Screening antimicrobial activity of various extracts of Urtica dioica
Amir Modarresi-Chahardehi1*, Darah Ibrahim1, Shaida Fariza-Sulaiman2* & Leila Mousavi2
*Dirección para correspondencia Abstract ]]>
Urtica dioica or stinging nettle is traditionally used as an herbal medicine in Western Asia. The current study represents the investigation of antimicrobial activity of U. dioica from nine crude extracts that were prepared using different organic solvents, obtained from two extraction methods: the Soxhlet extractor (Method I), which included the use of four solvents with ]]>
Streptococcus sp., miconazole nitrate (30µg/mL) as positive control for fungi and yeast, and pure methanol (v/v) as negative control. The disc diffusion assay was used to determine the sensitivity of the samples, whilst the broth ]]>
Bacillus cereus, MRSA and Vibrio parahaemolyticus. A selection of extracts that showed some activity was further tested for the MIC and minimal bactericidal concentrations (MBC). MIC values of Bacillus subtilis and ]]>
Staphylococcus aureus (MRSA) using butanol extract of extraction method II (BE II) were 8.33 and 16.33mg/mL, respectively; while the MIC value using ethyl acetate extract of extraction method II (EAE II) for Vibrio parahaemolyticus was 0.13mg/mL. Our study showed that 47.06% of extracts inhibited Gram-negative (8 out of 17), and 63.63% of extracts also inhibited Gram-positive bacteria (7 out of 11); besides, statistically the frequency of antimicrobial activity was 13.45% (35 out of 342) which in ]]>
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Key words: Urtica dioica, antimicrobial activity, disc diffusion method, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC).
Resumen
Urtica dioica u ortiga se utiliza tradicionalmente como medicina herbaria en el oeste de Asia. En esta investigación se estudia la actividad antimicrobiana de nueve extractos crudos de U. dioica, los cuales fueron preparados utilizando diferentes disolventes orgánicos y obtenidos a partir de dos métodos de ]]>
Streptococcus sp., ]]>
Bacillus cereus, MRSA y
Vibrio parahaemolyticus. Una selección de extractos que mostraron algún tipo de actividad se probó para el CIM y las concentraciones mínimas bactericidas (CMB). Los valores de CIM de Bacillus subtilis y de Staphylococcus aureus resistentes a la meticilina (MRSA) usando extracto de butanol mediante el método de extracción II ]]>
Vibrio parahaemolyticus fue 0.13mg/mL. Nuestro estudio mostró que el 47.06% de los extractos inhibieron bacterias Gram-negativas (8 de 17), y el 63,63% de los extractos también inhibieron bacterias Gram-positivas (7 de 11), además que estadísticamente la frecuencia de la actividad antimicrobiana fue de 13.45% (35 de 342), que de este porcentaje un ]]>
Palabras clave:Urtica dioica, actividad antimicrobiana, método de difusión en disco, concentración mínima ]]>
Currently, research and development of new drugs from natural resources in a systematic and strategic manner has become the global trend. Natural product derived medicines are widely used and account for more than 30% of therapeutic agents presently prescribed in clinics (Yang et ]]>
2008). The activities have been selected because of their great medicinal relevance. Within the recent years, infections have increased to a great extent and resistance against antibiotics becomes an ever-increasing therapeutic problem (Austin et al. 1999). Because of the need for new antimicrobial agents and strategies for their use in the treatment of serious Gram-negative and Gram-positive infections is evident (Shah 2005, Gülçin et al. 2008, Gülçin ]]>
et al. 2010) and is greater than ever because of the emergence of multidrug resistance in common pathogens, the rapid emergence of new infections, and the potential for use of multidrug-resistant agents in bioweapons (Spellberg et al. 2003).
Urtica L. Stinging ]]>
et al., 2006). Among Urtica species, Urticadioica and Urtica urens have already been known and therefore consumed for a ]]>
U. dioica herbs are used to treat stomachache in Turkish folk medicine (Gulçin et al. 2004), and in Iran (Pourmorad et al. 2006, Monfared et al. 2011). Besides, this herb is used to treat rheumatic pain and for colds and cough (Sezik et al. ]]>
Plants are important sources of potentially useful structures for the development of new chemotherapeutic agents. The first step towards this goal is the in vitro antimicrobial activity assay (Saklani & Chandra 2012). The main purpose of this study was ]]>
U. dioica and compare two methods of extraction and the ability of different kinds of U. dioica crude extracts to inhibit various pathogenic microorganisms.
