固相萃取用于中草药金属离子及活性成份分析方法的研究

【作者】 杨亚玲；

【导师】 林强；

【作者基本信息】 昆明理工大学 ， 矿物加工工程， 2006， 博士

【摘要】 本文选择了杨梅、鱼腥草、云南甜茶几种中草药为研究对象，进行了其几种重要无机微量元素及黄酮、糖有效成份分析方法的研究。利用固相萃取技术对中草药样品进行前处理，进行了“功能试剂的合成、筛选及结构性能间关系”的研究；建立了“固相萃取光度法测定中草药中钴、钒、银”；“固相萃取富集，高效液相色谱法测定中药中镍、锡、铜、铅、镉、汞”；“固相萃取富集，高效液相色谱法测定中药中铁、钴、镍、铜、锌、锰”；“固相萃取和高效液相色谱法测定杨梅、甜茶、鱼腥草中的黄酮类物质”；“固相萃取-高效液相色谱法测定杨梅、甜茶、鱼腥草中的糖类物质”几个新分析方法体系。并进行了“方法应用—甜茶、鱼腥草中黄酮分离、制备、抗氧化及抗过敏活性的研究”。（一） 2-喹啉偶氮类试剂的合成及在中草药微量元素测定中有应用首次合成了6个2-喹啉偶氮类试剂，其中有2-喹啉偶氮苯酚类试剂、2-喹琳偶氮苯胺类试剂和2-喹啉偶氮苯甲酸类试剂，利用红外光谱、质谱、核磁共振鉴定所合成为目标产物。研究试剂与金属离子的显色反应，并比较了几个试剂的分析性能，发现该类试剂的灵敏度比相应的吡啶偶氮类试剂，噻唑偶氮类试剂，苯并噻唑偶氮类试剂等杂环偶氮类试剂有提高：根据配位基-OH、-NH₂、-COOH与所测金属离子形成配合物稳定性、灵敏度、选择性等方面的影响判断2-喹啉偶氮类试剂结构与分析性能的关系。将其应用在中草药微量元素的测定中，具体为：1固相萃取光度法测定中草药中的钴、钒、银研究合成的2-喹啉偶氮类试剂与金属离子的显色反应，在适当的掩蔽剂存在下，2-喹啉偶氮苯酚类试剂对钒和2-喹啉偶氮苯胺类试剂对银的显色反应是特效的，且生成络合物具有一定的疏水性，可被固相萃取柱富集，基于上述特点，我们在国内首次把反相键合硅胶固相萃取技术用于无机元素光度分析，建立了2-（2-喹啉偶氮）-5-二申（乙）氨基苯酚固相萃取光度法测定钒、2-（2-喹啉偶氮）-5-二甲（乙）氨基苯胺固相萃取光度法测定银和2-（2-喹啉偶氮）-5-二甲（乙）氨基苯甲酸固相萃取光度法测定钴六个方法。由于上述方法采用了固相萃取富集，富集倍数达100倍，远远高于LLE；而且还克服了LLE易乳化，相分离慢的缺点；方法有机溶剂消耗少。通过固相萃取对分析物的高倍数富集，在一定程度上弥补了吸光光度法灵敏度不够高的缺点，且方法对环境的污染小，符合绿色化学的要求。2固相萃取富集，高效液相色谱法测定中草药中镍、锡、铜、铅、镉、汞根据卟啉类试剂在碱性条件下能和镍、锡、铜、铅、镉、汞等多种重金属元素生成稳定的络合物，络合物具有很高的灵敏度，但用于光度分析试剂选择性差的特点，我们研究了用四-（对-氨基苯基）-卟啉（T₄APP）柱前衍生，固相萃取富集，高效液相色谱测定中草药中的镍、锡、银、铅、镉、汞。由于本实验采用了特殊填料的Waters Xterra ^TNRP₁₈色谱柱（pH范围为1～12）为固定相，用pH为10的碱性缓冲液为流动相分离卟啉重金属络合物；解决了卟啉重金属络合物分离只能用碱性流动相洗脱（卟啉重金属络合物在弱酸性和中性条件下稳定性差，用弱酸性或中性流动相洗脱会造成络合物分解），而普通色谱柱在碱性（pH＞8）条件下不稳定的问题，首次实现了镍、锡、银、铅、镉、汞卟啉络合物的分离测定。3固相萃取富集，高效液相色谱法测定中药中铁、钴、镍、铜、锌、锰我们合成的2-喹啉偶氮酚类试剂，研究发现由于该类试剂共轭体系扩大，灵敏度有所提高。该类试剂作为光度分析试剂选择性差，但是它们能络合离子多的特点恰恰适合用于液相色谱分析。鉴于该类试剂能与铁、钴、镍、铜、锌、锰生成稳定的有色络合物，我们研究了2-（2-喹啉偶氮）-5-二乙氨基苯酚（QADEAP）与铁、钴、镍、铜、锌、锰的合理显色反应条件和色谱分离条件，将QADEAP作为铁、钴、镍、铜、锌、锰的衍生试剂。结合微波消解样品技术，建立了2-（7-喹啉偶氮）-5-二乙氨基苯酚柱前衍生，固相萃取富集，高效液相色谱分离测定中药样品中的铁、钴、镍、铜、锌、锰的方法，方法灵敏度高，能同时测定多种元素。4方法相关性研究将所建立的高效液相色谱法测定中草药中的过渡金属与重金属元素方法与《中国药典》2005年版一部中方法进行相关性研究。研究表明，两种方法的相关性较好，12种微量元素测定的两种方法的相关系数均在0.9以上，所建立的方法具有一定的可靠性。（二）固相萃取和高效液相色谱法测定中草药中的黄酮类物质研究了用固相萃取预分离，高效液相色谱法测定中草药中黄酮类物质的方法。1固相萃取和高效液相色谱法测定杨梅中的黄酮类物质杨梅和杨梅树叶的黄酮用90％甲醇加热回流提取，提取液用WatersSep-Pak-C₁₈固相萃取小柱预分离脱脂，以Waters Nova-Pak-C₁₈（3.9×150mm，5μm）色谱柱为固定相，0.05mol／L磷酸二氢钾缓冲溶液和甲醇为流动相，在该色谱条件下，杨梅中主要的黄酮成分均达到基线分离；用紫外二极管矩阵检测器检测，并作了色谱峰纯度分辨。方法标准回收率为96％～106％，相对标准偏差为1.5％～2.3％。2固相萃取和高效液相色谱法测定甜茶中的黄酮类物质甜茶中的黄酮用80％乙醇溶液、固液比20:1、在80℃时回流提取3次，60分钟，提取液用Waters Sep-Pak-C₁₈固相萃取小柱预分离脱脂，以WatersNova-Pak-C₁₈（3.9×150mm，5μm）色谱柱为固定相，以0.05 mol·L^-1磷酸二氢钾缓冲溶液和甲醇的比例为40:60（V/V）为流动相，在该色谱条件下，甜茶中主要的黄酮成分均达到基线分离；用紫外二极管矩阵检测器检测，并作了色谱峰纯度分辨。方法标准回收率为97％～103％，相对标准偏差为0.87％～2.2％。3固相萃取和高效液相色谱法测定鱼腥草中的黄酮鱼腥草根和鱼腥草叶中的黄酮用80％乙醇溶液、固液比50:1、在80℃时回流提取3次，60分钟，提取液用Waters Sep-Pak-C₁₈固相萃取小柱预分离脱脂，以Waters Nova-Pak-C₁₈（3.9×150mm，5μm）色谱柱为固定相，以0.05 mol·L^-1磷酸二氢钾缓冲溶液和甲醇的比例为40:60（V/V）为流动相，在该色谱条件下，鱼腥草中主要的黄酮成分均达到基线分离；用紫外二极管矩阵检测器检测，并作了色谱峰纯度分辨。方法标准回收率为98％～101％，相对标准偏差为0.85％～2.2％。（三）固相萃取和高效液相色谱法测定中草药中的糖类物质研究了高效液相色谱法测定鱼腥草甜茶杨梅中的糖类物质，包含水溶性糖的成分含量、多糖的组成。