Antioxidant Defense and Hepatoprotection by Procyanidins from

Article pubs.acs.org/JAFC

Antioxidant Defense and Hepatoprotection by Procyanidins from Almond (Prunus amygdalus) Skins Van-Long Truong,† Min-Ji Bak,† Mira Jun,‡ Ah-Ng Tony Kong,§ Chi-Tang Ho,|| and Woo-Sik Jeong*,† †

Department of Smart Foods and Drugs, Department of Food and Life Sciences, College of Biomedical Science and Engineering, Inje University, Gimhae 621-749, Korea ‡ Department of Food Science and Nutrition, Dong-A University, Busan 604-714, Korea § Department of Pharmaceutics, Ernest Mario School of Pharmacy, , Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, United States || Department of Food Science, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08903, United States S Supporting Information *

ABSTRACT: Procyanidins, polymeric flavan-3-ols, are known to possess antioxidant, antiatherogenic, and anticarcinogenic properties. In the present study, we investigated the role of almond (Prunus amygdalus) skin procyanidins (ASP) in regulating the protein expression of phase II detoxifying and antioxidant enzymes in HepG2 cells and acetaminophen (APAP)-treated hepatotoxic mice. Treatments of ASP significantly induced the expression of phase II enzymes including NAD(P)H:quinoneoxidoreductase 1, catalase, glutathione peroxidase, and superoxide dismutase in the cells and mice. ASP also potently enhanced the expression of nuclear factor-E2-related factor 2 (Nrf2) and antioxidant response element (ARE)-reporter gene activity in vitro. APAP-induced hepatotoxic markers including AST and ALT in mice were inhibited by ASP administration. However, regulation of upstream kinases by ASP was different between in vitro and in vivo models. Collectively, ASP could induce the activation of Nrf2/ARE-mediated phase II detoxifying/antioxidant enzymes but with differential regulation on upstream kinases between in vitro and in vivo. KEYWORDS: Prunus amygdalus, almond skin, procyanidins, Nrf2, ARE, antioxidant enzymes, MAPKs, cytoprotection, acetaminophen, hepatotoxicity

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INTRODUCTION One of the important strategies of chemopreventive phytochemicals against cancer is the activation of cellular defensive mechanisms to block or prevent cells/tissues from exposure to carcinogens including chemical carcinogens and reactive oxygen species (ROS).1,2 This could be achieved from the induction of the phase II detoxifying and/or antioxidant enzymes such as heme oxygenase-1 (HO-1), NAD(P)H:quinoneoxidoreductase 1 (NQO1), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione S-transferase (GST). Most of these enzymes are known to contain a specific nucleotide sequence called antioxidant response element (ARE) in their 5′-upstream promoter region and regulated by the nuclear factor-E2-related factor 2 (Nrf2), a member of CNC family of bZIP transcription factor.3,4 Activation of Nrf2 by stimuli leads to the liberation of Nrf2 from Keap1 (a cytosolic repressor, Kelch-like ECH-associated protein 1) and facilitates Nrf2 translocation to the nucleus, where it heterodimerizes with small Maf proteins and transcriptionally activates ARE-mediated phase II detoxifying and antioxidant genes.5 Many studies have indicated that the mechanism of signal transduction for the activation of Nrf2 and Nrf2/AREdriven detoxifying and antioxidant enzymes is related to the activation of the upstream protein kinases including mitogenactivated protein kinases (MAPKs), protein C kinase (PCK), and phosphatidylinositol-3-kinase (PI3K) via phosphorylation.6,7 © XXXX American Chemical Society

Acetaminophen (APAP) is a well-known and widely used analgesic and antipyretic drug that is regarded safe at therapeutic doses.8 However, an overdose of APAP can lead to severe liver damage resulting from the formation of the highly reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI) due to saturation and incompletion of detoxifying metabolism of APAP by glucuronidation and sulfation.9 The covalent binding of NAPQI to cellular macromolecules can lead to series of molecular events, including protein alkylation, membrane lipid peroxidation, and formation of ROS and reactive nitrogen species (RNS), which are responsible for acute liver failure.10 These events lead to a decline of intracellular defense system consisting of various antioxidant enzymes, which results in cell death caused by oxidative stress.11 Procyanidins are phenolic polymers mainly composed of the monomeric flavan-3-ol units such as (+)-catechin and (−)-epicatechin, and they are found in various fruits, vegetables, nuts, seeds, and grains.12 Biological activities of procyanidins may depend on their isomerization, monomer bonding type, and degree of polymerization. Recent reports support that the consumption of dietary procyanidins is an Received: June 7, 2014 Revised: July 26, 2014 Accepted: July 31, 2014

