含甲氧基黄酮类化合物UGT代谢特征及其机理研究
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摘要
黄酮类化合物(Flavonoids)是一类多酚类物质,广泛存在于自然界的豆类、谷类、水果、蔬菜及许多药用植物中。大量研究报道表明黄酮类化合物可以清除自由基,具有抗氧化、抗炎、保护心血管等多种生物活性、药理作用,其抗肿瘤作用更是受到广泛关注。但是,黄酮类化合物口服生物利用度低,阻碍了具有实用价值的化学预防治疗制剂研发。因而,如果想把该类化合物研发为疾病预防治疗制剂,寻找提高其生物利用度的有效途径是关键需解决的问题。
黄酮类化合物在体内的代谢情况复杂,广泛存在的葡萄糖醛酸化代谢、硫酸化代谢,是导致其生物利用度低的重要原因；其中,经尿苷二磷酸葡萄糖苷酸转移酶(UDP-Glucuronosyl transferases, UGTs)催化的葡萄糖醛酸化反应是主要的代谢途径。以往研究表明,黄酮类化合物的葡萄糖醛酸化代谢具有一定的底物特异性,该代谢受到其复杂化学结构变化如甲氧基、羟基变化影响,其中羟基变化对于黄酮类化合物葡萄糖醛酸化代谢的影响研究较多；此外,最近发现含甲氧基黄酮所具有的化学预防能力往往优于非甲氧基黄酮类化合物。因此,本论文重点研究含甲氧基黄酮类化合物的葡萄糖醛酸化代谢特征及机理。
为了研究含甲氧基黄酮类化合物的葡萄糖醛酸化代谢规律,选定了两个单甲氧基5,7-二羟基黄酮化合物：汉黄芩素(Wogonin)和千层纸黄素(Oroxylin A),以及三个含甲氧基5-单羟基黄酮化合物：杨芽黄素(Tectochrysin,5-hydroxy-7-methoxy flavone,5H7MF)、5-羟基-7,8-二甲氧基黄酮(5-hydroxy-7,8-dimethoxyflavone,5H7,8MF)、5-羟基-6,7,8,4’-四甲氧基黄酮(5-hydroxy-6,7,8,4'-tetramethoxy flavone,5H6,7,8,4'MF)做为模型化合物。其中,前两个化合物含两个酚羟基,结构中甲氧基位置有微小变化；而后三个化合物含单个酚羟基,A环上所含甲氧基数量依次递增。拟用12种商品化人源重组UGT酶以及人体肝、肠微粒体,在体外对这五个化合物进行系统葡萄糖醛酸化代谢研究,寻找出其代谢规律、代谢特征,并拟达到以下目的：1、对这些化合物进行葡萄糖醛酸化代谢特征研究,得到其UGT酶特异性代谢指纹图谱(UGT-isoform specific metabolic fingerprint, GSMF)。2、分析UGT酶代谢行为特征,寻找其与人体主要代谢器官肝、肠的葡萄糖醛酸化代谢情况之间的相关性,并运用UGT酶催化的葡萄糖醛酸化代谢特征对肝、肠特异性葡萄糖醛酸化代谢情况进行预测。3、分析化学结构变化如甲氧基位置、数量变化,对模型化合物UGT酶特异性代谢特征的影响。
主要研究方法如下：
一、人肝、肠微粒体和人源UGT酶的酶活性实验
为了得到各模型黄酮化合物的GSMF,重要UGT的特异性代谢动力学情况及器官特异性葡萄糖醛酸化代谢情况,在体外运用器官微粒体和UGT酶测定酶活性。酶孵育实验步骤如下：(1)混合微粒体或UGT酶(反应最佳终浓度范围是0.0053～0.053毫克蛋白每毫升)、氯化镁0.88 mM、葡糖二酸单内酯4.4 mM、丙甲菌素0.022 mg/ml;含有不同浓度底物的50 mM磷酸氢二钾溶液(pH 7.4);以及UDPGA (3.5mM)。(2)将混合后终体积为680μl的混合物平均分以得三等份,每份200 pl,置于37。C按预设时间(10至60 min)同时孵育。(3)加入内含90μM苯乙酮的94%乙腈6%醋酸溶液(作内标,其中汉黄芩素、千层纸黄素、5H6,7,8,4'MF用苯乙酮,而5H7MF、5H7,8MF换为苯丙酮)终止反应。终止反应后的混合液于13,000 rpm离心15分钟,取上清液进行UPLC分析。
测定GSMF时,仅采用了高、中、低三个底物浓度2.5,10以及35μM。描绘UGT代谢动力学情况时,如UGT 1A1,1A3,1A7-1A10对汉黄芩素、千层纸黄素代谢情况,采用了1.25-35μM(0.5-35μM)范围内9-11个底物浓度。
二、UPLC分析方法
为了对各模型化合物及代谢产物进行分析定量,采用了以下UPLC方法：对于汉黄芩素、千层纸黄素及其相应葡萄糖醛酸化代谢物,分析系统为Waters Acquity UPLC,采用光二极管阵列检测器(DAD)及Empower软件。分离柱为BEHC18,1.7μm,2.1×50mm。流动相B为100%乙腈,流动相A为0.1%(v／v)甲酸水溶液(pH 2.5)。流速为0.4 ml/min,采用梯度洗脱,0～1.5分钟时,流动相B占30-40%；1.5～2.5分钟时,流动相B占40-70%;2.5～3.0分钟时,流动相B占70-30%。检测波长280 nm,注入样品体积10μl。对于杨芽黄素、5-羟基-7,8-二甲氧基黄酮、5-羟基-6,7,8,4'-四甲氧基黄酮及其代谢物的UPLC分析定量方法与以上方法大体一致,不同之处仅在于梯度洗脱方法变为：0-1.5分钟时,流动相B占30-40%；1.5～3.0分钟时,流动相B占40-90%;3.0～4.0分钟时,流动相B占90-30%。
为了确保各模型化合物在实验过程中稳定,还进行了稳定性实验。在高、中、低三个浓度40μM,10μM和1.25μM(2.5μM)条件下,为每个化合物的分析方法做了日内及日间差异验证。
三、所选黄酮化合物葡萄糖醛酸化代谢物转化因子测定方法
为了准确地测定转化因子K值,从而进一步准确定量各葡萄糖醛酸化代谢产物,本研究采用了以下方法测定转化因子K值：(1)对于5,7-二羟基黄酮：汉黄芩素、千层纸黄素,选用了活性最强的UGT酶对底物进行葡萄糖醛酸化反应。其它5位单羟基黄酮则采用了大鼠微粒体来进行此过程。(2)用二氯甲烷萃取两次除去苷元(水相样品／二氯甲烷为2：5,v／v),葡萄糖醛酸苷存在于水相样品中。(3)将水相样品等分为两份,其中一份直接进行UPLC分析,另一份经β-葡萄糖醛酸苷酶(800 units/ml)在37℃水解。水解时间分别是：汉黄芩素水相样品1小时,千层纸黄素样品10小时,杨芽黄素、5-羟基-7,8-二甲氧基黄酮样品各12小时。将水解前后两份样品相比较,用苷峰面积的减少量除以苷元峰面积的增加量,而间接测得各转化因子值。
为了进一步考察所测定的转化因子K值是否准确,直接利用汉黄芩素及汉黄芩苷商品,配制同时含有相同浓度汉黄芩素及其苷的一系列浓度由低至高的标准溶液,则在各个标准溶液中两者浓度由低至高分别为：0.625,1.25,2.5,5,10,20,40μM。将这一系列标准溶液样品分别进UPLC进行分析,得到汉黄芩素峰面积(wogonin peak area, Aw)和汉黄芩苷峰面积(wogonoside peak area,AWG)。从这一系列标准曲线所测得的AWG:AW比值在各浓度下的平均值,即为汉黄芩苷转化因子值。将此直接方法所测得的结果与以上间接方法测得的转化因子结果进行比较,可以考察间接测定方法的准确性。
四、考察各个分析条件对转化因子K值的影响。
为了控制分析过程中各个分析条件对转化因子测定准确性的影响,进一步考察了实验过程中下列分析条件对转化因子K值测定的影响：流动相pH值、离子强度、样品介质pH值、检测波长。利用上述配制的汉黄芩素、汉黄芩苷系列标准溶液,逐个改变上述分析条件,并在改变某个条件时保持其它条件不变,观察AWG:AW比值的变化情况。五、应用LC-MS/MS推测所选黄酮化合物对应葡萄糖醛酸化代谢物的结构
为了进一步推测所选模型黄酮化合物经代谢后产生葡萄糖醛酸化代谢物的结构,采用超高效液相-电喷雾-质谱／质谱(UPLC-ESI-MSn)方法对黄酮及其葡萄糖醛酸化代谢物进行分离、检测以及分析,运用正离子方式检测,各黄酮及其苷的分离色谱条件与UPLC相同。质谱仪的主要质谱工作参数如下：仪器名称,三重四极杆质谱(Waters Micromass Quattro Premier XE);扫描模式,正离子模式；毛细管电压(capillary voltage) 3KV;锥孔电压(cone voltage) 35V;离子源温度(ion source temperature) 100℃;去溶剂化温度(desolvation temperature)350℃；锥孔气流(cone gas flow)50升／小时；去溶剂化温度气流(desolvation temperature gas flow)600升／小时；数据的采集及分析采用MassLynx V4.1软件(Waters Corp, Milford, MA, USA)。
六、酶动力学数据分析
为了得出汉黄芩素、千层纸黄素的UGT代谢动力学情况及两者在肝、肠的葡萄糖醛酸化动力学情况,将酶活性实验所得动力学数据进行分析。UGT酶、人类肝肠微粒体葡萄糖醛酸化代谢速率可以每分钟每毫克蛋白催化代谢反应所形成代谢物的量表示(nmol/min/mg)。动力学参数可以进一步根据Eadie-Hofstee图情况获得：如果Eadie-Hofstee图为线性或表现出非典型动力学(atypical kinetics)特点时,则在各个底物浓度(C)下黄酮葡萄糖醛酸苷的形成速率(V)变化情况会符合标准Michaelis-Menten方程或其它非典型动力学如autoactivation和biphasic动力学方程。数据拟合时采用ADAPTⅡ程序。而且,为了找出最合适的模型,采用阿开克(氏)信息判据(Akaike's information criterion, AIC)对各个侯选模型进行辨认,并应用了简约性原则(the rule of parsimony)。
七、运用UGT酶预测人肝、肠微粒体中黄酮葡萄糖醛酸化反应规律
为了预测模型黄酮化合物在人肝、肠微粒体中葡萄糖醛酸化代谢情况,结合UGT在各组织中表达情况及各UGT对化合物代谢的贡献大小,采用了以下方法：先经研究得出代谢黄酮化合物的主要UGT酶；接着参考各UGT在肝、肠中表达水平将几种起主要代谢作用的UGT联合起来,即运用加权平均方法计算这几种UGT葡萄糖醛酸化反应速率的平均值,用以预测该化合物在肝、肠微粒体中的代谢情况。再接下来,画出对应Eadie-Hofstee图以及纵轴为葡萄糖醛酸化反应联合速率、横轴为黄酮化合物浓度的葡萄糖醛酸化反应速率变化情况图,并获得各个表观动力学参数。此外,应用线性回归方法,得到起主要代谢作用联合UGT的葡萄糖醛酸化反应速率与肝、肠微粒体葡萄糖醛酸化反应速率间的相关系数。
八、统计学分析
标准曲线采用线性回归分析；以UGT数据与肝微粒体数据拟合采用线性相关分析；其他数据采用SPSS13.0统计软件分析处理,实验数据用均数±标准差(Mean±SD)表示；独立样本数据用One-Way ANOVA检验。组间多重比较采用Tukey法,方差不齐的采用Tamhane's T2法,显著性标准为α=0.05。