Materials and Methods
Preparation of the extract: The aerial parts of U. dioica including leaves and stems were collected in Iran from Salmanshahr city in Mazandaran province (the geographical coordinates given by GPS: latitude: 36º42’34” N-51º08’57” E and altitude: 21m) and Tehran city in Tehran province (the geographical coordinates given by GPS: latitude: 35º50’21” N-51º25’22” E ]]>
The second method (Method II) ]]>
]]>
Antimicrobial activity test: Antimicrobial activity was determined by using Disc Diffusion following the method described by the National Committee for Clinical Laboratory Standard (NCCLS) (2002). All bacterial strains including American Type Culture Collection (ATCC) and clinical were used in the study. The test bacteria was removed aseptically with an inoculating loop and transferred to a test tube containing 5mL of sterile distilled water. Sufficient inoculums were added until the turbidity equaled 0.5 McFarland (108cfu/ mL) standards (bioMerieux, Marcy d’Etoile, France). ]]>
Streptococcus sp. or miconazole nitrate (30µg/mL) ]]>
]]>
To calculate of percentage of antimicrobial activity, number of effective crude extract or number of tested microorganism were divided on total of selected microorganisms. We compared both methods of extraction (Method I and II), and between Gram-negative and Grampositive bacteria.
Determination of the minimum inhibitory concentration (MIC): MIC was determined by the liquid dilution method. Dilution series were set up with 0.13, 0.26, 0.52, 1.04, 2.08, 4.16, 8.33, 16.66, 33.33, 66.66 and 133.33mg/ mL of nutrient broth medium. Each test tube was added with 0.5mL of standardized suspension of bacteria and incubated at 37°C for 24 hours. The lowest dilution of the extract that retained its inhibitory effect resulting in no growth (absence of turbidity) of a microorganism was recorded as the MIC value of the extract. The microorganism growth was indicated by the turbidity. A ]]>
Determination of the minimum bactericidal concentration (MBC): The minimum bactericidal concentration of the plant extract on the clinical bacterial isolates was done according to the method highlighted in NCCLS (2002). Briefly, one loopful of the inoculums from each of the non-turbid tubes was ]]>
Results
The results of the antimicrobial activity tests of crude extracts are shown in table 1. It was found that nine crude extracts of Urtica dioica at 100mg/mL concentration exhibited various antibacterial and antimycotic activities but no antifungal activity.
Urtica dioica extracts gave large inhibition zone to Acinetobacter calcoaceticus,Bacillus cereus, B. spizizenii ATCC 6633, Micrococcus sp. and Vibrio parahaemolyticus. According to
table 1, BE II was effective against Escherichia coli, B. subtilis and methicillin resistant Staphylococcus aureus (MRSA). Among the Gram-negative bacteria tested, Klebsiellapneumonia, E. coli, Salmonella paratyphy B showed exhibited ]]>
B. cereus, B. spizizenii ATCC 6633 and V. parahaemolyticus exhibited zones of inhibition between 10 and 14mm in diameter. The mold, Saccharomyces cerevisiae was found with zones of inhibition from 9 to 10-14mm. Nevertheless, the results seem to indicate that method I (Soxhlet extractor) may be more ]]>
All the extracts failed to inhibit the mold, except all crude extract from extraction method I against S. cerevisiae. Also Aspergillus niger USM A1 and ]]>
Trichophyton rubrum showed reduced of UFC, but nor hypha. In addition, pure methanol (control) had no inhibitory effects on pathogenic microbes tested.
The MIC values of the crude extracts of U. dioica were determined against bacterial species that ]]>
table 2. The agar dilution method showed MIC values of 0.13 to 66.66mg/mL. The broader spectrum of activities could be due to synergistic effects of the various components in the U. dioica extract. EAE II and CE I showed the lowest concentration of MIC values of 0.13 and 4.16mg/mL, respectively, against V. parahaemolyticus. The antimycotic activity against S. ]]>
of ethyl acetate extract from method I (EAE I) was also strong with MIC and MBC values of 2.08 and 8.33mg/mL, respectively.
In this study a 47.06% of extracts inhibited Gram-negative (eight out of 17) and 63.63% of extracts also inhibited Gram-positive bacteria (seven out of 11) and the frequency of antimicrobial activity statically was 13.45% (35 out of 342) which in ]]>
According to antimicrobial activity of Urtica dioica as shown in figure 1, EAE I>HE I>CE I>ME ]]>
U. dioica, ethyl acetate extract from extraction method I (EAE I) showed the highest antimicrobial activity with more than 25%. In fact, 10.46% (16 out of 153) and 22.22% (22 out of 99) of extracts inhibited of Gram-negative and Grampositive bacteria, respectively. However, EAE I showed high frequency of inhibition of growth of bacteria. Crude ]]>
U. dioica exhibited poor antibacterial activity against Pseudomonas and Staphylococcus spp. and fungicidal activity was not observed.
