Optimization Of Extraction Process For Rhizoma Paridis By Mass Trial And Study On Its Anti-tumor Mechanism

Objective:We selected Rhizoma Paridis as research object to optimize extraction process by mass trial,separated and purificated two anti-tumor active components PCB5-9 and PCB4—5 respectively,to investigate the mechanisms of saponins-induced apoptosis on SGC-7901 cells,in order to find new drug action target and establish theory foundation of new method of treating human poorly differentiated gastric adenocarcinoma.Methods:(1) We applied orthogonal design to optimize the extraction process for the saponins from Rhizoma Paridis.We used ethyl alcohol as extracting solvent to gain Chinese medical concrete,used petroleum ether,ethyl acetate and n-Butanol to extract saponins from it;and used macoropore resin,gelica gel,ODS,RP-HPLC to separate saponins.(2) Applied MTT assay to detect the inhibition effect of saponins from Rhizoma Paridis against human erythroleukemia cell line K562 and human poorly differentiated gastric adenocarcinoma cell line SGC-7901 culturing in vitro, and determined effective dose and median inhibition concentration(IC₅₀) of saponins to SGC-7901 cells and K562 cell after being treated with it.(3) Saponins from Rhizoma Paridis were marked by fluorescent reagent 5(6) -TAMRA-5-maleimide,applied MTT assay to detect the inhibition effect of saponins from Rhizoma Paridis,saponins marked by TAMRA and TAMRA against SGC-7901culturing in vitro,and to investigated cell-stained process of the fluorescent labeling saponins by fluorescence microscope.(4) Investigated the mechanisms of saponins-induced apoptosis on SGC-7901 cells by the way of DNA gel electroplhoresis.(5) The concentration of Ca²⁺ intra-cellular([Ca²⁺]i) was measured by the laser scanning confocal microscope(LSCM).We treated SGC-7901 with different concentration of saponins for 24hours,used the fluorescent probe Ca²⁺ to mark the intra-cellular Ca²⁺ to observe its statical effect on concentration of Ca²⁺.We marked the intra-cellular Ca²⁺,then gave SGC-7901 with different concentration of saponins for 10 minutes to observe its dynamic effect on concentration of Ca² immediately.Results:(1) The optimum contraction conditions of saponins from Rhizoma Paridis were as follows:70%ethyl alcohol,5hours,8 times dosage liquor ratio.According to this condition,we abstracted and separated saponins PCB 5-9(83-93) and PCB4-5 (39-48),their purification were 97.63%and 91.80%respectively by HPLC.Their structures needed further verification.(2) Studies in vitro indicated that two compositions could inhibit growth of K562,SGC—7901 and their inhibition rate had dose-time depended relationship.The highest inhibition rate of PCB 5-9(83-93) on K562,SGC—7901 were 88.54%and 75.08%respectively,IC₅₀ at 72h 6.55μg/mL and 20.48μg/mL respectively;PCB 4-5(39-48) respectively 88.58%and 75.68%,IC₅₀ at 72h 5.69μg/mL,4.83μg/mL respectively.(3) Studies of fluorescent labeling saponins on SGC-7901 in vitro showed fluorescent labeling saponins could inhibit growth of SGC-7901,when the concentration was 0.03mmol/L,its inhibition rate was 52.28%.5(6)-TAMRA-5- maleimide had no inhibition action on SGC-7901, red fluorescence appeared in cell by the way of fluorescence microscope after SGC-7901 had been treated by fluorescent labeling saponins for 2.5h.(4) Agarose gel electrophoresis showed typical DNA ladder strip after cell SGC-7901 had been treated with PCB 5-9(83-93) for 24h,and brightness of strip increased along with the concentration of it raises.(5) The concentration of Ca²⁺ intra-cellular([Ca²⁺]i) measured by LSCM after different concentration of saponins treated SGC-7901 for 24h showed significant variation(P＜0.05) compared with control;[Ca²⁺]i had no change when saponins treated SGC-7901 for 10min.Conclusion:The optimum contraction conditions of saponins from Rhizoma Paridis by the way of orthogonal experiment design could be applied in industry.Saponins could inhibit the proliferation of the SGC-7901 and K562,and induce cell SGC-7901 apoptosis.The increased concentration of Ca²⁺ suggested calcium-calmodulin pathway might be one of it's mechanism.Fluorescent labeling saponins had inhibition action on SGC-7901,and it could get into cells successfully.