food and bioproducts processing 9 0 ( 2 0 1 2 ) 748–754

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Food and Bioproducts Processing

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

Extraction of phenolic compounds from Vitex agnus-castus L.

Mohamed Latoui a,b , Bahar Aliakbarian b , Alessandro A. Casazza b , Mongi Seffen a ,

Attilio Converti b , Patrizia Perego b,∗
a Laboratory of Chemistry, Higher Institute of Agronomy, Chott Meriam 4042, Sousse, Tunisia
b Department of Chemical and Process Engineering “G.B. Bonino”, Genoa University, via Opera Pia 15, I-16145 Genoa, Italy

a b s t r a c t

The primary objective of this study was to valorized Vitex agnus-castus residues in terms of phenolic com-
pounds. The effects of extraction time (30–360 min), solid to liquid ratio (0.1–0.3 gDryBiomass /mlSolvent ), type of
solvent and different tissue types (leave, roots and seeds) on total polyphenols, o-diphenols, total flavonoids
and anthocyanins were evaluated. The highest total polyphenol (31.5 mgCaffeicAcidEquivalent /gDryBiomass ) and o-
diphenol (12.4 mgCaffeicAcidEquivalent /gDryBiomass ) contents were obtained from methanolic extract of leaves after
180 min using a solid/liquid ratio of 0.1 gDryBiomass /mlSolvent , while total flavonoids, reached a maximum value of
19.4 mgCatechinEquivalent /gDryBiomass after 360 min under the same conditions. Roots of V. agnus-castus were found to
be a good source of anthocyanins with the highest yield of 0.62 mgMalvidinEquivalent /gDryBiomass using ethanol as a sol-
vent (180 min and 0.2 gDryBiomass /mlSolvent ). The maximum antiradical power (178.5 ␮lextract /␮gDPPH ) was exhibited by
the methanolic leave extract obtained after 360 min at solid/liquid ratio of 0.3 gDryBiomass /mlSolvent .
© 2012 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Keywords: Vitex agnus-castus; Antioxidant recovery; Solid–liquid extraction; DPPH

1. Introduction 2009). These metabolites were detected for multiple uses in

Vitex agnus-castus (VAC), also note as chaste tree, is an impor-
tant medicinal plant belonging to the Verbenaceae family, that
grows naturally in Mediterranean area and is widespread in
South, West and North Turkey (Şarer and Gökbulut, 2008).
In Turkish folk medicine, VAC is used as diuretic, diges-
tive, antifungal, anti-anxiety, early birth and stomachache
(Honda et al., 1996). Ripe fruit of VAC is used in folk medicine
for the treatment of various obstetric and gynecological
disorders, and in ancient Greek times it was also for the treat-
ment of menstrual problems, pain, swelling, inflammation,
headaches, rheumatism, and sexual dysfunction (Webster
et al., 2006).
Every plant is a complex of wide range of chemical moi-
eties, among which alkaloids, polyphenols, terpenes, steroids,
essential oils and glycosides. These secondary metabolites
are synthesized by various metabolic pathways, which can be
used by plants for protection against yeast and moulds as well
as resistance to insect attack, and in some of them even for
protection against ultraviolet and drought conditions (Dweck,
herbal medicine, pharmacology, cosmetics, food, etc. Their
composition may vary according to the area of growth, soil
and weather conditions, harvest period, cultivation process
and, of course, to the plant part (leaves, seeds, etc.) (Gairola
et al., 2010; Salick et al., 2009).
The storage conditions, extraction time and type of sol-
vent are considered to have a significant impact on the final
chemical composition. For instance, the content of natural
preservatives produced by plants to protect the fruit and
the leaves will fall dramatically once they have been sepa-
rated from the main plant (Dweck, 2009). The productivity of
secondary metabolites may be also enhanced by optimizing
cultivation conditions, cell selection and genetic transforma-
tion (Kovalenko et al., 2004).
In the last few years, the search for non-aggressive, alter-
native hormonal therapies with little or no side effects has
become of clinical importance, and the influence that VAC
exerts on the female hormonal system has received consider-
able attention (Lucks et al., 2002). Most of the clinical studies
have been performed using alcoholic extracts of the mature

