Biological activities and phenolic compounds of olive oil mill wastewater from Abani, endemic Algerian variety

Ethics statement

All experiments were conducted in accordance with the guidelines of the Declaration of the World Medical Association of Helsinki. The experiments were approving by the scientific committee of the Faculty of Nature and Life Sciences, Abbes Laghrour University of Khenchela, Algeria. They were performed in accordance with relevant guidelines and regulations. The blood donation for this in vitro study was approved by the Ethics Committee of the Central Laboratory of Ahmed Ben Bella Hospital Khenchela, Algeria. Written consent to participate and approval for publication was obtained from each volunteer. Informed consent was obtained from all subjects.

Physicochemical properties

The olive oil mill wastewater (OMW) from Abani variety was obtained from a modern olive mill situated in Khenchela, eastern Algeria, in November 2019. It was collected directly from the decanter, frozen immediately and kept at − 20 °C until use. Standard Methods⁴⁴ were used to measure pH, electrical conductivity (EC), dry matter (DM), total suspended solids (TSS), organic matter (OM), mineral matter (MM), biological oxygen demand (BOD₅), and chemical oxygen demand (COD). The pH level was measured using a pH meter (AdwaAD1000). Electrical Conductivity (EC) was determined by conductivity meter type (inoLab WTW). Dry matter content (DM) was measured by drying at 105 °C for 24 h. Organic matter (OM) was calculated by the difference between the dry weight of the OMW and its weight after the calcination. Mineral matter (MM) was determined by weighing after ignition in a muffle furnace type (Nabertherm) at 550 °C, for 24 h. The chemical oxygen demand (COD) was determined using potassium dichromate, as described by BOD₅ (biological oxygen demand) is determined by the respirometric method. Analyzes were carried out in triplicate.

Polyphenol extraction methods

The phenolic compounds were extracted using the maceration method. 1 g of OMW powder was mixed with 10 mL of pure methanol. Then, vortexed for 15 min and kept to macerate overnight at 4 °C in the dark. After maceration, filtering using filter paper is performed. The macerate was collected and added to 10 mL of methanol (90%) for a second time; the combination was vortexed for 15 min before being left to macerate for 1 h. The two filtrates are mixed and filtered through sodium sulfate-containing cellulose paper. The solution was condensed in a rotary evaporator (HAHNVAPOR) at 40 °C, and the dry material was stored.

Total phenolic content (TPC)

The total phenolic content was determined following the Folin–Ciocalteu method⁴⁵. Taking 125 µL of the extract diluted 100 times is put in the presence of 500 µL of distilled water and 125 µL of the FCR After stirring and standing for 3 min, 1250 µL of a 7% CO₃Na₂ solution was added to the mixture. The volume of the mixture was adjusted to 3 mL with ultrapure water and then left in the dark at room temperature for 90 min. The results were expressed as milligrams of gallic acid equivalents per milliliter of extract (mg GAE/mL). Gallic acid calibration curve was used to quantify the total phenolic content (TPC) of extracts (y = 0.0046 x + 0.0108, R² = 0.9967).

Total flavonoids content (TFC)

The quantification of total flavonoids content was performed by the method of⁴⁶. To a dose of 250 µL of the extract diluted 100 times with methanol, is added 75 µL of a 5% NaNO₂ solution. After 6 min of incubation at room temperature, 150 µL of an aluminum chloride solution AlCl₃ was added to the mixture. After 5 min of incubation at room temperature, 500 µL of sodium hydroxide was added to the mixture and then the volume was adjusted to 2500 µL with distilled water. The results were expressed as milligrams rutin equivalents per milliliter of extract (mg RE/mL). The total flavonoids content (TFC) was calculated following the calibration curve prepared from rutin (y = 0.0103 x + 0.0061, R² = 0.9963).

Tannin condensed content (TCC)

The quantification of condensed content was performed according to the method of⁴⁷ by reaction with vanillin in the presence of sulfuric acid. A volume of 0.5 mL intake of the suitably diluted extract is mixed with 2 mL of 1% vanillin and then added with 2 mL of concentrated sulfuric acid. After homogenization, the mixture is incubated at room temperature. The results were expressed as milligrams of tannic acid equivalent per milliliter of extract (mg TAE/mL). The tannins condensed content (TCC) was calculated using tannic acid calibration curve (y = 0.0066 x + 0.0113, R² = 0.9969).

