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Comparison of different methods for the extraction of cannabinoids from

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Article  in  Natural Product Research · April 2019

DOI: 10.1080/14786419.2019.1601194

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 Natural Product Research
Formerly Natural Product Letters

ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20

Comparison of different methods for the

extraction of cannabinoids from cannabis

Daniela De Vita, Valentina Noemi Madia, Valeria Tudino, Francesco Saccoliti,

Alessandro De Leo, Antonella Messore, Patrizia Roscilli, Alfonso Botto, Ivano
Pindinello, Gianluigi Santilli, Luigi Scipione, Roberta Costi & Roberto Di Santo

To cite this article: Daniela De Vita, Valentina Noemi Madia, Valeria Tudino, Francesco Saccoliti,
Alessandro De Leo, Antonella Messore, Patrizia Roscilli, Alfonso Botto, Ivano Pindinello, Gianluigi
Santilli, Luigi Scipione, Roberta Costi & Roberto Di Santo (2019): Comparison of different methods
for the extraction of cannabinoids from cannabis, Natural Product Research

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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1601194

Comparison of different methods for the extraction

of cannabinoids from cannabis
Daniela De Vitaa, Valentina Noemi Madiaa, Valeria Tudinoa, Francesco Saccolitia,
Alessandro De Leoa, Antonella Messorea, Patrizia Roscillib, Alfonso Bottob,
Ivano Pindinelloa, Gianluigi Santillia, Luigi Scipionea , Roberta Costia and
Roberto Di Santoa
a
Dipartimento di Chimica e Tecnologie del Farmaco, Istituto Pasteur-Fondazione Cenci Bolognetti,
Universita di Roma La Sapienza, Roma, Italy; bExenia group srl Albignasego, Padova, Italy

ABSTRACT ARTICLE HISTORY

Cannabis oils, namely concentrated cannabis extracts, are getting Received 19 January 2019
plenty of attention because of their therapeutic potential for treatment Accepted 22 March 2019
of patients with cancer, HIV, multiple sclerosis and several other pathol-
ogies. Here we propose the use of ultrasound-assisted extraction (UAE) KEYWORDS
and microwave-assisted extraction (MAE) as alternative methods to the Cannabis extraction;
industrial hemp; medicinal
current protocols followed by pharmacists, the only authorized to cannabis; ultrasound-
manipulate standardized Cannabis. A third method, consisting of the assisted extraction;
use of Tween 20 as surfactant, was considered. Our best extraction microwave-
methodology for commercial hemp extraction was applied to medi- assisted extraction
cinal cannabis. Here we report the results obtained for ‘Eletta campana’,
‘Carmagnola selezionata’, BediolV, FM2V and BedrocanV.
R R R

1. Introduction
Until recently, Cannabis sativa L., (Cannabaceae) was exclusively considered as a
substance of abuse. Since its therapeutic effects have been investigated for the

CONTACT Roberta Costi roberta.costi@uniroma1.it

Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1601194.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
2 D. DEVITA ET AL.

Table 1. Extraction with sonication. Values are expressed in mg/mL.

CARMAGNOLA SELEZIONATA CBD-A CBD CBN THC THC-A CBD TOT THC TOT
Referencea 4.90 4.79 0.25 0.49 0.97 9.30 1.40
0.5 g in 12.5 þ 12.5 mL of EtOH, 25min þ 25minb 4.79 4.56 0.32 0.58 0.92 8.78 1.39
0.5 g in 25 mL of EtOH, 50 minb 4.78 4.90 0.32 0.58 0.94 8.88 1.41
0.5g in 25 mL of EtOH, 120 minb 4.78 4.62 0.32 0.54 0.98 8.86 1.41
0.5g in 25 mL of olive oil, 120 minb 1.66 1.06 0.26 0.22 0.26 2.53 0.45
a
Extraction in ethanol under reflux, at 90  C for 50 min.
b
The extraction was performed at 60  C.

treatment of several medical conditions, the potential of Cannabis is changing

(Whiting et al. 2015). Increasing evidences suggested that cannabinoids can be used
in different pathological conditions, such as symptoms of muscle problems associated
to multiple sclerosis, nausea and vomiting caused by chemotherapy, and anorexia in
cancer or HIV patients (Cressey 2015; Abrams and Guzman 2015). Two synthetic can-
nabinoids (Marinol, AbbVie Inc and Cesamet, Meda Pharmaceuticals Inc) and the
extract of Cannabis sativa, used as oromucosal spray (Sativex, GW Pharma Ltd.)
obtained approval for pharmaceutical uses (Badowski and Yanful 2018; Nielsen et al.
2018; Giacoppo et al. 2017) In addition, several standardized cannabinoid preparations
from dried female flower tops of the plant, which must be handled and prepared by
the pharmacist upon medical prescription, have been authorized. As different studies
suggested, the use of the extract as a complex of cannabinoids and terpenes, instead
of pure synthetic cannabinoids, allows taking advantage of the so called entourage
effects. (Russo 2011). The cannabis plant contains more than one hundred cannabi-
noids (Fellermeier et al. 2001). The main cannabinoids are D9-Tetrahydrocannabinol
(THC) and its structurally analogue, although not psychoactive, Cannabidiol (CBD).
They are present in the plant as inactive acids (THC-A and CBD-A) and they can be
converted in the corresponding non-acidic forms via thermal decarboxylation (Meng
et al. 2018). Besides of pharmaceutical cannabis, several European countries, including
Italy, allow “cannabis light” containing low level of the psychoactive component THC
and its precursor THC-A. Here we describe different extraction procedures of commer-
cially available industrial hemp and medicinal cannabis, and how the extracted canna-
binoids composition changes depending on the used method. Chemical structures of
compounds described in this study are shown in Chart 1.

2. Results and discussion

This work aimed at identifying alternative methods to those currently used by pharma-
cists for the extraction of standardized Cannabis with a consistent composition of can-
nabinoids and terpenes. The most common methods consist in using olive oil or
ethanol, both of pharmaceutical grade, at room temperature or under heating
(Romano and Hazekamp 2013; Citti et al. 2016). To meet the raising demand of reli-
able and easy to handle cannabis extraction methods, we analyzed different proce-
dures that could be easily performed by pharmacists. Therefore, this study aims at
comparing the extraction yield of the principal cannabinoids (CBD-A and CBD, THC,
THC-A and CBN, the latter coming from the oxidation of THC) obtained using different
extraction methods of commercial available varieties of Italian Hemp. Particularly, the
 NATURAL PRODUCT RESEARCH 3

Table 2. Ultrasound-assisted extraction. Values are expressed in mg/mL. The extraction was
performed with 0.5 g of plant material in 25 mL of EtOH for 50 min at 60  C.
THC TOT CBD TOT
BEDIOL (6.5 THC; 8 CBD)a 2.00 4.80
FM2 (5–8 THC; 7.5–12 CBD)a 1.90 8.10
BEDROCAN (18–22 THC; 1 CBD)a 5.60 0.10
a
The data in brackets represent the content of THC or CBD in medicinal cannabis expressed in %w/w.

Chart 1. Cannabinoids described in this study.

composition of the extract obtained by the reference method consisting in heating

the plant material under reflux at 90  C for 50 min, was compared with that from son-
ication, microwave-assisted extractions and tween 2%, in water or in ethanol. In par-
ticular, we evaluated the extraction yield of UAE at 60  C on the variety ‘Carmagnola
selezionata’ by analysing the influence of both solvent and extraction time (Table 1).
Indeed, we set the extraction time at 50 or 120 min, considering ethanol or olive oil as
solvents. Thereafter, the sonicated samples were filtered through filter paper, followed
by evaporation of the solvent (in the case of ethanol), in order to produce a concen-
trated residue that was reconstituted with a known amount of solvent. The HPLC ana-
lysis of each sample shows that the extraction yield of cannabinoids is up to three/
four times higher if ethanol is used.
The best UAE conditions in terms of extraction yield of industrial hemps, obtained
with EtOH for 50 min at 60  C, were applied to medicinal cannabis (BediolV, FM2V,
R R

BedrocanV) at Pharmacy S. Vittorio, Brescia, Italy.

R

As shown in Table 2, even though the total amount of THC and CBD is lower than
control, it is worthy of note that sonication allowed to extract 8.1 mg/mL of total CBD
from FM2, a content comparable to control, at milder temperature and in a more inex-
pensive, safe, environmental-friendly and energy-efficient way.
The microwave-assisted extraction (MAE) yield of the variety ‘Eletta Campana’ was
evaluated by analyzing four different parameters: time, temperature, ramping time
and solvent. As shown in Table 3, the amount of CBD increases by increasing both
time and temperature of at least 4 times under microwave conditions in respect to ref-
erence extraction. The solvent also affects the content of CBD as a better result is
obtained by using olive oil instead of ethanol with a shorter ramping time.
Noteworthy, an opposite trend of the extracted CBD-A content is observed because it
4 D. DEVITA ET AL.

Table 3. Microwave assisted extraction. Values are expressed in mg/mL.

ELETTA CAMPANA CBD-A CBD CBN THC THC-A CBD TOT THC TOT
Referencea 5.80 1.14 0.11 0.18 0.16 6.28 0.32
3 min, 90  C, T ramping 1.4 minb 0.70 4.30 0.70 0.90 0.30 4.92 1.16
10 min 90  C, T ramping 2.15 minb 0.43 5.90 0.79 1.00 0.39 6.29 1.34
3 min, 120  C, T ramping 4.15 minb 0.03 6.40 0.79 1.10 0.56 6.42 1.50
10 min, 120  C, T ramping 1.53 minb 0.02 6.50 0.79 0.10 0.40 6.52 0.45
0.1 g in 5 mL of MCT oil, 3 min, 90  C, T ramping, 3 min 0.03 5.30 0.90 0.90 0.40 5.33 1.25
0.1 g in 5 mL of olive oil, 3 min, 90  C, T ramping 1.4 min 1.00 6.50 0.70 1.30 0.40 6.58 1.65
a
Extraction in ethanol under reflux, at 90  C for 50 min.
b
The extraction was performed with 0.1 g of plant material in 5 mL of EtOH.

Table 4. Microwave assisted extraction. Values are expressed in mg/mL. The extraction was
performed with 0.1g of plant material in 5 ml of olive oil for 3 min at 90  C, T ramping 1.4 min.
THC TOT CBD TOT
BEDIOL (6.5 THC; 8 CBD)a 1.20 2.35
FM2 (5–8 THC; 7.5–12 CBD)a 1.60 3.06
BEDROCAN (18–22 THC; 1 CBD)a 4.10 0.10
a
The data in brackets represent the content of THC or CBD in medicinal cannabis expressed in %w/w.

Table 5. Extraction with 2% Tween solution. Values are expressed in mg/mL.

ELETTA CAMPANA CBD-A CBD CBN THC THC-A CBD TOT THC TOT
Referencea 5.80 1.14 0.11 0.18 0.16 6.28 0.32
5 mL of 2% Tween 20 in H2O, 2 h, RTb 0.67 2.70 0.90 0.60 0.30 3.29 0.86
5 mL of 2% Tween 20 in EtOH, 2 h, RTb 1.20 2.40 0.70 0.70 0.30 3.46 0.96
5mL of 2% Tween 20 in H2O, 2 h, 90 Cb 0.88 2.40 0.70 0.70 0.30 3.18 0.96
5 mL of 2% Tween 20 in EtOH, 2 h, 90 Cb 0.56 2.80 0.70 0.70 0.30 3.29 0.96
5 mL of 2% Tween 20 in H2O, 50 min, 90 Cb 1.10 2.50 0.70 0.70 0.30 3.47 0.96
5 mL of 2% Tween 20 in EtOH, 50 min, 90 Cb 0.93 2.70 0.70 0.70 0.30 3.52 0.96
a
Extraction in ethanol under reflux, at 90  C for 50 min.
b
The extraction was performed with 0.1 g of plant material.

