On a global scale, people have used plants to treat diseases and infections, and this has raised interest on the plant biodiversity potencial in the search of antimicrobial principles. In this work, 75 crude n-hexanes, dichloromethane and methanol extracts from the aerial parts of 25 plants belonging to four botanical families (Asteraceae, Euphorbiaceae, Rubiaceae and Solanaceae), collected at the Natural Regional Park Ucumari (Risaralda, Colombia), were evaluated for their antibacterial and antifungal activities by the agar well diffusion method. The antibacterial activities were assayed against two Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis, and three Gram-negative ones named, Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa. In addition, the same plant extracts were tested against the yeast Candida albicans and the fungi Aspergillus fumigatus and Fusarium solani. Overall, the plant extracts examined displayed better bactericide rather than fungicide activities. In general, the best antibacterial activity was showed by the plant extracts from the Rubiaceae family, followed in order by the extracts from the Euphorbiaceae and Solanaceae ones. It is important to emphasize the great activity displayed by the methanol extract of Alchornea coelophylla (Euphorbiaceae) that inhibited four out of five bacteria tested (B. Subtilis, P. aeruginosa, S. aureus and E. coli). Furthermore, the best Minimal Inhibitory Concentration for the extracts with antifungal activities were displayed by the dichloromethane extracts from Acalypha diversifolia and Euphorbia sp (Euphorbiaceae). The most susceptible fungus evaluated was F. Solani since 60% and 20% of the dichloromethane and methanol extracts evaluated inhibited the growth of this phytopathogenic fungus. The antimicrobial activity of the different plant extracts examined in this work could be related to the secondary metabolites contents and their interaction and susceptibility of pathogenic microorganism evaluated.
Alrededor del mundo, la gente ha usado las plantas para tratar enfermedades e infecciones, este potencial ha hecho que se incremente el interés en la biodiversidad vegetal como fuente de principios antimicrobianos. En este trabajo, se evaluaron 75 extractos crudos de n-hexano, diclorometano y metanol, obtenidos a partir de la parte aérea de 25 especies de plantas proveniente de cuatro familias botánicas (Asteraceae, Euphorbiaceae, Rubiaceae y Solanaceae), colectadas en el Parque Regional Natural Ucumari (Risaralda, Colombia); los cuales fueron evaluados por sus actividades antibacteriana y antifúngica a través del método de difusión en pozo. La actividad antibacteriana fue ensayada frente a las bacterias Gram-positivas Staphylococcus aureus y Bacillus subtilis, y las g-negativas Klebsiella pneumoniae, Escherichia coli y Pseudomonas aeruginosa. Adicionalmente, las mismas plantas fueron evaluadas frente a la levadura Candida albicans y los hongos Aspergillus fumigatus y Fusarium solani. En general, las plantas ensayadas mostraron mejor actividad antibacteriana que antifúngica; donde la familia Rubiaceae fue la que presentó mayor actividad antibacteriana, seguida por las familias Euphorbiaceae y Solanaceae. El extracto metanólico de Alchornea coelophylla (Euphorbiaceae) fue el que presentó mejor actividad antibacteriana al inhibir cuatro de las bacteria ensayadas (B. Subtilis, P. aeruginosa, S. aureus y E. coli); y los extractos de diclorometano de Acalypha diversifolia y Euphorbia sp. (Euphorbiaceae) fueron los que tuvieron la menor Concentración Mínima Inhibitoria en la actividad antifúngica. El hongo evaluado más susceptible fue F. Solani, el cual fue inhibido por el 60% y el 20% de los extractos de diclorometano y metanol, respectivamente. Se considera que la actividad biológica de estos extractos, se relaciona con los metabolitos secundarios que ellos contienen y las diferentes susceptibilidades de los microorganismos patogénicos evaluados.
Palabras clave: actividad antimicrobial, bactericida, bioprospección, ensayo por difusión en pozo, fungicida, MIC.
