Research Article

Received: 14 December 2015 Revised: 22 March 2016 Accepted article published: 23 April 2016 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jsfa.7774

Seed oil extraction from red prickly pear using

hexane and supercritical CO2: assessment of
phenolic compound composition, antioxidant
and antibacterial activities
Mohamed Koubaa,a* Houcine Mhemdi,a Francisco J Barba,b Armel
Angelotti,a Fatma Bouaziz,c Semia Ellouz Chaabounic and Eugène Vorobieva

Abstract
BACKGROUND: Investigating Opuntia species for their seed oil content is of much importance owing to their potential use
for food and in cosmetic applications. These oils have an important content in unsaturated fatty acids as well as antioxidant
compounds (e.g. polyphenols, vitamin E), which have been associated with the prevention of some chronic diseases. Moreover,
Opuntia stricta oils possess important antimicrobial activities. For instance, the main focus of this study was to compare the
effectiveness of conventional (hexane extraction) and novel (supercritical (SC)-CO2 ) extraction methods for the recovery of oil
and phenolic compounds from O. stricta seeds. The oil yield of both extracts was then compared and the polyphenol content
and composition of both extracts were determined by liquid chromatography–high-resolution mass spectrometry. Additionally,
antioxidant (DPPH assay) and antimicrobial activities (disc diffusion method) of O. stricta seed oils were determined.

RESULTS: The oil yield (based on Soxhlet’s method) of O. stricta seeds was determined using SC-CO2 (49.9 ± 2.2%), and hexane
(49.0 ± 1.5%). Although obtaining similar oil extraction yields using the two methods, the extracted oil using SC-CO2 was more
enriched in polyphenols (172.2 ± 11.9 𝛍g gallic acid equivalents (GAE) g−1 oil) than that extracted using hexane (76.0 ± 6.9 𝛍g
GAE g−1 of oil). Polyphenol profiles showed that the SC-CO2 process led to the yield of more compounds (45) than that using
hexane extraction (11). Moreover, the antioxidant and antimicrobial activities of SC-CO2 extract showed a high percentage of
inhibition.

CONCLUSION: SC-CO2 extraction of O. stricta seed oil led to extraction of oil with a similar yield to that with hexane extraction, but
with higher polyphenol content. The extract containing polyphenols exhibited high antioxidant and antibacterial properties,
demonstrating their great potential as feedstock for high-oil quality.
© 2016 Society of Chemical Industry

Keywords: red prickly pear; seed oil; supercritical CO2 ; hexane extraction; polyphenols

INTRODUCTION
Opuntia cactus plants can be fully exploited in an integrated
manner since their bioactive components can be extracted from
the different parts of its anatomy: flowers, fruits, cladodes, roots
and seeds.1 Opuntia seed extracts show several phytochemical
properties.2,3 In fact, there has been an increased interest in the
antioxidant activity and health-improving capacity of cactus seed
polyphenols.4 – 8 The seeds contain 70–150 g kg−1 (w/w) of oil that
is rich in vitamins, minerals,9 as well as in active polyphenols
known for their antioxidant and antibacterial properties.10,11 This
oil is mainly destined for cosmetic products as organic oil for
anti-ageing and anti-wrinkle purposes. Moreover, the fatty acid
(FA) composition, rich in unsaturated FAs, indicates the potential
of Opuntia seed oils as feedstock for the food, pharmaceutical and
cosmetic markets.12,13
It has been shown that, due to the hard coating of Opuntia seeds,
their pressing allows only the partial extraction of seed oil content.
In fact, it takes approximately 1 ton of cactus fruit to obtain 1 L
of oil, which explains the high price of this oil (∼$1500 L−1 ).1 The
use of organic solvents such as hexane may lead to increased oil
extraction yields from these seeds. However, their use is associated
with health and environmental issues. Therefore, there is a need
∗ Correspondence to: M Koubaa, Laboratoire Transformations Intégrées de la
Matière Renouvelable (UTC/ESCOM, EA 4297 TIMR), Département Génie des
Procédés Industriels, Centre de Recherche de Royallieu, Université de Technolo-
gie de Compiègne, Sorbonne Universités, BP 20529, 60205 Compiègne Cedex,
France. E-mail: koubaa.mohamed@gmail.com
a Laboratoire Transformations Intégrées de la Matière Renouvelable, Départe-
ment Génie des Procédés Industriels, Centre de Recherche de Royallieu, Univer-
sité de Technologie de Compiègne, 60205 Compiègne, France
b Faculty of Pharmacy, Nutrition and Food Science Area, Universitat de València,
s/n 46100 Burjassot, València, Spain
c Enzyme Bioconversion Unit, National School of Engineering, 3038, Sfax Univer-
sity, Tunisia

