Characterization of Phenolic Compounds and ... - ACS Publications

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Characterization of Phenolic Compounds and Antioxidant and Antiinflammatory Activities from Mamuyo (Styrax ramirezii Greenm.) Fruit Michael A. Timmers,† Jorge L. Guerrero-Medina,†,§ Debora Esposito,† Mary H. Grace,† Octavio Paredes-López,# Pedro A. García-Saucedo,§ and Mary Ann Lila*,† †

Plants for Human Health Institute, North Carolina State University, NCRC, 600 Laureate Way, Kannapolis, North Carolina 28081, United States § Universidad Michoacana de San Nicolás de Hidalgo, Uruapan, Michoacán, Mexico # Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional, Irapuato, Guanajuato, Mexico S Supporting Information *

ABSTRACT: Extracts of Styrax ramirezii Greenm., a fruit traditionally valued for health and wellness in Mexico, were analyzed phytochemically and evaluated for antioxidant and anti-inflammatory activity. Six norneolignans were identified by HPLC-TOFMS, and the two major compounds were isolated for further evaluation. The effects of the isolated norneolignans, egonol and homoegonol, on lipopolysaccharide (LPS)-induced nitric oxide (NO) production, reactive oxygen species (ROS) production, and biomarkers of inflammation were evaluated. Of the tested compounds, egonol potently inhibited the production of NO and also significantly reduced the release of ROS. Consistent with these observations, the mRNA expression levels of inducible nitric oxide synthase (iNOS) (0.668 ± 0.108), cyclooxygenase-2 (COX-2) (0.553 ± 0.007), interleukin-1β (IL-1β) (0.093 ± 0.005), and interleukin-6 (IL-6) (0.298 ± 0.076) were reduced by egonol. The activity for both egonol and homoegonol increased in a concentration-dependent manner. These results suggest the potential of S. ramirezii Greenm. fruit to contribute to a healthy diet, rich in antioxidant and anti-inflammatory compounds. KEYWORDS: LC-TOF-MS, Styrax ramirezii, antioxidant activity, anti-inflammatory activity, norneolignan

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INTRODUCTION The association between fruit consumption and the reduced risk of chronic diseases such as cancer, diabetes, cardiovascular disease, and arthritis has been well established.1,2 Fruits and vegetables are a major source of antioxidants, mainly due to a high polyphenolic content. Berries are a particularly valuable source of polyphenolics such as tannins, stilbenes, flavonoids, and phenolic acids. These phytoactive classes have been studied extensively to determine the molecular level mechanisms of action.3 There is an ever-increasing interest in the discovery of different, novel fruits that may possess unique polyphenolic profiles.4−7 The genus Styrax (Styracaceae) comprises 130 species of trees and shrubs widespread in Asian, American, and the Mediterranean regions.8,9 Some species, such as Styrax japonica, have primarily been ornamental inclusions within gardens,10 whereas in Asia and South America Styrax has been used in traditional medicines for treatment of fever, inflammation, and stomach diseases.11 Previous studies on Styrax extracts have identified classes of compounds such as sterols, saponins, terpenoids, and benzofuran derivatives.11−16 These extracts have been identified as a means to control plant and animal diseases and to inhibit human carcinogenesis.17,18 In Mexico, edible fruits of the endemic species Styrax ramirezii Greenm., known locally as “mamuyo”, are of interest because of their many functional properties. At full ripeness, the small edible drupes have a sweet taste and develop an intense © XXXX American Chemical Society

purple color, which, when extracted, has been used as a natural colorant.8,9 Current research on S. ramirezii is limited, and the composition and bioactivity of these fruits have not been characterized. To gain further insight into the activity of S. ramirezii, fruit extracts were analyzed to establish phytochemical characterization as well as antioxidant and anti-inflammatory activities.

