Abstract: Objective: This study aimed to analyze the bioactive components of garlic-derived extracellular nanovesicles (GENs) and explore their anti-inflammatory activity against colitis in mice. Methods: GENs were isolated from garlic juice by ultracentrifugation and sucrose density gradient centrifugation, and the morphology and structure of GENs were characterized. Lipids and proteins were detected by liquid chromatography tandem mass spectrometry (LC-MS/MS) and analyzed by online databases. Meanwhile, RNA sequencing was carried out for sequence identification and target gene prediction of microRNAs (miRNAs). Caco-2 cells were cultured as a model of monolayer intestinal epithelial cells and were determined for apparent permeability coefficient by fluorescence spectroscopy after lipopolysaccharide (LPS) stimulation. The level of nitric oxide (NO) in the supernatant was measured by a commercial assay kit, and interleukin-6 (IL-6), interleukin-1β (IL-1β), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) by enzyme-linked immunosorbent assay (ELISA). C57BL/6J mice were orally administrated with GENs (20, 100 and 500 mg/(kg mb·d)) to evaluate the toxicity of GENs. The effect of GENs on the disease activity index (DAI) score and colon length of mice with 3% dextran sulphate sodium salt (DSS)-induced colitis was investigated. Colonic tissue sections were stained with hematoxylin and eosin (H&E) and observed. The levels of IL-6, IFN-γ and TNF-α as well as NO in the serum were measured by ELISA and a commercial assay kit, respectively. Results: GENs were goblet nanovesicles containing 26 lipids, 62 proteins and 317 microRNAs. Phosphatidylcholine (PC) was the most abundant lipid component. Five miRNAs targeting human genes encoding inflammatory factors were predicted. GENs significantly reduced LPS-induced damage to the permeability of Caco-2 cell monolayers, inhibited the production of NO, and regulated the secretion of IL-6, IL-1β, IFN-γ and TNF-α. GENs were non-toxic towards mice at all doses tested. GENs at 100 mg/(kg mb·d) significantly decreased the DAI score of mice (P < 0.05), improved shortening of colon length and pathological damage induced by DSS and repressed the increase in serum IL-6, IFN-γ and NO levels. Conclusion: GENs exert an anti-inflammatory activity probably through their bioactive molecules such as phosphatidylcholine and specific miRNAs. GENs have great potential for alleviating intestinal inflammation and preventing DSS-induced colitis in mice.

Key words: garlic; extracellular nanovesicles; bioactive components; anti-inflammatory activity; colitis