Food Chemistry 132 (2012) 406412

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Food Chemistry
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Phenolic prole and antioxidant activities of olive mill wastewater

Abdelilah El-Abbassi, Hajar Kiai, Abdellatif Hadi
Food Science Laboratory, Department of Biology, Faculty of Sciences-Semalia, Cadi Ayyad University, P.O. Box 2390, 40090 Marrakech, Morocco

a r t i c l e i n f o a b s t r a c t

Article history: Olive trees play an important role in the Moroccan agro-economy, providing both employment and export
Received 3 July 2011 revenue. However, the olive oil industry generates large amounts of wastes and wastewaters. The disposal
Received in revised form 25 September 2011 of these polluting by-products is a signicant environmental problem that needs an adequate solution. On
Accepted 2 November 2011
one hand, the phytotoxic and antimicrobial effects of olive mill wastewaters are mainly due to their pheno-
Available online 10 November 2011
lic content. The hydrophilic character of the polyphenols results in the major proportion of natural phenols
being separated into the water phase during the olive processing. On other hand, the health benets arising
Keywords:
from a diet containing olive oil have been attributed to its richness in phenolic compounds that act as nat-
Olive mill wastewaters
Phenolic compounds
ural antioxidants and are thought to contribute to the prevention of heart diseases and cancers. Olive mill
Antioxidant activity wastewater (OMW) samples have been analysed in terms of their phenolic constituents and antioxidant
Radical scavenging activities. The total phenolic content, avonoids, avanols, and proanthocyanidins were determined. The
Iron(II) chelating activity antioxidant and radical scavenging activity of phenolic extracts and microltred samples was evaluated
Lipid peroxidation using different tests (iron(II) chelating activity, total antioxidant capacity, DPPH assays and lipid peroxida-
tion test). The obtained results reveal the considerable antioxidant capacity of the OMW, that can be con-
sidered as an inexpensive potential source of high added value powerful natural antioxidants comparable to
some synthetic antioxidants commonly used in the food industry.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
Olive mill wastewaters (OMW) are the main liquid efuents
generated by the olive oil production industry. The annual world
OMW production is estimated from 10 to over 30 million m3
(Eroglu, Eroglu, Gndz, Trker, & Ycel, 2006). Although the
quantity of the waste produced is still much smaller than other
types of waste and its production is seasonal, the contribution of
OMW to environmental pollution is important. In terms of pollu-
tion effect, 1 m3 of OMW is reported to be equivalent to 200 m3
of domestic sewage (Tsagaraki, Lazarides, & Petrotos, 2007). Its dis-
posal in water reservoirs (ground water reservoirs, surface aquatic
reservoirs, seashores, and sea) without pretreatment, leads to se-
vere problems for the whole ecosystem. OMW also show a toxic
action to some plants and microorganisms since they exhibit a sub-
stantial concentration of phenolic compounds. These latter are the
largest family of naturally occurring antioxidants in plants that in-
clude a wide variety of structures with a common motif, the phenol
molecule. They expand from the simplest structures, such as phe-
nolic acids and alcohols, to the most complex oligomeric ones such
as proanthocyanidins.
According to several studies, phenolic compounds from olives
have signicant health benets. They possess cancer chemopreven-
tive, cardioprotective, and neuroprotective activities (Tsagaraki
Corresponding author. Tel.: +212 524 434 649x515; fax: +212 524 436 769.
E-mail addresses: a.hadi@ucam.ac.ma, hadi.abdellatif@googlemail.com (A. Hadi).
et al., 2007). Epidemiological studies have correlated the low inci-
dence of coronary heart disease, atherosclerosis, and some types of
cancer (colorectal and breast cancer) with olive oil consumption in
the Mediterranean diet (Feki, Allouche, & Sayadi, 2005). Natural phe-
nols from olive and its by-products are now recognised as potential
targets for the food, cosmetic and pharmaceutical industries. Nowa-
days, interest in novel sources of natural antioxidants is steadily
growing. Since OMW is available in huge quantities and exhibits
high concentrations of phenolic compounds, they may turn into a
natural source of valuable and powerful antioxidants within the
few coming years.
As a part of a comprehensive study of the nature and functional-
ity of OMW and its phenolic extract, we investigate here the pheno-
lic prole and the antioxidant activity of OMW samples generated
by two different olive oil processing techniques (olive press and de-
canter centrifugation systems) using four tests with different acting
mechanisms. We have also investigated the effect of storage on
OMW phenolic content and on the antioxidant capacity.
2. Materials and methods
2.1. Chemicals
10 -10 Diphenyl-20 picrylhydrazyl (DPPH), 2-thiobarbituric acid,
potassium ferricyanide, tyrosol, ferrozine, FolinCioucalteu
reagent, ferric chloride, ethylenediaminetetraacetic acid (EDTA), L-
ascorbic acid, sodium hydroxide, and trichloroacetic acid were pur-
chased from Sigma Aldrich (Germany). Ascorbic acid and butylated

