黄酮类化合物和乌头碱代谢机理研究
摘要
来源于植物的天然产物在预防和治疗疾病方面发挥了重要作用,随着药物特别是化学药物的开发进入瓶颈期,植物来源的天然药物的研制越来越受到重视。黄酮类和乌头碱类化合物是植物来源中具有药用价值的两类重要化合物。约有20%的中草药中均含有具有抗癌、抗氧化等多种药理活性的黄酮类化合物；而乌头碱类双酯二萜型生物碱具有较强抗炎、镇痛作用,其在临床上出现的不良反应和中毒事件使其应用受到极大限制。从药物的吸收、分布、代谢和排泄(ADME)等方面探求这两类化合物的体内特征及机理可为其作用机制和毒性监控提供依据。本论文从药物代谢关键环节上深入阐明黄酮类化合物和乌头碱在ADME上的代谢机理,为这两类化合物的药代特征及机理和毒性预警及临床安全应用提供理论依据。
一、有效成分黄酮类化合物(flavonoids)的UGT代谢机理研究
黄酮类(包括异黄酮)化合物广泛存在于植物界,包括多种水果、蔬菜和药用植物。大量研究表明黄酮类化合物具有多种生物活性,如抗肿瘤,抗炎,预防心血管疾病等。然而,大多数(异)黄酮类化合物还没有被开发为化学预防或治疗药物,最大的挑战来自于其体内极低的生物利用度(<10%)；包括葡萄糖醛酸转移酶(UGTs)和磺酸转移酶(SULTs)在内的Ⅱ相代谢酶引起的首过效应是其生物利用度低的重要原因之一。
本论文首次引入(异)黄酮类UGT特异性代谢指纹图谱的概念,并将UGT特异性代谢指纹图谱应用于药物相互反应、基因多态性预测和底物结构-代谢活性相关性(SMR)等研究。黄酮类化合物UGT特异性代谢指纹图谱的建立能为其它UGT底物的代谢指纹图谱提供示范性研究,从而为提高通过葡萄糖醛酸化途径代谢的药物的安全性与有效性提供理论支持。鉴于此,我们采用12种人重组UGT亚酶进行以下两方面的研究：
1.异黄酮类化合物(isoflavones) UGT代谢机理及其UGT特异性指纹图谱的研究
运用12种重组人UGT亚酶和人肝、肠微粒体对6种异黄酮类化合物：染料木黄酮(genistein)、鹰嘴豆芽素A(biochanin A)、芒柄花黄素(formononetin)、樱黄素(prunetin)、大豆黄酮(daidzein)和黄豆黄素(glycitein)的UGT代谢机理进行研究。结果显示这6种化合物主要由UGT1A1、UGT1A8、UGT1A9和UGT1A10等4种UGT亚酶代谢。Glycitein总是其中代谢最快的化合物,而prunetin代谢最慢(p<0.05)。UGT1A1对其他5种异黄酮代谢都较快,除了prunetin(p<0.05),其原因与prunetin吡喃环上的7-OH密切相关,此位点可能就是UGT1A1的活性识别位点。以6个化合物被12个UGT亚酶代谢的速率均值建立UGT特异性指纹图谱。我们发现不同的化合物有特定的UGT特异性指纹图谱,此指纹谱受底物结构和浓度的影响。Genistein和prunetin的4种UGT亚酶的酶代动力学参数显示,三种不同的代谢模式由化合物的Km和Vmax值决定。
与此同时,我们运用UGT特异性代谢指纹图谱预测了异黄酮类化合物在人肝、肠微粒体中的代谢情况。除formononetin外,其余5个化合物在肝微粒体中的代谢能被很好的预测,其在肠微粒体中的代谢却不能预测,这可能与在肠道中新发现的可代谢黄酮等酚类化合物的UGT3A家族有关,而UGT3A家族的亚酶目前尚未被商品化。
2.黄酮类化合物(flavones)UGT代谢机理及其UGT特异性指纹图谱的研究
运用12种重组人UGT亚酶及人肝、肠微粒体对7种单羟基黄酮类化合物：2'-,3'-,4'-,3-,5-,6-和7-hydroxyflavone(HF)及3种双羟基化合物：3,7-,3,5-和3,4'-dihydroxyflavone(diHF)的UGT代谢特征进行研究。3个双羟基黄酮的葡萄糖醛酸结合位点可由其对应的单羟基黄酮的紫外扫描光谱判断。研究结果显示,UGT1A1, UGT1A7, UGT1A8, UGT1A9, UGT1A10和UGT2B7为代谢黄酮类化合物最多的6种UGT亚酶。3-HF是7种化合物中代谢最快的化合物,而5-HF代谢最慢(p<0.05)。5-HF代谢缓慢的原因可能与其5-OH和4-羰基形成了分子内氢键从而阻碍葡萄糖醛酸结合反应有关。黄酮类化合物的UGT特异性代谢指纹图谱也与特定UGT亚酶,底物羟基位置及浓度密切相关。黄酮类化合物的UGT特异性代谢与其在人肝、肠微粒体中的代谢具有良好的相关性。
二、有毒成分乌头碱(aconitine)毒代动力学和CYP代谢机理,及黄酮化合物(flavonoids)对其代谢的影响研究
乌头碱类化合物(aconite alkaloids)具有较强药理活性,但其毒性反应亦十分明显,其主要毒性反应表现为神经、心脏和肝肾毒性。我们选择最为常见的单体乌头碱(aconitine)作为研究对象。采用小鼠醋酸扭体法和毒性评价实验测定了乌头碱的安全窗,其范围是0.06879-0.08708mg/kg(ED95-LD5),安全窗相当窄。这种高毒性和狭窄的安全治疗窗预示代谢酶的代谢作用对毒性起重要的调控作用。本章以乌头碱的毒代动力学研究入手,研究其固有毒性特点,同时从影响毒代动力学特征的关键环节药物代谢酶上深入阐明乌头碱致毒和解毒的分子机理。同时,本文选择了在食物及药用植物中广泛分布的染料木黄酮(genistein)和大豆黄酮(daidzein)作为异黄酮中的代表性成分,初步研究其对乌头碱代谢的影响,从而研究两类成分的相互作用,为寻找乌头碱的减毒方法和途径提供初步探索。
1.乌头碱在大鼠体内的毒代动力学研究
建立灵敏的UPLC/MS/MS检测方法,检测口服中毒剂量(0.504mg/kg)下的乌头碱血药浓度,计算大鼠体内的毒代动力学参数。乌头碱在大鼠体内吸收迅速,血药浓度最高峰出现在0.5 h左右,浓度达最大值(Cmax7.43±3.84ng/ml)后迅速降低。我们推测其血药浓度的迅速降低与药物代谢酶对其的代谢作用密切相关。
2.乌头碱代谢产物的结构鉴定及其CYP酶代谢机理研究
运用液质联用(UPLC/MS/MS)及高分辨质谱(HRMS)分析方法,研究乌头碱在人、大鼠、小鼠、狗肝微粒体中的代谢产物,并鉴定了四种微粒体中相同的11种代谢产物,同时,在四种不同动物的肝微粒体中代谢产物无显著性差异。为了找到参与乌头碱代谢的P450亚型,考察了各种CYP亚酶的选择性抑制剂对乌头碱代谢产物相对生成量的影响。研究显示,CYP2D6不代谢乌头碱；CYP3A的抑制剂酮康唑在5μM可强烈的抑制乌头碱代谢产物M2 (16-O-deme thyl-aconitine)的生成；同时观察到CYP1A2抑制剂也在一定程度上抑制了乌头碱的代谢,而CYP2C也产生明显的抑制效应。CYP1A2、CYP2C8、CYP2C9是代谢M1 (O-demethyl-aconitine)及M3 (O-didemethyl-aconitine)的共同CYP亚酶。乌头碱的CYP代谢途径是去甲基化,羟化及脱氢反应,参与乌头碱代谢的主要CYP亚型是CYP3A4、CYP1A2及CYP2C。
3.黄酮类化合物(flavonoids)对乌头碱体外代谢的影响
本文选择了染料木黄酮(genistein)和大豆黄酮(daidzein)为受试药品,研究其对乌头碱代谢的影响。结果表明genistein可明显地影响乌头碱类在肝微粒体中的代谢,而与genistein相比,只在A环5位上相差一个羟基的daidzein对乌头碱代谢的影响明显减弱。结果提示黄酮类化合物中羟基取代对乌头碱代谢有影响作用,A环上的5位羟基可能是其影响乌头碱代谢的活性位点之一。
结论：本文首次建立了16种黄酮类化合物的UGT特异性代谢指纹图谱,阐明了其UGT代谢机理以及UGT亚型与肝肠微粒体代谢的相关性；首次证明乌头碱在人及3种动物肝微粒体中的代谢途径相同,并阐明了其在人肝微粒体中的CYP代谢机理,同时证明黄酮类化合物对乌头碱代谢有影响,因此,本文系统深入研究了黄酮类化合物和乌头碱的代谢,为这两类天然产物的药代特征及机理和毒性预警及临床安全应用提供理论依据。
It is well known that natural products are the main sources of chemo-preventive and chemo-therapeutic agents, since the chemical drug development is much more difficult than before. The use of flavonoids and aconite alkaloids, two kinds of efficient natural products, was limited because of low bioavailability or toxicity. Flavonoids are widely distributed in plants and Chinese herbs. Aconitine, which is used to cause side effects and clinical adverse reactions, is the typical ingredient of aconite with anti-inflammatory and analgesic effect, as well as toxicity. In order to develop and enlarge the usage of flavonoids and aconite alkaloids, it is necessary to do research on the absorption, distribution, metabolism and excretion (ADME) in vivo and in vitro for a better understand of the metabolic characterizations of flavonoids and aconitine. The aim of this thesis is to elucidate the metabolic mechanism of flavonoids and aconitine, which is helpful to the evaluations of drug therapy, drug toxicity and clinical drug safety.
