In vitro Antimicrobial Activity of Methanolic Extract from Varthemia iphionoides Leaves

In the Mediterranean region, Varthemia iphionoides is commonly used in folk medicine for the treatment of gastrointestinal disorders. The present study described the antimicrobial activity of the methanolic extract of V. iphionoides leaves. The extract was assayed against a panel of pathogenic bacteria and fungi using agar well diffusion method. The antibacterial activity of the methanolic extract was investigated against six standard bacterial species and was found to exhibit high antibacterial activity. The most sensitive bacterium was Klebsiella pneumoniae ATCC 13883 followed by Proteus vulgaris ATCC 13315 and methicillin-resistant Staphylococcus aureus ATCC 95047. The least sensitive bacterium to V. iphionoides methanolic extract was Escherichia coli O157:H7 ATCC43895. Antifungal susceptibility of 13 fungal species was tested against V. iphionoides methanolic extract. Among the fungal species studied, Fusarium lini was the most sensitive and Beauveria bassiana was the most resistant to the extract. Good antifungal activity has been displayed by the methanolic extract of V. iphionoides against Aspergillus brasiliensis, Aspergillus niger, and Aspergillus alliaceus, Aspergillus flavus, Cunninghamella echinulata, Gibberella fujikuroi, Macrophomina phaseolina, Cephalosporum aphidicola, Rhizoupus stolonifer, Curvularia lunata and Cunninghamella elegans. The observed antimicrobial potential of V. iphionoides indicated that this plant possesses bioactive compounds that are able to combat pathogenic microorganisms and support its traditional use in the treatment of pathogen infection.


Introduction
The misuse of antimicrobial drugs is often associated with drug resistance in pathogenic microorganism (Cowan, 1999).Drug resistance resulted in critical health problems during the treatment of infectious disease (Mahady, 2005).The increasing public awareness of hazards associated with conventional antimicrobial drugs and the public trend of adopting natural antimicrobial agents have accelerated the use of plants and their extracts to combat microbial infections (Abreu, McBain, & Simoes, 2012;Zhang et al., 2013;Catteau, Van Bambeke, & Quetin-Leclercq, 2015).
To protect themselves against microbial and herbivory attacks, plants produce a large variety of secondary metabolites, such as, quinones, tannins, flavonoids, terpenoids, and alkaloids.These metabolites have been found to possess antimicrobial activities (González-Lamothe et al., 2009;Savoia, 2012).The concentrations of these phytochemicals are particularly very high in medicinal and aromatic plants.These plants are widely used in ethnomedicine around the world.The antimicrobial activity of medicinal plants has been reported by an enormous number of studies worldwide (Shahid et al., 2009;Wangchuk et al., 2011).
The aim of the present study was to evaluate the antimicrobial effects of V. iphionoides methanolic extract on various bacterial and fungal species.

Plant Material and Preparation of Plant Extract
The leaves of Varthemia iphionoides were collected at the flowering stage in April 2014 from wild population found in Al-Salt governorate (20 km west Amman, Jordan; semi-arid region; mean annual rainfall; 350 mm).The collected leaves were thoroughly washed with distilled water and were air dried in an aerated place at room temperature.The dried leaves were stored at -18 °C in vacuum-sealed package.
Twenty-gram portions of finely-powdered leaves were extracted for 2 days with 100 ml of absolute methanol at room temperature under constant shaking of 120 rpm.The extract was then filtered twice (Whatman no. 1) and the solvent was evaporated to dryness in a rotary evaporator at 40 °C.The dried plant extract was dissolved in 0.05% dimethyl sulfoxide to a final concentration of 30 mg ml -1 and sterilized by filtration through 0.45 µm millipore filters.

Standard Drugs Used for Antimicrobial Assay
Ampicillin (10 µg/disc) and vancomycin (30 µg/disc) were used as reference antibiotics against the tested bacteria.For the fungal strains, cycloheximide (250 µg/disc) and nystatin (10 µg/disc) were employed as reference antifungal agents.

Inhibition Zone Determination by Agar-Well Diffusion Assay
The antibacterial and antifungal assays of the methanolic extract of V. iphionoides were carried out by the agar-well diffusion method (Perez, Paul, & Bazerque, 1990).Briefly, 100 µl of bacterial and fungal suspensions were spread uniformly over agar plates.Small wells (5 mm in diameter) were cut in the agar plates using a sterile cork borer.Fifty microliter containing 30 mg ml -1 of the plant extract were added into each well.The plates were incubated for 24 h at 37 °C for the bacterial culture, and for 48 h at 30 °C for the fungal culture.Antimicrobial activity was evaluated by measuring the diameter of the inhibition zone around the well.

Statistical Analysis
The data were expressed as mean ± standard error (SE) of five independent replicates.One-way ANOVA test (LSD) was used to evaluate the significant differences among means at P < 0.05.