所得实验结果如下：一、对于汉黄芩素、千层纸黄素,所得代谢产物只有一个,且都为7位葡糖醛酸化物。GSMF实验表明,对汉黄芩素代谢速率最快的几个UGT为UGT1A3、1A8、1A9以及1A10,对于千层纸黄素,除了这几种UGT之外还有1A7。UGT酶特异性代谢研究结果表明UGT1A3、UGT1A7-1A10催化的动力学反应情况,都有已知动力学模型与之相符。而人肝、肠微粒体催化两黄酮化合物葡萄糖醛酸化代谢,则呈现简单Michaelis-Menten动力学特点。经过对动力学参数及动力学反应类型进行比较,表明UGT1A9可能是负责两黄酮化合物肝代谢的主要UGT酶。相反,两化合物的肠代谢则可能由UGT1A亚家族酶联合介导,其中UGT1A10占主导作用。相关性研究结果则明确表明,UGT酶可以用来对该两黄酮化合物在人体肝、肠微粒体中的代谢情况进行预测。总体来说,随甲氧基由8位变至6位,多数UGT代谢速率明显降低,特别是起主要代谢作用的UGT1A3,1A8, 1A9。
二、5-羟基-6,7,8,4'-四甲氧基黄酮经代谢后未发现任何代谢物。5H7MF和5H7,8MF经代谢后则各发现了一个单葡糖醛酸化代谢产物。GSMF实验表明,对于5H7MF和5H7,8MF,几乎所有UGT酶都对代谢有较低水平贡献,对5H7MF代谢明显最快的UGT酶为UGT1A1,其次为1A3：而对于5H7,8MF,代谢最快的UGT酶为UGT1A3,其次为位于同一外显子簇的UGT1A7-1A10,特别是UGT1A9(底物浓度为2.5,10μM时)和UGT1A8(底物浓度为35μM时),再次是UGT1A1及UGT2B15、2B17。相关性研究结果表明对于三个含甲氧基黄酮5H7MF、5H7、8MF、5H6,7,8,4'MF,联合UGT反应速率与肝微粒体反应速率之间有明显相关性。随着模型化合物甲氧基数量变化,各UGT特异性代谢特征也在不断发生变化。
三、为所选各化合物的分析方法做了日内及日间差异验证,测得五个化合物的精密度和准确度范围分别在可接受的0.02%-6.75%和86.73%-110.84%范围内。说明对所选模型化合物采用了可靠的方法进行分析,其稳定性研究结果显示该五个黄酮化合物在24小时时间范围内稳定,稳定性满足本研究所需。
四、为所选黄酮化合物的葡萄糖醛酸化代谢物进行转化因子测定,测得汉黄芩素、千层纸黄素、杨芽黄素、5-羟基-7,8-二甲氧基黄酮对应葡萄糖醛酸化代谢物的转化因子值分别为1.09±0.04、1.29±0.05、2.05±0.18、1.25±0.09。对于汉黄芩素和汉黄芩苷,两化合物都有商品提供,配制同时含有同浓度此两化合物的标准溶液,直接测得转化因子值为1.03±0.02,与以上间接方法测得的转化因子结果1.09±0.04非常接近,偏差在可接受范围之内。说明各黄酮葡萄糖醛酸化代谢物转化因子值能准确地进行测定。
五、考察实验过程中各个分析条件对转化因子K值测定的影响,发现随着流动相pH值由2.5增至8.5,汉黄芩素的出峰时间在pH值为3.5、4.5、6.5时相互接近,pH 8.5时前移39.43%；汉黄芩苷出峰时间前移21.88～41.47%,且所出峰接近溶剂峰,影响分析准确性。流动相pH 8.5与pH 2.5时测得各浓度下AWG:AW平均值接近,pH 3.5、4.5、6.5较pH 2.5时测得的AWG:AW平均值升高10.7-14.6%。在所选检测波长254、274、280、342nm条件下,所测得各浓度下AWG:AW平均值较接近,仅342nm波长条件下测得的AWG:AW平均值与280nm条件下所测值偏差较大,达9.71%。流动相离子强度、样品介质pH值变化时,各浓度下AWG:AW平均值无明显变化。
根据上述实验结果,本论文的结论如下：
一、对单甲氧基5,7二羟基黄酮汉黄芩素、千层纸黄素进行GSMF研究表明两化合物主要由UGTIA亚家族酶代谢,其中对代谢贡献最大的是UGT1A3及UGT1A7-1A10; GSMF及UGT酶特异性代谢情况,可用于预测人体肝、肠微粒体催化的葡萄糖醛酸化反应速率及代谢情况；甲氧取代基在A环上的位置变化可明显影响两化合物的代谢特征。
二、对含甲氧基5位单羟基黄酮杨芽黄素(5H7MF)、5-羟基-7,8-二甲氧基黄酮(5H7,8MF)和5-羟基-6,7,8,4'-四甲氧基黄酮(5H6,7,8,4'MF)进行GSMF研究表明5H6,7,8,4'MF检测不到代谢物,其它化合物主要由UGT1A亚家族酶代谢,其中对代谢贡献相对较大的是UGT1A1、UGT1A3及UGT1A8-1A10;初步发现运用GSMF可预测含甲氧基5位单羟基黄酮在肝的葡萄糖醛酸化代谢情况；模型化合物甲氧基数量变化会明显影响其UGT特异性代谢特征。
三、对所选五个黄酮化合物,采用了可靠的分析方法进行分析,稳定性研究结果显示该五个黄酮化合物在24小时时间范围内稳定,稳定性满足本研究所需。
四、为所选黄酮化合物的葡萄糖醛酸化代谢物进行转化因子值测定,间接测得汉黄芩素、千层纸黄素、杨芽黄素、5-羟基-7,8-二甲氧基黄酮对应葡萄糖醛酸化代谢物的转化因子值分别为1.09±0.04、1.29±0.05、2.05±0.18、1.25±0.09。采用汉黄芩素和汉黄芩苷商品配制标准溶液直接测得汉黄芩苷转化因子值与以上结果非常接近,因此本研究中各代谢物转化因子值能较好地进行测定。
五、利用含有汉黄芩素及其苷的系列标准溶液,考察各个分析条件对转化因子K值的影响,发现流动相pH值对汉黄芩苷、汉黄芩素的出峰时间有明显影响,特别pH 8.5时,两者前移明显,影响分析准确性；偏酸性条件有利于出峰的稳定,流动相pH值<7时测得AWG:AW平均值变化不明显。不同检测波长条件下测得AWG:AW平均值接近。流动相离子强度、样品介质pH值变化对转化因子值无明显影响。
综上所述,本研究在体外测定得到五个含甲氧基化合物的UGT酶特异性代谢指纹图谱,即参与两个5,7-二羟基黄酮化合物汉黄芩素、千层纸黄素代谢的UGT主要为UGT1A3及UGT1A7-1A10;在三个5-羟基黄酮化合物之中,5H6,7,8,4'MF未发现代谢物,参与5H7MF、5H7,8MF代谢的UGT主要为UGT1A1、1A3及UGT1A7-1A10。GSMF及UGT酶特异性代谢情况可以用来预测两个5,7-二羟基黄酮化合物在人体肝、肠,以及5H7,8MF、5H7MF、5H6,7,8,4'MF在人体肝的葡萄糖醛酸化代谢情况。同时,发现随着汉黄芩素、千层纸黄素之间微小的甲氧基取代位置变化,即由8位变至6位,多数UGT代谢速率明显降低；5H7MF、5H7,8MF、5H6,7,8,4'MF之间存在的甲氧取代基数量差别,也对黄酮类化合物葡萄糖醛酸化代谢特征有明显影响。
Flavonoids, characterized as polyphenolics, are widely distributed in our daily diets, beverages, medicinal plants and herbal remedies. The diverse biological effects of flavonoids have attracted great interests of scientists. The major reported pharmacological activities of flavonoids include antioxidative effects, protection against cardiovascular disease, and anticancer effects, etc. Despite of the above reported beneficial properties and demonstrated preclinical activities, it's a serious conern that the oral bioavailabilites of flavonoids were reported to be low (<10%), which is a big challenge to develop flavonoids into chemo-preventive and chemo-therapeutic agents. Therefore, a key issue in the development of flavonoids as disease prevention and therapy agents is to find a way to increase their bioavailabilities.
The metabolic pathways of flavonoids were diverse and complicated because of their complex structures. It was reported that extensive first-pass metabolism by phaseⅡenzymes including UGTs and SULTs was suggested to be the major causes for their low bioavailabilities; also the glucuronidation of flavonoids is the major metabolic pathway resposnsible for their low bioavailabilities. The majority of UGTs displayed broad and overlapping substrate specificities. Many studies including our own have shown that glucuronidation of flavonoids were strongly influenced by their structures. Furthermore, recent studies demonstrated that the methoxylated flavones, which may have chemopreventive properties superior to the more common unmethylated flavonoids or polyphenols. Therefore, the aim of this thesis is to investigate the mechanism on the UGT metabolism of methoxylated flavonoids.
