| Forsythia named LianQiao in china which is included in the part one ofthe pharmacopoeia of the people's republic of china published in2010is thedry fruits of Forsythia suspense (Thunb.) Vahl. It belongs to the family ofOleaceae. The herb of Forsythia suspense always grows thickly in mountainand hillsideis, it's a perennial herb widely distributed in the province ofLiaoNing, HeBei, et al. In the past few years, the chemical research of theherb of Forsythia has become a popular domain for its variety of bioactivitiessuch as lignans, phenylethyl alcohol, volatile oil and so on. Forsythia is atraditional Chinese medicine commonly used as heat antidote. In recent years,domestic and foreign reports on the phytochemical of Forsythia gradualincreaseed, which contained antibacterial activity, antiviral, inflam mation,erysipedas, inhibition of elastase, cAMP phosphodiesterase activity, et al. Tothe best of our knowledge, Forsythia was investigated almost limited to thequalitation and quantitation in vitro, while its physiological disposition in vivoof bioactive constituents has not yet been investigated. The Forsythia is usedmore and more, therefore, the value of studing on Forsythia and itsmetabolism in vitro and vivo is much more important.LC-MS technology has showed its value in analyzing complex mixturesand has become a powerful tool in the online structural characterization andquantitation of various natural compounds owing to its high sensitivity, goodseparation efficacy and our considerable knowledge regarding its structure. Inthe present study, HPLC-MS was performed to analyze on Forsythia and itsmetabolites in vitro and vivo. First, a novel sensitive and selectiveHPLC-ESI-MS method was developed and validated to simultaneouslydeterminate and identify5constituents of Forsythia suspensa in rat bile afteroral administration of Forsythia suspensa extract. Second, phillyrin and its metabolites in rat intestinal flora were firstly identified by LC-MS/MS. Thechemical structures of three metabolites were identified by1H-NMR andESI-MS, which inferred possible microbial transformation pathways ofphillyrin by rat intestinal flora. Third, a rapid analysis method for phillyrin andits two metabolites in rat plasma by HPLC-MS was firstly developed andvalidated to analyze plasma samples and its application to pharmacokineticstudy of the three analytes after oral administration of phillyrin. At last,HPLC-ESI-MS method was developed and validated to simultaneouslydeterminate and identify phillyrin and its three metabolites in rat bile,excrement and urine after oral administration of phillyrin.Part one HPLC-ESI-MS/MS quantitative method for simultaneousanalysis of five bioactive constituents of Forsythia suspensain rat bile after oral administration of Forsythia suspensaextractObjective: To develop a sensitive, specific and rapid LC-MS method andvalidate for the simultaneous determination of5constituents of Forsythiasuspensa in rat bile.Methods: Bile samples were collected after single oral administration ofForsythia suspensa extract (20mg/kg). HPLC-MS method MRM mode wasused for the quantification of the five bioactive constituents of Forsythiasuspensa in rat bile. Acetaminophen was used as the internal standard (IS).The chromatographic separation was carried out on a C18column withgradient elution. The detection of analytes was performed on a tandem masssystem equipped with a turbo ion spray interface in positive and negativemode at the same time. The optimized mass transition ion-pairs (m/z) forquantitation were (+)-pinoresinol-β-D-glucoside (519.3/357.2),matairesinol-4'-O-glucoside (519.3/357.2), hyperin (463.2/300.1), phillyrin(533.5/371.2), phillygenin (371.2/356.1), IS (150.1/170.0). The total run timewas15min between injections.Results: The accumulation of the five analytes excreted lackly before10h after the Forsythia suspensa extract was administrated, and a great quantity was eliminated in the period of10-12h, then a little was discharged within36h, the average percentages of the five analytes excreted in the bile over thedose administered were0.002%,0.234%,0.116%,0.288%and12.700%,respectively.Conclusion: A selective LC-ESI-MS method was developed andvalidated for the simultaneous determination of the five bioactive constituentsof Forsythia suspensa in rat bile. The results showed that this method is robust,specific and sensitive and it can successfully fulfill the requirements of thisexcretion study.Part Two The metabolism of phillyrin in vitroObjective: In