Accepted Manuscript

Menthol-based hydrophobic deep eutectic solvents: towards greener and efficient

extraction of phytocannabinoids

Tomáš Křížek, Miroslava Bursová, Rachel Horsley, Martin Kuchař, Petr Tůma,
Radomír Čabala, Tomáš Hložek

PII: S0959-6526(18)31407-0

DOI: 10.1016/j.jclepro.2018.05.080

Reference: JCLP 12937

To appear in: Journal of Cleaner Production

Received Date: 18 January 2018

Revised Date: 27 April 2018

Accepted Date: 09 May 2018

Please cite this article as: Tomáš Křížek, Miroslava Bursová, Rachel Horsley, Martin Kuchař, Petr
Tůma, Radomír Čabala, Tomáš Hložek, Menthol-based hydrophobic deep eutectic solvents:
towards greener and efficient extraction of phytocannabinoids, Journal of Cleaner Production
(2018), doi: 10.1016/j.jclepro.2018.05.080

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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 ACCEPTED MANUSCRIPT

Menthol-based hydrophobic deep eutectic solvents: towards greener and efficient

extraction of phytocannabinoids

Tomáš Křížek a, Miroslava Bursová a,b, Rachel Horsley c, Martin Kuchař c,d, Petr Tůma e,
Radomír Čabala a,b, Tomáš Hložek a,b*

a Charles University, Faculty of Science, Department of Analytical Chemistry, Albertov 6, 128

43 Prague 2, Czech Republic
b Charles University and General University Hospital, First Faculty of Medicine, Institute of
Forensic Medicine and Toxicology, Ke Karlovu 2, 121 08 Prague 2, Czech Republic
c National Institute of Mental Health, Topolová 748, 250 67 Klecany, Czech Republic
d Forensic Laboratory of Biologically Active Substances, Department of Chemistry of Natural
Compounds, Faculty of Food and Biochemical Technology, University of Chemistry and
Technology Prague, Technická 5, 166 28 Prague 6, Czech Republic
e Charles University, Third Faculty of Medicine, Department of Hygiene, Ruská 87, 100 00
Prague 10, Czech Republic

∗ Corresponding author: Tel.: +420 224 967 191

E-mail address: tomas.hlozek@vfn.cz

Keyword: Cannabis sativa, extraction, hydrophobic deep eutectic solvent, menthol,

phytocannabinoids
 ACCEPTED MANUSCRIPT

Abstract
As the demand for medical cannabis preparations increases, so does the use of the
common organic solvents that are used in the extraction and quantification of
phytocannabinoids. Since common organic solvents are typically hazardous to the environment
and to human health, it is vital to identify safer, greener, and more efficient alternatives. The
aim of the present research was to develop a series of hydrophobic deep eutectic solvents
(DESs) based on terpenes and natural organic acids and to establish whether these might be
potential substitutes for the extraction of phytocannabinoids (tetrahydrocannabinol,
cannabidiol, and their carboxylated homologues) from raw cannabis plant material.
Data were obtained using capillary electrophoresis with diode array detection (DAD).
Initial screening showed that the DES composed of a menthol: acetic acid (1:1 molar ratio)
mixture showed the greatest extraction efficiency (of all the DESs that were tested), with yields
ranging from 118.6 % to 132.6 % (compared to a methanol: chloroform mixture). In conclusion,
menthol: acetic acid DES extraction is efficient, as well as non-toxic and biodegradable. As
such it has applications within the pharmaceutical industry and represents a greener alternative
organic solvent for the extraction of phytocannabinoids.
 ACCEPTED MANUSCRIPT

