https://doi.org/10.15414/agrobiodiversity.2019.2585-8246.014-024 AGROBIODIVERSITY FOR IMPROVING NUTRITION , HEALTH AND LIFE QUALITY 2019 ANTIBACTERIAL PROPERTIES OF ETHANOLIC EXTRACTS OBTAINED FROM LEAVES OF SOME THYMUS L. (LAMIACEAE) REPRESENTATIVES AGAINST ACINETOBACTER BAUMANNII Honcharenko Vitaliy1, Tkachenko Halyna*2, Nachychko Viktor1,3, Prokopiv Andriy1,3, Osadowski Zbigniew2 1Department of Botany, Faculty of Biology, Ivan Franko National University in Lviv, Lviv, Ukraine 2 3Botanic Garden of Ivan Franko National University in Lviv, Lviv, Ukraine Institute of Biology and Earth Sciences, Pomeranian University in Słupsk, Poland Received: 15. 11. 2019 Revised: 18. 11. 2019 Published: 28. 11. 2019 The Thymus L. is one of the most widely used genera in folk medicine, where it is popular for its stimulatory action on all organism functions. The purpose of this study was to evaluate the antimicrobial Thymus L. representatives (Thymus serpyllum L., Th. pannonicus All., Th. × porcii Borbás, Th. pulegioides L., Th. alpestris effectsagainst ofAcinetobacter five ethanolic baumannii extracts obtained from leaves of some Tausch ex A. Kern.) complex isolate, resistant to gentamicin and ciprofloxacin (specimen antimicrobial3680, UK NEQAS). activity. Freshly Antimicrobial leaves were activity washed, was weighed, determined crushed, using andthe homogenizedagar disk diffusion in 96% assay. ethanol The (in proportion 1 : 19) at room temperature. The extracts were then filteredThymus and species investigated inhibited for theirmild activity against A. baumannii Th. serpyllum mm,present for studyTh. pannonicus has shown that ethanolic extracts obtainedThymus × from porcii leaves of Th. pulegioides Th.. alpestrisThe mean diameter of inhibition zone for was (10.45 ±0.81) effective plant against Acinetobacter (10.82 ±0.63) baumannii mm, for was Thymus pannonicus (9.57 ±0.75). The mm, present for study lays the basis(10.54 for ±0.52) future mm, research, and for to validate the (10.62 possible ±0.54) use mm. of Thymus It should species be noted as thata candidate the most in antimicrobially the treatment of bacterial infections. The knowledge about the chemical profile of the extract will help in explaining the observed activity and designing experiments for activity fractionation for isolation of the active principle. The identification of precise molecular mechanisms addressing how these extracts inhibit bacterialKeywords: growth Thymus needs to be explored. species, leaf extracts, agar disk diffusion assay, antibacterial activity, inhibition zone diameter *Corresponding author: Halyna Tkachenko, Institute of Biology and Earth Sciences, Pomeranian University in Słupsk, Arciszewski 22b, 76-200 Słupsk, Poland * [email protected] CC BY-NC-ND 4.0 – 14 – Honcharenko, V., Tkachenko, H., Nachychko, V., Prokopiv, A., Osadowski, Z. Agr.bio.div. Impr. Nut., Health Life Qual., 2019, 14–24 Introduction Acinetobacter baumannii multidrug-resistant microorganisms in hospitals worldwide responsible for hospital- is an opportunistic pathogen and one of the six most important is responsible for a vast array of infections, i.e. ventilator-associated as well as bloodstream acquired nosocomial infections (Antunes et al., 2014; Lee et al., 2017). This human pathogen The spread of multidrug-resistant Acinetobacter strains is cause for concern (Harding et al., infections in critically ill patients, and mortality rates can reach 35% (Antunes et al., 2014). A. baumannii, few antibiotics are effective for treating infections caused by this pathogen (Lee 2018). Due to the prevalence of infections and outbreaks caused by multi-drug resistant etBecause al., 2017). of the limited number of available antibiotics, we used medicinal plants with an from leaves of Thymus methicillin-resistantantibacterial effect. In Staphylococcus our previous study, aureus we have revealed that the ethanolic extracts obtained L. representatives exhibited different activity against clinical isolatesThymus of pulegioide Th.. Resultspannonicus obtained for antibacterial activityTh. serpyllum exhibited L. various degrees of the zone of inhibition and significantTh. × porcii activity Borbás was and observed Th. alpestris for Tausch s L. (21–26 mm) followed by All. (14–18 mm) and (13–18 mm), respectively while some extracts ( ex A. Kern.) showed mild activity at the same concentration. The above results revealed that potentialthe selected of plantsthese plantscan be asfurther novel utilized antimicrobial for isolation agents, of thetargeting active ingredientsthe multidrug-resistant as the crude extracts were found good for inhibition of MRSA. It is therefore important to investigate the Th. pulegioides Salmonellabacteria and enteritidis clinical importance (Honcharenko et al., 2018a). Additionally, the ethanolic extract obtained from the leaves of wasTh. pulegioidesthe most effective followed plant by extractsTh. pannonicus against studiedTh. and alpestris presented in the work by Th.Honcharenko × porcii et al. (2018b). The thenantibacterial by Th. serpyllum activity of extracts was greatest for (13.1 ±0.85 mm) followed by Thymus (12.6 ±0.25 mm), (12.2 ±0.55 mm), and against β-lactamase producing (10.5 ±0.23 Pseudomonas mm) (Honcharenko aeruginosa et al., 2018b). Moreover, the ethanolic extracts obtained from leaves of Th. serpyllum representatives exhibited intermediateTh. pannonicus activity Th. × porcii Th. pulegioides (Honcharenko et al., 2018c). The Th.mean alpestris diameter of inhibition zone for was (11.3 ±0.3) mm, for Thymus It(11.5 should ±0.5) be mm,noted for that the most (9.8antimicrobial ±0.5) mm, effective for plant against (11.2 β-lactamase ±0.7) mm, producing and for P. aeruginosa (12.8 was Th. ±0.8) alpestris mm. The effects varied significantly according to the taxa. Th. alpestris followed by Th. pannonicus, being highly followed active by with Th. theserpyllum ethanolic and extract then by (mean Th. pulegioides diameter of inhibition zone was 12.8 ±0.8 mm). The antibacterial activity of extracts was greatest for (HoncharenkoOur previous studies et al., 2018c). lay the basis for future research, to validate the possible use of Thymus species as a candidate in the treatment of bacterial infections and a potential target for the and relevant uses in the medicine and veterinary. Recent studies have shown that Thymus activity-guided isolation of active constituents in order to explore the mechanism of action species have strong antimicrobial and antioxidant activities (Jia et al., 2010). The thyme – 15 – Honcharenko, V., Tkachenko, H., Nachychko, V., Prokopiv, A., Osadowski, Z. Agr.bio.div. Impr. Nut., Health Life Qual., 2019, 14–24 essential oil has also remarkable antibacterial effects, which are associated with the presence carvacrol had the most powerful effect against Listeria monocytogenes, followed by thymol, eugenol,of their phenolic cinnamaldeyde, components, and isoeugenol. carvacrol, andIn comparative thymol. In astudies study byincluding Yamazaki the etoregano al. (2004), and thyme essential oils, the two oils demonstrated remarkable antibacterial activity. Thymol is a naturally occurring phenol monoterpene derivative of cymene and isomer of Thymus vulgaris pharmacy,carvacrol and food, is oneand ofcosmetic the major industry constituents due to several(10–64%) studies of the that essential have evaluated oils of thyme the potential ( therapeutic L.). Nowadays,uses of this thymol compound and thyme for the present treatment a wide of range disorders of functional affecting possibilities the respiratory, in the nervous, and cardiovascular systems. Moreover, this compound also exhibits antimicrobial, antioxidant, anti-carcinogenesis, anti-inflammatory, and antispasmodic activities, as well as a potential as a growth enhancer and immunomodulatorThymus (Salehi vulgaris et al., 2018). inhibited the growth of Staphylococcus epidermidis and Escherichia coli Schelzof orange, et al. eucalyptus, (2006) observed fennel, thatgeranium, the essential cedar, oilginger, of turpentine, rosemary, and tea tree, whereas of 15 essential oils used against foodborne more (Listeria efficiently monocytogenes than the essential, Salmonella oils typhimurium, E. coli Brochothrix thermosphacta and Pseudomonas fluorescens 0157:H7) and food spoilage bacteria ( ), the oregano, thyme, and cinnamon oils demonstrated the Acinetobactermost powerful baumannii antibacterial, E. coli effect, Klebsiella (Mith et pneumoniae al., 2014). ,Additionally, and Pseudomonas the oregano, aeruginosa thyme, (Sakkas and basil oils exhibited sufficient to moderate effects against multi-resistant clinical isolates of etThese al., 2016). data have prompted us to verify the antibacterial effects of four species and one Thymus genus sampled in the western part of Ukraine against Acinetobacter baumanii. Therefore, the aim of this study was to evaluate the antimicrobial interspecific hybrid of Thymus representatives (Thymus serpyllum, Th. pannonicus., Th. × porcii, Th. pulegioides, Th. alpestris Acinetobacter effectsbaumanii of five strain. ethanolic extracts obtained from leaves of some ) against Material and methodology Collection of Plant Materials Thymus serpyllum were collected among the grass on sandy soil in the edge of a pine forest (Baymaky village, Bilohirya district, Samples were harvested in June–August, 2016. Leaves of Th. pannonicus were harvested among grass in the roadside between the two cultivated Khmelnytsky region, Ukraine; N 50° 03´ 58.9´´, E 26° 13´ 37,5´´, 257 m a.s.l.). Leaves of Th. pulegioides