Study On Antimicrobial Activity Of Secondary Metabolites From Plant Endophytic Fungi

The morbidity and mortality of bacteria,fungi and other microbial infections are high in clinic,which seriously threaten human health and life.In the course of treatment,due to the abuse of antibiotics,methicillin-resistant Staphylococcus aureus,extended-spectrumβ-lactamase-producing Enterobacter,multi-drug-resistant Pseudomonas aeruginosa,multi-drug-resistant Mycobacterium tuberculosis and other drug-resistant bacteria showed extensive resistance toβ-lactams,macrolides,aminoglycosides,quinolones and rifamycin.It has caused great hindrance to the treatment of clinical infection.Therefore,the development of new and efficient antibiotics is extremely urgent.In recent years,due to the widespread use of antibiotics,immunosuppressants and antineoplastic drugs,the development of diagnosis and treatment techniques such as organ transplantation,hematopoietic stem cell transplantation and intervention,basic diseases such as diabetes,AIDS,cancer,and the aging of the population,resulting in an increase in the number of people with low immune function,the incidence of fungal infection is increasing,and the fatality rate is high.The diagnosis and treatment of fungal infection has been paid more and more attention.Due to the few kinds of antifungal drugs in clinic,and often accompanied by toxic side effects on liver,kidney and other organs,it is difficult to treat clinically and seriously affect the prognosis.Therefore,there is an urgent need to find new,effective and low-toxic antifungal drugs to reduce clinical mortality.Plant endophytic fungi are fungi that parasitize in various tissues and cells of healthy plants during one or all periods of their life cycle,and do not show obvious symptoms of disease to the host plants.Plant endophytic fungi are widely distributed and varied,which can be found in all kinds of plants,and there are many kinds of bioactive products,including alkaloids,peptides,organic acids,steroids,terpenoids,phenols,quinones,flavonoids and so on.it has many pharmacological properties,such as antibacterial,anti-tumor,anti-oxidation,anti-inflammation,immune regulation and so on.Compared with the development of medicinal plants,there are some disadvantages,such as long growth cycle,harsh culture conditions and high cost,while endophytic fungi have the advantages of short growth cycle,simple culture conditions and low cost.To a certain extent,as a new microbial resource,plant endophytic fungi can replace medicinal plants to become a huge treasure house for screening natural active compounds and leading compounds of new drugs.secondary metabolites have extensive application prospects and research value in drug research and biological control.In this study,21 healthy plant samples were collected from different areas,87strains of endophytic fungi were isolated and purified,which were identified as 11genera,of which Aspergillus spp.（25.3%）had the highest isolation rate.Followed by Fusarium spp.（17.2%）and Alternaria spp.（14.9%）.Seven strains with good antibacterial activity were screened by Agar diffusion method and microdilution method.Among them,the Annulohypoxylon stygium YC16-2 has the best antibacterial activity,and its metabolites against Candida albicans,Candida krusei,Candida parapsilosis,Candida tropicalis and Aspergillus fumigatus have MIC values in the range of 62.5-125μg/m L.The MIC values of drug-resistant bacteria,including Staphylococcus aureus,Klebsiella pneumoniae,Escherichia coli and Coagulase-negative staphylococci（CNS）,ranged from 62.5 to 125μg/m L,indicating that the metabolites of the bacteria had good antibacterial and antifungal activity.The metabolites of strain YC16-2 were extracted and the toxicity test was carried out by using Caenorhabditis elegans.The results showed that its LC₅₀to nematodes was 19.52 mg/m L,and its effective bacteriostatic concentration was lower than LC₅₀,indicating that the metabolites of the strain were safe.In order to increase the yield of metabolites,the fermentation conditions of the strain were optimized by single factor.The results showed that the optimum inoculation amount of spore suspension was 1.6×10⁸CFU/m L,the optimum p H was6.0,the optimum culture temperature was 28℃,the best nutrients were potato powder and yeast extract,and the optimum liquid volume was 100 m L medium（250m L conical flask）.Using the optimized system for fermentation,the yield of metabolite increased by 24.21%compared with that before optimization.The metabolites of the strain were isolated and purified by silica gel column chromatography in order to obtain antibacterial substances.Eight components were separated by gradient elution with different ratios of dichloromethane and methanol.The antibacterial activity of component C,D and H was the best.The inhibitory concentration of component C to CNS was 25μg/m L,that of component D to C.albicans was 12.5μg/m L,and that of component H to A.fumigatus was 12.5μg/m L,and had a certain inhibitory effect on the proliferation of lung cancer tumor cells.According to the GC-MS chromatographic analysis of the active components,it is inferred that component C may be benzoic acid,component D may be amides,and component H is a multi-component mixture,which needs further separation.In this study,the secondary metabolites of 87 endophytic fungi isolated from plants from different regions were used to detect their antibacterial activity against common pathogenic fungi and drug-resistant bacteria in clinical infection.After screening,a strain,Annulohypoxylon stygium,with high antibacterial activity was found.Through the optimization of fermentation conditions,the dry weight of the metabolite of this strain could be increased by 24.21%.The active metabolites were isolated and purified by silica gel column chromatography.It was found that components C,D and H had the best antibacterial activity and had certain activity of inhibiting tumor cell proliferation.Through GC-MS chromatography,it is inferred that components C and D may be benzoic acid and amide derivatives,and component H is a mixture.The purpose of this study is to find the secondary metabolites of plant endophytic fungi with antibacterial and antifungal activity.The results can provide some experimental basis for the development of new antimicrobials.