ABSTRACT
Objective: To investigate the anti-hepatocarcinoma effect and mechanisms of the alkali hydrolysate of total saponins from Pulsatilla chinensis (PAHS). Methods: MTT assay was used to evaluate the effect of PAHS on proliferation of human liver cancer cell line SMMC-7721 in vitro; Cell morphology was observed by Giemsa staining. The effects of PAHS on apoptosis, cell cycle, and mitochondrial membrane potential of SMMC-7721 were detected by Hoechst 33258 staining and flow cytometry assay; Western blotting was employed to detect the protein expression of Cytochrome C, Caspase-3, cleaved Caspase-3, and Bcl-2 in SMMC-7721 cells. An in vivo liver cancer model was established using ICR mice subcutaneously received H22 hepatoma carcinoma cells to detect tumor growth inhibitory rate. Morphological changes of the tumor samples were observed by HE staining and transmission electron microscope. Results: MTT assay showed that PAHS could inhibit proliferation and increase apoptosis of SMMC-7721 cells in dose-and time-dependent manners in vitro, block the cell cycle in S phase, and decrease mitochondria membrane potential; PAHS could significantly increase the expression of Cytochrome C and cleaved Caspase-3 and decrease the expression of Bcl-2 and Caspase-3. PAHS dramatically decreased the weight of tumor tissue in a dose-dependent manner. Histopathological examination showed that large necrosis area and apoptotic cells were found in tumor tissues of mice in PAHS administrated group. Conclusion: PAHS exerts antitumor activity in vitro and in vivo by inducing apoptosis, and its mechanism is related to regulation of the mitochondrial pathway.
ABSTRACT
Objective: To investigate the antirumour activity of saponins from Pulsatilla chinensis against the Bel-7402 human hepatocellular carcinoma xenograft in nude mice, and to explore the mechanism of energy metabolism. Methods: The antitumor activity in vivo was estimated using a Bel-7402 xenograft model. The tumor volume was measured, the tumor inhibitory rate was calculated, the levels of lactic acid and ATP were assessed in Bel-7402 xenograft in nude mice, energy metabolic enzymes in Bel-7402 xenograft in nude mice were detected by ELISA assay, and hypoxia inducible factor-1 (HIF-1α) was detected by Western blotting assay. Results: The sapoinins from P. chinensis could inhibit the growth of Bel-7402 xenograft in a dose-depedent manner. The tumor inhibitory rates at the dose of 400, 200, and 100 mg/kg were 42.8%, 31.5%, and 14.9%. They had no side effects on white blood cell (WBC) in blood; The saponins from P. chinensis could decrease the contents of lactic acid and ATP. They also could decrease the levels of glycolytic enzymes phosphofructokinase (PFK), hexokinase-II (HK-II), and pyruvate kinase (PK), while increase the level of succinatedehydrogenase (SDH) that was the key enzyme during tricarboxylic acid cycle. Western blotting indicated that the expression of HIF-1α protein was significantly decreased in Bel-7402 xenograft in nude mice. Conclusion: The saponins from P. chinensis could inhibit the growth of Bel-7402 xenograft in nude mice in vivo, the mechanism is related with regulating energy metabolism through the HIF-1α pathway.
ABSTRACT
Objective: To develop an HPLC method for the simultaneous determination of eight saponins in alkali hydrolysate of total saponins from Pulsatilla chinensis. Methods: HPLC was performed on a Kromasil C18 analytical column (250 mm × 4.6 mm, 5.0 μm) at 35°C with MeOH-0.2% HCOOH solution as the mobile phase by gradient elution and the step gradients were as follows: 0-30 min, 70%-100% MeOH; The flow rate was 1.0 mL/min; ELSD gasification chamber temperature was 40°C; Gas pressure of carrier gas N2 was 350 kPa. Results: The linear response (the log values of peak areas with corresponding log values of sample introducing amounts) ranges were 0.799-4.568 μg for pulsatilla saponin D, 0.563-6.756 μg for hederagenin 3-O-β-D-glucopyranosyl-(1→4)-α-L-arabinopyranoside, 0.431-2.683 μg for pulsatilla saponin A, 0.894-7.826 μg for hederacolchiside A1, 0.643-7.504 μg for pulsatilla saponin F, 1.351-7.822 μg for oleanolic acid 3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl - (1→3)-α-L-rhamnopyranosyl-(1→2) - α-L-arabinopyranoside, 0.629-2.515 μg for oleanolic acid 3-O-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl - (1→2)-α-L-arabinopyranoside, and 0.698-2.794 μg for oleanolic acid 3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside, respectively (n = 5). The average recoveries of the eight saponins were between 99.0% and 101.0%, and RSD values were less than 1.5%. Conclusion: The results demonstrate that the established method has the adequate accuracy and selectivity for the quality control of alkali hydrolysate of total saponins from P. chinensis.