紫芝糖蛋白化学结构及抗肿瘤活性的研究
摘要
采用水提醇沉得到四个不同浓度的乙醇沉淀,通过对其理化性质的研究得知其中主要含有中性糖和蛋白质,而药理结果证明醇沉淀淀部分是紫芝抗肿瘤的主要成分。醇沉淀通过除蛋白、超滤法证明了其活性成分是大分子的糖蛋白部分。采用还原乙酰化测定了他们的单糖组成,发现他们均含有Fuc、Ara、Xyl、Man、Gal、Glu。通过离子交换柱和凝胶柱层析分离纯化出纯化的糖蛋白。并用Hakomori法研究了纯化的紫芝糖蛋白的中性多糖部分的平均结构,结果表明紫芝糖蛋白中糖部分是以(1-3)连接的Glu为主链。
GanodermaSinensis or Chinese Ganoderma, is the sporophore of Ganoderma sinense in the Polyporaceae. For its great value in nutrition, health maintenance, and medical treatment, it has been accepted as a precious traditional Chinese medicine. It is demonstrated in the modern pharmaceutics and clinics that Ganoderma has the function in resisting oncosis and senility together with decreasing the blood glucose and the blood fat. Confirmed by the scientific research, it is the polysaccharides in Ganoderma that plays the above functions. In order to illustrate the structure of a specific saccharide, it is required to be characterized in the following six aspects: (1) Molecular mass; (2) the compositive glycose; (3) the location of the glucosidic bond within the glycose residues; (4) the ligation way of the glycose residues; (5) the kind of the circular structure; (6) the configuration of the glucosidic bond.
In this research we analyzed the supernatant and sediments by the means of ethanol gradient precipitation. The supernatant is mainly composed by the matters of smaller molecules, such as the glycose, oligose and ect, however, the sediments are mainly the polysaccharides. From the previous reports, the main pharmaceutical component is also the polysaccharide, which is the reason why we applied detailed research in the sediments. Firstly, we take the assaying of the neutral saccharide, protein and the glycuronate, the result of which showed that the former two components respectively occupied 40% and 20 % with the lowest contents of the glycuronate. Through the ultrafiltration and the deproteinization, the polysaccharide mainly contains the glycoprotein, the larger one of which is the main components of GanodermaSinensis used as resistance of the oncosis. To further analysis, we adopted the chemical components and molecular mass research for the different concentration ethanol-precipitated sediments. Final result showed all the sediments contained the fucose, arabinose, xylose, mannose, galactose and glucose which proved they belong to the homologous series of the glycoprotein, only with distinct mole ratios and different molecular mass. For deeper analysis of the complicate structure of the glycoprotein, we got the purified glycoprotein through the process of isolation by DEAE- Sephadex A-50 and subsequent purification by Sephadex G-150. Finally, using the PMP derivatization and methylation, we understand, in its principal chain, it is (1-3) glucose composition.
GanodermaSinensis or Chinese Ganoderma, is the sporophore of Ganoderma sinense in the Polyporaceae. For its great value in nutrition, health maintenance, and medical treatment, it has been accepted as a precious traditional Chinese medicine. It is demonstrated in the modern pharmaceutics and clinics that Ganoderma has the function in resisting oncosis and senility together with decreasing the blood glucose and the blood fat. Confirmed by the scientific research, it is the polysaccharides in Ganoderma that plays the above functions. In order to illustrate the structure of a specific saccharide, it is required to be characterized in the following six aspects: (1) Molecular mass; (2) the compositive glycose; (3) the location of the glucosidic bond within the glycose residues; (4) the ligation way of the glycose residues; (5) the kind of the circular structure; (6) the configuration of the glucosidic bond.
