Study On Antibacterial Natural Products From Endophytic Fungi Of Three Medicinal Plants

Endophytic fungi are a kind of special microbial population living in plant tissues without adverse effects on host plants.Due to their special living environment,they can produce abundant secondary metabolites with novel structures and extensive biological activities in the long-term complex symbiotic relationship with host plants.Especially in recent years,many researchers have made some progress in the discovery of natural products with antibacterial activities.With the increasingly serious problem of bacterial resistance and serious shortage of antibiotics,we have reason to believe that endophytic fungi are one of the effective ways to discover new antibiotic lead compounds.This article selects the seven kinds of medicinal value of plants as experimental material to separate the endophytic fungi in the plant tissue and screen their antibacterial activities.Then three selected active target strains were fermented and cultured on a large scale,and the chemical constituents and biological activities of their secondary metabolites were preliminarily studied.The research contents and results are as follows:Seven medicinal plants were isolated and purified from endophytic fungi,which include Panax notoginseng,Dendrobium nobile Lindl,Verbena officinalis,Stemona japonica,Epimedium acuminatum Franch,Artemisia argyi and Taxus madia,and a total of 84 strains of endophytic fungi were isolated.Three strains of endophytic fungi,Leptosphaeria sp.,Penicillium ochrochloron and Fusarium oxysporum,with strong anti-bacterial activity and rich secondary metabolites were screened through the combination of anti-bacterial activity screening and HPLC chemical screening.We fermented these three active strains in a large-scale way.The secondary metabolites of these strains were separate,purified and identified by various modern separation techniques and spectral techniques.We studied the secondary metabolites of endophytic fungus Leptosphaeria sp.and found that fifteen compounds were identified as known compounds,including six nitrogen compounds?1-6?,one quinone compounds?7?,three small molecule compounds?8-10?,one trimer of ethyl terephthalate?11?,and four diterpenoids?12-15?.Fourteen compounds were found in the study of endophytic fungus Penicillium ochrochloron,of which 16-18 were new compounds and the rest were known compounds,including three?-pyrones?16-18?,one octahydronaphthalene?19?,five small molecular compounds?20-22,28,29?and five steroids?23-27?.A study on endophytic fungi Fusarium oxysporum showed that twelve compounds were known,including four small molecular compounds?30,33,34,40?,one?-pyrone compound?31?,two steroids?35,36?,three esters?32,37,39?,one cyclopeptide compound?38?,and one flavonoid compound?41?.Finally,we evaluated the anti-human pathogenic bacterial activity,anti-plant pathogenic fungal activity,anti-tumor cytotoxic activity and adipocyte sugar consumption activity of the isolated monomer compounds.Antibacterial activity evaluation found that the MIC of compound 7 and 13-15 against Bacillus cereus reached 3.12,12.5,6.25,6.25?g/mL,and that of compound 14 resist Bacillus subtilis and Pseudomonas aeruginosa and its MIC reached 12.5?g/mL,respectively.The evaluation of antifungal activity illustrate that the MIC of compounds 13 and 15 reached12.5?g/mL against Rhizoctonia cerealis,that of compounds 16,17 and 19 reached 12.5?g/mL against Fusarium graminearum,and that of compound 38 against Botryosphaeria berengeriana,Verticillium dahliae Kleb,Bipolaris carbonum Wilson,Alteraria alternata?Fries?Keissler,Botrytis cinerea Pers.reached 25,25,12.5,12.5,12.5?g/mL,respectively,which had significant antifungal activity.The antitumor cytotoxic activity evaluation showed that compound 19 had inhibitory effect on five cancer cell lines?A549,LN229,MGC,LOVO,MDA231?,and IC₅₀0 was 51.45,23.43,39.16,46.97,42.85?M,respectively.The glucose consumption of compound 38 to3T3-L1 adipocyte was 8.536233?mmol/L?,which was obviously larger than that of normal group 5.820297?mmol/L?,indicating that compound 38 was a potential hypoglycemic active molecule.In summary,three strains of endophytic fungi with antibacterial activity were screened and 41 monomeric compounds including three new compounds were obtained through fermentation,extraction,isolation,purification and structural identification in this paper.We evaluated biological activities of these monomers.We found that compounds 7 and 13-15 had significant antibacterial activity,compound 19 had moderate antitumor activity,and compound 38 had obvious activity of increasing sugar consumption in adipocytes.These results indicate that endophytic fungi have the potential to produce natural products with significant antimicrobial activity.At the same time,it can be seen that the secondary metabolites are abundant and the biological activity is diverse,which is an important microbial resource for discovering new antibiotic lead compounds.