The Isolation And Identification Of Natural Total Saponins In Three Herbs And The Research Of Anti-cancer Activity

Objective:To explore primarily methods of extraction and isolation of total saponins in Rhizoma pardis, Ardisia pusilla A.DC. and Entada phaseoloides.To compare the inhibitory effect of rhizoma pardis total saponins(RPTS) with its alcohol extracts and polyphyllin I on human gastric cancer cell BGC823 in vitro. Meanwhile,The aim of this study was to assess the natural interaction between RPTS and chemotherapeutic agents, such as 5-fluorouracil(5-Fu), oxaliplatin(Oxa) widely used in gastric cancer treatment.Methods:Aqueous or alcohol extracts of Rhizoma paridis, Ardisia pusilla A.DC. and Entada phaseoloides were obtained using heat reflux extraction(HRE) techniques. The extracts were isolated by the macroporous and produced total saponins. To select the best method of extraction and isolation with the inhibition rate of the anti-tumor in vitro, MTT staining method (MTT) determined repressive effect of different extracts or component part of total saponins on cell proliferation. Foam reaction,Liebermann-Burchard reaction and high performance liquid chromatography (HPLC) served as identification methods of total saponins. MTT assay and colony formation assay(FCM) were employed for detection of effect of rhizoma paridis total saponins, alcohol extract and polyphyllin I on human gastric cancer cell BGC823 proliferation and colony formation. The influence of the RPTS on chemotherapeutic agents 5-Fu or Oxa was measured using MTT assay and FCM.Results:The component parts of rhizoma paridis extracts at the concentration of 70% ethanol had the strongest inhibition of cell proliferation, where total saponins was. Content of polyphyllinⅠ,ⅡandⅥin total saponins by HPLC were 8.86%,2.63%,4.40% respectively. Similarly,we concluded that Ardisia pusilla A.DC total saponins was extracts at the concentration of 90% ethanol and Entada phaseoloides total saponins was extracts at the concentration of 70% ethanol. Both of them had weak antitumor activity,we continued to study RPTS only. The IC50 doses of BGC823 exposured to rhizoma pardis total saponins, alcohol extracts and Polyphyllin I were (8.63±0.30),(2.61±0.03), (1.97±0.01)μg/mL respectively. At the concentrations of 3.13,6.25,12.5μg/mL, the inhibition rate of colony forning were all 100% for RPTS and 30.84%,53.27%,100% for its alcohol extracts. MTT assay and FCM were employed for detection of the synergistic effect between RPTS and 5-Fu or Oxa on human gastric cancer cell BGC823 proliferation and apoptosis.Conclusion:According to antineoplastic activity in vitro and results of identification,heat reflux extraction techniques and macroporous adsorptive resins techniques were suitable for extraction and isolation of total saponins in rhizoma pardis,but not suitable for Ardisia pusilla A.DC. or Entada phaseoloides. To improve or change the methods of extraction and isolation of total saponins in Ardisia pusilla A.DC. and Entada phaseoloides maybe can enhance the anti-tumor activity. The RPTS had stronger activity than the alcohol extract in inhibition of proliferation and colony formation of human gastric cancer cell BGC823 and had weaker activity than PolyphyllinⅠ.However,the IC50 concentration of RPTS was low and near to Polyphyllin I. These results confirmed correctness and necessity of the methods of extraction and isolation of total saponins in rhizoma pardis. RPTS could significantly increase the antineoplastic activity of 5-Fu or Oxa in a synergistic manner in vitro. It is clear that RPTS has considerable potential as anti-cancer drug and is worthy studing further more.