Assessment of antimicrobial activity of essential oils using agar well diffusion method
The antimicrobial activity of six essential oils, viz eugenol, rosemary, rosehip, lavender, clary sage, and lemongrass were evaluated using the agar well diffusion method against selected microbial strains. Zones of inhibition (ZOI) were measured in millimetres after a 24-hour incubation at 37 °C. All tests were performed in triplicate (n = 3), and the results are expressed as mean ± standard deviation (Table 1). Each essential oil was tested at concentrations of 10, 25, 50, 75, and 100 µg/mL. A dose-dependent increase in antimicrobial activity was observed, with higher concentrations yielding larger zones of inhibition. Eugenol demonstrated the highest overall efficacy, showing strong inhibitory activity across all tested organisms, including polymicrobial cultures (25.0 ± 0.8 mm at 100 µg/ml). It also exhibited large inhibition zones against K. pneumoniae (23.0 ± 0.8 mm), E. coli (21.7 ± 0.5 mm), and A. baumannii (21.3 ± 0.5 mm), likely due to its phenolic structure that disrupts bacterial membranes and interferes with cellular processes.

Table (1):
Antimicrobial activity of individual essential oils against clinically relevant bacterial pathogens assessed by the agar well diffusion method

EU: Eugenol, RM: Rosemary oil, RH: Rosehip oil, LV: Lavender oil, CS: Clary sage oil, LG: Lemongrass oil, PC: Positive Control

Rosemary oil showed significant antimicrobial activity, particularly against MRSA (22.7 ± 0.5 mm), E. coli (21.3 ± 0.9 mm), and A. baumannii (22.3 ± 0.5 mm). Its bioactivity is attributed to components like 1,8-cineole and a-pinene.⁶ Rosehip oil displayed moderate activity, especially against E. coli (22.3 ± 1.2 mm) and K. pneumoniae (22.0 ± 0.8 mm), with slightly reduced efficacy against P. aeruginosa and A. baumannii. Its effects are possibly linked to its high content of polyunsaturated fatty acids and antioxidants.¹⁶ Lavender oil was effective against both Gram-negative and Gram-positive strains, notably E. coli (23.0 ± 0.8 mm), A. baumannii (22.3 ± 0.9 mm), and MRSA (20.7 ± 0.9 mm), attributed to its main constituents namely linalool and linalyl acetate.^6,18 Clary sage exhibited moderate efficacy against monocultures such as MRSA (22.0 ± 0.8 mm) and A. baumannii (21.0 ± 0.8 mm) but showed no inhibition against polymicrobial cultures at concentrations above 25 µg/ml, suggesting potential antagonistic effects or degradation in complex environments.²¹ Lemongrass oil showed selective activity, particularly against MRSA (21.3 ± 1.2 mm), S. pyogenes (21.3 ± 0.5 mm), and E. coli (21.7 ± 0.5 mm), while its effect on polymicrobial cultures was relatively weak (17.3 ± 0.5 mm at 100 µg/ml). This may be due to the variable stability or efficacy of citral, its principal active compound.¹⁷

Among the tested essential oils, eugenol exhibited the most potent and consistent broad-spectrum antimicrobial activity, including against polymicrobial cultures, likely due to its phenolic structure that disrupts bacterial membranes and cellular functions. Rosemary oil also showed strong efficacy, particularly against multidrug-resistant strains, attributable to bioactive monoterpenes such as 1,8-cineole and a-pinene. Lavender and rosehip oils demonstrated moderate, strain-dependent activity, reflecting their mixed phytochemical composition and indirect antimicrobial mechanisms. In contrast, clary sage and lemongrass oils displayed selective or reduced efficacy in polymicrobial settings, possibly due to limited stability or antagonistic interactions in complex microbial environments. Overall, these findings highlight the importance of selecting essential oils based on infection type and microbial complexity rather than relying on uniform antimicrobial performance.

Determination of minimum inhibitory concentration of essential oils
The minimum inhibitory concentrations (MICs) of six essential oils, viz, eugenol, rosemary, rosehip, lavender, clary sage, and lemongrass were evaluated against selected clinically relevant pathogens using the broth microdilution method, with a concentration range from 1000 to 1 µg/mL. The MIC values are presented in Table 2.

