金线莲多糖的分离纯化、结构表征及其抗肿瘤活性
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摘要
本论文采用超声法提取金线莲多糖(A. roxburghii Polysaccharide, ARPS)并测定其含量;同时应用高速逆流色谱( High-speed counter current chromatography,HSCCC)分离纯化ARPS,并对其结构进行分析和表征;应用柱前衍生化高效液相色谱法(High performance liquid chromatography, HPLC)分析ARPS的单糖组成及其摩尔比。体外实验检测ARPS对5种肿瘤细胞增殖的抑制率,并研究对抑制作用最强的K562细胞的形态学影响,初步探讨其作用机制。
本实验分为四部分:
(一)金线莲多糖苯酚-硫酸法测定条件的优化用超声法提取ARPS,以苯酚-硫酸法进行含量测定,葡萄糖为标准品,检测波长为490 nm,利用正交设计方法对测定条件进行优化,最佳测定条件为1.0 mL样液,加5 %苯酚液0.6 mL、硫酸6.0 mL,100℃水浴保温15 min,测得金线莲超声提取物中粗多糖含量为12.09%。
(二)高速逆流色谱分离纯化金线莲多糖及其结构表征采用HSCCC分离纯化ARPS。两相溶剂系统为:12.5% PEG 1000-20 %磷酸钾(pH = 6.8)双水相体系,逆流色谱仪转速为850 r/min,流动相流速1.0 mL/min,固定相保留率30.3 %。从100 g金线莲粉末中分离纯化得到2 g ARPS。经紫外光谱分析、凝胶柱层析法分析示ARPS是分子量分布均一的多糖组分。测定得其分子量为18197,柱前衍生化高效液相色谱法分析其单糖是由甘露糖、鼠李糖、半乳糖、阿拉伯糖和岩藻糖组成,且这5种单糖的摩尔比值为4.43: 14.04: 1.00: 2.28: 6.58。
(三)金线莲多糖抗肿瘤作用体外实验ARPS对人慢性白血病粒细胞K562、肝癌HepG-2、胃癌SGC-7901、胃癌MGC-803和大肠癌HCT-116细胞株生长的影响,采用CCK-8法检测ARPS对这5种肿瘤细胞增殖的抑制率,发现ARPS对胃癌MGC-803和大肠癌HCT-116细胞株抑制效果并不理想,实验测得对前三种细胞的增殖抑制率的IC50分别是19.28、34.45和53.93 nmol/mL, ARPS对K562细胞生长影响最强。初步探讨ARPS对K562作用机制。
(四)综述:中草药多糖提取分离纯化研究进展综述了多糖制备常用的提取与纯化工艺与新技术的应用进展,分析了各种方法的优缺点并探讨了发展前景。
In this paper, polysaccharide from Anoectochilus roxburghii (ARPS) was obtained with the ultrasound extraction, the content determination of them were studied. The High-speed counter current chromatography(HSCCC)was successlly applied to separate and purify ARPS. The structural identification of ARPS was confirmed. HPLC method was described for the quantitative analysis and molar ratio of component monosaccharides of ARPS. The anticancer effects of ARPS on five tumor cells and morphology effect of K562 which had the strongest reaction with ARPS were investigated. The antitumor activity on K562 and its mechanism of ARPS was approached initially.
This thesis includes four parts as follows:
PartⅠ: Optimization of Phenol-Sulfuric Acid Determination Conditions of Polysaccharide in Anoectochilu roxburghii(wall) Lindl by Orthogonal Test
Polysaccharide was obtained with the ultrasound extraction and alcohol precipitation method. Taking phenol-sulfuric acid method to the content determination, and glucose as parameters. The absorbance was measured at 490 nm. L9(34) Orthogonal Design optimum determined conditions. When the sample solution used 1.0 mL, 0.6 mL of 5% solution of phenol, sulfuric acid 6.0 mL, 100℃water bath for 15 min reaction conditions determination, the results is best. The content of crude polysaccharide was 12.09% in A. roxburghii.
