Ellagic Acid Identified through Metabolomic Analysis Is an Active

Nov 7, 2013 - These results demonstrate that ellagic acid from strawberry 'Seolhyang' is the major component playing a crucial role in inflammation ...
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Ellagic Acid Identified through Metabolomic Analysis Is an Active Metabolite in Strawberry (‘Seolhyang’) Regulating Lipopolysaccharide-Induced Inflammation Jaehoo Lee,†,∥ Sugyeong Kim,†,∥ Hyeju Namgung,† Young-Hee Jo,† Cheng Bao,† Hyung-Kyoon Choi,‡ Joong-Hyuck Auh,*,† and Hong Jin Lee*,† †

Department of Food Science and Technology, Chung-Ang University, Anseong 456-756, South Korea College of Pharmacy, Chung-Ang University, Seoul 156-756, South Korea

‡

S Supporting Information *

ABSTRACT: This study employed the metabolomic approach to identify the key constituent exerting anti-inflammatory activity in murine macrophage RAW 264.7 cells. Among the six different fractions (SF1−SF6) of the strawberry ‘Seolhyang’, SF4 showed more significant inhibition on iNOS expression than SF3, and ellagic acid was determined as the most significant different component between SF4 and SF3 using orthogonal partial least-squares discriminant analysis. Ellagic acid (0.3 and 1.0 μM) and SF4 (100 μg/mL) were found to regulate the same inflammatory mediators, inhibitory κB (IκB) and mitogen-activated protein kinases (MAPKs), which led to the reduction of tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), and iNOS expressions. These results demonstrate that ellagic acid from strawberry ‘Seolhyang’ is the major component playing a crucial role in inflammation, suggesting the possible application of metabolomic analysis to determining the key ingredients having biological functions in the complicated food matrix. KEYWORDS: strawberry, ellagic acid, metabolomics, inflammation, murine macrophage cells

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a useful tool for phenotyping in food science.20 A variety of foods such as black raspberry,21 tomato,22 rice,23 and wheat24 have been analyzed on the basis of metabolomics. A mass spectrometry (MS)-coupled method is widely used for metabolite profiling due to its good sensitivity and resolution, although a longer time is needed for analysis.25 Metabolomics applies multivariate statistical tools on a data set acquired from the MS-coupled method to discriminate systematic variation using principal component analysis (PCA), and a subsequent supervised statistical analysis such as partial least-squares discriminatory analysis (PLS-DA) or orthogonal PLS-DA (OPLS-DA) can clearly separate a data set into different groups, finally screening candidate metabolite for variation.26 Therefore, it is worthwhile to employ metabolomic analysis to profile the whole metabolite in the strawberry fractions and identify the key constituents exerting health-promoting beneficial effects. In this study, we separated strawberry powder into different fractions using organic solvents and compared the effect on regulating lipopolysaccharide (LPS)-induced inflammation in murine macrophage RAW 264.7 cells. In addition, we identified the component, ellagic acid, that plays a critical role in the antiinflammatory activity of fraction 4 (F4) and confirmed that both F4 and ellagic acid exerted the same regulation in

INTRODUCTION A significant number of studies including in vitro, in vivo, and clinical trials have been performed to demonstrate the physiological efficacy of strawberry consumption on regulating oxidative stress,1,2 inflammation,3,4 cardiovascular diseases,5−7 obesity,4,8 diabetes,9−11 and cancer.12−16 Although there is strong evidence of the strawberries for their health-promoting activities, most of the studies were performed with strawberry powder or extracts, questioning the critical constituents playing an important role in their biological functions. To identify the active constituents, many studies have tried to fractionate through extraction with different solvents and separate them into single compounds using instrumental analysis. For example, Zhang et al. identified 10 different phenolic constituents from strawberry extract including pelargonidin, cyanidin-3-glucoside, and ellagic acid and investigated their antioxidant properties and antiproliferative activity in human oral, colon, and prostate cancer cells.17 However, it takes a great deal of time to keep subfractionating, and it is also hard to collect a significant amount of the single compound to pursue the experiments to demonstrate the physiological efficacy of the active components from complex resources. Therefore, it is necessary to employ a new approach such as metabolomics to accelerate the identification of the constituents responsible for the biological activities. Metabolomics is for nontargeted profiling and identification of the whole metabolome within an organism or system, under a given set of conditions.18 It measures all or a substantial portion of all metabolites within a sample and analyzes the whole set of metabolites according to their molecular weights.19 It is one of the fastest growing technologies and getting focus as © XXXX American Chemical Society

Special Issue: 2013 Berry Health Benefits Symposium Received: August 29, 2013 Revised: November 6, 2013 Accepted: November 7, 2013

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Figure 1. Principal component analysis for the metabolites in extracted fractions analyzed by LC-ESI-ion trap MS/MS in negative (left) and postive (right) ion modes. SF−SF6: ‘Seolhyang’ strawberry fractions extracted as described under Materials and Methods.

restoring the inhibitory κB (IκB) and inhibiting cytokine expression.