Table (2):
Minimum Inhibitory Concentration (MIC) of different Essential Oils against Test Microorganisms

The results revealed notable variations in antimicrobial efficacy among the oils. Eugenol demonstrated the most potent and consistent activity, exhibiting MIC values of 62.5 µg/mL against MRSA, Streptococcus pyogenes, and E. coli, and 125 µg/mL against A. baumannii, P. aeruginosa, K. pneumoniae, and polymicrobial cultures. This broad-spectrum effectiveness can be attributed to eugenol’s phenolic structure, which enables it to disrupt bacterial membranes, inhibit enzymes, and interfere with quorum sensing.

Rosemary oil displayed comparable antimicrobial potency, with MICs of 62.5 µg/mL for MRSA, Streptococcus, E. coli, P. aeruginosa, and K. pneumoniae, and 125 µg/mL for A. baumannii and polymicrobial cultures. Its efficacy is likely due to active constituents such as 1,8-cineole, camphor, and a-pinene, which are known to compromise microbial cell wall integrity and metabolic processes. Lemongrass oil showed selective activity, with MICs of 62.5 µg/mL against MRSA, 125 µg/mL against P. aeruginosa, and 250 µg/mL for the remaining organisms. The presence of citral, a key bioactive compound, may explain its effectiveness, although reduced efficacy against certain strains suggests limitations in complex infections.

Lavender oil exhibited variable antimicrobial activity, with MIC values ranging from 125 µg/mL (MRSA, Pseudomonas) to 500/ µg/mL (E. coli). Its inconsistent performance may stem from variability in its phytochemical composition and relatively lower concentrations of antimicrobial constituents like linalool and linalyl acetate, or possible interference by antagonistic components. Rosehip oil showed uniform inhibitory effects at 250 µg/mL for most pathogens, with reduced efficacy against K. pneumoniae (MIC: 500 µg/mL). Despite the higher MICs, its activity across a wide range of organisms suggests potential therapeutic value, possibly owing to its rich profile of flavonoids, phenolic acids, and volatile compounds.²² Clary sage oil demonstrated the weakest antimicrobial activity, with MICs of 250 µg/mL against MRSA, Streptococcus, E. coli, and A. baumannii, and 500 µg/mL for P. aeruginosa, K. pneumoniae, and polymicrobial cultures. The diminished performance may reflect either a low concentration of active agents or the presence of constituents that counteract antimicrobial action.^6,18

Among the tested strains, P. aeruginosa generally showed higher resistance, consistent with its well-established intrinsic mechanisms such as efflux pumps and biofilm formation. Interestingly, despite the complexity of polymicrobial cultures, eugenol and rosemary oil maintained appreciable activity, indicating their potential in managing mixed-pathogen infections.^18,23 The lower MIC values observed for eugenol and rosemary oil against MDR organisms indicate high antimicrobial potency at reduced concentrations, which is clinically advantageous for minimizing toxicity, lowering effective doses, and reducing resistance pressure. Their sustained activity against polymicrobial cultures further supports their potential translational application as adjunct or alternative agents in managing complex and drug-resistant infections. In summary, the MIC findings confirm the superior antimicrobial efficacy of eugenol, followed closely by rosemary oil, across a spectrum of Gram-positive and Gram-negative bacteria. The differential susceptibility patterns highlight the need for careful selection of essential oils based on the target organism and the intended clinical application.

Determination of antioxidant potential of essential oils by dpph scavenging activity
The antioxidant potential of the selected essential oils and their combination was assessed using the DPPH radical scavenging assay. The IC₅₀ values, representing the concentration required to inhibit 50% of DPPH radicals, were calculated for each group (n = 3), and the results are summarized in Figure 1. Statistical analysis using one-way ANOVA indicated significant differences among the treatment groups (F_7,16 = 121.271, p < 0.001). Levene’s test confirmed homogeneity of variances (F_7,16 = 0.873, P = 0.548).

The mean IC₅₀ values (µg/mL ± SD) were as follows: Combination (11.78 ± 0.70), Control (18.39 ± 0.56), Eugenol (19.12 ± 1.08), Rosemary (19.59 ± 0.75), Lavender (20.92 ± 0.79), Rosehip (21.78 ± 0.67), Clary sage (24.58 ± 1.30), and Lemongrass (33.61 ± 1.52). The overall mean IC₅₀ across treatments was 21.22 ± 5.98/ µg/mL, with values ranging from 11.05 to 34.92/ µg/mL.

