Abstract

The abusive use of antibiotics causes the destruction of intestinal flora and the proliferation of antibiotic-resistant pathogens. Date palm is used in traditional medicine in the Saharan regions due to its biological properties.
The study aimed to identify the phytochemical composition and assess the antibacterial activity of the methanolic extracts of three date cultivars from Algeria. Their total phenolic, flavonoid, and flavonol contents were measured spectrophotometrically. The phytochemical screening was conducted by HPLC fingerprinting using twenty-three pure phenolic compounds as standards. The antibacterial activity against pathogenic bacterial species was assessed using the disk diffusion method.
The colorimetric methods showed that the total phenolic, flavonoid, and flavonol contents ranged from 2.13 ± 0.09 to 2.67 ± 0.02 mg GAE/100 g DW, 1.33 ± 0.21 to 1.55 ± 0.13 mg CEQ/100 g DW, and 0.41 ± 0.23 to 0.47 ± 0.05 mg REQ/100 g DW, respectively. HPLC fingerprinting showed that the extracts of date cultivars served as an excellent source of bioactive compounds (gallic acid, tannic acid, ferulic acid, vanillin, caffeine, quercetin, luteolin, rutin, aspegenin, isorhamnetin, and hesperidin). They also exhibited an antibacterial potential with an inhibition zone diameter ranging from 8.40 to 12.50 mm.
The results clearly demonstrate the antibacterial potency of date palm fruits, which could be attributed to their considerable content of phenolic compounds such as gallic acid, rutin, quercetin, and luteolin.

Keywords

Phoenix dactylifera L., high-performance liquid chromatography fingerprinting, phenolic compounds, flavonoids, flavonol, secondary metabolites

INTRODUCTION

The date palm (Phoenix dactylifera L.) is an important fruit crop for the populations of the Middle East and North Africa. This fruit has great nutritional and economic importance [1, 2].

Since antiquity, the date and its pits have been used in traditional medicine in the Saharan regions, more precisely in the oases, where the date palm was cultivated [3]. Dates have high energy values and are rich in reductive and easily assimilated sugars, minerals (selenium, potassium, calcium, magnesium, manganese, and iron) and vitamins (A, B, and C). In addition, this fruit is endowed with numerous health benefits resulting from the mixture of secondary metabolites (polyphenols, anthocyanins, carotenoids, tannins, procyanidins, sterols, flavonols, flavones, anthocyanidins, isoflavones, phytoestrogens, phenolic acids, cinnamic acid derivatives, and volatile compounds) [4]. The date palm has been the subject of phytochemical, pharmacological, and nutritional research [5–8]. Various studies have determined the physicochemical composition of dates but scarce field research has only focused on phenolic components and involved only a few varieties of this fruit. These bioactive compounds are gaining increasing interest, given their important biological properties [9, 10].

This study was part of a program to valorize the Algerian flora through the search for new compounds or active ingredients. We aimed to determine phenolic compounds and to assess the antibacterial effect of methanolic extracts from three cultivars (Hamraya, Figheth, and Tamajort) of the date palm (P. dactylifera) fruits growing in the El-Oued region (Algeria).

STUDY OBJECTS AND METHODS

Plant material collection. Three date palm fruit cultivars (Phoenix dactylifera L.) were collected from the El-Oued region (South-East of Algeria) at the final stage of fruit ripeness, at the beginning of the 2012 harvest season. They are locally named Hamraya (33’34’30,09N, 6’49’43,11E), Figheth (33’35’15,56N, 6’49’36,83E), and Tamajort (33’33’39,99N, 6’47’27,73E).

Preparation of extracts. The preparation of methanolic extracts was performed according to the research by Biglari et al. [11]. In particular, 100 mg of the edible parts of each cultivar were macerated in 300 mL of methanol-water (4:1, v/v) at room temperature under continuous shaking for 5 h. The mixture was filtered and concentrated to obtain crude extracts, which were kept at 4°C until used.

Total phenolic content assay. The total phenolic content was determined by the spectrophotometric method described by Al-Farsi et al. [12]. For this, 200 μL of each extract was added to 1.5 mL of the Folin-Ciocalteu reagent. The solutions were mixed and incubated in the dark for 5 min. Then, 1.5 mL of sodium bicarbonate (60 g/L) was added to the reaction medium. After 90 min of incubation at room temperature, the absorbance of all extracts was measured with a UVvisible spectrophotometer at 725 nm against the blank without extract. The phenolic content was expressed as milligrams of gallic acid equivalent per 100 g dry weight (mg GAE/100 g DW) based on a calibration line constructed from the standard solution of gallic acid.

