Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (PS) and trypsin-EDTA were purchased from Gibco (Grand Island, NY, USA). 2′,7′-Dichlorodihydrofluorescein diacetate (H₂DCF-DA) was obtained from Molecular Probes (Carlsbad, CA, USA). 2,2-Diphenyl-1-picrylhydrazyl (DPPH), H₂O₂ and Hoechst 33342 were purchased from Sigma Biochemical (St. Louis, MO, USA). All other chemicals were of analytical grade.

An ethanol extract of the mulberry fruits (15 kg) was acquired by extraction with 70% ethanol (36 liters) for 24 h at room temperature 3 times. The solvent was filtered and evaporated under a vacuum to afford 2,200 g crude ethanol extracts (yield, 14.6%). The lyophilized crude ethanol extracts were successively fractionated with ethyl-acetate (EtOAc, MAE extract), n-butanol (BuOH, MAB extract) and water (H₂O, MAH extract). Each fraction was evaporated in vacuo. The yields of the extracts and the fractions were as follows: 1.59% (EtOAc fraction), 10.23% (BuOH fraction) and 83.63% (H₂O fraction).

Mouse insulin-producing MIN6N pancreatic β-cells were derived from a mouse pancreatic islet cell line. The MIN6N β-cells were provided by Professor H.Y. Kwon (College of Medicine, Hallym University, Chuncheon, Korea). The cells were cultured in DMEM (11 mM glucose; Gibco) supplemented with 10% inactivated FBS and 1% PS and maintained at 37°C in a humidified 5% CO₂ incubator. The cells were cultured up to approximately 85% confluence and harvested with 0.25% trypsin-EDTA. The cells were harvested and subcultured for an additional 48 h in DMEM. The cells were maintained in these culture conditions for all the experiments.

The effects of the fractionated extracts (MAB, MAE and MAH) on DPPH radicals was estimated according to the Blois method (1). MAB, MAE and MAH were dissolved in EtOH over a concentration range of 10 to 500 μg/ml. Each sample solution (400 μl each) was mixed with DPPH solution (600 μl) and incubated for 30 min. to allow reactions. The absorbance of the resulting solution was measured at 520 nm using an ELISA microplate reader (Model 550; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The cytotoxic effects of the fractionated extracts in pancreatic MIN6N β-cells were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which is based on the reduction of a tetrazolium salt by mitochondrial dehydrogenase in viable cells (23). The MIN6N β-cells were seeded in 12-well plates and incubated for 24 h. The cells were then treated with the fractionated extracts and incubated for 20 h at 37°C; 500 μg/ml MTT working solution were then added to each well followed by incubation for 2.5 h at 37°C. The violet formazan crystal in each well was dissolved in isopropyl alcohol and the absorbance of each well was measured at 570 nm using an ELISA microplate reader (model 550; Bio-Rad Laboratories, Inc.).

To investigate the protective effects of the fractionated extracts against H₂O₂-induced cell death, the cells were first incubated with DMEM containing 0.5% FBS for 24 h. Following this, the indicated dose of the fractionated extract was added for 20 h, prior to treatment with 0.7 mM H₂O₂ for 4 h. Cell viability was measured by MTT assay. In brief, 500 μg/ml MTT solution were added to each well followed by incubation for 2.5 h at 37°C. The absorbance of each well was then measured at 570 nm using an ELISA microplate reader (model 550; Bio-Rad Laboratories, Inc.).

To determine the effects of fractionated extracts on H₂O₂-induced intracellular ROS generation, the MIN6N β-cells were seeded in 12-well plates the day prior to treatment with the extracts. The β-cells were treated with various concentrations of fractionated extracts for 20 h, followed by the addition of 0.7 mM H₂O₂ to each well. After 4 h of incubation, 5 μM H₂DCF-DA solution in phosphate-buffered saline (PBS, pH 7.38) was added and the fluorescence was measured at excitation and emission wavelengths of 485 and 535 nm, respectively, using a microplate spectrofluorometer. For image analysis of the production of intracellular ROS, the MIN6N β-cells were seeded in cover slip-loaded 12-well plates, which were then treated with various concentrations of fractionated extracts for 20 h, followed by the addition of 0.7 mM H₂O₂ to each well. After 4 h of incubation, H₂DCF-DA solution was added to each well of the plate, which was incubated for 2 h at 37°C. Images of the stained cells were collected using a fluorescence microscope (Nikon, Tokyo, Japan).

Lipid peroxidation was assayed using the thiobarbituric acid (TBA) reaction as previously described (24). In brief, the cells were seeded in 12-well plates and incubated for 24 h. Subsequently, the cells were treated with various concentrations of fractionated extracts for 20 h, followed by the addition of 0.7 mM H₂O₂ to each well. After 4 h of incubation, the cells were washed with cold PBS, harvested with 0.25% trypsin-EDTA and homogenized in cold 1.15% potassium chloride (KCl). A 100-ml aliquot of homogenized cells was then mixed with 0.2 ml of 8.1% sodium dodecyl sulfate, 1.5 ml of 20% acetic acid (pH 3.6) and 1.5 ml of 0.8% TBA. The solution was then heated to 95°C for 2 h. After cooling at room temperature, an BuOH/pyridine mixture (15:1, v/v) was added and the mixture was shaken for 5 min and then centrifuged at 1,000 × for 10 min. The supernatant was isolated and the absorbance was measured at 535 nm.

