Isolation And Purification Of Melittin And The Study On Mechanism Of Its Radiation-proof Effect

The melittin used in the dissertation was isolated and purified through polydextran gel from honeybee (Apis mellifera lijustica) worker venom which was collected by QF-1 Super Auto Electronic BV-Collector at Teaching Apiary of Bee Science College in Fujian Agriculture and Forest University. The separated component was approved by electrophoresis to be at electrophoretic purification. To evaluate its radiation-proof effect on mice in our experiment, the melittin was injected into the abdomental cavity of the mice before exposed to the Co⁶⁰ . The survival time, the micronucleus rate in bone marrow cell, the chromosome aberrance and the result of single cell gel electrophoresis (SCGE) were observed. The transformtion efficiency of mice spleen cells in each group was also investigated by MTT assay. The mechanism of melittin's protection effect was explored by Pyrogallol Autoxidation and Fenton system in eliminating super-oxide anion and hydroxyl free radical. The results were as follow.1．Melittin's radiation-proof effectThe results showed that melittin could availably improve the survival rate and average survival time of the radiated mice. When injected 0．6, 0．8, or 1．0mg/kg of melittin before radiation, the mice survived 3．2, 12．4 and 5．7 days more and the survival rate increased 21．9%, 52．6% and 28．3% than that in control group injected only 0．9% brine, respectively. The micronucleus rate decreased 3．36‰, 8．09‰, and 8．15‰and the rate of chromosome aberrance decreased 7．8%, 17．1% and 17．4%. The best result was abtained when injecting 0．8mg/kg of melittin into mice 24h before radiation: 12．4d and 52．6% upgrading in survial time and survival rate, 8．09‰and 17．1% downgrading in micronucleus rate and chromosome aberrance.The SCGE Test showed that the comet percentage in melittin-injected marrow cells of the irradiated mice was significantly higher than that in control group, melittin made comet percentage of marrow cells decreased 32．5%, 57．6%, 44．81%. And the comet tail length in control group was significantly higher than that in melittin-injected group in which the comet tail length decreased 34．2%, 40．3%, 37．2%. The results suggested that melittin could protect the DNA in marrow cell of the irradiated mice from irradiation damage.2．The mechanism of melittin's radiation-proof effect.After irradiated for 24 hours, the proliferation rate of spleen cells cultivated in vitro was measured by MTT assay. The results showed that the injection of melittin into mice before irradiation could increase the proliferation rate of spleen cells of irradiated mice. The proliferative rate of spleen cells could increase 9．1% , 12．9% and 15% higher when mice was injected melittin by 0．6mg/kg, 0．8mg/kg and 1．0 mg/kg weight, respectively than that in control. One of the mechanisms of melittin radiation-proof effect is its improving the immunity ability in mice by spleen cells proliferation. The mechanism of melittin's protection effect was explored by Pyrogallol Autoxidation and Fenton system in eliminating super-oxide anion and hydroxyl free radical. The results were that melittin could eliminate 31．23%, 46．02%, 67．39% of the super-oxidized anion and 35．21%, 65．48%, 61．97% of hydroxyl free radical when added 10μl at 10mg/ml, 20mg/ml, 30mg/ml to the reaction system. Melittin had a good elimination effect on super-oxide anion and hydroxyl free radical.The experimental results had approved that the melittin had such functions as the radiation-proof effect and rehabilitating damnification to tissue in the appects of molecular cell biology, immune ability and elimination effect on free radical, which not only provided us the basical research results but also afforded clinical apitherapy the theoretic instruction.