Extraction_of_proanthocyanidins_from_gra

Accepted Manuscript
Title: Extraction of proanthocyanidins from grape marc by
supercritical fluid extraction using CO 2 as solvent and
ethanol-water mixture as co-solvent
Author: Carla Da Porto Andrea Natolino Deborha Decorti
PII:
S0896-8446(13)00417-8
DOI:
http://dx.doi.org/doi:10.1016/j.supflu.2013.12.013
Reference: SUPFLU 2880
To appear in:
J. of Supercritical Fluids
Received date: 25-8-2013
Revised date: 20-12-2013
Accepted date: 21-12-2013
Please cite this article as: C. Da Porto, A. Natolino, D. Decorti, Extraction of
proanthocyanidins from grape marc by supercritical fluid extraction using CO 2 as
solvent and ethanol-water mixture as co-solvent, The Journal of Supercritical Fluids
(2014), http://dx.doi.org/10.1016/j.supflu.2013.12.013
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.

Extraction of proanthocyanidins from grape marc by supercritical fluid
extraction using CO 2 as solvent and ethanol-water mixture as co-solvent
Carla Da Porto*, Andrea Natolino, Deborha Decorti,
Department of Food Science, University of Udine, via Sondrio 2/A, 33100 Udine, Italy
Accepted Manuscript
_____________________________
* Corresponding author. Tel.: +39 0432 558141; fax: +39 0432 558130 ; e-mail:carla.daporto@uniud.it
Abstract
1
Page 1 of 22

The extraction of phenolic compounds from grape marc using supercritical CO 2 containing 15%
ethanol-water mixture (57% v/v) (EtW) as co-solvent, at 8, 10, 20 and 30 MPa/313.15 K suggested
8 MPa as the most suitable pressure. At 8MPa/313.15K, different co-solvent percentages (15, 10,
7.5%) at CO 2 flow rate of 6 and 4 kg/h were investigated for proanthocyanidins (PAs) the
extraction. The highest extraction yields were obtained at 4kg/h CO 2 -7.5% EtW flow rate (2600
mg GAE 100 g DM -1 ) and 6kg/h CO 2 - 10%EtW (2527 mg GAE 100 g DM -1 ). At 6kg/h CO 2 - 10%EtW
flow rate the amounts of monomeric (188 mg catechin 100 g -1 DM ) and oligomeric (154 mg catechin 100 g
DM -1 ) fractions of PAs, as well as their antioxidant activity (809 and 546 mg α-tocopherol 100 g -1 DM )
were higher than at 4kg/h CO 2 -7.5% EtW- flow rate. SC-CO 2 methods were compared with
conventional methanol extraction.
Keywords: supercritical fluid extraction, co-solvent ethanol-water mixture, phenolic compounds,
antioxidant activity, grape marc
Introduction
The wine production industries generate a large quantities of waste, including grape marc and wine
lees. Grape marc has a heavy environmental impact for the high content of organic residues that
considerably increase chemical and biochemical oxygen demands. This biomass could be disposed
and valorized by extraction of residues phenols which represent added-value biOECtive compounds
useful in pharmaceutical, cosmetics and food industry.
Grape’s polyphenols include flavonoids and non-flavonoids [1]. Proanthocyanidins (PAs), also
known as condensed tannins, are oligomeric and polymeric flavonoids of high complexity; their
biOECtive properties are determinate by molecular composition and size [1-3]. PAs subunits are
differenced by their substitutions and the stereochemistry of their structures. The most common
monomers are (+)-catechin, (-)-epicatechin, (-)-epicatechin gallate and (-)-epigallocatechin [2].
Accepted Manuscript
Recent research on the role of PAs as plant-based health-beneficial components in the human diet
reported potential health beneficial effects including antioxidant, anti-diabetic, anti-carcinogenic,
and anti-inflammatory activities. The healthy properties of PAs largely depend on their structure
and especially on their degree of polymerization. Cos et al. [4] reported that at least monomers and
smaller oligomeric proanthocyanidins are absorbed.
Supercritical fluid extraction (SFE) is an environment-friendly technology that represents an
alternative to conventional extraction methods and offers several advantages over classical solvent
extraction methods. In fact, supercritical fluids have a high diffusivity and low density, viscosity
and surface tension. Supercritical carbon dioxide (SC-CO 2 ) is the most commonly used solvent in
2
Page 2 of 22