1. Introduction
The Cannabis sativa plant is known to contain more than five hundred compounds, of
which more than one hundred and thirteen are phytocannabinoids, however only a few have
been studied for their biological activity. Cannabinoids are biosynthesised as prenylated
aromatic carboxylic acids; almost no neutral cannabinoids can be found in raw cannabis plant
material, however, they may convert to their neutral homologues by spontaneous
decarboxylation in the presence of light or heat. The first cannabinoid in the biosynthetic
pathway is cannabigerolic acid, which is transformed either into tetrahydrocannabinolic acid
(THCA) or cannabidiolic acid (CBDA), each by a particular synthase (Hanuš et al., 2016). The
two major cannabinoids (and those best known for their therapeutic potential) are
tetrahydrocannabinol (THC) and cannabidiol (CBD), i.e., the neutral homologues of THCA and
CBDA, respectively (Fig. 1). Whilst THC is the main psychoactive agent of cannabis and is
responsible for the euphoric effects (the ‘high’) sought by recreational users, CBD lacks
significant psychotomimetic effects; rather, it seems to counteract the psychotomimetic and
behavioural effects of THC, as well as showing anxiolytic and antipsychotic properties in and
of itself (Hložek et al., 2017).
Extraction methods for quantifying phytocannabinoids in raw plant material are essential
for potency monitoring of seized cannabis products as well as for assessing the ratio of
phytocannabinoids in medical cannabis strains. The importance of the latter is evidenced by the
growing list of confirmed medical indications and by the rising number of countries where
cannabis use (in some form) for medicinal purposes is legal (Abuhasira et al., 2018). As the
demand for cannabis increases, so too does the use of the common organic solvents that are
used in contemporary analytical extraction processes. Historically, the extraction solvent
commonly used for the analysis of phytocannabinoids was a mixture of methanol: chloroform
(9:1; v/v), with chloroform being used to dissolve the internal standard di-n-octyl phthalate
(Smith and Vaughan, 1976). Chlorinated solvents are known to be of generally high toxicity to
humans as well as damaging to the environment. Although commercially available deuterated
cannabinoid standards can be used instead, the use of chloroform continues (Swift et al., 2013;
Aizpurua-Olaizola et al., 2016; De Backer et al., 2012; ElSohly et al., 2016; Gul et al., 2015).
The removal of chloroform from the extraction solvent would improve laboratory safety, whilst
also reducing the environmental impact of chlorinated solvent usage.
In the search for a safer, greener solvent for use in phytocannabinoid extraction, recent
research has been focused on alcohols, i.e., methanol and ethanol (Patel et al., 2017; Brighenti
et al., 2017). In this context, ionic liquids have also received considerable attention as extraction
 ACCEPTED MANUSCRIPT

solvents owing to their unique physical and chemical characteristics (Hosseini et al., 2017;
Rostamnia et al., 2016) however, their application has been limited by the high costs of
synthesis and purification, and because of the toxicity of some of their ingredients (Abdus
Salam et al., 2016). A novel medium – deep eutectic solvent (DES) – with properties similar to
those of ionic liquids (ILs) has been reported (Abbott et al., 2003). According to theory, a DES
is created as an adequate mixture of hydrogen bond acceptors (HBA) and hydrogen bond donors
(HBD) which can self-associate through hydrogen-bonding interactions; this results in a strong
depression of the freezing-melting point of the mixture (Zainal-Abidin et al., 2017). The
majority of eutectic mixtures proposed so far are based on renewable resources, such as
carboxylic and amino acids, sugars, amines, and polyols. Therefore, in comparison to traditional
ILs, DESs have a number of important advantages including low toxicity, low precursor cost,
simple synthesis, negligible volatility, and high biodegradability (Zainal-Abidin et al., 2017).
From an analytical point of view, the utilisation of DES for extraction has become an
area of intense research interest. There are numerous articles detailing the use of DESs as
extraction media to obtain bioactive compounds (such as phenolic acids, flavonoids and
polyphenols) from various plant materials (Bajkacz and Adamek, 2017; Khezeli et al., 2016).
To date, the majority of studies have used DESs with hydrophilic characteristics, which narrows
the range of target extractable compounds. To extend the usage of DES for the extraction of
non-polar compounds, the need to develop and test new hydrophobic DESs is obvious. Two
different families of hydrophobic DESs have been prepared and studied so far. The first one,
introduced by Marrucho´s group, was based on menthol (HBA) and natural organic acids
(HBD). The extraction potential of these DES was confirmed on model biomolecules in
aqueous media and in neonicotinoid removal from wastewater (Ribeiro et al., 2015; Florindo et
al., 2017). The second used quaternary ammonium salts (HBA) and organic acids and alcohols
(HBD), and was introduced by Kroon´s group (van Osch et al., 2015). Later, these hydrophobic
DESs were used for the first time to extract bioactive compounds from plant material:
artemisinin from Artemisia annua leaves and polyprenyl acetates from Ginkgo biloba leaves
(Cao et al., 2017a; Cao et al., 2017b). However, given the toxicity of the starting material,
namely methyl trioctylammonium chloride (Lewis, 2008), there may be a need for further
purification of the resulting extracts, particularly if they are for pharmaceutical usage.
In this work, we aimed (i) to evaluate (for the first time) hydrophobic DESs based on
menthol and natural carboxylic acids and the efficiency with which they extract
phytocannabinoids (THC, CBD, THCA, CBDA) from cannabis plant material, and (ii) to
assess their extraction potential in comparison to common organic solvents (methanol, ethanol,
 ACCEPTED MANUSCRIPT

