ABSTRACT
Objective To investigate the effects of Pulsatilla saponin D and sorafenib on the metastasis of human hepa?toma cell line. Methods The human hepatoma cell line BEL-7402 cells were divided into Pulsatilla saponin D group (con?centration of 11.9 mg/L), sorafenib group (concentration of 2.15μmol/L), the combined group (Pulsatilla saponin D 11.9 mg/L+Sorafenib 2.15μmol/L) and the control group (ordinary broth). The inhibition effects of Pulsatilla saponin D and sorafenib monotherapy and combination therapy on BEL-7402 cell migration were detected by MTT assay, Transwell chamber experi?ment and cell scratch experiment. Western blot assay was used to detect the expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 gene protein. Results MTT assay showed that Pulsatilla saponin D (11.9 mg/L), sorafenib (2.15μmol/L) monotherapy and combination therapy had inhibitory effects on BEL-7402 cell proliferation, and the 24-h inhibi?tion rate was<15%. Results of Transwell chamber experiment and cell scratch test showed that the migration inhibitory rate was significantly higher in combination group than that of monotherapy group (P<0.01). The combined effect of madicine was the addition (0.85≤Q≤1.15). Western blot detection showed that there was a higher effect of down-regulation on MMP-2 and MMP-9 in combined group than that of monotherapy group. Conclusion Pulsatilla saponin D and sorafenib synergis?tically inhibit the metastasis of BEL-7402 cells. The joint effects are superior to monotherapy.
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Objective: To prepare the solid dispersion of Pulsatilla saponin D (PSD-SD) and evalution its in vivo and in vitro drug release behavior. Methods: The PSD-SD was prepared by solvent method. Three carriers were used in the PSD-SD. Infrared spectroscopy (IR), differential thermal analysis (DSC), and X-ray diffraction (XRD) were used to determine the PSD-SD. Dissolution rates and pharmacokinetic parameters were evaluated in vitro and in vivo characteristics of the PSD-SD. Results: When the PEG 6000 was used as carrier, the solubility of PSD was increased from 2.39 to 7.06 mg/mL, and the cumulative release rate of PSD reached 90% in 60 min, and the bioavailability of PSD was increased to 2.24 times. Conclusion: The solid dispersion prepared PSD can increase the solubility, dissolution rate, and bioavailability.
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Objective: To identify the metabolites of Pulsatilla saponin D (PSD) in the intestinal microflora of rats in vitro. Methods: The sample of PSD incubated in intestinal microflora of rats in vitro was analyzed by a ultraperformance liquid chromatography/hybrid triple quadrupole linear ion trap mass spectrometry (UPLC-Q-trap-MS). The data were processed with Analyst 1.5 software, and the metabolites of PSD were confirmed by comparing the retention time and MS/MS spectra. The possible structures of metabolites were proposed by analyzing the proposed fragmentation pathway. Results: PSD (M0, parent) and seven metabolites were identified, including hederagenin 3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinosyl (M1), hederagenin 3-O-α-L-glucopyranosyl-(1→4)-α-L-arabinosyl (M2), hederagenin (M3), hydroxylation of PSD (M4), methylation of PSD (M5 and M6), and dehydrogenation of PSD (M7). Conclusion: PSD has a wide range of metabolic in the intestinal microflora of rats in vitro.
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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.
ABSTRACT
Objective: To investigate the intestinal absorption characteristics of Pulsatilla saponin D in rats. Methods: In situ single-pass intestinal perfusion model was used to inspect the absorption of Pulsatilla saponin D in the intestinal tract of rats. HPLC was used to determine the concentration of Pulsatilla saponin D in intestinal perfusion fluid samples. Absorption rate constant (Ka) and apparent permeability coefficient (Papp) were used as indexes to investigate the effects of absorption sites, drug concentration, different pH values, and P-glycoprotein (P-gp) inhibitor on Pulsatilla saponin D absorption. Results: There was the significant difference (Pileum>jejunum>duodenum. With the pH value increasing, the Ka and Papp values also increased and both of them had significant differences (P0.05); There was significant difference (P<0.05) in Ka and Papp values with and without P-gp inhibitor. Conclusion: Pulsatilla saponin D could be well absorbed in whole intestinal segments of rats, and the best intestinal absorption site is colon; The drug concentration in a certain range has no effect on Ka and Papp values, which preliminarily comfirms that the obsorption mechanism of Pulsatilla saponin D could be passive diffusion; Pulsatilla saponin D may be a substrate of P-gp and possess the saturation phenomenon of transporters.