Formulation and Evaluation of Anti-Inflammatory Ointment Using Ficus Religiosa Bark

These limitations have encouraged researchers to explore safer and more effective alternatives derived from natural sources. Medicinal plants have gained considerable attention because they contain diverse phytochemicals with significant therapeutic activities and generally produce fewer side effects than synthetic drugs. Herbal medicines have been used for centuries in traditional healthcare systems and continue to play an important role in modern medicine. They are rich sources of biologically active constituents such as flavonoids, tannins, phenolic compounds, terpenoids, saponins, and alkaloids. These phytochemicals possess antioxidant, antimicrobial, anti-inflammatory, wound-healing, and immunomodulatory properties. Owing to their natural origin, wide availability, affordability, and favorable safety profile, herbal formulations are increasingly being investigated for the development of topical therapeutic products. Among various medicinal plants, Ficus religiosa Linn. (Family: Moraceae), commonly known as the Peepal tree, is one of the most valuable medicinal plants used in traditional Indian medicine. Different parts of the plant, including the bark, leaves, fruits, roots, and latex, have been reported to possess several pharmacological activities such as anti-inflammatory, antioxidant, antimicrobial, antidiabetic, analgesic, wound-healing, and antiulcer effects. The bark is particularly rich in flavonoids, tannins, polyphenols, phytosterols, and terpenoids, which are believed to contribute significantly to its anti-inflammatory potential. Topical drug delivery systems such as ointments offer several advantages over oral dosage forms in the treatment of localized inflammatory conditions. Ointments provide direct delivery of the active constituents to the affected site, minimize systemic drug exposure, prolong the contact time of the drug with the skin, improve patient compliance, and reduce systemic adverse effects. In addition, the occlusive nature of ointment bases enhances hydration of the skin and facilitates better penetration of active phytoconstituents. The effectiveness of a topical herbal formulation depends not only on the therapeutic activity of the plant extract but also on its physicochemical properties, stability, and quality. Therefore, appropriate formulation development and systematic evaluation are essential to ensure product safety, efficacy, and reproducibility. Important quality parameters include colour, odour, homogeneity, pH, viscosity, spreadability, extrudability, diffusion characteristics, stability, and skin compatibility. Phytochemical screening also plays a significant role in confirming the presence of bioactive constituents responsible for the therapeutic activity of the formulation.  In the present study, herbal anti-inflammatory ointment containing Ficus religiosa bark extract was formulated using the fusion method. The extract was obtained by Soxhlet extraction using a hydroalcoholic solvent system and incorporated into an oleaginous ointment base to prepare different formulations. The prepared formulations were evaluated for their physicochemical characteristics and anti-inflammatory activity using the protein denaturation assay. The study was undertaken to develop a stable, effective, safe, and economical herbal topical formulation that could serve as a promising natural alternative for the management of inflammatory conditions. Medicinal plants have been utilized for centuries in traditional healthcare systems and continue to serve as an important source of bioactive compounds for modern drug discovery. Herbal medicines are increasingly accepted because of their natural origin, cost-effectiveness, accessibility, and reduced incidence of adverse reactions These phytoconstituents exert their pharmacological effects by scavenging reactive oxygen species, suppressing oxidative stress, and inhibiting inflammatory mediators such as cyclooxygenase (COX), lipoxygenase (LOX), tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). 

MATERIALS AND METHODS

2.1 MATERIALS

Ficus religiosa bark powder was procured from SGM Hospital, Mahagaon,

Kolhapur, Maharashtra, India. Hard paraffin, cetostearyl alcohol, wool fat (lanolin), white soft paraffin, methyl salicylate, methyl paraben, and propyl paraben were used for the preparation of the herbal anti-inflammatory ointment. Ethanol and distilled water were used for the extraction of the plant material. All chemicals and reagents employed in the study were of analytical grade and used without further purification.

