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

This research was supported by the Institute of Cannabis Research at Colorado State University-Pueblo, the Korea Innovation Foundation grant funded by the Korean government (MSIT) (2021-DD-UP-0379), and Chuncheon city (Hemp R&#38;D and industrialization, 2020-2021).

1. Plant material preparation
<ol>
	<li>Obtain Cherry Wine inflorescences from plants grown in the field, planted in a south-to-north configuration, with plants 1 m apart in center and rows 1.2 m apart (cultivation located in Longmont, Colorado, USA).</li>
	<li>Air-dry the inflorescences at 35 &#176;C for 48 h. Grind the inflorescences using a grinding machine set a 177 &#181;m.</li>
	<li>Pass the pulverized material through the No. 80 mesh sieve. Store the resulting powder in a sealed bag at room temp...

<ol>
	<li>Hemphill, J. K., Turner, J. C., Mahlberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoid+content+of+individual+plant+organs+from+different+geographical+strains+of+Cannabis+sativa+L.">Cannabinoid content of individual plant organs from different geographical strains of Cannabis sativa L.</a> Journal of Natural Products. 43 (1), 112-122 (1980).</li><li>Baldino, L., Scognamiglio, M., Reverchon, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supercritical+fluid+technologies+applied+to+the+extraction+of+compounds+of+industrial+interest+from+Cannabis+sativa+L.+and+to+their+pharmaceutical+formulations:+A+review.">Supercritical fluid technologies applied to the extraction of compounds of industrial interest from Cannabis sativa L. and to their pharmaceutical formulations: A review.</a> Journal of Supercritical Fluids. 165, 104960 (2020).</li><li>Daniel, R. 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P., 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=Ultrasound+assisted+maceration+for+improving+the+aromatization+of+extra-virgin+olive+oil+with+rosemary+and+basil.">Ultrasound assisted maceration for improving the aromatization of extra-virgin olive oil with rosemary and basil.</a> Food Research International. 135, 109305 (2020).</li><li>Kshitiz, K., 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=Ultrasound+assisted+extraction+(UAE)+of+bioactive+compounds+from+fruit+and+vegetable+processing+by-products:+A+review.">Ultrasound assisted extraction (UAE) of bioactive compounds from fruit and vegetable processing by-products: A review.</a> Ultrasinics Sonochemistry. 70, 105325 (2017).</li><li>Mudge, E. M., Murch, S. J., Brown, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leaner+and+greener+analysis+of+cannabinoids.">Leaner and greener analysis of cannabinoids.</a> Analytical and Bioanalytical Chemistry. 409 (12), 3153-3163 (2017).</li><li>De Vita, D., 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=Comparison+of+different+methods+for+the+extraction+of+cannabinoids+from+cannabis.">Comparison of different methods for the extraction of cannabinoids from cannabis.</a> Natural Product Research. 34 (20), 2952-2958 (2020).</li><li>Ro&#382;anc, J., 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=Different+Cannabis+sativa+extraction+methods+result+in+different+biological+activities+against+a+colon+cancer+cell+line+and+healthy+colon+cells.">Different Cannabis sativa extraction methods result in different biological activities against a colon cancer cell line and healthy colon cells.</a> Plants. 10 (3), 566 (2021).</li><li>Kar&#287;ili, U., Ayta&#231;, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supercritical+fluid+extraction+of+cannabinoids+(THC+and+CBD)+from+four+different+strains+of+cannabis+grown+in+different+regions.">Supercritical fluid extraction of cannabinoids (THC and CBD) from four different strains of cannabis grown in different regions.</a> The Journal of Supercritical Fluids. 179, 105410 (2022).</li><li>Sushma, 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=Optimization+of+ultrasound-assisted+extraction+(UAE)+process+for+the+recovery+of+bioactive+compounds+from+bitter+gourd+using+response+surface+methodology+(RSM).">Optimization of ultrasound-assisted extraction (UAE) process for the recovery of bioactive compounds from bitter gourd using response surface methodology (RSM).</a> Food and Bioproducts Processing. 120, 120-122 (2022).</li><li>David, J. P., 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=Potency+of+&#916;9-THC+and+Other+Cannabinoids+in+Cannabis+in+England+in+2005:+Implications+for+Psychoactivity+and+Pharmacology.">Potency of &#916;9-THC and Other Cannabinoids in Cannabis in England in 2005: Implications for Psychoactivity and Pharmacology.</a> Journal of Forensic Sciences. 11, 129 (2008).</li><li>Agarwal, C., M&#225;th&#233;, K., Hofmann, T., Cs&#243;ka, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-assisted+extraction+of+cannabinoids+from+Cannabis+Sativa+L.+optimized+by+response+surface+methodology.">Ultrasound-assisted extraction of cannabinoids from Cannabis Sativa L. optimized by response surface methodology.</a> Journal of Food Science. 83 (3), 700-710 (2018).</li><li>Oroian, M., Ursachi, F., Dranca, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+ultrasonic+amplitude,+temperature,+time+and+solvent+concentration+on+bioactive+compounds+extraction+from+propolis.">Influence of ultrasonic amplitude, temperature, time and solvent concentration on bioactive compounds extraction from propolis.</a> Ultrasonics Sonochemistry. 64 (2020), 105021 (2020).</li><li>Garrett, E. R., Hunt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiochemical+properties,+solubility,+and+protein+binding+of+&#916;9-tetrahydrocannabinol.">Physiochemical properties, solubility, and protein binding of &#916;9-tetrahydrocannabinol.</a> Journal of Pharmaceutical Sciences. 63 (7), 1056-1064 (1974).</li><li>Metcalf, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+Abstracts.">Chemical Abstracts.</a> United States patent. , (2020).</li><li>Lazarjani, M. 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. 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The polarity of a solvent plays a critical role in the effective extraction of compounds. Since acidic cannabinoids are slightly polar in nature, due in large part to the carboxylic acid moiety, it was assumed that a polar solvent such as methanol or ethanol would be most effective. Garrett and Hunt19, in their study using THC, demonstrated that solubility in aqueous ethanol was based on percent ethanol in the solution and ionic strength of the solution. While ionic strength was not examined in th...

