Nitric oxide (NO) scavenging capacity of natural antioxidants

Food Chemistry 129 (2011) 866–870

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Nitric oxide (NO) scavenging capacity of natural antioxidants Yoshimi Sueishi a,⇑, Masashi Hori a, Masakazu Kita b, Yashige Kotake c a

Department of Chemistry, Faculty of Science, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan Graduate School of Education, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan c Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA b

a r t i c l e

i n f o

Article history: Received 27 December 2010 Received in revised form 11 March 2011 Accepted 5 May 2011 Available online 11 May 2011 Keywords: Nitric oxide Hydrophilic antioxidants Kinetic study EPR PTIO NO-scavenging capacity

a b s t r a c t Nitric oxide (NO)-scavenging capacities of several hydrophilic antioxidants were determined by using the PTIO method, a competitive NO-scavenging method with 2-phenyl-4,4,5,5-tetramethylimidazoline-1oxyl 3-oxide (PTIO). Relative NO-scavenging rates of antioxidants were measured with respect to PTIO and the scavenging rate constants were calculated based on PTIO’s rate constant. Results indicated that NO-scavenging rate constants of the antioxidants were: uric acid (2.5) > caffeic acid (1.2) > trolox (1.0) > genistein (0.19) > glutathione (0)  N-acetylcysteine (0), where the numbers are expressed in trolox equivalent unit. The oxidation potentials of these antioxidants were measured and the order in the magnitude of oxidation potential was in good accordance with NO-scavenging capacity. Based on the results, we have suggested that the primary chemical process of the antioxidant reaction with NO can be characterised with the electron transfer from NO to the antioxidant. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Antioxidants (AOx) like vitamin C or vitamin E are abundant in plant-derived food. They are recognised to react with free radicals and thought to protect biological systems from free radical damage (Huang, Ou, & Prior, 2005). Various methods have been developed to measure the AOx’s ability to scavenge free radicals. For the so called oxygen radicals, the assay called oxygen radical absorbance capacity (ORAC) method is widely used to measure the antioxidant activity in food (Huang et al., 2005; Kohri et al., 2009; Prior et al., 2003; Tabart, Kevers, Pincemail, Defraigne, & Dommes, 2009). Although it is well-known that free radicals to be scavenged by antioxidants include nitric oxide (NO), NO has never been a subject for scavenging capacity measurement in ORAC-like methods (Al-Laith, 2010; Cook, Wink, Blount, Krishna, & Hanbauer, 1996; Nishibayashi, Asanuma, Kohno, Gomez-Vargas, & Ogawa, 1996; Poderoso et al., 1999; Rubbo et al., 2000; Sreejayan & Rao, 1997). Recently, using the chemiluminescence-based method with the multisyringe flow injection analysis (MSFIA), the NO scavenging capacity for some antioxidants was evaluated; however the MSFIA method has a few experimental disadvantages such as the low selectivity for NO and the requirement of careful choice of experimental conditions (Miyamoto et al., 2007; Ribeiro, Magalhases, Segundo, Reis, & Lima, 2010). In living biological systems, a high level of NO and its oxidised derivatives such as peroxynitrite are

⇑ Corresponding author. Fax: +81 862517853. E-mail address: [email protected] (Y. Sueishi). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.05.036

known to be toxic (Szabo & Thiemermann, 1994), but the ability of antioxidants to scavenge NO has been hardly evaluated. In biological systems, NO is generated by catalytic action of nitric oxide synthase (NOS) on L-arginine and the role of NO such as endothelium-derived relaxation factor (EDRF) has become an important subject of research (Moncada & Higgs, 1993; Palmer, Ferrige, & Moncada, 1987; Tsuchiya et al., 1999). However, in pathological situations NO is also known to injure cells and tissues at relatively high concentrations (Szabo & Thiemermann, 1994). Although in the detection of short lived free radicals EPR spin trapping technique has been widely used, common spin-trapping agents seem to be incapable of trapping NO (Arroyo & Kohno, 1991; Janzen, 1971). Alternatively, as a method to detect and quantify NO, the reaction of NO with nitronyl compounds has been utilised to form stable nitroxide. The compound called PTIO (2phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide) has been used for such a purpose (Scheme 1) (Akaike et al., 1993). The formation of PTI in Scheme 1 is the evidence of NO presence. In the present study, using PTIO as an NO detection agent, we measured NO-scavenging capacity and applied it to various hydrophilic antioxidants: i.e., uric acid, caffeic acid, genistein, glutathione, N-acetylcysteine (NAC), and trolox. The amount of NO was quantified and the decrease of NO level by the presence of the antioxidant was converted into NO-scavenging capacity. We speculate that NO-scavenging reaction proceeds through a redox mechanism but not free radical mechanism. Thus, oxidation potentials for the antioxidants were measured. The results indicated that NO reacts with the hydrophilic antioxidants depending on their chemical properties such as redox potential.

