Elucidation of the Chemical Structure and Determination of the

Jan 21, 2015 - Xiu-Min Chen , Yue Dai , and David D. Kitts. Journal of Agricultural and Food Chemistry 2016 64 (47), 9072-9077. Abstract | Full Text H...
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Elucidation of the Chemical Structure and Determination of the Production Conditions for a Bioactive Maillard Reaction Product, [5(5,6-Dihydro‑4H‑pyridin-3-ylidenemethyl)furan-2-yl]methanol, Isolated from a Glucose−Lysine Heated Mixture Xiu-Min Chen,† Gang Chen,‡ Hongwen Chen,‡ Yilin Zhang,‡ and David D. Kitts*,† †

Food, Nutrition, and Health, Faculty of Land and Food Systems, University of British Columbia, 2205 East Mall, Vancouver, British Columbia V6T 1Z4, Canada ‡ Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia V5A 1S6, Canada ABSTRACT: We previously isolated a bioactive molecule, named F3-A, from an aqueous glucose (Glc) and lysine (Lys) Maillard reaction (MR) model system. Herein, F3-A was verified as [5-(5,6-dihydro-4H-pyridin-3-ylidenemethyl)furan-2yl]methanol (5) and was subsequently synthesized for confirmation of bioactivity. Using Taguchi and factorial designs, we determined that the conditions which best increased the yield of F3-A were at pH 6 with a sugar:amino acid ratio of 2:1 and heating time of 12 h at 100 °C. The MR mixtures containing glucose produced highest yield, compared to fructose, lactose, and sucrose. Both the F3-A recovered from Glc-Lys MR mixture and the synthesized product exhibited significant (P < 0.05), dose dependent, nitric oxide (NO) inhibitory activity in Caco-2 cells that was comparable to aminoguanidine (AG) and pyrrolidine dithiocarbamate (PDTC), respectively. Finally, an additional inhibitory effect of F3-A was determined when coincubated with AG in cytokine-induced Caco-2 cells. This bioactivity points to a potential role in preventing intestinal inflammation. KEYWORDS: Maillard reaction, glucose, lysine, nitric oxide, anti-inflammation

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INTRODUCTION A typical western diet comprised of baked bread, roasted meat, and coffee is a source of Maillard reaction (MR) products (MRPs), generated when reducing sugars react with amino acids, peptides, or proteins during a thermal process. The MR not only influences food sensory perception but also may impact specific value-added health benefits of some food systems.1 Although many studies have reported pro-inflammatory activities of MRPs, there are other studies which have indicated that MRPs exhibit anti-inflammatory capacity. For example, 3-methyl-1,2-cyclopentanedione (3-MCP), a MRP present in roasted coffee, effectively decreases pro-inflammatory gene expression, such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression and inactivated nuclear factor-kappa B (NF-κB).2 More recently, Jin et al.3 found that 2,4-bis(p-hydroxyphenyl)-2-butenal, a MRP isolated from fructose-tyrosine MR model, inhibited iNOS and COX-2 expression as well as activation of astrocytes and microglial cells in an Alzheimer’s disease mouse model. Our previous studies were the first to show that a fraction of MRPs comprised of three small molecular weight compounds isolated from glucose-lysine (Glc-Lys) MR model possessed nitric oxide (NO) inhibitory activity in differentiated Caco-2 cells, which were induced by interferon-gamma (IFN-γ) and phorbol 12-myristate 13-acetate (PMA) cocktail.4,5 Two of these compounds were identified as 5-hydroxymethyl-2-furfural (HMF) and 5-hydroxymethyl-2-furoic acid (HMFA).6 The chemical structure of the most bioactive compound, referred to as F3-A, was not elucidated at that time. Herein, we report results of a series of experiments designed to characterize the chemical structure of F3-A and also to chemically synthesize © 2015 American Chemical Society

this compound for use in verifying its anti-inflammatory bioactivity. Moreover, we also optimized the reaction conditions for F3-A production in Glc-Lys mixtures using Taguchi and factorial designs. It is well-known that excessive and prolonged nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) can be an underlying cause for many inflammatory conditions that result in increased mucosal permeability, the consequence of which can trigger inflammatory bowel diseases (IBD).7,8 IBD, such as Crohn’s disease and ulcerative colitis, affect 0.5−1% of the population in Western countries.9 The precise etiology of IBD is presently not clear; however, growing experimental and clinical evidence show that excessive NO synthesis from upregulated iNOS has an important role in the development of intestinal inflammation and in the pathogenesis of IBD.10 The human colon adenocarcinoma cell line, Caco-2, has been used extensively to study intestinal inflammation, and we have used this cell model to show that NO levels can be increased when cells are treated with a mixture of IFN-γ and PMA.11 The purpose of our study was to examine the NO inhibitory effects of the isolated and synthesized F3-A on inflamed differentiated Caco-2 cells and to compare activities with two known NO inhibitors, including a selective iNOS inhibitor, aminoguanidine (AG), and pyrrolidine dithiocarbamate (PDTC), a known inhibitor of nuclear factor-kappa B (NF-κB). Received: Revised: Accepted: Published: 1739

