Food Chemistry 132 (2012) 406–412

Contents lists available at SciVerse ScienceDirect

Food Chemistry

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

Phenolic profile and antioxidant activities of olive mill wastewater

Abdelilah El-Abbassi, Hajar Kiai, Abdellatif Hafidi ⇑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:Received 3 July 2011Received in revised form 25 September 2011Accepted 2 November 2011Available online 10 November 2011

Keywords:Olive mill wastewatersPhenolic compoundsAntioxidant activityRadical scavengingIron(II) chelating activityLipid peroxidation

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.11.013

⇑ Corresponding author. Tel.: +212 524 434 649x51E-mail addresses: a.hafidi@ucam.ac.ma, hafidi.abdella

Olive trees play an important role in the Moroccan agro-economy, providing both employment and exportrevenue. However, the olive oil industry generates large amounts of wastes and wastewaters. The disposalof these polluting by-products is a significant environmental problem that needs an adequate solution. Onone hand, the phytotoxic and antimicrobial effects of olive mill wastewaters are mainly due to their pheno-lic content. The hydrophilic character of the polyphenols results in the major proportion of natural phenolsbeing separated into the water phase during the olive processing. On other hand, the health benefits arisingfrom a diet containing olive oil have been attributed to its richness in phenolic compounds that act as nat-ural antioxidants and are thought to contribute to the prevention of heart diseases and cancers. Olive millwastewater (OMW) samples have been analysed in terms of their phenolic constituents and antioxidantactivities. The total phenolic content, flavonoids, flavanols, and proanthocyanidins were determined. Theantioxidant and radical scavenging activity of phenolic extracts and microfiltred samples was evaluatedusing 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 tosome synthetic antioxidants commonly used in the food industry.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction et al., 2007). Epidemiological studies have correlated the low inci-

Olive mill wastewaters (OMW) are the main liquid effluentsgenerated by the olive oil production industry. The annual worldOMW production is estimated from 10 to over 30 million m3

(Eroglu, Eroglu, Gündüz, Türker, & Yücel, 2006). Although thequantity of the waste produced is still much smaller than othertypes of waste and its production is seasonal, the contribution ofOMW 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 aquaticreservoirs, seashores, and sea) without pretreatment, leads to se-vere problems for the whole ecosystem. OMW also show a toxicaction to some plants and microorganisms since they exhibit a sub-stantial concentration of phenolic compounds. These latter are thelargest family of naturally occurring antioxidants in plants that in-clude a wide variety of structures with a common motif, the phenolmolecule. They expand from the simplest structures, such as phe-nolic acids and alcohols, to the most complex oligomeric ones suchas proanthocyanidins.

According to several studies, phenolic compounds from oliveshave significant health benefits. They possess cancer chemopreven-tive, cardioprotective, and neuroprotective activities (Tsagaraki

ll rights reserved.

5; fax: +212 524 436 769.tif@googlemail.com (A. Hafidi).

dence of coronary heart disease, atherosclerosis, and some types ofcancer (colorectal and breast cancer) with olive oil consumption inthe Mediterranean diet (Feki, Allouche, & Sayadi, 2005). Natural phe-nols from olive and its by-products are now recognised as potentialtargets for the food, cosmetic and pharmaceutical industries. Nowa-days, interest in novel sources of natural antioxidants is steadilygrowing. Since OMW is available in huge quantities and exhibitshigh concentrations of phenolic compounds, they may turn into anatural source of valuable and powerful antioxidants within thefew 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 profile and the antioxidant activity of OMW samples generatedby two different olive oil processing techniques (olive press and de-canter centrifugation systems) using four tests with different actingmechanisms. We have also investigated the effect of storage onOMW phenolic content and on the antioxidant capacity.

2. Materials and methods

2.1. Chemicals

10-10Diphenyl-20picrylhydrazyl (DPPH), 2-thiobarbituric acid,potassium ferricyanide, tyrosol, ferrozine, Folin–Cioucalteureagent, ferric chloride, ethylenediaminetetraacetic acid (EDTA), L-ascorbic acid, sodium hydroxide, and trichloroacetic acid were pur-chased from Sigma Aldrich (Germany). Ascorbic acid and butylated

A. El-Abbassi et al. / Food Chemistry 132 (2012) 406–412 407

hydroxytoluene (BHT) were procured from Merck (France). Othersolvents were obtained from Sigma Aldrich Germany and were ofanalytical grade.

2.2. Physicochemical characterisation of OMW samples

Olive mill samples were collected from two different olive oilmills in the area of Marrakech during the season of 2008/2009.The two mills used different milling techniques, which weresemi-modern (OMW1) and modern (OMW2) three-phase pro-cesses. The processed olive fruits are from the Moroccan Picholinevariety. The physicochemical characterisation of OMW sampleswas carried out as follow:

- Total organic carbone (TOC) was determined using EUROGLASTOC analyser (Thermo Scientific, Germany).

