| Objective: To select the crude lyophilized powder of Cobra venom in the southern part of Anhui Province,extract and purify the analgesic active component(CVAF)and to explore the effect of external analgesia and its possible mechanism of action,then to study the time-effect relationship,dose-effect relationship of CVAF analgesia and the tolerance and addiction of CVAF medication.Methods: 1.Separation,purification and molecular weight determination of CVAF:CVAF was purified by SP-Sephadex C-50 cation exchange chromatography and Sephadex G-50 size exclusion chromatography,and molecular weight was determined by SDS-PAGE gel electrophoresis.2.Preparation of topical gel,The lyophilized CVAF lyophilized powder is purified and formulated into a topical gel preparation having a pH of 6.5-7.0 with a gel matrix and an antiseptic treatment agent.3.Analgesic effect test of CVAF,40 SD rats were modeled in inflammatory model with complete Freund's adjuvant.The experimental group was divided into 5 groups,(1)saline control group(NS group),(2)complete Freunds Inflammatory group(CFA group),(3)diclofenac sodium analgesic positive control group(CFA+SL group),(4)flurbiprofen analgesicpositive control group(CFA+FB group),(5)CVAF analgesic experimental group(CFA+CVAF)Group),using hot tingling instrument and mechanical tenderness instrument to measure the daily heat pain reaction time and the weight of tenderness reaction of each group of rats after treatment,and compare the topical treatment of cobra venom active component CVAF gel.The effects of heat pain and mechanical pain on rats in each group.The contents of IL-1 and TNF-? in the spinal cord homogenate of each group were detected by ELISA.The pathological changes of rat toe tissues were observed by HE staining.4.CVAF time-effect experiment: 20 mice were divided into two groups,CVAF group and NS group,the heat pain reaction time before and after administration was measured at 30 min,1 h,2 h,4 h,6 h,8 h,12 h.Observe the CVAF time effect relationship.5.Dose-effect experiment of CVAF,50 mice were divided into5 groups.Based on the range of drug safety doses demonstrated by the LD50 experiment,five doses of 1/8,1/6,1/4,1/3,and 1/2 LD50 were taken;that is,0.08mg/Kg,0.1 mg/Kg,and 0.16.The dose-effect relationship of CVAF was observed by mg/Kg,0.2 mg/Kg,and 0.3 mg/Kg,before and after 2 hours of administration.6.CVAF drug tolerance test,30 mice were randomly divided into 3 groups,CVAF group(0.3 mg/Kg),morphine group(5 mg/Kg),NS(0.2 ml/10 g)group,the same selection The heat pain reaction time before and 2 hours after the administration of the rats was measured for 8 consecutive days,and the tolerance to the drugs was observed.7.Addiction test of CVAF medication,24 mice were randomly divided into 3 groups,8 in each group,CVAF group,morphine group and saline group,administered twice a day(9:30,14:30).For a total of 3 days.The CVAF group received 6 doses of 0.1,0.2,0.2,0.3,0.3,0.4 mg/Kg intraperitoneally,the total dose was 1.5 mg/Kg,and the morphine group was injected intraperitoneally 6 times.The doses of 6 times were 5,10,and 10,respectively.15,15 and 20 mg / kg,the total dose was 75 mg / kg;saline group,intraperitoneal injection of normal saline,6 times were 0.2ml / 10 g.Naloxone 6 mg/kg was intraperitoneally injected 2 hours after the last administration,and the number ofmice with withdrawal symptoms in each group was recorded.Results: 1.After SP-Sephadex C-50 cation exchange chromatography and Sephadex G-50 size exclusion chromatography were separated twice,lyophilized to obtain dry powder of analgesic active component of Weinan cobra venom,and molecular weight of CVAF determined by SDS-PAGE electrophoresis.It is 6.5KDa.2.The gel preparation made of CVAF is a transparent gel with a pH between 6.5 and 7.0.3.In the analgesic effect experiment,the body weight of the rats in the CFA group was the slowest,and the weight gain in the NS group was stable.Compared with the CFA group,the rats in the CVAF analgesia group were 7 after the administration.The difference in body weight between day and day 14 was statistically significant(P<0.05,P<0.01).From the hot tingling and mechanical tenderness experiments,the thermal pain threshold of the CVAF analgesic group was significantly higher than that of the CFA group from the fourth day of treatment(P<0.01);the mechanical mechanism of the CVAF group The tenderness threshold was improved compared with each group,and was significantly higher than that of CFA group(P<0.01).The concentration of IL-1and TNF-? in spinal cord homogenate was lower than that of CFA group.The significance of learning(P<0.01),in HE staining,the edema of inflammatory pain in the CFA group was obvious.The neutrophils and lymphocytes and macrophage infiltration were observed under the microscope,while the toe tissues of the CVAF group were observed.Edema was significantly reduced,and inflammatory infiltration was relatively insignificant.4.The comparison of thermal pain reaction time at different time points showed that CVAF started to take effect after 0.5h of administration(P<0.05),the analgesic effect continued until 4h(P<0.05),and the thermal pain reaction time prolonged after 2h.The most significant difference was statistically significant(P<0.01).5.The thermal pain reaction time of different doses after 2 hours was measured.The heat pain reaction time of the 0.1 mg/Kg and 0.2 mg/Kg dose groups was reduced compared with the basic heat pain reaction time(P<0.05).There was a statisticallysignificant difference between the 0.3 mg/Kg dose group and the basic heat pain response(P<0.01).6.In the tolerability experiment,it was found that the basic pain threshold of each mouse in different groups was determined by continuous eight days.Compared with the NS group,the basic pain threshold of the CVAF analgesia group was in the fourth,sixth,and seventh consecutive medications.The daily difference was statistically significant(P<0.05).7.In the addiction experiment,8 mice in the MF group showed withdrawal symptoms and vertical tail reaction,but none of the NS group and CVAF mice showed the above reaction.Conclusion: The relative molecular weight of cobra venom active component CVAF is6500 Da.The topical gel preparation made of lyophilized powder can improve the thermal pain threshold and mechanical tenderness threshold of rats with inflammatory pain.The mechanism may be related to reducing the release of inflammatory mediators and inhibiting inflammatory response.The optimal dose of CVAF intraperitoneal injection was 0.3mg/Kg,and the best effect was after 2 hours of treatment.No tolerance was observed after 7 days of treatment.No addiction was found after 6 consecutive administrations. |
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