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Extraction Kinetics of Phenolics from Carob (Ceratonia siliqua L.) Kibbles Using Environmentally Benign Solvents


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A series of non-toxic and environmentally benign solvent systems, composed of water/ethanol, acidified with either acetic or citric acid, were tested for their efficiency in extracting polyphenols from dried carob kibbles. 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 polyphenol 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-rhamnoside. The detection of the last compound in carob extracts is reported for the first time.
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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
dw Dry weight
GAE Gallic acid equivalents
TP Total polyphenols
TRE Trolox equivalents
List of symbols
Antiradical activity
kExtraction rate constant
T Temperature (C)
tTime (h)
Extraction yield in total polyphenols (mg GAE
Extraction yield in total polyphenols at saturation
(mg GAE g
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
´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
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.
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
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Determination of the Extraction Yield in Total
Polyphenols (Y
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
was calculated using gallic acid as the calibration standard
(C =951 9A
-1.49). Yield in total polyphenols
) was expressed in mg GAE per gram of dry weight
(dw), using the following equation:
YTP mg GAE g1dw
¼ð951 A750 1:49ÞV
where V is the volume of the extraction medium (mL) and
mthe dw of carob kibbles (g).
Determination of the Antiradical Activity (A
A previously reported methodology, based on the stable
radical DPPH, was used [15]. Results were expressed as
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
, 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
12.0 and
Microsoft Excel
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
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
and A
. 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
values and t(Fig. 2), was a
hyperbola described by the equation:
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 [1618], when the boundary
conditions t=0totand Y
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
and A
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
Acidification Y
(mg GAE g
(mM TRE g
30 9.32 ±0.78 0.50 ±0.02
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
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
0.27 ±0.01
90 1 % Acetic acid 3.67 ±0.28
0.18 ±0.00
90 1 % Citric acid 4.44 ±0.50
0.20 ±0.01
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
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
remained constant. Based on such a simplification, the
integrated law for the extraction kinetics would be:
YTP tðÞ¼Y2
1þYTPðsÞkt ð3Þ
where Y
and krepresent the TP yield at saturation and
the extraction rate constant, respectively. Transformation
of Eq. (3) yields the following linearized form:
when tapproaches 0, the initial extraction rate, h, given as
/t, is defined as:
Plotting t/Y
versus t, would give a straight line in the
form of y =ax ?b (Fig. 3), where a =1/Y
b=1/h. Thus for each temperature tested, Y
hcould be determined graphically. Y
represents the
maximum (optimal) Y
attained under the given set of
experimental conditions.
The non-linear regression between Y
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;
 ð6Þ
Thus using the Eq. (6), Y
can be very reliably predicted
as a function of T, within the range 20–60 C.
Time (h)
0 2 4 6 8 10121416
(mg GAE g
T = 20 oC
T = 40 oC
T = 60 oC
Fig. 2 Time course of Y
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
(h g mg
T = 20
T = 40
T = 60
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 (
(mg GAE g
Fig. 4 The 3-parameter exponential growth model representing the
dependence of Y
on temperature
Table 2 Y
values and statistical parameters (square correlation
and pvalues) calculated after implementing non-
linear regression analysis between Y
and tvalues, at the various
temperatures tested
20 40 60
(mg GAE g
10.63 ±0.74 14.16 ±0.99 53.76 ±3.76
0.983 0.959 0.997
p0.0086 0.0210 0.0015
Regressions were established at least at a 95 % significance level
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After rearrangement of Eq. (3), the yield of the extrac-
tion in TP at any time, t, can be calculated:
Combining Eqs. (5), (6) and (7):
kð10:28 þ0:0308 1:1285TÞ2þt
10:28 þ0:0308 1:1285Tð8Þ
This empirical equation represents the evolution model of
during extraction of polyphenols from carob kibbles
with 30 % (v/v) aqueous ethanol and provides the values
for Y
at any time tand any temperature T.
