Abstract
As part of a program oriented towards the discovery of bioactive natural products, 44 methanol extracts from 37 Brazilian traditional medicinal plants, most of them commonly used for treating conditions likely to be associated with microorganisms, were evaluated for their antibacterial activity and toxicity to brine shrimp. The agar-well diffusion method was used against Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhimurium, Shigella sonnei, Klebsiella pneumoniae, Escherichia coli, and Bacillus cereus. The active extracts were subjected to serial dilution assay for determination of the minimal inhibitory concentration. Phytochemical analysis of the extracts for their major groups of phytoconstituents is also reported. Extracts of Baccharis dracunculifolia, Cajanus cajan, Eugenia uniflora, Solanum palinacanthum and Solanum concinnum presented strong antibacterial activity with MIC values below 10 μg/mL for some bacterial strains. The extracts of Mikania glomerata and Leonurus sibiricus showed significant toxicity against brine shrimp with LC50 values of 63 and 86 μg/mL, respectively.
Introduction
Bacteria cause important human diseases, especially in tropical regions and in immunocompromised or immunodeficient patients. Despite the existence of potent antibiotic agents, resistant or multi-resistant strains are continuously appearing, imposing the need for permanent search and development of new drugs (CitationArora & Kaur, 2007; CitationAl-Fatimi et al., 2008). Many efforts have been made to discover new antimicrobial compounds from various kinds of sources such as microorganisms, animals, and plants. One such resource is folk medicine. Systematic screening of them may result in the discovery of novel effective compounds (CitationTomoko et al., 2002). It has been estimated that 77% of the pharmacologically active, plant-derived components were discovered after the ethnomedical uses of the plants started to be investigated (CitationCordell, 2000). Further, while it is estimated that there are 250,000 to 500,000 species of plants on Earth, only a small percentage (1 to 10%) of these are used as food by both humans and other animal species, leaving a huge potential for medicinal plant product development (CitationHeinrich & Gibbons, 2001). Contrary to synthetic drugs, antimicrobials of plant origin are not associated with many side effects and have an enormous therapeutic potential to heal many infectious diseases (CitationIwu et al., 1999).
It is believed that plants which are rich in a wide variety of secondary metabolites belonging to chemical classes such as tannins, terpenoids, alkaloids, polyphenols are generally superior in their antimicrobial activities (CitationCowan, 1999). This suggests that the strength of biological activities of a natural product is dependent on the diversity and quantity of such constituents. Therefore, simultaneous determination of the compounds which are possibly responsible for any biological activity would facilitate the decision-making process as to the selection of the plants for in-depth future investigation (Geyid et al., 2005). In view of this, we have also undertaken chemical screening of all the plants that were subjected to biological screening.
In this paper, we report the antibacterial and cytotoxic activity, and also phytochemical screening of 44 extracts from 37 species, most of them reported in Brazilian traditional medicine as useful in treating infectious diseases. In vitro antibacterial screening permits the selection of crude plant extracts with potentially useful properties to be used for further chemical and pharmacological studies (Machado et al., 2003). So, it is also hoped that the study will facilitate the selection, for further investigation, of plants with a relatively high level of potency and/or with wide range of biological activities.
Materials and Methods
Plant material
Specimens of 37 species () were collected in Juiz de Fora, Minas Gerais, Brazil, between March and May, 2004. A voucher specimen was deposited at the Herbarium Leopoldo Krieger (CESJ) of Federal University of Juiz de Fora.
Table 1. Ethnomedical data on medicinal plants.
Preparation of plant extracts
The dried parts of the plant (50 g each) were powdered and macerated with methanol (3 3 200 ml) for five days at room temperature. After evaporation of the solvent under reduced pressure, the respective methanol extracts were obtained. All the extracts were kept in tightly stoppered bottles under refrigeration (4°C) until used for the biological testing and phytochemical analysis.
Antibacterial assay
Test organisms
The test organisms used were Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 15442), Salmonella typhimurium (ATCC 13311), Shigella sonnei (ATCC 11060), Klebsiella pneumoniae (ATCC 13866), Escherichia coli (ATCC 10536) and Bacillus cereus (ATCC 11778).
Agar-well diffusion method
Qualitative antibacterial activity of the extracts was evaluated using the agar plate diffusion test (CitationPerez et al., 1990). An inoculum of each bacterial strain was suspended in Mueller Hinton broth and incubated overnight at 37°C. Standard Mueller-Hinton agar was used as a medium for bacteria cultivation. The extract was dissolved in sterile DMSO at a concentration of 100 mg/mL; 0.1 mL of diluted inoculum (105 CFU/mL) of tested organism was spread on agar plates. Wells of 6 mm diameter were punched into the agar medium and filled with 50 μL of plant extract. The plates were incubated for 24 h at 37°C. Chloramphenicol (30 μg) was used as the positive control. The diameters of the resulting inhibition zones were measured and, as the well was 6 mm, inhibition zones less than 7 mm were not considered. All assays were carried out in triplicate.