0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.11.013
 A. El-Abbassi et al. / Food Chemistry 132 (2012) 406412
hydroxytoluene (BHT) were procured from Merck (France). Other
solvents were obtained from Sigma Aldrich Germany and were of
analytical grade.
2.2. Physicochemical characterisation of OMW samples
Olive mill samples were collected from two different olive oil
mills in the area of Marrakech during the season of 2008/2009.
The two mills used different milling techniques, which were
semi-modern (OMW1) and modern (OMW2) three-phase pro-
cesses. The processed olive fruits are from the Moroccan Picholine
variety. The physicochemical characterisation of OMW samples
was carried out as follow:
- Total organic carbone (TOC) was determined using EUROGLAS
TOC analyser (Thermo Scientic, Germany).
- Chemical oxygen demand (COD) was determined by the dichro-
mate method. The appropriate amount of wastewater samples
was diluted up to 100 times and introduced into a lab-prepared
digestion solution containing potassium dichromate, sulphuric
acid and mercuric sulphate and the mixture was then incubated
for 120 min at 150 C in a COD reactor (Model WTW CR3000,
Germany). COD concentration was measured colorimetrically
at 600 nm using a MultiLab P5 (WTW, Germany). The standard
solutions of 1, 2, 3, 4 g of O2 per litre were prepared using the
potassium biphthalate.
- Dissolved chemical oxygen demand (DCOD) was measured on
ultraltrated (on membrane of 50 kDa MWCO; Microdyn-Nadir
GmbH, Germany) samples using the same protocol as for COD.
- Total suspended solids was determined after ultraltration of the
olive mill wastewater samples through a membrane of 50 kDa
(MWCO). The dry residue was then determined by drying the
permeate at 105 C overnight and expressed as g of TSS per litre.
2.3. OMW microltration
A polyethersulfone membrane (MicrodynNadir, Germany) with
0.05 lm pore size was used. Microltration was carried out at a
room temperature in a stirred ultraltration cell (AMICON 8200,
Millipore USA) with a 200 ml volume. The effective surface area
of the membrane was 28.7 cm2. The transmembrane pressure
was applied with pressurised nitrogen gas. Microltration was
conducted under a transmembrane pressure of 4 bars and the cell
was stirred at 250 rpm using a magnetic stirrer. The obtained per-
meate was directly used for antioxidant assays and compared to
the phenolic extract from OMW.
2.4. OMW extracts
The phenolic extract was obtained by a liquidliquid extraction
of the OMW. First, the pH of OMW samples (5 ml) was adjusted to
pH 2 using HCl (2 M). After defatting with n-hexane, extractions
with ethyl acetate were performed thrice, and the three extracts
were brought to dryness by vacuum evaporation at 40 C, and then
recuperated in 5 ml methanol. The resulting extract is called phe-
nolic extract.
2.5. Total phenolic content, avonoids, avanols, and proanthocyanidi
ns determinations
- Total phenolic content (TPC) was determined following the
FolinCiocalteu spectrophotometeric using tyrosol as a standard.
The phenolic extract (0.1 ml) in a volumetric ask was diluted
with distilled water (3.4 ml). FolinCiocalteu reagent (0.5 ml)
was added and the contents of ask were mixed thoroughly. After
3 min 1 ml of a 20% anhydrous sodium carbonate solution (w/v)
407
was added, and then the mixture was allowed to stand for 1 h in
the dark. The optical density of the blue-coloured samples was
measured at 765 nm. The total phenolic content was determined
as tyrosol equivalents (TYE) and values are expressed as g of tyro-
sol/l of olive oil mill wastewaters.
- For the total avonoids, a modied method from Kim, Chun,
Kim, Moon, and Lee (2003) was used. A 0.2 ml aliquot of extract
appropriately diluted was mixed with 0.8 ml distilled water in a
5 ml assays tube, 0.06 ml 5% NaNO2 was added, and allowed to
react for 5 min. Afterwards, 0.04 ml 10% AlCl3 was added and
the mixture stood for further 5 min. Finally, 0.4 ml 1 M Na2CO3
and 0.5 ml distilled water were added to the reaction mixture,
and the absorbance at 510 nm was obtained against a similarly
prepared blank, by replacing the extract with distilled water.
Total avonoid content was calculated from a calibration curve
using catechin as a standard, and expressed as mg catechin
equivalents (CTE) per litre of the extract.
- Flavanols were determined after derivatisation with p-(dimeth-
ylamino)-cinnamaldehyde (DMACA), using the optimised pro-
tocol established by Nigel and Glories (1991). The extract
(0.2 ml), suitably diluted with methanol, was introduced into
a 5 ml assays tube and 0.5 ml HCl (0.24 N in methanol) and
0.5 ml DMACA solution (0.2% in methanol) were added. The
mixture was allowed to react for 5 min at room temperature,
and the absorbance was determined at 640 nm. The control
was prepared by replacing sample with methanol. The concen-
tration of total avanols was calculated from a calibration
curve, using catechin as a standard. The results are expressed
as mg of catechin equivalents (CTE) per litre of the extract.
- Proanthocyanidins were analysed by the method described by
Waterman and Mole (1994). Butanol reagent was prepared by
mixing 70 mg ferrous sulphate (FeSO4) with 5 ml concentrated
HCl and made to 100 ml with n-butanol. An aliquot of 0.1 ml
sample was mixed thoroughly with 1.4 ml butanol reagent
and heated at 95 C in a water bath for 45 min. The sample
was than cooled, 0.5 ml n-butanol was added and the absor-
bance was measured at 550 nm. Results were expressed as
cyanidin equivalents (CYE) per litre of the extract using a molar
extinction coefcient of e = 26,900 and MW = 449.2.
2.6. Antioxidant and radical scavenging activity of phenolic extracts
2.6.1. Determination of the total antioxidant capacity
The total antioxidant capacity (TAC) was determined according
to the method described by Pan et al., 2008. The phenolic extract
(0.5 ml) was combined with 1.5 ml of a reagent solution (0.6 M sul-
phuric acid, 28 mM sodium phosphate and 4 mM ammonium
molybdate). The reaction mixture was incubated at 95 C for
150 min. Once the mixture cooled to room temperature, the absor-
bance of the mixture was measured at 695 nm against a blank. The
readings were taken every 30 min. The antioxidant activity was
expressed as the absorbance of the sample. The antioxidant activ-
ity of BHT (0.5 mg/ml) and a-tocopherol (0.5 mg/ml) were also
assayed for comparison.
2.6.2. Free radical-scavenging ability
Free radical-scavenging ability of different phenolic extracts
was determined using a stable 2,2-diphenyl-2-picrylhydrazyl rad-
ical (DPPH). The free radical working solution was prepared by
dissolving 4 mg of DPPH in 100 ml of ethanol. A 100 ll aliquot
of the sample, adequately diluted with ethanol, was placed in a
cuvette and reacted with 3 ml of DPPH working solution. The mix-
ture was shaken vigorously and left to stand for 60 min at room
temperature in the dark. The decrease in absorbance was measured
at 517 nm after 60 min, against ethanol as a blank. Low absorbance
of the reaction mixture indicates high free radical-scavenging
408 A. El-Abbassi et al. / Food Chemistry 132 (2012) 406412

