Formulation, Characterization and Anti-Inflammatory Evaluation of Barbaloin Loaded Topical Gel

Inflammation is a complex protective biological response initiated in response to tissue injury, infection, or other harmful stimuli. It plays an essential role in eliminating injurious agents and initiating tissue repair. Although acute inflammation is generally beneficial and self-limiting, persistent or dysregulated inflammation may result in progressive tissue damage and contribute to the development of chronic inflammatory disorders, including dermatitis, psoriasis, rheumatoid arthritis, and osteoarthritis [1]. Non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids are widely used for the management of inflammatory conditions because of their established therapeutic efficacy. However, prolonged or inappropriate use of these agents may be associated with adverse effects such as gastrointestinal irritation, renal impairment, cardiovascular complications, immunosuppression, and skin-related effects. These limitations have encouraged the investigation of alternative therapeutic agents with improved safety and the potential for localized drug delivery [2,3]. Natural products have attracted considerable interest in pharmaceutical research because of their diverse pharmacological activities and potential therapeutic benefits. Aloe vera (Aloe barbadensis Miller) is a medicinal plant widely investigated for its anti-inflammatory, antioxidant, antimicrobial, and wound-healing properties. Barbaloin (aloin), a major anthraquinone C-glycoside associated with Aloe vera, is one of its important bioactive constituents. Experimental studies have reported that Barbaloin can modulate inflammatory responses through effects on inflammatory mediators, oxidative stress, and cellular signalling pathways, supporting its potential as a natural anti-inflammatory agent [4–9]. Despite its pharmacological potential, the effective topical delivery of Barbaloin presents formulation challenges. The therapeutic performance of a topical drug depends not only on the intrinsic activity of the active compound but also on its physicochemical characteristics, stability, drug release, and ability to remain available at the site of application. Conventional administration may also result in inadequate drug concentration at the target site. Therefore, development of an appropriate topical delivery system may improve local drug availability and provide more controlled drug release [10]. Topical drug delivery is particularly advantageous for the treatment of localized skin disorders because it allows the active pharmaceutical ingredient to be delivered directly to the affected area while potentially reducing systemic exposure. Among various topical dosage forms, gels are widely used because of their ease of application, non-greasy nature, good patient acceptability, and ability to provide uniform drug distribution. Hydrophilic polymeric gels can also be designed to control drug release by modifying the viscosity and structure of the gel matrix [11–14]. The selection and concentration of gelling polymers are important determinants of the performance of topical formulations. Carbopol 934 and hydroxypropyl methylcellulose (HPMC) are commonly used hydrophilic polymers that can provide suitable viscosity, consistency, spreadability, and drug-release characteristics. Changes in polymer concentration can alter the microstructure of the gel matrix and consequently influence drug diffusion, drug availability, and overall formulation performance [15–18]. Hence, systematic optimization of polymer concentration is essential for developing a topical formulation with desirable physicochemical and therapeutic properties. The skin consists principally of the epidermis, dermis, and subcutaneous tissue and acts as an important barrier against external substances. While this barrier protects the body, it can also restrict the penetration of many therapeutic agents. For localized inflammatory skin conditions, an appropriately designed topical formulation may help maintain the active compound at or near the site of inflammation while minimizing unnecessary systemic exposure. Therefore, formulation strategies that provide satisfactory spreadability, skin compatibility, stability, and controlled drug release are important for successful topical therapy [19–21]. Although the pharmacological properties of Aloe vera and Barbaloin have been extensively investigated, comparatively limited work has focused on the systematic development and optimization of a Barbaloin-loaded topical gel using different concentrations of Carbopol 934 and HPMC, followed by comprehensive physicochemical, drug-release, kinetic, stability, and anti-inflammatory evaluation. In particular, establishing an appropriate polymer concentration that provides a balance between formulation characteristics and biological performance is important for the development of an effective topical dosage form [22–25]. Therefore, the present study was undertaken to formulate and evaluate Barbaloin-loaded topical gel formulations using Carbopol 934 and HPMC as gelling polymers. Nine formulations (F1–F9) were prepared with varying polymer concentrations and evaluated for their physicochemical properties, drug content, in-vitro drug release, release kinetics, stability, and in-vitro anti-inflammatory activity using the protein denaturation inhibition assay. Based on the overall evaluation, the formulation demonstrating the most desirable characteristics and anti-inflammatory performance was selected as the optimized formulation. The study was aimed at developing a stable and patient-acceptable topical gel capable of providing controlled Barbaloin delivery and promising localized anti-inflammatory activity.

