Multicomponent Pharmacokinetics And Antioxidation Pharmacodynamics Of Tea Polyphenols In Rats As Well As Their Correlations

Objective:To develop a LC-MS/MS assay for simultaneous determina-tion of 4 main active catechins of tea polyphenols (TP), including (-)epigallocatechin-3-gallate (EGCG), (-)epicatechin-3-gallate(ECG), (-) epigallocatechin (EGC) and (-)epicatechin(EC), in rat plasma and thereby to study its multi-component pharmacokinetics and bioavailability in rats. Using a method of serum pharmacology, antioxidation pharmacodynamics of Tea Polyphenols was studied in rat. To study the correlations between the multicomponent pharmacokinetics and the antioxidation pharmacodynamics of tea polyphenols, the integrated pharmacokinetics of multiple effective components contained in tea polyphenols was analyzed.Methods:Following a single step liquid-liquid extraction of plasma samples with ethyl acetate, the 4 catechins were separated on a Hypersil ODS C18 column using a isocratic mobile phase composed of MeOH-H2O (30:70). The detection using a mass spectrometer was performed under negative ESI in the MRM mode using target ions at m/z 457.0→168.8(EGCG),441.0→168.8(ECG),305.1→124.8(EGC),289.1→108.8(EC) and 150.9→135.8 (Vanillin as an internal standard). Four groups of 5 rats each were administered orally and intravenously, respectively, with two doses (50 mg/kg and 100 mg/kg) of tea polyphenols formulated in sterile saline solution. Plasma samples were collected at different time post dosing and measured for concentrations of catechins. The 3p97 program was used to calculate respective pharmacokinetic parameters of catechins of tea polyphenols administered intravenously. In order to obtain the bioavail-ability of the above 4 catechins, the pharmacokinetic parameters of catechins of tea polyphenols administered orally were calculated by using non-compartment models. To compare the pharmacokinetic of EGCG administered in the way of tea polyphenols with the one of EGCG, rats were administered orally and intravenously respectively the above doses and the pharmacokinetic parameters were calculated respectively. The enterohepatic circulation fractions of 4 catechins were then calculated from the biliaryfistula model analysis in rat. The inhibitory effect on lipid peroxidantion of medicinal serum was the indexe of antioxidation pharmacodynamics of tea polyphenols. The pharmacodynamics parameters of tea polyphenols were calculated by imitating pharmacokinetic. A approach of self-defined weighting coefficient based on the area under the curve from zero to infinity (AUC0-∞) has been created to obtain the holistic pharmacokinetic profiles of tea polyphenols. The inhibition ratio of lipid peroxidantion versus holistic plasma concentration of tea polyphenols were plotted to obtain therelated coefficient by linear regression.Results:The analytes were identified by reference to both MRM and tR values (1.70,3.53,1.18,2.29 and 2.86 min for EGCG, ECG, EGC, EC, and IS, respectively), and quantified using peak area method of internal standard. The calibration curves had good linearity over the range of 10～10000 ng·mL-1 for EGCG and 5～5000 ng·mL-1 for ECG, EGC and EC with weighed linear regression equation(r> 0.9966). The intra-and inter-day precision (R.S.D.), obtained by measuring QC samples at high, middle and low concentrations in a set of 5 replicates on a within-run precision and between-run precision was both better than 10%, and the average accuracy, expressed as relative recovery and matrix effects calculated based on linear regression equation of calibration curve was 90%～110%and 88%～106%, respectively. The extraction recovery of EGCG, ECG, EGC and EC were all better than 72%. The plasma samples were stable for at least 8 h at room temperature,30 days at-20℃, and the EtoAc-extracted plasma samples 24 h at 4℃. The C-T curves of 4 catechins could be best fitted to three-compartment model for intravenous dosing. Based on iv C-T curve, the EGCG, ECG, EGC and EC were characterized by t1/2γof 651～705 min, 605～621.min,144～159 min and 101～104 min, as well as Vd of 24～28 L·kg-1,16～17 L·kg-1,4.2～7.8 L·kg-1 and 7.1～7.3 L·kg-1, respectively, and CL of 0.026～0.029 L·min-1·kg-1,0.018～0.019 L·min-1·kg-1,0027～0.038 L·min-1·kg-1 and 0.041～0.046 L·min-1·kg-1. After oral administration of TP, the EGCG, ECG, EGC and EC concentrations were detectable at as early as 2 min postdosing and a double peak or even a multiple peak phenomenon was evident. However there was not the phenomenon in rats of biliaryfistula model. The absolute bioavailability(F) was calculated as 3.29～9.93%(EGCG),3.27-8.85%(ECG),6.58～8.07%(EGC) and 9.24-14.3%(EC). The enterohepatic circulation fractions of 4 catechins were between 6.1%and 21.8%Conclusion:The method, because of its high sensitive, high specific serological, good Precision, high coefficient of recovery, negligible matrix effects, developed in the present study is validated to fully meet requirements for pharmacokinetics study of TP injection in rats. The 4 catechins derived from TP showed a rapid distribution and solw elimination from deep peripheral compartment with EC having shortest t1/2 p and t1/2Y compared to others. A rapid absorption but poor bioavailability was evident. The double or even multiple peak C-T curves exist, suggesting the possible presence of enterohepatic circulation after oral administration. Statistical analysis showed that the antioxidation pharmacodynamics of tea polyphenols was in good accordance with the multicomponent pharma-cokinetics. The antioxidation effect-time curves of tea polyphenols could be best fitted to three-compartment model for intravenous dosing. Instead of representational pharmacokinetics of a single component, integrated pharmacokinetics of multiple effective components could reflect the characteristic of herbal medicines with multiple-components and targets better.