Maceration and Liquid-liquid extractions of phenolic compounds and antioxidants from Algerian olive oil mill wastewater

Abstract

Purpose
Olive oil mill wastewater (OMW) is a major waste stream from the olive oil industry. It is highly polluted due to phenolic compounds. The present study focused on the physicochemical properties of OMW as well as the quantitative and qualitative effects of two methods of extraction (maceration and liquid-liquid extraction) of phenolic compounds.

Methods
Liquid-liquid extraction and maceration methods were used for the extraction of phenolics. Spectrophotometry and High-Performance Liquid Chromatography-Electrospray Ionization–Mass Spectrometry (HPLC-ESI-MS) were adopted to quantify the phytochemical contents and the phenolic compounds. The antioxidant potentials were evaluated using three assays DPPH, ABTS + and FRAP radical scavenging activities.

Results
The findings showed that the OMW was an acidic effluent (pH = 5.05) loaded with mineral and organic matter expressed in terms of a high value of electrical conductivity (EC = 13.51 mS/cm). Indeed, the OMW showed high dry matter, chemical oxygen demand (COD) and biological oxygen demand (BOD5). The extract obtained by the maceration method showed the highest yields of total polyphenol, flavonoid, and tannins contents than the liquid-liquid extraction method. The LC-MS results revealed the presence of 16 phenolic compounds in the extract obtained by the maceration method and only 12 phenolic compounds were found in the extract obtained by the second method. Quinic acid was identified as the most abundant compound. Moreover, the macerated extracts possessed the highest antioxidant potential as evidenced by their strong DPPH, ABTS and FRAP radical scavenging activities compared to the liquid-liquid extracts.

Conclusions
The maceration methods seemed to be the most effective method for extracting phenolic compounds from OMW than liquid-liquid extraction. The OMW constitute a rich source of natural phenolic compounds that could be used as a potential source of natural antioxidants.

Full text

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Maceration and Liquid-liquid extractions of phenolic
compounds and antioxidants from Algerian olive oil
mill wastewater
Zakia Gueboudji
Universite Abbes Laghrour Khenchela
Kenza Kadi
Universite Abbes Laghrour Khenchela
Maher Mahmoudi (  mahmoudi.maher@fst.utm.tn )
University of Gabes: Universite de Gabes https://orcid.org/0000-0002-4114-2959
Hédia Hannachi
University of Tunis El Manar: Universite de Tunis El Manar
Kamel Nagaz
Institut des Regions Arides
Dalila Addad
Universite Abbes Laghrour Khenchela
Leila Ben Yahya
Institut des Regions Arides
Belgacem Lachehib
Institut des Regions Arides
Kamel Hessini
Taif University College of Science
Research Article
Keywords: Olive oil mill wastewater, LC-MS, extraction methods, phenolic compounds, antioxidant activity
Posted Date: April 13th, 2022
DOI: https://doi.org/10.21203/rs.3.rs-1423045/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
Read Full License