- Chemical oxygen demand (COD) was determined by the dichro-mate method. The appropriate amount of wastewater sampleswas diluted up to 100 times and introduced into a lab-prepareddigestion solution containing potassium dichromate, sulphuricacid and mercuric sulphate and the mixture was then incubatedfor 120 min at 150 �C in a COD reactor (Model WTW CR3000,Germany). COD concentration was measured colorimetricallyat 600 nm using a MultiLab P5 (WTW, Germany). The standardsolutions of 1, 2, 3, 4 g of O2 per litre were prepared using thepotassium biphthalate.

- Dissolved chemical oxygen demand (DCOD) was measured onultrafiltrated (on membrane of 50 kDa MWCO; Microdyn-NadirGmbH, Germany) samples using the same protocol as for COD.

- Total suspended solids was determined after ultrafiltration of theolive mill wastewater samples through a membrane of 50 kDa(MWCO). The dry residue was then determined by drying thepermeate at 105 �C overnight and expressed as g of TSS per litre.

2.3. OMW microfiltration

A polyethersulfone membrane (MicrodynNadir, Germany) with0.05 lm pore size was used. Microfiltration was carried out at aroom temperature in a stirred ultrafiltration cell (AMICON 8200,Millipore USA) with a 200 ml volume. The effective surface areaof the membrane was 28.7 cm2. The transmembrane pressurewas applied with pressurised nitrogen gas. Microfiltration wasconducted under a transmembrane pressure of 4 bars and the cellwas stirred at 250 rpm using a magnetic stirrer. The obtained per-meate was directly used for antioxidant assays and compared tothe phenolic extract from OMW.

2.4. OMW extracts

The phenolic extract was obtained by a liquid–liquid extractionof the OMW. First, the pH of OMW samples (5 ml) was adjusted topH 2 using HCl (2 M). After defatting with n-hexane, extractionswith ethyl acetate were performed thrice, and the three extractswere brought to dryness by vacuum evaporation at 40 �C, and thenrecuperated in 5 ml methanol. The resulting extract is called ‘phe-nolic extract’.

2.5. Total phenolic content, flavonoids, flavanols, and proanthocyanidins determinations

- Total phenolic content (TPC) was determined following theFolin–Ciocalteu spectrophotometeric using tyrosol as a standard.The phenolic extract (0.1 ml) in a volumetric flask was dilutedwith distilled water (3.4 ml). Folin–Ciocalteu reagent (0.5 ml)was added and the contents of flask were mixed thoroughly. After3 min 1 ml of a 20% anhydrous sodium carbonate solution (w/v)

was added, and then the mixture was allowed to stand for 1 h inthe dark. The optical density of the blue-coloured samples wasmeasured at 765 nm. The total phenolic content was determinedas tyrosol equivalents (TYE) and values are expressed as g of tyro-sol/l of olive oil mill wastewaters.

- For the total flavonoids, a modified method from Kim, Chun,Kim, Moon, and Lee (2003) was used. A 0.2 ml aliquot of extractappropriately diluted was mixed with 0.8 ml distilled water in a5 ml assays tube, 0.06 ml 5% NaNO2 was added, and allowed toreact for 5 min. Afterwards, 0.04 ml 10% AlCl3 was added andthe 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 similarlyprepared blank, by replacing the extract with distilled water.Total flavonoid content was calculated from a calibration curveusing catechin as a standard, and expressed as mg catechinequivalents (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 intoa 5 ml assays tube and 0.5 ml HCl (0.24 N in methanol) and0.5 ml DMACA solution (0.2% in methanol) were added. Themixture was allowed to react for 5 min at room temperature,and the absorbance was determined at 640 nm. The controlwas prepared by replacing sample with methanol. The concen-tration of total flavanols was calculated from a calibrationcurve, using catechin as a standard. The results are expressedas mg of catechin equivalents (CTE) per litre of the extract.

- Proanthocyanidins were analysed by the method described byWaterman and Mole (1994). Butanol reagent was prepared bymixing 70 mg ferrous sulphate (FeSO4) with 5 ml concentratedHCl and made to 100 ml with n-butanol. An aliquot of 0.1 mlsample was mixed thoroughly with 1.4 ml butanol reagentand heated at 95 �C in a water bath for 45 min. The samplewas than cooled, 0.5 ml n-butanol was added and the absor-bance was measured at 550 nm. Results were expressed ascyanidin equivalents (CYE) per litre of the extract using a molarextinction coefficient 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 capacityThe 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 ammoniummolybdate). The reaction mixture was incubated at 95 �C for150 min. Once the mixture cooled to room temperature, the absor-bance of the mixture was measured at 695 nm against a blank. Thereadings were taken every 30 min. The antioxidant activity wasexpressed as the absorbance of the sample. The antioxidant activ-ity of BHT (0.5 mg/ml) and a-tocopherol (0.5 mg/ml) were alsoassayed for comparison.

