这项工作由美国心脏协会（14GRNT20180028）和佛罗里达州立大学研究和创意理事会（COFRS）资助。

1.试剂的制备<ol><li>通过混合80 mL无水乙醇（分子生物学级）和20 mL细胞培养级无菌水，制备80％乙醇（100mL）。 </li><li>为了制备多酚提取物（10mg / mL），称量10mg CE或PE。在细胞培养罩下加入1mL平原Dulbecco&#39;s Modified Eagle Medium（DMEM）。涡流解决方案。等分于200μL，储存于-20°C。 </li><li>准备裂解缓冲液<ol><li>加入5 mL 1M HEPES储备溶液（pH 7.4; 50mM最终），1mL 5M NaCl储备溶液（最终50mM），1mL 0.5M EDTA储备溶液（最终5mM），2mL 0.5 M NaF储备溶液（10mM最终），286μL700mM Na 3 VO 4储备溶液（最终2mM），5mL 200mM Na 4 P 2 O 7储备溶液（最终10mM）和5mL在水中制备20％Triton-X-100储备溶液（最终为1％）。加水至100 mL。保持在4°C。 </li><li&gt;加入10μL/ mL蛋白酶抑制剂鸡尾酒。当细胞准备好溶解时加入新鲜物质。 </li></ol></li><li>准备Laemmli样品缓冲液（4x）。 <ol><li>加入31.25mL 2M Tris储备溶液（pH6.8），100mL甘油，50mL在水中制备的20％十二烷基硫酸钠（SDS）溶液和50mL的β-巯基乙醇。加水至250 mL。保持在4°C。 </li></ol></li><li>为了制备Tris缓冲盐水（TBS）缓冲液，称重3g Tris（25mM最终），0.18g KCl（2.5mM最终）和8.76g NaCl（最终150mM）。将pH调节至7.4，加水至1 L。保持在室温。 </li><li>为了制备TBS-T，加入997.5mL TBS和2.5mL在水中制备的20％Triton-X-100溶液。保持在RT。 </li></ol> 2.黑莓提取物的制备<ol><li>从冷冻干燥的浆果粉中制备粗提物。 <ol><li>称取10克冷冻干燥的黑莓粉末（或5克新鲜粉末）。如果使用冷冻水果，切断它在开始提取前非常小。 </li><li>将水果粉末与100mL的80％含水乙醇混合在1L圆底锥形瓶中。 </li><li>使用25℃的超声波浴在42kHz，135W下将混合物超声处理20分钟，两者之间没有任何时间间隔。在柔和的光线下连续吹氮，进行超音波处理。对于冷冻水果，将培养时间增加至4小时。 </li><li>通过＃2滤纸过滤混合物，使用带有真空抽吸的冷藏布氏漏斗。 注意:在此步骤结束时，样品残留物出现在滤纸上;这就是所谓的滤饼。 </li><li>用50mL的100％乙醇冲洗滤饼。保存滤液并将滤饼加入到含有100mL 80％乙醇水溶液的1L圆底烧瓶中。 </li><li>对残留物重复提取过程（步骤2.1.2 - 2.1.4）。 </li><li>将两个滤液合并到另外50 mL的圆底烧瓶中的80％乙醇水溶液。 注意:最终体积应约为300 mL。 </li><li>使用旋转蒸发器在62°C和50 rpm下蒸发溶剂。继续这个过程，直到乙醇不再蒸发。 注意:此过程大约需要45分钟。 </li><li>将样品转移到50mL锥形管中。通过在管的顶部注入氮气10分钟来蒸发乙醇。 注意:需要这一步来确保乙醇的完全蒸发。该步骤中样品的体积约为20mL。 </li><li>在-80℃下将样品冷冻至少24小时。 注意:需要此步骤才能进行更有效的冷冻干燥过程。 </li><li>将样品在-50℃下冷冻约8小时。将样品储存在-20°C。 注意:冷冻干燥器在室内在-50°C下产生强大的真空，以干燥样品，但保留大多数化合物，如息肉烷醇。 </li></ol></li><li>从粗提取物制备纯化的多酚提取物。 <ol><li>称取冷冻干燥的提取样品。 注意:该步骤中提取物的体积约为10mL。 </li><li>向粗提取物中加入两卷（约20mL）氯仿，并在搅拌板中摇动溶液5分钟。 </li><li>将混合物倒入分液漏斗。使粗提取物在RT下倾析1小时以获得纯化的级分。 注意:在此步骤中，将形成两相混合物。 </li><li>从分液漏斗中弃去底层（氯仿相），并将水层收集到干净的烧杯中。 </li><li>加入两倍体积的乙酸乙酯至含水部分，并使用磁力搅拌棒将混合物在室温下搅拌5分钟。 </li><li>用＃2滤纸过滤混合物，使用冷藏的布氏漏斗进行真空抽吸。 </li><li>收集过滤的样品并将其转移到圆底烧瓶中，作为我们用旋转蒸发器。 </li><li>将旋转蒸发器设置为62°C和50 rpm，并蒸发乙酸乙酯约30分钟。 </li><li>将样品在-50℃下冷冻约8小时。将样品转移到50mL锥形管中，并将其储存在-20°C。 </li></ol></li></ol> 3.用浆果提取物处理VSMC <ol><li> VSMCs文化。 <ol><li>如前所述，通过进行酶消化从Sprague-Dawley大鼠胸主动脉分离VSMC。培养VSMC如29所述。 </li><li>使用含有1g / L葡萄糖和补充有10％胎牛血清（FBS），100U / mL青霉素，100mg / mL链霉素和2mM L-谷氨酰胺的DMEM制备用于培养VSMC的完全培养基。将完整的培养基储存在4°C。 </li></ol></li><li> VSMCs与多酚提取物的孵育。 <ol><li>拆分VSMC，丢弃文化重新培养并用磷酸盐缓冲盐水（PBS）洗涤细胞两次。加入4mL PBS和2mL胰蛋白酶EDTA 0.25％。在37℃的CO 2培养箱中孵育细胞5分钟。 <ol><li>收集细胞，将其与2mL完全培养基在15mL离心管中混合，并以1,100×g离心5分钟。弃去上清液，并用1 mL PBS重悬细胞沉淀。使用血细胞计数器计数细胞。 </li><li>在6孔培养板中每孔培养50,000个VSMC，并在含有10％FBS的完全培养基中生长，直到达到90％融合（约3天）。在37℃的湿润5％CO 2培养箱中维持VSMC。 </li></ol></li><li>使用含有1g / L葡萄糖，100U / mL青霉素，100mg / mL链霉素，2mM L-谷氨酰胺和0.5％FBS的DMEM培养基准备处理培养基。将处理介质储存在4°C。 注意:多酚提取物和Ang II使用我直接添加到细胞dium。 </li><li>通过将10毫克提取物溶解在1毫升纯DMEM培养基中，制备10毫克/毫升粗提物（CE）或多酚提纯物（PE）的储备液。将储存液在-20°C下保存在200μL等分试样中。 </li><li>用含有0.5％FBS的2mL处理培养基孵育VSMC，加入CE或PE提取物达到50-500μg/ mL的浓度。通过添加新鲜的多酚提取物每24小时更换培养基。处理细胞3 d。 </li><li>对于用Ang II或其他激素和生长因子治疗，在加入Ang II（100nM）之前，通过在含有0.5％FBS的治疗培养基中孵育细胞使细胞饥饿至少24小时。 注意:建议在添加Ang II之前，在0.5％FBS处理介质中培养和不使用CE和PE。 <ol><li>饥饿24小时后，加入100 nM Ang II。每24小时更换新鲜的CE，PE或Ang II的治疗培养基3 d。 </li></ol></li></ol></li><li>细胞样品的制备<ol><li>在冷PBS中洗涤经过处理的细胞两次，并在冰上用200μL裂解缓冲液裂解。 </li><li>使用细胞刮刀收集细胞裂解物，并将裂解物转移到1.5 mL微量离心管中。在冰上孵育总细胞裂解物20分钟，每5分钟涡旋。 </li><li>以125 W的三次突发超声波处理10秒，每次间隔2秒。通过超声处理将样品保持在冰上。 </li><li>使用595nm的蛋白质测定试剂测量蛋白质浓度。 </li><li>将样品储存在-20°C进行Western印迹分析。 </li></ol></li><li>蛋白质印迹。 <ol><li>从对照未处理和多酚处理或Ang II处理的样品中混合等量的蛋白质（约50μg），用裂解缓冲液，以获得最大总体积为50μL。加入16μL的4x Laemmli样品缓冲液。在75℃下将样品加热5分钟。 </li><li>分离样品在10％SDS-PAGE凝胶中，并在半干转移系统中将其在10V下转移至PDVF膜75分钟，以用特异性抗体进行蛋白质印迹分析。 </li><li>在TBS 0.05％Triton-X100缓冲液（TBS-T）中用2％牛奶封闭膜20分钟。 </li><li>通过用TBS洗涤膜至少三次（每次5分钟），并在室温下用1次抗体孵育1小时或4℃孵育除去牛奶。 </li><li>用TBS-T洗涤膜三次，每次10分钟，并与HRP连接的二级抗体孵育45分钟。 </li><li>用TBS-T洗涤膜三次，每次10分钟，并通过增强的化学发光（ECL）显影。 </li></ol></li></ol>