November 24, 2014 January 20, 2015 January 21, 2015 January 21, 2015 DOI: 10.1021/jf505579m J. Agric. Food Chem. 2015, 63, 1739−1746

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Journal of Agricultural and Food Chemistry Scheme 1. Synthesis of F3-A

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mixture was heated in a warm water bath (∼50 °C) for 2 h and then cooled in an ice/water bath overnight. The mixture was diluted with water to dissolve solids, and the aqueous layer was then extracted twice with MTBE (100 mL). The combined organic phases were dried with Na2SO4 and concentrated by rotary evaporation to yield an oily, pale yellow residue, 1,2-dehydropiperidine (dHP, 3, ∼4 g). This crude material was stored in a −20 °C freezer and used in the next step without further purification. [5-(5,6-Dihydro-4H-pyridin-3-ylidenemethyl)-furan-2-yl]methanol (5). HMF (4, 160 mg, 1.3 mmol) was dissolved in MeOH (500 μL) in a BioTage 0.5−2 mL microwave vial, and dHP (3, 300 mg, 3.6 mmol) was added. The vessel was sealed and irradiated using microwave set at 100 °C for 5 min. In a separate reaction, HMF (4, 260 mg, 2.1 mmol) and dHP (3, 500 mg, 6.0 mmol) were reacted in the same manner. The two runs were combined and purified by dryloading flash chromatography on a SiliCycle SiliaSep-HP 12 g cartridge performed on a BioTae Isolera instrument using a 2−20% MeOH/ DCM linear gradient as the eluent. Fractions with significant 370 nm absorption were pooled and concentrated to thick oil, which solidified upon standing. Trituration in MTBE (∼2 mL) gave a crystalline powder that was filtered and washed with MTBE (total ∼10 mL in 3 portions). Drying on high vacuum at room temperature gave F3-A (5) as light brown powder, 186 mg (yield 29.2%). The synthesized F3-A was named F3-AS. 1H NMR (500 MHz, DMSO) δ: 7.92 (s, 1 H), 6.62 (d, J = 3.3 Hz, 1 H), 6.53 (s, 1 H), 6.43 (d, J = 3.3 Hz, 1 H), 5.31 (s, 1 H), 4.42 (s, 2 H), 3.54 (td, J = 5.6, 1.8 Hz, 2 H), 3.33 (s, 4 H), 2.66 (td, J = 6.8, 1.9 Hz, 2 H), 1.74−1.49 (m, 2 H) ppm. 13C NMR (126 MHz, DMSO) δ: 161.85, 156.97, 150.97, 128.29, 122.29, 114.23, 109.47, 55.77, 48.67, 24.28, 20.78 ppm. Determination of Optimal Reaction Conditions for F3-A Production Using Taguchi Orthogonal Design. A four-factor /four-level (L16 (44)) Taguchi orthogonal array (Table 1) was chosen

MATERIALS AND METHODS

Preparation and Separation of [5-(5,6-Dihydro-4H-pyridin3-ylidenemethyl)furan-2-yl]methanol (F3-A). We repeated our original MR protocol to recover F3-A from a heated aqueous Glc-Lys MR mixture.4 Briefly, D-glucose (0.8 mol/L) was mixed with L-lysine monohydrochlide (0.8 mol/L) in 100 mL of ddH2O and adjusted to pH 7. The mixtures were autoclaved (Laboratory VMP, Cliton, NJ) at 121 °C for 60 min in screw-capped glass bottles and then rapidly chilled on ice and centrifuged at 3400×g. In a preliminary experiment to increase the yield of F3-A on extraction, MR mixtures were adjusted to pH 2, 6, and 12, respectively, and then extracted with chloroform. We found that F3-A was recovered in the chloroform fraction at a higher amount when the pH was adjusted to 12. Therefore, in the following experiment, the supernatant was adjusted to pH 12 using sodium hydroxide, followed by extraction with chloroform, three times using a 2:1 (v/v) ratio. Pooled chloroform fractions were collected and evaporated under reduced pressure using a rotary evaporator. The concentrated sample (around 1 g) was redissolved in 10 mL of dichloromethane (DCM) and loaded onto a glass column (3 × 25 cm) packed with 50 g of silica gel (32−63 μm, 230−400 mesh, Selecto Scientific, Georgia). The column was sequentially eluted with 200 mL of DCM, 90% DCM/10% MeOH, 80% DCM/20% MeOH, 70% DCM/30% MeOH, and 60% DCM/40% MeOH, respectively, using flash chromatography. Eluents (20 mL/per tube) were collected, and the presence of F3-A in the eluent was detected using both analytical thin layer chromatography (TLC) and HPLC. TLC was performed using aluminum plates precoated with silica gel 60F-254 (EMD Millipore Corporation, Billerica, MA). Plates were developed in DCM:MeOH (8:2, v/v) mixture and air-dried. F3-A with a retention factor (Rf) of 0.53 on the plate was visualized using a KMnO4 staining solution, followed by air drying. The eluent containing F3-A collected from the silica column was combined and evaporated under vacuum. The concentrated crude extract was reloaded onto a newly prepared silica gel column and eluted with 100 mL of dichloromethane, 200 mL of 90% DCM/10% MeOH, 300 mL of 80% DCM/20% MeOH, and 100 mL of 70% DCM/30% MeOH, respectively. TLC analysis identified tube 19 containing F3-A which was used for NMR and highresolution mass spectrometry analysis. This fraction was named F3AC19. Purity of F3-A in the F3-AC19 fraction was greater than 95%, as determined by NMR. Synthesis of [5-(5,6-Dihydro-4H-pyridin-3-ylidenemethyl)furan-2-yl]methanol (F3-A). Scheme 1 shows the procedures to synthesize intermediate compounds and F3-A. N-Chloropiperidine (2). In a 500 mL, 3-neck round-bottom flask was added methyl tert-butyl ether (MTBE, 160 mL), piperidine (5 mL, 50 mmol), and t-BuOH (2.4 mL, 25 mmol). The solution was cooled to 0 °C in an ice/water bath. Bleach (active ingredient NaOCl, nominal 5% active chlorine, 0.5 M, 110 mL) and acetic acid (3.2 mL, diluted with 3.2 mL of water) were added simultaneously from two dropping funnels, at a rate that maintained internal temperature below 10 °C. The mixture was stirred at 0 °C for 1 h, and the organic phase was separated leaving the aqueous phase which was extracted with MTBE (100 mL). The combined organic fractions were dried with Na2SO4 and filtered. This solution was used in the next step without isolation or purification. 1,2-Dehydropiperidine (3). To the above filtrate kept at 0 °C in an ice/water bath was added solid t-BuOK (11.2 g, 100 mmol) in one batch, followed by MeOH (80 mL, in 10−20 mL portions). The

