inactivation-pathogens-via-visible-light-photolysis-riboflavin-5

Inactivation of Pathogens <em>via</em> Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can ...

Ming Chuan University

The micronutrients in many cellular processes, riboflavin (vitamin B(2)), FMN, and FAD are photo-sensitive to UV and visible light to generate reactive oxygen species (ROS). The riboflavin photochemical treatment with UV light has been applied for the inactivation of microorganisms to serve as an effective and safe technology. Ultra-violet or high-intensity radiation is, however, considered as a highly risky practice. This study was working on the application of visible LED lights to riboflavin photochemical reactions to development an effective antimicrobial treatment. The photosensitization of bacterial genome with riboflavin was investigated in vitro and in vivo by light quality and irradiation dosage. The riboflavin photochemical treatment with blue LED light was proved to be able to inactivate E. coli by damaging nucleic acids with ROS generated. Riboflavin is capable of intercalating between the bases of bacterial DNA or RNA and absorbs lights in the visible regions. LED light illumination could be a more accessible and safe practice for riboflavin photochemical treatments to achieve hygienic requirements in vitro.

Blue light induced free radicals from riboflavin on E. coli DNA damage.

The Folin-Ciocalteu method is widely applied for the determination of the total phenolic contents in natural products. This method is significantly affected by the addition of sodium carbonate. The currently applied Folin-Ciocalteu methods may have been modified without any validation in the quantitative standards and the order of processes. In this study, serial experiments were performed to investigate the effect of phenolic calibrations based on the classic Folin-Ciocalteu method. Esterification condensations were observed in the assays with prior basification for gallic acid and catechin used as quantitative standards. The phenolic contents obtained in the samples differed depending on when basification occurred compared with the gallic acid calibration. The bias of the classic Folin-Ciocalteu method derived from cross-linkage of molecules was first defined in this study. The performance of the Folin-Ciocalteu method is optimised and validated again. 

Effect of esterification condensation on the Folin-Ciocalteu method for the quantitative measurement of total phenols.

The micronutrients in many cellular processes, riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) are photo-sensitive to UV and visible light for generating reactive oxygen species (ROS). Produced from phosphorylation of riboflavin, FMN is more water-soluble and rapidly transformed into free riboflavin after ingestion. This study investigated the application of visible blue light with FMN to development of an effective antimicrobial treatment. The photosensitization of bacterial viability with FMN was investigated by light quality, intensity, time, and irradiation dosage. The blue light-induced photochemical reaction with FMN could inactivate Escherichiacoli by the generated ROS in damaging nucleic acids, which was validated. This novel photodynamic technique could be a safe practice for photo-induced inactivation of environmental microorganism to achieve hygienic requirements in food processing. 

Investigations of blue light-induced reactive oxygen species from flavin mononucleotide on inactivation of E. coli.

Determination of the superoxide dismutase activity is an important issue in the fields of biochemistry and the medical sciences. In the riboflavin/nitro blue tetrazolium (B2/NBT) method, the light sources used for generating superoxide anion radicals from light-excited riboflavin are normally fluorescent lamps. However, the conditions of B2/NBT experiments vary. This study investigated the effect of the light source on the light-excitation of riboflavin. The effectiveness of the photolysis was controlled by the wavelength of the light source. The spectra of fluorescent lamps are composed of multiple colour lights, and the emission spectra of fluorescent lamps made by different manufacturers may vary. Blue light was determined to be the most efficient for the photochemical reaction of riboflavin in visible region. The quality of the blue light in fluorescent lamps is critical to the photo-decomposition of riboflavin. A blue light is better than a fluorescent lamp for the photo-decomposition of riboflavin. The performance of the B2/NBT method is thereby optimized. 

Investigations of riboflavin photolysis via coloured light in the nitro blue tetrazolium assay for superoxide dismutase activity.

