Research article Received: 25 June 2014,
Accepted: 3 September 2014
Published online in Wiley Online Library: 6 November 2014
(wileyonlinelibrary.com) DOI 10.1002/bio.2785
Studies on the antioxidant activity of some chromonylrhodanine derivatives Irena Kruk,a Teresa Piechowska,a Paweł Berczyński,a Aleksandra Kładna,b Oya Bozdağ-Dündar,c Meltem Ceylan-Unlusoyc and Hassan Y. Aboul-Eneind* ABSTRACT: Fifteen chromonylrhodamine derivatives (CRs) were synthesized and the antioxidant activity levels were evaluated for the first time. The antioxidant activity potencies of these chromone derivatives were evaluated towards superoxide anion radicals, hydroxyl radicals and 2,2-diphenyl-1-picrylhydrazyl radicals. Also, the total antioxidant capacity of the tested compounds was measured using the ferric-ferrozine assay. The antioxidant activities were investigated using a chemiluminescence (CL) assay, spectrophotometry measurements, direct electron paramagnetic resonance (EPR) and the EPR spin-trapping technique. The 5,5-dimethyl- 1-pyrroline-1-oxide (DMPO) was applied as spin trap. Eleven of the 15 chromone compounds exhibited a decrease in the CL accompanying the superoxide anion radical produced in anhydrous dimethylsulfoxide (DMSO), ranging from 71–94% at concentration of 1 mmol /L; four of these compounds enhanced light emission in the range 231–672%. Similarly, these compounds caused 28–58% inhibition in the intensity of the DMPO-OOH radical EPR signal and the DMPO-OH radical (from 12–48%). Furthermore, three of these compounds showed very good antioxidant response towards the DPPH radical (EC50: 0.51–0.56 μmol/L) and the high reduction potentials. These findings demonstrate that the chromone compounds tested may be considered as effective free radicals scavengers, a finding that is of great pharmacological importance. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Chromonylrhodamine derivatives; Radical scavenging activity; Chemiluminescence; EPR study
Introduction Over the past 4–5 decades, oxidative stress has been implicated in oxidative biomolecule damage resulting in deleterious changes and loss of nutrition value (1–3), as well as in the pathogenesis of several human diseases (chronic inflammation, neurodegenerative disorders, atherosclerosis, diabetes mellitus, cancer) (4,5). Oxidative stress is defined as an imbalance between reactive oxygen species (ROS) such as superoxide anion radical (O•2 ); hydroxyl radical (HO•), peroxyl radical (ROO•); hydrogen peroxide H2O2; singlet oxygen (1O2) generation and defence and repair of antioxidant systems. The search for a antioxidant i.e., ‘a substance that when present at low concentrations compared with that of an oxidizable substrate significantly delays or prevents oxidation of that substrate’ (1) has received much attention and is the topic of much recent research (6,7). A recent review (6), as well as studies from our group (8,9), provide evidence that compounds containing the chromone skeleton can act as suitable antioxidants. In this study, we have undertaken for the first time an evaluation of the ROS scavenging activity of some chromonylrhodanine derivatives (CRs), (Fig. 1) to explain the origin of antidiabetic properties.
Experimental Reagents
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All the chemicals and the reagents were of analytical grade. The compounds CR1–CR15 were synthesized by using chromone carboxaldehyde and the rhodanine ring (10). The structure of synthesized chromone derivatives (CRs) was elucidated by
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elementary analysis, 1H NMR and mass spectral data. All reagents were purchased from E. Merck (Darmstadt, Germany) and Aldrich (Milwaukee, MI, USA) except potassium superoxide (KO2) and ammonium ferrous sulfate hexahydrate that were purchased from Fluka (Buchs, Switzerland). The CR compounds, trolox and tiron were dissolved in DMSO, which is suitable for dissolving both water-soluble and water-insoluble chemicals.
Free radicals scavenging assays Superoxide anion radical scavenging activity. Superoxide anion radicals were generated using the reaction between 18-crown-
* Correspondence to: Hassan Y. Aboul-Enein. Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Cairo 12311, Egypt. E-mail: [email protected] a
Institute of Physics, Faculty of Mechanical Engineering and Mechatronics, West Pomeranian University of Technology in Szczecin, Al. Piastów 48/49, 70-311 Szczecin, Poland
b
Department of History of Medicine and Medical Ethics, Pomeranian Medical University, Rybacka 1, 70-204 Szczecin, Poland
c
Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Tandoğan, Ankara, Turkey
d
Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Cairo 12311, Egypt Abbreviations: EPR, electron paramagnetic resonance; ROS, reactive oxygen species.
Copyright © 2014 John Wiley & Sons, Ltd.
Antioxidant activity of some chromonylrhodanine derivatives
Figure 1. Chemical structures of chromonylrhodanine derivatives (CRs).
6-ether and KO2 (11). The reaction mixture contained a 60 mg aliquot of 18-crown-6-ether dissolved in 10 mL of dry DMSO and 7 mg of KO2 was added quickly, to avoid contact with air humidity. This reaction was stirred for 60 min to give a pale yellow solution of 10 mmol/L superoxide anion radical. The concentration of radical was determined using the absorbance at λmax ¼ 251nm ; ε ¼ 2686±29 mol cm-1 : For the measurement, a superoxide anion radical was used as a 1 mmol/L solution in DMSO. Two assays were used to evaluate the activity of CRs towards superoxide anion radical, chemiluminescence (CL) and electron paramagnetic resonance (EPR).
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EPR spectroscopy and spin trapping. The superoxide anion radical scavenging was measured using the previously described spin-trapping methodology (13). The method is based on the reaction of DMPO with superoxide anion radical to yield the DMPO-OOH spin adduct having the half-time about 91 s at pH5 in aprotic solvents. The reaction mixture used consisted of the following reagents: 0.1 mol/L DMPO, 1 mmol/L superoxide anion radical, 1 mmol/L a CR compound. All reagents were dissolved in DMSO. Because the scavenger samples were made up in DMSO, the control reaction also contained an equivalent
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The assay is based on superoxide anion radical-induced light emission in DMSO and has been described previously (12). Briefly, the light emitting reaction was carried out in a thermostated glass cuvette placed in a light-tight chamber. The light intensity was measured using an EMI9553Q photomultiplier (Photek, East Sussex, UK) with an S20 cathode sensitive in the range 200–800 nm, interfaced with Chemiluminescence assay.
a computer for data acquisition and handling. The assay was performed at 294 ± 1 K. The CL signals from the superoxide anion radical/DMSO reaction or that influenced by the presence in it of a test compound was monitored as the kinetic curve of the CL decay. The well known inhibitor of superoxide anion radical – tiron – was used as positive control. The scavenging potency was calculated as the percentage inhibition of the CL intensity Q(%) = [(I∘ I)/I∘] × 100 %, where Io is the light intensity measured in the absence of an inhibitor and I is the light intensity measured in the presence of the inhibitor.
I. Kruk et al. amount of DMSO. The spectra were recorded using a quartz cuvette with an optical path length of 0.25 mm at 293 ± 1 K. The DMPO-OOH signal was analyzed approximately after 1 min from the beginning of the reaction. The scavenging ability was calculated as the percentage inhibition of the EPR signal amplitude Q(%) = [(H∘ H)/H∘] × 100 %, where Ho and H are the relative height of the second peak in the spectrum of the control and H in the presence of a CR compound, respectively, EPR spectra were recorded using a standard X-band spectrometer operating at 9.3GHz with a modulation frequency of 100 Hz of a steady magnetic field. The instrumental conditions were as follows: microwave power 20 mW, modulation amplitude 0.25 mT, time constant 0.3 s, and receiver gain 3.2 × 104. Hydroxyl radical scavenging activity. For the hydroxyl radical generation, the Fenton reaction was used Fe(II) + H2O2 → HO° + HO– + Fe(III) (14,15). As the hydroxyl radical, similar to the superoxide radical, is too short–lived to be detected directly, the spintrapping assay with DMPO as the trap was used. The assay was performed at room temperature. The reaction mixtures in the sample contained the following final concentration of reactants: 0.5 mmol/L H2O2, 10 mmol/L sodium trifluoroacetate buffer pH 6.15, 62.5 μmol/L FeSO4(NH4)SO4 and 25 mmol/L DMPO without a CR compound (control) or with 2.5 mmol/L of a test compound dissolved in DMSO (25% v/v). The scavenging activity was calculated as the percentage inhibition of the DMPO-OH spin adduct amplitude of the second peak in the EPR spectrum. The spectra were analyzed approximately after 4 min from the start of reaction. The conditions of EPR measurement were as follows: microwave power 20 mW, modulation amplitude 0.5 mT, time constant 0.3 s, receiver gain 2.5 × 104.
