ANTIMICROBIAL ACTIVITY OF FLUORESCENT BENZANTHRONE IN AQUEOUS SOLUTION AND IN POLYLACTIC ACID FILM

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International Journal of Pharmaceutical, Biological and Chemical Sciences (IJPBCS) | JUL-SEPT 2014 | VOLUME 3 | ISSUE 3 | 66-74 www.ijpbcs.net or www.ijpbcs.com

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ANTIMICROBIAL ACTIVITY OF FLUORESCENT BENZANTHRONE

IN AQUEOUS SOLUTION AND IN POLYLACTIC ACID FILM

M.S.I. Makki1, D. Staneva2, E. Vasileva-Tonkova3, T.R. Sobahi1, R.M. Abdеl-Rahman1, A.M. Asiri1, 4, I. Grabchev1, 5*

1

Chemistry Department, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia

2

University of Chemical Technology and Metallurgy, 1756 Sofia, Bulgaria

3

Institute of Microbiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria

4

Center of Excellence for Advanced Materials Research (CEAMR), King Abdulaziz University, Jeddah 21589, Saudi Arabia

5_{Sofia University “St. Kliment Ohridski”, Faculty of Medicine, 1407 Sofia, Bulgaria}

*Corresponding Author Email: [email protected]

INTRODUCTION

One of the main tasks faced by the mankind in the

course of its long history is the improvement of quality

of life where the level of medical knowledge has

always had a particular role. This public health

problem has increased especially for the last years.

Due to this fact, the search for new effective bioactive

agents with novel chemical structures is a great

worldwide concern and is the basis of the

investigations of researchers from many countries.

In the recent years dye chemistry is one of the key

research domains because the dyes have found

different non traditional applications in high

technology including in medicine and biology [1]. It is

known that the cationic compounds and especially the

quaternary ammonium salts are important biocides and

they can be used as effective antiseptic and disinfectant

agents in different areas [2,3]. They are active against a

broad spectrum of microorganisms such as

Gram-positive and Gram-negative bacteria, fungi and certain

types of viruses [4]. The microbial agents damage the

cell wall or alter cell membrane permeability, denature

proteins, inhibit enzyme activity or inhibit lipid

synthesis, all of them are essential for cell survival.

An interesting area of research is a combination of the

properties of substances with dyeing capacity with

antibacterial properties. This requires the introduction

of specific groups in the chromophores systems to give

other antibacterial properties without changing its color

performance. Dyes with quaternary ammonium group

can be prepared by incorporating long alkyl chain

quaternary ammonium salts into the chromophores

through covalent bonds. The obtained cationic dyes

show excellent color and antimicrobial functions in

solutions [5-8]. Various cationic compounds have been

ABSTRACT:

Тhe antimicrobial activity of a new benzanthrone dye (B) with biocide quaternary ammonium group has been investigated against Gram-positive and Gram-negative indicator bacteria and yeasts using agar well diffusion method and Minimum inhibitory concentration. Thin polylactic acid film with compound B into the polymer matrix has been prepared and its antimicrobial ability in aqueous solution has also been evaluated. The results showed that the polymer film has very good antibacterial activity against P. aeruginosa, E. coli and B. subtilis which is due to the release of B from the polymer.

KEYWORDS:

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I. Grabchev* et al;ANTIMICROBIAL ACTIVITY OF FLUORESCENT BENZANTHRONE IN AQUEOUS SOLUTION...

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tested such as the polyelectrolyte layers and the

dendrimers [9, 10].

Polylactide acid (PLA) is a biodegradable and

biocompatible polymer produced from renewable

resources such as corn, potatoes, sugar etc. Due to

transparently and low toxicity the PLA is one of the

most promising biopolymers able to replace the

petroleum-derived polymers for different industrial

applications [11, 12]. PLA ﬁber is most commonly

dyed with disperse dyes [13].

A new benzanthrone dye was recently synthesized by

incorporating biocidal quaternary ammonium group

into the chomophore structure [14]. This compound

has exhibited very good dyeing ability. It has been

shown that this dye changes its color parameter in

different pH media and demonstrated that can be used

as a sensor for metal ions and ammines [15,16 ].

In this work we investigate the antimicrobial properties

of a benzanthrone dye with biocidal quaternary

ammonium group. Its antimicrobial activity against

bacteria and yeasts and minimum inhibitory

concentration are discussed. The antibacterial

efficiency of the thin PLA-B film in aqueous solution

was also evaluated against positive and

Gram-negative bacteria.

MATERIALS AND METHODS

The synthesis and characterization of compound B has

been described recently [17] (Figure 1).

Fig. 1: Chemical structure of compound B

Preparation of polylactic acid film

Pure polylactic acid (PLA) and antimicrobial PLA

films were prepared by solvent casting method.

