我们感谢已故的博士。规范泰勒，系植物与土壤科学在肯塔基大学，允许我们使用的样品从他的红三叶草地块为这项研究。该项目是由农业部美国农业部。

1。植物提取物的制备<ol><li>见卡根和Flythe 10从红车轴草提取CV酚类化合物。 Kenland。 </li><li>要提取其他化合物在其它植物中，检查（很多）里描述的植物化学分析文献植物或代谢物的具体提取方法，或者寻找协议，如那些KHURRAM 等。7,8的其中分离许多化合物具有广泛极性范围。 </li></ol> 2。薄层板的制备<ol><li>清洁TLC板通过在一个或多个极性，中性溶剂，以便移动吸附的污染物远离发展的区域。 <ol><li>在通风橱中，准备足够的清洁溶剂（ 例如 15-100的乙酸乙酯-甲醇2点01毫升，V / V）以覆盖薄层显影腔室的底部，以及一个TLC板的下边缘时，集内部腔室中。 </li><li>使用COMMER上出售的玻璃薄层色谱显影室（不同的尺寸可供选择，有盖）或金属箔覆盖耐热玻璃烧杯或保存罐。 </li><li>用剪刀剪下铝或塑料背（柔性）硅胶板，它有不同的大小（20厘米×20厘米，小），以符合可用显影室。 （注意:二氧化硅可导致肺损伤，如果吸入在通风橱中工作，并处理TLC板的手套，以避免皮肤的油脂在二氧化硅上。） </li><li>插入板进入腔室，具有顶端靠在腔室壁。板不应相互接触。覆盖所述腔室，并让通过毛细作用的溶剂向上移动的板。 </li><li>当溶剂到达板的顶部，从腔室取出板并在通风橱内以站立位置安排，直至溶剂已蒸发。 </li><li>检查是否有杂质，通过寻找下一个可见的黄色带迁移附近的薄层板的顶部光或紫外线下的荧光波段（UV）光（见"杂质前沿"，或者如果在图2B）。如果大多数板仍具有微黄色调，重复清洗过程。 </li><li>从所述腔室移出TLC板后，弃去溶剂。允许残留溶剂来使用室2协议之前完全蒸发。 </li><li>以除去残留的水分，可以影响在硅胶16的化合物的迁移，直立支撑板在干燥炉中于100℃（10〜15分钟为20×20cm的板，和5分钟7厘米×10厘米板）。 </li><li>如果100℃的干燥炉中不可用，热板为一个较长的时间周期在较低温度下（ 即 40分钟，在60℃）。 </li><li>后板是干的，让他们冷却到加载前环境温度。 </li></ol></li></ol> 3。钱伯斯开发的提取分离制备<oL&gt; <li>用剪刀剪下一张滤纸略低于室高度，大约一半的宽度腔周长。本文作为一个灯芯画溶剂起来室壁和饱和与溶剂蒸气室，从而提高分离1的重现性。 </li><li>在通风橱中，混合溶剂（乙酸乙酯 - 甲醇4:1，V / V，这项研究）。倒入溶剂混合气进入室内并覆盖。等到整个灯芯是用溶剂湿润，说明室饱和度，把板进入室内。 </li></ol> 4。加载中TLC板与发展<ol><li>轻轻地用铅笔标注的产地。如果薄层板的吸附剂是软的，容易损坏，使商标在边缘。化合物应该是当板被插入到腔室TLC展开溶剂的表面之上。 </li><li>溶解提取物在足够的有机溶剂（在这种情况下，甲醇），具有浓缩溶液而不是浑浊停牌。 </li><li>加载样品和标准品作为窄频带用微升注射器或毛细管微量，留下1厘米的边界在板的两侧。允许带干（呈扇形散开板或加载它在通风橱帮助）。 </li><li>如果样品的浓度较大时在干燥的乐队需要放在盘子里，"overspot"通过加载采样一次。 </li><li>用镊子或钳子，饱和TLC室内设置板（S）。板不应该接触的灯芯，因为它可以提供溶剂板在接触点，从而改变化合物的迁移的路径。盖上腔，让板发展。 </li></ol> 5。用于制备生物测定板的<ol><li>从TLC罐除去板（次）之前，溶剂前沿到达板的顶部，并标记溶剂前沿，用铅笔的高度。让板在干燥通风橱。 <ol><li>开发任何剩余的TLC板在同一薄层腔室，这是戈nerally可用一整天，如果保持关闭。对于TLC室重拍溶剂混合物如溶剂室中的量显着减少。 </li></ol></li><li>后板是干燥的，带可视化在可见光或紫外光，并划定乐队用铅笔。与便携式UV灯的观察室中很方便，尤其是当灯可以在两个254纳米（短波UV）和365纳米（长波紫外）检测化合物。 注:在这一点上或下一个步骤之后，板可包上保鲜膜，用铝箔纸覆盖，并储存在-20°C。最大存储时间取决于化合物的稳定性。因为二氧化硅是不是中性的，一些化合物可能会降低，而在TLC板上。 </li><li>拍摄或绘制板。 <ol><li>使用凝胶photodocumentation系统如果一个配有顶部紫外线和/或可见光是可用的。 </li><li>如果没有商业photodocumentation系统是可用的，一盒里面的照片板，内衬深色纸或布，无线TH在三脚架上的摄像机设置和便携式UV光钳位在一个环站17。当拍摄有没有荧光指示板，用蓝色光过滤器在相机镜头以提高乐队的外观17。 </li></ol></li><li>以有助于表征带，通过测量由化合物和溶剂前面行驶的距离，并除以前者由后者（ 图2A），计算保留因子（R f）值。 </li></ol> 6。细菌培养和检测<ol><li>准备和厌氧条件下接种媒体。在这种情况下使用的文化是梭状芽孢杆菌sticklandii应变SR，这是从詹姆斯·B·罗素，康奈尔大学，纽约州Ithaca的培养物保藏中心。 <ol><li>见Flythe和卡根13和材料清单HAB介质的描述。 </li></ol></li><li>成长的文化指数或固定相。使用无菌无氧techniquES与厌氧微生物工作时。 </li><li> （1％V / V）接种培养到琼脂（0.75％重量/体积）下降到低于60℃的温度下熔融后的琼脂轻轻混匀并立即带入厌氧室（95％的CO 2 -5％H 2气氛民政事务局媒体）倒。 </li><li>倒入分成15毫米×100毫米的塑料培养皿中，并允许凝固。 </li><li>用剪刀，剪薄层板为含利息波段（S）区。用绳索捆绑面朝下放置到琼脂平板上，标志​​着对烫画带跟踪原稿方向薄层板上。放置一个未使用的TLC条上的琼脂板作为对照。 </li><li>培养琼脂平板上，琼脂面朝下，在厌氧室（24小时，39℃）。 </li></ol> 7。的生物测定琼脂平板可视化<ol><li>用钳子，从琼脂平板上取下乐队，而他们在厌氧室。 </li><li>添加1％（W / V）TEtrazolium红色，在水中制备，滴加到琼脂平板的表面上。允许的颜色发展为至少20分钟，或直至所述控制板完全变成红色，在厌氧室中取出之前。厌氧菌将开始移离厌氧室后立即失去活力，但颜色是稳定超过24小时。 </li><li>在可见光下拍摄。 </li></ol>

