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INTRODUCTION
Inflammation is a protective immune response against infection, tissue injury, and irritants, characterized by redness, swelling, pain, and loss of function (Kim and Lee, 2024). Although synthetic anti-inflammatory agents such as NSAIDs and corticosteroids are effective, their prolonged use is associated with adverse effects including gastric irritation, renal toxicity, and cardiovascular complications (Stone et al., 2024). Therefore, the development of safer plant-based topical therapies has gained increasing interest.
Tinospora cordifolia (Giloy), a medicinal plant belonging to the family Menispermaceae, possesses well-documented anti-inflammatory, antioxidant, immunomodulatory, and antimicrobial properties due to the presence of alkaloids, glycosides, diterpenoids, phenolics, and polysaccharides (Saha and Ghosh, 2012; Sharma et al., 2019). Previous studies have reported that T. cordifolia extract can modulate inflammatory cytokines and immune responses, supporting its potential application in inflammatory skin disorders such as psoriasis and dermatitis.
Topical gels are widely used for dermal drug delivery because of their ease of application, better patient compliance, and prolonged retention at the site of action (Patil et al., 2019). Carbopol 934 is commonly employed as a gelling polymer for topical formulations, while propylene glycol acts as a humectant and penetration enhancer, and Triethanolamine (TEA) facilitates gel formation (Shiehzadeh et al., 2023).
Design of Experiments (DoE) using Box-Behnken Design (BBD) provides a systematic and efficient approach for formulation optimization by evaluating the influence of multiple formulation variables with fewer experimental runs (Barad, 2014; Abdallah, 2014). Therefore, the present study aimed to develop and optimize a Tinospora cordifolia extract-loaded topical gel using BBD and evaluate its physicochemical properties, extract release behavior, and antimicrobial activity.
MATERIALS AND METHODS
Materials
Giloy stem extract (Tinospora cordifolia) was procured from an authenticated online platform. Carbopol 934 was obtained from Loba Chemie Pvt. Ltd., Mumbai. Triethanolamine, methylparaben, propylene glycol, isopropyl alcohol, sodium hydroxide, and buffer salts were procured from Thermo Fisher Scientific India Pvt. Ltd., Mumbai. Distilled water was prepared in-house. Microbial strains Staphylococcus epidermidis (MTCC 441), Escherichia coli (MTCC 1687), and Candida albicans were obtained from Microbial Type Culture Collection and Gene Bank (MTCC), India.
Preparation of Giloy Stem Extract
50 g of powdered stem of T. cordifolia was Soxhlet-extracted using 480 mL ethanol (Ezhilarasu et al., 2023) at approximately 50ºC for 6-8 hr (7 cycles) until the siphon tube became colorless. The extract was concentrated by rotary evaporation and the dried residue was stored for gel preparation.
Formulation of Giloy Herbal Gel
The gel (100 g) was prepared by slowly dispersing Carbopol 934 in distilled water with continuous stirring (Yetukuri et al., 2024) followed by homogenization using Ultra-Turrax (30 min) to form the gel base. Giloy extract (1 g) was dissolved in propylene glycol (15 mL) and isopropyl alcohol (2 mL); methylparaben (0.150 g) was incorporated into this solution. The extract solution was gradually added to the Carbopol dispersion with continuous stirring. Volume was adjusted to 100 mL with distilled water, followed by dropwise addition of TEA to neutralize Carbopol and achieve the desired gel consistency.
Screening of Critical Formulation Variables
Preliminary trials established feasible ranges for the three independent variables: Carbopol 934 (0.5-1.5% w/w), TEA (0.3-1.0% w/w), and propylene glycol (5.0-15.0% w/w). These ranges were selected to ensure adequate gel formation and skin compatibility.
ATR-FTIR Analysis
ATR-FTIR analysis of the Giloy extract and herbal gel formulation was performed in the range of 4000-400 cm⁻¹ using ATR-FTIR spectroscopy. The spectra showed characteristic peaks at 3289.71 cm⁻¹ corresponding to O-H stretching vibrations and at 1633.06 cm⁻¹ corresponding to C=C stretching and N-H bending vibrations, confirming the presence of major phytoconstituents in the formulation (Kulkarni et al., 2018).
GC - MS Analysis
Gas Chromatography - Mass Spectroscopy (GC-MS) analysis of the Giloy stem powder extract was performed after stabilizing the instrument and ensuring proper helium carrier gas flow (Figure 1). One microliter of the prepared sample was injected, and compounds were separated in the GC column followed by mass spectral detection. The obtained spectra were compared with the NIST library, and compounds were identified based on retention time and spectral matching.
