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A comparative study on chemical composition, antibiofilm and biological activities of leaves extracts of four Tunisian olive cultivars

Olea europaea L. is one of the most important fruit trees in Tunisia because of its content of many potentially bioactive compounds. The aim of this study was to evaluate the chemical composition, antibiofilm, antiradical and acethylcholinesterase inhibitory activities from four Tunisian cultivars of Olea europaea L., i.e. ‘Chetoui’, ‘Meski’, ‘Oueslati’ and ‘Jarboui’. By means of standardized methods, total phenols were determined and some of them characterized by HPLC. The total phenols and flavonoids contents were found to be the highest in the leaves of Chetoui cultivar. The Chetoui cultivar exhibited an important antioxidant and anticholinesterasic activity and an important anti-biofilm activity against Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, Candida albicans, Enterococcus faecalis and Escherichia coli, with percentages of inhibition comprised between 83 and 93% at 2xMIC values. Olive leaves extracts could be used in the control of bacterial biofilms in food and food-related environments.


Introduction
The study of biologically active compounds from natural sources has always been of great interest to scientists looking for new sources of drugs to treat infectious diseases. Indeed, disorders caused by microorganisms remain a major threat to public health, despite the considerable progress of medicine (Morse, 2004;Edziri et al., 2010).
Olea europaea L. is a species of the Oleaceae family and it is an important source of nutritients and biologically active principles throughout the history of civilization. It is an evergreen tree or shrub native to the Mediterranean region. Typically, it is a short tree, that rarely exceeds 10-15 meters in height. In Tunisia, O. europaea is one of the most important cultivated fruit crop, with approximately 70 million trees, covering 1600 thousand hectares. Olive leaves extracts have reported to have health benefits, such as increasing energy levels, lowering blood pressure, and supporting the cardiovascular and immune systems (Khayyal et al., 2002;Covas, 2007;El and Karakaya, 2009).
Olives contain many potentially bioactive compounds having antioxidant, anti-inflammatory, antiviral, and hypocholesterolemic properties (Covas, 2007). O. europaea is widely studied in nutrition research, whereas the leaves are important for their content in secondary metabolites, in particular the secoiridoid derivatives oleacein and oleuropein, the former being responsible for the hypotensive and hypoglycemic activities (Gonzalez et al., 1992).
It has been shown that the qualitative and quantitative phenolic composition of the olive tree strongly differs among cultivars, plant parts and environmental conditions (Techathuvanan et al., 2014).
The cultivar Chetoui, together with three other minor ones, Meski, Oueslati and Jarboui, are cultivated in Tunisia. The olive cultivar, Chemlali, accounts for about 80% of the national olive oil production and is grown in central and southern Tunisia. The second most cultivated one, Chetoui is mainly widespread in the north of the country, and provides about 20% of the national production. Jarboui is mainly grown in the North-West of Tunisia (Teboursouk), while Oueslati in central part of the country (Kairouan). No scientific report about the chemical composition, antibiofilm and acethylcholinesterase inhibitory activities concerning the leaves extracts of these four Tunisian four cultivars is present in the literature.
So the present study aims to compare the chemical composition, antibiofilm, antioxidant and anticholinesterase activities of the methanol leaves extracts obtained from these four Tunisian cultivars grown in the north and center of Tunisia.

Plant and sample preparation
Olea europaea L. leaves of the four Tunisian cultivars Chetoui, Meski, Oueslati and Jarboui were respectively collected from the North (Beja and Tunis), the center (Kairouan) and the Sahel region (Sousse) of Tunisia. Collection was performed in February, when the leaves completed their growth cycle. The leaves were manually isolated to obtain a weight of 100 g. Leaves were washed under running tap water to remove dust particles, insects and plankton, and then dried under the shade at room temperature. The dried plant materials were milled into fine powder using mechanical grinder and stored at 4 C. Voucher specimens (Rus.1212) have been deposited in the Faculty of Pharmacy of Monastir University.

Preparation of methanol extracts
Extracts were prepared according to the methodology proposed by Casas-Sanchez et al. (2007), with minor modifications. Briefly, 100 g of dried leaves at 70 C temperature for 24 hrs, were soaked in 500 mL of methanol for 24 h at room temperature (25 AE 2 C). Extraction was repeated three times, and the obtained extracts were combined and filtered through number 1 Whatman filter paper n 1(Sigma Aldrich, France). The solutions were concentrated to dryness under reduced pressure using a rotary evaporator at 45 C. Extracts were stored at 4 C until further experiments.