LC–MS separation and identification of phenolic compounds

The OMW extracts were analysed using a Shimadzu UFLC XR system consisted of an electrospray ionization source (ESI) equipped with two LC-20ADXR solvent delivery units, a SIL-20AXR autosampler, an SCL-10A system controller, a CTO-20 AC column oven, a DGU-20AS degasser (Shimadzu, Kyoto, Japan). A volume of 5 μL each extract was injected at 0.5 mL/min to a Discover BIO Wide Pore C18 column (150 mm × 3 mm, 3 μm) at 40 °C and separated with two mobile phases: A (0.1% formic acid in water v/v) and B (0.1% formic acid in methanol v/v) following the programmed linear gradient elution : 0–14 min, from 10 to 20% B; 14–27 min, from 20 to 55% B; 27–37 min, from 55 to 100% B; 37–45 min, 100% B; and 45–50 min 10% B^48,49. The ionization mode was negative and The ESI conditions were set as follows: capillary voltage of − 3.5 v, a nebulizing gas flow of 1.5 L/min, a dry gas flow rate of 15 L/min, a DL (dissolving line) temperature of 280 °C, a block source temperature of 400 °C, and a voltage detector of 1.35 V. Compounds were identified by comparing their retention time and mass spectra with those of reference standards. The validation of the method was determined as detailed in⁵⁰.

Antioxidant assays

DPPH free radical-scavenging activity

The antioxidant activity of different extractions was evaluated following⁵¹ method using the free radical DPPH (2,2-diphenyl-1-picrylhydrazyl) with some adjustment. A test sample of 0.5 mL of the extract at different concentrations is mixed with 0.5 mL of a solution of DPPH (0.2 mM in methanol). After vigorous shaking of the mixture, it is left to stand for 30 min in the dark. The results were given as 50% inhibition concentration (IC₅₀) and compared with the antioxidant standards (BHT, Ascorbic acid and rutin).

ABTS⁺ free radical scavenging activity

The ABTS (2,2-Azino-bis-3-ethyl benzothiazoline-6-sulfonic acid) scavenging activity was determined according to the method of⁵¹ with some adjustment. A volume of 10 μL of the extract is added to a volume of 990 μL of ABTS solution. The discoloration relative to the control, containing ABTS⁺ and the solvent (ethanol), is measured with a spectrophotometer at 734 nm after 30 min of incubation in the dark. The results were given as 50% inhibition concentration (IC₅₀) and compared with the antioxidant standards (BHT, Ascorbic acid, and rutin).

FRAP ferric reducing antioxidant power

The FRAP activity was evaluated following⁵¹ with some adjustment. It consists of mixing 1 mL of each solution of extracts or of the standard antioxidant at different concentrations with 1 mL of phosphate buffer (0.2 M, pH 6, 6) and 1 mL of a 1% solution of potassium ferricyanide[K₃Fe (CN)₆]). The mixture obtained is incubated at 50 °C for 20 min, and then 1 mL of 10% trichloroacetic acid (CCl₃COOH) is added to stop the reaction. The mixture is centrifuged at 2000g for 10 min. To 1 mL of the supernatant are added 1 mL of distilled water and 0.5 mL of 0.1% iron chloride (FeCl₃). The reaction medium is incubated at room temperature for 10 min. The results were given as 50% inhibition concentration (IC₅₀) and compared with the antioxidant standards (BHT, Ascorbic acid, and rutin).

