【作者】 彭游；

【导师】 邓泽元；

【作者基本信息】 南昌大学 ， 食品科学， 2009， 博士

【摘要】 沙棘、葛根和大豆中含有的黄酮类物质:槲皮素、大豆苷元和染料木素等具有抗氧化活性、心血管保护作用、抗癌、抗炎作用、抗溃疡作用、抗过敏作用、胃保护作用、抗血小板作用及雌激素等多种药理功能,但这些化合物含有多个极性基团羟基,亲脂性弱,同时由于羟基在分子间形成氢键,晶格能较高,亲水性也较差（槲皮素在水中溶解度为7ug/mL）,进入体内迅速被吸收并代谢,与糖分子结合转化为葡萄糖苷酸等而失活,存在强烈的首过效应,生物利用度极低,如槲皮素的生物利用度小于3.6%,大豆苷元约为12%,染料木素约为30%,导致这些物质生物活性低,至今难以在临床上作为药物广泛使用。本论文主要进行了沙棘、大豆、葛根中黄酮的微波干法辅助提取及机理研究,类黄酮的前药分子设计与合成,前药的大鼠体内药物动力学试验,以及部分黄酮化学修饰物的活性测试与构效关系研究。植物中黄酮的提取主要采用常规加热回流溶剂提取法,经济与环境成本均很高,本论文尝试建立了微波光波组合法用于辅助提取沙棘、葛根、大豆中的黄酮,并初步阐述了微波提取黄酮的机理。研究表明,原料粉碎至0.022～0.056mm,加入9.4%的DMF调匀,通过微波光波组合方式加热,功率为800W（微波55%与光波45%）,加热8min或6min后,用200mL乙醇分两次萃取总黄酮,纯化后提取率与常规方法提取相当。为了优化槲皮素、大豆苷元和染料木素的药物动力学性质,阻断首过效应以提高口服生物利用度和药效,以槲皮素、大豆苷元和染料木素为先导化合物设计并成功合成了未见文献报道的黄酮苯磺酸酯三个系列共17个化合物,分别是:槲皮素衍生物3个,大豆苷元和染料木素衍生物各7个。所有的新化合物均经¹HNMR、MS、IR及元素分析确证,并报道了其中三个衍生物:化合物1、11、13的晶体结构。晶体结构表明:每个化合物的中S均处于不规则四面体的中间。化合物1,11分子内存在π-πstacking作用,化合物11,13分别通过分子间氢键而稳定。建立了类黄酮苯磺酸酯的前药研究方法并研究了水解反应机理,主要包括溶解度、表观脂水分配系数LogP、水解动力学常数的测定。在对选定化合物1、12、13的前药研究中,发现三个化合物的脂溶性与水溶性均有显著的提高,化合物1、12、13的表观脂水分配系数LogP分别为2.04、3.57和1.97,符合“规则-5”。化合物1、12、13的水解反应符合假一级反应,水解速率常数分别为:k=5×10^-5s^-1、9×10^-5s^-1和1×10^-5s^-1,半衰期分别为:3.85、2.14、19.25h。化合物12的水解反应主要4′-酯键优先水解,生成化合物13,并可进一步水解成大豆苷元。根据前药研究结果结合计算机辅助药物设计软件ChemAxond的计算结果发现:类黄酮苯磺酸酯化修饰后,其影响药物动力学性质的一些理化特征得以优化,有可能有相对较好的过膜吸收与转运分布性以及良好的口服生物利用度。分别以染料木素或大豆苷元为内标,建立了测定血浆中游离大豆苷元及其前药,或染料木素及其前药的液相色谱分析方法,并进行了完整的方法确证。其条件为:色谱柱为Diamonsil C18柱（200×4.6mm,LD.,5μm）,流动相为CH₃OH:甲酸溶液（0.1%）（1:1,v/v）,流速为0.5 mL/min,进样量为10μL,检测波长λ=248nm或262nm,柱温25℃。该方法灵敏、准确,选择性强。被测药物的日内和日间精密度（RSD）均小于14%,准确度（RE）在士13%范围内,大豆苷元及其前药和染料木素及其前药在大鼠血浆中的提取回收率均高于66.1%,只需50μL血浆即可满足大豆苷元及其前药或染料木素及其前药血药浓度的测定要求。建立了类黄酮苯磺酸酯衍生物的前药筛选与药物动力学方法。经大鼠体内代谢表明,合成的17个新的黄酮类苯磺酸酯衍生物有7个为类黄酮的前药。大豆苷元前药12、13、15的相对生物利用度分别为:42.9%、21.5%和26.8%;染料木素前药6、7、9、10的相对生物利用度分别为:198.6%、110.9%、159.2%和253.8%,均超过100%。说明通过对染料木素羟基的结构修饰来优化药物动力学性质、阻断首过代谢从而提高口服生物利用度的思路得到证实。选择部分大豆苷元修饰物12,13进行体外抑制血管平滑肌细胞增殖实验和抗肿瘤筛选,根据实验结果并结合计算机辅助药物设计软件ChemAxond进行初步的构效关系研究。体外活性测试表明:大豆苷元化学修饰物12、13的抗HL-60的活性明显增强,达1个数量级。大豆苷元化学修饰物12、13的抑制血管平滑肌细胞（VSMC）增殖活性增强分别达100和10³倍,化合物13在10^-7mol/L仍有56.06%的抑制率。从对先导化合物大豆苷元的结构优化角度来看,可能是苯磺酸酯基的引入优化了空间结构、增加了分子的可极化率和变形性,改变了药物的电荷分布,有利于药物更有效的与靶标的识别作用而导致药物药效性质的根本性改变。表明大豆苷元经苯磺酸酯化后结构得以优化,其苯磺酸酯衍生物的药理活性需要进一步研究。更多还原