Polyphenols are associated with a wide range of physiological properties. Catechin is a flavan-3-ol with five phenolic hydroxyl groups. After blue light illumination, the transparent solution of catechin became yellowish. The effects of visible light illumination (400-800nm) were investigated on molecular structures and antioxidant capacities of catechin. Under the neutral or alkaline aqueous with the illumination of blue light, the photolysis and polymerization of catechin were observed in this study. A chromogenic catechin dimer was separated and identified as a proanthocyanidin by the chromatographic technique and mass spectrometry. For quantitative evaluation, the signal intensities of the catechin and the photochemical product show a negative correlation in the liquid chromatograms. The oligomer of flavan-3-ols (catechin dimer) is suggested as a dimeric B type proanthocyanidin, which has the molecular formula CHO and 578.14g/mol in exact mass. The mass spectrum of catechin dimer had characteristic ion signals in m/z 577, 560, 439Da. However, the total phenolic contents and scavenging O activity of catechin treated by blue light illumination are not changed significantly at the neutral or alkaline aqueous. Our results of photocatalytic oligomers of catechin provide a novel way to explain the sensory changes of green tea and a biochemical mechanism under the irradiation environments.

Photo-catalytic polymerization of catechin molecules in alkaline aqueous.

The light sensitive compound riboflavin-5'-phosphate (or flavin mononucleotide, FMN) generates reactive oxygen species (ROS) upon photo-irradiation. FMN is required by all flavoproteins because it is a cofactor of biological blue-light receptors. The photochemical effects of FMN after irradiation by blue or violet light on the inactivation of Staphylococcus aureus strains, including a methicillin-resistant strain (MRSA), were investigated in this study. Upon blue- or violet-light photo-treatment, FMN was shown to inactivate S. aureus due to the generated ROS. Effective bacterial inactivation can be achieved by FMN photolysis without an exogenous electron provider. Inactivation rates of 94.9 and 95.2% in S. aureus and MRSA, respectively, can be reached by blue light irradiation (2.0mW/cm) with 120μM FMN for 120min. A lower FMN concentration and a shorter time are required to reach similar effects by violet light irradiation. Inactivation rates of 96.3 and 97.0% in S. aureus and MRSA, respectively, can be reached by violet light irradiation (1.0mW/cm) with 30μM FMN for 30min. The sensitivity of the inherent photosensitizers is lower under blue-light irradiation. A long exposure photolytic treatment of FMN by blue light is required to inactivate S. aureus. Violet light was found to be more efficient in S. aureus inactivation at the same radiant intensity. FMN photolysis with blue or violet light irradiation enhanced the inactivation rates of S. aureus and MRSA. FMN photochemical treatment could be a supplemental technique in hygienic decontamination processes.

Effects of blue or violet light on the inactivation of Staphylococcus aureus by riboflavin-5'-phosphate photolysis.

Crystal violet (CV) is applied in daily use mainly as a commercial dye and antimicrobial agent. Waste water containing CV may affect aquatic ecosystems. Riboflavin, also known as vitamin B, is non-toxic and an essential vitamin required for the functions of the human body. Riboflavin is photosensitive to UV and visible light in terms of generating reactive oxygen species. This study investigated the potential application of blue light on riboflavin, so as to come up with an effective way of degrading CV during its treatment. Photosensitivity of CV leading to degradation in the presence of riboflavin was investigated by light intensity, exposure time, and irradiation dosage. The degradation of CV during riboflavin photolysis treatment was studied by a UV/vis spectrometry and chromatography. The effects of CV degradation on microbial viability are relevant when considering the influences on the ecosystem. This study proved that riboflavin photochemical treatment with blue light degrades CV dye by ROS formation. The riboflavin photolysis-treated CV solution appeared to be transparent during conformational transformations of the CV that was rearranged by free radical species generated from riboflavin photolysis. After riboflavin photolysis, colony-forming units (CFUs) were determined for each CV solution. CFU preservation was 85.2% for the CV dissolved riboflavin solution treated with blue light irradiation at 2.0mW/cm for 120min. Degradation of CV by riboflavin photochemical procedures can greatly reduce antimicrobial ability and serve as an environmental friendly waste water treatment method. Our results presented here concerning riboflavin photolysis in degradation of CV provide a novel technique, and a simple and safe practice for environmental decontamination processes.

Blue light induced free radicals from riboflavin in degradation of crystal violet by microbial viability evaluation.

Catechin is a flavan-3-ol, a derivative of flavans, with four phenolic hydroxyl groups, which exhibits a wide range of physiological properties. Chromatographic analyses were employed to examine the effects of blue light irradiation on the changes of catechin hydrate in an alkaline condition. In particular, the detection of a superoxide anion radical (O₂•), a reactive oxygen species (ROS), and the inactivation of  ()—including a carbapenem-resistant  (CRAB)—was investigated during the photoreaction of catechin hydrate. Following basification with blue light irradiation, the transparent solution of catechin hydrate turned yellowish, and a chromogenic catechin dimer was separated and identified as a proanthocyanidin. Adding ascorbic acid during the photolytic treatment of catechin hydrate decreased the dimer formation, suggesting that ascorbic acid can suppress the photosensitive oxidation of catechin. When catechin hydrate was irradiated by blue light in an alkaline solution, O₂• was produced via photosensitized oxidation, enhancing the inactivation of  and CRAB. The present findings on the photon-induced oxidation of catechin hydrate provides a safe practice for the inactivation of environmental microorganisms.

Effects of Blue-Light-Induced Free Radical Formation from Catechin Hydrate on the Inactivation of  Including a Carbapenem-Resistant Strain.

The adaptability of bacterial resistance to antibiotics contributes to its high efficiency during evolution. Tetracycline (TC) is a broad-spectrum antimicrobial agent. Chromatographic analyses and mass spectrometry were used to study the effects of the light illumination of a 462 nm light-emitting diode (LED) on the conformational changes of TC in a phosphate buffer solution (PBS, pH 7.8). Especially, the inactivation of superoxide anion radicals (O₂•) and  (), including that of a multidrug-resistant  (), were investigated during the photolysis of TC. A photolysis product of TC (PPT) was generated in an alkaline solution after the illumination of a blue light. The mass spectra of PPT had characteristic ion signals in m/z 459, 445, and 249.1 Da. The PPT has the molecular formula of CHN₂O₉, and the exact mass is 458.44 g/mol. The inactivation of  is not significant with TC treatment. The drug-resistant ability of  has a less significant effect on PPT, and the changed conformation of TC retained the inactivation ability of  upon blue light photoreaction. With TC, illuminated by a blue light in a pH 7.8 PBS, O₂• was generated from TC photolysis, which enhanced the inactivation of  and . A 96.6% inactivation rate of  was reached with TC under 2.0 mW/cm² blue light illumination at 25 ± 3 °C for 120 min, and the effects of the TC-treated photoreaction on  viability repressed the growth of  by 4 to 5 logs. The present study of the blue light photoreaction of TC offers a new approach to the inactivation of 

Effects of 462 nm Light-Emitting Diode on the Inactivation of  and a Multidrug-Resistant by Tetracycline Photoreaction.

Catechins belonging to polyhydroxylated polyphenols are the primary compounds found in green tea. They are associated with many physiological properties. Epicatechin (EC) is a non-gallate-type catechin with four phenolic hydroxyl groups attached. The changes in EC treated with color light illumination in an alkaline condition were investigated by chromatographic and mass analyses in this study. In particular, the superoxide anion radical (O₂•) was investigated during the EC photolytic process. EC is unstable under blue light illumination in an alkaline solution. When EC was treated with blue light illumination in an alkaline solution, O₂• was found to occur via a photosensitive redox reaction. In addition, the generation of monomeric, dimeric, and trimeric compounds is investigated. On the other hand, epigallocatechin gallate (EGCG), which is a gallate-type catechin, is stable under blue light illumination in an alkaline solution. Adding EGCG, during the blue light illumination treatment of EC decreased photolytic formation, suggesting that gallate-type catechins can suppress the photosensitive oxidation of EC. Gallate-type catechins are formed via the esterification of non-gallate-type catechins and gallic acid (GA). The carbonyl group on the gallate moiety of gallate-type catechins appears to exhibit its effect on the stability against the photosensitive oxidation caused by blue light illumination.