In order to explore the metabolic characterization of methoxylated flavonoids, two 5,7-dihydroxyflavone, wogonin and oroxylin A, as well as three 5-monohydroxyflavone, tectochrysin (5-hydroxy-7-methoxyflavone,5H7MF),5-hydroxy-7,8-dimethoxyflavone (5H7,8MF),5-hydroxy-6,7,8,4'-tetramethoxy-flavone (5H6,7,8,4'MF) were selected as the model compounds. For the first two dihydroxyflavone, there exist minor structural differences of methoxyl position between them. For the later three monohydroxyflavone, the number of methoxyl is increased in order. A set of commercially available expressed human UGTs and human intestinal and liver microsomes were used to make detailed and systematic metabolic profiling studies for the five flavonoids in vitro. Firstly, the objective of the studies is to obtain the UGT-isoform specific metabolic fingerprint (GSMF) for the five model compounds. Another objective is to determine if GSMF and isoform-specific metabolism profiles might be used to predict glucuronidation rates and profiles in microsmes derived from human intestine and liver, two major organs responsible for first-pass metabolism as well as recycling via enteric and enterohepatic schemes. Finally, the third objective is to analyze the effect of methoxyl changes on the characterization of UGT-isoform specific metabolic of model compounds.
Methods.
1. Enzymatic activities of human expressed UGTs and organ microsomes
For the experiments of GSMF, isoform-specific metabolism profiles and human organ-specific metabolism profiles, the incubation procedures for measuring enzyme's activities using microsomes or UGTs were performed and essentially the same as the previous publications from University of Houston. Briefly, the procedures were as follows:(1) mix microsomes/supersomes (final concentration≈in range of 0.0053-0.053 mg protein per ml as optimum for the reaction), magnesium chloride (0.88 mM), saccharolactone (4.4 mM), alamethicin (0.022 mg/ml); different concentrations of substrates in a 50 mM potassium phosphate buffer (pH 7.4); and UDPGA (3.5 mM, add last) to a final volume of 680μl; (2) separate the 680μl mixture to obtain three equal portions with the volume of 200μl, and incubate them at 37℃simultaneously for a predetermined period of time (10 to 60 min); and (3) stop the reaction by the addition of 100μl of 94%acetonitrile/6%glacial acetic acid containing 90μM acetophenone (propiophenone for 5H7MF,5H7,8MF) as the internal standard. The reaction mixture was centrifuged at 13,000 rpm for 15min and the supernatant was directly subjected to UPLC for analysis. To profile UGT's activities, three substrate concentrations,2.5,10 and 35μM were used. To profile kinetics of wogonin and oroxylin A by UGT 1A1,1A3 and 1A7-1A10,9-11 substrate concentrations in the range of 1.25-35μM (0.5-35μM) were used.
2. UPLC analysis of five flavones and their glucuronides
To make precise analysis for all model compounds and their metabolites, a common method was applied for wogonin, oroxylin A as well as their corresponding glucuronides:system, Waters Acquity UPLC with photodiode array detector and Empower software; column, BEH C18,1.7μm,2.1×50 mm; mobile phase B,100% acetonitrile, mobile phase A,100%aqueous buffer (0.1%,v/v formic acid, pH 2.5); flow rate 0.4 ml/min; gradient,0 to 1.5min,30-40%B,1.5 to 2.5 min,40-70%B, 2.50 to 3.0 min,70-30%B, wavelength,280 nm for flavones and their respective glucuronides and acetophenone; and injection volume,10μl. For 5H7MF,5H7,8MF, 5H6,7,8,4'MF as well as their corresponding glucuronides, analysis method was similar to that of wogonin, oroxylin A with minor modification as follows:gradient,0 to 1.5min,30-40%B,1.5 to 3.0 min,40-90%B,3.0 to 4.0 min,90-30%B.
Stability studies were made to ensure all model compounds keep stable during the course of experiments. Analytical methods for each compound were validated for inter-day and intra-day variation using 6 samples at three concentrations (40,10 and 1.25μM).
3. Determination of conversion factor for four flavone glucuronides
To quanify glucuronides more precisely, following experiment procedures were operated to obtain the conversion factor (K) for the glucuronides of the four flavones: (1) glucuronidate substrates with the most active UGT isoform using the incubation procedures described previously; for 5H7MF,5H7,8MF with only one phenolic hydroxyl group, rat liver microsomes were used; (2) extract the aqueous samples containing glucuronides with dichloromethane (sample/dichloromethane=2:5, v/v) twice to remove the aglycones; (3) divide one extracted aqueous sample into two equal portions, where one portion was subjected to UPLC for analysis directly, and the other was analyzed after hydrolysis by P-glucuronidase (800 units/ml) at 37℃for 1 h (10 h for oroxylin A,12 h for 5H7MF,5H7,8MF). The conversion factor of wogonin was also directly derived using the standard curve of both wogonin and wogonoside.
