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
Olive trees play an important role in the Moroccan agro-economy, providing both employment and export revenue. However, the olive oil industry generates large amounts of wastes and wastewaters. The disposal of these polluting by-products is a signicant environmental problem that needs an adequate solution. On one hand, the phytotoxic and antimicrobial effects of olive mill wastewaters are mainly due to their phenolic 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 from a diet containing olive oil have been attributed to its richness in phenolic compounds that act as natural antioxidants and are thought to contribute to the prevention of heart diseases and cancers. Olive mill wastewater (OMW) samples have been analysed in terms of their phenolic constituents and antioxidant activities. The total phenolic content, avonoids, avanols, and proanthocyanidins were determined. The antioxidant and radical scavenging activity of phenolic extracts and microltred samples was evaluated using different tests (iron(II) chelating activity, total antioxidant capacity, DPPH assays and lipid peroxidation test). The obtained results reveal the considerable antioxidant capacity of the OMW, that can be considered 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.

Article history: Received 3 July 2011 Received in revised form 25 September 2011 Accepted 2 November 2011 Available online 10 November 2011 Keywords: Olive mill wastewaters Phenolic compounds Antioxidant activity Radical scavenging Iron(II) chelating activity Lipid peroxidation

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 pollution effect, 1 m3 of OMW is reported to be equivalent to 200 m3 of domestic sewage (Tsagaraki, Lazarides, & Petrotos, 2007). Its disposal in water reservoirs (ground water reservoirs, surface aquatic reservoirs, seashores, and sea) without pretreatment, leads to severe problems for the whole ecosystem. OMW also show a toxic action to some plants and microorganisms since they exhibit a substantial concentration of phenolic compounds. These latter are the largest family of naturally occurring antioxidants in plants that include a wide variety of structures with a common motif, the phenol molecule. They expand from the simplest structures, such as phenolic 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 chemopreventive, cardioprotective, and neuroprotective activities (Tsagaraki

et al., 2007). Epidemiological studies have correlated the low incidence 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 phenols from olive and its by-products are now recognised as potential targets for the food, cosmetic and pharmaceutical industries. Nowadays, 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 functionality of OMW and its phenolic extract, we investigate here the phenolic prole and the antioxidant activity of OMW samples generated by two different olive oil processing techniques (olive press and decanter 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), Lascorbic acid, sodium hydroxide, and trichloroacetic acid were purchased from Sigma Aldrich (Germany). Ascorbic acid and butylated

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). 0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.11.013

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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 processes. 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 dichromate 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 permeate 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 phenolic 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)

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 tyrosol/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-(dimethylamino)-cinnamaldehyde (DMACA), using the optimised protocol 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 concentration 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 absorbance 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 sulphuric 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 absorbance 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 activity 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 radical (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 mixture 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

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

2.7. HPLC analysis of OMW extracts The analysis was performed on a JASCO HPLC system, equipped by a JASCO UV detector (UV-975) operating at 280 nm. The column used to analyse polyphenols was a reversed phase Lichrosphere C18 (4 250 mm i.d 5 lm), and the column was washed with acetonitrile 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 3.1. Physicochemical characterisation of OMW samples Table 1 shows the main physicochemical characteristics of OMW samples. OMW samples are slightly acidic. OMW from the semi-modern (OMW1) unit showed high electrical conductivity, which was more than three times that of the OMW2. Traditionally Moroccan farmers preserve olive fruits during storage by salt addition. In modern milling units more water is used, and for this reason, almost all parameters values were reduced in comparison to samples from the semi-modern unit (OMW1). 3.2. OMW microltration Microltration of OMW exhibits a reasonable ux (350 l/hm2) and the obtained permeate was less coloured (80% less at 465 nm) compared to the feed. The chemical oxygen demand, the dry residue and the TPC were also reduced by 62%, 30% and 7%, respectively. Different dilutions were prepared from the microltrate (permeate) for the estimation of its antioxidant activity using different methods. 3.3. The OMW total phenolic content composition OMW1 showed a higher phenolic content (9.8 g/l) compared to OMW2 (6.1 g/l). The main components of the total phenolic content are avonoids (Table 2), a group of natural substances with antioxidant, anti-inammatory, antiallergic, antiviral and anticarcinogenic properties (Leopoldini et al., 2011). This phenolic group corresponds to 66.8% of OMW1 phenolic content, but lower amounts (44.3%) have been revealed for avonoids in OMW2 (Table 2). Besides their antioxidant activities, avonoids are able
Table 1 The main physicochemical characteristics of olive mill wastewater samples. Parameters pH EC Dry residue Ash TOC TPC Sugar COD DCOD TSS Sodium Potassium Calcium Unit mS/cm g/l g/l g/l g of TYE/l g/l g of O2/l g of O2/l g/l g/l g/l g/l OMW1 5.2 0.1 43 0.9 173 13 47 2.5 31 4.3 9.82 0.3 23 1.7 113 9.8 10 0.2 87 5.9 2.17 0.18 1.34 0.11 1.92 0.14 OMW2 5.1 0.1 13 0.5 128 8 15 1.2 14 1.9 6.11 0.2 12 1.1 51 7.6 8 0.2 48 4.5 1.68 0.13 0.75 0.06 1.22 0.11

