The Metabolism Studies Of6-gingerol In Rat Liver Microsomes

Objective6-gingerol is the main composition from Ginger’s spicy flavor that possesses a wide range of biological activities in gingerols. The current research demonstrates that it has very good effect in antioxidant and anticancer. The prior pharmacokinetic study of6-gingerol in vivo indicate that the absorption of6-gingerol is fast after oral administer to rats, mainly distributed in the gastrointestinal tract tissue and liver, but low blood concentration, its speculate that the first effect after oral administration. Liver is the main place of drug metabolism, though some studies found the metabolites of6-gingerol in rat’s urine and feces after oral administration, the condition of6-gingerol after metabolism in liver, the effects on metabolic enzymes in liver and what the effect to enzymes in liver has not been reported.In order to study the metabolism and enzyme kinetics of6-gingerol in rat liver microsomal incubation in vitro system, therefore the paper established a rat liver microsomal incubation system in vitro and detection method of HPLC-UV;To investigate the metabolites and their metablic pathways through establishing the LC-MS conditions;To investigate the effect of6-gingerol on the main liver enzymes isoforms that involve in drug metabolism in vivo through administering the6-gingerol to rat by oral administration.The Through the above research explore the possible metabolic pathways of6-gingerol, the effect of liver enzyme on metabolism of6-gingerol and the effect of6-gingerol on cytochrome p450enzyme, the result can provid the experimental data for the metabolism of6-gingerol and the evidence of drug-drug interaction with others, it is very significance in indicating the clinical rational medication, as well as providing the envidece of theory and experiment for the further research of6-gingerol and development and utilization of ginger. Method1. The rat liver microsomes were prepared by calcium precipitation method and the concentration of the protein and CYP450enzyme determined by the method of Lowry and carbon monoxide reduction of differential spectrum respectively;6-gingerol was incubated in200u L incubation system that contained NADPH (1mM), rat liver microsomes(1mg· mL-1), potassium phosphate buffer(0.1M, PH=7.4)for30min at37℃. Reaction was terminated by adding150μ L acetonitrile, and then add50μL interior label, The tubes were then vortexed and centrifuged at12, OOOXg for10min,200μL of the supernatant was transferred into a1.5ml EP tube and an aliquot (20μ L) was injected into HPLC system for analysis. Chromatography analysis was performed on a C18reversed phase column(4.6mm×250mm,5μm, Kcrosmasil) with a C18security guard column, The mobile phase was mehanol-water (65:35, v/v) at a flow rate of1ml· min-1. The analytes were separated by the analytical column set at room temperature. Detection was set at wavelength of280nm.2. Time for microsomal incubation, protein concentration and substrate concentration were used as investigating objects. When one of them was changed after the immobilization of the other, the conditions for microsomal incubation could be optimized by observing the metabolic rate of6-gingerol;NADPH and NADH were used as helper factor to investigate the effect of6-gingerol on metabolism;Substrate deletion approach was used to measure kinetic parameters (Vmax, Km, CLint) for microsomal enzyme.3. For studying the inhition of6-gingerol metabolism, a CYP-specific inhibtor was chose to confirm CYP involvement. To determine the CYP isoforms involved, we investigated the inhibitory effects of CYP-selective inhibitors including α-naphthoflavone (CYP1A), Cimetidine (CYP2C), Quinine (CYP2D), Ticlopidine hydrochloride(CYP2B),4-Methylpyrazole(CYP2E), and Ketoconazole (CYP3A) on6-gingerol metabolism in untreated rat liver microsomes, the inhibitor concentration were range was0-100μmol· mL-1,6-gingerol was added and incubated with inhibitors in rat liver microsomes, the ratio of6-gingerol in samples with and without inhibitor was calculated and effect of different inhibitors on the metabolism of6-gingerol was evaluated.4. The metabolites of6-gingerol in rat liver mircosomes were indentied by ACQUITY UPLC BEH C18(2.1×50mm,1.7μm), column temperature was set at30℃.The initial mobile phase condition consisted of acetonitrile and water both fortified with0.1%of formic acid and was maintained at a ratio of5:95from0to2min. From2to12min a linear gradient was applied and a ratio of80:20to5:95from12to16min, at last a ratio of5:95from16to18min. The flow rate was fixed at200μ L·min-1,20μ L aliquots were analyzed. The mass spectrometer was operating in full-sacn MS[100-600] with a positive mode. The ESI capillary cone was set to3000V, sample cone was set to30V, source temperature was set to100℃.The nebulization was assisted