【作者】 渠琛玲；

【导师】 张寒琦；

【作者基本信息】 吉林大学 ， 分析化学， 2009， 博士

【摘要】 本文研究了西洋参中的人参皂苷和氨基酸随参龄和部位的变化以及西洋参有效成分人参皂苷与生物分子的相互作用。采用高压微波辅助提取技术提取西洋参中的人参皂苷,并用高效液相色谱(HPLC)-蒸发光散射检测器(ELSD)对12种人参皂苷进行含量测定,其中包括西洋参的特征性成分F11。使用高压微波水解西洋参中的蛋白,并用2, 4-二硝基氟苯微波衍生,采用HPLC-UV对3-5年生西洋参各部位中西洋参中18种氨基酸进行测定。建立了快速测定氨基酸的方法,大大缩短了氨基酸分析时间,并为氨基酸的在线衍生奠定了基础。用电喷雾质谱法研究了人参皂苷与溶菌酶的相互作用。用质谱峰强度直接计算人参皂苷-溶菌酶形成非共价复合物的解离常数。并研究了不同竞争体系下解离常数的差别。用电喷雾质谱法研究了药物分子与18种氨基酸的相互作用,得到了药物-氨基酸复合物的解离常数;并基于理论计算的结果,提出了两种结合模式,为找到药物与蛋白的结合位点及结合方式提供了依据。用质谱法研究了18种α-氨基酸的碎裂,得到了18种氨基酸在正离子模式下和负离子模式下的裂解规律。更多还原

【Abstract】 American ginseng was widely studied for its so many pharmacological actions because it can enhance immunologic function of organism, has anti-weary, anti-oxygen deficit and cardiac muscle protective function, has anti-heart rate abnormal, anti-tumor, anti-atherosclerosis function, and can reduce the blood sugar et al.. The main effective constituents in American ginseng are ginsenosides. There are more than 20 kinds of amino acids in American ginseng, and some of them can not be synthesized by human body itself. So the functions of amino acids in American ginseng can not be ignored. The investigations of the contents of ginsenosides and amino acids in different ages and differents parts of American ginseng can provides the foundation for reasonably planting, expanding medicinal parts of American ginseng and producting medicine.The interactions of drug and protein are of great significance in pharmacokinetics and clinical pharmacology, which made it an active research area. In this thesis the noncovalent interactions of lysozyme and ginsenosides were investigated. In order to evaluate the interactions of ginsenosides and proteins which are composed ofα-amino acids, electrospray ionization mass spectrometry was employed to study the noncovalent interactions between ginsenosides and 18 kinds ofα-amino acids.In the second chapter, high pressure microwave-assisted extraction(HPMAE) was applied for extracting ginsenosides from American ginseng. The ginsenosides Rg1, Re, F11, Rf, Rg2, Rh1, Rb1, Rc, Rb2, Rb3, Rd, Rh2 were determined by high pressure liquid chromatography coupled with a evaporative high scattering detector(HPLC-ELSD). Two extraction methods, microwave-assisted extraction and Soxlet extraction were compared. Except that the extraction efficiencies of ginsenosides Rg2,Rb1 and Rc obtained by HPMAE were lower than those obtained by Soxlet extraction, the extraction efficiencies of other ginsenosides obtained by HPMAE were all higher than those obtained by Soxhlet extraction. HPMAE required only 10 min, but Soxhlet extraction required 5 h. The results showed that the contents of ginsenosides in the leaf were higher than those in other parts, such as root, rhizome, stem. So the leaf of American ginseng can be a new resouce for ginsenosides. The total content of these 12 kinds of ginsenosides in main root increases with years and that in leaf is not related to years.In the third chapter, the proteins in American ginseng was hydrolyted by the high pressure microwave-assisted hydrolyzation(HPMAH). The contents of 18 kinds of amino acids(Asp, Glu, Ser, His, Gly, Pro, Ala, Val, Met, Cys, Ile, Leu, Trp, Phe, Lys, Tyr) in different parts of 3-5 year old American ginseng were determined by HPLC-UV. Microwave-assisted hydrolytion and microwave-assisted derivation were compared with traditional methods. To get the same results, microwave-assisted hydrolytion required only 15 min but traditional hydrolyzation required 24 h. The microwave-assisted derivation cost only 30 s but traditional derivation required 1 h. A new determinion method for amino acids was established. The analysis time was greatly reduced and the online derivation for determination of amino acids should be possible.In the fourth chapter, the noncovalent binding of lysozyme (Ly) and ginsenoside Rg1,Re,Rd,Rh2 was studied by electrospray ionization mass spectrometry. The dissociation constants of the noncovalent complexes were directly calculated based on the peak intensities of the lysozyme and the complexes of lysozyme and ginsenoside in mass spectra. The dissociation constants in different systems were in the main consistent, which shows that this method was reliable. It can be concluded that the stronger the peak of the complex, the better the precision. Based on the results given in the fifth chapter (the acidic and basic amino acids, including Asp, Glu, Lys and Arg, can be bound to ginsenosides more strongly than other amino acids), and the structure of lysozyme (the molecular surface has a deep, long and narrow scoop channel, whose size can hold 6 monosaccharide unit of the polysaccharide substrate exactly), we deduced that ginsenosides interacts with the two active sites GLu35 and Asp52 of lysozyme.In the fifth chapter, electrospray ionization mass spectrometry was employed to study the noncovalent interactions between ginsenosides (Rb2, Rb3, Re, Rg1 and Rh1) and 18 kinds ofα-amino acids (Asp, Glu, Asn, Phe, Gln, Thr, Ser, Met, Trp, Val, Gly, Ile, Ala, Leu, Pro, His, Lys and Arg). The 1:1 and 2:1 noncovalent complexes of ginsenosides and amino acids were observed in the mass spectra. The dissociation constants for the noncovalent complexes were directly calculated based on peak intensities of ginsenosides and the noncovalent complexes in the mass spectra. Based on the dissociation constants, it can be concluded that the acidic and basic amino acids, Asp, Glu, Lys and Arg, can be bound to ginsenosides more strongly than other amino acids. The experimental results were verified by theoretical calculations of parameters of noncovalent interaction between ginsenoside Re and Arg which served as a representative example. Two kinds of binding forms,“head-tail”(“H-T”) and“head-head”(“H-H”), were proposed to explain the interaction between ginsenosides and amino acids. And the interaction in“H-T”form was stronger than that in“H-H”form.In the sixth chapter, electrospray ionization tandem mass spectrometry (MS/MS) was applied to study fragmentation pathways of 18 kinds ofα-amino acids, including Asp, Asn, Glu, Gln, Gly, Ala, Ser, Thr, His, Met, Val, Leu, Ile, Phe, Trp, Lys, Arg and Pro in positive and negative ion mode. The group–NH2 and–COOH which joined toα-C were found to be more inclined to be fragmented, but not the side chain. The peak intensities of molecular ions in positive ion mode are stronger than these in negative mode.更多还原