Evaluation of antioxidant, antihemolytic and antibacterial potential of six Moroccan date fruit (Phoenix dactylifera L.) varieties

2.1 Preparation of rich polyphenol extracts

The rich Phenolic compound extract was prepared according to the method of Bouhlali et al. (2015c). Briefly, 30 g of pitted and crushed date fruit was extracted with 150 mL methanol–water (4:1, v/v), at 35 °C for 12 h using an orbital shaker-incubator. The mixture was then filtered and the filtrate was concentrated under reduced pressure at 40 °C until the total evaporation of solvent using a rotary evaporator. The results of methanolic crude extract were kept at −20 °C in dark glass bottles until use.

2.2 Measurement of total polyphenolic contents

The total phenolic contents of date fruit extract were measured according to the method of Bouhlali et al. (2015b). Briefly, 500 μL of Folin–Ciocalteu reagent (10-fold diluted with water) was mixed with 100 μL of date fruit solution, and then 400 μL of aqueous sodium carbonate solution (7.5% w/v) was added. The mixture was allowed to stand for 60 min at room temperature and the absorbance measured at 765 nm. The calibration curve was prepared using Gallic acid. The total phenolic compounds were expressed as Gallic acid equivalent in mg/100 g dry weight (DW) of date fruit.

2.3 Measurement of flavonoid content

The total flavonoid content of date fruit extract was determined by the method of Kim et al. (2003). One mL of date fruit solution was mixed with 4 mL of distilled water. Then 300 μL of aqueous sodium nitrite solution (5%) was added, followed by 300 μL of aqueous aluminum chloride solution (10%). Test tubes were incubated for 5 min at room temperature, and then 2 mL of sodium hydroxide (1 M) was added to the mixture and then the final volume was made upto 10 mL with distilled water. The mixture was vortexed and the absorbance was determined at 510 nm. The absorbance was calibrated to a standard curve of Rutin solution and the results were expressed as mg Rutin equivalents (RE)/100 of dry weight (DW) of date fruit.

2.4 Measurement of total condensed tannins

The total condensed tannins were determined using the method described by Heimler et al. (2006). Concisely 400 μL of the date fruit extract was mixed with 3 mL of methanolic solution of vanillin (4%) and 1.5 mL of concentrated hydrochloric acid. The absorbance was measured at 500 nm after incubation at room temperature for 15 min. Catechin was used to prepare a calibration curve, and the results were expressed as mg CE (catechin equivalents)/100 g of dry weight (DW) date fruit.

2.5 ABTS radical scavenging assay

The ABTS assay was done using the method of Re et al. (1999). The ABTS radical cations (ABTS⁺) were produced by reacting aqueous solution of ABTS (7 mM) with an aqueous solution of potassium persulphate (2.45 mM). The mixture was left at an ambient temperature in the dark for 12–16 h before use, the resulting solution was diluted with distilled water to obtain 0.700 ± 0.005 at 734 nm in the absorbance. 30 μL of the sample added to 3 mL of the ABTS solution was allowed at an ambient temperature. After 6 min the absorbance at 734 nm was recorded immediately. Trolox solution was used to prepare the standard curve and the total antioxidants were expressed as mmol of Trolox equivalents per 100 g of dry weight (DW) of date fruit.

2.6 DPPH radical scavenging activity

Radical scavenging activity of the date fruit extract against stable DPPH was evaluated as described by Blois (1958) method with slight modifications. The reaction mixture contained 100 μL of date fruit extract at a different concentration and 1.9 mL of methanolic DPPH (0.3 mM). The resultant mixtures were left at room temperature for 20 min and the absorbance was measured at 517 nm. IC₅₀ (concentration providing 50 % inhibition) values were calculated from the plotted graph of scavenging activity against the concentrations of the samples. $$\%\text{inhibition}=\frac{\left(\text{Abs}\left(\text{control}\right)-\text{Abs}\left(\text{sample}\right)\right)}{\text{Abs}\left(\text{control}\right)}\times 100$$ Abs control is the absorbance without extract; Abs sample is the absorbance of the extract or standard.