% 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 concentration 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 radicalscavenging ability of ascorbic acid and BHT was also evaluated and 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 modication. The adequately diluted phenolic extract (0.1 ml) was mixed with methanol (2.6 ml) and 2 mM FeCl2 (0.1 ml) and then 5 mM ferrozine (0.2 ml). The mixture was shaken vigorously and left to stand at room temperature in the dark for 10 min. Absorbance of the resulting solution was measured spectrophotometrically at 562 nm. A low absorbance of the resulting solution indicated a strong Fe2+-chelating ability. The ability to chelate ferrous ion (ICA) and prevent formation of ferrous ion-ferrozine complex, was calculated using the following equation:

ICA % 1 Asample =Acontrol 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). Measurements were achieved for dilutions up to 40 times against distilled water for the microltred samples and against methanol for the phenolic extracts. All analyses were run in triplicate and averaged. Sample concentration providing 50% inhibition (IC50) was calculated from the graph plotting inhibition percentage against extract concentration. EDTA calibration solutions (8, 16, 24, 32, 40, and 48 lM) were prepared and their ICA were determined following the same protocol. 2.6.4. Lipid peroxidation index To assess the ability of OMW to inhibit lipid peroxidation, the method described by Singh, Singh, Kumar, and Arora (2007) was adopted. The 2-thiobarbituric acid (TBA) reacts with malondialdehyde (MDA) to form a diadduct, a pink chromogen, which can be detected spectrophotometrically at 532 nm. Normal male Wistar rats (250 g) were used for the preparation of liver homogenate. The perfused 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 15 min, and clear cell-free supernatant was used for the study of in vitro lipid peroxidation. Different dilutions of microltred OMW samples and their methanolic extracts were taken in test tubes. One millilitre of 0.15 M KCl and 0.5 ml of rat liver homogenate were added to the test tubes. Peroxidation was initiated by adding 100 ll of 0.2 mM ferric chloride. After incubation at 37 C for 30 min, the reaction was stopped by adding 2 ml of icecold HCl (0.25 N) containing 15% trichloroacetic acid (TCA), 0.38% TBA, and 0.5% BHT. The reaction mixtures were heated at 80 C for 60 min. The samples were cooled and centrifuged, and the absorbance of the supernatants was measured at 532 nm. The percentage of lipid peroxidation inhibition (LPI) was calculated by the following formula:

LPI% 1 Asample =Ablank 100

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 determined by following the same protocol as for the samples.

Values are the average of three measurements standard deviation. Abbreviations: EC, electrical conductivity; TOC, total organic compounds; TPC, total phenolic content; COD, Chemical oxygen demand; DCOD, dissolved chemical oxygen 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.

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to inhibit lipid peroxidation and platelet aggregation, as well as improve increased capillary permeability and fragility (Leopoldini et al., 2011). Main avonoid subgroups in olive mill wastewaters are avanols and proanthocyanidins. These phenolics were positively 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.

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 concentrations 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 phenolic extracts were measured and compared to that of BHT. According to our results, OMW1-lF and OMW2-lF showed significant TACs, which were two times higher than that of their respective 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 wellknown 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 hydrogen donor. Results reported in Table 4 demonstrate that the OMW2lF 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 scavenging 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 OMW1PhE against time at different dosages are shown in Fig. 2, respectively. 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 approximated 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, especially 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-

3.4. HPLC prole of the total phenolic content of OMW To determine and compare the phenolic proles, an HPLC analysis was performed and the phenolic compounds of OMW were identied (Fig. 1 and Table 3). The TPC as revealed by the HPLC quantication represent only 55% and 63.7% of the depicted spectrophotometric 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 detected 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 concentration 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, & Robards, 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 concentrations (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 concentrations (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 OMW1 OMW2 TPC TYE g/l 9.82 0.53 (100%) 6.11 0.2 (100%) Flavonoids CAE g/l 6.56 0.21 (66.8%) 2.71 0.14 (44.3%) Flavanols CAE mg/l 2.8 0.04 (28.5%) 1.9 0.03 (31.1%) Proanthocyanidins CYE mg/l 17.03 0.14 (0.17%) 9.02 0.85 (0.15%)

The values between brackets are the relative proportion for each constituent compared to the total phenolic content (TPC).

