Food Chemistry 172 (2015) 640–649

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Hydroxyl radical reactions and the radical scavenging activity of b-carboline alkaloids Tomás Herraiz ⇑, Juan Galisteo Instituto de Ciencia y Tecnología de Alimentos y Nutrición (ICTAN), Spanish National Research Council (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain

a r t i c l e

i n f o

Article history: Received 8 May 2014 Received in revised form 2 August 2014 Accepted 16 September 2014 Available online 28 September 2014 Chemical compounds studied in this article: Norharman (PubChem CID: 64961) Harman (PubChem CID: 5281404) 1,2,3,4-Tetrahydro-beta-carboline (PubChem CID: 107838) 1,2,3,4-Tetrahydro-beta-carboline-3carboxylic acid (PubChem CID: 98285) 1-Methyl-1,2,3,4-tetrahydro-betacarboline-3-carboxylic acid (PubChem CID: 73530) 1-Methyl-1,2,3,4-tetrahydro-beta-carboline (PubChem CID: 91522) Melatonin (PubChem CID: 896) Pinoline (PubChem CID: 1868) 6-Hydroxydopamine (PubChem CID: 4624) Ascorbic acid (PubChem CID: 54678501)

a b s t r a c t b-Carbolines are bioactive pyridoindole alkaloids occurring in foods, plants and the human body. Their activity as hydroxyl radical (OH) scavengers is reported here by using three different methods: deoxyribose degradation, hydroxylation of benzoate and hydroxylation of 20 -deoxyguanosine to give 8-hydroxy20 -deoxyguanosine (8-OHdG) as assessed by RP-HPLC (MS). Fenton reactions (Fe2+/Fe3+ plus H2O2) were used for OH generation, and the radical increased in the presence of ascorbic acid or 6-hydroxydopamine as pro-oxidants. b-Carbolines were scavengers of OH in the three assays and in the presence of pro-oxidants. Tetrahydro-b-carboline-3-carboxylic acids were active against the hydroxylation of 20 -deoxyguanosine. b-Carbolines reacted with hydroxyl radicals (OH) affording hydroxy-b-carbolines, whereas tetrahydro-b-carbolines gave oxidative and degradation products. On the basis of IC50 and reaction rates (k), b-carbolines (norharman and harman), and tetrahydro-b-carbolines (tetrahydro-b-carboline, 1-methyltetrahydro-b-carboline and pinoline) were good OH radical scavengers and their activity was comparable to that of the indole, melatonin, which is an effective hydroxyl radical scavenger and antioxidant. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Hydroxyl radical scavengers Antioxidants b-Carboline alkaloids Tetrahydro-b-carbolines Pyridoindoles Indoles Melatonin Deoxyribose Benzoate 8-Hydroxy-20 -deoxyguanosine

1. Introduction Reactive oxygen species (ROS) are involved in human diseases (Halliwell & Gutteridge, 1999). A major source of ROS within cells is the mitochondria where molecular oxygen (O2) is incompletely  reduced to superoxide anion (O 2 ). The superoxide anion (O2 ) is dismutated by superoxide dismutase (SOD) to form H2O2 that ⇑ Corresponding author. Fax: +34 915644853. E-mail addresses: [email protected], [email protected] (T. Herraiz). http://dx.doi.org/10.1016/j.foodchem.2014.09.091 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

interacts with transition metals, such as Fe2+ or Cu+, to produce hydroxyl radicals (OH). Hydroxyl radicals (OH) cause much oxidative damage in biomolecules. These radicals subtract hydrogen atoms from lipids, which after reaction with oxygen give peroxyl radicals (ROO) and initiate lipid peroxidation. They also react with DNA and cause chemical modifications in sugars, purines and pyrimidines, resulting in DNA mutations, as occur with the formation of 8-hydroxydeoxyguanosine (8-OHdG) from 2-deoxyguanosine (Halliwell & Gutteridge, 1999). The cell uses enzymatic defenses against ROS, and also endogenous and dietary

