Journal of Photochemistry and Photobiology B: Biology 143 (2015) 44–51
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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Synthesis, photophysical and antimicrobial activity of new water soluble ammonium quaternary benzanthrone in solution and in polylactide film Desislava Staneva a, Evgenia Vasileva-Tonkova b, Mohamad Saleh I. Makki c, Tariq Rashad Sobahi c, Reda Mohamed Abdel-Rahman c, Abdullah M. Asiri c,d, Ivo Grabchev c,e,⇑ a
University of Chemical Technology and Metallurgy, 1756 Sofia, Bulgaria Institute of Microbiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria Chemistry Department, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia d Center of Excellence for Advanced Materials Research (CEAMR), King Abdulaziz University, Jeddah 21589, Saudi Arabia e Sofia University ‘‘St. Kliment Ohridski’’, Faculty of Medicine, 1407 Sofia, Bulgaria b c
a r t i c l e
i n f o
Article history: Received 8 July 2014 Received in revised form 30 October 2014 Accepted 27 December 2014 Available online 6 January 2015
a b s t r a c t The synthesis of a new cationic water soluble fluorescent 1-[(7-oxo-7H-benzo[de]anthracen-3-ylcarbamoyl)-methyl]-triethylammonium chloride (B) has been described. Due to the presence of the quaternary amino group, the compound is soluble in water. Its photophysical characteristics in aqueous solution and organic solvents with different polarity have been determined using absorption and fluorescence spectroscopy. The photostability of compound B has been investigated in aqueous media. The newly synthesized compound has been tested in vitro for its antimicrobial activity against eight bacterial and two yeasts cultures. The results obtained suggest that the newly synthesized compound is effective in treating the relevant pathogens and is suitable in designing new effective antimicrobial preparations. The incorporation of the compound into thin polylactic acid film and its release into water solution has been also investigated. It was demonstrated that the compound released from the polymer polylactic acid matrix exhibited a prolonged good antibacterial activity. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Derivatives of benzo[de]anthracen-7-one (benzanthrone) are well known as fluorophores emitting fluorescence from yellowgreen to orange-red. Their excellent color characteristics conditioned by the emitted fluorescence and high photo stability make them preferable for surface or structural coloration of polymers [1,2]. Recent studies have shown that 3-oxy-and 3-aminosubstituted benzanthrone derivatives can be used in some non-traditional applications as components in liquid–crystalline systems for electro-optical displays [3–7] or as sensors for biological important metal ions and amines [8–11]. The amidinobenzanthrone derivatives have showed potential as biomedical probes for proteins, lipids, and cells. Such dyes can be also utilized as suitable sensing probes for checking solvent polarity [12,13]. In the last century one of the progresses of modern medicine is related to the invention and application of antibiotics, and a number of effective antimicrobial preparations have been created including antibacterial, antifungal, antiparasitic and antiviral ⇑ Corresponding author at: Sofia University ‘‘St. Kliment Ohridski’’, Faculty of Medicine, 1407 Sofia, Bulgaria. Tel.: +359 2 8161319. E-mail address: [email protected]fia.bg (I. Grabchev). http://dx.doi.org/10.1016/j.jphotobiol.2014.12.024 1011-1344/Ó 2015 Elsevier B.V. All rights reserved.
[14,15]. The fast mutation of genes of the microorganisms which on its turn leads to hampering their elimination and their resistance against the common antibiotics has become a serious problem in the resent years. Moreover, many antimicrobial drugs are difficult to apply because of their low water solubility, cytotoxicity to healthy tissues or rapid degradation. On the other hand, their antimicrobial activity against intracellular microbes may be also severely limited by poor permeability. That is why in the last years the search of new antibacterial and antifungal chemotherapeutics has become an important part of medicinal chemistry as actively explored new structures. Currently, a variety of chemical structures and their metal complexes with biological important metal cations were tested intensively [16–18]. Some of the known structures with microbiological activity are the compounds containing quaternary nitrogen atom which are used in many fields, such as water treatment, medicine and healthcare products, food applications, and textile products. In this context, cationic compounds have attracted a great attention due to their water solubility and high antimicrobial activity [19–21]. The presence of a quaternary ammonium group enables the biostatic activity of B because of the positive charge at the N-atom inflicts a variety of detrimental effects on microbes, including damage to cell membranes, denaturation of proteins and disruption to the cell structure [22,23].
D. Staneva et al. / Journal of Photochemistry and Photobiology B: Biology 143 (2015) 44–51
Various cationic architectures have been tested also such as the polyelectrolyte layers and the dendrimers [24,25]. Polylactic acid (PLA) as an aliphatic polyester is one of the most promising biodegradable and biocompatible synthetic material because it is thermoplastic, having high strength, high modulus and good processability. PLA and its copolymers have high potential for use in a wide range of applications such as food packaging for fresh products, for production of fibers and fabrics, materials with biomedical and antibacterial activity. In the last years, different nanocomposites based on PLA with nanoclays or active additives have been developed and reported [26,27]. They are limited investigation on the antibacterial activity of compounds incorporated into PLA film [28–30]. To our knowledge they are not such investigation with additives combining antimicrobial and dyeing properties. In this paper we report the synthesis and photophysical characteristics of a new water soluble cationic benzanthrone derivative (1-[(7-oxo-7H-benzo[de]anthracen-3-ylcarbamoyl)-methyl]-triethylammonium chloride) in organic solvents of different polarity designed to exhibit antimicrobial activity. Also, the objective of this study was to incorporate the new benzanthrone into PLA thin film and to investigate its antimicrobial activity. 2. Experimental part 2.1. Materials and methods The synthesis of 2-chloro-N-(7-oxo-7H-benzo[de]anthracen-3yl)-acetamide as a precursor for the synthesis has been synthesized according to a method described previously [9]. PLA was obtained from Sigma Aldrich (Mw 18,000, Tg 46–50 °C). 2.1.1. Synthesis of 1-[(7-oxo-7H-benzo[de]anthracen-3-ylcarbamoyl)methyl]-triethylammonium chloride (B) 2-Chloro-N-(7-oxo-7H-benzo[de]anthracen-3-yl)-acetamide (3.21 g, 0.01 mol) was dissolved in 30 ml dioxin, then 3.0 ml of triethylamine was added and the solution was stirred for 5 h at 60 °C. After cooling to room temperature, the precipitate was filtered off, washed with acetone and dried in vacuum at 40 °C. Yield: 73%. FT-IR cm1: 3077, 2980, 2943, 1680, 1650, 1603, 1542, 1456, 1311, 1161, 1014, 949, 773, 698. 1 H NMR (DMSO-d6, ppm): d 11.45 (br s, 1H, NH), 8.81–8.78 (d, J = 5.34 Hz, 2H), 8.70–8.68 (d, J = 6.46 Hz, 1H), 8.59–8.42 (d, J = 8.40 Hz, 1H), 8.35–832 (d. J = 6.6 Hz, 2H), 8.09–8.06 (d, J = 8.17 Hz, 1H,), 7.79 (t, J = 7.68, Hz, 1H), 8.87 (t, J = 6.92, Hz, 1H), 7.63 (t, J = 7.65 Hz, 1H), 4.57 s, 2H), 3.64–3.55 (2, J = 7.15 Hz, 6H), 1.30 (t, J = 7.06 Hz, 9H). NH2
NHCCH2Cl O
Cl
Cl O
O
O
N(CH2CH3)3
+ NHCCH2N(CH2CH3)3 O Cl-
O Scheme 1. Synthesis of cationic 1-[(7-oxo-7H-benzo[de]anthracen-3-ylcarbamoyl)-methyl]-triethylammonium chloride.