<ol>
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C., 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=Polyphenols+purified+from+the+Brazilian+aroeira+plant+(Schinus+terebinthifolius,+Raddi)+induce+apoptotic+and+autophagic+cell+death+of+DU145+cells.">Polyphenols purified from the Brazilian aroeira plant (Schinus terebinthifolius, Raddi) induce apoptotic and autophagic cell death of DU145 cells.</a> Anticancer Res. 26 (1A), 379-387 (2006).</li><li>Griendling, K. K., Taubman, M. B., Akers, M., Mendlowitz, M., Alexander, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+phosphatidylinositol-specific+phospholipase+C+from+cultured+vascular+smooth+muscle+cells.">Characterization of phosphatidylinositol-specific phospholipase C from cultured vascular smooth muscle cells.</a> J Biol Chem. 266 (23), 15498-15504 (1991).</li><li>Vuong, T., 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=Role+of+a+polyphenol-enriched+preparation+on+chemoprevention+of+mammary+carcinoma+through+cancer+stem+cells+and+inflammatory+pathways+modulation.">Role of a polyphenol-enriched preparation on chemoprevention of mammary carcinoma through cancer stem cells and inflammatory pathways modulation.</a> J Transl Med. 14, (2016).</li></ol>

作者没有什么可以披露的。

从浆果中分离的多酚含有不同的成分。这里描述的基于乙醇的提取方案允许鉴定存在于粗制和纯化的多酚提取物中的不同水平的酚酸和类黄酮（ 表1 ）。 CE富含没食子酸，阿魏酸，4-O-咖啡酰奎宁酸和5-O-咖啡酰奎宁酸。纯化方法没有显着改变没食子酸和对香豆酸的含量。然而，它提高了3-0-椰油酰奎宁酸的水平，从170.5-235.3ppm和槲皮素从24.5-95ppm。相比之下，在EDTA的纯化过程中阿魏酸和芦丁被丢失。 用50 - 500μg/ mL CE和PE处理VSMC显示，两种提取物均有效减少ERK1 / 2的基础磷酸化。然而，PE显示出在较低浓度下该激酶活性更强的下调:100＆＃181;对于PE，g / mL相对于CE为400-500μg/ mL。这些结果可能反映了在PE中发现的3- O-花青酰奎宁酸或槲皮素的浓度较高。需要在培养细胞中使用单个酚类化合物来鉴定负责ERK1 / 2磷酸化下调的具体化合物。 由BL，RB和BRB 21分离的纯化多酚以及由BL CE引起的ERK1 / 2引起的信号激酶（包括Akt，ERK1 / 2和p38MAPK）的活性降低与本文所示的一致以前的报告。例如，从蓝莓分离的多酚提取物降低了乳腺癌细胞的肿瘤生长，部分通过降低Akt和ERK1 / 2 30的活性。如前所述，这些激酶还参与Ang II 21诱导细胞衰老，表明来自BL，RB和BRB的多酚d减少血管老化和与CVD相关的功能障碍。 正如我们之前报道的，过氧化氢酶和SOD2的表达没有被PE上调，甚至浓度高达500μg/ mL。由于CE在提高过氧化氢酶和SOD2表达方面也无效，这些数据表明在纯化方案中损失的酚类化合物不参与这些抗氧化酶的调节。这些数据还表明，这里显示的纯化方案有效地将酚类化合物与Ang II信号调节，氧化应激和细胞衰老相关。例如，代表性的结果显示，PE强烈地下调Ang II诱导的ERK1 / 2磷酸化。这种激酶的磷酸化的评估是相关的，因为ERK1 / 2活性的抑制阻止Ang II诱导的细胞衰老21 。 在t对以前的方案进行了修改，在这里添加了超声处理，以提高CE的提取率。另外，为了纯化多酚而不是使用C18 Cartridge，如Kim和Lee 27所述 ，这是Queires 等人的一种更传统的方法。 28在这里被采纳。在多酚部分的纯化中加入过滤步骤，使用滤纸除去杂质。在限制方面，超音波和蒸发步骤应根据所用仪器的类型进行仔细监测，因为超声和蒸发的持续时间和温度是本协议中的关键步骤。与本实验室中使用的先前方法27,28 （数据未显示）相比，添加到该方法中的修饰导致多酚的最高产率，这很可能是由于加入超声处理步骤。作为指在方案部分中，该方案可用于各种浆果的冷冻干燥粉末以及冷冻水果。该方法已成功应用于从树莓和黑莓21以及蓝莓和草莓中提取和纯化多酚（数据未显示）。因此，该方法也可用于从其他类型的食物（包括蔬菜）中提取多酚。 总之，这项工作详细介绍了一种快速，经济有效且易于重现的方法，用于从多种酚类中分离出多酚，这样可以保持和浓缩在VSMC中保护氧化应激的化合物。

