6-姜酚在大鼠肝微粒体中的代谢研究
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摘要
研究背景与目的
6-姜酚是生姜辣味成分姜辣素中的主要成分,具有广泛的生物活性,目前研究证明其在抗氧化和抗肿瘤方面具有很好的效果。前期对6-姜酚大鼠体内药代动力学研究表明：6-酚口服给药后在大鼠体内吸收迅速,主要分布在肝脏、胃肠道组织中,而血药浓度较低,推测其口服给药存在首过效应,而肝脏是药物代谢的主要场所,虽然目前有研究确认了6-姜酚经口服给药后在大鼠尿及粪便中的代谢产物,但6-姜酚经肝脏代谢后的情况、肝脏中药酶对其代谢影响以及其本身对肝药酶有何影响还未见报道。
因此本文拟通过建立大鼠肝微粒体体外孵育体系及其在大鼠肝微粒孵育体系中的HPLC-UV检测方法,研究6-姜酚在肝微粒体中的代谢情况,考察其在体外孵育体系中的酶动力学以及选择性细胞色素P450抑制剂对其代谢的影响；通过建立LC-MS条件考察6-姜酚在大鼠肝微粒中的代谢产物及其代谢途径；通过给予大鼠灌服6-姜酚,考察6-姜酚是否对参与体内药物代谢的主要肝药酶亚型活性有影响。通过上述研究探究6-姜酚在体内的可能代谢途径,肝药酶对其代谢影响以及6-姜酚对CYP450的影响,从而提供6-姜酚代谢实验资料以及与其他药物相互作用的依据,这对临床合理用药具有十分重要的意义,也为6-姜酚的进一步研究及姜的开发利用提供了理论和实验依据。
方法
1.采用钙沉淀法制备大鼠肝微粒体,Lowry法和一氧化碳还原光谱示差法分别测其蛋白含量和CYP450酶含量；6-姜酚在总体积为200μL孵育体系中于37℃水浴振荡孵育30mim,反应体系包含NADPH (1mM),肝微粒体蛋白(1mg·mL-1), PBS缓冲液(0.1M, PH=7.4),反应结束后150μL乙腈终止反应,加50μL内标,12000g离心10min,取上清液20μL进样。肝微粒体样品采用KcrosmasiL C18柱(4.6mm×250mm,5μm)色谱柱,以甲醇-水(65：35)为流动相,流速1mL·min-1,检测波长280nm，进样量20μL,进样时间为20min,检测温度为室温进行分析测定。
2.以孵育时间、大鼠肝微粒体蛋白浓度和底物浓度为考察对象,固定其中两个孵育条件,观察第三条件对6-姜酚代谢率的影响从而优化了孵育条件；考察了辅助因子NADPH和NADH对6-姜酚代谢的影响；用底物消除法计算了6-姜酚在大鼠肝微粒中的酶促动力学参数(最大速率Vmax、米氏常数Km和清除率CLint)。
3.采用CYP450酶专属性抑制剂：α-萘黄酮(CYP1A)、西米替丁(CYP2C)、奎宁(CYP2D)、噻氯匹定(CYP2B)、4-甲基吡唑(CYP2E)、酮康唑(CYP3A),各抑制剂浓度范围为0-100μmol· L-1,分别加入大鼠肝微粒体中与6-姜酚共同孵育,测定6-姜酚的消除量,以阴性样品中6-姜酚消除量的百分比定量,评价不同抑制剂对6-姜酚代谢的影响。
4.6-姜酚在大鼠肝微粒体中代谢产物的鉴定采用ACQUITY UPLC BEH C18(2.1×50mm,1.7μm)色谱柱,柱温30℃。以0.1%甲酸水溶液(A)-乙腈(B)梯度洗脱,洗脱程序如下：0～2min,95%A;2～12min;95%～20%A;12～16min;20%～95%A;16～18min;95%A。进样量为20μL,流速为0.2mL·min-1.模式检测为ESI正离子模式,质谱采集范围m／z：100-600。离子源参数：Capillary cone:3000V, Sample cone:30V, Source Temp:100℃,去溶剂化温度：350℃,去溶剂气(N2)体积流量：500L·h-1,雾化气(N2)体积流量：50L·h-1,二级质谱碰撞能量为20V。
