超高效液相色谱法分析稻米酚酸化合物组分及其含量

doi:10.3864/j.issn.0578-1752.2015.09.05

摘要/Abstract

摘要： 【目的】稻米酚酸化合物是天然抗氧化物的重要来源，本研究建立糙米和精米中酚酸化合物的超高效液相色谱的定性定量方法，并分析其在籽粒中的分布特征。【方法】以没食子酸、原儿茶酸、龙胆酸、对羟基苯甲酸、绿原酸、香草酸、咖啡酸、丁香酸、对香豆酸、阿魏酸、芥子酸、异阿魏酸、2-羟基肉桂酸和反式肉桂酸14种酚酸化合物标准品，对糙米和精米中酚酸进行定性和定量分析。色谱柱为Agilent ZORBAX Eclipse Plus C18（2.1 mm×50 mm，1.8 μm-Micron），流动相为0.1%乙酸水溶液（A）/乙腈（B），柱温30℃，流速为0.5 mL·min^-1，采用梯度洗脱，洗脱程序为0—1 min，8%—10%B；1—2.5 min，10%—13%B；2.5—5.5 min，13%B；5.5—6 min，13%—21%B ；6—6.5 min，21%—27%B；6.5—7.5 min，27%—50%B；7.5—9 min，50%—100%B；9—12 min，100%B；12—12.5 min，100%—8%B。检测波长为280和325 nm。【结果】14种酚酸在8 min内完全分离，经质谱鉴定，精米及糙米中检测出的物质共有11种，分别是没食子酸、龙胆酸、对羟基苯甲酸、香草酸、咖啡酸、丁香酸、对香豆酸、阿魏酸、芥子酸、异阿魏酸和反式肉桂酸。可进行定量分析的酚酸共8种，分别是对羟基苯甲酸、香草酸、丁香酸、对香豆酸、阿魏酸、芥子酸、异阿魏酸和反式肉桂酸，线性范围为5—220 μg·mL^-1 (R²=0.9994—0.9999)，检出限为0.002—0.03 μg·mL^-1，定量限为0.004—0.08 μg·mL^-1，回收率为84.11%—114.43%。对羟基苯甲酸、香草酸、丁香酸、对香豆酸、阿魏酸、芥子酸、异阿魏酸和反式肉桂酸在精米和糙米中的含量差异显著，其中对羟基苯甲酸、香草酸、丁香酸、对香豆酸、阿魏酸、芥子酸和反式肉桂酸集中存在于胚乳外层中，精米/糙米含量百分比变幅为4.52%—16.73%。而异阿魏酸在稻米籽粒中分布较均匀，其精米/糙米含量百分比达到45.86%。【结论】该方法简便、快速、准确、可靠，不仅适合稻米中酚酸化合物的含量测定，而且对于其他谷物中酚酸化合物含量的测定也具有一定参考价值。

关键词: 酚酸, 超高效液相色谱, 糙米, 精米

Abstract: 【Objective】 Phenolic acids in rice grain are the main source for natural antioxidants. This study aims to develop an ultra-high performance liquid chromatography method for the determination of phenolic acids in brown rice and milled rice, and analyze the tissue distribution of phenolic acids in rice grain. 【Method】Standard compounds of the target phenolic acids, including gallic acid, protocatechuic acid, gentisic acid, 2,5-dihydroxybenzoic acid, 4-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, ρ-coumaric acid, ferulic acid, sinapic acid, isoferulic acid, 2-hydroxycinnamic acid, and trans-cinnamic acid, were purchased from Sigma-Aldrich. The liquid-chromatographic separation was performed on Agilent ZORBAX Eclipse Plus C18 (2.1 mm×50 mm, 1.8 μm-Micron) column kept at 30℃, using acetonitrile/acetic acid/water mixture as the mobile phase with a flow rate of 0.5 mL·min^-1 through gradient elution: 0-1 min, 8%-10%B; 1-2.5 min, 10%-13%B; 2.5-5.5 min, 13%B; 5.5-6 min, 13%-21%B; 6-6.5 min, 21%-27%B; 6.5-7.5 min, 27%-50%B; 7.5-9 min, 50%-100%B; 9-12 min, 100%B; 12-12.5 min, 100%-8%B. And the detection wavelength was set at 280 nm and 325 nm. 【Result】 Results showed that a good separation of 14 phenolic acids was achieved within 8 min. Eleven phenolic acids were detected in brown and milled rice by mass spectrometry including gallic acid, gentisic acid, 4-hydroxybenzoic acid, vanillic acid, syringic acid, ρ-coumaric acid, ferulic acid, sinapic acid, isoferulic acid, trans-cinnamic acid. With eight of them can be used for quantitative analysis, including 4-hydroxybenzoic acid, vanillic acid, syringic acid, ρ-coumaric acid, ferulic acid, sinapic acid, isoferulic acid, trans-cinnamic acid. The proposed method exhibited a linear range of 5-220 μg·mL^-1(R²=0.9994-0.9999), with limits of quantification ranging from 0.002 to 0.03 μg·mL^-1, recovery rates from 84.11% to 114.43%. A significant difference existed in the distribution of 4-hydroxybenzoic acid, vanillic acid, syringic acid, ρ-coumaric acid, ferulic acid, sinapic acid, isoferulic acid, trans-cinnamic acid between brown (B) and milled rice (M). With 4-hydroxybenzoic acid, vanillic acid, syringic acid, ρ-coumaric acid, ferulic acid, sinapic acid, trans-cinnamic acid mainly concentrated in the outer endosperm, and the ratio of M to B was 4.52%-16.72%. By contrast, a considerable part of isoferulic acid accumulated in the endosperm, having a M/B value of 45.86%. 【Conclusion】 It was proved that the proposed method is simple, rapid, accurate, reliability, and should be used for the determination of phenolic acids in rice grain as well as other cereal grains.

