The antibacterial activity of the ethanolic leaf extract of Ficus vasta Forssk. (moraceae) against aeromonas spp. strains

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The antibacterial activity of the ethanolic leaf extract of ficus vasta forssk. (moraceae) against aeromonas spp. strains

Recently, there is an increased frequency of multiple drug resistance in fish pathogenic microorganism due to indiscriminate use of commercial antimicrobial drugs commonly used to treat various diseases [25]. Consequently, the use of antibiotics and chemotherapeutics in aquaculture has received considerable attention because their accumulation both in the environment and in fish can be potentially risky to consumers and the environment [1]. Therefore, the use of natural products is considered as an alternative to control infections in aquaculture. Some plants are reported to have antimicrobial activity against several pathogenic bacteria. In this study, we tested the ethanolic extract obtained from Ficus vasta Forssk. leaves for antimicrobial activity against Aer- omonas strains (A. sobria, A. hydrophila, A. salmonicida subsp. salmonicida).

Among 37 genera of Moraceae comprising 1050-1100 species in total, Ficus L. is the largest one with ca 750 species of tropical and subtropical distribution worldwide. Its characteristic features include the presence of waxy glands on vegetative plant parts, heterostyly, and prolonged protogyny, i.e., the anthesis of staminate flowers in already mature fruits [5, 10]. Ficus trees have widely been used by humans over their history in a variety of industries and fields of activity. Virtually all parts of their body are utilized by local people in various medicinal practices to cure wounds, sores, stomach and eye problems, headaches and toothaches, and even tumors and cancer, etc. A number of species are known helpful in healing disorders of digestive and respiratory systems, parasitic infections, and also as painkillers, tonics, and ecbolics [16].

Ficus vasta Forssk. is a monoecious deciduous tree reaching up to 25 m in height, terrestrial, hemi-epiphytic or epilithic, with yellowish to whitish or brownish hirsute leafy twigs. The species is native to northeastern Africa and usually grows in relatively dry habitats or in riverine vegetation. Its leaves reach 8-35 cm in length and 425 cm in width; they are cordiform to suborbicular or ovate to reniform, coriaceous and densely puberulous to almost glabrous, with shortly acuminate to rounded apex, cordate base, and long petiole. The pedunculate puberulous syconia of subglobose to ellipsoid shape are born in pairs or solitary in the leaf axils or just below the leaves and reach 22.5 cm in diameter, at maturity green with paler spots [6].

The leaves of F. vasta are traditionally used for the treatment of rheumatisms, pains and intestinal worms [22].

Recently, Taviano and co-workers (2018) evaluated the antioxidant and antimicrobial properties of a hydroalcoholic extract obtained from the leaves of F. vasta. In this study, the phenolic profile of the F. vasta leaf extract was characterized by HPLC-PDA/ESI-MS. The antioxidant properties were examined by different in vitro systems: DPPH test, reducing power and metal chelating activity assays. Moreover, the ability of the extract to inhibite Escherichia coli growth and survival from H2O2- induced oxidative stress was evaluated. By HPLC-PDA/ESI-MS analysis 12 compounds belonging to the groups of phenolic acids and flavonoids were identified. The extract exhibited bacteriostatic activity against almost all the bacteria tested (MICs: 250-62.5 pg/mL) [27].

Fig. 1. Specimen of Ficus vasta, cultivated at Botanic Garden of Ivan Franko Lviv National University (Lviv, Ukraine) (A), and leaf sample of F. vasta (B).

Also, qualitative preliminary phytochemical analysis of F. vasta leaves, undertaken by Rashed with coauthors (2015), revealed the presence of carbohydrates, tannins, flavonoids, coumarins, and triterpenes [22]. Moreover, various phytoconstituents such as P-sitosterol, stigmasterol, lupeol, ursolic acid, and some flavonoids were isolated and identified from F. vasta aerial parts [23]. As for biological activities of F. vasta, very few studies have been carried out on the leaves of this species [19, 21].

We have reported already data on the antioxidant and antibacterial effects of extracts from various plant belonged to the Ficus genus [28-39]. Our current scientific project undertaken in the frame of cooperation programme between Institute of Biology and Environmental Protection (Pomeranian University in Slupsk, Poland), M. M. Gryshko National Botanic Gardens of National Academy of Sciences of Ukraine (Kyiv, Ukraine), and Ivan Franko Lviv National University (Lviv, Ukraine) directed to assessment of medicinal properties of tropical plants.