2.7 Ferric reducing antioxidant power assay

The ferric reducing activity of the date fruit extract was estimated based on the method of Benzie and Strain (1999). The FRAP reagent was prepared by mixing 50 mL of acetate buffer (0.3 M) at (pH 3.6), 5 mL tripyridyltriazine (TPTZ) solution 10 mM prepared in HCl (40 mM) and 5 mL of ferric chloride solution (FeCl₃) (20 mM). 2 mL of the freshly prepared FRAP reagent was added to 10 μL of extract. Then the absorbance was measured at 593 nm against the blank after 10 min at ambient temperature. Trolox was used to prepare the standard curve. The result was expressed as Trolox equivalent in mmol/100 g of dry weight (DW) of date fruit.

2.8 Evaluation of protective effect date fruit extract against AAPH induced erythrocyte oxidative hemolysis

The anti-hemolytic activity induced by a peroxyl radical initiator, AAPH was measured according to the method established by Bouhlali et al. (2015a). Two hundred microliters of Rabbit blood collected in heparin bulbs were mixed with 10 μL of date fruit extract, and then 600 μL of AAPH (10%) was added. The mixture was incubated at 37 °C. The absorbance of the mixture was measured at 450 nm every 5 min. The date fruit extract was replaced by Trolox and saline (0.9% NaCl) in the positive and the negative control respectively. The Protective effects of date fruit extract on free radical induced hemolysis of erythrocytes were estimated from the time required for half-hemolysis.

2.9 Antibacterial activity

2.10 Statistical analysis

Statistical analysis was performed using StatView 5.0 software. The experimental results were reported as mean ± SE (standard error) (n = 6) on a dry weight for all experiments except antibacterial activity where the results were reported as mean ± SD (standard deviation) (n = 3). The analysis of variance (ANOVA) and the post hoc Bonferroni. (p < 0.0018) tests were used to compare the experimental groups. Pearson’s correlation coefficient (r) was used to measure the association between two variables. Differences at p < 0.05 were considered significant.

3.1 Phenolic content

The total phenolic contents of date fruit varieties were estimated using Folin–Ciocalteu’s colorimetric method, which is a simple, rapid, reproducible and low-cost method. This method relies on the reduction of phosphomolybdic–phosphotungstic acid in an alkaline medium in the presence of phenolic compounds (Fu et al., 2011). Table 1 showed significant differences within the analyzed date fruit varieties (p < 0.05). The highest value of total polyphenols was determined in Bousrdon (537.07 mg GAE/100 g DW) and the lowest level was found in Bouskri (331.86 mg GAE/100 g DW). The polyphenolic content in this study was higher compared to the study of Hasnaoui et al. (2012) for the Moroccan varieties who found the total phenolic content ranged between 171.4 and 353.92 mg GAE/100 g DW. Our results confirm previous results established by Lemine et al. (2014) (405.5–661.1 mg GAE/100 g DW) for Mauritanian date cultivars and Benmeddour et al. (2013) (226–955 mg GAE/100 g DW) for Algerian date cultivars. These variations in the total phenolic contents may depend on varieties, maturity growing condition, storage conditions, fertilizer, soil type, season, geographic origin and amount of sunlight received may also be critical in this respect (Al-Farsi et al., 2007; Besbes et al., 2009).

3.2 Flavonoids content

Table 1 summarized that the flavonoid content in analyzed date fruits varied widely and ranged from 68.88 to 208.53 mg RE/100 g DW. Jihl cultivar showed the highest amount of flavonoid contents and Bouskri cultivar showed the lowest level. Our results are largely in good concordance with those previously announced by Benmeddour et al. (2013) and Lemine et al. (2014) who established that the total flavonoid content on dried matter of date fruit at the same stage of maturation “Tamr” ranged from 15.22 to 299.74 mg QE/100 g and 39.5 to 112.5 mg QE/100 g respectively. However, our observed results are very higher than those reported by Biglari et al. (2008) who found that the total flavonoid content in the Iranian date cultivars varied from 1.62 to 81.79 mg CE/100 g DW.