with nitrogen gas heated to350℃and set to a flow rate of500L· h-1, the second mass spectrometer crash energy was set to20V.5.6-gingerol was purify by polyamide column. Male SD rats were divided into six groups by weight randomly, each group has ten rats. Water was administered by orally to blank group, peanut oil was administered by orally to solvent group at doses of2mL·100g-1,6-gingerol was administered by orally to rats at doses of200,100,50mg· kg-1body weight as the high, middle, low dose group respectively, Phenobarbitol sodium injection was injected by intraperitoneally to phenobarbitol sodium group at doses of80mg· kg-1.All of groups were administered by orally for7days except phenobarbitol sodium group for5days.The liver microsomes were prepared after administering ending. Compare to blank group, the liver index, protein concentration, content of CYP450and CYPb5, ERD and ADM enzyme as the observing objects to identify the effect of6-gingerol on the enzymatic activity of CYP450.Result1.The content of pretein and CYP450in rat liver microsomes were20.66mg· mL-1and0.64nmol· mg-1protein respectively. The recovery of this method was100.6-104.2%, and the precisions of intra-and inter-day were1.6-7.5%, it’s accordance with the requirements of the biological drug determination. The calibration curve was linear in the range from1-100μ g· mL-1,6-gingerol were found to be stable in room temperature and in freeze for48h.2. Icubation time of10min, protein concentration of1mg· mL-1and substrate concentration of17μ M were found to be optimal for the metabolism of6-gingerol in rat liver mirosomes. There is no effect on the metabolism of6-gingerol when using NADPH with NADH compare to using NADPH separately, but 6-gingerol was not be metabolized when using the NADH separately as the helper factor. The Km, Vmax and CLint for6-gingerol were5.64μ mol· L-1、3.13nmol· min-1· mg-1, CLint为0.55mL· min-1· mg-1respectively in rat liver mirosomes.3. The inhitition ratios for α-naphthoflavone, Cimetidine, Ticlopidine hydrochloride,4-Methylpyrazole were all<20%, So they had no inhibitory effects; quinine had a moderate inhibitory effect with an inhibition ratio of20.2%, and ketoconazole had a strong inhibitory effect with an inhibition ratio of65.7%. This suggested that the CYP3A and CYP2D6-selectiove inhibitors contributed to the in vitro metabolism of6-gingerol. CYP2C, CYP2B, and CYP2E inhibitors did not show obvious inhibitory effects on6-gingerol metabolism on6-gingerol in rat liver microsomes.4.6-gingerol was metabolized and two metabolites were identified in rat liver mirosomes, which were6-shogaol and dehydro-6-zingerone.5. The purify of6-gingerol is more than90%through polyamide column. The6-gingerol have no effects on the contents of rat liver index and microsomal protein in rat treated with6-gingerol (200、100、50mg· kg-1· d-1)(P>0.05) but can remarkable decrease the contents of CYP450and CYPb5respectively (P<0.01); the activity of ADM was inhibited in rat treated with6-gingerol(200、100、50mg· kg-1· d-1)(P<0.05) and the effect of inhibition become stronger with the dose of gingerol. The activity of ERD was inhibited in rat treated with6-gingerol (200mg· kg-1· d-1)(P<0.05).Conclusion1.The HPLC-UV method was very convenient and efficient for detecting the6-gingerol in rat liver mirosomes, detection range, recovery, precision and stability can satify the requirement of biological sample analysis. The developed method was suitable for the research of6-gingerol in rat liver microsomes, as well as providing the reference for detecting6-gingerol in other organisms matrix.2. The metabolism of6-gingerol was dependent on NADPH;Compare to traditional product produce method, if it can choose the suitable substrate concentration and data processing method, the substrate deletion approach was a reliable and convenient method for determining the kinetic parameters, which can provid the important parameters for the futher research of6-gingerol.3. CYP3A was the main CYP450isoforms involving in metabolism of6-gingerol in rat liver microsomes, CYP2D was the second charge for the metabolism of6-gingerol, CYP1A, CYP2B, CYP2E, CYP2C were not participate in the metabolism of6-gingerol.4. The metabolites of6-gingerol in rat liver mirosomes were6-shogaol and dehydro-6-zingerone possibly, the metablized pathway was dehydration and oxidative dehydrogenation.5. The high, middle, low doses of6-gingerol can decrease the contents of CYP450and CYPb5demonstrate that6-gingerol can inhibit the activity of CYP450enzyme, the high, middle,low doses of6-gingerol have the effect of inhibiting to activity of ADM;the high dose of6-gingerol can inhibite the activity of ERD, which predicted that6-gingerol may have the side effect on the drug that metabolize by CYP3A or CYP2E1combined using with6-gingerol or ginger, especially under the long-term and big dose.