多酚是在其结构中含有至少一个酚环的化合物，并且大量存在于植物界1中 。人类已经花了数千年的时间消耗植物用于药用，而没有意识到这种化合物的存在2 。许多水果和蔬菜具有一些共享的多酚化合物，尽管量不同，包括类黄酮，二苯乙烯和酚酸3 。虽然多酚通常与五颜六色的水果和蔬菜有关，但这并不是完全正确的。例如，玉米黄质和黄嘌呤存在于不是高度多彩的蔬菜中，例如洋葱和大蒜，其来自the the家庭并且与许多健康益处相关4 。除了与几种健康益处相关5 ，多酚还通过保护它们免受昆虫的影响而为植物服务d紫外线辐射2 。多酚通常在人类饮食中发现，被认为是强大的抗氧化剂，因为它们可以清除活性氧物质（ROS） 6,7,8 。它们还具有抗炎9 ，抗菌10 ，抗高血压11 ，抗致癌12,13属性。 流行病学研究表明，类黄酮和心血管疾病（CVD）发生率16,17和死亡率14,15之间呈负相关。浆果在美国广泛消费，并含有大量的多酚，包括类黄酮。例如，消费黑莓（BL）果汁（300<html> mL / d）8周，血脂异常患者的收缩压明显降低18 。 Jeong et al。 19报道，与服用安慰剂的人相比，每天摄入2.5克黑莓（BRB）提取物的高血压男性和女性的24小时和夜间血压较低。覆盆子（RB）降低血压，同时增加自发性高血压大鼠超氧化物歧化酶（SOD）的表达20 。最近已经显示，BL，RB和BRB降低血管平滑肌细胞（VSMC）中血管紧张素II（Ang II）诱导的ROS和衰老水平21 。另外，来自BL提取物的花色素苷分数降低了诱导型一氧化氮合酶（iNOS）的表达，并抑制了脂多糖（LPS）刺激下的核因子κB（NF-κB）和细胞外信号调节激酶（ERK）的活性J774细胞ass ="xref"&gt; 22。 BRB提取物在体外降低NF-κB活化和环氧合酶2（COX-2）表达23 ，改善脂质分布，并预防喂食高脂肪饮食的小鼠动脉粥样硬化病变形成24 。被认为是浆果中最丰富的类黄酮的花青素通过减少肿瘤坏死因子α（TNF-α）产生25并减少VSMC 26的增殖和迁移来调节LPS刺激的RAW 264.7巨噬细胞中的炎症反应。 由于越来越多的兴趣了解多酚在人体健康和疾病中的作用，重要的是优化提取方法。溶剂萃取广泛用于该目的，因为其具有成本效益且易于重现。在本研究中，用乙醇和超声辅助提取物一起进行溶剂萃取n方法，改编自Kim和Lee 27 。使用氯仿和乙酸乙酯对粗提物（CE）进行纯化和分级，得到从Queires 等 28获得的纯化提取物（PE）级分。此外，比较了在降低ERK1 / 2的基础磷酸化时，来自BL的粗制品与纯化多酚提取物的功效，并提供了纯化的BL多酚提取物对血管紧张素II诱导的VSMC信号降低的抑制作用的代表性实例。

<table border="0" cellpadding="0" cellspacing="0" width="971">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Angiotensin II</td>
			<td style="width:157px;">Sigma-Aldrich, Inc.</td>
			<td style="width:168px;">A9525-10MG</td>
			<td style="width:343px;">Treatment of VSMCs</td>
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			<td height="21" style="height:21px;width:303px;">&#946;-actin</td>
			<td style="width:157px;">Sigma-Aldrich, Inc.</td>
			<td style="width:168px;">A2228</td>
			<td>Primary antibody (1:5000)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Blackberry fruit</td>
			<td style="width:157px;">Mercer Foods</td>
			<td style="width:168px;"></td>
			<td>Freeze-dried blackberry powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Catalase&#160;</td>
			<td style="width:157px;">Calbiochem</td>
			<td style="width:168px;">219010</td>
			<td>Primary antibody (1:1000)</td>
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			<td height="21" style="height:21px;">Chloroform</td>
			<td style="width:157px;">Biotech Grd, Inc.</td>
			<td style="width:168px;">97064-678</td>
			<td>Preparation of purified polyphenol extracts</td>
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			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:157px;">Mediatech, Inc.</td>
			<td style="width:168px;">10-014-CV</td>
			<td>Culture of VSMCs</td>
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			<td height="21" style="height:21px;">Ethanol (absolute molecular biology grade)</td>
			<td style="width:157px;">Sigma-Aldrich, Inc.</td>
			<td style="width:168px;">E7023-500ML</td>
			<td>Preparation of polyphenol extracts&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Ethylacetate</td>
			<td style="width:157px;">Sigma-Aldrich, Inc.</td>
			<td style="width:168px;">439169</td>
			<td>Preparation of purified polyphenol extracts</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:303px;">ERK1/2</td>
			<td style="width:157px;">Cell Signaling Technology, Inc.</td>
			<td style="width:168px;">9102S</td>
			<td>Primary antibody (1:500)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">EDTA, 500 mM, pH 8.0</td>
			<td style="width:157px;">Teknova, Inc.</td>
			<td style="width:168px;">E0306</td>
			<td>Lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Freeze-Dryer</td>
			<td style="width:157px;">Labconco</td>
			<td style="width:168px;">VirTis Benchtop K</td>
