Antioxidant activity against methanol extraction of eucheuma cotonii and e. s...
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1. ORIGINAL ARTICLE
Ultrasound-Assisted Green Extraction of Eggplant Peel
(Solanum melongena) Polyphenols Using Aqueous
Mixtures of Glycerol and Ethanol:
Optimisation and Kinetics
Katiana Philippi1
& Nikos Tsamandouras1
&
Spyros Grigorakis2
& Dimitris P. Makris1
Received: 16 November 2015 /Accepted: 17 February 2016
# Springer International Publishing Switzerland 2016
Abstract Eggplant peels were used to evaluate glycerol and ethanol for their ability to recover
polyphenolic antioxidants. The evaluation was based on optimisation by a Box-Behnken
experimental design and kinetics. The results showed that extraction with water/ethanol and
water/glycerol mixtures, under optimised conditions, afforded virtually equal yield in total
polyphenols, which was 13.40 and 13.51 mg caffeic acid equivalents per g dry weight,
respectively. The extraction kinetics revealed that diffusion of phenolics in water/glycerol
mixtures was slower (0.85× 10−12
m2
s−1
) compared with the one attained with water/ethanol
(2.23× 10−12
m2
s−1
), yet the ability of both systems to recover essentially the same levels of
total polyphenols was confirmed. The determination of total chlorogenates, total flavonoids
and total pigments indicated that water/glycerol might be a more effective solvent system, but
controversies were observed with regard to the antiradical activity and reducing power. The
analytical polyphenolic profile of both extracts was dominated by chlorogenic acid and no
major differences were recorded, a finding indicating that none of the solvent systems
displayed selectivity. The results suggested that glycerol may be an ideal candidate for use
in eco-friendly extraction processes.
Keywords Antioxidants.Eggplantpolyphenols.Glycerolasextractionsolvent.Greensolvents
. Ultrasound-assisted extraction
Environ. Process.
DOI 10.1007/s40710-016-0140-8
* Dimitris P. Makris
dmakris@aegean.gr
1
School of Environment, University of the Aegean, Mitr. Ioakim Street, Myrina 81400 Lemnos,
Greece
2
Food Quality & Chemistry of Natural Products, International Centre for Advanced Mediterranean
Agronomic Studies (CIHEAM), P.O. Box 85, Chania 73100, Greece
2. 1 Introduction
The agri-food sector generates a high burden of by-products and wastes, as a result of plant
food processing. This residual material is composed principally of rejected plant tissues,
including peels, seeds, husks etc., and may cause severe environmental problems, if not
managed properly (Santana-Méridas et al. 2012). The increasingly tighter regulations regard-
ing organic waste handling, as well as the demand for sustainable food processing procedures,
have shifted the agri-food industry towards eco-friendly strategies to improve cost-
effectiveness and meet customers’ demand (Arancon et al. 2013).
One of the higher-value options is food waste valorisation, which over the past few years
has gained a great attention as a potential alternative to the disposal of residues in landfill sites.
Valorisation of food processing residues is an intriguing concept, based on the recognition that
this waste biomass is in fact an inexpensive and abundant source rich in bioactive phytochem-
icals, which could be used in the manufacturing of high value-added products, such as food
additives, nutritional supplements, cosmetics and pharmaceuticals (Galanakis 2012). In spite
the diversity of biologically important constituents occurring in plant food wastes, particular
emphasis has been given to polyphenolic compounds, which may possess a spectrum of
beneficial properties, such as antioxidant, anti-inflammatory, cardioprotective and anticarcino-
genic (Babbar et al. 2015).
The recovery of polyphenolic substances from residues of the plant food industry has been a
major concern towards the development of highly efficient methodologies and a great deal of
research has been devoted to techniques pertaining to solid–liquid extraction. The combination of
an appropriate solvent, along with physico-chemical treatments involving microwave heating,
ultrasounds and high pressure (pressurised liquids), have become the tools of preference,
displaying high recovery yields (Baiano 2014). However, several extraction procedures devel-
oped on a laboratory scale have inherently serious shortcomings, which would preclude them
from being implemented on an industrial level, owed to the toxicity of solvents frequently used
and the need for recycling (acetone, methanol), strict control by State laws (ethanol), as well as
increased cost and questionable efficiency (pressurised liquids, supercritical fluids).
