CC BY
42
UDK 639.3:615.32:615.38
12 3 1
Halyna Tkachenko , Lyudmyla Buyun , Elzbieta Terech-Majewska , Zbigniew Osadowski
institute of Biology and Environmental Protection, Pomeranian University in Slupsk,
Arciszewski Str. 22b, 76-200 Slupsk, Poland, e-mail: tkachenko@apsl.edu.pl 2M.M. Gryshko National Botanical Garden, National Academy of Sciences of Ukraine,
Timiryazevska Str. 1, Kyiv, Ukraine, e-mail: buyun@nbg.kiev.ua 3Department of Epizootiology, University of Warmia and Mazury in Olsztyn, Poland
SCREENING FOR ANTIMICROBIAL ACTIVITIES OF THE ETHANOLIC EXTRACT DERIVED FROM FICUS HISPIDA L.F. LEAVES (MORACEAE) AGAINST
FISH PATHOGENS
Use of natural products has been considered as an alternative to antibiotics in fish health management to control bacterial infections in aquaculture. Many plants were shown to have potential for being effective herbal drugs against the fish and other aquaculture pathogens. Therefore, the aim of this study was to test the efficacy of ethanolic extract prepared from Ficus hispida leaves against fish pathogens, Aeromonas hy-drophila, Citrobacter freundii, Pseudomonas fluorescens to evaluate the possible use of this plant in preventing infections caused by these bacteria in aquaculture. 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 200 and 400 ¡L of standardized inoculum (108 CFU/mL) of bacterium and spread with sterile swabs. Aeromonas hydrophila (strain E 2/7/15) isolated locally from gill of rainbow trout (Oncorhynchus mykiss Walbaum) and Pseudomonas fluorescens (strain E 1/7/15) isolated locally from internal organs of rainbow trout with clinical features of furunculosis (kidneys were gray, liver was pale and fragile, enlarged spleen with exudate in the body cavity), as well as Citrobacter freundii isolated locally from gill of eel (Anguilla anguilla L.) with clinical features of disease were used as test organisms. Our results from the disc diffusion assay indicated that the A. hydrophila revealed intermediate susceptibility concerning to ethanolic extract obtained from leaves of F. hispida (inhibition zone diameters were ranged from 8 to 12 mm). The most effective at least causing a zone of inhibition 14-16 mm was ethanolic extract from F. hispida against P. fluorescens both in 200 ¡L of standardized inoculum (108 CFU/mL) of bacterium (inhibition zone diameters were ranged from 15 to 16 mm) and 400 ¡L (14-15 mm). Our results demonstrated that the C. freundii revealed intermediate susceptibility F. hispida (inhibition zone diameters were ranged between 11 and 15 mm). Thus, the preliminary screening assay indicated that F. hispida leaves extract possess great potential for the therapy of bacterial infections and may be used as a natural antiseptic and antimicrobial agent. Further investigation needs to be focused on isolation and identification of those bioactive compounds, which would be a platform for further pharmacological studies, in vivo tests and practical applications in fish health management.
Key words: Ficus hispida L.f., antimicrobial activity, Aeromonas hydrophila, Citrobacter freundii, Pseudomonas fluorescens, Kirby-Bauer disk diffusion susceptibility test.
