CC BY
96Труды ВНИРО
Аквакультура
2016 г. Том 162
УДК 615. 012. 1: 582.949.2: 581 . 3
The antimicrobial activity of some ethanolic extracts obtained from Ficus spp. leaves against Aeromonas hydrophila
Halyna Tkachenko1, Lyudmyla Buyun2, Elxbieta Terech-Majewska3, Zbigniew Osadowski1, Yevgenii Sosnovskyi4, Vitaliy Honcharenko5, Andriy Prokopiv4,5-
1 Department of Zoology and Animal Physiology, Institute of Biology and Environmental Protection, Pomeranian University in Slupsk, Poland; Arciszewski Str. 22B, 76—200 Slupsk, Poland;
2 M . Gryshko National Botanical Garden, National Academy of Sciences of Ukraine, Kyiv, Ukraine
3 Department of Epizootiology, University of Warmia and Mazury in Olsztyn, Poland
4 Botanical Garden of Ivan Franko Lviv National University, Lviv, Ukraine
5 Ivan Franko Lviv National University, Lviv, Ukraine E-mail: tkachenko@apsl .edu .pl, biology. apsl@gmail .com
Nowadays numerous medicinal plants possessing antiviral, antibacterial, antifungal and antiparasitic activities have a significant role in aquaculture as prophylactic and therapeutic agents against fish pathogens . This prompted us to determine the in vitro antimicrobial activity of ethanolic extracts from leaves of various Ficus species (Moraceae) against the bacterial strain of Aeromonas hydrophila isolated locally from infected rainbow trout (Oncorhynchus mykiss) with the aim of providing a scientific proof for the use of the plants in the treatment of bacterial infections induced by Aeromonas spp in fish The antimicrobial susceptibility testing was done on Muller-Hinton agar by disc diffusion method . Our results indicated that extracts offer a promising alternative to the use of antibiotics in controlling A- hydrophila growth. In our study, most ethanolic extracts obtained from Ficus spp . proved effective against the bacterial strain of Gram-negative A- hydrophila tested, with 10—12 mm zones of inhibition being observed . A- hydrophila demonstrated the highest susceptibility against F- pumila. Among various species of Ficus with moderate activity against A- hydrophila, the highest antibacterial activity for F- benghalensis, F- benjamina, F-deltoidea, F- hispida, F- lyrata was displayed . Thus, Ficus spp . leaves possess great medicinal potential for the therapy of bacterial and fungal infections and may be used as a natural antiseptic and antimicrobial agent in veterinary. It is concluded from the present study that these products can be used in aquaculture as therapeutic and prophylactic agents against fish pathogens, with antimicrobial and immunostimulant properties . Further investigation is necessary to identify those bioactive compounds, which will be a platform for further pharmacological studies and clinical applications
Key words: antibacterial activity, Aeromonas hydrophila, infected rainbow trout Oncorhynchus mykiss, Ficus spp , ethanolic extracts, zone of growth inhibition
Introduction.
The genus Aeromonas of the family Aero-monadaceae includes Gram-negative facultative anaerobes that have worldwide distribution in
aquatic environments and are pathogenic to fish and other animals [Cipriano, Austin, 2011] . A. hydrophila is an autochthonous species in freshwater environments and a member of the normal
microflora in the fish intestinal tract [Cipriano, 2001] . On the other hand, A. hydrophila causes diverse pathologic conditions that include acute, chronic, and latent infections . Aeromonas bacteria are also the etiologic agents responsible for a variety of infections in both immunocompetent and immunocompromised humans . Severity of disease is influenced by a number of interrelated factors, including bacterial virulence, the kind and degree of stress exerted on a population of fish, as well as the resistance and physiological condition of the host . Pathologic conditions attributed to members of the A. hydrophila complex include dermal ulceration, hemorrhagic septicemia, red sore disease, red rot disease, and scale protrusion disease [Cipriano, 2001] . In salmonids, A. salmonicida causes furunculosis, a disease characterized by skin ulcers and septicemia . Moreover, other Aeromonas species are involved in similar pathological diseases [Austin, Austin, 2007]. Findings of Zepe-da-Velázquez and co-workers [2015] confirmed that the Aeromonas species, A. hydrophila, A. salmonicida, and A. veronii, are associated with septicemia and dermal lesions in rainbow trout Our previous study revealed that cytoplasmic vac-uolation of hepatocytes, loss of normal basophilia by cytoplasm, presence of pigment deposition in the parenchyma are a signs of liver deterioration in trout affected by A. hydrophila Impaired circulation in the liver manifested in oedema and an occurrence of a large number of erythrocytes in the sinusoids and the intercellular space [Tkachenko et al . , 2014] .