The DPPH radical scavenging activity was measured by the method described by Nanjo et al. (16) with slight modification, i.e. using the solvent mixture DMSO (25% v/v)/C2H5OH (75% v/v). Reaction mixture contained 0.125 mmol/L DPPH dissolved in ethanol and the tested compounds at various concentrations, dissolved in DMSO. The trolox and vitamin C were used as positive controls. The tested compounds were allowed to react with a very stable radical DPPH showing a characteristic EPR spectrum. The reduction of the DPPH radical was followed by monitoring the decrease in its EPR signal intensity. Antiradical activity was calculated using the following equation R (%) = [(Ho-H) / Ho] × 100%, where H is the relative height of the third peak in the radical EPR spectrum in the presence of the tested compound, Ho is the relative height of the third peak in the spectrum of the control. The R (%) value was plotted against the test compounds concentration. The concentration of the CRs samples, trolox and vitamin C necessary to decrease the initial DPPH° concentration by 50% (IC50) was obtained from the graphs. EPR spectra were recorded at room temperature. The conditions of EPR measurements were as follows: microwave power 20 mW, modulation amplitude 0.2 mT, time constant 0.3 s, and receiver gain 1 · 104. 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH°) scavenging assay.
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Ferric reducing potency assay. The total antioxidant activity measurement was based on an electron transfer from an antioxidant to ferric ion of the Fe (III)–ferrozine complex which is reduced to Fe (II) exhibiting an absorbance at 562 nm. (17). The Fe (III)–ferrozine aqueous complex consisted of 2 mmol/L Fe (III) and 10 mmol/L ferrozine. The reaction mixture contained 0.024 g of NH4Fe(SO4)2 · 12H2O dissolved in 1 mL of 1 mol/L HCl
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and 0.123 g ferrozine dissolved in 3 mL water. After mixing, the mixture was diluted to 25 mL with distilled water. The final concentration of Fe(III) was 2 mmol/L and 0.1 mmol/L ferrozine. The complex was kept in a stoppered dark colored bottle at room temperature. After 30 min standing time the absorbance at 562 nm was measured. Working solutions of reagents were as follows: 1.5 mL of the complex added to 1 mL of antioxidant solution at 1 mmol/L concentration and mixed with 2 mL of buffer (0.2 mol/L acetic acid/sodium acetate, pH 5.5). After 1.5 h standing at room temperature an increase in absorbance at 562 nm was monitored. Trolox (water-soluble vitamin E analog) was used as a positive control. Absorption spectra were acquired using a Carl Zeiss Technology M-40 with Win-Aspect Software (Jena, Germany).All experiments were conducted at least three times and the data are expressed as means ± SD.
Results and discussion Fifteen chromone derivatives, previously synthesized, were examined for their antioxidant activity, i.e. the ability to deactivating superoxide anion radical, hydroxyl radical, and DPPH radical. Also, the total antioxidant capacity was determined using ferrozine as reagent. Two methods were applied to evaluate activity of CR compounds towards superoxide anion radical: chemiluminescence and EPR. Figure 2 summarizes the results obtained using CL measurements. Typical CL kinetics from the superoxide anion radical/DMSO system alone (blank), and in the presence of representative compounds CR2 and CR6 are shown in Fig. 2A. The solution of superoxide anion radical in DMSO emits weak CL that originates from singlet oxygen as follows (18,19) 2O•2 þ 2ðCH3 Þ2 SO→1 O2 þ ðCH3 Þ2 SO2 þ CH3 SOðCHÞ þ OH O•2 →1 O2 þ electron O•2 þ O2 →O•4 O•4 →ð1 O2 Þ þ O•2 21 O2 →ð1 O2 Þ2 →23 O2 þ hυ The superoxide/DMSO reaction connected with CL technique had been successfully adopted for the first time in our laboratory to assess antioxidative properties several antioxidants against superoxide radical in hydrophobic medium (8,9). Compound CR6, similarly as compound CR1, CR3, CR4, CR7, CR9, CR10 and CR12-CR15 (kinetics not shown) showed inhibitory effects. Conversely, compounds CR2, CR5, CR8, CR11 enhanced the light emission (Fig. 2B). The scavenging effect of the CR derivatives was compared to the reference compound tiron (data not shown) that reacts with superoxide anion radical with a high rate constant (~108 L mol–1 s–1(20)). We found that tiron exhibited 96% quenching of CL under our experimental conditions, whereas CR compounds showed a significant decrease (ranging from 71–94%) in the CL intensity. The specificity of the reaction between 18-crown-6-ether and KO2 in DMSO as a source of O2• was also checked by adding 400 U• mL–1 SOD at the start of the reaction and observing a 98% suppression CL (data not shown). The superoxide anion radical is known to promote proton transfer from much weaker acids than water acting as an oxidant, can act as a reducing agent in aprotic solvents, and shows a high nucleophilicity toward SN2 substitution reaction
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Figure 2. Superoxide anion radical scavenging activity of the CRs compounds. (A) Time course of Cl decay in the superoxide anion radical/DMSO reaction (blank) and the CL decay monitored under the same condition as the blank but in the presence of representative chromone derivatives CR2 and CR6 (1 mmol/L). (B) Inhibitory effect of the evaluated CR compounds on the CL intensity. (C) EPR spectrum of the DMPO-OOH spin adduct arising in superoxide anion radical (1 mmol/L) in DMSO and inhibitory effect of CR compounds (1 mmol/L) exerted on this adduct.
(21). According to chemistry of superoxide anion radical and to mechanisms documented in the literature through which compound may act as antioxidant by a radical scavenging (6), the following reactions should be considered: reduction of superoxide anion radical CR þ O•2 → CR•þ þ O2 2 followed by the stabilization of the O2–2 species by DMSO (18) þ O2 2 þ 2H → H2 O2
and donate a hydrogen atom from the ring B of a CR molecule to superoxide anion radical CRðHÞ þ O•2 → CR• þ HO 2
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2HO•2 → 1 O2 þ H2 O2 Compounds CR5 and CR8 reached a very strong light emission enhancing effect at 1 mmol/L concentration. Compounds CR2 and CR11 reached a weaker enhancing effect. In general, compounds CR that exerted the enhancing effect on the CL contain in their structure an ester group linked to the ring B. The observed differences in the CL intensity enhancing potency (CR2 < CR11 < CR5 < CR8) may be due to presence of methyl group/groups in the chromone core. The substituent could change the stabilization of the products formed from CR derivatives, thus their antioxidant potency. This suggestion is in accordance with the finding of Phosrithong et al. (22) that chromone skeleton of an antioxidant is responsible for the stabilization of the antioxidant molecule during electron and/or hydrogen atom transfer. In turn, an electrophilic substituent NO2 (CR13, CR14, CR15) pulls a pair electrons from the ring A. This effect is associated with the positive charge of ring
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Both the above processes may be responsible for the quenching of the light emission directly (compounds CR1, CR3, CR4, CR6, CR7, CR9, CR10 and CR12-CR15). In turn, donation of proton from compounds CR to superoxide anion radical might lead to increasing of the light emission (CR2, CR5, CR8, CR11) as follows:
CRðHÞ þ O•2 → CR þ HO•2
I. Kruk et al. A and the possibility of SN2 substitution reaction occurrence, in accordance with high nucleophilicity of superoxide anion radical. The second method used to elucidate the superoxide anion radical scavenging capacity of CRs was based on their ability to inhibit the DMPO-OOH free radical formation. The DMPO trap forms with superoxide anion radical the adduct nitroxide free radical formed by covalent bonding of the radical with the spin trap. The DMPO-OOH adduct is unstable (the half-life is 91 s at pH5) and decays into nonradical species and DMPO-OH adduct (23,24) having long half-life (over 2 h) and showing the spectrum completely distinct from the DMPO-OH spectrum. For this reason, the DMPO-OOH spectra were recorded after 1 min. The spectrum of DMPO-OOH adduct, consisting of a 12-lines, formed in the 18-crown-6-ether/KO2/DMSO system is shown in Fig. 2C. We observed hyperfine couplings aN = 14.3G, aH = 11.5G and aγH ¼ 1:2G similarly to other (24). At 1 mmol/L CRs at least 28% inhibition in the intensity of the EPR signal was detected, and adding SOD (200 U/mL) decreased the EPR signal by a 98% (data not shown). All the tested CRs were able to scavenge hydroxyl radicals generated in the Fenton reaction in the presence of a spin trap DMPO (Figure 3). The rate constant for hydroxyl radical trapping by DMPO is very high (about 3.4 × 109 L mol–1 s–1) (24). The EPR spectra of the DMPO-OH spin adduct consisted of a quartet signal with hyperfine couplings aN = aH = 14.9 G. In the presence of CRs, the EPR signal decreased with time. The order of the scavenging activities measured for these chromones was CR14 > CR13 > CR3 > CR10 > CR15 > CR6 > CR9 > CR4 = CR8 > CR12 > CR5 > CR2 > CR1 > CR7 > CR11. As is illustrated in Fig. 3 compounds CR5, CR2, CR1, CR7 and CR11 revealed to be less potent providing the hydroxyl radicals scavenging from about 12–18%. We have checked the specificity of the Fenton reaction as a generator hydroxyl radicals using the positive control catalase, an enzyme that decomposes hydrogen peroxide to water. In the presence of 190 μg/mL catalase, the DMPO-OH spin adduct was not observed. These data confirm that the observed EPR spectrum was attributed to the nitroxide formed by hydroxyl radical trapping by DMPO. Since the concentration of DMSO in the reaction mixture was relatively high and its high rate constant of reaction with hydroxyl radical (7 × 109 L mol–1 s–1) (24) were greater than the DMPO concentration and rate constant
(2.1 × 109 M–1 s–1) (25), most hydroxyl radicals would have react with DMSO resulting in relatively poor hydroxyl radical scavenging activity observed for all the tested CRs. Indeed, we found that the observed spectra were a combination of the hydroxyl and CH3 radical adducts with the DMPO trap (data not shown). This finding is consistent with the findings of Ghersi-Egea et al. (25) and others that a secondary radical CH3 derived from DMSO forms of the DMPO-CH3 adduct. Therefore, care should be taken to distinguish between the DMPO-OH signal and the signal of the DMPO adduct of methyl radical. Concerning hydroxyl radical activity, the species is one of the strongest oxidizing reagents able to react with the first encountered biomolecule (1,3,5). This species can act by: (i) adding to a double bond; (ii) reaction with hydrogen–containing molecules followed by H-abstraction, and (iii) rapid electron transfer reaction. The antioxidant activity of the tested compounds was also measured against DPPH free radical (Figure 4, Table 1). The DPPH radical can react via two mechanisms (26,27): (i) a direct abstraction of hydrogen atom, and (ii) an electron transfer process from a molecule to DPPH•. The extent of one or the second pathway is dependent on the redox potential of the compounds involved in the reaction and the solvent polarity (27). In polar solvents one can expect an electron transfer process from the CR compound to DPPH•. Thus this process is predominant under our experimental conditions. A typical spectrum of the DPPH radical and the kinetic behaviour of the EPR amplitude of the DPPH radical signal is shown in Fig. 4. The spectrum monitored is very similar to that reported by other authors (26). Three types of kinetic behaviour were observed. Only compounds CR13-CR15 and trolox–the well known radical scavenger, reacted rapidly with the DPPH radical reaching a steady state in less than 1 min. For compounds CR3 and CR1, the steady state was reached after approximately 3 and 5 min, respectively. The remaining compounds tested exhibited slower kinetics of hyperbolic shape taking from 9 min to 1 h (CR8) to reach a steady state. As indicated in the methods section, the antiradical reactivity of CRs was evaluated from the plot of the percentage DPPH radical remaining against the CR concentration. The amount of a CR compound necessary to decrease the initial DPPH radical concentration by 50% (Table 1) was evaluated after 2 min of reaction time. In Fig. 4 we can observe that in the case
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Figure 3. EPR spectrum of the DMPO-OH spin adduct formed in the Fenton reaction and inhibitory effect of CRs compounds exerted on this adduct.
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Figure 4. Kinetic curves of the DPPH free radical reduction measured in the presence of CR compounds. Inside EPR spectrum of DPPH free radical in DMSO/ethanol mixture of solvents.
Table 1. DPPH free radical scavenging activity of tested chromone derivatives Compound CR1 CR2 CR3 CR4 CR5 CR6 CR7 CR8 CR9
IC50 (μmol L- 1) Compound 229 ± 4 236 ± 3 183 ± 3 495 ± 15 258 ± 4 249 ± 5 841 ± 17 889 ± 21 387 ± 11
CR10 CR11 CR12 CR13 CR14 CR15 Trolox Vitamin C
IC50 (μmol L- 1) 427 ± 14 248 ± 4 253 ± 3 52.3 ± 3.0 51.2 ± 2.1 56.5 ± 2.8 18 ± 1.2 352 ± 19
DPPH: 2,2-diphenyl-1-picrylhydrazyl, IC50: inhibition concentration. IC50 is the mean value of three measurements.
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of those compounds exhibiting rapid kinetic behaviour like trolox and compounds CR13–CR15 the precision of the method in the IC50 determination was high. In the case of the CR compounds where a steady state was achieved later than 2 min, the IC50 inhibition concentrations will be erroneous because reactions were still continued and degradation products were involved. From Table 1 we can note that 10 of 15 CRs examined exhibited higher activity than vitamin C, but less activity than trolox. This means that majority of CRs are more potent radical scavengers than that known good antioxidant – vitamin C. The DPPH radical scavenging activities of these compounds are diverse and comes as follows, starting from highest to lowest: trolox > CR14 = CR13 > CR15 > CR3 > CR1 > CR2 > CR11 = CR6 > CR5 > CR12 > Vitamin C > CR9 > CR10 > CR4 > CR7 > CR8. As mentioned above, medium is an important factor that influences on the reaction CRs with the DPPH radical, and this makes difficult to compare results with literature data. However,
in the case of trolox the IC50 value measured is in good accordance with some authors (4,28), but higher than those reported by Parejo et al. (29) found for tiron and vitamin C. To confirm the antioxidant property of CR compounds the assay of total antioxidant power using ferrozine as reagent was applied. The results of these measurements are shown in Fig. 5. In this method, the examined chromone derivatives were able to reduce Fe(III) ion, in the presence of ferrozine, to the Fe(II)– ferrozine complex. The complex exhibits a very high absorbance at 562 nm. This method is a very sensitive for evaluating antioxidant activity compounds having even a weak reducing potential (17). From the data listed in Fig. 5, it can be supposed that compounds CR3 and CR13–CR15 posses the highest reducing power, higher than trolox. These observations are in accordance with the lowest values of IC50 (Table 1) and the highest scavenging ratios of hydroxyl radical (Fig. 3), measured using the EPR technique. All these compounds inhibited high abilities to scavenge superoxide anion radical. The presence of the 2,3-double bond in conjugation with the 4-oxo function of a carbonyl group in the C-ring in chromone core enables the CR compounds to an electron transfer reaction. The presence of NO2 groups in chromone core (compounds CR13–CR15) may stabilize the products arising from those compounds and their reducing abilities. In conclusion, several methods are used to study the antioxidant activity of various samples, reviewed recently in (30). In the present paper, we have applied to the chromone derivatives tested the techniques which are commonly used in the subject literature, and found to be reproducible and comparable. It is noteworthy that the CL technique is often more sensitive than other assays and exhibits the high stability, rapidity, instrument simplicity. For this reason, the CL method has recently received much attention of researchers which proved useful in biotechnology, pharmacology or bioanalysis (31,32). Our findings confirm the high sensitivity this method in evaluation of CRs reactivity towards superoxide anion radical. We found that the
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Figure 5. Reduction of Fe (III) in the Fe (III) - ferrozine complex to Fe (II) in the presence of CR compounds and the standard antioxidant–trolox.