2. MATERIALS AND METHODS

2.1 MATERIALS

Barbaloin was procured from Yucca Enterprises, Mumbai, India, and used as the active pharmaceutical ingredient. Carbopol 934 and hydroxypropyl methylcellulose (HPMC) were selected as gelling polymers for the development of topical gel formulations. Propylene glycol was incorporated as a humectant and penetration-enhancing agent [26], while methyl paraben served as the preservative. Triethanolamine was used as a neutralizing agent for pH adjustment and development of the desired gel consistency. Purified water was used as the aqueous vehicle. Methanol, phosphate buffer components, hydrochloric acid, and other reagents required for the analytical and experimental procedures were of analytical grade. Bovine serum albumin (BSA) was used for the in-vitro protein denaturation assay. All materials were used as received without further purification.

2.2 Preparation of Barbaloin Topical Gel

Barbaloin topical gel formulations were prepared by the dispersion method using Carbopol 934 or HPMC as the gelling polymer. The required quantity of polymer was gradually dispersed in purified water under continuous stirring and allowed to hydrate adequately. Propylene glycol and methyl paraben were subsequently incorporated into the hydrated polymer dispersion. Barbaloin was then added gradually with continuous stirring to obtain a uniform drug dispersion. Triethanolamine was added slowly to adjust the pH and obtain the required gel consistency. The prepared formulations were kept undisturbed to allow entrapped air to escape and were subsequently transferred into suitable containers for further evaluation. Nine formulations (F1–F9) were developed by varying the concentration of Carbopol 934 or HPMC, while the quantities of Barbaloin, propylene glycol, methyl paraben, and other formulation components were maintained constant. The composition of the prepared formulations is presented in Table 1.

Time (hr)	Cumulative Drug release (%±SD)
0.5	14.28±0.42
1	27.64±0.56
2	42.85±0.61
3	57.92±0.54
4	70.48±0.47
5	81.73±0.52
6	89.64±0.49
7	94.82±0.45
8	98.21±0.62

Figure 3.1 Higuchi modelrelease kinetic (F1-F4)

Figure 3.1Higuchi modelrelease kinetic (F5-F9)

Figure 3.5(A) Zero-order Release Plot of Optimized Barbaloin Gel (F3)

Figure 3.5(B) First-order Release Plot of Optimized Barbaloin Gel (F3)

Figure 3.5(D) Korsmeyer–Peppas Release Plot of Optimized Barbaloin Gel (F3)

Figure3.6 Higuchi Model Release Plot

Figure 3.7-Higuchi Release Comparison of Optimized Barbaloin Gel (F3) and Standard Ketoprofen Gel.

Figure 3.8 In-vitro anti -inflammatory acticity of barbaloin topical gel

Discussion

The variation in protein denaturation inhibition among the formulations indicates that the type and concentration of the gelling polymer influenced the anti-inflammatory performance of the Barbaloin-loaded gels. The comparatively higher inhibition observed with Carbopol 934-based formulations may be related to differences in gel structure and drug availability. F3, containing 1.5% Carbopol 934, showed the highest inhibition (76.84 ± 0.88%), whereas a slight reduction was observed following further increase in polymer concentration, as demonstrated by F4 (73.96 ± 1.05%). This reduction may be associated with increased polymer concentration and viscosity, which can restrict drug mobility and release. A similar trend was observed with HPMC, where activity increased up to F8 and subsequently decreased in F9. These findings suggest that an optimum polymer concentration is important for achieving desirable anti-inflammatory performance. Accordingly, F3 was selected as the optimized formulation for further evaluation.