Table 1. Taguchi Orthogonal Array and F3-A and HMF Yielda exp. no.

S:AA ratio

pH

T (°C)

t (min)

F3-A (μg/g)

HMF (μg/g)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

0.5:1 0.5:1 0.5:1 0.5:1 1:1 1:1 1:1 1:1 2:1 2:1 2:1 2:1 3:1 3:1 3:1 3:1

5 6 7 8 5 6 7 8 5 6 7 8 5 6 7 8

70 80 90 100 80 70 100 90 90 100 70 80 100 90 80 70

30 60 90 120 90 120 30 60 120 90 60 30 60 30 120 90

NDb ND 0.3 ± 0.0 ND ND ND ND 0.9 ± 0.3 5.7 ± 2.5 11.1 ± 3.3 ND ND 0.7 ± 0.3 ND 0.9 ± 0.2 ND

ND ND 3.1 ± 1.8 ND ND ND 0.7 ± 0.2 8.5 ± 2.3 9.1 ± 3.9 4.9 ± 1.6 0.4 ± 0.1 ND 2.5 ± 0.9 0.1 ± 0.0 4.1 ± 0.8 1.8 ± 0.5

a

S:AA ratio, sugar:amino acid ratio; T, temperature (°C); t, heating time (min). bND, not detectable.