			<td>Preparation of polyphenol extracts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">FBS</td>
			<td style="width:157px;">Seradigm</td>
			<td style="width:168px;">1400-500</td>
			<td>Cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">HEPES</td>
			<td style="width:157px;">Sigma-Aldrich, Inc.</td>
			<td style="width:168px;">H3375</td>
			<td>Lysis buffer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">NaCl</td>
			<td style="width:157px;">EMD Millipore, Inc.</td>
			<td style="width:168px;">7760</td>
			<td>Lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">NaF</td>
			<td style="width:157px;">J.T.Baker, Inc.</td>
			<td style="width:168px;">3688-01&#160;</td>
			<td>Lysis buffer</td>
		</tr>
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			<td height="25" style="height:25px;width:303px;">Na3VO4</td>
			<td style="width:157px;">Sigma-Aldrich, Inc.</td>
			<td style="width:168px;">450243</td>
			<td>Lysis buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:303px;">Na4P2O7 , decahydrate</td>
			<td style="width:157px;">Sigma-Aldrich, Inc.</td>
			<td style="width:168px;">S-9515</td>
			<td>Lysis buffer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:303px;">phospho ERK1/2&#160;</td>
			<td style="width:157px;">Cell Signaling Technology, Inc.</td>
			<td style="width:168px;">9101S</td>
			<td>Primary antibody (1:1000)</td>
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			<td height="21" style="height:21px;">Protease inhibitor cocktail</td>
			<td style="width:157px;">Sigma-Aldrich, Inc.</td>
			<td style="width:168px;">P8340-5ml</td>
			<td>Lysis buffer</td>
		</tr>
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			<td height="41" style="height:41px;width:303px;">Protein assay dye reagent</td>
			<td style="width:157px;">Bio-Rad Laboratories, Inc.</td>
			<td style="width:168px;">500-0006</td>
			<td>Protein concentration Measurement</td>
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			<td height="21" style="height:21px;width:303px;">PVDF transfer membrane</td>
			<td style="width:157px;">Thermo Scientific, Inc.</td>
			<td style="width:168px;">88518</td>
			<td>Western blots</td>
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			<td style="width:157px;">Buchi Labortechnik</td>
			<td style="width:168px;">Rotavapor 
			R3000</td>
			<td>Preparation of polyphenol extracts</td>
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			<td height="21" style="height:21px;">Sterile water</td>
			<td style="width:157px;">Mediatech, Inc.</td>
			<td style="width:168px;">25-055-CV</td>
			<td>Preparation of polyphenol extracts</td>
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			<td height="21" style="height:21px;width:303px;">Sonicator</td>
			<td style="width:157px;">QSonica, LLC</td>
			<td style="width:168px;">Q125</td>
			<td>Preparation of cell extracts</td>
		</tr>
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			<td height="21" style="height:21px;width:303px;">SOD2</td>
			<td style="width:157px;">Enzo Life Sciences, Inc.</td>
			<td style="width:168px;">ADI-SOD-110-F</td>
			<td>Primary antibody (1:1000)</td>
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			<td style="width:157px;">Sigma-Aldrich, Inc.</td>
			<td style="width:168px;">X100</td>
			<td>Western blots</td>
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			<td style="width:157px;">GE Healthcare, Inc.</td>
			<td style="width:168px;">28317-241</td>
			<td>Preparation of polyphenol extracts</td>
		</tr>
	</tbody>
</table>

