Development and Evaluation of Essential Oil-Enriched Topical Hydrogels for Antimicrobial Activity Against Anaerobic Pathogens Associated with Gas Gangrene

Gas gangrene is a severe and rapidly progressing necrotizing infection characterized by extensive tissue destruction, gas production, and systemic toxicity. The disease is primarily associated with anaerobic bacterial pathogens such as Clostridium perfringens and Clostridium septicum, which produce potent exotoxins capable of causing rapid myonecrosis and vascular damage. The condition frequently develops in traumatic wounds, ischemic tissues, or post-surgical infections where oxygen tension is significantly reduced, creating a favourable environment for clostridial proliferation (Ghanouni et al., 2022; Huang et al., 2022; Jia et al., 2025; Wu et al., 2025).

Despite advances in antimicrobial therapy and surgical management, gas gangrene remains associated with considerable morbidity and mortality. Conventional treatment strategies primarily involve aggressive surgical debridement combined with systemic antibiotic therapy. However, several limitations are associated with these approaches. Antibiotic penetration into necrotic tissue is often inadequate due to impaired vascularization, while the anaerobic environment reduces the effectiveness of many antimicrobial agents. Furthermore, the emergence of antimicrobial resistance and biofilm-associated infections has complicated the management of anaerobic wound infections (Huang et al., 2022; Jia et al., 2025; Woog & Destro, 2022; Wu et al., 2025).

Biofilm formation represents a major challenge in chronic and necrotizing infections. Biofilms are structured microbial communities enclosed within a self-produced extracellular polymeric matrix that protects bacteria from antibiotics and host immune responses. This protective mechanism contributes to persistent infections, delayed healing, and increased resistance to treatment. Therefore, there is an urgent need for alternative therapeutic systems capable of targeting anaerobic pathogens and disrupting biofilm structures (Huang et al., 2022; Jia et al., 2025; Su et al., 2026; Woog & Destro, 2022; Wu et al., 2025).

In recent years, plant-derived essential oils have gained considerable attention as potential antimicrobial agents due to their broad-spectrum activity and multi-target mechanisms of action. Essential oils contain complex mixtures of terpenoids, phenolics, and aromatic compounds capable of disrupting microbial cell membranes, interfering with metabolic processes, and inducing oxidative stress. Among various essential oils, eucalyptus oil, clove oil, and neem oil have demonstrated promising antimicrobial and wound-healing properties.

Eucalyptus oil, rich in 1,8-cineole, possesses strong antimicrobial and penetration-enhancing properties. Clove oil contains eugenol, a phenolic compound known for potent bactericidal and anti-inflammatory activity. Neem oil exhibits antimicrobial, antioxidant, and wound-healing effects due to the presence of bioactive compounds such as azadirachtin and nimbidin. The synergistic combination of these oils may enhance antimicrobial efficacy while reducing the required concentration of individual components (Qi et al., 2026; Sha et al., 2026; Sharma et al., 2026; Shi et al., 2026; Trinh et al., 2026; Vater et al., 2026; Vaz et al., 2026; Wang et al., 2026).

However, the practical application of essential oils is limited by their poor aqueous solubility, volatility, and instability. To overcome these limitations, incorporation into suitable drug delivery systems is necessary. Hydrogels have emerged as effective topical delivery platforms due to their high water content, biocompatibility, controlled release behaviour, and ability to maintain a moist wound environment. The incorporation of essential oils into hydrogel matrices can enhance stability, improve retention at the application site, and facilitate sustained drug release (Farruggia et al., 2024; Fernandes et al., 2024; Filipe et al., 2022; Fincheira et al., 2024). Therefore, the present study aimed to develop and optimize a topical hydrogel formulation enriched with a synergistic blend of eucalyptus, clove, and neem essential oils for enhanced antimicrobial activity against anaerobic pathogens associated with gas gangrene.

