CN108498486B - 一种bsa-egcg-tps纳米颗粒的制备方法 - Google Patents
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Abstract
本发明公开了一种BSA‑EGCG‑TPS纳米颗粒的制备方法,包括如下步骤:将BSA溶于水中,制备BSA水溶液,将无水乙醇逐滴滴入BSA溶液中,搅拌,静置,逐滴滴入戊二醛,搅拌,离心,水洗涤沉淀,将沉淀重新悬浮到水中,得到BSA纳米颗粒悬浮液;将EGCG溶于水中制备EGCG水溶液,并逐滴滴入到BSA纳米颗粒悬浮液中,搅拌,得到BSA‑EGCG纳米颗粒悬浮液;将茶多糖TPS溶于水中,制备茶多糖水溶液,并逐滴滴入到BSA‑EGCG纳米颗粒悬浮液中,搅拌,离心,水洗,冻干,得到BSA‑EGCG‑TPS纳米颗粒BETN。制备过程简便,无毒,安全,得到的纳米颗粒稳定性高,抗氧化活性好。
Description
技术领域
本发明涉及一种BSA-EGCG-TPS纳米颗粒的制备方法,属于纳米颗粒技术应用领域。
背景技术
EGCG即表没食子儿茶素没食子酸酯,是绿茶茶多酚中的主要组成成分,是一种黄烷醇-3-醇。EGCG具有抗氧化、抗肿瘤、抗糖尿病等多种作用。然而,EGCG在人体内的吸收率低于5%,口服后低于1%;而且,EGCG非常容易被氧化。稳定性差、生物利用度低和毒性大的缺点限制了EGCG的充分利用。茶多糖是茶叶中的另一重要活性成分,也是一种潜在功能性食品。
BSA即牛血清白蛋白,具有无毒、非免疫原性、低成本、生物相容性好和生物可降解性的特点,它可以在药物传递系统中作为多功能蛋白载体。此外,BSA还可以将不同疏水性或不稳定的大分子或小分子药物转运到靶位点。
目前,纳米技术在许多领域特别是医学和生物技术领域的应用越来越受到人们的关注。纳米的概念已经从1纳米到100纳米扩展到1纳米到1000纳米。纳米技术已被应用于包括癌症在内的多种疾病的诊断和治疗。高溶解度、超小体积、可生物降解、毒副作用低、高稳定性、靶向性和高效性等是纳米颗粒的优点。近些年来,蛋白-多糖复合纳米颗粒在食品、保健、医药等领域越来越受欢迎,特别是作为小分子多酚的包封载体。蛋白质、多糖和多酚之间会相互作用,其相互作用力主要是非共价键,主要包括氢键、疏水作用力、亲水作用力和范德华力。蛋白质-多酚-多糖复合物因其在开发新的多功能食品材料和药物传递系统方面的巨大潜力而备受关注。
发明内容
本发明的目的是克服现有技术的不足,提供一种BSA-EGCG-TPS纳米颗粒的制备方法。
本发明的第二个目的是提供一种BSA-EGCG-TPS纳米颗粒。
本发明的技术方案概述如下:
一种BSA-EGCG-TPS纳米颗粒的制备方法,包括如下步骤:
(1)制备茶多糖:取绿茶粉末制备得到粗茶多糖,再经过聚酰胺柱色谱纯化,超滤,得到分子量为30~80KDa的茶多糖,所述茶多糖简称TPS;
(2)按比例,将40~55mL的乙醇滴入15~25mL浓度为1.5~2.5mg/mL的牛血清白蛋白水溶液,所述牛血清白蛋白简称BSA,20~30℃,搅拌10~20min;再滴入0.75~1.5mL戊二醛,20~30℃,搅拌45~75min;在21100×g离心10~20min,用去离子水洗涤沉淀2~4次,将沉淀重新悬浮到18~25mL水中,得到BSA纳米颗粒悬浮液;
