This study systematically explored the antidiabetic potential of Coix lacryma-jobi (Job’s tears) fruit extract through various in vitro assays. The extract demonstrated potent inhibition of key glucose-metabolising enzymes: α-glucosidase (50% inhibitory concentration [IC50] = 4.31 ± 0.19 mg/ml), α-amylase (IC50 = 11.02 ± 0.11 mg/ml), and dipeptidyl peptidase-4 (DPP-4) surpassing the effects of insulin and sitagliptin at optimal concentrations. Glucose uptake assays in L6 myoblasts showed enhanced absorption (122.89% at 62.5 μg/ml), akin to insulin’s activity, likely via GLUT4 modulation. The extract’s antioxidant capacity (IC50 = 7.18 ± 0.12 mg/ml 2,2-diphenyl-1-picrylhydrazyl scavenging) was attributed to its phenolic (5.3 ± 0.62 mg gallic acid equivalent per gram) and flavonoid content (1.219 ± 0.15 mg quercetin equivalent per gram), aiding in oxidative stress mitigation. Polymerase chain reaction (PCR) array analysis indicated upregulation of insulin signalling genes (GLUT4, IRS1, and PIK3R1) and downregulation of inflammatory markers (TNF and IL6). Minimal cytotoxicity (IC50 > 6 mg/ml) in L6 and 3T3-L1 cells highlighted its safety. Coix lacryma-jobi shows promise as both a therapeutic agent and a functional food ingredient, offering new insights into DPP-4 inhibition and multifaceted antidiabetic mechanisms.
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