2. MATERIALS AND METHODS

2.1 Materials

Eucalyptus oil (Eucalyptus globulus), clove oil (Syzygium aromaticum), and neem oil (Azadirachta indica) were procured from certified pharmaceutical suppliers and used as active antimicrobial agents in the formulation development process. Carbomer 934 and hydroxypropyl methylcellulose (HPMC) were used as polymeric gelling agents for hydrogel preparation. Tween 80 served as the surfactant for essential oil dispersion, while propylene glycol was used as a cosolvent and penetration enhancer. Triethanolamine was used for pH adjustment and gel neutralization. All solvents and reagents employed in the study were of analytical grade.

2.2 Preformulation Studies

2.2.1 Organoleptic Evaluation of Essential Oils

The procured essential oils were subjected to organoleptic evaluation to determine their appearance, colour, Odor, and clarity under normal daylight conditions (Adena et al., 2021; Ismail et al., 2021; Jain et al., 2021; Raj et al., 2026; Weimer et al., 2026).

2.2.2 Gas Chromatography–Mass Spectrometry (GC–MS) Analysis

GC–MS analysis was carried out to identify major phytochemical constituents present in the essential oils. The analysis was performed using a GC–MS system equipped with a capillary column under optimized chromatographic conditions. The obtained spectra were compared with standard spectral libraries for compound identification (Adena et al., 2021; Ismail et al., 2021; Jain et al., 2021; Raj et al., 2026; Weimer et al., 2026).

2.2.3 Fourier Transform Infrared (FT-IR) Compatibility Study

Compatibility studies between essential oils and selected excipients were performed using FT-IR spectroscopy. Samples were mixed with potassium bromide and compressed into pellets before analysis over a scanning range of 4000–400 cm⁻¹ (Adena et al., 2021; Ismail et al., 2021; Jain et al., 2021; Raj et al., 2026; Weimer et al., 2026).

2.3 Experimental Design

A three-factor, three-level Box–Behnken design was employed for optimization of the hydrogel formulations. The independent variables included essential oil concentration, surfactant concentration, and polymer concentration. The dependent variables selected for optimization included viscosity, spreadability, drug release, and antimicrobial activity. Design-Expert® software was used for statistical optimization and analysis of experimental responses (Adena et al., 2021; Ismail et al., 2021; Jain et al., 2021; Raj et al., 2026; Weimer et al., 2026).

2.4 Preparation of Essential Oil-Enriched Hydrogels

Hydrogel formulations were prepared using carbomer 934 and hydroxypropyl methylcellulose (HPMC) as polymeric bases. Carbomer was dispersed in purified water and allowed to hydrate overnight. HPMC solution was prepared separately under continuous stirring. The essential oil blend containing eucalyptus oil, clove oil, and neem oil was prepared in a fixed ratio and mixed with Tween 80 and propylene glycol to obtain a homogeneous oil phase. The oil phase was gradually incorporated into the hydrated polymeric dispersion with continuous stirring. Triethanolamine was added dropwise until a uniform gel consistency was obtained. The final weight of each formulation was adjusted with purified water (Koilpillai & Narayanasamy, 2024; Singh et al., 2026; Tang et al., 2024; Vemula et al., 2024).

2.5 Evaluation of Hydrogel Formulations

2.5.1 Determination of pH

The pH of the prepared hydrogel formulations was measured using a calibrated digital pH meter at room temperature (Alves et al., 2022; Beraldo-Araújo et al., 2022; Haimhoffer et al., 2021; Santonocito et al., 2022; Steele & Austin, 2016).

2.5.2 Viscosity Measurement

Viscosity measurements were carried out using a Brookfield viscometer fitted with appropriate spindle assembly at controlled rotational speed.

2.5.3 Spreadability Study

Spreadability of the formulations was determined using the glass slide method. The time required for separation of slides under applied weight was recorded and used for calculation (Ameur et al., 2022; Azevedo et al., 2022; Azizpour et al., 2025; Baccouri et al., 2008).

2.5.4 Drug Content Determination

Drug content uniformity was evaluated by dissolving a known quantity of hydrogel in ethanol followed by spectrophotometric analysis of extracted active components (Ameur et al., 2022; Azevedo et al., 2022; Azizpour et al., 2025; Baccouri et al., 2008).

