摘要
软枣猕猴桃Actinidia arguta (Sieb.et Zucc.) Planch. ex Miq.又名软枣子、猕猴梨、藤梨,是猕猴桃科、猕猴桃属多年生落叶藤本植物。软枣猕猴桃以其耐寒性而著称,果实很小,如枣般大,表皮光滑无毛,可直接食用,甜酸比适中,风味极佳。它富含多种营养成分,如高Vc含量(200-400mg/100g)、高活性的蛋白酶、种类齐全的微量元素、丰富的不饱和脂肪酸含量等。其果实、种子及根茎均可入药,是理想的绿色食品和食疗食品。
目前对软枣猕猴桃的生物活性研究主要集中在根和茎上,而对于果实生物活性的研究仅限于果汁或果实提取物,无法确定其中某一具体物质的生物活性。多糖是软枣猕猴桃中的主要成分之一,如能确定其相关生物活性,并通过科学的生产方法将它提取出来,制成软枣猕猴桃多糖的相关产品,或作为食品添加剂,广泛添加于各类食品中,从而提高食品的营养价值,那不仅对于软枣猕猴桃产业的发展有极大的带动作用,而且还可以产生巨大的经济效益和社会效益。
本研究以软枣猕猴桃鲜果为原料,对软枣猕猴桃多糖进行了提取、纯化,对其结构进行了初步鉴定,并对其体外抗氧化活性和体内免疫调节作用进行了详细的研究,为今后软枣猕猴桃多糖的开发利用奠定了理论基础。研究结果如下：
(1)在水提工艺之前首先确定了软枣猕猴桃的除杂工艺：乙醇浓度80%,料液比1：2g：mL,温度20℃,时间60min,通过该工艺,可以在几乎不损失多糖的前提下最大程度的除去色素、还原糖等杂质；然后分别采用高温水浴法、低温摇床法、微波法提取软枣猕猴桃多糖,并通过正交试验优化提取工艺,得到三种提取方法的最佳工艺条件。高温水提工艺：料水比1：9g：mL,温度100℃,时间120min,多糖提取率为2.19%；低温水提工艺：料水比1：9g：mL,温度40℃,时间120min,多糖提取率为1.54%；微波提取工艺：料水比1：12g：mL,微波功率500W,微波处理时间10min,多糖提取率为1.41%。
(2)采用高温水提工艺提取得到的软枣猕猴桃多糖AAP-1的得率为2.9%,颜色为白色,不含酚羟基和还原糖,含有淀粉,其多糖含量、糖醛酸含量和蛋白质含量分别为71.3%、15.1%、2.51%；红外光谱分析可知AAP-1在400-4000cm-1范围具有糖类的特征吸收峰,但它不含有果胶多糖中的GalA在指纹区的特征吸收峰；单糖组成测定结果表明：它由甘露糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖和阿拉伯糖7种单糖组成,各单糖的摩尔百分含量依次为：1.85%、0.11%、0.37%、0.24%、94.72%、2.45%、0.26%,其中葡萄糖的摩尔百分含量很高,推测是由于含有淀粉的缘故。
(3)采用低温水提工艺提取得到的软枣猕猴桃多糖AAP-2的得率为1.8%,颜色为白色,不含酚羟基、还原糖和淀粉,其多糖含量、糖醛酸含量和蛋白质含量分别为32.7%、46.2%、3.14%；红外光谱分析可知AAP-2在400～4000cm-1范围具有糖类的特征吸收峰并且证明它为果胶多糖；单糖组成测定结果表明它也由甘露糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖和阿拉伯糖7种单糖组成,各单糖的摩尔百分含量依次为：14.35%、4.16%、1.84%、6.64%、9.89%、24.75%、38.37%,其中甘露糖、半乳糖和阿拉伯糖的含量较高。
(4)采用微波辅助工艺提取得到的软枣猕猴桃多糖AAP-3的得率为2.6%,颜色为白色,不含酚羟基、还原糖和淀粉,其多糖含量、糖醛酸含量和蛋白质含量分别为30.8%、59.3%、1.06%；红外光谱分析可知AAP-3在400-4000cm-1范围具有糖类的特征吸收峰,并且证明它也为果胶多糖；单糖组成测定结果表明它也由甘露糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖和阿拉伯糖7种单糖组成,各单糖的摩尔百分含量依次为：13.16%、5.05%、1.53%、16.05%、21.97%、18.89%、23.35%,其中甘露糖、半乳糖醛酸、半乳糖和阿拉伯糖的含量较高。
(5)体外抗氧化实验的结果表明：AAP-1具有一定的还原力,它清除DPPH自由基和羟基自由基的能力相对较强,清除超氧阴离子自由基的能力很弱；AAP-2的总还原力和清除DPPH自由基的能力略高于AAP-1,清除羟基自由基的能力显著高于AAP-1,清除超氧阴离子自由基的能力与AAP-1相近；AAP-3的总还原力、清除DPPH自由基和羟基自由基的能力显著高于AAP-2,但清除超氧阴离子自由基的能力依然很弱。
