Mechanism of Zushima in treating rheumatoid arthritis based on network pharmacology and molecular docking / 中国药理学通报

Aim To investigate the potential mechanism of Zushima in the treatment of rheumatoid arthritis (RA). Methods Through PubMed literature mining, combined with tcmsp, TCMIP, Batman TCM, Pub- Chem, Swiss target prediction, GeneCards and other databases, the active ingredients, action targets and RA targets of Zushima were obtained. The active ingredient RA target pathway network was constructed by u- sing Cytoscape 3. 7. 2 software. The protein-protein interaction network was constructed by string database. David online tool was used to analyze the enrichment of GO and KEGG pathway. The molecular docking was verified in autodocking Vina and other software. Results Through screening, 22 chemical constituents were obtained, and 273 potential targets were identi fied. GO (P <0.05) enriched 1 376 GO items, mainly involving cell surface receptor connection signal transduction, plasma membrane part, nucleotide binding and so on. KEGG ( P < 0. 05) pathway analysis showed that 58 signaling pathways were involved in the focal adhesion pathway, chemokine signaling pathway, MAPK signaling pathway and so on. Conclusions The multi-component and multi-target mechanism of Zushima in treating rheumatoid arthritis can be studied by network pharmacology method, which provides scientific basis for experimental research and clinical application of Zushima.

Effect of Process Change on in Vitro Release and Percutaneous Penetration of Zushima Guanjie Zhitong Gao / 中国实验方剂学杂志

Objective:To investigate the effect of the production process of Zushima Guanjie Zhitong Gao from solvent method to hot-pressed method on <italic>in vitro</italic> kinetic behavior of this preparation. Method:Solvent and hot-pressed methods were used to prepare three batches of samples above pilot scale， and <italic>in vitro</italic> release and percutaneous penetration of the index components （7，8-dihydroxycoumarin and methyl salicylate） in Zushima Guanjie Zhitong Gao were investigated by modified Franz diffusing cells. Result:The contents of 7，8-dihydroxycoumarin and methyl salicylate in Zushima Guanjie Zhitong Gao prepared by solvent method were 73.72， 494.67 μg/patch， and their contents in hot-pressed method samples were 159.21， 2 638.99 μg/patch， respectively. In the solvent method samples， the average cumulative release amounts of 7，8-dihydroxycoumarin and methyl salicylate in 24 h were 2.04， 12.21 μg， and their average cumulative release amounts in 24 h of hot-pressed method samples were 2.16， 36.24 μg， respectively. In the solvent method samples， the average cumulative permeation amounts of 7，8-dihydroxycoumarin and methyl salicylate in 24 h were 0.38， 2.79 μg， and they were 0.40， 7.49 μg in hot-pressed method samples. The cumulative release and permeation amounts in 24 h of 7，8-dihydroxycoumarin in the hot-pressed method samples were basically the same as those of the solvent method samples， but the cumulative release and permeation amounts in 24 h of methyl salicylate in the hot-pressed method samples were significantly higher than those of the solvent method samples （<italic>P</italic><0.05）. Conclusion:The retention of 7，8-dihydroxycoumarin and methyl salicylate by hot-pressed method is better than that of the solvent method. The process change has no significant effect on the <italic>in vitro</italic> kinetics of 7，8-dihydroxycoumarin in Zushima Guanjie Zhitong Gao， however， after the change from the solvent method to the hot-pressed method， the methyl salicylate in this preparation has a higher cumulative release and permeation amounts.

Real-time cell analysis combined with pattern recognition to evaluate anaphylaxis of Zushima Injection / 中国中药杂志

This paper was aimed to establish a new method for evaluating the anaphylactoid reaction of 15 batches of Zushima Injection from different manufacturers in vitro. Basophilic leukemia cell line RBL-2 H3 cells were cultured in vitro and Compound 48/80 was selected as positive drug. Real-time cell analysis(RTCA) system was used to detect the changes of cell index(CI) value after drug intervention. The degranulation of RBL-2 H3 cells was verified with the toluidine blue staining technology by observing the changes of cell morphology and skeleton. Clustering method was used to analyze the CI values of 15 batches of Zushima Injection on RBL-2 H3 cells. The results showed Compound 48/80(20 μg·mL~(-1)) significantly changed the cell morphology and cytoskeleton, with obvious degranulation. After adding Compound 48/80, CI value decreased rapidly within 30 minutes, then decreased slowly, suggesting that RTCA system can be used for rapid and sensitive evaluation of RBL-2 H3 cell degranulation. The results of cluster analysis showed that Zushima Injection from different manufacturers had different effects on RBL-2 H3 cells. S1-S8 and Compound 48/80 groups were grouped into one cluster, which suggesting that the sample might have potential clinical anaphylaxis. S9-S15 and the normal control group were grouped into one cluster, suggesting there was no anaphylactoid reaction in the sample. In this study, a rapid in vitro anaphylaxis evaluation technique based on RTCA system and pattern recognition method was established, which can be used for rapid in vitro evaluation of anaphylaxis for traditional Chinese medicine injection.

