利用中压制备液相色谱从桑葚中快速制备矢车菊素-3-葡萄糖苷单体
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摘要
单体花色苷的快速大量制备长久以来是花色苷产业化中的难题,而中压制备液相色谱在产业应用中有着很大的开发空间。选取花色苷组分较单一的桑葚为实验原料,经提取分离总花色苷后使用填装有反相C18填料的耐压玻璃柱作为中压制备液相色谱柱,纯化制备矢车菊素-3-葡萄糖苷单体。结果显示:3个色谱分离峰中目的峰(峰2)经高效液相色谱和质谱确证为由矢车菊素-3-葡萄糖苷(cyanidin-3-glucoside,C3G)和矢车菊素-3-芸香糖苷(cyanidin-3-rutinoside,C3R)组成,采用峰面积归一化法计算得到C3G纯度为73.56%;通过对峰2采用切割方式进行收集,C3G纯度达到98%以上,单次收集到C3G单体溶液650 mL。中压制备液相色谱法单次上样量大、步骤简洁、成本低廉,可为矢车菊素-3-葡萄糖苷单体的规模化生产提供一定的参考。
Rapid large-scale preparation of monomeric anthocyanins has always been a problem in the industrialization of anthocyanins. Preparative medium pressure liquid chromatography(MPLC) has great potential for application in the food industry. Cyanidin-3-glucoside(C3G) was puri?ed by preparative MPLC using a C18 column from the anthocyanins extracted from mulberry fruit, containing only a few kinds of anthocyanins. The results showed that a total of three chromatographic peaks were separated. The second peak was con?rmed to be composed of cyanidin-3-rutinoside(C3 R) and C3G by high performance liquid chromatography(HPLC) and mass spectrometry(MS), and C3G accounted for 73.56% of the total amount as calculated by the peak area normalization method. The purity of C3G was over 98% when the peak was collected in several fractions, and a total of 650 mL of C3G was obtained in one operation. The preparative MPLC method has the advantages of large sample loading volume, simple operation and low cost and can provide a useful tool for rapid and large-scale production of C3G.
Rapid large-scale preparation of monomeric anthocyanins has always been a problem in the industrialization of anthocyanins. Preparative medium pressure liquid chromatography(MPLC) has great potential for application in the food industry. Cyanidin-3-glucoside(C3G) was puri?ed by preparative MPLC using a C18 column from the anthocyanins extracted from mulberry fruit, containing only a few kinds of anthocyanins. The results showed that a total of three chromatographic peaks were separated. The second peak was con?rmed to be composed of cyanidin-3-rutinoside(C3 R) and C3G by high performance liquid chromatography(HPLC) and mass spectrometry(MS), and C3G accounted for 73.56% of the total amount as calculated by the peak area normalization method. The purity of C3G was over 98% when the peak was collected in several fractions, and a total of 650 mL of C3G was obtained in one operation. The preparative MPLC method has the advantages of large sample loading volume, simple operation and low cost and can provide a useful tool for rapid and large-scale production of C3G.
引文
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[26]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.
[27]刘静波,陈晶晶,王二雷,等.蓝莓果实中花色苷单体的色谱分离纯化[J].食品科学,2017,38(2):206-213.DOI:10.7506/spkx1002-6630-201702033.
[28]白猛猛.桑葚中花色苷和西番莲中黄酮苷的分离纯化和NMR鉴定[D].重庆:西南大学,2010:30-31.
[29]CHEN L,XIN X L,LAN R,et al.Isolation of cyanidin 3-glucoside from blue honeysuckle fruits by high-speed counter-current chromatography[J].Food Chemistry,2014,152(2):386-390.DOI:10.1016/j.foodchem.2013.11.080.
[30]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.DOI:10.7506/spkx1002-6630-201618012.
[31]汪礼洋.树莓中花色苷的提取、分离、纯化及抗氧化活性研究[D].郑州:河南工业大学,2016:32-33.
[2]MIGUEL M G.Anthocyanins:antioxidant and/or anti-inflammatory activities[J].Journal of Applied Pharmaceutical Science,2011,1(6):7-15.
[3]HASSELLUND S S,FLAA A,KJELDSEN S E,et al.Effects of anthocyanins on cardiovascular risk factors and inflammation in prehypertensive men:a double-blind randomized placebo-controlled crossover study[J].Journal of Human Hypertension,2013,27(2):100-106.DOI:10.1038/jhh.2012.4.
[4]SUN J X,XU W,ZHU C J,et al.Cyanidin-3-O-glucoside protects against 1,3-dichloro-2-propanol-induced reduction of progesterone by upregulation of steroidogenic enzymes and cAMP level in leydig cells[J].Frontiers in Pharmacology,2016,7:1-10.DOI:10.3389/fphar.2016.00399.
[5]凌文华.膳食花色苷与健康[M].北京:科学出版社,2014:25-26.
[6]陈亮,辛秀兰,袁其朋.野生桑葚中花色苷成分分析[J].食品工业科技,2012,33(15):307-310.DOI:10.13386/j.issn1002-0306.2012.15.091.