(3)将15~25mL浓度为0.75~1.5mg/mL的表没食子儿茶素没食子酸酯水溶液,滴入到8~12mL BSA纳米颗粒悬浮液中,20~30℃,搅拌20~40min,得到BSA-EGCG纳米颗粒悬浮液,所述表没食子儿茶素没食子酸酯简称EGCG,所述BSA-EGCG纳米颗粒悬浮液简称BEN悬浮液;
(4)将步骤(1)获得的茶多糖加水配成浓度为1~2.5mg/mL茶多糖水溶液,取5mL滴入到15~25mL BEN悬浮液中,20~30℃,搅拌20~40min,在21100×g条件下离心10~20min,用去离子水洗涤沉淀2~4次,冻干,得到BSA-EGCG-TPS纳米颗粒,所述BSA-EGCG-TPS纳米颗粒简称BETN。
步骤(1)优选为:制备茶多糖:取绿茶粉末,用相当于绿茶粉末质量4~6倍的体积浓度为75%~85%的乙醇水溶液浸提20~28h,过滤,残渣干燥,用相当于残渣质量15~25倍的90~100℃水浸提残渣1.5~2.5h,过滤,浸提2~4次,合并水提滤液,浓缩至合并水提滤液体积的1/10~1/5得浓缩液,加入相当于浓缩液体积4倍的体积浓度为95%乙醇水溶液,沉淀,4℃,放置8~12h,离心,固体冻干,得到粗茶多糖;再经聚酰胺柱色谱纯化,超滤,得到分子量为30~80KDa的茶多糖,所述茶多糖简称TPS。
步骤(2)优选为:将50mL的乙醇滴入20mL浓度为2mg/mL的牛血清白蛋白水溶液,所述牛血清白蛋白简称BSA,25℃,搅拌15min;再滴入1mL戊二醛,25℃,搅拌60min;在21100×g离心15min,用去离子水洗涤沉淀3次,将沉淀重新悬浮到20mL水中,得到BSA纳米颗粒悬浮液。
步骤(3)优选为:将20mL浓度为1mg/mL的表没食子儿茶素没食子酸酯水溶液,滴入到10mL BSA纳米颗粒悬浮液中,25℃,搅拌30min,得到BSA-EGCG纳米颗粒悬浮液,所述表没食子儿茶素没食子酸酯简称EGCG,所述BSA-EGCG纳米颗粒悬浮液简称BEN悬浮液;
步骤(4)优选为:将步骤(1)获得的茶多糖加水配成浓度为2mg/mL茶多糖水溶液,取5mL滴入到20mL BEN悬浮液中,25℃,搅拌30min,在21100×g条件下离心15min,用去离子水洗涤沉淀3次,冻干,得到BSA-EGCG-TPS纳米颗粒,所述BSA-EGCG-TPS纳米颗粒简称BETN。
上述方法制备的BSA-EGCG-TPS纳米颗粒。
本发明的优点:
本发明采用了茶多糖和牛血清白蛋白作为EGCG的复合载体,不仅保护了EGCG,而且发挥了茶多糖和牛血清白蛋白作为天然产物大分子具有的安全、绿色等优势,还可充分发挥茶多糖作为活性成分的生物功能。针对蛋白-多酚复合物,茶多糖的加入还能够克服蛋白-多酚复合物因较强的非共价相互作用力易形成沉淀的缺点,大大提高了BSA-EGCG-TPS纳米颗粒的稳定性。本发明的方法制备的BSA-EGCG-TPS纳米颗粒具有稳定性高,可持续释放,抗氧化性好的特点。本发明的方法制备过程简便,绿色、环保、安全,包封率和载药量高,所得纳米颗粒生物活性高。
附图说明
图1为BSA-EGCG-TPS纳米颗粒扫描电镜SEM照片。
具体实施方式
下面结合具体实施例对本发明作进一步的说明。
本发明的原料为商品,例如:
EGCG:购于大连美仑生物技术有限公司;
牛血清白蛋白:购于北京索来宝科技有限公司。
上述原料的介绍是为了使本领域的技术人员能够更好地理解本发明,但并不对本发明作任何限制。
实施例1
一种BSA-EGCG-TPS纳米颗粒的制备方法,包括如下步骤:
