Study On The Pharmacokinetics Of Tea Polyphenols Injection In Rats

Part 1 The Plasma Pharmacokinetics Study of Tea Polyphenols Injection in RatsObjective: To develop a simple and specific reversed-phase HPLC-UV method for simultaneous determination of (-) Epigallocatechin-3-gallate (EGCG) and (-) Epicatechin-3-gallate (ECG) as the main antioxidant active ingredients of tea polyphenols (TP), and thereby to study their plasma Pharmacokinetics (PK) in rats.Methods: EGCG and ECG in plasma were eluted on a Kromasil C18 analytical column (4.6×150 mm, 5μm) protected by a C18 pre-column (4.6×20 mm,10μm) with a linear gradient mobile phase composed of CH3CN (A)-0.1% citric acid (B) and a gradient flow rate. The UV detector was set at 280 nm. An aliquot 200μL of plasma sample spiked with IS and in presence of a small volume of 20% Vit C solution was prepared by liquid-liquid extraction procedure with more than double volumes of EtoAc twice and then evaporation of organic phase under N2 stream to dry, followed by reconstitution with 100μL of 20% CH3CN aqueous solution, 60μL of supernatant was injected onto chromatograph. The peak area ratios of analytes to vanillin as internal standard vs concentration of analytes to construct calibration curves.The method validation including specificity, linearity, precision, accuracy, recovery and stability was conducted by routine procedures as described in guiding principles of PK study issued by SFDA. Fifteen rats were randomly divided into 3 groups (each group containing 5 animals) receiving iv administration via caudal vein of TP 150, 100 and 50 mg·kg^-1, respectively. Plasma samples were collected from orbital sinus prior to dosing and different time (2, 5, 10, 20, 40, 60, 90, 120, 180, 240, 300 min) postdosing.Results: Under the specified HPLC and plasma samples preparation conditions, the chromatographic peaks of EGCG, ECG and IS were resolved well from each other with retention time (t_r) of 11.2 min, 18.3 and 16.4 min, respectively;there was no interference from endogenous substances, meta- bolites and impurities of TP raw material. The calibration curves had good linearity over the range of 0.25～300μg·mL^-1 for EGCG and 0.1～60μg·mL^-1 for ECG,with weighed linear regression equation of Y (peak area ratio of analyte / IS) = 0.1404X (analyte concentration,μg·mL^-1) + 0.005059 (r²=0.9996, n=3) and Y=0.1393X + 0.0007530 (r²=0.9997, n=3) respectively; 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 single assay day and 5 separate days, was better than 7.97% and 12.99%, respectively, and the average accuracy,exprested as relative recovery calculated based on linear regression equation of calibration curve, was between 86.12%～112.42%. The extraction recovery of EGCG and ECG was 79.80%～84.64% and 76.59%～87.27%, respectively. The plasma samples were stable for at least 30 days at -20℃and 8 h at room temperature; EGCG, ECG and IS stock solutions 2 months at -20℃, and the EtoAc-extracted plasma samples 24 h at 4℃. The PK behavior of EGCG and ECG in plasma after iv administration could be described by two- compartment model and first-order kinetics, with t1/2β112.39～145.20 min and 46.63～61.48 min, Vd 4.59～5.95 L·kg^-1 and 2.01～2.75 L·kg^-1, CL 0.025～0.033 L·(kg·min) -1 and 0.024～0.034 L·(kg·min) -1 for EGCG and ECG, respectively.Conclusion: The method developed in the present study is validated to fully meet requirements for PK study of TP injection in rats. The plasma data demonstrated a rapid elimination from body of EGCG and ECG evidenced by a short t1/2 of 1～2 h, and follow linear pharmacokinetics in rats.The two tea catechins are distributed wildly in the body, reflected by much larger Vd than total body water. When the comparison is made between the two, EGCG eliminates less rapidly