∗
Corresponding author at: Via Opera Pia 15, 16154 Genoa, Italy. Tel.: +39 010 3532916; fax: +39 010 3532586.
E-mail address: p.perego@unige.it (P. Perego).
Received 16 August 2011; Received in revised form 16 January 2012; Accepted 19 January 2012
0960-3085/$ – see front matter © 2012 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.fbp.2012.01.003
 food and bioproducts processing 9 0 ( 2 0 1 2 ) 748–754
fruits (Batchelder and Scalzo, 1995); therefore, a considerable
amount of researches has been carried out to verify the phar-
macological activity and to investigate the modality of VAC
extract action. Extract with non-particular side effects became
one of the most popular alternative therapies for women who
do not respond to or tolerate the hormonal or psychotropic
drugs (Schellenberg, 2001; Webster et al., 2006).
In the last decades, several successful clinical trials
supported the use of VAC extracts for the treatment of pre-
menstrual syndrome (PMS) (Schellenberg, 2001), as the likely
result of its opiate activity (Webster et al., 2006). Hirobe et al.
(1994) have also reported that the extract of VAC fruits, ripened
in Israel, exerts an antitumor effect on the Chinese ham-
ster lung carcinoma cells line V-79. Moreover, VAC methanol
extracts were shown to contain eight flavonoids, seven of
which having cytotoxic activity against mouse lymphocytic
leukemia P388.
Since VAC is a medicinal plant, it is essential to quantify
and qualify its active compounds that contribute to thera-
peutic and medicinal activities. Several studies have reported
that VAC contains a lot of different secondary metabolites,
among which iridoids (Hänsel and Winde, 1959), flavonoids
(Hirobe et al., 1997), diterpenoids (Li et al., 2002), essential oils
(Kuruüzüm-Uz et al., 2003), ketosteroids (Šaden-Krehula et al.,
1990).
Since there is no report, to the best of our knowledge, on
phenolic compounds extraction from VAC grown naturally in
Tunisia and their characterization, an accurate study to qual-
ify and quantify alcoholic extracts from such a medicinal plant
would be of great industrial concern. Based on these con-
siderations and with the aim of exploiting the potential of
Tunisian VAC residues from the pharmaceutical industry, the
present study was addressed to conventional phenolic extrac-
tion from different parts (leaves, roots and seeds) of this plant,
selecting the extraction time (30–360 min), solid to liquid ratio
(0.1–0.3 gDB /mlS ) and type of solvent (ethanol or methanol) as
the independent variables, and the levels of total polyphenols,
o-diphenols, flavonoids and anthocyanins as the responses.
The antioxidant activity of leave extracts was also investigated
in terms of their ability to reduce the radical 2,2-diphenyl-1-
picrylhydrazyl (DPPH).
2. Materials and methods
2.1. Reagents
Analytic grade methanol and ethanol, Folin–Ciocalteau
reagent and pure standards of caffeic acid, catechin, and mal-
vidin were employed. All these reagents were purchased from
Sigma Chemical Co. (St. Louis, MO, USA). Standard stock solu-
tions were prepared with methanol or ethanol, wrapped in
aluminium foil and stored at −20 ◦ C prior to analyses.
2.2. Raw material
In this work, we used VAC grown naturally in the Tunisian
regions of Oued Barkoukech (east of Tabarka) and Ichkeul Lake
(near city of Bizerte). Different parts of VAC (leaves, roots and
seeds) were collected from fresh plants. Samples were dried
in room temperature (20 ± 1 ◦ C), then ground to a fine powder
(particle size of 0.8 mm) using a laboratory mixer, and stored
at 4 ◦ C. All samples were analyzed within 3 months after col-
lection.
749
2.3. Extraction method
Dried and milled samples of seeds, leaves or roots were
extracted in glass test tubes with screw caps, and the sus-
pension was continuously mixed by a magnetic stirrer, model
Mr 2002 (Heidolph, Kelheim, Germany). The extractions were
performed in the dark and at room temperature using a
solid–liquid ratio of 0.10, 0.20 and 0.30 grams of dried biomass
per milliliter of solvent (gDB /mlS ), and the extraction time was
30, 60, 90, 180, 360 and 420 min.
Tests were performed in duplicate varying the amount of
dry samples, while the solvent volume was kept constant to
maintain the same head space in the test-tubes in order to pre-
vent oxidation reactions. The extract was separated from solid
fraction by centrifugation at 6000 × g for 10 min (ALC PK131
Centrifuges, Alberta, Canada) and then subjected to quantita-
tive analyses. The solid/liquid ratio (S/L) is usually considered
a factor having a large impact on the extraction of phytochem-
icals from various plant matrices, and the range selected in
the present study was based on values frequently reported in
the published literature (Casazza et al., 2010). Methanol and
ethanol were selected as extraction solvents as they have been
frequently used to extract phenolic compounds from different
plants (Bukhari et al., 2008; Webster et al., 2006).
In order to compare the yield of phenolic compounds
extracted from seeds, leaves or roots at room temperature with
the extraction at higher temperatures, additional extractions
were performed using Soxhlet apparatus (Sarikurkcu et al.,
2009). The dried samples (2 g) were extracted by using a Soxh-
let extractor for 5 h with 100 ml of deionized water. The water
extracts where then filtered through 0.20 ␮m membranes (Sar-
torius Stedim Biotech GmbH, Goettingen, Germany) and kept
at 4 ◦ C for further analysis. Extractions were performed in
duplicate.
2.4. Total polyphenols
Total polyphenol (TP) concentration was measured by the col-
orimetric Folin–Ciocalteau assay (Swain and Hillis, 1959) using
a UV–Vis spectrophotometer, model Lambda 25 (Perkin Elmer,
Wellesley, MA, USA) at a wavelength of 725 nm. TP concen-
tration was calibrated (R2 = 0.999) using standard methanolic
or ethanolic solutions of caffeic acid (10–1000 ␮g/ml), and
expressed as milligrams of caffeic acid equivalent per gram of
dry biomass (mgCAE /gDB ) by means of the linear relationship:
ABS725 = 0.002TP + 0.004 (1)
In fact, caffeic acid has been widely used as a reference
when working with TP extracts (Aliakbarian et al., 2008, 2009;
Ranalli et al., 2001). Analysis were performed in triplicate.
2.5. o-Diphenols
The concentration of o-diphenols (OD) in the alcoholic extract,
also expressed as mgCAE /gDB , was determined by the molyb-
date method (Gutfinger, 1981). Analysis were performed in
triplicate. The calibration straight line (R2 = 0.999) was made
with standard alcoholic solutions of caffeic acid in the range
10–250 ␮g/ml:
ABS350 = 0.004OD + 0.001 (2)
750 food and bioproducts processing 9 0 ( 2 0 1 2 ) 748–754

