汉黄芩苷和汉黄芩素肠局部循环新处置机制
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摘要
药物的处置过程包括了药物的分布、代谢和排泄,与药物的疗效密切相关。药效的起始时间和药效的强度主要由药物分布到治疗部位的速度和量决定,药物作用的持续时间则主要由药物的代谢和排泄决定。
许多具有葡萄糖醛酸化和磺酸化等二相代谢途径的药物在体内可以进行循环,如肝肠循环(Enterohepatic cycle)和肠肠循环(Enteric cycle),这类处置机制使药物在体内具有较长的保留时间,对延长药物的作用时间有非常重要的意义。
早在半个世纪之前人们就认识到有些药物在体内可形成肝肠循环,许多药物以葡萄糖醛酸苷或磺酸苷等二相代谢结合物形式从肝脏随胆汁被排入肠道,在肠道细菌的β-葡萄糖醛酸苷酶(Glucuronidase, GUS)或磺基转移酶(Sulphotransferase, SULT)作用下水解成苷元,苷元易被肠道黏膜细胞吸收,在细胞内的二相代谢结合酶如葡萄糖醛酸转移酶(UDP-glucuronosyltranferase, UGT)等酶作用下再形成药物的葡萄糖醛酸苷或磺酸苷,这些苷进入循环系统后,可经肝脏从胆汁再次被排入肠道,从而形成完整的肝肠循环。
肝肠循环对体内必需物质的利用和许多药物发挥疗效都是非常重要的。机体内这种肝肠循环可以有效地利用体内必需物质,例如帮助消化的胆酸,一般进食一次,胆酸至少要经历2-3次的肝肠循环；其他具有肝肠循环的身体必需物质有维生素D3、维生素B12、叶酸和雌激素等。在体内具有肝肠循环的药物有很多,如地高辛、洋地黄毒苷、氨苄青霉素、吗啡、美沙酮、苯妥因钠、华法令、氯丙嗪等。
药物的肠肠循环则是肝肠循环之外的另一种体内循环,直到近来才被人们注意到。肠肠循环是指苷元类药物在十二指肠、空肠、回肠和结肠各段均易被吸收进入肠黏膜细胞,在UGT等酶作用下转化成葡萄糖醛酸苷或磺酸苷,部分苷被肠黏膜细胞直接外排至肠腔,此后,在肠道后段被细菌分泌的β-GUS酶和SULT酶作用下水解成苷元,再次被吸收进入肠黏膜细胞的过程。肠肠循环与肝肠循环的主要区别在于药物的葡萄糖醛酸苷或磺酸苷不是经肝脏随胆汁被排入肠道,而是直接被黏膜细胞外排至肠道的。
我们在研究汉黄芩素(Wogonin)的吸收代谢过程中,发现汉黄芩苷(汉黄芩素-7-0-葡萄糖醛酸苷Wogonoside)与汉黄芩素在体内相互转化,因而发现在肠道易葡萄糖醛酸化的化合物具有第三种循环,也可能是最重要的一种循环：肠细胞局部循环。在局部循环中,药物在的葡萄糖醛酸苷被肠黏膜细胞外排,随后可被黏膜细胞外主要来源于肠道细胞分泌的β-GUS酶水解为苷元,苷元再被此肠黏膜细胞吸收,经细胞内UGT酶作用重新形成药物的葡萄糖醛酸苷,部分苷将再被肠黏膜细胞外排至肠腔,因此苷元及其葡萄糖醛酸苷可在十二指肠、空肠、回肠和结肠整个肠道某一肠黏膜细胞及此细胞外侧肠道局部不断循环。肠局部循环与肠肠循环主要区别有两点：一是酶的来源不同,二是药物重吸收的部位不同。肠肠循环中的β-GUS酶由细菌分泌,细菌主要集中在肠道后端,故药物葡萄糖醛酸苷主要在肠道后段水解,药物的重吸收应主要在肠道后段。局部循环中的β-GUS酶主要来源于肠细胞,整个肠道的黏膜细胞均可重吸收药物并形成局部循环,这对肠道疾病的治疗具有极为重要的意义。
我们选择汉黄芩素和汉黄芩苷为模型化合物研究肠细胞局部循环机制是基于以下理由：
一、汉黄芩素具有非常重要的药理活性,如用于抗炎和抗肿瘤。汉黄芩素能恢复肿瘤坏死因子受体细胞凋亡诱导配体(Tumor necrosis factor receptor apoptosis-inducing ligand, TRAIL)失活的癌细胞中TRAIL的敏感性；在人结肠癌HCT116细胞,汉黄芩素可通过p53依赖的PUMA诱导作用诱导细胞凋亡。
二、汉黄芩素在体内生物利用度很低,可能与其在体内存在广泛的葡萄糖醛酸化反应生成其葡萄糖醛酸苷——汉黄芩苷有关。
三、汉黄芩素和汉黄芩苷存在于多数处方药物中,已广泛运用于人体,例如BZL101,一种含有汉黄芩素的植物水提物,对乳腺癌细胞株有效,Ⅰ期临床试验表明它毒性低,显示良好的临床效应。一些含中药黄芩的组方,具有抑制肠道炎症的功效。
四、多种黄酮类化合物都对胃肠道疾病有效,例如水飞蓟宾可以抑制丙型肝炎病毒,而绿茶中黄酮类化合物均对结肠癌有活性。汉黄芩素和汉黄芩苷可能是用于胃肠道疾病的潜在药物,且药物的肠局部循环可能对肠道疾病的治疗极为有利。
五、汉黄芩素和汉黄芩苷经济易得,大量价廉的黄芩等植物可为二者提供丰富的来源。
研究的方法和结果如下：
一、汉黄芩苷与汉黄芩素相互转化研究
我们采用了在体大鼠灌流模型来考察两种药物的吸收代谢。雄性SD大鼠(80-110天),280-350 g,选用大鼠十二指肠、空肠、回肠末端和结肠四个肠段进行在体灌流实验。结果表明汉黄芩素和汉黄芩苷在大鼠肠道内相互转换。灌流两个浓度的汉黄芩素(5μM和20μM)时,在四个肠段灌流液中都能发现汉黄芩素的葡萄糖醛酸苷——汉黄芩苷。灌流两种浓度(5μM和20μM)的汉黄芩苷时,在四个肠段灌流液中同样都能发现其苷元汉黄芩素；令人惊讶的是,小肠上段(十二指肠、空肠)灌流液中也有汉黄芩素出现。传统观念认为,肠道内水解黄酮萄萄糖醛酸苷的β-GUS酶来源于细菌,而十二指肠和空肠中细菌数量远远低于回肠末端和结肠,缺少来源于细菌的β-GUS酶,汉黄芩苷在十二指肠和空肠内应不会被水解为汉黄芩素,也没有萄萄糖醛酸苷在小肠上段被水解的文献报道。
汉黄芩素比汉黄芩苷的极性小,在肠道更容易渗透,它的膜渗透系数P*eff值比汉黄芩苷的高约10倍。和预期一样,当灌流苷元汉黄芩素,在四段肠都有良好吸收,例如灌注5μM时苷元在四段肠的百分吸收率分别为：66.87%、66.73%、67.00%、74.62%；灌流汉黄芩苷时,它在每一个肠段对应的吸收率都比汉黄芩素低。
我们收集了四段肠的空白灌流液,分别加入汉黄芩素和汉黄芩苷,结果发现汉黄芩苷在肠空白灌流液中不稳定,20μM的汉黄芩苷24小时内几乎完全被水解为汉黄芩素。这表明空白灌流液中存在能水解汉黄芩苷的酶,但这种水解酶是不是β-GUS酶,或是否由细菌分泌仍不清楚,这种水解酶的种类和来源都有待更进一步的研究。此外,汉黄芩素在肠空白灌流液中稳定,不会转化为汉黄芩苷。这表明空白灌流液中没有UGT酶,在灌流汉黄芩素时,灌流液中出现的汉黄芩苷应来源于肠黏膜细胞的外排。
二、汉黄芩苷与汉黄芩素溶解性、稳定性和吸附性考察
为了防止药物溶解性、稳定性和吸附性影响实验数据的准确性,我们考察了汉黄芩苷与汉黄芩素的这些性质。结果发现二者在HBSS溶液中160-0.3125μM浓度范围内完全溶解,稳定性也完全满足吸收代谢实验研究的需要,且实验中使用到的硅胶管和EP管对二者没有吸附性。这些结果显示,汉黄芩苷在肠道和灌流液中水解与其物化稳定性无关,也与溶解性和吸附性无关。
三、水解酶种类研究
我们先采用空白灌流液来研究汉黄芩苷的水解酶种类。
为了探明水解酶的种类,我们首先寻找了一种化学终止剂(94%乙腈和6%甲酸组成),以便能按需要求随时终止酶的水解反应,考察任一酶水解反应进行的程度。实验证明,加入反应终止液后,能完全终止水解酶的水解作用,汉黄芩苷在加有终止液的空白灌流液中24小时保持稳定。在药物灌流实验中,收集样品前均加入此终止液,使药物在灌流液从肠道导管出口流出后酶水解作用就被终止,保证了水解过程只发生在肠道,在收集管中药物不会再发生水解,以此测定药物在肠道内水解的程度。
最早我们考虑到水解作用可能是受细菌的影响,故采用13000rpm离心30min后,用0.22μm无菌过滤的方式去除空白灌流液中的细菌,结果发现空白灌流液除菌对汉黄芩苷的水解没有影响,这显示引起药物水解的可能是游离的水解酶。
因为乳酸根皮苷水解酶(Lactasc Phlorizin Hydrolase, LPH)在体内对葡萄糖苷的水解过程中起重要作用,它是否也能水解葡萄糖醛酸苷仍不清楚,故加入其特异性抑制剂——20mM的葡萄糖酸内酯,结果发现抑制LPH酶对汉黄芩苷的水解无明显影响。
当我们加入P-GUS酶的特异性抑制剂——0.1mM葡萄糖二酸单内酯时,发现空白灌液中汉黄芩苷的水解被显著抑制,当加入4.4mM葡萄糖二酸单内酯时(4.4mM为UGT酶药物代谢实验中用于抑制葡萄糖醛酸苷水解时的葡萄糖二酸单内酯浓度),能完全抑制汉黄芩苷的水解。
随后我们进行了大鼠在体灌流实验,结果显示在小肠上段(十二指肠和空肠)汉黄芩苷的吸收和其苷元的转化率会受β-GUS酶的抑制剂——葡萄糖二酸单内酯(0.1 mM)抑制,但不会受LPH酶的抑制剂——葡萄糖酸内酯抑制。这表明水解汉黄芩苷的是β-GUS酶。
四、水解酶的来源研究
肠道中β-GUS酶的可能有三种来源：第一种可能是来源于肝脏,分泌的酶可随胆汁排入肠道；第二种可能是肠道菌群分泌；第三种可能是来源于肠道细胞。但传统观念认为动物的P-GUS酶只存在于细胞内,肠道菌群是肠道中β-GUS酶的主要来源。
我们将汉黄芩素和汉黄芩苷加入胆汁溶液,发现二者在胆汁中的稳定性较好,且在灌流实验时,胆汁被收集另存,没有进入肠道,这否定了β-GUS酶主要来源于肝脏或胆汁的可能性。
我们收集了不同肠段不同时间段的新鲜空白灌流液。我们取一部分新鲜空白灌流液进行细菌培养后做总菌落计数,比较灌流液样品中的细菌数量；另一部分加入汉黄芩苷,考查其水解速率。若肠道菌落是GUS酶的主要来源,那么肠道灌流液中的细菌数将会与其对苷的水解能力成正比。然而,实验结果表明细菌的数量与汉黄芩苷的水解速率不相关。
我们采用大鼠在体灌流实验,并对大鼠肠道做急性抗生素处理,即灌液前在20gM汉黄芩苷的HBSS溶液中加入混合抗生素(100单位青霉素和链霉素为0.1mg/毫升),同样,经过半小时灌流平衡之后再收集灌流液样品检测,结果显示混合抗生素不会影响汉黄芩苷的吸收或代谢。这些结果都与认为肠道β-GUS酶来源于细菌的观点不一致,至少说明肠道菌落不是肠道内β-GUS酶的主要来源。在以上实验结果基础上,我们推测大鼠肠道细胞可能是肠道中β-GUS酶的一种主要来源。
五、GUS酶来源的酶代动力学验证
由于缺乏能区分β-GUS酶是来源于细菌或是大鼠的商业性单克隆抗体,我们只能采用酶代动力学参数用来区分他们。为此,我们制备了肠道S9微粒,因为S9中含有光滑内质网,包含了大部分细胞内酶。以肠道S9中的β-GUS酶作为大鼠肠细胞来源的对照。另外,我们还制备了大鼠粪上清液,提取粪便微生物中的酶类,以粪上清液中的β-GUS酶作为细菌来源的对照。
我们选择了细菌数量差距大于2700倍的高、中、低宽浓度范围内的四个灌流液样品进行比较,发现空肠90-120min的肠灌流液样品中细菌量最少,但有最高的水解速率。相比之下,结肠60-90min灌流液样品中细菌量最多,但其水解速率最低。结果显示细菌量最多的样品并不是具有最快的水解速率。
我们测定了汉黄芩苷在同一个时间段(60-90min)四段肠的空白灌流液中的水解速率,结果再次表明具有细菌数最多的结肠灌流液并没有最快的水解速率。
我们测定了汉黄芩苷在空肠中5个时间段的空白灌流液中的水解速率,结果显示灌流液中β-GUS酶的量(体现在Vmax值上)随时间呈下降趋势,表明β-GUS酶的量随着灌流时间延长在减少。
我们还测量灌流液的总蛋白浓度,考察总蛋白浓度与水解活性的关系,但令人奇怪的是它们也不相关。此外,我们发现灌流液中总蛋白含量没有随着灌注时间减少,这显示蛋白可能是在灌流过程中的脱落的肠黏膜细胞或(和)粘液组织。最后,发现灌流液中的细菌量与其总蛋白浓度也没有相关性。
我们测定灌流液样品、肠道S9和粪上清液中β-GUS酶的动力学参数,得到Eadie-Hofstee图。结果表明汉黄芩苷在不同的介质中的酶催化水解都遵循典型的米氏动力学规律(Eadie-Hofstee图呈线性)。三段小肠S9的Km值都相似(25-30μM),但它们均明显高于粪上清的Km(7.7μM)。再比较肠道灌流液样本的Km值(15.8-25.6μM),除了回肠末端的两个灌流液样品Km值(6.3-6.9μM)接近粪上清液的Km值外,其他灌流液样品的Km值都与来源于肠道S9的Km值很相接近。这些结果都显示空白灌流液中的β-GUS酶应主要来源于肠道细胞。
六、汉黄芩苷及汉黄芩素的药代动力学研究
我们采用大鼠灌胃给药后,眼眶取血方式检测血药浓度来研究汉黄芩苷及汉黄芩素的药动学特征。采用3P97的统计矩法计算药时曲线下面积(AUC0-24h)等参数。结果显示汉黄芩苷与汉黄芩素灌胃给药后,药血浓度曲线均呈双峰,曲线相似,血中药物主要以汉黄芩苷形式存在,汉黄芩素的量极低,这显示汉黄芩素在体内存在广泛的葡萄糖醛酸化。
从药动学参数来看,二者Tmax1、Tmax2、Cmax2、t1/2、MRT0_T、AUC0-T、AUC0-∞值都非常接近。这些参数显示二者在体内具有相同的动力学模式。这与实验预期一样,可能因为汉黄芩苷与汉黄芩素在体内都会出现局部循环过程,应有相同的动力学模式。二者的平均滞留时间MRT0-T值都较大,分别为11.53±1.27h和9.36±9.95h,这可能与二者在肠道中的局部循环处置过程延长了它们在体内的滞留时间有关。
根据上述实验结果,本论文的结论如下：
第一,汉黄芩苷与汉黄芩素在体内相互转化,汉黄芩苷在肠道能迅速水解成汉黄芩素,并且被快速吸收。
第二,汉黄芩苷主要是在β-GUS酶作用下水解的。因为空白灌流液中的水解可以被其特异性抑制剂——葡萄糖二酸单内酯抑制,此外,当汉黄芩苷与葡萄糖二酸单内酯同时灌流时,汉黄芩苷的吸收显著减少,而LPH酶抑制剂或抗生素与汉黄芩苷一起灌流时,不会影响其吸收。
第三,肠道中β-GUS酶的活性是以肠来源的β-GUS酶占主导,因为在小肠上段(如：空肠)酶的水解活性比结肠强,而在不同肠道的空白灌流液细菌计数显示,结肠中细菌数是最高的。
第四,小肠上段的空白灌流液中P-GUS酶的活性更强(图-9B),且汉黄芩苷在十二指肠和空肠的空白灌流液的水解Km值与它们对应肠道S9的Km值几乎相同。从浓度与速率比的图形趋势和Eadie-Hofstee图来看,它们的动力学模式也非常相似。
第五,汉黄芩素与汉黄芩苷大鼠口服给药后,二者药物动力学参数非常相似,它们的平均滞留时间MRT0-T值都较大,药血浓度曲线均呈双峰,曲线相似趋势。
第六,肠道中没有游离的UGT酶,此酶只存在于黏膜细胞内。汉黄芩素在空白灌流液非常稳定,在体灌流汉黄芩素时,肠灌流液中的汉黄芩苷均来源于黏膜细胞的外排。
在以上实验结果的支持下,因此我们得出黄酮类葡萄糖醛酸化合物可以在肠道形成局部循环的结论。