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10 min, then returned to the initial conditions (5% B) in 5 min and conditioning at 5% B over 10 min, finished at 55 min. The column temperature was 36 °C, and the UV spectra were measured between 180 and 800 nm. Acquisition was performed in the negative and positive modes by electrospray ionization (ESI) source, and the m/z range was 100−1000 Da. The capillary temperature was 275 °C, and the source voltage was set to 5 kV for ionization. Data Processing and Metabolite Identification. Peak detection, alignment, and identification were performed using SIEVE software (Thermo Fisher Scientific, Waltham, MA, USA). The MS/ MS fragmentation patterns were used for informative nontargeted metabolic profiling of LC-MS data. The acquired LC-MS/MS spectrum was identified after comparison with those proposed by the Massbank database (www.massbank.jp), KEGG database (www. kegg.co.jp), Metlin (http://masspec.scripps.edu), and related reports. Statistical Analysis. Samples were run in five replicates. All of these statistical analyses were performed using SIMCA (Umetrics, Sweden). Unsupervised multivariate statistical analysis was used to detect possible differences among groups by PCA, and the S-plot generated by OPLS-DA was applied to detect maximal separation of distinctive metabolites in each fraction. For cell proliferation assay, Western blot, and quantitative RT-PCR, the Student t test was used. Cell Culture. RAW 264.7 cells were obtained from the Korean Cell Line Bank (Seoul, Korea) and were cultured in DMEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% HEPES at 37 °C, 5% CO2. The RAW 264.7 cells were stimulated with LPS (100 ng/mL) and treated with different fractions (SF1−SF6) or ellagic acid at the concentrations as shown in the figure captions. Cell Proliferation Assay. RAW 264.7 cells were plated (1 × 104 cells/well) in a 96-well plate overnight and then treated with different concentrations of strawberry fractions (20, 50, and 100 μg/mL) for 72 h in the growth medium. Before 4 h of harvest, 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) solution (200 μL) was added to each well. The dark blue formazan crystals formed were dissolved in DMSO, and the absorbance was measured with an ELISA reader at 570 nm (Molecular Devices, Sunnyvale, CA, USA). Cell proliferation was described as the relative cell viability compared to the control. Western Blot Analysis. After RAW 264.7 cells were cultured overnight, they were treated with strawberry fractions and ellagic acid for 15 min or 24 h. The cells were washed with PBS and then harvested with radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris-HCl, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.1 mM Na3VO4, 1% phenylmethanesulfonyl fluoride, 1% protease inhibitor) to extract the cellular proteins. After

MATERIALS AND METHODS

Materials. ‘Sheolhyang’ strawberry (Fragaria × anananssa Duch.) cultivated in Asan, South Korea, was purchased in May 2012 and used for the multisolvent extraction. Elliagic acid, chlorogenic acid, pcoumaric acid, kaempferol, quercetin hydrate, quercetin 3-O-glucoside, (+)-catechin hydrate, pelargonidin 3-O-glucoside, pelargonidin 3-Orutinoside, cyanidin 3-O-glucoside chloride, and formic acid were obtained from the Sigma-Aldrich Corp. (St. Louis, MO, USA). Ethyl acetate, methanol, and acetonitrile were from DUCKSAN (Ansan, South Korea), SAMCHUN (Yeosu, South Korea), and Burdick & Jackson (Muskegon, MI, USA), respectively. Metabolite Extraction. Strawberries were cleaned immediately after harvest and freeze-dried, and dried powders were stored at −80 °C until extraction. Polyphenols in the strawberry were prepared by multisolvent extraction with ethyl acetate and methanol according to the method of Mäaẗ tä-Riihinen et al. with modification.27 Freeze-dried strawberry powders (2.5 g) were mixed with 10 mL of ethyl acetate and stirred for 30 min at room temperature, and the supernatant was collected. The supernatant was divided in two groups as fraction 1 (SF1), and the other part was washed with 0.1 M sodium acetate (pH 7.0) and distilled water and prepared as fraction 2 (SF2). Solvents in SF1 and SF2 were evaporated and resuspended with methanol for LCMS/MS analysis. The residue of ethyl acetate extraction was extracted with acidic methanol, and the supernatant was divided into two groups. Supernatant was indicated as fraction 3 (SF3), and fraction 4 (SF4) was prepared after acidic hydrolysis of fraction 3. The residue after acidic methanol extraction was further treated with acidic methanol, and the supernatant was collected as fraction 6 (SF6); then fraction 5 (SF5) was prepared after more acidic hydrolysis of the residues. LC-MS/MS Analysis. Samples were analyzed using an Accela HPLC system with a PDA detector (Accela 80 Hz PDA detector) and an LTQ-Velos ion trap mass spectrometer fitted with a heat electrospray ionization interface (Thermo Fisher Scientific, San Jose, CA, USA). Separation was carried out using an UPLC BEH C18 column (1.7 μm, 100 × 2.1 mm i.d., Acquity, Waters, Milford, MA, USA). The elution gradient was carried out with a binary solvent system consisting of 0.1% formic acid in water (phase A) and 0.1% formic acid in acetonitrile (phase B) at a constant flow rate of 0.3 mL/ min. The linear gradient profile was as follows: 5−10% B over 5 min, held at a 10% B over 5 min, 10−40% B over 20 min, 40−90% B over B

