【作者】 梁健钦；

【导师】 邓家刚；

【作者基本信息】 广西医科大学 ， 药理学， 2011， 博士

【摘要】 1目的与意义芒果苷（Mangiferin,MGF）是一种具有Xanthone结构的葡萄糖C-糖苷（分子式为C19H18O11,分子量为422.34）,存在于漆树科植物芒果（Mangifera indica L.）的果实、叶、树皮,百合科植物知母（Anemarrhena asphodeloides Bge.）的根茎、地上部分,鸢尾科植物射干（Belamcanda chinensis（L.）DC.）的花、叶等植物中。MGF分子具有较大的平面结构,溶解度较差（在纯水及pH 1.3⁷.4的缓冲溶液中平均溶解度<1mg·mL-1）,而经换算人的口服有效剂量为630mg,溶解单剂量MGF需要的胃肠道的液体体积>250mL,根据生物药剂学分类系统（Biopharmaceutics classification system,BCS）的定义MGF属于一种溶解性差的药物。此外,MGF在正丁醇-水系统中分配系数（Oil/water partition coefficient,P）非常小（pH1⁴时,P值约为2.3,当pH值大于6.86时,P值仅为0.01）,通常P值与胃肠通透性相关性较好,P值在100¹000时,被动吸收较好,推测MGF胃肠通透性较差。再者,MGF在小鼠体内肠道吸收效果较差,证实了MGF通透性差。综合MGF上述三方面的性质,MGF的低油水分配系数可能与其在小鼠体内低生物利用度直接相关,按照生物药剂学分类系统（BCS）原则,MGF可归类为BCS第4类药物（低溶解性-低通透性）,此类药物一般存在严重的生物利用度问题。MGF具有丰富的药理作用,包括抗炎、抗氧化、抗肿瘤、免疫调节作用、抗糖尿病作用等。因此,课题组拟将MGF开发成为一种具有抗炎作用的新药,但研发过程中发现MGF存在严重的吸收问题。MGF在人体中的抗炎剂量为630mg/次,一日3次,作为一个纯度在90%以上的单体化合物,服用剂量显然很大。将MGF开发成新药,必须解决MGF低吸收的问题,如果提高了MGF吸收的生物利用度,药效提高,最终能调整MGF的给药量。目前,课题组已经开展提高MGF溶解性的研究（应用的技术包括成盐、磺酸盐、金属离子螯合物、制剂手段提高溶解度等）,而在提高MGF通透性方面的研究尚属空白。将低脂溶性药物制备成酯类化合物或前药,从而提高药物的油水分配系数,是提高该类药物胃肠通透性（即被动吸收）的最常用的前药设计策略。MGF具有黄酮类结构,还有多个酚羟基及糖基上的多个羟基,可以对这些羟基进行酰化修饰,增加药物分子整体脂溶性。采用化学法对黄酮类化合物进行酰化,空间选择性不高,并且将活性酚羟基酰化,导致抗氧化活性降低。本文利用脂肪酶的酯化反应或酯交换作用,可选择性将化合物酯化修饰,不但提高黄酮类化合物的脂溶性,还能提高其稳定性及抗氧化活性。脂肪酶（Lipase,EC 3.1.1.3）,即三酰基甘油酯水解酶,该酶催化天然底物油脂的水解,生成的产物包括脂肪酸、甘油和甘油单酯或二酯。脂肪酶被广泛用于酯、硫羟酸酯、酰胺类化合物、多元醇酯、多元酸酯三酰甘油酯及一些疏水性酯类的水解、醇解、酯化、转酯化及酯类的逆向合成反应。在非水有机溶剂中,脂肪酶Novozym 435对葡萄糖基6位羟基专属性很高,可以催化葡萄糖的单酰化。为此,本论文针对MGF低油水分配系数,从提高MGF通透性角度出发,采用酶法合成MGF酯类前药,尝试解决MGF生物利用度问题,达到提高药效的目的。理论上,还可为BCS 4类药物的研发提供思路。2方法和结果全文对MGF进行结构修饰,合成酯类前药,并对产物结构及相关理化性质进行测定,最后测定该前药的抗炎活性。方法与结果如下:（1）该部分对MGF溶解度及油水分配系数进行研究。采用HPLC法测定MGF在pH为1.32,2.52,3.32,4.50,5.00,6.86,7.40,8.01缓冲溶液中的平衡溶解度。结果显示MGF的溶解度随pH提高而增加。在pH 1.3⁵.0的溶解度约为0.16 mg·mL^-1;在pH 6.86⁷.4时溶解度增加至0.82 mg·mL^-1;当pH 8.01时溶解度为1.44 mg·mL^-1。采用摇瓶-HPLC法测定MGF在正辛醇-缓冲溶液（pH 1.32,2.52,3.32,4.50,5.00,6.86,7.40,8.01）体系中的表观油水分配系数（P）。MGF的油水分配系数随pH增加而降低,P值从2.313降低至0.05。根据生物药剂学分类系统（BCS）,推测MGF属于BCS 4类药物（低溶解度-低通透性药物）。提示,可以通过合成MGF脂溶性前药,提高脂溶性达到提高MGF通透性的目的。（2）本部分对MGF糖酯衍生物的非水相酶促合成的可行性进行研究。以MGF为酰基受体,以甲酸乙酯、乙酸乙酯、丙酸乙酯为酰基供体,利用商品脂肪酶Novozym 435为催化剂,在二氧六环中经过转酯化反应,合成了3种酰化的MGF糖酯衍生物,经HPLC-MS分析鉴定3种产物为甲酰基MGF、乙酰基MGF、丙酰基MGF。从HPLC图谱及TLC图谱分析,衍生物的脂溶性均明显提高,达到目标化合物预期目的。（3）本部分对6′-O-丙酰基芒果苷（PMGF）合成、分离、结构鉴定进行研究,并对合成条件进行优化。首先,以MGF为酰基受体,丙酸乙酯为酰基供体,利用脂肪酶在有机溶剂中的酯交换作用,在MGF糖基6′-OH选择性酰化。其次,采用制备液相色谱法分离得到高纯度PMGF。然后,测定产物的MS谱、一维¹H-NMR和¹³C-NMR确定分子量及分子结构,并测定（g）-HMBC及（g）-HMQC对C、H进行归属,确定酰化位置,最后确证产物结构为6′-O-丙酰基芒果苷。最后,优化酶促反应条件,优化后的酶促反应条件为:在8 mL二氧六环中加入30 mg Novozym 435、芒果苷30mg、丙酸乙酯1mL,在恒温水浴振荡摇床中60℃下反应20h。（4）对PMGF水溶解度、油水分配系数、体外水解稳定性进行考察,并与MGF做对比研究。首先,采用摇瓶法测定PMGF在纯水（37℃）的溶解度,结果显示PMGF溶解度为0.166±0.02μg·mL^-1,与同条件下MGF的溶解度接近,PMGF并未由于脂溶性提高而导致水溶解度下降。其次,采用HPLC法测定PMGF在正辛醇-缓冲溶液中的油水分配系数,结果显示PMGF在pH 1.3⁸.01范围内油水分配系数由21.104降低至0.108,比MGF均显著增加4.33⁹2.36倍,其中在中性及碱性环境下提高倍数均在10倍以上,尤其在模拟肠液的pH（pH7.4）环境中增加了93.36倍,预测PMGF在肠道通透性将有很大程度提高。然后,采用HPLC法测定PMGF在pH 2及7.4溶液中的水解稳定性。