Name: a generalized method for determining free soluble phenolic acid composition and antioxidant capacity of cereals and legumes
Uploaded: 2022-06-10T13:00:00
Description: Watch this Scientific Journal Video about A Generalized Method for Determining Free Soluble Phenolic Acid Composition and Antioxidant Capacity of Cereals and Legumes at JoVE.com

作者感谢Alison Ser女士和Hannah Oduro-Obeng女士的技术支持，以及Janice Fajardo女士和Miguel del Rosario先生的视频编辑支持。

1. 样品类型
<ol><li>本研究使用五个全谷物样品，包括两种谷物（例如硬粒小麦和黄玉米）和三种豆类（例如黑眼豇豆，大豆和红芸豆）。</li>
	<li>使用咖啡研磨机将50克每种谷物一式三份研磨成面粉，并通过500μm筛子。</li>
	<li>将它们储存在-20°C。</li>
</ol>2. 样品制备
<ol><li>
以干物质含量的测定和干重表达为基础 注：根据AOAC （2000）<sup ...

<ol>
	<li>Huitu, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silicon,+endophytes+and+secondary+metabolites+as+grass+defenses+against+mammalian+herbivores.">Silicon, endophytes and secondary metabolites as grass defenses against mammalian herbivores.</a> Frontiers in Plant Science. 5, 478 (2014).</li><li>Joshi, J. R., Burdman, S., Lipsky, A., Yariv, S., Yedidia, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+phenolic+acids+affect+the+virulence+of+Pectobacterium+aroidearum+and+P.+carotovorum+ssp.+brasiliense+via+quorum+sensing+regulation.">Plant phenolic acids affect the virulence of Pectobacterium aroidearum and P. carotovorum ssp. brasiliense via quorum sensing regulation.</a> Molecular Plant Pathology. 17 (4), 487-500 (2016).</li><li>Dykes, L., Rooney, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenolic+compounds+in+cereal+grains+and+their+health+benefits.">Phenolic compounds in cereal grains and their health benefits.</a> Cereal Foods World. 52 (3), 105-111 (2007).</li><li>Xiang, J., Apea-Bah, F. B., Ndolo, V. U., Katundu, M. C., Beta, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profile+of+phenolic+compounds+and+antioxidant+activity+of+finger+millet+varieties.">Profile of phenolic compounds and antioxidant activity of finger millet varieties.</a> Food Chemistry. 275, 361-368 (2019).</li><li>Qiu, Y., Liu, Q., Beta, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidant+properties+of+commercial+wild+rice+and+analysis+of+soluble+and+insoluble+phenolic+acids.">Antioxidant properties of commercial wild rice and analysis of soluble and insoluble phenolic acids.</a> Food Chemistry. 121 (1), 140-147 (2010).</li><li>Beverly, R. L., Motarjemi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Encyclopedia of Food Safety. 3, 309-314 (2014).</li><li>FAO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food+Outlook+-+Biannual+report+on+global+food+markets.">Food Outlook - Biannual report on global food markets.</a> Food and Agriculture Organization. , (2020).</li><li>Erbersdobler, H. F., Barth, C. A., Jahreis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Legumes+in+human+nutrition.+Nutrient+content+and+protein+quality+of+pulses.">Legumes in human nutrition. Nutrient content and protein quality of pulses.</a> Ernahrungs Umschau. 64 (9), 134-139 (2017).</li><li>Due&#241;as, M., Hern&#225;ndez, T., Estrella, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+in+vitro+antioxidant+capacity+of+the+seed+coat+and+the+cotyledon+of+legumes+in+relation+to+their+phenolic+contents.">Assessment of in vitro antioxidant capacity of the seed coat and the cotyledon of legumes in relation to their phenolic contents.</a> Food Chemistry. 98 (1), 95-103 (2006).</li><li>Khang, D. T., Dung, T. N., Elzaawely, A. A., Xuan, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenolic+profiles+and+antioxidant+activity+of+germinated+legumes.">Phenolic profiles and antioxidant activity of germinated legumes.</a> Foods. 5 (2), 27 (2016).</li><li>Hefni, M. E., Amann, L. S., Witth&#246;ft, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+HPLC-UV+method+for+the+quantification+of+phenolic+acids+in+cereals.">A HPLC-UV method for the quantification of phenolic acids in cereals.</a> Food Analytical Methods. 