Polyphenolic Composition
As can be seen in Table 2, maximum Y
was found for
the extraction performed at 60 C, reaching 53.76 mg GAE
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
Dried carob
30 % Ethanol, 60 C 53.76 This
Carob kibbles Boiling 80 %
13.51 [19]
Chopped dried
carob kibbles
0.1 % HCl in
(60/30/10, v/v/v)
13.83 [15]
Carob kibbles 80 % Acetone 9.28 [6]
Carob kibbles Supercritical CO
27.10 [11]
Carob kibbles Water, 98.5 C 39.50 [3]
Roasted carob
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
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.
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
and tvalues
revealed that polyphenol extraction obeyed 2nd-order
kinetics. On such a basis, for each temperature tested,
the saturation Y
values (Y
) could be determined
Non-linear regression between Y
and T showed
that polyphenol extraction followed a 3-parameter
exponential growth as a function of T. The optimal
, obtained at 60 C, was 53.76 mg GAE g
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–
[M ?H]
Other ions Tentative
1 3.92 266 171 Gallic acid
2 14.23 256,
465 318, 487
[M ?Na]
3 15.41 252,
569 303, 475 Quercetin p-
benzoyl 3-O-
4 17.07 260,
449 303, 471
[M ?Na]
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-
Waste Biomass Valor
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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... It is mostly grown and produced in Spain, Italy, Portugal, Northern Africa, the larger Mediterranean islands, Iran, the Canary Islands, and Macao, where it is employed in the food and pharmaceutical industries because of its chemical composition [44]. Major polyphenols found in carob kibbles have been tentatively identified, including gallic acid, myricetin 3-O-rhamnoside, quercetin p-hydroxy benzoyl 3-O-rhamnoside, and quercetin 3-O-rhamnoside [45]. Every year, 50,000 tons of carob fruit are grown in Portugal's Algarve region, which makes it difficult to dispose of the waste generated by this sector [46]. ...
... The results of this research differ to some extent from previous studies where various biosorbents were applied and indicated that boron adsorption can be favored at lower or higher temperatures. For instance, the adsorption efficiency of boron onto olive bagasse decreased as temperature increased (25,45, and 55 • C), showing that the sorption process is promoted at low temperatures [37]. The opposite results were obtained by Bursali et al. (2009) for the boron sorption using marine seaweed-Caulerpa racemosa var. ...
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The release of boron into the environment as a result of anthropogenic activity modifies sustainable natural conditions, thus affecting ecosystems. To meet water quality regulations, commercial and natural boron adsorbents are available to reduce its concentrations in industrial effluents, with the former being not only more expensive but also less sustainable. In the publication, the biosorption parameters of carob kibbles (Ceratonia siliqua L.) were optimized in order to remove boron from aqueous solutions using batch experiments. The biosorbent used in the present research was agro-waste biomass provided by the local locust-beam gum industry. Boron removal by carob kibbles was favored at high initial pH values, and this capacity was found to be a function of boron initial concentration, biosorbent content in the solution, and particle size. The change in temperature did not affect the potential of biomass to remove boron. The highest boron removal efficiency (55.1%) was achieved under the following optimal conditions: 50 g/L biosorbent dose (Cs), with particle size range 0.025–0.106 mm, for the initial concentration (C0) of boron in the solution of 100 mg/L, at an initial pH of 11.5, for 5 h at 25 °C. This investigation suggests that carob kibble agro-waste can be valorized as a biosorbent to remove boron from wastewater, and the boron-loaded residue may eventually be explored as a new boron-fertilizer.
... Even though carob seeds represent only a small part of the total weight of the fruit, they are of great industrial interest, as they are used to produce locust bean gum (LBG, E410), a natural food thickening and stabilizing agent [1,3]. The pulp remaining after the industrial exploitation of the seeds, also referred to as carob kibble, is considered an agri-food waste [4,5]. Carob pulp has, so far, been used as livestock feed, while recently the worldwide market has turned its attention to carob pulp as it is considered a promising functional and nutraceutical food component [3,6,7]. ...
