Study On The Sedative And Hypnotic Activities And Chemical Constituents Of Diaphragma Juglandis Fructus

With the accelerated pace of life and work,insomnia and its related disease have become serious threat to the public health.Traditional chemical synthetic drugs can not meet the insomniac's requirement for many adverse reactions arised during the treatment of insomnia.At present,traditional Chinese medicine which could improve sleep disorders has been highly regarded and some of them have been verified the sedative and hypnotic activities.Diaphragma juglandis Fructus,the wooden diaphragma of Juglans regia L.,has long been used to reinforce the kidney and control noctural emission.In recent years,Diaphragma juglandis Fructus has been applied to improve sleep disorders and acquired certain therapeutic effect.However,few related research has been done and there is no report on the sedative and hypnotic components of Diaphragma juglandis Fructus.In order to expand its application,sedative and hypnotic activities as well as the chemical constituents of Diaphragma juglandis Fructus have been investigated in this thesis.Objective:?1?To study the sedative and hypnotic activities of Diaphragma juglandis Fructusa.?2?To investigate the chemical constituents of Diaphragma juglandis Fructusa to clarify its material basis of biological activity.?3?To explore the possible sedative and hypnotic activities of isolated components based on molecular docking technology and Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform?TCMSP?,which can provide reference for elaborating the material basis of sedative and hypnotic activity as well as screening for sedative and hypnotic components from Diaphragma juglandis Fructus.Methods:?1?Spontaneous locomotor activity and sleep induced by threshold dosage of pentobarbital sodium in mice were used to evaluate the sedative and hypnotic activities of Diaphragma juglandis Fructus.The further research at a molecular level was explored by measuring related neurotransmitters such as gamma-aminobutyric acid?GABA?,dopamine?DA?and norepinephrine?NE?in mice brains with enzyme-linked immuno sorbent assay?ELISA?.?2?The chemical constituents of Diaphragma juglandis Fructus were investigated on the basis of previous research.The compounds from n-butanol extract were isolated and purified by using of SiO₂,Sephadex LH-20,ODS column chromatography,medium pressure liquid chromatography and high performance liquid chromatography.The structures of these compounds were determined on the basis of physical properties,spectra and et al.?3?The possible sedative and hypnotic components in Diaphragma juglandis Fructus which expected to be high efficient and low toxic were presumed by docking with hOX₂R,the target protein of a new insomnia drug Suvorexant,as well as the data mining from TCMSP.Results:?1?Pharmacological studies revealed that the extract of Diaphragma juglandis Fructus could shorten the sleeping latency,prolong their sleeping duration in pentobarbital sodium treated mice and indicate the trend of reducing the spontaneous activities in normal mice.These preliminary results proved the sedative and hypnotic activities of Diaphragma juglandis Fructus.A trend of increasing GABA,decreasing NA and DA content in the brains of mice indicated the sedative and hypnotic activity of Diaphragma juglandis Fructus at a molecular level.?2?A phytochemical investigation on the n-butanol extract of Diaphragma juglandis Fructus resulted in the isolation of eighteen compounds.Their structures as well as two isomeric compounds isolated from EtOAc extract were identified as?2S,3S?-taxifolin-3-O-?-D-arabinofuranoside?1?,?2S,3S?-taxifolin-3-O-?-L-arabinofuranoside?2?,Leeaoside?3?,dihydrovomifoliol-9-O-?-D-glucopyranoside?4?,vomifoliol-9-O-?-D-glucopyranoside?5?,4?-dihydrophaseic acid?-glucopyranose ester?6?,lyoniside?7?,nudiposide?8?,?-?-5?-methoxyisolariciresinol-9?-O-?-D-xylopyranoside?9?,?+?-lyoniresinol-3?-O-?-D-glucopyranoside?10?,akeqintoside A?11?,kaempferol?12?,catechin?13?,naringenin?14?,kaempferol-3-O-?-L-rhamnoside?15?,4-hydroxy-?-tetralone-4-O-?-D-glucopyranoside?16?,4,5-dihydroxy-?-tetralone-4-O-?-D-glucopyranoside?17?,4,5,8-trihydroxy-?-tetralone-5-O-?-D-glucopyranoside?18?,leonuriside A?19?and daucosterol?20?.?3?The hOX2R was applied to dock with the compounds isolated from Diaphragma juglandis Fructus.The results showed that parts of flavonoid glycoside,lignan glycoside and abscisic acid derivatives isolated from Diaphragma juglandis Fructus achieved relatively high total score,indicating the potential activity of orexin receptor antagonist.Among them,abscisic acid derivatives were also found to be closely related to the GABA receptor subunits??1??2??3??6?through data mining from TCMSP.Conclusion:According to the behavioral pharmacology experiments,a certain sedative and hypnotic activities of Diaphragma juglandis Fructus was confirmed.Further research on the regulation of neurotransmitter verified the sedative and hypnotic activities at a molecular level.Phytochemical study on the extract of Diaphragma juglandis Fructus yielded twenty compounds.Among them,compound 1 was a new compound,compounds 2-12 and 15-19were isolated from the plant for the first time.Molecular docking and TCMSP data mining results indicated somg flavonoid glycoside,lignan glycoside and abscisic acid derivatives might be the sedative and hypnotic active ingredients.Thereinto,the abscisic acid derivatives showed great potential sedative and hypnotic activities.This study provided theoretical basis on the application of Diaphragma juglandis Fructus in insomnia and reference for elaborating the material basis of sedative and hypnotic activity as well as screening for sedative and hypnotic components from Diaphragma juglandis Fructus.