Name: extraction and analysis of taiwanese green propolis
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Extraction and Analysis of Taiwanese Green Propolis at JoVE.com

这份手稿是由华莱士学术编辑编辑。

1. 乙醇提取化合物的制备
<ol><li>重量10克冷冻台湾绿色蜂胶, 这是从台湾蜂箱收集的 5月至7月, 并使用香料研磨机磨碎。确认整片台湾绿色蜂胶被磨成细粉, 没有任何大颗粒。</li>
	<li>加入100毫升的各种浓度的乙醇 (60%, 70%, 80%, 95% 和 99.5%) 和水分离烧瓶, 并混合每个浓度的10克地面蜂胶。</li>
	<li>在25°c 下孵化, 并在250转/分的温度下摇瓶48小时。</li>
	<li>通过孔径为25μm 的滤纸过滤乙醇提取物。</li>
	<li...

<ol>
	<li>Zabaiou, N., 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=Biological+properties+of+propolis+extracts:+Something+new+from+an+ancient+product.">Biological properties of propolis extracts: Something new from an ancient product.</a> Chemistry and Physics of Lipids. 207 (Pt B), 214-222 (2017).</li><li>Bankova, V. B., De Castro, S. L., Marcucci, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propolis:+Recent+advances+in+chemistry+and+plant+origin.">Propolis: Recent advances in chemistry and plant origin.</a> Apidologie. 31 (1), 3-15 (2000).</li><li>Park, Y. K., Alencar, S. M., Aguiar, C. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seasonal+effect+on+Brazilian+propolis+antibacterial+activity.">Seasonal effect on Brazilian propolis antibacterial activity.</a> Journal of Ethnopharmacology. 73 (1-2), 243-249 (2000).</li><li>Chen, Y. W., 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=Characterization+of+Taiwanese+propolis+collected+from+different+locations+and+seasons.">Characterization of Taiwanese propolis collected from different locations and seasons.</a> Journal of the Science of Food and Agriculture. 88 (3), 412-419 (2008).</li><li>Grange, J. M., Davey, 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=Antibacterial+properties+of+propolis+(bee+glue).">Antibacterial properties of propolis (bee glue).</a> Journal of the Royal Society of Medicine. 83 (3), 159-160 (1990).</li><li>Kujumgiev, A., 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=Antibacterial,+antifungal+and+antiviral+activity+of+propolis+from+different+geographic+origins.">Antibacterial, antifungal and antiviral activity of propolis from different geographic origins.</a> Journal of Ethnopharmacology. 64 (3), 235-240 (1999).</li><li>Krol, W., Scheller, S., Shani, J., Pietsz, G., Czuba, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+effect+of+ethanol+extract+of+propolis+and+antibiotics+in+the+growth+of+Staphylococcus+aureus.">Synergistic effect of ethanol extract of propolis and antibiotics in the growth of Staphylococcus aureus.</a> Drug Research. 43 (5), 607-609 (1993).</li><li>Lu, L. C., Chen, Y. W., Chou, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibacterial+and+DPPH+free+radical-scavenging+activities+of+the+ethanol+extract+of+propolis+collected+in+Taiwan.">Antibacterial and DPPH free radical-scavenging activities of the ethanol extract of propolis collected in Taiwan.</a> Journal of Food and Drug Analysis. 11 (4), 277-282 (2003).</li><li>Tosi, B., Donini, A., Romagnoli, C., Bruni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+activity+of+some+commercial+extracts+of+propolis+prepared+with+different+solvents.">Antimicrobial activity of some commercial extracts of propolis prepared with different solvents.</a> Phytotherapy Research. 10 (4), 335-336 (1996).</li><li>Cunha, I. B. S., 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=Factors+that+influence+the+yield+and+composition+of+Brazilian+propolis+extracts.">Factors that influence the yield and composition of Brazilian propolis extracts.</a> Journal of the Brazilian Chemical Society. 15 (6), 964-970 (2004).</li><li>Woo, S. O., Hong, I. P., Han, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+properties+of+propolis+with+ethanol+concentration.">Extraction properties of propolis with ethanol concentration.</a> Journal of Apicultural Research. 30 (3), 211-216 (2015).</li><li>Park, Y. K., Ikegaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+water+and+ethanolic+extracts+of+propolis+and+evaluation+of+the+preparations.">Preparation of water and ethanolic extracts of propolis and evaluation of the preparations.</a> Bioscience, Biotechnology, and Biochemistry. 62 (11), 2230-2232 (1998).</li><li>Ramanauskien&#279;, K., Ink&#279;nien&#279;, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propolis+oil+extract:+quality+analysis+and+evaluation+of+its+antimicrobial+activity.">Propolis oil extract: quality analysis and evaluation of its antimicrobial activity.</a> Natural Product Research. 25 (15), 1463-1468 (2011).</li><li>Machado, B. A., 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=Chemical+composition+and+biological+activity+of+extracts+obtained+by+supercritical+extraction+and+ethanolic+extraction+of+brown,+green+and+red+propolis+derived+from+different+geographic+regions+in+brazil.">Chemical composition and biological activity of extracts obtained by supercritical extraction and ethanolic extraction of brown, green and red propolis derived from different geographic regions in brazil.</a> PLoS One. 11 (1), e0145954 (2016).</li><li>Monroy, Y. M., 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=Brazilian+green+propolis+extracts+obtained+by+conventional+processes+and+by+processes+at+high+pressure+with+supercritical+carbon+dioxide,+ethanol+and+water.">Brazilian green propolis extracts obtained by conventional processes and by processes at high pressure with supercritical carbon dioxide, ethanol and water.</a> The Journal of Supercritical Fluids. 130, 189-197 (2017).</li><li>Popova, M., Chen, C. N., Chen, P. Y., Huang, C. Y., Bankova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+validated+spectrophotometric+method+for+quantification+of+prenylated+flavanones+in+pacific+propolis+from+Taiwan.">A validated spectrophotometric method for quantification of prenylated flavanones in pacific propolis from Taiwan.</a> Phytochemical Analysis. 21 (2), 186-191 (2010).</li><li>Chen, L. H., 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=Taiwanese+green+propolis+ethanol+extract+delays+the+progression+of+type+2+diabetes+mellitus+in+rats+treated+with+streptozotocin/high-fat+diet.">Taiwanese green propolis ethanol extract delays the progression of type 2 diabetes mellitus in rats treated with streptozotocin/high-fat diet.</a> Nutrients. 10 (4), e503 (2018).</li></ol>

