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37Труды ВНИРО
Аквакультура
2017 Г. Том 167
УДК 615. 012. 1: 582.949.2: 581 . 3
Antibacterial screening of ethanolic extracts obtained from leaves of various Ficus species (Moraceae) against Citrobacter freundii
Halyna Tkachenko1, Lyudmyla Buyun2, Elzbieta Terech-Majewska3, Zbigniew Osadowski1
1 Dep . of Zoology and Animal Physiology, Institute of Biology and Environmental Protection, Pomeranian University in Slupsk, Poland
2 M . Gryshko National Botanical Garden, NASU, Kyiv, Ukraine
3 Dep . of Epizootiology, University of Warmia and Mazury, Olsztyn, Poland
Medicinal herbs play an alternative role to antibiotic therapy in aquaculture . Ficus species (Moraceae) 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. Present study aimed to investigate the in vitro antimicrobial activity of the ethanolic leaf extracts of various Ficus species against fish pathogen, Citrobacter freundii. The leaves of Ficus species were collected in M . Gryshko National Botanical Garden (Kyiv, Ukraine) . Freshly crushed leaves were washed, weighted, and homogenized in 96 % ethanol (in proportion 1:10) at room temperature . Citrobacter freundii was isolated locally from gill of eel (Anguilla anguilla) with clinical features of disease . The antimicrobial susceptibility testing was done on Muller-Hinton agar by disc diffusion method (Kirby-Bauer disk diffusion susceptibility test protocol) . Muller-Hinton agar plates were inoculated with 400 |J of standardized inoculum (108 CFU/mL) of bacterium and spread with sterile swabs . Our results demonstrate that various species of Ficus had mild antibacterial in vitro activity against C. freundii. The results proved that the ethanolic extracts obtained from F. pumila, F. binnendijkii 'Amstel Gold', F. carica, F. erecta, F. hispida, F. mucuso, F. palmeri, F. religiosa possess considerably sufficient antibacterial potential against C. freundii
Key words: Ficus species, ethanolic extracts, antibacterial in vitro activity, zone of growth inhibition, Citrobacter freundii, eel Anguilla anguilla.
Introduction The parasitic, bacterial, and fungal diseases act as major limiting factors for fish farming, meaning that massive amounts of antibiotics, disinfectants, and pesticides in order to control mortality and avoid huge economic losses have to make use [Valladao et al . , 2015] . However, most chemical products are toxic towards fish and compromise their tissues . On the other hand, such chemother-apeutics are potential xenobiotics in the nature and may give rise to serious risks to human health and
cause environmental pollution [Malheiros et al . , 2016] .
The herbal extracts can be used in fish culture as alternatives to vaccines, antibiotics or chemo-therapeutic agents [Galina et al . , 2009] . Herbal medicines appear as sustainable and effective alternatives for conventional drugs for disease control in aquaculture . Plant-based products have shown promise, in that it is natural and biodegradable and has antimicrobial activity against various pathogens relating to fish [Valladao et al . , 2015] .
Many medicinal plants and their extracts act as immunostimulants . The use of immunostimulants has been considered to be the most environmentally friendly method of preventing piscine disease [Carbone and Faggio, 2016] . One group of immunostimulants that has shown numerous beneficial effects in aquatic animals, is tropical herbs . Indeed, Chinese herbs can stimulate the development of organs directly associated with the immune response, such as the thymus and spleen as well as increase antibody production [Galina et al . , 2009] . Moreover, one of the therapeutic strategies in ayurvedic medicine (plant-derived therapeutics) is to increase the body's natural resistance to the disease-causing agent rather than to directly neutralize the agent itself [Devasagayam and Sainis, 2002] . Successful results on controlling diseases in shrimp and fish by herbs have been reported in Mexico, Thailand, Japan and Turkey [Galina et al , 2009]
One of the potential plants that can be used as antimicrobial to enhance survival and immune competence is Ficus genus . Recently, researchers have reported promising effects from many species from Ficus genus for treating parasitic diseases and broad activity against bacteria and fungi [Salem et al ., 2013] . Ficus trees have a number of uses in various industries and fields of human activity. Virtually all parts of their body are utilized in ethnomedicine to cure disorders of digestive and respiratory systems, skin diseases, parasitic infections, etc Some species have been cited to have analgesic, tonic, and ecbolic effects [Lansky and Paavilainen, 2011] .
The pantropical genus Ficus L ., with its approximately 750 species, is the largest within the family Moraceae and one of the most speciose genera of flowering plants . Among all Moraceae, it is characterized by the presence of waxy glands on vegetative organs, heterostyly, and prolonged protogyny, that is the anthesis of staminate flowers in already mature fruits These features are functionally linked to the unique pollination mode in Ficus involving mutualistic relationships with agaonid wasps (order Hymenoptera). The closed urceolate inflorescences provide a shelter for the development of wasps, which, in turn, are the only pollinators of these plants ensuring their reproductive propagation [Cook, Rasplus, 2003; Berg, Corner, 2005] .
