Isolation And Identfication Berberine In The Endophytic Fungi Of Coptis Chinensis Franch

Berberine has extensive pharmacological action and main extracted from Coptis chinensis Franch. However, because of predatory excavation, the long growing period, special growing environment and other reasons, the number of wild coptis are retained decreases in recent years. Even a few species threatened with extinction. Therefore, it is significant to explore new ways to produce berberine. Due to the fact that endophytic fungi have the potential to produce active compounds as the host plant, to screen endophytic fungi from normal Coptis chinensis which can produce berberine will provide new medicinal source for berberine.In this experiment, a way named tissue print culture was used to isolate endophytic fungi from the tissue berberine of plant. At first, in order to screen berberine-producing endophytic fungi, the way of thin layer chromatography(TLC) and high performance liquid chromatography(HPLC) were used; then we used mass spectrography(MS) and nuclear magnetic resonance(NMR) to check its structure. Finally, their species were identificated by the method combining the morphological and molecular biology. The results are as follows:1. A total of 15 strains of endophytic were obtained from tissue of coptis plant.2. The result of TLC found that the strain HL-Y-3 mycelium liquid possesses the same spot of colour and Rf value consistent with reference substance, and HPLC showed that the retention time is 4.598 min that is close to 4.626 min of reference substance. Preliminary determine the strain HL-Y-3 produce berberine. The MS and NMR validation results showed that the berberine of HL-Y-3 hyphae is agreement with the reference substance structure, further determination the HL-Y-3 hyphae can produce berberine, calculate the content of 9.313 μg/g.3. The central of HL-Y-3 colony is sag olive, surrounding white villous hyphae, under the microscope indicated that hyphae of the 3-6 μm. At last, the colony produce spores; several spores series into a chain, single spore is a rod or spindle shape, each spore was divided into 5 ~ 10 cells, according to HL-Y-3 morphological features, strain of HL-Y-3 preliminary identified Alternaria sp. The 546 bp DNA obtained by amplifying ITS1 fragments; compared Alternaria lini(KF 293969) ITS1 sequence whith ITS1 of HL-Y-3 in NCBI database, the result show included homology was 99%, combination with characteristics of morphology and molecular biology, identification of strains HL-Y-3 is Alternaria sp.