| Objective:To determine and quantitate the ingredients of Hedyotis diffusa Willd (HD),adoptting Ultra Performance Liquid Chromatography (UPLC) method;to detect absorbed chemical constituents in plasma of SD rats intragastric administrated with Hedyotis diffusa decoction; to inquire the therapeutic effects of the absorbed ingredient in the Collagen-Induced Arthritis (CIA) model of SD rats,using the absorbed ingredient as an intervention.Method:(1) UPLC-PDA method was adopted to determine the content of the ingredients in Hedyotis diffusa. Then drawed the standard curves of the various components and the detection limit, precision, recovery, sample stability and reproducibility of the experimental method were examined in this study;(2) By comparing UV spectra and chromatograms with plasma of the Control group, ferulic acid and p-coumaric acid reference substance, two components were measured out from the plasma of SD rats intragastric administrated with Hedyotis diffusa decoction. Which suggested that ferulic acid and p-coumaric acid from Hedyotis diffusa could be absorbed into the SD rats blood.(3) According to the level of the TNF-alpha and IL-lbeta content in the serum of CIA rat models measured by enzyme-linked immunosorbent assay (ELISA), screened the appropriate dosage of ferulic acid.(4)Using the appropriate dosage as an intervention, researched the effect of ferulic acid on rat model of CIA,via observing general condition, arthritis index, paw swelling, synovial tissue pathological changes and joint X-ray and levels of IL-1β,TNF-α in groups. Further validated the role of ferulic acid in the treatment of rheumatoid arthritis.Result:(1) Ferulic acid and p-coumaric acid were seprarated from Hedyotis diffusa,with UPLD-PDA method; The calibration curve showed linear regression within the ranges of Ferulic acid (0.005-0.08mg/ml), R2>0.9994. The average recovery of ferulic acid was100.4%, with RSD value1.26%. The calibration curve showed linear regression within the ranges of p-coumaric acid (0.0028-0.045mg/ml), R2>0.9998. The average recovery of p-coumaric acid was100.4%, with RSD value1.26%. Hedyotis diffusa contenteded233ng of ferulic acid,529ng of p-coumaric acid in one milligram.(2) Ferulic acidN p-coumaric ingredients were measured out in the rat plasma intragastric administrated with Hedyotis diffusa; (3)Low, medium, high dosages of Ferulic acid could down regulat the level of IL-1β、 TNF-α in serum of the CIA rats. but the high dosage resulted better effect.(4)①General condition observation:Listlessness, poor appetite and activity were shown in the model group rats. After2weeks of treatment with Ferulic acid in the treatment group, these symptoms were improved,There were not these symptoms in the normal group.②Changes of weight-growth:The weight of the model group and treatment group rats increased slowly compared with the normal group; After3weeks treatmented, the weight of the treated group rats compared with model group increased significantly(P<0.05).③Arthritis index scoring evaluation:In the first four weeks of the experiment, arthritis index score increased gradually in model group and treated group, reached a peak about on2Id; Arthritis index score in treated group was significantly lower than that in the model group on35d,42d separately (P<0.01).④Swelling 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 model group after3weeks of treatment(P<0.01).⑤Observation 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 and bone damage were significantly reduced in the treated group rats,after4weeks of treatment.⑥Pathological observation of syno vials Many new vasculars, inflammatory cell and thicken synovials were observed in the model group42days after immunization,The treatment group showed a few new vasculars, inflammatory cell and mild thicken synovial after4weeks of treatment.⑦The test of IL-1β TNF-a levels in serum:The IL-1β TNF-a level in model group and treatment group is notably higher than that in the normal group(P<0.01), but the IL-1β、TNF-a level in treatment group is notably lower than that in model group on28d,42d separately (P <0.01).Conclusion:For the first time, ferulic acid was found to be absorbed into the blood circulation as one of ingredient in Hedyotis diffusa,by adopting UPLC-PDA method. The animal experiment suggested ferulic acid could alleviate the inflammatory response, and down regulate the levels of cytokines(IL-1beta, TNF-alpha)in CIA modle. Ferulic acid could be used for treating rheumatoid arthritis, Hedyotis diffusa active ingredient might be one of the material basis for the efficacy of BZXD in the treatment of rheumatoid arthritis. |
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