| 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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