Study On The Purification Of Huperzine A And Huperzine B And Endophytic Fungus In Huperzia Serrata

Huperzine A (HupA) is a natural alkaloid existed in the herbHuperzia serrata, which is proved to be a potent and reversible inhibitorof acetylcholinesterase (AChEI). It can be used to treat Alzheimer'sdisease (AD) and other memory diseases, thus becomes the focus issue ofresearchers. Huperzine B (HupB) is similar with HupA in both structureand AChEI activity. Natural HupA, HupB and their skeletons are currentlybeing widely applied to pharmacological and clinical studies. However,the content of HupA and HupB in Huperziaceae plants are very low, thislead to a limitation of the development of new AD drugs. For the purposeof use of limited plant resources sustainably, we aimed to develop a rapidand low-cost method to separate and purify HupA and HupB. Differentextraction and purification methods were compared, also a precisedetection and isolation method was established, in which the key factor ofmodifiers has been studied. At last, we optimized the preparative RPCprocess. In addition, this study also conducted the screening and preliminary activity study of endophytic fungus within Huperzia serrataplants.1. A fast and accurate analytical method of detecting HupA and HupBwas developed. The mobile solvent was consisted of0.1%TFA within18%methanol, the flow rate was1mL/min. This method confirmed the contentdifferences of various origin of Huperzia serrata. We found that adding amodifier can effectively improve the separation of HupA and HupB, thetailing was also reduced. Acidic modifiers in the mobile phase can work aspH stabilizer, it has a significant impact on the retention time andseparation resolution, trifluoroacetic acid (TFA) was proved to be the bestacid modifier.2. The effect of the traditional extraction, ultrasonic extraction andflash extraction, and the differences between purification by the resins andliquid-liquid extraction method were evaluated in this research. The yieldof ultrasonic extraction was up to75%, it was simple to be operated andmagnified to industrial scale; Weak polar macroporous resin SP850performed best in both adsorption and desorption effects among all theresins, it can concentrate the origin extract to more than100times, therecovery of HupA and HupB reaches more than90%.3. The injection volume and flow rate of preparative RPC wereOptimized, the best condition was:0.1%TFA, and flow rate at8mL/min,with sample amount of40mg, HupA and HupB were simultaneously obtained with the purity of above98%, the total recovery for them was83%and81.8%respectively.4. Fifty-two endophytic funguses were isolated from Huperziaserrata collected in Anhui Huangshan, and Fujian Sanming area. Soakingwith75%ethanol for3min, and treating for15-18min by15%sodiumhypochlorite can achieve the best disinfection efficiency. A kind ofpenicillium tested may produce antibacterial substances and the analoguesof Huperzia serrata alkaloids.