Anti-Inflammatory Activity and Free Radical Scavenging... : Journal of Pharmacy and Bioallied Sciences (2025)

INTRODUCTION

Tridax procumbens (TP) leaves are reportedly used in traditional medicine to treat diarrhea, dysentery, and bronchial catarrh as well as to restore hair. Since ancient times, alternative medicine has made use of therapeutic compounds obtained from plants. The tribal healers apply the whole plant extract as a wound-healing ailment.^[1] The anti-inflammatory, immunomodulatory, wound-healing, hepatoprotective, anti-leishmanial, hypoglycemic, anti-hyperglycemic, and hair growth promoting properties of TP have been documented in earlier pharmacological studies.^[2] The phytochemical analysis documents the separation of sterols, flavonoids, polysaccharides, lipid components, and derivatives of bergenin from TP.^[3]

Chitosan is a polymer that contains small quantities of N-acetylglucosamine. It is an analog of chitin which is the world’s second most abundant biopolymer behind cellulose.^[4] Substances with antioxidant and anti-inflammatory qualities efficiently lower reactive oxygen species levels, decreasing oxidative stress and the inflammatory response while accelerating the healing of wounds.^[5] The present study aims to evaluate the anti-inflammatory activity and free radical scavenging activity of TPC gel.

MATERIALS AND METHODS

Preparation of TP leaves-based chitosan gel

A total of 50 mL chitosan solution is prepared by dissolving 0.5 g of medium molecular weight chitosan in 49 mL of distilled water and 1 mL of glacial acetic acid. To guarantee perfect homogeneity, the mixture was agitated constantly for 24 hours with a magnetic stirrer. Following this initial preparation, the TP leaf extract was added to the solution and stirred for a further 24 hours to form a homogeneous gel. The resulting gel was then tested for anti-inflammatory and antioxidant properties.

Evaluation of anti-inflammatory activity

Bovine serum albumin (BSA) denaturation assay

To assess the anti-inflammatory properties of TP chitosan (TPC) gel, a modified version of the Muzushima and Kabayashi method was used. Initially, 0.05 mL of the test gel in varying volumes (10 μL, 20 μL, 30 μL, 40 μL, and 50 μL) was added to 0.45 mL of a 1% aqueous bovine serum albumin solution. To preserve the experiment’s ideal conditions, the pH of the liquid was adjusted to 6.3 with a little amount of 1N hydrochloric acid. The samples produced were incubated at room temperature for 20 minutes to ensure appropriate interaction of the components. Following incubation, the samples were heated in a water bath at 55 degrees Celsius for 30 minutes, which is an important step in causing protein denaturation. After heating, the samples were left to cool to room temperature. The absorbance of each sample was determined using spectroscopy at a wavelength of 660 nm. Diclofenac sodium was employed as the standard reference for anti-inflammatory activity, with dimethyl sulfoxide (DMSO) acting as a control. The proportion of protein denaturation inhibition was estimated using the following formula: To calculate percentage inhibition, divide the absorbance of the control by the absorbance of the sample and multiply by 100. This method accurately measures the TPC gel’s anti-inflammatory capability by comparing its ability to inhibit protein denaturation to standard and control samples.

Egg albumin (EA) denaturation assay

A total of 2.8 mL of newly prepared phosphate-buffered saline (pH 6.3) and 0.2 mL of egg albumin retrieved from hens’ eggs were combined to form a mixture of 5 mL. The test gel was prepared with concentrations of 10 μL, 20 μL, 30 μL, 40 μL, and 50 μL. Diclofenac sodium served as a positive control in the experiment. The combinations were heated in a water bath at 37°C for 15 minutes to denaturate then cooled to room temperature. After cooling, each sample’s absorbance was measured using spectroscopy at 660 nm. This setting enabled for the evaluation of the test gel’s anti-inflammatory effects by comparing the levels of protein denaturation inhibition to those obtained with the positive control, diclofenac sodium.

Evaluation of antioxidant activity

DPPH method

The DPPH assay was used to assess the antioxidant activity of the synthesized test gel. The gel was combined with 1 mL of 0.1 mM DPPH in methanol and 450 μL of 50 mM Tris HCl buffer (pH 7.4). The combination was incubated for 30 minutes at different concentrations of the test gel (10 μL, 20 μL, 30 μL, 40 μL, and 50 μL). After incubation, the absorbance at 517 nm was measured to assess the reduction of DPPH free radicals. Ascorbic acid was employed as the standard reference. This approach precisely determines the antioxidant capacity of the test gel by comparing its ability to neutralize DPPH free radicals to the standard ascorbic acid.

Hydroxyl radical scavenging assay

Fresh solutions were employed for best results. The reaction mixture (1.0 mL) included various components:

• 100 μL of 28 mM 2-deoxy-2-ribose dissolved in 7.4 pH phosphate buffer
• 500 μL of test gel at different concentrations (10 μL, 20 μL, 30 μL, 40 μL, and 50 μL)
• Mixture of 200 μL of 200 μM FeCl3 with 1.04 mM EDTA in a 1:1 volume ratio
• 100 μL of 1.0 mM hydrogen peroxide (H₂O₂)
• 100 μL 1.0 mM ascorbic acid.