12 (12), 2802-2812 (2019).</li><li>AOAC. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Official Methods of Analysis. 17th edn. , (2000).</li><li>Apea-Bah, F. B., Head, D., Scales, R., Bazylo, R., Beta, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrothermal+extraction,+a+promising+method+for+concentrating+phenolic+antioxidants+from+red+osier+dogwood+(Cornus+stolonifer)+leaves+and+stems.">Hydrothermal extraction, a promising method for concentrating phenolic antioxidants from red osier dogwood (Cornus stolonifer) leaves and stems.</a> Heliyon. 6 (10), 05158 (2020).</li><li>Apea-Bah, F. B., Minnaar, A., Bester, M. J., Duodu, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sorghum-cowpea+composite+porridge+as+a+functional+food,+Part+II:+Antioxidant+properties+as+affected+by+simulated+in+vitro+gastrointestinal+digestion.">Sorghum-cowpea composite porridge as a functional food, Part II: Antioxidant properties as affected by simulated in vitro gastrointestinal digestion.</a> Food Chemistry. 197, 307-315 (2016).</li><li>Robbins, R. J., Bean, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+quantitative+high-performance+liquid+chromatography-photodiode+array+detection+measurement+system+for+phenolic+acids.">Development of a quantitative high-performance liquid chromatography-photodiode array detection measurement system for phenolic acids.</a> Journal of Chromatography A. 1038 (1-2), 97-105 (2004).</li><li>Singleton, V. L., Rossi, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetry+of+total+phenolics+with+phosphomolybdic-phosphotungstic+acid+reagents.">Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents.</a> American Journal of Enology and Viticulture. 16, 144-158 (1965).</li><li>Prior, R. L., Wu, X., Schaich, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+methods+for+the+determination+of+antioxidant+capacity+and+phenolics+in+foods+and+dietary+supplements.">Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements.</a> Journal of Agricultural and Food Chemistry. 53 (10), 4290-4302 (2005).</li><li>Ainsworth, E. A., Gillespie, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+total+phenolic+content+and+other+oxidation+substrates+in+plant+tissues+using+Folin-Ciocalteu+reagent.">Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin-Ciocalteu reagent.</a> Nature Protocols. 2 (4), 875-877 (2007).</li><li>Waterhouse, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+total+phenolics.">Determination of total phenolics.</a> Current Protocols in Food Analytical Chemistry. 1, 1-8 (2002).</li><li>Huang, D., Ou, B., Prior, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemistry+behind+antioxidant+capacity+assays.">The chemistry behind antioxidant capacity assays.</a> Journal of Agricultural and Food Chemistry. 53 (6), 1841-1856 (2005).</li><li>Esterbauer, H., W&#228;g, G., Puhl, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+peroxidation+and+its+role+in+atherosclerosis.">Lipid peroxidation and its role in atherosclerosis.</a> British Medical Bulletin. 49 (3), 566-576 (1993).</li><li>Esterbauer, H., Gebicki, J., Puhl, H., J&#252;rgens, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+lipid+peroxidation+and+antioxidants+in+oxidative+modification+of+LDL.">The role of lipid peroxidation and antioxidants in oxidative modification of LDL.</a> Free Radical Biology and Medicine. 13 (4), 341-390 (1992).</li><li>Apea-Bah, F. B., Serem, J. C., Bester, M. J., Duodu, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenolic+composition+and+antioxidant+properties+of+Koose,+a+deep-fat+fried+cowpea+cake.">Phenolic composition and antioxidant properties of Koose, a deep-fat fried cowpea cake.</a> Food Chemistry. 237, 247-256 (2017).</li></ol>

全谷物被选为具有代表性的谷物和豆类，在世界范围内具有广泛的食品应用。虽然每种谷物的品种之间可能存在差异，但本研究的重点是展示一种用于全谷物的游离酚酸提取和分析的广义方法。通过大幅减少样品和溶剂的量来修改提取方法，以减少进行此类实验时释放到环境中的化学物质的量。这种改性还可以从毫克量的全谷物中提取酚醛。
HPLC分析产生样品中组成酚酸的色?...