45
13
C NMR: 181.8, 162.3, 137.2, 134.1, 130.5, 129.7, 128.5, 127.3, 126.7, 125.4, 123.9, 123.3, 56.4, 54.2, 7.6. Elemental analysis: C25H27N2O2Cl (426.56): calcd. C 70.33, H 6.33, N 6.56; Found C 70.68, H 6.29, N 6.69. 2.1.2. Spectral investigations UV–Vis spectrophotometric investigations were performed using ‘‘Thermo Spectronic Unicam UV 500’’ spectrophotometer. Emission spectra were taken on a ‘‘Cary Eclipse’’ spectrofluorometer. All spectra were recorded using 1 cm path length synthetic quartz glass cells. Absorption and fluorescence measurements of the benzanthrone compound B were carried out at 105 mol l1 concentration. The quantum yield of B was determined on the basis of its absorption and fluorescence spectra according to Eq. (1):
UF ¼ Ust
Su Ast n2Du Sst Au n2Dst
ð1Þ
where the UF is the fluorescence quantum yield of the sample, Ust is the fluorescence quantum yield of the standard, Ast and Au represent the absorbance of the standard and sample at the excited wavelength respectively, while Sst and Su are the integrated emission band areas of the standard and sample respectively, and nDst and nDu is the solvent refractive index of the standard and sample. Subscripts u and s refer to the unknown and standard respectively. Rhodamine 6G was used as a reference (Ust = 0.88) [31]. 1H (600.13 MHz) and 13C (150.92 MHz) spectra were acquired on an AVANCE AV600 II + NMR spectrometer. The measurements were carried out in CDCl3 solution at ambient temperature. The chemical shifts were referenced to a tetramethylsilane (TMS) standard. The photodegradation was carried out using a solar light simulator (Suntest CPS+, heraus) equipped with a 1.5 kW xenon arc lamp. Thin layer chromatographic (TLC) analysis of the dyes was followed on silica gel (Fluka F60 254 20 20; 0.2 mm) using the solvent system n-heptane/acetone (1:1) as an eluent. 2.1.3. Preparation of PLA film Pure PLA and antimicrobial PLA films were prepared by solvent casting method. 0.5 g polylactic acid was dissolved in 10 ml chloroform and 0.0005 g compound B was added. After 30 min stirring, the homogeneous mixture was poured into a Petri dish and the solvent was evaporated slowly. Thus was obtained a stable polymer film having a thickness of 80 lm. The same method has been used to produce pure PLA film. 2.1.4. Antimicrobial activity test The newly synthesized compound B was investigated for antimicrobial activity by the conventional agar diffusion method [32] against the following pathogenic indicator cultures: Gram-positive bacteria Bacillus subtilis, Bacillus cereus, Sarcina lutea and Micrococcus luteus, and Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli, Acinetobacter johnsonii and Xanthomonas oryzae, and yeasts Candida lipolytica and Saccharomyces cerevisiae. The compound was dissolved in distilled H2O to obtain 0.5% stock solution. The sample solution (20 ll) was spotted on filter paper discs and applied to peptone-yeast extract-agar plates with each freshly grown indicator culture. The effect of the sample concentration on the growth of P. aeruginosa and B. cereus was studied using well agar-diffusion assay by adding into the wells of 10, 20, 30 and 50 ll of the sample solution. Commercial discs with gentamicin (10 mg/disc) and nystatin (100 units/disc) were used as reference standards for antibacterial and antifungal activity, respectively. Two replicates were performed for each treatment. After incubation of the plates for 24–48 h at 28 ± 2 °C, the resulting clearing zones, if any, were measured.
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Table 1 Photophysical characteristics of 1-[(7-oxo-7H-benzo[de]anthracen-3-ylcarbamoyl)-methyl]-triethylammonium chloride in different solvents. Solvent
kA (nm)
e (l mol1 cm1)
kF (nm)
mA–mF (cm1)
f
UF
Water Ethanol Methanol N,N-dimethilformamide Acetonitrile Chloroform Tetrahydrofuran
411 410 410 419 417 426 428
10,600 11,200 11,000 10,300 10,400 12,300 12,500
560 552 554 540 544 537 535
6474 6274 6340 5348 5598 4852 4673
0.213 0.298 0.265 0.302 0.312 0.341 0.349
0.012 0.011 0.011 0.094 0.120 0.245 0.298
2
4
3
600
7
6
λF
520 480 440
1
2
3
4
5
λA
6
7
400
600
Fl. Intensity
λA / λF [nm]
1
5
Fluorescence Intensity
560
500 400
540
480
0
80
160
240
Time / min
300 200 100
35
40
45
50
55
E T (30) kcal
60
65
mol -1
0
500
550
600
650
700
Wavelength / nm Fig. 1. Dependence of absorption (kA) and fluorescence (kF) maxima of compound B on the empirical parameter of solvent polarity ET(30): 1 – tetrahydrofuran, 2 – chloroform, 3 – acetonitrile, 4 – N,N-dimethilformamide, 5 – ethanol, 6 – methanol, 7 – water.
6800
/ cm-1
6000
νA - νF
5600
6
5
6400
7
Fig. 3. Fluorescent spectra of compound B (c = 1 105 mol l1) obtained in aqueous solution at pH = 7.0 during the irradiation of 240 min. Inset show change in the fluorescence intensity at kF = 560 nm of B on the time.
visible inhibition (clear) zone around the well was observed (compared with the control) after incubation of the plates for 48–72 h at 25 °C. An inhibition zone P 10 mm indicated the presence of an inhibitory activity. The MIC value was determined as the zero intercept of a linear regression of the squared size of the inhibition zones, plotted against the logarithm of the sample concentration.
4 3
5200 2 4800
1 36
40
44
48
52
56
60
64
-1
ET (30) / kcal mol
Fig. 2. Dependence of Stokes shift (mA–mF) of compound B on the empirical parameter of solvent polarity ET(30): 1 – tetrahydrofuran, 2 – chloroform, 3 – acetonitrile, 4 – N,N-dimethilformamide, 5 – ethanol, 6 – methanol, 7 – water.
