A. distichum Nakai [voucher no. Park1001(ANH)] was collected from the Misun-Hyang Theme Park. An extract from air-dried A. distichum was obtained by immersion in 80% methanol for 3 days and a using a sonicator (40 KHz; 700 W; Hwashin Technology Co., Ltd.). The methanol-soluble extracts were filtered and concentrated using an N-1110S vacuum evaporator (Eyela). The extracts were sequentially fractionated three times with 1:1 ratio of petroleum ether, 1:1 ratio of chloroform and 1:1 ratio of ethyl acetate. The ethyl acetate fraction from A. distichum was harvested and refrigerated at 4°C until use.

Acteoside was isolated from the ethyl acetate fraction (500 g) of A. distichum using an Isolera™ Spektra Accelerated Chromatographic Isolation system at 25°C with SNAP KP-SIL (100 g; 170×40 mm) and SNAP Ultra (25 g; 80 × 35 mm) cartridges (both from Biotage AB). The mobile phases consisted of chloroform (solvent A) and methanol (solvent B). The flow rate was kept at 50 ml/min for a total run volume of 1,600 ml.

The system was run with a gradient program: 0–32 min, 6–50% B and the peaks of interest were monitored at 335 nm by a photodiode array (PDA) detector with SNAP KP-SIL cartridges. Subsequently, using the same mobile phases, the flow rate was kept constant at 75 ml/min for a total run volume of 600 ml. Then the system was run with a gradient program: 0–32 min, 6–50% B and the peaks of interest were monitored at 335 nm by a PDA detector using SNAP Ultra cartridges. Lastly, 2.5 g AAD was further analyzed by high-performance liquid chromatography (HPLC)-PDA and liquid chromatography-mass spectrometry (LC-MS) for further analysis and identification.

AAD (1 mg/ml) was analyzed in a Waters 2695 HPLC system equipped with Waters 2996 PDA using an Xbridge-C18 column (250×4.6 mm; 5 µm; all from Waters Corporation). The binary mobile phase consisted of acetonitrile (solvent A) and water containing 1% acetic acid (solvent B). Both solvents were filtered through a 0.45-µm filter prior to use. The flow rate was kept constant at 1.0 ml/min for a total run time of 40 min at 25°C. The system was run with a gradient program: i) 0–5 min, 10–15% A: 90–85% B; ii) 5–15 min, 15% A: 85% B; iii) 15–40 min, 15–40% A: 85–60% B. The sample injection volume was 10 µl. The peaks of interest were monitored at 335 nm by a PDA and compared with an acteoside standard (V4015-10MG; Sigma-Aldrich; Merck KGaA).

LC-MS analysis was performed using a Nexera X2 Ultra high-performance liquid chromatograph/mass selective detector at 25°C, equipped with an LCMS-8050 quadrupole mass spectrometer (all from Shimadzu Corporation). The liquid chromatographic system consisted of a binary pump, on-line vacuum degasser and thermostatic column compartment, connected in-line to the mass spectrometer. Data acquisition and analysis were carried out using Shimadzu LabSolutions 5.0 (Shimadzu Corporation). Samples (3 µl) were injected into the HPLC system using a binary gradient of deionized water (solvent A) and acetonitrile (solvent B) and isocratically eluted in 50% solvent B for 1 min at a flow rate of 0.3 ml/min. The mass spectrometer was fitted to an atmospheric pressure electrospray ionization source operated in negative ion mode. The electrospray capillary voltage was set to 4.0 kV, with a nebulizing gas (10.7 bar) flow rate of 3 l/min and a drying gas temperature of 300°C. MS data were acquired in the Scan mode (mass range m/z 300–800) and the Product ion scan mode.

