ABSTRACT
OBJECTIVE： To study the effects of benzoyl aconitine （BAC） on autophagy and apoptosis of human lung cancer A549 cells， and to investigate its mechanism in anti-non-small cell lung cancer. METHODS： A549 cells were treated with different doses of BAC （10， 50， 100， 200， 400 μmol/L）， and then cell morphology was obtained； the proliferation inhibition rate of the cell was determined by CCK-8 assay. The cells were divided into control group （without drug）， BAC low-dose and high-dose groups （200， 400 μmol/L）. After treated with relevant drugs， the apoptosis rate of cells was determined by flow cytometry. The gene and protein expression of apoptosis-related factors Bcl-2， Bax， Caspase-3 as well as autophagy-related factors Beclin1， LC3， P62 were determined by RT-PCR and Western blotting assay. RESULTS： After treated with different doses of BAC， the cells were shrunken and sparsely arranged； inhibitory rate of cell proliferation was increased significantly in BAC 100， 200， 400 μmol/L groups （P＜0.05 or P＜0.01）. Results of flow cytometry showed that the apoptotic rates of cells were increased to different extents in BAC low-dose and high-dose groups after treated for 24 and 48 h， in a concentration and time-dependent manner. Compared with control group， mRNA and protein expression of Bcl-2 and P62 were decreased to different extents in BAC groups； mRNA expression of Bax， Caspase-3， Beclin1 and LC3 as well as protein expression of Bax， Active caspase-3， P62， Beclin1， LC3Ⅱ/Ⅰ were increased to different extent； there was statistical significance in mRNA expression of Caspase-3， and protein expression of Bcl-2， Active Caspase-3， Beclin1， LC3Ⅱ/Ⅰ and P62 in BAC low-dose group as well as all target mRNA and protein expression in BAC high-dose group （P＜0.05 or P＜0.01）， in dose-dependent manner. CONCLUSIONS： BAC can inhibit the proliferation and promote the apoptosis of A549 cells， promote Beclin1， LC3（LC3Ⅱ/Ⅰ），Bax and Caspase-3 （Active Caspase-3） gene and their protein expression， but inhibit P62 and Bcl-2 gene and their protein expression. The mechanism may be related to BAC inducing apoptosis by promoting excessive autophagy of cells.
ABSTRACT
Objective: An HPLC-MS/MS method was established for determination of 10 kinds of components in Xiaojin Pills such as ligustilide, protocatechuic acid, inosine, oleanolic acid, benzoyl neatidine, benzoyl aconitrate, benzoyl aconitine, neaconitine, hypaconitine, aconitine, and combined with cluster analysis, principal component analysis (PCA) and other chemical metrology methods to assess the quality consistency of different batches of Xiaojin Pills from different manufacturers. Methods: The HPLC-MS/MS method was established for the simultaneous determination of 10 active ingredients in Xiaojin Pills. The Agilent Technologies Zorbax Eclipse XDB C18 column (150 mm × 4.6 mm, 5 μm) was used; 0.1% formic acid aqueous solution-acetonitrile as mobile phase; The mass spectrum was scanned by ESI+ multiple reaction monitoring (MRM) mode. And the content analysis was carried out by cluster analysis and principal component analysis, and the difference of the quality of commercially available Xiaojin Pills was evaluated comprehensively. Results: The methodological validation results showed that the linear range of 10 compounds was good (R2 > 0.990 0). The limit of quantification was 0.01-4.49 ng/mL, and the average recovery was 94.82%-104.33%. The results showed that the quality of Xiaojin Pills in a factory was different from others. Benzoyl aconitine, benzoyl aconitine, aconitine and inosine were the main components of classification. The load chart after PCA reduced dimension processing showed that the content of benzoyl neatidine, benzoylthioate, aconitine and inosine were the most distant from the origin, and the difference in content was the main cause of the difference in quality consistency. Conclusion: Considering the poor quality consistency of Xiaojin Pills, the quality control of Aconiti Kusnezoffii Radix and Lonicerae Radix should be strengthened in the production of Xiaojin Pills.
ABSTRACT
Objective To study the effect of Radix Astragali and Aconiti Lateralis Radix Preparata on the intestinal absorption of the three kinds of monoester-type alkaloids (benzoylthioate, benzoyl neostearine, and benzoyl aconitine) and three kinds of diester-type alkaloids (hypaconitine, neaconitine, and aconitine) in Aconiti Lateralis Radix Preparata. Methods The rat duodenum, the jejunum, and the ileum were selected to study the intestinal segment. The apparent permeability coefficient (Papp) was used to evaluate the effect of Radix Astragali on the Papp of six kinds of alkaloids in Aconiti Lateralis Radix Preparata. Results When Aconiti Lateralis Radix Preparata and Radix Astragali was 3:1, Radix Astragali can significantly reduce the dipeptide alkaloid Papp in the duodenum and ileum and reduce the monoester-type alkaloid Papp in three kinds of intestinal segments; When Aconiti Lateralis Radix Preparata and Radix Astragali was 1:1, except in the ileum, Radix Astragali can significantly reduce the Papp of the diester-type alkaloid; When Aconiti Lateralis Radix Preparata and Radix Astragali was 1:3, Radix Astragali can significantly reduce the Papp of the monoester-type alkaloid (except the parenteral) and significantly reduced the Papp of the three diester-type alkaloids. Conclusion Radix Astragali can inhibit the absorption of aconite alkaloids, and its inhibition effect is different due to different compatibility ratios, types of alkaloids and intestinal segments.