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In recent years, carob and its derived products have gained wide attention due to their health-promoting effects, which are mainly attributed to their phenolic compounds. Carob samples (carob pulps, powders, and syrups) were analyzed to investigate their phenolic profile using high-performance liquid chromatography (HPLC), with gallic acid and rutin being the most abundant compounds. Moreover, the antioxidant capacity and total phenolic content of the samples were estimated through DPPH (IC50 98.83–488.47 mg extract/mL), FRAP (48.58–144.32 μmol TE/g product), and Folin–Ciocalteu (7.20–23.18 mg GAE/g product) spectrophotometric assays. The effect of thermal treatment and geographical origin of carobs and carob-derived products on their phenolic composition was assessed. Both factors significantly affect the concentrations of secondary metabolites and, therefore, samples’ antioxidant activity (p-value < 10−7). The obtained results (antioxidant activity and phenolic profile) were evaluated via chemometrics, through a preliminary principal component analysis (PCA) and orthogonal partial least square-discriminant analysis (OPLS-DA). The OPLS-DA model performed satisfactorily, differentiating all samples according to their matrix. Our results indicate that polyphenols and antioxidant capacity can be chemical markers for the classification of carob and its derived products.
... The concentration of polyphenols in carob fruits ranges between 45-5376 mg Gallic-acid equivalents per 100 g and is influenced by extraction methods, environmental and genetic factors. [45] According to Novotni et al. [48] carob phenolics are covalently bonded to dietary fiber, whereas some are present as free or soluble conjugated forms. Almanasrah et al. [49] suggested that carob germ and seeds are a good source of phenolics extracted using different methods. ...
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Carob (Ceratonia siliqua) is one of Asia and Africa's popular nutritional and medicinal crops. This unique plant has an outstanding functional properties and nutritional profile. Carob has high sugar content, drought resistance and is very economical. Carob fruit consists of pulp and seed that are rich sources of different bioactive components. Carob has wide applications in various industries (food, pharmaceuticals and cosmetics) as an anti-oxidant, thickener, stabilizer, lactic acid production and emulsions. The trend of moving towards natural products further highlights the use of carob in different fields due to its excellent nutritional and therapeutic profile. Carob bean gum is widely used in the food industry. The current review has highlighted the nutritional composition, bioactive profile, functional properties, and recent findings on the subject.
... Phenolic compounds in the CP include primarily hydroxybenzoic acids, flavonols, flavan-3-ols and gallotannins. In addition, Gallic acid and its derivatives such as methyl gallate comprise the [17,34,35]. These compounds have bioactive effects and health benefits in various therapeutic areas, as well as the prevention of certain diseases such as cancer, diabetes and diarrhea. ...
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Recently, the trend of many studies to replace synthetic antifungal commonly used during storage with natural antifungal; Plant extracts has been one of the most promising strategies for this purpose. Therefore, this study aimed to evaluate the aqueous extract of the carob pulp (AECP) at 1.5, 2.5, and 5 mg/ml on the growth of some toxigenic Aspergillus species, and its effectiveness as an anti-mycotoxigenic (aflatoxins (AFs) and ochratoxin A (OTA) during storage red peanut kernels (RPKs) for 90 days. The results indicate that the increased concentration of AECP, the area of inhibition zone increased with all treatments. As well, the strain of Aspergillus ochraceus was more affected by AECP than Aspergillus aflatoxiformans followed by Aspergillus parasiticus which recorded (15.7 ± 0.4 mm), (13.4 ± 0.5 mm), and (11.9 ± 0.4 mm) at 5 mg/ml. Respectively. While, the lowest inhibition was shown with Aspergillus flavus at three concentrations of AECP. The impact of AECP on the accumulation of total AFs and OTA during storage of RPKs infected by A. parasiticus and A. ochraceus , respectively, for 90 days. The results showed that the active compounds in the AECP at 5 mg/ml can protect RPKs for 45 days without detecting any amounts of AFs and OTA. After 90 days, the inhibition percentages for the four types of aflatoxins (AFB 1 , AFG 1 , AFB 2 , AFG 2 ) and OTA compared with positive control samples were 85.1, 76.5, 86.5, 84.2, and 87.5%, respectively. Finally, AFB 1 recorded the lowest inhibition percentages of all treatments; on the contrary, it was OTA.
