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Contents lists available at ScienceDirect

The Journal of Supercritical Fluids

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

Antioxidant capacity of Colombian seaweeds: 1. Extracts obtained

from Gracilaria mammillaris by means of supercritical uid extraction
Mnica Ospina, Henry I. Castro-Vargas, Fabin Parada-Alfonso
Laboratorio de uidos presurizados, Departamento de Qumica, Universidad Nacional de Colombia, Bogot D.C., Colombia

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

Article history: Red seaweed Gracilaria mammillaris from the Colombian Caribbean coast were investigated as source
Received 2 December 2016 of extracts with antioxidant activity (AA), by means of supercritical CO2 extraction with ethanol as co-
Received in revised form 24 February 2017 solvent. A central composite design was used to investigate the effects of pressure (10, 20 and 30 MPa),
Accepted 25 February 2017
temperature (40, 50 and 60 C), and co-solvent concentration (2, 5 and 8%) on the extraction yield, AA, and
Available online xxx
total content of phenols and carotenes. The obtained extracts were compared with those produced using
Soxhlet extraction at reduced pressure. The AA of samples was evaluated by determining their capacity
Keywords:
for protecting an edible oil against oxidation, upon accelerated oxidation trials. The extracts obtained
Gracilaria mammillaris
Seaweed
at 30 MPa, 60 C and 8% co-solvent showed the highest AA, inhibiting in 42.1% the formation of TBARS
Supercritical uid extraction after six days of accelerated oxidation. These results indicate that red seaweed G. mammillaris could be a
Antioxidant activity promissory source of extracts with AA.
Oxidation of oils 2017 Published by Elsevier B.V.
Response surface analysis

1. Introduction Di-tert-butyl-4-hydroxytoluene), TBHQ (tert-Butylhydroquinone),

Marine macro algae, or seaweeds, exhibit high stability against
oxidation, despite being exposed to the marine stress conditions,
such as light, rapid uctuations in temperatures, osmotic stress
and desiccation [1]. These marine organisms synthesize and accu-
mulate compounds to protect themselves against the high levels
of ultraviolet energy radiation, including mycosporine-type amino
acids, carotenoids ( and -carotene) and xanthophylls (astaxan-
thin, fucoxanthin and zeaxanthin), phenolic compounds such as
phenolic acids, cinnamic acids, orotanins, catechins, bromophe-
nols, vitamins E and C, and polysaccharides. These molecules can
capture free radicals, chelate pro-oxidant metals, accept and donate
electrons, disrupt the lipid peroxidation, and inactivate reactive
oxygen and nitrogen species [25]. Specically, red seaweeds are
reported to contain halogenated phenolic compounds, sulfated
polysaccharides, and carotenoids [3,6,7]. Thus, they can be consid-
ered as promissory sources of compounds with antioxidant activity
(AA), and could be used as protecting agents against oxidative pro-
cesses in different systems.
Lipid oxidation is the main cause of deterioration of foods [8].
It generates rancidity, and loss of nutritional value and sensorial
quality. For this reason, synthetic antioxidants such as BHT (3,5-
Corresponding author.
E-mail address: fparadaa@unal.edu.co (F. Parada-Alfonso).
or gallates (i.e. gallic acid) are widely used in the edible oil indus-
try. However, the use of these antioxidants has been restricted,
because of their high volatility and thermal instability, and since
some animal studies have showed that they could promote can-
cer [9,10]. Consequently, research studies on the obtaining natural
antioxidants are acquiring special relevance.
Gracilaria mammillaris (Montagne) M.A. Howe is a red seaweed
of the Gracilariaceae family geographically distributed in various
coasts of North, Central and South America, Africa, Vietnam and
Indonesia, as well as in some Caribbean islands. It is used as food
in Hawaii, Indonesia, Malaysia, Philippines, and Vietnam, among
other countries [11], and employed as raw material with other
species of the same genus for the production of agar-agar for bac-
teriological cultures, and for different applications in the food and
cosmetic industries [12].
Supercritical uid extraction (SFE) may be considered as a
green extraction process, characterized by using GRAS-type sol-
vents (generally recognized as safe). It has some advantages over
other conventional extraction techniques, such as less extraction
times and solvent consumption, low or no toxicity, and free-solvent
and non-thermal-degraded extracts [13]. Moreover, the extraction
selectivity can be tuned by adjusting the pressure, temperature and
the type of solvent.
The objective of this work was to evaluate the use of supercrit-
ical CO2 extraction with and ethanol as co-solvent, for obtaining
extracts from red seaweed G. mammillaris having protective effects