2.6.2. Free radical-scavenging abilityFree 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 bydissolving 4 mg of DPPH� in 100 ml of ethanol. A 100 ll aliquotof the sample, adequately diluted with ethanol, was placed in acuvette and reacted with 3 ml of DPPH�working solution. The mix-ture was shaken vigorously and left to stand for 60 min at roomtemperature in the dark. The decrease in absorbance was measuredat 517 nm after 60 min, against ethanol as a blank. Low absorbanceof the reaction mixture indicates high free radical-scavenging

Table 1The main physicochemical characteristics of olive mill wastewater samples.

Parameters Unit OMW1 OMW2

pH – 5.2 ± 0.1 5.1 ± 0.1EC mS/cm 43 ± 0.9 13 ± 0.5Dry residue g/l 173 ± 13 128 ± 8Ash g/l 47 ± 2.5 15 ± 1.2TOC g/l 31 ± 4.3 14 ± 1.9TPC g of TYE/l 9.82 ± 0.3 6.11 ± 0.2Sugar g/l 23 ± 1.7 12 ± 1.1COD g of O2/l 113 ± 9.8 51 ± 7.6DCOD g of O2/l 10 ± 0.2 8 ± 0.2TSS g/l 87 ± 5.9 48 ± 4.5Sodium g/l 2.17 ± 0.18 1.68 ± 0.13Potassium g/l 1.34 ± 0.11 0.75 ± 0.06Calcium g/l 1.92 ± 0.14 1.22 ± 0.11

Values are the average of three measurements ± standard deviation.Abbreviations: EC, electrical conductivity; TOC, total organic compounds; TPC, totalphenolic content; COD, Chemical oxygen demand; DCOD, dissolved chemical oxy-gen demand; TSS, total suspended solids; OMW1 and OMW2 are olive millwastewater samples issued from semi-modern and modern three-phase oilextraction processes, respectively.

408 A. El-Abbassi et al. / Food Chemistry 132 (2012) 406–412

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

% Inhibition ¼ 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 thegraph plotting inhibition percentage against extract concentration.The obtained results were expressed as mg TYE/l of phenolic extractneeded to reduce DPPH radical signal by 50%. The Free radical-scavenging ability of ascorbic acid and BHT was also evaluatedand compared to our extracts.

2.6.3. Iron(II) chelating activity (ICA)The chelating of ferrous ions by the sample was estimated using

the method described by Yen, Duh, and Chuange (2000) with mod-ification. The adequately diluted phenolic extract (0.1 ml) wasmixed with methanol (2.6 ml) and 2 mM FeCl2 (0.1 ml) and then5 mM ferrozine (0.2 ml). The mixture was shaken vigorously andleft to stand at room temperature in the dark for 10 min. Absor-bance of the resulting solution was measured spectrophotometri-cally at 562 nm. A low absorbance of the resulting solutionindicated a strong Fe2+-chelating ability. The ability to chelate fer-rous ion (ICA) and prevent formation of ferrous ion-ferrozine com-plex, was calculated using the following equation:

ICA ð%Þ ¼ 1� ðAsample=AcontrolÞ� �

� 100

where Acontrol was the absorbance of a mixture of methanol (2.7 ml),2 mM FeCl2 (0.1 ml) and 5 mM ferrozine (0.2 ml). Measurementswere achieved for dilutions up to 40 times against distilled waterfor the microfiltred samples and against methanol for the phenolicextracts. All analyses were run in triplicate and averaged. Sampleconcentration providing 50% inhibition (IC50) was calculated fromthe graph plotting inhibition percentage against extract concentra-tion. EDTA calibration solutions (8, 16, 24, 32, 40, and 48 lM) wereprepared and their ICA were determined following the same protocol.

2.6.4. Lipid peroxidation indexTo assess the ability of OMW to inhibit lipid peroxidation, the

method described by Singh, Singh, Kumar, and Arora (2007) wasadopted. The 2-thiobarbituric acid (TBA) reacts with malondialde-hyde (MDA) to form a diadduct, a pink chromogen, which can be de-tected spectrophotometrically at 532 nm. Normal male Wistar rats(250 g) were used for the preparation of liver homogenate. The per-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 for15 min, and clear cell-free supernatant was used for the study ofin vitro lipid peroxidation. Different dilutions of microfiltred OMWsamples and their methanolic extracts were taken in test tubes.One millilitre of 0.15 M KCl and 0.5 ml of rat liver homogenate wereadded to the test tubes. Peroxidation was initiated by adding 100 llof 0.2 mM ferric chloride. After incubation at 37 �C for 30 min, thereaction was stopped by adding 2 ml of icecold HCl (0.25 N) contain-ing 15% trichloroacetic acid (TCA), 0.38% TBA, and 0.5% BHT. Thereaction mixtures were heated at 80 �C for 60 min. The samples werecooled and centrifuged, and the absorbance of the supernatants wasmeasured at 532 nm. The percentage of lipid peroxidation inhibition(LPI) was calculated by the following formula:

LPI% ¼ 1� Asample=Ablank� �

� 100

The sample concentration providing 50% inhibition (IC50) wascalculated from the graph plotting inhibition percentage againstextract 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.