5.利用聚酰胺柱纯化6-姜酚；雄性SD大鼠60只按体重随机分组6组,每组10只。空白组给予蒸馏水,溶媒组给予花生油2mL·100g-1,6-姜酚高、中、低剂量组分别给予花生油助溶的6-姜酚200,100,50mg·kg-1,苯巴比妥钠组给予苯巴比妥钠注射液80mg.kg-‘。空白组、溶媒组以及6-姜酚高、中、低剂量组灌胃给药7天,苯巴比妥钠组腹腔给药5天,给药结束后制备肝微粒体。通过比较实验组与空白组的肝指数、肝微粒体蛋白浓度、CYP450酶含量、CYPb5含量以及红霉素N-脱甲基酶(Erythromycin N-demethylase, ERD)和氨基比林N-脱甲基酶(Aminopyrence N-demethylase,ADM)活性,确定6-姜酚对CYP450酶活性的影响。
结果
1.大鼠肝微粒蛋白浓度为20.66mg·mL-1, CYP450含量为0.64nmol·mg-1protein;所建立的HPLC-UV检测方法的回收率为100.6～104.2%,日内和日间精密度RSD为1.6-7.5%,符合生物样品测定要求；6-姜酚在1～100μg.mL-1范围内呈良好的线性关系；6-姜酚在室温以及4℃下放置48h内稳定性符合实验要求。
2.6-姜酚在大鼠肝微粒体中孵育的最佳时间为10min,肝微粒体蛋白浓度为1.0mg·mL-1,底物浓度为17.0μM; NADPH和NADH合用时与单独使用NADPH相比,对6-姜酚代谢程度没有明显影响,而单独使用NADH作为辅助因子时6-姜酚未有明显代谢；6-姜酚在大鼠肝微粒体中的表观米氏常数Km和Vmax分别为5.64μmol·L-1、3.13nmol·min-1·mg-1, CLint为0.55mL·min-1·mg-1。
3.α-萘黄酮、噻氯匹定、西米替丁、4-甲基吡唑抑制率小于20%,所以它们无抑制作用；奎宁抑制率为20.2%,为中度抑制作用；酮康唑抑制率为65.7%,故为强抑制剂。这表明在大鼠肝微粒体中参与6-姜酚代谢的CYP450酶亚型主要是CYP3A,其次是CYP2D,而CYP1A、2B、2C、2E作用不明显。
4.在大鼠肝微粒体孵育液发现了6-姜酚原型药(MO)以及2个可能代谢产物(M1、M2),经分析发现M1、M2分别为6-姜酚脱水生成的6-姜稀酚以及氧化脱氢产生的脱氢姜酮。
5.通过聚酰胺柱纯化后6-姜酚的纯度大于90%；6-姜酚经口服给药后与空白组比较发现：低、中、高剂量组对肝指数、微粒体蛋白浓度没有影响(P＞0.05)；能显著降低CYP450酶以及CYPb5含量(P<0.01)；低、中、高剂量组均能抑制ADM酶的活性(P<0.01),高剂量组能抑制ERD酶的活性(P<0.01)。
结论
1.所建立的6-姜酚在大鼠肝微粒体中的HPLC-UV测定法方便快捷,其检测范围、回收率、精密度和稳定性基本符合生物样品分析的要求,可应用于6-姜酚在大鼠肝微粒中的代谢研究,也为6-姜酚在其他生物基质样品的测定提供了参考依据。
2.6-姜酚在大鼠肝微粒体中的代谢是依赖NADPH进行的；与传统的产物生成法相比较,在选择合适的底物浓度及数据处理方法下,底物消除法是一种可靠、简便测定肝微粒体酶动力学参数的方法,所得到的酶动力学参数为6-姜酚进一步研究提供了重要参数。
3. CYP3A是在大鼠肝微粒体中参与6-姜酚代谢的主要CYP450酶亚型,CYP2D次之,CYPIA、CYP2B、CYP2E、CYP2C没有参与其代谢。
4.6-姜酚在大鼠肝微粒体中可能代谢产物为6-姜稀酚和脱氢姜酮,代谢途径为脱水与氧化脱氢。
5.6-姜酚高、中、低剂量均能降低CYP450总蛋白和CYPb5含量,表明6-姜酚能抑制CYP450酶活性,对CYP450主要酶亚型影响上,6-姜酚高、中、低剂量能抑制ADM酶的活性；6-姜酚高剂量能抑制ERD酶的活性。这预示着6-姜酚或姜制品在临床上与其他药物特别是经CYP3A与CYP2E1代谢的药物合用时,要注意在长期大剂量使用下,可能影响这些药物的代谢。
Objective
6-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.
Method
1. 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.
Result
1.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).
Conclusion
1.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.
6-姜酚是生姜辣味成分姜辣素中的主要成分,具有广泛的生物活性,目前研究证明其在抗氧化和抗肿瘤方面具有很好的效果。前期对6-姜酚大鼠体内药代动力学研究表明：6-酚口服给药后在大鼠体内吸收迅速,主要分布在肝脏、胃肠道组织中,而血药浓度较低,推测其口服给药存在首过效应,而肝脏是药物代谢的主要场所,虽然目前有研究确认了6-姜酚经口服给药后在大鼠尿及粪便中的代谢产物,但6-姜酚经肝脏代谢后的情况、肝脏中药酶对其代谢影响以及其本身对肝药酶有何影响还未见报道。
因此本文拟通过建立大鼠肝微粒体体外孵育体系及其在大鼠肝微粒孵育体系中的HPLC-UV检测方法,研究6-姜酚在肝微粒体中的代谢情况,考察其在体外孵育体系中的酶动力学以及选择性细胞色素P450抑制剂对其代谢的影响；通过建立LC-MS条件考察6-姜酚在大鼠肝微粒中的代谢产物及其代谢途径；通过给予大鼠灌服6-姜酚,考察6-姜酚是否对参与体内药物代谢的主要肝药酶亚型活性有影响。通过上述研究探究6-姜酚在体内的可能代谢途径,肝药酶对其代谢影响以及6-姜酚对CYP450的影响,从而提供6-姜酚代谢实验资料以及与其他药物相互作用的依据,这对临床合理用药具有十分重要的意义,也为6-姜酚的进一步研究及姜的开发利用提供了理论和实验依据。
方法
1.采用钙沉淀法制备大鼠肝微粒体,Lowry法和一氧化碳还原光谱示差法分别测其蛋白含量和CYP450酶含量；6-姜酚在总体积为200μL孵育体系中于37℃水浴振荡孵育30mim,反应体系包含NADPH (1mM),肝微粒体蛋白(1mg·mL-1), PBS缓冲液(0.1M, PH=7.4),反应结束后150μL乙腈终止反应,加50μL内标,12000g离心10min,取上清液20μL进样。肝微粒体样品采用KcrosmasiL C18柱(4.6mm×250mm,5μm)色谱柱,以甲醇-水(65：35)为流动相,流速1mL·min-1,检测波长280nm，进样量20μL,进样时间为20min,检测温度为室温进行分析测定。
2.以孵育时间、大鼠肝微粒体蛋白浓度和底物浓度为考察对象,固定其中两个孵育条件,观察第三条件对6-姜酚代谢率的影响从而优化了孵育条件；考察了辅助因子NADPH和NADH对6-姜酚代谢的影响；用底物消除法计算了6-姜酚在大鼠肝微粒中的酶促动力学参数(最大速率Vmax、米氏常数Km和清除率CLint)。
3.采用CYP450酶专属性抑制剂：α-萘黄酮(CYP1A)、西米替丁(CYP2C)、奎宁(CYP2D)、噻氯匹定(CYP2B)、4-甲基吡唑(CYP2E)、酮康唑(CYP3A),各抑制剂浓度范围为0-100μmol· L-1,分别加入大鼠肝微粒体中与6-姜酚共同孵育,测定6-姜酚的消除量,以阴性样品中6-姜酚消除量的百分比定量,评价不同抑制剂对6-姜酚代谢的影响。
4.6-姜酚在大鼠肝微粒体中代谢产物的鉴定采用ACQUITY UPLC BEH C18(2.1×50mm,1.7μm)色谱柱,柱温30℃。以0.1%甲酸水溶液(A)-乙腈(B)梯度洗脱,洗脱程序如下：0～2min,95%A;2～12min;95%～20%A;12～16min;20%～95%A;16～18min;95%A。进样量为20μL,流速为0.2mL·min-1.模式检测为ESI正离子模式,质谱采集范围m／z：100-600。离子源参数：Capillary cone:3000V, Sample cone:30V, Source Temp:100℃,去溶剂化温度：350℃,去溶剂气(N2)体积流量：500L·h-1,雾化气(N2)体积流量：50L·h-1,二级质谱碰撞能量为20V。
