CN109628523A - 枸杞多糖提取分离和纯化方法、枸杞糖肽及降血糖药物 - Google Patents
枸杞多糖提取分离和纯化方法、枸杞糖肽及降血糖药物 Download PDFInfo
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Abstract
本发明属于提取;分离;纯化技术领域,公开了一种枸杞多糖提取分离和纯化方法、枸杞糖肽及降血糖药物;枸杞多糖具有增强免疫力、防衰老、增强造血功能、防止遗传损伤等作用;枸杞多糖(LBP)是酸性杂多糖同多肽或蛋白质构成的的复合性多糖,且存在Glycan‑O‑Ser的糖肽结构;枸杞糖肽的抗肿瘤作用通过极其复杂的调控方式完成:通过干预相关酶和蛋白质的表达,抑制肝糖原的生成,从而达到降血糖的作用。LBP对于胰岛素和胰岛细胞有积极作用,从而间接达到降血糖的效果,并不是通过促进胰腺分泌胰岛素达到降糖效果,可以说是治疗糖尿病非常好的一类产品。
Description
技术领域
本发明属于提取;分离;纯化技术领域,尤其涉及一种枸杞多糖提取分离和纯化方法、枸杞糖肽及降血糖药物。
背景技术
目前,业内常用的现有技术是这样的:枸杞,枸杞子的性味甘、平;枸杞叶的性味为苦、甘;性凉。功能上枸杞子可以养肝,滋肾,润肺。枸杞叶具体补虚益精,清热明目的功效。而枸杞多糖是一种水溶性多糖,是枸杞中最主要的活性成分,相对分子质量为68-200;枸杞多糖是枸杞中主要有效成分之一,具有增强免疫力、防衰老、增强造血功能、防止遗传损伤等作用,大多数现有技术利用的都是枸杞多糖的粗品;目前现有工艺的提纯方法都是用传统的醇沉水提工艺,导致提纯枸杞类产品的产率不足1%,而且混合物结构复杂,并不能得到满足功效的产品。现有技术的工艺不稳定,每次得到的产品的结构均不确定,每次的测试结果千差万别,而且产率非常低;因此,对于枸杞多糖的提取分离和纯化方法以及其组成成分和结构的分析研究报道甚少。
综上所述,现有技术存在的问题是:
(1)现有工艺的提纯方法都是用传统的醇沉水提工艺,导致提纯枸杞类产品的产率不足1%,而且混合物结构复杂,并不能得到满足功效的产品;
(2)现有技术的工艺不稳定,每次得到的产品的结构均不确定,每次的测试结果千差万别,而且产率非常低。
发明内容
针对现有技术存在的问题,本发明提供了一种枸杞多糖提取分离和纯化方法、枸杞糖肽及降血糖药物。
本发明是这样实现的,一种枸杞多糖提取分离和纯化方法,所述枸杞多糖提取分离和纯化方法包括以下步骤:
步骤一:收获后,枸杞果实经干燥处理,备用;
步骤二:干果碎粉用氯仿:甲醇(2:1)混合物回流,在80℃中用80%乙醇回流提取低聚糖和小分子;在特定装置中,用石油醚在90℃下得到低聚糖;
步骤三:在亚临界条件下,使用加压水结合超声辅助处理得到低聚糖,在100℃下用5mPa压力和26mL×g·1的液固比,在超声功率下53分钟,得到粗LBP;
步骤四:根据不同的方法对粗LBP提取物进行去蛋白处理;
步骤五:采用亲和性和反相高效液相色谱的组合对产品进行纯化,纯化后将获得的过滤浓缩的水提取物加入四体积95%乙醇,沉淀多糖组分;
步骤六:利用无水乙醇和丙酮洗涤,将过滤的粗LBP沉淀冻干并储存。
进一步,所述步骤二中,将超声波80W与纤维素酶56℃结合,培养时间20分钟后在进行处理;
所述步骤四中,粗LBP提取物采用酶解、SEVAG法或三嵌段共聚物盐双水相萃取和透析膜方法进行去蛋白处理;
所述步骤五中,沉淀多糖组分在4℃下过夜培养。
本发明的另一目的在于提供一种由所述枸杞多糖提取分离和纯化方法得到的枸杞多糖,所述枸杞多糖分离纯化得到枸杞糖肽;
