Characterization of nitrated phenolic compounds for their anti-oxidant, pro-oxidant, and nitration activities

Archives of Biochemistry and Biophysics 513 (2011) 10–18

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Characterization of nitrated phenolic compounds for their anti-oxidant, pro-oxidant, and nitration activities Yusuke Iwasaki a,⇑, Maki Nomoto a, Momoko Oda a, Keisuke Mochizuki a, Yuki Nakano a, Yuji Ishii b, Rie Ito a, Koichi Saito a, Takashi Umemura b, Akiyoshi Nishikawa b, Hiroyuki Nakazawa a a b

Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan

a r t i c l e

i n f o

Article history: Received 11 May 2011 and in revised form 15 June 2011 Available online 24 June 2011 Keywords: Chlorogenic acid Caffeic acid Sodium nitrite Anti-oxidant Pro-oxidant

a b s t r a c t Coffee is one of the most widely consumed beverages worldwide. Evidence of the health benefits and the important contribution of coffee brew to the intake of anti-oxidants in the diet has increased coffee consumption. Chlorogenic acid (ChA) and caffeic acid (CaA) are the major phenolic compounds in coffee. However, phenolic compounds, which are generally effective anti-oxidants, can become pro-oxidants in the presence of Cu2+ to induce DNA damage under certain conditions. On the other hand, sodium nitrite (NaNO2) is widely used as a food additive to preserve and tinge color on cured meat and fish. It is possible that phenolic compounds react with NaNO2 under acidic conditions, such as gastric juice. In this study, we identified compounds produced by the reaction between ChA or CaA in coffee and NaNO2 in artificial gastric juice. The identified phenolic compounds and nitrated phenolic compounds were assessed for their anti-oxidant, pro-oxidant, and nitration activities by performing an in vitro assay. The nitrated phenolic compounds seemed to show increased anti-oxidant activity and decreased prooxidant activity. However, one nitrated CaA compound that has a furoxan ring showed the ability to release NO 2 in the neutral condition. Ó 2011 Elsevier Inc. All rights reserved.

Introduction Coffee is one of the most widely consumed beverages worldwide. During the past few years, evidence of the health benefits [1] and the important contribution of coffee brew to the intake of anti-oxidants in the diet [2–4] has increased coffee consumption. Many studies have examined the association between coffee consumption and health, particularly cardiovascular morbidity. The question of whether or not coffee intake increases the risk of coronary heart disease remains unanswered. Several studies have shown that caffeine in coffee induces various acute cardiovascular effects, including effects on blood pressure, circulating catecholamines, arterial stiffness, and endothelium-dependent vasodilation [5,6]. On the other hand, coffee consumption is also associated with the decreased risk of type 2 diabetes [7], Alzheimer’s disease [8], and cancer [9]. It is said that these effects are produced by polyphenols, which have anti-oxidant activity. Anti-oxidant activity refers to the ability of polyphenol compounds to prevent damage caused by reactive oxygen species (ROS) (such as by radical scavenging) or to prevent the generation of these species (by binding iron) [10]. Chlorogenic acid (ChA) and caffeic acid (CaA) are the major phenolic

⇑ Corresponding author. Fax: +81 3 5498 5765. E-mail address: [email protected] (Y. Iwasaki). 0003-9861/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2011.06.009

compounds in coffee and act as anti-oxidants. However, phenolic compounds, which are generally effective anti-oxidants, can become pro-oxidants in the presence of Cu2+ to induce DNA damage under certain conditions. It has been demonstrated that compounds bearing o-dihydroxyl groups (i.e., ChA and CaA) are the most active in inducing plasmid pBR322 DNA strand breakage in the presence of Cu2+ [11]. The major dietary source of nitrite includes cured meat and cereals, but approximately 90% of nitrite ingested by humans is accounted for by the reduction of nitrate in the oral cavity via the action of nitrate reductase produced by microorganisms present in the oral cavity [12]. Sodium nitrite (NaNO2) is widely used as a food additive to preserve and tinge color on cured meat and fish [13]. Nitrite concentrations in vivo are 0.5–3.6 lM in plasma [14], 15 lM in respiratory tract lining fluid [15], and 30–210 lM in saliva [16], making nitrous acid formation likely in many tissue compartments. Excessive nitrite production is noted particularly in inflammation. Humans, therefore, ingest nitrite from both exogenous and endogenous sources. At the acidic pH of the stomach, nitrite yields nitric oxide (NO) and NO-derived species that may exert a biological impact locally in terms of antimicrobial effects, blood flow, mucus secretion, and gastric motility. It has been reported that the co-administration of catechol and NaNO2 to rats increased 8-hydroxy-20 -deoxyguanosine (8-OHdG) levels in forestomach epithelium DNA [17].

