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 temperature for future use.</li>
</ol>
2. Ultrasound extraction
<ol>
	<li>Weigh 0.5 g of the Cannabis inflorescence powder into a 50 mL conical tube. Add 40 mL of the solvent (e.g., 50% ethanol in deionized water) to the vessel.</li>
	<li>Place the extraction vessel in the ultrasonic bath set at 40 kHz and at room temperature (sonication power is 100 W).</li>
	<li>Perform the extraction in the ultrasonic bath for 30 min, increasing the temperature of the bath from 25 &#176;C to 30 &#176;C.</li>
	<li>Decant the extraction fluid into a centrifuge tube.</li>
	<li>Centrifuge the fluid at 3,000 x g at 15 &#176;C for 15 min. Filter the supernatant under vacuum through an 8 &#181;m filter paper.</li>
</ol>
3. High-performance liquid chromatography (HPLC) quantitative analysis
<ol>
	<li>Dilute seven cannabinoid standards: cannabichromene (CBC), CBD, CBDA, cannabinol (CBN), tetrahydrocannabinolic acid (THCA), &#916;8-THC, and &#916;9-THC&#160;to operating concentrations of 100, 50, 25, and 12.5 &#181;g/mL in 100% methanol. Mix and sonicate for 5 min in an ultrasonic bath set at 40 kHz and sonication power of 100 W</li>
	<li>Filter the standards through a 0.45 &#181;m polytetrafluoroethylene (PTFE) syringe filter. Filter the sample supernatant (from step 2.5) through a 0.45 &#181;m PTFE syringe filter.</li>
	<li>Put the sample to be analyzed in a 1.5 mL vial into the HPLC autosampler and load 10 &#181;L at a time.</li>
	<li>Run HPLC as per the conditions and parameters provided in Table 1. Derive cannabinoid concentrations in 50-200 &#181;g/mL from the generated standard curve.</li>
	<li>Multiply with the volume of the solvent (40 mL) used in the extraction process to obtain &#181;g of cannabinoid. Convert the &#181;g of cannabinoid to mg of cannabinoid by dividing it by 1000.</li>
	<li>Divide with the original weight of the plant material (0.5 g) used in the extraction to obtain mg/g dry weight.</li>
</ol>
4. Optimization using response surface methodology
<ol>
	<li>Establish the model, consisting of 15 experimental runs with 12 factorial points and three center points as shown in Table 4 using a data analysis tool.</li>
	<li>Optimize the extraction parameters, extraction time (T), solvent concentration (S), and sample/solvent ratio (R) using response surface methodology and central composite design. Set the range of variables T, S, and R as 5-30 min, 20%-100%, and 60-100 (1:X), respectively.</li>
	<li>Select the total yield of lipophilic extract and the yields of extracted CBD and CBDA as response factors (RF).</li>
</ol>

<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. G., 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=Supercritical+extraction+strategies+using+CO2+and+ethanol+to+obtain+cannabinoid+compounds+from+cannabis+hybrid+flowers.">Supercritical extraction strategies using CO2 and ethanol to obtain cannabinoid compounds from cannabis hybrid flowers.</a> Journal of CO2 Utilization. 30, 241-248 (2019).</li><li>Azmir, 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=Techniques+for+extraction+of+bioactive+compounds+from+plant+materials:+A+review.">Techniques for extraction of bioactive compounds from plant materials: A review.</a> Journal of Food Engineering. 117 (4), 426-436 (2013).</li><li>Ohl, C. D., Kurz, T., Geisler, R., Lindau, O., Lauterborn, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bubble+dynamics,+shock+waves+and+sonoluminescence.">Bubble dynamics, shock waves and sonoluminescence.</a> Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences. 357 (1751), 269-294 (1999).</li><li>Castro-Puyana, M., Marina, M. L., Plaza, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water+as+green+extraction+solvent:+Principles+and+reasons+for+its+use.">Water as green extraction solvent: Principles and reasons for its use.</a> Current Opinion in Green and Sustainable Chemistry. 5, 31-36 (2017).</li><li>Herrera, M. C., De Castro, M. 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+phenolic+compounds+from+strawberries+prior+to+liquid+chromatographic+separation+and+photodiode+array+ultraviolet+detection.">Ultrasound-assisted extraction of phenolic compounds from strawberries prior to liquid chromatographic separation and photodiode array ultraviolet detection.</a> Journal of Chromatography A. 1100 (1), 1-7 (2005).</li><li>Mason, T. J., Paniwnyk, L., Lorimer, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+uses+of+ultrasound+in+food+technology.">The uses of ultrasound in food technology.</a> Ultrasonics Sonochemistry. 3 (3), 253-260 (1996).</li><li>Soares, V. 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. 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>. FDA Available from: <a target="_blank" href="https://www.cfsanappsexternal.fda.gov/scripts/fdcc/?set=SCOGS">https://www.cfsanappsexternal.fda.gov/scripts/fdcc/?set=SCOGS</a> (2021)</li><li>Rae, 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=Estimation+of+ultrasound+induced+cavitation+bubble+temperatures+in+aqueous+solutions.">Estimation of ultrasound induced cavitation bubble temperatures in aqueous solutions.</a> Ultrasonics Sonochemistry. 12, 325-329 (2005).</li><li>Moreno, T., Montanes, F., Tallon, S. J., Fenton, T., King, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+cannabinoids+from+hemp+(Cannabis+sativa+L.)+using+high+pressure+solvents:+An+overview+of+different+processing+options.">Extraction of cannabinoids from hemp (Cannabis sativa L.) using high pressure solvents: An overview of different processing options.</a> Journal of Supercritical Fluids. 161, 104850 (2020).</li><li>Zhang, Q. W., Lin, L. G., Ye, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+extraction+and+isolation+of+natural+products:+a+comprehensive+review.">Techniques for extraction and isolation of natural products: a comprehensive review.</a> Chinese Medicine. 13 (20), 1-26 (2018).</li><li>Fathordoobady, F., Singh, A., Kitts, D. D., Singh, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemp+(Cannabis+sativa+L.)+extract:+Anti-microbial+properties,+methods+of+extraction,+and+potential+oral+delivery.">Hemp (Cannabis sativa L.) extract: Anti-microbial properties, methods of extraction, and potential oral delivery.</a> Food Reviews International. 35 (7), 664-684 (2019).</li></ol>