PartⅠ
The UGT-mediated Metabolism of Flavonoids
Flavonoids are widely distributed in a variety of natural plants and edible foods including fruits and vegetables. Scientific researches have demonstrated their obvious biological activities. Despite of these claimed health benefits and demonstrated preclinical activities, there are significant challenges associated with development of isoflavones and flavones into chemo-preventive and chemo-therapeutic agents. The major challenge currently is their low bioavailabilities (<10%), as the result of extensive first-pass metabolism by phaseⅡenzymes including UGTs and SULTs. So we chose two main kinds of flavonoids:isoflavones and flavones to better understand the metabolic mechanism of flavonoids.
The present studies represent a detailed and systematic study of UGT isoform-specific metabolism of 6 isoflavones and 10 flavones. We have shown for the first time that the glucuronidation of flavonoids were all UGT isoform-, substrate chemical structure-and concentration-dependent, which can be captured by metabolic "fingerprint" using expressed human UGT isoforms. We demonstrated for the first time the multifaceted utilities of UGT metabolic "fingerprint" in defining for drug interactions, genetic polymorphism, major organs for metabolism, and SMR. We believe that the approach developed here may be of general utilities in defining the metabolic "fingerprint" of other UGT substrates, which in turn could improve the safety as well as efficacy of drugs that are inactivated or eliminated by the glucuronidation pathway.
1. The UGT-mediated Metabolism of Isoflavones and the Characterization of UGT Metabolic Fingerprint of Isoflavones
We characterized the isofonm-specific glucuronidation of six isoflavones: genistein, daidzein, glycitein, formononetin, biochanin A and prunetin using 12 expressed human UGTs and human intestinal and liver microsomes. The results indicated that these isoflavones were metabolized most rapidly by one of these four UGT isoforms:UGT1A1, UGT1A8, UGT1A9 and UGT1A10. Furthermore, glycitein was usually metabolized the fastest whereas prunetin the slowest. Because prunetin was the only compound not metabolized by UGT1A1, the hydroxyl group at position 7 in the pyrane ring of isoflavones is a structural feature important for active site recognition. Using the rates of metabolism by 12 UGT isoforms as a means to establish the metabolic "fingerprint", we found that each isoflavone had distinctive concentration-dependent patterns. Determination of kinetic parameters of glucuronidation using genistein and prunetin indicated that the distinct concentration-dependent metabolic patterns were the result of differences in Km and Vmax values. We then measured how well metabolic "fingerprinting" predicted metabolism of these isoflavones by human intestinal and liver microsomes. We found that the prediction was rather successful for five isoflavones in the liver microsomes, but not successful in the intestinal microsomes. We propose that a newly discovered UGT3A1 isoform capable of metabolizing phenols and estrogens may be responsible for the metabolism of isoflavones such as formononetin in humans. In conclusion, the first systematic study of metabolic "fingerprinting" of six common isoflavones showed that each isoflavone has UGT isoform-specific metabolic patterns that are concentration-dependent and predictive of metabolism of the isoflavones in liver microsomes.
2. The UGT-mediated Metabolism of Flavones and the Characterization of UGT Metabolic Fingerprint of Flavones
The present study aims to predict the region-specific glucuronidation of three dihydroxyflavones and seven mono-hydroxyflavones in human liver and intestinal microsomes using recombinant UGT isoforms. Seven mono-hydroxyflavones (HFs), 2'-,3'-,4'-,3-,5-,6-, and 7-hydroxyflavone, and three di-hydroxyflavones (diHFs), 3,7-,3,5-and 3,4'-dihydroxyflavone were chosen. The results indicated that the position of glucuronidation of three diHFs could be determined by using the UV spectra of relevant HFs. The results also indicated that UGT1A1, UGT1A7, UGT1A8, UGT1A9, UGT1A10 and UGT2B7 were the most important six UGT isoforms for metabolizing the chosen flavones. Regardless of isoforms used,3-HF was always metabolized the fastest whereas 5-HF was usually metabolized the slowest, probably due to the formation of an intra-molecular hydrogen bond between 4-carbonyl and 5-OH group. Relevant UGT isoform-specific metabolism rates were found to correlate well with the rates of glucuronidation in human intestinal and liver microsomes, except for 3'-HF. In conclusion, the glucuronidation "fingerprint" of seven selected mono-hydroxyflavones was affected by UGT isoforms used, positions of the-OH group, and the substrate concentrations, and the rates of glucuronidation by important recombinant UGTs correlated well with those obtained using human liver and intestinal microsomes.
PartⅡ
Aconitine Toxicokinetics and Its CYP-mediated Metabolism,and the Effect of Flavonoids on the Metabolism of Aconitine
Aconitine, a famous aconite alkaloid, is well known for its high toxicity that induces severe arrhythmias leading to death. Our previous research has found that the security window of aconitine was 0.06879-0.08708 mg/kg (ED95-LD50). And it was suggested that the high toxicity and narrow security window were correlated with the metabolism by enzyme in vivo. In order to understand the molecular mechanism on the toxicity, we paid attention to the toxicokinetics of aconitine and how the phase I reaction affect the toxicokinetics, which could give more evidence to the evaluation of clinical safety of aconitine. In the end, we chose two isoflavones, genistein and daidzein, to investigate if flavonoids could affect the metabolism of aconitine.
1. Toxicokinetics of Aconitine in Rat
In order to investigate the pharmacokinetic behaviors of aconitine in rats, a sensitive and fast UPLC-MS/MS method was successfully developed. Aconitine was absorbed fleetly after oral administration at 0.504 mg/kg. The concentration reached the highest level in rat plasma (Cmax 7.43±3.84 ng/ml) around 0.5 hour. We presumed it related to the effect of metabolic enzyme in vivo.