http://dx.doi.org/10.1016/j.supu.2017.02.023
0896-8446/ 2017 Published by Elsevier B.V.

Please cite this article in press as: M. Ospina, et al., Antioxidant capacity of Colombian seaweeds: 1. Extracts obtained from Gracilaria
mammillaris by means of supercritical uid extraction, J. Supercrit. Fluids (2017), http://dx.doi.org/10.1016/j.supu.2017.02.023
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against oxidative deterioration of edible oils, as well as to deter-

mine the effects of the processing variables (pressure, temperature
and concentration of co-solvent) on the extraction yield, AA, and
total content of phenolic compounds (TCP) and carotenes (TCC) of
the obtained extracts. To the best of our knowledge, studies related
to the antioxidant activity of extracts of this seaweed have not been
reported.

2. Materials and methods

2.1. Materials and reagents

Red seaweeds G. mammillaris were collected in Santa Marta

(Colombia) in June 2014. The edible oil used for determining
the antioxidant activity was a blend of 70% soybean oil and 30%
RBD-type palm olein (rened, blanched, and deodorized) free of
antioxidants (Team Foods, Colombia). The reagents used were
CO2 (99.95% purity) (Linde Colombia S.A.), Folin-Ciocalteu reagent,
isooctane, trichloroacetic acid, thiobarbituric acid (Panreac), gallic
acid, dichloromethane (Merck), TBHQ (TCI-Tokyo Kasei), 1,1,3,3-
tetraethoxypropane-TEP, and beta-carotene type I synthetic 93%
UV (SigmaAldrich). All the solvents used were analytical or HPLC
grade.

2.2. Preparation of samples

The salt, sand and epiphytes of the seaweeds were removed with
tap water. The clean seaweeds were dried at 45 C in a vacuum oven
until constant weight. After grinding, the material was sieved using
a vertical vibratory stirrer with sieves of pore diameters ranging
from 0.15 to 0.60 mm. The samples were packaged in dark plastic
bags and stored in desiccators at room temperature, until their fur-
ther extraction processes. Seaweeds with particle sizes between 0.3
and 0.5 mm were used for the SFE trials [14,15], and those having
sizes lower than 0.3 mm were employed for the Soxhlet extraction
at reduced pressure.

2.3. Supercritical uid extraction (SFE)

The SFE experiments were carried out in o laboratory scale

equipment working on dynamic mode at a ux of 0.4 kg/h CO2 , with
ethanol as co-solvent. For each extraction trial, samples of about
14.0 g were used. The sample depletion time was 240 min deter-
minate according to previous kinetics assays (extraction curve) not
reported. Then, extractions were carried out at three different levels Fig. 1. Standardized effect of the SFE parameters on (a) Extraction yield, (b) TCP, and
of pressure, temperature and concentration of co-solvent, accord- (c) TCC of extracts from red seaweed G. mammillaris. and indicate
ing to the experimental design shown in Table 1. The extracts were positive and negative effects of the operation parameter on the evaluated variable,
concentrated by vacuum rotoevaporation, and stored at 20 C respectively.
until further analyses.

