Study On Separation And Preparation Of High-purity Huperzine A By Medium Pressure Preparative Chromatography

Huperzine A is an alkaloid extracted from huperzia serrata.It is an efficient reversible acetylcholinesterase(AChE)inhibitor,and has been used for treating benign memory disorders,Alzheimer's disease and myasthenia gravis.Therefore,the demand for huperzine A is on the increase.In this thesis,aiming at the shortage of current extraction and purification process,the novel process was proposed and optimized.From the point of view of industrial production,some low-efficiency process routes are discarded.At the same time,the medium pressure preparative chromatography is used to replace the high pressure preparative chromatography for reducing production costs and improving productivity.The main creative results were achieved as follows:(1)Several extraction methods of huperzine A from huperzia serrata were compared,and it was found that the ultrasonic extraction was simple and efficient.Then a high performance liquid chromatography(HPLC)method for the determination of huperzine A in huperzia serrata was established and validated.The results indicated that the proposed HPLC method were accurate and reliable.(2)The extraction conditions of huperzia serrata were determined.The extract of huperzia serrata was obtained by soaking with 1%aqueous H2SO4.The repeatability of the extraction process of huperzia serrata was investigated.The average extraction rate of the batch was 90.00%with RSD 1.9%,and the average extraction rate was 89.60%with RSD1.1%as different batches of huperzia serrata raw materials were used.(3)The conditions for the enrichment of huperzine A was studied.The enrichment effects of ion exchange resin and the pH of the extract solution were investigated by static adsorption experiment.It was confirmed that FPC14 was the best medium for capturing huperzine A in the extract solution.The elution conditions were determined by dynamic experiments.The eluent was a mixed solution of 1 mol/L NH4C1 solution(pH 8.0):ethanol(1:1,v/v).According to the method,Huperzine A extract was obtained,and the percentage of huperzine A was 2.3%and the recovery was 91.3%.(4)High-purity huperzine A was prepared by two-step preparative chromatography.UniPSN 30-300 and UniSil 30 C18 chromatographic packing were used in series,and the final product with purity of 99.68%was obtained in two steps.The comprehensive recovery rate was 88.3%.(5)As a result,a new process for the preparation of huperzine A was developed.A strong cation exchange resin was used to enrich huperzine A in the extract,and a novel polymeric RPC media was used in the first preparative chromatography.The loading capacity of polymeric RPC media was 41 mg/ml,far more than that of C18 chromatographic packing.And moreover,because of its good acid and alkali resistance,the polymeric RPC media can accept acidic and alkaline solvent regeneration cleaning,its service life is significantly increased with a significant cost advantage.The respective characteristics of polymer fillers and C18 fillers were utilized to provide an efficient,green and economical way to obtain pharmaceutical grade huperzine A through technological linkage.