Study On The Mechanism Of Baihe Dihuang Decoction In The Treatment Of Insomnia

Insomnia is a common sleep disorder disease,which has a serious impact on people’s lives.Many traditional Chinese medicines have good efficacy in the treatment of insomnia.Baihe Dihuang Decoction originated from the Synopsis of the Golden Chamber,is a prescription for the treatment of insomnia,but its mechanism is still unclear.This research will focus on the mechanism of Baihe Dihuang Decoction in the treatment of insomnia.This project will optimization the best extraction technology of Baihe Dihuang Decoction,on this basis,in order to study the mechanism of Baihe Dihuang Decoction in treating insomnia,and screen out the best effective site of Baihe Dihuang Decoction.This project carried on the following experiment:(1)The orthogonalexperiment was designed with ethanol concentration,solvent multiple,soaking time and extraction time as factors,three levels were selected for each factor.The content of catalpol and extraction rate in Baihe Dihuang Decoction was used as evaluation index,and comprehensive weighted scoring method was used to analyze the the experimental results The result showed that the best extraction technology of Baihe Dihuang Decoction is to soak for 90 minutes in 12-fold dose of 50%ethanol and extract for 60 minutes.The best extraction technology was proved that the content of Catalpol and extraction rate were high,and the extraction technology was stable and feasible,and the best extraction technology can be used for the following study.(2)Insomnia model rats were induced by intraperitoneal injection of PCPA,successful rats were randomly divided into model group,diazepam group,Baihe Dihuang Decoction low,medium and high dose group,additional one blank group,continuous administration for 7d.The behavior score was measured by open field test,and the effect of Baihe Dihuang Decoction on sleep time of rats was evaluated by reversal reflex experiment.Themethod of HPLC-ECDwas established to determine monoamine neurotransmitters in rat brain,and the contents of DA,5-HIAA,HVA and 5-HT in cortex,brainstem and hippocampus of rats were determined by this method.Compared with the blank group,the body mass and open field scores of rats in model group were decreased significantly(p<0.05).The residence time,modification times,fecal particles number and sleep latency of the central compartment were significantly increased(p<0.05).The content of DA in different brain regions wasincreased significantly,and the content of 5-HT was decreased significantly of rats in model group(p<0.05).Compared with the model group,The central residence time,modification times and fecal granules of rats in each treatment group were decreased significantly(p<0.05).Sleep latency was Shorten,and sleep duration was prolonged significantly of rats in Baihe Dihuang Decoction group(p<0.05).The content of DA in cortex of rats in Baihe Dihuang Decoction group was significantly decreased(p<0.05).The content of 5-HT in cortex,brainstem and hippocampus of rats in diazepam group and Baihe Dihuang Decoction low dose group was increased significantly(p<0.05).The content of 5-HT in brainstem and hippocampus of rats in middle and high dosage groups of Baihe Dihuang Decoction was increased significantly(p<0.05).The content of DA in cortex of rats in blank group,model group and diazepam group was more higher,so HVA could be detected.HVA was not detected in the other groups.The result showed that Baihe Dihuang Decoction can improve the ethology and sleep of insomnia rats.The regulating effect of Baihe Dihuang Decoction on monoamine neurotransmitters in rat brain may be the mechanism of its treatment of insomnia.(3)The method of LC-MS/MS was established to determine the amino acid neurotransmitters in rat serum by LC-MS/MS,and the contents of Glu and GABA in rat serum were determined by this method.Compared with the blank group,there were no significant changes of GABA and Glu in serum of rats in model group,the ratio of Glu and GABA was increased significantly(p<0.01).Compared with the model group,the ratio of Glu and GABA in Diazepam group,Baihe Dihuang Decoction medium dose group and Baihe Dihuang Decoction high dose group was decreased significantly in each treatment group(p<0.05).The result showed that the role of treating insomnia is closely connected to the regulation of expression level of inhibitory neurotransmitter GABA and excitatory neurotransmitter Glu in rats.(4)Baihe Dihuang Decoction were firstly extracted with petroleum ether,dichloromethane,ethyl acetate and water saturated n-butanol successivelyto obtain different polarity extraction part.The best sedative-hypnotic effective sitewas screened by Drosophila insomnia model,and the besteffective site were further evaluated through the monitoring of expression level on DA,5-HI A A,HVA and 5-HT in Drosophila brain.Compared with the blank group,sleep time of Drosophila in model group was significantly shortened(p<0.05).The content of DA in the brain of Drosophila was increased significantly(p<0.05),and thecontents of 5-HIAA and 5-HT were decreased significantly in model group(p<0.05).Compared with the model group,the sleep time of Drosophila was obviously prolonged in Baihe Dihuang Decoction group,low-dose group of petroleum ether extract,medium-dose group of dichloromethane extract,medium-dose group of ethyl acetate extract and low-dose group of water-saturated n-butanol extract(p<0.05).The contents of DA and HVA in the brain of Drosophilawere decreased in previously screened functional groups,while the contents of 5-HT and 5-HIAA were increased in those groups.The effect of dichloromethane extract on Drosophila sleep and monoamine neurotransmitters in Drosophila brain is most obvious.In this study,Drosophila melanogaster was used as a model organism to screen the effective parts of Baihe Dihuang Decoction in the treatment of insomnia for the first time,The results showed that Petroleum ether,dichloromethane,ethyl acetate and water saturated n-butanol extracts from Baihe Dihuang Decoction have the treatment of insomnia effects.Their treatment of insomnia effects may be connected to the regulation of expression level of monoamine neurotransmitters in the brain.