Anti-Obesity and Anti-Steatotic Effects of Bixin (Apocarotenoid from Bixa orellana L. Seeds): Dose-Dependent Correlations with Bioactivity

Annatto, a tropical shrub from Central and South America and parts of India, contains Bixin, an apocarotenoid pigment. Bixin is conventionally used as a natural food colorant and is now receiving attention for its health-promoting nutraceutical properties, particularly in chronic diseases (metabolic syndrome—MetS—and cancers). This study investigates the dose-dependent anti-obesity and anti-steatotic effects of Bixin in in vitro cell culture models. The anti-adipogenic and anti-steatotic effects of Bixin were examined in well-established in vitro models of obesity and non-alcoholic fatty liver disease (NAFLD/steatosis) using 3T3-L1 preadipocytes (by a differentiation protocol) and HepG2cells (steatosis-induced with oleic acid), respectively. Bixin was administered in the following concentration range: 1 μg mL−1–20 μg mL−1 (obesity model) and 2.5 μg mL−1–10 μg mL−1 (NAFLD/steatosis model). The neutral lipid content was estimated by Oil Red O staining; ROS/RNS were quantified by 2′,7′-dichlorofluorescein diacetate (DCFDA) and nitrite assays; and malondialdehyde (MDA), the biochemical marker of lipid peroxidation, was assessed by TBARS assay. At lower concentrations, 5 μg mL−1 in steatotic cells and 10 μg mL−1 in matured adipocytes, Bixin significantly reduced lipid accumulation in both hepatocytes and adipocytes (p < 0.05), demonstrating its potential as an anti-steatotic and anti-obesity agent. This beneficial effect was correlated with a reduction in oxidative stress levels (decreased MDA and ROS/RNS levels). Strikingly, at higher concentrations (>10 μg mL−1), Bixin showed increased lipid accumulation and oxidative stress. Bixin exhibits anti-obesity and anti-steatotic effects at lower doses, which correlates with its antioxidant properties. However, its bioactivity is dose-dependent, meaning that at higher concentrations, it ceases to inhibit adipogenesis. This opposing response is accompanied by elevated oxidative stress levels, indicating a pro-oxidant effect at higher doses, which suggests its anti-cancer potential. The present study highlights the significance of dosage optimization of nutraceuticals and dietary ingredients with respect to their intended biological applications, such as MetS and cancer treatment.