Determination of pigments content
The procedure was carried out in the dark at 4 C. A leaf sample (0.25 g) was mashed using a mortar and pestle with 80% acetone (v/v). The extract was filtered and centrifuged in sealed tubes at 15000 g for 5 min. The supernatant was collected and the absorbance was read at 663 and 647 nm for determination chlorophyll a and chlorophyll b, respectively, and at 470 nm for the carotenoid content using an UV-VIS spectrophotometer (Spectro Double Beam PC UVD-2950, Labomed). The concentrations of chlorophyll a, chlorophyll b, and the sum of leaf carotenoids (xanthophylls and carotenes) were given in μg ml À1 of extract solution, according to the equations of Lichtenthaler and Buschmann (2001): Total phenols were determined using a slightly modified Folin-Ciocalteu procedure as described by Montedoro et al. (1992). Briefly, 0.125 ml of extract solution were mixed with 0.5 ml distilled water and 0.125 ml of Folin-Ciocalteu reagent. After 3 min, 1.25 ml of 7% Na 2 CO 3 solution was added to the mixture. The final volume was adjusted to 2.5 ml with distilled water and thoroughly mixed. The absorbance of the resulting blue complex was then measured at 760 nm after incubation for 90 min at 23 C in dark (Spectro UV-VIS, Double Beam PC UVD-2950, Labomed). The total phenols content was expressed as mg gallic acid equivalents per gram of dry weight (mg GAE/g DW).
The calibration curve range for gallic acid was 0-400 μg/ml. Triplicate measurements were taken for all the samples.

Total flavonoids
Total flavonoids contents were measured by the colorimetric assay developed by Zhishen et al. (1999). An aliquot of suitable diluted samples or standard solution of catechin was mixed with 0.075 ml of (5% w/v) NaNO 2 solution and kept at rest for 6 min. Afterwards, 0.15 ml of a fresh (10%) AlCl 3 solution was added to the mixture, followed by 0.5 ml of (1 M) NaOH solution after 5 min. The final volume was adjusted to 2.5 ml with distilled water and thoroughly mixed. The absorbance of the mixture was determined at 510 nm (Spectro UV-VIS, Double Beam PC UVD-2950, Labomed) against the same mixture without the sample (blank). Total flavonoids of plant organs were expressed as mg catechin equivalents/g dry weight (mg CE/g DW).

Identification of phenolic compounds using HPLC
The presence and amount of phenolic compounds in the extracts were studied by reversed-phase HPLC analysis by means of a binary gradient elution using an Agilent Technologies 1100 series liquid chromatography (HPLC, Palo Alto, CA, USA) coupled with an UV-VIS multi-wavelength detector. The separation was carried out at room temperature on a Eurospher-100 C 18 reversed-phase column. The mobile phase consisted of acetonitrile (solvent A) and water containing 0.2 % sulfuric acid (solvent B). The flow rate was 0.5 ml min -1 . The gradient program was as follows: 15% A/85% B, 0-12 min; 40% A/60% B, 12-14 min; 60% A/ 40% B, 14-18 min; 80% A/20% B, 18-20 min; 90% A/10% B, 20-24 min; 100% A, 24-28 min. The injection volume was 20 μl, and peaks were monitored at 280 nm. Samples were filtered through a 0.45 mm membrane filter before injection. Peaks were identified by congruent retention times compared with standards. Quantification of phenolic compounds was achieved by High-performance liquid chromatography (HPLC) comparing peak areas with those of resorcinol used as internal standard. Data were expressed as mg of phenols/100 g of dry weight (DW).
2.9. Antimicrobial activity 2.9.1. Micro-well determination of MIC, MBC and MFC The MIC (Minimum Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) were determined on the plant extracts that showed antimicrobial activity, by a broth microdilution method proposed by Novy et al. (2015) with minor modifications. Briefly, 100 μL of Mueller-Hinton Broth (Difco) plus different concentrations of plant extracts were prepared and transferred to each microplate well to obtain serial dilutions of the active extract, ranging from 4 to 512 mg/mL. Then, 10 μL of a fresh culture (final concentration of 1 Â 10 6 CFU/mL) of test organisms was added. Microplates were incubated at 37 C for 24 h. Wells with no added plant extract were used as a positive growth control. Wells without added bacteria were used as a negative growth control. MIC was defined as the lowest concentration of the extract that restricted the visible growth of microorganism tested.
To determine MBC and MFC (Minimum Fungicidal Concentration), 100 μL from each well that showed no visible growth were re-inoculated on Müller-Hinton agar (MH) agar plates incubated at 37 C for 24 h. MBC and MFC was defined as the lowest extract concentration showing no bacterial and fungi growth.

