Open Veterinary Journal, (2026), Vol. 16(4): 2095-2113
Research Article
10.5455/OVJ.2026.v16.i4.14
Superior efficacy of 2% Olea europaea leaf extract ointment compared with Fucidin® in accelerating wound closure and resolving infection in rat models
Ahmed Saeed Kabbashi1*, Taher Issa Shailabi2, Yasmeen Nouh Amrajaa3, Namarq Moftah Bokhairallah1,
Noor Al. Houda Nasser Rages1, Shahd Ezedeen Al. Shhome1, Shimaa Osama Bosallom1, Nehal Moftah Al. Daikh1 and Bushray Salih Jabullah4
1Department of Biomedical Science, Faculty of Pharmacy, Omar Al-Mukhtar University, Al-Bayda, Libya
2Department of Pharmacology and Toxicology, Faculty of Pharmacy, Omar Al-Mukhtar University, Al-Bayda, Libya
3Department of Pharmaceutics, Faculty of Pharmacy, Omar Al-Mukhtar University, Al Bayda, Libya
4Al-Jabal Al-Akhdar Diabetes Center, Ministry of Health, Al-Bayda, Libya
*Corresponding Author: Ahmed Saeed Kabbashi. Department of Biomedical Science, Faculty of Pharmacy, Omar Al-Mukhtar University, Al Bayda, Libya. Email: ahmed.saeed [at] omu.edu.ly
Submitted: 18/11/2025 Revised: 26/02/2026 Accepted: 15/03/2026 Published: 30/04/2026
© 2025 Open Veterinary Journal
This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.
ABSTRACT
Background: Impaired wound healing, intensified by microbial infection and increasing antibiotic resistance, underscores the urgent need for novel therapeutic agents. Olea europaea L. (olive) leaf, which is rich in bioactive polyphenols, is a promising candidate for its traditional medicinal use.
Aim: This study aimed to evaluate the wound-healing efficacy of a topical O. europaea leaf extract (OLE) ointment in clean and infected wound models by directly comparing its performance with that of a standard topical antibiotic.
Methods: The phytochemical characterization and in vitro antibacterial activity of 75% ethanolic OLE were assessed. Ointments containing 1% and 2% (w/w) OLE were developed and characterized physicochemically. Their efficacy was evaluated in vivo using full-thickness excisional wounds in rats and compared with untreated, vehicle, and Fucidin®-treated controls. Wound models infected with Staphylococcus aureus and Pseudomonas aeruginosa were also established. Wound closure was monitored planimetrically for 21 days.
Results: OLE was rich in phenols, flavonoids, and tannins and exhibited potent in vitro antibacterial activity against both pathogens, showing superior inhibition of S. aureus compared with that of Fucidin®. In the clean-wound model, 2% OLE ointment induced the most rapid healing, achieving complete closure by day 13, significantly outperforming Fucidin® (day 18; p < 0.01). In the infected models, both OLE ointments significantly accelerated healing and infection resolution compared with the controls. Notably, the 1% OLE formulation demonstrated superior early activity in infected wounds, reaching 100% closure by days 12 (S. aureus) and 13 (P. aeruginosa), while outperforming Fucidin® (days 18–20).
Conclusion: The 2% OLE ointment possesses potent dose-dependent wound-healing and broad-spectrum antimicrobial activities, outperforming a standard antibiotic in clean wounds and showing high efficacy in infected models. Its multi-target action, derived from a complex phytochemical profile, validates its traditional use and highlights its potential as a leading natural formulation for advanced wound management, particularly in the era of antimicrobial resistance.
Keywords: Olea europaea, Phytochemical, Polyphenols, Rat model, Wound healing.
Introduction
Skin wounds represent a significant and growing challenge in global healthcare. While acute wounds typically follow a predictable healing trajectory, chronic wounds, which are often associated with conditions such as diabetes and vascular insufficiency, frequently stagnate in a state of pathological inflammation (Malone et al., 2017; Hurlow et al., 2025). This impairment is profoundly exacerbated by microbial colonization and biofilm formation, which disrupt the highly coordinated cascade of healing, involving hemostasis, inflammation, proliferation, and remodeling (Maheswary et al., 2021; Uberoi et al., 2024). The resulting dysbiosis of the wound microbiome leads to persistent infection, increased morbidity, prolonged hospitalization, and soaring treatment costs (Zielińska et al., 2023; Bin Ahmed Bakri, 2025).
Conventional wound management relies on antimicrobials, debridement, and advanced dressings. However, the rising threat of antimicrobial resistance (AMR) and the potential adverse effects of synthetic drugs have revitalized interest in natural product-based therapies (Atanasov et al., 2021; Jangra et al., 2025). Medicinal plants offer a vast repository of bioactive secondary metabolites with multifaceted mechanisms of action. Polyphenols (including flavonoids and tannins) and terpenoids are particularly notable for promoting healing through potent antimicrobial, antioxidant, and anti-inflammatory activities (Bharadvaja et al., 2023; Chintada and Golla, 2025; El-Saadony et al., 2025). Specifically, their antioxidant capacity mitigates oxidative stress in the wound bed, while their anti-inflammatory action modulates key cytokines, creating a conducive microenvironment for tissue repair (Criollo-Mendoza et al., 2023; Trepa et al., 2024).