水溶性糖样品用超声震荡浸取，浸取液用Waters Sep-pakC₁₈固相萃取小柱预分离，以Waters carbohydrate高效糖柱为固定相，乙腈：水=70:30为流动相分离，蒸发光散射仪为检测器检测，实现了8种水溶性单糖和二糖的同时测定；鱼腥草多糖、杨梅多糖用80℃水浴中加热提取3次，每次2h、液固比30:1，甜茶多糖用80℃水浴中加热提取3次，每次3h、液固比20:1，再进行醇沉得粗多糖。除蛋白、上凝胶层析分离柱得多糖纯品，用Waters Sep-pak C₁₈固相萃取小柱预分离，以Waters carbohydrate高效糖柱为固定相，乙腈：水=70:30为流动相分离，蒸发光散射仪为检测器检测，得到鱼腥草中鼠李糖:木糖:阿拉伯糖:果糖:半乳糖=1.075:1.000:1.952:1.965:3.619，甜茶中鼠李糖:木糖:阿拉伯糖:葡萄糖:半乳糖=1.000:1.317:1.739:3.254:2.725，杨梅中鼠李糖:木糖:阿拉伯糖:果糖葡:萄糖:半乳糖=1.000:1.507:1.993:12.635:1.642:1.394。（四）方法应用—甜茶、鱼腥草中黄酮分离、制备以固相萃取和高效液相色谱法测定甜茶和鱼腥草中的黄酮类物质的研究为基础，在此研究提供可靠、准确分析方法的基础上，以大孔树脂作为固相萃取法分离甜茶、鱼腥草黄酮的固定萃取剂，对甜茶、鱼腥草提取物进行预分离除去油脂类物质，蜡质，色素等弱极性物质，再进行制备色谱分离，得到黄酮单体。（五）甜茶、鱼腥草抗氧化抗过敏活性研究采用超氧阴离子自由基（O₂^-）、羟基自由基体系对鱼腥草、甜茶中的黄酮单体及总黄酮进行抗氧化活性的研究；采用透明质酸酶体外抑制实验和毛细血管通透性体内实验来分别对鱼腥草和甜茶黄酮单体及总黄酮进行了抗过敏活性研究。更多还原

【Abstract】 In this thesis, the study objects are several kinds of Chinese herbs, such as myricarubra, Houttuynia cordata and Yunnan Lithhocarpus polgstachrch Rehd, and studiedthrough analyzing their efficient compositions of several important abio-microelements,flavone and sugar. Solid phase extraction was used in the analysis of natural products,the study of reactivity,, and syntheses of a few agents. Through solid phase extractionlight intensity method, some new systems of analyzing methods are set up, such as thedetermination of cobalt, vanadium and silver; the determination of nickel, tin, copper,lead, cadmium and Hg; the determination of Fe, Cobalt, nickel, copper, Zn andmanganese; the determination of flavones and sugars in myrica rubra, Lithhocarpuspolgstachrch Rehd and Houttuynia cordata. The method is used in the study ofseparating flavone, reparation, anti-oxidation and the antiallergic activeites inLithhocarpus polgstachrch Rehd and Houttuynia cordata.（一） The synthesis of 2-quinoline azo agents and the application in measuringabio-microelements in Chinese herbsSix 2-quinoline azo agents were synthesized for the first time: 2-quinoline azophenol agent, 2-quinoline azo anlin agent and 2-quinoline azo benzoic acid agent.These have been proved to be the targeted products with infrared ray spectrum, massspectrum and core magnetism resonance. Through the developing feedback between thestudy reagents and metal hydronium, comparing the performances of the reagents, itwas found that the sensitivity of this kind of reagents was improved more than pyridineazo agent, thiazole azo agent, benzothiazole azo agent etc, heterocyclic azo agent. Therelationship between the structure and analyzed performances was estimated accordingto the stability and sensitivity and selectivity of the synthesis of ligand-OH、-NH₂、-COOH and metal hydronium. In the application in measuring abio-microelements inChinese herbal medicine, they are:1. The determination of cobalt, vanadium, silver in herbs with solid phaseextraction methodIn the Presence of masking agent, color reactions of 2-quinoline