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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

A peanut sprout is known to contain a significant level of resveratrol, which was reported to have beneficial effects in our body due to its antioxidant activities. The purpose of this study was to evaluate the cytoprotective activity of ethanol extract of peanut sprout (EPS) from ultraviolet B (UVB)-induced oxidative stress in human dermal fibroblasts (HDF). EPS was revealed to contain 54.2 μg g−1 of trans-resveratrol. The DCF-DA-positive reactive oxygen species level was increased by 50 mJ cm−2 of UVB irradiation (2150 ± 450% of nonirradiated control), which was markedly suppressed by EPS treatment (180 ± 42% of control). Annexin V-positive apoptotic cell death induced by UVB irradiation (16.4 ± 4.5%) was also significantly inhibited by EPS treatment (6.7 ± 2.5%). EPS induced up-regulation and nuclear translocation of Nrf2, a transcription factor for antioxidant and detoxifying enzymes, in HDF as a dose-dependent manner. UVB irradiation up-regulated Nrf2-dependent enzymes of heme oxygenase-1, NAD(P)H:quinine oxidoreductase-1 and glutathione-S-transferase pi, and they were further stimulated by EPS treatment. Taken together, EPS is an efficient cytoprotective agent against UVB-induced oxidative stress by activation of Nrf2 and upregulation of Nrf2-relating antioxidant and detoxifying enzymes in HDF.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Peanut sprout (PS) contains various amino acids, sugars, proteins, fatty acids and vitamins. It also has a wide variety of functions, such as anti-inflammation, antioxidation, antiaging and anticancer [1]. The budding process of some plants requires very active cellular activities with high energy consumption, and therefore intracellular oxidative stress becomes extremely high. In consequence, many plants including peanut products have been reported to express high levels of antioxidants in sprout products compared to their original plants, which play an essential role in protecting themselves from oxidative damage during germination [1, 2]. For example, resveratrol content of PS was ca 11.7–25.7 μg g−1, but that of peanut was ca 2.3–4.5 μg g−1 [1]. Total polyphenol content of PS was increased up to ca 3.6 times (22.4 mg g−1) higher than that of peanut (6.3 mg g−1) [2]. Ethanol extract of PS (EPS) is expected to function as a cytoprotective agent, as it contains a relatively high content of resveratrol. To date, resveratrol has been detected in many plants of almost 70 species, including grapes, red wines, peanuts, berries and others. Many studies show that resveratrol possesses cytoprotective activities of antioxidant, anti-inflammatory, immunomodulatory and chemopreventive properties [3-6].

As for the cytoprotective mechanisms to eliminate harmful oxidants in cells, a variety of antioxidants such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) have been widely studied. Among them, a group of antioxidant and detoxifying enzymes have been classified into phase II enzymes, which are actively modulated by NF-E2-related factor 2 (Nrf2) and antioxidant-related element (ARE) pathway [7]. These group of enzymes, including heme oxygenase-1 (HO-1), NAD(P)H:quinine oxidoreductase-1 (NQO-1) and glutathione-S-transferase (GST), can protect our body from harmful oxidants from both exogenous environmental factors and endogenous xenobiotic metabolism [8]. They are commonly mediated by Nrf2, which is negatively regulated by a kelch-like ECH-associated protein 1 (Keap1). In normal status, Nrf2 is associated with Keap-1 in the cytoplasm, which prevents migration of Nrf2 to the nucleus. When oxidative stress is imposed to the cells, in contrast, Nrf2 is dissociated from Keap1, leading to migration of Nrf2 to the nucleus to bind with ARE, which is located in the promoter region of those enzyme family [9].

With these backgrounds, we evaluated the cytoprotective activity of EPS from oxidative stress in human dermal fibroblasts (HDF). We imposed oxidative stress to HDF by UVB irradiation, and the cytoprotective activity of EPS was then evaluated by studying cell viability and reactive oxygen species (ROS)-scavenging activity in UVB-irradiated HDF. EPS-mediated cytoprotective mechanism was unraveled by studying Nrf2 activation as well as modulation of Nrf2-related antioxidant and detoxifying enzymes of HO-1, NQO-1 and GSTpi.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
Preparation of EPS stock and EPS solution

Dried PS was steamed, dried for grinding and prepared for PS powder. Ten percent of PS powder was dissolved with 70% of pure water and 30% of ethyl alcohol. The solution was then centrifuged to remove peanut oil, followed by ethanol removal, and then concentrated at low temperature to prepare an EPS stock. The EPS stock was further dissolved with PBS solution to prepare EPS solution of different doses from 0.005% to 2.5% from the stock.