总的来说,我们发现一个新的肠局部循环机制,它将提高黄酮类化合物如汉黄芩素的局部生物利用度和作用时间。这种局部循环机制可能对在肠道具有广泛葡萄糖醛酸化的其他药物和食物成分的生物利用度产生重大影响。由于有大量酚类物质在肠道中可以葡萄糖醛酸化,这种新发现的循环处置机制对酚类物质的生理作用可能有非常大的影响,可能改变它们的局部生物利用度和滞留时间,最终影响这类重要化合物的药效和毒性。
Drug disposition involving distribution, metabolism, and excretion, has great relation with drug's effects. The rate and amount of drug reaches the target site may influence the initial time and the degree of drug affecting. Moreover, the duration time of drug affecting last is determined primarily by the metabolism and excretion.
The amount of drug distribution differs in various organs, and distribution characterization varied for diverse drugs. Similarly, metabolism and excretion characterization varied for diverse drugs. Many drugs proceed enteric and enterhepatic recycling in body by the means of phase II metabolism, such as glucuronidation and sulfation, which play a central role in prolonging the effecting time of drugs.
Enterhepatic recycling scheme was recognized more than half a century ago. It involves the action of liver to excrete the conjugated phase II metabolites and action of the microflora or bacterialβ-glucuronidase or sulfatases to release the aglycones (from the conjugated phase II metabolites). Released aglycones can enter the body again and form glucuronides or sulfates again, thereby completing the recycling loop.
Enterhepatic recycling scheme may help body utilize endogenous necessary material effectively. For example, during the bile digesting, cholic acid would experience enterhepatic recycling two or three times after administration once. Other similar necessary materials are vit D3, vit B12, folic acid etc. Many drugs like digoxin, cardigin, diethylstilbestrol, ampicillin etc, would also experience recycling scheme of this kind. Therefore, enterhepatic recycling is of great importance to the utilization of endogenous necessary material and exerting of drug effects.
Enteric recycling has only been termed more recently. In this recycling scheme, the phase II conjugates are excreted by the enterocytes, and once again, the action of bacterialβ-glucuronidase or sulfatases is required to release aglycone for reabsorption.
In our study, we have identified the third and perhaps the most important recycling scheme for compounds that are extensively glucuronidated in the gut:the local recycling. In the local recycling scheme, the phase II conjugates are excreted by the enterocytes, and also once again, the action of bacterial P-glucuronidase or sulfatases is required to release aglycone for reabsorption. After reabsorption, drug was again glucuronidated by UDP-glucuronosyltranferase (UGT), and one part of the glucuronides was excreted by the enterocytes. In this way, drug and its glucuronide could form a complete recycling loop at one enterocyte and gut outside it.
Wogonin, the aglycone of wogonoside(wogonin-7-O-glucuronide), was a kind of dihydroxylflavone. It has attracted a lot of attention for its anti-tumor and anti-inflammatory activity in the gut and elsewhere and was used here as the model compound to prove the presence of this recycling scheme. Wogonin was selected because it is known to be extensively glucuronidated. It was also chosen because of its important pharmacological activities, including its ability to restore the sensitivity of tumor necrosis factor receptor apoptosis-inducing ligand (TRAIL) in TRAIL-resistant cancer cells. In human colon cancer HCT116 cells, it was shown that wogonin-induced apoptosis was via p53-dependent PUMA induction. Wogonin was also selected because it has been used widely in humans, mostly in the form of herbal formulation. For example, BZL 101, an aqueous herbal extract active against breast cancer cell lines, was shown to have a favorable toxicity profile in a phase I clinical trial and demonstrated encouraging clinical activity. Hange-shashin-to (HST), a combination of seven herbs including Scutellaria baicalensis, was found to suppress inflammatory bowel disease. Another reason to study wogonin was several flavonoids were shown to be active in management of the gastrointestinal diseases. For example, silibinin was shown to inhibit hepatitis C virus, whereas green tea flavonoids were shown to be active against colon cancer. Lastly, wogonin was also selected because large quantities of wogonoside were available commercially at a reasonable cost.
Methods and Results.
1. The conversion study of wogonin and wogonoside.
A "four site perfusion model" was used to investigate the transport and metabolism of wogonin or wogonoside. Male Sprague-Dawley rats (80-110 days old) weighing from 280 to 350 g were obtained to operate the experiments, and four segments of the intestine (duodenum, upper jejunum, terminal ileum and colon) were perfused simultaneously with perfusate containing the compound of interest. The result showed that wogonin and wogonoside could convert to each other in the rat intestine individually. When wogonin of 5μM and 20μM concentrations was perfused, wogonoside was respectively found in all four segments of intestine. On the contrary, when wogonoside of 5μM and 20μM concentrations was perfused, wogonin was respectively found in all four segments of intestine. To our surprise, wogonin was also found in the upper segments of intestine (duodenum, jejunum). According to classical view,β-glucuronidase that hydrolyse glucuronides comes from bacteria. However, bacteria count in duodenum, jejunum was much lower than that of ileum and colon.In addition, wogonoside couldn't be converted into wogonin in duodenum and jejunum, for lack of bacterialβ-glucuronidase.
Compared with wogonoside, wogonin was smaller and less polar, and therefore it penetrated the intestinal membrane more rapidly. Moreover, P*eff of wogonin is 10 times higher than that of wogonoside. As expected, when wogonin or aglycone was perfused, it was well absorbed in all four segments of intestine. When wogonin of 5μM concentration was perfused, its absorption percentage was 66.87%,66.73%, 67.00%,74.62% in order at the four segments of intestine. When wogonoside was perfused, its absorption percentage was lower than that of wogonin at respective region of intestine.
