Phytochemical profile, cytotoxic, antioxidant, and allelopathic potentials of aqueous leaf extracts of Olea europaea

Abstract Although bioactivities of Olea europaea (OE) have been widely described, most of them were related to its methanolic extracts or its essential oils, While data related to aqueous extracts still very scarce. Thus, in this study, the phytochemical composition, the antioxidant activity, the cytotoxic potential, and the allelopathic potential of aqueous leaf extracts from two varieties of Olea europaea were investigated and compared. High‐performance liquid chromatography (HPLC) was used to identify and quantify the constituents of the tested plants, and spectrophotometric methods to evaluate antioxidant activities. The cytotoxic potential was investigated using murine oligodendrocytes (158N) while germination seeds’ test was used for allelopathic activity. HPLC analysis showed the presence of 10 phenolic compounds in both extracts. Chemlali variety showed the highest antioxidant and allelopathic activities. Regarding the cytotoxicity effect, a significant increase in cell viability was observed with both of our extracts compared to untreated cells. These results confirm that aqueous extracts from OE produce a range of substances with potential antioxidant, antifungal, and allelopathic effects without toxic effects. Thus, they could be used as an alternative of chemical compounds.

Olive leaf is one among large amounts of by-products generated by olive oil production processes (Nunes et al., 2016). This agro-industrial material has also been widely used in traditional remedies in Mediterranean and European countries (Abd El-Rahman, 2016).
It is generally used as energy biomass or animal feed and considered low-cost raw materials (Sofo et al., 2008). These aerial parts were also chosen due to their beneficial effects on human health and their important antioxidant potentials (Abd El-Rahman, 2016;Guex et al., 2019). A study performed by Candar, Demirci, Baran, and Akpınar (2018) showed that cinnamon, black cumin, and olive leaves were the most commonly favored plant products among diabetic patients.
Bioactivities of olive leaf compounds have been reported (El & Karakaya, 2009;Khaliq et al., 2015). Several data showed that they are active against a wide range of microorganisms, have in vitro and in vivo antioxidant activity, antiproliferative effect against cancer and endothelial cells and radioprotective activity (Abd El-Rahman, 2016;Guex et al., 2019). The potential of olive leaves for the prevention of many human diseases such as hypertension, cardiovascular, and diabetes, has been performed (Orak, Karamac, & Amarowicz, 2019).
Recently, the photoprotective potential of olive leaves extract has been also evaluated (da Silva et al., 2019), whereas only minor studies report the bioactivities of the two varieties of leaves of Olea europaea sampled in genotypes grown Tunisia (Khlif et al., 2015). To the best of our knowledge, no data are available regarding the cytotoxicity and the allelopathic activities of these varieties.
Due to a high interest on environmental protection and organic agriculture, attention has been focused on allelopathy research. In fact, allelopathy is a biological phenomenon by which an organism produces one or more biochemicals that influence the germination, reproduction, growth, and survival of other organisms (Shengpeng, Zhangshun, Shanyun, Wan, & Han, 2015). Additionally, excessive use of weed herbicides has resulted in a significant emergence of new strains resistant to chemical herbicides which have negative impact on human health and the environment (Mardi & El-Darier, 2018).
Thus, biological control method using natural plants as herbicides will be a better alternative to chemical weedicides. Aqueous olive leaf extracts will be candidate for such assays.
Accordingly, the aims of the current work were to evaluate, for the first time, the photochemical composition, the antiradical potential, as well as the cytotoxicity, and allopathic activities of the aqueous extract of two varieties of Olea europaea leaf harvested in the Sahel region of Tunisia Chemlali and Meski.

| Plant materials
Fresh and healthy leaves of two varieties (Meski and Chemlali) of olive trees (Olea europaea L.) were obtained from the Sahel of Tunisia (M'saken, Tunisia). The trees of each variety were grown under the same climatic and soil conditions and were collected in May. The taxonomic identification of all plant samples is identified/authenticated by the forest engineer of Bou-Hedma Natural Park, and a voucher specimen was deposited at the herbarium of the Laboratory of Medicinal Plants (INAT). Fresh leaves were transported to the laboratory, air-dried under room temperature, pulverized in a mortar to particles with sizes <0.8 mm, and finely powdered with an electric mill and kept for the extraction process.

| Extract preparation: Preparation of the infusion
To prepare infusion extracts, 100 ml of boiled distilled water was added to the sample (5 g) and was stored at room temperature for 5 min. Samples were then filtered under reduced pressure and finally lyophilized. They were re-dissolved in water to obtain a stock of solution of 1 mg/ml.

| Total Phenolic Content (TPC)
The TPC of olive leaf extracts was performed using Folin-Ciocalteu's procedure and gallic acid as the standard. 0.25 ml of the sample was combined with 1.25 ml of Folin-Ciocalteu's reagent (diluted tenfold), and 1 ml of Na 2 CO 3 (75 mg/ml). After incubation at 40°C for 40 min, the absorbance of the mixture was measured at 765 nm. All determinations were prepared in triplicate, and quantification was done on the basis of the standard curve of gallic acid. The TPC was expressed as mg gallic acid equivalents (GAE) per g of extract.

| Total flavonoids
The amount of flavonoids was determined according to the method of Jia, Tang, and Wu (1999). The extracts (250 μl), distilled water (1,250 μl), and sodium iodide Na 2 NO 2 (75 μl 5%) were shaken. Then, 150 μl of AlCl 3 (10%) was added and allowed to stand for 6 min before adding 500 μl of NaOH (1 M) and 250 μl of distilled water. The mixture was left to ambient temperature for 15 min; absorbance was then recorded at 510 nm. The total flavonoid content was expressed in milligrams of catechin equivalent (CE) per gram of samples. Analysis of each sample was carried out in triplicate.

| Flavonol content
The content of flavonols was determined by AlCl 3 method as described by (Miliauskas, Venskutonis, & Van Beek, 2004). Briefly, 500 μl of the plant extract was mixed with 500 μl aluminum trichloride (2%) and 1,500 μl of acetate of sodium (5%). The mixture was shaken and allowed at room temperature in obscurity for 2 hr 30 min. The absorption at 440 nm was then noted. Standard rutin samples were carried out from 0.05 g rutin. All determinations were performed in triplicate. The amount of flavonols in plant extracts was determined in milligrams of rutin equivalents (RUE) per 100 g of extracts.
Samples were filtered through a 0.45-mm membrane filter before injection.
Identification of peaks was performed by congruent retention times compared with standards. HPLC was used for the quantification of phenolic compounds by comparing peak areas with those of resorcinol used as internal standard. Data were expressed as mg of phenols/100 g of dry weight (DW).

| Cytotoxicity analysis by the MTT assay
Murine oligodendrocytes (158N) were used as a cell model to evaluate the cytotoxicity of the infusions extracted from two varieties of Olea Europaea leaves (Chemlali and Meski). Dulbecco's modified Eagle medium (DMEM) (Lonza, Amboise, France) supplemented with 5% (v/v) heat-inactivated fetal bovine serum (Dutscher, Brumath, France) and 1% antibiotics (penicillin, streptomycin) (Dutscher) were used for cell culture. The MTT assay was used to evaluate the effects of treatments on cell viability. MTT salt is reduced to formazan by the mitochondrial enzyme succinate dehydrogenase in the metabolically active cells and was performed on 158N cells as previously described (Nury et al., 2014).
Cells were plated in 96-well culture plates at a density of 1.5 × 10 4 cells/ well and treated for 24 hr with different concentrations of two varieties of Olea Europaea leaf infusion (Chemlali and Meski; 5-400 μg/ml). Vitamin E (α-tocopherol, 400 µM = 172 µg/ml) was used as positive control. Absorbance of plates was determined at 570 nm with a microplate reader (Mindray, Hamburg, Germany).

| Evaluation of antioxidant activity
Three different in vitro assays were carried out for the antioxidant activity of our extracts using solutions prepared by serial dilution of stock solution: scavenging effects on DPPH (2, 2-diphenyl-1-picrylhydrazyl) radicals, ferric-reducing antioxidant power assay (FRAP), and ABTS•+ radical scavenging activity.

Determination of DPPH radical scavenging activity
The antiradical activity of olive leaf extracts toward DPPH (1,1-diphenyl-2-picrylhydrazyl radical) was performed according to the method described by Kartal et al with some modifications to adapt the procedure using 96-well microplates (Kartal et al., 2007). Briefly, 180 μl of various concentrations of extracts (0.009-10 mg/ml) was added to 1,620 μl of DPPH, prepared daily, and kept in the dark when not used. The absorbance was measured at 517 nm. The scavenging activity was expressed as IC50, and the extract dose needed to induce a 50% inhibition. A lower IC50 value refers to a higher an-

Determination of ferric-reducing antioxidant power (FRAP)
Ferric-reducing antioxidant power of infusion extract experiment was conducted according to Li et al. (2007) Method. Samples were mixed with 2.5 ml sodium phosphate buffer (pH 6.6) and 1% potassium ferricyanide (2.5 ml). After incubation at 50°C for 20 min, 10% trichloroacetic acid (2.5 ml) was added to 2.5 ml of distilled water and 0.1 ml of ferric chloride (0.1%) and the mixture was centrifuged.
The absorbance was measured at 700 nm. Results were expressed on (EC 50 ) (Li et al., 2007).

| Allelopathic bioassay
Germination test was used to study the possible allelopathic effects of two different studied varieties of Olea europaea (Meski and Chemlali) on seeds of Triticum aestivum and Linum usitatissimum.
Healthy and uniform seeds were sterilized with 95% chloric acid and washed with distilled water more than two times. Twenty seeds were placed with suitable amount of different concentrations of the infusions (0.25, 0.5 and 1 mg/ml) in sterilized Petri dishes provided with two layers of filter paper. Three replicates were prepared from each concentration, and distilled water (0%) was used as a control.
To avoid reduction of moisture content of the blotting paper, equal volume of distilled water was added to the Petri dishes. Seeds incubated at 25ºC and were observed daily and considered germi-

| Statistical analysis
The experiment results were statistically analyzed by SPSS Statistics for Windows version 21,0, and Student's t test and Duncan's multiple range test were used.

| In vitro Toxicity of Extracts against Murine Oligodendrocytes (158N)
As far as we know, few reports are available on the cytotoxicity of plant extracts before proceeding to their biological activities. It is worth mentioning that the present study was also the first endeavor

| Total polyphenol, flavonoid, and flavonol contents
The screening of phytochemical of the tested plants showed that the presence of polyphenols, flavonoids, and flavonols in high concentration was observed in both Chemlali and octobri extracts. The **Indicate significant difference between values at p < .001 level (Student's t test). and the type of solvent used through the extraction protocol (Edziri et al., 2019;Zairi et al., 2018).  Figure 1 showed the HPLC separation of the phenolic compounds of olive leaf extract. It permitted the identification of ten compounds including hydroxytyrosol, 4HOBenz and tyrosol (substituted phenol), catechin hydrate (flavan-3-ols), lute-7-rutinoside (Flavonoids), verbascoside and oleuropein (oleuropeosides), luteolin-7-Glu; apigenin 7Glu, and quercetin (flavones). As described in Table 1 (Edziri et al., 2019). These results are also in agreement with (Brahmi, Mechri, Dhibi, & Hammami, 2012), who showed that the olive organs from the southern cultivars (Chemlali) had the highest level of oleuropein. Based on these results, we can conclude that polyphenol compounds are more abundant in methanolic than aqueous extracts.

| Identification of active biomolecules by HPLC
Furthermore, as far as we know, few studies are conducted to determine the phenolic composition of aqueous extracts of Olea europaea in Tunisia. The evaluation of these extracts is very limited, only few plants have been performed to date, and no data have been described before to determine the polyphenol composition of decoction or infusion extracts. Almost all the available studies in the literature reported mostly on methanolic extracts or essential oils (Brahmi et al., 2012;Edziri et al., 2019;Khlif et al., 2015).
The differences seen in the phenolic profiles of extracts from various origins might be related to the growing and the environmental conditions (soil composition, climate, altitude, rainfall). They can directly interfere with the content of chemical components (e.g., phenolic compounds) and as a consequence in their therapeutic effects (Edziri et al., 2019). In our work, some of these factors were removed: Olive trees were grown under the soil conditions and same climatic and leaves' collection was done within one month. These results show that each extraction method enhanced the recovery of specific phenolic groups with different performance.

