Name: extraction and purification of polyphenols from freeze-dried berry powder for the treatment of vascular smooth muscle cells in vitro
Uploaded: 2017-07-05T14:00:00.000+00:00
Duration: 12 min
Description: 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

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>

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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?...

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><tbody><tr><td>Angiotensin II</td><td>Sigma-Aldrich, Inc.</td><td>A9525-10MG</td><td>Treatment of VSMCs</td></tr><tr><td>&amp;beta;-actin</td><td>Sigma-Aldrich, Inc.</td><td>A2228</td><td>Primary antibody (1:5000)</td></tr><tr><td>Blackberry fruit</td><td>Mercer Foods</td><td></td><td>Freeze-dried blackberry powder</td></tr><tr><td>Catalase&amp;nbsp;</td><td>Calbiochem</td><td>219010</td><td>Primary antibody (1:1000)</td></tr><tr><td>Chloroform</td><td>Biotech Grd, Inc.</td><td>97064-678</td><td>Preparation of purified polyphenol extracts</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM )</td><td>Mediatech, Inc.</td><td>10-014-CV</td><td>Culture of VSMCs</td></tr><tr><td>Ethanol (absolute molecular biology grade)</td><td>Sigma-Aldrich, Inc.</td><td>E7023-500ML</td><td>Preparation of polyphenol extracts&amp;nbsp;</td></tr><tr><td>Ethylacetate</td><td>Sigma-Aldrich, Inc.</td><td>439169</td><td>Preparation of purified polyphenol extracts</td></tr><tr><td>ERK1/2</td><td>Cell Signaling Technology, Inc.</td><td>9102S</td><td>Primary antibody (1:500)</td></tr><tr><td>EDTA, 500 mM, pH 8.0</td><td>Teknova, Inc.</td><td>E0306</td><td>Lysis buffer</td></tr><tr><td>Freeze-Dryer</td><td>Labconco</td><td>VirTis Benchtop K</td><td>Preparation of polyphenol extracts</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Seradigm</td><td>1400-500</td><td>Cell culture</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich, Inc.</td><td>H3375</td><td>Lysis buffer&amp;nbsp;</td></tr><tr><td>NaCl</td><td>EMD Millipore, Inc.</td><td>7760</td><td>Lysis buffer</td></tr><tr><td>NaF</td><td>J.T.Baker, Inc.</td><td>3688-01&amp;nbsp;</td><td>Lysis buffer</td></tr><tr><td>Na&lt;sub&gt;3&lt;/sub&gt;VO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich, Inc.</td><td>450243</td><td>Lysis buffer</td></tr><tr><td>Na&lt;sub&gt;4&lt;/sub&gt;P&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt; , decahydrate</td><td>Sigma-Aldrich, Inc.</td><td>S-9515</td><td>Lysis buffer</td></tr><tr><td>phospho ERK1/2&amp;nbsp;</td><td>Cell Signaling Technology, Inc.</td><td>9101S</td><td>Primary antibody (1:1000)</td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma-Aldrich, Inc.</td><td>P8340-5ml</td><td>Lysis buffer</td></tr><tr><td>Protein assay dye reagent</td><td>Bio-Rad Laboratories, Inc.</td><td>500-0006</td><td>Protein concentration Measurement</td></tr><tr><td>PVDF transfer membrane</td><td>Thermo Scientific, Inc.</td><td>88518</td><td>Western blots</td></tr><tr><td>Rotatory Evaporator</td><td>Buchi Labortechnik</td><td>Rotavapor&lt;br/&gt; R3000</td><td>Preparation of polyphenol extracts</td></tr><tr><td>Sterile water</td><td>Mediatech, Inc.</td><td>25-055-CV</td><td>Preparation of polyphenol extracts</td></tr><tr><td>Sonicator</td><td>QSonica, LLC</td><td>Q125</td><td>Preparation of cell extracts</td></tr><tr><td>SOD2</td><td>Enzo Life Sciences, Inc.</td><td>ADI-SOD-110-F</td><td>Primary antibody (1:1000)</td></tr><tr><td>Triton-X-100</td><td>Sigma-Aldrich, Inc.</td><td>X100</td><td>Western blots</td></tr><tr><td>Whatman #2 filter paper</td><td>GE Healthcare, Inc.</td><td>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?...

