| Saponin, as a class of compounds with higher content in Traditional Chinese Medicine, have various activities, such as anti-tumor, antimicrobial, the treatment of cardiovascular diseases, regulating body metabolism and immunity, so they are the index component to evaluate the quality of Traditional Chinese Medicine. However, due to the special structure of saponin compounds, the lack of characteristic UV absorption groups, they reveal end absorption, and are difficult to determine, as one of the difficulties of quality control for Traditional Chinese Medicine.In recent years, derivatization technology develop rapidly, has been widely used in the analysis of amino acids, fatty acids, alkaloids, esters, alcohols, aldehydes, sugars, enantiomers and so on, it was also used in the analysis of saponins for Traditional Chinese Medicine. For saponins are main active component in Radix polygalae, but they are difficult to determine using existing methods, so we selected Radix polygalae as the inspection object, senegenin which is the hydrolysis products of Saponins in hydrochloric acid as the basic object, use existing equipment, preliminarily study the pre-column derivatization HPLC-UV method for determinating senegenin, the method offer available reference for the determination of saponins.The main research content and methods as follows:1. we selected Radix polygalae as the basic object of study, investigated the conditions of degreasing, extraction, hydrolization for Radix polygalae. Then we studied the optimum conditions of preparing senegenin by macroporous resin column chromatography, silica gel column chromatography, MPLC. At last, we made it and identified by ultraviolet UV, MS,'H NMR, determinated the purity of senegenin by HPLC-UV area normalization method.2. We selected senegenin as the basic object of study, studied on the derivatization reaction, investigated the derivatization reagent, reaction monitoring method, the ways of derivatization reaction, solvent, derivative reactant ratio, reaction time and so on. Finally we selected the optimum conditions of reaction conditions:magnetic stirring, methylene chloride, ice water bath,4-dimethylaminopyridine (DMAP) as catalyst, vpyridine:msenegenin = 50:1, mbenzoyl chloride:msenegenin= 60:1, reacting for 1 hour. Under these conditions, senegenin is completely reacted.3. Senegenin was the basic object of study, senegenin derivatives was synthesized, based on the optimal reaction conditions, studied the method for preparing derivative of senegenin through silica gel column chromatography, gel column chromatography, semi-preparative HPLC, and made sterling, characterized derivative by UV, MS,1H NMR, named it 2,3-bis(benzoyloxy)-senegenin, finally we determinated the purity of senegenin by HPLC-UV area normalization method, its purity conforms to chemical reference substance, so providing chemical reference substance for pre-column derivatization HPLC-UV determination.4. We established the method of Pre-column derivatization HPLC-UV for the determination of senegenin in Radix polygalae, based on the optimal reaction conditions of senegenin, chromatographic conditions:Column: Diamonsil (diamond) C18 (200 mm×4.6 mm,5μm); mobile phase:methanol-0.05% phosphoric acid (80:20); flow rate:1.0 ml·min-1; detection wavelength:230 nm; column Temperature:room temperature; injection volume:20μl. Results:The linear equation was Y=2431 OX+2.116 (correlation coefficient r=0.9995), linear range:0.0165～0.33 ml·min-1, the minimum detectable concentration was 0.12μg·ml-1, the minimum detection limit was 2.3 ng. The method is accurate, sensitive.The method of pre-column derivatization HPLC-UV for the determination of senegenin in Radix polygalae, avoid the problem of end absorption. The method is sensitive, however, it needs further studies on some aspects. |
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