(1)制备茶多糖:取绿茶粉末,用相当于绿茶粉末质量5倍的体积浓度为80%的乙醇水溶液浸提24h,过滤,残渣干燥,用相当于残渣质量20倍的95℃水浸提残渣2h,过滤,浸提3次,合并水提滤液,浓缩至合并水提滤液体积的1/8得浓缩液,加入相当于浓缩液体积4倍的体积浓度为95%乙醇水溶液,沉淀,4℃,放置10h,离心,固体冻干,得到粗茶多糖;再经聚酰胺柱色谱纯化,超滤,得到分子量为30~80KDa的茶多糖,所述茶多糖简称TPS。
(2)将50mL的乙醇滴入20mL浓度为2mg/mL的牛血清白蛋白水溶液,所述牛血清白蛋白简称BSA,25℃,搅拌15min;再滴入1mL戊二醛,25℃,搅拌60min;在21100×g离心15min,用去离子水洗涤沉淀3次,将沉淀重新悬浮到20mL水中,得到BSA纳米颗粒悬浮液。
(3)将20mL浓度为1mg/mL的表没食子儿茶素没食子酸酯水溶液,滴入到10mL BSA纳米颗粒悬浮液中,25℃,搅拌30min,得到BSA-EGCG纳米颗粒悬浮液,所述表没食子儿茶素没食子酸酯简称EGCG,所述BSA-EGCG纳米颗粒悬浮液简称BEN悬浮液。
(4)将步骤(1)获得的茶多糖加水配成浓度为2mg/mL茶多糖水溶液,取5mL逐滴加入到20mL BEN悬浮液中,25℃,搅拌30min,在21100×g条件下离心15min,用去离子水洗涤沉淀3次,冻干,得到BSA-EGCG-TPS纳米颗粒(见图1),所述BSA-EGCG-TPS纳米颗粒简称BETN。
包封率和载药量分别为47.54%和45.14%。DPPH清除率的IC50值为217.04μg/mL。
实施例2
一种BSA-EGCG-TPS纳米颗粒的制备方法,包括如下步骤:
(1)制备茶多糖:取绿茶粉末,用相当于绿茶粉末质量4倍的体积浓度为85%的乙醇水溶液浸提20h,过滤,残渣干燥,用相当于残渣质量15倍的100℃水浸提残渣1.5h,过滤,浸提2次,合并水提滤液,浓缩至合并水提滤液体积的1/10得浓缩液,加入相当于浓缩液体积4倍的体积浓度为95%乙醇水溶液,沉淀,4℃,放置8h,离心,固体冻干,得到粗茶多糖;再经聚酰胺柱色谱纯化,超滤,得到分子量为30~80KDa的茶多糖,所述茶多糖简称TPS。
(2)将55mL的乙醇滴入15mL浓度为2.5mg/mL的牛血清白蛋白水溶液,所述牛血清白蛋白简称BSA,20℃,搅拌20min;再滴入0.75mL戊二醛,20℃,搅拌45min;在21100×g离心10min,用去离子水洗涤沉淀2次,将沉淀重新悬浮到18mL水中,得到BSA纳米颗粒悬浮液。
(3)将15mL浓度为1.5mg/mL的表没食子儿茶素没食子酸酯水溶液,滴入到8mL BSA纳米颗粒悬浮液中,20℃,搅拌20min,得到BSA-EGCG纳米颗粒悬浮液,所述表没食子儿茶素没食子酸酯简称EGCG,所述BSA-EGCG纳米颗粒悬浮液简称BEN悬浮液。
(4)将步骤(1)获得的茶多糖加水配成浓度为1mg/mL茶多糖水溶液,取5mL滴入到25mL BEN悬浮液中,20℃,搅拌20min,在21100×g条件下离心10min,用去离子水洗涤沉淀2次,冻干,得到BSA-EGCG-TPS纳米颗粒(粒径与实施例1相似),所述BSA-EGCG-TPS纳米颗粒简称BETN。
包封率和载药量分别为40.12%和38.24%。DPPH清除率的IC50值为205.17μg/mL。
实施例3
一种BSA-EGCG-TPS纳米颗粒的制备方法,包括如下步骤:
