| Purpose:Bombyx batryticatus,also known as celestial worms and worms,are dry bodies of the larvae of Bombyx mori Linnaeus 4 to 5 instars infected(or artificially inoculated)with Beauveria bassiana(Bals.)Vuilknt.In the application of commonly used Chinese patent medicines,the stiff silkworm accounts for about 9.5%,which is widely used in clinical practice.As the animal medicine in traditional Chinese medicine,protein and peptides(polypeptides)are the main chemical components unique to the dead silkworm.Therefore,this experiment mainly focuses on the protein components of the dead silkworm.The protein components are added to the dead silkworm as a quality evaluation index.In the quality evaluation system of the processing technology,at the same time further explore the content of the cyclic peptide compound beauvericin contained in the dead silkworm and the new pharmacological effects.Use orthogonal experiments to optimize the processing technology of bran-fried silkworm,optimize the extraction,separation and purification of silkworm protein by response surface method to determine the optimal protein extraction process,and use the purified protein to prevent and treat in vitro neurotoxicity molecules.The mechanism is studied,and the effect of beauvericin on glioma is initially explored.Method:The range of material-to-liquid ratio,frying temperature,and frying time was investigated by single factor.After determining the range,orthogonal experiment was used to determine the total protein concentration by BCA method,and quercetin,kaempferol,and Beauveria bassiana were determined by HPLC method.And use the above detection items as evaluation indicators to comprehensively evaluate the OD value to determine the best processing technology for bran-fried rigid silkworm;through the single-factor investigation of p H,extraction time,and material-to-liquid ratio,the response surface method is used to design and optimize Protein extraction technology,and determine the best extraction conditions;through the investigation of protein isoelectric point and ammonium sulfate precipitation concentration of the preliminary separation and purification of the protein,determine the molecular weight of the protein by SDS-PAGE,and further study the crude silkworm protein extraction The protective effect of glutamate on PC-12 cytotoxicity induced by glutamate,to explore the inhibitory effect of the cyclic peptide compound Beauvericin on U251 glioma cells and its molecular mechanism.Results:The optimal processing conditions of the stiff silkworm orthogonal processing were as follows: material-liquid ratio 1:10,frying temperature 200℃,and frying time 5min.The best extraction process for stiff silkworm protein was material-liquid ratio 1:62,extraction time 41 min,p H 8.0,The protein content obtained by using the best extraction process is 139.9mg/g.The isoelectric point of stiff silkworm protein is about p H5.0.The molecular weight of isolated and purified protein is mainly concentrated in 20-35 k Da,and the maximum protein molecular weight is about 70 k Da.After processing,the protein content is significantly reduced,and the electrophoresis band of bran fried product is significantly lighter than that of raw product,Or disappear.Stiff silkworm protein has a certain protective effect on PC-12 cells induced by glutamate,and shows a quantitative dependence.The expression of BAX is significantly lower than that of the model group,BCL-2 is significantly higher than that of the model group,and NMDAr is higher than that of the model group.There was a significant decrease.The intracellular calcium ion was stained and observed with a fluorescence microscope.The intracellular calcium ion concentration of the treatment group was significantly lower than that of the model group.Beauvericin can inhibit the proliferation of U251 cells and promote cell apoptosis by up-regulating Bax,Caspase-3 and down-regulating Cyclin D1,Cyclin E,Cyclin B and Bcl-2,reducing S phase,and proliferating U251 glioma cell lines Produce an inhibitory effect.Conclusion:The processing technology of bran-fried silkworm was optimized through multi-index comprehensive evaluation.Quercetin,kaempferol,beauvericin,and total protein were used as evaluation indicators to comprehensively evaluate the processing technology,which further demonstrated the scientific rationality of the processing technology.Using response surface to optimize the extraction process of the dead silkworm protein can extract the protein components of the dead silkworm to the maximum extent.The ammonium sulfate precipitation method can further effectively separate and purify the dead silkworm protein.The purified protein can better explain its pharmacological effects.Reduce interference.Through the study of the toxicity of the crude protein on PC-12 cells induced by glutamate,it can further explain the effective substance and molecular mechanism of the dead silkworm in the treatment of convulsions and epilepsy.The rigid silkworm protein can regulate NMDA receptors.Inhibit the excitotoxicity of nerve caused by glutamate,and show a certain amount of dependence.The experiment also proved that Beauverin can inhibit the proliferation of glioma cells and promote apoptosis,which provides a drug experimental basis for the auxiliary application of Beauverin in the clinical treatment of glioma. |
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