Chemical Components Of Rhizoma Anemarrhenae-Phellodendri Chinesis Herb-pair In Vivo And Pharmacokinetic Studies

Objective1.To study the Chemical components of Rhizoma Anemarrhenae-Phellodendri Chinesis(RA-PC)herb-pair in vivo on normal and diabetic rats.2.To develop and evaluate a concentation detection method for mangiferin,neomangiferin,timosaponin C,timosaponin N,timosaponin BII,phellodendrine and magflorine in rats plasma.Investigated the difference pharmacokinetic parameters of the 7 components between normal and diabetic rats3.To study the metabolites and metabolic pathways of phellodendrine in ratsMethods1.Ultra-high performance liquid phase and linear track orbital ion trap combined with high resolution mass spectrometry were used to analyze the components in normal and type 2 diabetic rats(incuced by feeding with high fat and high glucose diet for one month and then injected with 35 mg·Kg-1 streptozotocin(STZ))after the administration of RA-PC herb-pair.Chromatographic conditions:The column was ZORBAX Eclipse XDB-C18(150 mm × 2.1 mm,3.5 ?m).The mobile phase A was 0.1%formic acid aqueous solution,B was acetonitrile;gradient elution:0?40 min,5%?14%B;40?60 min,14%?30%B;60-80min,30%?50%B;80?90 min,50%?95%B;90?95 min,95%B;The first 3 min mobile phase was to the waste.The flow rate was 0.2 mL·min-1.The column temperature was 30 ?.PDA detection wavelength range 200?600nm.Mass spectrometry conditions:(CID)mode;positive ion mode electrospray voltage was 4 kV;negative ion mode spray voltage was-3.5 kV;sheath gas(ShG)was 45 arb;atomization temperature was(GC)was 5 arb;sweep gas(SwG)0 arb;The first-class mass spectrometry using Fourier transform mass spectrometry(FTMS)scanning mode,a resolution of 30000;The second-class mass spectra were scanned by data dependency scanning;the resolution was 15,000;the collision energy was 35 eV.Identification method:By comparing Chromatographic and Mass spectrometry information of water decoction of RA-PC herb-pair on the same experimental conditions,comparing Chromatographic and Mass spectrometry information between test sample and blank sample or Reference substance,through access to literature and other methods to be confirmed.2.Ultra-high performance liquid phase and linear orbital ion trap combined with high resolution mass spectrometry and Shimadzu liquid chromatography-API4000+triple quadrupole mass spectrometer(LC-MS/MS)were used to compeleted the determination of the plasma concentration of mangiferine,neomangiferin,timosaponin C,timosaponin N,timosaponin BII,phellodendrine and magflorine in normal and diabetic Rats.Normal and diabetic rats were respective 6,afforded the CMC-Na Suspension of RA-PC herb-pair on freeze-dried powder of water extract(mangiferin 9.36 mg·Kg-1,neomangiferin 18.592 mg·Kg-1,timosaponin BII 85.84 mg·Kg-1,timosaponin N 3.68 mg·Kg-1,timosaponin C 5.104 mg·Kg-1,phellodendrine 3.856 mg·Kg-1 and magflorine 0.885 mg·Kg-1)by single oral administration.The plasma concentrations of the components in normal and diabetic rats were measured after administration at 0.083?0.25?0.5?1?1.5?2?3?4?6?8?11?24h.The pharmacokinetic parameters of each component were calculated by DAS software,the differences of statistical parameters between the two groups were compared by SPSS.Determination of phellodendrine and magflorine:(LC-MS/MS)with Turbo Ionspray ion source(ESI)and Analyst 1.6 data processing system(US AB):SHIMADZU LC-20A type HPLC(Shimadzu Corporation).The mobile phase A was 0.1%formic acid aqueous solution,the mobile phase B was acetonitrile.The target components phellodnedrine,magflorine and the inner standard aconitine were elurated by gradient elution with phase B at 0-1min,10%to 60%;1-6 min 60%.The flow rate was 0.2 mL·min-1.The column was ZORBAX Eclipse XDB-C18 column(2.1 X 150 mm,5 ?m).Column temperature at 30 ? Injection volume was 10?L.Ionization source was ESI source,Ions spray voltage(IS)was 4000 V.Atomization temperature was 450 ?;curtain gas(CUR)was 40;collision gas(CAD)was 10;atomization gas(GAS1)was 50;detection method was positive ion multi-ion reaction monitoring(MRM),the ion pairs used for quantitative analysis were 342.3? 297.2(magflorine),342.3?192.1(phellodendrine).Determination of mangiferin,neomangiferin,timosaponin C,timosaponin N,timosaponin BII:Ultra-high pressure liquid phase and linear track ion trap combination high resolution mass spectrometry(Thermo fisher,USA)with electrospray ionization source(ESI)and Xcalibur 2.0.7 data processing system.The column was CORTECS(?)UPLC(?)C18(2.1X100 mm,1.6? m).The mobile phase A was 0.1%formic acid aqueous solution,phase B was acetonitrile,the flow rate was 0.2 mL·min-1.The detection of mangiferine,neomangiferin,timosaponin C,timosaponin N,timosaponin BII and inner standaerd ginsenoside Rg2 were conducted at a gradient elution with phase B from 0-10 min,6-30%;10-18 min,30-40%;18-20 min,40-90%;The first 2.5 min mobile phase.Column temperature 30 ?,injection volume 10 ?L,analysis time 20 min.Ion source was electrospray ESI source,crack induced by collision(CID)mode detection.Electrospray voltage of-3.5 KV,sheath gas(ShG)Was 45 arb,the atomization temperature was 325 ?,the auxiliary gas(AG)was 5 arb,the sweep gas(SwG)0 