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Some dietary phenolics with anti-oxidant activity can reduce nitrite to NO under acidic conditions. As coffee contains ChA and CaA, it is possible that these compounds reduce nitrous acid to NO when NaNO2 is mixed with gastric juice after drinking coffee. In fact, it has been reported that ChA and CaA were nitrated by NaNO2 under acidic conditions, and the nitration could give rise to several nitrated ChA and CaA compounds in vitro [18]. However, there are no reports of the characteristics of the nitrated compounds. In the present study, we identified compounds produced by the reaction between ChA or CaA in coffee and NaNO2 in artificial gastric juice. The identified phenolic compounds and the nitrated phenolic compounds were assessed for their anti-oxidant, pro-oxidant, and nitration activities by performing an in vitro assay.

Materials and methods Reagents and chemicals Chlorogenic acid (ChA) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Caffeic acid (CaA), nuclease P1, and DNA Extractor Kit were purchased from Wako Pure Chemical Industries (Tokyo, Japan). Copper(II) sulfate pentahydrate and sodium nitrite (NaNO2) were purchased from Kanto Chemical (Tokyo, Japan). a-(4-Pyridyl-1-oxide)-N-tert-butylnitrone (POBN) was purchased from Labotech Co. (Tokyo, Japan). Deoxyribonucleic acid sodium salt

HO

O

from calf thymus, phosphatase alkaline from bovine intestinal mucosa, deoxyguanosine (dG), and 8-hydroxy-20 -deoxyguanosine (8OHdG) were obtained from Sigma (Tokyo, Japan). Water was purified using a Milli-Q gradient A10 system (Millipore, MA, USA). Other chemicals and solvents were obtained from Wako Pure Chemical Industries.

Synthesis of nitrated chlorogenic acid and caffeic acid Nitrated ChA and CaA were synthesized according to the method reported by Napolitano and d’Ischia [18]. Briefly, ChA or CaA (5 mmol) was dissolved in 0.05 M acetate buffer, pH 4 (500 mL) and NaNO2 (25 mmol) and the mixture was continuously stirred at room temperature. Then, the mixture was extracted with ethyl acetate (3  150 mL) and the combined organic layers were dried over sodium sulfate to dryness. The target substance was removed from the reaction solution by silica gel chromatography using toluene–ethyl acetate 80:20 (eluent A), 60:40 (eluent B) or 40:60 (eluent C). Purified nitrated ChA and CaA were identified by comparison of their spectral properties (1H NMR, 13C NMR, and LC-PDA-ESI-MS) with those reported [18,19]. Their chemical structures are shown in Fig. 1. Nitrochlorogenic acid (ChA-NO). UV: kmax (MeOH) 276, 339, 430 nm. 1H NMR (CD3OD) d (ppm): 2.12 (m, 4H), 3.74 (dd, J = 10.0, 3.2 Hz, 1H), 4.20 (m, 1H), 5.42 (m, 1H), 6.31 (d, J = 15.6 Hz, 1H), 7.05 (s, 1H), 7.57 (s, 1H), 8.15 (d, J = 15.6 Hz, 1H).

OH

HO

O

OH

HO

HO

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O COOH OH

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HO

COOH OH

NO2

Nitrochlorogenic acid (ChA-NO)

Chlorogenic acid (ChA)

O

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HO OH

N OH

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HO

2-(3,4-Dihydroxyphenyl)-2-oxoethanaloxime (CaA-NO-1)

Caffeic acid (CaA)

-O

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+ N

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6,7-Dihydroxy-1,2-(4H)-benzoxazin-4-one (CaA-NO-2)

HO

2-Oxy-3-(3,4-dihydroxyphenyl)-1,2,5-oxadiazole (CaA-NO-3)

Fig. 1. Structures of ChA, CaA, and nitrated phenolic compounds.

