【作者】 李学坚；

【导师】 邓家刚；

【作者基本信息】 广西医科大学 ， 药理学， 2012， 博士

【摘要】 1研究背景芒果苷(Mangiferin)系纯天然化合物,具有广泛的生物活性,植物来源广,生产技术成熟,市售产品纯度高达95%以上。芒果苷至今仍未被开发为临床药物的原因之一是其药理作用泛而弱,定位不突出。有学者指出,芒果苷属于BCS-4类药物,溶解性和跨膜通透性均很弱,生物利用度低,从而制约了芒果苷药理作用的发挥。为了提高芒果苷的生物利用度从而提高其药理活性,大多研究从提高芒果苷的水溶性入手,但收效不明显；另有一些研究从提高芒果苷的跨膜通透性入手,这些研究全部集中在制备高脂溶性衍生物方面,虽有一些成效,但也不甚理想,所制备的衍生物至今均未得到开发应用。本论文也从提高芒果苷的跨膜通透性入手,制备高脂溶性的芒果苷酯化衍生物。目前尚未见到与芒果苷酯化衍生物有关的文献报道,本论文从这一空白点切入,预计会得到一些较有意义的研究结果。2方法与结果本论文的研究,涵盖化学合成、分离纯化、化合物结构确证、理化性质表征、药理学研究等内容。(1)化学合成。以芒果苷为起始化合物,以酸酐为溶剂,以H+为催化剂,使芒果苷分别与乙酸酐、丙酸酐和丁酸酐反应,生成芒果苷酯化衍生物。反应的选择性不强,反应产物中包含3-5个酯化程度不同的“多酯化衍生化合物”。(2)分离纯化和结构确证。采用硅胶柱层析方法从反应产物中分离得到7个新结构化合物(化合物Ⅰ-Ⅳ、PAM、HPM、HBM),依据1HNMR图谱、13C NMR图谱和HMBC图谱,全部确证其化学结构。其中3个产率最高的分别为：(1)7,2’,3’,4’,6’-五乙酯化芒果苷衍生物,或7,2’,3’,4’,6’-penta-acetyl-mangiferin (PAM),分子式：C29H28O16;(2)3,6,7,2’,3’,4’,6’-七丙酯化芒果苷衍生物,或3,6,7,2’,3’,4’,6’-hepta-propionyl-mangiferin (HPM),分子式：C40H46O18；(3)3,6,7,2’,3’,4’-六丁酯化芒果苷衍生物,或3,6,7,2’,3’,4’-hexa-butyryl-mangiferin (HBM),分子式：C43H54017。论文对这3个化合物进行了重点研究。(3)理化性质。PAM、HPM和HBM不溶于水而易溶于醋酸乙酯；在pH<9水中稳定,在碱水中有少部分分解；熔点明显降低,较芒果苷低近100℃；紫外吸收特征明显。(4)表观油/水分配系数。分别测定芒果苷、PAM、HPM和HBM在正辛醇和水组成的液—液互萃两相中的浓度,计算出油/水分配系数。芒果苷的油/水分配系数随pH值的减小而增大,但绝对数值较小(小于6)；PAM、HPM和HBM的油/水分配系数均大于100,最大值出现在pH7(均大于270),并随溶液酸度或碱度的增大而减小。(5)采用浓氨水致咳的小鼠模型研究止咳作用。与空白对照组比较,芒果苷高、中剂量组(1.0、0.5 mmol·kg-1)均有显著的止咳作用,可显著延长小鼠的咳嗽潜伏期(P<0.05),并能显著减少咳嗽次数(P<0.01)。PAM、HPM和HBM虽有一定的止咳作用趋势,引咳潜伏期也有所延长,但未显示出统计学显著性差异(P>0.05)；咳嗽次数也有所减少,但未显示出统计学显著性差异(P>0.05)。(6)采用小鼠气管排泌酚红模型研究化痰作用。与空白对照组比较,芒果苷高、中剂量组(1.0、0.5 mmol·kg-1)均能显著促进小鼠排泌酚红(P<0.05或P<0.01)；PAM、HPM和HBM虽然也有一定的促进排泌作用,但未显示出统计学显著性差异(P>0.05)。(7)采用组胺—胆碱联合引喘法研究平喘作用。与空白对照组比较,芒果苷高、中剂量组(1.0、0.5mmol·kg-1)组均能显著延长豚鼠的引喘潜伏期(P<0.05或P<0.01)；PAM、HPM和HBM虽然也显示出一定的延长作用趋势,但未显示出统计学显著性差异(P>0.05)。(8)采用体外细菌平皿培养法研究抑菌作用。PAM、HPM和HBM除在最大浓度(75 mg·mL-1)时对金黄色葡萄球菌敏感株(G+)和乙型副伤寒杆菌(G-)有抑制作用外,对其它菌株均未表现出抑制作用。(9)采用体外抗HSV-1研究抗单纯疱疹病毒作用。芒果苷、PAM、HPM和HBM均表现出一定的细胞毒性,但毒性很低；对疱疹病毒HSV-1的抑制作用不明显,CPE抑制率均不超过25%。(10)通过测定小鼠耳廓肿胀度及其腹腔毛细血管通透性来研究抗炎作用。与空白对照组比较,芒果苷高中剂量组(1.0.0.5mmol·kg-1)、PAM高中剂量组(0.25、0.125mmol·kg-1)、HPM高中低剂量组(0.25、0.125、0.063mmol·kg-1)和HBM高中低剂量组(0.25、0.125、0.063mmol·kg-1)均能显著抑制小鼠耳廓肿胀(P<0.05)；芒果苷低剂量组(0.25mmol·kg-1),PAM低剂量组(0.063mmol·kg-1)虽然有抑制肿胀的趋势,但未显示出统计学显著性差异(P>0.05)。腹腔通透性试验,芒果苷高中低剂量组(1.0、0.5、0.25mmol·kg-1)、PAM高中剂量组(0.25、0.125mmol·kg-1)、HPM高中低剂量组(0.25、0.125、0.063mmol·kg-1)和HBM高中剂量组(0.25、0.125mmol·kg-1)均能显著抑制毛细血管的通透性,减少腹腔液渗出,显示出显著的抗炎作用(P<0.01或P<0.05)；PAM低剂量组(0.063mmol·kg-1)和HBM低剂量组(0.063mmol·kg-1)也有抑制毛细血管通透性的作用趋势,但未显示出统计学显著性差异(P>0.05)。(11)采用MTT法和荷瘤动物研究抑制肿瘤作用。MTT法结果表明,PAM、HPM和HBM能使7901、hella和7404细胞株的活细胞数量减少,提示药物可促使肿瘤细胞死亡。与空白对照组比较,HBM高剂量组(1.0mmol·kg-1)对小鼠荷瘤(S180)有一定的抑制作用(P<0.05)；中剂量组(0.5mmol·kg-1)有一定的作用趋势,但未显示出统计学显著性差异(P>0.05)。(12)降血糖活性及降血糖作用机制研究。①采用链脲佐菌素所致糖尿病小鼠模型研究降血糖作用。