Determination of resveratrol content in EPS

An amount of 1 mL of PS concentration was dissolved in 9 mL of ethanol, then filtered with 0.45 um Teflon syringe filters before injecting into high-performance liquid chromatography (HPLC) (pump: Jasco PU-98; detector: Jasco UV-975; column: phenomenex [250 × 4.6 × 4 μm] C18, injected volume: 20 μL, detected at 306 nm, flow rate: 0.6 mL min−1). A standard trans-resveratrol (Sigma-Aldrich Co., St Louis, MO) was used for a standard material and run under same condition for a reference for quantification.

Primary culture of HDF and UV irradiation

Intact human skin from circumcised foreskin was obtained after informed consent according to the guidelines of the local Ethics Committee of Chonnam National University Hospital Institutional Review Board and the Declaration of Helsinki Principles (IRB no. 1-2009-11-136). The skin was cut into small pieces, then treated with 2.4 U mL−1 dispase (Boehringer Mannheim, Ingelheim, Germany) for 30 min at 37°C. The dermal tissues were separated from the epidermis, minced into small pieces with iris scissors; primary HDF were then obtained by culturing in DMEM supplemented with 10% fetal bovine serum and antibiotics (100 U mL−1 penicillin and 100 μg mL−1 streptomycin) in a 5% CO2 incubator. The second to ninth passage HDF were used for all experiments.

For UV irradiation, HDF of 70–80% confluency were washed three times with PBS after removing culture media. Just after washing, HDF were irradiated under the UV radiator equipped with a bank of six UVB sunlamps (FSX72T 12/UVB-HO; National Biological Corp., Twinburg, OH). The tubes were designed to emit UVB light between 290 and 320 nm in wavelengths with a peak emission at 300 nm. When the intensity of UV lights was measured with an IL 700 (International Light, Newburyport, MA), UV bulbs were detected to emit 0.64 mW cm−2 s−1 of UVB ray and 0.19 mW cm−2 s−1 of UVA ray. Despite minor contamination of UVA ray in those bulbs, we regarded that UV-mediated oxidative stress in our experiments was mediated by UVB, in considering that the biological potency of UVB to induce skin inflammation is 1000 times stronger than that of UVA. After UV irradiation, HDF were further cultured with fresh culture medium in the presence or absence of EPS. For EPS experiments in UVB-irradiated HDF, EPS was treated for 24 h after cells were irradiated, except the ROS study.

MTT assay

To determine cell viability, HDF were seeded into 96-well plates and treated with different concentrations of EPS for 24 h. We used a colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) kit (Chemicon International, Inc., Billerica, MA) for cell survival and proliferation, according to the manufacturer's instructions. Briefly, 10 mL of MTT solution was added to each well (0.5 mg mL−1), and plates were incubated at 37°C for 2 h. The resulting formazan crystals were dissolved with 100 mL of dimethylsulfoxide and absorbance was read at 570 nm in an ELISA reader (Molecular Devices, Sunnyvale, CA).