PartⅡ: High Speed Countercurrent chromatographic purification of polysaccharides from Anoectochilu roxburghii (wall) Lindl with an aqueous two-phase system and the structure representation of ARPS
Polysaccharides were purified from A. roxburghii by HSCCC. The two phase system consisted of an aqueous polymer two-phase system composed of 12.5% (w/w) polyethylene glycol (PEG) 1000, 10% (w/w) potassium dihydrogen phosphate, and 10% (w/w) dipotassium hydrogen phosphate buffer at pH 6.8. An orthogonal design was used for optimizing the revolution speed of the separation column, flow rate of the mobile phase and separation temperature, which were 850 r/min, 1.0 mL/min and 25℃, respectively. UV Spectrum and Sephadex G-100 analysis of the fractions collected by preparative HSCCC showed that ARPS was homogeneous fractions related to molecular weight. The preparative separation of A. roxburghii (100 g) was successfully performed, yielding 2 g of polysaccharides at high purity. The structural identification of ARPS was confirmed by FT-IR and 1HNMR. The average molecular weight of ARPS was 18197 which determined by gel chromatography. HPLC method was described for the quantitative analysis of component monosaccharides of ARPS which was carried out on a RP-C18 column using precolumn derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP), and the result showed that ARPS was a typical heteropolysaccharide and was composed of rhamnose, xylose, galactose, fucose and arabinose. As to their peak area, the corresponding molar ratio was 4.43: 14.04: 1.00: 2.28: 6.58, respectively.
PartⅢ: Anticancer activities of the polysaccharide from A.roxburghii.
We investigated the anticancer effects of ARPS on human chronic leukemia K562 cells, liver carcinoma HepG-2 cells, gastric carcinoma SGC-7901 cells, MGC-803 cells and colon carcinoma HCT-116 cells. Results showed that the IC50 on the anterior three cells were 19.28,34.45 and 53.93 nmol/mL, respectively. The strongest effect on the mentioned cells is K562. The antitumor activity on K562 and its mechanism of ARPS was approached initially.
PartⅣ: A review of the research about the extract method and technology of polysaccharide from Chinese herbal medicine was given.
本实验分为四部分:
(一)金线莲多糖苯酚-硫酸法测定条件的优化用超声法提取ARPS,以苯酚-硫酸法进行含量测定,葡萄糖为标准品,检测波长为490 nm,利用正交设计方法对测定条件进行优化,最佳测定条件为1.0 mL样液,加5 %苯酚液0.6 mL、硫酸6.0 mL,100℃水浴保温15 min,测得金线莲超声提取物中粗多糖含量为12.09%。
(二)高速逆流色谱分离纯化金线莲多糖及其结构表征采用HSCCC分离纯化ARPS。两相溶剂系统为:12.5% PEG 1000-20 %磷酸钾(pH = 6.8)双水相体系,逆流色谱仪转速为850 r/min,流动相流速1.0 mL/min,固定相保留率30.3 %。从100 g金线莲粉末中分离纯化得到2 g ARPS。经紫外光谱分析、凝胶柱层析法分析示ARPS是分子量分布均一的多糖组分。测定得其分子量为18197,柱前衍生化高效液相色谱法分析其单糖是由甘露糖、鼠李糖、半乳糖、阿拉伯糖和岩藻糖组成,且这5种单糖的摩尔比值为4.43: 14.04: 1.00: 2.28: 6.58。
(三)金线莲多糖抗肿瘤作用体外实验ARPS对人慢性白血病粒细胞K562、肝癌HepG-2、胃癌SGC-7901、胃癌MGC-803和大肠癌HCT-116细胞株生长的影响,采用CCK-8法检测ARPS对这5种肿瘤细胞增殖的抑制率,发现ARPS对胃癌MGC-803和大肠癌HCT-116细胞株抑制效果并不理想,实验测得对前三种细胞的增殖抑制率的IC50分别是19.28、34.45和53.93 nmol/mL, ARPS对K562细胞生长影响最强。初步探讨ARPS对K562作用机制。
(四)综述:中草药多糖提取分离纯化研究进展综述了多糖制备常用的提取与纯化工艺与新技术的应用进展,分析了各种方法的优缺点并探讨了发展前景。
In this paper, polysaccharide from Anoectochilus roxburghii (ARPS) was obtained with the ultrasound extraction, the content determination of them were studied. The High-speed counter current chromatography(HSCCC)was successlly applied to separate and purify ARPS. The structural identification of ARPS was confirmed. HPLC method was described for the quantitative analysis and molar ratio of component monosaccharides of ARPS. The anticancer effects of ARPS on five tumor cells and morphology effect of K562 which had the strongest reaction with ARPS were investigated. The antitumor activity on K562 and its mechanism of ARPS was approached initially.