与空白对照组比较,PAM高中剂量组(0.25,0.125mmol·kg-1), HPM高中剂量组(0.25,0.125mmol·kg-1)和HBM高中低剂量组(0.25,0.125,0.063mmol·kg-1)显示显著的降血糖作用(P<0.01)；芒果苷高中剂量组(1.0, 0.5mmol·kg-1), PAM低剂量组(0.063mmol·kg-1)和HPM低剂量组(0.063mmol·kg-1)显示一定的降血糖作用(P<0.05)；芒果苷低剂量组(0.25mmol·kg-1)有降血糖的作用趋势,但未显示出统计学显著性差异(P>0.05)。②采用肾上腺素所致高血糖小鼠模型研究对抗急剧血糖升高的作用。芒果苷高剂量组(1.0mmol·kg-1)、PAM高剂量组(1.0mmol·kg-1)、HPM高剂量组(1.0mmol·kg-1)和HBM高剂量组(1.0mmol·kg-1)均没有对抗肾上腺素的升血糖作用(P>0.05)。③在体外抑制蛋白酪氨酸磷酸酶1B(PTP1B)活性的试验中,在5μg·mL-1浓度时,PAM、HPM和HBM就显示出较高的PTP1B酶抑制作用；在20μg·mL-1浓度时,PTP1B酶产生明显沉淀,表明PAM、HPM和HBM具有极强的抑制作用。3结论(1)合成方法简单易行,可产生多种新化合物。采用酸酐直接酰化(酯化)法,很容易从芒果苷制备芒果苷酯化衍生物；采用常规的硅胶柱层析法可方便、较大量地分离和纯化芒果苷酯化衍生物,得到化合物单体。本文所得7个酯化衍生物均为新结构化合物,其结构为首次报道。(2)酯化衍生物PAM、HPM和HBM的为高脂溶性物质,其理化性质、油/水分配系数的数值大小和变化趋势完全不同于芒果苷；油／水分配系数远大于芒果苷的。(3)酯化衍生物的药理作用有明显改变。芒果苷在止咳、化痰、平喘、抑菌、抗病毒等方面有一定的活性,而酯化衍生物]PAM、HPM和HBM却没有,提示PAM、HPM和HBM因化学结构改变而丧失了这些药理活性。PAM、HPM和HBM在体外细胞培养中,能使7404、hella和7901细胞株的活细胞数量减少,提示其具有促进肿瘤细胞死亡的作用；HBM对荷瘤小鼠的肿瘤细胞(S180)也有较弱的抑制生长作用,这些作用与芒果苷相似,提示PAM、HPM和HBM保持了芒果苷的这一活性。PAM、HPM和HBM抗炎和降血糖作用的效价强度比芒果苷的高,只需1/4的摩尔剂量,即可产生与芒果苷相似的抗炎和降血糖效果,提示其抗炎和降血糖活性比芒果苷的高；此外,PAM、HPM和HBM能明显抑制蛋白酪氨酸磷酸酶1B(PTP1B),提示PAM、HPM和HBM因化学结构改变而使抗炎和降血糖活性大大增强。(4)抗炎和降血糖作用显示一定的结构一效应关系。从芒果苷、PAM(乙酯化)、HPM(丙酯化)到HBM(丁酯化),酯化基团大小递增,脂溶性也递增,在相同剂量时,抗炎和降血糖作用强度呈递增变化。(5)酯化衍生物的降糖作用机制。芒果苷及其酯化衍生物PAM、HPM、HBM不具有胰岛素样作用,也不能刺激胰岛细胞在短时间内分泌大量胰岛素。PAM、HPM和HBM的降血糖作用机制可能是对PTPIB的抑制和促进受损胰岛细胞的恢复：一方面,通过抑制PTPIB,提高胰岛素受体浓度,从而提高胰岛素的利用率；另一方面,药物的高脂溶性使跨膜通透性增加,使更多药物进入胰岛细胞,使胰岛细胞得到更好地恢复,从而使胰岛细胞的分泌量增加。(6)化学结构对药理作用的影响大。从结构变化与药理活性变化的关系分析,芒果苷苷元上-OH基团的有无及数量,决定了止咳、化痰、平喘、抑菌、抗病毒活性的有无；而抗炎、抑制PTPIB活性和降血糖作用,只与芒果苷母核的化学结构和结构特点有关,与母核上链接的其它基团无关。(7)芒果苷苷元比芒果苷本身更具有研究开发价值。去除葡萄糖基后的芒果苷苷元,在相同剂量下其抗炎、降血糖作用可能更加显著；如进行酯化衍生,衍生物的抗炎、降血糖作用可能会得到飞跃式提升4创新点(1)用BCS药剂分类原则作为指导思想,对芒果苷进行结构修饰,提高其脂溶性,从而提高其跨膜通透性,增加其生物利用度,最终提高其药理活性；药理研究结果表明,抗炎和降血糖作用达到了预期的目的。(2)首次采用化学方法制备芒果苷酯化衍生物；所制备的7个酯化衍生物(化合物Ⅰ-Ⅳ、PAM、HPM、HBM)均具有新的化学结构,为首次报道。更多还原

【Abstract】 1 BackgroundMangiferin, a naturally occurring xanthone glucoside, is widely distributed in herbals such as Mangifera indica L., Anemarrhena asphodeloides Bge and Swertia mussotii Franch and has recently attracted much attention due to its anti-diabetic, anti-inflammatory, expectorant, antitussive and anti-asthma activities.Mangiferin now can be produced in large scale and with very high purity (more than 95%). Unfortunately, to date mangiferin has not yet been developed to clinical drug, just because it has too many activities to focus on a stronger one.According to scientists, mangiferin has certain bio-activities; its weak pharmacological activity is due to its low solubility, low permeability and low bio-availability. In other words, mangiferin is a BCS-4 drug.In order to improve the bio-availability of mangiferin so as to improve its pharmacological activity, some studies were involved in improvement of solubility, and had little effects. Improvement of permeability was another way, but only few studies were involved. These studies all focused on preparation of highly lipid-soluble derivatives, which were still not good enough to be put into application even though they showed certain pharmacological activities.In this paper we tried to prepare lipid-soluble derivatives too. But one thing was important:we prepared esterified-derivatives, which have not yet been mentioned in existing literatures. These novel esterified-derivatives may lead to some breakthroughs.2 Methods and resultsThis paper reported the chemical synthesis, separation and structure confirmation of 7 novel mangiferin esterified-derivatives. In addition, the physicochemical properties and pharmacological activities of 3 main target compounds (PAM, HPM and HBM) were studied.(1) Synthesis. Mangiferin as original compound, H+ as catalyst, anhydride as solvent, mangiferin respectively reacted with acetic anhydride, propionic anhydride and butyric anhydride to produce esterified-derivatives. The reaction selectivity was so low that the reaction yielded 3 to 5 multi-acylation compounds.(2) Separation, purification and structure confirmation. Silica gel chromatography was employed to separate target compounds from the reaction product.7 novel compounds were separated and confirmed by 1H NMR,13C NMR and HMBC spectrum, their chemical structure seen in the following figure. Out of these 7 novel compounds,3 main target compounds were as follow: (a)7,2’,3’,4’,6’-penta-acetyl-mangiferin(PAM), formula:C29H28O16; (b)3,6,7,2’,3’,4’,6’-hepta-propionyl-mangiferin(HPM), formula:C40H46O18; (c)3,6,7,2’,3’,4’-hexa-butyryl-mangiferin(HBM), formula:C43H54O17. These 7 compounds were reported for the first time.(3) Physicochemical properties. PAM, HPM and HBM were easily soluble in ethyl acetate, hardly soluble in water, stable in aq. solution at pH＜9; they partly decomposed in aq. solution at pH＞10, especially at high temperature. Their melting points decreased sharply with a rate of about 100℃. They had obvious features of UV absorption. (4) O/w distribution coefficient. The concentrations of mangiferin, PAM, HPM and HBM in water and n-octanol, by which to form a liquid-liquid extraction system, were determined respectively so as to calculate the o/w distribution coefficient. The o/w distribution coefficient of mangiferin was smaller than that of PAM, HPM and HBM, and it increased when pH decreased. The distribution coefficients of PAM, HPM and HBM were all bigger than 100, with a max at pH7 (＞270); and with the increase of acidity or alkalinity, they decreased.(5) Coughing mice induced by strong ammonia were employed to study the antitussive activity. Mangiferin’s high- and mid-dose group (1.0,0.5 mmol·kg-1) showed certain antitussive effect because they could significantly decrease the cough frequency(P＜0.01) and prolong the cough incubation period(P＜0.05). The antitussive effect of PAM, HPM and HBM was not significant (P＞0.05) even though they had the trend to decrease the cough frequency and to prolong the cough incubation period.(6) Phenol red secreting mice were used to study the expectorant activity. Mangiferin’s high- and mid-dose group (1.0,0.5 mmol·kg-1) showed certain expectorant effect because they could significantly increase the secretory of phenol red (P＜0.05 or P＜0.01); the expectorant effect of PAM, HPM and HBM was not significant (P＞0.05) even though they had the trend to increase the secretory of phenol red.(7) Asthma guinea pigs induced by histamine-Ach were used to study the anti-asthma activity. Mangiferin’s high-and mid-dose group (1.0,0.5 mmol·kg-1) showed certain anti-asthma effect because they could significantly prolong the asthma incubation period (P＜0.05 or P＜0.01); the anti-asthma effect of PAM, HPM and HBM was not significant (P＞0.05) even though they had the trend to prolong the asthma incubation period.(8) Bacteria culture in vitro was employed to study the bacteriostatic activity. Although PAM, HPM and HBM showed bacteriostatic effect for MRSA(sensitive) and Bacillus paratyphosus at the max concentration (75 mg·mL-1) they didn’t show that for other bacteria, or at other concentration levels.(9) HSV-1 inhibition test was used to study the anti-virus activity. Cytotoxicity of mangiferin, PAM, HPM and HBM was all not so strong; they could not obviously inhibit HSV-1, and the CPE inhibition rate was less than 50%.(10) Swelling of mice ear and permeability of mice celiac capillary were used to evaluate the anti-inflammatory activity. Mangiferin’s high-and mid-dose group (1.0,0.5 mmol·kg-1), PAM’s high- and mid-dose group (0.25,0.125 mmol·kg-1), HPM’s high-, mid- and low-dose group (0.25,0.125, 0.063mmol·kg-1) and HBM’s high-, mid- and low-dose group (0.25,0.125, 0.063mmol·kg-1) could significantly inhibit the ear swelling, showed obvious anti-inflammatory effect; although mangiferin’s low-dose group (0.25mmol·kg-1) and PAM’s low-dose group (0.063mmol·kg-1) had the trend to inhibit the ear swelling, the inhibition effect was not significant (P＞0.05).In another test, mangifrin’s high-, mid- and low-dose group(1.0,0.5, 0.25mmol·kg-1), PAM’s high-and mid-dose group(0.25,0.125mmol·kg-1), HPM’s high-, mid- and low-dose group(0.25,0.125,0.063mmol·kg-1) and HBM’s high- and mid-dose group(0.25, 0.125mmol·kg-1) could significantly decrease the permeability of mice celiac capillary (P＜0.01 or P＜0.05), showed obvious anti-inflammatory effect; although PAM’s low-dose group (0.063mmol·kg-1) and HBM’s low-dose group (0.063mmol·kg-1) had the trend to decrease the permeability of mice celiac capillary their anti-inflammatory effect was not significant (P>0.05).