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dx.doi.org/10.1021/jf5027247 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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Guyot et al.21 and Porter et al.22 Two and half milliliters of acidic reagent [5% (v/v) concd HCl in BuOH] and 100 μL of iron reagent [2% (w/v) solution of NH4Fe(SO4)212H2O in 2 M HCl] were added to about 5 mg of the procyanidin isolate in a glass screw-capped test tube. The tube was sealed with a screw cap, agitated vigorously, and heated for 40 min at 95 °C. To determine the presence of procyanidin and the degree of polymerization of ASP isolate, the vanillin assay in glacial acetic acid was performed by the method of Butler et al.23 The isolate was dissolved in a minimum volume of methanol and filled up to 1 mL with glacial acetic acid. The isolate solution was reacted with 5 mL of vanillin reagent consisting of 0.5% vanillin and 4% concd HCl in glacial acetic acid (1:1, v/v) at room temperature for 20 min. The absorbance of the reaction mixture was measured at 510 nm. Catechin was used as a standard. Depolymerization of Procyanidins with Phloroglucinol. Depolymerization of ASP was performed according to the method of Kennedy and Jones24 with slight modification. ASP (10 mg), phloroglucinol (20 mg), and ascorbic acid (5 mg) were dissolved in 1 mL of 0.1 N HCl in methanol. The mixture was shaken vigorously and reacted at 50 °C for 20 min. After reaction, the solvent was evaporated by a stream of nitrogen gas and dissolved in 500 μL of distilled water. The solution was extracted 5 times with 1 mL of ethyl acetate. The ethyl acetate extracts were combined, and the solvent was removed by nitrogen gas. The residue was dissolved in 500 μL of methanol and analyzed by HPLC-MS. HPLC-Mass Spectrometry (MS) Analyses. HPLC-MS was carried out using a VG Platform II mass spectrometer (Micromass, Beverly, MA) equipped with atmospheric pressure chemical ionization (APCI) probe in the positive ion mode. The HPLC system consisted of a Varian 9012 HPLC equipped with Varian 9050 UV detector and a Supelcosil LC-18 column (250 × 4.6 mm, particle size 5 μm). Detection wavelength was set at 280 nm, and injection volume was 20 μL. The mobile phase included 1% acetic acid (A) and methanol (B) as follows; isocratic: 0% B, 5 min; gradient: 0−15% B, 5−30 min; 15− 60% B, 30−65 min; 60−90% B, 65−90 min. The mass was scanned between 50 and 2000 amu. The source temperature was set at 150 °C, and the probe temperature was 450 °C. The cone voltage and the corona discharge were 15 V and 3 kV, respectively. The scan rate and interscan delay were 2 and 0.3 s, respectively. Cell Culture and Treatment. The human hepatoma cell line, HepG2, purchased from American Type Culture Collection (Rockville, MD), was maintained at 37 °C, humidified atmosphere of 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), streptomycin (100 μg/mL), penicillin (100 units/mL), 1% essential amino acids, and 1% glutamax. The culture medium was renewed once after 2 days. HepG2 cells were allowed to grow about 80−90% confluency and then starved overnight with serum-free DMEM medium prior to treatment with either vehicle (DMSO, 0.1%) or various concentrations of ASP for the indicated periods. Cell Viability Assay. The viability of HepG2 cells in medium containing ASP was determined by MTT assay, which is based on the metabolism of mitochondrial succinate dehydrogenase in viable cells. The cells were seeded onto 24-well plates at a density of 5 × 105 cells/ well and incubated at 37 °C, 5% CO2 for 24 h in a complete medium. After starvation, the cells were treated with a series of ASP concentrations for 24 h and followed by incubation with 50 μL of MTT solution (5 mg/mL) at 37 °C for 4 h in dark. A product from MTT metabolism, formazan crystal, was dissolved in DMSO (500 μL/ well). The absorbance was read at 570 nm with a microplate reader (BioTek, Winooski, VT). The levels of cell survival were expressed as a percentage of that in 0.1% DMSO-treated control. Preparation of Cytosolic and Nuclear Extract. The cytosolic and nuclear proteins were prepared by using a Nuclear and Cytoplasmic Extraction Reagents kit (Pierce Biotechnology Inc., Rockford, IL) according to the manufacturer’s instructions. Protein concentrations were measured by BCA protein assay (Pierce Biotechnology). Assay of Reporter Gene Activity. The HepG2-C8 cell line containing pARE-T1-luciferase reporter gene was previously de-

important strategy to promote health due to their beneficially biological properties, such as prevention of cardiovascular diseases and cancer.13,14 Procyanidins have also been wellknown as a strong anti-inflammatory agent both in vitro and in vivo by modulating nuclear factor κB (NF-κB) and MAPKs pathways.15,16 Almond skin has been reported to contain abundant phenolic compounds including catechins and the polymer procyanidins with different degrees of polymerization. Existence of catechins, B-type procyanidin dimers, procyanidin trimers, tetramers and A- and B-type oligomers has been determined in almond skins.17,18 A roasted almond skin has recently been reported to contain a series of procyanidin polymers with up to 11-mer.19 Although the composition of almond skin procyanidins has been relatively well-characterized, the biological roles of almond skin procyanidins have not been extensively studied. Previous findings on biological properties of almond skin procyanidins have been limited to radical scavenging antioxidant activity, α-amylase inhibition, and metabolic profiles in human.17−20 However, chemopreventive and cytoprotective roles of almond skin procyanidins (ASP) have been poorly characterized. In this study, we isolated ASP and evaluated their cytoprotective properties and the underlying mechanism in regulation of Nrf2/ARE pathway in the human hepatomaHepG2 cell line as well as in the APAP-induced hepatotoxic mouse model.

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MATERIALS AND METHODS

Reagents. The skins of almond (Prunus amygdalus) resulting from the blanching of almonds were a courtesy gift of Almond Board of California (Modesto, CA). Phlorogucinol and Toyopearl HW-40F were supplied by Sigma Chemical Co (St. Louis, MO). Solvents for column chromatography and HPLC analysis were of HPLC grade and purchased from Fisher Scientific (Springfield, NJ). MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], sulforaphane (SFN), DMSO, acetaminophen, and silymarin were purchased from Sigma-Aldrich (St. Louis, MO). The colorimetric aspartate transaminase (AST) and alanine transaminase (ALT) assay kits were supplied by Young-Dong Co. (Seoul, Korea). Anti-Nrf2, anti-NQO1, anti-SOD2, anti-GPx, and horseradish-peroxidase-conjugated antigoat immunoglobulin IgG antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-p-ERK, anti-p-JNK, anti-pp38, anti-p-Akt, anti-CAT, and horseradish-peroxidase-conjugated antirabbit immunoglobulin IgG were purchased from Cell Signaling Technology (Beverly, MA). U0126 (MEK1/2 inhibitor), SP600125 (JNK inhibitor), SB202190 (p38 inhibitor), and LY294002 (PI3K inhibitor) were purchased from Cell Signaling Technology. All other reagents used in this study were of the highest grade commercially available. Preparation of ASP. ASP was purified from aqueous extracts of almond skins. Briefly, almond skins (100 g) were milled to fine particles and extracted in 70% aqueous acetone for 48 h at room temperature. After the extraction, the solution was filtered twice through Whatman No.1 filter paper, and the acetone was evaporated using a rotary evaporator with vacuum at 35 °C. After removing the lipids by hexane, the aqueous residue was lypophilized to yield a dry powder. The powder sample was dissolved in 50% methanol and loaded onto a size exclusion chromatographic column packed with Toyopearl TSK HW-40-F (Sigma), which was preconditioned by eluting with 50% methanol (three times of column volume). After preconditioning, the loaded sample was washed with 50% methanol to remove monomeric phenolic compounds, and then eluted with 66% acetone to obtain procyanidins isolate. The eluted ASP isolate was further analyzed. BuOH-HCl Hydrolysis and Vanillin Assay in Glacial Acetic Acid. To determine the presence of interflavan units of ASP isolate, a butanol-acid hydrolysis was carried out according to the methods of B