2.1.5. Determination of the minimal inhibitory concentration (MIC) The MIC of B compound against the studied bacteria was determined using agar-well diffusion method [33,34]. Petri plates containing nutrient agar medium were inoculated with suspensions of the indicator microorganisms (in exponential growth phase), and the surface of the agar was punched with 7-mm in diameter wells. The tested B compound with starting concentration 0.5% was further diluted in dist. H2O to obtain the following eight concentrations for its MIC determination: 0.05, 0.10, 0.15, 0.20, 0.25, 0.40, 0.50 and 0.75 mg/ml. 50 ll of each sample dilution was added into the wells of each plate. The solution without B sample was used as a control. The MIC value was defined as the lowest concentration at which a
2.1.6. Test of film antimicrobial activity The antimicrobial effect of the obtained B-PLA film was investigated against Gram-negative bacteria E. coli and P. aeruginosa as test microorganisms. For antimicrobial tests, square shape specimens of 6 mm were cut from the PLA and B-PLA films under aseptic conditions. The test tubes with 2.5 ml sterile nutrient broth medium were inoculated with overnight bacterial cultures and left at room temperature for 15 min. Then, the specimens were inserted into the test tubes. Test tubes without inserted film specimens were also prepared for each bacterial culture. After 24 h incubation at 25 °C under shaking at 240 rpm, the specimens were removed and the bacterial growth was determined by measuring the optical density of the medium at 570 nm (OD570). In parallel, specimens were placed onto nutrient agar surface in Petri plates seeded with the test cultures. After incubation of the plates for 48 h at 25 °C, the diameter of the formed zones, if any, was measured. 3. Results and discussion 3.1. Chemistry 3.1.1. Synthesis of 1-[(7-oxo-7H-benzo[de]anthracen-3-ylcarbamoyl)methyl]-triethylammonium chloride (B) The synthetic route for the preparation of cationic compound B is outlined in Scheme 1. The starting 3-amino-benzo[de]antracen-
D. Staneva et al. / Journal of Photochemistry and Photobiology B: Biology 143 (2015) 44–51
47
0,25
Absorbance
0,20 t = 120 min
0,15
t = 10 min
0,10
0,05
300
350
400
450
500
Wavelength / nm Fig. 4. Absorption release profile of B from B–PLA matrix in aqueous solution at pH = 7 and 25 °C for 120 min.
75 Fl. Intensity
Fluorescence intensity
75
60
45
60 45 30 15 0 0
30
60
90
120
Time / min
30
15
0
450
500
550
600
650
700
Wavelength / nm Fig. 5. Fluorescent release profile of B from B–PLA matrix in aqueous solution at pH = 7 at 25 °C for 120 min. Inset show the change of fluorescence intensity with the time.
7-one has been obtained by nitration and reduction of nitro group of the commercially available benzanthrone. Acylation of the primary amino group was carried out with chloracetylchloride at 50 °C for 2 h in dioxan solution. The obtained product 2-chloroN-7(oxo-7H-benzo[de]antracene-3-yl-acetamid was isolated by pouring the reaction mixture into ice water and filtering the precipitate. The compound has an activated chlorine atom, which react with triethylamine to obtain the final product 1-[(7-oxo7H-benzo[de]anthracen-3-ylcarbamoyl)-methyl]-triethylammonium chloride as yellow precipitate after cooling, which was filtrated and washed by acetone and dried under vacuum. 3.1.2. Photophysical characteristics of 1-[(7-oxo-7Hbenzo[de]anthracen-3-ylcarbamoyl)-methyl]-triethylammonium chloride The absorption and fluorescence properties of the benzanthrone derivatives are related to the extent of polarisation of the chromophoric system upon irradiation, resulting from the interaction between electron-donor substituent at C-3 position and electronacceptor carbonyl group within the benzanthrone molecule [7]. Clearly, the bands of absorption and emission of the benzanthrones
Fig. 6. Antibacterial activity of B compound (10 ll), inhibition zone (mm in diameter). Gentamicin (G, 10 ll/disc).
largely depend on the electron-donating power of the substituents at C-3 position. Furthermore, their spectral properties depend on the nature of the surrounding environment (polarity, viscosity, and formation of hydrogen bonds or other intermolecular interactions). Table 1 presents the spectral characteristics of the benzanthrone compound B in organic solvents with different polarity: the absorption (kA) and fluorescence (kF) maxima, the extinction coefficient e, Stokes shift (mA–mF), oscillator strength (f) and quantum yield of fluorescence (UF). The empirical parameter of solvent polarity (ET(30) kcal mol1) was used to characterized the polarity of the medium [35]. It was shown that the polarity of the solvents has a counterproductive effect on the position of the absorption and fluorescence maxima (Fig. 1). As can be seen, the solvent polarity influences the position of the absorption maxima, DkA = 17 nm. In comparison with the fluorescent maxima in non-polar solvents, a bathochromic shift of DkF = 25 nm for those in polar solvents was observed. The greater bathochromic shift can be explained by enhancing the dipole moment of the molecule upon excitation due to the electron density redistribution. The excited molecule is more stable in polar solvents due to stronger interactions with the solvents dipole. These results confirm a conformational change occurred in the singlet excited state S1 upon excitation of S0. Similar results have been achieved with benzanthrone derivatives with different substituents at C-3 position [7]. Stokes shift (mA–mF) and oscillator strength (f) are important parameters of the fluorescent compounds. The Stokes shift is a parameter which indicates the difference in properties and structure of the dyes between the ground S0 and the first excited state S1. Fig. 2 presents that the Stokes shift depends on the media polarity and it is larger in the case of polar solvents when the hydrogen bond formation or dipole–dipole interactions are favored in comparison to nonpolar media. The Stokes shift range obtained in this work is in the region mA–mF = 4673–6474 cm1. The results are very similar to those obtained for the monomer benzanthrone derivatives with different substituent at C-3 position, which indicated that the nature of the substituent has effect upon the Stokes shift. The oscillator strength (f), shows the effective number of electrons whose transition from ground to excited state, gives the absorption area in the electron spectrum. Values of the oscillator strength can be calculated using the Eq. (1):
f ¼ 4:32 109 Dm1=2 e max
ð2Þ
where Dm1/2 is the width of the absorption band (in cm1) at 1/2 emax. The values of the oscillator strength depend on the solvent
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Fig. 7. Growth inhibition of P. aeruginosa and B. cereus by different amounts (10, 20, 30 and 50 ll) of sample B.
