Electron Spin Resonance Spin-trapping Method Research Articles

Analysis of Compounds of Curcuma Rhizome Using Mass Spectrometry and Investigation of the Antioxidant Activity of Rhizome Extracts

The medicinal plant garden of Hoshi University located in southern Tokyo is home to many medicinal plants, and analysis of odor and measurement of antioxidant activity of cultivated plant have been carried out. In this study compounds originated from Curcuma rhizomes, i.e., Curcuma longa, Curcuma aromatica, Curcuma zedoaria, Curcuma xanthorrhiza, were investigated and antioxidant activities of rhizome extracts were measured. Volatile compounds originating from the Curcuma rhizomes were analyzed using thermal desorption (TD)-GC-MS with solid-phase micro extraction fiber as an adsorption device. p-Cymene, 1,8-cineol, β-elemene, and β-caryophyllene were the predominant constituents in most cases. Curcuminoids, which were not identified by TD-GC-MS, were detected using direct analysis in real time time-of-flight MS. Curcumin and demethoxycurcumin were detected from both C. longa and C. xanthorrhiza. The antioxidant activity of each Curcuma species rhizome was confirmed using the electron spin-resonance spin-trapping method with potent scavenging activity against superoxide anion radicals. Extracts from Curcuma rhizomes cultivated in the medicinal plant garden exhibited antioxidant activities, and the order of the activity of methanol extracts was: C. longa>C. xanthorrhiza>C. aromatica>C. zedoaria. Phenolic compounds, particularly curcumin, are known to possess potent antioxidant activity and are extracted with methanol, thus it reflects the intensity of the antioxidant activity.

Mechanism of UV-driven Photoelectrocatalytic Degradation of Berberine Chloride Form Using the ESR Spin-trapping Method.

Photoelectrocatalytic (PEC) system greatly improves the migration of photoexcited charges, retards the fast recombination of electron-hole and increases the lifetime of photogenerated holes. In this article, we constructed a novel PEC system to degrade berberine chloride form (BCF). XRD patterns indicated aging time was an important condition for the crystal type of TiO2, and the best proportion of anatase/rutile was 80/20. The band gap energy of the TiO2 was 3.01 eV. We demonstrated that a synergistic effect existed between photocatalysis (PC) and electrocatalysis (EC). Besides, we discussed the influence of pH, current density, the amount of catalyst and initial BCF concentration on the degradation of BCF. Electron spin resonance (ESR) through spin trap 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) and the scavenging experiments suggested that the reactive oxygen species (ROS) were superoxide radicals (O2·-), hydroxyl radicals (·OH) and sulfate radical (SO4·-) in PEC system. Furthermore, we compared the two pathway of formation DMPO-SO4 adducts and found that SO4·- was the most major oxidized species in degrading BCF. We proposed a plausible reaction mechanism. Intriguingly, even under lower current and weaker light conditions, the PEC process maintained its effectiveness, demonstrating the feasibility of the PEC approach.

Antimicrobial Intervention by Photoirradiation of Grape Pomace Extracts via Hydroxyl Radical Generation.

The annual production of grape worldwide amounts to almost 70 million tons, and around 80% is used for winemaking. The two major wastes from winemaking process, pomace and lees account for 20 and 7% of the grapes, respectively. They have been expected as a valuable resource to be recycled because they are rich in polyphenols. Polyphenols possess prooxidatve activity as well as antioxidative one just like a two sides of a coin. A typical example of the prooxidative activity is antibacterial activity of catechins. The activity is exerted through oxidation of phenolic hydroxyl moiety coulpled with reduction of dissolved oxygen leading to hydrogen peroxide (H2O2) generation. In addition, once the oxidation of phenolic hydroxyl moiety is augmented by photoirradiation, highly reactive hydroxyl radical (·OH) is generated. Accordingly, there have been several reports showing that photoirardiation of polyphenols exerts bactericidal activity via ·OH generation. This review focuses mainly on antimicrobial intervention by photoirradiation of grape pomace extract in relation to ·OH generation analyzed by an electron spin resonance-spin trapping method.