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 ...

extraction-purification-polyphenols-from-freeze-dried-berry-powder

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

Colletotrichum fioriniae Development in Water and Chloroform-based Blueberry and Cranberry Floral Extracts

To accurately monitor the phenology of the bloom period and the temporal dynamics of floral chemical cues on fungal fruit rotting pathogens, floral extraction methods and coverslip bioassays were developed utilizing Colletotrichum fioriniae. In blueberry and cranberry, this pathogen is optimally controlled by applying fungicides during the bloom period because of the role flowers play in the initial stages of infection. The protocol detailed here describes how floral extracts (FE) were obtained using water-, chloroform-, and field rainwater-based methods for later use in corresponding glass coverslip bioassays. Each FE served to provide a different set of information: response of C. fioriniae to mobilized floral chemical cues in water (water-based), pathogen response to flower and fruit surface waxes (chloroform-based), and field-based monitoring of collected floral rainwater, moving in vitro observations to an agricultural setting. The FE is broadly described as either water- or chloroform-based, with an appropriate bioassay described to compensate for the inherent differences between these two materials. Rainwater that had run off flowers was collected in unique devices for each crop, alluding to the flexibility and application of this approach for other crop systems. The bioassays are quick, inexpensive, simple, and provide the ability to generate spatiotemporal and site-specific information about the presence of stimulatory floral compounds from various sources. This information will ultimately better inform disease management strategies, as FE decrease the time needed for infection to occur, thus providing insight into changing risks for pathogen infection over the growing season.

Colletotrichum fioriniae Development in Water and Chloroform-based Blueberry and Cranberry Floral Extracts

Here, bioassays designed to monitor the development of a fungal pathogen, Colletotrichum fioriniae, in the presence of blueberry or cranberry floral extracts on glass coverslips are described. Water-, chloroform-, and field rainwater- based floral extraction techniques are detailed as well as insight into how this information can be applied.

Fioriniae水和氯蓝莓和越橘花提取物的研究进展

To accurately monitor the phenology of the bloom period and the temporal dynamics of floral chemical cues on fungal fruit rotting pathogens, floral ...

科研

环境科学

Semi-Targeted Ultra-High-Performance Chromatography Coupled to Mass Spectrometry Analysis of Phenolic Metabolites in Plasma of Elderly Adults

A group of 23 elderly persons was given functional meals (a beverage and a muffin) specially formulated for the prevention of sarcopenia (age-related loss of muscle mass). Plasma samples were taken at the beginning of the intervention and after 30 days of consuming the functional meals. A semi-targeted ultra-high-performance chromatography coupled with tandem mass (UPLC-MS/MS) analysis was carried out to identify phenolic compounds and their metabolites. Plasma proteins were precipitated with ethanol and the samples were concentrated and resuspended in the mobile phase (1:1 acetonitrile: water) before injection into the UPLC-MS/MS instrument. Separation was carried out with a C18 reverse-phase column, and compounds were identified using their experimental mass, isotopic distribution, and fragment pattern. Compounds of interest were compared to those of data banks and the internal semi-targeted library. Preliminary results showed that the major metabolites identified after the intervention were phenylacetic acid, glycitin, 3-hydroxyphenylvaleric acid, and gomisin M2.

The goal of this protocol is to detect phenolic metabolites in plasma using a semi-targeted chromatography-mass spectrometry method.

老年人血浆中酚类代谢物的半靶向超高效色谱耦合质谱分析

A group of 23 elderly persons was given functional meals (a beverage and a muffin) specially formulated for the prevention of sarcopenia (age-related loss ...

生物化学

基于多元醇的微波辅助萃取，可获得酚类和抗氧化剂的收率

Microwave-Assisted Extraction of Phenolic Compounds and Antioxidants for Cosmetic Applications Using Polyol-Based Technology

