Abstract This study presents a novel antifungal topical delivery approach through the development of a nano-emulgel incorporating Cullen corylifolium seed extract, a medicinal herb known for its broad-spectrum antimicrobial activity. Bioactive constituents were extracted using ethanolic Soxhlet extraction, followed by phytochemical screening and GC-MS profiling to identify key antifungal compounds. Six nano-emulgel formulations (CN1–CN6) were prepared using Carbopol 940 as a gelling agent, along with Tween 80, glycerin, and liquid paraffin. Physicochemical evaluations including pH, viscosity, spreadability, and extrudability confirmed their suitability for topical application. ATR-FTIR analysis confirmed no chemical incompatibility between the extract and excipients. Among all, formulation CN3 demonstrated optimal physical and mechanical properties. Scanning Electron Microscopy, which was simply employed for surface morphology analysis only, revealed well-defined emulsion droplets dispersed within gel matrix, and particle studies, which was used to understand the hydrodynamic size, showed nanoparticles with average particle size of 737.82 nm with zeta potential of –3.72 mV, indicating modest colloidal stability. In-vitro drug release studies exhibited a sustained release profile, with a cumulative release of 92.9% over 240 minutes, best fitting Higuchi and Korsmeyer–Peppas kinetic models. Antifungal testing against Candida albicans demonstrated a significant inhibition zone (17 mm) at a concentration of 200 µg/mL. Accelerated stability studies over three months confirmed the retention of physical integrity and antifungal efficacy. These findings suggest that the developed nano-emulgel system offers a promising natural, stable, and efficacious strategy for topical antifungal therapy with enhanced skin penetration and prolonged drug release.

Keywords: Cullen corylifolium, herbal nano-emulgel, antifungal activity, skin targeting

Citation:  Rajappa, M.C., Venkatasubramaniam, N., Muthumani, K., Sivaji, R., Ganamoorthi, S., and Vezhaventhan, V. 2026. Design, characterization and antifungal activity of Cullen corylifolium seed extract-loaded nanoemulgel with enhanced dermal penetration. Natural and Life Sciences Communications. 25(3): e2026045.

Graphical Abstract:

INTRODUCTION

Superficial fungal infections such as candidiasis, dermatophytosis, and tinea infections represent a significant portion of dermatological conditions affecting people worldwide. These infections are becoming increasingly difficult to manage due to the emergence of drug-resistant fungal strains, limited skin penetration of conventional topical agents, and associated side effects. Moreover, many antifungal treatments suffer from drawbacks like poor patient adherence, irritation, and inadequate drug retention at the infection site. These limitations highlight the need for advanced drug delivery systems that can improve therapeutic outcomes while minimizing systemic exposure (Chanyachailert et al., 2023).

Among novel drug delivery strategies, nano-emulgel systems have garnered attention for their dual benefits: the superior skin permeation capability of nanoemulsions and the convenience, spreadability, and retention characteristics of gels. Nanoemulgels can enhance the solubility, stability, and penetration of lipophilic or poorly soluble drugs through the stratum corneum, the primary barrier of the skin (Lal et al., 2023). Antifungal nanoemulgels has expressed enhanced permeation profile compared to marketed gels (Donthi et al., 2023) because nanogel-based platforms improve antifungal activity through targeted delivery and increased cellular uptake, resulting in effective penetration of fungal biofilms and minimization of side effects. (Chawalke et al., 2025; Wu et al., 2025) This formulation is better than nanoemulsion itself. Research proved enhanced antifungal activity of Naftitine nanoemulgel than its nanoemulsion form against Microsporum canis and Trichophyton rubrum. (Phalak et al., 2024)

Cullen corylifolium (commonly known as Babchi) is a medicinal plant used in traditional medicine for treating various skin ailments. The seed extract of this plant is particularly rich in bioactive constituents such as flavonoids, terpenoids, coumarins, and phenolic compounds. Notably, (+)-Bakuchiol, a predominant compound, has demonstrated potent antifungal, antibacterial, and antioxidant properties. These pharmacological effects support its use as a natural antifungal agent in topical formulations (Nizam et al., 2023).

The present study focuses on the formulation, optimization, and evaluation of a nano-emulgel loaded with the ethanolic extract of Cullen corylifolium. The objective is to improve the antifungal efficacy and skin permeability of the herbal extract through a stable and effective nano-emulgel system. A series of formulations were prepared using various excipients, and the most suitable one was identified through systematic evaluation of physicochemical properties, drug release, antifungal performance, and stability. This study serves as a foundation for future in vivo investigations to establish clinical utility (Duangjit et al., 2024).

MATERIALS AND METHODS

Materials

Cullen corylifolium seeds were sourced from an authenticated herbal supplier (AUT/VMU/297). Ethanol [CAS: 64-17-5; 99% pure], petroleum ether [CAS: 8032-32-4], and distilled water were used as solvents. Excipients included Carbopol 940 (gelling agent) [CAS: 9003-01-4], Tween 80 (surfactant) [CAS: 9005-65-6], glycerin (humectant) [CAS: 56-81-5], liquid paraffin (oil phase) [CAS: 8012-95-1], methyl paraben (preservative) [CAS: 99-76-3], and triethanolamine (neutralizer) [CAS: 102-71-6], all of analytical grade and obtained from certified chemical laboratories.

Extraction and phytochemical screening
Dried seeds of Cullen corylifolium were coarsely powdered and extracted using Soxhlet apparatus with selected solvent. The extract was filtered, concentrated under reduced pressure, and stored at 4°C until use. Drying under reduced pressure using rotary vacuum evaporator (Rotavap Junior, Medico, India) helps in preserving the thermolabile compounds of the extract. Preliminary phytochemical screening was performed using standard procedures to identify constituents such as alkaloids, flavonoids, tannins, phenols, and terpenoids (Zhang et al., 2023).

