Potential of Ko-Klan Traditional Thai Remedy for Evaluation of Antioxidant and RT-PCR Anti-Inflammatory Activities.

Ko-klan is a traditional Thai herbal medicine that is officially recognized for treating muscle pain; however, its mechanism of action has not been characterized. This study investigated the antioxidant and anti-inflammatory properties of three Ko-klan remedy formulations and identified potential bioactive markers using molecular docking. Ko-klan formulations were extracted using decoction, maceration, ultrasound-assisted extraction (UAE), and microwave-assisted extraction (MAE), with ethanol as the solvent. Phytochemical analyses were performed to determine the total phenolic content (TPC) and total flavonoid content (TFC), with antioxidant activity evaluated using the DPPH, ABTS, and FRAP assays. The anti-inflammatory effects were assessed by gene expression analysis (TNF-α, iNOS, COX-2, and IL-1β) and inhibition of nitric oxide production, and lipoxygenase (LOX) activity. LC-ESI-QTOF-MS/MS identified 175 compounds, with molecular docking performed against 5-LOX. Formulation-3 exhibited notable bioactivity, with a TPC of up to 1.08 g GAE/g and a TFC of 7.30 mg QE/g, and IC50 values of 51.8 μg/mL for DPPH and 92.3 μg/mL for ABTS assays, 7 to 10 fold than the Trolox standard. Anti-inflammatory activity showed comparable inhibition of nitric oxide production to the L-NAME standard and effective LOX inhibition at 62.7 μg/mL. The MAE-based Ko-klan remedy extract significantly suppressed LPS-induced inflammatory gene expression, comparable to dexamethasone. Molecular docking showed that caffeoyl quinic acid and brazilin were potent 5-LOX inhibitors with binding energies of -10.14 and -10.24 kcal/mol, respectively. Results demonstrate that Ko-klan remedies, particularly formulation-3, possessed significant antioxidant and anti-inflammatory activities, with phytochemical richness, effective suppression of inflammatory mediators, and potential bioactive markers such as caffeoyl quinic acid and brazilin, thereby supporting their traditional use and providing a scientific basis for further therapeutic development.