and distributes wider than ECG, but has almost same CL as that of ECG.Part 2 The Tissue Distribution Study of Tea Polyphenols Injection in RatsObjective: To develop a simple and specific reversed-phase HPLC-UV method for simultaneous determination of EGCG and ECG, the main active ingredients of tea polyphenols (TP), and thereby to study their tissue distribution in rats.Methods: The separation of tea catechins was achieved using the same chromatographic conditions as that described under section 1"plasma Pharmacokinetics study". A preparative procedure consisting of homoge- nization of tissues with saturated saline at a ratio of 1 g:4 mL (heart, liver, brain) and 1 g:9 mL (kidney) and subsequently liquid-liquid extraction with double volumes of EtoAc, followed by evaporation of organic phase to dry, then reconstitution as described under section 1 was developed. The peak area ratios of analytes to vanillin as internal standard vs concentration of analytes to construct calibration curves.The validation of the method including specificity, linearity, precision, accuracy, recovery and stability was conducted by routine procedures as described in guiding principles of PK study issued by SFDA. Fifteen rats were randomly divided into 3 groups (each group containing 5 animals), based on 3 different time point of tissues collection postdosing and received iv administration of TP 100 mg·kg^-1.Results: Under the above specified HPLC conditions and preparative procedure, the chromatographic peaks of EGCG, ECG and IS were resolved well from each other with retention time (t_r) of 11.2 min, 18.3 and 16.4 min, respectively; there was no interference from endogenous substances, metabolites and impurities of TP raw material. The calibration curves had good linearity over the range 0.25～20μg·mL^-1 for EGCG and 0.125～10μg·mL^-1 for ECG. The heart, liver, kidney and brain tissues homogenate samples'weighed linear regression equation of EGCG and ECG are as follows: Y (peak area ratio of analyte / IS) = 0.7252X (analyte concentration,μg·mL^-1) + 0.001502 (r²=0.9995, n=3), Y=0.7170X– 0.001043 (r²=0.9988, n=3); Y= 0.7609X + 0.06228 (r²=0.9996, n=3), Y=0.6051X– 0.02370 (r²=0.9990, n=3); Y= 1.1210X + 0.004801 (r²=0.9989, n=3), Y=1.1852X– 0.05144 (r²=0.9959, n=3) and Y= 0.8366X– 0.02721(r²=0.9999, n=3), Y=0.8303X– 0.02856 (r²=0.9999, n=3), respectively. The intra- and inter-day precision (R.S.D.) was better than 15%. The average accuracy was between 96.13%～104.19%. The extraction recovery of EGCG and ECG was 51.90%～75.54% and 53.13%～80.65%, respectively. The tissues homogenate samples were stable for at least 30 days at -20℃and 8 h at room temperature. The tissue distribution of TP was investigated at 20, 60 and 120 min postdosing in rats. When 100 mg·kg^-1 of TP was administered iv to rats, EGCG and ECG were rapidly distributed into the most of tissues, and kidney tissue exhibited the highest exposure, while the tissue with the lowest exposure was brain.Conclusion: The method developed in the present study is validated to fully meet requirements for TP injection tissue distribution study in rats. The tissues data indicate a rapid distribution of EGCG and ECG in rats into the most of tissues, and kidney tissue exhibited the highest exposure, while brain with the lowest.Part 3 The Excretion of Tea Polyphenols Injection from Urine, Bile and Feces of RatsObjective: To develop a simple and specific reversed-phase HPLC-UV method for simultaneous determination of EGCG, ECG, EGC and EC, derived from tea polyphenols (TP), and thereby to study their excretion from urine, bile and feces of rats after iv administration of TP.Methods: The urine and bile levels of the above stated 4 tea catechins were quantified by HPLC-UV methods based on the same