Biological Activities Of Endophytic Fungi And Their Metabolites From The Original Plants Of FRITILLARIAE CIRRHOSAE BULBUS

Endophytic fungi from medicinal plants can produce a large number of secondary metabolites with novel structures,unique activities,and some of them showed the same or similar structures to the host plants.It not only becomes an important way to find new natural compounds,but also protects endangered medicinal plants and reduces the destruction of medicinal plant diversity and shows ecological significance.FRITILLARIAE CIRRHOSAE is a famous medicinal plant which located in Sichuan province of China.Its bulbs are used as drug-injecting parts,and have good efficacy value and various pharmacological activities.However,the pharmacological importance of endophytic fungi from Fritillaria species,has not been extensively explored.In this study,to lay a foundation and provide scientific basis for further development and utilization of endophytic fungi from FRITILLARIAE CIRRHOSAE,the antimicrobial,antioxidative and whitening activities of 18 endophytic fungal strains isolated from Fritillaria cirrhosa,Fritillaria wabuensis,Fritillaria unibracteata,and Fritillaria przewalskii?the original plants of FRITILLARIAE CIRRHOSAE?and their secondary metabolites were systematic studied.The main findings are as follows:?1?Screening and evaluation of strains with antagonistic effectsUsing the five-point confrontation test,five endophytic fungi from F.wabuensis with good antagonism against plant pathogens were screened from the 18 strains of endophytic fungi.Subsequently,the inhibitory spectrum of 5 endophytic fungal strains against 23 plant pathogenic bacteria was determined by the two-point method.Of them,1WBY2 and3WBY3 displayed the widest anti-plant pathogens spectra,and their inhibition rates to Sclerotinia fuckeliana,Thielaviopsis basicola,Phyricularia grisea and Colletotrichum gloeosporioides were more than 60%.At the same time,the antagonistic indices of the 9physiological races of P.grisea were all over 50%.The experiment also found that the rice seed after soaking in 1WBY2 fermentation broth had significantly higher seedling penetration rate than that of the clear water control at 96h,and the average root length was significantly higher after 7d than that of the clear water control.In addition,the potting experiment was conducted to control the P.grisea.The result showed that the control effect of fermentation solution of 1WBY2 on P.grisea could reach 66.69%,which ranked only second to Tricyclazole.Then the volatile components of 1WBY2 endophytic fungi had no inhibitory effect on plant pathogenic bacteria using plate snap method.Besides,the liquid culture products of the strain were studied.The results showed that the antagonistic index of the fermented liquid sterilized by the membrane filtration was higher than that of the autoclaved fermentation broth,while the anti-plant pathogens activities of the sterile fermented liquid from 1WBY2 and 3WBY3 were evaluated,and the sterile fermentation liquid of 1WBY2showed a strong inhibitory activity against multiple pathogens.In addition,the qualitative analysis and antibacterial test of the chemical components of 1WBY2 mycelia and fermentation broth were tested.The results showed that there existed many active substances such as phenols in the 1WBY2 fermentation broth,and its antibacterial components mainly existed at the ethyl acetate extraction site.?2?Screening and evaluation of strains with whitening activeIn order to evaluate the whitening activities of secondary metabolic products from the18 endophytic fungi,the inhibitory activity of the fungi against to tyrosinase was measured.The results showed that the inhibition abilities of strain WBS020,1WBY2 and 7WBY2against to tyrosinase were significantly higher than the other stains,and the inhibition rates were 79.86±0.50?%?,87.48±1.30?%?and 87.31±1.05?%?,respectively.And the stability was maintained after high temperature treatment.The ethyl acetate and n-butanol extracts of WBS020,1WBY2 and 7WBY2 displayed a significantly higher inhibition activity against to tyrosinase than the petroleum ether.And the n-butanol fraction from strain WBS020 showed the strongest activity,while ethyl acetate fractions from 1WBY2 and7WBY2 showed the strongest activities.In addition,the inhibition rates value of Vc is significantly higher than the other extracts,but the ethyl acetate extracts from strain1WBY2 and 7WBY2 still reach to 90.86±0.81?%?and 88.92±7.13?%?at 8 mg/mL,respectively.?3?Screening and evaluation of strains with antioxidant activityThe purpose of this experiment was to study the antioxidant activities of sterile fermented liquid of single culture and co-culture of five endophytic fungi from F.wabuensis using DPPH radical scavenging,ABTS radical scavenging and the ability of FRAP iron reduction.The antioxidant activities of the petroleum ether extracts?30-60??,ethanol absolute extracts,n-butyl alcohol extracts were tested,respectively.Vitamin C was used as positive control.The results showed that the oxidation method of three combination,including 4WBY1+WBS019,6WBY3+WBS019 and WBS019+WBS020sterile fermented liquid antioxidant activity at least in two oxidation methods were significantly higher than that of single culture evaluation.It was also found that the antioxidant activities of ethyl acetate and n-butanol extractions of previous three combinations were stronger than petroleum ether extracts.The DPPH free radical scavenging rate of ethyl acetate extracts?1.8 mg/mL?from WBS019+WBS020 co-culture combination could reach to 74.38%±0.65%.The ABTS radical scavenging efficiency of n-butyl alcohol extracts(IC₅₀=0.25 mg/mL)of 6WBY3+WBS019 co-culture combination was near to the Vc(IC₅₀=0.07 mg/mL).The Fe³⁺reduction abilities of all kinds of the extracts from previous three combinations were significant weaker than the positive control.The results show that the endophytic fungi co-culture has potential value in use under appropriate conditions.