Key words: phenolic acids, ultra-high performance liquid chromatography, brown rice, milled rice

张娜，王国祥，Abacar Jose Daniel，刘正辉，丁承强，唐设，李刚华，王绍华，丁艳锋. 超高效液相色谱法分析稻米酚酸化合物组分及其含量[J]. 中国农业科学, 2015, 48(9): 1718-1726.

ZHANG Na, WANG Guo-xiang, Abacar Jose Daniel, LIU Zheng-hui, DING Cheng-qiang,TANG She, LI Gang-hua, WANG Shao-hua, DING Yan-feng. Determination of Phenolic Acids in Rice by Ultra-High Performance Liquid Chromatography[J]. Scientia Agricultura Sinica, 2015, 48(9): 1718-1726.

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参考文献

[1] Kahkonen M P, Hopia A I, Vuorela H J, Rauha J P, Pihlaja K, Kujala T S, Heinonen M. Antioxidant activity of plant extracts containing phenolic compounds. Journal of Agricultural and Food Chemistry, 1999, 47: 3954-3962.

[2] Slavin J L. Mechanisms for the impact of whole grain foods on cancer risk. Journal of the American College of Nutrition, 2000, 19: 300-307.

[3] Halvorsen B L, Carlsen M H, Phillips K M, Bohn S K, Holte K, Jacobs D R, Blomhoff R. Content of redox-active compounds (ie, antioxidants) in foods consumed in the United States. American Journal of Clinical Nutrition, 2006, 84: 95-135.

[4] Adom K K, Sorrells M E, Liu R H. Phytochemicals and antioxidant activity of milled fractions of different wheat varieties. Journal of Agriculture and Food Chemistry, 2005, 53: 2297-2306.

[5] Zielinski H, Kozlowska H. Antioxidant activity and total phenolics in selected cereal grains and their different morphological fractions. Journal of Agricultural and Food Chemistry, 2000, 48: 2008-2016.

[6] Hall C. Antioxidants in Food: Practical Applications. England: Woodhead Publishing Limited, 2001: 159-209.

[7] Hao M L, Beta T. Qualitative and quantitative analysis of the major phenolic compounds as antioxidants in barley and flaxseed hulls using HPLC/MS/MS. Journal of the Science of Food and Agriculture, 2012, 92: 2062-2068.

[8] Sani I M, Iqbal S, Chan K W, Ismail M. Effect of acid and base catalyzed hydrolysis on the yield of phenolics and antioxidant activity of extracts from germinated brown rice (GBR) . Molecules, 2012, 17: 7584-7594.

[9] Jun H I, Song G S, Yang E I, Youn Y, Kim Y S. Antioxidant activities and phenolic compounds of pigmented rice bran extracts. Journal of Food Science, 2012, 77: 759-764.

[10] Gratacós-Cubarsí M, Ribas-Agustí A, García-Regueiro J A, Castellari M. Simultaneous evaluation of intact glucosinolates and phenolic compounds by UPLC - DAD-MS/MS in Brassica oleracea L. var. botrytis. Food Chemistry, 2010, 121: 257-263.

[11] Lou Z, Wang H, Zhu S, Zhang M, Gao Y, Ma C, Wang Z. Improved extraction and identification by ultra performance liquid chromatography tandem mass spectrometry of phenolic compounds in burdock leaves. Journal of Chromatography A, 2010, 1217: 2441-2446.

[12] Yao Y, Cheng X Z, Wang L X, Wang S H, Ren G. Major phenolic compounds, antioxidant capacity and antidiabetic potential of rice bean(Vigna umbellata L.) in China. International Journal of Molecular Sciences, 2012, 13: 2707-2716.

[13] Irakli M N, Samanidou V F, Biliaderis C G, Papadoyannis I N. Development and validation of an HPLC-method for determination of free and bound phenolic acids in cereals after solid-phase extraction. Food Chemistry, 2012, 134: 1624-1632.

[14] Zhang Y, Wang L, Yao Y, Yan J, He Z. Phenolic acid profiles of Chinese wheat cultivars. Journal of Cereal Science, 2012, 56: 629-635.

[15] Zhou Z, Robards K, Helliwell S, Blanchard C. The distribution of phenolic acids in rice. Food Chemistry, 2004, 87: 401-406.

