| Yinhuang preparation, an antimicrobial and antipyretic herbal preparation, is a combined herbal remedy comprised of two herbs:Lonicera japonica Thunb and Scutellariae baicalensis Georgi. Lonicera japonica Thunb and Scutellariae baicalensis Georgi are two commonly used traditional Chinese medicines (TCM). The two materials contain many active ingredients. They have antioxidant, scavenging free radicals, anti-inflammatory and other effects. They are principally used in the treatment more than 40 kinds of diseases such as a variety of infections, all kinds of inflammation, heat disease, cancer radiotherapy, erysipelas. Therefore, the active ingredients in Yinhuang preparation was studied. It can be effectively applied to the quality control.In this paper, the extract methods of Lonicera japonica Thunb and Scutellariae baicalensis Georgi were screened, extraction conditions were optimized, and the liquid chromatographic condition was studied for the separation of mixed extract. Finally, the off-line two-dimensional liquid chromatography and LC-MS technique were applied for the separation and analysis of chemical components in the mixed extract.This paper is composed of six chapters as follows:In Chapter 1, the background and significance of thesis were introduced briefly. Including the chemical constituents and pharmacological activities of Lonicera japonica Thunb and Scutellariae baicalensis Georgi, principle and advances in studies on multi-dimensional HPLC. The important significance of development of two-dimensional liquid chromatography was elaborated for the separation of complex samples, especially herbal medicine.In Chapter 2, the extraction method of Lonicera japonica Thunb was developed and the extraction condition was optimized. The optimization of factors is composed of the extraction solvent, the extraction method, the extraction time, the ratio of liquids to solvents and extraction power. The optimum extraction conditions were as follows:ratio of 70% methanol solution to material 40; the extraction time,1 min; extraction four times; microwave power 400 W.In Chapter 3, the extraction method of Scutellariae baicalensis Georgi was developed and the extraction condition was optimized. The optimization of factors is composed of the extraction solvent, the extraction method, the extraction time, the ratio of liquids to solvents and extraction power. The optimum extraction conditions were as follows:ratio of 70% methanol solution to material 80; extraction time, 1min; extraction three times; microwave power 640 W.In Chapter 4, the chromatographic condition was optimized for the separation of the Lonicera japonica Thunb and Scutellariae baicalensis Georgi mixed extract.11 representative peaks were selected for the study of methodology. The optimized chromatographic condition is:column:250 mm×4.6 mm i.d.; mobile phase:methanol (A) and water with 0.1% (v/v) phosphoric acid (B); gradient conditions:0-10 min,20%A-35%A; 10-24 min,35%A-55%A; 24-35 min,55%A-70%A; 35-40 min,70%A-70%A; 40.01-45 min,20%A-20%A; detection wavelength:254 nm. The flow rate was set at 1.0 mL/min, and the injection volume was 10μL.The mixed extract of Lonicera japonica Thunb and Scutellariae baicalensis Georgi was separated well in the above condition. The result demonstrated that the method is simple, rapid and accurate. It might be used for the comprehensive quality control of the preparation.In Chapter 5, an approach of two-dimensional liquid chromatography was established for the separation compounds in the mixed extract of Lonicera japonica Thunb and Scutellariae baicalensis Georgi. A CN column was used as first dimensional column and a Ci8 column was used as second dimensional column. The results demonstrated that high peak capacity and high resolution were achieved.In Chapter 6, LC-MS technique was used in the identification of components in the mixed extract of Lonicera japonica Thunb and Scutellariae baicalensis Georgi.In conclusion, LC-MS and 2D-HPLC technique were successfully used in the separation on the mixed extract of Lonicera japonica Thunb and Scutellariae baicalensis Georgi. Component analysis was carried out by liquid chromatography-mass spectrometry. It showed that the method established is suitable for the separation and analysis of traditional Chinese medicine and other complex samples. |
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