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Figure 1. Experimental setup for supercritical CO2 extraction of O. stricta seed oil. Ex, extractor; PM, plant material; C, condenser; E, heat exchanger; S1 and
S2 , separators; P, pressure sensor; T, temperature probe; GC-FID, gas chromatography–flame ionization detector; UPLC-HRMS, ultra-performance liquid
chromatography–high-resolution mass spectrometry; SC-CO2 , supercritical CO2 .
to establish environmentally friendly technologies for extraction
in full correspondence with the green extraction concept, which
can reduce solvent consumption, extraction time and toxicity.14,15
In this regard, supercritical (SC)-CO2 constitutes a potential
tool in traditional solvent and mechanical pressing extraction
methods.16 The advantages of using SC-CO2 for such applications
are its low toxicity, non-inflammability, and relatively low critical
points of pressure and temperature.17 Although several studies
have been published over the last years evaluating the potential
of SC-CO2 to extract valuable compounds from different Opuntia
species in the presence of organic solvent,5,18 none of them have
been focused on Opuntia stricta (red prickly pear) seed oils, and
there is a lack of information in the literature concerning the com-
position of these oils in phenolic compounds.
For this purpose, this work was devoted to evaluating and
comparing the efficiency of using conventional (hexane) and
emerging (SC-CO2 ) extraction techniques to recover O. stricta
seed oils rich in phenolic compounds. Gas chromatography and
ultra-performance liquid chromatography–high-resolution mass
spectrometry (UPLC-HRMS) were used for the identification and
quantification of the FA composition and polyphenol profile,
respectively. Additionally, the antioxidant and the antibacterial
activities of the extracts were evaluated.
MATERIALS AND METHODS
Chemicals
Hexane, methanol, toluene and sodium bisulfite were obtained
from Fisher Scientific (Illkirch, France). Methanolic HCl (3 mol L−1 )
and DPPH (2,2-diphenyl-1-picrylhydrazyl) were purchased from
Sigma-Aldrich (Saint-Quentin Fallavier, France). Sodium chloride
(NaCl) was purchased from Loba Chemie (India). Tryptone was
obtained from Suvchem (India), agar agar from Chemi-Pharma
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M Koubaa et al.
(Tunisia), yeast extract from Biokar Diagnostics (France) and
ciprofloxacin discs from Fisher Scientific (France).
Plant materials
Ripe spineless red prickly pear (Opuntia stricta, Haworth variety
(Haw.)) fruits were harvested in the suburb of Sfax city (Tunisia) in
February 2014. The seeds, representing ∼100 g kg−1 of the fruit’s
fresh weight, were separated manually from the pulp and peel,
washed and dried for 5 days at 40 ∘ C. The seed moisture content,
determined using an infrared desiccator (Scaltec, Germany) was
50 ± 2 g kg−1 . Dried seeds were then ground in liquid nitrogen,
giving an average particle diameter of 360 μm, determined using
a granulometer (Mastersizer 2000, Malvern Instruments GmbH,
Germany).
Seed oil content
Seed oil content was determined by Soxhlet’s method.19 Briefly, 5 g
of ground seeds were washed with a reflux of hexane for 24 h at
100 ∘ C. The oil content (in g.kg−1 fresh weight) was determined
as the difference of the round flask masses, before and after
extraction, divided by the total amount of plant material.
Seed oil extraction processes
Supercritical fluid extraction
Oil was extracted from ground prickly pear seeds with pilot-scale
equipment (Separex, France), using SC-CO2 (Fig. 1), as previously
described,20 with slight modifications. Twenty grams of ground
seeds (PM) were introduced into the extraction chamber (Ex) and
pre-heated at 40 ∘ C. Owing to the rigid structure of the plant
material, 10 min conditioning time with SC-CO2 at 35 MPa was
required, thus allowing the diffusion of CO2 inside the cells. After
conditioning, the backpressure and recycling valves were opened,
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Opuntia stricta seed oil: extraction and phenolic compounds analysis
