Endophytic Fungus From Agastache Rugosa (Fisch Et Mey) O.kuntze.: Separation, Purification And Bioactivity Study On The Ethyl Acetate Extract Of Secondary Metabolites

In recent years,endophytic fungi of medicinal plants are potential resources for obtaining new natural products and developing new drugs,and have important research and development value.Endophytic fungi have become the focus of research in various disciplines such as plant microbiology,plant medicine,plant breeding,Chinese medicine and medicinal botany.The medicinal plants in Gansu Province are widely distributed,rich in resources,and diverse in variety.There are many studies on the chemical constituents of various medicinal plants,but there are few reports on the endophytic fungi of medicinal plants.Therefore,using the medicinal material Agastache rugosus of Gansu Province as the research material,this thesis studies the antioxidation activity and the chemical constituents of secondary fungi of endophytic fungi from Agastache rugosus.The research content and results are as follows:1.Isolation and purification of endophytic fungi from Agastache rugosus:11endophytic fungi were isolated from the roots and leaves of Agastache rugosa by tissue block method.Five strains were obtained from roots,namely HXG-1 to HXG-5,and six strains were obtained from leaves,which were HXY-2 to HXY-7.According to their morphology and microscopic observation,it was identified as Penicillium sp.,Didymopsis sp.,Alternaria sp.,Tubercularia sp.and Stemphylium sp..Stemphylium sp.is the dominant genus.2.Antioxidant activity:There are 44 samples from the endophytic fungi in the Agastache rugosus root,52 samples of endophytic fungi in the Agastache rugosus flower part of the laboratory and 32 samples of endophytic fungi from roots and bulbs of Lanzhou Lilium.Evaluating the antioxidant activity of the above 128 samples by TLC-DPPH method.The spot of the positive control was set to 100%and the result showed:a total of 7 out of 44 samples of endophytic fungi in musk root and leaf showed significant antioxidant activity,of which Y-2A had the strongest antioxidant activity.The anti-oxidation spot gray value percentage is 124.67%,and its antioxidant activity is stronger than the positive drug Vc.The other 6 parts are Y-6A 75.51%,Y-5A 26.59%,G-1A 20.21%,Y-5C 17.30%,Y-3A 11.77%and G-4A 11.66%.A total of 9 out of 52samples of endophytic fungi in the musk flower area had significant antioxidant activity,of which H-9A had the strongest antioxidant activity,and its gray value percentage was70.15%.The remaining 8 parts were H-13A 48.84%,H-7A 46.45%,H-12A 40.04%,H-11A 31.08%,H-4A 18.84%,H-9B 16.94%,H-7B 15.24%,H-7C 9.21%.Among the32 samples of endophytic fungi in roots and stems of Lanzhou Lilium,6 samples showed antioxidant activity,and the highest activity was BHJ-1A,and the gray value percentage was 18.13%.The rest were BHJ-1-1A 16.71%,BHG-9A 13.63%,BHJ-2A11.04%,BHJ-5B 7.37%,and BHG-5A 6.68%.?A-ethyl acetate fraction,B-n-butanol fraction,C-ethanol fraction?3.Chemical composition research:he anti-oxidation activity of HXY-2 was fermented,the mycelium and the fermentation broth were separated,and the fermentation broth was extracted with ethyl acetate to obtain an ethyl acetate extract.The above sample was subjected to various chromatographic methods such as silica gel column chromatography,gel column chromatography and TLC thin layer chromatography,and 2 compounds was separated from the endophytic fungus HXY-2secondary metabolite ethyl acetate extract.The results were analyzed by ¹H-NMR,¹³C-NMR and ESI-MS and other modern spectroscopic techniques to identify the structure of the compounds,respectively.4.HXY-2 strain identification:Identificate the strain HXY-2 with the best antioxidant activity by morphological characteristics combined with molecular biology methods.The strain was identified as Alternaria alternate.