... Dichloromethane Acetone [39,40]. Notably, during the first hour of the extraction, there was a rapid extraction of capsaicin after the extraction slowed down and attained a steady state. ...
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Capsaicin is a valuable compound found in Capsicum annuum. The present study aimed to explore the efficiency of different solvents and surfactants on its extraction by maceration. Ethyl acetate was found to be the best solvent followed by dichloromethane and acetone, respectively. Overall order of efficiency of the solvents used was this: ethyl acetate > dichloromethane > acetone > glycerol > acetonitrile > methanol > acetic acid > toluene. Extractability of ethyl acetate for capsaicin remained unaffected by the surfactants. Tween-80 had very positive effect on the extraction efficiency of dichloromethane (DCM) and acetone. Kinetics of the extraction with the most efficient solvent ethyl acetate showed extraction of capsaicin to follow a pseudo-second order kinetic model. In conclusion, for extraction of capsaicin from green chili, ethyl acetate was the most powerful amongst the solvents used in the present work and tween-80 had a notable positive effect on the efficiency of DCM and acetone.
... Nonetheless, the different results (ash content) of carob powder could be affected by the location of its cultivation and other factors such as mineral content of the soil, climate conditions, and variety of carob (cultivated and wild species). These influencing factors are supported by [41], [42], who highlighted that the nutritional compounds of carob products are impacted by the genetic profile, environmental conditions, and other cultivating factors. Hence, the 46.1% margin of ash content of WCP to the control sample could be due to the high mineral content of cocoa beans, especially in phosphorus, potassium, magnesium [43]. ...
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This study used Cypriot Wild Carob Powder to serve as an alternative for cocoa powder. The study conducted various physiochemical experiments, encompassing milling yield, water activity, colour analysis and proximate analysis. We noted that the milling yield, water activity, and dietary fibre content of WCP are higher than cocoa powder. Conversely, cocoa powder had moisture content (2.1 ± 0.37), ash (3.42 ± 0.05), protein (4.66 ± 0.78), fat (not detected), carbohydrate (46.7 ± 0.87) and energy (205 Cal), that were higher than WCP (6.56 ± 0.24), (4.63 ± 0.03), (24.3 ± 0.66) & (14.5 ± 0.15), (426 Cal) respectively. Hence, it is evident that the high dietary fibre and low energy values of WCP make it a suitable substitute for cocoa powder-based products to alleviate the concern of obesity.
... The extraction kinetics data were approximated by a second-order model according to [29,30]. Such an approach was successfully applied to describe extraction kinetics in NADES [31]. ...
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Chamaenerion angustifolium (L.) Scop. (fireweed) is a perennial herbaceous plant of the Onagraceae family widely used in folk and scientific medicine. It is a promising source of bioactive components. One of the modern trends in extraction is the use of natural deep eutectic solvents (NADESs) combined with ultrasound-assisted extraction (UAE). However, works devoted to the extraction of biologically active substances from C. angustifolium using NADESs are scarce. The aim of this work is a comprehensive study of UAE of bioactive components from C. angustifolium using NADESs based on choline chloride and malonic, malic, tartaric, and citric acids. The antioxidative properties, total phenols, and flavonoids content were estimated for NADES-based extracts. The reference solvents were water and 90% v/v ethanol. Volatile extracted components were identified using GC-MS. The kinetics of the UAE were studied at 45 °C for 20–180 min with water added to 30 wt% NADES. The power of the ultrasound was 120 W, and the frequency was 40 kHz. It was found that NADES choline chloride + citric acid is more effective for the extraction of bioactive components. For this, NADES UAE conditions were optimized following a Box–Behnken design of the experiment and a response surface methodology. The temperature ranged from 30 to 60 °C, the time of extraction ranged from 20 to 60, and the addition of water ranged from 30 to 70 wt%. We established the optimal extraction conditions: temperature 58 °C, time of extraction 35 min, and 70 wt% water. The obtained results expand the knowledge about the use of NADES for the extraction of biologically active compounds from cheap and available plant raw materials.