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在文章中商品名称或商品的一提的是，仅用作提供特定信息之目的，并不意味着推荐或认可由美国农业部。作者宣称没有竞争的经济利益。

这个协议描述了一种用于提取物中分离到的化合物的子集，并通过接触生物自显影法测定的那些子集的简单方法。该方法非常类似于1使用Chomnawang 等人 15来筛选植物代谢产物抑制到淋病的细菌。生物自显影来屏幕抗菌植物化合物的类型取决于许多因素，包括微生物检验，实验室的设置，而该人（次）进行生物测定的喜好。联系生物自显影，在这项研究中所使用的方法，已经被批评为根据的化合物扩散到接种媒体12的能力。抑制过度的化合物在低浓度时，或者几乎没有能力扩散到介质的区域，如果生长在渗出下面琼脂发生可能无法检测到。此外，抑制由一个带创建一个区域可能扩散到乐队的初始边界，从而可能掩盖邻国化合物14中创建的抑制区。然而，接触生物自显影技术是一种简单的方法来使用，并且堆叠培养皿的能力，最大限度地减少所需的空间量。另外，敷设薄层频带到琼脂消除了带的溶解和扩散时TLC板上覆盖有接种的琼脂或喷洒试验微生物，可以发生的风险。 从上述任何类型的生物自显影的获取信息依赖于上述色谱条件下，测试微生物浓度，和文化/孵化条件。抗菌乐队的色谱分辨率是可有可无的，但灵敏度可能为一个良好的解决了带6更大。乐队可以通过发展再上板或多种溶剂体系由开发前90度转弯TLC板隔开在一维（ 图2B），或在两个维度在第二溶剂16,19。极性化合物可能很难在硅胶上分离，而无需使用含有酸或碱的溶剂混合物。这些可能是致死的试验微生物12，虽然使用的少量的氨基酸已有报道14,15。替代溶剂系统可包括水或甲醇作为部件20,21。极性化合物也可以对不同类型的TLC板，如涂有C 18的吸附剂或微晶纤维素板的解决。然而，生长12或灵敏度的显色剂14可被带负上一些这些吸附剂的影响。 可用于生物自显影试验微生物和媒体的许多不同组合。但是，选择微生物和媒体时，几个因素必须加以考虑。媒体应该进行测试，以确定有没有组件，降低了TETrazolium盐，并导致非生物的颜色变化。琼脂也应该是足够轻的颜色，以提供染色细胞和抑制未染色区之间的对比度。一种微生物，只能生长琼脂表面上可随TLC乐队或删除染色过程中冲洗掉。此外，微生物/媒体组合，最大限度地减少天然气产量的选择将减少气泡的琼脂。瘤胃HAB生长在碳酸盐缓冲的，碳水化合物的培养基与氨基酸或肽作为生长衬底的混合物。该琼脂平板是浅金色和透明。当C。 sticklandii是生长在培养基中，大部分的产品都溶于（挥发性脂肪酸，氨）。很少的气体产生时，这导致了平稳的琼脂板上，以无气泡。 该生物测定有几个潜在的应用。抑菌圈的大小，可作为一个粗略的估计抑制性化合物的量，抑制带的半径，因为是成正比的化合物的量的对数使所述抑制17,22。也许最常见的使用薄层板上，生物测定是缩小可能的抗菌化合物的范围在植物提取物。后抑制区域由生物测定确定，在一个重复的TLC板上的相应区域可以用溶剂洗脱，例如甲醇或乙酸乙酯中，并通过HPLC用紫外光谱检测到开始表征生物活性化合物10,14进行分析。