After preparing the reaction mixture, it was incubated at 37°C for one hour. During this time, the hydroxyl radicals in the mixture would begin the breakdown of deoxyribose.

The degree of deoxyribose breakdown was then quantified using a spectrophotometer to determine absorbance at 532 nm, with a blank solution acting as the baseline reference. Vitamin E was utilized as a positive control to assess the test gel’s ability to scavenge hydroxyl radicals. This experiment will clearly indicate the test gel’s antioxidant activity by demonstrating its ability to neutralize hydroxyl radicals and prevent the oxidative destruction of deoxyribose.

RESULTS

Evaluation of anti-inflammatory activity

TP leaves extract-based chitosan gel demonstrated a stronger anti-inflammatory effect compared to the standard in both assays, as indicated by higher % of inhibition across all concentrations. The test gel showed a gradual increase in % of inhibition as the concentration increased, reaching close to 100% at 50 μL. Both assays showed that increasing the concentration of test gel leads to greater inhibition of inflammation, suggesting a dose-dependent response. The trends in both assays are consistent, indicating that TP leaves extract-based chitosan gel is effective in reducing inflammation in both BSA and EA assays [Figure 1].

Evaluation of antioxidant activity

DPPH assay

TPC gel showed a consistent % of inhibition across all concentrations, indicating a stable antioxidant capacity and demonstrated a similar trend with % of inhibition closely matching that of the standard. Both the standard and the test gel exhibited significant antioxidant activity, as indicated by high % of inhibition values across all concentrations [Figure 2]. There is no significant increase in % of inhibition with increasing concentrations, suggesting that both treatments may reach a saturation point in their antioxidant activity.

Hydroxyl radical scavenging assay

TPC gel showed an increase in % of inhibition, with values approaching those of the standard at higher concentrations. Both the standard and the test gel effectively scavenge hydrogen peroxide, as indicated by increasing % of inhibition with higher concentrations. While the standard showed slightly higher % of inhibition at lower concentrations, the test gel demonstrated a stronger response, particularly at higher concentrations. The results indicated a dose-dependent response for both the test and the standard, with higher concentrations leading to greater inhibition of H₂O₂.

DISCUSSION

Overall, the present study indicated that TP leaves-based chitosan could be a promising candidate for anti-inflammatory applications, outperforming the standard treatment in both assays, and the antioxidant capacity of TPC gel is comparable to that of the standard, indicating its potential as an effective antioxidant agent. Both assays demonstrate that TPC gel possesses significant antioxidant properties, comparable to the standard in the DPPH assay and effective in scavenging hydrogen peroxide in the H₂O₂ assay. The findings suggest that TPC gel could be a valuable natural antioxidant, with potential applications in food preservation, cosmetics, or therapeutic formulations.

Joushan Ara et al.^[6] concluded that the methanolic extract has outstanding antioxidant activity. The methanolic leaf extract has mild anti-inflammatory properties as well. TP (Linn.) has distinct elements in each section of the plant, and the pharmacological effects of the plant differ depending on which part is studied. In contrast, the present study showed potent anti-inflammatory activity, whereas antioxidant activity was similar. Pooja Singh et al.^[7] concluded that the ethanol extracts showed greater antioxidant activity than methanol and aqueous extracts, according to the results of in vitro antioxidant activity studies. These findings suggest that the extracts have antioxidant properties and may account for some of TP’ medicinal use, whereas the present study showed similar antioxidant activity.

Prakash et al.^[8] concluded that rats treated with an ethanolic leaf extract of TP exhibit substantial anti-inflammatory properties, whereas the present study showed similar anti-inflammatory activity. J D Habila et al.^[9] concluded that the TP possesses antioxidant activity, which was found to be correlated with the plant samples’ overall phenolic content, whereas the present study showed similar antioxidant activity.

The current study does not concentrate on enhancing the bioavailability and effectiveness of chitosan gels by refining the formulation with TP leaves extract. In the future, research may concentrate on paving the path for therapeutic applications in oral inflammatory diseases like periodontal diseases by verifying laboratory findings in human and animal models.^[10,11] The limitations of the current study were that the chitosan gel may not be as effective as they could be due to stability issues, such as deterioration with time, and inconsistent outcomes may arise from variations in the concentration and activity of bioactive chemicals found in TP.

CONCLUSION

TPC gel has tremendous potential for treating inflammatory oral disorders because of its potent anti-inflammatory and antioxidant properties. The gel’s antioxidant activity can help neutralize harmful free radicals, lowering oxidative stress and improving overall oral health. Its use may offer a natural and effective alternative for enhancing periodontal health by lowering inflammation. The gel’s combination of anti-inflammatory and antioxidant properties makes it an attractive option for therapeutic application in oral healthcare.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Acknowledgment

The authors would like to thank Saveetha Dental College, Chennai, India for providing the platform and support system to carry out this work.

REFERENCES