One of the overriding considerations is how disease may be controlled The application of vaccine technology to fish diseases started with A. salmonicida in the 1940s and now commercial products based on iron-regulated outer membrane proteins (IROMPs) of typical strains are available . Motile aeromonads have attracted less interest in vaccine development . However, these pathogens have been the focus of probiotics, im-munostimulants and medicinal plants, some of which stimulate immune memory Finally, the use of genetically disease-resistant stock has already been considered for A. salmonicida [Cipriano, Austin, 2011]
The benefits of medicinal plants have been the focus in many studies originating from Asia in controlling infections induced by Aeromonas
[Cipriano, Austin, 2011] . The active compounds of herbs possess characteristics that could be useful in fish and shrimp culture; various herbs can stimulate growth and appetite, enhance immune system responses, and have broad spectrum of antimicrobial activity [Friedman et al . , 2002] .
Resistance against antibiotics of many bacteria is accumulating . Therefore, searches for new substances with antimicrobial activity have become an urgent necessity . Medicinal plants involved in traditional medicine are potential sources of antimicrobial compounds . Recently, there has been increasing interest in Ficus spp . (Moraceae) due to its chemical composition and the potential health benefits . Ficus spp . have been extensively used in traditional medicine for a wide range of ailments of the central nervous system, endocrine system, gastrointestinal tract, reproductive system, respiratory system and infectious disorders [Kumar, Au-gusti, 1989; Cherian, Augusti, 1993; Kirana et al . , 2009, 2011; Usman et al . , 2009; Ahmad et al . , 2011; Ilyanie et al . , 2011; Dangarembizi et al . , 2012; Arunachalam, Parimelazhagan, 2013; Gul-e-Rana et al ., 2013; Farsi et al ., 2014] . However, although many species within the genus Ficus have been encompassed by phytochemical and pharmacological investigations in previous years, there are many species that have not been studied and whose ethnobotanical relevance is yet to be investigated This prompted us to determine the in vitro antimicrobial activity of ethanolic extracts from leaves of various Ficus species against the bacterial strain of A. hydrophila isolated locally from infected rainbow trout (Oncorhynchus my-kiss (Walbaum, 1792)) with the aim of providing a scientific rationale for the use of the plant in the treatment of bacterial infections induced by Aero-monas spp in fish
Materials and methods.
Collection of Plant Material. The leaves of F. aspera G .Forst, F. benghalensis L., F. benjam-ina L., F. benjamina 'Reginald', F. binnendijkii (Miq .) Miq ., F. binnendijkii 'Amstel Gold', F. binnendijkii 'Amstel King', F. binnendijkii 'Amstel Gold', F. carica L., F. craterostoma Mildbr . & Burret, F. cyathistipula Warb., F. deltoidea Jack, F. drupacea Thunb ., F. drupacea 'Black Velvet', F. elastica Roxb ., F. elastica 'Variegata', F. erecta Thunb., F. erecta var . sieboldii (Miq .)
