| The purpose of this study was to investigate the factors and dynamic varitation rule for the accumulation of Huperzine A(Hup A) from Huperzia serrata(H.serrata) thallus which could produce Hup A, an anti-Alzheimer’s disease drug candidate. Designed five medium(B5、6,7-V、MS、N6,、White) to find which was the best one for thallus. The optimal concentration of plant growth substances for the growth of H.serrata thallus were screened using orthogonal design method, and designed concentration gradient(g/L)(10、15、20、25、30) to find the best sucrose concentration. Studied the optimum light through light wave(blue、white、red)、light intensity(0 lx、1000 lx、1500 lx、2000 lx) and light time(12 h/d、15 h/d). Through digestion time and volume of tartaric acid,studied the optimal extraction process of Hup A. Identified the Hup A by Thin-layer Chromatography(TLC) and High Performance Liquid Chromato-graphy(HPLC) of tartaric acid extracts of thallus and stems or leaves of Huperzia serrata. The kinetics curve of thallus growth was described using Logistic growth model, and analysised the genetic stability of thallus through comparing with the different stage thallus POD. All these outcomes could give a basis for establishing the suspension cellus culture of Huperzia serrata and production of the natural active components in large-scale. All the paper results were as follow:1.The thallus multiplication test showed that 1/4 MS+20 g/L without any hormone was the optimum culture medium for thallus of Huperzia serrata.2.Light condition was the major factor in the progress of plant growth and chemical synthesis in cell. The light regulation test showed that the thallus in 12 h/d 1500 lx white light growed and accumulated Hup A best.The relative growth rate was 3174.5%,and the content of huperzine A in the thallus was 72.45 ug/L3.The test finded the the suitable way to extract and determinat Hup A.Firstly,10 mL2% tartaric acid soaked the dry powder for 24 h, then ultrasonic machine handled the power 30 min, repeated 3 times. Secondly, chloroform extracted Hup A 5 times, then natural air dried chloroform. Thirdly,used methanol to purificat Hup A. The best developing solvent of Hup A is chloroform / acetone isopropanol / water(4:4:2:0.1, v/v/v/v) had in thin-layer chromatography.The high performance liquid chromategraphy separation effect of the best conditions is the mobile phase of methanol ammonium acetate(0.08 mol/L; =3:7 pH=6), flow rate: 0.8 mL /min, detection wavelength: 308 nm, column temperature: 25 ℃, sample size: 20 μL.4.According to the growth curve of H.settata thallus like “S” shape, the best successive generation date of callus culture of H.serrata was 50 days. The concentration of Hup A increased obviously after 60 days when the thallus reached the stable period and the maximal value of Hup A was 7.453 μg/g at 85 days, which meant that the rapid growth of thallus was priority to the accumulation of Hup A. The relational model between the accumulation of Hup A from thallus and its growth was non-growth coniugation. In addition,the growth law of thallus was consistent with Logistic growth model and the maximum specific growth rate( μmax) was 0.071/ d.5.The POD isozyme analysis test for the different culture time of thallus from a showed that the isozyme staining of growth time of 70 d thallus is deeper than others. It could say that thallus after growing 70 d are metabolically active. |
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