Rapid identification of components in Wuzhuyu Decoction using UHPLC Q-Exactive Orbitrap MS~n and molecular network technology / 中国中药杂志

Wuzhuyu Decoction, the classical formula recorded in the Treatise on Febrile Diseases(Shang Han Lun), has been included in the Catalogue of Ancient Classic Prescriptions(the First Batch). Consisting of Euodiae Fructus, Ginseng Radix et Rhizoma, Zingiberis Rhizoma Recens, and Jujubae Fructus, it is effective in warming the middle, tonifying deficiency, dispelling cold, and descending adverse Qi, and is widely applied clinically with remarkable efficacies. For a classical formula, the chemical composition is the material basis and an important premise for quantity value transfer. This study aimed to establish a rapid identification method of chemical components in Wuzhuyu Decoction by high-resolution mass spectrometry(HR-MS) and molecular network. AQUITY UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 μm) was used for sample separation, and acetonitrile-0.1% formic acid in water was used as mobile phases for gradient elution. Q-Exactive Orbitrap MS data were collected in positive and negative ion modes, and GNPS molecular network was plotted according to the similarity of MS/MS fragmentation modes. Cytoscape 3.6.1 was used to screen molecular clusters with similar structures. Finally, the chemical components of Wuzhuyu Decoction were rapidly identified according to the controls, as well as the information of retention time, accurate relative molecular weight of HR-MS, and MS/MS multistage fragments. A total of 105 chemical components were identified in Wuzhuyu Decoction. This study can provide data for the follow-up quality control, standard substance research, and pharmacodynamic material research on Wuzhuyu Decoction, as well as references for the rapid qualitative analysis of the chemical components of Chinese medicine.

Effect of Wuzhuyu Decoction on serum metabolic spectrum in rats with deficiency cold and vomiting based on UPLC-MS / 中草药

Objective: To observe the effect of Wuzhuyu Decoction on the serum metabolic profile of the model rats with deficiency cold, and explore its possible mechanism for treating deficiency cold and vomiting syndrome. Methods: A rat model of deficiency cold and vomiting was prepared by a composite method (Rhei Radix et Rhizoma + cisplatin). The high-throughput metabolome data of the serum of rats with deficiency-cold and vomiting was obtained by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS), and multivariate analysis combinated with database retrieval was used to analyzed the endogenous metabolites in rat serum from different groups and discover differential metabolites. Besides, metabolites were analyzed by Metabo Analyst software for possible metabolic pathways. Results: The serum metabolic spectrum of control group and model group were completely separated. The Wuzhuyu Decoction group was close to the control group, indicating that the horizontal model of endogenous material changes was successfully replicated, and Wuzhuyu Decoction could interfere with the symptoms of deficiency cold and vomiting in rats, suggesting that the rat body had a tendency to return to normal state. In total, modular analysis of 35 serum differential metabolites, and deficiency cold and vomiting syndrome was related to abnormal lipid metabolism and amino acid metabolism pathways and Wuzhuyu Decoction may have a regulatory effect on its pathways; Metabolites revealed close relationship among seven modules; Pyroglutamic acid, arachidonic acid, serotonin, isocitric acid, triglyceride and other biological targets can thus be used as biomarkers of deficiency cold and vomiting. Conclusion: Wuzhuyu Decoction can improve the physiological characteristics of the model of deficiency-cold and vomiting. The mechanism may be related to the regulation of abnormal amino acid metabolism, energy metabolism, and lipid metabolism in rats with deficiency-cold vomiting.

Effect of Wuzhuyu Decoction on urine metabolic spectrum in rats with deficiency cold and vomit based on UHPLC-MS / 中草药

Objective: To observe the changes of urine metabolic profile of Wuzhuyu Decoction in rats with deficiency cold and vomit, and explore its possible mechanism of treatment of deficiency cold and vomit syndrome. Methods: A rat model of deficiency cold and vomit was prepared by a composite method (rhucax + cisplatin). Rats were randomly divided into control group, model group, and Wuzhuyu Decoction group. UPLC-MS/MS combined with principal component analysis and partial least squares analysis were used to analyze urine data and identify potential biomarkers. Diversified ROC curves were used to validate differential metabolites; Pathway Analysis database was used for topological analysis of differential metabolites; R and Cytoscape were used for correlation analysis and modular analysis of metabolites. Results: The urine metabolic spectrum of control group and model group were completely separated. Wuzhuyu Decoction group was close to the control group, indicating that the model was successfully replicated; And Wuzhuyu Decoction can interfere with the symptoms of deficiency cold and vomit in rats, suggesting that the rat body had a tendency to return to normal state. Through modular analysis of 34 urine differential metabolites, the deficiency cold and vomit treatment was revealed to affect biosynthesis of valine, leucine and isoleucine, phenylalanine, tyrosine and tryptophan biosynthesis, arginine and proline metabolism, tryptophan metabolism, tyrosine metabolism, arachidonic acid metabolism, citric acid cycle (TCA cycle) pathways. Modularity analysis revealed that there was close relationship between 10 modules; Alanine, leucine, tyrosine, tryptophan, isoleucine, succinic acid, alanine, fumaric acid, malic acid, isocitrate, and other biological targets can thus be used as markers of deficiency cold and vomit. Conclusion: Wuzhuyu Decoction can improve the physiological characteristics of the model of deficiency cold and vomit. The mechanism may be related to the regulation of amino acid metabolism, energy metabolism and lipid metabolism in rats.

