铁皮石斛多糖提取及对羟基自由基诱导的SH-SY5Y细胞凋亡的抑制作用

摘要/Abstract

摘要： 以酶解超声提取法提取铁皮石斛多糖，通过对料液比、超声时间、复合酶浓度的单因素和正交实验，确定铁皮石斛多糖的最佳提取条件；以高效液相色谱法（High Performance Liquid Chromatography，HPLC）对铁皮石斛多糖进行组成成分分析；以香豆素-3-羧酸 (Coumarin-3-carboxylic acid，CCA)荧光法测定铁皮石斛多糖的羟基自由基清除能力；以噻唑蓝(methyl thiazoly1 tetrazolium，MTT)实验和细胞流式实验研究铁皮石斛多糖提取物对SH-SY5Y细胞的保护作用。结果表明，最优铁皮石斛多糖提取条件为：料液比1:100（g/mL）、超声时间2 h、复合酶（纤维素酶和果胶酶1:1等量混合）加酶量为50 %（复合酶质量：铁皮石斛质量），最优提取条件下铁皮石斛多糖的得率为25.69 %，其主要单糖组分为甘露糖和葡萄糖。在浓度为20-80 μg/mL范围内铁皮石斛多糖提取物能有效清除Cu2+催化H2O2产生的羟基自由基并有效抑制SH-SY5Y细胞的凋亡（凋亡率从56.97 %下降到17.97 %），且其作用效果成浓度依赖性。因此，铁皮石斛多糖可通过清除羟基自由从而抑制Cu2+催化H2O2产生的羟基自由基诱导的SH-SY5Y细胞凋亡。

关键词: 铁皮石斛多糖, 提取, 羟基自由基, 神经母细胞瘤细胞（SH-SY5Y）

Abstract: Enzymatic hydrolysis and ultrasonic method were used for polysaccharides extraction from Dendrobium officinale. The optimum extraction conditions of polysaccharides from Dendrobium officinale were determined by single factor and orthogonal experiments with material-liquid ratio, ultrasonic time, and enzyme concentration. High performance liquid chromatography (HPLC) was used for analyzing the composition of Dendrobium officinale polysaccharides. Coumarin-3-carboxylic acid (CCA) fluorescence method was employed for the antioxidant activity determination of Dendrobium officinale polysaccharides. Methyl thiazolyl tetrazolium (MTT) and cell flow cytometry experiments were employed for investigating the inhibition effect of polysaccharides for SH-SY5Y apoptosis which induced by hydroxyl radical. The results showed that the optimum extraction conditions were as follows: the ratio of material to liquid was 1:100, the ultrasonic time was 2 hours, the amount of complex enzymes (cellulase and pectinase mixed equally) was 50 %, and the yield of polysaccharides was 25.69 %. The main monosaccharide composition of Dendrobium officinale polysaccharides is mannose and glucose. At the concentration rang of Dendrobium officinale polysaccharides was from 20 to 80 ug/mL, they can effectively scavenge the hydroxyl radicals produced by Cu2+ catalyzing hydrogen peroxide and effectively inhibit the apoptosis of SH-SY5Y cells (the apoptotic rate decreased from 56.95 % to 17.97 %). The cell apoptosis inhibition effect of the polysaccharides is concentration-dependent. Therefore, Dendrobium officinale polysaccharides can inhibit SH-SY5Y cell apoptosis induced by hydroxyl radicals, which produced by Cu2+ catalyzed hydrogen peroxide reaction.

Key words: dendrobium officinale polysaccharide, extraction, hydroxyl radical, SH-SY5Y cell

中图分类号:

TS201.4

张雅丹赵梦倩周彬彬杨煜佼徐友志王梓郡王焱君许迪雅张琳. 铁皮石斛多糖提取及对羟基自由基诱导的SH-SY5Y细胞凋亡的抑制作用[J]. 食品科学, 0, (): 0-0.

Ya-Dan ZHANG Meng-Qian ZHAO 琳 zhanglin张. Extraction of polysaccharides from Dendrobium officinale, and their inhibition effect for apoptosis of SH-SY5Y cells induced by hydroxyl radical[J]. FOOD SCIENCE, 0, (): 0-0.

