Exploring the Antimicrobial Capabilities of Date Palm Seed Essential Oil

Introduction

The swift rise of antibiotic-resistant bacteria has emerged as one of the most significant pressing challenges in modern healthcare. As conventional antibiotics lose their effectiveness against multidrug-resistant (MDR) pathogens, the search for alternative antimicrobial agents has intensified [1]. Natural sources, particularly plant-based compounds, has gained significant attention due to their ability to address resistant bacterial strains while reducing the likelihood of promoting additional resistance [2]. Plants have long been a cornerstone of traditional medicine, offering a rich repository of bioactive compounds with therapeutic properties. Among these, essential oils—volatile, aromatic liquids extracted from plants—have shown remarkable antimicrobial, antioxidant, and anti-inflammatory activities[3]. Their complex chemical composition, which includes phenolics, terpenes, and aldehydes, contributes to their broad-spectrum efficacy against various pathogens. Importantly, essential oils are considered safer and more sustainable alternatives to synthetic drugs, as they are less likely to promote resistance and have lower toxicity profiles[4].

The date palm (Phoenix dactylifera L.), often referred to as the “tree of life,” is one of the most economically and medicinally significant plants in arid and semi-arid regions while the fruit is widely consumed for its nutritional value, the seeds are often discarded as agricultural waste[5]. However, recent studies have revealed that date palm seeds are rich in bioactive compounds, including phenolics, flavonoids, and essential oils, which exhibit potent antimicrobial and antioxidant properties[6]. These results indicate that date palm seeds have the potential to be a significant source of natural antimicrobial agents.

Despite the growing interest in plant-derived antimicrobials, there is limited research on the specific bioactive components of date palm seed essential oil and their mechanisms of action[7]. Furthermore, comparative studies evaluating the efficacy of date palm seed extracts against conventional antibiotics are scarce[14]. This research seeks to tackle these gaps by investigating the antibacterial activity of essential oil extracted from date palm seeds against a panel of gram-positive and gram-negative bacterial pathogens [25].Additionally, the study compares the efficacy of the essential oil with that of commonly used antibiotics, providing insights into its potential as an alternative or adjunctive therapy for bacterial infections[13]. The results of this research have significant significance for the creation of natural antimicrobial solutions, particularly in the context of combating antibiotic resistance. By elucidating the antibacterial properties of date palm seed essential oil, this study contributes to the growing body of research on the therapeutic potential of plant-derived compounds and underscoresthe importance of exploring natural resources in the search for novel antimicrobial agents[22].

Materials and Methods

Collection and Preparation of Plant Material

Date palm seeds (Phoenix dactylifera L.) were procured from a local market in Benghazi, Libya, in 2019. The seeds, chosen for their uniform size and high quality, were meticulously cleaned to remove debris. After air-drying at ambient temperature, the seeds were finely ground using a mechanical grinder and then stored in airtight containers to maintain their integrity until further use[19].

Bacterial Strains and Culture Conditions

The antibacterial properties of the essential oil were tested against a diverse group of clinically relevant bacterial strains, including both Gram-positive and Gram-negative organisms. The strains used were:

• Staphylococcus aureus
• Staphylococcus hemolytic
• Micrococcus spp.
• Escherichia coli
• Pseudomonas aeruginosa
• Klebsiella pneumoniae
• Proteus vulgaris

These strains were sourced from the Microbiology Unit of the Faculty of Applied Science. Identification was conducted using standard microbiological methods, such as colony morphology, Gram staining, and biochemical profiling[17].

Extraction of Essential Oil

Essential oil was extracted from 100 grams of powdered date palm seeds using a Soxhlet apparatus and light petroleum ether as a solvent (boiling point range: 40–60°C). The process lasted approximately four hours to ensure complete oil recovery. After extraction, the solvent was removed under reduced pressure using a rotary evaporator at 40°C. The extracted oil was subsequently dried using anhydrous sodium sulfate to remove moisture and then stored in amber-colored airtight containers at 7°C to prevent oxidation and photodegradation[11].

Preparation of Essential Oil Dilutions

To evaluate concentration-dependent antibacterial activity, the essential oil was diluted using dimethyl sulfoxide (DMSO). Final concentrations of 25%, 50%, 75%, and 100% (v/v) were prepared. DMSO was used as a negative control to verify that the observed antibacterial effects were attributable solely to the oil[20].

Quantification of Total Phenolic Content (TPC)

The phenolic content of the essential oil was measured via the Folin-Ciocalteu colorimetric assay. Samples of the oil, prepared at concentrations of 100–500 µg/mL, were mixed with Folin-Ciocalteu reagent and sodium carbonate solution [5]. After adjusting the mixture’s final volume to 1 mL with distilled water, it was incubated for 10 minutes at room temperature. The absorbance at 725 nm was measured using a UV-visible spectrophotometer[9]. Pyrogallol was used as the standard for the calibration curve, and the phenolic content was expressed as pyrogallol equivalents (µg/mL) [16]. All experiments were conducted in triplicate, and data were analyzed statistically using ANOVA.

