Isolation And Identification Of Endophytic Fungi From Cornus Officinalis Producing Loganin And Their Culture Conditions

Endophytic fungi is such a kind of fungi that survive inside of the healthy plants'tissues and organs during its parts of life time or all stages of life cycle,causing no discernible symptoms of disease.It was found that the endophytic fungi can be commonly found inside of the plants' tissues.During the long-term symbiosis and interplay with its host plants,some endophytic fungi strains can produce biological active ingredient similar or identical to their hosts.Thus,the endophytic fungi of the medicinal plant have become great resources to obtain the natural active products.Cornus officinalis Sieb.et Zucc.is a kind of deciduous arbor belong to Cornaceae and Cornus,and mainly distributed in Henan,Zhejiang,Shaanxi,Shanxi,Anhui and Sichuan of our country.The air-dried fruits without kernel of C.officinalis are one of the three precious woody herb in the world.Modern Applied Pharmacy research have indicated that,C.offcinalis has diverse biological activities,such as immune regulation,anti-bacterial,antioxidant,anti-tumor and cardiac functions,etc,which is listed in the Food combination traditional Chinese medicine(TCM)directory.Since flowering C.officinalis vulnerable to damage during the late spring and other natural disasters,the impact of man-made cut burning,cutting branches,in addition to changing ecological environment,in recent years,and so on have caused some damage to C.officinalis resources.Hence,to fully develope C.officinalis endophytic fungi,looking for similar or identical biological active compounds as their hosts may provide new ideas for the protection,sustainable development and utilization of C.officinalis natural resources.In this paper,in order to provide a reference for the research of C.officinalis alternative resources,the endophytic fungi of the medicinal plants C.officinalis were isolated and purified.Besides the endophytic fungi which can produce the active ingredient loganin were selected.And the main results are as follows.(1)Adopting routine separation methods of microorganism,in all,27 strains of th endophytic fungi were isolated from the symptomless tissues of Cornus officinalis.Among which,8 from stems,leaves-6,flowers-3,fruits-10.Strains were numbered:J1-J8,Y1-Y6,H1-H3,G1-G10.(2)Using filter paper method,with Bacills subtilis(A99),Staphlococcus aureus(26003),Escherichia coli(A70)ect as indicator bacteria,the antimicrobial activity of the 27 endophytic fungi strains' fermentation products were detected.The results indicated that,22 isolates were tested inhibitory activity on at least one kind of bacteria,besides,G3,G5,G9 represented stronger antibacterial activity.(3)TLC result shows that,the mycelia extracts of isolate G9 had a constituent with the same RF value and a same color spots as the authentic loganin.Also HPLC shows that isolate G9 had a retention time same as the authentic loganin.Besides,HPLC-MS presents that the identical ion fragment peak generated by isolate G9 isolate is very consistent with the authentic loganin's molecular-ion peak.Therefore,the results indicated that,isolate G9 strains is capable of producing the bioactive ingredient-loganin like its host.(4)According to the morphological characteristics microscopic observation combined with ITS nucleotide sequences analysis,it was identified the G9 strains should be Alternaria eichhorniae.(5)To enhance high production,the optimization tests are carried out to the culture medium component and condition of isolate G9.Through the comparison of mycelial growth and active compounds accumulation cultivated on several kinds of different basic culture medium in common use,and the best basal medium was bean sprouts medium.By the ANOVA analysis of optimized culture medium component,the best corbin and the best nitrogen resource were glucose and yeast extract.According to the optimized by orthogonal experiment,the best ferment condition for mycelial growth and loganin accumulation of isolate G9 turned out to be that:bean sprouts medium as based use,glucose6%,yeast extract 0.3%,PH6.5.Cultured on the optimized medium,the mycelial dry weight of isolate G9 increased by 35.19%and loganin yield improved by 21.57%.