Study On Epoxy Monoterpene Eucalyptol Of Mongolian Medicine Sugmul-3 Passing Through The Blood-Brain Barrier And Its Pharmacokinetics In Brain

Objective The aim of this study was to investigate the effects of Sugmul-3 essential oil and its epoxy monoterpene on the permeability of blood-brain barrier（BBB）,to establish a method for the determination of eucalyptol in the brain tissue of rats,and to validate the method.Based on the established in vivo content determination method,the pharmacokinetic study in the brain was completed to explore the material basis of the effect of Mongolian medicine Sugmul-3 on the nervous system,and to reveal the potential medicinal value of eucalyptol,an epoxy monoterpene in Sugmule-3.Methods 1.SD rats were randomly divided into 8 groups:blank control group,PCPA model group,low dose（13 mg/kg）,medium dose（130 mg/kg）,high dose（260 mg/kg）of Sugmule-3 essential oil groups and eucalyptol extract low dose（6.5 mg/kg）,medium dose（65mg/kg）and high dose（130 mg/kg）groups.Except the control group,the rats in other groups were given 300mg/kg 4-Chloro-DL-phenylalanine（PCPA）for 2 consecutive days to establish insomnia model.After the establishment of the model,the rats were given corresponding intervention drugs for 7 days.After the last administration,Evans blue（EB）dye was injected into the tail vein,and the brain tissue of rats was collected and the EB content in brain tissue was determined by fluorescence spectrophotometry to evaluate the opening effect of BBB.The ultrastructural changes of BBB in rat cerebral cortex were observed by transmission electron microscope（TEM）to reveal the effect of Sugmul-3 essential oil and its epoxy monoterpene eucalyptol on the ultrastructure of BBB.The GC-MS/MS analysis technique was used to establish a method for the determination of eucalyptol in the brain tissue of rats,and the methodology was verified.Chromatographic conditions:Agilent J&WHP-5ms column（30m×0.25 mm,0.25?m）,carrier gas is high purity helium with a flow rate of 2.25 m L/min,and collision gas is high purity nitrogen with a flow rate of 1.5 m L/min.Heating program:100C 1.5min;heating up to 280℃,1.5 min at 25℃/min;total running 23.7 min.The forward sample port temperature is 250℃,the split ratio is 5:1 and the injection volume is 1?L.The conditions of mass spectrometry were as follows:ion source temperature 230℃,quadrupole temperature150℃,electron ionization（EI）voltage 70e V,solvent delay time 9min.Multiple reaction monitoring（MRM）mode was used for data acquisition.Eugenol was used as internal standard to quantify eugenol and its methodology was verified.3.After a single dose of 32.5 mg/kg,the brains of SD rats were collected at 0.05、0.5、1、2.5、5、10 and 15 h respectively to complete the brain pharmacokinetics of eucalyptol.Results 1.The amount of EB exudation in brain tissue of rats in each dose group of Mongolian medicine Sugmul-3 essential oil was higher than that in the control group,and the amount of EB exudation in the middle dose of eucalyptol group was significantly higher than that in the control group.TEM showed that the ultrastructure of BBB in the control group was normal,while the ultrastructure of BBB in PCPA group,SHG group and AG group had obvious morphological changes.In PCPA group,the capillary lumen of cerebral cortex was irregular,the connection of endothelial cells was loose,and the astrocytes outside the basement membrane were swollen.SHG group and AG group significantly increased the loose tight junction between endothelial cells and the number of swallowing vesicles.2.Acetonitrile protein precipitation method was used as the pre-treatment method for the determination of brain tissue content.The eucalyptol in rat brain tissue has a good linear relationship within the concentration range of 1～50000 ng/m L,the correlation coefficient is r>0.999,the lower limit of quantification is 1 ng/m L,the extraction recovery is 66.7%-73.4%,the intra-day precision is less than 6.33%,and the inter-day precision is less than 11.03%.At 4℃,the stability RSD within24 hours is less than 5.33%,and the stability RSD of the automatic injector within 12 hours is less than 9.15%.Under the condition of 20℃freezing for 30 days,the stability RSD is less than 9.21%.Under the condition of three freeze-thaw cycles,the stability RSD is less than8.63%.The results show that the stability is good,and the stability RSD is less than 10%.3.After the establishment of a method for the determination of cineole in rat brain tissue,a single dose of eucalyptol was given by intragastric administration to complete the study of brain pharmacokinetics.The pharmacokinetic parameters of eucalyptus oil essence brain were as follows:C_max:3702±400.39 ng/m L;AUC_（0^t）:10238±516.69;t _max:3.06±0.25 h;t_1/2:2:0.5±0.0 h.Conclusions This study confirmed that the essential oil of Sugmul-3 and its epoxy monoterpene eucalyptol can improve the permeability of BBB.The method established in this study for the determination of eucalyptol GC-MS/MS in brain tissue is simple,accurate and sensitive,and provides an experimental basis for the determination of monoterpene essential compounds at a later stage.Eucalyptol has the characteristics of rapid distribution and metabolism in the brain after oral administration.This characteristic reveals the potential medicinal value of eucalyptol and provides experimental data for the development of drug candidates for central nervous system diseases.