Figure 1. Soxhlet apparatus method extraction for Cullen corylifolium seed extract.

GC-MS analysis

GC–MS A Shimadzu GC 2010 PLUS instrument equipped with a QP2020 mass spectrometer was used for the analysis. The injections (1 µL) were made in split mode (10:5) with an injector temperature of 280°C on the SH-Rxi-5Sil MS capillary column (30 m × 0.25 mm, 0.25 µm). Helium was used as the carrier gas at a constant flow rate of 1.0 mL/min. The oven program was 80°C (4 min) followed by a ramp at 10°C/min to 240°C (2 min), then 8°C/min to 280°C with an 8-min cool down and hold, for a total run time of 35 min.

Peak purity of each chromatographic peak was evaluated by comparing mass spectra at leading edge, apex, and trailing edge. Peaks with consistent ion profiles and stable library match scores at all three points were seen as pure. Any changes in characteristic ion ratios or spectral similarity indicated co-elution. Spectra were further analyzed using the software’s deconvolution function to confirm accurate peak assignment and identification of components, when necessary. (Hassan et al., 2019; Wang et al., 2023)

Formulation of nano-emulgel

Nanoemulsion was prepared by blending the extract and liquid paraffin (oil phase) with an aqueous phase containing Tween 80, glycerin, and preservatives using high-speed homogenization. A gel base was prepared separately using Carbopol 940 and neutralized with triethanolamine. The nanoemulsion was gradually incorporated into the gel with continuous stirring to produce nano-emulgel formulations (CN1–CN6). A total of 30 gm of nanoemulgel was formulated with gel concentration varying between 0.5%w/w to 1.5%w/w. (Hosny et al., 2021)

Table 1. Formulation optimization of nanoemulgel containing Cullen corylifolium (CN1-CN6) containing 30 gm.

Physicochemical properties

Formulations were evaluated for pH using a digital pH meter (Systronics µ-362), viscosity (Brookfield viscometer), and spreadability (parallel glass slide method). These tests ensured topical application suitability (Gumadoh et al., 2024).

Spreadability

A glass plate was placed with calculated quantity of nanoemulgel (350 mg) and another glass plate was placed parallel hand wash dropped from a height of 5 cm. Spreadability of the nanoemulgel was calculated by the following formula: (Gangadharappa et al., 2017)

Where,

M- Weight applied on the upper plate

L- Distance travelled by the slide

T- Time for separation of the glass plates

Extrudability

The prepared nanoemulgel was filled into the aluminum collapsible tubes. A weight of about 500 grams was applied externally on the filled tube and the extruded amount was measured. The extrudability was measured by the following formula: (Shukla et al., 2022)

Compatibility analysis

ATR-FTIR spectroscopy (Bruker Alpha-II) was performed to check for chemical compatibility between the extract and excipients. Spectral analysis (4,000–400 cm⁻¹) was used to detect any interaction-related shifts (Altamimi et al., 2022).

Particle size and zeta potential

Dynamic Light Scattering (DLS) using a Zetasizer (Malvern v8.02) was employed to measure droplet size and zeta potential of nanoemulsion, which was diluted from nanoemulgel. The polydispersity index (PDI) was used to assess uniformity, while zeta potential values provided insight into colloidal stability (Sadler et al., 2025).

Surface morphology

With minor modifications, Scanning Electron Microscopy (Carl Zeiss EVO 18) was used to analyze the surface morphology and apparent shape of the emulgel as described in the literature. Utilizing double-sided sticky tape, the emulgels were adhered to metal stubs to create the samples for SEM, which were then vacuum-casted to dry. After using a sputter-coater to apply a 10 nm thick gold coating, the sample was examined under a high-resolution SEM (Cavallaro et al., 2021).

In-vitro drug diffusion

The drug diffusion study of nanoemulgel formulations (CN1–CN6) was performed using a Franz diffusion cell with an eggshell membrane. Fresh membranes were cleaned, hydrated in phosphate buffer (pH 7.4) for 30 minutes, and mounted between the donor and receptor compartments. The receptor chamber was filled with 20 mL of phosphate buffer pH 7.4 + 5-10% ethanol, maintained at 37 ± 0.5 °C, and stirred continuously. Sink conditions were achieved by adding 5% ethanol to the buffer, as it does not affect the buffer and simultaneously improve the solubility of the API. Each formulation (1 g; equivalent to 39.3 mg of API) was placed in the donor compartment. Samples (1 mL) were withdrawn at intervals of 0, 15, 30, 45, 60, 120, 180, and 240 minutes, and replaced with fresh buffer. The samples were analyzed using a UV-Visible spectrophotometer at 262 nm. This wavelength was finalized based on the absorption maxima of active phytochemical constituent observed in relevant literature. Cumulative drug release was calculated using a standard calibration curve and plotted against time to compare the release profiles of all formulations (Bashir et al., 2021; Grzelecka et al., 2025).

Drug release kinetics

Release Kinetic Profiling was performed using Microsoft Excel. The release profiles were fitted to mathematical models: zero-order, first-order, Higuchi, Korsmeyer-Peppas, and Hixson-Crowell. The model with the highest correlation coefficient (R²) indicated the dominant release mechanism. The results are shown in Table S2 and Figure S2-S6. (Jafari et al., 2022)

Antifungal activity

Antifungal efficacy was evaluated against Candida albicans using the agar well diffusion method. Different concentrations (50–200 µg/mL) of the nano-emulgel were tested, and zones of inhibition were measured post incubation. Fluconazole (1%w/v) and ethanol extract free nanoemulgel were used as the positive control and negative control respectively. (Ahmad et al., 2023)

Stability studies

The optimized nanoemulgel formulation (CN3) was subjected to accelerated stability testing under ICH conditions (40°C ± 2°C / 75% RH ± 5%) for three months using a stability chamber (Rashmi Technologies). Critical Quality Attributes (CQA) included particle size, pH, viscosity, spreadability, extrudability, and visual appearance at monthly intervals to assess stability (Mahtab et al., 2016; Akhter et al., 2024).