SFE. It is inert, non-toxic, and allows extraction at lower temperature and relatively low pressure.
Furthermore, the extracts obtained by SFE are of high quality [5].
SFE with SC-CO 2 has been applied on wine by-products for the recovery of grape seed oil [6-10],
and with the addition of a co-solvent, usually ethanol or methanol, for the recovery of phenols [11-
16]. However, to the best of our knowledge, there has been no work, thus far, on the extraction of
phenols from grape marc using SC-CO 2 with ethanol-water mixture as co-solvent.
In this study, the extraction of phenolic compounds from grape marc using SC-CO 2 extraction at
different pressure, and two CO 2 flow rates modified with different percentages of ethanol-water
mixture (57% v/v) as co-solvent were tested . The effect of pressure on the extraction of
polyphenols at four different pressures in the range of 8-30 MPa at 313.15 K, as well as the effect
of 4 and 6 kg/ h CO 2 flow rates modified with 7.5, 10 and 15% ethanol-water, at 8 MPa and 313.15
K have been investigated. The performance of SC-CO 2 methods were checked by evaluation of
phenolic yield, proanthocyanidins content and antioxidant activity. SC-CO 2 methods were
compared with conventional methanol extraction.
2. Material and methods
2.1 Materials and reagents
Grape marc from white grape (Vitis vinifera L.) varieties was collected during September 2012 in
Friuli Venezia- Giulia region (Italy).
Carbon dioxide (mass fraction purity 0.999 in the liquid phase) was supplied by Sapio s.r.l (Udine,
Italy). Free stable DPPH radical (DPPH • ), Folin–Ciocalteau reagent, gallic acid, (± )-catechin, (+)-
α-tocopherol and vanillin 99% were purchased from Sigma-Aldrich (Milan, Italy). Sep-Pak Plus
tC18cartridge WAT 036810 and WAT 036800 were purchased from Waters (Milan, Italy). Other
reagents were of analytical grade or higher available purity.
2.2 Grape marc preparation
Accepted Manuscript
Grape marc was air dried at room temperature (moisture 14.3% ± 0.3 w/w) and stored at 277.15 K
until use. Grinding of grape marc was carried out on a domestic mill, and particles characterized by
size classification in a standard sifter with several mesh sizes (<0.5, 0.8-1.0, 1.0-1.25, 1.25-1.50,
1.50-1.75, 1.75-2.0 > 2.0 mm). An average particle diameter d p =0.83±0.05 mm was adopted, being
calculated by Sauter’s equation [17] to a set of fractions within the previous mesh sized:
d p = m t / Σ i=1 k m i /d pi
3
Page 3 of 22

where m i is the mass of particles retained below mesh size d pi, m t is the total mass of milled seeds
and k is the number of mesh sized
2.3 Conventional solvent extraction
Ground grape marc (25 g) was continuously extracted with 300 mL n-hexane for 6 h at a maximum
temperature of 343.15 K in a Soxhlet apparatus to extract lipids. Subsequently, 1 g of defatted grape
marc with 5 mL methanol were mixed and shaken at room temperature for 90 min to extract
phenolic compounds [18].
2.4 Supercritical fluid extraction (SFE)
SFE pilot-plant (SCF100 serie 3 PLC-GR-DLMP, Separeco S.r.l, Pinerolo, Italy ) equipped with 1
L extraction vessel (E 1 ), two 0.3 L separators in series (S 1, S 2 ), and a tank (B 1 ) where CO 2 is stored
and recycled was used. The solvent used was carbon dioxide (Sapio s.r.l ,Udine, Italy). The flow
sheet of SFE pilot plant is given in Figure 1.
Ground grape marc was defatted by SC-CO 2 extraction. The extractor was filled with 0.480 kg of
grape marc (density 600 kg m -3 ). As suggested by Sovova et al. [7] pressure was 28 MPa and
temperature 318.25 K, while CO 2 flow rate was 10 kg/h and 3 h the total extraction time,
corresponding to 62.5 Q (kg CO 2 /kg feed).
Subsequently, to extract polyphenols from the defatted grape marc, due to the polarity of
polyphenols, the addition of a co-solvent to the SC-CO 2 was needed. Different percentages (15, 10
and 7.5%) of ethanol aqueous mixture at 57% (v/v) ethanol were used as co-solvent. Mixtures of
alcohols and water have revealed to be more efficient in extracting phenolic constituents than the
corresponding mono-component solvent system [19]. The ethanol-water mixture at 57% (v/v)
ethanol was chosen as co-solvent because Makris et al. [20] reported that efficient extraction of
Accepted Manuscript
phenolics from all white vinification solid by-products was achieved, using conventional extraction
procedure, employing this solvent system, and the extracts so obtained highlighted the highest
antioxidant activity.
The extractor was filled with 0.1 kg of ground defatted grape marc distributed in glass beads (0.005
m). The true density of grape marc, determined by picnometry with helium gas (Pycnomatic ATC,
Thermo electron corporation, Milan, Italy), was 1411 ± 21 kg m -3 . The apparent bed density was
750 kg m -3 and the total porosity on the bed particles was calculated to be 0.47.
After a preliminary study on the effect of different pressures (8, 10, 20 and 30 MPa) at 313.15 K on
the extraction of polyphenols, the extractions were carried out at 8 MPa. The solvent flow rates used
4
Page 4 of 22