酚酸是植物中研究的最重要植物化学物质之一，因为它们在植物防御食草动物和真菌感染以及维持植物组织中的结构支撑和完整性方面发挥着至关重要的作用1，2。它们在谷物的麸皮和豆类的种皮中含量丰富3.在结构上，它们分为两组：羟基苯甲酸（图1）和羟基肉桂酸（图2）。谷物和豆类中常见的羟基苯甲酸包括没食子酸、 对羟基苯甲酸、2，4-二羟基苯甲酸、原儿茶酸、香草酸和丁香酸，而常见的羟基肉桂酸包括咖啡酸、 对香豆酸、阿魏酸和芥子酸3。酚酸还具有抗氧化特性，因为它们能够清除导致脂肪氧化酸败的自由基，并在生理系统中启动和传播自由基诱导的氧化应激4，5。由于作为抗氧化剂的这种重要的生理作用，它们是最近研究的主题。这是因为当作为植物性食物的成分食用时，它们可以发挥抗氧化保护作用。
谷物和谷物制品是全世界人类和动物的主要碳水化合物食物来源6.谷物包括小麦，大米，玉米（玉米），大麦，小黑麦，小米和高粱。其中，玉米是利用量最大的，2019/2020年全球利用率估计为11.357亿吨，其次是小麦，同期全球估计利用率为7.575亿吨。谷物食品是消费者的重要能量来源，因为它们是碳水化合物的丰富来源。它们还提供一些蛋白质，脂肪，纤维，维生素和矿物质6。除了它们的营养价值外，谷物还是植物化学抗氧化剂的良好来源，特别是酚酸，它们具有保护生理系统免受自由基诱导的氧化损伤的潜力3。豆类也是营养物质的良好来源，蛋白质含量通常高于谷物。它们还含有维生素和矿物质，用于制备各种食物8。此外，豆类是各种植物化学抗氧化剂的良好来源，包括酚酸，类黄酮，花青素和原花青素9，10。不同品种的谷物和豆类可能具有不同的酚酸组成。因此，有必要研究谷物和豆类的酚酸组成及其品种，以了解它们在酚类抗氧化剂方面的潜在健康益处。
已经报道了许多用于测量谷物和豆类谷物中酚酸含量并确定其抗氧化活性的测定方法。全谷物酚酸最常用的分析方法是分光光度法和液相色谱法11。这项工作的目的是展示一种用于测定游离可溶性酚酸组成的广义高压液相色谱方法，以及用于测定某些全谷物谷物和豆类的总酚含量和抗氧化能力的分光光度法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL Falcon conical centrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-527-90</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Amber glass ID Surestop vial</td>
			<td>Thermo Scientific</td>
			<td>C5000-2W</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Amber microcentrifuge tubes</td>
			<td>VWR</td>
			<td>20170-084</td>
			<td></td>
		</tr>
		<tr>
			<td>2,2&#8242;-Azobis(2-amidinopropane) dihydrochloride (AAPH)</td>
			<td>Sigma-Aldrich</td>
			<td>440914-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>2,2&#39;-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) (C18H18N4O6S4) &#8805;98%,</td>
			<td>Sigma Aldrich</td>
			<td>A1888-2G</td>
			<td></td>
		</tr>
		<tr>
			<td>2,2-Diphenyl-1pikrylhydrazyl (DPPH) (C18H12N5O6)</td>
			<td>Sigma Aldrich</td>
			<td>D913-2</td>
			<td></td>
		</tr>
		<tr>
			<td>6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (C14H18O4), &#8805;98%</td>
			<td>Fluka Chemika</td>
			<td>56510</td>
			<td></td>
		</tr>
		<tr>
			<td>9 mm Autosampler Vial Screw Thread Caps</td>
			<td>Thermo Scientific</td>
			<td>60180-670</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well flat bottom plates</td>
			<td>Fisher Scientific</td>
			<td>12565501</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent BioTek ELx800 microplate reader</td>
			<td>Fisher Scientific</td>
			<td>BT-ELX800NB</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent BioTek Precision 2000 96/384 Automated Microplate Pipetting System</td>
			<td>Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent BioTek FLx800 Microplate Fluorescence Reader</td>
			<td>Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical balance SI-114</td>
			<td>Denver Instrument</td>
			<td>SI-114.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Autosampler, Waters 717 Plus</td>
			<td>Waters</td>
			<td>WAT078900</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 3 mL syringe Luer-Lok Tip</td>
			<td>BD</td>
			<td>309657</td>
			<td></td>
		</tr>
		<tr>
			<td>Bransonic ultrasonic cleaner, Branson 5510</td>
			<td>Millipore Sigma</td>
			<td>Z245143</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning LSE Vortex Mixer</td>
			<td>Corning</td>
			<td>6775</td>
			<td></td>
		</tr>
		<tr>
			<td>Durapore Filter (0.45 &#181;m PVDF Membrane)</td>
			<td>Merck Millipore Ltd</td>
			<td>HVLP04700&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Durapore Membrane Filters (0.45 &#181;m HV)</td>
			<td>Merck Millipore Ltd</td>
			<td>HVHP04700</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Research&#160;plus, 0.5-10 &#181;L</td>
			<td>Eppendorf</td>
			<td>3123000020</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Research&#160;plus, 0.5-5 mL</td>
			<td>Eppendorf</td>
			<td>3123000071</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Research&#160;plus, 100-1000 &#181;L</td>
