Abstract The aim of the current study is to evaluate the antioxidant, antibacterial, and phagocytic activities of the hydroethanolic extract of Phoenix dactylifera fruit variety Mech degla. The antioxidant activity was determined through DPPH assay, ferrous ions chelating assay, and CUPRAC assay. The antibacterial activity against four bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae), was evaluated using the disc diffusion method, while the in vivo phagocytic activity was measured using carbon clearance method. The results showed that the extract exhibited good antioxidant activity with the IC₅₀ values of 0.89 ± 0.3 g/l and 0.56 ± 0.24 g/l for DPPH and ferrous ions chelating activities, respectively, and the A_0.5 value of 1.45 ± 0.15 g/l in the CUPRAC assay. Moreover, the extract showed notable antibacterial activity against all tested strains with the highest zone of inhibition (23.3 ± 0.3 mm) against S. pneumonia. Besides the extract did not show any significant toxicity, it was able to stimulate the phagocytic activity with a significantly faster carbon clearance rate at the dose of up to 100 mg/kg compared to the control group. Our results suggested that the Phoenix dactylifera fruit represents a promising source for the development of safe and effective immunomodulators as remedies to prevent and treat various microbial and immune-related diseases.

Keywords: Antibacterial activity, Antioxidant activity, Immunomodulators, Phagocytic activity, Phoenix dactylifera.

Citation: Sakhri, A., Sakhri, F. Z., and Bensouici, C. 2024. Antioxidant, antibacterial and immunomodulatory properties of algerian Phoenix dactylifera. Natural and Life Sciences Communications. 23(4): e2024056.

INTRODUCTION

Fruits are universally known as a source of health benefits due to their richness of active constituents such as antioxidant compounds which can reduce the risks of free-radical mediated diseases such as cancer, coronary heart diseases, and neurodegenerative diseases including Parkinson’s and Alzheimer’s diseases (Kim et al., 2015; Al-Alawi et al., 2017). Bioactive compounds presented in fruits have received great attention through their ability to modify the activity of the immune system, which can provide new and potent immunomodulators for the treatment of several diseases including infectious diseases (Alhazmi et al., 2021).

Phoenix dactylifera (PD, Arecaceae family), commonly known as date palm is one of the most popular and traditionally cultivated fruit trees distributed mainly in arid regions of the Middle East and North Africa (Gantait et al., 2018; Al-Shwyeh, 2019; Echegaray  et al., 2020). The date fruit is considered as an ideal source for human nutrition due to its composition of essential nutrients, including carbohydrates, minerals, proteins, and fibers. In addition, its richness in bioactive compounds such as anthocyanins, phenolics, carotenoids, procyanidins, vitamins and flavonoids may elucidate its common use in folk medicine for the treatment of many diseases (Al-Alawi et al., 2017; Alshwyeh, 2020; Ourradi et al., 2021).

Epidemiological evidence, coupled with clinical studies strongly support the assertion that dietary consumption of date fruit can favorably modulate serum glucose and lipid level, as well as markers associated with inflammation, diabetes, and cardiovascular diseases (Rock et al.,2009; Ali Haimoud et al.,2016; Alalwan et al., 2020). Additionally, it has been shown to reduce the need for labor intervention (Nasiri et al., 2019). In the same context, in vitro and in vivo studies have revealed a wide range of biological activities of date fruit including gastroprotective (Al-Qarawi et al., 2005), nephroprotective (El Arem et al., 2014), , antiparasitic (Albakhit  et al., 2016), antimicrobial (Qadoos et al., 2017), cardioprotective  (Alhaider et al., 2017), anti-inflammatory (El Hilaly et al., 2018), antiviral (Allahyari et al., 2021), hepatoprotective (Fatani et al., 2022) and anticancer effects (Abdelbaky et al., 2023).

As for its health benefits, date fruit has an important socio-economic role in many countries. In terms of world production, Algeria is the fourth-largest producer of dates after Egypt, Saudi Arabia and Iran with 1.152 million tons in 2020 (FAO, 2020). In addition, the exploitation of dates in diverse areas of food and health processed products such as jams, jellies, and syrups is rapidly increasing (Echegaray et al., 2020; Farag et al., 2021).

