*Dirección para correspondencia

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Abstract

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.

Key words: antimicrobial activity, bactericide, bioprospection, fungicide, MIC, well diffusion assay.

Resumen

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.

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Crude  plant  extracts  have  been  used  in folk medicine for treatment of abscesses, insect bites, mycosis, inflammations, intestinal helminths,  diarrhoea  among  others  ills  (Holetz 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 patients with bacteremia nowadays is becoming more complicated, because the increasing microbial resistance against the limited number of available commercial antimicrobial agents (Davies 2007). In addition, pathogenic strains of bacteria and fungi are especially prevalent in immunecompromised patients, causing many deceases annually. Hence, alternative therapies are urgently needed to treat patients affected by pathogenic microorganisms.

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.,  a temperature average 14°C  and  an  rainfall  average  of  2 700mm/ year. The plants were identified taxonomically by  Dr. F.J.  Roldán  and  a  voucher  specimen for each plant material was deposited at the University of Antioquia Herbarium, Medellín, Colombia (Table 1).

Each  plant  sample  collected  was  oven dried at 50ºC with forced air for 72h. The dry materials were ground to a fine powder and aliquots of 300g were extracted by maceration successively with 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 pressure and the extracts were stored at -10ºC until further processing (Niño 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 pneumoniae (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 for 18h; while, the fungal strains were subcultured on a Sabouraud dextrose agar (Becton, Dickinson and Co. Sparks, USA) at 25ºC for 72h.

The antibacterial activities of crude extracts were  evaluated  through the agar-well  diffusion assay (Ríos et al. 1998). The n-hexanes, dichloromethane and methanol extracts were resuspended in ethanol and tested at concentrations of 4.00, 2.00, 1.00, 0.50 and 25mg/ml; for the different bacteria assayed, cefotaxime at 25mg/ml was used as positive control, with the exception of P. aeruginosa, for which 0.5mg/ml concentration was employed. In all antimicrobial assays ethanol was used as negative control.

The procedures for the antimycotic assays were described by Niño 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 following chromogenic agents: Dragendorff, anisaldehyde-sulfuric acid, vainillin  (1%)  in sulfuric acid-ethanol, ferric chloride (1%) and aluminum trichloride (2%) in ethanol, in order to search for: alkaloids, sterols, terpenes, saponins, phenols, tannins and flavonoids, respectively. All determinations were done in triplicate and standards for the respective natural product assayed were used.

The results for both the antibacterial and antimycotic assays were recorded for each plant extract by determining the concentration in which the inhibition zones near each well were visualized. All tests were carried out in triplicate  and  repeated  twice. Then, the minimum inhibitory concentration (MIC) was obtained.

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 antibacterial activities investigated were displayed mainly by Gram-positive bacteria, where 36% of the methanol extracts showed antibacterial activity against 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 plant extracts displayed activity  against  the  Gram-negative  bacteria, 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 species related to the Euphorbiaceae family where the extracts from 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 addition, the 60% of the methanol extracts from members of the Solanaceae family showed activities against the bacteria tested. 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 of the Asteraceae family; however, the dichloromethane and 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. solani, 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 Indian methanol plant extracts (Mahida & Mohan 2006). In addition, these results are related to the outstanding activity displayed by the crude extracts of some Indian Euphorbiaceae weeds against P. aeruginosa (Parmesha et al. 2008).

Results displayed by the Solanaceae family correlate very well with those obtained in the antibacterial assay performed with the alkaloids dimissidine, dihydrosolacongestidine and solasodine isolated from 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 methanol extract of 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 pygmaea (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 antibacterial activity was showed by the methanol extracts of the species evaluated followed by the 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 extracted by 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  and  may  not  be the consequence of their action on a single target. In addition, the phenomenon of membrane bleeding has been observed with several antimicrobial agents (Epand 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 be throughout intercalation with cell wall and/or DNA constituents; while, terpenoids display their action through membrane disruption mechanisms (Cowan 1999).

Antifungal activities: These results correlate  very  well  with  those  displayed  by  the crude extracts of Andrachne cordifolia Muell (Euphorbiaceae) (Ahmad et al. 2007), as well as the root extracts of 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 extraction procedures (Mahida & Mohan 2006).

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 that originate food spoilage due to microbial resistance to some antimicrobial agents used nowadays in food preservation. These results also provides valuable information for further isolation and characterization studies of active phytocompounds, necessary for the development of new drugs.

Phytochemical screening: In general, the most active plant extracts from the Euphorbiaceae family displayed the presence of tannins and flavonoids; these facts are in consonance with the polyphenols content of some plants from the Euphorbiaceae family that showed condensate and hydrolysable tannins, flavonoids, among others, responsible for their biological activities (Abdulladzhanova 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 collection.

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*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

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