After blank intestinal perfusate of four segments were collected, wogonin and wogonoside was put in it individually. It was found that wogonoside was unstable in blank intestinal perfusate, wogonoside of 20μM concentration was almost hydrolysis into its aglycone within 24 hours. It proved that enzymes hydrolyzing wogonoside did exist in the blank intestinal perfusate, and further research should be carried on to determine the kinds and sources of the hydrolysis enzymes. Wogonin was found stable in blank intestinal perfusate, and wouldn't convert into its glucuronide. It showed that no UGT exists in the blank intestinal perfusate, and the glucuronide found in the perfusate when wogonin was perfused, should be released from the enterocytes.
2. The dissolubility, stability, adsorbability study of wogonin and wogonoside.
The dissolubility, stability, adsorbability were investigated individually during experiment to assure the accuracy of all data. It was found that two compounds dissolve completely in HBSS buffer in the concentration range of 0.3125-160μM, and stability of the two chemicals could meet the requirement of absorption and metabolism study, all silica tube and EP tube used in the experiment didn't show adsorbability to the two chemicals. All above results indicate that the wogonoside hydrolysis in gut and perfusate has no relation with their chemical stability, or dissolubility and adsorbability.
3. The kinds study of the hydrolysis enzymes of wogonoside.
Blank intestinal perfusate was collected to investigate the kinds of wogonoside hydrolysis enzymes. A stop solution (consisting of 94% acetonitrile and 6% glacial acetic acid) was first developed to ensure the enzyme hydrolysis could be ended at any time. It was demonstrated that wogonoside is stable in blank intestinal perfusate during 24 hours after the addition of the stop solution. Hence, the stop solution was added into all samples before their collections to stabilize the drug in samples.
The influence of bacteria was first considered, and experiments were performed to determine the effects of bacteria presence. The results indicated that removal of bacteria via centrifugation (13,000 rpm,30 min) and aseptic filtration did not improve stability of wogonoside. Therefore, free hydrolysis enzymes might be the main reason for the wogonoside hydrolysis.
Lactasc phlorizin hydrolase (LPH) is a major contributor to the glucoside hydrolysis in the intestine. After addition of gluconolactone, a specific inhibitor of the LPH, it was found that the hydrolysis of wogonoside was not inhibited.
When 0.1 mM saccharolactone, the specific inhibitor of the glucuronidase was investigated, it was found that the hydrolysis of wogonoside was inhibited significantly. When the concentration rose from 0.1 mM to 4.4 mM, the hydrolysis of wogonoside was found inhibited thoroughly.
The later perfusion experiment showed that the wognoside absorption and its aglycone conversion rate on the upper intestine would be inhibited by the specific inhibitor of the glucuronidase,0.1 mM saccharolactone, not by the specific inhibitor of LPH, gluconolactone. All the results showed thatβ-glucuronidase is exactly the enzyme responsible for the wogonoside hydrolysis.
4. The source study of the hydrolysis enzymes of wogonoside.
The P-glucuronidase in intestinal gut may come from three sources. The first source may be the liver, and the enzymes excreted there may enter gut with bile. The second source may be the intestinal microflora, this is the most obvious source and has been widely accepted. The third source may be the enterocytes.
After adding wogonin and wogonoside into bile, both of the compounds were found stable. Moreover, a bile duct cannulation was made in perfusion experiments to avoid the bile entering the intestinal gut. Therefore, it's impossible for theβ-glucuronidase to come from liver in this study.
Sequential perfusate samples were collected from all four regions, and amount of bacteria in every sample was determined and then plotted against hydrolysis reaction rates measured using 20μM wogonoside. The results indicated that there was no correlation between the numbers of bacteria present in the perfusate and rate of wogonoside hydrolysis.
To make acute antibiotic treatment, an antibiotic mixture of penicillin and gentamicin was added into the HBSS buffer containing 20μM wogonoside before perfusion. The results indicated that absorption or metabolism of wogonoside was not impacted. All these results didn't accordance with the opinion that the P-glucuronidase in intestinal gut supplied by the intestinal microflora, and the intestinal bacteria couldn't be the sole source for the intestinal gut. Based on above results, we conclude that enterocytes in rat intestine gut might be the main source ofβ-glucuronidase in intestinal gut.
5. The kinetic validation of the source of GUS enzyme
Because of the lack of commercial monoclonal antibody to distinguish bacteriaβ-glucuronidase from mammalian one, kinetic parameters were used to distinguish them. To do so, rat intestine S9 fractions were prepared for S9 contains the smooth endoplasmic reticulum with most enterocyte enzymes in it, and the P-glucuronidase in S9 fractions could be referred, when the source of intestinal gut was considered. In addition, rat feces extract was also prepared, and the P-glucuronidase in feces extract could be referred, when the source of bateria was considered.
We also selected four perfusate samples with drastically different amounts of bacteria (difference of greater than 2,700-fold), and found that 90-120 min jejunal perfusate sample had the highest Vmax value even though it has the lowest bacteria count. In contrast,60-90 min colon perfusate sample with the highest bacteria count had the lowest Vmax value.
The above results showed that samples with higher bacteria count did not have faster hydrolysis rates. To confirm this finding, we also determine the metabolism of wogonoside in different perfusate collected at the same time, and the results again indicated that samples with the highest bacteria count (colon) did not have faster hydrolysis rates. However, the amounts of enzyme (as represented by the Vmax values) present in the perfusate appeared to decrease with time, indicating amount of enzymes present in the perfusate was less after continuous single-pass perfusion.
To determine if protein concentration in the perfusate could correlate with the hydrolysis activities, the amounts of protein in the perfusate were plotted against hydrolysis rates, and surprisingly, there was no correlation either. In addition, we found that protein concentration did not decrease with perfusion time, indicating that protein in the perfusate may be due to sloughing off from the enterocytes or mucus or both. Lastly, there was no correlation between bacteria counts and protein concentration in the perfusate.
The kinetic parameters were determined and Eadie-Hofstee plots were generated forβ-glucuronidase derived from intestinal S9 fractions, microbial enzymatic extract, and perfusate samples. The results indicated that enzyme-catalyzed hydrolysis of wogonoside in different sample matrix all followed a classic Michaelis-Menten kinetic profile. The Km values of intestinal S9 fractions from all three small intestinal segment were similar (25-30μM), but they were significantly higher than those derived from microbial enzymatic extract (7.7μM). When comparing to the Km values derived from the intestinal perfusate samples (15.8-25.6μM), they were all close to the Km values derived from the intestinal S9 fractions, except for two terminal ileal perfusate samples, which displayed Km values (6.3-6.9μM) closely resembled that of the microbial enzymatic extract. All the results showed that (3-glucuronidase in blank perfusion should mainly come from intestinal gut.
6. The pharmacokinetic study of wogonin and wognoside
To study the pharmacokinetic characterization of wogonin and wognoside, blood samples were collected from fossa orbitalis to determine drug concentrations in blood after rat intragastric administration. Pharmacokinetic parameters such as AUCo-24h were obtained using 3P97 statistical moment to calculate. It was found that the blood drug concentration curves of the two compounds both displayed double peaks, and their curve profiles were similar. Most chemicals in blood were wogonoside, only little amount of wogonin exist, this indicated that wognonin was glucuronidated extensively in vivo, and exist in the form of wogonoside in blood. For pharmacokinetic parameters, the MRTo-T values of both wogonin and wogonoside were 11.53±1.27 and 9.36±0.95hour respectively, this might have relation with the local recycling in the gut.
Conclusions.
1. Wogonin and wogonoside were found to be converted to each other in the rat intestine, wogonoside could be hydrolyzed into its aglycone, which was then absorped rapidly.
2. Wogonoside is mainly hydrolyzed byβ-glucuronidase. The result comes from the following facts. The wogonoside hydrolysis could be inhibited by the specific inhibitor of glucuronidase, saccharolactone. Moreover, the wogonoside absorption was found decrease significantly when saccharolactone was used to co-perfuse with wogonoside, and the absorption didn't decrease when the specific inhibitor of LPH or antibiotics was used to co-perfuse with wogonoside.
3. The activities ofβ-glucuronidase in the gut were mediated by enteric. The result comes from the fact that the enzyme hydrolysis activity in upper intestinal segments (ie.jejunum) was higher than that in colon, while the bacteria counts result showed that colon perfusate samples with the highest bacteria count than other three segments of the intestine.
4. Theβ-glucuronidase activities were higher in upper small intestinal blank perfusate and that the Km values of hydrolysis reaction in duodenal and jejunal blank perfusate were nearly identical to those of their intestinal S9 fraction, respectively. Their kinetic profiles were also very similar as shown by both regular concentration versus rate plots and Eadie-Hofstee plots.
5. After rat intragastric administration, it was found that the MRTo-T values of both wogonin and wognoside were rarely high, their blood drug concentration curves displayed double peaks, and curve profiles were similar.
6. No free glucuronidation enzyme exist in the gut, and they only exists in the enteroytes. Wogonin was very stable in blank intestinal perfusate, the glucuronide found in the perfusate when wogonin was perfused, should be released from the enterocytes.
Our conclusion that flavonoid glucuronides can be recycled locally was well-supported by the above results. In conclusion, we have discovered a novel local recycling mechanism that will significantly enhance the local bioavailability and residence time of flavonoids such as wogonin. This local recycling mechanism has the potential to significantly impact the bioavailabilities of other drugs and dietary chemicals that also undergo extensive glucuronidation in the gut. Because a large numbers of phenolics are glucuronidated significantly in gut, this newly discover recycling mechanism should have a significant physiological role in governing the local bioavailability and residence time of phenolics, which will ultimate impact the pharmacodynamic and toxicological effects of this class of important compounds, many of which are showing promise as chemopreventive agents.