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The carob (Ceratonia siliqua L.), a plant that grows widely in the Mediterranean climate, has a very dry and hard fruit that contains substantial amounts of sugar and phenolic compounds. This study aimed to determine the optimum extraction conditions for obtaining pure sugar extract with the least undesired compounds from the dried carob pod, and exclude the phenolic compounds that contribute to bitterness, astringency, turbidity, and color. In this study, the effect of extraction parameters as the fruit particle size (X1: 0.30–3.67 mm or 50.0–5.0 mesh), water: fruit ratio (liquid/solid ratio, X2: 2.3–5.7 ml g-1), extraction temperature (X3: 15–75 °C), and extraction time (X4: 44–196 min) on sugar composition and phenolic contents of carob extract was evaluated and an experimental design was developed using response surface methodology central composite design. When the effects of the interactions between the factors on the sugar concentration of carob extract were examined; the most significant effect (p<0.01) was shown by the extraction temperature and extraction time interaction (X3 × X4). The interactions of particle size and water: fruit ratio (X1 × X2) and interactions of particle size and extraction time (X1 × X4) were statistically significant (p<0.05). The optimum extraction conditions were determined as X1: 4.0 mm (5.0 mesh), X2: 4 ml g–1, X3: 20 °C, and X4: 200 minutes. In this study, the individual effects of the factors (X1, X2, X3, and X4) on the extraction were examined and the interaction effects of factors on responses to the extract. Consequently, regression equations showed the relations between the responses and the tested factors.
Morphological characterization was investigated by agro‐morphological criteria related to carob seed size in four different moroccan regions collected in 2018 and 2019. There was no significant difference (p ≤ 0.05) on the seeds lengths and widths. However, a significant difference between seeds thickness and total seeds weight per pod (p ≤ 0.05) were observed between these four populations. The fatty acid, sterol, tocopherol, hydrocarbon, and the unoxygenated composition of carob seed extracts (Ceratonia siliqua L.) were studied. The mean fat yield of the seeds obtained is 1.53%–2.17%, 2.14%–2.15%, 1.61%–1.62%, 1.71%–1.75% for, respectively, the P1 (Meknes), P2 (Fez), P3 (Khemisset), and P4 (Marrakech) in 2018 and 2019. The seed oil was extracted with hexane and the analysis of the fatty fraction was performed by gas chromatography–mass spectrometry (GC–MS). Results show that the major fatty acids for 2018 and 2019 are linoleic acid (61.48%–61.52%, 52.12%–52.14%, 57.76%–58.15%, 61.33%–61.52%), palmitic acid (15.78%–15.81%, 16.44%–16.45%, 19.11%–18.37%, 20.24%–20.32%), oleic acid (11.03%–11.04%, 8.72%–8.82%, 8.51%–8.61%, 8.41%–8.53%), stearic acid (4.35%–3.14%, 5.40%–5.43%, 3.12%–3.13%, 0.96%–1.56%), and cerotic acid (0.62%–0.53%, 4.51%–4.52%, 4.03%–4.06%, 3.84%–3.87%). The unsaturated fatty acids (69.39% in 2018 and 69.68% in 2019) are the most dominant in the four seed extracts compared to the saturated fatty acids. In addition, the oil carob seeds analysis revealed the presence of γ‐tocopherol, α‐tocopherol and four sterols that included campesterol, stigmasterol, and β‐sitosterol. Moreover, the determination of hydrocarbon and un‐oxygenated compounds confirmed the existence of major compounds such as heptadecane, 2‐methyltriacontane, 1‐iodo hexadecane and 1‐iodo octadecane. The hierarchical analysis based on the morphological and chromatographic characterization of the seeds allowed the identification of three groups. Consequently, the first group consisted of populations from Marrakesh (P4) and Khemisset (P3), the second group consisted of the P1 from Meknes, and the P2 from Fez constituted the third group.