2.7. HPLC analysis of OMW extracts

The analysis was performed on a JASCO HPLC system, equippedby a JASCO UV detector (UV-975) operating at 280 nm. The columnused 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/wateracidified with acetic acid was chosen as the optimal mobile phase.The flow rate was 0.8 ml/min and the injection volume was 20 ll.The identification of phenolic compounds was fulfilled on the basisof their retention time in comparison with phenolic standards.

3. Results and discussion

3.1. Physicochemical characterisation of OMW samples

Table 1 shows the main physicochemical characteristics ofOMW samples. OMW samples are slightly acidic. OMW from thesemi-modern (OMW1) unit showed high electrical conductivity,which was more than three times that of the OMW2. TraditionallyMoroccan farmers preserve olive fruits during storage by salt addi-tion. In modern milling units more water is used, and for this rea-son, almost all parameters values were reduced in comparison tosamples from the semi-modern unit (OMW1).

3.2. OMW microfiltration

Microfiltration of OMW exhibits a reasonable flux (350 l/h�m2)and the obtained permeate was less coloured (80% less at465 nm) compared to the feed. The chemical oxygen demand, thedry residue and the TPC were also reduced by 62%, 30% and 7%,respectively. Different dilutions were prepared from the microfil-trate (permeate) for the estimation of its antioxidant activity usingdifferent methods.

3.3. The OMW total phenolic content composition

OMW1 showed a higher phenolic content (9.8 g/l) compared toOMW2 (6.1 g/l). The main components of the total phenoliccontent are flavonoids (Table 2), a group of natural substances withantioxidant, anti-inflammatory, antiallergic, antiviral and anticar-cinogenic properties (Leopoldini et al., 2011). This phenolic groupcorresponds to 66.8% of OMW1 phenolic content, but loweramounts (44.3%) have been revealed for flavonoids in OMW2(Table 2). Besides their antioxidant activities, flavonoids are able

A. El-Abbassi et al. / Food Chemistry 132 (2012) 406–412 409

to inhibit lipid peroxidation and platelet aggregation, as well as im-prove increased capillary permeability and fragility (Leopoldiniet al., 2011). Main flavonoid subgroups in olive mill wastewatersare flavanols and proanthocyanidins. These phenolics were posi-tively associated to an increased plasma antioxidant activity inthe rats (Facino et al., 1999). Flavanols and proanthocyanidinsshowed more or less the same proportions compared to the TPCof the two OMW samples (Table 2). However, a minute differencebetween the two samples can considerably affect its antioxidantactivity since these compounds act at very low concentrations.The differences in phenolic composition between the two OMWmay be ascribed to any or all of the olive ripeness degree and/orprocessing and farming practices.

3.4. HPLC profile of the total phenolic content of OMW

To determine and compare the phenolic profiles, an HPLC analy-sis was performed and the phenolic compounds of OMW were iden-tified (Fig. 1 and Table 3). The TPC as revealed by the HPLCquantification 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 analysisleading to an overestimation of the phenolic content. OMW1 andOMW2 showed different phenolic profiles in terms of concentrationand also in terms of composition since oleuropein aglycone was de-tected only in OMW2 (Fig. 1 and Table 3). The results showed thathydroxytyrosol was the most abundant phenolic compound inOMW and represents about 70% and 55% of the total phenolic con-centration of OMW1 and OMW2, respectively. Hydroxytyrosol hasbeen an important focus of research since its discovery (Ragazzi &Veronese, 1973). Hydroxytyrosol-4-b-glucoside, hydroxytyrosoland caffeic acid are hydrolysis products of verbascoside and thehydroxytyrosol-secoiridoid, and they show a powerful antioxidantactivities (Cofrades et al., 2011; Obied, Bedgood, Prenzler, & Ro-bards, 2007). Hydroxytyrosol inhibits human LDL oxidation, inhibitsplatelet aggregation and exhibits anti-inflammatory and anticancerproperties (Bouallagui et al., 2011; Obied et al., 2007). The caffeicacid also was found in OMW samples (Table 3), but at very low con-centrations (0.06–0.09 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 0.3–0.6 g ofTYE/l was also found to be a strong antioxidant in lipid systems andexhibits the antihyperglycaemic and antioxidant properties(Punithavathi, Prince, Kumar, & Selvakumari, 2011). Gallic acid isused in processed food, cosmetics and food packing materials toprevent 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), isreported 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 2The total phenolic content and its constituents of different OMW samples.