在植物中测定抗微生物化合物需要分离的植物提取物的组分，露出试验微生物的那些部件，并确定由任何所述化合物的微生物的生长是否受到抑制。分离由纸或薄层色谱法（PC或TLC）是方便的，因为很多化合物可在平面表面上进行分离。分离是基于极性，与一些化合物的结合紧密的吸附剂（纤维素在PC机的情况下，以及各种在薄层的情况下，吸附剂）和迁移小于其他1， 图1提供的相对位置的一个例子在硅胶TLC板上分离后极性和非极性的酚类化合物。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51411/51411fig1highres.jpg" src="/files/ftp_upload/51411/51411fig1.jpg" /> 图1。示出在硅胶薄层色谱（TLC）板分离后不同极性的化合物的分布。红三叶草（ 红三叶 L.）的酚类化合物被用作一个例子。极性化合物，如clovamide，有一种极性吸附剂像二氧化硅有很强的亲和力，并保持靠近原点（OR），而极性较小的化合物，如靠近溶剂前沿的3异黄酮（SF），分区更容易进入溶剂（除非水，酸，碱或包含它们比二氧化硅极性较小）和迁移更远了板。 的提取物在TLC板上分离后，试验微生物可以暴露于板上的所有化合物中，从而加速的提取物2的活性成分的鉴定。如果是真菌或细菌培养物暴露于色谱，会发生微生物生长无处不在，除了以上与生长抑制剂领域Ÿ化合物。抑制区则可以通过观察菌丝生长和生长无区域之间的对比，如果真菌已经应用3或者通过与该改变颜色由活细胞4降低或水解时的化合物喷雾可视化。虽然使用纸或薄层色谱的抗菌试验首先被施加到抗生素5和杀菌剂3,6，植物提取物，现在经常筛选出抗微生物化合物用这种方法，通常被称为生物自显影。本文中所描述的协议适用于薄层色谱生物自显影。 TLC被广泛使用，因为它是相对快速的，并且可以在不同的吸附剂（ 如硅胶，淀粉，氧化铝），以及提供良好的分辨率和灵敏度1进行。 植物提取物可用于TLC以多种方式来制备。常用的方法包括提取植物材料的ALCohol -水的混合物，如80％乙醇7,8，可能与另外的酸或碱9。以下在这些溶剂，其中含有一些水，并且可能是酸性或碱性的提取，提取物，必须进行浓缩，以便它们可以应用到TLC板在最小的体积。的醇-水提取物中的浓度可以通过与水不混溶的有机溶剂的8或与此类溶剂，如乙酸乙酯-乙醚（1:1，V / V）10,11的混合物隔开来实现。异植物代谢产物提取到不同的有机溶剂，这取决于它们的极性。以确保植物有机的酸或碱都在此阶段萃取到有机溶剂中，醇 - 水提取物的pH可以升高或降低与水溶性酸或碱解离的分析物转化为它们的nondissociated的形式，然后将它们溶于中性有机溶剂9。然后将有机相可以通过电子邮件在减压或在氮气氛下vaporated并调整为薄层所需的音量。该提取物的pH不太可能是致命的，因为分析物的分割成中性溶剂，小终体积，并在TLC板上分离前的提取物蒸发生物测定微生物。 这两种真菌和细菌被用作试验的微生物在植物生物自显影提取2。一些真菌，如黑星病的孢子发芽在TLC板（除了与抑制性化合物的区域），如果在一个营养液喷洒到板并孵育在潮湿的环境中几天3。 C.暗菌丝体星病的noninhibitory区提供了一个鲜明的对比，免费菌丝生长的区域。虽然细菌已被施加到薄层色谱（TLC）板以相同的方式4,12，细菌也被倾倒在TLC板面在琼脂覆盖13,14。酵母，如白色念珠菌 ，可在琼脂覆盖层被应用，以及14。此外，TLC板可以向下放置脸上琼脂上接种细菌10,15或酵母8，称为接触性生物自显影2的方法。 我们描述了一种接触生物自显影筛选抗菌酚类化合物从红三叶草（ 红三叶品种。Kenland）。该测试微生物是梭菌sticklandii，一个超瘤胃氨产生菌（HAB）和专性厌氧菌。尽管所使用的分离不能解决提取物的所有组成部分，它们有助于确定抗菌活性的区域，从而缩小可能的抗菌化合物的游泳池。该协议采用标准程序TLC 1。该协议还介绍了一些需要培养obli技术门厌氧菌对这样的测定，接触生物自显影15和可视化方法的具有四唑鎓盐，其染色活细胞2,4的 ​​使用。