allowing oil recovery in the separators and recycling of CO2 .
This separation was performed by decreasing the CO2 pressure
below the critical point (6 MPa) to allow return to the gas phase,
the release of oil in the separators (maintained at 40 ∘ C) and
the recycling of CO2 in the system. The recycled CO2 was then
compressed by the CO2 pump before injection into the extractor
in its supercritical form. The extraction time was 120 min under
a continuous flux of CO2 (8.5 kg h−1 ). The extracted oil was then
stored at −20 ∘ C until analysis.
Hexane extraction
Hexane extraction was performed as previously described,21 with
slight modifications. In brief, 10 g of ground seeds were mixed with
100 mL hexane and stirred at 250 g for 120 min at 40 ∘ C (Fig. 1).
The mixture was then centrifuged for 3 min at 4000 × g and the
supernatant was evaporated using a rotary evaporator system
under vacuum at 60 ∘ C. The extracted oil was quantified and stored
at −20 ∘ C until analysis.
Oil extraction yield
For all experiments, the oil yield (OY) was calculated as follows:
Extracted oil mass (g)
Oil yield (OY) , % = × 100 (1)
Total oil mass in the sample (g)
Fatty acid composition analysis
FA composition of the different extracted oils was determined
using gas chromatography–flame ionization detection (GC-FID),
as previously described,22 with slight modifications. First, fatty acid
methyl esters (FAMEs) were prepared as follows: 5 mg extracted
oil was solubilized in 150 μL toluene, then 0.5 mL methanolic
HCl (3 mol L−1 ) was added. The mixture was flushed for 5 s with
nitrogen, vortexed for 5 s, then incubated for 2 h at 80 ∘ C. Reaction
tubes were vortexed every 30 min to maximize the reaction yield.
The transesterification was stopped by adding 250 μL sodium
bisulfite (50 g L−1 in water) and FAMEs were extracted by adding
2 mL hexane. The mixture was vortexed rigorously for 1 min, then
centrifuged for 2 min at 10 000 × g, allowing separation of the
aqueous and organic phases. 100 μL was taken from the upper
phase containing FAMEs and diluted 10 times in hexane. The
composition was analysed using a Shimadzu GC-FID (2010-Plus)
instrument equipped with a BPX-70 (Supelco) capillary column
(30 m length, 0.25 mm diameter and 0.25 μm film thickness). The
GC oven temperature was first set at 140 ∘ C, then increased to
230 ∘ C at 19 ∘ C min−1 and held for 5 min. The helium carrier gas
flow was set to 1 mL min−1 and the injection volume was 1 μL with
split 10. FAME standards were injected in order to identify the
different eluted molecules.
Extraction, quantification and identification of seed oil
polyphenols
Total phenolic compounds
Phenolic compounds were extracted from the different oils as pre-
viously described.23 The phase containing the extracted phenolics
was then quantified using Folin–Ciocalteu (FC) reagent, as pre-
viously described,24 with slight modifications. In brief, 40 μL phe-
nolic compounds extract was mixed with 160 μL water and 1 mL
FC reagent, diluted 10 times in water. After 5 min incubation at
room temperature, 800 μL Na2 CO3 (75 g L−1 ) was added, and the
mixture was vortexed and incubated for 10 min at 50 ∘ C. Phe-
nolic compound concentration was determined after measuring
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the absorbance at 750 nm, using a spectrophotometer (Libra S32,
Biochrom, France). The results were expressed in micrograms of
gallic acid equivalent (GAE) per gram of oil using a pre-established
standard curve.
UPLC-HRMS identification of polyphenols
The composition of the extracted phenolic compounds was deter-
mined using UPLC-HRMS as described previously,23 with slight
modifications. Analysis was performed using a UPLC 1290 Infinity
(Agilent Technologies) equipped with a Thermo Hypersyl Gold col-
umn (4.6 mm × 150 mm × 5 μm). After injection (20 μL), the com-
pounds were separated using a flow rate of 0.7 mL min−1 . The
mobile phase used in this work was prepared using 1 g L−1 formic
acid in water (solvent A) and acetonitrile (solvent B). The gradi-
ent was defined as follows as a percentage of solvent B: from 0
to 20 min, 6%; from 20 to 35 min, 18%; from 35 to 45 min, 28%;
from 45 to 48 min, 60 %; from 48 to 55 min, 90%. After separa-
tion, the column was cleaned using 90% of solvent B for 5 min.
Molecule identification was performed using a mass spectrome-
ter (Q-TOF UHD 6538, Agilent Technologies), acquiring the mass
spectra in positive ion mode, using dual electrosparay ionization.
The nebulization gas was adjusted to 30 psi, and the ion spray volt-
age was set to 3.5 kV. The temperature of the ion source was fixed
at 300 ∘ C. All data were acquired and processed using MassHunter
B.05.01 software. The identification of phenolic compounds was
performed based on a home-generated database containing 500
compounds and 30 injected standard molecules, as previously
described.23
DPPH free radical scavenging activities
DPPH free radical assay was used to evaluate the antioxidant
capacity of phenolic compounds extracted from O. stricta seed
oil. The method was adapted from previously published work,25
with modifications. Different phenolic compound concentrations
(from 1 to 35 μg GAE mL−1 ) were prepared in methanol. 500 μL
from each solution was mixed with 375 μL methanol and 125 μL
DPPH (0.2 g.L−1 in methanol). The mixtures were vortexed for 5 s,
then incubated for 1 h in the dark at room temperature. A control
solution, without phenolic compounds, was prepared under the
same conditions. The absorbance was measured at 517 nm, using
a Biochrom Libra S32 spectrophotometer, and the DPPH reducing
capacity of the samples was evaluated compared to butylated
hydroxyanisole (BHA) and ascorbic acid reference curves. The
reducing capacity was calculated as follows:
Acontrol − Asample
DPPH scavenging activity, % = × 100 (2)
Acontrol
where Asample and Acontrol represent the absorbance of the sample
and the control solution, respectively.
Antibacterial activities of seed oil phenolic compounds
The ability of phenolic compounds, extracted from seed oils, to
inhibit bacterial growth was evaluated against seven pathogenic
strains (American Type Culture Collection (ATCC), USA). Both
Gram-positive (Bacillus subtilis (ATCC 26633), Enterococcus faecalis
(ATCC 29212), Bacillus thuringiensis (ATCC 10792) and Klebsiella
pneumoniae (ATCC 13883)) and Gram-negative (Escherichia coli
(ATCC 25922), Salmonella typhimurium (ATCC 19430) and Enter-
obacter sp.) bacteria were tested. All activities were determined
using the disc diffusion’s method.26,27 A culture suspension (100 μL
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Figure 2. Fatty acid composition of O. stricta seed oil obtained using extractions with SC-CO2 and hexane. Analyses were performed using a GC-FID
instrument after transesterification of oils. FAMEs, fatty acid methyl esters.
of 106 UFC (Unit Forming Colony) mL−1 ) was spread over the sur-
face of agar plates prepared with solid Luria broth (LB) medium
(tryptone, 10 g L−1 ; NaCl, 10 g L−1 ; yeast extract, 5 g L−1 ; and agar
agar, 18 g L−1 ). Two sterile filter papers (6 mm diameter) were
aseptically placed on the inoculated agar plates, then imbibed
with 10 μg and 20 μg seed oil phenolic compounds, respectively.
A ciprofloxacin disc (5 μg per disc) was placed in each plate and
was used as a positive control. The plates were incubated at 37 ∘ C
for 24 h to allow bacterial growth. Antimicrobial activities were
measured as the diameter of the clear zone of growth inhibition
using ImageJ software (Wayne Rasband).
Statistical analysis
All experiments were performed in triplicate, except for antibacte-
rial activities, which were performed in duplicate. The values are
expressed as the average of the replicates ± standard deviations.
The error bars presented on the figures correspond to the standard
deviation. Significant differences between the results were calcu-
lated by multiple sample comparison of the means (ANOVA) using
the software SPSS Version 22 (IBM® SPSS® Statistics, USA) and the
LSD test, with a significance level of P < 0.05.
RESULTS AND DISCUSSION
Seed oil extraction