Inhibition of biofilm formation
The inhibition of biofilm formation was tested only against seven type strains including B. subtilis, S. aureus MR, S. aureus MTR, E. coli, P. aeroginosa, C. albicans, E. foecalis, was examined by using the modified micro dilution method (Changwei et al., 2008). Prevention of biofilm formation was examined by microdilution, similar to the MIC assay for planktonic cells ((Montedoro et al., 1992). Overnight cultures grown in Brain Heart Infusion (BHI) were diluted to 10 6 CFU/mL in BHI supplemented with 2% glucose (w/v). A 200 μl aliquot was Transferred to a 96-well microtiter plate, and 100 μl of extracts with final concentration corresponding to MIC, 2xMIC, 4xMIC was added. The medium without extract was used as a control. Each assay was repeated three times. After incubation at 37 C for 24 h, the culture supernatant was discarded, and the wells were washed twice with phosphate-buffered saline (PBS) to remove non adherent cells. The plates were air-dried, and the surface-attached cells were stained with 200 μl of 0.1% crystal violet for 30 min. then, the crystal violet was removed and the plate was washed with water. Crystal violet stained biofilm cells were determined at 570 nm with with a Multiskan reader (BioRad, Tokyo, Japan). In order to access the ability of the extract to prevent biofilm formation, the percentage of biofilm inhibition was calculated using the equation: ½ðODðgrowth controlÞ À ODðsampleÞÞ=ODðgrowth controlÞ Â 100 where OD (growth control) refers to the absorbance of the bacteria growth without extract, and OD(sample) refers to the absorbance of the extract with bacteria.
2.11. Antiradical activities 2.11.1. DPPH radical-scavenging assay The DPPH method (Jacob and Shenbagaraman, 2011) was used to determine the antioxidant activity of the olive extracts. A 20 μl sample from the stock solution was dissolved in absolute ethanol to a final volume of 1 ml and then added to a 1 ml of 0.1 mM DPPH (in absolute ethanol). The reaction mixture was kept at room temperature. The optical density (OD) of the solution was measured after 20 min at 517 nm using a Spectro UV-VIS, Double Beam PC UVD-2950 (Labomed) spectrophotometer. The optical densities of the samples in the absence of DPPH were subtracted from the corresponding OD in presence of DPPH. The percent reduction values were determined and compared with appropriate standards. The percent inhibition of the free radical DPPH (I %) was calculated using the equation: where A blank is the absorbance of the control reaction containing all reagents except the tested compound), and A sample is the absorbance of the tested compound. Butylated hydroxytoluene (BHT) was used as positive control.

ABTS radical-scavenging activity
For the determination of the antiradical activity, a protocol based on the ABTS þ free radical decolorization assay was used, according to the method of Lavelli (2002). In brief, 5.0 ml of a 7.0 mM ABTS solution were treated overnight in the dark with 88.0 μl of a 140 mM potassium persulfate solution to yield the ABTS radical cation. Prior to use in the assay, the ABTS radical cation was diluted with ethanol to an initial absorbance of about 0.700 (ratio of 1:88) at 734 nm and 30 C. Free radical-scavenging activity was assessed by mixing 1.0 ml of diluted ABTS þ radical cation with 10 μl of sample. Absorbance was measured after 20 minutes after addition of the antioxidant at 520 nm using a Spectro UV-VIS, Double Beam PC UVD-2950 (Labomed) spectrophotometer. Results were expressed as percent inhibition. All tests were carried out in triplicate. Trolox was used as positive control.