activity. All determinations were performed in duplicate. The afn-
ity of the test material to quench DPPH radicals (% inhibition of
DPPH) was calculated according to the following equation:

by a JASCO UV detector (UV-975) operating at 280 nm. The column
% Inhibition 1  Asample =Acontrol  100
where Acontrol was measured as the absorbance of DPPH in ethanol
(3 ml) plus ethanol (100 ll) instead of samples. The sample concen-
tration providing 50% inhibition (IC50) was calculated from the
graph plotting inhibition percentage against extract concentration.
The obtained results were expressed as mg TYE/l of phenolic extract
needed to reduce DPPH radical signal by 50%. The Free radical-
scavenging ability of ascorbic acid and BHT was also evaluated
and compared to our extracts.
2.7. HPLC analysis of OMW extracts
The analysis was performed on a JASCO HPLC system, equipped
used to analyse polyphenols was a reversed phase Lichrosphere C18
(4  250 mm i.d 5 lm), and the column was washed with acetoni-
trile 100% before and after analysis. A mixture of acetonitrile/water
acidied with acetic acid was chosen as the optimal mobile phase.
The ow rate was 0.8 ml/min and the injection volume was 20 ll.
The identication of phenolic compounds was fullled on the basis
of their retention time in comparison with phenolic standards.
3. Results and discussion