<table border="0" cellpadding="0" cellspacing="0" width="739">
	<tbody>
		<tr>
			<td>Silica F254 TLC plates, aluminum-backed, 0.2 mm thickness, 20 &#215; 20 cm</td>
			<td>EMD Chemicals&#160;</td>
			<td>5554/7</td>
			<td>These plates are coated with silica that contains an indicator fluorescing at 254 nm.&#160; Compounds absorbing at that wavelength appear dark on a fluorescent green background.&#160; Alternative sources include Analtech, Selecto Scientific, Fluka.&#160; Adsorbents other than silica may be needed.&#160; Plastic-backed plates may be suitable, depending on the solvents to be used.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Sharp, heavy-duty scissors&#160;</td>
			<td>any sewing supply company</td>
			<td>similar to Fiskars&#160; 175800-1002</td>
			<td>For cutting TLC plates.&#160; A paper cutter with a sharp blade can be used as well.&#160; Do not inhale silica dust.</td>
		</tr>
		<tr>
			<td>Drying oven at 100 &#176;C (mechanical convection)</td>
			<td>Thermo Scientific</td>
			<td>PR305225M</td>
			<td>Quincy Lab, Inc, Chicago, IL (www.quincylab.com); Cascade Technical Sciences, Hillsboro, OR (www.cascadetek.com)</td>
		</tr>
		<tr>
			<td>TLC chamber</td>
			<td>Kimble Chase&#160;</td>
			<td>416180-0000</td>
			<td>Alternative sources:&#160; Aldrich. Pyrex beakers or preserving jars can be used for small plates (i.e. 5 &#215; 10 cm).&#160; Cover with aluminum foil (jar lids may contain material extractable by solvent vapors).</td>
		</tr>
		<tr>
			<td>50-&#181;L syringe with flat needle tip</td>
			<td>Hamilton</td>
			<td>80965</td>
			<td>For loading amounts of standard or sample exceeding 5-10 &#181;L.&#160; Alternative sources are equivalent.</td>
		</tr>
		<tr>
			<td>micropipets</td>
			<td>Drummond</td>
			<td>2-000-001</td>
			<td>For loading small amounts of standards or samples.&#160; Alternative sources:&#160; VWR.&#160; Also, Pasteur pipets can be stretched to a thinner diameter with a butane torch.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Filter paper (#1 grade)</td>
			<td>Whatman</td>
			<td>1001 917</td>
			<td>Serves as a chamber wick.&#160; Other grades of filter paper are OK.&#160; This size can be trimmed for the chambers holding 20 &#215; 20 cm plates.&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr>
			<td>Beaker tongs</td>
			<td>Fisher Scientific</td>
			<td>15-186</td>
			<td>For putting plates in and out of a large TLC chamber.&#160; Alternate sources: VWR&#160;</td>
		</tr>
		<tr>
			<td>Flat-edge forceps&#160;</td>
			<td>Fisher Scientific</td>
			<td>10-275</td>
			<td>For putting plates in and out of a small chamber.&#160;&#160; Alternate sources: VWR&#160;</td>
		</tr>
		<tr>
			<td>Small portable UV lamp with 4-Watt or 6-Watt bulbs for short- and long-wave UV light illumination (254 and 365 nm, respectively)</td>
			<td>Ultraviolet Products&#160;</td>
			<td>95-0271-01</td>
			<td>Alternate sources: Spectronics Corporation (www.spectroline.net)</td>
		</tr>
		<tr>
			<td>Viewing cabinet for use with hand-held UV lamp</td>
			<td>Ultraviolet Products&#160;</td>
			<td>Chromato-Vue C-10E</td>
			<td>UV-active bands are more easily circled if plates can be set in here.&#160; Alternate sources: Spectronics Corporation.&#160;</td>
		</tr>
		<tr>
			<td>Photodocumentation system with overhead UV lamp and visible lamp</td>
			<td>Kodak&#160;</td>
			<td>Gel Logic 200</td>
			<td>&#160;Alternate sources: Ultraviolet Products (www.uvp.com).&#160; See protocol for homemade alternative.</td>
		</tr>
		<tr>
			<td>Anaerobic Chamber, Type A, Vinyl</td>
			<td>Coy&#160;</td>
			<td>7150000</td>