PMGF在该溶液中均能缓慢水解为MGF。PMGF在pH2的酸性溶液中,按照一级速度过程水解为MGF,半衰期为69.3h;在pH 7.4偏碱性溶液中,水解为MGF的比例及速度较慢,部分PMGF变成3种脂溶性高于MGF的未知产物（推测是丙酰基转移到其他羟基后形成的产物,脂溶性仍然比MGF有所提高）,导致PMGF浓度下降较快,半衰期为34.6h。最后,PMGF浓度在血浆中按照非线性速度过程水解变成MGF,半衰期为11.7²0.6h。综合上述内容,与原型MGF相比较,PMGF在不降低溶解度前提下脂溶性显著提高,在胃肠pH环境下能缓慢水解（水解半衰期>30h）为MGF,主要在血浆中水解为原型MGF。推测将MGF修饰为PMGF能显著提高MGF的胃肠通透性,在吸收的时间段内,PMGF比例较大,吸收入血后,能顺利水解还原为MGF,提示,PMGF很有潜力作为MGF的前药。（5）本部分研究了人肠道菌群对MGF和PMGF的代谢作用。采用离体代谢研究的方法,用富含人肠道菌群的培养液与MGF/PMGF在厌氧条件下共同孵育,采用大孔树脂、制备液相色谱法分离代谢产物,经HPLC-MS、一维¹H-NMR和¹³C-NMR确证代谢产物结构为MGF苷元（Norathyriol）。结果如下:PMGF在孵育开始0²h内全部水解为MGF,孵育第4⁸h时间段PMGF和MGF逐步代谢为Norathyriol,第7h的Norathyriol浓度达到最高,随后Norathyriol浓度持续下降,第12h仍然有明显代谢产物。证实,PMGF转化为MGF的主要部位是肠道,暴露在肠道菌群环境中仅需要2h就能将PMGF分解完全;MGF及PMGF在肠道菌群作用下转化为Norathyriol,时滞4h。在肠道菌群作用下,PMGF完全水解需要2h,推测在此水解阶段,未被水解的高脂溶性的PMGF持续吸收,净效应就是PMGF的吸收速度与程度会比原型MGF的要高。此外,代谢产物Norathyriol吸收的特征,有待进一步研究。（6）对比研究了PMGF的体内、体外抗炎活性。首先,利用小鼠巨噬细胞株RAW264.7在脂多糖刺激下产生炎性细胞因子,观察PMGF和MGF对炎性细胞因子生成的影响,结果显示低浓度(5μg?mL^-1)MGF及PMGF体外对NO、IL^-1、TNF-α无显著抑制作用（抑制率<7%）。其次,小鼠二甲苯致耳肿胀试验表明,MGF和PMGF均显示显著的抗炎作用（抑制率>30%,P<0.05）,PMGF中、低剂量组的抗炎效果高于MGF中、低剂量组但无显著性差异（P>0.05）,提示,PMGF中、低剂量组药效具有高于同剂量的MGF的趋势,出现以上现象,可能由于提高MGF脂溶性改善膜通透性,增加了MGF经小鼠胃肠道的生物利用度,达到增效的目的。3结论针对MGF溶解度低且油水分配系数低的特性,围绕MGF结构修饰主题,通过酶催化反应,最终找到高活性的高脂溶性的MGF前药—PMGF,PMGF的溶解度与原型MGF相当,但PMGF脂溶性显著提高,抗炎药效有优于MGF的趋势,有理由认为PMGF作为MGF前药是有效的。以上研究,为MGF前药的研究提供了新的化合物结构类型,为有目的的开发MGF新药奠定了基础。本文对难溶性药物的开发研究亦有一定的指导意义。本文从通透性方面入手,提高MGF脂溶性,动物药效证明酯类前药PMGF抗炎效果有优于MGF的趋势。可见,从通透性方面改善BCS 4类药物的生物利用度是行之有效的。4创新点（1）首次利用固定化脂肪酶Novozym 435在非水相中合成了3种MGF脂溶性衍生物甲酰基MGF、乙酰基MGF、丙酰基MGF。（2）提高MGF脂溶性,动物实验证实达到增效的目的。（3）首次将BCS指导原则应用到MGF研究中,以“增加脂溶性来改善膜通透性”为指导思想,指导整个MGF前药的设计,并取得良好的效果。更多还原

【Abstract】 1 Purpose and significanceMangiferin [2-C-d-gluco-pyranosyl-1, 3, 6, 7-tetrahydroxyxantone; C₁₉H₁₈O₁₁; Mw, 422.34] is a kind of active flavonoids, a xanthone C-glycoside, and has been reported in various parts of Mangifera indica: fruits, leaves, stem bark; Anemarrhena asphodeloides Bge: leaves, stem bark, roots; Belamcanda chinensis: flower, leaves.Mangiferin （MGF） has poor solubility (<1 mg·mL^-1) over the pH range 1.3 to 7.4, due to the large plane molecular structure. The dose of anti-inflammatory for human is about 630mg. It need more than 250 mL volume of aqueous media to dissolve the amount of mangfiferin according Biopharmaceutics Classification System （BCS, abbr.） guidance. BCS is a framework for classifying drug substances based on their aqueous solubility and intestinal permeability. The partition coefficient （P, abbr.） in octanol/water system of MGF is ranging from 0.01 to 2.3, while pH value is from 6.86 to 4. At the same time, the bioavailability of MGF in rats is poor which was evaluating by in situ intestinal perfusion model. Permeability is believed to be the key factors of absorption. Partition coefficient of drug has good correlation with gastrointestinal permeability. Typically, partition coefficient