Phenolic Contents and Antioxidant Activities of Date Palm (Phoenix dactylifera L.) Leaves

Abstract

The antioxidant capacity and phenolic content of three varieties of Phoenix dactylifera leaves, namely Deglet Nour, Medjhoul, and Barhee, were studied. The antioxidant activities of extracts of different leaf varieties obtained with solvents of different polarity were investigated using assays of 2, 2-diphenyl-2-picrymhydrasyl hydrate radical-scavenging activity, total phenolics and flavonoids amount, condensed tannins, reducing power, and total antioxidant capacity. The results showed that all the extracts exhibited antioxidant and radical-scavenging activities at different magnitudes and potency. The decreasing order of antioxidant and radical-scavenging activities among the extracts assayed were found to be methanol (MeOH) fraction > ethyl acetate fraction > hexane fraction > water extract. Correlation analysis indicated that there is a linear relationship between antioxidant potency, free radical-scavenging ability, and the content of phenolic and flavonoids compounds of Phoenix dactylifera leaf extracts. These results showed that Phoenix dactylifera leaf extracts are a valuable natural antioxidant, which can be applied in both healthy medicine and food industry and biotechnology.

Keywords:

• Antioxidant
• Date palm
• Flavonoid
• Phenolic
• Radical scavenging capacity

INTRODUCTION

The date palm (Phoenix dactylifera L.) is a tropical and subtropical tree that belongs to the palmaceae (Arecaceae) family and plays an essential ecological role in Arabian countries. In Tunisia, oases cover almost 40,000 ha and represent an original form of human development in very harsh climatic conditions.[1] Actually, each year after the fruit harvesting, the upkeep of the date palm trees results in the production of a huge renewable amount of palm leaves and floral stems supporting date fruits. These renewable resources are essentially used as biofuel for domestic purposes, animal feed, as well as shelter building.[2] Chemical composition of some varieties of date palm roots and fruits[3,4] and its important antioxidant activities have been extensively investigated[5,6] due to the presence of water-soluble compounds with potent free radical-scavenging effects. Based on our knowledge, no studies have been done on the determination of phenolic content and antioxidant capacity of Phoenix dactylifera leaves. However, a thorough investigation of the chemical composition of this abundant renewable resource can lead to a new exploitation for medicine, food applications, and biotechnology.

In this context, phytochemicals extracted from natural plants were reported to have antioxidant and radical-scavenging activities.[7,8] Furthermore, enzymatic oxidation as well as auto-oxidation of lipids during storage and processing is the major reaction responsible for the deterioration of food quality affecting the colour, flavour, texture, and nutritive value of the foods. Antioxidants are often added to foods to prevent the radical chain reactions of oxidation by inhibiting the initiation and propagation step leading to the termination of the reaction and a delay in the oxidation process. However, the commonly used synthetic antioxidants, such as butylated hydroxyanisole and butylated hydroxy toluene (BHT), are restricted by legislative rules because they are suspected to have some toxic effects and possible carcinogens.[9] Therefore, there has been a considerable interest by the industry and a growing trend in consumer preferences for natural antioxidants over synthetic compounds, and elimination of synthetic antioxidants in food applications has given more impetus to explore natural source of antioxidants. Thus, antioxidants are of interest to both food scientists and health professionals and there has been a convergence of interest among researchers in these fields as the role of antioxidants in the diet and their impact on human health has come under attention.

Furthermore, the effect of genotype imposes one of the greatest constraints in the process of date palm regeneration by tissue culture technique. This effect is largely dependent on the richness of tissues in phenolic compounds, which once emitted and oxidized in the culture media, become toxic, which blocks the cell proliferation process and impedes the strain establishment. The determination of tissue richness in antioxidants and phenolic compounds before any approach of multiplication allows to modulate at time the nature and the amount of organic additives (adsorbent, antioxidant) to add to the culture media so that cell proliferation can take place and the formation of new tissue can be ensured. Hence, a good knowledge of the phenolic compounds/antioxidants balance before any attempt of date palm regeneration via tissue culture technique is of interest.

