Chemical Profiles and Antioxidant Activities of Leaf, Pulp, and Stone of Cultivated and Wild Olive Trees (Olea Europaea L.)

ABSTRACT

In this study, the phytochemical profile including the fatty acids composition and the minerals, the polyphenols, and the proteins contents were quantified on leaves, pulp, and stone of two oleasters trees from southern Tunisia. Two olive cultivars (Zarrazi and Chemlali) were used for comparisons. The antioxidant activities were conducted using the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and the 2,2-diphenylpicrylhydrazyl (DPPH) assays. Results showed that the pulp and the stone of oleasters (wild olives) were richer in total lipid having, respectively, 16.30% and 9.42% of Dry Weight basis (DW). Qualitatively, the fatty acids profile of pulp and stone from cultivars and oleasters was identical. The oleic acid is the major fatty acid having the highest percentage in the oils from the stone of oleaster1 (69.08%) followed by linoleic acid. However, quantitatively, significant differences in fatty acids composition were noted. The oleaster stones were richer in storage proteins showing that the globulin is the major fraction (50.47%), followed by albumin (25.72%), prolamin (17.61%), and glutelin (6.19%) fractions. Concerning the minerals, results showed that the pulp is richer in potassium (862.87 mg/100 g DW) than leaves (526.50 mg/100 g DW) and stone (136.50 mg/100 g DW). In contrast, the leaves have the highest phosphorus content (42.74 mg/100 g DW). The highest polyphenols content was 711.55 mg/100 g DW in the pulp followed by the leaves and stone. The leaves, pulp, and stone extracts showed high antioxidant activities using DPPH and ABTS assays. The oleasters seem to be a potential source of edible oil, protein, and antioxidants. They could be used as supplements and/or ingredients in oil and fat industries.

KEYWORDS:

• Olea europaea
• wild olive
• protein
• minerals
• lipid
• polyphenols
• antioxidant

Introduction

The cultivated olive tree (Olea europaea L. Var. europaea) and the wild olive tree or oleaster (Olea europaea L Var. sylvestris) are two botanical varieties belonging to the Oleaceae family and dispread in Mediterranean Basin (Green, 2002) where it is indigenous and in other regions with a Mediterranean climate where it has been introduced (Costa, 1998). Historically, it has been reported that the wild olive trees are native in Tunisia (Camps-Fabrer, 1997) and are dispread in natural and agro-ecosystems (Hannachi et al., 2008). The olive oil used in food as a frying medium, and in cosmetic formulations as an emollient. It is a combination of monounsaturated and polyunsaturated fatty acids with low-saturated fatty acid levels. The olive oil is the most useful edible oil in the world due to its stability, nutriment contents, and beneficial effects (Lopez-Miranda et al., 2007). The olive oil is loaded with polyphenols as endogenous antioxidants protecting the lipid from oxidation. Researchers are continuously seeking natural antioxidants that will sufficiently protect fats and oils from oxidation (Loliger et al., 1996; Wiczkowski et al., 2016). There is a rising interest for natural antioxidants as bioactive components of foods. The antioxidant capacity and the oxidative stability were substantially improved by adding olive leaves extract to the commercially available oils, olive, sunflower, and palm oils (Salta et al., 2007). The protective effects of diets rich in fruit and vegetables against cardiovascular diseases and some cancers have been attributed partly to the antioxidant compounds contained therein, particularly to the polyphenol compounds (Castañer et al., 2011). Flavonoids are a widely distributed group of polyphenol compounds and are identified as antioxidants in various biological systems (Chu et al., 2000). Researchers have focused on antioxidant compounds derived from leaves and fruit of olive trees, numerous fruits, and vegetables, as well as aromatic plants. In traditional remedies, the olive tree leaves have been widely used as a diuretic, hypotensive, emollient, febrifuge and tonic, for urinary and bladder infections and for headaches (Cherif et al., 1996; Hutchings et al., 1996). Due to their richness in phenolic compounds, olive leaves extracts from oleaster have reduced colon cancer (Zeriouh et al., 2017). Olive stone is rich in cellulose, hemicelluloses, lignin, minerals, oil, unsaturated fatty acids, proteins, and all essential amino acids (Hannachi et al., 2013a; Martín et al., 2003; Ranalli et al., 2002; Rodríguez et al., 2008). Due to its exfoliation properties, olive stones have been used as a component to aid in skin exfoliation in cosmetic products formulations (Rodríguez et al., 2008). Despite the large amount of olive oils produced and their confirmed nutritional values, little is known about the chemical composition of different parts of olive (pulp, stone, and leaves) of wild olive trees or oleaster (Var sylvestris) in Tunisia.

Structurally, the skin, called epicarp, the pulp or flesh, called mesocarp and the stone, called the woody endocarp or stone containing the seed. These different parts constitute the olive fruit.

The objective of this study is to carry out the chemical profiling of leaves, pulp, and stone of four olive trees: two cultivars, ‘Zarrazi’ and ‘Chemlali’, and wild olive trees (oleasters) from natural ecosystems (Oleaster1 and Oleaster2). Phytochemical profile of leaves, pulp, and stone was conducted including: (i) the oil content and fatty acids composition of pulp and stone; (ii) the soluble protein content and the protein fractions of stone; (iii) the mineral content of leaves, pulp and stone and; (iv) the polyphenols content the antioxidant activities of leaves, pulp, and stone extracts.

Materials and Methods

Plant Materials and Morphological Characterization

Olives were picked at maturity time at December month from two olive cultivars called ‘Zarrazi’ and ‘Chemlali’, cultivated in situ and two oleaster trees from the natural ecosystem in Tunisian southern. Pulps were separated from their stones. Olive leaves, pulps, and stones were kept at 75°C to obtain the dry matter. Dried pulps, stones, and leaves were powdered. The obtained dry matter was conserved in glass bottles for further analyses.

Thirteen olives, leaves, and stones from each sample were used to the morphological characterization: length, diameter, and weight (Table 1).

Table 1. Morphological characteristics of olive, stone, and leaves from studied olive trees.

