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ORIGINAL PAPER
Extraction Kinetics of Phenolics from Carob (Ceratonia siliqua L.)
Kibbles Using Environmentally Benign Solvents
Melita Cavdarova •Dimitris P. Makris
Received: 18 October 2013 / Accepted: 17 February 2014
Springer Science+Business Media Dordrecht 2014
Abstract A series of non-toxic and environmentally
benign solvent systems, composed of water/ethanol, acid-
ified with either acetic or citric acid, were tested for their
efficiency in extracting polyphenols from dried carob kib-
bles. The best solvent system (30 % ethanol) was then used
in a kinetic study, attempted by deploying non-linear
regression. The leaching of polyphenols was found to obey
2nd-order kinetics, while the correlation of the total poly-
phenol yield at saturation with temperature obeyed a
3-parameter exponential growth model. The highest yield
of total polyphenols at saturation was 53.76 mg gallic acid
equivalents per gram of dry weight, achieved at 60 C. The
analysis of this extract using liquid chromatography–diode
array–mass spectroscopy led to the tentative identification
of gallic acid, myricetin 3-O-rhamnoside, quercetin 3-O-
rhamnoside and quercetin p-hydroxy benzoyl 3-O-rham-
noside. The detection of the last compound in carob
extracts is reported for the first time.
Keywords Antioxidants Carob Kinetics Liquid
chromatography–mass spectrometry Polyphenols
Abbreviations
dw Dry weight
GAE Gallic acid equivalents
TP Total polyphenols
TRE Trolox equivalents
List of symbols
A
AR
Antiradical activity
kExtraction rate constant
T Temperature (C)
tTime (h)
Y
TP
Extraction yield in total polyphenols (mg GAE
g
-1
dw)
Y
TP(s)
Extraction yield in total polyphenols at saturation
(mg GAE g
-1
dw)
Introduction
Carob bean is the fruit of the carob tree (Ceratonia siliqua
L.), which is an endemic species of the Mediterranean
regions. The fruit is mostly used in the food industry for the
recovery of bean gum, which is a polysaccharide (galac-
tomannan) contained in the endosperm of the seeds [1,2].
Thus carob kibbles (deseeded chopped pods) may be
regarded as an agri-food waste material, which is rich in
sugars (48–56 %), but it may also contain a significant
amount of polyphenols [3,4].
Some early reports indicated that carob pod extracts might
possess high antioxidant potency [5,6] but more recent studies
also demonstrated antimicrobial effects exerted by carob pod
extracts [7]. In spite of these findings, which could presumably
be attributed to the presence of polyphenolic substances, there
have been a few detailed investigations pertaining to the
analytical polyphenolic composition of carob pods [8,9].
Furthermore, although recent efforts have focused on the
identification of parameters that might affect polyphenol
extractability from carob pods [3,10,11], critical factors of the
M. Cavdarova
Food Quality and Chemistry of Natural Products, Centre
International de Hautes Etudes Agronomiques
Me
´diterrane
´ennes, Mediterranean Agronomic Institute of Chania
(M. A. I. Ch.), P.O. Box 85, 73100 Chania, Greece
D. P. Makris (&)
School of Environment, University of the Aegean, Mitr. Ioakim
Street, 81400 Myrina, Lemnos, Greece
e-mail: dmakris@aegean.gr
123
Waste Biomass Valor
DOI 10.1007/s12649-014-9298-3
extraction process are yet to be identified. Because carob pods
have been shown to contain important amounts of gallic acid
derivatives and gallotannins, factorssuchasthesolvent
composition and temperature or extraction time might play
prominent role, as evidence by previous examinations on plant
materials with similar composition [12,13].
Ethanol is a bio-solvent, produced via fermentation of
various carbohydrate-containing raw materials. Unlike
other solvents such as methanol or acetone, ethanol is non-
toxic and can be reused following its recovery after
removal from the extract through distillation, thus gener-
ating practically zero wastes. Therefore, ethanol can be
considered as an environmentally benign solvent. On such
a consideration, the study presented herein is an approach
in evaluating the effect of temperature on the extraction of
polyphenols from pulverised carob kibbles. Following an
initial screening using various, environmentally compatible
(non-toxic) solvents, kinetics and non-linear regression
analysis were employed as a first step to define a set of
conditions that may be used for further engineering the
extraction process. Some principal polyphenols detected in
the richest extract obtained, were tentatively identified
using liquid chromatography–mass spectroscopy.