In this research we analyzed the supernatant and sediments by the means of ethanol gradient precipitation. The supernatant is mainly composed by the matters of smaller molecules, such as the glycose, oligose and ect, however, the sediments are mainly the polysaccharides. From the previous reports, the main pharmaceutical component is also the polysaccharide, which is the reason why we applied detailed research in the sediments. Firstly, we take the assaying of the neutral saccharide, protein and the glycuronate, the result of which showed that the former two components respectively occupied 40% and 20 % with the lowest contents of the glycuronate. Through the ultrafiltration and the deproteinization, the polysaccharide mainly contains the glycoprotein, the larger one of which is the main components of GanodermaSinensis used as resistance of the oncosis. To further analysis, we adopted the chemical components and molecular mass research for the different concentration ethanol-precipitated sediments. Final result showed all the sediments contained the fucose, arabinose, xylose, mannose, galactose and glucose which proved they belong to the homologous series of the glycoprotein, only with distinct mole ratios and different molecular mass. For deeper analysis of the complicate structure of the glycoprotein, we got the purified glycoprotein through the process of isolation by DEAE- Sephadex A-50 and subsequent purification by Sephadex G-150. Finally, using the PMP derivatization and methylation, we understand, in its principal chain, it is (1-3) glucose composition.
引文
1. 张树政,糖生物学:生命科学中的新前沿,生命的化学,1999,19(3):103-106。
2. 张惟杰,糖复合物生化研究技术。浙江大学出版社,1999,第二版。
3. 李建武,萧能赓,余瑞元等。生物化学实验原理方法。北京:北京大学出版社。1994,160-176。
4. 齐慧玲,Sevag 法去除白及多糖中蛋白的研究。天津化工,2000,3:20。
5. Thomas M.Jones.,Peter Albersheim, a gas chromatographic method for the determination of aldose and uronic acid consitituents of plant cell wall polysaccharides, Plant Physiol.,1972,49:926-936
6. Thomas J.Waeghe,Alan G.Darvill,Michel Mcneil,et.al,determin-
7. ation,by metylation analysis,of the glycosyllinkage compsition of microgram quantities of complex carbohydrates,Carbohydrate Research, 1983, 123:281-304.
8. 姜瑞芝,高其品,焦连庆等,多糖甲基化方法研究,长白山中医药研究与开发,1994,3(2):39-42。
9. Kalyan Rao Anumula,Paul B.Taylor,Acomprehensive proced-ure for preparation of partially methylated alditol acetates from glycoprotein carbohydrates,Analytical Biochemistry,1992,203:101-108.
10. Hakomori,S.a rapid permethylation of glycolipid, and polysaccharide catalyzed by methylsulfinyl cabanion in dimethyl sulfoxide,J Biochem.(Tokyo),1964,55(22):205-208.
2. 张惟杰,糖复合物生化研究技术。浙江大学出版社,1999,第二版。
3. 李建武,萧能赓,余瑞元等。生物化学实验原理方法。北京:北京大学出版社。1994,160-176。
4. 齐慧玲,Sevag 法去除白及多糖中蛋白的研究。天津化工,2000,3:20。
5. Thomas M.Jones.,Peter Albersheim, a gas chromatographic method for the determination of aldose and uronic acid consitituents of plant cell wall polysaccharides, Plant Physiol.,1972,49:926-936
6. Thomas J.Waeghe,Alan G.Darvill,Michel Mcneil,et.al,determin-
7. ation,by metylation analysis,of the glycosyllinkage compsition of microgram quantities of complex carbohydrates,Carbohydrate Research, 1983, 123:281-304.
8. 姜瑞芝,高其品,焦连庆等,多糖甲基化方法研究,长白山中医药研究与开发,1994,3(2):39-42。
9. Kalyan Rao Anumula,Paul B.Taylor,Acomprehensive proced-ure for preparation of partially methylated alditol acetates from glycoprotein carbohydrates,Analytical Biochemistry,1992,203:101-108.
10. Hakomori,S.a rapid permethylation of glycolipid, and polysaccharide catalyzed by methylsulfinyl cabanion in dimethyl sulfoxide,J Biochem.(Tokyo),1964,55(22):205-208.