Isolation,Purification,Structure Characterization And Anti-Inflammatory Activity Of Polysaccharides From Lonicera Japonica Thunb

Honeysuckle belongs to the genus Lonicera in the family Lonicerae and is widely cultivated.It belongs to the medicinal food group of plants and has a variety of biological activities such as anti-inflammatory,immune,antioxidant and anti-tumour.Previous studies on Lonicera japonica have mainly focused on volatile oils,organic acids,flavonoids and other small molecules,with less research on polysaccharides and their structural properties and biological activities.Therefore,this paper took Lonicera japonica as the research object to extract polysaccharide and optimize the extraction process.Through separation and purification,the physicochemical properties,structural characteristics and anti-inflammatory activities of purified polysaccharide components were studied.The main research contents are as follows:（1）Single factor combined with response surface methodology to determine the hot water extraction process of polysaccharides from Lonicera japonica.The specific process parameters were material-to-liquid ratio of 1∶30（g/m L）,extraction time of 120 min,extraction temperature of 70℃,and polysaccharide yield of 6.45%±0.15%.（2）The crude polysaccharides of Lonicera japonica were purified by column chromatography,and the structure of the polysaccharide fractions of Lonicera japonica were characterized by spectroscopic analysis,chromatographic analysis,spectral analysis and electron microscopic observation.The crude polysaccharide from Lonicera japonica（LJP）was purified by DEAE-52 cellulose column chromatography to obtain three fractions:LJP-1,LJP-2 and LJP-3.The results of HPAEC and HPGPC-MALLS/RID showed that LJP-1mainly consisted of arabinose,glucose and galactose in the ratio of 43.57∶26.97∶21.61,with an average molecular weight of 153 k Da;LJP-2 had the highest proportion of glucose,followed by galacturonic acid and galactose,with an average molecular weight of 63.54 k Da;LJP-3 contained mainly glucose,ribose and galactose,with an average molecular weight of27.52 k Da.The presence of glycosidic bonds ofβ-configuration andα-configuration in LJP-1;D-glucopyranose in LJP-2,and D-glucopyranose in LJP-3.The main chain of LJP-2 was→5)-α-L-Araf（1→and→4）-α-L-Galp A（1→and→2）-α-L-Rhap(1→,terminating in residue T-β-D-Glcp.Electron microscopic observation of polysaccharides from Lonicera japonica mostly in the form of flakes and blocks,polysaccharide particles in LJP-1 are chain-like and interact in a helical structure,LJP-2 and LJP-3 show a larger amorphous form.（3）Anti-inflammatory activity of polysaccharide from Lonicera japonica was evaluated by cellular and zebrafish models.The anti-inflammatory results in an in vitro macrophage model showed that polysaccharides from Lonicera japonica may exert anti-inflammatory effects by down-regulating the expression levels of key inflammatory factors and inflammatory proteins in the NF-κB signaling pathway,and that the polysaccharides significantly inhibited the secretion of NO,TNF-α,IL-1βand IL-6 with a dose-dependent effect.The maximum inhibition rates of the three polysaccharide fractions on NO were87.45%,89.17%and 91.45%,respectively;the inhibition of TNF-αwas the most obvious with 55.18%～60.28%,followed by IL-1βand IL-6 with 42.50%～43.87%and38.25%～41.06%,respectively.LJP-2 was most effective in inhibiting the expression of the three inflammatory proteins p-IκBα,COX-2 and i NOS,with maximum inhibition rates of79.24%,74.57%and 83.09%,respectively.The anti-inflammatory results in an in vivo zebrafish model showed that polysaccharide from Lonicera japonica could effectively alleviate the inflammatory response induced by Cu SO₄by blocking the activation of NF-κB/MAPK signaling pathway through down-regulating the expression levels of inflammation related gene expression levels.LJP-2 and LJP-3 were the most effective in reducing the lateral migration of macrophages,with the maximum reduction in migration rate of 95.87%and 86.78%.Differences in polysaccharide fractions and doses result in different rates of inhibition of expression levels of relevant inflammatory factors,with the maximum inhibition rates being:Iκbα（50.51%）,My D88（38.81%）,COX-2（39.31%）,PTGES（22.48%）,NF-кB（48.19%）,IL-6（65.87%）,AP-1（12.89%）,IL-1β（73.22%）,MAPK（8.89%）and p-38（24.14%）.The polysaccharides from Lonicera japonica possess in vivo and in vitro anti-inflammatory activities and can reduce the secretion of inflammatory factors,the overexpression of inflammatory proteins and inflammation related gene expression levels,providing theoretical support for the application of polysaccharides from Lonicera japonica in the development of nutraceuticals and natural drugs.