许多具有葡萄糖醛酸化和磺酸化等二相代谢途径的药物在体内可以进行循环,如肝肠循环(Enterohepatic cycle)和肠肠循环(Enteric cycle),这类处置机制使药物在体内具有较长的保留时间,对延长药物的作用时间有非常重要的意义。
早在半个世纪之前人们就认识到有些药物在体内可形成肝肠循环,许多药物以葡萄糖醛酸苷或磺酸苷等二相代谢结合物形式从肝脏随胆汁被排入肠道,在肠道细菌的β-葡萄糖醛酸苷酶(Glucuronidase, GUS)或磺基转移酶(Sulphotransferase, SULT)作用下水解成苷元,苷元易被肠道黏膜细胞吸收,在细胞内的二相代谢结合酶如葡萄糖醛酸转移酶(UDP-glucuronosyltranferase, UGT)等酶作用下再形成药物的葡萄糖醛酸苷或磺酸苷,这些苷进入循环系统后,可经肝脏从胆汁再次被排入肠道,从而形成完整的肝肠循环。
肝肠循环对体内必需物质的利用和许多药物发挥疗效都是非常重要的。机体内这种肝肠循环可以有效地利用体内必需物质,例如帮助消化的胆酸,一般进食一次,胆酸至少要经历2-3次的肝肠循环；其他具有肝肠循环的身体必需物质有维生素D3、维生素B12、叶酸和雌激素等。在体内具有肝肠循环的药物有很多,如地高辛、洋地黄毒苷、氨苄青霉素、吗啡、美沙酮、苯妥因钠、华法令、氯丙嗪等。
药物的肠肠循环则是肝肠循环之外的另一种体内循环,直到近来才被人们注意到。肠肠循环是指苷元类药物在十二指肠、空肠、回肠和结肠各段均易被吸收进入肠黏膜细胞,在UGT等酶作用下转化成葡萄糖醛酸苷或磺酸苷,部分苷被肠黏膜细胞直接外排至肠腔,此后,在肠道后段被细菌分泌的β-GUS酶和SULT酶作用下水解成苷元,再次被吸收进入肠黏膜细胞的过程。肠肠循环与肝肠循环的主要区别在于药物的葡萄糖醛酸苷或磺酸苷不是经肝脏随胆汁被排入肠道,而是直接被黏膜细胞外排至肠道的。
我们在研究汉黄芩素(Wogonin)的吸收代谢过程中,发现汉黄芩苷(汉黄芩素-7-0-葡萄糖醛酸苷Wogonoside)与汉黄芩素在体内相互转化,因而发现在肠道易葡萄糖醛酸化的化合物具有第三种循环,也可能是最重要的一种循环：肠细胞局部循环。在局部循环中,药物在的葡萄糖醛酸苷被肠黏膜细胞外排,随后可被黏膜细胞外主要来源于肠道细胞分泌的β-GUS酶水解为苷元,苷元再被此肠黏膜细胞吸收,经细胞内UGT酶作用重新形成药物的葡萄糖醛酸苷,部分苷将再被肠黏膜细胞外排至肠腔,因此苷元及其葡萄糖醛酸苷可在十二指肠、空肠、回肠和结肠整个肠道某一肠黏膜细胞及此细胞外侧肠道局部不断循环。肠局部循环与肠肠循环主要区别有两点：一是酶的来源不同,二是药物重吸收的部位不同。肠肠循环中的β-GUS酶由细菌分泌,细菌主要集中在肠道后端,故药物葡萄糖醛酸苷主要在肠道后段水解,药物的重吸收应主要在肠道后段。局部循环中的β-GUS酶主要来源于肠细胞,整个肠道的黏膜细胞均可重吸收药物并形成局部循环,这对肠道疾病的治疗具有极为重要的意义。
我们选择汉黄芩素和汉黄芩苷为模型化合物研究肠细胞局部循环机制是基于以下理由：
一、汉黄芩素具有非常重要的药理活性,如用于抗炎和抗肿瘤。汉黄芩素能恢复肿瘤坏死因子受体细胞凋亡诱导配体(Tumor necrosis factor receptor apoptosis-inducing ligand, TRAIL)失活的癌细胞中TRAIL的敏感性；在人结肠癌HCT116细胞,汉黄芩素可通过p53依赖的PUMA诱导作用诱导细胞凋亡。
二、汉黄芩素在体内生物利用度很低,可能与其在体内存在广泛的葡萄糖醛酸化反应生成其葡萄糖醛酸苷——汉黄芩苷有关。
三、汉黄芩素和汉黄芩苷存在于多数处方药物中,已广泛运用于人体,例如BZL101,一种含有汉黄芩素的植物水提物,对乳腺癌细胞株有效,Ⅰ期临床试验表明它毒性低,显示良好的临床效应。一些含中药黄芩的组方,具有抑制肠道炎症的功效。
四、多种黄酮类化合物都对胃肠道疾病有效,例如水飞蓟宾可以抑制丙型肝炎病毒,而绿茶中黄酮类化合物均对结肠癌有活性。汉黄芩素和汉黄芩苷可能是用于胃肠道疾病的潜在药物,且药物的肠局部循环可能对肠道疾病的治疗极为有利。
五、汉黄芩素和汉黄芩苷经济易得,大量价廉的黄芩等植物可为二者提供丰富的来源。
研究的方法和结果如下：
一、汉黄芩苷与汉黄芩素相互转化研究
我们采用了在体大鼠灌流模型来考察两种药物的吸收代谢。雄性SD大鼠(80-110天),280-350 g,选用大鼠十二指肠、空肠、回肠末端和结肠四个肠段进行在体灌流实验。结果表明汉黄芩素和汉黄芩苷在大鼠肠道内相互转换。灌流两个浓度的汉黄芩素(5μM和20μM)时,在四个肠段灌流液中都能发现汉黄芩素的葡萄糖醛酸苷——汉黄芩苷。灌流两种浓度(5μM和20μM)的汉黄芩苷时,在四个肠段灌流液中同样都能发现其苷元汉黄芩素；令人惊讶的是,小肠上段(十二指肠、空肠)灌流液中也有汉黄芩素出现。传统观念认为,肠道内水解黄酮萄萄糖醛酸苷的β-GUS酶来源于细菌,而十二指肠和空肠中细菌数量远远低于回肠末端和结肠,缺少来源于细菌的β-GUS酶,汉黄芩苷在十二指肠和空肠内应不会被水解为汉黄芩素,也没有萄萄糖醛酸苷在小肠上段被水解的文献报道。
汉黄芩素比汉黄芩苷的极性小,在肠道更容易渗透,它的膜渗透系数P*eff值比汉黄芩苷的高约10倍。和预期一样,当灌流苷元汉黄芩素,在四段肠都有良好吸收,例如灌注5μM时苷元在四段肠的百分吸收率分别为：66.87%、66.73%、67.00%、74.62%；灌流汉黄芩苷时,它在每一个肠段对应的吸收率都比汉黄芩素低。
我们收集了四段肠的空白灌流液,分别加入汉黄芩素和汉黄芩苷,结果发现汉黄芩苷在肠空白灌流液中不稳定,20μM的汉黄芩苷24小时内几乎完全被水解为汉黄芩素。这表明空白灌流液中存在能水解汉黄芩苷的酶,但这种水解酶是不是β-GUS酶,或是否由细菌分泌仍不清楚,这种水解酶的种类和来源都有待更进一步的研究。此外,汉黄芩素在肠空白灌流液中稳定,不会转化为汉黄芩苷。这表明空白灌流液中没有UGT酶,在灌流汉黄芩素时,灌流液中出现的汉黄芩苷应来源于肠黏膜细胞的外排。
二、汉黄芩苷与汉黄芩素溶解性、稳定性和吸附性考察
为了防止药物溶解性、稳定性和吸附性影响实验数据的准确性,我们考察了汉黄芩苷与汉黄芩素的这些性质。结果发现二者在HBSS溶液中160-0.3125μM浓度范围内完全溶解,稳定性也完全满足吸收代谢实验研究的需要,且实验中使用到的硅胶管和EP管对二者没有吸附性。这些结果显示,汉黄芩苷在肠道和灌流液中水解与其物化稳定性无关,也与溶解性和吸附性无关。
三、水解酶种类研究
我们先采用空白灌流液来研究汉黄芩苷的水解酶种类。
为了探明水解酶的种类,我们首先寻找了一种化学终止剂(94%乙腈和6%甲酸组成),以便能按需要求随时终止酶的水解反应,考察任一酶水解反应进行的程度。实验证明,加入反应终止液后,能完全终止水解酶的水解作用,汉黄芩苷在加有终止液的空白灌流液中24小时保持稳定。在药物灌流实验中,收集样品前均加入此终止液,使药物在灌流液从肠道导管出口流出后酶水解作用就被终止,保证了水解过程只发生在肠道,在收集管中药物不会再发生水解,以此测定药物在肠道内水解的程度。
最早我们考虑到水解作用可能是受细菌的影响,故采用13000rpm离心30min后,用0.22μm无菌过滤的方式去除空白灌流液中的细菌,结果发现空白灌流液除菌对汉黄芩苷的水解没有影响,这显示引起药物水解的可能是游离的水解酶。
因为乳酸根皮苷水解酶(Lactasc Phlorizin Hydrolase, LPH)在体内对葡萄糖苷的水解过程中起重要作用,它是否也能水解葡萄糖醛酸苷仍不清楚,故加入其特异性抑制剂——20mM的葡萄糖酸内酯,结果发现抑制LPH酶对汉黄芩苷的水解无明显影响。
当我们加入P-GUS酶的特异性抑制剂——0.1mM葡萄糖二酸单内酯时,发现空白灌液中汉黄芩苷的水解被显著抑制,当加入4.4mM葡萄糖二酸单内酯时(4.4mM为UGT酶药物代谢实验中用于抑制葡萄糖醛酸苷水解时的葡萄糖二酸单内酯浓度),能完全抑制汉黄芩苷的水解。
随后我们进行了大鼠在体灌流实验,结果显示在小肠上段(十二指肠和空肠)汉黄芩苷的吸收和其苷元的转化率会受β-GUS酶的抑制剂——葡萄糖二酸单内酯(0.1 mM)抑制,但不会受LPH酶的抑制剂——葡萄糖酸内酯抑制。这表明水解汉黄芩苷的是β-GUS酶。
四、水解酶的来源研究
肠道中β-GUS酶的可能有三种来源：第一种可能是来源于肝脏,分泌的酶可随胆汁排入肠道；第二种可能是肠道菌群分泌；第三种可能是来源于肠道细胞。但传统观念认为动物的P-GUS酶只存在于细胞内,肠道菌群是肠道中β-GUS酶的主要来源。
我们将汉黄芩素和汉黄芩苷加入胆汁溶液,发现二者在胆汁中的稳定性较好,且在灌流实验时,胆汁被收集另存,没有进入肠道,这否定了β-GUS酶主要来源于肝脏或胆汁的可能性。
我们收集了不同肠段不同时间段的新鲜空白灌流液。我们取一部分新鲜空白灌流液进行细菌培养后做总菌落计数,比较灌流液样品中的细菌数量；另一部分加入汉黄芩苷,考查其水解速率。若肠道菌落是GUS酶的主要来源,那么肠道灌流液中的细菌数将会与其对苷的水解能力成正比。然而,实验结果表明细菌的数量与汉黄芩苷的水解速率不相关。
我们采用大鼠在体灌流实验,并对大鼠肠道做急性抗生素处理,即灌液前在20gM汉黄芩苷的HBSS溶液中加入混合抗生素(100单位青霉素和链霉素为0.1mg/毫升),同样,经过半小时灌流平衡之后再收集灌流液样品检测,结果显示混合抗生素不会影响汉黄芩苷的吸收或代谢。这些结果都与认为肠道β-GUS酶来源于细菌的观点不一致,至少说明肠道菌落不是肠道内β-GUS酶的主要来源。在以上实验结果基础上,我们推测大鼠肠道细胞可能是肠道中β-GUS酶的一种主要来源。
五、GUS酶来源的酶代动力学验证
由于缺乏能区分β-GUS酶是来源于细菌或是大鼠的商业性单克隆抗体,我们只能采用酶代动力学参数用来区分他们。为此,我们制备了肠道S9微粒,因为S9中含有光滑内质网,包含了大部分细胞内酶。以肠道S9中的β-GUS酶作为大鼠肠细胞来源的对照。另外,我们还制备了大鼠粪上清液,提取粪便微生物中的酶类,以粪上清液中的β-GUS酶作为细菌来源的对照。
我们选择了细菌数量差距大于2700倍的高、中、低宽浓度范围内的四个灌流液样品进行比较,发现空肠90-120min的肠灌流液样品中细菌量最少,但有最高的水解速率。相比之下,结肠60-90min灌流液样品中细菌量最多,但其水解速率最低。结果显示细菌量最多的样品并不是具有最快的水解速率。
我们测定了汉黄芩苷在同一个时间段(60-90min)四段肠的空白灌流液中的水解速率,结果再次表明具有细菌数最多的结肠灌流液并没有最快的水解速率。
我们测定了汉黄芩苷在空肠中5个时间段的空白灌流液中的水解速率,结果显示灌流液中β-GUS酶的量(体现在Vmax值上)随时间呈下降趋势,表明β-GUS酶的量随着灌流时间延长在减少。
我们还测量灌流液的总蛋白浓度,考察总蛋白浓度与水解活性的关系,但令人奇怪的是它们也不相关。此外,我们发现灌流液中总蛋白含量没有随着灌注时间减少,这显示蛋白可能是在灌流过程中的脱落的肠黏膜细胞或(和)粘液组织。最后,发现灌流液中的细菌量与其总蛋白浓度也没有相关性。
我们测定灌流液样品、肠道S9和粪上清液中β-GUS酶的动力学参数,得到Eadie-Hofstee图。结果表明汉黄芩苷在不同的介质中的酶催化水解都遵循典型的米氏动力学规律(Eadie-Hofstee图呈线性)。三段小肠S9的Km值都相似(25-30μM),但它们均明显高于粪上清的Km(7.7μM)。再比较肠道灌流液样本的Km值(15.8-25.6μM),除了回肠末端的两个灌流液样品Km值(6.3-6.9μM)接近粪上清液的Km值外,其他灌流液样品的Km值都与来源于肠道S9的Km值很相接近。这些结果都显示空白灌流液中的β-GUS酶应主要来源于肠道细胞。
六、汉黄芩苷及汉黄芩素的药代动力学研究
我们采用大鼠灌胃给药后,眼眶取血方式检测血药浓度来研究汉黄芩苷及汉黄芩素的药动学特征。采用3P97的统计矩法计算药时曲线下面积(AUC0-24h)等参数。结果显示汉黄芩苷与汉黄芩素灌胃给药后,药血浓度曲线均呈双峰,曲线相似,血中药物主要以汉黄芩苷形式存在,汉黄芩素的量极低,这显示汉黄芩素在体内存在广泛的葡萄糖醛酸化。
从药动学参数来看,二者Tmax1、Tmax2、Cmax2、t1/2、MRT0_T、AUC0-T、AUC0-∞值都非常接近。这些参数显示二者在体内具有相同的动力学模式。这与实验预期一样,可能因为汉黄芩苷与汉黄芩素在体内都会出现局部循环过程,应有相同的动力学模式。二者的平均滞留时间MRT0-T值都较大,分别为11.53±1.27h和9.36±9.95h,这可能与二者在肠道中的局部循环处置过程延长了它们在体内的滞留时间有关。
根据上述实验结果,本论文的结论如下：
第一,汉黄芩苷与汉黄芩素在体内相互转化,汉黄芩苷在肠道能迅速水解成汉黄芩素,并且被快速吸收。
第二,汉黄芩苷主要是在β-GUS酶作用下水解的。因为空白灌流液中的水解可以被其特异性抑制剂——葡萄糖二酸单内酯抑制,此外,当汉黄芩苷与葡萄糖二酸单内酯同时灌流时,汉黄芩苷的吸收显著减少,而LPH酶抑制剂或抗生素与汉黄芩苷一起灌流时,不会影响其吸收。
第三,肠道中β-GUS酶的活性是以肠来源的β-GUS酶占主导,因为在小肠上段(如：空肠)酶的水解活性比结肠强,而在不同肠道的空白灌流液细菌计数显示,结肠中细菌数是最高的。
第四,小肠上段的空白灌流液中P-GUS酶的活性更强(图-9B),且汉黄芩苷在十二指肠和空肠的空白灌流液的水解Km值与它们对应肠道S9的Km值几乎相同。从浓度与速率比的图形趋势和Eadie-Hofstee图来看,它们的动力学模式也非常相似。
第五,汉黄芩素与汉黄芩苷大鼠口服给药后,二者药物动力学参数非常相似,它们的平均滞留时间MRT0-T值都较大,药血浓度曲线均呈双峰,曲线相似趋势。
第六,肠道中没有游离的UGT酶,此酶只存在于黏膜细胞内。汉黄芩素在空白灌流液非常稳定,在体灌流汉黄芩素时,肠灌流液中的汉黄芩苷均来源于黏膜细胞的外排。
在以上实验结果的支持下,因此我们得出黄酮类葡萄糖醛酸化合物可以在肠道形成局部循环的结论。
总的来说,我们发现一个新的肠局部循环机制,它将提高黄酮类化合物如汉黄芩素的局部生物利用度和作用时间。这种局部循环机制可能对在肠道具有广泛葡萄糖醛酸化的其他药物和食物成分的生物利用度产生重大影响。由于有大量酚类物质在肠道中可以葡萄糖醛酸化,这种新发现的循环处置机制对酚类物质的生理作用可能有非常大的影响,可能改变它们的局部生物利用度和滞留时间,最终影响这类重要化合物的药效和毒性。
Drug disposition involving distribution, metabolism, and excretion, has great relation with drug's effects. The rate and amount of drug reaches the target site may influence the initial time and the degree of drug affecting. Moreover, the duration time of drug affecting last is determined primarily by the metabolism and excretion.
The amount of drug distribution differs in various organs, and distribution characterization varied for diverse drugs. Similarly, metabolism and excretion characterization varied for diverse drugs. Many drugs proceed enteric and enterhepatic recycling in body by the means of phase II metabolism, such as glucuronidation and sulfation, which play a central role in prolonging the effecting time of drugs.