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containing PTIO (0.2 mM) and antioxidant (for example [trolox] = 32 lM) was prepared in phosphate buffer. The sample solution was mixed with NONOate solution (final pH = 7.4) and after 1 min incubation period, EPR signals were recorded at 298 K: Sweep time 30 s, time constant 0.1 s, microwave power 5 mW. Using an attached computer program (WIN-RAD system, Radical Research, Inc. (Hino, Japan)), we calculated the relative abundance (concentration) of two components with a computer-assisted double integration routine of the first-derivative signal of each component. The concentration of PTIO for the observed EPR signals was determined based on the calibration line of PTIO. Subsequently, relative NO-scavenging rates of PTIO and AOx were calculated using the concentration of PTIO and antioxidant following the procedure described in Section 2.4.

Scheme 1.

2. Materials and methods 2.1. Reagents 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) for an assay of NO was purchased from Tokyo Chemical Industry Co. (Tokyo, Japan) (Fig. 1). Six natural antioxidants used in this study are shown in Fig. 1. Trolox, reduced glutathione, 3,4-dihydroxycinnamic acid (caffeic acid) and N-acetylcysteine (NAC) were obtained from Wako Pure Chemicals (Osaka, Japan). Genistein and uric acid were purchased from Tokyo Chemical Industry Co. and Nacalai Tesque (Kyoto, Japan), respectively. As a source of NO, diethylamine NONOate sodium salt (NONOate) was obtained from Aldrich Chemical Company, Inc. (Milwaukee WI, USA). Water was purified by distillation and 100 mM sodium phosphate buffer (pH = 7.4) was used as a solvent.

2.3. Electrochemical measurements for hydrophilic antioxidants Electrochemical measurements for antioxidants were carried out by rotating disk electrode voltammetry (RDE) on phosphate buffer (pH = 7.4, [AOx] = 1.0  103 M and [NaClO4] = 1 M) at 298 ± 1 K by using a RRDE-3A rotating ring disk electrode (BAS) and an electrochemical analyser model 701 D (BAS). A grassy-carbon rotating-disk (2000 rpm), a platinum-wire, and Ag/Ag+ electrode were used as the working, counter, and reference electrodes, respectively. The oxidation wave of K3Fe(CN)6 was observed at +210 mV vs. Ag/Ag+. 2.4. Kinetic analysis

2.2. EPR measurement and procedure The reaction of NO with PTIO was monitored with EPR spectrometry. A JEOL-FE3XG spectrometer (Akishima, Japan) equipped with a 100 kHz field modulator was used for EPR measurements. A typical experimental procedure was as follows. NONOate was dissolved in basic aqueous solution to make a stock solution: [NONOate] = 50 mM [NaOH] = 0.1 M (M = mol dm3). A solution

A competitive reaction against NO between PTIO and antioxidant was used to determine relative NO-scavenging rates (Buettner & Mason, 1987; Kohri et al., 2009). Kinetic formulation of the competition reaction between the radical scavenging reagent (PTIO in this case) and antioxidant has been published elsewhere (Kohri et al., 2009). In the presence of PTIO and antioxidants, the following NO-scavenging reactions should occur:

PTIO þ NO ! PTI þ NO2 ; O N

O -

O

N O

The relative rate for NO-scavenging can be expressed as follows:

N

N

d½AOx=dt kAOx ½AOx ¼ d½PTIO=dt kPTIO ½PTIO

NONOate

PTIO

O

HO O

HS

COOH

NH2

OH

d½AOx=dt Sb  Sa ¼ d½PTIO=dt S0  Sb

N-Acetylcysteine Trolox HO

O

CH CHCO2H

OH O

Caffeic acid

HOOC H2 N

N H

Glutathione

OH

Genistein

HS O

H N O

COOH

O

H N N H

ð2Þ

In this study, the concentrations of PTIO and AOx were kept low so that the reaction between them could be neglected, and thus excess amounts of PTIO and AOx for the NO radical were not present. The concentrations of AOx and PTIO at a given time can be expressed by

(NAC)

HO

ð1Þ

When S0, Sa, and Sb are the computer-mediated double-integrated values for EPR signals of PTIO alone, PTIO + NO, and PTIO + AOx + NO, respectively, the relative NO-scavenging rates for PTIO and AOx can be given by:

Antioxidants (AOx)

HO

rate constant kPTIO

AOx þ NO ! EPR silent product; rate constant kAOx

O NH N H

O

Uric acid

Fig. 1. Structures of PTIO, NONOate and various hydrophilic antioxidants.

½AOx ¼ ½AOx0  aðSb  Sa Þ; ½PTIO ¼ aSb ;

ð3Þ ð4Þ

where [AOx]0 denotes the initial concentration of AOx. The constant a was obtained from the calibration line of PTIO. Using Eqs. (2)–(4), we can rewrite Eq. (1) as:

Sb  Sa kAOx ½AOx0  aðSb  Sa Þ ¼ S0  Sb kPTIO aSb

ð5Þ

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Eq. (5) is the final equation and the plot of ðSb  Sa Þ=ðS0  Sb Þ against f½AOx0  aðSb  Sa Þg=aSb will give a zero-crossing line with the slope kAOx/kPTIO. Using the kAOx/kPTIO values, the results for the NO-scavenging capacities will be expressed in trolox-equivalent units.

3. Results and discussion 3.1. EPR spectrum of PTIO First, we confirmed that NO-scavenging rate constants for AOx and PTIO did not depend on the NONOate concentrations, suggesting that the direct reaction between NONOate and PTIO is negligible. Fig. 2a shows that PTIO (AN = 0.82 mT) was the sole EPR active species before mixing with NONOate. After the addition of NONOate, PTIO’s EPR intensity decreased and the EPR signal from the product PTI (AN = 0.98 and 0.43 mT) appeared (Fig. 2b). When all PTIO, NONOate and AOx were mixed, the EPR peak intensity of PTI was decreased, as compared with that in the absence of AOx (Fig. 2c), indicating part of NO was scavenged by adjusting the relative intensities of PTIO and PTI by the antioxidant. The EPR spectral patterns were readily reproduced by computer spectrum simulation (Fig. 2b and c). PTIO radical concentration was calculated using the simulated spectra, from which relative NO-scavenging rates for PTIO and AOx were calculated according to Eq. (5).

3.2. Evaluation of NO-scavenging ability Fig. 3 shows a typical plot in the trolox/PTIO system. As predicted by Eq. (5), the plot of the relative NO-scavenging rates gives a straight line that passes through the origin, indicating that the reaction scheme and the calculation procedures of relative NOscavenging rate constants (kAOx/kPTIO) using Eq. (5) are justified. The ratios of NO-scavenging rate constants of six hydrophilic antioxidants against PTIO are listed in Table 1. The kAOx/kPTIO value for each antioxidant is normalised using the standard antioxidant trolox, and the results are presented in trolox equivalent unit (Table 1) together with AAPH-derived oxygen radical scavenging capacities (Kohri et al., 2009). The results are also illustrated in a bar graph in Fig. 4. Among the antioxidants tested, uric acid showed the highest NO-scavenging capacity and genistein showed the lowest. Glutathione and NAC did not show NO-scavenging ability. The order of the NO-scavenging capacity is uric acid > caffeic acid > trolox > genistein (>glutathione  NAC = 0). Oxygen radical scavenging capacity (ORAC) for those antioxidants that was determined with the ORAC-EPR method was in the order of uric acid > caffeic acid > trolox > glutathione > genistein  NAC (Kohri et al., 2009). It is of interest to note that uric acid showed the highest values  for both NO and oxygen radical (RO radical). Serum uric acid concentration in healthy human is relatively high (average 40 mg/l (i.e. 0.24 mM) (Haldane, 1955)); therefore, it is possible that endogenous uric acid plays a patho-physiological role by scavenging both NO and oxygen radical.

Fig. 2. (a) EPR spectrum of PTIO (0.2 mM) before mixing NONOate. (b) EPR spectrum obtained after mixing NONOate (1 min incubation period) and computer-simulated spectra. (c) EPR spectrum obtained after mixing trolox (32 lM) and NONOate, and computer-simulated spectra.

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0.6

0.2 0

0.02 0.04 0.06 0.08 0.1 {[AOx] 0- (Sb-Sa)}/ Sb

Fig. 3. A plot of ðSb  Sa Þ=ðS0  Sb Þ against f½AOx0  aðSb  Sa Þg=aSb according Eq. (5). The slope represents the ratio of NO-scavenging rate constants kAOx/kPTIO.