In Algeria, most Algerian palm oases are located in the regions south of the Saharan Atlas Mountains. Several studies have focused on identifying the main properties of different Algerian date fruit varieties from the provinces of Ghardaia (Mansouri et al., 2005; Tassoult et al., 2021), Ouargla (Zineb et al., 2012), Biskra (Benmeddour et al., 2013), and Adrar (Abdul-Hamid et al., 2018). Whereas, none of the existing data reported about the date varieties of M’doukal region (gate of the desert). Moreover, while most studies were focused on evaluating the antioxidant activities of different date fruit varieties (Al-Jasass et al., 2015; Assirey, 2021; Bano et al., 2022), the exploration of their immunomodulatory properties has not been extensively conducted. The present study was performed to evaluate the antioxidant, antimicrobial and immunomodulatory activities of the date palm fruit Mech Degla variety from the M'Doukal region.

MATERIAL AND METHODS

Materials

Plant material

Fresh samples of date fruit, Mech Degla variety, were collected at their mature stage (Tamr stage) from different fully grown date palms in a private farm located in M’doukal, Batna Province, North-Eastern Algeria with coordinates of 35°7′18″N 5°10′56″E. Date fruits were packaged, transported to the laboratory, and stored at 4°C. Identification of the samples was determined based on the information provided by experienced farmers and confirmed at the department of Biology, University of Batna 2.

Microorganisms test

Four clinically isolated bacteria, including two Gram negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and two Gram positive bacteria (Staphylococcus aureus and Streptococcus pneumoniae), were obtained from the laboratory of Clinical Microbiology, University Hospital Centre Benflis Touhami Batna- Algeria. 

Experimental animals

Adult albino male mice, 2–2.5 months old, with a weight range between 25 and 33 g were provided from the animal stock facility of Pasteur Institute in Algiers-Algeria. The animal studies were approved by the ethics committee of institutional animals and conducted according to Executive Decree n°10–90 completing the Executive Decree n°04–82 of the Algerian Government, and the ethical principles and guidelines for the purpose of control and supervision of experiments on animals (OECD, 2002).

Methods

Sample preparation and extraction

PD fruits were rinsed in tap water, pitted, air-dried under shadow then ground to a fine powder using a grinder. A quantity of 50 g was extracted with 70%v/v ethanol for 24 h at room temperature (25°C) using a mechanical shaker. The macerate was filtered using Whatman No.1 to discard the precipitate. After completing three repeated extraction processes, the filtrate was concentrated on a rotary evaporator. The obtained PD extract was stored at -20°C for further analysis.

Antioxidant activity

DPPH assay

The antioxidant activity of PD extract was evaluated using 2,2-diphenyl-1-picryl hydrazyl (DPPH) assay as reported by  Majhenič et al. (2007) with slight modifications. Different concentrations of PD extract (dissolved in methanol) were prepared. A volume of 2,800 µl of methanolic solution of DPPH (6x10^-5 M) was mixed with 200 µl of each concentration and incubated in the darkness at 37°C. Twenty minutes later, the absorbance was measured at 517 nm using a spectrophotometer against blank. Ascorbic acid was used as a standard. The percentage of the DPPH scavenging activity was calculated using the following equation:

% inhibition = [(A₀ –A₁)/A₀] x 100

Where A₀ represents the absorbance of the blank sample, and A₁ represents the absorbance of the extract/standard

IC₅₀ (the concentration required to scavenge 50% of free radicals) was calculated from the regression equation, prepared from the concentration of samples and percentage inhibition of free radical formation.

Ferrous ions chelating capacity

The chelating activity of the extract on Fe²⁺ was measured in accordance with Tel et al. (2013). In brief, 40 μl of FeCl₂ reagent (0.2 mM) was added to 80 μl of the extract solution (dissolved in ethanol at different concentrations). The reaction was initiated by the addition of 80 μl ferene (0.5 mM). The mixture was shaken vigorously and left at room temperature. After 10 min, the absorbance was measured at 593 nm. Ethylenediamine tetra-acetic acid (EDTA) was used as an antioxidant standard.

The ability of the PD extract to chelate ferrous ion was calculated using the formula:

Fe²⁺chelating activity (%) = [(A₀−A₁) ∕A₀] x100

In which, A₀ is the absorbance of the blank sample and A₁ is the absorbance of the PD extract/standard.

