Cytotoxic and Pro‐Apoptotic Effects of Cold‐Pressed Hemp Seed Oil on Lung Cells: Cannabinoid and Fatty Acid Profiling via LC–MS/MS and GC–FID

ABSTRACT This study aimed to evaluate the cytotoxic and pro‐apoptotic effects of cold‐pressed hemp seed oil on human lung carcinoma (A549) and normal bronchial epithelial (BEAS‐2B) cells. Cell viability was assessed using MTS assay across a concentration range of 1–50 µg/mL, and a dose‐dependent decline was observed in both cell types. At the highest concentration (50 µg/mL), cell viability decreased to 27% in A549 and 22% in BEAS‐2B cells, indicating limited selectivity. Flow cytometry analysis showed that treatment with 25 µg/mL hemp seed oil resulted in a total late apoptotic/necrotic cell population of 67.13% in A549 cells, with minimal early apoptosis observed. LC–MS/MS analysis confirmed the presence of cannabidiol (CBD, 24.55 µg/mL) and cannabinol (CBN, 11.91 µg/mL), with excellent recovery rates (CBD: 96.10 ± 2.38%, CBN: 98.75 ± 1.76%) and a validated linear range of 1–1000 µg/mL ( R 2 > 0.99). GC–FID analysis identified a favorable fatty acid profile dominated by linoleic acid (55.7%), α‐linolenic acid (18.19%), and oleic acid (14.12%), which may contribute synergistically to the observed bioactivity. While these in vitro findings suggest that hemp seed oil may possess anticancer activity, further in vivo studies are warranted to clarify its mechanisms of action, bioavailability, and therapeutic applicability.