蓝莓果实中花色苷单体的色谱分离纯化
|
摘要
为充分开发蓝莓果实的潜在应用价值,主要采用柱色谱法及半制备高效液相色谱法系统研究蓝莓果实中花色苷(花青素的糖苷形式)单体的制备技术。蓝莓花色苷粗提液经超声辅助浸提、乙酸乙酯萃取2次,能够促进花色苷类物质的溶出,并有效去除溶液中的黄酮类杂质。经Amberlite XAD-7HP大孔树脂层析、Sep-Pak C18固相萃取,所得蓝莓花色苷粗品的纯度为62.49%。经Sephadex LH-20凝胶色谱柱分离,获得的3种花色苷纯化组分纯度在65%~75%之间。运用半制备型高效液相色谱技术从3种花色苷纯化组分中制备出两种蓝莓果实中含量较低的半乳糖苷化的花色苷单体,经分析型高效液相色谱鉴定为飞燕草素-3-O-半乳糖苷和锦葵色素-3-O-半乳糖苷,纯度分别为96.98%和95.63%。本研究为花色苷单体的规模化生产提供了技术参考,同时为实现蓝莓花青素高附加值产品的生产提供良好理论依据。
To fully develop the potential application of blueberry fruits, this paper is focused on the preparation of anthocyanin monomers from blueberry fruits using column chromatography and semi-preparative high performance liquid chromatography(HPLC). Crude blueberry anthocyanins were ultrasonically extracted with acidified 70% ethanol and further subjected to two cycles of ethyl acetate extraction for the purpose of facilitating the dissolution of anthocyanins and removing non-anthocyanin flavonoids. The purity of crude blueberry anthocyanins reached 62.49% after Amberlite XAD-7HP column chromatography separationand Sep-Pak C18 solid-phase extraction. Three anthocyanin fractions were obtained by Sephadex LH-20 column chromatography with purities ranging from 65% to 75%. Using semi-preparative HPLC, two pure anthocyanin monomers were successfully isolated from the three purified anthocyanin fractions, which were identified as delphinidin-3-O-galactoside and malvidin-3-O-galactoside by analytical HPLC, with purities of 96.98% and 95.63% respectively. The present study can provide technical references for large-scale production of anthocyanin glycoside monomers, and also provide a good theoretical basis for the production of high value-added products of blueberry anthocyanins.
To fully develop the potential application of blueberry fruits, this paper is focused on the preparation of anthocyanin monomers from blueberry fruits using column chromatography and semi-preparative high performance liquid chromatography(HPLC). Crude blueberry anthocyanins were ultrasonically extracted with acidified 70% ethanol and further subjected to two cycles of ethyl acetate extraction for the purpose of facilitating the dissolution of anthocyanins and removing non-anthocyanin flavonoids. The purity of crude blueberry anthocyanins reached 62.49% after Amberlite XAD-7HP column chromatography separationand Sep-Pak C18 solid-phase extraction. Three anthocyanin fractions were obtained by Sephadex LH-20 column chromatography with purities ranging from 65% to 75%. Using semi-preparative HPLC, two pure anthocyanin monomers were successfully isolated from the three purified anthocyanin fractions, which were identified as delphinidin-3-O-galactoside and malvidin-3-O-galactoside by analytical HPLC, with purities of 96.98% and 95.63% respectively. The present study can provide technical references for large-scale production of anthocyanin glycoside monomers, and also provide a good theoretical basis for the production of high value-added products of blueberry anthocyanins.
引文
[1]YOU Q,WANG B W,CHEN F,et al.Comparison of anthocyanins and phenolics in organically and conventionally grown blueberries in selected cultivars[J].Food Chemistry,2011,125(1):201-208.DOI:10.1016/j.foodchem.2010.08.063.
[2]SPECIALE A,CIMINO F,SAIJA A,et al.Bioavailability and molecular activities of anthocyanins as modulators of endothelial function[J].Genes and Nutrition,2014,9(4):1-19.DOI:10.1007/s12263-014-0404-8.
