Studies On Separation Of The Metabolites Of Alantolactone And Isoalantolactone In Vitro And Identification Of In Vivo And In Vitro Metabolites Of The Two Compounds In Rats

Two natural products,alantolactone(AL)and isoalantolactone(i AL),were mainly isolated from Inula Helenium.In Chinese Pharmacopoeia,Inula Helenium was reported that can strong spleen and stomach,relieve pain by making the gas smoothly and placate the fetus.Modern pharmacological studies on AL and iAL showed that the two compounds had antibacterial,anthelmintic,anti-inflammatory,antineoplastic activities and other biological activities.In recent study,the primary research was focus on their biological activities of inhibiting proliferation of cancer cell,inducing cancer cell apoptosis and anti-inflammatory activities,and their mechanism of action.In the past,the bottleneck of metabolite identification was attributed to the low levels metabolites which were often affected by the endogenous components and background noise.Therefore,there were no systematic study on the metabolites and metabolic pathways of AL and iAL.In recent study,the problem has been successfully solved by the multiple mass defect filter(MMDF)and the dynamic background subtraction(DBS)on-line data acquisition methods,which could trigger an information dependent acquisition scan.This technology has a good sensitivity and a high-resolution capability.Moreover,it can accurately determine the molecular weight for all of the probable metabolites,and then select to trigger the MS/MS scan on the basis of the experimental data.In our study,we used the above method on a UPLC-Triple TOF 5600+ to identify the metabolites of AL and iAL in vivo and to detect their samples of the rat urine,the rat bile,the rat faeces and the biotransformation,and then deduced the metabolic pathways according to the metabolites.The metabolites obtained from AL and iAL incubated with rat intestinal bacteria provided a theoretical basis for the identification of metabolites in rats.The experimental results indicated that this method can provide a template basis for the detection of metabolites in vivo of other ?-methylene-?-lactones,and the result can be a theoretical foundation for the metabolic processes and pharmacological effects of AL and iAL in vivo.Objective: Establishing a generally applicabile identification method of metabolites in vivo by combined UPLC-Triple TOF-MS technology with the structure of the metabolites obtained from AL and i AL incubated with rat intestinal bacteria,and using it to identify the metabolites of AL and iAL in rat,including urine,bile,faeces samples,and the biotransformation samples.In addition,the metabolic pathway can be deduced according to the metabolites of AL and iAL.Methods:1 Preparation of intestinal bacteria biotransformation metabolites: combined the fresh rat faeces with anaerobic cultural medium for 30 min,then AL or iAL was mixed with the culture solution of intestinal bacteria.After incubating for 48 hours with anaerobic culture at 37 ?C,the mixture was extracted with an equal volume of ethyl acetate for three times.The extract was evaporated to dryness,and then subjected to silica gel column chromatography.The fractions with similar components were combined under the guidance of TLC analysis.The combined fractions were further separated by HPLC to yield pure compounds.The structures of these compounds were identified by spectroscopic methods with HRMS,1H-NMR,13C-NMR,1H-1H COSY,HSQC,HMBC and NOESY.2 Identification of metabolites of AL(iAL)in vivo and in vitro: The pretreatment samples were detected by the UPLC-Triple TOF-MS method,and then the software Metabolitepilot 1.5,including extracted ion chromatography,mass defect filter,product ion filter,neutral loss filter,were used for post-acquisition data mining.In addition,the MasterView in Peak View 2.0 was used to acquire the exact molecular mass,accurate formula and the MS/MS spectrum.The metabolites in vivo were identified by comparing the retention time and the MS/MS spectrum with the pure metabolites obtained above.The metabolites with no standards were speculated by comparing with the Chemspider database or analyzing the product ions.Results: A total of 44 metabolites,in intestinal bacteria biotransformation samples and in rat urine,bile and faeces after the oral administration of AL,were identified,including 11 metabolites in urine,10 metabolites in bile,38 metabolites in feaces and 13 metabolits in biotransformation sample.For iAL,there were 46 metabolites containing 10 metabolites in urine,10 metabolites in bile,39 metabolites in feaces and 13 metabolits in biotransformation sample.It's worth noting that 44 novel sulfur-containing dimer metabolites,which have never been reported before,were identified.However,there only two types of phase II metabolites,Cysteine conjugation and N-acetylcysteine conjugation,were found in AL and iAL samples.The metabolic pathways were deduced according to the metabolites of AL and iAL.Conclusion: The metabolites of AL and iAL in vivo were successfully detected and identified by using the method of UPLC-Triple TOF-MS,and then the result can be a theoretical foundation for the metabolic processes and pharmacological effects of AL and iAL in vivo.Simultaneously,the experimental results indicated that this method can be used for the detection of metabolites in vivo of other sesquiterpene containing ?-methylene-?-lactone moiety.