绿原酸的定量、类似物及衍生物的制备和生物活性研究

发布时间：2018-06-19 05:51

本文选题：UPLC-MS/MS + 茵陈　；参考：《内蒙古大学》2017年博士论文

【摘要】：绿原酸是自然界广泛存在并具有多种生物活性的天然产物,其含量是中药茵陈质量控制的重要指标。茵陈为菊科植物茵陈蒿或滨蒿的地上干燥部分,始载于《神农本草经》,具有清热利湿、利胆退黄的功效,现代药理研究证实其还具有抗癌、抗炎和解热镇痛作用。中药茵陈中含有绿原酸类、黄酮类及香豆素类等多种化学成分。本文建立了超高效液相色谱-串联四极杆质谱联用法(UPLC-MS/MS)以同时快速分析茵陈中包括绿原酸在内的6种成分的含量,并比较了不同采收时间、不同采集部位及不同干燥方式处理后样品中6种成分含量的变化,结果表明:茵陈药材中绿原酸的含量显著高于其它黄酮化合物。此外,在不同月份采集的样品中,绿原酸含量的分布规律均呈现:叶茎根,冷冻干燥处理荫干处理,其中4月采集样品经冷冻干燥处理后叶中绿原酸含量最高,为10.823 μg/mg。其他5种黄酮成分的含量变化如3,5,3',4'-四羟基-6,7-二甲氧基黄酮的含量在6月冷冻干燥样品叶中含量最高;3'-甲氧基蓟黄素及蓟黄素在茵陈叶中的含量随着时间的推移逐渐升高;而金圣草黄素和鼠李柠檬素在茵陈药材中的含量很低。此次建立的同时快速分析茵陈中包括绿原酸在内的6种成分的方法灵敏度和分辨率高、稳定性强,为茵陈药材质量控制提供了可借鉴的分析方法。由于绿原酸分子结构中存在5个酚羟基和一个羧基,因此具有很高的亲水性和较低的亲脂性。本实验采用生物转化技术在改善绿原酸的亲脂性方面做了研究。利用猪胰脂肪酶催化完成了绿原酸乙酰化的反应,并摸索出该酶催化绿原酸乙酰化反应的最适反应条件,即选取甲基叔丁基醚作为反应溶剂,乙酸乙烯酯作为酰基供体,在绿原酸:乙酸乙烯酯=1:6,脂肪酶100 mg,50 ℃条件下,绿原酸的乙酰化产率最高。此外,将酶催化反应混合物用ODS进行分离纯化,通过UPLC-MS/MS定性分析以及NMR结构确定,最终得到绿原酸的两种乙酰化物即1-乙酰绿原酸和1,5-二乙酰绿原酸。多年研究证明绿原酸具有很强的生物活性,但是由于其结构特殊性——酯键的存在,使其口服后仅有一小部分可以被完全吸收,而很大一部分在体内水解,水解后的绿原酸许多生物活性消失。因此,迫切需要研究合成稳定性更高的绿原酸类似物。本研究以奎尼酸二缩丙酮为起始原料,经过氧化、羟氨化、催化氢化、酰化等大量实验研究,合成了稳定性更高的绿原酸类似物,经过包括NOE在内的NMR分析、以及高分辨质谱的检测,鉴定为3α-咖啡酰奎尼酸酰胺,该化合物不仅保留了绿原酸的抗氧化活性和抗HCV病毒活性,同时还有较高的稳定性和较低的毒性。为了验证绿原酸类似物及衍生物的生物活性,找到活性更优的天然产物的类似物及衍生物,本文比较了6种化合物在清除DPPH自由基活性、抑制细胞内活性氧活性、过氧游离基诱导DNA氧化损伤的保护作用、抑制α-葡萄糖苷酶活性以及抑制二肽基肽酶活性等五个方面的活性。研究结果表明:绿原酸类似物3α-咖啡酰奎尼酸酰胺在抑制细胞内活性氧活性方面优于绿原酸,同时3α-咖啡酰奎尼酸酰胺、1-乙酰绿原酸和1,5-二乙酰绿原酸对过氧游离基诱导的DNA氧化损伤的保护作用也强于绿原酸。尽管上述化合物在离体实验中均呈现出良好的生物活性,但能否被小肠吸收进入血液循环,需要进一步验证。本实验采用国内外公认的,在口服药物吸收转运研究中应用广泛的人源结肠癌Caco-2细胞单层模型研究其吸收转运特性,结果表明:以绿原酸为母核,经过将酯键转变为酰胺键或酚羟基乙酰化后,化合物在Caco-2细胞单层模型中的转运能力增强,为新药的开发以及临床用药提供理论依据。
[Abstract]:Chlorogenic acid is a natural product which exists widely in nature and has a variety of biological activity. Its content is an important indicator of the quality control of Chinese herbal medicine. The dry part of the Artemisia Artemisia or Artemisia Artemisia is carried in the "Shennong herbal medicine", which has the effect of clearing heat and dampness and taking advantage of the yellowing, and the modern pharmacological study confirmed its anti-cancer. The antiinflammatory and antipyretic analgesic effects. Chinese herbal medicine contains a variety of chemical constituents such as chlorogenic acid, flavonoids and coumarins. In this paper, a super high performance liquid chromatography tandem quadrupole mass spectrometry (UPLC-MS/MS) was established for the simultaneous rapid analysis of 6 components, including chlorogenic acid, and the different harvest time. The results showed that the content of chlorogenic acid in Chinese medicinal herbs was significantly higher than that of other flavonoids. In addition, the distribution rules of chlorogenic acid content in the samples collected in different months were all: leaf stem root, lyophilization treatment and shady treatment, in which the samples were collected in April. The content of chlorogenic acid in the leaves of freeze-dried products was the highest, and the content of other 5 kinds of flavonoids in 10.823 g/mg., such as 3,5,3', 4'- four hydroxyl -6,7- two methoxy flavonoids, was the highest in the leaves of frozen dry samples in June, and the content of 3'- methoxy thistle and thistemin in the leaf of Artemisia gradually increased with the time. At the same time, the determination of 6 components, including chlorogenic acid, is high sensitivity, high resolution and strong stability, which provides a useful analytical method for the quality control of rhin medicinal materials. 