Name: ultrasonic-assisted extraction of cannabidiolic acid from cannabis biomass
Uploaded: 2022-05-27T13:30:00
Description: Watch this Scientific Journal Video about Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass at JoVE.com

这项研究得到了科罗拉多州立大学普韦布洛分校大麻研究所，韩国政府资助的韩国创新基金会赠款（MSIT）（2021-DD-UP-0379）和春川市（大麻研发和工业化，2020-2021）的支持。

1. 植物材料制备
<ol><li>从田间种植的植物中获得樱桃酒花序，以南北向的配置种植，植物中心相距1米，行距1.2米（种植位于美国科罗拉多州朗蒙特）。</li>
	<li>在35°C下风干花序48小时。使用177μm的研磨机研磨花序。</li>
	<li>将粉碎后的物料通过80号筛网。将产生的粉末储存在室温下的密封袋中，以备将来使用。</li>
</ol>2. 超声提取
<ol><li>称取0.5g 大?...

<ol>
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P., Young, O., Kebede, L., 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=Processing+and+extraction+methods+of+medicinal+cannabis:+a+narrative+review.">Processing and extraction methods of medicinal cannabis: a narrative review.</a> Journal of Cannabis Research. 3 (1), 1-15 (2021).</li><li>Lewis-Bakker, M. M., Yang, Y., Vyawahare, R., Kotra, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extractions+of+medical+cannabis+cultivars+and+the+role+of+decarboxylation+in+optimal+receptor+responses.">Extractions of medical cannabis cultivars and the role of decarboxylation in optimal receptor responses.</a> Cannabis and Cannabinoid Research. 4 (3), 183-194 (2019).</li><li>Brighenti, V., Pellati, F., Steinbach, M., Maran, D., Benvenuti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+new+extraction+technique+and+HPLC+method+for+the+analysis+of+non-psychoactive+cannabinoids+in+fibre-type+Cannabis+sativa+L.(hemp).">Development of a new extraction technique and HPLC method for the analysis of non-psychoactive cannabinoids in fibre-type Cannabis sativa L.(hemp).</a> Journal of Pharmaceutical and Biomedical Analysis. 143, 228-236 (2017).</li><li>. 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溶剂的极性在化合物的有效提取中起着至关重要的作用。由于酸性大麻素在性质上略带极性，这在很大程度上是由于羧酸部分，因此假设甲醇或乙醇等极性溶剂最有效。Garrett和Hunt19在他们使用THC的研究中证明，乙醇水溶液中的溶解度基于溶液中的乙醇百分比和溶液的离子强度。虽然在目前的研究中没有检查离子强度，但可以假设它在提高50%乙醇的提取效率方面发挥了重要作用。...

工业大麻（大麻 属）在各种植物组织（花，叶和茎）中产生酸性大麻素，在花中发现的浓度最高1。 大麻 行业利用几种方法来提取这些化合物。其中一种方法是利用非极性和/或极性溶剂的溶剂萃取，其中乙醇是最常用的。然而，单独使用溶剂萃取的能力有限;因此，增强提取技术，如微波辅助提取（MAE）和超声辅助提取（UAE），旨在提高产量。此外，可以使用超临界流体技术提取高浓度大麻二酚（CBD）2.
萃取是一个动态过程，有几个因素会影响其效率，即水分含量，粒度和溶剂3。具体而言，对于阿联酋技术，效率由温度，压力，频率和时间4控制。
超声波辅助提取是超声波通过液体搅拌颗粒的过程。在搅拌过程中，植物材料经历声空化，压缩和膨胀循环，形成气泡，在溶液中坍塌，导致产生极端温度和压力5。压力和温度的变化改变了溶剂的物理性质，这可导致提取6的效率增加。此外，空化可以破坏分子相互作用，导致有机和无机化合物从植物基质7中浸出。该过程涉及两种主要类型的物理现象：（1）扩散到细胞壁上，以及（2）在打破细胞壁8后冲洗细胞内容物。然而，阿联酋的使用并非没有陷阱;有几份报告称，阿联酋可以降解化合物9，10。此外，在空化部位产生的温度高于大麻素脱羧所需的温度。然而，Mudge等人使用阿联酋 ，没有观察到CBD或四氢大麻酚（THC）的大量脱羧，从而证明阿联酋是提取大麻素的有效和绿色方法，因为它们可以使用低能量快速提取。
De Vita等人12 专门检查了MAE和UAY方法的使用，发现当对每种方法应用最佳条件时，UAE提取了更多存在于植物材料中的酸性和中性CBD和THC。同样，Rožanc等人13 比较了多种提取方法（阿联酋，索氏，浸渍和超临界流体），并检查了提取物的生物活性。Rožanc证明所有方法都有效提取大麻素;然而，超临界流体和阿联酋在提取大麻二酸（CBDA）方面最有效。此外，当通过2，2-二苯基-1-苦味酰肼（DPPH）测定时，阿联酋提取具有最高的生物活性。Rožanc的研究还表明，虽然提取过程在生产粗提取物方面是有效的，但仍有一部分非大麻素化合物会影响提取物的生物活性。此外，这些化合物可以使从粗提取物13中分离和纯化单个大麻素化合物变得复杂。
超临界流体萃取（SFE）技术已被用于提取中性大麻素。几项研究表明，SFE加上有机溶剂，如乙醇，导致中性大麻素2，3的提取效率更高。当压力增加到能够提取酸性大麻素的水平时，非大麻素含量也增加。因此，这些高压对于工业加工是不切实际的，因为SFE对大麻素的选择性降低，需要额外的后处理。因此，脱羧必须在SFE之前进行，这可能导致大麻素损失高达18%2。为了提高SFE中的效率，已经与固相萃取等技术相结合，以增加最终萃取物14的纯度。然而，尽管具有高纯度作为最终产品，但只获得中性大麻素。
传统上，在分析实验室中，大麻素在9：1甲醇：氯仿混合物中提取。然而，Mudge等人11 证明，在使用阿联酋时，可以使用单一溶剂进行有效的提取。研究表明，80%甲醇与传统的9：1甲醇：氯仿提取一样有效，从而表明更环保的溶剂可以同样有效。因此，由于具有几个好处，包括低资本成本，减少提取时间以及降低能源使用和溶剂量，因此对阿联酋的潜在用途进行了检查。然而，在阿联酋的情况下，当使用极性溶剂时，可以提取叶绿素和其他非大麻素，这可能会导致颜色7的问题。因此，为了研究在商业规模上获得酸性大麻素的潜力，阿联酋使用工业大麻品种樱桃酒。Cherry Wine是 C. sativa 和 C. indica的杂交种，是The Wife和Charlotte's Cherries品种之间的杂交种。樱桃酒品种是一种高CBDA生产菌株（15%至25%CBD），具有低水平的四氢大麻酸（THCA）。该品种是籼稻为主的 C. indica菌株，开花7至9周。
为了建立最佳的阿联酋提取方案，采用了两种方法：传统的一次一个因素（OFT）优化和使用中央复合材料设计（CCD）的实验设计（DoE）方法15。对于DoE，基于样品/溶剂比，提取时间和溶剂浓度作为因素优化了CBDA / CBD提取，并通过响应表面方法（RSM）分析了结果数据。总之，所描述的方案概述了提取最大量的CBDA / CBD的最佳方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetonitrile</td>
			<td>J.K.Baker</td>
			<td>9017-88</td>
			<td>solvent</td>
		</tr>
		<tr>
			<td>Cannabichromene</td>
			<td>Cerilliant</td>
			<td>C-143</td>
			<td>Cannabinoids standard</td>
		</tr>
		<tr>
			<td>Cannabidiol</td>
			<td>Cerilliant</td>
			<td>C-045</td>
			<td>Cannabinoids standard</td>
		</tr>
		<tr>
			<td>Cannabidiolic acid</td>
			<td>Cerilliant</td>
			<td>C-144</td>
			<td>Cannabinoids standard</td>
		</tr>
		<tr>
			<td>Cannabidivarin</td>
			<td>Cerilliant</td>
			<td>C-140</td>
			<td>Cannabinoids standard</td>
		</tr>
		<tr>
			<td>Cannabigerol</td>
			<td>Cerilliant</td>
			<td>C-141</td>
			<td>Cannabinoids standard</td>
		</tr>
		<tr>
			<td>Cannabinol</td>
			<td>Cerilliant</td>
			<td>C-046</td>
			<td>Cannabinoids standard</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Hanil Scientific Inc</td>
			<td>Supra 22K</td>
			<td>Centrifuge</td>
		</tr>
		<tr>
			<td>Cherry Wine hemp</td>
			<td>CFH, Ltd.</td>
			<td>-</td>
			<td>Flower extraction material</td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>TEDIA</td>
			<td>WS2211-001</td>
			<td>solvent</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>TEDIA</td>
			<td>ES1431-001</td>
			<td>solvent</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Whatman</td>
			<td>#2</td>
			<td>Filtering</td>
		</tr>
		<tr>
			<td>Grinder</td>
			<td>Daesung Artlon</td>
			<td>DA280-S</td>
			<td>Milling</td>
		</tr>
		<tr>
			<td>HPLC</td>
			<td>Shimadzu</td>
			<td>LC-10 system</td>
			<td>Analysis of Cannabinoid</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>TEDIA</td>
			<td>MS1922-001</td>
			<td>solvent</td>
		</tr>
		<tr>
			<td>Minitab 16.2.0</td>
			<td>Minitab Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filters</td>
			<td>Whatman</td>
			<td>6779-1304</td>
			<td>Filtering</td>
		</tr>
		<tr>
			<td>Tetrahydrocannabivarin</td>
			<td>Cerilliant</td>
			<td>T-094</td>
			<td>Cannabinoids standard</td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Sigma-aldrich</td>
			<td>302031-1L</td>
			<td>HPLC flow solvent</td>
		</tr>
		<tr>
			<td>Untrasonic bath</td>
			<td>Jinwoo</td>
			<td>4020P</td>
			<td>Ultrasonic extraction</td>
		</tr>
		<tr>
			<td>Zorbax Eclipse plus C18 HPLC column</td>
			<td>Agilent</td>
			<td>9599990-902</td>
			<td>HPLC column</td>
		</tr>
		<tr>
			<td>&#916;8 - Tetrahydrocannabinol</td>
			<td>Cerilliant</td>
			<td>T-032</td>
			<td>Cannabinoids standard</td>
		</tr>
		<tr>
			<td>&#916;9 - Tetrahydrocannabinol</td>
			<td>Cerilliant</td>
			<td>T-005</td>
			<td>Cannabinoids standard</td>
		</tr>
		<tr>
			<td>&#916;9 - Tetrahydrocannabinolic acid</td>
			<td>Cerilliant</td>
			<td>T-093</td>
			<td>Cannabinoids standard</td>
		</tr>
	</tbody>
</table>

ultrasonic-assisted extraction of cannabidiolic acid from cannabis biomass

工业大麻（大麻属）具有许多具有潜在医疗益处的感兴趣化合物。在这些化合物中，大麻素已成为人们关注的焦点，特别是酸性大麻素。由于缺乏精神活性，重点正在转向酸性大麻素。大麻植物产生酸性大麻素，大麻植物产生低水平的精神药物大麻素。因此，利用大麻进行酸性大麻素提取将消除提取前作为大麻素来源的脱羧需求。使用溶剂萃取是获得酸性大麻素的理想选择，因为它们在超临界CO2等溶剂中的溶解度受到限制，因为达到其溶解度常数所需的高压和温度。另一种旨在增加溶解度的方法是超声辅助提取。在该方案中，研究了溶剂极性（乙腈0.46，乙醇0.65，甲醇0.76和水1.00）和浓度（20%，50%，70%，90%和100%）对超声辅助提取效率的影响。结果表明，水是最有效的，乙腈是最有效的溶剂。乙醇被进一步检查，因为它具有最低的毒性，并且通常被认为是安全的（GRAS）。令人惊讶的是，水中50%乙醇是从大麻中提取最大量大麻素的最有效乙醇浓度。与100%乙醇相比，大麻二酸浓度增加28%，与100%乙腈相比增加23%。虽然确定50%乙醇是我们应用中最有效的浓度，但该方法也被证明对替代溶剂有效。因此，所提出的方法被认为有效且快速地提取酸性大麻素。

使用的溶剂范围从极性指数的中间（0.460 - ACN）到极性（1.000 - 水）。从表2可以看出，水没有成为大麻素的有效提取剂，这并不意外，因为大麻素由于其疏水性而在水中的溶解度有限13。与水相比，其他溶剂具有相似的CBD和CBDA的提取值，与两种醇相比，极性最低的溶剂乙腈（ACN）具有更高的提取率。虽然统计上较低，但乙醇能够分别提取ACN和甲醇提取的主要大麻素CBD...