Brine shrimp cytotoxicity test

The brine shrimp lethality bioassay was performed using the method of²⁹. Artemia nauplii were obtained by hatching brine shrimp eggs (Artemia salina) in artificial seawater (3.8% NaCl solution) for 48 h. The dissolution of 30 mg of OMW was carried out in 3 mL of artificial seawater containing 20% ​​DMSO to give a concentration of 10 μg/mL. From this solution 0.1, 5, 10, 20, 40, and 80 μL were transferred to each 10 mL vial and using artificial seawater the volume was adjusted to 10 mL per artificial seawater. Artemia nauplii were cultivated in these solutions and their mortality was observed after 24 h. The number of surviving larvae is counted in each tube and the mortality is calculated at each concentration as follows:

The control mortality should not exceed 15%. The artificial seawater medium containing DMSO used for the analysis was used as a negative control. K₂Cr₂O₇ was used as a standard in this test.

The 50% Death Concentration (DC50) reflecting the toxicity of the products is estimated as: (−)Toxicity when DC50 ≥ 100 μg/mL, (+) Toxicity when 100 μg/mL > DC50 ≥ 50 μg/mL, (++) Toxicity when 50 μg/mL > DC50 ≥ 10 μg/mL, and (+++) Toxicity when DC50 < 10 μg/mL.

The number of dead and alive nauplii was recorded after 24 h. Nauplii were considered dead if no internal or external movement was observed within 30 s. The percent mortality of salted shrimp and the LC 50 (median lethal concentration) was then calculated. This was done by plotting the percent mortality against the logarithm of the extract concentration. The LC 50 value was derived from the regression equation²⁹.

Cytotoxicity test of the phenolic extract of OMW on human neutrophils

Isolation of neutrophils by the Dextran–Ficoll method

The purpose of this method is to separate blood cells from fresh blood taken on an anticoagulant. The principle is based on the separation of blood components according to their density. The number of neutrophils recovered from a 400 mL blood bag after isolation ranges from 6 to 10 × 10⁸ neutrophils. Isolation of Polynuclear Neutrophils begins with mixing one volume of blood with one volume of 2% Dextran-T500 (prepared in advance in 0.9% NaCl and filtered). After 30 min incubation at room temperature, the majority of erythrocytes precipitate at the bottom of the tube. The upper phase contains the white blood cells. The latter is collected in clean tubes and the red blood cells of the lower phase are discarded. The phase containing the white blood cells is centrifuged for 8 min at 400 g at 22 °C. The pellet is suspended a second time in phosphate buffer, then deposited on a Ficoll cushion and centrifuged for 30 min at 400g and at 22 °C. After this step, three phases are distinguished, the pellet corresponds to the granulocytes (eosinophils, neutrophils and basophils) with the contaminating erythrocytes, the mononuclear cells (monocytes and lymphocytes) form a ring between the pellet and the supernatant is a mixture of plasma, buffer phosphate and Ficoll. Lysis of residual erythrocytes is carried out by adding a cold 0.2% NaCl hypotonic solution to the pellet, mixing for 40 s, isotonicity is restored by adding the same volume of 1.6% NaCl. The solution is buffered by adding cold phosphate buffer. The neutrophils are recovered in a small volume of phosphate buffer, after centrifugation for 8 min at 400g and 4 °C. Neutrophils are stored at 4 °C until use.

Neutrophil viability and purity test

The neutrophil viability test is performed by the Trypan blue exclusion test, which stains dead cells blue. While the purity of Polynuclear Neutrophils is checked with another dye, crystal violet stains the nuclei of cells, making it possible to distinguish the polylobed nucleus characteristic of neutrophils from other cell nuclei. The count is done on a Malassez slide by light microscopy at magnification × 40. Their effect has been verified on the viability of neutrophils, this test is very important if one plans to use these molecules in humans. To test the purified extracts, the purity of the freshly isolated neutrophils is checked with crystal violet (dye-based on gentian violet and acetic acid) and they are counted. The cells are resuspended in phosphate buffer and stored at 4 °C before using them. Before testing the extracts of the polyphenols on the neutrophils, it was been checked the cytotoxicity of the product, by incubating the neutrophils in the presence of high concentrations of polyphenols ranging from 0 to 300 µg/mL for 30 min, the dead cells allow the blue of Trypan, which stains them blue, unlike living cells which remain transparent.