Assessment of Antibacterial Activity

1. Preparation of Bacterial Inoculum Bacterial strains were cultured in nutrient broth at 37°C for 24 hours to ensure active growth. Cultures were refreshed every 3–4 days to maintain viability. The bacterial suspensions were standardized to an optical density of 0.1 at 600 nm, equivalent to approximately 10810^8 CFU/mL [24].
2. Agar Ditch Method The antibacterial activity of the essential oil was tested using the agar ditch method. Wells of 8 mm diameter were prepared aseptically in Mueller-Hinton agar plates. Each well was filled with 100 µL of the essential oil at concentrations of 25%, 50%, 75%, and 100%. Plates were inoculated with bacterial suspensions and incubated at 37°C for 24 hours. Zone diameters of inhibition were measured in millimeters using a digital caliper. Experiments were performed in triplicate, and results were reported as mean ± standard deviation [18].
3. Minimum Inhibitory Concentration (MIC) The MIC was determined using well diffusion. Serial dilutions of the essential oil (25%, 50%, 75%, and 100%) were introduced into wells on agar plates pre-inoculated with bacterial strains. Plates were incubated at 37°C for 24 hours, and MIC was identified as the lowest concentration that inhibited visible bacterial growth. [23].

Comparative Analysis with Commercial Antibiotics

The antibacterial efficacy of the essential oil was compared against conventional antibiotics, including imipenem (10 µg), ampicillin (10 µg), erythromycin (15 µg), vancomycin (30 µg), and ceftriaxone (30 µg). Antibiotic discs were placed on Mueller-Hinton agar plates inoculated with bacterial strains. After 24 hours of incubation at 37°C, inhibition zones were measured and compared to those produced by the essential oil[21].

Statistical Analysis

All experiments were conducted in triplicate. Data were presented as mean ± standard deviation and analyzed statistically using one-way ANOVA, followed by Tukey’s post-hoc test for group comparisons. Significance was determined at p<0.05p < 0.05. SPSS software (version 25.0) was used for analysis.

Results

Total Phenolic Content (TPC) of Essential Oil

The total phenolic content (TPC) of the essential oil derived from date palm seeds was assessed using the Folin-Ciocalteu assay. Findings revealed a direct relationship between oil concentration and phenolic content. At 100 µg/mL, the phenolic content was 1.16 µg/mL, increasing to 2.31 µg/mL at 500 µg/mL. This growth pattern signifies the abundance of phenolic compounds in the essential oil, underscoring their potential role in antimicrobial properties. Table 1 and Figure 1 illustrate these trends.

The essential oil exhibited strong antibacterial activity across a spectrum of Gram-positive and Gram-negative bacterial strains, with variations based on concentration and bacterial type.

1. Gram-Positive Bacteria Gram-positive bacteria demonstrated significant sensitivity to the essential oil.
   • Staphylococcus aureus: Inhibition zones ranged from 17.3 mm at 25% concentration to 22 mm at 100%.
   • Staphylococcus hemolyticus: Zones varied from 15.6 mm at 25% to 17.6 mm at 100%.
   • Micrococcus spp.: Demonstrated inhibition zones of 17.6 mm at 25% and 22 mm at 100%.
2. Gram-negative bacteria Activity against Gram-negative strains varied considerably.
   • Escherichia coli: Inhibition zones increased from 8.3 mm at 25% to 20.3 mm at 100%.
   • Klebsiella pneumoniae: Zones ranged from 2.3 mm at 25% to 15 mm at 100%.
   • Pseudomonas aeruginosa: Showed complete resistance at all concentrations.

Antibacterial activity of essential oil extracted from seed powder of Dates Each assay was performed in triplicate, and the results, expressed as the zone of inhibition (in millimeters), were presented as mean values ± standard deviation. The mean diameter of the inhibition zone (in mm) was measured following 24 hours of incubation. Bacterial strains that exhibited no significant inhibition were classified as resistant (denoted as “R”).

Comparison with Conventional Antibiotics

The essential oil’s antibacterial efficacy surpassed that of several conventional antibiotics, particularly against Gram-positive bacteria. Staphylococcus aureus and Micrococcus spp.Showed resistance to antibiotics like ampicillin, ceftriaxone, and imipenem, but were sensitive to the essential oil. Gram-negative strains such as Pseudomonas aeruginosa, which exhibited resistance to all tested antibiotics, remained unaffected by the essential oil. These findings highlight the oil’s potential as a natural alternative. Tables 3 and 4, along with Figures 9–15, depict this comparative analysis.

Minimum Inhibitory Concentration (MIC)

The MIC values of the essential oil varied depending on the bacterial strain.

