Yoshimi Niwano

Photolysis of hydrogen peroxide, an effective disinfection system via hydroxyl radical formation.

ABSTRACT The relationship between the amount of hydroxyl radicals generated by photolysis of H2O2 and bactericidal activity was examined. H2O2 (1 M) was irradiated with laser light at a wavelength of 405 nm to generate hydroxyl radicals. Electron spin resonance spin trapping analysis showed that the amount of hydroxyl radicals produced increased with the irradiation time. Four species of pathogenic oral bacteria, Staphylococcus aureus, Aggregatibacter actinomycetemcomitans, Streptococcus mutans, and Enterococcus faecalis, were used in the bactericidal assay. S. mutans in a model biofilm was also examined. Laser irradiation of suspensions in 1 M H2O2 resulted in a >99.99% reduction of the viable counts of each of the test species within 3 min of treatment. Treatment of S. mutans in a biofilm resulted in a >99.999% reduction of viable counts within 3 min. Other results demonstrated that the bactericidal activity was dependent on the amount of hydroxyl radicals generated. Treatment of bacteria with 200 to 300 μM hydroxyl radicals would result in reductions of viable counts of >99.99%.

Extensive Screening for Edible Herbal Extracts with Potent Scavenging Activity against Superoxide Anions

To search for edible herbal extracts with potent antioxidant activity, we conducted a large scale screening based on the superoxide scavenging activity. That is, scavenging activity against superoxide anions were extensively screened from ethanol extracts of approximately 1,000 kinds of herbs by applying an electron spin resonance (ESR)-spin trapping method. Among them we chose four edible herbal extracts with prominently potent ability to reduce the signal intensity of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)–OOH, a spin adduct formed by DMPO and superoxide anion. They are the extracts from Punica granatum (Peel), Syzygium aromaticum (Bud), Mangifera indica (Kernel), and Phyllanthus emblica (Fruit), and are allowed to be used as foodstuffs according to the Japanese legal regulation. The ESR-spin trapping method coupled with steady state kinetic analysis showed that all of the four extracts directly scavenge superoxide anions, and that the superoxide scavenging potential of any of the extracts was comparable to that of L-ascorbic acid. Furthermore, polyphenol determination indicates that the activity is at least in part attributable to polyphenols. These results with such large scale screening might give useful information when choosing a potent antioxidant as a foodstuff.

Fracture resistance of monolithic zirconia molar crowns with reduced thickness

Abstract Objectives. The purpose of the present study was to analyze the relationship between fracture load of monolithic zirconia crowns and axial/occlusal thickness and to evaluate the fracture resistance of monolithic zirconia crowns with reduced thickness in comparison with that of monolithic lithium disilicate crowns with regular thickness. Materials and methods. Monolithic zirconia crowns (Lava Plus Zirconia, 3M/ESPE) with specified axial/occlusal thicknesses and lithium disilicate crowns (IPS e.max press, Ivoclar/Vivadent) with regular thickness were fabricated using a dental CAD/CAM system and a press technique, respectively. The crowns cemented onto dies were loaded until fracture. Based on measurements of the crown thickness made by micro-CT and the fracture load, multiple regression analysis was performed. Results. It was revealed that the occlusal thickness significantly affected the fracture load (p < 0.01), but the axial thickness did not (p = 0.2828). Although the reduction of the occlusal thickness decreased the fracture resistance of the monolithic zirconia crowns, the fracture load of the zirconia crowns with the occlusal thickness of 0.5 mm (5558 ± 522 N) was significantly higher than that of lithium disilicate crowns with an occlusal thickness of 1.5 mm (3147 ± 409 N). Conclusion. Within the limitations of the present study, it is suggested that monolithic zirconia crown with chamfer width of 0.5 mm and occlusal thickness of 0.5 mm can be used in the molar region in terms of fracture resistance.

Reevaluation of analytical methods for photogenerated singlet oxygen

The aim of the present study is to compare different analytical methods for singlet oxygen and to discuss an appropriate way to evaluate the yield of singlet oxygen photogenerated from photosensitizers. Singlet oxygen photogenerated from rose bengal was evaluated by electron spin resonance analysis using sterically hindered amines, spectrophotometric analysis of 1,3-diphenylisobenzofuran oxidation, and analysis of fluorescent probe (Singlet Oxygen Sensor Green®). All of the analytical methods could evaluate the relative yield of singlet oxygen. The sensitivity of the analytical methods was 1,3-diphenylisobenzofuran < electron spin resonance < Singlet Oxygen Sensor Green®. However, Singlet Oxygen Sensor Green® could be used only when the concentration of rose bengal was very low (<1 µM). In addition, since the absorption spectra of 1,3-diphenylisobenzofuran is considerably changed by irradiation of 405 nm laser, photosensitizers which are excited by light with a wavelength of around 400 nm such as hematoporphyrin cannot be used in the 1,3-diphenylisobenzofuran oxidation method. On the other hand, electron spin resonance analysis using a sterically hindered amine, especially 2,2,6,6-tetramethyl-4-piperidinol and 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide, had proper sensitivity and wide detectable range for the yield of photogenerated singlet oxygen. Therefore, in photodynamic therapy, it is suggested that the relative yield of singlet oxygen generated by various photosensitizers can be evaluated properly by electron spin resonance analysis.

