Open Chem., 2019; 17: 1244–1251 Research Article Desislava Staneva, Stanislava Yordanova*, Evgenia Vasileva-Tonkova, Stanimir Stoyanov, Ivo Grabchev Photophysical and antibacterial activity of light- activated quaternary eosin Y https://doi.org/10.1515/chem-2019-0135 received December 3, 2018; accepted May 9, 2019. Keywords: eosin Y, photophysics, antimicrobial activity, antibacterial textile Abstract: The functional characteristics of a new eosin dye with biocidal quaternary ammonium group (E) were studied in aqueous solution and in organic solvents of 1 Introduction different polarity. The spectral properties depend on the nature and polarity of the respective solvents. The Fluorescent compounds are often used in medicine, antimicrobial activity of compound E has been tested in pharmacy, biology and environmental protection [1,2]. vitro against Gram-negative bacteria (Escherichia coli, Among the known fluorophore structures used in these Acinetobacter johnsoni and Pseudomonas aeruginosa), fields, the eosin Y and its derivatives are very important. Gram-positive bacteria (Sarcina lutea and Bacillus cereus) They belong to the group of xanthene fluorescent dyes and the antifungal activity was tested against the yeasts with a wide range of photophysical and biological Candida lipolytica in solution and after treated on cotton applications, due to their low toxicity in vivo, and high fabric. Broth dilution test has been used for quantitative water solubility [3]. The utility of eosin derivatives is evaluation of the antimicrobial activity of compound E associated to their good spectral characteristics and the against the model strains. The ability of compound E to possibility to interact with different type of biomolecules inhibit the growth of model Gram-negative P. aeruginosa [4-8]. Photophysical properties of eosin in solution strain was assessed after 16 h of incubation in presence strongly depend on the solvents polarity and possibility and absence of light. These experiments were conducted of hydrogen bond formations. Depending on pH eosin Y in planktonic format in solution and on cotton fabric. exhibit tautomeric structures with different proteolytic The results suggest that the new compound is effective forms, and its colour depends on the respective forms in treating the relevant pathogens with better results [9,10]. In recent years, eosin Y was also successfully used being obtained by irradiation with light. In this case the as photoredox catalyst in organic synthesis [11,12]. quaternary ammonium group promotes the binding of A new scientific area of research is the combination eosin Y moiety to the bacterial cell wall thus accelerating of dyeing process with antibacterial properties in one bacterial photo inactivation. compound [13]. This can be achieved through the introduction of specific groups into the dye chromophore systems to give antibacterial properties without changing their colour characteristics. In this case quaternary ammonium group can be bonded to the fluorophores by incorporating alkyl chain into the chromophore *Corresponding author: Stanislava Yordanova, Sofia University “St. system through covalent bonds. The cationic dyes thus Kliment Ohridski”, Faculty of Chemistry and Pharmacy, 1164 Sofia, Bulgaria, E-mail: [email protected] obtained show good colour characteristics and high Desislava Staneva, University of Chemical Technology and antibacterial activity in solutions [14-18]. In the last years Metallurgy, 1756 Sofia, Bulgaria in our laboratory, fluorophores with different chemical Evgenia Vasileva-Tonkova, The Stephan Angeloff Institute of structure having ammonium quaternary groups have been Microbiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria synthesized and their antibacterial and antifungal activity Stanimir Stoyanov, Sofia University “St. Kliment Ohridski”, Faculty of Chemistry and Pharmacy, 1164 Sofia, Bulgaria were investigated in solution or after their incorporation Ivo Grabchev, Sofia University “St. Kliment Ohridski”, Faculty of into polylactide matrix, or on the textile fabrics [19-21]. The Medicine, 1407 Sofia, Bulgaria relevance of such studies is due to the fact than in the last Open Access. © 2019 Desislava Staneva et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution alone 4.0 License. Photophysical and antibacterial activity of light-activated quaternary eosin Y 1245 Br Br ethanol (EtOH), dimethylsulfoxide (DMSO), N,N- O O O O dimethylformamide (DMF), dichloromethane (CH Cl ), Cl 2 2 1,4-dioxane and ethylacetate (EtOAc) used in this study Br Br N were of spectroscopic grade. Fluorescence quantum HOOC yield was determined on the basis of the absorption and fluorescence spectra, using Rhodamine 6G as reference (Φst = 0.94 in ethanol [28]). Scheme 1: Chemical structure of quaternary eosin Y (E). 