(6)软枣猕猴桃多糖AAP-3经过DEAE-52纤维素柱层析,分别用蒸馏水、0.1mol/LNaCl、0.2mol/L NaCl、0.3mol/LNaCl洗脱得到四个组分,依次命名为AAP-3a、AAP-3b、AAP-3c、AAP-3d,四个组分的得率分别为13.8%、47.0%、24.4%、8.4%,总回收率为93.6%；这四个组分再经过SephadexG-100凝胶柱层析后,都是只得到一个洗脱峰且峰形比较对称。经检测,每个组分中均含有极微量的蛋白；将SephadexG-100凝胶层析柱纯化后的四个多糖组分再经过SephadexG-200凝胶柱层析,依然只得到一个洗脱峰且峰形对称,说明经离子交换柱分离得到的每一部分,在凝胶柱上不再分离。经检测,每个组分中均不含有蛋白质。
(7)软枣猕猴桃多糖四个纯化组分的抗氧化活性测定结果表明：软枣猕猴桃多糖纯品也具有清除DPPH自由基和羟基自由基的能力,但对超氧阴离子自由基的清除能力依然很弱；盐洗组分的抗氧化活性明显优于水洗组分；三个盐洗组分中AAP-3b的抗氧化活性最强,AAP-3d次之,AAP-3c最弱；AAP-3b为软枣猕猴桃多糖中主要的抗氧化活性组分。
(8)紫外光谱扫描结果表明：AAP-3a、AAP-3b、AAP-3c、AAP-3d均不含蛋白、多肽及核酸,但四个组分的峰型表现出极大的差异,最大吸收波长也略有不同,表明四个组分在构成上有显著差异。
(9)采用PMP柱前衍生高效液相色谱法测定AAP-3a、AAP-3b、AAP-3c、AAP-3d的单糖组成,结果表明四个组分在单糖组成上差异显著。AAP-3a和AAP-3b由甘露糖、鼠李糖、半乳糖醛酸、葡萄糖、半乳糖和阿拉伯糖6种单糖组成；AAP-3c和AAP-3d由甘露糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖和阿拉伯糖7种单糖组成。AAP-3a以葡萄糖为主,AAP-3b以甘露糖和半乳糖醛酸为主,AAP-3c以半乳糖醛酸、半乳糖和阿拉伯糖为主,AAP-3d以甘露糖、半乳糖醛酸、半乳糖和阿拉伯糖为主。
(10)红外光谱扫描结果表明：AAP-3a为中性多糖,它以α-糖苷键为主,并含有一定量的β-糖苷键；AAP-3b为果胶多糖,它以p-糖苷键为主,并含有一定量的α-糖苷键；AAP-3c也为果胶多糖,其中p-糖苷键的数量多于α-糖苷键。
(11)采用高效凝胶渗透色谱测定AAP-3b和AAP-3c的分子量,结果表明AAP-3b和AAP-3c仍然不是单一组分,AAP-3b中主要组分的重均分子量(Mw)为7,298Da,AAP-3c中主要组分的重均分子量(Mw)为14,146Da。
(12)通过体内实验研究了AAP-3b的免疫活性,结果表明：软枣猕猴桃多糖AAP-3b高剂量组可以促进大鼠的生长,并显著提高大鼠的脾脏指数；AAP-3b中剂量组和高剂量组可以显著提高大鼠的胸腺指数、吞噬指数和ConA诱导的大鼠脾淋巴细胞转化指数。确证了软枣猕猴桃多糖可以通过促进免疫器官生长、增强细胞免疫功能、增强单核巨噬细胞吞噬能力等方式来提高机体的免疫力,具有一定的免疫活性。
Actinidia arguta Sieb.et Zucc belongs to Actinidiaceae, Actinidia is perennial and deciduous liana. Actinidia arguta is famous for its cold resistance, the fruit is very small, like jujube, skin smooth and glabrous, direct edible. Its flavor is excellent, sweet and sour is moderate. It is rich in many nutrients, such as high content of Vc(200-400mg/100g), high activity of protease, variety of trace elements and unsaturated fatty acid. It is an ideal green food and health food, its root, stem and fruit are all useful for us because of their medical value and healthy function.