2. The Characterization of Metabolites and CYP-mediated Metabolic Mechanism of Aconitine
An UPLC/MS/MS and a high resolution mass spectrogram (HRMS) method were developed to determine the characteristics of metabolism of aconitine. We found 11 metabolites of aconitine in phase I reaction system in human, rat, mice and dog liver microsomes. In order to distinguish the specific-CYP isoforms responsible for metabolising aconitine, six selective CYP-inhibitors were used in phase I reaction system. The results indicated that CYP2D6 could not metabolize aconitine. CYP3A4 produced 16-O-demethyl-aconitine. CYP1A2 and CYP2C could metabolize aconitine moderately.
3. The Effect of Flavonoids on the Metabolism of Aconitine
We determined the effect of two famous flavonoids, genistein and daidzein, on the metabolism of aconitine in CYP reaction system. The results indicated that genistein could affect the metabolism of aconitine distinctly in vitro while daidzein did not.
In summary, we established the UGT fingerprints of sixteen flavonoids and elucidated the metabolic mechanism by UGT for the first time. Also we demonstrated the metabolic pathways of aconitine in human, rat, mice and dog liver microsomes are similar and elucidated the CYP-mediated metabolic mechanism. In the end, we demonstrated the obvious effect of flavonoids on the metabolism of aconitine.
一、有效成分黄酮类化合物(flavonoids)的UGT代谢机理研究
黄酮类(包括异黄酮)化合物广泛存在于植物界,包括多种水果、蔬菜和药用植物。大量研究表明黄酮类化合物具有多种生物活性,如抗肿瘤,抗炎,预防心血管疾病等。然而,大多数(异)黄酮类化合物还没有被开发为化学预防或治疗药物,最大的挑战来自于其体内极低的生物利用度(<10%)；包括葡萄糖醛酸转移酶(UGTs)和磺酸转移酶(SULTs)在内的Ⅱ相代谢酶引起的首过效应是其生物利用度低的重要原因之一。
本论文首次引入(异)黄酮类UGT特异性代谢指纹图谱的概念,并将UGT特异性代谢指纹图谱应用于药物相互反应、基因多态性预测和底物结构-代谢活性相关性(SMR)等研究。黄酮类化合物UGT特异性代谢指纹图谱的建立能为其它UGT底物的代谢指纹图谱提供示范性研究,从而为提高通过葡萄糖醛酸化途径代谢的药物的安全性与有效性提供理论支持。鉴于此,我们采用12种人重组UGT亚酶进行以下两方面的研究：
1.异黄酮类化合物(isoflavones) UGT代谢机理及其UGT特异性指纹图谱的研究
运用12种重组人UGT亚酶和人肝、肠微粒体对6种异黄酮类化合物：染料木黄酮(genistein)、鹰嘴豆芽素A(biochanin A)、芒柄花黄素(formononetin)、樱黄素(prunetin)、大豆黄酮(daidzein)和黄豆黄素(glycitein)的UGT代谢机理进行研究。结果显示这6种化合物主要由UGT1A1、UGT1A8、UGT1A9和UGT1A10等4种UGT亚酶代谢。Glycitein总是其中代谢最快的化合物,而prunetin代谢最慢(p<0.05)。UGT1A1对其他5种异黄酮代谢都较快,除了prunetin(p<0.05),其原因与prunetin吡喃环上的7-OH密切相关,此位点可能就是UGT1A1的活性识别位点。以6个化合物被12个UGT亚酶代谢的速率均值建立UGT特异性指纹图谱。我们发现不同的化合物有特定的UGT特异性指纹图谱,此指纹谱受底物结构和浓度的影响。Genistein和prunetin的4种UGT亚酶的酶代动力学参数显示,三种不同的代谢模式由化合物的Km和Vmax值决定。
与此同时,我们运用UGT特异性代谢指纹图谱预测了异黄酮类化合物在人肝、肠微粒体中的代谢情况。除formononetin外,其余5个化合物在肝微粒体中的代谢能被很好的预测,其在肠微粒体中的代谢却不能预测,这可能与在肠道中新发现的可代谢黄酮等酚类化合物的UGT3A家族有关,而UGT3A家族的亚酶目前尚未被商品化。
2.黄酮类化合物(flavones)UGT代谢机理及其UGT特异性指纹图谱的研究
运用12种重组人UGT亚酶及人肝、肠微粒体对7种单羟基黄酮类化合物：2'-,3'-,4'-,3-,5-,6-和7-hydroxyflavone(HF)及3种双羟基化合物：3,7-,3,5-和3,4'-dihydroxyflavone(diHF)的UGT代谢特征进行研究。3个双羟基黄酮的葡萄糖醛酸结合位点可由其对应的单羟基黄酮的紫外扫描光谱判断。研究结果显示,UGT1A1, UGT1A7, UGT1A8, UGT1A9, UGT1A10和UGT2B7为代谢黄酮类化合物最多的6种UGT亚酶。3-HF是7种化合物中代谢最快的化合物,而5-HF代谢最慢(p<0.05)。5-HF代谢缓慢的原因可能与其5-OH和4-羰基形成了分子内氢键从而阻碍葡萄糖醛酸结合反应有关。黄酮类化合物的UGT特异性代谢指纹图谱也与特定UGT亚酶,底物羟基位置及浓度密切相关。黄酮类化合物的UGT特异性代谢与其在人肝、肠微粒体中的代谢具有良好的相关性。
二、有毒成分乌头碱(aconitine)毒代动力学和CYP代谢机理,及黄酮化合物(flavonoids)对其代谢的影响研究
乌头碱类化合物(aconite alkaloids)具有较强药理活性,但其毒性反应亦十分明显,其主要毒性反应表现为神经、心脏和肝肾毒性。我们选择最为常见的单体乌头碱(aconitine)作为研究对象。采用小鼠醋酸扭体法和毒性评价实验测定了乌头碱的安全窗,其范围是0.06879-0.08708mg/kg(ED95-LD5),安全窗相当窄。这种高毒性和狭窄的安全治疗窗预示代谢酶的代谢作用对毒性起重要的调控作用。本章以乌头碱的毒代动力学研究入手,研究其固有毒性特点,同时从影响毒代动力学特征的关键环节药物代谢酶上深入阐明乌头碱致毒和解毒的分子机理。同时,本文选择了在食物及药用植物中广泛分布的染料木黄酮(genistein)和大豆黄酮(daidzein)作为异黄酮中的代表性成分,初步研究其对乌头碱代谢的影响,从而研究两类成分的相互作用,为寻找乌头碱的减毒方法和途径提供初步探索。
1.乌头碱在大鼠体内的毒代动力学研究
建立灵敏的UPLC/MS/MS检测方法,检测口服中毒剂量(0.504mg/kg)下的乌头碱血药浓度,计算大鼠体内的毒代动力学参数。乌头碱在大鼠体内吸收迅速,血药浓度最高峰出现在0.5 h左右,浓度达最大值(Cmax7.43±3.84ng/ml)后迅速降低。我们推测其血药浓度的迅速降低与药物代谢酶对其的代谢作用密切相关。
2.乌头碱代谢产物的结构鉴定及其CYP酶代谢机理研究
运用液质联用(UPLC/MS/MS)及高分辨质谱(HRMS)分析方法,研究乌头碱在人、大鼠、小鼠、狗肝微粒体中的代谢产物,并鉴定了四种微粒体中相同的11种代谢产物,同时,在四种不同动物的肝微粒体中代谢产物无显著性差异。为了找到参与乌头碱代谢的P450亚型,考察了各种CYP亚酶的选择性抑制剂对乌头碱代谢产物相对生成量的影响。研究显示,CYP2D6不代谢乌头碱；CYP3A的抑制剂酮康唑在5μM可强烈的抑制乌头碱代谢产物M2 (16-O-deme thyl-aconitine)的生成；同时观察到CYP1A2抑制剂也在一定程度上抑制了乌头碱的代谢,而CYP2C也产生明显的抑制效应。CYP1A2、CYP2C8、CYP2C9是代谢M1 (O-demethyl-aconitine)及M3 (O-didemethyl-aconitine)的共同CYP亚酶。乌头碱的CYP代谢途径是去甲基化,羟化及脱氢反应,参与乌头碱代谢的主要CYP亚型是CYP3A4、CYP1A2及CYP2C。
3.黄酮类化合物(flavonoids)对乌头碱体外代谢的影响
本文选择了染料木黄酮(genistein)和大豆黄酮(daidzein)为受试药品,研究其对乌头碱代谢的影响。结果表明genistein可明显地影响乌头碱类在肝微粒体中的代谢,而与genistein相比,只在A环5位上相差一个羟基的daidzein对乌头碱代谢的影响明显减弱。结果提示黄酮类化合物中羟基取代对乌头碱代谢有影响作用,A环上的5位羟基可能是其影响乌头碱代谢的活性位点之一。
结论：本文首次建立了16种黄酮类化合物的UGT特异性代谢指纹图谱,阐明了其UGT代谢机理以及UGT亚型与肝肠微粒体代谢的相关性；首次证明乌头碱在人及3种动物肝微粒体中的代谢途径相同,并阐明了其在人肝微粒体中的CYP代谢机理,同时证明黄酮类化合物对乌头碱代谢有影响,因此,本文系统深入研究了黄酮类化合物和乌头碱的代谢,为这两类天然产物的药代特征及机理和毒性预警及临床安全应用提供理论依据。
It is well known that natural products are the main sources of chemo-preventive and chemo-therapeutic agents, since the chemical drug development is much more difficult than before. The use of flavonoids and aconite alkaloids, two kinds of efficient natural products, was limited because of low bioavailability or toxicity. Flavonoids are widely distributed in plants and Chinese herbs. Aconitine, which is used to cause side effects and clinical adverse reactions, is the typical ingredient of aconite with anti-inflammatory and analgesic effect, as well as toxicity. In order to develop and enlarge the usage of flavonoids and aconite alkaloids, it is necessary to do research on the absorption, distribution, metabolism and excretion (ADME) in vivo and in vitro for a better understand of the metabolic characterizations of flavonoids and aconitine. The aim of this thesis is to elucidate the metabolic mechanism of flavonoids and aconitine, which is helpful to the evaluations of drug therapy, drug toxicity and clinical drug safety.
