Valorization of <i>Juglans regia</i> L. (Walnut) green husk from Jordan: Analysis of fatty acids, phenolics, antioxidant, and cytotoxic activities

Abstract This study evaluated the extraction methods for optimizing lipid and phenolic yields from Juglans regia L. (walnut) green husk and assessed the extracts’ cytotoxic effects on cancer cells (liver cancer cell line, HepG2) and normal human cells (HUVEC). Among the various lipid extraction techniques, the Bligh and Dyer method was most efficient (3.4% dry weight), followed by Soxhlet extraction with n -hexane (2.4%), Folch (2.3%), and sonication (1.7%). For phenolic extraction, sonication with 70% ethanol achieved the highest yield (18.8%), with Soxhlet extraction close behind (17.7%). Total phenolic content varied, with reflux extraction yielding 0.07 ± 0.05 mg GAE/g and hexane Soxhlet extraction at 0.023 ± 0.03 mg GAE/g. Cytotoxicity assays showed dose-dependent inhibitory effects on HepG2 cells for both 70% ethanol maceration and Bligh and Dyer extracts, with IC 50 values of 277.3 and 237.5 μg/mL, respectively. The Bligh and Dyer extract had an IC 50 of 185.3 μg/mL on HUVEC cells. Microscopy and DAPI staining indicated apoptotic changes in HepG2 cells, while DCFH-DA staining revealed elevated ROS levels, suggesting oxidative stress. Gas Chromatography-Mass Spectrometry identified unique compounds with potential bioactivity, and molecular docking highlighted 5-hydroxy-1-tetralone as a strong binder with protein 3TZM (−8.0 kcal/mol). This study underscores the influence of extraction methods on yield and bioactivity.