5 phenols exist in the molecular structure of chlorogenic acid. The hydroxyl group and a carboxyl group have high hydrophilicity and lower lipophilicity. In this experiment, bioconversion technology was used to improve the lipophilicity of chlorogenic acid. The reaction of chlorogenic acid acetylation was catalyzed by pig pancreas lipase, and the optimum reaction conditions for the enzyme catalyzed chlorogenic acid acetylation were found out. Methyl tert butyl ether was used as reaction solvent and vinyl acetate as an acyl donor. Under the conditions of Lv Yuan acid vinyl acetate =1:6, lipase 100 mg, 50 C, the yield of Lv Yuan acid was the highest. In addition, the enzyme catalytic reaction mixture was separated and purified with ODS, UPLC-MS/MS qualitative analysis and NMR structure were determined, and Lv Yuan was finally obtained. Two acetyl compounds of acid, 1- acetyl chlorogenic acid and 1,5- two acetyl chlorogenic acid, have proved that chlorogenic acid has very strong biological activity for many years. But because of its structural specificity, ester bond, only a small portion of the ester bond can be absorbed completely, and a large part is hydrolyzed in the body and many organisms of chlorogenic acid after hydrolysis. The activity disappears. Therefore, there is an urgent need to study the synthesis of more stable chlorogenic acid analogues. In this study, a large amount of chlorogenic acid analogues with higher stability were synthesized by a large number of experimental studies, such as oxidation, aminohydrylation, catalytic hydrogenation and acylation with two acetone, quinine acid, through NMR analysis including NOE, and high resolution mass spectrometry. The test, identified as 3 alpha caffeoquinic acid amide, not only preserves the antioxidant activity of chlorogenic acid and anti HCV virus activity, but also has high stability and lower toxicity. In order to verify the bioactivity of chlorogenic acid analogues and derivatives, we find analogues and derivatives of natural products with better activity. The activities of the 6 compounds in the removal of DPPH free radical activity, inhibition of active oxygen activity in cells, protection of DNA oxidative damage induced by peroxy free radicals, inhibition of the activity of alpha glucosidase and inhibition of the activity of two peptidylpeptidase, the results showed that the chlorogenic acid like substance 3 alpha cofylquinic acid amide was inhibited in the cells. Active oxygen activity is superior to chlorogenic acid, while 3 alpha cofylquinic acid amides, 1- acetyl chlorogenic acid and 1,5- two acetyl chlorogenic acid are more protective than chlorogenic acid induced DNA oxidative damage induced by peroxy free radicals. Although the above compounds have good bioactivity in the isolated experiments, they can be absorbed into the blood by the small intestine. In this experiment, we need further verification. This experiment uses a widely recognized human source colon cancer Caco-2 cell monolayer model, which is widely accepted in the study of oral drug absorption and transport, to study the absorption and transport properties of human colon cancer cells. The results show that chlorogenic acid is the parent nucleus, after the ester bond is transformed into an amyl amine bond or a phenolic hydroxyl acetylation, the compound is in Caco-2 cells. The transport capacity of monolayer model is enhanced, which provides a theoretical basis for the development of new drugs and clinical medication.
【学位授予单位】：内蒙古大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R284;R285

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