Western blot analysis

Whole cell extracts were prepared by sonication in RIPA lysis buffer (50 mm Tris-HCl [pH 7.4], 150 mm NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mm EDTA [pH 8.0], and protease inhibitor cocktail), followed by centrifugation (18 000 g for 15 min at 4°C). Nuclear extracts were prepared with the NE-PER nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL). Briefly, cells were scraped from culture plates, suspended in ice-cold cytoplasmic extraction reagent I (CER I) and incubated on ice for 10 min. Subsequently, cytoplasmic extraction reagent II (CER II) was added to the cell suspension, mixed by vortexing for 5 s, and the cytosolic protein fraction was collected by centrifugation at 18 000 g for 5 min at 4°C. The pellet was re-suspended in ice-cold nuclear extraction reagent for 1 h on ice with shaking. The nuclear extract was collected by centrifugation at 14 000 g at 4°C for 10 min. Protein amounts of whole and nuclear extracts were measured using a BCA protein assay kit. The cell extract (30 μg) was separated by 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands were probed with the following primary antibodies overnight at 4°C: anti-NQO1 (ab28947, 1:1000; Abcam, Cambridgeshire, UK); anti-HO-1 (SPA-896, 1:2000; Stressgen Bioreagents, Ann Arbor, MI); anti-GSTpi (ab65977, 1:1000; Abcam); anti-Nrf2 (sc-722, 1:1000; Santa Cruz Biotechnology, CA); anti-lamin B (sc-6216, 1:200; Santa Cruz Biotechnology) and anti-β-actin (ab-6276, 1:1000; Abcam). HRP-conjugated goat anti-rabbit IgG antibody (1:5000; Jackson ImmunoResearch, West Grove, PA), HRP-conjugated rabbit anti-goat IgG (1:5000; Jackson ImmunoResearch) and HRP-conjugated antimouse IgG antibody (1:5000; Jackson ImmunoResearch) were used for secondary antibodies. Immune complexes were visualized using an ECL kit (Millipore, Billerica, MA). β-Actin from total extract and lamin B from nuclear extract were used for internal standards to standardize the protein amounts loaded in each lane. All data are representative of three experiments.

Measurement of intracellular ROS levels

Fluorogenic 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA; Invitrogen, Carlsbad, CA) was reacted with intracellular ROS, and was detected by fluorescence-activated cell sorting (FACS) analysis. Briefly, HDF were seeded in each well of a 60 mm culture dish at a density of 6 × 104 cells per well and treated with EPS for 1 h. Instead of using a 60 mm culture dish for FACS experiments, four-well slide chambers were used for confocal experiments. For ROS experiments, EPS was pretreated before UV irradiation, as ROS rapidly produced and disappeared during UV irradiation. After washing with PBS, HDF were labeled with 10 μm of DCF-DA at 37°C for 30 min, washed with PBS and then were exposed to UVB irradiation at a dose of 50 J cm−2. After washing, HDF were suspended in 500 μL PBS. The cell suspension was analyzed by a FACScalibur flow cytometer (FACSCalibur; Becton Dickinson, CA) with an excitation wavelength of 488 nm and emission wavelength of 530 nm. Data analyses were based on 10 000 detected events using the Cell Quest software. At the same time, UVB-induced intracellular ROS produced was detected with a confocal microscopy using DCF-DA dye. Counter staining was performed with 4′,6-diamidino-2-phenylindole, and fluorescence signals were detected with wavelengths of 492–495 nm for excitation and 517–527 nm for emission. Images were visualized using a confocal microscopy with a 40× objective lens on the laser scanning microscope (LSM 510; Carl Zeiss, Jena, Germany), analyzed by the LSM 5 browser imaging software.

Measurement of apoptotic cell death

To analyze apoptotic cell death by UVB irradiation, HDF were labeled with Annexin V (AV)-FITC and propidium iodide (PI) dyes using the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, Mountain View, CA). The reaction mixture was incubated for 10 min in the dark at room temperature. FACS analysis was performed using the FACScalibur flow cytometer (Becton Dickinson), and was analyzed by CellQuest Software Version 5.2.1. This dye-exclusion test for the integrity of cell membrane allows to discriminate necrotic cells from AV-positively stained apoptotic cells. The cells lacking AV-FITC and PI signals were regarded as live cells. Among AV-positively stained cells, PI-negative cells were regarded as apoptotic cells, and PI uptake cells were regarded as necrotic cells.

Determination of Nrf2 activation

HDF were seeded in each well of the slide chamber at a density of 3 × 104 cells per well and treated with 0.1% EPS solution for 24 h. After washing with PBS, cells were fixed in 4% paraformaldehyde in PBS for 10 min, then permeabilized with PBS containing 0.5% TritonX-100 (PBS-T) for 15 min. The cells were incubated with PBS-T containing 1% BSA for 1 h, and stained with anti-Nrf2 antibody (sc-722, 1:100; Santa Cruz Biotechnology) overnight at 4°C, followed by incubation for 1 h with Alexa Fluor 488 goat anti-rabbit IgG (A11034, H+L, 1:500; Invitrogen). Images were visualized using confocal microscopy with a 20× objective on a laser scanning microscope (LSM 510; Carl Zeiss), and analyzed using a LSM 5 browser imaging software.