• Staphylococcus hemolyticus: MIC of 2.5%.
• Staphylococcus aureus: MIC of 5%.
• Micrococcus spp.: MIC of 10%.
• Klebsiella pneumoniae: MIC of 50%.
• Other strains, including Escherichia coli, Pseudomonas aeruginosa, and Proteus vulgaris, exhibited complete resistance at all tested concentrations.

Discussion

The global rise of multidrug-resistant (MDR) bacterial strains necessitates urgent exploration of alternative antimicrobial agents. This study highlights the antibacterial capabilities of essential oil extracted from date palm (Phoenix dactylifera L.) seeds, which demonstrated remarkable efficacy against a variety of Gram-positive and Gram-negative bacteria[12].The findings strongly suggest that this essential oil could serve as a natural substitute for traditional antibiotics, providing a promising strategy in the fight against antibiotic resistance.

Antibacterial Activity of Essential Oil The essential oil showcased pronounced antibacterial effects, particularly against Gram-positive bacteria such as Staphylococcus aureus and Staphylococcus hemolyticus, with inhibition zones spanning 15.6 mm to 22 mm. Interestingly, these results indicate superior performance compared to conventional antibiotics like ampicillin and ceftriaxone. The elevated levels of phenolic compounds detected in the essential oil are likely responsible for its potent activity, as these compounds are known for their membrane-disrupting and enzyme-inhibiting properties. This observation aligns with existing studies on the antimicrobial potential of plant-derived oils rich in phenolics[10]. Conversely, the oil exhibited variable effectiveness against Gram-negative bacteria. While it effectively targeted Escherichia coli and Klebsiella pneumonia, it was entirely ineffective against Pseudomonas aeruginosa. The resistance observed in P. aeruginosa could be attributed to structural factors such as its protective outer membrane or inherent resistance mechanisms like efflux pumps.

Comparison with Conventional Antibiotics The comparative analysis highlights the essential oil’s superior antibacterial potential against Gram-positive pathogens when contrasted with standard antibiotics. For instance, while Staphylococcus aureus showed resistance to antibiotics such as imipenem, ampicillin, and ceftriaxone, it remained sensitive to the essential oil across all concentrations. Similarly, Micrococcus spp.Demonstrated resistance to erythromycin, ampicillin, and ceftriaxone but was inhibited by the essential oil. This reinforces the oil’s role as a viable alternative for combating antibiotic-resistant Gram-positive bacteria[7]. However, the inability of the oil to combat certain Gram-negative strains underscores the need for further research to enhance its efficacy.

Phenolic Compounds and Antimicrobial Activity Phenolic compounds in the oil, quantified in this study, correlate directly with its antibacterial activity[9]. These compounds, known for disrupting bacterial cell functions, increase in concentration within the oil, enhancing its antimicrobial potential. The concentration-dependent antimicrobial activity observed further validates the critical role of phenolics in this essential oil’s efficacy.

Implications for Future Research This study highlights the untapped potential of date palm seeds, often considered agricultural waste, as a cost-effective and sustainable source of bioactive antimicrobial compounds. By focusing on their application, the global reliance on synthetic antibiotics could be reduced[8]. Future studies should aim to isolate the active components of this essential oil, assess their safety profiles, and explore potential synergistic effects with existing antibiotics. Such investigations could pave the way for novel treatment strategies to mitigate the pressing issue of antibiotic resistance.

Conclusion

This research underscores the significant antibacterial efficacy of essential oil derived from date palm (Phoenix dactylifera L.) seeds, particularly in combating Gram-positive bacterial strains. The oil displayed robust antimicrobial activity, with inhibition zones ranging from 15.6 mm to 22 mm against pathogens like Staphylococcus aureus [6]. The high concentration of phenolic compounds, which increases with the oil’s concentration, plays a critical role in its ability to disrupt bacterial membranes and inhibit enzyme activity. Notably, the oil outperformed several conventional antibiotics such as ampicillin and ceftriaxone when used against Gram-positive bacteria, suggesting its potential as a natural alternative [3]. However, its effectiveness against Gram-negative bacteria varied, with pathogens like Pseudomonas aeruginosa exhibiting complete resistance due to the protective outer membrane characteristic of such bacteria these findings hold promise for addressing the growing issue of antibiotic resistance. Additionally, the potential of date palm seeds, often discarded as waste, to serve as a sustainable source of therapeutic agents adds further value to this natural resource. Future studies should delve into identifying the exact bioactive components contributing to its antimicrobial properties, as well as assessing its clinical safety and efficacy. Exploring its synergistic use with traditional antibiotics may further enhance its effectiveness and help mitigate resistance development. By leveraging bioactive compounds like date palm seed essential oil, new pathways in antimicrobial therapy can be explored to tackle multidrug-resistant bacterial infections.

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