(1)制备茶多糖:取绿茶粉末,用相当于绿茶粉末质量6倍的体积浓度为75%的乙醇水溶液浸提28h,过滤,残渣干燥,用相当于残渣质量25倍的90℃水浸提残渣2.5h,过滤,浸提4次,合并水提滤液,浓缩至合并水提滤液体积的1/5得浓缩液,加入相当于浓缩液体积4倍的体积浓度为95%乙醇水溶液,沉淀,4℃,放置12h,离心,固体冻干,得到粗茶多糖;再经聚酰胺柱色谱纯化,超滤,得到分子量为30~80KDa的茶多糖,所述茶多糖简称TPS。
(2)将40mL的乙醇滴入25mL浓度为1.5mg/mL的牛血清白蛋白水溶液,所述牛血清白蛋白简称BSA,30℃,搅拌10min;再滴入1.5mL戊二醛,30℃,搅拌75min;在21100×g离心20min,用去离子水洗涤沉淀4次,将沉淀重新悬浮到25mL水中,得到BSA纳米颗粒悬浮液。
(3)将25mL浓度为0.75mg/mL的表没食子儿茶素没食子酸酯水溶液,滴入到12mLBSA纳米颗粒悬浮液中,30℃,搅拌40min,得到BSA-EGCG纳米颗粒悬浮液,所述表没食子儿茶素没食子酸酯简称EGCG,所述BSA-EGCG纳米颗粒悬浮液简称BEN悬浮液。
(4)将步骤(1)获得的茶多糖加水配成浓度为2.5mg/mL茶多糖水溶液,取5mL逐滴加入到15mL BEN悬浮液中,30℃,搅拌40min,在21100×g条件下离心20min,用去离子水洗涤沉淀4次,冻干,得到BSA-EGCG-TPS纳米颗粒(粒径与实施例1相似),所述BSA-EGCG-TPS纳米颗粒简称BETN。
包封率和载药量分别为42.35%和40.95%。DPPH清除率的IC50值为250.74μg/mL。
实施例4
一种BSA-EGCG-TPS纳米颗粒的制备方法,包括如下步骤:
(1)制备茶多糖:取绿茶粉末,用相当于绿茶粉末质量5倍的体积浓度为80%的乙醇水溶液浸提24h,过滤,残渣干燥,用相当于残渣质量20倍的95℃水浸提残渣2h,过滤,浸提3次,合并水提滤液,浓缩至合并水提滤液体积的1/8得浓缩液,加入相当于浓缩液体积4倍的体积浓度为95%乙醇水溶液,沉淀,4℃,放置12h,离心,固体冻干,得到粗茶多糖;再经聚酰胺柱色谱纯化,超滤,得到分子量为30~80KDa的茶多糖,所述茶多糖简称TPS。
(2)将40mL的乙醇滴入25mL浓度为1.5mg/mL的牛血清白蛋白水溶液,所述牛血清白蛋白简称BSA,25℃,搅拌15min;再滴入1mL戊二醛,25℃,搅拌60min;在21100×g离心15min,用去离子水洗涤沉淀3次,将沉淀重新悬浮到20mL水中,得到BSA纳米颗粒悬浮液。
(3)将25mL浓度为1mg/mL的表没食子儿茶素没食子酸酯水溶液,滴入到10mL BSA纳米颗粒悬浮液中,25℃,搅拌30min,得到BSA-EGCG纳米颗粒悬浮液,所述表没食子儿茶素没食子酸酯简称EGCG,所述BSA-EGCG纳米颗粒悬浮液简称BEN悬浮液。
(4)将步骤(1)获得的茶多糖加水配成浓度为2.5mg/mL茶多糖水溶液,取5mL滴入到15mL BEN悬浮液中,25℃,搅拌30min,在21100×g条件下离心15min,用去离子水洗涤沉淀3次,冻干,得到BSA-EGCG-TPS纳米颗粒(粒径与实施例1相似),所述BSA-EGCG-TPS纳米颗粒简称BETN。
包封率和载药量分别为44.77%和43.15%。DPPH清除率的IC50值为243.62μg/mL。
Claims (6)