DISCUSSION

The present study was focused on the formulation and evaluation of Barbaloin-loaded topical gel formulations using Carbopol 934 and HPMC as gelling polymers. Nine formulations (F1–F9) were prepared with varying polymer concentrations and evaluated for their physicochemical characteristics, drug content, drug-release behaviour, release kinetics, irritation potential, stability, and in-vitro anti-inflammatory activity. All prepared formulations exhibited satisfactory physical appearance, with clear to translucent appearance, smooth texture, and good homogeneity. No visible grittiness, particulate matter, or phase separation was observed. The pH values of the formulations ranged from 6.10 ± 0.03 to 6.82 ± 0.04, indicating that the prepared gels possessed pH values suitable for topical application [28]. A gradual increase in pH was observed with increasing polymer concentration. The spreadability of the formulations was also satisfactory, although formulations containing higher polymer concentrations showed comparatively lower spreadability. This behaviour may be attributed to the formation of a denser polymeric network and increased gel consistency. The viscosity of the formulations ranged from 8,900 to 10,180 cP. Carbopol 934-based formulations showed viscosity values ranging from 8,900 to 9,345 cP, whereas HPMC-based formulations exhibited values ranging from 9,498 to 10,180 cP. The overall increase in viscosity with increasing polymer concentration indicates that polymer concentration influenced the consistency and internal structure of the gel matrix. Higher viscosity may also affect drug mobility and consequently influence the release characteristics of the formulation [29]. The drug content of the prepared formulations ranged from 97.45 ± 0.82% to 99.98 ± 0.54%, demonstrating satisfactory and relatively uniform incorporation of Barbaloin into the gel formulations. Among the investigated formulations, F3 exhibited the highest drug content of 99.98 ± 0.54%. The high drug content observed across the formulations indicates that the preparation method was suitable for obtaining relatively uniform drug distribution within the gel matrix. The optimized F3 formulation was subjected to an in-vitro drug-release study using a Franz diffusion cell. The cumulative drug release increased progressively from 14.28 ± 0.42% at 0.5 h to 98.21 ± 0.62% at 8 h. The progressive release profile indicates that the gel matrix provided a controlled environment for Barbaloin release over the investigated period. The polymeric network may have influenced the movement of the drug through the hydrated gel matrix and thereby contributed to the observed release behaviour. The release data of F3 were fitted to Zero-order, First-order, Higuchi, and Korsmeyer–Peppas kinetic models. The Higuchi model exhibited the highest regression coefficient (R² = 0.994), followed by the Korsmeyer–Peppas model (R² = 0.989), First-order model (R² = 0.986), and Zero-order model (R² = 0.975). The comparatively higher R² value for the Higuchi model suggests that diffusion was an important contributor to Barbaloin release from the gel matrix. The good fit obtained with the Korsmeyer–Peppas model further indicates the possible contribution of both drug diffusion and polymer relaxation/swelling. The release exponent (n = 0.689), when interpreted using the appropriate release-data region and geometry, is consistent with anomalous or non-Fickian transport, suggesting the involvement of more than one release mechanism. The comparative kinetic analysis showed that F3 exhibited higher regression coefficients than the standard Ketoprofen gel for all the investigated models. The Higuchi model showed the highest R² for both F3 and Ketoprofen gel, with values of 0.994 and 0.971, respectively. These findings indicate that diffusion was an important component of drug release in both formulations. However, the difference in R² values represents model fit and should not be interpreted as evidence that F3 has greater clinical efficacy than Ketoprofen. The in-vitro anti-inflammatory activity of the formulations was evaluated using the protein denaturation inhibition assay. All formulations demonstrated inhibition of protein denaturation, with values ranging from 58.73 ± 1.26% to 76.84 ± 0.88%. Carbopol 934-based formulations generally showed higher inhibition values than HPMC-based formulations. Among the Carbopol formulations, the inhibition increased from 63.42 ± 1.12% in F1 to 69.15 ± 0.94% in F2 and reached the highest value of 76.84 ± 0.88% in F3 containing 1.5% Carbopol 934. Further increase in Carbopol 934 concentration to 2.0% resulted in a decrease in inhibition to 73.96 ± 1.05% in F4. Among the HPMC-based formulations, inhibition increased from 58.73 ± 1.26% in F5 to 68.35 ± 1.14% in F8, followed by a decrease to 65.87 ± 1.19% in F9. The observed variation indicates that both polymer type and concentration influenced the anti-inflammatory performance of the formulations. The higher activity observed with F3 may be associated with an optimum balance between polymer concentration, gel structure, and Barbaloin availability. The decrease observed at higher polymer concentrations may be related to increased viscosity and polymer network density, which can restrict drug mobility and release. The HET-CAM study showed an irritation score of 0.0 for the tested formulations, with no visible signs of hemorrhage, vascular lysis, or coagulation during the observation period. These findings indicate a favourable irritation profile under the experimental conditions. However, HET-CAM findings should be considered as an in-vitro preliminary assessment and should not be interpreted as confirmation of clinical skin safety. The optimized F3 formulation was further evaluated for stability. Only minor changes were observed in appearance, pH, viscosity, and drug content after three months of storage under the selected study conditions. The drug content decreased from 99.98 ± 0.54% to 99.36 ± 0.58%, while pH changed from 6.24 ± 0.03 to 6.22 ± 0.04 and viscosity changed from 9188 to 9146 cP. These observations indicate that F3 maintained its evaluated physicochemical characteristics during the investigated stability period. Overall, the findings demonstrate that the type and concentration of gelling polymer played an important role in determining the physicochemical properties, drug-release behaviour, and anti-inflammatory performance of the Barbaloin-loaded topical gels. Among the investigated formulations, F3 containing 1.5% Carbopol 934 provided the most favourable overall performance and was therefore selected as the optimized formulation.

