PURPOSE: Oxidative damage to the corneal epithelium may be involved in dry eye disease. The bioavailability and efficacy of antioxidants in human corneal limbal epithelial (HCLE) cells were measured to determine whether antioxidants might be beneficial constituents of lubricant eye drops. METHODS: The activity of antioxidants was evaluated using a cellular antioxidant activity assay in which, cells were loaded with the reactive oxygen species (ROS)-sensitive fluorescent indicator, 2',7'-dichlorofluorescin diacetate (DCFH-DA), and an antioxidant compound. ROS were then generated intracellularly using 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP) or extracellularly using xanthine oxidase, and the ability of an antioxidant to inhibit ROS-generated fluorescence was measured. RESULTS: When ROS were generated by ABAP, EC50 values for quercetin, epigallocatechin gallate (EGCG), n-propyl gallate, and gallic acid were 2.98, 3.41, 6.30, and 50.7 μM, respectively. When ROS were generated extracellularly by xanthine oxidase, EC50 values for quercetin, EGCG, n-propyl gallate, and gallic acid were 41.3, 56.5, 70.5, and 337.5 μM. These values were reduced significantly when an antioxidant was present both in the medium with the xanthine oxidase and within the cells. CONCLUSIONS: The antioxidants were effective at quenching ROS in HCLE cells, indicating that they are bioavailable and might be effective in protecting the corneal epithelium from oxidative damage if included in a lubricant eye drop.
PURPOSE: Oxidative damage to the corneal epithelium may be involved in dry eye disease. The bioavailability and efficacy of antioxidants in human corneal limbal epithelial (HCLE) cells were measured to determine whether antioxidants might be beneficial constituents of lubricant eye drops. METHODS: The activity of antioxidants was evaluated using a cellular antioxidant activity assay in which, cells were loaded with the reactive oxygen species (ROS)-sensitive fluorescent indicator, 2',7'-dichlorofluorescin diacetate (DCFH-DA), and an antioxidant compound. ROS were then generated intracellularly using 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP) or extracellularly using xanthine oxidase, and the ability of an antioxidant to inhibit ROS-generated fluorescence was measured. RESULTS: When ROS were generated by ABAP, EC50 values for quercetin, epigallocatechin gallate (EGCG), n-propyl gallate, and gallic acid were 2.98, 3.41, 6.30, and 50.7 μM, respectively. When ROS were generated extracellularly by xanthine oxidase, EC50 values for quercetin, EGCG, n-propyl gallate, and gallic acid were 41.3, 56.5, 70.5, and 337.5 μM. These values were reduced significantly when an antioxidant was present both in the medium with the xanthine oxidase and within the cells. CONCLUSIONS: The antioxidants were effective at quenching ROS in HCLE cells, indicating that they are bioavailable and might be effective in protecting the corneal epithelium from oxidative damage if included in a lubricant eye drop.