polarity. As can be seen in Table 1, the values for the oscillator strength were in the range of 0.213–0.349. The ability of the photoactive molecules to emit the absorbed light energy is characterized quantitatively by the fluorescence quantum yield. The calculated UF were in the region 0.011– 0.298. It is seen than the quantum yield increases more than ten times in non-polar solvents compared with the polar. This variation arises from the solvent dependence of the nonradiative rate constant [36,37] or by possible formation of hydrogen bonds which favor less radiation transition causing a decrease of the fluorescence quantum yield of B. 3.1.3. Photostability of compound B in aqueous solution The photostability of water soluble antimicrobial compounds is very important in the cases when they are use as biocides. Photodegradation of compound B has been measured comparing the fluorescence maxima of the solutions at concentrations 1 105 mol l1 before and after irradiation at pH = 7.0. Fig. 3 plots the change of the fluorescence intensity with the time It is seen that the reduction on the fluorescence intensity at kF = 560 nm was negligible during the irradiation of first 120 min, which integrates that the stability of the compound B in aqueous solution is excellent. Also there is not any change in the fluorescence maxima before and after the irradiation. This fact demonstrates that the products of photodestruction neither absorb nor fluoresce in the spectral region where the compound B is photoactive. 3.1.4. Absorption and fluorescence investigation of colored PLA film in aqueous media It was of interest to study the antibacterial activity of the newly synthesized compound B after its incorporation into a thin polymer film of PLA. We used a solution casting method for the formation of PLA film and we prepared film from a solution of 5 g/100 ml chloroform solution and 0.05 g/100 ml of compound B. The obtained film was stable, flexible and transparent with intensive yellow color and thickness of 80 lm. In order to explore the possibility to use the PLA-B as antimicrobial film the release of B from the PLA-B matrix has been investigated in aqueous media at pH = 7 by dropping method. The PLA film is hydrophobic while B compound is more hydrophilic and as a result the release of B from the polymer matrix is possible. We conducted an experiment to investigate the possibility to release of compound B from the PLA film in the aqueous solution in the interval up to 120 min at pH = 7 by dropping method. During the contact of PLA-B film with aqueous media the compound B releases from the PLA matrix and the aqueous solution becomes
yellow in color. This process has been followed by using absorption and fluorescence spectroscopy. Figs. 4 and 5 showed that the absorption and respective fluorescence emission of compound B increased with time. It is seen that initially the PLA film releases a large amount of B, and accordingly the absorption and fluorescence are higher, and with time, this effect gradually decreases. Perhaps in this case, the compound B more easily released from the surface of the PLA film and its inner layers and passed into the aqueous solution. The absorption and fluorescence maxima of B in water are kA = 413 nm and kF = 557 nm, respectively. It is seen that no change of the position of the absorption and fluorescence maxima, but the intensity was increased. This is a new and interesting characteristic of PLA-B system, indicating that the compound B can be released slowly into the water solution and to exhibit a prolonged antibacterial activity. 3.2. Biological activity 3.2.1. Antimicrobial activity of 1-[(7-oxo-7H-benzo[de]anthracen-3ylcarbamoyl)-methyl]-triethylammonium chloride The newly synthesized compound was tested in vitro for antimicrobial activity against seven bacterial and two yeasts cultures. The sample exhibited comparable or better zones of inhibition of the growth of most indicator cultures than standard drug gentamicin (Fig. 6). Highest inhibition activity was observed against tested yeast strains (zones of clearance 23-24 mm) followed by the activity against B. cereus (zone of clearance 21 mm). The compound showed moderate activity against five bacterial cultures (zones of inhibition 13–15 mm), and weak activity towards M. luteus (zone of inhibition 12 mm). Highest inhibition of the growth of B. cereus and P. aeruginosa was observed using 30 and 50 ll of the sample solution (zone of clearance 24 and 14 mm, respectively) (Fig. 7). 3.2.2. Minimum inhibitory concentration determination (MIC) The MIC of B sample against the studied microbial cultures was determined by the agar diffusion method. As can be seen in Fig. 8 and Table 2, the MIC of the compound B varied in the range 48– 155 lg/ml indicating good antimicrobial potential. It was most effective (the lowest MIC) in inhibiting the growth of B. subtilis at 48 lg/ml followed by Sarcina lutea (MIC 58 lg/ml), and least effective (the highest MIC) towards B. cereus strain. M. luteus and X. oryzae strains were found to be resistant towards the compound at the used concentration. Cationic compounds have emerged as promising candidates for development of antimicrobial agents. Quaternary ammonium compounds with attached positively charged quaternary
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Fig. 8. Well-agar diffusion assay for MIC determination of the B compound against some indicator bacteria and yeasts. Concentration range: 1 – 0.05 mg/mL; 2 – 0.10 mg/mL; 3 – 0.15 mg/mL; 4 – 0.20 mg/mL; 5 – 0.25 mg/mL; 6 – 0.40 mg/mL; 7 – 0.50 mg/mL; 8 – 0.75 mg/mL.
ammonium groups are membrane active agents with a target site predominantly at the cytoplasmic (inner) membrane in bacteria or the plasma membrane in yeasts. Various cationic architectures have been tested such as the polyelectrolyte layers [24,38] and the dendrimers have also been used [39,40]. The following processes occur with microorganisms exposed to cationic agents [41,42]: (i) adsorption and penetration of the agent into the cell wall; (ii) reaction with the cytoplasmic membrane (lipid or protein) followed by membrane disorganization; (iii) leakage of intracellular low-molecular-weight material; (iv) degradation of proteins and nucleic acids; and (v) wall lysis caused by autolytic enzymes. There would be a loss of structural organization and integrity of the cytoplasmic membrane in bacteria, together with
Table 2 MIC of -[(7-oxo-7H-benzo[de]anthracen-3-ylcarbamoyl)-methyl]triethylammonium chloride towards some indicator bacteria and yeasts. Strains
MIC (lg/mL)
Bacillus subtilis Bacillus cereus Sarcina lutea Pseudomonas aeruginosa Escherichia coli Acinetobacter johnsonii Saccharomyces cerevisiae Candida lipolytica
48 155 58 124 73 105 96 82
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the water solution and to exhibit a prolonged good antibacterial activity.
Acknowledgements This paper was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, under Grant No. 17-130-35-HiCi. The authors, therefore, acknowledge with thanks DSR technical and financial support.
References
Fig. 9. Effect of PLA film (KS), and B-PLA film on the growth of P. aeruginosa and E. coli strains tested in liquid nutrient broth (NB). KO, NB without added film.
other damaging effects of the bacterial cell [43]. The antibacterial effect of the compound B reported here was higher than that of recently reported other benzanthrone derivatives [44]. The results obtained suggest that the newly synthesized compound B might be used as antimicrobial additive for biomedical devices and in the agriculture for plant protection.
3.2.3. Antimicrobial activity of B compound incorporated into PLA thin film The antimicrobial activity of the solid film PLA-B agains P. aeruginosa and E. coli has been investigated in aqueous solution and the results are presented in Fig. 9. As seen, the inhibition efficiency of the B-PLA film was higher than that of the PLA film against both tested strains. The B-PLA composite film causes a significant decrease in the optical densities of growth media of tested strains P. aeruginosa and E. coli (around 31% and 25%, respectively), while in the presence of the pure PLA film this decrease was much lower (7% and 19% respectively). The antimicrobial effect is due to release of the hydrophilic B compound from the PLA matrix by diffusion. No zones of inhibition were observed in nutrient agar tests which is probably due to insufficient degree of hydrophilicity of the film surface that is difficult to be achieved on the agar surface, which prevent the release of the B from the film. It has been reported that some other factors such as the level of immobilized antimicrobial activity retained at film surfaces and the surface area of films may also have effect the release profile of the immobilized activity [45].