Discussion ]]>
The results suggest that U. dioica exhibits bactericidal and mycoticidal activities. The resistance of fungal species against the crude extracts of U. dioica could be due to their morphological structure; fungi have thicker cell walls and contain higher percentage of chitin (Madigan & Martinko 2006). Furthermore, the pathogenic microorganisms showed relatively lower MIC values ranging from ]]>
Bacillus cereus, Micrococcus sp.), while relatively less antibacterial activity was associated with Gram-negative bacteria. The review by Pellecuer et al. (1976) showed that phenolics are the predominant active compound in some family plants, with Gram-positive ]]>
The different crude extract preparations revealed different spectrums of activities, especially with the use of the disc diffusion method. Otshudi et al. (1999) indicated that diethyl ether extracts of plants were inactive against bacteria compared to aqueous ]]>
et al. 2000). This reason may slightly explain why the broad inhibitory activity of diethyl ether extract on Method II (DEE II) was poor when compared with the two kinds of methanol extracts from both methods (ME I and ME II). According to our results, ethyl acetate, hexane and chloroform ]]>
et al. 2009).
A large zone of inhibition was observed against A. calcoaceticus ]]>
U. dioica also could be used to treat V. parahaemolyticus, B. cereus, S. aureus and methicillin-resistant S. aureus (MRSA) infections. As it is well known, ]]>
E. coli and Bacillus species, especially B. cereus, are agents of food poisoning. The most interesting area of application for plant extracts is the inhibition of growth and reduction in numbers of the more serious food-borne pathogens (Burt 2004). B. cereus and B. subtilis are responsible for a minority of foodborne illnesses causing ]]>
Gulçin et al. (2004) found some antimicrobial activity of the U. dioica water extract against E. coli, Enterobacter aerogenes, S. epidermidis and Candida albicans, while there was no activity against Pseudomonas aeruginosa. Their ]]>
et al. (2008) found that methanol extract of Forrskalea tinacissima other species in this family had low antibacterial activity against S. aureus and Microccus flavus, but no activity against B. cereus and E. coli. As the same result from Pilea microphylla (Urticaceae family) had low ]]>
S. aureus by using mixture of acetone and ethyl acetate (Facey et al. 1999), and recent study have reported on the antibacterial activity of extracts from P. microphylla on Gram-positive bacteria such as B. cereus, B. subtilis and Methicillin resistant S. aureus (Modarresi-Chahardehi et al. ]]>
A study reported that Urera baccifera from Urticaceae showed no antimicrobial activity against C. albicans, E. coli, P. aeruginosa and S. aureus. Also the extracts of U. dioica showed no ]]>
et al. 1987). The 80% ethanol extract of U. dioica did not show any inhibition zone against the tested bacteria (Dulger & Gonuz 2004). In other study, Steenkamp et al. (2004) confirmed that various studies support their results which U. urens possess no antibacterial activity (Rabe & van Staden 1997, Dulger & Gonuz ]]>
Salmonella typhi, antimicrobial equivalence from U. urens obtained 15.5±0.0mm. The antimicrobial activity was evaluated by measuring the inhibition zone diameter observed at 48hrs. The mean values were interpolated by comparison to a reference concentration-response curve of ampicillin. Antimicrobial ]]>
U. urens was still low compared to other plant tested in other family (ranging from 9.8±0.4 to 73.4±19.6mm).
In contrast to the Rabe & van Staden (1997) report, methanol and also water extracts from root part of Pouzolzia ]]>
, that belongs to Urticaceae family, showed less antibacterial activity against S. aureus, S. epidermis and B. subtilis. However, MIC values against these bacteria were 4.0, 2.0 and 2.0mg/mL, respectively.
Generally, crude ]]>
et al. 1997, Gao et al. 1999). Grampositive bacteria, B. cereus, MRSA, were most susceptible to the crude extract of Method I, but it can be ]]>
et al. 2003, Darah et al. 2011). To state that the reason Grampositive bacteria are susceptible to the crude extract is related to the extraction method. It is a proven ]]>
Extracts of ethyl acetate, hexane from extraction method I and butanol from extraction method II of U. dioica were found to be effective against some of strains studied. In ]]>
in vivo evaluation of antimicrobial activity, along with toxicity assays, present in the two extracts before it is used for commercialization in the form of pharmaceutical medicine.
Acknowledgments
This material is based upon work supported by a grant and fellowship obtained from Universiti Sains Malaysia (USM). ]]>
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*Correspondecia:Amir Modarresi-Chahardehi: Industrial Biotechnology Research Laboratory, School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia; amirmch@yahoo.com Darah Ibrahim: Industrial Biotechnology Research Laboratory, School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia; darah@usm.my ]]>
Shaida Fariza-Sulaiman: Phytocemistry Laboratory, School of Biological sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia; shaida@usm.my Leila Mousavi: Phytocemistry Laboratory, School of Biological sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia; leila_mosavi12007@yahoo.com 1. Industrial Biotechnology Research Laboratory, School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia; amirmch@yahoo.com, darah@usm.my 2. Phytocemistry Laboratory, School of Biological sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia; shaida@usm.my, leila_mosavi12007@yahoo.com
Received 30-IX-2011.Corrected 20-IV-2012.Accepted 21-V-2012.
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