Optimizing health benefits of walnut (Juglans regia L.) agricultural by-products: Impact of maceration and Soxhlet extraction methods on phytochemical composition, enzyme inhibition, antioxidant, antimicrobial, and cytotoxic activities

Jııglans regia L. is a species from Juglandaceae family that draws attention due to its nutritional and health benefits worldwide. In this study, significant agricultural by-products of the walnut such as leaves and green husks were investigated in terms of enzyme inhibition, antioxidant capacity, antimicrobial and cytotoxic activity based on the phytochemical composition. They were compared with extraction methods named maceration and Soxhlet extraction. For this aim, the enzyme inhibition activity was tested with α-amylase, α-glucosidase, acetylcholinesterase, butyrylcholinesterase and tyrosinase enzyme inhibition methods, the antioxidant activity was investigated with DPPH●, ABTS●+ and FRAP tests. However, the antimicrobial activity was investigated with the microdilution method and cytotoxic activity with the MTT method against HeLa and RL95-2 cell lines. The phytochemical content of the active extract was determined by TPC, TFC and the major compounds were determined with LC-HRMS methods. As a result, the highest anti-cholinesterase activity even from positive control was found in leaf extract by maceration method (IC50 = 162.225 ± 2.656 and 108.634 ± 6.691 μg/mL, respectively), as well as antioxidant activity. Moreover, in contrast to a moderate antimicrobial effect, the highest cytotoxic activity was found in leaf extract against RL95-2 cell line with 86.12 μg/mL IC50 values. Additionally, ellagic and chlorogenic acids were revealed as major compounds on the results of phytochemical analyses of active leaf extract. Thus, it was concluded that the leaf extract of the walnut by the maceration method could be chosen for industrial applications spanning the food, pharmaceutical, and cosmetic sectors.