Nrf2 knock-down experiment

Transfection was carried out using OligofectamineTM (Invitrogen) transfection reagent. We purchased a commercial primer for Nrf2 siRNA from Santa Cruz Biotechnology. HDF in six-well plates at 50–60% confluency were incubated with 3 mL of culture media lacking antibiotics and containing 10 mL of OligofectamineTM with or without 10 mm of Nrf2 siRNA. After 6 h, the cells were washed with PBS, and HDF were further cultured in new medium lacking supplements at 37°C in a CO2 incubator for 24–72 h.

Statistics

All experiments were carried out in triplicate, and the results are expressed as mean ± standard deviation. Comparisons between samples were carried out using Student's t-test with statistical significance considered to be < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

EPS contains relatively high amount of resveratrol

In HPLC, EPS stock was revealed to contain 54.2 ± 0.85 mg g−1 of trans-resveratrol from standard calibration measurement with a trans-resveratrol (Fig. 1). Besides resveratrol, phenolic compounds of catechin gallate (CG), epicatechin gallate (ECG), epigallocatechin (EGC), and epicatechin (EC) were detected to be contained in EPS at concentration of 28.7 ± 0.46, 72.9 ± 0.56, 19.5 ± 0.46 and 55.6 ± 0.51 µg g−1, respectively. The original stock of EPS was serially diluted from 2.5% up to 0.005% with HDF culture media of DMEM solution to find out nontoxic concentrations for further experiments. In the MTT assay, lower than 0.625% concentrations of EPS solution showed almost 100% cell survival rate (data not shown). Under the microscopy, however, cell morphology of HDF, which were treated with EPS of greater than 0.2% concentrations, was slightly changed harboring elongated dendrites. Therefore, all following experiments except dose-related ones were performed with 0.1% EPS in treating HDF.

image

Figure 1. Quantification of trans-resveratrol level in EPS. To quantify the content of resveratrol, 1 mL of EPS stock was dissolved in 9 mL of ethanol, filtered and analyzed with HPLC. trans-Resveratrol, purchased from Sigma-Aldrich Co., was used for a standard material. Arrow indicates the trans-resveratrol peak among EPS components.

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EPS protects HDF from UVB-induced cell death

We evaluated the protective activity of EPS against UVB-induced cell death in HDF. In control experiments, UVB nonirradiated HDF showed a few number of AV- and PI-positive cells (2.2 ± 1.5% of AV-positive cells, 1.3 ± 0.5 of PI-positive cells, n = 3) (Fig. 2A). To induce cell death, HDF were irradiated with 50 mJ cm−2 of UVB, and cells were harvested at 24 h of postirradiation. In FACS analysis, the number of AV-positive cells indicating apoptotic cell death was increased to 16.4 ± 4.5% (n = 3) in UVB-irradiated HDF (Fig. 2B). When EPS was treated for 24 h after UVB irradiation, the number of AV-positive cells was markedly suppressed in EPS-treated, UVB-irradiated HDF (6.7 ± 2.5%, n = 3) (Fig. 2C), indicating the anti-apoptotic activity of EPS (< 0.01).

image

Figure 2. Effect of EPS on UVB-induced cell death of HDF. After HDF were irradiated with 50 mJ cm−2 of UVB to induce cell death, cells at 24 h of postirradiation were labeled with AV/PI dyes for FACS analysis. (A) Control HDF without UVB irradiation; (B) UVB-irradiated HDF; and (C) EPS-treated, UVB-irradiated HDF. FL1-H indicates AV-positive cells and FL2-H indicates PI-positive cells.

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EPS suppresses UVB-induced ROS production in HDF

To unravel the cytoprotective mechanism of EPS, we tested whether EPS had a potential to inhibit UVB-induced ROS production in HDF. For experiments, HDF were irradiated with 50 mJ cm−2 of UVB, and then intracellular ROS levels were checked immediately by DCF-DA-positive reactivity by confocal microscopy and cell sorting analysis. In the confocal microscopic study, DCF-DA-positive signals were not detected in nonirradiated control HDF (Fig. 3A-a). The DCF-DA-positive signals, which were strongly detected from UVB-irradiated HDF (Fig. 3A-b), were barely detected from EPS-treated, UVB-irradiated HDF (Fig. 3A-c), indicating a strong ROS-scavenging activity of EPS. To quantify the levels of intracellular ROS, FACS analysis for DCF-DA-positive cells was performed. When the average number of DCF-DA-positive cells of nonirradiated control HDF (Fig. 3B-a) was designated as 100%, that of UVB-irradiated HDF was increased to 2150 ± 450% of control level (n = 3, < 0.001) (Fig. 3B-b). However, DCF-DA-positive cells were markedly decreased to 180 ± 42% of control level in EPS-treated, UVB-irradiated HDF (n = 3) (Fig. 3B-c).