Cupric reducing antioxidant capacity (CUPRAC)

The CUPRAC was measured according to Apak et al. (2004) with slight modifications. In each well of 96 well plates, a mixture of 50 μl of copper (II) chloride solution (10 mM), 50 µl of neocaproine solution (7.5 mM), and 60 μl of ammonium acetate buffer (1 M, pH 7.0) was added. Then, 40 μl of the sample extracts dissolved in methanol at different concentrations were added. The well plates were shaken vigorously and left at room temperature. After one hour, the absorbance of the mixed solution was recorded at 450 nm. Butylated hydroxyanisole (BHA) was used as antioxidant standard. The presence of antioxidant compounds reduced the cupric ions to cuprous ions, resulting in the formation of a stable complex between neocuproine and cuprous ions. The results were given as A_0.50 (g/l) corresponding to the concentration indicating 0.50 absorbance intensity. A higher absorbance indicates a higher reducing capacity of antioxidants.

Antibacterial assay

The disc diffusion method was used to evaluate the antibacterial activity of PD extract as described by Islam et al. (2013) with slight modifications. Sterilized paper discs (6 mm) were impregnated with 10 μl of PD extract dissolved in DMSO (200 mg/ml) and dried aseptically. The impregnated discs were placed on the surface of plates that were previously inoculated with bacterial suspensions (0.5 McFarland standards) and incubated at 35 ± 2°C for 24 h. Chloramphenicol (10 µg/disc) was used as a positive control. While, discs impregnated with 10 μl of DMSO and clean discs (without solvent) were used as negative controls. After incubation, the antibacterial activity was evaluated by measuring the diameter of the inhibition zone surrounding the discs.

Acute toxicity study

The oral acute toxicity study of PD extract was carried out as per the OECD guideline 423 (OECD, 2002). In brief, a single animal was administered a high dose of the extract (1,000 mg/kg) and observed for up to 48 h. Following the absence of toxicity signs (body weight variation, salivation, tremors, convulsions, diarrhea, death.) an additional four animals were administered the same dose and observed for comparison with the normal group. A control group of mice (n = 5) received distilled water in the same volume as that of the treated group. All animas were closely observed at a regular interval for 14 days.

In vivo phagocytic activity

The phagocytic activity of PD extract was evaluated in vivo according to Sahu et al. (2010) with slight modifications. Mice were divided into seven groups of five animals each. Animal in the control group (CON) received the vehicle (0.9%w/v NaCl), whereas animals in other groups received i.p of PD extract suspended in 0.9%w/v normal saline at doses of 30, 50, 70, 100, 150, and 200 mg/kg body weight, respectively. After 5 days of treatment, mice have injected with carbon ink suspension (10 µl/g body weight) through the tail vein. A blood sample from each mouse (25 μl) was drawn from the orbital vein at 0 and 15 min and immediately lysis in 2 ml of sodium carbonate solution (0.1%). The absorbance of the mixture was measured at 675 nm.

The phagocytic activity is expressed by the phagocytic index K which is calculated by the following equation:  K = (ln (OD₁) – ln (OD₂)) /(t₂-t₁)

Where, OD₁ and OD₂ are the optical densities at t₁=0 min and t₂=15min, respectively.

Statistical analysis

All data are presented as the mean ± SD with each in vitro assay conducted in triplicate samples. One-way analysis of variance (ANOVA) followed by a Tukey's multiple comparison test was used to assess statistical significance using SPSS software (version 25.0). The values of P< 0.05 were considered statistically significant.

RESULTS

Plant extraction yield

The date fruits were extracted with 70%v/v ethanol and the obtained yield from 50 g was 44.10%.

Antioxidant activities of PD extract

The antioxidant activity of the PD extract evaluated through the three previously mentioned methods, is presented in Table 1. In all assays, antioxidant activity increased in a dose dependent manner.  The PD extract displayed robust free-radical-scavenging activity, ranging from 60.51% to 70.17% of DPPH radicals scavenged. Moreover, the extract exhibited efficient chelation of ferrous ions and demonstrated notable reducing activity of copper ions. However, the PD extract showed less antioxidant activities than those observed with the standard solutions.

Table 1. Antioxidant activity of Phoenix dactylifera fruit extract.

Note: Values are means ± standard deviation of three parallel measurements. a: Phoenix dactylifera extract, b: butylated hydroxyanisole, c: ethylenediamine tetra-acetic acid.

Antibacterial activity

The antibacterial activity of PD extract against four strains of human pathogenic bacteria is presented in Table 2. The extract exhibited an inhibitory growth activity against all tested strains at a concentration of 200 mg/ml. The results showed maximum inhibition against Gram positive bacteria, with the highest zone of inhibition recorded against S. pneumonia (23.3 ± 0.3 mm). The lowest zone of inhibition was observed against E.coli with 14.1 ± 0.3 mm.

