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OBJECTIVE To study the antifungal activity of Huangqin decoction （HQD） against Trichophyton mentagrophytes and explore its mechanism. METHODS Minimal inhibitory concentration （MIC）， minimal fungicidal concentration （MFC）， mycelial length， spore germination rate， biomass and mycelium ultrastructure observation were performed to evaluate the antifungal activity of HQD against T. mentagrophytes. The effects of HQD on the cell wall of T. mentagrophytes were detected through sorbitol protection experiment. By measuring the content of ergosterol and the activities of squalene epoxide （SE） and lanosterol 14α-demethylase （CYP51）， the activity of HQD on the cell membrane of T. mentagrophytes was investigated. The effects of HQD on T. mentagrophytes mitochondria were investigated by determining the activities of malate dehydrogenase （MDH）， succinate dehydrogenase （SDH）， and ATPases （including sodium potassium ATPase， calcium magnesium ATPase， and total ATPase）. RESULTS HQD exhibited significant antifungal activity against T. mentagrophytes with MIC of 3.13 mg/mL and MFC of 25 mg/mL. After intervention with HQD， the mycelial length of T. mentagrophytes was significantly shortened （P＜0.05）； spore germination rate， biomass， the content of ergosterol in the cell membrane， the activities of SE and CYP51 in the cell membrane and MDH， SDH and ATPase in mitochondria were all decreased significantly （P＜0.05）； cell structure had been ；damaged to a certain extent， but the integrity of the cell wall had not been affected. CONCLUSIONS HQD shows significant antifungal activity against T. mentagrophytes， the mechanism of which may be associated with reducing the 0791- content of ergosterol in the cell membrane and the activities of SE， CYP51， and mitochondria-related enzymes.
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OBJECTIVE To establish the fingerprint of Huangqin decoction （HQD）， to separate the phase states and screen the active phase states of antidermatophytic activity so as to study the spectrum-effect relationship. METHODS HPLC method was adopted using baicalin as reference， the fingerprints of 10 batches of HQD were drawn and the similarity evaluation was carried out using the Similarity Evaluation System of Chromatographic Fingerprint of TCM （2012 edition） to determine the common peak； the phase states of HQD were separated and characterized by high-speed centrifugation and membrane dialysis. The minimum inhibitory concentrations （MIC） of HQD and its different phase states against Trichophyton mentagrophytes were determined simultaneously. Using the peak area of 37 common peaks as independent variable， MIC as dependent variable， Pearson correlation analysis was performed by using SPSS 21.0 software. RESULTS A total of 37 common peaks were obtained in HPLC fingerprints of 10 batches of HQD， with the similarity higher than 0.99. Ten components were identified， such as albiflorin， paeoniflorin， liquiritin apioside， baicalin， melaleuca glycoside A， wogonoside， baicalein， glycyrrhizic acid， wogonin and oroxylin A. HQD was split into 3 phase states， such as precipitation phase （HQD-P）， solution phase （HQD-S） and nano phase （HQD-N）. The morphology of HQD-P was irregular granular， and the average particle size was 4.670-91.522 μm. The morphology of HQD-S was uniform flakes， and no particle size was detected. HQD-N was spherical in shape and the particle size was （129.0±12.9） nm. MIC values of each phase state of HQD against T. mentagrophytes in different phase states were HQD-N （4.64 mg/mL） ＜HQD （5.85 mg/mL） ＜HQD-P （7.37 mg/mL） ＜HQD-S （12.89 mg/mL） at the same dosage. Pearson correlation analysis showed that the peak area of 25 of the 37 common peaks （including identified components） was significantly negatively correlated with MIC （absolute values of correlation coefficient＞0.95 and P＜0.05）. CONCLUSIONS The chemical composition of 10 batches of HQD is consistent； HQD-N is the active phase state of HQD. Ten components such as paeoniflorin， liquiritin apioside and baicalin may be the main active components of HQD. The antidermatophytic effect of HQD is closely related to its component content and physical phase state.
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Objective To establish the fingerprint spectrum and assay three active components (hesperidin, salvianolic acid B and chrysophanol) in Shenshuaining granule by HPLC method. Methods The chromatographic separation was achieved on SunFireTM C18 column with acetonitrile-0.1% formic acid aqueous solution as mobile phase. Gradient elution program was applied with flow rate of 1.0 ml/min, detection wavelength at 254 nm and the column temperature at 25 ℃. The fingerprint spectrum was established and three active components in Shenshuaining granule were assayed. Results There were 22 common peaks on the fingerprints after analyzing chromatograms from 10 batches of Shenshuaining granules. Good fingerprint similarities (≥0.9) between different batches and the control chromatogram were found. This method has great repeatability, stability and precision, which meets all the assay requirements. Conclusion A simple and reliable HPLC method was developed, which is suitable for the fingerprint establishment of Shenshuaining granules. It provides a method for the quality control of Shenshuaining granules.