Current biological activity research of Actinidia arguta focuses on the root and stem, the biological activity research of fruit is limited to fruit juice or fruit extract, unable to determine the biological activity of a specific substance. Polysaccharide is one of the main components of Actinidia arguta, if its relevant biological activity can be determined, and extracted by scientific method, then make kinds of polysaccharide products, or as a food additive apply to variety of foods with improving the nutritional value of food, it will not only promote the development of Actinidia arguta industry, but also produce huge economic benefit and social benefit.
This paper, taking Actinidia arguta fresh fruit as raw material, extracted, purified and indentified the polysaccharides and carried out a detailed study on its antioxidant activity in vitro and immunoregulation role in vivo. This study has laid a theoretical basis for future in-depth research and utilization of Actinidia arguta polysaccharides. The results are as follows:
(1) Decontaminate process of Actinidia arguta was determined firstly:ethanol concentration80%, ratio of solid to liquid1:2g:mL, temperature20℃, time60min, through this process, without loss much of polysaccharide, pigment, reducing sugar and other impurities were removed maximumly; then Actinidia arguta polysaccharide were extracted by hot water method, warm water method and microwave method, and the optimum extraction process were confirmed by orthogonal design. The optimum process of hot water method:the ratio of material to water1:9g:mL, temperature100℃, time120min, extraction rate of polysaccharide is2.19%; the optimum technology of warm water method:the ratio of material to water1:9g:mL, temperature40℃, time120min, extraction rate of polysaccharide is1.54%; microwave extraction process:the ratio of material to water1:12g:mL, microwave power500W, time10min, extraction rate of polysaccharide is1.41%.
(2) The yield of AAP-1extracted by hot water is2.9%, white, no containing phenolic hydroxyl and reducing sugar, containing starch, and the content of polysaccharide, uronic acid and protein are71.3%,15.1%and2.51%respectively; infrared spectroscopic analysis showed that AAP-1has the characteristic absorption peaks of polysaccharide in400～4000cm-1, but it dose not contain the characteristic absorption peaks of GalA belongs to pectic polysaccharide in the fingerprint area. Monosaccharide composition results showed that it is composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, the molar percentage of simple sugars are1.85%,0.11%,0.37%,0.24%,94.72%,2.45%,0.26%respectively, in which the molar percentage of glucose is highest, presumably because of the existence of starch.
(3) The yield of AAP-2extracted by warm water is1.8%, white, no containing phenolic hydroxyl, reducing sugar and starch, the content of polysaccharide, uronic acid and protein are32.7%,46.2%and3.14%respectively; infrared spectroscopic analysis showed AAP-2has the characteristic absorption peaks of polysaccharide in400-4000cm-1, and it is pectic polysaccharide. Monosaccharide composition results showed that it is composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, the molar percentage of simple sugars are14.35%,4.16%,1.84%,6.64%,9.89%,24.75%,38.37%respectively, in which the molar percentage of mannose, galactose and arabinose are higher.