image

Figure 3. Effect of EPS on ROS production in UVB-irradiated HDF. (A) After HDF were irradiated with 50 mJ cm−2 of UVB, cells were labeled with DCF-DA dye to detect intracellular ROS by a confocal microscope. (a) Control HDF without UVB irradiation; (b) UVB-irradiated HDF; and (c) EPS-treated, UVB-irradiated HDF. Scale bar indicates 20 μm. (B) Under the same condition, FACS analysis to detect the DCF-DA-positive cells was performed. (a) Control HDF; (b) UVB-irradiated HDF; and (c) EPS-treated, UVB-irradiated HDF. The average number of DCF-DA-positive cells shown in (a) is designated as 100%.

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EPS activates Nrf2 in HDF

As EPS showed cytoprotective activity in UVB-irradiated HDF, we tested whether EPS could activate Nrf2 in HDF. In Western blot analysis, Nrf2 of the nuclear extract was upregulated by EPS in a dose-dependent manner (Fig. 4A). In confocal microscopy, Nrf2-positive fluorescence was translocated to the nucleus from the cytoplasm by EPS, indicating EPS-induced activation of Nrf2 in HDF (Fig. 4B, lower panel). In EPS nontreated control HDF, Nrf2-positive signals were detected weakly from the cytoplasm (Fig. 4B, upper panel).

image

Figure 4. Effect of EPS on Nrf2 activation in HDF. In relation to the induction of antioxidant and detoxifying enzymes in EPS-treated HDF, we tested whether EPS could activate Nrf2. After HDF were treated with EPS at concentrations of 0.001–0.1% for 24 h, cell extracts were prepared for immunoblots. (A) Western blot analysis for Nrf2 levels in the nuclear extracts from EPS-treated HDF (0.001–0.1%) and nontreated control HDF (0). Lamin-B was used for an internal standard of protein loading from nuclear extracts. (B) Confocal microscopic detection of Nrf2-positive fluorescence in EPS-treated HDF (lower panel) and nontreated control HDF (upper panel). Scale bar indicates 20 μm.

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Nrf2-dependent expression of HO-1, NQO-1 and GSTpi in HDF

To evaluate which enzymes are targets of the Nrf2 transcriptional factor, we performed Nrf2 knockdown experiments by transfecting Nrf2 siRNA in HDF. In RT-PCR, transfection with Nrf2 siRNA induced the suppression of Nrf2 mRNA levels at 24–72 h after transfection, compared to nontransfected control HDF (Fig. 5A). In RT-PCR and densitometric analysis, HO-1, NQO-1 and GSTpi were all downregulated in Nrf2 siRNA-transfected HDF at 24–72 h after transfection, indicating Nrf2-dependent expression of HO-1, NQO-1 and GSTpi in HDF (Fig. 5A,B).

image

Figure 5. Effect of Nrf2 knockdown on Nrf2-relating antioxidant and detoxifying enzymes in HDF. After Nrf2 siRNA was transfected, RT-PCR experiments for Nrf2 and its relating antioxidant and detoxifying enzymes were performed. (A) Representative results on mRNA levels of Nrf2 and relating enzymes in Nrf2 siRNA-transfected HDF (+) and Nrf2 siRNA nontransfected HDF (−) at 24–72 h after transfection are shown. GAPDH was used for an internal standard of mRNA loading. (B) Relative enzyme levels from three independent experiments are quantified by densitometric analysis (n = 3). *< 0.05, **< 0.01.