Enterhepatic recycling scheme was recognized more than half a century ago. It involves the action of liver to excrete the conjugated phase II metabolites and action of the microflora or bacterialβ-glucuronidase or sulfatases to release the aglycones (from the conjugated phase II metabolites). Released aglycones can enter the body again and form glucuronides or sulfates again, thereby completing the recycling loop.
Enterhepatic recycling scheme may help body utilize endogenous necessary material effectively. For example, during the bile digesting, cholic acid would experience enterhepatic recycling two or three times after administration once. Other similar necessary materials are vit D3, vit B12, folic acid etc. Many drugs like digoxin, cardigin, diethylstilbestrol, ampicillin etc, would also experience recycling scheme of this kind. Therefore, enterhepatic recycling is of great importance to the utilization of endogenous necessary material and exerting of drug effects.
Enteric recycling has only been termed more recently. In this recycling scheme, the phase II conjugates are excreted by the enterocytes, and once again, the action of bacterialβ-glucuronidase or sulfatases is required to release aglycone for reabsorption.
In our study, we have identified the third and perhaps the most important recycling scheme for compounds that are extensively glucuronidated in the gut:the local recycling. In the local recycling scheme, the phase II conjugates are excreted by the enterocytes, and also once again, the action of bacterial P-glucuronidase or sulfatases is required to release aglycone for reabsorption. After reabsorption, drug was again glucuronidated by UDP-glucuronosyltranferase (UGT), and one part of the glucuronides was excreted by the enterocytes. In this way, drug and its glucuronide could form a complete recycling loop at one enterocyte and gut outside it.
Wogonin, the aglycone of wogonoside(wogonin-7-O-glucuronide), was a kind of dihydroxylflavone. It has attracted a lot of attention for its anti-tumor and anti-inflammatory activity in the gut and elsewhere and was used here as the model compound to prove the presence of this recycling scheme. Wogonin was selected because it is known to be extensively glucuronidated. It was also chosen because of its important pharmacological activities, including its ability to restore the sensitivity of tumor necrosis factor receptor apoptosis-inducing ligand (TRAIL) in TRAIL-resistant cancer cells. In human colon cancer HCT116 cells, it was shown that wogonin-induced apoptosis was via p53-dependent PUMA induction. Wogonin was also selected because it has been used widely in humans, mostly in the form of herbal formulation. For example, BZL 101, an aqueous herbal extract active against breast cancer cell lines, was shown to have a favorable toxicity profile in a phase I clinical trial and demonstrated encouraging clinical activity. Hange-shashin-to (HST), a combination of seven herbs including Scutellaria baicalensis, was found to suppress inflammatory bowel disease. Another reason to study wogonin was several flavonoids were shown to be active in management of the gastrointestinal diseases. For example, silibinin was shown to inhibit hepatitis C virus, whereas green tea flavonoids were shown to be active against colon cancer. Lastly, wogonin was also selected because large quantities of wogonoside were available commercially at a reasonable cost.
Methods and Results.
1. The conversion study of wogonin and wogonoside.
A "four site perfusion model" was used to investigate the transport and metabolism of wogonin or wogonoside. Male Sprague-Dawley rats (80-110 days old) weighing from 280 to 350 g were obtained to operate the experiments, and four segments of the intestine (duodenum, upper jejunum, terminal ileum and colon) were perfused simultaneously with perfusate containing the compound of interest. The result showed that wogonin and wogonoside could convert to each other in the rat intestine individually. When wogonin of 5μM and 20μM concentrations was perfused, wogonoside was respectively found in all four segments of intestine. On the contrary, when wogonoside of 5μM and 20μM concentrations was perfused, wogonin was respectively found in all four segments of intestine. To our surprise, wogonin was also found in the upper segments of intestine (duodenum, jejunum). According to classical view,β-glucuronidase that hydrolyse glucuronides comes from bacteria. However, bacteria count in duodenum, jejunum was much lower than that of ileum and colon.In addition, wogonoside couldn't be converted into wogonin in duodenum and jejunum, for lack of bacterialβ-glucuronidase.
Compared with wogonoside, wogonin was smaller and less polar, and therefore it penetrated the intestinal membrane more rapidly. Moreover, P*eff of wogonin is 10 times higher than that of wogonoside. As expected, when wogonin or aglycone was perfused, it was well absorbed in all four segments of intestine. When wogonin of 5μM concentration was perfused, its absorption percentage was 66.87%,66.73%, 67.00%,74.62% in order at the four segments of intestine. When wogonoside was perfused, its absorption percentage was lower than that of wogonin at respective region of intestine.
After blank intestinal perfusate of four segments were collected, wogonin and wogonoside was put in it individually. It was found that wogonoside was unstable in blank intestinal perfusate, wogonoside of 20μM concentration was almost hydrolysis into its aglycone within 24 hours. It proved that enzymes hydrolyzing wogonoside did exist in the blank intestinal perfusate, and further research should be carried on to determine the kinds and sources of the hydrolysis enzymes. Wogonin was found stable in blank intestinal perfusate, and wouldn't convert into its glucuronide. It showed that no UGT exists in the blank intestinal perfusate, and the glucuronide found in the perfusate when wogonin was perfused, should be released from the enterocytes.
2. The dissolubility, stability, adsorbability study of wogonin and wogonoside.
The dissolubility, stability, adsorbability were investigated individually during experiment to assure the accuracy of all data. It was found that two compounds dissolve completely in HBSS buffer in the concentration range of 0.3125-160μM, and stability of the two chemicals could meet the requirement of absorption and metabolism study, all silica tube and EP tube used in the experiment didn't show adsorbability to the two chemicals. All above results indicate that the wogonoside hydrolysis in gut and perfusate has no relation with their chemical stability, or dissolubility and adsorbability.
3. The kinds study of the hydrolysis enzymes of wogonoside.
Blank intestinal perfusate was collected to investigate the kinds of wogonoside hydrolysis enzymes. A stop solution (consisting of 94% acetonitrile and 6% glacial acetic acid) was first developed to ensure the enzyme hydrolysis could be ended at any time. It was demonstrated that wogonoside is stable in blank intestinal perfusate during 24 hours after the addition of the stop solution. Hence, the stop solution was added into all samples before their collections to stabilize the drug in samples.