Table 2. Antibacterial activity of Phoenix dactylifera fruit extract.

Note: Values are the means of three replicates ± SD, a: Phoenix dactylifera extract, b: Chloramphenicol (10 µg/disc).

 Acute toxicity study

The extract was found to be safe up to 1,000 mg/kg body weight, and all mice were free of any toxicity signs during the observation period of 14 days.

In vivo phagocytic activity
As shown in Figure 1, the phagocytic index exhibited a notable increase in a dose-dependent manner. Mice pretreated with PD extract at a dose of 150 mg/ml demonstrated a significantly higher phagocytic index (P<0.001) compared to CON and the groups treated with 30, 50, and 70 mg/kg doses.  Notably, there was no significant difference observed between the 150 mg/kg and 200 mg/kg doses. Accordingly, the half-time carbon in the bloodstream significantly decreased in a dose-dependent manner (Figure 2), with the shortest durations recorded at the dose of 150 mg/mg, with the corresponding value of 8.2 min. This value was significantly different from that recorded for the control group (25.4 min) with P<0.001.

Figure 1. Effect of Phoenix dactylifera fruit extract on phagocytic activity.
n = 5, values are the mean ± SD, *P˂ 0.05, **P˂ 0.01, ***P˂ 0.001 vs control group. ##P˂ 0.01, ###P˂ 0.001 vs 150 mg/kg group.

Figure 2. Effect of Phoenix dactylifera fruit extract on the half time of carbon in the bloodstream. n = 5, values are the mean ± SD, *P˂0.05, **P˂0.01 vs control group.

DISCUSSION

The association between healthy consumption of fruit and low risk of chronic diseases has drawn increased interest. Numerous studies have underscored the natural compounds' ability to suppress free-radical generation and reducing oxidative stress (Hussain et al., 2020). They are also seen as promising sources for novel medicines intended to enhance the immune function and resist microbial growth. Therefore, our study was undertaken to provide insight into a less-studied variety of date fruits with potential antioxidant, antimicrobial, and immunomodulatory activities.

The date fruit was extracted with 70%v/v ethanol to obtain a higher extraction which yielded a higher extraction rate and a diverse array of compounds known for their versatile biological activities (Kadum et al., 2019).

Due to the extensive biodiversity among plant varieties, antioxidant molecules may act through three principal mechanisms: scavenging or reducing free radicals and chelation of transition metals. Thus, it is recommended to evaluate the in vitro antioxidant capacity of tested samples using various analytical methods (Mamache et al., 2020). In our study, the antioxidant activity of the PD extract was evaluated using three different methods (DPPH, Ferrous chelating and CUPRAC assays). The DPPH assay is easy, rapid, inexpensive and commonly applied to determine the antioxidant capacity of the natural products with the advantage of being unaffected by side reactions (Zihad et al., 2021; Marliani et al., 2022). It is based on the measurement of the scavenging capacity of antioxidants found in the extract towards DPPH radical free radicals (Jadid et al., 2017). Ferrous ion chelating activity is very important since it reduces the metal concentration and the subsequent oxidative damage. Chelating agents could reduce redox potential thereby stabilizing the oxidized metal ion (Gulcin and Elwasel, 2022). In the CUPRAC method, the CUPRAC reagent (chromogenic), bis(neocuproine)copper(II) chloride (Cu(II)-Nc) is reduced by antioxidants present in the plant extract generating a colored product (cuprak chromophore) bis(neocuproine) copper(I) cation (Cu(I)-Nc) (Özyürek et al., 2011). Furthermore, the CUPRAC assay provides a better evaluation of in vitro antioxidant ability (Hachani et al., 2018). Even though these methods were based on different mechanisms of the antioxidant assay, the PD extract showed an important antioxidant activity in all the tests. This result was in agreement with Mansouri et al. (2005), Benmeddour et al. (2013), and Zhang et al. (2017) who reported that PD extract has a proton donating ability and could serve as free radical inhibitors or scavengers activity. Numerous studies have linked the presence of phenolics, carotenoids, and anthocyanins in date fruit with its antioxidant capacity (Benmeddour et al., 2013; Al-Mamary et al., 2014; Zihad et al., 2021). Mansouri et al. (2005) demonstrated the presence of p-coumaric, ferulic and sinapic acids and some cinnamic acid derivatives in different varieties of Algerian date fruits. Another study of Kadum et al. (2019) correlated the strongest antioxidant activity of Piyarom and Rabbi varieties to the higher concentration of ascorbic acid, epicatechin, citric acid, and gallic acid in these varieties compared to Deglet Nour, Ajwa, and Anbara varieties. Hence, we propose that the antioxidant activity of date fruit is due to the synergistic and additive effects of multiple compounds present in the extract.