were 6kg/h CO 2 modified with 15 and 10% co-solvent (EtW) (namely 6CO 2 -15%EtW and 6CO 2 -
10% EtW ) and 4 kg/h CO 2 modified with 15 and 7.5% EtW (namely 4CO2-15%EtW and 4CO 2 -
7.5%EtW) .
Aliquots of grape extract were collected during extractions in volumetric flask at intervals of about
30 min, to assess several data points for the overall extraction curves (OECs). The ethanol aqueous
mixture was then removed from the extracts with rotary evaporator (Buchi, B465, Switzerland) at
318.15 K. After removal of solvent the extracts were weighted and analyzed. All experiments were
conducted in duplicate.
2.5 Total phenolic content
Purification by C 18 cartridge was carried out for the samples to eliminate the interference of sugars,
non volatile acids and amino acids in total phenols determination. The total phenolic content (TPC)
values of the grape marc extracts were measured using the Folin–Ciocalteau reagent, according to
Yu et al. [21]. Briefly, the reaction mixture contained 100 µL of extract or solvent, 500 µL of the
Folin-Ciocateau reagent, 1.5 mL of 20% sodium carbonate, and 1.5 mL of pure water. After 2 h of
reaction at ambient temperature, absorbance was read at 765 nm using a UV–Vis spectrophotometer
(Shimadzu UV 1650, Italy) to calculate TPC. Gallic acid was employed as the standard. A
calibration curve was made with standard solutions of gallic acid in the range 0.2–10 mg mL -1 and
measures were carried out at 765 nm (R 2 =0.99). All analyses were performed in triplicate. Results
were expressed as milligrams of equivalent gallic acid per 100 gram of dried matter (mg GAE 100 g
DM -1 )
2.6 Fractionation of proanthocyanidins
Grape marc extracts were fractionated as reported by Sun et al [22]. Briefly, 5 mL of grape marc
extracts was concentrated to dryness in a rotary evaporator at <303.15 K. The residue was dissolved
Accepted Manuscript
in 20 mL of 67 mM phosphate buffer, pH 7.0. The pH of the resulting solution was adjusted to 7.0
with NaOH or HCl. Two C 18 Sep-Pak cartridges were assembled (WAT 36800 on the top and WAT
36810 at the bottom) and conditioned sequentially with 10 mL of methanol, 20 mL of deionized
water and 10 mL of phosphate buffer, pH 7.0. Samples were passed through the cartridges at flow
rate not higher than 2 mL min -1 , and phenolic acids were then eliminated by elution with 10 mL of
67 mM phosphate buffer at pH 7.0. The cartridges were dried with nitrogen flow and eluted
sequentially with 25 mL of ethyl acetate (fraction FI + FII, containing monomeric and oligomeric
flavan-3-ols) and with 15 mL of methanol (fraction FIII, containing polymeric proanthocyanidins).
The ethyl acetate eluate was taken to dryness under vacuum using a rotary evaporator (Rotavapor
5
Page 5 of 22