2.2 Preliminary antimicrobial screening years the antimicrobial resistance of different pathogens The antibacterial activity of the investigated compound has become a major problem in medicine and clinical E was determined against Gram-positive bacteria practice. This encouraged many research laboratories (Sarcina lutea and Bacillus cereus), Gram-negative to start searching for and investigating novel and more bacteria (Acinetobacter johnsonii, Escherichia coli and active antibacterial drugs [22-24]. Pseudomonas aeruginosa) and the antifungal activity was In the last few years, photodynamic therapy has been tested against the yeasts Candida lipolytica. Microbial used against the resistance of pathogens to the medicines cultures were maintained at 4°C on Mueller-Hinton agar administered in practice. In this case, the microbiological (MHA) slants and transferred monthly. activity of the preparations used is due to the generation of reactive oxygen species upon irradiation with visible light, which kill the bacteria by oxidative burst [25]. It 2.3 In vitro antimicrobial assay has been reported that eosin Y has some antibacterial photoactivity [26]. Broth dilution test was used for quantitative evaluation In this paper we present the results on photophysical of the antimicrobial activity of compound E against the characterization of a new ammonium quaternary eosin Y model strains. Тhe concentration of compound E dissolved in organic solvents of different polarity and evaluation of in water was 5 mg/ml (0.623 µM) and was further diluted its antimicrobial activity against different pathogens. The in each consecutive test tube in sterile meat-peptone broth effect of visible light on the antimicrobial activity of the (MPB, pH 7.0) to final concentrations of 0.018, 0.038, 0.077, new eosin Y derivative has also been tested in solution 0.156, 0.249 and 0.312 and 0.623 mM. After inoculation and after its deposition on cotton fabric. with 2% (v/v) of each standardized cell suspension, the tubes were incubated at appropriate temperature for 24 h under shaking (at 240 rpm). The microbial growth 2 Experimental part was assessed by measuring the optical density of the medium at 600 nm (OD600). The growth control, sterility control and control of the compound E were used. The % 2.1 Materials and methods survival of the test cultures was determined on the basis of the positive control which was considered as 100%. The The synthesis and application of a quaternary ammonium experiments were conducted in triplicate. The light source eosin Y (E) as photoinitiator of polymerization of acrylate used for irradiation was with the parameters: HL 8325, 25 monomers has been described recently (Scheme 1) [27]. w, 1230 Lumen, 6400 K, Horoz [27]. The light source of lamp used for irradiation was with the parameters: HL 8325, 25 w, 1230 Lumen, 6400 K, Horoz. Absorption spectra were performed using “Thermo 2.4 Antimicrobial activity of cotton fabrics Spectronic Unicam UV 500” spectrophotometer. The fluorescence spectra were taken on a “Cary Eclipse” The antimicrobial effectiveness of cotton fabrics treated spectrophotometer. All spectra were recorded using 1 with 0.5% solution of the compound E was investigated cm path length synthetic quartz glass cells. Absorption by the shaking flask method. C. lipolytica, B. cereus and fluorescence measurements of the eosin compound and A. johnsonii were used as model strains. Test tubes E were carried out at 10-6 mol.L-1 concentration. Organic containing sterile MPB (1.0 ml) and square specimens solvents: acetonitrile (MeCN), methanol (MeOH), (10 mm x 10 mm) were inoculated with each overnight 1246 Desislava Staneva et al. grown microbial culture. Tubes with untreated cotton and Table 1: Photophysical characteristics of compound E in solvents without specimens were also prepared as controls. After 24 with different polarity. h incubation at appropriate temperature under shaking, Solvent l l n - n ε l mol-1 F microbial growth was determined by measuring OD . A F A F F 600 nm nm cm-1 cm-1 To evaluate the antimicrobial activities of the samples, the reduction in cell density between the untreated and Water 515 535 726 110000 0.22 treated samples after incubation was compared. All MeOH 522 540 639 110000 0.59 antimicrobial activity tests were done in triplicate. EtOH 529 546 589 120000 0.54 DMF 538 552 471 110000 0.36 2.5 Treatment of cotton fabrics with E MeCN 538 556 602 98000 0.71 DMSO 545 562 555 95100 0.68 In 5 ml of water the compound E (5 mg) was dissolved CH2Cl2 543 561 590 95100 0.22 and 1g of cotton fabric (weight 140 g/m2) was added in the solution for 30 min at 25°C. Then cotton sample was dried EtOAc 540 556 533 94200 0.40 at ambient temperature. Dioxane 540 555 501 92100 0.20 Ethical approval: The conducted research is not related to either human or animal use. 1.0 1.0 0.8 0.8 3 Results and Discussion 0.6 0.6 3.1 Photophysical characteristics of 0.4 0.4 ammonium quaternary eosin Y (E) Photophysical properties of the eosin Y derivatives as a 0.2 0.2 part of xanthene dyes are characterized by heterocyclic Normalized absorption (a.u.) 0.0 0.0 Normalized fluorescence (a.u.) system containing a dibenzo-1,4-pyran ring. Its basic 400 450 500 550 600 650 700 spectral characteristics depend from the solvent polarity Wavelength, nm and for formation of hydrogen bonds with solvents.