Study On Pharmacodynamics Substance Foundation Of Huangqigegen Decoction

Objective: To screen the pharmacodynamics substances which modulate of the development diabetic nephropathy(DN) and establish a “Pharmacodynamics-HPLC Fingerprint” of Huangqigegen decoction.Methods: The Transwell co-culture system with Macrophage and glomerular mesangial cellswas used to observe the effects on AGEs-induced inflammation in mouse mesangial cells.The optimal analytic conditions of HPLC Fingerprint Chromatography of Huangqigegen decoction were investigated including wave detection wavelength, the ingredient of mobile phase, chromatographic column and elute mode. According with the technical demand of fingerprint about injection of TCM, Chromatographic Fingerprint Analysis of TCM(Version 2004A) was used for similarity evaluation we formulated the fingerprint’s examination standard of the injection, as the methodological study was examined for precision, exclusivity and durability. Combination of the model of cell membrane chromatography(CMC) and the HPLC fingerprint to optimize experiment conditions, such as sample pre-treatment, washing times of unreacted components, etc. The pharmacological effect was proved by anti-inflammatory mesangial cells model induced by AGEs.Components were qualitative analysis and identified by HPLC Fingerprint-ESI MSn including unreacted, membrane reattached and intracellular screened by CMC,Chemical structure of pharmacodynamics substances was confirming by mass spectra data.Results: Data of high, middle, low dosage groups showed significantly anti-inflammatory activity on mesangial cells induced by AGEs with satisfied dose-effect relationship. The HPLC fingerprint of Huangqigegen decoction wereexclusive, reproducible, precise, and reliable. The similarities of 10 samples were greater than 0.97 which meet the requirements. Also can be used for the quality control of Huangqigegen decoction. 10 times of the cell disruption sample showed anti-inflammatory activity in pharmacodynamics study. The anti-inflammation activity substances in Huangqigegen decoction could be screened by CMC. Puerarin was the component binding to cell membrane were identified during the matching search of HPLC Fingerprint-MS, as Puerarin, 3’-Methoxy Puerarin, puerarin-xyloside daidzein, Ononin, daidzin transduced into cells.Conclusion: The reasrch achieved our goal to preliminarily clarify the anti-inflammation activity substances in Huangqigegen decoction by CMC and HPLC fingerprint-MS Hyphenated Technique, also laid a foundation for the development of new drug and quality control of Huangqigegen decoction.