一项研究报告说, 使用不同浓度的乙醇进行浸渍可用于巴西蜂胶提取17;然而, 这一过程是耗时的17。从巴西蜂胶17中提取酚类生物活性化合物 (如酚类化合物) 至少需要10天。另外, 乙醇萃取结合加热在 37°c, 50°c, 或 70°c 30分钟, 已被建议提取巴西蜂胶19,21,22.根据乙醇<sup class="xre...

蜂胶是由蜜蜂品种阿皮斯·梅莱夫生产的一种天然树脂混合物。蜂胶自古以来就被广泛用于民间医药。最近的一项研究报告说, 蜂胶有利于预防微生物感染和炎症1。大量研究表明, 蜂胶中的主要生物活性化合物是黄酮类化合物、酚酸酯、婚前性p-香豆酸和二戊酸 2,3。通过高效液相色谱法 (hplc)4、5、6、7, 测定了台湾绿蜂胶中10种婚前黄酮衍生物。其中最丰富的是丙二醇 c、d、f和 g 5、7。台湾绿色蜂胶具有相当大的生物效应, 与其高含量的丙氨酸8有关。
蜂胶中生物活性化合物的浓度因获得蜂胶的季节和地理位置而有很大差异。欧洲蜂胶主要含有黄酮类和酚酸 9.巴西蜂胶中的主要生物活性化合物是婚前的p-香豆酸, 如青蒿素 c10.一项研究表明, 该季节是决定台湾绿色蜂胶 11中总丙氨酸含量的关键因素。台湾绿色蜂胶中的丙蛋白含量在夏季 (5月至7月) 最高, 在冬季最低。蜂胶的抗菌性能被广泛认为是生物活性的指标。一般来说, 从各地区采集的蜂胶样品具有类似的抗菌性能;例如, 它通常对几乎所有革兰氏阳性细菌有效, 对革兰氏阴性菌10、12、13 具有有限的抗菌作用。结果表明, 蜂胶中黄酮类化合物之间的协同作用具有抗菌作用.同样, 据报道, 台湾绿色蜂胶对革兰氏阳性菌有抗菌作用。此外, 一项研究还确定了在台湾绿色蜂胶8中, 丙氨酸相互作用的抗菌作用。
蜂胶中生物活性化合物的特性是困难的, 因为其化学成分可能因其来源而异。因此, 有必要建立一种可行的、可重复的方法来确定台湾绿色蜂胶的质量。然而, 台湾绿色蜂胶提取和随后的功能分析还没有建立标准程序。提出了几种不同应用有机和无机溶剂的蜂胶提取方法 16、17、18、19、20.由于蜂胶是一种亲脂性混合物, 研究表明, 有机萃取比无机萃取物的提取物好于无机萃取物8、18、19.台湾绿色蜂胶中的总丙蛋白浓度及其抗菌性能是衡量台湾绿色蜂胶质量的关键指标。因此, 本研究的目的是提出一种利用乙醇作为溶剂提取和表征台湾绿色蜂胶抗菌性能的方案。