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Journal of Agricultural and Food Chemistry to investigate the effects of four different reaction conditions including sugar:amino acid ratio, pH, heating temperature, and heating time on the yield of F3-A in a Glc-Lys MR mixture. The experiments were performed in triplicate over three concessive days. Glucose was mixed with L-lysine (0.8 mol/L) at sugar to amino acid ratios (mole ratio) of 0.5:1, 1:1, 2:1, or 3:1 in ddH2O, and pH was adjusted to 5, 6, 7, or 8, respectively, using concentrated HCl. The mixtures (100 mL) were put in capped glass bottles and heated in an oven (Blue M electronic company, Blue Island, IL) at 70, 80, 90, or 100 °C, for 30, 60, 90, or 120 min time periods, respectively, after which they were rapidly chilled on ice. An aliquot (15 mL) of sample was stored at −20 °C for further analysis, and the remainder was freeze-dried. Effects of Reactants and Concentration and Heating Time on the Yield of F3-A. According to the optimal conditions from Taguchi design, glucose was mixed with L-lysine at sugar to amino acid (S:AA) mole ratios of 0.2:0.1, 0.4:0.2, 0.8:0.4, or 1.6:0.8 in distilled deionized water, and the pH was adjusted to 6 using 1 N HCl. The mixtures were heated at 100 °C for 1.5, 3, 6, 9, 12, and 24 h, respectively, in an oven (Blue M electronic company, Blue Island, IL) and then rapidly chilled on ice. The yield of F3-A was measured using HPLC, and F3-AS was used as standard. To determine if specific sugars produced relatively greater F3-A, we also compared yield derived from fructose, glucose, sucrose, and lactose (1.6 mol/L), respectively, mixed with lysine (0.8 mol/L) at an adjusted pH 6. The mixtures were heated at 100 °C for 4 h, and the yield of F3-A was measured using HPLC. HPLC Analysis. F3-A was detected by analytical reverse phase HPLC using an Agilent 1100 LC system (Agilent, Santa Clara, CA) equipped with a Zorbax SB-C18 column (4.6 × 150 mm, 3.5 μm, Agilent, U.S.A.) and monitored with a diode array detector (DAD) at 370 and 385 nm. A linear gradient comprised of 5−20% MeOH/0.2% formic acid delivered at a flow rate of 1 mL/min for 6 min was used to separate the sample components. The column temperature was set at 30 °C. FTIR and UV−Vis Spectrophotometry Analysis. F3-AC19 and F3-AS were dissolved in methanol, and Fourier transform infrared spectroscopy (FTIR) spectrum on both samples was measured by PerkinElmer Spectrum 100 FTIR spectrometer with a horizontal attenuated total reflectance (HATR) sampling accessory. For ultraviolet−visible (UV−vis) spectrophotometry, F3-AC19 and F3-AS were dissolved in MeOH or 0.002% formic acid/ddH2O at a concentration of 0.02 mg/mL, respectively. UV-vis spectrum 200− 700 nm was recorded using a spectrophotometer (Multiskan Ascent, Thermo-Labsystems, Finland). HRMS. High-resolution mass spectrometry (HRMS) of F3-AC19 was conducted using a Bruker maXis Impact Ultra-High Resolution tandem time-of-flight (UHR-Qq-TOF) mass spectrometer, and the data was acquired using Compass 1.5 software. The MS condition was set as follows: ionization mode, positive electrospray ionization (+ESI), gas temperature, 180 °C; gas flow, 8 L/min, nebulizer, 2 bar; capillary voltage: 3800 V; mass scanning range, 50−1500 Da. For MS/MS, nitrogen was used as the collision gas, collision energy was 35 eV, and sodium formate was the accurate mass calibrant. NMR. 1H and 13C NMR spectra were recorded with a Bruker Avance III 600 MHz spectrometer using a QNP Cryoprobe. NMR data processing was performed with Topspin3.1 software The spectra were referenced to the corresponding solvent signals.12 Cell Culture. Caco-2 cells (HTB-37, ATCC) were cultured in complete Minimum Essential Medium (MEM) containing Earle’s salts and supplemented with 10% FBS (Invitrogen, Canada) and 100 units/ mL of penicillin and 100 μg/mL of streptomycin (Sigma, St. Louis, MO). Cells were incubated at 37 °C under a 5% CO2 atm. The medium was changed every 2−3 days. Caco-2 cells were subcultured weekly. NO Inhibitory Activity in Caco-2 Cell. NO in Caco-2 cells was measured using the conditions reported by Chen and Kitts (2008).11 Caco-2 cells seeded on 96-well plates (Sarstedt, Nü mbrecht, Germany) at a density of 105 cells/cm2 in complete MEM (100 μL) were cultured for 3 weeks. The fully differentiated Caco-2 cells were supplied with fresh MEM without FBS and incubated with, or without,

experimental samples for 1 h followed by a treatment of inducer which consisted of 8000 U/mL IFN-γ + 0.1 μg/mL PMA for 48 h. NO levels in the culture medium were determined using the Griess reagent (50 μL of 1% sulfanilamide in 5% phosphoric acid and 50 μL of 0.1% N(1-naphthyl)ethylenediamine dihydrochloride) after reducing nitrate to nitrite with nitrate reductase (Sigma, St. Louis, MO). The color change was measured by a spectrophotometer (Multiskan Ascent, ThermoLabsystems, Finland) at 540 nm. Nitrite and nitrate levels were determined using sodium nitrate as the standard. Percent inhibition was calculated using the following equation (NOst − NOsample)/(NOst − NOblank ) × 100% where NOst is the NO concentration of the cell supernatant incubated with inducer; NOblank is the NO concentration of the cell supernatant without samples and inducer; NOsample is the NO concentration of cells incubated with samples and inducer. Cell Viability. The cell viability was tested using MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma, St. Louis, MO) assay described by Chen and Kitts.4 After cells were treated with sample and inducer, they were then incubated with 0.5 mg/mL MTT in complete MEM for 4 h, followed by an overnight incubation with 10% sodium dodecyl sulfate (SDS). Absorbance was read at 570 nm using a spectrophotometer (Multiskan Ascent, Thermo Labsystems, Helsinki, Finland).