Optimization of gel formulation using DoE
A three-factor, three-level BBD was employed using Design-Expert® software. The independent variables Carbopol 934 (A), TEA (B), and propylene glycol (C) were evaluated at three levels (low: −1, medium: 0, high: +1) as shown in Table 1. Seventeen experimental runs including five center points were generated. Dependent responses were viscosity (cP), retention time (min), and phenolic content (%). Data were fitted to a quadratic polynomial model and analyzed by ANOVA. Numerical optimization was performed using the desirability function approach.
| Factor | Independent Variable | Low (−1) | Medium (0) | High (+1) |
|---|---|---|---|---|
| A | Carbopol 934 (% w/w) | 0.5 | 1 | 1.5 |
| B | TEA (% w/w) | 0.3 | 0.65 | 1 |
| C | Propylene glycol (% w/w) | 5 | 10 | 15 |
Evaluation tests
The optimized Giloy herbal gel was evaluated for physicochemical and stability parameters including physical appearance, pH, viscosity, retention time, phenolic content, spreadability, extrudability, homogeneity, skin compatibility, washability, and short-term stability. Physical characteristics such as colour, odour, consistency, homogeneity, and grittiness were assessed visually (Inamdar and Bhise, 2023; Prabu et al., 2017). The pH was determined using a calibrated digital pH meter after dispersing the gel in distilled water (Dantas et al., 2016). Viscosity was measured using a Brookfield viscometer at 10 rpm, while retention time and spreadability were evaluated using the glass slide method (Prabu et al., 2017). Total phenolic content was estimated by the Folin-Ciocalteu method and expressed as gallic acid equivalents (Upadhyay et al., 2013). Extrudability was determined using a collapsible tube method, whereas homogeneity and washability were assessed by visual and manual examination. Skin irritation potential was evaluated through a preliminary qualitative observation for visible signs of redness, itching, or irritation following topical application of the formulation (Yadav et al., 2024). Short-term stability studies were performed at 4±2ºC, 25±2ºC, and 40±2ºC for 24 hr, and the formulations were evaluated for changes in appearance, pH, and viscosity.
In vitro extract release study
In vitro extract release of the Giloy herbal gel was evaluated using a Franz diffusion cell with a hydrated dialysis membrane (Dereli et al., 2021). Franz Cells are a widely used methodology to evaluate in vitro drug permeation (Salamanca et al., 2018). The receptor compartment was filled with phosphate buffer (pH 6.8/7.4) maintained at 37±0.5ºC with continuous stirring. 1 g of gel was placed in the donor compartment, and samples were withdrawn at predetermined time intervals and replaced with fresh buffer to maintain sink conditions. The samples were analysed using a UV spectrophotometer, and cumulative extract release was calculated using a calibration curve prepared from Tinospora cordifolia extract. These values were expressed relative to the total extract content incorporated into the formulation.
Antimicrobial studies
Antimicrobial activity of the Giloy herbal gel was evaluated using the agar well diffusion and broth dilution methods. Brain-heart infusion agar plates inoculated with Staphylococcus epidermidis, Escherichia coli, and Candida albicans were used to determine the zones of inhibition after incubation at 37ºC for 24 hr. The Minimum Inhibitory Concentration (MIC) was determined by serial broth dilution using Giloy extract concentrations ranging from 5000 to 0.31 µg/mL (Kowalska-Krochmal and Dudek-Wicher, 2021). MIC was recorded as the lowest concentration showing no visible microbial growth after incubation under aseptic conditions (Vanegas et al., 2021).
RESULTS
Optimization of Giloy gel formulation using Box-Behnken design
The Box-Behnken Design (BBD) comprising 17 experimental runs (Table 2) demonstrated significant effects of Carbopol 934, Triethanolamine (TEA), and propylene glycol on the formulation responses. The quadratic models showed good statistical significance (p<0.05) with acceptable R² values (Table 3). Carbopol 934 and TEA significantly influenced viscosity and retention time, while propylene glycol markedly affected phenolic content. The 3D response surface plots indicated that increasing Carbopol 934 and TEA improved viscosity and retention behavior (Figure 2), whereas higher propylene glycol concentration enhanced phenolic content. Numerical optimization identified an optimal formulation containing approximately 1.5% Carbopol 934, 1% TEA, and 15% propylene glycol with an overall desirability of 0.849, confirming a robust and optimized formulation design.