PartⅠ
The UGT-mediated Metabolism of Flavonoids
Flavonoids are widely distributed in a variety of natural plants and edible foods including fruits and vegetables. Scientific researches have demonstrated their obvious biological activities. Despite of these claimed health benefits and demonstrated preclinical activities, there are significant challenges associated with development of isoflavones and flavones into chemo-preventive and chemo-therapeutic agents. The major challenge currently is their low bioavailabilities (<10%), as the result of extensive first-pass metabolism by phaseⅡenzymes including UGTs and SULTs. So we chose two main kinds of flavonoids:isoflavones and flavones to better understand the metabolic mechanism of flavonoids.
The present studies represent a detailed and systematic study of UGT isoform-specific metabolism of 6 isoflavones and 10 flavones. We have shown for the first time that the glucuronidation of flavonoids were all UGT isoform-, substrate chemical structure-and concentration-dependent, which can be captured by metabolic "fingerprint" using expressed human UGT isoforms. We demonstrated for the first time the multifaceted utilities of UGT metabolic "fingerprint" in defining for drug interactions, genetic polymorphism, major organs for metabolism, and SMR. We believe that the approach developed here may be of general utilities in defining the metabolic "fingerprint" of other UGT substrates, which in turn could improve the safety as well as efficacy of drugs that are inactivated or eliminated by the glucuronidation pathway.
1. The UGT-mediated Metabolism of Isoflavones and the Characterization of UGT Metabolic Fingerprint of Isoflavones
We characterized the isofonm-specific glucuronidation of six isoflavones: genistein, daidzein, glycitein, formononetin, biochanin A and prunetin using 12 expressed human UGTs and human intestinal and liver microsomes. The results indicated that these isoflavones were metabolized most rapidly by one of these four UGT isoforms:UGT1A1, UGT1A8, UGT1A9 and UGT1A10. Furthermore, glycitein was usually metabolized the fastest whereas prunetin the slowest. Because prunetin was the only compound not metabolized by UGT1A1, the hydroxyl group at position 7 in the pyrane ring of isoflavones is a structural feature important for active site recognition. Using the rates of metabolism by 12 UGT isoforms as a means to establish the metabolic "fingerprint", we found that each isoflavone had distinctive concentration-dependent patterns. Determination of kinetic parameters of glucuronidation using genistein and prunetin indicated that the distinct concentration-dependent metabolic patterns were the result of differences in Km and Vmax values. We then measured how well metabolic "fingerprinting" predicted metabolism of these isoflavones by human intestinal and liver microsomes. We found that the prediction was rather successful for five isoflavones in the liver microsomes, but not successful in the intestinal microsomes. We propose that a newly discovered UGT3A1 isoform capable of metabolizing phenols and estrogens may be responsible for the metabolism of isoflavones such as formononetin in humans. In conclusion, the first systematic study of metabolic "fingerprinting" of six common isoflavones showed that each isoflavone has UGT isoform-specific metabolic patterns that are concentration-dependent and predictive of metabolism of the isoflavones in liver microsomes.
2. The UGT-mediated Metabolism of Flavones and the Characterization of UGT Metabolic Fingerprint of Flavones
The present study aims to predict the region-specific glucuronidation of three dihydroxyflavones and seven mono-hydroxyflavones in human liver and intestinal microsomes using recombinant UGT isoforms. Seven mono-hydroxyflavones (HFs), 2'-,3'-,4'-,3-,5-,6-, and 7-hydroxyflavone, and three di-hydroxyflavones (diHFs), 3,7-,3,5-and 3,4'-dihydroxyflavone were chosen. The results indicated that the position of glucuronidation of three diHFs could be determined by using the UV spectra of relevant HFs. The results also indicated that UGT1A1, UGT1A7, UGT1A8, UGT1A9, UGT1A10 and UGT2B7 were the most important six UGT isoforms for metabolizing the chosen flavones. Regardless of isoforms used,3-HF was always metabolized the fastest whereas 5-HF was usually metabolized the slowest, probably due to the formation of an intra-molecular hydrogen bond between 4-carbonyl and 5-OH group. Relevant UGT isoform-specific metabolism rates were found to correlate well with the rates of glucuronidation in human intestinal and liver microsomes, except for 3'-HF. In conclusion, the glucuronidation "fingerprint" of seven selected mono-hydroxyflavones was affected by UGT isoforms used, positions of the-OH group, and the substrate concentrations, and the rates of glucuronidation by important recombinant UGTs correlated well with those obtained using human liver and intestinal microsomes.
PartⅡ
Aconitine Toxicokinetics and Its CYP-mediated Metabolism,and the Effect of Flavonoids on the Metabolism of Aconitine
Aconitine, a famous aconite alkaloid, is well known for its high toxicity that induces severe arrhythmias leading to death. Our previous research has found that the security window of aconitine was 0.06879-0.08708 mg/kg (ED95-LD50). And it was suggested that the high toxicity and narrow security window were correlated with the metabolism by enzyme in vivo. In order to understand the molecular mechanism on the toxicity, we paid attention to the toxicokinetics of aconitine and how the phase I reaction affect the toxicokinetics, which could give more evidence to the evaluation of clinical safety of aconitine. In the end, we chose two isoflavones, genistein and daidzein, to investigate if flavonoids could affect the metabolism of aconitine.
1. Toxicokinetics of Aconitine in Rat
In order to investigate the pharmacokinetic behaviors of aconitine in rats, a sensitive and fast UPLC-MS/MS method was successfully developed. Aconitine was absorbed fleetly after oral administration at 0.504 mg/kg. The concentration reached the highest level in rat plasma (Cmax 7.43±3.84 ng/ml) around 0.5 hour. We presumed it related to the effect of metabolic enzyme in vivo.
