Protective Effect of Dealcoholized Persimmonwine on H 2 O 2-Induced Oxidative Injury in H 9 c 2 Cardiomyocytes

In this study, we investigated the antioxidant capacity of persimmon wine (PW) and dealcoholized persimmon wine (DPW). Both PW and DPW showed radical scavenging activity in the DPPH (1-diphenyl-2-picrylhydrazyl) assay. We next analyzed the phenolic content and major compounds present in PW using high-performance liquid chromatography (HPLC). Phenolic compounds, including gallic acid, catechin, and epicatechin, were found in PW. Gallic acid was the most abundant phenolic compound (157.5 μg/ml) in PW. In addition, the protective effects of DPW and gallic acid against H2O2-induced cell injury in H9c2 cardiomyocytes were investigated. Pretreatment with DPW or gallic acid strongly inhibited H2O2-induced cell death in a dose-dependent manner. These results suggested that PW and its major phenolic component, gallic acid, were effective inhibitors of oxidative stress and oxidative stress-induced cardiomyocyte injury.


Introduction
Persimmon is widely grown in oriental countries, such as China, Japan, and Korea.Persimmon fruit showed some lipid lowering effects in animal models (Matsumoto, Watanabe, Ohya, & Yokoyama, 2006).Furthermore, it is abundant in nutrients, including vitamins A, B, and C; carotenoids; glucose, and fructose.Persimmon also contains other active compounds, such as polyphenols, which have been reported to have protective effects against oxidative stress, and exerted benefits on diabetes, obesity, cardiovascular disease, and even cancer (Matsumoto, Watanabe, Ohya, & Yokoyama, 2006;Wojcik, Burzynska-Pedziwiatr, & Wozniak, 2010).Phenolic composition is an important factor affecting the functionality of natural ingredients or products (Cabrera, Artacho, & Giménez, 2006).A number of studies have identified a correlation between the phenolic components and physiological activities of natural ingredients or products (Kılıçgün & Altıner, 2010).
Reactive oxygen species (ROS) have been reported to be associated with the development of various diseases, including cardiovascular disease.In particular, heart ischemia and reperfusion lead to the generation of ROS, thereby resulting in cellular injury (Varela, Rolo, & Palmeira, 2011).A number of studies have suggested that antioxidants have protective effects on ischemia-reperfusion-induced cell death.For example, administration of antioxidant protected against ischemia-reperfusion injury in cardiomyocytes (Braunersreuther & Jaquet, 2012).Several antioxidants found in food sources also exerted protective effects against ROS-induced cellular injury in various cell types (Rodrigo, Prieto, & Castillo, 2013;López-Miranda et al., 2012).
Persimmon wine (PW) contains various chemical compounds, including polyphenols.Although PW has bioactive compounds and is believed to influence various physiological functions, its ability to protect against oxidative stress has not yet been investigated.To determine the antioxidant activity of PW in vitro, either PW or dealcoholized persimmon wine (DPW) were used.Dealcoholized persimmon wine (DPW) was used to investigate cardioprotection in H9c2 cells because the alcohol in PW can affect the cell system and was therefore not added directly to the cells.

Wine Making and Sample Preparation
PW was produced by Yangchon Persimmon Wine Farm Corporation (Choosi wine, 2010).Briefly, harvested persimmons were randomly crushed and incubated with 1.5% citric acid, 2.5% tartaric acid, and 250 ppm sulfite in sterile conditions.After incubation, 0.2% Monascus (KCCM 60170, Korean Culture Center of Microorganisms, Seoul, South Korea) was added and the temperature was maintained at approximately 26 °C for 10 days.Thereafter, the temperature was increased to 50 °C for an additional 3 days to induce alcoholic fermentation.Once fermentation was complete, the fermented liquid was transferred to a new tank, inoculated with red wine yeast and pectin lyase (Pascal Biotech, Paris, France), and maintained at 26 °C for 14 days.
Cellulase (Lot No. CTD1150106, Amano Enzyme Inc., Nagoya, Japan) was added for 30 days, and thereafter, batonnage was performed for 50 days to remove the yeast.Finally, PW was filtered (Papeleradel Besós Placas Filtrantes Sl, Barcelona, Spain) and stored at 18 °C for 1 year.DPW was prepared by evaporation in a rotary evaporator (Laborota 4000, Heidolph Instruments Inc., Schwabach, Germany), and freeze-drying to remove the alcoholic and aqueous phases.The resulting wine powder was re-suspended in distilled water prior to the cell culture experiments.

DPPH (1-diphenyl-2-picrylhydrazyl) Radical-Scavenging Assay
DPPH radical-scavenging activity was investigated according to the method of Hou et al (Hou et al., 2001).DPPH was dissolved in methanol (spectrophotometric grade) at various concentrations.PW and DPW samples (0.1 ml) were mixed with 0.1 ml of 300 µM DPPH solution for 30 min in the dark.The absorbance at 517 nm (A517) was determined, using methanol as the blank.DPPH radical-scavenging activity was calculated according to the following equation: scavenging activity (%) = 100 × (A517blank -A517sample)/A517blank.IC50 values denote the concentration of sample required to scavenge 50% of DPPH free radicals.

Quantitative Analysis of Phenolic Compounds
Analysis was conducted using a high-performance liquid chromatography (HPLC) system (Jasco, Japan) with a Bondapak C18 (10 µm, 3.9 × 300 mm) column, and a mobile phase of distilled water containing 2% acetic acid (solvent A) and 50% acetonitrile containing 0.5% acetic acid (solvent B).Gradient elution of 10-80% solvent B over 70 min was used at a flow rate of 0.8 mL/min.The column temperature was maintained at 40 °C, and the signal was detected at 280 nm.Gallic acid, catechin, epicatechin, gallocatechin, gallocatechingallate, and catechingallate were used as standards for quantification and were purchased from Sigma (Sigma-Aldrich, St. Louis, Mo., U.S.A.).

ROS Measurement
Cellular ROS were measured using a fluorescence microscope as described previously (Hwang et al., 2006).Briefly, cells were pre-treated with DPW for 1 h, and then exposed to 500 µM H 2 O 2 for 4 h.After an additional 30-min treatment with 10 µM 2',7′-dichlorofluorescin diacetate (DCFH-DA), cells were washed with PBS and the fluorescence was analyzed by fluorescence microscopy.

Statistical Analysis
All data are presented as means ± SD.Statistical analysis by one-way ANOVA was carried out using SPSS 9.0 (SPSS Inc., Chicago, IL).All experiments were performed in triplicate and repeated at least 3 times.

Results and Discussion
We analyzed thephenolic contents of PW (Figure 1A) using HPLC.As shown in Table 1, we analyzed several ) www.ccsen

Conclusion
The present study demonstrated that DPW exhibited antioxidant effects and protected against H 2 O 2 -stimulated cell death in H9c2 cells.Moreover, our findings implicated gallic acid as a key antioxidant constituent in DPW.Further studies should investigate the precise mechanism by which DPW and gallic acid exert protective effects against oxidative stress and cardiac injury.
Figure ormance liquid avenging activ