The influence of bacteria was first considered, and experiments were performed to determine the effects of bacteria presence. The results indicated that removal of bacteria via centrifugation (13,000 rpm,30 min) and aseptic filtration did not improve stability of wogonoside. Therefore, free hydrolysis enzymes might be the main reason for the wogonoside hydrolysis.
Lactasc phlorizin hydrolase (LPH) is a major contributor to the glucoside hydrolysis in the intestine. After addition of gluconolactone, a specific inhibitor of the LPH, it was found that the hydrolysis of wogonoside was not inhibited.
When 0.1 mM saccharolactone, the specific inhibitor of the glucuronidase was investigated, it was found that the hydrolysis of wogonoside was inhibited significantly. When the concentration rose from 0.1 mM to 4.4 mM, the hydrolysis of wogonoside was found inhibited thoroughly.
The later perfusion experiment showed that the wognoside absorption and its aglycone conversion rate on the upper intestine would be inhibited by the specific inhibitor of the glucuronidase,0.1 mM saccharolactone, not by the specific inhibitor of LPH, gluconolactone. All the results showed thatβ-glucuronidase is exactly the enzyme responsible for the wogonoside hydrolysis.
4. The source study of the hydrolysis enzymes of wogonoside.
The P-glucuronidase in intestinal gut may come from three sources. The first source may be the liver, and the enzymes excreted there may enter gut with bile. The second source may be the intestinal microflora, this is the most obvious source and has been widely accepted. The third source may be the enterocytes.
After adding wogonin and wogonoside into bile, both of the compounds were found stable. Moreover, a bile duct cannulation was made in perfusion experiments to avoid the bile entering the intestinal gut. Therefore, it's impossible for theβ-glucuronidase to come from liver in this study.
Sequential perfusate samples were collected from all four regions, and amount of bacteria in every sample was determined and then plotted against hydrolysis reaction rates measured using 20μM wogonoside. The results indicated that there was no correlation between the numbers of bacteria present in the perfusate and rate of wogonoside hydrolysis.
To make acute antibiotic treatment, an antibiotic mixture of penicillin and gentamicin was added into the HBSS buffer containing 20μM wogonoside before perfusion. The results indicated that absorption or metabolism of wogonoside was not impacted. All these results didn't accordance with the opinion that the P-glucuronidase in intestinal gut supplied by the intestinal microflora, and the intestinal bacteria couldn't be the sole source for the intestinal gut. Based on above results, we conclude that enterocytes in rat intestine gut might be the main source ofβ-glucuronidase in intestinal gut.
5. The kinetic validation of the source of GUS enzyme
Because of the lack of commercial monoclonal antibody to distinguish bacteriaβ-glucuronidase from mammalian one, kinetic parameters were used to distinguish them. To do so, rat intestine S9 fractions were prepared for S9 contains the smooth endoplasmic reticulum with most enterocyte enzymes in it, and the P-glucuronidase in S9 fractions could be referred, when the source of intestinal gut was considered. In addition, rat feces extract was also prepared, and the P-glucuronidase in feces extract could be referred, when the source of bateria was considered.
We also selected four perfusate samples with drastically different amounts of bacteria (difference of greater than 2,700-fold), and found that 90-120 min jejunal perfusate sample had the highest Vmax value even though it has the lowest bacteria count. In contrast,60-90 min colon perfusate sample with the highest bacteria count had the lowest Vmax value.
The above results showed that samples with higher bacteria count did not have faster hydrolysis rates. To confirm this finding, we also determine the metabolism of wogonoside in different perfusate collected at the same time, and the results again indicated that samples with the highest bacteria count (colon) did not have faster hydrolysis rates. However, the amounts of enzyme (as represented by the Vmax values) present in the perfusate appeared to decrease with time, indicating amount of enzymes present in the perfusate was less after continuous single-pass perfusion.
To determine if protein concentration in the perfusate could correlate with the hydrolysis activities, the amounts of protein in the perfusate were plotted against hydrolysis rates, and surprisingly, there was no correlation either. In addition, we found that protein concentration did not decrease with perfusion time, indicating that protein in the perfusate may be due to sloughing off from the enterocytes or mucus or both. Lastly, there was no correlation between bacteria counts and protein concentration in the perfusate.
The kinetic parameters were determined and Eadie-Hofstee plots were generated forβ-glucuronidase derived from intestinal S9 fractions, microbial enzymatic extract, and perfusate samples. The results indicated that enzyme-catalyzed hydrolysis of wogonoside in different sample matrix all followed a classic Michaelis-Menten kinetic profile. The Km values of intestinal S9 fractions from all three small intestinal segment were similar (25-30μM), but they were significantly higher than those derived from microbial enzymatic extract (7.7μM). When comparing to the Km values derived from the intestinal perfusate samples (15.8-25.6μM), they were all close to the Km values derived from the intestinal S9 fractions, except for two terminal ileal perfusate samples, which displayed Km values (6.3-6.9μM) closely resembled that of the microbial enzymatic extract. All the results showed that (3-glucuronidase in blank perfusion should mainly come from intestinal gut.
6. The pharmacokinetic study of wogonin and wognoside
To study the pharmacokinetic characterization of wogonin and wognoside, blood samples were collected from fossa orbitalis to determine drug concentrations in blood after rat intragastric administration. Pharmacokinetic parameters such as AUCo-24h were obtained using 3P97 statistical moment to calculate. It was found that the blood drug concentration curves of the two compounds both displayed double peaks, and their curve profiles were similar. Most chemicals in blood were wogonoside, only little amount of wogonin exist, this indicated that wognonin was glucuronidated extensively in vivo, and exist in the form of wogonoside in blood. For pharmacokinetic parameters, the MRTo-T values of both wogonin and wogonoside were 11.53±1.27 and 9.36±0.95hour respectively, this might have relation with the local recycling in the gut.
Conclusions.
1. Wogonin and wogonoside were found to be converted to each other in the rat intestine, wogonoside could be hydrolyzed into its aglycone, which was then absorped rapidly.
2. Wogonoside is mainly hydrolyzed byβ-glucuronidase. The result comes from the following facts. The wogonoside hydrolysis could be inhibited by the specific inhibitor of glucuronidase, saccharolactone. Moreover, the wogonoside absorption was found decrease significantly when saccharolactone was used to co-perfuse with wogonoside, and the absorption didn't decrease when the specific inhibitor of LPH or antibiotics was used to co-perfuse with wogonoside.
3. The activities ofβ-glucuronidase in the gut were mediated by enteric. The result comes from the fact that the enzyme hydrolysis activity in upper intestinal segments (ie.jejunum) was higher than that in colon, while the bacteria counts result showed that colon perfusate samples with the highest bacteria count than other three segments of the intestine.
4. Theβ-glucuronidase activities were higher in upper small intestinal blank perfusate and that the Km values of hydrolysis reaction in duodenal and jejunal blank perfusate were nearly identical to those of their intestinal S9 fraction, respectively. Their kinetic profiles were also very similar as shown by both regular concentration versus rate plots and Eadie-Hofstee plots.
5. After rat intragastric administration, it was found that the MRTo-T values of both wogonin and wognoside were rarely high, their blood drug concentration curves displayed double peaks, and curve profiles were similar.
6. No free glucuronidation enzyme exist in the gut, and they only exists in the enteroytes. Wogonin was very stable in blank intestinal perfusate, the glucuronide found in the perfusate when wogonin was perfused, should be released from the enterocytes.
Our conclusion that flavonoid glucuronides can be recycled locally was well-supported by the above results. In conclusion, we have discovered a novel local recycling mechanism that will significantly enhance the local bioavailability and residence time of flavonoids such as wogonin. This local recycling mechanism has the potential to significantly impact the bioavailabilities of other drugs and dietary chemicals that also undergo extensive glucuronidation in the gut. Because a large numbers of phenolics are glucuronidated significantly in gut, this newly discover recycling mechanism should have a significant physiological role in governing the local bioavailability and residence time of phenolics, which will ultimate impact the pharmacodynamic and toxicological effects of this class of important compounds, many of which are showing promise as chemopreventive agents.
引文
[1]Manwaring, W.H., Enterohepatic Circulation of Estrogens. Cal West Med, 1943.59(5):p.257.
[2]魏树礼主编,生物药剂学与药物动力学.1997.p.36.
[3]Chen, J., H. Lin, and M. Hu, Metabolism of flavonoids via enteric recycling: role of intestinal disposition. J Pharmacol Exp Ther,2003.304(3):p.1228-35.
[4]Hu, M., Commentary:bioavailability of flavonoids and polyphenols:call to arms. Mol Pharm,2007.4(6):p.803-6.
[5]杨娟,傅军鹏.黄芩活性成分及药效研究近况.实用医药杂志(山东),2004.21(3)：p.271-273.
[6]宋琳莉,孟庆刚.黄芩的药理作用研究进展.中华中医药学刊,2008.26(8)：p.1676-1678.
[7]梁英,韩鲁佳.黄芩中黄酮类化合物药理学作用研究进展.中国农业大学学报,2003.8(6)：p.9-14.
[8]张保国,梁晓夏,刘庆芳.黄岑汤现代药效学研究与临床运用.中国中药杂志,2008.33(22)：p.2587-2590.
[9]王孝先.黄芩汤，白头翁汤，葛根芩连汤对肠道菌株抑菌作用的实验观察.中国中医基础医学杂志,2001.7(1)：p.42-44.
[10]姚干,何宗玉,方泰惠.岑栀胶囊抗病毒和抗菌作用的实验研究.中成药,2006.28(002)：p.225-228.
[11]刘菊福,卢长安,廖福龙,等.不同产地黄芩提取物主要药效作用的比较.中国实验方剂学杂志,2002(S1)：p.-
[12]应群芳,张慧华.黄岑不同炮制品的体外抑菌作用研究.山东中医杂志,2007.26(10)：p.711-712.
[13]孔晋亮,刘晓岚,陈一强,等.黄芩水煎液对生物被膜的抑制作用及对左氧氟沙星的增效作用.中华微生物学和免疫学杂志,2007(03).
[14]孔晋亮,刘晓岚,陈一强,等.黄芩水煎液联合左氧氟沙星对铜绿假单胞菌生物被膜的影响.天津医药,2008(05).
[15]栾耀芳,孔祥山，吴国英.黄芩等8味中药对mrs的抑菌性研究.山东医药,2005(11).
[16]王连心,孟庆刚.黄岑及其提取物治疗细菌性痢疾研究进展.中华中医药学刊,2008.26(8)：p.1697-1700.
[17]鲁晓睛,王斌,阎志勇.三种中草药制剂对肠道致病菌体外抗菌作用的研究.中国消毒学杂志,2008.25(3)：p.270-272.
[18]吴静,胡东,王克霞.黄岑和黄芩苷对幽门螺杆菌的体外抗菌活性研究.中药材,2008.31(5)：p.707-710.
[19]曹翠萍,宁海强,孙丽,等.中药对大肠杆菌抑制作用及耐药性诱导作用的研究.西南农业学报,2007.20(5)：p.1101-1104.
[20]滕兰菊,桂芳,黄刚,等.黄芩等中草药对52株伤寒杆菌药敏试验研究.怀化医专学报,2002.1(2)：p.22-23.
[21]刘云波,郭丽华,邱世翠.黄芩体外抑菌作用研究.时珍国医国药,2002.13(3)：p.596.
[22]栾耀芳,张凤花,吴国英.黄芩等8种中药对产β-内酰胺酶大肠埃希菌的敏感性研究.山东中医杂志,2005.24(10)：p.629-631.