R210, Buchi, Flawil, Switzerland), redissolved in 3 mL of phosphate buffer at pH 7.0 and reloaded
onto the same series of cartridges, that had been conditioned as described above. The cartridges
were dried with nitrogen flow and eluted sequentially with 25 mL of diethyl ether (fraction FI,
containing monomers) and 15 mL of methanol (fraction FII, containing oligomers). The fractions
FI, FII and FIII were evaporated to dryness under vacuum in 3 mL of methanol. Sample
fractionation was performed in duplicate. The total flavan-3-ol content of each fraction was
determined by vanillin assay according to the method described by Sun et al [22]. Results were
expressed as milligrams of equivalent catechin acid per 100 gram of dried matter (mg catechin 100 g
DM -1 )
2.7 Antioxidant activity
The antioxidant activity of phenolic extracts and proanthocyanidins fractions was evaluated by the
total free radical scavenger capacity (RSC) following the methodology described by Espin et al.
[23] with slight modification. In brief, 10 μL of methanolic extract, previously diluted 1:10, was
added with 1990 μL of fresh methanol DPPH solution (93μM). Then the mixture was shaken
vigorously and left in darkness for 60 min. Finally, the absorbance of the mixture was measured
against pure methanol (blank) at 515 nm using a UV–Vis spectrophotometer, (Shimadzu UV 1650,
Italy). The RSC is the difference of the concentration of DPPH free radical (C DPPH•, i ) previously
dissolved in methanol, after 60 min of reaction with the samples (C DPPH•, f ). The antioxidant
activity of the samples was expressed as milligrams of α-tocopherol per 100 gram of dried matter
(mg α-tocopherol 100 g -1 DM ) A calibration curve was made with standard solutions of α-tocopherol in
the range 5.8 ⋅10 –5 – 2.3 ⋅10 –3 mol L -1 (R 2 =0.98). All analyses were performed in triplicate.
3. Results and Discussion
3.1 Effect of pressure
Accepted Manuscript
The effect of pressure on the extraction of polyphenols was studied at 8, 10, 20 and 30 MPa/
313.15 K, at 6CO 2 -15%EtW flow rate for 240 min, as shown in Figure 2. The concentration of
phenols increases with decreasing extraction pressure, with faster extraction kinetic observed at 8
MPa. At 8 MPa the extraction of phenols (1768 mg GAE 100 g -1 DM is higher than at 30 MPa (340
mg GAE 100 g -1 DM ). As suggested by Farìas-Compomanes et al. [16] the low mass-transfer rates at
the high pressure may be partially due to the low dispersion coefficient of the modified SC-CO 2
which accounts for the axial and radial diffusion mechanisms, the not-homogeneous characteristics
of the raw material (skins and seeds), and the high porosity of the extraction bed . These results
6
Page 6 of 22

suggest 8 MPa was the most suitable pressure for supercritical carbon dioxide extraction of grape
marc.
3.1 Effect of solvent flow rate
In Figure 3 the overall extraction curves (OECs) (phenols content vs. time) obtained for grape marc
extracted at 8 MPa/ 313.15 K, 6CO 2 -15%EtW and 4CO 2 -15% EtW solvent flow rate for 300 min
are plotted to evaluate the effect of solvent flow rate on the extraction of total phenols.
Both OECs exhibit a constant-extraction rate period (CER) of 150 min, and a diffusion-controlled
period (DC) follows. An intermediary falling extraction rate (FER) period cannot be observed [24]
The initial linear period (CER) corresponds about the 84 and 65% of the final extracted phenols at
6CO 2 -15%EtW and 4CO 2 -15%EtW flow rate, respectively. It is worth noting that the OECs
overlapping up to 150 min. This indicates that the extraction velocity is independent of solvent
flow rates and corresponds to the extract solubility. The slopes of these lines, as they are related to
the extract solubility, only depend on pressure and temperature. After 150 min, the OECs diverge,
exhibiting a diffusion-controlled period (DC) and here the slopes depend on particle size and
solvent flow rate. Such trends corroborates the hypothesis of the broken plus intact cells model
proposed by Sovová [25] . The kinetic parameters calculated from the adjustment of the OECs at
6CO 2 -15%EtW and 4CO 2 -15%EtW flow rate for the diffusion controlled period (DC) are reported
in Table 1. The results indicate that as the flow rate decreases the mass transfer increases. This
could be attributed to the fact that 4CO 2 -15%EtW flow rate is slower than 6CO 2 -15%EtW and
thus the contact time between the solvent and the compounds to be extracted is increased. This
may have positively affected the extraction efficiency.
3.2 Effect of co-solvent percentages
In Figure 4 the OECs are plotted to evaluate the effect on the extraction of total phenols at 8 MPa/
Accepted Manuscript
13.15 K, of 7.5 and 15% EtW at 4 kg/h CO 2 flow rate, and 10 and 15% EtW at 6kg/h CO 2 flow
rate. By the comparison of the OECs, it is possible to observe that the highest values of phenols
extraction are obtained at 4CO 2 -7.5%EtW flow rate (2600 mg GAE 100 g DM -1 ) and 6CO2-10%EtW-
flow rate (2527 mg GAE 100 g -1 DM ).
Carbon dioxide, ethanol and water are solvents with different polarities that, when mixed in
different proportions allow to obtain homogeneous solvent mixtures, depending on the particular
temperature and pressure and the individual solvents molar fractions. Table 2 reports the individual
solvent molar fractions calculated at 4 and 6 kg/h CO 2 flow rate with different percentage of the
ethanol-water mixture at 57% (v/v) ethanol as co-solvent. It is worth to note that the highest
7
Page 7 of 22

