Abstract
Sixty-five different extracts from 22 plants belonging to 6 of the most representative families in the Regional Natural Park Ucumarí (RNPU) were investigated for their in vitro antibacterial activity through the plate-well diffusion assay against the following bacteria: Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. The plant extracts were obtained by Soxhlet in n-hexanes, dichloromethane, and methanol. Of these, 32.31% of the extracts were active against B. subtilis, 10.77% exhibited activity against K. pneumoniae, 7.69% of the plant extracts showed antibacterial activity against P. aeruginosa, 9.23% were bioactive to S. aureus, and 4.61% were found to be active against E. coli. The methanol extracts from the plants studied exhibited the highest antibacterial activity.
Introduction
According to Hamburger and Hostettmann (Citation1991), the potential of higher plants as a source for new drugs is still largely unexplored. Among the estimated 250,000 to 500,000 plant species, only a small percentage has been phytochemically investigated, and the fraction submitted to biological or pharmacological screening is even smaller. Moreover, Rodriguez and West (Citation1995) considered that tropical plant species contain three- to four-times more active chemical constituents than those from the temperate zones.
The screening of plant extracts and natural products for antimicrobial activity has revealed the potential of higher plants as a novel source of agents to treat infectious diseases (Mitsher et al., Citation1987).
According to Brantner and Grein (Citation1994), in many areas around the world there is a rich tradition in the use of herbal medicine for the treatment of many infectious diseases. Recently, there has been an increased interest in antimicrobial agents from plant origin due to the resistance that pathogenic microorganisms have developed against synthetic antibiotics and to the rapid rate of plant species extinction (Recio et al., Citation1989; Essawi & Srour, Citation2000).
A literature review concerning the antibacterial activity of plant extracts and pure secondary metabolites constituents has shown that many studies have been carried out on this field by Olano et al. (Citation1996), Sanabria-Galindo et al. (Citation1998), Murphy (Citation1999), Essawi and Srour (Citation2000), Ali et al. (Citation2001), Lopez et al. (Citation2001), Moreno-Murillo et al. (Citation2001), and Cimanga et al. (Citation2002). Therefore, it is of great importance to carry out plant extracts screening from Regional Natural Park Ucumari (RNPU) in order to determine their antibacterial activity.
In this work, we report the results of the antibacterial screening of 22 plants (65 extracts) from RNPU flora.
Materials and Methods
Plant material
The plants used for this study were collected in February 2000 in different places at RNPU. They belong to the Asteraceae, Euphorbiaceae, Melastomataceae, Podocarpaceae, Rubiaceae, and Solanaceae families and were identified taxonomically by Dr. Francisco Javier Roldan. A voucher specimen of each plant was deposited at the Herbarium of Universidad de Antioquia, (Medellín, Colombia).
Aerial plant samples were oven dried at 40°C, grounded, and Soxhlet extracted successively with the following organic solvents: n-hexanes, dichloromethane, and methanol. The different extracts were concentrated at reduced pressure to dryness and stored at 4°C until assayed.
The plants collected at RNPU and studied for their preliminary phytochemical screening were reported by Niño et al. (Citation2003).
Antibacterial in vitro assays
The following bacteria strains were employed in the screening: Gram-positive Bacillus subtilis (ATCC 21556), Staphylococcus aureus (ATCC 6538), and the Gram-negative Klebsiella pneumoniae (ATCC 10031), Pseudomonas aeruginosa, (ATCC 27853) and Escherichia coli (ATCC 9637).
Antibacterial activity from the crude plant extracts was determined by the well diffusion method according to Rios et al. (Citation1988). Each plant extract was dissolved in 95% ethanol and tested at 5 different concentrations (10,000, 5000, 2500, 1250, and 625 ppm).
The microorganisms were cultured overnight at 37°C in a brain heart infusion (BHI) broth. Suspensions of each microorganism with a concentration of 106 colonies/ml were made in isotonic sodium chloride solution. Under sterile conditions and with homogenization, 1 ml of the respective bacterial suspension and 20 ml of the sterile Mueller Hinton agar were added to each Petri dish. After solidification, seven wells of 7-mm diameter were made in each plate also under sterile conditions. Each well was filled with 20 µl of one of the five different concentrations for each extract. Twenty microliters of the cefotaxime (250 ppm) and 20 µl of ethanol were used as positive and negative controls, respectively, for the different bacteria assayed; the exception was with P. aeruginosa where 20 μl of cefotaxime (500 ppm) was added on each positive control well. All plates were incubated at 37°C for 24 h. Inhibition zone diameter around each well was measured and recorded at the end of the incubation time.
Data analysis
The average for the inhibition zone was obtained from three replicates for each plant extract. The degree of inhibition for each extract was evaluated as weak, if the extract was capable to inhibit the growth of a bacteria assayed at one or two of the concentrations tested, and as moderate or strong if the extract was able to inhibit the growth of a bacteria at three or four out of five of the concentrations tested, respectively.
Results and Discussion
The 65 different crude plant extracts (n-hexanes, dichloromethane, and methanol) were screened for their antibacterial activity against five bacteria (two Gram-positive and three Gram-negative). The results are shown in .
Table 1 Antibacterial activity of crude plant extracts from RNUP.
As can be deduced from :
The crude methanol extracts gave the strongest effect against Gram-positive bacteria than those against gram-negative. Comparatively, the crude dichloromethane extracts gave equal activities as those from n-hexanes.
Miconia lehmannii Cong. and Tibouchina grossa (L. f.) Cong. (Melastomataceae) were the only active plant extracts against the five bacteria used in this research.
The three different extracts from Witheringia coccoloboides (Damn) Hunz (Solanaceae) exhibited strong activity against B. subtilis while the methanol extract from Miconia lehmannii Cong. gave strong activity against E. coli.
The dichloromethane extract from Hyeronima macrocarpa Muell. Arg. (Euphorbiaceae) gave moderate activity against B. subtilis and the methanol extract from Tibouchina grossa (L.f.) Cong. showed moderate activity against E. coli and K. pneumoniae.
The most active extracts against the five bacteria tested belong to the Melastomataceae family. This result correlates very well with those found for the antimycotic activity from the same plant extracts (Niño et al., Citation2003).
According to the preliminary phytochemical screening on the methanol crude extracts, it can be deduced that the phytocompounds related to the antibacterial activity could be alkaloids, phenols, tannins, lactones, or steroids.
Conclusions
The 65 extracts from the 22 plants related to 6 botanical families showed significant activity against the bacteria strains assayed, as follows: 11 were active against B. subtilis, 6 against S. aureus, 6 against K. pneumoniae, 5 against P. aeruginosa, and 3 against E. coli.
Under the experimental conditions of this research, the Melastomataceae family was the one with the higher antibacterial activity (36.6%), followed by the Euphorbiaceae (22.2%), Solanaceae (15.5%), Asteraceae (5%), and the least sensitive ones were those from Rubiaceae (1%) and Podocarpaceae (0%).
From this study, we can infer that the flora from RNPU has great potential in the search for new natural compounds with antibacterial activity and perhaps novel mechanism of action. A complete study should be conducted on the extracts with the strongest activities in order to isolate and identify the phytocompounds related to these properties.
Acknowledgments
The authors are very grateful to the Universidad Tecnológica de Pereira for financial support of the project. We also wish to offer thanks to the CARDER corporation for their assistance in granting the permission to collect the specimens used in this research.
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