Analysis of chemical constituents in ethyl acetate extract of Taxilli Herba by UPLC-Q-Exactive-MS and screening of potential xanthine oxidase inhibitors / 中国中药杂志

The present study analyzed and identified the chemical constituents from ethyl acetate(EA) extract of Taxilli Herba with UPLC-Q-Exactive-MS and screened active xanthine oxidase(XO) inhibitors with HPLC. The analysis was performed on an Hypersil GOLD C_(18) reversed-phase column(2.1 mm×50 mm, 1.9 μm), with the mobile phase of water containing 1% formic acid(A) and methanol(B) under gradient elution, the flow rate of 0.3 mL·min~(-1), and the injection volume of 5 μL. ESI source was used for MS and the compounds were collected in positive and negative ion modes. Xcalibur 4.1 was used to analyze the retention time, accurate relative molecular weight, and fragmentation of the compounds. The inhibitory activity of some known compounds on XO was screened by HPLC. Thirty chemical constituents were identified, including phenolic acids and flavonoids by experimental data combined with information of standards, data reported previously, and databases, such as MzCloud and ChemSpider. The activities of 10 chemical components were screened. Gallic acid and naringenin chalcone had strong inhibitory activities on XO with IC_(50) of 57 μg·mL~(-1) and 108 μg·mL~(-1). UPLC-Q-Exactive-MS allows the accurate, rapid, and comprehensive identification of main chemical constituents from Taxilli Herba. Gallic acid and naringenin chalcone may be the active components of XO inhibitors.

Effect of Stemona tuberosa Alkaloids on Apoptosis of SMMC-7721 Cells and Expression of Bcl-2， Bax， and Cleaved Caspase-3 / 中国实验方剂学杂志

Objective:To investigate the effect of<italic> Stemona tuberosa</italic> alkaloids on the apoptosis of human hepatoma SMMC-7721 cells and the expression of apoptosis-related proteins including B lymphocytoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， and cleaved cysteinyl aspartate-specific protease-3 （cleaved Caspase-3）. Method:SMMC-7721 cells were routinely cultured， passaged， and treated with various concentrations （50， 75， 112， 167， and 250 mg·L^-1） of <italic>S. tuberosa </italic>alkaloids， while those in the blank control group were only treated with 10% fetal bovine serum. The cell proliferation was determined by tetrazolium bromide （MTT） colorimetry and colony assay and the cell apoptosis by Hoechst 33258 staining. The protein expression levels of Bcl-2， Bax， and cleaved Caspase-3 were detected by Western blot. Result:<italic>S. tuberosa</italic> alkaloids inhibited the proliferation of SMMC-7721 cells， and the inhibition rate was significantly increased in comparison with that in the blank control group （<italic>P</italic><0.01）， with the half maximal inhibitory concentrations （IC₅₀） at 24 h， 48 h， and 72 h being （173.36±8.75）， （112.14±16.50）， and （96.41±2.60）mg·L^-1， respectively. The cell colony-inhibitory activity was significantly increased in a dose-dependent manner （<italic>P</italic><0.01）. Compared with the blank control group， <italic>S. tuberosa</italic> alkaloids promoted the apoptosis of SMMC-7721 cells， manifested as increased number of apoptotic cells and elevated apoptotic rate （<italic>P</italic><0.01）. The typical morphological changes such as brightly blue-fluorescent condensed nuclei， cytoplasmic shrinking， and karyopyknosis were found under the upright fluorescence microscope. As revealed by comparison with the blank control group， the expression of Bcl-2 was significantly down-regulated （<italic>P</italic><0.01）， while the protein expression levels of pro-apoptotic protein Bax and cleaved Caspase-3 in the 75， 112， 167， and 250 mg·L^-1 <italic>S. tuberosa</italic> alkaloids groups were significantly up-regulated （<italic>P</italic><0.01）. Conclusion:<italic>S. tuberosa </italic>alkaloids inhibit the proliferation of SMMC-7721 cells and promote their apoptosis possibly by inhibiting Bcl-2 protein expression and promoting Bax and cleaved Caspase-3 protein expression.