Screening And Identification Of The Huperzine A Produced Endophyte Fungi From Huperzina Serrata

Alzheimer disease (AD) is a very important disease influencing old people’s life quality, and usually prevented and cured by Acetylcholinesterase inhibitor (AChEI) drugs efficiently. Huperzine A(Hup A), a natural alkaloid, extracted from a traditional Chinese herb Huperzia serrata(H. serrata), as a particular medicine used for AD disease, is more efficient, less side effect than any other drugs to deal with this disease improvement due to its high effective, special and reversable AChEI activity. Now Hup A product used for AD in the market mostly are from two reliable sources:first one is extracted natural products without a sufficient wild plants resources and aseptic seedling in hand. Second one is synthesized compounds which are limited by more complicated process, higher cost, and lower activities. H. serrata have rich endophytes which live in the healthy plant in a long term. The theroical and practical researches of H. serrata’s endophytes showed that they are capable to produce Hup A. Given the H. serrata’s endophytes brand-new ability, it has been considered as a new Hup A resource and has attracted world wide medical interesting especially from Chinese herb researches’.This study was trying to screen out new producing Hup A endophytes from Huperzia serrata which can provide fundamental theory for Hup A drug exploration from endophyte. Separated from healthy H. serrata’s different tissues, endophytes were purified with PDA medium. After expanding the endophytic fungi’ fermentation, alkaloid were extracted from the mycelium by acid-water method and condense the alkaloid by High Speed Counter Current Chromatography (HSCCC). Through High Performance Liquid Chromatography (HPLC) method, every strain was screened by checking the Hup A’s presence comparing with standard Hup A. Then it’s necessary to identify the Hup A which has merely certificated by HPLC from endophyte by Thin Layer Chromatography (TLC), Liquid Chromatography- Mass (LC-MS), and confirmed its AChEI activity by DTNB method. At last strains was classified the by morophology and molecular method. At last this study came to these conclusions as follow:164 endophytic fungi were separated from H. serrata’s different tissue. Firstly it was arranged to collecte the wild plant from the Enshi city Hubei province and separated the fungi from surface sterilized healthy plant.41% endophytes were separated from plant leaves, while 53% endophytes were from stem.64 endophytes were purified and classified by their morphology and numbered them before stored in refrigerator at -70℃.2 One H. serrata’s endophytes strain ES026A producing Hup A was screened out. By acid-water alkaloid extraction method 48 strain H. serrata’s endophytes curde alkaloids were extracted and detected the presence of Hup A comparing with standard Hup A by HPLC. H. serrata endophyte ES026A was fermented and extracted crude alkaloid from the mycelium. Crude alkaloid was purifed by HSCCC and detected by LC-MS experiment, whose results demonstrated that the purified alkaloid had the same Mass chromatography as the standard Hup A. Its crude alkaloid extract was also detected by TLC which indicated the same removing speed as standard sample.3 H. serrata’s endophytic fugus ES026A’s Hup A yield have achieved 1μg per gram dried mycelium and the crude alkaloid has an AChEI IC50:0.23313μg/mL. Hup A yield has achieved 1μg per gram dried mycelium by HPLC detection and concentration-peak area linear relationship. By DTNB method, ES026A’s crude alkaloid has AChEI activity. The crude compound’s AChEI activity appeared more higher activity more higher concentration. This crude alkaloid had an IC50 value 0.23313μg/mL which was three times bigger than the standard Hup A’s.4 Identification of H. serrata’s endophytes ES026A which could produce Hup A: By morophology and molecular method according to correspounding references, these conclusion were that the ES026A strain belongs to Colletotrichum gloeosporioides through the spore’s microscope picture, strains’ITS sequence which have submitted to GENNBANK, and unique rDNA sequcence for Colletotrichum gloeosporioides. Now the ITS sequence has formally got an accession number HQ876466.1 from GENEBANK.5 H. serrata.’s endophyte ES052BA can produce Hup A analogues by LC-MS. H. serrata.’s endophyte ES052BA’s crude alkaloid were detected by LC-MS, which showed that the alkaloid had different MS spectrum but same MS/MS spectrum as standard Hup A. This revealed that a new producing Hup A analogues H. serrata endophyte strain ES052BA had discovered.