所述枸杞多糖分子质量为1.524×105,由阿拉伯糖、鼠李糖、木糖、甘露糖、半乳糖和葡萄糖6种中性单糖和半乳糖醛酸及18种氨基酸组成。
进一步,所述枸杞多糖是酸性杂多糖同多肽或蛋白质构成的复合性多糖;存在Glycan-O-Ser的糖肽结构。
本发明的另一目的在于提供一种包含所述枸杞糖肽的降血糖药物。
本发明的另一目的在于提供一种由所述枸杞糖肽制备的降血糖胶囊。
本发明的另一目的在于提供一种由所述枸杞糖肽制备的降血糖片剂。
综上所述,本发明的优点及积极效果为:枸杞多糖具有增强免疫力、防衰老、增强造血功能、防止遗传损伤等作用;枸杞多糖(LBP)是酸性杂多糖同多肽或蛋白质构成的复合性多糖,且存在Glycan-O-Ser的糖肽结构;枸杞糖肽在治疗糖尿病方面是降血糖部分,LBP的降血糖作用也是通过细胞调控来实现的,一方面,通过对于大小鼠的干预实验,可以得出LBP具有直接降血糖的效果,并且具有剂量依赖性,作用机制主要是通过干预相关酶和蛋白质的表达,抑制肝糖原的生成,达到降血糖的作用;一方面,LBP对于胰岛素和胰岛细胞有积极作用,间接达到降血糖的效果,不是通过促进胰腺分泌胰岛素达到降糖效果,可以说是治疗糖尿病非常好的一类产品。
枸杞多糖具有增强免疫力、防衰老、增强造血功能、防止遗传损伤等作用;枸杞多糖(LBP)是酸性杂多糖同多肽或蛋白质构成的复合性多糖,且存在Glycan-O-Ser的糖肽结构;枸杞糖肽的抗肿瘤作用通过极其复杂的调控方式完成:诱导肿瘤细胞的凋亡表达;增强了身体对于肿瘤细胞的免疫能力。枸杞多糖能显著改善荷瘤小鼠的状况,使腹腔积液型荷瘤小鼠的生命延长41%~85%,并显著地抑制这两种实体瘤的生长,抑制率达81.0%和72.9%。
本发明可以将产品提纯的产率提高8倍,而且产品的纯度得以保证,功效得以保证。
附图说明
图1是本发明实施例提供的枸杞多糖提取分离和纯化方法流程图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
针对现有技术存在利用的都是枸杞多糖的粗品;对于枸杞多糖的提取分离和纯化方法以及其组成成分和结构的分析研究报道甚少的问题;枸杞多糖具有增强免疫力、防衰老、增强造血功能、防止遗传损伤等作用。
下面结合附图对本发明的应用原理作详细的描述。
如图1所示,本发明实施例提供的枸杞多糖提取分离和纯化方法包括以下步骤:
S101:收获后,枸杞果实经干燥处理,备用;
S102:干果碎粉后,用氯仿:甲醇(2:1)混合物回流,在80℃中用80%乙醇回流提取低聚糖和小分子;在特定装置中,用石油醚在90℃下得到低聚糖;
S103:在亚临界条件下,使用加压水结合超声辅助处理得到低聚糖,在100℃下用5mPa压力和26mL×g·1的液固比,在超声功率下53分钟,得到粗LBP;
S104:根据不同的方法对粗LBP提取物进行去蛋白处理;
S105:采用亲和性和反相高效液相色谱(RPHPLC)的组合对产品进行纯化,纯化后将获得的过滤浓缩的水提取物加入四体积95%乙醇,沉淀多糖组分;
S106:利用无水乙醇和丙酮洗涤,将过滤的粗LBP沉淀冻干并储存。
步骤S102中,本发明实施例提供的超声波(80W)与纤维素酶(中温)(56℃)结合,培养时间20分钟后在进行处理。
步骤S104中,本发明实施例提供的粗LBP提取物根据不同的方法进行去蛋白处理,如酶解、SEVAG法、或三嵌段共聚物盐双水相萃取和透析膜。
步骤S105中,本发明实施例提供的沉淀多糖组分,通常在4℃下过夜培养。