2.6 In Vitro Drug Release Study

In vitro drug release studies were carried out using Franz diffusion cells fitted with dialysis membranes. The receptor compartment was filled with phosphate buffer (pH 6.8) maintained at 37 ± 0.5°C under continuous stirring conditions. Aliquots were withdrawn at predetermined time intervals and replaced with fresh receptor medium. Samples were analyzed spectrophotometrically to determine cumulative drug release (Ameur et al., 2022; Azevedo et al., 2022; Azizpour et al., 2025; Baccouri et al., 2008).

2.7 Drug Release Kinetics

The release data obtained from in vitro drug release studies were fitted into various kinetic models, including (Ameur et al., 2022; Azevedo et al., 2022; Azizpour et al., 2025; Baccouri et al., 2008):

• Zero-order model
• First-order model
• Higuchi diffusion model
• Korsmeyer–Peppas model

Correlation coefficients (R² values) were used to determine the best-fit model and release mechanism.

2.8 Antimicrobial Activity

2.8.1 Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

Antimicrobial activity was evaluated against anaerobic pathogens associated with gas gangrene, including Clostridium perfringens and Clostridium septicum. MIC and MBC values were determined using broth microdilution techniques under anaerobic incubation conditions (Ahmed Khan & van Vuuren, 2021; Wu et al., 2025; Xu et al., 2026; Yang et al., 2026; Zhang et al., 2026).

2.8.2 Time–Kill Kinetics Study

Time–kill studies were performed by exposing bacterial suspensions to optimized hydrogel formulations at concentrations equivalent to 1× MIC and 2× MIC. Viable bacterial counts were determined at predetermined intervals (Xu et al., 2026; Yang et al., 2026; Zhang et al., 2026).

2.8.3 Checkerboard Assay and Synergy Analysis

Synergistic interaction among essential oils was evaluated using the checkerboard assay. Fractional inhibitory concentration index (FICI) values were calculated to determine the nature of interaction (Xu et al., 2026; Yang et al., 2026; Zhang et al., 2026).

2.9 Antibiofilm Activity

Biofilm inhibition and eradication studies were performed using crystal violet staining methods. The ability of hydrogel formulations to inhibit biofilm formation and disrupt preformed biofilms was evaluated spectrophotometrically (Xu et al., 2026; Yang et al., 2026; Zhang et al., 2026).

2.10 Ex Vivo Permeation Study

Ex vivo permeation studies were carried out using Franz diffusion cells fitted with excised biological membranes. The receptor compartment contained phosphate buffer maintained at controlled temperature and stirring conditions. Samples were collected at predetermined intervals and analyzed to determine cumulative permeation and flux values.

2.11 Cytocompatibility Study

The cytocompatibility of the developed hydrogel formulations was evaluated using the MTT assay. Cells were exposed to hydrogel samples for specified durations, followed by assessment of cell viability based on mitochondrial reduction of MTT reagent.

2.12 Stability Study

The optimized hydrogel formulation was subjected to stability studies under controlled storage conditions for three months. Physicochemical parameters including pH, viscosity, and drug content were periodically evaluated.

2.13 Statistical Analysis

All experiments were carried out in triplicate and results were expressed as mean ± standard deviation. Statistical analysis was performed using Design-Expert® software and GraphPad Prism software. Analysis of variance (ANOVA) was used to determine statistical significance, with p < 0.05 considered significant.

3. RESULTS AND DISCUSSION

3.1 Preformulation Studies

Preformulation studies were carried out to evaluate the physicochemical characteristics and compatibility of essential oils with selected excipients prior to formulation development.

3.1.1 Organoleptic Evaluation

The essential oils exhibited characteristic appearance, odor, and clarity, confirming acceptable quality and purity.