Acetylcholinesterase (AChE) inhibition
The enzymatic activity was measured using a slightly modified version of the method described by Bahloul et al. (2016). Briefly, 50 μL of buffer 0.1 M (pH 8), 25 μL of the methanol extract dissolved in ethanol at different concentrations, then 25 μL of 0.22 U/mL of AChE enzyme were mixed. After 20 min incubation at 37 C, 25 μL of 15 mM acetylthiocholine iodide (AChI) and 125 μL of 3 mM 5,5 0 -dithiobis-(2-nitrobenzoic acid) were added, and the resulting mixture incubated for 30 min at room temperature. The absorbance of the mixture was measured at 405 nm using an UV-VIS SpectraMax 190 Microplate Reader. The inhibitory effect of the test compound was determined by comparison to the negative control: inhibition percentage (I%) ¼ ((A 0 À A 1 )/A 0 )) Â 100, where A0 refers to the absorbance of the blank sample, and A1 to the absorbance of the sample. The test was repeated three times. The inhibition of the enzyme activity was expressed as IC 50 (the concentration of the sample required to inhibit 50% of enzyme), which was calculated by a linear regression analysis.

Statistical analysis
Data are expressed as mean AE standard deviation (SD). Statistical analysis involved a one-way analysis of variance (ANOVA). A value of P less than 0.05 (p < 0.05) was considered statistically significant.

Total phenols, flavonoids and chlorophyll contents
The results of the total phenols, flavonoids and chlorophyll contents are listed in Table 1. The methanol extracts of the leaves of the Chetoui cultivar is characterized by the highest total phenols (47.47 mg GAE/g), flavonoids (7.29 mg CE/g) and chlorophyll (38.5 μg/ml) amounts, followed by the Meski, Oueslati and Jarboui cultivars. Statistical analysis of the total phenols and flavonoids contents showed significant differences between the four cultivars, whilst the total chlorophyll amounts were not statistically different between cultivars. It can be hypothesized that the significant differences observed for total phenols and flavonoids may be related both to genetics and to the geographical origin (Kallithraka et al., 2004;Felhi et al., 2016).
The results on total phenols and flavonoids contents support the observed activity of the extracts against free radicals (Felhi et al., 2016). In other scientific report Blasi et al. (2016) demonstrated that Phenolic contents from four Italian cultivars increased from December to March (P 0.01) and from March to June (P 0.01), and they reported that the highest total phenols values (P 0.05) were found in June for all cultivars. so we can conclude that phenol content showed marked variations with plant growth, in fact probably the phenol storage in the leaves is a time dependent regulated process, according to the life cycle of olive leaves. In addition € Ozcan et al. (2019) demonstrated that fatty acid composition and phenols contents of the olive oils showed differences depending on the olive variety. Akbaş et al. (2017) reported that The highest total phenol contents were found in olive leaves heated in microwave 540 W and athmospheric air.

Identification and quantification of phenolic compounds by HPLC
The HPLC analysis of the olive leaves extracts of the four cultivars allowed the identification of nine phenolic compounds (Table 2). Hydroxytyrosol, tyrosol, 4-hydroxybenzoic acid, rutin, luteolin-7-Oglucoside, apigenin-7-O-glucoside, oleuropein, apigenin and catechin hydrate. All these compounds were previously characterized in olive leaves (Meirinhos et al., 2005;Sahin and Bilgin, 2012). The major phenolic components common to all the four cultivars were oleuropein and apigenin-7-O-glucoside, with oleiropein as the main one. This component was present in highest amounts in the Meski cultivar. As shown in Table 2, Chetoui and Meski cultivars had the highest phenolic content compared to Oueslati and Jarboui ones. Apigenin was the least represented derivative.
These differences are not surprising because of the influence of both genetic and geographic factors (North, Sahel and Center Tunisia). These results are in agreement with Brahmi et al. (2013), who found that the olive organs from the northern cultivars (Chetoui) had the highest level of hydroxybenzoic acids, hydroxycinnamic acids and flavonoids than the southern ones (Chemlali).
Additionally, these results are also in good agreement with those reported by Benavente-Garcia et al. (2000), which showed that the polyphenols in olive leaves consists of oleuropeosides (oleuropein and verbascoside), flavones (luteolin, diosmetin, apigenin-7-O-glucoside, luteolin-7-O-glucoside, and diosmetin-7-O-glucoside), flavonols (rutin), flavan-3-ols (catechin), and substituted phenol (tyrosol, hydroxytyrosol, vanillin, vanillic acid, and caffeic acid). Rocchetti et al. (2019) reported that homogenizer-assisted extraction using methanol 100% produced an extract of M. oleifera leaves with the highest amounts of phenolic compounds. These findings demonstrate that each extraction method promoted the recovery of specific phenolic subclasses with different efficiencies. So we can conclude that extraction methods and conditions (solvent, time, temperature, ...) influence the type of phenolic compounds of leaves extracts. Table 3 shows the MIC values for the four olive cultivars obtained using the microdilution method. The values varied between 32 and 128 μg/ml, without large difference between the four olive cultivars extracts.

Antibacterial activity
The Chetoui methanol extract had the best antibacterial activity against all gram negative bacteria and against all S. aureus strains, with a MIC and MBC values of 32 μg/ml. In addition, this extract showed a good antifungal activity against Candida strains, with a MIC between 32 and 64 μg/ml Techathuvanan et al. (2014) found that Olea europaea leaf extracts have antimicrobial activities against foodborne pathogens, such as S. aureus, E. coli, Salmonella spp., and L. monocytogenes, with MIC values ranging between 1.4 and 5.2 mg/ml. This good antibacterial activity of Tunisian olive varieties could be attributed to oleuropein, which is the major compound identified in the present study or this is may be due to the climatic difference between tunisian varieties and turkich variety. Liu et al. (2017) demonstrated that at 62.5 mg/ml, ethanolic extracts of olive leaves almost completely inhibited the growth of Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella enteritidis. In addition, they observed that ethanol extracts were able to destroy the flagella of L. monocytogenes and to reduce the motility of the pathogens. Furthermore they found that the extracts inhibited biofilm formation in L. monocytogenes and S. enteritidis. According to Masoko and Makgapeetja (2015), the methanol extract of Olea africana have a good antibacterial activity against E. coli, P. aeruginosa, E. faecalis and S. aureus, with MIC values comprised between 0.24 and 0.63 mg/ml. The differences observed between the present study results and these findings are probably due to differences between the two olive species. Owen et al. (2003) also reported that olive leaves have antimicrobial activity against E. coli, S. aureus, B. cereus and S. typhi. The results reported in the present study show similarity with previous ones (Markin et al., 2003;Pereira et al., 2007;Sudjana et al., 2009;Lee and Lee, 2010;G€ okmen et al., 2014) about the antimicrobial activity of olive leaves extracts. According to Pereira et al. (2007), the antimicrobial mechanism of the extract consists in the denaturation of the proteins and increasing cell membrane permeability. Similarly, Lee and Lee (2010) reported that the combined phenols mixture prepared from an olive leaves extract showed inhibition effects against B. cereus and S. enteritidis. In addition, G€ okmen et al. (2014) showed that the MICs of olive leaves extract against L. monocytogenes, E. coli O157, E. sakazakii and P. aeruginosa was !32 μg mL À1 , while the MIC against B. cereus, S. aureus, E. faecalis, P. vulgaris, E. coli, Salmonella typhimurium was !16 mg mL À1 .

Antibiofilm activity
The four olive cultivars showed variable effects on the development of a preformed biofilm (Fig. 1. Chetoui and Meski extracts showed the best antibiofilm activity against P. aeroginosa, E. coli, B. cerus, E. faecalis, S. aureus, S. aureus MTR and C. albicans, with inhibition values of >50% at MIC doses. In detail, they all inhibit the biofilm of all the tested strains between 72 and 89.8% at doses of 2MIC, and the difference between these two concentrations was statistically significant. The Chetoui cultivar showed important anti-biofilm activities against P. aeruginosa, B. cereus, S. aureus, C. albicans, E. faecalis and E. coli, with percentage of Means with different letters were significantly different at the level of p < 0.05. GAE: Gallic acid equivalent, CE: Catechin equivalent, Galanthamine: positive control with IC50 of 0.4 μg/ml). inhibition comprised between 83 and 93% at 2MIC values (Fig. 1). Also the methanol extracts from Jarboui and Oueslati showed a good antibiofilm activity against all the tested strains, with percentages of inhibition ranging from 54.5 to 83.8% at a concentration of 2 MIC ( Fig. 1 C  D). Only the Jarboui extract presented lesser level of biofilm inhibition activity (29.3%) against P. aeruginosa at MIC dose. For Jarboui and Oueslati cultivars, a significant difference was observed between the two concentrations (MIC and 2MIC). To the best of our knowledge, the present study is the first one about the antibiofilm activity of methanol extract of olive leaves obtained from these four Tunisian olive cultivars on the selected microorganisms. In a previous study, it was shown that the olive mill waste was able to reduce biofilm formation in E. coli (Carraro et al., 2014). On the contrary, according to Liu et al. (2017), olive leaves extracts slightly inhibited the biofilm formation in L. monocytogenes at 7.8 mg/ml. In the same study, the biofilm formation in S. enteritidis, was more inhibited (74% at 15.6 mg/ml).
The observed antibiofilm activity is generally attributable to the high concentrations of phenolic compounds, sucha as oleuropein, which antibiofilm activity was previously tested (Carraro et al., 2014) or may be due to a synergistic effect of some phenols contained in the olive extracts.
The mechanisms of antibacterial and antibiofilm activities of oleuropein are not completely understood. However, phenolic compounds have the ability to increase the permeability of cell membranes, thus facilitating their rupture (Taweechaisupapong et al., 2012). Casas- Sanchez et al. (2007) reported the interaction of oleuropein with phosphatidylglycerol at the surface of the bacterial cell membrane, causing changes that lead to the disruption of the cell envelope. The observed antimicrobial activities could be due, at least in part, to these properties of the phenol derivatives contained in the four olive leaves extracts.

Antioxidant and acetylcholinesterase (AChE) inhibition activity
DPPH and ABTS are free radicals that provide information about the potential of the extract components to inhibit oxidative cell damages by preventing reactive radical species from attacking key biomolecules in biological and food systems. They are also frequently used by the food industry and agricultural researchers to measure the antioxidant Table 3 Determination of MIC (μg/mL) and MBC (μg/mL) of methanol extracts of olive leaves of four different cultivars. capacities of foods. The results on the antioxidant activity using the DPPH assay of the four olive cultivars are shown in Fig. 2. It is apparent that the Chetoui and Oueslati methanol extracts have the highest antioxidant activity (87.05 and 70.47%, respectively). Noteworthy, the Chetoui extract exhibited a better DPPH scavenging activity than the reference antioxidant BHT (81.5%). Instead, a lesser activity was observed for the methanol extracts of Meski and Jarboui cultivars (47.3 and 61.6%, respectively). These differences were statistically significant and in good agreement with Brahmi et al. (2013) that reported a greater activity for the methanol extracts of various olive organs cultivated in the North of Tunisia than those obtained from the southern cultivars (see Fig. 3).
The antioxidant activity was evaluated using also the ABTS methods, obtaining results that substantially confirmed the trend observed with the DPPH assay. Fig. 2 shows that Chetoui and Meski extracts had the best antioxidant activity by this method compared to other cultivars and the reference antioxidant Trolox. These high antioxidant activities could be attributed to their high levels of Total phenolic content (TPC) and flavonoids, especially can be due to oleuropein, which was known by its antioxidant activity ( € Ozcan and Matthaus, 2017). As demonstrated by Cheung et al. (2003), free radical scavenging activity is strongly related to the presence of polyphenol compounds. Indeed, the results of the HPLC analysis demonstrated the abundance of oleuropein, a wellknown antioxidant derivative (Fukumoto and Mazza, 2000;Stamatopoulos et al., 2014). The antioxidant activity was correlated with the presence of phenolic compounds also in other studies (Fukumoto and Mazza, 2000;Brahmi et al., 2013).
Generally, it can be observed that all olive leaf extracts exhibited high radical scavenging activity even if it is well known that antioxidant capacity is influenced by several factors, among which harvesting period and cultivar (Yorulmaz et al., 2012;Brahmi et al., 2015). The results reported by Blasi et al. (2016) showed that the antioxidant activity of four Italian cultivars was the highest in March, when the leaves had completed their growth, while it decreased slightly until September, when started the ripening of the fruit. They demonstrated that The lowest values had been found for all cultivar samples harvested in December (from 40.9 of Frantoio to 67.1% of Dolce Agogia), while olive leaves from Leccino cultivar harvested in March exhibited the highest activity (86.1%). Further more the antioxidant activity determined by ABTS showed significant differences also between Moraiolo cultivar and Frantoio and between Moraiolo and Leccino (P ¼ 0.05) with regard to September harvest.
Furthermore, the results reported by Abaza et al. (2011) showed that the DPPH radical scavenging activity increased with the extract concentration (at 0.5 mg/mL the activity was 59.74%, using 70% EtOH). But Urbani et al. (2015) reported that all methanolic saffron samples (Italy) showed very low radical scavenging activity with DPPH method while higher values were observed with hydrolysed sample but saffron methanolic extracts exhibited higher antioxidant activity with ABTS test than the respective hydrolyzed, they also demonstrated a strong correlation between antioxidant activity and secondary metabolit content (Urbani et al., 2015). Akbaş et al. (2017) tested the effect of drying on antioxidant activity of Turkich olive leaves they demonstrated that the antioxidant activity of olive leaves heated in microwave 180 W change between 76.99% (Akdeniz Yerli) and 82.26% (Sarı Ulak) and they reported that antioxidant activity values of leave samples heated in microwave 360 W varied between 81.20% (Yagl ık) and 82.24% (Gemlik).
We can conclude that antioxidant activity depend on cultivars, extraction and drying methods and influenced by chemical composition in the tested plants.
The methanol extracts of Chetoui and Meski also showed the highest rates of acethylcholinesterase inhibition capacity (IC 50 ¼ 500 μg/ml) (Table 1). Results were higher than galanthamine (positive control; IC 50 ¼ 0. 4 μg/ml), a drug approved for the treatment of the Alzheimer's disease. Crowch and Okello (2009) found that galanthamine was 500 times more potent than the aqueous extracts of Rhamnus prinoides (IC 50 ¼ 0.2 mg/ml). Ferreira et al. (2006) reported the extract obtained from Hypericum undulatum as a powerful inhibitor, showing 81.7% inhibition at 5 mg/ml. To the best of our knowledge, the present study is the first to report the inhibition of acetylcholinesterase of extracts obtained from these olive cultivars. A positive correlation was observed between the IC 50 values and the total phenols and flavonoids contents.
Acetylcholinesterase is the principal enzyme involved in the hydrolysis of acetylcholine. The great reduction of this neurotransmitter in the cerebral cortex is a significant factor in Alzheimer's disease. Therefore, the research of new compounds with the capacity of inhibiting that enzyme has been considered as a promising strategy for the treatment of neurological disorders in which an insufficient cholinergic neurotransmission is involved.

Conclusions
In summary, the present study evaluated the differences in total phenols and flavonoids contents of four different leaves extracts obtained from Tunisian olive cultivars. Moreover, by means of HPLC analyses, the Fig. 2. Free radical scavenging capacities of methanolic extracts from the leaves of Chetoui, Meski, Ouslati and Jarboui cultivars by the ABTS assay. Values are expressed as means AE standard deviation (n ¼ 3). Means with different letters were significantly different at the level of p < 0.05. Fig. 3. Free radical scavenging capacities of methanolic extracts from leaves of Chetoui, Meski, Ouslati and Jarboui cultivars by the ABTS assay. Values are expressed as means AE standard deviation (n ¼ 3). Means with different letters were significantly different at the level of p < 0.05. differences in type and amounts of each compound were assessed for every olive cultivar. The Chetoui cultivar was the richest in flavonoids and polyphenols. Furthermore, in this cultivar oleuropein was the main phenol. Chetoui and Meski showed the highest antioxidant and acetylcholinesterase inhibition activities. Significant differences were observed for antioxidant and acetylcholinesterase inhibition activities for the extracts of the four olive cultivars. It has been shown that Chetoui and Meski methanol extracts exhibit a good antibacterial activity against Gram positive and negative strains and were able to inhibit the biofilm formation on polystyrene surface, with inhibition values of >50% with at MIC doses.
In addition, this study provides data for supporting the use of Tunisian olive leaves extracts as natural antioxidant agents, and confirms that these extracts represent a significant source of phenolic compounds. Therefore, it is suggested that further works should be performed on the isolation and identification of the antimicrobial, antioxidant, antbiofilm and anticholinesterasic active constituents and their possible synergistic/ antagonistic interactions.

Author contribution statement
Hayet Edziri: Conceived and designed the experiments; Wrote the paper.
Guido Flamini: Performed the experiments. Maha Mastouri: Conceived and designed the experiments.

Funding statement
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.