azo phenol agent with vanadium, 2-quinoline azo anlin agent with silver are very efficient. The complexproduced from these reactions was found hydrophobic and can be concentrated by solidphase column extractor. For the first time in China, the reverse key silica gel techniquewas applied in luminosity analysis of abio-elements. Accordingly, there are sixmethods-using solid phase extraction luminosity analysis of 2-（2-quinoline azophenol）-5-dimethylin （lignocaine） phenol to measure vandadium, solid phase extractionluminosity analysis of 2-（2-quinoline azo phenol）-5-dimethylin （lignocaine） anilin tomeasure silver, solid phase extraction luminosity analysis of 2-（2-quinoline azophenol）-5-dimethylin （lignocaine） benzoic acid to measure cobalt. As solid phaseextraction concentration is adopted in these methods, the amount being concentrated（100 times） is much geater than that of LLE and without the disadvantages of LLE.Using these methods, less organic solvent was consumed and the lower sensitivity ofabsorbance method can also be compensated. They are also environment-friendly.2. Solid phase extraction concentration followed by high performance liquidchromatography for the detetmination of nickel, tin, copper, lead, cadmium,mercury in Chinese herbsPorphine agent and Nickel, tin, silver, lead, cadmium, and mercury can becomestable complex compound, with high sensitivity, under the condition of alkalescence.Nickel, tin, silver, lead, cadmium, and mercury are derived from Terra-（4-aminophenyl）-porphine （T₄-APP） pre-column and determined with methods of solid phase extractionconcentration and high performance liquid chromatogaphy. Waters Xterra^TM RP₁₈column （pH1～12） is adopted as solid phase, alkaline buffer solution with pH value of 10is adopted as mobile phase to separate porphine heavy metal complex: This solves theproblem that such complex can only be separated by alkaline mobile phase and normalcolumn is unstable when pH value is greater than 8. The separation measurement ofnickel, tin, silver, lead, cadmium and mercury porphine complex were achieved for thefirst time.3. Solid phase extraction concentration followed by high performance liquidchromatography for the measurement of iron, cobalt, nickel, copper, zinc and manganese in Chinese herbsSensitivitY of the synthesized 2-quinoline azo and phenol has been expandedbecause of their conjugated system. They are poor in selectivity as luminosity analysisagent but suitable for liquid chromatography analysis. Conditions of color reaction andchromatography separation of 2-（2-quinoline azo phenol）-5-dimethylin （lignocaine）phenol with iron, cobalt, nickel, copper, zinc and manganese have been studied.2-（2-quinoline azo phenol）-5-dimethylin （lignocaine） phenol pre-column deriving, solidphase extraction and concentration, high performance liquid