Post hoc analysis using Tukey’s HSD test revealed five distinct homogeneous subsets, confirming that the combination treatment had significantly greater antioxidant activity than all other groups (P < 0.001). Lemongrass exhibited the weakest antioxidant potential. Based on IC₅₀ values, the antioxidant potency of the treatments can be ranked as:

Combination > Control > Eugenol > Rosemary > Lavender > Rosehip > Clary sage > Lemongrass.

Further comparisons using Fisher’s LSD test supported most pairwise differences and identified additional significant contrasts not captured by the more conservative Tukey’s test. These findings underscore the substantial variability in antioxidant capacity among the tested essential oils. Eugenol and rosemary oils demonstrated relatively strong antioxidant activity, with IC₅₀ values of 19.12 µg/mL and 19.59 µg/mL, respectively. Eugenol, a phenolic compound abundant in clove oil, is well-known for its ability to donate hydrogen atoms via its hydroxyl group, thereby neutralizing free radicals.²⁴ Similarly, rosemary essential oil contains potent antioxidants such as rosmarinic acid, carnosic acid, and other phenolic diterpenes that enhance its radical scavenging capability.²⁵

Lavender (IC₅₀ = 20.92 µg/mL) and rosehip oil (21.78 µg/mL) exhibited moderate antioxidant activity, consistent with their reported phenolic content and traditional medicinal use. These oils are rich in monoterpenes and select phenolic compounds, though at lower levels than those found in eugenol or rosemary.²⁶ On the other hand, clary sage (24.58 µg/mL) and lemongrass (33.61 µg/mL) showed comparatively weaker antioxidant performance. The lower activity of Lemongrass can be attributed to its citral-based composition, which, although possessing antioxidant properties, is less effective than phenolic constituents in scavenging free radicals. The reduced antioxidant potential of these oils likely stems from their chemical profiles, which are dominated by monoterpene hydrocarbons rather than phenolic structures.²⁰

Notably, the combination of essential oils displayed the lowest IC₅₀ value (11.78 µg/mL), indicating synergistic antioxidant effects. This superior activity suggests that blending essential oils may enhance radical scavenging through complementary or additive mechanisms involving multiple bioactive compounds. Previous studies have reported similar synergism in essential oil mixtures, where the total antioxidant activity exceeds that of the individual components.²⁷ In conclusion, the DPPH assay results indicate that essential oils vary significantly in their antioxidant capacities, influenced by their phytochemical composition. While eugenol and rosemary show strong individual performance, combining oils may offer an effective strategy to maximize antioxidant potential for therapeutic applications.

Determination of anti-inflammatory activity by hrbc membrane stabilization assay
The anti-inflammatory activity of the selected essential oils and their combination was evaluated using the human red blood cell (HRBC) membrane stabilization assay. The IC₅₀ values, representing the concentration required to inhibit 50% hemolysis, were determined for each treatment group (n = 3). The results are presented in Figure 2, with statistical analysis conducted using one-way ANOVA followed by Tukey’s HSD post hoc test.

The mean IC₅₀ values (µg/mL ± SD) ranged from 28.19 ± 0.75 µg/mL (Combination) to 49.67 ± 0.56 µg/mL (Clary sage). The control group exhibited an IC₅₀ of 28.48 ± 0.62 µg/mL, indicating that lower IC₅₀ values correlated with higher membrane stabilization potential and, hence, greater anti-inflammatory efficacy. Levene’s test confirmed the assumption of homogeneity of variances (F_7,16 = 0.854, P = 0.561), validating the ANOVA, which revealed highly significant differences among the treatments (F_7,16 = 386.778, P < 0.001), with an effect size of ח² = 0.994, indicating that 99.4% of the variance was due to treatment differences. Tukey’s HSD post hoc test identified five homogeneous subsets. The Combination treatment (28.19 µg/mL) and Control (28.48 µg/mL) formed one subset with no significant difference (P = 1.000), suggesting equivalent membrane-stabilizing activity. Eugenol (36.18 µg/mL), Rosemary (38.67 µg/mL), and Lemongrass (41.70 µg/mL) formed distinct subsets, demonstrating moderate potency. The least effective treatments were Rosehip (43.11 µg/mL), Lavender (47.94 µg/mL), and Clary sage (49.67 µg/mL), which required higher concentrations to achieve comparable stabilization effects.

