Isolation And Identification Of The Antioxidant,Sedative And Hypnotic Active Components Of Diaphragma Juglandis Fructus And Mechanism Research

Diaphragma juglandis Fructus(DJF)is the wooden diaphragm of walnut fruit,referred to as distracting wood,also known as walnut diaphragm and walnut valve.The main components of DJF include phenols(2.39%?3.54%),saponins(1.86%?5.98%)and other substances,and its biological activities include strengthening the spleen and kidney,anti-oxidation,antibacterial,and anti-tumor.As the pace of life accelerates and social pressures intensify,the spectrum of human diseases has undergone major changes.Chronic diseases and sub-health have become the main factors endangering human health which were closely related to oxidative stress and sleep disorders.Therefore,this article focused on clarifying the active ingredients in DJF that have antioxidant,sedative-hypnotic functions and preliminary exploration of their related mechanisms.The specific content was as follows:1.Determine the optimal extraction conditions for the extract of DJF and evaluate the antioxidant,sedative and sedative-hypnotic activities of the crude extract of DJF.The optimal extraction process for the crude extracts of DJF was determined:the material-liquid ratio(m/v)was 1?10,and the extraction time was 4 h,extraction temperature was 60°C,ethanol concentration was 70%,the number of extractions was 2 times.The above conditions were adopted to extract the alcohol extract of DJF(yield 21.75%);the filter residue was extracted with deionized water to obtain an aqueous extract of DJF(yield 17.80%).The aqueous extract was treated with alcohol precipitation to prepare an alcohol-soluble phase and an alcohol precipitation phase.The alcohol-soluble phase and the alcohol extract were mixed to obtain a mixed extract,and then obtained three components by loading the AB-8 macroporous adsorption resin column,which were the water eluting component,the 30%ethanol eluting component,and the 70%ethanol eluting component.The results of free radical scavenging experiments showed that the 30%ethanol eluted fraction had in vitro antioxidant activity(IC_50-DPPH=7.89?g/m L;IC_50-ABTS=50.59?g/m L).The experimental results of the mouse behavioral model showed that the water-eluted fraction had sedative-hypnotic activity:it could reduce the mouses'movement speed and time(p<0.05),increase the staying time in the central area;increase the natural sleep time(p<0.01),shorten the sleep latency induced by sodium pentobarbital by 24.62%(p<0.05),and prolong sleep duration by 61.49%(p<0.05);the trends of the two were consistent with the positive control.2.Separation,purification and structure identification of anti-oxidant active components of DJF.According to the results of antioxidant experiments in vitro,the 30%alcohol elution component separated from the mixed extract by macroporous resin was separated by MCI column and ODS column in sequence.Finally,four monomer compounds were obtained and identified by nuclear magnetic resonance(NMR):(1'-methyl-2'-hydroxy)propane-O-?-D-glucopyranoside(1),(4'-hydroxyphenyl)methylene-O-?-D-glucopyranosyl-?-L-arabinopyranoside(2),p-hydroxycinnamicacid(3),2-carboxy-5,7-dihydroxy-3-naphthyl-?-D-glucopyranoside(CDG)(4).In view of the results of in vitro antioxidant experiments,the order of antioxidant capacity was:compound4>compound 3>compound 2>compound 1.3.The biological effects of CDG on the antioxidant activity in Caenorhabditis elegans were analyzed by multiple indicators,and its antioxidant mechanism was explored based on metabonomic analysis methods.Basic experiment results of nematodes showed that CDG could significantly extend the lifespan of nematodes,reduce the accumulation of lipofuscin,and increase the ability to resist stress in a dose-dependent manner.This was due to the increase of the activity of superoxide dismutase(SOD)and catalase(CAT)and the decrease in ROS accumulation and MDA content.The metabolomics results showed that compared with the control group,the metabolism of nematodes changed significantly after CDG treatment,and the levels of multiple metabolites were significantly up-regulated or down-regulated,indicating that CDG could affect the metabolic activities of the nematodes.Enrichment of metabolic pathways found significant changes in amino acid metabolism,glycerolipid metabolism,starch and sucrose metabolism,aminoacyl-t RNA biosynthesis and other pathways after CDG treatment.4.Separation,purification and identification of active ingredients in the sedative and hypnotic of DJF.The water-eluted component separated from the mixed extract by the macroporous resin were separated through MCI column.Through the mouse behavior model,it was concluded that the water-eluted component of the MCI column had the effect of sedation and hypnosis.The eluted fractions were separated and purified by ODS column,and finally four monomer compounds were obtained,which were identified by NMR as follows:1,5-anhydro-D-glucitol(5),3-hydroxy-4-iminobutyric acid(6),(1'-methyl-2'-hydroxy)propane-O-?-D-glucopyranoside(7),6-hydroxy-1,2-naphthyl(2-O-?-D-glucopyranoside)(8).The results of animal behavior experiments showed that compound 5 significantly prolonged sleep time(natural sleep duration increased by 19.87%;pentobarbital sodium induced sleep duration increased by 87.00%(p<0.05));compound 6 could not only prolong the sleep time and shorten the sleep latency(p<0.05),but also reduce the speed and time of mice movement and increase the residence time in the central area(p<0.05);compound 7 and 8 have no obvious difference..5.Multi-index preliminary analysis of the sedative and hypnotic mechanism of monomer compounds.UPLC-MS measurement of neurotransmitters showed that:compounds 5 and 6significantly reduced the levels of serotonin(5-HT)and glutamic acid(Glu)in serum or brain tissue(p<0.05),significantly increased the content of?-aminobutyric acid(GABA)(p<0.05);the result of GC-TOF/MS determination of fecal short-chain fatty acids(SCFAs)showed that after intragastric administration of compounds 5 and 6,the total content of SCFAs in the feces of mice increased significantly(p<0.05),especially acetic acid and propionic acid.The mechanism of sedation and hypnosis might be mediating 5-HT and GABA ergic systems to affect the body's sleep-wake rhythm,and might be closely related to intestinal microbes and their metabolites.