extraction of phenols are obtained at lowest co-solvent percentages used, 4CO 2 -7.5%EtW and
6CO2-10%EtW flow rates, when the molar fractions of water and ethanol are the lowest and those
of carbon dioxide the highest. This could be due to two main factors: a) the increase of co-solvent
percentage to 15% may induce the saturation of CO 2 with ethanol-water, with consequent formation
of two or three phases for the specific conditions of temperature and pressure of the system [26, 27
]; b) solvent-solvent interactions in competition for the solvation of solutes occur at 15 % cosolvent,
reducing extraction and decreasing the process yield.
3.3 Yield, fractionation of proanthocyanidins and antioxidant activity
Chemical composition of grape marc extracts obtained by conventional methanol extraction and
SC-CO 2 at 4CO 2 -7.5%EtW and 6CO2-10%EtW flow rate is reported in Table 3. The process
efficiency is quantitatively related to extraction yield. The global yields of SC-CO 2 at 4CO 2 -
7.5%EtW and 6CO2-10%EtW flow rates are 13.1 and 14.6 % w/w, respectively, lower than
classical extraction with methanol, which was 15.6% w/w. The results obtained indicate that both
SC-CO 2 modified with EtW and methanol extract a large number of soluble compounds.
-
The highest value of phenols concentration is found for methanol extraction (2813 mg GAE 100 g DM 1 ). Pinelo et al. [18] reported methanol as the most selective organic solvent for extracting phenolic
compounds from grape marc. However, it is interesting to note that phenols extracted by SC-CO 2 ,
both at 6CO2-10%EtW (2527 mg GAE 100 g -1 DM ) and 4CO 2 -7.5%EtW (2600 mg GAE 100 g -1 DM )
flow rate give yields, ranging about 90-92% of methanol extraction yield.
Phenolic yields resulted similar to that (3169 mg GAE 100 g -1 DM ) reported by Aliakbarian et al. [28]
using subcritical water, and much higher (198.4 and 173.1 g kg -1 of extract, respectively at4CO 2 -
7.5%EtW and 6CO2-10%EtW flow rate) than 23 g kg -1 of extract reported by Farìas-Campomones
et al [16] .
The highest total antioxidant activity for grape marc extracts is obtained at 6CO2-10%EtW flow
rate (8703 mg α–tocopherol 100 g DM -1 ). Under these operating conditions, total antioxidant activity
Accepted Manuscript
increased by 20% compared to 4CO 2 -7.5%EtW flow rate (7187 mg α–tocopherol 100 g DM -1 ) and
it is about 13- folds that of methanol extract (678 mg α–tocopherol 100 g DM -1 ). This suggests that
different CO 2 flow rate and percentage of co-solvent affect the SC-CO 2 extraction of phenols
responsible for the antioxidant activity of the extracts, as well as the extraction methods.
Both the extracts obtained at 6CO2-10%EtW (703.7 mg catechin 100 g -1 DM ) and 4CO 2 -7.5%EtW
(630.2 mg catechin 100 g -1 DM ) flow rate, compared to methanol extract (159.0 mg catechin 100 g -1 DM )
present high level of total proanthocyanidins (PAs).
8
Page 8 of 22

However, it is interesting to note that at 6CO2-10%EtW flow rate the amount of monomeric
fraction of PAs, as well as its contribution to the total antioxidant activity of grape marc extract, is
about 2-folds than at 4CO 2 -7.5%EtW , while the amount of oligomeric fraction results slightly
higher and the amount of the polymeric fraction lower. This suggests that supercritical fluid
extraction of PAs from grape marc carried out at 8 MPa, 313.15 K using 6 kg/h CO 2 flow rate plus
10% EtW is more selective in extracting proanthocyanidins fractions beneficial for human health
than 4 kg/h CO 2 flow rate plus 7.5% EtW. It is possible to observe from Table 2 that polymeric
fractions of PAs always show the highest antioxidant activity. This can be attributed to the structure
of polymeric flavan-3-ols characterized by the presence of several hydroxyl functions exhibiting a
higher ability to donate a hydrogen atom and to support the unpaired electron as compared to the
low molecular weight phenols [29]. Finally, comparing supercritical carbon dioxide extraction to
methanol extraction, it is worth to note that about 60% of the total antioxidant activity results
explained by PAs in SFE, and 97% in the conventional extraction. This indicates that the
supercritical operating conditions developed are able to extract not only selectively the PAs, but a
great amount of other antioxidant compounds, not extractable with the conventional method.
Conclusions
Supercritical fluid extraction using CO 2 as solvent at 6 kg/h CO 2 flow rate and 10% ethanol-water
mixture (57% v/v) as co-solvent at 313.15 K and 8MPa, proved to be an efficient extraction
methodology to achieve grape marc extracts rich in PAs.
CO 2 flow rate and co-solvent concentration affected extraction kinetics, extraction yields, and
composition and antioxidant activity of extracts. At constant CO 2 flow rate (4 and 6 kg/h), low
concentration of ethanol-water co-solvent (7.5 and 10%) favored the extraction of phenolic
compounds with high antioxidant activity. The most remarkable obtained results are the
supercritical operating conditions developed, able to extract selectively the PAs and to obtain a
Accepted Manuscript
great amount of other antioxidant compounds, not extractable with the conventional method.
Acknowledgements
The authors wish to thank "AGER - Agroalimentare e Ricerca", for financial support of this
investigation. Project AGER, grant n° 2011-0283"
References
[1] M.D. Bourzeix, D. Weyland, N. Heredia, Etude des catechins et des procyanidols de la grappe
de raisin, du vin et d’autres derives de la vigne, Bulletin de l’OIV 669-670 (1986) 1171-1254
9
Page 9 of 22