Statistical evaluation

The statistical analyses were performed using Jamovi 2.6.44 statistical software. The number of replicates for each mentioned tests are three in number (n=3). One way ANOVA was performed for pH, viscosity, spreadability, extrudability followed by Tukey’s HSD test was employed as a post-hoc studies. Two-way ANOVA with replication assessed the effect of formulation and time on drug release (P < 0.05). Tukey’s post hoc test was used for pairwise comparisons among formulations. (Shenoy et al., 2021)

RESULTS

Phytochemical screening of Cullen corylifolium seed extracts

The products obtained after extraction with different solvent systems were subjected to various chemical tests to identify the active phytoconstituents present in the extracts.

Table 2. Phytochemical tests for Cullen corylifolium seed extracts.

Note: “-”= Absence, “+” =slight presence, “++” = strong presence, “+++” = More strong presence.

The ethanolic and aqueous extracts of Cullen corylifolium exhibited a broader range of phytoconstituents compared to the Petroleum ether extract. These bioactive compounds, such as flavonoids, phenols, tannins and terpenoids, are known for their antifungal and therapeutic properties, making these extracts suitable candidates for topical Nano-emulgel formulations.

GC-MS analysis

The GC-MS results confirm the presence of several antifungal and bioactive compounds in the ethanol extract of Cullen corylifolium seeds. It shows the presence of (+)-Bakuchiol acetate, 9,12-Octadecadienoic acid, undec-10-ynoic acid nonyl ester.

Figure 2. GC-MS peak report of Cullen corylifolium seeds ethanol extract.

Table 3. GC-MS analysis report of Cullen corylifolium seeds ethanol extract.

Compatibility studies (ATR-FTIR)

ATR-FTIR analysis was performed to assess any possible chemical interactions between the extract and excipients. The characteristic peaks of functional groups from Cullen corylifolium extract were retained in all excipient-loaded spectra without any significant shift or disappearance. This confirmed the absence of incompatibilities between the herbal extract and formulation components, indicating a stable and compatible system.

Figure 3. ATR- FTIR Spectrum of Cullen corylifolium (A) seed extract, (B) extract + all excipients, (C) comparision of extract + all excipients.

Table 4. ATR-FTIR peak interpretation of Cullen corylifolium seed extract + all excipients.

Organoleptic properties

The colour, odour, appearance of the formulations CN3 visual inspection was observed. Based on the observation it was found satisfactory for the formulation of Cullen corylifolium nanoemulgel form good consistency and no discomfort likely to arise in patient compliance. This is shown Table S1 (Supplementary data).

Physicochemical evaluation of Cullen corylifolium nanoemulgel

The prepared formulations (CN1–CN6) were subjected to evaluation for pH, viscosity, spreadability, and extrudability. The compiled results are presented in Table 5

Table 5. Physicochemical properties of Cullen corylifolium nanoemulgel formulations.

The ethanolic extract was incorporated into a nanoemulgel system for topical delivery. Six formulations (CN1 to CN6) were prepared using varying concentrations of gelling agents and surfactants. The physical properties such as appearance, color, odor, pH, viscosity, spreadability, and extrudability of all formulations were evaluated. Among these, formulation CN3 was found to exhibit ideal characteristics with a pH of 4.9 (within skin pH range), viscosity of 5,200 cps, and spreadability of 18.70 g.cm/sec, indicating a smooth and user-friendly topical formulation.

Particle size and zeta potential

The optimized nanoemulgel formulation (CN3) demonstrated a particle size distribution was calculated to be 737.82 ± 53.26 nm as measured by dynamic light scattering (DLS), suggesting efficient skin permeation. The polydispersity index of 0.265 indicated good stability and absence of agglomeration between particles. Besides, there was absence of notable peak intensity beyond 1,000 nm, excluding the possibility of large population of micro-sized particles (>1,000 nm). The zeta potential of -3.72 mV indicated modest physical stability of the emulsion system, as electrostatic repulsion among particles can reduce aggregation. The nanometric size and appropriate surface charge enhanced the dermal penetration of antifungal phytoconstituents.

Figure 4. (A) Particle size distribution & (B) Zeta potential of Cullen corylifolium extract loaded nanoemulgel.

Surface morphology (SEM)

Scanning Electron Microscopy (SEM) of the optimized formulation revealed spherical to near-spherical structures with sizes between 1 µm and 10 µm. The particles were uniformly distributed, confirming consistent emulsification in nanoemulgel matrix. The smooth morphology is advantageous for skin adhesion and enhances permeation by forming a stable film over the application site.

Figure 5. Scanning electron microscopy (SEM) of optimized formulation CN3 showing uniform spherical particles magnified at (A) 12.97 kX (1 µm) and (B) 8.83 kX (2µm).

In-vitro drug diffusion study

Franz diffusion studies using an eggshell membrane were conducted over a 4-hour period to evaluate the drug release profiles of the nanoemulgel formulations. Among all formulations, CN3 exhibited the highest cumulative drug release of 92.9% at 240 minutes, other formulation exhibited release above 85%. The enhanced release profile of CN3 is attributed to its optimized composition of Carbopol 940, Tween 80, and glycerin, which collectively promoted better solubilization, emulsification, and diffusion of the bioactive compounds. Although all formulations demonstrated sustained drug release, CN3 emerged as the most efficient formulation in terms of permeability and drug release performance. The flux and permeability coefficient were calculated to be 0.06513 mg/cm²/min and 0.001657 cm/min respectively. 