In the present study, the effects of the ripening stage, processing method, and geographical origin were investigated through the analysis of carobs and derived products by use of high-performance liquid chromatography-electrospray ionization high-resolution mass spectrometry (HPLC-ESI-HRMS), in combination with chemometric analysis. The distribution of secondary metabolites across the fruit was also established through the characterization of the phenolic pattern of different carob parts. It was observed that carob seeds represent a better potential source of polyphenols since significantly higher levels were demonstrated. The content of polyphenols of both pulp and seeds was diminished upon maturation. Therefore, unripe pods were considerably more enriched in polyphenols. Noticeable changes were also observed during carob processing, especially over the thermal treatment of carob products. In regard to the effect of geographical origin, a multivariate statistical approach was employed to study the relationship between the phenolic composition of carobs and their growing regions.
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Carob tree (Ceratoniasiliqua) is widely cultivated in the Mediterranean area and is considered to be an important component of vegetation for economic and environmental reasons. The possibility of utilizing carob podsas a source of polyphenol antioxidants was examined by performing extractions with various solvent systems, in order to evaluate and optimize the conditions for the recovery of polyphenols and proanthocyanidins., such as water, ethanol, acetone and ethyl acetate have been used for the extraction of phenolics from plant materials, often with different proportions of water. Maximum quantities of polyphenol components were found in 10 % aqueous ethanol, as evaluated by measuring total polyphenol and maximum content of proanthocyanidins was found in 70% aqueous acetone. By contrast, ethyl acetate was inefficient in extracting polyphenols. The assessment of the antioxidant potency of carob pod extracts employing DPPH assay showed that carobs contain polyphenols with appreciable radical scavenging properties. A simple method for the separation of proanthocyanidins from other polyphenols was accomplished by sequential liquid extraction using pure ethanol and 70% aqueous acetone, this method needs further procedures for the purification of separated proanthocyanidins.
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Purpose To study the composition of extracts of Ginja cherries stems and leaves obtained after extraction using different solvents and to evaluate their antioxidant activities. Stems and leaves are by-products of the Ginjinha cherry liquor production; extracting valuable compounds with the most appropriate solvent would valorise these wastes. Method The extraction was performed using different solvents (acetone, ethanol, methanol, ethyl acetate, water, 2-propanol); liquid chromatography-electro spray ionization-mass spectrometry (LC-ESI/MS) was utilized to identify the predominant phenolic acids and flavonoids present in the resulting extracts. The Total Phenolic Content was determined with the Folin-Ciocolteau method. The antioxidant activity was also tested using the ABTS+ essay. Results Stems extracts showed a higher concentration in polyphenols than those from leaves. The solvent affected remarkably the extracts compositions: considering the polyphenols content ethanol and water gave the best results for stems and leaves, respectively. A good correlation was established between the antioxidant activity of the extracts and their polyphenolic composition.
