Yoshimi Niwano, phd Research Center for Functional Food Materials Sunny Health Co., Ltd. Saito Biotechnology Incubator, 7-7-15 Saito-Asagi, Ibaraki Osaka 567-0085 Japan E-mail: firstname.lastname@example.org
Ciclopirox olamine (CPO), a hydroxypyridone derivative, belongs to antifungal agents used for the treatment of superficial fungal infections. Recently, it has been reported that CPO acts as a potential chelating agent and influences some cellular processes of the fungus by chelating metal irons.1 Therefore, CPO likely inhibits Fenton-type reaction in which hydroxyl radicals are formed from the hydrogen peroxide and Fe ions. In the present study, we examined the effect of CPO on the hydroxyl radical formation through the Fenton reaction and ultrasound irradiation to water by using electron spin resonance (ESR) analyses. In addition, direct scavenging activities of CPO against hydroxyl radicals and superoxide anions were examined.
Materials and Methods
Test material and reagents
Ciclopirox olamine and hypoxanthine were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Xanthine oxidase (XOD from cow milk) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were purchased from Labotec Co., Ltd. (Tokyo, Japan). All the other reagents used were of analytical grade.
ESR analyses for hydroxyl radical from the Fenton reaction
Ciclopirox olamine was dissolved in pure water to be a concentration of 10 mg/mL, and then twofold dilutions were made by adding pure water. Fifty microliter of 2 mm hydrogen peroxide dissolved in 0.1 m phosphate buffer (PB), 50 µL of 8.9 mm DMPO dissolved in pure water, 50 µL of each CPO solution or solvent alone, and 50 µL of 0.2 mm FeSO4 dissolved in pure water were placed in a test tube and mixed. Each mixture was transferred to the ESR spectrometry cell, and the DMPO-OH spin adduct was quantified 113 s after the addition of FeSO4. Signal intensities were evaluated from the peak height of the second signal of the DMPO-OH spin adduct. Measurement conditions of ESR (JES-FA-100, JEOL, Tokyo, Japan) were as follows: field sweep, 330.50–340.50 mT; field modulation frequency, 100 kHz; field modulation width, 0.1 mT; amplitude, 250; sweep time, 2 min; time constant, 0.1 s; microwave frequency, 9.420 GHz; microwave power, 4 mW.
ESR analyses for hydroxyl radical from an ultrasound device
A schematic drawing of ultrasound device operated at 1650 kHz for hydroxyl radical generation is illustrated in Fig. 1. A glass tube (Φ15 × 85 mm) with the reaction mixture which consisted of 880 µL of pure water, 100 µL of different concentrations of CPO dissolved in pure water, and 20 µL of 111.25 mm DMPO dissolved in pure water was set in the device. Then the reaction mixture was exposed to sonication for 2 min. The reaction mixture obtained after the exposure was immediately transferred to the ESR spectrometry cell for the ESR analysis. The signal intensity of the DMPO-OH spin adduct was recorded as is the case with the Fenton reaction. Measurement conditions of ESR were the same as in those in the Fenton reaction except that amplitude was 300.
ESR analyses for superoxide anion from a hypoxanthine-XOD reaction system
Assay for superoxide scavenging activity was essentially identical to that described in the previous paper.2–4 Fifty microliter of 2 mm hypoxanthine dissolved in 0.1 m phosphate buffered saline (PBS), 50 µL of different concentrations of CPO dissolved in pure water or solvent (pure water) alone, 30 µL of dimethylsulfoxide (DMSO), 20 µL of 4.5 m DMPO dissolved in pure water, and 50 µL of XOD dissolved to be 0.4 U/mL in 0.1 m PB were placed in a test tube and mixed. Each mixture was transferred to the ESR spectrometry cell, and the DMPO-OOH spin adduct was quantified 97 s after the addition of XOD. Signal intensities were evaluated from the peak height of the first signal of the DMPO-OOH spin adduct. Measurement conditions of ESR were as follows: field sweep, 330.50–340.50 mT; field modulation frequency, 100 kHz; field modulation width, 0.07 mT; amplitude, 200; sweep time, 2 min; time constant, 0.1 s; microwave frequency, 9.420 GHz; microwave power, 4 mW.
Results and Discussion
Figure 2 shows the representative ESR spectra of DMPO-OH as a spin adduct formed by DMPO and hydroxyl radical obtained from the Fenton reaction, which was assigned by hyperfine coupling constants, aN = aH = 1.49 mT, indicating that the amount of hydroxyl radicals was clearly reduced by CPO. Figure 3 summarizes the concentration effect of CPO on the signal intensities of DMPO-OH from the Fenton reaction. CPO reduced potently the signal intensity of DMPO-OH, and the IC50 (the concentration which inhibits the formation of DMPO-OH by 50%) was as low as 1.29 × 10 µg/mL (4.81 × 10−5m). If CPO interferes with the Fenton reaction as a Fe-chelater, the signal intensity is supposed to be reduced. Therefore, to further examine the hydroxyl radical–scavenging activity of CPO, an ultrasound technology was applied as a selectable generation system for hydroxyl radicals. Figure 4 shows the representative ESR spectra of DMPO-OH obtained from the reaction mixtures containing different concentrations of CPO, indicating that CPO has an ability to scavenge directly hydroxyl radicals. From the experiment, the IC50 and the rate constant were 1.09 × 10 µg/mL (4.06 × 10−5m), which coincided with that obtained in the Fenton reaction. In both of the experiments, we also calculated the rate constants by using the equation and, 2.1 × 109/m/s (the rate constant between hydroxyl radical and DMPO) as reported previously.5,6 The calculated constants in the experiments using the Fenton reaction and the ultrasound technology were similar to each other (9.71 × 1010/m/s in the Fenton reaction, and 1.15 × 1011/m/s in the ultrasound technology). We checked the references describing the rate constant between hydroxyl radical and scavengers. As a result of our survey, the fastest rate constant was found between hydroxyl radical and CoQ10 in which the rate constant was 6.25 × 1010/m/s.7 Therefore, the rate constant obtained in this study is considered to be high. One of the reason is CPO is a Fe-cheleter, so that the rate constant became 1010 order in the Fenton reaction. However, in the experiment in which an ultrasound technology was used as a hydroxyl radical generator, the rate constant still became 1011 order. Since we cannot explain the reason for such fast rate constant at the present time, further study is required on the point of view of reaction kinetics between hydroxyl radical and CPO.
Figure 5 shows the representative ESR spectra of DMPO-OOH (an adduct formed by DMPO and superoxide anion) obtained from the hypoxanthine-XOD reaction system, indicating that the amount of superoxide anions was not potently reduced by the addition of CPO even at the highest concentration.
The present study clearly revealed that CPO has a potent capacity to scavenge directly hydroxyl radical but not superoxide anion. Although generation of reactive oxygen species (ROS) through myeloperoxidase and NADPH oxidase by phagocytic cells such as neutrophils is known to be an important host defense mechanism directed toward killing of invading microorganisms,8,9 ROS have potential deleterious effects on the biological system since they can damage proteins, lipids, and nucleic acids.10–12 In the case of NADPH oxidase, superoxide anion is generated and dismutation of superoxide anion then generates hydrogen peroxide that gives rise to hydroxyl radical formation through a Fenton-type reaction.13–16 More recently, it has been found that hydroxyl radical is produced through XOD in the mouse skin treated with lipopolysaccharide.17 Since CPO is known to exert anti-inflammatory activity,18 the scavenging effect of CPO against hydroxyl radicals may contribute to the improvement of the skin inflammation caused by invading fungi.