The main objectives of this study were (i) to determine the polyphenolic content (total phenolics, total flavonoid, and condensed tannin), (ii) to evaluate their antioxidant capacity (ascorbic acid equivalent antioxidant capacity, reducing power and scavenging ability on 2, 2-diphenyl-2-picrymhydrazyl hydrate (DPPH) radicals), and (iii) to examine the efficiency of different solvent systems for the extraction of polyphenols from three varieties of Phoenix dactylifera palm leaves.

MATERIAL AND METHODS

Plant Material

Fresh leaves of three Phoenix dactylifera varieties (Deglet Nour, Medjhoul, and Barhee) were harvested in March from the garden of Sciences Faculty, Sfax, Tunisia. Collected leaves were washed with distilled water and allowed to air dry at room temperature for 15–20 days. After complete drying, the leaves were ground to a fine powder using a laboratory grinder and kept at 4°C until further use.

Extraction of Antioxidants from Phoenix dactylifera Leaves

The dried leaves of Phoenix dactylifera varieties (10 g) were extracted sequentially (by a maceration method) by adding solvents of increasing polarity: hexane, ethyl acetate, methanol, and water. The powder was first extracted by stirring with 100 mL of hexane at room temperature for 24 h. The extract was filtered through Whatman No. 1 filter paper in a Buchner funnel. The filtrate was concentrated to dryness with a rotary evaporator at 40°C. The remaining residues were successively extracted with ethyl acetate, methanol, and water under the same conditions. The water extract was freeze dried. All dried samples of each extract were kept in the dark at 4°C, after determining their yield prior to analysis.The dried sample of each extract was weighed and the yield of soluble constituents was calculated from the equation below:

where W ₁ was the weight of extract after evaporation of different solvents and W ₂ was the dry weight of the fresh plant sample.

Determination of Antioxidant Contents in Phoenix dactylifera Leaf Extracts

Estimation of total phenolic contents

Total phenols were determined according to the method described by Julkunen-Titto.[10] An aliquot (50 μl) of each extract or standard solution was mixed with 1 ml of dd H₂O and 0.5 ml of Folin–Ciocalteu's phenol reagent. Afterwards 2.5 ml of 20% Na₂CO₃ solution were added to the mixture followed by incubating at ambient temperature in the dark for 20 min. The absorbance against a blank was measured at 735 nm (Jenway 3305, Essex, England). Caffeic acid was used to prepare a standard curve (0.05–0.5 mg/ml; y = 2.5904x − 0.0091; r ² = 0.998; where y is the absorbance and x is the standard concentration). The results were expressed as mg caffeic acid equivalents (CAE/g extract).

Estimation of total flavonoid contents

Flavonoid contents were determined according to the method of Zhishen et al.[11] An aliquot (250 μl) of each extract or standard solution was mixed with 1.25 ml of dd H₂O and 75 μl of 5% NaNO₂ solution. After 6 min, 150 μl of 10% AlCl₃ solution was added. An aliquot of 0.5 ml of NaOH (1 M) solution was added 5 min later and then the total volume was made up to 2.5 ml with dd H₂O. Quercetin was used for standard curve construction (0.05–0.5 mg/ml; y = 0.752x + 0.0023; r ² = 0.99; where y is the absorbance and x is the standard concentration). The results were expressed as mg quercetin equivalents (QE)/g extract.

Estimation of condensed tannin contents

Condensed tannins were determined according to the method of Julkunen-Titto.[10] An aliquot (50 μl) of each extract or standard solution was mixed with 1.5 ml of 4% vanillin (prepared with MeOH) and then 750 μl of HCl (12 M) were added. The well-mixed solution was incubated at ambient temperature in the dark for 20 min. The absorbance against blank was determined at 500 nm. Catechin was used to make the standard curve (0.05–0.5 mg/ml; y = 0.5825x; r ² = 0.9277; where y is the absorbance and x is the standard concentration). The results were expressed as mg catechin equivalents (CE)/g extract.

