Screening Of The Huperzine A Produced Endophytic Fungi From Huperzia Serrata And Analysis Of Its Secondary Metabolites

Huperzia serrata（Thunb.）Trev.（H.serrata）is a perennial native plant with high medicinal value.It is often used for bruises,blood circulation and blood stasis in folks.Recent studies have shown that Huperzine A in this plant has a good effect on Alzheimer’s disease.However,global resources of H.serrata are in short supply,and there are technical barriers in artificial breeding.The chemical activity of Huperzine A is far less than that of natural Huperzine A,which makes it urgent to find new sources of Huperzine A.H.serrata contains a lot of endophytic fungi,and the secondary metabolites of endophytic fungi are very abundant.In this study,the endophytic fungi in H.serrata were isolated in order to find the endophytic fungi that produce Huperzine A.The fermentation characteristics of the strain were studied,which provided a theoretical basis for expanding cultivation.And its secondary metabolites were isolated and the active ingredients were further searched via cytotoxic activity experiments.The research contents and results are as follows:（1）A total of 57 endophytic fungi were isolated from H.serrata in this study.Theseendophytic fungi were distributed in 20 genera and 29 species by ITS.Among them,there are 9 endophytic fungi belonging to the Fusarium sp.,8 endophytic fungibelonging to the Chaetomiun sp.,4 endophytic fungi belonging to the collectotrichumsp.,4 endophytic fungi belonging to the Trichoderma sp.,4 endophytic fungi belongingto the Arthrinium sp.,3 endophytic fungi belonging to the Pezicula sp.,and theremaining 20 strains of endophytic fungi distributed in other 14 genera.Among theseendophytic fungi,39 were from the leaves of H.serrata,12 were from the stem,and 6were from the root.The results showed that the endophytic fungi were the mostabundant in the leaves,followed by the stems and the least in the roots.（2）The recovery rates of Huperzine A under different conditions were compared,themethod of extracting total alkaloids was optimized.The endophytic fungi isolated werefermented and cultured,and the total alkaloids in the endophytic fungus hypha wereinitially detected by HPLC.Sample 68 was found to have the same retention time asthe Huperzine A standard.The MS and MS/MS of the sample were consistent withHuperzine A standard by LC-MS.Therefore,it was confirmed that the strain 68 couldproduce Huperzine A and named it NWUHS001.NWUHS001 was sent to the ChinaGeneral Microbial Culture Collection Management Center（CGMCC）to save andidentification（CGMCC NO.:16786）.The strain was identified as Colletotrichumgloeosporioides.The fermentation characteristics showed that the highest Huperzine Ayield was 1.46μg/g CDW on the sixth day of fermentation.In addition,it was foundthat the Huperzine A production of endophytic fungi gradually decreased withsubculture and degeneration.（3）The crude extract activity of NWUHS001 was tested by acetylcholinesterase inhibitionexperiment with IC₅₀of 0.055mg/m L.According to the detection of the sample by theHPLC,it is known that the secondary metabolites of NWUHS001 have othercomponents besides Huperzine A.In order to further study the secondary metabolitesof NWUHS001,a total of 239.2804 g of dry mycelium was collected by fermentation.Compound A was obtained by pre-HPLC.Compound A was identified as pterosin B via1H-NMR,13C-NMR,DEPT,HSQC,HMBC combined with mass spectrometry data.（4）The cytotoxic activity of pterosin B was tested by the CCK-8.Four representativehuman tumor cells were selected,the human liver cancer cell line Hep G2,the humanlung cancer cell line H226,the human epidermal cancer cell line A431,and the humanpancreatic cancer cell line PANC1.The vitro inhibitory effect of the compound onthese tumor cells was tested,and the results showed that pterosin B had a goodinhibitory effect on Hep G2,with an IC₅₀ of 0.1705μmol/m L,but showed no stronginhibitory effect on other cell lines...