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Figure 1. HPLC chromatogram of depolymerization products from ASP (A) and APCI-MS spectrum of major peak (peak at 16.27 min) (B). scribed.25 ARE-luciferase activity was measured by using luciferase kit (Promega, Madison, WI) following the manufacturer’s instruction. Briefly, after treatment with ASP for 12 h, the cells were collected in 1× cell lysis buffer containing 25 mM Tris (pH 7.8 with H3PO4), 2 mM EDTA, 2 mM DTT, 10% glycerol, 1% triton X-100. After centrifugation, a 10 μL aliquot of supernatant was assayed for luciferase activity by using GloMax 20/20 luminometer (Promega). Luciferase activity was normalized against total protein concentration, determined by BCA protein assay, and expressed as a fold of induction over that in vehicle-treated control. Assay for Antioxidant Enzymes Activity. For samples from cells, after exposure to ASP for 12 h, the cells were homogenized in ice-cold 50 mM phosphate buffer (pH 7.4) by sonication, followed by centrifugation at 10 000g for 20 min to separately obtain supernatant and pellet for further experiments. For samples from mice, liver tissues were collected and homogenized in phosphate buffer (pH 7.0). The protein content was determined using the BCA protein assay (Pierce). CAT activity was determined by the method of Abei26 following the decrease in absorbance of H2O2 at 240 nm. One unit of CAT was defined as the amount of enzymes that decomposes 1.0 μM of H2O2 in a minute. GPx activity was performed according to the method of Bongdanska et al.27 A unit of GPx was defined as an amount of enzyme that oxidized 1 nM of NADPH per minute. SOD activity was determined by the method of Oyanagui.28 The amount of protein required for 50% inhibition in the absorbance at 550 nm was defined as one unit of SOD activity. Animals and Experimental Design. Male Balb/c mice (6 weeks old, 20−25 g) used in the study were obtained from Hyochang Science (Daegu, Korea). The mice were housed in an environmentally controlled room at 25 ± 2 °C, 55−60% humidity, 12 h light/dark cycle and allowed free access to standard laboratory diet and distilled water ad libitum for at least 1 week prior to the experiments. The mice were assigned to six groups (n = 5) and treated as follows: Group1 (control group), animals received only 50% polyglycol as vehicle. Group 2 (ASP group), animals were orally gavaged with 10 mg/kg ASP for three times per week. Group 3 (APAP group), mice were received 50% polyglycol for three times per week and then injected intraperitoneally (ip) with a single dose of APAP (300 mg/kg) in 10 mL/kg saline. Groups 4 and 5 (APAP + ASP group), animals were orally administered with 1 or 10 mg/kg of ASP, per os three times per week and then were exposed to APAP (300 mg/kg, ip) 2 h after the last dose of ASP. Group 6 (APAP + silymarin group), silymarin (50 mg/kg) was administered for three times per week and followed by single dose of APAP (300 mg/kg) once only. Silymarin has been wellknown as a natural hepatoprotective agent in treatment of toxic liver diseases and is therefore considered as a positive control in this experiment.29 Eight hours after APAP administration, the mice were

sacrificed to obtain their blood and livers for further biochemical analysis. Measurement of Serum ALT and AST Levels in APAPTreated Mice. The level of liver damage was determined by detecting the enzymatic activities of plasma ALT and AST. Blood samples from mice were centrifuged at 3000g at 4 °C for 10 min, and the ALT and AST activities in serum supernatant were measured using commercially available kits (Youngdong Tech Co., Ltd). Western Blot Analysis. For samples from cells, HepG2 cells were washed with ice-cold PBS (pH 7.4) after treatments, and cells were lysed in 200 μL of cold cell lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/mL leupeptin, 1 mM phenylmethanesulfonyl fluoride) on ice, for 1 h. To analyze protein expression, the proteins (40 μg) of each sample, normalized by BCA protein assay kit (Pierce), were resolved on SDSPAGE gels and then transferred onto polyvinylidene fluoride membranes using semidry transfer system (Bio-Rad, Hercules, CA). The membranes were then blocked in PBST (0.1% Tween 20 in phosphate-buffered saline) containing 5% nonfat dried milk for 2 h at 4 °C and followed by incubation with specific primary antibodies overnight at 4 °C. After hybridization with primary antibodies, the membranes were washed with PBST four times, incubated with horseradish-peroxidase-conjugated antirabbit or antigoat immunoglobulin IgG secondary antibodies for 3 h at 4 °C, and washed with PBST four times. Finally, blots were visualized using enhanced chemiluminescence Western blotting reagents (Santa Cruz Biotechnology). For samples from mice, liver tissues were collected and homogenized in cell lysis buffer. Liver tissue homogenates were then centrifuged at 13 000 rpm for 10 min, 4 °C to obtain supernatants. The analytical procedure was the same as that mentioned above. Statistical Analysis. Data were expressed as means ± SD. Statistical comparisons were evaluated using analysis of variance (ANOVA) followed by Tukey’s test. P < 0.05 was considered as significant.

