高速逆流色谱结合半制备型液相色谱分离茶树紫芽花色苷研究
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摘要
花色苷是植物中一类具有重要生物活性的次生代谢产物,因其含量较低,使得分离制备花色苷高纯品难度较大。本研究以茶树紫芽为实验材料,以花色苷得率与纯度为考察指标,优化筛选了应用高速逆流色谱结合半制备型液相色谱分离纯化花色苷高纯度的方法。结果表明,茶样经提取、脱脂、阳离子交换树脂分离等前处理后,总花色苷得率28.18 mg/g,纯度为20.38%;以高速逆流色谱分离的最优溶剂体系乙酸乙酯∶正丁醇∶乙腈∶0.1%三氟乙酸水溶液=8∶35∶13∶60(v/v/v/v),除去部分杂质后,总花色苷得率达到160.59 mg/g,纯度为43.64%,再结合半制备型液相色谱分离出5种花色苷组分,纯度最高达90.61%,其优化色谱条件为C18制备柱、波长280 nm、柱温30℃、流动相1%醋酸水溶液(A)-乙腈(B)、流速5 mL/min、进样量1 mL、梯度洗脱(0～55 min,10%～40%B)。研究表明高速逆流色谱结合半制备型液相色谱可有效地分离纯化茶树紫芽花色苷及其组分。
Anthocyanins are a class of secondary metabolites in plants with important biological activities.it is difficult to separate and prepare high-purity anthocyanins from plants because of the low-content.the purple bud tea was used as experimental material in this study,taking the yield and the purity of anthocyanins as an indicators,the method of separation and purification of anthocyanins by high-speeed counter-current chromatography(HSCCC) coupled with semipreparative high performance liquid chromatography(PHPLC) were optimized and screened.The results showed that total anthocyanin yield was 28.18 mg/g and the purity of anthocyanin was 20.38% after sample pretreatment by extraction,degreasing and cation exchange resin.The optimal solvent system in high-speed counter-current chromatography was ethyl acetate: n-butanol: acetonitrile: 0.1% trifluoroacetic acid = 8:35:13:60(v/v/v/v),with most of the impurities were removed from samples,total anthocyanin yield reached 160.59 mg/g,the purity of anthocyanin was 43.64%,then,the five kinds of anthocyanin components was separated by semi-preparative high performance liquid chromatography with a purity of up to 90.61%,the optimized conditions were C18 preparative column,280 nm wavelength,column temperature was fixed at30 ℃,the mobile phase was 1% acetic acid(A)-acetonitrile(B),flow rate was 5 mL/min,injection volume was 1 mL with headspace sampling,condition of gradient elution: 0～55 minutes,B10% → 40%.These data indicated that high-speed counter-current chromatography coupled with semi-preparative high performance liquid chromatography can effectively separate and purify the anthocyanins and their components.
Anthocyanins are a class of secondary metabolites in plants with important biological activities.it is difficult to separate and prepare high-purity anthocyanins from plants because of the low-content.the purple bud tea was used as experimental material in this study,taking the yield and the purity of anthocyanins as an indicators,the method of separation and purification of anthocyanins by high-speeed counter-current chromatography(HSCCC) coupled with semipreparative high performance liquid chromatography(PHPLC) were optimized and screened.The results showed that total anthocyanin yield was 28.18 mg/g and the purity of anthocyanin was 20.38% after sample pretreatment by extraction,degreasing and cation exchange resin.The optimal solvent system in high-speed counter-current chromatography was ethyl acetate: n-butanol: acetonitrile: 0.1% trifluoroacetic acid = 8:35:13:60(v/v/v/v),with most of the impurities were removed from samples,total anthocyanin yield reached 160.59 mg/g,the purity of anthocyanin was 43.64%,then,the five kinds of anthocyanin components was separated by semi-preparative high performance liquid chromatography with a purity of up to 90.61%,the optimized conditions were C18 preparative column,280 nm wavelength,column temperature was fixed at30 ℃,the mobile phase was 1% acetic acid(A)-acetonitrile(B),flow rate was 5 mL/min,injection volume was 1 mL with headspace sampling,condition of gradient elution: 0～55 minutes,B10% → 40%.These data indicated that high-speed counter-current chromatography coupled with semi-preparative high performance liquid chromatography can effectively separate and purify the anthocyanins and their components.
引文
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[20]王维茜,邓洁红,刘永红.半制备型高效液相色谱法分离刺葡萄花色苷单体[J].食品科学,2016,37(18):71-76.
[21]刘林峰,周阳,林玲,等.茶树紫芽中多种花色苷组分同时检测方法的建立[J].食品与机械,2018,34(02):67-72.
[22]顾子杨,崔梦迪,李玉华,等.黑枸杞花色苷纯化工艺的优化与评价[J].中国药理学通报,2018,34(10):1373-1379.
[23]谭亮,董琦,曹静亚,等.黑果枸杞中花色苷的提取与结构鉴定[J].天然产物研究与开发,2014,26(11):1797-1802,1760.
[24]Escribano-Bailón,M.T.,Santos-Buelga,C.,&RivasGonzalo,J.C.(2004).Anthocyanins in cereals.Journal of Chromatography A,1054(1-2),129-141.
[25]Chang-Ling Zhao,Yu-Qi Yu,Zhong-Jian Chen,et al.Stabilityincreasing effects of anthocyanin glycosyl acylation[J].Food Chemistry,2017,214.
[26]沈晓佳.天然红色茶叶的化学分析[D].上海:华东理工大学,2013.