The rational exploitation of waste material with the view of recovering polyphenolic
compounds should embrace processes that generate far less or even zero further waste;
otherwise no concept of Bgreen^ or Bsustainable^ could be substantiated. Thus, research on
such a field should uphold principles pertaining to green extraction, including reduced energy,
alternative, cheap and non-toxic solvents etc., without compromising extraction yield and
extract quality (Chemat et al. 2012). This challenge, launched mainly by the cost effectiveness
and environment protection, strongly suggests that technological innovations in the direction
of utilising novel extraction media and techniques are imminent.
Glycerol is a material largely unexploited for purposes of phytochemical extraction,
although it possesses features that would match those of a green solvent, because it is non-
toxic, non-flammable, non-volatile, and inexpensive, as it is a biodiesel industry by-product.
Recent examinations support that water/glycerol mixtures may be very effective in extracting
polyphenols (Apostolakis et al. 2014; Karakashov et al. 2015a, b), yet the information
provided is rather limited to fully assess its potential with regard to a process destined for
efficient polyphenol recovery from plant material.
To this prospect, this study was undertaken to optimise extraction of polyphenolics from
eggplant peels, a food waste that possesses a range of substances that cover a spectrum of
polarities. Eggplant is a worldwide diffused vegetable and it is considered to be one of the top
K. Philippi et al.
3. ten vegetables displaying high oxygen radical absorbance capacity. Polyphenols from eggplant
fruit, mainly the skin, exhibit health benefits and several pharmacological properties besides
antioxidant activity, such as hepatoprotective, anti-inflammatory, hypolipidemic, antiallergic
and anticancer (Salerno et al. 2014). The extractions carried out were assisted by ultrasounds
(Katsampa et al. 2015) and the variables taken into consideration for the optimisation included
glycerol concentration, liquid-to-solid ratio and temperature. Extractions with ethanol were
also performed for comparison, because ethanol is the most common bio-solvent employed for
polyphenol recovery. As a further step, kinetics was performed to assess extraction with both
glycerol and ethanol quantitatively, by estimating basic kinetic parameters. The extracts
obtained were evaluated for antioxidant activity and the principal phenolics were tentatively
identified by liquid chromatography-diode array-mass spectrometry.
2 Materials and Methods
2.1 Chemicals and Reagents
Glycerol (>99 %) and absolute ethanol were from Fisher Scientific (New Jersey, U.S.A.). The
solvents used for liquid chromatography were HPLC grade. Ascorbic acid, Folin-Ciocalteu
reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), caffeic acid, and 2,4,6-tripyridyl-s-triazine
(TPTZ) were from Sigma-Aldrich (Steinheim, Germany). Ferric chloride hexahydrate and
aluminium chloride were from Acros Organics (New Jersey, U.S.A.).
2.2 Eggplant Peels
Fresh purple-skinned eggplants (Solanum melongena L.), with no apparent damages
and infections, were purchased from a local grocery store (Myrina, Lemnos). The
plant material was transferred to the laboratory and peeled manually with a sharp
cutter. The peels were placed immediately in an oven and dried at 70 °C for 48 h.
The dried peels were pulverised in a laboratory mill into a fine powder with particle
size diameter of approximately 0.2 mm. This powder was used in the extraction
processes implemented.
2.3 Ultrasound-Assisted Extraction
A suitable aliquot of dried peel powder was introduced in a 15-mL screw-cap tube, with 10 mL
of solvent of varying amounts of either aqueous glycerol or aqueous ethanol. The liquid-to-
solid ratio (RL/S) and the solvent composition (Csolv) were predetermined according to the
experimental design (Table 1). The tube was vortexed for 5 s to form slurry and the mixture
was extracted in a temperature-controlled, sonication bath (Elma P70, Singer, Germany), at
fixed sonication conditions (power of 140 W, a frequency of 37 kHz, and an acoustic energy
density (AED) of 35 W L−1
) for 90 min. The extractions were performed within a temperature
(T) range of 50–80 °C, as dictated by the experimental design (Table 1). After the completion
of the extraction, samples were centrifuged for 10 min in a table centrifugator (Hermle,
Wehingen, Germany), at 15,000 rpm. The clear centrifugate was used for further analysis.