<table>
	<tbody>
		<tr>
			<td>ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman no. 4 filter paper</td>
			<td>Sigma-Aldrich</td>
			<td>WHA1004125</td>
			<td></td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>reverse phase RP-18 column</td>
			<td>Phenomenex Inc.</td>
			<td>00G-0234-E0</td>
			<td></td>
		</tr>
		<tr>
			<td>Staphylococcus aureus</td>
			<td>ATCC</td>
			<td>BCRC 10780</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli</td>
			<td>ATCC</td>
			<td>BCRC 10675</td>
			<td></td>
		</tr>
		<tr>
			<td>tryptic soy broth</td>
			<td>Sigma-Aldrich</td>
			<td>22092</td>
			<td></td>
		</tr>
		<tr>
			<td>nutrient broth</td>
			<td>Sigma-Aldrich</td>
			<td>70122</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>spice grinder</td>
			<td>Waring</td>
			<td>WSG60K</td>
			<td></td>
		</tr>
		<tr>
			<td>microplate Reader</td>
			<td>Molecular Devices</td>
			<td>EMAX PLUS</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC system</td>
			<td>Agilent</td>
			<td>1200 Series</td>
			<td></td>
		</tr>
		<tr>
			<td>vacuum evaporation</td>
			<td>B&#220;CHI</td>
			<td>Rotavapor R-215</td>
			<td></td>
		</tr>
	</tbody>
</table>

extraction and analysis of taiwanese green propolis

台湾绿色蜂胶富含前乳化类黄酮类化合物, 具有广泛的生物活性, 如抗氧化剂、抗菌药和抗癌类。台湾绿色蜂胶的生物活性化合物为丙二醇, 即 c、d、f 和 g。台湾绿色蜂胶中的蜂胶浓度因季节和地理位置而异。因此, 建立一个标准的、可重复的程序来确定台湾绿色蜂胶的质量是至关重要的。在这里, 我们提出了一个协议, 使用乙醇为基础的提取, 高效液相色谱, 和抗菌活性分析, 以表征台湾绿色蜂胶质量。该方法表明, 95% 和99.5% 乙醇提取物可实现台湾绿色蜂胶的最大干物质产量, 从而产生具有抗菌性能的最高浓度的丙氨酸。根据这些发现, 本协议被认为是确定台湾绿色蜂胶质量的可靠和可重复的。

乙醇提取的积极代表性数据见表 1。台湾绿蜂胶的干物质产量与乙醇的浓度呈正相关。95% 和99.5% 乙醇提取物的干物质产量最高。以水为萃取溶剂, 台湾绿色蜂胶的干物质产率最低。这些结果表明, 乙醇等有机溶剂在台湾绿色蜂胶提取中表现最佳。使用高效液相色谱法 (图 1a) 识别和量化标准丙氨酸 c、d、f 和 g 的信号。用单独的丙氨酸标准?...

Watch this Scientific Journal Video about Extraction and Analysis of Taiwanese Green Propolis at JoVE.com

Extraction and Analysis of Taiwanese Green Propolis

台湾绿蜂胶的提取与分析

我们提出了一个协议, 使用乙醇作为溶剂提取和表征台湾绿色蜂胶, 具有抗菌活性。

Taiwanese green propolis is rich in prenylated flavonoids and exhibits a broad range of biological activities, such as antioxidant, antibacterial, and ...