CR3, CR13–CR15 compounds have shown a high activity as scavengers of DPPH free radical, hydroxyl and superoxide anion radicals and possess high reducing power (IC50 values were only three times higher than that found for trolox). Yet, the antioxidant power of CR3 and CR13–CR15 group of compounds is in good accordance with the hierarchy of their reducing powers, using the ferric-ferrozine method. A unique aspect of the present study was the comprehensive analysis of both antioxidant and reducing powers of the chromone derivatives examined for the first time. This preliminary findings show that at least few of the evaluated CRs are promising for treating diabetes as the β-cells dysfunction is dependent on hydroxyl radical and superoxide anion radical levels (4). Due to overwhelming evidence that antioxidants prevent the occurrence of diseases mediated by ROS, it would be important to identify the reaction intermediates and products, using chromatography.
References
562
1. Aruoma OI. Free radicals and antioxidants strategies in sports. J Nutr Biochem 1995;5:370–81. 2. Namiki M Antioxidants/antimutagens in food. Critical Rev Food Sci Nutr 1990;29:273–300. 3. Sanchez-Moreno C. Review: Methods used to evaluate in free radical scavenging activity in food and biological systems. Food Sci Tech Int 2001;8:121–37. 4. Valko M, Leibfritz D, Moncol J, Cronin MTD, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007;39:44–84. 5. Halliwell B, Gutteridge IMC. Free radicals in Biology and Medicine. New York: Oxford University Press, 1999. 6. Quideau S, Deffieux D, Douat-Casassus C, Pouységu L. Plant polyphenols: chemical properties, biological activities, and synthesis. Angew Chem Int Ed 2011;50:586–622. 7. Guo X-F, Yue Y-D, Tang F, Wang J, Yao X. Antioxidant properties of major flavonoids and subfractions of the extract of Phyllostachys pubescens leaves. J Food Biochem 2013;37:501–9. 8. Berczyński P, Duchnik E, Kruk I, Piechowska T, Aboul-Enein HY, Bozdağ-Dϋndar O, Ceylan-Unluosoy M. 6-Methyl 3-chromonyl 2,4thiazolidinedione/2,4–imidalidinedione/2-thioxo-imidazolidine4-one compounds: novel scavengers of reactive oxygen species. Luminescence 2013; DOI 10.1002/bio.2554.
wileyonlinelibrary.com/journal/luminescence
9. Berczyński P, Kładna A, Piechowska T, Kruk I, Bozdağ-Dϋndar O, Aboul-Enein HY, Ceylan-Unluosoy M, Ertan R. Studies on the antioxidant activity of some thiazolidinedione, imidazolidinedione and rhodanine derivatives having a flavone care. Luminescence 2014, DOI 10.1002/bio.2667. 10. Research Organization of Ankara University (09B3336003) Project, Studies on the synthesis and antidiabetic activity of some new thiazolidinedione derivatives, 2009–2012, Oya Bozdağ-Dündar (Project coordinator). 11. Valentine JS, Miksztal AR, Sawyer DT. Methods for study of superoxide chemistry in nonaqueous solution. Methods Enzymol 1984;105:71–81. 12. Kruk I, Michalska T, Kładna A, Berczyński P, Aboul-Enein HY. Chemiluminescence investigations of antioxidative activities of some antibiotics against superoxide anion radical. Luminescence 2011;26:598–603. 13. Finkelstein E, Rosen GM, Rauckman EJ. Spin trapping of superoxide and hydroxyl radical: practical aspects. Arch Biochem Biophys 1980;200:1–16. 14. Goldstein S, Meyerstein D, Czapski G. The Fenton reagents. Free Radic Biol Med 1993;15:435–45. 15. Walling C. Fenton’s reagents revisited. Acc Chem Res 1975;8:125–31. 16. Nanjo F, Goto K, Seto R, Suzuki M, Sakai M, Hara Y. Scavenging effects of tea catechins and their derivatives on 1,1-diphenyl-2picrylhydrazyl radical. Free Radic Biol Med 1996;21:895–952. 17. Berker KI, Güçlü K, Demirata B, Apak R. A novel antioxidant assay of ferric reducing capacity measurement using ferrozine as the colour forming complexation reagent. Anal Methods 2010;2:1770–8. 18. Gampp H, Lippard SJ. Reinvestigation of 18-crown-6-ether/potassium superoxide solutions in Me2SO. Inorg Chem 1983;22:357–8. 19. Khan AU, Kasha M. Chemiluminescence arising from simultaneous transition in pairs of singlet oxygen molecules. J Am Chem Soc 1970;92:3293–300. 20. Greenstock CL, Miller RW. The oxidation of tiron by superoxide anion. Kinetics of the reaction in aqueous solution and in chloroplasts. Biochim Biophys Acta 1975;396:11–16. 21. Sawyer DT, Gibian MJ, Morrison MM, Seo ET. On the chemical reactivity of superoxide ion. J Am Chem Soc 1978;100:627–8. 22. Phosrithong N, Samee W, Nunthanavanit P, Ungwitayatorn J. In vitro antioxidant activity study of novel chromone derivatives. Chem Biol Drug Des 2012;79:981–9. 23. Kalyanaraman B, Perez-Feyes E, Mason RP. Spin-trapping and direct spin resonance investigations of the redox metabolism of quinone anticancer drugs. Biochim Biophys Acta 1980;630:119–30. 24. Finkelstein E, Rosen GM, Rauckman EJ. Spin trapping. Kinetic of the reaction of superoxide and hydroxyl radicals with nitrones. J Am Chem Soc 1980;102:4494–9. 25. Ghersi-Egea J-F, Maupoil V, Ray D, Rochette L. Electronic spin resonance detection of superoxide and hydroxyl radicals during the
Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2015; 30: 556–563
Antioxidant activity of some chromonylrhodanine derivatives
26. 27. 28. 29.
reductive metabolism of drugs by rat brain preparations and isolated cerebral microvessels. Free Radic Biol Med 1998,28;1074–81. Tang Y-Z, Liu Z-Q. Free-radical scavenging effect of carbazole derivatives on DPPH and ABTS radicals. J Am Oil Chem Soc 2007;84:1095–100. Foti MC, Daquino C, Geraci C. Electron-transfer reaction of cinnamic • acids and their methyl esters with the DPPH radical in alcoholic solutions. J Org Chem 2004;69:2309–14. Villaño D, Fernández-Pachón MS, Moyá ML, Troncoso AM, GarciaParrilla MC. Radical scavenging ability of polyphenolic compounds towards DPPH free radical. Talanta 2007;71:230–5. Parejo I, Codina C, Petrakis C, Kefalas P. Evaluation of scavenging activity assessed by Co(II)/EDTA-induced luminol chemiluminescence
and DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical assay. J Pharm Toxicol Methods 2000;44:507–12. 30. Alam MN, Bristi NJ, R Md. Review on in vivo and in vitro methods evaluation of antioxidant activity. Saudi Pharm J 2013;21:143–52. 31. Khan MN, Jan MR, Shah J, Lee SH. A validated silver-nanoparticleenhanced chemiluminescence method for the determination of citalopram in pharmaceutical preparations and human plasma. Luminescence 2014;29:266–74. 32. Tao X, Wang W, Wang Z, Cao X, Zhu J, Niu L, Wu X, Jiang H, Shen J. Development of a highly sensitive chemiluminescence enzyme immunoassay using enhanced luminol as substrate. Luminescence 2014;29:301–6.