Crude plant extracts have been used in folk medicine for ]]>
et al. 2002). This medicinal potential has led the pharmaceutical industry to search for more effective agents, with the aim to discover potentially useful active constituents that can serve as new medicinal molecules or templates for the synthesis of new drug entities (Pretorius et al. 2003; Newman & Cragg 2007). ]]>
In an effort to discover new lead compounds many research groups have screened plant extracts to detect secondary metabolites with relevant biological activities (Harish et al. 2007). The search for new antimicrobial compounds is particularly important, since bacteremia remains a significant cause of morbidity and mortality in many nosocomial infections worldwide. The treatment of ]]>
]]>
Colombia is a biodiverse rich country although, many plant species are at risk of extinction due to factors such as inadaptability problems associated to climate change conditions as well as anthropic actions, among others. The aim of this work was to investigate the bactericide and fungicide activities of 75 plant extracts belonging to 25 species associated to four botanical families, collected at The Natural Regional Park Ucumarí (NRPU), located in the Central Colombian Andean Mountain region (Pereira, Colombia). ]]>
Materials and Methods
Plant material: The adult aerial plant samples (2kg, leaves and branches) used for this study were collected in May-June 2006 in NRPU with an altitudinal range between 2 100-2 450m.a.s.l., ]]>
Table 1).
Each plant sample ]]>
n-hexanes, dichloromethane and methanol (thrice with portions of 900ml for each solvent). In this work the solvents used were analytical grade from Mallickrodt (Phillipsburg, NJ, USA). In all cases, solvents were pulled out and separately concentrated to dryness in a rotary evaporator at 45ºC under reduced ]]>
et al. 2006).
Test organisms: The following Gram-positive bacteria were assayed Staphylococcus aureus (ATCC 6538) and Bacillus subtilis (ATCC 21556); while, the Gram-negative Klebsiella ]]>
(ATCC 10031), Escherichia coli (ATCC 9637) and Pseudomonas aeruginosa (ATCC 27853) were used. In addition, for antimycotic screening the yeast Candida albicans (ATCC 18804) and the fungi Aspergillus fumigatus (ATCC 1022) and Fusarium solani (ATCC 11712) were tested. The bacteria were first subcultured in a Müller Hinton nutrient broth (Oxoid, Basingstoke, England) and incubated at 37ºC ]]>
The antibacterial activities of crude extracts were evaluated through the agar-well diffusion assay (Ríos et al. 1998). The n-hexanes, dichloromethane and ]]>
P. aeruginosa, for which 0.5mg/ml concentration was employed. In all antimicrobial assays ethanol was used as negative control.
The procedures for ]]>
et al. (2003). For these assays, the three different extracts for each plant species were dissolved and tested at the same concentrations employed for the antibacterial tests. Ketokonazole at 0.25mg/ml was used as positive control.
Phytochemical screening of plant extracts: Plant extract phytochemical screening was performed by using thin layer chromatography (TLC) on Silica Gel 60 F254 sheets (Merck, Darmstadt, Germany) following the procedure described by Wagner & Bladt (1996). The systems chloroform-ethyl acetate-methanol (2:2:1) was used for methanol extracts elution; while, n-hexanes-ethyl acetate (7:3) was employed for dichloromethane and n-hexanes extracts elution, respectively. After development, the phytocompounds were visualized through the use of the ]]>
The results for both the ]]>
Results
Antimicrobial activities: The results from the antibacterial and antifungal activities investigated from the collected plant extracts are reported in table 1.
The results from the ]]>
S. aureus; while, 32% of the n-hexanes extracts displayed antibacterial activity against B. subtilis. In addition, 28% of the methanol and 20% of the n-hexanes ]]>
P. aeruginosa.
The 83% of the n-hexanes extracts of species belonging to the Rubiaceae family examined in this work displayed great bactericidal activity. The antibacterial activity of the previous family was followed by the 78% of the methanol extracts of ]]>
Acalypha platyphylla Müll. Arg., Alchornea coelophylla Pax & K. Hoffm., Hyeronima macrocarpa Müll. Arg. and Mabea montana Müll. Arg., displayed MIC values ranging from 1.0 to 4.0mg/ml against B. subtilis, S. aureus, E. Coli and P. Aeruginosa. In ]]>
Solanum leucocarpum Dunal and Solanum deflexiflorum Bitter resulted two of the most active species in this work.
