【作者】 谢升谷；

【导师】 曾苏；

【作者基本信息】 浙江大学 ， 药物分析学， 2007， 硕士

【摘要】 黄酮是一类小分子多酚化合物,广泛存在于植物源食物中,如水果、蔬菜、豆类、谷类、药用植物、香科、花、茶以及红酒当中,具有相当丰富的含量。黄酮的化学母核结构,为两个具有取代基团的苯环通过中间三个碳原子连接而成的一系列化合物,分为A、B、C三个环。环上不同的取代基导致了不同的药理与药物代谢的活性。根据其C环的不同,可以分为:（异）黄酮类、黄酮醇类、二氢（异）黄酮类、查耳酮类、花色素类、黄烷类等。到2002年底,已鉴定化学结构的黄酮大约有8000多个。作为一种食物源黄酮,不同区域与生活习惯的人们每天摄入黄酮量约为50-1000mg不等。现代研究表明,该类化学物质具有十分广泛的药理作用,尤其对心血管系统和抗肿瘤方面具有明显的活性。科学家逐渐认识到:蔬菜、水果、中草药中的植物黄酮对人体健康有着无可替代的重要作用。黄酮作为一类多酚类化合物,具有多个羟基,易发生Ⅱ相代谢结合反应,其在体内的葡醛酸化是主要的代谢途径之一。染料木素、芹菜素、槲皮素、山奈酚、黄芩素等黄酮在大鼠或人尿液中均发现有葡醛酸苷结合物。大多数黄酮在生物体内都易发生葡醛酸化,因此研究其代谢途径及其与重组UGTs的结构-代谢关系对于生物黄酮的合理利用和此类药物的合理设计具有十分重要的意义。鉴于UGT在黄酮化合物代谢中所起的重要作用,我们采用人重组UGTs进行以下三个方面的研究。一、芹菜素UGTs体外代谢以及主要代谢物活性鉴定有文献报道芹菜素在大鼠体内外的主要代谢途径之一为葡醛酸化。本节实验通过五种UGT重组酶与芹菜素的代谢作用研究,认为代谢物M1主要由UGT1A6介导催化,代谢物M2主要由UGT1A3和UGT1A9催化,UGT1A6和UGT2B7也起着较弱的作用,代谢物M3主要由UGT2B7介导催化,其中UGT1A4几乎不起催化作用。UGT1A3.1,UGT1A3.4和UGT1A3.5对代谢物M2的催化作用显著高于另外二种UGTA3变异体（UGT1A3.2和UGT1A3.3）。芹菜素代谢活性主要由β-葡醛酸水解酶、HPLC-MS和化学诊断试剂结合UV数据进行确认。根据以上数据,鉴定代谢物M1为芹菜素-5-葡醛酸苷,M2为芹菜素-7-葡醛酸苷,M3为芹菜素-4′-葡醛酸苷。不同实验动物与人肝微粒体的代谢结果表明,在四个种属的肝微粒体当中,芹菜素与兔肝微粒体具有最高的亲和力(Km=36.31μmol·L^-1),对犬肝微粒体具有最低的亲和力(Km=120.85μmol·L^-1)。不同种属的芹菜素的代谢速率（Vmax/Km）大小分别为:兔＞犬＞大鼠＞人。抗氧化实验表明,代谢物M2比底物具有更好的抗氧化能力。而Bac-to-Bac系统表达的UGT重组酶对不同有机溶剂与其它表达系统相比具有更好的耐受性。结合文献数据认为,UGT1A6对黄酮的化学结构具有十分严格的选择性,只有同时具备5-,7-,4′-羟基,且在3-位羟基的黄酮才易被其催化。二、UGT1A3和UGTA9催化黄酮结构-代谢关系研究在人肝中表达的UGT1A家族同工酶主要有:UGT1A1、UGT1A3、UGT1A4、UGT1A6和UGT1A9。现有的文献表明,UGT1A1、UGT1A3和UGT1A9主要催化黄酮化合物,而UGT1A4主要催化胺类,UGT1A6主要催化平面酚类化合物。本节考察了UGT1A3和UGT1A9与黄酮化合物的效构关系。结果表明,具有7位羟基的黄酮是UGT1A3、UGT1A9的良好底物,B环的位置和糖基对其活性具有显著的影响,而其它的化学结构也对活性有较小的影响。尽管UGT1A3和UGT1A9的底物库各自交叉重叠,但又互不相同,呈无规律性,但其黄酮类底物在本实验的研究中呈现良好的一致性,除了槲皮素-3-O-呋喃阿拉伯糖苷。经酶动力学参数的定量研究以及western-blot分析,可以看出UGT1A9与UGT1A3相比,对于黄酮具有更高的代谢活性。这二种UGT对黄酮高效的代谢活性,可能提示了其在体内的重要作用。三、UGT1A3催化黄酮化合物2D-QSMR研究由于定量结构-代谢关系在药理活性方面研究甚广,而在药物代谢领域主要集中于CYP的研究,如CYP同工酶的底物选择性与抑制剂特异性研究均取得了较大的进展。相对于CYP在代谢预测和底物特异性描述方面的发展,UGT则相对落后。通过建立人UGT1A3催化黄酮类化合物定量结构-代谢关系,考察不同结构参数对其葡醛酸结合反应的影响。方法为采用半经验量子化学计算法MOPAC-AM1及逐步回归方法对11个具有类似结构黄酮化合物的结构参数和UGT1A3催化葡醛酸反应的Km值进行定量结构-代谢关系研究。所建立的QSMR模型(pKm=-2.20707+0.00256HOF+3.21290 Q_C5)具有较好的拟合性。由定量结构-代谢关系结果可知,分子生成热（HOF）和5位C上的静电荷二个结构参数是影响其葡醛酸结合活性的主要结构因素。更多还原

【Abstract】 Flavonoids are a broad class of low molecular weight,widely distributed in fruits, vegetables,nuts,seeds,herbs,spices,stems,flowers,as well as tea and red wine. Flavonoids are benzo-γ-pyrone derivatives with phenolic and pyrane rings and are classified according to substitutions.The chemical structure of flavonoids has three phenolic rings referred to as A,B,and C rings.The biochemical activities and metabolism of flavonoids depend on their chemical structures and the relative orientation of various moieties on the molecule.Flavonoids are classified according to their chemical structures.The major flavonoid classes include flavonols,flavones, flavanones,flavanols,anthocyanidins,isoflavones,dihydroflavonols,and chalcones. Over 8000 flavonoids have been