Simultaneous determination of seven effective components in Shenkui Tongmai granules based on UHPLC-MS/MS.

OBJECTIVES: The objective of the study was to establish the quality standard of the formulation of Shenkui Tongmai granules (SKTM) from the perspective of safety and effectiveness. METHODS: A sensitive and specific method to simultaneously detect seven effective components by ultra-high performance liquid chromatography-tandem-mass spectrometry (UHPLC-MS/MS) in SKTM, including calycosin 7-O-ß-d-glucopyranoside, icariin, sodium danshensu, hyperoside, astragaloside IV, hesperidin, and salvianolic acid, was developed and validated. A Kromasil 100-3.5 C18 column with a mobile phase of 6.5 mmol/l ammonium acetate in acetonitrile was used to separate these above-listed components. Gradient programming was used with a flow rate of 0.2 ml/min, and the components were achieved in 13 min. Multiple reaction monitoring (MRM) in positive/negative mode was applied for the MS/MS detection. KEY FINDINGS: The analytical method was satisfactorily validated for linearity, accuracy and precision, repeatability and stability. The developed UHPLC-MS/MS method had high repeatability and accuracy and it was in a good linear relationship within their respective ranges (r = 0.9999) with the RSD value of the sample recovery of less than 5%. CONCLUSIONS: The current method established here is suitable for use in determining seven effective components in SKTM simultaneously, which may provide a new reliable method for overall quality control of SKTM.

HPLC specific chromatogram spectrum-effect relationship for Shuanghuanglian on MDCK cell injury induced by influenza A virus (H1N1) / 中国中药杂志

To establish HPLC specific chromatogram and its correlation with the protection effect of Shuanghuanglian on MDCK (Madin-Darby canine kidney) cell injury induced by influenza A virus( H1N1). Nine recipes of Shuanghuanglian based on the official prescription were prepared according to orthogonal test for HPLC analysis and MDCK cells protection experiment separately (cytopathic effect (CPE) method was used for observing the virus infectivity and MTT staining results were used as the determining indexes for drug concentration selection and analyzing cell viability). The results suggested that all the other Shuang-Huang-Lian recipes except recipe1 demonstrate protecting effect on MDCK cell injury induced by influenza A virus (P < 0.01, P < 0.001). Stepwise regression analysis was used for analyzing the relationships between HPLC fingerprint and the protecting effect of Shuanghuanglian on influenza A virus induced MDCK cell injury. Peak 2, 3, 6, 8 and 12 were found to be strongly related with anti-influenza A virus efficacy. Stepwise regression analysis of recipes data and efficacy data showed that Lonicerae Japonicae Flos and Forsythiae Fructus were positively associated with the protecting effect of cells injury. From HPLC fingerprints, we found that peak 2, 3, 12 were from Lonicerae Japonicae Flos and peak 6, 8 were from Forsythiae Fructus. Four peaks were identified through comparing the retention time between the standard and Shuanghuanglian recipes, and they were chlorogenicacid, cryptochlorogenic acid, forsythoside B and 3,4-dicaffeoylquinic acid respectively. Caffeic acid derivatives in Lonicerae Japonicae Flos and Forsythiae Fructus were found to be greatly correlated with anti-influenza A virus efficacy and maybe the substance basis of Shuanghuanglian.

Effects of guanxinping tablet containing serum on H2O2-induced apoptosis and NF-kappaB expressions in vascular endothelial cells / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effects of Guanxinping Tablet (GT) containing serum on H2O2-induced apoptosis and the nuclear factor kappa B (NF-kappaB) expression in vascular endothelial cells (VECs).</p><p><b>METHODS</b>Rabbits were randomly divided into the normal control group (treated with normal saline, 10 mL/kg), the verapamil group (0. 02 g/kg, 10 mL/kg), the small dose GT group (2; 8 g/kg, 10 mL/kg), the middle dose GT group (5.6 g/kg, 10 mL/kg), and the large dose GT group (11.2 g/kg, 10 mL/kg), 3 in each group. The medication was given to rabbits by gastrogavage for 3 successive days. The gastrogavage was performed twice on the last day with an interval of 2 h. One h after the last medication the peripheral blood was sampled from the vein of the ear edge. The blood was put for 1 h and centrifuged at 2 500 r/min for 30 min. The serum was extracted and deactivated at 56 degrees C for 30 min to prepare the drug containing serum. The apoptosis injury model was established using 100 micromol/L H2O2 induced VECs in the log phase growth. After modeling they were divided into 6 groups, 5 samples in each group, i. e., the normal group (10% vehicle serum culture solution), the model group (10% vehicle serum culture solution +100 micromol/L H2O2), the verapamil group (10% verapamil serum culture solution +100 micromol/L H2O2), the low dose GT group (10% low dose GT culture solution +100 micromol/L H2O2), the middle dose GT group (10% middle dose GT culture solution + 100 micromol/L H2O2), and the high dose GT group (10% high dose GT culture solution + 100 micromol/L H2O2). THE VEC apoptotic rate was detected using flow cytometry. The protein expression of NF-kappaB was detected using Western blot.</p><p><b>RESULTS</b>The VEC apoptosis rate (9.00% +/- 1.18%) and the protein expression of NF-kappaB (0.39% +/- 0.06%) increased more in the model group than in the normal control group (P<0.01). Compared with the model group, the VEC apoptosis rate of the verapamil group (6.00% +/- 0.18%), the large dose GT group (5.30% +/- 0.08%), and the middle dose GT group (6.83% +/- 0.51%) were obviously lower. The expression of NF-kappaB of each treatment group significantly decreased (the verapamil group: 0.28% +/- 0.03%; the small dose GT group: 0.33% +/- 0.03%; the middle dose GT group: 0.30% +/- 0.03%; the large dose GT group: 0.28% +/- 0.04%, P<0.01, P<0.05).</p><p><b>CONCLUSIONS</b>GT could fight against H2O2-induced VEC cell apoptosis. Its mechanism might be correlated with regulating the expression of NF-kappaB protein.</p>