arb,the resolution was 30000,the mass scanning range m/z was 200-1200.3.The metabolites contained in plasma,bile,urine and feces of the rats were determined by ultra-high performance liquid phase with linear ion trap orbital ion trap after oral administration of 80 mg·Kg-1 phellodendrine.The mobile phase A was 0.1%formic acid aqueous solution,phase B was acetonitrile.Gradient elution was conducted with phase B from 0-40 min,5%-14%.The first 3 min mobile phase was to the waste.Flow rate was 0.2 mL min-1.Column.temperature was 30?.The ion source was ESI,the detection mode was in positive ion with(CID)mode.Electrospray voltage was 4.5 KV.Sheath gas(ShG)was 45 arb.Atomization temperature was 325 ?,auxiliary gas(AG)was 5 arb,purge gas(SwG)0 arb,collision energy of 30 eV.MS1 used Fourier transform mass spectrometry(FTMS)with a resolution of 30000 and a mass scanning range of 215-900.MS2 and MS3 used data dependency scanning(DDS)with a resolution of 15000.Identification of metabolites:Using UHPLC-LTQ-Orbitrap XL,comparing the chromatogram of samples after administration with the chromatogram of blank sample,preliminary screening of possible metabolite peaks,and according to the existing metabolic biotransformation type and the structure of phellodendrine reverse speculation that its possible metabolites molecular weight and molecular structure,and then,check for presence in chromatograms and mass spectra,if it exists,determine whether it was phellodnedrine metabolites by examining the debris informationResults1.Through this study,a total of 16 prototype components(10 of which were flavonoids,saponins and organic acids components derived from RA.6 of which were alkaloids that were origined from PC)and 11 metabolites(6 metabolites generated from mangiferin,5 metabolites were transferred from corresponding alkaloids)were identified in the normal and diabetic rats plasma.There was no difference between the two groups.2.Liquid chromatography-mass spectrometry was used to the methodological study of plasma concentration of mangiferin,neomangiferin,timosaponin BII,timosaponin N,timosaponins C,phellodendrine and magflorine.The accurency was between(80.48 ± 6.58)%-(108.54 ± 5.71)%,and the intra-day and inter-day precision(RSD)were ?10.54%.The stability results were between(87.92 ± 2.24)%-(110,52 ± 3.24)%,the specificity is good.After a single dose of RA-PC herb pair extract(freeze-dried powder)0.16 g·Kg-1 in normal and diabetic rats respectively.Measured by the established quantitative method that the AUC0~t of the timosaponin N and phellodendrine in the diabetic model group were significantly larger than those in the normal group,while the Cmax of timosaponin C and phellodendrine in the diabetic model group were significantly higher than the normal group.3.In this study,25 metabolites of phellodendrine in the body were identified by the use of high-resolution mass spectrometry and LC-MSn technology.Its metabolic forms were mainly glucuronidation,hydroxylation,dehydrogenation,demethylation,methylation,etc.The metabolites of glucuronidation were mainly identified in bile,urine,plasma,hydroxylation and Methylated metabolites are mainly identified in feces samples.ConclusionIn this paper,the UHPLC-LTQ-Orbitrap XL high-resolution multi-stage liquid chromatography system were used to identify 16 prototype components and 11 metabolites in the plasma of normal and diabetic rats after a single dose of RA-PC herb pair.And the components species in normal and diabetic rats were no different.Indicating that the normal and diabetic pathology model had no effect on the type of components absorbed into the blood However,the LC-MS technology was used to determine the 7 components(mangiferine,neomangiferin,et al.)after a single dose of RA-PC herb pair and the pharmacokinetic parameters were calculated.The mean values of AUC0~t?and Cmax,of the neomangiferin,timosaponin BII,timosaponin N,timosaponin C and phellodendrine in the diabetic group were higher than those in the normal group,but not statistically significant.The mean values of Cmax with timosaponin C and phellodendrine in the diabetic model group were significantly higher than those in the normal group,so as the mean values of AUC0~t with timosaponin N and phellodendrine.Indicating that the pathological model of diabetes on the absorption and utilization of some components had an impact,which might be associated with hypoglycemic Pharmacological effects.As pharmacokinetic behavior of phellodendrine in the two physiological models were significant differences,This article used UHPLC-LTQ-Orbitrap XL high resolution multi-stage liquid chromatography mass spectrometry system to study the metabolites of phellodendrine(the representative ingredient of RA-PC herb pair)for the first time.A total of 25 metabolites of phellodendrine were identified in rats' plasma,bile,urine and feces samples.Its metabolites were mainly generated from glucuronidation,hydroxylation,dehydrogenation,demethylation,methylation,et al.In conclusion,this study provided some scientific basis for the in vivo constituents of RA-PC herb pairs and the pharmacokinetics of the components and the pharmacological metabolism of the constituents.