identified to 2002.And flavonoids are an integral part of the human diet.The amount of daily flavonoids consumed would be about 50-1000 mg per day according to different region and living habits.The flavonoid natural products exert a wide range of biochemical and pharmacological properties,in particular cardiovascular disease and cancer.As the further research,we know that flavonoids in our daily diet play an important role in human health and disease protection.As a class of plant phenols phenolics,flavonoids have more than one free hydroxyl group,are easy to be conjugated with UDPGA.Conjugation reactions with glucuronic acid seem to be the most common type of metabolic pathways for the flavonoids.In particular,there are now strong evidences for extensive phaseⅡmetabolism of the aglycones such as genistein,apigenin,quercetin,kaempferol and baicalein to glucuronides in rats or human urine.Glucuronidation of flavonoids via UGTs may be the most common metabolic pathways,and studies on flavonoids glucuronidation and structure-metabolism relationships are meaningful,do a great favor to bioflavonoids development and rational drug design.For the importance of UGTs in flavonoids glucuronidation,we try to elucidate the UGTs role in flavonoids metabolism and structure-metabolism relationships between UGTs and flavonoids using human recombinant UGTs.1.In vitro glucuronidation of apigenin by human liver microsomes and its main metabolite bioactivityIn the previous study,potential metabolic pathways of apigenin in rats were found. Incubation in the presence of human recombinant UGTs demonstrated that M1 is almost exclusively catalyzed by UGT1A6,M2 is formed by UGT1A3,UGT1A9 and only to a minor extent by UGT1A6 and UGT2B7,whereas M3 is mainly produced by UGT2B7,while UGT1A4 have no contribution to apigenin glucuronidation. UGT1A3.1,UGT1A3.4 And UGT1A3.5 had much higher glucuronidation efficiency to the 7-hydroxyl group of apigenin than other two UGT1A3 variants（UGT1A3.2 and UGT1A3.3）.Apigenin glucuronides were isolated from a reaction mixture consisting of apigenin and UGTs fortified with UDPGA and identified by hydrolysis reaction withβ-glucuronidase,HPLC-MS and UV analysis.On the basis of these results,M1 was identified as 5-glucuronide apigenin,M2 as 7-glucuronide apigenin and M3 as 4’-glucuronide apigenin.There were species differences in glucuronidation of apigenin with liver microsomes obtained from human,rabbit,rat and dog.Kinetic analysis indicated apigenin had the highest affinity for the rabbit liver microsomal enzyme(Km=36.31μmol·L^-1)and the lowest affinity for the dog liver mcirosomal enzyme(Km=120.85μmol·L^-1)in four species.The glucuronidation metabolic activity （Vmax/Km）of apigenin was in the following order:rabbit＞dog＞rat＞human.The results showed that 7-glucuronide apigenin had a stronger antioxidant