The transition from a biologically active plant extract to a safe, stable, and effective therapeutic agent is non-trivial and requires a sophisticated formulation. Topical preparations are particularly advantageous for wound management because of their localized delivery, ease of application, and ability to maintain a moist wound environment conducive to healing (Dhivya et al., 2015). Traditional semi-solid vehicles, such as ointments and creams, provide a foundational platform that offers occlusive or moisturizing properties that can enhance lipophilic active delivery )Bhalekar et al., 2004b, Ribeiro et al., 2024).
However, the inherent challenges of many natural compounds, such as poor solubility, chemical instability, and limited skin penetration, necessitate the development of advanced drug delivery systems. Contemporary pharmaceutical research has focused on these advanced systems to enhance the efficacy of natural agents. For instance, cinnamaldehyde-loaded self-nanoemulsifying drug delivery systems have demonstrated superior burn healing acceleration in animal models, mechanistically linked to improved bioactive penetration and synergistic antimicrobial, antioxidant, and anti-inflammatory effects (Qureshi et al., 2022). Similarly, other formulations, such as eucalyptol ointment, underscore the importance of pharmaceutical vehicles in achieving stable release and time-dependent therapeutic effects (Mohammed et al., 2022). Therefore, developing an optimal delivery system, such as a traditional ointment or a nanotherapeutic formulation, is a critical step in translating in vitro-proven bioactivity into robust in vivo clinical efficacy.
Olea europaea L. (olive tree), a cornerstone of Mediterranean ethnobotany, has been revered for centuries owing to its health benefits. Its leaves are an exceptionally rich source of bioactive phenols, with oleuropein being the predominant secoiridoid glycoside, acclaimed for its potent antioxidant, anti-inflammatory, and antimicrobial properties, along with other major phenolics, such as hydroxytyrosol, verbascoside, and luteolin-7-glucoside (Boss et al., 2016; Elhrech et al., 2024). Although the systemic benefits of for conditions such as hypertension and hyperlipidemia are well documented, its targeted application in topical wound care remains relatively underexplored.
A significant gap exists in the preclinical literature regarding a direct, head-to-head comparison of a standardized O. europaea leaf extract (OLE) formulation with a first-line standard-of-care topical antibiotic. Most existing studies have either focused on in vitro activity or evaluated OLE in simple wound models without a robust clinical comparator (Al-Warhi et al., 2022). Simultaneous evaluation of both clean and clinically relevant infected wound models is lacking. This limitation limits the translational understanding of the dual therapeutic potential of OLE: direct antimicrobial action and intrinsic tissue regenerative capacity.
This study was designed to bridge these gaps through an integrated phytochemical, formulation, and in vivo efficacy investigation. Our specific objectives were as follows: (1) to perform comprehensive phytochemical and in vitro biological profiling (antibacterial and cytotoxicity) of a hydroalcoholic OLE; (2) to develop and physicochemically characterize stable ointment formulations containing OLE at two concentrations; and (3) to systematically evaluate their efficacy in standard and bacterially infected (Staphylococcus aureus and Pseudomonas aeruginosa) full-thickness wound models in rats, with a direct, concentration-matched comparison to the standard topical antibiotic Fucidin® (2% fusidic acid). This study contributes to the field by evaluating the efficacy of ointment while providing a clear foundation for future comparisons with more advanced nanotechnological delivery systems. Our work provides a rigorous scientific foundation for the traditional use of olive leaves and evaluates their potential as multi-target therapeutics for complex wound management by addressing these aims.
Materials and Methods
Chemicals, reagents, and plant materials
All chemicals and solvents were of analytical or pharmaceutical grade. Ethanol (75% and 99.9%), methanol, and phytochemical screening reagents (Mayer’s reagent, FeCl₃, and Folin-Ciocalteu reagent) were procured from Sigma-Aldrich (USA) and Merck KGaA (Germany), respectively. Pharmaceutical-grade ointment bases (white soft paraffin, liquid paraffin, and beeswax) were obtained from Fisher Scientific (USA). Microbiological media (Mueller-Hinton agar/broth) were obtained from HiMedia Laboratories (India). The reference antibiotic ointment Fucidin® (2% fusidic acid) was purchased from a local pharmacy. Fresh O. europaea L. leaves (Fig. 1) were collected from Al-Bayda, Libya, in July 2024.
Fig. 1. Morphological stages of the O. europaea L. (olive tree) leaves used for extraction. (A) Mature tree, (B) fresh leaves, (C) shade-dried leaves, and (D) powdered leaf material.
Plant authentication and extraction
Authentication
Taxonomic authentication of the plant material was performed by Dr. Ensaf Dakhil, a senior botanist at Omar Al-Mukhtar University. A voucher specimen (OE-2024-023) was deposited in the herbarium of the university. Permission to collect plants was obtained before sampling.
Extraction procedure
Fresh O. europaea L. leaves were collected from Al-Bayda, Libya, in July 2024. The collected leaves were shade-dried at room temperature (approximately 25°C) until completely dry and then pulverized using a mechanical grinder into a fine powder (Fig. 1). Specifically, a sample weighing approximately 50 g was immersed in 500 ml of 75% ethanol as the extraction solvent at a ratio of 1:5 (w/v), that is, one part plant material to five parts solvent, for 72 hours with occasional shaking. The selection of this hydroalcoholic solvent aligns with established practices for optimal phenolic extraction from O. europaea (Asmaey et al., 2024; Elhrech et al., 2024). The filtrate was concentrated using a rotary evaporator under reduced pressure at 55°C (Heidolph, Germany). The dried extract (OLE) was stored in an airtight, light-protected container at 4°C. The extraction yield was 13.82% (w/w).
