Solid Phase Extraction For Herbal Metal Ions And Active Ingredients Analytical Methods

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.