2.6.3. Iron(II) chelating activity (ICA) 3.1. Physicochemical characterisation of OMW samples
The chelating of ferrous ions by the sample was estimated using
the method described by Yen, Duh, and Chuange (2000) with mod- Table 1 shows the main physicochemical characteristics of
ication. The adequately diluted phenolic extract (0.1 ml) was OMW samples. OMW samples are slightly acidic. OMW from the
mixed with methanol (2.6 ml) and 2 mM FeCl2 (0.1 ml) and then semi-modern (OMW1) unit showed high electrical conductivity,
5 mM ferrozine (0.2 ml). The mixture was shaken vigorously and which was more than three times that of the OMW2. Traditionally
left to stand at room temperature in the dark for 10 min. Absor- Moroccan farmers preserve olive fruits during storage by salt addi-
bance of the resulting solution was measured spectrophotometri- tion. In modern milling units more water is used, and for this rea-
cally at 562 nm. A low absorbance of the resulting solution son, almost all parameters values were reduced in comparison to
indicated a strong Fe2+-chelating ability. The ability to chelate fer- samples from the semi-modern unit (OMW1).
rous ion (ICA) and prevent formation of ferrous ion-ferrozine com-
plex, was calculated using the following equation:
 3.2. OMW microltration
ICA % 1  Asample =Acontrol  100
Microltration of OMW exhibits a reasonable ux (350 l/hm2)
where Acontrol was the absorbance of a mixture of methanol (2.7 ml),
and the obtained permeate was less coloured (80% less at
2 mM FeCl2 (0.1 ml) and 5 mM ferrozine (0.2 ml). Measurements
465 nm) compared to the feed. The chemical oxygen demand, the
were achieved for dilutions up to 40 times against distilled water
dry residue and the TPC were also reduced by 62%, 30% and 7%,
for the microltred samples and against methanol for the phenolic
respectively. Different dilutions were prepared from the microl-
extracts. All analyses were run in triplicate and averaged. Sample
trate (permeate) for the estimation of its antioxidant activity using
concentration providing 50% inhibition (IC50) was calculated from
different methods.
the graph plotting inhibition percentage against extract concentra-
tion. EDTA calibration solutions (8, 16, 24, 32, 40, and 48 lM) were
prepared and their ICA were determined following the same protocol. 3.3. The OMW total phenolic content composition

OMW1 showed a higher phenolic content (9.8 g/l) compared to

2.6.4. Lipid peroxidation index
OMW2 (6.1 g/l). The main components of the total phenolic
To assess the ability of OMW to inhibit lipid peroxidation, the
content are avonoids (Table 2), a group of natural substances with
method described by Singh, Singh, Kumar, and Arora (2007) was
antioxidant, anti-inammatory, antiallergic, antiviral and anticar-
adopted. The 2-thiobarbituric acid (TBA) reacts with malondialde-
cinogenic properties (Leopoldini et al., 2011). This phenolic group
hyde (MDA) to form a diadduct, a pink chromogen, which can be de-
corresponds to 66.8% of OMW1 phenolic content, but lower
tected spectrophotometrically at 532 nm. Normal male Wistar rats
amounts (44.3%) have been revealed for avonoids in OMW2
(250 g) were used for the preparation of liver homogenate. The per-
(Table 2). Besides their antioxidant activities, avonoids are able
fused liver was isolated, and 10% (w/v) homogenate was prepared at
4 C with 0.15 M KCl. The homogenate was centrifuged at 800g for Table 1
15 min, and clear cell-free supernatant was used for the study of The main physicochemical characteristics of olive mill wastewater samples.
in vitro lipid peroxidation. Different dilutions of microltred OMW Parameters Unit OMW1 OMW2
samples and their methanolic extracts were taken in test tubes.
pH 5.2 0.1 5.1 0.1
One millilitre of 0.15 M KCl and 0.5 ml of rat liver homogenate were EC mS/cm 43 0.9 13 0.5
added to the test tubes. Peroxidation was initiated by adding 100 ll Dry residue g/l 173 13 128 8
of 0.2 mM ferric chloride. After incubation at 37 C for 30 min, the Ash g/l 47 2.5 15 1.2
reaction was stopped by adding 2 ml of icecold HCl (0.25 N) contain- TOC g/l 31 4.3 14 1.9
TPC g of TYE/l 9.82 0.3 6.11 0.2
ing 15% trichloroacetic acid (TCA), 0.38% TBA, and 0.5% BHT. The Sugar g/l 23 1.7 12 1.1
reaction mixtures were heated at 80 C for 60 min. The samples were COD g of O2/l 113 9.8 51 7.6
cooled and centrifuged, and the absorbance of the supernatants was DCOD g of O2/l 10 0.2 8 0.2
measured at 532 nm. The percentage of lipid peroxidation inhibition TSS g/l 87 5.9 48 4.5
Sodium g/l 2.17 0.18 1.68 0.13
(LPI) was calculated by the following formula:
 Potassium g/l 1.34 0.11 0.75 0.06
LPI% 1  Asample =Ablank  100 Calcium g/l 1.92 0.14 1.22 0.11

Values are the average of three measurements standard deviation.