			<td>This chamber is appropriate for anaerobic bacteria, like Clostridium sticklandii, as described. &#160;However, growth conditions must be tailored to organism used in the assay.&#160; A biosafety cabinet and other precautions should be taken if pathogenic organisms are used. Alternate sources: Anaerobe Systems, BioRad, Plas Labs, others&#160;</td>
		</tr>
		<tr>
			<td>Tetrazolium red</td>
			<td>Sigma-Aldrich</td>
			<td>T8877</td>
			<td>Alternate sources: MP Biomedicals, Santa Cruz Biotechnology, Alfa Aesar</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Ingredients for HAB media</td>
		</tr>
		<tr>
			<td>Pyridoxamine &#183; 2 HCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9380</td>
			<td>For this and for all the other reagents in this table, alternative sources are equivalent.</td>
		</tr>
		<tr>
			<td>Riboflavin</td>
			<td>Sigma-Aldrich</td>
			<td>R4500</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine HCl</td>
			<td>Sigma-Aldrich</td>
			<td>T3902</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N3376</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium D-Pantothenate</td>
			<td>Sigma-Aldrich</td>
			<td>C8731</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipoic Acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T5625</td>
			<td></td>
		</tr>
		<tr>
			<td>p-Aminobenzoic acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A9878</td>
			<td></td>
		</tr>
		<tr>
			<td>Folic acid</td>
			<td>Sigma-Aldrich</td>
			<td>F8798</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Sigma-Aldrich</td>
			<td>B4639</td>
			<td></td>
		</tr>
		<tr>
			<td>Cobalamine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C3607</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridoxal HCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9130</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridoxine</td>
			<td>Sigma-Aldrich</td>
			<td>P5669</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron sulfate &#183; 7 H2O</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;F8263</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc sulfate &#183; 7 H2O</td>
			<td>Sigma-Aldrich</td>
			<td>Z0251</td>
			<td></td>
		</tr>
		<tr>
			<td>Manganese chloride &#183; 4 H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M8054</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sigma-Aldrich</td>
			<td>B6768</td>
			<td></td>
		</tr>
		<tr>
			<td>Cobalt chloride &#183; 6 H2O</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;C8661</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper chloride &#183; 2 H2O</td>
			<td>Sigma-Aldrich</td>
			<td>459097</td>
			<td></td>
		</tr>
		<tr>
			<td>Nickel chloride &#183; 6 H2O</td>
			<td>Sigma-Aldrich</td>
			<td>203866</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium molybdate &#183; 2 H2O</td>
			<td>Sigma-Aldrich</td>
			<td>331058</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Trypticase (Pancreatic digest of casein)</td>
			<td>Thermo Fisher</td>
			<td>B11921</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic anhydrous</td>
			<td>Thermo Fisher</td>
			<td>P284</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium carbonate &#183; H2O&#160;</td>
			<td>Thermo Fisher</td>
			<td>S636</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Thermo Fisher</td>
			<td>50841063</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate &#183; 6 H2O</td>
			<td>Thermo Fisher</td>
			<td>7791-18-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride &#183; 2 H2O</td>
			<td>Thermo Fisher</td>
			<td>BP510</td>
			<td></td>
		</tr>
		<tr>
			<td>Cysteine HCl</td>
			<td>Thermo Fisher</td>
			<td>19464780</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic anhydrous</td>
			<td>Thermo Fisher</td>
			<td>P290</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Thermo Fisher</td>
			<td>BP358</td>
			<td></td>
		</tr>
	</tbody>
</table>

