CN108997510A - 一种高良姜多糖的抗氧化应用及其分离纯化方法 - Google Patents
一种高良姜多糖的抗氧化应用及其分离纯化方法 Download PDFInfo
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- CN108997510A CN108997510A CN201810904675.7A CN201810904675A CN108997510A CN 108997510 A CN108997510 A CN 108997510A CN 201810904675 A CN201810904675 A CN 201810904675A CN 108997510 A CN108997510 A CN 108997510A
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- galangal
- polysaccharide
- distilled water
- thick many
- many candies
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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
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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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
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Sustainable Development (AREA)
- Toxicology (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
本发明涉及一种高良姜多糖的抗氧化应用及其分离纯化方法,本申请涉及高良姜粗多糖、高良姜精制多糖的抗氧化应用,所述高良姜粗多糖、高良姜精制多糖通过对高良姜进行预处理、分级提取和醇沉步骤的结合,获得高良姜粗多糖,并进一步的利用阴阳离子树脂串联柱、离子交换凝胶柱和凝胶色谱柱法对高良姜粗多糖进一步了分离纯化,得到六种高良姜精制多糖。本发明提取率高、纯度高的高良姜多糖的提取分离方法有利于提高高良姜多糖的产率,避免因提取方法不当导致的资源浪费,增加高良姜的附加值,精制获得的高良姜多糖及其混合物相对于高良姜粗多糖具有明显更优的抗氧化活性,也有利于后续针对于高良姜多糖其他药理活性的深入研究,促进高良姜多糖产业的发展。
Description
技术领域
本发明属于药物领域,具体涉及一种高良姜多糖的抗氧化应用及其分离纯化方法。
背景技术
高良姜作为一种常用中药,用药历史悠久,为姜科山姜属植物高良姜(Alpiniaofficinarum Hance)的干燥根茎,主产于广东、广西、海南与中国台湾等,又称风姜、高凉姜,良姜,未辛,性热,归脾、胃经,具有温胃止呕,散寒止痛的功效,常用于脘腹冷痛,胃寒呕吐,嗳气吞酸等症的治疗,如经典的二姜丸即为高良姜与炮姜组成,可用于胃寒、脘腹冷痛的治疗。
近年来对于高良姜所含化学成分及其药理作用的研究逐渐深入,现代药理学研究显示,高良姜具有抗菌、抗病毒、抗癌、抗氧化、胃肠道保护等作用。植物化学研究显示,高良姜主要含有挥发油类、黄酮类、二芳基庚烷类、苯丙素类、糖苷类及多糖类成分,其中针对于黄酮类和二芳基庚烷类成分的提取分离及药理活性研究比较广泛,但针对于高良姜多糖的研究仍然较少。
