Research On The Effect And Mechanism Of Zhizi Houpu Decoction In The Treatment Of Insomnia

Insomnia is a common sleep disorder in clinic,which affects people's daily life and work.In addition,it can also induce cardiovascular and cerebrovascular diseases,mental diseases and so on.The pathogenesis of insomnia is complex.The brain structures involved include cerebral cortex,brainstem,hippocampus,hypothalamus,suprachiasmatic nucleus,etc.They are closely related to sleep-wake rhythm and neurotransmitters in the central nervous system,including monoamine neurotransmitters such as 5-hydroxytryptamine(5-HT),5-hydroxyindole acetic acid(5-HIAA),dopamine(DA),homovanillic acid(HVA)and amino acid neurotransmitters such as gamma-aminobutyric acid(GABA),glutamic acid(Glu).At present,most of the western drugs used to treat insomnia have side effects such as drug resistance and addiction.Chinese traditional medicine has a long history in the treatment of insomnia.Many single Chinese herbs and Chinese herbal compound prescriptions have good effects in the treatment of insomnia,and are favored by people.Zhi-Zi-Hou-Pu decoction originated from Shang Han Lun,is one of the classical prescriptions for insomnia,but its mechanism is still unclear.Based on the above,this study intends to establish a mice model of insomnia induced by p-chlorophenylalanine(PCPA)on the basis of optimizing the extraction process of Zhi-Zi-Hou-Pu decoction.The contents of monoamine neurotransmitters in cerebral cortex,brain stem and hippocampus of mice were determined by high performance liquid chromatography,the content of amino acid neurotransmitters in serum of mice were determined by liquid chromatography-mass spectrometry,to analyze the relationship between monoamine neurotransmitters,amino acid neurotransmitters and Zhi-Zi-Hou-Pu decoction in the treatment of insomnia,and to explore the effect and mechanism of Zhi-Zi-Hou-Pu decoction in the treatment of insomnia.Taking Drosophila melanogaster as the research object,PySolo activity monitoring system was used to analyze the effects of Zhi-Zi-Hou-Pu decoction and its different extracts on Drosophila melanogaster sleep,to screen the effective fractions of Zhi-Zi-Hou-Pu decoction in the treatment of insomnia.The content of monoamine neurotransmitters in the head of Drosophila melanogaster were determined in order to further explore the mechanism of Zhi-Zi-Hou-Pu decoction in treating insomnia.1.Optimizing the Extraction Technology of Zhi-Zi-Hou-Pu decoction by Orthogonal TestIn order to lay the foundation for the follow-up study of this subject,an orthogonal experiment was used to establish a method for simultaneous determination of geniposide,naringin,hesperidin,neohesperidin,honokiol and magnolol in Zhi-Zi-Hou-Pu decoction by HPLC.The extraction process of Zhi-Zi-Hou-Pu decoction was optimized with the extraction amount of each component and the yield of extract as indicators.The optimum extraction process was soaked in ethanol with a volume fraction of 70%at 12 times for 60 minutes,then heated and refluxed for 90 minutes.The validation experiment showed that Zhi-Zi-Hou-Pu decoction was extracted by heating reflux under the optimum technological conditions,the content of the main components and the yield of the extract were both high and stable.2.Effect of Zhi-Zi-Hou-Pu decoction on Behavior and Neurotransmitters in Tissues of Insomnia Model MiceThe model of insomnia mice induced by intraperitoneal injection of PCPA was established,the macroscopic signs,body mass changes and righting reflex tests were used as indicators to evaluate the model.The contents of monoamine neurotransmitters 5-HT,DA and their metabolites 5-HIAA and HVA in cerebral cortex,brainstem and hippocampus of mice were determined by HPLC-ECD,the contents of amino acid neurotransmitters GABA and Glu in serum of mice were determined by LC-MS/MS.The results showed that PCPA could successfully replicate the insomnia model by intraperitoneal injection for two consecutive days,and the sleep condition of the insomnia model mice was improved after the treatment of Zhi-Zi-Hou-Pu decoction.The amount of DA in cortex,brainstem and hippocampus decreased significantly,the amount of 5-HT increased significantly,and the level of Glu/GABA in serum decreased significantly.3.Screening of effective fractions and mechanism of Zhi-Zi-Hou-Pu decoction for Insomnia Based on Drosophila melanogaster as model organismIn this study,7-day-old virgin Drosophila melanogaster was used to establish a sleep deprivation model by repeated light stimulation at night.Using PySolo activity monitoring system and Drosophila activity as index,the effective parts of Zhi-Zi-Hou-Pu decoction for insomnia were screened,the contents of monoamine neurotransmitters DA,5-HIAA,HVA and 5-HT in the head of Drosophila melanogaster were determined by HPLC-ECD.The results showed that the sleep deprivation model could be successfully established by light stimulation.A certain dose of Zhi-Zi-Hou-Pu decoction and its petroleum ether fraction,dichloromethane fraction,ethyl acetate fraction and n-butanol fraction could improve sleep of sleep deprivation Drosophila melanogaster.It can also reduced the content of DA and HVA in the brain of Drosophila melanogaster and increased the content of 5-HT and 5-HIAA.The effect of dichloromethane fraction is especially obvious.In conclusion,the effect of Zhi-Zi-Hou-Pu decoction on insomnia is definite and it can significantly improve the sleep status of insomnia model mice and sleep-deprived Drosophila melanogaster.The effective fractions include petroleum ether fraction,dichloromethane fraction,ethyl acetate fraction and n-butanol fraction,among which dichloromethane fraction may be the main effective fraction.It is speculated that it may play a role in improving sleep by regulating the levels of monoamine neurotransmitters and amino acid neurotransmitters in the body,i.e.reducing the contents of DA and HVA,increasing the contents of 5-HT and 5-HIAA,and reducing the amount of Glu/GABA.