Ficus spp . has 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 [Usman et al . , 2009; Ahmad et al , 2011; Dangarembizi et al , 2012; Arunachalam and Parimelazhagan, 2013] 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 Therefore, this study assessed the antibacterial activity of ethanolic extracts of various Ficus species against Citrobacter freun-dii, a fish pathogen isolated locally from gill of eel (Anguilla anguilla L .) with clinical features of disease
Materials and methods Plant Materials. The leaves of F. aspera G . Forst, F. benghalensis L ., F. benjamina L., F. benjamina 'Reginald', F. binnendijkii Miq ., F. binnendijkii 'Amstel Gold', F. binnendijkii 'Amstel King', F. carica L ., F. craterostoma Warb . ex 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. luschnathiana (Miq .) Miq., F. lyrata Warb., F. macrophylla Desf ex Pers , F. mucuso Welw ex Ficalho, F. natalensis Hochst. subsp . natalen-sis, 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. sagit-tata J Koenig ex Vahl, F. septica Burm f , F. sur Forssk ., F. sycomorus L., F. vasta Forssk ., F. vil-losa Blume were collected in M . Gryshko National Botanical Garden (NBG, Kyiv, Ukraine) . The whole collections of tropical and subtropical plants at NBG (including Ficus spp . plants) have the status of a National Heritage Collections of Ukraine The species author abbreviations were followed by Brummitt and Powell (1992)
Preparing 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 . The extracts were then filtered and investigated for their antimicrobial activity. All extracts were stored at 4 °C until use .
Method of Culturing Pathological Sample and identification Method of the Bacteria. Citrobacter freundii isolated locally from gill of eel (Anguilla anguilla L . ) with clinical features of disease . Samples of internal organs (kidney, spleen, liver) were taken and homogenized before preincubation in TSB broth (Tripticase Soya Broth, Oxoid) for 24 hrs . After preincubation, bacterial culture was transferred to two different cultivation mediums: TSA (Tripticase Soya Agar, Oxoid) and BHIA (Brain Heart Infusion Agar, Oxoid) supplemented with 5% of sheep blood (OIE Fish Diseases Commission 2000) . After 48 hrs of incubation at 27 °C, characteristic round, smooth colonies were selected for further examination They were not pigment and did not induce haemolysis on blood agar Bacterial species was identified on the basis of key phenotypic characters and with the use of the oxidase test and API E test kit (Biomerieux, France) . The results of test were interpreted in accordance with the manufacturer's protocol, after 24 hrs of incubation at 37 °C .
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 suspension of microorganisms were suspended in sterile PBS and the turbidity adjusted equivalent to that of a 0 . 5 McFarland standard . Muller-Hinton agar plates were inoculated with 400 ^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) [Bauer et al . , 1966] . The C. freundii isolates were individually tested against 4 antibiotics The results were determined using the disk diffusion method The tested antibiotics were as follows: gentamicin, tet-racycline, enrofloxacin, and sulfonamide A negative control disc impregnated by sterile ethanol was used in each experiment The sensitivity of strain was also studied to the commercial prepara-
tion 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 assessment of antimicrobial activity was based on measurement of the diameter of the inhibition zone formed around the disks
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 bacterium to the phytochemicals tested: Susceptible (S) > 15 mm, Intermediate (I) = 11—14 mm, and Resistant (R) < 10 mm [Okoth et al . , 2013] .
Results
The results of antimicrobial activity of ethano-lic extracts obtained from leaves of various Ficus species are presented in Table 1 and Figs 1—5 .A comparison of susceptibility categories, i e intermediate and resistant, for the disk diffusion technique is demonstrated in Table 1 .
Our results revealed that C. freundii (400 ^l of standardized inoculum) had intermediate susceptibility (diameter of inhibition zone) concerning to ethanolic extracts obtained from leaves of F. binnendijkii 'Amstel Gold' (mean of inhibition zone diameter was 11 mm), F. carica (13 mm), F. erecta (12 mm), F. hispida (14 mm), F. mucu-so (14 mm), F. palmeri (13 mm), F. religiosa (12 mm) . The highest anti-Citrobacter activity was demonstrated for F. pumila (15 mm) . C. freundii was resistant against ethanolic extracts from F. aspera, F. benghalensis, F. benjamina, F. benjam-ina 'Reginald', F. binnendijkii, F. binnendijkii 'Amstel King', F. craterostoma, F. cyathistipula, F. deltoidea, F. drupacea, F. drupacea 'Black Velvet', F. elastica, F. elastica 'Variegata', F. erecta var . sieboldii, F. luschnathiana, F. lyrata, F. mac-rophylla, F. natalensis subsp . natalensis, F. na-talensis subsp . leprieurii, F. platypoda, F. rubiginosa, F. sagittata, F. septica, F. sur, F. sycomorus, F. vasta, F. villosa (inhibition zone diameters were ranged from 6 to 10 mm) (Table 1) .
