Abstract
Background/Objectives: Obesity, a metabolic disorder resulting from an energy imbalance, often leads to excess fat and related diseases. Browning of white adipose tissue, which increases energy expenditure, is a promising anti-obesity strategy. Herbal medicines are considered safer than conventional drugs, but their fat browning mechanisms remain unclear. Therefore, this study aims to examine the effects of Coix lacryma-jobi L. and Raphanus sativus L., alongside their active compounds, coixol and sinigrin. Methods: Cytotoxicity in 3T3-L1 cells was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Lipid accumulation was quantified by the Oil Red O (ORO) staining. Immunofluorescence staining was employed to evaluate mitochondrial activity and uncoupling protein 1 (UCP1). Protein and mRNA expressions were analysed using western blot and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. Results: In 3T3-L1 adipocytes, ORO staining showed reduced lipid accumulation and droplet size after treatment. qRT-PCR, western blot, and immunostaining revealed that coixol and sinigrin upregulated browning markers (UCP1, PGC-1α, PRDM16) and beige fat genes (Cd137, Cidea, Cited, Fgf21, Tbx1, Tmem26). They also upregulated mitochondrial biogenesis genes (Cox4, Nrf1, Tfam), downregulated lipogenic genes (Fasn, Lpl, Srebf1, Acaca), and increased lipolytic (Atgl, Hsl, Plin1) and fatty acid oxidation genes (Aco1, Cpt1, Ppara). Mechanistic studies revealed that fat browning was associated with β3-adrenergic receptor activation and AMPK phosphorylation. Conclusions: Overall, coixol and sinigrin promote fat browning and metabolic improvement, highlighting their potential as natural anti-obesity agents.