| Run | A (%) | B (%) | C (%) | Viscosity (cP) | Retention Time (Sec) | Phenolic Content (%) |
|---|---|---|---|---|---|---|
| 1 | 1 | 0.3 | 15 | 42 | 118 | 22 |
| 2 | 1 | 0.65 | 10 | 48.5 | 120 | 19 |
| 3 | 0.5 | 0.3 | 10 | 40 | 116 | 17 |
| 4 | 1.5 | 1 | 10 | 56 | 126 | 18 |
| 5 | 1 | 0.65 | 10 | 48.5 | 119 | 19 |
| 6 | 1.5 | 0.3 | 10 | 53 | 124 | 17 |
| 7 | 1 | 0.65 | 10 | 48 | 120 | 19 |
| 8 | 1.5 | 0.65 | 15 | 51 | 123 | 22 |
| 9 | 0.5 | 1 | 10 | 47.3 | 117 | 17 |
| 10 | 1 | 0.3 | 5 | 44 | 118 | 16 |
| 11 | 0.5 | 0.65 | 5 | 39.2 | 115 | 16 |
| 12 | 0.5 | 0.65 | 15 | 42 | 117 | 22 |
| 13 | 1 | 1 | 15 | 50 | 124 | 23 |
| 14 | 1 | 0.65 | 10 | 48.5 | 120 | 19 |
| 15 | 1.5 | 0.65 | 5 | 57 | 127 | 16 |
| 16 | 1 | 1 | 5 | 52 | 125 | 16 |
| 17 | 1 | 0.65 | 10 | 48 | 120 | 19 |
| Source | Viscosity (F, p-value) | Retention Time (F, p-value) | Phenolic Content (F, p-value) |
|---|---|---|---|
| Model | 27.65, <0.0001 (sig) | 11.51, 0.0020 (sig) | 278.86, <0.0001 (sig) |
| A (Carbopol 934) | 86.06, <0.0001 (sig) | 11.19, 0.0183 (sig) | 3.50, 0.1036 (ns) |
| B (TEA) | 78.56, <0.0001 (sig) | 3.53, 0.0788 (ns) | 14.00, 0.0072 (sig) |
| C (Propylene glycol) | 5.99, 0.0455 (sig) | 1.35, 0.4708 (ns) | 2187.50, <0.0001 (sig) |
| AB | 4.21, 0.0794 (ns) | 0.13, 0.7299 (ns) | 0.00, 1.0000 (ns) |
| AC | 1.76, 0.2044 (ns) | 4.05, 0.0690 (ns) | 0.00, 1.0000 (ns) |
| BC | 0.00, 1.0000 (ns) | 0.13, 0.7299 (ns) | 7.00, 0.0331 (sig) |
| A² | 1.11, 0.3277 (ns) | 0.02, 0.8869 (ns) | 117.89, <0.0001 (sig) |
| B² | 1.21, 0.3196 (ns) | 0.09, 0.7689 (ns) | 66.32, <0.0001 (sig) |
| C² | 96.01, 0.0001 (sig) | 21.05, 0.0052 (sig) | 117.89, <0.0001 (sig) |
| Lack of Fit | 32.86, 0.0028 (sig) | 21.25, 0.0064 (sig) | - |
ATR-FTIR Analysis
The ATR-FTIR spectra of the Giloy extract and herbal gel formulation exhibited characteristic peaks corresponding to major functional groups present in the formulation (Figure 3). Broad peaks observed around 3329-3272 cm⁻¹ indicated O-H stretching of phenols and alcohols, while peaks near 2974 and 2912 cm⁻¹ represented C-H stretching of alkyl groups. The peak around 1715-1730 cm⁻¹ corresponded to C=O stretching of Carbopol and methyl paraben. Additional peaks at 1645, 1328, 1279, and 1043 cm⁻¹ confirmed the presence of carbonyl, phenolic, and glycosidic functional groups, indicating compatibility of the extract with formulation excipients and retention of major phytoconstituents in the gel formulation.
Evaluation Studies
The optimized gel showed satisfactory physicochemical properties suitable for topical application. The formulation exhibited a skin-compatible pH (6.52±0.09), good viscosity (52,381±1037 cP), and adequate retention time (146.3±4.1 sec), indicating desirable consistency and adhesive characteristics. The gel also demonstrated satisfactory phenolic content (18.53%), good spreadability (25.0±4.3 g·cm/sec), easy extrudability, homogeneity without phase separation, and good washability. Short-term stability studies conducted at 4±2ºC, 25±2ºC, and 40±2ºC showed no significant changes in appearance, pH, or viscosity, confirming formulation stability under different storage conditions (Table 4).