姜科植物多糖,如春砂仁、莪术等植物多糖具有广泛的药理活性,如抗肿瘤、调节免疫、抗氧化等,但仅有少量文献报道了高良姜多糖具有抗氧化活性、对酪氨酸激酶的抑制作用,目前对于高良姜多糖的研究仍然较少的原因之一是现有的高良姜多糖的提取、分离、纯化工艺存在诸多的不足和缺点,如现有的溶剂提取法、酸提法、碱提法、酶解法、超滤法、超声法、微波法、超临界流体萃取法等提取率都偏低,缺乏有效的分离纯化方法,导致提取得到高良姜多糖纯度不高,从而阻碍了对于高良姜多糖的进一步研究,也不利于高良姜产业的发展。
本发明发明人在充分调研现有技术的基础上,力图提供一种高纯度的高良姜多糖及其分离纯化方法和应用,从而促进高良姜产业的发展壮大。
发明内容
本发明的目的是提供一种高良姜多糖的抗氧化应用及其分离纯化方法。
本申请的一个目的是提供了一种高良姜多糖再制备抗氧化药物组合物、保健品、食品中的用途。
本申请的再一个目的是提供了一种高良姜粗多糖的提取分离方法,包括如下步骤:
(1)将高良姜药材粉碎,得到高良姜粗粉;乙醇浸泡加热回流预处理高良姜粗粉,然后将预处理后的药渣晾干;
(2)将晾干后的药渣室温蒸馏水浸泡,滤过,合并滤液,滤液减压浓缩,经醇沉、透析、冷冻干燥得高良姜冷浸多糖(A);
(3)冷水浸提后的药渣热水煎煮,冷却后滤过、合并滤液。滤液减压浓缩,经醇沉、透析、冷冻干燥得高良姜热提多糖(B);
(4)热提后的药渣再加入氢氧化钠溶液,室温浸提,滤过,合并滤液,用盐酸调pH值至中性,上清液减压浓缩,经醇沉、透析、冷冻干燥得高良姜碱提多糖(C);
(5)将步骤(2)-(4)得到的高良姜多糖分级提取液在45℃减压浓缩,加入95%乙醇使乙醇终浓度为80%,抽滤,得沉淀,将沉淀用无水乙醇和丙酮交替洗涤2次,再用去离子水复溶,透析,冷冻干燥,即得本发明的高良姜粗多糖。
优选的,步骤(1)中所述加热回流的温度为50-90℃;所述的乙醇浸泡时间为5-20h;回流提取的时间为1-5h;乙醇回流提取的次数为1-5次;乙醇的体积分数为70-95%;
步骤(2)所述蒸馏水浸泡时间为5-20h;浸泡次数为1-5次;
步骤(3)中所述热水温度为80-100℃;煎煮时间为2-5h;煎煮次数1-5次;
步骤(4)中所述的氢氧化钠的浓度为1mol/L;盐酸的浓度为0.5mol/L;
步骤(2)-(4)中所述的减压浓缩温度为45℃;醇沉中的沉淀剂为95%乙醇,并使乙醇终浓度为80%;将沉淀用无水乙醇和丙酮交替洗涤2次,再用去离子水复溶,透析,冷冻干燥;干燥温度为45℃。
更优选的,步骤(1)中所述加热回流的温度为75℃;所述的乙醇浸泡时间为12h;回流提取的时间为3h;乙醇回流提取的次数为3次;乙醇的体积分数为95%;
步骤(2)所述蒸馏水浸泡时间为12h;浸泡次数为3次;
步骤(3)中所述热水温度为100℃;煎煮时间为3h;煎煮次数3次;
本申请的再一个目的是提供一种高良姜粗多糖,其通过以上方法制备得到。
本发明的再一个目的是提供一种高良姜粗多糖在制备抗氧化药物组合物、保健品或食品中的用途,所述高良姜粗多糖通过以上方法制备得到。
本发明的再一个目的是提供一种高良姜多糖的分离纯化方法,包括以下步骤:
(1)将高良姜多糖B组分配制成终浓度为5%的多糖溶液,转入Amberlite FPA90-Cl+Amberlite IRC-84阴阳离子树脂串联柱中,首先以蒸馏水作为洗脱液,流速10mL/min,,苯酚一硫酸法跟踪检测洗脱液,至无糖组分流出,改用0.5M NaCl洗脱,至无糖组分流出为止,分别得到水洗脱和0.5M NaCl洗脱组分,将这两个组分分别装入透析袋用蒸馏水透析,透析后袋内液减压浓缩,冷冻干燥,分别得到水和0.5M NaCl洗脱部位Fr.A和Fr.B;