[2] J.M. Souquet, V. Cheynier, F. Brossaud, M. Moutounet, Polymeric proanthocyanidins from
grape skins, Phytochemistry 43 (1996) 509-512
[3] S.B. Lotito, L. Actis-Goretta, M.L. Renart, M. Caligiuri, D. Rein, H.H. Schmitz, F. M.
Steinberg, C.L. Keen, C.G. Fraga, Influence of oligomer chain length on the antioxidant activity
of procyanidins, Biochemical and Biophysical Research Communications 276 (2000) 945-951
[4] P. Cos, T. De Bruyne, N. Hermans, S. Apers, D.V. Berghe, A.J. Vlietinck, Proanthocyanidins in
health care: current and new trends, Current Medicinal Chemistry 11 (2004) 1345-59
[5] G. Brunner, Gas extraction: An introduction to fundamentals of supercritical fluids and the
application to separation processes. H. Baumgurtel , H. Franck , E.U. (Eds.), Topic in physical
chemistry Springer, New York, 1994
[6] A.M. Gomez, C.P. Lopez, E.M. De la Ossa, Recovery of grape seed oil by liquid and
supercritical carbon dioxide extraction: a comparison with conventional solvent extraction,
Chemical Engineering J. 61 (1996) 227–231
[7] H. Sovová, M. Zarevucka, M. Vacek, K. Stransky, Solubility of two vegetable oils in
supercritical CO 2, The J. of Supercritical Fluids 20 (2001) 15–28.
[8] L. Fiori, Grape seed oil supercritical extraction kinetic and solubility data: critical apprOECh
and modeling, The J. of Supercritical Fluids 43 (2007) 43–54.
[9] C.P. Passos, R.M. Silva, F.A. Da Silva, M.A. Coimbra, C.M. Silva, Enhancement of the
supercritical fluid extraction of grape seed oil by using enzymatically pre-treated seed, The J. of
Supercritical Fluids 48 (2009) 225–229.
[10] C.P. Passos, R.M. Silva, F.A. Da Silva, M.A. Coimbra, C.M. Silva, Supercritical fluid
extraction of grape seed (Vitis vinifera L.) oil. Effect of the operating conditions upon oil
composition and antioxidant capacity, Chemical Engineering J. 160 (2010) 634-640.
[11] R. Murga, R. Ruiz, S. Beltran, J.L. Cabezas, Extraction of natural complex phenols and tannins
from grape seeds by using supercritical mixtures of carbon dioxide and alcohol, J. Agriculture
Food Chemistry 48 (2000) 3408–3412.
Accepted Manuscript
[12] L. Casas, C. Mantell, M. Rodríguez, M., E. J. M. D. L. Ossa, A. Roldán, I.D. Ory, Extraction
of resveratrol from the pomace of Palomino fino grapes by supercritical carbon dioxide, J. of
Food Engineering 96 (2010) 304–308.
[13] M. Pinelo, R.A. Ruiz, J. Sineiro, F. J. Senorans, G. Reglero, M.J. Nunez, Supercritical fluid
and solid–liquid extraction of phenolic antioxidants from grape pomace: A comparative study.
European Food Research Technology 226 (2007) 199–205.
10
Page 10 of 22