considerably decreases by increasing time and temperature of extraction. This trend

may suggest a conversion of the CBD-A into the decarboxylated form under micro-
wave conditions. Regarding the THC and THC-A contents in ethanol extract, the former
seems to be overall influenced, even though to a less extent, in a similar fashion as
CBD, while the latter slightly increases by increasing the temperature and decreases
by decreasing the ramping time. Even though the microwave-assisted extraction
allows better results in terms of extracted amount of THC and THC-A in respect to the
reference method regardless of the used solvent, the extraction rate of total THC and
CBD was higher in olive oil compared to ethyl alcohol.
Similarly to UAE, we decided to apply our MAE best conditions of industrial hemps
to medicinal cannabis (BediolV, FM2V, BedrocanV) at Pharmacy S. Vittorio, Brescia, Italy.
R R R

Also in this case, before performing the extraction, the plant material underwent
decarboxylation. As observed with industrial hemp, also for the medicinal one the UAE
proved to be more satisfactory than MAE in terms of extraction yield of both total
THC and CBD amount. Moreover, differently from UAE, the total CBD content extracted
from FM2 resulted lower than control, being 3.06 mg/mL (Table 4) instead of 8.1 mg/
mL obtained by UAE.
In Table 5 are reported the results obtained for the variety ‘Eletta Campana’
extracted with 2% solution of tween in ethanol or water. As general trend, the
 NATURAL PRODUCT RESEARCH 5

contents of total THC in presence of surfactant is lower if compared to the reference

procedure. Among the experiment with tween 20, the total THC amount was about
the same for all the conditions. On the contrary, the total CBD values were up to 2
times lower than the reference content. Interestingly, while in the reference extract
the CBD-A is 5 times higher than the neutral form, the content of CBD in the extract
with surfactant is always higher than CBD-A one.

3. Experimental
3.1 Materials
Industrial hemp (Eletta campana, Carmagnola selezionata) is available in the Italian
market for legal sales. The extraction of medicinal cannabis (BediolV, FM2V and
R R

BedrocanV) was performed by the pharmacist, according to the best experimental

R

procedure of industrial hemps, and analyzed for the cannabinoid titration as required
by Italian law. Certified reference solutions (1 mg/mL) of Cannabidiol (CBD),
Cannabidiolic acid (CBDA), D9-Tetrahydrocannabinol (THC), Cannabinol (CBN),
D9-Tetrahydrocannabinolic acid (THCA) were purchased from Sigma-Aldrich (Milan,
Italy). Organic solvents HPLC grade were purchased from Sigma-Aldrich (Milan, Italy)
and Carlo Erba, and used without further purification. Tween 20, MCT (medium chain
triglycerides) and olive oils, all of pharmaceutical grade, were obtained from
Farmalabor Srl. Water was purified before use by a Milli-Q plus 185 system (Milford,
MA, USA). Ultrasonic-assisted extraction was performed with an Elmasonic S 60 H
(Elmasonic) sonicator. CEM Discover Microwave was used for microwave-assisted
extraction (MAE). Method development for analysis of the extract was carried out with
a HPLC system consisting of a Shimadzu LC-10AD pump, a SIL-10AD autosampler
using a 200 lL sample loop, a CTO-10AC column oven, a SPD-10A detector. The
detector was set at 220 nm. Data analyses were performed using the LC solutions soft-
ware. The HPLC analysis were performed using an analytical column Waters Symmetry
C18 (150 mm  4.6 mm, 3.5 lm) by isocratic elution with a flow rate of 1.0 mL min1
at 30  C. The mobile phase composition was acetonitrile:water 80:20 (0.1% TFA) v/v.
The calibration curve was constructed with standard solutions of CBD-A, CBD, CBN,
THC-A and THC in ethanol ranging from 0.01 to 1 mg/mL. Peak area of each com-
pounds were plotted vs. actual concentrations. Linearity was assessed through evalu-
ation of the coefficient of determination (R2). The total CBD content, expressed as mg/
mL, is intended as the sum of CBD and (0.887  CBD-A) and the total THC content,
expressed in mg/mL, results from the sum of THC and (0.887  THC-A)

3.2 Sample preparation

3.2.1. Decarboxylation
The decarboxylation of the raw material (medicinal cannabis) was performed in a pre-
heated oven at 120–130  C. The raw material was ground, spread on an aluminum foil
and placed in a Pyrex crystallizer. To avoid the dispersion of volatile components, a
further aluminum foil was used to cover the container. After 30 min, it was allowed to
reach room temperature before removing the aluminum cover.
6 D. DEVITA ET AL.

3.2.2. Extraction with Ethanol under reflux

A mixture of 1 g of the sample of industrial hemp and 50 mL of ethanol (96%) in a
round bottom flask equipped with a condenser was placed in an oil bath and heated
at 90  C under magnetic stirring for 50 min. Subsequently, the suspension was cooled
down to room temperature, filtered by using filter paper and the solvent removed
under vacuum. The residue was then reconstituted with 5 mL of ethanol and 10 lL of
this solution were diluted to 1 mL before to injected into HPLC system.

3.2.3. Extraction with sonication

0.5 g of powder was weighed, placed in a round bottom flask and a proper volume of solv-
ent (ethanol or oil) was added. Then the mixture was placed into the ultrasonic cleaning
equipment for extraction process at 60  C for 50 or 120 min. All extracts were paper filtered
and the opportune volume was injected into HPLC system for the analysis.

3.2.4. Microwave-assisted extraction

0.5 g of sample was placed into the lined extraction vessel for microwave sample prep-
aration covered with 5 mL of solvent (ethanol or oil) and extracted for 3 or 10 min at
90 or 120  C. Then the vessels were allowed to reach room temperature before open-
ing and the extract was filtered using paper filter and analyzed.

3.2.5. Extraction with Tween 20 solution

A mixture of 100 mg of plant material and 5 mL of a 2% tween solution in water or etha-
nol was stirred in a round bottom flask for 50 min or 2 h, at room temperature or 90  C.
When heat was used, after filtrating, the solvent was evaporated under vacuum and the
residue was reconstituted with a known volume of solvent (ethanol) and analyzed.

4. Conclusion and future perspective

The content of cannabinoids CBD-A, CBD, THC, THC-A and CBN was determined by
HPLC after extraction with different procedures from two variety of ‘cannabis light’. We
considered for this study a conventional procedure of extraction with ethanol under
reflux that was compared with sonication, microwave-assisted extraction and, eventu-
ally, with ethanol or water solution of a surfactant. All the procedures are very handy
and allow to drastically minimize the extraction time, as for microwave extraction, or to
work at mild temperature, as in the case of sonication. The best conditions for extraction
by sonication or microwaves were used to extract standardized cannabinoid prepara-
tions. Therefore, our future goal is to optimize our method in order to define the best
extraction conditions that could be applied to cannabis-based medicinal extracts and
easily performed by pharmacists. This will allow us to deeply evaluate the cannabinoid
composition of medicinal cannabis extract by using the new protocols here proposed.

Acknowledgements
We are indebted to the pharmacists Drs. Piero Lussignoli, Adalberto Fabbriconi, Pietro Siciliano
and Mario Marcucci for providing the cannabis-based medicinal extracts and for their continued
help and valuable advice.
 NATURAL PRODUCT RESEARCH 7

Disclosure statement
No potential conflict of interest was reported by the authors.

ORCID
Luigi Scipione http://orcid.org/0000-0002-2006-7005
Roberta Costi http://orcid.org/0000-0002-1314-9029
Roberto Di Santo http://orcid.org/0000-0002-4279-7666

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