[27]张宏宝.茶树红紫色芽叶中花青素组分的分离及鉴定[D].泰安:山东农业大学,2009.
[28]李雨浩,张楠,向珊珊,等.黑果腺肋花楸花色苷提取工艺及其稳定性[J].食品工业科技,2019,40(3):120-126.
[29]罗娅君,边清泉,张琦,等.高速逆流色谱分离与鉴定牡荆化学成分[J].分析测试学报,2011,30(09):1044-1049.
[30]于丽丽,刘巧,陈丹,等.制备型高效液相色谱法在天然药物分离中的应用[J].中华中医药学刊,2009,27(07):1465-1467.
[2]阎芙洁.桑葚花色苷对糖代谢的调控作用及其机制研究[D].杭州:浙江大学,2018.
[3]刘玉晨,李敬双,于洋.蓝莓花色苷对小鼠淋巴细胞和腹腔巨噬细胞免疫调节作用的研究[J].食品工业科技,2018,39(19):296-301.
[4]陈焱坤,刘晓玲,李敬双,等.紫薯花色苷对小鼠淋巴细胞免疫功能的调节作用[J].食品工业科技,2018,39(03):305-310.
[5]申芮萌,杨岚,于宁,等.葡聚糖凝胶Sephadex LH-20柱层析分离纯化蓝莓花色苷的研究[J].食品工业科技,2016,37(9)58-63
[6]曹少谦,潘思轶,姚晓琳,等.柱层析法分离纯化血橙花色苷[J].中国农业科学,2009,42(5):1728-1736.
[7]于泽源,赵剑辉,李兴国,等.大孔树脂-中压柱层析联用分离纯化蓝莓花色苷[J].食品科学,2018,(1):118-123.
[8]刘雪辉,王振,吴琪,等.高速逆流色谱法分离玫瑰茄中的花色苷[J].现代食品科技,2014,(1):190-194.
[9]陆英,李佳银,罗晋,等.高速逆流色谱制备分离紫甘薯花色苷[J].分析化学,2011,39(6):851-856.
[10]Saito T,Honma D,Tagashira M,et al.Anthocyanins from new red leaf tea'Sunrouge'.[J].Journal of Agricultural and Food Chemistry,2011,59(9):4779-4782.
[11]Gonzalezparamas A M,Silva F L,Martinlopez P,et al.Flavanol-anthocyanin condensed pigments in plant extracts[J].Food Chemistry,2006,94(3):428-436.
[12]王秋霜,凌彩金,刘淑媚,等.红紫芽茶叶花青素提取分离及p H稳定性初探[J].中国农学通报,2014,30(9):291-296.
[13]费旭元.紫娟茶中花青素的提取分离及抗氧化活性研究[D].北京:中国农业科学院,2012.
[14]罗正飞,梁名志,王立波.“紫娟”茶花青素提取工艺研究[J].滇西科技师范学院学报,2011,33(4):111-114.
[15]马淑青,吕晓玲,范辉,等.阳离子交换树脂二次纯化紫甘薯花色苷的研究[J].离子交换与吸附,2010,26(3):218-225.
[16]周育辉,黄秋香,向柏霖,等.改性阳离子交换树脂应用研究进展[J].化工技术与开发,2016,45(6):43-48.
[17]董占波.吸附分离法制备茶叶儿茶素类和茶氨酸的研究[D].杭州:浙江大学,2012.
[18]胡金奎,戴军,张九勋,等.树脂法分离纯化桑葚花色苷[J].食品与发酵工业,2012,38(8):190-196.
[19]陈田,商婷婷,何珍,等.辣椒生物碱的高速逆流色谱分离及抗炎活性测定[J].湖南农业大学学报(自然科学版),2018,1):56-61.
[20]王维茜,邓洁红,刘永红.半制备型高效液相色谱法分离刺葡萄花色苷单体[J].食品科学,2016,37(18):71-76.
[21]刘林峰,周阳,林玲,等.茶树紫芽中多种花色苷组分同时检测方法的建立[J].食品与机械,2018,34(02):67-72.
[22]顾子杨,崔梦迪,李玉华,等.黑枸杞花色苷纯化工艺的优化与评价[J].中国药理学通报,2018,34(10):1373-1379.
[23]谭亮,董琦,曹静亚,等.黑果枸杞中花色苷的提取与结构鉴定[J].天然产物研究与开发,2014,26(11):1797-1802,1760.
[24]Escribano-Bailón,M.T.,Santos-Buelga,C.,&RivasGonzalo,J.C.(2004).Anthocyanins in cereals.Journal of Chromatography A,1054(1-2),129-141.
[25]Chang-Ling Zhao,Yu-Qi Yu,Zhong-Jian Chen,et al.Stabilityincreasing effects of anthocyanin glycosyl acylation[J].Food Chemistry,2017,214.
[26]沈晓佳.天然红色茶叶的化学分析[D].上海:华东理工大学,2013.
[27]张宏宝.茶树红紫色芽叶中花青素组分的分离及鉴定[D].泰安:山东农业大学,2009.
[28]李雨浩,张楠,向珊珊,等.黑果腺肋花楸花色苷提取工艺及其稳定性[J].食品工业科技,2019,40(3):120-126.
[29]罗娅君,边清泉,张琦,等.高速逆流色谱分离与鉴定牡荆化学成分[J].分析测试学报,2011,30(09):1044-1049.
[30]于丽丽,刘巧,陈丹,等.制备型高效液相色谱法在天然药物分离中的应用[J].中华中医药学刊,2009,27(07):1465-1467.