龙眼肉多糖硫酸酯化修饰及修饰前后体外抗肿瘤活性初步研究
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摘要
目的:从龙眼肉中提取、纯化得到龙眼肉多糖,对其进行硫酸酯化结构修饰,并测定修饰前、后龙眼肉多糖的体外抗肿瘤活性。
方法:通过热水浸提法提取龙眼肉多糖,Sevag-木瓜蛋白酶法除龙眼肉多糖蛋白,H_2O_2法除龙眼肉多糖色素,苯酚-硫酸法测龙眼肉多糖的多糖含量,DEAE-纤维素柱层析纯化龙眼肉多糖,龙眼肉多糖A的纯度用醋酸薄膜纤维电泳和比旋光度法检测,采用浓硫酸法对龙眼肉多糖A进行硫酸酯化结构修饰,L9(3~4)正交设计对浓硫酸法的反应温度、反应时间及正丁醇与浓硫酸的反应试剂等条件进行优选,硫酸钡-比浊法测定其硫酸基含量,DEAE-纤维素柱层析纯化硫酸酯化龙眼肉多糖(LYRP4-1),通过凝胶色谱法测定龙眼肉多糖A和LYRP4-1的分子量,MTT法体外测定龙眼肉多糖A和LYRP4-1对鼻咽癌HONE1细胞的抑制作用,苏木素-伊红染色和流式细胞仪观察鼻咽癌HONE1细胞凋亡情况。
结果:热水浸提法的龙眼肉多糖得率为6.30%,龙眼肉总糖含量为58.72%。龙眼肉多糖清除蛋白率为52.30%。DEAE-纤维素柱层析纯化流分龙眼肉多糖A得率为64.13%、龙眼肉多糖B得率为4.85%和龙眼肉多糖C得率为15.43%。醋酸薄膜纤维电泳和比旋光度法检测龙眼肉多糖A为均一多糖。硫酸酯化反应经L9(3~4)正交设计得到当反应时间为3h,反应温度为10℃,反应酯化试剂比为3:1时,浓硫酸酯化反应可以得到较高的取代度,取代度为2.03。LYRP4的pH值为4.63,显示为弱酸性。LYRP4-1在浓度5-80μg·ml~(-1)范围内对鼻咽癌HONE1的抑制率与浓度呈现依赖关系,当浓度达到80μg·ml~(-1)时最大抑制率为31.07%,龙眼肉多糖A在浓度5-80μg·ml~(-1)范围内对鼻咽癌HONE1的抑制率随剂量的升高而逐步升高,当浓度为80μg·ml~(-1)时最大抑制率为16.32%。HE染色显示随LYRP4~(-1)浓度的升高细胞出现较明显的凋亡现象。流式图显示LYRP4~(-1)的最高凋亡率为31.98%,龙眼肉多糖A的最高凋亡率为11.63%。
结论:经浓硫酸法可得到硫酸酯化多糖LYRP4;LYRP4~(-1)对鼻咽癌HONE1的抑制率有低敏作用,但高于龙眼肉多糖A; LPRP4~(-1)体外对鼻咽癌细胞HONE1有一定的细胞凋亡作用。
Objective: To sulfat the purified polysaccharide from Arillus Longan and investigate the antitumor activities of the primitive polysaccharide and its sulfated derivative in vitro.
Methods:?The polysaccharide was extracted with hot water from Arillus longan. The total sugar content of crud polysaccharide was determined by using the phenol-sulfuric acid method. The polysaccharide was purified by the deproteinizing of enzyme-sevag method and the decolouring by H2O2.?The polysaccharide was further purified by DEAE-cellulose laminar analysis. The purity of polysaccharide-A was detected by testing with cellulose acetate membrance electrophoresis and polarimetry. The polysaccharide-A was sulfated with concentrated sulfuric acid. The content of LYRP4 was investigated by opacity analysis of permanent white sulfate precipitation. Reagent proportion, reaction temperature and reaction time were selected with substitute degree and yield as index by L9(34) orthogonal test. The LYRP4 was further purified by DEAE-cellulose so as to gain LYRP4~(-1). Both the molecuar weights of polysaccharide-A and LYRP4-1 were respectively determined through GPC. The antitumor activities of polysaccharide-A and LYRP4-1 were assayed by MTT method in vitro. The apoptotic cells were observed by using the Hematoxylin-eosin staining and FCM.