Table 6. Data for the in-vitro diffusion study (Mean ± SD; n=3) for various extract loaded nanoemulgel formulations (CN1-CN6). The timepoints having different letters are statistically different to each other within the row (P < 0.05, Tukey’s test).

Tukey’s HSD test (critical value = 2.44%) revealed that the optimized formulation CN3 exhibited significantly higher drug release compared to all others, particularly CN1 (6%), CN2 (4.2%), and CN4 (3.4%). These differences indicate the superior release characteristics of CN3 over other formulations. The pairwise comparisons are tabulated in Table S7.

Figure 6. In-vitro diffusion profiles of extract-loaded nanoemulgels (CN1–CN6).

Drug release kinetics

The drug release kinetics followed the Higuchi model and Zero-order release pattern. This confirms that the drug release was driven by both diffusion and erosion mechanisms. All the formulations (CN1-CN6) followed Fickian Diffusion since the n-value (0.20) was less than 0.45. The result was shown in Table S8 and Figure S1-S6.

Antifungal activity

Agar well diffusion assays were used to assess antifungal activity against Candida albicans. The zone of inhibition was dose-dependent and ranged from 10 mm (50 µg/mL) to 17 mm (200 µg/mL). The optimized formulation (CN3) showed strong antifungal activity at higher concentrations, comparable to the positive control (24 mm). This activity is attributed to the presence of bioactive compounds such as (+)-Bakuchiol acetate, 9,12-Octadecadienoic acid, and other phytochemicals confirmed through GC-MS analysis. The results support the traditional use of Cullen corylifolium as an antifungal agent and confirm its enhanced efficacy when delivered via a nanoemulgel system. Within CN3, increasing the dose from 50 to 100, 150 or 200 µg/mL significantly enhanced antifungal activity (all P < 0.001), and further increases from 100 to 150 µg/mL and from 150 to 200 µg/mL also produced significant, though smaller, improvements (P < 0.01). 

Table 7. Zone of inhibition obtained by sample against C. albicans. Values having different letters are statistically different to each other within the row (P < 0.05, Tukey’s test).

Note: Mean ± SD; n=3; PC-Positive Control (Fluconazole 1%w/v); NC-Negative Control (Ethanol extract free nanoemulgel).

Figure 7. Antifungal activity observed in terms of zone of inhibition (mm) for Candida albicans (A) in culture medium and (B) graphical representation of zone of inhibition (mm) for different sample concentration with positive control (* P < 0.05; ** P < 0.01; *** P < 0.001, Tukey’s test)

Stability studies

Accelerated stability testing was performed on CN3 for three months under ICH guidelines (40°C ± 2°C / 75% RH ± 5%). The formulation retained its physical characteristics (color, odor, consistency), with minimal changes in pH, viscosity, spreadability, and extrudability. The results confirm that CN3 remains stable under accelerated conditions, ensuring shelf-life reliability and patient safety. Particle size remained within the same range of 700 to 800 nm, even after 3 months.

Table 8. Stability studies of optimized formulation (CN3) for the ethanolic extract of Cullen corylifolium nanoemulgel performed at 40 ± 2°C / 75 ± 5% RH as per ICH guidelines. Values of an individual parameter having different letters are statistically different to each other within the row (P <0.05, Tukey’s test).

DISCUSSION

The phytochemical screening highlights the novel potential of Cullen corylifolium seed extracts, particularly the ethanolic extract, which exhibited a rich profile of bioactive constituents such as flavonoids, phenols, tannins, and terpenoids compounds with well-documented antifungal and skin-healing properties. This broad spectrum of activity, absent in the petroleum ether extract, underscores ethanol’s superiority as a solvent for extracting therapeutically relevant compounds. Notably, the aqueous extract showed a high presence of alkaloids and steroids, indicating its potential role in immunomodulatory and regenerative applications. These findings support the innovative use of ethanolic extract in nano-emulgel systems, offering enhanced penetration and synergistic antifungal action through phytoconstituent-rich delivery. (Opaleke et al., 2022) 

Further characterization was done using Gas Chromatography-Mass Spectrometry (GC-MS), which confirmed the presence of major phytocompounds such as (+)-Bakuchiol acetate, 9,12-Octadecadienoic acid, 3-Trifluoromethylbenzoic acid undec-2-enyl ester, and Undec-10-ynoic acid nonyl ester. These compounds were reported to possess antifungal and antibacterial activities. (Konappa et al., 2020)

The ATR-FTIR spectral analysis was employed to assess the molecular-level compatibility between Cullen corylifolium seed extract and the formulation excipients. The extract exhibited characteristic peaks corresponding to various functional groups such as hydroxyl (~3,270 cm⁻¹), aliphatic C–H (~2,920 cm⁻¹), carbonyl (~1,735 cm⁻¹), aromatic C=C (~1,610 cm⁻¹), and ether linkages (~1,040–1,150 cm⁻¹), indicating the presence of key phytoconstituents like flavonoids, bakuchiol derivatives, and glycosides. Comparatively, the excipient spectra also showed overlapping peaks in similar regions—O–H (~3,260–3,300 cm⁻¹), C–H (~2,920–2,850 cm⁻¹), and C=O (~1,730–1,740 cm⁻¹)—without any significant peak shift, broadening, or disappearance. This confirms that no new chemical bonds or interactions were formed, thereby suggesting the absence of incompatibility between the bioactive extract and formulation components. (Bunaciu et al., 2025)

The physicochemical characterization detailed below clearly indicates that formulation CN3 possesses the most suitable combination of pH, viscosity, spreadability, and extrudability, making it a stable and skin-compatible nanoemulgel. (Rathee et al., 2023)

pH values across formulations ranged between 3.8 and 7.3, all within acceptable topical ranges. CN3 had a pH of 4.9, aligning closely with natural skin pH (4.5–6), thus ensuring minimal irritation potential. (Lukić et al., 2021)

Viscosity was found to depend on the gelling agent concentration. CN1, though having the highest viscosity (6,850 cps), exhibited poor spreadability. In contrast, CN3 showed an optimal viscosity of 5,200 cps, offering a balance between structural stability and application ease. The ideal viscosity range for topical gels typically lies between 2,000–6,000 cps, which supports ease of spreading while maintaining formulation integrity (Cao et al., 2024).