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RESULTS AND DISCUSSION Effects of Reaction Conditions and Type of Reactants on the Yield of F3-A. In order to increase the yield of F3-A, Table 2. Yield (μg/g) of F3-A in Glc-Lys with Different Concentration and Heating Timea S:AAb(mol/L: mol/L) time (h)

0.2:0.1

0.4:0.2

0.8:0.4

1.6:0.8

1.5 3 6 9 12 24

NDAα 2 ± 1Aα 5 ± 1Aα 28 ± 4Bα 43 ± 8Cα 96 ± 1Dα

NDAα 6 ± 0Aα 25 ± 5Aα 80 ± 5Bα 226 ± 33Cα 389 ± 19Dα

NDAα 50 ± 8Aα 147 ± 7Aα 527 ± 49Bα 3558 ± 369Bβ 11807 ± 154Cβ

9 ± 0Aβ 392 ± 90Aβ 4560 ± 602Aβ 9682 ± 395Dβ 11115 ± 484Eγ 6789 ± 672Cβ

All samples were adjusted to pH 6 and heated at 100 °C. Superscripts with different letters (A−E) denote significantly different (P < 0.05) in the same column. Superscripts (αβγ) with different letters denotes significantly different (P < 0.05) in the same row. bS:AA ratio, sugar:amino acid ratio. a

Table 3. Effects of Type of Sugar on the Yield (μg/g) of F3Aa models

yield (μg/g)b

Lac-Lys Suc-Lys Fru-Lys Glu-Lys

104 32 363 2286

± ± ± ±

7B 1A 1C 26D

a

Sugar (1.6 mol/L) and lysine (0.8 mol/L) mixtures were adjusted to pH 6 and heated at 100 °C for 4 h. bSuperscripts (A−D) with different letters denotes significantly different (P < 0.05) in the same column.

two steps were performed to optimize the reaction conditions. First, a Taguchi design was used to determine the sugar to amino acid ratio, pH, temperature, and heating time. Previously, we have used Taguchi design to successfully optimize the inducers for NO production in differentiated Caco-2 cells11 and to define the coculture conditions for Caco2 and HT29-MTX cells.13 Herein, a four-factor/four-level 1741

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Figure 1. HPLC profile of Glc-Lys heated mixture monitored at 370 nm.

Figure 3. UV−vis spectrum of F3-A in MeOH and 0.002% formic acid at a concentration of 0.02 mg/mL.

Figure 2. HRMS spectrum of F3-A.

Table 4. NMR Data of F3-A position

δc

δH (m, J in Hz)

1 2 3 4 5 7 8 9 10 11 13 14

48.72 20.84 24.61 128.45 163.35 124.23 151.78 114.61 110.20 156.35 57.23

3.65 (2H, t, J = 5.94) 1.77 (2H, m) 2.75 (2H, t, J = 6.61)

Figure 4. FTIR spectrum of F3-A.

Since the yield of F3-A was still low using conditions obtained from Taguchi design, in the second step, we held constant temperature, pH, and reactant ratio conditions that were obtained from the Taguchi design and varied the reactant concentration and the heating time, using a factorial design. The yield of F3-A from Glc-Lys mixture decreased as the reactant concentration was lowered, when keeping the molar ratio the same (Table 2). In general, more F3-A was produced when heating time was increased to 24 h. One exception existed when the sugar:amino acid ratio of 1.6:0.8 (mol/L: mol/L) was used; however, we attribute this to the gel like precipitates that were formed when reactants at these concentrations were heated for 24 h. Therefore, the optimal conditions used are Glc:Lys ratio set at 1.6:0.8 (mol/L: mol/L) and a heating duration of 12 h. The yield of F3-A increased more than 1000fold when the Glu-Lys mixture (1.6:0.8, mol/L: mol/L) was heated for 12 h, compared to shorter heating times (e.g., 1.5 h). In order to show the effects of various sugar types on the yield of F3-A, we conducted F3-A recovery experiments using fructose, glucose, lactose, and sucrose, respectively. Hence, hexoses or disaccharides comprised of hexose, known to generate HMF during heating and involving MR or caramelizing, were found to be necessary precursors to produce F3-A. Monosaccharides had a significantly (P < 0.05) greater affinity to produce F3-A compared to disaccharides containing hexose

7.90 (1H, s) 6.46 (1H, d, J = 2.08) 6.54 (1H, d, J = 3.36) 6.44 (1H, d, J = 3.42) 4.66 (2H, s) 4.01 (1H, s)

(L16) Taguchi design (Table 1) which includes 16 experiments in the orthogonal array was used to investigate the effects of four levels of the four reaction conditions (control factors) including S:AA ratio, pH, temperature, and heating time on F3A production in glucose and lysine aqueous mixtures. Within the multiple conditions tested, F3-A concentration ranged from undetectable amounts to 11.1 ± 3.3 (μg/g) (Table 1). From the ANOVA table, the relative impact of the different factors tested to affect production of F3-A followed in descending order: sugar:amino acid (S:AA) ratio < temperature (T) < heating time (t) < pH. The optimal conditions for producing F3-A were determined to be S:AA ratio = 2:1, T = 100 °C, t = 90 min, pH = 6. The production of F3-A precursor, HMF, was also measured. The amount of HMF was positively significantly (P = 0.023) correlated with the yield of F3-A. 1742

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Figure 5. NO inhibitory (A) and cell viability (B) effects of F3-AS, F3-AC19, AG, and PDTC on Caco-2 cells. F3-AS: synthetic F3-A; F3-AC19: isolated F3-A from Glu-Lys MR mixture.