thin-layer chromatographic (tlc) separations and bioassays of plant extracts to identify antimicrobial compounds

对植物的抗微生物化合物的常用屏幕包括由纸或薄层色谱法（PC或TLC）分离的植物提取物，露出色谱微生物悬浮液（ 例如，在液体培养基真菌或细菌或琼脂），允许时间为微生物生长中的潮湿环境中，并且可视区与无微生物生长。该筛选方法，被称为生物自显影的效果，取决于色谱分离的质量和用微生物培养条件下所采取的照顾。本文介绍了一种新型的应用氨基酸发酵菌的TLC和接触生物自显影技术的标准协议。将提取物上分离柔性（铝背）硅胶TLC板，并频带下的紫外线（UV）光可视化。区域被切出并孵育面朝下于琼脂接种试验微生物。抑制带被染色的琼脂平板可视化s的四氮唑红。该方法适用于红三叶草（ 红三叶品种。Kenland）分离酚类化合物及其筛查对梭状芽孢杆菌sticklandii，一个超氨产生菌（HAB）是原产于牛瘤胃活动。在TLC的方法适用于许多类型的植物提取物和其它的细菌菌种（好氧或厌氧），以及真菌的，可以作为测试生物体，如果培养条件进行修改，以适应植物的生长要求。

红三叶草（ 红三叶品种。Kenland）提取物，含有酚类化合物，有代表性的二氧化硅薄层色谱分离见图2。红车轴草提取物中乙酸乙酯-正己烷分离（9:1，V / V），超过850厘米，导致五阶，一是从原点不完全解析（ 图2A）。然而， 图2B表明，大约两倍多的条带，表明当红三叶草提取物（来自同一品种，但在一个单独的情节在同一农场种植）不同的样本中分离了一个较大的距离（而不是15厘米8.5厘米），并用在不同的溶剂系统的两个连续的发展。 图2B的色谱图中，用乙酸乙酯-己烷首先研制（9:1，V / V），然后使之干燥，并在乙酸乙酯-甲醇中分离（78:22，V / V）。这些结果表明，溶剂系统（或一系列的溶剂S的两个板的尺寸和选择度提供意見）会影响分离的色谱化合物的数量。在这种特定情况下，洗脱，并从相同的萃取的其它TLC分离频带的高效液相色谱法（HPLC）证实，其余在或接近原点的化合物主要包括缀合的极性基团的酚类化合物（酚羧酸或异黄酮）如氨基酸衍生物，糖类，或苹果酸。与此相反，未缀合的极性部分异黄酮迁移更远了TLC板10。 与梭菌sticklandii，一个超瘤胃氨生产菌（HAB）， 如图2A所示，在薄层板上的生物测定的结果示于图3。添加1％（W / V）的四氮唑红到24小时培养后的琼脂平板水溶液产生了鲜红的颜色，当活细胞进行染色。带1的潜伏期（鹰嘴豆芽素A）和Ba的一部分ND 2（芒柄 ​​花素）对细菌接种的琼脂导致了一个明确的抑制区（ 图3A）。然而，温育的带2的其余部分，随着条带3和4中，没有抑制细菌生长（ 图3B）。这些结果表明，鹰嘴豆芽素A是抑制到C。 sticklandii增长，但芒柄花素不是。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51411/51411fig2highres.jpg" src="/files/ftp_upload/51411/51411fig2.jpg" /> 中红车轴草提取物CV图2。硅胶薄层色谱（TLC）分离。 Kenland，在（A）的乙酸乙酯-己烷（9:1，V / V），或（B）在随后的发展在乙酸乙酯-甲醇中相同的溶剂（78:22，V / V）的距离迁移由开发原点（OR）和溶剂前沿（SF）之间的溶剂是表示靠近括号这两个边界之间。在图2B中，"杂质前沿"（IF，杂质的位置向上移动的板通过预洗涤在极性溶剂中）被视为邻近所述板的顶部上的暗带。提取，从180-250毫克冻干红三叶草的叶子编制及茎，溶于甲醇，并提取物（A）或32％（B）的10％加载到硅胶板。加载标准是芒柄花素（F，7.1纳摩尔），鹰嘴豆芽素A（BA，7.4纳摩尔），染料木素（G，9.8纳摩尔），和clovamide（以Cl， 图2B只，8.7纳摩尔）。条带，盘旋而下，手持紫外灯波长为254 nm（短波紫外线）和365纳米（长波紫外线）查看。数码相机是用来在短波紫外光拍摄的板块。的编号，在图2A中的三叶草提取物条带指的是区域的右切出用于生物测定。保留因子（R F，距离traveleD由乐队/距离通过顺丰速行驶）是在括号中图2A带数字之后。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51411/51411fig3highres.jpg" src="/files/ftp_upload/51411/51411fig3.jpg" /> 图3中的（A）波段1和波段2从图2A，和（B）带2-4从同一薄层板的薄层板的不完全解决的上部薄层板上，生物测定结果。乐队被裁减下来的脸民政事务局上中等接种梭菌sticklandii，并在39℃下厌氧培养24小时在此温育期结束时，带被除去，并且琼脂平板上进行染色的1％的水溶液（重量/体积）的四唑和红色可见光下拍摄的。

Watch this Scientific Journal Video about Thin-layer Chromatographic TLC Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds at JoVE.com

Thin-layer Chromatographic TLC Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

方法描述为薄层色谱（TLC）分离植物提取物和生物自显影接触，以确定抗菌代谢产物。该方法适用于红三叶草酚类化合物抑制超氨生产菌（HAB），原产于牛瘤胃的筛选。

薄层色谱（TLC）分离和植物提取物的生物测定识别抗菌化合物

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the ...

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Nanyang Technological University

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Analytical Chemistry

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Unit 1

Thin-layer Chromatographic (TLC) Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

Principles of Chromatography

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67.6K 次观看.  United States Department of Agriculture. 方法描述为薄层色谱（TLC）分离植物提取物和生物自显影接触，以确定抗菌代谢产物。该方法适用于红三叶草酚类化合物抑制超氨生产菌（HAB），原产于牛瘤胃的筛选。 

视频: Thin-layer Chromatographic TLC Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

Profiling the Triacylglyceride Contents in Bat Integumentary Lipids by Preparative Thin Layer Chromatography and MALDI-TOF Mass Spectrometry

The mammalian integument includes sebaceous glands that secrete an oily material onto the skin surface. Sebum production is part of the innate immune system that is protective against pathogenic microbes. Abnormal sebum production and chemical composition are also a clinical symptom of specific skin diseases. Sebum contains a complex mixture of lipids, including triacylglycerides, which is species-specific. The broad chemical properties exhibited by diverse lipid classes hinder the specific determination of sebum composition. Analytical techniques for lipids typically require chemical derivatizations that are labor-intensive and increase sample preparation costs. This paper describes how to extract lipids from mammalian integument, separate broad lipid classes by thin-layer chromatography, and profile the triacylglyceride contents using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This robust method enables a direct determination of the triacylglyceride profiles among species and individuals, and it can be readily applied to any taxonomic group of mammals.

Mammalian integument contains solvent-extractable lipids that can provide chemical compositions characteristic of individual species. This paper presents a routine method for separating broad lipid classes isolated from integumentary tissues using thin layer chromatography and determining the triacylglyceride profile by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

用制备薄层色谱和MALDI-TOF质谱分析的三酸甘油酯含量的蝙蝠外皮血脂

The mammalian integument includes sebaceous glands that secrete an oily material onto the skin surface. Sebum production is part of the innate immune ...

