| OBJECTIVE: In this project,Mongolian medicine sugmel—3 volatile oil was selected as the research object.Based on the determination of chemical components and main index components in vivo and in vitro,combined with pharmacodynamic screening in the treatment of insomnia,the effective substance basis of Mongolian medicine sugmel—3 volatile oil was revealed and its quality control was studied,so as to provide scientific basis for further development and research of this prescription.METHODS : 1.The essential oils of Mongolian medicine sugmel—3,cardamom,caryophyllum and Piper longum were prepared and identified by GC-MS.Gas phase conditions: HP-5 capillary column(30mm × 0.25 mm × 0.25 μm),injection port temperature 270℃,carrier gas: helium,carrier gas: flow rate 1 ml/min,split ratio:10∶ 1,injection volume: 1μL.Temperature programming: the column temperature was 40 ℃,maintained for 1 min,increased to 60℃ at the rate of 5℃/ min,increased to 100℃at the rate of 8℃/ min,maintained for 8 min,increased to 280℃ at the rate of 10 ℃/ min,maintained for 3 min.Mass spectrometric conditions: ionization source(EI),temperature of ion source: 230℃,solvent delay: 5 min,ionization energy: 70 EV,scanning mass: 40-650 M/Z.chemical constituents were identified by NIST library search system.On the basis of studying the chemical constituents in vitro,rats were divided into five groups: blank group,sugmel—3 group,and volatile oil group.The rats were given 40 times of the clinical dose.The blank serum,containing serum and brain tissue were taken 30 min,60 min and 90 min after administration.Serum and brain tissue fluid were pretreated,and the above chromatographic conditions were used to determine the components of sugmel—3 volatile oil into blood and brain.2.On the basis of the identification of the essential oil of sugmel—3,the contents of eucalyptol and cuminaldehyde were determined by GC.The chromatographic conditions were as follows: Dimensions SH-Rtx-capillary column(30 mm×0.25 mm×0.25μm),carrier gas: high purity helium,injection port temperature: 270℃,carrier gas flow rate: 1 ml / min,temperature programmed: 80℃(1 min)→ 200℃(30℃/ min-15 min),injection volume: 1μL,flow rate: 1.0 ml/ min.3.The mice were randomly divided into blank group,positive control group,high-dose and low-dose sugmel—3 groups,high-dose and low-dose sugmel—3groups,high-dose and low-dose sugmel—3 groups,and high-dose and low-dose sugmel—3 groups.The mice were put into the autonomic activity tester to adapt for 2min.1h after administration,the number of animal activities and standing times in05 min were recorded.One hour after the last administration,each rat was intraperitoneally injected with pentobarbital sodium 50 mg/kg.The sleep latency(righting reflex disappearance time)and sleep duration(righting reflex disappearance to recovery time)were observed and recorded.the rats were randomly divided into blank group,model group,positive control group,high and low dose groups of sugmel—3 Decoction volatile oil,high and low dose groups of single drug volatile oil and high and low dose groups of single drug pair volatile oil to prepare PCPA insomnia model.The contents of 5-HT,GABA,IL-1β,SOD and MDA in brain tissue of rats in each group were detected by ELISA.The data were analyzed by SPSS.22.0Learn software for data analysis.The measurement data were expressed as(mean ±standard deviation).The differences among the groups were analyzed by one-way ANOVA,and the differences between the groups were analyzed by t-test.RESULTS: 1.A total of 19 chemical components were identified from the essential oil of sugemule-3,and the main components were monoterpenoids.These include α-Pinene β-Pinene,myrcene,o-isopropyltoluene,eucalyptol γ-Terpinene,linalool,terpineol α-Terpineol,3-p-menthen-7-al,cuminaldehyde α-Terpinene-7-al,1,4-p-menthylene-7-aldehyde α-Sandalene,caryophyllene,humus,gimacline D β-There are 19 chemical constituents,such as red myrrhene,8-heptadecene,etc.The essential oil of Mongolian medicine sugemule-3 absorbed into blood were eucalyptol and its metabolites 2-oxabicyclo [2,2,2] octanol,1.3.3-trimethyl-,benzoic acid,4-(1-methylethyl)-,methyleste and methylone,(1-hydroxycyclohexyl)phenyl-.Two chemical constituents were identified from brain tissue characteristic maps,mainly eucalyptol and its metabolites 2-oxabicyclo [2,2,2] octanol,1.3.3-trimethyl-.2.Eucalyptoly=869874x-10366(r=0.9999),Cuminaldehyde y = 821871x-4001.8(r = 0.999 9).The linear range of Eucalyptol was39~635 μg/ml and Cuminaldehyde was136~2187μg/ml.The average recoveries were 98.51% ~ 110.11%(RSD = 3.87%,n = 6),95.7% ~ 107,82%(RSD = 4.12%,n = 6),5.4885mg/g for Eucalyptol and1.0690mg/g for Cuminaldehyde.3.The influence on mice’s independent activity: each single drug and volatile oil of sugmel—3 decoction can significantly reduce the activity time and standing times of mice.In the influence of hypnotic dose of pentobarbital sodium on the sleep time of mice: each single drug and volatile oil of sugme—3 decoction can shorten the sleep latency and prolong the sleep time.In the aspect of the effect on brain tissue neurotransmitters of PCPA insomnia rats: the single drug and volatile oil group can increase the content of GABA and 5-HT in PCPA insomnia model rats.In the aspect of the effect on the related cytokines in the brain tissue of PCPA insomnia rats: the compound,single drug and drug pair groups of sugmel—3 volatile oil had obvious effect on IL-1 β and other cytokines.The content of SOD was increased and the content of MDA was decreased in the three groups,but the difference was not obvious.CONCLUTION: 1.Sugmel—3 volatile oil mainly contains terpenoids represented by eucalyptol and cuminaldehyde.Judging from the absorption components of serum and brain tissue,eucalyptol and cuminaldehyde may be the basis for the potential effective substances of Sugmel—3 volatile oil.2.The content of eucalyptus essential oil of Sugmel—3 is 5.4885mg/10 mg,and3.the content of cuminaldehyde is 1.0690mg/10 mg.4.Sugmel—3 volatile oil has sedative and hypnotic effects.Its hypnotic mechanism may be related to central neurotransmitters and regulating cytokines. |
PDF Full Text Request