| Phloretin is a polyphenolic compound of chalcone.In recent years,it has been found that phloretin has a variety of pharmacological effects,such as oxidation,immunosuppression,anti-inflammatory,anti-tumor,anti-bacterial and hypoglycemic effect.2',4',6',4-tetra-acetylphloretin is a prodrug of phloretin,it was metabolized to phloretin to exert the pharmacological effects in vivo.In this study,we examined and compared the pharmacokinetics of 2',4',6',4-tetra-acetylphloretin with those of phloretin in dogs and rats by taking advantage of LC-MS/MS.Meanwhile,the method was applied to investigate and compare tissue distribution of 2',4',6',4-tetra-acetylphloretin and phloretin in rats.Our research will provide helpful information for the development of the drug and a basis knowledge for clinical application.? Synthesis of 2',4',6',4-tetra-acetylphloretin2',4',6',4-tetra-acetylphloretin was synthesized from phloretin and acetic anhydride,acetic anhydride is used as a reactant and also solvent.The mixture was heated in 140? for 3 hours.The materials are simple,accessible and relatively cheap,the yield is 83% with purity greater than 98%.? Compare pharmacokinetics between 2',4',6',4-tetra-acetylphloretin and phloretin 1.Establishment of LC-MS/MS method for the determination of phloretin in plasma and tissueA simple,sensitive and rapid liquid chromatography-tandem mass spectrometry(LC-MS/MS)method has been developed and validated for determination of phloretin in dog and rat plasma and tissue using resveratrol as internal standard.The phloretin was separated by the Agilent EC C18 column(100 mm × 4.6 mm,3.5 ?m)and determined by LC-MS/MS.The eletctrospray ionization(ESI)source was operated in negative ionization mode for phloretin and resveratrol(internal standard,IS).The multiple reaction monitoring(MRM)transitions were chosen to m/z 273.0?m/z 166.8 for phloretin,m/z 227.0?m/z 142.91 for IS.The method was validated for accuracy,precision,linearity,range,selectivity,lower limit of quantification(LLOQ),recovery,and matrix effect.All validation parameters met the acceptance criteria according to regulatory guidelines.2.Investigation on the absorption kinetics of 2',4',6',4-tetra-acetylphloretin and phloretin in SD ratsWe studied pharmacokinetics of phloretin and 2',4',6',4-tetra-acetylphloretin in Sprague-Dawley(SD)rats.The concentration of PH was determined at different time after oral administration at three dosages of phloretin(100 mg,200 mg,300 mg)and 2',4',6',4-tetra-acetylphloretin(160 mg,320 mg,480 mg)respectively.Pholretin can be detected in 2 mintues and rapidly absorption within 30 min,then reached the maximum plasma concentration(Cmax),and the concentration decreased sharply.AUC and Cmax increased among the three dosages,the results indicated that the concentration-time(C-T)curves of different dosages accorded with first-order linear equation.There were no significant differences in other pharmacokinetic parameters such as t1/2,and tmax among the three dosages.After phloretin was transformed into 2',4',6',4-tetra-acetylphloretin,the drug rapidly hydrolyzed in vivo.The original drug can be detected in 5 min.AUC and t1/2 increased among the three dosages which declared that the concentration-time(C-T)curves also accorded with first-order linear equation.There is almost no difference in t1/2 of 2',4',6',4-tetra-acetylphloretin and phloretin in rats,indicating that rapid absorption and distribution of 2',4',6',4-tetra-acetylphloretin in rats.There were no differences in tmax among the three dosages.After administration of 2',4',6',4-tetra-acetylphloretin at low,medium and high dose,the drug rapidly hydrolyzed in vivo.The original drug can be detected in 5 min.AUC and Cmax were increased with dose,the trend showed that the C-T curves of different doses of blood were consistent with the first order linear equation.There is almost no difference in t1/2 of 2',4',6',4-tetra-acetylphloretin and phloretin in rats,indicating that 2',4',6',4-tetra-acetylphloretin absorption and distribution rapidly in vivo.Three doses of AUC were increased,indicating that as a prodrug of phloretin,2',4',6',4-tetra-acetylphloretin increased the bioavailability in rats.3.Investigation on the distirbution kinetics of 2',4',6',4-tetra-acetylphloretin and phloretin in SD ratsAfter administration of 100 mg · kg-1 phloretin in rats,the data showed that the drug was rapidly distributed but not very broadly in rats tissue.The concentration of drug in the heart,spleen,kidney and genital organs is lower than in lungs.It can be speculated that most of the phloretin has been transformed into other substances in the body by excretion of urine.The concentration of phloretin in the brain is almost undetectable,which indicated that phloretin had difficulty in crossing the blood-brain barrier.After administration of 160 mg · kg-1 2',4',6',4-tetra-acetylphloretin in rats,the results showed the drug rapidly distribution in vivo.The concentrations of phloretin in the stomach and intestine are decreased,but the concentrations and retention time in other tissues are increased,indicating that the distribution of 2',4',6',4-tetra-acetylphloretin is more balanced in vivo.Meanwhile,2',4',6',4-tetra-acetylphloretin was same as phloretin had difficulty in crossing the BBB(blood-brain barrier).4.Investigation on the excrection of 2',4',6',4-tetra-acetylphloretin and phloretin in SD ratsAfter administration of 2',4',6',4-tetra-acetylphloretin and phloretin in SD rats,The excretion of phloretin in urine and feces was 1.28% and 0.6%,1.91% and 2.01%,respectively.Data show that after acetylation,2',4',6',4-tetra-acetylphloretin hydrolyzed in vivo,a small amount of excretion in the form of phloretin,most of the drugs excreted in other forms;compared with the phloretin,acetylated phloretin's excretion in form of phloretin was significantly increased.Literature reports showed that the excrection of phloretin not only by prototype but other metabolites,such as: phloroglucino,phloretic acid,dehydrogenation products,?-oxidation products and glycine conjugate products,phlorizin sulfate Salt and other metabolites.5.Investigation on the absorption kinetics of 2',4',6',4-tetra-acetylphloretin and phloretin in beagle dogsWe studied pharmacokinetics of phloretin and 2',4',6',4-tetra-acetylphloretin in dogs.The concentrations of phloretin was determined at different time after oral administration at three dosages of phloretin(300mg,600 mg,1200mg)and 2',4',6',4-tetra-acetylphloretin(480mg,960 mg,1920mg).It turned out that the concentration-time(C-T)curves among the three dosages accorded with first-order linear equation.Accompanying dose increase,AUC,Cmax and t1/2 showed an increasing trend.The tmax increases significantly among low and medium dose of phloretin.After phloretin was transformed into 2',4',6',4-tetra-acetylphloretin,the drug rapidly hydrolyzed in vivo.The original drug can be detected in 10 min.AUC and Cmax increased among the three dosages which revealed that the concentration-time(C-T)curves of different dosages accorded with first-order linear equation.There were no differences in tmax and t1/2 among the three dosages.Pholretin rapidly absorption within 30 min,then reached the maximum plasma concentration(Cmax),and the concentration decreased sharply.After phloretin converted to 2',4',6',4-tetra-acetylphloretin,the AUC increased 2.5 times among the three doses,indicating that 2',4',6',4-tetra-acetylphloretin increased the bioavailability of phloretin.At the same time,its t1/2 increased 5.6,4.1 and 2.7 times among the three dosage and tmax increased 1.3 times in the low dosage group.The Cmax decreased 0.5 times among the low and medium dosages,indicating that the absorption and distribution of 2',4',6',4-tetra-acetylphloretin was more stable than those of phloretin. |
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