2.4. Soxhlet extraction at reduced pressure (SEPr)
The SEPr trials were achieved using three Soxhlet extraction
units connected in series to a vacuum pump equipped with a
vacuum regulator. Seaweed samples (5.07.0 g) were mixed with
250 mL of the following solvents at the indicated temperature and
pressure conditions: hexane (35 C, 0.30 atm), ethyl acetate (AcOEt)
(38 C, 0.23 atm), and ethanol (EtOH) (40 C, 0.18 atm). The extrac-
tions were carried out during 7 h, following the sequence described
below: a seaweed sample (M1) was rst extracted with hexane
for obtaining the extract S1. Subsequently, the residual cake of M1
was extracted with AcOEt for producing the extract S2, and the
residual cake was extracted with EtOH for getting the extract S3. A
second seaweed sample (M2) was extracted with AcOEt for obtain-
ing the extract S4. The residual cake was further extracted with
EtOH which produced the extract S5. A third seaweed sample (M3)
was extracted only with EtOH for producing the extract S6. All the
extract samples were concentrated by vacuum rotoevaporation and
stored at 20 C until further analyses.
2.5. Total content of phenolic compounds (TCP)
The TCP of the extracts was determined by means of the colori-
metric method of Folin-Ciocalteu, with some modications [16,17].
Extracts (100 L) diluted in EtOH (8 mg/mL) were mixed with
the Folin-Ciocalteu reagent (750 L, 10%, w/v aqueous solution),
and maintained in darkness. After 5 min, 750 L of an aqueous
sodium carbonate solution (6%, w/v) were added, and the solution
was allowed to stand for 90 min in darkness. Subsequently, the
absorbance at 765 nm was measured using a spectrophotometer
UV-Vis (Genesys 10, Thermo-Scientic). The results were expressed
in mg gallic acid equivalents per gram of seaweed in dry basis (mg

Please cite this article in press as: M. Ospina, et al., Antioxidant capacity of Colombian seaweeds: 1. Extracts obtained from Gracilaria
mammillaris by means of supercritical uid extraction, J. Supercrit. Fluids (2017), http://dx.doi.org/10.1016/j.supu.2017.02.023
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Table 1
Extraction yield, total contents of phenolic compounds (TCP) and total content of carotenoids (TCC) of SFE and SEPr extracts from red seaweed G. mammillaris.

Extract Pressure (MPa) Temperature ( C) EtOH (%w/w) Extraction yield (%w/w db.) TCP (mg GAE/g) TCC (mg C/g)

1 10 40 2 0.10 0.368 0.002a 0.597 0.037a

2 30 40 2 0.88 1.872 0.018bc 1.511 0.072b
3 10 60 2 0.36 0.720 0.007d 0.447 0.000ja
4 30 60 2 0.53 1.749 0.011e 1.479 0.047b
5 10 40 8 1.51 2.668 0.021f 2.119 0.063c
6 30 40 8 1.71 3.641 0.032g 2.158 0.075c
7 10 60 8 1.40 2.862 0.025h 0.920 0.071d
8 30 60 8 3.03 3.791 0.035i 2.214 0.073c
9 10 50 5 0.42 1.692 0.016e 0.919 0.041d
10 30 50 5 0.74 2.701 0.024f 5.038 0.087e
11 20 40 5 0.51 1.907 0.015b 2.687 0.075f
12 20 60 5 0.63 2.464 0.022j 3.987 0.067g
13 20 50 2 0.34 1.565 0.014k 2.505 0.061f
14 20 50 8 1.03 3.272 0.032l 2.670 0.069f
15.1 20 50 5 0.54 2.421 0.020j 3.582 0.059h
15.2 20 50 5 0.60
15.3 20 50 5 0.81
15.4 20 50 5 0.58
15.5 20 50 5 0.73
S1 0.24 0.02a 0.286 0.003a 0.937 0.088d
S2 0.64 0.03b 2.619 0.024f 4.583 0.072i
S3 6.75 0.08f 1.772 0.032ce 0.266 0.027j
S4 0.81 0.03g 3.919 0.065m 3.969 0.072g
S5 6.62 0.09f 2.622 0.081f 0.282 0.027j
S6 5.12 0.03h 3.486 0.053n 1.134 0.055k

Means in columns followed by the same letter are not signicantly different by Tukey test at the 5% level.
For extracts 15.115.5 shown the average value of TPC and TCC.
S1 extract obtaining with hexane from a seaweed sample (M1). S2 extract obtaining with AcOEt from residual cake of M1. S3 extract obtaining with EtOH from residual cake
of S2 obtaining. S4 extract obtaining with AcOEt from seaweed sample (M2). S5 extract obtaining with EtOH from residual cake of M2. S6 extract obtaining with EtOH from
a seaweed sample (M3).

