| Tadehaginoside i,a natural eompound isolated and purified from Tadehagi triquetrum.It is called 3,5-dihydroxyphenyl-6-O-trans-p-hydroxy cinnamoyl-?-D-glucopyranoside,which is a phenylpropanoid glycoside.In recent years,tadehaginoside has been proved to have the biological activities of hypoglycemic and anti-hepatitis.However,the pharmacokinetics of the compound was not well studied.In present study,we extracted,isolated,identified and purified tadehaginoside from Tadehagi triquetrum,and then applied it to the study of pharmacokinetics.A specific and sensitive liquid chromatography-tandem mass spectrometry(LC-MS/MS)method was established for the quantitative determination of tadehaginoside and its main metabolites in rat plasma,and for the determination of tadehaginoside in tissues.Its pharmacokinetic behavior in SD rats was investigated to provide scientific basis for the further research.Firstly,a LC-MS/MS method for the determination of tadehaginoside in SD rat plasma was established.Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique,operating in multiple reaction monitoring(MRM)and negative ion mode.The precursor to product ion transitions monitored for tadehaginoside and IS were m/z 433.3?m/z 125.2,and m/z 301.1?m/z 151.0,respectively.The chromatographic column was SynergiTW Fusion-RP 80A C18(4?m,i.d x 50 mm,2.10 mm Phenomenex).The column temperature was set at 40?.The mobile phase water(containing 0.1‰ formic acid)and methanol(containing 0.1‰ formic acid).The mode was gradient elution and the flow rate was 0.5 mL/min.Methodological validation showed that endogenous substances in plasma did not interfere with the determination of tadehaginoside and quercetin;The assay was validated with linear range of 1-2000 ng/mL for tadehaginoside(r>0.99);and the lower limit of quantification was 1 ng/mL.The accuracy,precision,matrix effect,extraction recovery and stability were good,which met the requirements for bioanalytical method validation.The pharmacokinetics of tadehaginoside in plasma of SD rats after intragastric(i.g.)and intravenous(i.v.)administration was determined by DAS 3.2.8 software.The results of pharmacokinetics showed that after a single i.g.administration of tadehaginoside(dose of 25 mg/kg),the t1/2Z,Tmax and Cmax in plasma were(2.51 ±2.21)h,(0.25±0.08)h and(6.01 ±2.15)ng/mL,respectively,and after a single i.v.administration of dose 5 mg/kg,the t1/2Z,Tmax and Cmax in plasma were(1.27±1.19)h5 0.08 h and(109.77±4.29)ng/mL.Then a LC-MS/MS method was established to determine the concentration of tadehaginoside in tissues.The endogenous substances in brain,heart,liver,spleen,lung,kidney,stomach,small intestine,skeletal muscle,body fat and testis did not interfere with the determination.The linear calibration curves were obtained in the concentration range of 5-2000 ng/mL(r>0.99).The lower limit of quantification was 5 ng/mL.The precision,accuracy,matrix effect and recovery of liver and kidney tissues were met the requirements of bioanalytical method validation.The results showed that tadehaginoside was mainly distributed in kidney,heart,spleen,lung,liver,small intestine and skeletal muscle.Tadehaginoside was not detected in all tissues 2 hours after administration.Finally,a LC-MS/MS method was established for the determination of p-hydroxycinnamic acid,a tadehaginoside metabolite in plasma.the linear calibration curves were obtained in the concentration range of 10-2000 ng/mL(r>0.99),and the lower limit was 10 ng/mL.The precision,accuracy,recovery and stability of the method met the requirements of bioanalytical method validation.The concentration of p-hydroxycinnamic acid was treated with DAS3.2.8.The pharmacokinetic results showed that after a single intravenous administration of 5 mg/kg tadehaginosido,the t1/2Z,Tmax and Cmax were(0.86+0.58)h?0.08h,and(1536.45±193.93)ng/mL,respectively.After a single intragastric administration of tadehaginoside 20 and 25mg/kg,the t1/2Z,Tmax,Cmax and AUC0-t of p-hydroxycinnamic acid were(1.24?1.67)h,(1.15?1.25)h,(452.98?837.75)ng/mL,(1138.75?2027.58)h·ng/mL,respectively.The above experimental results show that:(1)The purity of tadehaginoside is more than 95%from Tadehagi triquetrum extract,which can be used in pharmaceutical study.(2)LC-MS/MS method for the determination of tadehaginoside in SD rat plasma is accurate,reliable,stable and repeatable.The half-life of end-elimination of tadehaginoside in SD rats was(2.51 ±2.21)h,while that of intravenous administration was(1.27±1.19)h.(3)The method is sensitive,accurate and reliable for the determination of tadehaginoside in SD rat tissues.Tadehaginoside was mainly distributed in the lungs,kidneys,heart,liver,spleen,skeletal muscle and small intestine,and tadehaginoside was detected in brain hardly.After 30 minutes of administration,the concentration of tadehaginoside in different tissues ranged from high to low were kidney,spleen,lung,heart,skeletal muscle,liver and small intestine.Tadehaginoside was not detected in all tissues 2 hours after administration,which indicated that tadehaginoside did not accumulate in SD rats.(4)LC-MS/MS is a feasible and stable method for the determination of p-hydroxycinnamic acid,the primary metabolite of tadehaginoside in SD rat plasma.After i.g.(does of 25mg/kg)and i.v.(does of 5mg/kg)administration of tadehaginoside,the t1/2Z of p-hydroxycinnamic acid was(1.24?1.67)h and(0.86±0.58)h,respectively.The Cmax and AUC0-t of p-hydroxycinnamic acid in plasma increased with the dose range of 20-25mg/kg by a single intragastric administration of tadehaginoside.The established LC-MS/MS determined method,and the pharmacokinetic results of tadehaginoside in this study provided a basis for evaluation of tadehaginoside in vivo and in vitro metabolism,which laid a foundation for its further development and clinical application. |
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