Bioavailability of Antioxidants Applied to Stratified Human Corneal Epithelial Cells

Abstract

Purpose: : Dry eye disease is caused by deficient tear production and inflammation. If the tear film is abnormal, the cornea is irritated, inflamed, and may be subject to oxidative damage. Adding an antioxidant to a lubricant eye drop could potentially be of benefit in treatment of dry eye disease. For an antioxidant to be effective it must be bioavailable and quench reactive oxygen species (ROS) in the cell. The goal of this study was to determine the bioavailability of quercetin, epigallocatechin gallate (EGCG), n-propyl gallate, and gallic acid, and measure their effectiveness in reducing reactive oxygen species (ROS) generated by 2,2'-azobis (2-amidinopropane) dihydrochloride (ABAP) in stratified human corneal limbal epithelial (HCLE) cells in culture.

Methods: : HCLE cells were loaded with 2’-7’-dichlorofluorescin diacetate (DCFH-DA). Inside the cell, DCFH-DA is cleaved by esterases to form the more polar molecule 2’-7’-dichlorofluorescin (DCFH). HCLE cells were then incubated in HBSS with an antioxidant for one hour. Cells were then exposed to ABAP and fluorescence was measured immediately and every twenty minutes for 60 minutes. When ROS oxidize DCFH, the fluorescent molecule dichlorofluorescin (DCF) is formed. Therefore, oxidation occurring in the HCLE cells can be monitored by following the change in fluorescence over time. An effective antioxidant reduces the fluorescence caused by ABAP. An EC₅₀ was calculated for each antioxidant by measurement of the area under the curve for a range of antioxidant concentrations.

Results: : All of the compounds tested were determined to be effective antioxidants. Quercetin reduced ROS with an EC₅₀ of 3.34 µM. EGCG was also able to reduce ROS generated by ABAP, with an EC₅₀ of 3.41 µM. n-Propyl gallate had an EC₅₀ of 6.34 µM. Gallic acid was a less effective antioxidant with an EC₅₀ of 60.89 µM.

Conclusions: : This study demonstrates that quercetin, epigallocatechin gallate, n-propyl gallate, and gallic acid are effective antioxidant compounds in HCLE cells. When applied directly to corneal cells in culture, each compound was able to quench ROS generated intracellularly by ABAP. These compounds have good bioavailability and might be effective in protecting the corneal epithelium from oxidative damage.

Keywords: cornea: tears/tear film/dry eye • cornea: epithelium • antioxidants