[3]WU T,JIANG Z,YIN J,et al.Anti-obesity effects of artificial planting blueberry(Vaccinium ashei)anthocyanin in high-fat diet-treated mice[J].International Journal of Food Sciences and Nutrition,2016,67(3):257-264.DOI:10.3109/09637486.2016.1146235.
[4]陈文超,刘回民,刘景圣.花青素抗肿瘤活性的研究进展[J].食品研究与开发,2016,37(1):211-215.DOI:10.3969/j.issn.1005-6521.2016.01.051.
[5]NORBERTO S,SILVA S,MEIRELES M,et al.Blueberry anthocyanins in health promotion:a metabolic overview[J].Journal of Functional Foods,2013,5(4):1518-1528.DOI:10.1016/j.jff.2013.08.015.
[6]许相雯.微波辅助萃取蓝莓中花青素及纯化的研究[D].哈尔滨:东北农业大学,2012:14-36.
[7]LI D N,MENG X J,LI B.Profiling of anthocyanins from blueberries produced in China using HPLC-DAD-MS and exploratory analysis by principal component analysis[J].Journal of Food Composition and Analysis,2015,47:1-7.DOI:10.1016/j.jfca.2015.09.005.
[8]BUNEA A,RUGIN?D,SCON?A Z,et al.Anthocyanin determination in blueberry extracts from various cultivars and their antiproliferative and apoptotic properties in B16-F10 metastatic murine melanoma c e l l s[J].P h y t o c h e m i s t r y,2 0 1 3,9 5:4 3 6-4 4 4.D O I:1 0.1 0 1 6/j.phytochem.2013.06.018.
[9]CANUTO G A,OLIVEIRA D R,da CONCEI??O L S,et al.Development and validation of a liquid chromatography method for anthocyanins in strawberry(Fragaria spp.)and complementary studies on stability,kinetics and antioxidant power[J].Food Chemistry,2016,192:566-574.DOI:10.1016/j.foodchem.2015.06.095.
[10]GALLOWAY K R,BRETZ S L,NOVAK M.Paper chromatography and UV-Vis spectroscopy to characterize anthocyanins and investigate antioxidant properties in the organic teaching laboratory[J].Journal of Chemical Education,2014,92(1):183-188.DOI:10.1021/ed400520n.
[11]CORREA-BETANZO J,PADMANABHAN P,CORREDIG M,et al.Complex formation of blueberry(Vaccinium angustifolium)anthocyanins during freeze-drying and its influence on their biological activity[J].Journal of Agricultural and Food Chemistry,2015,63(11):2935-2946.DOI:10.1021/acs.jafc.5b00016.
[12]ARACELI C,MADELOURDES P H,MAELENA P,et al.Chemical studies of anthocyanins:a review[J].Food Chemistry,2009,113(4):859-871.DOI:10.1016/j.foodchem.2008.09.001.
[13]潘利华,王建飞,叶兴乾,等.蓝莓花青素的提取工艺及其免疫调节活性[J].食品科学,2014,35(2):81-86.DOI:10.7506/spkx1002-6630-201402015.
[14]董周永,阿嘎,赵国建,等.大孔树脂对红心萝卜花色苷的纯化[J].食品科学,2011,32(4):21-24.
[15]王二雷.蓝莓花青素高纯提取物的制备技术及诱导肿瘤细胞凋亡作用研究[D].长春:吉林大学,2014:34-93.
[16]刘均玉,许晨,许建中,等.高效半制备液相色谱法从橘皮中分离制备黄酮类化合物[J].中国食品添加剂,2009(2):157-160.
[17]王二雷,陈晶晶,赵叶辉,等.小叶女贞果实花青素组分鉴定及色谱纯化技术[J].农业工程学报,2016,32(4):302-308.DOI:10.11975/j.issn.1002-6819.2016.04.043.
[18]郭丹妮,向灿辉,陈阳,等.葡聚糖凝胶色谱结合高速逆流色谱提取蓝莓中花青素[J].食品工业,2016,37(2):181-183.