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Accepted: 3 September 2014
Published online in Wiley Online Library: 6 November 2014
(wileyonlinelibrary.com) DOI 10.1002/bio.2785
Studies on the antioxidant activity of some chromonylrhodanine derivatives Irena Kruk,a Teresa Piechowska,a Paweł Berczyński,a Aleksandra Kładna,b Oya Bozdağ-Dündar,c Meltem Ceylan-Unlusoyc and Hassan Y. Aboul-Eneind* ABSTRACT: Fifteen chromonylrhodamine derivatives (CRs) were synthesized and the antioxidant activity levels were evaluated for the first time. The antioxidant activity potencies of these chromone derivatives were evaluated towards superoxide anion radicals, hydroxyl radicals and 2,2-diphenyl-1-picrylhydrazyl radicals. Also, the total antioxidant capacity of the tested compounds was measured using the ferric-ferrozine assay. The antioxidant activities were investigated using a chemiluminescence (CL) assay, spectrophotometry measurements, direct electron paramagnetic resonance (EPR) and the EPR spin-trapping technique. The 5,5-dimethyl- 1-pyrroline-1-oxide (DMPO) was applied as spin trap. Eleven of the 15 chromone compounds exhibited a decrease in the CL accompanying the superoxide anion radical produced in anhydrous dimethylsulfoxide (DMSO), ranging from 71–94% at concentration of 1 mmol /L; four of these compounds enhanced light emission in the range 231–672%. Similarly, these compounds caused 28–58% inhibition in the intensity of the DMPO-OOH radical EPR signal and the DMPO-OH radical (from 12–48%). Furthermore, three of these compounds showed very good antioxidant response towards the DPPH radical (EC50: 0.51–0.56 μmol/L) and the high reduction potentials. These findings demonstrate that the chromone compounds tested may be considered as effective free radicals scavengers, a finding that is of great pharmacological importance. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Chromonylrhodamine derivatives; Radical scavenging activity; Chemiluminescence; EPR study
Introduction Over the past 4–5 decades, oxidative stress has been implicated in oxidative biomolecule damage resulting in deleterious changes and loss of nutrition value (1–3), as well as in the pathogenesis of several human diseases (chronic inflammation, neurodegenerative disorders, atherosclerosis, diabetes mellitus, cancer) (4,5). Oxidative stress is defined as an imbalance between reactive oxygen species (ROS) such as superoxide anion radical (O•2 ); hydroxyl radical (HO•), peroxyl radical (ROO•); hydrogen peroxide H2O2; singlet oxygen (1O2) generation and defence and repair of antioxidant systems. The search for a antioxidant i.e., ‘a substance that when present at low concentrations compared with that of an oxidizable substrate significantly delays or prevents oxidation of that substrate’ (1) has received much attention and is the topic of much recent research (6,7). A recent review (6), as well as studies from our group (8,9), provide evidence that compounds containing the chromone skeleton can act as suitable antioxidants. In this study, we have undertaken for the first time an evaluation of the ROS scavenging activity of some chromonylrhodanine derivatives (CRs), (Fig. 1) to explain the origin of antidiabetic properties.
Experimental Reagents
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All the chemicals and the reagents were of analytical grade. The compounds CR1–CR15 were synthesized by using chromone carboxaldehyde and the rhodanine ring (10). The structure of synthesized chromone derivatives (CRs) was elucidated by
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elementary analysis, 1H NMR and mass spectral data. All reagents were purchased from E. Merck (Darmstadt, Germany) and Aldrich (Milwaukee, MI, USA) except potassium superoxide (KO2) and ammonium ferrous sulfate hexahydrate that were purchased from Fluka (Buchs, Switzerland). The CR compounds, trolox and tiron were dissolved in DMSO, which is suitable for dissolving both water-soluble and water-insoluble chemicals.
Free radicals scavenging assays Superoxide anion radical scavenging activity. Superoxide anion radicals were generated using the reaction between 18-crown-
* Correspondence to: Hassan Y. Aboul-Enein. Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Cairo 12311, Egypt. E-mail: [email protected] a
Institute of Physics, Faculty of Mechanical Engineering and Mechatronics, West Pomeranian University of Technology in Szczecin, Al. Piastów 48/49, 70-311 Szczecin, Poland
b
Department of History of Medicine and Medical Ethics, Pomeranian Medical University, Rybacka 1, 70-204 Szczecin, Poland
c
Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Tandoğan, Ankara, Turkey
d
Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Cairo 12311, Egypt Abbreviations: EPR, electron paramagnetic resonance; ROS, reactive oxygen species.
Copyright © 2014 John Wiley & Sons, Ltd.
Antioxidant activity of some chromonylrhodanine derivatives
Figure 1. Chemical structures of chromonylrhodanine derivatives (CRs).
6-ether and KO2 (11). The reaction mixture contained a 60 mg aliquot of 18-crown-6-ether dissolved in 10 mL of dry DMSO and 7 mg of KO2 was added quickly, to avoid contact with air humidity. This reaction was stirred for 60 min to give a pale yellow solution of 10 mmol/L superoxide anion radical. The concentration of radical was determined using the absorbance at λmax ¼ 251nm ; ε ¼ 2686±29 mol cm-1 : For the measurement, a superoxide anion radical was used as a 1 mmol/L solution in DMSO. Two assays were used to evaluate the activity of CRs towards superoxide anion radical, chemiluminescence (CL) and electron paramagnetic resonance (EPR).
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EPR spectroscopy and spin trapping. The superoxide anion radical scavenging was measured using the previously described spin-trapping methodology (13). The method is based on the reaction of DMPO with superoxide anion radical to yield the DMPO-OOH spin adduct having the half-time about 91 s at pH5 in aprotic solvents. The reaction mixture used consisted of the following reagents: 0.1 mol/L DMPO, 1 mmol/L superoxide anion radical, 1 mmol/L a CR compound. All reagents were dissolved in DMSO. Because the scavenger samples were made up in DMSO, the control reaction also contained an equivalent
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The assay is based on superoxide anion radical-induced light emission in DMSO and has been described previously (12). Briefly, the light emitting reaction was carried out in a thermostated glass cuvette placed in a light-tight chamber. The light intensity was measured using an EMI9553Q photomultiplier (Photek, East Sussex, UK) with an S20 cathode sensitive in the range 200–800 nm, interfaced with Chemiluminescence assay.
a computer for data acquisition and handling. The assay was performed at 294 ± 1 K. The CL signals from the superoxide anion radical/DMSO reaction or that influenced by the presence in it of a test compound was monitored as the kinetic curve of the CL decay. The well known inhibitor of superoxide anion radical – tiron – was used as positive control. The scavenging potency was calculated as the percentage inhibition of the CL intensity Q(%) = [(I∘ I)/I∘] × 100 %, where Io is the light intensity measured in the absence of an inhibitor and I is the light intensity measured in the presence of the inhibitor.
I. Kruk et al. amount of DMSO. The spectra were recorded using a quartz cuvette with an optical path length of 0.25 mm at 293 ± 1 K. The DMPO-OOH signal was analyzed approximately after 1 min from the beginning of the reaction. The scavenging ability was calculated as the percentage inhibition of the EPR signal amplitude Q(%) = [(H∘ H)/H∘] × 100 %, where Ho and H are the relative height of the second peak in the spectrum of the control and H in the presence of a CR compound, respectively, EPR spectra were recorded using a standard X-band spectrometer operating at 9.3GHz with a modulation frequency of 100 Hz of a steady magnetic field. The instrumental conditions were as follows: microwave power 20 mW, modulation amplitude 0.25 mT, time constant 0.3 s, and receiver gain 3.2 × 104. Hydroxyl radical scavenging activity. For the hydroxyl radical generation, the Fenton reaction was used Fe(II) + H2O2 → HO° + HO– + Fe(III) (14,15). As the hydroxyl radical, similar to the superoxide radical, is too short–lived to be detected directly, the spintrapping assay with DMPO as the trap was used. The assay was performed at room temperature. The reaction mixtures in the sample contained the following final concentration of reactants: 0.5 mmol/L H2O2, 10 mmol/L sodium trifluoroacetate buffer pH 6.15, 62.5 μmol/L FeSO4(NH4)SO4 and 25 mmol/L DMPO without a CR compound (control) or with 2.5 mmol/L of a test compound dissolved in DMSO (25% v/v). The scavenging activity was calculated as the percentage inhibition of the DMPO-OH spin adduct amplitude of the second peak in the EPR spectrum. The spectra were analyzed approximately after 4 min from the start of reaction. The conditions of EPR measurement were as follows: microwave power 20 mW, modulation amplitude 0.5 mT, time constant 0.3 s, receiver gain 2.5 × 104.
The DPPH radical scavenging activity was measured by the method described by Nanjo et al. (16) with slight modification, i.e. using the solvent mixture DMSO (25% v/v)/C2H5OH (75% v/v). Reaction mixture contained 0.125 mmol/L DPPH dissolved in ethanol and the tested compounds at various concentrations, dissolved in DMSO. The trolox and vitamin C were used as positive controls. The tested compounds were allowed to react with a very stable radical DPPH showing a characteristic EPR spectrum. The reduction of the DPPH radical was followed by monitoring the decrease in its EPR signal intensity. Antiradical activity was calculated using the following equation R (%) = [(Ho-H) / Ho] × 100%, where H is the relative height of the third peak in the radical EPR spectrum in the presence of the tested compound, Ho is the relative height of the third peak in the spectrum of the control. The R (%) value was plotted against the test compounds concentration. The concentration of the CRs samples, trolox and vitamin C necessary to decrease the initial DPPH° concentration by 50% (IC50) was obtained from the graphs. EPR spectra were recorded at room temperature. The conditions of EPR measurements were as follows: microwave power 20 mW, modulation amplitude 0.2 mT, time constant 0.3 s, and receiver gain 1 · 104. 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH°) scavenging assay.
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Ferric reducing potency assay. The total antioxidant activity measurement was based on an electron transfer from an antioxidant to ferric ion of the Fe (III)–ferrozine complex which is reduced to Fe (II) exhibiting an absorbance at 562 nm. (17). The Fe (III)–ferrozine aqueous complex consisted of 2 mmol/L Fe (III) and 10 mmol/L ferrozine. The reaction mixture contained 0.024 g of NH4Fe(SO4)2 · 12H2O dissolved in 1 mL of 1 mol/L HCl
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and 0.123 g ferrozine dissolved in 3 mL water. After mixing, the mixture was diluted to 25 mL with distilled water. The final concentration of Fe(III) was 2 mmol/L and 0.1 mmol/L ferrozine. The complex was kept in a stoppered dark colored bottle at room temperature. After 30 min standing time the absorbance at 562 nm was measured. Working solutions of reagents were as follows: 1.5 mL of the complex added to 1 mL of antioxidant solution at 1 mmol/L concentration and mixed with 2 mL of buffer (0.2 mol/L acetic acid/sodium acetate, pH 5.5). After 1.5 h standing at room temperature an increase in absorbance at 562 nm was monitored. Trolox (water-soluble vitamin E analog) was used as a positive control. Absorption spectra were acquired using a Carl Zeiss Technology M-40 with Win-Aspect Software (Jena, Germany).All experiments were conducted at least three times and the data are expressed as means ± SD.
Results and discussion Fifteen chromone derivatives, previously synthesized, were examined for their antioxidant activity, i.e. the ability to deactivating superoxide anion radical, hydroxyl radical, and DPPH radical. Also, the total antioxidant capacity was determined using ferrozine as reagent. Two methods were applied to evaluate activity of CR compounds towards superoxide anion radical: chemiluminescence and EPR. Figure 2 summarizes the results obtained using CL measurements. Typical CL kinetics from the superoxide anion radical/DMSO system alone (blank), and in the presence of representative compounds CR2 and CR6 are shown in Fig. 2A. The solution of superoxide anion radical in DMSO emits weak CL that originates from singlet oxygen as follows (18,19) 2O•2 þ 2ðCH3 Þ2 SO→1 O2 þ ðCH3 Þ2 SO2 þ CH3 SOðCHÞ þ OH O•2 →1 O2 þ electron O•2 þ O2 →O•4 O•4 →ð1 O2 Þ þ O•2 21 O2 →ð1 O2 Þ2 →23 O2 þ hυ The superoxide/DMSO reaction connected with CL technique had been successfully adopted for the first time in our laboratory to assess antioxidative properties several antioxidants against superoxide radical in hydrophobic medium (8,9). Compound CR6, similarly as compound CR1, CR3, CR4, CR7, CR9, CR10 and CR12-CR15 (kinetics not shown) showed inhibitory effects. Conversely, compounds CR2, CR5, CR8, CR11 enhanced the light emission (Fig. 2B). The scavenging effect of the CR derivatives was compared to the reference compound tiron (data not shown) that reacts with superoxide anion radical with a high rate constant (~108 L mol–1 s–1(20)). We found that tiron exhibited 96% quenching of CL under our experimental conditions, whereas CR compounds showed a significant decrease (ranging from 71–94%) in the CL intensity. The specificity of the reaction between 18-crown-6-ether and KO2 in DMSO as a source of O2• was also checked by adding 400 U• mL–1 SOD at the start of the reaction and observing a 98% suppression CL (data not shown). The superoxide anion radical is known to promote proton transfer from much weaker acids than water acting as an oxidant, can act as a reducing agent in aprotic solvents, and shows a high nucleophilicity toward SN2 substitution reaction
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Figure 2. Superoxide anion radical scavenging activity of the CRs compounds. (A) Time course of Cl decay in the superoxide anion radical/DMSO reaction (blank) and the CL decay monitored under the same condition as the blank but in the presence of representative chromone derivatives CR2 and CR6 (1 mmol/L). (B) Inhibitory effect of the evaluated CR compounds on the CL intensity. (C) EPR spectrum of the DMPO-OOH spin adduct arising in superoxide anion radical (1 mmol/L) in DMSO and inhibitory effect of CR compounds (1 mmol/L) exerted on this adduct.
(21). According to chemistry of superoxide anion radical and to mechanisms documented in the literature through which compound may act as antioxidant by a radical scavenging (6), the following reactions should be considered: reduction of superoxide anion radical CR þ O•2 → CR•þ þ O2 2 followed by the stabilization of the O2–2 species by DMSO (18) þ O2 2 þ 2H → H2 O2
and donate a hydrogen atom from the ring B of a CR molecule to superoxide anion radical CRðHÞ þ O•2 → CR• þ HO 2
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2HO•2 → 1 O2 þ H2 O2 Compounds CR5 and CR8 reached a very strong light emission enhancing effect at 1 mmol/L concentration. Compounds CR2 and CR11 reached a weaker enhancing effect. In general, compounds CR that exerted the enhancing effect on the CL contain in their structure an ester group linked to the ring B. The observed differences in the CL intensity enhancing potency (CR2 < CR11 < CR5 < CR8) may be due to presence of methyl group/groups in the chromone core. The substituent could change the stabilization of the products formed from CR derivatives, thus their antioxidant potency. This suggestion is in accordance with the finding of Phosrithong et al. (22) that chromone skeleton of an antioxidant is responsible for the stabilization of the antioxidant molecule during electron and/or hydrogen atom transfer. In turn, an electrophilic substituent NO2 (CR13, CR14, CR15) pulls a pair electrons from the ring A. This effect is associated with the positive charge of ring
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Both the above processes may be responsible for the quenching of the light emission directly (compounds CR1, CR3, CR4, CR6, CR7, CR9, CR10 and CR12-CR15). In turn, donation of proton from compounds CR to superoxide anion radical might lead to increasing of the light emission (CR2, CR5, CR8, CR11) as follows:
CRðHÞ þ O•2 → CR þ HO•2
I. Kruk et al. A and the possibility of SN2 substitution reaction occurrence, in accordance with high nucleophilicity of superoxide anion radical. The second method used to elucidate the superoxide anion radical scavenging capacity of CRs was based on their ability to inhibit the DMPO-OOH free radical formation. The DMPO trap forms with superoxide anion radical the adduct nitroxide free radical formed by covalent bonding of the radical with the spin trap. The DMPO-OOH adduct is unstable (the half-life is 91 s at pH5) and decays into nonradical species and DMPO-OH adduct (23,24) having long half-life (over 2 h) and showing the spectrum completely distinct from the DMPO-OH spectrum. For this reason, the DMPO-OOH spectra were recorded after 1 min. The spectrum of DMPO-OOH adduct, consisting of a 12-lines, formed in the 18-crown-6-ether/KO2/DMSO system is shown in Fig. 2C. We observed hyperfine couplings aN = 14.3G, aH = 11.5G and aγH ¼ 1:2G similarly to other (24). At 1 mmol/L CRs at least 28% inhibition in the intensity of the EPR signal was detected, and adding SOD (200 U/mL) decreased the EPR signal by a 98% (data not shown). All the tested CRs were able to scavenge hydroxyl radicals generated in the Fenton reaction in the presence of a spin trap DMPO (Figure 3). The rate constant for hydroxyl radical trapping by DMPO is very high (about 3.4 × 109 L mol–1 s–1) (24). The EPR spectra of the DMPO-OH spin adduct consisted of a quartet signal with hyperfine couplings aN = aH = 14.9 G. In the presence of CRs, the EPR signal decreased with time. The order of the scavenging activities measured for these chromones was CR14 > CR13 > CR3 > CR10 > CR15 > CR6 > CR9 > CR4 = CR8 > CR12 > CR5 > CR2 > CR1 > CR7 > CR11. As is illustrated in Fig. 3 compounds CR5, CR2, CR1, CR7 and CR11 revealed to be less potent providing the hydroxyl radicals scavenging from about 12–18%. We have checked the specificity of the Fenton reaction as a generator hydroxyl radicals using the positive control catalase, an enzyme that decomposes hydrogen peroxide to water. In the presence of 190 μg/mL catalase, the DMPO-OH spin adduct was not observed. These data confirm that the observed EPR spectrum was attributed to the nitroxide formed by hydroxyl radical trapping by DMPO. Since the concentration of DMSO in the reaction mixture was relatively high and its high rate constant of reaction with hydroxyl radical (7 × 109 L mol–1 s–1) (24) were greater than the DMPO concentration and rate constant
(2.1 × 109 M–1 s–1) (25), most hydroxyl radicals would have react with DMSO resulting in relatively poor hydroxyl radical scavenging activity observed for all the tested CRs. Indeed, we found that the observed spectra were a combination of the hydroxyl and CH3 radical adducts with the DMPO trap (data not shown). This finding is consistent with the findings of Ghersi-Egea et al. (25) and others that a secondary radical CH3 derived from DMSO forms of the DMPO-CH3 adduct. Therefore, care should be taken to distinguish between the DMPO-OH signal and the signal of the DMPO adduct of methyl radical. Concerning hydroxyl radical activity, the species is one of the strongest oxidizing reagents able to react with the first encountered biomolecule (1,3,5). This species can act by: (i) adding to a double bond; (ii) reaction with hydrogen–containing molecules followed by H-abstraction, and (iii) rapid electron transfer reaction. The antioxidant activity of the tested compounds was also measured against DPPH free radical (Figure 4, Table 1). The DPPH radical can react via two mechanisms (26,27): (i) a direct abstraction of hydrogen atom, and (ii) an electron transfer process from a molecule to DPPH•. The extent of one or the second pathway is dependent on the redox potential of the compounds involved in the reaction and the solvent polarity (27). In polar solvents one can expect an electron transfer process from the CR compound to DPPH•. Thus this process is predominant under our experimental conditions. A typical spectrum of the DPPH radical and the kinetic behaviour of the EPR amplitude of the DPPH radical signal is shown in Fig. 4. The spectrum monitored is very similar to that reported by other authors (26). Three types of kinetic behaviour were observed. Only compounds CR13-CR15 and trolox–the well known radical scavenger, reacted rapidly with the DPPH radical reaching a steady state in less than 1 min. For compounds CR3 and CR1, the steady state was reached after approximately 3 and 5 min, respectively. The remaining compounds tested exhibited slower kinetics of hyperbolic shape taking from 9 min to 1 h (CR8) to reach a steady state. As indicated in the methods section, the antiradical reactivity of CRs was evaluated from the plot of the percentage DPPH radical remaining against the CR concentration. The amount of a CR compound necessary to decrease the initial DPPH radical concentration by 50% (Table 1) was evaluated after 2 min of reaction time. In Fig. 4 we can observe that in the case
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Figure 3. EPR spectrum of the DMPO-OH spin adduct formed in the Fenton reaction and inhibitory effect of CRs compounds exerted on this adduct.
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Antioxidant activity of some chromonylrhodanine derivatives
Figure 4. Kinetic curves of the DPPH free radical reduction measured in the presence of CR compounds. Inside EPR spectrum of DPPH free radical in DMSO/ethanol mixture of solvents.
Table 1. DPPH free radical scavenging activity of tested chromone derivatives Compound CR1 CR2 CR3 CR4 CR5 CR6 CR7 CR8 CR9
IC50 (μmol L- 1) Compound 229 ± 4 236 ± 3 183 ± 3 495 ± 15 258 ± 4 249 ± 5 841 ± 17 889 ± 21 387 ± 11
CR10 CR11 CR12 CR13 CR14 CR15 Trolox Vitamin C
IC50 (μmol L- 1) 427 ± 14 248 ± 4 253 ± 3 52.3 ± 3.0 51.2 ± 2.1 56.5 ± 2.8 18 ± 1.2 352 ± 19
DPPH: 2,2-diphenyl-1-picrylhydrazyl, IC50: inhibition concentration. IC50 is the mean value of three measurements.
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of those compounds exhibiting rapid kinetic behaviour like trolox and compounds CR13–CR15 the precision of the method in the IC50 determination was high. In the case of the CR compounds where a steady state was achieved later than 2 min, the IC50 inhibition concentrations will be erroneous because reactions were still continued and degradation products were involved. From Table 1 we can note that 10 of 15 CRs examined exhibited higher activity than vitamin C, but less activity than trolox. This means that majority of CRs are more potent radical scavengers than that known good antioxidant – vitamin C. The DPPH radical scavenging activities of these compounds are diverse and comes as follows, starting from highest to lowest: trolox > CR14 = CR13 > CR15 > CR3 > CR1 > CR2 > CR11 = CR6 > CR5 > CR12 > Vitamin C > CR9 > CR10 > CR4 > CR7 > CR8. As mentioned above, medium is an important factor that influences on the reaction CRs with the DPPH radical, and this makes difficult to compare results with literature data. However,
in the case of trolox the IC50 value measured is in good accordance with some authors (4,28), but higher than those reported by Parejo et al. (29) found for tiron and vitamin C. To confirm the antioxidant property of CR compounds the assay of total antioxidant power using ferrozine as reagent was applied. The results of these measurements are shown in Fig. 5. In this method, the examined chromone derivatives were able to reduce Fe(III) ion, in the presence of ferrozine, to the Fe(II)– ferrozine complex. The complex exhibits a very high absorbance at 562 nm. This method is a very sensitive for evaluating antioxidant activity compounds having even a weak reducing potential (17). From the data listed in Fig. 5, it can be supposed that compounds CR3 and CR13–CR15 posses the highest reducing power, higher than trolox. These observations are in accordance with the lowest values of IC50 (Table 1) and the highest scavenging ratios of hydroxyl radical (Fig. 3), measured using the EPR technique. All these compounds inhibited high abilities to scavenge superoxide anion radical. The presence of the 2,3-double bond in conjugation with the 4-oxo function of a carbonyl group in the C-ring in chromone core enables the CR compounds to an electron transfer reaction. The presence of NO2 groups in chromone core (compounds CR13–CR15) may stabilize the products arising from those compounds and their reducing abilities. In conclusion, several methods are used to study the antioxidant activity of various samples, reviewed recently in (30). In the present paper, we have applied to the chromone derivatives tested the techniques which are commonly used in the subject literature, and found to be reproducible and comparable. It is noteworthy that the CL technique is often more sensitive than other assays and exhibits the high stability, rapidity, instrument simplicity. For this reason, the CL method has recently received much attention of researchers which proved useful in biotechnology, pharmacology or bioanalysis (31,32). Our findings confirm the high sensitivity this method in evaluation of CRs reactivity towards superoxide anion radical. We found that the
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Figure 5. Reduction of Fe (III) in the Fe (III) - ferrozine complex to Fe (II) in the presence of CR compounds and the standard antioxidant–trolox.