Fig. 2. Inhibition in the production of HPL and TBARS of the extracts obtained by means of SFE and SEPr in comparison to synthetic antioxidants. The control sample exhibited
no inhibition.

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Fig. 3. Effect of SFE operation parameters on the inhibition of HPL of G. mammillaris extracts.

Fig. 4. Effect of SFE operation parameters on the inhibition of TBARS of G. mammillaris extracts.

GAE/g db.). Gallic acid was used as standard for the calibration curve
(y = 4.897x + 0.027, R2 = 0.996), with concentrations between 0.02
and 0.19 mg/mL.
2.6. Total content of carotenes (TCC)
The TCC of the extracts was evaluated following the method pro-
posed by Parsons and Strickland with some modications [18,19].
Acetone at 90% (2 mL) was added to 5 mg extract, and the slurry
was centrifuged for 10 min at 4 C and 14,000 rpm. The precip-
itate was washed successively with acetone until discoloration,
and all the supernatants were poured in a volumetric ask, whose
volume was completed with acetone. The absorbance at 480 nm
was determined using a spectrophotometer UV-Vis (Genesys 10,
Thermo-Scientic). The results were expressed as mg carotenes per
gram of seaweed in dry basis (mg C/g db.). -Carotene type I was

Please cite this article in press as: M. Ospina, et al., Antioxidant capacity of Colombian seaweeds: 1. Extracts obtained from Gracilaria
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used for the calibration curve (y = 70.524x 0.0053, R2 = 0.996) in
concentrations varying from 0.0005 to 0.0035 mg/mL.
2.7. Antioxidant activity evaluation
The AA of the obtained extracts was evaluated by measuring
their protecting capacity against the accelerated oxidation of one
edible oil, through the analyses of hydroperoxides of linoleic acid
(HPL) and thiobarbituric acid reactive species (TBARS).
The accelerated oxidation experiments were carried out on the
basis of previous reports [2022]. For that, 20 g of oil samples were
poured in amber asks, and an ethanolic solution of ferrous chloride
was added until reach a Fe2+ concentration of 3.5 ppm. The obtained
extracts and the commercial antioxidants (BHT, THBQ, and gallic
acid (GA)) were dissolved in ethanol and added separately to the
oil samples until reach a concentration of 200 ppm [23,24]. Control
samples were prepared similarly, but ethanol was added, instead
antioxidants. All samples were kept at 70 C during 6 days, bubbling
air during 5 min, four times per day.
HPL determinations were performed after 2, 4 and 6 days of
accelerated oxidation, according to the method described previ-
ously [25,26]. The absorbance of oil samples diluted in isooctane
(1 mg/L) was measured at 234 in a spectrophotometer UV-
Vis (Genesys 10, Thermo-Scientic). The concentration of HPL,
expressed as mmol linoleic acid/kg oil, was calculated using the
molar extinction coefcient () of hydroperoxides of linoleic acid
(26,000 M1 cm1 ). The results were also expressed in percentage
of inhibition, according to Eq. (1) [27]:
[HPL]c [HPL]e
HPL inhibition (%) = 100
[HPL]c
where [HPL]c and [HPL]e are the concentrations of hydroperoxides
in the control and oil samples, respectively.
The concentration of some secondary and nal species of lipid
oxidation was followed through the reactivity method of these
species with thiobarbituric acid (TBARS), after 2, 4 and 6 days of
accelerated oxidation, with some modications [28,29]. 50 mg of
the oil samples were added with an ethanolic solution of TBHQ
(23 mM), 5 mL of trichloroacetic acid (0.30 M in HCl 0.2 N), and one
aqueous solution of thiobarbituric acid (26 mM). After heated at
8085 C in a water bath during 40 min, they were cooled down
at 04 C for 30 min. A 3 mL aliquot was taken, mixed with 3 mL
of dichloromethane, and centrifuged at 5500 rpm during 10 min
at room temperature. The absorbance of supernatants was mea-
