CN103265643B - 白僵蚕抗癌活性多糖bbpw-1的分离纯化制备方法 - Google Patents
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Abstract
本发明涉及一种白僵蚕抗癌活性多糖BBPW-1的分离纯化制备方法。白僵蚕粉经丙酮-石油醚回流脱脂和80%乙醇回流除去苷类和生物碱后,用蒸馏水于80~100°C下提取出粗多糖。粗多糖经Sevag法去除蛋白质后,用DEAE琼脂糖凝胶离子交换层析分离出中性多糖组分,再经丙烯葡聚糖凝胶S-300层析,取第1个洗脱峰,减压浓缩后经丙烯葡聚糖凝胶S-500层析进一步纯化,冷冻干燥得到白僵蚕抗癌活性多糖BBPW-1。本发明制备的产物,纯度均一,分子量为3.67×106 Da,对人宫颈癌细胞Hela和人肝癌细胞HepG2的生长均有抑制作用,对正常细胞人胚胎肾细胞HEK293和小鼠巨噬细胞RAW264.7生长均无不良影响,可用于抗癌产品开发。
Description
技术领域
本发明涉及一种白僵蚕抗癌活性多糖BBPW-1的分离纯化制备方法。
背景技术
天然高分子化合物多糖,主要来自高等动植物细胞膜和微生物细胞壁,是构成生命活动的4大基本物质之一。多糖具有多种生物活性,与蛋白质形成的糖蛋白以及与脂类形成的脂多糖,在细胞的识别、分泌以及蛋白的加工和转运方面等发挥着不可忽视的重要作用。自上世纪50年代末菌多糖被发现具有抗肿瘤活性以来,从细菌、真菌、藻类、植物和动物中提取出多种抗癌多糖并应用于临床,由于天然多糖有效的抗癌作用和较低的毒性,受到人们越来越广泛的重视。
白僵蚕是家蚕(Bombyx mori)幼虫感染病原白僵菌(Beauveria bassiana)后致死的干燥菌虫体,为我国名贵的传统中药材,其药用目前仍十分广泛,还常年出口外销。白僵蚕性平,味咸、辛,具有祛风解痉、消痰散结、抗惊厥、抗凝、催眠、抑菌、抗癌、降血糖、降血脂等药理作用。但白僵蚕药用机理特别是抗癌机理还不清楚,这阻害了白僵蚕药用水平提高及药用范围扩大。分离纯化制备白僵蚕抗癌活性成分,对于推进白僵蚕药用开发具有积极的现实意义。
发明内容
本发明的目的在于提供一种白僵蚕抗癌活性多糖BBPW-1的分离纯化制备方法,是采用回流提取、DEAE-Sepharose Fast Flow离子交换层析柱和Sephacryl S-300层析柱分离,Sephacryl S-300层析柱纯化的方法,制备白僵蚕抗癌活性多糖BBPW-1。
为了达到上述目的,本发明采用的技术方案如下:
1)白僵蚕于-50℃下真空干燥36 h,于-15℃下超微粉碎5 min,过80~100目筛,得到白僵蚕粉;
2)取白僵蚕粉,按W:V为1:3加入丙酮-石油醚混合液,丙酮与石油醚体积比为1:1,在60°C下回流脱脂1 h,重复2次,抽滤,残渣用5倍体积质量浓度为80%乙醇在95°C下回流1 h,除去苷类和生物碱,重复3次,抽滤,残渣于40°C 烘干;
3)烘干残渣按W:V为1:5加入蒸馏水,超声波处理40~50 min后,于80~100°C下提取1~1.5 h,从白僵蚕中提取出粗多糖;
4)粗多糖按W:V为1:10加入蒸馏水溶解,用体积比为4:1的氯仿与正丁醇除蛋白6次,碘颜色反应和茚三酮反应均检测不到蛋白质,于50°C下旋蒸以除去氯仿与正丁醇,加入质量浓度为80%乙醇,使乙醇质量浓度达到80%,充分搅拌后置冰箱过夜,沉淀得到多糖;
5)多糖用蒸馏水溶解配制成20 mg/mL溶液,8000 rpm 离心10 min,取上清液,过0.45 μm滤膜,上DEAE-Sepharose Fast Flow离子交换层析柱,用蒸馏水以3.0~3.5 mL/min流速洗脱,自动收集器收集,苯酚-硫酸法检测,收集有显色反应的洗脱液,减压浓缩,-50℃下真空干燥,得到中性多糖组分,取名BBPW;
