Formulation, Optimization and Evaluation of Thermoresponsive In-Situ Gel of Centella Asiatica Leaf Extract

Pharmaceutical formulation is the process of combining an active pharmaceutical ingredient (API) with suitable excipients to produce a stable, safe, and effective dosage form. Depending on the route of administration, pharmaceutical formulations are broadly classified as oral, parenteral, and topical dosage forms. Among these, topical drug delivery systems are extensively used for the treatment of skin disorders because they deliver the drug directly to the affected site, minimize systemic adverse effects, and improve patient compliance. Common topical dosage forms include ointments, creams, gels, lotions, pastes, and transdermal patches ^[1,2]. Topical drug delivery offers several advantages over conventional oral therapy, including avoidance of first-pass hepatic metabolism, reduced gastrointestinal side effects, improved bioavailability at the target site, and ease of application. Drugs applied topically penetrate the skin mainly through the stratum corneum, the outermost layer of the epidermis, which serves as the primary barrier to drug absorption. Drug permeation may also occur through hair follicles and sweat glands, although these pathways contribute minimally compared to intercellular diffusion through the stratum corneum. The extent of drug penetration depends on several factors, including the physicochemical properties of the drug, the formulation, and the physiological condition of the skin ^[3,4]. Despite the advantages of conventional topical formulations, they often suffer from limitations such as poor retention at the site of application, frequent dosing, inadequate drug penetration, and patient inconvenience due to greasiness or poor cosmetic acceptability. To overcome these drawbacks, advanced drug delivery systems such as thermoresponsive in-situ gels have been developed. These formulations exist as free-flowing liquids at room temperature but undergo rapid transformation into a gel when exposed to body or skin temperature. This unique property increases the residence time of the formulation at the site of application and provides sustained and controlled drug release ^[5]. Thermoresponsive in-situ gels are mainly prepared using temperature-sensitive polymers such as Poloxamer 407, Poloxamer 188, and other biodegradable polymers. These polymers exhibit reversible sol-gel transition at physiological temperatures (32–37°C). The gel formed after application acts as a drug reservoir, allowing prolonged release of the therapeutic agent while maintaining an effective drug concentration at the wound site. Such systems reduce the frequency of application, improve patient compliance, and minimize systemic drug absorption and associated adverse effects ^[6,7]. In-situ gel systems may undergo gelation through different mechanisms, including temperature-sensitive, pH-sensitive, ion-activated, swelling-induced, diffusion-controlled, and chemically cross-linked processes. Among these, thermally triggered systems are the most suitable for topical administration because they undergo gelation under physiological conditions without requiring external stimuli. Thermoresponsive hydrogels also maintain a moist wound environment, facilitate autolytic debridement, reduce scar formation, protect the wound from microbial contamination, and enhance tissue regeneration ^[8]. The skin is the largest organ of the human body and functions as a protective barrier against physical injury, microbial invasion, ultraviolet radiation, and excessive water loss. It consists of three principal layers: the epidermis, dermis, and subcutaneous tissue. The epidermis provides the major barrier to drug permeation, whereas the dermis contains connective tissue, blood vessels, lymphatics, nerve endings, sebaceous glands, sweat glands, and hair follicles that support wound healing and tissue repair. A thorough understanding of skin anatomy and the mechanisms of drug penetration is essential for the successful design of topical drug delivery systems ^[9]. The development of an optimized topical formulation requires systematic evaluation of formulation variables and their influence on product performance. Statistical optimization techniques such as Response Surface Methodology (RSM) are widely employed in pharmaceutical research to establish relationships between formulation variables and critical quality attributes. Among these techniques, the Box–Behnken Design (BBD) is one of the most commonly used experimental designs because it requires fewer experimental runs while effectively studying the interaction between independent variables. This approach facilitates optimization of parameters such as polymer concentration, gelation temperature, viscosity, spreadability, drug release, and stability, thereby improving formulation quality and reducing development time ^[10,11]. Thus, thermoresponsive in-situ gel systems represent a promising advancement in topical drug delivery. Their ability to transform into a gel at physiological temperature, prolong residence time, provide sustained drug release, improve patient convenience, and enhance therapeutic efficacy makes them highly suitable for wound healing applications. Furthermore, optimization using statistical experimental designs ensures the development of safe, effective, and reproducible formulations with improved pharmaceutical performance.

