Luteolin, luteolin 7-glucoside, quercetin, quercitrin and resveratrol were obtained from Sigma-Aldrich (St. Louis, MO, USA), dissolved in ethanol and stored at −20°C until required. In addition, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 3-morpholinosydnonimine hydrochloride (SIN-1) and carboxy-H₂DCFDA were obtained from Sigma-Aldrich. Dulbeccos modified Eagles medium (DMEM), fetal bovine serum (FBS) and penicillin-streptomycin were purchased from GE Healthcare (HyClone; Logan, UT, USA). Rabbit polyclonal antibodies against phospho-AMPKα1/2 (Thr 172; cat. no. sc-33524), SIRT1 (cat. no. sc-15404) transcription factor IIB (cat. no. sc-225) and mouse monoclonal β-actin (cat. no. sc-47778), and goat anti-rabbit IgG-horseradish peroxidase (HRP)-conjugated (cat. no. sc-2004) and anti-mouse IgG-HRP-conjugated (cat. no. sc-2031) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). 2,7-Dichlorodihydrofluorescein diacetate (H₂DCFDA) was purchased from Invitrogen Life Technologies (Eugene, OR, USA).

Fresh leaves of Acanthopanax sessiliflorus, Rubus crataegifolius (Bunge), Vitis thunbergii var. sinuata and H. japonicus were collected in Busan (Korea) and authenticated by Professor JS Choi at Pukyong National University (Busan, Korea). These plant specimens were deposited in Professor Hae Chung's laboratory (Pusan National University, Busan, Korea). The fresh leaves of these plants were dried, chopped into small pieces and refluxed with absolute ethanol (EtOH). The extract of each plant was separated from the residues through Whatman No. 1 filter paper (GE Heathcare Life Sciences, Pittsburgh, PA, USA), then concentrated to dryness to render the EtOH extract. The extract was subsequently suspended in EtOH and stored at −20°C until required.

A BY4742 yeast strain (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0; EUROSCARF, Frankfurt, Germany) was used for chronological lifespan (CLS) measurements, as described previously (17). Yeast was grown to exponential phase in rich yeast, peptone, dextrose medium or in synthetic defined minimal medium (Sigma-Aldrich), both containing 2% glucose, which were prepared as described by Sherman (18). CLS measurements were performed as previously described (19).

The scavenging activity of the agents under investigation was assessed using H₂DCFDA, a fluorescent oxidative stress indicator. For the measurement of ROS-scavenging activity in a cell-free system, H₂DCFDA was mixed with esterase (pH 7.4) and incubated for 20 min at 37°C. The mixture was then placed on ice in the dark until immediately prior to measurement. H₂DCFDA was hydrolyzed to non-fluorescent 2,7′-dichlorodihydrofluorescein (DCFH) by esterase (Sigma-Aldrich) and subsequently oxidized to highly fluorescent 2,7-dichlorofluorescein by the ROS, ·O₂⁻ (20). The fluorescence intensity of the oxidized DCFH was quantified using a GENios fluorescence microplate reader (Tecan Group Ltd., Männedorf, Switzerland) at excitation and emission wavelengths of 485 and 530 nm, respectively. Measurement was performed for 30 min, with or without the addition of SIN-1 as an ·O₂⁻ donor. In addition, a similar experiment was performed using Trolox as a positive control to compare for antioxidant capacity.

Human fibroblast Hs27 (CRL-1634) cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in DMEM containing 10% FBS, penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37°C in a humidified atmosphere of 5% CO₂ in air. The fibroblast cells were plated at 90–95% confluency for all the experiments.

Intracellular ROS generation was measured using carboxy-H₂DCFDA, a cell-permeable dye. This compound is oxidized intracellularly by ROS to form fluorescent DCF. Briefly, the Hs27 cells were incubated for 24 h in a 96-well plate. After one day, the medium was replaced with fresh serum-free medium containing HJE or flavinoids. The cells were pretreated with HJE or flavonoids for 1 h and were then exposed to ultraviolet B (UVB), according to designated experimental conditions. UVB irradiation was carried out using a UV Crosslinker (CL-1000; UVP, LLC, Upland, CA, USA) at the desired intensity (100 J/m²). Prior to UVB exposure, the cells were washed with phosphate-buffered saline (PBS) and resusupended in fresh PBS. Subsequently, the cells were incubated with 10 µM carboxy-H₂DCFDA for 10 min at 37°C, and washed twice with PBS. Modulations in fluorescence intensity were measured every 5 min for 30 min using a GENios fluorescence plate reader, at excitation and emission wavelengths of 485 and 530 nm, respectively.

Cells were washed with ice-cold PBS and harvested. A buffer containing 10 mM Tris (pH 8.0), 1.5 mM MgCl₂, 1 mM DTT, 0.1% Nonidet P-40 and protease inhibitors was used to extract the cytosolic fractions by centrifugation at 14,000 × g for 15 min at 4°C. Nuclear fractions were extracted from the resulting pellets using a buffer containing 10 mM Tris (pH 8.0), 50 mM KCl, 100 mM NaCl and protease inhibitors. Aliquots of the cytosolic or nuclear extracts were boiled in gel loading buffer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) for 5 min.

In order to determine the expression levels of the proteins under investigation, cell extracts were prepared and western blot analysis was conducted. In brief, cell extracts containing equal quantities of proteins (20 µg) were subjected to 8–10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were probed with the primary antibodies (1:1,000 dilutions) overnight at 4°C, followed by the HRP-conjugated secondary antibodies (1:5,000 dilutions) for 1 h at room temperature. Signals were detected using an enhanced chemiluminescence reagent (AbFrontier Co., Ltd., Seoul, Korea).