4. Conclusion In this study we report the synthesis and characterization of a new cationic water soluble benzanthrone derivative having quaternary triethylammonium group. Photophysical characteristics of the compound have been determined in different organic solvents and they have demonstrated good dependence from the solvent polarity. It has been shown that the new benzanthrone compound B was very photostable in aqueous solution in low molar concentration. The results showed good inhibitory activity of the novel benzanthrone compound B against the tested bacterial and yeasts cultures. Antimicrobial activity observed against phytopathogenic strain X. oryzae suggested potential application of the compound B for plant protection. The gradual release of the substance with antimicrobial activity, which has previously been incorporated into the PLA thin film, has been investigated. The results showed that the compound B may be released slowly into
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Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Synthesis, photophysical and antimicrobial activity of new water soluble ammonium quaternary benzanthrone in solution and in polylactide film Desislava Staneva a, Evgenia Vasileva-Tonkova b, Mohamad Saleh I. Makki c, Tariq Rashad Sobahi c, Reda Mohamed Abdel-Rahman c, Abdullah M. Asiri c,d, Ivo Grabchev c,e,⇑ a
University of Chemical Technology and Metallurgy, 1756 Sofia, Bulgaria Institute of Microbiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria Chemistry Department, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia d Center of Excellence for Advanced Materials Research (CEAMR), King Abdulaziz University, Jeddah 21589, Saudi Arabia e Sofia University ‘‘St. Kliment Ohridski’’, Faculty of Medicine, 1407 Sofia, Bulgaria b c
a r t i c l e
i n f o
Article history: Received 8 July 2014 Received in revised form 30 October 2014 Accepted 27 December 2014 Available online 6 January 2015
a b s t r a c t The synthesis of a new cationic water soluble fluorescent 1-[(7-oxo-7H-benzo[de]anthracen-3-ylcarbamoyl)-methyl]-triethylammonium chloride (B) has been described. Due to the presence of the quaternary amino group, the compound is soluble in water. Its photophysical characteristics in aqueous solution and organic solvents with different polarity have been determined using absorption and fluorescence spectroscopy. The photostability of compound B has been investigated in aqueous media. The newly synthesized compound has been tested in vitro for its antimicrobial activity against eight bacterial and two yeasts cultures. The results obtained suggest that the newly synthesized compound is effective in treating the relevant pathogens and is suitable in designing new effective antimicrobial preparations. The incorporation of the compound into thin polylactic acid film and its release into water solution has been also investigated. It was demonstrated that the compound released from the polymer polylactic acid matrix exhibited a prolonged good antibacterial activity. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Derivatives of benzo[de]anthracen-7-one (benzanthrone) are well known as fluorophores emitting fluorescence from yellowgreen to orange-red. Their excellent color characteristics conditioned by the emitted fluorescence and high photo stability make them preferable for surface or structural coloration of polymers [1,2]. Recent studies have shown that 3-oxy-and 3-aminosubstituted benzanthrone derivatives can be used in some non-traditional applications as components in liquid–crystalline systems for electro-optical displays [3–7] or as sensors for biological important metal ions and amines [8–11]. The amidinobenzanthrone derivatives have showed potential as biomedical probes for proteins, lipids, and cells. Such dyes can be also utilized as suitable sensing probes for checking solvent polarity [12,13]. In the last century one of the progresses of modern medicine is related to the invention and application of antibiotics, and a number of effective antimicrobial preparations have been created including antibacterial, antifungal, antiparasitic and antiviral ⇑ Corresponding author at: Sofia University ‘‘St. Kliment Ohridski’’, Faculty of Medicine, 1407 Sofia, Bulgaria. Tel.: +359 2 8161319. E-mail address: [email protected]fia.bg (I. Grabchev). http://dx.doi.org/10.1016/j.jphotobiol.2014.12.024 1011-1344/Ó 2015 Elsevier B.V. All rights reserved.
[14,15]. The fast mutation of genes of the microorganisms which on its turn leads to hampering their elimination and their resistance against the common antibiotics has become a serious problem in the resent years. Moreover, many antimicrobial drugs are difficult to apply because of their low water solubility, cytotoxicity to healthy tissues or rapid degradation. On the other hand, their antimicrobial activity against intracellular microbes may be also severely limited by poor permeability. That is why in the last years the search of new antibacterial and antifungal chemotherapeutics has become an important part of medicinal chemistry as actively explored new structures. Currently, a variety of chemical structures and their metal complexes with biological important metal cations were tested intensively [16–18]. Some of the known structures with microbiological activity are the compounds containing quaternary nitrogen atom which are used in many fields, such as water treatment, medicine and healthcare products, food applications, and textile products. In this context, cationic compounds have attracted a great attention due to their water solubility and high antimicrobial activity [19–21]. The presence of a quaternary ammonium group enables the biostatic activity of B because of the positive charge at the N-atom inflicts a variety of detrimental effects on microbes, including damage to cell membranes, denaturation of proteins and disruption to the cell structure [22,23].
D. Staneva et al. / Journal of Photochemistry and Photobiology B: Biology 143 (2015) 44–51
Various cationic architectures have been tested also such as the polyelectrolyte layers and the dendrimers [24,25]. Polylactic acid (PLA) as an aliphatic polyester is one of the most promising biodegradable and biocompatible synthetic material because it is thermoplastic, having high strength, high modulus and good processability. PLA and its copolymers have high potential for use in a wide range of applications such as food packaging for fresh products, for production of fibers and fabrics, materials with biomedical and antibacterial activity. In the last years, different nanocomposites based on PLA with nanoclays or active additives have been developed and reported [26,27]. They are limited investigation on the antibacterial activity of compounds incorporated into PLA film [28–30]. To our knowledge they are not such investigation with additives combining antimicrobial and dyeing properties. In this paper we report the synthesis and photophysical characteristics of a new water soluble cationic benzanthrone derivative (1-[(7-oxo-7H-benzo[de]anthracen-3-ylcarbamoyl)-methyl]-triethylammonium chloride) in organic solvents of different polarity designed to exhibit antimicrobial activity. Also, the objective of this study was to incorporate the new benzanthrone into PLA thin film and to investigate its antimicrobial activity. 2. Experimental part 2.1. Materials and methods The synthesis of 2-chloro-N-(7-oxo-7H-benzo[de]anthracen-3yl)-acetamide as a precursor for the synthesis has been synthesized according to a method described previously [9]. PLA was obtained from Sigma Aldrich (Mw 18,000, Tg 46–50 °C). 2.1.1. Synthesis of 1-[(7-oxo-7H-benzo[de]anthracen-3-ylcarbamoyl)methyl]-triethylammonium chloride (B) 2-Chloro-N-(7-oxo-7H-benzo[de]anthracen-3-yl)-acetamide (3.21 g, 0.01 mol) was dissolved in 30 ml dioxin, then 3.0 ml of triethylamine was added and the solution was stirred for 5 h at 60 °C. After cooling to room temperature, the precipitate was filtered off, washed with acetone and dried in vacuum at 40 °C. Yield: 73%. FT-IR cm1: 3077, 2980, 2943, 1680, 1650, 1603, 1542, 1456, 1311, 1161, 1014, 949, 773, 698. 1 H NMR (DMSO-d6, ppm): d 11.45 (br s, 1H, NH), 8.81–8.78 (d, J = 5.34 Hz, 2H), 8.70–8.68 (d, J = 6.46 Hz, 1H), 8.59–8.42 (d, J = 8.40 Hz, 1H), 8.35–832 (d. J = 6.6 Hz, 2H), 8.09–8.06 (d, J = 8.17 Hz, 1H,), 7.79 (t, J = 7.68, Hz, 1H), 8.87 (t, J = 6.92, Hz, 1H), 7.63 (t, J = 7.65 Hz, 1H), 4.57 s, 2H), 3.64–3.55 (2, J = 7.15 Hz, 6H), 1.30 (t, J = 7.06 Hz, 9H). NH2
NHCCH2Cl O
Cl
Cl O
O
O
N(CH2CH3)3
+ NHCCH2N(CH2CH3)3 O Cl-
O Scheme 1. Synthesis of cationic 1-[(7-oxo-7H-benzo[de]anthracen-3-ylcarbamoyl)-methyl]-triethylammonium chloride.