Antimicrobial activity of ethanolic extracts obtained from leaves of various Ficus species are shown in Figs 1—5 .
Table 1. Susceptibility or resistance of Citrobacter freundii against ethanolic extracts obtained from leaves of various
Ficus species
Ficus species Susceptibility or resistance of Citrobacter freundii Susceptible (S) > 15 mm 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. luschnathiana +
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 +
Fig. 1. Antimicrobial activity of ethanolic extracts obtained from leaves of F. hispida (1), F. villosa (2), F. mucuso (3), F. benghalensis (4), F. carica (6) (A), F. pumila (7), F. macrophylla (8), F. sycomorus (9), F. luschnathiana (10) F. elastica 'Variegata' (11), F. aspera (12) (B) against C. freundii.
Fig. 2. Antimicrobial activity of ethanolic extracts obtained from leaves of F. religiosa (1З), F. cyathistipula (14), F. lyrata (15), F. binnendijkii (18) (A), F. elastica (19), F. benjamina 'Reginald' (20), F. binnendijkii 'Amstel King'
(2З), F. binnendijkii (24) (B) against C. freundii.
Fig. 3. Antimicrobial activity of ethanolic extracts obtained from leaves of F. luschnathiana (26), F. craterostoma (27), F. drupacea 'Black Velvet' (28), F. elastica (29), F. drupacea (30) (A), F. septica (31), F. natalensis subsp. leprieurii (34), F. binnendijkii 'Amstel Gold' (35), F. deltoidea (36) (B) against C. freundii.
Fig. 4. Antimicrobial activity of ethanolic extracts obtained from leaves of F. erecta var. sieboldii (37), F. rubiginosa (40), F. erecta (41), F. sagittata (42) (A), F. lyrata (45), F. vasta (48) (B) against C. freundii.
Fig. 5. Antimicrobial activity of ethanolic extracts obtained from leaves of F. palmeri (50), F. natalensis subsp. natalensis (52), F. platypoda (54), F. sagittata (42) (A), F. pumila (55) (B) against C. freundii.
Fig. 6. Antimicrobial activity of different concentrations of garlic (Allium sativum) extract (A), and tested antibiotics
(B) against C. freundii.
Discussion
Researchers are in continuous search for the exploration of novel sources of more effective compounds for the treatment of infectious diseases in aquaculture [Das et al . , 2008; Manivasagan et al ., 2013]. As previously reported, plants possess antibacterial secondary metabolites which are getting more and more importance due to their negligible toxicity and adverse effects for aquatic organisms and environment [Galina et al . , 2009; Hai, 2015] . The aim of the current study is to take a step towards the achievement of novel natural antimicrobial agents against fish pathogen, Citrobacter freundii. Results of the current study revealed that F. pumila possess high antibacterial results against C. freundii. This significance is in the terms of therapeutic applications against various disorders, i e skin diseases, enlargement of liver and spleen, dysentery, diarrhea, diabetes, leprosy, lung complaints, leucorrhoea, heart diseases, cough, asthma, piles, ulcers, gonorrhea and rheumatism [Kaur, 2012] . Similarly, the antimicrobial activity of ethanolic extracts from leaves of F. binnendijkii 'Amstel Gold', F. carica, F. erecta, F. hispida, F. mucuso, F. palmeri, F. religiosa is also dominant . It was also observed that the F. hispida and F. mucuso were the most active one against C. freundii.
