**6. Screening method for antioxidants that suppress the photosensitizer reaction** *in vitro*

We have proposed an *in vitro* assay using cytotoxicity and hydrogen peroxide as detection indicators in a screening method for compounds that suppress cell


#### **Table 1.**

*The classification of typical antioxidants.*

*Photocatalysis in the Skin Related to UVA Photoaging DOI: http://dx.doi.org/10.5772/intechopen.107445*

#### **Figure 4.**

*Efficacy of ergothioneine against the production of hydrogen peroxide through UVA-photosensitization: Samples were added to HBSS containing 1 μmol/L riboflavin, 30 μmol/L folic acid, and 100 μmol/L tryptophan. Hydrogen peroxide is detected in UVA irradiation control in this assay. For samples, each dose of ergothioneine (EGT) was used. A lamp (Toshiba Lighting & Technology Corporation, Yokosuka, Japan) emitting a UVA spectrum (340–410 nm) was used as the UVA source and was adjusted to an intensity of 1.0 mW/cm<sup>2</sup> . Each sample solution was irradiated for 1 hour in an ice box for temperature control (3.6 J/cm<sup>2</sup> ). The amount of hydrogen peroxide generated in each solution after UVA irradiation with 3.6 J/cm<sup>2</sup> was measured by ADHP/HRP methods. Data are expressed as means ± S.D. statistical analysis was performed by Student's t-test with p-value <0.010 (\*\*p < 0.010) considered statistically significant differences.*

damage caused by UVA-photosensitization. This is because past studies have suggested that hydrogen peroxide generated in the solvent by the UVA photosensitization reaction is the main cause of cell damage [52, 79]. Furthermore, by coexisting with the target compound during UVA exposure, it is possible to evaluate the photosensitization reaction between the target active ingredients and other components.

As an interesting example, histidine, which is used as a singlet oxygen quencher, has not been shown as an effective compound in this *in vitro* assay. This was because histidine enhances the photosensitizing response to riboflavin during UVA exposure [51]. These indicate that they may not be useful depending on the conditions of amino acids and vitamins in which existing antioxidants that are expected to have a photosensitizing effect coexist. Ergothioneine is a powerful antioxidant that has been reported to eliminate singlet oxygen, superoxide, and hydrogen peroxide. A previous study reported that ergothioneine has a protective ability against hydrogen peroxide and other ROS [76, 80]. It was indicated that ergothioneine has an anti-photosensitization efficacy because ergothioneine was treated only during UVA irradiation [81]. In our *in vitro* assay, using hydrogen peroxide as an index, the amount of hydrogen peroxide produced was suppressed in a concentration-dependent manner, without increasing hydrogen peroxide production at any concentration (**Figure 4**). These findings suggest that ergothioneine may prevents the progression of photoaging in the skin.