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EPS upregulates Nrf2-relating antioxidant and detoxifying enzymes in HDF

From the result that EPS activated Nrf2 in HDF, we evaluated whether UVB irradiation could modulate Nrf2-relating enzymes. For experiments, HDF were irradiated with 50 mJ cm−2 of UVB, and cells were harvested at 24 h of postirradiation. As for testing the effect of EPS, UVB-irradiated HDF were treated with EPS for 24 h after irradiation. In Western blot analysis, NQO-1 and GSTpi were upregulated in UVB-irradiated HDF. The UVB-induced upregulation of NQO-1 and GSTpi was further stimulated by EPS treatment (Fig. 6A). Thereafter, when UVB nonirradiated HDF were treated with EPS for 24 h, all HO-1, NQO-1 and GSTpi were upregulated in a dose-dependent manner at concentrations of 0.001–0.1% (Fig. 6B), suggesting that EPS has the potential to induce antioxidant and detoxifying enzymes without oxidative stress. Relative expression levels of those enzymes from three independent experiments were analyzed by densitometry (Fig. 6C).

image

Figure 6. Effect of EPS on Nrf2-relating antioxidant and detoxifying enzymes in UVB-irradiated or nonirradiated HDF. (A) Effect of EPS on antioxidant and detoxifying enzymes in UVB-irradiated HDF. After HDF were irradiated with 50 mJ cm−2 of UVB, they were treated with EPS for 24 h. Western blot analysis for HO-1, NQO-1, and GSTpi in nonirradiated HDF (−, −), UVB-irradiated HDF (+, −) and EPS-treated, UVB-irradiated HDF (+, +), respectively. β-Actin was used for an internal standard for protein loading. (B) Effect of EPS on antioxidant and detoxifying enzymes in nonirradiated HDF. For experiments, HDF were treated with EPS at concentrations of 0.001–0.1% for 24 h, and then cell extracts were prepared for immunoblots. Representative results on expression levels of HO-1, NQO-1 and GSTpi are shown. (C) Relative expression levels from three independent experiments are quantified by densitometric analysis (n = 3). (a) Densitometric analysis for (A). (b) Densitometric analysis for (B). *< 0.05, **< 0.01.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

In this study, EPS has been shown to have a potent cytoprotective activity against UVB-induced oxidative stress in HDF. The protective mechanism of EPS might be related to the antioxidant activity by modulating a group of enzymes or direct sunscreen activity through absorption of UV light. Among the two possibilities, we could confirm the antioxidant activity of EPS, in that EPS could upregulate HO-1, NQO-1 and GSTpi in UVB nonirradiated HDF. Also, our result that NQO-1 and GSTpi were upregulated in EPS-treated, UVB-irradiated HDF, when EPS was treated after UVB irradiation, strongly supports the antioxidant role of EPS rather than its sunscreen effect. Interestingly, HO-1 was not induced by UVB irradiation even in the presence of EPS. In a previous study, HO-1 was reported to be upregulated by 2.5–10 mJ cm−2 of UVB irradiation, but was downregulated by 25 mJ cm−2 of UVB irradiation, suggesting the UVB-labile characteristic of HO-1 [10]. Taken together, our result that EPS cannot induce HO-1 expression in UVB-irradiated HDF might be related to the high dose of UVB irradiation (50 mJ cm−2).

UVA-induced oxidative stress was reported to activate Nrf2, as it increases antioxidant activities in human keratinocytes [11]. In that study, UVA irradiation to HaCaT cells activated Nrf2, and as a result, the activities of SOD and GST were increased. The protective mechanism was further reinforced by resveratrol treatment. Consistently, we could observe that UVB irradiation activated Nrf2-relating antioxidant and detoxifying enzymes, and EPS treatment further stimulated the protective mechanism in HDF. Among enzymatic antioxidants, EPS was revealed to mainly activate Nrf2-dependent antioxidants and detoxifying enzymes of HO-1, NQO-1 and GSTpi from our Nrf2 siRNA experiments. In our previous study, HO-1 and GSTpi were independent on Nrf2, but NQO-1 was dependent on Nrf2, in human epidermal keratinocytes [12]. Therefore, different regulatory mechanisms are supposed to work depending on the cell types in modulating antioxidant and detoxifying enzymes, even if Nrf2 is one of the key molecules to modulate a group of those enzymes. In other studies, resveratrol induced CAT, SOD, GPx, NQO and GST for scavenging ROS in the primary rat hepatocytes [13, 14]. Oral administration of resveratrol increased NQO-1 and CAT activities in guinea pigs [15]. Resveratrol induced NQO-1 and GST in cardiomyocytes in a concentration and time-dependent manner [16]. Based on our previous studies that a group of those phase II enzymes were ubiquitously expressed in the skin [12, 17], the present results imply that EPS can be an efficient cytoprotective agent against UV-induced oxidative stress in the skin.