[7]HASSIMOTTO N M,GENOVESE M I,LAJOLO F M.Absorption and metabolism of cyanidin-3-glucoside and cyanidin-3-rutinoside extracted from wild mulberry(Morus nigra L.)in rats[J].Nutrition Research,2008,28(3):198-207.DOI:10.1016/j.nutres.2007.12.012.
[8]GERASOPOULOS D,STAVROULAKIS G.Quality characteristics of four mulberry(Morus sp)cultivars in the area of Chania,Greece[J].Journal of the Science of Food and Agriculture,1997,73(2):261-264.
[9]曹少谦,刘亮,张超,等.桑葚花色苷的分离纯化及其热降解动力学研究[J].中国食品学报,2015,15(5):54-62.DOI:10.16429/j.1009-7848.2015.05.008.
[10]李燕丽,罗琼仙,杨雪梅,等.‘紫娟’茶花色苷的分离鉴定[J].食品科学,2017,38(12):125-130.DOI:10.7506/spkx1002-6630-201712019.
[11] 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 cells[J].Phytochemistry,2013,95(6):436-444.DOI:10.1016/j.phytochem.2013.06.018.
[12]CHEN L,XIN X L,LAN R,et al.Isolation of cyanidin-3-glucoside from bule honeysuckle fruits by high-speed countercurrent chromatography[J].Food Chemistry,2014,152(2):386-390.DOI:10.1016/j.foodchem.2013.11.080.
[13]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.
[14]CHENG Y J,LIANG Q L,HU P,et al.Combination of normal-phase medium-pressure liquid chromatography and high-performance countercurrent chromatography for preparation of ginsenoside-Ro from Panax ginseng with high recovery and efficiency[J]. Separation and Purification Technology,2010,73(3):397-402.DOI:10.1016/j.seppur.2010.04.029.
[15]WANG Y,ZHANG H C,LIANG H,et al.Purification,antioxidant activity and protein-precipitating capacity of punicalin from pomegranate husk[J].Food Chemistry,2013,138(1):437-443.DOI:10.1016/j.foodchem.2012.10.092.
[16]LU X,ZHENG Z C,MIAO S,et al.Separation of oligosaccharides from lotus seeds via medium-pressure liquid chromatography coupled with ELSD and DAD[J].Scientific Reports,2017,7:1-16.DOI:10.1038/srep44174.
[17]林丹,李春苗,鲜殊,等.中压制备液相色谱快速分离制备儿茶素单体[J].天然产物研究与开发,2013,25(1):92-95;100.DOI:10.16333/j.1001-6880.2013.01.022.
[18]RAMAN G,CHO M,BRODBELT J S,et al.Isolation and purification of closely related Citrus,limonoid glucosides by flash chromatography[J].Phytochemical Analysis,2005,16(3):155-160.DOI:10.1002.pca.835.
[19]李婷,钟英,王芝,等.芦荟苷A、B以及异芦荟色苷D的同时分离纯化[J].天然产物研究与开发,2011,23(5):878-881;945.DOI:10.16333/j.1001-6880.2011.05.017.
[20]WANG Y,LIU H,SHEN L F,et al.Isolation and purification of six iridoid glycosides from gardenia jasminoides fruit by mediumpressure liquid chromatography combined with macroporous resin chromatography[J].Journal of Separation Science,2015,38(23):4119-4126.DOI:10.1002/jssc.201500705.
[21]凌文华.一种从黑米中分离制备高纯度花色苷单体的方法:200910194366.6[P].2010-06-09[2017-12-08].
[22]FR?YTLOG C,SLIMESTAD R,ANDERSEN?M.Combination of chromatographic techniques for the preparative isolation of anthocyanins:applied on blackcurrant(Ribes nigrum)fruits[J].Journal of Chromatography A,1998,825(1):89-95.DOI:10.1016/S0021-9673(98)00673-6.
[23]孙建霞.高压脉冲电场对矢车菊素-3-葡萄糖苷和矢车菊素-3-槐糖苷的稳定性影响研究[D].北京:中国农业大学,2010:28-29.
[24] 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,2016,47(6):1-7.DOI:10.1016/j.jfca.2015.09.005.
[25] WU X L, PRIOR R L. Systematic identification and characterization of anthocyanins by HPLC-ESI-MS/MS in common foods in the United States:fruits and berries[J].Journal of Agricultural and Food Chemistry,2005,53(7):2589-2599.DOI:10.1021/jf048068b.
[26]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.
[27]刘静波,陈晶晶,王二雷,等.蓝莓果实中花色苷单体的色谱分离纯化[J].食品科学,2017,38(2):206-213.DOI:10.7506/spkx1002-6630-201702033.
[28]白猛猛.桑葚中花色苷和西番莲中黄酮苷的分离纯化和NMR鉴定[D].重庆:西南大学,2010:30-31.
[29]CHEN L,XIN X L,LAN R,et al.Isolation of cyanidin 3-glucoside from blue honeysuckle fruits by high-speed counter-current chromatography[J].Food Chemistry,2014,152(2):386-390.DOI:10.1016/j.foodchem.2013.11.080.
[30]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.DOI:10.7506/spkx1002-6630-201618012.
[31]汪礼洋.树莓中花色苷的提取、分离、纯化及抗氧化活性研究[D].郑州:河南工业大学,2016:32-33.