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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.
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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.
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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.
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Purpose To compare pharmacokineics of puerarin and crude extract in rats.Methods Rats received 500 mg/kg puerarin and puerarin crude extract by oral administration respectively.Hydroxybenzoic acid was selected as internal standard and the plasma concentration of the puerarin and crude extract was analyzed by HPLC.The pharmacokinetics parameters were calculated with DAS2.0.Results The pharmacokinetics of puerarin and puerarin crude extract was both best fitted with two-compartment models in rats after oral administration,and the pharmacokinetics main parameters of the two formulations were different:the AUC_(0-t) and C_(max) of puerarin were much greater than those of puerarin crude extract,but T_(max),t_(1/(2z)),CL/F and V_z/F were much lesser than those of puerarin crude extract.Conclusion The complex components in pueraria crude extract can affect the pharmacokinetics of puerarin in rat in vivo.
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AIM To investigate whether there is vasodilator nerve innervation in rat hind limb and what the nature of vasodilator nerve is. METHODS Wistar rats were treated with reserpine 1 mg?kg -1 ip at 24 h before experiment. The rats were pithed and the hind limb vascular bed was perfused with Krebs-Henseleite solution containing 1 mmol?L -1 phenylephrine at 2 ml?min -1 speed. The hind limb perfused pressure (HPP) as a main index was continuously recorded. Spinal cord electrical stimulation (SES) was repeatedly applied via an electrode at L 1～2 level of lumber vertebra. The various tool drugs were administered by iv or infusion by added to persusion solution. The data is expressed as decrease percentages of HPP increased by continuous infusion of phenylephrine. RESULTS HPP was increased from (5 7?1 5) to (21 6?3 7) kPa ( n =37) after phenylephrine perfusion. SES caused a fall of HPP in frequency dependent and voltage dependent manner. An optimum parameters of SES (10 Hz, 50 V and 1 msec) was selected to observe effects of various tool drugs on depressor response of HPP to SES. Tetrotodoxin (0 3 ?mol?L -1 ) abolished the effect completely. L NAME (10 ?mol?L -1 ), a NO synthase inhibitor, had no effect. Ganglion blocker arfonad (110 mg?kg -1 , iv, M R blocker atropine (10 ?mol?L -1 ), ? receptor blocker propranolol (1 ?mol?L -1 ) and P 1 receptor blocker aminophylline (10 ?mol?L -1 ) had also no effect. Glibenclamide (0 1 mmol?L -1 ), an ATP sensitive K + channel blocker, markedly abolished and slightly reversed the effect. CONCLUTION The nonadrenergic noncholinergic vasodilator nerve exists in rat hind limb and is not nitroxidergic or purinergic nerve. This nerve may be peptidergic nerve which releases some peptide such as CGRP and the major mechanism of vasodilatation probably activates the ATP sensitive K + channel.