Study On Chemical Constituents And In Vivo Metabolism Of Caesalpinia Sappan L

Sappan Lignum,the dried heartwood of Caesalpinia sappan L,was used for dispel stasis and relieve pain,protect the liver and promote blood regeneration in Traditional Chinese Medicine;Modern research have found that it show many pharmacological activities,such as:anti-tumor,anti-cancer,anti-inflammatory,anti-oxidation,vasodilatation,immune suppression,nervous system protection,hypoglycemic,etc.ObjectiveIn order to further explore the material basis and identify the possible metabolic pathway of Caesalpinia sappan L,the chemical constituents of the extract of Sappan Lignum was analysis,and the metabolism of the extract were studied.After the separation of the main constituents,an efficient,rapid and accurate method for the qualitative analysis was developed by using ultra performance liquid chromatography combined with time of flight mass spectrometry?UPLC-TOF-MS/MS?,which enable us to analyze systematically the chemical constituents of the extract of Caesalpinia sappan L.This provide scientific experimental basis for further exploration of the chemical composition and pharmacological research.On the basis of qualitative research,the possible in vivo constituents and metabolites of Caesalpinia sappan L were analyzed,which provided the scientific basis for the study of the pharmacodynamic constituents.The possible metabolites and metabolic pathways of protosappanin B were also analyzed,and the possible metabolism of protosappanins compound in vivo was deduced.Methods:1.Study on the main chemical constituents of Caesalpinia sappan LThe ethyl acetate part of the alcohol extract of the Caesalpinia sappan L was studied by the traditional extraction and separation methods.After separation and purification,NMR,HR-ESI-MS/MS were used to analyze and identify the structure of the compounds.2.Qualitative Study on the chemical constituents of the alcohol extract from Caesalpinia sappan LBased on a single compound,we analyzed extracts from Caesalpinia sappan L by UPLC-TOF-MS/MS.The chromatographic separation was performed on a C₁₈ column with a mobile phase consisted of acetonitrile and 0.1%formic acid in a gradient elution mode,the flow rate was 0.35mL/min.The mass spectral analysis was conducted in a negative electrospray ionization mode.3.Study on the metabolism of alcohol extract of Caesalpinia sappan L in SD ratsBased on the qualitative results,we used the same analysis method as above to study the metabolism of in vivo.4.Study on the metabolism of protosappanin B in SD ratsThe metabolism of protosappanin B in vivo was analyzed by using the same instrumentation as above.The chromatographic separation was performed on a C₁₈ column with a mobile phase consisted of methanol and0.1%formic acid in a gradient elution mode,the flow rate was 0.3 mL/min.The mass spectral analysis was conducted in a negative electrospray ionization mode.Results:1.12 compounds were isolated from ethyl acetate extraction parts of Caesalpinia sappan L,and their structures were identified by using modern spectroscopy technology(HR-ESI-MS,¹H-NMR,¹³C-NMR).The isolated compounds were identified as Brazilin?1?,3`-O-methylbrazilin?2?,Brazilein?3?,Protosappanin A?4?,Sappanone B?5?,Sappanchalcone?6?,Protosappanin B?7?,Sappanol?8?,Caesalpin J?9?,Syringate?10?,Epiatechin?11?,Sappanone A?12?.2.UPLC-TOF-MS/MS was usded for qualitative analysis of extraction of Caesalpinia sappan L.With mass spectrum information and elements analysis and comparison with related literatures and reference substance data,108 compounds from the extraction of Caesalpinia sappan L were identified,including 11 Brazilins and 2 of which were not reported;24 Protosapanins,12 unreported;6 Chalcones,1 unreported;22Homoisoflavonoids,3 unreported;21 Dimers,18 unreported;10Flavonoids and derivatives,1 unreported;14 other phenolic acids.3.Based on qualitative analysis,chemical constituents analysis of the Caesalpinia sappan L in vivo revealed 101 components,60 of which were metabolites,41 prototype chemical compounds were detected in rats,19 of which were found in blood,17 in bile,and 39 in excreta.4.The metabolism of protosappanin B in vivo was explored and 35components were found,9 of which were detected in blood samples,25 in bile and 23 in excreta.Conclusion:This thesis was based on the main chemical constituents of Caesalpinia sappan L.By using UPLC-TOF-MS/MS,the chemical compositions of it were detected quickly and comprehensively,and to its in vivo metabolism was study.It provides scientific experimental data for the modernization of TCM and the clinical experimental research.