45
13
C NMR: 181.8, 162.3, 137.2, 134.1, 130.5, 129.7, 128.5, 127.3, 126.7, 125.4, 123.9, 123.3, 56.4, 54.2, 7.6. Elemental analysis: C25H27N2O2Cl (426.56): calcd. C 70.33, H 6.33, N 6.56; Found C 70.68, H 6.29, N 6.69. 2.1.2. Spectral investigations UV–Vis spectrophotometric investigations were performed using ‘‘Thermo Spectronic Unicam UV 500’’ spectrophotometer. Emission spectra were taken on a ‘‘Cary Eclipse’’ spectrofluorometer. All spectra were recorded using 1 cm path length synthetic quartz glass cells. Absorption and fluorescence measurements of the benzanthrone compound B were carried out at 105 mol l1 concentration. The quantum yield of B was determined on the basis of its absorption and fluorescence spectra according to Eq. (1):
UF ¼ Ust
Su Ast n2Du Sst Au n2Dst
ð1Þ
where the UF is the fluorescence quantum yield of the sample, Ust is the fluorescence quantum yield of the standard, Ast and Au represent the absorbance of the standard and sample at the excited wavelength respectively, while Sst and Su are the integrated emission band areas of the standard and sample respectively, and nDst and nDu is the solvent refractive index of the standard and sample. Subscripts u and s refer to the unknown and standard respectively. Rhodamine 6G was used as a reference (Ust = 0.88) [31]. 1H (600.13 MHz) and 13C (150.92 MHz) spectra were acquired on an AVANCE AV600 II + NMR spectrometer. The measurements were carried out in CDCl3 solution at ambient temperature. The chemical shifts were referenced to a tetramethylsilane (TMS) standard. The photodegradation was carried out using a solar light simulator (Suntest CPS+, heraus) equipped with a 1.5 kW xenon arc lamp. Thin layer chromatographic (TLC) analysis of the dyes was followed on silica gel (Fluka F60 254 20 20; 0.2 mm) using the solvent system n-heptane/acetone (1:1) as an eluent. 2.1.3. Preparation of PLA film Pure PLA and antimicrobial PLA films were prepared by solvent casting method. 0.5 g polylactic acid was dissolved in 10 ml chloroform and 0.0005 g compound B was added. After 30 min stirring, the homogeneous mixture was poured into a Petri dish and the solvent was evaporated slowly. Thus was obtained a stable polymer film having a thickness of 80 lm. The same method has been used to produce pure PLA film. 2.1.4. Antimicrobial activity test The newly synthesized compound B was investigated for antimicrobial activity by the conventional agar diffusion method [32] against the following pathogenic indicator cultures: Gram-positive bacteria Bacillus subtilis, Bacillus cereus, Sarcina lutea and Micrococcus luteus, and Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli, Acinetobacter johnsonii and Xanthomonas oryzae, and yeasts Candida lipolytica and Saccharomyces cerevisiae. The compound was dissolved in distilled H2O to obtain 0.5% stock solution. The sample solution (20 ll) was spotted on filter paper discs and applied to peptone-yeast extract-agar plates with each freshly grown indicator culture. The effect of the sample concentration on the growth of P. aeruginosa and B. cereus was studied using well agar-diffusion assay by adding into the wells of 10, 20, 30 and 50 ll of the sample solution. Commercial discs with gentamicin (10 mg/disc) and nystatin (100 units/disc) were used as reference standards for antibacterial and antifungal activity, respectively. Two replicates were performed for each treatment. After incubation of the plates for 24–48 h at 28 ± 2 °C, the resulting clearing zones, if any, were measured.
46
D. Staneva et al. / Journal of Photochemistry and Photobiology B: Biology 143 (2015) 44–51
Table 1 Photophysical characteristics of 1-[(7-oxo-7H-benzo[de]anthracen-3-ylcarbamoyl)-methyl]-triethylammonium chloride in different solvents. Solvent
kA (nm)
e (l mol1 cm1)
kF (nm)
mA–mF (cm1)
f
UF
Water Ethanol Methanol N,N-dimethilformamide Acetonitrile Chloroform Tetrahydrofuran
411 410 410 419 417 426 428
10,600 11,200 11,000 10,300 10,400 12,300 12,500
560 552 554 540 544 537 535
6474 6274 6340 5348 5598 4852 4673
0.213 0.298 0.265 0.302 0.312 0.341 0.349
0.012 0.011 0.011 0.094 0.120 0.245 0.298
2
4
3
600
7
6
λF
520 480 440
1
2
3
4
5
λA
6
7
400
600
Fl. Intensity
λA / λF [nm]
1
5
Fluorescence Intensity
560
500 400
540
480
0
80
160
240
Time / min
300 200 100
35
40
45
50
55
E T (30) kcal
60
65
mol -1
0
500
550
600
650
700
Wavelength / nm Fig. 1. Dependence of absorption (kA) and fluorescence (kF) maxima of compound B on the empirical parameter of solvent polarity ET(30): 1 – tetrahydrofuran, 2 – chloroform, 3 – acetonitrile, 4 – N,N-dimethilformamide, 5 – ethanol, 6 – methanol, 7 – water.
6800
/ cm-1
6000
νA - νF
5600
6
5
6400
7
Fig. 3. Fluorescent spectra of compound B (c = 1 105 mol l1) obtained in aqueous solution at pH = 7.0 during the irradiation of 240 min. Inset show change in the fluorescence intensity at kF = 560 nm of B on the time.
visible inhibition (clear) zone around the well was observed (compared with the control) after incubation of the plates for 48–72 h at 25 °C. An inhibition zone P 10 mm indicated the presence of an inhibitory activity. The MIC value was determined as the zero intercept of a linear regression of the squared size of the inhibition zones, plotted against the logarithm of the sample concentration.
4 3
5200 2 4800
1 36
40
44
48
52
56
60
64
-1
ET (30) / kcal mol
Fig. 2. Dependence of Stokes shift (mA–mF) of compound B on the empirical parameter of solvent polarity ET(30): 1 – tetrahydrofuran, 2 – chloroform, 3 – acetonitrile, 4 – N,N-dimethilformamide, 5 – ethanol, 6 – methanol, 7 – water.