[14] K. Ghafoor, J. Park, Y.H. Choi, Optimization of supercritical fluid extraction of biOECtive
compounds from grape (Vitis labrusca B.) peel by using response surface methodology, Food
Science Emerging Technology 11 (2010), 485–490.
[15] E.E. Yilmaz, E.B. Ozvural, H. Vural, Extraction and identification of proanthocyanidins from
grape seed (Vitis Vinifera) using supercritical carbon dioxide, The J. of Supercritical Fluids 55
(2011), 924–928
[16] A.M. Farias-Campomanes, M.A. Rostagno, A.M. Meireles, Production of polyphenol extracts
from grape bagasse using supercritical fluids: Yield, extract composition and economic
evaluation, The J. of Supercritical Fluids 77 (2013) 70-78
[17] N.P. Povh, M.O.M. Marques, M.A.A. Meireles, Supercritical CO 2 extraction of essential oil
and oleoresin from chamomile (Chamomilla recutetia L. Rauschert), The J. of Supercritical
Fluids 21 (2001) 245-256
[18] M. Pinelo, M. Rubilar, M. Jerez, J., Sineiro, M.J. Nunez, Effect of solvent to-solid ratio on the
total phenolic content and antiradical ativity of extracts from different components of grape
marc, J. Agriculture Food Chemistry 53 (2005) 2111-2117.
[19] Y. Yilmaz, R.T. Toledo, Oxygen radical absorbance capacities of grape/wine industry
byproducts and effect of solvent type on extraction of grape seed polyphenols, J. of Food
Composition and Analysis 19, (2006), 41-44
[20] D.P. Makris, G. Boskou, N.K. Andrikopoulos, Recovery of antioxidant phenolics from white
vinification solid by-products employing water/ethanol mixtures, Bioresource Techology 98
(2007) 2963-2967.
[21] L. Yu, J. Perret, M. Harris, J. Wilson, S. Haley, Antioxidant properties of bran extracts from
‘‘Akron’’ wheat grown at different locations, J. Agriculture Food Chemistry 51 (2003) 1566–
1570
[22] B. Sun, P. Belchior, M.J. Ricardo-da-Silva, M. I. Spranger, M.I., Isolation and purification of
dimeric and trimeric procyanidins from grape seeds, J. Chromatography A. 841 (1999) 115-121.
Accepted Manuscript
[23] J. C. Espìn, C. Soler-Rivas, H.J. Wichers, Characterization of the total free radical scavenger
capacity of vegetable oil fractions using 2,2-diphenyl-1-picrylhydrazyl radical, J. Agriculture
Food Chemistry 48 (2000) 648-656
[24] G. Pereira, M.A. Meireles, Supercritical fluid extraction of bioactive compounds:
fundamentals, application and economic perspectives. Food and Bioprocess Technology 3(2010)
340-372].
[25] H. Sovová, Mathematical model for supercritical fluid extraction of natural products and
extraction curve evaluation., The J. of Supercritical Fluids 33 (2005) 35-52.
11
Page 11 of 22

[26] J.H. Yoon, H. Lee, B.H. Chung BH, High pressure three-phase equilibria for the carbon
dioxide-ethanol-water system, Fluid Phase Equilibria 102 (1994) 287-292
[27] S.J. Yao, Y.X. Guan, Z.Q. Zhu, Investigation of phase-equilibrium for ternary-systems
containing ethanol, water and carbon dioxide at elevated pressure, Fluid Phase Equilibria 99
(1994), 249-259
[28] B. Aliakbarian, A. Fathi , P. Perego, F. Dehghani, Extraction of antioxidants from winery
wastes using subcritical water, The J. of Supercritical Fluids J. Sup. Fluids 65 (2012) 18-24
[29] N. Saint-Cricq de Gaulejac, C. Provost, N. Vivas, Comparative study of polyphenols
scavenging activities assessed by different methods, J. Agriculture Food Chemistry J. Agric.
Food Chem. 47 (1999), 425-431
Accepted Manuscript
12
Page 12 of 22

*Graphical Abstract (for review)
Accepted Manuscrip
Page 13 of 22

*Highlights (for review)
Highlights
CO 2 flow rates modified with 15% EtOH-W (57% v/v)
Effect of pressure on phenols extraction studied at 8-30MPa/313.15 K
Effect of co-solvent percentage studied at 7.5, 10 and 15%
High phenols yield and antioxidant activity at low co-solvent percentage
High selectivity for proanthocyanidins fractions at 6 kg/h CO2-10% EtOH-W
Accepted Manuscript
Page 14 of 22

Figure(s)
Figure 1.
Accepted Manuscript
Page 15 of 22

Figure(s)
Figure 2.
2000
1800
1600
1400
mg GAE/100 g DM
1200
1000
800
600
400
200
0
8 MPa
10 MPa
20 MPa
30 MPa
0 50 100 150 200 250 300
Time (min)
Accepted Manuscript
Page 16 of 22

