Antitumor Effect And Mechanisms Of Alkali Hydrolysate Of Total Saponins From Pulsatilla Chinensis

Aim: In our previous study, we found that total saponins from Pulsatilla chinensisinhibited the growth of tumors in vivo and in virtro. Compared with total saponins, Alkalihydrolysate of total saponins had better antitumor effect. In this study, we will investigateantitumor effect and mechanisms of alkali hydrolysate of total saponins from Pulsatillachinensis.Methods: The HPLC method was used to determination the contents of eightsaponins in alkali hydrolysate of total saponins from Pulsatilla chinensis; In vitro, MTTand clone formation test were used to examine the growth of human carcinomaSMMC-7721and A549. Additionally, Giemsa staining was also used to observe themorphology of tumour cells; In vivo, ICR mice bearing H22and S180cells were used, C57mice transplanted with Lewis tumor cells were also used. Blood extracted from tumourbearing mice was used to make the blood examination. Tumor tissue was stained withhematoxylin and eosin, and then observed the change of cell morphology; Ultrastructure oftumor tissue was examined with transmission electron microscope; Flow cytometry wasused to detect the cell cycle and apoptosis of tumor cells; Hoechst33258assay kit wereperformed to observe the morphology of apoptosis cells; Important signal molecules ofmitochondrial pathway and PI3K pathway were detected by western blot.Results: The total content of eight saponins (pulsatilla saponin D, hederagenin3-O-β-D-glucopyranosyl-(1→4)-α-L-arabinopyranoside, pulsatilla saponin A,hederacolchiside A1, pulsatilla saponin F, oleanolic acid3-O-β-D-glucopyranosyl-(1→4)-β-D-gluco-pyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside, oleanolic acid3-O-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L- arabinopyranoside, oleanolic acid3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopy-ranoside) in alkali hydrolysate of total saponins from Pulsatilla chinensis was more than85%. In vitro, PAHS (0.80-25.00μg/ml) exhibited an inhibited effect on SMMC-7721andA549cells growth in concentration and time-dependent manners by MTT and cloneformation test; In vivo, PAHS (50，100，200mg/kg) could inhibit the tumor weight of H22,S180and Lewis in concentration manners, when treated with200mg/kg, the inhibitionrate were49.8%,43.7%and70.6%. The HE staining of all three tumors were presentedwith wide ranges of necrosis tissue. The results of transmission electronic microscopeshowed that PAHS (50，100，200mg/kg) groups exhibited remarkable ultrastructuralcharacteristics of apoptotic programmed cell death such as nuclei degenerated, cytoplasmshrinkage, chromatin condensated, and decreases of cell volume. Compared with CTXgroup, the number of WBC, spleen index, thymus index were imprpove. These resultsindicated that PAHS had little toxicity. PAHS (6.25,12.50,25.00μg/ml) could induceapoptosis on SMMC-7721and A549cells. Additionally, SMMC-7721cells were arrestedin S phase and A549cells in G0/G1phase. Western blotting showed that PAHS could upregulate the expression of Cyt-C、Cleaved Caspase-3、Bax, and down regulate theexpression of PI3Kp85、p-Akt、p-mTOR、p-p70S6K.Conclusions: PAHS could inhibit the growth of liver and lung cancer, the mechanismmight be that PAHS induce apoptosis through PI3K/AKT/mTOR pathway.