Anti-inflammatory activity

Inhibition of protein denaturation (IPD)

It is determined by the method described by⁵² with slight modifications. The concept is that the phenolic extract of OMW inhibits denaturation of BSA induced by heat (72 °C) 0.1 mL of each concentration of extract added to 1 mL of 0.2% BSA solution prepared in Tris–HCl pH 6.6, then incubated at 37 °C for 15 min then in a water bath at 72 °C for 5 min. After cooling, the turbidity is measured at 600 nm in a cell spectrophotometer (SPECORD 210 plus). Diclofenac sodium standard (injectable form) was produced using the same technique in ultra-pure distilled water from a 500-ppm mother solution, with distilled water serving as a negative control.

Membrane stabilizing potential (MSP)

It was measured according to⁵³. An equivalent volume of blood was collected from healthy human volunteers who had not taken any NSAIDs for two weeks before blood collection and combined with an equal volume of sterile Alsever solution. This blood solution was centrifuged for 10 min at 3000 rpm, the packed cells were separated and washed with iso-saline solution, and a 10% (v/v) suspension was made using an iso-saline solution.

1 mL phosphate-buffered saline, 0.5 mL 10% blood suspension, 0.5 mL phenolic extract of OMW with various concentrations, and 2 mL hypotonic saline make up the dose combination. All test mixtures were incubated at 37 °C for 30 min and then centrifuged at 3000 rpm for 20 min. The hemoglobin concentration was measured using a spectrophotometric measurement at 560 nm after the supernatant was separated.

The negative control was distilled water and the positive control was diclofenac sodium at the final concentration. The IC₅₀ was measured once again using a graph that showed inhibition at various doses.

Anticoagulant activity in vitro

Endogenous coagulation pathway (APTT)

Activating partial thromboplastin time (APTT) was determined according to²⁹. A platelet plasma pool comprises a plasma combination from ten healthy, untreated individuals with normal APTT and PT.

The activity of the phenolic extract was established on a volume of 100 μL whose plasma is 90 μL was mixed with 10 μL of extract. After 15 min of incubation at 37 °C 100 μL cephalin kaolin was added to the mixture, which was re-incubated for 3 min with agitation at 37 °C. Using a coagulometer, the coagulation time was measured by adding 100 µL of warmed calcium chloride (0.025 M). In parallel, a positive control of calciparine (unfractionated heparin) and a negative control test (substituting the samples with a 0.9% NaCl solution) were performed under identical circumstances. An increase in APTT in the presence of polyphenols compared to the negative control implies an anticoagulant impact at this route level. Clotting time was determined by an automatic coagulation analysis system (Coa DATA 4004).

Exogenous coagulation pathway (PT)

Prothrombin time (PT) was determined according to the protocol described by⁵⁴. The coagulation time of citrated plasma in the presence of an excess of calcium thromboplastin is measured in this activity using platelet-poor plasma in the presence of calcium thromboplastin. The phenol extract (90 and 10 µL, respectively) was combined with 100 µL of platelet-poor plasma that had been warmed for 2 min at 37 °C. After 15 min of incubation at 37 °C, 200 µL of calcium thromboplastin was added to the mixture, which had been warmed for at least 15 min at 37 °C. Coagulation time was determined by an automatic coagulation analysis system (CoaDATA 4004).

Study of antioxidant activity on a cell model

Hemolysis test

The anti-hemolytic effect of plant extracts is evaluated in vitro using the Erythrocyte model. The latter is easily isolated from blood and its membrane shows similarities to other cell membranes⁵⁵.

Preparation of the erythrocyte suspension

The blood used to prepare the erythrocyte suspensions was taken from healthy people in heparin tubes. Serological analyses were carried out to exclude any risk of contamination of any pathology. After centrifugation of the blood at 3000 rpm/5 min, the recovered pellet is washed 3 times with the Phosphate Buffered Saline (PBS) solution formed from 10 mM potassium phosphate buffer, pH = 7.4 and 154 mM of NaCl. Each wash consists of a suspension of the cells in iso-saline PBS and centrifugation at 3000 rpm/5 min. After the last centrifugation, the pellet is suspended for the second time again in a solution of iso-saline PBS at the rate of 1 volume of the pellet and 9 volumes of PBS, thus obtaining a hematocrit at 10%⁵⁶.