The least active plant extracts in this work was associated to members ]]>
n-hexanes extracts from Calea angosturana Hieron. and Vernonia canescens Kunth, displayed good activity against Gram-positive bacteria.
With regards to the antifungal assays, the most susceptible one was F. ]]>
, where 15 (60%) out of 25 dichloromethane and 5 (20%) out of 25 of the methanol extracts assayed showed activities against this phytopathogenic fungus. The most active dichloromethane plant extracts against F. solani were those from the Euphorbiaceae and Solanaceae families with 6 and 5 active extracts, respectively. Acalypha diversifolia Jacq. and Euphorbia sp., displayed the lowest MIC values against this fungus.
Phytochemical screening: The results from the phytochemical screening for the 75 plant extracts examined are listed on table 1, and they showed the presence of sterols, saponins, alkaloids, tannins and flavonoids.
]]>
Discussion
Antibacterial activities: The results reached in this work are related to those obtained for S. aureus (68%) and B. subtilis (36%) in a screening performed on some ]]>
P. aeruginosa (Parmesha et al. 2008).
Results displayed by the Solanaceae family correlate very well with those obtained in the antibacterial ]]>
Solanum leucocarpum that displayed MIC values of 250, 125 and 62.5µg/ml, respectively against S. aureus; the same isolated alkaloids also showed MIC values higher to 1 000µg/ml against B. subtilis, P. Aeruginosa, E. coli and K. pneumoniae (Niño et al. 2009). ]]>
At the same time, the results showed by the Euphorbiaceae family correlate very well with those reported for Alchornea cordifolia (Euphorbiaceae), which methanol and ethanol extracts displayed MIC values of 1.5 and 2.6mg/ml, respectively against S. aureus (Ajali 2000). Also, the results from this research were in concordance with those reported for ]]>
Putranjiva roxburghii Wall (Euphorbiaceae) that displayed MIC values ranging from 0.5-4.0mg/ml, against most of the organisms evaluated (Mahida & Mohan 2006). Additionally, other extracts, such as the aqueous extract from Euphorbia tirucalli L., at 10mg/ml showed antibacterial activity against 11 human pathogenic bacteria (Mohana et al. 2008); furthermore, the ether, hexane and chloroform extracts from ]]>
Acalypha indica Linn displayed antibacterial activity against E. coli, Aeromonas hydrophila and S. aereus (Muthuvelan & Balaji Raja 2008).
Results displayed by the members of the Rubiaceae family studied were opposite to those found for the ]]>
Borreria hispida (Linn.) K. Schum., (Rubiaceae) that displayed strong antibacterial activity in the range of 0.25 to 50mg/ml (Muthu et al. 2010).
Results related to extracts from the Asteraceae family evaluated here were consistent with those from the petroleum ether extract of Evax ]]>
(L.) Brot. (Asteraceae) that was the most active against S. aureus; while, the chloroform extract resulted less potent against the Gram-positives S. epidermidis and Micrococcus luteus (Boussaada et al. 2008).
In general, the best ]]>
n-hexanes ones. The antimicrobial effect of methanol extracts against the microorganisms tested may be due to the capability of methanol like solvents to extract some of the active secondary constituents from these plants, such as phenols, alkaloids, saponins, which are reported to have antimicrobial properties (Okwu & Josiah 2006, Mothana et al. 2010). Although the low polarity of phytocompounds ]]>
n-hexanes, they also had phytocompounds that interact in some way with the bacteria assayed and displayed the second order of activities showed in this work.