5.利用聚酰胺柱纯化6-姜酚；雄性SD大鼠60只按体重随机分组6组,每组10只。空白组给予蒸馏水,溶媒组给予花生油2mL·100g-1,6-姜酚高、中、低剂量组分别给予花生油助溶的6-姜酚200,100,50mg·kg-1,苯巴比妥钠组给予苯巴比妥钠注射液80mg.kg-‘。空白组、溶媒组以及6-姜酚高、中、低剂量组灌胃给药7天,苯巴比妥钠组腹腔给药5天,给药结束后制备肝微粒体。通过比较实验组与空白组的肝指数、肝微粒体蛋白浓度、CYP450酶含量、CYPb5含量以及红霉素N-脱甲基酶(Erythromycin N-demethylase, ERD)和氨基比林N-脱甲基酶(Aminopyrence N-demethylase,ADM)活性,确定6-姜酚对CYP450酶活性的影响。
结果
1.大鼠肝微粒蛋白浓度为20.66mg·mL-1, CYP450含量为0.64nmol·mg-1protein;所建立的HPLC-UV检测方法的回收率为100.6～104.2%,日内和日间精密度RSD为1.6-7.5%,符合生物样品测定要求；6-姜酚在1～100μg.mL-1范围内呈良好的线性关系；6-姜酚在室温以及4℃下放置48h内稳定性符合实验要求。
2.6-姜酚在大鼠肝微粒体中孵育的最佳时间为10min,肝微粒体蛋白浓度为1.0mg·mL-1,底物浓度为17.0μM; NADPH和NADH合用时与单独使用NADPH相比,对6-姜酚代谢程度没有明显影响,而单独使用NADH作为辅助因子时6-姜酚未有明显代谢；6-姜酚在大鼠肝微粒体中的表观米氏常数Km和Vmax分别为5.64μmol·L-1、3.13nmol·min-1·mg-1, CLint为0.55mL·min-1·mg-1。
3.α-萘黄酮、噻氯匹定、西米替丁、4-甲基吡唑抑制率小于20%,所以它们无抑制作用；奎宁抑制率为20.2%,为中度抑制作用；酮康唑抑制率为65.7%,故为强抑制剂。这表明在大鼠肝微粒体中参与6-姜酚代谢的CYP450酶亚型主要是CYP3A,其次是CYP2D,而CYP1A、2B、2C、2E作用不明显。
4.在大鼠肝微粒体孵育液发现了6-姜酚原型药(MO)以及2个可能代谢产物(M1、M2),经分析发现M1、M2分别为6-姜酚脱水生成的6-姜稀酚以及氧化脱氢产生的脱氢姜酮。
5.通过聚酰胺柱纯化后6-姜酚的纯度大于90%；6-姜酚经口服给药后与空白组比较发现：低、中、高剂量组对肝指数、微粒体蛋白浓度没有影响(P＞0.05)；能显著降低CYP450酶以及CYPb5含量(P<0.01)；低、中、高剂量组均能抑制ADM酶的活性(P<0.01),高剂量组能抑制ERD酶的活性(P<0.01)。
结论
1.所建立的6-姜酚在大鼠肝微粒体中的HPLC-UV测定法方便快捷,其检测范围、回收率、精密度和稳定性基本符合生物样品分析的要求,可应用于6-姜酚在大鼠肝微粒中的代谢研究,也为6-姜酚在其他生物基质样品的测定提供了参考依据。
2.6-姜酚在大鼠肝微粒体中的代谢是依赖NADPH进行的；与传统的产物生成法相比较,在选择合适的底物浓度及数据处理方法下,底物消除法是一种可靠、简便测定肝微粒体酶动力学参数的方法,所得到的酶动力学参数为6-姜酚进一步研究提供了重要参数。
3. CYP3A是在大鼠肝微粒体中参与6-姜酚代谢的主要CYP450酶亚型,CYP2D次之,CYPIA、CYP2B、CYP2E、CYP2C没有参与其代谢。
4.6-姜酚在大鼠肝微粒体中可能代谢产物为6-姜稀酚和脱氢姜酮,代谢途径为脱水与氧化脱氢。
5.6-姜酚高、中、低剂量均能降低CYP450总蛋白和CYPb5含量,表明6-姜酚能抑制CYP450酶活性,对CYP450主要酶亚型影响上,6-姜酚高、中、低剂量能抑制ADM酶的活性；6-姜酚高剂量能抑制ERD酶的活性。这预示着6-姜酚或姜制品在临床上与其他药物特别是经CYP3A与CYP2E1代谢的药物合用时,要注意在长期大剂量使用下,可能影响这些药物的代谢。
Objective
6-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.
Method
1. 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.
Result
1.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).
Conclusion
1.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.
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