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Agrobiodiversity.2019.2585-8246.361-372 https://doi.org/10.15414/agrobiodiversity.2019.2585-8246.361-372 AGROBIODIVERSITY FOR IMPROVING NUTRITION , HEALTH AND LIFE QUALITY 2019 ANTIBACTERIAL ACTIVITIES OF ETHANOLIC EXTRACT OBTAINED FROM RHODODENDRON MYRTIFOLIUM SCHOTT & KOTSCHY LEAVES AGAINST CLINICALLY ISOLATED BACTERIAL STRAINS Honcharenko Vitaliy1, Tkachenko Halyna2, Prokopiv Andriy1, 3, Nachychko Viktor1, 3, Sosnovsky Yevhen3, Góralczyk Anna2, 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: 3. 12. 2019 Revised: 8. 12. 2019 Published: 10. 12. 2019 The plants belonging to the genus Rhododendron L. and isolated compounds demonstrate diverse cytotoxic activity, as well as antibacterial activity. Our study was designed the effects of Rhododendron myrtifoliumbiological activities Schott &including Kotschy anti-inflammatory, leaf extract against analgesic, bacterial anti-microbial, strains were monitored anti-diabetic, in vitro insecticidal by the disk and diffusion method. The aim of this study was to assess possible antibacterial effects of an ethanolic extract derived from Rhododendron myrtifolium leaves against Citrobacter freundii, Enterobacter cloacae, Klebsiella pneumoniae, and Escherichia coli the study contributes to on-going investigations on the bioactivity potential of plant species such as the Rhododendron. Leaves of Rhododendron myrtifolium strain locally were isolated harvested from on human the side biological of the road fluids. between Thus, the Menchul valley and Rogneska valley (Kvasy village, Rakhiv district, Zakarpattia region, Ukraine). The results revealed that extract exerts antibacterial activity against Citrobacter freundii. However, the Enterobacter cloacae, Klebsiella pneumoniae, and Escherichia coli were resistant to R. myrtifolium leaf extract. Maximum in vitro inhibition was scored against Citrobacter freundii, followed by Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae, which presented inhibition zones of (12.1 ±0.9) mm, (9.1 ±0.5) mm, (7.5 ±0.6) mm, and (7.2 ±0.5) mm, respectively. In the case of the positive controls, 96% ethanol possesses a mild antibacterial effect, which presented inhibition zones of (6.5 ±0.7) mm. The results from the screening study performed by the disc diffusion method revealed that R. myrtifolium possesses a mild antibacterial activity against C. freundii. However, further investigation is needed to determine the bioavailability of the active compounds and to determine the dose and toxicity before it can be used as therapeutic agents. Keywords: Rhododendron, 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 – 361 – Honcharenko, V., Tkachenko, H., Prokopiv, A., Nachychko, V., Sosnovsky, Y., Góralczyk, A., Osadowski, Z. Agr.bio.div. Impr. Nut., Health Life Qual., 2019, 361–372 Introduction Antimicrobial resistance has become a pre-eminent concern in medicine and public health. This problem is widespread, and the causative factors are uncontrolled (Mah and Memish, 2000). Control of infections caused by multi-drug resistant Gram-positive and Gram-negative bacteria has become a major problem in various countries in the prevention of infectious diseases. Currently, the spread of multi-drug resistant bacteria is not only through nosocomial infections, but also occurs in the community (Radji et al., 2013). Several multi- drug resistant bacteria that are most commonly found, especially through nosocomial infections, are Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (Rice, 2008). In the past few decades, these strains become increasingly important pathogens in hospitals and play an important role in the colonization and infection of hospitalized patients by a variety of nosocomial infections including bacteremia, urinary tract infections, and nosocomial pneumonia (Radji et al., 2013). bacteria with a large group of antibiotics. Therefore, it seems reasonable to explore alternativeTreatment ofantimicrobial these infections agents is foroften control very multi-drugdifficult due resistant to the cross-resistancebacteria. Recently, of thesethere as an alternative approach to discover new antimicrobial compounds (Ríos and Recio, 2005; Radjihas been et al., growing 2013). interestsRhododendron to find species antimicrobial have been compounds proved to frompossess medicinal medicinal plant and extracts health promotion properties, including the ability to inhibit the growth of some types of pathogenic bacteria (Innocenti et al., 2010; Silici et al., 2010; Popescu and Kopp, 2013; Rezk et al., 2015; Wang et al., 2015; Li et al., 2016; Hakeem Said et al., 2017; Shrestha et al., 2017). The genus Rhododendron L. (Ericaceae) is one of the most species-rich among angiosperms, comprising over 1000 species spreading across the northern hemisphere and with the center of diversity in southeastern Asia (Irving and Hebda, 1993). These plants are morphologically diverse, including evergreen and deciduous shrubs, subshrubs, and trees, mostly terrestrial but sometimes chasmophytic or epiphytic, with greyish-brown striate bark, spirally arranged branches, and leaves, often in pseudo-whorls, and umbellate to pyramidal racemes of 1–30 conspicuous fragrant flowers. Because of its foliage and flowers, the genus Ais noteworthywell-known European in ornamental member cultivation of the genus and isgardening, R. myrtifolium with Schottnumerous & Kotschy, varieties an artificiallyevergreen clump-formingbred thanks to dwarfrhododendrons’ shrub up to natural 50 cm inability height, to occurringinterspecific in high-mountainhybridization (Cullen, habitats 2005). of the eastern and southern Carpathian Mountains and northern Balkans, largely within altitudes of 1,400–2,500 m. The species is featured in small narrowly elliptic to obovate coriaceous leaves abaxially covered with glandular scales containing essential oils, terminal inflorescences 2006;of tubular-campanulate Voloschuk and Prokopiv, pinkish 2011). flowers, Although and long-pedunculate endangered in countries dry multilocular of its distribution, capsules R.containing myrtifolium numerous has been diminutive used in folk seeds medicine (Cullen, for the 1980; preparation Mircea, of2005; herbal Boratyński teas (Dihoru et andal., Boruz, 2014; Nedelcheva and Draganov, 2014) and presents a major touristic attraction during – 362 – Honcharenko, V., Tkachenko, H., Prokopiv, A., Nachychko, V., Sosnovsky, Y., Góralczyk, A., Osadowski, Z. Agr.bio.div. Impr. Nut., Health Life Qual., 2019, 361–372 R. ferrugineum L. and R. hirsutum L. (e.g., Sosnovsky et al., 2017) have been shown to possess cytotoxic,its mass flowering antibacterial, period and in antiviral mountains effects (Rivers, of their 2017). extracts Its evolutionary(Louis et al., 2010;closest Gescher relatives, et al., 2011; Seephonkai et al., 2011; Rezk et al., 2015b), while the biochemical features and bioactive potentials of R. myrtifolium remain unexplored. Rhododendron species have been traditionally used in China, Nepal, Russia, and North America for treating human diseases like asthma and skin diseases. These species are known to be aagainst good sourceinflammation, of polyphenolic pain, skin plant ailments, secondary common plant cold, metabolites and gastrointestinal (Shrestha et al.,disorders 2017). Theand plant extracts belonging to the genus Rhododendron and isolated compounds demonstrated diabetic, insecticidal and cytotoxic activity (Popescu and Kopp, 2013), as well as antibacterial activitydiverse (Innocentibiological etactivities al., 2010; including Silici et al., anti-inflammatory, 2010; Popescu and analgesic, Kopp, 2013; anti-microbial, Rezk et al., 2015;anti- Wang et al., 2015; Li et al., 2016; Hakeem Said et al., 2017; Shrestha et al., 2017). The aim of this study was to assess possible antibacterial effects of an ethanolic extract derived from Rhododendron myrtifolium leaves against Citrobacter freundii, Enterobacter cloacae, Klebsiella pneumoniae, and Escherichia coli strain locally isolated from human biological plant species such as the Rhododendron. Hence, the effects of Rhododendron myrtifolium leaf extractfluids. Thus, against the bacterial study contributes strains were to monitored on-going investigations in vitro by the ondisk the diffusion bioactivity method. potential of Materials and methodology Collection of Plant Materials Leaves of Rhododendron myrtifolium were harvested on the side of the road between the Menchul valley and Rogneska valley (Kvasy village, Rakhiv district, Zakarpattia region, Ukraine; washed to remove all the attached materials and used to prepare the ethanolic extract. N 48˚ 09‘ 28.4“, E 24˚ 20‘ 05.6“, 1,485 m a.s.l.) (Figure 1). Plant samples were thoroughly Preparation of Plant Extracts Freshly leaves were washed, weighed, crushed, and homogenized in 96% ethanol (in for antimicrobial activity. proportion 1 : 19, w/w) at room temperature. The extract was then filtered and investigated Antimicrobial susceptibility testing Non-repetitive clinical strains of isolated from biological materials infected patients were
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