| Schisandra chinensis (Turcz.) Baill(Magnoliaceae, schisandra)is an edible Chinese herb mainlydistributed in Liaoning, Jilin, Heilongjiang provinces of China. Early researches about Schisandrachinensis were focused on lipid-soluble composition such as lignan, triterpenoid, and essential oil, etc. Andthe material is discarded after lipid-soluble components extraction. In recent years, researches found thatwater-soluble ingredients contain a lot of polysaccharides, which causes attentions of researchers. Theresidue of Schisandra chinensis from lipid-soluble components extraction is used as raw materials in thisarticle, the extraction, purification, physical and chemical properties, structure, antioxidant andimmunodulatory activities of polysaccharide were studied. And hope to lay the foundation for theSchisandra chinensis industrialized development. And the main results are as follows.1. Professional softwares Design Expert7.0and Minitab15were used to optimize the polysaccharidesextraction by hot water and extraction associated with ultrasonic and enzyme pretreatment by responsesurface method (RSM). The optimum conditions of extraction by hot water are ratio of material to water for1:40, temperature at90.3℃, time for5hours, and the yield of crude polysaccharide is (10.99±0.22)%. Theoptimum ultrasonic pretreatment conditions are ratio of material to water for1:40, power of ultrasonic at300W, temperature at30℃, and time for1hour. Afterultrasonic pretreatment, traditional extraction is doneand the yield of crude polysaccharide is (16.73±0.25)%. The optimum enzyme pretreatment conditions areratio of material to water for1:40, pectinase dosage of1.19%, temperature at30℃, time for4hours, andpH value at4.21. After the pretreatment, traditional extraction is done and the yield of crude polysaccharideis (20.82±0.25)%.2. Crude polysaccharides were purified by organic solvents, enzymatic treatments and columnchromatography and results show as follows: method of acetocaustin-n-butyl alcohol combining enzymepretreatment is the best way to get rid of protein. After enzymic hydrolysis pretreatment of adding1%protease to the polysaccharide solution at50℃with pH value of5for140min, acetocaustin-n-butyl alcoholis added with the same volume of polysaccharide solution. Treatment of acetocaustin-n-butyl alcohol isrepeated for four times to get rid of protein and the retention rate of polysaccharides is68.3%.Polysaccharides were decolored and separated by polyamide column chromatography after deproteinization,and three fractions SCP-A, SCP-B and SCP-C were got with accounts for67.3%,2.42%and1.35%of totalpolysaccharides respectively. SCP-0seprated from SCP-A through Sephacryl S-300HR gel column was identified as a homogeneous component by ultraviolet spectrum, Sephacryl S-300HR gel columnchromatography, cellulose acetate membrane electrophoresis method and gel chromatography, and thepurity of SCP-0is94.6%. SCP-0was further purified by HPLC then SCP-01was obtained. Gelchromatogram of SCP-01showed a single sharp symmetrical peak form indicating a high purity withmolecular weight distribution width of1.22.3. Results of physical and chemical properties researches are as follows.(1) The solubility ofpolysaccharide is affected by temperature intensively and dissolving slowly at low temperature, theappropriate temperature is60℃. The polysaccharidehas good solubility at different pH value and goodsolubility in salt solution with concentration commonly in food processing. The solubility is affected by saltsolution and pH value less and the polysaccharides dissolve faster in alkaline conditions.(2) The viscosityof polysaccharide solution is affected by concentration and temperature more. With the increasingconcentration, the viscosity increased significantly, as the temperature increases, viscosity falls. NaClsolution, pH value and heat treatment affect the viscosity of polysaccharide solution less and thepolysaccharide has good stability in processing.(3) The total sugar content of SCP-0is88.7%, the uronicacid content is12.7%, and the average molecular weight is394kDa.(4) Ultrasonic treatment will causepolysaccharide degradation and average molecular weight down.4. Chemical composition and structure of polysaccharide SCP-01was studied by high performanceliquid chromatography, gas chromatography, infrared spectrum, atomic force microscope and such as thecombination of the classical chemical analysis method. And the results show as follows.(1)Monosaccharide composition of SCP-01contains Man, Rha, Glc and Ara with molar ratio of3.6:2.25:9.08:1.(2) SCP-01has α-D-pyranoside and α-D-Man, no or few β configuration.(3)Monosaccharide residues linked by1→,1→6bond types accounts for31.68%of total residues, residueslinked by1→2,1→2,6,1→4,1→4,6bond types accounts for28.64%and residues linked by1→3,1→2,3,1→2,4,1→3,4,1→3,6,1→2,3,4bond types accounts for39.68%. There is a nonreducing end group1→or a1→6glycosidic bond per3.2residues.(4) Main chain of SCP-01is formed of Man, Rha, Glc and Aralinked by1→,1→6,1→4and1→4,6bond types, and a few Man and some Glc linked by1→3,1→2,3,1→2,4,1→3,4,1→3,6,1→2,3,4bond types exist in the main chain at intervals. SCP-01has manybranches which are formed of Glc, Man and few GlcA linked by1→3,1→2,3,1→2,4,1→3,4,1→3,6,1→2,3,4bond types.(5) The height of polysaccharide molecular chain is about0.35~0.7nm and spiral and coil structures are found in molecules. Polysaccharide aggregates in water are observed as clusters, spirals,gel network and particles.5. Antioxidant tests in vitro show that SCP extracted by hot water and associated with ultrasonic havestrong activities of·OH and DPPH· scavenging, the effect of scavenging DPPH· is more remarkable thanthat of·OH and scavenging rate increases with the concentration of polysaccharide. However,·O2-scavenging activity of SCP-01is hardly observed. Polysaccharides extracted associated with ultrasonichave stronger scavenging activity than that extracted by hot water.6. The results of cell culture in vitro are as follows.(1) SCP extracted by hot water and associated withultrasonic can promote mice splenic lymphocytes proliferation and the best is SCP-0, then the SCP-3.(2)Samples SCP-0and SCP-3can promote PBMC proliferation in some concentration range and also canenhance the proliferation induced by PHA and LPS. However, the promotion effects of other SCP sampleson PBMC are hardly observed.(3) SCP-0has effect on stimulation of cell factors secretion and canstimulate the secretion of IFN-γ, IL-4and TNF-α, promote the secretion of IFN-γ, IL-4and TNF-α inducedby PHA and has no effect on the secretion induced by LPS.(4) SCP-0can stimulate PBMC producing IgMand IgG alone or in combination with LPS and the stimulation of combination is more effective.7. Animal experiments indicate that SCP-0has good regulating action on immunity of the low immunemice confronting the inhibitory effect of cytoxan on nonspecific and specific immune function of mice.(1)SCP-0can enhance the carbon clearance index, the serum hemolysin level, the antibody-forming cell level,the splenic lymphocyte proliferation induced by ConA and LPS and the NK cell activity.(2) SCP-0canincrease the secretion of IFN-γ, IL-4and TNF-α in serum.(3) SCP-0can increase the activities of SOD,CAT and GSH-PX in hepatic tissue of mice meanwhile reduces the MDA production.
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