T. Herraiz, J. Galisteo / Food Chemistry 172 (2015) 640–649

antioxidants, such as vitamins, carotenoids, flavonoids and phenolic acids, among others (Halliwell & Gutteridge, 1999; Shahidi & Ho, 2007). In this regard, indole and pyridoindole molecules have attracted attention (Herraiz & Galisteo, 2003, 2004; Juranek, Horakova, Rackova, & Stefek, 2010; Matuszak, Reszka, & Chignell, 1997). Among them, the most studied is melatonin, which is a potent radical scavenger and antioxidant that improves a number of diseases related to oxidative stress (Galano, Tan, & Reiter, 2013; Poeggeler et al., 1994; Sanchez-Barcelo, Mediavilla, Tan, & Reiter, 2010; Singhal, Srivastava, Agrawal, Jain, & Singh, 2012). b-Carbolines (bCs) are naturally-occurring pyridoindole alkaloids (Fig. 1) produced from indoleamines and aldehydes or a-ketoacids (Herraiz, 2004b). These alkaloids occur in foods and plants, and also appear in biological tissues and fluids (Herraiz, 1998, 2004a,b; Herraiz, González, Ancín-Azpilicueta, Arán, & Guillén, 2010; Robinson, Anderson, Crosby, Nutt, & Hudson, 2003; Rommelspacher, May, & Susilo, 1991; Zhao et al., 2012). b-Carbolines exhibit a wide range of biological, pharmacological and toxicological activities. They inhibit monoamine oxidase (MAO), monoamine uptake, and bind to benzodiazepine and serotonin receptors (Cao, Peng, Wang, & Xu, 2007; Herraiz & Chaparro, 2006; Herraiz et al., 2010; Robinson et al., 2003; Rommelspacher et al., 1991). These compounds exert protective effects linked to enzyme inhibition and antioxidant actions (Bi, Cai, Liu, BaudyFloc’h, & Bi, 2007; El Gendy, Soshilov, Denison, & El-Kadi, 2012; Herraiz & Chaparro, 2006; Herraiz & Galisteo, 2003; Herraiz & Guillén, 2011; Kim, Jang, Han, & Lee, 2001; Moura, Richter, Boeira, Henriques, & Saffi, 2007; Wernicke et al., 2010). In this regard, this research investigates the antioxidant activity of b-carbolines as scavengers of hydroxyl radicals (OH) as well as the reactions of these compounds with these radicals. For this purpose, three different chemical assays were implemented and subsequently used to

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detect hydroxyl radicals (OH). These were based on the aromatic hydroxylation of benzoate, deoxyribose degradation and formation of 8-hydroxydeoxyguanosine (8-OHdG) from 20 -deoxyguanosine followed by HPLC-DAD-MS. As a result, it has been proven for the first time that the b-carboline alkaloids occurring in foods, plants and biological systems are good direct scavengers of hydroxyl radicals (OH).

2. Materials and methods 2.1. Reagents and chemicals Ascorbic acid, FeSO4, FeCl3 and benzoic acid were obtained from Merck (Darmstadt, Germany). 20 -deoxyguanosine (2dGua), 8hydroxy-20 -deoxyguanosine (8-OHdG), EDTA, 6-hydroxydopamine, thiobarbituric acid (TBA), 1,1,3,3-tetramethoxypropane (malondialdehyde) were from Sigma-Aldrich (St. Louis, MO, USA). 3Hydroxybenzoic acid, 4-hydroxybenzoic acid and 2-deoxy-D-ribose from Fluka (St. Louis, MO); dimethyl sulfoxide (DMSO) and ethanol from Scharlau (Spain), mannitol from Riedel-de Haen (St. Louis, MO); trichloroacetic acid and hydrogen peroxide (H2O2) from Panreac (Spain). The indole melatonin was obtained from Acros (Belgium), and the b-carboline alkaloids, harman (1-methyl-9H-pyrido[3,4-b]indole), norharman (9H-pyrido [3,4-b] indole or b-carboline), (1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid (MTCA), pinoline (6-methoxy-1,2,3,4-tetrahydro-b-carboline) and 1,2,3,4-tetrahydro-b-carboline (THbC) were obtained from Sigma–Aldrich (St. Louis, MO). 1,2,3,4-Tetrahydro-bcarboline-3-carboxylic acid (THCA) and 1-methyl-1,2,3,4-tetrahydro-b-carboline (MeTHbC) were synthesised by a Pictet–Spengler reaction (Herraiz, 1998). b-Carbolines and melatonin had purity

Fig. 1. Structures of b-carboline alkaloids occurring in foods, plants and biological systems. 1,2,3,4-Tetrahydro-b-carboline-3-carboxylic acid (THCA); 1-methyl-1,2,3,4tetrahydro-b-carboline-3-carboxylic acid (MTCA); 1,2,3,4-tetrahydro-b-carboline (THbC); 1-methyl-1,2,3,4-tetrahydro-b-carboline (MeTHbC), 6-methoxy-1,2,3,4-tetrahydrob-caboline (pinoline); 9H-pyrido[3,4-b]indole (norharman), 1-methyl-9H-pyrido[3,4-b]indole (harman) and N-acetyl-5-methoxytryptamine (melatonin).