The authors declare no competing interests.

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 ...

ultrasonic-assisted-extraction-cannabidiolic-acid-from-cannabis

Research

JoVE Journal

Biochemistry

5.9K Views.  Colorado State University-Pueblo. 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.

Video: Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass

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 the large-scale identification and quantification of the proteins present in complex biological systems.The knowledge gained from a proteomic approach can potentially lead to a better understanding of the pathogenic mechanisms underlying diseases, allowing for the identification of novel diagnostic and prognostic disease markers, and, hopefully, of therapeutic targets. However, the cardiac mitral valve represents a very challenging sample for proteomic analysis because of the low cellularity in proteoglycan and collagen-enriched extracellular matrix. This makes it challenging to extract proteins for a global proteomic analysis. This work describes a protocol that is compatible with subsequent protein analysis, such as quantitative proteomics and immunoblotting. This can allow for the correlation of data concerning protein expression with data on quantitative mRNA expression and non-quantitative immunohistochemical analysis. Indeed, these approaches, when performed together, will lead to a more comprehensive understanding of the molecular mechanisms underlying diseases, from mRNA to post-translational protein modification. Thus, this method can be relevant to researchers interested in the study of cardiac valve physiopathology.

The protein composition of the human mitral valve is still partially unknown, because its analysis is complicated by low cellularity and therefore by low protein biosynthesis. This work provides a protocol to efficiently extract protein for the analysis of the mitral valve proteome.

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 determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

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 chemical defense because it has antimicrobial activity and is considered a natural insecticide. Caffeine can also produce negative allelopathic effects that prevent the growth of surrounding plants. In addition, people around the world consume caffeine for its analgesic and stimulatory effects. Due to interest in the technological applications of caffeine, research on the biosynthetic pathway of this compound has grown. These studies have primarily focused on understanding the biochemical and molecular mechanisms that regulate the biosynthesis of caffeine. In vitro tissue culture has become a useful system for studying this biosynthetic pathway. This article will describe a step-by-step protocol for the quantification of caffeine and for measuring the transcript levels of the gene (CCS1) encoding caffeine synthase (CS) in cell suspensions of C. arabica L. as well as its activity.

This protocol describes an efficient methodology for the extraction and quantification of caffeine in cell suspensions of C. arabica L. and an experimental process for evaluating the enzymatic activity of caffeine synthase with the expression level of the gene that encodes this enzyme.

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 anticancer ones. The bioactive compounds of Taiwanese green propolis are propolins, namely C, D, F, and G. The concentration of propolins in Taiwanese green propolis varies depending on the season and geographic location. Thus, it is critical to establish a standard and repeatable procedure for determining the quality of Taiwanese green propolis. Here, we present a protocol that uses ethanol-based extraction, high-performance liquid chromatography, and an antibacterial activity analysis to characterize Taiwanese green propolis quality. This method indicates that 95% and 99.5% ethanol extractions achieve the maximum dry matter yields from Taiwanese green propolis, thereby yielding the highest concentrations of propolins that have antibacterial properties. According to these findings, the present protocol is deemed reliable and repeatable for determining the quality of Taiwanese green propolis.