| Evaluation of the antioxidant activity in vitro
Food industry and agricultural researchers use frequently DPPH, FRAP, and ABTS assays to evaluate the antioxidant activity of plants.
These free radicals give an idea about the potential of the extract compounds to delay oxidative cell damages. The chelating activity IC 50 obtained using three different methods is also shown in Table 2.
All tested extracts showed a high reducing power in a concentrationdependent manner ( Table 2). The lower the IC50 values, the higher the chelating capacity of the plant extract. Thus, the highest radical scavenging effectiveness was observed with aqueous extracts of Chemlali cultivar followed by Meski cultivar, thereby attesting its higher capacity in eliminating the formed reactive oxygen species (Table 3).
All the extracts had a promising scavenging effect attesting the presence of some compounds that are electron donors and convert free radicals into more stable products. showed that methanolic extracts of the cv. "chetoui" had greater antioxidant potential on DPPH radicals when compared to those reported for fruits and leaves of cv. "chemlali" (Brahmi et al., 2012). oleuropein, a well-known antioxidant derivative (Orak et al., 2019).

| Evaluation of allelopathic effect in vitro
The impact of allelopathy on composition and the structure of biological communities is relatively uninvestigated in olive extracts (Thiébaut, Thouvenot, & Rodríguez-Pérez, 2018). Thus, the aim of this study is to assess whether the aqueous leaf extracts (infusion) of the O. europaea had an allelopathic effect on seed germination of T aestivum and Linum usitatissimum. The results reported in Table 4 confirmed that Olea europaea leaves contain a naturally occurring allelopathic substance in dose-dependent manner.
Noticeably, germination percentage displayed a gradual decrease with the increase of all O. europaea aqueous extracts. As can be seen in Table 4, over 80% of the seeds germinated in control. However, response to olive leaves was high; thus, germination of Triticum aestivum and Linum usitatissimum seed was inhibited by our extracts.
Seeds irrigated by aqueous extract obtained from Chemlali cultivar exhibited the highest percentage of inhibition of the germination (70%) followed by Meski leaf extract (35%) compared to control.
These data are in disagreement with a recent study conducted by (Al-Samarai, Mahdi, & Al-Hilali, 2018), who showed that aqueous extracts of olive leaves from Iraq produced the lowest rate of inhibition (23%) of Cyperus rotundus L. The lack of the utilization of aqueous extracts makes it difficult to compare their allelopathic strength.
The inhibitory potential of allelopathy is complex, and different groups of chemicals can be involved such as phenolic compounds, terpenoids, flavonoids, alkaloids, amino acids, steroids, and carbohydrates (Al-Samarai et al., 2018). It can be due to the congenital effects of plant containment in soluble substances in water and to biotic and the fluctuations of abiotic parameters, such as climatic conditions (Petrussa et al., 2013), the presence of herbivores and/or pathogens (Gatti et al., 2014;Silva, Overbeck, & Soares, 2014) and stage in the life history of the plant (Lombardo, Mjelde, Källqvist, & Brettum, 2013;Santonja, Le Rouzic, & Thiébaut, 2018;Thiébaut et al., 2018). In recent data, it was reported that the inhibitory activity may be attributed to the accumulation of glucosinolate in the plant parts, especially the leaves (Al-Samarai et al., 2018). In this case, infusion samples might secrete allelopathic compounds to eliminate and destroy other plant species. In nature, four main ways are used to release allelochemicals, for instance, leaching, decomposition, volatilization, and exudation.

| CON CLUS ION
The results of the present data clearly showed that the use of aqueous extracts such as infusion could be a better and more promising source of antioxidant and allopathic agents than ethanolic or methanolic extracts. Devoid of toxicity and being rich in phenolic compounds, either phenolic acids or flavonoids, aqueous extracts could be benefic for health to overcome oxidative stress and reactive species production. Additionally, they could be a solution for inhibiting plant invasion. They could inhibit seedling growth, making them useful for eco-friendly, biocontrol program management.
However, further experiments are warranted in order to purify, explore the mechanisms of action involved, and elucidate the structure

CO N FLI C T O F I NTE R E S T S
The authors declare that there is no conflict of interest.