Phytochemical profiling
Crude OLE was qualitatively screened for major classes of secondary metabolites (phenols, flavonoids, tannins, alkaloids, saponins, coumarins, sterols, triterpenes, and anthraquinones) using standardized procedures (Yadav et al., 2011; Tessema and Molla, 2021) Figure 3.
Fig. 3. In vitro antibacterial activity of O. europaea leaf extract against (A) S. aureus and (B) P. aeruginosa.
In vitro biological assays
Antibacterial activity (agar well diffusion)
The antibacterial activity of OLE was evaluated against clinical isolates of S. aureus and P. aeruginosa. The same strains were subsequently used in an in vivo wound infection model. Bacterial suspensions were standardized to 0.5 McFarland turbidity (~ 1.5 × 108 CFU/ml) using the agar well diffusion method (Balouiri et al., 2016; Wayne, 2017), bacterial suspensions were standardized to 0.5 McFarland turbidity (~1.5 × 10⁸ CFU/ml). Wells with a diameter of 6 mm were punched into Mueller–Hinton agar plates and filled with 100 µl of OLE solution (100 mg/ml in 10% DMSO), Fucidin® ointment (2% fusidic acid), 10% DMSO (negative control), or sterile distilled water (negative control). The plates were incubated at 37°C for 18–24 hours, after which the diameters of the inhibition zones (including the well diameter) were measured in triplicate using a digital caliper.
Cytotoxicity screening (brine shrimp lethality bioassay)
A preliminary toxicity assessment was performed using a lethality bioassay of brine shrimp (Artemia salina) (Meyer et al., 1982). OLE stock solutions were prepared in methanol to achieve final test concentrations of 10, 100, and 1,000 µg/ml in artificial seawater. Ten nauplii were added to each vial. The controls were artificial seawater (negative control) and etoposide (positive cytotoxic control). After 24 hours, the lethality was recorded. The median lethal concentration (LD₅₀) with 95% confidence intervals was calculated using probit analysis (Kupchan et al., 1969; Ma et al., 1989). An extract with an LD₅₀ >1,000 µg/ml was considered nontoxic in this model (Meyer et al., 1982).
Formulation of OLE-loaded ointments
Rationale for concentration selection and formulation design
Two medicated ointments were developed by incorporating OLE into an oleaginous base: 1% w/w OLE ointment (F2) and 2% w/w OLE ointment (F1). A blank ointment base (F0) was used as the vehicle control. The selection of these specific concentrations was based on the following: (1) preliminary in vitro assessments indicating significant biological activity within this range; (2) safety considerations for topical application to wounded skin; and (3) the intentional equivalence of the 2% formulation to the concentration of the standard comparator, Fucidin® ointment (2% w/w), to enable a direct, concentration-matched efficacy comparison.
Preparation method
The ointments were prepared using the classical fusion method (Bhalekar et al., 2004a). The base components (soft paraffin, 52%; liquid paraffin, 25%; and beeswax, 3%) were weighed according to the proportions listed in Table 1, melted in a water bath (≤70°C), and stirred to form a homogeneous melt. Pre-weighed OLE was gradually incorporated into the molten base with continuous stirring for the medicated formulations. The mixture was cooled with slow stirring until it was sealed. All ointments were stored in airtight amber glass containers at room temperature (25℃ ± 2°C) until further evaluation.
Table 1. Composition of the OLE ointment formulations (%, w/w).
Physicochemical characterization and evaluation of stability
The prepared ointments (F0, F1, and F2) were evaluated according to the following parameters.
Organoleptic properties and homogeneity
The color, odor, texture, and homogeneity/phase separation were visually inspected. Figure 2 shows the macroscopic appearance and homogeneity.
Fig. 2. Macroscopic appearance and homogeneity test of the ointment formulations (A) Prepared ointments: (Left) blank base (F0), (Right) 2% OLE (F1), and 1% OLE (F2). (B) Results of the homogeneity test showing uniform texture and the absence of coarse particles upon smearing on a glass slide.
pH measurement
The ointment (0.5 g) was dispersed in 50 ml of distilled water, and the pH of the suspension was measured using a calibrated digital pH meter (Mettler-Toledo, Switzerland) at room temperature (25℃ ± 1°C) (Shaikh et al., 2018).
Spreadability test
Quantification using a modified slide method under a weight of 20 g. The spreadability (S) was calculated as follows:
S=(M × L) / T
where M is the weight (20 g), L is the distance traveled (6 cm), and T is the time (s) (Navitha et al., 2020).
In vitro diffusion
Preliminary assessment was conducted by packing the ointment (0.5 g) into a well (6 mm) in a nutrient agar plate and incubating at 32°C for 60 minutes (Navitha et al., 2020). The diffusion diameter was then measured.
Washability
The samples were subjected to subjective evaluation by rinsing them with water (Shaikh et al., 2018).