Analysis of Volatile Compounds of Curcuma longa (Turmeric) and Investigation of the Antioxidant Activity of Rhizome Extracts

Fragrances originating from plants are widely believed to have therapeutic properties. The volatile compounds originating from Curcuma longa (turmeric) plant cultivated in a medicinal plant garden located in southern Tokyo were investigated using thermal desorption-gas chromatography-mass spectrometry. Sampling from rhizomes of C. longa was performed at three different development stages, i.e., July (when young rhizomes emerge), September (flowers bloom), and November (ready for harvest). Using a polydimethylsiloxane (PDMS)-coated bar as an adsorption device for volatile compounds, 1,8-cineol, α-terpinolene, β-caryophyllene, and ar-curcumene were the predominant constituents in most cases. Additional volatile compounds such as α-terpinene, p-cymene, and (E)-β- farnesene were identified when PDMS/carboxene/divinylbenzene-coated fiber was used. ar-Turmerone was found from ripened rhizomes (in September and November). The leaves of C. longa yielded the same compounds as the rhizomes as well as compound characteristic of leaves such as 3-hexen-1-ol. The volatile compounds obtained from C. longa roots were the same as those from the rhizomes. The antioxidant activity of both water and methanol extracts of C. longa rhizomes collected from the medicinal plant garden was confirmed using electron spin-resonance spin-trapping method with potent scavenging activity against superoxide anion radical (O2A‹Â‘A‹Â‰). Extracts from ripe rhizomes ready for harvest exhibited greater antioxidant activity than those obtained from young rhizomes.

N-Vinylcarbazole as Versatile Photoinaddimer of Photopolymerization under Household UV LED Bulb (392 nm).

N-vinylcarbazole (NVK) can act simultaneously as a photoinitiator, an additive, and a mono-mer (photoinaddimer) of photopolymerization upon exposure to the household ultraviolet (UV) light-emitting diode (LED) bulb (emission wavelength centered at 392 nm). Even though the light absorption spectrum of NVK exhibits weak overlapping with the emission spectrum of the UV LED, the active species (i.e., radicals and cations) can be generated from the interaction between NVK and diphenyliodonium hexafluorophosphate (Iod) under irradiation of this LED device, which is investigated by steady state photolysis and electron spin resonance spin-trapping methods. Interestingly, the generated radicals and cations from the NVK/Iod system demonstrate high efficiency to initiate the free radical photopolymerization of (meth)acrylates and the cationic photopolymerization of epoxide and divinyl ether under the UV LED irradiation, and the one-step simultaneous catonic/radical photopolymerization of expoxide/acrylate blend can lead to the formation of tack free polyacrylate/polyether-based interpenetrated polymer network film within 10 min even when the polymerization process is exposed to the atmosphere highlighting the high efficiency of the system to reduce the oxygen inhibition effect. More interestingly, NVK/Iod system can also initiate the photopolymerization of NVK under the UV LED irradiation to produce polyvinylcarbazole, and NVK acts as both a photoinitiator and a monomer in the system.

Detection of High-frequency Ultrasound-induced Singlet Oxygen by the ESR Spin-trapping Method

Abstract Singlet oxygen (1O2) generated by high-frequency (1.65 MHz) ultrasonic irradiation of an aqueous solution was detected using the electron spin resonance (ESR) spin-trapping method.

Preparation of soluble stable C60/human serum albumin nanoparticles via cyclodextrin complexation and their reactive oxygen production characteristics

AimsThe stability of solutions of fullerene C60 with human serum albumin (C60/HSA) has not been studied in detail. In this study, we report on the preparation of stable C60/HSA solutions that are formed via the formation of C60/HP-β-CyD nanoparticles, i.e. by transferring C60 molecules from HP-β-CyD to HSA molecules, and an investigation of the reactive oxygen species on the behavior of the resulting C60/HSA. Main methodsStructural and functional properties were examined by spectroscopic techniques, including circular dichroism (CD), fluorescent spectra (FI), and electron spin resonance spin-trapping method, and by cell viability test using A549 cells. Key findingAqueous C60/HSA solutions with a small distribution size, excellent dispersion stability and a high dispersion concentration were obtained without the use of organic solvents. CD and FI spectra indicated that the HSA had undergone structural changes as the result of C60/HSA formation. Binding experiments indicated that C60/HSA had increased Site I and Site II-ligand binding capabilities. The HSA, in the form of C60/HSA was, therefore, conformationally altered, and its binding capabilities were enhanced. Radical scavenging studies using the 1,1′-diphenyl-2-picrylhydrazyl radical showed that C60/HSA had an increased antioxidant activity, compared to HSA alone. Further, C60/HSA efficiently generated not only superoxide anion radicals O2·− but also singlet oxygen 1O2 through photoirradiation. C60/HSA showed cell toxicity characteristics after light irradiation, but no toxicity was observed in the absence of irradiation. SignificanceC60/HSA not only has an excellent stability and antioxidant activity, but also has substantial phototoxicity properties. It thus appears that HSA can be used as a carrier of C60 in drug delivery systems for photodynamic therapy.