when sugars were heated 50 °C under pH 3.5. The presence of amino acid increased HMF formation rate for glucose but not sucrose. Our results indicated that the production of F3-A precursor, HMF, is faster in glucose and lysine MR models than that of fructose and sucrose in our experimental conditions, the ultimate outcome being a higher F3-A production from glucose models. Proposed Reaction Route for Synthesis of F3-A. We found that the chemical synthesis of F3-A favored acidic conditions and that both the alpha and epsilon amino groups of lysine were involved in the reaction.20 A possible route for F3-A production in Glc-Lys can therefore be related to the production of HMF and reaction with compound 3, 1,2dehydropiperidine.21 HMF is formed via various pathways, including the aldo−keto isomerization of aldohexose, followed by the dehydration of furanose tautomers,22 decomposition from 3-deoxyhexosone produced by MR or by carameliza-

(Table 3). Nonenzymatic browning of aldohexoses in the presence of lysine has been reported to occur faster than that of ketohexoses in similar heat-processed, aqueous model systems.14−16 This would explain the relatively higher production of F3-A from the glucose than the fructose MR model. Sucrose itself is a nonreducing sugar; hence, without hydrolysis there should not be a Maillard sugar−amino acid interaction. The fact that we detected a trace amount of F3-A from sucrose, compared to lactose, a reducing disaccharide, indicates sucrose hydrolyzed to glucose and fructose at 100 °C under pH 6. Results of Buera et al. (1987)17 confirm this with their report that 10% hydrolysis of sucrose occurred when heated at 55 °C at pH 6 and produced yellow-brownish products. Alternatively, degradation of sucrose occurring with caramelizing will also result in HMF formation.18 However, Lee and Nagy (1990)19 showed that the rate of HMF formation in fructose is much faster than in sucrose followed by glucose 1743

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in CMC-se software (version 2.0.10) for possible chemical structures which were ranked based on observed correlations and chemical shift predictions. The chemical shift and 1H and 13 C NMR spectrum are shown in Table 4. After manual verification, F3-AC19 was identified as [5-(5,6-dihydro-4Hpyridin-3-ylidenemethyl)-furan-2-yl]methanol. The compound has a root-mean-square (rms) deviation of 4.6 ppm, indicating a good match between both proposed and actual chemical structures. This compound was also synthesized as described previously, and the 1H and 13C spectra were compared with the isolated compounds. The results confirmed that F3-AS and F3AC19 had the same structure. F3-A is easily soluble in MeOH and acidic ddH2O. When dissolved in MeOH, F3-A gave two major absorbance peaks at 315 and 370 nm, with maximum absorbance peak occurring at 315 nm (Figure 3). Under acidic condition, the maximum absorbance (λmax) of F3-A shifted to a higher wavelength (370 nm). This result indicated that protonation increased the solubility of F3-A in H2O and therefore might have also caused the rearrangement of double bond resulting in the red shift of λmax. In previous work, Totsuka et. al28 and Miller et. al20 identified F3-A from HMF-Lys and Glc-Lys MR models, respectively. The physiochemical properties, FTIR (Figure 4), MS, and NMR data of separated and synthesized compounds were identical in our experiments and similar to previous work. NO Inhibitory Effects in Caco-2 Cells. The NO inhibitory effects of separated F3-AC19 and synthesized F3-AS were tested using Caco-2 cells. A NO inhibitor AG and a NF-κB inhibitor PDTC were chosen to compare the NO inhibitory effect with our samples. F3-AC19 and F3-AS both produced a dose dependent inhibition of NO production in Caco-2 cells (Figure 5 A) with an IC50 = 47.8 ± 2.8 and 71.1 ± 3.5 μmol/L, respectively. The cell viabilities of all treatments were comparable to the untreated cells (Figure 5 B). The IC50 of F3-AC19 was significantly (P < 0.05) lower than that of F3-AS, indicating that the isolated F3-AC19 had higher NO inhibitory affinity than the synthesized sample. We attribute this result to the possible trace amounts of other MRPs in F3-AC19 that could contribute to a higher NO inhibitory capacity. F3-A can exist at both Z and E configurations; thus, in aqueous conditions the isomers interconvert. It is possible that one of the configurations has a relatively higher activity than the other, and the production and separation process for F3-AC19 from Glc-Lys MR model possible tended to produce the isomer with a characteristically higher NO inhibitory activity. Of particular importance was the observation that the IC50 of F3-AS was similar to that of AG and that the IC50 of F3-AC19 was also equivalent to that of PDTC. AG is a selective iNOS inhibitor that prevents NO and iNOS synthesis in IFN-γ + PMA induced Caco-2 cells.11 PDTC, a NF-κB inhibitor, prevents NF-κB activation, thus inhibiting both NO and iNOS in activated Caco-2 cells.29 The results described herein indicate that F3-A has a similar NO inhibitory capacity to that of the iNOS and NF-κB inhibitors in inflamed Caco-2 cells. When the synthetic F3-A was coincubated with AG, it showed an additive NO inhibitory effect in Caco-2 cells (Figure 6), thereby further indicating that F3-A could be useful to increase activity of known anti-inflammation drugs. Previous studies from our laboratory have reported that a fraction (F3) composed of small molecular weight components derived from a Glc-Lys MR mixture was effective at inhibiting inflammatory reactions in Caco-2 cells induced by IFN-γ and PMA cocktail.5,6 Anti-inflammatory activity of F-3 was shown