| Parameter | Observed Value (Mean±SD) | Inference |
|---|---|---|
| pH | 6.52±0.09 | Skin-compatible |
| Viscosity (cP) | 52,381±1037 | Good consistency for topical use |
| Retention time (sec) | 146.3±4.1 | Good adhesive properties |
| Phenolic content (%) | 18.53% | Satisfactory phytoconstituent retention |
| Spreadability (g·cm/sec) | 25.0±4.3 | Adequate for topical application |
| Extrudability | 20 g | Easy extrusion from tube |
| Homogeneity | Homogeneous | No lumps or phase separation |
| Skin irritation potential | No visible irritation observed | Preliminary qualitative assessment |
| Washability (sec) | 23.3±5.1 | Easily washable with water |
Antimicrobial studies
The optimized gel exhibited antimicrobial activity against all tested microbial strains. The formulation showed zones of inhibition of 17 mm against Staphylococcus epidermidis, 12 mm against Escherichia coli, and 10 mm against Candida albicans, whereas the plain gel base showed no inhibition. The standard drug produced a zone of inhibition of 22 mm against Staphylococcus epidermidis, Escherichia coli, and Candida albicans under the experimental conditions employed. The Minimum Inhibitory Concentration (MIC) values were found to be 625 µg/mL for Staphylococcus epidermidis, 1250 µg/mL for Escherichia coli, and 2500 µg/mL for Candida albicans, indicating comparatively higher antimicrobial activity against the Gram-positive bacterium Staphylococcus epidermidis.
In vitro extract release study
The optimized formulation exhibited sustained extract release, with approximately 100% cumulative extract release achieved within 4 hr (Figure 4). Kinetic modeling demonstrated that the release profile showed the highest correlation with the zero-order model (R²=0.975), followed by the Higuchi model (R²=0.952), whereas the first-order model showed comparatively lower correlation (R²=0.743). These findings suggest that the formulation exhibited near-constant extract release with diffusion-controlled release characteristics from the polymeric gel matrix.
DISCUSSION
The present study successfully developed and optimized a Tinospora cordifolia extract-loaded topical gel using Box-Behnken Design (BBD). Carbopol 934, triethanolamine (TEA), and propylene glycol significantly influenced the physicochemical characteristics of the formulation, particularly viscosity, retention time, and phenolic content. Carbopol 934 acted as the primary gelling polymer, while TEA facilitated gel network formation through neutralization. Propylene glycol contributed to improved spreadability and consistency due to its humectant and penetration-enhancing properties. Similar observations have been reported in previous Carbopol-based topical gel formulations (Prabu et al., 2017).
The optimized formulation exhibited suitable physicochemical properties for topical application. The pH of the gel (6.52±0.09) was within the acceptable physiological skin range, indicating good skin compatibility and minimal irritation potential. The gel also demonstrated high viscosity, satisfactory retention time, good spreadability, and easy extrudability, which are important parameters for patient compliance and prolonged skin contact. Such properties are considered essential quality attributes for topical herbal formulations (Inamdar and Bhise, 2023).
In vitro extract release studies demonstrated sustained release behavior with approximately complete extract release within 4 hr. Release kinetics showed good fitting to both zero-order and Higuchi models, suggesting controlled diffusion-mediated release from the polymeric gel matrix. Sustained release of extract constituents from topical gels may prolong the availability of bioactive phytoconstituents at the site of application and enhance therapeutic efficacy. Similar diffusion-controlled release profiles have been reported for Carbopol-based hydrogels due to their crosslinked polymeric structure (Dantas et al., 2016).
ATR-FTIR analysis confirmed compatibility between the Giloy extract and formulation excipients. Characteristic peaks corresponding to hydroxyl, carbonyl, alkyl, and glycosidic functional groups were retained without significant peak shifting or disappearance, indicating the absence of major chemical interaction. These findings confirm successful incorporation of the extract into the gel matrix and support formulation stability (Kulkarni et al., 2018).
The optimized Giloy gel also demonstrated antimicrobial activity against Staphylococcus epidermidis, Escherichia coli, and Candida albicans, with comparatively higher activity against S. epidermidis. The antimicrobial activity may be attributed to phytoconstituents such as alkaloids, glycosides, diterpenoids, and phenolic compounds present in T. cordifolia (Upadhyay et al., 2013). Short-term stability studies showed no significant changes in appearance, pH, or viscosity under different storage conditions, indicating acceptable formulation stability. Overall, the developed Giloy herbal gel demonstrated promising physicochemical, release, and antimicrobial properties for potential topical anti-inflammatory application.
CONCLUSION
A Tinospora cordifolia extract-loaded topical gel was successfully optimized using Box-Behnken Design. The optimized formulation exhibited suitable physicochemical properties, good skin compatibility, satisfactory phenolic content, and sustained extract release behavior. ATR-FTIR analysis confirmed compatibility between the extract and excipients. The formulation also demonstrated notable antimicrobial activity against Staphylococcus epidermidis, Escherichia coli, and Candida albicans. These findings suggest that the Giloy herbal gel is a promising candidate for topical anti-inflammatory and antimicrobial therapy, warranting further in vivo and long-term stability studies.