Secondary Metabolites And Their Biological Activities Of Three Endophytic Fusarium Species From Chinese Herbal Medicines

Endophytic fungi are a class of fungi that live in the interior of healthy plant tissues for part or all of their life and do not cause obvious disease symptoms to plants.It has been found that endophytic fungi from medicinal plants may participate in the metabolism of active components of medicinal plants or produce same bioactive substances as host plants after long-term coevolution.Therefore,endophytes from medicinal plants are important sources of biologically active natural products with novel structures.Fusarium,is a large family of filamentous fungi widely distributed in soil and plants.At present,the secondary metabolites of this genus have been studied extensively and systematically,and a series of active substances with novel structures have been found.Based on previous work,the secondary metabolites of two Fusarium species,namely Fusarium sp.L-33 and L-46 were studied.As a result,30 compounds,including 8 new ones and a new natural product were isolated.Some of the compounds were preliminarily screened for cytotoxic activities.At the same time,the antifungal activity of equisetin,the main metabolite of Fusarium sp.Y-2 isolated from Hedyotis diffusa Willd was also studied.As our investigation on the secondary metabolites of Fusarium sp.L-33 from Dioscorea opposita Thunb.,a total of 17 compounds were obtained and identified,including 5 new compounds,namely compound 2(2a/2b)and Fusarilactones D-G(3,4,6,and 7).It is worth mentioning that the absolute configuration of the known compound biscognin A(5)was determined.The 10 known compounds were identified as phomapyrone C(8),phomapyrone D(9),chlamydosporol(10),fusarilactone A(11),acuminatopyrone(12),(-)-oxysporidinone(13),(-)-4,6′-anhydrooxysporidinone(14),5-hydroxy-7-(2’-hydroxypropyl)-2-methyl-chromone(15),phenylacetic acid(16),and P-hydroxyphenylacetic acid(17).The cytotoxic activity of 1,2,4,5,7,8,10,15 were evaluated against five human cancer cell lines.The results showed that,none of the above compounds showed significant cytotoxic activity.The secondary metabolites of Fusarium sp.L-46 from Dioscorea opposita Thunb.were studied,as a result,13 compounds were isolated and identified,including 3 new compounds,fusarilactones H-J(1-3)and a new natural product4-hydroxy-3,5,6-trimethyl-2H-pyran-2-one(4).Nine known compounds was identified as 4-hydroxygibepyrone A(5),neovasinin(6),neovasifuranone B(7),neovasifuranone A(8),fusarester C(9),ergosterol(10),2-(4-hydroxyphenyl)ethylacetate(11),m-hydroxyphenylacetic acid(12),and benzylalcohol(13).The antifungal activity of equisetin was studied.Carbendazim was used as a positive control.At the concentrations of 50 μg/m L and 100 μg/m L,to evaluate the antifungal activity of equisetin against 8 plant pathogenic fungi(Alternaria pluriseptata,Cercospora arachidicola Hori,Cercospora personata Berk.et Curt,Colletotrichum graminicolum,Fmoniliforme moniliforme,Curvularia lunata(Wakker)Boed,Bipolaris carbonum Wilson,Rhizoctonia cerealis).The results showed that when the concentration of equisetin was 50 μg/m L,the inhibition rates on plant pathogenic fungi ranged from 4.98% to 59.6%,and the inhibition rates on Cercospora arachidicola Hori,Colletotrichum graminicolum and Rhizoctonia cerealis were more than 50%.When the concentration was 100 μg/m L,the inhibition activity of equisetin on various plant pathogenic fungi was enhanced,the inhibition rates ranged from28.92% to 81.82%,and the inhibition rate on 5 kinds of plant pathogenic fungi were more than 50%.At the same concentration,compared with positive control,equisetin showed higher antifungal activity against Alternaria pluriseptata,Cercospora personata,Curvularia lunata and Rhizoctonia cerealis than carbendazim.At 100μg/m L,the inhibition rate on Rhizoctonia cerealis was 81.82 ± 2.00%,which was much higher than that of carbendazim at 28.54 ± 2.87%.On the basis of the activity study,Rhizoctonia cerealis was selected for further studies of the inhibitory mechanism of equisetin.Under the treatment of equisetin with the final concentration of 100 μg/m L,the effect on the morphology and structure of mycelia was observed through the microscope,the effect of equisetin on the cell membrane was determined by the conductivity method,and the effect of equisetin on the reducing sugar absorption of fungi was determined by the DNS method.The results showed that after the treatment of equisetin,the mycelium branch reduced and fractured.The permeability of the cell membrane increased,resulting in the increase of conductivity.Equisetin also affected the absorption of reducing sugar of Rhizoctonia cerealis.These results showed that equisetin had broad-spectrum activity against plant pathogens,which could provide a theoretical basis for the development of plant fungicides.