Spreadability is crucial for user comfort and uniform dosing. CN3 had a spreadability of 18.70 g·cm/sec, which falls within the ideal range of 17–25 g·cm/sec, ensuring efficient topical coverage without runoff or excessive shear. (Sharma and Bansal, 2023)

Extrudability, which indicates the ease of formulation delivery from collapsible containers, was found to be >90% for CN3, categorizing it as excellent according to pharmacopeial standards. This result suggests that CN3 has ideal rheological properties, likely due to the appropriate concentration of Carbopol 940, which imparts optimal viscosity and smooth texture. The excellent extrudability enhances patient compliance by allowing effortless application and uniform dosing. (Goyal et al., 2021; Bahman et al., 2025)

Particle size analysis revealed that the CN3 nanoemulgel contained nanometric particles ranging between 700–900 nm, with a polydispersity index (PDI) of 0.265, indicating a fairly uniform and narrow particle size distribution, as PDI values below 0.3 generally reflect homogeneity in colloidal systems. It should be noted that many nanoemulgel formulations exhibited PDI greater than 0.3 in spite of particle size >600 nm, which was greater than the current research. (Karri et al., 2024; Sghier et al., 2024) Furthermore, nanodroplets ranging within the 700 nm and 900 nm are helpful in efficient dermal permeation. This indicates the quality of mixing process during the formulation stage (Cavallaro et al., 2021). SEM imaging further confirmed the presence of spherical and smooth-surfaced particles under resolution of 1 µm, supporting the formation of well-defined emulsion droplets (Saravanan et al., 2025). The scanning electron microscopy exhibited microscopic particles which is due to removal of solvent and surfactant shells during sample preparation. This occasionally leads to particles aggregation. (Filippov et al., 2023) Similar condition was observed in research in which the surface morphology was analyzed in the resolution range where average particle size was 10 times lesser than this magnification scale adopted in the SEM. (Yetukuri et al., 2023; Anuradha et al., 2024) The zeta potential was recorded at -3.72 mV, suggesting modest physical stability of the formulation. Although zeta potential values above ±30 mV typically indicate strong electrostatic stabilization, values closer to neutral as seen here can still provide acceptable stability when combined with steric stabilization from surfactants and polymers such as Carbopol 940 (Algahtani et al., 2020; Bashir et al., 2021)

The in-vitro drug release profile showed that formulation CN3 achieved the highest release (92.9%) within 240 minutes, indicating controlled and sustained drug delivery suitable for prolonged antifungal action. The analysis revealed that both time and formulation type had a significant impact on the drug diffusion behavior, emphasizing the time-dependent nature of release and the distinct performance differences among formulations. The observed consistency across experimental replicates further strengthens the reliability of the findings, suggesting that the formulation matrix plays a crucial role in modulating release dynamics (Askarizadeh et al., 2023).

The in-vitro release data for the optimized formulation CN3 showed an appreciable correlation with the Zero-order plot (R² = 0.9675), indicating a sustained and controlled release profile. The data also exhibited good linearity with the Higuchi diffusion model, suggesting that the drug release was governed primarily by a diffusion mechanism without any significant swelling or erosion of the polymer matrix (Bashir et al., 2021).

The sustained release of the nanoemulgel by following zero order kinetics confirmed the absence of an immediate release of the API from the formulation. (Gaanapriya et al., 2024) The release of the API is governed by matrix diffusion. This is a common feature of nanoemulgel because they act as reservoirs and promote sustained release of the API. (Akhter et al., 2024; Ojsteršek et al., 2024) The formulations expressing Higuchi model generally followed Fickian diffusion (n < 0.45), which was consistent with existing literature. (Noor et al., 2023; Gaanapriya et al., 2024) This is due to the absence of polymer swelling in Higuchi following models. (Dave et al., 2023)

The antifungal activity of the ethanolic extract and nanoemulgel formulation was evaluated against Candida albicans using the agar well diffusion method. The extract and formulation exhibited significant zones of inhibition, with maximum antifungal effect observed at concentrations of 200 µg/mL and 150 µg/mL, respectively. (Buanpech et al., 2024) The optimized formulation CN3 was subjected to a short-term stability study for a period of 3 months under accelerated storage conditions (40 ± 2°C / 75 ± 5% RH). No significant changes were observed in terms of physical appearance, pH, viscosity, or drug content, confirming that the formulation remained stable throughout the study period (Modhave et al., 2024).

CONCLUSION

The developed Cullen corylifolium seed extract loaded nano-emulgel formulation exhibited excellent physicochemical properties, sustained drug release, and significant antifungal activity. Among the tested batches, CN3 showed superior performance and stability. This study highlights the potential of using nano-emulgel as an effective topical drug delivery system for herbal extracts, offering improved therapeutic efficacy, enhanced skin permeation, and prolonged shelf life. Future direction of this research should include in vivo studies to confirm clinical applicability before commercialization.

ACKNOWLEDGEMENTS

The authors are deeply grateful to the Principal of Vinayaka Mission’s College of Pharmacy, Vinayaka Mission’s Research Foundation, for giving us the support to accomplish this work. We sincerely thank CIMF-Periyar University,Salem (Funded by RUSA) for conducting GC-MS and SEM for this research. We also extend our sincere thanks to PSG Institute of Advanced Studies (Coimbatore) for the DLS and Zeta Potential studies. We would also acknowledge ProGen Molecular Biology Lab (Acme), Salem, for carrying out antifungal activity of the optimized formulation.