Watch this Scientific Journal Video about Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass at JoVE.com

Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass

超声波辅助提取（UAE）提高了溶剂的提取效率，当应用于 大麻 属生物质时，它减少了提取所需的时间。这降低了由于降解而导致的成本和潜在的大麻素损失。此外，由于溶剂使用率低，阿联酋被认为是一种绿色方法。

从 大麻 生物质中超声辅助提取大麻二酸

Industrial hemp (Cannabis spp.) has many compounds of interest with potential medical benefits. Of these compounds, cannabinoids have come to the center ...

该协议展示了CBD提取的整个过程，从制备材料，溶剂提取和测量CBD含量开始。通过建议优化的溶剂和溶剂浓度，该方法消除了不必要的过程，并有助于工业目的的高效溶剂提取。首先从田间种植的植物中获得樱桃酒花序。将花序在35摄氏度下风干48小时。使用177微米的研磨机研磨花序。将粉碎后的物料通过80目筛，并在室温下将粉末储存在密封袋中。在50毫升锥形管中，称量0.5克大麻花序粉末。在去离子水中加入40毫升50%乙醇。将提取容器置于室温设定为40千赫兹的超声波浴中。进行提取30分钟，将浴槽温度从25摄氏度提高到30摄氏度。超声处理后，将提取液倾析到离心管中。将流体在15摄氏度下以3000倍G离心15分钟。在真空下通过8微米滤纸过滤上清液。将大麻素标准品稀释至100%甲醇中每毫升100，50，25和12.5微克的工作浓度。在40千赫兹的超声波浴和100瓦的超声处理功率中混合并超声处理5分钟。通过0.45微米聚四氟乙烯注射器过滤器过滤标准品和大麻样品上清液。将样品放入1.5毫升小瓶中，然后将其放入HPLC自动进样器中。加载10微升样品并使用该表中所示的参数运行HPLC。生成标准曲线，并以每毫升50至200微克为单位得出大麻素浓度。通过乘以提取过程中使用的溶剂的体积来计算大麻素的重量（以微克为单位），通过将值除以1， 000来将其转换为毫克。将此值除以提取所用植物材料的重量，得到每克干重毫克数。HPLC分析表明，当使用100%溶剂时，乙腈最有利于提取单个大麻素。然而，由于毒性最低，乙醇被进一步检查。乙醇水对提取的大麻素浓度的影响的评估表明，在50%乙醇下观察到最大提取。与提取CBDA的无水乙醇相比，增加了39.7%。THCA水平也降低了20.3%，采用实验设计方法，优化了CBDA加CBD的提取。响应面方法分析证实了理想的参数，即30分钟提取，53.4%乙醇在水中以及1至100的样品溶剂比。与浸渍法相比，使用超声辅助提取方法获得更大量的CBDA。使用超声波方法提取的CBD量也增加了一倍，表明大麻素提取效率更高。重要的是要记住样品与溶剂的比例，提取时间和溶剂浓度。通过本研究进行优化。这个过程可以用于从大麻中提取其他大麻素。

Research

JoVE Journal

Biochemistry

Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

从人体二尖瓣提取蛋白质的优化方案

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

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 ...

Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions

植物细胞悬浮液中咖啡因的提取、酶活性及咖啡因合成酶基因的表达

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a ...

Extraction and Analysis of Taiwanese Green Propolis

台湾绿蜂胶的提取与分析

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

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

从鸡蛋黄中分离脂蛋白颗粒,研究细菌病原体脂肪酸并入膜磷脂

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

使用核酸探针进行核酸活性的动力学筛选

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC

使用SAX-HPLC从不同植物物种中提取和定量可溶性、放射性标记肌醇多磷酸盐

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant ...

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid (TGA)

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid TGA

使用硫二甘酸（TGA）估计植物生物质木质素含量

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls ...

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 ...

Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction

通过真空辅助吸附剂提取从人类相关样品中捕获活性产生的微生物挥发性有机化合物

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the ...

从 大麻 生物质中超声辅助提取大麻二酸

从大麻生物质中超声辅助提取大麻二酸