Effect of Acupuncture and Moxibustion plus Zushima on Serum VEGF and bF GF in Patients with Knee Osteoarthritis / 上海针灸杂志

Objective To investigate the effect of acupuncture and moxibustion plus Zushima on serum vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b FGF) in patients with knee osteoarthritis.Method Two hundred and thirty-six patients with knee osteoarthritis were allocated, using a random number table, to two groups, 118 cases each. The control group was treated with Celecoxib and the observation group, with acupuncture and moxibustion plus Zushima. The therapeutic effects, and pre-treatment and post-treatment VAS scores and WOMAC scores and serum VEGF and bFGF levels were compared between the two groups of patients. The adverse reaction incidences were compared between the two groups of patients. Result The total efficacy rate was 87.3％ in the control group and 94.9％ in the observation group and was significantly higher in the observation group than in the control group (P<0.05). There were no statistically significant pre-treatment differences in the VAS score and WOMAC score between the two groups (P>0.05). The VAS score and WOMAC score decreased significantly in both groups after treatment (P<0.05). After treatment, there was no statistically significant difference in the VAS score between the two groups (P>0.05) but the WOMAC score was significantly lower in the observation group than in the control group (P<0.05). There were no statistically significant pre-treatment differences in VEGF and bFGF between the two groups (P>0.05). After treatment, VEGF and bFGF decreased significantly in both groups (P<0.05) and were significantly lower in the observation group than in the control group (P<0.05). The adverse reaction incidence was3.4％ in the control group and 1.7％ in the observation group with no statistically significant difference between the two groups (P> 0.05). Conclusion Acupuncture and moxibustion plus Zushima can reduce inflammatory reactions, inhibit synovial angiogenesis, and effectively relive the pain, and improve articular functions in the treatment of knee osteoarthritis.

Interventional effects of Zushima Tablets on lipid metabolomic disorder of rheumatoid arthritis in rats based on UPLC-Q-Exactive Orbitrap MS / 中草药

Objective To explore the changed trend of endogenous lipid metabolites in rats with rheumatoid arthritis during different disease periods and the regulation of Zushima Tablets on differential metabolites based on UPLC-Q-Exactive Orbitrap MS. Methods The collagen-induced arthritis (CIA) rats were established using bovine type II collagen. The serum samples were collected before (0 d) and 14, 21, and 36 d after model construction from control, model, and Zushima Tablets groups [0.6 g/(kg•d)], respectively. The lipids were extracted and identified by bioinformatics analysis to find the lipid differential metabolites. Results It was obviously observed that lipid metabolism disordered in CIA rats compared with the control group. The content of three acylcarnitines was increased significantly while the level of TG showed a downward trend in the CIA model group when the TG carbon chain was shorter than 58 carbon atoms. To the opposite, the content of TG substances with atomic number greater than fifty-eight in the model group was significantly higher than that of the control group. Zushima tablet obviously regulated the metabolism levels of a phosphatidylserine, a ceramide, two acylcarnitines, four phosphatidylcholines, and four triglycerides. Conclusion This study demonstrated that rheumatoid arthritis caused the disorder of lipid metabolism while Zushima Tablets had regulatory effects on some lipid metabolites.