Results and Discussion
The present study investigated the in vitro antibacterial and antifungal activities of the methanolic extract of V. iphionoides using the agar well diffusion method.This method of testing the antimicrobial activity is a common standard method recommended by the Clinical and Laboratories Standards Institute (CLSI, 2009).Methanol was chosen in the present study as the extraction solvent, since it proved to be more effective than other solvents for the extraction of antimicrobial substances from plants (Chandrasekaran & Venkatesalu, 2004;Rhouma et al., 2009).
The antibacterial activity of the methanolic extract of this medicinal plant against six standard bacterial species is shown in Table 1.The methanolic extract of V. iphionoides displayed antibacterial effect against all tested bacterial species with inhibition zones ranging from 1.68 to 25.2 mm.The statistical analysis revealed significant differences among the different bacterial species.Klebsiella pneumonia (ATCC 13883), a Gram negative bacterium, showed the maximum sensitivity to the methanolic extract of V. iphionoides with mean diameter of inhibitory zone of 25.2 mm.
In addition, extract of V. iphionoides also exhibited strong antibacterial activity against Proteus vulgaris (ATCC 13315), methicillin-resistant Staphylococcus aureus (ATCC 95047), and Klebsiella oxytoca (ATCC 13182) with inhibition zone diameter of 21.4, 21.3, and 18.4 mm, respectively.Whereas, the methanolic extract has a fair bacteriostatic effect on Salmonella typhimurium (ATCC 19430) with inhibition zone diameter of 11.0 mm.Limited bactericidal activity against Staphylococcus aureus and Salmonella typhimurium were previously reported for V. iphionoides water extract (Afifi et al., 1991).
The antibacterial activity of the methanolic extract against Escherichia coli O157:H7 (ATCC43895) was not very promising.This bacterial species was resistant to both ampicillin and vancomycin antibiotics and was the least affected by the methanolic extract of V. iphionoides.The low sensitivity of Escherichia coli O157:H7 to V. iphionoides extract is in agreement with a previous study.It was reported that crude aqueous extract (Afifi et al., 1991) and ethanolic extract (Masadeh et al., 2013) of the aerial part of V. iphionoides exhibited limited antimicrobial activity against E. coli.Note. a Values are given as mean ± standard error of five replicates.Means followed by different letters are significantly different at P < 0.05 according to the LSD test.
b Ampicillin was tested at 10 µg and Vancomycin was tested at 30 µg.Bacterial strain is considered antibiotic resistant when the inhibition zone diameter is equal to or small than 11 mm for Ampicillin and 13 mm for Vancomycin.
The methanolic extract of V. iphionoides possessed antifungal activity against 13 fungal species with inhibition zones ranging from 2.0 to 19.6 mm (Table 2).Fusarium lini was significantly the most susceptible fungal species with inhibition zone measuring 19.6 mm in diameter.Good antifungal activity has been displayed by the methanolic extract of V. iphionoides against cycloheximide-and cystatin-sensitive fungi, namely, Cunninghamella echinulata, Gibberella fujikuroi, Macrophomina phaseolina, Cephalosporum aphidicola, Aspergillus flavus, and Rhizoupus stolonifer.Moreover, Curvularia lunata and Cunninghamella elegans were also susceptible to methanolic extract of V. iphionoides with inhibition zone diameter of 13.4 and 12.0 mm, respectively.In this regards, Afifi et al. (1991) stated that flavonoids isolated from V. iphionoides showed antifungal activities; the compound kumatakenin exhibited potent activity against Fusarium solani and Candida tropicalis, while 3ʹ,3ʹ-di-O-methylquercetin and xanthomicrol showed antifungal activities against Fusarium solani, Aspergillus parasiticus, and Candida tropicalis.
Three Aspergillus species, namely, Aspergillus brasiliensis, Aspergillus niger, and Aspergillus alliaceus, which showed resistance to both cycloheximide and cystatin, exhibited high sensitivity toward V. iphionoides methanolic extract.However, methanolic extract of V. iphionoides showed weak inhibition effect on the growth of Beauveria bassiana, because small inhibition zone (2.0 mm in diameter) was observed.Note. a Values are given as mean ± standard error of five replicates.Means followed by different letters are significantly different at P < 0.05 according to the LSD test.
b Cycloheximide was tested at 250 µg and Nystatin was tested at 10 µg.Fungal strain is considered antifungal-agent resistant when the inhibition zone diameter is equal to or small than 8 mm for both Cycloheximide and Nystatin.
The present results support the traditional use of V. iphionoides plant in treatment of pathogen infection.Different types of phenolic compounds have been previously identified in V. iphionoides (Abu-Romman et al., 2015).The strong antimicrobial activities of medicinal plants are associated in parts with high content of phenolic compounds (Rhouma et al., 2009).These compounds have been suggested to inhibit microbial growth by disturbing the cytoplasmic membrane, transport, electron flow, and microbial enzymes (Sikkema, De Bont, & Poolman, 1995;Gupta et al., 2012).

Conclusion
The present results showed the efficacy of the methanolic extract of V. iphionoides against a panel of bacteria and fungi, indicating that this plant possesses bioactive compounds that are able to combat pathogenic microorganisms.Our results, therefore, demonstrate a promising potential of including V. iphionoides in pharmaceutical industry for therapy of infectious diseases.However, additional studies are necessary to investigate the toxicity and side effects of the bioactive constituents of V. iphionoides.Further studies are required to determine the efficacy of different V. iphionoides extracts against various other pathogenic microorganisms.Moreover, V. iphionoides obtained from different geographical regions of Jordan could also be investigated; to evaluate better potential of this medicinal plant as a source of antimicrobial compounds.

Table 1 .
Antibacterial activity of methanolic extract of Varthemia iphionoides based on agar well diffusion method

Table 2 .
Antifungal activity of methanolic extract of Varthemia iphionoides based on agar well diffusion method.