Analysis of variance was used to analyze the differences between each group, and Dunnetts multiple comparison test was used to determine the differences between the mean values of the groups. All statistical analyses were conducted using GraphPad Prism version 5.02 (GraphPad Software, Inc., San Diego, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Effects of HJE on the lifespan of yeast were investigated. In traditional Korean medicine, plants with palmate (hand-shaped) leaves are considered to possess health benefits. Thus, extracts of A. sessiliflorus, a well-studied medicinal plant, V. thunbergii var. sinuata and R. crataegifolius (Bunge) were also examined. To evaluate the anti-aging effect of the plant extracts, yeast cells were cultivated with the extracts for 25 days and the CLS was measured by monitoring the number of colony-forming units (CFUs). The number of viable yeast cells that were able to reproduce and form colonies reduced in culture over time, regardless of the presence of plant extracts. As presented in Fig. 1A, the number of CFUs was higher in the HJE-treated cultures when compared with untreated control cultures between days 10 and 25. However, cultivation with the extracts from A. sessiliflorus, V. thunbergii var. sinuata or R. crataegifolius (Bunge) was shown to have no effect on the yeast lifespan.

Therefore, the HJE exhibited comparatively notable lifespan extension, and the effects of different concentrations of HJE on the yeast lifespan were subsequently examined. The results indicated that HJE increased the viability of yeast cells in a concentration-dependent manner (Fig. 1B). Collectively, these results indicated that HJE exerts a beneficial anti-aging effect.

A number of previous studies have demonstrated that AMPK and SIRT1 serve important functions in the aging process (21–23). As HJE was observed to exert a beneficial effect on the lifespan of yeast, the effect of HJE on AMPK and SIRT1 expression was subsequently examined. AMPK is the principal energy sensor in eukaryotic cells and functions to maintain cellular energy homeostasis and mitochondrial biogenesis (24). Aging is associated with a reduction in AMPK-induced mitochondrial biogenesis, and the activation of AMPK has been observed to increase the lifespan of fruit flies (25,26). The results of the present study indicated that HJE readily activated AMPK (Fig. 2).

SIRT1 is predominantly located in the nuclei and is responsible for oxidative stress. In addition, a decrease in nuclear SIRT1 levels has been previously reported in the hearts of aged mice (27,28). Therefore, in the present study, nuclear proteins were employed for the detection of SIRT1. The results demonstrated that HJE treatment increased nuclear SIRT1 levels in a concentration-dependent manner (Fig. 2), indicating that HJE may extend the lifespan by modulating the expression levels of AMPK and SIRT1. However, further mechanistic experiments are required at the molecular and organism levels.

The effect of HJE on ROS generation was evaluated to elucidate the mechanism underlying the HJE-mediated extension of yeast lifespan. An equivalent concentration of Trolox, a water-soluble vitamin E analog, was used as a positive control for comparison with the inhibitory effect of HJE on SIN-1-induced ROS in a cell-free system. As presented in Fig. 3A, HJE appeared to scavenge the ROS generated by SIN-1 in a concentration-dependent manner. However, the HJE-induced inhibition of ROS generation was lower compared with that of Trolox.

As the antioxidative capacity of the testing molecules may have differed between cell-free and intracellular systems, the antioxidative effect of HJE was examined in Hs27 human fibroblast cells. UVB radiation has been reported to induce ROS generation, resulting in cellular senescence, and the role of SIRT1 in UVB-induced skin aging is well-established (29,30). Thus, UVB was used to induce ROS generation in the Hs27 skin fibroblast cells. Pretreatment with HJE (5 µg/ml) resulted in a significant reduction (34.8%) in the generation of ROS when compared with the UVB-exposed control cells (Fig. 3B). These results indicated that HJE effectively scavenged ROS in the cell-free system and UVB-stimulated fibroblast cells.

H. japonicus is known to contain a number of flavonoids and phenolics that are responsible for various biological activities (8,11,12). In order to investigate whether the antioxidative capacity of HJE is mediated by the aforementioned active constituents, the effects of luteolin, luteolin 7-glycoside, quercetin and quercitrin (structures shown in Fig. 4A) on oxidative stress were determined. The ROS scavenging activities of the HJE-derived flavonoids, at concentrations of 0.2, 1 and 5 µM, on the cell-free system are presented in Fig. 4B. Among the four tested compounds from HJE, luteolin 7-glucoside exhibited the highest ROS scavenging capacity.

Next, the intracellular antioxidative effect of HJE-derived flavonoids was investigated. UVB radiation was used to induce ROS generation in order to assess the capacities of the HJE-derived flavonoids to inhibit intracellular ROS. The scavenging activities of the active constituents on UVB-induced ROS in pretreated Hs27 cells are shown in Fig. 5. In particular, luteolin was observed to exert a marked ROS scavenging effect on intracellular ROS, while luteolin 7-glucoside was most effective at scavenging ROS in the cell-free system. Thus, the results indicated clear differences in the antioxidative capacity among the HJE-derived flavonoids. The rank order was as follows: Luteolin > luteolin 7-glucoside = quercetin > quercitrin in the intracellular system. These results clearly demonstrated that the potent antioxidative properties of HJE may be mediated by flavonoids.