	Fruit (olive)			Stone			Leaf
	Length (mm)	Diameter (mm)	Weight (g)	Length (mm)	Diameter (mm)	Weight (g)	Length (mm)	Width (mm)
Zarrazi	21.48 ± 1.76^d	16.64 ± 1.40^d	2.39 ± 0.62^d	15.81 ± 1.65^d	8.60 ± 0.89^d	0.50 ± 0.13^d	68.90 ± 7.65^d	11.93 ± 1.41^c
Chemlali	13.53 ± 0.88^c	9.65 ± 0.53^c	0.80 ± 0.10^c	11.64 ± 0.92^c	5.01 ± 0.27^c	0.21 ± 0.03^c	52.93 ± 8.84^c	10.70 ± 1.76^b
Oleaster 1	10.05 ± 1.17^b	6.16 ± 1.06^b	0.49 ± 0.10^a	7.83 ± 0.97^b	4.45 ± 0.72^b	0.23 ± 0.03^b	44.57 ± 5.66^b	9.17 ± 1.32^a
Oleaster 2	7.68 ± 1.48^a	5.13 ± 0.87^a	0.34 ± 0.13^a	5.79 ± 0.73^a	3.63 ± 0.59^a	0.18 ± 0.02^a	38.00 ± 7.22^a	10.13 ± 1.66^b

*Each value is the mean ± Standard deviation; Means in the same column with different lowercase letters (a, b, c …etc.) represent significant difference variation (p<0.05) according to Duncan’s multiple range test.

Chemicals and Reagents

All solvents were of reagent grade without any further purification. Gallic acid, rutin, and Folin-Ciocalteu’s phenol reagent were purchased from Sigma Chemical Co. (USA). The analytical reagent grade methanol was obtained from Lab-Scan (Labscan Ltd, Dublin, Ireland). The water used in sampling was prepared with a Millipore Simplicity (Millipore S.A.S., Molsheim, France). All chemicals used in antioxidant activity assays were of chromatography grade and were purchased from Sigma Chemical Co. (Poole, Dorset). Spectrophotometric measurements were performed on Shimadzu UV-1600 spectrometer (Shimadzu, Kyoto, Japan).

Lipid Extraction and Fatty Acids Analysis

The oil content was determined by Soxhlet using 10 g dry weight (pulp or stone) and 200 mL of hexane at 70ºC for 6 h. The oil content was computed as a percentage per dry weight basis (Nasri and Triki, 2007).

Oil extracted in chloroform and methanol by Allen and Good (1975) protocol was used to conduct fatty acids composition using 5 g of plant materials (pulp or stone) that were previously treated to neutralize lipase activities. Then, the plant materials were crushed in a mortar with a pestle in the presence of chloroform and methanol (v/v), and then saltwater NaCl 1% (v) was added. The obtained homogenate was centrifuged at 3000×g for 15 min. The chloroform phase containing lipids was recuperated. The lipid fraction was obtained after evaporation of chloroform in a rotary evaporator at 50°C and was conserved in (1:4, v:v) toluene and ethanol at −20°C for transmethylation (Vorbeck and Marinetti, 1965).

Fats were transmethylated using boron trifluoride in methanol at 65°C for 5 min (Metcalfe et al., 1966). The obtained fatty acid methyl esters (FAMEs) were analyzed on a Hewlett Packard Model 5890 gas chromatograph (Palo Alto, CA, USA) fitted with a CPSIL-88 column (100 m × 0.25 mm i.d., film thickness 0.20 μm, Varian, Les Ulis, France). Hydrogen was used as a carrier gas (inlet pressure 210 kPa). The oven temperature was held at 60°C for 15 min, increased to 85°C at 3°C/min, then to 190°C at 20°C/min, and finally held at 190°C. The injector and the flame ionization detector were maintained at 250°C and 280°C, respectively. FAMEs were identified by comparison with standards (SupelcoTM37 FAME, Sigma–Aldrich Co., USA) and with references data of mass specter library Wiley 7.0 (HPMass Spectral Libraries, Palo Alto, USA). The data were computed using the Galaxie software (Varian, France) and reported as a percentage of the total fatty acids. All analyses were made in triplicate.

Storage Proteins Extraction from Olive Stones

Storage proteins from olive stones were extracted using the fractionation protocol of protein categories basing on their solubility differences in various solvents (Osborne, 1924): albumin (water-soluble), globulin (saline soluble), prolamin (alcohol soluble) and glutelin (residue fraction). Five hundred-milligram dry weight of stone was extracted with 10 mL distilled water. The suspension was stirred at laboratory temperature for 20 min and then centrifuged at 11200×g for 15 min. The filtrated supernatant was used as the first extract containing the albumin fraction. The remaining insoluble sample was mixed with 10 mL of aqueous NaCl 5% (w/v) solution. The extraction procedure was repeated, and the second extract was collected including the globulin fraction. The third extract containing the prolamin fraction was obtained by aqueous 70% (v/v) ethanol. The latest extract (glutelin fraction) was obtained using an aqueous 0.2% NaOH solution.

Storage Proteins Determination Using Bradford Assay

The protein content of each sample was quantified using the method described by Bradford (1976). One hundred-milligram Coomassie Brilliant Blue G-250 (Sigma-Aldrich Co) was dissolved in 50 mL ethanol (95%), then 100 mL of phosphoric acid (85%) was added. The obtained solution was diluted, filtered, and used as the color reagent for protein quantification. Two hundred microliter of protein extract from olive stone were added to 200 μL distilled water and 2 mL Coomassie Brilliant Blue previously prepared. The obtained solution was adjusted to the volume of 1000 mL and was filtered. After 5 min, the absorbance was measured at 595 nm. The standard solutions of reagent grade BSA (Equitech-Bio, Inc., Kerrville, TX) were prepared having a concentration of 0–150 mg/L. After 5 min, the absorbance was measured at 595 nm. The protein content of each olive stone extract was determined by fitting a least-squares regression curve of the standard protein concentrations versus absorbance.