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MATERIALS AND METHODS

Chemicals. All solvents used were of HPLC grade purchased from Fisher Scientific (Pittsburgh, PA, USA) unless specified. HPLC-MS grade solvents acetonitrile, water, and 0.1% formic acid in water (Honeywell B&J) were purchased from VWR International (Suwanee, GA, USA). Folin−Ciocalteu (F−C) reagent, 4-dimethylaminocinnamaldehyde (DMAC), gallic acid, hydrochloric acid, potassium chloride, sodium acetate, sodium hydroxide, sodium nitrite, aluminum chloride, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 6-hydroxy2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), as well as the analytical standards catechin, epicatechin, and chlorogenic acid, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cyanidin3-O-β-glucoside and procyanidins A2, B1, and B2 were purchased from ChromaDex Inc. (Irvine, CA, USA). The mouse macrophage cell line RAW 264.7 (ATCC TIB-71, obtained from American Type Culture Collection, Livingstone, MT, USA) was maintained in Received: August 15, 2015 Revised: November 16, 2015 Accepted: November 17, 2015

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DOI: 10.1021/acs.jafc.5b04781 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

0.25, and 0.5 mg/mL were used to form a standard curve, and the value determined from the extract was expressed as total anthocyanin content. Total flavonoid content was measured using a modification of the methodology reported by Kim et al.22 Briefly, 0.6 mL of crude extract was diluted with 2.4 mL of 50% methanol to make three aliquots of 1 mL. Each aliquot or standard (dilutions of 20, 40, 60, 80, and 100 mg of catechin/L) was mixed with 4 mL of water in a 10 mL volumetric flask. At 0 min, 0.3 mL of 5% sodium nitrite (NaNO2) solution was added. After 5 min, 0.3 mL of 10% aluminum chloride (AlCl3) was added. One minute later, 2 mL of 1 M sodium hydroxide solution was added, and immediately the mix was diluted to the marked volume, and the absorbance was measured in triplicate at 510 nm. Samples were then compared to the standard curve of catechin as milligrams per gram of DW catechin equivalents. HPLC-TOF-MS Analysis. HPLC-TOF-MS analyses of the extracts were conducted on an Agilent Eclipse XDB-C18 column (3.0 × 250 mm, 5 μm) (Agilent) using a gradient method (0−2 min, 10% CH3CN/H2O + 0.1% formic acid; 14−24 min, 75%; 26−30 min, 100%; 32−40 min, 10%). TOF parameters included a drying gas of 10 L/min, nebulizer pressure of 45 psi, capillary voltage of 3.5 kV, fragmentor voltage of 80 V, and a mass range of m/z 200−1000. Antioxidant Activity. Antioxidant capacity (DPPH assay) was measured as micromolar 6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid (Trolox) equivalent per gram of dry weight (μM TE/g DW) using the methodology reported by Truong et al.23 Aliquots of the sample extracts, in triplicate, were mixed with a 0.08 M solution of DPPH in 80% methanol, the absorbance was measured at 515 nm after 3 h in the dark, and results were obtained with a standard curve made with dilutions of 100, 200, 300, 400, and 500 μmol of Trolox diluted in 95% ethanol. Cell Viability Assay. RAW 264.7 cells were seeded in a 96-well plate (5 × 104 cells/well) incubated overnight at 37 °C for the viability assay. Cells were charged with 180 μL of treatment medium (DMEM supplemented with 100 IU/mL penicillin/100 μg/mL streptomycin and 10% fetal bovine serum). Subsequently, 20 μL of MTT reagent ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] at 5 mg/mL) was added, and the mixture was incubated for 4 h at 37 °C. Cell viability was measured after 4 h of exposure by the MTT assay in triplicate and quantified spectrophotometrically at 550 nm using a microplate reader SynergyH1 (BioTek, Winooski, VT, USA).24 The concentrations of test reagents that showed no changes in cell viability compared with that vehicle (ethanol at a final concentration of 0.1%) were selected for further studies. Reactive Oxygen Species (ROS) Assay. For determining in vitro ROS, a fluorescent dye protocol was utilized.25 RAW 264.7 macrophage cells were seeded at a concentration of 5 × 105 cells/ well) into a 24-well plate and incubated overnight at 37 °C. Cells were charged with 500 μL of 50 μM 2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (H2DCFDA) (Molecular Probes, Eugene, OR, USA), prepared fresh daily in sterile phosphate-buffered saline (PBS) for 30 min. Fluorescent medium was aspirated, and cells were exposed to 25 μL of extract/fraction (100 μg/mL final concentration) and 1 μL of lipopolysaccharide (LPS; from Escherichia coli 026:B6) and incubated for 24 h, after which the fluorescence of 2′,7′dichlorofluorescein (DCF) was measured at 485 nm (excitation) and 515 nm (emission) on a microplate reader. The known antioxidant dexamethasone (DEX) was used as a positive control. The experiments were performed with three independent replications, each replication assayed at least in duplicate. Nitric Oxide Radical Inhibition Assay. The abilities of test samples to inhibit nitric oxide radical formation were determined according to the procedure of Oliveira et al.26 Cells were seeded in 96well plates (4 × 105 cells/well) 24 h prior to treatment. Next, 100 μL of 20 mM sodium nitroprusside was incubated with 100 μL of sample (prepared at 1 mg/mL in 80% methanol) for 60 min at room temperature, under light. Subsequently, 100 μL of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine in 2% phosphoric acid) was added, and the mixture was incubated at room temperature for 10 min and then read on a microplate reader at 562 nm.

Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Carlsbad, CA, USA), supplemented with 100 IU/mL penicillin/100 μg/mL streptomycin (Fisher) and 10% fetal bovine serum (Life Technologies) at a density not exceeding 5 × 105 cells/mL and maintained at 37 °C in a humidified incubator with 5% CO2. General Experimental. All HPLC analyses were conducted using a 1200 series HPLC (Agilent Technologies, Santa Clara, CA, USA), equipped with a DAD detector. HPLC-TOF-ESI-MS analysis was performed using an Agilent 6220a TOF-MS, equipped with a 1200 series HPLC (Agilent Technologies). Plant Material. S. ramirezii Greenm. is a 5−15 m tall evergreen tree that grows in the forests of midwestern Mexico, which produces edible drupes that have an ellipsoid shape approximately 8−13 mm long and 4−9 mm in diameter, with one seed approximately 27% of the total weight of fruit.9 Fruits were collected in March 2012 in Michoacán, Mexico (19° 36′ 21.16” N, 100° 38′ 40.96” W; 2570 m) and freeze-dried. The seeds were then separated from flesh, and the flesh and skins were ground to a powder and stored at −60 °C until analysis. Fruit contained approximately 82.6 ± 1.2% water prior to lyophilization. Plant identification was conducted by Dr. Sergio Zamudio Ruiz from the Instituto de Ecologı ́a A.C. in Pátzcuaro, Michoacán, Mexico; a voucher sample of the plant is deposited in the Instituto de Ecologı ́a Herbarium under code Col 32. Extraction and Polyphenol Analyses. For qualitative and isolation purposes, lyophilized fruit (30 g) was extracted with acidified methanol (0.1% acetic acid) (800 mL). This extracted material was reextracted with acidified methanol (800 mL) and combined with the first extract for complete extraction of material. A portion of the acidified methanol extract (10 g) was sequentially partitioned via liquid−liquid extraction into petroleum spirits, dichloromethane, ethyl acetate, and methanol fractions. Isolation of norneolignans from the petroleum spirits fraction was conducted using a Grace (Columbia, MD, USA) Reveleris preparative purification system in preparative mode, equipped with a DAD and ELSD for detection, and automatic fraction collection. The semipreparative column used was an Alltech Econosil C18 column (10 × 250 mm, 10 μm) (Grace), using an isocratic method (60% CH3CN/H2O) at a flow rate of 5 mL/min and detection at 311 nm. For quantitative analysis, 5 mg of the lyophilized powdered fruit was mixed with 1 mL of 50% methanol, stirred for 3 min in a vortex at 845g, and then placed in a sonic bath for 15 min before centrifugation at 1503g for 10 min. The supernatant (crude extract) was collected and filtered using a 2 μm Teflon (PTFE) filter, and the filtered crude extract was stored at −20 °C until analysis. Total phenolic concentration was measured as milligrams of gallic acid equivalents per gram of dry weight (mg GAE/g DW) using the methodology reported by Singleton, Orthofer, and LamuelaRaventós.19 Aliquots of crude extract (0.5 mL) were diluted with water (1:8), 0.5 mL of F− reagent was added and mixed, and 5 mL of sodium carbonate solution (0.5 M) and 2.5 mL of water were added and mixed. Solutions were incubated for 2 h in the dark. Absorbance was measured at 765 nm in a Shimadzu UV−visible spectrophotometer (Tokyo, Japan). Values were obtained with a gallic acid standard curve made with dilutions of 50, 100, 250, and 500 mg/L. Total proanthocyanidin (PAC) content was measured colorimetrically in the crude extract as milligrams of procyanidin A2 equiv per gram of dry weight (mg/g DW) using the DMAC method in a 96-well plate as previously described by Wallace and Giusti.20 A series of dilutions of standard procyanidin A2 were prepared in 80% ethanol. Aliquots of crude extract, blank, and standards (63 μL) were mixed with DMAC solution reagent (189 μL). The plate reader protocol was set to read the absorbance (640 nm) of each well in the plate every minute for 30 min (SpectraMax M3, Sunnyvale, CA, USA). Absorbance was compared with a standard curve of procyanidin A2 standards at 3.125, 6.25, 12.5, 25, 50, or 100 μg/L. Quantitative determination of anthocyanin content was performed using the HPLC system described in the previous section with an RPSupelcosil-C18 column, (4.6 × 250 mm, 5 μm) (Supelco, Bellefonte, PA, USA) and the method as described in Grace et al.21 Cyanidin-3-Oβ-glucoside standards at concentrations of 0.03125, 0.0625, 0.125, B