on the range of 100–1000 is required for efficient passive transcellular transport. So it is presume that low bioavailability of MGF may be owing to low permeability. Therefore, MGF is classified as belonging to BCS-4 class, low solubility and low permeability, and mostly encounter serious bioavailability problems. MGF has been reported to have multiple biological effects, including anti-inflammatory, antidiabetic, antioxidant, antitumor, immunomodulatory, anti-allergic, antiviral, antibacterial, etc. The R&D members intended to development MGF as a new drug with anti-inflammatory effect, but we found that MGF had serious absorption problems. To exploit MGF for a new drug, it is essential to ameliorate bioavailability after oral administration. After enhanced the bioavailability of MGF, the given dose of MGF would be reduce ultimately. Currently, some techniques, including salt formation, sulfonate formation, preparation technique to improve solubility, have been carried out to improve the solubility of MGF. But there is no any report on the increase of permeability of MGF.To improve drug’s permeability by increasing partition coefficient, acylation is one of the useful and conventional methods for the synthesis of prodrug. The chemical acylation of flavonoids is notregioselective and produce some phenolic-hydroxyl-mask by-products. For shielded the functional hydroxyl group which are responsible for the antioxidant activity of flavonoids, the activity would decrease more or less. However, the enzymatic acylation of flavonoids by lipases with phenolic acids is more regioselective than chemical acylation and may enhance not only their solubility in various media, but also their stability and their antioxidant activity. Lipase is reported to use for the structure modification such as arbutin, isoquercetin, phloridzin, rutin, narigin, etc.Lipase （Lipase, EC 3.1.1.3）, three acyl ester hydrolase, is a kind of enzyme catalyzed hydrolysis of natural substrate oil to obtain fatty acids, glycerol and glycerol ester or diester. Lipase is widely used for the reactions of hydrolysis; alcoholysis, esterification, transesterification and ester reverse reaction for substrates （sulfur carboxylates, amides, polyol esters, multi-glycerol triglyceride ester and some hydrophobic ester involved）. Lipase Novozym 435 （a form of Candida Antarctica lipase B） is found to be an effective biocatalyst for the acylation of glucose alone with high regioselective to 6-hydroxyl of glucose.Therefore, the enzymatic synthesis of MGF ester, as a kind of prodrug, was investigated on the aim of bioavailability improvement resulted in the enhancing the permeability. In theory, it will provide some ideas for the R&D of BCS 4 drugs.2 Methods and resultsThis work could be divided into several sections, structure modification, Structure identification, Determination of Physical and Chemical Properties and Anti-inflammatory activity assay.