2.6. Total flavonoids
The total flavonoids (TF) content of the alcoholic extracts was
determined using the colorimetric method described by Jemai
et al. (2009) with some modifications. After mixing 0.25 ml of
each dilute extract with 1.25 ml of deionized water, we added
0.075 ml of 5% sodium nitrite solution, after 5 min of reac-
tion 0.15 ml of 10% aluminium chloride, after additional 6 min
0.5 ml of 1.0 M NaOH, and finally deionized water until a final
volume of 3 ml. The absorbance of the mixture was deter-
mined at 510 nm using the same spectrophotometer as above.
Analysis were performed in triplicate. The calibration straight
line (R2 = 0.991) was made using standard alcoholic solutions
of catechin in the range 0.01–0.50 ␮g/ml:
ABS510 = 0.002TF + 0.012 (3)
2.7. Anthocyanins
In order to determine the anthocyanins (AN) content of
the alcoholic extracts, the colorimetric method described
by Di Stefano et al. (1989) was used. According to this
method, 1 ml aliquot of extract was diluted with a 70:30
promoted by oxygen present in the empty space of test tubes
(Liyana-Pathirana and Shahidi, 2005; Yap et al., 2009).Also, the
yield of phenolic compounds decreased with increasing S/L
(0.10, 0.20 and 0.30 gDB /mlS ) in all fractions, because the larger
the volume of solvent, the higher the phenolic compounds
fraction solubilized (Prasad et al., 2009).
3.1. Total polyphenols and o-diphenols
Figs. 1 and 2a–c show the results of the quantitative determi-
nations of the various fractions of TP and OD.
Making reference to the optimal extraction conditions,
we can see in Fig. 1a, that TP extraction from leaves was
more effective with methanol (31.5 mgCAE /gDB ) than with
ethanol (20.5 mgCAE /gDB ), although both occurred after the
same extraction time (180 min) and S/L (0.10 gDB /mlS ). These
values are higher than the TP contents of acetone extracts
from star fruit (Averrhoa carambola L.) residues (9.6 mg/gDB
expressed as gallic acid-GA) (Yap et al., 2009) and methano-
lic extract from olive oil (0.6 mgCAE /gDB ) (Aliakbarian et al.,
a 0.1 ethanol
35 0.2 ethanol
Total Polyphenols (mgCAE/gDB)