The oil content (dry basis) of O. stricta seeds was determined
using Soxhlet (235 ± 22 g kg−1 ), showing a higher value than that
reported by Ennouri and co-workers (110.5 g kg−1 ).28 The oil con-
tent found by Soxhlet’s method was taken as a reference to
determine the oil extraction yields using SC-CO2 (49.9% ± 2.2%)
and hexane (49% ± 1.5%). Although obtaining the same oil yield,
SC-CO2 extraction presents many advantages compared to con-
ventional hexane extraction. In fact, with hexane, an oil–hexane
mixture is obtained. Hexane should then be evaporated, thus con-
suming a lot of energy, and the risk of degradation of oil quality by
thermal hydrolysis and/or oxidation.29 On the other hand, SC-CO2
extraction allows us to obtain pure oil without residual organic sol-
vent traces, which is of great importance for the cosmetics market.
In addition, concerns about solvent residues in the oleoresin prod-
ucts, new regulations regarding volatile organic solvent emissions
in the air, and the extent of further refining that is required after
the extraction step, restrain the use of organic solvents such as
hexane.30
FA composition of O. stricta seed oils obtained using conven-
tional hexane and SC-CO2 extraction processes (Fig. 2), shows that
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M Koubaa et al.
the major FA is C18:2 (linoleic acid) (∼70% of total FAs), and unsatu-
rated FAs represent ∼85% of the total FAs. This FA profile is similar
to that obtained by Ennouri and co-workers.28 Some differences
have been recorded between FAMEs of oils extracted using SC-CO2
and hexane. In fact, higher content of C18:1 and C18:3, and lower
content of C18:2 were observed in oil obtained by SC-CO2 com-
pared to that obtained by hexane. Similar differences were already
observed when comparing the FA profiles of hazelnuts obtained
using supercritical fluid and organic solvent extractions.31
Quantification of seed oil polyphenols and identification
using UPLC-HRMS
Phenolic compound content was determined for the extracted
oils from O. stricta seeds, using SC-CO2 and hexane. One-way
ANOVA analysis showed that polyphenol concentration is greatly
influenced by the extraction method. Therefore, the results
obtained show that the amount of phenolic compounds extracted
using SC-CO2 (172.2 ± 11.9 μg GAE g−1 oil) was higher than that
obtained by hexane extraction (76.1 ± 6.9 μg GAE g−1 oil). These
results concur with those previously reported for grape seed
oil.32 Increasing the oil’s total phenolic compound (TPC) content
leads to enhancement of the resistance to oxidation, proving that
the SC-CO2 process provides the highest oil quality. Increased
amounts of polyphenols obtained by the SC-CO2 process is a
result of the improved drainage of the viscosity-lowered oil. In
fact, the oil deposited on the solid material contains most of the
polyphenols.20 This behaviour concurs with that reported for the
comparison between the quality of canola oil obtained using
conventional and supercritical fluid extractions.33
The composition of the extracted phenolic compounds was
determined using UPLC-HRMS, and the identification was per-
formed according to a home-generated database containing 500
compounds as well as to 30 injected standards. Results (Table 1)
show that the oil extracted using SC-CO2 was more enriched in
individual phenolic compounds, with 45 compounds, than that
obtained using hexane, with 11 compounds. The molecules iden-
tified according to the injected standards were vanillin, vanillic
acid, gallic acid, ferulic acid, syringic acid, sinapic acid, caffeic acid,
butein, cyanidin, catechin, enterodiol, pinoresinol and apigenin.
These results were concordant with those previously reported.23
Antioxidant and antibacterial activities of extracted phenolic
compounds
Antioxidant activities of the extracts obtained from oils (SC-CO2
and hexane) were assessed through reduction of the DPPH free
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Table 1. Phenolic compound content in Opuntia stricta seed oil obtained using SC-CO2 and hexane, and identified UPLC-HRMS