DPPH radical scavenging activity was measured as previously described (30), with minor modifications. A 300 µM DPPH solution (Sigma-Aldrich; Merck KGaA) in 95% alcohol was prepared. The solution was adjusted to an absorbance value of 1.00 at 515 nm. A 40-µl volume of sample was mixed with 760 µl DDPH solution, then incubated for 20 min in the dark at 25°C. L-ascorbic acid was used as a positive control. Absorbance was measured at 515 nm using an ultraviolet (UV)/visible spectrophotometer (Xma-3000PC; Human Corporation). The radical scavenging activity was measured as a decrease in the absorbance of DPPH and calculated using the following formula:

DPPH radical scavenging activity (%) = [1-(A_Sample-A_Blank)/A_Control] ×100 where ‘A_Sample’ = Absorbance values of DPPH radicals following treatment with sample, ‘A_Blank’ = Absorbance values of ethanol and ‘A_Control’ = Absorbance values of DPPH radicals.

ABTS radical scavenging activity was measured as previously described (31), with some modifications. A 7.4 mM ABTS and 2.6 mM potassium persulfate solution (both from Sigma-Aldrich; Merck KGaA) in distilled water was prepared 24 h prior to use. The solution was adjusted to an absorbance value of 0.70 at 732 nm. A 40 µl volume of sample was mixed with 760 µl ABTS solution, then incubated for 20 min in the dark at 25°C. L-ascorbic acid was used as a positive control. Absorbance was measured using a UV/visible spectrophotometer at 732 nm. The radical scavenging activity was measured as a decrease in the absorbance of ABTS and calculated using the following formula: ABTS radical scavenging activity (%) = [1-(A_Sample-A_Blank)/A_Control] ×100 where ‘A_Sample’ = Absorbance values of ABTS radicals after treatment with sample, ‘A_Blank’ = Absorbance values of H₂O and ‘A_Control’ = Absorbance values of ABTS radicals.

The inhibitory effect on oxidative DNA damage was measured as previously described (32). Conversion of supercoiled plasmid DNA to open circular forms has been used as an index of DNA damage (32). In the absence of OH⁻ radicals and Fe²⁺ ions, plasmid DNA mainly exists in a supercoiled form. However, in the presence of OH⁻ radicals and Fe²⁺, plasmid DNA is cleaved, converting its supercoiled form into open-circular forms. AAD (0–200 µM) was pre-incubated with 5 mM FeCl₂ or 0.4 mM FeSO₄ + 0.4 mM H₂O₂ for 15 min at 25°C. The ΦX-174 RF I plasmid DNA (50 µg/ml; Promega Corporation) was then added to the mixtures. A solution containing 50% glycerol (v/v), 40 mM EDTA, and 0.05% bromophenol blue (Sigma-Aldrich; Merck KGaA) was added to stop the reaction. Reaction mixtures were analyzed by agarose gel electrophoresis on 1% gels with DNA SafeStain (Lamda Biotech). DNA in the gel was visualized using the Chemi-Doc imaging system (Bio-Rad Laboratories, Inc.) controlled by Image Lab software 6.0 (Bio-Rad Laboratories, Inc.).

The murine skin fibroblast cell line NIH 3T3 was purchased from American Type Culture Collection. Cells were cultured under sterile conditions at 37°C in a humid incubator with 5% of CO₂, in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum (Biowest), 1% antibiotics (penicillin/streptomycin; HyClone; Cytiva) and 0.2% anti-mycoplasma agents (Plasmocin prophylactic; InvivoGen). The oxidative damage was induced by 150 µM FeSO₄ + 600 µM H₂O₂ for the cell viability, western blotting, and PCR assays.

Cells (1.0×10⁴ cells/ml) were treated with AAD (0–200 µg/ml) and incubated at 37°C for 24 h. After 24 h, cells were treated with 10 µl alamarBlue^® cell viability reagent (Thermo Fisher Scientific, Inc.). After 2 h, cell viability was measured using a UV/Visible spectrophotometer at 570 nm.

