CN115141288A - 知母活性多糖、知母粗多糖及制备方法和应用 - Google Patents
知母活性多糖、知母粗多糖及制备方法和应用 Download PDFInfo
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- CN115141288A CN115141288A CN202210895667.7A CN202210895667A CN115141288A CN 115141288 A CN115141288 A CN 115141288A CN 202210895667 A CN202210895667 A CN 202210895667A CN 115141288 A CN115141288 A CN 115141288A
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- Prior art keywords
- polysaccharide
- rhizoma anemarrhenae
- water
- active
- crude polysaccharide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
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- A—HUMAN NECESSITIES
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- General Health & Medical Sciences (AREA)
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- Alternative & Traditional Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了一种知母活性多糖、知母粗多糖及其制备方法和应用。该知母活性多糖由阿拉伯糖、半乳糖和甘露糖三种单糖聚合而成,该活性多糖的化学式如式(Ⅰ)所示。本发明的知母活性多糖能够通过上调AGEs损伤成骨细胞模型GPX4和SLC7A11蛋白表达,抑制细胞脂质过氧化物产生,减少细胞氧化应激损伤,最终达到促进成骨细胞增殖的目的;表明该知母活性多糖、含有该知母活性多糖的知母粗多糖或知母本身均能够用于制备治疗、延缓或减轻AGEs相关疾病的药物或保健食品,达到消除或减轻AGEs造成的细胞损伤的目的。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种知母活性多糖、知母粗多糖及其制备方法和应用。
背景技术
晚期糖基化终末产物(AGEs)是体内蛋白质、脂质或核酸类大分子暴露于还原糖中生成的一组复杂的异质性物质,AGEs会直接或间接与细胞表面受体结合而发挥细胞毒性作用;如大量研究表明,AGEs与骨质疏松症的发生发展高度相关,特别是会抑制成骨细胞增殖,进而减少骨形成。
为消除或减轻AGEs产生的细胞毒性,申请号为201880042349.8的中国发明专利申请公开了一种越橘或其提取物在制备治疗、延缓或减轻AGEs相关疾病的药物或保健食品中的应用,该研究发现越橘提取物能够抑制AGEs生成,从而有助于延缓和减轻糖尿病、动脉粥样硬化、慢性肾病、肝病、神经退行性疾病等多种疾病的发生和发展。
然而,越橘的获取成本较高,导致采用越橘提取物制备的相关药物的成本也较高。
知母是临床常用的传统中药材,也是常用中药复方的组成部分,在我们国内广泛分布。知母的药用部分通常是其干燥根状茎,性苦寒,有滋阴降火、润燥滑肠、利大小便之效,主治温热病、高热烦渴、咳嗽气喘、燥咳、便秘、骨蒸潮热、虚烦不眠、消渴淋浊,属清热下火药。