and methanol: chloroform (9:1; v/v) mixture), and (iii) to substitute menthol for other abundant
terpenes to explore the possibility of tailoring of this class of hydrophobic DES.

Place for Figure 1.

2. Materials and methods

2.1. Chemicals and reagents
Cannabidiol (CBD), cannabidiolic acid (CBDA), tetrahydrocannabinol (THC), and
tetrahydrocannabinolic acid (THCA) standards were purchased from Cerilliant (USA), each
with a purity of at least 98 %. Menthol, terpineol, linalol, geraniol, borneol, ethanol, chloroform,
formic acid, acetic acid, propionic acid, butyric acid, hexanoic acid, octanoic acid, dodecanoic
acid, lactic acid, phenylacetic acid, mandelic acid, oxalic acid, malonic acid, tartaric acid,
phthalic acid, glycolic acid, malic acid, hippuric acid, pyruvic acid and aspartic acid were
purchased from Sigma Aldrich (Germany), each with a purity of at least p. a. or 97 %. Sodium
hydroxide (p. a., Penta, Czech Republic), phosphoric acid (85 %, Lach-Ner, Czech Republic)
and methanol (> 99.9 %, Honeywell, France) were used as purchased. Deionised water,
prepared by a water purification system (Premier MFG’D, USA), was used for the preparation
of all aqueous solutions.

2.2. Plant material

The plant material Cannabis sativa) was kindly donated by the Forensic Laboratory of
Biologically Active Substances, and it originated from a seizure (made by Czech Police) of
clandestine, indoor-cultivated cannabis. The plant material exhibited THC/CBD balanced strain
inflorescences. Inflorescences were first ground using a laboratory-grade knife mill, followed
by further homogenisation in a mortar. The resulting powder was sampled without any
additional sieving.

2.3. Hydrophobic DESs preparation

Menthol was chosen as a starting HBA to prepare hydrophobic DESs. The respective
HBDs were the different aliphatic, aromatic and hydroxy carboxylic acids (see Table 1).
Hydrophobic DESs were prepared by heating the various molar mixtures of HBA and HBD to
80 °C, with constant stirring, until a homogenous liquid was formed.

Place for Table 1.

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2.4. DES extraction procedure

For the preliminary extraction screening, 20 mg of ground plant material was mixed
with 0.8 mL of each DES in 2 mL Eppendorf tubes. After vortexing briefly, the plant material
was extracted for 10 min in an ultrasonic bath (water temperature at 30 °C) and then centrifuged
at 10 000 rpm for 3 min. The resulting liquid supernatant was withdrawn and diluted with a
background electrolyte. Each experiment was repeated three times for each DES and the
respective cannabinoids were quantified using capillary electrophoresis with DAD.