Minerals Contents

One gram of powdered dry matter (leaves, pulp, and stone) was placed in a porcelain capsule and was calcined by the muffle furnace (Vulcan TM-500) at 550°C during 4 h. After cooling, ashes were attacked by 5 mL of deionized water and 1 mL of hydrochloric acid and were kept at boiling under agitation. The capsule content was filtered and the filtrate was adjusted by deionized water to a volume of 100 mL. This solution was used for all mineral analyses. The combined concentration of calcium and magnesium ions was determined by complexometric titration. The calcium, magnesium, sodium, and potassium contents were determined using the flame photometer (Sherwood 410, Sherwood Scientific Ltd, Cambridge, UK); and the total phosphorus was determined using a Spectrophotometer (Secomam 1000, French).

Total Polyphenol (TP) and Total Flavonoid (TF) Contents

The TP and TF were extracted by maceration process under agitation during 24 h at 25°C and using methanol as extraction solvent. The obtained solution was filtered and then centrifuged at 11200×g for 15 mn. The obtained methanolic extracts from different olive organs were used for TP and TF contents determination.

The TP contents in methanol extract from olive organs were estimated, in triplicate, by the Folin–Ciocalteu method (Hannachi et al., 2011; Nasri et al., 2011). In the tube, 0.5 mL of Folin–Ciocalteu (Prolabo) reagent were added to 0.5 mL methanolic extract from leaves, pulp, and stone dry matter followed by 4 mL of sodium carbonate 1 M solution. The tubes were placed for 5 min at 45°C in a water bath and then put in a cold water bath. Absorbance was measured at 765 nm. Gallic acid was used to make the calibration curve. The TP contents were expressed as mg gallic acid equivalents per 100 g dry weight (mg GAE/100 g DW).

The TF contents of olive leaves, pulp, and stone were measured spectrophotometrically, in triplicate (Elfalleh et al., 2009; Hannachi et al., 2011). This method is based on the formation of a complex flavonoid–aluminium having the maximum absorbance at 430 nm. One milliliter of methanolic extract was mixed with 1 mL of 2% AlCl₃ methanolic solution. After incubation at room temperature for 15 min, the absorbance of the reaction mixture was measured at 430 nm. The Rutin was used to make a calibration curve. The TF contents were expressed as mg rutin equivalents per 100 g dry weight (mg RE/100 g DW).

Total Carotenoid Content (TC)

Carotenoids were extracted from olives tissues including fresh leaves, pulp, and stone (Gitelson et al., 2003). Fifty milligrams of each sample were put into a tube and grounded for 3 min in 1 mL extraction buffer (80% acetone: Tris-HCl [1%, w/v]). After the complete pigments extraction, an additional of 1 mL extraction buffer was used to wash the pestle. All extraction solutions were combined and debris was removed by centrifugation. A volume of 1 mL of the supernatant was diluted to obtain a 3 mL final solution. The absorbance of the final solution was measured at 470 647 and 663 nm. The concentrations of carotenoids were calculated as described by Lichtenthaler (1987). All experiments were conducted in triplicate and the TC contents were reported as mg per kg of fresh weight (mg/Kg FW).

DPPH Radical Scavenging Activity

The radical scavenging activity on DPPH radical of methanolic extracts from leaves, pulp, and stone was determined, in triplicate (Okonogi et al., 2007). Two milliliters of 100 μM methanolic solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) were mixed with an aliquot of 100 μL of each dilution (1:1000). The solution was shaken and kept in darkness at room temperature for 30 min, and then the absorbance was measured at 517 nm to determine the concentration of remaining DPPH. Results were expressed as mM trolox equivalent antioxidant capacity (TEAC mM).

The radical scavenging activity on DPPH radical was determined by the following formula:

$\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{r}\mathrm{e}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{a}\mathrm{t}517\mathrm{n}\mathrm{m}=\left[\frac{Ab{s}_{t0}-Ab{s}_{ti}}{Ab{s}_{t0}}\right]\times 100$

With Abs _t0: control absorbance in the presence of DPPH and Abs_ti: Absorbance measured in the presence of related concentration of Trolox with DPPH.

The curve Absorbance reduction at 517 nm function of Trolox concentration (mM) was used to determine the DPPH radical scavenging activity of each sample.

ABTS Radical Scavenging Activity

The radical scavenging activity on ABTS radical of each olive extract was determined (Nasri et al., 2011). The ABTS+ radical was generated by mixing 7 mM ABTS solution with 2.45 mM K₂S₂O₈ in the dark for 24 h, at room temperature. Before usage, the ABTS⁺ solution was diluted with ethanol to get an absorbance of 0.700 ± 0.020 at 734 nm. Twenty-five microliters of extract sample or Trolox standard was added to 1 ml of the diluted ABTS⁺ solution. The reaction mixture was homogenized for 20 s and then the absorbance was recorded at 734 nm up to 5 min. Results were expressed as mM trolox equivalent antioxidant capacity (TEAC mM).

The radical scavenging activity on ABTS radical was determined by the following formula:

$\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{r}\mathrm{e}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{a}\mathrm{t}734\mathrm{n}\mathrm{m}=\left[\frac{Ab{s}_{t0}-Ab{s}_{ti}}{Ab{s}_{t0}}\right]\times 100$

With Abs _t0: control absorbance in the presence of ABTS and Abs_ti: Absorbance measured in the presence of related concentration of Trolox with ABTS.

The curve Absorbance reduction at 734 nm function of Trolox concentration (mM) was used to determine the ABTS radical scavenging activity of each sample.

Statistical and Chemometric Methods

Analysis of variance (ANOVA) and Duncan’s multiple range test were performed using Statistica software (version 8), to evaluate the significance of differences between individual chemical contents and morphological parameters of cultivated and wild olive trees and between different studied organs (pulp, stone, and leaves) at the level of p < .05.

Results and Discussion

Morphological Parameters

Statistical analysis applied on morphological parameters of leaves, olive, and stone showed significant differences reflecting the variability of studied samples (Table 1). It has been reported that the morphological parameters of olive tree were influenced by the genotype and the environmental factors (Boucheffa et al., 2019; Hannachi et al., 2007) and they varied significantly between cultivated and wild olive trees (Boucheffa et al., 2019; Hannachi et al., 2008, 2016).