近年来国内外学者对知母活性成分之一——糖类化合物开展研究,发现知母多糖具有降血糖作用、抗炎作用和抗氧化功能。目前已有关于知母糖的研究主要包括:(1)将知母采用70%乙醇进行醇沉后,收集0.05MNaCl洗脱部位,再用Sephacryl S-100HR columns进行纯化,最后得到分子量为2720g/mol的多糖,该多糖的主要单糖组成为甘露糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖、木糖、阿拉伯糖和海藻糖。这种多糖能够抑制CoCl2造成SH-SY5Y(人神经母细胞瘤细胞)凋亡,抑制脂多糖诱导RAW264.7(小鼠单核巨噬细胞)分泌炎症因子水平;(2)把知母糖经羧甲基化、硫酸化后得到的多糖具有清除DPPH自由基的能力;(3)采用80%乙醇沉淀后,分别收集水和0.5mol/LNaCl洗脱部位,获得的两种多糖组分的分子量均在130000-576500g/mol之间,其单糖组成是鼠李糖、阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖,这两种多糖能够抑制肿瘤细胞(AGS、MKN-28和MKN-45)的增殖活性;(4)获得的分子量为5800Da、单糖组成是葡萄糖和甘露糖的知母多糖能够增加肝癌细胞(HepG2)的葡萄糖消耗量。
目前为止尚没有知母或其提取物在制备治疗、延缓或减轻AGEs相关疾病的药物或保健食品中的应用的相关报道。
发明内容
本发明的发明目的是提供一种知母活性多糖、知母粗多糖及其制备方法和应用,该知母活性多糖能够治疗、延缓或减轻AGEs相关疾病。
为实现上述发明目的,本发明的技术方案为:
一种知母活性多糖,由阿拉伯糖、半乳糖和甘露糖三种单糖聚合而成,该活性多糖的化学式如式(Ⅰ)所示:
本发明研究发现,该知母活性多糖能够显著提高AGEs损伤成骨细胞的增殖活性,降低AGEs损伤成骨细胞中活性氧、脂质过氧化物及脂质氧化合物的含量、促进AGEs损伤成骨细胞中谷胱甘肽过氧化物酶、胱氨酸转运蛋白的表达。表明该知母活性多糖能够通过上调AGEs损伤成骨细胞模型GPX4和SLC7A11蛋白表达,抑制细胞脂质过氧化物产生,减少细胞氧化应激损伤,最终达到促进成骨细胞增殖的目的。
进一步地,该知母活性多糖中,阿拉伯糖、半乳糖和甘露糖的摩尔比为6:1:3;该知母活性多糖的重均分子量为14KDa。
进一步地,该知母活性多糖的糖链组成摩尔比为:T-Araf:1,5-Araf:1,3,5-Araf:1,4-Manp:T-Manp:1,6-Galp:1,3,6-Galp:T-Galp=0.129:0.224:0.132:0.195:0.051:0.044:0.088:0.087。
本发明还提供了上述的知母活性多糖的制备方法,该制备方法依次包括以下步骤:
(1)将知母粉碎后加入10倍量乙醇,回流提取,过滤,取沉淀;
(2)向沉淀中加入20倍量水,回流提取,过滤,取滤液并浓缩,获得浓缩液;
(3)向浓缩液中加入95%乙醇,使混合液中含醇量为70%,静置过夜后过滤,取沉淀并将沉淀溶于水中,获得水溶液;
(4)采用Sevage试剂对水溶液进行萃取,取水相,浓缩并干燥后获得一级粗多糖;
(5)将所述的一级粗多糖溶于水中,获得一级粗多糖水溶液;
(6)将一级粗多糖水溶液上样到含DEAE-52填料的层析柱中,加入3倍柱体积的水洗脱,收集馏分,用纯水透析该馏分,截留分子量为3500Da,而后浓缩、冻干,获得二级粗多糖;
(7)将所述的二级粗多糖溶于水中,获得二级粗多糖水溶液;
(8)将二级粗多糖水溶液上样至含有Sephacryl S-200HR填料的层析柱中,加入水洗脱,1mL/min的流速,收集550-900min馏分,获得所述的知母活性多糖。
在上述的知母活性多糖的制备方法中,步骤(4)中所述的Sevage试剂由氯仿和正丁醇按4:1的体积比混合而成。
在上述的知母活性多糖的制备方法中,步骤(4)中,Sevage试剂和水溶液的体积比为1:3。
本发明还提供了一种知母粗多糖,该知母粗多糖即含有上述的知母活性多糖以及其他杂质。
本发明还提供了上述的知母粗多糖的制备方法,该制备方法依次包括以下步骤:
(1)将知母粉碎后加入10倍量乙醇,回流提取,过滤,取沉淀;
(2)向沉淀中加入20倍量水,回流提取,过滤,取滤液并浓缩,获得浓缩液;
(3)向浓缩液中加入95%乙醇,使混合液中含醇量为70%,静置过夜后过滤,取沉淀并将沉淀溶于水中,获得水溶液;
(4)采用Sevage试剂对水溶液进行萃取,取水相,浓缩并干燥后获得所述的一级粗多糖,该一级粗多糖即为所述的知母粗多糖;
或者,还包括:
(5)将所述的一级粗多糖溶于水中,获得一级粗多糖水溶液;
(6)将一级粗多糖水溶液上样到含DEAE-52填料的层析柱中,加入3倍柱体积的水洗脱,收集馏分,用纯水透析该馏分,截留分子量为3500Da,而后浓缩、冻干,获得二级粗多糖,该二级粗多糖即为所述的知母粗多糖。
本发明还提供了知母、所述的知母活性多糖或所述的知母粗多糖在制备治疗、延缓或减轻AGEs相关疾病的药物或保健食品中的应用,该AGEs相关疾病至少包括脂质过氧化损伤、骨质疏松症。
与现有技术相比,本发明的有益效果体现在:
本发明的知母活性多糖能够通过上调AGEs损伤成骨细胞模型GPX4和SLC7A11蛋白表达,抑制细胞脂质过氧化物产生,减少细胞氧化应激损伤,最终达到促进成骨细胞增殖的目的;表明该知母活性多糖、含有该知母活性多糖的知母粗多糖或知母本身均能够用于制备治疗、延缓或减轻AGEs相关疾病的药物或保健食品,达到消除或减轻AGEs造成的细胞损伤的目的。
附图说明
图1为本发明从知母中提取的各多糖样品对AGEs损伤成骨细胞增值活性的影响;
其中,AGEs表示晚期糖基化终末产物,Cell viability(%)表示细胞活力(百分率),**表示P<0.01,*表示P<0.05,下同;
图2为本发明的知母活性多糖PAAP-1B对AGEs损伤成骨细胞中活性氧含量的影响;
其中,Mitosox(MFI,of control)表示线粒体ROS自由基(相对于空白对照组的平均荧光强度);
图3为本发明的知母活性多糖PAAP-1B对AGEs损伤成骨细胞中脂质氧化物含量的影响;
其中,C11 BODIPY(MFI,of control)表示脂质过氧化物含量相对于空白对照组的平均荧光强度;
图4为本发明的知母活性多糖PAAP-1B对AGEs损伤成骨细胞中脂质氧化物标记物4-Hydroxynonenal含量的影响;
其中,4-HNE/GAPDH表示4-羟基壬醛相对于GAPDH的含量;
图5为本发明的知母活性多糖PAAP-1B对AGEs损伤成骨细胞中谷胱甘肽过氧化物酶4表达水平的影响;
其中,GPX4/GAPDH表示谷胱甘肽过氧化物酶4相对于GAPDH的表达水平;
图6为本发明的知母活性多糖PAAP-1B对AGEs损伤成骨细胞中胱氨酸转运蛋白表达水平的影响;
其中,SLC7A11/GAPDH表示胱氨酸转运蛋白相对于GAPDH的表达水平。
具体实施方式
下面结合附图和具体实施方式对本发明的技术方案作进一步详细说明。
实施例1
一、知母活性多糖的制备和结构表征
1、知母活性多糖的制备
本实施例一种知母活性多糖的制备方法,依次包括以下步骤:
(1)取知母药材500g,将其粉碎后,过40目筛,而后加入10倍量乙醇,回流提取2h,过滤,取沉淀;
(2)向沉淀中加入20倍量水,回流提取,过滤,取滤液并浓缩,获得浓缩液;
步骤(2)可重复多次,将所有滤液合并后浓缩;
(3)向浓缩液中加入95%乙醇,使混合液中含醇量为70%,4℃静置过夜后过滤,取沉淀并将沉淀溶于水中,5000×g离心15min,去除不溶物,获得水溶液;
(4)采用Sevage试剂对水溶液进行萃取,取水相,浓缩并干燥后获得一级粗多糖;
其中,该Sevage试剂由氯仿和正丁醇按4:1的体积比混合而成,Sevage试剂和水溶液的体积比为1:3;
(5)将一级粗多糖溶于水中,获得浓度为20mg/mL的一级粗多糖水溶液;
