Contributed equally
The perennial herb,
Aging is associated with the deterioration of a number of physiological processes, leading to a decline in functional capabilities, which ultimately impacts the health and overall function of an organism. Among humans and other mammals, these deteriorations occur primarily in the immune system, and result in an increased susceptibility to various conditions, including chronic inflammation, muscle loss, cancer and age-associated degenerative disorders (
It is widely acknowledged that the limitation of calorie intake, also known as calorie restriction (CR), may increase the mean lifespan of an organism by up to 60%, while reducing the incidence of degenerative disease (
Medicinal plants have been used for millennia in numerous cultures to prevent and treat of a variety of diseases. However, the active constituents of these medicinal plants and their precise mechanisms of action are not fully understood.
To the best of our knowledge, the potential of HJE to extend the lifespan and its effect on the aging process have not yet been investigated. Thus, the aim of the present study was to investigate the effect of HJE on lifespan, and to elucidate the signaling pathways and active constituents involved in lifespan extension. In addition, the antioxidant capacities of HJE and its active constituents were evaluated, since reactive oxygen species (ROS) are a major contributing factor to the aging process.
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
A BY4742 yeast strain (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0; EUROSCARF, Frankfurt, Germany) was used for chronological lifespan (CLS) measurements, as described previously (
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− (
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
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 (
A number of previous studies have demonstrated that AMPK and SIRT1 serve important functions in the aging process (
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 (
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
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 (
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
The present study aimed to investigate the effects of HJE on yeast lifespan. Furthermore, the effect of HJE on the expression levels of SIRT1 and AMPK, which are involved in lifespan modulation, was determined. In addition, as oxidative stress is a major contributing factor in the aging process, the antioxidative capacities of HJE and its active constituents were assessed.
To the best of our knowledge, the present study is the first to propose that HJE may be able to increase the life span and delay the detrimental health effects associated with aging. Previous studies have hypothesized that medicinal plants, such as
In the present study, HJE was observed to activate AMPK in human fibroblast cells. AMPK has been demonstrated to serve a key function in the process of aging and the determination of lifespan (
An additional possible mechanism for the life-extending effect of HJE involves SIRT1. The function of sirtuins in lifespan modulation in yeast was recognized over a decade ago; however, the capacity of sirtuins to extend lifespans in other organisms remains controversial. There are seven sirtuin homologs (SIRT1–7) in mammals, of which SIRT1 is the most extensively studied. In mammals, the anti-aging mechanism underlying CR has been shown to involve the activation of SIRT1 in numerous tissues (
A recognized mechanism underlying the aging process is the accumulation of oxidative damage; a hypothesis that has been widely accepted (
In conclusion, the results obtained in the present study demonstrated that pretreatment with HJE enhanced the lifespan of yeast. Furthermore, HJE was shown to exert antioxidant activities in a cell-free system and in human fibroblast cells. In addition, the active constituents of
The study was supported by grants from the Research and Development Program of the Ministry of Trade, Industry and Energy (MOTIE)/Korea Institute for Advancement of Technology (no. N0000697; Establishment of Infrastructure for Anti Aging Industry Support) and the Research and Development Program of MOTIE/Korea Evaluation Institute of Technology (no. 10040391; Development of Functional Food Materials and Device for Prevention of Aging Associated Muscle Function Decrease). The authors thank the Aging Tissue Bank (Busan, Korea) for providing research information.
Effect of
Effects of
Effects of HJE on ROS generation. The inhibitory effect of HJE on ROS production was evaluated using a 2′,7′-dichlorodihydrofluorescein diacetate assay to detect the ROS. Trolox, a well-known scavenger of ROS, was used as positive control. (A) Scavenging activity of HJE on ROS generation induced by 10 µM SIN-1 was measured
Effects of active constituents from