The results of our previous reports are in line with the findings of our current study which indicate that like other species of Ficus genus, F. pumila, F. binnendijkii 'Amstel Gold', F. carica, F. erecta, F. hispida, F. mucuso, F. palmeri, F. religiosa are also prominent candidates of this genus as alternative antimicrobial agents in aquaculture . In our previous study, we determined the in vitro antimicrobial activity of ethanolic extracts from the leaves of various Ficus species against the bacterial strain of A. hydrophila isolated locally from infected rainbow trout (Oncorhynchus my-kiss Walbaum) with the aim of providing scientific rationale for using these plants in the treatment of bacterial infections induced by Aeromonas spp in fish (Tkachenko et al . 2016a, b) . Interestingly, ethanolic extracts obtained from Ficus species demonstrated different antibacterial activity against A. hydrophila inoculated with 200 ^l and 400 ^l of standardized inoculum (108 CFU ml-1) of bacterium strain . Previous study using 200 ^l of A. hydrophila inoculum revealed that F. ben-
ghalensis, F. benjamina, F. binnendijkii, F. cy-athistipula, F. deltoidea, F. erecta, F. erecta var . sieboldii, F. hispida, F. luschnathiana, F. lyrata, F. macrophylla, F. mucuso, F. natalensis subsp . leprieurii, F. natalensis subsp . natalensis, F. palmeri, F. platypoda, F. pumila, F. rubiginosa, F. sur, F. sycomorus, and F. villosa possessed good antibacterial activity (diameters of inhibition zones ranged from 10 to 14 mm), while A. hydrophila was resistant against ethanolic exstracts from F. aspera, F. benjamina 'Reginald', F. binnendijkii 'Amstel Gold', F. binnendijkii 'Amstel King', F. carica, F. craterostoma, F. drupacea, F. drupacea 'Black Velvet', F. elastica, F. elastica 'Variegata', F. religiosa, F. sagittata, F. septica, and F. vasta [Tkachenko et al . , 2016a] . On the other hand, in study with 400 ^l of A. hydrophi-la inoculum, bacterium demonstrated the highest susceptibility to F. pumila leaves extract (diameters of inhibition zones ranged from 16 to 18 mm), while intermediate susceptibility was demonstrated to ethanolic extracts obtained from F. bengha-lensis, F. benjamina, F. benjamina 'Reginald', F. binnendijkii, F. carica, F. cyathistipula, F. deltoidea, F. drupacea, F. drupacea 'Black Velvet', F. elastica, F. erecta, F. erecta var . sieboldii, F. hispida, F. lyrata, F. macrophylla, F. natalensis subsp . leprieurii, F. natalensis subsp . na-talensis, F. palmeri, F. religiosa, F. rubiginosa, F. septica, and F. sycomorus leaves [Tkachenko et al . , 2016b] . In other our study, most ethanolic extracts obtained from Ficus spp . , proved effective against the bacterial strain of Gram-negative P. fluorescens (strain E1/7/15) isolated locally from internal organs of rainbow trout (Oncorhynchus mykiss Walbaum) with clinical features of furunculosis, with 8 — 15 mm zones of inhibition being observed Among various species of Ficus the most effective against P. fluorescens (400 ^l of standardized inoculum) were the ethanolic extracts of the leaves of ten Ficus species: F. craterostoma, F. cyathistipula, F. drupacea 'Black Velvet', F. hispida, F. macrophylla, F. mucuso, F. pumila, F. villosa (Tkachenko et al . 2016c) . Various species of Ficus had mild antibacterial in vitro activity against C. freundii isolated locally from infected eel (Anguilla anguilla L . ) in dissolved to 200 ^l of standardized inoculum (Kir-by-Bauer disk diffusion susceptibility test protocol) Our results proved that the extracts from
F. drupacea, F. septica, F. deltoidea as well as F. hispida, F. mucuso, F. pumila, F. craterosto-ma exhibit a favorable antibacterial activity against C. freundii [Tkachenko et al ., 2016d] .
The results obtained in the current study can be correlated with the previous publications . Other species of Ficus genus also possess effective potential against microbes i . e . , F. religiosa have been reported to possess strong antibacterial and antifungal activities [Preethi et al . , 2010; Gayath-ri, Kannabiran, 2009; Solomon-Wisdom et al . , 2011; Rajiv, Rajeshwari, 2012; Al Askari et al . , 2013; Essien et al ., 2016] . In a study of Preethi and co-workers (2010), aqueous extract of F. religiosa leaves compared to the ethanol and methanol extracts showed the strongest inhibitory activity against bacteria (Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhi) as evaluated by the agar diffusion method . Rajiv and Rajeshwari (2012) screened antimicrobial activity of F. religiosa bark, leaf, stem, and fruit aqueous extracts against a number of major pathogens (Aeromonas hydrophila, Enterobacter aerogenes, E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes, Aspergillus niger, and Candida albicans) and conducted their phytochemical analysis All tested extracts appeared active against the pathogens at concentrations 25 — 100 mg/ml, the widest inhibition zone (15 — 16 mm) resulting from the highest concentration . Fruit extract showed generally the weakest activity and only the leaf extract affected the whole set of tested organisms at maximal concentration Antibacterial properties of the extracts were generally better pronounced than untifungal ones [Rajiv, Rajeshwari, 2012] . Gayathri and Kannabiran (2009) reported quite high antibacterial activity of F. benghalensis bark aqueous extract against Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. Phytochemical screening of the extracts showed tannins and saponins to significantly prevail over other chemical classes, suggesting their substantial contribution to the antimicrobial properties of the species [Gayathri, Kannabiran, 2009] . Solomon-Wisdom and co-workers (2011) tested leaf and stem bark methanolic extracts from F. sur against several bacterial and fungal pathogens (Bacillus subtilis, E. coli, P. aeruginosa, Salmonella typhimorium, S. aureus, and
Candida pseudotropicalis) and carried out their phytochemical screening . Leaf and bark extracts showed inhibition of all tested organisms except P. aeruginosa and 5. typhimorium. Phytochemical analysis showed leaf extract to have richer content compared to stem bark extract . Saponins, steroids, and tannins were present in both extracts . Leaf extract additionally contained volatile oils and phenols, while stem bark extract was distinct in containing saponin glycosides [Solomon-Wisdom et al . , 2011] .
