| Rhizoma Panacis Japonici, a specialized resource of tujia nation in HuNan province,belongs to Araliaceae plant.It contains various trierpenoid saponins,flavoxate, and contains some active substances such as amino acids,traceelements, and so on.Being a natural plant used as both medicines and foods,it has been applied widely on clinical treat and health protection, having both the nutrition effects of ginseng and the effects of notoginseng that can activate blood circulation to dissipate blood stasis.At present, the research aimed to total saponins of Rhizoma Panacis Japonici (TsPJ) is concentrated mainly on exploitation of drug to treat rheumatoid arthritis, and systematic research on extraction and purification for its active substance remains unreported, systematic research about its antineoplasmic activity is rare.The article studied majorly technology conditions of extracting and purifying total saponins, established content determination method of TsPJ, posed the best technology parameters suitable to extract and purify for total saponins.In addition, studied initially and tried on its antineoplasmic activity in vitro. The research work and experiment results are as follows:1,Content determination method of TsPJBy selecting ginsenoside Re as control article, colorating with 5% HClO4 and vanillin-CH3COOH, determined absorbability in the Point of 552nm with ultraviolet-visible absorption speetroscopy and calculated content of TsPJ. The results showed that the method is linear in the range of 40.48～202.40μg (R2=0.9997), absorb ability stability with in 90min, RSD 1.42%, moreover, the average recovery 100.46%,RSD 1.06%(n=5). The conclusion is that the method is sensitive and reliable, which may be used for the quality conirol of TsPJ.2,The optimization of extraction conditions for TsPJOn the basis of single factor experiment, designed L9(34)orthogonal test with the extraction rate of total saponins as index, to determine the rational microwave extraction process;then comparing it with heating reflux extraction and transonic extraction. The optimum condition of microwave extraction was as follows:the microwave power was 400 W, the solvent was 90% ethanol,the proportion of raw material to solvent was 1:18,extracting 4 times and 5 min at each time.Under the optimal condition, the extraction content of TsPJ was up to 97.77%.The microwave extraction has a series of advantages, such as higher efficiency, shorter extracting time, lower exhausting energy, environmental protection, etc.It provided new evidence for industrializing production of Panax japonicus and developing new drug.3,The optimization of purification process for total saponins of TsPJ5 kinds of macroporous resin were selected from different companys, according to their different polarity. Static absorption indicated that D101 had a good absorptive character of TsPJ. The optimum technic showed as follows:the optimal concentration of the rawmaterial was 0.2 g·mL-1,PH was 6,washed with 3 BV of 70% ethano, and with the absorption and elution velocity of flow 1 mL·min-1.The experimental results showed that TsPJ in the solid extract could reach 82.53%,well above unpurified of 24.36%,their refine ratio and diversion ratio reached respectively 338.81% and 88.02%.On the whole,the optimal conditions are suitable for separation and purification of total saponinsin.4,Apoptosis of hepatocellular carcinoma HepG2 cells induced by TsPJThe HepG2 cells were treated with different doses of TsPJ.Cell proliferation was measured by MTT assay. Cell cycle and apoptosis rate were analyzed by flow cytometry. The results showed that TsPJ inhibited proliferation of HepG2 cells in concentration dependent and time dependent manner. FCM assay showed that TsPJ induced apoptosis of HepG2 cells after treated with 50,100,200 and 400μg·mL-1 TsPJ for 48h. The apoptosis rates were increased (P<0.01).TsPJ probably inhibits the proliferation of HepG2 cells by inducing apoptosis. |
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