| Different medicinal parts of Poria come from the dried sclerotium of the fungus Poria cocos(Schw.)Wolf(Polyporaceae),and usually include Poriae Cutis,Rubra Poria,White Poria,Poria cum Radix Pini due to the different medicinal parts and functions.Poriae Cutis,the skin of Poria,has the effects of inducing diuresis to alleviating edema;Rubra Poria,the pink part near the skin,is particularly good at clearing water passage,removing heat and dampness,nourishing the heart and moistening the lung;White Poria,the white part of the middle of Poria,has been used for inducing diuresis and excreting dampness,invigorating the spleen and normalizing function of the stomach;Poria cum Radix Pini is the white central part with pine root and has the effects of tranquilizing the mind and inducing diuresis.Along with the different parts from the outside to the inside,the efficacy of tranquilizing the mind is enhanced,while the efficacy of inducing diuresis is weakened.The chemical component studies indicated that triterpene compounds are the main effective components in different medicinal parts of Poria.Systematic studies have not been reported about the pharmacokinetics of triterpene compounds in different medicinal parts of Poria,so the effective components and mechanisms which make the differences among different medicinal parts of Poria are still unclear.In the present study,UHPLC-Q-TOF-MS/MS was used to develop a method for the simultaneous quantitative some triterpene compounds in blood samples of rats,and compare the difference of the pharmacokinetics of different medicinal parts of Poria.Moreover,metabolites of triterpene compounds in rats were identified after gavage administration of Poriae Cutis extract and possible metabolite pathways were supposed.Part one Pharmacokinetics comparison of triterpene compounds from different medicinal parts of PoriaObjective: To develop an UHPLC-Q-TOF-MS/MS method for the simultaneous determination of 8 trieterpene components in rats plasma and to compare the pharmacokinetic characteristics of different medicinal parts of Poria.Methods:1 Preparation of 4 extracts: different medicinal parts of Poria were heating-refluxed by 90% ethanol individually,and the extracts were filtered and evapotated under reduced pressure.The residue was dissolved in water to get the Poria cum Radix Pini extract(2.5 g/m L of the Poria cum Radix Pini),White Poria extract(2.5 g/m L of the White Poria),Rubra Poria extract(2.0 g/m L of the Rubra Poria),Poriae Cutis extract(2.0 g/m L of the Poriae Cuti).2 Sample collection and pretreatment: 24 healthy male Sprague-Dawley rats(250±30 g)were randomly divided into four groups(6 rats/group,Poria cum Radix Pini group,White Poria group,and Rubra Poria group,Poriae Cutis group)and administrated four extracts respectively(25 g/kg).Blood samples of Poria cum Radix Pini,White Poria,and Rubra Poria were collected at 5 min,10 min,0.5 h,1 h,2 h,4 h,8 h,12 h,24 h,36 h,48 h while Poriae Cutis were collected at 10 min,30 min,1 h,2 h,4 h,8 h,12 h,24 h,36 h,48 h,72 h from the posterior orbital veinusing after administration.Plasma samples were precipitated with acetonitrile.3 Sample analysis conditions: The chromatographic separation was carried out on an ACQUITY UPLC? BEH C18(2.1×100 mm,5 ?m)column using mobile phase consisted of 0.05% formic acid-0.5 mmol/L ammonium acetate and acetonitrile with a linear gradient elution.Detection and quantification was performed by MS/MS using electrospray ion source(ESI)and product ion scan mode.All of the targeted components were detected under negative ion mode.The targeted product ions was used to obtain the peak area of analytes,and internal standard method and weighted(1/x~2)simple linear regression were employed for the determination of the concentration of the analytes in plasma samples.4 Pharmacokinetic studies and analysis: Based on the results of concentration-time curves,the pharmacokinetic parameters were calculated by DAS3.0 non-compartmental model.Then,the pharmacokinetic differences of the four herbs were compared.Results: The eight analytes in plasama samples showed good linearity over the detected concentration range and the correlation cofficients were no less than 0.9927.The RSD of intra-and inter-batch were no more than 10.3%,RE was in the range of-6.1% and 7.7%.The mean matrix effect and extraction recoveries of all analytes were in the ranges of 91.4 %~112.3% and 72.6%~102.6%,respectively.Poricoic acid AM was not detected in each group,and other 7 components have the most absorption in Poriae Cutis.Conclusion: The developed UHPLC-Q-TOF-MS/MS method is rapid,sensitive,specific and stable and it can be applied to determine 8 triterpene constituents from different medicinal parts of Poria in plasma.The results of pharmacokinetics are useful for the further study of pharmocological mechanism of Poria.Part two Identification of the absorbed constituents and metabolites in rats after gavage administration of Poriae Cutis extractObjective: To characterize the metabolites of triterpene compounds in rats after gavage administration of Poriae Cutis extract.Methods:1 Preparation of Poriae Cutis extract: Poriae Cutis were heating-refluxed by 90% ethanol individually,and the extracts were filtered and evapotated under reduced pressure.The residue was dissolved in water to get the Poriae Cutis extract(2.0 g/m L of the Poriae Cutis).2 Sample collection and pretreatment: 12 healthy male Sprague-Dawley rats(250±30 g)were randomly divided into four groups(3 rats/group).Blood samples were collected at 1,2,4,8,12,24 and 36 h after administration(25 g/kg),24 hours bile samples and 72 hours urine samples were separately collected after administration(25 g/kg).All the plasma,bile and urine samples were merged and extracted with ethyl acetate,respectively.The upper solution was collected and dried under reduced pressure.The residuary was dissolved with methanol.3 Sample analysis conditions: UHPLC-Q-TOF-MS/MS was used in the analysis.The chromatographic separation was carried out on Phenomenex Luna C18(2)100A(150×2.0 mm,3 ?m)column using mobile phase consisted of 0.1% formic acid-2 mmol/L ammonium acetate and acetonitrile solution with a gradient elution.Negative ion electrospray mode was operated in the high-resolution mass spectrometer.A full scan was employed in MS1 and DBS was used.MS/MS data were generated using powerful IDA(Information-Dependent Acquisition)algorithms.For the IDA criteria,eight of the most intense candidate ions of per cycle were selected to do a production scan.4 Analytical strategy: First,build an information database of the triterpene compounds in Poriae Cutis according to literatures.Secondly,use Peakview 2.0 software to find the main absorbed components in blood samples,prototype compounds in bile and urine samples.Then these components were classified into several groups according to their structures,and the typical compounds which can be identified with standards and have good absorption in vivo in each group were found out.Thirdly,these screened compounds were analyzed by Metabolitepilot 1.5 software to find out the main metabolites in biological samples.Finally,the structure of the metabolites were identified based on the cleavage pathway of parents and the polarity of the metabolites.Besides,the possible metabolite pathway were deduced according to the metabolites.Results: 15 main absorbed constituents were found in the plasma samples by Peakview 2.0 software,and classified into four groups according to the structure characteristics.Dehydrotumulosic acid,poricoic acid A and pachymic acid were found as the parent compounds.By Metabolitepilot 1.5 software,102 main absorbed constituents and metabolites were tentatively characterized,and the main possible metabolite pathway include methylation,oxidation,hydrogenation,hydrolysis and glucuronide conjugation.Conclusion: By UHPLC-Q-TOF-MS/MS method,the main metabolites of Poriae Cutis in vivo are successfully screened and indentified,which would be helpful for the better understanding of the metabolism of Poriae Cutis in vivo. |
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