Sample TPC Flavonoids Flavanols ProanthocyanidinsTYE 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 constituentcompared to the total phenolic content (TPC).

antioxidant activity than ascorbic acid and BHT when using DPPHtest (Fig. 2 and Table 4). p-Coumaric acid, which is known to be aweak antioxidant compound (Terpinc et al., 2011), showed concen-trations of 0.5–0.8 g of TYE/l.

Finally, oleuropein aglycone, which is a hydrolysis product ofoleuropein, was found to be useful in the treatment of variousinflammatory diseases (Impellizzeri et al., 2011). This compoundwas identified only in OMW2 at a low concentration (0.12 g ofTYE/l).

3.5. Antioxidant activities of OMW extracts and microfiltred samples

To compare the antioxidant capacity of an extract, one test doesnot appear to be sufficient since various mechanisms are involvedin the antioxidant action. A multidimensional evaluation of theantioxidant activity is required (Obied et al., 2007). Our sampleswere subjected to four antioxidant assays, representing differentantioxidant mechanisms. The assay of the TAC is based on thereduction of Mo (VI) to Mo (V) and the subsequent formation ofa green phosphate/Mo (V) complex at acid pH. High absorbanceindicates a significant antioxidant activity. In this assay, the TACsof OMW1 and OMW2 microfiltres (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 microfiltred OMW showed a13% 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 solutiondisplays a deep purple colour with a maximum absorbance at517 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 lowestIC50), which is higher than the activity of ascorbic acid, followedby OMW1-lF and OMW2-PhE (with comparable activity to ascorbicacid) and finally the OMW1-PhE showed the lowest radical scaveng-ing activity (Table 4). All samples showed lower activity comparedto BHT in term of radical scavenging activity. Decreases of DPPHabsorbances 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 appreciableDPPH radical scavenging ability. However, the tested antioxidantsshowed different DPPH inhibition kinetics. The different reactionkinetics depend on the nature of the antioxidant reacted with theDPPH radical. Three types of behaviour were observed. In Fig. 2a,an example of rapid kinetic behaviour is shown. The ascorbic acidshowed the first kinetic type of DPPH inhibition. It reacted rapidlywith the DPPH reaching a steady state in less than 1 min. The secondtype of behaviour which can be considered as an intermediate typewas shown by OMW1 phenolic extract (Fig. 2). Such behaviour mustbe the result of the different individual contributions, of the phenoliccompounds in the sample. For this OMW1-PhE, the steady state wasreached after approximately 20 min. The third kinetic type is shownby the tyrosol (Fig. 2) which is a slow reaction and did not reach thesteady state within one hour of reaction time. As can be approxi-mated by the line plotting the DPPH inhibition after 60 min againsttyrosol concentration, the concentration needed to reach 50% ofDPPH inhibition at 60 min of reaction time is found to be 14 g/l oftyrosol.

The ability of phenolic extracts and microfiltred samples tocompete with ferrozine for chelating iron(II) ions was measured.The results showed that a high content of phenol compounds doesnot 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 phenoliccompounds (Leopoldini et al., 2011). The metal-chelating proper-

050

100150200250300350400450500550

0 5 10 15 20 25 30

mV

Retention time (min)

1 2

3

4 5

67

OMW2-PhEd:1/6

050

100150200250300350400450500550

mV

1

2

3

45

6

OMW1-PhEd:1/6

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-Coumaricacid. (7) Oleuropein aglycone. Peaks 3, 4, 5 and 6 were identified by use of standards. The remaining peaks were tentatively identified by comparison with scientific literaturedata.

Table 3OMW 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 ± 0.57Hydroxytyrosol-4-b-glucoside 0.168 ± 0.012 3.10 ± 0.22 0.226 ± 0.015 5.79 ± 0.39Hydroxytyrosol 3.766 ± 0.243 69.64 ± 4.49 2.127 ± 0.150 54.65 ± 3.85Tyrosol 2.491 ± 0.017 4.61 ± 0.31 0.246 ± 0.014 6.32 ± 0.36Cafeic acid 0.092 ± 0.007 1.70 ± 0.12 0.057 ± 0.004 1.48 ± 0.10Para-coumaric acid 0.549 ± 0.038 10.16 ± 0.70 0.785 ± 0.055 20.16 ± 1.41Oleuropein aglycone 0 0 0.121 ± 0.008 3.10 ± 0.21

Total 5.407 ± 0.358 100 3.893 ± 0.268 100

410 A. El-Abbassi et al. / Food Chemistry 132 (2012) 406–412

ties of phenolic compounds are attributed to specific structural fea-tures, requiring two points of coordination between the metal andthe phenolic compound. Thus, o-diphenol (30,40-diOH-) in ring Band ketol (3-OH-4-keto or 5-OH-4-keto) structures in ring C showgood chelating activity (Leopoldini et al., 2011).