在这里，我们提出了使用乙醇作为溶剂来提取和描述台湾绿色蜂胶抑制抗菌活性的规程。该协议是确定台湾绿色蜂胶质量的可靠和可重复的方法。演示程序将是陈春丁和易贤，我的实验室博士生。首先，称出100克台湾绿色蜂胶。使用香料研磨机，研磨蜂胶，确保碎片研磨成细粉，没有任何大颗粒。设置五个烧瓶，并加入100毫升的各种浓度乙醇在水中。将 10 克地蜂胶混合到每个烧瓶中的乙醇溶液中，在 25 摄氏度下孵育，在 250 RPM 下摇晃 48 小时。之后，使用25微米孔径的滤纸过滤乙醇提取物。使用体积烧瓶，用95%乙醇将滤瓶重新构成其原始体积为100毫升。将重组后的提取物存放在零下 20 摄氏度，直到准备好使用。当准备为 HPLC 准备提取物时，使用真空蒸发将每个提取物的 10 毫升在 40 摄氏度下浓缩 10 分钟 10 分钟。在45摄氏度下烘烤干物质24小时。然后，用10毫升95%乙醇重新制备每个干物质样品。使用 0.22 微米孔径的注射器过滤器，重新过滤每个提取物，并收集滤物，使用 HPLC 进行直接分析。要开始建立标准曲线，请以 88.8 比 11.2 的比例准备一升移动相甲醇与水溶液。使用移动相溶液作为溶剂，准备如文本协议中概述的丙烯标准浓度的连续稀释。将每个标准浓度的 20 微升按顺序从最低浓度注入反向相列到最高浓度。然后，将 HPLC 柱设置为 30 摄氏度，将紫外线检测器的豁免长度设置为 280 纳米，将流速设置为每分钟 1 毫升，将记录器时间设置为 20 分钟。至少分析三次标准。在此之后，使用计算表软件将测量响应与浓度堵塞，从而形成具有已确定方程和 R 平方值的标准曲线。要开始分析提取，请将每个萃取的 20 微升注入反向相位列。将 HPLC 柱设置为 30 摄氏度的温度，流量为每分钟 1 毫升。将紫外线检测器的豁免长度设置为 280 纳米，将记录器时间设置为 20 分钟。分析数据提取至少三次。然后，使用标准曲线确定每个乙醇提取物中丙烯的浓度。在制备测试生物体和乙醇提取物后，将10微升稀释乙醇提取物加入96井板的井中，每毫升0.156至640微克。使用汤将每井的体积调整为 100 微升，确保所有稀释中保持 5%DMSO。接下来，将100微升的细菌培养剂接种到96井板的每个井中。在37摄氏度的高温下进行48小时。使用 590 纳米的微孔板读卡器根据浊度分析细菌生长，并确定最低抑制浓度。在此之后，从每一个井中接种10微升的液体培养剂，这些井表现出生长到阿加板上。在37摄氏度下孵育24小时。确定未显示可见细菌生长的最低浓度，以确定细菌活性。完全消除细胞生长的浓度被认为是最小的细菌浓度。在这项研究中，用乙醇提取台湾绿色蜂胶。当使用高浓度乙醇时，干物质的产量似乎是最高的，而使用水时，干物质的产量是最低的。这些结果表明，有机溶剂在萃取过程中表现最佳，产量与乙醇浓度呈正相关。同样，丙烯的浓度似乎在提取过程中与乙醇浓度呈正相关，在95%和99.5%乙醇提取物中获得最高产量的丙烯。然后对乙醇提取物对大肠杆菌和大肠杆菌的抗菌影响进行调查。S aureus 的平均最低抑制浓度似乎在每毫升 10 到 20 微克之间，细菌的平均浓度似乎是每毫升 20 微克。然而，水提取物对古雷乌斯没有任何抗菌作用。使用水或乙醇提取物时，对大肠杆菌没有抗菌影响。关键的实验过程之一是在研磨过程中回避台湾绿色蜂胶的均匀性。

Research

JoVE Journal

Biochemistry

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到<em&gt;取值</em&gt; -citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

科研

生物化学

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

一种简单的分级提取方法，用于从单个样品中综合分析代谢物，脂质和蛋白质

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

大肠杆菌中异构表达对膜迁移者的特性与膜囊泡的产生

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein

使用亚细胞室特异性红氧敏感绿色荧光蛋白评估细胞氧化

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. ...

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

大鼠胰腺组织快速、经济高效的RNA提取

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective ...

Ganglioside Extraction, Purification and Profiling

神经节苷脂提取、纯化和分析

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are ...

Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver

从猪肾和小鼠肝脏中提取和纯化FAHD1蛋白

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as ...

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope

使用200 KV透射电子显微镜常规收集高分辨率冷冻电镜数据集

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ...

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

纳米巴支持的脂质双层系统，用于体外研究膜曲率传感蛋白

Membrane curvature plays important roles&#160;in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It ...