Separation Of Polysaccharide From Blue Honeysuckle And Exploration Of Its Chemical Stucture And Immunomodulate Activity

ObjectivesBlue honeysuckle,which are berry fruits obtained from Lonicera caerulea L.belonging to genus Lonicera,family Caprifoliaceae,are a sort of local food resources.In China,Lonicera caerulea L.plants are cultivated in cold-temperate and mid-temperate regions such as northeast,north and northwest area.Till now,the immunomodulatory activity of Blue Honeysuckle polysaccharides has not been reported yet.Therefore,this study intends to extract and purify the polysaccharide from Blue Honeysuckle,analyze the chemical structure of the purified polysaccharide component THP-2,and explore whether THP-2 has a regulatory effect on the functions of DC2.4 and Raw264.7.Methods1.The raw materials were decolorized by the ethanol immersion and the crude polysaccharide was extracted by the "water extraction and alcohol precipitation"method and purified using DEAE-Sepharose FF and Sephadex-G100 gel chromatography,then the sugar content and protein content of crude polysaccharides and purified components THP-2 were detected.2.The molecular weight,chemical bond and functional group,monosaccharide composition and glycosidic bond type of THP-2 were detected using HPGPC,FTIR,ion chromatography,periodate oxidation and smith degradation and NMR spectroscopy,respectively.3.The THP-2 was used to intervene DC2.4 to test the effects in cell proliferation ability,costimulatory molecules and MHC-Ⅱ molecules expression,cytokine expression,and ROS and NO contents after intervention.Moreover,the content of protein p-p65、pIκB、p-Akt in cells after intervention were also observed.4.The THP-2 was used to intervene Raw264.7 to test the effects of the intervention on cell proliferation ability,costimulatory molecules expression level,cytokine expression level,ROS content and NO content.The effects on protein p-p65 and p-IκB are also detected.Results1.The "water extraction and alcohol precipitation" method exhibit a high performance to extract crude polysaccharides from Blue Honeysuckle.After DEAE-Sepharose-FF gel chromatography purification,four components are separated,named THP-1,THP2,THP-3,THP-4.there was still only one fraction after THP-2 purified by Sephadex G-100 gel chromatography.THP-2 has higher saccharides contents and lower protein contents than crude polysaccharides and the peak of proteins characteristic absorption optical wavelength in THP-2 is also markly lower than crude polysaccharides.2.THP-2 is a polysaccharide with an average molecular weight of 123.09 kDa and is composd of galactose,arabinose,glucose,mannose,xylose,glucuronic acid and galacturonic acid with molar ratio of 27.84:52.28:8.47:6.05:2.75:0.90:0.88:0.83,respectively.The infrared spectrum reveals the existence of carboxyl and mannose.The glycosidic bonds in THP-2 include(1→5),(1→3),(1→4),(1→6),(1→3,4),(1→6,4)bond.Gongo red assay indicates that THP-2 may has a non-triple helix structure able to combine with Gongo red.3.THP-2 increased the expression of costimulatory molecules,MHC-II molecules,IL1β,IL-6,TNF-α and raised the contents of ROS in DC2.4 cells.Moreover,THP-2 promoted NO secretion by enhancing iNOS expression.The upregulation of P-p65、P-IκB、P-Akt protein content as well as the mRNA level of iNOS were also found.4.THP-2 could stimulate the expression of CD80,CD86,CD40 and IL-1β,IL-6,TNFa and raise the ROS contents in Raw264.7 cells.Besides,increased iNOS expression induced by THP-2 promoted NO secretion.An elevated p-IκB content and the decrease of p65 total protein content were also observed.Conclusion1.Methods using for blue honeysuckle polysaccharide separation and purification is workable.Meanwhile,the monosaccharide composition and glycosidic bond composition are rich,implying that THP-2 may possess a complex structure.2.THP-2 can promote the maturation and differentiation of DC2.4 in more modest way comparing to LPS.The promotion ability may attribute to the activation of NF-κB and PI3K/Akt signal pathway and may associated with the increased ROS content.3.THP-2 can also enhanced the M1 type differentiation of Raw264.7 cells in gentler way comparing to LPS.This differentiation ablilty may work through the activation of NF-κB signal pathway and may related to the raise of ROS content.