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.

Scavenging or Quenching Effect of Melanin on Superoxide Anion and Singlet Oxygen

Although photoprotective properties of skin melanin have been well documented, a few studies on the effect of melanin on reactive oxygen species (ROS) generated by ultraviolet (UV) irradiation have been reported. To study the interaction of melanin with ROS, scavenging or quenching effect of melanin on O2•− and 1O2 was examined by electron spin resonance (ESR)-spin trapping methods and a spectrophotometric method, respectively. Melanin potently interacted with O2•− generated in a hypoxanthine (HPX)-xanthine oxidase (XOD) reaction, and with 1O2 generated from a peroxidase, H2O2, and halide system. In the HPX-XOD reaction, it was proved that melanin doses not interfere with the enzyme reaction. It is confirmed that one of the mechanisms by which melanin protects UV-induced skin damage is likely scavenging or quenching activity against ROS such as O2•− and 1O2.

Bactericidal Action of Photogenerated Singlet Oxygen from Photosensitizers Used in Plaque Disclosing Agents

Background Photodynamic therapy (PDT) has been suggested as an efficient clinical approach for the treatment of dental plaque in the field of dental care. In PDT, once the photosensitizer is irradiated with light of a specific wavelength, it transfers the excitation energy to molecular oxygen, which gives rise to singlet oxygen. Methodology/Principal Findings Since plaque disclosing agents usually contain photosensitizers such as rose bengal, erythrosine, and phloxine, they could be used for PTD upon photoactivation. The aim of the present study is to compare the ability of these three photosensitizers to produce singlet oxygen in relation to their bactericidal activity. The generation rates of singlet oxygen determined by applying an electron spin resonance technique were in the order phloxine > erythrosine ≒ rose bengal. On the other hand, rose bengal showed the highest bactericidal activity against Streptococcus mutans, a major causative pathogen of caries, followed by erythrosine and phloxine, both of which showed activity similar to each other. One of the reasons for the discrepancy between the singlet oxygen generating ability and bactericidal activity was the incorporation efficiency of the photosensitizers into the bacterial cells. The incorporation rate of rose bengal was the highest among the three photosensitizers examined in the present study, likely leading to the highest bactericidal activity. Meanwhile, the addition of L-histidine, a singlet oxygen quencher, cancelled the bactericidal activity of any of the three photoactivated photosensitizers, proving that singlet oxygen was responsible for the bactericidal action. Conclusions It is strongly suggested that rose bengal is a suitable photosensitizer for the plaque disclosing agents as compared to the other two photosensitizers, phloxine and erythrosine, when used for PDT.

The influence of low-temperature degradation and cyclic loading on the fracture resistance of monolithic zirconia molar crowns

The present study analyzed the kinetics of low-temperature degradation (LTD) in zirconia, and evaluated the influence of LTD and cyclic loading on the fracture resistance of monolithic zirconia molar crowns. Bar-shaped zirconia specimens were divided into nine groups and autoclaved at 134°C for 0-200h to induce LTD. The surface fraction and penetration depth of the monoclinic phase were examined using X-ray diffraction and scanning electron microscopy. Monolithic zirconia molar crowns were prepared for crown fracture testing. The crowns were autoclaved for 0-100h (n=6) and cemented to dies. Six crown-die samples that were not autoclaved and six samples that were autoclaved for 100h were subjected to cyclic loading with a load of 300N for 240,000 cycles. All samples were tested in a load-to-failure test. The monoclinic fraction on the surface increased with autoclaving time and reached a plateau after 50h. The depth of the monoclinic phase increased without reaching a plateau. The fracture load of the crowns significantly decreased from 5683N (SD: 342) to 3975N (SD: 194) after 100h of autoclaving. Cyclic loading did not significantly affect the fracture resistance of the crowns in all cases. Kinetic analysis showed no linear correlation between the surface fraction and depth of the monoclinic phase after 50h of autoclaving. Even though LTD increased the monoclinic phase, resulting in lower strength, the fracture resistance of the monolithic zirconia crowns was still sufficient to withstand the loading conditions in the molar regions.

Bactericidal action of photoirradiated gallic acid via reactive oxygen species formation.

It is known that gallic acid shows antimicrobial activity. In the present study, photoirradiation induced reactive oxygen species formation was investigated for augmentation of the antimicrobial activity of gallic acid. Staphylococcus aureus suspended in 4 mmol/L gallic acid was exposed to blue light of a LED at 400 nm. This treatment killed the bacteria, and a >5-log reduction of the viable counts was observed within 15 min. By contrast, neither the LED treatment alone nor the treatment with gallic acid alone showed substantial bactericidal effect. When hydroxyl radical scavengers were added to the suspension, the bactericidal effect of photoirradiated gallic acid was attenuated. Furthermore, electron spin resonance analysis demonstrated that hydroxyl radicals were generated by the photoirradiation of gallic acid. Thus, the present study suggests that the photo-oxidation can enhance the antimicrobial activity of gallic acid via hydroxyl radical formation.

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.