bioactivity than apigenin assayed by MDA method.The recombinant UGTs expressed by Bac-to-Bac system showed different organic solvent inhibitions compared with UGTS expressed other cell lines.According to flavonoids UGT1A6 substrate reported,we concluded that 5-,7-,4’-position hydroxyl groups,without 3-hydroxyl group may be the essential chemical elements.2.Glucuronidation of flavonoids by human UGT1A3,UGT1A9 and structure-metabolism relationshipsA recent study has suggested that only UGT1A1,1A3,1A4,1A6 and 1A9 are expressed in human liver of the UGT1 gene complex.Previously,we consider that UGT1A1,1A3,and 1A9 are the main enzymes catalyzing the glucuronidation of flavonoids in human liver while human UGT1A4 mainly catalyze amines and UGT1A6 preferentially the glucuronidation of planar phenols.This study proposes features of the flavonoid structure necessary to confer it as a substrate for human UGT1A3 and UGT1A9.The preferred substrates for UGT1A3 and UGT1A9 contain the hydroxyl group at C7-position.The glycon and the position of B ring have conspicuous influences on the glucuronidation activity,and other chemical structures of flavonoids have minor affects.Generally speaking,the substrates for UGT1A3 and UGT1A9 are different,the result shows that the two UGTs have mostly overlapped flavonoids substrates,except quercetin-3-O-arabinoside.From the quantitative study, UGT1A9 in general has higher glucuronidation efficiency than UGT1A3.The high glucuronidation efficiency toward many flavonoids observed suggests that the contribution of UGTs in the metabolism of these flavonoids may be significant.3.QSMR studies of flavonoids and glucuronidation involved in human UGT1A3QSAR（quantitative structure-activity relationships）is wildly used in pharmacology and pharmaceutical chemistry.And in drug metabolism,QSMR is mainly focus on CYP450,such as,pharmacophore and 3D-QSMR models have been developed that can be used to infer the active site binding requirements of substrates and inhibitors for numerous CYP450 isoforms and that can predict apparent Km for substrates and apparent Ki for inhibitors.However,the development of models for predicting metabolism and for characterizing structural features of substrates for UGT isoforms is less advanced relative to CYP450.In order to explore the 2D-QSMR model for flavonoids substrates of human UGT1A3,the parameters which are closely related with the flavonoids activity.The relationship between flavonoid structures and values of Km for human UGT1A3 was studied for 11 flavonoids compounds by the calculation of quantum chemistry at MOPAC-AM1.Combining their calculated results with experimental data,a QSMR (pKm=-2.20707+0.00256HOF+3.21290 Q_C5)was obtained with stepwise regress. The results show the heat of formation（HOF）and Q_C5have conspicuous contributions to the values of Km for UGT1A3.更多还原