The MIN6N β-cells were labeled using the cell-permeable, DNA-specific fluorescent dye, Hoechst 33342 as previously described (25). Cells with homogeneously stained nuclei were considered viable, whereas the presence of chromatin fragmentation was indicative of H₂O₂-induced apoptosis (26). The MIN6N β-cells were seeded in 12-well plates. Twenty-four hours later, the cells were treated with various concentrations of fractionated extracts for 20 h, followed by further incubation for 4 h prior to exposure to 0.7 mM H₂O₂. Hoechst 33342 working solution was then added to each well, followed by 15 min of incubation at room temperature. Images of the stained cells were collected using a fluorescence microscope (Nikon), in order to examine the degree of DNA fragmentation.

All measurements are from at least 3 independent experiments and the values are expressed as the means ± SD. Statistical analysis was performed using the Student’s t-test. A value of p<0.05 was considered to indicate a statistically significant difference.

To determine the radical scavenging activity of the fractionated extracts, we measured scavenged DPPH radicals in the presence or absence of fractionated extracts (MAB, MAE and MAH). As shown Fig. 1, the extracts scavenged the DPPH radicals in a dose-dependent manner, with a 400 μg/ml dose of each extract inducing a reduction in DPPH radicals between 59 and 67%. MAE exhibited the most potent DPPH radical scavenger activity, inducing reductions of 51, 67 and 69% at concentrations of 100, 400 and 500 μg/ml, respectively. These results suggest that fractionated mulberry fruit extracts have good antioxidant properties.

To determine the range of doses over which fractionated extracts of mulberry were non-toxic, MIN6N β-cell viability was measured by MTT assay (Fig. 2). Cell viability was >90% in the presence of all extracts, up to a concentration of 200 μg/ml and in the case of MAE, viability was still above this value (92%) at a 400 μg/ml dose.

To measure the cytotoxicity of H₂O₂, we treated the cells with various doses of H₂O₂ for 4 h. As shown in Fig. 3A, the concentrations of H₂O₂ at ≥0.7 mM markedly reduced cell viability, with cell death reaching approximately 57% at 0.7 mM H₂O₂. This dose of H₂O₂ also induced cell death in a time-dependent manner (Fig. 3B). We thus selected 0.7 mM H₂O₂ and an exposure time of 4 h for the subsequent experiments.

The protective effects of MAB, MAE and MAH in the H₂O₂-treated MIN6N β-cells were measured by MTT assay. The cells were pre-treated with various concentrations of MAB, MAE and MAH for 20 h, followed by treatment with H₂O₂ for 4 h. In these experiments, cell viability decreased to approximately 55% following treatment with H₂O₂ in the controls. Pre-treatment with 100 μg/ml MAB, MAE and MAH restored cell viability to 62, 75 and 63% of the control (Fig. 4), respectively. MAE was particularly effective in preventing H₂O₂-induced cell death. Thus, we conclude that all 3 extracts are capable of preventing cell death induced by oxidative stress.

Intracellular ROS levels in the H₂O₂-treated MIN6N β-cells were determined using the ROS-sensitive fluorescent probe, H₂DCF-DA, which is cleaved by intracellular esterases into its non-fluorescent form, DCFH. This form, which is no longer membrane permeable, can be further oxidized by H₂O₂ to its fluorescent form, DCF (27). MAB, MAE and MAH were able to scavenge intracellular ROS in a dose-dependent manner. Consistent with its protective effects on cell viability, MAE was a much more effective suppressor of intracellular ROS compared with the other fractions. The ROS scavenging activity increased up to approximately 34% with 100 μg/ml MAE compared with the cells treated only with H₂O₂ (Fig. 5A).

In the fluorescent microscopic images, the fluorescence intensity of the H₂DCF-DA stain was enhanced in the H₂O₂-treated MIN6N β-cells. As a result, the fractionated mulberry extracts reduced the green fluorescence intensity, compared with the cells treated only with H₂O₂, indicating a reduction in ROS generation. Among the fractionated extracts, MAB and MAE markedly decreased the intracellular fluorescence intensity at 100 μg/ml (Fig. 5B). These data suggest that MAB and MAE possessed greater ROS scavenging activity than MAH.

Based on the above results, we hypothesized that the antioxidant properties of the extracts would reduce ROS-induced cellular damage, such as lipid peroxidation and DNA fragmentation.

The inhibitory effect of the fractionated extracts on lipid peroxidation in H₂O₂-treated MIN6N β-cells was determined by measuring TBA reactive substances (TBARS), lipid peroxidation products, as previously described (28). As shown in Fig. 6, the cells exposed to H₂O₂ showed an increase in lipid peroxidation as indicated by the generation of TBARS, whereas, the fractionated extracts inhibited lipid peroxidation. The inhibitory effect of 100 μg/ml MAB, MAE and MAH was 22, 29 and 22%, respectively compared with the cells treated only with H₂O₂. These results indicate that the ability of the fractionated extracts to block on H₂O₂-induced TBARS formation may be due to their intracellular ROS scavenging activity.

In order to confirm the DNA fragmentation induced by H₂O₂ in MIN6N β-cells, we stained their DNA with Hoechst 33342. The microscopic images in presented Fig. 7 illustrate that treatment the of the cells with H₂O₂ induced DNA fragmentation, a characteristic feature of apoptosis. However, pre-treatment with 100 μg/ml of each fractionated extract decreased the level of DNA fragmentation when compared with the cells treated with H₂O₂ alone. These results confirmed that MAB, MAE and MAH inhibited oxidative stress-induced DNA fragmentation in MIN6N β-cells.