Therefore, the aim of the present study was to evaluate the antibacterial efficacy of ethanolic extracts derived from F. vasta against three Aeromonas strains (Aeromonas sobria, Aeromonas hydrophila, Aeromonas salmonicida subsp. salmonicida).

Materials and methods. Collection of plant material and preparing plant extract. The leaves of F. vasta were collected in M.M. Gryshko National Botanic Garden (NBG, Kyiv, Ukraine) and Botanic Garden of Ivan Franko Lviv National University (Lviv, Ukraine). The whole collections of tropical and subtropical plants both at NBG and Botanical Garden of Ivan Franko Lviv National University (including Ficus spp. plants) have the status of a National Heritage Collection of Ukraine. The species author abbreviations were followed by Brummitt and Powell (1992).

The sampled leaves were brought into the laboratory for antimicrobial studies. Then, freshly collected leaves were weighed and homogenized in 96 % ethanol (in proportion 1:10) at room temperature, and centrifuged at 3,000 g for 5 minutes. Supernatants were stored at -20°C in bottles protected with the laminated paper until required.

Method of culturing pathological sample and identification method of the Aeromonas strains. Three Aeromonas strains: Aeromonas sobria (K825) and Aer- omonas hydrophila (K886), as well as Aeromonas salmonicida subsp. salmonicida (St30), originated from freshwater fish species such as common carp (Cyprinys carpio L.) and rainbow trout (Oncorhynchus mykiss Walbaum), respectively, were isolated in Department of Fish Diseases, The National Veterinary Research Institute in Pu- lawy (Poland). Bacteria were collected from fish exhibiting clinical disorders. Each isolate was inoculated onto trypticase soy agar (TSA) (BioMerieux) and incubated at 27°C±2°C for 24 h. Pure colonies were used for biochemical identifications, according to the manufacturer's instructions, except the temperature of incubation, which was at 27°C ± 1 °C. The following identification systems were used in the study: API 20E, API 20NE, API 50CH (BioMerieux). Presumptive Aeromonas isolates were further identified to the species level by restriction analysis of 16S rDNA genes amplified by polymerase chain reactions (PCR) [12].

Bacterial growth inhibition test of plant extracts by the disk diffusion method. Antimicrobial susceptibility of the tested Aeromonas isolates was performed by the Kir- by-Bauer disc diffusion method according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI, 2014) [9]. Each inoculum of bacteria in the density of 0.5 Mc McFarland was cultured on Mueller-Hinton agar for 24 h at 28±2 °C. Seven drugs representing different antimicrobial classes as quinolones, tetracyclines, sul- phonamides, and phenicols were used. After incubation, the inhibition zones were measured. Interpretation criteria have been adopted from that available for Aeromonas salmonicida [8].

Statistical analysis. Statistical analysis of the data obtained was performed by employing the mean ± standard error of the mean (S.E.M.). All variables were randomized according to the phytochemical activity of strains tested [40]. The following zone diameter criteria were used to assign susceptibility or resistance of bacteria to the phytochemicals tested: Susceptible (S) > 15 mm, Intermediate (I) = 10-15 mm, and Resistant (R) < 10 mm [18].

Results and discussion. The results of antimicrobial activity of an ethanolic extract derived from F. vasta leaves against three Aeromonas strains are presented in Fig. 2.

The largest inhibition zone diameter (20.63±1.44 mm) was obtained against Aeromonas sobria (K825) growth, while the smallest inhibition diameter (13.38±0.42 mm) and (13.0±0.94 mm) was obtained against Aeromonas hydrophila (K886) and Aeromonas salmonicida subsp. salmonicida (St30) strains, respectively (Fig. 2). The ethanolic extract of F. vasta exhibited the intermediate activity against Aeromonas hydrophila and Aeromonas salmonicida subsp. salmonicida (St30), while Aeromonas sobria exhibited high susceptibility activity.

Species of Aeromonas are short, Gram-negative, facultative anaerobic, nonspore-forming, motile bacilli with a single flagellum, and can ferment glucose with or without the production of gas. They are 0.3-1.0 pm in diameter and 1.0-3.5 pm in length. They occur ubiquitously and autochthonously in aquatic environments. The genus Aeromonas is within the family Aeromonadaceae, which comprises of 14 different species; among which, Aeromonas hydrophila is known to infect fish, reptiles, amphibians, and humans. Some diseases caused by A. hydrophila can spread from animals to humans and vice versa (Pachanawan et al., 2008).