Antioxidant Capacity Assay of the Phoenix dactylifera Leaf Extracts

It is important to select and employ a stable and rapid method to determine antioxidant activity of many samples in time of consuming. Several methods have been developed to assay free radical scavenging capacity and total antioxidant activity of plant extracts. The most common and reliable method involves the determination of the disappearance of free radicals using a spectrophotometer. In this work, three methods were used: (i) 1,1-diphenyl picrylhydrazyl (DPPH) scavenging activity for estimation the free radical scavenging properties, (ii) the reducing power, and (iii) phosphomolybdenum method used for measuring the total antioxidant capacity.

Scavenging ability toward DPPH radical (DPPH^• assay)

The DPPH^• assay was performed as described by Shirwaikar et al.[12] This method depends on the reduction of purple DPPH^• to a yellow-coloured diphenyl picrylhydrazine and the remaining DPPH^•, which showed maximum absorption at 517 nm, was measured. About 2 ml of various concentrations of each extract was added to 2 ml solution of 0.1 mM DPPH^•. An equal amount of methanol and DPPH^• served as the control. After 20 min of incubation at 37°C in the dark, the absorbance was recorded at 517 nm. The experiment was performed in triplicate. The DPPH radical scavenging activity was calculated according to the following equation:

where A _sample and A _control are absorbance of sample and control. The concentration of sample required to scavenge 50% of DPPH^• (IC₅₀) were determined. Decreasing of the DPPH^• solution absorbance indicates an increase of the DPPH radical scavenging activity. Gallic acid was utilised for comparison.

Reducing power

Reducing power was determined according to the method of Oyaizu.[13] An aliquot of each sample or standard solution prepared with MeOH (250 μl) was mixed with 250 μl of sodium phosphate buffer (0.2 M, pH 6.6) and 250 μl of 1% K₃Fe (CN)₆ incubated at 50°C for 20 min. After adding 250 μl of 10% trichloroacetic acid, the mixture was centrifuged at 3750 g for 10 min. The supernatant (100 μl) was then taken out and immediately mixed with 100 μl of MeOH and 25 μl of 0.1% ferric chloride. After incubation for 10 min, the absorbance against blank was determined at 700 nm. The EC50 value is the concentration at which the absorbance is 0.5. Ascorbic acid was used for comparison.

Determination of total antioxidant capacity by phosphomolybdenum method

The total antioxidant capacity of the extracts was evaluated by the phosphomolybdenum method as described by Prieto et al.[14] The assay is based on the reduction of Mo(VI) to Mo(V) by the extract and subsequent formation of a green phosphate/Mo(V) complex at acid pH. Each sample solution (0.3 ml) and ascorbic acid (100 μg/ml) were combined with 3 ml of reagent (0.6 M sulphuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). A typical blank solution contained 3 ml of reagent solution and the appropriate volume of the same solvent used for the sample. All tubes were capped and incubated in a boiling water bath at 95°C for 90 min. After the samples had been cooled to room temperature, the absorbance of the solution of each sample was measured at 695 nm against the blank using a UV–Vis spectrophotometer. The experiment was performed in triplicate. The antioxidant activity is expressed as the number of equivalents of ascorbic acid/g of extract.

Statistical Analysis

The data were analysed using the statistical package program Stat view 5 Soft Ware for Windows (SAS Institute, Berkley, CA, USA). Statistical analysis was performed using one-way analysis of variance (ANOVA). All values were executed in triplicate and the mean values were calculated. Correlation analysis of the antioxidant capacity versus the total phenolic and flavonoid contents were carried out using the correlation and regression coefficients, which were calculated using the correlation matrix and regression analysis by the Stat view program. Differences were considered significant if p < 0.05.