Results: The yield of hot-water extraction method was 6.30% and the total sugar content of Arillus Longan was 58.72%. The deproteinate rate of polysaccharide from Arillus Longan was 52.30%. The yield of polysaccharide-A by DEAE-cellulose laminar analysis was 64.13%, the yield of polysaccharide-B was 4.85% and polysaccharide-C was 15.43%. The polysaccharide-A was a homogeneous polysaccharide. When the reaction time was 3h, the reaction temperature was 10℃, and the reagent ratio between butyl alcolho and concentrated sulfuric acid was 3:1, the concentrated sulfuric acid esterification could get a high degree from substitution of sulfation which was 2.03. The pH of LYRP4 was 4.63. The LYRP4-1’s growth inhibition ratio of nasopharyngeal carcinoma HONE1 cells was gradually increasing in the concentration range of 5-80μg·ml~(-1), the inhibition ratio could reach to 31.07% when its concentration was 80μg·ml~(-1), while the polysaccharide-A’s growth inhibition ratio of nasopharyngeal carcinoma HONE1 cells at the same concentration was 16.32%. Hematoxylin-eosin staining showed that nasopharyngeal carcinoma HONE1 cells appeared apoptosis phenomenon with the LYRP4-1 concentration elevating. The apoptosis rate in high dose group of LYRP4-1 was 31.98% and the polysaccharide-A’s was 11.63%, detected by FCM.
Conclusion: The polysaccharide-A can get the polysaccharide-A’s sulfated derivative by using the concentrated sulfuric acid esterification. The LYRP4-1’s growth inhibition ratio of nasopharyngeal carcinoma HONE1cells is higher than the polysaccharide-A’s. The LYRP4-1 has apoptotic effect on the nasopharyngeal carcinoma HONE1 cells.
方法:通过热水浸提法提取龙眼肉多糖,Sevag-木瓜蛋白酶法除龙眼肉多糖蛋白,H_2O_2法除龙眼肉多糖色素,苯酚-硫酸法测龙眼肉多糖的多糖含量,DEAE-纤维素柱层析纯化龙眼肉多糖,龙眼肉多糖A的纯度用醋酸薄膜纤维电泳和比旋光度法检测,采用浓硫酸法对龙眼肉多糖A进行硫酸酯化结构修饰,L9(3~4)正交设计对浓硫酸法的反应温度、反应时间及正丁醇与浓硫酸的反应试剂等条件进行优选,硫酸钡-比浊法测定其硫酸基含量,DEAE-纤维素柱层析纯化硫酸酯化龙眼肉多糖(LYRP4-1),通过凝胶色谱法测定龙眼肉多糖A和LYRP4-1的分子量,MTT法体外测定龙眼肉多糖A和LYRP4-1对鼻咽癌HONE1细胞的抑制作用,苏木素-伊红染色和流式细胞仪观察鼻咽癌HONE1细胞凋亡情况。
结果:热水浸提法的龙眼肉多糖得率为6.30%,龙眼肉总糖含量为58.72%。龙眼肉多糖清除蛋白率为52.30%。DEAE-纤维素柱层析纯化流分龙眼肉多糖A得率为64.13%、龙眼肉多糖B得率为4.85%和龙眼肉多糖C得率为15.43%。醋酸薄膜纤维电泳和比旋光度法检测龙眼肉多糖A为均一多糖。硫酸酯化反应经L9(3~4)正交设计得到当反应时间为3h,反应温度为10℃,反应酯化试剂比为3:1时,浓硫酸酯化反应可以得到较高的取代度,取代度为2.03。LYRP4的pH值为4.63,显示为弱酸性。LYRP4-1在浓度5-80μg·ml~(-1)范围内对鼻咽癌HONE1的抑制率与浓度呈现依赖关系,当浓度达到80μg·ml~(-1)时最大抑制率为31.07%,龙眼肉多糖A在浓度5-80μg·ml~(-1)范围内对鼻咽癌HONE1的抑制率随剂量的升高而逐步升高,当浓度为80μg·ml~(-1)时最大抑制率为16.32%。HE染色显示随LYRP4~(-1)浓度的升高细胞出现较明显的凋亡现象。流式图显示LYRP4~(-1)的最高凋亡率为31.98%,龙眼肉多糖A的最高凋亡率为11.63%。
结论:经浓硫酸法可得到硫酸酯化多糖LYRP4;LYRP4~(-1)对鼻咽癌HONE1的抑制率有低敏作用,但高于龙眼肉多糖A; LPRP4~(-1)体外对鼻咽癌细胞HONE1有一定的细胞凋亡作用。
Objective: To sulfat the purified polysaccharide from Arillus Longan and investigate the antitumor activities of the primitive polysaccharide and its sulfated derivative in vitro.