In the same way, essential oils from leaves of Ficus species have been reported to possess antimicrobial potential . Essien and co-workers (2016) obtained essential oils from leaves of F. mucuso (misspelled as "Ficus mucoso") and Casuarina equisetifolia and screened them for chemical content as well as cytotoxic (against human cancer cells) and antimicrobial activities . Microorganisms tested included Gram-positive (Bacillus ce-reus ATCC14579 and 5. aureus ATCC29213) and Gram-negative (E. coli ATCC254922 and P. aeruginosa ATCC27853) bacteria and fungi (Aspergillus niger ATCC16401 and Candida albicans ATCC10231). In F. mucuso, identified were 35 constituents representing 100 % of the leaf essential oil content The leaf oil was found to be rich in terpenoids (89 6% of its content) Gram-positive bacteria appeared more sensitive to the treatments than Gram-negative ones (Essien et al . 2016) . Kubmarawa and co-workers (2007) carried out an antimicrobial and phytochemical screening of 50 Nigerian plant species ethanolic extracts, among which were five species of Ficus (i . e ., F. abutifolia (Miq .) Miq ., F. platyphyl-la Del ., F. polita Vahl, F. sycomorus L ., and F. thonningii Blume) . Microbial strains used in the study were Bacillus subtilis NCTC8236, E. coli ATCC9637, P. aeruginosa ATCC27853, 5. aureus ATCC13709, and C. albicans ATCC10231. Ficus stem bark extracts demonstrated comparatively low antimicrobial activity, with the broadest activity spectrum being of F. thonningii extract (active against all microorganisms except P. aeruginosa and 5. aureus). Extracts from F. polita and F. sycomorus showed no activity at all P. aeruginosa was in general moderately susceptible compared to other bacteria tested, although no Ficus extract was active against it Phytochemical analysis revealed the presence
of only saponins and volatile oil in F. thonningii extract and saponins and flavonoids in F. polita extract, while richer chemical content was found in F. abutifolia (tannins, alkaloids, and volatile oil), F. platyphylla (saponins, flavonoids, alkaloids, and volatile oil), and F. sycomorus (gly-cosides, tannins, flavonoids, and volatile oil) extracts (Kubmarawa et al . 2007) . Al Askari and co-workers (2013) screened aqueous and ethan-olic leaf extracts of F. carica from different regions of Morocco against 16 bacterial strains (Acine-tobacter baumannii, Escherichia coli, Hafnia alveie, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, P. fluorescens, Salmonella arizonae, S. entericatyphimurium, S. enteritidis, Staphylococcus aureus, S. aureus meticillin-resistant, S. epidermidis, Streptococcus pyogenes, S. sanguins, and Yersinia enterocoliti-ca) and 8 yeasts (Candida famata, C. glabrata, C. parapsilosis, C. tropicalis, two strains for each species). In general, aqueous extract was found more active against Gram-positive bacteria than Gram-negative ones and it was not active against yeasts . Ethanolic extract demonstrated stronger inhibitory activity compared to aqueous extract and inhibited growth of both bacteria and fungi [Al Askari et al . , 2013] .