(11) MTT and tumor bearing mice were used to evaluate the anti-tumor activity. In MTT test, PAM, HPM and HBM showed the effect to enhance the apoptosis of 7901, hella and 7404 tumor cell. In another test, HBM high-dose group (1.0mmol·kg-1) could significantly inhibit the S180 tumor cell in vivo (P＜0.05). Although HBM mid-dose group (0.5 mmol·kg-1) had the trend to inhibit tumor cell its inhibition effect was not significant (P＞0.05).(12) PTP1B inhibition test and hyperglycemia model mice were used to evaluate the hypoglycemic activity and to investigate the hypoglycemic mechanism.In hyperglycemia mice induced by STZ, the mangiferin’s high- and mid-dose group (1.0,0.5 mmol·kg-1) and all dose levels group (0.25,0.125, 0.063 mmol·kg-1) of PAM, HPM and HBM significantly showed hypoglycemic effect(P＜0.01 or P＜0.05). Although mangiferin’s low-dose group (0.25 mmol·kg-1) had the trend to decrease the mice glucose level its effect was not significant (P＞0.05).In hyperglycemia mice induced by adrenaline, high-dose group (1.0 mmol·kg-1) of PAM, HPM and HBM could not supress the sharp increase of mice glucose level (P＞0.05). In PTP1B inhibition test in vitro, at 5\ig-mL’1, PAM, HPM and HBM showed obvious PTP1B inhibition effect; precipitates appeared at 20μg·mL-1, this indicated that PAM, HPM and HBM had very high PTP1B inhibition activity.3 Conclusions(1) Esterified-derivatives not only can be easily prepared from mangiferin by direct acylation reaction, but also can be easily, efficiently separated and purified from the reaction product. Novel compound IⅠ～Ⅳ, PAM, HPM and HBM in this paper are reported for the first time.(2) The physicochemical properties and the o/w distribution coefficient of PAM, HPM and HBM are different from those of mangiferin.(3) PAM, HPM and HBM do not show any antitussive, expectorant, anti-asthma, bacteriostatic and antiviral effect even though mangiferin certainly showes these activities. This indicates that esterified-derivatives lose these activities because of being acylated. On the other hand, PAM, HPM and HBM can slightly enhance the apoptosis of 7901, hella and 7404 tumor cell in vitro, and have slight inhibition effect on S180 tumor cell in vivo. This indicates that esterified-derivatives inherite the slight tumor inhibition activity from mangiferin. Third, in the case of showing similar anti-inflammatory and hypoglycemic effect, mangiferin requires four times the molar dose that derivatives neede; in addition, PAM, HPM and HBM can significantly inhibit PTP1B. This indicates that esterified-derivatives have stronger hypoglycemic activity than mangiferin.