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2,2'-Azobis(isobutyronitrile)-derived alkylperoxyl radical scavenging activity assay of hydrophilic antioxidants by employing EPR spin trap method.

Kohri S, Fujii H - J Clin Biochem Nutr (2013)

Bottom Line: The alkylperoxyl radical species was generated by the photolysis of azo-radical initiator 2,2'-azobis(isobutyronitrile), in which the radical generation rate and period were controlled by the illumination light.The scavenging activities toward alkylperoxyl radical and alkoxy radical species were evaluated in six antioxidants.Although quercetin showed the highest activity toward both radicals, the order of the relative activities in the other antioxidants was different mutually between the alkylperoxyl radical and the alkoxyl radical.

View Article: PubMed Central - PubMed

Affiliation: Center for Medical Education, Sapporo Medical University, South-1 West-17, Chuo-ku, Sapporo, Hokkaido 060-8556, Japan.

ABSTRACT
As interest in the study of antioxidant intake from foods and other agricultural products increases, methods for performing radical scavenging activity assays based on the electron paramagnetic resonance spectroscopic method, in which there is no interference from the sample color and turbidity, are required. In this study, we have developed a rapid and simple electron paramagnetic resonance based assay to evaluate the alkylperoxyl radical scavenging activity of several antioxidants. The alkylperoxyl radical species was generated by the photolysis of azo-radical initiator 2,2'-azobis(isobutyronitrile), in which the radical generation rate and period were controlled by the illumination light. The relative alkylperoxyl radical scavenging activity was obtained by a simple formula of competing reaction of antioxidant and spin trap toward the oxygen radical. The scavenging activities toward alkylperoxyl radical and alkoxy radical species were evaluated in six antioxidants. Although quercetin showed the highest activity toward both radicals, the order of the relative activities in the other antioxidants was different mutually between the alkylperoxyl radical and the alkoxyl radical. This alkylperoxyl radical scavenging activity assay based on electron paramagnetic resonance spectroscopy is useful for evaluation of colored and turbid food samples.

No MeSH data available.


Related in: MedlinePlus

EPR spectrum of spin adduct of CYPMPO trapping the radical generated from AIBN after termination of illumination. A 72 µl solution containing 11 mM AIBN, 67% (w/w) of 100 mM phosphate buffer, and 33% (w/w) of acetonitrile was illuminated by UV light for 30 s on an ice bath, followed by addition of an 8 µl solution of 100 mM CYPMPO after termination of illumination.
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Figure 2: EPR spectrum of spin adduct of CYPMPO trapping the radical generated from AIBN after termination of illumination. A 72 µl solution containing 11 mM AIBN, 67% (w/w) of 100 mM phosphate buffer, and 33% (w/w) of acetonitrile was illuminated by UV light for 30 s on an ice bath, followed by addition of an 8 µl solution of 100 mM CYPMPO after termination of illumination.

Mentions: The EPR spectra of the spin adduct of CYPMPO when coexisting AIBN was photolyzed is shown in Fig. 1A, and its simulated spectrum is shown in Fig. 1B. The EPR spectrum pattern was same as the reported spectrum generated by the photolysis of AIBN.(4) The HFCCs were estimated as follows: A(H) = 1.11, A(N) = 1.33, A(P) = 4.68. This spectrum was assigned to ROO• adduct of CYPMPO [R is -C(CH3)2-CN]. The EPR spectrum of radical adduct of CYPMPO by the photolysis of AAPH and its simulation spectrum are shown in Fig. 1C and D, respectively. The relative differences of the estimated HFCCs [A(H) = 1.21, A(N) = 1.35, A(P) = 4.78] were less than 3% compared to the previously reported values.(4) This spectrum was assigned to RO• adduct of CYPMPO [R is -C(CH3)2-C(NH2)NH2+]. The same spectrum pattern was also obtained in experiments where an AIBN solution cooled in an ice bath during illumination was mixed with CYPMPO solution after termination of illumination (Fig. 2). The estimated hyperfine coupling constants were as follows: A(H) = 1.03, A(N) = 1.30, A(P) = 4.62. The EPR spectra of the ROO• adduct in the presence of various concentrations of PG are shown in Fig. 3. As the concentration of PG increased, the peak height decreased. The value of (IB – I)/I was plotted as a function of [Antioxidant]/[CYPMPO] in Fig. 4 in which the antioxidant was caffeic acid, and PG. The theoretical relationship is represented as a line passing through the origin by the equation, (IB – I)/I = kA/kC [Antioxidant]/[CYPMPO], derived from Equation 6. The results were well fitted to the theoretical line. The relative RSAs of the antioxidants toward ROO• and RO• are summarized in Table 1. The orders of the relative RSAs were different between ROO• and RO• but quercetin which was the top in both two radicals.