RESULTS AND DISCUSSION

Extractable Total Phenols, Flavonoids, and Anthocyanins Content

It is well known that plant phenolics, in general, are highly effective free radical scavengers and antioxidants. Consequently, the antioxidant activities of plant/herb extracts are often explained with respect to their total phenolic and flavonoid contents, with good correlation. Table 1 presents polyphenols, flavonoids, and condensed tannins contents obtained for all the leaf extracts. The content phenolic compounds in extracts, determined from regression equation of calibration curve (y = 2.5904x − 0.0091; r ² = 0.998) and expressed in caffeic acid equivalents (CAE), varied between 1.41 mg and 146.46 mg/g plant extract. These amounts were comparable with results described in the literature for other extracts of plant products.[15,16] The highest content of total phenolic compounds was found in extracts obtained with methanol for the three varieties: Deglet Nour, Medjhoul, and Barhee (69.06 ± 0.41, 79.71 ± 2.95, and 146.46 ± 2.61 mg/g plant extract, respectively), whereas the contents obtained with ethyl acetate were much smaller for the three varieties cited above (10.69 ± 0.09, 6.51 ± 1.70, and 4.03 ± 0.47 mg/g plant extract, respectively). These results are in agreement with Skerget et al.'s[17] study. Overall, the order of the phenolic contents of the test samples was methanol extract > ethyl acetate extract > water extract > hexane extract.

Table 1 Total amount of phenolic, flavonoid, and condensed tannin contents of leaf extracts from different solvent extraction systems

Varieties	Solvents	Total phenolics (mg CAE/g plant extract)^a	Total flavonoids (mg QE/mg plant extract)^a	Condensed tannins (mg CE/mg plant extract)^a
Deglet Nour	Methanol	69.06 ± 0.41^a	176.76 ± 2.04^a	59.56 ± 2.93^a
	Ethyl acetate	10.69 ± 0.09^a	300.38 ± 24.32^a	9.52 ± 2.36^a
	Water	5.39 ± 1.33^a	22.84 ± 4.54^a	<1
	Hexane	2.78 ± 0.74^a	89.44 ± 17.35^a	<1
	Total	87.92 ± 2.57	589.42 ± 48.25	69.08 ± 5.29
Medjhoul	Methanol	79.71 ± 2.95^b	321.71 ± 25.34^b	117.82 ± 5.44^b
	Ethyl acetate	6.51 ± 1.70^a,b	302.75 ± 9.28^a	1.09 ± 0.78^b
	Water	10.59 ± 0.67^b	47.55 ± 0.31^b	<1
	Hexane	1.41 ± 0.28^a	69.93 ± 5.48^a,b	<1
	Total	98.22 ± 5.6	741.94 ± 40.41	118.91 ± 6.22
Barhee	Methanol	146.46 ± 2.61^c	149.45 ± 8.55^c	326.467 ± 24.25^c
	Ethyl acetate	4.03 ± 0.47^b	312.96 ± 26.59^a	33.46 ± 3.28^c
	Water	29.21 ± 5.41^c	24.62 ± 1.81^a,c	23.67 ± 9.08
	Hexane	2.28 ± 0.58^a	91.55 ± 1.99^a,c	<1
	Total	181.98 ± 9.07	578.58 ± 38.94	383.59 ± 36.91
Values are means ± SD (n = 3). Values with the same superscript letter (a–c) are not statistically significant at p < 0.05.

The content of flavonoids (mg/g plant extract) in the different extracts, determined from regression equation of calibration curve (y = 0.752x + 0.0023; r ² = 0.99) and expressed in quercetin equivalents, varied from 22.84 to 321.71 mg/g plant extract. The results given in Table 1 showed that ethyl acetate and methanol extracts contained high levels for all varieties (Deglet Nour, Medjhoul, and Barhee) indicating high correlation between total phenolics and flavonoids contents. Our results are in agreement with other studies,[18,19] which demonstrated that both flavonoid and phenolic compounds from plant leaves are known to have diverse biological activities and may also be responsible for the pharmacological actions. Moreover, our data indicate that high flavonoids and phenolic compounds in methanol and ethyl acetate extracts may account for their strong antioxidant activities compared with hexane and water extracts.