Oil Content and Fatty Acids Composition of Olive Pulp and Stone

Qualitatively the fatty acid profiles of pulp and stone oils were identical (Table 2). The oleic acid (C18:1n-9) is the major fatty acid, followed by the linoleic (C18:2n-6) and the palmitic acids (C16:0) in oils extracted from both stone and pulp of cultivars and oleasters. The highest oleic acid content is 69.08% in oil from extracted Oleaster 1 pulp and 64.39% in oil extracted from Chemlali stone. Quantitatively, the fatty acids composition showed significant differences among studied oils and between oils extracted from pulp and stone (Table 2).

Table 2. Fatty acid composition (% of total fatty acid esters) of oil extracted from stone and pulp of studied olive trees.

		16:00	16:1n-9	16:1n-7	18:00	18:1n-9	18:1n-7
Stone	Zarrazi	9.65 ± 0.01^c	0.20 ± 0.00^a	0.10 ± 0.00^c	4.46 ± 0.01^a	60.73 ± 0.02^d	0.89 ± 0.02^a
	Chemlali	10.29 ± 0.11^b	0.09 ± 0.01^b	0.56 ± 0.01^b	4.36 ± 0.00^b	64.39 ± 0.01^a	0.98 ± 0.02^a
	Oleaster 1	12.33 ± 0.06^a	nd	0.50 ± 0.01^a,b	3.10 ± 0.01^d	63.65 ± 0.02^b	1.50 ± 0.00^b
	Oleaster 2	12.01 ± 0.08^a	0.09 ± 0.01^b	0.48 ± 0.04^b	3.52 ± 0.01^c	61.97 ± 0.14^c	1.45 ± 0.02^b
Pulp	Zarrazi	9.62 ± 0.02^d	0.16 ± 0.00^a	0.26 ± 0.00^d	3.21 ± 0.01^a	61.58 ± 0.06^c	0.97 ± 0.02^d
	Chemlali	19.47 ± 0.06^a	0.09 ± 0.00^d	2.55 ± 0.01^a	3.02 ± 0.00^b	46.66 ± 0.03^d	3.04 ± 0.09^a
	Oleaster 1	12.57 ± 0.03^b	0.14 ± 0.01^b	0.73 ± 0.01^c	2.00 ± 0.00^c	69.08 ± 0.06^a	1.93 ± 0.001^c
	Oleaster 2	14.7 ± 0.10^c	0.10 ± 0.00^c	1.30 ± 0.01^b	2.00 ± 0.01^c	65.00 ± 0.10^b	2.50 ± 0.10^b
Mean	Stone	11.07 ± 1.21^b	0.09 ± 0.01^b	0.41 ± 0.19^b	3.86 ± 0.61^a	62.68 ± 1.53^a	1.21 ± 0.03^b
	Pulp	14.09 ± 3.84^a	0,13 ± 0.03^a	1.21 ± 0.91^a	2.56 ± 0.60^b	60.57 ± 3.04^b	2.12 ± 0.08^a
		18:2n-6	18:3n-3	20:00	20:1n-9	22:00	C24:0
Stone	Zarrazi	20.05 ± 0.01^a	0.45 ± 0.00^a	0.74 ± 0.001^a	0.88 ± 0.01^a	0.32 ± 0.01^a	0.16 ± 0.01^b
	Chemlali	14.76 ± 0.04^d	0.34 ± 0.00^b	0.67 ± 0.001^b	0.40 ± 0.001^c	0.32 ± 0.04^a	0.16 ± 0.01^b
	Oleaster 1	15.93 ± 0.02^c	0.50 ± 0.00^a	0.58 ± 0.01^d	0.39 ± 0.01^c	0.40 ± 0.07^a	0.17 ± 0.00^a,b
	Oleaster 2	17.80 ± 0.01^b	0.46 ± 0.00^a	0.61 ± 0.001^c	0.44 ± 0.01^b	0.34 ± 0.01^a	0.18 ± 0.01^a
Pulp	Zarrazi	21.37 ± 0.04^b	0.61 ± 0.01^c	0.45 ± 0.07^d	0.59 ± 0.001^b	0.19 ± 0.01^b	0.09 ± 0.00^d
	Chemlali	21.94 ± 0.01^a	0.83 ± 0.00^b	0.56 ± 0.01^c	0.23 ± 0.001^e	0.17 ± 0.00^b	0.10 ± 0.00^d
	Oleaster 1	11.09 ± 0.03^h	0.86 ± 0.06^a,b	0.45 ± 0.001^d	0.38 ± 0.04^c	0.33 ± 0.00^a	0.13 ± 0.00^c
	Oleaster 2	12.00 ± 0.01^g	0.93 ± 0.01^a	0.40 ± 0.01^d	0.32 ± 0.01^d	0.16 ± 0.01^b	0.10 ± 0.00^d
Mean	Stone	17.13 ± 2.14^a	0.44 ± 0.06^b	0.65 ± 0.07^a	0.53 ± 0.22^a	0.34 ± 0.05^a	0.16 ± 0.01^a
	Pulp	16.60 ± 5.41^b	0.81 ± 0.13^a	0.47 ± 0.06^b	0.38 ± 0.14^b	0.21 ± 0.08^b	0.11 ± 0.02^b

Each value is the mean ± Standard deviation, Means in the same column with different lowercase letters (a, b, c …etc.) represent significant difference variation (p<0.05) according to Duncan’s multiple range test.

The cultivars were distinguished by their high oil content in both pulp and stone compared to the studied oleasters. The oil content of pulp and stone ranged from 14.00 (Zarrazi) to 16.30% DW (Chemlali) and from 7.26 (Oleaster 1) to 9.42% DW (Chemlali), respectively. The pulp showed the highest oil content (15.04% DW) compared to the stone (8.11%DW) (Table 3). It has been reported that the oil content of stone from Spain olive cultivars has an average of 5.53% DW (Heredia et al., 1987). Differences would be explained by genetic, environmental factors. The olive stones represent about 10% by weight of the olive fruit with considerable amounts of fat and protein (Rodríguez et al., 2008).