 MATERIALS AND METHODS

Centella asiatica extract was prepared in lab for the study. Calendula officinalis extract was obtained from Raasa Oil. Poloxamer 407 was procured from Sai Mirra Innopharm Pvt. Ltd. Carbopol 934, ethanol, and glycerine were purchased from Chennai Chemicals, Chennai. All the chemicals and reagents used in the study were of analytical grade.

Preparation of Centella asiatica Extract

Fresh leaves of Centella asiatica were collected, washed thoroughly with tap water followed by distilled water, and shade-dried at room temperature for 7–10 days. The dried leaves were powdered using a mechanical grinder and stored in an airtight container. About 50 g of the powdered material was extracted with 500 mL of ethanol (1:10, w/v) by hot maceration at 50–60°C for 2–3 h with occasional stirring. The extract was filtered through Whatman No.1 filter paper and concentrated on a water bath below 50°C until a semisolid mass was obtained. The dried extract was weighed to determine the percentage yield and stored at 4°C until further use ^[12,13].

Figure 1: Preparation of Centella Asiatica Extract

Determination of λmax

A stock solution of Centella asiatica extract was prepared by dissolving 10 mL of the extract in 100 mL of phosphate buffer (pH 7.2). From this stock solution, 10 mL was further diluted to 100 mL with the same buffer. The solution was scanned over the wavelength range of 200–400 nm using a UV–Visible spectrophotometer to determine the wavelength of maximum absorbance (λmax)

Preparation of Calibration Curve

The stock solution was serially diluted with phosphate buffer (pH 7.2) to obtain concentrations ranging from 5–25 μg/mL. The absorbance of each solution was measured at 299 nm using a UV–Visible spectrophotometer, and a calibration curve was constructed by plotting absorbance against concentration

Optimization of Thermoresponsive In-situ Gel Using Box–Behnken Design

The formulation of the thermoresponsive in-situ gel containing Centella asiatica extract and Calendula officinalis oil was optimized using a three-factor, three-level Box–Behnken Design (BBD) generated by Design-Expert® software (Version 13). The independent variables selected were Poloxamer 407 concentration (18–22%), Carbopol 940 concentration (0.2–0.6%), and glycerine concentration (2–4%), while gelation temperature (R1) and viscosity (R2) were chosen as the dependent responses. A total of 13 experimental runs were performed to evaluate the individual, interaction, and quadratic effects of the formulation variables. The experimental data were fitted to a second-order polynomial equation, and statistical analysis was carried out using analysis of variance (ANOVA). The optimized formulation was selected based on the desirability function, and the predicted responses were validated by comparing them with the experimental values to determine the prediction error and model accuracy. Independent and dependent variables are listed in Table 1. The Polynominal equation generated by this experimental design is as follows,

Y = β0+ β1X1+ β2X2+ β3X3+ β12X1X2 + β13X1X3+ β23X2X3 + β11X12+ β22X22 + β33X32

Where Y is the dependent variable, β0 is the intercept, β1 to β33 are the regression coefficient and X1, X2 and X3 are the independent variables selected from preliminary experiments. 

Figure 2: Absorption spectrum of Centella asiatica extract.

Figure 3: Calibration curve of Centella asiatica extract.

S. No	Formulation Code	Factor 1 A: Poloxamer 407 %	Factor 2 B: Carbopol 940 %	Factor 3 C: Glycerin %	Response 1 Gellation Temp 0c	Response 2 Viscosity Cps
1	F1	22	0.6	3	31	1354
2	F2	20	0.2	4	30	1440
3	F3	18	0.2	3	32	1561
4	F4	18	0.4	4	41	1250
5	F5	20	0.6	4	33	1169
6	F6	20	0.4	3	30	977
7	F7	22	0.4	4	33	1280
8	F8	22	0.2	3	32	1369
9	F9	18	0.6	3	41	1280
10	F10	22	0.4	2	33	1124
11	F11	20	0.2	2	31	996
12	F12	20	0.6	2	32	880
13	F13	18	0.4	2	33	1487

Figure 4: Formulation of Thermoresponsive Insitu Gel

Figure 5: Perturbation plot for Response Y1.

 

Figure 6: 3D Response surface plot for Response Y1

Fig.7: Perturbation plot for Response Y2.

  

Fig.8: 3D Response surface plot for Response Y2.