2. The Characterization of Metabolites and CYP-mediated Metabolic Mechanism of Aconitine
An UPLC/MS/MS and a high resolution mass spectrogram (HRMS) method were developed to determine the characteristics of metabolism of aconitine. We found 11 metabolites of aconitine in phase I reaction system in human, rat, mice and dog liver microsomes. In order to distinguish the specific-CYP isoforms responsible for metabolising aconitine, six selective CYP-inhibitors were used in phase I reaction system. The results indicated that CYP2D6 could not metabolize aconitine. CYP3A4 produced 16-O-demethyl-aconitine. CYP1A2 and CYP2C could metabolize aconitine moderately.
3. The Effect of Flavonoids on the Metabolism of Aconitine
We determined the effect of two famous flavonoids, genistein and daidzein, on the metabolism of aconitine in CYP reaction system. The results indicated that genistein could affect the metabolism of aconitine distinctly in vitro while daidzein did not.
In summary, we established the UGT fingerprints of sixteen flavonoids and elucidated the metabolic mechanism by UGT for the first time. Also we demonstrated the metabolic pathways of aconitine in human, rat, mice and dog liver microsomes are similar and elucidated the CYP-mediated metabolic mechanism. In the end, we demonstrated the obvious effect of flavonoids on the metabolism of aconitine.
引文
[1]Kurzer MS, Xu X. Dietary phytoestrogens. Annu Rev Nutr 1997;17:353-381.
[2]Lin LZ, He XG, Lindenmaier M, et al. LC-ESI-MS study of the flavonoid glycoside malonates of red clover (Trifolium pratense). J Agric Food Chem 2000;48(2):354-365.
[3]Lukas SE, Penetar D, Berko J, et al. An extract of the Chinese herbal root kudzu reduces alcohol drinking by heavy drinkers in a naturalistic setting. Alcohol Clin Exp Res 2005;29(5):756-762.
[4]Barnes S. Soy isoflavones--phytoestrogens and what else? J Nutr 2004;134 (5):1225S-1228S.
[5]Thomasset SC, Berry DP, Garcea G, et al. Dietary polyphenolic phytochemicals--promising cancer chemopreventive agents in humans? A review of their clinical properties. Int J Cancer 2007;120(3):451-458.
[6]Zubik L, Meydani M. Bioavailability of soybean isoflavones from aglycone and glucoside forms in American women. Am J Clin Nutr 2003;77(6): 1459-1465.
[7]Hu M. Commentary:bioavailability of flavonoids and polyphenols:call to arms. Mol Pharm 2007;4(6):803-806.
[8]Benavente-Garcia O, Castillo J. Update on uses and properties of citrus flavonoids:new findings in anticancer, cardiovascular, and anti-inflammatory activity. J Agric Food Chem 2008;56(15):6185-6205.
[9]Wang X, Morris ME. Pharmacokinetics and bioavailability of the flavonoid 7,8-benzoflavone in rats. JPharm Sci 2008;97(10):4546-4556.
[10]Walle T, Otake Y, Brubaker JA, et al. Disposition and metabolism of the flavonoid chrysin in normal volunteers. Br J Clin Pharmacol 2001;51(2):143-146.
[11]Liu X, Tam VH, Hu M. 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):873-882.
[12]Boutin JA, Meunier F, Lambert PH, et al. In vivo and in vitro glucuronidation of the flavonoid diosmetin in rats. Drug Metab Dispos 1993;21(6):1157-1166.
[13]Ross JA, Kasum CM. Dietary flavonoids:bioavailability, metabolic effects, and safety. Annu Rev Nutr 2002;22:19-34.
[14]de Vries JH, Hollman PC, Meyboom S, et al. Plasma concentrations and urinary excretion of the antioxidant flavonols quercetin and kaempferol as biomarkers for dietary intake. Am J Clin Nutr 1998;68(1):60-65.
[15]Moon JH, Nakata R, Oshima S, et al. Accumulation of quercetin conjugates in blood plasma after the short-term ingestion of onion by women. Am J Physiol Regul Integr Comp Physiol 2000;279(2):R461-467.
[16]Liu Y, Dai Y, Xun L, et al. Enteric disposition and recycling of flavonoids and ginkgo flavonoids. J Altern Complement Med 2003;9(5):631-640.
[17]Baker JR, Satarug S, Edwards RJ, et al. Potential for early involvement of CYP isoforms in aspects of human cadmium toxicity. Toxicol Lett 2003;137 (1-2):85-93.
[18]Guengerich FP. Update information on human P450s. Drug Metab Rev 2002;34(1-2):7-15.
[19]Guengerich FP, Rendic S. Update information on drug metabolism systems-2009, part Ⅰ. Curr Drug Metab;11(1):1-3.
[20]Rendic S, Guengerich FP. Update Information on Drug Metabolism Systems-2009, Part Ⅱ:Summary of Information on the Effects of Desases and Environmental Factors on Human Cytochrome P450 (CYP) Enzymes and Transporters. Curr Drug Metab.
[21]Ingelman-Sundberg M. Pharmacogenetics of cytochrome P450 and its applications in drug therapy:the past, present and future. Trends Pharmacol Sci 2004;25(4):193-200.
[22]Le Marchand L, Franke AA, Custer L, et al. Lifestyle and nutritional correlates of cytochrome CYP1A2 activity:inverse associations with plasma lutein and alpha-tocopherol. Pharmacogenetics 1997;7(1):11-19.
[23]Yao D, Ding S, Burchell B, et al. Detoxication of vinca alkaloids by human P450 CYP3A4-mediated metabolism:implications for the development of drug resistance. JPharmacol Exp Ther 2000;294(1):387-395.
[24]Huang Z, Roy P, Waxman DJ. Role of human liver microsomal CYP3A4 and CYP2B6 in catalyzing N-dechloroethylation of cyclophosphamide and ifosfamide. Biochem Pharmacol 2000;59(8):961-972.
[25]Ekhart C, Doodeman VD, Rodenhuis S, et al. Influence of polymorphisms of drug metabolizing enzymes (CYP2B6, CYP2C9, CYP2C19, CYP3A4, CYP3A5, GSTA1, GSTP1, ALDH1A1 and ALDH3A1) on the pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide. Pharmacogenet Genomics 2008;18(6):515-523.
[26]Taniguchi R, Kumai T, Matsumoto N, et al. Utilization of human liver microsomes to explain individual differences in paclitaxel metabolism by CYP2C8 and CYP3A4. J Pharmacol Sci 2005;97(1):83-90.
[27]Santos A, Zanetta S, Cresteil T, et al. Metabolism of irinotecan (CPT-11) by CYP3A4 and CYP3A5 in humans. Clin Cancer Res 2000;6(5):2012-2020.
[28]Nakamura H, Nakasa H, Ishii I, et al. Effects of endogenous steroids on CYP3A4-mediated drug metabolism by human liver microsomes. Drug Metab Dispos 2002;30(5):534-540.
[29]Lee SJ, Kim WY, Kim H, et al. Identification of new CYP2C19 variants exhibiting decreased enzyme activity in the metabolism of S-mephenytoin and omeprazole. Drug Metab Dispos 2009;37(11):2262-2269.
[30]Fu M, Wu M, Wang JF, et al. Disruption of the intracellular Ca2+ homeostasis in the cardiac excitation-contraction coupling is a crucial mechanism of arrhythmic toxicity in aconitine-induced cardiomyocytes. Biochem Biophys Res Commun 2007;354(4):929-936.
[31]Xiao K, Wang L, Liu Y, et al. Study of aconitine toxicity in rat embryos in vitro. Birth Defects Res B Dev Reprod Toxicol 2007;80(3):208-212.
[32]艾路,孙莹,张宏桂.复方中药中乌头生物碱在人体内的代谢产物.北京中医药大学学报 2007；6(30)：955-958.