The microfiltrated OMW samples showed the highest iron(II)chelating activity (ICA) with an IC50 lower than that obtained forEDTA (Fig. 3). Besides the simple phenolic compounds, theobtained high ICA can be attributed to the flavonoids and tannin con-tents of microfiltrated 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 hydrolysableand condensed tannins in OMW were reported to be around 7 and2.3 g/l, respectively (Hamdi, Khadir, & Garcia et al., 1991).

Iron-induced lipid peroxidation is a well-validated system forgenerating reactive oxygen species (Jomova & Valko, 2011). Inbiological systems, lipid peroxidation, which refer to the oxidativedegradation 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 oflipid 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 significant difference (p > 0.05) was observedbetween the microfiltrate 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 andexceed 1000 lg/ml, whereas in case of DPPH, the IC50 values didnot exceed 263 lg/ml. Iron, a transition metal, is capable ofgenerating free radicals from peroxides by the Fenton reactionand is implicated in many human diseases (Jomova & Valko,2011). Fe2+ has also been reported to produce radicals and lipidperoxidation, so the reduction of Fe2+ concentrations in the Fentonreaction would protect from the oxidative damage.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 10 20 30 40 50 60

Abs

orba

nce

at 5

17nm

Reaction time (min)

Tyrosol (mg/ml)Ascorbic acid (mg/ml)

TPC of OMW1-PhE (mg/ml)

0.5 1 20.1 0.15 0.3172 218 288

Fig. 2. Time course of the decrease of the DPPH absorbance when using differentconcentrations of tyrosol, ascorbic acid and OMW1-PhE.

Table 4Antioxidant activities of different phenolic extracts compared to some referenceantioxidants.

TAC* IC50 (lg/ml)

DPPH ICA LPI

OMW1-PhE 0.124a ± 0.020 263a ± 2.5 238a ± 0.24 1069a ± 56OMW2-PhE 0.121a ± 0.002 169b ± 5.2 136b ± 0.19 860b ± 25OMW1-lF 0.249b ± 0.005 161b ± 4.8 13c ± 0.02 1103a ± 77OMW2-lF 0.262b ± 0.008 123c ± 3.7 15cd ± 0.03 213c ± 18EDTA – – 17d ± 0.03 –Ascorbic acid – 158b ± 14.3 – –BHT 0.302d ± 0.019 15.2d ± 1.1 – 43d ± 3.5

Values are mean ± standard deviation, n = 3.Means with superscripts having the same letter are not significantly different.* Determined for a concentration of 0.1 g per litre.

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

Fig. 4. Lipid peroxidation inhibition by different OMW samples and BHT standardat different concentrations.

A. El-Abbassi et al. / Food Chemistry 132 (2012) 406–412 411

From these results, it appears clearly that all the antioxidantactivity of the OMW cannot be ascribed exclusively to the phenoliccontent. Most likely, some non-phenolic compounds contribute tothe overall antioxidant activity of the OMW or at least enhance theantioxidant activity of the phenolic compounds especially the totalantioxidant capacity, the Iron(II) chelating activity and the radicalscavenging activity. Since the ethyl acetate liquid–liquid extractionis more selective for low and medium molecular weight phenols(Visioli et al., 1999) and it is not appropriate for extracting heaviermolecules that remain in the water phase, the high antioxidant

activity in microfiltrated OMW can be most likely attributed tothe difference in phenolic compositions between the methanolicextracts and the microfiltrated OMW. Furthermore, the antioxidantactivity of various OMW extracts was reported to be directly corre-lating with percentage of free hydroxytyrosol and their antioxidantproperties was found to be the result of their phenol compositionrather than their phenol content (Leonardis et al., 2009).

The obtained results show a great potential of OMW aqueousextract (microfiltrated OMW) to be used as antioxidants in food.Studies on the safety and efficacy of olive polyphenols (Christianet al., 2004) show that polyphenols from olive fruit and itsby-product can be considered as safe and non-toxic for humanconsummation. Christian et al. (2004) reported that no mortalityor clinical signs of toxicity were noted after 29 days of consecutiveadministration of an aqueous olive pulp extract (5 g/kg/day) toCrl:CD� Sprague Dawley rats suggesting that the LD50 of theextract must be greater than 5 g/kg.

3.6. Effect of storage time on the OMW phenolic content and itsantioxidant activity

OMW samples from semi-modern milling unit were stored atroom temperature in dark during 1 year. Samples were taken peri-odically and were analysed in terms of their total phenolic contentand radical scavenging activity (DPPH assay). Fig. 5 shows the evo-lution of TPC and IC50 against time. The TPC increased considerablyduring the 4 first months and started to decrease slightly beyondthe fifth month (Fig. 5). The highest antioxidant activity however(lowest IC50 value), was observed during the third month of storage(IC50 = 150 lg/ml). The evolution of IC50 does not seem to be corre-lated to the evolution of total phenolic content during storage(Fig. 5). These results are in agreement with the finding of Atanass-ova, Kefalas, and Psillakis (2005) who reported that the antioxidantactivity of OMW is not directly linked to the total phenols whenestimating 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, someantioxidants 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 activityseems to be also affected by many factors such the initial physico-chemical parameters of OMW samples, temperature of storageand, finally, the bacterial and fungal flora existing in OMW.