Table 3. Oil content, Saturated (SFA), monounsaturated (MUFA), polyunsaturated (PUFA), and unsaturated (UFA) fatty acids of oils extracted from the stone and the pulp of olive cultivars and oleasters.

		Oil content (%DW)	SFA	MUFA	PUFA	UFA
Stone	Zarrazi	8.23 ± 0.77^b	15.50 ± 0.01^d	62.89 ± 0.01^d	20.50 ± 0.01^a	83.39 ± 0.01^a
	Chemlali	9.42 ± 0.58^a	15.95 ± 0.13^c	66.41 ± 0.02^a	15.25 ± 0.04^d	81.66 ± 0.02^d
	Oleaster1	7.26 ± 0.26^d	16.57 ± 0.01^b	66.03 ± 0.04^b	16.43 ± 0.02^c	82.46 ± 0.06^b,c
	Oleaster2	7.57 ± 1.33^c	16.86 ± 0.08^a	64.42 ± 0.06^c	18.26 ± 0.01^b	82.67 ± 0.07^b
Pulp	Zarrazi	14.00 ± 1.00^c	13.60 ± 0.08^d	63.61 ± 0.03^c	21.97 ± 0.04^b	85.58 ± 0.01^a
	Chemlali	16.30 ± 1.30^a	23.37 ± 0.06^a	52.56 ± 0.06^d	22.77 ± 0.01^a	75.33 ± 0.07^d
	Oleaster1	14.40 ± 1.03^c	15.54 ± 0.04^c	72.25 ± 0.04^a	11.95 ± 0.03^d	84.20 ± 0.01^b
	Oleaster2	15.40 ± 1.03^b	17.43 ± 0.10^b	68.07 ± 1.69^b	12.95 ± 0.01^c	81.02 ± 1.68^c
Mean	Stone	8.11 ± 0.90^b	16.22 ± 0.57^b	64.93 ± 1.50^a	17.61 ± 2.12^a	82.54 ± 0.66^a
	Pulp	15.04 ± 0.97^a	17.48 ± 3.91^a	64.12 ± 7.87^b	17.41 ± 5.32^b	81.53 ± 4.26^b

Each value is the mean ± Standard deviation, DW: dry weight, Means in the same column for each olive organs with different lowercase letters (a, b, c …etc.) represent significant difference variation (p<0.05) according to Duncan’s multiple range test.

The MUFA (monounsaturated fatty acids) of stone oils ranged from 62.89 (Zarrazi cultivar) to 66.41% (Chemlali). The oil extracted from oleaster 1 pulp showed the highest monounsaturated fatty acids (MUFA) content (72.25%). These results showed that the oils extracted from oleaster pulp and stone oils were richer in MUFA as the two studied olive cultivars (Table 3). The oil extracted from the pulp was richer in PUFA (polyunsaturated fatty acids) having 22.77 (Chemlali) and 21.97% (Zarrazi). The PUFA have a percentage of 20.50 in oil extracted from Zarrazi stone, followed by the oleaster 2 stone (18.26%). All studied oils have high-unsaturated fatty acids (UFA) and lower saturated fatty acids (SFA) contents.

The main source of vegetable fats in the Mediterranean diet is olive oil. The composition of this oil differs from other vegetable oils that are currently consumed in many countries. Olive oil contains high amounts of oleic acid constituting one of the parameters characterizing olive oil. Oils extracted from olives having oleic acid higher than 55% are categorized as extra virgin olive oil based on International Olive Oil Council norms (IOOC, 1992) as expected for Algerian oleaster oils showing an important richness on oleic acid (Boucheffa et al., 2019). In the other hand, the beneficial health properties of olive oil have been attributed to the high oleic acid content (Chu et al., 2000). In addition, the preventive superiority of olive oil is attributed to its antioxidant components (Delgado-Adámz et al., 2014). As expected, our results showed that the oleic acid is the major fatty acids in oleaster oils extracted separately from both stone and pulp as the olive cultivars oils. Results were in accordance with Ranalli et al. (2002) showing that the oleic acid was the major fatty acids (73.01%) of oils extracted from the pulp of Italian olive varieties from single crop year. Consequently, pulp and stone oils were richer MUFA essentially due to higher contents of oleic acid constituting the major fatty acid component. Consequently, the oleaster oils from pulp and stone would be a source of essential fatty acids required for human health as expected on Algerian oleasters, the oils showed an important richness on oleic acid (Boucheffa et al., 2019).

Fatty acid composition is a major determinant of oil quality and its differences are mainly due to the genetic control, soil, and climatic conditions (Breene et al., 2007; Hannachi et al., 2007). In this study, the fatty acids profile varied according to the tissues (pulp, stone) and according to the olive types (cultivars or oleasters). Results showed that the studied olive trees (cultivars and oleasters) and olive organs (pulp and stone) factors affected the quantitative but not the qualitative fatty acid composition. These results were in accordance with previous studies conducted on the characterization of wild and cultivated olive oils (Bouarroudj et al., 2016; Boucheffa et al., 2019; Dabbou et al., 2011; Hannachi et al., 2013b). The quantitative differences would be explained by genetic and environmental factors (Bouarroudj et al., 2016; Boucheffa et al., 2019; Breene et al., 2007; Hannachi et al., 2007).

The PUFA are well documented to have protective effects against lipid peroxidation (Kratz et al., 2002). In this study, all studied oils were loaded with unsaturated fatty acids. Results showed that the PUFA and SFA of oils extracted from stone were 17.61% and 16.22%, respectively. Ranalli et al. (2002) showed that the PUFA and SFA of oil extracted from Italian olive stone had a percentage of 8.61% and 16.67%, respectively.

The high level of unsaturated fatty acids makes the studied oils desirable in terms of nutrients; because the unsaturated fatty acids amounts is a criterion used to valorize new vegetable oils (Nehdi, 2011). It has been reported that the consumption of oil extracted from Algeria oleaster decreased significantly the plasma triglyceride concentration, total cholesterol, and low-density lipoprotein-cholesterol (LDL-C) and increased the high-density lipoprotein-cholesterol (HDL-C) concentrations showing the improvement of the plasma lipid profile of healthy volunteers (Belarbi et al., 2011).

