Study On The Total Secondary Saponin From Liangtoujian With The Anti-breast Cancer Activity Against To MCF-7 Cells

According to Chinese Pharmacopeia,Liangtoujian is the rhizome of Anemone raddeana Regel.Extensive phytochemical and pharmacological studies of A.raddeana demonstrated that triterpenoid saponins were the main components to contribute antitumor activities.In our previous study,TSS showed potential antitumor activity in vitro tests on various cancer cells.In this work,we verificated the antitumor activity of TSS by breast cancer MCF-7 cell line,and conducted the further studies,concluding the pharmacodynamics effect in vivo,the mechanism of inducing apoptosis,chemical components,and pharmacokinetic study.1.Screening of potential fraction and triterpenoid saponins from Anemone raddeana Regel against MCF-7 cell lineIn vitro,MTT assay was used to examine effect on the growth of MCF-7 breast tumor cells with ATS,TSS or Rs-10 of various concentrations after 48 h.IC50 value was 19.649μg/ml,6.559 μg/ml,and 4.407±0.12 μg/ml,respectively.To determine whether the in vitro effects extend to an in vivo xenograft model,the effect of ATS,TSS and Rs-10 was tested in vivo against tumors derived from MCF-7 cells injected into the backside of nude mice.The inhibitory rates were 40.32%,60.00%and 48.45%at the same dose equal to the same dose of crude drug.The TSS had the lower IC50value in breast tumor cells viability and the more vigorous inhibitory effect on growth of transplanted tumor MCF-7 in mice.An HPLC-ESI-Q/TOF-MS method was established for the screening and identification of the main chemical components in ATS and TSS.A total of 33 compounds from ATS and 23 compounds from TSS were identified preliminarily based on retention time,MS spectra and comparing with literatures.Combined with the previous biological studies,it could be speculated that the antitumor activity was related to the structural features of 28-carboxylic acid and the lateral chain of C-3.2.Acute and sub-acute(28 days)oral toxicity evaluation of TSSFor acute toxicity study,after a single dose of oral administration of TSS,the mice were observed for mortality and clinical signs for 14 days.The acute toxicity study showed that the LD50 value was 879.461 mg/kg for mice,and no difference in gross pathology was observed between control and treatment groups.That was implying TSS a slight toxic in mice.In the sub-acute toxicity study,TSS was administered orally at doses of 50,100 and 200 mg/kg/day for 28 days to male and female mice.The sub-acute toxicity study showed that daily oral administration of TSS induced no significant difference in body weight,food consumption,water intake,and hematological parameters,organ weight,gross pathology.In biochemical analysis,compared with the control group,CREA and UREA levels rose at dose of 200 mg/kg in male mice.However,no histological changes were observed in kidney.The results of this study provided evidence that the doses of TSS(50,100 and 200 mg/kg)might be nephrotoxicity potential with male mice and should be safe with female mice.3.The effect of TSS on nude mice transplanted tumor induced by human breast cancer MCF-7 cells and the study on the mechanism of TSS inducing apoptosis effect.Thirty nude mice bearing human MCF-7 breast cancer cells were randomly divided into 5 groups.According to the dosing regimen,mice in vechicle(0 mg/kg)group and TSS(50,100,200 mg/kg)groups were with intragastric administration,cisplatin(10 mg/kg)group were with intraperitoneal injection for 28 days.Cisplatin and TSS obviously inhibited the growth of transplanted tumor.The inhibit rate cisplatin was 48.70%,while TSS at dose of 50 mg/kg,100 mg/kg,200 mg/kg group was 33.7%,44.28%,54.03%respectively,and the treatment with TSS exhibited inhibit capacity in a dose-dependent manner.There is no significant adverse reaction of TSS on nude mice.For the in-depth study of its anticancer mechanism,cell cycle profile of cells treated with TSS was determined using PI staining assay.TSS and Rs-10 could induce MCF-7 cell cycle arrest at G0/G1,but it appeared no dose-dependent.Apoptosis induction by TSS was examined using Hoechst 33258 staining assay and Annexin V-FITC/PI double staining assay.The expression levels of signaling molecules associated with apoptosis were analyzed by Western Blot.The mechanistic study showed that TSS induced apoptosis mainly through mitochondrial apoptotic signaling pathway,supported by the increase of the ratio Bax/Bcl-2,the activation of Caspase-9 and Caspase-3.Besides,TSS has no influence on the expression of Fas.The result of Rs-10 was consistent with TSS.4.The isolation of TSS by and the determination of chemical constituents in TSS by HPLCBy means of the silica gel column chromatography,ODS column chromatography and preparative liquid chromatography,9 components were isolated from TSS.The structures of these compounds were identified on the basis of NMR spectral data and physicochemical characters.An HPLC method was developed for the simultaneous determination of 7 triterpenoid saponins in TSS.Methodology validation(precision,accuracy,recoveries and stability)was in accordance with relevant requirements.The content of Rs-10 was 45.10%,while EK was 6.61%.The content of other compounds was in the range of 0.67-3.71%.The total content of 7 saponins in TSS was more than 60%.5.Pharmacokinetic studies of active triterpenoid saponins and TSSA rapid and validated HPLC-MS/MS method was developed to simultaneously determine the active compounds(Rs-10 and EK).Quantitative analysis was performed using the multiple-reaction monitoring(MRM)mode.The result of the assay had good reproducibility with acceptable accuracy and precision.Under the developed analytical conditions,the obtained values of main pharmacokinetic parameters(Cmax and AUC0-t)indicated that the pure compounds were more efficient than the TSS extract in Rs-10 and EK absorption.The t1/2 values of Rs-10 and EK were 0.8 h and 8.8 h separately,demonstrating the longer retention time of Rs-10 in rat plasma In addition,pharmacokinetic studies of two individual compounds demonstrated their poor oral absorption in rat(aF%,0.019-1.521).