| Background Apple polyphenol is the common name of the apple polyphenolic substances, is the secondary metabolite produced during fruit growth and development, including the hydroxycinnamic acids, and polymers of catechins, flavonols, dihydro chalcone ketone. It has strong antioxidant and antimicrobial activity, ability of inhibition of lipid peroxidation, anti-tumor, anti-mutagenic, anti-atherosclerosis, coronary heart disease and stroke prevention, prevention of dental caries, radiation protection. It has high efficacy on the application of relaxation in cardiovascular, atherosclerosis, cartilage disease, deterioration of eyesight, allergies, tooth decay and stroke. It plays a significant role both in medicine and chemical industry. Its excellent pharmacological activities will make it a star in the field of drug research and development, and the pharmacokinetic studies before the development of new drugs is very important.Objective To detect the plasma concentrations of procyanidin B2, catechin and epicatechin simultaneously, which are the main active ingredients of apple polyphenols, by HPLC-MS/MS method. To test pharmacokinetic parameters to see the absorption, distribution and elimination in rats, in order to provide relevant data for the report for approval of new drug and clinical researchs.Methods 168 Wistar male rats of fasting before the test day for 12 hours weighed were randomly divided into low, medium and high dose group with 56 of each group, seting of 14 blood points, each sampling point with 4 rats, then intragastric administrated with 0.20g/mL apple polyphenol solution in accordance with the high (2g/kg), in (1.0g/kg), low (0.5g/kg) administered dose, timing, removing 1.50mL blood from the carotid sinus with heparin, respectively at 0,0.083,0.167,0.25,0.5, 0.75,1,1.5,2,3,4,6,8,12h,2000g centrifugation for 10min. Then the plasma separated is stored at-80℃. 100μL plasma sample added 10μL 100ng·mL-1 bergenin solution which is the internal standard is vortexed for lmin, mixing, added 0.5mL methanol, vortexed for 2min, 10000rpm centrifuged for 5min.Then the upper clear liquid was bathed in 40℃water, drying by N2, redissolved with 100μL mobile phase solution. We detect plasma concentrations by HPLC-MS/MS method, and calculate the pharmacokinetic parameters of procyanidins B2, catechin and epicatechin of high, medium and low doses in rats with DSA2.0 pharmacokinetic program.Results 1. The retention time of procyanidin B2, catechin and epicatechin is 4.406min,4.951min,6.188min respectively, bergenin's is 4.9min, separating baseline between the three, not interferenced by blank plasma and metabolism materials and other components of AP. Matrix effect is 85.76%-100.86%; absolute recovery of the three is 85.48%～99.35%, relative recovery is 75.65%～111.00%; The linear range of procyanidinB2 and catechin is 1～100ng·mL-1, epicatechin's is 1～500ng·mL-1; linear regression equation:procyanidin B2 is y=0.0982x-0.0116 (R2=0.99241149); catechin is y=0.0168x-0.0043 (R2=0.99198981); epicatechin is y=0.0050x +0.0028 (R2=0.99220807); the limit of quantification is lng·mL-1, the relative deviation is less than 15%;days and day variations are less than 11%;plasma samples placed in the -20℃for 24h and 7 days showes good stability, the relative deviation is less than 7.79%.2. The pharmacokinetic parameters of procyanidin B2 of high, medium and low dose were:t1/2:(1.18±0.41) h, (1.18±0.41) h and (1.09±0.12) h. The fist Tmax is 0.5h, the second Tmax is 2h,3h,2h respectively. Cmax is (64.78±3.15) ng-mL-1, (30.21±7.89) ng-mL-1 and (3.80±0.46) ng-mL"1 respectively. AUC0-t is (194.07±15.22) ng-mL-'-h, (77.20±14.04) ng·mL-1·h and (9.31±0.64) ng·mL-1±h respectively. AUC0～∞is (194.54±15.27) ng·mL-1·h, (77.70±14.19) ng·mL-1·h and (9.76±0.58) ng·mL-1·h respectively.The pharmacokinetic parameters of catechin of high, medium and low dose were:t1/2:(1.22±0.15) h, (1.27±0.12) h and (1.29±0.41) h. The fist Tmax is 0.5h, the second Tmax is 3h. Cmax is (125.32±20.37) ng·mL-1, (35.42±4.84) ng·mL-1 and (13.49±3.90) ng·mL-1 respectively. AUCO-t is (374.98±44.54) ng·mL-1·h, (120.91±27.60) ng·mL-1·h and (38.17±8.39) ng·mL-1·h respectively. AUC0～∞is (376.50±44.86) ng·mL-1·h, (121.40±27.73) ng·mL-1·h and (39.40±7.75) ng-mL-1·h respectively.The pharmacokinetic parameters of epicatechin of high, medium and low dose were:t1/2:(0.87±0.19) h, (1.04±0.35) h and (0.87±0.19) h. The fist Tmax are 0.5h all, the second Tmax is 3h,2h,2h respectively. Cmax is (827.31±74.43) ng·mL-1, (322.55±38.23) ng·mL-1 and (77.43±12.83) ng-mL-1 respectively.AUCO-t is (2016.20±97.39) ng·mL-1·h, (420.07±14.29) ng-mL-1·h and (122.64±14.25) ng·mL-1·h respectively. AUC0～∞is (2017.26±98.06) ng-mL-1·h, (421.86±14.21) ng·mL-1·h and 123.04±14.08) ng·mL-1·h respectively.Conclusions 1. In this paper, the simultaneous determination of procyanidin B2, catechin and epicatechin which is the main active ingredient of apple polyphenol in plasma samples with HPLC-MS/MS method is set up. The methodology verified that the method is simple, sensitive, specific, reproducible, can be used for animal and human pharmacokinetic study.2. The metabolism and elimination of the procyanidins in rats in vivo are of linear dynamics characteristic, follow the two-compartment kinetic model.3. Procyanidins in the blood of rats showed a two peak concentrations, indicating that the three substances of procyanidins may exist enterohepatic circulation.4. Absorption of the three substances of procyanidins was in no difference in speed. Epicatechin is well absorbed. Procyanidins are removed similarly. The three substances are widely distributed in rats, and may accumulate in some tissues. |
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