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Figure 1. Compounds identified from Styrax ramirezii Greenm. Absorbances were compared against a calibration curve created with serial dilutions of sodium nitrite (R2 = 0.998). Biomarkers of Inflammation by Gene Expression Analysis. Cells were subcultured by TrypLE (Life Technologies) when dishes reached to 90% confluence with a 1:5 ratio in fresh medium. Cells were seeded in 24-well plates (5 × 105 cells/well) 24 h prior to treatment. The cells were then treated with berry extracts at predetermined doses for 1 h before elicitation with LPS at 1 μg/mL for an additional 4 h. For every experiment, one positive control (cells treated with DEX, at 3.92 μg/mL) and one negative control (cells treatment with vehicle) were included. Three replicates were made for both the treatments and the controls. At the end of the treatment period, cells were harvested in TRIzol reagent (Life Technologies) for subsequent cellular RNA extraction. Total RNA Extraction, Purification, and cDNA Synthesis. The total RNA was isolated from RAW macrophages using TRIzol reagent (Life Technologies) following the manufacturer’s instructions. RNA was quantified spectrophotometrically using the SynergyH1/Take 3 (BioTek). The cDNAs were synthesized using 2 μg of RNA for each sample using a commercially available high-capacity cDNA reverse transcription kit (Life Technologies), following the manufacturer’s protocol on an ABI GeneAMP 9700 (Life Technologies). Quantitative PCR Analysis. The resulting cDNA was amplified by real-time quantitative PCR using SYBR green PCR Master Mix (Life Technologies). To avoid interference due to genomic DNA contamination, only intron-overlapping primers were selected using Primer Express version 2.0 software (Applied Biosystems, Foster City, CA, USA) as follows: β-actin, forward primer 5′-AAC CGT GAA AAG ATG ACC CAG AT-3′, reverse primer 5′-CAC AGC CTG GAT GGC TAC GT-3′; COX2, forward primer 5′-TGG TGC CTG GTC TGA TGA TG-3′, reverse primer 5′-GTG GTA ACC GCT CAG GTG TTG-3′; iNOS, forward primer 5′-CCC TCC TGA TCT TGT GTT GGA-3′, reverse primer 5′-TCA ACC CGA GCT CCT GGA A3′; IL-6, forward primer 5′-TAG TCC TTC CTA CCC CAA TTT CC-3′, reverse primer 5′-TTG GTC CTT AGC CAC TCC TTC-3′; and IL-1β, forward primer 5′-CAA CCA ACA AGT GAT ATT CTC CAT G-3′, reverse primer 5′-GAT CCA CAC TCT CCA GCT GCA-

3′. Quantitative PCR (qPCR) amplifications were performed on an ABI 7500 Fast Real Time PCR (Life Technologies) using 1 cycle at 50 °C for 2 min and 1 cycle of 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The dissociation curve was completed with 1 cycle of 1 min at 95 °C, 30 s at 55 °C, and 30 s at 95 °C. mRNA expression was analyzed using the ΔΔCT method and normalized with respect to the expression of the β-actin housekeeping genes using 7500 Fast System SDS software v1.3.0 (Life Technologies). A value of 1.0 imply overexpression of the particular gene in excess of LPS stimulation. Amplification of specific transcripts was further confirmed by obtaining melting curve profiles. All samples were run in duplicate. Significance was set at p < 0.05; (∗) significant compared with LPS. Values are reported as means. Statistical Analysis. Statistical analyses were performed using Prism 4.0 (GraphPad Software, San Diego, CA, USA). Data were analyzed by one-way ANOVA with treatment as a factor. Post hoc analyses of differences between individual experimental groups were made using Dunnett’s multiple-comparison tests.

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RESULTS AND DISCUSSION Phytochemical Analysis. A portion of the petroleum spirits extract (5 mg) was diluted in methanol and subjected to HPLC-TOF-MS analysis as described previously. The major component eluted at 15.31 min and displayed an m/z of 343.1600 [M + H]+ (calcd for C20H23O5, m/z 343.1545), which, based on extracted UV maxima of 312 nm and a literature search for compounds from this family of plants, was identified as the norneolignan, homoegonol (1) (Figure 1).27,28 The second major norneolignan eluted at 16.36 min and displayed an m/z of 327.1255 [M + H]+ (C19H19O5, m/z 327.1232) and on the basis of extracted UV maxima of 316 nm, C


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Figure 2. Effects of homoegonol (1) and egonol (2) on cell viability of murine RAW 264.7 macrophage cells. Cells were treated with test agents at 5−100 μg/mL concentration for 1 h, stimulated with LPS (1 μg/mL), and incubated for 24 h. Cell viability was quantified spectrophotometrically by the MTT assay. Vehicle values were obtained in the absence of LPS or test samples. Dexamethasone (DEX) was used as positive control at a concentration of 3.92 μg/mL. All samples were assayed in triplicate. Results are expressed as means ± SE, n = 2 experiments. (∗) p < 0.05 versus the LPS-treated group.

