Riboflavin (Rb) and its biotransformed metabolites including flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) have an isoalloxazine ring as a common photoactive moiety. In this study, photostability and photosensitizing properties of Rb, FMN, and FAD were evaluated under a regular fluorescent light and different LEDs (blue, red, green, and white), and the characteristics of their photochemical reaction were also investigated during the photooxidation processes. The photostability of the isoalloxazine derivatives showed a significant difference depending on the type of light source and its illuminance, as well as the alkyl group structure of each derivative bound to the isoalloxazine ring and their concentrations. Among the derivatives, FAD was most stable under irradiation of all types of light. Among the lights irradiated, Blue LED induced the most rapid photodegradation of the derivatives; their half-lives of Rb, FMN, and FAD at 50 μM under 50 lx Blue LED were 0.88, 0.38, and 5.04 h, respectively. HPLC analyses for the degradation products from the derivatives under Blue LED irradiation indicated that lumichrome (LC) was a common photooxidation product from each derivative. Rb and FMN showed more potent photosensitizing activities than FAD under irradiation of all types of light. Blue LED also induced the strongest photosensitizing activities of all Rb derivatives. Enhancement of DCF fluorescence by Rb was most pronounced under light, indicating that ROS generation due to its photosensitizing activity was most prominent. Rb and FMN significantly enhanced lipid peroxidation up to 4 μM, whereas the extent of lipid oxidation rather decreased at higher concentrations. FAD promoted lipid oxidation more potently under light with a concentration-dependent manner up to 32 μM. Photo-degraded Rb and FMN showed a potent Fe (II) chelating activity, whereas the activity of FAD was not observed. All the derivatives displayed an antioxidant potential, showing ABTS radical scavenging activity and reducing activity of Fe (III) to Fe (II) only when irradiated with light. Rb and FMN induced the most potent phototoxicity in both keratinocyte (HaCaT) and human lens epithelial cells (HLE B-3). In particular, HLE B-3 were much more sensitive to the phototoxicity induced by the derivatives than HaCaT.
The cytotoxicity and H₂O₂ generation induced by Rb under light showed a big difference in different culture media including PBS, HBSS, and phenol red free DMEM (RPF-D). In particular, Rb induced the greatest response in RPF-D containing basal amino acids. Therefore, a potential for the interactions between Rb derivatives and different amino acids under light was evaluated in the nitro blue tetrazolium (NBT) reduction system, and the consequent effects through these interactions on the Rb-induced photosensitizing reactions including ROS generation and phototoxicity werealso analyzed. NBT-reducing properties of Rb, LC, lumiflavin (LF), and FMN under blue LED irradiation were investigated in the presence of different 21 amino acids. The NBT reduction occurred effectively in the combined reaction system of Rb derivatives with 6 amino acids including His, Met, Cys, Arg, Hyp, and Asn. Among the Rb derivatives, Rb and FNM displayed the most potent NBT reduction activity in the presence of the amino acids. The NBT reduction was induced by Cys even without Rb, regardless of the light irradiation. SOD strongly inhibited the NBT reduction induced Rb combined with the 5 amino acids except Cys. Rb-induced ROS generation decreased in the combined reaction with Met, His, and Cys during light irradiation. However, the amount of H₂O₂ produced in each combined reaction increased as the concentrations of Rb and each amino acid increased. The phototoxicity of Rb in skin cells was enhanced amino acid concentration-dependently in the presence of Rb under light. In addition, the interaction of Rb derivatives with iron ions in the O/W system was also analyzed. Lipid peroxidation induced by the derivatives was more pronounced in the presence of metal ions under light. However, all three derivatives significantly inhibited the Fenton reaction-induced lipid peroxidation in a concentration-dependent manner.
The present study indicates that the Rb and its derivatives could play dual roles as pro- and anti-oxidant and cause photo-damages on the living system in light-stimulated conditions. These results also suggest that Rb and its isoalloxazine derivatives may cause deterioration of food quality or biological changes in vivo through interactions with various food ingredients and biomolecules, including amino acids and iron ion under light. Therefore, when Rb and its derivatives are used as ingredients in various foods and cosmetics, it needs to be carefully considered photosensitizing properties of these derivatives and their consequent effects on quality degradation of the products as well as physiological actions in body.