These findings highlight significant variability in the anti-inflammatory properties of essential oils. Among the individual treatments, Eugenol exhibited the highest potency, likely due to its phenolic hydroxyl group enabling efficient interaction with phospholipid bilayers through hydrogen bonding and hydrophobic interactions. These mechanisms stabilize the red blood cell membrane and reduce hemolysis, aligning with eugenol’s known anti-inflammatory and antioxidant properties.²⁸

Rosemary and Lemongrass showed moderate efficacy. Their activity is attributed to monoterpenes such as 1,8-cineole and citral, respectively, which protect cellular membranes by reducing lipid peroxidation and protein denaturation.^9,17 In contrast, Clary sage, Lavender, and Rosehip demonstrated weaker membrane-stabilizing capacity. Their higher IC₅₀ values suggest either a lower concentration of effective bioactives or different modes of action that are less suited to direct membrane stabilization.¹⁶ Notably, the combination treatment, although yielding the lowest mean IC₅₀ (28.19 µg/mL), was statistically indistinguishable from the control. This suggests that anticipated synergistic effects were absent. Instead, antagonistic interactions or dilution effects within the multi-component blend may have masked the individual potencies of its constituents, a finding contrary to previous expectations for enhanced efficacy in essential oil mixtures.

The substantial F-statistic and high effect size strongly indicate that treatment type has a decisive impact on anti-inflammatory activity, with minimal variability within groups. These results underscore the therapeutic promise of Eugenol, Rosemary, and Lemongrass as effective membrane stabilizers and support their further exploration in inflammation models. Additionally, the findings warrant deeper investigation into the dose-dependent interactions in essential oil combinations to optimize formulations for synergistic rather than antagonistic outcomes.

Phytochemical profiling of essential oil by gc-ms analysis: insights into bioactive constituents
Gas Chromatography-Mass Spectrometry (GC-MS) analysis was employed to identify and quantify the chemical constituents of the essential oil sample. A total of 40 compounds were detected, encompassing a wide range of monoterpenes, sesquiterpenes, oxygenated derivatives, and fatty acid esters, with retention times spanning from 3.594-47.175 minutes. The identified compounds, along with their retention times and relative peak area percentages, are detailed in Table 3 and visualized in the chromatogram in Figure 3.

Table (3):
GC-MS Profile of Essential Oil Constituents

The analysis revealed a complex and pharmacologically rich composition, with eucalyptol (1,8-cineole) being the most abundant compound at 30.22% (RT = 8.247 min), followed by a-pinene at 26.17% (RT = 5.38 min). Together, these two monoterpenes accounted for over 56% of the total oil content, signifying their central role in the oil’s bioactivity. Other notable constituents included isoborneol (10.36%), eugenol (7.05%), camphene (4.96%), and β-pinene (3.89%). Minor constituents comprised D-limonene (2.74%), 3-cyclohexene-1-methanol (2.70%), linalool (1.17%), p-cymene (1.44%), and (+)-3-carene (0.91%). Fatty acids such as oleic acid (0.57%) and 6-octadecenoic acid (0.61%) were also identified, indicating the presence of lipid-derived components.

The dominance of eucalyptol is particularly significant, given its well-established antimicrobial, anti-inflammatory, and mucolytic activities, which make it a promising agent for wound healing and respiratory applications.²⁹ a-Pinene, the second major constituent, is a monoterpene hydrocarbon known for its broad-spectrum antimicrobial and anti-inflammatory effects, and its ability to enhance skin permeability, thus improving the topical delivery of active compounds.

Isoborneol, a monoterpenoid alcohol (10.36%), contributes to the oil’s antiviral and antibacterial properties, while eugenol (7.05%) a phenolic compound commonly found in clove oil adds antioxidant, antimicrobial, and analgesic properties.⁶ Additional compounds such as camphene, b-pinene, and D-limonene are known for promoting transdermal penetration and supporting tissue regeneration.^24,27

The oxygenated monoterpenes, which form the most pharmacologically significant fraction, are dominated by eucalyptol, followed by camphor (0.44%) and isoborneol. These components are associated with anti-inflammatory, bronchodilatory, and analgesic activities. Linalool (1.17%) and linalyl acetate (0.31%), although present in smaller amounts, are known to exhibit antibacterial, sedative, and antioxidant effects.¹¹ The identification of bornyl acetate (0.25%) also suggests a contribution to both aroma and therapeutic functionality.

Phenolic compounds, particularly eugenol, were identified at two retention times (0.44% and 7.05%), reaffirming its key role in the oil’s synergistic antimicrobial and analgesic effects.¹ Despite its lower abundance, the presence of caryophyllene (0.09%), a sesquiterpene, adds further value due to its anti-inflammatory and cannabinoid receptor-binding capabilities.