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550 500 450 400 350 300 250 200 150 100 50 0
550 500 450 400 350 300 250 200 150 100 50 0

OMW1-PhE d:1/6

mV

1 2 4 5 6

OMW2-PhE d:1/6

mV

1 2

6 4 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) Concentration (g of TYE/l) Gallic acid Hydroxytyrosol-4-b-glucoside Hydroxytyrosol Tyrosol Cafeic acid Para-coumaric acid Oleuropein aglycone Total 0.583 0.041 0.168 0.012 3.766 0.243 2.491 0.017 0.092 0.007 0.549 0.038 0 5.407 0.358 Proportion (%) 10.79 0.76 3.10 0.22 69.64 4.49 4.61 0.31 1.70 0.12 10.16 0.70 0 100 OMW2 phenolic extract (OMW2-PhE) Concentration (g of TYE/l) 0.331 0.022 0.226 0.015 2.127 0.150 0.246 0.014 0.057 0.004 0.785 0.055 0.121 0.008 3.893 0.268 Proportion (%) 8.51 0.57 5.79 0.39 54.65 3.85 6.32 0.36 1.48 0.10 20.16 1.41 3.10 0.21 100

ties of phenolic compounds are attributed to specic structural features, 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 contents 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 malondialdehyde (MDA), and is found to be an important cause of cell membrane 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 compared 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 compared 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.

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1.4 1.2

Tyrosol (mg/ml) Ascorbic acid (mg/ml) TPC of OMW1-PhE (mg/ml)

0.5 0.1 172

1 0.15 218

2 0.3 288

1 0.8 0.6 0.4 0.2 0

0 10 20 30 40 50 60

Reaction time (min)

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

activity in microltrated OMW can be most likely attributed to the difference in phenolic compositions between the methanolic extracts and the microltrated OMW. Furthermore, the antioxidant activity of various OMW extracts was reported to be directly correlating with percentage of free hydroxytyrosol and their antioxidant properties was found to be the result of their phenol composition rather than their phenol content (Leonardis et al., 2009). The obtained results show a great potential of OMW aqueous extract (microltrated OMW) to be used as antioxidants in food. Studies on the safety and efcacy of olive polyphenols (Christian et al., 2004) show that polyphenols from olive fruit and its by-product can be considered as safe and non-toxic for human consummation. Christian et al. (2004) reported that no mortality or clinical signs of toxicity were noted after 29 days of consecutive administration of an aqueous olive pulp extract (5 g/kg/day) to Crl:CD Sprague Dawley rats suggesting that the LD50 of the extract must be greater than 5 g/kg.

Absorbance at 517nm

Table 4 Antioxidant activities of different phenolic extracts compared to some reference antioxidants. TAC* IC50 (lg/ml) DPPH OMW1-PhE OMW2-PhE OMW1-lF OMW2-lF EDTA Ascorbic acid BHT 0.124a 0.020 0.121a 0.002 0.249b 0.005 0.262b 0.008 0.302d 0.019 263a 2.5 169b 5.2 161b 4.8 123c 3.7 158b 14.3 15.2d 1.1 ICA 238a 0.24 136b 0.19 13c 0.02 15cd 0.03 17d 0.03 LPI 1069a 56 860b 25 1103a 77 213c 18 43d 3.5

3.6. Effect of storage time on the OMW phenolic content and its antioxidant activity OMW samples from semi-modern milling unit were stored at room temperature in dark during 1 year. Samples were taken periodically and were analysed in terms of their total phenolic content and radical scavenging activity (DPPH assay). Fig. 5 shows the evolution of TPC and IC50 against time. The TPC increased considerably during the 4 rst months and started to decrease slightly beyond the fth 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 correlated to the evolution of total phenolic content during storage (Fig. 5). These results are in agreement with the nding of Atanassova, Kefalas, and Psillakis (2005) who reported that the antioxidant 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 compounds. Different reactions may take place during storage, some antioxidants may disappear and/or new molecules can be produced 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 physicochemical 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-

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

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 molecules that remain in the water phase, the high antioxidant

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

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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 studies investigated the effect of storage on the phenolic content of OMW. Feki, Allouche, Bouaziz, Gargoubi, and Sayadi (2006), reported a signicant accumulation of hydroxytyrosol after 5 months of storage. The corresponding concentration increased by 257 302%. However, the concentrations of the other phenolic compounds were markedly decreased. Furthermore, it was reported that the OMW storage facilitates the liquidliquid extraction procedure 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 differing reaction mechanisms is necessary to give an overall understanding of the mechanisms of action of an antioxidant. Signicant differences between phenolic extracts and micro-ltrates 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 mechanisms 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 Sciences (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. References
Atanassova, D., Kefalas, P., & Psillakis, E. (2005). Measuring the antioxidant activity of olive oil mill wastewater using chemiluminescence. Environment International, 31(2), 275280. Bouallagui, Z., Bouaziz, M., Lassoued, S., Engasser, J. M., Ghoul, M., & Sayadi, S. (2011). Hydroxytyrosol Acyl Esters: Biosynthesis and Activities. Applied Biochemistry and Biotechnology, 163(5), 592599.