Elucidating the Biological Role of Fenugreek Seed-Derived Nanovesicles: Isolation, Physicochemical Characterization, and In Vitro Evaluation

Plant-derived nanovesicles (PDNVs) have gained prominence as effective drug delivery vehicles owing to their biocompatibility, nutritional source, and therapeutic efficacy. The present study isolated nanovesicles from Trigonella foenum-graecum (fenugreek) seeds using ultracentrifugation, utilizing Tween 80 (0.5% and 1%) to improve stability and inhibit aggregation. Morphological characterization by SEM and HR-TEM validated that the fenugreek-derived nanovesicles (FDNs) were spherical and had a bilayer membrane structure. FDNs showed no cytotoxic effects on RAW 264.7 macrophage cells at concentrations up to 62.5 μg/mL and maintained structural stability at −20 °C for four months. Cellular uptake investigations revealed total internalization within 12 h. Functionally, FDNs demonstrated significant antioxidant activity across various assays, including DPPH, hydrogen peroxide, superoxide anion, nitric oxide, and ROS scavenging. Antibacterial screening demonstrated inhibitory effects on Bacillus subtilis and Pseudomonas aeruginosa, with minimum inhibitory concentrations (MICs) of 90.45 and 868.5 μg/mL, respectively. A comprehensive analysis of the FDN cargo by metabolomics and proteomics identified a diverse array of bioactive compounds, such as flavonoids, alkaloids, triterpenoids, and fatty acids, in addition to proteins associated with metabolism, defense, and enzymatic activity. The results collectively emphasize the multifunctional potential of FDNs as strong antioxidant and antibacterial agents, establishing them as promising candidates for future anti-inflammatory and therapeutic formulations.