DPPH Radical Scavenging Activity

The bleaching of DPPH absorption (517 nm) by a test compound is representative of its capacity to scavenge free radicals, generated independent of any enzymatic or transition metal-based systems. Figure 1 illustrates a significant (p < 0.05) decrease in the concentration of DPPH radical due to the scavenging ability of the extracts gallic acid, as a positive compound, which presents the highest activity at all concentrations. The IC₅₀ values (the concentration with scavenging activity of 50%) of scavenging activities on DPPH radical in methanol extract of three varieties (Deglet Nour, Medjhoul, and Barhee) were found to be 2.08, 2.11, and 2.14 mg/ml, respectively (Table 2). The DPPH radical-scavenging activity was found to be in the order of Barhee > Medjhoul > Deglet Nour. The water and hexane extracts showed the most potent activity and indicated that compounds have the strongest radical-scavenging activity in these varieties. This activity of extracts could be related to the nature of phenolics, thus, contributing to their electron transfer/hydrogen donating ability. Similar results were reported by others.[20]

Figure 1 DPPH scavenging activity of leaf extracts from different solvent extraction systems: (a) Deglet Nour variety; (b) Medjhoul variety; (c) Barhee variety. Each value is expressed as mean ±SD (n = 3).

Table 2 Yield and free radical scavenging activity of leaf extract from different solvent extraction systems

	Yield and DPPH free radical scavenging activity (IC50) ^a
	Methanol leaf extract		Ethyl acetate leaf extract		Hexane leaf extract		Water leaf extract
Varieties	Yield (%)	IC50 (mg/ml)	Yield (%)	IC50 (mg/ml)	Yield (%)	IC50 (mg/ml)	Yield (%)	IC50 (mg/ml)
Deglet Nour	26.4	2.08 ± 0.00	10.6	2.15 ± 0.02	6.6	2.53 ± 0.01	21.2	3.30 ± 0.05
Medjhoul	21.2	2.11 ± 0.11	16.6	2.32 ± 0.03	10.6	5.09 ± 0.19	20.6	3.81 ± 0.18
Barhee	30	2.14 ± 0.00	11.7	2.34 ± 0.00	12.7	3.29 ± 0.01	25	3.80 ± 0.04
Values of EC50 are expressed as mean of triplicate determinations ±SD.
^aIC50, concentration in mg/ml required scavenging the DPPH radical (100 mg/ml) by 50%.

Total Antioxidant Capacity

The phosphomolybdenum method is based on the reduction of Mo(VI) to Mo(V) by the antioxidant compound and the formation of a green phosphate/Mo(V) complex with a maximal absorption at 695 nm. Increase of the absorbance indicated the increase of the total antioxidant capacity. The antioxidant capacities of the extracts of Phoenix dactylifera were measured spectrophotometrically through this method and BHT was used as a positive compound. Potent antioxidant capacity has been found in all samples. The results showed that methanol extract exhibited the highest activity in the three varieties of Phoenix dactylifera: Deglet Nour, Medjhoul, and Barhee (244.79 ± 6.37, 285.65 ± 13.75, and 249.82 ± 34.46 mg Eq ascorbic acid/g extract). Published reports on the total antioxidant activity of these varieties of leaf extracts are not available. However, a total antioxidant activity of 245–376 mg ascorbic acid/g extract has been reported in higher plant extracts of Phillanthus species.[21] Higher activity in methanol extract may be due to the interferences of other compounds present in this extract, and it has also been reported that solvents used for extraction have a dramatic effect on the chemical species.[22]

Reducing Power

Fe (III) reduction is often used as an indicator of electron-donating activity, which is an important mechanism of phenolic antioxidant action, and can be strongly correlated with other antioxidant properties.[23] The reducing power of the extracts was measured by direct electron donation in the reduction of [Fe(CN)₆]³⁻ to [Fe(CN)₆]⁴⁻. The production was visualized by the addition of free Fe³⁺ ions after the reduction reaction, by forming the intense Prussian blue colour complex (Fe³⁺)₄[Fe²⁺(CN−)6]₃ and quantified by an absorbance measurement at 700 nm. Increased absorbance of the reaction mixture indicates increased reducing power.

