【作者】 孙清华；

【导师】 柳仁民；

【作者基本信息】 聊城大学 ， 分析化学， 2006， 硕士

【摘要】 高速逆流色谱(High-Speed Counter-Current Chromatography, HSCCC)是利用螺旋管方向性与高速行星式运动相结合产生的一种独特的流体动力学现象,使样品中的各组分在相对移动的互不相溶的两相(一相为固定相,另一相由恒流泵连续输入,为流动相)中实现高效的接触、混合、分配和传递,并按照分配系数的不同依次洗脱而获得分离的一种液-液色谱技术[1]。它消除了由于使用载体而带来的样品吸附、污染、峰形变形等不良情况,此外,它还具有高效、快速、进样量大和回收率高等优点,已渐渐从最初的分析型色谱技术发展到具有较大容量的制备型色谱技术,特别适合天然药物中有效成分的分离制备[2],成为在中药有效成分的分离纯化领域最有优势的分离方法之一。中药化学成分较为复杂,所以想研究和应用其中的有效成分,必须将它们从中药中提取出来并进一步加以分离和精制,得到有效单体。由于中药所含成分种类繁多,含量有高有低,所以中药有效成分的提取、分离和精制是一项十分艰巨而细致的工作。而HSCCC非常适合天然药物中有效成分的分离制备。目前,应用HSCCC法在提取分离天然药物中的黄酮、生物碱、香豆素、皂甙和蒽醌类衍生物等有效成分方面已获得满意结果[3]。本文对HSCCC在中草药有效成分的制备性分离纯化方面的应用进行了研究,建立了分离纯化秦皮、乌药、知母、木蝴蝶和槐果皮中的有效成分的新方法,并利用紫外光谱(UV)、高效液相色谱(HPLC)、核磁共振波谱(NMR)等对目标产物的纯度和化学结构进行了分析和鉴定。1. HSCCC分离纯化秦皮中的香豆素类化合物建立了HSCCC一次性分离、纯化秦皮中多种香豆素类化合物的新方法。溶剂体系为正丁醇-甲醇-0.5%醋酸(5:1:5,v/v)。从150 mg秦皮粗提物中一步分离纯化出14.3 mg秦皮苷(fraxin),26.5 mg七叶苷(aesculin),5.8 mg秦皮素(fraxetin),32.4 mg七叶素(aesculetin);纯度分别为97.6%,99.5%, 97.2%和98.7%。化合物结构由其1H-NMR和13C-NMR数据来确定。2. HSCCC分离纯化乌药中的倍半萜类化合物应用制备型HSCCC分离、纯化乌药中的倍半萜类化合物,所用溶剂体系为石油醚-乙酸乙酯-甲醇-水(5:5:6:4,v/v)。从450 mg乌药粗提物中一步分离纯化得到40.2 mg乌更多还原