Experimental
Chemicals
All solvents used for chromatographic analyses were of
HPLC grade. Folin–Ciocalteu phenol reagent, ascorbic
acid and absolute ethanol were from Fluka (Steinheim,
Germany). Gallic acid, Trolox
and 2,20-diphenyl-pic-
rylhydrazyl (DPPH) stable radical were from Sigma
Chemical Co (St. Louis, MO, USA). Acetic acid and citric
acid were from Merck (Darmstad, Germany).
Plant Material
Dry carob kibbles (C.siliqua L.) were obtained from a
carob-processing factory (Chania, Crete). The material was
pulverised into a fine powder immediately after receipt
(Fig. 1) in a domestic blender and stored at -40 C.
Batch Extraction Procedure for Solvent Assay
Preliminary extractions were performed to assess the effi-
ciency of various solvent systems in extracting total poly-
phenols (TP) from carob pod powder. The solvent systems
used are given in Table 1. Extractions of powdered carob
pod (3 g) were performed with 50 mL of solvent in a
100-mL conical flask. The mixture was stirred at 400 rpm
with magnetic stirrer for 30 min at room temperature
(23 ±1C) and then filtered through filter paper. The
filtrate was passed through 0.45-lm syringe filters and this
solution was used for further analysis.
Extraction Procedure for the Kinetic Study
Extractions were performed as above using 30 % (v/v)
aqueous ethanol for predetermined time intervals. For the
extractions carried out at 20 C, stirring was performed in a
temperature-controlled chamber. Extractions at 40 and 60 C
were performed in a temperature-controlled water bath.
Fig. 1 Plant material
(pulverised carob kibbles) used
in this study
Waste Biomass Valor
123
Determination of the Extraction Yield in Total
Polyphenols (Y
TP
)
Analysis was carried out employing the Folin–Ciocalteu
methodology [14]. Briefly, concentration in TP of the
extracts obtained in mg gallic acid equivalents (GAE) L
-1
was calculated using gallic acid as the calibration standard
(C =951 9A
750
-1.49). Yield in total polyphenols
(Y
TP
) was expressed in mg GAE per gram of dry weight
(dw), using the following equation:
YTP mg GAE g1dw
¼ð951 A750 1:49ÞV
mð1Þ
where V is the volume of the extraction medium (mL) and
mthe dw of carob kibbles (g).
Determination of the Antiradical Activity (A
AR
)
A previously reported methodology, based on the stable
radical DPPH, was used [15]. Results were expressed as
Trolox
equivalents (mM TRE) per gram of dw.
Liquid Chromatography–Mass Spectrometry
A Finnigan MAT Spectra System P4000 pump was used
coupled with a UV6000LP diode array detector and a Finn-
igan AQA mass spectrometer. Analyses were carried out on a
Superspher RP-18, 125 92 mm, 4 lm, column (Macherey-
Nagel, Germany), protected by a guard column packed with
the same material, and maintained at 40 C. Analyses were
carried out employing electrospray ionization at the positive
ion mode, with acquisition set at collision energies of 12 and
80 eV, capillary voltage 3.5 kV, source voltage 28 V,
detector voltage 650 V and probe temperature 350 C.
Eluent (A) and eluent (B) were 2.5 % acetic acid and MeOH,
respectively. The flow rate was 0.33 mL min
-1
, and the
elution programme used was as follows: 0–5 min, 20 % B;
5–25 min, 80 % B; 25–30 min, 80 % B.
Statistical Analysis
All determinations were carried out at least in triplicate and
values were averaged and given along the standard devia-
tion (±SD). All correlations were established using non-
linear regression, at least at a 95 % significance level
(p\0.05). For all statistics, Sigma Plot
TM
12.0 and
Microsoft Excel
TM
2010 were used.