（1） MGF was classified as belonging to BCS-4 class via research of biopharmaceutics properties of MGF. The solubility and octanol/water partition coefficient of MGF were very low over the pH range 1.3 to 8.0, solubility changed from 0.16 to 1.44 mg·mL^-1, partition coefficient was ranging from 2.313 to 0.05, while pH value was from 6.86 to 4. Moreover, the bioavailability of MGF in rats was low. The permeability of MGF could be increased by synthesis the fat-soluble derivatives.（2） In this part, novel sugar ester prodrugs （formyl MGF, acetyl MGF, propionyl MGF） of MGF, were synthesized by transesterification in non-aqueous medium using commercial immobilized lipase （Novozym 435） as biocatalyst. The HPLC method for reaction process monitoring was established. The molecular weight of these prodrugs was confirmed by HPLC-MS. The enzymatic synthesis was an attractive and economic way for preparation of mono-ester of glycoside, and provided a promising way for grafting acyl group onto glycoside directly.（3） In this part, novel sugar ester prodrug （6′-O-propionyl MGF,PMGF） was synthesized by transesterification in non-aqueous medium using commercial immobilized lipase （Novozym 435） as biocatalyst MGF as acyl acceptor, ethyl propionate as acyl donor without the need of vinyl ester. The HPLC method and purification process of the prodrug was established. The chemical structure of this prodrug was confirmed by HPLC-MS, 1H-NMR, 13C-NM, （g）-HMBC and （g）-HMQC. The effects of the substrate amount, temperature, the nature of the solvent, reaction time and the initial water content were investigated. Novozym 435 retained the highest activity in dioxane. The optimal conditions were the follow: In the 8 mL dioxane containing 30mg MGF and 30mg lipase, adding 1mL ethyl propionate, then the synthesis was performed at 60℃for 20h. Novozym 435 had a high stability. After the lipase continuously used for 15 times, the concentration of product was not changed significantly compared with the first time. Novozym 435 could catalyze the regioselective acylation of 6’ hydroxyl group at glucose moiety through the transesterification.（4） In this part, the physicochemical and hydrolysis were evaluated in vitro. Firstly, the equilibrium solubility of PMGF was measured by shake-flask method. The solubility was 0.166±0.02μg·mL^-1, and did not decline as a result of fat-soluble increase. Secondly, the octanol/water partition coefficient of PMGF was very low over the pH range 1.3 to 8.0, and was ranging from 21.104 to 0.08. The partition coefficient of PMGF was increased significantly for 4.33 92.36 times compared with MGF. The prodrug was significantly more hydrophilic than MGF. Thirdly, the hydrolysis of MGF and PMGF were studied in