This thesis includes four parts as follows:
PartⅠ: Optimization of Phenol-Sulfuric Acid Determination Conditions of Polysaccharide in Anoectochilu roxburghii(wall) Lindl by Orthogonal Test
Polysaccharide was obtained with the ultrasound extraction and alcohol precipitation method. Taking phenol-sulfuric acid method to the content determination, and glucose as parameters. The absorbance was measured at 490 nm. L9(34) Orthogonal Design optimum determined conditions. When the sample solution used 1.0 mL, 0.6 mL of 5% solution of phenol, sulfuric acid 6.0 mL, 100℃water bath for 15 min reaction conditions determination, the results is best. The content of crude polysaccharide was 12.09% in A. roxburghii.
PartⅡ: High Speed Countercurrent chromatographic purification of polysaccharides from Anoectochilu roxburghii (wall) Lindl with an aqueous two-phase system and the structure representation of ARPS
Polysaccharides were purified from A. roxburghii by HSCCC. The two phase system consisted of an aqueous polymer two-phase system composed of 12.5% (w/w) polyethylene glycol (PEG) 1000, 10% (w/w) potassium dihydrogen phosphate, and 10% (w/w) dipotassium hydrogen phosphate buffer at pH 6.8. An orthogonal design was used for optimizing the revolution speed of the separation column, flow rate of the mobile phase and separation temperature, which were 850 r/min, 1.0 mL/min and 25℃, respectively. UV Spectrum and Sephadex G-100 analysis of the fractions collected by preparative HSCCC showed that ARPS was homogeneous fractions related to molecular weight. The preparative separation of A. roxburghii (100 g) was successfully performed, yielding 2 g of polysaccharides at high purity. The structural identification of ARPS was confirmed by FT-IR and 1HNMR. The average molecular weight of ARPS was 18197 which determined by gel chromatography. HPLC method was described for the quantitative analysis of component monosaccharides of ARPS which was carried out on a RP-C18 column using precolumn derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP), and the result showed that ARPS was a typical heteropolysaccharide and was composed of rhamnose, xylose, galactose, fucose and arabinose. As to their peak area, the corresponding molar ratio was 4.43: 14.04: 1.00: 2.28: 6.58, respectively.
PartⅢ: Anticancer activities of the polysaccharide from A.roxburghii.
We investigated the anticancer effects of ARPS on human chronic leukemia K562 cells, liver carcinoma HepG-2 cells, gastric carcinoma SGC-7901 cells, MGC-803 cells and colon carcinoma HCT-116 cells. Results showed that the IC50 on the anterior three cells were 19.28,34.45 and 53.93 nmol/mL, respectively. The strongest effect on the mentioned cells is K562. The antitumor activity on K562 and its mechanism of ARPS was approached initially.
PartⅣ: A review of the research about the extract method and technology of polysaccharide from Chinese herbal medicine was given.
引文
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