Study On Analgesic Activity Of Total Alkaloids From Ethnic Medicine Cynanchum Komarovii Al.Iljinski

Objective:In this study,crude extracts of Cynanchum komarovii Al.Iljinski was extracted by ethanol,and the total alkaloids of Cynanchum komarovii Al.Iljinski?TACKI?was prepared by the classic method of acidic dissolution.The analgesic effect of TACKI was evaluated by classical analgesic model.The effect TACKI on chronic inflammatory pain and its mechanism were evaluated by collagen?-induced arthritis model.Methods:1.Preparation of TACKI:the ground part of the Cynanchum komarovii Al.Iljinski was dried and crushed,and 75%ethanol was extracted by ultrasonic method.Liposoluble constituents were removed by extraction with petroleum ether,and TACKI was prepared by the classic method of acidic dissolution.2.Acute toxicity study:According to the preliminary experiment,lowest dose of 2457.6mg/kg and the highest dose of 6000 mg/kg were intragastrically administered once in ICR male mice?0.2 mL/10g weight?,continuous observation for 14 days.3.Acute pain study:Physical stimulation?thermal stimulation?and chemical stimulation?formalin and acetic acid?were used to induce nociceptive pain in mice.The analgesic effect of TACKI was evaluated by paw withdrawal latency,licking time,and writhing times and latent period of pain in mice in acute heat stimulating test,formalin test,and acetic acid writhing test,respectively.4.Chronic inflammatory pain study:The analgesic effect of TACKI on chronic inflammatory pain induced by bovine collagen type II-induced rheumatoid arthritis?CIA?rats model by paw withdrawal latency and paw withdrawal threshold measured with radiant heat stimulation and von Frey filaments.5.Analgesic mechanism study:The expression of GFAP,Iba-1,and NMDAR2B protein in rats'brain tissue were detected by western blot and immunohistochemistry.6.The contents of inflammatory mediators TNF-?,IL-17 and IL-6 in serum of rats were determined by ELISA.7.Toxicity of kidney and liver:The hepatorenal toxicity of TACKI to rats was evaluated by HE staining of liver and kidney.Results:1.Preparation of TACKI:After extraction,TACKI 638.00 g was obtained,and extracting rate was 3.83%2.Acute toxicity study:The acute toxicity test showed that the LD₅₀ of TACKI in mice was 2960.88 mg/kg,and some toxic symptoms such as dyspnea,tremor,opisthotonus,ataxia,exophthalmos,excessive secretion of saliva and watery stool.3.Acute pain studyAcute heat stimulating test:Compared with model group,TACKI 200 mg/kg prolonged the paw withdrawal latency in mice after administration for 1 h and 2 h,but after administration 4 h,TACKI 400 mg/kg prolonged paw withdrawal latency in mice?P<0 05?.Formalin test:Compared with the model group,TACKI significantly reduced the licking paw time in I phase with dose-dependently.TACKI 400 mg/kg and TACKI 200 mg/kg significantly reduced the licking paw time in II phase in mice?P<0.01,P<0.05?.Acetic acid writhing test:Compared with the model group,TACKI 400 mg/kg reduced the number of writhing in mice?P<0.05?and prolonged latent period of pain?P<0.001?4.Chronic inflammatory pain study:There was no significant difference among each group of by paw withdrawal latency and paw withdrawal threshold?P>0.05 for all?berore establishing model.After administration for 25 days,the paw withdrawal latency in the TACKI 300 mg/kg group was prolonged?P<0.001?,and the paw withdrawal threshold in the TACKI 300 mg/kg,TACKI 150 mg/kg group and the TACKI 75 mg/kg group was significantly increased?P<0.01,P<0.01,P<0.05?.5.Effect of TACKI on the expression of GFAP,Iba-1,and NMDAR2B protein in rats'brain:Compared with control group,the expression of GFAP protein in brain tissue of model group was significantly increased?P<0.01?,while that of TACKI 300 mg/kg and TACKI150 mg/kg group was significantly lower than that of model group?P<0.01,P<0.05?.Compared with control group,the expression of Iba-1 protein in brain tissue of model group was significantly increased?P<0.05?.Compared with model group,Iba-1 protein showed a decreasing tendency in TACKI administration groups.Compared with control group,the expression of NMDAR2B protein in brain tissue of model group was significantly increased?P<0.05?,while that of TACKI 300 mg/kg group was significantly lower than that of model group?P<0.05?.6.Compared with control group,the contents of TNF-?,IL-17 and IL-6 in serum of model group were significantly increased?P<0.01,P<0.05,P<0.05?;Compared with model group,TACKI 300 mg/kg decreased the contents of TNF-?and IL-6?P<0.05,P<0.05?,TACKI 150 mg/kg could decreased the content of IL-17?P<0.05?.7.Toxicity of kidney and liver:Pathological examination of liver tissue showed that there was no significant difference among groups.Pathological examination of renal tissue showed that the number of eosinophils in the kidneys decreased and the basophils increased in the TACKI administration groups compared with the control group.Conclusion:1.The extraction rate of TACKI was 3.83%?.2.The LD₅₀ of TACKI was 2960.88 mg/kg in mice,and there may be kidney toxicity in rats.3.TACKI had significant analgesic effect on both acute pain and chronic inflammatory pain.4.The analgesic mechanism of TACKI may be related to the inhibition of the activation of astrocytes to reduce the content of inflammatory mediators.