(4) The yield of AAP-3extracted by microwave method is2.6%, white, no containing phenolic hydroxyl, reducing sugar and starch, the content of polysaccharide, uronic acid and protein are30.8%,59.3%,1.06%respectively; infrared spectroscopic analysis showed AAP-3has the characteristic absorption peaks of polysaccharide in400-4000cm-1, and it is also pectic polysaccharide. Monosaccharide composition results showed that it is composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, the molar percentage of simple sugars are13.16%,5.05%,1.53%,16.05%,21.97%,18.89%,23.35%respectively, in which the molar percentage of mannose, glucuronic acid, galactose and arabinose are higher.
(5) Antioxidant experiment results showed that AAP-1has a certain reducing power and its ability of scavenging DPPH·,·yOH are relatively strong, the ability of scavenging O2-·is weak; the reducing power and the ability of scavenging DPPH·,·OH of AAP-2are stronger than that of AAP-1, the ability of scavenging O2-·is similar to AAP-1; the reducing power and the ability of scavenging DPP·,·OH of AAP-3are significantly higher than that of AAP-2, but the ability of scavenging O2-·is still weak.
(6) AAP-3was purified by DEAE-52cellulose column chromatography, four components were eluted by distilled water,0.1mol/L NaCl,0.2mol/L NaCl,0.3mol/L NaCl, named AAP-3a, AAP-3b, AAP-3c, AAP-3d successively, the yield of four components are13.8%,47.0%,24.4%,8.4%, respectively, the total recovery is93.6%. The four components were separated by SephadexG-100column chromatography, only one elution peak is found and the shape is symmetrical, each component contains trace amounts of protein; then four purified components were separated by SephadexG-200column chromatography, still get one elution peak and the shape is symmetrical, the components separated by ion exchange column no longer separate on the Sephadex column, and each component does not contain protein.
(7) The results of antioxidant activity of four purified polysaccharide components showed that purified components have the ability of scavenging DPPH·and·OH, but the ability of scavenging02-·are still weak; the antioxidant activity of salt eluting components are superior to the washing component; in the three salt eluting components, AAP-3b has the strongest antioxidant activity, AAP-3c has the weakest antioxidant activity; AAP-3b is the main antioxidant component.
(8) Ultraviolet spectrum scanning results showed that AAP-3a, AAP-3b, AAP-3c, AAP-3d do not contain protein, peptides and nucleic acids, the shape of the four spectrograms are different and the maximum absorption wavelength are also slightly different, the results indicate that the structure of the four components are different.
(9) The monosaccharide composition of AAP-3a, AAP-3b, AAP-3c, AAP-3d were determined by PMP pre-column derivative HPLC, the results showed that the monosaccharide composition of four components are significantly different. AAP-3a and AAP-3b are composed of mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose; AAP-3c and AAP-3d are composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose. Glucose is the predominant sugar in AAP-3a; mannose and galacturonic acid are the predominant sugars in AAP-3b; galacturonic acid, galactose and arabinose are the predominant sugars in AAP-3c; mannose, galacturonic acid, galactose and arabinose are the predominant sugars in AAP-3d.
(10) Infrared spectrum scanning results showed that AAP-3a is neutral polysaccharide, in which the content of a-glycosidic bond is predominant; AAP-3b is pectic polysaccharide, in which the content of β-glycosidic bond is predominant; AAP-3c also is pectic polysaccharide, in which the content of β-glycosidic bond is more than a-glycosidic bond.
(11) The molecular weight of AAP-3b, AAP-3c were determined by gel permeation chromatography, the results showed that AAP-3b and AAP-3c are still not a single component, the molecular weight (Mw) of main component in AAP-3b is7298Da, the molecular weight (Mw) of main component in AAP-3c is14146Da.
(12) The immunomodulation of AAP-3b was studied in vivo, the results showed that the high dose group of AAP-3b can promote the growth of rats, and significantly improve the spleen index of rats; the medium dose group and high dose group can significantly increase the thymus index, phagocytic index and the spleen lymphocyte transformation index induced by ConA. It was confirmed Actinidia arguta polysaccharide can improve the body's immunity by promoting the growth of immune organs, enhancing the cellular immune function and the phagocytosis of mononuclear macrophage.
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