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13

C NMR (CD3OD) d (ppm): 39.5 (CH2), 41.3 (CH2), 73.2 (CH), 73.3 (CH), 75.3 (C), 78.2 (CH), 113.2 (CH), 116.0 (CH), 121.2 (CH), 125.6 (C), 140.7 (C), 144.0 (CH), 150.2 (C), 156.2 (C), 169.1 (C), 181.8 (C). ESI-MS: m/z 398 [MH]. 2-(3,4-Dihydroxyphenyl)-2-oxoethanaloxime (CaA-NO-1). 1H NMR (acetone-d6) d (ppm): 6.91 (d, 1 H, J = 8.8 Hz), 7.61 (dd, 1H, J = 8.8, 2.0 Hz), 7.62 (d, 1H, J = 2.0 Hz), 7.91 (s, 1H). 13C NMR (acetone-d6) d (ppm): 115.2 (CH), 117.1 (CH), 124.5 (CH), 129.2 (C), 141.7 (C), 145.3 (C), 148.7 (CH), 187.1 (C). ESI-MS: m/z 180 [MH]. 6,7-Dihydroxy-1,2-(4H)-benzoxazin-4-one (CaA-NO-2). 1H NMR (CD3OD) d (ppm): 6.95 (s, 1H), 7.33 (s, 1H), 8.13 (s, 1H). ESI-MS: m/z 178 [MH]. 2-Oxy-3-(3,4-dihydroxyphenyl)-1,2,5-oxadiazole (CaA-NO-3). UV: kmax (CH3OH) 247, 316 nm. 1H NMR (CD3OD) d (ppm): 6.89 (d, J = 8.4 Hz, 1H), 7.32 (dd, J = 8.4, 2.0 Hz, 1H), 7.54 (d, J = 2.0 Hz, 1H), 8.92 (s, 1H). 13C NMR d (ppm): 113.9 (CH), 115.4 (C), 116.2 (C), 117.3 (CH), 120.1 (CH), 146.4 (CH), 147.5 (C), 149.7 (C). ESIMS: m/z 193 [MH]. Analytical conditions for ChA, CaA, and nitrated phenolic compounds High-performance liquid chromatography was performed with a SHIMADZU (Shimadzu, Tokyo, Japan) system that consisted of an LC10ADVP pump, an SIL-HTC autosampler, a CTO-10AVP thermostated column compartment, a DGU-14AM vacuum degasser, and a SPD-M10AVP photodiode array detector, and was connected to a SHIMADZU LCMS-2010 A mass spectrometer. Separation of the analytes

The modified DPPH method was used for the determination of anti-oxidant activity [20]. DPPH radical solution (2 mM) was prepared in methanol and the phenolic compounds were diluted in methanol to concentrations ranging from 0.1 to 2 mM. In a 1.5 mL disposable tube, the prepared DPPH (250 lL) solution was added to a sample of diluted phenolic compound (50 lL) and methanol (200 lL). The mixed samples were incubated for 30 min at 37 °C. Absorbance was monitored at 540 nm with a BIO-RAD Model 550 microplate reader. Then, the effect of phenolic compounds on DPPH absorbance was estimated. DPPH scavenging activity was determined with the following equation: % scavenging activity = [Acontrol  Asample]/[Acontrol  Aascorbic acid]  100. DPPH plus ascorbic acid (10 mM) was used as positive control. Data are means ± SD of three determinations.

1.5

ChA (100 µM)

50

0.5 0 300

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Intensity (×10)

Intensity (×100)

DPPH radical scavenging activity

(mAU)

(300 nm)

1.0

was accomplished on an Xbridge C18 column (3.5 lm, 2.1  150 mm; Waters, Japan). Column oven temperature was maintained at 40 °C. Mobile phase was 0.1% aqueous formic acid and 0.1% formic acid in acetonitrile (90:10, v/v) and the flow rate was constant at 0.2 mL/min. The photodiode array detector was set at 300 nm. The analytes were detected in the electrospray negative ionization mode using the scan ion monitoring mode. Curved desolvation line and heat block temperatures for the analysis were set at 250 and 200 °C, respectively. Nebulizer gas flow was set at 1.5 L/min and detector voltage was set at 1.3 eV.

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Fig. 2. UV chromatograms and spectra of ChA, CaA, and nitrated phenolic compounds. Separation of the analytes was accomplished on an XBridge C18 column (3.5 lm, 2.1  150 mm; Waters, Japan). Mobile phase was 0.1% aqueous formic acid and 0.1% formic acid in acetonitrile (90:10, v/v).

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A

Intensity (×100)

Acidic condition (0.1 M HCl) Peak B ChA-NO (60.2 µM)

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(300 nm)

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Acidulous condition (pH 4.0) Peak D CaA-NO-1 Peak F (40.5 µM) Peak E CaA-NO-3 CaA-NO-2 (18.3 µM) (64.5 µM)

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+ NaNO2 180 min

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0.0 3.0 2.0 1.0 0.0 0

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Retention time (min) Fig. 3. Reaction between phenolic compound and sodium nitrite under the acidic condition. (A) UV chromatograms of the reaction between phenolic compound and sodium nitrate. (B) Time course of generated compounds under the acidic or acidulous condition.

Electron spin resonance measurement of hydroxyl radical The ESR method was used for the determination of hydroxyl radical (OH) [21]. Instead of directly trapping OH, this method traps and measures CH3 by using POBN that is formed during the interaction of DMSO with OH. Moreover, this method can detect OH, which is not affected by Cu2+. The analysis of CH3 was carried out with an ESR spectrometer (JES–RE1X, JEOL Co., Tokyo, Japan). The ESR spectrum was measured at a microwave frequency of 9.43 GHz, a magnetic field of 335.5 ± 5 mT, a microwave power of 9 mW, a modulation of 100 kHz, a time constant of 0.03 s, and a sweep time of 30 s, using the ESR spectrometer.

The spectra of the samples were scanned to record the signal intensities (peak-to-peak heights). A typical reaction mixture for incubation consisted of phosphate buffer (50 mM, pH 7.4), POBN (10 mM), DMSO (10%), phenolic compound (2 mM), and copper(II) sulfate pentahydrate (1 mM) in a final volume of 0.3 mL. Samples were used in the reaction that was performed at 37 °C for 30 min. DNA digestion and determination of dG and 8-OHdG In order to prevent the formation of oxidative by-products during DNA isolation, DNA was digested by using the slightly modified

Y. Iwasaki et al. / Archives of Biochemistry and Biophysics 513 (2011) 10–18

Concentration of analyte (µM)

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Fig. 3 (continued)

Neutral condition (PBS) 100 80 ChA 60

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Fig. 4. Sample stability of phenolic and nitrated phenolic compounds under acidic and neutral conditions. ⁄P < 0.05 and

method of Ishii et al. [22]. Calf thymus DNA (2 mg/mL) at 250 lL was incubated at 37 °C for 30 min after adding 50 lL of phenolic compound (2 mM) and 50 lL of copper(II) sulfate pentahydrate (1 mM) in 0.15 mL of phosphate buffer (50 mM, pH 7.4). Treated calf thymus DNA was immediately centrifuged at 10,000g for 5 min at 10 °C after the total volume was adjusted to 1.5 mL by adding NaI and 2-propanol. After washing with ethanol, the pellet was dissolved in 0.2 mL of 20 mM sodium acetate buffer, pH 4.8.

** **

CaA-NO-1

⁄⁄

P < 0.01 vs. 0 min).

DNA was enzymatically hydrolyzed by adding 5.0 lL of nuclease P1 to obtain a concentration of 500 U/mL. The mixture was incubated at 60 °C for 15 min. After the addition of 20 lL of 1.0 M Tris–HCl buffer (pH 8.0), 5.0 lL of alkaline phosphatase was added to give a final concentration of 20 U/mL. The mixture was passed through a 3000 NMWL filter (Millipore, Tokyo, Japan) after incubating at 37 °C for 60 min. Then, the digested solution was injected into the HPLC–UV–ECD instrument for 8-OHdG and dG analysis.

Y. Iwasaki et al. / Archives of Biochemistry and Biophysics 513 (2011) 10–18

Statistical analysis

100

Anti-oxidant activity (%)

15

All results are expressed as means ± SD. Statistics were analyzed using one-way analysis of variance (ANOVA) and if statistically significant, post hoc analysis using the Dunnett method was conducted for multiple comparisons among groups. Values of P < 0.05 and 0.01 were considered statistically significant.

75

50

Results 25

ChA

Characterization of products formed in the reaction of phenolic compounds with NaNO2

ChA-NO 0 0

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Anti-oxidant activity (%)

100

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Concentration of analyte (µM) Fig. 5. Anti-oxidant activities of phenolic compounds as assessed by DPPH assay. DPPH concentration was 1 mM. Data points represent means ± SD (n = 6).

HPLC–UV–ECD conditions for dG and 8-OHdG analysis In the assay for dG and 8-OHdG, the UV detector and the ECD instrument used were a Shimadzu SPD10A (Tokyo, Japan) and an ESA Coulochem III (Tokyo, Japan), respectively. A Shimadzu 10Avp pump (Tokyo, Japan) was used to induce flow through the analytical column. An Inertsil ODS3 (4.6 mm  150 mm, 3.0 lm, GL Sciences, Tokyo, Japan) was used for separation. An aliquot (20 lL) of the sample was injected into the ODS column whose temperature was maintained at 40 °C. The mobile phase was a mixture of 10 mM sodium dihydrogen phosphate/methanol (97/ 3, v/v). The compounds were eluted isocratically at a flow rate of 1.0 mL/min. The wavelength of the UV detector was set at 290 nm for the detection of dG. The Coulochem III ECD instrument was used with a guard cell (Model 5020; 350 mV) and an analytical cell (Model 5011; electrode 1, 150 mV; electrode 2, 300 mV).

Analysis of nitrate by the Griess assay Solutions (1 mM) of all compounds were prepared at 37 °C using sodium phosphate buffer (pH 7.4) or 0.1 M HCl. After incubation, an aqueous solution of NO 2 was mixed with Griess reagent (25 mM sulfanilamide and 2.5 mM N-(1-naphthyl)ethylenediamine in 2.5% phosphoric acid) and the whole was incubated at room temperature for 10 min. Absorbance was monitored at 540 nm with a BIO-RAD Model 550 microplate reader. Acidification of the buffered solution resulted in an absorbance band at 540 nm, which was indicative of the corresponding azo dye [23,24].

In order to identify compounds produced by the reaction between ChA or CaA and NaNO2 in artificial gastric juice, both standards and synthesized compounds were prepared and analyzed (Fig. 2). In the first series of experiments, ChA or CaA (1 mmol) was reacted with NaNO2 (2 mmol) in the acidic condition (0.1 M HCl) or the acidulous condition (0.05 M acetate buffer; pH 4.0). To identify the substrates and the products, HPLC was performed after incubation of ChA or CaA in the presence of NaNO2. HPLC of the reaction mixture after 3 h incubation indicated complete substrate consumption and the presence of a complex pattern of products (Fig. 3A). Peaks B, D, E, and F corresponded to the major components after incubation. Peaks A and C, which are ChA and CaA, respectively, showed a marked decrease in intensity in the presence of NaNO2. Peaks B, D, E, and F showed an increase in intensity and corresponded to the major components after incubation (Fig. 3B). The complexity of the reaction mixture was confirmed by MS and NMR measurements. Peaks B, D, E, and F each gave a deprotonated ion peak [MH] in their ESI/MS spectra, which appeared at m/z 398, 180, 178, and 193, respectively, suggesting the formation of nitrated derivatives of ChA-NO, CaANO-1, CaA-NO-2, and CaA-NO-3. The chemical stability of the analytes was assessed under several conditions and the results are shown in Fig. 4. All compounds were stable under the acidic condition. On the other hand, compounds CaA-NO-2 and CaA-NO-3, which are nitrated CaA, decomposed under the neutral condition. These data suggest that nitrated phenolic compounds were formed by reacting each phenolic compound (ChA or CaA) with NaNO2 and could exist stably under the gastric acid condition. DPPH radical scavenging activity of phenolic and nitrated phenolic compounds The anti-oxidant activities of the phenolic compounds were measured by the DPPH method, which is one of the oldest and the most frequently used methods for evaluating anti-oxidant activity. The scavenging effects of phenolic compounds on DPPH radicals are shown in Fig. 5. All the phenolic and nitrated phenolic compounds exhibited anti-oxidant activity. The DPPH radical scavenging activities of nitrated phenolic compounds, such as ChA-NO, CaA-NO-1, CaA-NO-2, and CaA-NO-3, were stronger than those of the non–nitrated phenolic compounds. Measurement of hydroxyl radical and oxidative damage in calf thymus DNA To investigate ROS generated by the reaction of phenolic compounds with Cu2+ we performed electron spin resonance (ESR) measurements and determined whether these compounds have the ability to generate ROS. The pro-oxidant activities induced by the reaction of phenolic compounds with copper are shown in Fig. 6A. The results indicated that ChA and CaA significantly gener-

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A

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PBS + Cu2+

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Relative intensity (signal / Mn2+)

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CaA-NO-2 CaA-NO-3

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Blank Blank + Cu2+ ChA + Cu2+ ChA-NO + Cu2+ CaA + Cu2+ CaA-NO-1 + Cu2+ CaA-NO-2 + Cu2+ CaA-NO-3 + Cu2+

332

334

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Field (mT)

Fig. 6. Pro-oxidant activities produced by the reaction between phenolic compounds and Cu2+. Data represent means ± S.D. (n = 6). ⁄P < 0.05 and ⁄⁄P < 0.01 vs. phenolic compound + copper).  P < 0.05 and   P < 0.01 vs. phenolic compound alone). (A) ESR. (B) HPLC–UV–ECD. (C) Representative ESR spectra of the interaction between ChA, CaA, or nitrated phenolic compounds and copper.

ated ROS in the presence of Cu2+. Meanwhile, nitrated phenolic compounds generated much less ROS in the same conditions. In the next set of experiments, we examined the effects of the reaction between phenolic or nitrated phenolic compounds and

Cu2+ on 8-OHdG formation using the same ESR conditions. When calf thymus DNA was treated with phenolic compounds in the presence of CuSO4, 8-OHdG was formed. ChA and CaA induced DNA oxidation in the presence of Cu2+ (Fig. 6B). Meanwhile,

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Acidic condition (0.1 M HCl)

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150

180

Reaction time (min)

Fig. 7. Concentration of NO 2 as assessed by Griess assay. Data points represent means ± SD (n = 6).

nitrated phenolic compounds obviously showed reduced pro-oxidant activity in the presence of Cu2+. Thus, nitrated phenolic compounds do not have pro-oxidant activity.

Measurement of nitric oxide by Griess assay The most famous and frequently used method of analysis of nitrite and nitrate is the Griess assay. To examine whether or not NO 2 is generated from phenolic or nitrated phenolic compounds, we performed the Griess assay. As shown in Fig. 7, all the compounds did not generate NO 2 under the acidic condition and only CaA-NO-3 was found to generate a significant amount of NO 2 in the neutral condition. These data suggest that CaA-NO-3 decomposed while generating NO 2 under the neutral condition. Discussion We have identified nitrated phenolic compounds generated by the reaction between ChA or CaA and NaNO2 in artificial gastric juice and assessed their anti-oxidant, pro-oxidant, and nitration activities by an in vitro assay. The mean pH of gastric juice is 1.2. However, it is significantly increased by an H2 receptor antagonist [25]. In our first series of experiments, ChA or CaA was reacted with NaNO2 in the acidic condition (0.1 M HCl) or the acidulous condition (0.05 M acetate buffer; pH 4.0). The reaction after 3 h indicated complete substrate consumption and the presence of a complex pattern of products (Fig. 3A). In particular, several compounds, such as ChA-NO and CaA-NO, were generated at high concentrations under the acidulous condition. The chemical stability of the analytes was assessed under several conditions and the results are shown in Fig. 4. All compounds existed stably under the acidic condition. On the other hand, compounds CaA-NO-2 and CaA-NO-3, which are nitrated CaA compounds, decomposed under the neutral condition. This result indicated that

