Comparative UHPLC-MS/MS-based untargeted metabolomics analysis, antioxidant, and anti-diabetic activities of six walnut cultivars

Walnuts are important stone fruits that are commonly consumed as food or used in food products. However, most studies have focused on specific compounds found in walnuts. Therefore, this study aimed to compare untargeted UHPLC-MS/MS metabolite profiles and determine the antioxidant and antidiabetic properties of six walnut cultivars. A total of 135 metabolites belonging to fourteen different classes were identified including amino acids, lipids, carboxylic acids and amides, phenyl alcohols and aldehydes, ketones, flavonoids, oxygenated hydrocarbons, glycosides, phenolic acids, hydrocarbons, sugar alcohols and acids, and vitamins. The main classes of metabolites were amino acids, lipids, oxygenated hydrocarbons, carboxylic acids and amides, phospholipids, and phenolic acids. The six cultivars were divided into two groups based on PCA, and PLS-DA score with significant differences in the number of distinct metabolites between the two groups. Hierarchical cluster analysis further confirmed these groups by comparing the similarities and differences of all metabolites in the six cultivars. Pathway enrichment analysis revealed that the most prominent metabolic pathways in walnuts were related to amino acids, followed by flavonoids and vitamins. The antioxidant and antidiabetic effects of walnuts varied significantly among the cultivars. Group A, which had higher levels of upregulated metabolites, showed greater IC₅₀ values for ABTS, and DPPH scavenging, as well as α-glucosidase activities. On the other hand, walnuts 4, 5, and 6 in group B had more downregulated metabolites and higher IC₅₀ values for α-amylase inhibition. These findings suggest that walnuts contain key metabolites with antioxidant and antidiabetic properties, which occur at cultivar-dependent levels.