Study On Crocus Sativus L. Anti-Insomnia Active Ingredients And Molecular Mechanism Based In Situ Synthesis Membrane Protein Affinity Chromatography(iSMAC)Technology

Objective:The research team found that the stigma and petal extracts of Crocus sativus L.can improve 5-hydroxytryptamine（5-HT）.Based on the above findings,this study used the model of insomnia induced by p-chlorophenylalanine（PCPA）in rats,cell-free synthesis（CFPS）technology and in situ synthesis membrane protein affinity chromatography（iSMAC）technology to investigate the effect of Crocus sativus L.on insomnia and preliminarily explore the relevant mechanism of action,providing a basis for the clinical application of Crocus sativus L.in the treatment of insomnia.Methods:（1）Using trait identification to identify the differences in Crocus sativus L.from Bozhou,Anhui,Chongming,Hangzhou,Zhejiang,Tehran,Abbasgang,and Assaruye regions,the contents of Crocin Ⅰ,Crocin Ⅱ,and Picrocrocin in six regions were determined by High-performance liquid chromatography（HPLC）,and the components of Crocus sativus L.from Bozhou,Anhui and Assaruye regions were analyzed using quadrupole tandem time-of-flight mass spectrometry（UPLC-QTOF-MS/MS）;（2）The rat model of insomnia was constructed by intraperitoneal injection of PCPA,and the Crocin Ⅰ,Crocin Ⅱ,Picrocrocin,Crocetin,Crocetindial were investigated by elevated cross maze test,open field test and pentobarbital sodium sleep test and the effects of Crocus sativus L.components on 5-HT,glucocorticoid（ACTH）,adrenocorticotropin releasing hormone（CRH）,corticosterone（CORT）,dopamine（DA）,norepinephrine（NE）in the hippocampus and hypothalamus of rats,as well as on the expression of 5-HT_1A mRNA.（3）Using CFPS and iSMAC technology,a 5-HT_1A-silica-liposome complex was synthesized,and a 5-HT_1A-silica-liposome-packed column was constructed.The methodology was investigated to screen the chemical components in Crocus sativus L.that bind to 5-HT_1A receptor.The binding sites and binding forces were determined using by frontier chromatography（FAC）and molecular docking technology.Results:（1）Through the determination of the characteristics of Crocus sativus L.from different regions,the content of Crocin Ⅰ,Crocin Ⅱ,and Picrocrocin,as well as the investigation of different components,it is shown that the quality of domestic Crocus sativus L.is generally better than that of foreign Crocus sativus L..The content of Crocin Ⅰ,Crocin Ⅱ,and Picrocrocin in Bozhou is the highest,and the content of Crocus sativus L.in Assaruye is the highest.（2）The improvement effect of Crocin Ⅰ,Crocin Ⅱ,Picrocrocin,Crocetin,Crocetindial on insomnia in rats:Crocin Ⅰ,Crocin Ⅱ,Picrocrocin,Crocetin,Crocetindial can increase the total distance of open-field experiment,reduce the duration,increase the percentage of open-arm residence time and open-arm residence times in the elevated cross maze experiment,and effectively improve the insomnia behavior of rats.Regulate the levels of 5-HT,ACTH,CRH,CORT,DA,NE in the brain and serum of rats,and promote the expression of 5-HT_1AmRNA.（3）Using CFPS and iSMAC technology to screen the chemical components in Crocus sativus L.that bind to the 5-HT_1A.Protein immunoblotting technology,fluorescence microscopy,electron microscopy,and surface energy spectrum technology show that the 5-HT_1A-silica-liposome complex could be successfully synthesized,the effectiveness of 5-HT_1A-silica-liposome-packed column was investigated by bio-chromatography,and the Picrocrocin that binds to the 5-HT_1A was screened.FAC verified that the 5-HT_1A target had a good affinity with Picrocrocin,and the K_D value was 11.61μmol/L,molecular docking results showed good docking activity.Conclusions:Through PCPA-induced insomnia experiment in rats,it was found that Crocin Ⅰ,crocin II,Picrocrocin,Crocetin,Crocetindial can effectively improve insomnia in rats.The iSMAC technology has achieved efficient in situ synthesis,and has been successfully applied to screen the affinity compounds of crocus sativus L..The screened Picrocrocin can bind to the 5-HT_1A and have strong affinity,providing a new direction for the development of Crocus sativus L.as a drug to improve insomnia.The iSMAC method also provides a practical method for the rapid preparation of MP-immobilized and other biological solid-phase materials.