| Arctium lappa L.(burdock),one kind of traditional Chinese medicinal and edible homologous plant,is rich in phytochemicals with nutrition and medicinal value.Fengxian county in Xuzhou City,Jiangsu Province of China is one of the main planting places of burdock and has the reputation of"hometown of A.lappa L.".Burdock is an indispensable food on the Japanese table,which is mostly exported to Japan,Korea,and other places.However,the cognition of burdock in China is relatively little.The reason for this phenomenon may be that the understanding of the material composition,nutritional value and physiological activity of burdock is relatively shallow,and there is no correct and comprehensive knowledge.Polysaccharides,as one of thebioactive substances in burdock,have attracted much attention in recent years.In this study,polysaccharides were extracted from burdock root and their biological activities,including antioxidant,and anti-inflammatory,were investigated.The research results and the main conclusions are as follows:(1)In the present study,Arctium lappa L.polysaccharides(ALP)was extracted by hot water,and the extraction conditions were optimized with Box-Behnken response surface design of the effects of liquid-to-solid ratio(10:1-30:1),extraction temperature(60-100℃),and extraction time(1-3 h)on the yield of ALP were analyzed.The data was fitted to polynomial response model and multiple regression analysis,with applied with a high coefficient of determination value(R~2=0.9754).These results indicated that the the extraction parameters significantly affected the polysaccharides yield,and the optimal extraction conditions were obtained:extraction temperature71.12℃,extraction time 2.51 h,and liquid-to-solid ratio 16.20:1.Derringer’s desirability prediction tool obtained the highest extraction yield of polysaccharides(9.73%),which was confirmed through validation experiments.In addition,ALP antioxidant capacity was investigated.The 1,1-diphenyl-2-trinitrophenylhydrazine(DPPH),2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS),and oxygen padical absorbance capacity(ORAC)values of optimized ALP were 232.23±4.19,407.84±1.48,and 2932.54±145.29μmol Trolox/g,respectively.These results indicated that ALP has potential antioxidant capacity.(2)In this study,lipopolysaccharide-stimulated RAW264.7 cells were used to investigate the immunoregulation function of ALP.The toxicity of ALP and lipopolysaccharide on RAW264.7cells was detected by cell counting kit-8,and the concentrations of ALP and lipopolysaccharide were determined.The phagocytic activity of RAW264.7 cells was detected by neutral red assay.The results showed that ALP could enhance the phagocytosis of mouse macrophages.In addition,it was found that ALP possessedboth antioxidant and anti-inflammatory effects by down-regulating nuclear factor-E2-related factor 2 pathway.Griess reagent was used to detect the effect of ALP on nitric oxide secretion of RAW264.7 cells,and the relative level of inducible nitric oxide synthase messenger RNA(m RNA)was detected by quantitative reverse transcription-polymerase chain reaction.Arctium lappa L.polysaccharides could inhibit the increase of nitric oxide level in RAW264.7 induced by lipopolysaccharide,indicating that ALP had potential anti-inflammatory activity.Western bloting,enzyme linked immunosorbent assay,quantitative reverse transcription-polymerase chain reaction were used to explore the possible signaling pathway of ALP’s immunomodulatory activity.It was found that ALP inhibited lipopolysaccharide-induced proinflammatory cytokines(interleukin-8,interleukin-6,interleukin-1β,and tumour necrosis factor-α)m RNA and protein relative levels in RAW264.7 cells by down-regulating the key genes and proteins of Toll-like receptor 4/nuclear factor-kappa B signaling pathway(cluster of differentiation14,Toll-like receptor 4,myeloid differentiation protein-2,tumor necrosis factor-associated factor 6,myeloid differentiation factor 88,and nuclear factor-kappa B).(3)In this study,interleukin-1βwas used to stimulate Caco-2 cells to explore the inhibitory effect of ALP on intestinal inflammation.The cell counting kit-8 was used to detect the toxicity of ALP and interleukin-1βon Caco-2 cells,and the concentrations of ALP and interleukin-1βwere determined.In addition,it was explored that ALP also played an antioxidant role through nuclear factor-E2-related factor 2 signaling pathway and protected the intestine cells by slowing down oxidative stress.Western bloting,enzyme linked immunosorbent assay,quantitative reverse transcription-polymerase chain reaction,and immunofluorescence were used to investigate whether ALP also inhibited intestinal inflammation through the Toll-like receptor 4/nuclear factor-kappa B signaling pathway.The results showed that ALP also could inhibit interleukin-1β-induced m RNA and protein expression of downstream inflammatory factors(monocyte chemoattractant protein-1,intercellular adhesion molecule-1,and vascular cell adhesion molecule-1)by down-regulating key genes and proteins of Toll-like receptor 4/nuclear factor-kappa B signaling pathway(Toll-like receptor 4,myeloid differentiation protein-2,tumor necrosis factor-associated factor 6,myeloid differentiation factor 88,and nuclear factor-kappa B).These results indicate that ALP has a significant immune regulatory and anti-inflammatory effects on macrophages and intestinal immune system,and there is a certain relationship between antioxidant and anti-inflammatory.These results lay a foundation for further research and application of ALP as functional foods or nutraceuticals to improve immunue system and prevent/treat inflammation in the future. |
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