4. Effects of UPLC analysis conditions on the conversion factor
In order to observe whether various analysis conditions, such as pH value and ionic strength of mobile phase, detection wavelength as well as pH value of sample medium, have influences on the conversion factor, a series of standard working solutions with seven concentrations in the range of 0.625 to 40μM for wogonin and its glucuronide were prepared. Then, analysis conditions were altered respectively and observed the variance of AWG:AW.When one of above analysis conditions changed, other analysis conditions were consistent with described in common method. Briefly, the procedures of varying analysis conditions were performed as follows:(1) prepare a series of mobile phase A with different pH value:3.5,4.5,6.5 and 8.5, via adding 0.1%aqueous ammonia into 0.1%methanoic acid solution (pH 2.5); (2) adjuste pH value of the series of solutions back to 2.5 by 1%methanoic acid, then ionic strength of these solutions were different from one another notwithstanding their pH values were same; (3) change the detection wavelentgh to 254,274,342nm; (4) pH value of sample medium were changed from 2.5 to 3.5,6.0,7.4 with 0.1% phosphoric acid.
5. Confirmation of flavone glucuronide structure by LC-MS/MS
To further confirm the identity of the metabolite as mono-glucuronides of their parent compounds, LC-MS/MS was applied. The separation, detection and analysis of flavones and their glucuronides were achieved by Waters Micromass Quattro Premier XE, operated in the positive ion mode. The main mass working parameters for the mass spectrometers were set as follows:capillary voltage,3KV; cone voltage,35V; ion source temperature,100℃; desolvation temperature,350℃; cone gas flow,501/hr; desolvation temperature gas flow,6001/hr. Data acquisition and analysis were performed using a MassLynx V4.1 software (Waters Corp, Milford, MA, USA).
6. Kinetic of glucuronidation
To obtain the kinetics of wogonin and oroxylin A glucuronidation by major UGT isoforms, human liver and intestinal microsomes, data from incubation experiments were used for analysis. Rates of flavone metabolism by expressed human UGT isoforms, human liver and intestine microsomes were expressed as amounts of metabolites formed per min per mg protein (nmol/min/mg). Kinetic parameters were then obtained according to the profile of Eadie-Hofstee plots. If the Eadie-Hofstee plot was linear or showed characteristic profiles of atypical kinetics, formation rates (V) of flavone glucuronides at respective substrate concentrations (C) were fit to the standard Michaelis-Menten equation or other atypical kinetics equations, using the ADAPTⅡprogram. To determine the best-fit model, the model candidates were discriminated using the Akaike's information criterion (AIC), and the rule of parsimony was applied.
7. Use of expressed UGTs to predict flavone glucuronidation in human intestinal and liver microsomes
To predict flavone glucuronidation in human intestinal and liver microsomes, tissue expression of different UGTs and the contribution of isoforms to flavone glucuronidation were considered. Because each tissue expressed different UGTs and/or different quantities of the same UGTs, we first determined the main isoforms responsible for the metabolism of each flavone and then use a combination of several major active isoforms to predict substrate metabolism profiles in human intestinal and liver microsomes. The combination was achieved based on weighted mean average expression levels. Subsequently, the glucuronidation profiles obtained using the combined glucuronidation rates versus flavone concentrations were used to obtain the apparent kinetic parameters after corresponding Eadie-Hofstee plots were generated. Moreover, linear regression was applied to derive apparent correlations between rates of reaction obtained using a combination of main UGT isoforms and those obtained using human intestinal and liver microsomes.
8. Statistical Analysis
One-way ANOVA with or without Tukey-Kramer multiple comparison (posthoc) tests were used to evaluate statistical differences. Differences were considered significant when p values were less than 0.05.
Results
1. For wogonin and oroxylin A, their metabolites were found to be wogonin-7-glucuronide and oroxylin A-7-glucuronide respectively, GSMF experiments indicated that they were metabolized mainly by UGTIAs, with major contributions from UGT1A3 and UGT1A7-1A10. Isoform-specific metabolism showed that kinetic profiles obtained using expressed UGT1A3 and UGT1A7-1A10 could fit to known kinetic models. Glucuronidation of both flavonoids in human intestinal and liver microsomes followed simple Michaelis-Menten kinetics. A comparison of the kinetic parameters and profiles suggests that UGT1A9 is likely the main isoform responsible for liver metabolism. In contrast, a combination of UGT1As with a major contribution from UGT1A10 contributed to their intestinal metabolism. Correlation studies clearly showed that UGT isoforms could predict metabolism of these two compounds in human intestinal and liver microsomes. With the change of methoxyl from 8-position to 6-position, the glucuronidation rates decreased for most isoforms, especially for UGT1 A3,1A8,1A9.
2. No metabolite was found for 5H6,7,8,4'MF. For 5H7MF and 5H7,8MF, metabolites were both found to be mono-glucuronides, almost all isoforms contribute to their glucuronidation at a low level. GSMF experiments indicated that UGT1A1 was found to be the most important isoform for 5H7MF, followed by UGT1A3. For 5H7,8MF, UGT1A3 was found to be the most important, followed by UGT1A7-1A10 and UGT1A1, especially UGT1A9 (at substrate concentration of 2.5μM,10μM) or UGT1A8 (at substrate concentration of 35μM). Correlation studies clearly showed that the glucuronidation rates of human liver microsomes have an apparent correlation with those derived from a combination of UGT 1A1,1A3 and 1A9. In addition, the UGT isoform-specific metabolic pattern changed with the increase of methoxyl number.
3. Analytical methods for each compound were validated for inter-day and intra-day variation, and found that precision and accuracy for five compounds were in the acceptable range of 0.02%-6.75%and 86.73%-110.84%respectively. The result suggests that the analytical methods for model compounds were credible, and all model flavones keep stable during the course of experiments.
4. The list of the conversion factor measured was as follows:wogonin,1.09±0.04; oroxylin A,1.29±0.05; 5H7MF,2.05±0.18; 5H7,8MF,1.25±0.09. The conversion factor of wogonoside was also derived directly using the standard curve of both wogonin and wogonoside, and the result was 1.03±0.02. Therefore, the two conversion factors of wogonoside derived from different methods were similar, showing that the accuracy of the conversion factor obtained for all glucuronides is acceptable.
5. When the effects of various analysis conditions on the deterimination of conversion factor were investigated, it was found that the retention time of wogonin and wogonoside moved forward 39.43%,41.47%respectively, when the pH value of mobile phase A increased to 8.5, and the ratio of AWG:AW obtained under pH 3.5,4.5, 6.5 was individually 10.7-14.6%higher than that obtained under pH 2.5, whereas the ratios determined under pH 2.5,8.5 were close to each other. With the variance of ionic strength of mobile phase A and pH value of sample medium, the ratios determined were similar. Under the predetermined detection wavelength of 254,274, 280 and 342 nm, the ratios of AWG:AW obtained were found similar, and the ratio obtained under 342 nm deviated 9.71%from those obtained under 280 nm.
Conclusions
1. For wogonin and oroxylinA, GSMF experiments indicated that both flavonoids were metabolized mainly by UGT1As, with major contributions from UGT1A3 and UGT1A7-1A10. GSMF and isoform-specific metabolism profiles can be used to predict glucuronidation rates and profiles in human tissue microsomes. Minor structural differences of methoxyl position between wogonin and oroxylin A significantly impacted their metabolism
2. For 5H7MF,5H7,8MF,5H6,7,8,4'MF, GSMF experiments indicated that no metabolite was found for 5H6,7,8,4'MF, other flavonoids were metabolized mainly by UGT1 As, with major contributions from UGT1A1, UGT1A3 and UGT1A8-1A10. GSMF could be used to predict the three methoxylated flavones metabolism in liver microsomes. Structural differences of methoxyl number significantly impacted their UGT isoform-specific metabolic pattern.
3. The selected five flavones, wogonin, oroxylin A as well as 5H7MF,5H7,8MF, 5H6,7,8,4'MF could be analyzed accurately, and keep their stability during the course of experiments.
4. For wogonin, the conversion factor derived directly from the standard curve of both wogonin and wogonoside, was very similar with that from the determination method used for all glucuronides of model flavone, so the accuracy of the conversion factor obtained for all glucuronides is acceptable.
5. Among all UPLC analysis conditions, pH value variance of mobile phase would lead to the change of retention time of wogonin, wogonoside and impact the determination of conversion factor, and pH values<7 were more suitable for the determination. In addition, results measured under various detection wavelength were found similar, ionic strength variance of mobile phase, pH value variance of the sample medium was found had no influences on the determination of conversion factor.
In conclusion, the GSMF were obtained for five methoxylated flavonoids. For two 5,7-dihydroxyflavone, wogonin and oroxylin A, they were metabolized mainly by UGT 1 A3 and UGT1A7-1A10. For three 5-monohydroxyflavone, no metabolite was found for 5H6,7,8,4'MF, UGT1A1, UGT1A3 and UGT1A7-1A10 were found to be the main isoforms contributing to the glucuronidation of 5H7MF and 5H7,8MF. GSMF and isoform-specific metabolism profiles, could be used to predict glucuronidation rates and profiles of wogonin, oroxylin A in human liver and intestine microsomes and predict glucuronidation rates of 5H7MF,5H7,8MF and 5H6,7,8,4'MF in human liver microsomes. For wogonin and oroxylin A, the glucuronidation rates of most isoforms were found decreased with the change of methoxyl from 8-position to 6-position. For 5H7MF,5H7,8MF and 5H6,7,8,4'MF, the UGT isoform-specific metabolic pattern were found changed with the vaiance of methoxyl number.