sured at 532 nm in a spectrophotometer UV-Vis (Genesys 10,
Thermo-Scientic). The results were expressed in mg MDA/kg oil,
through the calibration curve (y = 0.001x + 0.010, R2 = 0.997) with
hydrolyzed TEP at concentrations ranging between 103.133 and
773.499 M. The inhibition of TBARS was calculated according to
Eq. (2) [30]:
[TBARS]c [TBARS]e
TBARS inhibition (%) = 100
[TBARS]c
where [TBARS]c and [TBARS]e are the TBARS concentrations in the
control and oil samples, respectively.
2.8. Experimental design and statistical analysis
The Soxhlet extractions were carried out following a completely
randomized design, while for the SFE a central composite design
( = 1) with 5 repetitions in the central point, was used. The exper-
imental region was delimited by three factors at three levels each
one, as presented in Table 1: Pressure at 10 MPa (), 20 MPa (0), and
30 MPa (+); temperature at 40 C (), 50 C (0), and 60 C (+); and
concentration of co-solvent 2% (), 5% (0), and 8% (+). The inuence
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mammillaris by means of supercritical uid extraction, J. Supercrit. Fluids (2017), http://dx.doi.org/10.1016/j.supu.2017.02.023
5
of the extraction parameters on the investigated variables (extrac-
tion yield, AA, TCP, and TCC) was evaluated by means of the Pareto
charts and response surface analyses, using the software STAT-
GRAPHICS XVI.II v. 16.02.0004. All measurements were made in
triplicate. Statistical differences were determined by means of anal-
ysis of variance (ANOVA), and mean comparisons by the Tukey test
(p < 0.05) were performed for the TCP and TCC. In the case of HPL and
TBARS, an analysis of variance with repeated measures (RANOVA),
and means comparisons by the Bonferroni test (p < 0.05), were used.
The statistical tests were performed using the software IBM SPSS
Statistics 23.
3. Results and discussion
3.1. Extraction yields
Table 1 presents the extraction yields obtained using the two
extraction techniques studied. As noticed, among the extracts pro-
duced by means of SFE, the extract number 8 showed the highest
yield (3.03%). As far as the SEPr are concerned, the highest yields
were achieved when using ethanol (S3: 6.75%, S5: 6.62%, and S6:
5.12%). These results indicate that the high the solvent polarity,
the higher the extraction yields. However, it is important to note
that the extraction yield of S6 was almost twice than the super-
critical extract number 8. In spite of this, the yields obtained in
this study were higher than those reported recently by Sivagnanam
et al. [31] for brown seaweed Saccharina japnica (1.09%) and Sar-
gassun horneri (1.41%), using CO2 -EtOH (9010%), at 25 MPa and
45 C. Eq. (3) presents the quadratic equation modelling the effect
of the operation variables on the extraction yield from seaweed G.
(1)
mammillaris:
Extraction yield (%) = 6.49659 0.197761 T 0.107614
P 0.436919 [EtOH] + 0.00162577 T 2 + 0.001025 T
P + 0.00541667 T [EtOH] + 0.00172577 P 2 + 0.00366667
P [EtOH] + 0.0308419 [EtOH]2 (3)
R2 : 73.97%; Standard error: 0.34
The Pareto chart presented in Fig. 1a, shows that the concen-
tration of co-solvent was the variable with most inuence on the
extraction yield, followed by the pressure. These results can be
explained because at pressures higher than 15 MPa, the increase
in temperature enhances the extraction yield, due to the rise in
the vapor pressure of solutes, which is known as retrogradation
[26,32]. Moreover, the co-solvent at the highest concentration, led
to an increase in the solubility, possibly due to (i) the alteration
and swelling of the matrix structure, causing changes that favour
the solvent access to the solutes [15]; (ii) an increase in the polarity
and density of the solvent, which allows more interactions with the
solutes [3234]; (iii) the rupture of the matrix-solute interactions,
which promotes the incorporation of the solvent to the active sites