2.2 Experimental Design

The present investigation was designed to formulate and evaluate herbal anti-inflammatory ointment containing hydroalcoholic extract of Ficus religiosa bark. Three formulations (F1, F2, and F3) were prepared by varying the proportions of the ointment base constituents while maintaining a constant concentration of Ficus religiosa extract and other active ingredients. The prepared formulations were evaluated for physicochemical characteristics, stability, and in vitro anti-inflammatory activity. 

2.3 Procurement of Plant Material

The powdered bark of Ficus religiosa was procured from SGM Hospital, Mahagaon, Kolhapur, Maharashtra, India. The plant powder was stored in an airtight container at room temperature in a dry place, protected from moisture and direct sunlight until further use for extraction.

2.4 Preparation of Hydroalcoholic Extract

The hydroalcoholic extract of Ficus religiosa bark was prepared by the Soxhlet extraction technique. Approximately 50 g of powdered bark was accurately weighed and packed into a cellulose extraction thimble.  The thimble was placed inside the Soxhlet apparatus and extracted using 250 mL of hydroalcoholic solvent (ethanol: distilled water, 30:70 v/v). The extraction was carried out at a temperature of 40–70°C for 72 hours, allowing repeated extraction cycles until the solvent in the siphon tube became colorless, indicating complete extraction of the phytoconstituents. After completion of the extraction process, the solvent was removed by evaporation to obtain a concentrated hydroalcoholic extract. The concentrated extract was collected in an amber-coloured container and stored under refrigerated conditions until further use for formulation development.

Figure 1: Soxhlet Extraction Apparatus

2.5 Preliminary Phytochemical Screening

The hydroalcoholic extract of Ficus religiosa bark was subjected to preliminary qualitative phytochemical screening using standard procedures to detect the presence of various secondary metabolites. The extract was examined for alkaloids, flavonoids, tannins, phenolic compounds, saponins, terpenoids, glycosides, carbohydrates, proteins, and phytosterols based on characteristic colour changes or precipitate formation.

2.6 Formulation of Herbal Ointment

Three herbal ointment formulations (F1, F2, and F3) were prepared by the fusion method. Hard paraffin, cetostearyl alcohol, wool fat (lanolin), and white soft paraffin were accurately weighed according to the formulation composition and melted in descending order of their melting points using a water bath maintained at 70–75°C. Methyl paraben and propyl paraben were dissolved in the molten base, followed by the gradual incorporation of the hydroalcoholic extract of Ficus religiosa with continuous stirring to ensure uniform distribution. Methyl salicylate was added after slight cooling of the base to minimize volatilization. Continuous stirring was maintained until a smooth and homogeneous semisolid ointment was obtained. The prepared formulations were transferred into sterile ointment containers, labelled, and stored at room temperature for further evaluation.

Table no.1 formulation table for herbal ointment

2.7 Evaluation of Herbal Ointment

The prepared formulations were evaluated for various physicochemical parameters according to standard pharmaceutical procedures.

2.7.1 Organoleptic Evaluation

The formulations were visually examined for colour, odour, appearance, texture, consistency, and homogeneity.

2.7.2 Determination of pH

The pH of each formulation was determined using a calibrated digital pH meter. Approximately 1 g of ointment was dispersed in 10 mL of distilled water and allowed to stand for 2 hours before measurement. The pH was recorded in triplicate, and the average value was calculated.

2.7.3 Homogeneity

Homogeneity was evaluated by visual inspection after the ointment had been set. The formulations were examined for uniformity, grittiness, and phase separation.

2.7.4 Viscosity

The viscosity of the prepared formulations was determined using a Brookfield digital viscometer under appropriate spindle and rotational speed conditions. Measurements were performed at room temperature, and the average values were recorded.

2.7.5 Spreadability

Spreadability was determined using the parallel glass slide method. A known quantity of ointment was placed between two glass slides, and a standard weight was applied. The time required for the upper slide to move a specified distance under the applied weight was recorded.

Spreadability was calculated using the standard formula: S = (M × L) / T where:

• S = Spreadability (g cm/s) 
• M = Weight tied to the upper slide (g) 
• L = Length moved by the slide (cm) 
• T = Time taken (s) 

2.7.6 Extrudability

Extrudability was evaluated by measuring the force required to extrude the ointment from a collapsible tube under standardized conditions.

