A Study On The Effect Characteristics Of Anti-depressive Bioactive Compments Of Chaihu-Shugan-San On Female And Male Stress Rats

Objective:To observe the effect of Chaihu-Shugan-San(CSS)and its absorbed bioactive compounds(ABCs)on depressive behaviors,pharmacokinetics,the blood oxygen level in the brain region depends on the influence of functional magnetic resonance imaging(BOLD-fMRI)and antidepressant-related biochemical indicators of acute(chronic)stress female rats.Based on the parallel comparison of the corresponding male data,the difference antidepressant effects of CSS on male and female rats were discussed.It would provide the theoretical evidence for elucidating the pharmacodynamic basis of the antidepressant effect of CSS and the clinical medication standards of different genders.Method:1.Quantitative detection of 28 chemicals in CSS by UPLC-qTOF-MS,and the standard curve of each component was drawn.The indicators of specificity,linear range,detection limit,limit of quantitation,repeatability and stability of the method and sample recovery were determined.Analytical column was a Waters BEH C18(100 × 2.1 mm,1.7 μm)column,maintained at 35℃;the mobile phases were 0.1%formic acid water(A)and acetonitrile(B),gradient elution:0～1 min,90%～90%A;1～3.5min,90%～80%A;3.5～5.5min,80%～74%A;5.5～21min,74%～5%A;21～25min,5%～5%A;25～25.1min,5%～90%A,flow rate was 0.4 mL/min;injection volume was 2μL;mass spectrometry used electrospray ionization source(ESI)for positive ion mode detection,the extracted ion current monitoring mode is used for quantitative determination.2.Four Sprague Dawley(SD)rats weighing 215.20±3.41g were randomly divided into the blank group(treated with 1mL/100g saline)and CSS 30g/kg group,(n=2,in each group).Bloods were taken from the posterior orbital venous plexus of rats 30 minutes after administration.After precipitating protein with methanol,UPLC-qTOF-MS method was used to analyze the main absorbed components in bloods of the SD rats gavaged by CSS.3.Antidepressive behavior experiment:The experiment was divided into 2 groups,acute stress group(AS group)and chronic unpredictable mild stress group(CUMS group).In the acute stress group,48 female rats and 48 male rats were randomly divided into 8 groups,respectively:blank control group,model group,positive control group(fluoxetine group),CSS low-dose group,CSS medium Dose Group,CSS high-dose group,10ABCs group and 22ABCs group,(n=6,in each group).In CUMS group,30 female rats and 30 male rats were randomly divided into 5 groups,respectively:blank control group,model group,positive control group(fluoxetine group),CSS normal dose group and 10 ABCs group,(n=6,in each group).The indexes such as the immobility time of swimming for 5 minutes and the moving distance of open field were detected and analyzed statistically.4.Six male and six female SD rats weighing 223.20±4.50 g were fasted for 12 hours before forced swimming for 15 minutes to build the acute stress model,then CSS 30g/kg were gavaged.Approximately 0.3 mLml of blood was drawn through the retro-orbital venous plexus at 5,10,15,20,30,45,60,90,120,180,240,360,480,600,and 720 minutes after intragastric administration.The sulfamethoxazole and schisandra alcohol were used as double internal standards.After protein precipitation by methanol,the plasma concentration of each time point was determined by UPLCqTOF-MS method.The pharmacokinetic parameters were calculated by WinNonlin 6.4 software.5.BOLD-fMRI experiment:The experiment was divided into healthy group,forced swimming group(FST 组)and FST+30g/kg dose of CSS group,with 6 male and 6 female rats in each group.Using the Bruke 7.0 nuclear magnetic instrument of the Molecular Imaging Center of Southeast University to take nuclear magnetic images),and the spmratIHEP tool kit was used for data image analysis.To observe the changes of blood oxygenation level in the brain regions of rats before and after the modeling and the intervention of Chaihu-Shugan-San.In addition,to analysis the changes in hippocampus and thalamus accurately.6.Enzyme-linked immunosorbent assay:After the behavioral experiments,the rats were anesthetized to take whole blood,hippocampus and other samples.They were stored in a refrigerator at-80℃,after pretreatment.In serum and hippocampus samples,CRH,ACTH,E2,5-HT,DA,NE,GHR,BDNF were measured.The differences among the healthy group,the model group,the Chinese medicine group,and the 10 ABCs group and the differences between male and female were compared and analyzed.Result:28 components of Chaihu Shugan San Decoction were quantitatively analyzed,and the chromatographic peaks of 28 monomer components were well separated without