Biological Activity Of An Endophytic Vitexin-Producing Fungus Dichotomopilus Funicola Y3 From Pigeon Pea(Cajanus Cajan[L.] Millsp.)

Endophytic fungi are known as potential resources for producing active compounds.Sometimes,endophytic fungi have been found to produce same or similar chemical compounds to those produced by its host,and also have been recognized as a repository of novel compounds of immense value in agriculture,industry,and medicine.Vitexin is the main active ingredient in the soybean plant,which has good anti oxidation,anti-tumor and blood pressure.Clinically used for the treatment of cardiovascular and cerebrovascular diseases,has a good protective effect on ischemic myocardial injury.Its mechanism is the increase of coronary blood flow,reduce the ejection resistance and plasma viscosity,improve red blood cell deformability,inhibit the formation of thrombus.The main natural source of vitexin is found in the medicinal plants that limited distribution and exists in extremely low concentrations.This experiment was made of endophytic fungusi isolated from Pigeon pea in our laboratory,our study was prompted by screening and identificating of vitexin producing endophytic fungus Y3,this has led us to discover novel vitexin sources for its more available and some basic researches.We carry out further work to increase vitexin yield by optimizing media for large scale vitexin production.At the same time,the antioxidation effect of endophytic fungus vitexin and the proliferation of osteoblast MC3T3-E1 were studied.The results are as follows:1.Basing on our observation of the morphological characteristics of Y3,it was preliminarily identified as Chaetomidium sp.The 558 bp DNA were obtained by amplifying ITS fragments.The result shows that ITS sequence homology of Y3 with NCBI Dichotomopilus funicola(EF017204)can reach 99%.Strain Y3 was identified as Dichotomopilus.funicola by its characteristic culture morphology and ITS r DNA sequence analysis,and its Genbank MF800960.2.First of all,based on the original medium,by single factor experiment,selected for vitexin synthesis carbon source(glucose),nitrogen source(ammonium chloride),temperature(30 ?)and pH(7),and preliminarily determines the fermentation culture of vitexin basic condition,and through the cultivation of the two weeks to determine the best training time for 8 days.Then,the Plackett-Burman design was used to screen out the significant inducible factors that could induce the increase in the yield of vitexin.Finally,the multivariate regression relationship between yield and significant factors is proposed by using the central composite design(CCD)experiment,and the theoretical extreme value is predicted and verified by experiments.The induction factors added in the optimized medium were 1-phenylalanine 0.06 g/L,salicylic acid 0.21 g/L,CuSO4·5H2O 0.19 g/L.It was predicted that the yield of the vitex side was 79.49 mg/L,which was verified by the above optimization culture conditions,and the yield of the vitex side was 79.15 mg/L,which was 4.61 times the original level.3.The endophytic fungus vitexin were evaluated for their antioxidant activities by OH scavengging activity test and DPPH scavenging activity test.The results are concluded as follows:Endophytic fungus vitexin have higher DPPH radical scavenging ability,clearance rate of up to 94.42%,and higher than that of the same concentration of Vc under clearance,calculated the IC50 ? 164 ?g/mL.At the same time,it also has strong OH scavenging ability,up to 53.12%,and is higher than the Vc scavenging ability under the same concentration,and its IC50=5.32 mg/mL is calculated.4.The MTT method is to detect cell proliferation by cell metabolism.This experiment for the research of endophytic fungi produce vitex nucleoside strains extract osteoblast proliferation of MC3T3 E1-promoting effect,vitex nucleoside standard as the positive control,dal crude extractings from endophytic fungi of producing for the samples under test to explore the different concentration,the influence of different culture time on color shades,established the optimum testing conditions.Results are as follows:endophytic fungi producing vitexin have varying degrees of impact to osteoblast MC3T3-E1.Based on the results of the above studies,the active strains of vitexin were selected from the endophytic fungi,and the species were identified by morphological and molecular identification.Through the optimization of fermentation can improve the yield of vitexin,and studies show that the Vitexin not only has strong antioxidant activity,but also has the proliferation of osteoblast MC3T3-E1 in mice.The research results of this paper have enriched the extraction sources and methods of vitexin,very worthy of development and utilization.