(v/v) ethanol/distilled water solution, and 1 ml of 1.0 M HCl 0.3 ethanol

0.1 methanol
was added. The solution was mixed thoroughly, and the 30 0.2 methanol
0.3 methanol
absorbance of the mixture determined at 540 nm. The AN con- 25
tent was calculated by the following equation:
20

15
AN = 16.17ABS540 D (4)
10
where D is the dilution factor, and expressed as milligrams 5
of malvidin equivalent per gram of dry biomass (mgME /gDB ).
0
Analysis were performed in triplicate. 0 60 120 180 240 300 360 420 480
Time (min)
2.8. Antioxidant activity
b 25 0.1 ethanol
The antioxidant activity of the extracts was measured in terms 0.2 ethanol
Total Polyphenols (mgCAE/gDB)

0.3 ethanol
of hydrogen-donating or radical-scavenging ability by means 20 0.1 methanol
0.2 methanol
of the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH• ) method 0.3 methanol

(Brand-Williams et al., 1995), which is widely used to describe 15

the antiradical power of different matrices (Aliakbarian et al.,
2009). For each sample, seven different dilutions ranging from 10
1:2 to 1:128 in methanol or ethanol were prepared. For each
extract, 0.10 ml of diluted sample was mixed with 3.90 ml 5

of DPPH• methanolic solution (9.15 × 10−5 mol/l). The reac-

0
tion mixtures were shaken and incubated for 1 h in the dark 0 60 120 180 240 300 360 420
at room temperature, and then the absorbance was read at Time (min)
515 nm using the same spectrophotometer as above. The anti-
radical power (ARP ␮gDPPH /␮lextract ) was expressed as 1/EC50, c 4 0.1 ethanol
where EC50 is the amount of extract necessary to decrease the 0.2 ethanol
Total Polyphenols (mgCAE/gDB)

0.3 ethanol
initial DPPH• concentration by 50%. Analysis were performed 0.1 methanol

in triplicate. 3 0.2 methanol

0.3 methanol

3. Results and discussion 2

The results of the quantitative analyses of TP, OD, TF and AN 1

in the leave, seed and root extracts from VAC, which are illus-
trated in Figs. 1–4, show that the yields of these chemical
0
compounds were greatly influenced either by the extraction 0 60 120 180 240 300 360 420
time or the solid/liquid ratio (S/L). Time (min)
In general, the yield of any class of compounds increased
with the extraction time up to a maximum value and then Fig. 1 – Results of total polyphenols extraction as function
decreased with further prolonging the extraction time. This of time (min) using ethanol and methanol as solvents: (a)
was the likely result of well-known oxidative phenomena leaves; (b) seeds; (c) roots.
 food and bioproducts processing 9 0 ( 2 0 1 2 ) 748–754 751

a 0.1 ethanol 0.2 ethanol a 25 0.1 ethanol

14 0.3 ethanol 0.1 methanol 0.2 ethanol

Total Flavonoids (mgCE/gDB)

0.2 methanol 0.3 methanol 0.3 ethanol
o-diphenols (mgCAE/gDB)

