| Huperzine A(Hup A)is an alkaloid isolated from Huperzia serrata(Thunb.)Trev.Studies have found that Hup A has a significant effect on Alzheimer’s disease by inhibiting acetylcholinesterase(ACh E),so Hup A is in high demand.H.serrata grows slowly and the extraction rate of Hup A is low.The biological activity and safety of synthetic Hup A are much lower than that of natural Hup A.In recent years,the production of Hup A by the culture of endophytic fungi of Huperzia serrata has become a hot research topic.However,the main problem is that the content of Hup A in endophytic fungi is too low,and the synthesis pathway of Hup A is not completely clear.In this study,starting from the isolation of the Hup A produced endophytic fungi,we tried to prove that CYP450 is the key point of regulating the metabolic pathway of Hup A by using Honokiol,which has inhibitory effect on CYP450,so as to study the biosynthesis of Hup A and provide data for the metabolic regulation of Hup A production by fungi.The main research contents and results are as follows:(1)From the H.serrata preliminary separation filter out 6 strains colony morphology of different color endophytes,fermentation cultivation,the 6 endophytes to extract the endophytic fungi bacteria fermentation metabolites,high performance liquid chromatography(HPLC)test,found the S-C-6 strains of metabolic extract is consistent with Hup A standard product retention time,in order to further confirm,through LC-MS detection analysis,found that the sample of MS and MS/MS were consistent with that of Hup A a standard product,Therefore,it was confirmed that strains S-C-6 could produce Hup A,and the strain was named NWUHS003.Based on morphological identification,ITS identification and phylogenetic tree analysis,NWUHS003 was identified as Fusarium sp.,with a yield of 5.74 g/g CDW per unit.The Hupa yield of NWUHS003(Colletotrichum gloeosporioides),another Hup A producing fungus isolated in the laboratory,was compared with that of NWUHS001.The Hup A yield of NWUHS001(10.16μg/ GDCW)was about 1.77 times that of NWUHS003(5.74 μg/ GDCW).Therefore,NWUHS001 was selected as the strain for subsequent experiments to reduce the proportion of errors caused by experimental operations in experimental results.(2)in this study,100μM and honokiol were added into the fermentation broth of the endophytic fungus-NWUHS001,which produced Hupa.After 7 days of fermentation,for the sake of the various angles of screening suitable concentration of honokiol,observe NWUHS001 morphological characteristics,detecting NWUHS001 activity(methylene blue staining)unit of analysis,comparative analysis of NWUHS001 dry weight,NWUHS001 Hup A production(HPLC),finally it is concluded that honokiol processing NWUHS001 bacteria(7 d)is 100μM,the appropriate concentration under the concentration,Magnolol and Honokiol had no significant effect on the activity and growth of NWUHS001,but inhibited the synthesis of Hupa of NWUHS001,and reduced the yield of Hupa per unit of NWUHS001 mycelia.(3)In this study,100μM and honokiol were added into the fermentation broth of the endophytic fungus-NWUHS001,which produced Hupa.After 7 days of fermentation,microsomes of NWUHS001 were prepared and CYP450 activity and content of the fungus were detected by CO binding differential spectroscopy.The results showed that 100 μM honokiol could significantly inhibit the CYP450 activity of NWUHS001,and the P450 content of this fungus was as low as 0.0156 nmol/mg after the treatment with honokiol,which was about 1/47 of the P450 content of the control with methanol,indicating that honokiol had a significant inhibition effect on the P450 activity of this fungus. |