these compounds were stable in gastric juice but decomposed after being absorbed by the body (i.e., blood and liver). Fig. 5 shows the results of determination of the anti-oxidant activity of the phenolic compounds by measuring DPPH radical scavenging activity. Our results indicated that nitrated phenolic compounds have stronger anti-oxidant activity than non-nitrated phenolic compounds. Sroka and Cisowski [26] reported the antioxidant activity of phenolic acid. The free radical scavenging activity of phenolic acids is correlated with the number of hydroxyl groups bound to the aromatic ring and/or the position of the hydroxyl groups (ortho or para) in the chemical structure. On the other hand, Kawabata et al. [27] investigated iron complexes of catechol and nitrocatechol derivatives. In the Fenton-like reaction, iron-catechol generated more OH than iron–nitrocatechols. Nitrocatechol derivatives having a conjugated structure can sequester chelated iron more effectively than catechol. These results indicated that nitrated phenolic compounds can bind to metal ion and prevent the generation of a large amount of OH. In general, phenolic compounds can switch from being anti-oxidants to pro-oxidants in the presence of Cu2+ to induce ROS production and subsequently DNA damage. It is said that ortho-dihydroxyl groups that can chelate with Cu2+ induce the greatest pro-oxidant activity. The initial electron-transfer oxidation of phenolic compounds by Cu2+ generates the corresponding semiquinone radical and the radical undergoes a second electron-transfer reaction with  O2 to form ortho-quinone and superoxide anion (O 2 ). O2 reacts with Cu+ to give hydrogen peroxide (H2O2), which is readily converted via a Fenton-like reaction into OH [11]. Pro-oxidant experiments (Fig. 6) have shown that ChA and CaA generate OH when mixed with Cu2+. On the other hand, nitrated phenolic compounds showed decreased pro-oxidant activity. These results corresponded to the results of anti-oxidant activity measurements. A number of NO donors, namely, molecules that are able to release NO in the physiological condition, such as furoxans and organic nitrates, were proven to display cytotoxic and cytostatic effects on viruses and microbial agents [28–30]. Furoxan (1,2,5-oxa-

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diazole-2-oxide) is a heterocyclic system known to chemists for its intriguing chemistry and structure. It is known that furoxans are able to release NO at physiological pH, in the presence of thiol cofactors. The mechanism of this release appears to be complex and may involve more than one redox form of NO [30]. Hybrid NO donor furoxan-based drugs are novel drugs that retain the pharmacological activity of the parent compound yet also has the biological activities of NO. To examine whether NO is generated from phenolic or nitrated phenolic compounds, we performed the Griess assay. CaA-NO-3 was found to significantly increase NO 2 concentration in the neutral condition (Fig. 7), because CaANO-3 has a furoxan ring. Conclusions We identified the characteristics of compounds generated by reacting ChA or CaA contained in coffee with NaNO2 in artificial gastric juice. All the compounds existed stably under the acidic condition. In addition, ChA, CaA, and the nitrated phenolic compounds were assessed for their anti-oxidant, pro-oxidant, and nitration activities by an in vitro assay. The nitrated phenolic compounds seemed to show an increase in anti-oxidant activity and a decrease in pro-oxidant activity. However, CaA-NO-3, which has a furoxan ring, significantly increased NO 2 concentration in the neutral condition. Further studies are required to reveal the chemical mechanisms underlying the anti-oxidant, pro-oxidant, and nitration activities of phenolic compounds. Acknowledgment This work was supported by a Grant-in-Aid from the Ministry of Health, Labour and Welfare, Japan. References [1] J.G. Dórea, T.H. da Costa, British Journal of Nutrition 93 (2005) 773–782. [2] R. Pulido, M. Hernández-García, F. Saura-Calixto, European Journal of Clinical Nutrition 57 (2003) 1275–1282. [3] N. Pellegrini, M. Serafini, B. Colombi, Journal of Nutrition 133 (2003) 2812–2819. [4] A. Svilaas, A.K. Sakhi, L.F. Andersen, T. Svilaas, E.C. Ström, Journal of Nutrition 134 (2004) 562–567. [5] A. Mahmud, J. Feely, Hypertension 38 (2001) 227–231.

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