For the kinetic assay, extractions were accomplished as described above for 90 min and
samples were obtained at predetermined intervals (5 to 90 min).
Green Solvent Extraction of Eggplant Peel Phenolics
4. 2.4 Determinations of Total Polyphenol Yield (YTP), Total Flavonoid Yield (YTFn)
and Total Pigment Yield (YTPm)
YTP was determined using the Folin-Ciocalteu reagent and expressed as mg gallic acid
equivalents (GAE) per g of dried material (Karakashov et al. 2015a, b). YTFn was determined
using the AlCl3 reagent as described previously (Karvela et al. 2009) and expressed as mg
rutin equivalents (RtE) per g of dried material. YTPm was determined following a previously
published protocol (Katsampa et al. 2015) and expressed as mg cyanidin 3-O-glucoside
equivalents (CyE) per g of dried material.
2.5 Determination of Total Chlorogenate Yield (YTCg)
An aliquot of centrifuged, clear extract was diluted 1:10 with methanol, placed in a 1-cm
quartz cell and the absorbance was obtained at 325 nm. The YTCg was determined as
chlorogenic acid equivalents (mg CGAE g−1
dw) using as MW=354 and ε= 18,130 M−1
cm−1
(Dao and Friedman 1992), as follows:
YTCg mg CGAE g‐1
dw
À Á
¼
19:53 Â A325 Â V
m
ð1Þ
where V is the volume of the extraction medium (L) and m the dry weight of the plant material
(g).
2.6 Antioxidant Assays
Ferric reducing power (PR) was estimated using the TPTZ methodology (Karakashov et al.
2015a, b). The antiradical activity (AAR) was measured with the DPPH probe, using a well-
established methodology (Karvela et al. 2012). Briefly, 0.025 mL extract was mixed with
0.975 mL DPPH solution (100 μM in methanol) and the absorbance at 515 nm was read
immediately after mixing (A515(i)) and after exactly 30 min (A515(f)). The AAR was determined
as μmol DPPH per g of dry weight, using the following equation (Alluis and Dangles 2001):
AAR μmol DPPH g‐1
dw
À Á
¼
CDPPH
CTP
 1−
A515 fð Þ
A515 ið Þ
 YTP ð2Þ
where CDPPH is the initial molar concentration of DPPH (μmol L−1
) and CTP is the total
polyphenol concentration of the extract, expressed as mg CAE L−1
.
Table 1 Experimental values and coded levels of the independent variables used for the 23
full-factorial design
Independent variables Code units Coded variable level
−1 0 1
Csolv (%) X1 0 45 90
RL/S (mL g−1
) X2 20 60 100
T (°C) X3 50 65 80
K. Philippi et al.
5. 2.7 Qualitative Liquid Chromatography-Diode Array-Mass Spectrometry
(LC-DAD-MS)
The equipment used was a Finnigan MAT Spectra System P4000 pump, coupled with a
UV6000LP diode array detector and a Finnigan AQA mass spectrometer. Analyses were
carried out as described elsewhere (Paleologou et al. 2016).
2.8 Experimental Design
A 23
-full factorial design (Box-Behnken) was used, as described previously (Paleologou et al.
2016). The three independent variables considered were Csolv (X1, varying between 0 and
90 %), RL/S (X2, varying between 20 and 100 mL g−1
) and T (X3, varying between 50 and
80 °C). Each variable was coded at three levels, −1, 0 and 1 (Table 1), according to the
following equation:
xi ¼
Xi−X0
ΔXi
; xi ¼ 1; 2; 3 ð3Þ
where xi and Xi are the dimensionless and the actual value of the independent variable i, X0 the
actual value of the independent variable i at the central point, and ΔXi the step change of Xi
corresponding to a unit variation of the dimensionless value.
Data from the experimental design were subjected to regression analysis using least square
regression methodology to obtain the parameters of the mathematical models. Analysis of
variance (ANOVA) was used to assess the significance of the model. Contour plots were
obtained using the fitted model.