[33]张宏桂,史向国,孙莹,et al.兔血液中乌头碱代谢产物的研究.吉林大学学报(理学版)2006(02).
[34]Kaneko R, Hattori S, Furuta S, et al. Sensitive analysis of aconitine, hypaconitine, mesaconitine and jesaconitine in human body fluids and Aconitum tubers by LC/ESI-TOF-MS. J Mass Spectrom 2006;41(6):810-814.
[35]Lu G, Dong Z, Wang Q, et al. Toxicity Assessment of Nine Types of Decoction Pieces from the Daughter Root of Aconitum carmichaeli (Fuzi) Based on the Chemical Analysis of their Diester Diterpenoid Alkaloids. Planta Med 2009.
[36]Wang Y, Wang S, Liu Y, et al. Characterization of metabolites and cytochrome P450 isoforms involved in the microsomal metabolism of aconitine. J Chromatogr B Analyt Technol Biomed Life Sci 2006;844(2):292-300.
[37]Lasser KE, Allen PD, Woolhandler SJ, et al. Timing of new black box warnings and withdrawals for prescription medications. JAMA 2002;287(17): 2215-2220.
[38]Piscitelli SC, Burstein AH, Chaitt D, et al. Indinavir concentrations and St John's wort. Lancet 2000;355(9203):547-548.
[39]Bone KM. Potential interaction of Ginkgo biloba leaf with antiplatelet or anticoagulant drugs:what is the evidence? Mol Nutr Food Res 2008;52(7): 764-771.
[40]Williamson EM. Interactions between herbal and conventional medicines. Expert Opin Drug Saf 2005;4(2):355-378.
[41]Li P, Callery PS, Gan LS, et al. Esterase inhibition by grapefruit juice flavonoids leading to a new drug interaction. Drug Metab Dispos 2007;35(7): 1203-1208.
[42]Birt DF, Hendrich S, Wang W. Dietary agents in cancer prevention:flavonoids and isoflavonoids. Pharmacol Ther 2001;90(2-3):157-177.
[43]Wang SW, Chen J, Jia X, 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):1837-1848.
[44]Wong YC, Zhang L, Lin G, et al. Intestinal first-pass glucuronidation activities of selected dihydroxyflavones. Int J Pharm 2009;366(1-2):14-20.
[45]Zhang L, Lin G, Zuo Z. Position preference on glucuronidation of mono-hydroxylflavones in human intestine. Life Sci 2006;78(24):2772-2780.
[46]Tang L, Singh R, Liu Z, et al. Structure and Concentration Changes Affect Characterization of UGT Isoform-Specific Metabolism of Isoflavones. Mol Pharm 2009.
[47]Lee HS, Ji HY, Park EJ, et al. In vitro metabolism of eupatilin by multiple cytochrome P450 and UDP-glucuronosyltransferase enzymes. Xenobiotica 2007;37(8):803-817.
[48]Tang L, Ye L, Singh R, et al. Use of Glucuronidation Fingerprinting to Describe and Predict mono-and di-Hydroxyflavone Metabolism by Recombinant UGT Isoforms and Human Intestinal and Liver Microsomes. Mol Pharm.
[49]Hu M, Chen J. Nucleobase-and p-glycoprotein-mediated transport of AG337 in a Caco-2 cell culture model. Mol Pharm 2004;1(3):194-200.
[50]Liu Y, Hu M. Absorption and metabolism of flavonoids in the caco-2 cell culture model and a perused rat intestinal model. Drug Metab Dispos 2002;30(4):370-377.
[51]Hutzler JM, Tracy TS. Atypical kinetic profiles in drug metabolism reactions. Drug Metab Dispos 2002;30(4):355-362.
[52]Houston JB, Kenworthy KE. In vitro-in vivo scaling of CYP kinetic-data not consistent with the classical Michaelis-Menten model. Drug Metab Dispos 2000;28(3):246-254.
[53]D Argenio DZ, Schumitzky A. Biomedical simutations resource;University of Southern California:Los Angeles 1997.
[54]Yamaoka K, Nakagawa T, Uno T. Application of Akaike's information criterion (AIC) in the evaluation of linear pharmacokinetic equations. J Pharmacokinet Biopharm 1978;6(2):165-175.
[55]Prasain JK, Barnes S. Metabolism and bioavailability of flavonoids in chemoprevention:current analytical strategies and future prospectus. Mol Pharm 2007;4(6):846-864.
[56]Joseph TB, Wang SW, Liu X, et al. Disposition of flavonoids via enteric recycling:enzyme stability affects characterization of prunetin glucuronidation across species, organs, and UGT isoforms. Mol Pharm 2007;4(6):883-894.
[57]Huang SM, Strong JM, Zhang L, et al. New era in drug interaction evaluation: US Food and Drug Administration update on CYP enzymes, transporters, and the guidance process. J Clin Pharmacol 2008;48(6):662-670.
[58]Murray M. Role of CYP pharmacogenetics and drug-drug interactions in the efficacy and safety of atypical and other antipsychotic agents. J Pharm Pharmacol 2006;58(7):871-885.
[59]Singh SS. Preclinical pharmacokinetics:an approach towards safer and efficacious drugs. Curr Drug Metab 2006;7(2):165-182.
[60]Doerge DR, Chang HC, Churchwell MI, et al. Analysis of soy isoflavone conjugation in vitro and in human blood using liquid chromatography-mass spectrometry. Drug Metab Dispos 2000;28(3):298-307.
[61]Frank D, Jaehde U, Fuhr U. Evaluation of probe drugs and pharmacokinetic metrics for CYP2D6 phenotyping. Eur J Clin Pharmacol 2007;63(4):321-333.
[62]Mackenzie PI, Rogers A, Treloar J, et al. Identification of UDP glycosyltransferase 3A1 as a UDP N-acetylglucosaminyltransferase. J Biol Chem 2008;283(52):36205-36210.
[63]Ohno S, Nakajin S. Determination of mRNA expression of human UDP-glucuronosyltransferases and application for localization in various human tissues by real-time reverse transcriptase-polymerase chain reaction. Drug Metab Dispos 2009;37(1):32-40.
[64]Chen J, Lin H, Hu M. Absorption and metabolism of genistein and its five isoflavone analogs in the human intestinal Caco-2 model. Cancer Chemother Pharmacol 2005;55(2):159-169.
[65]Gibson DT, Hensley M, Yoshioka H, et al. Formation of (+)-cis-2,3-dihydroxy-1-methylcyclohexa-4,6-diene from toluene by Pseudomonas putida. Biochemistry 1970;9(7):1626-1630.
[66]Nakamura A, Nakajima M, Yamanaka H, et al. Expression of UGT1A and UGT2B mRNA in human normal tissues and various cell lines. Drug Metab Dispos 2008;36(8):1461-1464.
[67]Fallon JK, Harbourt DE, Maleki SH, et al. Absolute quantification of human uridine-diphosphate glucuronosyl transferase (UGT) enzyme isoforms 1A1 and 1A6 by tandem LC-MS. Drug Metab Lett 2008;2(3):210-222.
[68]Zhang L, Zuo Z, Lin G Intestinal and hepatic glucuronidation of flavonoids. Mol Pharm 2007;4(6):833-845.
[69]Zhang L, Lin G, Zuo Z. Involvement of UDP-glucuronosyltransferases in the extensive liver and intestinal first-pass metabolism of flavonoid baicalein. Pharm Res 2007;24(1):81-89.
[70]Boersma MG, van der Woude H, Bogaards J, et al. Regioselectivity of phase II metabolism of luteolin and quercetin by UDP-glucuronosyl transferases. Chem Res Toxicol 2002;15(5):662-670.
[71]Day AJ, Mellon F, Barron D, et al. Human metabolism of dietary flavonoids: identification of plasma metabolites of quercetin. Free Radic Res 2001;35(6): 941-952.