【Abstract】 Flavonoids,quercetin,daidzein and genistein,are a class of naturally occurring polyphenolic compounds that have been isolated from various vascular plants such as hippophae rhamnoides L.,kudzuvine root and soybean et al.Despite their in vitro biological activity,such as antioxidant,antiinflammatory,antiviral,antiproliferative, and anticarcinogenic effects,quercetin,daidzein and genistein have not been employed widely in therapeutic medicine as drugs because their poor lipophilicity, hydrophilicity and their first pass effect,which result from their polar hydroxyls in molecules,will result in weak bioavailability（quercetin 3.6%,daidzein 12%,genistein 30%）.In this paper,we focus on investigating the microwave radiation extraction method and extraction mechanism of three flavones,design and synthesis of prodrugs of flavones and their pharmacokinetics,some derivatives’ activities.The microwave radiation extraction method of three flavones（quercetin,daidzein and genistein） from three plants,hippophae rhamnoides L.,kudzuvine root and soybean respectively were investigated,and microwave radiation extraction mechanism were also studied systematically with IR,FM etc.The sample was comminuted to powder of 0.022～0.056mm,9.4%DMF were added into sample and put it into microwave oven.The sample was heated up with microwave and light wave combination（55:45） by 6min or 8 min durativly.Flavones were extracted twice by 200mL ethanol.After recycling ethanol,flavones were obtained.To optimize pharmacokinetics of three flavones which resulted in increasing their oral bioavailability,the seventeen new sulfonic acid esters of three flavones were designed according to principles of prodrugs and synthesised in high yield with excellent regioselectivity.Among them there are 3 quercetin derivatives,7 daidzein derivatives and 7 genistein derivatives respectively.Their structures were characterized by IR,MS,elemental analysis and ¹H NMR spectra.The crystal structures of compound 1,11and13 were reported respectively.As expected,each S atom locates at the center of the tetrahedral geometry.In the crystal structure of 11 and 13,molecules are linked through intermolecular hydrogen bonds.In addition, relatively shorter centroid distances among the rings are observed,implying the existence ofπ-πstacking interactions in the compounds 1 and 11.The prodrug studies method of benzene sulfonic acid esters was set up and reaction pathway of hydrolysis was also investigated.It contains mensuration of solubility,partition coefficients and hydrolysis constants of quercetin,daidzein and their analogs 1,12 and 13.The solubility of the prodrugs increased in all examined solvents compared with their technicalmaterial.The apparent lipid/water partition coefficients of compound 1,12 and 13 are 2.04,3.57 and 1.97 respectively.All prodrugs hydrolysis are modeled as a pseudo first-order reaction under constant conditions of pH and temperature,and their hydrolysis constants are 5×10^-5s^-1, 9×10^-5s^-1and 1×10^-5s^-1 respectively.Their half lives T_1/2 are 3.85,2.14 and 19.25h, respectively.The 4’-OH in compound 12 was hydrolysised firistly to obtain compound 13,and farther to get daidzein.The experiment results and counts of ChemAxon of these compounds indicate the oral bioavailabilities are improved comparing with their technicalmaterials.An HPLC procedure was developed and validated for the determination of free daidzein（genistein） and its prodrugs in biological samples with genistein（daidzein） as the internal standard.The analyte and internal standard （genistein or daidzein） were extracted from plasma samples by CH₃COOC₂H₅,and chromatographed on a Diamonsil Cog column（200×4.6 mm,I.D.,5μm） with the mobile phase consisting of water:formic acid（1:1,v/v） at a flow rate of 0.5 mL/min. The intra-and inter-day precision in terms of RSD were both under 14%,and the accuracy in terms of RE ranged from-10%to 13%.The extraction recoveries of daidzein,genistein and their prodrugs all exceed 66.1%.Prodrug screening and prodrug pharmacokinetics experiments were operated using Wistar rats.Among 17 sulfonic acid esters there are 7 prodrugs of these flavones.The pharmacokinetics of these prodrugs were systematically investigated. The relative bioavailabilities of prodrug12,13 and 15 compared with daidzein at the same molar dose were calculated to be 42.9%,21.5%and 26.8%,respectively;and the relative bioavailabilities of prodrug6,7,9 and 10 compared with genistein at the same molar dose were calculated to be 198.6%,110.9%,159.2%and 253.8%, respectively.Higher relative bioavailabilities of prodrug10 and prodrug 7 compared with genistein indicated great improvements of their pharmacokinetic behaviors after structural modifications.Prodrug 10 and 7 were new drug candidates of genistein with better oral bioavailability.The anticancer and inhibiting the viability of vascular smooth muscle cells activities of some sulfonic acid ester derivatives of daidzein were screened in vitro, their structure-activity relationship were investigated by results and counts of computer-assisted drug design software ChemAxon.Compounds 12 and 13 showed better anti-HL-60 activities than daidzein（P＜0.01）.Conpound 12 and 13 showed better inhibiting the viability of vascular smooth muscle cells activities than daidzein. The compound 12 shows about 100 times inhibiting activity than daidzein and compound 13 is 10³ times.Inhibitory rate of the compound 13 is 56.06%at 10^-7mol/L in vitro.更多还原