Hodyna D., Trush M., Metelytsia L., Tarasyuk O., Rogalsky S.

Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Science of Ukraine

Murmanska St., 1, Kyiv 02094, Ukraine

A comparative study of imidazolium and imidazolinium ionic liquids: antimicrobial activity and acute toxicity

Ionic liquids (ILs) are low temperature molten salts composed of bulky organic cations and various anions. Due to unusual combination of extremely low vapour pressure, high thermal stability and ionic conductivity ILs have found numerous applications such as alternative “green” solvents in organic synthesis, polymer chemistry, enzyme catalysis, drug delivery systems etc. [1]. Recently imidazolium ILs comprising bulky 1,3-dialkylimidazolium cations with long aliphatic chains were found to possess broad range of antimicrobial activity against both Gram-negative and Gram-positive bacteria, fungi and algae, as well as pronounced anti-biofilm activity [2-4]. It was indicated that alkyl substituted imidazolium cations interact with the negatively charged bacterial cell membrane and disrupt it, causing leakage of cell. Due to their lipophilic nature, the alkyl chains of imidazolium cations may interact with the cell membrane constituents, thereby increasing membrane permeability [2].

However, potential toxicity of ILs is one of the key factors determining their availability for practical applications, especially in medical devices, protective coatings etc. From this point of view, water insoluble ILs can be very promising antimicrobial additives due to their high washing resistance and therefore potentially higher durability, as well as lower environmental impact.

The aim of this research was to evaluate antimicrobial activity and acute toxicity of two water immiscible ILs comprising tetrafluoroborate anion and such cations as 1-dodecyl-3-methylimidazolium (DMIM-BF₄) and 2-dodecylamino-imidazolinium (DAIM-BF₄). It should be noted that there are no data in the literature concerning the biological activity of imidazolinium based ILs.

ILs were synthesized according to following schemes:

Antimicrobial properties of the ILs were tested by using standard agar disk diffusion method [5]. The tested bacterial strains were Staphylococcus aureus (ATCC-25923), Escherichia coli (ATCC-25922), Pseudomonas aeruginosa (ATCC-27853) and fungi strains were Candida albicans (M 885 ATCC 10231) and clinical isolates Candida albicans, Candida glabrata and Candida krusei.

Bacterial strains were subcultured on Mueller Hinton Agar and fungal strains were subcultured on Sabouraud agar in Petri plates according to the manufacturer’s instructions. All ILs were investigated in concentrations of 1.0; 0.1 % and were dissolved in DMSO. 0,02 ml of test compounds were applied on the sterile paper discs (6 mm diameter). The microbial culture was evenly poured onto the surface of agar plates into a volume of 0,2 ml of sterile saline solution to produce final concentration of 1∙10⁵ CFU /ml. The plates were incubated at +37 ° C for 24 hr.

The antimicrobial activity of ILs was assessed by measuring zone diameter of the growth inhibition (in mm).

Acute toxicity testing was carried out on Zebrafish (Danio rerio) as an aquatic model. The maximum allowable concentration of ILs (LD₅₀) was taken 5.5 mg/l [6].

The results of antimicrobial activity of ILs are presented in Table. 1

Table 1. Antimicrobial activity of DAIM-BF₄ and DMIM-BF₄ by zone diameters of growth inhibition of tested bacterial and fungal strains

№	Tested microorganisms	Conc., %	Inhibition zone of ILs, mm
			DAIM-BF4	DMIM-BF4
1	Е. coli ATCC	1,0	35	22
		0,1	20	10
2	St. aureus ATCC	1,0	24	32
		0,1	20	23
3	Ps. aeruginosa ATCC	1,0	21	30
		0,1	16	21
4	C. albicans ATCC	1,0	30	30
		0,1	17	16
5	C. albicans	1,0	25	28
		0,1	13	22
6	C. glabrata	1,0	>40	30
		0,1	25	20
7	C. krusei	1,0	>40	>40
		0,1	30	22

According to the obtained data both ILs have high antimicrobial activity, showing broad inhibition zones for both bacterial and fungi test-cultures. It can be suggested that as in the case for imidazolium ILs, the antimicrobial properties of DAIM-BF₄ are also caused by the presence of imidazolinium cation with delocalized charge, as well as hydrophobic alkyl tail.

The investigation of acute toxicity (LD₅₀) of ILs on the model hidrobiont Zebrafish (Danio rerio) showed that DAIM-BF₄ is three times less toxic (LD₅₀ was 20 mg/l) than DMIM-BF₄ (LD₅₀ was 7 mg/l). Figure 1 and Figure 2 presents summary data for antimicrobial activity and acute toxicity of the investigated ILs.

Thus, the results of our research indicate on availability of imidazolinium salts as potential antimicrobial agents with broad range of biological activity and reduced toxicity.

Figure 1. Antimicrobial activity of ILs Figure 2. Acute toxicity of ILs

References

1. Welton, T. (1999) Room-Temperature Ionic Liquids. Solvents for Synthesis and Catalysis. Chem Rev 99 (8), 2071-2084.

2. Pernak, J., Sobaszkiewicz, K., Mirska, I. (2003) Anti-microbial activities of ionic liquids. Green Chem 5, 52-56.

3. Docherty, K., Kulpa, C. (2005) Toxicity and antimicrobial activity of imidazolium and pyridinium ionic liquids. Green Chem 7, 185-189.

4. Carson, L., Chau, P., Earle, M., Gilea, M., Gilmore, B., Gorman, S., McCann, M., Seddon, K. (2009) Antibiofilm activities of 1-alkyl-3-methylimidazolium chloride ionic liquids. Green Chem 11, 492–497.

5. Bauer A., Kirby W., Sherris J., Turck M. Antibiotic susceptibility testing by a standardized single disk method // Am. J. Clin. Pathol. - 1966. - 45, No 4. - P. 493-496.

6. Шаляпин Г. Исследование действия биоцидов (на примере полигексаметиленгуанидинов) на гидробионтов различных систематических групп // Рыбохозяйственная токсикология. - 2010. - 11, №1 (41). - с. 199-206.