Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass

Summary

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.

Abstract

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.

Introduction

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 compounds^9,10. 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, Rožanc et al.¹³ compared multiple methods of extraction (UAE, soxhlet, maceration, and supercritical fluid) and examined the extracts' biological activity. Rožanc 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. Rožanc'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' 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 cannabinoids^2,3. 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'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.

Protocol

1. Plant material preparation

1. 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).
2. Air-dry the inflorescences at 35 °C for 48 h. Grind the inflorescences using a grinding machine set a 177 µm.
3. Pass the pulverized material through the No. 80 mesh sieve. Store the resulting powder in a sealed bag at room temperature for future use.

2. Ultrasound extraction

1. 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.
2. Place the extraction vessel in the ultrasonic bath set at 40 kHz and at room temperature (sonication power is 100 W).
3. Perform the extraction in the ultrasonic bath for 30 min, increasing the temperature of the bath from 25 °C to 30 °C.
4. Decant the extraction fluid into a centrifuge tube.
5. Centrifuge the fluid at 3,000 x g at 15 °C for 15 min. Filter the supernatant under vacuum through an 8 µm filter paper.

3. High-performance liquid chromatography (HPLC) quantitative analysis

1. Dilute seven cannabinoid standards: cannabichromene (CBC), CBD, CBDA, cannabinol (CBN), tetrahydrocannabinolic acid (THCA), Δ⁸-THC, and Δ⁹-THC to operating concentrations of 100, 50, 25, and 12.5 µg/mL in 100% methanol. Mix and sonicate for 5 min in an ultrasonic bath set at 40 kHz and sonication power of 100 W
2. Filter the standards through a 0.45 µm polytetrafluoroethylene (PTFE) syringe filter. Filter the sample supernatant (from step 2.5) through a 0.45 µm PTFE syringe filter.
3. Put the sample to be analyzed in a 1.5 mL vial into the HPLC autosampler and load 10 µL at a time.
4. Run HPLC as per the conditions and parameters provided in Table 1. Derive cannabinoid concentrations in 50-200 µg/mL from the generated standard curve.
5. Multiply with the volume of the solvent (40 mL) used in the extraction process to obtain µg of cannabinoid. Convert the µg of cannabinoid to mg of cannabinoid by dividing it by 1000.
6. Divide with the original weight of the plant material (0.5 g) used in the extraction to obtain mg/g dry weight.

4. Optimization using response surface methodology

1. 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.
2. 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.
3. Select the total yield of lipophilic extract and the yields of extracted CBD and CBDA as response factors (RF).

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetonitrile	J.K.Baker	9017-88	solvent
Cannabichromene	Cerilliant	C-143	Cannabinoids standard
Cannabidiol	Cerilliant	C-045	Cannabinoids standard
Cannabidiolic acid	Cerilliant	C-144	Cannabinoids standard
Cannabidivarin	Cerilliant	C-140	Cannabinoids standard
Cannabigerol	Cerilliant	C-141	Cannabinoids standard
Cannabinol	Cerilliant	C-046	Cannabinoids standard
Centrifuge	Hanil Scientific Inc	Supra 22K	Centrifuge
Cherry Wine hemp	CFH, Ltd.	-	Flower extraction material
Distilled water	TEDIA	WS2211-001	solvent
Ethanol	TEDIA	ES1431-001	solvent
Filter paper	Whatman	#2	Filtering
Grinder	Daesung Artlon	DA280-S	Milling
HPLC	Shimadzu	LC-10 system	Analysis of Cannabinoid
Methanol	TEDIA	MS1922-001	solvent
Minitab 16.2.0	Minitab Inc.
Syringe filters	Whatman	6779-1304	Filtering
Tetrahydrocannabivarin	Cerilliant	T-094	Cannabinoids standard
Trifluoroacetic acid	Sigma-aldrich	302031-1L	HPLC flow solvent
Untrasonic bath	Jinwoo	4020P	Ultrasonic extraction
Zorbax Eclipse plus C18 HPLC column	Agilent	9599990-902	HPLC column
Δ8 - Tetrahydrocannabinol	Cerilliant	T-032	Cannabinoids standard
Δ9 - Tetrahydrocannabinol	Cerilliant	T-005	Cannabinoids standard
Δ9 - Tetrahydrocannabinolic acid	Cerilliant	T-093	Cannabinoids standard