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higher than 95% and were dissolved in milli-Q water, buffered media or a slightly acidic solution. Benzoic acid was dissolved in a slightly alkaline solution. 2.2. Aromatic hydroxylation of benzoate by hydroxyl radicals (OH) and scavenging by b-carbolines The hydroxylation of benzoate by hydroxyl radicals (OH) generated in the Fenton reaction (Gutteridge, 1987; Halliwell & Gutteridge, 1999) was studied and the products analysed by HPLC and HPLC-MS. The optimal conditions were set as follows: Eppendorf tubes (final reaction volume 1 ml) containing (in this order) FeSO4 (50 lM), EDTA (30 lM), benzoate (150 lM), antioxidant substance (b-carboline or melatonin) in a concentration ranging from 0 to 1 mM, phosphate buffer (pH 7.2) (4 mM), and H2O2 (500 lM), were incubated at 37 °C for 60 min, and then frozen until injected into the HPLC or HPLC-MS, the same day. Assays were performed at least in quadruplicate. In addition, assays were also carried out with ethanol, DMSO and mannitol as radical scavengers instead of b-carbolines. In a modification of the method, incubation media also included a pro-activating agent (i.e. species reducing Fe3+ to Fe2+ or generating H2O2) (Nappi & Vass, 1997), which was added before the buffer. These agents were ascorbic acid (25–100 lM) or 6-hydroxydopamine (5–15 lM). The antioxidant concentration at which the reduction of hydroxylation by OH radicals was a 50% (IC50) was calculated by fitting to a non-linear regression of percentage inhibition for the aromatic hydroxylation against concentration using GraphPad Prism 4.0. The reaction rate constants (k) of the compounds with OH were calculated indirectly (Halliwell & Gutteridge, 1999) by a linear regression of the representation of 1/A (response) versus the concentration of antioxidant, allowing a straight line with slope: k/(ko[S]Ao), where k is the apparent reaction constant of the antioxidant with OH radicals, [S] is the concentration of the substrate (benzoate), 1/A0 is the intercept of the line, and k0 is a constant of reaction of OH with benzoate (3.3  109 M1 s1) (Gutteridge, 1987). The reaction rates calculated with this method are valid for the experimental conditions used and afford acceptable values (Gutteridge, 1987). 2.3. Degradation of 2-deoxyribose by hydroxyl radicals (OH) and scavenging by b-carbolines Hydroxyl radicals (OH) react with 2-deoxyribose, and the degradation products can be determined by absorbance (Halliwell & Gutteridge, 1999). The generation of degradation products is proportional to the oxidation of 2-deoxyribose by OH and decreases in presence of antioxidants (Gutteridge, 1987; Halliwell & Gutteridge, 1999). Hydroxyl radicals were generated by the Fenton reaction in 1 ml final volume, containing in order: phosphate buffer (pH 7.2) (25 mM), EDTA (100 lM), FeCl3 (100 lM), 2-deoxyribose (3 mM), antioxidant substance (b-carboline or melatonin) at concentrations ranging from 0 (absence) to 1 mM, and ascorbic acid (100 lM). The reaction was initiated upon addition of H2O2 (1 mM), and the samples were incubated at 37 °C for 60 min. Alternatively, in some experiments, ascorbic acid was replaced by 6-hydroxydopamine (100 lM). Assays were performed in quadruplicate and controls were carried out for the absence of reactive species (Fe3+, H2O2 or 2-deoxyribose). Assays were also carried out with ethanol and mannitol as radical scavengers instead of b-carbolines. After incubation, the degradation products of 2-deoxyribose were determined by colorimetric reaction with thiobarbituric acid (TBA). For this, 0.5 ml was taken off the incubation mixture and added to 0.25 ml of TBA (1% solution in 50 mM NaOH), 0.25 ml of trichloroacetic acid (2.8% solution) and 2 ml of Milli-Q water. The tubes were sealed and heated at 100 °C for 15 min; then, cooled on ice and the absorbance measured at 532 nm. Absorbance