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centrifugation at 12000g for 15 min, the same amount of protein (30 μg) was loaded in 10% polyacrylamide gel and electrophoretically separated and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The primary antibodies against iNOS, COX2, IκBα, JNK (Santa Cruz Biotech, Santa Cruz, CA, USA), phospho-ERK, phospho-p38, phospho-JNK, phospho-Stat1 (Cell Signaling Technology, Boston, MA, USA), β-actin (Sigma), and secondary antibodies (Santa Cruz) were used. The expression levels of proteins were visualized with EZ capture MG (ATTO, Tokyo, Japan) and quantified with CS Analyzer (ver.3.0, ATTO). RNA Extraction and Quantitative RT-PCR Analysis. RAW 264.7 cells (7 × 105 cells/10 cm dish) were treated with strawberry fraction (SF4) and ellagic acid for 15 h. Total cellular RNA was isolated by Trizol reagent (Sigma) and reverse-transcribed to cDNA using a RevertAid First Strand cDNA Synthesis Kit (Theromo Scientific, Lafayette, CO, USA) in a 96-well format Atlas Thermal Cycler (Astec, Fukuoka, Japan). The cDNA was amplified using TaqMan Fast Advanced Master Mix (Applied Biosystems, Foster City, CA, USA) on an ABI Prism 7500 real-time PCR system (Applied Biosystems). The temperature cycling conditions used for amplification were one cycle of 50 °C for 2 min, one cycle of 95 °C for 20 s, and 40 cycles of 95 °C for 3 s and 60 °C for 30 s. Labeled TaqMan primers including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) were purchased from Applied Biosystems. The relative quantification of target mRNA was calculated using the comparative 2−ΔΔCT method after normalization with the internal control, GAPDH.