We present a protocol for using ethanol as a solvent to extract and characterize Taiwanese green propolis that exhibits antibacterial activity.

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 majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

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

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 physiology. Due to their negative charge, low abundance and susceptibility to hydrolytic activities, the detection and quantification of these molecules is challenging. This is particularly the case for highly phosphorylated forms containing &#8216;high-energy&#8217; diphospho bonds, also termed inositol pyrophosphates (PP-InsPs). Due to its high sensitivity, strong anion exchange high-performance liquid chromatography (SAX-HPLC) of plants labeled with [3H]-myo-inositol is currently the method of choice to analyze these molecules. By using [3H]-myo-inositol to radiolabel plant seedlings, various InsP species including several non-enantiomeric isomers can be detected and discriminated with high sensitivity. Here, the setup of a suitable SAX-HPLC system is described, as well as the complete workflow from plant cultivation, radiolabeling and InsP extraction to the SAX-HPLC run and subsequent data analysis. The protocol presented here allows the discrimination and quantification of various InsP species, including several non-enantiomeric isomers and of the PP-InsPs, InsP7 and InsP8, and can be easily adapted to other plant species. As examples, SAX-HPLC analyses of Arabidopsis thaliana and Lotus japonicus seedlings are performed and complete InsP profiles are presented and discussed. The method described here represents a promising tool to better understand the biological roles of InsPs in plants.

Here we describe strong anion exchange high-performance liquid chromatography of [3H]-myo-inositol-labeled seedlings which is a highly sensitive method to detect and quantify inositol polyphosphates in plants.

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)

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 and is an aromatic heteropolymer primarily composed of three monolignols with significant industrial importance. Lignin plays an important role in plant growth and development, protects&#160;from biotic and abiotic stresses, and in the quality of animal fodder, the wood, and industrial lignin products. Accurate estimation of lignin content is essential for both fundamental understanding of the lignin biosynthesis and industrial applications of biomass. The thioglycolic acid (TGA) method is a highly reliable method of estimating the total lignin content in the plant biomass. This method estimates the lignin content by forming thioethers with the benzyl alcohol groups of lignin, which are soluble in alkaline conditions and insoluble in acidic conditions. The total lignin content is estimated using a standard curve generated from commercial bamboo lignin.

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid TGA

Here, we present a modified TGA method for estimation of lignin content in herbaceous plant biomass. This method estimates the lignin content by forming specific thioether bonds with lignin and presents an advantage over the Klason method, as it requires a relatively small sample for lignin content estimation.

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 abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

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 host&#39;s microbial community. To disentangle the origin of microbial VOCs, volatile headspace analysis of bacterial mono- and co-cultures of Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, and stable isotope probing in biological samples of feces, saliva, sewage, and sputum were performed. Mono- and co-cultures were used to identify volatile production from individual bacterial species or in combination with stable isotope probing to identify the active metabolism of microbes from the biological samples.
Vacuum-assisted sorbent extraction (VASE) was employed to extract the VOCs. VASE is an easy-to-use, commercialized, solvent-free headspace extraction method for semi-volatile and volatile compounds. The lack of solvents and the near-vacuum conditions used during extraction make developing a method relatively easy and fast when compared to other extraction options such as tert-butylation and solid phase microextraction. The workflow described here was used to identify specific volatile signatures from mono- and co-cultures. Furthermore, analysis of the stable isotope probing of human associated biological samples identified VOCs that were either commonly or uniquely produced. This paper presents the general workflow and experimental considerations of VASE in conjunction with stable isotope probing of live microbial cultures.

This protocol describes the extraction of volatile organic compounds from a biological sample with the vacuum-assisted sorbent extraction method, gas chromatography coupled with mass spectrometry using the Entech Sample Preparation Rail, and data analysis. It also describes culture of biological samples and stable isotope probing.

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

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

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as oxaloacetate decarboxylase in mitochondria. This article presents a series of methods for the extraction and purification of FAHD1 from swine kidney and mouse liver. Covered methods are ionic exchange chromatography with fast protein liquid chromatography (FPLC), preparative and analytical gel filtration with FPLC, and proteomic approaches. After total protein extraction, ammonium sulfate precipitation and ionic exchange chromatography were explored, and FAHD1 was extracted via a sequential strategy using ionic exchange and size-exclusion chromatography. This representative approach may be adapted to other proteins of interest (expressed at significant levels) and modified for other tissues. Purified protein from tissue may support the development of high-quality antibodies, and/or potent and specific pharmacological inhibitors.

This protocol describes how to extract fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) from swine kidney and mouse liver. The listed methods may be adapted to other proteins of interest and modified for other tissues.