The sample concentration providing 50% inhibition (IC50) was
calculated from the graph plotting inhibition percentage against
extract concentration. BHT calibration solutions (0.05, 0.1, 0.2,
0.3, 0.4, and 0.5 mg/ml) were prepared and their ICA was deter-
mined by following the same protocol as for the samples.
Abbreviations: EC, electrical conductivity; TOC, total organic compounds; TPC, total
phenolic content; COD, Chemical oxygen demand; DCOD, dissolved chemical oxy-
gen demand; TSS, total suspended solids; OMW1 and OMW2 are olive mill
wastewater samples issued from semi-modern and modern three-phase oil
extraction processes, respectively.
 A. El-Abbassi et al. / Food Chemistry 132 (2012) 406412
to inhibit lipid peroxidation and platelet aggregation, as well as im-
prove increased capillary permeability and fragility (Leopoldini
et al., 2011). Main avonoid subgroups in olive mill wastewaters
are avanols and proanthocyanidins. These phenolics were posi-
tively associated to an increased plasma antioxidant activity in
the rats (Facino et al., 1999). Flavanols and proanthocyanidins
showed more or less the same proportions compared to the TPC
of the two OMW samples (Table 2). However, a minute difference
between the two samples can considerably affect its antioxidant
activity since these compounds act at very low concentrations.
The differences in phenolic composition between the two OMW
may be ascribed to any or all of the olive ripeness degree and/or
processing and farming practices.
3.4. HPLC prole of the total phenolic content of OMW
To determine and compare the phenolic proles, an HPLC analy-
sis was performed and the phenolic compounds of OMW were iden-
tied (Fig. 1 and Table 3). The TPC as revealed by the HPLC
quantication represent only 55% and 63.7% of the depicted spec-
trophotometric estimation in Table 2. A drawback of the Folin
Ciocalteu assay is that reducing agents can interfere in the analysis
leading to an overestimation of the phenolic content. OMW1 and
OMW2 showed different phenolic proles in terms of concentration
and also in terms of composition since oleuropein aglycone was de-
tected only in OMW2 (Fig. 1 and Table 3). The results showed that
hydroxytyrosol was the most abundant phenolic compound in
OMW and represents about 70% and 55% of the total phenolic con-
centration of OMW1 and OMW2, respectively. Hydroxytyrosol has
been an important focus of research since its discovery (Ragazzi &
Veronese, 1973). Hydroxytyrosol-4-b-glucoside, hydroxytyrosol
and caffeic acid are hydrolysis products of verbascoside and the
hydroxytyrosol-secoiridoid, and they show a powerful antioxidant
activities (Cofrades et al., 2011; Obied, Bedgood, Prenzler, & Ro-
bards, 2007). Hydroxytyrosol inhibits human LDL oxidation, inhibits
platelet aggregation and exhibits anti-inammatory and anticancer
properties (Bouallagui et al., 2011; Obied et al., 2007). The caffeic
acid also was found in OMW samples (Table 3), but at very low con-
centrations (0.060.09 g of TYE/l). Obied, Prenzler, and Robards
(2008) reported that caffeic acid shows a higher antioxidant activity
(DPPH test) than hydroxytyrosol and oleuropein with an IC50 of 1.7,
2.3 and 7.6 lg/ml, respectively.
Gallic acid which accounts in our OMW samples for 0.30.6 g of
TYE/l was also found to be a strong antioxidant in lipid systems and
exhibits the antihyperglycaemic and antioxidant properties
(Punithavathi, Prince, Kumar, & Selvakumari, 2011). Gallic acid is
used in processed food, cosmetics and food packing materials to
prevent rancidity induced by lipid peroxidation and spoilage
(Yen, Duh, & Tsai, 2002). Tyrosol, which showed similar concentra-
tions (0.25 g of TYE/l) in the both OMW samples (Table 3), is
reported to be effective in preserving cellular anti-oxidant defenses
(Samuel, Thirunavukkarasu, Penumathsa, Paul, & Maulik, 2008).
However, according to our results, tyrosol showed a much lower
Table 2
The total phenolic content and its constituents of different OMW samples.
Sample TPC Flavonoids Flavanols Proanthocyanidins
TYE g/l CAE g/l CAE mg/l CYE mg/l
OMW1 9.82 0.53 6.56 0.21 2.8 0.04 17.03 0.14
(100%) (66.8%) (28.5%) (0.17%)
OMW2 6.11 0.2 2.71 0.14 1.9 0.03 9.02 0.85