Results and Discussion
Solvent Assay
A series of acidified and non-acidified, hydro alcoholic
solvent systems were tested for their efficiency to recover
polyphenolics from carob powder (Table 1). All systems
containing 90 % ethanol were shown to yield statistically
low TP values, while their A
AR
levels were also signifi-
cantly low. Solutions with 30 and 60 % ethanol, acidified
with either acetic or citric acid, displayed comparable
yields, which were not statistically different. Thus the
solvent system composed of 30 % ethanol was chosen for
the kinetic study, since it provided extracts with both high
Y
TP
and A
AR
. This outcome is in line with recent studies,
which showed that mixtures of ethanol/water (10 %) were
the most efficient in extracting polyphenols from carob
pods, compared with aqueous acetone or methanol [4].
Kinetics of TP Extraction
The model fitted to the extraction kinetics using non-linear
regression between Y
TP
values and t(Fig. 2), was a
hyperbola described by the equation:
y¼ax
1þbx ð2Þ
For all temperatures tested, fitting was statistically signif-
icant (Table 2), suggesting that extraction yield as a
function of tcan be adequately predicted by the Eq. (2).
This equation actually describes a 2nd-order extraction
rate, as previously reported [16–18], when the boundary
conditions t=0totand Y
TP(t)
=0toY
TP(t)
are consid-
ered. The 2nd-order extraction rate might indicate that
there are two phases implicated in the leaching of poly-
phenols from the solid carob particles; first, there is a high
Table 1 Y
TP
and A
AR
values of the carob pod extracts obtained by
employing various extracting media, composed of varying amounts of
ethanol and acidified with either acetic or citric acid
Extraction media Yield and antiradical activity
% EtOH
(v/v)
Acidification Y
TP
(mg GAE g
-1
)
A
AR
(mM TRE g
-1
)
30 – 9.32 ±0.78 0.50 ±0.02
a
30 1 % Acetic acid 9.48 ±0.44 0.39 ±0.01
30 1 % Citric acid 11.30 ±0.67 0.40 ±0.03
60 – 8.37 ±0.39 0.50 ±0.01
a
60 1 % Acetic acid 8.25 ±0.70 0.35 ±0.00
60 1 % Citric acid 11.31 ±0.43 0.40 ±0.01
90 – 4.30 ±0.50
b
0.27 ±0.01
c
90 1 % Acetic acid 3.67 ±0.28
b
0.18 ±0.00
a
90 1 % Citric acid 4.44 ±0.50
b
0.20 ±0.01
a
Values represent means of triplicate determination (±SD)
Superscripted Greek letters a,band cdenote statistical difference at a
99.9, 99 and 95 % significance level, respectively
Waste Biomass Valor
123
rate of extraction, possibly owed to the most polar poly-
phenols; afterwards, the rate is significantly slowed down,
suggesting extraction of less polar substances.
These considerations were made on the concession that
(1) polyphenols leached from the solid parts into the
solution through diffusion, and (2) at saturation conditions
Y
TP
remained constant. Based on such a simplification, the
integrated law for the extraction kinetics would be:
YTP tðÞ¼Y2
TPðsÞkt
1þYTPðsÞkt ð3Þ
where Y
TP(s)
and krepresent the TP yield at saturation and
the extraction rate constant, respectively. Transformation
of Eq. (3) yields the following linearized form:
t
YTPðtÞ¼1
kY2
TPðsÞþt
YTPðsÞð4Þ
when tapproaches 0, the initial extraction rate, h, given as
Y
TP(t)
/t, is defined as:
h¼kY2
TPðsÞð5Þ
Plotting t/Y
TP(t)
versus t, would give a straight line in the
form of y =ax ?b (Fig. 3), where a =1/Y
TP(s)
and
b=1/h. Thus for each temperature tested, Y
TP(s)
and
hcould be determined graphically. Y
TP(s)
represents the
maximum (optimal) Y
TP
attained under the given set of
experimental conditions.