2.9 Statistical Analyses and Extraction Kinetics
All extractions were carried out twice and all determinations in triplicate. Values reported are
averages. Response surface design and associated statistics were performed with JMP™ 10.
Kinetics was estimated by non-linear regression between YTP and t, using SigmaPlot™ 12.0,
at least at a 95 % significance level.
3 Results and Discussion
3.1 Extraction Optimisation
The experimental screening aimed at evaluating the effect of Csolv, RL/S and T on the extraction
process. The values of the response (YTP) determined experimentally were analysed by
multiple regression and by omitting the non-significant factors (p 0.05), the mathematical
models obtained are shown in Table 2. The significance of model fitting was evaluated using
the square coefficient of correlation (R2
), which was greater than 0.88 (p≤0.0339). This
finding suggested a satisfactory match between observed and predicted responses, and that
both models given in Table 2 can reliably predict the set of the experimental conditions that
optimized the response. Values of the independent process variables (X1, X2 and X3), as well
as measured and predicted values for the response are analytically given in Tables 3 and 4, for
Green Solvent Extraction of Eggplant Peel Phenolics
6. the extractions performed with water/glycerol and water/ethanol mixtures, respectively. The
variation in YTP as a function of simultaneous variation in the process variables were given as
contour plots (Figs. 1 and 2).
For the extractions performed with water/glycerol mixtures, the significant terms of the
model were only Csolv and RL/S, which strongly suggested that the process is largely temper-
ature-independent. On the other hand, cross term of Csolv with T, but also quadratic effects of
both Csolv and RL/S were significant in the extraction with water/ethanol mixtures. This
outcome stressed that the dependence of the extraction on the variables was considerably
different from that with water/glycerol. The use of the mathematical models enabled the
determination of the set of optimal conditions, under which the maximum YTP can be attained.
The determination was based on the maximisation of the desirability (Figs. 1 and 2).
As can be seen in Table 5, the maximum YTP of 13.51±1.85 mg CAE g−1
dw that could be
achieved using water/glycerol mixtures, would require a Csolv = 90 % (w/v), RL/S = 100 mL g−1
and T=50 °C. The pattern concerning the extraction with water/ethanol was substantially
different, requiring Csolv =40 % (v/v), RL/S =82 mL g−1
and T=80 °C, but the theoretical
maximal value of YTP (13.40±0.61 mg CAE g−1
dw) was virtually equal to that attained with
water/glycerol mixtures. These values are close to 10.03 mg GAE g−1
dw reported for
microwave-assisted extraction of eggplant peels with 50 % (v/v) ethanol (Salerno et al.
2014) and significantly higher than 7.16 mg GAE g−1
dw reported for the extraction with
50 % (v/v) ethanol (Chatterjee et al. 2013). However, much lower values of 0.14 mg GAE g−1
dw were found in eggplant peels extracts prepared with 70 % (v/v) ethanol (Boulekbache-
Makhlouf et al. 2013).
The optimal Csolv determined for the extraction with water/ethanol mixtures matched
exactly the one found for polyphenol extraction from pomegranate husks (Amyrgialaki et al.
2014) and it was also very close to 48 % (v/v) found for polyphenol extraction from Citrus
limon (Dahmoune et al. 2013) and 35 % (v/v) for ultrasound-assisted polyphenol extraction
from Laurus nobilis L. (Muñiz-Márquez et al. 2013). On the other hand, the optimal Csolv
found for the extraction with water/glycerol mixtures was 9-fold higher than the ones
previously used (Apostolakis et al. 2014; Karakashov et al. 2015a, b), but in these studies
no optimisation was carried out and thus no credible comparison could be made.
Ethanol possesses lower dielectric constant (ϵ= 25.2) than glycerol (ϵ = 42.5), hence a
lower proportion could reduce water polarity, rendering the solvent system appropriate for
enhanced polyphenol solubilisation. The differences in the optimal concentration found
between water/glycerol and water/ethanol mixtures are thus possibly attributed to their differ-
ent polarity. In any case, both glycerol and ethanol have dielectric constants lower than that of
water (Karakashov et al. 2015a, b; Bazykina et al. 2002), and aqueous mixtures containing
either solvent would dissolve higher amounts of polyphenols, most of which are rather
sparingly water-soluble (Kassing et al. 2010). Nevertheless, the influence of hydrogen bonding
Table 2 Polynomial equations and statistical parameters describing the effect of the independent variables
considered on the response (YTP), calculated after implementation of a central composite experimental design