【Abstract】 High-speed counter-current chromatography is a support-free liquid-liquid partitionchromatographic technique. The method provides an advantage over the conventional columnchromatography by eliminating the use of a solid support where an amount of stationaryphase is limited and dangers of irreversible adsorption from the support are inevitably present.In addtion, it has many other advantages such as high efficency, high loading capacity andhigh recovery, so it is very suitable for separation and purification of fragile cpmpounds fromtraditional Chinese herbs and other natural products. It has become one of the mostpreponderant separating and analytical techniques.The constituents are complexive in natural drugs. In order to study and apply the activecomponent in use, the separation and purification of these components are very important.Because the natural drugs contain all kinds of constituents and the content of the compoundsis different, so the separation and purification of these componets is an arduous andmeticulous work. As an advanced separation technique, HSCCC has been widely used for theseparation of active components from traditional Chinese herbs and other natural products inrecent years. Successful application of HSCCC has been reported for the purification offlavones, alkaloids, coumarins, anthraquinones and saponins.The present article developed a HSCCC method for the separation and purification ofcoumarins from Cortex Fraxinus;sesquiterpene lactone compounds from Radix Linderae;mangiferin, neomangiferin, cis-hinkiresinol and monoethyl-cis-hinkiresinol from RhizomaAnemarrhenae;flavones from Oroxylum Iindicum and Sophora japonica L.1. Preparative isolation and purification of coumarin compounds from Cortex Fraxinusby HSCCCHSCCC was successfully used for the isolation and purification of coumarin compoundsfrom Cortex Fraxinus, the Chinese herbal drug, n-butanol-methanol-0.5% acetic acid(5:1.5:5,v/v) was used as the two-phase solvent system. 14.3 mg of fraxin, 26.5 mg ofaesculin, 5.8 mg of fraxetin and 32.4 mg of aesculetin with the purity of 97.6%, 99.5%,97.2% and 98.7% respectively were obtained from 150 mg of crude extracts of cortexfraxinus in a single run. The structures of the isolated compounds were identified by 1H-NMRand 13C-NMR.2. Preparative isolation and purification of linderalactone and lindenenol from RadixLinderae by HSCCCHSCCC method for isolation and purification of sesquiterpene lactone compounds fromChinese traditional medicinal herb Radix Linderae (Wuyao in Chinese) was successfullyestablished by using light petroleum-ethyl acetate-methanol-water (5:5:6:4,v/v) as thetwo-phase solvent system. The upper phase of the two-phase solvent system was used as thestationary phase of HSCCC. 40.2 mg of linderalactone and 64.8 mg of lindenenol wereobtained from 450 mg of crude extracts in one-step separation. The purity of linderalactoneand lindenenol was 99.7% and 98.2%, respectively, as determined by HPLC. The structuresof linderalactone and lindenenol were identified by 1H-NMR and 13C-NMR.3. Preparative isolation and purification of four compounds from the Chinese medicinalherb Rhizoma Anemarrhenae by HSCCCHSCCC was applied to the separation and purification of mangiferin, neomangiferin,cis-hinkiresinol and (-)-4’-O-methylnyasol from the Chinese medicinal herb RhizomaAnemarrhenae. 500 mg of crude extracts were separated by using n-butanol-acetic acid (1%)(1:1,v/v) as the two-phase solvent system and yielded 35.3 mg of neomangiferin and 245.4mg of mangiferin. During this separation, cis-hinkiresinol and (-)-4’-O-methylnyasol werestill maintained in the stationary phase. The stationary phase was collected, evaporated todryness and separated with light petroleum-ethyl acetate-methanol-water (1:1:1.2:0.8,v/v) and1:1:1.4:0.6 (v/v) in gradient elution, which yielded 17.2 mg of cis-hinkiresinol and 12.4 mgof (-)-4’-O-methylnyasol. The purity of mangiferin, neomangiferin, cis-hinkiresinol and(-)-4’-O-methylnyasol was 96.3%, 98.0%, 97.3% and 98.2%, respectively, as determined byHPLC. The chemical structures of these components were identified by 1H-NMR and13C-NMR4. Preparative isolation and purification of baicalein and chrysin from the extracts ofOroxylum indicum by HSCCCHSCCC was successfully used for the isolation and purification of baicalein and chrysinfrom the extracts of Oroxylum indicum. From 300 mg of the crude extracts, 25.5 mg ofbaicalein and 36.6 mg of chrysin were obtained with a purity of 99.2% and 100%,respectively.HSCCC conditions: two-phase solvent system: light petroleum-ethyl acetate-methanol-water (5:5:5:5,v/v) and (5:5:7:3,v/v);solid phase: the upper phase of (5:5:5:5,v/v);mobilephase: lower phase of (5:5:5:5,v/v) and (5:5:7:3,v/v) used in gradient elution mode as follows:0-50 min, the volume ratio of (5:5:5:5,v/v) and (5:5:7:3,v/v) system was continuouslychanged from 100:0 to 0:100;flow rate: 2.0 ml min-1;revolution speed: 850 rpm;detectionwavelength: 254 nm;separation temperature: 25°C.HPLC conditions: Column: YWG ODS C18 column (200×4.6 mm ID. 10 μm);mobilephase: methanol-phosphoric acid (0.1%) used in gradient elution mode as follows: 0-30 min,the volume ratio of methanol and phosphoric acid (0.1%) was continuously changed from40:60 to 70:30;flow rate: 1.0 ml min-1;detection wavelength: 254 nm.5. Preparative isolation and purification of flavone compounds from Sophora japonica L.by HSCCCHSCCC and D-101 macroporous resin column were applied to the separation andpurification of genistein-7,4’-di-O-β-D-glucoside (I), genistein-7-O-β-D-glucopyranosde-4’-O-[(α-L-rhamnopyransoyl)-(1-2)-β-D-glucopyranosede] (II), kaempferol-3-O-β-D-sophoroside (III), quercetin-3-O-β-L-ramnopyranosyl-(1-6)-β-D-glucopyranoside (IV),genistein-4’-β-L-rhamnopyransoyl-(1-2)-α-D-glucopyranoside (V) and kaempferol-3-O-β-L-ramnopyranosyl-(1-6)-β-D-glucopyranoside (VI) from the Chinese medicinal herbSophora japonica L. 35 milligrams of sample 1 were separated by using n-butanol-acetic acid(1%) (1:1.v/v) as the two-phase solvent system and yield 7.7 mg compound I, 4.3 mgcompound II. One hundred milligrams of sample 2 was separated by using ethylacetate-n-butanol-acetic acid (1%) (1:0.16:1,v/v) as the two-phase solvent system and yielded4.5 mg compound III, 2.7 mg compound IV, 6.4 mg compound V and 1.2 mg compound VIwith the purity of 97%, 99%, 98%, 99%, 99% and 90%. The chemical structures of thesecomponents were identified by 1H-NMR and 13C-NMR.更多还原