Evaluation of Photodynamic Activity of C60/2-Hydroxypropyl-β-Cyclodextrin Nanoparticles

The objective of this study is to evaluate the ability of C(60)/2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) naonparticles to generate reactive oxygen species (ROS) and to induce cell toxicity by the photoirradiation. C(60) nanoparticles were prepared by cogrinding with HP-β-CyD for 3 h at 4°C under reduced pressure. The photodynamic activity of C(60)/HP-β-CyD nanoparticles was evaluated by spectroscopic methods, including the electron spin resonance spin-trapping method, and by the cell viability test using Hela cells. C(60)/HP-β-CyD nanoparticles efficiently generated not only superoxide anion radical (O(2)(·-)) and hydroxyl radical (·OH), but also singlet oxygen ((1)O(2)) through photoirradiation. The ROS generation was enhanced by decreasing the mean particle diameter of C(60) nanoparticles, and the particle size smaller than 90 nm showed a high generation of ·OH and (1)O(2). In addition, HP-β-CyD enhanced the generation of (1)O(2), compared with polyvinylpyrrolidone (an effective solubillizer for C(60)), due to partial disposition of C(60) in the hydrophobic CyD cavity. Furthermore, C(60) /HP-β-CyD nanoparticles showed cell toxicity after the light irradiation, but no toxicity was observed without the light irradiation. Therefore, HP-β-CyD is useful for the preparation of stable C(60) nanoparticles with high ROS generation ability, and C(60)/HP-β-CyD nanoparticles are a promising photosensitizer for photodynamic therapy.

Free radical formation from sonolysis of water in the presence of different gases

In the present study by applying electron spin resonance-spin trapping method, when a high frequency (1650 kHz) ultrasound was irradiated to water dissolved with different gas molecules (O2, N2, Ar, Ne, He, and H2) at 25°C of water bulk temperature, free radical generation pattern differed dependently on the dissolved gas molecules. Only •OH was detected in the O2-dissolved water sample, and the amount of the radical was much greater than that determined in any of other gas-dissolved water samples. One of the possible reasons to explain why the •H radical was not detected in the O2-dissolved water is that the •H reacts with O2 to form •OOH. However, no electron spin resonance signals related to the adduct of not only 5,5-dimethyl-1-pyrroline-N-oxide but 5-(2,2-Dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide and •OOH were observed. In the H2-dissolved water, only •H was detected, suggesting that H2 reduces or neutralizes •OH. In the N2-disolved water, both •OH and •H were detected at comparable level. In the water samples dissolved with rare gases (Ar, Ne, and He), the amount of •H was almost double as compared with that of •OH, and both •OH and •H yields increased in the order Ar > Ne > He.

Scavenging Effects of Metal Complexes of Water-Soluble Thiacalix[4]arenetetrasulfonate on Superoxide Anion Radicals

