Analysis of Antidepressant Quality Markers of Bupleuri Radix Before and After Vinegar-processing Based on UPLC-Q-TOF-MS and Network Pharmacology / 中国实验方剂学杂志

ObjectiveTo analyze the antidepressant quality markers（Q-Marker） of Bupleuri Radix（BP） before and after vinegar-processing by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS）， multivariate statistical analysis and network pharmacology. MethodUPLC-Q-TOF-MS was used to analyze the chemical basis of raw and vinegar-processed products of BP， and principal component analysis（PCA） orthogonal partial least squares-discriminant analysis（OPLS-DA） were used to identify the differential components in BP that changed significantly before and after vinegar-processing， which were regarded as candidate quality markers（Q-Marker）. Then the disease-drug-component-target network related to antidepressant effect of BP was constructed by network pharmacology， and the antidepressant Q-Marker of raw and vinegar-processed products of BP was determined. Rats were randomly divided into blank group， model group， fluoxetine group（2.67 mg·kg-1） and total saponin group（0.72 mg·kg-1）， except the blank group， rats in the other groups were subjected to chronic unpredictable mild stress（CUMS）. Three weeks after the start of modeling， rats in each administration group were given the corresponding dose of drugs once a day for 4 weeks， and rats in the blank and model groups were given normal saline with dose of 10 mL·kg-1. At 1 day before modeling， 21 days and 28 days after administration， body mass weighing， sucrose preference test and open field test were performed on each group . After 28 days of administration， real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was used to detect the mRNA expression levels of phosphatidylinositol 3-kinase（PI3K）， protein kinase B（Akt）， mammalian target of rapamycin（mTOR）， glycogen synthase kinase-3β（GSK-3β）， forkhead box transcription factor O3a（FoxO3a） and β-catenin in hippocampal tissues of rats in each group， while protein expression levels of PI3K， Akt， mTOR and FoxO3a in hippocampal tissues of rats in each group were detected by Western blot. ResultThere were 19 components in BP showed significant changes before and after vinegar-processing， and 9 components such as saikosaponin A， saikosaponin B1， saikosaponin B2， saikosaponin C and saikosaponin D were identified as potential Q-Marker through S-plot differential marker screening. Combined with the disease-drug-component-target network， saikosaponin A， saikosaponin B1， saikosaponin B2 and saikosaponin D were identified as antidepressant Q-Marker of raw and vinegar-processed products of BP. According to the results of pharmacodynamic tests， after 28 d of administration， compared with the blank group， the body mass， sucrose preference index and open field total score of rats in model group， fluoxetine group and total saponin group decreased significantly（P<0.01）. Compared with the model group， the body mass， sucrose preference index and open field total score in total saponin group increased significantly（P<0.01）. Compared with the blank group， mRNA expression levels of PI3K， Akt， mTOR and β-catenin in hippocampus of rats in the model group decreased significantly（P<0.05）， while mRNA expression levels of GSK-3β and FoxO3a increased significantly（P<0.05）. Compared with the model group， mRNA expression levels of PI3K， Akt， mTOR and β-catenin in hippocampus of rats in the total saponin group were increased significantly（P<0.05）， while mRNA expression levels of GSK-3β and FoxO3a decreased significantly（P<0.05）. Compared with the blank group， the protein expression levels of Akt and mTOR in hippocampus of the model group decreased significantly（P<0.01）， while the protein expression levels of PI3K and FoxO3a increased significantly（P<0.01）. Compared with the model group， the expression level of Akt in hippocampus of the total saponin group increased significantly（P<0.01）， the mTOR expression level was increased but not statistically significant， while the protein expression levels of PI3K and FoxO3a decreased significantly（P<0.01）. ConclusionThe chemical constituents of BP changed greatly after vinegar-processing， and the antidepressant Q-Marker of raw and vinegar-processed products of BP was determined by chemical basis， pharmacodynamics， network pharmacology and signaling pathway， which provided a reference for further research on quality control， pharmacodynamic substance basis and processing mechanism of BP.

Analysis of Quality Markers Related to Expelling Phlegm and Arresting Coughing of Trichosanthis Fructus Based Pharmacokinetics and Multivariate Statistical Methods / 中国实验方剂学杂志

Objective:Based on pharmacokinetics， the antitussive and expectorant related quality markers （Q-marker） of Trichosanthis Fructus were screened from diosmetin-7-<italic>O</italic>-glucopyranoside， diosmetin， apigenin， vanillic acid and cinnamic acid， and the candidate Q-marker was evaluated by multivariate statistical method. Method:Six healthy rats were randomly selected and the 70% ethanol extract of Trichosanthis Fructus （dose of 20 g·kg^-1） was given by intragastric administration. Blood was collected from the orbital vein at different time points， and the plasma concentrations of 5 components （diosmetin-7-<italic>O</italic>-glucopyranoside， diosmetin， apigenin， vanillic acid and cinnamic acid） from Trichosanthis Fructus were detected simultaneously by high performance liquid chromatography-triple quadrupole tandem mass spectrometry （HPLC-QqQ-MS/MS）. The main detection conditions were as following：mobile phase of 0.2% formic acid aqueous solution （A）-acetonitrile （B） for gradient elution （0-4 min， 6%-23%B； 4-5 min， 23%-59.5%B； 5-10 min， 59.5%-60%B）， flow rate of 0.5 mL·min^-1， the detection wavelength at 254 nm， electrospray ionization （ESI）， positive ion mode detection， multiple reaction monitoring （MRM） mode scanning， scanning range of <italic>m</italic>/<italic>z</italic> 50-1 500. Diosmetin-7-<italic>O</italic>-glucopyranoside， diosmetin， apigenin and vanillic acid with clear pharmacokinetic behaviors were selected as candidate Q-marker about antitussive and expectorant of Trichosanthis Fructus. The contents of these components in 9 batches of medicinal materials were determined and the main detection conditions were the same as the pharmacokinetic study. SPSS 21.0 was used for cluster analysis and principal component analysis （PAC） based on the results of determination. Result:The pharmacokinetic results showed that the area under concentration-time curve （AUC_0-<italic>_t</italic>） of 4 components （diosmetin-7-<italic>O</italic>-glucopyranoside， diosmetin， apigenin and vanillic acid） were （111.28±9.94）， （27.08±2.76）， （1 376.12±101.86）， （631.32±64.72） μg·h·L^-1， respectively. The 9 batches of Trichosanthis Fructus samples were clustered into 3 groups by systematic cluster analysis. The clustering results were related to the variety of Trichosanthis Fructus and also affected by the origin. The PCA results showed that the comprehensive scores of Gaotang Trichosanthis Fructus， Shanxi Trichosanthis Fructus， Hebei Ben Trichosanthis Fructus were 1.919， 1.356 and 0.299， respectively， ranking in the top 3 among all samples. The comprehensive scores of Nongkeyuan No. 1， Hebei Trichosanthis Fructus and Nongkeyuan No. 2 were -0.804， -1.085， -1.120， respectively， which were in the last 3 positions among all samples. Conclusion:The pharmacokinetic characteristics and quality evaluation of diosmetin-7-<italic>O</italic>-glucopyranoside， diosmetin， apigenin and vanillic acid meet the requirements about antitussive and expectorant related Q-marker of Trichosanthis Fructus.