(2) of the matrix for an easier solubilization of the solutes [35].
3.2. Total content of phenolic compounds (TCP)
Table 1 presents the TCP of the extracts for the two extraction
techniques investigated. As shown in this table, among the extracts
obtained using SFE, the number 8 presented the highest TCP
(3.79 mg GAE/g), which could be attributed to the increase in the
solvent polarity due to the increase in the concentration of ethanol
in the solvent mixture. On the other hand, the extract S4, pro-
duced by means of SEPr with AcOEt as solvent, displayed the highest
content of TCP (3.92 mg GAE/g), being similar to that obtained by
using SFE. Sivagnanam et al. [31] found TCP of 0.64 0.01 and
0.60 0.05 mg EGA/g in extracts from the seaweeds S. japnica
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and S. horneri, respectively, using SFE with CO2 -EtOH as solvent, at
25 MPa and 45 C. Eq. (4) presents the quadratic equation modelling
the effect of the operation variables on the TCP:
TCP (mg GAE/g) = 5.56253 + 0.152613 P + 0.260041 [EtOH]
+0.164353 T 0.00131474 P 2 0.00262917 P [EtOH]
0.00064875 P T + 0.0100584 [EtOH]2 + 0.000479167
[EtOH] T 0.00142474 T 2 (4)
R2 :98.63%; Standard error: 0.14
From the Pareto diagram depicted in Fig. 1b, it can be appre-
ciated that the concentration of co-solvent was the parameter
affecting the most the TCP, followed by the pressure and the tem-
perature. The increase in the levels of pressure and concentration of
co-solvent led to a rise in the TCP, because there is an increase in the
polarity and density of the solvent mixture CO2 -EtOH, due to the
formation of hydrogen bonds and dipole-dipole interactions, which
stimulate the extraction of polar compounds with high molecu-
lar weight [33,36]. However, the increase of the temperature at
its highest level, led to a slight decrease in the TCP, possibly due
to polymerization or oxidation reactions of some phenolic com-
pounds at temperatures above 50 C [33].
3.3. Total content of carotenes (TCC)
The TCC of the extracts is presented in Table 1. The highest con-
centration of carotenes (5.04 mg C/g) was found in the extract 10,
obtained with SFE at 30 MPa, 50 C and 5% co-solvent. As far as
the SEPr is concerned, the extracts S2 and S4 showed the highest
values of TCC (4.58 and 3.97 mg C/g, respectively). These extracts
were obtained using AcOEt as solvent, which indicates that the
extracted compounds have afnity by solvents of moderate polar-
ity. The results of TCC reported in this study for the both extraction
techniques, were higher than those recently published [3739].
Eq. (5) presents the quadratic equation modelling the effect of the
operation variables on the TCC:
TCC (mg C/g) = 12.9067 + 0.288048 P + 1.60115 [EtOH]
2
+0.345095 T 0.00717862 P 0.00255875 P [EtOH]
+0.00171725 P T 0.123176 [EtOH]2 0.00400369
[EtOH] T 0.00359671 T 2 (5)
R2 :80.46%; Standard error: 0.80.
As indicated by the Pareto diagram shown in Fig. 1c, the pressure
was the parameter inuencing the most the TCC, since the increase
in the solvent density as the pressure augmented, led to higher
extraction of carotenoids.
3.4. Antioxidant activity
The AA of the extracts comprised the evaluation for protecting
an edible oil against oxidation in both, the initial and the intermedi-
ate stages, through HPL and TBARS analyses. In general, all extracts
and antioxidants used as reference, displayed a reduction in the oil
protection during the accelerated oxidation trials. All the concen-
trations of HPL and TBARS in the oil samples were lower than those
found in the control sample, presenting signicant differences, as
shown in Appendix 1 (Tables A.1A.4). These results indicate that
the extracts from seaweed G. mammillaris effectively protected the
edible oil against oxidation in both the initial and nal stages. How-
ever, this protection was lower than that offered by the antioxidants