433.25), (+)-catechin hydrate (RT 2.12, m/z 291.13), 4′-Omethylglucoliquiritigenin (RT 9.27, m/z 433.18), pelargonidin 3-O-(6-O-malonyl-β-D-glucoside) (RT 14.66, m/z 519.29), and quercetin malonylhexose (RT 20.68, m/z 551.31) were mainly found in positive ion mode. Strawberry Fraction 2 (SF2). SF2 was washed SF1, and epicatechin (RT 5.42, m/z 289.69), baicalein-7-O-glucoside (RT 8.16, m/z 431.33), and proanthocyanidin B4 (RT 4.71, m/ z 577.30) were found in negative ion mode, whereas pelargonidin 3-O-glucoside (RT 7.89, m/z 433.25), (+)-catechin hydrate (RT 5.37, m/z 291.06), luteolin (RT 19.10, m/z 287.73), kaempferol (RT 20.65, m/z 287.80), and 1-O-galloylβ-D-glucose (RT 13.83, m/z 333.17) were identified under positive ion mode. Strawberry Fraction 3 (SF3). SF3, as a simple methanol extract, proanthocyanidin dimer (RT 4.71, m/z 577.30), (+)-catechin hydrate (RT 5.42, m/z 289.69), ellagitannin (RT 10.93, m/z 935.22), kaempferol coumaroyl hexose (RT 22.41, m/z 593.29), quercetin 3-O-glucuronide (RT 15.42, m/z 477.16), and p-coumaroyl glucoside (RT 5.90, m/z 325.19) were identified in negative ion mode. Pelargonidin 3-Oglucoside (RT 7.89, m/z 433.25) and pelargonidin 3-O-(6malonyl-β-D-glucoside) (RT 14.66, m/z 519.29) were found in positive ion mode. Strawberry Fraction 4 (SF4). SF4 was prepared through the acidic hydrolysis step of SF3. In negative mode of analysis, pelargonidin 3-O-glucoside (RT 6.91, m/z 431.16), delphinidin (RT 15.29, m/z 301.67), ellagic acid (RT 13.16, m/z 301.73), quercetin 3-methyl ether 7-rhamnoside (RT 3.35, m/z 461.16), and 5,2′-dihydroxy-6,7,8,6′-tetramethoxyflavone (RT 18.22, m/ z 373.27) were found, whereas pelargonidin 3-O-glucoside (RT 7.89, m/z 433.25), pelargonidin (RT 15.90, m/z 271.64), gallic acid (RT 14.17, m/z 171.32), and petunidin (RT 14.24, m/z 317.14) were identified in positive ion mode. Strawberry Fraction 5 (SF5). Delphinidin (RT 15.29, m/z 301.67), kaempferol 7,4′-dimethyl ether 3-O-sulfate (RT 26.06, m/z 393.18), bis-HHDP-glucose (RT 2.94, m/z 739.42), and ellagic acid (RT 13.16, m/z 301.73) were detected in negative ion mode, whereas pelargonidin 3-O-glucoside (RT 7.89, m/z 433.25), pelargonidin (RT 15.90, m/z 271.64), and diosmetin and (RT 13.51, m/z 301.18) were found in positive ion mode. Strawberry Fraction 6 (SF6). Ellagic acid (RT 17.302, m/z 301.66), delphinidin (RT 15.29, m/z 301.67), and cyanidin 3glucoside-7-rhamnoside (RT 24.75, m/z 593.31) were the main compounds in negative ion mode. Pelargonidin 3-O-glucoside (RT 7.89, m/z 433.25), pelargonidin (RT 12.52, m/z 271.53), diosmetin (RT 13.51, m/z 301.18), and cyanidin 3-O-(6-O-pcoumaroyl)-glucoside (RT 22.02, m/z 595.25) were identified in positive ion mode of analysis. Strawberry Fractions Exerted Anti-inflammatory Activity in RAW 264.7 Cells without Cytotoxicity. To evaluate the cytotoxic effect of strawberry, RAW 264.7 cells were treated with different concentrations (20, 50, and 100 μg/ mL) of strawberry fractions (SF1−SF6), and cell viability was determined by the MTT assay. As shown in Figure 2A, none of the strawberry fractions showed significant cytotoxicity at the concentrations tested. To investigate the regulatory activity of strawberry fractions on inflammation, we evaluated the expression level of the well-known inflammatory markers, iNOS and COX-2, after the induction LPS. The murine macrophage RAW 264.7 cells were treated with the strawberry fractions (20 μg/mL) for 24 h. Among the fractions, SF4 exerted the strongest inhibition of iNOS protein expression,

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RESULTS Metabolomic Phenotyping of Multistep Solvent Extracts from Strawberry. Through multisolvent extraction, strawberry metabolites were fractionated into six groups. Fractions 1 (SF1) and 2 (SF2) were extracted with ethyl acetate, and fractions 3−6 (SF3−SF6) were prepared by methanol extracts. The yield of each fraction from SF1 to SF6 was determined as 0.01, 2-fold of ellagic acid was recovered in the ileal fluid from volunteers with an ileostomy after raspberry consumption, indicating the role of the stomach and/or the small intestine for the release of ellagic acid from ellagitannins.46 In our study, ellagitannins in SF3 and ellagic acid in SF4 were detected because a more acidic condition was applied in SF4. These results suggest that the extraction conditions of SF4 may mimic the physiological conditions of the gastrointestinal tract in terms of the hydrolysis of ellagitannins.