3.3. NO-scavenging rate and mechanism The rate constants of PTIO reaction with NO was determined to be 5.15  103 M1 s1 and this value was used to calculate the NOscavenging rate constants for the present antioxidants (Table 1) (Akaike et al., 1993). Thus, the rate constants of NO-scavenging reaction by the antioxidants ranged from 7.21  103 (genistein) to 9.58  104 (uric acid) M1s1. The order of oxygen radical scavenging capacity (ORAC) having a free radical mechanism indicates that the agreement with the NO-scavenging capacity is only fair. In Table 1, the ORAC values of glutathione and genistein became smaller with increasing the bond dissociation energies of glutathione S–H (367 kJ mol1) and genistein O–H (369 kJ mol1) (Rauk et al., 1999; Zielonka, Gebicki, & Grynkiewicz, 2003), conversely the NO-scavenging capacity of genistein is larger than that of glutathione. We assumed that NO scavenging reactions by the phenolic antioxidants are via redox reactions but not free radical mechanism. However, nitric oxide is operative as the electron-donor and accepter in the chemical behaviour because of the redox flexibility (Hughes, 1999). In such a case, the investigation of oxidation potentials of antioxidants may be helpful in understanding the reaction processes of NO with antioxidants. We measured oxidation potential E1/2(ox) of the antioxidants and the data are listed in Table 1. It appears that 200 mV is a threshold E1/2(ox) and the compound with E1/2(ox) smaller than 200 mV showed negligible NO-scavenging ability. The rate constants for NO scavenging reactions increase with an increase in the E1/2(ox) values, suggesting that the NO’s reactivity can be characterised with reduction of AOx.The primary chemical process can be expressed by the following electron-transfer reaction from NO to AOx (Hughes, 1999):

NO-scavenging capacity (vs.trolox)

Fig. 4. A bar graph for NO scavenging capacities (in PTIO and trolox equivalent unit) and ORAC-EPR values for the six antioxidants.

3 2 1 0

3

4

5 6 E -E1/2(ox) / eV

Fig. 5. The relationship between trolox-equivalent NO-scavenging capacities and DE  E1=2 ðoxÞ values of hydrophilic antioxidants.

DE  E1=2 ðoxÞ, where DE is transition energy obtained from the absorption band in the UV region (Dodsworth & Lever, 1986; Okuno, Kita, Kashiwabara, & Fujita, 1989). Therefore, a smaller value of DE  E1=2 ðoxÞ means an easier electron-transfer to the antioxidant. The DE  E1=2 ðoxÞ in hydrophilic antioxidants calculated from the present data are listed in the far right column of Table 1. The plot of the NO-scavenging capacity vs. DE  E1=2 ðoxÞ values shows a good correlation (Fig. 5), supporting the notion that antioxidants’ NO scavenging reaction is based on the redox interaction.

NO þ AOx ! NOþ þ reduced AOx !! : Though in the present study the reduced form of antioxidant could not be detected because of the rapid reaction with other species present in the medium, Galano et al. suggested the AOx-reduced form for the antioxidant reaction as intermediate species (Edge, El-Agamey, Land, Navaratnam, & Truscott, 2007; Galano, Vargas, & Martinez, 2010). We hypothesised that antioxidant’s electron acceptance capacity may be expressed in terms of Table 1 Relative NO radical scavenging rate constants and ability as measured with PTIO. Antioxidant

Uric acid Caffeic acid Trolox Genistein Glutathione NAC a

ROa

NO 1

kAOx/kPTIO (vs. PTIO)

kAOx/M

18.6 ± 0.6 8.8 ± 0.4 7.3 ± 0.3 1.4 ± 0.2 0 0

9.58  104 4.53  104 3.76  104 7.21  103 0 0

s

1

vs. trolox

vs. trolox

2.5 ± 0.2 1.2 ± 0.1 1.0 0.19 ± 0.03 0 0

2.2 1.6 1.0 0.43 0.57 0.40

Alkoxyl radical generated from the AAPH decomposition (Kohri et al., 2009).

E1/2(ox) (mV)

kmax (nm)

DE (eV)

DE  E1=2 ðoxÞ (eV)

470 310 230 195 10 <10

291 311 288 264 205 207

4.257 3.987 4.301 4.692 6.042 5.984

3.79 3.67 4.07 4.50 6.05 >5.99

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