Total flavonoids assay. Total flavonoids were determined by the method described by Biglari et al. [11]. For this, 4 mL of distilled water and 1 mL of each extract were added to 0.3 mL of 5% sodium nitrite (NaNO₂) and 0.3 mL of 2% aluminum chloride (AlCl₃) in methanol. After incubation for 5 min at room temperature, 2 mL of 1% sodium hydroxide (NaOH) in methanol was added. The mixture was diluted to 10 mL with distilled water. The absorbance of the resulting mixture was measured directly with a UV-visible spectrophotometer at 510 nm against the blank. The flavonoid contents were expressed as milligrams of catechin equivalent per 100 g dry weight (mg CEQ/100 g DW) based on a calibration line constructed from the standard solution of catechin.

Total flavonols assay. To determine total flavonols, 500 μL of each methanolic extract was added to 500 μL of 2% aluminum chloride (AlCl₃) and 500 μL of 5% sodium acetate (CH₃COONa). The absorbance was determined at 440 nm after incubation for 2.5 h at room temperature. The flavonol contents were expressed as milligrams of rutin equivalent per 100 g dry weight (mg REQ/100 g DW) based on a calibration line constructed from the standard solution of rutin [13]. 

HPLC analysis of phenolic compounds. To determine phenolic compounds, 10 mg of powdered extracts of P. dactylifera were dissolved in 10 mL of methanol to a final concentration of 1 mg/mL. The solution was filtered through а 0.45-μm syringe filter for sterilization. Then, 1 mg of each standard was dissolved individually in 1 mL of methanol and sterile-filtered through a 0.45-μm syringe filter before subjecting to high-performance liquid chromatography (HPLC). The analysis of phenolic compounds in various extracts was carried out using HPLC coupled with a visible UV multi-wavelength detector under the following operating conditions:
– steel column: 25×0.46 cm; 
– stationary phase: C18; 
– elution solvent: methanol:acetonitrile (30:70 v/v); 
– wavelengths: 220, 280, 300, and 365 nm; and 
– injection loop capacity: 20 μL.

To identify the compounds, 23 pure phenolic compounds were used as standards, namely rutin, naringin, quercetin, luteolin, isorhamnetin, 2,5-dimethyl hydroxycinnamic acid, 3,4,5-trimethoxybenzoic acid, 3,4,5- trimethoxycinnamic acid, ferulic acid, gallic acid, m-anisic acid, o-anisic acid, syringic acid, transcinnamic acid, 3,4-dimethoxycinnamic acid, 2,5-dihydroxycinnamic acid, apigenin, caffeine, vanillin, tannic acid, naringenin-7-O-glucoside, hesperidine, and caffeic acid. The peaks were identified by comparing the retention time of the standard compounds with that of different peaks obtained in the HPLC analysis of the extracts [13].

Antibacterial activity. The bacterial strains used to evaluate the antibacterial potentials of the date extracts included three Gram-positive (Staphylococcus aureus ATCC25223, Bacillus spizizenii ATCC6633, and Listeria monocytogenes ATCC15313) and three Gram-negative (Pseudomonas aeruginosa ATCC27853, Escherichia coli ATCC8739, and Salmonella typhimurium ATCC14028) strains. The bacterial strains were obtained from the Pasteur Institute of Algiers (Algeria). All the bacteria were grown on nutrient agar at 4°C.

The antibacterial potentials of the methanolic date extracts were determined using the agar disc diffusion method [14]. The bacterial strains were cultured in a nutrient broth for 24 h and diluted with sterilized peptone water. Suspensions of the tested microorganisms (0.5 McFarland units) were spread onto the media plates. Sterile filter paper disks of 6 mm in diameter were impregnated with 20 μL of each extract. Methanol was used as a negative control, while pure standard antibiotics (streptomycin and cefazolin) at a concentration of 1 mg/mL were used as a positive control. The plates were incubated at 37°C for 24 h. The zones of inhibition appearing around the disks were measured and recorded in mm. All the tests were performed in triplicate.

Statistical analysis. The results were given as mean ± standard deviation (SD). The analysis of variance (ANOVA) was used to look at the differences in mean values. Turkey’s test was used to determine statistically significant differences (p < 0.05).

RESULTS AND DISCUSSION

Bioactive content. The results shown in Table 1 correspond to the yield and bioactive contents (phenolic, flavonoid, and flavonol amounts) of the methanolic extracts of three date cultivars (Phoenix dactylifera L.) from Algeria. As we can see, the Figheth and Tamajort cultivars had the highest yields. There was no difference in the total phenolic content (p > 0.05) between the extracts of Tamajort, Figheth, and Hamraya. The Hamraya extract had the maximum flavonoid amount, compared to the Figheth and Tamajort cultivars (Table 1).

Finally, we found no significant differences (p > 0.05) between the flavonol contents of the Hamraya, Figheth, and Tamajort varieties.

