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

Generation Mechanism of Radical Species by Tyrosine-Tyrosinase Reaction

Alleviated melanin formation in the skin through inhibition of tyrosine-tyrosinase reaction is one of the major targets of cosmetics for whitening ability. Since melanin has a pivotal role for photoprotection, there are pros and cons of inhibition of melanin formation. This study applying electron spin resonance (ESR)-spin trapping method revealed that •H and •OH are generated through tyrosine-tyrosinase reaction. When deuterium water was used instead of H2O, the signal of 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-H (a spin adduct of DMPO and •H) greatly decreased, whilst DMPO-OH (a spin adduct of DMPO and •OH) did not. Thus, it is suggested that •H was derived from H2O, and •OH through oxidative catalytic process of tyrosine to dopaquinone. Our study suggests that tyrosinase inhibitors might contribute to alleviate the oxidative damage of the skin by inhibiting •OH generation via the enzyme reaction

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

Myristic Acid, A Side Chain of Phorbol Myristate Acetate (PMA), Can Activate Human Polymorphonuclear Leukocytes to Produce Oxygen Radicals More Potently than PMA

Myristic acid (MyA), which is a saturated fatty acid (C14:0) and a side chain of phorbol 12-myristate 13-acetate (PMA), was examined if MyA stimulates human polymorphonuclear leukocytes (PMNs) to release oxygen radicals comparable to PMA by applying electron paramagnetic resonance (EPR)-spin-trapping method. When MyA was added to isolated human PMNs, spin adducts of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-OH and DMPO-OOH were time-dependently observed. The amounts of these spin adducts were larger than those of PMNs stimulated by PMA. These results clearly show that MyA is more potent agent to prime human PMNs than PMA, in a point of view of not only O2·− but also ·OH production. This fact calls attention that too much intake of MyA that is known to be contained vegetable oils can lead to crippling effect through uncontrolled production of reactive oxygen species

Fungicidal Action of Hydroxyl Radicals Generated by Ultrasound in Water

It is well known that hydroxyl radicals are generated by ultrasound in water. This study with an electron spin resonance spin-trapping technique showed that hydroxyl radical generation was positively correlated with ultrasound duration and water temperature. The clear fungicidal action against Trichophyton spp. evident by studying cultured cells and the degradation of cytoplasmic and surface structures observed by transmission and scanning electron microscopy suggest that ultrasound in hot water is effective for sterilization of dermatophyte contamination and could be effective for the treatment of tinea infection

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

Evaluation of the potential biological toxicities of aqueous extracts from red tide phytoplankton cultures in in vitro and in vivo systems

The biological toxic potentials of aqueous extracts from the dinophycean flagellates Gymnodinium impudicum and Alexandrium affine and the raphidophycean flagellate Chattonella ovata were examined in both in vitro and in vivo systems. Interestingly, the extract from A. affine was the only one that showed potent cytotoxicities towards HeLa, Vero, and Neuro-2a cells in a concentration- dependent manner. Mice given intraperitoneal injections of the extracts revealed that none of the extracts exhibited serious toxicities in mice. However, temporal body weight loss was observed in the mice injected with the extract from A. affine during the early stage, and the dramatic enlargement of spleens was also observed in the mice on the 7th day after injection. Since A. affine extract showed potent hemolytic activity in vitro towards mouse erythrocytes, hemolytic anemia may be a possible mechanism responsible for the splenomegaly in the mice injected with A. affine extract. Similar marginal effects were observed in the mice injected with the extract from C. ovata; however, no significant toxic or detrimental effects were detected in the mice injected with the extract from G. impudicum. These results suggest that the extract from G. impudicum may not be contaminated with detectable levels of biologically hazardous compounds and may be relatively safe compared with the other two extracts

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

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., 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.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