measurements were converted into concentration of malondialdehyde (MDA) by using a calibration curve obtained with known concentrations of MDA. The incubation media was also analysed by HPLC to follow the reaction of antioxidants. IC50 values (concentration of antioxidant which inhibits the degradation of 2-deoxyribose by 50%) were calculated with the program GraphPad prism. The reaction rate (k) was determined indirectly by using the same procedure described above for the aromatic hydroxylation of benzoate, using a linear regression 1/absorbance (532 nm) versus concentration of the antioxidant (Halliwell & Gutteridge, 1999). The rate constant k0 of OH with 2-deoxyribose was 1.9  109 M1 s1 (Gutteridge, 1987). 2.4. Reaction of 20 -deoxyguanosine (2dGua) with hydroxyl radicals (OH) and scavenging by b-carbolines The reaction of 20 -deoxyguanosine (2dGua) with hydroxyl radicals (OH) was studied and the formation of 8-hydroxy-20 -deoxyguanosine (8-OHdG) determined by HPLC and HPLC-MS. 8-OHdG is considered a good biomarker for the oxidative damage of DNA by hydroxyl radicals (Seet et al., 2010). The experimental conditions for the reaction of 2dGua with OH radicals were set as follows: Eppendorf tubes with a 1 ml final volume, containing phosphate buffer pH 7.2 (25 mM), 20 -deoxyguanosine (2dGua) (150 lM), the antioxidant compound (b-carboline or melatonin) in concentrations ranging from 0 (absence) to 1 mM, FeCl3 (100 lM), EDTA (100 lM), and ascorbic acid (200 lM), or instead 6-hydroxydopamine (200 lM). To initiate the reaction, H2O2 (1 mM) was added and the samples incubated at 37 °C for 60 min. Assays were also carried out with ethanol and mannitol as radical scavengers instead of b-carbolines. Assays were performed in triplicate, and after incubation the samples were frozen until injection into HPLC the same day. The IC50 values were calculated by non-linear regression of the percentage of inhibition of 8OHdG versus the concentration of antioxidant. 2.5. Analysis by RP-HPLC and RP-HPLC-MS (ESI) 2.5.1. Assay of aromatic hydroxylation of benzoate The chromatographic analyses of the hydroxylation products of benzoate, and the oxidation products of b-carbolines and melatonin, were performed by RP-HPLC. The apparatus was a Hewlett–Packard HPLC 1050 series with a diode array detector (DAD) series 1100 (Agilent) and 1046A fluorescence detector. For chromatographic separation, a 150 mm  3.9 mm, 4 lm, Nova-pak C18 column (Waters, Milford, MA, USA) was used. The chromatographic conditions were as follows: eluent A: 50 mM ammonium phosphate buffer, pH 3; and eluent B, 20% of eluent A in acetonitrile. A linear gradient was programmed from 100% A (0% B) to 32% B in 8 min and then 90% B at 18 min. The flow rate was 1 ml/min, the temperature was 40 °C and the injection volume was 20 ll. The products of hydroxylation of benzoate (i.e. 3- and 4-hydroxybenzoic acid) were detected by absorbance (DAD) at 254 nm (Fig. 2A). Under the conditions used, the retention time was 6.4 and 5.5 min for 3-hydroxybenzoic acid and 4-hydroxybenzoic acid, respectively. Absorbance (254 and 280 nm) and fluorescence were used for the detection of tetrahydro-b-carbolines and melatonin (excitation at 270 nm, emission at 343 nm) and aromatic b-carbolines (excitation at 300 nm, emission at 433 nm). The concentrations of hydroxybenzoates in the reaction media were determined from calibration curves of absorbance (254 nm) as a function of the concentration of 3-hydroxybenzoic or 4-hydroxybenzoic acid standards. Reaction media of benzoate and hydroxyl radicals (OH) were also analysed by liquid chromatography coupled to mass spectrometry (HPLC-MS). For that, a Hewlett-Packard (HP) 1100 series with a diode array detector (DAD) and a quadrupole mass analyser

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4

2

4

3-OH-Benzoate

8

6

2´-deoxyguanosine

0

6

mAU 500

10

12 min

10

12

Benzoate

30

(B)

2

(b)

3-OH-Benzoate

mAU

4-OH-Benzoate

(a)

30

4-OH-Benzoate

(A)

Benzoate

mAU

8

mAU 50

min

8-OHdG

40 30 20 10 0

220 240 260 280 300 320 340 360 380 nm

0

0

2

4

6

8

min

Fig. 2. (A) RP-HPLC chromatograms (absorbance 254 nm) corresponding to the aromatic hydroxylation of benzoate by hydroxyl radicals (OH) generated in Fenton reactions (Fe2+, H2O2): control reaction (a); and in presence of 6-hydroxydopamine (100 lM) (b). (B) RP-HPLC chromatogram (absorbance at 254 nm) corresponding to hydroxylation of 20 -deoxyguanosine and formation of 8-hydroxy-20 -deoxyguanosine (8-OHdG) by hydroxyl radicals (OH) generated in the Fenton reaction (Fe3+, ascorbic acid, H2O2) as indicated in experimental.

(Hewlett–Packard) (electrospray ionisation) were used. The chromatographic separation was performed on a 150  3.9 mm, 4 lm, Nova-pak C18 column (Waters), using Milli-Q water with 0.5% formic acid as eluent A, and acetonitrile with 0.5% formic acid as eluent B. A linear gradient from 0% to 60% B in 20 min was used. The flow rate was 0.8 ml/min, the column temperature was 40 °C and the injection volume was 50 ll. The drying gas temperature of the mass analyser was 340 °C with a flow of 10 l/min, the nebuliser pressure was 40 psi, the capillary voltage was 4000 V, and fragmentation voltage was 60 V with acquisition from 50 to 1000 amu, working under the negative electrospray mode of ionisation for the detection of hydroxybenzoates (m/z 137) and positive ion mode for b-carbolines.

appeared with 8OHdG and, in these cases, an isocratic elution (100%, 50 mM phosphate buffer, pH 3) was used instead, with 8OHdG eluting at 14 min. The concentration of 8-OHdG was determined from the corresponding calibration curve obtained with a standard of 8-OHdG (concentration range from 0 to 25 lM). Confirmation of the identity of 8-OHdG was done by its characteristic UV–VIS spectrum (DAD) (Fig. 2B), and by co-elution with the appropriate standard. Identification of 8-OHdG was also accomplished by HPLC-MS (electrospray) using the same system and chromatographic conditions as for hydroxylation of benzoate but with electrospray working under positive ion mode of ionisation (m/z 284 (M+H)+, m/z 168 (M+H-116).