(4) For the mangiferin esterified-derivatives, there exists a trend that the longer esterification moieties (non→acetyl→propionyl→butyryl), the higher lipid-solubility, and the stronger anti-inflammatory and hypoglycemic activity. In other words, there exists a certain structure-activity relationship.(5) Mangiferin, PAM, HPM and HBM neither have the insulin-like activity nor have the activity to stimulate the islet cell to secrete insulin heavily in short time. The hypoglycemic mechanism of PAM, HPM and HBM may lie in PTP1B inhibition and islet cell repair. First, PTP1B inhibition can increase the concentration of insulin receptor as to increase the availability of insulin; on the other way, high lipid-solubility means high transmembrane permeability, more drugs get into the islet cell and make the cell recover from the STZ damage as to increase the insulin secretion.(6) By analyzing the corresponding relation of the chemical structure changes and the pharmacological activity changes, it indicates that-OHs on the aglycon exist or not would directly make the compound to have antitussive, expectorant, anti-asthma, bacteriostatic and antiviral activity or not; in addition, PTP1B inhibition, anti-inflammatory and hypoglycemic activity only get related with the chemical structure and the structure features of the aglycon, or to say, these activities have nothing to do with other moieties on the aglycon.(7) The aglycon is more valuable than mangiferin itself. The pharmacological activity of mangiferin depends on its aglycon; and the aglycon reasonably has stronger activity than mangiferin, for example, the anti-inflammatory and hypoglycemic activity. It would be certain that acylated aglycon of mangiferin had very strong anti-inflammatory and hypoglycemic activity.4 Innovations(1)Took BCS principles as guidelines, the chemical structure of mangiferin was modified to increase the lipid-solubility of the target compounds. High lipid-solubility led to high transmembrane permeability, so that more drugs got into the cell to increase the bio-availability, and as a result, the pharmacological activity significantly strengthened. The study results in this parer indicated that the anti-inflammatory and hypoglycemic activity of mangiferin esterified-derivatives (PAM, HPM and HBM) achieved the expected purpose.(2)For the first time, esterified-derivatives of mangiferin were chemically prepared. The esterified-derivatives (compoundⅠ～Ⅳ, PAM, HPM and HBM) mentioned in this paper were novel compounds. The anti-inflammatory and hypoglycemic activities of PAM, HPM and HBM were far more stronger than these of mangiferin, their parent compound.更多还原