The yield varies according to several parameters: the plant material studied (particle size), the physicochemical characteristics of the solvents used, and the solvents’ polarity. It also depends on storage conditions, duration, harvest period, the method, and extraction conditions [13].

To the best of our knowledge, there are no publications on the phenolic and flavonoid compounds of the date cultivars under study analyzed by using HPLC coupled with a visible UV multi-wavelength detector (220, 280, 300, and 365 nm). However, our findings were in accordance with those of Biglari et al. who showed that the total polyphenol contents of dates from Iran ranged from 2.89 to 6.64 mg GAE/100 g DW [11]. In addition, the study by Alam et al. on the extracts of 26 date varieties from the United Arab Emirates and Pakistan found total phenols ranging from 46 to 397 mg GAE/100 g fresh weight (FW) [15].

Kadum et al. tested the ethanolic extracts of five date varieties (Ajwa, Anbara, Piyarom, Rabbi, and Deglet Nour) from Malaysia [16]. They found that the flavonoid amounts varied from 38.63 to 57.07 mg RE/100g DW.

The results in this study were consistent with our previous study, where flavonoids and flavonols of the dates from Algeria varied from 1.06 ± 0.12 to 4.23 ± 0.29 mg CEQ/100 g DW and from 0.44 ± 0.10 to 1.43 ± 0.15 mg REQ/100 g DW, respectively [17].

In the study by Benmeddour et al., the flavonol amounts of date cultivars from Biskra region (Algeria) ranged between 6.73 and 36.64 mg REQ/100 g DW [13].

Several factors can affect the bioactive content of plants. Indeed, studies have shown that these are extrinsic factors (such as geographical and climatic factors), genetic factors, and the degree of the plant’s maturation [12, 18].

HPLC analysis of phenolic compounds. The phenolic compounds of the date methanolic extracts were detected by HPLC at 220, 280, 300, and 365 nm. The retention times of the standard phenolic compounds were compared with the peaks of the chromatograms of the extracts. At 220 nm, 3,4,5-trimethoxybenzoic acid and m-anisic acid were detected only in the methanolic extracts of Figheth and Hamraya. At 280 nm, gallic acid, tannic acid, caffeine, naringenin-7-o-glucoside, transcinnamic acid, 2,5-dimethyl hydroxycinnamic acid, and hesperidin were detected in the methanolic extracts of Hamraya, Figheth, and Tamajort (Table 2).

The chromatographic analyses at 300 nm identified caffeic acid, vanillin, ferulic acid, 3-hydroxy-4-methoxycinnamic acid, 3,4,5-trimethoxycinnamic acid, and 3,4-dimethoxycinnamic acid in all the date extracts. The qualitative analysis at 365 nm revealed the presence of rutin, o-anisic acid, luteolin, quercetin, aspegenin, and isorhamnetin in all the extracts studied (Table 3).

Numerous studies have identified polyphenolic compounds in P. dactylifera extracts by HPLC analysis. Indeed, the study by Souli et al. identified and quantified trans-ferulic and syringic acids as major phenolic compounds in most Tunisian date cultivars [19].

The chromatographic analyses of the extracts of Al-Qasim cultivated in Saudi Arabia revealed the presence of 3,30-di-O-methyl ellagic acid, 7-methoxyquercetin- O-hexose isomers, caffeic acid, ferulic acid, isomers of quercentin-rutinoside, kaempferol methylether, p-hydroxybenzoic acid, phytol, punicalagin, and quercetin-3-O-glucoside (isoquercitrin) [20].

Dhaouadi et al. quantified phenolic compounds in the aqueous extract of the Deglet-Nour cultivar grown in Tunisia [21]. In their study, the amounts of coumaric, gallic, vanillic, cinnamic, 3,4-dicaffeoylquinic, 5-Ocaffeoyl shikimic, caffeoyl-quinic, and caffeic acids were 23.03, 6.79, 2.55, 35.79, 13.86, 17.70, 285, and 9.81 mg/100 g, respectively. Similarly, Kchaou et al. established a phenolic profile of the hydroacetonic extracts of three Tunisian date cultivars, including gallic, vanillic, caffeic, syringic, coumaric, ferulic, and sinapic acids, as well as rutin [22]. El Sohaimy et al. tested four Omani date varieties by HPLC. The separation showed the presence of gallic, coumaric, caffeic, vanillic, and syringic acids with respective contents of 19.14, 1.67, 1.75, 0.27, and 0.37 mg/100 g [23].

In a study by Shahdadi et al., aqueous and ethanolic extracts from Egyptian date cultivars contained 7.51 and 5.28 μg/g of gallic acid, 2.85 and 1.79 μg/g of tannic acid, and 0.15 and 0.22 μg/g of ferulic acid, respectively [24]. In contrast, the study reported the absence of cinnamic acid. The differences between our results and those reported in literature can be attributed to the geographic origin of the fruits. The phenolic compounds in P. dactylifera extracts may also differ because of the extraction solvent [22, 25, 26].