Г.М. Ткаченко, Л.И. Буюн, Э. Терех-Маевская, 3. Осадовский
СКРИНИНГ АНТИМИКРОБНОЙ АКТИВНОСТИ ЭТАНОЛЬНОГО ЭКСТРАКТА, ВЫДЕЛЕННОГО ИЗ ЛИСТЬЕВ FICUS HISPIDA L.F. (MORACEAE) ОТНОСИТЕЛЬНО ПАТОГЕНОВ РЫБ
Использование продуктов природного происхождения рассматривается как альтернатива антибиотикам для борьбы с бактериальными инфекциями в аквакультуре. Было показано, что многие растения обладают терапевтическим потенциалом для эффективного их применения относительно микробных возбудителей. Поэтому целью этого исследования было проверить антимикробную эффективность этанольного экстракта, полученного из листьев Ficus hispida L.f., относительно таких патогенов рыб, как Aeromonas hydrophila, Citrobacter freundii, Pseudomonas fluorescens для оценки
возможного использования этого растения для предотвращения инфекций, вызванных этими бактериями в аквакулътуре. Исследование антимикробной чувствительности определяли с помощью диско-диффузионного метода Байера-Кирби. В чашки с агаром Muller-Hinton инокулировали 200 и 400 мкл стандартизированного инокулята (108 КОЕ/мл) бактерии и распределяли его стерильными тампонами. Aeromonas hydrophila (штамм E 2/7/15,) изолированный локально с жабр радужной форели (Oncorhynchus mykiss Walbaum), и Pseudomonas fluorescens (штамм E 1/7/15), выделенный из внутренних органов радужной форели с клиническими признаками фурункулеза, а также Citrobacter freundii, выделенный из жабр угря (Anguilla anguilla L.) с клиническими признаками заболевания, использовали в качестве тестовых микроорганизмов. Наши результаты показали, что A. hydrophila выявил посредственную восприимчивость относительно этанольного экстракта, полученного из листьев F. hispida (диаметры зон ингибирования варьировались от 8 до 12 мм). Наиболее эффективным, по крайней мере, вызывающим зону ингибирования роста 14-16 мм, оказался экстракт F. hispida относительно P. fluorescens как при нанесении 200 мкл стандартизированного посевного материала (диаметр зон варьировался от 15 до 16 мм ), так и 400 мкл (14-15 мм). Наши результаты также показали, что C. freundii показал посредственную восприимчивость к экстракту F. hispida (диаметры зон варьировались от 11 до 15 мм). Таким образом, предварительный скрининг-анализ выявил, что экстракт листьев F. hispida обладает антибактериальным потенциалом в терапии бактериальных инфекций in vitro и может использоваться в качестве природного антисептика и антимикробного агента. Дальнейшие наши исследования будут сосредоточены на выделении и идентификации биологически активных соединений, которые станут платформой для дальнейших фармакологических исследований in vivo и практических применений в аквакулътуре лососевых рыб.
Ключевые слова: Ficus hispida L.f., антимикробная активность, Aeromonas hydrophila, Citrobacter freundii, Pseudomonas fluorescens, диско-диффузионный метод Байера-Кирби.
Introduction
Fish farming is now a sufficiently large and a mature industry, contributing 17 percent of the global animal-based protein supply in 2010 [20], that have justified the development of an effective range of antimicrobial agents [1]. Currently, bacterial infections in aquaculture are mainly controlled by antibiotics. However, recently, the use of antibiotics in aquaculture has received considerable attention because their use can lead to the development of drug resistant bacteria, thereby reducing drug efficacy. Moreover, the accumulation of antibiotics both in the environment and in fish can be potentially risky to consumers and the environment [11]. Consequently, there is an urgent need for development of alternative therapies against bacterial pathogens in aquaculture.
Use of natural products has been considered as an alternative to antibiotics in fish health management to control bacterial infections in aquaculture. Many plants were shown to have potential for being effective herbal drugs against the fish and other aquaculture pathogens [12]. Additionally, it is an attractive method for increasing the protective capabilities of fish [11]. Ficus (Moraceae) species are reported to have antimicrobial activity against several pathogenic bacteria and have been used as traditional medicines for the treatment of human diseases [2, 6, 13, 14]. Moreover, in line with the growing interest in the antibacterial properties of different plants, in our previous researches, we have used ethanolic extracts derived from leaves of various Ficus species to assess antibacterial activity against harmful fish pathogens, Aeromonas hydrophila, Citrobacter freundii, Pseudomonas fluorescens [15-18].
