葡萄果皮花青素提取工艺优化研究
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摘要
利用高效液相色谱(HPLC)对提取葡萄果皮中花青素的提取液、提取方法和色谱条件进行比较,以探讨不同条件对花青素的提取效果的影响。采用正交试验设计选择最佳的提取工艺,利用5种花青素峰面积分析‘夏黑’葡萄果皮所含花青素的含量。试验结果表明,提取液为甲醇:水=1:1、磷酸调至p H2.0、提取超声辅助提取30 min为提取夏黑葡萄果皮花青素含量的最佳工艺。色谱条件为流动相A:水/乙腈/甲酸=40/50/10(v/v/v),流动相B:水/乙腈/甲酸=87/3/10(v/v/v),柱温45℃,进样量20μL,检测波长520 nm。梯度洗脱条件为:(1)0～15 min,6%A→30%A;(2)15～30 min,30%A→50%A;(3)30～35 min,50%A→60%A;(4)35～40 min,60%A～6%A,呈现的色谱图分离度更好,总体效果更佳。
To study the effects of different conditions on the extraction of anthocyanins, an orthogonal experimental design L9(33) was used to optimize the condition of the high performance liquid chromatography(HPLC) assay of anthocyanin composition and content in peel of summer black grape. Three factors affecting extraction process of anthocyanin including, extraction solution, method and three chromatographic conditions were evaluated with an aim to establish a high-throughput extracting process of assay in anthocyanin composition and content in peel of grape. The results showed that the optimal process of assay in anthocyanin composition and content consisted of the extract solution of methanol: water=1:1, phosphoric acid pH value of2.0, the extraction time of 30 min. The chromatographic condition included mobile phase A: water/acetonitrile/formic acid=40/50/10(v/v/v) and mobile phase B: water/acetonitrile/formic acid=87/3/10(v/v/v), column temperature of 45℃, injection volume of 20 μL, detection wavelength of 520 nm, and the gradient elution conditions were:(1) 0-15 min, 6%A→30%A;(2)15-30 min, 30%A→50%A;(3)30-35 min, 50%A→60%A;(4)35-40 min, 60%A→6%A. Under these conditions, elphinidin-3-O-glucoside, cyanidin-3-O-glucoside,pelargonidin-3-O-glucoside, peonidin-3-O-glucoside and malvidin-3-gluconside chloride were separated successfully.
To study the effects of different conditions on the extraction of anthocyanins, an orthogonal experimental design L9(33) was used to optimize the condition of the high performance liquid chromatography(HPLC) assay of anthocyanin composition and content in peel of summer black grape. Three factors affecting extraction process of anthocyanin including, extraction solution, method and three chromatographic conditions were evaluated with an aim to establish a high-throughput extracting process of assay in anthocyanin composition and content in peel of grape. The results showed that the optimal process of assay in anthocyanin composition and content consisted of the extract solution of methanol: water=1:1, phosphoric acid pH value of2.0, the extraction time of 30 min. The chromatographic condition included mobile phase A: water/acetonitrile/formic acid=40/50/10(v/v/v) and mobile phase B: water/acetonitrile/formic acid=87/3/10(v/v/v), column temperature of 45℃, injection volume of 20 μL, detection wavelength of 520 nm, and the gradient elution conditions were:(1) 0-15 min, 6%A→30%A;(2)15-30 min, 30%A→50%A;(3)30-35 min, 50%A→60%A;(4)35-40 min, 60%A→6%A. Under these conditions, elphinidin-3-O-glucoside, cyanidin-3-O-glucoside,pelargonidin-3-O-glucoside, peonidin-3-O-glucoside and malvidin-3-gluconside chloride were separated successfully.
引文
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[14]李琪,李广,金丽琼,等.HPLC法测定甘肃地产不同品种酿酒葡萄中的花色苷[J].中国酿造,2014(3):132-136.
[15]Kamiloglu O.Influence of some cultural practices on yield,fruit quality and individual anthocyanins of table grape cv.‘Horoz Karasi’[J].Journal of Animal and Plant Sciences,2011,21(2):240-245.
[16]李杨.烟73葡萄皮中七种花色苷单体的制备及分析应用[D].无锡:江南大学,2012.
[17]刘笑宏,郭淑华,牛彦杰,等8个葡萄品种的花色苷组分及含量分析[J].果树学报,2017,34(4):444-453.
[18]刘冰,葛谦,张艳,等.HPLC法同时测定葡萄和葡萄酒中6种基本花色苷[J].中国酿造,2017,36(2):162-165.
[19]赵权,王军,韩富亮.不同品种山葡萄酒中花色苷的成分分析[J].西北农林科技大学学报:自然科学版,2013(6):195-201.
[20]陈曦,周小平,韩舜愈,等.HPLC测定葡萄和葡萄酒中花色苷[J].光谱实验室,2013,30(4):1704-1709.
[21]杜月娇,贺阳,文连奎.山葡萄花青素超高压提取工艺优化及其组分分析[J].食品科学,2017,38(10):258-263.
[22]李双石,李浡,张虎成,等.超声波提取条件对酿酒葡萄皮渣花色苷的影响[J].食品工业科技,2013,34(18):294-297.