黄酮类化合物在体内的代谢情况复杂,广泛存在的葡萄糖醛酸化代谢、硫酸化代谢,是导致其生物利用度低的重要原因；其中,经尿苷二磷酸葡萄糖苷酸转移酶(UDP-Glucuronosyl transferases, UGTs)催化的葡萄糖醛酸化反应是主要的代谢途径。以往研究表明,黄酮类化合物的葡萄糖醛酸化代谢具有一定的底物特异性,该代谢受到其复杂化学结构变化如甲氧基、羟基变化影响,其中羟基变化对于黄酮类化合物葡萄糖醛酸化代谢的影响研究较多；此外,最近发现含甲氧基黄酮所具有的化学预防能力往往优于非甲氧基黄酮类化合物。因此,本论文重点研究含甲氧基黄酮类化合物的葡萄糖醛酸化代谢特征及机理。
为了研究含甲氧基黄酮类化合物的葡萄糖醛酸化代谢规律,选定了两个单甲氧基5,7-二羟基黄酮化合物：汉黄芩素(Wogonin)和千层纸黄素(Oroxylin A),以及三个含甲氧基5-单羟基黄酮化合物：杨芽黄素(Tectochrysin,5-hydroxy-7-methoxy flavone,5H7MF)、5-羟基-7,8-二甲氧基黄酮(5-hydroxy-7,8-dimethoxyflavone,5H7,8MF)、5-羟基-6,7,8,4’-四甲氧基黄酮(5-hydroxy-6,7,8,4'-tetramethoxy flavone,5H6,7,8,4'MF)做为模型化合物。其中,前两个化合物含两个酚羟基,结构中甲氧基位置有微小变化；而后三个化合物含单个酚羟基,A环上所含甲氧基数量依次递增。拟用12种商品化人源重组UGT酶以及人体肝、肠微粒体,在体外对这五个化合物进行系统葡萄糖醛酸化代谢研究,寻找出其代谢规律、代谢特征,并拟达到以下目的：1、对这些化合物进行葡萄糖醛酸化代谢特征研究,得到其UGT酶特异性代谢指纹图谱(UGT-isoform specific metabolic fingerprint, GSMF)。2、分析UGT酶代谢行为特征,寻找其与人体主要代谢器官肝、肠的葡萄糖醛酸化代谢情况之间的相关性,并运用UGT酶催化的葡萄糖醛酸化代谢特征对肝、肠特异性葡萄糖醛酸化代谢情况进行预测。3、分析化学结构变化如甲氧基位置、数量变化,对模型化合物UGT酶特异性代谢特征的影响。
主要研究方法如下：
一、人肝、肠微粒体和人源UGT酶的酶活性实验
为了得到各模型黄酮化合物的GSMF,重要UGT的特异性代谢动力学情况及器官特异性葡萄糖醛酸化代谢情况,在体外运用器官微粒体和UGT酶测定酶活性。酶孵育实验步骤如下：(1)混合微粒体或UGT酶(反应最佳终浓度范围是0.0053～0.053毫克蛋白每毫升)、氯化镁0.88 mM、葡糖二酸单内酯4.4 mM、丙甲菌素0.022 mg/ml;含有不同浓度底物的50 mM磷酸氢二钾溶液(pH 7.4);以及UDPGA (3.5mM)。(2)将混合后终体积为680μl的混合物平均分以得三等份,每份200 pl,置于37。C按预设时间(10至60 min)同时孵育。(3)加入内含90μM苯乙酮的94%乙腈6%醋酸溶液(作内标,其中汉黄芩素、千层纸黄素、5H6,7,8,4'MF用苯乙酮,而5H7MF、5H7,8MF换为苯丙酮)终止反应。终止反应后的混合液于13,000 rpm离心15分钟,取上清液进行UPLC分析。
测定GSMF时,仅采用了高、中、低三个底物浓度2.5,10以及35μM。描绘UGT代谢动力学情况时,如UGT 1A1,1A3,1A7-1A10对汉黄芩素、千层纸黄素代谢情况,采用了1.25-35μM(0.5-35μM)范围内9-11个底物浓度。
二、UPLC分析方法
为了对各模型化合物及代谢产物进行分析定量,采用了以下UPLC方法：对于汉黄芩素、千层纸黄素及其相应葡萄糖醛酸化代谢物,分析系统为Waters Acquity UPLC,采用光二极管阵列检测器(DAD)及Empower软件。分离柱为BEHC18,1.7μm,2.1×50mm。流动相B为100%乙腈,流动相A为0.1%(v／v)甲酸水溶液(pH 2.5)。流速为0.4 ml/min,采用梯度洗脱,0～1.5分钟时,流动相B占30-40%；1.5～2.5分钟时,流动相B占40-70%;2.5～3.0分钟时,流动相B占70-30%。检测波长280 nm,注入样品体积10μl。对于杨芽黄素、5-羟基-7,8-二甲氧基黄酮、5-羟基-6,7,8,4'-四甲氧基黄酮及其代谢物的UPLC分析定量方法与以上方法大体一致,不同之处仅在于梯度洗脱方法变为：0-1.5分钟时,流动相B占30-40%；1.5～3.0分钟时,流动相B占40-90%;3.0～4.0分钟时,流动相B占90-30%。
为了确保各模型化合物在实验过程中稳定,还进行了稳定性实验。在高、中、低三个浓度40μM,10μM和1.25μM(2.5μM)条件下,为每个化合物的分析方法做了日内及日间差异验证。
三、所选黄酮化合物葡萄糖醛酸化代谢物转化因子测定方法
为了准确地测定转化因子K值,从而进一步准确定量各葡萄糖醛酸化代谢产物,本研究采用了以下方法测定转化因子K值：(1)对于5,7-二羟基黄酮：汉黄芩素、千层纸黄素,选用了活性最强的UGT酶对底物进行葡萄糖醛酸化反应。其它5位单羟基黄酮则采用了大鼠微粒体来进行此过程。(2)用二氯甲烷萃取两次除去苷元(水相样品／二氯甲烷为2：5,v／v),葡萄糖醛酸苷存在于水相样品中。(3)将水相样品等分为两份,其中一份直接进行UPLC分析,另一份经β-葡萄糖醛酸苷酶(800 units/ml)在37℃水解。水解时间分别是：汉黄芩素水相样品1小时,千层纸黄素样品10小时,杨芽黄素、5-羟基-7,8-二甲氧基黄酮样品各12小时。将水解前后两份样品相比较,用苷峰面积的减少量除以苷元峰面积的增加量,而间接测得各转化因子值。
为了进一步考察所测定的转化因子K值是否准确,直接利用汉黄芩素及汉黄芩苷商品,配制同时含有相同浓度汉黄芩素及其苷的一系列浓度由低至高的标准溶液,则在各个标准溶液中两者浓度由低至高分别为：0.625,1.25,2.5,5,10,20,40μM。将这一系列标准溶液样品分别进UPLC进行分析,得到汉黄芩素峰面积(wogonin peak area, Aw)和汉黄芩苷峰面积(wogonoside peak area,AWG)。从这一系列标准曲线所测得的AWG:AW比值在各浓度下的平均值,即为汉黄芩苷转化因子值。将此直接方法所测得的结果与以上间接方法测得的转化因子结果进行比较,可以考察间接测定方法的准确性。
四、考察各个分析条件对转化因子K值的影响。
为了控制分析过程中各个分析条件对转化因子测定准确性的影响,进一步考察了实验过程中下列分析条件对转化因子K值测定的影响：流动相pH值、离子强度、样品介质pH值、检测波长。利用上述配制的汉黄芩素、汉黄芩苷系列标准溶液,逐个改变上述分析条件,并在改变某个条件时保持其它条件不变,观察AWG:AW比值的变化情况。五、应用LC-MS/MS推测所选黄酮化合物对应葡萄糖醛酸化代谢物的结构
为了进一步推测所选模型黄酮化合物经代谢后产生葡萄糖醛酸化代谢物的结构,采用超高效液相-电喷雾-质谱／质谱(UPLC-ESI-MSn)方法对黄酮及其葡萄糖醛酸化代谢物进行分离、检测以及分析,运用正离子方式检测,各黄酮及其苷的分离色谱条件与UPLC相同。质谱仪的主要质谱工作参数如下：仪器名称,三重四极杆质谱(Waters Micromass Quattro Premier XE);扫描模式,正离子模式；毛细管电压(capillary voltage) 3KV;锥孔电压(cone voltage) 35V;离子源温度(ion source temperature) 100℃;去溶剂化温度(desolvation temperature)350℃；锥孔气流(cone gas flow)50升／小时；去溶剂化温度气流(desolvation temperature gas flow)600升／小时；数据的采集及分析采用MassLynx V4.1软件(Waters Corp, Milford, MA, USA)。
六、酶动力学数据分析
为了得出汉黄芩素、千层纸黄素的UGT代谢动力学情况及两者在肝、肠的葡萄糖醛酸化动力学情况,将酶活性实验所得动力学数据进行分析。UGT酶、人类肝肠微粒体葡萄糖醛酸化代谢速率可以每分钟每毫克蛋白催化代谢反应所形成代谢物的量表示(nmol/min/mg)。动力学参数可以进一步根据Eadie-Hofstee图情况获得：如果Eadie-Hofstee图为线性或表现出非典型动力学(atypical kinetics)特点时,则在各个底物浓度(C)下黄酮葡萄糖醛酸苷的形成速率(V)变化情况会符合标准Michaelis-Menten方程或其它非典型动力学如autoactivation和biphasic动力学方程。数据拟合时采用ADAPTⅡ程序。而且,为了找出最合适的模型,采用阿开克(氏)信息判据(Akaike's information criterion, AIC)对各个侯选模型进行辨认,并应用了简约性原则(the rule of parsimony)。
七、运用UGT酶预测人肝、肠微粒体中黄酮葡萄糖醛酸化反应规律
为了预测模型黄酮化合物在人肝、肠微粒体中葡萄糖醛酸化代谢情况,结合UGT在各组织中表达情况及各UGT对化合物代谢的贡献大小,采用了以下方法：先经研究得出代谢黄酮化合物的主要UGT酶；接着参考各UGT在肝、肠中表达水平将几种起主要代谢作用的UGT联合起来,即运用加权平均方法计算这几种UGT葡萄糖醛酸化反应速率的平均值,用以预测该化合物在肝、肠微粒体中的代谢情况。再接下来,画出对应Eadie-Hofstee图以及纵轴为葡萄糖醛酸化反应联合速率、横轴为黄酮化合物浓度的葡萄糖醛酸化反应速率变化情况图,并获得各个表观动力学参数。此外,应用线性回归方法,得到起主要代谢作用联合UGT的葡萄糖醛酸化反应速率与肝、肠微粒体葡萄糖醛酸化反应速率间的相关系数。
八、统计学分析
标准曲线采用线性回归分析；以UGT数据与肝微粒体数据拟合采用线性相关分析；其他数据采用SPSS13.0统计软件分析处理,实验数据用均数±标准差(Mean±SD)表示；独立样本数据用One-Way ANOVA检验。组间多重比较采用Tukey法,方差不齐的采用Tamhane's T2法,显著性标准为α=0.05。
所得实验结果如下：一、对于汉黄芩素、千层纸黄素,所得代谢产物只有一个,且都为7位葡糖醛酸化物。GSMF实验表明,对汉黄芩素代谢速率最快的几个UGT为UGT1A3、1A8、1A9以及1A10,对于千层纸黄素,除了这几种UGT之外还有1A7。UGT酶特异性代谢研究结果表明UGT1A3、UGT1A7-1A10催化的动力学反应情况,都有已知动力学模型与之相符。而人肝、肠微粒体催化两黄酮化合物葡萄糖醛酸化代谢,则呈现简单Michaelis-Menten动力学特点。经过对动力学参数及动力学反应类型进行比较,表明UGT1A9可能是负责两黄酮化合物肝代谢的主要UGT酶。相反,两化合物的肠代谢则可能由UGT1A亚家族酶联合介导,其中UGT1A10占主导作用。相关性研究结果则明确表明,UGT酶可以用来对该两黄酮化合物在人体肝、肠微粒体中的代谢情况进行预测。总体来说,随甲氧基由8位变至6位,多数UGT代谢速率明显降低,特别是起主要代谢作用的UGT1A3,1A8, 1A9。
二、5-羟基-6,7,8,4'-四甲氧基黄酮经代谢后未发现任何代谢物。5H7MF和5H7,8MF经代谢后则各发现了一个单葡糖醛酸化代谢产物。GSMF实验表明,对于5H7MF和5H7,8MF,几乎所有UGT酶都对代谢有较低水平贡献,对5H7MF代谢明显最快的UGT酶为UGT1A1,其次为1A3：而对于5H7,8MF,代谢最快的UGT酶为UGT1A3,其次为位于同一外显子簇的UGT1A7-1A10,特别是UGT1A9(底物浓度为2.5,10μM时)和UGT1A8(底物浓度为35μM时),再次是UGT1A1及UGT2B15、2B17。相关性研究结果表明对于三个含甲氧基黄酮5H7MF、5H7、8MF、5H6,7,8,4'MF,联合UGT反应速率与肝微粒体反应速率之间有明显相关性。随着模型化合物甲氧基数量变化,各UGT特异性代谢特征也在不断发生变化。
三、为所选各化合物的分析方法做了日内及日间差异验证,测得五个化合物的精密度和准确度范围分别在可接受的0.02%-6.75%和86.73%-110.84%范围内。说明对所选模型化合物采用了可靠的方法进行分析,其稳定性研究结果显示该五个黄酮化合物在24小时时间范围内稳定,稳定性满足本研究所需。
四、为所选黄酮化合物的葡萄糖醛酸化代谢物进行转化因子测定,测得汉黄芩素、千层纸黄素、杨芽黄素、5-羟基-7,8-二甲氧基黄酮对应葡萄糖醛酸化代谢物的转化因子值分别为1.09±0.04、1.29±0.05、2.05±0.18、1.25±0.09。对于汉黄芩素和汉黄芩苷,两化合物都有商品提供,配制同时含有同浓度此两化合物的标准溶液,直接测得转化因子值为1.03±0.02,与以上间接方法测得的转化因子结果1.09±0.04非常接近,偏差在可接受范围之内。说明各黄酮葡萄糖醛酸化代谢物转化因子值能准确地进行测定。
五、考察实验过程中各个分析条件对转化因子K值测定的影响,发现随着流动相pH值由2.5增至8.5,汉黄芩素的出峰时间在pH值为3.5、4.5、6.5时相互接近,pH 8.5时前移39.43%；汉黄芩苷出峰时间前移21.88～41.47%,且所出峰接近溶剂峰,影响分析准确性。流动相pH 8.5与pH 2.5时测得各浓度下AWG:AW平均值接近,pH 3.5、4.5、6.5较pH 2.5时测得的AWG:AW平均值升高10.7-14.6%。在所选检测波长254、274、280、342nm条件下,所测得各浓度下AWG:AW平均值较接近,仅342nm波长条件下测得的AWG:AW平均值与280nm条件下所测值偏差较大,达9.71%。流动相离子强度、样品介质pH值变化时,各浓度下AWG:AW平均值无明显变化。
根据上述实验结果,本论文的结论如下：
一、对单甲氧基5,7二羟基黄酮汉黄芩素、千层纸黄素进行GSMF研究表明两化合物主要由UGTIA亚家族酶代谢,其中对代谢贡献最大的是UGT1A3及UGT1A7-1A10; GSMF及UGT酶特异性代谢情况,可用于预测人体肝、肠微粒体催化的葡萄糖醛酸化反应速率及代谢情况；甲氧取代基在A环上的位置变化可明显影响两化合物的代谢特征。
二、对含甲氧基5位单羟基黄酮杨芽黄素(5H7MF)、5-羟基-7,8-二甲氧基黄酮(5H7,8MF)和5-羟基-6,7,8,4'-四甲氧基黄酮(5H6,7,8,4'MF)进行GSMF研究表明5H6,7,8,4'MF检测不到代谢物,其它化合物主要由UGT1A亚家族酶代谢,其中对代谢贡献相对较大的是UGT1A1、UGT1A3及UGT1A8-1A10;初步发现运用GSMF可预测含甲氧基5位单羟基黄酮在肝的葡萄糖醛酸化代谢情况；模型化合物甲氧基数量变化会明显影响其UGT特异性代谢特征。
三、对所选五个黄酮化合物,采用了可靠的分析方法进行分析,稳定性研究结果显示该五个黄酮化合物在24小时时间范围内稳定,稳定性满足本研究所需。
四、为所选黄酮化合物的葡萄糖醛酸化代谢物进行转化因子值测定,间接测得汉黄芩素、千层纸黄素、杨芽黄素、5-羟基-7,8-二甲氧基黄酮对应葡萄糖醛酸化代谢物的转化因子值分别为1.09±0.04、1.29±0.05、2.05±0.18、1.25±0.09。采用汉黄芩素和汉黄芩苷商品配制标准溶液直接测得汉黄芩苷转化因子值与以上结果非常接近,因此本研究中各代谢物转化因子值能较好地进行测定。
五、利用含有汉黄芩素及其苷的系列标准溶液,考察各个分析条件对转化因子K值的影响,发现流动相pH值对汉黄芩苷、汉黄芩素的出峰时间有明显影响,特别pH 8.5时,两者前移明显,影响分析准确性；偏酸性条件有利于出峰的稳定,流动相pH值<7时测得AWG:AW平均值变化不明显。不同检测波长条件下测得AWG:AW平均值接近。流动相离子强度、样品介质pH值变化对转化因子值无明显影响。
综上所述,本研究在体外测定得到五个含甲氧基化合物的UGT酶特异性代谢指纹图谱,即参与两个5,7-二羟基黄酮化合物汉黄芩素、千层纸黄素代谢的UGT主要为UGT1A3及UGT1A7-1A10;在三个5-羟基黄酮化合物之中,5H6,7,8,4'MF未发现代谢物,参与5H7MF、5H7,8MF代谢的UGT主要为UGT1A1、1A3及UGT1A7-1A10。GSMF及UGT酶特异性代谢情况可以用来预测两个5,7-二羟基黄酮化合物在人体肝、肠,以及5H7,8MF、5H7MF、5H6,7,8,4'MF在人体肝的葡萄糖醛酸化代谢情况。同时,发现随着汉黄芩素、千层纸黄素之间微小的甲氧基取代位置变化,即由8位变至6位,多数UGT代谢速率明显降低；5H7MF、5H7,8MF、5H6,7,8,4'MF之间存在的甲氧取代基数量差别,也对黄酮类化合物葡萄糖醛酸化代谢特征有明显影响。
Flavonoids, characterized as polyphenolics, are widely distributed in our daily diets, beverages, medicinal plants and herbal remedies. The diverse biological effects of flavonoids have attracted great interests of scientists. The major reported pharmacological activities of flavonoids include antioxidative effects, protection against cardiovascular disease, and anticancer effects, etc. Despite of the above reported beneficial properties and demonstrated preclinical activities, it's a serious conern that the oral bioavailabilites of flavonoids were reported to be low (<10%), which is a big challenge to develop flavonoids into chemo-preventive and chemo-therapeutic agents. Therefore, a key issue in the development of flavonoids as disease prevention and therapy agents is to find a way to increase their bioavailabilities.
The metabolic pathways of flavonoids were diverse and complicated because of their complex structures. It was reported that extensive first-pass metabolism by phaseⅡenzymes including UGTs and SULTs was suggested to be the major causes for their low bioavailabilities; also the glucuronidation of flavonoids is the major metabolic pathway resposnsible for their low bioavailabilities. The majority of UGTs displayed broad and overlapping substrate specificities. Many studies including our own have shown that glucuronidation of flavonoids were strongly influenced by their structures. Furthermore, recent studies demonstrated that the methoxylated flavones, which may have chemopreventive properties superior to the more common unmethylated flavonoids or polyphenols. Therefore, the aim of this thesis is to investigate the mechanism on the UGT metabolism of methoxylated flavonoids.
In order to explore the metabolic characterization of methoxylated flavonoids, two 5,7-dihydroxyflavone, wogonin and oroxylin A, as well as three 5-monohydroxyflavone, tectochrysin (5-hydroxy-7-methoxyflavone,5H7MF),5-hydroxy-7,8-dimethoxyflavone (5H7,8MF),5-hydroxy-6,7,8,4'-tetramethoxy-flavone (5H6,7,8,4'MF) were selected as the model compounds. For the first two dihydroxyflavone, there exist minor structural differences of methoxyl position between them. For the later three monohydroxyflavone, the number of methoxyl is increased in order. A set of commercially available expressed human UGTs and human intestinal and liver microsomes were used to make detailed and systematic metabolic profiling studies for the five flavonoids in vitro. Firstly, the objective of the studies is to obtain the UGT-isoform specific metabolic fingerprint (GSMF) for the five model compounds. Another objective is to determine if GSMF and isoform-specific metabolism profiles might be used to predict glucuronidation rates and profiles in microsmes derived from human intestine and liver, two major organs responsible for first-pass metabolism as well as recycling via enteric and enterohepatic schemes. Finally, the third objective is to analyze the effect of methoxyl changes on the characterization of UGT-isoform specific metabolic of model compounds.
Methods.
1. Enzymatic activities of human expressed UGTs and organ microsomes
For the experiments of GSMF, isoform-specific metabolism profiles and human organ-specific metabolism profiles, the incubation procedures for measuring enzyme's activities using microsomes or UGTs were performed and essentially the same as the previous publications from University of Houston. Briefly, the procedures were as follows:(1) mix microsomes/supersomes (final concentration≈in range of 0.0053-0.053 mg protein per ml as optimum for the reaction), magnesium chloride (0.88 mM), saccharolactone (4.4 mM), alamethicin (0.022 mg/ml); different concentrations of substrates in a 50 mM potassium phosphate buffer (pH 7.4); and UDPGA (3.5 mM, add last) to a final volume of 680μl; (2) separate the 680μl mixture to obtain three equal portions with the volume of 200μl, and incubate them at 37℃simultaneously for a predetermined period of time (10 to 60 min); and (3) stop the reaction by the addition of 100μl of 94%acetonitrile/6%glacial acetic acid containing 90μM acetophenone (propiophenone for 5H7MF,5H7,8MF) as the internal standard. The reaction mixture was centrifuged at 13,000 rpm for 15min and the supernatant was directly subjected to UPLC for analysis. To profile UGT's activities, three substrate concentrations,2.5,10 and 35μM were used. To profile kinetics of wogonin and oroxylin A by UGT 1A1,1A3 and 1A7-1A10,9-11 substrate concentrations in the range of 1.25-35μM (0.5-35μM) were used.
2. UPLC analysis of five flavones and their glucuronides
To make precise analysis for all model compounds and their metabolites, a common method was applied for wogonin, oroxylin A as well as their corresponding glucuronides:system, Waters Acquity UPLC with photodiode array detector and Empower software; column, BEH C18,1.7μm,2.1×50 mm; mobile phase B,100% acetonitrile, mobile phase A,100%aqueous buffer (0.1%,v/v formic acid, pH 2.5); flow rate 0.4 ml/min; gradient,0 to 1.5min,30-40%B,1.5 to 2.5 min,40-70%B, 2.50 to 3.0 min,70-30%B, wavelength,280 nm for flavones and their respective glucuronides and acetophenone; and injection volume,10μl. For 5H7MF,5H7,8MF, 5H6,7,8,4'MF as well as their corresponding glucuronides, analysis method was similar to that of wogonin, oroxylin A with minor modification as follows:gradient,0 to 1.5min,30-40%B,1.5 to 3.0 min,40-90%B,3.0 to 4.0 min,90-30%B.
Stability studies were made to ensure all model compounds keep stable during the course of experiments. Analytical methods for each compound were validated for inter-day and intra-day variation using 6 samples at three concentrations (40,10 and 1.25μM).
3. Determination of conversion factor for four flavone glucuronides
To quanify glucuronides more precisely, following experiment procedures were operated to obtain the conversion factor (K) for the glucuronides of the four flavones: (1) glucuronidate substrates with the most active UGT isoform using the incubation procedures described previously; for 5H7MF,5H7,8MF with only one phenolic hydroxyl group, rat liver microsomes were used; (2) extract the aqueous samples containing glucuronides with dichloromethane (sample/dichloromethane=2:5, v/v) twice to remove the aglycones; (3) divide one extracted aqueous sample into two equal portions, where one portion was subjected to UPLC for analysis directly, and the other was analyzed after hydrolysis by P-glucuronidase (800 units/ml) at 37℃for 1 h (10 h for oroxylin A,12 h for 5H7MF,5H7,8MF). The conversion factor of wogonin was also directly derived using the standard curve of both wogonin and wogonoside.
4. Effects of UPLC analysis conditions on the conversion factor
In order to observe whether various analysis conditions, such as pH value and ionic strength of mobile phase, detection wavelength as well as pH value of sample medium, have influences on the conversion factor, a series of standard working solutions with seven concentrations in the range of 0.625 to 40μM for wogonin and its glucuronide were prepared. Then, analysis conditions were altered respectively and observed the variance of AWG:AW.When one of above analysis conditions changed, other analysis conditions were consistent with described in common method. Briefly, the procedures of varying analysis conditions were performed as follows:(1) prepare a series of mobile phase A with different pH value:3.5,4.5,6.5 and 8.5, via adding 0.1%aqueous ammonia into 0.1%methanoic acid solution (pH 2.5); (2) adjuste pH value of the series of solutions back to 2.5 by 1%methanoic acid, then ionic strength of these solutions were different from one another notwithstanding their pH values were same; (3) change the detection wavelentgh to 254,274,342nm; (4) pH value of sample medium were changed from 2.5 to 3.5,6.0,7.4 with 0.1% phosphoric acid.
