Integrated In Silico and Experimental Evaluation of the Anti-Inflammatory Activity of Tithonia Diversifolia Root Extract against Cyclooxygenase-2

Objective: In the current research, the possible anti-inflammatory effects of the ethanolic root extract of Tithonia diversifolia have been investigated by means of an integrated in silico and experimental approach focusing on cyclooxygenase-2 (COX-2) as the target molecule. Methods: The phytochemical composition of the ethanolic root extract was assessed by GC–MS analysis. Molecular docking of the identified phytochemicals, such as nepetin, chlorogenic acid, diversified, luteolin, germacrene D, and 6-acetyl-7-hydroxy-2,3-dimethyl-4H-chromen-4-one, on COX-2 (PDB ID: 3LN1) have been done using Schrödinger Glide XP molecular docking software. Ligand–protein binding energies have been estimated using Prime/MM-GBSA approach. Additionally, ADMET prediction has been carried out to assess drug-likeness, absorption, permeability, and metabolism of the compounds. The anti-inflammatory activity of the ethanolic root extract has been evaluated in in vitro protein denaturation inhibition assay and in vivo carrageenan-induced paw edema test in Wistar rats using 200 and 400 mg/kg doses. Results: Luteolin possessed the highest COX-2 docking score (-10.113 kcal/mol) amongst all identified phytoconstituents while nepetin possessed the next best COX-2 docking score (-10.095 kcal/mol) as compared with the negative value of diclofenac (-8.036 kcal/mol). The prime/MM-GBSA calculations revealed that all four phytoconstituents (luteolin, 6-acetyl-7-hydroxy-2,3-dimethyl-4H-chromen-4-one, nepetin, and germacrene D) had favorable binding energies (-38.16,-36.14,-30.51, and-27.45 kcal/mol, respectively). The ethanolic root extract showed significant inhibition of protein denaturation along with significant reduction in carrageenan-induced paw edema with maximum effect seen at higher dose of 400 mg/kg. Conclusion: It can be concluded from the above results that the ethanolic root extract of Tithonia diversifolia shows considerable anti-inflammatory effects. The activity could be due to the interactions of its bioactive constituents, namely, luteolin and nepetin, with the COX-2 protein. Both the results from the computer study and the laboratory experiments suggest further exploration of T. diversifolia as a possible anti-inflammatory agent.