The scavenging effects of metal complexes of thiacalix[4]arenetetrasulfonate (Me-TCAS[4], Me=H₂, Fe³(+), Mn³(+), Mn²(+), Cu²(+), and Zn²(+)) on superoxide anion radicals (O₂⁻) generated from the xanthine-xanthine oxidase system were investigated by the nitroblue tetrazolium (NBT) method and electron spin resonance (ESR) spin-trapping method using 5,5-dimethyl-1-pyrroline-N-oxide as a trapping reagent. As a reference, calix[4]arenetetrasulfonate (H₂)-CAS[4]), calix[6]arenehexasulfonate (H₂-CAS[6]) and calix[8]areneoctasulfonate (H₂-CAS[8]) were also examined. The results by the NBT method indicated that Fe³(+)- and Mn³(+)-TCAS[4] exhibited the highest O₂⁻ scavenging activity among Me-TCAS[4] and H₂-CAS[n] (n = 4, 6, 8) in this study. The IC₅₀ values of Fe³(+)- and Mn³(+)-TCAS[4] for O₂⁻ scavenging activity were estimated to be 5.3 and 7.8 µM, respectively, and were almost the same as those of tannin acid, catechin and their derivatives, which are known as very effective scavengers of O₂⁻. Scavenging activities were in the order of Fe³(+)- and Mn³(+)-TCAS[4]>>Mn²(+)-, Cu²(+)-, and Zn(2+)-TCAS[4]>>H(2)-TCAS[4] and H₂-CAS[n] (n=4, 6, 8). Each activity of Me-TCAS[4] (Me=Fe³(+), Mn³(+), Mn²(+), Cu²(+), and Zn²(+)) was higher than that of the corresponding metal ion, indicating that H₂-TCAS[4] has the ability to raise the activity of the metal ion itself by forming a complex. Also, the ESR spin-trapping method revealed that Fe³(+)- and Mn³(+)-TCAS[4] showed high O₂⁻ scavenging activities, similarly to the results by the NBT method.

Applications of Electron Spin Resonance Spectrometry for Reactive Oxygen Species and Reactive Nitrogen Species Research

Electron spin resonance (ESR) spectroscopy has been widely applied in the research of biological free radicals for quantitative and qualitative analyses of reactive oxygen species (ROS) and reactive nitrogen species (RNS). The ESR spin-trapping method was developed in the early 1970s and enabled the analysis of short-lived free radicals. This method is now widely used as one of the most powerful tools for free radical studies. In this report, some of the studies that applied ESR for the measurement of ROS and RNS during oxidative stress are discussed.

Extensive screening for herbal extracts with potent antioxidant properties

This paper summarizes our research for herbal extracts with potent antioxidant activity obtained from a large scale screening based on superoxide radical (O2•−) scavenging activity followed by characterization of antioxidant properties. Firstly, scavenging activity against O2•− was extensively screened from ethanol extracts of approximately 1000 kinds of herbs by applying an electron spin resonance (ESR)-spin trapping method, and we chose four edible herbal extracts with prominently potent ability to scavenge O2•−. They are the extracts from Punica granatum (Peel), Syzygium aromaticum (Bud), Mangifera indica (Kernel), and Phyllanthus emblica (Fruit). These extracts were further examined to determine if they also scavenge hydroxyl radical (•OH), by applying the ESR spin-trapping method, and if they have heat resistance as a desirable characteristic feature. Experiments with the Fenton reaction and photolysis of H2O2 induced by UV irradiation demonstrated that all four extracts have potent ability to directly scavenge •OH. Furthermore, the scavenging activities against O2•− and •OH of the extracts of P. granatum (peel), M. indica (kernel) and P. emblica (fruit) proved to be heat-resistant.The results of the review might give useful information when choosing a potent antioxidant as a foodstuff. For instance, the four herbal extracts chosen from extensive screening possess desirable antioxidant properties. In particular, the extracts of the aforementioned three herbs are expected to be suitable for food processing in which thermal devices are used, because of their heat resistance.

Stimulatory Effects of Low-concentration Reactive Oxygen Species on Calcification Ability of Human Dental Pulp Cells

The present study was conducted to investigate the effects of reactive oxygen species (ROS) on the calcification ability of human dental pulp (HDP) cells. HDP cells were treated with 100 μmol/L hydrogen peroxide (H 2O 2) for 5 or 10 minutes (5-min ROS group and 10-min ROS group) to investigate the mechanism of transmission to cells. Untreated cells were used as controls. Generation of free radicals was quantified by the electron spin resonance spin-trapping method and found to be increased by treatment with ROS. Formation of calcified nodules was also investigated by von Kossa staining and alizarin red S staining. Twenty-eight days after exposure, calcified nodules were present in cell cultures that had been treated with ROS for 5 or 10 minutes. Expression of mRNAs for osteopontin (OPN) and osteocalcin (OCN) was significantly greater in 10-min ROS group 6 and 9 days, respectively, after exposure than in controls. Production of OPN and OCN by 10-min ROS group was also greater 12 and 18 days, respectively, after exposure than in controls. These results suggested that calcification of HDP cells was stimulated by H 2O 2 and by the ROS it generated.

Investigation of the generation of hydroxyl radicals and their oxidative role in the presence of heterogeneous copper catalysts

The presence and impact of hydroxyl radicals generated via the catalytic decomposition of H 2O 2 over heterogeneous copper catalysts were investigated by using two detection methods, an electron spin resonance-spin trapping method and a chemical probe method. Detection of the (5,5-dimethyl-1-pyrroline-N-oxide)–OH adduct signal and formation of 4-chlorocatechol during the oxidation of a 4-chlorophenol substrate demonstrated that the three heterogeneous copper catalysts employed here (CuO, Cu/Al 2O 3 and CuO · ZnO/Al 2O 3) were capable of generating hydroxyl radicals in combination with H 2O 2. The oxidative mechanism of the hydroxyl radical in the presence of heterogeneous copper catalysts is discussed with regard to the further oxidation of the (5,5-dimethyl-1-pyrroline-N-oxide)–OH adduct and hydroxylated products of 4-chlorophenol oxidation. Interestingly, integration of the 5,5-dimethyl-1-pyrroline-N-oxide–OH adduct signal could not be used to reliably measure the total amount of hydroxyl radicals generated as a result of oxidative attack on the adduct. This may be as a result of locally higher hydroxyl radical concentrations in the presence of a heterogeneous catalyst leading to further unwanted oxidation of the (5,5-dimethyl-1-pyrroline-N-oxide)–OH.

The Roles of Human-Inducible Nitric Oxide Synthase and Xanthine Oxidase in Regulating Chronic Inflammation in Radicular Cysts

The immunohistochemical activities of nitric oxide synthase (NOS), xanthine oxidase(XO) and nitrotyrosine(NT) in human radicular cysts were measured. The molecular interactions of nitric oxide (NO) or peroxynitrate(ONOO-) and superoxide(O2- •) generated in the xanthine/XO system were kinetically investigated in vitro to clarify the mechanism of tissue injury. To investigate the interaction between NO • and O2- • generated in radicular cysts, the localization of NOS and XO was evaluated by an immunohistochemical staining method. Furthermore, the presence of ONOO- was immunohistochemically estimated. The molecular interactions of O2- •, NO • and ONOO- were analyzed by using the electron spin resonance spin-trapping method. In an area of inflammatory reaction, human-inducible NOS (iNOS), XO and NT were positive compared with a normal area, although only iNOS production was confirmed an area of fibrous reaction. Kinetic studies showed that 1-hydroxy-2-oxo-3 (N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC-7) reacted directly with O2- •, whereas ONOO- did not react with O2- • but inhibited XO directly. The close involvement of iNOS production in the inflammatory region and the influence of the inflammatory reaction with both NO • production from iNOS and O2- • production from XO were suggested. The presence of iNOS in cysts suggests that NO • produced in periapical lesions plays an important role in the regulation of chronic lesions via the reaction with O2- • produced catalyzed by XO. From the reaction with O2- •, and NO • generated by the iNOS system, the formation of ONOO- and its direct inhibition of XO activity decreased the amount of O2- • produced and as a result inhibited the continued formation of ONOO-.

Distinguishing the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-OH radical quenching effect from the hydroxyl radical scavenging effect in the ESR spin-trapping method

The spin-trapping method has been widely accepted to measure the hydroxyl radical scavenging activity in electron spin resonance (ESR) spectroscopy, however, the disappearance of the trapped signal of the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-OH radical may lead to an erroneous conclusion, as the decreased signal must result from the rapid quenching of the DMPO-OH formed. The DMPO-OH quenching activity of a compound can be measured by tracing the signal decay resulting from its addition after the cessation of DMPO-OH formation. It was found that Fe(II) ions could quench the DMPO-OH radical quite rapidly, and phosphate ions could enhance the DMPO-OH radical quenching activity of the Fe(II) ions.

Superoxide scavenging activities of sixty Chinese medicines determined by an ESR spin-trapping method using electrogenerated superoxide.

Superoxide-scavenging activities of 60 kinds of Chinese herbal medicines were determined accurately by an electron spin resonance (ESR) spin-trapping technique using 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) as a spin-trapping reagent. As a source of superoxide in this method, superoxide generated by one-electron reduction of the oxygen molecule in dimethyl sulfoxide solution was used. As a result of these studies, very powerful scavenging activity was found in Chinese medicines for inflammation, diseases of blood circulation and for tumors.

Determination of Superoxide Scavenging Activity of a Sample Containing Xanthine Oxidase Inhibitor by ESR Spin Trapping

The objective of this research was to determine the true superoxide (O2-) scavenging activity of antioxidants in a sample containing xanthine oxidase (XOD) inhibitor. A previous method involving electron spin resonance (ESR) spin-trapping has generally used 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) as a spin trap and the O2- generation system of hypoxanthine (HPX)-xanthine oxidase (XOD). However, this method has a problem in that the ESR signal intensity of the DMPO spin adduct of O2- (DMPO-OOH) is decreased not only by the reaction of a sample with O2-, but also by the inhibition of XOD. Therefore, the ESR spin-trapping method combined with the SOD addition method was developed as a new experimental technique in this study. The results indicate that the O2- scavenging activity of a sample containing allopurinol as an XOD inhibitor could be determined by using this method.

Detection of free radicals generated from hydrogen peroxide, gallic acid and haemoprotein chemiluminescence system by electron spin resonance spectroscopy.

Low-level chemiluminescence is produced in a hydrogen peroxide (H(2)O(2))/gallic acid/haemoprotein system with single broad peaks around 520 nm, regardless of the biological role of the haemoprotein. The free haem iron systems (haemin and haematin systems) gave a higher photon intensity (1.5 x 10(4) and 2.0 x 10(4) cps) than that of the H(2)O(2)/gallic acid/haematoporphyrin system. These results indicated that haem iron plays a significant role in the photon emission of haemoprotein systems. A free radical with a g value of 2. 0058 was detected through a direct electron spin resonance (ESR) method. The photon intensity of the H(2)O(2)/gallic acid/haemoprotein system decreased in the order: HRP > cytochrome c > myoglobin > haemoglobin, and this corresponded to the decrease in radical intensity. These results indicated that the formation of the free radical with a g value of 2.0058 may be the key step for chemiluminescence in the H(2)O(2)/gallic acid/haemoprotein system. A quartet line similar to DMPO-OH adducts and uncomplexed free radicals (g = 2.0058) was detected using the ESR spin-trapping method in the H(2)O(2)/gallic acid/cytochrome c system.

Mechanisms of active oxygen species reduction by non-steroidal anti-inflammatory drugs

Many forms of active oxygen have been suggested to participate in the course of inflammation. Anti-inflammatory drugs have been considered to function as active oxygen inhibitors. However, detailed mechanisms for such inhibitory activity remain unclear because of little well established methods to study inhibitory effect of anti-inflammatory drugs on active oxygen species. In this report, the author investigated four non-steroidal anti-inflammatory drugs, including acetaminophen, sodium salicylate, naproxen and flurbiprofen, their elimination and inhibition ability of active oxygen, using of the electron spin resonance spin-trapping method and the horseradish peroxidase method. In this experiment as active oxygen models, superoxide was evolved from a hypoxanthine-xanthine oxidase reaction system, and hydrogen peroxide by the spontaneous dismutation reaction. The data here show that the amount of superoxide was reduced in the manner of concentration of non-steroidal anti-inflammatory drugs in the reaction. Kinetic studies for these reaction showed that acetaminophen and sodium salicylate reacted with superoxide competitively, whereas naproxen and flurbiprofen did not. Analysis of generation of hydrogen peroxide formed by the spontaneous dismutation of superoxide derived from the reaction system revealed that hydrogen peroxide was increased by acetaminophen and decreased by sodium salicylate, naproxen and flurbiprofen.