5. Confirmation of flavone glucuronide structure by LC-MS/MS
To further confirm the identity of the metabolite as mono-glucuronides of their parent compounds, LC-MS/MS was applied. The separation, detection and analysis of flavones and their glucuronides were achieved by Waters Micromass Quattro Premier XE, operated in the positive ion mode. The main mass working parameters for the mass spectrometers were set as follows:capillary voltage,3KV; cone voltage,35V; ion source temperature,100℃; desolvation temperature,350℃; cone gas flow,501/hr; desolvation temperature gas flow,6001/hr. Data acquisition and analysis were performed using a MassLynx V4.1 software (Waters Corp, Milford, MA, USA).
6. Kinetic of glucuronidation
To obtain the kinetics of wogonin and oroxylin A glucuronidation by major UGT isoforms, human liver and intestinal microsomes, data from incubation experiments were used for analysis. Rates of flavone metabolism by expressed human UGT isoforms, human liver and intestine microsomes were expressed as amounts of metabolites formed per min per mg protein (nmol/min/mg). Kinetic parameters were then obtained according to the profile of Eadie-Hofstee plots. If the Eadie-Hofstee plot was linear or showed characteristic profiles of atypical kinetics, formation rates (V) of flavone glucuronides at respective substrate concentrations (C) were fit to the standard Michaelis-Menten equation or other atypical kinetics equations, using the ADAPTⅡprogram. To determine the best-fit model, the model candidates were discriminated using the Akaike's information criterion (AIC), and the rule of parsimony was applied.
7. Use of expressed UGTs to predict flavone glucuronidation in human intestinal and liver microsomes
To predict flavone glucuronidation in human intestinal and liver microsomes, tissue expression of different UGTs and the contribution of isoforms to flavone glucuronidation were considered. Because each tissue expressed different UGTs and/or different quantities of the same UGTs, we first determined the main isoforms responsible for the metabolism of each flavone and then use a combination of several major active isoforms to predict substrate metabolism profiles in human intestinal and liver microsomes. The combination was achieved based on weighted mean average expression levels. Subsequently, the glucuronidation profiles obtained using the combined glucuronidation rates versus flavone concentrations were used to obtain the apparent kinetic parameters after corresponding Eadie-Hofstee plots were generated. Moreover, linear regression was applied to derive apparent correlations between rates of reaction obtained using a combination of main UGT isoforms and those obtained using human intestinal and liver microsomes.
8. Statistical Analysis
One-way ANOVA with or without Tukey-Kramer multiple comparison (posthoc) tests were used to evaluate statistical differences. Differences were considered significant when p values were less than 0.05.
Results
1. For wogonin and oroxylin A, their metabolites were found to be wogonin-7-glucuronide and oroxylin A-7-glucuronide respectively, GSMF experiments indicated that they were metabolized mainly by UGTIAs, with major contributions from UGT1A3 and UGT1A7-1A10. Isoform-specific metabolism showed that kinetic profiles obtained using expressed UGT1A3 and UGT1A7-1A10 could fit to known kinetic models. Glucuronidation of both flavonoids in human intestinal and liver microsomes followed simple Michaelis-Menten kinetics. A comparison of the kinetic parameters and profiles suggests that UGT1A9 is likely the main isoform responsible for liver metabolism. In contrast, a combination of UGT1As with a major contribution from UGT1A10 contributed to their intestinal metabolism. Correlation studies clearly showed that UGT isoforms could predict metabolism of these two compounds in human intestinal and liver microsomes. With the change of methoxyl from 8-position to 6-position, the glucuronidation rates decreased for most isoforms, especially for UGT1 A3,1A8,1A9.
2. No metabolite was found for 5H6,7,8,4'MF. For 5H7MF and 5H7,8MF, metabolites were both found to be mono-glucuronides, almost all isoforms contribute to their glucuronidation at a low level. GSMF experiments indicated that UGT1A1 was found to be the most important isoform for 5H7MF, followed by UGT1A3. For 5H7,8MF, UGT1A3 was found to be the most important, followed by UGT1A7-1A10 and UGT1A1, especially UGT1A9 (at substrate concentration of 2.5μM,10μM) or UGT1A8 (at substrate concentration of 35μM). Correlation studies clearly showed that the glucuronidation rates of human liver microsomes have an apparent correlation with those derived from a combination of UGT 1A1,1A3 and 1A9. In addition, the UGT isoform-specific metabolic pattern changed with the increase of methoxyl number.
3. Analytical methods for each compound were validated for inter-day and intra-day variation, and found that precision and accuracy for five compounds were in the acceptable range of 0.02%-6.75%and 86.73%-110.84%respectively. The result suggests that the analytical methods for model compounds were credible, and all model flavones keep stable during the course of experiments.
4. The list of the conversion factor measured was as follows:wogonin,1.09±0.04; oroxylin A,1.29±0.05; 5H7MF,2.05±0.18; 5H7,8MF,1.25±0.09. The conversion factor of wogonoside was also derived directly using the standard curve of both wogonin and wogonoside, and the result was 1.03±0.02. Therefore, the two conversion factors of wogonoside derived from different methods were similar, showing that the accuracy of the conversion factor obtained for all glucuronides is acceptable.
5. When the effects of various analysis conditions on the deterimination of conversion factor were investigated, it was found that the retention time of wogonin and wogonoside moved forward 39.43%,41.47%respectively, when the pH value of mobile phase A increased to 8.5, and the ratio of AWG:AW obtained under pH 3.5,4.5, 6.5 was individually 10.7-14.6%higher than that obtained under pH 2.5, whereas the ratios determined under pH 2.5,8.5 were close to each other. With the variance of ionic strength of mobile phase A and pH value of sample medium, the ratios determined were similar. Under the predetermined detection wavelength of 254,274, 280 and 342 nm, the ratios of AWG:AW obtained were found similar, and the ratio obtained under 342 nm deviated 9.71%from those obtained under 280 nm.
Conclusions
1. For wogonin and oroxylinA, GSMF experiments indicated that both flavonoids were metabolized mainly by UGT1As, with major contributions from UGT1A3 and UGT1A7-1A10. GSMF and isoform-specific metabolism profiles can be used to predict glucuronidation rates and profiles in human tissue microsomes. Minor structural differences of methoxyl position between wogonin and oroxylin A significantly impacted their metabolism
2. For 5H7MF,5H7,8MF,5H6,7,8,4'MF, GSMF experiments indicated that no metabolite was found for 5H6,7,8,4'MF, other flavonoids were metabolized mainly by UGT1 As, with major contributions from UGT1A1, UGT1A3 and UGT1A8-1A10. GSMF could be used to predict the three methoxylated flavones metabolism in liver microsomes. Structural differences of methoxyl number significantly impacted their UGT isoform-specific metabolic pattern.
3. The selected five flavones, wogonin, oroxylin A as well as 5H7MF,5H7,8MF, 5H6,7,8,4'MF could be analyzed accurately, and keep their stability during the course of experiments.
4. For wogonin, the conversion factor derived directly from the standard curve of both wogonin and wogonoside, was very similar with that from the determination method used for all glucuronides of model flavone, so the accuracy of the conversion factor obtained for all glucuronides is acceptable.
5. Among all UPLC analysis conditions, pH value variance of mobile phase would lead to the change of retention time of wogonin, wogonoside and impact the determination of conversion factor, and pH values<7 were more suitable for the determination. In addition, results measured under various detection wavelength were found similar, ionic strength variance of mobile phase, pH value variance of the sample medium was found had no influences on the determination of conversion factor.
In conclusion, the GSMF were obtained for five methoxylated flavonoids. For two 5,7-dihydroxyflavone, wogonin and oroxylin A, they were metabolized mainly by UGT 1 A3 and UGT1A7-1A10. For three 5-monohydroxyflavone, no metabolite was found for 5H6,7,8,4'MF, UGT1A1, UGT1A3 and UGT1A7-1A10 were found to be the main isoforms contributing to the glucuronidation of 5H7MF and 5H7,8MF. GSMF and isoform-specific metabolism profiles, could be used to predict glucuronidation rates and profiles of wogonin, oroxylin A in human liver and intestine microsomes and predict glucuronidation rates of 5H7MF,5H7,8MF and 5H6,7,8,4'MF in human liver microsomes. For wogonin and oroxylin A, the glucuronidation rates of most isoforms were found decreased with the change of methoxyl from 8-position to 6-position. For 5H7MF,5H7,8MF and 5H6,7,8,4'MF, the UGT isoform-specific metabolic pattern were found changed with the vaiance of methoxyl number.
引文
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