In the current study, extracellular vesicles from pomegranate juice (PgEVs) were isolated for the first time using size exclusion chromatography (SEC). This method permitted us to obtain highly enriched EV samples without most of the non-EV co-isolated proteins. The characterization of PgEVs through nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) allowed the determination of vesicles’ concentration/volume, size, and morphology. It was confirmed from the analytical data that PgEVs contain a homogeneous population of vesicles, with a dimension and structure comparable to plant-derived EVs. Proteomic analyses by LC-MS/MS led to the characterization of 131 proteins, and several of them were related commonly to the biogenesis and transport of EVs, and/or proposed as EV markers. PgEVs exerted anti-inflammatory, antioxidant and wound-healing effects when added to the in vitro cultures of monocytic (THP-1) and intestinal (Caco-2) cell lines, respectively.

Plant-derived nanovesicles (PDNVs) are increasingly recognized as mediators of intercellular communication in plants, where they play roles in defense, signaling, and cell wall remodeling. In addition, PDNVs are gaining increasing attention for their biomedical potential, both as natural delivery systems and as bioactive entities, with promising applications in inflammatory disorders and cancer. In this study, we isolated carob nanovesicles (CbNVs) from the apoplastic fluid of carob pods (Ceratonia siliqua L.) using vacuum infiltration centrifugation followed by tangential flow filtration and size-exclusion chromatography. Morphological and biophysical analyses revealed spherical vesicles, while proteomic profiling identified 197 proteins, including suggested PDNV markers such as annexin, HSP70, GAPDH, elongation factors, malate dehydrogenase, and TET-8. These proteins were enriched in metabolic processes, stress responses, and cell wall modification pathways. Protein–protein interaction analysis further linked HSP70 to pectinesterases, reinforcing their role in cell wall remodeling and stress adaptation. Functionally, CbNVs were non-cytotoxic to human macrophages, keratinocytes, and intestinal cells. Notably, CbNVs significantly reduced LPS-induced NF-κB expression in macrophages and promoted wound closure in keratinocytes, with superior efficacy compared to the carob bioactive compound D-pinitol. These findings suggest that CbNVs harbor a synergistic cargo of bioactive molecules with anti-inflammatory and wound-healing properties, highlighting their potential as natural nanotherapeutics.