The Effect of Milling on the Ethanolic Extract Composition of Dried Walnut (Juglans regia L.) Shells

This study investigates the ethanolic extract of dried walnut (Juglans regia L.) shells upon hammer milling (HM) and ball milling (BM) grinding processes. Marked differences were observed in the attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectra. The two extracts were investigated by reversed-phase liquid chromatography coupled with electrospray ionization and high-resolution mass spectrometry (RPLC-ESI-HRMS). Following enzymatic digestion, the fatty acids (FAs) were examined, and tandem MS of epoxidized species was applied to establish the C-C double bond position; the most abundant species were FA 18:2 Δ^9,12, FA 18:1 Δ⁹, and FA 18:3 Δ^9,12,15. However, no significant qualitative differences were observed between FAs in the two samples. Thus, the presence of potential active secondary metabolites was explored, and more than 30 phenolic compounds, including phenols, ellagic acid derivatives, and flavonoids, were found. Interestingly, the HM samples showed a high concentration of ellagitannins and hydrolyzable tannins, which were absent in the BM sample. These findings corroborate the greater phenolic content in the HM sample, as evaluated by the Folin-Ciocalteu test. Among the others, the occurrence of lanceoloside A at m/z 391.1037 [C₁₉H₂₀O₉-H]^-, and a closely related benzoyl derivate at m/z 405.1190 (C₂₀H₂₂O₉-H]^-), was ascertained. The study provides valuable information that highlights the significance of physical pre-treatments, such as mill grinding, in shaping the composition of extracts, with potential applications in the biorefinery or pharmaceutical industries.