King, F. hispida L .f., F. luschanthiana (Miq .) Miq ., F. lyrata Warb., F. macrophylla Desf. ex Pers ., F. mucuso Welw . ex Ficalho, F. natalensis Hochst. subsp . natalensis, F. natalensis Hochst . subsp . leprieurii (Miq .) C .C . Berg, F. palmeri S .Watson, F. platypoda (Miq .) A .Cunn . ex Miq ., F. pumila L ., F. religiosa L., F. rubiginosa Desf. ex Vent ., F. sagittata J .Koenig ex Vahl, F. septica Burm . f., F. sur Forssk., F. sycomorus L., F. vasta Forssk ., F. villosa Blume were collected in M . Gryshko National Botanical Garden (Kyiv, Ukraine) and Botanical Garden of Ivan Franko Lviv National University (Lviv, Ukraine) during March and September, 2015 . The whole collection of tropical and subtropical plants both at NBG and Botanical Garden of Ivan Franko Lviv National University (including Ficus spp . plants) has the status of a National Heritage Collection of Ukraine The species author abbreviations were followed by Brummitt and Powell [1992] .
Preparation of Plant Extracts. The sampled leaves of Ficus spp were brought into the laboratory for antimicrobial studies Freshly crushed leaves were washed, weighted, and homogenized in 96% ethanol (in proportion 1:10) at room temperature
Method of Culturing Pathological Sample. Aeromonas hydrophila (strain E 2/7/15) isolated locally from gill of rainbow trout (Oncorhynchus mykiss Walbaum) with clinical features of furunculosis (kidneys were gray, liver was pale and fragile, enlarged spleen with exudate in the body cavity) was used as test organism Fish infection had a mixed character (Pseudomonas fluorescens, A. hydrophila complex, Shewanel-la putrefaciens). The increased mortality among trout has been occurred Fish with clinical signs of disease were euthanized with Propiscin (Inland Fisheries Institute, Poland), anesthetic designed for fish, in immersion using 2 mL per liter of water Samples of internal organs (kidneys, spleen, liver) weighting 2 g were taken and homogenized before preincubation in TSB broth (Tripticase Soya Broth, Oxoid) for 24 hrs.
Identification Method of the Bacteria. After preincubation, bacterial culture was transferred to two different cultivation media: TSA (Tripticase Soya Agar, Oxoid) and BHIA (Brain Heart Infusion Agar, Oxoid) supplemented with 5% of sheep blood [OIE Fish Diseases Commission
2000, Fredrichs, 1993]. After 48 hrs of incubation at 27 °C, characteristic pink colonies were selected for further examination . Bacterial species were identified with the oxidase test and API E test kit (Biomerieux, France) . The results of the test were interpreted in accordance with the manufacturer's protocol, after 24 hrs of incubation at 27 °C . Codes ++V-V--+V+++--+-VV in API E test were identified as A- hydrophila.
Bacterial Growth Inhibition Test of Plant Extracts by the Disk Diffusion Method. Strain tested was plated on TSA medium (Tryptone Soya Agar) and incubated for 24 hrs at 25 °C. Then the suspensions of microorganisms were suspended in sterile PBS and the turbidity adjusted equivalent to that of a 0 . 5 McFarland standard . Antimicrobial activity of extracts was evaluated by using agar well diffusion method [Bauer et al . , 1966] . Muller-Hinton agar plates were inoculated with 200 ^l of standardized inoculum (108 CFU/mL) of bacterium and spread with sterile swabs
Sterile filter paper discs impregnated by extract were applied over each of the culture plates, 15 min after bacteria suspension was placed The antimicrobial susceptibility testing was done on Muller-Hinton agar by disc diffusion method (Kirby-Bauer disk diffusion susceptibility test protocol). The A- hydrophila isolates were individually tested against 4 antibiotics The tested antibiotics were as follows: oxytetracycline (30 ^g), enrofloksacin (5 ^g), gentamicin (10 ^g); sulphamethoxazole/trimethoprim (25 ^g) . A negative control disc impregnated by sterile ethanol was used in each experiment The sensitivity of strain was also studied to the commercial preparation with extracts of garlic (in dilution 1:10, 1:100 and 1:1000) . After culturing bacteria on Muel-ler-Hinton agar, the disks were placed on the same plates and incubated for 24 hrs at 25 °C The diameters of the inhibition zones were measured in millimeters, and compared with those of the control and standard susceptibility disks Activity was evidenced by the presence of a zone of inhibition surrounding the well Each test was repeated six times The following zone diameter criteria were used to assign susceptibility or resistance of bacteria to the phytochemicals tested: Susceptible (S) > 15 mm, Intermediate (I) = 11—14 mm, and Resistant (R) < 10 mm [Okoth et al . , 2013] .