Phenolic compound name Formula Molecular mass (g mol−1 ) SC-CO2 Hexane

Catechol C6 H6 O2 110.0376 x x
Cinnamic acid C9 H8 O2 148.0533 x x
3-Phenylpropionic acid C9 H10 O2 150.0684 x x
Vanillin C8 H8 O3 152.0486 x ND
m-Coumaric acid C9 H8 O3 164.0482 x ND
Vanillic acid C8 H8 O4 168.0406 x ND
Gallic acid C7 H6 O5 170.0214 x ND
Juglone C10 H6 O3 174.0327 x ND
Caffeic acid C9 H8 O4 180.0437 x ND
Syringaldehyde C9 H10 O4 182.0589 x ND
3-O-Methylgallic acid C8 H8 O5 184.0359 x ND
Psoralen C11 H6 O3 186.03 x x
Ferulic acid C10 H10 O4 194.0596 x ND
Hydroxycaffeic acid C9 H8 O5 196.0385 x ND
Syringic acid C9 H10 O5 198.0518 x x
Sinapaldehyde C11 H12 O4 208.0754 x x
Sinapic acid C11 H12 O5 224.0692 x ND
Piceatannol C14 H12 O4 244.0749 x ND
Isopimpinellin C13 H10 O5 246.052 x ND
Apigenin C15 H10 O5 270.0534 x ND
Butein C15 H12 O5 272.0705 x ND
Phloretin C15 H14 O5 274.0857 x ND
p-Coumaroyl malic acid C13 H12 O7 280.0573 x ND
Cyanidin C15 H10 O6 286.0464 x ND
Catechin C15 H14 O6 290.0807 x ND
Enterodiol C18 H22 O4 302.1515 x ND
Taxifoline C15 H12 O7 304.0553 x ND
3′ -O-Methylcatechin C16 H16 O6 304.0946 x x
(+)-Gallocatechin C15 H14 O7 306.0719 x x
Bisdemethoxycurcumin C19 H16 O4 308.1079 x x
Sinapine C16 H23 NO5 309.1592 x ND
Pinoresinol C20 H22 O6 358.1437 x ND
3,4′ ,5-Trihydroxysylbene-3-b-D-glucopyranoside C20 H22 O8 390.1279 x ND
Astringin C20 H22 O9 406.1235 x ND
Isorhapontin C21 H24 O9 420.1434 x ND
4′ -O-Methyl-(−)-epicatechin 3′ -O-glucuronide C22 H24 O12 480.1255 x x
(−)-Epigallocatechin 3-O-glucuronide C21 H22 O13 482.1069 x ND
Viscutin 1 C27 H26 O11 526.1475 x x
Cyanidin 3-O-(6′′ -malonyl-glucoside) C24 H23 O14 535.1103 x ND
Procyanidin B2 C30 H26 O12 578.1381 x ND
Naringin C27 H32 O14 580.1735 x ND
Cyanidin 3-O-rutinoside C27 H31 O15 595.1649 x ND
Cyanidin 3-O-(3′′ ,6′′ -O-dimalonyl-glucoside) C27 H25 O17 621.1042 x ND
Pelargonidin 3 C27 H31 ClO15 630.132 x ND
Cyanidin 3-O-diglucoside-5-O-glucoside C33 H41 O21 773.2169 x ND

x, detected; ND, not detected.