The non-linear regression between Y
TP(s)
and Tvalues
(Fig. 4) was shown to obey a 3-parameter exponential
growth function. This function was described by the fol-
lowing equation:
YTPðsÞ¼10:28 þ0:0308 1:1285T;
R2¼1:000;p\0:0001
ð6Þ
Thus using the Eq. (6), Y
TP(s)
can be very reliably predicted
as a function of T, within the range 20–60 C.
Time (h)
0 2 4 6 8 10121416
Y
TP
(mg GAE g
-1
dw)
0
10
20
30
40
50
60
T = 20 oC
T = 40 oC
T = 60 oC
Fig. 2 Time course of Y
TP
during the extraction of carob kibbles
using 30 % (v/v) aqueous ethanol. Extractions were carried out at 20,
40 and 60 C, under continuous magnetic stirring at 400 rpm
t (h)
0 2 4 6 8 10121416
t/Y
TP
(h g mg
-1
)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
T = 20
o
C
T = 40
o
C
T = 60
o
C
Fig. 3 Second-order extraction kinetics of TP from pulverised carob
kibbles, using 30 % aqueous ethanol. Extractions were carried out at
20, 40 and 60 C, under continuous magnetic stirring at 400 rpm
T (
o
C)
020406080
Y
TP(s)
(mg GAE g
-1
dw)
0
10
20
30
40
50
60
Measured
Fitted
Fig. 4 The 3-parameter exponential growth model representing the
dependence of Y
TP(s)
on temperature
Table 2 Y
TP(s)
values and statistical parameters (square correlation
coefficient—R
2
and pvalues) calculated after implementing non-
linear regression analysis between Y
TP
and tvalues, at the various
temperatures tested
T(C)
20 40 60
Y
TP(s)
(mg GAE g
-1
dw)
10.63 ±0.74 14.16 ±0.99 53.76 ±3.76
R
2
0.983 0.959 0.997
p0.0086 0.0210 0.0015
Regressions were established at least at a 95 % significance level
(p\0.05)
Waste Biomass Valor
123
After rearrangement of Eq. (3), the yield of the extrac-
tion in TP at any time, t, can be calculated:
YTPðtÞ¼t
1
hþt
YTPðsÞð7Þ
Combining Eqs. (5), (6) and (7):
YTPðt;TÞ¼t
1
kð10:28 þ0:0308 1:1285TÞ2þt
10:28 þ0:0308 1:1285Tð8Þ
This empirical equation represents the evolution model of
Y
TP
during extraction of polyphenols from carob kibbles
with 30 % (v/v) aqueous ethanol and provides the values
for Y
TP
at any time tand any temperature T.
Polyphenolic Composition
As can be seen in Table 2, maximum Y
TP(s)
was found for
the extraction performed at 60 C, reaching 53.76 mg GAE
g
-1
dw. This value is almost 1.1–5.8 times higher than
those reported in the literature, obtained under various
extraction conditions (Table 3). Although TP content in
carobs may vary largely during ripening [21], the unripe
fruits being very rich in polyphenols, the values reported
are likely to reflect the content of fully ripe fruits, since
unripe ones are not used for processing. Therefore the
differences observed are most possibly attributed to vari-
etal differences, as well as pre-processing of the raw
material and the extraction conditions employed.
The liquid chromatography–diode array–mass spec-
trometry (LC–DAD–MS) analysis of the optimally
obtained extract (Fig. 5) revealed the presence of gallic
acid as the predominant phenolic, while three other com-
pounds could be tentatively identified on the basis of their
mass spectra. Compound (2) exhibited a pseudo-molecular
ion at m/z =465 (Table 4), adduct with Na
?
at m/z =487
and a fragment corresponding to the aglycone at m/
z=319. This compound was assigned to myricetin 3-O-
rhamoside [8]. Likewise, compound (4) showed a pseudo-
molecular ion at m/z =449, adduct with Na
?
