The hydroxyl radical scavenging activity was measured using the 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) spin adduct generated in an iron-containing xanthine oxidase (XO)/hypoxanthine (HX) (Fe-XO/HX) system using the spin-trapping method. The sampling procedure is as follows. All reagents were dissolved in 20mM Tris–HCl buffer solution (pH 7.4). Approximately 50 μL of 1.2 mM HX, 50 μL of 1.2 mM EDTA, 50 μL of 0.06 mM FeCl3, and 50 μL of 0.9 M KCl were well mixed in a test tube; subsequently, 3 μL of DMPO and 50 μL of test sample were added. After 50 μL of 0.30 U/mL XO was added to initiate the reaction, a 200 μL aliquot of the mixture was immediately placed into a flat quartz cell to scan and record the ESR spectra. ESR observations were performed using a JES-RE1X spectrometer (JEOL, Tokyo, Japan) using the following settings: modulation width, 0.2 mT; time constant, 0.03 s; and microwave frequency, 9.405 GHz.
Jes re1x spectrometer
Manufactured by JEOL
Sourced in Japan
The JES-RE1X spectrometer is a compact and versatile electron spin resonance (ESR) instrument designed for a wide range of analytical applications. It provides a reliable and efficient means of detecting and characterizing paramagnetic species in various sample types. The core function of the JES-RE1X is to generate and detect electron spin resonance signals, enabling the analysis of molecular and material properties.
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3 protocols using jes re1x spectrometer
1
Hydroxyl Radical Scavenging Activity Assay
2
Quantification of TEMPOL by EPR
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Exactly 2.86 mg of TEMPOL was weighed into a 10 ml measuring flask filled with Milli-Q water to obtain 1,660 μM standard TEMPOL solution, and stocked in a sealed plastic tube at 4°C. A concentration series (1.66, 2.28, 3.24, 4.84, 7.69, 13.3, 25.9, 61.5, 106, 208, 338, 492, 756, 1,250, and 1,660 μM) of TEMPOL water solution was prepared by diluting the standard solution before each measurement. A 100-μl aliquot of the sample TEMPOL solution was loaded into PTFE tubing and measured by an X-band EPR spectrometer 3 times for each concentration. In total, 19, 18, or 8 measurements were performed for 1.66–25.9 μM, 61.5–1,250 μM, or 1,660 μM samples during a 2-year period. The stock 1,660 μM solution was prepared twice during a 2-year period. The JES-RE1X spectrometer (JEOL, Tokyo, Japan) was used for only this experiment and the center peak of TEMPOL was noted.
3
ESR Analysis of Radical Scavenging by TQ and THQ
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The radical scavenging activities of TQ and THQ were evaluated using ESR spectroscopy. THQ was synthesized according to a previously reported procedure. 8) (link) The ESR assay was conducted with galvinoxyl radical (GO•) in the presence of test compounds. These reagents were * To whom correspondence should be addressed. e-mail: [email protected]; [email protected] Fig. 1. Thymoquinone (TQ) and Its Reduced Metabolites, Semiquinone and Thymohydroquinone (THQ) dissolved in acetonitrile. Predetermined concentrations of test compounds were mixed with 50 µM GO• and then immediately introduced into a flat quartz cell, which was then placed in the ESR sample cavity. ESR observations were performed with a JES-RE1X spectrometer (JEOL, Tokyo, Japan) at the following instrument settings: modulation width, 0.2 mT; time constant, 0.03 s; and microwave frequency, 9.405 GHz.
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