Figure(s)
Figure 3.
3000
2500
2000
mg GAE/100 g DM
1500
1000
500
0
Accepted Manuscript
4CO2-15%EtW
6CO2-15%EtW
0 50 100 150 200 250 300 350
Time (min)
Page 17 of 22

Figure(s)
Figure 4.
3000
2500
2000
mg GAE /100 g DM
1500
1000
500
0
6CO2-10%EtW
6CO2 -15%EtW
4CO2 -7.5% EtW
4CO2-15% EtW
0 50 100 150 200 250 300 350
Time (min)
Accepted Manuscript
Page 18 of 22

Figure(s)
Figure captions
Figure 1. SFE pilot plant flow sheet. (B 1 ) storage tank; (E 1 ) Extraction vessel; (S 1 ,S 2 ) Separators;
(H#) Heater exchangers; (C 1 ) Condenser; (HV#) Hand valves; (MV 1 ) membrane valve; (NVR#) No
return valves; (P) Diaphragm pumps; (F 1 ) Flowmeter; (M#) Manometers; (k) Safety devices; (FL 1 )
Coriolis mass flowmeter; (D) Co-solvent storage tank; (X#) Mixer
Figure 2. Extraction curves of polyphenols from grape marc at 313.15 K, 6 kg/h CO 2 flow rate
modified with 15% ethanol-water.
Figure 3. Overall extraction curves of grape marc extracted at 8 MPa/313.15 K, 4 and 6 kg/h CO 2
flow rate modified with 15% ethanol-water.
Figure 4. Overall extraction curves for the SFE of grape marc at 8 MPa/ 313.15 K, 4 kg/h CO2
flow rate modified with 7.5 and 15% ethanol-water and 6 kg/h CO 2 flow rate modified with 10 and
15% ethanol-water.
.
Accepted Manuscript
Page 19 of 22

Table(s)
Table 1. Kinetic parameters calculated from the adjustment of the OACs for the diffusioncontrolled
period (DC)
Flow rate
4 kg/h CO2-15% EtW 6 kg/hCO2-15% EtW
t DC (min) 150 150
M DC (kg s -1 ) 2.69 x 10 -7 1.96 x 10 -7
Y DC (kg extract kg CO 2 -1 ) 2.42 x 10 -4 1.76 x 10 -4
R DC (mg GAE 100 g DM -1 ) 2428 1768
Accepted Manuscript
Page 20 of 22

Table(s)
Table 2. Individual solvent molar fractions calculated at different flow rates.
Accepted Manuscript
Page 21 of 22

Table(s)
Table 3. Chemical composition of grape marc extracts obtained by methanol and SFE extraction methods
Classical Extraction
Methanol 4 kg/h CO 2 -7.5% EtW 6 kg/h CO 2 -10% EtW
Global Yield (% w/w) 15.6 ± 1.2 13.1 ± 0.9 14.6 ± 1.5
Total Phenols (mg GAE 100 g -1 DM ) 2813 ± 10.8 2600 ± 9.8 2527 ± 11.5
Phenolic Yield (g GAE kg -1 DM ) 180.3 ± 0.4 198.4 ± 0.7 173.1 ± 0.5
Phenolic Yield (% SFE/methanolic yield) 100 92 90
Total Antioxidant Activity (mg α-tocopherol 100 g -1 DM ) 678 ± 15.5 7187 ± 16.9 8703 ± 17.5
Proanthocyanidins (mg catechin 100 g -1 DM )
Monomeric fraction (FI) 1.2 ± 0.2 88.9 ± 2.2 188.0 ± 3.8
Oligomeric fraction (FII) 4.1 ± 0.1 99.6 ± 2.6 154.2 ± 5.8
Polymeric fraction (FIII) 153.7 ± 0.2 441.7 ± 3.6 361.5 ± 18.6
Proanthocyanidins (%)
Monomeric fraction (FI) 1 14 27
Oligomeric fraction (FII) 3 16 22
Polymeric fraction (FIII) 97 70 51
Antioxidant Activity (mg α-tocopherol 100 g -1 DM )
Monomeric fraction (FI) 28.1 ± 1.2 351.2 ± 9.6 808.7 ± 10.2
Oligomeric fraction (FII) 30.1 ± 2.4 393.5 ± 6.4 545.8 ± 7.3
Polymeric fraction (FIII) 600.5 ± 2.9 3587.1 ± 7.2 3675.5 ± 6.8
Accepted Manuscript
SC-CO 2 Extraction
Antioxidant Activity (% on total AA)
Monomeric fraction (FI) 4 5 9
Oligomeric fraction (FII) 4 5 6
Polymeric fraction (FIII) 88 50 42
Page 22 of 22