Industrial hemp (Cannabis spp.) produces acidic cannabinoids in various plant tissues (flowers, leaves, and stems), with the highest concentration found in the flower1. The Cannabis industry utilizes several methods to extract these compounds. One such method is solvent extraction that utilizes a non-polar and/or polar solvent, of which ethanol is the most commonly used. However, solvent extraction alone is limited in its ability; therefore, augmentative extraction techniques, such as microwave-assisted extraction (MAE) and ultrasonic-assisted extraction (UAE), are designed to increase the yield. In addition, high concentration cannabidiol (CBD) can be extracted using supercritical fluid technologies2.
Extraction is a dynamic process, and several factors influence its efficiency, namely moisture content, particle size, and solvent3. Specifically, for the UAE technique, efficiency is governed by temperature, pressure, frequency, and time4.
Ultrasonic-assisted extraction is the process where ultrasonic waves are passed through a liquid to agitate particles. During the agitation process, plant materials experience acoustic cavitation, cycles of compression and expansion which form bubbles that collapse in solution resulting in the generation of extreme temperature and pressure5. The pressure and temperature changes alter the physical properties of the solvents, which can result in increased efficacy of extraction6. Additionally, cavitation can disrupt molecular interactions leading to organic and inorganic compounds leaching from the plant matrix7. The process involves two main types of physical phenomena: (1) diffusion across the cell wall, and (2) rinsing of the cellular contents after breaking the wall8. However, the use of UAE is not without its pitfalls; there are several reports that UAE can degrade compounds9,10. Additionally, the temperatures generated at the cavitation sites are above those necessary for decarboxylation of cannabinoids. However, Mudge et al.11 used UAE and did not observe large decarboxylation of CBD or tetrahydrocannabinol (THC), thereby demonstrating that UAE is an efficient and green method for the extraction of cannabinoids since they can be extracted quickly using low energy.
De Vita et al.12 examined the use of MAE and UAE methods specifically and found that when applying the optimal conditions for each method, UAE extracted more of the acidic and neutral CBD and THC present in the plant material. Similarly, Ro&#382;anc et al.13 compared multiple methods of extraction (UAE, soxhlet, maceration, and supercritical fluid) and examined the extracts&#39; biological activity. Ro&#382;anc demonstrated that all the methods were effective at extracting cannabinoids; however supercritical fluid and UAE were most effective at extracting cannabidiolic acid (CBDA). Additionally, the UAE extraction had the highest biological activity when measured by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Ro&#382;anc&#39;s study also showed that while the extraction processes are effective at producing crude extracts, there remains a portion of non-cannabinoid compounds that influence the extracts&#39; biological activity. Additionally, these compounds can complicate the isolation and purification of individual cannabinoid compounds from the crude extracts13.
Supercritical fluid extraction (SFE) techniques have been used to extract neutral cannabinoids. Several studies demonstrated that SFE plus an organic solvent, such as ethanol, resulted in higher extraction efficiencies of neutral cannabinoids2,3. When the pressure was increased to levels capable of extracting the acidic cannabinoids, non-cannabinoid content also increased. As such, these high pressures are not practical for industrial processing as the selectivity of SFE for cannabinoids decreased and additional post-processing is required. Consequently, decarboxylation must be done prior to SFE, which can result in cannabinoid losses of up to 18%2. To increase efficiencies in SFE, it has been combined with techniques such as solid-phase extraction to increase the purity of the final extract14. However, despite having high purity as the final product, only neutral cannabinoids are obtained.
Traditionally, in the analytical laboratory, cannabinoids were extracted in a 9:1 methanol:chloroform mixture. However, Mudge et al.11 demonstrated that effective extraction can be carried out with single solvents when employing UAE. The study showed that 80% methanol was as effective as the traditional 9:1 methanol:chloroform extraction, thereby indicating that greener solvents can be as effective. As such, UAE was examined for its potential use due to having several benefits, including low capital cost, reduced extraction time, and lower energy use and solvent volumes. However, in the case of UAE, when polar solvents are used, chlorophyll and other non-cannabinoids can be extracted, which may cause a problem in color7. Consequently, to examine the potential for obtaining acidic cannabinoids at a commercial scale, UAE was employed using the industrial hemp variety Cherry Wine. Cherry Wine is a hybrid of C. sativa and C. indica, a cross between the varieties of The Wife and Charlotte&#39;s Cherries. The Cherry Wine varietal is a high CBDA producing strain (15% to 25% CBD) with low levels of tetrahydrocannabinolic acid (THCA). The varietal is a C. indica-dominate strain that has 7 to 9 weeks of flowering.
In order to establish the optimal UAE extraction protocol, two approaches were taken: the traditional one factor at a time (OFT) optimization and a Design of Experiment (DoE) approach using a Central Composite Design (CCD)15. For the DoE, CBDA/CBD extraction was optimized based on the sample/solvent ratio, extraction time, and solvent concentration as factors, and the resulting data was analyzed by Response Surface Methodology (RSM). In conclusion, the protocol described outlines the optimal method for extracting the highest amount of CBDA/CBD.