Selection of optimized batch

The selection of optimized batch for Thermoresponsive Insitu gel was done by making various trials with the goal of least Gellation temp (30⁰C - 41⁰C) and least Viscosity (880 cps – 1561 cps). The selection of the batch was done by numeric optimization with a desirability function. The constraints for Gellation temp (Y1), and Viscosity (Y2) was minimized. The optimized batch given by the software was prepared and analyzed. The optimized batch consisted of Polaxomer 407 conc (A=21.39%), Carbopol 940 conc (B=0.3714 %), and Glycerin conc (C= 2%) with Gellation temp and Viscosity predicted to be 32⁰C and 949.476 Cps respectively. The Gellation temp observed was 31⁰C and Viscosity of 1346 Cps. Thus, the batch giving minimum Gellation temp and minimum Viscosity was selected as the optimized batch.

Fig.9: Optimized Thermoresponsive Insitu gel formulation

Characterization of Thermoresponsive Insitu Gel:

1. Determination of sol-gel temperature (Tsol-gel)

The solution to gelling temperature of optimized In-situ gel formulation was found to 31ºC, which shows the optimized formulation converted to gel on the surface of the skin after application.

2. Determination of viscosity

The viscosity of optimized formulation was 116 cps this viscosity offer easy to administration the formulation in solution state by using spraying system. The gelling state viscosity of optimized formulation was 1346 cps.

3. Clarity of formulation

The clarity of optimized formulation was found to be very clear (+++).

4. Gelling capacity

The gelling capacity of optimized formulation was (+++) immediate gelation remains for nearly an hour

5. Determination of pH

The pH of optimized In-situ gel formulation was found to be 5.57, which shows the optimized formulation was compatible with skin pH and the drug molecule more active at this pH level of the formulation.

6. Spreadability

The spreadability of optimized Mupirocin In-situ gel formulation was found to be 5.7cm .The optimized formulation majorly and evenly covering the affected area.

7. Drug Content

The drug content of optimized formulation was determined by UV-visible spectrophotometric method the drug content was found to be 94.76%.

8. In-vitro diffusion studies of the gel

In-vitro Release profile of optimized formulation of mupirocin In-situ gel and market formulation shown in figure 10 The results of optimized formulation shows (72.32%) of drug released upto 8 hrs. The In-vitro drug release results shows the drug released from the In-situ gel polymeric system sustained manner and expected to release remaining medicaments some extend period of time.

Fig.10: In vitro % drug release

9. In-vitro antibacterial study

The zone of inhibition for the best formulation was compared with the zone of inhibition of the gentamicin (positive control). The zone of inhibition for gentamicin (S) was found to be 25 mm and for best formulation (T) 23.5 mm, indicating that the OPT formulation was sensitive to the microorganism s. aureus.

Fig.11: zone of inhibition of OPT-Formulation

CONCLUSION:

Conventional topical formulations often exhibit limitations such as poor retention at the application site, frequent dosing, and inadequate drug release. To overcome these drawbacks, a thermoresponsive in-situ gel system was developed using Poloxamer 407 and Carbopol 940, which undergo sol-to-gel transition at skin temperature, thereby enhancing residence time and sustained drug release. Centella asiatica extract was prepared by ethanolic extraction and characterized using UV-visible spectrophotometry. The maximum absorption wavelength (λmax) was observed at 299 nm, and a calibration curve was established for quantitative analysis. The formulation variables were optimized using a three-factor, three-level Box–Behnken design with Design-Expert® software. Thirteen formulations were prepared and evaluated for gelation temperature, viscosity, pH, spreadability, clarity, gelling capacity, drug content, in-vitro drug diffusion, and antibacterial activity. Among the prepared formulations, the optimized batch exhibited a gelation temperature close to physiological skin temperature, appropriate viscosity, satisfactory spreadability, acceptable drug content, and sustained drug release. The optimized formulation also demonstrated significant antibacterial activity and showed promising wound-healing potential due to the combined therapeutic effects of Centella asiatica and Calendula officinalis. In conclusion, the developed thermoresponsive in-situ gel proved to be a stable, effective, and patient-friendly topical drug delivery system. The formulation enhanced drug retention, reduced the frequency of application, and improved therapeutic efficacy. Therefore, it represents a promising herbal alternative for wound management and warrants further preclinical and clinical studies for future commercialization.

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