extraction and purification of polyphenols from freeze-dried berry powder for the treatment of vascular smooth muscle cells in vitro

流行病学研究表明，增加的类黄酮摄入与美国（美国）和欧洲的心血管疾病（CVD）死亡率降低相关。浆果在美国广泛消费，多酚含量高。已经显示多酚与许多分子靶标相互作用并发挥许多积极的生物功能，包括抗氧化剂，抗炎和心脏保护作用。从黑莓（BL），覆盆子（RB）和黑莓（BRB）分离的多酚可以减少血管紧张素II（Ang II）的氧化应激和细胞衰老。这项工作提供了从冻干浆果制备多酚提取物的方案的详细描述。使用80％乙醇水溶液和超声波辅助提取方法，从冷冻干燥的浆果粉中进行多酚提取。粗提物进一步纯化，用氯仿和乙酸乙酯分级分离，分别。在培养物中的血管平滑肌细胞（VSMC）上测试粗制和纯化提取物的作用。

以前已经证明，从BL，RB和BRB分离的多酚提取物降低VSMC响应Ang II 21的衰老。已经显示这些纯化的多酚提取物通过降低Akt，p38丝裂原活化蛋白激酶（MAPK）和ERK1 / 2的磷酸化来调节Ang II信号传导。 BL通过降低NADPH氧化酶（Nox）1的表达来防止衰老，NADPH氧化酶（Nox）1是产生超氧化物阴离子并被Ang II强烈上调的酶。相比之下，RB和BRB通过Nox1非依赖性机制防止衰老，因为它们增加抗氧化酶SOD1，SOD2和谷胱甘肽过氧化物酶1（GPx-1）的表达。 BL不能增加SOD2表达，而没有提取物减弱Ang II 21对过氧化氢酶的下调。 BL的重点是确定BL对SOD2和过氧化氢酶表达的影响是否缺乏可以通过纯化过程中多酚化合物的损失或提取物中某些多酚化合物浓度不足来解释。将VSMC与含有0.5％FBS的培养基中的50-500μg/ mL CE（ 图1A ）或PE（ 图1B ）孵育三天。 CE或PE均不会在任何测试浓度下增加SOD2或过氧化氢酶水平。作为阳性对照，测量了ERK1 / 2磷酸化，发现CE降低了高于300μg/ mL浓度的激酶的磷酸化。相比之下，PE在约100μg/ mL时有效（ 图1B ）。在浓度为200-500μg/ mL时观察到低磷酸化水平。这些数据支持以前的观察结果，显示200μg/ mL BL PE足以减少Ang II信号21 。这些结果表明较高浓度的多酚cPE含量与CE相比，可以解释该提取物在降低ERK1 / 2磷酸化方面的效率。为了测试这个想法，比较了两种提取物的多酚组成。如前所述，通过HPLC进行CE中多酚化合物的鉴定和定量（ 表1 ）。在CE中，与CE相比，3- O-花红醌和槲皮素在较高水平存在，而阿魏酸和芦丁仅在CE中发现。接下来，在加入100nM Ang II之前，用200μg/ mL BL PE处理VSMC 24小时（ 图1C ）。如先前报道的21 ，BL多酚提取物降低Ang II诱导的ERK1 / 2磷酸化，但对过氧化氢酶和SOD2表达没有影响。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55605/55605fig1.jpg" /> <strong&gt;图1:黑莓多酚减少VSMC中的基底和Ang II诱导的ERK1 / 2磷酸化。在10％FBS中以约90％汇合培养的VSMC以50-500μg/ mL BL粗提物（CE）（A），纯化提取物（PE）（ B ）或200μg/ mL PE（ C ）在0.5 ％FBS DMEM培养基3 d。 C ）用BL PE孵育24小时后，加入Ang II（100nM），将细胞孵育3天。新鲜提取物和Ang II的培养基每天更换。然后将细胞洗涤并裂解，并将总细胞提取物在10％PAGE-SDS凝胶中分离。用针对磷酸化ERK1 / 2（Thr202 / Tyr204），ERK1 / 2，过氧化氢酶和SOD2的兔抗体和针对β-肌动蛋白的小鼠抗体测试Western印迹。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55605/55605fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <table border="1" fo:keep-together.within-page ="1"fo:keep-with-next.within-page ="always"&gt; <tbody><tr><td> 分析物（ppm） </td><td> PE </td><td> CE </td></tr><tr><td colspan="3"> 酚酸 </td></tr><tr><td>没食子酸</td><td> 243.5 </td><td> 321.9 </td></tr><tr><td>对香豆酸</td><td> 32.9 </td><td> 46.5 </td></tr><tr><td>阿魏酸</td><td> - </td><td> 236.5 </td></tr><tr><td> 绿原酸 </td><td></td><td></td></tr><tr><td> 3-O-咖啡酰奎宁酸</td><td> 235.3 </td><td> 170.5 </td></tr><tr><td> 4-O-咖啡酰奎宁酸</td><td> 13 </td><td> 76.9 </td></tr><tr><td> 5-O-咖啡酰奎宁酸</td><td> 14.1 </td><td> 49.9 </td></tr><tr><td colspan="3"> 类黄酮 </td></tr><tr><td colspan="3"> 黄酮 </td></tr><tr><td>槲皮素</td><td> 95 </td><td> 24.5 </td></tr><tr><td colspan="3"> 黄烷酮 </td></tr><tr><td>芦丁</td><td> - </td><td> 37.8 </td></tr></tbody></table> 表1: 粗多酚纯化提取物中黑莓多酚组成的分析。使用高效液相色谱（HPLC）分析粗提物（CE）和纯化提取物（PE）中的酚酸和类黄酮。分析物的浓度用ppm表示。 BL PE中多酚的组成最近出版21，并添加到表中以与CE进行比较。

Watch this Scientific Journal Video about Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro at JoVE.com

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

本工作详细介绍了从冷冻干燥的浆果粉中制备富含多酚的提取物的逐步方法。此外，它提供了在使用血管平滑肌细胞（VSMC）的肽激素血管紧张素II（Ang II）存在下如何在细胞培养物中使用这些富含多酚的提取物的详细描述。

从冷冻干燥的浆果粉中提取和纯化多酚用于治疗血管平滑肌细胞<em&gt;体外</em

Epidemiological studies indicate that increased flavonoid intake correlates with decreased mortality due to cardiovascular diseases (CVD) in the United ...

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Nanyang Technological University

Research

JoVE Journal

Chemistry

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

19.0K 次观看.  University of Arkansas for Medical Sciences. 本工作详细介绍了从冷冻干燥的浆果粉中制备富含多酚的提取物的逐步方法。此外，它提供了在使用血管平滑肌细胞（VSMC）的肽激素血管紧张素II（Ang II）存在下如何在细胞培养物中使用这些富含多酚的提取物的详细描述。 

视频: Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in starch. Iodine forms complexes with both amylose and long chains within amylopectin. After the addition of iodine to a starch sample, the maximum absorption of amylose and amylopectin occurs at 620 and 550 nm, respectively. The amylose/amylopectin ratio can be estimated from the ratio of the 620 and 550 nm absorbance values and comparing them to a standard curve in which specific known concentrations are plotted against absorption values. This high throughput, inexpensive method is reliable and reproducible, allowing the evaluation of large populations of potato clones.&#160;

快速高通量直链淀粉测定冻干马铃薯块茎样品

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in ...

科研

化学

Folding and Characterization of a Bio-responsive Robot from DNA Origami

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both stimuli and cargo can be tailored according to specific needs. Here we describe the DNA nanorobot fabrication protocol, with the use of the DNA origami technique. The procedure initiates by mixing short single-strand DNA staples into a stock mixture which is then added to a long, circular, single-strand DNA scaffold in presence of a folding buffer. A standard thermo cycler is programmed to gradually lower the mixing reaction temperature to facilitate the staples-to-scaffold annealing, which is the guiding force behind the folding of the nanorobot. Once the 60 hr folding reaction is complete, excess staples are discarded using a centrifugal filter, followed by visualization via agarose-gel electrophoresis (AGE). Finally, successful fabrication of the nanorobot is verified by transmission electron microscopy (TEM), with the use of uranyl-formate as negative stain.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM).