Polylactic acid (0.5 g) was dissolved in 10 ml

chloroform and 5 x 10-4 g compound B was added

After 30 minutes stirring, the homogeneous mixture

was poured into a Petri dish and the solvent was

evaporated slowly. Thus a stable polymer film with a

thickness of 80 m was obtained. The same method

has been used to produce pure PLA film.

Preliminary microbiological screening

Preliminary microbiological screening test for

determination of the antimicrobial activity of the newly

synthesized B compound was accomplished by the

agar well diffusion method with slight modification

[17]. The following indicator microbial strains were

used: Gram-positive bacteria Bacillus subtilis, Bacillus

cereus, Sarcina lutea and Micrococcus luteus,

Gram-negative bacteria Pseudomonas aeruginosa,

Escherichia coli, Acinetobacter johnsonii and

Xanthomonas oryzae, and yeasts Candida lipolytica

and Saccharomyces cerevisiae. Sterile Petri plates with

nutrient agar medium (pH = 7.2) were prepared with 4

mm agar layer thickness. The investigated compound

was dissolved in dist. H2O to obtain stock solution with

concentration 10 mg/mL. Suspensions of overnight

grown indicator cultures were prepared and smeared

onto the surface of nutrient agar in Petri plates. Wells

(7 mm in diameter) were punched aseptically in the

agar medium, to which 100 µL of the sample solution

(1.5 mg/ml) were added. Commercial discs with

gentamicin (10 µg) and nystatin (100 units) were used

as reference standards for antibacterial and antifungal

activity, respectively. After incubation of the plates at

25°C for 48 h, the diameter (in mm) of the resulting

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Determination of Minimum Inhibitory

Concentration (MIC)

The MIC of the tested B compound against the

indicator bacteria and yeasts was determined by the

agar serial diffusion method. After inoculation of Petri

plates containing nutrient agar with suspensions of

exponentially grown indicator strains, wells (7-mm in

diameter) were made in the agar surface. The solution

of B compound with starting concentration 10 mg/mL

was further diluted in dist. H2O to obtain the following

concentrations (mg/ml): 0.1, 0.2, 0.3, 0.5, 0.8, 1.0 and

1.5. Then, 100 µL of each sample dilution were added

into the wells. The MIC of B against each indicator

strain was defined as the lowest concentration that

inhibited the visible growth of the strains in the form of

zone of inhibition around the wells after incubation of

the plates for 48 h at 25°C. An inhibition zone of

greater than 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 B concentration.

Test of films antimicrobial activity

The antimicrobial effect of the obtained PLA-B film

was investigated against Gram-negative Escherichia

coli and Pseudomonas aeruginosa, and Gram-positive

Bacillus subtilis as test microorganisms. For

antimicrobial tests, square shape speciments of 6 mm

were cut from the PLA and PLA-B 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 speciments 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 turbidity of the medium

at 570 nm (OD570). The speciments were placed also

onto Petri plates containing nutrient agar 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.

Spectral measurements

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

concentration 105 mol/l.

RESULTS AND DISCUSSION

Release of B from PLA matrix

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.

Absorption and fluorescence spectroscopy have been

used for detection the colour change of the aqueous

solution. In Figures 2 and 3 are presented in vitro

release profiles of compound B from the PLA matrix

investigated by both spectral methods. During the

contact of the PLA-B film with water the compound B

releases from the PLA matrix and the aqueous solution

becomes yellow in color. A rapid release of B has been

observed in the first 60 min and continued up to 180

min. Due to the strong hydrophobic interaction

between compound B and PLA, the efficiency of the

release of B increased with the time indicating that the

compound B has been released from the polymer matrix by diffusion. The absorption and fluorescence

maxima in aqueous solution were determined A = 414

nm and F = 555 nm respectively, which are the same

as compound B investigated in aqueous media [17].

This means that no changes in the structure of

compound B occurred after its introduction into the

polymer matrix or after the release thereof. As can be

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and fluorescence maxima remained unchanged but the

intensity increased.

250 300 350 400 450 500

0,0 0,2 0,4 0,6 0,8 1,0

t = 180 min

A

bsor

ban

ce

Wavelength, nm

t = 10 mim

Fig. 2: Absorption release profile of B from PLA-B matrix in aqueous solution at pH = 7 and 25oC for 180 min

500 550 600 650 700

0 200 400 600 800 1000

0 40 80 120 160 200 0

200 400 600 800 1000

F

l.

in

te

ns

ity

Time / min

F

luor

escen

ce int

ensity

Wavelength / nm

Fig. 3: Fluorescent release profile of B from PLA-B matrix in aqueous solution at pH = 7 at 25oC for 180 min.

