血耳多糖的提取工艺、微观结构及其抗炎症作用
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摘要
采用单因素法和响应曲面法优化热水提取法提取血耳总糖的工艺条件,得到最佳提取条件:液料比124∶1、提取时间120 min和提取温度100℃,其总糖提取率可达到35.347%±0.551%。利用Sevage法、冻融法、透析和DEAE-Sepharose CL-6B柱层析等方法对血耳粗多糖进行分离纯化,得到均一组分(命名为TSP-Ⅱ)。TSP-Ⅱ的回收率为31.56%,相对分子质量是350 ku,糖含量和蛋白质含量分别为97.3%和1.05%。红外光谱显示TSP-II具有糖类物质的特征吸收峰。透射电子显微镜观察TSP-II的微观结构,显示TSP-II是无分支结构的多糖,糖链之间相互缠绕。通过1μg/mL脂多糖诱导小鼠巨噬细胞RAW264.7产生炎症因子(TNF-α、IL-6和COX-2)。经脂多糖诱导的细胞添加TSP-II(75,150μg/mL)后,3种炎症因子的m RNA表达量均明显降低,说明血耳多糖TSP-II具有良好的抗炎症作用。
To obtain the optimal processing conditions for maximum total sugar yield, single factor investigation and response surface methodology(RSM) were employed. The optimal processing conditions were as follows: liquid to solid ratio 124 mL/g, extraction time 120 min, extraction temperature 100 ℃. The experimental yield was 35.347%±0.551%. A polysaccharide fraction from Tremella samguinea Peng(named TSP-II) was purified by Sevag, frozen-thawed, and DEAE-Sepharose CL-6 B column chromatography. The yield of TSP-Ⅱ was 31.56%. Its relative molecular weight was 350 ku, the content of total sugar was 97.3%, and protein was 1.05%. Characteristic peaks of polysaccharide TSP-II appeared on it fourier transform infrared spectrum(FTIR). Transmission electron microscopy(TEM) analysis revealed a primary non-branched and entangled state in its microstructure. TSP-II(75, 150 μg/mL) showed significant inhibitory activities on TNF-α, IL-6, and COX-2 gene expressions in 1 μg/mL LPS-stimulated RAW264.7 mouse macrophage cells.These results suggested the potential effects of TSP-II on anti-inflammatory properties.
To obtain the optimal processing conditions for maximum total sugar yield, single factor investigation and response surface methodology(RSM) were employed. The optimal processing conditions were as follows: liquid to solid ratio 124 mL/g, extraction time 120 min, extraction temperature 100 ℃. The experimental yield was 35.347%±0.551%. A polysaccharide fraction from Tremella samguinea Peng(named TSP-II) was purified by Sevag, frozen-thawed, and DEAE-Sepharose CL-6 B column chromatography. The yield of TSP-Ⅱ was 31.56%. Its relative molecular weight was 350 ku, the content of total sugar was 97.3%, and protein was 1.05%. Characteristic peaks of polysaccharide TSP-II appeared on it fourier transform infrared spectrum(FTIR). Transmission electron microscopy(TEM) analysis revealed a primary non-branched and entangled state in its microstructure. TSP-II(75, 150 μg/mL) showed significant inhibitory activities on TNF-α, IL-6, and COX-2 gene expressions in 1 μg/mL LPS-stimulated RAW264.7 mouse macrophage cells.These results suggested the potential effects of TSP-II on anti-inflammatory properties.
引文
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[19] CHEN L, CHEUNG P C K. Mushroom dietary fiber from the fruiting body of pleurotus tuber-regium:fractionation and structural elucidation of nondigestible cell wall components[J]. Journal of Agricul tural and Food Chemistry, 2014, 62(13):2891-2899.
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[22] WANG K, LI W, RUI X, et al. Chemical modification, characterization and bioactivity of a released exopolysaccharide(r-EPS1)from Lactobacillus plantarum 70810[J]. Glycoconjugate Journal, 2015, 32(7):17-27.
[2]卯晓岚.中国大型真菌[M].郑州:河南科学技术出版社, 2000, 10(2):11-13.
