1. Introduction

Sophora flavescens Ait. (Leguminosae) is a traditional herbal medicine and has been widely used as functional food ingredient because of its potential health benefits, such as antihelminthic, anti-inflammatory, and antimicrobial activities ^{[1, 2, 3]}. However, most of previous research work focused on small molecular compounds from S. flavescens, such as alkaloids and flavonoids. Untilnow, there have been few reports regarding the polysaccharides from S. flavescens and their potential biological activities.

Antioxidants are substances that may protect cells from the damage caused by unstable molecules known as free radicals. Antioxidants interact with and stabilize free radicals. Also, may prevent some of the damage free radicals might otherwise cause ^[4]. These free radicals have been implicated in more than a hundred diseases, including inflammatory diseases ^[5], cancer ^[6], atherosclerosis ^[7], type 1 and 2 diabetes (diabetes mellitus) ^[8], malaria ^[9], neurodegenerative diseases ^[10], and aging ^[11]. Antioxidants can act at different levels in an oxidative sequence. This may be illustrated by considering one of the many mechanisms by which oxidative stress can cause damage, stimulation. Many synthetic antioxidants, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and propyl gallate (PG) may be used to retard lipid peroxidation in many fields ^[12]. However, the use of synthetic antioxidants is under increasing scrutiny due to the potential health risks associated with such compounds ^[13]. Therefore, the search for natural antioxidants, as alternatives to synthetic compounds, is of great interest among researchers ^[14]. Our results suggest that ESSA could have potential applications as a natural antioxidant supplement in the food industry.

2. Materials and Methods

3-[4,5-dimethy-thiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay kit were procured from Sigma Aldrich (St. Louis, MO, USA). Culture plates and culture dishes were purchased from Nunc, Inc. (North Aurora Road, IL, USA). Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Hyclone (Logan, UT, USA). All other reagents were of the highest grade available commercially.

PC12 cell line obtained from American Type Culture Collection, were cultured in DMEM supplemented with penicillin, streptomycin and 10% heat-inactivated FBS in 5% CO₂, 95% air and humidified atmosphere at 37°C.

For evaluating the cytotoxicity of ESSA, the cells were harvested using phosphate buffered saline (PBS) containing 0.15% trypsin and 0.08% EDTA, Cells were incubated in well plates at a density of 5 x 10⁵ cells/well. MTT solution was added to each well. Following incubation for 4 h at 37°C in 5% CO₂, the supernatant was removed. The medium was removed and the cells were washed by PBS twice, and the formazan crystals produced in viable cells were solubilized in 200 μL DMSO. The absorbance was measured using a microplate reader (Tecan Trading AG, Männedorf, Switzerland) at 550 nm. All experiments were performed with three replicates ^[15].

For sub-G1 and cell cycle analysis, B16 cells were suspended in ethanol with 0.5% Tween-20 and left for 24 h at 4°C. The cells were then harvested by centrifugation and resuspended in 1.0 mL of phosphate-buffered saline containing 0.05 mg/mL of PI and 10 μg/mL of RNase A, and incubated at 37°C for 30 min. Analysis of apoptotic cell death was performed by measuring the hypodiploid DNA contents using a flow cytometer (FACS-Caliber; Becton Dickinson; Franklin Lakes, NJ, USA). The cells in the sub-G1 population were considered apoptotic cells, and the percentage of cells in each phase of the cell cycle was determined ^[16].

To obtain the total cell lysate, 50 μL of radioimmunoprecipitation assay (RIPA) buffer was added to the changed cells (3 x 10⁵ cells/mL) cultured in 6-well plates. The cells were harvested, incubated for 10 min on ice, and centrifuged at 14,000 g for 10 min at 4°C. The protein concentration was determined using a DC protein assay from Bio-Rad, and 20 μg of whole cell lysate was separated on 12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). Electrophoresis was performed and the proteins were transferred to Polyvinylidene difluoride (PVDF) membranes using an electro blotting apparatus. The membranes were blocked for 1 h in a mixture of Tris-Buffered Saline and Tween 20 (TBS-T), containing 0.1% Tween-20 and 5% dry skim milk; the membranes were incubated overnight with primary antibodies (1:1000) followed by incubation for 1 h with horseradish peroxidase-conjugated secondary antibodies (1:1000). The optical densities of the antibody-specific bands were analyzed by a Luminescent Image Analyzer, LAS-3000 (Fuji, Tokyo, Japan) ^[17].