Protein Content of Olive Stone

The contents of storage proteins extracted from olive stone have values of 199.50 and 193.61 mg/g DW in two oleasters, and 144.68 and 138.98 mg/g DW in cultivars. In all studied stones, the globulin is the major fraction (50.47%), followed by albumin (25.72%), prolamin (17.61%), and glutelin (6.19%) fractions. The stone of oleasters was richer in protein content compared to the cultivars (Table 4).

Table 4. Soluble protein contents and protein fractions from olive stone (mg/g DW).

		Oleasters (wild olive trees)		Cultivars (cultivated olive trees)
Solvants	Proteinic fraction	Oleaster 1	Oleaster 2	Zarrazi	Chemlali	Mean mg/g DW	Proteins %
H20 pH = 6.5	Albumin	64.88 ± 21.21^a	53.40 ± 37.65^b	28.73 ± 0.11^c	27.11 ± 0.48^c	43.53 ± 18.63	25.72
NaCl/H20 5%	Globulin	97.09 ± 0.05^a	88.01 ± 0.05^c	67.20 ± 0.32^d	89.29 ± 0.05^b	85.40 ± 12.78	50.47
Ethanol/H20 70%	Prolamin	23.93 ± 0.11^c	30.83 ± 0.00^b	44.89 ± 0.05^a	19.54 ± 0.05^d	29.79 ± 11.08	17.61
NaOH/H20 0.2%	Glutelin	13.61 ± 0.27^b	21.38 ± 0.11^a	3.86 ± 0.05^c	3.04 ± 0.16^d	10.47 ± 8.71	6.19
Total		199.50 ± 38.48^a	193.61 ± 29.63^a	144.68 ± 26.70^b	138.98 ± 37.73^b	169.19 mg/g DW = 16.92%

Each value is the mean ± Standard deviation; DW: dry weight; Means in the same line with different lowercase letters (a, b, c …etc.) represent significant difference variation (p<0.05) according to Duncan’s multiple range test.

The stone of wild olive trees was richer in protein content as noted previously (Hannachi et al., 2013a). The seed storage proteins are important because they ensure feeding the germinating embryo that enables it to attain the autotrophy. They are also important for the human and animal nutrition providing more than half of daily protein requirement (Cheftel et al., 1985). Most of the physiologically active proteins (enzymes) are found in the albumin or globulin groups. Nutritionally, the albumins and globulins have a very good amino acid balance. They are relatively richer in lysine, tryptophan, and methionine and in all essential amino acids are present in olive stone proteins (Ranalli et al., 2002).

Globulin, prolamin, and glutelin as storage reserves, are not present systematically in the seeds of all plant species. In the present study, we noted that the globulin is the major protein fraction constituent for studied cultivars and oleaster olive stones, followed by the albumin, prolamin, and glutelin (Table 4). The differences between cultivars and oleaster stones suggested that the genotype is a factor influencing the protein fractions, which are in agreement with those for most legumes (Vasconcelos et al., 2010). Indeed, its richness in oil, the olive stones were, also, a good source of protein with globulins as the major fraction accounting 50.47% of total storage proteins. The high level of proteins confirms that the olive stone is a good nutritional complement for the animal food formulation as reported previously (Martín et al., 2003).

Mineral Contents of Leaves, Pulp, and Stone

The mineral content of different organs (leaves, pulp, and stone) was expressed as mg per 100 g dry weight basis (Table 5). The leaves of cultivar and oleaster olive trees were richer in phosphorus (42.74 mg/100 g DW) than the pulp (27.50 mg/100 g DW) and stone (22.15 mg/100 g DW). Conversely, the pulp was richer in potassium (862.87 mg/100 g DW) than leaves (526.50 mg/100 g DW) and stone (136.50 mg/100 g DW). Statistically, the differences between organs (leaves, pulp, and stone) were significant. The oleaster pulps were distinguishable by their richness in potassium (1170.00 and 1267.50 mg/100 g DW) and in sodium (25.30 and 25.31 mg/100 g DW). The cultivars leaves were richer in potassium (663.00 and 585.00 mg/100 g DW) which is in agreement with results obtained on olive leaves from Tunisian cultivars having 446.6 to 931.6 mg/100 g DW (Bahloul et al., 2009). The mineral content determination of olives is getting interested to evaluate the link between their nutritional status and the olive oil quality. It has been reported that the mineral components of the olive pulp are quantitatively more important than the olive stone (Rayon and Robards, 1998).

Table 5. Mineral contents (mg/100 g DW) from leaves, pulp, and stone of olive cultivars and oleaster.