In Morocco, OMW are only produced during 4–5 months of theyear, and, consequently, storage may be needed if high-added-

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.

412 A. El-Abbassi et al. / Food Chemistry 132 (2012) 406–412

value compounds are to be recovered from these effluents duringextended periods out of the olive oil production season. Few stud-ies investigated the effect of storage on the phenolic content ofOMW. Feki, Allouche, Bouaziz, Gargoubi, and Sayadi (2006), re-ported a significant accumulation of hydroxytyrosol after 5 monthsof storage. The corresponding concentration increased by 257–302%. However, the concentrations of the other phenolic com-pounds were markedly decreased. Furthermore, it was reportedthat the OMW storage facilitates the liquid–liquid 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.Significant differences between phenolic extracts and micro-fil-trates of OMW were observed in terms of antioxidant potential.This finding may be attributed to the fact that not all phenolicsare extracted by ethyl acetate, and besides some non-phenoliccompounds existing in microfiltrates may exhibit an antioxidantactivity. Furthermore, some phenolic subgroups such as flavonoidsshow higher antioxidant activity in the aqueous phase.

Further studies are needed for the isolation and characterisationof the non-phenolic fraction and elucidate its antioxidant mecha-nisms and/or the existence of possible synergism, if any, with thephenolic compounds.

OMW seems promising as a source for natural high-added-valuecompounds. An appropriate extraction method should be developedand an integrate treatment and valorisation process can be established.

Acknowledgments

The authors acknowledge the International Foundation for Sci-ences (IFS) for the financial support of this work under the GrantNumber W4749. In addition, the authors greatly thank Prof. MohaTaourirte and Mr. Mounsef Neffa from the Faculty of Sciences andTechnologies – Gueliz (Marrakech, Morocco) for their collaborationin the HPLC analysis.

References

Atanassova, D., Kefalas, P., & Psillakis, E. (2005). Measuring the antioxidant activityof olive oil mill wastewater using chemiluminescence. EnvironmentInternational, 31(2), 275–280.

Bouallagui, Z., Bouaziz, M., Lassoued, S., Engasser, J. M., Ghoul, M., & Sayadi, S.(2011). Hydroxytyrosol Acyl Esters: Biosynthesis and Activities. AppliedBiochemistry and Biotechnology, 163(5), 592–599.

Christian, M., Sharper, V., Hoberman, A., Seng, J., Fu, L., Covell, D., et al. (2004). Thetoxicity profile of hydrolyzed aqueous olive pulp extract. Drug and ChemicalToxicology, 27, 309–330.

Cofrades, S., Salcedo Sandoval, L., Delgado-Pando, G., López-López, I., Ruiz-Capillas,C., & Jiménez-Colmenero, F. (2011). Antioxidant activity of hydroxytyrosol infrankfurters enriched with n-3 polyunsaturated fatty acids. Food Chemistry,129(2), 429–436.

Eroglu, E., Eroglu, _I., Gündüz, U., Türker, L., & Yücel, M. (2006). Biological hydrogenproduction from olive mill wastewater with two-stage processes. InternationalJournal of Hydrogen Energy, 31, 1527–1535.

Facino, R. M., Carini, M., Aldini, G., Berti, F., Rossoni, G., Bombardelli, E., et al. (1999).Diet enriched with procyanidins enhances antioxidant activity and reducesmyocardial post-ischaemic damage in rats. Life Sciences, 64, 627–642.

Feki, M., Allouche, N., Bouaziz, M., Gargoubi, A., & Sayadi, S. (2006). Effect of storageof olive mill wastewaters on hydroxytyrosol concentration. European Journal ofLipid Science and Technology, 108, 1021–1027.

Feki, I., Allouche, N., & Sayadi, S. (2005). The use of polyphenolic extract, purifiedhydroxytyrosol and 3, 4- dihydroxyphenyl acetic acid from olive millwastewater for the stabilization of refined oils: a potential alternative tosynthetic antioxidants. Food Chemistry, 93, 197–204.

Hamdi, M., Khadir, A., & Garcia, J. L. (1991). The use of Aspergillus niger for thebioconversion of olive mill waste-waters. Applied Microbiological Biotechnology,34, 828–831.

Impellizzeri, D., Esposito, E., Mazzon, E., Paterniti, I., Di Paola, R., Bramanti, P., et al.(2011). The effects of oleuropein aglycone, an olive oil compound, in a mousemodel of carrageenan-induced pleurisy. Clinical Nutrition, doi:10.1016/j.clnu.2011.02.004.