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RESULTS Isolation and Detection of Procyanidins. Isolation and purification of ASP was carried out by conventional isolation methods as described in the Materials and Methods section. The presence of procyanidins in ASP isolate was determined by hydrolytic reactions such as BuOH-HCl and vanillin assays. The BuOH-HCl hydrolysis reaction of the ASP isolate in the presence of iron(III) salts and heat (95 °C) produced a deep red color (data not shown). Accordingly, these results indicate the presence of interflavan bonds procyanidins in ASP.21,22 The C



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Figure 2. Induction of phase II detoxifying and antioxidant enzymes by ASP in HepG2 cells. (A) Protein expression of NQO1. (B) Protein expression of GPx, CAT, and SOD-2. The cells were treated with either vehicle (DMSO 0.1%) or indicated concentrations of ASP for 12 h. Equal amounts of total protein were subjected to Western blotting to analyze the expression of each protein. The results were quantitated by densitometry, and the levels of proteins were normalized with β-actin. (C) Enzyme activity of GPx, CAT, and Mn-SOD. After treatment with indicated concentration of ASP, the activity of CAT, GPx, and Mn-SOD enzymes was determined, as described in Materials and Method section. Sulforaphane (SFN, 25 μM) was used as a positive control. Data represent the average ± SD of three independent experiments with the similar results. (*) p < 0.05 compared to the vehicle-treated control.

approximate polyphenol content of the ASP isolate by vanillin assay in glacial acetic acid was also carried out. As a result, the ASP isolate showed about 24.1% polyphenol content when catechin, one of major monomeric flavan-3-ol units of procyanidins, was used to standardize this assay. The vanillin assay is a sensitive and simple method to determine flavan-3ols, dihydrochalcones, and procyanidins. Although the ASP isolate might contain other compounds, the values could be underestimated because the assay has the limitation that only the terminal units of procyanidins react with vanillin in glacial acetic acid. However, this characteristic of the vanillin assay makes it possible to estimate the degree of polymerization. The degree of polymerization can be estimated as the ratio of absorbance of monomer to polymer when measured as the same amount.23 As a result, the polymerization degree of ASP was found to be 4.2. HPLC-MS Analysis of ASP. The ASP was directly analyzed by HPLC with C18 column, and a broad hump peak at 280 nm was observed (see the Supporting Information Figure S1), which strongly demonstrated the presence of polymeric procyanidins as major constituents in the isolate. Moreover, depolymerization reaction of the ASP isolate with phloroglucinol permitted the evaluation of ASP composition. The interflavan bonds of procyanidins can be cleaved under acidic condition, releasing extension and terminal subunits. The releasing extension subunit intermediates (electrophiles) can be

trapped by phloroglucinols (nucleophiles) to produce detectable adducts. 24 The HPLC-MS analysis permitted the determination of cleavage products from the polymeric almond skin procyanidins. The depolymerization of procyanidins yielded several peaks (Figure 1A). However, except for the peaks before 7 min attributed to solvent, ascorbic acid and phloroglucinol, only one major peak at 16.27 min was observed from the HPLC chromatogram. The mass spectrum of the major peak showed m/z = 415 [M + H]+ as base peak, m/z = 437 [M = Na]+, m/z = 289 [M − H]+, m/z = 245 [289 − CO2], and m/z = 127 [phloroglucinol + H]+ (Figure 1B). Therefore, the major peak was assigned as phloroglucinol adduct resulting from the reaction between phloroglucinol and flavanol extension unit of polymeric procyanidins. These findings suggest that there is only one predominant flavanol as extension subunits in the polymer, which is either (+)-catechin or (−)-epicatechin. A polymeric procyanidin fraction from unripe almond fruit flesh has been shown to contain only procyanidins presenting (−)-epicatechin in their extension units but with both catechin and epicatechin as terminal units (ratio of 3:2).30 (+)-Catechin (peak at 20.4 min) and (−)-epicatechin (peak at 24.32 min) as terminal subunits were found in the depolymerization reaction mixture by the HPLC-MS analysis. These compounds were identified by the interpretation of their mass spectrum as well as by comparison of their retention time with standard materials and with the D



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Figure 3. Induction of ARE-reporter gene activity and Nrf2 expression by ASP in HepG2-C8/HepG2 cells. (A) ARE-luciferase acidity. HepG2-C8 cells stably transfected with pARE-luciferase reporter gene were treated with either vehicle (DMSO 0.1%) or ASP (10, 25, and 50 μg/mL) for 12 h. Luciferase activity was normalized with total protein content and expresses as fold induction of vehicle-treated control. (B) Nrf2 expression. HepG2 cells were exposed to either vehicle or indicated concentrations of ASP for 2 h. Equal amounts of total protein were subjected to Western blot analysis using specific antibodies for Nrf2 and β-actin. (C) Nuclear localization of Nrf2. Cytosolic and nuclear fractions were prepared and analyzed by Western blotting. The results were quantitated by densitometry, and Nrf2 protein levels were normalized with β-actin (cytosolic fraction) or Lamin B (nuclear fraction). Sulforaphane (SFN, 25 μM) was used as a positive control. All data represent means ± SD of three independent experiments. (*) p < 0.05 compared to the vehicle-treated control.

literature.24,31 The amount of these terminal subunits was quite small compared to the extension subunits, suggesting that the almond skin polymeric procyanidins might have a very high degree of polymerization. The HPLC-MS analysis also showed compounds with MW 576, m/z = 577 [M + H]+ (peak at 29.99 min). The molecular ion at 577 is two units lower than the (epi)catechin dimer with C−C interflavan bond, indicating that the presence of A-type double interflavan linkage.30 A- and Btype procyanidins have been found in almond skin with polymerization degrees from dimer to 11-mer.18,19,30 Expression and Activity of Phase II/Antioxidant Enzymes by ASP in HepG2 Cells. To determine the appropriate concentrations of ASP in HepG2 cells, cell viability was determined after treatments with various concentrations of ASP (0−200 μg/mL) using the MTT assay. Over 90% of the cells survived after treatments with up to 100 μg/mL ASP for 24 h and 80% at the dose range 100−200 μg/mL (see the Supporting Information Figure S2). Based on the cell viability data, doses below 50 μg/mL of ASP were used for further experiments. Expression of NQO1, a phase II detoxifying enzyme, in HepG2 cells was examined by Western blotting after exposure of cells to 10, 25, and 50 μg/mL of ASP for 12 h. As illustrated in Figure 2A, ASP treatment significantly increased the NQO1 protein expression in a dose-dependent manner. The maximal induction of NQO1 (1.8 fold) was obtained at 50 μg/mL ASP, whereas sulforaphane displayed more potent induction of the protein expression (2.3-fold). To determine the effects of ASP on the expression of antioxidant enzymes, the protein levels of SOD-2, CAT, and

GPx were measured after incubating HepG2 cells with ASP. The expressions of all three antioxidant enzymes were significantly and concentration-dependently upregulated by ASP treatment in the cells (Figure 2B). Especially, the protein expression levels of SOD-2 induced by ASP at the indicated dose range were greater than that induced by SFN (25 μM). These inductions of antioxidant enzymes by ASP have been found to be correlated with the activity of these enzymes. Correlation coefficients (R2) between protein expressions and enzyme activities of CAT, GPx, and Mn-SOD are 0.66, 0.83, and 0.82, respectively. As displayed in Figure 2C, ASP treatments resulted in the enhancement of the enzymes’ activity, although Mn-SOD activity was most potently induced by ASP at 50 μg/mL. Induction of ARE-Reporter Gene Activity and Nuclear Translocation of Nrf2 by ASP in Cells. To elucidate whether the induction of phase II detoxifying and antioxidant enzymes by ASP was mediated by activation of Nrf2/ARE pathway, the HepG2-C8 cells stably transfected with a pARETI-luciferase reporter gene were treated with ASP for 12 h. As illustrated in Figure 3A, the luciferase activity dramatically increased in response to ASP treatments at the indicated doses. The cells exposed to 10, 25, and 50 μg/mL of ASP displayed the ARE-luciferase activity to 6-, 40-, 55-fold compared to untreated control, respectively. The induction of ARE-luciferase activity by ASP at above 25 μg/mL was much greater than that induced by SFN (18-fold). Transcription factor Nrf2 is known to play a crucial role in the expression of ARE-mediated phase II detoxifying/antioxidant enzymes such as NQO1, SOD, CAT, and GPx.32,33 Therefore, we further evaluated whether the E



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Figure 4. Effects of ASP on phosphorylations of MAPKs (A) and Akt (B) in HepG2 cells. The cells incubated in the absence or presence of ASP (10, 25, and 50 μg/mL) for 1 h were subjected to Western blot analysis using specific antibodies for p-ERK, p-JNK, p-p38, p-Akt, and β-actin. The intensity of indicated proteins was detected by densitometry and expressed as fold of vehicle-treated control. Sulforaphane (SFN, 25 μM) was used as a positive control. Each data represents the means ± SD of three independent experiments. (*) p < 0.05 compared to the vehicle-treated control.

Figure 5. Effect of MAPK inhibitors on ASP-induced Nrf2 expression (A) and ARE-luciferase activity (B) in HepG2/HepG2-C8 cells. Cells were pretreated with or without U0126 (MEK1/2 inhibitor), SP600125 (JNK inhibitor), SB202190 (p38 inhibitor), and LY294002 (PI3K inhibitor) for 1 h and then incubated in the absence or presence of ASP (50 μg/mL) for another 1 h (A) or 12 h (B). The Nrf2 expression was determined by Western blotting, and ARE-luciferase activity was evaluated as described in the Materials and Methods section. The intensity of indicated proteins was detected by densitometry and expressed as fold of vehicle-treated control. Each data represents the means ± SD of three independent experiments. (*) p < 0.05 compared to the vehicle-treated control. (#)p < 0.05 indicates difference from the ASP alone group.

enhanced expressions of ARE-mediated phase II enzymes by ASP were associated with the induction of Nrf2. As expected, the ASP-treated HepG2 cells exhibited an increased expression of Nrf2 protein in a dose-dependent manner (Figure 3B).

Moreover, ASP treatment resulted in the translocation of Nrf2 protein from cytosol to the nucleus (Figure 3C). These data strongly suggest that the induction of phase II detoxifying/ antioxidant enzymes by ASP may be mediated by the enhanced F



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expression and nuclear translocation of Nrf2 in cooperation with ARE. Phosphorylations of Upstream Protein Kinases by ASP in Cells. In order to elucidate whether ASP participates in the Nrf2/ARE-related upstream signaling pathways, we examined the phosphorylations of three MAPK subfamilies including ERK, JNK, and p38 as well as PI3K/Akt. As shown in Figure 4, ASP dramatically and dose-dependently stimulated the phosphorylation levels of all three MAPKs as well as Akt. The phosphorylation levels of ERK, JNK, p38, and Akt by ASP at the highest concentration of 50 μg/mL reached approximately 8.6-, 7.6-, 3.1-, and 4.6-fold, respectively, compared to that by vehicle-treated control. We further analyzed which upstream kinases were more responsible for the ASP-mediated Nrf2/ARE activation, using specific inhibitors for each kinase: U0126 (MEK1/2 inhibitor), SP600125 (JNK inhibitor), SB202190 (p38 inhibitor) and LY294002 (PI3K inhibitor). Pretreatment with U0126 or LY294002 prior to ASP treatment completely blocked the ASP-induced Nrf2 expression to near basal level, whereas SP600125 and SB202190 were not as effective as U0126 or LY294002 in the blockade of the ASPinduced Nrf2 expression (Figure 5A). Similarly, the ASPinduced ARE-luciferase activity was more effectively suppressed by the inhibitors of ERK and Akt (Figure 5B). Taken together, these results indicate that the ASP-induced activation of Nrf2/ ARE-pathway might be at least in part through the activation of ERK and PI3K/Akt pathways in HepG2 cells. Effects of ASP on Serum ALT and AST Levels in APAPTreated Mice. In the present study, APAP treatment (300 mg/kg) resulted in a dramatic increase in activity of serum ALT and AST, biomarkers of hepatotoxicity,34 compared to vehicle treatment (Figure 6). Plasma ALT and AST levels increased 9and 7-fold after an 8 h APAP challenge, respectively, suggesting a significant damage in the hepatocytes by APAP. Preadministrations with ASP at 1 mg/kg and 10 mg/kg, however, significantly attenuated the levels of the ALT and AST activity. Silymarin (50 mg/kg), reported to possess protective capacity against APAP-mediated liver necrosis, similarly inhibited the APAP-elevated activity of ALT and AST. The administration of ASP alone did not affect the basal activity of these transaminases. Induction of Nrf2 and Phase II Enzyme Expression by ASP in the Liver of APAP-Treated Mice. The activation of Nrf2 and phase II enzyme NQO1 in the cell line was further evaluated in APAP-treated animal model. The injection of APAP dramatically lowered the expression of Nrf2 as well as that of NQO1; however, administrations of ASP at both 1 mg/ kg and 10 mg/kg restored the reduced expression levels of Nrf2 by APAP to near basal level, whereas administration of ASP alone did not change the basal protein levels (Figure 7). Moreover, the level of NQO1 expression induced by ASP was higher than that induced by the positive control silymarin. Our study is the first in vivo report demonstrating that procyanidins from almond skins have hepatoprotective activity through activation of Nrf2 and Nrf2/ARE-mediated antioxidant defense mechanism. Effects of ASP on the Expression and Activity of Antioxidant Enzymes in the Liver of APAP-Treated Mice. The expression and activity of antioxidant enzymes including SOD-2, GPx, and CAT by ASP were also determined in the APAP-treated mice. Similar to the data observed in Figure 7, the injection of APAP to mice resulted in significant reduction in the expressions of all three antioxidant enzymes. However,

Figure 6. Effects of ASP administration on serum ALT and AST levels in APAP-treated mice. Vehicle or ASP (1 or 10 mg/kg) or silymarin (SIL, 50 mg/kg) was orally administered three times per week before intraperitoneal injection of APAP (300 mg/kg) injection. Activities of plasma ALT and AST were measured 8 h after the APAP injection, as described in the Materials and Methods. The data are expressed by means ± SD for five mice. (*) p < 0.05, significantly different from the vehicle control group. (#) p < 0.05, significantly different from the APAP-treated group.

Figure 7. Effects of ASP administration on Nrf2 and NQO1 expression in APAP-treated mice liver. Liver tissues were collected at 8 h following APAP challenge to analyze Nrf2 and NQO1. Silymarin (SIL, 50 mg/kg) was used as a positive control. The data are expressed by means ± SD for five mice. (*) p < 0.05, significantly different from the vehicle control group. (#) p < 0.05, significantly different from the APAP-treated group.

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Figure 8. Effects of ASP administration on the expression (A) and activity (B) of antioxidant enzymes in APAP-treated mice liver. Liver tissues were collected at 8 h following APAP challenge to analyze antioxidant enzymes. Silymarin (SIL, 50 mg/kg) was used as a positive control. The data are expressed by means ± SD for five mice. (*) p < 0.05, significantly different from the vehicle control group. (#) p < 0.05, significantly different from the APAP-treated group.

Figure 9. Effects of ASP administration on upstream kinases in APAP-treated mice liver. Liver tissues were collected at 8 h following APAP challenge to analyze phosphorylations of ERK, JNK, p38 (A) and Akt (B). Silymarin (SIL, 50 mg/kg) was used as a positive control. The data are expressed by means ± SD for five mice. (*) p < 0.05, significantly different from the vehicle control group. (#) p < 0.05, significantly different from the APAPtreated group.

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metabolizing and eliminating chemicals and their metabolites.1,33 In this study, our results indicate that ASP possesses an ability to stimulate these phase II detoxifying and antioxidant enzymes in vitro and in vivo. The phase II detoxifying enzyme NQO1 catalyzes the two-electron reductive metabolism and detoxification of quinones and their derivatives resulting in protection of cells against chemical-oxidative stress and cancer.38 Moffit et al. have shown enhanced levels of hepatic NQO1 may reduce the reactive NAPQI availability by converting it to the parent APAP compound.39 Role of cellular defensive phytochemicals against oxidative stress can be roughly classified into two categories; direct scavenger of ROS/free radicals or indirect stimulator of cellular antioxidant defense systems including antioxidant enzymes. The major ROS in the mechanism of APAP-induced hepatotoxicity in mice has been superoxide anion radical (O2−), which dismutates to molecular oxygen and hydrogen peroxide or reacts with nitric oxide (NO) to form peroxynitrite (ONOO−), a potent oxidant and nitrating species.10 Our results clearly show that ASP significantly induces the expression and activity of SOD in cell and APAP-treated mice, indicating cytoprotection of liver cells/tissues from APAP damage by ASPmediated SOD activation. Our findings suggest that ASP potentiates the cellular defense system through activation of phase II and antioxidant enzymes both in vitro and in vivo. Induction of phase II/antioxidant enzymes has been positively associated with transcription factor Nrf2 expression and its nuclear translocation.33 Therefore, we assume that the induction of the phase II/antioxidant enzymes and cytoprotection by ASP may be due to the activation of Nrf2/ARE pathway. Our previous study has also proclaimed that procyanidins from wild grape (Vitis amuresis) seed is a potential chemopreventive agent that activates the phase II enzymes through the regulation of Nrf2/ARE pathway.40 Stress-response kinases have been involved in the regulation of cellular signaling pathways in response to a variety of stresses from both external and internal environments.1 Previous studies have shown that activation of PI3K/Akt and MAPKs including ERK, JNK and p38 MAPK has been shown to participate in the activation of Nrf2 and induction of AREmediated gene expressions in various in vitro and in vivo models.41 Yu et al. have indicated that the induction of phase II detoxifying enzymes by SFN and tert-butyl hydroquinone is mediated by activation of ERK pathway.25 Also, it has been shown that JNK implicates in the induction of phase II enzymes by phenethyl isothiocyanate, whereas p38 MAPK activates Nrf2 in response to quercetin in HepG2 cells.42,43 Although we have found similar effects of ASP in the activation of Nrf2 and phase II/antioxidant genes both in vitro and in vivo models, however, the modulation of upstream kinases by ASP seems to be different between cell line and mouse model. In the HepG2 cell model, ASP potently upregulated the phosphorylations of all three upstream MAPKs and Akt. Moreover, blockade of ERK and Akt using the corresponding specific inhibitor resulted in the inhibition of ASP-induced Nrf2-ARE activation, indicating involvement of these kinases in ASP-mediated stimulation of cytoprotective mechanisms. However, data on upstream kinases from mice were quite different from those observed in cells. ASP administration did not alter the basal phosphorylation levels of these kinases in the liver of mice while overdose of APAP significantly changed the phosphorylation levels. In addition, APAP seemed to differentially regulate each MAPK in mice; it induced phosphorylation of ERK and JNK but

ASP preadministration significantly restored the APAP-reduced protein levels of GPx and SOD-2 at doses of both 1 and 10 mg/ kg, although the restoration of CAT expression by ASP was not statistically significant (p < 0.05) (Figure 8A). The activity of these antioxidant enzymes was also measured in the differentially treated mouse groups. Similar to the data of protein levels, APAP challenge caused reductions in the enzymes activities; however, only decreases in GPx and Mn-SOD activity were significant but not in CAT activity (Figure 8B). Restoration of GPx and Mn-SOD activity was observed only in ASP 10 mg/kg group. The positive control silymarin was not significantly effective in the restoration of antioxidant enzyme activity decreased by APAP treatment in this mouse model system. These data demonstrated that the enhancement of both expression and activity of hepatic antioxidant enzymes by ASP might contribute to attenuate the acute hepatotoxic effect caused by APAP in mice. Effects of ASP on Upstream Kinases in APAP-Treated Mice. Based on the regulatory roles of ASP on MAPKS observed in cells, we further examined the modulation of MAPKs by ASP in APAP-treated mice. In the present study, exposure of mice to 300 mg/kg of APAP resulted in strong increases in the phosphorylation levels of ERK and JNK (2.3− 2.7-fold) (Figure 9A). However, APAP treatment significantly downregulated the phosphorylation level of p38 in the mice. Preadministration of ASP (1 or 10 mg/kg) or silymarin (50 mg/kg) inhibited the APAP-induced phosphorylations of the hepatic ERK and JNK. However, the decreased p38 phosphorylations by APAP were significantly restored by ASP or silymarin preadministrations. The phosphorylation level of Akt was not significantly changed by APAP but significantly lowered in ASP treatment at 10 mg/kg (Figure 9B). Overdose of APAP-caused liver damage in rodents has been related to an early oxidative stress and the involvement of JNK activation.11 The inhibitory effect on the JNK phosphorylation plays vital role in protection of cell/tissue against APAP toxicity. Saito et al. suggested that the JNK inhibitor SP600125 protects mice against APAP-induced liver injury mainly by preventing the mitochondrial oxidant stress and peroxynitrite formation.35 A recent study also has demonstrated sustained activation of JNK and ERK by APAP in mice and possible protection by ROS scavenging antioxidant.36 Additionally, PI3K/Akt pathway has been demonstrated to be involved in hepatotoxicity caused by toxicants such as carbon tetrachloride.37 However, effects of APAP toxicity on the phosphorylation of Akt have not been fully understood yet.

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DISCUSSION In the present study, various analytical tools confirmed the existence of procyanidins in almond skins with the average polymerization degree about 4.2. Although the exact structures of ASP still remain unclear due to the structural complexity as well as the variety differences. In spite of the efforts for the structural analyses on almond skin procyanidins, their biological roles and the underlying molecular mechanisms have been largely unknown. Our present study shows the cytoprotective activity of ASP in cells as well as in APAP-induced mice. Protection of cells/tissues from endogenous and/or exogenous carcinogens has been regarded as a key defense mechanism of cancer chemoprevention by natural products. This can be mainly achieved by the induction of phase II detoxifying/antioxidant enzymes such as NQO1, GST, CAT, GPx, and SOD, which are capable of scavenging ROS, I



