【作者】 崔永明；

【导师】 余龙江； 敖明章；

【作者基本信息】 华中科技大学 ， 生物制药工程， 2008， 博士

【摘要】 随着对甘草药理作用的进一步认识,全世界对甘草的需求日益增长,甘草资源急剧减少,合理开发甘草资源已十分必要。甘草的植物化学成分十分复杂,以往人们只对甘草中的三萜类化合物作了大量的研究,对其质量评价也仅以甘草酸的含量作为标准,难以全面地评价甘草的质量。近年来,对甘草药理作用的深入研究表明,甘草黄酮类化合物具有多种药理作用,是一类非常有效的活性成分。因此,本课题对甘草中总黄酮的分离纯化及重要单体甘草查尔酮A和光甘草定的分离鉴定和药效进行研究,同时建立胀果甘草黄酮类化合物的HPLC指纹图谱,为甘草黄酮资源的有效、合理开发和质量评价提供依据。本论文首先建立了甘草中总黄酮的含量测定方法,测定胀果甘草、光果甘草和乌拉尔甘草中总黄酮平均含量分别为:4.476 %、2.478 %和1.372%。对比研究了甘草总黄酮的几种不同提取方法,结果超声辅助法提取甘草总黄酮在提取时间和提取效率方面均优于其他几种方法。利用响应面分析法优化甘草总黄酮超声辅助提取的工艺参数,得到超声辅助提取甘草总黄酮的最佳工艺,以此工艺进行超声辅助提取甘草总黄酮一次提取率达到80.56 %。通过比较,选择AB-8大孔树脂纯化甘草总黄酮,优化了AB-8大孔树脂纯化甘草总黄酮的实验条件,经过纯化的甘草总黄酮含量达到60 %以上,收率80 %以上。建立了基于生物信息学的甘草黄酮类化学成分抗乙酰胆碱酯酶活性虚拟评价的新方法,在光果甘草几种主要化合物成分中,光甘草定与乙酰胆碱酯酶的对接能和结合能均较小,结合“吻合度”很好,与三种乙酰胆碱酯酶抑制剂(加兰他敏、石杉碱甲和美曲膦脂)的对接能和结合能接近。通过分子对接分析,将光甘草定A环或B环上的氢原子用氟原子取代进行对接分析,得到对接能和结合能与光甘草定相似的小分子5’-氟光甘草定。根据分子对接结果,从光果甘草中提取分离并鉴定了光甘草定,还建立了甘草中光甘草定的含量测定方法,测定了光果甘草中光甘草定的平均含量为0.199 %,而胀果甘草和乌拉尔甘草中没有检测到光甘草定。以东莨宕碱所致的小鼠认知功能障碍为模型,研究了光甘草定对小鼠学习记忆功能的影响,在“Y”型电迷宫实验和跳台实验中,光甘草定组(2, 4 mg/kg)与模型组相比,学习记忆成绩均有显著提高,且光甘草定大剂量组(4 mg/kg)获得了与阳性组脑复康(400 mg/kg)相当的结果。中高剂量的光甘草定(2,4 mg/kg)和阳性药美曲膦脂(50 mg/kg)能使小鼠大脑乙酰胆碱酯酶的活性分别下降14.4%, 19.0%和21.3%。本论文还从胀果甘草中提取分离了甘草查尔酮A,并运用LC-MS和NMR对其结构进行了鉴定。并建立了甘草中甘草查尔酮A的含量测定方法,测定了胀果甘草和光果甘草中甘草查尔酮A的平均含量分别为0.411 %和0.054 %,而乌拉尔甘草中没有检测到甘草查尔酮A。以二甲苯所致的小鼠耳肿胀和角叉菜胶所致的大鼠足肿胀研究了甘草查尔酮A的抗炎效果,结果表明甘草查尔酮A (5,10 mg/kg)能显著抑制二甲苯所致的小鼠耳肿胀。三个剂量2.5,5和10 mg/kg的甘草查尔酮A均能显著抑制角叉菜胶所致的大鼠足肿胀。甘草查尔酮A (0.1, 0.5和1μg/mL )能显著抑制COX-2的合成和活性,且高剂量(1μg/mL)的甘草查尔酮A对COX-2合成的抑制作用要强于0.5μg/mL的阳性药吲哚美辛。采用HPLC方法,以乙腈-0.1%冰醋酸为流动相进行梯度洗脱,对10批胀果甘草样品中黄酮类化合物进行了分析研究,并采用计算机软件对色谱峰数据进行评价,建立了胀果甘草黄酮类成分的HPLC指纹图谱,可利用其特征峰有效的评价甘草药材的质量。更多还原

【Abstract】 The demands for licorice in the world are growing with the further research on pharmacological effects of licorice. Thus, it is necessary to exploit rationally resources of licorice due to its sharp decline. The phytochemical components of licorice are so complex. However, only the terpenoids such as glycyrrhizic acid were investigated in-depth previously. It is not reasonable to assess the quality of licorice in general only by referring to content of glycyrrhizic acid. In recent years, the in-depth study of pharmacological effects of licorice indicated that flavonoids from licorice were effective active ingredients, possessing a variety of pharmacological effects, and have become research hotspots in herbs. In this study, the isolation, identification and pharmacodynamics of total flavonoids of licorice (TFL), licochalcone A and glabridin were investigated. Meanwhile, the HPLC fingerprint of flavonoids of Glycyrrhiza inflata was established for the effective and reasonable exploitation and evaluation of flavonoids of licorice.The method for the determination of TFL was established. The concents of TFL in Glycyrrhiza inflata Bat., Glycyrrhiza glabra L. and Glycyrrhiza uralensis Fisch were 4.476 %, 2.478 % and 1.372 %, respectively..The extraction techniques for TFL were investigated by contrast. The results showed that ultrasonic-assisted extraction was better than other extractions. The extracting parameters of TFL through ultrasonic-assisted extraction were determined using Response Surface Methododlogy. The extraction of TFL was 80.56 % under the obtained better process conditions. The optimum purification conditions of TFL with macroporous resin AB-8 was obtained. With macroporous resin to adsorb and purify, the yield of TFL was higher than 80 % and the content was higher than 60 %.Based on numerator docking, a new method was established for evaluating the anti-acetylcholinesterase activity of flavonoids from licorice. The results showed that the docking and binding energy of glabridin were lower than those of others components, and were considerable with those of acetylcholinesterase inhibitors (galanthamine, huperzine and metrifonate). The formed compounds are steady. Based on molecular docking analysis, 5’-fluglabridin was obtained with equivalent energy with glabridin when replacing H atom or hydroxy in the A-ring or B-ring of glabridin with F atom.According to molecular docking evaluation, glabridin was isolated from Glycyrrhiza glabra and its structure was identified. The method for the determination of glabridin was established. The content of glabridin in Glycyrrhiza glabra L. was 0.199 %, but it could not be detected in Glycyrrhiza inflata Bat. or Glycyrrhiza uralensis Fisch.The effects of glabridin on scopolamine-induced learning and memory impairment were studied in mice. In Y-electronic maze test and step-down test, glabridin (2, 4 mg kg-1, p.o.) could significantly enhance the learning and memory ability of mice compared with the model group in mice. The effect exhibited by glabridin at higher dose (4 mg/kg) was considerable with that of piracetam (400 mg kg-1, i.p.), a clinically used nootropic agent. Furthermore, both glabridin (2, 4 mg kg-1) and metrifonate (50 mg kg-1, i.p.), used as a standard drug, can reduced the brain cholinesterase activity to 14.4 %, 19.0 % and 21.3 %, respectively, compared with control group.Licochalcone A was isolated from the roots of Glycyrrhiza inflata and identified with LC-MS and NMR. The method for determination of licochalcone A was established. The contents of licochalcone A in Glycyrrhiza inflata Bat. and Glycyrrhiza glabra L. were 0.411 % and 0.054 %, respectively, but it could not be detected in Glycyrrhiza uralensis Fisch.Furthermore, the anti-inflammatory activity of licochalcone A was evaluated in xylene-induced mice ear edema and carrageenan-induced paw edema tests. At the concentrations of 5 and 10 mg/ear, licochalcone A showed remarkable effect against acute inflammation induced by xylene, and at the doses of 2.5, 5, 10 mg/kg (p.o.), licochalcone A reduced significantly paw edema induced by carrageenan compared with the control group. Both COX-2 activity and expression were significantly inhibited by licochalcone A at all tested doses. The inhibitory effect of glabridin on COX-2 expression exhibited by glabridin at higher dose (1μg/mL) was higher than that of indomethacin (0.5μg mL-1).The flavonoids from 10 samples of Glycyrrhiza inflata were analysized with HPLC using a step gradient of acetonitrile-0.1 % acetic acid. The HPLC fingerprint chromatogram of flavonoids from Glycyrrhiza inflata was established using computer software to evaluate the data of chromatographic peaks. It is effective to evaluate the quality of licorice in virtue of the characteristic peaks of HPLC fingerprint chromatogram更多还原