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Berberine is widely used in traditional Iranian medicine to treat diabetes and inflammatory conditions. This study was aimed at developing a method for the preparation of Berberine nanoparticles (Nano-Ber) in order to improve its aqueous-phase solubility and its complex formation with human serum albumin (HSA) and holo-transferrin (HTF) from the viewpoint of interaction behavior. Nano-Ber was prepared with olive oil as the oil phase, Tween 80 as the surfactant and Span 60 as the co-surfactant. Nano-Ber was obtained with a spherical shape and a mean particle size of 43.7 ± 3.6 nm, with an optimal oil:surfactant:co-surfactant ratio of 1:2:2, w/w/w. The antioxidant activity of Nano-Ber in comparison with Berberine was tested using DPPH and it was found that Nano-Ber had a large antioxidant activity. The cytotoxicity effects of Nano-Ber and Berberine on HepG2 were compared by MTT assay and detected in …