Response Polynomial equation R2
p
Water/glycerol 11.30 + 1.03Csolv + 1.08RL/S 0.88 0.0339
Water/ethanol 12.18–1.08Csolv + 0.91RL/S + 0.32CsolvT–2.98Csolv
2
–0.64RL/S
2
0.99 0.0001
K. Philippi et al.
8. and steric effects, which may be considerably implicated in the solubility of polyphenols in
water/glycerol and water/ethanol mixtures, should not be overlooked (Galanakis et al. 2013).
Fig. 1 Contour plots and desirability function, describing the effect of the three independent variables considered
(Csolv, RL/S, T) on the YTP, upon simultaneous variation. Data obtained using water/glycerol mixtures, under
sonication (140 W, 37 kHz, 35 W L−1
), for 90 min
Fig. 2 Contour plots and desirability function, describing the effect of the three independent variables considered
(Csolv, RL/S, T) on the YTP, upon simultaneous variation. Data obtained using water/ethanol mixtures, under
sonication (140 W, 37 kHz, 35 W L−1
), for 90 min
K. Philippi et al.
9. Differences were also seen with regard to the optimal RL/S and T. Since solid–liquid
polyphenol extraction is a process governed by diffusional phenomena, it could be argued
that the differences observed reflected the influence of these variables (RL/S and T) on the
diffusion rate of polyphenols from the solid particles into the liquid phase. The general
mathematical expression that correlates viscosity (η), diffusion (D) and temperature (T) is
the Stokes-Einstein equation (Karakashov et al. 2015b):
D ¼
kBT
6πηrs
ð4Þ
where kB is the Boltzmann’s constant, rs the effective radius of the diffusing molecule and η the
viscosity. Equation (7) dictates that the higher the η, the lower the D. Glycerol is much more
viscous than ethanol (d =1.261 and 0.789 g cm−3
at 25 °C, respectively), and therefore, the
higher RL/S determined for the extraction with water/glycerol could be justified. It has been
supported that higher RL/S promotes higher extraction yield, because during mass transfer, the
concentration gradient between the solid and the bulk of the liquid is greater when a higher
solvent-to-solid ratio is used (Rakotondramasy-Rabesiaka et al. 2010). If the amount of the
dispersed phase is not adequately lower compared with the liquid phase, then there may be a
non-negligible resistance to mass transfer. Thus higher RL/S could putatively compensate for
the slow-down of the extraction, due to increased viscosity.
On the other hand, the higher optimal temperature found for the extraction with water/
ethanol mixtures would appear paradox. Based on the Eq. (4), it would be reasonably
anticipated that the efficient extraction with water/glycerol mixtures would require significant-
ly higher temperature, to compensate for the increased viscosity. To explain such a phenom-
enon, in addition to solubility effects ascribed to the polarity of the water/glycerol mixture, as
discussed above, the effect of ultrasonication at this point should also be considered. Ultrasonic
power is known to provoke voids in a liquid, characterised as cavitation bubbles, which are
responsive of the ultrasonic effect. During ultrasonication, these bubbles are able to grow up to
a critical point, beyond which they collapse releasing large amounts of energy. The combina-
tion of high temperature/high pressure involved in such a process disrupts the solid particle
integrity, resulting in the release of the solute in the liquid phase (Chemat and Khan 2011).
Liquids with high vapour pressure, such as 40 % (v/v) ethanol cavitate at lower intensity
and it would be expected that ultrasonication would be more efficient with this solvent system,
giving higher YTP. The fact that maximum YTP with water/ethanol was recorded at 80 °C
would be in concurrence, because cavitation bubbles are more easily produced as temperature
raises. Nevertheless, the effects resulting from cavitational collapse are also reduced as
temperature increases. In other words, lower temperatures and solvents with low vapour
pressure (water/glycerol) are required to get the maximum sonochemical benefit (Mason and
Lorimer 2002). This is presumably the reason why 90 % (w/v) glycerol at 50 °C was as
effective as 40 % (v/v) ethanol at 80 °C, in spite of the high difference in viscosity.