[19]BARNES J S,NGUYEN H P,SHEN S,et al.General method for extraction of blueberry anthocyanins and identification using high performance liquid chromatography-electrospray ionization-ion traptime of flight-mass spectrometry[J].Journal of Chromatography A,2009,1216(23):4728-4735.DOI:10.1016/j.chroma.2009.04.032.
[20]WANG E L,YIN Y G,XU C N,et al.Isolation of high-purity anthocyanin mixtures and monomers from blueberries using combined chromatographic techniques[J].Journal of Chromatography A,2014,1327:39-48.DOI:10.1016/j.chroma.2013.12.070.
[21]宁德生,梁小燕,方宏,等.半制备高压液相色谱法制备罗汉果苷Ⅴ标准品[J].食品科学,2010,31(12):137-140.
[22]龚金炎,毛建卫,黄伟素,等.竹茹中苜蓿素对照品的高效液相半制备色谱制备研究[J].中草药,2012,43(5):919-921.
[23]王秋霜,凌彩金,刘淑媚,等.花青素分离纯化及其组分鉴定研究进展[J].食品工业科技,2013,34(3):358-360.DOI:10.13386/j.issn1002-0306.2013.03.054.
[24]CORRALES M,TOEPFL S,BUTZ P,et al.Extraction of anthocyanins from grape by-products assisted by ultrasonics,high hydrostatic pressure or pulsed electric fields:a comparison[J].Innovative Food Science and Emerging Technologies,2008,9(1):85-91.DOI:10.1016/j.ifset.2007.06.002.
[25]BURAN T J,SANDHU A K,LI Z,et al.Adsorption/desorption characteristics and separation of anthocyanins and polyphenols from blueberries using macroporous adsorbent resins[J].Journal o f F o o d E n g i n e e r i n g,2 0 1 4,1 2 8:1 6 7-1 7 3.D O I:1 0.1 0 1 6/j.jfoodeng.2013.12.029.
[2]SPECIALE A,CIMINO F,SAIJA A,et al.Bioavailability and molecular activities of anthocyanins as modulators of endothelial function[J].Genes and Nutrition,2014,9(4):1-19.DOI:10.1007/s12263-014-0404-8.
[3]WU T,JIANG Z,YIN J,et al.Anti-obesity effects of artificial planting blueberry(Vaccinium ashei)anthocyanin in high-fat diet-treated mice[J].International Journal of Food Sciences and Nutrition,2016,67(3):257-264.DOI:10.3109/09637486.2016.1146235.
[4]陈文超,刘回民,刘景圣.花青素抗肿瘤活性的研究进展[J].食品研究与开发,2016,37(1):211-215.DOI:10.3969/j.issn.1005-6521.2016.01.051.
[5]NORBERTO S,SILVA S,MEIRELES M,et al.Blueberry anthocyanins in health promotion:a metabolic overview[J].Journal of Functional Foods,2013,5(4):1518-1528.DOI:10.1016/j.jff.2013.08.015.
[6]许相雯.微波辅助萃取蓝莓中花青素及纯化的研究[D].哈尔滨:东北农业大学,2012:14-36.
[7]LI D N,MENG X J,LI B.Profiling of anthocyanins from blueberries produced in China using HPLC-DAD-MS and exploratory analysis by principal component analysis[J].Journal of Food Composition and Analysis,2015,47:1-7.DOI:10.1016/j.jfca.2015.09.005.
[8]BUNEA A,RUGIN?D,SCON?A Z,et al.Anthocyanin determination in blueberry extracts from various cultivars and their antiproliferative and apoptotic properties in B16-F10 metastatic murine melanoma c e l l s[J].P h y t o c h e m i s t r y,2 0 1 3,9 5:4 3 6-4 4 4.D O I:1 0.1 0 1 6/j.phytochem.2013.06.018.
[9]CANUTO G A,OLIVEIRA D R,da CONCEI??O L S,et al.Development and validation of a liquid chromatography method for anthocyanins in strawberry(Fragaria spp.)and complementary studies on stability,kinetics and antioxidant power[J].Food Chemistry,2016,192:566-574.DOI:10.1016/j.foodchem.2015.06.095.