Plasma metabonomic study on Zushima Gancao Tablet in treatment of rheumatoid arthritis based on UPLC/LTQ-Orbitrap-MS / 中草药

Objective: To observe the regulating action of Zushima Gancao Tablet on abnormal metabolites by analyzing the changes of endogenous metabolites in plasma of rheumatoid arthritis (RA) rats, and to explore the mechanism of Zushima Gancao Tablet in treating RA from the perspective of metabonomics. Methods: Rats were divided into normal group (NG), model group (MG), positive medicine group (PMG), and Zushima Gancao Tablet group (ZGG). Adjuvant arthritis (AA) rats were established with freund complete adjuvant, rats in PMG and ZGG groups were ig administered with tripterygium glycosides and Zushima Gancao Tablet respectively for a month. Plasma samples were collected on days 1, 10, 20, and 28 before and after administration. UPLC/LTQ-Orbitrap-MS was applied to analyzing the metabolic profile changes of each group in different durations of the disease and mechanism of drug intervention. Results: The related pathways of AA mainly involved metabolisms of amino acids, lipids, nucleic acids, and vitamins. Zushima Gancao Tablet had great effect on AA by regulating 12 biomarkers. Conclusion: Small molecule metabolites in AA rats are deviated from the normal ones. The effects of Zushima Gancao Tablet and positive medicine (tripterygium polyglycoside) on AA are similar, but the mechanisms were different. Zushima Gancao Tablet down-regulates the level of LPC in secondary lesions while tripterygium polyglycoside tablets could effectively inhibit the metabolisms of a variety of amino acids during acute inflammation.

Optimization of Preparation Technology for Zushima Gel Cream / 中国中医药信息杂志

Objective To optimize preparation process of Zushima Gel Cream. Methods The comprehensive evaluation set sensory evaluation, initial adhesive force, viscous force, and peeling strength score as indexes. The mixing time, refining temperature, mixing speed, and powder adding sequence were investigation factors. Orthogonal experiment was used to optimize forming process. Results Conditions of optimized preparation process were as following: add Zushima powder in Viscomate NP-700 and glycerol; mixing time was 5 min; refining temperature was 40 ℃; mixing speed was 100 r/min. Conclusion The preparation process is good and optimized Zushima Gel Cream has a good adhesive force, good glossiness and excipients. The preparation process is good.

A Study on Quality Standard Improvement forZushima Plaster / 中国中医药信息杂志

Objective To improve the quality standard ofZushima Plaster.Methods TLC was used to do qualitative identification of daphnetin and 7-hydroxycoumarin inZushima Plaster. The contents of daphnetin and 7-hydroxycoumarin inZushima Plaster were determined by HPLC. The chromatographic column was Agilent Eclipse Plus C18 (4.6 mm×250 mm, 5μm), and the mobile phase consisted of acetonitrile-0.1% phosphoric asid (15∶85, V/V) at a flow rate of 1.0 mL/min. The column temperature was maintained at 30℃, and the detection wavelength was set at 327 nm. Results Characteristic spots of daphnetin and 7-hydroxycoumarin could be detected by TLC from the samples, and the spots were clear and specific without interference from negative samples. The calibration curve of daphnetin and 7-hydroxycoumarin were linear in the ranges of 0.180-1.800μg and 0.100-1.00μg (r=0.999 1,r=0.999 2), and the average recovery rates were 98.99% and 101.48%, RSDwere1.43% and 1.32%, respectively (n=9).Conclusion The established method appears easy to use, accurate and specific, and therefore can be used for the quality control ofZushima Plaster.

Extraction Process of Zushima Gel Cream / 中国中医药信息杂志

Objective To optimize the extraction process of Zushima Gel Cream.Methods Setting the content of daphnetin as an investigating index, and the amount of solvent, soak time and the extraction time as investigating factors, the orthogonal experiment was used to optimize the extraction process.Results The best extraction process was as follows:adding 30 mL water to degreasing Zushima Gel Cream;soaking for half an hour;extracting one time for two hours. The average content of daphnetin was 9.83 mg/g.Conclusion The extraction process is stable, reliable, and energy-saving. The study provided experimental evidence for the preparation process of Zushima Gel Cream.

Determination of Three Kinds of Phenylpropanoids in Stem Rind of Daphne giraldii by HPLC / 中国中医药信息杂志

Objective To establish a HPLC method for the determination of daphnetin, 7-OH coumarin and syringing in Zushima. Methods The separation was performed on YMC-Pack ODS (4.6 mm?250 mm, 5 ?m) column with a mobile phase consisting of methanol-0.5% acetic acid-water (19︰8︰1) as gradient elution at the flow rate of 1.0 mL/min. The dual detective wavelength was set at 327 nm and 266 nm. The column temperature was 30 ℃. Results Baseline separation was achieved for the analysis of the three index components. The average recoveries were 98.4%～100.2%. Conclusion This method is convenient, rapid and accurate. It can be used for quality control in production of Zushima.