Table 5 Optimal, predicted conditions and theoretically calculated maximal values for the response (YTP)
Solvent system Maximal predicted value (mg CAE g−1
dw) Optimal conditions
Csolv (%) RL/S (mL g−1
) T (°C)
Water/glycerol 13.51 ± 1.85 90 100 50
Water/ethanol 13.40 ± 0.61 40 82 80
Green Solvent Extraction of Eggplant Peel Phenolics
10. 3.2 Kinetic Assay
Too ascertain the above hypothesis, a kinetic assay was conducted to produce quantitative data
concerning the rapidity and efficacy of the extractions performed under the conditions
presented in Table 5. Correlation of YTP values with t using non-linear regression was highly
significant (R2
0.996), pointing to a second-order model (Cavdarova and Makris 2014;
Tzima et al. 2014) (Fig. 3):
YTP tð Þ ¼
Y2
TP sð Þkt
1 þ YTP sð Þkt
ð5Þ
YTP(s) and k represent the TP yield at saturation and the extraction rate constant, respec-
tively. Equation (5) may be transformed as follows:
t
YTP tð Þ
¼
1
kY2
TP sð Þ
þ
t
YTP sð Þ
ð6Þ
When t approaches 0, the initial extraction rate, h, given as YTP(t)/t, can be determined as:
h ¼ kY2
TP sð Þ ð7Þ
A plot of t/YTP(t) against t yields a straight line with a slope=1/YTP(s) and intercept=1/h
(Fig. 4). Hence YTP(s) and k could be determined graphically (Table 6). Correlation of YTP and
t (min)
0 20 40 60 80 100
YTP
(mgCAEg-1
dw)
0
2
4
6
8
10
12
14
90% (w/v) glycerol, RL/S = 100 mL g
-1
, 50
o
C
40% (v/v) ethanol, RL/S = 82 mL g-1
, 80 o
C
Fitted model
Fig. 3 Non-linear regression between YTP and t values, during extraction of TP from dried EPP, using water/
glycerol and water/ethanol mixtures. Extractions were carried under sonication (140 W, 37 kHz, 35 W L−1
), for
90 min
K. Philippi et al.
11. the effective diffusion of the solute (polyphenols) can be given by the equation below
(Karakashov et al. 2015a; Pinelo et al. 2008):
YTP tð Þ
YTP sð Þ
¼ 1−
6
π2
X ∞
n¼1
1
n
e−Den2π2t
r2
ð8Þ
where De is the effective diffusion coefficient (m2
s−1
) and r the radius of the solid particles.
After the extraction has proceeded for beyond the initial stage (washing), only the first term of
the series solution becomes significant, hence:
1−
YTP tð Þ
YTP sð Þ
¼
6
π2
e−Deπ2t
r2
ð9Þ
The linearised form of Eq. (9) would be:
ln
YTP sð Þ
YTP sð Þ− YTP tð Þ
¼ ln
π2
6
þ
Deπ2
t
r2
ð10Þ
t (min)
0 20 40 60 80 100
t/YTP(mingmg-1
)
0
2
4
6
8
10
90% (w/v) glycerol, RL/S = 100 mL g-1
, 50 o
C
40% (v/v) ethanol, RL/S = 82 mL g-1
, 80 o
C
Fitted line
Fig. 4 Second-order kinetics of TP extraction from dried EPP using water/glycerol and water/ethanol mixtures.
Extractions were carried under sonication (140 W, 37 kHz, 35 W L−1
), for 90 min
Table 6 Parameters of second-order kinetics, determined for the extraction of TP from EPP, using the two
different extraction media and the corresponding optimal conditions, as given in Table 5
Extraction medium Kinetic parameters
k (g mg−1
min−1
) De (m2
s−1
) × 10−12
YTP(s) (mg CAE g−1
dw)
Water/glycerol 0.107 0.85 11.67
Water/ethanol 0.326 2.23 12.80
Green Solvent Extraction of Eggplant Peel Phenolics
12. If ln
YTP sð Þ
YTP sð Þ− YTP tð Þ
is plotted as a function of t, then De can be determined graphically, from
the slope of the straight line (slope ¼ Deπ2
r2 ) (Table 6). The points on the plot are scattered along
two intersecting straight lines. Because the first line corresponds to the fast stage of extraction
(washing), which can be considered as the non-limiting stage, De coefficients were estimated
by determining the slope of the slow stage of diffusion process (line with the shallow slop),
which might be more indicative for the effect of the solvents tested and the conditions
employed.