[10]GALLOWAY K R,BRETZ S L,NOVAK M.Paper chromatography and UV-Vis spectroscopy to characterize anthocyanins and investigate antioxidant properties in the organic teaching laboratory[J].Journal of Chemical Education,2014,92(1):183-188.DOI:10.1021/ed400520n.
[11]CORREA-BETANZO J,PADMANABHAN P,CORREDIG M,et al.Complex formation of blueberry(Vaccinium angustifolium)anthocyanins during freeze-drying and its influence on their biological activity[J].Journal of Agricultural and Food Chemistry,2015,63(11):2935-2946.DOI:10.1021/acs.jafc.5b00016.
[12]ARACELI C,MADELOURDES P H,MAELENA P,et al.Chemical studies of anthocyanins:a review[J].Food Chemistry,2009,113(4):859-871.DOI:10.1016/j.foodchem.2008.09.001.
[13]潘利华,王建飞,叶兴乾,等.蓝莓花青素的提取工艺及其免疫调节活性[J].食品科学,2014,35(2):81-86.DOI:10.7506/spkx1002-6630-201402015.
[14]董周永,阿嘎,赵国建,等.大孔树脂对红心萝卜花色苷的纯化[J].食品科学,2011,32(4):21-24.
[15]王二雷.蓝莓花青素高纯提取物的制备技术及诱导肿瘤细胞凋亡作用研究[D].长春:吉林大学,2014:34-93.
[16]刘均玉,许晨,许建中,等.高效半制备液相色谱法从橘皮中分离制备黄酮类化合物[J].中国食品添加剂,2009(2):157-160.
[17]王二雷,陈晶晶,赵叶辉,等.小叶女贞果实花青素组分鉴定及色谱纯化技术[J].农业工程学报,2016,32(4):302-308.DOI:10.11975/j.issn.1002-6819.2016.04.043.
[18]郭丹妮,向灿辉,陈阳,等.葡聚糖凝胶色谱结合高速逆流色谱提取蓝莓中花青素[J].食品工业,2016,37(2):181-183.
[19]BARNES J S,NGUYEN H P,SHEN S,et al.General method for extraction of blueberry anthocyanins and identification using high performance liquid chromatography-electrospray ionization-ion traptime of flight-mass spectrometry[J].Journal of Chromatography A,2009,1216(23):4728-4735.DOI:10.1016/j.chroma.2009.04.032.
[20]WANG E L,YIN Y G,XU C N,et al.Isolation of high-purity anthocyanin mixtures and monomers from blueberries using combined chromatographic techniques[J].Journal of Chromatography A,2014,1327:39-48.DOI:10.1016/j.chroma.2013.12.070.
[21]宁德生,梁小燕,方宏,等.半制备高压液相色谱法制备罗汉果苷Ⅴ标准品[J].食品科学,2010,31(12):137-140.
[22]龚金炎,毛建卫,黄伟素,等.竹茹中苜蓿素对照品的高效液相半制备色谱制备研究[J].中草药,2012,43(5):919-921.
[23]王秋霜,凌彩金,刘淑媚,等.花青素分离纯化及其组分鉴定研究进展[J].食品工业科技,2013,34(3):358-360.DOI:10.13386/j.issn1002-0306.2013.03.054.
[24]CORRALES M,TOEPFL S,BUTZ P,et al.Extraction of anthocyanins from grape by-products assisted by ultrasonics,high hydrostatic pressure or pulsed electric fields:a comparison[J].Innovative Food Science and Emerging Technologies,2008,9(1):85-91.DOI:10.1016/j.ifset.2007.06.002.
[25]BURAN T J,SANDHU A K,LI Z,et al.Adsorption/desorption characteristics and separation of anthocyanins and polyphenols from blueberries using macroporous adsorbent resins[J].Journal o f F o o d E n g i n e e r i n g,2 0 1 4,1 2 8:1 6 7-1 7 3.D O I:1 0.1 0 1 6/j.jfoodeng.2013.12.029.