3. Results and discussion 2.5.2. Assay of hydroxylation of 20 -deoxyguanosine (2dGua) The analysis of 8-hydroxy-20 -deoxyguanosine (8-OHdG) generated during the reaction of 2-dGua with hydroxyl radicals (OH) was carried out by RP-HPLC-DAD (Fig. 2B). The same equipment, eluents and chromatographic conditions as for hydroxylation of benzoate were employed. 8-OHdG eluted at 3.7 min and was detected at 254 nm. However, in some samples, an interference

3.1. Aromatic hydroxylation of benzoate by hydroxyl radical (OH) and radical scavenging by b-carbolines Hydroxyl radicals (OH) generated in a Fenton reaction (Fe2+ + H2O2) were reacted with benzoate to give hydroxybenzoate isomers. Two products were identified by HPLC-MS, DAD and

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coelution with standards as 3-hydroxybenzoic acid (UVmax at 235 and 296 nm, ESI() at m/z 137 (MH)) and 4-hydroxybenzoic acid (UVmax 255 nm, ESI() at m/z 137 (MH)) (Fig. 2A). Hydroxylation depended on the presence of Fe2+ and H2O2, and 3-hydroxybenzoate concentration was slightly higher than 4hydroxybenzoate, although both products followed the same trend regarding activation/inhibition. Ethanol, mannitol and DMSO inhibited hydroxylation (70%, 50% and 90% inhibition, at 1, 3, and 0.1 mM, respectively) and is in agreement with the involvement of hydroxyl radicals (Halliwell & Gutteridge, 1999; Nappi & Vass, 1997). Aromatic hydroxylation of benzoate by OH radicals was substantially reduced by b-carbolines and melatonin (Fig. 3A–D). Thus, norharman, harman, THbC, MeTHbC, pinoline and melatonin were scavengers of OH, and inhibited hydroxylation in a concentration dependent manner. In contrast, THCA and MTCA showed no activity in the range used. The lowest IC50 values (i.e. best radical scavengers) corresponded to melatonin, harman and norharman, followed by tetrahydro-b-carbolines (Table 1). The apparent second order rate constants (k) reached values of 1010 M1 s1, suggesting that the compounds were good radical scavengers. The presence of ascorbic acid or 6-hydroxydopamine in the Fenton reaction increased the reaction rate of benzoate with OH (Figs. 2A and 3E, G). This pro-oxidant effect arises from increasing OH level by reduction of Fe3+ to Fe2+ (Halliwell & Gutteridge, 1999; Nappi & Vass, 1997) and/or formation of H2O2 (Nappi & Vass, 1997). b-Carbolines and melatonin were radical scavengers also in presence of ascorbic acid and 6-hydroxydopamine as pro-oxidants (Fig. 3F, H). Therefore, they differ from other antioxidants like phenols that can be pro-oxidants in OH-generating systems in presence of transition metals (Dorman & Hiltunen, 2011; Macakova et al., 2012). The antioxidant activity of b-carbolines and indoles as radical scavengers suggests a reaction of these compounds with the radical. This was confirmed here for melatonin as it was degraded to N-acetyl-N-formyl-5-methoxykynuramine (AFMK) and oxindole which is in agreement with other results (Horstman, Wrona, & Dryhurst, 2002) (Fig. 4Aa). Aromatic b-carbolines (norharman and harman) reacted with OH affording hydroxylation products that were identified as hydroxy-b-carbolines by HPLC-MS (ESI+) and DAD (e.g. hydroxynorharman, m/z 185 (M+H)+ and hydroxyharman m/z 199 (M+H)+), with a main product being the corresponding 6-hydroxy-b-carboline (Fig. 4Ab). Remarkably, these products are the same as those found from metabolism by cytochrome P450 enzymes (Herraiz, Guillén, & Arán, 2008). Tetrahydro-b-carbolines reacted with OH to give oxidative and degradation products that were not characterised further, whereas THCA and MTCA were oxidised to aromatic b-carbolines (norharman and harman, respectively) (Fig. 4Ac) that were identified by HPLC-MS (ESI+) (M+H)+ at 169 for norharman and 183 for harman), UV spectrum and co-elution with standards. This latter oxidation was not accompanied with radical scavenging activity. Fig. 4B gives a scheme of these reactions. 3.2. Degradation of 2-deoxyribose by hydroxyl radicals (OH) and radical scavenging by b-carbolines Hydroxyl radicals (OH) generated in the Fenton reaction (Fe3+ + ascorbic acid + H2O2) reacted with 2-deoxyribose to give degradation products that were determined by absorbance as TBA-reaction products. In this system, b-carbolines and melatonin inhibited the degradation of 2-deoxyribose by OH (Fig. 5A). Radical scavengers such as ethanol and mannitol also inhibited this reaction although they required higher concentrations (5-10 mM for inhibition higher than 50%). The highest activity (i.e. lowest IC50) (Table 1) corresponded to melatonin, tetrahydro-b-carbolines, harman and norharman, and the lowest to THCA and MTCA. The calculated rate constants reached values of 1010 M1 s1. When