Methods:?The polysaccharide was extracted with hot water from Arillus longan. The total sugar content of crud polysaccharide was determined by using the phenol-sulfuric acid method. The polysaccharide was purified by the deproteinizing of enzyme-sevag method and the decolouring by H2O2.?The polysaccharide was further purified by DEAE-cellulose laminar analysis. The purity of polysaccharide-A was detected by testing with cellulose acetate membrance electrophoresis and polarimetry. The polysaccharide-A was sulfated with concentrated sulfuric acid. The content of LYRP4 was investigated by opacity analysis of permanent white sulfate precipitation. Reagent proportion, reaction temperature and reaction time were selected with substitute degree and yield as index by L9(34) orthogonal test. The LYRP4 was further purified by DEAE-cellulose so as to gain LYRP4~(-1). Both the molecuar weights of polysaccharide-A and LYRP4-1 were respectively determined through GPC. The antitumor activities of polysaccharide-A and LYRP4-1 were assayed by MTT method in vitro. The apoptotic cells were observed by using the Hematoxylin-eosin staining and FCM.
Results: The yield of hot-water extraction method was 6.30% and the total sugar content of Arillus Longan was 58.72%. The deproteinate rate of polysaccharide from Arillus Longan was 52.30%. The yield of polysaccharide-A by DEAE-cellulose laminar analysis was 64.13%, the yield of polysaccharide-B was 4.85% and polysaccharide-C was 15.43%. The polysaccharide-A was a homogeneous polysaccharide. When the reaction time was 3h, the reaction temperature was 10℃, and the reagent ratio between butyl alcolho and concentrated sulfuric acid was 3:1, the concentrated sulfuric acid esterification could get a high degree from substitution of sulfation which was 2.03. The pH of LYRP4 was 4.63. The LYRP4-1’s growth inhibition ratio of nasopharyngeal carcinoma HONE1 cells was gradually increasing in the concentration range of 5-80μg·ml~(-1), the inhibition ratio could reach to 31.07% when its concentration was 80μg·ml~(-1), while the polysaccharide-A’s growth inhibition ratio of nasopharyngeal carcinoma HONE1 cells at the same concentration was 16.32%. Hematoxylin-eosin staining showed that nasopharyngeal carcinoma HONE1 cells appeared apoptosis phenomenon with the LYRP4-1 concentration elevating. The apoptosis rate in high dose group of LYRP4-1 was 31.98% and the polysaccharide-A’s was 11.63%, detected by FCM.
Conclusion: The polysaccharide-A can get the polysaccharide-A’s sulfated derivative by using the concentrated sulfuric acid esterification. The LYRP4-1’s growth inhibition ratio of nasopharyngeal carcinoma HONE1cells is higher than the polysaccharide-A’s. The LYRP4-1 has apoptotic effect on the nasopharyngeal carcinoma HONE1 cells.
引文
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