Bacterial infections have been of major concern to public health due to the spread of antimicrobial resistance. Regarding their ability to produce a variety of bioactive compounds, many medicinal plants have been tested and identified as good sources of natural antimicrobial substances and as a potential treatment of infections by resistant pathogens (Manandhar et al., 2019). The PD extract showed an inhibitory growth activity against all tested strains (E. coli, P. aeruginosa, S. aureus, and S. pneumoniae). This was in line with prior studies (Al-Dhaihan and Bhat, 2012; El Sohaimy et al., 2015; Sani et al., 2017) reported the sensitivity of both Gram positive and Gram negative bacteria to date fruit extract. In addition, several studies (Bonilla et Sobral, 2017; Likittrakulwong et al., 2023) reported that Gram negative bacteria are more resistant to plant extract which is in agreement with our findings. The variation on susceptibility to PD extract might be attributed to the presence of an outer complex membrane in Gram negative bacteria which blocked the passage of active components present in the extract such as phenolics, flavonoids and terpenoids (Mai-Prochnow et al., 2016; Tangjitjaroenkun et al., 2017). Moreover, phenolic compounds may interact with proteins of the microbial cell membrane and/or enzymes causing cell lysis (Mostafa et al., 2018).

Despite decades that 35% of modern drugs are based on natural products such as Aspirin (Ephedrine), and Paclitaxel (Artemisinin) (Calixto, 2019), natural immunomodulators are less studied. Immunomodulators stimulate the body’s natural defense against pathogens and maintain immune-system homeostasis. The reticuloendothelial system (RES, also known as the mononuclear phagocytic system, or MPS) is a diffuse system of phagocytic cells that provides the first body’s line of defense. The main role of RES is to capture and destroy unwanted microorganisms and enhance the innate (nonspecific) immune response. In our study, the carbon clearance test was realized to evaluate the effect of PD extract on RES. When ink containing colloidal carbon is administered intravenously, macrophages engulf the carbon particles of the ink, and the rate at which the carbon particles are cleared from the bloodstream is known as the phagocytic index (George et al., 2014). The increase in the phagocytic index after injection of PD extract reflects the enhancement of the phagocytosis activity of the RES. Our results are in line with prior studies that showed an increment in the phagocytic index when animals were pretreated with medicinal plants (Patel and Asdaq, 2010; Chan-Zapata et al., 2018; Alves et al., 2020; Sakhri et al., 2021). In addition, a study conducted by Karasawa et al. (2011) demonstrated that the hot water extract of date fruit stimulates IL-12 produced by macrophages, which leads to the activation of NK cells and differentiation of naive T cells into Th1 cells thereby enhancing the cellular immune system. The immunomodulatory activity of PD extract could be attributed to various phytoconstituents. Elhazmi et al, (2021) reported that polysaccharides, terpenoids, flavonoids, alkaloids, glycosides, and lactones are the most important phytochemicals, which provide the immunomodulation activity of medicinal plants. Therefore, date fruit could be used as an effective immunomodulator when the immune system is compromised to improve both innate and cell-mediated immune response or for preventing some diseases.

CONCLUSION

The scientific results obtained from this study enhance our knowledge of the health-promoting effects of date fruit in the protection against the oxidation of biological macromolecules and indicate potential antibacterial effects against microbial infections. In addition, these results lead us to believe that the PD extract represents a promising tool for the development of safe and effective immunomodulators as remedies to prevent and treat inflammatory and immune-related diseases. Further purification and isolation of the bioactive compounds in PD extract would provide possible identification of the mechanism of action and possible lead compounds for the development of new drugs.

ACKNOWLEDGEMENTS

The authors would like to thank Dr. Hadjira Sakhri for the contribution in collecting fruit samples.

AUTHOR CONTRIBUTIONS

Afaf Sakhri designed the study, Afaf Sakhri, Fatma Zahra Sakhri and Chawki Bensouici performed the experiments, Afaf Sakhri analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.

CONFLICT OF INTEREST

We declare that we have no conflict of interest.

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