步骤S106中,本发明实施例提供的冷冻时间一般为90小时以上。
步骤S106中,本发明实施例提供的用无水乙醇和丙酮洗涤后,将过滤的粗LBP沉淀冻干并储存,然后用热风干燥、真空干燥和喷雾干燥进行处理粗品LBP,方法选用喷雾干燥。
本发明进一步从枸杞多糖(LBP)中分离纯化得到5种免疫活性较强的糖缀合物,统称为枸杞糖肽(简称LbGp),LBP纯品含有4个级分,LBP的分子质量约为1.524×105,由阿拉伯糖(Ara),鼠李糖(Rha),木糖(Xyl),甘露糖(Man),半乳糖(Gal)和葡萄糖(Glc)6种中性单糖和半乳糖醛酸及18种氨基酸组成。枸杞多糖(LBP)是酸性杂多糖同多肽或蛋白质构成的复合性多糖,且存在Glycan-O-Ser的糖肽结构。
本发明进一步从枸杞多糖(LBP)中分离纯化得到5种免疫活性较强的糖缀合物,统称为枸杞糖肽(简称LbGp),LBP纯品含有4个级分,LBP的分子质量约为1.524×105,由阿拉伯糖(Ara),鼠李糖(Rha),木糖(Xyl),甘露糖(Man),半乳糖(Gal)和葡萄糖(Glc)6种中性单糖和半乳糖醛酸及18种氨基酸组成。枸杞多糖(LBP)是酸性杂多糖同多肽或蛋白质构成的的复合性多糖,且存在Glycan-O-Ser的糖肽结构。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (8)
1.一种枸杞多糖提取分离和纯化方法,其特征在于,所述枸杞多糖提取分离和纯化方法包括以下步骤:
步骤一:收获后,枸杞果实经干燥处理,备用;
步骤二:干果碎粉用氯仿:甲醇2:1混合物回流,在80℃中用80%乙醇回流提取低聚糖和小分子;在特定装置中,用石油醚在90℃下得到低聚糖;
步骤三:在亚临界条件下,使用加压水结合超声辅助处理得到低聚糖,在100℃下用5mPa压力和26mL×g·1的液固比,在超声功率下53分钟,得到粗LBP;
步骤四:根据不同的方法对粗LBP提取物进行去蛋白处理;
步骤五:采用亲和性和反相高效液相色谱的组合对产品进行纯化,纯化后将获得的过滤浓缩的水提取物加入四体积95%乙醇,沉淀多糖组分;
步骤六:利用无水乙醇和丙酮洗涤,将过滤的粗LBP沉淀冻干并储存。
2.如权利要求1所述的枸杞多糖提取分离和纯化方法,其特征在于,所述步骤二中,将超声波80W与纤维素酶56℃结合,培养时间20分钟后在进行处理;
所述步骤四中,粗LBP提取物采用酶解、SEVAG法或三嵌段共聚物盐双水相萃取和透析膜方法进行去蛋白处理;
所述步骤五中,沉淀多糖组分在4℃下过夜培养。
3.如权利要求1所述的枸杞多糖提取分离和纯化方法,其特征在于,所述步骤六中,冷冻时间为90小时以上;
所述步骤六中,用无水乙醇和丙酮洗涤后,将过滤的粗LBP沉淀冻干并储存,然后采用热风干燥、真空干燥和喷雾干燥进行处理粗品LBP。
4.一种由权利要求1所述枸杞多糖提取分离和纯化方法得到的枸杞多糖,其特征在于,所述枸杞多糖分离纯化得到枸杞糖肽;
所述枸杞多糖分子质量为1.524×105,由阿拉伯糖、鼠李糖、木糖、甘露糖、半乳糖和葡萄糖6种中性单糖和半乳糖醛酸及18种氨基酸组成。
5.如权利要求4所述的枸杞多糖,其特征在于,所述枸杞多糖是酸性杂多糖同多肽或蛋白质构成的复合性多糖;存在Glycan-O-Ser的糖肽结构。
6.一种包含权利要求4所述枸杞糖肽的降血糖药物。
7.一种由权利要求4所述枸杞糖肽制备的降血糖胶囊。
8.一种由权利要求4所述枸杞糖肽制备的降血糖片剂。
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