Several classes of secondary metabolites such as alkaloids, triterpenes, sterols, flavonoids, polyphenols and saponins have been reported to have antibacterial properties [Cowan, 1999; Kuete et al . , 2008; Mbaveng et al . , 2015; Voukeng et al . , 2016] . Their presence in the studied plant extracts could explain the antibacterial effects of the tested samples Antibacterial activity of tannins and saponins isolated from plant species are well documented [Gayathri, Kannabiran, 2009; Us-man et al , 2009] The presence of flavonoids and polyphenols is the basis for the analgesic and anti-inflammatory activities of various parts of Ficus including the fruit, latex, bark, roots, and leaves [Modi et al . , 2012; Eteraf-Oskouei et al . , 2015] . The antimicrobial activity of purified flavonoids may result in susceptibility differences against species with different origins and background [Taguri et al , 2004] This could explain the difference in sensitivity to Ficus extracts used in this work and a previously tested other species of Ficus [Tkachenko et al . , 2016a-d] . The highest antibacterial potential of Ficus could be explained by the
amount of flavonoids present . However, the activity showed by ethanolic extracts of Ficus species may result from the interactions of different polyphenols . Most studies on the antimicrobial potential of polyphenols have focused on the inhibitory activity of individual components . The inhibitory effect of phenolics could be explained by absorption across cell membranes, interactions with enzymes, substrate and metal ion deprivation [Scal-bert, 1991] . Direct interaction between the two compounds may result in changes of the structural conformation thus reducing the inhibitory activity [Mandalari et al . , 2010] . Antibacterial flavonoids might be having multiple cellular targets, rather than one specific site of action [Kumar, Pandey, 2013] . 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 [Cowan, 1999; Kumar, Pandey, 2013] . Moreover, many flavonoids are shown to have antioxidative activity, free radical scavenging capacity, coronary heart disease prevention, hepatoprotective, anti-inflammatory, and anticancer activities, while some flavonoids exhibit potential antiviral activities [Kumar, Pandey, 2013] .
Conclusions The study reveals that among the plants of Ficus species screened, F. pumila, F. binnendijkii 'Amstel Gold', F. carica, F. erecta, F. hispida, F. mucuso, F. palmeri, F. religiosa were rich in antimicrobial agents Secondly it may also be implied that the ethanolic fraction was rich in such secondary metabolites which confer antimicrobial potential to these plants . Based on our current investigation it can be concluded that F. pumila, F. binnendijkii 'Amstel Gold', F. carica, F. erecta, F. hispida, F. mucuso, F. palmeri, F. religiosa possess considerably antibacterial potential against C. freundii. It may also be concluded that antimicrobial potential of ethanolic extracts obtained from leaves of various Ficus species might be due to wide variety of compounds present in these plants Moreover, we observed that specifically the ethanolic fraction of F. pumila possesses consider-
ably high activity against C. freundii. Therefore, these species are potent targets to be subjected to bio-guided isolation and exploration of novel natural antimicrobials for application in aquaculture .
Referenses
Ahmad S., Rao H, Akhtar M, Ahmad I., Hayat M.M., Iqbal Z., Nisar-ur-Rahman 2011. Phytochemical composition and pharmacological prospectus of Ficus bengalensis Linn . (Moraceae) // J . Med . Plants Res . ,
5: 6393-6400. Al Askari G., Kahouadji A., Khedid K., Ouaffak L., Mousaddak M, Charof R, Mennane Z. 2013 . In vitro antimicrobial activity of aqueous and ethanolic extracts of leaves of Ficus carica collected from five different regions of Morocco // J .of Materials and Environmental Science, 4(1): 33-38. Arunachalam K., Parimelazhagan T. 2013. Antiinflammatory, wound healing and in-vivo antioxidant properties of the leaves of Ficus amplissima Smith . // J . Eth-nopharmacol , 145(1): 139-145 Bauer A.W., Kirby W.M, Sherris J.C., Turck M. 1966. Antibiotic susceptibility testing by a standardized single
disk method // Am . J . Clin . Pathol . , 45(4): 493-496. Berg C.C., Corner E.J.H. 2005. Moraceae (Ficus). Flora Malesiana / Noteboom H.P. (ed . ) . Ser. 1, Vol . 17, Part 2 . National Herbarium Nederland, Leiden, pp 1-730
Brummit R.K., Powell C.E.P. (Eds.) 1992. Authors