In relation to anticancer activity, resveratrol is known to modulate not only phase II enzymes but also phase I of cytochrome (CYP) P450 enzymes [18-20]. Resveratrol inhibited CYP 1A1 and 1A2 enzyme activities in human mammary carcinoma cells [18]. Administration of resveratrol to mice could suppress CYPA1 expression, as it inhibited benzo[a]pyrene-induced DNA adduct formation [19]. Daily oral administration of resveratrol to healthy human volunteers could inhibit the activities of phase I (CYP3A4, 2D6 and 2C9) and yet increase phase II enzyme of GSTpi [20]. Instead of EPS, methanol extracts of PS were previously reported to have a potent antioxidant activity of 1,1-diphenyl-2-picryl-hydrazyl scavenging activity as well as antioxidant activity against linoic acid oxidation [1]. Methanol extract of resveratrol was reported to have a cytoprotective activity in neurons against glutamate-induced neurotoxicity [21].

This study has shown that EPS contains a relatively high amount of resveratrol. The antioxidant activity of EPS becomes more potent than that of peanuts from our study in that EPS contains a variety of phenolic compounds of CG, ECG, EGC and EC. As resveratrol-containing foods or beverages have been reported to be beneficial to our health, studies were focused to detect an ideal source for resveratrol from many candidate natural plants of low cost and high resveratrol concentration. So far, grapes and red wines have been widely studied, demonstrating that the resveratrol content of grape skin was 35.6–170.6 mg kg−1, and that of red wine was 0.05–10.9 μg mL−1 as a trans-form and 0.04–8.71 μg mL−1 as a cis-form [22, 23]. In another study, resveratrol content in red wines was found to be 6.5 μg mL−1 as cis- and trans-forms [24]. PS can be an alternative source for resveratrol-containing plant products, in that PS contains a relatively high content of resveratrol [2, 21]. Of interest, the resveratrol content in EPS was 54.2 μg g−1, which is a far higher level of resveratrol compared with the present reported PS products so far [1]. We believe that EPS has several advantages among other candidate natural plants such as grapes, in that EPS can be easily obtained without any limitations in climates, seasons and locations. Peanut kernels are available worldwide.

EPS shows potent antioxidant activity against UVB-induced oxidative stress through its strong inhibitory activity for ROS formation in UVB-irradiated HDF. Resveratrol as a polyphenolic compound is known to have strong antioxidant activity, which could be applied in the prevention of and therapy for many diseases and cancers [25, 26]. Besides antioxidant and antitumor effects, resveratrol was studied for its anti-inflammatory activity. Resveratrol suppressed NF-κB signaling, which led to the downregulation of the expression of a set of inflammatory cytokines [27]. Resveratrol inhibited the activation and expression of various inflammatory mediators, such as interleukin (IL)-6, 8, matrix metalloproteinases (MMPs)-1, 3, 13, tumor necrosis factor-α (TNF-α), cyclooxygenase-2, and inducible nitric oxide synthase [27-29]. Although the beneficial effects of resveratrol are well known in cells, there is an irrelevance between in vitro effects and in vivo activities, when resveratrol was administered orally [26, 30]. The poor bioavailability of resveratrol might be related to the rapid metabolism in our body, demonstrating a short initial half-life of 8–14 min [26, 31]. Therefore, topical delivery of resveratrol through the skin can be an alternate strategy in developing new resveratrol-derived antioxidant products. Percutaneous delivery of resveratrol was reported to achieve therapeutic effects without any serious adverse reactions in the nude mouse skin in vivo [30]. In conclusion, EPS is revealed to be an efficient cytoprotective agent containing a relatively high concentration of resveratrol, which can be developed as a novel antioxidant to protect the skin from UV-induced oxidative stress.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

This research was supported by High Value-added Food Technology Development Program (109156-3), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
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    Wang, K. H., Y. H. Lai, J. C. Chang, T. F. Ko, S. L. Shyu and R. Y. Chiou (2005) Germination of peanut kernels to enhance resveratrol biosynthesis and prepare sprouts as a functional vegetable. Agric. Food Chem. 53, 242246.
  • 2
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