2.5. Instrumentation and equipment

Capillary electrophoresis experiments were carried out on an Agilent 1600 CE
instrument (Agilent Technologies, Germany) with DAD at 220 nm. Other equipment consisted
of a Grindomix GM 200 laboratory-grade knife mill (Retsch Haan, Germany), an Enetron
ultrasonic bath equipped with a timer and temperature control (Sonic, China), an Arex Digital
magnetic stirrer with heating plate (Velp Scientifica, Italy).

2.6. Electrophoretic conditions

An unmodified fused-silica capillary, 50 μm I.D., 375 μm O.D., with 50.0 cm total
length and 8.5 cm effective length (Polymicro Technologies, USA), was used for
electrophoretic separation. Prior to the first measurement of the day, the capillary was activated
by rinsing with 1 mol/L NaOH for 10 min, followed by water for 10 min (water pressure at
93 kPa). Between individual runs, the capillary was flushed for 1.5 min with a background
electrolyte. The background electrolyte solution consisted of phosphate buffer (10 mmol/L
phosphoric acid, adjusted to pH 12.5 with 10 mol/L NaOH) and methanol in ratio 60: 40 (v/v).
The analysis was performed in the short-end injection format. Samples were introduced to the
outlet end of the capillary using a pressure of 5 kPa for 3 s. The separation voltage was set to -
30 kV, inducing an electric current of approximately 25 µA. The temperature of the capillary
cassette was maintained at 25 °C. The content of phytocannabinoids was based on a standard
addition calibration method
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3. Results and discussion

3.1. Hydrophobic DES preparation
There are various methods reported for the preparation of DESs, including methods
based on heating, freeze-drying or evaporation. For convenience, the heating method was
employed here, since this procedure is simple and means that multiple DESs can be prepared
simultaneously. Menthol was chosen (according to previous research; Ribeiro et al., 2015) as
the non-toxic HBA to prepare a range of hydrophobic DESs using different carboxylic acid
HBDs at 1:1 HBA/HBD molar ratio. This was also based on previous empirical findings
indicate that the molar ratio of the eutectic components in a binary system should be near unity
because the lattice binding a single component depends on involvement with other components
of the lattice (Liu et al., 2018). Ten menthol-based DESs were prepared successfully as
homogenous liquids, without any crystal precipitation at normal ambient laboratory
temperature (Table 1). The ionic nature and physicochemical properties of some menthol based
DESs (menthol: acetic; butyric; hexanoic; octanoic; dodecanoic; lactic acid) have been already
characterized by means of H1 NMR, FTIR and differential scanning calorimetry (Ribeiro et al.,
2015; Florindo et al., 2017). The remainder of DESs reported here (i.e., menthol: formic;
propionic; phenylacetic; mandelic acid) were used for extraction without further
physicochemical characterization. On the other hand, across menthol: acid 1:1 molar ratio, other
carboxylic acids did not form DESs with menthol (expressed as transparent liquid), even when
heated to higher temperatures (Table 1). Each of the prepared DESs formed a two-phase system
with water at any ratio.

3.2. Effects of DES composition on extraction

The ten successfully prepared DESs were screened for the efficiency with which they
extracted phytocannabinoids from cannabis inflorescences. As can be seen (Fig. 2), extraction
efficiency is greatly affected by the HBD used. Formic acid produced the highest THC yields
of all the DESs that were tested. Regarding the total sum of the mass of phytocannabinoids
(THC, THCA, CBD and CBDA) that were extracted, acetic acid (C2) had the highest overall
phytocannabinoid extraction efficiency. The extraction yields of THC and THCA steadily
declined in a homologous series towards hexanoic acid (C2-C6), although with further increases
in alkyl chain length (C8-C12), extraction yields rose. This effect could be accounted for by
two opposing processes: firstly, with increasing viscosity, extraction efficiency might decrease
owing to limited mass transfer; secondly, the increasing chain length might facilitate extraction
of substances with higher log P values (for instance, the log P value for THC is 6.97; log P for
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CBD is 5.79). The aforementioned trends were also valid for CBD and CBDA extraction
efficiencies, although the lower log P values of these analytes resulted in less pronounced
differences in the homologous series. Alternatively, extraction efficiency might be affected by
the capacity of the DES to cause rapid rupture and destruction of the plants’ physical structures,
similar to the effects of long-term ultrasound irradiation (Jeong et al., 2017). Therefore,
increases in extraction efficiency might be attributable to this, independently of any obvious
log P correlation.
To determine the designability of this class of hydrophobic DES, we explored the
extraction efficiencies of four abundant terpenes (terpineol, linalool, geraniol and borneol) as
substitute HBAs for menthol. Based on the results of our preliminary DES screening, acetic
acid (with the greatest overall extraction efficiency) was the selected HBD, to be combined
with the different terpenes, each at a 1:1 molar ratio. The resulting DESs were prepared as
transparent liquids. However, none of the four terpene-based DESs that were tested achieved
an extraction efficiency higher than that achieved with menthol as the HBA (Fig. 3).

Place for Figure 2.

Place for Figure 3.

Place for Figure 4.

3.3. Evaluation of the extraction efficiency of DESs in comparison to organic solvents

Historically, phytocannabinoids have been extracted using a methanol: chloroform
mixture but recently, extraction methods using alcohols without the inclusion of toxic
chloroform have been developed and evaluated (Patel et al., 2017; Brighenti et al., 2017). Under
the same extraction conditions (see chapter 2.4.), the extraction yields of menthol: acetic acid
were compared with three organic solvents that are typically used for phytocannabinoid
extraction (ethanol, methanol and methanol: chloroform mixture, see Fig. 4. Table 2 shows the
relative extraction efficiencies of these solvents (based on the assumption that the extraction
efficiency of a methanol: chloroform mixture is 100 %). The extraction efficiencies achieved
with menthol: acetic acid range between 118.6 % – 132.6 %, exceeding the performance of the
methanol: chloroform mixture and the alcohols across each of the phytocannabinoids. However,
given the number of repeated measurements (three) for each extraction, inferential statistical
comparison of solvents was not possible.
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Place for Table 2.

4. Conclusion
To date, the preparation of DESs, in particular hydrophobic DESs with rational
analytical applications is still under-explored. We evaluated, for the first time, the
phytocannabinoid extraction efficiency of hydrophobic DESs based on menthol and natural
carboxylic acids. Given the terpene nature of phytocannabinoids and their corresponding high
log P, they were successfully extracted by a hydrophobic menthol: acetic acid DES which,
compared to commonly-used organic solvents, had superior extraction efficiencies for all of the
phytocannabinoids that were evaluated. The menthol: acetic acid-based extraction developed
here is therefore efficient; and owing to its biodegradability and pharmaceutically acceptable
toxicity, it is a safer, more sustainable and greener alternative to common organic solvents for
phytocannabinoid extraction. Further DES tailoring might focus on other common secondary
plant metabolites as these are abundant and inexpensive; combined with their ease of
preparation, this yields promising candidate HBDs for use in non-toxic, menthol-based
hydrophobic DESs.

Acknowledgement
This work was supported by Charles University (project GAUK 968216), Specific
University Research (SVV), Charles University Research Centre program No. UNCE/SCI/014,
the Czech Science Foundation (project 17-12648S) and project MICR VI20172020056. TH
would like to thank Dominika Ochodnická for helpful and inspiring comments during the
manuscript preparation
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CH3 CH3
A B
OH OH O
H H
OH
H H
H3C H 3C
O CH3 O CH3
H3C H 3C

CH3 CH3
C D
OH OH O
H H

OH
H H
H 3C H3C
HO CH3 HO CH3
H 2C H2C

Fig. 1. Structures of THC (A), THCA (B), CBD (C) and CBDA (D).
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THC
80 THCA
CBD
Extraction yield (mg/g)

CBDA

60

40

20

0
A

A
A

AA
A

A
A

:M
:F

:A

:P

:B

:H

:O

:D

:L

:P
M
M

M
M

M
M
Fig. 2. Comparison of the extraction yields (mg/g) of THC, THCA, CBD and CBDA obtained
using different DESs based on menthol as the HBA and different organic acids as HBDs (n=3).
M…menthol, FA…formic acid, AA…acetic acid, PA…propionic acid, BA…butyric acid,
HA…hexanoic acid, OA…octanoic acid, DA…dodecanoic acid, LA…lactic acid,
PAA…phenylacetic acid, MA…mandelic acid.
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THC
80
THCA
CBD
Extraction yield (mg/g)

CBDA
60

40

20

0
M:AA T:AA L:AA G:AA B:AA

Fig. 3. Comparison of the extraction yields (mg/g) of THC, THCA, CBD and CBDA obtained
using different DESs based on different terpenes (M…menthol, T…terpenol, B...borneol,
G…geraniol, L…linalol) as HBAs and acetic acid (AA) as the HBD (n = 3).
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THC
THCA
80 CBD
Extraction yield (mg/g)

CBDA

60

40

20

0
M:AA MeOH EtOH MeOH:CHCl3

Fig. 4. Comparison of the extraction yields (mg/g) of THC, THCA, CBD and CBDA obtained
using different organic solvents and menthol: acetic acid (M:AA) hydrophobic deep eutectic
solvent (n=3). M…menthol, AA…acetic acid, MeOH...methanol, EtOH…ethanol, MeOH:
CHCl3… methanol: chloroform mixture (9:1; v/v).
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Highlights

1. A series of menthol-based deep eutectic solvents (DESs) were prepared.

2. Hydrophobic DESs were used to extract phytocannabinoids from plant material.

3. Menthol:acetic acid DES showed superior extraction yields to conventional organic

solvents.

4. Menthol-based DESs are greener alternatives for phytocannabinoid extraction.

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Table 1
Overview of the terpene-based hydrophobic DESs that were tested.
Abbreviation HBA HBDs Molar ratio Heating temperature Aspect at room temperature
M:FA Menthol Formic acid 1:1 80°C Transparent liquid
M:AA Acetic acid 1:1 80°C Transparent liquid
M:PA Propionic acid 1:1 80°C Transparent liquid
M:BA Butyric acid 1:1 80°C Transparent liquid
M:HA Hexanoic acid 1:1 80°C Transparent liquid
M:OA Octanoic acid 1:1 80°C Transparent liquid
M:DA Dodecanoic acid 1:1 80°C Transparent liquid
M:LA Lactic acid 1:1 80°C Transparent liquid
M:PAA Phenylacetic acid 1:1 80°C Transparent yellow liquid
M:MA Mandelic acid 1:1 80°C Transparent liquid
Menthol Oxalic acid 1:1 80 – 95 °C Solid liquid mixture
Malonic acid 1:1 80 – 95 °C Solid liquid mixture
Tartaric acid 1:1 80 – 95 °C Solid liquid mixture
Phthalic acid 1:1 80 – 95 °C Solid liquid mixture
Glycolic acid 1:1 80 – 95 °C Two-phase liquid
Malic acid 1:1 80 – 95 °C Solid liquid mixture
Hippuric acid 1:1 80 – 95 °C Solid liquid mixture
Pyruvic acid 1:1 80 – 95 °C Two-phase liquid
Aspartic acid 1:1 80 – 95 °C Solid liquid mixture
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Table 2
Comparison of the phytocannabinoid (THC, THCA, CBD and CBDA) extraction efficiencies
(%) of common organic solvents and a menthol: acetic acid hydrophobic DES. Methanol:
chloroform mixture (9:1; v/v) was used as a reference and was assumed to be 100 % efficient.
Extraction efficiency (%)
THC THCA CBD CBDA
Menthol: acetic acid 132.6 ± 5.3 128.8 ± 2.4 118.6 ± 5.7 122.7 ± 2.2
Methanol 112.0 ± 5.1 107.8 ± 2.7 97.7 ± 0.4 103.3 ± 1.6
Ethanol 118.4 ± 2.7 110.1 ± 2.4 102.3 ± 2.0 106.3 ± 1.9
Methanol: chloroform (9:1; v/v) 100 100 100 100