6-hydroxydopamine replaced ascorbic acid in the reaction, a higher degradation of 2-deoxyribose (i.e. higher production of  OH) was observed, but the results regarding scavenging by b-carbolines and melatonin remained (Fig. 5Ac). These data confirmed again that b-carbolines and melatonin were hydroxyl radical (OH) scavengers. 3.3. Reaction of 20 -deoxyguanosine with hydroxyl radicals (OH) and scavenging by b-carbolines A new assay was implemented here to investigate the radical scavenging activity of b-carbolines. It was based on the reaction of OH radicals generated in a Fenton system (Fe3+ + ascorbic acid + H2O2) with the nucleoside 20 -deoxyguanosine (2dGua). In this reaction, 8-hydroxy-20 -deoxyguanosine (8-OHdG) was detected as a main product (Fig. 2B). This compound was identified by HPLC-MS (ESI+) (m/z 284 (M+H)+, 322 (M+K)+, and 168 (M+H116), DAD (kmax at 248 and 295 nm), and co-elution with an authentic standard of 8-OHdG. The formation of 8-OHdG from 2dGua under the experimental conditions averaged 15.3 lM (10% conversion). b-Carbolines (norharman and harman), tetrahydrob-carbolines (THbC and MeTHbC), and melatonin reduced the formation of 8-OHdG (i.e. reaction with OH) in a concentration dependent manner (Fig. 5B). The IC50 values were similar for b-carbolines and melatonin (Table 1). In this case, THCA and MTCA decreased the formation of 8-OHdG to the same extent as other b-carbolines and melatonin. Classical radical scavengers such as mannitol and ethanol were inhibitors of the reaction (70% inhibition at 2 mM). When 6-hydroxydopamine replaced ascorbic acid in the Fenton reaction, qualitative data were similar although b-carbolines provided a slightly higher inhibition (Fig. 5Bc,d and Table 1). Therefore, b-carbolines and melatonin were OH radical scavengers in the presence of 2-deoxyguanosine confirming previous results with deoxyribose and benzoate. b-Carbolines and melatonin reacted with OH giving the products of Fig. 4, as determined by chromatographic analysis. The results described above indicate that b-carbolines are active as hydroxyl radical (OH) scavengers. This conclusion was achieved with hydroxyl radicals generated in Fenton systems by using three different chemical methods of detection: aromatic hydroxylation of benzoate, deoxyribose degradation and hydroxylation of 20 -deoxyguanosine. Detection of OH radicals is a difficult task (Halliwell & Gutteridge, 1999; Li, 2013). Here, three different methods were employed and two (i.e. 20 -deoxyguanosine and benzoate) used HPLC-DAD-MS, allowing for a more specific analysis of radical-reaction products. Deoxyribose degradation and aromatic hydroxylation followed by spectrophotometry have been employed for this purpose (Halliwell & Gutteridge, 1999; Li, 2013; Stoyanova, Geuns, Hideg, & Van den Ende, 2011) whereas the formation of 8-hydroxy-20 -deoxyguanosine (8-OHdG) from 2deoxyguanosine is currently used as a biomarker of oxidative stress and genotoxicity (Seet et al., 2010). In this study, OH radicals were generated in various Fenton systems (Fe2+/H2O2 and Fe3+/ ascorbic acid or 6-hydroxydopamine/H2O2). In those systems, ascorbic acid and 6-hydroxydopamine reduced Fe3+ to Fe2+ exhibiting pro-oxidant effects by increasing OH radicals. In addition, 6hydroxydopamine produces semiquinones, superoxide radicals and H2O2, increasing OH radicals as a result (Nappi & Vass, 1997). b-Carbolines and tetrahydro-b-carbolines decreased the reaction of OH radicals with the test compounds (benzoate, 2deoxyribose and 20 -deoxyguanosine) both in the presence or absence of ascorbic acid or 6-hydroxydopamine as pro-oxidants. MTCA and THCA were poor OH scavengers in the benzoate assay but protected 20 -deoxyguanosine suggesting a different behaviour. As suggested by IC50 values and reaction rates (k), b-carbolines and melatonin reacted with OH radicals in a competitive manner as

T. Herraiz, J. Galisteo / Food Chemistry 172 (2015) 640–649

(A)

(B)

(C)

(D)

(E)

(F)

(G)

(H)

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Fig. 3. Graphs (A–D): scavenging of OH radicals by b-carbolines and melatonin determined as the inhibition of the aromatic hydroxylation of benzoate to give 3hydroxybenzoate (A and B) or 4-hydroxybenzoate (C and D) in presence of increasing concentrations of compounds. Values higher than 15 lM of scavenger were significantly different from the control in the absence of scavengers (p < 0.05), except for THCA and MTCA. Graphs (E and G): increased OH radicals (hydroxylation of benzoate to 4hydroxybenzoate) generated in the Fenton reaction in presence of ascorbic acid (E), or 6-hydroxydopamine (G) as pro-oxidants. Graphs (F and H): scavenging of OH radicals by aromatic b-carbolines or melatonin (100 lM) in presence of ascorbic acid (25 lM) (F), or 6-hydroxydopamine (5 lM) (H). ⁄Values significantly different from control in absence of scavenger (p < 0.05). Values are from quadruplicates. 3-Hydroxybenzoate followed the same trend than 4-hydroxybenzoate shown in graphs.

good radical scavengers. Results obtained with the three assays were qualitatively similar and differences could be attributed to the distinct conditions and chemical characteristics of the assays.

Interestingly, the antioxidant activity of b-carbolines as hydroxyl radical (OH) scavengers was qualitatively and quantitatively comparable to that of melatonin. The rate constant (k) determined

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Table 1 IC50 and reaction rate (k) values determined for b-carbolines and melatonin as OH radicals scavengers. Benzoate hydroxylation IC50 (lM) Norharman Harman Melatonin THbC MeTHbC Pinoline THCA MTCA

s

*

IC50 (asc) (lM)

)

10

26.1 ± 4.6 22.1 ± 3.0 20.5 ± 2.3 44.9 ± 5.0 29.7 ± 3.9 41.0 ± 3.0 – –

1.3  10 2.2  1010 3.7  1010 1.6  1010 1.5  1010 7.0  109

k (M

320 ± 3 301 ± 8 185 ± 1 246 ± 5 278 ± 6

1

1

IC50 (6OHD) (lM)

IC50 (asc)* (lM)

IC50 (6OHD)* (lM)

9

540 ± 23 405 ± 12 463 ± 12

433 ± 66 421 ± 56 405 ± 11 580 ± 26 495 ± 47

253 ± 24 253 ± 23 333 ± 30 294 ± 31 550 ± 16

314 ± 16 243 ± 19

436 ± 38 488 ± 38

s

*

)

9.0  10 1.1  1010 1.7  1010 1.1  1010 1.1  1010

>500 >500

60

0

2

4

Benzoate

AFMK

Oxindole

Benzoate

3-OH-Norharman

OH-Norharman 3-OH-Benzoate

8

6

10

min

Benzoate

(c)

6

10 min

8

harman

4

mtca

2

3-OH-Benzoate

0

6

Norharman

4

(b)

60

mAU

2

4-OH-Benzoate

0

3-OH-Benzoate

(a)

60

mAU

4-OH-Benzoate

mAU

6-OH-Norharman

(A)

Melatonin

Ascorbic acid (asc); 6-hdroxydopamine (6OHD).

4-OH-Benzoate

*

k (M

20 -dGua hydroxylation (8-OHdG)

Deoxyribose degradation

1 1

8

10

min

(B)

Fig. 4. (A) RP-HPLC chromatograms (absorbance at 254 nm) of reaction media generating OH radicals (Fe2+/H2O2/benzoate) in the presence of melatonin (a), norharman (b), or MTCA (c). (B): Reactions of b-carbolines (R: H or CH3) and melatonin with hydroxyl radicals (OH).

T. Herraiz, J. Galisteo / Food Chemistry 172 (2015) 640–649

(A)

647

(B)

Fig. 5. (A) Scavenging of OH radicals by b-carbolines and melatonin as determined by the inhibition of coloured products (A532nm) measured as MDA (malondialdehyde) in deoxyribose degradation. Assays containing ascorbic acid (100 lM) (a and b), or 6-hydroxydopamine (100 lM) (c). Values higher than 50 lM of scavenger were significantly different from control in absence of compound (p < 0.05), except for THCA and MTCA that were higher than 250 lM (p < 0.05). Values are from quadruplicates. (B) Scavenging of OH radicals by b-carbolines and melatonin as determined by inhibition of the hydroxylation of 20 -deoxyguanosine (i.e. formation of 8-OHdG) in presence of ascorbic acid 200 lM (a and b) or 6-hydroxydopamine 200 lM (c and d). Values higher than 250 lM of scavenger were significantly different from control in absence of compound (p < 0.05). Values are from triplicates.