Development of in vitro induced hemolysis tests

The exposure of red blood cells (RBC) to certain physicochemical parameters such as the hypotonic medium, the use of a membrane disruptor such as detergents or reactive oxygen species, causes a rupture of its cytoplasmic membrane thus causing the release of the hemoglobin, which will then be determined by visible absorbance spectrophotometry at 540 nm. To test the anti-hemolytic effect of the phenolic extracts of OMW, tests on an erythrocyte model with hemolysis induced by three different agents (hypotonic medium, salicylic acid and H₂O₂) were carried out.

Induction with salicylic acid

In test tubes each containing 4.5 mL of hypotonic NaCl (4.5 mg/mL), 50 μL of salicylic acid in different concentrations (0.1, 0.2, 0.3, 0.4 and 0.5 mg/mL) were added. The control tube receives the same volume of PBS buffer, and then a volume of 500 μL of the erythrocyte suspension is added to each tube. After that, the tubes are homogenized, incubated at 37 °C for 30 min in a water bath, and centrifuged at 3000 rpm for 5 min. The absorbance is then measured at 540 nm³⁹.

Hypotonic induction

To determine the concentration of NaCl, which causes the lysis of red blood cells, 100 μL of the erythrocyte suspension (10%) were added to 5 mL of NaCl at different concentrations (2, 3, 4, 5, 6 and 7 mg/mL), as well as a negative (Isotonic NaCl 9 mg/mL) and positive (distilled water) control. After incubation for 30 min at room temperature, the mixture was centrifuged at 3000 rpm/10 min and the DO was read at 540 nm⁵⁷.

Induction by hydrogen peroxide H₂O₂

A volume of 500 μL of H₂O₂ at different dilutions (0, 1, 2, 5 and 10 mM) were mixed with a volume of 250 μL of the suspension of red blood cells. After 3 h of incubation at 37 °C, PBS was added and then the mixture was subjected to centrifugation for 10 min with a speed of 3000 rpm. The absorbance of the supernatant was read at 540 nm. Controls were prepared by replacing H₂O₂ with distilled water for the positive control and with PBS for the negative control⁴⁸.

The percent hemolysis for all tests was calculated using the following formula⁵⁵:

Measurement of the oxidative explosion of polynuclear neutrophils by the cytochrome c reduction technique

The production of O₂^·− by activated neutrophils is measured by the cytochrome c reduction technique⁴⁹. The principle of this test is based on the use of oxidized cytochrome c “Fe³⁺” in the presence of a source producing superoxide anions (O₂^·−) such as neutrophils or by an acellular system composed of xanthine/hypoxanthine and xanthine oxidase. In the presence of this very reactive and unstable O₂^·− radical, cytochrome c is reduced to Fe²⁺. This test exclusively detects the extracellular superoxide anion because cytochrome c does not cross the cell membrane; the reduced cytochrome is measured by spectrophotometry at 550 nm. In practice, neutrophils at 10⁶ cells/mL in phosphate buffer are pretreated with increasing concentrations of polyphenols (0, 50, 100, 200 and 300) µg/mL for 10 min at 37 °C. The cells are incubated in the presence of cytochrome c at 1 mg/mL final and then stimulated with Phorbol Myristate Acetate (PMA) (100 ng/mL) and the absorbance is measured at 550 nm using a thermostated spectrophotometer brand UVIKON 860. The production of O₂^·− is measured for 10 min and the results are expressed in nanomoles of O₂^·− produced per minute and per million neutrophils using Beer Lambert’s law: A = ε. C. l, where ε is the molar extinction coefficient (l. Mol⁻¹. Cm⁻¹), A being the absorbance (without unit), C is the concentration of the solute (mol/L).

Statistical study

Data obtained were presented as mean ± SD of three dependent determinations. Significant differences between means of total phenolic, total flavonoids, tannins and LC–MS analysis results were determined by Student t-test, and p values (< 0.05) were regarded as significant. Results of antioxidant, anti-inflammatory and anticoagulant activities were subjected to statistical analysis of variance (ANOVA) using ECXEL STAT (version 2014) package at p < 0.05 significant levels.