Flavonoids are known to be synthesized by plants in response to microbial infection; hence, it should not be astonishing that they have been found in vitro to be effective antimicrobial substances against a wide array of microorganisms (Kumar and Pandey, 2013). Several scientific investigations showed that flavonoids possess a wide range of health benefits, which include oxidative stress such as anti-allergic, anti-inflammatory, analgesic, and cardio protective, hepatoprotective, anticancer, antiviral activities. This variability of therapeutic potentials depend on flavonoid structure (Agrawal, 2011; Kumar and Pandey, 2013).

3.3 Condensed tannin content

The determination of condensed tannin content was done using the vanillin–HCl assay, which is based on the measurement of red color that results when vanillin reacts with the meta-substituted ring of flavonols. However, the fact that any substituted monomeric flavanol can react with the vanillin makes this assay nonspecific for condensed tannin determination (Schofield et al., 2001). The results of condensed tannin content which showed significant differences among analyzed date fruit varieties is illustrated in Table1. Bousrdon cultivar showed the highest level of condensed tannins (92.141 mg CE/100 g DW) while the lowest level was observed in Bouskri cultivar (57.564 mg CE/100 g DW). Our results are lower than those reported by Benmeddour et al. (2013) who found that condensed tannin content varied between 82.81 and 525.06 mg CE/100 g DW.

3.4 Antioxidant activities

Date fruit contains a wide range of phenolic compounds, which have diverse antioxidant capacities. To better examine their antioxidant capacities, different assays are required. Hence, in this study the ferric reducing ability (FRAP) and free radical scavenging activity assay DPPH were used in this respect.

Simplicity, rapidity, reproducibility as well as the excellent relationship between the molar concentration of antioxidant and the ferric reducing ability, make FRAP the better assay to assess the antioxidant potential of food polyphenol compound (Müller et al., 2010). This method is based on the ability of electron-donating antioxidants to reduce at an acidic medium, a colorless ferric complex (Fe³⁺–tripyridyltriazine) to blue-colored ferrous complex (Fe²⁺–tripyridyltriazine) which had a maximum absorbance at 593 nm (Dudonne et al., 2009).

The result showed that all cultivars exhibited a good reducing power which is varied significantly (p < 0.05) from 406.61 to 860 μmol TE/100 g DW for Jihl and Bouskri, respectively. As shown in this study (Table 4), there is a strong positive relationship between the antioxidant activity measured by FRAP assay and the total polyphenol contents (r = 0.871), flavonoids content (r = 0.948), as well as condensed tannins (r = 0.952). Therefore, flavonoids are the principal provider to the antioxidant potential of date fruit extract.

DPPH (1,1-diphenyl-2-picrylhydrazyl) assay is one of the well-known and widely used methods for estimating antioxidant activity which is easy to perform and has an acceptable accuracy (Zhou and Yu, 2004). DPPH is a stable free radical, when it accepts a hydrogen radical or an electron from an antioxidant compound it becomes a stable diamagnetic molecule and loses its purple coloration which was measured at 517 nm.

The averages of free radical scavenging activity of date fruit cultivars using DPPH assay are displayed in Table 1, which showed a significant difference between examined date fruit varieties. Among these varieties Jihl cultivar (2.046 g of date/L) exhibited the highest level of scavenging activity based on DPPH assay with a lower IC₅₀ value (IC₅₀ value is the concentration of the sample required to inhibit 50% of radical) while Bouskri cultivar possessed the lowest level of antioxidant activity with a high IC₅₀ (6.255 g of date/L). The scavenging activity of date fruit is still very low compared to the synthetic antioxidant (Trolox) which possessed an DPPH IC₅₀ (0.0101 g of Trolox /L).