for this indole is consistent with that reported previously (i.e. 2.6–3.7  1010 M1 s1) (Matuszak et al., 1997). Melatonin is a potent OH radical scavenger, and it is effective in numerous in vitro and in vivo studies in which oxidative stress is involved, such as inflammatory diseases, radiation, and neurodegenerative diseases (Sanchez-Barcelo et al., 2010; Singhal et al., 2012). It reacts with OH radicals yielding N-acetyl-N-formyl-5-methoxykynurenamine (AFMK) by cleavage of the indole ring, and affords new products which are also radical scavengers and antioxidants (Galano et al., 2013; Horstman et al., 2002). As melatonin, tetrahydro-b-carbolines also contain an indole ring and were good hydroxyl radical (OH) scavengers whereas aromatic b-carbolines (norharman and harman) scavenged OH radicals giving rise to hydroxy-b-carbolines (Fig. 4). Iron-mediated hydroxyl radicals are involved in diseases linked to oxidative stress (Halliwell & Gutteridge, 1999). Indeed, neurotoxins like 6-hydroxydopamine or N-methyl-4-phenylpyridinium (MPP+) trigger neurodegeneration with hydroxyl radicals involved in their toxic effects (Lin, Meng, Liu, & Zheng, 2013; Saito et al., 2007). In animals models of neurodegeneration, melatonin was protective against OH radicals generated by 6-hydroxydopamine (Borah & Mohanakumar,

2009; Singhal et al., 2012). As seen here, both b-carbolines and melatonin were OH radical scavengers in the presence of 6-hydoxydopamine as a pro-oxidant agent. b-Carboline alkaloids occur in foods and plants (Herraiz, 1998, 2004a; Herraiz et al., 2010), and also in the human body where they appear to accumulate in some regions, and exert biological, pharmacological and toxicological effects (Fekkes & Bode, 1993; Robinson et al., 2003; Rommelspacher et al., 1991; Östergren, Annas, Skog, Lindquist, & Brittebo, 2004). b-Carbolines are monoamine oxidase (MAO) inhibitors and in this regard they may protect against oxidative stress, as the inhibition of MAO results in lower levels of toxic products (aldehydes, ammonia and H2O2) (Herraiz & Chaparro, 2006; Herraiz & Guillén, 2011; Lieu, Chinta, Rane, & Andersen, 2013). In addition, these compounds exert protective effects that could be linked to antioxidant actions (Herraiz & Galisteo, 2003; Herraiz & Guillén, 2011; Kim et al., 2001; Moura et al., 2007; Wernicke et al., 2010). In this regard, these pyridoindoles are good scavengers of hydroxyl radicals (OH) as shown here. Then, if accumulated in tissues, b-carboline alkaloids arising from exogenous sources (i.e. foods and plants) could behave as hydroxyl radical scavengers and antioxidants. Nevertheless, in

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order to be effective as scavengers, b-carbolines need to accumulate and be close to the site of radical generation. b-Carbolines appear to accumulate in some brain regions (Fekkes & Bode, 1993; Östergren et al., 2004), but further studies will be needed to confirm any antioxidant effects. On the other hand, as described here, tetrahydro-b-carbolines and b-carbolines afford new oxidative products by reacting with hydroxyl radicals, and they are also bioactive substances (Herraiz & Chaparro, 2006; Schott, Decker, Rommelspacher, & Lehmann, 2006).

4. Conclusions Three chemical methods were implemented for detection of hydroxyl radicals (OH) generated in Fenton systems. These methods were based on the degradation of deoxyribose, and the aromatic hydroxylation of benzoate and hydroxylation of 20 -deoxyguanosine to 8OHdG as assessed by HPLC-DAD(MS). Subsequently, the activity of naturally-occurring b-carboline alkaloids as hydroxyl radical (OH) scavengers was studied as well as the reactions of these compounds with the radicals. It is shown that aromatic b-carbolines (norharman and harman) and tetrahydrob-carbolines (THbC, MeTHbC and pinoline) were good scavengers of hydroxyl radicals. Tetrahydro-b-carboline-3-carboxylic acids (THCA and MTCA) were active against the hydroxylation of 2-deoxyguanosine to 8-OHdG. b-Carbolines were active scavengers in the presence of 6-hydroxydopamine and ascorbic acid as pro-oxidants in Fenton systems. b-Carbolines reacted with hydroxyl radicals (OH) affording hydroxy-b-carbolines, whereas tetrahydro-bcarbolines gave oxidative and degradation products. The activity of b-carbolines (pyridoindoles) as hydroxyl radical scavengers was qualitatively and quantitatively comparable to that of melatonin that is an effective in vitro and in vivo indole antioxidant and radical scavenger.

Acknowledgements The authors are grateful to CSIC (Spain) (Project 200470E658) and Spanish government (MICINN, Spain) (Project AGL201018448) for supporting this work.

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