Results. comparison of susceptibility categories, i . e . sus-
The results of screening study of antimicrobial ceptible, intermediate, and resistant, for the disk
activity of ethanolic extracts obtained from Ficus diffusion method is shown in Table 1 spp . leaves are presented in Table 1, Figs 1—5 .A
Fig. 1 . Antimicrobial activity of ethanolic extracts obtained from F. benghalensis (4), F. benjamina (5) (A), F. cyathistipula (14), and F. lyrata (15) against A. hydrophila (B) measured as inhibition zone diameter .
Fig. 2 . Antimicrobial activity of ethanolic extracts obtained from F. natalensis subsp . leprieurii (34) and F. deltoidea (36) (A), as well as F. palmeri (50), F. hispida (51), and F. natalensis subsp . natalensis (52) against A. hydrophila
(B) measured as inhibition zone diameter
Fig. 3 . Antimicrobial activity of ethanolic extracts obtained from F. pumila (55, 60) against A. hydrophila (A), and
tested antibiotics measured as inhibition zone diameter
Table 1. Susceptibility or resistance of A. hydrophila against ethanolic extracts obtained from Ficus spp . leaves
Ficus spp . Susceptibility or resistance of A. hydrophila
Intermediate (I) = 11—14 mm Resistant (R) < 10 mm
F. aspera +
F. benghalensis +
F. benjamina +
F. benjamina 'Reginald' +
F. binnendijkii +
F. binnendijkii 'Amstel Gold' +
F. binnendijkii 'Amstel King' +
F. carica +
F. craterostoma +
F. cyathistipula +
F. deltoidea +
F. drupacea +
F. drupacea 'Black Velvet' +
F. elastica +
F. elastica 'Variegata' +
F. erecta +
F. erecta var sieboldii +
F. hispida +
F. luschanthiana +
F. lyrata +
F. macrophylla +
F. mucuso +
F. natalensis subsp . leprieurii +
F. natalensis subsp . natalensis +
F. palmeri +
F. platypoda +
F. pumila +
F. religiosa +
F. rubiginosa +
F. sagittata +
F. septica +
F. sur +
F. sycomorus +
F. vasta +
F. villosa +
Our results showed that the A. hydrophila revealed intermediate susceptibility (diameter of inhibition zone) concerning to ethanolic extracts obtained from F. benghalensis, F. benjamina, F. binnendijkii, F. cyathistipula, F. del-toidea, F. erecta, F. erecta var . sieboldii, F. hispida, F. luschanthiana, F. lyrata, F. macrophylla, F. mucuso, F. natalensis subsp . leprieurii, F. natalensis subsp . natalensis, F. palmeri, F. platypo-da, F. pumila, F. rubiginosa, F. sur, F. sycomorus, and F. villosa. A. hydrophila was resistant against ethanolic exstracts from F. aspera, F. benjamina 'Reginald', F. binnendijkii 'Amstel Gold', F. binnendijkii 'Amstel King', F. carica, F. craterosto-ma, F. drupacea, F. drupacea 'Black Velvet', F. elastica, F. elastica 'Variegata', F. religiosa, F. sagittata, F. septica, and F. vasta (Table 1) .
Discussion.
