万寿菊多糖的纯化、组成分析及其体外抗氧化和抗肿瘤活性研究

Purification and composition analysis of polysaccharide from Tagetes erecta L.and its antioxidant and antitumor activities in vitro

对万寿菊多糖(Tagetes erecta L.polysaccharides,TEPs)提取工艺进行优化并对粗多糖进行分离纯化,对得到的多糖组分进行单糖组成及体外抗氧化和抗肿瘤活性分析。响应面法优化万寿菊多糖最佳提取工艺为:提取温度80℃、提取时间2 h、料液比1∶21(g∶mL),此条件下提取率为3.7%。利用DEAE-52纤维素和Sephadex G-150柱层析分离纯化TEPs得2种均一多糖(TEPs-1和TEPs-2);利用高效体积排阻色谱法测定2种均一多糖重均分子量分别为4.69和5.73 kDa;利用1-苯基-3-甲基-5-吡唑啉酮(1-phenyl-3-methyl-5-pyrazolone,PMP)-柱前衍生HPLC法测定其单糖组成,TEPs两种组分均由7种单糖组成,其中甘露糖、半乳糖、葡萄糖和阿拉伯糖4种单糖含量较高。1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基、羟基自由基、超氧阴离子、2,2′-联氮-双-3-乙基苯并噻唑啉-6-磺酸(2,2′-azino-bis 3-ethylbenzthiazoline-6-sulphonate acid,ABTS)自由基清除实验结果表明,TEPs-1和TEPs-2均有明显的抗氧化活性,且呈现良好的浓度依赖关系,质量浓度越大清除率越高。MTT实验结果表明,TEPs两个组分对MCF-7细胞均有一定生长抑制作用,TEPs-1和TEPs-2对MCF-7细胞的IC 50分别为319.76和134.04μg/mL。万寿菊多糖具有良好的体外抗氧化和抑瘤活性,可为万寿菊多糖产品开发提供参考。

The preparation process of Tagetes erecta L.polysaccharide(TEPs)was optimized,and the polysaccharide was separated and purified.The obtained polysaccharide components were analyzed for monosaccharide composition,antioxidant and antitumor in vitro.The results showed that the optimal extraction process of TEPs by response surface method was:extraction condition was 80℃for 2 h,solid-liquid ratio 1∶21(g∶mL)and the extraction rate of this strip was 3.7%.Moreover,two homogeneous polysaccharides(TEPs-1 and TEPs-2)were isolated and purified from TEPs by DEAE-52 cellulose and Sephadex G-150 column chromatography.The average molecular weight of TEPs-1 and TEPs-2 were 4.69 kDa and 5.73 kDa measured by high performance size exclusion chromatography(HPSEC),respectively.Furthermore,monosaccharide composition was analyzed by precolumn derivatization with 1-phenyl-3-methyl-5-pyrazolone(PMP)coupled to HPLC.The results indicated that the two components of TEPs were composed of seven monosaccharides and the contents of mannose,galactose,glucose and arabinose were high.Besides,1,1-diphenyl-2-trinitrophenylhydrazine radical(DPPH·),hydroxyl radical,superoxide anion,2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate)radical(ABTS·)scavenging experimental results indicated that TEPs-1 and TEPs-2 had obvious antioxidant activities,good concentration dependence,greater the mass concentration and high clearance rate.Meanwhile,the MTT results showed that TEPs had a certain growth inhibition effect on MCF-7 cells,and the IC 50 of TEPs-1 and TEPs-2 on MCF-7 cells were 319.76 and 134.04 g/mL,respectively.TEPs has good antioxidant and tumor suppressive activity in vitro,which could provide references for marigold polysaccharide product development.