(6)将一级粗多糖水溶液上样到含DEAE-52填料的层析柱中,依次加入3倍柱体积的水、7.5倍柱体积0.2M NaCl、3倍柱体积0.5M NaCl、3倍柱体积1.0M NaCl进行梯度洗脱,分别收集馏分,用纯水透析各馏分,截留分子量为3500Da,而后浓缩、冻干,获得三种二级粗多糖样品,分别是纯水为洗脱液的AAP-1,0.2MNaCl为洗脱液的AAP-2和1.0MNaCl为洗脱液的AAP-3;
(7)将AAP-1溶于水中,获得浓度为5mg/mL的二级粗多糖水溶液;
(8)将二级粗多糖水溶液上样至含有Sephacryl S-200HR填料的层析柱中,加入水洗脱,在1mL/min的流速下,收集0-550min馏分获得精多糖PAAP-1A,收集550-900min馏分获得精多糖PAAP-1B;其中,PAAP-1B即为本实施例的知母活性多糖。
2、知母活性多糖的结构表征
(1)分子量
经检测,本发明知母活性多糖PAAP-1B的重均分子量Mw为14KDa。
(2)单糖组成
经检测,本发明知母活性多糖PAAP-1B由阿拉伯糖(简称Araf)、半乳糖(简称Galp)和甘露糖(简称Manp)组成,且Araf、Galp和Manp的摩尔比为6:1:3。
(3)糖链组成
经检测,本发明知母活性多糖PAAP-1B的糖链组成摩尔比为:T-Araf:1,5-Araf:1,3,5-Araf:1,4-Manp:T-Manp:1,6-Galp:1,3,6-Galp:T-Galp=0.129:0.224:0.132:0.195:0.051:0.044:0.088:0.087。
(4)核磁共振谱
本发明知母活性多糖PAAP-1B的核磁表征结果如表1所示。
表1明知母活性多糖PAAP-1B的核磁表征结果
1 | 2 | 3 | 4 | 5 | 6 | |
T-α-Manp | 5.16 | 4.16 | 3.78 | 3.52 | 3.68 | 3.91 |
A | 107.0 | 70 | 72.1 | 70.8 | 72.7 | 60.5 |
T-α-Araf | 5.13 | 4.11 | 3.82 | 3.98 | 3.63 | |
B | 107.1 | 82.4 | 81.3 | 83.7 | 62.5 | |
1,3,5-α-Araf | 5.10 | 4.27 | 4.07 | 4.26 | 3.82 | |
C | 107.4 | 82.3 | 84.1 | 81.4 | 66.5 | |
1,5-α-Araf | 5.07 | 4.11 | 3.94 | 4.27 | 3.91 | |
D | 107.5 | 83.9 | 76.8 | 79.2 | 69.6 | |
1,4-β-Manp | 4.74 | 4.16 | 3.93 | 3.54 | 3.59 | 3.75 |
E | 100.1 | 70.5 | 73.4 | 76.5 | 74.5 | 60.8 |
T-β-Galp | 4.62 | 3.66 | 3.61 | 3.77 | 3.89 | 3.78 |
F | 104.3 | 74.8 | 74.4 | 73.3 | 76.5 | 62.5 |
1,6-β-Galp | 4.43 | 3.51 | 3.62 | 3.86 | 3.85 | 3.72 |
G | 103.6 | 75 | 71.8 | 76.5 | 76.4 | 66.5 |
1.3,6-β-Galp | 4.44 | 3.53 | 3.85 | 3.84 | 3.64 | 3.76 |
H | 103.7 | 75.1 | 82.2 | 71.4 | 69.9 | 66.2 |
T-α-Manp | 5.16 | 4.16 | 3.78 | 3.52 | 3.68 | 3.91 |
A | 107.0 | 70 | 72.1 | 70.8 | 72.7 | 60.5 |
(5)化学结构表征
