Anti-inflammatory And Analgesic Effects Of Total Saponin Of Dioscorea And Its Effect On CPLA2/COX Signaling Pathway

Objective:The extraction method of Total Saponin of Dioscorea?TSD?and an HPLC method for the content determination of dioscin in Dioscoreae Hypoglaucae polibin were established.Determination of the content of TSD by UV spectrophotometry were adopted.the anti-inflammatory and analgesic effects of TSD and its effects on the air pouch inflammation of mice and c PLA2?cytosolic phospholipases A2?/COX?cyclooxygenase?signaling pathway were studyed.Methods:1.The extract was obtained by ethanol reflux extraction,spectrophotometer was used to determine the total saponins with perchloric acid for colorimetric reaction and diosgenin as the reference substance at 409±1 nm as measurement wavelength.The chromatographic separation was performed on an ACE EXCEL C18 column?4.6mm×250mm,5?m?with mobile phase of methanol-water solution with gradient elution;the detection wavelength was 208 nm;the column temperature was 35?;the injection volume was 20?L.2.A model of acute gouty arthritis induced by monosodium urate?MSU?in rats.Observing the gait of rats and measuring the degree of joint swelling.ELISA method was used to detect the inflammation factor interleukin-1??IL-1??,Interleukin-18?IL-18?level,and joint histopathological examination.Acetic acid and hot plate method were used to induce pain in mice,Observing the writhing response and pain threshold of mice.3.Sixty KM male mice were randomly divided into normal group,model group,high,medium and low TSD?600,200,60 mg/kg.d?group and dexamethasone group?0.30mg/kg.d?.To replicate the subcutaneous air pouch inflammation model,we injected the monosodium urate into mice and then administer it by intragastric administration for 1hour,2 times a day for 2 consecutive days.Collect the air pouch lavage fluid,and measure the amount of exudate,white blood cells,IL-1?and prostaglandin E₂?PGE₂?;RT-PCR detection of c PLA2,COX-1 and COX-2 m RNA expression in air pouch lining tissues;Western Blot and immunohistochemical detection of c PLA2,COX-1 and COX-2 protein expression in air pouch tissues.Results:1.The best extraction process was as follows:ten times 95%alcohol,to reflux for 1 hour and extracted for 3 times.The organic solvent extraction was extracted by isometric petroleum ether and water saturated n-butanol for twice.The linearity range of diosgenin was 25.4-152.4?g?r=0.9992?.The average recovery was99.39%?RSD=1.62%?.Dioscin showed a good linear relationship among the range of1.0-8.0?g?r=0.9994?,with the average recovery of 99.22%?RSD=1.56%?.The contents of total saponins of Dioscorea in three batches of herbs were 61.74%,58.78%and 60.47%,respectively,and the contents of dioscin were 33.9%,33.15%and 32.26%,respectively.2.TSD can significantly reduce the degree of joint swelling in rats with acute gouty arthritis?P<0.01 or P<0.05?.Meanwhile,TSD markedly reduced the levels of IL-1?and IL-18 in synovial fluid exudates?P<0.01 or P<0.05?,Reduced inflammation of joint pathology.In addition,TSD remarkably suppressed acetic acid-induced writhing response and prolonged pain threshold in hot plate assay?P<0.01 or P<0.05?.3.Compared with the normal group,the exudate volume,white blood cell count,PGE₂and IL-1?levels of the model group mice were significantly increased?P<0.01 or P<0.05?.CPLA2,COX-1,COX-2 m RNA and protein expression in air pouch tissue were significantly up-regulated?P<0.01 or P<0.05?in air pouch inflammation model.Compared with the model group,the exudate volume,white blood cell count,PGE₂ and IL-1?levels of the TSD high,medium and low dose groups and dexamethasone group decreased significantly?P<0.01 or P<0.05?.The expressions of c PLA2,COX-2m RNA and protein in air pouch tissues were significantly down-regulated?P<0.01 or P<0.05?.Conclusions:1.As the quantitative analysis of TSD and Dioscin,the method of extraction is simple,accurate and reproducible.The content of total saponins and dioscin in the extract can meet the requirements of pharmacodynamic experiments.2.TSD has a strong anti-inflammatory and analgesic effect,and its anti-inflammatory mechanism may be related to inhibition of c PLA2/COX signaling pathway and reduction of related inflammatory factors.