obvious peak interference.The linear regression in the linear detection range(r>0.99),the repeatability and sample recovery rate were good.The relative standard deviations were small(RSD<4.70%),and the decoction samples were stable within 24 hours.In the anti-depressive experiment,the immobility time of the model group increased when compared with the normal group,it proved that the acute stress model was built successfully(P<0.01).while the female rats showed lower activity in the CUMS model experiment(P<0.05);Compared with the model group,immobility time was inversely proportional to the dose of CSS.When different concentrations of CSS decoction were administered,the immobility time decreased in this concentration range in a dosedependent manner,under the acute stress model,CSS Decoction had the best curative effect on reducing the immobility time of rats when it was administered at 30g/kg(P<0.01),At the same time,from the level of acute and chronic stress experiments,it reveals to a certain extent that ABC can represent more than 90%of the therapeutic effect of CSS.In addition,the pharmacokinetics of ferulic acid,naringin,hydrated orange lactone,nobiletin and hesperidin in rats after acute stress stimulation showed significant differences in male and female.In BOLD-fMRI experiment,we found that after stress modeling,blood oxygen levels in the hippocampus and thalamus of male rats were increased,while decreased in female.However,after CSS administration,the blood oxygen level in the hippocampus of male rats increased while the blood oxygen level in the thalamus decreased;the blood oxygen level in hippocampus and thalamus of female rats increased.In the depression-related biochemical experiments,compared with the healthy group we found that after the acute and chronic stress modeling,the CRH and ACTH contents in the male and female rats in the model group increased significantly in serum and hippocampus of both the male and female rats,while E2,DA,NE,5-HT,GHR,BDNF and TrkB were decreased.However,the content of various substances in serum and hippocampus of male and female rats were changed after the treatment with CSS decoction and its representative pharmacodynamics 10 ABCs.The opposite trends appeared in the above data,and the substance content in different sex rats also showed significant differences.The experimental data above provided a reference basis for the clinical male and female drug specifications of CSS.Conclusion:1.This experiment successfully established a UPLC-qTOF-MS analytical method for simultaneous determination of 28 phtochemicals such as ferulic acid and hydrated orange lactone in Chaihu Shugan San.The method is simple,rapid,and sensitive,which provides a basis of gavage dose for pharmacodynamic studies.2.The 22 components of plasma in SD rats were successfully separated and characterized after the administration of Chaihu-Shugan-San(30g/kg),which provided the evidence for the pharmacodynamic study.3.The degree of depression in male and female rats was significantly different after stress.At the same time,10ABCs exerted anti-depressive effect similar to its parent prescription ChaihuShugan-San,and the pharmacodynamics of Chaihu-Shugan-San were preliminarily elucidated.4.This experiment successfully established a UPLC-qTOF-MS analysis method for the determination of l0ABCs in plasma of rats with acute stress in rats with Chaihu Shugan San.This method had strong specificity and high sensitivity and could be used for the pharmacokinetic study of Chaihu-Shugan-San after intragastric administration.At the same time,the pharmacokinetic parameters of some absorbed components in blood were found to be statistically differences in gender,which provided the data support for further revealing the gender differences in the antidepressant efficacy of Chaihu-Shugan-San.5.This experiment used the BOLD-fMRI technology,visually reflected the influence of ChaihuShugan-San on the changes of blood oxygen levels in hippocampus and thalamus in male and female rats after stress.Moreover,the effect of Chaihu-Shugan-San on emotion regulation was verified.This technology had great potential in the pathological localization of depression,the monitoring of depression status,the efficacy of antidepressant therapy and prognosis.6.In this part of the experiment,the method of enzyme-linked immunosorbent assay was used to verify the antidepressant effect of Chaihu-Shugan-San and 10ABCs from the perspective of hormone secretion and changes of brain proteins and their receptors,and it strongly proved that there was a significant difference in the biochemical indicators of depression between males and females.