(2)将Fr.A和Fr.B分别溶解于蒸馏水配成浓度为5%的糖溶液,离心,滤过,滤液分别经DEAE-Cellulose DE-52离子交换柱,Fr.A依次用蒸馏水、0.1M NaCl、0.2M NaCl溶液作为洗脱液,流速2mL/min,分别收集各流份,苯酚一硫酸法跟踪检测洗脱液,测定490nm下的吸光度,绘制洗脱曲线,同一吸收峰内的洗脱液合并,用蒸馏水透析,透析袋内液减压浓缩,冷冻干燥,分别得到两者洗脱部位Fr.A-1和Fr.A-2,Fr.B分别以水、0.1M、0.2M、0.4M的NaCl溶液进行梯度洗脱,流速2mL/min,流出液10mL/管自动部分收集器分管收集,隔管苯酚-硫酸法显色,每管溶液紫外分光光度计490nm测定,以洗脱管数-光密度值绘制洗脱曲线,合并相同峰位的组分,用蒸馏水透析,透析袋内液减压浓缩,冷冻干燥,得水洗脱组分Fr.B-1,Fr.B-2,Fr.B-3,Fr.B-4;
(3)Fr.A-1,Fr.A-2,Fr.B-1,Fr.B-2,Fr.B-3和Fr.B-4分别溶解于蒸馏水配成浓度为5%的糖溶液,离心,滤过,滤液经过Sephacryl S-400凝胶色谱柱,以蒸馏水作为洗脱剂,0.5mL/min,5mL/管分管收集,根据苯酚-硫酸法检测结果,测定490nm下的吸光度,绘制洗脱曲线,合并相同峰位的流分,用蒸馏水透析,透析袋内液减压浓缩,冷冻干燥,得到Fr.A-1-1,Fr.A-2-1,Fr.B-1-1、Fr.B-2-1、Fr.B-3-1和Fr.B-4-1六个组分,分别命名为WN-A1,WN-A2,WN-B1,WN-B2,WN-B3和WN-B4。
所述的六种高良姜精制多糖都为均一组分,其Mn分子量分别为:WN-A1分子量为243.36Da,WN-A2分子量为623.28Da,WN-B1分子量为3.81×103Da,WN-B2分子量为7.22×103Da,WN-B3分子量为3.68×106Da,WN-B4分子量为3.34×108Da。
本发明的再一个目的是提供一种高良姜精制多糖,其选自WN-A1,WN-A2,WN-B1,WN-B2,WN-B3、WN-B4中的一种或多种,所述WN-A1,WN-A2,WN-B1,WN-B2,WN-B3、WN-B4由以上方法制备得到,优选的所述高良姜精制多糖由WN-A1,WN-A2,WN-B1,WN-B2,WN-B3、WN-B4组成。
本发明的另一个目的是提供高良姜精制多糖在制备抗氧化药物、试剂、保健品中的应用,所述高良姜精制多糖选自WN-A1,WN-A2,WN-B1,WN-B2,WN-B3、WN-B4中的一种或多种,优选的,所述高良姜精制多糖由WN-A1,WN-A2,WN-B1,WN-B2,WN-B3、WN-B4组成。
本发明有益效果
本发明高良姜多糖提取及分离纯化方法通过预处理、分级提取及醇沉步骤使得高良姜多糖的提取率现对于现有提取方法获得明显提高。本发明分离纯化得到的六种高良姜精制多糖及其混合物相对于高良姜粗多糖具有明显更优的抗氧化活性。
本发明提取率高、纯度高的高良姜多糖的提取分离方法有利于提高高良姜多糖的产率,避免因提取方法不当导致的资源浪费,增加高良姜的附加值,精制获得的高良姜多糖也有利于后续针对于高良姜多糖药理活性的深入研究,促进高良姜多糖产业的发展。
附图说明
图1是实施例1的高良姜粗多糖提取分离流程图。
图2和图3是实施例3的离子交换柱层析洗脱曲线图。A:Fr.A B:Fr.B C:Fr.A-1 D:Fr.A-2 E:Fr.B-1 F:Fr.B-2 G:Fr.B-3 H:Fr.B-4
图4是实施例3制备得到的高良姜精制多糖(WN-A1、WN-A2、WN-B1、WN-B2、WN-B3和WN-B4)的紫外扫描光谱图。A:WN-A1,B:WN-A2 C:WN-B1 D:WN-B2 E:WN-B3 F:WN-B4
图5是实施例3制备得到的高良姜精制多糖(WN-A1、WN-A2、WN-B1、WN-B2、WN-B3和WN-B4)的红外光谱图。
图6是实施例3制备得到的高良姜精制多糖(WN-A1、WN-A2、WN-B1、WN-B2、WN-B3和WN-B4)的HPSEC图谱。
图7是实施例3制备得到的高良姜精制多糖(WN-A1、WN-A2、WN-B1、WN-B2、WN-B3)样品酸水解后PMP衍生物HPLC分离图谱。峰:1.甘露糖;3.鼠李糖;4.葡萄糖醛酸;5.半乳糖醛酸;7.半乳糖;9.阿拉伯糖。
具体实施方式
在下文中更详细地描述了本发明以有助于对本发明的理解。
实施例中未注明具体条件的实验方法,通常按照常规条件或按照制造厂商所建议的条件。
以下实施例所用试剂及器材:透析袋(MWCO:3500)Sigma公司;弱阴性阴离子交换树脂FPA90-Cl-、弱阳性阳离子交换树脂FPC3500 Amberlite公司;离子交换纤维素DEAE-Cellulose 52 Whatman(Maidstone,UK);2695高效液相色谱仪Waters公司;常规化学试剂均购自广东西陇精细化工有限公司。
实施例1:一种高良姜粗多糖的提取方法:
(1)高良姜药材0.8kg,粗粉碎后加95%乙醇浸泡过夜,加热回流提取3次,每次3h,除去高良姜中的挥发油类、黄酮类、二芳基庚烷类、色素和低聚糖,提取后的药渣晾干。
(2)将晾干后的药渣室温蒸馏水浸泡3次,每次12h,滤过后合并滤液。滤液减压浓缩,经醇沉、透析、冷冻干燥得高良姜冷浸多糖(A)32g。
(3)冷水浸提后的药渣热水煎煮3次,每次3h,冷却后滤过、合并滤液。滤液减压浓缩,经醇沉、透析、冷冻干燥得高良姜热提多糖(B)55g。
(4)热提后的药渣再加入1mol/L NaOH溶液,室温浸提3次,每次8h,滤过,合并滤液。滤液用0.5mol/L HCl调pH值至中性,上清液减压浓缩,经醇沉、透析、冷冻干燥得高良姜碱提多糖(C)35g。
(5)步骤(2)-(4)得到的高良姜多糖分级提取液在45℃减压浓缩,加入95%乙醇使乙醇终浓度为80%,抽滤,得沉淀,将沉淀用无水乙醇和丙酮交替洗涤2次,再用去离子水复溶,透析,冷冻干燥,得高良姜粗多糖120g(流程图见图1),经计算经醇沉除杂后本申请高良姜粗多糖的提取率依然达到了为15.0%,远高于现有的10%左右的提取率。
实施例2:高良姜多糖含量的测定
多糖含量采用硫酸一苯酚法进行检测。多糖在浓硫酸的作用下,脱水生成糖醛及其衍生物,与苯酚结合后产生黄色物质,该物质在490nm处有最大吸光值,吸光值的大小与多糖含量呈正比。
(1)标准曲线的制备:称取一定量的葡萄糖于105℃烘干至恒重后,准确称取10.00mg,纯水定容至100ml,即得葡萄糖标准储备液。精密量取0.1mg/mL的葡萄糖标准品溶液0.625、1.25、2.5、5.0mL分别置于10mL容量瓶中并定容至刻度得浓度依次为0.00625mg/mL、0.0125mg/mL、0.025mg/mL、0.05mg/mL的葡萄糖标准品溶液。取1.0mL上述各溶液分别置于10mL具塞试管中,再分别加6%苯酚试剂1.0mL摇匀,迅速滴加浓硫酸5.0mL,即刻摇匀,放置5min后,置沸水浴加热15min,取出冷却至室温。另以蒸馏水1.0mL,作为空白对照同上操作,于490nm处测定吸光度值。,以吸光度对葡萄糖浓度进行回归,得到回归方程y=16.282x+0.0346(相关系数R2=0.9998)。式中y为在波长处的吸光值,x为葡萄糖的浓度(mg/mL)。
(2)换算因子的测定:精密称取60℃干燥至恒重的除蛋白精制高良姜多糖10.00mg,置于100mL容量瓶中,加水溶解并稀释至刻度,取此多糖样品溶液1mL按测定标准曲线同样的方法操作,平行测定5次,由回归方程计算出5份高良姜多糖中葡萄糖含量和平均值,再计算出换算因子f=W/(C×D)=1.247(RSD 0.221%,n=5),式中W为多糖质量(mg),C为多糖溶液中葡萄糖浓度(mg/mL),D为多糖的稀释倍数。
(3)样品含量测定:精密称取60℃干燥至恒重的高良姜多糖A、B和C各5.00mg,分别溶解于双蒸水中,定容至25mL,得浓度为0.2mg/mL的样品溶液,分别取此多糖样品溶液1mL,按测定标准曲线同样的方法操作,平行测量3次,从回归方程中求出供试液中葡萄糖的含量,按下式计算样品中多糖含量:多糖含量(%)=C×D×f×100/W
(4)经含量测定得多糖得率依次为:4.0%、22.13%、3.75%。根据标准曲线测得高良姜多糖A、B和C的总糖含量分别为:75.82%、80.12%、74.56%。
实施例3:高良姜精制多糖(WN-A1、WN-A2、WN-B1、WN-B2、WN-B3和WN-B4)的制备:
(1)称取实施例1中制备得到的高良姜粗多糖(B)9.1g,配制成终浓度为5%的多糖溶液,转入Amberlite FPA90-Cl+Amberlite IRC-84阴阳离子树脂串联柱中。首先以蒸馏水作为洗脱液,流速10mL/min,,苯酚一硫酸法跟踪检测洗脱液,至无糖组分流出。改用0.5MNaCl洗脱,至无糖组分流出为止。分别得到水洗脱和0.5M NaCl洗脱组分。将这两个组分分别装入透析袋用蒸馏水透析,透析后袋内液减压浓缩,冷冻干燥,分别得到水和0.5M NaCl洗脱部位Fr.A和Fr.B。
(2)将Fr.A和Fr.B分别溶解于蒸馏水配成浓度为5%的糖溶液,离心,滤过,滤液分别经DEAE-Cellulose DE-52离子交换柱,Fr.A依次用蒸馏水、0.1M NaCl、0.2M NaCl溶液作为洗脱液,流速2mL/min,分别收集各流份,苯酚一硫酸法跟踪检测洗脱液,测定490nm下的吸光度,绘制洗脱曲线(图2),同一吸收峰内的洗脱液合并,用蒸馏水透析,透析袋内液减压浓缩,冷冻干燥,分别得到两者洗脱部位Fr.A-1和Fr.A-2。Fr.B分别以水、0.1M、0.2M、0.4M的NaCl溶液进行梯度洗脱,流速2mL/min,流出液10mL/管自动部分收集器分管收集,隔管苯酚-硫酸法显色,每管溶液紫外分光光度计490nm测定,以洗脱管数-光密度值绘制洗脱曲线(图2),合并相同峰位的组分,用蒸馏水透析,透析袋内液减压浓缩,冷冻干燥,得水洗脱组分Fr.B-1,Fr.B-2,Fr.B-3,Fr.B-4。
(3)Fr.A-1,Fr.A-2,Fr.B-1,Fr.B-2,Fr.B-3和Fr.B-4分别溶解于蒸馏水配成浓度为5%的糖溶液,离心,滤过,滤液经过Sephacryl S-400凝胶色谱柱,以蒸馏水作为洗脱剂,0.5mL/min,5mL/管分管收集,根据苯酚-硫酸法检测结果,测定490nm下的吸光度,绘制洗脱曲线(图3),合并相同峰位的流分,用蒸馏水透析,透析袋内液减压浓缩,冷冻干燥,得到Fr.A-1-1,Fr.A-2-1,Fr.B-1-1、Fr.B-2-1、Fr.B-3-1和Fr.B-4-1六个组分,分别命名为WN-A1,WN-A2,WN-B1,WN-B2,WN-B3和WN-B4。
实施例4:紫外扫描图谱和红外光谱扫描
(1)紫外光谱
分别取实施例1-3中制备得到的高良姜精制多糖(WN-A1、WN-A2、WN-B1、WN-B2、WN-B3和WN-B4)各10mg分别溶解于5mL容量瓶中,配制成2mg/mL的溶液,在200-400nm波长下扫描,记录UV图谱。
(2)红外光谱
分别取实施例1-3中制备得到的高良姜精制多糖(WN-A1、WN-A2、WN-B1、WN-B2、WN-B3和WN-B4)2mg,与KBr混合研细成均匀薄层状,在4000-400cm-1波数范围内进行红外扫描。
结果分析:
(1)多糖紫外光谱分析结果:紫外光谱在260nm处无吸收,说明不含核酸;在280nm处大部分多糖均有微弱的吸收,说明含有少量的蛋白质。(见图4)
(2)多糖红外光谱分析结果:实施例1-3中制备得到的高良姜精制多糖(WN-A1、WN-A2、WN-B1、WN-B2、WN-B3和WN-B4)在3400cm-1处强吸收峰是糖类分子间或分子内的O-H伸缩振动峰,在2900cm-1附近有稍弱吸收峰,有次甲基或甲基的C-H的伸缩振动,在波数1422cm-1和1383cm-1附近的双峰为C-H的变角振动,此为糖类化合物的特征吸收峰。六种多糖在波数1653cm-1-1635cm-1处明显吸收峰是羰基C=O的伸缩振动,,加之1249cm-1-897cm-1`的C-OH伸缩振动,,印证了WN-A1、WN-A2、WN-B1、WN-B2、WN-B3和WN-B4中含有糖醛酸,,进一步说明六种多糖均是酸性多糖。此外,六种多糖在1249cm-1-897cm-1处有2个吸收峰表示WN-A1、WN-A2、WN-B1、WN-B2、WN-B3和WN-B4糖环构型为呋喃型。(图5)
实施例5:WN-A1、WN-A2、WN-B1、WN-B2、WN-B3和WN-B4的分子量及纯度鉴定
采用高效液相色谱仪分别对实施例1-3中制备得到的高良姜精制多糖WN-A1、WN-A2、WN-B1、WN-B2、WN-B3和WN-B4的分子量及其纯度进行测定。色谱条件:waters高效液相色谱系统(2996泵,Alltech ELSD2000,Shodex sugar KS-805+保护柱KS-G),流动相为超纯水,流速0.5mL/min,柱温25℃。
结果分析:
实施例3制备得到的六种高良姜精制多糖(WN-A1、WN-A2、WN-B1、WN-B2、WN-B3和WN-B4)的HPSEC图谱显示为单一对称峰(见图6),表明以上六种高良姜多糖为均一性多糖。经HPLC-ELSD法测定WN-A1、WN-A2、WN-B1、WN-B2、WN-B3和WN-B4的分子量分别为2.34×104Da、5.88×106 Da、3.81×103 Da、7.22×103 Da、3.68×106 Da和3.34×108 Da。
实施例6 WN-A1、WN-A2、WN-B1、WN-B2、WN-B3和WN-B4的单糖组成分析
采用柱前衍生化HPLC法测定高良姜均多糖的单糖组成分析。分别吸取单糖标准溶液和混合单糖标准溶液各1400μL与700μL 0.5mol/L PMP甲醇溶液及700μL 0.3mol/L NaOH溶液至7mL离心管中,密封,涡旋混合30s,70℃水浴反应45min,冷却至室温后加入700μL0.3mol/L HCl中和,再加入乙酸异戊酯3mL涡旋混合30s,10000转离心10min,吸取上层液弃去,依法进行萃取两次,再加3mL氯仿涡旋混合30s,10000转离心10min,吸取上层水液过0.22μm微孔滤膜,进行HPLC分析。再按标品衍生化的方法对多糖样品水解液进行衍生化。
色谱条件:Waters2695型高效液相色谱系统(2996泵、breeze工作站),色谱柱TSKgel ODS-100V(4.6mm×25mm,5μm),流动相为0.2mol/L磷酸盐缓冲溶液-乙腈,流速1.0mL/min,柱温25℃,检测波长254nm。
结果分析:
WN-A1为酸性多糖,由D-甘露糖、L-鼠李糖、D-葡萄糖醛酸、D-半乳糖醛酸、D-半乳糖、L-阿拉伯糖组成。其摩尔百分比为4.2:4.5:1.7:6.1:45.8:37.8。WN-A2为酸性多糖,由L-鼠李糖、D-半乳糖醛酸、D-半乳糖、L-阿拉伯糖组成。其摩尔百分比为12.3:49.3:21.5:16.8。WN-B1为酸性多糖,由D-甘露糖、L-鼠李糖、D-半乳糖醛酸、D-半乳糖、L-阿拉伯糖组成。其摩尔百分比为:15.6:7.5:42.2:14.4:20.3。WN-B2为酸性多糖,由D-甘露糖、L-鼠李糖、D-葡萄糖醛酸、D-半乳糖醛酸、D-半乳糖、L-阿拉伯糖组成。其摩尔百分比16.3:4.0:2.9:13.4:21.7:41.7。WN-B3为酸性多糖,由D-甘露糖、L-鼠李糖、D-葡萄糖醛酸、D-半乳糖醛酸、D-半乳糖、L-阿拉伯糖组成。其摩尔百分比4.5:4.3:36.1:5.7:23.2:20.1。(见图7和表1)
表1实施例3制备得到的高良姜精制多糖(WN-A1、WN-A2、WN-B1、WN-B2、WN-B3)所含单糖摩尔百分比分析
效果例1:高良姜多糖精制多糖的抗氧化活性
1、测试物:本发明高良姜粗多糖、本发明高良姜精制多糖WN-A1,WN-A2,WN-B1,WN-B2,WN-B3、WN-B4,本发明高良姜精制多糖混合物(由WN-A1,WN-A2,WN-B1,WN-B2,WN-B3、WN-B4按照1:1:1:1:1:1的比例配制)、维生素C。
2、实验方法
准确称取DPPH·20mg,溶解于无水乙醇中,并定容与250mL棕色容量瓶中,从而制得浓度为2×10-4mol/L的DPPH·乙醇溶液,于0-4℃下避光保存。
将各测试物使用无水乙醇配置成20mg/L的溶液,取各测试无乙醇溶液2mL与DPPH·乙醇溶液2mL,加入到具塞试管中,混匀后于室温下静置30min,用无水乙醇做餐皮测定在517nm处的吸光度A1,同时测定DPPH·溶液于2mL无水乙醇混合液在517nm处的吸光度A2,以及2mL测试物乙醇容易与2mL无水乙醇混合液在517nm处的吸光度A3,计算各测试物的DPPH清除率,具体结果见表2,其中
测试物的DPPH清除率(简称清除率)=[(A2+A3)-A1]/A2×100%。
3、实验结果
表2高良姜精制多糖的DPPH清除率
表2实验结果显示了,本发明高良姜精制多糖WN-A1,WN-A2,WN-B1,WN-B2,WN-B3、WN-B4均具有明显优于高良姜粗多糖的DPPH清除率,表明本发明高良姜精制多糖WN-A1,WN-A2,WN-B1,WN-B2,WN-B3、WN-B4相对于高良姜粗多糖均具有明显更优的抗氧化能力,而本发明高良姜多糖混合物则具有更为优异的DPPH清除率,显示了本发明高良姜精制多糖WN-A1,WN-A2,WN-B1,WN-B2,WN-B3、WN-B4混合后抗氧化能力相对于单独高良姜精制多糖明显增强,具有协同增效作用。
以上描述了本发明优选实施方式,然其并非用以限定本发明。本领域技术人员对在此公开的实施方案可进行并不偏离本发明范畴和精神的改进和变化。
Claims (10)
1.高良姜多糖在制备治疗抗氧化药物组合物、保健品、食品中的应用。
2.一种高良姜粗多糖的提取分离方法,其特征在于包括如下步骤:
(1)将高良姜药材粉碎,得到高良姜粗粉;乙醇浸泡加热回流预处理高良姜粗粉,然后将预处理后的药渣晾干;
(2)将晾干后的药渣室温蒸馏水浸泡,滤过,合并滤液,滤液减压浓缩,经醇沉、透析、冷冻干燥得高良姜冷浸多糖(A);
(3)冷水浸提后的药渣热水煎煮,冷却后滤过、合并滤液。滤液减压浓缩,经醇沉、透析、冷冻干燥得高良姜热提多糖(B);
(4)热提后的药渣再加入氢氧化钠溶液,室温浸提,滤过,合并滤液,用盐酸调pH值至中性,上清液减压浓缩,经醇沉、透析、冷冻干燥得高良姜碱提多糖(C);
(5)将步骤(2)-(4)得到的高良姜多糖分级提取液在45℃减压浓缩,加入95%乙醇使乙醇终浓度为80%,抽滤,得沉淀,将沉淀用无水乙醇和丙酮交替洗涤2次,再用去离子水复溶,透析,冷冻干燥,即得高良姜粗多糖。
3.根据权利要求1所述的高良姜粗多糖的提取分离方法,其特征在于步骤(1)中所述加热回流的温度为50-90℃;所述的乙醇浸泡时间为5-20h;回流提取的时间为1-5h;乙醇回流提取的次数为1-5次;乙醇的体积分数为70-95%;步骤(2)所述蒸馏水浸泡时间为5-20h;浸泡次数为1-5次;步骤(3)中所述热水温度为80-100℃;煎煮时间为2-5h;煎煮次数1-5次;步骤(4)中所述的氢氧化钠的浓度为1mol/L;盐酸的浓度为0.5mol/L。
4.根据权利要求1所述的高良姜粗多糖的提取分离方法,其特征在于步骤(1)中所述加热回流的温度为75℃;所述的乙醇浸泡时间为12h;回流提取的时间为3h;乙醇回流提取的次数为3次;乙醇的体积分数为95%;步骤(2)所述蒸馏水浸泡时间为12h;浸泡次数为3次;步骤(3)中所述热水温度为100℃;煎煮时间为3h;煎煮次数3次。
5.权利要求1-3任一项所述方法制备得到的高良姜粗多糖。
6.一种高良姜粗多糖的分离纯化方法,其特征在于包括以下步骤:
(1)将权利要求1-3任一项所述的制备方法中步骤(3)所获得的高良姜多糖B组分配制成终浓度为5%的多糖溶液,转入Amberlite FPA90-Cl+Amberlite IRC-84阴阳离子树脂串联柱中,首先以蒸馏水作为洗脱液,流速10mL/min,,苯酚一硫酸法跟踪检测洗脱液,至无糖组分流出,改用0.5M NaCl洗脱,至无糖组分流出为止,分别得到水洗脱和0.5M NaCl洗脱组分,将这两个组分分别装入透析袋用蒸馏水透析,透析后袋内液减压浓缩,冷冻干燥,分别得到水和0.5M NaCl洗脱部位Fr.A和Fr.B;
(2)将Fr.A和Fr.B分别溶解于蒸馏水配成浓度为5%的糖溶液,离心,滤过,滤液分别经DEAE-Cellulose DE-52离子交换柱,Fr.A依次用蒸馏水、0.1M NaCl、0.2M NaCl溶液作为洗脱液,流速2mL/min,分别收集各流份,苯酚一硫酸法跟踪检测洗脱液,测定490nm下的吸光度,绘制洗脱曲线,同一吸收峰内的洗脱液合并,用蒸馏水透析,透析袋内液减压浓缩,冷冻干燥,分别得到两者洗脱部位Fr.A-1和Fr.A-2,Fr.B分别以水、0.1M、0.2M、0.4M的NaCl溶液进行梯度洗脱,流速2mL/min,流出液10mL/管自动部分收集器分管收集,隔管苯酚-硫酸法显色,每管溶液紫外分光光度计490nm测定,以洗脱管数-光密度值绘制洗脱曲线,合并相同峰位的组分,用蒸馏水透析,透析袋内液减压浓缩,冷冻干燥,得水洗脱组分Fr.B-1,Fr.B-2,Fr.B-3,Fr.B-4;
(3)Fr.A-1,Fr.A-2,Fr.B-1,Fr.B-2,Fr.B-3和Fr.B-4分别溶解于蒸馏水配成浓度为5%的糖溶液,离心,滤过,滤液经过Sephacryl S-400凝胶色谱柱,以蒸馏水作为洗脱剂,0.5mL/min,5mL/管分管收集,根据苯酚-硫酸法检测结果,测定490nm下的吸光度,绘制洗脱曲线,合并相同峰位的流分,用蒸馏水透析,透析袋内液减压浓缩,冷冻干燥,得到Fr.A-1-1,Fr.A-2-1,Fr.B-1-1、Fr.B-2-1、Fr.B-3-1和Fr.B-4-1六个组分,分别命名为WN-A1,WN-A2,WN-B1,WN-B2,WN-B3和WN-B4。
7.根据权利要求5所述的高良姜多糖的分离纯化方法,其特征在于六种高良姜精制多糖都为均一组分,其Mn分子量分别为:WN-A1分子量为243.36Da,WN-A2分子量为623.28Da,WN-B1分子量为3.81×103Da,WN-B2分子量为7.22×103Da,WN-B3分子量为3.68×106Da,WN-B4分子量为3.34×108Da。
8.一种高良姜精制多糖,其特征在于选自WN-A1,WN-A2,WN-B1,WN-B2,WN-B3、WN-B4中的一种或多种,所述WN-A1,WN-A2,WN-B1,WN-B2,WN-B3、WN-B4通过权利要求5或6所述的方法制备得到。
9.根据权利要求7所述的高良姜精制多糖,其特征在于由WN-A1,WN-A2,WN-B1,WN-B2,WN-B3、WN-B4组成,所述WN-A1,WN-A2,WN-B1,WN-B2,WN-B3、WN-B4通过权利要求5或6所述的方法制备得到。
10.权利要求7或8所述的高良姜精制多糖在制备抗氧化药物组合物、保健品或食品中的用途。
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CN113355166A (zh) * | 2021-06-30 | 2021-09-07 | 中国热带农业科学院热带作物品种资源研究所 | 一种抑制单增李斯特菌的高良姜精油的提取方法及高良姜精油 |
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