AUTHOR CONTRIBUTIONS

Margret Chandira Rajappa: Conceptualization (Equal), Validation (Lead), Resource (Equal), Supervision (Lead), Project Administration (Lead); Nagasubramanian Venkatasubramaniam: Data Curation (Lead), Formal Analysis (Lead), Methodology (Equal), Writing – Review & Editing (Lead), Investigation (Lead), Project Administration (Equal); Karthikeyan Muthumani: Writing – Original Draft (Equal), Writing – Review & Editing (Equal), Methodology (Equal); Ranjithkumar Sivaji: Data Curation (Equal), Formal Analysis (Equal), Writing – Original Draft (Equal), Writing – Review & Editing (Equal), Investigation (Equal); Sanjay Ganamoorthi: Methodology (Equal), Validation (Equal), Resource (Equal), Data Curation (Equal), Formal Analysis (Equal), Writing – Review & Editing (Equal), Investigation (Equal); Vignesh Vezhaventhan: Conceptualization (Equal), Methodology (Lead), Formal Analysis (Equal), Validation (Equal), Resource (Equal), Writing – Original Draft (Lead), Writing – Review & Editing (Equal), Investigation (Equal).

CONFLICT OF INTEREST

The authors declare that they hold no competing interests.

REFERENCES

Ahmad, I., Farheen, M., Kukreti, A., Afzal, O., Akhter, M.H., Chitme, H., Visht, S., Altamimi, A.S., Alossaimi, M.A., Alsulami, E.R., et al. 2023. Natural oils enhance the topical delivery of ketoconazole by nanoemulgel for fungal infections. ACS Omega. 8(31): 28233-28248. https://doi.org/10.1021/acsomega.3c01571

Akhter, A., Shirazi, J.H., Khan, S.H.M., Hussain, M.D., and Kazi, M. 2024. Development and evaluation of nanoemulsion gel loaded with bioactive extract of Cucumis melo var. agrestis: A novel approach for enhanced skin permeability and antifungal activity. Heliyon. 10: e35069. https://doi.org/10.1016/j.heliyon.2024.e35069

Algahtani, M.S., Ahmad, M.Z., and Ahmad, J. 2020. Nanoemulgel for improved topical delivery of retinyl palmitate: Formulation design and stability evaluation. Nanomaterials. 10(5): 848. https://doi.org/10.3390/nano10050848

Altamimi, M.A., Hussain, A., Mahdi, W.A., Imam, S.S., Alshammari, M.A., Alshehri, S., and Khan, M.R. 2022. Mechanistic insights into luteolin-loaded elastic liposomes for transdermal delivery: HSPiP predictive parameters and instrument-based evidence. ACS Omega. 7(51): 48202-48214. https://doi.org/10.1021/acsomega.2c06288

Anuradha, U., Bhavana, V., Chary, P.S., Kalia, N.P., and Mehra, N.K. 2024. Exploration of the topical nanoemulgel bearing with ferulic acid and essential oil for diabetic wound healing. Pathophysiology. 31(4): 680-698. https://doi.org/10.3390/pathophysiology31040049

Askarizadeh, M., Esfandiari, N., Honarvar, B., Sajadian, S.A., and Azdarpour, A. 2023. Kinetic modeling to explain the release of medicine from drug delivery systems. ChemBioEng Reviews. 10(6): 1006-1049. https://doi.org/10.1002/cben.202300027

Bahman, M., Topelius, N.S., and Viitala, T. 2025. Semi-solid extruded tablets for personalized pediatric use: Development, quality control and in-vitro assessment of enteral tube administration. European Journal of Pharmaceutical Sciences. 107122. https://doi.org/10.1016/j.ejps.2025.107122

Bashir, M., Ahmad, J., Asif, M., Khan, S.U., Irfan, M., Ibrahim, A.Y., Asghar, S., Khan, I.U., Iqbal, M.S., Haseeb, A., et al. 2021. Nanoemulgel, an innovative carrier for diflunisal topical delivery with profound anti-inflammatory effect: In vitro and in vivo evaluation. International Journal of Nanomedicine. 2021: 1457-1472. https://doi.org/10.2147/IJN.S294653

Buanpech, P., Chaijareenont, P., Wanachantararak, P., and Silthampitag, P. 2024. Antifungal effect and durability of chitosan oligosaccharide coating on heat-cured polymethylmethacrylate surface. Natural and Life Sciences Communications. 23(1): e2024004. https://doi.org/10.12982/NLSC.2024.004

Bunaciu, A.A., Hoang, V.D., and Aboul-Enein, H.Y. 2025. Fourier transform infrared spectroscopy used in drug excipients compatibility studies. Applied Spectroscopy Reviews. 60(5): 385-403. https://doi.org/10.1080/05704928.2024.2438747

Cao, H., Zhang, B., Wang, W., Li, Y., Jia, M., Yu, W., Liu, L., Huang, J., Chen, H., Lai, Y., et al. 2024. Development of high-flowability melt PPS-based composites through blending with g-C3N4. Polymer. 293: 126683. https://doi.org/10.1016/j.polymer.2024.126683

Cavallaro, S., Hååg, P., Viktorsson, K., Krozer, A., Fogel, K., Lewensohn, R., Linnros, J., and Dev, A. 2021. Comparison and optimization of nanoscale extracellular vesicle imaging by scanning electron microscopy for accurate size-based profiling and morphological analysis. Nanoscale Advances. 3(11): 3053-3063. https://doi.org/10.1039/D0NA00948B