(100%) (44.3%) (31.1%) (0.15%)
The values between brackets are the relative proportion for each constituent
compared to the total phenolic content (TPC).
409
antioxidant activity than ascorbic acid and BHT when using DPPH
test (Fig. 2 and Table 4). p-Coumaric acid, which is known to be a
weak antioxidant compound (Terpinc et al., 2011), showed concen-
trations of 0.50.8 g of TYE/l.
Finally, oleuropein aglycone, which is a hydrolysis product of
oleuropein, was found to be useful in the treatment of various
inammatory diseases (Impellizzeri et al., 2011). This compound
was identied only in OMW2 at a low concentration (0.12 g of
TYE/l).
3.5. Antioxidant activities of OMW extracts and microltred samples
To compare the antioxidant capacity of an extract, one test does
not appear to be sufcient since various mechanisms are involved
in the antioxidant action. A multidimensional evaluation of the
antioxidant activity is required (Obied et al., 2007). Our samples
were subjected to four antioxidant assays, representing different
antioxidant mechanisms. The assay of the TAC is based on the
reduction of Mo (VI) to Mo (V) and the subsequent formation of
a green phosphate/Mo (V) complex at acid pH. High absorbance
indicates a signicant antioxidant activity. In this assay, the TACs
of OMW1 and OMW2 microltres (lF) and of their respective phe-
nolic extracts were measured and compared to that of BHT.
According to our results, OMW1-lF and OMW2-lF showed signif-
icant TACs, which were two times higher than that of their respec-
tive phenolic extracts. However, the microltred OMW showed a
13% to 18% lower TAC compared to the BHT (Table 4).
Free radical scavenging ability by hydrogen donation is a well-
known antioxidation mechanism. A freshly prepared DPPH solution
displays a deep purple colour with a maximum absorbance at
517 nm, which gradually decreases in the presence of a good hydro-
gen donor. Results reported in Table 4 demonstrate that the OMW2-
lF showed the highest free radical-scavenging activity (the lowest
IC50), which is higher than the activity of ascorbic acid, followed
by OMW1-lF and OMW2-PhE (with comparable activity to ascorbic
acid) and nally the OMW1-PhE showed the lowest radical scaveng-
ing activity (Table 4). All samples showed lower activity compared
to BHT in term of radical scavenging activity. Decreases of DPPH
absorbances in the presence of ascorbic acid, tyrosol and OMW1-
PhE against time at different dosages are shown in Fig. 2, respec-
tively. It is clear from Fig. 2 that OMW1-PhE exhibited appreciable
DPPH radical scavenging ability. However, the tested antioxidants
showed different DPPH inhibition kinetics. The different reaction
kinetics depend on the nature of the antioxidant reacted with the
DPPH radical. Three types of behaviour were observed. In Fig. 2a,
an example of rapid kinetic behaviour is shown. The ascorbic acid
showed the rst kinetic type of DPPH inhibition. It reacted rapidly
with the DPPH reaching a steady state in less than 1 min. The second
type of behaviour which can be considered as an intermediate type
was shown by OMW1 phenolic extract (Fig. 2). Such behaviour must
be the result of the different individual contributions, of the phenolic
compounds in the sample. For this OMW1-PhE, the steady state was
reached after approximately 20 min. The third kinetic type is shown
by the tyrosol (Fig. 2) which is a slow reaction and did not reach the
steady state within one hour of reaction time. As can be approxi-
mated by the line plotting the DPPH inhibition after 60 min against
tyrosol concentration, the concentration needed to reach 50% of
DPPH inhibition at 60 min of reaction time is found to be 14 g/l of
tyrosol.
The ability of phenolic extracts and microltred samples to
compete with ferrozine for chelating iron(II) ions was measured.
The results showed that a high content of phenol compounds does
not positively correlate with a high Fe2+ chelating activity, espe-
cially in the case of the phenolic extracts (Table 4). Nevertheless,
previous studies have reported chelating properties for phenolic
compounds (Leopoldini et al., 2011). The metal-chelating proper-
410 A. El-Abbassi et al. / Food Chemistry 132 (2012) 406412

550
500 3 OMW1-PhE
450 d:1/6
400
350
300

mV
250
200
150
1
100
2 4 6
50
5
0

550
500 OMW2-PhE
450 d:1/6
400
350
300 3
mV

250
200
150
100 6
1 2 4 7
50 5
0

0 5 10 15 20 25 30
Retention time (min)
Fig. 1. HPLC chromatograms of OMW phenolic extracts: (1) Gallic acid. (2) Hydroxytyrosol-4-b-glucoside. (3) Hydroxytyrosol. (4) Tyrosol. (5) Caffeic acid. (6) p-Coumaric
acid. (7) Oleuropein aglycone. Peaks 3, 4, 5 and 6 were identied by use of standards. The remaining peaks were tentatively identied by comparison with scientic literature
data.

Table 3
OMW phenolic extracts composition as revealed by HPLC analysis.

Phenolic compound OMW1 phenolic extract (OMW1-PhE) OMW2 phenolic extract (OMW2-PhE)
Concentration (g of TYE/l) Proportion (%) Concentration (g of TYE/l) Proportion (%)
Gallic acid 0.583 0.041 10.79 0.76 0.331 0.022 8.51 0.57
Hydroxytyrosol-4-b-glucoside 0.168 0.012 3.10 0.22 0.226 0.015 5.79 0.39
Hydroxytyrosol 3.766 0.243 69.64 4.49 2.127 0.150 54.65 3.85
Tyrosol 2.491 0.017 4.61 0.31 0.246 0.014 6.32 0.36
Cafeic acid 0.092 0.007 1.70 0.12 0.057 0.004 1.48 0.10
Para-coumaric acid 0.549 0.038 10.16 0.70 0.785 0.055 20.16 1.41
Oleuropein aglycone 0 0 0.121 0.008 3.10 0.21
Total 5.407 0.358 100 3.893 0.268 100

ties of phenolic compounds are attributed to specic structural fea-
tures, requiring two points of coordination between the metal and
the phenolic compound. Thus, o-diphenol (30 ,40 -diOH-) in ring B
and ketol (3-OH-4-keto or 5-OH-4-keto) structures in ring C show
good chelating activity (Leopoldini et al., 2011).
The microltrated OMW samples showed the highest iron(II)
chelating activity (ICA) with an IC50 lower than that obtained for
EDTA (Fig. 3). Besides the simple phenolic compounds, the
obtained high ICA can be attributed to the avonoids and tannin con-
tents of microltrated OMW. The ability of tannins to chelate Fe(II)
and other metal ions, such as Cu(II) and Zn(II), was reported by Kar-
amac (2009) in selected edible nuts. Concentrations of hydrolysable
and condensed tannins in OMW were reported to be around 7 and
2.3 g/l, respectively (Hamdi, Khadir, & Garcia et al., 1991).
Iron-induced lipid peroxidation is a well-validated system for
generating reactive oxygen species (Jomova & Valko, 2011). In
biological systems, lipid peroxidation, which refer to the oxidative
degradation of polyunsaturated fatty acids in the cell membranes,
generates a number of degradation products, such as malondialde-
hyde (MDA), and is found to be an important cause of cell mem-
brane destruction and cell damage (Yoshikawa, Naito, & Kondo,
1997). MDA is one of the major products of lipid peroxidation,
which has been extensively studied and measured as an index of
lipid peroxidation and as a marker of oxidative stress (Janero,
1990). Fig. 4 shows that OMW2 exhibited more LPI activity as com-
pared to OMW1. No signicant difference (p > 0.05) was observed
between the microltrate and the phenolic extract of OMW1
(Table 4 and Fig. 5). The LPI and ICA assays are less sensitive com-
pared to the DPPH because their IC50 values were higher and
exceed 1000 lg/ml, whereas in case of DPPH, the IC50 values did
not exceed 263 lg/ml. Iron, a transition metal, is capable of
generating free radicals from peroxides by the Fenton reaction
and is implicated in many human diseases (Jomova & Valko,
2011). Fe2+ has also been reported to produce radicals and lipid
peroxidation, so the reduction of Fe2+ concentrations in the Fenton
reaction would protect from the oxidative damage.
 A. El-Abbassi et al. / Food Chemistry 132 (2012) 406412 411

1.4
Tyrosol (mg/ml) 0.5 1 2
activity in microltrated OMW can be most likely attributed to
Ascorbic acid (mg/ml) 0.1 0.15 0.3 the difference in phenolic compositions between the methanolic
1.2 TPC of OMW1-PhE (mg/ml) 172 218 288 extracts and the microltrated OMW. Furthermore, the antioxidant
activity of various OMW extracts was reported to be directly corre-
Absorbance at 517nm

1 lating with percentage of free hydroxytyrosol and their antioxidant

properties was found to be the result of their phenol composition
0.8
rather than their phenol content (Leonardis et al., 2009).
0.6
The obtained results show a great potential of OMW aqueous
extract (microltrated OMW) to be used as antioxidants in food.
0.4 Studies on the safety and efcacy of olive polyphenols (Christian
et al., 2004) show that polyphenols from olive fruit and its
0.2 by-product can be considered as safe and non-toxic for human
consummation. Christian et al. (2004) reported that no mortality
0 or clinical signs of toxicity were noted after 29 days of consecutive
0 10 20 30 40 50 60
administration of an aqueous olive pulp extract (5 g/kg/day) to
Reaction time (min) Crl:CD Sprague Dawley rats suggesting that the LD50 of the
extract must be greater than 5 g/kg.
Fig. 2. Time course of the decrease of the DPPH absorbance when using different
concentrations of tyrosol, ascorbic acid and OMW1-PhE.

