| Object:To separate and purify an analgesic active ingredients from scorpion venom.Methods:(1) Selecting the separation conditions:Superdex 75 pre-installed gel chromatography column were used to separate the analgesia active ingredients from the scorpion venom. The effects of pre-process methods, kinds of washing buffer and elution velocity, and the concentration of protein in samples on the separation results were surveyed.(2) Studied on the molecular weight of components and their pharmacological actions:The purity and molecular weights of components were checked by SDS-PAGE; The toxicity of scorpion venom and the components from the scorpion venom were determinated by the LD50; The analgesic or antitumor effects of the components from the scorpion venom were evaluated by the methods of hot plate and acetic acid twisting or transplantation of S180 sarcoma and H22 in mice.(3) Evaluation of the safety of SVF(the 5th of scorpion venom):The hemolytic, vascular and muscle irritant effect of SVF were also evaluated.(4) Studied the analgesia effect of SVF and its mechanism:The pharmacological action of SVF were evaluated by acetic acid induced twisting, hot-plate or formalin induced foot pain. The relationship between analgesic action of SVF and opioid receptors were explored by the naloxone antagonism experiment; The effect of SVF on the incubation, duration, times of depolarization and repolarization, negative after-potential, positive after-potential, amplitude and conduction velocity of action potential of rat sciatic nerve were measured conduction, To explore the analgesic mechanism of SVF, the effect of topical administration of SVF on the pain reaction induced by light and hot radiation or formalin were checked in mice.Results:(1)The protein content of scorpion venom is 85.51%, and most of the molecular weight were between 10 to 25KDa. The best condition of separating the active ingredients from scorpion venom are these:sample protein content 10mg/mL,0.05 M PBS,pH7.2 washing buffer flow at 1.0 ml/min.(2)The 5th components was a single strip showed in SDS-PAGE and had a good analgesic effect and its molecular weight is about 10KDa-15KDa; LD50 of Scorpion venom crude toxin was 1.3561 mg/kg, and LDsoof SVF was 4.3564 mg/kg; Scorpion venom components can obviously increase the pain threshold and decrease the times of twisting in mice, and SVF has the best effect.There is no significant effect on the body weight, thymus index, tumor weight in mice with S180 or the survival time in mice with H22.(3)SVF has no hemolysis or agglomeration to red blood cells of rabbits,no obvious stimulation to blood vessels or muscle of rabbit.(4)SVF 0.6mg/kg,0.3mg/kg and 0.15mg/kg can obviously increase the pain threshold induced by hot and decrease the times of twisting reaction induced by acetic acid in mice; SVF 0.6mg/kg,0.3mg/kg can significantly reduce the pain reaction during the first phase and second phase in formalin-induced mice. (P< 0.05 or P< 0.01). Naloxone can not antagonize the analgesic effect of SVF.SVF 1.112mg/ml,0.834mg/ml,0.626mg/ml,0.470mg/ml could decrease the amplitude and slow down the conduction velocity of action potentials of rat sciatic nerve. SVF 0.470mg/ml also could significantly shorten the duration and times of depolarization(P<0.05 or P<0.01). Topical administration of SVF could increase the pain thresholds induced by light and hot radiation, and lower the pain score of formalin response in mice.Conclusion:The results of this study showed that SVF has reached electrophoresis purity by gel filtration separation. SVF has good security,obvious analgesic effects. Its analgesic mechanism may be partly related to opioid receptors or blocking the nerve conduction. |
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