12 0.1 methanol
20 0.2 methanol
0.3 methanol
10
15
8

6 10
4
5
2

0 0
0 60 120 180 240 300 360 420 0 60 120 180 240 300 360 420 480
Time (min) Time (min)

b 7 0.1 ethanol 0.2 ethanol b 15 0.1 ethanol

0.3 ethanol 0.1 methanol

Total Flavonoids (mgCE/gDB)

0.2 ethanol
6
o-diphenols (mgCAE/gDB)

0.2 methanol 0.3 methanol 0.3 ethanol

0.1 methanol
0.2 methanol
5
10
4

3
5
2

1
0
0
0 60 120 180 240 300 360 420
0 60 120 180 240 300 360 420
Time (min)
Time (min)
c
c 2.5 0.1 ethanol 0.2 ethanol
2 0.1 ethanol
Total Flavonoids (mgCE/gDB)

0.3 ethanol 0.1 methanol

0.2 ethanol
o-diphenols (mgCAE/gDB)

0.2 methanol 0.3 methanol 0.3 ethanol

2 0.1 methanol
0.2 methanol
1,5 0.3 methanol

1.5
1
1

0,5
0.5

0 0
0 60 120 180 240 300 360 420 0 60 120 180 240 300 360 420
Time (min) Time (min)

Fig. 2 – Results of o-diphenols extraction as function of Fig. 3 – Results of total flavonoids extraction as function of
time (min) using ethanol and methanol as solvents: (a) time (min) using ethanol and methanol as solvents: (a)
leaves; (b) seeds; (c) roots. leaves; (b) seeds; (c) roots.

2008), but lower than that in ethanolic extracts of grape seeds
(73.7 mgGAE /gDB ) (Casazza et al., 2010) and methanolic extracts
of olive pomace (45.2 mgCAE /gDB ) (Aliakbarian et al., 2011).
The above behavior was qualitatively similar to that of OD
present as the only fraction of simple phenols (Fig. 2, panel a).
These compounds, which were found to have special antiox-
idant activity and were reported to play an important role in
extract stability (Aliakbarian et al., 2008), reached, under the
same conditions, maximum values of 7.4 mgCAE /gDB in ethanol
and 12.4 mgCAE /gDB in methanol, respectively, which consti-
tuted 29.4–46.9% of TP, with only a few differences depending
on the solvent used.
Surprisingly, ethanol behaved as a better solvent than
methanol to extract from seeds the same classes of com-
pounds (Figs. 1 and 2, panels b), whose concentrations
in the extract reached after only 30 min maximum values
(19.2 mgCAE /gDB TP and 5.4 mgCAE /gDB OD) about 3–5-fold those
obtained with methanol.
Because of the very low TP and OD contents of
roots, no significant variation in solvent selectivity was
detected, the highest values for both classes of compounds
(2.8–2.9 mgCAE /gDB TP and 1.3–1.9 mgCAE /gDB OD) having been
obtained again at S/L = 0.10 gDB /mlS (Figs. 1 and 2, panels c).
In general, the highest yields from all three plant parts were
obtained at the lowest S/L (0.10 gDB /mlS ), which means that the
solvent amounts employed at higher S/L were insufficient to
complete the extraction of these two classes of compounds.
Although the TP and OD yields followed the above-mentioned
general increase with time up to maximum values, and sub-
sequent decrease due to degradation, the time of maximum
yields seemed to strongly depend on the plant part. It did in
fact increase from 30 min with seeds to 180 min with leaves,
as the likely result of the different ability of solvent to reach
the inside of cells consequent to the different susceptibility of
these plant organs to the milling operation.
3.2. Flavonoids and anthocyanins
As is well known, the flavonoids are a class of plant sec-
ondary metabolites or yellow pigments having structures
similar to those of flavones. Total flavonoids in both ethanolic
and methanolic extracts represented the major fraction of TP,
752 food and bioproducts processing 9 0 ( 2 0 1 2 ) 748–754

a 0.40 0.1 ethanol

nature of this plant organ with respect to leaves. Finally,
0.35
0.2 ethanol although the very low TF concentrations in root extracts (close
Anthocyanins (mgME/gDB)