This study aimed to find a natural source, antimicrobial substance to replace antibiotics and chemotherapeutics for the treatment of Aeromonas-induced infections in fish aquaculture. According to our preliminary study and studies of other researchers [24, 26, 28-39], it has been demonstrated that a number of ethanolic extracts derived from various Ficus species and their cultivars showed antibacterial activity against pathogenic isolates as well as antibiotic-resistant bacteria. The plant extracts of the above plant species can be used as a source which could yield alternative drugs to improve the treatment of infection caused by these Aeromonas strains.

The documented pharmacological activities of Ficus plants in relation to their use as antimicrobial preparations are summarized below. Al-Fatimi and coworkers (2007) investigated antioxidant, antimicrobial, and cytotoxic activities of di- chloromethane, methanol and aqueous extracts of 30 plant species from Yemen. The extracts were tested against Gram-positive bacteria (Bacillus subtilis ATCC 6059, Micrococcus flavus SBUG 16, and Staphylococcus aureus ATCC 29213), Gram-negative bacteria (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853), Candida maltosa SBUG 17, and several opportunistic human fungal pathogens including yeasts (Candida albicans ATCC 90028 and C. krusei ATCC 90878) and hyphomy- cetes (Absidia corymbifera 100798, Aspergillus fumigatus 13550/99, and Trichophyton mentagrophytes 05/2004). S. aureus appeared the most susceptible among bacteria tested. Fruit extracts from F. vasta, the only Ficus species tested, generally were among the most active ones against bacteria, though they did not inhibit E. coli and fungal pathogens. The only methanol extract of F. vasta affected S. aureus, although with comparatively high inhibition efficacy: inhibition zone diameter of 18 mm and MIC of 50 pg/ml [2].

Aqueous extract of F. asperifolia Miq. stem bark assessed by Annan and Houghton (2008) for its activity against a number of bacteria species (Bacillus subtilis,coli, Micrococcus flavus, Pseudomonas aeruginosa, and Staphylococcus aureus) was found to have a generally weak antibacterial action. The minimal inhibition concentration of the extract for P. aeruginosa was 512 pg/ml, which was the highest value among the tested organisms [4].

Maregesi and co-workers (2008) screened n-hexane, methanol, and water extracts from 39 plant species of 22 families against a number of bacteria (Bacillus cereus ATCC 14579, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 15442, Klebsiella pneumoniae ATCC 13883, and Salmonella typhimurium ATCC 13311), fungi (Aspergillus niger ATCC 16404 and Candida albicans ATCC 10231), and viruses (Herpes simplex Virus type 1, Vesicular Stomatitis Virus T2, Semliki forest A7, and Coxsackie B2). Gram-negative bacteria generally appeared hardly sensitive to the extracts tested, with MIC values of 1000 pg/ml and (mostly) higher. Stem bark extracts of F. sycomorus, the only Ficus species examined, were active against Gram-positive bacteria and fungi with MIC 1000 pg/ml and higher, while they showed comparatively strong antiviral activity [17].

Kuete and co-workers (2008) conducted a detailed study on antimicrobial activity of crude methanolic extracts and isolated flavonoids and isoflavonoids from F. chla- mydocarpa root bark and F. cordata stem bark against 18 species of pathogenic microorganisms including mycobacteria, fungi, Gram-negative and Gram-positive bacteria. Some crude extracts and compounds were found active against all tested organisms with different level of inhibition. In respect to P. aeruginosa, F. chlamydocarpa crude extract was not active and F. cordata extract showed the weakest inhibition based on both disc diffusion and micro-dilution tests (with MIC 156,25 pg/ml). Particular compounds of these two species with generally the highest inhibitory activity, namely Luteolin (from F. chlamydocarpa) and Epiafzelechin (from F. cordata), similarly showed the weakest effect on P. aeruginosa at MICs 78,12 and >625 pg/ml respectively. In general, P. aeruginosa demonstrated the lowest susceptibility among all organisms tested [15].

