| Zhi-Zi-Hou-Po decoction is comprised of Gardenia jasminoides Ellis,Citrus aurantium L.and Magnolia officinalis Rehd.et Wils..In this study,the antidepressant-like effects of Zhi-Zi-Hou-Po decoction on depression rats were evaluated.Moreover,therapeutic constituents basis was identified in Zhi-Zi-Hou-Po decoction and in rat serum.In addtion,the metabolites related with depression were evaluated in rat plasma after treatment with Zhi-Zi-Hou-Po decoction,and the related neurotransmitter pathway was analyzed in rat hippocampus.Furthermore,the pharmacokinetics and pharmacodynamics(PK-PD)of iridoid glycosides in rat plasma was charaterized and applied to a comparative study in normal and depressive rats.In a word,the studies provide the theoretical foundation for therapeutic basis and antidepressant mechanism of Zhi-Zi-Hou-Po decoction.1.Antidepressant-like effect of Zhi-Zi-Hou-Po decoction in a rat model of depressionAntidepressant-like effects of Zhi-Zi-Hou-Po decoction(3.66,7.32 and 14.64 g/kg/day)on CUMS-induced rats were evaluated in a rat model of chronic unpredictable mild stress through behavioral tests,neuroendocrine system and hippocampal neurogenesis.The results demonstrated that Zhi-Zi-Hou-Po decoction significantly reversed the depressive-like behaviors,involving decrease in coat state test,increase in sucrose preference test,decrease in the duration of immobility in the forced swimming test,increase in the duration of center and non-periphery zone,reduction in the duration of periphery zone in the open-field test compared with the CUMS group.In addition,Zhi-Zi-Hou-Po decoction produced a significant increase in the percentage of dexamethasone induced plasma corticosterone suppression and significant decrease in plasma ACTH and CORT levels.Moreover,Zhi-Zi-Hou-Po decoction significantly increased the BDNF,DCX and BrdU/NeuN levels compared with the CUMS group.The above results revealed that Zhi-Zi-Hou-Po decoction exerted antidepressant like effects on CUMS-induced rats.2.Indentification of chemical components in vivo and in vitroAn ultra high performance liquid chromatography-Q Exactive mass spectrometer method was established to identify multi-components of Zhi-Zi-Hou-Po decoction and the constituents in rat plasma after oral administration of Zhi-Zi-Hou-Po decoction.93 compounds in Zhi-Zi-Hou-Po decoction were identified or tentatively characterized,among the identified compounds,35 compounds from Gardeniae Fructus,19 compounds from Aurantii Fructus Immaturus and 39 compounds from Magnoliae Officinalis Cortex.The compounds in the Gardeniae Fructus were mainly iridoid glycosides,monoterpenoids,glycosides of crocetin,organic acids and flavonoids.The compounds in the Aurantii Fructus Immaturus were mainly flavonoids and limonin.The compounds in the Magnoliae Officinalis Cortex were mainly lignans and phenylethanoid glycosides.On the basis of serum pharmacochemistry,the absorbed components and metabolites in rat plasma after administration of Zhi-Zi-Hou-Po decoction were identified.A total of 56 constituents,including 32 prototype compounds and 24 metabolites were identified or tentatively charaterized.Results indicated that the prototype compounds in the Gardeniae Fructus were mainly iridoid glycosides,monoterpenoids,the prototype compounds in the Aurantii Fructus Immaturus were mainly flavonoids and the prototype compounds in the Magnoliae Officinalis Cortex were mainly lignans and phenylethanoid glycosides.The metabolites were classified as iridoid glycoside-related metabolites,flavonoid-related metabolites and lignan-related metabolites.The above results preliminarily clarify the therapeutic basis and provide essential data for further studying the relationship between the chemical components and pharmacological activity of Zhi-Zi-Hou-Po decoction.3.The effect of Zhi-Zi-Hou-Po decoction on metabolites in a rat model of depressionA liquid chromatography-mass spectrometry(LC-MS)method was developed for simultaneous determination of metabolite targets:tryptophan(Trp),tyrosine(Tyr),phenylalanine(Phe),hippuric acid(HA),glutamic acid(Glu),γ-aminobutyric acid(GABA),creatinine(Cr),indole-3-acetic acid(IAA)and phenaceturic acid(PA),as well as to study the potential antidepressant mechanism of Zhi-Zi-Hou-Po decoction based on holistic view of an organism.After the addition of internal standard tinidazole,the plasma samples were subjected to protein precipitation with acetonitrile,and then evaporation to dryness and reconstitution.The analysis was performed on a CAPCELL PAK C18 column with methanol and 0.01%formic acid in water using gradient elution.The method showed a good linearity with the other validation parameters for specificity,extraction recovery,matrix effect,precision,accuracy and