Pharmacokinetic Studies Of Active Principles From Alpiniae Oxyphylla Fructus Extract In Rats

Objective:Alpinia oxyphylla Miq.?YiZhi?,belongs to the family Zingiberaceae,is usually used for the treatment of diarrhea,intestinal disorders and urinary incontinence symptoms.The ethanol extract of Yizhi had anti-diarrheal activities,which contains sesquiterpenes?i.e.nootkatone?,diarylheptanoids?i.e.yakuchinone A,yakuchinone B and oxyphyllacinol?and flavonoids?i.e.tectochrysin,chrysin,izalpinin,apigenin-7,4'-dimethylether and kaempferide?compounds.Pharmacology studies showed that these compounds have many biological activities,such as anti-cancer,neuroprotective and so on.However,the fates and bioavailability of these active compounds in the body after oral administration?p.o.?are still unknown.Thus,the present study was to research the exposure forms and levels,to clarify the characteristics of exposure,tissue distribution and excretion,and to reveal the main elimination route for influence the systemic exposure of the above active chemicals after oral administration of Yizhi extract in rat.Methods:For the qualitative analysis,protein precipitation method was used to extract the active components and related metabolites in biological samples.For the quantitative analysis,liquid-liquid extraction method was used to extract the active components in plasma and bile samples after treated by enzyme hydrolysis with a mixture of?-glucuronidase and sulfatase.And the tissue,urine and faeces samples were treated directly by liquid-liquid extraction.LC-MS-MS was used to determine the forms and concentrations levels of the active ingredients in the biological samples.Results:Diarylheptanoids and flavonoids formed mainly phase II metabolites in plasma and bile,parent chemicals were mainly found in urine and faeces.Almost all the targeted chemicals and some monoglucuronidated metabolites were measured in stomach,small intestine,large intestine and liver.In other tissues,these analytes had lower exposure levels or couldn't be detected.Moreover,kaempferol glucuronide was also observed in the plasma,which originated from the kaempferide via O-demethylation reaction and then catalyzed by UDP-glucuronyltransferase to form glucuronidated kaempferol metabolites.The free form of nootkatone was found in the above-mentioned samples.A rapid,specific and sensitive HPLC-MS-MS method was developed and validated for the determination of the active components in the above bio-samples.LC separation was performed on a Phenomenex Kinetex 2.6?m XB-C18 column?2.10 mm i.d.×50 mm?at 40?.The mobile phase consisted of water?contain 0.1‰formic acid?for eluent A and acetonitrile?contain 0.1‰formic acid?for eluent B under a gradient elution at a flow rate of 0.35 mL/min.An electrospray ionization with positive-ion and multiple reaction monitoring mode was applied.The ion suppression effect for all the analytes was overcome through combining a liquid-liquid extraction method for clean-up,elution gradient optimization for separation and inclusion of very low concentration of formic acid into the mobile phase,accuracy?84.7-112%?and precision?<13.6%?met the analysis requirements.After p.o.administration of YiZhi extract,the active compounds were rapidly absorbed from gastrointestinal tract,which reached their C_maxax at 0.5 h,and the decreased rapidly.Except for yakuchionone B,all the target analytes were detected in the plasma samples following p.o.dosing at a highest dose level?0.54 g solid extract/kg?.kaempferol has long retention time in vivo?MRT 28.9 h?.In addition,the plasma concentration-time curves of flavonoids and diarylheptanoids were bimodal.Power model was used to assess the dose linearity based on 90%Confidence Interval?90%CI?for the ration of dose normalized means within pre-specificed limits?0.8,1.25?.Except for apigenin-7,4'-dimethylether,a positive correlation between the area under the plasma concentration-time curve?AUC?of the active compounds and the dose of p.o.administration of YiZhi extract.Highest concentrations were found in gastrointestinal tissues and liver,with the overall exposure levels ranking as follows:stomach>small intestine>large intestine>liver.Nootkatone was the highest exposed compound of the active principles in all the 11 tested tissues.The concentration-time curves of flavonoids and diarylheptanoids were bimodal in stomach,small intestine and large intestine,and there is also a trend of bimodal in the liver.The diarylheptanoids and flavonoids were mainly excreted by bile with phase II metabolites,whereas the parent chemicals were excreted lower into urine.Nootkatone was excreted substantially into both urine and bile.Cumulative amounts of the target analytes excreted into urine were found to be well correlated with the dose?R²,0.4012-0.9339?.Conclusions:The pharmacokinetic parameters of Yizhi active compound in rat were firstly obtained after oral administration of Yizhi extract.Meanwhile,the characteristics of exposure,tissue distribution and excretion were clarified,and the main elimination route that influence the systemic exposure were revealed.The information gained from this study will be helpful for the study of the medicinal material basis and enlargement of clinical application of Yizhi.