Page 2/16
Abstract
Purpose
Olive oil mill wastewater (OMW) is a major waste stream from the olive oil industry. It is highly polluted
due to phenolic compounds. The present study focused on the physicochemical properties of OMW as
well as the quantitative and qualitative effects of two methods of extraction (maceration and liquid-liquid
extraction) of phenolic compounds.
Methods
Liquid-liquid extraction and maceration methods were used for the extraction of phenolics.
Spectrophotometry and High-Performance Liquid Chromatography-Electrospray Ionization–Mass
Spectrometry (HPLC-ESI-MS) were adopted to quantify the phytochemical contents and the phenolic
compounds. The antioxidant potentials were evaluated using three assays DPPH, ABTS + and FRAP
radical scavenging activities.
Results
The ndings showed that the OMW was an acidic euent (pH = 5.05) loaded with mineral and organic
matter expressed in terms of a high value of electrical conductivity (EC = 13.51 mS/cm). Indeed, the OMW
showed high dry matter, chemical oxygen demand (COD) and biological oxygen demand (BOD5). The
extract obtained by the maceration method showed the highest yields of total polyphenol, avonoid, and
tannins contents than the liquid-liquid extraction method. The LC-MS results revealed the presence of 16
phenolic compounds in the extract obtained by the maceration method and only 12 phenolic compounds
were found in the extract obtained by the second method. Quinic acid was identied as the most
abundant compound. Moreover, the macerated extracts possessed the highest antioxidant potential as
evidenced by their strong DPPH, ABTS and FRAP radical scavenging activities compared to the liquid-
liquid extracts.
Conclusions
The maceration methods seemed to be the most effective method for extracting phenolic compounds
from OMW than liquid-liquid extraction. The OMW constitute a rich source of natural phenolic
compounds that could be used as a potential source of natural antioxidants.
Introduction
The olive oil industry is an important sector of the economy, concentrated mainly in the Mediterranean
countries (Alique et al. 2020), including Algeria. In addition to the solid wastes that are called pomace, it

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generates very large amounts of liquid discharges and wastewater in olive oil presses. It consists of the
water found in the fruit (olive) and the water added during olive oil extraction processes named olive oil
mill wastewater (OMW) (Zakia et al. 2021). This industry is increasing annually with the cumulative
demand for olive oil consumption, due to its very benecial effect on health with its therapeutic, diet, and
nutritional properties. Consequently, this industrial sector leads to an increase in solid and liquid wastes
production that are a source of environmental pollution, especially in Mediterranean countries. The olive
oil mill wastewaters are high conductivity and dark acidic liquids that contain interesting compounds
including sugars, proteins, dietary bres and phenolics (Gueboudji et al. 2021a, Haddad et al. 2017,
Rocha et al. 2022). Moreover, this great soil damage is due to the slow degradation of these phenolic
compounds, which are the reason for their dark color (Gueboudji et al. 2021c). This situation is
exacerbated by the seasonality of olive oil production and the large quantities of vegetable water
produced (Koutrotsios &Zervakis 2014).
Phenolic compounds are products of the secondary metabolism of plants, widely distributed to many
phenolic groups and includes about 9 000 different known structures (Wan et al. 2021), ranging from
simple phenolic molecules with low molecular weight like phenolic acids for high-polymerized
compounds such as tannins. The phenolics possessed potential benecial effects on health such as the
prevention of ROS (reactive oxygen species)-related diseases such as aging, cancer, and chronic diseases
(Khan et al. 2020). Phenolic compounds have several biological properties and are used in many
industrial elds because of their antioxidant activity including the pharmaceutical, food, and cosmetic
industries (Gueboudji et al. 2021a, Soberón et al. 2019).
Some factors would inuence the phenolic compound's composition of OMW such as the olive varieties,
climatic conditions, olive oil extraction process and ripening degree of the fruit (Prazeres et al. 2021).
However, the selective recovery of phenolic substances from OMW represents a valid approach for the
reduction of their environmental toxicity and an opportunity to obtain high added-value molecules (De
Bruno et al. 2018). Many recovery studies of OMW polyphenols have been carried out on a small scale
and various techniques are used individually or in combination (Martins et al. 2021). These techniques
mainly include solvent extraction, adsorption, membrane separation, supercritical uid extractions,
ultrasound treatment, and chromatographic processes (Soberón et al. 2019). Phenol recovery processes
generally involve a condensation step before performing the sequential extraction steps with organic
solvents such as methanol, ethanol, or hydro-alcoholic solutions. These processes aim to recover either a
particular phenol in pure form or a mixture of phenol in the form of a crude product (Alonso-Riaño et al.
2020).
This study aimed to make a comparison between two methods of extraction of phenolic compounds
from OMW and to evaluate the most suitable method for the recovery of these phenolic compounds, to
maximize the phenolic yield and reduce the quantity of solvents used in extraction.
Experimental Work