Stability study
Stability testing was conducted for 4 weeks under two storage conditions: (1) refrigerated conditions (2℃ ± 1°C) and (2) room temperature (25℃ ± 2°C, 60% ± 5% relative humidity). The following properties were evaluated weekly: organoleptic properties (color, odor, and texture), homogeneity, pH value, spreadability, and physical stability. Quantitative re-evaluation of these parameters was performed at week 4 and compared with the initial (day 0) values to assess any statistically significant alterations. The stability of all ointment formulations was statistically analyzed using a paired t-test, with a p-value >0.05 considered not significant.
In vivo wound healing studies
Animals and their ethical approval
Seventy-two adult male albino Wistar rats (180–200 g) were obtained from the institutional animal facility and housed under standard conditions (22℃ ± 2°C; 12-h light/dark cycle) with ad libitum access to food and water.
Sampling, randomization, and blinding
After 1 week of acclimatization, we stratified the rats by body weight into blocks to ensure homogeneity. A computer-generated randomization sequence (www.randomizer.org) was employed for all subsequent allocation steps.
Animals were randomly assigned to two main cohorts: the excision wound model (n=24) and the infected wound model (n=48). The infected cohort was further divided into S. aureus-infected (n=24) and P. aeruginosa-infected (n=24) sub-cohorts. Each animal was assigned a unique identification number.
Following wound creation (and infection for relevant groups), the animals were allocated to their pre-defined treatment groups (n=6/group, as detailed in Table 2) by matching their ID numbers to the pre-generated randomization sequence.
Table 2. Experimental design of in vivo wound healing studies.
A randomization list was prepared and maintained by an independent researcher who was not involved in wound induction, treatment, or assessment to ensure allocation concealment. Throughout the study, the personnel responsible for daily wound measurements and clinical observations were blinded to the group assignments. Treatments were performed using identical, unlabeled containers coded only with the animal ID number.
Experimental groups and treatment protocol
Seventy-two adult male albino Wistar rats were assigned to three main wound models: a clean (non-infected) excision wound model, a S. aureus-infected wound model, and a P. aeruginosa-infected wound model. Each model consisted of four experimental groups (n=6 per group) for 12 groups.
The treatment groups were as follows:
Group I (negative control): No treatment was given to non-infected wounds.
Group II (F1): Noninfected wounds treated with 2% OLE ointment.
Group III (F2): Noninfected wounds treated with 1% OLE ointment.
Group IV (positive control): Non-infected wounds treated with Fucidin® ointment (2% fusidic acid).
Group V (negative control): No treatment was given to S. aureus-infected wounds.
Group VI (F1): S. aureus-infected wounds treated with 2% OLE ointment.
Group VII (F2): S. aureus-infected wounds treated with 1% OLE ointment.
Group VIII (positive control): wounds infected with S. aureus treated with Fucidin® ointment.
Group IX (negative control): P. aeruginosa-infected wounds received no treatment.
Group X (F1): P. aeruginosa-infected wounds treated with 2% OLE ointment.
Group XI (F2): P. aeruginosa-infected wounds treated with 1% OLE ointment.
Group XII (positive control): P. aeruginosa-infected wounds treated with Fucidin® ointment.
Topical treatments were applied twice daily using a sterile applicator. Treatment for the excision model commenced immediately after wound creation. Treatment began 24 hours after infection (following confirmation of successful infection) in the infected models.
Wound creation and treatment protocol
Anesthesia and surgery
Following anesthesia (ketamine/xylazine, 80/10 mg/kg, intraperitoneally), the dorsal fur was shaved, and the skin was disinfected. A single, full-thickness circular excisional wound of approximately 154 mm² (diameter ~14 mm) was created in the mid-dorsal region using sterile surgical scissors and forceps. The entire thickness of the skin was removed down to the underlying muscle fascia. This study employed a full-thickness excisional wound model in rats. This study employed a full-thickness excisional wound model in rats, including both clean (non-infected) and infected wound models using S. aureus and P. aeruginosa.
Postoperative analgesia
All animals received a single subcutaneous injection of meloxicam (1 mg/kg) immediately after surgery, which was repeated once daily for the next 48 hours (Hedenqvist and P, 2021).
Treatment
Topical treatments were applied twice daily using a sterile applicator, starting immediately after group assignment for the excision model and 24 hours post-infection for the infected models.
Infected wound model protocol
After standard wound creation, the wound bed was topically inoculated with 10 µl of a standardized bacterial suspension (1.5 × 10⁸ CFU/ml) of either S. aureus or P. aeruginosa. These pathogens were selected based on their high clinical prevalence in wound infections (Sisay et al., 2019; Guan et al., 2021). The inoculum was spread evenly and absorbed for 15 minutes. Twenty-four hours after inoculation, we confirmed successful infection by qualitative culture of wound swabs on Mueller–Hinton agar. Animals with confirmed infections were included in the treatment phase.
Wound measurement and analysis
The wound dimensions were assessed using a dual-method approach. The major and minor axes were measured directly on the animals using sterile calibrated digital calipers, and photographs were taken using a digital camera. A researcher blinded to the treatment groups conducted these measurements.