Effects of Epigallocatechin Gallate on the Stability of Epicatechin in a Photolytic Process.

Tetracycline (TC) is a broad-spectrum antibiotic compound. Wastewater with TC may have an adverse effect on ecosystems. Riboflavin-5'-phosphate (FMN or flavin mononucleotide) is a non-toxic product of the phosphorylation of vitamin B and is required for the proper functioning of the humans. FMN is sensitized to ultraviolet (UV) and blue light radiation, as evidenced by the generation of reactive oxygen species (ROS). This study inspects feasible applications of blue light on FMN so as to develop a valid way of degrading TC by FMN photolysis. We used the increased rate of bacterial survival as a practical indicator of antibiotic degradation. TC in the presence of FMN solution decomposed completely after 20 W/m of blue light irradiation (TCF treatment), and the degradation of TC (D-TCF) occurred after the photolytic process. After TCF treatment, colony-forming units (CFUs) of  ( were determined for the D-TCF solution. The CFU of  preservation was 93.2% of the D-TCF solution (50 μg/mL of TC in the presence of 114 μg/mL of FMN solution treated with 20 W/m of blue light irradiation at 25 °C for 1 h) cultivation. The mass spectrum of D-TCF showed diagnostic ion signals at m/z 431.0 and 414.0 Da. The molecular formula of D-TCF was CHNO, and the exact mass was 430.44 g/mol. TC degradation by FMN photolysis can significantly decrease the antimicrobial ability of TC. The results expressed here regarding the influence of FMN photolysis on TC degradation offer an environmentally sound wastewater treatment method.

The Influence of the Degradation of Tetracycline by Free Radicals from Riboflavin-5'-Phosphate Photolysis on Microbial Viability.

Tea is rich in catechins and aluminum. In this study, the process of catechin photolysis was applied as a model for examining the effects of aluminum chloride (AlCl) on the structural changes of catechin and the alteration of aluminum complexes under blue light irradiation (BLI) at pH 8 using liquid chromatography and mass spectrometry techniques. Additionally, the effects of anions on catechin upon the addition of AlCl and treatment with BLI were also studied. In this study, when 1 mM catechin was treated with BLI, a superoxide anion radical (O2•-) was generated in an air-saturated aqueous solution, in addition to forming a dimeric catechin (proanthocyanidin) via a photon-induced redox reaction. The relative percentage of catechin was found to be 59.0 and 95.7 for catechin treated with BLI and catechin upon the addition of 1 mM AlCl treated with BLI, respectively. It suggested that catechin treated with BLI could be suppressed by AlCl, while AlCl did not form a complex with catechin in the photolytic system. However, under the same conditions, it was also found that the addition of AlCl inhibited the photolytic formation of O2•-, and reduced the generation of proanthocyanidin, suggesting that the disconnection of proanthocyanidin was achieved by AlCl acting as a catalyst under treatment with BLI. The influence of 1 mM fluoride (F-) and 1 mM oxalate (C2O42-) ions on the photolysis of 1 mM catechin upon the addition of 1 mM AlCl and treatment with BLI was found to be insignificant, implying that, during the photolysis of catechin, the Al species were either neutral or negatively charged and the aluminum species did not form a complex with anions in the photolytic system. Therefore, aluminum, which is an amphoteric species, has an inherent potential to stabilize the photolysis of catechin in an alkaline conditions, while suppressing the O2•- and proanthocyanidin generation via aluminum ion catalysis in the catechin/Al system under treatment with BLI.

Catechin Photolysis Suppression by Aluminum Chloride under Alkaline Conditions and Assessment with Liquid Chromatography-Mass Spectrometry.