0.3 ethanol
0.1 methanol to the detection limit of the analytical procedure) do not allow
0.30 0.2 methanol
0.3 methanol highlighting any appreciable influence of the other two oper-
0.25
ating variables, it is clear that methanol behaved as a much
0.20 better solvent than ethanol, providing a maximum level in the
0.15 extract of 1.69 mgCE /gDB after180 min.
0.10 Anthocyanins are the most important pigments of the vas-
0.05 cular plants with potential antioxidant activity. Several reports
focused on the biological activity (Kong et al., 2003) and the
0.00
0 60 120 180 240 300 360 420 effect of this important class of flavonoids in the prevention
Time (min) of cardiovascular diseases and diabetes and cancer treatment
(Nichenametla et al., 2006). The results of extraction tests
b 0.1 ethanol made on all three plant parts (Fig. 4a–c) show that, contrary
0.12 0.2 ethanol
to the other classes of compounds, their concentration was
Anthocyanins (mgME/gDB)

0.3 ethanol
0.1 methanol
0.10 0.2 methanol the highest in the root extract (0.56, 0.62 and 0.48 mgME /gDB
0.3 methanol
with 0.3, 0.2 and 0.1 gDB /mlS , respectively, with ethanol and
0.08
0.36 mgME /gDB with methanol) and almost negligible in the
0.06 seed one (<0.12 mgME /gDB with both solvents).
0.04 This behavior might be due to the fact that antho-
cyanins are highly instable and very susceptible to degradation
0.02 induced by light, oxygen and temperature. In this aspect,
0.00 Laleh et al. (2006) found that exposure to the light accel-
0 60 120 180 240 300 360 420 erated the destruction of anthocyanins from four different
Time (min) Berberies species up to 26% when compared with dark con-
ditions. They also confirmed that temperatures up to 35 ◦ C
c 0.70 resulted to anthocyanins’ degradation. One interesting find-
Anthocyanins (mgME/gDB)

0.60
0.50
0.40
0.30
0.20
0.10
0.00
0 60 120 180 240
Time (min)
Fig. 4 – Results of anthocyanins extraction as function of
time (min) using ethanol and methanol as solvents: (a)
leaves; (b) seeds; (c) roots.
varying in the range 55.2–69.1% in leaves and 30.2–63.2%
in roots, thus confirming their abundance in V. agnus-castus
reported in the literature (Hoberg et al., 2000).
Consistently with the more hydrophobic structure of their
molecules, TF extraction from leaves required in methanol
longer extraction time compared to TP, reaching a maximum
value of their concentration in the extract (19.4 mgCE /gDB ) after
no less than 360 min, while ethanol ensured less efficient
extraction (12.8 mgCE /gDB after 180 min) (Fig. 3). From seeds,
likewise TP and OD, the maximum TF yield (14.6 mgCE /gDB )
with ethanol was about 3.5-fold that with methanol and was
detected only after 30 min, confirming the less recalcitrant
ing that can be gleaned from these results is that this family
of flavonoids constituted only a very little fraction of TF both
in leaves (1.17–4.02%) and seeds (0.77–6.99%) extracts, almost
irrespectively of the extraction solvent, whereas it accounted
for even 38.3 and 31.5% of TF extracted from roots with ethanol
0.1 ethanol
and methanol, respectively.
0.2 ethanol
0.3 ethanol
0.1 methanol
0.2 methanol 3.3. Soxhlet extraction
0.3 methanol
300 360 420 In order to ensure complete extraction of phenolic com-
pounds, the aqueous Soxhlet extraction was employed using
different parts of biomass. The results reported in Table 1
demonstrated the same trends for TP, OD and TF when using
leaves, roots and seeds. While in the case of AN, higher con-
tent was achieved when using seeds as biomass followed by
roots and leaves.
The comparative study showed that the highest TP and
OD contents (31.50 and 12.36 mgCAE /gDB ) which were obtained
using leaves, methanol and 180 min were lower than the Soxh-
let extract results (46.85 and 43.44 mgCAE /gDB for TP and OD,
respectively).
Using alcoholic extraction, in the same operative condi-
tions and longer extraction time (360 min) TF resulted to be
19.41 mgCE /gDB , this value was approximately the half that
obtained by Soxhlet apparatus (42.29 mgCE /gDB ).
In the case of roots, alcoholic extraction resulted to be more
efficient for AN recovery in comparison with aqueous Soxhlet
extraction.

Table 1 – Results of total polyphenols (TP), o-diphenols (OD), total flavonoids (TF) and anthocyanins (AN) obtained by
Soxhlet extraction of leaves, seeds and roots of Vitex agnus-castus using water.
TP (mgCAE /gDB ) OD (mgCAE /gDB ) TF (mgCE /gDB ) AN (mgME /gDB )

Leaves 46.85 ± 1.42 43.44 ± 2.10 42.29 ± 1.86 0.38 ± 0.01

Seeds 25.87 ± 0.91 18.42 ± 0.27 16.00 ± 0.81 0.60 ± 0.01
Roots 8.81 ± 0.47 6.45 ± 0.14 2.92 ± 0.28 0.52 ± 0.01
 food and bioproducts processing 9 0 ( 2 0 1 2 ) 748–754
200
180
160
ARP µgDPPH/µLextract
140
120
100
80
60
40
20
0 reactor. Food Chem. 128 (3), 704–710.
0 60 120 180 240 300
Time (min)
Fig. 5 – Antioxidant activity expressed as antiradical power
(ARP) of (a) ethanolic and (b) methanolic extracts of VAC
leaves.
These differences might be due to the fact that higher
temperatures (100 ◦ C) in the case of using Soxhlet enhanced
solvent solubility and consequently higher phenolic extrac-
tion yields.
3.4. Antioxidant activity
An evaluation of the antioxidant activity of V. agnus-castus
extracts was done using exclusively the methanolic extracts
of leaves, since they exhibited by far the highest total polyphe-
nols yield (31.5 mgCAE /gDM ). It is well known in fact that this
activity is directly related to the antiradical power of these
compounds (see Section 2).The results illustrated in Fig. 5
show that the extraction time considerably increased the
ARP up to a maximum value, beyond which it decreased
owing to oxidation. However, as far as the effect of S/L is
concerned, this operating variable had, as expected, an oppo-
site effect compared to the previous responses (TP, TF, etc.),
because the most concentrated the extracts (less efficient
extraction), the highest their ARP. In particular, the maximum
ARP (178.5 ␮lextract /␮gDPPH ) was exhibited by the methano-
lic leave extract obtained after 360 min at S/L = 0.3 gDB /mlS .
Such an ARP yield was 76.6% higher than the one detected
in the best ethanolic extract. This value is much higher than
those (0.27 ␮lextract /␮gDPPH ) obtained by Hajdú et al. (2007) for
VAC fruit extracts, either using ethylacetate (14.7 ␮l/␮g) or
methanol/water (0.3 ␮l/␮g) as solvent.
4. Conclusions
In this work we characterized for the first time different
parts (leaves, seeds and roots) of Tunisian V. agnus-castus in
terms of phenolic compounds (total polyphenols, o-diphenols,
total flavonoids and anthocyanins). The highest total polyphe-
nol, o-diphenol and flavonoid contents were obtained from
methanolic extract of leaves. Roots of Vitex were found to
be a good source of anthocyanins with the highest yield
using ethanol as solvent. Methanolic leave extracts exhibited
an interesting antioxidant activity. The preliminary results
achieved in this part of the study suggest that further complete
optimization of the proposed treatment, taking into account
the effects of extraction temperature, drying time and qualifi-
cation of the single phenolic compounds in the extracts, will
be necessary. Naturally grown VAC can be successfully used as
an inexpensive source of phenolic compounds, and the food
and pharmaceutical industries may be benefited.
753
0.1 ethanol Acknowledgment
0.2 ethanol
0.3 ethanol
0.1 methanol
0.2 methanol The authors are grateful to the Erasmus Mundus Program
0.3 methanol
IMAGEEN for the PhD fellowship of Mohamed Latoui.
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