Studies On Endophytic Fungi With Sipeimine Production Ability From A Vulnerable Medicinal Plant Fritillaria Ussuriensis Maxim

10 strains of endophytic fungi isolated from the bulbs of a vulnerable medicinal plant Fritillaria ussuriensis Maxim were studied. By the deposition reaction with Dragendorff's agent, and thin layer chromatography (TLC), a strain (P7) was screened out because of its ability to produce of isosteroidal alkaloid. It was found by the TLC detection that majority alkaloid P7 produced was located out of cell.The procedure of extraction of sipeimine was optimized. Through column chromatography (CO , six alkaloids were obtained from the Chinese pharmacal material Fritillaria ussuriensis. By comparing TLC results of the culture filtrate of P7 and the six alkaloids by column chromatography, we found that a spot of the P7 sample had the Rf value equal to one of the collection from CC, and the standard sample too. And then the three spots with same Rf value were scanned under UV and visible spectrum. The absorption curve of the extract was basically the same as that of the standard sample. And the results of HPLC-ELSD showed that the remaining time and shape of apex of voltage signal of the P7 sample was same as that of the standard sipeimine. So, the sample of P7 extract should be sipeimine or it's analogue.According to the growth rate of the colonies and their growth potential on different media, an optimum solid medium was selected. Through comparing the biomass and the yield of sipeimine, a basic liquid medium was also selected out. By changing different carbon source, nitrogen source and culture temperature, the optimized culture conditions were selected. The optimized conditions of both mycelia growth and sipeimine—production were glucose, urine and the temperature about 26°C, respectively.The fermentation conditions of P7 were then further optimized by the orthogonal experiment of L16 (4~4). The results showed that glucose 60g/L urine 0.5g/L, the liquid medium quantity were 100ml/300ml and pH about 7.0, shaking rate 80 times per minute were the optimum conditions for its mycelial growth; glucose 70g/L, urine 1.5g/L, liquid medium quantity 75ml/300ml and pH 6.5, shaking rate 80 times per minute were suitable for producing sipeimine.The strain P7 was classified mainly according to its microscopic morphological characters.P7 should be a strain belonging to the Fungi Imperfecti, Moniliales, Moniliaceae, Cephalosporium Corda.