EA was purchased Daum International (Hanam, Korea). The EA was washed and dried at room temperature for about 1 week. The dried EA was ground into a fine powder in a blender. About 10 g of EA powder was extracted with distilled water at 60℃. for 15 hours. The extract was filtered with filter paper. The filtrate was concentrated under reduced pressure at 45℃ using a rotary evaporator. The concentrated extract was lyophilized and quantified. The stock solution of the extract was stored frozen at a concentration of 1 mg/mL and used for further experimental analysis.

DPPH analysis was performed with modifications as previously described (Lee et al., 2017). Briefly, 100 μL of two-fold serial dilutions of extracts from various concentration ranges were placed in 96-well microplates. Then, 100 μL of 200 μM DPPH (Sigma, St. Louis, USA) in methanol was added to each sample. For blank samples, the extract and DPPH solutions were replaced with distilled water. Assay controls contained 100 μL ascorbic acid and 100 μL DPPH solution. After incubating the reaction mixture for 15 min at room temperature, absorbance was recorded at 515 nm.

HaCaT keratinocytes, a human keratinocyte cell line used in this study, were purchased from American Type Culture Collection (ATCC, VA, USA), and HaCaT cell lines were cultured in Dulbeccós modified Eagles medium (Gibco, Paisley, UK). A medium containing 10% fetal bovine serum (Gibco, Paisley, UK) and 1% penicillin (100 IU/mL)/streptomycin (100 μg/mL) (Gibco, Paisley, UK) was used. Culture conditions were maintained at 37℃ and 5% CO₂.

A methylthiazolyldiphenyl-tetrazolium bromide (MTT) colorimetric assay was performed to evaluate the cytotoxicity of the extract. Various concentrations of EA were exposed to HaCaT keratinocytes for 24 h. After removal of the medium, the cells were incubated with a 0.5 mg/mL MTT (Sigma, ST. Louis, USA) solution prepared in serum and phenol red-free culture medium for 4 hours. The resulting formazan crystals were dissolved in DMSO and absorbance was measured at 570 nm using a multi-plate reader.

The level of the lipid peroxidation product MDA in cell lysates was determined according to the instructions in a commercial assay kit (Cayman, MI, USA). MDA was analyzed using the thiobarbituric acid (TBA) assay, which is based on the release of color complexes resulting from the reaction of TBA with MDA. The level of the lipid peroxidation products were measured spectrophotometrically.

Data was expressed as mean ± standard error and compared by one-way analysis of variance. Significant differences between groups used Duncan's test. It was considered statistically significant when P < 0.05.

In this study, distilled water was used as the extraction solvent. After removing the extraction solvent using a rotary evaporator, the extract was lyophilized and quantified. The yield was 20.0%.

DPPH is a stable radical and is mainly used to determine the antioxidant potential of natural and synthetic compounds. First, the concentration of the substance that reduced the absorbance of the DPPH radical solution by 50% and the ascorbic acid standard was calculated. Then, the antioxidant capacity of the extract was shown graphically (Fig. 1). It was confirmed that the extract increased in a concentration-dependent manner compared to the ascorbic acid standard up to 800 g/mL.

Fig. 1. DPPH radical scavenging activity.

We determined the effect of EA on the viability of HaCaT keratinocytes according to the MTT colorimetric assay method. EA affected the viability of HaCaT cells in a dose-dependent manner. At concentrations up to 200 μg/mL, cell viability was greater than 90% (Fig. 2). Therefore, we limited the concentration of the extract to 200 μg/mL for further experimental analysis.

Fig. 2. Cytotoxic effects of EA extracts on HaCaT keratinocytes.

EA extract shows cytoprotective effect on HaCaT keratinocytes against oxidative damage caused by H₂O₂. Exposure of HaCaT keratinocytes to 0.4 mM H₂O₂ for 2 h significantly reduced cell viability. Pretreatment of HaCaT keratinocytes with EA extract at a concentration of 200 μg/mL significantly reduced the cytotoxicity induced by H₂O₂ exposure (Fig. 3).

Fig. 3. Protective Effect of EA extracts against H₂O₂-induced HaCaT keratinocytes.

MDA is considered as one of the key molecules in cellular antioxidant defense mechanisms. Thus, the production of MDA mainly depends on the amount of H₂O₂ produced by cells under stress conditions. In this study, we confirmed that pretreatment of cells with EA significantly rescued the H₂O₂-induced the production of MDA depletion (Fig. 4).

Fig. 4. Effect of EA extracts against H₂O₂-induced MDA depletion in HaCaT keratinocytes.

Oxidative stress results from overproduction of ROS or failure of cellular endogenous antioxidant defense mechanisms (Ray et al., 2012). In this respect, dietary supplementation of exogenous antioxidants such as plant extracts is very important to maintain a balanced cellular antioxidant status. In this study, we determined the antioxidant potential of EA using widely considered chemical radical scavenging assays such as DPPH. In addition, the antioxidant capacity of natural products is generally expressed as compared to the well-established standard antioxidant ascorbic acid. Furthermore, because even the same antioxidants have different effects in chemical and cell-based assays, we further investigated the antioxidant potential of EA extracts using a cell-based model. HaCaT keratinocytes, a human keratinocyte line, have a stable antioxidant defense system and are widely used to screen for beneficial effects of various antioxidants (Alía et al., 2006). The H₂O₂, a non-radical derivative of oxygen, is widely used as an exogenous oxidant to induce oxidative stress in cell-based antioxidant assays (Chen et al., 2007; Jiang et al., 2014; Han et al., 2017). Based on this background, in this study, the antioxidant efficacy of EA extract was confirmed by inducing oxidative stress in HaCaT keratinocytes using the H₂O₂.

In conclusion, the results of DPPH, a chemical antioxidant assay, clearly demonstrated the antioxidant capacity of EA extracted with distilled water. In addition, cell-based assays provide useful information on the protective effect of EA on oxidative stress-induced apoptosis.