Wound areas were measured on days 0, 3, 6, 9, 12, 15, 18, and 21 post-wounding (or post-infection for the infected models). Additional measurements were taken on days 13, 14, 19, and 20 as healing progressed. The wound area was calculated using the following formula for an ellipse:
Area=π × (major axis/2) × (minor axis/2)
The percentage of wound closure was then calculated as follows:
% closure=[(A₀ - Aₙ)/A₀] × 100
where: A₀= Initial wound area (mm²) on day 0: Aₙ= Wound area (mm²) on measurement day n.
Statistical analysis
Data are presented as the mean ± standard error of the mean. Intergroup comparisons of wound closure percentages at each time point were performed using two-way analysis of variance with treatment and time as factors, followed by Tukey’s honest significant difference post hoc test for all pairwise multiple comparisons. Statistical analyses were conducted using SPSS software (version 26.0; IBM Corp., USA) and GraphPad Prism (version 10.0; GraphPad Software, USA). Statistical significance was set at p < 0.05.
Ethical approval
All procedures were conducted in strict accordance with institutional guidelines and were approved by the Libyan National Committee for Biosafety and Bioethics (reference number: NBC.007). A. 24.24).
Results
Phytochemical composition of the OLE
Qualitative phytochemical screening revealed that the 75% ethanolic extract of O. europaea leaves was rich in several bioactive secondary metabolites. The extract was positive for phenols, flavonoids, tannins, coumarins, sterols, and triterpenes. No saponins, alkaloids, or anthraquinones were detected under these experimental conditions. The results are summarized in Table 3.
Table 3. Phytochemical screening of O. europaea leaf extract.
In vitro biological activities of crude OLE
Antibacterial activity
The agar well diffusion assay demonstrated the potent and direct antibacterial activity of crude OLE against the primary wound pathogens tested. As shown in Table 4, the extract (100 mg/ml) exhibited strong activity against both S. aureus and P. aeruginosa, with mean inhibition zone diameters of 25.0 ± 0.10 and 21.0 ± 0.01 mm, respectively. According to the adapted interpretative criteria for plant extracts, both values fall within the "Susceptible" (S) range. Notably, OLE demonstrated superior activity against S. aureus compared to the reference antibiotic Fucidin® ointment (22.5 ± 0.50 mm). Against P. aeruginosa, OLE showed significant activity (21.0 ± 0.01 mm), whereas Fucidin® exhibited no inhibition (0.00 ± 0.00 mm), classifying it as resistant. Figure 2 shows representative images of the inhibition zones.
Table 4. In vitro antibacterial activity of O. europaea leaf extract and reference antibiotic.
Cytotoxicity profile
The brine shrimp lethality bioassay indicated a favorable preliminary safety profile for OLE. Minimal lethality was observed at the highest tested concentration (1,000 µg/ml). No mortality was observed at concentrations of 100 and 10 µg/ml. The median LD₅₀ was greater than 1,000 µg/ml, which classified the extract as "non-toxic" according to the standard criteria for this model. In contrast, the positive control (etoposide) exhibited high toxicity, with an LD₅₀ of 7.46 µg/ml. Table 5 presents the results.
Table 5. Cytotoxicity evaluation of O. europaea leaf extract using the brine shrimp lethality bioassay:
Development and characterization of OLE-loaded ointments
Formulation and organoleptic properties
Simple oleaginous ointment base (F0) and medicated formulations containing 1% OLE (F2) and 2% OLE (F1) were successfully prepared using the fusion method. All the formulations were semi-solid and homogeneous, indicating no phase separation. The blank base was yellowish-white, while the incorporation of OLE imparted a characteristic yellowish-green to dark green color and a distinctive herbal odor. Figure 2A shows the macroscopic appearance, and Figure 2B shows the homogeneity test results.
Physicochemical evaluation
Table 6 presents the comprehensive physicochemical profiles of the formulations. All ointments demonstrated properties suitable for topical application.
Table 6. Physicochemical characterization of the ointment formulations.
pH value
The values ranged from 6.41 ± 0.10 to 6.82 ± 0.19, which is in accordance with the physiological skin pH.
Spreadability
The 2% OLE ointment (F1) exhibited the highest spreadability (39.20 ± 0.30 g·cm/s).
Washability
All formulations were easily washed with water.
Stability study
The physical and chemical stabilities of the prepared ointment formulations (F0, F1, and F2) were evaluated over 4 weeks under two storage conditions. Supplementary Tables S1–S3 provide the detailed stability profile, which includes data under both refrigerated (S1) and room temperature (S2) conditions, along with a statistical analysis of the stability parameters (S3).
No statistically significant changes were observed in pH (p > 0.87 for all formulations) or spreadability (p > 0.75 for all formulations) between weeks 4 and 0 under refrigerated storage (2°C). The organoleptic properties remained largely unchanged, with no alteration in color, homogeneity, or phase separation. A slight fading of the herbal odor was noted in OLE-containing formulations (F1 and F2) from week 3 onwards, but this was minimal.
All formulations exhibited high stability at room temperature (25°C). Minimal variations in the pH (<0.2% change) and spreadability (<0.2% change), which were not statistically significant (p >0.43 for all comparisons). The color of the blank formulation (F0) showed slight darkening by week 3, whereas the OLE-loaded formulations maintained their color. Slight fading of the herbal odor was observed in samples F1 and F2 from week 3, similar to that in the refrigerated samples. All formulations remained homogeneous, with no phase separation or signs of microbial growth throughout the study.
