Carthamus tinctorius
L. (Safflower) is widely used in traditional Japanese, Korean, Chinese, Arabian, and Persian herbal medicine to treat metabolic diseases. This study aimed to characterize
C. tinctorius
seed oil components and estimate its inhibitory effects on free radicals, porcine pancreatic lipase, α-amylase, and cytotoxic. To describe the phytochemical components of
C. tinctorius
seed oil, the Gas Chromatography-Mass Spectrometry (GC-MS) technique was performed, while reference biochemical analytical assays were utilized for biological testing. The results showed that seven fatty acids accounting for 100% of the total oil were identified, and the major fatty acid was linoleic acid (79.98 ± 0.79%), followed by oleic (11.20 ± 0.21%) and palmitic (5.71 ± 0.12%) acids. The biological tests revealed that
C. tinctorius
seed oil has potent inhibitory effects on free radicals, porcine pancreatic lipase, and α-amylase, with IC
50
values of 13.18 ± 0.07, 43.6 ± 0.09 and 31.62 ± 0.65 μg/ml, respectively, in comparison with positive controls commercial drugs Trolox, Orlistat, and Acarbose, which have IC
50
values of 4.1 ± 0.57, 12.88 ± 0.94, and 28.18 ± 1.22 μg/ml, respectively.
C. tinctorius
oil showed potential cytotoxic effects against tested cancer cells lines with a concentration-dependent effect on cancer cell viability. Given these findings, it is clear that
C. tinctorius
oil exhibits potent DPPH free radicals, antilipase, porcine pancreatic α-amylase inhibitory, and cytotoxic properties in comparison to the positive controls. Future in vivo research on
C. tinctorius
seed oil is warranted to elucidate the oil’s mechanism of action and to decipher the molecular pathways involved in its anti-obesity, antidiabetic, antioxidant, and cytotoxic activities.