used as reference (especially for the TBHQ and gallic acid).
The extracts S3, S5, and S6, which showed yields higher than
5%, displayed a low protection against lipid oxidation. The extract
Please cite this article in press as: M. Ospina, et al., Antioxidant capacity of Colombian seaweeds: 1. Extracts obtained from Gracilaria
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S4 presented the higher inhibition of HPL (24.68%), followed by
the synthetic antioxidant BHT (24.50%) and the extract number 6
obtained using SFE (20.86%). However, there were no signicant
differences between the inhibition of HPL offered by the extracts 6
and 8, and the BHT, as indicated in Table A.1.
It is worth mentioning that the inhibition to the formation of the
HPL offered by all extracts was low in comparison to the inhibition
to the formation of the TBARS. For the latter, the extracts 8 and 6,
obtained by means of SFE, presented inhibition values of 42.10%
and 36.18%, respectively, which were signicantly higher than the
inhibition caused by the extract S4 (30.33%) produced using SEPr.
Moreover, the results conrm the low protection against the sec-
ondary oxidation species offered by the synthetic BHT (20.71%).
Fig. 2 presents the inhibition of HPL and TBARS of the extracts
and commercial antioxidants after six days of accelerated oxida-
tion. As it can be noticed, the extract S4 displayed the highest
inhibition among the extracts obtained by means of SEPr. How-
ever, this inhibition was lower than that offered by the extracts 6
and 8, especially for the TBARS.
The inuence of the SFE processing variables on the inhibition of
HPL and TBARS after six days of accelerated oxidation is presented
in the surface responses depicted in Figs. 3 and 4, respectively,
which corresponds to Eqs. (6) and (7).
HPL Inhibition (%) = 83.1298 2.05692 T 3.90786 [EtOH]
1.55274 P + 0.0164555 T 2 + 0.0106175 T [EtOH]
+0.0146232 T P + 0.273013 [EtOH]2 + 0.0481879
[EtOH] P + 0.0232101 P 2 (6)
R2 : 87.12%; Standard error: 2.26.
TBARS Inhibition (%) = 57.9553 1.69337 T 0.276697
P 0.847353 [EtOH] + 0.0130863 T 2 + 0.0119273 T
P + 0.0344925 T [EtOH] 0.00752927 P 2 + 0.078564 P
[EtOH] + 0.0334582 [EtOH]2 (7)
R2 :90.48; Standard error: 3.31.
According to Figs. 3 and 4, the pressure was the operation vari-
able affecting the most the production of extracts capable to inhibit
the HPL (the high the operation pressure, the higher the inhibition
of HPL), while the concentration of co-solvent and the pressure
showed a direct correlation with the production of extracts having
protecting properties against the formation of TBARS.
Taking into account the above results, it is possible to produce
extracts from red seaweed G. mammillaris with protecting capacity
against lipid oxidation of edible oils, by means of green technique
such as SFE with CO2 -EtOH.
4. Conclusion
The extracts from red seaweed Gracilaria mammillaris (Mon-
tagne) M.A. Howe delayed the oxidation of the edible oil. The
operation parameters that inuenced the most the evaluated vari-
ables were the concentration of co-solvent and the pressure. The
extraction yields, TCP and the inhibition of TBARS after six days
of accelerated oxidation, were dependent on the concentration of
co-solvent, while the TCC was mainly inuenced by the operation
pressure. Taking into account that these extracts were composed of
various compounds having chemical afnity for the solvent used in
the extraction process, and that they were not puried molecules
such as TBHQ and BHT, their protection against oxidation was lower
than the commercial antioxidants at the added concentrations
(200 ppm). However, the results obtained in this study indicate that
the red seaweed G. mammillaris is a promissory source of extracts
G Model
SUPFLU-3867; No. of Pages 9 ARTICLE IN PRESS
M. Ospina et al. / J. of Supercritical Fluids xxx (2017) xxxxxx 7