of plant names A list of authors of scientific names of plants, with recommended standard forms of their names, including abbreviations . Kew, 732 p . Carbone D., Faggio C. Importance of prebiotics in aquaculture as immunostimulants Effects on immune system of Sparus aurata and Dicentrarchus labrax // Fish Shellfish Immunol . , 54: 172-178. Cook J.M., Rasplus J.-Y. 2003. Mutualists with attitude: coevolving fig wasps and figs // Trends in Ecology &
Evolution 18(5): 241-248. Cowan M.M. 1999 Plant products as antimicrobial agents
Clin Microbiol Rev 12(4): 564-582 Dangarembizi R., Erlwanger K.H., Moyo D., Chivandi E. 2012 Phytochemistry, pharmacology and ethnome-dicinal uses of Ficus thonningii (Blume Moraceae): a review // Afr J Tradit Complement Altern Med ,
10(2): 203-212 Das S., Ward L.R., Burke C. 2008 . Prospects of using marine actinobacteria as probiotics in aquaculture
// Appl . Microbiol. Biotechnol. , 81(3): 419-429. Devasagayam T.P.A., Sainis K.B. 2002 . Immune system and antioxidants, especially those derived from Indian medicinal plants // Indian J . Exp . Biol . , 40: 639-655. Essien E. E., Newby J.M., Walker T.M., Ogunwan-de I.A., Setzer W.N., Ekundayo O. 2016 Essential oil
constituents, anticancer and antimicrobial activity of Ficus mucosa and Casuarina equisetifolia leaves // Am . J . of Essential Oils and Natural Products, 4(1): 1—6 . Eteraf-Oskouei T., Allahyari S., Akbarzadeh-Atashkhos-row A, Delazar A., Pashaii M, Can S.H., Najafi M. 2015 . Methanolic Extract of Ficus carica Linn . Leaves Exerts Antiangiogenesis Effects Based on the Rat Air Pouch Model of Inflammation // Evid . Based Complement. Alternat. Med . , 2015: 760405. Calina J., Yin C., Ardo L., Jeney Z. 2009. The use of im-munostimulating herbs in fish . An overview of research
// Fish Physiol. Biochem, 35(4): 669-676. Cayathri M., Kannabiran K. 2009. Antimicrobial activity of Hemidesmus indicus, Ficus bengalensis and Ptero-carpus marsupium roxb . // Indian J . of Pharmaceutical
Sci. , 71(5): 578-581 . Hai N.V. 2015 . The use of medicinal plants as immuno-stimulants in aquaculture: A review // Aquaculture,
446: 88-96
Kaur J. 2012 Pharmacognostical and Preliminary Phy-tochemical Studies on the Leaf Extract of Ficus pumi-la Linn // J of Pharmacognosy and Phytochemistry,
1(4): 105-111 .
Kuete V., Ngameni B., Simo C.C., Tankeu R.K., Ngad-jui B.T, Meyer J.J, Lall N, Kuiate J.R. 2008. Antimicrobial activity of the crude extracts and compounds from Ficus chlamydocarpa and Ficus cordata (Mora-ceae) // J Ethnopharmacol , 120(1): 17-24 Kumar S., Pandey A.K. 2013 . Chemistry and biological activities of flavonoids: an overview // Scientific World Journal, 2013: 162750 . http://dx . doi . org/10 1155/2013/162750 Lansky E.P., Paavilainen H.M. 2011 . Figs: the genus Ficus // Traditional herbal medicines for modern times / Hardman R . (ed .) . Vol . 9. CRC Press, Boca Raton, pp 1-357
Malheiros D.F., Maciel P.O., Videira M.N., Tavares-Di-asa M. 2016 Toxicity of the essential oil of Mentha piperita in Arapaima gigas (pirarucu) and antiparasitic effects on Dawestrema spp . (Monogenea) // Aquaculture 455: 81-86. Mandalari C., Bisignano C., DArrigo M., Cinestra C., Arena A., Tomaino A., Wickham M.S. 2010 . Antimicrobial potential of polyphenols extracted from almond
skins // Lett. Appl. Microbiol. , 51(1): 83-89. Manivasagan P., Venkatesan J., Sivakumar K., Kim S.K. 2013 Marine actinobacterial metabolites: current status and future perspectives // Microbiol Res , 168(6):
311-332 .
Mbaveng T.A., Sandjo L.P., Tankeo S.B., Ndifor A.R., Pantaleon A., Nagdjui T.B., Kuete V. 2015. Antibacterial activity of nineteen selected natural products against multi-drug resistant Gram-negative phenotypes // Springer Plus 4: 823
Modi R.K., Kawadkar M., Sheikh S., Kastwar R., Ti-wari G. 2012. A review on: comparative studies on ethanolic extract of root and stem bark of Ficus carica for analgesic and antiinflammatory activities // International Journal of Pharmacy and Life Sciences, 3(8):
1930-1934.
Okoth D.A., Chenia H.Y., Koorbanally N.A. 2013 . Antibacterial and antioxidant activities of flavonoids from Lannea alata (Engl . ) Engl . (Anacardiaceae) // Phy-tochem. Lett. , 6: 476-481 .
Preethi R., Devanathan V.V., Loganathan M. 2010 . Antimicrobial and antioxidant efficacy of some medicinal plants against food borne pathogens // Advances in
Biol . Res . 4(2): 122-125.
Rajiv P., Rajeshwari S. 2012 . Screening for phytochemi-cals and antimicrobial activity of aqueous extract of Ficus religiosa Linn . // Int . J . of Pharmacy and Pharmaceutical Sci 4: 207-209
Salem M. Z.M., Salem A . Z. M. , Camacho L. M. , Ali H M 2013 Antimicrobial activities and phyto-chemical composition of extracts of Ficus species: An overview // Afr. J . Microbiol . Res . , 7(33): 42074219
Scalbert A. 1991 Antimicrobial properties of tannins // Phytochemistry, 12: 3875 -3883.