Doxycycline hyclate (DCH) and minocycline hydrochloride (MH) are tetracycline antibiotics and broad-spectrum antimicrobial agents. The changes in DCH and MH under blue light (λ = 462 nm) irradiation in alkaline conditions (BLIA) were investigated. Deactivation caused by superoxide anion radical (O) and deactivation from DCH and MH during photolysis on Staphylococcus aureus (S. aureus), including methicillin-resistant S. aureus (MRSA), were studied. DCH is relatively unstable compared to MH under BLIA. The level of O generated from the MH-treated photoreaction is lower than that from DCH photolysis, and the DCH-treated photoreaction is more efficient at inactivating S. aureus and MRSA at the same radiant intensity. DCH subjected to BLIA decreased the viability of S. aureus and MRSA by 3.84 and 5.15 log, respectively. Two photolytic products of DCH (PPDs) were generated under BLIA. The mass spectra of the PPDs featured molecular ions at m/z 460.8 and 458.8. The molecular formulas of the PPDs were CHNO and CHNO, and their exact masses were 462.44 and 460.44 g/mol, respectively. These results bolster the photolytic oxidation that leads to DCH-enhanced deactivation of S. aureus and MRSA. Photochemical treatment of DCH could be applied as a supplement in hygienic processes.

Effects of free radicals from doxycycline hyclate and minocycline hydrochloride under blue light irradiation on the deactivation of Staphylococcus aureus, including a methicillin-resistant strain.

Article	Year
Blue light induced free radicals from riboflavin on E. coli DNA damage. Journal of photochemistry and photobiology. B, Biology\| PubMed ID: 23347966	2013
Effect of esterification condensation on the Folin-Ciocalteu method for the quantitative measurement of total phenols. Food chemistry\| PubMed ID: 25306311	2015
Investigations of blue light-induced reactive oxygen species from flavin mononucleotide on inactivation of E. coli. Journal of photochemistry and photobiology. B, Biology\| PubMed ID: 25617617	2015
Investigations of riboflavin photolysis via coloured light in the nitro blue tetrazolium assay for superoxide dismutase activity. Journal of photochemistry and photobiology. B, Biology\| PubMed ID: 25985146	2015
Photo-catalytic polymerization of catechin molecules in alkaline aqueous. Journal of photochemistry and photobiology. B, Biology\| PubMed ID: 27776259	2016
Effects of blue or violet light on the inactivation of Staphylococcus aureus by riboflavin-5'-phosphate photolysis. Journal of photochemistry and photobiology. B, Biology\| PubMed ID: 28715781	2017
Blue light induced free radicals from riboflavin in degradation of crystal violet by microbial viability evaluation. Journal of photochemistry and photobiology. B, Biology\| PubMed ID: 28822287	2017
Effects of Blue-Light-Induced Free Radical Formation from Catechin Hydrate on the Inactivation of Including a Carbapenem-Resistant Strain. Molecules (Basel, Switzerland)\| PubMed ID: 29973539	2018
Effects of 462 nm Light-Emitting Diode on the Inactivation of and a Multidrug-Resistant by Tetracycline Photoreaction. Journal of clinical medicine\| PubMed ID: 30213146	2018
Effects of Epigallocatechin Gallate on the Stability of Epicatechin in a Photolytic Process. Molecules (Basel, Switzerland)\| PubMed ID: 30813243	2019
The Influence of the Degradation of Tetracycline by Free Radicals from Riboflavin-5'-Phosphate Photolysis on Microbial Viability. Microorganisms\| PubMed ID: 31661888	2019
Catechin Photolysis Suppression by Aluminum Chloride under Alkaline Conditions and Assessment with Liquid Chromatography-Mass Spectrometry. Molecules (Basel, Switzerland)\| PubMed ID: 33348758	2020
Effects of free radicals from doxycycline hyclate and minocycline hydrochloride under blue light irradiation on the deactivation of Staphylococcus aureus, including a methicillin-resistant s... Journal of photochemistry and photobiology. B, Biology\| PubMed ID: 34864528	2022

Ji-Yuan Liang

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