Statistical analysis confirmed that the differences between the initial and final pH and spreadability values under both storage conditions were not significant (p > 0.05), indicating robust physical and chemical stability of the ointments over the 4-week period.
Efficacy of in vivo wound healing
Excision (clean) wound model
The 2% OLE ointment significantly accelerated healing, resulting in complete wound closure by day 13. This was statistically earlier than the 1% OLE formulation, which reached full closure on day 14 (p < 0.05 for comparisons at days 12 and 13), and was markedly superior to both the Fucidin® group (day 18; p < 0.01) and the untreated control (day 21; p < 0.001). The temporal progression of wound closure percentages is detailed in Table 7, and the macroscopic progression of healing is illustrated in Fig. 4. An area under the curve (AUC) analysis is provided in Supplementary Table S4 for a quantitative assessment of overall healing efficacy across the entire observation period.
Table 7. Percentage wound contraction in the excision wound model (Mean ± S.E.M).
Fig. 4. Gross morphological progression of wound healing in a rat excision wound model treated with O. europaea leaf extract (OLE) ointments.
Staphylococcus aureus infection wound model
In S. aureus-infected wounds, both OLE ointments significantly accelerated healing compared with the untreated infected control (p < 0.001 for both formulations). A concentration-dependent response was observed: the 1% OLE formulation (F2) demonstrated superior early wound closure, achieving 80.00% ± 0.08% by day 6 compared to 66.66% ± 1.08% for the 2% OLE formulation. A statistically significant difference in a direct intergroup comparison (p < 0.05). The 1% OLE ointment achieved complete wound closure (100%) by day 12, whereas the 2% formulation achieved full closure by day 13. Both OLE treatments outperformed the Fucidin®-treated groups, which required 18 days for complete healing (Table 9, Fig. 5). The clinical signs of infection (edema and exudate) resolved more rapidly in OLE-treated animals. Supplementary Tables S5 and S6 present detailed statistical analyses of pairwise group comparisons and AUC assessment for cumulative wound healing in this model, respectively.
Table 9. Temporal progression of wound contraction in a S. aureus-infected excision wound model.
Fig. 5. Gross morphological progression of wound healing in a S. aureus-infected rat model treated with O. europaea leaf extract (OLE) ointments.
Pseudomonas aeruginosa infection wound model
Similarly, OLE ointments demonstrated potent therapeutic activity in P. aeruginosa-infected wounds. The 1% OLE formulation showed significantly better closure rates at intermediate time points (days 9 and 12, p < 0.05) than the 2% formulation, despite achieving 100% closure by day 13. The Fucidin® group healed more slowly, with complete closure on day 20. The infected control group exhibited markedly delayed healing and failed to achieve full closure within the 21-day observation period. The results are summarized in Table 10 and Figure 6. Comprehensive statistical evaluation and AUC analysis of P. aeruginosa-infected models are presented in Supplementary Tables S7 and S8.
Table 10. Temporal progression of wound contraction in a P. aeruginosa-infected excision wound model.
Fig. 6. Gross morphological progression of wound healing in a rat model of P. aeruginosa infection treated with O. europaea leaf extract (OLE) ointments.
Discussion
This study provides compelling evidence for the therapeutic potential of a topical ointment formulated from OLE for wound management. The choice of Fucidin® (2% fusidic acid) as a positive control was strategically based on (1) its proven clinical efficacy against S. aureus, the primary pathogen in one of our infection models, (2) its status as a first-line topical antibiotic in dermatological practice, and (3) the 2% concentration matched our OLE formulation, enabling a direct, concentration-matched efficacy comparison. This design allowed for a clinically meaningful assessment of OLE performance relative to established standard care.
The superior wound closure kinetics observed with the 2% OLE ointment, particularly its outperformance of the standard antibiotic Fucidin® in the excision model (Table 7, Fig. 4), suggest that OLE possesses intrinsic tissue-regenerative properties beyond antimicrobial action. In vitro testing of Fucidin® ointment revealed a strong inhibitory effect against S. aureus (22.5 mm), consistent with its known spectrum, but no activity against P. aeruginosa, which further explains its relatively slower healing in the Pseudomonas-infected model compared with the broad-spectrum OLE ointment. This aligns with the known pleiotropic bioactivities of olive polyphenols, such as oleuropein, which enhances fibroblast proliferation and collagen deposition in vitro (Boss et al., 2016; Liu et al., 2022). The finding that the 2% formulation excelled in clean wounds, whereas the 1% formulation showed superior early activity in infected models, points to a concentration-dependent modulation of distinct biological pathways. This may indicate that effective antimicrobial saturation is achieved at approximately 1% concentration for membrane disruption and anti-biofilm activity against the tested pathogens (Elhrech et al., 2024; Dermeche et al., 2025), whereas a higher concentration (2%) is required to maximally stimulate cellular proliferation and tissue remodeling processes (Boss et al., 2016).
The ability of OLE ointments, particularly the 2% formulation, to significantly enhance healing in wounds deliberately infected with both S. aureus and P. aeruginosa (Tables 8 and 9) underscores a critical dual advantage: robust antimicrobial activity coupled with direct tissue healing promotion. This broad-spectrum efficacy aligns with recent studies that have demonstrated the capacity of olive polyphenols to disrupt bacterial membranes and inhibit biofilm formation (Dermeche et al., 2025). This multi-targeted action profile simultaneously addresses microbial burden and impaired healing, positioning OLE as a potentially more advantageous therapeutic agent than single-target antibiotics, such as Fucidin®, in complex wound environments (Muteeb et al., 2023).
Table 8. Statistical analysis of OLE ointment efficacy in the non-infected excision wound model.
The significant acceleration of wound healing observed in this study aligns with and extends previous research on O. europaea. A direct comparative summary of OLE ointment performance against S. aureus and P. aeruginosa is listed in Supplementary Table S9.
Our phytochemical profile (Table 3) corroborates the typical composition of (Boss et al., 2016; Elhrech et al., 2024). The in vitro antibacterial results, particularly the superior activity against S. aureus (25.0 mm inhibition zone) compared to standard antibiotics (Table 4), are consistent with the efficacy of oleuropein against Gram-positive pathogens. Notably, our finding of substantial activity against P. aeruginosa (21.0 mm) at 100 mg/ml adds a valuable dimension, as many prior reports have focused primarily on Gram-positive bacteria or required higher concentrations for Gram-negative inhibition (El-Saadony et al., 2025). Regarding in vivo efficacy, our 2% OLE ointment achieved complete wound closure by day 13 in the excision model, outperforming both the timeline reported by Al-Warhi et al. (2022) (days 15–16) and the standard antibiotic Fucidin® (day 20) in a direct, head-to-head comparison.
The key advances presented here include: (1) the formulation and characterization of a stable ointment at two clinically relevant concentrations (1% and 2%); (2) a direct, concentration-matched efficacy comparison with a standard topical antibiotic (Fucidin®), revealing the superiority of OLE in tissue regeneration; and (3) the concurrent demonstration of potent, broad-spectrum in vitro antimicrobial activity and accelerated in vivo healing in infected wounds. This integrated approach bridges the gap between documented phytochemistry and comprehensive therapeutic validation.
Based on this comparative analysis, the superior performance of the 2% OLE ointment can be attributed to the fundamental differences in its mechanism of action compared with Fucidin®. Fucidin® acts primarily as a single-target protein synthesis inhibitor with a narrow spectrum and is largely inactive against Gram-negative bacteria like P. aeruginosa. Its role in wound healing is indirect. In contrast, OLE ointment embodies a multi-target, pleiotropic strategy: (1) broad-spectrum antimicrobial action likely through membrane disruption, (2) potent antioxidant and anti-inflammatory activities that mitigate oxidative stress, and (3) direct pro-proliferative and pro-angiogenic effects on repair cells (Boss et al., 2016; Liu et al., 2022; Dermeche et al., 2025).
Specifically, olive polyphenols, such as oleuropein, have been reported to enhance fibroblast proliferation and migration, increase collagen synthesis and deposition, modulate inflammatory cytokines (reducing IL-1β and TNF-α), promote angiogenesis through vascular endothelial growth factor (VEGF) upregulation, and exhibit potent antioxidant activity that reduces oxidative stress in the wound bed (Boss et al., 2016; Liu et al., 2022; Dermeche et al., 2025). This combination allows OLE to simultaneously attack infection, modulate the wound microenvironment, and actively promote tissue repair, which explains its superiority over a pure antibiotic in clean wounds and its high efficacy in infected wounds.
All formulations fall within the physiological pH range for the skin (approximately 4.5–7.0) and are notably close to neutral pH, which is beneficial for wound healing, as chronic wounds often have an alkaline pH that impairs healing. The 2% formulation (pH 6.82) is slightly more alkaline than the 1% formulation (pH 6.58); however, both remain well within the acceptable range and are unlikely to cause tissue damage induced by pH. However, this subtle pH difference could influence bacterial growth kinetics, enzyme activity, or cell behavior in the infected wound microenvironment, potentially contributing to the differential early-stage efficacy observed between the two formulations.
Furthermore, certain phenolic compounds may exhibit pro-oxidant rather than antioxidant effects at higher concentrations, potentially creating transient oxidative stress that could delay early healing events in the infected wound environment. This concentration-dependent shift from antioxidant to pro-oxidant activity has been documented for various polyphenols and may explain the biphasic dose-response observed in our infected wound models, where the 1% formulation provided optimal early healing compared with the 2% formulation. These hypotheses warrant dedicated investigation through dose-response studies across a wider concentration range with accompanying mechanistic endpoints.
Specific cellular and molecular mechanisms (e.g., fibroblast proliferation, collagen synthesis, and angiogenesis) were not directly measured in our study. The significant wound-healing activity observed provides a compelling rationale for future mechanistic investigations employing histological techniques (e.g., Masson’s trichrome staining) and molecular assays (e.g., quantification of TGF-β and VEGF) to validate and elucidate the precise pathways involved.
The promising in vitro cytotoxicity profile, in which the OLE extract was classified as non-toxic (LD₅₀ > 1,000 µg/ml) in the brine shrimp lethality bioassay (Table 5), provides preliminary evidence of a wide safety margin for its bioactive constituents. Furthermore, all animals were monitored daily for signs of local intolerance throughout the 21-day in vivo study period. No visible signs of irritation, erythema, or edema were observed. No visible signs of irritation, erythema, edema, itching, or sensitization were observed at the application sites in any of the treatment groups, including the 2% OLE formulation. The wounds in the OLE-treated groups exhibited healthy granulation tissue without excessive inflammation or adverse reactions. However, we acknowledge that formal skin irritation testing (e.g., the Draize test) and comprehensive histopathological examination of periwound tissue would further strengthen the safety profile and are recommended for future investigations to support clinical translation.
Although this study provides strong evidence for the wound-healing efficacy of OLE ointment, several methodological and mechanistic limitations must be acknowledged. The use of a non-standardized crude extract without quantitative phytochemical profiling via High-Performance Liquid Chromatography/Liquid Chromatography-Mass Spectrometry to identify key bioactive constituents, coupled with the absence of systematic pharmaceutical characterization (e.g., solubility, critical quality control parameters, and contact angle measurements for wettability), limits the formulation’s reproducibility and translational potential. Mechanistically, the lack of histopathological analysis, quantification of inflammatory mediators and angiogenesis markers, gene expression profiling, and advanced computational modeling (e.g., GCMC simulations of polyphenol–membrane interactions) restricts insights into the underlying biological pathways. Furthermore, the experimental models employing single-pathogen challenges in healthy rats and only two concentrations did not fully replicate the complex polymicrobial, biofilm-rich environment of chronic human wounds or define a complete therapeutic window; moreover, they did not explore the nano-formulation’s potential for enhanced delivery.
To address these gaps, we propose a multifaceted future agenda: (1) implementing HPLC/LC-MS-based phytochemical standardization; (2) incorporating advanced formulation science, including contact angle analysis and nano-delivery system development; (3) utilizing computational approaches (e.g., GCMC simulations) along with molecular techniques to elucidate mechanisms; and (4) expanding preclinical validation through broader concentration ranges and complex chronic wound models (e.g., diabetic rats with polymicrobial/biofilm infections). These analyses would confirm whether the accelerated closure observed with OLE treatment corresponds to histologically superior healing with well-organized collagen deposition and appropriate inflammatory resolution. This integrated, multi-scale strategy will bridge phytochemical composition with biological activity, transforming this promising lead into a mechanism-defined, optimized, and clinically viable therapeutic for advanced wound management.
Conclusion
This study provides strong preclinical evidence that a stable and well-characterized 2% topical ointment derived from OLE is an effective multi-target therapeutic agent for wound management, with significant antimicrobial activity, especially against S. aureus, as well as antioxidant, anti-inflammatory, and tissue-regenerative properties. The formulation not only outperformed standard antibiotic treatment in a clean-wound model but also maintained excellent physicochemical stability over 4 weeks under both refrigerated and room temperature conditions. These findings validate the traditional use of olive leaves and position 2% OLE ointment as a promising natural alternative in wound care, particularly in the context of AMR. Future work should focus on isolating key bioactive compounds, elucidating their molecular mechanisms, conducting long-term stability and safety studies, and evaluating their efficacy in chronic wound models to advance their clinical applications.
Acknowledgments
The authors extend their sincere gratitude to the Department of Biomedical Science and Pharmaceutics, Faculty of Pharmacy, Omar Al-Mukhtar University, for providing the necessary laboratory facilities and infrastructure to conduct this study. The authors would like to thank the Libyan National Committee for Biosafety and Bioethics for expedited review and ethical approval of the study protocol.
Conflict of interest
The authors have no conflicts of interest to declare.
Funding
This study received no external funding.
Authors' contributions
Conceptualization, A.S.K. and T.I.S.; Methodology, A.S.K., T.I.S., and Y.A.; Software, N.M.B. and S.O.B.; Validation, Y.A., N.A.N.R., and S.E.A.; Formal Analysis, A.S.K. and N.M.A.; Investigation, N.M.B., N.A.N.R., S.E.A., S.O.B., B.S.J., and N.M.A.; Resources, A.S.K. and T.I.S.; Data Curation, N.M.B. and S.O.B.; Writing – Original Draft Preparation, A.S.K., N.A.N.R., and S.E.A.; Writing – Review & Editing, T.I.S., Y.A., B.S.J., and N.M.A.; Visualization, S.O.B.; Supervision, A.S.K. and T.I.S.; Project Administration, A.S.K.; Funding Acquisition, A.S.K. All authors have read and agreed to the published version of the manuscript.
Data availability
The data supporting the findings of this study are available upon reasonable request from the corresponding author.
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Supplementary Materials
Part 1: Physicochemical stability profile of formulations
Supplementary Table S1. Stability at refrigerated conditions (2℃ ± 1°C)
Supplementary Table S2. Stability at room temperature (25℃ ± 2°C, 60% ± 5% RH).
Supplementary Table S3. Statistical analysis of stability data - comparison of week 4 versus Day 0 values.
Part 2: Wound healing efficacy and antimicrobial activity data
Supplementary Table S4. AUC analysis for the excision wound model.
Supplementary Table S5. Comprehensive statistical analysis of wound healing efficacy in S. aureus-infected model.
Supplementary Table S6. AUC analysis of cumulative wound healing in S. aureus-infected model.
Supplementary Table S7. Comprehensive statistical analysis of wound healing efficacy in P. aeruginosa-infected model.
Supplementary Table S8. AUC analysis of cumulative wound healing in P. aeruginosa-infected model.
Supplementary Table S9. Comparative efficacy of OLE ointments against different bacterial pathogens.