The utilization of polyols as green solvents for extracting bioactive compounds from plant materials has gained attention due to their safety and inert behavior with plant bioactive chemicals. This study explores the sustainable extraction of phenolic compounds and natural antioxidants from coffee silverskin using the microwave-assisted extraction (MAE) method with polyol-based solvents: glycerin, propylene glycol (PG), butylene glycol (BG), methylpropanediol (MPD), isopentyldiol (IPD), pentylene glycol, 1,2-hexanediol, and hexylene glycol (HG). A comparative analysis was conducted on conventional and non-conventional solvent extractions, focusing on their impact on the bioactive compounds of MAE, encompassing parameters such as total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activities like the 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay (DPPH), the 2,2&#8242;-azino-bis(-3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging assay (ABTS), and the ferric reducing antioxidant power assay (FRAP). The highest values were observed for TPC with aqueous-1,2-hexanediol extraction (52.0 &#177; 3.0 mg GAE/g sample), TFC with aqueous-1,2-hexanediol extraction (20.0 &#177; 1.7 mg QE/g sample), DPPH with aqueous-HG&#160;extraction (13.6 &#177; 0.3 mg TE/g sample), ABTS with aqueous-pentylene glycol extraction (8.2 &#177; 0.1 mg TE/g sample), and FRAP with aqueous-HG extraction (21.1 &#177; 1.3 mg Fe (II) E/g sample). This research aims to advance eco-friendly extraction technology through natural plant components, promoting sustainability by minimizing hazardous chemical use while reducing time and energy consumption, with potential applications in cosmetics.

作者聚焦：使用基于多元醇的微波辅助技术对生物活性化合物进行环保萃取

This protocol details the utilization of a polyol-based microwave-assisted extraction method for extracting phenolic compounds and natural antioxidants, representing a practical and environmentally sustainable approach to the development of ready-to-use extracts.

使用基于多元醇的技术微波辅助萃取用于化妆品应用的酚类化合物和抗氧化剂

The utilization of polyols as green solvents for extracting bioactive compounds from plant materials has gained attention due to their safety and inert ...

化学

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

鼠冠状动脉血管平滑肌细胞的分离

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

生物学

Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of vasoconstriction and vasorelaxation. After isolating the IPA and VSMCs, the characteristics of vascular responses in physiological and pathological conditions can be assessed in the absence of extrinsic factors such as nerve signals, hormones, cytokines, etc. Thus, the IPA and VSMCs serve as excellent models for studying vascular physiology/pathophysiology, along with various experimental investigations, such as modulation by pharmacological agents, patch-clamp electrophysiological analysis, calcium imaging, etc. Here, we have used a technique for isolating the IPA to investigate vascular responses in an organ bath setup. IPA segments were mounted on the organ bath chamber via intraluminal wires and stimulated by various pharmacological agents. The changes in IPA vascular tone (i.e., vasoconstriction and vasorelaxation), were recorded using an isometric force transducer and physiological data analysis software program. We implemented several experimental protocols, which can be adapted to investigate the mechanisms of vasorelaxation/vasoconstriction for studying the pharmacological activities of phytochemical or synthetic drugs. The protocols can also be used to evaluate drugs&#39; roles in modulating various diseases, including pulmonary arterial hypertension. The IPA model allows us to investigate the concentration-response curve, which is crucial in assessing drugs&#39; pharmacodynamic parameters.

Vascular responses of arterial pulmonary circulation can be explored using intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs). The present study describes the isolation of IPA in detail and the protocols used for investigating vasorelaxation in response to physiological stimuli.

分离肺内动脉和平滑肌细胞以研究血管反应

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of ...

医学

Network Pharmacology Prediction and Metabolomics Validation of the Mechanism of Fructus Phyllanthi against Hyperlipidemia

Hyperlipidemia has become a leading risk factor for cardiovascular diseases and liver injury worldwide. Fructus Phyllanthi (FP) is an effective drug against hyperlipidemia in Traditional Chinese Medicine (TCM) and Indian Medicine theories, however the potential mechanism requires further exploration. The present research aims to reveal the mechanism of FP against hyperlipidemia based on an integrated strategy combining network pharmacology prediction with metabolomics validation. A high-fat diet (HFD)-induced mice model was established by evaluating the plasma lipid levels, including total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). Network pharmacology was applied to find out the active ingredients of FP and potential targets against hyperlipidemia. Metabolomics of plasma and liver were performed to identify differential metabolites and their corresponding pathways among the normal group, model group, and intervention group. The relationship between network pharmacology and metabolomics was further constructed to obtain a comprehensive view of the process of FP against hyperlipidemia. The obtained key target proteins were verified by molecular docking. These results reflected that FP improved the plasma lipid levels and liver injury of hyperlipidemia induced by a HFD. Gallic acid, quercetin, and beta-sitosterol in FP were demonstrated as the key active compounds. A total of 16 and six potential differential metabolites in plasma and liver, respectively, were found to be involved in the therapeutic effects of FP against hyperlipidemia by metabolomics. Further, integration analysis indicated that the intervention effects were associated with CYP1A1, AChE, and MGAM, as well as the adjustment of L-kynurenine, corticosterone, acetylcholine, and raffinose, mainly involving tryptophan metabolism pathway. Molecular docking ensured that the above ingredients acting on hyperlipidemia-related protein targets played a key role in lowering lipids. In summary, this research provided a new possibility for preventing and treating hyperlipidemia.