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Material
The olive oil mill wastewaterswere taken from a modern olive oil cold extraction unit, located at Baghai-
Khenchela (eastern Algeria) and were obtainedafter olive oil extraction from Zlitni olives variety in
January 2020.
Physicochemical characteristics of OMW
Electrical conductivity (EC) and pH of OMW were directly measured using a conductivity meterandpH
meter.The dry matter (DM) was determined after sample drying at 105°C. Fatty matter (FM) was
determined by the chloroform/methanol method as described by (Aissam 2003). Chemical oxygen
demand (COD) and biological oxygen demand (BOD5) were evaluated, respectively by the potassium
dichromate andthe respirometric methods (Rodier et al. 1984).
Polyphenol extraction
Liquid-liquid extraction
The liquid-liquid extraction in ethyl acetate was done accordingto the method described by (De Marco et
al. 2007).A volume of20 mL of OMW were acidied at pH = 2 with a few drops of HCl and mixed with 30
mL of hexane. The solution was mixed vigorously and centrifuged at 3000 t/min for 5 min. After that, 20
mL of ethyl acetate were added, and the homogenate was shaken vigorously for 15 min and then
centrifuged at 3200 t/min for 10 min in (4 oC). The phases were separated, and the extraction was
repeated four times. The obtained ve phases of ethyl acetate were collected and combined and the
dissolved water was removed with sodium sulfate anhydrous, and the solvent was evaporated under
vacuum in a rotary evaporator at 40oC. The dryresidues were dissolved in methanol and stored at -18 °C.
The extraction process was carried out in triplicate.
Extraction by maceration
OMW powder (1 g of) were mixed with 10 mL of pure methanol. Then, the mixture was vortexed for 15
min, and let macerate at 4 °C in the dark overnight and ltered through lter paper. The macerate was
then collected and was added to 10 mL of methanol for a second time, the mixture was vortexed for 15
min and left to macerate for 1 hour. The two ltrates were combined and ltered through cellulose paper
containing sodium sulfate. The solvent was evaporated at 40°C in a rotating evaporator under a vacuum.
The dry residue was storedin 6 mL of methanol at -18 °C. The extraction was performed in triplicate.
Determination of total phenoliccontent (TPC)
The total phenolic content (TPC) of eachextract was determined following the Folin–Ciocalteu method
(Müller et al. 2010). The TPC of extracts was estimated according to the calibration curve prepared using
Gallic acid (y = 0.0048 x + 0.0027, R2 = 0.9982). The results were expressed as grams of Gallic acid
equivalents per 100 grams of dry matter of initial OMW (g GAE/100 g DM).

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Determination of totalavonoid content (TFC)
The quantication of the total avonoid content (TFC) of each extract was performed using the method
described previously(Topçu et al. 2007). The TFC was calculated following the calibration curve prepared
usingquercetin (y = 0.0034 x + 0.0311, R 2 = 0.9991). The results were expressed as gram quercetin
equivalents per 100 grams of dry matter of initial OMW (g QE/100g DM). 
Determination of total tannin content (TTC)
The quantication of the total tannin content (TTC) of each extract was performed by the method
described previously(Hagerman 2002). The TTC was estimated according to the calibration curve
prepared using catechin (y = 0.0037 x + 0.0681, R2= 0.9979). The results were expressed in grams of
catechin equivalent per 100 grams of dry matter of initial OMW (g CE/100g DM).
Liquid chromatography-mass spectrometry (LC-MS) analysis ofphenolic compounds
The identication of phenolic compounds in OMW extracts was determinedby LC-MS according to the
methodology described by Mahmoudi et al.(Mahmoudi et al. 2020, Mahmoudi et al. 2021a, Mahmoudi et
al. 2021b).The analysis of phenolic compounds was performed on a Shimadzu UFLC XR system (Kyoto,
Japan), equipped with a SIL-20AXR auto-sampler, a CTO-20 AC column oven, a LC-20ADXR binary pump
and a quadrupole 2020 detector system. This instrument was equipped with an Inertsil ODS-4 C18 3 µm
column (L150×3.0 mm i.d.). The column temperature was set at 40 °C and the injection volume was 20 µl
with a ow rate of 0.5 mL/min. Water 95% + MeOh5% + Acetic acid 0.2% and CAN 50% + H2O 50% +
Acetic acid 0.2% were used as mobile phases A and B, respectively. The analysis was performed using a
linear gradient programmed as follows: 0,01-14 min, from 10% to 20% B; 14-27 min, 0 from 20% to 55% B;
27-37 min, from 55% to 100% B ; 37-45 min, 100% B ; 45-50 min 10% B. Dissolving line temperature was
275°C, nebulizing gas ow 1,50 L/min, the drying gas was set at 15,00 L/min and temperature of Heat
block was 450°C. LC-ESI (-) MS mass spectra [M-H] - were acquired using Lab Solutions software. The
identication and the quantication of obtained pics were determined by comparison with the relative
retention times and UV spectra with those of standard phenolic compounds as detailed in(Mahmoudi et
al. 2021b).
Antioxidant assays
DPPH free radical-scavenging activity
The antioxidant activity of different extractions was evaluated using the free radical DPPH (2,2-diphenyl-
1-picrylhydrazyl) (Mahmoudi et al. 2021b).The results were given as 50% inhibition concentration (IC50)
and compared with the antioxidant standards (BHT, Ascorbic acid and Trolox).To assess this activity, 0.5
mL of extract at different concentrations was mixed with 0.5 mL of a solution of DPPH (0.2 mM in
methanol). After vigorous shaking, the mixture was left to stand at room temperature for30 minand the
absorbance was read at 517 nm

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ABTS+ free radical scavenging activity
The ABTS scavenging activity was determined as described (Arnao et al. 2001).The ABTS solution was
prepared by mixing ABTS (7 mM) with potassium persulfate and the mixture was incubated in the dark
before use. The prepared solution was diluted with methanol to have an absorbance of 0.7 ± 0.02. After
adding 25 µL of Trolox extract or standard to 2 mL of the diluted ABTS solution, the absorbance at 734
nm was measuredfor 5 min. The results were given as 50% inhibition concentration (IC50) and compared
with the antioxidant standards (BHT, Ascorbic acid and Trolox).
FRAP ferric reducing antioxidant power
The FRAP activity was evaluated according to (Kocak et al. 2016).A volume of 2 mL of FRAP reagent
was added to 0.3 mL of the extract samples in a 10 mL volumetric ask, adjusted to a nal volume of 10
mL with ultrapure water. The obtained solution was allowed to stand atroom temperature for 5 min and
then centrifuged for 10 min at 10 000 rpm to remove any kind of solid matter. Absorbance was measured
at 593 nm andthe results were given as 50% inhibition concentration (IC50) and compared with the
antioxidant standards (BHT, Ascorbic acid and Trolox).
Statistical analysis
Data obtained were presented as mean ± standard deviation of three dependent determinations.
Signicant differences between means of total phenolic, total avonoids, tannins contents, LC-MS
analysis andantioxidant activityresults were determined byanalysis of variance (ANOVA) and Duncan’s
multiple ranges. Differences considered signicant at p< 0.05. Statistical analyses were performed using
XLSTAT software (www.xlstat.com).
Results And Discussion
Physicochemical criteria
The physicochemical properties of the studied OMW were presented in Table1. The OMW obtained from
the Zlitni variety were acidic euents (pH = 5.05) loaded with mineral and organic matter expressed in
terms of a high value of electrical conductivity (EC = 13.51 mS/cm). Indeed, the OMW showed high dry
matter, chemical oxygen demand (COD) and biological oxygen demand (BOD5) and were found to be,
respectively, 110.67, 208 g/L and 75 g/L. However, the level of the fatty matter was low (0.99%). Results
of physicochemical criteria of OMW were in accordance with those obtained in the literature. OMW was
an acidic liquid, with pH values varying from 3 to 5 and with an electrical conductivity value of 16.79
mS/cm. Generally, it composed of water (83–94%), organic matter (4–16%), lipids (1 to 14%), COD (40–
220 g/L), BOD5 (35–110 g/L) (Alique et al. 2020, Değirmenbaşı &Takaç 2018). The quality and quantity
of OMW varied according to different factors such as production process type, olives verities, use of
pesticides and fertilizers, ripening stage, climatic conditions, and geographic area (El-Abbassi et al. 2017).

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Table 1
Physicochemical criteria of the studied Olive Mill
Wastewater (OMW)
Parameters Value
pH 5.05 ± 0.05
EC: electrical conductivity (mS/cm) 13.51 ± 0.07
FM: fatty matter (%) 0.99 ± 0.10
DM: dry matter (g/L) 110.67 ± 6.03
COD: chemical oxygen demand (g/L) 208.00 ± 10.00
BOD5: biological oxygen demand (g/L) 75.00 ± 4.36
Total Phenolic, Flavonoids And Tannins Contents
The total polyphenol, avonoid and tannin contents extracted by the two methods were presented in
Fig.1. Statistical analysis showed signicant differences between means of total polyphenols, total
avonoids and tannins contents extracted with two methods. The maceration was more ecient than the
liquid-liquid extraction method to obtain high total polyphenol (22.97 versus 6.47 g GAE/100g DM), total
avonoid (2.34 versus 1.1 g QE /100g DM) and tannin contents (2.47 versus 0.847 g CE /100g DM).
When compared to the liquid-liquid extraction technique, these contents increased by 255.02, 112.73, and
191.62 per cent, respectively. OMW was characterized by the richness in phenolic compounds. It has been
noted that the TPC of OMW has an amount of 788.96 ± 1.41 mg /100mL found by (Romeo et al. 2020)
and varying from 0.5 to 24 g/L (Değirmenbaşı &Takaç 2018). The results that we obtained are found in
this range.
Identication And Quantication Of Phenolic Compounds
By Lc-ms Analysis
The content of phenolic compounds obtained by LC-MS was shown in Table2. Thirty-one (31)
compounds were identied under the analytical conditions, of which 16 compounds were found in
extracts obtained through the maceration and only 12 compounds were found in the extract obtained by
liquid-liquid extraction.

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Table 2
LC-ESI-MS analysis of phenolic compounds from of OMW Zlitni variety obtained by two extraction
methods
Phenolic compounds formula Retention
time [M-
H] -
m/z
Liquid – liquid
extraction (ppm) Extraction by
maceration
(ppm)
Quinic acid C7H12O62.043 191 4.80 ± 4.5b35.0 ± 2.6a
Catechin (+) C15H14O612.45 289 0.03 ± 0.3a0.3 ± 0.6a
Caffeic acid C9H8O416.167 179 N.D. 2.44 ± 2.12
Rutin C27H30O16 25.283 609 0.02 ± 0.04b0.20 ± 0.10a
Hyperoside (quercetin-3-
O
-galactoside) C21H20O12 25.494 463 0.04 ± 0.07b0.17 ± 0.02a
Luteolin-7-
O
-glucoside C21H20O11 25.867 447 N.D. 0.10 ± 0.00
Naringin C27H32O14 27.413 579 0.02 ± 0.04b0.10 ± 0.00a
4,5-di-O-caffeoyquinic
acid C25H24O12 28.017 515 N.D. 0.20 ± 0.00
Quercetrin (quercetin-3-
O
-rhamonosid C21H20O11 28.35 447 0.04 ± 0.06b0.30 ± 0.10a
Apegenin-7-
O
-glucoside C15H10O528.061 431 N.D. 0.70 ± 0.00
Salviolinic acid C36H30O16 29.394 717 0.10 ± 0.10b0.40 ± 0.00a
Kampherol C15H10O633.292 285 1.60 ± 1.00b3.0 ± 0.20a
Quercetin C15H10O733.35 301 0.50 ± 0.70a0.3 ± 0.10b
Naringenin C15H12O535.317 271 0.40 ± 0.20a0.5 ± 0.70a
Apegenin C15H10O535.872 269 0.42 ± 0.18b0.7 ± 0.00a
Cirsiliol C17H14O736.97 329 2.30 ± 2.96a1.3 ± 0.10b
Total phenols content  - - 9.836 ± 0.323 45.112 ± 0.213
Each value is the mean ± Standard deviation (SD); N.D.: not determined. Means in the same line with
different letters differ signicantly (
p
< 0.05);
Phenolic compounds identied were quinic acid, catechin (+), caffeic acid, rutin, hyperoside (quercetin-3-
O
-galactoside), luteolin-7-
O
-glucoside, naringin, 4,5-di-
O
-caffeoyquinic acid, quercetrin, apegenin-7-
O
-
glucoside, salviolinic acid, kampherol, quercetin, naringenin, apegenin and cirsiliol. Four phenolic acids
were identied in the macerated extract and were caffeic acid, luteolin-7-
O
-glucoside, 4,5-di-
O
-

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caffeoyquinic acid and apigenin 7-
O
-glucoside. The results indicated that the maceration method gives
higher values than the liquid-liquid extraction method in quinic acid, rutin, hyperoside, luteolin-7glucoside,
4,5-di-
O
- caffeoyquinic acid and salviolinic acid. The two methods give the same values in terms of
catechin (+), caffeic acid, naringin, quercetrin (quercetin-3-
O
-rhamonosid), apegenin-7-
O
-glucoside,
salviolinic acid, kaempferol, quercetin, naringenin, apegenin and cirsiliol. The highest level of phenolic
compounds was recorded in the macerated extract with an average value of 45.112 ppm, compared with
extracts obtained by the liquid-liquid method (9.836 ppm). The quinic acid was the major phenolic
compound with an average value of 4.82 in extracts obtained by liquid-liquid followed, in decreasing
order by cirsiliol (2.3 ppm), kaempferol (1.6 ppm), quercetin (0.5 ppm), apigenin (0.42 ppm), naringenin
(0.4 ppm), catechin (+) (0.3 ppm), salviolinic acid (0.1 ppm), hyperoside (quercetin-3-
O
-galactoside (0.04
ppm), quercetrin (quercetin-3-
O
-rhamonosid) (0.04 ppm), rutin (0.02 ppm) and naringin (0.02 ppm). For
the maceration method, quinic acid was also the major phenolic compound with an average value of
(35.1 ppm) and followed by kaempferol (3 ppm), caffeic acid (2.44 ppm), cirsiliol (1.3 ppm), apegenin
(0.7 ppm), apegenin-7-
O
-glucoside (0.7 ppm), naringenin (0.5 ppm), salviolinic acid (0.4 ppm), quercetrin
(quercetin-3-o-rhamonosid (0.3 ppm), quercetin (0.3 ppm), Rutin (0.2 ppm), 4,5-di-
O
-caffeoyquinic acid
(0.2 ppm), hyperoside (quercetin-3-
O
-galactoside (0.17 ppm), luteolin-7-
O
- glucoside (0.1 ppm) and
naringin (0.1 ppm).
Results showed that quinic acid was the major compound in the extracts obtained by the two methods of
extraction. Several researchers were identied phenolic compounds by HPLC after liquid-liquid extraction
and several compounds were characterized such as gallic acid, hydroxytyrosol-4-β-glucoside,
hydroxytyrosol, tyrosol, caffeic acid, p-coumaric acid and oleuropein aglycone (El-Abbassi et al. 2012).
Moreover, Romeo et al. (Romeo et al. 2020) identied ten compounds including chlorogenic acid, vanillic
acid, caffeic acid, p-coumaric acid, verbascoside, luteolin and apigenin. Some phenolic compounds
frequently prevalent in OMW, such as gallic acid, p-coumaric acid, were not detected in our extracts.
These compounds are easily oxidizable and their transformation was possible (Belaid et al. 2002). The
presence of caffeic acid, luteolin-7-
O
-glucoside, 4, 5-di-O-caffeoyquinic acid, apigenin-7-
O
-glucoside only
in the extracts obtained by the maceration method would be explained by their oxidization and therefore
their rapid possible transformation.
It was noted that the difference in the quantity and quality of the phenolic compounds was due to the
loss of a percentage of them that remains trapped in hexane and ethyl acetate phases during the liquid-
liquid extraction. It was conrmed that not all phenolic compounds were extracted by ethyl acetate,
especially the phenolic compounds of high molecular weight as tannins (El-Abbassi et al. 2017). In
addition, Freeze-drying was recommended to preserve the phenolic fraction of the olive oil mill
wastewaters from any variation. According to Turkmen et al. (Turkmen et al. 2007), phenolic compounds
were generally extracted using suitable solvents such as methanol, ethanol and acetol, N, N-
dimethylformamide. Therefore, methanol was a polar solvent that allow the extraction of polyphenols.
The maceration method was simple, high eciency, and economical for polyphenols extraction. The
eciency of the method was mostly inuenced by the solvent, the pH of the extraction medium that

