Study On Chemicals And Absolute Bioavailability Of Sappan Lignum,Heartwood Of Caesalpinia Sappan L.

The dried heartwood of Caesalpinia sappan(Leguminous L.),named Sappan Lignum,has been widely used as an emmenagogue,hemostatic and anti-inflammatory agent in Traditional Chinese Medicine[1].The latest pharmacological studies suggest that Sappan Lignum possess rich biological activities.The chemicals from Sappan Lignum have been roughly divided into five major categories by skeletal differences,including brazilines,dibezoxocin derivatives,homoisoflavonoids,chalcones and phenylpropanoids[2].A simple and accurate method with high efficiency for separation and characterization of complex compounds in aqueous extract of Sappan Lignum was established by Ultra-High-Pressure Liquid Chromatography coupled with Linear Ion Trap-Orbitrap Mass Spectrometry(u-HPLC-LTQ-Orbitrap-MSn).An Thermo Hypersil Gold C18 column(2.1 mm×100 mm.,1.9 ?m)was used in this study with the mobile phase of methanol-water(0.2% formic acid)in gradient elution mode.MSn data with high resolution were obtained from LTQ-Orbitrap mass spectrometry.Based on the fragmentation pathways and major diagnostic fragment ions of main components which were clearly proposed,17 of 31 constituents in lyophilized powder of plant water extracts were rapidly and accurately characterized or tentatively identified by comparing with references while others still unknown.It costs 36 min in the whole analysis.This u-HPLC-LTQ-Orbitrap-MSn platform with high resolution turned out to be an effective approach for rapid qualitative analysis in the complicated mixtures of Chinese Traditional Medicine extracts.This study was also aim to develop an UPlC method to determine brazilin and protosappanin B in aqueous extract of Sappan Lignum,heartwood of Caesalpinia Sappan L.Two active components,brazilin and protosappanin B,were analyzed by UPLC method.An ACQUICTY UPLC HSS T3(2.1 mm×100 mm,1.8 ?m)column was used in this study with the mobile phase of methanol-water(0.2% glacial acetic acid)in gradient elution mode and it cost 7.5 minutes throughout the general courses.The column temperature was 40 ? with a flow rate of 0.3 mL/min and the detective wavelength was 280 nm.The injected volume was 10 ?L.The results showed that the peaks of these two active components were separated commendably at the same time.The chromatographic peak areas also had good correlation with concentrations for both of brazilin and protosappanin B in a linear range of 0.195-200 ?g/m L(R2=0.9998).The average recoveries of brazilin and protosappanin B were 103.89%(RSD=1.26%)and 99.83%(RSD=1.87%)respectively.Compared to HPLC,it apparently promoted efficiency by UPLC in analyzing brazilin and protosappanin B of Sappan Lignum with higher sensitivity,which could provide a reference for the establishment of new determination method of Sappan Lignum.Additionally,this study was aim to develop UPLC-MS/MS(Ultra Performance Liquid Chromatography-Mass Spectrum/Mass Spectrum)methods to determine absolute bioavailability of protosappanin B and brazilin in rat respectively and their pharmacokinetics parameters.Protosappanin B were detected in channel 303.26>231.13 by a MS detector in the negative mode with an ACQUICTY UPLC BEH Symmetry Shield RP-18(2.1 mm×100 mm,1.7 ?m)column by observing the changes of protosappanin B in blood after intravenous injection(3 mg·kg-1,I.V.)and Per Os(15 mg·kg-1,P.O.).While brazilin were detected in channel 285.23>163.21 in the negative mode,too,with an ACQUICTY UPLC HSS T3(2.1 mm×100 mm,1.8 ?m)column by observing the changes of brazilin in blood after intravenous injection(3 mg·kg-1,I.V.)and Per Os(30 mg·kg-1,P.O.).Both of the general courses costs 5 minutes with the mobile phase of acetonitrile-water(5 m M ammonium acetate)in gradient elution mode.The column temperature were both 25 ? with a flow rate of 0.3 mL·min?1.Each sample was injected by 10 ?L.The pharmacokinetic parameters were calculated by WinNonLin3.3 software and absolute bioavailability wascalculate based on AUC and dose.At last,excellent linear relationship of protosappanin B was obtained in a beamy range of 0.49-2000 ng·mL-1while it was 3.90-1000ng·m L-1(R2=0.998)for brazilin.Their average recovery rate,RSD of intra-day and inter-day all met the requirement of study.Metabolism of protosappanin B and brazilin all fitted in non-compartment model following intravenous and oral administration.The absolute bioavailability of protosappanin B and brazilin was 12.20% and19.40%,respectively.The absolute bioavailabilities of these two compounds were both relativelylow.All of these results suggested that UPLC-MS/MS methods were specific,rapid and sensitive,suitable for detection of concentration of protosappanin B or brazilin in rat plasma.