this compound was identified as egonol (2) (Figure 1). Four related norneolignans (3−6) and the lignin styraxin (7) were also identified on the basis of the mass and UV maxima (Figure 1). Norneolignans are benzofuran derivatives that have exhibited a wide range of biological activity, including cytotoxicity and acetylcholinesterase inhibition, as well as antibacterial, antifungal, anti-inflammatory, and antischistosomal activities.11,17,29−35 From the crude acidified methanol extract, four anthocyanins were identified as the glucosides and sambubiosides of cyanidin and delphinidin (8−11) on the basis of the mass and UV maxima (Figure 1). These four anthocyanins are common in other health-relevant functional fruits such as the maqui berry (Aristotelia chilensis),36 acai (Euterpe oleracea Mart.),37 blueberry (Vaccinium spp.),38 raspberry (Rubus spp.),39 and blackberry (Rubus spp.).40 Schreckinger et al. found that extracts from A. chilensis containing cyanidin sambubioside (9), delphinidin glucoside (10), and delphinidin sambubioside (11) have shown the ability to inhibit α-glucosidase and α-amylase, and it was determined that the concentrations of those anthocyanins in the extract ameliorated diabetes symptoms.4 Xie et al. demonstrated that an analytical standard of delphinidin-3glucoside (10) had the ability to neutralize effects of oxidative stress and apoptosis in endothelial cells for prevention of atherosclerotic lesions.41 Cyanidin-3-glucoside (8) has been one of the most highly studied anthocyanins, having exhibited anticancer,42 antiobesity, and antidiabetic activities.43 Cyanidin sambubioside (9) from peanut testae inhibited nitric oxide production associated with inflammatory problems.44 The two major norneolignans (1, 2) (6.1 and 5.2 mg, respectively) were isolated at approximately 85% purity from the petroleum spirits extract (50 mg) using the semipreparative HPLC method as described previously, with retention times of 10.5 min for homoegonol (1) and 13.0 min for egonol (2), and utilized for antioxidant and anti-inflammatory activity analyses. Total Phenolic Content. The total phenolic content of the fruit from S. ramirezii was 17.8 mg/g DW as gallic acid

Figure 3. Effects of homoegonol (1) and egonol (2) on LPS-induced radical oxygen species (ROS) production (A) and nitric oxide (NO) production (B) in RAW 264.7 macrophage cells. Cells were treated with test agents at 50 μg/mL concentration for 1 h, stimulated with LPS (1 μg/mL), and incubated for 24 h. Vehicle values were obtained in the absence of LPS or test samples. Dexamethasone (DEX) was used as positive control at a concentration of 3.92 μg/mL. All samples were assayed in triplicate. Results are expressed as means ± SE, n = 2 experiments. (∗) p < 0.05 versus the LPS-treated group.

equivalents (GAE). This value is similar to that obtained from commonly eaten fruits such as blackberries (20.55 mg/g GAE),3 red raspberries (13.46 mg/g GAE), and strawberries (10.33 mg/g GAE).45 Proanthocyanidins (4.80 mg/g DW) were the most prominent major polyphenolic group detected in the fruits, followed by anthocyanins (1.22 mg/g DW) and other flavonoids (1.12 mg/g DW). Effect of Homoegonol (1) and Egonol (2) on Cell Viability. The cell viability results of RAW macrophages is shown in Figure 2. Compounds 1 and 2 were evaluated at 5, 10, 50, and 100 μg/mL, in which none of the doses reduced cell viability below 85% of the control and, thus, were not considered cytotoxic at concentrations up to 100 μg/mL. Antioxidant Activity by Chemical Assay. Antioxidant activity of the crude extract (114.6 mg TE/g DW) was comparable to that of antioxidant-rich fruits such as the blueberry species Vaccinium corymbosum and Vaccinium ashei (107.2 and 136.2 μM TE/g DW, respectively).46 Some literature has shown a relationship between antioxidant activity and total phenolic content, including anthocyanins, as well as suggestions that the polyphenolic compounds interact to contribute to antioxidant potential.45,46 In Vitro ROS and NO Inhibition. Cellular antioxidant activities of compounds 1 and 2 were determined using an in vitro system to gauge reduction of radical-mediated oxidation in D


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Figure 4. Anti-inflammatory bioactivity of homoegonol (1) and egonol (2). Effects on the expression of the inflammatory biomarkers genes in the LPS-stimulated RAW264.7 macrophages. Genes involved in the acute-phase response, inflammatory response, and humoral immune responses are represented: IL-1β assay (A), IL-6 assay (B) COX-2 assay (C), and iNOS assay (D). Macrophages were pretreated with 5 or 50 μg/mL of individual compounds as specified, and inflammatory response was induced with 1 μg/mL LPS for 6 h. Changes in gene expression were measured by comparing mRNA quantity relative to LPS. A value of

Characterization of Phenolic Compounds and ... - ACS Publications

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