2,2'-Azobis(isobutyronitrile)-derived alkylperoxyl radical scavenging activity assay of hydrophilic antioxidants by employing EPR spin trap method.

Kohri S, Fujii H - J Clin Biochem Nutr (2013)

EPR spectrum of spin adduct of CYPMPO trapping the radical generated from AIBN after termination of illumination. A 72 µl solution containing 11 mM AIBN, 67% (w/w) of 100 mM phosphate buffer, and 33% (w/w) of acetonitrile was illuminated by UV light for 30 s on an ice bath, followed by addition of an 8 µl solution of 100 mM CYPMPO after termination of illumination.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3818267&req=5

Figure 2: EPR spectrum of spin adduct of CYPMPO trapping the radical generated from AIBN after termination of illumination. A 72 µl solution containing 11 mM AIBN, 67% (w/w) of 100 mM phosphate buffer, and 33% (w/w) of acetonitrile was illuminated by UV light for 30 s on an ice bath, followed by addition of an 8 µl solution of 100 mM CYPMPO after termination of illumination.
Mentions: The EPR spectra of the spin adduct of CYPMPO when coexisting AIBN was photolyzed is shown in Fig. 1A, and its simulated spectrum is shown in Fig. 1B. The EPR spectrum pattern was same as the reported spectrum generated by the photolysis of AIBN.(4) The HFCCs were estimated as follows: A(H) = 1.11, A(N) = 1.33, A(P) = 4.68. This spectrum was assigned to ROO• adduct of CYPMPO [R is -C(CH3)2-CN]. The EPR spectrum of radical adduct of CYPMPO by the photolysis of AAPH and its simulation spectrum are shown in Fig. 1C and D, respectively. The relative differences of the estimated HFCCs [A(H) = 1.21, A(N) = 1.35, A(P) = 4.78] were less than 3% compared to the previously reported values.(4) This spectrum was assigned to RO• adduct of CYPMPO [R is -C(CH3)2-C(NH2)NH2+]. The same spectrum pattern was also obtained in experiments where an AIBN solution cooled in an ice bath during illumination was mixed with CYPMPO solution after termination of illumination (Fig. 2). The estimated hyperfine coupling constants were as follows: A(H) = 1.03, A(N) = 1.30, A(P) = 4.62. The EPR spectra of the ROO• adduct in the presence of various concentrations of PG are shown in Fig. 3. As the concentration of PG increased, the peak height decreased. The value of (IB – I)/I was plotted as a function of [Antioxidant]/[CYPMPO] in Fig. 4 in which the antioxidant was caffeic acid, and PG. The theoretical relationship is represented as a line passing through the origin by the equation, (IB – I)/I = kA/kC [Antioxidant]/[CYPMPO], derived from Equation 6. The results were well fitted to the theoretical line. The relative RSAs of the antioxidants toward ROO• and RO• are summarized in Table 1. The orders of the relative RSAs were different between ROO• and RO• but quercetin which was the top in both two radicals.

Bottom Line: The alkylperoxyl radical species was generated by the photolysis of azo-radical initiator 2,2'-azobis(isobutyronitrile), in which the radical generation rate and period were controlled by the illumination light.The scavenging activities toward alkylperoxyl radical and alkoxy radical species were evaluated in six antioxidants.Although quercetin showed the highest activity toward both radicals, the order of the relative activities in the other antioxidants was different mutually between the alkylperoxyl radical and the alkoxyl radical.

View Article: PubMed Central - PubMed

Affiliation: Center for Medical Education, Sapporo Medical University, South-1 West-17, Chuo-ku, Sapporo, Hokkaido 060-8556, Japan.

ABSTRACT
As interest in the study of antioxidant intake from foods and other agricultural products increases, methods for performing radical scavenging activity assays based on the electron paramagnetic resonance spectroscopic method, in which there is no interference from the sample color and turbidity, are required. In this study, we have developed a rapid and simple electron paramagnetic resonance based assay to evaluate the alkylperoxyl radical scavenging activity of several antioxidants. The alkylperoxyl radical species was generated by the photolysis of azo-radical initiator 2,2'-azobis(isobutyronitrile), in which the radical generation rate and period were controlled by the illumination light. The relative alkylperoxyl radical scavenging activity was obtained by a simple formula of competing reaction of antioxidant and spin trap toward the oxygen radical. The scavenging activities toward alkylperoxyl radical and alkoxy radical species were evaluated in six antioxidants. Although quercetin showed the highest activity toward both radicals, the order of the relative activities in the other antioxidants was different mutually between the alkylperoxyl radical and the alkoxyl radical. This alkylperoxyl radical scavenging activity assay based on electron paramagnetic resonance spectroscopy is useful for evaluation of colored and turbid food samples.

No MeSH data available.


Related in: MedlinePlus