Cells were lysed using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.) supplemented with a protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). The protein concentration in the sample was determined using the Bradford protein assay (Bio-Rad Laboratories, Inc.). Proteins (20 µg) were separated by SDS-PAGE on 10% gels, then transferred to a PVDF (Bio-Rad Laboratories, Inc.). The membrane was blocked for 1 h at 20°C with 5% bovine serum albumin (BSA; Biosesang) in TBS containing 1% Tween-20 (TBS-T), then incubated with primary antibodies in 3% BSA overnight at 4°C. The following primary antibodies were used at 1:1,000 (all from Abcam): i) Anti-γH2AX (Ser139) polyclonal antibody (cat. no. ab11174); ii) anti-H2AX polyclonal antibody (cat. no. ab11175); iii) anti-phosphorylated-p53 (p-p53; Ser15) polyclonal antibody (cat. no. ab1431); iv) anti-p53 polyclonal antibody (cat. no. ab131442); and v) anti-GAPDH monoclonal antibody (cat. no. ab8245). After washing with TBS-T, membranes were incubated with anti-rabbit IgG monoclonal antibody (cat. no. 18-8816-33; Rockland Immunochemicals Inc.; 1:5,000) or anti-mouse IgG monoclonal antibody (cat. no. 18-8817-33; Rockland Immunochemicals Inc.; 1:5,000) horseradish peroxidase-conjugated secondary antibodies for 1 h. Immunoreactive bands were visualized using an ECL western blotting substrate and the Chemi-Doc imaging system (both from Bio-Rad Laboratories, Inc.). Bands were analyzed using the ImageJ software v1.51K (National Institutes of Health).

Total RNA was extracted from NIH 3T3 cells using the NucleoSpin™ RNA Plus kit (Macherey-Nagel; Thermo Fisher Scientific, Inc.). cDNA was synthesized using ReverTra Ace-α-™ (Toyobo Life Science), according to the manufacturer's protocol. qPCR was carried out using Quick Taq^® HS Dye Mix (Toyobo Life Science). Amplification was initiated at 94°C for 2 min, followed by 30 amplification cycles at 94°C for 30 sec (denaturation), then 54.2°C for 30 sec (annealing/extension) using Thermal cycler (T100™; Bio-Rad Laboratories, Inc.). Primer sequences using Primer 3 program 0.4.0 (http://bioinfo.ut.ee/primer3-0.4.0/) are presented in Table I. DNA density was visualized by 2% agarose gel (A1200; Biosesang) with 1% DNA SafeStain (C138; Lamda Biotech Corporation). Transcription levels were normalized to GAPDH. Density was analyzed using the ImageJ software 1.51K (National Institutes of Health).

cDNA samples were generated using the aforementioned method. qPCR was carried out using the Quanti Tect^® SYBR-Green PCR kit (Qiagen GmbH) on a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Primers were designed using the Primer 3 program 0.4.0 (http://bioinfo.ut.ee/primer3-0.4.0/; Table I). Thermocycling conditions consisted of a 15-min initial denaturation at 95°C, followed by 40 amplification cycles at 94°C for 15 sec (denaturation), 60°C for 30 sec (annealing) and 72°C for 30 sec (extension). Transcription levels were normalized to GAPDH. The 2^−ΔΔCq formula was used to analyze mRNA expression levels, where ΔΔCq = (Cq_target - Cq_GAPDH)_sample - (Cq_target - Cq_GAPDH)_control (33). Data were analyzed using the ABI 7500 Software 2.3 (Applied Biosystems; Thermo Fisher Scientific, Inc.).

γ-H2AX and p-p53 are markers for DNA damage and repair (34). NIH 3T3 cells were treated with AAD (0, 100 µM) and oxidative damage (150 µM FeSO₄ + 600 µM H₂O₂) at 37°C for 24 h. Following treatment, samples were prepared on sterilized glass coverslips (BD Biosciences). Cells were then fixed with 4% paraformaldehyde for 15 min at 20°C, then blocked for 1 h with 3% BSA (Biosesang) and 0.1% Triton X-100 for permeabilization at 20°C. Cells were incubated with anti-γ-H2AX (cat. no. ab11174; 1:500) and anti-p-p53 (cat. no. ab1431; 1:500) overnight at 4°C, then with Alexa Fluor 488 (cat. no. ab150077; 1:500) and Alexa Fluor 568-conjugated secondary antibodies (cat. no. ab175471; 1:500; All antibodies from Abcam) for an additional 1 h at 20°C in the dark. Nuclei were stained using DAPI (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C. Slides were mounted and images captured using a CKX53 fluorescence microscope (magnification, ×400; Olympus Corporation).