The detection of higher molecular weight constituents such as stigmast-5-en-3-ol (0.43%) – a phytosterol – suggests potential for wound healing, skin barrier repair, and improved oil stability.³⁰ Similarly, fatty acids like oleic acid (0.57%) not only stabilize the oil but may also enhance penetration and bioavailability of active constituents.³¹

Overall, the GC-MS chemical fingerprint illustrates a well-balanced profile dominated by eucalyptol and a-pinene, complemented by a diverse range of bioactive constituents with known antimicrobial, antioxidant, anti-inflammatory, and transdermal-enhancing effects. The combination of these compounds likely contributes to the observed synergistic therapeutic potential, supporting the oil’s traditional uses and its application in modern medicinal and topical formulations.

Synergistic antimicrobial efficacy of combinatorial essential oils using agar disc diffusion assay
The antimicrobial efficacy of a combinatorial essential oil (EO) formulation was evaluated against seven clinically significant bacterial strains using the agar disc diffusion method. Zones of inhibition were measured in millimetres and compared to control treatments, with results summarized in Figure 4. Statistical significance was determined using two-way ANOVA followed by Tukey’s post hoc test (P < 0.05). The EO combination exhibited significantly enhanced antimicrobial activity compared to controls for A. baumannii (24.67 ± 0.57 mm vs. 20.67 ± 0.57 mm), E. coli (24.00 ± 1.00 mm vs. 21.33 ± 1.15 mm), K. pneumoniae (24.00 ± 1.00 mm vs. 21.67 ± 0.57 mm), and MRSA (22.67 ± 1.15 mm vs. 20.67 ± 0.57 mm). These differences were statistically significant (P < 0.05), confirming the superior antibacterial potential of the EO blend.

Although increases in inhibition zones were also observed for P. aeruginosa, S. pyogenes, and polymicrobial cultures, these were not statistically significant (p > 0.05), likely due to greater response variability or inherent resistance mechanisms. The order of microbial susceptibility to the EO combination was as follows: A. baumannii > E. coli > K. pneumoniae = P. aeruginosa > MRSA = S. pyogenes > Polymicrobial. The two-way ANOVA confirmed that both treatment type and microbial strain had significant effects on antimicrobial outcomes (p < 0.05), with robust interaction effects validating the influence of EO combinations across multiple pathogens.

These findings demonstrate that the EO combination offers a broad-spectrum antimicrobial effect, particularly potent against multidrug-resistant Gram-negative bacteria, such as A. baumannii and E. coli, as well as the Gram-positive MRSA strain. This enhanced efficacy likely results from synergistic interactions among key bioactive components including eugenol, 1,8-cineole, geraniol, linalool, and citral, which are known to disrupt bacterial membranes, interfere with energy metabolism, and inhibit biofilm formation.³²

Consistent with prior studies,^3,28 the combination of phenolic-rich and monoterpene-based essential oils appears to result in additive or synergistic effects, improving efficacy over individual oil applications. In the case of A. baumannii, a pathogen known for efflux pump mechanisms and membrane impermeability, the EO combination may enhance permeability and exert cumulative stress on microbial targets.⁸

The enhanced antimicrobial activity observed with essential oil combinations underscores the importance of synergistic interactions in combating multidrug-resistant pathogens. Although only modest improvements were noted against highly resilient organisms such as Pseudomonas aeruginosa and polymicrobial cultures, these trends suggest that formulation limitations may restrict efficacy rather than intrinsic antimicrobial potential. Future formulation strategies offer promising avenues to overcome these constraints.³³ Nanoencapsulation methods, including polymeric nanoparticles, lipid-based nanocarriers, and nanoemulsions, can improve EO stability, protect volatile bioactive compounds from degradation, improve aqueous dispersibility, and enable sustained release at the site of infection. These systems can also enhance penetration across bacterial membranes and biofilms, which are major contributors to resistance in MDR pathogens. Additionally, integration of EOs into hydrogel-based delivery platforms could enable localized, controlled release while maintaining effective concentrations over prolonged periods, particularly for topical applications such as wound infections, burns, and diabetic ulcers. Hydrogels may further support synergistic interactions between EO components and improve biocompatibility and patient compliance. Therefore, the observed synergistic antimicrobial trends, combined with advances in nanotechnology and biomaterial-based delivery systems, support the future development of EO-based formulations as adjunct or alternative therapies for managing resistant and polymicrobial infections.