CONCLUSION

The present study successfully developed and evaluated Barbaloin-loaded topical gel formulations using Carbopol 934 and HPMC as gelling polymers. Nine formulations (F1–F9) were prepared and evaluated for their physicochemical properties, drug content, in-vitro drug release, release kinetics, irritation potential, stability, and anti-inflammatory activity. Among the prepared formulations, F3 containing 1.5% Carbopol 934 demonstrated the most favourable overall performance. It exhibited the highest drug content (99.98 ± 0.54%) and the highest inhibition of protein denaturation (76.84 ± 0.88%). The optimized F3 formulation showed progressive drug release, reaching 98.21 ± 0.62% after 8 h. The release data showed the best fit with the Higuchi model (R² = 0.994), indicating a major contribution of diffusion to the release process, while the Korsmeyer–Peppas model (R² = 0.989; n = 0.689) suggested the possible contribution of diffusion along with polymer relaxation/swelling. The optimized formulation also exhibited a favourable in-vitro irritation profile with an HET-CAM irritation score of 0.0 and maintained satisfactory physicochemical characteristics during the three-month stability study. The findings demonstrate that appropriate selection and concentration of the gelling polymer are important for achieving desirable formulation performance. In conclusion, the developed Barbaloin-loaded topical gel, particularly formulation F3, demonstrated satisfactory physicochemical characteristics, favourable drug-release behaviour, stability, and promising in-vitro anti-inflammatory activity. The formulation may therefore be considered a potential candidate for further investigation as a topical delivery system for Barbaloin. Further studies, including detailed skin permeation, in-vivo efficacy, and comprehensive safety evaluation, would be required to establish its therapeutic potential for topical inflammatory conditions.

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