[16] Ti H, Li Q, Zhang R, Zhang M, Deng Y, Wei Z, Chi J, Zhang Y. Free and bound phenolic profiles and antioxidant activity of milled fractions of different indica rice varieties cultivated in southern China. Food Chemistry, 2014, 159: 166-174.

[17] Min B, Gu L, McClung A M, Bergman C J, Chen M-H. Free and bound total phenolic concentrations, antioxidant capacities, and profiles of proanthocyanidins and anthocyanins in whole grain rice (Oryza sativa L.) of different bran colours. Food Chemistry, 2012, 133: 715-722.

[18] Adom K K, Liu R H. Antioxidant activity of grains. Journal of Agricultural and Food Chemistry, 2002, 50: 6182-6187.

[19] Nardini M, Ghiselli A. Determination of free and bound phenolic acids in beer. Food Chemistry, 2004, 84: 137-143.

[20] Wang C J, Zhao J B, Chen F, Cheng Y Q, Guo A H. Separation, identification, and quantitation of phenolic acids in Chinese waxberry (Myrica Rubra) juice by HPLC-PDA-ESI-MS. Journal of Food Science, 2012, 77: 272-277.

[21] Choi M Y, Chai C, Park J H, Lim J, Lee J, Kwon S W. Effects of storage period and heat treatment on phenolic compound composition in dried Citrus peels (Chenpi) and discrimination of Chenpi with different storage periods through targeted metabolomic study using HPLC-DAD analysis. Journal of Pharmaceutical and Biomedical Analysis, 2011, 54: 638-645.

[22] Vichapong J, Sookserm M, Srijesdaruk V, Swatsitang P, Srijaranai S. High performance liquid chromatographic analysis of phenolic compounds and their antioxidant activities in rice varieties. Lebensmittel- Wissenschaft Und-Technologie-Food Science and Technology, 2010, 43: 1325-1330.

[23] Tian S, Nakamura K, Cui T, Kayahara H. High-performance liquid chromatographic determination of phenolic compounds in rice. Journal of Chromatography A, 2005, 1063: 121-128.

[24] Tian S, Nakamura K, Kayahara H. Analysis of phenolic compounds in white rice, brown rice, and germinated brown rice. Journal of Agricultural and Food Chemistry, 2004, 52: 4808-4813.

[25] Wang L, Yao Y, He Z, Wang D, Liu A, Zhang Y. Determination of phenolic acid concentrations in wheat flours produced at different extraction rates. Journal of Cereal Science, 2013, 57: 67-72.

[26] Alrahmany R, Avis T J, Tsopmo A. Treatment of oat bran with carbohydrases increases soluble phenolic acid content and influences antioxidant and antimicrobial activities. Food Research International, 2013, 52: 568-574.

[27] Shao Y, Xu F, Sun X, Bao J, Beta T. Identification and quantification of phenolic acids and anthocyanins as antioxidants in bran, embryo and endosperm of white, red and black rice kernels (Oryza sativa L.). Journal of Cereal Science, 2014, 59: 211-218.

[28] Butsat S, Siriamornpun S. Antioxidant capacities and phenolic compounds of the husk, bran and endosperm of Thai rice. Food Chemistry, 2010, 119: 606-613.

[29] Liu R H. Whole grain phytochemicals and health. Journal of Cereal Science, 2007, 46: 207-219.

[30]Liu Z H, Wang H Y, Wang X E, Zhang G P, Chen P D, Liu D J. Phytase activity, phytate, iron, and zinc contents in wheat pearling fractions and their variation across production locations. Journal of Cereal Science, 2007, 45: 319-326.

[31] Liu Z H, Wang H Y, Wang X E, Xu H, Gao D, Zhang G, Chen P, Liu D. Effect of wheat pearling on flour phytase activity, phytic acid, iron, and zinc content. Lebensmittel-Wissenschaft Und-Technologie-Food Science and Technology, 2008, 41: 521-527.

[32] Antoine C, Peyron S, Lullien-Pellerin V, Abecassis J, Rouau X. Wheat bran tissue fractionation using biochemical markers. Journal of Cereal Science, 2004, 39: 387-393.

[33] Hemery Y, Lullien-Pellerin V, Rouau X, Abecassis J, Samson M F, Åman P, von Reding W, Spoerndli C, Barron C. Biochemical markers: efficient tools for the assessment of wheat grain tissue proportions in milling fractions. Journal of Cereal Science, 2009, 49: 55-64.

[34] Barron C, Samson M F, Lullien-Pellerin V, Rouau X. Wheat grain tissue proportions in milling fractions using biochemical marker measurements: application to different wheat cultivars. Journal of Cereal Science, 2011, 53: 306-311.

[35] Liu I M, Hsu F L, Chen C F, Cheng J T. Antihyperglycemic action of isoferulic acid in streptozotocin-induced diabetic rats. British Journal ofPharmacology, 2000, 129: 631-636.

[36] Liu I M, Chen W C, Cheng J T. Mediation of beta-endorphin by isoferulic acid to lower plasma glucose in streptozotocin-induced diabetic rats. Journal of Pharmacologyand Experimental Therapeutics, 2003, 307: 1196-1204.