radicals. The results obtained (Fig. 3a) showed that the DPPH inhi-
bition increased proportionally to the concentration of extract.
Similar curves were obtained using the two extracts, reaching
a maximum of inhibition with 25 μg GAE mL−1 . Total scaveng-
ing activities were higher than that obtained for O. joconostle
methanolic seed extract (49.76% ± 0.49%)34 and similar to O. ficus
indica acetonic seed extract (95%).35 The half inhibition concen-
tration (IC50 ) values, which are the concentrations required to
reduce the DPPH free radicals by 50%, were also determined. In
general, the lowest IC50 corresponds to the highest DPPH scaveng-
ing activity. The obtained IC50 values were 3, 3.5, 9 and 10 μg mL−1 ,
respectively, for ascorbic acid, BHA, extract from seed oil extracted
with hexane and extract from seed oil extracted with SC-CO2 .
These values were lower than that found for the extracts obtained
from O. ficus indica and O. stricta peels and pulps.36 Opuntia stricta
seed oil represents, therefore, a strong electron donor and could
react with free radicals to convert them to more stable products
and terminate the radical chain reaction.

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 www.soci.org M Koubaa et al.

Figure 3. (a) Antioxidant activities of the extracted phenolic compounds from O. stricta seed oils, obtained using SC-CO2 and hexane extractions. Butylated
hydroxyanisole (BHA) and ascorbic acid were used as reference antioxidant molecules. (b) Antibacterial activities of phenolics extracted from oil obtained
by SC-CO2 . CIP, ciprofloxacin. White arrows indicate the control discs.

On the other hand, in vitro antibacterial activities of phenolic
compounds extracted from O. stricta seed oil, using SC-CO2 extrac-
tion, were tested against seven pathogenic strains (Fig. 3b). Growth
inhibition was monitored by measuring the diameter (in mm) of
the clear zone around each disc. The results obtained (Fig. 3b and
Table 2) show that O. stricta seed oils are efficient against the tested
pathogenic strains. Ciprofloxacin, the antibiotic used as positive
control, was partially active against B. thuringiensis; however, clear
growth inhibition zone was observed when using O. stricta seed
oil phenolics. The diameters of the inhibition zones (Table 2) were
between 9.8 ± 1.0 and 12.9 ± 1.5 mm when using 20 μg pheno-
lics. These results concur with a previous work demonstrating the
antibacterial effect of O. stricta extracts,37 and other works per-
formed with O. ficus indica extracts showing potent antibacterial
activities.38 – 40
CONCLUSIONS
From the results obtained in this work, it can be concluded that
SC-CO2 allows us to obtain enriched oil in phenolic compounds

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Opuntia stricta seed oil: extraction and phenolic compounds analysis
Table 2. Diameters of the bacterial growth inhibition zone using the
extract obtained from O. stricta seed oils, using SC-CO2 extraction.
Gram-positive and Gram-negative bacteria were tested. Ciprofloxacin
was used as positive control
5 μg 20 μg
ciprofloxacin phenolics
Bacterial strain (mm) (mm)
Gram-positive Bacillus subtilis 21.5 ± 1.1 11.8 ± 1.2
Enterococcus faecalis 19.8 ± 0.9 12.1 ± 1.3
Bacillus thuringiensis 35.0 ± 1.0 12.4 ± 0.8
Klebsiella pneumoniae 36.5 ± 0.5 9.8 ± 1.0
Gram-negative Escherichia coli 50.1 ± 1.4 12.9 ± 1.5
Salmonella typhimurium 20.6 ± 0.7 11.0 ± 0.8
Enterobacter sp. 21.6 ± 1.0 12.8 ± 0.9
compared to that obtained using hexane, which demonstrates the
efficiency of SC-CO2 extraction as a green process. The phytochem-
ical properties of the seed oil extracts were demonstrated through
DPPH free radical scavenging assays and antibacterial activities,
showing great potential of O. stricta seed oil as a promising source
of bioactive compounds. Although these interesting results were
obtained, further investigations using other emerging technolo-
gies such as gas-assisted mechanical expression could be per-
formed in order to enhance the extraction yield and to obtain oil
with higher TPC content than that obtained using SC-CO2 .
ACKNOWLEDGEMENT
The authors thank Mr Franck Merlier, the head of the analytical
platform in the Enzyme and Cell Engineering Laboratory, Com-
piègne University of Technology (France), for his constructive feed-
back in analyzing the generated data of phenolic compounds.
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