			<td>Eppendorf</td>
			<td>3123000063</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Research&#160;plus, 10-100 &#181;L</td>
			<td>Eppendorf</td>
			<td>3123000047</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl acetate, HPLC grade</td>
			<td>Fisher Chemical</td>
			<td>E195-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferulic acid standard</td>
			<td>Sigma Aldrich</td>
			<td>128708-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein</td>
			<td>Fisher Scientific</td>
			<td>AC119245000</td>
			<td></td>
		</tr>
		<tr>
			<td>Folin &#38; Ciocalteu phenol reagent</td>
			<td>Sigma Aldrich</td>
			<td>F9252</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid, 99%</td>
			<td>Acros Organics, Janssen Pharmaceuticalaan 3a</td>
			<td>27048-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Gallic acid standard</td>
			<td>Sigma</td>
			<td>G7384</td>
			<td></td>
		</tr>
		<tr>
			<td>High performance liquid chromatograph (HPLC), Waters 2695</td>
			<td>Waters</td>
			<td>960402</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol, HPLC grade</td>
			<td>Fisher Chemical</td>
			<td>A452-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro pipet tips, 0.5-10 &#181;L</td>
			<td>Fisherbrand</td>
			<td>21-197-2F</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge Sorvall Legend Micro 21 centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75002435</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel micropipette, Proline Plus, 30-300 &#181;L</td>
			<td>Sartorius</td>
			<td>728240</td>
			<td></td>
		</tr>
		<tr>
			<td>Photodiode array detector, Waters 2996</td>
			<td>Waters</td>
			<td>720000350EN</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet tips, 1000 &#181;L</td>
			<td>VWR</td>
			<td>83007-382</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet tips, 1-5 mL</td>
			<td>VWR</td>
			<td>82018-840</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium persulfate (K2S2O8), &#8805;99.0%</td>
			<td>Sigma Aldrich</td>
			<td>216224-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic anhydrous (K2HPO4)</td>
			<td>Fisher Scientific</td>
			<td>P288-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Fisher Scientific</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr>
			<td>PYREX 250 mL Short Neck Boiling Flask, Round Bottom</td>
			<td>Corning</td>
			<td>4321-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Reversed phase C18 Analytical Column (100 x 3 mm) Accucore aQ</td>
			<td>Thermo Scientific</td>
			<td>17326-103030</td>
			<td></td>
		</tr>
		<tr>
			<td>Roto evaporator, IKA RV 10</td>
			<td>IKA</td>
			<td>&#160;0010005185</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium carbonate (NaCO3) anhydrous</td>
			<td>Fisher Chemical</td>
			<td>S263-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Mallinckrodt AR&#174;</td>
			<td>7581</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic anhydrous (Na2HPO4)</td>
			<td>Fisher Scientific</td>
			<td>BP332-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic anhydrous (NaH2PO4)</td>
			<td>Fisher bioreagents</td>
			<td>BP329-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Standardization pipet tips 0-200&#181;L</td>
			<td>Fisherbrand</td>
			<td>02-681-134</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Driven Filter unit (0.22 &#181;m)</td>
			<td>&#160;Millex&#174;-GV</td>
			<td>SLGVR04NL</td>
			<td></td>
		</tr>
		<tr>
			<td>Target micro-serts vial insert (400 &#181;L)</td>
			<td>Thermo Scientific</td>
			<td>C4011-631</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure water (Direct Q-3 UV system with pump)</td>
			<td>Millipore</td>
			<td>ZRQSVP030</td>
			<td></td>
		</tr>
	</tbody>
</table>

a generalized method for determining free soluble phenolic acid composition and antioxidant capacity of cereals and legumes

酚酸是一类同时具有酚基和羧基的有机化合物。它们存在于谷物中，并集中在谷物的麸皮或豆类的种皮中。它们具有抗氧化特性，近年来引起了人们对其潜在的抗氧化保护健康功能的研究兴趣。这项工作提出了一种从全谷物中提取游离可溶性酚酸的广义方法，并分析了它们的抗氧化能力。使用五个全谷物样本，包括两种谷物（小麦和黄玉米）和三种豆类（豇豆，芸豆和大豆）。将谷物碾磨成面粉，并使用甲醇水溶液提取其游离可溶性酚酸。然后使用高压液相色谱仪（HPLC）鉴定化合物。Folin-Ciocalteu方法测定其总酚含量，同时使用DPPH自由基清除能力，Trolox等效抗氧化能力（TEAC）和氧自由基吸收能力（ORAC）测定测定其抗氧化能力。鉴定出的酚酸包括香草酸、咖啡酸、对香豆酸和阿魏酸。香草酸仅在豇豆中鉴定，而咖啡酸仅在芸豆中鉴定。在黄玉米、豇豆和大豆中鉴定出对香豆酸，而在所有样品中鉴定出阿魏酸。阿魏酸是确定的主要酚酸。样品中酚酸的总浓度按以下顺序下降：大豆>豇豆>黄玉米=芸豆>小麦。总抗氧化能力（DPPH，TEAC和ORAC测定值的总和）下降如下：大豆>芸豆>黄玉米=豇豆>小麦。这项研究得出结论，HPLC分析以及DPPH，TEAC和ORAC测定提供了有关全谷物酚酸组成和抗氧化特性的有用信息。

表2 显示了在谷物和豆类谷物中鉴定的酚酸。根据现有的正品标准，在样品中鉴定出四种酚酸，它们是：香草酸，咖啡酸， 对香豆酸和阿魏酸。香草酸是一种羟基苯甲酸，而其他三种是羟基肉桂酸。香草酸仅在黑眼豇豆中鉴定，而咖啡酸仅在芸豆中鉴定。 在黄玉米，豇豆豆和大豆中鉴定出对香豆酸。按干重计算，其浓度范围在7.57至12.48微克/克面粉之间，黄玉米的最低值...

Watch this Scientific Journal Video about A Generalized Method for Determining Free Soluble Phenolic Acid Composition and Antioxidant Capacity of Cereals and Legumes at JoVE.com

A Generalized Method for Determining Free Soluble Phenolic Acid Composition and Antioxidant Capacity of Cereals and Legumes

酚酸是存在于全谷物中的重要植物化学物质。它们具有生物活性特性，如抗氧化保护功能。这项工作旨在报告HPLC鉴定的广义方法，总酚含量估计以及谷物和豆类中酚酸抗氧化能力的测定。

一种测定谷物和豆类游离可溶性酚酸组成和抗氧化能力的广义方法

Phenolic acids are a class of organic compounds that bear both a phenolic group, and a carboxylic group. They are found in grains and concentrate in the ...

一种测定谷物和豆类的游离可溶性酚酸组成和抗氧化能力的广义方法。全谷物，包括谷物和豆类，构成了人类饮食的重要组成部分。它们与人类的营养相关性早已得到认可。然而，最近，已经报道了它们的抗氧化保护健康益处。在谷物的外层和豆类的种皮中发现的酚酸有助于全谷物的抗氧化特性。它们清除对生物分子造成氧化损伤的自由基。在全谷物中发现的两类酚酸是羟基苯甲酸和羟基肉桂酸。本研究为提取全谷物酚酸和测定其体外抗氧化能力提供了一种简单的方法。本研究使用五个全谷物样品，硬质小麦，黄玉米，黑眼豇豆，大豆和红芸豆。将100毫克的全谷物面粉样品准确称取，直接放入琥珀色的两毫升容量微型离心管中。管子的深色有助于防止混合物暴露在光线下。向每个含有样品的管中加入一毫升80%甲醇水溶液。短暂涡旋以混合甲醇溶液和样品。将样品超声处理60分钟以提取游离可溶性酚类化合物。在超声处理期间在样品上盖上盖子，以增加对光的保护。超声处理后，将混合物以20， 000倍G离心5分钟，以沉淀固体残留物，使上清液位于顶部。游离酚类化合物在离心后将存在于上清液中。上清液在注入HPLC仪器之前需要过滤。要过滤上清液，请取出3 mL注射器的柱塞并连接注射器过滤器。过滤器的孔径应不大于0.22微米。将约0.4毫升上清液移入注射器顶部。重新插入柱塞并将液体通过过滤器推入含有小瓶插入物的HPLC小瓶中。一旦仪器被设置为运行手稿中概述的HPLC分析方法，将小瓶装入转盘中以与样品列表相对应。获得320纳米和280纳米的HPLC色谱图，显示代表不同酚类化合物的不同峰。使用适当的标准曲线，量化320纳米的羟基肉桂酸，因为它们在该波长处具有最大的吸光度。通过相同的原理，量化280纳米的羟基苯甲酸。使用Trolox（维生素E的水溶性类似物）作为标准来估计全谷物提取物的体外抗氧化能力。在猎鹰管中准确称量一毫克的Trolox。用四毫升50%甲醇水溶液溶解，制备每升1毫升的储备溶液，相当于每升1000微摩尔。准备六种浓度的Trolox，即每升50，100，200，400，600和800微摩尔，以绘制标准曲线，以估计DPPH自由基清除能力和Trolox的等效抗氧化能力，TEAC。类似地，制备每升6.25、12.5、25和50微摩尔的Trolox浓度，用于估计氧自由基吸收能力ORAC。每个浓度的总体积为500微升，如表一所示。分析前用甲醇稀释样品提取物。在这里，黄玉米和豇豆提取物被稀释了两倍。小麦和芸豆提取物稀释5次，大豆提取物用甲醇稀释10倍。称取8.23毫克ABTS放入一个干净的，容量为两毫升的琥珀色微量离心管中。接下来，称取1.62毫克的过硫酸钾放入一个干净的、容量为两毫升的琥珀色微量离心管中。通过涡旋将每个称重的化学品溶解在一毫升蒸馏水中。这导致16毫摩尔ABTS溶液和6毫摩尔过硫酸钾溶液。通过将ABTS和过硫酸钾溶液等体积混合来制备ABTS储备溶液。溶液将立即变为深色。让这种混合物在黑暗中孵育12至16小时。用200毫摩尔磷酸盐缓冲盐水稀释ABTS储备溶液30倍，形成ABTS工作溶液。为此，将58毫升200毫摩尔PBS添加到两毫升ABTS工作溶液中。工作溶液将含有0.27毫摩尔ABTS和0.1毫摩尔过硫酸钾。对于分析，将每种稀释的提取物的10微升置于96孔微孔板中。向每个孔中加入190微升ABTS工作溶液，孵育60分钟。在酶标仪中测量750纳米的反应混合物的吸光度。使用浓度范围为每升100至800微摩尔的Trolox标准品来绘制校准曲线。DPPH抗氧化测定需要一种产生自由基的化合物DPPH。称出1.2毫克DPPH到空的15毫升容量离心管中。将DPPH溶解在甲醇中以制备60微摩尔溶液。DPPH测定测试样品提取物清除DPPH产生的自由基的能力。将五微升样品提取物加入微孔板孔中。接下来，加入195微升60微摩尔DPPH甲醇溶液并孵育60分钟。测量515纳米处的吸光度。使用 Trolox 标准品绘制校准曲线。图一显示了在全谷物中发现的羟基苯甲酸的结构。在目前的这项研究中，香草酸是唯一确定的羟基苯甲酸。图二显示了在全谷物中发现的羟基肉桂酸的结构。在目前的研究中，在样品中鉴定了香豆酸，咖啡酸和阿魏酸。表二显示了样品中鉴定的酚酸。如前所述，香草酸是样品中唯一鉴定的羟基苯甲酸。它仅在豇豆提取物中鉴定出来。羟基肉桂酸咖啡酸仅在芸豆中鉴定，而对香豆酸在黄玉米，豇豆和大豆中鉴定。在所有样品中均鉴定出阿魏酸，并且是样品中的主要酚酸。酚酸的总浓度从大到低的顺序是大豆，豇豆，黄玉米和芸豆或同等物，其次是小麦。表三显示了样品的总酚类含量和抗氧化能力。抗氧化能力包括DPPH自由基清除能力，TEAC，ORAC和样品的总抗氧化能力。总抗氧化能力是DPPH，TEAC和ORAC值的总和。与表二类似，大豆的总抗氧化能力最高。然而，芸豆不是豇豆，而是芸豆的总抗氧化能力第二高，尽管豇豆的总酚酸含量第二高。这种异常与单个酚酸的结构以及羟基苯甲酸香草酸对豇豆中羟基肉桂酸的抗氧化作用的可能拮抗作用有关。这项研究得出的结论是，全谷物的酚酸成分不同。在所研究的全谷物中，大豆的酚酸总量最高，相应地，抗氧化能力也最高。

Research

JoVE Journal

Chemistry

Free Radicals in Chemical Biology: from Chemical Behavior to Biomarker Development

自由基化学生物学的化学行为，生物标记开发

The involvement of free radicals in life sciences has constantly increased with time and has been connected to several physiological and pathological ...

科研

化学

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle ...

Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation

朝仿生学木：纳米纤维素，木质素的装配式自由站立电影和合成聚阳离子

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and ...

Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, ...

Synthesis of a Water-soluble Metal&#8211;Organic Complex Array

Synthesis of a Water-soluble MetalÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œOrganic Complex Array

一个水溶性金属 - 有机络合物阵列合成

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as ...

Determining the Chemical Composition of Corrosion Inhibitor/Metal Interfaces with XPS: Minimizing Post Immersion Oxidation

确定缓蚀剂/金属界面的化学成分与XPS：最小化后浸入氧化

An approach for acquiring more reliable X-ray photoelectron spectroscopy data from corrosion inhibitor/metal interfaces is described. More specifically, ...

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

大肠杆菌无细胞蛋白合成: 一种强大、灵活和可访问的平台技术的协议

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

Synthesis Method for Cellulose Nanofiber Biotemplated Palladium Composite Aerogels

纤维素纳米纤维生物模板复合气凝胶的合成方法

Here, a method to synthesize cellulose nanofiber biotemplated palladium composite aerogels is presented. Noble metal aerogel synthesis methods often ...

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

基于NMR的活性测定,用于测定核苷裂解酶的化合物抑制、IC50值、伪影活性和全细胞活性

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides