J.

of Supercritical Fluids 149 (2019) 20–25

Contents lists available at ScienceDirect

The Journal of Supercritical Fluids

journal homepage: www.elsevier.com/locate/supflu

Analysis of the antitumor activity of bioactive compounds of Cannabis

flowers extracted by green solvents
Daniel Ribeiro Grijó a , Danielle Lazarin Bidoia b , Celso Vataru Nakamura b ,
Ignacio Vieitez Osorio c,∗ , Lúcio Cardozo-Filho a,d
a
Department of Chemical Engineering, Universidade Estadual de Maringá, Maringá, PR, Brazil
b
Department of Health Basic Sciences, Universidade Estadual de Maringá (UEM), Maringá, PR, Brazil
c
Departamento de Ciencia y Tecnología de Alimentos, Facultad de Química, Universidad de la República, Montevideo, Uruguay
d
Center Research, Centro Universitário da Fundação de Ensino Octávio Bastos (UNIFEOB), São João da Boa Vista, SP, Brazil

h i g h l i g h t s g r a p h i c a l a b s t r a c t

• Supercritical extraction of cannabi-

noids in a simple and sequential step.
• Research strategies to increase the
content of 9 -THC and CBD before
extraction.
• Supercritical extracts with high cyto-
toxicity for antitumor cells and low
cytotoxicity for non-tumor cells.
• Extracts with high concentration of
neutral cannabinoids have high anti-
tumor activity for cervical cells.

a r t i c l e i n f o a b s t r a c t

Article history: The inhibitory effects of Cannabis flower extracts, obtained by supercritical carbon dioxide (scCO2 ) with
Received 17 November 2018 and without modifier, on various human tumour cells and non-tumour cells were evaluated. Different
Received in revised form 20 March 2019 techniques were used to optimize the polarity interaction between solute and solvent before extraction.
Accepted 20 March 2019
An increase in the cannabinoid content of interest (CBD/THC) was evaluated by decarboxylation of the
Available online 21 March 2019
flowers at 110 and 140 ◦ C. Extractions with pure scCO2 were conducted in a single step at temperatures
of 50 and 70 ◦ C and pressures of 22 and 40 MPa, varying the time of prior decarboxylation by 0.5 or 2.0 h.
Keywords:
Sequential extractions were conducted in a first step at 35 ◦ C and 10 MPa and then in a second step at
Supercritical carbon dioxide
Cannabinoids
70 ◦ C and 40 MPa, with and without modifier. Contents of the cannabinoids were evaluated using HPLC.
Decarboxylation Antitumor activity of the extracts was evaluated using the MTT assay. The highest yields and highest
Antitumor activity cannabinoid contents in the extracts were obtained with high solvent density values. Extracts with high
MTT assay concentrations of neutral cannabinoids showed high antitumor activity for cervical cancer cell lines.
© 2019 Elsevier B.V. All rights reserved.

1. Introduction increasingly promoted scientific and industrial interest inter-

The pharmacological and medicinal potential of substances
called cannabinoids, specific to the genus Cannabis, have
∗ Corresponding author.
E-mail address: ivieitez@fq.edu.uy (I. Vieitez Osorio).
nationally. Among these substances, cannabidiol (CBD) and
9 -tetrahydrocannabinol (9 -THC) are those with the most
significant properties [1]. The highest concentrations of cannabi-
noids in Cannabis plants are cannabidiolic acid (CBDA) and
9 -tetrahydrocannabinoic acid (9 -THCA). The transformation of
these acidic cannabinoids into their respective neutral cannabi-
noids, CBD and 9 -THC, is possible by enacting a decarboxylation

https://doi.org/10.1016/j.supflu.2019.03.012
0896-8446/© 2019 Elsevier B.V. All rights reserved.
 D. Ribeiro Grijó et al. / J. of Supercritical Fluids 149 (2019) 20–25
reaction [2]. This reaction is favoured by several factors such as
storage time [3], heating [4], and the use of alkaline conditions
[5]. Controlled heating is the simplest technique used to pro-
mote decarboxylation, and it avoids the degradation of desirable
cannabinoids [2]. Upon degradation, CBD can be converted to
9 -THC [6] and/or cannabielsoin (CBE) [7], and 9 -THC can be
converted to cannabinol (CBN) and/or 8 -tetrahydrocannabinol
(8 -THC) [8].
The CBD and 9 -THC formed during decarboxylation exhibit
non-polar solute behaviour and have a higher solubility in super-
critical carbon dioxide (scCO2 ) as a solvent than the acidic
cannabinoids [9]. The extraction of bioactive compounds from
Cannabis flowers using scCO2 , both neat and with a modifier, results
in an extract with high concentrations of cannabinoids, and it is free
of organic solvent residues [10]. By changing the extraction temper-
ature and pressure, it is possible to fractionate the extract in order
to obtain high concentrations of different cannabinoids [11,12].
Studies with tumour cell lines are widely used in research. In
vitro assays with tumour cells allow the evaluation of a large num-
ber of compounds in a short period of time, promoting the discovery
of new drugs with anticancer potential. Positive results are extrap-
olated to the study of tumours in vivo. Thus in vitro assays are
transition studies recognized by the pharmaceutical industries [13].
Studies of in vitro antitumor activity using CBD and 9 -THC by MTT
assay have shown promising results for colorectal [14,15], prostate
[16,17], and cervical [18,19] cells.
The purpose of this work is to evaluate the antitumor capacity
of extracts from flowers of the genus Cannabis obtained by scCO2 .
Different techniques were used to optimize the polarity interac-
tion between solute and solvent before extraction. The techniques
include decarboxylation of the samples as well as the use of ethanol
as a co-solvent in the extraction process. The extraction conditions
were selected using solubility data of CBD and 9 -THC in scCO2
[20,21] as well as the operational limitation of the experimental
module. The extracts obtained were purified using the winteri-
zation process [9]. The chemical profile of the cannabinoids CBD,
9 -THC and CBN of the extracts and in fresh flowers was ana-
lysed by high performance liquid chromatography with a diode
array detector (HPLC/DAD) [22]. Antitumor activity of the extracts
obtained was evaluated using the MTT assay [23].
2. Materials and methods
2.1. Plant material
This study was carried out using fresh flowers of non-pollinated
female plants. Initially, they were milled in a manual shredder. The
percent relative humidity of 3 g samples was determined in tripli-
cate using an electrically heated drying oven (Kenton, DHG9070)
at 35 ◦ C with air circulation for approximately 20 h until the weight
of the sample remained constant.
2.2. Chemicals
Table 1 presents some information about the chemicals used in
this work. All chemicals were used without further treatment.
2.3. Calibration curves
The calibration curves for the concentrations of CBD, 9 -THC
and CBN were performed from six dilutions (0.001, 0.003, 0.005,
0.010, 0.050, and 0.100 mg mL−1 ) in methanol using an HPLC Chro-
matograph (Shimadizu, 20A) with a diode array detector at 220 nm
and an RP-8 column (SUPELCOSIL (TM): 250 × 4.5 mm, 5 ␮m) at
35 ◦ C. The mobile phase used was a solution of acetonitrile and
water (8:2 v/v) under isocratic conditions with a flow rate of 1 mL
21
min−1 for 10 min [22]. The calibration curves for analyte quantifi-
cation showed linearities (R2 ) higher than 0.9999.
2.4. Decarboxylation process
The decarboxylation process is well described in the literature
[2–5,9,22]. However, this analysis before the extraction process
can provide important data because the profiles of variations in
cannabinoid contents depended on the qualities or varieties of the
sample [24].
Samples of the flowers were heated in duplicate at tempera-
tures of 110 and 140 ◦ C for a period of six hours using an oven
(Kenton, DHG9070). The cannabinoid content of each sample was
determined at the intervals of 15, 30, 45, 60, 120, 180, 240, 300, and
360 min.
Analyses were conducted by adding 3 mL of a
methanol/chloroform mixture (9:1 v/v) to 150 mg of each milled
and decarboxylated flower sample. The solution was homogenized
with an ultrasonic agitator (Elmasonic, P) for 15 min at 37 Hz and
40 ◦ C. It was then centrifuged (Thermo Scientific Sorvall, ST8R)
for 10 min at 3000 rpm, and the supernatant was collected. For
analysis of the cannabinoid composition, 200 ␮L of the supernatant
was diluted in methanol to a concentration of 1 mg mL−1 [22].
2.5. Supercritical extracts
The equipment consisted of a syringe-type pump (Thar Tech-
nologies), a stainless-steel extractor with a capacity of 25 mL
(8.4 cm in height and 2.0 cm in diameter), and micrometric valves
for depressurizing. The porosity of the bed during the experiments
was 0.85 ± 0.03. The extraction temperature was maintained by a
thermostatic oven. The extraction flow was controlled by a flow
totalizer under normal conditions of temperature and pressure. The
experimental extractions were performed at Facultad de Química,
Universidad de la República (UdelaR) of Uruguay, and the method
has been described in several studies of our research group that are
in the literature [25].
The extraction conditions were defined from the solubility stud-
ies of the cannabinoids studied in scCO2 [20,21]. In order to increase
the cannabinoids of interest (CBD and 9 -THC), two different
extraction methodologies using scCO2 were evaluated. In the first
extraction methodology, the samples were initially submitted to
decarboxylation (heating) followed by extraction with pure scCO2
in a single step [9]. The second methodology used sequential extrac-
tion steps under different conditions (temperature and pressure)
[11] and a change of solute/solvent polarity (previous decarboxy-
lation [9], and 5% ethanol was used as co-solvent [10,11]) to obtain
a fraction with higher cannabinoid content.
2.5.1. Supercritical extraction at single step
Single-step extractions with pure scCO2 were conducted using
a total amount of 5.5 g of Cannabis flowers in each experiment. The
extractions were conducted by varying the pre-heat time of the
samples using an oven at 140 ◦ C for 0.5 and 2.0 h. The pressures
were 22 and 40 MPa and the temperatures were 50 and 70 ◦ C. In
total, five different experimental conditions were evaluated (#1,
#2, #3, #4, and #5). Each extraction was interrupted when the flow
meter totalled 90 L of CO2 at ambient conditions, and CO2 flow was
adjusted to an average value of 0.5 L min−1 .
2.5.2. Supercritical sequential extraction
Two experiments (#6 and #7) were conducted to evaluate the
sequential extraction methodology. For each experiment, 3.0 g of
Cannabis flower samples were used, and the extractor volume was
filled with glass beads. Each experiment was carried out in two
22 D. Ribeiro Grijó et al. / J. of Supercritical Fluids 149 (2019) 20–25

Table 1
Chemicals employed in this work.
a
Chemical IUPAC Nomenclature Molecular Molar mass Supplier (country) Minimum
formula (g mol−1 ) purity (%)

CBD Cannabidiol C21 H30 O2 314.47 99.0

Grace Davison Discovery
9 -THC 9 -tetrahydrocannabinol C21 H30 O2 314.47 91.0
Science (United States)
CBN Cannabinol C21 H26 O2 310.44 97.9
Methanol Methanol CH4 O 32.04 Sigma-Aldrich (United States) 99.9
Acetonitrile Acetonitrile C2 H6 N 41.05 Sigma-Aldrich (United States) 99.5
Water Water H2 O 18.02 Sartorius (Uruguay) Ultra-pure
Chloroform Chloroform CHCl3 119.37 Sigma-Aldrich (United States) 99.9
Carbon Dioxide Carbon Dioxide CO2 44.01 Linde (Uruguay) 99.995
Ethanol Ethanol C2 H6 O 46.07 Sigma-Aldrich (United States) 99.9
Hexane Hexane C6 H14 86.18 Sigma-Aldrich (United States) 99.9
Nitrogen Nitrogen N2 28.01 Linde (Uruguay) 99.995
MTT 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium C18 H16 BrN5 S 414.32 Amresco (United States) 98.0
bromide
DMSO Dimethyl Sulfoxide C2 H6 OS 78.13 Synth (Brasil) 99.9
a
Purities were provided by the manufacturers.

steps (example: #6 = #6.1 + #6.2). The first step for both experi-
ments (#6.1 and #7.1) was conducted with pure scCO2 under mild
supercritical conditions (near the critical point), at 35 ◦ C and 10 MPa
[11], to try to extract non-cannabinoid compounds. The second
step for both experiments (#6.2 and #7.2) was conducted at 70 ◦ C
[20] and 40 MPa to optimize cannabinoid extraction. However, for
the first experiment (#6), the second step (#6.2) was conducted
without the application of techniques for changing solute/solvent
polarity. While for the second experiment (#7), the second step
(#7.2) was conducted after the previous decarboxylation of the
sample [9] for 30 min at 140 ◦ C and the addition of 5% ethanol as
a co-solvent [10,11]. Extractions from each step were terminated
when the total CO2 flow meter indicated 60 L under normal tem-
perature and pressure conditions.
2.5.3. Winterization process
The purification of crude extracts by the winterization process
promotes the removal of waxes. The process consists in resuspend-
ing the extract in n-hexane [9] or ethanol [10] and then cooling.
n-Hexane was the solvent chosen because it exhibits a non-polar
behaviour like the neutral cannabinoids. To each one gram of crude
extract obtained was added 10 mL of n-hexane. Thereafter, each
mixture was refrigerated at −20 ◦ C in a freezer (Cuder) for 20 h.
The supernatant was filtered using a syringe filter. The n-hexane
was removed with a stream of nitrogen at 40 ◦ C.
2.5.4. Determination of the composition of the cannabinoids
fraction
An amount of 20 mL of methanol was added to 30 mg of each
winterized extract. The solution was then subjected to ultrasonic
shaking for 30 min at 37 Hz and 40 ◦ C. An aliquot of the homoge-
neous sample was diluted in methanol in the proportion of 1:10.
This aliquot was subjected to 20 min of ultrasonic agitation at 37 Hz
and 40 ◦ C, and then injected into an HPLC [10].
2.5.5. Antitumor activity
Cells from different tumour strains, including colorectal adeno-
carcinoma (Caco-2), prostate cancer (PC3), cervical cancer (HeLa,
SiHa and C33), and non-tumour cells, such as human keratinocytes
(HaCaT), African green monkey kidney epithelial cells (Vero) and
fibroblasts L929 (L929), were cultured. The cultures were per-
formed (2.5 × 105 cells mL−1 ) using DMEM (Dulbecco’s modified
Eagle’s medium; Gibco Invitrogen, New York, USA) or RPMI 1640
(Roswell Park Memorial Institute 1640; Gibco Invitrogen, New
York, USA) supplemented with 2 mM L-glutamine and 10% FBS
(fetal bovine serum). They were dispensed into a sterile 96-well
plate and incubated for 24 h at 37 ◦ C in a humid atmosphere
oven with 5% CO2 tension. Analyses of anti-tumour activity were
performed at the Laboratory of Technological Innovation in the
Development of Drugs and Cosmetics of the State University of
Maringá.
The antitumor activity of the extracts was evaluated from a col-
orimetric assay (MTT assay). This assay is based on the ability of
viable mitochondria to reduce MTT, (3-[4,5-dimethylthiazol-2-yl]-
2,5 diphenyl tetrazolium bromide), to a substance called purple
formazan [23]. The experiments were conducted by evaluating
the concentration values of the extracts required to inhibit 50%
cell growth (CC50 ). After the cell culture period, the supernatant
was withdrawn and increasing concentrations of the extracts were
added. After 48 h of incubation under the same culture conditions,
the cells were washed with 0.01 M phosphate-buffered saline (PBS),
and 50 ␮L MTT (2 mg mL−1 ) was added. They were then incubated
in the absence of light at 25 ◦ C, and after 4 h 150 ␮L of DMSO
were added to break the cells and to solubilise crystals of purple
formazan. The absorbance was evaluated in a spectrophotometer
(BIO-TEK Power WaveXS) at 570 nm.
Evaluation of the cytotoxicity of the extracts was also performed
by the MTT assay. Analyses were performed using the non-tumour
cells: HaCaT, Vero, and L929. The antitumor activity index of the
extracts, the ratio between the CC50 value of a non-tumour cell and
the CC50 value of a tumour cell, is an adequate way to evaluate
the selectivity (efficiency) of the extracts. High index values are
required and indicate a more selective antitumor activity [26].
3. Results and discussion
3.1. Decarboxylation
The initial contents of the cannabinoids studied were
0.09 ± 0.01% of CBD, 2.4 ± 0.2% of 9 -THC, and 0.06 ± 0.03% of CBN.
Fig. 1 shows the increase in the content of these cannabinoids that
is due to heating the flower samples at different temperatures.
The 9 -THC was the only analysed cannabinoid that presented
a significant variation of content in the flowers submitted to the
decarboxylation process (Fig. 1a and b). The content of 9 -THC
in flowers was maximized at 10.0 ± 0.8% after 30 min at 140 ◦ C
and 90 min at 110 ◦ C. The contents of CBD and 9 -THC had higher
changes at 140 ◦ C.
The initial relative humidity of the flower samples was
3.2 ± 0.4%. The dried flowers submitted to the decarboxylation pro-
cess at 140 ◦ C had a mass loss of 8.1 ± 0.8% after 30 min and of
10.2 ± 0.3% after 120 min. The additional mass loss of dried flowers
is mainly related to the volatilization of the essential oils [27]. The
 D. Ribeiro Grijó et al. / J. of Supercritical Fluids 149 (2019) 20–25 23

Fig. 1. Thermal behaviour of different cannabinoids (× CBD; 䊉 9 -THC; and 䊐 CBN) in flower samples at: (a) 140 ◦ C and (b) 110 ◦ C.

Table 2 Table 3
Analysis of the composition of winterized extracts obtained by decarboxylation Analysis of the composition of winterized oils obtained by fraction extraction
method. method.

Run Conditions Yield (%) Substances Total (%) Run Conditions Yield (%) Substances Total (%)

CBD 3.2 ± 0.4 CBD 0.26 ± 0.03

Pre-heating:
9 -THC 84 ± 1 ◦ 9 -THC 9.2 ± 0.4
140 ◦ C for 10 MPa; 35 C
CBN 2.9 ± 0.3 #6.1 1.3 CBN 0.30 ± 0.03
#1 30 min 8.3
◦ −1 Others 90 ± 1
22 MPa; 70 C Others 10.4 ± 0.5 ␳ = 0.713 g mL
␳ = 0.695 g mL−1
CBD 0.27 ± 0.02
CBD 3.1 ± 0.3 ◦ 9 -THC 21.4 ± 0.5
Pre-heating: 40 MPa; 70 C
9 -THC 75 ± 1 #6.2 5.4 CBN 0.66 ± 0.07
140 ◦ C for
CBN 2.9 ± 0.3
#2 30 min 9.4 −1 Others 77.6 ± 0.7
␳ = 0.857 g mL
40 MPa; 70 ◦ C Others 18.7 ± 0.5
CBD 2.0 ± 0.1
␳ = 0.857 g mL−1 Pre-heating:
9 -THC 61.0 ± 0.5
140 ◦ C for
CBD 0.25 ± 0.01 CBN 2.4 ± 0.2
Pre-heating: #7.1 30 min 3.6
9 -THC 54.4 ± 0.4
140 ◦ C for
CBN 12.1 ± 0.4 10 MPa; 35 ◦ C Others 34.7 ± 0.3
#3 120 min 5.9
␳ = 0.713 g mL−1
22 MPa; 70 ◦ C Others 33.2 ± 0.4
CBD 0.05 ± 0.01
␳ = 0.695 g mL−1
Co-solvent: 5% 9 -THC 0.84 ± 0.06
CBD 2.8 ± 0.2 EtOH CBN 1.5 ± 0.1
Pre-heating: #7.2 5.4
9 -THC 40.4 ± 0.5
140 ◦ C for
CBN 1.00 ± 0.09 40 MPa; 70 ◦ C Others 98 ± 1
#4 120 min 7.1
␳ = 0.857 g mL−1
40 MPa; 50 ◦ C Others 55.8 ± 0.4
␳ = 0.923 g mL−1

CBD 2.7 ± 0.2 performed at 40 MPa, runs #2 and #5, resulted in a higher yield for
Pre-heating:
9 -THC 68.3 ± 0.4
140 ◦ C for the higher temperature and lower density of the solvent. Possibly,
CBN 3.0 ± 0.1
#5 30 min 7.9 this behaviour is due to the higher solubility of 9 -THC in scCO2 at
40 MPa; 50 ◦ C Others 26 ± 0.2 70 ◦ C [20].
␳ = 0.923 g mL−1 The decarboxylation process at 140 ◦ C for 30 min was suffi-
cient to maximize 9-THC and CBD contents in the sample. When
the process was performed during 120 min under this tempera-
amount of mass lost may vary according to the initial composition
ture condition, it caused degradation of the products of interest.
of the sample [9].
Therefore, under these conditions, reduced decarboxylation times
significantly favoured the increase of 9 -THC and of CBD. The 9 -
3.2. Supercritical extraction at single step
THC content had the highest increase. The high content of 9 -THC
in the sample favoured the extractions at 70 ◦ C [20], while the low
Table 2 shows the mass percent yields and the cannabinoid com-
content of CBD was insufficient to have an advantage in the extrac-
position of supercritical extracts obtained with a single step relating
tions at 50 ◦ C [21]. Therefore, the conditions of higher mass percent
to the temperature, pressure and density conditions of the pure
yield, runs #1 and #2, were obtained using a lower decarboxylation
scCO2 used as the solvent. In all evaluated conditions, the decar-
time and a higher extraction temperature. However, the operating
boxylation process, at 140 ◦ C and different times, was applied for
conditions of run #1 are more economically viable due to the lower
the pre-treatment of flower samples.
value of operating pressure.
The extraction conditions with the highest values of pressure
and density resulted in higher percentage yields. The extraction
operating pressure promoted a significant effect on the mass per- 3.3. Supercritical sequential extraction
centage yield in runs #1 and #2 performed at 70 ◦ C. However, run
#2 had a higher concentration of interferents. The effect of the Table 3 shows the operating conditions for sequential extrac-
operating temperature on the mass percentage yield of the extract tion using pure scCO2 , scCO2 with modifier (5% ethanol) as well as
24 D. Ribeiro Grijó et al. / J. of Supercritical Fluids 149 (2019) 20–25

Table 4
Cytotoxic concentration of extracts (mg mL−1 ) to inhibit 50% of cells (CC50 ).

Tumor Cells Normal Cells

#
Caco-2 PC3 Hela SiHa C33 HaCat Vero L929

1 63 ± 4 80 ± 4 27.5 ± 4 31 ± 4 25 ± 1 106 ± 5 218 ± 2 146 ± 5

2 88 ± 4 92 ± 5 40 ± 5 42 ± 3 31 ± 3 128 ± 6 240 ± 4 166 ± 4
3 91 ± 6 122 ± 6 33 ± 4 44 ± 5 24 ± 3 131 ± 4 226 ± 4 188 ± 1
4 117 ± 5 137 ± 4 42 ± 5 55 ± 5 44 ± 5 139 ± 5 233 ± 6 194 ± 6
5 22 ± 1 32 ± 3 2.7 ± 0.1 3.1 ± 0.7 2.8 ± 0.4 80 ± 2 131.9 ± 0.9 121 ± 3
6.2 49.8 ± 0.9 54 ± 1 14 ± 3 15 ± 1 8.3 ± 0.9 94 ± 6 147 ± 5 113 ± 3
7.2 84 ± 4 96.3 ± 0.9 33 ± 3 37 ± 3 32 ± 5 103 ± 4 199 ± 5 131 ± 4

Table 5
Antitumor activity indexes of the extracts.

N1 N2 N3 N1 N2 N3 N1 N2 N3 N1 N2 N3 N1 N2 N3
#
Caco-2 PC3 Hela SiHa C33

1 1.7 3.5 2.3 1.3 2.7 1.8 3.9 7.9 5.3 3.4 7.1 4.7 4.2 8.7 5.8
2 1.5 2.7 1.9 1.4 2.6 1.8 3.2 6.0 4.2 3.0 5.7 3.9 4.1 7.8 5.4
3 1.4 2.5 2.1 1.1 1.9 1.5 4.0 6.9 5.7 3.0 5.2 4.3 5.6 9.6 8.0
4 1.2 2.0 1.7 1.0 1.7 1.4 3.3 5.5 4.6 2.5 4.2 3.5 3.1 5.3 4.4
5 3.7 6.1 5.6 2.5 4.1 3.8 29.8 48.9 44.9 25.9 42.5 39.1 28.7 47.1 43.3
6.2 1.9 2.9 2.3 1.7 2.7 2.1 6.7 10.5 8.1 6.4 10.0 7.7 11.3 17.7 13.6
7.2 1.2 2.4 1.6 1.1 2.1 1.4 3.2 6.1 4.0 2.8 5.4 3.6 3.2 6.1 4.0

with or without the application of the decarboxylation process to
potentiate the neutral cannabinoid content.
Experiment #6, as predicted by solubility data [20], showed that
the use of low temperature and pressure values in a sample of
fresh flowers (#6.1) provides a lower 9 -THC content in the extract
than when using high values (#6.2). However, even with favourable
temperature and pressure conditions, the 9 -THC content in the
extract will not be significantly high if the sample is not previ-
ously decarboxylated. The significant increase in yield of extract
#6.2 may be related to the increase of solvent density and conse-
quently the extraction of substances such as acidic cannabinoids
[10] and essential oils [11,12].
Analysing the first extraction step for each of the experiments,
runs #6.1 and #7.1, it can be seen that the yield of run #7.1 was
higher than that of run #6.1. This behaviour can be explained
because the application of the previous decarboxylation process
on sample #7 increased the 9 -THC content and consequently
the non-polar character, favouring the interaction with the solvent
used (pure CO2 ). For the decarboxylated sample (run #7.1), a high
content of 9 -THC was obtained even in conditions slightly above
the critical point of CO2 . Experiment #7 demonstrated that when
the sample was previously decarboxylated, increasing the polarity
of the solvent (#7.2) is not recommended for the extraction of the
neutral cannabinoids (CBD, 9 -THC and CBN). However, the use of
a co-solvent contributes significantly to the extraction yield.
3.4. Antitumor activity
The cytotoxic concentrations of seven extracts (mg mL−1 )
required for the 50% inhibition (CC50 ) of five tumour cells and three
non-tumour cells are described in Table 4. The compositions of the
respective extracts used are described in Tables 2 and 3.
The bioactive compounds contained in the extract from run #5
demonstrated a greater antitumor potential for all five tumour cell
types, because smaller concentrations were required for 50% inhibi-
tion of cells (CC50 ). The potential antitumor activities of extract #5
relative to cervical tumour cells (Hela, SiHa and C33) were similar
and more significant than for the other types of tumour cells evalu-
ated. The extracts showed, in general, better inhibition for cervical
tumour cells and worse for prostate tumour cells (PC3). Extracts
#2, #3, and #4 had the lowest cytotoxic effects for the three types
of non-tumour cells studied.
Table 5 shows the antitumor activity index, ratio between the
CC50 value of a non-tumour cell and the CC50 value of a tumour cell,
of the extracts analysed.
The most selective antitumor activities were related to cervical
cells (Hela, SiHa and C33). The high anti-tumour activity of CBD
and 9 -THC [14–19], as well as the other neutral cannabinoids like
CBN [28], canabigerol (CBG) and cannabinochron (CBC) [29], may
be related to the higher indexes of antitumor activity presented
by extract #5. All of these neutral cannabinoids can be increased
by controlled heating of a sample. Thus, by analysing the solubility
data of these compounds in CO2 [9], it was observed that CBD and
CBN had the best values at 50 ◦ C. In addition, the solubility of 9 -
THC and CBG may be optimal at this temperature when using high
pressures such as 40 MPa. This means that the other extracts had
worse indices of anti-tumour activity because they underwent an
excessive decarboxylation process and/or were not submitted to
optimal conditions (high solvent density) to obtain these neutral
cannabinoids during the extraction.
In general, it has been shown that neutral cannabinoids have
anti-tumour activity superior to acidic cannabinoids [29]. Stud-
ies have shown that CBD, CBG, BCC, and CBN may have higher
anti-tumour activity than 9 -THC [28–30]. In addition, the solubil-
ities of these four neutral cannabinoids at 50 ◦ C in CO2 are greater
than that of 9 -THC. Therefore, future studies may help to define
more detailed methodologies for obtaining extracts with high anti-
tumour activity.
4. Conclusion
The variety of Cannabis flowers studied presented a high poten-
tial of 9 -THC. Therefore, it was possible to obtain high yields at
the extraction temperature of 70 ◦ C, as predicted by the literature
solubility data [20], as well as by using the optimum conditions
of decarboxylation to maximize the content of this cannabinoid”.
Extraction of the decarboxylated samples using conditions close
to the critical point of CO2 also made it possible to obtain a high
content of 9 -THC. The high density of the solvent obtained under
a pressure of 40 MPa and at 50 ◦ C, allowed the extraction to pro-
vide a high yield and high 9 -THC content. This condition is also
favourable for the extraction of other neutral cannabinoids [9]
which possibly explains the values obtained for antitumor activity
towards cervical cells. Consequently, obtaining extracts of Cannabis
 D. Ribeiro Grijó et al. / J. of Supercritical Fluids 149 (2019) 20–25 25