2.7.7 Loss on Drying

Loss on drying was determined by weighing approximately 1 g of the formulation before and after drying at the prescribed temperature until a constant weight was obtained. The percentage loss on drying was calculated.

2.7.8 Diffusion Study

The diffusion characteristics of the formulations were evaluated using the agar diffusion method. A measured quantity of ointment was placed in a cavity prepared in agar gel, and the diameter of diffusion was measured after the specified incubation period.

2.8 In Vitro Anti-inflammatory Activity

The anti-inflammatory activity of the prepared formulations was evaluated using the protein denaturation assay with diclofenac sodium as the reference standard. Different concentrations of the formulations were mixed with bovine serum albumin solution and incubated under controlled conditions. Protein denaturation was induced by heating, followed by cooling to room temperature. The absorbance of the reaction mixture was measured using a UV–Visible spectrophotometer at the specified wavelength. The percentage inhibition of protein denaturation was calculated using the following equation:

% Inhibition = [(Absorbance of Control − Absorbance of Sample) / Absorbance of Control] × 100

The obtained values were compared with those of the standard drug.

2.9 Statistical Analysis

All experiments were performed in triplicate, and the results were expressed as mean ± standard deviation (SD). The experimental data were analysed using descriptive statistical methods to compare the physicochemical characteristics and anti-inflammatory activity of the formulations.

RESULTS

3.1 Preliminary Phytochemical Screening

Preliminary phytochemical analysis of the hydroalcoholic extract of Ficus religiosa bark confirmed the presence of several biologically active secondary metabolites. The extract showed positive results for flavonoids, tannins, phenolic compounds, saponins, terpenoids, and phytosterols. These phytoconstituents are known to possess antioxidant and anti-inflammatory activities and may contribute to the therapeutic efficacy of the formulated herbal ointment.

Table 2. Preliminary Phytochemical Screening Results

3.2 Physicochemical Evaluation of Herbal Ointment

Three herbal ointment formulations (F1, F2, and F3) were successfully prepared using the fusion method. All formulations exhibited smooth semisolid consistency, pale yellow colour, characteristic odour, satisfactory homogeneity, and absence of grittiness or phase separation.  The prepared ointments were aesthetically acceptable and suitable for topical application.

Table 3. Organoleptic Evaluation of Formulations

3.3 pH Determination

The pH values of all formulations were found to be within the acceptable range for topical preparations and were compatible with the physiological pH of the skin. This indicates that the formulations are unlikely to cause skin irritation and are suitable for external application.

Table 4. pH of Formulations

3.4 Viscosity

The viscosity of the formulations was determined using a Brookfield viscometer. All formulations exhibited appropriate viscosity, ensuring adequate consistency, stability, and ease of application. Minor variations in viscosity were observed due to differences in the composition of the ointment base.

Table 5. Viscosity of Formulations

3.5 Spreadability

The prepared formulations demonstrated satisfactory spreadability, indicating uniform distribution over the skin surface with minimal effort. The variation in the proportions of hard paraffin, cetostearyl alcohol, and wool fat influenced the spreadability of the formulations.

Table 6. Spreadability of Formulations

3.6 Extrudability, Diffusion Study and Loss on Drying

All formulations showed good extrudability from the collapsible tube without requiring excessive force. The diffusion study demonstrated satisfactory release of the active constituents from the ointment base, while the percentage loss on drying remained within acceptable limits, indicating adequate physical stability of the formulations.

3.7 In Vitro Anti-inflammatory Activity

The anti-inflammatory activity of the prepared formulations was evaluated by the protein denaturation assay using diclofenac sodium as the reference standard. The formulations exhibited concentration-dependent inhibition of protein denaturation. Among the prepared formulations, the optimized formulation demonstrated the highest inhibitory activity, suggesting improved anti-inflammatory potential. However, the standard drug exhibited greater inhibition than the herbal formulations.

Table 7. Absorbance & % Inhibition of formulation