Study On The Material Basis And Action Mechanism Of Nelumbinis Plumula To Clear Away The Heart-heat And Tranquillize

Nelumbinis Plumula is a genus of Nelumbo Nucifera Gaertn in the Family Nelumbinaceae.The dried young leaves and radicles in mature seeds have the effect of clearing away the heart-heat and tranquilizing,restoring normal coordination between heart and kidney,astringent essence and hemostasis.It is used for heat entering the pericardium,delirium,disharmony between heart and kidney,insomnia spermatorrhea,heat in blood and hematemesis.The pharmacological research of Nelumbinis Plumula mainly focuses on cardiovascular protection and anti-tumor,but there are few studies on the treatment of insomnia,and the mechanism of Nelumbinis Plumula treatment of insomnia has not been reported.The main active components of Nelumbinis Plumula were alkaloids and flavonoids,and isoquinoline alkaloids were the main structural types of alkaloids.Orexin receptor antagonistsare a new kind of sleeping drugs,one of which belongs to tetrahydroisoquinoline compound.Therefore,it is speculated that Nelumbinis Plumula may exert a sedative effect by inhibiting the activity of orexin receptor.In order to reveal the material basis and mechanism of Nelumbinis Plumulato clear away the heart-heat and tranquilize,this study mainly completed the following work:1.Molecular docking technique was used to screen the active compounds acting on orexin receptor in Nelumbinis Plumula.Molecular docking technology was used to evaluate of the interaction between isoquinoline alkaloids and orexin receptor in Nelumbinis Plumula.The results showed that these alkaloids interacted with orexin receptor.Among them,neferine,liensinine and isoliensinineto orexin receptor was equivalent to that of orexin receptor antagonist almorexant,which was significantly higher than other alkaloids.By analyzing the interaction between the three alkaloids and the orexin receptor 2,we found that they can form hydrogen bonds,ion-πandπ-πinteraction with amino acid residues in the receptor protein pocket.In addition,because there are many hydrophobic groups in the molecular skeleton of the three compounds,they are more likely to interact with the hydrophobic amino acid residues in the pocket.Therefore,it is speculated that the alkaloids in Nelumbinis Plumula can play the role of clearing away the heart-heat and tranquilizing by inhibiting the activity of orexin receptor,and tetrahydroisoquinoline alkaloids are the main active components.2.The extraction process of Nelumbinis Plumulaby heating reflux method was determined.Taking the extraction rate of neferine as the index,through single factor experiment and response surface test,it is determined that the best process of heating and reflux extraction of Nelumbinis Plumula is as follows:solid-liquid ratio of 1:15,reflux time of 1.5h,ethanol volume fraction of 70%,reflux times of 2 times,and the extraction rate of neferine was about 90%.3.The purification process of total alkaloid from Nelumbinis Plumula was determined.Taking the transfer rate of neferine as the index,the purification effects of acid solution-alkali precipitation method and macroporous resin adsorption method on total alkaloid from Nelumbinis Plumula were investigated.It was determined that the purification method of total alkaloids was acidsolution-alkali precipitation method.The specific process was as follows:take the water extract of Nelumbinis Plumula,extract with petroleum ether to remove fat soluble components,add 10%HCl to the aqueous phase to adjust to p H 3,filter,add saturated Na₂CO₃solution to the filtrate to adjust to p H 10,stand for 24 hours,filter and collect the filter residue.The filtrate is extracted with dichloromethane until colorless,combined with dichloromethane extract,evaporated to dryness under reduced pressure,and combined with the above filter residue to obtain the total alkaloids of Nelumbinis Plumula.4.Preparation of neferine.The total alkaloid of Nelumbinis Plumula was prepared by repeated silica gel column chromatography and ethanol-water recrystallization.According to the physicochemical properties and spectral characteristics,the obtained compound was identified as neferine.5.Tranquilizing effect of neferine.The hypnotic effect of neferine was suprathreshold dose and subthreshold dose of pentobarbital sodium combined with sleep experiment.The results showed that different doses of neferine could shorten the sleep latency,prolong the sleep duration and improve the incidence of sleep in mices.6.Effect of neferine on orexin receptor m RNA level in insomnia model mices.The mice model of insomnia was established by DL-4-chlorophenylalanine.Different doses of neferine were given by gavage.The orexin receptor gene expression in different tissues of mices was detected by real-time fluorescence quantitative PCR.The results showed that neferine could significantly reduce the levels of OX1R m RNA in the mice cardioid,adrenal gland,cardioid and pancreas,as well as the OX2R m RNA in cardioid,adrenal gland and cardioid.7.Effect of neferine on the content of sleep related neurotransmitters in serum of insomnia model mices.The insomnia mice model was established by DL-4-chlorophenylalanine.Different doses of neferine were given by gavage.The content of 5-HT,GABA,Glu and NE in mice serum were detected by enzyme-linked immunosorbent assay.The results showed that the contents of 5-HT and GABA in serum of mice were significantly increased,while the content of NE and the ratio of Glu/GABA were significantly decreased.