Abstract:The immunomodulatory effect of bioactive peptides from hemp seeds on RAW264.7 macrophages was investigated. The bioactive peptides from hemp seeds were prepared by enzymatic hydrolysis,ultrafiltration,and Sephadex G-15 gel filtration chromatography. Afterwards,the peptide sequences were identified by LC-MS/MS. The proliferation,morphology,phagocytosis,and secretion of nitric oxide （NO）,interleukin （IL）-1β,IL-6,and tumor necrosis factor（TNF）-α in RAW264.7 macrophages treated with the bioactive peptides were determined by the cell-counting kit 8,microscope observation,neutral red phagocytosis assay,nitrate reductase assay,and enzyme-linked immunosorbent assay. At the same time,the effect of bioactive peptides from hemp seeds on the distribution of macrophages in each phase of cell cycle was studied by flow cytometry. The results showed that the bioactive peptides from hemp seeds mainly consisted of 9 peptides,including DGDGSGFF,MPEDVIAN,and SYNNGDQQ,with the molecular weights ranging from 740 to 1 038 Da. The bioactive peptides promoted the proliferation and differentiation of RAW264.7 cells,demonstrating the strongest immunoenhancing effect at the concentration of 1 000 μg/mL. The number of cells in the G0/G1 phase increased by 56.76%,RAW264.7 cells phagocytic ability enhanced by 1.33 times,and the secretion levels of NO,IL-1β,IL-6,and TNF-α were increased by 1.84,3.94,3.19,and 2.52 times,respectively. It can be concluded that the bioactive peptides from hemp seeds had the potential role of activating macrophages and enhancing non-specific immunity.