| Bombyx batryticatus(BB)is a dry body of the 4th to 5th instar larvae of Bombyx mori infected(or artificially inoculated)with Beauveria bassiana.BB is often used in the treatment of epilepsy combination with other drugs,but its antiepileptic mechanism is not clear.The one of characteristic component different from Bombyx mori is Beauverianin,which has been reported to prolong the incubation period of isoniazidinduced convulsive model.However,whether it can be used as an anti-epileptic index component of Bombyx batryticatus needs further validation.Bombyx batryticatus is often used in treatment of epilepsy with other drugs,but its antiepileptic mechanism is not clear.In addition,there are few reports about Beauverianin,and its high market price and complex synthesis process are not conducive to the quality control of traditional Chinese medicine using Beauverianin as raw material.Based on the research status of the Bombyx batryticatus above,the following studies were carried out in this paper.Purpose:In order to further clarify the anti epileptic mechanism of Bombyx Batryticatus and the anti epileptic active components of Bombyx Batryticatus,and provide new ideas and methods for the quality control of Bombyx Batryticatus.Method:Firstly,the anti-convulsant effect of BB was taken as the starting point,and the acute and chronic models(EMS,MET,PTZ)related to epilepsy were established successively.The pathological conditions of mice hippocampus were detected by HE staining and Nissl staining;Vagus related factors DA,5-HT,γ-GABA and excitatory neurotransmitter Glu were detected by ELISA kit;In order to further study the antiepileptic mechanism of BB,immunohistochemistry was used to detect the optical densities of Cytochrome C,EAAT 1 and GAD 65 in mice.The m RNA and protein expressions of GABRA1,GAD-65,GAT-1,GLS,Ea AT1 and Cyt C were detected by RT-PCR and Western Blot.In addition,16 S r RNA sequencing technology was used to analyze the effects of BB on the relative abundance and diversity of intestinal microflora in mice with PTZ chronic ignition,and to explore the regulatory mechanism of BB on intestinal microflora.Finally,we synthesized and dematerialized beauvericin,a symbolic component of BB.First,the structure of the synthesized compounds were confirmed by the UV,IR,NMR and LC-MS;Second,set up the new content of identification and HPLC method;Third,through the on ignition residue,moisture,residual solvents investigation to determine the purity of beauvericin;Last,the stability of the beauvericin by accelerating the stability research of.Result:First of all,this study showed that there were differences in antiepileptic activity between Bombyx mori,Bombyx batryticatus and Beauvericin,and the antiepileptic activity of BB was stronger.Secondly,the mechanism studies showed that BB could decrease the m RNA and protein expressions of GAT 1,GLS,and Cyt C while upregulating the contents and protein expressions of GABRA1,GAD-65,and EAAT1,suggesting that BB could exert anti-epileptic activity by regulating the related pathway of the vagus nerve.16 S r RNA sequencing showed that BB could not only improve the disturbance of intestinal flora in epileptic mice,but also regulate the dominant flora to the normal group,indicating that BB could participate in the regulation of intestinal flora.In conclusion,BB plays an anti-epileptic role by regulating the "brain-gut axis" system.Finally,the four spectra of compound 13 showed that the synthesis of beauvericin was successful.In addition,the results of TLC identification,purity test and stability test showed that the purity of beauvericin was more than 99%,and its properties were stable,which could be used for the quality control of BB.Conclusion:In this study,three animal models of epilepsy were used to study the antiepileptic activity of BB through "brain-gut axis" system.Further,the identification of beauvericin provided a new method for the quality control of BB. |
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