In Vitro Anti-Inflammatory Potential of Orthosiphon Thymiflorus (Roth) Sleesen Extracts via Inhibition of Cyclooxygenase, Lipoxygenase, and Protein Denaturation

Inflammation is a key pathological factor in many chronic diseases. The genus Orthosiphon is traditionally used for inflammatory conditions, but O. thymiflorus remains underexplored. This study evaluated the in vitro anti-inflammatory efficacy of chloroform and ethanolic extracts of O. thymiflorus whole plant. Anti-inflammatory activity was assessed using three complementary in vitro assays: Cyclooxygenase (COX) inhibition assay using LPS-stimulated RAW 264.7 macrophage cell lysates; Lipoxygenase (LOX) inhibition assay using the same cell line; and heat-induced egg albumin denaturation assay. Diclofenac sodium was used as the standard reference drug. Both extracts exhibited concentration-dependent inhibition across all assays. In the COX assay, the ethanolic extract demonstrated superior activity (33.51% inhibition at 100µg/mL) compared to the chloroform extract (25.95%). A similar trend was observed in the LOX assay (Ethanolic: 31.58%; Chloroform: 26.04% at 100 µg/mL). The albumin denaturation assay confirmed the membrane-stabilizing potential of both extracts, with ethanolic extract showing greater protective effect at higher concentrations. The ethanolic extract of O. thymiflorus possesses significant anti-inflammatory activity, likely mediated through dual inhibition of COX and LOX pathways and stabilization of protein structures. These findings validate the traditional use of the plant and suggest its potential as a source for novel anti-inflammatory agents.