1.一种BSA-EGCG-TPS纳米颗粒的制备方法,其特征是包括如下步骤:
(1)制备茶多糖:取绿茶粉末制备得到粗茶多糖,再经过聚酰胺柱色谱纯化,超滤,得到分子量为30~80KDa的茶多糖,所述茶多糖简称TPS;
(2)按比例,将40~55mL的乙醇滴入15~25mL浓度为1.5~2.5mg/mL的牛血清白蛋白水溶液,所述牛血清白蛋白简称BSA,20~30℃,搅拌10~20min;再滴入0.75~1.5mL戊二醛,20~30℃,搅拌45~75min;在21100×g离心10~20min,用去离子水洗涤沉淀2~4次,将沉淀重新悬浮到18~25mL水中,得到BSA纳米颗粒悬浮液;
(3)将15~25mL浓度为0.75~1.5mg/mL的表没食子儿茶素没食子酸酯水溶液,滴入到8~12mL BSA纳米颗粒悬浮液中,20~30℃,搅拌20~40min,得到BSA-EGCG纳米颗粒悬浮液,所述表没食子儿茶素没食子酸酯简称EGCG,所述BSA-EGCG纳米颗粒悬浮液简称BEN悬浮液;
(4)将步骤(1)获得的茶多糖加水配成浓度为1~2.5mg/mL茶多糖水溶液,取5mL滴入到15~25mL BEN悬浮液中,20~30℃,搅拌20~40min,在21100×g条件下离心10~20min,用去离子水洗涤沉淀2~4次,冻干,得到BSA-EGCG-TPS纳米颗粒,所述BSA-EGCG-TPS纳米颗粒简称BETN。
2.根据权利要求1所述的方法,其特征是所述步骤(1)为:制备茶多糖:取绿茶粉末,用相当于绿茶粉末质量4~6倍的体积浓度为75%~85%的乙醇水溶液浸提20~28h,过滤,残渣干燥,用相当于残渣质量15~25倍的90~100℃水浸提残渣1.5~2.5h,过滤,浸提2~4次,合并水提滤液,浓缩至合并水提滤液体积的1/10~1/5得浓缩液,加入相当于浓缩液体积4倍的体积浓度为95%乙醇水溶液,沉淀,4℃,放置8~12h,离心,固体冻干,得到粗茶多糖;再经聚酰胺柱色谱纯化,超滤,得到分子量为30~80KDa的茶多糖,所述茶多糖简称TPS。
3.根据权利要求1所述的方法,其特征是所述步骤(2)为:将50mL的乙醇滴入20mL浓度为2mg/mL的牛血清白蛋白水溶液,所述牛血清白蛋白简称BSA,25℃,搅拌15min;再滴入1mL戊二醛,25℃,搅拌60min;在21100×g离心15min,用去离子水洗涤沉淀3次,将沉淀重新悬浮到20mL水中,得到BSA纳米颗粒悬浮液。
4.根据权利要求1所述的方法,其特征是所述步骤(3)为:将20mL浓度为1mg/mL的表没食子儿茶素没食子酸酯水溶液,滴入到10mL BSA纳米颗粒悬浮液中,25℃,搅拌30min,得到BSA-EGCG纳米颗粒悬浮液,所述表没食子儿茶素没食子酸酯简称EGCG,所述BSA-EGCG纳米颗粒悬浮液简称BEN悬浮液。
5.根据权利要求1所述的方法,其特征是所述步骤(4)为:将步骤(1)获得的茶多糖加水配成浓度为2mg/mL茶多糖水溶液,取5mL滴入到20mL BEN悬浮液中,25℃,搅拌30min,在21100×g条件下离心15min,用去离子水洗涤沉淀3次,冻干,得到BSA-EGCG-TPS纳米颗粒,所述BSA-EGCG-TPS纳米颗粒简称BETN。
6.权利要求1-5之一的方法制备的BSA-EGCG-TPS纳米颗粒。
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