6)BBPW用蒸馏水溶解配制成10 mg/mL溶液,过0.45 μm 滤膜,上Sephacryl S-300 层析柱,用蒸馏水以1.0~1.2 mL/min流速洗脱,自动收集器收集,苯酚-硫酸法检测,收集第1个洗脱峰的洗脱液,减压浓缩成浓度为10 mg/mL;
7)浓缩液上Sephacryl S-500层析柱,用蒸馏水以1.0~1.2 mL/min流速洗脱,自动收集器收集,苯酚-硫酸法检测,收集有显色反应的洗脱液,-50℃下真空干燥,得到均一的多糖组分,取名BBPW-1。
本发明具有的有益效果是:
本发明分离纯化制备的白僵蚕抗癌活性多糖BBPW-1,纯度均一,分子量为3.67×106 Da,对人宫颈癌细胞Hela和人肝癌细胞HepG2的生长均表现出明显的抑制作用,对正常细胞人胚胎肾细胞HEK293和小鼠巨噬细胞RAW264.7生长均无不良影响,可用于抗癌产品的开发,这对于提升白僵蚕药用价值,具有积极的社会效益和经济效益。
附图说明
图1是BBPW-1纯度检测的高效液相色谱图。
图2是BBPW-1对人宫颈癌细胞Hela和人肝癌细胞HepG2生长的抑制图。
具体实施方式
实施之前,先将白僵蚕于-50℃下真空干燥36 h,于-15℃下超微粉碎5 min,过80~100目筛,得到白僵蚕粉;
取白僵蚕粉,按W:V为1:3加入丙酮-石油醚混合液,丙酮与石油醚体积比为1:1,在60°C下回流脱脂1 h,重复2次,抽滤,残渣用5倍体积质量浓度为80%乙醇在95°C下回流1 h,除去苷类和生物碱,重复3次,抽滤,残渣于40°C烘干。
实施例1:
取过80目筛的白僵蚕粉,按上述方法脱脂、除苷类和生物碱后,按1:5料液比加入蒸馏水,超声波处理40 min,于100°C下提取1 h,提取出粗多糖;粗多糖用Sevag法(体积比氯仿:正丁醇=4:1)除蛋白后,上DEAE-Sepharose Fast Flow离子交换层析柱,用蒸馏水以3.0 mL/min流速洗脱,自动收集器收集,得到中性多糖组分BBPW;BBPW上Sephacryl S-300 层析柱,用蒸馏水以1.0 mL/min流速洗脱,自动收集器收集,收集第1个洗脱峰的洗脱液,减压浓缩;浓缩液上Sephacryl S-500层析柱,用蒸馏水以1.0 mL/min流速洗脱,自动收集器收集,得到多糖组分BBPW-1。BBPW-1纯度均一(如图1所示),分子量3.67×106 Da,在浓度2.5 mg/mL时对人宫颈癌细胞Hela和人肝癌细胞HepG2生长的抑制率分别为59.1%和50.2%(如图2所示),对正常细胞人胚胎肾细胞HEK293和小鼠巨噬细胞RAW264.7生长无不良影响。
实施例2:
取过100目筛的白僵蚕粉,按上述方法脱脂、除苷类和生物碱后,按1:5料液比加入蒸馏水,超声波处理50 min,于100°C下提取1.5 h,提取出粗多糖;粗多糖用Sevag法(体积比氯仿:正丁醇=4:1)除蛋白后,上DEAE-Sepharose Fast Flow离子交换层析柱,用蒸馏水以3.5 mL/min流速洗脱,自动收集器收集,得到中性多糖组分BBPW;BBPW上Sephacryl S-300 层析柱,用蒸馏水以1.2 mL/min流速洗脱,自动收集器收集,收集第1个洗脱峰的洗脱液,减压浓缩;浓缩液上Sephacryl S-500层析柱,用蒸馏水以1.2 mL/min流速洗脱,自动收集器收集,得到多糖组分BBPW-1。BBPW-1纯度均一(与实施例1中的图2相似),分子量3.67×106 Da,在浓度2.5 mg/mL时对人宫颈癌细胞Hela和人肝癌细胞HepG2生长的抑制率分别为59.0%和50.6%(与实施例1中的图2相似),对正常细胞人胚胎肾细胞HEK293和小鼠巨噬细胞RAW264.7生长无不良影响。