Our results indicated that extracts offer a promising alternative to the use of antibiotics in controlling A. hydrophila. In our study, most eth-anolic extracts obtained from Ficus spp proved effective against the bacterial strain of Gram-negative A. hydrophila tested, with 10 — 12 mm zones of inhibition being observed A. hydroph-ila demonstrated the highest susceptibility against F. pumila (Table 1, Fig . 3). Among various species of Ficus with moderate activity against A. hydrophila, the highest antibacterial activity for F. benghalensis, F. benjamina, F. deltoidea, F. hispida, F. lyrata was noted .
It is well documented that various Ficus spp . have been used against Gram-positive and Gram-negative bacteria [Salem et al ., 2013] . The scientific research on Ficus spp indicated that these plants have received increasing interest in recent years . For instance, F. deltoidea has been reported to have beneficial pharmaceutical uses as an antidiabetic, anti-inflammatory, antinociceptive, antimelanogenic, antiphotoaging, antioxidant, antiulcerogenic, and antibacterial agent with an-tioxidant, antihyperglycemic, anti-inflammatory, antiulcerogenic and antinociceptive activity [Bun-awan et al ., 2014; Dzolin et al ., 2015] . Adam and co-workers (2007) reported that almost all of the parts of F. deltoidea plant including the root, bark, leaf and fig have their own medicinal properties Abdsamah and co-workers (2012) studied the in vitro antimicrobial activity of the chloroform,
methanol and aqueous extracts of F. deltoidea against 2 Gram-positive [Staphylococcus aureus (IMR S-277), Bacillus subtilis (IMR K-1)], 2 Gram-negative [Escherichia coli (IMR E-940), Pseudomonas aeruginosa (IMR P-84)] and 1 fungal strain, Candida albicans (IMR C-44) . All the extracts showed inhibitory activity on the fungus, Gram-positive and Gram-negative bacteria strains tested except for the chloroform and aqueous extracts on B. subtilis, E. coli, and P. aeruginosa [Abdsamah et al ., 2012] . In line with these studies, in our investigation, ethanolic extract obtained from F. deltoidea showed intermediate antibacterial efficiency against A. hydrophila with 11—13 mm zone of growth inhibition (Fig . 2А) .
Similar antibacterial activity was shown for ethanolic exstract from F. benjamina (Fig . 1). F. benjamina also possesses high medicinal potential . The plant is also used as antimicrobial, antinociceptive, antipyretic, hypotensive and anti-dysentery remedy [Imran et al ., 2014] . Sirisha and co-workers (2010) suggested that the leaves, bark and fruits of F. benjamina contain various bioactive constituents like cinnamic acid, lactose, naringenin, quercetin, caffeic acid and stigmas-terol The fruit extract of F. benjamina possessed both antitumor and significant antibacterial activities Medicinal importance of this plant encouraged us to carry out the antimicrobial study of the ethanolic extract from the leaves of F. benjamina plants against A. hydrophila. Parveen and co-workers [2009] reported about the isolation and characterization of a new triterpene, (9,11), (18,19)-disecoolean-12-en-28-oic acid (1) along with ß-amyrin (2). The compound 1 exhibited significant antimicrobial activity against Salmonella typhimurium (MTCC-98), C. albicans (IA0-109), S. aureus (IAO-SA-22), E. coli (K-12) and low activity against A. niger (lab isolate ICAR) and A. brassicola.
Our results demonstrate that F. benghalensis has intermediate in vitro activity against A. hydrophila . Our study is in agreement with studies of other authors The bark of F. benghalensis exhibited significant antibacterial activity against S. aureus, P. aeruginosa and K. pneumoniae [Gayathri et al . , 2009] .