3.6. Effect of storage time on the OMW phenolic content and its
Table 4 antioxidant activity
Antioxidant activities of different phenolic extracts compared to some reference
antioxidants.
OMW samples from semi-modern milling unit were stored at
TAC* IC50 (lg/ml) room temperature in dark during 1 year. Samples were taken peri-
DPPH ICA LPI odically and were analysed in terms of their total phenolic content
and radical scavenging activity (DPPH assay). Fig. 5 shows the evo-
OMW1-PhE 0.124a 0.020 263a 2.5 238a 0.24 1069a 56
OMW2-PhE 0.121a 0.002 169b 5.2 136b 0.19 860b 25 lution of TPC and IC50 against time. The TPC increased considerably
OMW1-lF 0.249b 0.005 161b 4.8 13c 0.02 1103a 77 during the 4 rst months and started to decrease slightly beyond
OMW2-lF 0.262b 0.008 123c 3.7 15cd 0.03 213c 18 the fth month (Fig. 5). The highest antioxidant activity however
EDTA 17d 0.03 (lowest IC50 value), was observed during the third month of storage
Ascorbic acid 158b 14.3
BHT 0.302d 0.019 15.2d 1.1 43d 3.5
(IC50 = 150 lg/ml). The evolution of IC50 does not seem to be corre-
lated to the evolution of total phenolic content during storage
Values are mean standard deviation, n = 3. (Fig. 5). These results are in agreement with the nding of Atanass-
Means with superscripts having the same letter are not signicantly different.
* ova, Kefalas, and Psillakis (2005) who reported that the antioxidant
Determined for a concentration of 0.1 g per litre.
activity of OMW is not directly linked to the total phenols when
estimating the antioxidant activity using chemiluminescence.
OMW are a very complex medium with a mixture of phenolic com-
pounds. Different reactions may take place during storage, some
antioxidants may disappear and/or new molecules can be pro-
duced affecting the antioxidant activity assessed by DPPH test.
The variation of the phenolic content and its antioxidant activity
seems to be also affected by many factors such the initial physico-
chemical parameters of OMW samples, temperature of storage
and, nally, the bacterial and fungal ora existing in OMW.
In Morocco, OMW are only produced during 45 months of the
year, and, consequently, storage may be needed if high-added-

Fig. 3. Iron(II) chelating activity of different samples and EDTA standard against the
concentration.

From these results, it appears clearly that all the antioxidant

activity of the OMW cannot be ascribed exclusively to the phenolic
content. Most likely, some non-phenolic compounds contribute to
the overall antioxidant activity of the OMW or at least enhance the
antioxidant activity of the phenolic compounds especially the total
antioxidant capacity, the Iron(II) chelating activity and the radical
scavenging activity. Since the ethyl acetate liquidliquid extraction
is more selective for low and medium molecular weight phenols
(Visioli et al., 1999) and it is not appropriate for extracting heavier Fig. 4. Lipid peroxidation inhibition by different OMW samples and BHT standard
molecules that remain in the water phase, the high antioxidant at different concentrations.
412 A. El-Abbassi et al. / Food Chemistry 132 (2012) 406412
Fig. 5. Evolution of the total phenolic content (TPC) and the antioxidant activity
(IC50, DPPH test) of OMW during storage at room temperature over 1 year.
value compounds are to be recovered from these efuents during
extended periods out of the olive oil production season. Few stud-
ies investigated the effect of storage on the phenolic content of
OMW. Feki, Allouche, Bouaziz, Gargoubi, and Sayadi (2006), re-
ported a signicant accumulation of hydroxytyrosol after 5 months
of storage. The corresponding concentration increased by 257
302%. However, the concentrations of the other phenolic com-
pounds were markedly decreased. Furthermore, it was reported
that the OMW storage facilitates the liquidliquid extraction pro-
cedure and improves the extraction yield of hydroxytyrosol which,
increased from 85.5% to 96.8%. (Feki et al., 2006).
4. Conclusion
The use of several antioxidant activity determinations with dif-
fering reaction mechanisms is necessary to give an overall under-
standing of the mechanisms of action of an antioxidant.
Signicant differences between phenolic extracts and micro-l-
trates of OMW were observed in terms of antioxidant potential.
This nding may be attributed to the fact that not all phenolics
are extracted by ethyl acetate, and besides some non-phenolic
compounds existing in microltrates may exhibit an antioxidant
activity. Furthermore, some phenolic subgroups such as avonoids
show higher antioxidant activity in the aqueous phase.
Further studies are needed for the isolation and characterisation
of the non-phenolic fraction and elucidate its antioxidant mecha-
nisms and/or the existence of possible synergism, if any, with the
phenolic compounds.
OMW seems promising as a source for natural high-added-value
compounds. An appropriate extraction method should be developed
and an integrate treatment and valorisation process can be established.
Acknowledgments
The authors acknowledge the International Foundation for Sci-
ences (IFS) for the nancial support of this work under the Grant
Number W4749. In addition, the authors greatly thank Prof. Moha
Taourirte and Mr. Mounsef Neffa from the Faculty of Sciences and
Technologies Gueliz (Marrakech, Morocco) for their collaboration
in the HPLC analysis.
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