[72]de Pascual-Teresa S, Johnston KL, DuPont MS, et al. Quercetin metabolites downregulate cyclooxygenase-2 transcription in human lymphocytes ex vivo but not in vivo. JNutr 2004;134(3):552-557.
[73]Li R, Wu Z, Zhang F, et al. Differentiation of three pairs-of aconite alkaloid isomers from Aconitum nagarum var. lasiandrum by electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom 2006;20(2): 157-170.
[74]Sun Y, Zhang HG, Shi XG, et al. [Study on metabolites on aconitine in rabbit urine]. Yao Xue Xue Bao 2002;37(10):781-783.
[75]Ameri A. The effects of Aconitum alkaloids on the central nervous system. Prog Neurobiol 1998;56(2):211-235.
[2]Lin LZ, He XG, Lindenmaier M, et al. LC-ESI-MS study of the flavonoid glycoside malonates of red clover (Trifolium pratense). J Agric Food Chem 2000;48(2):354-365.
[3]Lukas SE, Penetar D, Berko J, et al. An extract of the Chinese herbal root kudzu reduces alcohol drinking by heavy drinkers in a naturalistic setting. Alcohol Clin Exp Res 2005;29(5):756-762.
[4]Barnes S. Soy isoflavones--phytoestrogens and what else? J Nutr 2004;134 (5):1225S-1228S.
[5]Thomasset SC, Berry DP, Garcea G, et al. Dietary polyphenolic phytochemicals--promising cancer chemopreventive agents in humans? A review of their clinical properties. Int J Cancer 2007;120(3):451-458.
[6]Zubik L, Meydani M. Bioavailability of soybean isoflavones from aglycone and glucoside forms in American women. Am J Clin Nutr 2003;77(6): 1459-1465.
[7]Hu M. Commentary:bioavailability of flavonoids and polyphenols:call to arms. Mol Pharm 2007;4(6):803-806.
[8]Benavente-Garcia O, Castillo J. Update on uses and properties of citrus flavonoids:new findings in anticancer, cardiovascular, and anti-inflammatory activity. J Agric Food Chem 2008;56(15):6185-6205.
[9]Wang X, Morris ME. Pharmacokinetics and bioavailability of the flavonoid 7,8-benzoflavone in rats. JPharm Sci 2008;97(10):4546-4556.
[10]Walle T, Otake Y, Brubaker JA, et al. Disposition and metabolism of the flavonoid chrysin in normal volunteers. Br J Clin Pharmacol 2001;51(2):143-146.
[11]Liu X, Tam VH, Hu M. 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):873-882.
[12]Boutin JA, Meunier F, Lambert PH, et al. In vivo and in vitro glucuronidation of the flavonoid diosmetin in rats. Drug Metab Dispos 1993;21(6):1157-1166.
[13]Ross JA, Kasum CM. Dietary flavonoids:bioavailability, metabolic effects, and safety. Annu Rev Nutr 2002;22:19-34.
[14]de Vries JH, Hollman PC, Meyboom S, et al. Plasma concentrations and urinary excretion of the antioxidant flavonols quercetin and kaempferol as biomarkers for dietary intake. Am J Clin Nutr 1998;68(1):60-65.
[15]Moon JH, Nakata R, Oshima S, et al. Accumulation of quercetin conjugates in blood plasma after the short-term ingestion of onion by women. Am J Physiol Regul Integr Comp Physiol 2000;279(2):R461-467.
[16]Liu Y, Dai Y, Xun L, et al. Enteric disposition and recycling of flavonoids and ginkgo flavonoids. J Altern Complement Med 2003;9(5):631-640.
[17]Baker JR, Satarug S, Edwards RJ, et al. Potential for early involvement of CYP isoforms in aspects of human cadmium toxicity. Toxicol Lett 2003;137 (1-2):85-93.
[18]Guengerich FP. Update information on human P450s. Drug Metab Rev 2002;34(1-2):7-15.
[19]Guengerich FP, Rendic S. Update information on drug metabolism systems-2009, part Ⅰ. Curr Drug Metab;11(1):1-3.
[20]Rendic S, Guengerich FP. Update Information on Drug Metabolism Systems-2009, Part Ⅱ:Summary of Information on the Effects of Desases and Environmental Factors on Human Cytochrome P450 (CYP) Enzymes and Transporters. Curr Drug Metab.
[21]Ingelman-Sundberg M. Pharmacogenetics of cytochrome P450 and its applications in drug therapy:the past, present and future. Trends Pharmacol Sci 2004;25(4):193-200.
[22]Le Marchand L, Franke AA, Custer L, et al. Lifestyle and nutritional correlates of cytochrome CYP1A2 activity:inverse associations with plasma lutein and alpha-tocopherol. Pharmacogenetics 1997;7(1):11-19.
[23]Yao D, Ding S, Burchell B, et al. Detoxication of vinca alkaloids by human P450 CYP3A4-mediated metabolism:implications for the development of drug resistance. JPharmacol Exp Ther 2000;294(1):387-395.
[24]Huang Z, Roy P, Waxman DJ. Role of human liver microsomal CYP3A4 and CYP2B6 in catalyzing N-dechloroethylation of cyclophosphamide and ifosfamide. Biochem Pharmacol 2000;59(8):961-972.
[25]Ekhart C, Doodeman VD, Rodenhuis S, et al. Influence of polymorphisms of drug metabolizing enzymes (CYP2B6, CYP2C9, CYP2C19, CYP3A4, CYP3A5, GSTA1, GSTP1, ALDH1A1 and ALDH3A1) on the pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide. Pharmacogenet Genomics 2008;18(6):515-523.
[26]Taniguchi R, Kumai T, Matsumoto N, et al. Utilization of human liver microsomes to explain individual differences in paclitaxel metabolism by CYP2C8 and CYP3A4. J Pharmacol Sci 2005;97(1):83-90.
[27]Santos A, Zanetta S, Cresteil T, et al. Metabolism of irinotecan (CPT-11) by CYP3A4 and CYP3A5 in humans. Clin Cancer Res 2000;6(5):2012-2020.
[28]Nakamura H, Nakasa H, Ishii I, et al. Effects of endogenous steroids on CYP3A4-mediated drug metabolism by human liver microsomes. Drug Metab Dispos 2002;30(5):534-540.
[29]Lee SJ, Kim WY, Kim H, et al. Identification of new CYP2C19 variants exhibiting decreased enzyme activity in the metabolism of S-mephenytoin and omeprazole. Drug Metab Dispos 2009;37(11):2262-2269.
[30]Fu M, Wu M, Wang JF, et al. Disruption of the intracellular Ca2+ homeostasis in the cardiac excitation-contraction coupling is a crucial mechanism of arrhythmic toxicity in aconitine-induced cardiomyocytes. Biochem Biophys Res Commun 2007;354(4):929-936.
[31]Xiao K, Wang L, Liu Y, et al. Study of aconitine toxicity in rat embryos in vitro. Birth Defects Res B Dev Reprod Toxicol 2007;80(3):208-212.
[32]艾路,孙莹,张宏桂.复方中药中乌头生物碱在人体内的代谢产物.北京中医药大学学报 2007；6(30)：955-958.
[33]张宏桂,史向国,孙莹,et al.兔血液中乌头碱代谢产物的研究.吉林大学学报(理学版)2006(02).
[34]Kaneko R, Hattori S, Furuta S, et al. Sensitive analysis of aconitine, hypaconitine, mesaconitine and jesaconitine in human body fluids and Aconitum tubers by LC/ESI-TOF-MS. J Mass Spectrom 2006;41(6):810-814.
[35]Lu G, Dong Z, Wang Q, et al. Toxicity Assessment of Nine Types of Decoction Pieces from the Daughter Root of Aconitum carmichaeli (Fuzi) Based on the Chemical Analysis of their Diester Diterpenoid Alkaloids. Planta Med 2009.