科研

化学

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic isotopes are typically incorporated metabolically to improve the sensitivity and allow for sequential resonance assignment. Reductive 13C-methylation is an alternative labeling method for proteins that are not amenable to bacterial host over-expression, the most common method of isotope incorporation. Reductive 13C-methylation is a chemical reaction performed under mild conditions that modifies a protein&#39;s primary amino groups (lysine &#949;-amino groups and the N-terminal &#945;-amino group) to 13C-dimethylamino groups. The structure and function of most proteins are not altered by the modification, making it a viable alternative to metabolic labeling. Because reductive 13C-methylation adds sparse, isotopic labels, traditional methods of assigning the NMR signals are not applicable. An alternative assignment method using mass spectrometry (MS) to aid in the assignment of protein 13C-dimethylamine NMR signals has been developed. The method relies on partial and different amounts of 13C-labeling at each primary amino group. One limitation of the method arises when the protein&#39;s N-terminal residue is a lysine because the &#945;- and &#949;-dimethylamino groups of Lys1 cannot be individually measured with MS. To circumvent this limitation, two methods are described to identify the NMR resonance of the 13C-dimethylamines associated with both the N-terminal &#945;-amine and the side chain &#949;-amine. The NMR signals of the N-terminal &#945;-dimethylamine and the side chain &#949;-dimethylamine of hen egg white lysozyme, Lys1, are identified in 1H-13C heteronuclear single-quantum coherence spectra.

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Two methods for assigning the &#945;- and &#949;-dimethylamine nuclear magnetic resonance signals of a reductively 13C-methylated N-terminal lysine are described. One method utilizes the pH-induced selectivity of the reductive methylation reaction, and the other uses aminopeptidase to selectively remove the N-terminal lysine.

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic ...

Analysis of Volatile and Oxidation Sensitive Compounds Using a Cold Inlet System and Electron Impact Mass Spectrometry

This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The analysis of volatile and oxidation sensitive compounds by mass spectrometry is not easily achieved, as all state-of-the-art mass spectrometric methods require at least one sample preparation step, e.g., dissolution and dilution of the analyte (electrospray ionization), co-crystallization of the analyte with a matrix compound (matrix-assisted laser desorption/ionization), or transfer of the prepared samples into the ionization source of the mass spectrometer, to be conducted under atmospheric conditions. Here, the use of a sample inlet system is described which enables the analysis of volatile metal organyls, silanes, and phosphanes using a sector field mass spectrometer equipped with an electron impact ionization source. All sample preparation steps and the sample introduction into the ion source of the mass spectrometer take place either under air-free conditions or under vacuum, enabling the analysis of compounds highly susceptible to oxidation. The presented technique is especially of interest for inorganic chemists, working with metal organyls, silanes, or phosphanes, which have to be handled using inert conditions, such as the Schlenk technique. The principle of operation is presented in this video.

This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The presented technique is especially of interest for inorganic chemists, working with metal organyls, silanes, or phosphanes which have to be handled using inert conditions, such as the Schlenk technique.

挥发和氧化敏感的化合物使用冷进气系统和电子轰击质谱分析

This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

On-line Analysis of Nitrogen Containing Compounds in Complex Hydrocarbon Matrixes

The shift to heavy crude oils and the use of alternative fossil resources such as shale oil are a challenge for the petrochemical industry. The composition of heavy crude oils and shale oils varies substantially depending on the origin of the mixture. In particular they contain an increased amount of nitrogen containing compounds compared to the conventionally used sweet crude oils. As nitrogen compounds have an influence on the operation of thermal processes occurring in coker units and steam crackers, and as some species are considered as environmentally hazardous, a detailed analysis of the reactions involving nitrogen containing compounds under pyrolysis conditions provides valuable information. Therefore a novel method has been developed and validated with a feedstock containing a high nitrogen content, i.e., a shale oil. First, the feed was characterized offline by comprehensive two-dimensional gas chromatography (GC &#215; GC) coupled with a nitrogen chemiluminescence detector (NCD). In a second step the on-line analysis method was developed and tested on a steam cracking pilot plant by feeding pyridine dissolved in heptane. The former being a representative compound for one of the most abundant classes of compounds present in shale oil. The composition of the reactor effluent was determined via an in-house developed automated sampling system followed by immediate injection of the sample on a GC &#215; GC coupled with a time-of-flight mass spectrometer (TOF-MS), flame ionization detector (FID) and NCD. A novel method for quantitative analysis of nitrogen containing compounds using NCD and 2-chloropyridine as an internal standard has been developed and demonstrated.

A method combining comprehensive two-dimensional gas chromatography with nitrogen chemiluminescence detection has been developed and applied to on-line analysis of nitrogen containing compounds in a complex hydrocarbon matrix.

在线含硫化合物在复杂油气矩阵氮分析

The shift to heavy crude oils and the use of alternative fossil resources such as shale oil are a challenge for the petrochemical industry. The ...