The present protocol describes an integrated strategy for exploring the key targets and mechanisms of Fructus Phyllanthi against hyperlipidemia based on network pharmacology prediction and metabolomics verification.

网络药理学预测及代谢组学验证 凤仙花抗高脂血症的机制

Hyperlipidemia has become a leading risk factor for cardiovascular diseases and liver injury worldwide. Fructus Phyllanthi (FP) is an effective drug ...

Using Multiple Light Scattering to Examine the Stability of Phyllanthus emblica L. Extracts Obtained with Different Extraction Methods

The extraction intermediate of traditional Chinese medicine is the key intermediate in the preparation process, and its stability has an important impact on the effectiveness and quality of the final product. However, existing stability evaluation methods are often time-consuming and labor-intensive, requiring long-term observation and the operation of complex equipment (such as high-performance liquid chromatography), and it is difficult to obtain more physical information about the instability of the system. Therefore, there is an urgent need to establish a fast and accurate stability analysis technology for traditional Chinese medicine. Multiple light scattering is a cutting-edge analytical method that can accurately and rapidly evaluate the stability of traditional Chinese medicines in an environment-friendly manner without changing the nature or state of the sample or using organic reagents.
In this work, using the precise scanning data of multiple light scattering, the present protocol rapidly acquired the variation curves for layer thickness, particle migration speed, and average particle size over time. This enabled the precise identification of the mechanism and crucial characteristics causing the system&#39;s instability in its early stages. Of note, the research period for the extraction process can be considerably shortened by the detailed quantification of the system stability, which also allows for a quick, accurate, and in-depth analysis of the effects of various extraction processes on the stability of Phyllanthus emblica L.

Using Multiple Light Scattering to Examine the Stability of Phyllanthus emblica L. Extracts Obtained with Different Extraction Methods

Here, we introduce a stability evaluation method based on multiple light scattering technology to evaluate the stability of traditional Chinese medicine extracts.

使用多重光散射检查不同提取方法获得的余 甘子 L.提取物的稳定性

The extraction intermediate of traditional Chinese medicine is the key intermediate in the preparation process, and its stability has an important impact ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

一种从小鼠中分离大脑围取体的高输出方法

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...

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

摘要

探索更多视频

此视频中的章节

Fioriniae水和氯蓝莓和越橘花提取物的研究进展

老年人血浆中酚类代谢物的半靶向超高效色谱耦合质谱分析

使用基于多元醇的技术微波辅助萃取用于化妆品应用的酚类化合物和抗氧化剂

使用基于多元醇的技术微波辅助萃取用于化妆品应用的酚类化合物和抗氧化剂

使用基于多元醇的技术微波辅助萃取用于化妆品应用的酚类化合物和抗氧化剂

鼠冠状动脉血管平滑肌细胞的分离

分离肺内动脉和平滑肌细胞以研究血管反应

网络药理学预测及代谢组学验证凤仙花抗高脂血症的机制

使用多重光散射检查不同提取方法获得的余甘子 L.提取物的稳定性

一种从小鼠中分离大脑围取体的高输出方法

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

摘要

探索更多视频

此视频中的章节

Fioriniae水和氯蓝莓和越橘花提取物的研究进展

老年人血浆中酚类代谢物的半靶向超高效色谱耦合质谱分析

使用基于多元醇的技术微波辅助萃取用于化妆品应用的酚类化合物和抗氧化剂

使用基于多元醇的技术微波辅助萃取用于化妆品应用的酚类化合物和抗氧化剂

使用基于多元醇的技术微波辅助萃取用于化妆品应用的酚类化合物和抗氧化剂

鼠冠状动脉血管平滑肌细胞的分离

分离肺内动脉和平滑肌细胞以研究血管反应

网络药理学预测及代谢组学验证 凤仙花抗高脂血症的机制

使用多重光散射检查不同提取方法获得的余 甘子 L.提取物的稳定性

一种从小鼠中分离大脑围取体的高输出方法

网络药理学预测及代谢组学验证凤仙花抗高脂血症的机制

使用多重光散射检查不同提取方法获得的余甘子 L.提取物的稳定性