Figure 6. NO inhibitory and cell viability effects of AG supplement with F3-AS in Caco-2 cell. F3-AS: synthetic F3-A.

tion,18,23 or methylglyoxal recombination with glyceraldehyde.24 Under acidic conditions, glucose is isomerized to fructose through Lobry de Bruyn−Alberda van Ekenstein transformation, followed by three dehydration steps to produce HMF.25 First, the furanose ring at C5 loses one H2O molecule to form fructofuranosyl oxocation; this is followed by enolization to the enol of 2,5-anhydro-D-mannose after loss of a proton. Second, the second H2O molecule is released to form a double bond in the furan ring. An additional H2O molecule is lost to form HMF.26 At pH 7, or acidic conditions, the Amadori product in Glc-Lys MR mixture undergoes 1,2enolization to produce HMF.27 L-Lysine also undergoes Strecker degradation during MR to produce 5-aminopentanal which can be cyclized to 1,2-dehydropiperidine. HMF and 1,2dehydropiperidine undergo aldo condensation and dehydration to form F3-A.21 Chemical Properties and Chemical Structure of [5(5,6-Dihydro-4H-pyridin-3-ylidenemethyl)-furan-2-yl]methanol. The HPLC profile of Glc-Lys heated mixture is shown in Figure 1. The retention time of F3-A was 5.15 min. The accurate mass data for F3-A (Figure 2) gave a signal that corresponded to the protonated form of the compound at m/z 192.1021 Da with major fragments including m/z 175.0994, 146.0966, 121.9663, 91.0542, and 62.9290 Da. Most of the error values are within ±5 ppm except for a few fragment ions, thus confirming a formula C11H13NO2. The unusual large number of fragment ions and the presence of multiple signals in one nominal mass such as m/z 123.0676 and 123.0441 are evidence of the m/z 192.1021 ion undergoing complex rearrangements in the fragmentation processes. Small amounts of F3-AC19 were dissolved in CD3OD; 1H, 13 C, COSY, 13C-APT, HSQC, and HMBC-NMR experiments were performed. The NMR data along with the molecular formula (C11H13NO2) derived from the MS results were input 1744

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Journal of Agricultural and Food Chemistry