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(2) Surh, Y.-J. Cancer chemoprevention with dietary phytochemicals. Nat. Rev. Cancer 2003, 3, 768−780. (3) Nguyen, T.; Nioi, P.; Pickett, C. B. The Nrf2-Antioxidant Response Element Signaling Pathway and Its Activation by Oxidative Stress. J. Biol. Chem. 2009, 284, 13291−13295. (4) Dhakshinamoorthy, D.; Jaiswal, A. K. Functional characterization and role of INrf2 in antioxidant response element-mediated expression and antioxidant induction of NAD(P)H:quinone oxidoreductase1 gene. Oncogene 2001, 20, 3906−3917. (5) Taguchi, K.; Motohashi, H.; Yamamoto, M. Molecular mechanisms of the Keap1−Nrf2 pathway in stress response and cancer evolution. Genes Cells 2011, 16, 123−140. (6) Yang, C.-M.; Huang, S.-M.; Liu, C.-L.; Hu, M.-L. Apo-8′lycopenal Induces Expression of HO-1 and NQO-1 via the ERK/p38Nrf2-ARE Pathway in Human HepG2 Cells. J. Agric. Food Chem. 2012, 60, 1576−1585. (7) Yeh, C.-T.; Yen, G.-C. Involvement of p38 MAPK and Nrf2 in phenolic acid-induced P-form phenol sulfotransferase expression in human hepatoma HepG2 cells. Carcinogenesis 2006, 27, 1008−1017. (8) Rumack, B. H. Acetaminophen misconceptions. Hepatology 2004, 40, 10−15. (9) Ishibe, T.; Kimura, A.; Ishida, Y.; Takayasu, T.; Hayashi, T.; Tsuneyama, K.; Matsushima, K.; Sakata, I.; Mukaida, N.; Kondo, T. Reduced acetaminophen-induced liver injury in mice by genetic disruption of IL-1 receptor antagonist. Lab. Invest. 2009, 89, 68−79. (10) Jaeschke, H.; Knight, T. R.; Bajt, M. L. The role of oxidant stress and reactive nitrogen species in acetaminophen hepatotoxicity. Toxicol. Lett. 2003, 144, 279−288. (11) McGill, M. R.; Williams, C. D.; Xie, Y.; Ramachandran, A.; Jaeschke, H. Acetaminophen-induced liver injury in rats and mice: comparison of protein adducts, mitochondrial dysfunction, and oxidative stress in the mechanism of toxicity. Toxicol. Appl. Pharmacol. 2012, 264, 387−394. (12) Jeong, W.-S.; Kong, A.-N. T. Biological Properties of Monomeric and Polymeric Catechins: Green Tea Catechins and Procyanidins. Pharm. Biol. 2004, 42, 84−93. (13) Corder, R.; Mullen, W.; Khan, N. Q.; Marks, S. C.; Wood, E. G.; Carrier, M. J.; Crozier, A. Oenology: Red wine procyanidins and vascular health. Nature 2006, 444, 566. (14) Gossé, F.; Guyot, S.; Roussi, S.; Lobstein, A.; Fischer, B.; Seiler, N.; Raul, F. Chemopreventive properties of apple procyanidins on human colon cancer-derived metastatic SW620 cells and in a rat model of colon carcinogenesis. Carcinogenesis 2005, 26, 1291−1295. (15) Bak, M. J.; Truong, V. L.; Kang, H. S.; Jun, M.; Jeong, W. S. Anti-inflammatory effect of procyanidins from wild grape (Vitis amurensis) seeds in LPS-induced RAW 264.7 cells. Oxid. Med. Cell. Longev. 2013, 2013 (Article ID 409321), 1−11. (16) Martinez-Micaelo, N.; González-Abuín, N.; Ardèvol, A.; Pinent, M.; Blay, M. T. Procyanidins and inflammation: Molecular targets and health implications. BioFactors 2012, 38, 257−265. (17) Sang, S.; Lapsley, K.; Jeong, W.-S.; Lachance, P. A.; Ho, C.-T.; Rosen, R. T. Antioxidative Phenolic Compounds Isolated from Almond Skins (Prunus amygdalus Batsch). J. Agric. Food Chem. 2002, 50, 2459−2463. (18) Monagas, M.; Garrido, I.; Lebrón-Aguilar, R.; Bartolome, B.; Gómez-Cordovés, C. Almond (Prunus dulcis (Mill.) D.A. Webb) Skins as a Potential Source of Bioactive Polyphenols. J. Agric. Food Chem. 2007, 55, 8498−8507. (19) Tsujita, T.; Shintani, T.; Sato, H. α-Amylase inhibitory activity from nut seed skin polyphenols. 1. Purification and characterization of almond seed skin polyphenols. J. Agric. Food Chem. 2013, 61, 4570− 4576. (20) Bartolome, B.; Monagas, M.; Garrido, I.; Gomez-Cordoves, C.; Martin-Alvarez, P. J.; Lebron-Aguilar, R.; Urpi-Sarda, M.; Llorach, R.; Andres-Lacueva, C. Almond (Prunus dulcis (Mill.) D.A. Webb) polyphenols: from chemical characterization to targeted analysis of phenolic metabolites in humans. Arch. Biochem. Biophys. 2010, 501, 124−33.

inhibited that of p38 and did not affect Akt phosphorylation. In any case, however, the pretreatment of ASP significantly changed the phosphorylation levels of these kinases toward basal levels. For example, ASP administration reduced the phosphorylations of ERK and JNK stimulated by overdose of APAP while it induced the phosphorylation of p38 reduced by APAP. The reason for this discrepancy in the regulation of the upstream kinases by ASP between cell line and mouse model is not clear at this moment. However, it might come from difference in bioavailability and/or pharmacokinetics between models in vitro and in vivo or the presence/absence of stimuli (APAP in this case). Despite of abundant data on biological activities of procyanidins, the in vivo bioavailability and efficacy of procyanidins have been continuously questioned. Procyanidins up to tetramers but not higher degrees of polymer have been shown to be absorbed and present in blood circulation.44−46 Therefore, ASP with average polymerization degree of 4.2 in our study might be able to be, at least in part, absorbed and exert its cytoprotective activity in the current mouse model. However, further studies on the exact molecular mechanisms and pharmacokinetics of ASP should be followed.

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1 shows HPLC chromatogram of almond skin procayanidins. Figure S2 shows the effect of almond skin procyanidins on viability of HepG2 cells. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +82-55-320-0691. Tel.: +8255-320-3238. Funding

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant No. 2013R1A1A2012599). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to the Almond Board of California for providing us the skins of almonds. We also thank to Dr. Paul A. Lachance for his guidance to initiate this research.

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ABBREVIATIONS USED ASP,almond skin procyanidins; ARE,antioxidant response element; ROS,reactive oxygen species; Nrf2,nuclear factor-E2related factor 2; CAT,catalase; GPx,glutathione peroxidase; SOD,superoxide dismutase; NQO1,NAD(P)H:quinoneoxidoreductase 1; MAPK,mitogen-activated protein kinase; ERK,extracellular regulated kinase; JNK,c-Jun Nterminal kinase; PI3K,phosphoinositide 3-kinase; HPLCMS,high-performance liquid chromatography− mass spectrometry

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REFERENCES

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Antioxidant Defense and Hepatoprotection by Procyanidins from

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