折叠的DNA折纸一个生物反应机器人与表征

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both ...

Synthesis and Purification of Iodoaziridines Involving Quantitative Selection of the Optimal Stationary Phase for Chromatography

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. Diiodomethyllithium is prepared by the deprotonation of diiodomethane with LiHMDS, in a THF/diethyl ether mixture, at -78 &#176;C in the dark. These conditions are essential for the stability of the LiCHI2 reagent generated. The subsequent dropwise addition of N-Ts aldimines to the preformed diiodomethyllithium solution affords an amino-diiodide intermediate, which is not isolated. Rapid warming of the reaction mixture to 0 &#176;C promotes cyclization to afford iodoaziridines with exclusive cis-diastereoselectivity. The addition and cyclization stages of the reaction are mediated in one reaction flask by careful temperature control.
Due to the sensitivity of the iodoaziridines to purification, assessment of suitable methods of purification is required. A protocol to assess the stability of sensitive compounds to stationary phases for column chromatography is described. This method is suitable to apply to new iodoaziridines, or other potentially sensitive novel compounds. Consequently this method may find application in range of synthetic projects. The procedure involves firstly the assessment of the reaction yield, prior to purification, by 1H NMR spectroscopy with comparison to an internal standard. Portions of impure product mixture are then exposed to slurries of various stationary phases appropriate for chromatography, in a solvent system suitable as the eluent in flash chromatography. After stirring for 30 min to mimic chromatography, followed by filtering, the samples are analyzed by 1H NMR spectroscopy. Calculated yields for each stationary phase are then compared to that initially obtained from the crude reaction mixture. The results obtained provide a quantitative assessment of the stability of the compound to the different stationary phases; hence the optimal can be selected. The choice of basic alumina, modified to activity IV, as a suitable stationary phase has allowed isolation of certain iodoaziridines in excellent yield and purity.

A protocol for the diastereoselective one-pot preparation of cis-N-Ts-iodoaziridines is described. The generation of diiodomethyllithium, addition to N-Ts aldimines and cyclization of the amino gem-diiodide intermediate to iodoaziridines is demonstrated. Also included is a protocol to rapidly and quantitatively assess the most appropriate stationary phase for purification by chromatography.

合成和Iodoaziridines纯化涉及定量选择最优固定相的色谱

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. ...

Supercritical Nitrogen Processing for the Purification of Reactive Porous Materials

Supercritical fluid extraction and drying methods are well established in numerous applications for the synthesis and processing of porous materials. Herein, nitrogen is presented as a novel supercritical drying fluid for specialized applications such as in the processing of reactive porous materials, where carbon dioxide and other fluids are not appropriate due to their higher chemical reactivity. Nitrogen exhibits similar physical properties in the near-critical region of its phase diagram as compared to carbon dioxide: a widely tunable density up to ~1 g ml-1, modest critical pressure (3.4 MPa), and small molecular diameter of ~3.6 &#197;. The key to achieving a high solvation power of nitrogen is to apply a processing temperature in the range of 80-150 K, where the density of nitrogen is an order of magnitude higher than at similar pressures near ambient temperature. The detailed solvation properties of nitrogen, and especially its selectivity, across a wide range of common target species of extraction still require further investigation. Herein we describe a protocol for the supercritical nitrogen processing of porous magnesium borohydride.

Nitrogen is an effective supercritical fluid for extraction or drying processes due to its small molecular size, high density in the near-liquid supercritical regime, and chemical inertness. We present a supercritical nitrogen drying protocol for the purification treatment of reactive, porous materials.

超临界氮处理的活性多孔材料的净化

Supercritical fluid extraction and drying methods are well established in numerous applications for the synthesis and processing of porous materials. ...

X-ray Powder Diffraction in Conservation Science: Towards Routine Crystal Structure Determination of Corrosion Products on Heritage Art Objects

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder diffraction (XRPD) is presented in detail via two case studies.
The first material under investigation was sodium copper formate hydroxide oxide hydrate, Cu4Na4O(HCOO)8(OH)2&#8729;4H2O (sample 1) which forms on soda glass/copper alloy composite historic objects (e.g., enamels) in museum collections, exposed to formaldehyde and formic acid emitted from wooden storage cabinets, adhesives, etc. This degradation phenomenon has recently been characterized as &#34;glass induced metal corrosion&#34;.
For the second case study, thecotrichite, Ca3(CH3COO)3Cl(NO3)2&#8729;6H2O (sample 2), was chosen, which is an efflorescent salt forming needlelike crystallites on tiles and limestone objects which are stored in wooden cabinets and display cases. In this case, the wood acts as source for acetic acid which reacts with soluble chloride and nitrate salts from the artifact or its environment.
The knowledge of the geometrical structure helps conservation science to better understand production and decay reactions and to allow for full quantitative analysis in the frequent case of mixtures.

Modern high resolution X-ray powder diffraction (XRPD) in the laboratory is used as an efficient tool to determine crystal structures of long-known corrosion products on historic objects.