Plant extracts are rich in many phytocompounds which are the cause of their bioactivities. The mechanism of action of many antimicrobials is complex ]]>
et al. 2008). For example, phenolic compounds make their actions through different mechanism, which includes membrane disruption, proteins binding, inhibition of proteins synthesis, enzymes inhibition, production of cell wall complexes, formation of disulfide bridges and intercalation with cell wall and/or DNA, among others (Bozdogan & Appelbaum 2004). In the same manner, the antimicrobial action of alkaloids could ]]>
Antifungal activities: These results correlate very well with those displayed by the crude extracts of Andrachne cordifolia Muell (Euphorbiaceae) (Ahmad et al. ]]>
Acalypha gaumeri Pax & K. Hoffm. and Croton chichenensis Lundell. which were active against the pathogenic fungi Alternaria tagetica, Colletotrichum gloeosporioides, Fusarium oxysporum and Rhizopus sp. (Gamboa-Angulo et al. 2008). ]]>
The antimicrobial activity evaluated in this work could be attributed to the presence of different phytocompounds in variable amounts in plant extracts (Table 1). The assayed antimicrobial activity from the plant species depend on the botanical species, the age, the part of the plant studied as well as the solvent used for ]]>
The fact that the most active extracts showed in this work were active against Gram-positive bacteria, is an important aspect, since many of the multidrug-resistant bacteria belong to this category where new chemotherapeutic agents are urgently needed to treat human diseases or to control foodborne microorganisms ]]>
Phytochemical screening: In general, the most active plant extracts from the Euphorbiaceae family displayed the presence of tannins and flavonoids; these facts are in ]]>
et al. 2003). The same way, the alkaloid isolated from the Solanaceae family may be the responsible for the biological activity displayed for these species in this work (Bruneton 2001, Niño et al. 2009). ]]>
Acknowledgments
The authors are very grateful to The Universidad Tecnológica de Pereira and COLCIENCIAS for the financial support of this project. In addition, the authors are also in debt with the CARDER corporation for granting permission to plant ]]>
References
Abdulladzhanova, N.G., S.M. Maviyanov & D.N. Dalimov. 2003. Polyphenols of certain plants of the Euphorbiaceae family. Chem. Nat. Compd. 39: 399-400. [ Links ]
Ahmad, B., S.M.H. Shah, S. Bashir, M. Nisar & M.I. Chaudry. 2007. Antibacterial and antifungal activities of Andrachne cordifolia Muell. J. Enzym. Inh. Med. Ch. 22: 726-729. [ Links ]
Ajali, U. 2000. Antibacterial activity of Alchornea cordifolia stem bark. Fitoterapia 71: 436-438. [ Links ]
Boussaada, O., J. Chriaa, R. Nabli, S. Ammar, D. Saidana, M.A. Mahjoub, I. Chraeif & A.N. Helal. 2008. Antimicrobial and antioxidant activities of methanol extracts of Evax pygmaea (Asteraceae) growing wild in Tunisia. World J. Microbiol. Biotechnol. 24:1289-1296. [ Links ]
Bozdogan, B. & P.C. Appelbaum. 2004. Oxazolidinones: activity, mode of action, and mechanism of resistance. Int. J. Antimicrob. Ag. 23: 113-119. [ Links ]
Davies, J. 2007. Microbes have the last word. EMBO Rep. 8: 616-621. [ Links ]
Epand, R.M., R.F. Epand & P.B. Savage. 2008. Ceragenins (cationic steroid compounds), a novel class of antimicrobial agents. Drug News Perspect. 21: 307-311. [ Links ]