参考文献

[1] 张琳.神经退行性疾病病理及潜在治疗药物研究[D]. 湖南: 中南大学, 2013. DOI: 10.7666/d.y2422594. [2] Puzzo D, Gulisano W, Arancio O, et al.The keystone of Alzheimers pathogensis might be sought in Aβ physiology[J].Neuroscience, 2015, 307:26-36 [3] Reddy P H.Abnormal tau, mitochondrial dysfunction, impaired axonal transport of mitochondria, and synaptic deprivation in Alzheimer’s disease[J].Brain Research, 2011, 1415:136-148 [4] Behl C, Davis J B, Lesley R, et al.Hydrogen peroxide mediates amyloid b-protein toxicity[J].Cell, 1994, 77(6):817-822 [5] Pecci L, Montefoschi G, Cavallini D.Some New Details of the Copper-Hydrogen Peroxide Interaction[J].Biochemical and Biophysical Research Communications, 1997, 235(1):264-267 [6] Jiang D, Li X, Liu L, et al.Reaction rates and mechanism of the ascorbic acid oxidation by molecular oxygen facilitated by Cu(II)-containing amyloid-beta complexes and aggregates[J].Journal of Physical Chemistry B, 2010, 114(14):4896-4903 [7]陈晓梅, 田丽霞, 单婷婷, 孙乐, 郭顺星.铁皮石斛种质资源和遗传育种研究进展[J].药学学报, 2018, 53(09):1493-1503 [8]宁英海, 顾敬文, 刘志萍, 银胜高.铁皮石斛活性成分及药理作用研究进展[J].亚太传统医药, 2016, 12(13):71-73 [9]林丽珍, 许仕锦, 杨永军, 杨辉, 雷胄熙, 沈伟, 魏刚.不同生长年限的仿野生铁皮石斛醇提物的抗肿瘤活性比较[J].中药新药与临床药理, 2018, 29(02):149-154 [10]谭青云, 袁永俊, 王丹, 蒋林利, 黄河, 彭洁.不同提取方式对铁皮石斛多糖及体外降血糖的影响[J].食品科技, 2019, 44(06):202-206 [11]余琪, 毛培江, 姜建民, 金祖汉.种药用石斛对增强小鼠免疫功能效果的比较研究[J].中国现代应用药学, 2017, 34(02):191-195 [12]Meng, L.Z.,Lv,GP,Hu,DJ,et alEffects of Polysaccharides from Different Species of Dendrobium (Shihu) on Macrophage Function[J].Molecules, 2013, 18(5):5779-5791 [13] 税维智.铁皮石斛多糖的分离纯化及调节糖代谢紊乱的机制研究[D]. 湖北: 华中科技大学, 2016. DOI: CNKI:CDMD:2.1016.781083. [14] 邱现创.铁皮石斛多糖提取工艺优化及其抗氧化活性研究[D]. 山西: 山西大学, 2017. DOI: CNKI:CDMD:2.1017.303258. [15]薛燕, 敢小双, 黄开丽, 李丽, 王巧娥, 董银卯.铁皮石斛多糖复合酶法提取工艺及其抗氧化性[J].食品工业科技, 2018, 39(03):215-219 [16]朱凯莉, 商玉萍, 张俊东, 周安, 潘见, 惠爱玲.超高压对石斛汁有效成分溶出及消化吸收的影响[J].食品工业, 2018, 39(08):82-86 [17]封毅, 黄茂康, 叶建保, 钟卫干, 乐宁.石斛多糖在饮料加工过程及货架期内的变化规律[J].南方农业学报, 2014, 45(07):1264-1267 [18]蔡兴, 王美娜, 梁权辉, 王玮, 李苑新.响应面法优化铁皮石斛叶闪式提取工艺[J].