Chanyachailert, P., Leeyaphan, C., and Bunyaratavej, S. 2023. Cutaneous fungal infections caused by dermatophytes and non-dermatophytes: An updated comprehensive review of epidemiology, clinical presentations, and diagnostic testing. Journal of Fungi. 9(6): 669. https://doi.org/10.3390/jof9060669

Dave, P.N., Macwan P.M., and Kamaliya, B. 2023. Biodegradable Gg-cl-poly(nipam-co-AA)/-o-MWCNT based hydrogel for combined drug delivery system of metformin and sodium Diclofenac: In vitro studies. RSC Advances. 13(33): 22875 -22885.c https://doi.org/10.1039/D3RA04728H

Donthi, M.R., Munnangi, S.R., Krishna, K.V., Saha, R.N., Singhvi, V., and Dubey, S.K. 2023. Nanoemulgel: A novel nano carrier as a tool for topical drug delivery. Pharmaceutics. 15(1): 164. https://doi.org/10.3390/pharmaceutics15010164

Duangjit, S., Jandum, S., Palakun, P., Rangsimawong, W., Sritananuwat, P., Samseethong, T., Jitsaeng, K., and Bumrungthai, S. 2024. Physicochemical characteristics and stability assessment of a topical formulation comprising Allium cepa and quercetin for cutaneous scar management. Natural and Life Sciences Communications. 23(4): e2024065. https://doi.org/10.12982/NLSC.2024.065

Filippov, S.K., Khusnutdinov, R., Murmiliuk, A., Inam, W., Zakharova, L.Ya., Zhang, H., and Khutoryanskiy, V.V. 2023. Dynamic light scattering and transmission electron microscopy in drug delivery: A roadmap for correct characterization of nanoparticles and interpretation of results. Materials Horizons. 10: 5354-5370. https://doi.org/10.1039/D3MH00717K

Gaanapriya, V., Sivakumar, V.M., and Thirumarimurugan, M. 2024. In-vitro studies of bioactive nanoemulgel from agro-waste and mathematical modeling of drug release. Indian Journal of Chemical Technology. 31: 135-142.

Goyal, G., Garg, T., Malik, B., Chauhan, G., Rath, G., and Goyal, A.K. 2021. Development and characterization of niosomal gel for topical delivery of benzoyl peroxide. Drug Delivery. 22(8): 1027-1042. https://doi.org/10.3109/10717544.2013.855277

Grzelecka, M., Kowalski, P., and Nowak, A. 2025. Evaluation of drug diffusion profiles in nanoemulgel formulations. Journal of Pharmaceutical Research. 18(2): 145-158.

Hassan, M.M., Mahmud, M.R.A., Islam, M.G. 2019. GC-MS analysis of bio-active compounds in ethanol extract of Putranjiva roxburghii Wall. fruit peel. Pharmacognosy Journal. 11(1): 146-149. https://doi.org/10.5530/pj.2019.1.24

Hosny, K.M., Khallaf, R.A., Asfour, H.Z., Rizg, W.Y., Alhakamy, N.A., Sindi, A.M., Alkhalidi, H.M., Abualsunun, W.A., Bakhaidar, R.B., Almehmady, A.M., et al. 2021. Development and optimization of cinnamon oil nanoemulgel for enhancement of solubility and evaluation of antibacterial, antifungal and analgesic effects against oral microbiota. Pharmaceutics. 13(7): 1008. https://doi.org/10.3390/pharmaceutics13071008

Jafari, S., Soleimani, M., and Badinezhad, M. 2022. Application of different mathematical models for further investigation of in vitro drug release mechanisms based on magnetic nano-composite. Polymer Bulletin. 79(2): 1021-1038. https://doi.org/10.1007/s00289-021-03537-9

Konappa, N., Udayashankar, A.C., Krishnamurthy, S., Pradeep, C.K., Chowdappa, S., and Jogaiah, S. 2020. GC-MS analysis of phytoconstituents from Amomum nilgiricum and molecular docking interactions of bioactive serverogenin acetate with target proteins. Scientific Reports. 10(1): 16438. https://doi.org/10.1038/s41598-020-73442-0

Kumadoh, D., Amekyeh, H., Archer, M.A., Kyene, M.O., Yeboah, G.N., Brew-Daniels, H., Adi-Dako, O., Osei-Asare, C., Adase, E., and Appiah, A.A. 2024. Determination of consistency in pH of some commercial herbal formulations in Ghana. Journal of Herbal Medicine. 45: 100876. https://doi.org/10.1016/j.hermed.2024.100876

Lal, D.K., Kumar, B., Saeedan, A.S., and Ansari, M.N. 2023. An overview of nanoemulgels for bioavailability enhancement in inflammatory conditions via topical delivery. Pharmaceutics. 15(4): 1187. https://doi.org/10.3390/pharmaceutics15041187

Lukić, M., Pantelić, I., and Savić, S.D. 2021. Towards optimal pH of the skin and topical formulations: From the current state of the art to tailored products. Cosmetics. 8(3): 69. https://doi.org/10.3390/cosmetics8030069

Mahtab, A., Anwar, M., Mallick, N., Naz, Z., Jain, G.K., and Ahmad, F.J. 2016. Transungual delivery of ketoconazole nanoemulgel for the effective management of onychomycosis. AAPS PharmSciTech. 17: 1477-1490. https://doi.org/10.1208/s12249-016-0488-0

Modhave, D., Vrielynck, S., and Roeleveld, K. 2024. Assessing drug product shelf life using the accelerated stability assessment program: A case study of a GLPG4399 capsule formulation. Pharmaceutics. 16(11): 1400. https://doi.org/10.3390/pharmaceutics16111400

Noor, A., Jamil, S., Sadeq, T.W., Mohammed Ameen, M.S., and Kohli, K. 2023. Development and evaluation of nanoformulations containing timur oil and rosemary oil for treatment of topical fungal infections. Gels. 9(7): 516. https://doi.org/10.3390/gels9070516