 

 

Table 2: GC–MS Analysis of Essential Oils

Essential Oil	Major Compound	Retention Time (min)	% Peak Area
Eucalyptus oil	1,8-Cineole	8.42	72.5
Clove oil	Eugenol	12.76	81.3
Neem oil	Azadirachtin	18.54	24.6

 

 

Figure 1A: GC–MS Chromatogram of Eucalyptus Oil

 

Figure 1B: GC–MS Chromatogram of Clove Oil

 

Figure 1C: GC–MS Chromatogram of Neem Oil

Table 3: FT-IR Peak Interpretation

Component	Characteristic Peak (cm⁻¹)	Functional Group
Eucalyptus oil	1050	C–O stretching
Clove oil	1510	Aromatic C=C
Carbomer	1700	C=O stretching
HPMC	3400	OH stretching

 

 

 

Figure 2: FT-IR spectra showing compatibility between essential oils and selected excipients

Table 4: Physicochemical Evaluation of Hydrogel Formulations

Batch	pH	Viscosity (cP)	Spreadability (g·cm/s)	Drug Content (%)
F1	5.62 ±0.02	4820 ±120	18.5 ±0.6	96.2 ±1.3
F2	5.58 ±0.03	4950 ±140	17.8 ±0.5	97.5 ±1.2
F3	5.65 ±0.02	5100 ±150	19.2 ±0.7	98.1 ±1.1
F4	5.60 ±0.02	5250 ±160	18.6 ±0.6	99.3 ±1.0
F5	5.67 ±0.01	4300 ±110	21.5 ±0.8	95.6 ±1.4
F6	5.61 ±0.02	4400 ±120	20.8 ±0.7	97.0 ±1.3
F7	5.63 ±0.02	5600 ±180	16.5 ±0.5	98.8 ±1.2
F8	5.59 ±0.02	5750 ±190	15.9 ±0.4	99.6 ±1.1
F9	5.64 ±0.02	4500 ±130	20.5 ±0.7	97.9 ±1.2
F10	5.60 ±0.03	4650 ±140	19.8 ±0.6	98.5 ±1.1
F11	5.62 ±0.02	5850 ±200	15.2 ±0.4	99.1 ±1.0
F12	5.58 ±0.02	6000 ±210	14.8 ±0.3	99.8 ±0.9
F13	5.61 ±0.01	5200 ±150	18.0 ±0.5	98.9 ±1.0

 

 

Figure 3: Effect of polymer concentration on viscosity of hydrogel formulations.

 

 

Figure 4: Relationship between viscosity and spreadability of hydrogel formulations.

 

 

Figure 5: In vitro drug release profile of selected hydrogel formulations.

Table 6: Drug Release Kinetics Model Fitting (R² Values)

Formulation	Zero-order	First-order	Higuchi	Korsmeyer–Peppas
F1	0.942	0.981	0.968	0.992
F5	0.955	0.987	0.972	0.995
F9	0.948	0.984	0.970	0.993
F13	0.951	0.985	0.971	0.994
F8	0.938	0.979	0.965	0.991

 

 

 

Figure 6: Korsmeyer–Peppas model plot.

Table 7: ANOVA for Viscosity Response

Source	Sum of Squares	df	Mean Square	F-value	p-value
Model	3.52E+06	9	3.91E+05	45.6	<0.0001
X₁	1.12E+05	1	1.12E+05	13.1	0.006
X₂	2.45E+05	1	2.45E+05	28.6	0.001
X₃	2.80E+06	1	2.80E+06	326.5	<0.0001

Table 8: Regression Statistics

Parameter	Value
R²	0.987
Adjusted R²	0.975
Predicted R²	0.962
Adequate Precision	18.5

 

 

 

Figure 7: Response surface plot showing effect of polymer and surfactant concentration on viscosity.

 

 

Figure 8: Contour plot showing combined effect of essential oil concentration and polymer concentration on drug release.

Table 9: MIC and MBC Values of Hydrogel Formulations

Formulation	MIC (µg/mL)	MBC (µg/mL)
F1	45	90
F5	30	60
F9	25	50
F13	18	36
F8	22	44

 

 

Figure 9: Comparative MIC values of hydrogel formulations against anaerobic pathogens.