Figure 2 shows the dose-response curves for the reducing power of the extracts from three varieties of Phoenix dactylifera leaves. The reducing power of the methanol extracts (Deglet Nour, Medjhoul, Barhee) increased from 0.105 ± 0.002, 0.145 ± 0.01, and 0.129 ± 0.01 at 0.25 mg/ml, respectively, to 1.23 ± 0.004, 1.63 ± 0.044, and 1.71 ± 0.040 at 4 mg/ml, respectively. The reducing power of ethyl acetate, hexane, and water extracts of the three varieties was slightly increased at a concentration of 0.25–4.00 mg/ml. For better comparison of reducing power among extracts from various leaves, the results were expressed as EC50 values, which is the effective concentration at which the absorbance was 0.5 and was obtained from interpolation from linear regression analysis. The EC50 value was very high for the methanol extract of the three varieties: Deglet Nour, Medjhoul, and Barhee (1.62 ± 0.01, 1.22 ± 0.03, and 1.16 ± 0.05 mg/ml, respectively), compared with those of ethyl acetate, hexane, and water extracts. EC50 values of the extracts in reducing power were significantly different (p < 0.05) with the EC50 values obtained for ascorbic acid (0.07 ± 0.003 mg/ml). Our results were in accordance with others who have reported that antioxidant properties are concomitant with the development of reducing power.[16,24]

Figure 2 Reducing power of leaf extract from different solvent extraction systems: (a) Deglet Nour variety; (b) Medjhoul variety; (c) Barhee variety. Each value is expressed as mean ±SD (n = 3).

Relationship between the Total Antioxidant Capacity and the Total Phenolic and Flavonoid Contents in Leaf Extracts

The correlation between the total content of phenolics and the antioxidant capacity were studied by many authors. Several studies established a linear correlation between the total phenolic content and the antioxidant capacity.[19,25] Others reported that there was no correlation.[26] The correlation coefficient between the total antioxidant capacity monitored by phosphomolybdenum method and the total phenolic and flavonoid contents of the methanol and ethyl acetate leaf extracts of the Phoenix dactylifera varieties were determined (Table 3). A linear correlation appeared between the total antioxidant capacity and the total phenolic and flavonoid contents of the methanol extract with excellent correlation coefficient of the three varieties of date palm (R ² = 0.687 for total phenolics and R ² = 0.773 for total flavonoids). Furthermore, a linear correlation appeared between the total antioxidant capacity and the total phenolic and flavonoid contents of the ethyl acetate leaf extract with good correlation coefficient (R ² = 0.713 for total phenolics and R ² = 0.712 for total flavonoids). The results are in good accordance with previous studies, which showed that high total phenolic content increases the antioxidant activity.[25,27]

Table 3 Correlations between the antioxidant activity IC₅₀/EC₅₀/TAS values and the antioxidant components (total phenolics, flavonoids, and condensed tannins) of the methanol and ethyl acetate leaf extracts of three active Phoenix dactylifera varieties

	Equations
Value	Total phenols	Total flavonoids	Condensed tannins
MeOH leaf extracts
TAS	Y = 0.211x + 25.01	Y = 0.279x − 19.55	Y = 0.021x − 0.9
	r ^2 = 0.687, F = 25.23, p < 0.001	r ^2 = 0.773, F = 17.55, p < 0.001	r ^2 = 0.521, F = 11.12, p < 0.01
IC50 of DPPH	Y = −1.442x + 40.12	Y = −1.772x + 5.77	Y = −0.889x + 9.82
radical-scavenging activity	r ^2 = 0.667, F = 13.21, p < 0.003	r ^2 = 0.659, F = 21.41, p < 0.001	r ^2 = 0.421, F = 7.22, p < 0.01
EC50 of reducing power	Y = −0.522x + 87.22	Y = −0.933x + 33.62	Y = −0.121x + 101.29
	r ^2 = 0.631, F = 22.68, p < 0.01	r ^2 = 0.751, F = 19.22, p < 0.002	r ^2 = 0.485, F = 13.02, p < 0.005
Ethyl acetate leaf extracts
TAS	Y = 0.155x + 73.44	Y = 0.722x + 67.08	Y = 0.141x + 13.98
	r ^2 = 0.713, F = 18.25, p < 0.001	r ^2 = 0.712, F = 31.55, p < 0.001	r ^2 = 0.463, F = 25.23, p < 0.001
IC50 of DPPH	Y = −0.412x + 55.02	Y = −0.823x + 72.91	Y = −0.365x + 41.9
radical-scavenging activity	r ^2 = 0.633, F = 26.38, p < 0.001	r ^2 = 0.631, F = 17.68, p < 0.001	r ^2 = 0.521, F = 11.75, p < 0.001
EC50 of reducing power	Y = −0.130x + 38.65	Y = −0.326x + 36.74	Y = −0.278x + 58.33
	r ^2 = 0.599, F = 15.38, p < 0.003	r ^2 = 0.568, F = 14.68, p < 0.004	r ^2 = 0.723, F = 22.68, p < 0.001