		Potassium (K)	Sodium (Na)	Phosphorus (P)	Mg+Ca	% Ash
Stone	Zarrazi	234.00 ± 14.00^a	11.50 ± 1.50^c	24.99 ± 1.73^b	10.00 ± 1.98^f,g	1.00 ± 0.13^f
	Chemlali	195.00 ± 5.00^b	18.40 ± 1.40^a	33.71 ± 2.71^a	5.00 ± 1.00^g	1.00 ± 0.23^f
	Oleaster1	54.60 ± 1.46^d	11.50 ± 1.50^c	20.79 ± 1.79^c	15.00 ± 1.00^ef	2.00 ± 0.33^f
	Oleaster2	62.40 ± 0.84^c	13.80 ± 1.70^b	10.10 ± 0.98^d	10.00 ± 11^a	1.00 ± 0.3^f
Pulp	Zarrazi	819.00 ± 19.00^c	20.70 ± 2.36^b	41.01 ± 1.10^a	20.00 ± 2.89^e	12.00 ± 1.89^c
	Chemlali	195.00 ± 5.00^d	16.10 ± 1.10^c	21.61 ± 2.61^c	15.00 ± 1,00^e,f	19.00 ± 2.00^a
	Oleaster1	1170.00 ± 70.00^b	25.30 ± 1.30^a	10.18 ± 1.09^d	5.00 ± 1.12^g	14.00 ± 2.10^b
	Oleaster2	1267.50 ± 37.50^a	25.31 ± 2.15^a	37.31 ± 2.16^b	20.00 ± 2.15^e	14.00 ± 2.00^b
Leaf	Zarrazi	663.00 ± 63.00^a	27.60 ± 3.60^a	50.88 ± 3.88^a	75.00 ± 5.00^b	6.00 ± 1.50^e
	Chemlali	585.00 ± 85.00^b	13.80 ± 1.97^c	50.66 ± 2.04^a	65.00 ± 3.45^c	10.00 ± 2.00^d
	Oleaster1	312.00 ± 12.00^d	27.60 ± 1.60^a	30.01 ± 2.01^d	10.00 ± 1.68^f,g	11.00 ± 2.13^c,d
	Oleaster2	546.00 ± 46.00^c	18.40 ± 2.40^b	39.54 ± 3.00^b	50.00 ± 2.00^d	6.00 ± 1.56^e
Mean	Stone	136.50 ± 83.03^c	13.80 ± 3.22^b	22.15 ± 8.95^c	10.00 ± 1.31^c	1.25 ± 0.50^c
	Pulp	862.87 ± 440.19^a	21.77 ± 4.27^a	27.50 ± 13.05^b	15.00 ± 6.50^b	14.75 ± 2.83^a
	Leaf	526.50 ± 145.30^b	21.70 ± 6.80^a	42.74 ± 9.26^a	50.00 ± 15.98^a	8.250 ± 2.53^b

Each value is the mean of triplicate analysis (mean ± Standard deviation); DW: dry weight; Means in the same column for each olive organs with different lowercase letters (a, b, c …etc.) represent significant difference variation (p<0.05) according to Duncan’s multiple range test.

Antioxidants Contents of Leaves, Pulp, and Stone

Among the studied olive organs, the pulp presents the highest contents of polyphenols (711.55 mg EGA/100 g DW), followed by the leaves (638.26 mg EGA/100 g DW) and finally the stone (319.08 mg EGA/100 g DW). Significant differences were noted between oleasters and cultivars for each studied organ including pulp, stone, and leaves (Table 6).

Table 6. Antioxidants from leaves, pulp, and stone of olive cultivars and oleaster.

		Polyphenols (mg EGA/100 g DW)	Flavonoids (mg ER/100 g DW)	Carotenoids (mg/kg FW)	DPPH (TEAC mM)	ABTS (TEAC mM)
Stone	Zarrazi	212.19 ± 6.56^b	13.14 ± 2.15^b	1.45 ± 0.21^c	14.11 ± 0.15^a	6.20 ± 0.00^a
	Chemlali	761.83 ± 1.97^a	22.33 ± 0.08^a	3.20 ± 0.46^a	10.18 ± 3.28^b	6.00 ± 0.00^b
	Oleaster1	178.77 ± 1.08^c	12.05 ± 0.07^c	1.07 ± 0.15^d	6.22 ± 0.31^d	6.00 ± 0.00^b
	Oleaster2	123.52 ± 0.71^d	11.46 ± 0.70^c	2.76 ± 0.39^b	8.15 ± 0.21^c	3.10 ± 0.00^c
Pulp	Zarrazi	466.24 ± 3.94^c	19.57 ± 1.39^d	8.49 ± 1.21^a	3.53 ± 0.04^d	9.10 ± 0.11^a
	Chemlali	925.17 ± 0.66^a	30.17 ± 3.35^a	4.43 ± 0.63^b	7.68 ± 0.26^c	4.00 ± 0.00^d
	Oleaster1	730.28 ± 0.66^b	23.25 ± 1.20^b	2.56 ± 0.37^d	14.13 ± 0.18^a	8.00 ± 0.00^b
	Oleaster2	724.53 ± 3.54^b	21.54 ± 1.45^c	3.91 ± 0.56^c	11.57 ± 0.09^b,c	7.80 ± 0.16^c
Leaf	Zarrazi	750.69 ± 0.66^b	22.95 ± 0.77^b	18.06 ± 2.58^a	7.09 ± 0.12^c	4.20 ± 0.00^c
	Chemlali	579.00 ± 0.66^c	17.62 ± 2.09^d	17.45 ± 2.49^a,b	13.62 ± 0.17^a	8.00 ± 0.00^b
	Oleaster1	391.92 ± 1.41^d	14.02 ± 2.13^e	14.43 ± 2.06^c	13.11 ± 0.16^b	12.00 ± 0.00^a
	Oleaster2	831.44 ± 0.66^a	24.39 ± 2.41^a	12.82 ± 1.83^d	3.54 ± 0.06^d	12.10 ± 0.01^a
Mean	Stone	319.08 ± 297.43^c	14.75 ± 0.75^c	2.12 ± 1.02^c	9.66 ± 3.36^a	5.30 ± 1.40^c
	Pulp	711.55 ± 188.27^a	23.63 ± 1.85^a	4.85 ± 2.55^b	9.23 ± 4.90^b	7.20 ± 2.00^b
	Leaf	638.26 ± 195.07^b	19.75 ± 1.85^b	15.69 ± 2.49^a	9.34 ± 4.51^ab	9.10 ± 3.50^a

Each value is the mean of triplicate analysis (mean ± Standard deviation), Means in the same column for each olive organs with different lowercase letters (a, b, c …etc.) represent significant difference variation (p<0.05) according to Duncan’s multiple range test.

DW: Dry Weight, FM: Fresh Weight, EGA: Equivalent Gallic Acid; ER: Equivalent Rutin; TEAC: Trolox equivalent antioxidant capacity.

The pulp has the highest flavonoids content with an average of 23.63 mg ER/100 g DW, whereas, the leaves and stone have 19.75 and 14.75 mg ER/100 g DW, respectively. The carotenoids content has an average of 15.69, 4.85 and 2.12 mg/kg fresh weight (FW), in leaves, pulp, and stone, respectively. Significant differences of polyphenols, flavonoids, and carotenoids contents were observed between pulp, stone, and leaves (Table 6). The leaves of oleaster 2 have the highest content on total polyphenols (831.44 mg EGA/100 g DW) and on total flavonoids (24.398 mg ER/100 g DW).