[3]谢好贵,陈美珍,张玉强.多糖抗肿瘤构效关系及其机制研究进展[J].食品科学, 2011, 32(11):329-333.
[4] CHEN B. Optimization of extraction of Tremella fuciformis polysaccharides and its antioxidant and antitumour activities in vitro[J]. Carbohydrate Polymers,2010, 81(2):420-424.
[5]胡庭俊,梁纪兰,程富胜,等.银耳多糖对小鼠脾脏淋巴细胞蛋白激酶C活性的影响[J].中草药,2005, 36(1):81-84.
[6] EUN J C, HYE J H, WOO K S, et al. Hypoglycemic effects of exopolysaccharides produced by mycelial cultures of two different mushrooms Tremella fuciformis and Phellinus baumii in ob/ob mice[J].Applied Microbiology&Biotechnology, 2007, 75(6):1257-1265.
[7]刘培勋,高小荣,徐文清,等.银耳碱提多糖抗氧化活性的研究[J].中药药理与临床, 2005, 21(4):35-37.
[8]汪虹,瞿伟菁,褚书地,等.金耳菌丝体多糖对小鼠高脂血症的防治作用[J].营养学报, 2002, 24(4):431-432.
[9]申建和,陈琼华.木耳多糖、银耳多糖和银耳孢子多糖对实验性血栓形成的影响[J].中国药科大学学报,1990, 21(1):39-42.
[10]刘西周,郭成金.采用L9(34)正交设计方法筛选血耳菌丝体液体培养基[J].中国食用菌, 2009, 28(1):36-38.
[11]王莜霖.黄芪多糖及其硫酸化修饰产物体外抗炎活性研究[D].杨凌:西北农林科技大学, 2014:30-34.
[12] LUO D. Optimization of total polysaccharide extraction from Dioscorea nipponica Makino using response surface methodology and uniform design[J]. Carbohydrate Polymers, 2012, 90(1):284-288.
[13] WANG Z, LUO D, CAI E. Optimization of polysaccharides extraction from Gynostemma pentaphyllum Makino using Uniform Design[J]. Carbohydrate Polymers, 2007, 69(2):311-317.
[14] LUO D, YUAN X, ZENG Y, et al. Structure elucidation of a major fucopyranose-rich heteropolysaccharide(STP-II)from Sargassum thunbergii[J]. Carbohydrate Polymers, 2016, 143(1):1-8.
[15] YUAN X, ZENG Y, NIE K, et al. Extraction optimization, characterization and bioactivities of a major polysaccharide from Sargassum thunbergii[J]. PLoS ONE, 2015, 10(12):e0144773.
[16] DUBOIS M, GILLIS K A, HAMILTON J K, et al.Colorimetric method for determination of sugars and related substances[J]. Analytical Chemistry, 1956,28(3):350-356.
[17] BRADFORD M M. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry, 1976, 72(1/2):248-254.
[18] CHEN L, LIU J, ZHANG Y, et al. A novel alkaline hemicellulosic heteroxylan isolated from alfalfa(Medicago sativa L.)stem and its thermal and antiinflammatory properties[J]. Journal of Agricultural and Food Chemistry, 2015, 63(11):2970-2978.
[19] CHEN L, CHEUNG P C K. Mushroom dietary fiber from the fruiting body of pleurotus tuber-regium:fractionation and structural elucidation of nondigestible cell wall components[J]. Journal of Agricul tural and Food Chemistry, 2014, 62(13):2891-2899.
[20]夏朝红,戴奇,房韦,等.几种多糖的红外光谱研究[J].武汉理工大学学报, 2007, 29(1):45-47.
[21] CHEN L, LIU J, ZHANG Y, et al. Structural,thermal, and anti-inflammatory properties of a novel pectic polysaccharide from alfalfa(Medicago sativa L.)stem[J]. Journal of Agricultural and Food Chemistry, 2015, 63(11):3219-3228.
[22] WANG K, LI W, RUI X, et al. Chemical modification, characterization and bioactivity of a released exopolysaccharide(r-EPS1)from Lactobacillus plantarum 70810[J]. Glycoconjugate Journal, 2015, 32(7):17-27.