Abstract:

Fluorescein leakage （FL） test and recombinant human corneal epithelial cell model eye stimulation （ET₅₀） test were both took to investigate the protective effects of three different molecular weight hyaluronic acids （HA） including high, low and oligomeric on eye irritation. Firstly, seven verification substances in different stimulation levels including benzalkonium chloride （5%）, 4-carboxy-benzaldehyde, promethazine hydrochloride, ethyl-2-methylaceto-acetate, glycerol, sodium lauryl sulfate （15%, w/%） and sodium hydroxide （10%, w/%） were used to verify the luciferin leakage test in our own laboratory. Then, fluorescein leakage test and in vitro artificial cornea model-eye stimulation ET₅₀ test were both conducted to evaluate the inhibitory effect of three hyaluronic acids on eye irritation by the index of fluorescence leakage rate as well as cell survival rate. Fluorescein leakage test method is established successfully and is confirmed that 10 g/L mass concentration of hyaluronic acid can significantly reduce the increase of fluorescence leakage rate caused by 0.25 g/L mass concentration of benzalkonium bromide. What’s more, incubating for another 24 h after exposure to 10 g/L mass concentration of hyaluronic acid and 0.25 g/L mass concentration of benzalkonium bromide for 30 min, the cell survival rate does not change, but the morphological change of the cells can be observed tending to be normal under the microscope, compared to the cells treated by 0.25 g/L mass concentration of benzalkonium bromide only. Meanwhile, in vitro artificial corneal model-eye stimulation test confirms that oligo hyaluronic acid can significantly inhibit the decrease of cell survival rate which causes by methyl acetate at mass concentration of 20 g/L rather than 10 g/L. Accordingly, in vitro artificial corneal model-eye stimulation test （ET₅₀） confirms that 10 g/L mass concentration of hyaluronic acid can significantly inhibit the decrease of cell survival rate and increase the ET₅₀ value which is induced by 0.5 g/L mass concentration of benzalkonium bromide, but it falls flat on serious irritation induced by 10% （w/%） content of Brij35 solution. In conclusion, these results show that high, low and oligomeric molecular weight hyaluronic acid can all significantly reduce the irritation of ocular stimulation induced by mild positive substances, which provides a basis for the protective effect of hyaluronic acid, reveals a new prospect of the application of hyaluronic acid in cosmetics and presents a new method of efficacy evaluation on cosmetic raw materials.

Key words: eye irritation, hyaluronic acid, eye irritation test, recombinant human corneal epithelium, fluorescein leakage