stability were within acceptance range.The validated method was applied to the comparative study of metabolite targets in control group,CUMS group,Zhi-Zi-Hou-Po decoction(3.66,7.32 and 14.64 g/kg)groups and fluoxetine group(0.015 g/kg).The results demonstrated Trp,Phe,Tyr,IAA,HA,Cr,Glu and SA levels were significant lower in model group,while PA and GABA were significant higher than those in control group.The rats treatment with Zhi-Zi-Hou-Po decoction showed a tendency of bringing the levels of all these metabolite targets to normal except Cr and Glu.The above results revealed that Zhi-Zi-Hou-Po decoction exerted antidepressant like effects possibly through modulating the disorders of amino acid metabolism related with neurotransmitters synthesis and gut microflora metabolism related with irritable bowel syndrome.4.The effect of Zhi-Zi-Hou-Po decoction on neurotransmitter pathway in a rat model of depressionAn ultra fast liquid chromatography-mass spectrometry(UFLC-MS/MS)method was developed for simultaneous determination of Trp,Tyr and 5-hydroxytryptamine(5-HT),and ELISA was used for determination of dopamine(DA),tryptophan hydroxylase(TPH)and tyrosine hydroxylase(TH),as well as to study the potential antidepressant mechanism of Zhi-Zi-Hou-Po decoction based on the pathway of neurotransmitter synthesis.After the addition of internal standard tinidazole,the hippocampus samples were subjected to protein precipitation with acetonitrile-methanol(75:25,v/v),and then separated on a CAPCELL CORE PC HILIC column with acetonitrile and 5 mM AcNH4/0.1%formic acid in water using gradient elution.The method showed a good linearity with the other validation parameters for specificity,extraction recovery,matrix effect,precision,accuracy and stability were within acceptance range.The validated method was applied to the comparative study of targets related with neurotransmitters synthesis in control group,CUMS group,Zhi-Zi-Hou-Po decoction(3.66,7.32 and 14.64 g/kg)groups and fluoxetine group(0.015 g/kg).The results demonstrated Trp,Tyr,5-HT,DA,TPH and TH were significant lower in model group than those in control group.The rats with Zhi-Zi-Hou-Po decoction treatment showed a tendency of bringing the levels of all these targets to normal.The above results revealed that Zhi-Zi-Hou-Po decoction exerted antidepressant like effects possibly through increasing Trp and Tyr levels,elevating TPH and TH activity,promoting 5-HT and DA synthesis,raising the levels of 5-HT and DA in the synaptic cleft.5.The study of pharmacokinetics and pharmacodynamics of iridoid glycosides from the Zhi-Zi-Hou-Po decoctionA liquid chromatography-mass spectrometry(LC-MS)method was developed to characterize the pharmacokinetics(PK)of geniposide,geniposidic acid,genipin-l-β-gentioboside,scandoside methyl ester and pharmacodynamics(PD)of tryptophan(Trp),tyrosine(Tyr),phenylalanine(Phe)in rat plasma.After the addition of salidroside as PK internal standard and tinidazole as PD internal standard,the plasma samples were subjected to protein precipitation with acetonitrile,and then evaporation to dryness and reconstitution.The analysis was performed on a CAPCELL PAK C18 column with methanol and 0.1%formic acid in water using gradient elution.The method showed a good linearity with the other validation parameters for specificity,extraction recovery,matrix effect,precision,accuracy and stability were within acceptance range.The validated method was applied to compare the PK and PD differences in control and chronic unpredictable mild stress(CUMS)induced depressive rats orally administrated with Zhi-Zi-Hou-Po decoction.Receiver operating characteristic(ROC)curve was used to evaluate the specificity and sensitivity of the PD markers.And the relationship of PK-PD was analyzed.The results indicated that the Tma,in geniposide and genipin-l-β-gentioboside were lower than that in geniposidic acid and scandoside methyl ester,suggesting the absorption of geniposide and genipin-l-β-gentioboside were slower than geniposidic acid and scandoside methyl ester.In addition,the AUC0-t,AUC0-∞ and Cmax of only genipin-l-β-gentioboside in the CUMS group significantly decrease,however,there were no significant difference for pharmacokinetics parameters of other iridoid glycosides.ROC results showed that Tyr was more sensitive than Trp and Phe as marker.The PK/PD profile provided the time courses of four iridoid glycosides and Tyr.The above results revealed that the structure and polarity of compound,the pathological state of depression might have an effect on the difference of PK-PD.The method will provide deeper insights into therapeutic basis and antidepressant mechanism to assist in clinical rational usage. |
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