Page 10/16
determined the compound solubility, the temperature, the extraction steps, and the solvent volume, as well
that the size and shape of the particles (Alonso-Riaño et al. 2020).
Antioxidant Potentials
The free radical scavenging activity determined by DPPH., ABTS, .and FRAP was widely used to estimate
the antiradical/antioxidant capacity of phenolic compounds of OMW extracted with two methods and
compared the data to many reference standards to obtain more useful and arguably essential results.
ANOVA one away analysis revealed a signicant difference of antioxidant potential depending on the
extraction methods (Table3). The results of DPPH radical scavenging activity showed that the macerated
extracts exhibited the highest antioxidant activity evidenced by a low IC50 value (7.55 µg/mL) higher than
that of BHT (11.11 µg/mL), ascorbic acid (12.28 µg/mL) and Trolox (16.12 µg/mL). Similarly, the
analysis data of the ABTS assay showed that the extract obtained from the maceration extraction
method give the best activity with (IC50:6.08) lower than that of ascorbic acid and BHT (1.52 and 2.2
µg/mL) respectively, and higher than that of Trolox and the liquid-liquid extract (9.06 and 13.51 µg/mL).
From the results of FRAP, extracts of the maceration extraction method were exhibited the highest
antioxidant activity (3.12 µg/mL) than ascorbic acid (9.94 µg/mL), and extracts from liquid-liquid
extraction (11.56), and much higher activity than Trolox (17.06 µg/mL) and BHT (20.05 µg/mL) (Table4).
This is supported by the ndings of Romeo et al. (Romeo et al. 2020) who reported that the olive mill
wastewater showed strong antiradical DPPH and ABTS scavenging activities (114.37 and 2569.19 mmol
TE/100mL. The obtained results showed that the total polyphenol content was highly and positively
correlated with the antioxidant capacity evaluated by the DPPH, ABTS and FRAP assays. According to De
Marco et al. (De Marco et al. 2007), the phenolic compounds of OMW were characterized by a strong
antioxidant potential. The
in vitro
antioxidant activity of natural extracts has received much more
attention. These methods involved the presence of oxidizing species such as free radicals and metal
complexes (Alam et al. 2013). Several studies have shown that the antioxidant activity depends on the
concentration of total polyphenols, the antioxidant structures, as well as the reaction time (Abramovič et
al. 2018, Gueboudji et al. 2021b, Leouifoudi et al. 2015). The antioxidant potential of the studied extracts
would be explained by the load of the total and the type of phenolic compounds and by the assembly of
three compounds found in high concentrations in the extracts tested that are quinic acid, kaempferol, and
cirsiol. Therefore, the present results of the antioxidant activity of phenolic extracts were in accordance
with their phenolic compounds composition. Indeed, the ability to reduce free radicals is largely
inuenced by the phenolic composition of the sample.