with AA, and they are an incentive to add value to the Colombian toria Nacional Jvenes Investigadores e Innovadores 2014). The
phycological sources. More studies are necessary to identify the authors thank Professor Luis-Felipe Gutirrez for his collaboration,
molecules responsible of such AA. and Universidad de Bogot Jorge Tadeo Lozano (Convocatoria de
investigacin 10-2013).

Acknowledgments
Appendix 1.

This project was fund by the Universidad Nacional de

Colombia (Project No. 201010023413) and Colciencias (Convoca-

Table A.1
Statistic differences in the inhibition of HPL of extracts from G. mammillaris obtained by means of SFE.

Extract 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 TBHQ AG BHT

*
2
3
*
4
5
* * *
6
*
7
* * * * * *
8
* * * *
9
* *
10
* *
11
* *
12
* *
13
* *
14
* * * * * *
15
* * * * * * * * * * * * * * *
TBHQ
* * * * * * * * * * * * * * * *
AG
* * * * * * * * * * * * * * *
BHT
* * * * * * * * * * * * * * * * * *
CONTROL
*
Signicant differences (p < 0.05) by Bonferroni test.

Table A.2
Statistic differences in the inhibition of TBARS of extracts from G. mammillaris obtained by means of SFE.

Extracts 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 TBHQ AG BHT

*
2
*
3
*
4
* * * *
5
* * * * *
6
* * * *
7
* * * * * *
8
* * * * *
9
* * * * * * *
10
* * * * * *
11
* * * * * * *
12
* * * * * * *
13
* * * * * * * * * * * *
14
* * * * * * * * * *
15
* * * * * * * * * * * * * * *
TBHQ
* * * * * * * * * * * * * * * *
AG
* * * * * * * * * * * *
BHT
* * * * * * * * * * * * * * * * * *
CONTROL
*
Signicant differences (p < 0.05) by Bonferroni test.

Table A.3
Statistic differences in the inhibition of HPL of extracts from G. mammillaris obtained by means of SEPr.

Extract 1 2 3 4 5 6 TBHQ AG
*
S2
* *
S3
* *
S4
* * *
S5
* * * * *
S6
* * * * * *
TBHQ
* * * * * * *
AG
* * * * * * * *
Control
*
Signicant differences (p < 0.05) by Bonferroni test.

Please cite this article in press as: M. Ospina, et al., Antioxidant capacity of Colombian seaweeds: 1. Extracts obtained from Gracilaria
mammillaris by means of supercritical uid extraction, J. Supercrit. Fluids (2017), http://dx.doi.org/10.1016/j.supu.2017.02.023
G Model
SUPFLU-3867; No. of Pages 9 ARTICLE IN PRESS
8 M. Ospina et al. / J. of Supercritical Fluids xxx (2017) xxxxxx
Table A.4
Statistic differences in the inhibition of TBARS of extracts from G. mammillaris obtained by means of SEPr.
Extract 1 2 3 4
*
S2
*
S3
* * *
S4
* *
S5
* * * *
S6
* * * *
TBHQ
* * * *
AG
* * * *
Control
*
Signicant differences (p < 0.05) by Bonferroni test.
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Please cite this article in press as: M. Ospina, et al., Antioxidant capacity of Colombian seaweeds: 1. Extracts obtained from Gracilaria
mammillaris by means of supercritical uid extraction, J. Supercrit. Fluids (2017), http://dx.doi.org/10.1016/j.supu.2017.02.023