It is known that fruit and vegetables play a key role against numerous cancer and cardiovascular disease (WCRF/AIC, 2007). This protective effect has generally been attributed to different antioxidant constituents, such as flavonoids and phenolic acids which are important antioxidants that protect the oil against oxygen radicals. Generally, the oxidative stability of the olive oil was mainly correlated with total polyphenols content (Ammar et al., 2014; Tura et al., 2007). The olive trees contained phenolic compounds that are potent antioxidants and are present in all olive tissues, including the pulp, leaves, and stone (Ranalli et al., 2002). Additionally, olive leaves have the most potent radical scavenging power of the different parts of olive trees due to its richness in polyphenols and were a useful source of high added value products (Briante et al., 2002). The medicinal properties of the oleaster tree are mostly attributed to its leaves richer on polyphenols (Arab and Bouchenak, 2013; Zeriouh et al., 2017). Results showed that olive leaves extracts have an average of 638.26 mg EGA/100 g DW. Kiritsakis et al. (2010) showed that the polyphenols content of Greek olive leaves using petroleum ether as solvent varied from 280 to 470 mg/100 g. These differences could be explained by genetic, environmental, and solvent factors.

The cultivar genotype is the most important factor influencing the antioxidant profile of the olive oil (Tura et al., 2007). The oleaster oils constitute a new edible oil source characterized richer in natural bioactive components (Boucheffa et al., 2019; Dabbou et al., 2011; Hannachi et al., 2013b).

Flavonoids are a group of polyphenol compounds with various chemical structures and characteristics. They occur in fruits, vegetables, and are an integral part of human diets. It is known that flavonoids exhibit many types of biological activities, including anti-inflammatory, antiviral, antibacterial activities, and they also act as antioxidants (Middleton et al., 2000).

The stone is richer in polyphenols and flavonoids as reported previously (Hannachi et al., 2013a; Martín et al., 2003; Rodríguez et al., 2008). It seems that they improve the organoleptic quality and oxidative stability of olive oil; although the olive seed contributes to the olive oil aroma during the virgin olive oil extraction (Luaces et al., 2003). Due to its richness by valuable components, the olive stone has been used as a metal biosorbent, a dietary animal supplementation, a renewable energy source, in cosmetic formulations and in industries (Carraro et al., 2005; Fiol et al., 2006; González et al., 2003).

Antioxidant Activities: ABTS and DPPH Scavenging Capacities

The antioxidant activities of the extracts from leaves, pulp, and stone were evaluated as mM trolox equivalent antioxidant capacity (TEAC mM) (Table 6). The free radical scavenging activity determined by DPPH varied from 6.22 (oleaster1) to 14.11 mM TEAC (Zarrazi cultivar) with an average of 9.66 TEAC mM in the stone extract. In pulp extract, the highest DPPH value is 14.13 mM TEAC (Oleaster1) and the lowest one is 3.53 TEAC mM (Zarrazi cultivar). The DPPH ranged from 3.54 (oleaster2) to 13.62 TEAC mM (Chemlali cultivar) in leaves extract. Statistically, the difference between DPPH average values of leaves, pulp, and stone was significant (Table 6).

The values determined by ABTS were higher in leaves with an average of 9.10 TEAC mM, followed by pulp (7.20 TEAC mM) and finally the stone extract (5.30 TEAC mM).

Free radical scavenging activity, determined by DPPH and ABTS was determined in several plant extracts (Delgado-Adámz et al., 2014; Elfalleh et al., 2011; Hannachi et al., 2011). Considering that multiple reaction characteristics and mechanisms involved in the estimation of the total antioxidants, no single method could accurately reflect all the antioxidants in a mixed system due to the complex nature of phytochemicals (Assadi et al., 2018; Elfalleh et al., 2011). Currently, the ABTS method was used to confirm the results obtained on the DPPH test since both methods are based on a similar antioxidant mechanism and the extracts used in both tests were methanol-soluble.

The antioxidant activity of polyphenols is mainly due to their redox properties which make them act as reducing agents, hydrogen donors, singlet oxygen quenchers and also may have a metallic chelating potential. It has been demonstrated that several components of fruits and vegetables are able to enclose high antioxidant activity, e.g., ascorbic acid, tocopherol, b-carotene, flavonoids, and polyphenols compounds (Krichene et al., 2007; Tura et al., 2007). A phenol extract with high hydroxytyrosol content obtained from olive leaves (Olea europaea L.) increased the oxidative stability of different food lipids (butter, lard, and cod liver oil) (Salta et al., 2007), showing the increasing interest in the use of natural antioxidants. It was previously anticipated that the chemical components and bioactivity of the African wild olive might differ from that of the European olive leaves due to the geographic differences (Chu et al., 2000). The antioxidant properties of the phenolic compounds are well known and continue to attract considerable scientific research effort. The results in the present study showed that all of the olive leaves, pulp, and stone extracts presented antioxidant activities reflecting their richness on natural antioxidants. Therefore, the oleasters would be valorized not only by its fatty acid profile but also by its richness on natural antioxidants; and would be used to improve the oxidative stability of food lipids. This work offers some indication of the chemical potentialities of oleasters as a new source of functional foods. As reported previously (Delgado-Adámz et al., 2014) the quality and the extending shelf life of olive oils were increased by the enrichment with olive leaves extracts. In this study, the leaves of oleasters seem to be a source of antioxidants and can be an attractive tool for industrial uses.

Conclusions

In this study, oleaster trees displayed good fatty acids profile as olive cultivars. This composition was in agreement with the International Olive Oil Council norms, as extra virgin oil. Therefore, they constitute important olive resources for nutritional oil quality. In spite of its lipid reserves, the oleaster stones as the cultivar ones were rich in soluble proteins. The globulin is the major fraction followed by the albumin, the prolamin, and the glutelin fractions. The oleaster is valued according to its oil as fatty acids composition, phenolic compounds, flavonoids, carotenoids, and mineral contents. The oleasters constitute natural antioxidants sources. All these parameters suggested qualifying the oleaster a valuable nutritional source.