Exploring the functional supplement potential of the Thai herbal formula Tri-Ka-Tuk via metabolomic and proteomic profiling in Drosophila

Tri-Ka-Tuk is a traditional Thai polyherbal formula composed of Zingiber officinale, Piper nigrum, and Piper retrofractum, historically used to promote digestive health, circulation, and systemic balance. Despite its long-standing traditional use, its molecular mechanisms remain poorly understood. This study aimed to elucidate the biological effects of Tri-Ka-Tuk using an integrative omics approach in Drosophila melanogaster, a well-established in vivo model. Adult male flies were fed with diets supplemented with Tri-Ka-Tuk at various concentrations. Toxicity and feeding behavior were evaluated, followed by in vivo oxidative stress assays using paraquat and hydrogen peroxide. Proteomic and metabolomic analyses were performed to investigate molecular changes induced by the formula. The formula was non-toxic at concentrations up to 20 mg/mL and did not significantly affect feeding behavior below 10 mg/mL. Supplementation significantly enhanced survival under oxidative stress, supporting its antioxidant activity. Proteomic analysis revealed differential expression of proteins associated with DNA repair, mitochondrial function, and cellular stress responses, including enrichment of Fanconi anemia and mTOR signaling pathways. Metabolomics profiling indicated a Warburg-like metabolic reprogramming, with altered levels of glycolytic and TCA cycle intermediates, suggesting improved metabolic efficiency and stress adaptation. These findings provide the first systems-level insight into the molecular mechanisms underlying the traditional use of Tri-Ka-Tuk formula. This research supports the application of spicy herbal formulations that include Tri-Ka-Tuk in functional food research focused on enhancing health and disease prevention.