hydrochloric acid buffer （pH 2）, phosphate buffer （pH 7.4） and plasma solution. MGF showed a high chemical stability in both the aqueous medium of pH 2, pH 7.4 and plasma PBS （pH 7.4）. But PMGF was hydrolysis to MGF in vitro. Moreover, three unknown substances were detected in PBS （pH 7.4） medium. It could be due to propionyl transfer to the adjacent hydroxyl of glucose. Summarily, the fat-soluble of PMGF was significantly increased. PMGF was hydrolyzed to prototype MGF in the plasma. It was speculated that the gastrointestinal permeability of PMGF could be increase significantly.（5） In this part, the metabolisms of PMGF and MGF in human intestinal flora in vitro were investigated. Human intestinal flora and PMGF/MGF were incubated under anaerobic conditions. The metabolite were separated and purified by preparative HPLC. PMGF was degraded to PMG within 2 h at the beginning of incubation. This characteristic could be benefit to the absorption of PMGF. Moreover, human intestinal flora could transform PMGF and MGF to aglycone of MGF （norathyriol）. Norathyriol was detected at 4h. The absorption mechanism of norathyriol would be planning to investigate.（6） In this part, the anti-inflammatory effect of PMGF was investigated in vitro and in vivo. In vitro, MGF and PMGF (5μg·mL^-1) were tested on TNFα, IL-1 and NO production in activated macrophages （RAW264.7 cell line） stimulated with LPS (5μg·ml^-1). All the inhibition effects of MGF and PMGF were below 10%. In vivo, the anti-inflammatory effect of PMGF was determined via mice auricular swelling model induced by dimethylbenzene. MGF and PMGF obviously relieved the mice auricular swelling. The effects of middle and low dose of PMGF were better than that of MGF probably due to a higher bioavailability of PMGF attributed to its high permeability.3 ConclusionIn conclusion, on the basis of these results obtain in this work, a kind of high lipophilic MGF ester, 6′-O-propionyl MGF, was obtained. The anti-inflammatory effect of PMGF was better than that of MGF probably due to a higher bioavailability of PMGF attributed to its high permeability. PMGF was demonstrated to be suitable for MGF prodrugs design. In theory, all these works could provide some ideas for the R&D of BCS 4 drugs.4 Innovation（1） For the first time, novel sugar ester prodrugs （formyl MGF, acetyl MGF, propionyl MGF） of MGF, were synthesized by transesterification in non-aqueous medium using commercial immobilized lipase （Novozym 435） as biocatalyst.（2） The fat-soluble MGF prodrug and synergism of anti-inflammatory are achieved.（3） During the course of structure modification of MGF, BCS guidance is applying into the MGF research. The enzymatic synthesis of MGF ester, as a kind of prodrug, was investigated on the aim of bioavailability improvement resulted in the enhancing the permeability.更多还原