Study On Extraction Technology Of The Huperzine A From Huperzia Serrata By Aqueous Two-phase System

Huperzine A, a sesquiterpene alkaloid isolated from the Chinese herbal medicine Huperzia serrata, which exhibits some pharmacological properties of neuroprotection, antinociception, antioxidant and antiapoptosis, has been used for treatment of Alzheimer’s disease, Vascular dementia, Myasthenia gravis, Mild cognitive impairment, Parkinson’s disease and so on. Aqueous two-phase extraction (ATPE), a new separation technique developed in recent years, has many advantages such as less separation procedure, milder operating condition, easier to scale-up and less residue of organic solvents in the products.In this paper, the method for huperzine A detection by HPLC was established, and the technologies for huperzine A extraction by enzyme-assisted ultrasound extraction and ethanol/ammonium sulfate aqueous two-phase system were optimized. The main results were as follows:(1) Establishment of HPLC method for detection of huperzine A.HPLC was performed on a PurospherRC18 column (4.6x250 mm, i.d.,5 μm) (Merck) with methanol/water (85/15, v/v) as the mobile phases,20 μL as sample volume,0.3 mL/min as flow rate and 308 nm as dection wavelength under column temperature of 30℃. Under these conditions, a good linear relationship between peak areas and huperzine A concentrations was obtained in the range from 1 μg/mL to 25 μg/mL of huperzine A with the correlation coefficient (R2) of 0.9948. The average recovery was 98.98% with the relative standard deviation (RSD) of 1.58%. All RSDs for precision, stability and repeatability were below 3%. The results demonstrated that this method is simple and accurate with good reproducibility, which could be used to guide the extraction of huperzine A from Huperzia serrata.(2) Optimization of enzyme-assisted ultrasound technology for huperzine A extraction.Enzyme-assisted ultrasound technology for the extraction of huperzine A from Huperzia serrata was optimized by the method of Duncan and Box-Behnken. The optimal conditions were cellulase of 6%, enzyme reaction time of 120 min, solid-liquid ratio of 1:65, ultrasonic time of 114 min and ultrasonic power of 760 W. Under the optimized conditions, the yield of huperzine A from the wild and micropropagated Huperzia serrata were 0.04087% and 0.05105%, respectively. The yield of huperzine A from the wild Huperzia serrata was 1.497-fold higher than that of ethanol refluxing method and 0.94-fold higher than that of acid extraction method.(3) Optimization of ethanol/ammonium sulfate aqueous two-phase system for huperzine A purification.Ethanol/ammonium sulfate aqueous two-phase system for huperzine A purification was optimized by the method of artificial neural networks (ANN).The optimal conditions were ethanol of 38%, ammonium sulfate of 20.6%, pH value of 8.85, temperature of 35.32℃. Under the optimized conditions, the extraction rate of huperzine A was 92.87%. The thermodynamics results indicated that the partition of huperzine A was controlled by entropy changes, the progress could occur spontaneously at the lower temperature. The yields of huperzine A which were extracted from the wild and micropropagated Huperzia serrata by the ethanol/ammonium sulfate aqueous two-phase system were 0.03742% and 0.04603%, the extraction rates were 91.32% and 90.16%, the purity were increased by 37.60% and 27.63%, respectively. The system had obviously effect on the purification of huperzine A.