| Subject: Resveratrol nanoemulsion was prepared and its quality was evaluated. The toxicity of resveratrol nanoemulsion on hepatoma carcinoma cell HepG2 and hepatocyte of rat in vitro was researched.Method: (1) To prepare the nanoemulsion with nonionic surfactant Polyoxyethylene castor oil ether, Cremophor RH-40, Tween80 and Span80. The effects of surfactants, cosurfactants and its mixing ratio, oil on nanoemulsion regions were examined and optimized by using the pseudo-ternary phase diagram. The physical characters and the stability of the nanoemulsion were assessed. (2) Choose isopropyl myristat as oil, ethanol as cosurfactants, select one kinds of surfactant from EL-40 and RH-40, using the pseudo-ternary phase diagram to prepare resveratrol nanoemulsion. Its average diameter, physical stability and content of resveratrol were evaluated by electron microscope, stability tests and UV spectrophotometry respectively. (3) MTT assay and cell clone were used to observe the effects of resvertrol namoemulsion on HepG2 cell growth, and the effect to growth curve was detected. Inverted microscope, HE staining and scanning microscope were used to reveal abnormal morphology of HepG2 cells treated with resvertrol namoemulsion. Cell cycle and apoptosis were detected with flow cytometry. The content of glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase and lactic acid dehydrogenase were tested in the HepG2 cell culture fluid. (4) Hepatocyte of rat was cultured, MTT assay were used to observe the effects of resvertrol namoemulsion on it. Three kinds of enzyme also tested on normal hepatocyte.Results: (1) To form namoemulsion was effected by surfactant, cosurfactant, oil and Km(proportionality of surfactant and cosurfactant). The blank nanoemulsion mean particle size was 10.7±1.2 nm. All particles were smaller than 100 nm. No phase separation was observed by centrifugation at 13,000 rpm for 30 minutes, storage at 40±2℃and relative humidity 75±5% for six months. (2) The nanoemulsion of region used Rh-40 as surfactant was larger than used EL-40 as surfactant. The results showed that the shape of resveratrol nanoemulsion was sphere under transmission electron microscope, with a mean diameter was 17.6±0.9 nm, well-distributed between 1100 nm. The content of resveratrol was 6.184±0.143 mg.mL-1. The absorbency and content of resveratrol was linearly dependent in 012 μg.mL-1, r=0.9993. The average recovery was 95.8%, and the RSD of precision with-day and between-day were all smaller than 0.25%. So, the resveratrol nanoemulsion prepared was stable and the means of UV spectrophotometry established was equal to analyse the content of nanoemulsion. Resveratrol was protected from been oxidized by the mechanism of nanoemulsion. (3) Treatment with resvertrol namoemulsion and resvertrol 4 mg.L-1 for 3 d respectively, cell proliferation degraded significantly (p<0.01), compared with control group. When the concentration of resvertrol was higher than 8mg.L-1, the rate of cell inhibition with resveratrol nanoemulsion was significant increased compared with resveratrol at 48 h, and IC50 were 6.5mg.L-1 and 24.2mg.L-1 respectively. HepG2 cells appeared apoptotic characteristics and stayed at S/G2 phase after treatment with 8 mg.L-1 resveratrol nanoemulsion. The content of ALT,AST,LDH were not changed. (4) Resvertrol namoemulsion and crude resveratrol have not significant toxicity to hepatocyte of rat. The content of ALT,AST,LDH also were not changed in the cell culture fluid.Conclusion: Resveratrol nanoemulsion was transparent and stable. The concentration and the accuracy of assaying of Resveratrol in Resveratrol nanoemulsion were high. Resveratrol was protected from been oxidized by the mechanism of nanoemulsion. The rate of cell inhibition on HepG2 treatment with resveratrol nanoemulsion was significant higher than resveratrol, and not has significant toxicity to hepatocyte.
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