ABSTRACT
The aim of the present study was to investigate the cell proliferation-inhibiting and anti-rheumatic activities of chemical components from Aconitum soongoricum Stapf. Chemical constituents of Aconitum soongoricum Stapf. were separated and purified by silica gel and Sephadex LH-20 chromatography. Structure was identified by spectroscopic technique, and physical/chemical properties were analyzed. The following four compounds were identified: i) Aconitine, ii) songorine, iii) 16, 17-dihydro-12ß, 16ß-epoxynapelline, and iv) 12-epi-napelline. Cell Counting kit-8 assay was performed to assess cell proliferation. ELISA was conducted to determine the cytokine contents, and reverse transcription-quantitative polymerase chain reaction and Western blot analysis were performed to detect the mRNA and protein expression levels. Compared with the lipopolysaccharide (LPS) group, the contents of IL-6, IL-1ß, TNF-α and PGE-2 in the culture supernatant were significantly declined in the leflunomide + LPS and intervention+LPS groups, as well as the mRNA expression levels of HIF-1α, VEGFA and TLR4. Treatments with songorine, benzoylaconine and aconitine (at different concentrations) significantly inhibited the proliferation of HFLS-RA cells. Compared with the LPS group, the contents of PGE-2, IL-6, IL-1ß and TNF-α in the culture supernatant were significantly decreased in the intervention groups, and the mRNA expression levels of TLR4, HIF-1α and VEGFA in the cells in the intervention groups. Songorine, benzoylaconine and aconitine from Aconitum soongoricum Stapf. have anti-rheumatic activities in vitro, which may inhibit the proliferation of HFLS-RA cells, and the underlying mechanisms may be associated with inhibiting the inflammatory cytokine production and downregulating the expression levels of HIF-1α, VEGF and TLR4.
ABSTRACT
Objective:To screen anti-EGFR components from Aconitum soongaricum Stapf. and investigate their inhibitory effect on HEK293 cells and EGFR/HEK293 high expression cell lines. Methods:A two-dimensional online system (EGFR/CMC-HPLC-MS) was built to screen anti-EGFR active compounds from Aconitum soongaricum Stapf.;molecular docking assay was performed to determine the binding affinity of the components to EGFR;MTT was used to confirm the inhibition effect of the screened compounds on HEK293 cells and EGFR/HEK293 high expression cell lines.Results:The screening results showed that aconitine,12-epi-napellin and songorine were the active components acting on EGFR like gefitinib; molecular docking assay showed the targeting components acting on EGFR in the similar manner with gefitinib used as a positive control drug; MTT assay confirmed the screened components had inhibitory effects on HEK293 cells and EGFR/HEK293 high expression cell lines in a dose-dependent manner within the dose range of 0.4- 50.0 μmol·L-1. Conclusion:The screening results are apparently consistent with the pharmacological effect. The online EGFR/CMC-HPLC-MS method has a good application prospect in the rapid screening of the active compounds in the complex system of traditional Chinese medicines,and fully demonstrates such advantages of cell membrane chromatography as simple and rapid with high efficiency.
ABSTRACT
Objective To optimize extraction process of Yinhuai Jiedu Granula.Methods On the basis of single factor experiments, effects of soaking time, solvent dosage and extracting time on contents of chlorogenic acid and total flavonoids were investigated by response surface methodology. HPLC was used to determine contents of chlorogenie acid and total flavonoids with mobile phase of acetonitrile-0.1% phosphoric acid for gradient elution (0–20 min, 13%A; 20–30 min, 13%–18%A; 30–55 min, 18%–20%A; 55–65 min, 20%–23%A) and detection wavelengthes of 327 nm and 256 nm for chlorogenic acid and total flavonoids respectively. Results Optimal extraction process was as following: soaked 60 minutes and extracted twice with 12 times the amount of water per time of 130 minutes. Concentrations of chlorogenic acid, rutin and baicalin were 51.497, 68.872, 25.763 μg/g respectively. The average extract yield was 33.105%. Conclusion The optimized extraction process is feasible and stable,which can provide data support for the further research on Yinhuaijiedu granula.