Inset shows the change of fluorescence intensity with the time

Antimicrobial screening and MIC

The antimicrobial efficiency depends strongly on the

concentration, which should not drop below the MIC.

This is why the antimicrobial activity of benzanthrone

B in aqueous solutions was tested by the minimum

inhibitory concentration (MIC).

It was found that compound B is biologically active

against the used eight indicator bacteria and two

yeasts. The results revealed that at the used conditions

the compound possesses good antibacterial and

antifungal potential. As seen in Figure 4, compound B exhibited better zones of inhibition against

Gram-positive bacteria (in the range 17-20 mm) than those

observed against the indicator Gram-negative bacteria

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The new compound exerted dose-dependent inhibition

activity (Figures 5, 6). The MIC values (mg/ml) of compound B against the indicator strains was

determined by the serial dilution method. As seen in

Figure 7, MIC values varied from 80 to 160 µg/ml.

The B was most effective (the lowest MIC) in

inhibiting the growth of S. lutea at about 80 µg/ml. The

highest MIC value 160 µg/ml was determined for the

yeast C. lipolytica followed by P. aeruginosa (150

µg/ml), and B. cereus (140 µg/ml). M. luteus was

found to be resistant to the used concentrations of B

compound.

Fig. 4: Inhibition zones (mm in diameter) of B compound (1.5 mg/ml) against the studied bacteria and yeasts. Controls: G, Gentamicin; NS, Nystatin

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Fig. 6. Inhibitory effects of compound B at different concentrations (mg/ml) against indicator bacteria and yeasts. An inhibition zone of greater than 10 mm indicates the presence of an inhibitory activity

Fig. 7: MIC values (µg/ml) of B compound against indicator bacteria and yeasts

Fig. 8: Effect of PLA film (KS), and PLA-B film on the growth of P. aeruginosa, E. coli and B. subtilis strains

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The following processes occur with microorganisms

exposed to cationic agents [18-20]: (i) adsorption and

penetration of the agent into the cell wall; (ii) reaction

with the components of the cytoplasmic membrane

leading to 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. The observed

antibacterial effect of the new B compound was better

than that of other recently reported benzanthrone

derivatives [21]. More pronounced antimicrobial effect

of B in the case of Gram-positive bacteria probably

due to the lack of additional permeability barriers. The

outer membrane in Gram-negative bacteria decreases

the outer membrane permeability impairing penetration

of antimicrobial agents in the cells [22, 23]. It has been

reported that other resistance mechanisms in

Gram-negative bacteria, such as efflux systems, have

increased effectiveness in the presence of the outer

membrane [24].

Antimicrobial activity of B incorporated films

In the resent years there is considerable interest in

biodegradable packaging due to environmental

concerns. Current innovations in food packaging

research include the development of active packaging

systems based on materials, which can include a

variety of additives such as antioxidants,

antimicrobials, vitamins, flavours and colorants with

the aim of improving their appearance and to extend

foodstuff shelf-life [25,26]. PLA is one of the most

important commercially available biobased and

biodegradable thermoplastic polyesters offering a

sustainable alternative for food packaging across a

wide range of commodity applications in response to

consumers' demands and market trends in the use of

renewable resources [27,28]. In the last years, different

nanocomposites based on PLA with nanoclays or

active additives have been developed and reported by

several authors [29,30].

The results of antimicrobial tests of the obtained film

are shown in Figure 8. As seen, the inhibition efficiency of the PLA film incorporated with B was

significantly higher than that of the PLA film against

both tested strains. The B-PLA film caused a

significant decrease in optical densities of growth

media of tested strains P. aeruginosa, E. coli and B.

subtilis (around 58%, 67% and 53%, respectively),

while in the presence of the pure PLA film this

decrease was much lower (7%, 19% and 9%,

respectively). The antimicrobial effect should be due to

release of the hydrophilic B compound from the

hydrophobic PLA matrix by diffusion. No zones of

inhibition were observed in nutrient agar tests 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 [31].

CONCLUSIONS

The antimicrobial screening of the newly synthesized

product showed its good antibacterial and antifungal

potential better pronounced in Gram-positive bacteria.

It could be concluded that the new benzanthrone

compound could be used as antimicrobial agent in

biomedical and agrochemical applications. Preliminary

tests showed the antimicrobial efficiency of the

obtained PLA films incorporated with the new B

compound suggesting its suitability for application as

new additive in the antimicrobial packaging

technology combining the dyeing with antimicrobial

effect.

ACKNOWLEDGEMENT

This paper was funded by the Deanship of Scientific

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grant no. (17-130-35-HiCi). The authors,

therefore, technical and financial support of KAU.

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