flowers at low temperatures and free of trace organic solvents has dysfunction and inhibits the apoptotic process induce by Clistridium difficile
proved to be an adequate and promising alternative process for toxin A in Caco-2 cells, United Eur. Gastroenterol. J. 5 (2017) 1108–1115,
http://dx.doi.org/10.1177/2050640617698622.
antitumor studies. [15] A. Greenhough, H.A. Patsos, A.C. Williams, C. Paraskeva, The cannabinoid
9 -tetrahydrocannabinol inhibits RAS-MAPK and PI3K-AKT survival
Acknowledgments signalling and induces BAD-mediated apoptosis in colorectal cancer cells, Int.
J. Cancer 121 (2007) 2172–2180, http://dx.doi.org/10.1002/ijc.22917.
[16] L. De Petrocellis, A. Ligresti, A.S. Moriello, M. Iappelli, R. Verde, C.G. Stott, L.
To Dr. Carlos García Carnelli (UdelaR) for providing the flower Cristino, P. Orlando, V. Di Marzo, Non-THC cannabinoids inhibit prostate
samples and cannabinoids standard and CAPES, CNPq, Fundação carcinoma growth in vitro and in vivo: pro-apoptotic effect and underlying
mechanisms, Br. J. Pharmacol. 168 (2013) 79–102, http://dx.doi.org/10.1111/j.
Araucária, and COMCAP for financial support and scholarships. 1476-5381.2012.02027.x.
[17] L. Ruiz, A. Miguel, I. Díaz-Laviada, 9 -tetrahydrocannabinol induces apoptosis
References in human prostate PC-3 cell via receptor-independent mechanism, FEBS Lett.
458 (1999) 400–404, http://dx.doi.org/10.1016/S0014-5793(99)01073-X.
[18] S.T. Lukhele, L.R. Motadi, Cannabidiol rather than Cannabis sativa extracts
[1] R.G. Pertwee, Pharmacological and therapeutic targets for
inhibit cell growth and induce apoptosis in cervical cancer cells, BMC
9 -tetrahydrocannabinol and cannabidiol, Euphytica 140 (2004) 73–82,
Complement. Altern. Med. 16 (2016) 335–351, http://dx.doi.org/10.1186/
http://dx.doi.org/10.1007/s10681-004-4756-9.
s12906-016-1280-0.
[2] M. Wang, Y.H. Wang, B. Avula, M. Radwan, A.S. Wanas, J. Van-Antwerp, J.F.
[19] W. Peschel, Quality control of traditional Cannabis tinctures: pattern, markers,
Parcher, M.A. Elsohly, I.A. Khan, Decarboxylation study of acidic
and stability, Sci. Pharma 84 (2016) 567–584, http://dx.doi.org/10.3390/
cannabinoids: a novel approach using ultra high performance supercritical
scipharm84030567.
fluid chromatography photodiode array mass spectrometry, Cannabis,
[20] H. Perrotin-Brunel, P.C. Perez, M.J.E. Van-Roosmalen, J. Van-Spronsen, G.J.
Cannabinoid Res. 1 (2016) 262–271, http://dx.doi.org/10.1089/can.2016.0020.
Witkamp, C.J. Peters, Solubility of 9 -tetrahydrocannabinol in supercritical
[3] A.N. Masoud, N.J. Doorenbos, Mississippi grown Cannabis sativa L. III.
carbon dioxide: experiments and modeling, J. Supercrit. Fluids 52 (2010)
Cannabinoid and cannabinoid acid content, J. Pharm. Sci. 62 (1973) 313–315,
6–10, http://dx.doi.org/10.1016/j.supflu.2009.12.001.
http://dx.doi.org/10.1002/jps.2600620229.
[21] H. Perrotin-Brunel, M.C. Kroon, M.J.E. Van-Roosmalen, J. Van-Spronsen, C.J.
[4] M. Kimura, K. Okamoto, Distribution of tetrahydrocannabinolic acid in fresh
Peters, G.J. Witkamp, Solubility of non-psychoactive cannabinoids in
wild Cannabis, Experientia 26 (1970) 819–820, http://dx.doi.org/10.1007/
supercritical carbon dioxide and comparison with psychoactive cannabinoids,
BF02114192.
J. Supercrit. Fluids 55 (2010) 603–608, http://dx.doi.org/10.1016/j.supflu.
[5] L. Grlić, A. Andrec, The content of acid fraction in Cannabis resin of various age
2010.09.011.
and provenance, Experientia 17 (1961) 325–326, http://dx.doi.org/10.1007/
[22] United Nations Office on Drugs and Crime, Recommended Methods for the
BF02158184.
Identification and Analysis of Cannabis and Cannabis Products, UNODC, 2009
[6] R. Adams, D.C. Pease, C.K. Cain, J.H. Clark, Isomerization of cannabidiol to
https://www.unodc.org/documents/scientific/ST-NAR-40-Ebook 1.pdf.
tetrahydrocannabinol, a physiologically active product. Conversion of
[23] T. Mosmann, Rapid colorimetric assay for cellular growth and survival:
cannabidiol to cannabinol, J. Am. Chem. Soc. 62 (1940) 2402–2405, http://dx.
application to proliferation and cytotoxicity tests, J. Immunol. Methods 65
doi.org/10.1021/ja01866a040.
(1983) 55–63, http://dx.doi.org/10.1016/0022-1759(83)90303-4.
[7] A. Shani, R. Mechoulam, Cannabielsoic acids. Isolation and synthesis by a
[24] D.R. Grijó, I. Vieitez, L. Cardozo-Filho, Supercritical extraction strategies using
novel oxidative cyclization, Tetraedron 30 (1974) 2437–2446, http://dx.doi.
CO2 and ethanol to obtain cannabinoid compounds from Cannabis hybrid
org/10.1016/S0040-4020(01)97114-5.
flowers, J. CO2 Util. 28 (2018) 174–180, http://dx.doi.org/10.1016/j.jcou.2018.
[8] E.R. Garret, J. Tsau, Stability of tetrahydrocannabinols I, J. Pharm. Sci. 63
12.015.
(1974) 1563–1574, http://dx.doi.org/10.1002/jps.2600631016.
[25] I. Vieitez, L. Maceiras, I. Jachmanián, S. Alborés, Antioxidant and antibacterial
[9] H. Perrotin-Brunel, Sustainable Production of Cannabinoids with Supercritical
activity of different extracts from herbs obtained by maceration or
Carbon Dioxide Technologies, Doctoral Thesis, Université de Technologie de
supercritical technology, J. Supercrit. Fluids 133 (2018) 58–64, http://dx.doi.
Compiègne, 2011 https://repository.tudelft.nl/islandora/object/
org/10.1016/j.supflu.2017.09.025.
uuid:c1b4471f-ea42-47cb-a230-5555d268fb4c/datastream/OBJ/download.
[26] M. Wardihan, G. Rusdi, M.A. Alam, Manggau, selective cytotoxicity evaluation
[10] L.J. Rovetto, N.V. Aieta, Supercritical carbon dioxide extraction of
in anticancer drug screening of Boehmeria virgate (forst) guill leaves to several
cannabinoids from Cannabis sativa L, J. Supercrit. Fluids 129 (2017) 16–27,
human cell lines: HeLa, WIDr, T47D and Vero, J. Pharm. Sci. 12 (2013)
http://dx.doi.org/10.1016/j.supflu.2017.03.014.
123–126, http://dx.doi.org/10.3329/dujps.v12i2.17615.
[11] J. Omar, M. Olivares, M. Alzaga, N. Etxebarria, Optimisation and
[27] L. Romano, A. Hazekamp, Cannabis oil: chemical evaluation of an upcoming
characterisation of marihuana extracts obtained by supercritical fluid
Cannabis - based medicine, Cannabinoids 1 (2013) 1–11 https://www.
extraction and focused ultrasound extraction and retention time locking
researchgate.net/publication/297707359 Cannabis oil Chemical evaluation
GC-MS, J. Sep. Sci. 36 (2013) 1397–1404, http://dx.doi.org/10.1002/jssc.
of an upcoming cannabis- based medicine/download.
201201103.
[28] A.E. Munson, L.S. Harris, M.A. Friedman, W.L. Dewey, R.A. Carchman,
[12] C. Da-Porto, D. Decorti, A. Natolino, Separation of aroma compounds from
Antineoplastic activity of cannabinoids, J. Natl. Cancer Inst. 55 (1975)
industrial hemp inflorescences (Cannabis sativa L.) by supercritical CO2
597–602, http://dx.doi.org/10.1093/jnci/55.3.597.
extractionand on-line fractionation, Ind. Crop Prod. 58 (2014) 99–103, http://
[29] A. Ligresti, A.S. Moriello, K. Starowicz, I. Matias, S. Pisanti, L. De-Petrocellis, C.
dx.doi.org/10.1016/j.indcrop.2014.03.042.
Laezza, G. Portella, M. Bifulco, V. Di-Marzo, Antitumor activity of plant
[13] R. Arantes-Rodrigues, A. Colaço, R. Pinto-Leite, P.A. Oliveira, In vitro and in vivo
cannabinoids with emphasis on the effect of cannabidiol on human breast
experimental models as tools to investigate the efficacy of antineoplastic
carcinoma, J. Pharmacol. Exp. Ther. 318 (2006) 1375–1387, http://dx.doi.org/
drugs on urinary bladder cancer, Anticancer Res. 33 (2013) 1273–1296
10.1124/jpet.106.105247.
https://pdfs.semanticscholar.org/edb0/
[30] J.F. Lesgards, N. Baldovini, N. Vidal, S. Pietri, Anticancer activities of essential
448a66682cecec85d04920fcf2180742b866.pdf.
oils constituents and synergy with conventional therapies: a review,
[14] S. Gigli, L. Seguella, M. Pesce, E. Bruzzese, A. D’alessandro, R. Cuomo, L.
Phytother. Res. 28 (2014) 1423–1446, http://dx.doi.org/10.1002/ptr.5165.
Steardo, G. Sarnelli, G. Esposito, Cannabidiol restores intestinal barrier