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DISCUSSION The metabolomics approach is an efficient way to open a whole set of metabolites and define unique metabolomic phenotyping through various statistical analyses along with subsequent annotation of the features. Although different techniques have been used to characterize and quantify active compounds in the berry family, just simple comparison and characterization have been attempted due to the complexity of the constituents and their dependence on the extraction methods.32 Major bioactive compounds in strawberry can be classified into five groups including phenolic acids, stilbenes, flavonoids, tannins, and lignans, and different features of each group make it difficult for the multidimensional characterization of bioactive compounds in a single step of analysis. However, successful application of metabolomics on the characterization of black raspberry demonstrated that the screening of bioactive compounds from strawberry would be possible through the correlation of metabolomic data with activity evaluation in vitro.33 In this study, we prepared six different fractions (SF1−SF6) of strawberry ‘Seolhyang’ using ethyl acetate and methanol, profiled the whole metabolites with HPLC-ESI-ion trap MS/ MS (Figure 1), and compared the inhibitory effects on iNOS protein expression (Figures 2 and 3). Among them, we chose the most active fractions, SF3 and SF4, and found the statistically unique components, kaempferol 7-O-glucoside and quercetin 3-O-glucoside in SF3 and ellagic acid in SF4, using OPLS-DA (Table 1). These results indicate that ellagic acid in SF4 may act as one of the key constituents exerting health beneficial efficacy when SF4 shows more significant physiological activities than SF3. Inflammation is the systematic process of the host defense mechanism to eliminate harmful stimuli such as bacterial infection and tissue injuries, and chronic inflammation is wellknown to be associated with the development of different chronic diseases including obesity, cardiovascular diseases, diabetes, and cancer.31 The cellular stimulus of the inflammation activated Toll-like receptors (TLRs), which transduce the signal through IKK/NFκB or MAPK/AP-1 signaling pathways and enhance gene expression of pro-inflammtory cytokines, iNOS, and COX-2.31,34 Strawberry juice, phenolic extracts, and polysaccharides in strawberry were reported to show antiinflammatory effects through reducing pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, as well as enhancing the anti-inflammtory cytokine IL-10.3,35,36 However, it has not been demonstrated which active component plays a key role in regulating inflammation in those studies. To investigate the role of strawberry fractions on regulating the inflammation and identify the crucial components in the fractions, we used murine macrophage RAW 264.7 cells, which have been widely applied and suggested as a useful model for the evaluation of inflammation and immune-related kinetics.37 In LPS-induced RAW 264.7 cells, SF4 exerted a more significant effect in G

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After the finding that neither ellagitannins nor ellagic acid was detected in urine,47 many studies have supported that ellagic acid is metabolized into urolithins, especially urolithin A and urolithin B,44,46,48,49 raising the question of whether ellagic acid can reach the target organ where the macrophage exists as its original form. Although it is a critical issue to identify the actual potent phytochemicals that can act in vivo system, most metabolites produced from colonic microflora or hepatic system are not frequently present in the natural resources. To overcome this hurdle, the process of metabolism of natural active components must be elucidated and the correlation of in vitro and in vivo physiological efficacies validated. Taken together, we employed the metabolomic approach to identify the key constituent showing physiological efficacy, that is, inflammation in this study. Ellagic acid was the significantly different component in SF4, compared to SF3, and we confirmed that ellagic acid possesses similar activity with SF4 in regulating NFκB and MAPK signaling pathways, which led to the reduction of cytokines (TNF-α and IL-1β) and iNOS expressions. These results demonstrate that ellagic acid from the strawberry ‘Seolhyang’ is the major component involved in suppressing inflammation. In addition, metabolomic analysis can be applied to determining the critical ingredients having biological functions in a complicated food matrix. It is, however, necessary to keep expanding the database to cover the whole metabolites in the mixture to reduce the possibility of missing potent unknown compounds.

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ASSOCIATED CONTENT

* Supporting Information Supplementary Tables 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Authors

*(J.-H.A.) Phone: +82-31-670-3079. Fax: +82-31-675-4853. Email: [email protected]. *(H.J.L.) Phone: +82-31-670-3030. Fax: +82-31-675-4853. Email: [email protected]. Author Contributions ∥

J.L. and S.K. contributed to this work equally.

Funding

This work was carried out with the support of the Cooperative Research Program for Agriculture Science and Technology Development (PJ008550), Rural Development Administration, Republic of Korea, and by Chung-Ang University Excellent Freshman Scholarship Grants. Notes

The authors declare no competing financial interest.

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REFERENCES

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ABBREVIATIONS USED

SF, ‘Seolhyang’ strawberry fraction; TNF-α, tumor necrosis factor-α; NFκB, nucelar factor κB; IκB, inhibitory κB; iNOS, inducible nitric oxide synthase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; ILs, interleukins; ELISA, enzyme-linked immunosorbent assay; LPS, lipopolysaccharide; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; PCA, principal component analysis; OPLS-DA, orthogonal partial least-squares discriminant analysis; ESI, electrospray ionization H

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Journal of Agricultural and Food Chemistry

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dx.doi.org/10.1021/jf4038503 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX