ISSN 2409-4943. Ukr. Biochem. J., 2017, Vol. 89, N 5 UDC 615.282.012:582.288:547.543 doi: https://doi.org/10.15407/ubj89.05.070 ETHYLTHIOSULFANILATE EFFECT ON Candida tropicalis L. B. ORIABINSKA1, S. О. Starovoitova2, S. V. VASYLYUK3, V. P. NOVIKOV3, v. І. LubenetS3* 1n ational technical university of ukraine “igor Sikorsky Kyiv Polytechnic institute”; 2n ational university of Food technologies, Kyiv, ukraine; 3Lviv Polytechnic national university, ukraine; е-mail: [email protected] t he problem of resistance of microorganisms to antibiotics stimulates the search for new chemothera- py drugs. ethylthiosulfanilate (etS, S-ethyl 4-amino-benzenesulfonothioate, S-ethyl ester of 4-aminobenze- nethiosulfoacid) as a structural analog of a number of phytoncides was synthesized based on thiosulfoacid salts. etS effect on opportunistic yeast fungi Candida tropicalis was studied. it was shown that etS in sub- fungicidal concentration (125 µg/ml) affected some aspects of a constructive and energy metabolism of fungi: inhibited endogenous respiration and reduced the pool of nucleic acids. additionaly the significant changes in lipogenesis of C. tropicalis were detected. it was established that etS in fungistatic and subfungicidal concentrations possessed membranotropic effect and provided a high degree of cooperativity of structural transitions of membranes. K e y w o r d s: Candida tropicalis, antimycotic action, ethylthiosulfanilate effect. he antimicrobial resistance to known drugs bles, effective pesticides, growth regulators, biocide is one of the challenges to modern medical additives, insecticides, radioprotectors [5]. T practice. Thus search for new substances Aryl esters of arylthiosulfoacids (p-tolyl- and development on their basis effective antimi- p-to luenethiosulfonate and p-methoxybenzene- crobial and antiviral drugs is an important task of p-methoxy-benzenethiosulfonate) possess high the modern pharmaceutical chemistry. Synthesis of insecticidal activity towards larvae anagasta kue- structural analogs of biologically active compounds hniella [6]. Some thiosulfoacid esters are promi- of natural origin is one of the known approaches that sing antithrombotic agents [7]. Alkyl esters of some allows creating new efficient agents for various pur- 4-acylaminobenzene- and heterocyclic thiosulfo- poses [1, 2]. S-Esters of thiosulfoacids are structural acids exhibit antifungal effect towards different analogs of volatile compounds from garlic (allium types of fungi (Candida albicans, Candida tropica- sativum L.) [2], onion (allium cepa L.) [3], various lis, Candida рarарsilosis, Candida krusei, Candida types of cabbage, especially cauliflower [1], deep- іпсоnsрісuа, Candida поrvegensis, Candida kеfyr, sea urchin echinocardium cordatum [4], etc. The Candida lusitaniae, Candida glabrata, Cryptococ- treatment with these plants has been considered as cus neoformans, trichosporon asahii, Geotrichum a promising approach for treatment of atherosclero- capitatum, blastoschizomyces capitatus) in low con- sis, coronary thrombosis, asthma as well as bacterial centrations [8]. infections. It is known that synthetic esters of thio- Alkylthiosulfanilates disrupt the normal sulfoacids exhibit a wide range of biological effects, physio logical functions of bacteria and fungi, es- and their antimicrobial activity often exceeds the ef- pecially in combination with rhamnolipid biosur- fectiveness of natural analogs. They are offered as factants. Bactericidal and fungicidal effect of thio- drug-candidates, preservatives of fruit and vegeta- sulfoesters in combination with rhamnolipids were © 2017 Oriabinska L. B. et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 70 L. b. oriabinska, S. О. Starovoitova, S. v. vasylyuk et al. tion in redox processes, contributing to disruption or of respiratory system of phytopathogenic microorga- nisms. ETS shows a stimulating effect on the growth of field pea and sorghum, and also enhances adap- tive capacity of plants to adverse conditions. This proves the prospects of use of ETS as efficient and environmentally safe agent to intensify the process of phytoremediation of contaminated soils [11]. Fol- lowing the wide range of possible applications and low toxicity of ethylthiosulfanilate an in-depth study of the mechanism of action of ETS on microorga- S cheme 1 nisms, especially on fungi is reasonable. Thus, the aim of this work was to study effect demonstrated on strains Pseudomonas aeruginosa, ethylthiosulfanilate action on the model of yeast bacillus subtilis, alcaligenes faecalis and rhizopus Candida tropicalis. nigricans [9]. Several synthesis protocols for ethylthiosulfa- Materials and Methods nilate were developed in Lviv Polytechnic National Methods of obtaining the ethylthiosulfanilate University (Ukraine) as outlined on Scheme 1 [9]. were presented in the previous work [9]. ETS stands out for its high fungicidal activity Yeast fungus C. tropicalis, obtained from the and low toxicity. It is an active substance of antifun- Museum of Cultures of Department of Industrial gal ointment “Esulan”™, which antimicrobial activi- Biotechnology, Faculty of Biotechnology and Bio- ty against various diseases of the skin, hair and nails technics NTUU “KPI” was used as a test culture. NHCOR NH NH was studied by G. N. NHCORPershin and others [10].2 Studies To2 study the effect of ethylthiosulfanilate on the Na S або of biological2 action of ETS haveMOH shown a wideC2H spec5Br - metabo lism of C. tropicalis, the cells were cultured trum of antimicrobialKHS activity [9, 10], it is active not on nutrient medium (beef-extract broth) with 2% only towards micromycetes, yeast cultures, but also glucose in the presence of subfungicidal concentra- SO Cl SO SM O S-S C H against gram-positiveSO andS M gram-negative bacteria, tions2 5of the test drug for 48 h at 37 °C [12]. O O O O including pathogenic, opportunistic and phytopatho- Determination of nucleic acids levels in yeast genic microorganisms. For instance clinical studies cells under the influence of ethylthiosulfanilate was R = -CH , -OCH NHCOCH revealed3 high3 efficiency of antifungal ointment3 [10]. + conducted spectrophotometrically using Spirin’s M = Na, K C H Br H Thiosulfoacid esters2 5 are highly reactive com- method, separation of DNAs and RNAs was per- pounds that interact with nucleophiles, electrophiles, formed by Schmidt’s and Tannhauser’s method [13]. and radicals. Nucleophilic substitutionO S-S reactionsC H oc- Yeast lipid composition was analyzed by thin cur with breaking of -S-S-bond due to the2 redistribu5 - O layer chromatography. To determine the total li- tion of electron density in thiosulfogroup that deter- pids we used the eluent system: benzene : ethyl- mines the direction of nucleophile attack [5]. acetate : acetic acid (85 : 15 : 1), and to determine phospholipids – system chloroform : methanol : wa- ter (65 : 25 : 4). Solution of sulfuric acid in metha- nol (10%) was used for the spots detection [14]. Percenta ge of distribution of total lipid fractions of yeast C. tropicalis influenced by subfungicidal con- ETS affects the metabolism of RNA and DNA, centration of ETS was conducted by densitometra- which contain amino groups, as well as proteins and tion of chromatographic plates (Laser densitometer aminoacids with disulfide and sulfide fragments, LKB Ultrascan XL, Sweden). which are components of plant cells and microor- Determination of endogenous respiration of ganisms. Pretreatment of seeds with ETS at low yeast was performed by using polarographic method concentrations improves germination of plants, in- (Polarograph LP 60) with open electrode of Clarke creases weight of seedlings, reducing the number of type at 37 °C. Respiration rate per unit of time for diseased plants [11]. The impact of ETS on plants is 1 mg of protein fractions was calculated [15]. closely related with its protective action, participa- 71 ISSN 2409-4943. Ukr. Biochem. J., 2017, Vol. 89, N 5 The investigation of influence of ethylthiosul- As shown in Table 1, ETS (125 µg/ml) inhibits fanilate on permeability of the cytoplasmic mem- the synthesis of DNA and RNA. The residual con- branes of yeast was determined photometrically by centration of DNA in the cells was 10.68 ± 0.01 and the release of low-molecular compounds having a 49.74 ± 0.05 µg/ml for RNA, that correspond to 27.52 maximum absorption at λ-260 nm [16]. and 39.13% of control, respectively. Namely, concen- All experiments were conducted at least three tration of nucleic acids in C. tropicalis cells under times using appropriate controls. Statistical analysis the influence of ETS decreased 3.6-fold for DNA and was performed using the software package of Micro- 2.56-fold for RNA. Simultaneous inhibition of both soft Excel, statistical significance was evaluated by types of nucleic acids – RNA and DNA usually oc- Student’s t-test. The difference was significant when curs when the chemotherapeutics act on the integrity P < 0.05. of the cell membranes [16]. A relatively small number of yeast species are Results and Discussion characterized by stable respiratory system. In most yeast, growth of yeast under condition of periodic Previous studies revealed that ETS is a com- cultivation is accompanied by changes in the struc- pound with broad spectrum of action and wide in- ture of the respiratory chain, especially at the ini-