Studys On The Coumarins Of Radix Angelicae Dahuricae And Its Metabolites In Vitro And In Vivo, And The Excretion Of Isodon Serra

Radix Angelicae dahuricae, is an important member of traditionalChinese medicine （TCM） originated from the roots of Angelicae dahuricae（Fisch. ex Hoffm.） Benth. et Hook. f. and Angelicae dahuricae （Fisch. exHoffm.） Benth. et Hook. f. var. formosana （Boiss.） Shan et Yuan. It is a wellknown TCM, which has been used in China for treatment of colds, headaches,toothache, coryza, trauma and leukorrhea, etc. A sensitive and selectiveHPLC-MS method was developed for simultaneous determination of the eightcoumarins in rat urine and bile and was applied to the excretion amount studyof the coumarins after oral administration of Radix Angelicae Dahuricaeextract. Cnidilin is an important content of Radix Angelicae dahuricae, aLC-MS method was established to study enzyme kinetics of cnidilinmetabolism in rat liver microsomes and tentative identification of newmetabolites of cnidilin.Isodon serra, is an important member of traditional Chinese medicine（TCM） originated from the dried entire plant of R. rubescens （Hemsl） Hara. Itis a well known TCM, which has been used in China for treatment of enteritis,jaundice, hepatitis, cholecystitis, cholelithiasis and Metabolic syndrome.Consequently, a sensitive and selective HPLC-ESI-MS/MS method wasdeveloped for simultaneous determination of the twelve diterpenoids in raturine and was applied to the excretion amount study of the diterpenoids afteroral administration of Isodon serra extract.Part one Simultaneous and sensitive determination of eight coumarinsin rat bile and urine after oral administration of RadixAngelicae Dahuricae extract by liquid chromatography-electrospray ionization mass spectrometry Objective: A selective and sensitive liquid chromatography–electrosprayionization mass spectrometry（HPLC-ESI-MS） method was developed andvalided for analysis Xanthotoxol, xanthotoxin, isoimpinellin, bergapten,oxypeucedanin, imperatorin, cnidilin and isoimperatorin in rat bile and urine.Methods: Separation on a Sapphire C₁8column （150mm×4.6mm,5μm）was achieved by water （0.1%aqueous formic acid） and methanol, isocraticelution. The mass spectrometer was operated in the positive modes bymonitoring the precursor–product combination in multiple reaction monitoring（MRM） mode. Internal standard （IS） was pimpinellin and the dosage of RadixAngelicae Dahuricae extract was8.0mL/kg. For bile, the samples werecollected during0-2,2-4,4-6,6-8,8-12,12-20,20-24h. For urine, the sampleswere collected during0-4,4-8,8-12,12-24,24-30,30-36,36-48,48-60,60-72h. The eight coumarins and IS were extracted twice by a simple liquid–liquidextraction （LLE） method by ethyl acetate.Results: The correlation coefficients were all higher than0.9932, theintra-and inter-day precisions （RSD） of these analytes all were less than14.5%. The extraction recoveries of the coumarins ranged from86.9to110.0%and the matrix effect values obtained for analytes ranged from86.8to110.0%. All the analytes in rat bile and urine were stable. In the bile samples,the eight coumarins excreted completely in twenty-four hours and the averagepercentages of coumarins excreted were0.045%,0.019%,0.177%,0.105%,0.337%,0.023%,0.024%,0.021%. In the urine samples, the eight coumarinsexcreted completely in seventy-two hours. The average percentages ofcoumarins （1-8） excreted were1.78%,0.095%,0.130%,0.292%,0.082%,0.008%,0.005%,0.004%.Conclusions: The method is robust and specific and it can successfullycompleted the requirements of the excretion study of the eight coumarins inRadix Angelicae Dahuricae.Part two Enzyme Kinetics of Cnidilin Metabolism in Rat LiverMicrosomesObjective: To study enzyme kinetics of cnidilin metabolism in rat liver microsomes, a LC-MS method was established for quantitative analysis of twometabolites M1（3",8-methoxy-isoimperatorin） and M2（5"-hydroxyl-8-methoxy-isoimperatorin）.Methods: Separation on a Sapphire C₁8column （150mm×4.6mm,5μm）was achieved by gradient elution with water （0.05%aqueous formic acid） andmethanol （0.05%aqueous formic acid）. The mass spectrometer was operatedin the positive modes in multiple reaction monitoring （MRM） mode. Internalstandard （IS） was pimpinellin. Cnidilin was added to the incubation liquid, andoscillated. And we obtained the best incubation time, protein concentration,substrate concentration. Lineweave-Burk graphic method was used tocalculate the maximum reaction rate （V_max） and K_m of enzyme kinetic.Results: The best incubation time, protein concentration, substrateconcentration were30min,1mg·mL^-1,46.35μmol·L^-1. The enzyme kineticsparameters of M1were V_max=1.1525μmol·（min·mg protein）^-1, K_m=133.1566μmol·L^-1 and the parameters of M2were V_max=1.6308μmol·（min·mg protein）^-1,K_m=292.8571μmol·L^-1.Conclusion: The method is simple, specific and reliable, which issuitable for the in vitro metabolism research of cnidilin.Part three Tentative identification of new metabolites of cnidilinObjective: To establish a HPLC-MS method to isolate and identifycnidilin metabolites products.Methods: Separation on a Sapphire C₁8column （150mm×4.6mm,5μm）was achieved by gradient elution with water （1mmol/L ammonium acetate）and methanol. The bile, urine and feces were collected. PREC-IDA-EPI andMRM-IDA-EPI were applied to analyses the metabolites products.Results: According to the fragmentation rules of furocoumarins and thecomparison of standards,18metabolites were identfied in rat after oraladministration of cnidilin, and14products were identified in bile,9productswere identified in urine,10products were identified in feces.Conclusion: The method is simple, specific and reliable, which issuitable for identification of new metabolites of cnidilin. Part four Simultaneous determination of twelve diterpenoids in raturine after oral administration of Isodon serra extract byliquid chromatography–electrospray ionization massspectrometryObjective: A selective HPLC-MS method operating both negative andpositive scanning modes in single analysis process was developed andvalidated to determinate twelve diterpenoids in rat urine.Methods: Separation on a Sapphire C₁8column （150mm×4.6mm,5μm）was achieved by gradient elution with water （0.01%aqueous formic acid） andmethanol （0.01%aqueous formic acid）. The mass spectrometer was operatedin both positive and negative modes in multiple reaction monitoring （MRM）mode. Internal standard （IS） was sulfamethoxazole （SMZ） and the dosage ofIsodon serra extract was8.0mL/kg. The samples were collected during0-4,4-8,8-12,12-24,24-30,30-36,36-48,48-60,60-72,72-84,84-96h. Thetwelve diterpenoids and IS were extracted by a simple liquid–liquid extraction（LLE） method by ethyl acetate.Results: The correlation coefficients were all higher than0.9923, theintra-and inter-day precisions （RSD） of these analytes all were less than10.8%. The extraction recoveries was all more than88.1%and the matrixeffect values obtained for analytes ranged from80.0to94.3%. The twelvediterpenoids excreted completely in ninety-six hours. The average percentagesof diterpenoids excreted were0.44%,1.73%,0.34%,5.71%,1.72%,0.11%,0.57%,1.13%,2.83%,4.74%,0.027%,0.43%.Conclusion: The method is robust and specific and it can successfullycompleted the requirements of the excretion study of the twelve diterpenoidsin Isodon serra.