亚太传统医药, 2016, 12(07):48-52 [19]王爽, 弓建红, 张寒娟, 许小华, 杨云.石斛多糖抗氧化活性研究[J].中国实用医药, 2009, 4(30):15-16 [20] 何铁光, 杨丽涛, 李杨瑞, 王灿琴, 黄岛平.铁皮石斛原球茎多糖DCPP1a-1的理化性质及抗肿瘤活性[J].天然产物研究与开发, 2007, 19(04):578-583 [21]LIU X F, ZHU J, REN F Z, et al.Orally administered Dendrobium officinale and its polysaccharides enhanceimmune functions in BALBc mice[J].Natural Product Communications, 2011, 6(6):867-870 [22]郑秋平, 邱道寿, 刘晓津, 蔡时可, 张蕾, 邓瑞云, 罗焕明, 张学武.铁皮石斛抗肿瘤活性成分的探究[J].现代食品科技, 2014, 30(05):12-17 [23]王杰, 葛颖华, 周萃, 童晔玲, 任泽明.鲜铁皮石斛提取物抗肺癌的机制研究[J].中国现代应用药学, 2014, 31(08):953-957 [24]劳梓钊, 廖沣, 吴茂勇, 陈立峰, 龙海珊, 吴依娜, 周瑞芳.铁皮石斛多糖延缓果蝇衰老与抗氧化作用研究[J].中药材, 2018, 41(07):1740-1742 [25]董增, 曹稳根, 段红, 张兴桃, 陈军, 张开.桔梗多糖提取、分离纯化以及生物活性研究[J].基因组学与应用生物学, 2018, 37(08):3534-3539 [26]盛家荣, 李志华, 易艳波, 等.铁皮石斛多糖的研究进展[J].广西科学院学报, 2011, 27(4):338-340 [27]马千里.铁皮石斛多糖含量测定不确定度分析[J].东莞理工学院学报, 2017, 24(01):63-67 [28]黄丽, 文凤娟, 李桂琼, 郭成鑫, 熊娇, 何丹, 周洋帆, 贺飞飞, 文国松.铁皮石斛多糖提取工艺及优化研究[J].云南农业大学学报自然科学, 2017, 32(05):884-888 [29]王培培, 鲁芹飞, 陈建南, 等.正交实验法优化铁皮石斛多糖的提取工艺[J].时珍国医国药, 2012, 23(11):2781-2782 [30]赖颖, 赵锦慧, 陈茹.响应面法优化超声波法提取红枣多糖工艺[J].生物加工过程, 2015, 13(05):42-46 [31]邱现创, 赵宁, 李晨, 王景雪.铁皮石斛多糖提取工艺优化及对果蝇抗氧化能力的影响[J].食品科学, 2018, 39(02):273-280 [32]刘静, 汪雨, 黄劲松, 郭军.西山焦枣多糖的超声辅助法提取工艺和抗氧化研究[J].池州学院学报, 2019, 33(03):24-28 [33]PRIOR R L, WU X L, SCHAICH K.Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements[J].Joumal of Agricultural and Food Chemistry, 2005, 53(10):4290-4302 [34]鲍素华, 查学强, 赫杰, 等.不同分子量铁皮石斛多糖体外抗氧化活性研究[J].食品科学, 2009, 30(21):123-127 [35]Lovell M A, Robertson J D, Teesdale W J, et al.Copper,iron and zinc in Alzheimer's disease senile plaques[J].J. Neurol. Sci., 1998, 158(1):47-52 [36] 蒋武.超氧化物歧化酶及过氧化氢酶模拟物的研究[D]. 湖北: 武汉工程大学, 2015. DOI: CNKI:CDMD:2.1015.667282. [37]Jakob R R, Jacobsen H.Alzheimer’s disease: From pathology to therapeutic approaches[J].Angew. Chem. Int. Ed. Engl., 2009, 48(17):3030-3059 [38]周怡昆, 薛耀明.高糖通过高渗透压改变基因表达抑制雪旺细胞增殖[J].昆明理工大学学报自然科学版, 2013, 38(04):60-64 [39]孟祥云, 汪永锋, 杨丽霞, 郭树明, 王永胜, 范强.中药多糖抗氧化作用及其机制研究进展[J].中华中医药杂志, 2018, 33(08):3504-3509