chromatograph), separationare first set up to measure iron, cobalt, nickel, copper, zinc and manganese in samples.This method is very sensitive and can measure many elements simultaneously.4. Relevant study with methodsThe established high performance liquid chromatography of measuring transitionmetal and heavy metal elements was related with the methods in the first part ofChinese Codex（published in 2005）. Relevant study was carried out. The study showsthat the two methods have good pertinency, with relevant modulus of 12 microelementsof over 0.9. The established method has certain reliability.（2） Solid phase extraction concentration followed by high performance liquidchromatography for the measurement of flavonoidThe method of Solid phase extraction concentration followed by high performanceliquid chromatography for the measurement of flavonoid was carried out.1 Measurement of flavonoid in myrica rubra by solid phase extraction andRP-HPLCThe flavonoid is extracted from the sample with 90% of methanol by heatingcircumfluence for 45 min. Then the extract was degreased by solid phase extractionwith Sep-Pak-C₁₈ cartridge. The flavonoid is separated on a waters Nova-Pak-C₁₈chromatogram column （3.9×150 mm, 5um）, with methanol and 0.05 mol/L potassiumdihydrogen phosphate buffer solution as mobile phase at a flow-rate of 0.5 ml/min, andmonitored with the photodiode array detector. The recoveries of the flavonoid are96%～106%, and relative standard deviations are 1.5%～2.3%. 2 Study on measurement of flavonoid in Lithhocarpus polgstachrch Rehd by solidphase extraction and RP-HPLCA high performance liquid chromatography （HPLC） method for the determinationof flavonoid in Lithhocarpus polgstaehrch Rehd has been studied. The flavonoid isextracted from the sample with 80% of ethanol, solid to liquid ratio 20:1, by refluxing 3times and 60 min. Then the extract was degreased by solid phase extraction withSep-Pak-C₁₈ cartridge. The flavonoid was separated on a Nova-Pak-C₁₈ chromatogramcolumn （3.9×150 mm, 5um）, with methanol and 0.05 mol/L potassium dihydrogenphosphate buffer solution （40:60） as mobile phase, and monitored with the photodiodearray detector. The recoveries of the flavonoid are 97%～103%, and relative standarddeviations are 0.87%～2.2%.3 Measurement of flavonoid in houttuynia by solid phase extraction and RP-HPLCA HPLC method for the determination of flavonoid in houttuynia has been studied.The flavonoid is extracted from the sample with 80% of ethanol, by heatingcircumfluence for 3 times and 60 min. Then the extract was degreased by solid phaseextraction with Sep-Pak-C₁₈ cartridge. The flavonoid is separated on a Nova-Pak-C₁₈chromatogram column （3.9×150 mm, Sum）, with methanol and 0.05 mol/L potassiumdihydrogen phosphate buffer solution （40:60） as mobile phase, and monitored with thephotodiode array detector. The recoveries of the flavonoid are 98%～101%, and relativestandard deviations are 0.85%～2.2%.