[23]刘平,叶惠芬,陈惠玲,等.5种中药对产酶菌的抑菌作用.中国微生态学杂志,2006(01).
[24]梅林,贺锡中,熊云,等.中药黄芩的血清抑菌活性试验研究.医学研究杂志,2009.38(1)：p.102-103,85.
[25]陈莉,朱凤静,张春枝.30种中药水提取物的体外抗菌活性试验.大连轻工业学院学报,2005.24(004)：p.269-271.
[26]吕小迅,周玉珍.黄岑，黄精抗真菌作用活性部位实验研究.广东药学院学报,1995.11(003)：p.180-180.
[27]赵铁华,陈四平,杨鹤松,等.黄芩茎叶活性部位抗病毒作用的研究.中国药科大学学报,2006.37(6)：p.544-547.
[28]徐珊,王乐,杨巧芳,等.黄岑抗病毒药理作用研究述评.中华中医药学刊,2007.25(7)：p.1355-1357.
[29]Ikemoto, S., et al., Antitumor effects of Scutellariae radix and its components baicalein, baicalin, and wogonin on bladder cancer cell lines. Urology,2000. 55(6):p.951-5.
[30]汤立建,赵良才,李庆林,等.葛根和黄芩乙醇提取物体外抗乳腺癌实验研究.安徽中医学院学报,2006.25(6)：p.23-27.
[31]张汉英,黎丹戎,李力,等.汉黄芩素对移植人卵巢癌SKOV3细胞裸鼠组织端粒酶蛋白hTERT影响与凋亡相关性研究.时珍国医国药,2008.19(8)：p.1987-1989.
[32]朴花子,崔弘,朴日龙,等.汉黄芩素对脂多糖诱导的促炎性因子的影响.西安交通大学学报：医学版,2008.29(2)：p.230-232.
[33]Fas, S.C., et al., Wogonin sensitizes resistant malignant cells to TNFalpha-and TRAIL-induced apoptosis. Blood,2006.108(12):p.3700-6.
[34]Rushworth, S.A. and O. Micheau, Molecular crosstalk between TRAIL and natural antioxidants in the treatment of cancer. Br J Pharmacol,2009.157(7): p.1186-8.
[35]Lee, D.H., et al., Role of p53, PUMA, and Bax in wogonin-induced apoptosis in human cancer cells. Biochem Pharmacol,2008.75(10):p.2020-33.
[36]Lee, D.H., J.G. Rhee, and Y.J. Lee, Reactive oxygen species up-regulate p53 and Puma; a possible mechanism for apoptosis during combined treatment with TRAIL and wogonin. Br J Pharmacol,2009.157(7):p.1189-202.
[37]Rugo, H., et al., Phase I trial and antitumor effects of BZL101 for patients with advanced breast cancer. Breast Cancer Res Treat,2007.105(1):p.17-28.
[38]Kawashima, K., et al., Pharmacological properties of traditional medicine (ⅩⅪⅩ):effect of Hange-shashin-to and the combinations of its herbal constituents on rat experimental colitis. Biol Pharm Bull,2004.27(10):p. 1599-603.
[39]Ahmed-Belkacem, A., et al., Silibinin and Related Compounds Are Direct Inhibitors of Hepatitis C Virus RNA-Dependent RNA Polymerase. Gastroenterology,2009.
[40]Ferenci, P., et al., Silibinin is a potent antiviral agent in patients with chronic hepatitis C not responding to pegylated interferon/ribavirin therapy. Gastroenterology,2008.135(5):p.1561-7.
[41]Polyak, S.J., et al., Inhibition of T-cell inflammatory cytokines, hepatocyte NF-kappaB signaling, and HCV infection by standardized Silymarin. Gastroenterology,2007.132(5):p.1925-36.
[42]Sukhthankar, M., et al., A green tea component suppresses posttranslational expression of basic fibroblast growth factor in colorectal cancer. Gastroenterology,2008.134(7):p.1972-80.
[43]Ghosal, A., et al., Identification of human UDP-glucuronosyltransferase enzyme(s) responsible for the glucuronidation of ezetimibe (Zetia). Drug Metab Dispos,2004.32(3):p.314-20.
[44]Kosoglou, T., et al., Ezetimibe:a review of its metabolism, pharmacokinetics and drug interactions. Clin Pharmacokinet,2005.44(5):p.467-94.
[45]45. Jeong, E.J., et al., Species-and disposition model-dependent metabolism of raloxifene in gut and liver:role of UGTIA10. Drug Metab Dispos,2005. 33(6):p.785-94.
[46]Levi, M.S., R.F. Borne, and J.S. Williamson, A review of cancer chemopreventive agents. Curr Med Chem,2001.8(11):p.1349-62.
[47]Logan, R.M., et al., Characterisation of mucosal changes in the alimentary tract following administration of irinotecan:implications for the pathobiology of mucositis. Cancer Chemother Pharmacol,2008.62(1):p.33-41.
[48]Nagano, T., et al., Antitumor activity of NK012 combined with cisplatin against small cell lung cancer and intestinal mucosal changes in tumor-bearing mouse after treatment. Clin Cancer Res,2009.15(13):p. 4348-55.
[49]Brill, S.S., et al., Glucuronidation of trans-resveratrol by human liver and intestinal microsomes and UGT isoforms. J Pharm Pharmacol,2006.58(4):p. 469-79.
[50]Walle, T., et al., High absorption but very low bioavailability of oral resveratrol in humans. Drug Metab Dispos,2004.32(12):p.1377-82.
[51]Jia, X., et al., Disposition of flavonoids via enteric recycling: enzyme-transporter coupling affects metabolism of biochanin A and formononetin and excretion of their phase II conjugates. J Pharmacol Exp Ther,2004.310(3):p.1103-13.
[52]Wang, S.W., et al., Disposition of flavonoids via enteric recycling:structural effects and lack of correlations between in vitro and in situ metabolic properties. Drug Metab Dispos,2006.34(11):p.1837-48.
[53]Liu, X., V.H. Tam, and M. Hu, Disposition of flavonoids via enteric recycling: determination of the UDP-glucuronosyltransferase isoforms responsible for the metabolism of flavonoids in intact Caco-2 TC7 cells using siRNA. Mol Pharm,2007.4(6):p.873-82.
[54]Houston, J.B. and K.E. Kenworthy, In vitro-in vivo scaling of CYP kinetic data not consistent with the classical Michaelis-Menten model. Drug Metab Dispos,2000.28(3):p.246-54.
[55]Hutzler, J.M. and T.S. Tracy, Atypical kinetic profiles in drug metabolism reactions. Drug Metab Dispos,2002.30(4):p.355-62.
[56]Sandanaraj, E., et al., Influence of UGT1A9 intronic I399C＞T polymorphism on SN-38 glucuronidation in Asian cancer patients. Pharmacogenomics J, 2008.8(3):p.174-85.
[57]Yamaoka, K., T. Nakagawa, and T. Uno, Application of Akaike's information criterion (AIC) in the evaluation of linear pharmacokinetic equations. J Pharmacokinet Biopharm,1978.6(2):p.165-75.
[58]Girard, H., et al., The novel UGT1A9 intronic 1399 polymorphism appears as a predictor of 7-ethyl-10-hydroxycamptothecin glucuronidation levels in the liver. Drug Metab Dispos,2006.34(7):p.1220-8.
[59]Dai, J.Y., J.L. Yang, and C. Li, Transport and metabolism of flavonoids from Chinese herbal remedy Xiaochaihu-tang across human intestinal Caco-2 cell monolayers. Acta Pharmacol Sin,2008.29(9):p.1086-93.
[60]Shi, R., et al., Influence of coptis Chinensis on pharmacokinetics of flavonoids after oral administration of radix Scutellariae in rats. Biopharm Drug Dispos, 2009.30(7):p.398-410.
[61]Akao, T., et al., Baicalin, the predominant flavone glucuronide of scutellariae radix, is absorbed from the rat gastrointestinal tract as the aglycone and restored to its original form. J Pharm Pharmacol,2000.52(12):p.1563-8.
[62]Xing, J., X. Chen, and D. Zhong, Absorption and enterohepatic circulation of baicalin in rats. Life Sci,2005.78(2):p.140-6.
[63]Lai, M.Y., et al., Comparison of metabolic pharmacokinetics of baicalin and baicalein in rats. J Pharm Pharmacol,2003.55(2):p.205-9.
[64]He, J.X., et al., Interaction between Shaoyao-Gancao-Tang and a laxative with respect to alteration of paeoniflorin metabolism by intestinal bacteria in rats. Phytomedicine,2007.14(7-8):p.452-9.
[65]Lu, T., et al., Comparative pharmacokinetics of baicalin after oral administration of pure baicalin, Radix scutellariae extract and Huang-Lian-Jie-Du-Tang to rats. J Ethnopharmacol,2007.110(3):p.412-8.
[66]Liu, Y., et al., Enteric disposition and recycling of flavonoids and ginkgo flavonoids. J Altern Complement Med,2003.9(5):p.631-40.
[67]Ausbubel FM, et al., Current Protocols in Molecular Biology, Vol 1. John Wiley & Sons, New York,1988, pp 1.3.1-4.1988.1.
[68]Yi, J.H., et al., Intestinal flora translocation and overgrowth in upper gastrointestinal tract induced by hepatic failure. World J Gastroenterol,1999. 5(4):p.327-329.
[69]China Parmacopeia,2005, the second edition,Appendix XI J,93-5.2005.
[70]Marshall, T. and K.M. Williams, Comparison of Coomassie Brilliant Blue protein dye-binding assays for determination of urinary protein concentration. Clin Chem,1987.33(4):p.577-8.
[71]Lin, Y.T., et al., Degradation offlavonoid aglycones by rabbit, rat and human fecal flora. Biol Pharm Bull,2003.26(5):p.747-51.
[2]魏树礼主编,生物药剂学与药物动力学.1997.p.36.
[3]Chen, J., H. Lin, and M. Hu, Metabolism of flavonoids via enteric recycling: role of intestinal disposition. J Pharmacol Exp Ther,2003.304(3):p.1228-35.
[4]Hu, M., Commentary:bioavailability of flavonoids and polyphenols:call to arms. Mol Pharm,2007.4(6):p.803-6.
[5]杨娟,傅军鹏.黄芩活性成分及药效研究近况.实用医药杂志(山东),2004.21(3)：p.271-273.
[6]宋琳莉,孟庆刚.黄芩的药理作用研究进展.中华中医药学刊,2008.26(8)：p.1676-1678.
[7]梁英,韩鲁佳.黄芩中黄酮类化合物药理学作用研究进展.中国农业大学学报,2003.8(6)：p.9-14.
[8]张保国,梁晓夏,刘庆芳.黄岑汤现代药效学研究与临床运用.中国中药杂志,2008.33(22)：p.2587-2590.
[9]王孝先.黄芩汤，白头翁汤，葛根芩连汤对肠道菌株抑菌作用的实验观察.中国中医基础医学杂志,2001.7(1)：p.42-44.
[10]姚干,何宗玉,方泰惠.岑栀胶囊抗病毒和抗菌作用的实验研究.中成药,2006.28(002)：p.225-228.
[11]刘菊福,卢长安,廖福龙,等.不同产地黄芩提取物主要药效作用的比较.中国实验方剂学杂志,2002(S1)：p.-
[12]应群芳,张慧华.黄岑不同炮制品的体外抑菌作用研究.山东中医杂志,2007.26(10)：p.711-712.