[23]Clifford M N.Anthocyanins-nature,occurrence and dietary burden[J].Journal of the Science of Food and Agriculture,2000,80(7):1063-1072.
[24]Kong J M,Chia L S,Goh N K,et al.Analysis and biological activities of anthocyanins[J].Phytochemistry,2003,64(5):923-933.
[25]李丹,沈才洪,马懿,等.夏黑葡萄花色苷的提取及抗氧化活性研究[J].应用化工,2016,45(8):1418-1423.
[26]李小娟,聂钰洪,刘琦琦,等.鲜食葡萄品种多酚类物质含量及抗氧化活性分析[J].北方园艺,2017(21):37-42.
[2]De La Cruz A A,Hilbert G,Rivière C,et al.Anthocyanin identification and composition of wild Vitis spp.accessions by using LC-MS and LC-NMR[J].Analytica chimica acta,2012,732:145-152.
[3]Nayak C A,Srinivas P,Rastogi N K.Characterisation of anthocyanins from Garcinia indica Choisy[J].Food chemistry,2010,118(3):719-724.
[4]Woodall G,Stewart G.Do anthocyanins play a role in UVprotection of the red juvenile leaves of Syzygium?[J].Journal of experimental botany,1998,49:1447-1450.
[5]Antonelli F,Bussotti F,Grifoni D,et al.Oak(Quercus robur L.)seedling responses to a realistic increase in UV-B radiation under open space conditions[J].Chemosphere,1998,36(4):841-845.
[6]Dixon R A,Achnine L,Kota P,et al.The phenylpropanoid pathway and plant defence-a genomics perspective[J].Molecular plant pathology,2002,3(5):371-390.
[7]Chalker-Scott L.Environmental significance of anthocyanins in plant stress responses[J].Photochemistry and photobiology,1999,70(1):1-9.
[8]Satué-Gracia M T,Heinonen M,Frankel E N.Anthocyanins as antioxidants on human low-density lipoprotein and lecithinliposome systems[J].Journal of Agricultural and Food Chemistry,1997,45(9):3362-3367.
[9]李颖畅,孟宪军,孙靖靖,等.蓝莓花色苷的降血脂和抗氧化作用[J].食品与发酵工业,2008,34(10):44-48.
[10]Kowalczyk E,Krzesi?ski P,Kura M,et al.Anthocyanins in medicine[J].Polish journal of pharmacology,2003,55(5):699-702.
[11]Chen P N,Chu S C,Chiou H L,et al.Mulberry anthocyanins,cyanidin 3-rutinoside and cyanidin 3-glucoside,exhibited an inhibitory effect on the migration and invasion of a human lung cancer cell line[J].Cancer letters,2006,235(2):248-259.
[12]孙建霞,张燕,胡小松,等.花青素的提取,分离以及纯化方法研究进展[J].食品与发酵工业,2008,34(8):111-117.
[13]Fraige K,Pereira-Filho E R,Carrilho E.Fingerprinting of anthocyanins from grapes produced in Brazil using HPLC-DAD-MS and exploratory analysis by principal component analysis[J].Food chemistry,2014,145:395-403.
[14]李琪,李广,金丽琼,等.HPLC法测定甘肃地产不同品种酿酒葡萄中的花色苷[J].中国酿造,2014(3):132-136.
[15]Kamiloglu O.Influence of some cultural practices on yield,fruit quality and individual anthocyanins of table grape cv.‘Horoz Karasi’[J].Journal of Animal and Plant Sciences,2011,21(2):240-245.
[16]李杨.烟73葡萄皮中七种花色苷单体的制备及分析应用[D].无锡:江南大学,2012.
[17]刘笑宏,郭淑华,牛彦杰,等8个葡萄品种的花色苷组分及含量分析[J].果树学报,2017,34(4):444-453.
[18]刘冰,葛谦,张艳,等.HPLC法同时测定葡萄和葡萄酒中6种基本花色苷[J].中国酿造,2017,36(2):162-165.
[19]赵权,王军,韩富亮.不同品种山葡萄酒中花色苷的成分分析[J].西北农林科技大学学报:自然科学版,2013(6):195-201.
[20]陈曦,周小平,韩舜愈,等.HPLC测定葡萄和葡萄酒中花色苷[J].光谱实验室,2013,30(4):1704-1709.
[21]杜月娇,贺阳,文连奎.山葡萄花青素超高压提取工艺优化及其组分分析[J].食品科学,2017,38(10):258-263.
[22]李双石,李浡,张虎成,等.超声波提取条件对酿酒葡萄皮渣花色苷的影响[J].食品工业科技,2013,34(18):294-297.
[23]Clifford M N.Anthocyanins-nature,occurrence and dietary burden[J].Journal of the Science of Food and Agriculture,2000,80(7):1063-1072.
[24]Kong J M,Chia L S,Goh N K,et al.Analysis and biological activities of anthocyanins[J].Phytochemistry,2003,64(5):923-933.
[25]李丹,沈才洪,马懿,等.夏黑葡萄花色苷的提取及抗氧化活性研究[J].应用化工,2016,45(8):1418-1423.
[26]李小娟,聂钰洪,刘琦琦,等.鲜食葡萄品种多酚类物质含量及抗氧化活性分析[J].北方园艺,2017(21):37-42.