2.1.5. Determination of the minimal inhibitory concentration (MIC) The MIC of B compound against the studied bacteria was determined using agar-well diffusion method [33,34]. Petri plates containing nutrient agar medium were inoculated with suspensions of the indicator microorganisms (in exponential growth phase), and the surface of the agar was punched with 7-mm in diameter wells. The tested B compound with starting concentration 0.5% was further diluted in dist. H2O to obtain the following eight concentrations for its MIC determination: 0.05, 0.10, 0.15, 0.20, 0.25, 0.40, 0.50 and 0.75 mg/ml. 50 ll of each sample dilution was added into the wells of each plate. The solution without B sample was used as a control. The MIC value was defined as the lowest concentration at which a
2.1.6. Test of film antimicrobial activity The antimicrobial effect of the obtained B-PLA film was investigated against Gram-negative bacteria E. coli and P. aeruginosa as test microorganisms. For antimicrobial tests, square shape specimens of 6 mm were cut from the PLA and B-PLA films under aseptic conditions. The test tubes with 2.5 ml sterile nutrient broth medium were inoculated with overnight bacterial cultures and left at room temperature for 15 min. Then, the specimens were inserted into the test tubes. Test tubes without inserted film specimens were also prepared for each bacterial culture. After 24 h incubation at 25 °C under shaking at 240 rpm, the specimens were removed and the bacterial growth was determined by measuring the optical density of the medium at 570 nm (OD570). In parallel, specimens were placed onto nutrient agar surface in Petri plates seeded with the test cultures. After incubation of the plates for 48 h at 25 °C, the diameter of the formed zones, if any, was measured. 3. Results and discussion 3.1. Chemistry 3.1.1. Synthesis of 1-[(7-oxo-7H-benzo[de]anthracen-3-ylcarbamoyl)methyl]-triethylammonium chloride (B) The synthetic route for the preparation of cationic compound B is outlined in Scheme 1. The starting 3-amino-benzo[de]antracen-
D. Staneva et al. / Journal of Photochemistry and Photobiology B: Biology 143 (2015) 44–51
47
0,25
Absorbance
0,20 t = 120 min
0,15
t = 10 min
0,10
0,05
300
350
400
450
500
Wavelength / nm Fig. 4. Absorption release profile of B from B–PLA matrix in aqueous solution at pH = 7 and 25 °C for 120 min.
75 Fl. Intensity
Fluorescence intensity
75
60
45
60 45 30 15 0 0
30
60
90
120
Time / min
30
15
0
450
500
550
600
650
700
Wavelength / nm Fig. 5. Fluorescent release profile of B from B–PLA matrix in aqueous solution at pH = 7 at 25 °C for 120 min. Inset show the change of fluorescence intensity with the time.
7-one has been obtained by nitration and reduction of nitro group of the commercially available benzanthrone. Acylation of the primary amino group was carried out with chloracetylchloride at 50 °C for 2 h in dioxan solution. The obtained product 2-chloroN-7(oxo-7H-benzo[de]antracene-3-yl-acetamid was isolated by pouring the reaction mixture into ice water and filtering the precipitate. The compound has an activated chlorine atom, which react with triethylamine to obtain the final product 1-[(7-oxo7H-benzo[de]anthracen-3-ylcarbamoyl)-methyl]-triethylammonium chloride as yellow precipitate after cooling, which was filtrated and washed by acetone and dried under vacuum. 3.1.2. Photophysical characteristics of 1-[(7-oxo-7Hbenzo[de]anthracen-3-ylcarbamoyl)-methyl]-triethylammonium chloride The absorption and fluorescence properties of the benzanthrone derivatives are related to the extent of polarisation of the chromophoric system upon irradiation, resulting from the interaction between electron-donor substituent at C-3 position and electronacceptor carbonyl group within the benzanthrone molecule [7]. Clearly, the bands of absorption and emission of the benzanthrones
Fig. 6. Antibacterial activity of B compound (10 ll), inhibition zone (mm in diameter). Gentamicin (G, 10 ll/disc).
largely depend on the electron-donating power of the substituents at C-3 position. Furthermore, their spectral properties depend on the nature of the surrounding environment (polarity, viscosity, and formation of hydrogen bonds or other intermolecular interactions). Table 1 presents the spectral characteristics of the benzanthrone compound B in organic solvents with different polarity: the absorption (kA) and fluorescence (kF) maxima, the extinction coefficient e, Stokes shift (mA–mF), oscillator strength (f) and quantum yield of fluorescence (UF). The empirical parameter of solvent polarity (ET(30) kcal mol1) was used to characterized the polarity of the medium [35]. It was shown that the polarity of the solvents has a counterproductive effect on the position of the absorption and fluorescence maxima (Fig. 1). As can be seen, the solvent polarity influences the position of the absorption maxima, DkA = 17 nm. In comparison with the fluorescent maxima in non-polar solvents, a bathochromic shift of DkF = 25 nm for those in polar solvents was observed. The greater bathochromic shift can be explained by enhancing the dipole moment of the molecule upon excitation due to the electron density redistribution. The excited molecule is more stable in polar solvents due to stronger interactions with the solvents dipole. These results confirm a conformational change occurred in the singlet excited state S1 upon excitation of S0. Similar results have been achieved with benzanthrone derivatives with different substituents at C-3 position [7]. Stokes shift (mA–mF) and oscillator strength (f) are important parameters of the fluorescent compounds. The Stokes shift is a parameter which indicates the difference in properties and structure of the dyes between the ground S0 and the first excited state S1. Fig. 2 presents that the Stokes shift depends on the media polarity and it is larger in the case of polar solvents when the hydrogen bond formation or dipole–dipole interactions are favored in comparison to nonpolar media. The Stokes shift range obtained in this work is in the region mA–mF = 4673–6474 cm1. The results are very similar to those obtained for the monomer benzanthrone derivatives with different substituent at C-3 position, which indicated that the nature of the substituent has effect upon the Stokes shift. The oscillator strength (f), shows the effective number of electrons whose transition from ground to excited state, gives the absorption area in the electron spectrum. Values of the oscillator strength can be calculated using the Eq. (1):
f ¼ 4:32 109 Dm1=2 e max
ð2Þ
where Dm1/2 is the width of the absorption band (in cm1) at 1/2 emax. The values of the oscillator strength depend on the solvent
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Fig. 7. Growth inhibition of P. aeruginosa and B. cereus by different amounts (10, 20, 30 and 50 ll) of sample B.