Solomon-Wisdom G.O., Shittu G.A., Agboola Y. A. 2011 Antimicrobial and phytochemical screening activities of Ficus sur (Forssk) // New York Science Journal, 4(1): 15-18
Taguri T., Tanaka T., Kouno I. 2004. Antimicrobial activity of 10 different plant polyphenols against bacteria causing food-borne disease // Biol . Pharm . Bull . ,
27(12): 1965-1969
Tkachenko H., Buyun L., Terech-Majewska E., Osad-owski Z., Sosnovskyi Y., Honcharenko V., Prokopiv A. 2016 a The antimicrobial activity of some ethanolic extracts obtained from Ficus spp. leaves against Aer-
omonas hydrophila // Труды ВНИРО, 162: 172— 183 .
Tkachenko H., Buyun L., Terech-Majewska E., Osad-owski Z. 2016 b . In vitro antimicrobial activity of ethanolic extracts obtained from Ficus spp . leaves against the fish pathogen Aeromonas hydrophila // Arch . Pol .
Fish. , 24: 219-230.
Tkachenko H., Buyun L., Terech-Majewska E., Osad-owski Z., Sosnovskyi Y., Honcharenko V., Prokopiv A. 2016 c . In vitro antibacterial efficacy of various eth-anolic extracts obtained from Ficus spp leaves against fish pathogen, Pseudomonas fluorescens // Globalisation and regional environment protection. Technique, technology, ecology / ed . Tadeusz Noch, Wioleta Mikolajczewska, Alicja Wesolowska. Gdansk, Wy-dawnictwo Gdanskiej Szkoly Wyzszej . P. 265-286 .
Tkachenko H., Buyun L., Terech-Majewska E., Osad-owski Z. 2016 d Antibacterial activity of ethanolic leaf extracts obtained from various Ficus species (Moraceae) against the fish pathogen, Citrobacter freundii. Baltic Coastal Zone // J .of Ecology and Protection of the Coastline, 20: 117-136
Usman H., Abdulrahman F., Usman A. 2009. Qualitative phytochemical screening and in vitro antimicrobial effects of methanol stem bark extract of Ficus thonnin-gii (Moraceae) // Afr. J . Tradit . Complement . Altern .
Med . , 6(3): 289-295.
Valladao G.M, Gallani S.U., Pilarski F. 2015 . Phyto-therapy as an alternative for treating fish disease //
J . Vet. Pharmacol . Ther. , 38(5): 417-428.
Voukeng I.K., Beng V.P., Kuete V. 2016 . Antibacterial activity of six medicinal Cameroonian plants against Gram-positive and Gram-negative multidrug resistant phenotypes // BMC Complement Altern Med , 16(1): 388
Поступила в редакцию 15.03.2017 г.
Принята после рецензии 10.05.2017 г.
Антимикробный скрининг спиртовых экстрактов, полученных из листьев разных видов Ficus в отношении Citrobacter freundii
Г.М. Ткаченко1, Л.И. Буюн2, Э. Терех-Маевская3, З. Осадовский1
1 Институт биологии и охраны среды, Поморская Академия, Слупск, Польша
2 Национальный ботанический сад им . Н .Н . Гришко НАНУ, Киев, Украина
3 Варминско-Мазурский Университет, Ольштын, Польша
Лекарственные растения играют альтернативную роль в антибиотикотерапии в аквакультуре Виды Ficus (Moraceae) обладают большим лечебным потенциалом в терапии бактериальных и грибковых инфекций, и могут быть использованы в качестве антисептиков и антимикробных агентов в ветеринарии Настоящее исследование предназначено для изучения in vitro антимикробной активности этанольных листовых экстрактов различных видов Ficus против возбудителя Citrobacter freundii Листья были собраны в Национальном ботаническом саду им . Н. Н. Гришко (Киев, Украина) . Свежесрезанные листья промывали, взвешивали и гомогенизировали в 96% этаноле (в пропорции 1:10) при комнатной температуре . Citrobacter freundii выделяли локально из жабр угря (Anguilla anguilla) с клиническими признаками заболевания . Испытание на антимикробную чувствительность проводили методом Кирби-Бауэра. В чашки с агаром Muller-Hinton инокулировали 400 мкл стандартизированного инокулята (108 КОЕ/мл) бактерии и распределяли равномерно стерильными тампонами Наши результаты показывают, что различные виды фикусов имели слабую антибактериальную активность in vitro против C. freundii Результаты также показали, что этанольные экстракты, полученные от F. pumila, F. binnendijkii 'Amstel Gold', F. carica, F. erecta, F. hispida, F.mucuso, F. palmeri, F. religiosa обладают значительным антибактериальным потенциалом против C. freundii
Ключевые слова: Ficus spp , листья, спиртовые экстракты, антибактериальная активность, зона ингибирования роста, Citrobacter freundii, угорь Anguilla anguilla.
