ABSTRACT
OBJECTIVE To improve the quality standard of Yi medicine Gynura japonica， and to evaluate its quality. METHODS Using 15 batches of G. japonica from different producing areas as samples， the contents of water， total ash， acid- insoluble ash and water-soluble extract were determined according to the method stated in part Ⅳ of Chinese Pharmacopoeia （2020 edition）. The contents of total alkaloid （calculated by senecionine） was determined by UV spectrophotometry. The contents of senecionine and seneciphylline were determined by HPLC. Using above 7 indexes as evaluation indexes， cluster heat map analysis， principal component analysis （PCA） and entropy weight approximation ideal ranking （TOPSIS） were used to evaluate the quality of medicinal material comprehensively. RESULTS Among 15 batches of G. japonica， the moisture contents were 8.88%-12.60%， the total ash contents were 4.43%-11.02%， the acid-insoluble ash contents were 0.56%-3.45%， the water-soluble extract contents were 21.71%-53.91%， the total alkaloid contents （calculated by senecionine） were 0.15%-0.39%， and the contents of senecionine and seneciphylline were 0.01% -0.05% and 0.01%-0.06% respectively. According to the results of various indicators， it was preliminarily proposed that the water content in the sample of G. japonica should not exceed 13.00%， the total ash content should not exceed 11.50%， the acid-insoluble ash content should not exceed 3.70%， the water-soluble extract should not be less than 20.70%， the total alkaloid content should not be less than 0.15%， the contents of senecionine and seneciphylline should not be less than 0.01% both. The results of cluster heat map analysis showed that the 15 batches of samples could be divided into four categories； the results of PCA and TOPSIS showed that the samples with high-quality ranking were jsq-2， jsq-5， jsq-6 and jsq-10， and the samples with low-quality ranking were jsq-4， jsq-13 and jsq-14. CONCLUSIONS In this study， the quantitative analysis method of total alkaloids （calculated by senecionine）， senecionine and seneciphylline in G. japonica is established， and the limits of each index are preliminarily determined. Among 15 batches of samples， the qualities of medicinal material collected from Linza Village of Ganluo County of Liangshan Yi Autonomous Prefecture， Machangping Village of Luojishan Town of Puge County of Liangshan Yi Autonomous Prefecture and other places are better.
ABSTRACT
@#The purpose of this study was to explore the effects of seneciphylline on the proliferation and autophagy of cervical cancer HeLa and Caski cells and the possible mechanisms of autophagy. MTT assay was used to evaluate the effect of seneciphylline on the proliferation of cervical cancer cells. Immunofluorescence assay was applied to investigate the formation of autophagosomes in GFP-LC3/HeLa and GFP-LC3/Caski cells. The effect of seneciphylline on the expression of autophagy-related proteins was checked by Western blotting. In addition, fluorescence colocation assay was used to detect the fusion of autophagosomes and lysosomes. Human cervical cancer subcutaneous xenografts in nude mice were used to evaluate the effect of seneciphylline on the growth of the tumor in vivo. Results showed that HeLa cells proliferation was inhibited by seneciphylline in a dose- and time- dependent manner. Seneciphylline could induce formation of autophagosomes, increase the expression of LC3-II and decrease the expression of P62, suggesting that seneciphylline induced autophagy in HeLa and Caski cells. Compared with seneciphylline alone, seneciphylline combined with later-stage autophagy inhibitor chloroquine significantly increased the expression of LC3-II and P62. Moreover, and fluorescence colocation assay showed that autophagosomes induced by seneciphylline could colocate with lysosomes, indicating that seneciphylline could induce the complete autophagy flux. Compared with seneciphylline alone, seneciphylline combined with earlier-stage autophagy inhibitor 3MA significantly increased the expression of LC3-II and significantly decreased HeLa and Caski cells viability, suggesting that seneciphylline induced protective autophagy. Compared with seneciphylline alone, seneciphylline combined with MEK inhibitor significantly decreased the expression of P-ERK1/2 and formation of autophagosomes, suggesting that autophagy induced by seneciphylline activated MEK/ERK1/2 signal pathway. In addition, seneciphylline showed a significant inhibitory effect on growth of human cervical cancer cells subcutaneous xenografts.