Further studies [14] focused on antimicrobial evaluation of methanolic extracts, hexane-ethyl acetate, and ethyl acetate-methanol extract fractions, and isolated compounds from stem bark of F. ovata Vahl., testing a range of microbe clinical isolates, including Gram-positive bacteria (methicillin-resistant Staphylococcus aureus LMP805, Streptococcus faecalis LMP806, and Bacillus cereus LMP716), Gram-negative bacteria (P-lactamase positive Escherichia coli LMP701, ampicillin-resistant Klebsiella pneumoniae LMP803, carbenicillin-resistant Pseudomonas aeruginosa LMP804, chloramphenicol-resistant Salmonella typhi LMP706, and chloramphenicol-resistant Citrobacter freundii LMP802), and fungi (Candida albicans LMP709U and Micro- sporum audouinii LMP725D). The crude extracts and certain fractions and compounds were found active against all organisms tested. P. aeruginosa was most strongly inhibited (MIC 156 pg/ml) by hexane 100 % and Hex-EtOAc 25 % fractions and two compounds of isoflavonoid and phenolic nature. The crude extract showed a MIC value of 312 pg/ml and other fractions and compounds produced MIC of 312 to 625 pg/ml against P. aeruginosa. Among all organisms tested, P. aeruginosa was moderately susceptible to the evaluated antimicrobial agents. In general, the most inhibitory-active compounds appeared to be 2'-hydroxyisoprunetin (isoflavonoid) and protocatechuic (phenolic) acid, affecting (nearly) all pathogens tested with relatively low MIC values. Antimicrobial activity of flavonoids and isoflavonoids was suggested to come from their ability to complex with the bacterial cell wall, resulting in the microbial growth inhibition [13, 14]. It should be also noted that in the study of Anani and co-workers (2000), evaluating plant antimicrobial activity by disc diffusion essay with the addition of UVA exposure, F. ovata root and bark methanolic extracts had no effect on the growth of P. aeruginosa [3].

Assessment of antimicrobial activity of the methanolic extract, its fractions and isolated compounds from F. polita Vahl. roots against a number of microbe strains (Candida albicans ATCC9002, Escherichia coli ATCC8739 and AG100, Klebsiella pneumoniae ATCC11296, Providencia smartii ATCC29916, Pseudomonas aeruginosa PA01, Salmonella typhi ATCC6539, and Staphylococcus aureus ATCC25922) revealed moderate susceptibility of P. aeruginosa [13]. Crude extract demonstrated relatively weak inhibition of this pathogen (MIC 128 pg/ml). Hexane fraction of the extract and one isolated compound (namely,(E)-3,5,4'-trihydroxy-stilbene-3,5-O-Я-D-diglucopyranoside C₂₆H₃₂O₁₃) showed the strongest inhibitory activity against P. aeruginosa with MIC 64 pg/ml, the value equal to that of chloramphenicol used as reference antibiotic. Ethyl acetate and hexane-ethyl acetate fractions appeared ineffective [13].

In a study by Harikrishnan and Balasundaram (2008), the antimicrobial potency of aqueous and ethanolic decoction (individual extract) and concoction (mixed extract) of three common medicinal herbs, turmeric Curcuma longa, Tulsi plant Ocimum sanctum, and neem Azadirachta indica, against the A. hydrophila growth was evaluated. Among the decoctions, A. indica exhibited the most potent antibacterial property (P <05) against A. hydrophila. Among the concoctions, both the aqueous and ethanolic triherbal extracts mixed in the ratio of 1:1:1 had higher antibacterial activity (P < 0.05) than the other concoctions and decoctions [11].

Conclusions

The ethanolic extracts of Ficus vasta were found to exhibit a strong antibacterial growth inhibitory effect against three Aeromonas strains. The active compounds involved in the anti-Aeromonas activity have yet to be identified. The evaluation of the benefit/risk balance for the use of these plants in the treatment of Aer- omonas-induced infections in the aquaculture could be better documented in in vivo study. A bioassay-guided fractionation study of the active extract of these plants is underway to identify the compound(s) responsible for this activity. Thus, the findings of this research contribute to an increase in knowledge about F. vasta, demonstrating the potential of F. vasta leaves as a source of plant-derived antimicrobial compounds to be used in fisheries.

Acknowledgments. The study was supported by a grant from the International Visegrad Fund, and we thank them for financial assistance for our study.

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