Importance of the Determination of Polyphenol Content and Antioxidant Capacity in In Vitro Culture

The data presented in Tables 1 and 4 show that the DN variety is significantly more rich in antioxidant amounts than the Barhee one with values of 353 and 304 (mg Eq ascorbic acid/g extract), respectively. However, the two varieties have very similar flavonoid contents but they differ in other classes of polyphenols. The Barhee variety displays a double dose of phenolic substances when compared to the DN variety, whereas it contains 5.5 times more in tannins. Although, for more antioxidants that are present in DN, there are much higher values of polyphenols in Barhee. Therefore, the balance between antioxidants and polyphenols is far in favor of the DN variety, which can explain the better behavior of this variety in in vitro culture.[28] Date palm tissue browning is the consequence of the phenolic compounds oxidation and quinines formation, which are toxic to the tissues.[29,30] This study can be considered of high interest in the prevention of explants browning via the preliminary determination of their content in polyphenol compounds and in antioxidant capacity. Consequently, more conduction of the in vitro cultures for many recalcitrant species especially for some date palm cultivars is related to the significant reduction of the emissions and of the polyphenol oxidations in the culture media. That is why the previous determination of antioxidant/polyphenols balance would be very useful. In fact, based on the obtained results and to assure a better tissue culture process by reducing explant browning, it can be evaluated to choose either the addition of appropriate adsorbent doses to the culture media (33), or the treatment of the explants with an antioxidant solution[31–33 or else the combination of both protocols when it is necessary.[34]

Table 4 Reducing power (EC₅₀) and total antioxidant capacity of leaf extract from different solvent extraction systems

	Methanol leaf extract		Ethyl acetate leaf extract		Hexane leaf extract		Water leaf extract		Total
	EC50 (mg/ml)	TAS (mg Eq ascorbic acid/g extract)	EC50 (mg/ml)	TAS (mg Eq ascorbic acid/g extract)	EC50 (mg/ml)	TAS (mg Eq ascorbic acid/g extract)	EC50 (mg/ml)	TAS (mg Eq ascorbic acid/g extract)	TAS (mg Eq ascorbic acid/g extract)
Deglet Nour	1.62 ± 0.01	244.79 ± 6.37	3.85 ± 0.23	73.15 ± 0.51	20.23 ± 4.45	12.73 ± 2.39	13.58 ± 5.60	22.74 ± 2.08	353.41 ± 11.35
Medjhoul	1.22 ± 0.03	285.65 ± 13.75	6.90 ± 1.03	42.01 ± 3.47	21.16 ± 0.15	13.88 ± 2.05	7.18 ± 0.05	22.22 ± 0.87	363.76 ± 20.14
Barhee	1.16 ± 0.05	249.82 ± 34.46	22.35 ± 0.52	22.11 ± 2.46	31.81 ± 2.14	7.84 ± 0.63	9.43 ± 0.01	24.89 ± 0.51	304.66 ± 38.06

CONCLUSION

The results obtained in this study clearly demonstrated that all the tested extracts of the three varieties of Phoenix dactylifera showed antioxidant and radical-scavenging activities at different magnitude and potency. The decreasing order of antioxidant and radical-scavenging activities among the extracts essayed through all methods is similar to the total phenolics and flavonoids contents of the extracts. Extract of Phoenix dactylifera leaves might be a valuable natural antioxidant source and seemed to be applicable in healthy medicine, the food industry, and biotechnology. However, the components responsible for the antioxidant activities of its extracts are currently unclear. Further work should be performed on the isolation and identification of the components in the extracts, especially in the MeOH extract. In addition, the in vivo safety needs to be thoroughly investigated in experimental rodent models prior to its possible application.

Notes

Hamadi Fetoui and Mohamed Makni contributed equally to this study.