经分析,本发明知母活性多糖PAAP-1B的化学结构如式(Ⅰ)所示:
二、各多糖样品的活性测试
采用晚期糖基化终末产物(AGEs,0.1mg/mL)损伤成骨细胞,制备AGEs损伤成骨细胞模型。而后分别称取AAP-1、AAP-2、AAP-3、PAAP-1A、PAAP-1B,用细胞培养液溶解稀释,调整至最终给药浓度为400μg/L。
将药物与AGEs损伤成骨细胞模型共培养48h后,采用MTT法检测本发明制备的多糖对细胞增殖活性的影响,检测结果见图1。
由图1可见,与模型组相比,空白组的细胞增殖活性显著较高(P<0.01),AAP-1及PAAP-1B给药组的细胞增殖活性也显著较高(P<0.01)。和AAP-1给药组相比,PAAP-1B给药组的细胞增殖活性显著较高(P<0.05)。
由此可见,AAP-1具有显著提高AGEs损伤成骨细胞增殖活性的作用,而AAP-2和AAP-3都没有促进成骨细胞增殖的作用。将AAP-1进一步通过凝胶层析柱分离纯化后得到PAAP-1A和PAAP-1B,其中PAAP-1A不能显著促进AGEs损伤成骨细胞增殖,而PAAP-1B能够显著促进AGEs损伤成骨细胞增殖;且与AAP-1相比,PAAP-1B的活性更强。
在此基础上,继续对PAAP-1B的活性进行分析。
三、知母活性多糖PAAP-1B的活性分析
(1)PAAP-1B对细胞活性氧含量的影响
采用Mitosox(invitrogen,#M36008)分别孵育空白组、模型组和PAAP-1B给药组细胞,孵育10min后用PBS清洗细胞两次以除去多余染料,而后采用流式细胞术检测本发明的知母活性多糖PAAP-1B对AGEs损伤成骨细胞中活性氧含量的影响,检测结果见图2。
由图2可见,和模型组相比,空白组和PAAP-1B给药组的活性氧含量显著偏低(P<0.01),说明本发明的知母活性多糖PAAP-1B具有显著的抗氧化活性。
(2)PAAP-1B对细胞脂质氧化物含量的影响
采用C11 BODIPY(invitrogen,#D3861)分别孵育空白组、模型组和PAAP-1B给药组细胞,孵育20min后用PBS清洗细胞两次以除去多余染料,而后采用流式细胞术检测各组细胞的C11 BODIPY荧光强度,以考察各组细胞中脂质氧化物的含量,检测结果见图3。
由图3可见,和空白组相比,模型组的脂质过氧化物含量显著偏高(P<0.05);而与模型组相比,PAAP-1B给药组的脂质过氧化物含量显著偏低(P<0.01)。表明本发明的知母活性多糖PAAP-1B能够拮抗脂质过氧化物的形成。
(3)PAAP-1B对细胞脂质氧化物标记物4-Hydroxynonenal含量的影响
采用免疫印迹法检测本发明的知母活性多糖PAAP-1B对AGEs损伤成骨细胞中脂质氧化物标记物4-Hydroxynonenal(4-HNE)含量的影响,检测结果见图4。
由图4可见,与空白组相比,模型组的4-HNE含量显著升高(P<0.01);而与模型组相比,PAAP-1B给药组的4-HNE含量显著降低(P<0.01),表明本发明的知母活性多糖PAAP-1B能够拮抗脂质过氧化物标记物4-HNE的生成。
(4)PAAP-1B对细胞谷胱甘肽过氧化物酶4表达的影响
采用免疫印迹法检测本发明的知母活性多糖PAAP-1B对AGEs损伤成骨细胞中谷胱甘肽过氧化物酶4(GPX4)的影响,检测结果见图5。
由图5可见,与空白组相比,模型组的GPX4表达水平显著降低(P<0.01);而与模型组相比,PAAP-1B给药组的GPX4的表达水平显著升高(P<0.01);表明本发明的知母活性多糖PAAP-1B能够提高细胞中GPX4的表达水平,从而分解细胞中因AGEs产生的过氧化物。
(5)PAAP-1B对细胞中胱氨酸转运蛋白表达的影响
采用免疫印迹法检测本发明的知母活性多糖PAAP-1B对AGEs损伤成骨细胞中胱氨酸转运蛋白(SLC7A11)的影响,检测结果见图6。