CR3, CR13–CR15 compounds have shown a high activity as scavengers of DPPH free radical, hydroxyl and superoxide anion radicals and possess high reducing power (IC50 values were only three times higher than that found for trolox). Yet, the antioxidant power of CR3 and CR13–CR15 group of compounds is in good accordance with the hierarchy of their reducing powers, using the ferric-ferrozine method. A unique aspect of the present study was the comprehensive analysis of both antioxidant and reducing powers of the chromone derivatives examined for the first time. This preliminary findings show that at least few of the evaluated CRs are promising for treating diabetes as the β-cells dysfunction is dependent on hydroxyl radical and superoxide anion radical levels (4). Due to overwhelming evidence that antioxidants prevent the occurrence of diseases mediated by ROS, it would be important to identify the reaction intermediates and products, using chromatography.
References
562
1. Aruoma OI. Free radicals and antioxidants strategies in sports. J Nutr Biochem 1995;5:370–81. 2. Namiki M Antioxidants/antimutagens in food. Critical Rev Food Sci Nutr 1990;29:273–300. 3. Sanchez-Moreno C. Review: Methods used to evaluate in free radical scavenging activity in food and biological systems. Food Sci Tech Int 2001;8:121–37. 4. Valko M, Leibfritz D, Moncol J, Cronin MTD, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007;39:44–84. 5. Halliwell B, Gutteridge IMC. Free radicals in Biology and Medicine. New York: Oxford University Press, 1999. 6. Quideau S, Deffieux D, Douat-Casassus C, Pouységu L. Plant polyphenols: chemical properties, biological activities, and synthesis. Angew Chem Int Ed 2011;50:586–622. 7. Guo X-F, Yue Y-D, Tang F, Wang J, Yao X. Antioxidant properties of major flavonoids and subfractions of the extract of Phyllostachys pubescens leaves. J Food Biochem 2013;37:501–9. 8. Berczyński P, Duchnik E, Kruk I, Piechowska T, Aboul-Enein HY, Bozdağ-Dϋndar O, Ceylan-Unluosoy M. 6-Methyl 3-chromonyl 2,4thiazolidinedione/2,4–imidalidinedione/2-thioxo-imidazolidine4-one compounds: novel scavengers of reactive oxygen species. Luminescence 2013; DOI 10.1002/bio.2554.
wileyonlinelibrary.com/journal/luminescence
9. Berczyński P, Kładna A, Piechowska T, Kruk I, Bozdağ-Dϋndar O, Aboul-Enein HY, Ceylan-Unluosoy M, Ertan R. Studies on the antioxidant activity of some thiazolidinedione, imidazolidinedione and rhodanine derivatives having a flavone care. Luminescence 2014, DOI 10.1002/bio.2667. 10. Research Organization of Ankara University (09B3336003) Project, Studies on the synthesis and antidiabetic activity of some new thiazolidinedione derivatives, 2009–2012, Oya Bozdağ-Dündar (Project coordinator). 11. Valentine JS, Miksztal AR, Sawyer DT. Methods for study of superoxide chemistry in nonaqueous solution. Methods Enzymol 1984;105:71–81. 12. Kruk I, Michalska T, Kładna A, Berczyński P, Aboul-Enein HY. Chemiluminescence investigations of antioxidative activities of some antibiotics against superoxide anion radical. Luminescence 2011;26:598–603. 13. Finkelstein E, Rosen GM, Rauckman EJ. Spin trapping of superoxide and hydroxyl radical: practical aspects. Arch Biochem Biophys 1980;200:1–16. 14. Goldstein S, Meyerstein D, Czapski G. The Fenton reagents. Free Radic Biol Med 1993;15:435–45. 15. Walling C. Fenton’s reagents revisited. Acc Chem Res 1975;8:125–31. 16. Nanjo F, Goto K, Seto R, Suzuki M, Sakai M, Hara Y. Scavenging effects of tea catechins and their derivatives on 1,1-diphenyl-2picrylhydrazyl radical. Free Radic Biol Med 1996;21:895–952. 17. Berker KI, Güçlü K, Demirata B, Apak R. A novel antioxidant assay of ferric reducing capacity measurement using ferrozine as the colour forming complexation reagent. Anal Methods 2010;2:1770–8. 18. Gampp H, Lippard SJ. Reinvestigation of 18-crown-6-ether/potassium superoxide solutions in Me2SO. Inorg Chem 1983;22:357–8. 19. Khan AU, Kasha M. Chemiluminescence arising from simultaneous transition in pairs of singlet oxygen molecules. J Am Chem Soc 1970;92:3293–300. 20. Greenstock CL, Miller RW. The oxidation of tiron by superoxide anion. Kinetics of the reaction in aqueous solution and in chloroplasts. Biochim Biophys Acta 1975;396:11–16. 21. Sawyer DT, Gibian MJ, Morrison MM, Seo ET. On the chemical reactivity of superoxide ion. J Am Chem Soc 1978;100:627–8. 22. Phosrithong N, Samee W, Nunthanavanit P, Ungwitayatorn J. In vitro antioxidant activity study of novel chromone derivatives. Chem Biol Drug Des 2012;79:981–9. 23. Kalyanaraman B, Perez-Feyes E, Mason RP. Spin-trapping and direct spin resonance investigations of the redox metabolism of quinone anticancer drugs. Biochim Biophys Acta 1980;630:119–30. 24. Finkelstein E, Rosen GM, Rauckman EJ. Spin trapping. Kinetic of the reaction of superoxide and hydroxyl radicals with nitrones. J Am Chem Soc 1980;102:4494–9. 25. Ghersi-Egea J-F, Maupoil V, Ray D, Rochette L. Electronic spin resonance detection of superoxide and hydroxyl radicals during the
Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2015; 30: 556–563
Antioxidant activity of some chromonylrhodanine derivatives
26. 27. 28. 29.
reductive metabolism of drugs by rat brain preparations and isolated cerebral microvessels. Free Radic Biol Med 1998,28;1074–81. Tang Y-Z, Liu Z-Q. Free-radical scavenging effect of carbazole derivatives on DPPH and ABTS radicals. J Am Oil Chem Soc 2007;84:1095–100. Foti MC, Daquino C, Geraci C. Electron-transfer reaction of cinnamic • acids and their methyl esters with the DPPH radical in alcoholic solutions. J Org Chem 2004;69:2309–14. Villaño D, Fernández-Pachón MS, Moyá ML, Troncoso AM, GarciaParrilla MC. Radical scavenging ability of polyphenolic compounds towards DPPH free radical. Talanta 2007;71:230–5. Parejo I, Codina C, Petrakis C, Kefalas P. Evaluation of scavenging activity assessed by Co(II)/EDTA-induced luminol chemiluminescence
and DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical assay. J Pharm Toxicol Methods 2000;44:507–12. 30. Alam MN, Bristi NJ, R Md. Review on in vivo and in vitro methods evaluation of antioxidant activity. Saudi Pharm J 2013;21:143–52. 31. Khan MN, Jan MR, Shah J, Lee SH. A validated silver-nanoparticleenhanced chemiluminescence method for the determination of citalopram in pharmaceutical preparations and human plasma. Luminescence 2014;29:266–74. 32. Tao X, Wang W, Wang Z, Cao X, Zhu J, Niu L, Wu X, Jiang H, Shen J. Development of a highly sensitive chemiluminescence enzyme immunoassay using enhanced luminol as substrate. Luminescence 2014;29:301–6.
563
Luminescence 2015; 30: 556–563
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