<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

Industrial hemp (Cannabis spp.) has many compounds of interest with potential medical benefits. Of these compounds, cannabinoids have come to the center of attention, specifically acidic cannabinoids. The focus is turning toward acidic cannabinoids due to their lack of psychotropic activity. Cannabis plants produce acidic cannabinoids with hemp plants producing low levels of psychotropic cannabinoids. As such, utilization of hemp for acidic cannabinoid extraction would eliminate the need for decarboxylation prior to extraction as a source for the cannabinoids. The use of solvent-based extraction is ideal for obtaining acidic cannabinoids as their solubility in solvents such as supercritical CO2 is limited due to the high pressure and temperature required to reach their solubility constants. An alternative method designed to increase solubility is ultrasonic-assisted extraction. In this protocol, the impact of solvent polarity (acetonitrile 0.46, ethanol 0.65, methanol 0.76, and water 1.00) and concentration (20%, 50%, 70%, 90%, and 100%) on ultrasonic-assisted extraction efficiency has been examined. Results show that water was the least effective and acetonitrile was the most effective solvent examined. Ethanol was further examined since it has the lowest toxicity and is generally regarded as safe (GRAS). Surprisingly, 50% ethanol in water is the most effective ethanol concentration for extracting the highest amount of cannabinoids from hemp. The increase in cannabidiolic acid concentration was 28% when compared to 100% ethanol, and 23% when compared to 100% acetonitrile. While it was determined that 50% ethanol is the most effective concentration for our application, the method has also been demonstrated to be effective with alternative solvents. Consequently, the proposed method is deemed effective and rapid for extracting acidic cannabinoids.

The solvents utilized range from the middle of the polarity index (0.460 - ACN) to polar (1.000 - water). From Table 2, it can be seen that water did not make an effective extractant for cannabinoids, which is not unexpected, as cannabinoids have limited solubility in water due to their hydrophobicity13. In contrast to water, the other solvents had similar extracted values of CBD and CBDA, with the least polar solvent acetonitrile (ACN) having higher extraction when compared to th...

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

Ultrasonic-assisted extraction (UAE) increases extraction efficiency of solvents and when applied to Cannabis spp. biomass it reduces the time required for extraction. This decreases the cost and potential cannabinoid loss due to degradation. Additionally, UAE is considered a green method due to low solvent use.

Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass

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

Research

JoVE Journal

Biochemistry

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

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

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

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

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

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

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

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

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

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

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

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