实施例3:
取过90目筛的白僵蚕粉,按上述方法脱脂、除苷类和生物碱后,按1:5料液比加入蒸馏水,超声波处理40 min,于100°C下提取1.2 h,提取出粗多糖;粗多糖用Sevag法(体积比氯仿:正丁醇=4:1)除蛋白后,上DEAE-Sepharose Fast Flow离子交换层析柱,用蒸馏水以3.2 mL/min流速洗脱,自动收集器收集,得到中性多糖组分BBPW;BBPW上Sephacryl S-300 层析柱,用蒸馏水以1.1 mL/min流速洗脱,自动收集器收集,收集第1个洗脱峰的洗脱液,减压浓缩;浓缩液上Sephacryl S-500层析柱,用蒸馏水以1.1 mL/min流速洗脱,自动收集器收集,得到多糖组分BBPW-1。BBPW-1纯度均一(与实施例1中的图1相似),分子量3.67×106 Da,在浓度2.5 mg/mL时对人宫颈癌细胞Hela和人肝癌细胞HepG2生长的抑制率分别为59.5%和49.8%(与实施例1中的图2相似),对正常细胞人胚胎肾细胞HEK293和小鼠巨噬细胞RAW264.7生长无不良影响。
Claims (1)
1.一种白僵蚕抗癌活性多糖BBPW-1的分离纯化制备方法,其特征在于:
1)白僵蚕于-50℃下真空干燥36 h,于-15℃下超微粉碎5 min,过80~100目筛,得到白僵蚕粉;
2)取白僵蚕粉,按W:V为1:3加入丙酮-石油醚混合液,丙酮与石油醚体积比为1:1,在60℃下回流脱脂1 h,重复2次,抽滤,残渣用5倍体积质量浓度为80%乙醇在95℃下回流1 h,除去苷类和生物碱,重复3次,抽滤,残渣于40℃烘干;
3)烘干残渣按W:V为1:5加入蒸馏水,超声波处理40~50 min后,于80~100℃下提取1~1.5 h,从白僵蚕中提取出粗多糖;
4)粗多糖按W:V为1:10加入蒸馏水溶解,用体积比为4:1的氯仿与正丁醇除蛋白6次,碘颜色反应和茚三酮反应均检测不到蛋白质,于50℃下旋蒸以除去氯仿与正丁醇,加入质量浓度为80%乙醇,使乙醇质量浓度达到80%,充分搅拌后置冰箱过夜,沉淀得到多糖;
5)多糖用蒸馏水溶解配制成20 mg/mL溶液,8000 rpm 离心10 min,取上清液,过0.45 μm滤膜,上DEAE-Sepharose Fast Flow离子交换层析柱,用蒸馏水以3.0~3.5 mL/min流速洗脱,自动收集器收集,苯酚-硫酸法检测,收集有显色反应的洗脱液,减压浓缩,-50℃下真空干燥,得到中性多糖组分,取名BBPW;
6)BBPW用蒸馏水溶解配制成10 mg/mL溶液,过0.45 μm 滤膜,上Sephacryl S-300 层析柱,用蒸馏水以1.0~1.2 mL/min流速洗脱,自动收集器收集,苯酚-硫酸法检测,收集第1个洗脱峰的洗脱液,减压浓缩成浓度为10 mg/mL;
7)浓缩液上Sephacryl S-500层析柱,用蒸馏水以1.0~1.2 mL/min流速洗脱,自动收集器收集,苯酚-硫酸法检测,收集有显色反应的洗脱液,-50℃下真空干燥,得到均一的多糖组分,取名BBPW-1;
8)采用配有TSK PWXL G5000糖分析柱的1525型高效液相色谱系统,以示差和紫外检测器平行检测BBPW-1,呈现单一对称峰,对照标准曲线计算分子量为3.67×106 Da,为一种高度聚合的大分子多糖;MTT法检测BBPW-1对人宫颈癌细胞Hela和人肝癌细胞HepG2的生长均表现出明显的抑制作用,在浓度2.5 mg/mL时的抑制率分别为59.1%和50.2%,对正常细胞人胚胎肾细胞HEK293和小鼠巨噬细胞RAW264.7生长均无不良影响。
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