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

利用毛细管电泳从植物组织量化有机酸:一个测试用例检查<em&gt;小粒种咖啡</em&gt;种子

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

Synthesis of Plant Phenol-derived Polymeric Dyes for Direct or Mordant-based Hair Dyeing

Effective hair dyeing through in situ incubation of keratin hair with the products of fungal laccase-catalyzed polymerization of plant phenols has been previously demonstrated. However, the dyeing process takes a long time to complete compared to commercial hair-dyeing products. To overcome this bottleneck, pre-synthesized polymeric products of the oxidative reaction of Trametes versicolor laccase on catechin and catechol, either with or without mordant agents (e.g., FeSO4), were here employed to achieve permanent keratin hair dyeing in various colors and shades. The laccase action in acidic sodium acetate buffer led to a deep black coloration after coupling reactions between the plant phenols. The colored dye products were then desalted and concentrated with ultrafiltration. The dyes, with or without mordant agents, caused a significant increase in &#916;E values (i.e., color difference value) in gray human hair within 2.5 hours. In addition, different keratin colors and shades were induced depending upon the mordanting and pH changes. The dyed hair also exhibited a strong resistance to detergent treatments, indicating that our methods can give rise to permanent hair dyeing.&#160;Overall, our work has provided novel insight into developing eco-friendly hair-dyeing methods as alternatives to commercial toxic diamine-based dyes.

Here, we present a protocol to use pre-synthesized polymeric products derived from fungal laccase-catalyzed polymerization of plant phenols, either with or without mordant agents (e.g., FeSO4), to induce detergent-resistant keratin hair dyeing within 2.5 hours.

对于直接或媒介为基础的头发染色厂苯酚衍生的高分子染料的合成

Effective hair dyeing through in situ incubation of keratin hair with the products of fungal laccase-catalyzed polymerization of plant phenols has been ...

Raman and IR Spectroelectrochemical Methods as Tools to Analyze Conjugated Organic Compounds

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule on the vibrational level of energy. Raman and IR spectroelectrochemistry can be used for advanced characterization of the structural changes in the organic electroactive compounds. Here, the step-by-step analysis by means of Raman and IR spectroelectrochemistry is shown. Raman and IR spectroelectrochemical techniques provide complementary information about structural changes occurring during an electrochemical process, i.e. allows for the investigation of redox processes and their products. The examples of IR and Raman spectroelectrochemical analysis are presented, in which the products of the redox reactions, both in solution and solid state, are identified.

A protocol of step-by-step Raman and IR spectroelectrochemical analysis is presented.

拉曼光谱和红外光谱电化学法作为分析共轭有机化合物的工具

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule ...

Using Cyclic Voltammetry, UV-Vis-NIR, and EPR Spectroelectrochemistry to Analyze Organic Compounds

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance (EPR) or ultraviolet-visible and near-infrared (UV-Vis-NIR) spectroscopies, we obtain useful information such as electron affinity, ionization potential, band-gap energies, the type of charge carriers, and degradation information that can be used to synthesize stable organic electronic devices. In this study, we present electrochemical and spectroelectrochemical methods to analyze the processes occurring in active layers of an organic device as well as the generated charge carriers.

In this article, we describe electrochemical, electron paramagnetic resonance, and ultraviolet-visible and near-infrared spectroelectrochemical methods to analyze organic compounds for application in organic electronics.

利用循环伏安法、紫外-近红外光谱和 EPR 光谱电化学对有机化合物进行分析

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance ...

Electrophoretic Crystallization of Ultrathin High-performance Metal-organic Framework Membranes

We report the synthesis of thin, highly intergrown, polycrystalline metal-organic framework (MOF) membranes on a wide range of unmodified porous and non-porous supports (polymer, ceramic, metal, carbon, and graphene). We developed a novel crystallization technique, which is termed the ENACT approach: the electrophoretic nuclei assembly for the crystallization of highly intergrown thin films (ENACT). This approach allows for a high density of heterogeneous nucleation of MOFs on a chosen substrate via the electrophoretic deposition (EPD) directly from the precursor sol. The growth of well-packed MOF nuclei leads to a highly intergrown polycrystalline MOF film. We show that this simple approach can be used for the synthesis of thin, intergrown zeolite imidazole framework (ZIF)-7 and ZIF-8 films. The resulting 500 nm-thick ZIF-8 membranes show a considerably high H2 permeance (8.3 x 10-6 mol m-2 s-1 Pa-1) and ideal gas selectivities (7.3 for H2/CO2, 15.5 for H2/N2, 16.2 for H2/CH4, and 2655 for H2/C3H8). An attractive performance for C3H6/C3H8 separation is also achieved (a C3H6 permeance of 9.9 x 10-8 mol m-2 s-1 Pa-1 and a C3H6/C3H8 ideal selectivity of 31.6 at 25 &#176;C). Overall, the ENACT process, owing to its simplicity, can be extended to synthesize intergrown thin films of a wide range of nanoporous crystalline materials.

A simple, reproducible, and versatile approach for the synthesis of intergrown, polycrystalline metal-organic framework membranes on a wide range of unmodified porous and non-porous supports is presented.

超薄高性能金属-有机骨架膜的电泳结晶

We report the synthesis of thin, highly intergrown, polycrystalline metal-organic framework (MOF) membranes on a wide range of unmodified porous and ...