| Objective:To develop a Ultra performance liquid chromatography method for quantitative determination of Paeoniflorin in Baishao and qualitative determination of active ingredientin in plasma of SD rat after taking-in Baishao decoction,then explore the therapeutic effect of active ingredientin on collagen-induced arthritis(CIA) rats,look for the monomer in BZXD which has an effective treatment to RAMethods:1The quantitative analysis on Paeoniflorin in Baishao,the analysis was perform on UPLC system with BEH (R) C18column (50mm×2.1mm,1.7μm).The mobile phase is consist of acetonitrile (A) and1%acetic acid (B),Flow rate and detection wavelength was set at0.25ml/min and190-480nm.Gragh the standard line、Precision、recovery、sample stability and reproducibility was examined in this study.2Use the ultra-high performance liquid chromatography method to qualitatively analyze the active ingredientin in plasma of SD rats after taking-in Baishao decoction. 3The Collagen induced arthritis in rats model was established by subcutaneous injection of bovine type II collagen mixed Freund’s adjuvant.Treated CIA rats with standard paeoniflorin and observe the influence of paeoniflorin on IL-1β、TNF-α levels closely related to the development of RA in serum,in the low、medium、high dosage of Paeoniflorin selected the optimal drug dosage of paeoniflorin treating collagen-induced arthritis in rats.Then,assessed the general situation, changes in weight-gain,arthritis index score and paw-swelling,observed pathological changes of paw’s x-ray image and the synovials. Tested therapeutic effects of paeoniflorin to CIA rats.Results:1Paeoniflorin,the active ingredient in Baishao was separated successfully in five minutes.Determined the paeoniflorin content in Baishao,dataed0.73mg/g. The calibration curve showed good linear regression within test ranges of Paeoniflorin,R2>0.9993.The relative standard deviations (RSD) of intra-day and inter-day Precision were from0.98%to3.65%,indicating good Precision.The sample of Baishao decoction has good stability in24h,with RSD value<2.64%.Baishao decoction has good reproducibility in the reproducibility test,with RSD value<2.68%.The recoveries of Paeoniflorin ranged from98.5%to103.5%,with RSD value<2.7%,showing good recoveries. 2The Paeoniflorin was detected successfully in plasma of SD rat after taking-in Baishao decoction in30minutes.3Low, medium, high dosage of Paeoniflorin could lower the IL-1β、TNF-α level in serum of the CIA rats,but the medium dosage (0.197mg/d) results better effect.4Treated CIA rats with medium dosage.(1) General condition observation:Listlessness,poor appetite and activity were shown in the model group rats.There were not these symptoms in the normal group and the paeoniflorin group latterly,for example,dall hair,but the model group.(2) Changes of weight-growth:The weight of the model group and treatment group rats increased slowly compared with the normal group; The weight of the treated group rats compared with model group increased significantly3weeks after the treatment(P<0.01).(3) Arthritis index scoring evaluation:Arthritis of rats was found after5-7days injection. With the extension of the course,arthritis index score increased gradually in model group and treated group, reached a peak about on21d; Arthritis index score in treated group was significantly lower than that in the model group on35d,42d separately(P<0.05).(4) Swelling degree evaluation in claws and feet:The thickness of claws and feet in model group was thicker than that in the normal group,swelling gradually increased with the extension of the course;The thickness of claws and feet in treated group was much thinner than that in the normal group after two weeks of treatment(P<0.05).(5) Observation of joint X-ray images: swelling of tissue around the joints was observed in model group rats clearly about14days after immunization. With the extension of the course, there were symptoms such as bone density decreased,obvious bone erosion lesions,joint space narrowing or even disappearing.The degree of swelling of tissue around the joints was significantly reduced, bone damage was not found in the treated group rats obviously after4weeks of treatment.(6) Pathological observation of synovium:many new vasculars,inflammatory cell and hyperplasia of synovium were observed in the model group42days after immunization,the treatment group showed a few new vasculars,inflammatory cell and mild thicken synovium after4weeks of treatment.(7) The test of IL-1|3,TNF-a level in serum:the IL-1β、TNF-α level in model group and treatment group is notably higher than that in the normal group(P<0.01);but the IL-1β,TNF-α level in treatment group is notably lower than that in model group on28d,42d separately (P<0.05).Conclusion:1Developed the Ultra performance liquid chromatography method for the determination of Paeoniflorin in plasma through the stomach.2Low, medium, high dosage of Paeoniflorin could lower the IL-lβ、TNF-α level in serum of CIA rat. but the medium dosage results better effect. 3Paeoniflorin could decrease the levels of inflammatory cytokines in serum significantly, reduce the inflammatory response and inhibit destruction of bone, delay the progression of the disease,and the test certifies that Paeoniflorin is the effective monomer in treating collagen-induced arthritis.Suggested that Baishao is the valid ingredient of BZXD in treating of rheumatoid arthritis. |
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