Antibacterial activity. The methanolic extracts of date fruits were screened for their antibacterial activities against six pathogenic bacterial species (Table 4). Theantibacterial activity was recorded when the inhibition zone was greater than 6 mm. The results of antibacterial screening revealed significant antibacterial activity. The inhibition zone diameters ranged from 08.40 ± 0.00 to 12.50 ± 1.00 mm. Streptomycin and cefazolin, which were used as positive experimental controls against all bacterial strains assayed, produced an inhibition zone diameter ranging from 19.16 ± 0.76 to 34.20 ± 0.00 mm, while no inhibitory effect could be observed for methanol used as a negative control.

Among the three extracts, Figheth was the most effective against Bacillus spizizenii ATCC6633, with the largest zone of inhibition (12.50 ± 1.00 mm), while Hamraya showed the smallest inhibition zone diameter (08.40 ± 0.00 mm) against Staphylococcus aureus ATCC638P. Table 4 shows that the methanolic extract of Tamajort had no effect on Listeria monocytogenes ATCC15313, Pseudomonas aeruginosa ATCC27853, and Escherichia coli ATCC8739.

Among natural substances widespread in medicinal plants, flavonoids and organic acids belong to the promising groups of bioactive compounds with strong antibacterial potency [27].

Alshwyeh tested the methanolic extracts of Saudi Arabian native date palm (Ajwa, Khalas Alkharj, and Al-Qasim) cultivars against Streptococcus pnuemoniae, Bacillus subtilis, Klebsiella pneumonia, S. aureus, and E. coli [20]. They found that the extracts displayed a broad spectrum of inhibitory effects against these pathogenic bacteria.

The inhibitory activity of plant extracts against the growth of microorganisms was attributed to the presence of phenolic compounds [28]. These antibacterial compounds act essentially by enzyme inhibition of DNA gyrase and disturb the function of bacterial cell membranes, retarding the growth and multiplication of bacteria.

Different antibacterial mechanisms of plant flavonoids were reported by Faegheh et al. and Górniak et al. [29, 30]. In particular, flavonoids inhibit nucleic acid synthesis, cytoplasmic membrane function, energy metabolism, biofilm attachment, and porin on the cell membrane, as well as alter membrane permeability and attenuate pathogenicity.

Abdullah et al. tested the antibacterial potential of hot aqueous and methanolic extracts of Ajwa date fruit against Gram-negative bacteria (Salmonella typhi, E. coli, Vibrio cholera, and Shigella flexneri) [31]. They found that the methanolic extract showed a higher antibacterial activity than the aqueous extract, suggesting that different extraction methods yield different phytochemicals producing the bactericidal effect.

Flavonoids cause increased permeability of the internal bacterial membrane and disruption of membrane potential. According to Usman Amin et al., the ring of flavonoids may play a role in intercalation or hydrogen binding with nucleic acid base stacks, which may explain their inhibitory action on the synthesis of DNA and mRNA [32].

Flavonoids are known for their powerful antioxidant power. Indeed, they could potentially have an effect on iron chelation, which prevents the intracellular penetration of the co-factor Ca²⁺ into the bacterial cell [33].

CONCLUSION

Our study was designed to determine the phytochemical composition and the antibacterial potential of the methanolic extracts of three cultivars of date palm fruits. HPLC revealed the presence of gallic acid, tannic acid, caffeine, naringenin-7-o-glucoside, trans-cinnamic acid, 2,5-dimethyl hydroxycinnamic acid, hesperidin, caffeic acid, vanillin, ferulic acid, 3-hydroxy-4- methoxycinnamic acid, 3,4,5-trimethoxycinnamic acid, 3,4-dimethoxycinnamic acid, rutin, o-anisic acid, luteolin, quercetin, aspegenin, and isorhamnetin in all the date cultivars. The results showed that the extracts serve as an excellent source of bioactive compounds (polyphenols, flavonoids, and flavonols) and exhibit antibacterial potency with an inhibition zone diameter ranging from 8.40 ± 0.00 to 12.50 ± 1.00 mm. The results clearly demonstrate the antibacterial activity of date palm fruits, which could be attributed to their considerable content of natural compounds. Thus, they can replace the antibiotics which are restricted because of several side effects.

Contribution

S. Ali Haimoud and R. Allem conceived and designed the analysis; contributed data and analysis tools; and performed the analysis. S. Ali Haimoud collected the data and wrote the paper.

CONFLICTS OF INTEREST

The authors declare that there is no conflict of interests related to the publication of this article.

ACKNOWLEDGEMENTS

The authors thank Dr. Mehdi Selmane (Department of Biology, University of Badji Mokhtar, Annaba, Algeria) for his help during the collection of plant material.

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