Priliminary Study Report On The Theraputic Material Basis Of Zhi-zi-hou-po Decoction In The Treatment Of Depression

Objective:To develop an ultra-fast liquid chromatography with tandem mass spectrometry(UFLC-MS/MS)method for simultaneous determination of geniposide,genipin gentiobioside,honokiol,magnolol,isonaringin,naringin,hesperidin,neohesperidin,naringenin and hesperetin in Zhi-Zi-Hou-Po Decoction(ZZHPD),and provide basis for quality control of ZZHPD.To establish an ultra high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UHPLC-Q-TOF-MS/MS)method was applied to com-prehensively detect and identify the chemical components of ZZHPD in vitro,an-d preliminary classify the in vitro chemical composition spectrum for its efficacy.The absorbed components and their metabolites in rat plasma and the brain of rats after oral administration of ZZHPD were identified and characterized through the above analysis method,and to preliminarily characterize of the chemical basis in vivo for the treatment of depression.Methods1.The contents of ten components were determined by UFLC-MS/MS.ch-romatographic separation was performed on Shim-Pack XR-ODS column(75 mm×3.0 mm,i.d.2.2 μm).The mobile phase consisted of 0.1%formic acid(A)and acetonitrile(B)in water at a flow rate of 0.5 mL/min with gradient elution(0-10 min,10%B→30%B;10-12 min,30%B→60%B;12-18 min,60%B→85%B)at 254 nm,and the equilibration time was 5 min.The column temperature was maintained at 30℃.Detection was performed on 3200 QTRAP mass spectrometry equipped with electrospray ionization source in negative ionization mode.Quantification was performed using multiple reactions monitoring(MRM)mode.2.The chemical compositions of ZZHPD were determined by UHPLC-Q-TOF-MS/MS.Chromatographic separation was performed on Shim-Pack XR-ODS column(75 mm×3.0 mm,i.d.2.2 μm).The mobile phase consisted of 0.1%formic acid(A)and acetonitrile(B)in water at a flow rate of 0.5 mL/min with gradient elution(0～10 min,10%B→30%B;10～12 min,30%B→60%B;12～18 min,60%B→75%B;18～25 min,75%B→90%B)at 254 nm,and the equilibration time was 15 min,the column temperature was maintained at 30℃.Mass spectrometric detection was performed with electrospray ionization source(ESI)in both positive and negative modes.The parameters of the mass spectrometer under the ESI mode were as follows:ion spray voltage:+5500 V/-4500 V;source temperature 550℃;curtain gas 35 psi;declustering potent-ial 40 V.Full-scan MS data were collected from 100 to 1250 Da in positiveand negative ion mode with collision energy of 20-60 eV.3.The chemical constituents of ZZHPD were studied using the same analytical method as in vitro studies,and the absorbed components and their metabolites in rat plasma after oral administration of ZZHPD were identified and characterized by comparing the chromatograms of ZZHPD,blank plasma and dosed plasma at different sampling time.4.The chemical components of ZZHPD into brain were studied using the same analytical method as in vivo studies,and the absorbed components and metabolites in the brain of rats after oral administration of ZZHPD were identified and characterized by comparing the chromatograms of ZZHPD,blank brain tissue and dosed brain tissue at different sampling time.Results1.The linear ranges were 1～40 μg/mL(r=0.9996)for geniposide,0.2～8 μg/mL(r=0.9997)for genipin gentiobioside,0.1～4 μg/mL(r=0.9993)for honokiol,0.1～4μg/mL(r=0.9997)for magnolol,0.2～8 μg/mL(r=0.9995)for isonaringin,1～40μg/mL(r=0.9997)for naringin,0.2～8 μg/mL(r=0.9985)for hesperidin,1～40μg/mL(r=0.9997)for neohesperidin,0.1～4 μg/mL(r=0.9992)for naringenin and 0.02～0.8 μg/mL(r=0.9994)for hesperetin.The average recovery(n=9)of the ten components were 98.0%,98.5%,96.9%,98.8%,98.4%,99.3%,99.4%,98.7%,98.9%and 97.9%,respectively.The content ranges of ten components in six batches were 4.51%-4.83%for geniposide,0.805%-0.841%for genipin gentiobioside,0.125%-0.134%for honokiol,0.133%-0.144%for magnolol,0.403%-0.435%for isonaringin,5.54%-5.84%for naringin,0.210%-0.222%for hesperidin,3.98%-4.30%for neohesperidin,0.0592%～0.0660%for naringenin and 0.0213%～0.0223%for hesperetin.2.A total of 88 compounds were identified or tentatively characterized.2 iridoid glycosides,6 flavonoids,2 lignans and 1 Organic acids were identified by comparing the known primary and secondary component information information of the reference substance.The structure of 77 unknown compounds except for reference.3.40 compounds were detected in dosed plasma after intragastric administration of ZZHPD,including 29 prototypical components and 11 metabolites.9 compounds were identified by comparing the known primary and secondary component information of the reference substance.The structure of 31 unknown compounds were speculated except for reference.4.14 compounds were detected in dosed brain tissue after intragastric administration of ZZHPD,including 10 prototypical components and 4 metabolites.6 compounds were identified by known primary and secondary component information of the reference substance.The structure of 8 unknown compounds were speculated except for reference.Conclusion:To develop firstly an ultra-fast liquid chromatography with tandem mass spectrometry(UFLC-MS/MS)method for simultaneous determination of 10 components by modern analytical techniques,and the chemical composition of ZZHPD was analyzed,the absorbed components and their metabolites in rat plasma and the brain of rats after oral administration of ZZHPD were identified,and to preliminarily elucidate of the chemical basis in vivo for the treatment of depression.This study can provide basis for quality evaluation and clinical application of ZZHPD in the future.