[13]孔晋亮,刘晓岚,陈一强,等.黄芩水煎液对生物被膜的抑制作用及对左氧氟沙星的增效作用.中华微生物学和免疫学杂志,2007(03).
[14]孔晋亮,刘晓岚,陈一强,等.黄芩水煎液联合左氧氟沙星对铜绿假单胞菌生物被膜的影响.天津医药,2008(05).
[15]栾耀芳,孔祥山，吴国英.黄芩等8味中药对mrs的抑菌性研究.山东医药,2005(11).
[16]王连心,孟庆刚.黄岑及其提取物治疗细菌性痢疾研究进展.中华中医药学刊,2008.26(8)：p.1697-1700.
[17]鲁晓睛,王斌,阎志勇.三种中草药制剂对肠道致病菌体外抗菌作用的研究.中国消毒学杂志,2008.25(3)：p.270-272.
[18]吴静,胡东,王克霞.黄岑和黄芩苷对幽门螺杆菌的体外抗菌活性研究.中药材,2008.31(5)：p.707-710.
[19]曹翠萍,宁海强,孙丽,等.中药对大肠杆菌抑制作用及耐药性诱导作用的研究.西南农业学报,2007.20(5)：p.1101-1104.
[20]滕兰菊,桂芳,黄刚,等.黄芩等中草药对52株伤寒杆菌药敏试验研究.怀化医专学报,2002.1(2)：p.22-23.
[21]刘云波,郭丽华,邱世翠.黄芩体外抑菌作用研究.时珍国医国药,2002.13(3)：p.596.
[22]栾耀芳,张凤花,吴国英.黄芩等8种中药对产β-内酰胺酶大肠埃希菌的敏感性研究.山东中医杂志,2005.24(10)：p.629-631.
[23]刘平,叶惠芬,陈惠玲,等.5种中药对产酶菌的抑菌作用.中国微生态学杂志,2006(01).
[24]梅林,贺锡中,熊云,等.中药黄芩的血清抑菌活性试验研究.医学研究杂志,2009.38(1)：p.102-103,85.
[25]陈莉,朱凤静,张春枝.30种中药水提取物的体外抗菌活性试验.大连轻工业学院学报,2005.24(004)：p.269-271.
[26]吕小迅,周玉珍.黄岑，黄精抗真菌作用活性部位实验研究.广东药学院学报,1995.11(003)：p.180-180.
[27]赵铁华,陈四平,杨鹤松,等.黄芩茎叶活性部位抗病毒作用的研究.中国药科大学学报,2006.37(6)：p.544-547.
[28]徐珊,王乐,杨巧芳,等.黄岑抗病毒药理作用研究述评.中华中医药学刊,2007.25(7)：p.1355-1357.
[29]Ikemoto, S., et al., Antitumor effects of Scutellariae radix and its components baicalein, baicalin, and wogonin on bladder cancer cell lines. Urology,2000. 55(6):p.951-5.
[30]汤立建,赵良才,李庆林,等.葛根和黄芩乙醇提取物体外抗乳腺癌实验研究.安徽中医学院学报,2006.25(6)：p.23-27.
[31]张汉英,黎丹戎,李力,等.汉黄芩素对移植人卵巢癌SKOV3细胞裸鼠组织端粒酶蛋白hTERT影响与凋亡相关性研究.时珍国医国药,2008.19(8)：p.1987-1989.
[32]朴花子,崔弘,朴日龙,等.汉黄芩素对脂多糖诱导的促炎性因子的影响.西安交通大学学报：医学版,2008.29(2)：p.230-232.
[33]Fas, S.C., et al., Wogonin sensitizes resistant malignant cells to TNFalpha-and TRAIL-induced apoptosis. Blood,2006.108(12):p.3700-6.
[34]Rushworth, S.A. and O. Micheau, Molecular crosstalk between TRAIL and natural antioxidants in the treatment of cancer. Br J Pharmacol,2009.157(7): p.1186-8.
[35]Lee, D.H., et al., Role of p53, PUMA, and Bax in wogonin-induced apoptosis in human cancer cells. Biochem Pharmacol,2008.75(10):p.2020-33.
[36]Lee, D.H., J.G. Rhee, and Y.J. Lee, Reactive oxygen species up-regulate p53 and Puma; a possible mechanism for apoptosis during combined treatment with TRAIL and wogonin. Br J Pharmacol,2009.157(7):p.1189-202.
[37]Rugo, H., et al., Phase I trial and antitumor effects of BZL101 for patients with advanced breast cancer. Breast Cancer Res Treat,2007.105(1):p.17-28.
[38]Kawashima, K., et al., Pharmacological properties of traditional medicine (ⅩⅪⅩ):effect of Hange-shashin-to and the combinations of its herbal constituents on rat experimental colitis. Biol Pharm Bull,2004.27(10):p. 1599-603.
[39]Ahmed-Belkacem, A., et al., Silibinin and Related Compounds Are Direct Inhibitors of Hepatitis C Virus RNA-Dependent RNA Polymerase. Gastroenterology,2009.
[40]Ferenci, P., et al., Silibinin is a potent antiviral agent in patients with chronic hepatitis C not responding to pegylated interferon/ribavirin therapy. Gastroenterology,2008.135(5):p.1561-7.
[41]Polyak, S.J., et al., Inhibition of T-cell inflammatory cytokines, hepatocyte NF-kappaB signaling, and HCV infection by standardized Silymarin. Gastroenterology,2007.132(5):p.1925-36.
[42]Sukhthankar, M., et al., A green tea component suppresses posttranslational expression of basic fibroblast growth factor in colorectal cancer. Gastroenterology,2008.134(7):p.1972-80.
[43]Ghosal, A., et al., Identification of human UDP-glucuronosyltransferase enzyme(s) responsible for the glucuronidation of ezetimibe (Zetia). Drug Metab Dispos,2004.32(3):p.314-20.
[44]Kosoglou, T., et al., Ezetimibe:a review of its metabolism, pharmacokinetics and drug interactions. Clin Pharmacokinet,2005.44(5):p.467-94.
[45]45. Jeong, E.J., et al., Species-and disposition model-dependent metabolism of raloxifene in gut and liver:role of UGTIA10. Drug Metab Dispos,2005. 33(6):p.785-94.
[46]Levi, M.S., R.F. Borne, and J.S. Williamson, A review of cancer chemopreventive agents. Curr Med Chem,2001.8(11):p.1349-62.
[47]Logan, R.M., et al., Characterisation of mucosal changes in the alimentary tract following administration of irinotecan:implications for the pathobiology of mucositis. Cancer Chemother Pharmacol,2008.62(1):p.33-41.
[48]Nagano, T., et al., Antitumor activity of NK012 combined with cisplatin against small cell lung cancer and intestinal mucosal changes in tumor-bearing mouse after treatment. Clin Cancer Res,2009.15(13):p. 4348-55.
[49]Brill, S.S., et al., Glucuronidation of trans-resveratrol by human liver and intestinal microsomes and UGT isoforms. J Pharm Pharmacol,2006.58(4):p. 469-79.
[50]Walle, T., et al., High absorption but very low bioavailability of oral resveratrol in humans. Drug Metab Dispos,2004.32(12):p.1377-82.
[51]Jia, X., et al., Disposition of flavonoids via enteric recycling: enzyme-transporter coupling affects metabolism of biochanin A and formononetin and excretion of their phase II conjugates. J Pharmacol Exp Ther,2004.310(3):p.1103-13.
[52]Wang, S.W., et al., Disposition of flavonoids via enteric recycling:structural effects and lack of correlations between in vitro and in situ metabolic properties. Drug Metab Dispos,2006.34(11):p.1837-48.
[53]Liu, X., V.H. Tam, and M. Hu, Disposition of flavonoids via enteric recycling: determination of the UDP-glucuronosyltransferase isoforms responsible for the metabolism of flavonoids in intact Caco-2 TC7 cells using siRNA. Mol Pharm,2007.4(6):p.873-82.
[54]Houston, J.B. and K.E. Kenworthy, In vitro-in vivo scaling of CYP kinetic data not consistent with the classical Michaelis-Menten model. Drug Metab Dispos,2000.28(3):p.246-54.
[55]Hutzler, J.M. and T.S. Tracy, Atypical kinetic profiles in drug metabolism reactions. Drug Metab Dispos,2002.30(4):p.355-62.
[56]Sandanaraj, E., et al., Influence of UGT1A9 intronic I399C＞T polymorphism on SN-38 glucuronidation in Asian cancer patients. Pharmacogenomics J, 2008.8(3):p.174-85.
[57]Yamaoka, K., T. Nakagawa, and T. Uno, Application of Akaike's information criterion (AIC) in the evaluation of linear pharmacokinetic equations. J Pharmacokinet Biopharm,1978.6(2):p.165-75.
[58]Girard, H., et al., The novel UGT1A9 intronic 1399 polymorphism appears as a predictor of 7-ethyl-10-hydroxycamptothecin glucuronidation levels in the liver. Drug Metab Dispos,2006.34(7):p.1220-8.
[59]Dai, J.Y., J.L. Yang, and C. Li, Transport and metabolism of flavonoids from Chinese herbal remedy Xiaochaihu-tang across human intestinal Caco-2 cell monolayers. Acta Pharmacol Sin,2008.29(9):p.1086-93.
[60]Shi, R., et al., Influence of coptis Chinensis on pharmacokinetics of flavonoids after oral administration of radix Scutellariae in rats. Biopharm Drug Dispos, 2009.30(7):p.398-410.
[61]Akao, T., et al., Baicalin, the predominant flavone glucuronide of scutellariae radix, is absorbed from the rat gastrointestinal tract as the aglycone and restored to its original form. J Pharm Pharmacol,2000.52(12):p.1563-8.
[62]Xing, J., X. Chen, and D. Zhong, Absorption and enterohepatic circulation of baicalin in rats. Life Sci,2005.78(2):p.140-6.
[63]Lai, M.Y., et al., Comparison of metabolic pharmacokinetics of baicalin and baicalein in rats. J Pharm Pharmacol,2003.55(2):p.205-9.
[64]He, J.X., et al., Interaction between Shaoyao-Gancao-Tang and a laxative with respect to alteration of paeoniflorin metabolism by intestinal bacteria in rats. Phytomedicine,2007.14(7-8):p.452-9.
[65]Lu, T., et al., Comparative pharmacokinetics of baicalin after oral administration of pure baicalin, Radix scutellariae extract and Huang-Lian-Jie-Du-Tang to rats. J Ethnopharmacol,2007.110(3):p.412-8.
[66]Liu, Y., et al., Enteric disposition and recycling of flavonoids and ginkgo flavonoids. J Altern Complement Med,2003.9(5):p.631-40.
[67]Ausbubel FM, et al., Current Protocols in Molecular Biology, Vol 1. John Wiley & Sons, New York,1988, pp 1.3.1-4.1988.1.
[68]Yi, J.H., et al., Intestinal flora translocation and overgrowth in upper gastrointestinal tract induced by hepatic failure. World J Gastroenterol,1999. 5(4):p.327-329.
[69]China Parmacopeia,2005, the second edition,Appendix XI J,93-5.2005.
[70]Marshall, T. and K.M. Williams, Comparison of Coomassie Brilliant Blue protein dye-binding assays for determination of urinary protein concentration. Clin Chem,1987.33(4):p.577-8.
[71]Lin, Y.T., et al., Degradation offlavonoid aglycones by rabbit, rat and human fecal flora. Biol Pharm Bull,2003.26(5):p.747-51.
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