at m/z =471
and a fragment corresponding to the aglycone at m/
z=303. This compound was assigned to quercetin 3-O-
rhamoside. Compound (3) displayed a pseudo-molecular
ion at m/z =569 and a fragment corresponding to the
aglycone at m/z =303. The fragment at 475 was attributed
to loss of a p-hydroxy benzoyl moiety with retention of the
carboxyl group on the sugar. Therefore, this compound was
assigned to quercetin p-hydroxy benzoyl 3-O-rhamnose
(Fig. 6). However, this tentative identification is reported
Table 3 Comparative bibliographic data illustrating the efficiency of
the extraction of TP from carob kibbles, under the conditions
described in this study
Material Extraction conditions Y
TP
(mg
GAE g
-1
)
Reference
Dried carob
powder
30 % Ethanol, 60 C 53.76 This
study
Carob kibbles Boiling 80 %
methanol
13.51 [19]
Chopped dried
carob kibbles
0.1 % HCl in
methanol/acetone/
water
(60/30/10, v/v/v)
13.83 [15]
Carob kibbles 80 % Acetone 9.28 [6]
Carob kibbles Supercritical CO
2
27.10 [11]
Carob kibbles Water, 98.5 C 39.50 [3]
Roasted carob
powder
Water, 99 C 15.69 [20]
Dried ground
carob kibbles
10 % Ethanol 49.60 [4]
Fig. 5 Liquid chromatography trace of the carob kibble extract obtained at 60 C, after 15 h. Monitoring of the eluate was carried out at 270 nm.
For peak assignment see Table 4
Waste Biomass Valor
123
with reservation, since this compound has not been previ-
ously identified in carob extracts and further investigation
is needed for full structural elucidation.
Phenolics identified in carob extracts included gallo-
tannins and gallic acid glucose esters [9], as well as some
flavanols [7,11]. Gallic acid and some gallotannins along
with quercetin and myricetin rhamnosides are principal
constituents of carob pods [8]. However, derivatives of
gallic acid were not detected in this study. This discrepancy
could be attributed to the conditions under which the
optimal extract was obtained (60 C), which might have
favoured hydrolysis of gallic acid esters with glucose,
hence their absence. Moreover, the role of the solvent
composition in facilitating gallotannin hydrolysis could not
be ruled out.
Conclusions
The most important finding of this study may be summa-
rised as follows:
•A solvent system composed of 30 % ethanol was found
to extract polyphenols from dried carob pods more
efficiently than solutions containing much higher
ethanol levels. Acidification with either acetic or citric
acid did not provoke any statistically significant
difference in the extraction yield.
•Non-linear regression between Y
TP
and tvalues
revealed that polyphenol extraction obeyed 2nd-order
kinetics. On such a basis, for each temperature tested,
the saturation Y
TP
values (Y
TP(s)
) could be determined
graphically.
•Non-linear regression between Y
TP(s)
and T showed
that polyphenol extraction followed a 3-parameter
exponential growth as a function of T. The optimal
Y
TP(s)
, obtained at 60 C, was 53.76 mg GAE g
-1
dw.
•Some of the major polyphenols detected in the
optimally obtained extract were tentatively identified
Table 4 Spectral characteristics of the polyphenols tentatively
identified in the carob kibble extract obtained at 60 C, using LC–
DAD–MS
Peak
#
Rt
(min)
UV–
Vis
[M ?H]
?
Other ions Tentative
identity
1 3.92 266 171 – Gallic acid
2 14.23 256,
352
465 318, 487
[M ?Na]
?
Myricetin
3-O-
rhamnoside
3 15.41 252,
286,
366
569 303, 475 Quercetin p-
hydroxy
benzoyl 3-O-
rhamnoside
4 17.07 260,
354
449 303, 471
[M ?Na]
?
Quercetin
3-O-
rhamnoside
Fig. 6 Chemical structures of the
polyphenols tentatively identified,
using liquid chromatography–diode
array–mass spectroscopy.
Assignments: (1), gallic acid; (2),
myricetin 3-O-rhamnoside; (3),
quercetin p-hydroxy benzoyl 3-O-
rhamnoside; (4) quercetin 3-O-
rhamnoside
Waste Biomass Valor
123
as gallic acid, myricetin 3-O-rhamnoside, quercetin
3-O-rhamnoside and quercetin p-hydroxy benzoyl 3-O-
rhamnoside. Gallic acid esters with glucose, as well as
gallotannins previously reported to occur in carob pod
extracts were not detected.
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