Nizam, N.N., Mahmud, S., Ark, S.A., Kamruzzaman, M., and Hasan, K.M. 2023. Bakuchiol, a natural constituent and its pharmacological benefits. F1000Research. 12: 29. https://doi.org/10.12688/f1000research.129072.2

Ojsteršek, T., Vrecer, F., and Hudovornik, G. 2024. Comparative fitting of mathematical models to carvedilol release profiles obtained from hypromellose matrix tablets. Pharmaceutics. 16: 498. https://doi.org/10.3390/pharmaceutics16040498

Opaleke, D.O., Salami, L.I., Uko-Aviomoh, E.E., Ijabadeniyi, O.A., and Arise, A.K. 2022. Determination of proximate composition, phytochemical contents, amino acid profile, and functional characteristics of Landolphia togolana root bark as a potential functional food. Chiang Mai University Journal of Natural Sciences. 21(1): e2022020. https://doi.org/10.12982/CMUJNS.2022.020

Phalak, S.D., Babu, R.H., Kaur, P., Pawar, H.R., Nagarani, B., Sangeetha, J., Kiran, K., and Begam, T. 2024. Development and characterization of naftifine- loaded nanoemulsion based nanogels: A promising approach for antifungal drug delivery. Frontiers in Health Informatics. 13(3): 9909-9926.

Rathee, J., Malhotra, S., Pandey, M., Jain, N., Kaul, S., Gupta, G., and Nagaich, U. 2023. Recent update on nanoemulsion impregnated hydrogel: A gleam into the revolutionary strategy for diffusion-controlled delivery of therapeutics. AAPS PharmSciTech. 24(6): 151. https://doi.org/10.1208/s12249-023-02611-x

Sadler, C.J., Sandler, J.P., Shamsabadi, A., Frenette, L.C., Creamer, A., and Stevens, M.M. 2025. Signal enhancement in immunoassays via coupling to catalytic nanoparticles. ACS Sensors. 10(6): 4622-4633. https://doi.org/10.1021/acssensors.5c00995

Saravanan, M., Mahalakshmi, B., Afeeza, K.L.G., Subriya, S., Sridevi, M., and Dilipan, E. 2025. Formulation and characterization of sodium alginate-based nanoparticle doped with seagrass (Syringodium isoetifolium) extract: In vitro anti-inflammatory, antioxidant and anti-diabetic activities. Natural and Life Sciences Communications. 24(2): e2025035. https://doi.org/10.12982/NLSC.2025.035

Sghier, K., Mur, M., Veiga, F., Paiva-Santos, A.C., and Pires, P.C. 2024. Novel therapeutic hybrid systems using hydrogels and nanotechnology: A focus on nanoemulgels for the treatment of skin diseases. Gels. 10(1): 45. https://doi.org/10.3390/gels10010045

Sharma, S. and Bansal, V. 2023. Development and characterization of Clitoria ternatea and Salvia officinalis anti-inflammatory gel. PEXACY International Journal of Pharmaceutical Science. 2(11): 36-56.

Shenoy, M., Raju, P.V., and Prasad, J. 2021. Optimization of physical schemes in WRF model on cyclone simulations over Bay of Bengal using one-way ANOVA and Tukey's test. Scientific Reports. 11(1): 24412. https://doi.org/10.1038/s41598-021-02723-z

Wang, H., Chen, K., Xue, R., Turghun, C., and Han, B. 2023. Identification of the chemical constituents in Cullen corylifolium ethanolic extract by LC-MS/MS and GC-MS. Natural Product Research. 37(8): 1392-1396. https://doi.org/10.1080/14786419.2021.2007911

Wu, Q., Cen, F., Xie, Y., Ning, X., Wang, J., Lin, Z., and Huang, J. 2025. Nanoparticle-based antifungal therapies innovations mechanisms and future prospects. PeerJ. 13: e19199. https://doi.org/10.7717/peerj.19199

Yetukuri, K., Umashankar, M.S., and Raghavamma, S.T.V. 2025. Development of Bergamot nanoemulgel using central composite design and its evaluation for antimicrobial activity. Journal of Pharmaceutical Science. 15(11): 123-135. https://doi.org/10.7324/JAPS.2025.236869

Zhang, M., Zhao, J., Dai, X., and Li, X. 2023. Extraction and analysis of chemical compositions of natural products and plants. Separations. 10(12): 598. https://doi.org/10.3390/separations10120598

OPEN access freely available online

Natural and Life Sciences Communications

Chiang Mai University, Thailand. https://cmuj.cmu.ac.th

Suplementary data

Table S1. Qualitative estimation of organoleptic properties of Cullen Corylifolium seed extract loaded nano-emulgel.

Table S2. ANOVA for pH.

ANOVA for viscosity

Table S3. ANOVA for viscosity.

ANOVA for spreadability

Table S4. ANOVA for spreadability.

ANOVA for extradubility

Table S5. ANOVA for extradubility.

ANOVA for in vitro diffusion study

Table S6. ANOVA for in vitro diffusion study.

Post hoc test

Table S7. Tukey HSD for formulation comparisons in in vitro drug release studies.

Table S8. Release kinetics data for Emulgel formulation CN1-CN6.

Figure S1. Post Hoc Tukey HSD graph in in vitro drug release studies.

Drug Release Kinetics

Figure S2. Zero order Kinectics data for formulation CN1-CN6 of Emulgel.

Figure S3. First order Kinectics data for formulation CN1-CN6 of Emulgel.

Figure S4. Higuchi Diffusion data for formulation CN1-CN6 of Emulgel.

Figure S5. Hixson-Crowel Plot data for formulation CN1-CN6 of Emulgel.

Figure S6. Korsmeyer Peppas Plot data for formulation CN1-CN6 of Emulgel.