polarity. As can be seen in Table 1, the values for the oscillator strength were in the range of 0.213–0.349. The ability of the photoactive molecules to emit the absorbed light energy is characterized quantitatively by the fluorescence quantum yield. The calculated UF were in the region 0.011– 0.298. It is seen than the quantum yield increases more than ten times in non-polar solvents compared with the polar. This variation arises from the solvent dependence of the nonradiative rate constant [36,37] or by possible formation of hydrogen bonds which favor less radiation transition causing a decrease of the fluorescence quantum yield of B. 3.1.3. Photostability of compound B in aqueous solution The photostability of water soluble antimicrobial compounds is very important in the cases when they are use as biocides. Photodegradation of compound B has been measured comparing the fluorescence maxima of the solutions at concentrations 1 105 mol l1 before and after irradiation at pH = 7.0. Fig. 3 plots the change of the fluorescence intensity with the time It is seen that the reduction on the fluorescence intensity at kF = 560 nm was negligible during the irradiation of first 120 min, which integrates that the stability of the compound B in aqueous solution is excellent. Also there is not any change in the fluorescence maxima before and after the irradiation. This fact demonstrates that the products of photodestruction neither absorb nor fluoresce in the spectral region where the compound B is photoactive. 3.1.4. Absorption and fluorescence investigation of colored PLA film in aqueous media It was of interest to study the antibacterial activity of the newly synthesized compound B after its incorporation into a thin polymer film of PLA. We used a solution casting method for the formation of PLA film and we prepared film from a solution of 5 g/100 ml chloroform solution and 0.05 g/100 ml of compound B. The obtained film was stable, flexible and transparent with intensive yellow color and thickness of 80 lm. In order to explore the possibility to use the PLA-B as antimicrobial film the release of B from the PLA-B matrix has been investigated in aqueous media at pH = 7 by dropping method. The PLA film is hydrophobic while B compound is more hydrophilic and as a result the release of B from the polymer matrix is possible. We conducted an experiment to investigate the possibility to release of compound B from the PLA film in the aqueous solution in the interval up to 120 min at pH = 7 by dropping method. During the contact of PLA-B film with aqueous media the compound B releases from the PLA matrix and the aqueous solution becomes
yellow in color. This process has been followed by using absorption and fluorescence spectroscopy. Figs. 4 and 5 showed that the absorption and respective fluorescence emission of compound B increased with time. It is seen that initially the PLA film releases a large amount of B, and accordingly the absorption and fluorescence are higher, and with time, this effect gradually decreases. Perhaps in this case, the compound B more easily released from the surface of the PLA film and its inner layers and passed into the aqueous solution. The absorption and fluorescence maxima of B in water are kA = 413 nm and kF = 557 nm, respectively. It is seen that no change of the position of the absorption and fluorescence maxima, but the intensity was increased. This is a new and interesting characteristic of PLA-B system, indicating that the compound B can be released slowly into the water solution and to exhibit a prolonged antibacterial activity. 3.2. Biological activity 3.2.1. Antimicrobial activity of 1-[(7-oxo-7H-benzo[de]anthracen-3ylcarbamoyl)-methyl]-triethylammonium chloride The newly synthesized compound was tested in vitro for antimicrobial activity against seven bacterial and two yeasts cultures. The sample exhibited comparable or better zones of inhibition of the growth of most indicator cultures than standard drug gentamicin (Fig. 6). Highest inhibition activity was observed against tested yeast strains (zones of clearance 23-24 mm) followed by the activity against B. cereus (zone of clearance 21 mm). The compound showed moderate activity against five bacterial cultures (zones of inhibition 13–15 mm), and weak activity towards M. luteus (zone of inhibition 12 mm). Highest inhibition of the growth of B. cereus and P. aeruginosa was observed using 30 and 50 ll of the sample solution (zone of clearance 24 and 14 mm, respectively) (Fig. 7). 3.2.2. Minimum inhibitory concentration determination (MIC) The MIC of B sample against the studied microbial cultures was determined by the agar diffusion method. As can be seen in Fig. 8 and Table 2, the MIC of the compound B varied in the range 48– 155 lg/ml indicating good antimicrobial potential. It was most effective (the lowest MIC) in inhibiting the growth of B. subtilis at 48 lg/ml followed by Sarcina lutea (MIC 58 lg/ml), and least effective (the highest MIC) towards B. cereus strain. M. luteus and X. oryzae strains were found to be resistant towards the compound at the used concentration. Cationic compounds have emerged as promising candidates for development of antimicrobial agents. Quaternary ammonium compounds with attached positively charged quaternary
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Fig. 8. Well-agar diffusion assay for MIC determination of the B compound against some indicator bacteria and yeasts. Concentration range: 1 – 0.05 mg/mL; 2 – 0.10 mg/mL; 3 – 0.15 mg/mL; 4 – 0.20 mg/mL; 5 – 0.25 mg/mL; 6 – 0.40 mg/mL; 7 – 0.50 mg/mL; 8 – 0.75 mg/mL.
ammonium groups are membrane active agents with a target site predominantly at the cytoplasmic (inner) membrane in bacteria or the plasma membrane in yeasts. Various cationic architectures have been tested such as the polyelectrolyte layers [24,38] and the dendrimers have also been used [39,40]. The following processes occur with microorganisms exposed to cationic agents [41,42]: (i) adsorption and penetration of the agent into the cell wall; (ii) reaction with the cytoplasmic membrane (lipid or protein) followed by membrane disorganization; (iii) leakage of intracellular low-molecular-weight material; (iv) degradation of proteins and nucleic acids; and (v) wall lysis caused by autolytic enzymes. There would be a loss of structural organization and integrity of the cytoplasmic membrane in bacteria, together with
Table 2 MIC of -[(7-oxo-7H-benzo[de]anthracen-3-ylcarbamoyl)-methyl]triethylammonium chloride towards some indicator bacteria and yeasts. Strains
MIC (lg/mL)
Bacillus subtilis Bacillus cereus Sarcina lutea Pseudomonas aeruginosa Escherichia coli Acinetobacter johnsonii Saccharomyces cerevisiae Candida lipolytica
48 155 58 124 73 105 96 82
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the water solution and to exhibit a prolonged good antibacterial activity.
Acknowledgements This paper was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, under Grant No. 17-130-35-HiCi. The authors, therefore, acknowledge with thanks DSR technical and financial support.
References
Fig. 9. Effect of PLA film (KS), and B-PLA film on the growth of P. aeruginosa and E. coli strains tested in liquid nutrient broth (NB). KO, NB without added film.
other damaging effects of the bacterial cell [43]. The antibacterial effect of the compound B reported here was higher than that of recently reported other benzanthrone derivatives [44]. The results obtained suggest that the newly synthesized compound B might be used as antimicrobial additive for biomedical devices and in the agriculture for plant protection.
3.2.3. Antimicrobial activity of B compound incorporated into PLA thin film The antimicrobial activity of the solid film PLA-B agains P. aeruginosa and E. coli has been investigated in aqueous solution and the results are presented in Fig. 9. As seen, the inhibition efficiency of the B-PLA film was higher than that of the PLA film against both tested strains. The B-PLA composite film causes a significant decrease in the optical densities of growth media of tested strains P. aeruginosa and E. coli (around 31% and 25%, respectively), while in the presence of the pure PLA film this decrease was much lower (7% and 19% respectively). The antimicrobial effect is due to release of the hydrophilic B compound from the PLA matrix by diffusion. No zones of inhibition were observed in nutrient agar tests which is probably due to insufficient degree of hydrophilicity of the film surface that is difficult to be achieved on the agar surface, which prevent the release of the B from the film. It has been reported that some other factors such as the level of immobilized antimicrobial activity retained at film surfaces and the surface area of films may also have effect the release profile of the immobilized activity [45].
4. Conclusion In this study we report the synthesis and characterization of a new cationic water soluble benzanthrone derivative having quaternary triethylammonium group. Photophysical characteristics of the compound have been determined in different organic solvents and they have demonstrated good dependence from the solvent polarity. It has been shown that the new benzanthrone compound B was very photostable in aqueous solution in low molar concentration. The results showed good inhibitory activity of the novel benzanthrone compound B against the tested bacterial and yeasts cultures. Antimicrobial activity observed against phytopathogenic strain X. oryzae suggested potential application of the compound B for plant protection. The gradual release of the substance with antimicrobial activity, which has previously been incorporated into the PLA thin film, has been investigated. The results showed that the compound B may be released slowly into
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