Janero, D. R. (1990). Malondialdehyde and thiobarbituric acid reactivity asdiagonostic indices of lipid peroxidation and peroxidative tissue injury. FreeRadical Biology and Medecine, 9, 515–540.

Jomova, K., & Valko, M. (2011). Advances in metal-induced oxidative stress andhuman disease. Toxicology, 283, 65–87.

Karamac, M. (2009). Chelation of Cu(II), Zn(II), and Fe(II) by tannin constituents ofselected edible nuts. International Journal of Molecular Sciences, 10, 5485–5497.

Kim, D. O., Chun, O. K., Kim, Y. J., Moon, H. Y., & Lee, C. Y. (2003). Quantification ofpolyphenolics and their antioxidant capacity in fresh plums. Journal ofAgricultural and Food Chemistry, 51, 6509–6515.

Leonardis, A., Macciola, V., & Nag, A. (2009). Antioxidant activity of various phenolextracts of olive-oil mill wastewaters. Acta Alimentaria, 38(1), 77–86.

Leopoldini, M., Russo, N., & Toscano, M. (2011). The molecular basis of workingmechanism of natural polyphenolic antioxidants. Food Chemistry, 125, 288–306.

Nigel, C. W., & Glories, Y. (1991). Use of a modified dimethylaminocinnamaldehydereagent for analysis of flavanols. American Journal of Enology and Viticulture, 42,364–366.

Obied, H. K., Bedgood, D. R., Jr., Prenzler, P. D., & Robards, K. (2007). Bioscreening ofAustralian olive mill waste extracts: Biophenol content, antioxidant,antimicrobial and molluscicidal activities. Food and Chemical Toxicology, 45,1238–1248.

Obied, H. K., Prenzler, P. D., & Robards, K. (2008). Potent antioxidant biophenolsfrom olive mill waste. Food Chemistry, 111, 171–178.

Pan, Y., Wang, K., Huang, S., Wang, H., Mu, X., He, C., et al. (2008). Antioxidantactivity of microwave-assisted extract of longan (Dimocarpus Longan Lour.)peel. Food Chemistry, 106, 1264–1270.

Punithavathi, V. R., Prince, P. S. M., Kumar, R., & Selvakumari, J. (2011).Antihyperglycaemic, antilipid peroxidative and antioxidant effects of gallicacid on streptozotocin induced diabetic Wistar rats. European Journal ofPharmacology, 650(1), 465–471.

Ragazzi, E., & Veronese, G. (1973). Research on the phenolic components of oliveoils. La Rivista Italiana delle Sostanze Grasse, 50, 443–452.

Samuel, S. M., Thirunavukkarasu, M., Penumathsa, S. V., Paul, D., & Maulik, N. J. (2008).Akt/FOXO3a/SIRT1-mediated cardioprotection by tyrosol against ischemic stress inrat in vivo model of myocardial infarction: switching gears toward survival andlongevity. Journal of Agricultural and Food Chemistry, 56, 9692–9698.

Singh, R., Singh, S., Kumar, S., & Arora, S. (2007). Evaluation of antioxidant potentialof ethyl acetate extract/fractions of Acacia auriculiformis A. Cunn. Food andChemical Toxicology, 45, 1216–1223.

Terpinc, P., Polak, T., Šegatin, N., Hanzlowsky, A., Ulrih, N. P., & Abramovic, H. (2011).Antioxidant properties of 4-vinyl derivatives of hydroxycinnamic acids. FoodChemistry, 128, 62–69.

Tsagaraki, E., Lazarides, H. N., & Petrotos, K. B. (2007). Olive mill wastewater. In V.Oreopoulou & W. Russ (Eds.), Utilisation of by-products and treatment of waste inthe food industry (pp. 133–157). Springer.

Visioli, F., Romani, A., Mulinacci, N., Zarini, S., Conte, D., Vincieri, F. F., et al. (1999).Antioxidant and other biological activities of olive mill waste waters. Journal ofAgricultural and Food Chemistry, 47(8), 3397–3401.

Waterman, P. G., & Mole, S. (1994). Analysis of phenolic plant metabolites. Oxford:Blackwell Scientific Publications.

Yen, G. C., Duh, P. D., & Chuange, D. Y. (2000). Antioxidant activity ofanthraquinones and anthrone. Food Chemistry, 70, 437–441.

Yen, G.-C., Duh, P.-D., & Tsai, H.-L. (2002). Antioxidant and pro-oxidant properties ofascorbic acid and gallic acid. Food Chemistry, 79, 307–313.

Yoshikawa, T., Naito, Y., & Kondo, M. (1997). Food and diseases. In M. Hiramatsu, T.Yoshikawa, & M. Inoue (Eds.), Free radicals and diseases (pp. 11–19). New York:Plenum press.