The k values determined for the extraction with water/glycerol and water/ethanol were
0.107 and 0.326 g mg−1
min−1
, respectively. This outcome pointed to a faster extraction rate
for the latter solvent system (Table 6). Furthermore, k determined for the water/glycerol
mixture was almost 9- to 34-fold fold higher than those determined for polyphenol extraction
from other plant material, using water/glycerol mixtures (Karakashov et al. 2015a, b;
Apostolakis et al. 2014). De for water/glycerol and water/ethanol were 0.85×10−12
m2
s−1
and 2.23× 10−12
m2
s−1
, respectively, indicating that polyphenol diffusion with the latter
solvent system is significantly faster. De determined for the water/glycerol system were
comparable to 0.14–1.6× 10−12
m2
s−1
for extraction of lignans from flaxseed (Ho et al.
2008) and 1.1×10−12
m2
s−1
for polyphenols extraction from red grape pomace with 50 %
ethanol (Pinelo et al. 2005).
The data discussed above clearly illustrate that polyphenol diffusion in the water/ethanol
system was faster compared with water/glycerol. However, the kinetic study demonstrated that
YTP(s) achieved with either solvent system displayed no statistical difference (Table 6) and that
they were virtually equal to those estimated by the mathematical models derived from the
response surface methodology, since they fell within the limits of standard deviation (Table 5).
3.3 Composition and Antioxidant Properties
In order to have a deeper insight into the extractability of the major classes of polyphenols that
occur in EPP, as well as on the antioxidant characteristics, the extracts obtained under optimal
Csolv, RL/S and T conditions, following a 90-min extraction, were analysed with regard to the
yield in total chlorogenates (YTCg), total flavonoids (YTFn) and total pigments (YTPm), but also
antiradical activity (AAR) and reducing power (PR). The two extracts exhibited marginal and
non-significant differences in YTCg (Fig. 5, upper plot), the values being 9.43±0.93 and 9.12
±0.07 mg CGAE g−1
dw, for the water/glycerol and water/ethanol extract, respectively. To the
contrary, the corresponding values were 5.86±0.00 and 3.39±0.08 mg RtE g−1
dw for YTFn,
and 885.1 ±62.0 and 314.8±11.0 μg CyE g−1
dw for YTPm, which strongly suggested that the
water/glycerol system had higher capacity in extracting flavonoids and pigments.
On the other hand, the AAR was significantly higher in the water/ethanol extract compared
with the water/glycerol extract, the corresponding values being 72.17 ± 1.30 and 66.04
±0.00 μmol DPPH g−1
dw (Fig. 6, upper plot). Likewise, the water/ethanol extract had a PR
value of 47.51 ± 1.26 μmol AAE g−1
dw, which was significantly different than 40.48
±3.2 μmol AAE g−1
dw displayed by the water/glycerol extract (Fig. 6, lower plot). Appar-
ently, higher antioxidant activity did not coincide with higher YTCg, YTFn and YTPm. In
eggplant extracts, stronger antioxidant activity has been correlated with higher total polyphe-
nol levels (Kaur et al. 2014; Nisha et al. 2009; Somawathi et al. 2014), but there has been no
sound evidence for correlation with specific polyphenol classes. Moreover, in spite the
consolidated concept that the higher polyphenol content is accompanied by a proportionally
K. Philippi et al.
13. high antioxidant capacity, several previous investigations highlighted that correlation between
the polyphenolic content and the antioxidant activity is not always statistically significant
Fig. 5 Comparative diagrams
showing the levels of YTCg
(upper), YTFn (middle) and YTPm
(lower), following 90 min extrac-
tion, under sonication (140 W,
37 kHz, 35 W L−1
) and optimised
conditions (see Table 5)
Green Solvent Extraction of Eggplant Peel Phenolics
14. (Khiari et al. 2009; Mylonaki et al. 2008). The differences in AAR and PR may reflect
differences in the total amount of polyphenols and interactions amongst them (synergism
and/or antagonism), which may affect the antioxidant activity of the extracts (Karvela et al.
