Preparation, Characterization, And In Vitro Activity Of Rapeseed Polysaccharides

Rapeseed meal is the byproduct of rapeseed after oil extraction.The study and exploitation of rapeseed polysaccharides is beneficial to the comprehensive utilization of rapeseed meal.There have been several research results on the structure of rapeseed polysaccharides,but their bioactivity has not been reported up to now.In this paper,the components stuctural characterization of rapeseed polysaccharides isolated and purified from Huaza No.4 rapeseed meal was identified by Fourier transform infrared spectrometry(FT-IR),gas chromatography(GC),Gas chromatography-mass spectrometry(GC-MS),high performance liquid chromatography(HPLC),and nuclear magnetic resonance(NMR).The chemiluminescence(CL) method was used to investigate the free radical scavenging activity of rapeseed polysaccharides.The immunomodulatory activity was investigated by MTT colorimetric assay and semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR).The major content and results are as follows:1 Preparation and characterization of rapeseed polysaccharides.There are some reports on pareparation and characterization of rapeseed polysaccharide abroad.The emphasis of this research is on optimization of extraction technology,environmentally friendly purification process,and characterization methods of polysaccharides from Huaza No.4 rapeseed Meal.1.1 The optimized extraction technology,of water-soluble polysaccharides derived from the pretreated rapeseed meal was performed by the central combinational design(CCD). the extraction technology of water-soluble polysaccharides composed of 94℃,2.9 h,and 28:1(mL/g) ratio of water to rapeseed meal.After extraction of three times,The insoluble residue on the filter paper was then resuspended and further treated(1 g/20 mL) with 5%NaOH and heated at 60℃for 2 h.Alkali-insoluble material was removed by filtration,and the filtrate was acidified to pH=5 with 4 mol/L HCl to yield an alkali-soluble polysaccharide fraction.Two major polysaccharide fractions,WPS-1 and APS-2,were isolated from water-soluble and alkali-soluble extracts of Huaza No.4 rapeseed meal with a stepwise procedure of Special No.1 macroporous adsorption resin column chromatography,ethanol precipitation and DE-52 cellulose column chromatography.WPS-1 and APS-2 were both relative homogeneous fractions,which were identified by Sephadex G-200 chromatography and gel permeation chromatography (GPC).1.2 Both WPS-1 and APS-2 were non-starch polysaccharides with white and flocculent appearance.UV,FT-IR,GC and HPLC were used to analyze the components of WPS-1 and APS-2.It was found that the weight-average molecular weight(Mw) of WPS-1 and APS-2 were 7.20×10⁵ and 1.61×10⁵,respectively.The number-average molecular weight(Mn) of WPS-1 and APS-2 were 2.45×10⁵ and 6.79×10⁵,respectively. It was found that WPS-1 and APS-2 were 2 kinds of polysaccharides containing proteins and uronic acids.The contents of neutral sugar,uronic acid,and protein of them were 83.2%and 66.8%;6.1%and 9.3%;3.8%and 8.7%,respectively.The associated protein portions in both polysaccharide fractions consisted of 13 different amino acids,including aspartic acid,glutamic acid,serine,glycine,arginine,threonine,alanine,tyrosine,valine, cystine,isoleucine,leucine and phenylalanine.To their monosaccharide composition, WPS-1 consisted primarily of Ara(66.6 mol%) and Gal(18.3 mol%) accompanied by some Glc(12.6 mol%) and GlcA(2.5 mol%);APS-2 consisted primarily of Ara(52.9 mol%) and Gal(21.7 mol%) accompanied by some Glc(10.6 mol%),Man(5.0 mol%), GalA(4.8 mol%),GlcA(3.6 mol%),and Rha(1.4 mol%).The structural characterization of WPS-1 was further investigated by methylation linkage analysis and 1-dimensional NMR(including ¹³C NMR and ¹H NMR).On the basis of these study results and considering other relevant studies,it was concluded that WPS-1 consisted manily of arabinan fragment,which was mainly(1→5) and(1→2) linked.2 In vitro activity of rapeseed polysaccharides.The bioactivity of rapeseed polysaccharides has been investigated preliminary by our lab.However,research on the bioactivity of rapeseed polysaccharides has received little attention up to now.The emphasis of this research is on the in vitro activity of polysaccharides at the cellular and molecular levels2.1 The chemiluminescence(CL) method was used to investigate the free radical scavenging activity of rapeseed polysaccharides.The scavenging ability of WPS-1 and APS-2 for O₂^·-,HO^·,and H₂O₂ were determined by a pyrogallol-luminol system, CuSO₄-phenanthroline-ascorbate-H₂O₂ system,and luminol-H₂O₂ system,respectively, on a BPCL Ultra-weak luminescence analyzer.The results indicated both WPS-1 and APS-2 have prominent ability of scavenging free radical in a concentration-dependent manner.The maximum inhibition rates of WPS-1 and APS-2 were 90.4%and 89.6%at the 2000μg/mL concentration,respectively.Their half-maximal inhibitory concentration (IC₅₀) values were 400±44μg/mL for WPS-1 and 450±64μg/mL for APS-2.WPS-1 was slightly more effective than APS-2 at scavenging O₂^·-.Both WPS-1 and APS-2 could scavenge HO^·and the maximum inhibition of CL by WPS-1 and APS-2 were 93.8%and 86.7%,respectively.The IC₅₀ value of WPS-1 was 240±18μg/mL,while that of APS-2 was 293±24μg/mL.WPS-1 was more effective than APS-2 at scavenging HO^·.The maximum inhibition by WPS-1 was 84.2%and by APS-2 was 85.2%.The IC₅₀ value of WPS-1 was 10.0±0.8μg/mL,while that of APS-2 was 6.1±0.5μg/mL.It was concluded that APS-2 was more effective than WPS-1 at scavenging H₂O₂.Compared with APS-2,WPS-1 was more effective at scavenging superoxide radical(O₂^·-) and hydroxyl radical(HO^·),but less effective at scavenging hydrogen peroxide(H₂O₂).In decreasing order,free radical scavenging activity of WPS-1 and APS-2 towards reactive oxygen species(ROS) was:H2O2＞HO^·＞O₂^·-.2.2 A nylon wool column was used to separate of spleen T and B lymphocytes. Through the MTT assay,rapeseed polysaccharides were found to significantly enhance proliferation of totol spleen lymphocytes and T lymphocytes.The optimal concentration of both WPS-1 and APS-2 to promote proliferation were 40μg/mL,but WPS-1 was more effective than APS-2 at promoting cell proliferation.Semi-quantitative RT-PCR was performed to determine changes in cytokine mRNA expression.The influence of WPS-1 to cytokine interleukin-2(IL-2),IL-4,IL-6,and interferonγ(IFN-γ) mRNA expression in T lymphocytes were investigated.First,the relation between time of T lymphocytes cells incubated with WPS-1 and the cytokine mRNA expression was studied.The best time of WPS-1 to promote IL-2,IL-4,IL-6 mRNA of expression were all 24 h.The best time to that of IFN-γwas 12 h.The best time of the cytokines mRNA expression were adopted,to further investigate the effect of the concentration of WPS-1 to cytokine mRNA expression.The results showed the best concentration of WPS-1 to promote IL-2,IL-6, and IFN-γmRNA expression were all 40μg/mL,to that of IL-4 was 20μg/mL.The results indicates that rapeseed polysaccharide can enhance the immune regulation of spleen T lymphocyte.2.3 Rapeseed polysaccharides promote proliferation macrophage cells,in a dose-dependent manner.The optimal concentration of both WPS-1 and APS-2 to promote proliferation were 40μg/mL,but WPS-1 was more effective than APS-2 at promoting cell proliferation,which similar to lymphocytes proliferation.Semi-quantitative RT-PCR was performed to determine changes in cytokine mRNA expression.The influence of WPS-1 to IL-6,tumor necrosis factor-α(TNF-α),and inducible nitric oxide synthase(iNOS) mRNA expression were all 40μg/mL.The results indicates that rapeseed polysaccharide can enhance the immune regulation of peritoneal macrophages.