（3） Measurement of sugars in Chinese herbal medicine by solid phase extractionand RP-HPLCDetermined sugars in Lithhocarpus polgstachrch Rehd and Houttuynia with HPLCconsists of water soluble sugars and the component of polysaccharides. The watersoluble sugars chromatography were extracted with water by ultrasonic oscillation andby solid phase extraction with Sep-pak C₁₈ cartridge. Sugars were separated on a Watershigh performance carbohydrate column as solid phase, with acetonitrile: water=70:30as mobile phase, and detected with evaporative light-scatter detector. The method wasapplied to the determination of water soluble monosaccharides and polysaccharides at the same time. The polysaccharides of Houttuynia and myrica rubra are extracted fromthe sample with water, by heating circumfluence of 80℃for 3 times and 2h and theratio of liquid and solid is 30:1. The polysaccharides of Lithhocarpus polgstachrch Rehdare extracted from the sample with water, by heating circumfluence of 80℃for 2 timesand 3h and the ratio of liquid and solid is 20:1. The samples were precipitated byethanol and eliminated protein and separated by gel chromatogaphy. The samples wereextracted with water by ultrasonic oscillation and by solid phase extraction withSep-pak C₁₈ cartridge. Sugars were separated on a Waters high performancecarbohydrate column as solid phase, with acetonitrile: water=70:30 as mobile phase,and detected with evaporative light-scatter detector. The components of polysaccharidesof Houttuynia are rhamnose:xylose:arabinose:fructose:galactose=1.075:1.000:1.952:1.965:3.619, in Lithhocarpus polgstachrch Rehd arerhamnose:xylose:arabinose:glucose:galactose=1.000:1.317:1.739:3.254:2.725, in myricarubra are rhamnose:xylose:arabinose:fructose:glucose:galactose=1.000:1.507:1.993:12.635:1.642:1.394.（4） Applications-flavonoid separation, preparation studyBased on the measurements of flavonoid by solid phase extraction and RP-HPLC,when preparing chromatography, first macroporous resin was adopted for pre-separationof flavonoid extract, then chromatography oil, waxiness, pigment etc, weak polarmaterial are separated from Lithhocarpus polgstachrch Rehd and Houttuynia. Thenchromatogram separation was prepared and flavonoid monocase was got.（5）The study of antioxidation and antiallergic activities of Lithhocarpuspolgstaehrch Rehd and HouttuyniaThe system ofsuperoxide anions redical （O₂^-）, hydroxyl radical is used to studyantioxidation activities of flavonoid monocase and flavone in Lithhocarpus polgstachrchRehd and Houttuynia through the experiments of outside experiment of measruig theirinhibitory effects on hyaluronidase and the inside experiment of mice’s blood capillarypenatrativity increased.更多还原