2012).
3.4 Polyphenol Identification
The LC-DAD-MS analysis showed that the composition of the extracts obtained with both
water/ethanol and water/glycerol was very similar (Fig. 7), with the predominant compound
being peak no 3 (Table 7). This peak (13.73 min) was tentatively assigned to a caffeoylquinic
acid and presumably corresponded to chlorogenic acid (5-O-caffeoylquinic acid), which is the
major eggplant phenolic (García-Salas et al. 2014). Peak 2 (13.33 min) gave almost identical
spectral characteristics, but this compound was not detected in the water/glycerol extract. It
could be postulated that this peak might represent the cis- isomer of peak 3 and this raises
concerns on plausible solvent effects. However, this assumption merits profounder investiga-
tion. Peak 1 (9.23 min) was also assigned to a caffeoylquinic acid while peak 4 was identified
Fig. 6 Comparative diagrams
showing the levels of AAR (upper)
and PR (lower), following 90 min
extraction, under sonication
(140 W, 37 kHz, 35 W L−1
) and
optimised conditions (see Table 5)
K. Philippi et al.
15. as a feroyl analogue and peak 6 (20.57 min) as a caffeoyl amide. On the other hand, peaks 5
(19.29 min) and 8 (26.03 min) were assigned to an anthocyanin (delphinidin 3-O-rutinose) and
a flavonol glycoside (kaempferol 3-O-rutinoside). All of the above mentioned constituents
have been tentatively identified in eggplant extracts by recent detailed investigations (García-
Salas et al. 2014; Sun et al. 2015).
The high similarity between the two extracts obtained under optimised conditions sug-
gested that there is no notable difference in the extraction selectivity. This is most probably
because EPP contain relatively polar phenolics, which can be very effectively extracted by the
solvents used. However, modification of selectivity by switching solvent composition is an
important issue, especially in the light of previous studies, which showed that water/glycerol
0 5 10 15 20 25 30 35 40 45 50 55
Time (min)
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
110000
120000
130000
140000
uAU
13.73
1.50
13.33
17.98
3.66 22.6312.39 15.79 18.73 58.755.9354.1926.034.24 52.689.20
50.10
0 5 10 15 20 25 30 35 40 45 50 55
Time (min)
-15000
-10000
-5000
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
60000
65000
70000
uAU
13.79
1.42
3.77 55.2354.72 55.5752.22
49.7514.319.23 13.03 18.014.76 47.42
Fig. 7 Chromatographic profiles of the water/ethanol (upper) and water/glycerol (lower) extracts, obtained
following 90 min extraction, under sonication (140 W, 37 kHz, 35 W L−1
) and optimised conditions (see
Table 5). Eluents were monitored at 320 nm
Green Solvent Extraction of Eggplant Peel Phenolics
16. mixtures might preferentially dissolve more polar compounds than water/ethanol, a phenom-
enon ascribed to solvent polarity (Apostolakis et al. 2014).
4 Conclusions
The examination herein constitutes the first analytical comparison between two green solvents,
glycerol and ethanol, with regard to their potency in recovering polyphenols under
ultrasonication. It was demonstrated that by using different combinations of extraction condi-
tions, including solvent composition, liquid-to-solid ratio and temperature, extraction of
polyphenols from eggplant peels may be equally effective, in spite of the significant differ-
ences pertaining to the extraction kinetics. Moreover, the analytical polyphenolic composition
of the extracts obtained indicated that there is no selectivity issue between the solvents used.
On the ground of these data, it can be said that a solvent such as ethanol, which has been used
in numerous investigations concerning polyphenol extraction, could be replaced by glycerol
without compromising the extraction efficiency. The lack of flammability and toxicity, the high
boiling point, the abundance and the low cost would make glycerol an ideal candidate for use
in processes where ethanol should be precluded. On the other hand, the incorporation of
glycerol extracts in cosmetics and pharmaceutical and nutraceutical formulations might be
simpler and straight forward, since glycerol is extensively used as a constituent in such
products.
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