ABSTRACT
ObjectiveTo compare the effects of total alkaloids， matrine， and sophoridine extracted from Sophora alopecuroides on the activity of pheochromocytoma cells （PC12 cells）. MethodThe effect of S. alopecuroides total alkaloids， matrine， and sophoridine at concentrations of 2， 1， 0.5， 0.25， 0.125， and 0.062 5 g·L-1 on the proliferation of PC12 cells was evaluated using the 3-（4，5-dimethylthiazol-2-yl）-2，5-diphenyl tetrazolium bromide （MTT） assay. The lactate dehydrogenase （LDH） leakage rate was measured by enzyme-linked immunosorbent assay （ELISA）. Flow cytometry was used to assess cell apoptosis rate， cell cycle distribution， and intracellular Ca2+ levels. Real-time quantitative polymerase chain reaction （Real-time PCR） was performed to determine the mRNA transcription levels of B-cell lymphoma-2 （Bcl-2） and Bcl-2-associated X protein （Bax）. Protein expression levels of apoptosis-related proteins Caspase-3， Caspase-8， Bcl-2， and Bax were detected by Western blot. ResultCompared to the control group， S. alopecuroides total alkaloids， matrine， and sophoridine inhibited the proliferation of PC12 cells， increased LDH leakage rate， enhanced intracellular Ca2+ fluorescence intensity， and induced cell apoptosis in concentration-dependent manner （P<0.05， P<0.01）. Among them， S. alopecuroides total alkaloids had the strongest inhibitory effect on cell proliferation and induction of apoptosis in PC12 cells （P<0.01）. After treatment with S. alopecuroides total alkaloids， matrine， and sophoridine， the cell cycle progression of PC12 cells was arrested at G1/G0 in the S. alopecuroides total alkaloids group， and at G1/S in the matrine and sophoridine groups. The expression levels of Bax mRNA were significantly increased （P<0.05， P<0.01）， while the expression levels of Bcl-2 mRNA were significantly decreased （P<0.05， P<0.01）. All treatments significantly downregulated the expression of the anti-apoptotic protein Bcl-2 （P<0.05， P<0.01） and upregulated the expression of the pro-apoptotic proteins Bax， Caspase-3， and Caspase-8 （P<0.05， P<0.01）， with the most significant protein expression changes observed in the S. alopecuroides total alkaloids group. ConclusionAmong the S. alopecuroides total alkaloids， matrine， and sophoridine， S. alopecuroides total alkaloids exhibit the strongest inhibitory effect on the activity of PC12 cells， and its mechanism of action may be related to the inhibition of anti-apoptotic protein expression， upregulation of pro-apoptotic protein expression， and activation of the mitochondrial Caspase-8 apoptotic pathway.
ABSTRACT
Aims: Purified saponin fraction was obtained from butanol extract of the aerial parts of Astragalus monspessulanus var. monspessulanus. The fraction was analyzed by HPLC and six saponins were determined. The saponin mixture was investigated for possible cytotoxicity on HepG2 cell line. Study Design: HPLC, in vitro study, biochemical analysis - lactate dehydrogenase leakage in medium Place and Duration of Study: Laboratory of Drug metabolism and drug toxicity, between September and November 2013. Methodology: The saponin mixture was studied on HepG2 cell line in 3 different concentrations –1, 2, 4 mg/ml for 24, 48 and 72 hours. The release of lactate dehydrogenase in the medium was measured spectrophotometrically. Results: It was found that the saponins have statistically significant cytotoxicity only in the highest concentration 4 mg/ml on 48 and 72 hours. At this concentration the sample increased the level of LDH (lactate dehydrogenase) with 37 % on 48 h and with 52 % on 72 h. Conclusion: Purified saponin fraction, isolated from Astragalus monspessulanus was found to be cytotoxic at the highest concentration 4 mg/ml on 48 and 72 hours in HepG2 cell line.