| Lilium lancifolium Thunb.has been used in traditional Chinese medicine,It have abundantly nutritional value and healthy beneficial function.Water-soluble polysaccharide is one of the main active ingredients in its scale,with anti-tumor,anti-oxidant,immunomodulatory and hypoglycemic activities.In this study,we purified and characterized the water-soluble polysaccharide fraction(LLPS)from L.lancifolium.In addition,we investigated the immune-enhancing activity of LLPS on macrophages in-vitro,and further studied the molecular mechanism of immunomodulatory activity of LLPS.Research results provide the scientific basis for the nutrition health care treatment of water-soluble polysaccharides from Lilium,and provide the theory basis for its application in food,medicine and other industries,and lay the foundation for rational development of lily resources.The main research results in this study are as follows:1.The extraction process of LLPS was optimized using single factor experiments and response surface analysis method,the optimum extraction technology of LLPS: extraction temperature 71?,extraction time 65 min,ratio raw material to water 1:21,and extraction two times,the extraction yield was 7.86%.2.The protein in crude polysaccharide solution was removed by Sevag method,then LLPS was isolated and purified using anion exchange chromatography(DEAE cellulose52),resulting in three fractions(LLPS-1,LLPS-2 and LLPS-3).The there fractions were further purified by sephadex G-100 exclusion chromatography,results showed that the three components were single symmetrical chromatographic peak,and indicated that their were homogeneous.3.The chemical characteristics of three polysaccharide fractions were determined by Gas chromatography(GC),permeation chromatography(GPC)and Fourier transform infrared spectra(TF-IR).Results showed that the total sugar contents of the three components(LLPS-1,LLPS-2 and LLPS-3)were 91.06±0.05%?83.47±0.11% and86.92±0.08%,the uronic acid content were 10.01±0.18%?2.61±0.04% and18.04±0.28 %,the monosaccharide composition were mainly predominantly of D-mannoseand D-glucose with the molar ratio of 1.77:1,1:16.06 and 1:1.04.the average molecular weight mass were 78610 Dal,5360Dal and 4790 Dal,respectively.Fourier transform infrared spectra(FT-IR)of three polysaccharide fractions also revealed typical characteristics of a polysaccharide containing uronic acid,pyranose rings.4.We investigated the immune-enhancing activity of LLPS-1 on macrophages RAW264.7 cels in-vitro by detecting phagocytic activity and the expression of cellular activity factor.Results showed that LLPS-1 was not cytotoxic to RAW264.7 cells up to the concentration of 600 ?g/ml;LLPS-1 enhanced the phagocytic activity and induced the production of NO in does-dependent manner in RAW 264.7 cells.LLPS-1 robust the expression of IL-6,MCP-1,TNF-?,IL-1?.5.We further studied the molecular mechanism of immunomodulatory activity of LLPS-1 using quantitative real-time PCR(qRT-PCR)and Western Blotting.Results showed that LLPS-1 stimulation enhanced the expression of pro-inflammatory cytokine of TNF-?,IL-1?,IL-6 and MCP-1 at the transcriptional level.In addition,LLPS-1 increased protein expression of Toll-like receptor 4 and the phosphorylation of IKK,I?B? and NF-?B/p65 in RAW 264.7 cells.In conclusion,LLPS-1 significantly up-regulated the expression of immune reactive cytokines in macrophages through TLR4-mediated NF-?B signal pathway.The results suggest that LLPS could potentially be used as a natural immunomodulatory. |
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