铁棒锤化学成分的HSCCC制备分离及抗氧化活性研究

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	铁棒锤化学成分的HSCCC制备分离及抗氧化活性研究
	刘永玲
学位类型	硕士
导师	李玉林
	2014-05
学位授予单位	中国科学院研究生院
学位授予地点	北京
学位专业	中药学
关键词	铁棒锤化学成分高效液相色谱-1 1-二苯基-2-三硝基苯肼高速逆流色谱响应面法
摘要	铁棒锤来源于毛茛科乌头属植物铁棒锤Aconitum pendulum Busch和伏毛铁棒锤Aconitum flavum Hand.-Mazz.的干燥块根和幼苗，现代研究表明铁棒锤具有解热、消炎、镇痛、局麻和抑菌作用，在西北地区民间广泛应用于治疗跌打肿痛、风湿关节痛、外伤出血等症状，发挥了良好的治疗作用。铁棒锤根和根茎中含生物碱类和黄酮类活性成分，具有很高的药用价值。本学位论文主要进行了铁棒锤（Aconitum pendulum Busch）化学成分的高速逆流色谱（HSCCC）分离制备、提取工艺优化及HPLC-DPPH抗氧化活性研究，取得了以下研究成果和结论： 1. 在单因素实验的基础上，选择液料比、提取温度、提取时间3个因素为自变量，以铁棒锤根中一种咪唑生物碱（ICABE）的提取率作为响应值，采用3因素3水平的响应面分析法，优化了铁棒锤乙醇超声提取工艺：液料比23.0 mL/g，提取温度为58 ℃，提取时间为51 min。 2. 采用HSCCC技术从铁棒锤根的氯仿萃取部分分离得到1个化合物，经1H-NMR、13C-NMR等波谱学数据分析鉴定为一种咪唑生物碱1H-Imidazole-2- carboxylic acid, butyl ester（ICABE）。 3. 采用大孔树脂粗分结合HPLC-DPPH方法快速筛选铁棒锤地上部分的抗氧化活性部位及成分。结果表明：大孔树脂水、10 %乙醇、30 %乙醇和50 %乙醇四个洗脱部位中，30 %乙醇部位对DPPH˙的抑制率最高，其中化合物（4）抗氧化性显著。此方法省时省力、操作简单易控、响应快速准确，具有良好的重现性。 4. 采用大孔树脂富集、HSCCC分离制备结合半制备色谱（semi-prep-HPLC）从铁棒锤地上部分粗提物大孔树脂预处理30 %乙醇洗脱部分中分离纯化了8种单体化合物，经1H-NMR和13C-NMR等波谱学数据分析鉴定为：木兰花碱（1）、槲皮素-7-O-α-L-鼠李糖基-3-O-β-D-葡萄糖基-(1→2)-β-D-葡萄糖苷（2）、槲皮素-7-O-α-L-鼠李糖基-3-O-(6′′′′-反式咖啡酰基)-β-D-葡萄糖基-(1→2)-β-D-葡萄糖苷（4）、山奈酚-7-O-α-L-鼠李糖基-3-O-(6′′′′-反式咖啡酰基)-β-D-葡萄糖基-(1→2)-β-D-葡萄糖苷（5）、槲皮素-7-O-α-L-鼠李糖基-3-O-(6′′′′-反式对香豆酰基)-β-D-葡萄糖基-(1→2)-β-D-葡萄糖苷（6）、山奈酚7-O-α-L-鼠李糖基-3-O-(6′′′′-反式对香豆酰基)-β-D-葡萄糖基-(1→2)-β-D-葡萄糖苷（7）、山奈酚-7-O-α-L-鼠李糖基-3-O-β-D-葡萄糖基-(1→2)-β-D-葡萄糖苷（8），羟基酪醇-1-O-(6′-反式阿魏酰基-(4′′→1′′′)-β-D-葡萄糖基)-β-D-葡萄糖苷（9）。经HPLC检测纯度分别为97. 2 %（1）、95.7%（2）、96.4 %（4）、97.2%（5）、98.6 %（6）、96.8 %（7）、98.8 %（8）和93.9 %（9）。该方法简便、快速，所得产物纯度高，适合于铁棒锤地上部分化学成分的制备分离。 5. 通过DPPH˙分光光度计法测定了铁棒锤地上部分分离得到的黄酮苷类化合物2、4、5、6、7和8的清除自由基能力。结果表明：化合物4和5清除DPPH˙自由基能力较强，其IC50值为16.92 μg/mL和27.23 μg/mL。
其他摘要	Radix Aconiti comes from the root and seedling of Aconitum pendulum Busch and Aconitum flavum Hand.-Mazz., which belong to the family of Ranunculaceae. Modern studies have demonstrated that Radix Aconiti has good effect on antipyretic, antiphlogistic, analgesic, local anesthesia and bacteriostasis. A. pendulum, as one medicinal resource of Radix Aconiti, has been widely used for treating injuries pain, rheumatism arthritis pain, trauma, external bleeding and other symptoms. Previous phytochemical studies have showed that A. pendulum is rich in alkaloids and flavonoids, which have high medicinal value. In this research, we have studied on the separation of chemical constitutes by high-speed counter-current chromatography (HSCCC), optimization of extraction process and evalution of antioxidant activity by HPLC-DPPH. The main achievements and conclusions are as follows. 1、The ultrasound-assisted extraction process parameters of ICABE from A. pendulum were optimized by the quadratic general spinning design on the ratio of solvent to solid, extraction temperature and extraction time. On the basis of single-factor test, the response surface methodology with 3 factors and 3 levels was adopted. The extraction rate of ICABE was used as the responsive values. The results indicate that the optimum conditions for ICABE are as follows: the ratio of solvent to solid is 23.0 mL/g, extracting temperature is 58 ℃ and extraction time is 51 min. 2、An imidazole alkaloid named 1H-Imidazole-2-Carboxylic acid, butyl ester (ICABE) was successfully separated from the chloroform extract of A.pendulum by HSCCC. The chemical structure was confirmed by UV, 1H and 13C NMR. 3、An efficient method was successfully established by HPLC-DPPH combined macroporous resin column chromatography (MRCC) for rapid screening the active site and chemical components from the crude extract of aerial parts of A. pendulum. As a resut, four fractions were obtained including water, 10 % ethanol, 30 % ethanol and 50 % ethanol fraction, in which 30 % ethanol fraction exhibits the highest antioxidant activity. Besides, compound 4 exhibits higher antioxidant activity than other compounds in 30 % ethanol fraction. The results show that the established method is simple, accurate and has good reprodueibility. 4、In this section, macroporous resin column chromatography (MRCC) was firstly used to treat the crude extract of aerial parts of A. pendulum. HSCCC and semi-prep-HPLC were then applied for separation of chemical constitutes in the 30 % ethanol elution fraction. As a result, eight compounds were obtained and their strucures were identified by UV and NMR as follows: magnoflorine (1); quercetin-7- O-α-L-rhamnopyranosyl-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside (2); quercetin-7-O-α-L-rhamnopyranosyl-3-O-(6′′′′-trans-caffeoyl)-β-D-glucopyranosyl- (1→2)-β-D-glucopyranoside (4); kaempferol-7-O-α-L-rhamnopyranosyl-3-O-(6′′′′- trans-caffeoyl)-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside (5); quercetin-7-O- α-L-rhamnopyranosyl-3-O-(6′′′′-trans-p-coumaroyl)-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside (6); kaempferol-7-O-α-L-rhamnopyranosyl-3-O-(6′′′′-trans-p- coumaroyl)-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside (7); kaempferol-7-O-α -L-rhamnopyranosyl-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside (8); hydroxytyrosol-1-O-(6′-trans-feruloyl-(4′′→1′′′)-O-β-D-glucopyranosyl)-β-D- glucopyranoside (9). HPLC analysis showed that the purity of each of the target compounds were 97.2 % (1)、95.7 % (2)、96.4 % (4)、97.2 % (5)、98.6 % (6)、96.8 % (7)、98.8 % (8)、93.9 % (9). The established method is simple, highly efficient and suitable for separation of compounds from the aerial parts of A. pendulum. 5、The activities of scavenging DPPH˙ free radicals of the flavonoid glycosides (2, 4, 5, 6, 7 and 8) isolated from the aerial parts of A. pendulum were evaluated. The results showed that compounds 4 and 5 had higher DPPH˙ radical scavenging activity with an IC50 value of 16.92 μg/mL and 27.23 μg/mL.
文献类型	学位论文
条目标识符	http://210.75.249.4/handle/363003/4034
专题	中国科学院西北高原生物研究所
推荐引用方式 GB/T 7714	刘永玲. 铁棒锤化学成分的HSCCC制备分离及抗氧化活性研究[D]. 北京. 中国科学院研究生院,2014.

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