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A new approach to the extraction of bioactive phenolic compounds from carob kibbles has been proposed. Carob pulp kibbles, a by-product of the carob bean gum production, were subject to supercritical fluid extraction (SFE) and to ultrasound assisted (UAE) and conventional solid-liquid extractions with two different solvent systems (100% H2O and 70% acetone), in order to obtain phenolic rich extracts with biological activities. These extracts were characterized for total phenolics content, antioxidant activity and their phenolic profile was qualitatively evaluated by HPLC-DAD. Chromatographic profile of SFE extract showed a diversity of phenolic compounds while ultrasound and conventional extracts contained mainly gallic acid. The highest phenolics concentration and antioxidant capacity was also found in the SFE extract. Preliminary screening of the extracts antiproliferative activity on rat N1E-115 neuroblastoma cells, and on human HeLa cervical and MCF-7 breast cancer cell lines revealed that carob SFE extract exhibited a much higher antiproliferative effect in the studied tumor cells, indicating its greater potential as a source of natural antitumor compounds. Supercritical fluid extraction revealed to be a more selective and efficient method of extraction. Thus, the potential association of two environmentally clean processes (UAE and SFE) for obtaining polyphenols from carob kibbles will enable bioactive compounds within an integrated and sustainable recovery process. Also, the solid residue after extractions could be used as dietary fiber.
Carob (Ceratonia siliqua L.) kibbles, a by-product of the carob bean gum production, were extracted by decoction and evaluated for its phenolic profile and potential use as an added-value antioxidant-rich product. The effect of decoction time (8–20 min) and temperature (80–100 °C) on the polyphenols content and overall antioxidant capacity expressed as percentage inhibition of the DPPH and ABTS free radicals, was performed using a Doehlert experimental design to assess the antioxidants extraction efficiency. It was observed that temperature had the most significant effect. The best conditions for total polyphenols and tannins extraction were 98.5 °C and 17 min, where ca. 39.5 mg GAE/g kibbles and 25.8 mg GAE/g kibbles on a dry basis were obtained, respectively. Under these conditions, inhibition of DPPH and ABTS radicals were 85% and 90%, respectively, showing the promising use of this biomass as an antioxidant source, which could be used, for example, in functional beverages.
Solid–liquid extraction was performed in a batch extractor, from aerial parts of Fumaria officinalis, in order to obtain an extract containing protopine. This study relates the influences of the temperature and nature of the solvent on the kinetics and rates of extraction. A method to quantify the protopine contents by Reverse Phase-High Pressure Liquid Chromatography was initially perfected. Then, a batch-extraction model based on the assumption of a second-order mechanism was developed to predict the rate constant of extraction, the saturated extraction capacity and the initial extraction rate with various temperatures in two solvents, water and ethanol 44% (w/w) in water. Furthermore, the activation energies were determined as based on the second-order rate constants of extraction used for the model building. The values resulting from these calculations and experiments were compared and discussed.
In recent years, there has been great development in the search for new natural compounds for food preservation aimed at a partial or total replacement of currently popular antimicrobial chemicals. Carob (Ceratonia siliqua) offers a natural promising alternative for food safety and bioconservation. In this work, the methanolic extract of carob leaves (MECL) was tested for the ability to inhibit the growth of a range of microorganisms. MECL inhibited the growth of Listeria monocytogenes at 28.12 μg/mL by the broth microdilution method. The effect of this bacteriostatic concentration on the growth of this bacterium revealed a pattern of inhibition characterized by (a) a resumed growth phase, which showed a lower rate of growth if compared with controls; and (b) first a lag and then a stationary phase at a lower bacterium concentration. The study of the chemical composition of MECL by high-performance liquid chromatography and liquid chromatography/mass spectrometry showed the presence of gallic acid, (-)-epigallocatechin-3-gallate, myricitrin, isoquercitin, catechin, chlorogenic acid, and malic acid. L. monocytogenes growth inhibition was recorded for myricitrin and gallic acid at 450 μg/mL and for (-)-epigallocatechin-3-gallate and isoquercitin, respectively, at 225 and 112.5 μg/mL. Taking into account that proline is a ligand of proline dehydrogenase (PDH), the use of this compound leads us to hypothesize the mode of action of MECL constituents.