Difference in intestinal absorption of Wuzhuyu decoction between normal and migraine model rats / 中国中药杂志

To compare the intestinal absorption of Wuzhuyu decoction(WZYD) between normal rats and migraine model rats, and investigate the optimized WZYD from aspect of absorption. The rat single pass intestinal perfusion test(SPIP) was adopted for optimized sample and un-optimized sample in normal and migraine model rats induced by nitroglycerin and reserpine. The contents of 8 ingredients were determined by high performance liquid chromatography(HPLC), and 4 absorption parameters for each ingredient were calculated and compared: unit area absorption(Mper area), absorption rate constant(Ka), apparent coefficient(Papp) and relative absorption rate(RA). The results showed that there was a great difference between normal rats and model rats in the intestinal absorption of the same WZYD. As compared with normal rats, the absorption parameters of most ingredients in optimized sample were increased in migraine model rats induced by nitroglycerin; Similar phenomena were also found in migraine model rats induced by reserpine. However, the absorption parameters of most ingredients were decreased in un-optimized sample. Therefore, pathological model rats shall be used for effective ingredient recognition based on the correlation between intestinal absorption spectra and pharmacological effects. As compared with the un-optimized samples, the absorption of effective ingredients was faster, easier and more adequate in the optimized samples, revealing their mechanism on better efficacy from the aspect of absorption.

Efficacy of Modified Wuzhuyu Decoction Granule ( ) for Migraine Patients with Cold and Stasis Obstructing Meridian Syndrome: A Randomized, Double-Blind, Placebo-Controlled Trial / 中国结合医学杂志

<p><b>OBJECTIVE</b>To study the efficacy of modified Wuzhuyu Decoction Granule (, MWDG) in the treatment of migraine patients with cold and stasis obstructing meridian syndrome.</p><p><b>METHODS</b>This study was a randomized, double-blind, placebo-controlled trial. A total of 78 migraine patients with cold and stasis obstructing meridian syndrome were recruited and randomly assigned by a ratio of 2:1 into a treatment group (51 cases) and a placebo group (27 cases). Patients in the treatment group were treated with MWDG while placebo granules were applied in the control group. The treatment course lasted for 12 weeks with a follow-up of 4 weeks. The primary outcome measures included frequency and days of migraine attacks and the secondary outcome measures were analgesics consumption and visual analogue scale (VAS) scores. All outcome assessments were conducted respectively at baseline, the 4th, 8th and 12th week, and the end of follow-up.</p><p><b>RESULTS</b>In the treatment group, significant decrease in frequency of migraine attacks were observed since the 4th week and that of analgesics consumption since the 8th week (both P<0.05). While, in the placebo group, significant decrease in frequency of migraine attacks were observed since the 8th week and that of analgesics consumption since the 12th week (both P<0.05). No significant decrease in days of migraine attacks and VAS scores of migraine pain were observed in both groups. Between the two groups, there were significant differences in VAS scores and intensity of pain appeared in the 8th week (P<0.05). However, no significant differences were found in days and frequency of migraine attacks and analgesics consumption (P>0.05).</p><p><b>CONCLUSIONS</b>MWDG was probably effective in the treatment of migraine especially for alleviating pain intensity. Furthermore, MWDG could reduce the frequency of migraine attacks and analgesics consumption sooner than the placebo.</p>

Correlation between absorption ingredients in Wuzhuyu Decoction and uptake amount in everted intestinal sac / 中草药

Objective: To explore the influence of the contents of nine ingredients in Wuzhuyu decoction on its own uptake amount into the everted intestinal sac, in order to blend Wuzhuyu decoction samples to improve the index of migraine model rats. Methods: The everted intestinal sac model was established to determine the absorption samples of 10 kinds of Wuzhuyu decoction. HPLC-DAD method was used to analyze the contents of nine ingredients in Wuzhuyu decoction and absorption samples quantitatively and semi-quantitatively. The partial least squares regression method was used to correlate the contents in the decoction and the total absorption quantity of nine ingredients. Results: In the experimental concentration range, the higher the concentration of limocitrin-3-O-β-D-glucoside, ginsenoside Rg1, 6-gingerol, and ruteavin in the decoction were, the more they were absorbed into the intestine sac, while ginsenosides Re, Rb1, limonin, evodiamine, and rutaecarpine were contrary. The interaction of different ingredients on each other's absorption was complicated. Conclusion: The above relationship should be considered to obtain the satisfactory samples of Wuzhuyu decoction.