Coixol and Sinigrin from Coix lacryma-jobi L. and Raphanus sativus L. Promote Fat Browning in 3T3-L1 Adipocytes

<b>Background/Objectives</b>: 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 <i>Coix lacryma-jobi</i> L. and <i>Raphanus sativus</i> L., alongside their active compounds, coixol and sinigrin. <b>Methods</b>: 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. <b>Results</b>: 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 (<i>Cd137</i>, <i>Cidea</i>, <i>Cited</i>, <i>Fgf21</i>, <i>Tbx1</i>, <i>Tmem26</i>). They also upregulated mitochondrial biogenesis genes (<i>Cox4</i>, <i>Nrf1</i>, <i>Tfam</i>), downregulated lipogenic genes (<i>Fasn</i>, <i>Lpl</i>, <i>Srebf1</i>, <i>Acaca</i>), and increased lipolytic (<i>Atgl</i>, <i>Hsl</i>, <i>Plin1</i>) and fatty acid oxidation genes (<i>Aco1</i>, <i>Cpt1</i>, <i>Ppara</i>). Mechanistic studies revealed that fat browning was associated with β3-adrenergic receptor activation and AMPK phosphorylation. <b>Conclusions</b>: Overall, coixol and sinigrin promote fat browning and metabolic improvement, highlighting their potential as natural anti-obesity agents.