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Table 3
The mean squares of the antioxidant activities
Source DF DPPH
(IC50 µg/mL)
ABTS
(IC50 µg/mL)
FRAP
(IC50 µg/mL)
Effect of extraction Methods 4 58.9* 74.38* 129.89*
Error 10 0.6 0.36 0.63
* : signicant effect
Table 4
Antioxidant’s activity of extracts by DPPH, ABTS and FRAP
DPPH IC50µg/mL ABTS IC50µg/mL FRAP IC50µg/mL
Extracts from the liquid-liquid extraction 18.93 ± 1.58a13.51 ± 0.02a11.56 ± 1.71c
Extracts from the
maceration
7.55 ± 0.49d6.08 ± 0.82c3.12 ± 0.3e
BHT 11.11 ± 0.31cd 2.2 ± 0.14d20.05 ± 0.19a
Ascorbic acid 12.28 ± 0.34c1.52 ± 0.19e9.94 ± 0.19d
Trolox 16.12 ± 0.3b9.06 ± 0.15b17.06 ± 0.2b
Each value is the mean ± Standard deviation (SD). Means in the same column with different letters
differ signicantly (
p
< 0.05)
Conclusion
The phytochemical contents and the antioxidant potentials of olive mill wastewater were deeply
investigated. Phenolic compounds and antioxidant activities were highly and signicantly dependent on
the extraction method of polyphenols. A total of 16 phenolic compounds were mostly predominated by
the quinic acid. Furthermore, DPPH·, ABTS·+ and FRAP antioxidant activities showed a signicant
variation between the two different methods and the maceration extraction gives the highest
concentration of phenolic compounds and the highest free radical scavenging activities. The maceration
extraction is a cheap, simple, and easy method. Finally, it should be noted that OMW constitutes a very
complex and fragile matrix to handle because it presented a natural antioxidant that can be used in
various industries such as food and pharmaceutical. The recovery of polyphenols offers the double
opportunity to obtain biomolecules with high additive value on the one hand and to reduce the pollutant
nature of OMW on the other hand, particularly present in the countries bordering the Mediterranean Sea.
Declarations

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Ethics Approval
Not applicable.
Consent to Participate
All authors consent to participate in the works of the manuscript.
Consent to Publish
All authors consent to submit and publish the manuscript.
Availability of data and materials
Data and materials are available upon request.
Competing Interests
The authors declare no competing interests.
Funding
No funding received for this work.
Authors’ Contributions
All authors contributed to the study conception and design
.
The rst draft of the manuscript was written
byZakia Gueboudji and Maher Mahmoudi. The supervision of the work was performed by Kenza Kadi,
Kamel Nagaz and Kamel Hessini. The laboratory experiments

and data analysis

wereperformed by Dalila
Addad Hedia Hannachi: Leila Ben Yahya, and Belgacem Lachehib contribute to the laboratory
experiments. All authors have read and approved the manuscript.
Acknowledgement
The authors would like to acknowledge Taif University Researchers Supporting Project number (TURSP-
2020/94), Taif University, Taif, Saudi Arabia. The authors are grateful to the IRA of Medenine, Tunisia for
their material support and their help in LC-MS analysis.
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Figures

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Figure 1
Total polyphenols, avonoids, and tannins contents of Zlitni OMW extracts by two methods of extraction
(TPC: total polyphenols content; TFC: total avonoids content; TTC: total tanins content; GAE: gallic acid
equivalent; QE: quercetin equivalent; catechin equivalent); for each content with different letters differ
signicantly (
p
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Supplementary Files
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