Verma and co-workers [2012, 2013] confirmed immunostimulatory role of F. benghalensis (prop-roots) and L. leucocephala (pod seed) in
Clarias gariepinus when supplemented in artificial feed . They evaluated the antibacterial activity of methanolic extracts of F. benghalensis (prop-root) by measurement of zone of inhibition against pathogenic bacteria, E. coli and A. hydrophila. Moreover, juvenile C. gariepinus were fed with 5% powder of F. benghalensis with respective feeds for 20 days prior to the experiment . Immu-nomodulatory response of supplementary feed was studied by challenging the fish intraperitoneal-ly at weekly intervals, with A. hydrophila. One set of fish, not challenged with A. hydrophila was used as a negative control, to analyze any detrimental effect of supplementary feed, while positive control, comprised of challenged fish fed with non-supplemented feed . Other two groups of fish were challenged with A. hydrophila and fed with respective supplementary feeds . Fish fed with supplementary feed showed increased lysozyme activity and phagocytic index indicating an increase in non-specific immune response . Moreover, serum lysozyme, tissue superoxide dismutase, percentage phagocytosis, phagocytotic index, nitric oxide (NO), total serum protein and immunoglobulin increased significantly in the treated fish compared to control fish
In our study, ethanolic extracts of F. lyrata and F. hispida leaves inhibited growth of A. hydrophila (Fig . 1B, 2B). In similar studies considerable antimicrobial activity of F. lyrata also has been found . Ethyl acetate extract of F. lyra-ta latex comprises compounds with antibacterial and anticandidal properties which can be used as antimicrobial agents in new drugs for therapy of infectious diseases The methanolic extract had no effect against bacteria except for Proteus mirabilis while the ethyl acetate extract had inhibition effect on the multiplication of five bacteria species (E. faecalis, Citobacter freundii, P. aeruginosa, E. coli and Proteus mirabilis) [Bidarigh et al ., 2011] . F. lyrata has been reported to have numerous bioactive compounds such as arabi-nose, P-amyrins, P-carotenes, glycosides, P-se-tosterols and xanthotoxol [Jeong and Lachance, 2001; Vaya and Mahmood, 2006] . Findings of Rizvi and co-workers [2010] suggest that Ursolic acid from F. lyrata has excellent antibacterial activity against several problematic bacteria like MRSA and ESBL producing bacteria, Pseudomonas, Salmonella, Shigella and Vibrio cholerae
and other known pathogens with drug resistance Ursolic acid and Acacetin-7-O-neohesperido-side contribute significantly to the antimicrobial activities of the crude extract of F. lyrata [Rizvi et al . , 2010] . Ahmad and Beg [2001] revealed that glycosides and saponins extracted from leaves using alcohol had biological effects but they had no effects on C. albicans, S. aureus and E. coli.
F. hispida was chosen for its abundance of alkaloids, carbohydrates, proteins and amino acids, sterols, phenols, flavonoids, gums and mucilage, glycosides, saponins, and terpenes [Ghosh et al , 2004] The broad antibacterial activities of this extract, apparently, could be explained as a result of the plant secondary metabolites Previously have been reported [Salem et al . , 2013], that the therapeutic properties of Ficus species may be attributed to the presence of a wide range of phyto-chemical compounds In general, Ficus species are rich sources of polyphenolic compounds In particular, flavonoids and isoflavonoids are responsible for the extract's strong antioxidant activity that may be useful in preventing diseases involving oxidative stress [Sirisha et al ., 2010] . Ali and Chaudhary [2011] have reported that F. hispida contains wide varieties of bioactive compounds from different phytochemical groups like alkaloids, carbohydrates, proteins and amino acids, sterols, phenols, flavonoids, gums and mucilage, glyco-sides, saponins, and terpenes Two substantial phenanthroindolizidine alkaloids, 6-O-methylty-lophorinidine and 2-demethoxytylophorine, and a novel biphenylhexahydroindolizine hispidine from stem and leaves of F. hispida were isolated by Venkatachalam and Mulchandani [1982] . Recently, hispidin has been reported to have anticancer activity [Ali and Chaudhary, 2011] . All the detected phenolic acids are known to have antimicrobial and antioxidant properties [Jaafar et al . , 2012] . The antimicrobial property of F. hispida extract may be due to its constituents