[36]Wang Y, Wang S, Liu Y, et al. Characterization of metabolites and cytochrome P450 isoforms involved in the microsomal metabolism of aconitine. J Chromatogr B Analyt Technol Biomed Life Sci 2006;844(2):292-300.
[37]Lasser KE, Allen PD, Woolhandler SJ, et al. Timing of new black box warnings and withdrawals for prescription medications. JAMA 2002;287(17): 2215-2220.
[38]Piscitelli SC, Burstein AH, Chaitt D, et al. Indinavir concentrations and St John's wort. Lancet 2000;355(9203):547-548.
[39]Bone KM. Potential interaction of Ginkgo biloba leaf with antiplatelet or anticoagulant drugs:what is the evidence? Mol Nutr Food Res 2008;52(7): 764-771.
[40]Williamson EM. Interactions between herbal and conventional medicines. Expert Opin Drug Saf 2005;4(2):355-378.
[41]Li P, Callery PS, Gan LS, et al. Esterase inhibition by grapefruit juice flavonoids leading to a new drug interaction. Drug Metab Dispos 2007;35(7): 1203-1208.
[42]Birt DF, Hendrich S, Wang W. Dietary agents in cancer prevention:flavonoids and isoflavonoids. Pharmacol Ther 2001;90(2-3):157-177.
[43]Wang SW, Chen J, Jia X, 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):1837-1848.
[44]Wong YC, Zhang L, Lin G, et al. Intestinal first-pass glucuronidation activities of selected dihydroxyflavones. Int J Pharm 2009;366(1-2):14-20.
[45]Zhang L, Lin G, Zuo Z. Position preference on glucuronidation of mono-hydroxylflavones in human intestine. Life Sci 2006;78(24):2772-2780.
[46]Tang L, Singh R, Liu Z, et al. Structure and Concentration Changes Affect Characterization of UGT Isoform-Specific Metabolism of Isoflavones. Mol Pharm 2009.
[47]Lee HS, Ji HY, Park EJ, et al. In vitro metabolism of eupatilin by multiple cytochrome P450 and UDP-glucuronosyltransferase enzymes. Xenobiotica 2007;37(8):803-817.
[48]Tang L, Ye L, Singh R, et al. Use of Glucuronidation Fingerprinting to Describe and Predict mono-and di-Hydroxyflavone Metabolism by Recombinant UGT Isoforms and Human Intestinal and Liver Microsomes. Mol Pharm.
[49]Hu M, Chen J. Nucleobase-and p-glycoprotein-mediated transport of AG337 in a Caco-2 cell culture model. Mol Pharm 2004;1(3):194-200.
[50]Liu Y, Hu M. Absorption and metabolism of flavonoids in the caco-2 cell culture model and a perused rat intestinal model. Drug Metab Dispos 2002;30(4):370-377.
[51]Hutzler JM, Tracy TS. Atypical kinetic profiles in drug metabolism reactions. Drug Metab Dispos 2002;30(4):355-362.
[52]Houston JB, Kenworthy KE. In vitro-in vivo scaling of CYP kinetic-data not consistent with the classical Michaelis-Menten model. Drug Metab Dispos 2000;28(3):246-254.
[53]D Argenio DZ, Schumitzky A. Biomedical simutations resource;University of Southern California:Los Angeles 1997.
[54]Yamaoka K, Nakagawa T, Uno T. Application of Akaike's information criterion (AIC) in the evaluation of linear pharmacokinetic equations. J Pharmacokinet Biopharm 1978;6(2):165-175.
[55]Prasain JK, Barnes S. Metabolism and bioavailability of flavonoids in chemoprevention:current analytical strategies and future prospectus. Mol Pharm 2007;4(6):846-864.
[56]Joseph TB, Wang SW, Liu X, et al. Disposition of flavonoids via enteric recycling:enzyme stability affects characterization of prunetin glucuronidation across species, organs, and UGT isoforms. Mol Pharm 2007;4(6):883-894.
[57]Huang SM, Strong JM, Zhang L, et al. New era in drug interaction evaluation: US Food and Drug Administration update on CYP enzymes, transporters, and the guidance process. J Clin Pharmacol 2008;48(6):662-670.
[58]Murray M. Role of CYP pharmacogenetics and drug-drug interactions in the efficacy and safety of atypical and other antipsychotic agents. J Pharm Pharmacol 2006;58(7):871-885.
[59]Singh SS. Preclinical pharmacokinetics:an approach towards safer and efficacious drugs. Curr Drug Metab 2006;7(2):165-182.
[60]Doerge DR, Chang HC, Churchwell MI, et al. Analysis of soy isoflavone conjugation in vitro and in human blood using liquid chromatography-mass spectrometry. Drug Metab Dispos 2000;28(3):298-307.
[61]Frank D, Jaehde U, Fuhr U. Evaluation of probe drugs and pharmacokinetic metrics for CYP2D6 phenotyping. Eur J Clin Pharmacol 2007;63(4):321-333.
[62]Mackenzie PI, Rogers A, Treloar J, et al. Identification of UDP glycosyltransferase 3A1 as a UDP N-acetylglucosaminyltransferase. J Biol Chem 2008;283(52):36205-36210.
[63]Ohno S, Nakajin S. Determination of mRNA expression of human UDP-glucuronosyltransferases and application for localization in various human tissues by real-time reverse transcriptase-polymerase chain reaction. Drug Metab Dispos 2009;37(1):32-40.
[64]Chen J, Lin H, Hu M. Absorption and metabolism of genistein and its five isoflavone analogs in the human intestinal Caco-2 model. Cancer Chemother Pharmacol 2005;55(2):159-169.
[65]Gibson DT, Hensley M, Yoshioka H, et al. Formation of (+)-cis-2,3-dihydroxy-1-methylcyclohexa-4,6-diene from toluene by Pseudomonas putida. Biochemistry 1970;9(7):1626-1630.
[66]Nakamura A, Nakajima M, Yamanaka H, et al. Expression of UGT1A and UGT2B mRNA in human normal tissues and various cell lines. Drug Metab Dispos 2008;36(8):1461-1464.
[67]Fallon JK, Harbourt DE, Maleki SH, et al. Absolute quantification of human uridine-diphosphate glucuronosyl transferase (UGT) enzyme isoforms 1A1 and 1A6 by tandem LC-MS. Drug Metab Lett 2008;2(3):210-222.
[68]Zhang L, Zuo Z, Lin G Intestinal and hepatic glucuronidation of flavonoids. Mol Pharm 2007;4(6):833-845.
[69]Zhang L, Lin G, Zuo Z. Involvement of UDP-glucuronosyltransferases in the extensive liver and intestinal first-pass metabolism of flavonoid baicalein. Pharm Res 2007;24(1):81-89.
[70]Boersma MG, van der Woude H, Bogaards J, et al. Regioselectivity of phase II metabolism of luteolin and quercetin by UDP-glucuronosyl transferases. Chem Res Toxicol 2002;15(5):662-670.
[71]Day AJ, Mellon F, Barron D, et al. Human metabolism of dietary flavonoids: identification of plasma metabolites of quercetin. Free Radic Res 2001;35(6): 941-952.
[72]de Pascual-Teresa S, Johnston KL, DuPont MS, et al. Quercetin metabolites downregulate cyclooxygenase-2 transcription in human lymphocytes ex vivo but not in vivo. JNutr 2004;134(3):552-557.
[73]Li R, Wu Z, Zhang F, et al. Differentiation of three pairs-of aconite alkaloid isomers from Aconitum nagarum var. lasiandrum by electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom 2006;20(2): 157-170.
[74]Sun Y, Zhang HG, Shi XG, et al. [Study on metabolites on aconitine in rabbit urine]. Yao Xue Xue Bao 2002;37(10):781-783.
[75]Ameri A. The effects of Aconitum alkaloids on the central nervous system. Prog Neurobiol 1998;56(2):211-235.