ABSTRACT
Objective To study the effects of different transport protein on the transport of 7,4'-dihydroxyflavone (7,4'-DHF) and its metabolite (7,4'-DHF-S) in Caco-2 cell model.Methods Ultra performance liquid chromatography was employed to determinethe content of 7,4'-DHF and 7,4'-DHF-S incubation buffer,their structures were identified by LC-MS/MS.Bidirectional transport of Caco-2 cells model was used to investigate the influence of ko143 (the inhibitor of BCRP) and MK571 (the inhibitor of MRP2) on the transport of 7,4'-DHF and 7,4'-DHF-S,respectively.Results Metabolic product of 7,4'-DHF in Caco-2 monolayer cell was identified as one monosulfate;PDR of 7,4'-DHF was (1.43 ± 0.11),PDR of ko143 and MK571 on the apparent permeability of 7,4'-DHF was (1.59 ± 0.04) and (1.48 ± 0.07) (P ＞ 0.05);PDR of 7,4'-DHF-S was (1.60 ± 0.06);ko143 could significantly reduce the apparent permeability of 7,4'-DHF-S,and the PDR was (0.23 ±0.03) (P ＜ 0.01);MK571 had no significant effect on the apparent permeability of the 7,4'-DHF-S,and the PDR was (1.51±0.04) (P ＞ 0.05).Conclusion Caco-2 cells can mediate the suffonated reaction of 7,4'-DHF;7,4'-dihydroxyflavone sulfonated combination product may be a substrate for BCRP.
ABSTRACT
Objective To investigate the kinectics characteristics of sulfation of apigenin mediated by SULTIA3.Methods After incubation of apigenin using in vitro SULT1A3 system, high-performance liquid chromatography was utilized to determine the sulfates of apigenin.Mass spectrum(MS) were employed to elucidate the structure of metabolite.The program GraphPad Prism 5 was used to perform the kinetic characterization of SULT1A3 catalyzed metabolism of apigenin.Results A liner calibration curve for the assay of apigenin was validated in the range of 0.15625 ~30 μM with the recoveries of at least 80% and intra-day and inter-day RSD of less than 15%.Metabolic product of apigenin and SULT1A3 in the incubated system was identified one monosulfate.The metabolic behavior of apigenin in SULT1A3 was followed substrate inhibition kinetics.Apparent kinetic parameters of metabolism of apigenin by SULT1A3, Kmwas(0.355 ±1.04) μM and Ksi was(23.62 ±0.06) μM,Vmax was(65.71 ±1.30) nmol/(min? mg),Vmax/Km was 185.10 mL/(min? mg).Conclusion SULT1A3 can mediate the binding of apigenin sulfonated reaction, and the character of enzymatic kinetics shows substrate inhibition.Sulfation of apigenin mediated by SULTIA3 may play an important role in phaseⅡmetabolic in vivo.
ABSTRACT
Objective To investigate the metabolic profile of chrysin in SULT1A3 and human small intestine S9.Methods After incubation of chrysin using in vitro SULT1A3 and human small intestine S9 system, high-performance liquid chromatography was utilized to determine the sulfates of chrysin.Mass spectrum(MS) were employed to elucidate the structures of metabolite.Results In the SULT1A3 with PAPS, Km were (3.06 ±1.04) and (0.41±0.06) μM, Vmax were (12.13 ±1.30) and (6.72 ±1.61) nmol/(min· mg), Vmax/Km were 3.96 and 16.39 mL/(min· mg), respectively.In the human small intestine S9 with PAPS, Km were (1.92 ±0.35) and (0.01 ±0.00) μM, Vmax were (0.52 ±0.02) and (0.08 ± 0.02) nmol/(min· mg), Vmax/Km were 0.27 and 8.00 mL/(min· mg).The metabolic behavior of chrysin in SULT1A3 and human small intestine S9 both were followed biphasic kinetics.The sulfation of chrysin in SULT1A3 showed a significant correlation with that in human small intestine S9(R2 =0.985).Conclusion The result indicates that SULT1A3 is the major enzyme to the metabolism of chrysin, human small intestine may be the main metabolic organs of chrysin.