The analysis of correlation (Table 4) between phenolic, flavonoid and scavenging activity indicated a strong negative correlation. Phenolic content/DPPH (r = −0.90) and DPPH/flavonoid content (r = −0.842) as well as DPPH/condensed tannins (r = −0.942). These strong negative correlations suggest that the phenolic compounds, especially flavonoid may be the main provider of the scavenging ability observed on date fruit.

3.5 Antihemolytic activity

The high membrane concentrations of polyunsaturated fatty acids (PUFA) and the hemoglobin redox reactions associated with O₂ transport, which are the potent promoters of reactive oxygen species make Erythrocytes the first targets for free radical attack (Nabavi et al., 2011), hence the need for their membrane protection. Table 2 illustrated the protective effect of date fruit extract on erythrocytes treated with a free radical generator (AAPH), which enhances membrane lipid peroxidation a key factor for cell lysis. Among analyzed date fruit varieties, Jihl exhibited considerable antihemolytic activity with a half time of hemolysis (210.99 min) more than the control (Trolox) with a half time of hemolysis (175.84 min), while Bouskri exhibited the lowest activity with a half time of hemolysis (158.70 min). The high positive correlation as shown in the Table 4 between flavonoid content and half time of hemolysis (r = 0.887) on the one hand, and phenolic content and the increase in half time of hemolysis (r = 0.690) on the other hand, justified the involvement of flavonoids and other polyphenols on the erythrocyte membrane stability as mentioned by various authors (De Freitas et al., 2008; Chaudhuri et al., 2007). Scavenging of lipid peroxyl radicals generated by AAPH seems to play a considerable part in antihemolytic activities as shown by a high correlation between antioxidant assays and antihemolytic activity (Table 4). The non-significant hemolysis observed when erythrocytes were treated only with date fruit extract as shown in the Table 3 can be justified as these extracts are nontoxic and harmless for the cells. However it possessed a membrane stabilizing effect that varied between 33.84% for Jihl and 0.44% for Bouskri compared to the control.

3.6 Antibacterial activity

The antibacterial screening of six Moroccan date fruit cultivars (Bousrdon, Jihl, Boufgous, Bousthammi, Majhoul and Bouskri) against S. aureus, B. cereus, B. subtilis, P. aeruginosa, E. coli, S. abony is shown in the Table 5. The antimicrobial activity of the samples was determined using the minimal inhibitory concentration (MIC) values for the tested bacteria.

All the tested date fruit extracts showed antibacterial activity except Majhoul and Bouskri extracts but this antibacterial effect is still lower compared to gentamicin (10 μg/disc) used as a reference antibiotic. This effect is varied between varieties and depends also on the tested bacterial strains.

Bousrdoun extract showed a higher antibacterial activity among analyzed varieties. The highest antibacterial activity of Bousrdoun was recorded against S. abony and against B. cereus with a MIC value of 2.5 mg mL⁻¹, the same antibacterial activity against B. cereus was shown using Jihl extract. The Majhoul and Bouskri varieties showed no activity against all bacterial strains tested (see Table 6).

The difference in the sensitivity of the studied bacterial strain against date fruit extract may be due to the change in their inherent structural or functional characteristics, which allows them to tolerate date fruit compounds. The nature and the combination of polyphenolic compounds present in the date fruit extract may also be critical in this respect. Several studies attributed the inhibitory effect of plant extracts against pathogenic bacteria to their polyphenolic compounds, precisely the ability of phenolic compounds to bind with the bacterial cell wall and then inhibit the bacterial growth (Sellam et al., 2013; Barbary et al., 2010; Baydar et al., 2004). These phenolics compounds may act through their ability to precipitate protein and inhibit enzymes of microorganisms (Naz et al., 2007). Results showed a good association between the concentrations of polyphenols compounds and the antimicrobial activity.