A wide range of chemical compounds have been also isolated and characterized from F. deltoidea, particularly from the leaves and figs [Bunawan et al ., 2014] . An initial comprehensive study on volatile compounds produced by the fruits was conducted by Grison-Pige and co-workers [2002] . The volatile compounds isolated and identified are mainly products of the shikimic acid pathway, terpenoids, and aliphat-
ic groups, generally present as floral fragrances in plants . Mohd Lip and co-workers [2009] have isolated and identified moretenol from F. deltoidea leaves . Later on, an antibacterial compound known as lupeol (C30H50O) was also isolated from the leaves of F- deltoidea and exhibited toxicity against S- aureus, B- subtilis, and E-coli [Suryati et al ., 2011] . Ong and co-workers [2011] demonstrated enhancement of flavonoid compounds (rutin, quercetin, and naringenin) in cell cultures of F- deltoidea influenced by different carbon sources as well as plant growth regulators A comprehensive study on flavonoid compounds of aqueous extract of F- deltoidea was conducted by Omar and co-workers [2011], and more than 25 compounds were identified Currently, two bioactive constituents known as vitexin and isovitexin have been isolated, identified, and evaluated to show a-glucosidase inhibition [Choo et al ., 2012] . The antibacterial activity of this extract is possibly linked to the presence of flavonoid compounds Antibacterial activities of flavonoid compounds isolated from plant species are well documented [Hendrich, 2006; Ferrazzano et al , 2011; Farzaei et al ., 2013] . A high amount of epi-catechin found in F- deltoidea may be responsible for the strong radical scavenging activities found in the extract A positive correlation was observed between flavonoid constituents present and radical scavenging activities of the aqueous extracts of F. deltoidea [Dzolin et al ., 2015] . Chemical analysis found four phenolic compounds (chlorogenic, p-coumaric, ferulic and syringic acids) in roots, three (chlorogenic, p-coumaric and ferulic acids) in stem and only one (caffeic acid) in leaves of F- benjamina [Imran et al ., 2014] . A glucoside, bengalenoside, was isolated from F- benghalensis and evaluated for hypoglycemic activity [Garg, Paliwal, 2011] . The phytochemical screening of F- benghalensis revealed the presence of saponins, tannins and flavonoids in aqueous and methanolic extract [Aswar et al , 2008] Levels of total phenolic, total flavonol and total flavonoid compounds in aerial roots in 70 mg/g of extract, 3 mg/g quercetin equivalent and 5 mg quercetin equivalent/g extract have also been reported [Sharma et al ., 2009] . Some natural compounds, i . e . glucoside, 20-tetratriaconthene-2-one, 6-heptatria-contene-10-one, pentariacontan-5-one, P-sitos-terol-a-D-glucose and meso-inositol have been
isolated from the bark [Subramanian and Misra,
1978]
Antibacterial flavonoids might be having multiple cellular targets, rather than one specific site of action One of their molecular actions is to form complex with proteins through nonspecific forces such as hydrogen bonding and hydrophobic effects, as well as by covalent bond formation Thus, their mode of antimicrobial action may be related to their ability to inactivate microbial adhesins, enzymes, cell envelope transport proteins, and so forth . Lipophilic flavonoids may also disrupt microbial membranes [Kumar, Pandey, 2013] .
It is desirable that antibiotic use in fish cultures will be reduced and replaced by natural medicines to prevent the emergence of bacterial resistance in aquatic animals and their environment . Antibiotics are widely used in fish farms to prophy-lactically treat bacterial infections and as a growth promoter Despite its widespread use, there is no regulation on this drug class in fish [Rigos, Troi-si, 2005] . Therefore, use of plant extracts can be more effective for preventive and therapeutically aims in aquaculture . In our study, the ethanolic extracts obtained from various species of Ficus leaves showed varying inhibitory activities against A- hydrophila
Conclusions.
Present study aimed to investigate the in vitro antimicrobial activity of the ethanolic leaf extracts of various Ficus spp , respectively using the disc diffusion method, against A- hydrophila- Our results demonstrate that various species of Ficus had intermediate antibacterial in vitro activity against A- hydrophila The highest antibacterial activity against A- hydrophila was noted for F- benghalensis, F- benjamina, F- deltoidea, F- hispida, F- lyra-ta Further studies should be conducted to verify this activity against other pathogenic bacteria of interest in aquaculture and to confirm immune response involvement or its potential as a virulence factors inhibitor Thus, Ficus spp leaves possess great medicinal potential for the therapy of bacterial and fungal infections and may be used as a natural antiseptic and antimicrobial agent in veterinary . These products can be used in aquaculture as therapeutic and prophylactic agents against fish pathogens, with antimicrobial and/or immu-nostimulant properties Further investigation is
necessary to identify those bioactive compounds, which will be a platform for further pharmacological studies and clinical applications
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Поступила в редакцию 04.04.16 г. Принята после рецензии 25.07.16 г.
Антимикробная активность некоторых спиртовых экстрактов, полученных из листьев видов Ficus, в отношении Aeromonas hydrophila
Ткаченко Г.М.1, Буюн Л.И.2, Терех-Маевская Э.3, Осадовский З.1, Сосновский Е.В.4,
Гончаренко В.И.5, Прокопив А.И.4,5
1 Кафедра зоологии и физиологии животных, Институт биологии и охраны среды, Поморская Академия в Слупске, Польша
2 Национальный ботанический сад им . Н . Н .Гришко НАН Украины, Киев, Украина
3 Кафедра эпизоотологии, Варминско-Мазурский Университет в Ольштыне, Польша
4 Ботанический сад Львовского национального университета имени Ивана Франко, Львов, Украина
5 Львовский национальный университет имени Ивана Франко, Львов, Украина
В настоящее время многочисленные лекарственные растения, обладающие противовирусными, антибактериальными, противогрибковыми и противопаразитарными свойствами, играют важную роль в аквакультуре в качестве профилактических и терапевтических агентов в лечении паразитозов рыб . Это послужило основанием для сравнения антимикробной активности спиртовых экстрактов из листьев различных видов фикусов (Moraceae) в отношении бактериального штамма Aeromonas hydrophila, изолированного локально из инфицированной радужной форели (Oncorhynchus mykiss) с целью выявления научных доказательств для использования этих растений в лечении бактериальных инфекций рыб, вызванных Aeromonas. Наши результаты показали, что использование исследованных экстрактов является перспективной альтернативой применению антибиотиков с целью контроля роста А. hydrophila. Полученные результаты свидетельствуют о том, что большинство спиртовых экстрактов, полученных из фикусов, являются эффективными в отношении роста бакте-
риального штамма A. hydrophila, с 10—12 мм зонами ингибирования роста . А. hydrophila оказался наиболее восприимчивым в отношении экстрактов листьев F. pumila. Среди различных видов фикуса с умеренной активностью в отношении А. hydrophila, высокая антибактериальная активность отмечена у F. benghalensis, F. benjamina, F. deltoidea, F. hispida, F. lyrata. Таким образом, экстракты из листьев фикусов обладают большим терапевтическим потенциалом в лечении бактериальных и грибковых инфекций и могут быть использованы в качестве природных антисептиков и проти-вомикробных агентов в ветеринарии . Из данного исследования сделан вывод, что эти продукты могут быть использованы в лечебно-профилактических мероприятиях в аквакультуре в качестве антимикробных средств . Дальнейшие исследования необходимы для идентификации биологически активных соединений, которые послужат платформой для дальнейших фармакологических исследований и клинического применения
Ключевые слова: антибактериальная активность, Aeromonas hydrophila, радужная форель Oncorhynchus mykiss, Ficus spp . , спиртовые экстракты, зона ингибирования роста.