chromatographic conditions as described under section 1, but feces levels based on a mild modified chromatographic conditions. The peak area ratios of analytes to vanillin as internal standard vs concentration of analytes to construct calibration curves.The method validation including specificity, linearity, precision, accuracy, recovery and stability was conducted by routine procedures as described in guiding principles of PK study issued by SFDA. Five-fold diluted urine, bile samples and 1 g : 9 mL feces homogenate samples spiked with IS extracted twice in presence of a small volume of 20% Vit C solution with EtoAc. The collected EtoAc fraction was evaporated under N2 stream to dry, followed by reconstitution with 20% CH3CN aqueous solution. Ten rats were randomly assigned into 2 groups (5 animals each), and given TP 100 mg·kg^-1 as a iv bolus injection. Bile samples were collected at different time of period postdosing in one group of rats; urine and feces samples were from the other.Results: The chromatographic peaks of EGCG, ECG, EGC, EC and IS were resolved well from each other with retention time (tr) of 11.2,18.3, 6.0, 9.6 and 16.4 min, for urine, bile respectively, and with 15.25,21.63, 7.72, 13.77 and 17.69 min for feces respectively; there was no interference from endogenous substances, metabolites and impurities of TP raw material. The calibration curves of urine had good linearity over the range0.50～10μg·mL^-1 for EGCG, 0.125～2.50μg·mL^-1 for ECG, 2.50～50μg·mL^-1 for EGC and 1.00～20μg·mL^-1 for EC. The calibration curves of bile had good linearity over the range0.2～5μg·mL^-1 for EGCG, 0.2～5μg·mL^-1 for ECG, 0.8～20μg·mL^-1 for EGC and 0.4～10μg·mL^-1 for EC. The calibration curves of feces homogenate had good linearity over the range1～25μg·mL^-1 for EGCG, 0.8～20μg·mL^-1 for ECG, 4～100μg·mL^-1 for EGC and 2～50μg·mL^-1 for EC. The urine, bile and feces homogenate samples'weighed linear regression equation of EGCG are as follows: Y(peak area ratio of analyte / IS)= 0.4505X (analyte concentration,μg·mL^-1) + 0.0485 (r²=0.9972, n=3),Y= 0.4347X+ 0.03650 (r²=0.9985, n=3) and Y= 0.1475X + 0.05908 (r²=0.9997, n=3); ECG are Y=0.3839X– 0.0489(r²=0.9962, n=3),Y=0.3512X + 0.03819 (r²=0.9994, n=3) and Y=0.2209X + 0.1219 (r²=0.9986, n=3) ;EGC are: Y= 0.0587X+ 0.1084 (r²=0.9954, n=3), Y = 0.04993X + 0.02456 (r²=0.9990, n=3) and Y=0.01933 X + 0.07218 (r²=0.9975, n=3),and EC are: Y= 0.207X + 0.2283 (r²=0.9972, n=3), Y=0.2220 X + 0.07966 (r²=0.9988, n=3) and Y=0.07538 X + 0.04321 (r²=0.9982, n=3), respectively. The intra- and inter-day precision (R.S.D.) was better than 10%. The average accuracy was between 90.07%～109.17%.The extraction recovery of EGCG, ECG , EGC and EC was 65.84%～91.60% , 82.53%～99.56% , 59.74%～93.00% and 77.88%～101.62%, respectively. The urine, bile and feces homogenate samples were stable for at least 30 days at -20℃and 8 h at room temperature. The excretion experiments showed that only about 1.45,0.84,7.88 and 10.73% of administrated dose for unchanged EGCG, ECG, EGC and EC were excreted in urine within 8 h, respectively, only 0.12,1.16,0.45 and 0.053% of administered dose was excreted in bile within 8 h, respectively, only 0.07 and 9.99% for EGCG and EGC in feces within 24 h, no ECG and EC were found in feces.Conclusion: The method developed in the present study is validated to sufficiently meet requirements for TP excretion study in rats. The EGCG, ECG,EGC and EC were excreted as unchanged form only in a very small percentage of administered dose from urine, bile and feces; among the 3 excretory routes urinary excretion predominated, biliary excretion was poor, feces excretion was negligible except for EGC. These findings suggest the afore said 4 tea catechins undergo extensive metabolism in body of rats.