The Studies On Isolation, Identification And Its Secondary Metabolites Bioactivity Of Endophytic Fungi From Narcissus L.

Endophytic fungi is a class of special habitat microbial resources, widely distributed in the moss, ferns, gymnosperms, and angiosperms,with long-term cooperative coevolution, they can produce the same or similar compounds with their host, and most compounds have novel structure. In view of this characteristic, In this thesis , the resources of endophytic fungi from Narcissus tazetta L. var. chinensis Roem and their bioactives were investigated. The results were as follows:1. Through selection of surface disinfectant and disinfection method of Narcissus L. tissues, we used the following two kinds of schemes:①the 75% ethanol for 60 s→sterile water rinse 3 times→5% NaClO for 5 min→sterile water rinse 3 times;②the 75% ethanol for 60 sec→sterile water rinse 3 times→0.1% HgCl for 1 min→sterile water rinse 3 times. By tissue surface-sterilized, isolation medium was PDA and cultured at 28℃in the incubator, 18 strains endophytic fungi were isolated from health Narcissus L. In this 18 strains endophytic fungi , 16 strains from root, 2 strains from bulb, 0 strain from leaf. These endophytic fungi have various colony morphology, through observation of their morphology, the most of endophytic fungi preliminary identified as Fungi Imperfecti, a few of them identified as Ascomycota.2. Crude fermentation extraction of each strain was tested by three different bioactive screening programs including anti-tumor activity, anti-microbial activity and acetylcholinesterase inhibitory(AChEI) activity. The result of the bioactive tests showed that 4 strains(22.2%) of endophytic fungi displayed anti-tumor activity on HepG2 cells, the strain number were SX-R201,SX-R202,SX-R203 and SX-R204 with inhibition rate 86.0%, 89.8%, 62.9% and 90.6% respectively. 15 strains(83.3%) displayed anti-microbial activities on one or more than one of four indicator organisms. Of those, 5 strains(27.8%) displayed inhibition activity to Staphloccocus aureus Rosenbach; 8 strains(44.4%) displayed inhibition activity to Escherichia coli; 2 strains(11.1%) displayed inhibition activity to Klebsiella pneumoniae; 8 strains(44.4%) displayed inhibition activity to Pseudomonas aeruginosa. 11 strains(61.1%) of endophytic fungi showed the acetylcholinesterase inhibitory activity at concentration of 1 mg/mL. Three strains(SX-R101, SX-S101 and SX-S102) have strong AChE inhibitory activity with IC50 value 0.4562mg/mL, 0.3897 mg/mL and 0.4122 mg/mL, respectively. 3. The identification of active strain(SX-S101) with stronger AChE inhibitory activity by ITS rDNA assay and molecular biology techniques indicated that SX-S101 was classified as Rhinocladiella sp. LA60. Two compounds R.sp-cp-1(77.6mg)and R.sp-cp-2(7.5mg)were isolated from SX-S101 fermented liquor, but they have no acetylcholinesterase inhibitory activity.