由图6可见,与空白组相比,模型组的SLC7A11表达水平显著降低(P<0.01);而与模型组相比,PAAP-1B给药组的SLC7A11表达水平显著升高(P<0.05)。表明本发明的知母活性多糖PAAP-1B能够提高细胞中胱氨酸转运蛋白SLC7A11的表达水平,从而向细胞内转运更多的胱氨酸以参与谷胱甘肽过氧化物酶的合成。
以上分析结果表明,本发明的知母活性多糖PAAP-1B能够通过上调AGEs损伤成骨细胞模型GPX4和SLC7A11蛋白表达,抑制细胞脂质过氧化物产生,减少细胞氧化应激损伤,最终达到促进成骨细胞增殖的目的。
Claims (10)
2.如权利要求1所述的知母活性多糖,其特征在于,阿拉伯糖、半乳糖和甘露糖的摩尔比为6:1:3,重均分子量为14KDa。
3.如权利要求1所述的知母活性多糖,其特征在于,糖链组成摩尔比为:T-Araf:1,5-Araf:1,3,5-Araf:1,4-Manp:T-Manp:1,6-Galp:1,3,6-Galp:T-Galp=0.129:0.224:0.132:0.195:0.051:0.044:0.088:0.087。
4.一种知母粗多糖,其特征在于,含有如权利要求1-3中任意一项所述的知母活性多糖。
5.如权利要求1-3中任意一项所述的知母活性多糖的制备方法,其特征在于,依次包括以下步骤:
(1)将知母粉碎后加入10倍量乙醇,回流提取,过滤,取沉淀;
(2)向沉淀中加入20倍量水,回流提取,过滤,取滤液并浓缩,获得浓缩液;
(3)向浓缩液中加入95%乙醇,使混合液中含醇量为70%,静置过夜后过滤,取沉淀并将沉淀溶于水中,获得水溶液;
(4)采用Sevage试剂对水溶液进行萃取,取水相,浓缩并干燥后获得一级粗多糖;
(5)将所述的一级粗多糖溶于水中,获得一级粗多糖水溶液;
(6)将一级粗多糖水溶液上样到含DEAE-52填料的层析柱中,加入3倍柱体积的水洗脱,收集馏分,用纯水透析该馏分,截留分子量为3500Da,而后浓缩、冻干,获得二级粗多糖;
(7)将所述的二级粗多糖溶于水中,获得二级粗多糖水溶液;
(8)将二级粗多糖水溶液上样至含有Sephacryl S-200HR填料的层析柱中,加入水洗脱,1mL/min的流速下收集550-900min馏分,获得所述的知母活性多糖。
6.如权利要求5所述的知母活性多糖的制备方法,其特征在于,步骤(4)中所述的Sevage试剂由氯仿和正丁醇按4:1的体积比混合而成。
7.如权利要求5所述的知母活性多糖的制备方法,其特征在于,步骤(4)中,Sevage试剂和水溶液的体积比为1:3。
8.如权利要求4所述的知母粗多糖的制备方法,其特征在于,依次包括以下步骤:
(1)将知母粉碎后加入10倍量乙醇,回流提取,过滤,取沉淀;
(2)向沉淀中加入20倍量水,回流提取,过滤,取滤液并浓缩,获得浓缩液;
(3)向浓缩液中加入95%乙醇,使混合液中含醇量为70%,静置过夜后过滤,取沉淀并将沉淀溶于水中,获得水溶液;
(4)采用Sevage试剂对水溶液进行萃取,取水相,浓缩并干燥后获得所述的一级粗多糖,该一级粗多糖即为所述的知母粗多糖;
或者,还包括:
(5)将所述的一级粗多糖溶于水中,获得一级粗多糖水溶液;
(6)将一级粗多糖水溶液上样到含DEAE-52填料的层析柱中,加入3倍柱体积的水洗脱,收集馏分,用纯水透析该馏分,截留分子量为3500Da,而后浓缩、冻干,获得二级粗多糖,该二级粗多糖即为所述的知母粗多糖。
9.如权利要求1-3中任意一项所述的知母活性多糖或如权利要求4所述的知母粗多糖在制备治疗、延缓或减轻AGEs相关疾病的药物或保健食品中的应用,其特征在于,所述的AGEs相关疾病至少包括脂质过氧化损伤、骨质疏松症。
10.知母在制备治疗、延缓或减轻AGEs相关疾病的药物或保健食品中的应用,其特征在于,所述的AGEs相关疾病至少包括脂质过氧化损伤、骨质疏松症。
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