Ficus L. is one of the largest genera of angiosperms, with about 750 species of terrestrial trees, shrubs, hemi-epiphytes, climbers and creepers oCcurring in the tropics and subtropics of the world [4]. Ficus hispida L.f., commonly known as the hairy fig or the rough-leafed stem fig, is a shrub or tree that can grow up to 15 m tall [10]. It is a dioecious, bat-dispersed species [7], distributed from Sri Lanka to India, and from South China across Southeast Asia to Australia [4]. This species is used for treatment of several disorder, e.g., ulcers, psoriasis, anemia, piles jaundice, vitiligo, hemorrhage, diabetes, convulsion, hepatitis, dysentery, biliousness, and as lactagogue and purgative agents [2]. In India, it is commonly cultivated for its pharmacological properties such as antidiar-rheal activity, as well as neuroprotective and hepatoprotective effects. It was reported that almost all
parts of this plant are used as a folklore remedy for the treatment of various ailments by the Indian traditional healers, but the leaves are of particular interest, among other parts, from a medicinal point of view. Additionally, the fruit is known to be active as aphrodisiac, tonic, lactagogue and emetic [2]. F. hispida was chosen for its abundance of alkaloids, carbohydrates, proteins and amino acids, sterols, phenols, flavonoids, gums and mucilage, glycosides, saponins, and terpenes [6]. Therefore, the aim of this study was to test the efficacy of ethanolic extract prepared from F. hispida leaves against fish pathogens, Aeromonas hydrophila, Citrobacter freundii, Pseudomonas fluorescens to evaluate the possible use of this plant in preventing infections caused by these bacteria in aquaculture.
Materials and methods
Collection of Plant Material. The leaves of F. hispida were sampled in M.M. Gryshko National Botanical Garden (Kyiv, Ukraine). The whole collection of tropical and subtropical plants at M.M. Gryshko National Botanical Garden (Kyiv, Ukraine) (including Ficus spp. plants) has the status of a National Heritage Collection of Ukraine. 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, and centrifuged at 3,000 g for 5 minutes. Su-pernatants were stored at -20 °C in bottles protected with laminated paper until required.
Method of Culturing Pathological Sample. Aeromonas hydrophila (strain E 2/7/15) isolated locally from gill of rainbow trout (Oncorhynchus mykiss Walbaum) and Pseudomonas fluorescens (strain E 1/7/15) isolated locally from internal organs of rainbow trout with clinical features of furunculosis (kidneys were gray, liver was pale and fragile, enlarged spleen with exudate in the body cavity), as well as Citrobacter freundii isolated locally from gill of eel (Anguilla anguilla L.) with clinical features of disease were used as test organisms. 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. 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). After 48 hrs of incubation at 27°C, characteristic pink colonies were selected for further examination. Bacterial species were identified with the use of 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. hydro-phila. The strain was obtained from Diagnostics Laboratory of Fish and Crayfish Diseases, Department of Veterinary Hygiene, Provincial Veterinary Inspectorate in Olsztyn (Poland).
Bacterial Growth Inhibition Test of Plant Extracts by the Disk Diffusion Method. Strains tested were plated on TSA medium (Tryptone Soya Agar) and incubated for 24 hrs at 25 °C. Then the suspension of microorganisms was suspended in sterile PBS and the turbidity adjusted equivalent to that of a 0.5 McFarland standard. The disc diffusion assay (Kirby-Bauer Method) was used to screen for antibacterial activity [3]. Muller-Hinton agar plates were inoculated with 200 and 400 |iL 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). 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 Mueller-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 and the average values of antimicrobial activity were calculated. The following zone diameter criteria were used to assign susceptibility or resistance of bacte-
ria to the phyt oChemicals tested: Susceptible (S) > 15 mm, Intermediate (I) = 11-14 mm, and Resistant (R) < 10 mm.