2,4-bis(p-hydroxyphenyl)-2-butenal in Tg2576 Alzheimer’s disease mice model. J. Neuroinflammation 2013, 10, 2. (4) Chen, X. M.; Kitts, D. D. Antioxidant and anti-inflammatory activities of Maillard reaction products isolated from sugar−amino acid model systems. J. Agric. Food Chem. 2011, 59, 11294−11303. (5) Chen, X. M.; Kitts, D. D. Characterization of antioxidant and anti-inflammatory activities of bioactive fractions recovered from a glucose−lysine Maillard reaction model system. Mol. Cell. Biochem. 2012, 364, 147−157. (6) Kitts, D. D.; Chen, X. M.; Jing, H. Demonstration of antioxidant and anti-inflammatory bioactivities from sugar−amino acid Maillard reaction products. J. Agric. Food Chem. 2012, 60, 6718−6727. (7) Alican, I.; Kubes, P. A critical role for nitric oxide in intestinal barrier function and dysfunction. Am. J. Physiol.: Gastrointest. Liver Physiol. 1996, 270, G225−G237. (8) Marion, R.; Coeffier, M.; Leplingard, A.; Favennec, L.; Ducrotte, P.; Dechelotte, P. Cytokine-stimulated nitric oxide production and inducible NO-synthase mRNA level in human intestinal cells: Lack of modulation by glutamine. Clin. Nutr. 2003, 22, 523−528. (9) Romier, B.; Van De Walle, J.; During, A.; Larondelle, Y.; Schneider, Y. J. Modulation of signalling nuclear factor−kappa B activation pathway by polyphenols in human intestinal Caco-2 cells. Br. J. Nutr. 2008, 100, 542−551. (10) Cirillo, C.; Sarnelli, G.; Esposito, G.; Grosso, M.; Petruzzelli, R.; Izzo, P.; Cali, G.; D’Armiento, F. P.; Rocco, A.; Nardone, G.; Iuvone, T.; Steardo, L.; Cuomo, R. Increased mucosal nitric oxide production in ulcerative colitis is mediated in part by the enteroglial-derived S100B protein. Neurogastroenterol. Motil. 2009, 21, 1209−e112. (11) Chen, X. M.; Kitts, D. D. Determining conditions for nitric oxide synthesis in Caco-2 cells using Taguchi and factorial experimental designs. Anal. Biochem. 2008, 381, 185−192. (12) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. NMR chemical shifts of common laboratory solvents as trace impurities. J. Org. Chem. 1997, 62, 7512−7515. (13) Chen, X. M.; Elisia, I.; Kitts, D. D. Defining conditions for the co-culture of Caco-2 and HT29-MTX cells using Taguchi design. J. Pharmacol. Toxicol. Methods 2010, 61, 334−342. (14) Baxter, J. H. Free amino-acid stability in reducing sugar systems. J. Food Sci. 1995, 60, 405−408. (15) Hayashi, T.; Namiki, M. Role of sugar fragmentation in an early stage browning of amino−carbonyl reaction of sugar with amino-acid. Agric. Biol. Chem. 1986, 50, 1965−1970. (16) Jing, H.; Kitts, D. D. Comparison of the antioxidative and cytotoxic properties of glucose−lysine and fructose−lysine Maillard reaction products. Food Res. Int. 2000, 33, 509−516. (17) Buera, M. D.; Chirife, J.; Resnik, S. L.; Wetzler, G. Nonenzymatic browning in liquid model systems of high water activityKinetics of color changes due to Maillard reaction between different single sugars and glycine and comparison with caramelization browning. J. Food Sci. 1987, 52, 1063−1067. (18) Kroh, L. W. Caramelisation in food and beverages. Food Chem. 1994, 51, 373−379. (19) Lee, H. S.; Nagy, S. Relative reactivities of sugars in the formation of 5-hydroxymethylfurfural in sugar−catalyst model systems. J. Food Process. Preserv. 1990, 14, 171−178. (20) Miller, R.; Olsson, K.; Pernemalm, P. A. Formation of aromaticcompounds from carbohydrates. 9. Reaction of D-glucose and L-lysine in slightly acidic, aqueous-solution. Acta Chem. Scand., Ser. B 1984, 38, 689−694. (21) Miller, R. Synthesis and stereochemistry of (E)-5-(3,4,5,6tetrahydropyrid-3-ylidenemethyl)-2-furanmethanol, a product of the reaction between D-glucose and L-lysine. Acta Chem. Scand., Ser. B 1987, 41, 208−209. (22) Mednick, M. L. Acid-base-catalyzed conversion of aldohexose into 5-(hydroxymethyl)-2-furfural. J. Org. Chem. 1962, 27, 398−&. (23) Richards, E. L. Non-enzymic browningReaction between Dglucose and glycine in the dry state. Biochem. J. 1956, 64, 639−644.

to be mediated through iNOS inhibition and down regulation of genes in NF-κB signaling;5 these activities correspond to NO inhibition. This study has elucidated the structure of the bioactive component present in F3, being [5-(5,6-dihydro-4Hpyridin-3-ylidenemethyl)-furan-2-yl]methanol, which was active at inhibiting NO in inflamed Caco-2 cells. We also found that process variables that influenced the yield of F3-A from a heated aqueous Glc-Lys reaction were lower pH and higher reactant concentration. Moreover, MR models comprised of aldohexose produce a higher amount of F3-A compared to ketohexose and disaccharides. The relative anti-inflammatory potency of F3-A corresponded to similar activity observed with AG, a selective iNOS inhibitor, and also PDTC, a Nf-κB inhibitor, both of which are involved in iNOS and NO inhibition.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 1-604-822-5560. Fax: 1-604-822-5143. E-mail: david. [email protected]. Funding

This work was supported by a NSERC-discovery grant to D.D.K. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED AG, aminoguanidine; APT, attached proton test; COSY, correlation spectroscopy; COX-2, cyclooxygenase-2; DAD, diode array detector; DCM, dichloromethane; dHP, 1,2dehydropiperidine; ESI, electrospray ionization; FTIR, Fourier transform infrared spectroscopy; Glc, glucose; HATR, horizontal attenuated total reflectance; HMBC, heteronuclear multiple bond correlation; HMF, 5-hydroxymethyl-2-furfural; HMFA, 5-hydroxymethyl-2-furoic acid; HPLC, high-performanc liquid chromatography; HRMS, high-resolution mass spectrometry; HSQC, heteronuclear single quantum coherence; IBD, inflammatory bowel diseases; IFN-γ, interferon-γ; iNOS, inducible nitric oxide synthase; Lys, lysine; 3-MCP, 3methyl-1,2-cyclopentanedione; MEM, minimum essential medium; MR, Maillard reaction; MRPs, Maillard reaction products; MTBE, methyl tert-butyl ether; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Nf-κB, nuclear factor-kappa B; NMR, nuclear magnetic resonance; NO, nitric oxide; PDTC, pyrrolidine dithiocarbamate; PMA, phorbol 12-myristate 13-acetate; rms, root-mean-square; RT, room temperature; SDS, sodium dodecyl sulfate; S:AA, sugar:amino acid; TLC, thin layer chromatography; UHRQq-TOF, ultrahigh-resolution tandem time-of-flight; UV−vis, ultraviolet−visible

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REFERENCES

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