CN109160954B - 一种香瓜茄酸性多糖及其提纯方法和用途 - Google Patents
一种香瓜茄酸性多糖及其提纯方法和用途 Download PDFInfo
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- CN109160954B CN109160954B CN201811148644.XA CN201811148644A CN109160954B CN 109160954 B CN109160954 B CN 109160954B CN 201811148644 A CN201811148644 A CN 201811148644A CN 109160954 B CN109160954 B CN 109160954B
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- 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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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了一种香瓜茄酸性多糖及其提纯方法和用途,所述酸性多糖为均一酸性杂多糖,其化学结构含有吡喃环和糖醛酸,由包括鼠李糖、阿拉伯糖、半乳糖、葡萄糖和半乳糖醛酸的单糖组成;包括将香瓜茄经过(1)烘干、粉碎和除杂和(2)水提醇沉得水提粗多糖的粗多糖提取;和将所述水提粗多糖经过(ⅰ)去除蛋白,透析,真空冷冻干燥;和(ⅱ)大孔树脂吸附;和(ⅲ)阴离子交换层析纯化;和/或(ⅳ)凝胶柱层析纯化的分离纯化处理,所得香瓜茄酸性多糖具有很好的抗氧化能力,可以作为新型抗氧化剂用于食品,化妆品和医药等产业。
Description
技术领域
本发明涉及生物提纯技术领域,具体涉及一种具有抗氧化作用的香瓜茄酸性多糖及其提纯方法和用途。
背景技术
香瓜茄(Solanum muricatum Ait)为茄科茄属多年生草本植物,原产于南美的安第斯山北麓,20世纪80年代引入国内,广泛分布于云南、甘肃等地,是当地农民增收的支柱产业,对其进行深入研究,能够促进对香瓜茄的综合开发利用,提升香瓜茄的经济效益。香瓜茄又名人参果,香瓜梨、艳果、香艳茄、香艳梨等,果实呈梨状、椭圆形、乳黄色浆果,未成熟时果皮呈绿色,在成熟期变为金黄色,覆盖着紫色或紫红色条纹。香瓜茄果实中富含丰富的钙,钾和磷等微量元素以及抗坏血酸,烟酸,核黄素和硫胺等维生素。
近年来,多糖研究逐渐成为生命科学领域的新热点,国内外研究发现植物多糖具有抗病毒、抗肿瘤、抗糖尿病和抗氧化等生理活性。目前,对香瓜茄的研究主要侧重于各种溶剂提取物以及小分子化合物,对香瓜茄中多糖的研究尚未有报道。
发明内容
本发明的目的在于填补香瓜茄多糖研究空白,提供一种具有抗氧化作用的香瓜茄酸性多糖及其提纯方法和用途,通过阴离子交换层析和凝胶柱层析纯化粗多糖,得到具有抗氧化活性的均一酸性多糖SMP-3a,用于制备食品、化妆品和医药用抗氧化剂。
为实现本发明的上述目的,采用如下技术方案:
第一方面,香瓜茄酸性多糖,为均一酸性杂多糖SMP-3a,其化学结构含有吡喃环和糖醛酸,重均分子量为6.03×104~6.11×104Da,由包括鼠李糖、阿拉伯糖、半乳糖、葡萄糖和半乳糖醛酸的单糖组成。
所述鼠李糖、阿拉伯糖、半乳糖、葡萄糖和半乳糖醛酸的摩尔比为19.16:19.60:24.25:6.42:21.32。
所述酸性多糖微观结构呈片状或碎屑状,表面光滑或有皱纹,质地致密。
第二方面,上述香瓜茄酸性多糖的提纯方法,包括将香瓜茄经过粗多糖提取和分离纯化处理的步骤;其中,
所述粗多糖提取过程包括将所述香瓜茄经过
(1)烘干、粉碎和除杂;和
(2)水提醇沉得水提粗多糖的子步骤;
所述分离纯化处理过程包括将所述水提粗多糖经过
(ⅰ)去除蛋白,透析,真空冷冻干燥;和
(ⅱ)大孔树脂吸附;和
(ⅲ)阴离子交换层析纯化;和/或
(ⅳ)凝胶柱层析纯化的子步骤。
在某些优选实施方案中,所述步骤(ⅱ)、步骤(ⅲ)和步骤(ⅳ)均包括所述步骤(ⅰ)中透析和真空冷冻干燥处理。在某些优选实施方案中,所述透析的透析袋截留分子量为3500~8000;所述真空冷冻干燥为在-50~-84℃条件下真空冷冻干燥。
在某些优选实施方案中,所述粗多糖提取过程包括:
将新鲜香瓜茄水洗、切片,50℃恒温烘干,粉碎成粉末,60~80目过筛,再用95%乙醇于80~90℃回流提取5~6h,去除所述粉末中脂质、色素和小分子化合物,烘干;
将除杂后的香瓜茄粉末浸泡于水中于80~90℃提取为2~5h,过滤得滤液,滤渣重复提取两次,合并滤液,真空减压浓缩,得香瓜茄提取液;
加入无水乙醇进行沉淀,其中所述香瓜茄提取液和无水乙醇的体积比为1:3,静置过夜,12000rpm离心10min,得沉淀物;
将所得沉淀物分别用无水乙醇、丙酮、乙醚洗涤,-50~-84℃真空冷冻干燥,得水提粗多糖。
在某些优选实施方案中,所述分离纯化过程包括:
将所述水提粗多糖溶于浓度为5.25%的三氯乙酸,静置去除蛋白,60℃真空减压蒸馏除去所述三氯乙酸,真空浓缩,透析,真空冷冻干燥,得除蛋白粗多糖;
将上述除蛋白粗多糖配成质量浓度为30mg/mL的水溶液,过0.45μm滤膜,上样,经大孔树脂纯化,收集滤液,透析,真空冷冻干燥;
将上述冷冻干燥产品配成质量浓度为40mg/mL的水溶液,过0.45μm滤膜,上样,用阴离子交换层析柱纯化,分别用200mL超纯水、0.05mol/L、0.1mol/L和0.3mol/L的NaCl溶液梯度洗脱,流速为1mL/min,每管8mL,收集并合并各洗脱部位产物,透析,真空冷冻干燥;
将上述收集产物配成质量浓度为50mg/mL的水溶液,过0.45μm滤膜,上样,用凝胶柱层析纯化,超纯水洗脱,流速为0.2mL/min,收集合并洗脱液,透析,真空冷冻干燥,即得。
在某些优选实施方案中,步骤(ⅲ)所述阴离子交换层析柱纯化过程中,分别用200mL超纯水、0.05mol/L、0.1mol/L和0.3mol/L的NaCl溶液梯度洗脱,流速为1mL/min,每管8mL,收集0.3mol/L的NaCl溶液洗脱部位产物,并经步骤(ⅳ)所述凝胶柱层析纯化处理。
第三方面,上述香瓜茄酸性多糖在制备食品、化妆品和医药用抗氧化剂的用途。
本发明的有益效果在于:
本发明首次从香瓜茄中提取粗多糖,并通过纯化分离得到中性多糖SMP-0和3种酸性多糖SMP-1、SMP-2、SMP-3,对SMP-3进行凝胶柱层析纯化首次得到具有抗氧化活性的均一酸性多糖SMP-3a,填补香瓜茄多糖研究空白,用于制备食品、化妆品和医药用抗氧化剂。
附图说明
图1为实施例1中香瓜茄酸性多糖SMP-3a的提纯方法流程图。
图2为DEAE-52纯化洗脱曲线图。
图3为SephadexG-100纯化洗脱曲线图。
图4为实施例1中香瓜茄酸性多糖SMP-3a高效凝胶排阻色谱色谱图。
图5为实施例1中香瓜茄酸性多糖SMP-3a以及标准单糖的高效阴离子色谱图。
图6为实施例1中香瓜茄酸性多糖SMP-3a紫外光谱图。
图7为实施例1中香瓜茄酸性多糖SMP-3a傅里叶红外光谱图。
图8为实施例1中香瓜茄酸性多糖SMP-3a扫描电镜图。
图9为实施例1中香瓜茄酸性多糖SMP-3a对羟自由基清除效果图。
图10为实施例1中香瓜茄酸性多糖SMP-3a对DPPH自由基清除效果图。
图11为实施例1中香瓜茄酸性多糖SMP-3a对ABTS自由基清除效果图。
具体实施方式
下面结合附图对本发明做进一步的详细说明,但不作为对本发明的限定。
以下实例中所用香瓜茄为甘肃武威香瓜茄,阴离子交换层析柱为DEAE-52阴离子交换层析柱,凝胶层析柱为SephadexG-100凝胶层析柱。
实施例1
参见图1,香瓜茄酸性多糖SMP-3a的提纯方法包括:(一)粗多糖提取和(二)分离纯化两个步骤,其中,
(一)粗多糖提取过程为:
(1)原料预处理:选择新鲜的香瓜茄,用水洗净后,切片,置于鼓风干燥箱,50℃恒温烘干,用粉碎机粉碎成粉末,过60目筛后备用。
(2)除杂:将上述香瓜茄粉用90%乙醇于90℃回流提取6h,以除去脂质、色素、单糖和小分子物质,烘干后备用。
(3)热水提取:将除杂后的香瓜茄粉末浸泡于水中,80℃提取4h,过滤得滤液,滤渣重复提取两次,合并滤液,真空减压浓缩,得香瓜茄提取液。
(4)乙醇沉淀:加入上述香瓜茄提取液3倍体积的无水乙醇进行沉淀,静置过夜,离心收集沉淀。
(5)清洗:分别用无水乙醇、丙酮、乙醚清洗沉淀,-84℃真空冷冻干燥,得水提粗多糖。
(二)分离纯化过程为:
(1)去除蛋白:将1.5g上述水提粗多糖溶解于5mL超纯水中,加入35mL6%的三氯乙酸(TCA),振荡均匀,置于4℃冰箱18h。
(2)透析:将上述溶液于60℃真空减压浓缩后,装入截留分子量为3500的透析袋,并置于2L烧杯中,每6h更换水,连续透析4天,并于-84℃真空冷冻干燥,得除蛋白粗多糖。
(3)AB-8大孔树脂吸附:将除蛋白粗多糖配成质量浓度为30mg/mL的水溶液,过0.45μm滤膜,上样,经AB-8大孔树脂纯化,去除一部分色素,收集滤液,并于-84℃真空冷冻干燥。
(4)DEAE-52阴离子交换层析:将上述冷冻干燥后的多糖配成质量浓度为40mg/mL的水溶液,过0.45μm滤膜,上样,分别用200mL超纯水,0.05mol/L、0.1mol/L、0.3mol/LNaCl溶液梯度洗脱,流速为1mL/min,收集洗脱液(每管8mL)。
采用苯酚-硫酸法检测每管洗脱液在490nm处的OD值。以管号为横坐标,每管洗脱液对应OD值为纵坐标绘制洗脱曲线,根据洗脱曲线合并单一峰的试管。浓缩后,装入截留分子量为3500的透析袋透析,并于-84℃真空冷冻干燥,得到一种中性多糖SMP-0,三种酸性多糖SMP-1、SMP-2和SMP-3,其中SMP-3含量远大于其他三种多糖,如附图2所示。
(5)SephadexG-100凝胶柱层析:称100mg上述(4)中纯化出的酸性多糖SMP-3,溶于2mL超纯水,上样,用超纯水洗脱,流速为0.2mL/min,收集洗脱液(每管4mL)。
采用苯酚-硫酸法检测每管洗脱液在490nm处的吸光度值。以管号为横坐标,每管洗脱液对应吸光度值为纵坐标绘制洗脱曲线,根据洗脱曲线合并单一峰的试管。浓缩后,装入截留分子量为3500的透析袋透析,并于-84℃真空冷冻干燥,得到均一酸性多糖SMP-3a,如附图3所示。
效果例1:SMP-3a分子质量的测定
采用高效凝胶排阻色谱法(HPSEC),结合多角度激光散色仪和折光示差检测仪测定SMP-3a的相对分子质量为6.07×104Da,如图4所示。
效果例2:单糖组成分析
以标准岩藻糖、鼠李糖、阿拉伯糖、半乳糖、葡萄糖、木糖、甘露糖、果糖、氨基葡萄糖、半乳糖醛酸和葡萄糖醛酸为对照,采用高效阴离子色谱法(HPAEC)测定SMP-3a的单糖组成,如图5所示,SMP-3a的水解产物主要有5个峰,保留时间分别为:9.52min,10.06min,12.93min,15.01min,38.4min。与标准单糖的保留时间比对后,得到SMP-3a主要由鼠李糖、阿拉伯糖、半乳糖、葡萄糖和半乳糖醛酸组成,其摩尔比为19.16:19.60:24.25:6.42:21.32。
效果例3:紫外光谱分析
把2mgSMP-3a溶于2mL蒸馏水,在波长220-400nm处进行紫外扫描,如图6所示,可以发现SMP-3a在260nm处没有吸收峰,表明SMP-3a中不含核酸;在280nm处SMP-3a具有微弱的吸收峰,表明SMP-3a中存在微量蛋白质。
效果例4:傅里叶红外光谱分析
将2mgSMP-3a与KBr粉末混合,压片后进行红外扫描,如图7所示,可以发现SMP-3a在4000~400cm-1范围内具有明显的多糖特征吸收峰。在3364.52cm-1处有一个很强且宽的峰,这是O-H的伸缩振动峰,在2974.26cm-1处为饱和C-H伸缩振动峰,1603.91cm-1处为酯化羧基COO-的伸缩振动峰。此外,在1418.87cm-1处有吸收峰,是C-H键的变形振动,表明SMP-3a中含有糖醛酸;1100~1010cm-1范围内存在3个吸收峰,表明SMP-3a具有吡喃环。
效果例5:扫描电子显微镜分析
采用S3400N扫描电子显微镜观察SMP-3a的微观结构,如图8所示,SMP-3a微观结构呈片状或碎屑状,表面光滑或有皱纹,质地致密。
效果例6:抗氧化活性测定
(1)羟自由基清除实验
配制200μL不同浓度(0.3125,0.625,1.25,2.5,5,10,20mg/mL)的SMP-3a水溶液,加入100μL的FeSO4(6mM),水杨酸(6mM)和H2O2(6mM),振荡均匀,37℃水浴加热30分钟,在510nm处测定吸光度值。清除率计算公式为:
清除率(%)=[1-(A1-A2)/A0]×100;其中,
A0是空白对照(超纯水)的吸光度值,
A1是不同浓度SMP-3a的吸光度值,
A2是用超纯水代替H2O2测得的不同浓度SMP-3a的本底吸光度值,如图9所示,SMP-3a对羟自由基的清除能力呈浓度依赖性,随着浓度的提高而增强,在质量浓度为20mg/ml时,SMP-3a对羟自由基的清除率达到85.75%,其IC50为3.08mg/ml。
(2)DPPH自由基清除实验
配制200μL不同浓度(0.3125,0.625,1.25,2.5,5,10,20mg/mL)的SMP-3a水溶液,加入200μL0.2mM的DPPH溶液,充分振荡均匀,黑暗处放置30min,在波长517nm处测定吸光度值。清除率计算公式为:
清除率(%)=[1-(A1-A2)/A0]×100;其中,
A0是空白对照(超纯水)的吸光度值,
A1是不同浓度SMP-3a的吸光度值,
A2是用超纯水代替DPPH溶液测得的不同浓度SMP-3a的本底吸光度值,如图10所示,SMP-3a对DPPH自由基的清除能力呈浓度依赖性,随着浓度的提高而增强,在质量浓度为20mg/ml时,SMP-3a对DPPH自由基的清除率达到57.35%,具有较强的清除DPPH自由基的能力。
(3)ABTS自由基清除实验
取10mLABTS(7mM)溶液与10mL过硫酸钾(2.45mM)混合,置于黑暗中反应15h以产生ABTS自由基。将混合溶液用pH=7.4的磷酸盐缓冲液稀释,直至稀释后的溶液在734nm处的吸光度为0.7±0.02。配制200μL不同浓度(0.3125,0.625,1.25,2.5,5,10,20mg/mL)的SMP-3a水溶液,加入3.8mL稀释过后的ABTS溶液,在波长734nm处测定吸光度值。清除率计算公式为:
清除率(%)=[1-(A1-A2)/A0]×100;其中:
A0是空白对照(超纯水)的吸光度值,
A1是不同浓度SMP-3a的吸光度值,
A2是用超纯水代替ABTS溶液测得的不同浓度SMP-3a的本底吸光度值,如图11所示,SMP-3a对ABTS自由基的清除能力呈浓度依赖性,随着浓度的提高而增强,在质量浓度为20mg/ml时,SMP-3a对ABTS自由基的清除率达到46.63%,具有一定的ABTS自由基清除能力。
以上详细描述了本发明的一种较佳具体实施例,凡无需创造性劳动就可以根据本发明的构思得到的技术方案,皆应在权利要求书所确定的保护范围内。
Claims (8)
1.香瓜茄酸性多糖,其特征在于,所述酸性多糖为均一酸性杂多糖,其化学结构含有吡喃环,重均分子量为6.03×104~6.11×104Da,由包括摩尔比为19.16:19.60:24.25:6.42:21.32的鼠李糖、阿拉伯糖、半乳糖、葡萄糖和半乳糖醛酸的单糖组成。
2.根据权利要求1所述的香瓜茄酸性多糖,其特征在于,所述酸性多糖微观结构呈片状或碎屑状,表面光滑或有皱纹,质地致密。
3.根据权利要求1或2所述的香瓜茄酸性多糖,其特征在于,其提纯方法包括将香瓜茄经过粗多糖提取和分离纯化处理的步骤;其中,
所述粗多糖提取过程包括将所述香瓜茄经过
(1)烘干、粉碎和除杂;和
(2)水提醇沉得水提粗多糖的子步骤;
所述分离纯化处理过程包括将所述水提粗多糖经过
(ⅰ)去除蛋白,透析,真空冷冻干燥;和
(ⅱ)大孔树脂吸附;和
(ⅲ)阴离子交换层析纯化;和/或
(ⅳ)凝胶柱层析纯化的子步骤。
4.根据权利要求3所述的香瓜茄酸性多糖,其特征在于,所述步骤(ⅱ)、步骤(ⅲ)和步骤(ⅳ)均包括所述步骤(ⅰ)中透析和真空冷冻干燥处理。
5.根据权利要求3或4所述的香瓜茄酸性多糖,其特征在于,
所述透析的透析袋截留分子量为3500~8000;
所述真空冷冻干燥为在温度为在-50~-84℃条件下真空冷冻干燥。
6.根据权利要求3所述的香瓜茄酸性多糖,其特征在于,所述粗多糖提取过程包括:
将新鲜香瓜茄水洗、切片,50℃恒温烘干,粉碎成粉末,60~80目过筛,再用95%乙醇于80~90℃回流提取5~6h,去除所述粉末中脂质、色素和小分子化合物,烘干;
将除杂后的香瓜茄粉末浸泡于水中于80~90℃提取为2~5h,过滤得滤液,滤渣重复提取两次,合并滤液,真空减压浓缩,得香瓜茄提取液;
加入无水乙醇进行沉淀,其中所述香瓜茄提取液和无水乙醇的体积比为1:3,静置过夜,12000rpm离心10min,得沉淀物;
将所得沉淀物分别用无水乙醇、丙酮、乙醚洗涤,-50~-84℃真空冷冻干燥,得水提粗多糖。
7.根据权利要求3或4所述的香瓜茄酸性多糖,其特征在于,所述分离纯化过程包括:
将所述水提粗多糖溶于浓度为5.25%的三氯乙酸,静置去除蛋白,60℃真空减压蒸馏除去所述三氯乙酸,真空浓缩,透析,真空冷冻干燥,得除蛋白粗多糖;
将上述除蛋白粗多糖配成质量浓度为30mg/mL的水溶液,过0.45μm滤膜,上样,经大孔树脂纯化,收集滤液,透析,真空冷冻干燥;
将上述冷冻干燥产品配成质量浓度为40mg/mL的水溶液,过0.45μm滤膜,上样,用阴离子交换层析柱纯化,分别用200mL超纯水、0.05mol/L、0.1mol/L和0.3mol/L的NaCl溶液梯度洗脱,流速为1mL/min,每管8mL,收集并合并各洗脱部位产物,透析,真空冷冻干燥;
将上述收集产物配成质量浓度为50mg/mL的水溶液,过0.45μm滤膜,上样,用凝胶柱层析纯化,超纯水洗脱,流速为0.2mL/min,收集合并洗脱液,透析,真空冷冻干燥,即得。
8.权利要求1或2所述的香瓜茄酸性多糖在制备食品、化妆品和医药用抗氧化剂的用途。
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