Results and discussion
Our results from the disc diffusion assay indicated that the A. hydrophila (200 and 400 ^l of standardized inoculum) revealed intermediate susceptibility concerning to ethanolic extract obtained from leaves of F. hispida (inhibition zone diameters were ranged from 8 to 12 mm) (fig. 1).
Fig. 1. Antimicrobial activity of ethanolic extracts obtained from F. hispida (1) against Aeromonas hydrophila. Muller-Hinton agar plates inoculated with 200 (A) and 400 |iL (B) of standardized inoculum
(108 CFU/mL) of bacterium (B)
The most effective at least causing a zone of inhibition 14-16 mm was ethanolic extract from F. hispida against Pseudomonas fluorescens both in 200 ^L of standardized inoculum (10 CFU/mL) of bacterium (inhibition zone diameters were ranged from 15 to 16 mm) and 400 ^L (1415 mm) (fig. 2).
Fig. 2. Antimicrobial activity of ethanolic extracts obtained from F. hispida (1) against Pseudomonas fluorescens. Muller-Hinton agar plates inoculated with 200 (A) and 400 |iL (B) of standardized inoculum
(108 CFU/mL) of bacterium (B)
Our results demonstrated that the C. freundii (200 and 400 ^l of standardized inoculum) revealed intermediate susceptibility F. hispida (inhibition zone diameters were ranged between 11 and 15 mm) (fig. 3).
Fig. 3. Antimicrobial activity of ethanolic extracts obtained from F. hispida (1) against Citrobacter freundii. Muller-Hinton agar plates inoculated with 200 (A) and 400 ^L (B) of standardized inoculum (108 CFU/mL)
of bacterium (B)
Chatterjee and co-workers (2015) screened leaf methanol extract from F. hispida for chemical content, antioxidant and antibacterial activity. Bacteria tested included 5 strains of Gram-positive Salmonella typhi (NCTC-74, B-111, C-145, E-3404, and A-2467) and 5 strains of Gram-negative Staphylococcus aureus (ML-357, ML-15, ML-366, ML-276, and ML-145.). Results showed S. aureus strains to have generally lower susceptibility to the extracts compared to S. typhi. Among the former, S. aureus ML-145 was not affected by any concentration tested (1-25 mg/ml), whereas strains ML-357, ML-366, and ML-276 were inhibited with MIC 25 mg/ml, and strain ML-15 was inhibited with MIC 10 mg/ml. Phytochemical analysis of the extract showed the presence of fla-vonoids, tannins, steroids, glycosides, and saponins, as well as absence of alkaloids and amino acids. Total phenolic content of the extract was almost twice as high as total flavonoid content [5].
The broad antibacterial activities of this extract, apparently, could be explained as a result of the plant secondary metabolites. Previously it has been reported [13, 14], that the therapeutic properties of Ficus species may be attributed to the presence of a wide range of phytochemical compounds. In general, Ficus species were reported to have the rich array 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 [13]. 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, glycosides, saponins, and terpenes [2]. Two substantial phenanthroin-dolizidine alkaloids, 6-O-methyltylophorinidine and 2-demethoxy-tylophorine, and a novel bi-phenylhexahydroindolizine hispidine from stem and leaves of F. hispida were isolated by Venkatachalam and Mulchandani (1982) [19]. Recently, hispidin has been reported to have anticancer activity [2]. All the detected phenolic acids are known to have antimicrobial and antioxidant properties [8]. Consequently, the antimicrobial property of F. hispida extract may be due to its constituents. Antibacterial flavonoids might be having multiple cellular targets, rather than one specific
A
B
site of action [9]. 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 [9].
Conclusions
The ethanolic extract obtained from F. hispida leaves showed varying inhibitory activities against all the test organisms. Thus, the preliminary screening assay indicated that F. hispida leaves extract possess great potential for the therapy of bacterial infections and may be used as a natural antiseptic and antimicrobial agent. Further investigation needs to be focused on isolation and identification of those bioactive compounds, which would be a platform for further pharmacological studies, in vivo tests and practical applications in fish health management.
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