All data were analyzed using GraphPad Prism 5.0 (GraphPad Software, Inc.) and are presented as the mean ± standard deviation. All experiments were performed ≥3 times. Data of DPPH and ABTS radical scavenging activity were analyzed using paired Student's t-test. Other data were analyzed by One-way ANOVA followed by Tukey's post hoc test were used to compare the means across multiple groups. P<0.05 was considered to indicate a statistically significant difference.

Acteoside standard and AAD extract were analyzed using HPLC-PDA. The standard (Fig. 1A) and the sample (Fig. 1B) displayed similar retention times (20.3 min) and UV spectra. The LC-MS data (Fig. 1C) also exhibited 623 m/z (M-H⁻). Based on these observations, the AAD sample was identified as an acteoside (Fig. 1D). The purity of AAD was >95%, based on a calibration curve (y = 16089× − 93989, R₂ = 0.9971).

AAD eliminated DPPH radicals in a dose-dependent manner (Fig. 2A). DPPH radicals were scavenged at all AAD concentrations, except 0.32 µM, although not to the same extent as with L-ascorbic acid. The half-maximal inhibitory concentration (IC₅₀) values of AAD and L-ascorbic acid were 8.81 and 5.08 µg/ml, respectively. Similarly, AAD also eliminated ABTS radicals in a dose-dependent manner (Fig. 2B). The IC₅₀ values of AAD and L-ascorbic acid were 6.47 and 10.49 µg/ml, respectively. Furthermore, at concentrations of 8 and 40 µM, the scavenging activity of AAD was significantly higher compared with L-ascorbic acid. These results indicated that AAD effectively scavenged DPPH and ABTS radicals in vitro.

The protective effects of AAD on oxidative DNA damage were evaluated in a DNA cleavage assay using ΦX-174 RF I DNA. The untreated control group (lane 1) was used as a positive control and assigned an open circular (OC) DNA band density value of 100%. In lane 2, the groups receiving Fe²⁺ or OH⁻ without AAD treatment were used as a negative control and given a value of 0%.

AAD inhibited Fe²⁺-induced oxidative DNA damage in a dose-dependent manner, as suggested by the significant OC band density increase at all concentrations, compared with the negative control (47.14±1.64% at 0.32 µM AAD vs. 97.66±3.41% at 200 µM; Fig. 3A). Additionally, AAD also significantly protected plasmid DNA from OH- radicals-induced oxidative damage in a dose-dependent manner (7.09±0.25% at 0.32 µM AAD vs. 88.05±3.09% at 200 µM; Fig. 3B). These results suggested that AAD could protect plasmid DNA from oxidative damage.

To determine whether AAD had any cytotoxic effect, NIH 3T3 cells were cultured in AAD concentrations ranging between 25 and 200 µM, and cell viability was then assessed following 24-h incubation. AAD had no effect on cell viability. In oxidative damage conditions, AAD increased cell viability compared with untreated cells (Fig. 4).

Western blotting assays suggested that, in the absence of AAD, oxidative damage significantly increased phosphorylation of p-53 and H2AX in NIH 3T3 cells. However, under oxidative damage conditions, the levels of p-p53 and γ-H2AX were significantly reduced by treatment with AAD. This reduction in p-p53 and γ-H2AX was dose-dependent (Fig. 5A and B). Additionally, gene expression levels of p53 and H2AX followed the same pattern, as indicated by RT-semi-qPCR (Fig. 5C and D) and RT-qPCR (Fig. 5E).

Immunofluorescence staining of p-p53 and γ-H2AX was also performed (Fig. 6). The levels of p-p53 and γ-H2AX increased following oxidative damage. However, p-p53 and γ-H2AX levels were reduced in the presence of AAD compared with untreated oxidative damaged cells. These results suggested that AAD could regulate the expression of p-p53 and γ-H2AX.