Statistical analyses were performed 3 times for all the experiments. The data are expressed as the mean ± one standard error of mean (SEM). Statistical analyses were assessed by Student’s t-test for paired data. Graph Pad Prism software version 4.00 (Graph Pad Software Inc., San Diego, CA) was used. Significant differences (p < 0.05) between the mean values of the triplicate samples were determined for various assays.

3. Results and Discussion

The effect of ESSA that the viability of PC12 cells were examined by MTT assay. PC12 cells were treated with ESSA at various concentrations (0, 10, 20, 40, 80, 160 and 320 μg/mL, respectively) for 18 h. As shown in Figure 1A, ESSA at up to 160 μg/mL showed cytostatic activity against PC12 cells proliferation. We further investigated neuroprotective effects on hydrogen peroxide-induced damage of EGCG. Cell apoptosis analysis showed the distribution of apoptotic cells throughout the cell cycle, as shown in Figure 1B, where the percentage of apoptotic cells in non-treated cells was 5.20%. The percentages of apoptotic cells observed was 36.3% at 1.0 mM hydrogen peroxide, while the percentage of apoptosis in ESSA-treated cells was 4.4% (only ESSA) and 12.5% (ESSA with hydrogen peroxide), respectively. These results indicate that the ESSA protect neuronal cells against hydrogen peroxide-induced oxidative stress. Bcl-2 and Bax are well-known protein of anti- and pro-apoptotic proteins (mitochondrial apoptotic pathway). In this study, the expression of Bax on cytosolic fraction tends to increase hydrogen peroxide-treated group compared with control group (Figure 2A). Contrary to the hydrogen peroxide group with ESSA group showed lower expression of Bax compared with hydrogen peroxide-treated group. In addition, expression of Bcl-2 tends to decrease on hydrogen peroxide-treated group compared with control group. On the other hand, hydrogen peroxide-treated with ESSA group showed higher expression of Bcl-2 than oxidative stress group (Figure 2B). Sophora flavescens is a medical herb whose roots are used as a diuretic and to treat pyogenic infection, jaundice, dysentery, and fever in traditional Asia medicine ^{[18, 19]}. In addition, Sophora flavescens combined with other herbs could improve or treat inflammatory diseases, including asthma and atopic dermatitis ^{[20, 21]}. In the present study, we focused on natural antioxidants from Sophora flavescens, which prepared by extracts. The neuro-protective effect of ESSA was determined by apoptosis analysis using a flow cytometer.

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Figure 1. Effect of ESSA on the cell viability of PC12 cells using the MTT assay. PC12 cells were incubated with 10, 20, 40, 80, 160 and 320 μg/mL of ESSA for 18 h. The results are shown as percentages of control samples (A). Cell apoptosis of PC12 cells after treatment with ESSA prior to hydrogen peroxide treatment (B). Data are presented as the mean ± S.E.M. (n = 3) for three independent experiments

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Figure 2. Change in apoptosis related proteins in PC12 cells after hydrogen peroxide treatment. Equal amounts of whole cell lysates were subjected to electrophoresis and analyzed for Bax (A) and Bcl-2 (B) by Western blot. Data are presented as the mean ± S.E.M. (n = 3) for three independent experiments

4. Conclusions

Utilization of the natural antioxidant properties of bioactive substances may help provide the food and pharmaceutical industries novel alternatives to control disease, since ROS are known to cause widespread cellular damage. In this study, we have experimentally demonstrated the protective effect of ESSA against hydrogen peroxide-induced damage in neuronal cells, resulting from the inhibition of free radicals and ROS activation responses. This study was conducted to investigate the hypothesis that the antioxidant activities and neuroprotective effects of ESSA can suppress hydrogen peroxide-induced injury to neuronal PC12 cells. However, the regulatory effect of ESSA in molecular mechanisms of cancer in animal will be needed.

Acknowledgments

This work was supported by a 2016 research grant from Youngsan University, Republic of Korea.

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