X射线粉末衍射在保护科学:迈向文物艺术品腐蚀产物的常规晶体结构测定

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

蛋白质生物缀合物的合成<em&gt;通过</em&gt;半胱氨酸马来酰亚胺化学

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Microfluidic Pneumatic Cages: A Novel Approach for In-chip Crystal Trapping, Manipulation and Controlled Chemical Treatment

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. Herein, we introduce a microfluidic-based technology, employing a double-layer microfluidic device, which can trap and localize in situ and ex situ synthesized structures on microfluidic channel surfaces. Crucially, we show how such a device can be used to conduct controlled chemical reactions onto on-chip trapped structures and we demonstrate how the synthetic pathway of a crystalline molecular material and its positioning inside a microfluidic channel can be precisely modified with this technology. This approach provides new opportunities for the controlled assembly of structures on surface and for their subsequent treatment.

Herein, we describe the fabrication and operation of a double-layer microfluidic system made of polydimethylsiloxane (PDMS). We demonstrate the potential of this device for trapping, directing the coordination pathway of a crystalline molecular material and controlling chemical reactions onto on-chip trapped structures.

微流控气动笼:在芯片水晶捕获，处理的新方法和控制化学处理

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. ...

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. 
Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

We present a convenient solid-phase extraction coupled to high-pressure liquid chromatography (HPLC) with electrochemical detection (ECD) for simultaneous determination of three monoamine neurotransmitters and two of their metabolites in infants' urine. We also identify the metabolite MHPG as a potential biomarker for the early diagnosis of brain damage for infants.

用高压液相色谱法提取和分析儿茶酚胺神经递质及其代谢物的简便方法

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and ...

Nanothermite with Meringue-like Morphology: From Loose Powder to Ultra-porous Objects

The goal of the protocol described in this article is to prepare aluminothermic compositions (nanothermites) in the form of porous, monolithic objects. Nanothermites are combustible materials made up of inorganic fuel and an oxidizer. In nanothermite foams, aluminum is the fuel and aluminum phosphate and tungsten trioxide are the oxidizing moieties. The highest flame propagation velocities (FPVs) in nanothermites are observed in loose powders and FPVs are strongly decreased by pelletizing nanothermite powders. From a physical standpoint, nanothermite loose powders are metastable systems. Their properties can be altered by unintentional compaction induced by shocks or vibrations or by the segregation of particles over time by settling phenomena, which originates from the density differences of their components. Moving from a powder to an object is the challenge that must be overcome to integrate nanothermites in pyrotechnic systems. Nanothermite objects must have both a high open porosity and good mechanical strength. Nanothermite foams meet both of these criteria, and they are prepared by dispersing a nano-sized aluminothermic mixture (Al/WO3) in orthophosphoric acid. The reaction of aluminum with the acid solution gives the AlPO4 &#34;cement&#34; in which Al and WO3 nanoparticles are embedded. In nanothermite foams, aluminum phosphate plays the dual role of binder and oxidizer. This method can be used with tungsten trioxide, which is not altered by the preparation process. It could probably be extended to some oxides, which are commonly used for the preparation of high performance nanothermites. The WO3-based nanothermite foams described in this article are particularly insensitive to impact and friction, which makes them far safer to handle than loose Al/WO3 powder. The fast combustion of these materials has interesting applications in pyrotechnic igniters. Their use in detonators as primers would require the incorporation of a secondary explosive in their composition.

This manuscript describes the synthesis of combustible aluminophosphate matrices by the reaction of orthophosphoric acid (H3PO4) with aluminum nanopowder. When this reaction is carried out with excess aluminum in the presence of tungsten trioxide nanopowder, it leads to a solid, porous nanothermite foam.

Nanothermite 与蛋白酥样形态学: 从松散的粉末到超多孔物体

The goal of the protocol described in this article is to prepare aluminothermic compositions (nanothermites) in the form of porous, monolithic objects. ...

Separation of Aldehydes and Reactive Ketones from Mixtures Using a Bisulfite Extraction Protocol

The purification of organic compounds is an essential component of routine synthetic operations. The ability to remove contaminants into an aqueous layer by generating a charged structure provides an opportunity to use extraction as a simple purification technique. By combining the use of a miscible organic solvent with saturated sodium bisulfite, aldehydes and reactive ketones can be successfully transformed into charged bisulfite adducts that can then be separated from other organic components of a mixture by the introduction of an immiscible organic layer. Here, we describe a simple protocol for the removal of aldehydes, including sterically-hindered neopentyl aldehydes and some ketones, from chemical mixtures. Ketones can be separated if they are sterically unhindered cyclic or methyl ketones. For aliphatic aldehydes and ketones, dimethylformamide is used as the miscible solvent to improve removal rates. The bisulfite addition reaction can be reversed by basification of the aqueous layer, allowing for the re-isolation of the reactive carbonyl component of a mixture.

Here, we present a protocol to remove aldehydes and reactive ketones from mixtures by a liquid-liquid extraction protocol directly with saturated sodium bisulfite in a miscible solvent. This combined protocol is rapid and facile to perform. The aldehyde or ketone can be re-isolated by the basification of the aqueous layer.

用亚硫酸氢萃取协议分离混合物中的醛和反应性酮

The purification of organic compounds is an essential component of routine synthetic operations. The ability to remove contaminants into an aqueous layer ...