| Ginseng(Panax ginseng C.A. Meyer) is a Perennial herbs Araliaceae, Which have pharmacological activities such as anti-tumor, remove free radicals in the body, increase the content of intracellular cytokines to improve immunity, Has attracted widespread attention in the research of traditional Chinese medicines in our country. Ginsenosides and ginseng polysaccharide is the main active components in ginseng, in the recent years, for the development of ginsenosides, ginseng polysaccharides were concentrated, the other ginseng component has not been fully utilized. The growth period of ginseng is commonly 4-6 years, so the use of ginseng plants as materials extraction is restricted. Using Agrobacterium rhizogenes transformation of ginseng root, obtained the hair root, it grows fast and low cost comparison, suitable for extraction and separation on a large scale. This experiment is mainly to other ginseng component of ginseng hair root secondary metabolites, ginseng hair root oligosaccharides by extraction and purification, study the activity of ginseng hair root oligosaccharides and structures, finally determine the best active ingredient of ginseng hair root oligosaccharides. For the ginseng root hair oligosaccharides pharmacological effects to provide basic data, application and development status of its processing has important value.The main contents and conclusions in the research were as follows:1.To screen high-yielding clones and expand ginseng hairy root culture as materials,the use of hot water extraction,80℃,10 the hour and 80℃,8小时twice ginseng hairy root adequate extraction.D101 removed ginseng hairy root water extract of saponins by macroporous resin,a flow rate of 10ml/min,column volume of 200ml,and finally by TLC eluent was not detected in the saponin.To ginseng hairy root water extract was added 95%ethanol,ethanol concentration reached 40%,50%,60%,70%,80%,90%,the pellet is discarded by centrifugation,the supernatant was collected 90 percent.The collected supernatant was added Sevage solution,the supernatant was removed by centrifugation and the protein.After the vacuum recovery,namely ginseng hairy root oligosaccharides.2. Send ginseng root oligosaccharides by sephadex G25, G15 gel chromatography, five kinds of oligosaccharides components was isolated, end group analysis method to determine the number average molecular weight were 2852.72, 1579.21, 1179.93, 587.29, 474.91 Da.3. Through the pre-column derivatization process before five oligosaccharide constituents, HPLC analysis. Results show that the five kinds of ginseng hair root oligosaccharides were composed of Man、Rha、Glc、Gal、Ara, the largest levels is Glc. Ginseng hair root oligosaccharides A Man: Rha: Glc: Gal: Ara = 0.89︰1.23︰4.63︰1.14︰0.85; ginseng hair root oligosaccharides B1 Man:Man:Rha:Glc:Gal:Ara=0.63︰1.08︰3.74︰0.6︰0.95; Ginseng hair root oligosaccharides B2 Man:Rha : Glc : Gal : Ara=0.76 ︰ 0.74 ︰ 3.26 ︰ 0.90 ︰ 0.63; Ginseng hair root oligosaccharides C1 Man:Rha:Glc:Gal:Ara=0.57︰0.88︰2.94︰0.71︰0.20; Ginseng hair root oligosaccharides C2 Man:Rha:Glc:Gal:Ara=0.40︰0.95︰2.83︰0.54︰0.08;4. Periodate oxidation method to determine the five oligosaccharide glycosidic bond connecting method and content. Ginseng hair root oligosaccharides A: 1'2or1'2,6、1'4、1'4,6 glycosidic bond of 34.64%,1'or1'6 glycosidic bond of 12.69%,1'3、1'3,6、1'2,3、1'2,4、1'3,4、1'2,3,4 glycosidic bond of 52.67%; Ginseng hair root oligosaccharides B1: 1'2or1'2,6 、 1'4 、 1'4,6 glycosidic bond of 30.92%,1'or1'6 glycosidic bond of 20.78%,1'3、1'3,6、1'2,3、1'2,4、1'3,4、1'2,3,4 glycosidic bond of 48.3% Ginseng hair root oligosaccharides B2: 1'2or1'2,6、1'4、1'4,6 glycosidic bond of 38.74%,1'or1'6 glycosidic bond of 25.64%,1'3、1'3,6、1'2,3、1'2,4、1'3,4、1'2,3,4 glycosidic bond of 35.62% Ginseng hair root oligosaccharides C1: 1'2or1'2,6、1'4、1'4,6 glycosidic bond of 44.58%,1'or1'6 glycosidic bond of 29.45%,1'3、1'3,6、1'2,3、1'2,4、1'3,4、1'2,3,4 glycosidic bond of 25.97% Ginseng hair root oligosaccharides C2: 1'2or1'2,6 、 1'4 、 1'4,6 glycosidic bond of 50.37%,1'or1'6 glycosidic bond of 32.76%,1'3、1'3,6、1'2,3、1'2,4、1'3,4、1'2,3,4 glycosidic bond of 16.87%.5. Based on body surface area method, to calculate fed the mice of the five components of oligosaccharides, each component make up 40 mg/kg/d, 30 mg/kg/d, 20 mg/kg/d, 10 mg/kg/d, 5 mg/kg/d, with positive control group, negative control group together to feeding mice, for the experiment of pharmacological activity. Cytokines in mice serum for ELISA method.Results show that ginseng hair root oligosaccharides B2 on the level of 30 mg/kg/d, IL- 2, Ig G, T- AOC has more significant differences,show that ginseng root hair oligosaccharides with immune activity and antioxidant activity,illustrates the ginseng root hair the size of the oligosaccharide biological activity and molecular weight.6.Quantitative PCR in mouse spleen IL-2, IL-4 gene and liver SOD, GSH-Px, P450 gene, results showed that in addition to IL-4 gene, the gene expression levels of the remaining four were significantly increased, indicating mice fed ginseng hairy root after oligosaccharides stimulate the expression of related genes upregulated, resulting in immune function and antioxidant effects. |
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