order to find the metabolites of phillyrin, the method of therat intestinal flora and HPLC-MS were used for studying the metabolism ofphillyrin in vitro. And the metabolites were obtained by the preparative.Methods: The phillyrin was cultured with the rat intestinal flora in vitro.The metabolites were discovered by comparing the HPLC-PDAchromatograms of intestinal flora samples with the corresponding blanks, andthe metabolites were identified by1H-NMR and ESI-MS, and the chemicalstructures of them were confirmed.Results: Three metabolites were found and prepared by preparative highperformance liquid and the chemical structures of them were confirmed by1H-NMR and ESI-MS. They are2-[(3',4'-hydroxy) phenyl]-6-[(3'',4''-dihydroxy) phenyl] parallel pairs of tetrahydrofuran (M1);2-[(3',4'-hydroxy)phenyl]-6-[(3'',4''-dimethoxy) phenyl] parallel pairs of tetrahydrofuran(M2);2-[(3',4'-dihydroxy) phenyl]-3-(hydroxymethyl)-4-[(3'',4''-dimethoxy) benzyl] tetrahydrofuran (M3), respectively.Conclusion: The intestinal flora could be used for the metabolism ofphillyrin in vitro, which was helpful to study the transformation pathways ofForsythia suspensa.Part Three Pharmacokinetic studies of the phillyrin and its twometabolites in rat plasma after oral administration ofphillyrin Objective: To establish a method to determination the phillyrin and itstwo metabolites in rat plasma after the orally administrating of phillyrin,HPLC-MS was used and validated to study the pharmacokinetics.Methods: Plama samples were collected from the rat retro-orbital vein,then acetone was added as protein precipitant, the samples were analyzed withgradient elution consisted of0.1％formic acid and methanol (containing0.1％formic acid) in the positive and negative mode. Sulfamethoxazole (SMZ) wasused as internal standard. The optimized mass transition ion-pairs (m/z) forquantitation were phillyrin (552.10/249.20) in positive mode, IS(254.10/156.00); M1(328.90/136.90), M2(357.00/151.00) in negative mode,IS (252.00/155.90). The total run time was7.0min between injections.Results: Phillyrin and its two metabolites were separated successfullywithin7min. A good precision, accuracy, linearity, detection limit andquantitation limit were found in the method, respectively. Pharmacokineticexperiments showed that original drug phillyrin eliminated faster, the peaktimes of the M1and M2were both later than phillyrin.Conclusion: The operation of the method is accurate, quick and sensitiveand can be used as a new method for simultaneous determination of phillyrinand its metabolites in rat plasma. With this method, not only the pathway ofthe biological transformation could be obtained, but also the pharmacokineticparameters of the medicine could be studied, and both of them were helpful tothe study of the metabolism of phillyrin.Part Four HPLC-ESI-MS/MS quantitative method for simultaneousanalysis of phillyrin and its three metabolites in rat bile,excrement and urine after oral administration of phillyrinObjective: To develop a sensitive, specific and rapid LC-MS method forthe simultaneous determination of phillyrin and its three metabolites in rat bile,excrement and urine.Methods: Samples were collected after single oral administration ofphillyrin (20mg/kg). HPLC-MS method MRM mode was used for thequantification of phillyrin and its three metabolites in the samples. SMZ was used as the internal standard (IS). The chromatographic separation was carriedout on a C18column with gradient elution. The detection of analytes wasperformed on a tandem mass system equipped with a turbo ion spray interfacein positive and negative mode at the same time. The optimized mass transitionion-pairs (m/z) for quantitation were phillyrin (552.10/249.20) in positivemode, IS (254.10/156.00); M1(328.90/136.90), M2(357.00/151.00), M3(359.00/146.80) in negative mode, IS (252.00/155.90). The total run time was7.0min between injections.Results: The accumulation of phillyrin and its three metabolites excretedwithin48h, and the phillyrin excreted by bile, while its metabolites excretedalmost by urine. The average percentages of phillyrin and its three metabolitesover the dose administered were less than12％.Conclusion: A selective LC-ESI-MS method was developed andvalidated for the simultaneous determination of phillyrin and its threemetabolites in rat bile, excrement and urine. The results showed that thismethod is robust, specific and sensitive and it can successfully fulfill therequirements of this excretion study. |
PDF Full Text Request