Table 10: Time–Kill Kinetics Data for Optimized Formulation (F13)

Time (h)	Log CFU/mL (1× MIC)	Log CFU/mL (2× MIC)
0	6.00 ±0.05	6.00 ±0.05
2	5.20 ±0.07	4.80 ±0.06
4	4.30 ±0.06	3.60 ±0.05
8	3.10 ±0.05	2.00 ±0.04
12	2.20 ±0.04	1.20 ±0.03
24	1.10 ±0.03	0.00 ±0.00

 

 

 

Figure 10: Time–kill kinetics curve of optimized hydrogel formulation.

Table 11: FICI Values for Essential Oil Combinations

Combination	FICI Value	Interpretation
Eucalyptus + Clove	0.42	Synergistic
Clove + Neem	0.48	Synergistic
Eucalyptus + Neem	0.55	Additive
Triple Combination	0.36	Strong synergy

 

 

 

Figure 11: FICI interaction plot showing synergistic effect among essential oil combinations.

Table 12: Biofilm Inhibition Activity of Hydrogel Formulations

Formulation	Biofilm Inhibition (%)
F1	52.4 ±1.8
F5	65.2 ±2.1
F9	72.8 ±2.4
F13	85.6 ±2.7
F8	78.3 ±2.5

 

Table 13: Biofilm Eradication Activity of Hydrogel Formulations

Formulation	Biofilm Eradication (%)
F1	45.6 ±1.6
F5	58.3 ±1.9
F9	66.5 ±2.2
F13	80.2 ±2.6
F8	72.4 ±2.3

 

 

 

Figure 12: Biofilm inhibition activity of hydrogel formulations

.

 

Figure 13: Biofilm eradication activity of hydrogel formulations.

Table 14: Ex Vivo Permeation Parameters

Formulation	Flux (µg/cm²/hr)	Permeability Coefficient (Kp)
F1	45.2 ±1.5	0.045
F5	58.6 ±1.8	0.058
F9	65.3 ±2.0	0.065
F13	78.4 ±2.4	0.078
F8	70.2 ±2.2	0.070

 

 

 

Figure 14: Ex vivo permeation profile of selected hydrogel formulations.

The slight reduction in viscosity observed during storage may be attributed to gradual relaxation of the polymeric network and minor changes in hydration behaviour of the hydrogel matrix. Similarly, the minimal reduction in drug content could be associated with limited volatilization or oxidation of essential oil constituents over time. However, the observed changes remained within acceptable pharmaceutical limits, confirming that the optimized formulation retained satisfactory stability under storage conditions. The stable pH profile further indicated that no significant chemical degradation or interaction occurred during the study period. These findings support the suitability of the developed hydrogel formulation for topical therapeutic application and potential long-term storage.

CONCLUSION

The present study successfully developed and optimized a topical hydrogel formulation enriched with a synergistic blend of eucalyptus, clove, and neem essential oils for enhanced antimicrobial activity against anaerobic pathogens associated with gas gangrene. The hydrogel formulations exhibited acceptable physicochemical properties, including appropriate pH, viscosity, spreadability, and drug content uniformity. The optimized formulation (F13) demonstrated sustained drug release over 12 hours and followed Korsmeyer–Peppas release kinetics, indicating a non-Fickian diffusion mechanism. Significant antimicrobial activity was observed against anaerobic pathogens, with the optimized formulation exhibiting the lowest MIC and MBC values. Time–kill studies confirmed concentration-dependent bactericidal activity, while checkerboard assays demonstrated strong synergistic interaction among the essential oils. The formulation also showed excellent antibiofilm activity, enhanced ex vivo permeation, acceptable cytocompatibility, and good stability over three months. The incorporation of essential oils into a hydrogel matrix effectively enhanced their stability, release behaviour, and antimicrobial efficacy. Overall, the findings indicate that the developed essential oil-enriched hydrogel represents a promising topical therapeutic system for the management of anaerobic wound infections and gas gangrene-associated complications.

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