Gamboa-Angulo, M.M., J. Cristóbal-Alejo, I.L. Medina-Baizabal, F. Chí-Romero, R. Méndez-González, P. Simá-Polanco & F. May-Pat. 2008. Antifungal properties of selected plants from the Yucatan peninsula, Mexico. World J. Microbiol. Biotechnol. 24: 1955-1959. [ Links ]
Harish, B.G., v. Krishna, R. Sharath, H.M. Kumar Swamy, H. Raja Naik & K.M. Mahadevan. 2007. Antibacterial activity of Celapanin, a sesquiterpene isolated from the leaves of Celestrus Paniculatus Wild Intl. J. Biomed. Pharma. Sci. 1: 65-68. [ Links ]
Holetz, F.B., G.L. Pessini, N.R. Sanches, D.A.G. Cortez, C.v. Nakamura & B.P. Dias Filho. 2002. Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases. Mem. Inst. Oswaldo Cruz 97: 1027-1031. [ Links ]
Mahida, Y. & J.S.S. Mohan. 2006. Screening of Indian plant extracts for antibacterial activity. Pharm. Biol. 44: 627-631. [ Links ]
Mohana, D.C., S. Satish & K.A. Raveesha. 2008. Antibacterial evaluation of some plant extracts against some human pathogenic bacteria. ABR. 3-4: 49-55. [ Links ]
Mothana, R.A.A., S.A.A. Abdo, S. Hasson, F.M.N. Althawab, S.A.Z. Alaghbari & U. Lindequist. 2010. Antimicrobial, antioxidant and cytotoxic activities and phytochemical screening of some Yemeni medicinal plants. eCAM. 7: 323-330. [ Links ]
Muthu, A.K., P. Sravanthi, D.S. Kumar, A.A. Smith & R. Manavalan. 2010. Evaluation of antibacterial activity of various extracts of whole plant of Borreria hispida (linn). IJPSR 1: 127-130. [ Links ]
Muthuvelan, B. & R. Balaji Raja. 2008. Studies on the efficiency of different extraction procedures on the anti microbial activity of selected medicinal plants. World J. Microbiol. Biotechnol. 24: 2837-2842. [ Links ]
Newman, D.J. & G.M. Cragg. 2007. Natural products as sources of new drugs over the last 25 years. J. Nat. Prod. 70: 461-477. [ Links ]
Okwu, D.E. & C. Josiah. 2006. Evaluation of the chemical composition of two Nigerian medicinal plants. Afr. J. Biotechnol. 5: 257-361. [ Links ]
Parmesha, M., S. Raghvendra, C.K. Ramesh, K.S. Manjunatha & G. Prakash. 2008. Antibacterial activity of some Euphorbiaceae weeds against pathogens. The Icfal. J. Life Sci. 2: 36-40. [ Links ]
Pretorius, J.C., S. Magama & P.C. Zietsman. 2003. Growth inhibition of plant pathogenic bacteria and fungi by extracts from selected South African plants species. S Afr. J. Bot. 20: 188-192. [ Links ]
Ríos, J.L., M.C. Recio & A. villar. 1998. Screening methods for natural products with antimicrobial activity: A review of the literature. J. Ethnopharm. 23: 127-149. [ Links ]
Wagner, H. & S. Bladt. 1996. Plant Drug Analysis. A thin layer chromatography atlas. Springer-verlag, Berlin, Germany. [ Links ]
*Correspondencia:Jaime Niño: Grupo de Biotecnología-Productos Naturales, Escuela de Tecnología Química, Universidad Tecnológica de Pereira, A.A. 97, Pereira, Colombia; janino@utp.edu.co Oscar M. Mosquera: Grupo de Biotecnología-Productos Naturales, Escuela de Tecnología Química, Universidad Tecnológica de Pereira, A.A. 97, Pereira, Colombia; omosquer@utp.edu.co Yaned M. Correa: Grupo de Biotecnología-Productos Naturales, Departamento de Química, Universidad de Caldas, A.A. 275, Manizales, Colombia; yaned.correa@ucaldas.edu.co 1. Grupo de Biotecnología-Productos Naturales, Escuela de Tecnología Química, Universidad Tecnológica de Pereira, A.A. 97, Pereira, Colombia; janino@utp.edu.co, omosquer@utp.edu.co 2. Grupo de Biotecnología-Productos Naturales, Departamento de Química, Universidad de Caldas, A.A. 275, Manizales, Colombia; yaned.correa@ucaldas.edu.co
Received 21-X-2011.Corrected 25-V-2012.Accepted 26-IV-2012.