Serial dilution assay for determination of the minimal inhibitory concentration
The minimal inhibitory concentration (MIC) of the extracts was determined by using broth microdilution techniques as described by the National Committee for Clinical Laboratory Standards (CitationNCCLS, 2002) in microtiters of 96 wells. MIC values were determined in Mueller Hinton broth (MHB). Bacteria were cultured overnight at 37ºC for 24 h in Mueller Hinton agar. Extract stock solutions were two-fold diluted from 5 to 0.005 mg/mL (final volume = 80 μL) and a final DMSO concentration ≤ 1%. Then, 100 μL of MHB were added onto microplates. Finally, 20 μL of 106 colony forming units (CFU/mL) (according to McFarland turbidity standards) of standardized bacterial suspensions were inoculated onto microplates and the test was performed in a volume of 200 μL. Plates were incubated at 37°C for 24 h. The same tests were performed simultaneously for growth control (MHB + bacteria) and sterility control (MHB + extract). Chloramphenicol was used as reference compounds with concentrations ranging from 500 to 0.24 μg/mL. The MIC was calculated as the highest dilution showing complete inhibition of tested strain. Only extracts that showed antimicrobial activity from agar-well diffusion were tested for MIC.
Quantitative evaluation of antibacterial activity
There are different ways of expressing the antibacterial activity of plant extracts based on the technique used. The agar diffusion method measures the width of the inhibition zone (mm or cm), while the micro-dilution method yield MIC values, the minimum concentration at which inhibition is observed (mg/mL). In this work, new ways of expressing antibacterial efficiency as comparative numerical values are used. Total antibacterial activity of the most active extracts (MIC below 100 μg/mL) was determined (CitationEloff, 2004):
Cytotoxicity assay
A brine shrimp lethality bioassay (Meyer, 1992) was carried out to investigate the cytotoxicity of the extracts. Brine shrimp (Artemia salina Leach) eggs were hatched in a beaker filled with seawater under constant aeration. After 48 h the nauplii were collected by pipette and were counted macroscopically in the stem of the pipette against a lighted background. Solutions of the extracts were made in DMSO at varying concentrations (10 to 1000 μg/mL) and incubated in triplicate vials with the brine shrimp larvae. Ten brine shrimp larvae were placed in each of the triplicate vials. After 24 h of incubation, the nauplii were examined against a lighted background, with a magnifying glass and the number of survivors in each vial were counted and noted. The resulting data were transformed to the Probit analysis (CitationFinney, 1971) for the determination of LC50 values for the extracts. Crude extracts with LC50 values lower than 250 μg/mL were considered significantly active (CitationRieser et al., 1996). Both positive (thymol) and negative (sea water containing 1% DMSO) control assays were carried out in order to verify the susceptibility of A. salina under assay conditions employed.
Phytochemical studies
A portion of each extract that was subjected for the biological screening was used for the identification of the major secondary metabolites employing the protocols described by (Matos, 1987).
Results
The paper describes the antibacterial, cytotoxic activity and phytochemical profile of 44 methanol extracts belonging to 37 Brazilian traditional medicinal plants, most of them commonly used for treating conditions likely to be associated with microorganisms. shows the botanical name, local name, voucher specimen number and major popular uses of the plants tested.
The phytochemical profile and the results of the brine shrimp assay and antibacterial activity of the extracts, using the agar diffusion method, are summarized in .
Table 2. Cytotoxic and antibacterial activity of methanol extracts of the selected medicinal plants and their phytocompounds.
According to the results of the agar diffusion assay, antibacterial activity against all bacteria tested were found for the following extracts: Pyrostegia venusta, Plantago lanceolata, Polygonum hydropiperoides, Ocimum gratissimum and Rosmarinus officinallis. It was also observed that Pseudomonas aeruginosa and Bacillus cereus were susceptible to all extracts tested and that 19 extracts were active against Staphylococcus aureus, 40 extracts against Salmonella thyphimurium, 36 extracts against Shigella sonnei, 21 extracts against Klebsiella pneumoniae and 12 extracts against Escherichia coli. The extracts that presented any antibacterial activity in this assay were subjected to the MIC evaluation and the results are presented in . Because the MIC value is inversely related to the quantity of antibacterial compounds present, an arbitrary measure of the quantity of antibacterial compounds present was calculated by dividing the quantity extracted in mg from 1 g plant material by the MIC value in mg/mL. This total activity () value indicates the volume to which the biologically active compound present in 1 g of the dried plant material can be diluted and still inhibits the growth of bacteria (CitationEloff, 2004).
Table 3. MIC of methanol extracts of selected medicinal plants.
Table 4. Total activity of the methanol extracts with MIC values below 100 μg/mL.
Discussion
According to CitationCos et al. (2006), plant extracts with MIC values below 100 μg/mL are very promising. Significant antibacterial effects against Bacillus cereus, Pseudomonas aeruginosa, Staphylococcus aureus and Shigella sonnei were presented by 20 extracts at the concentrations of 5 to 78 μg/mL. Baccharis dracunculifolia, Cajanus cajan, Eugenia uniflora, Solanum palinacanthum and Solanum concinnum were the most active with the MIC values less than 10 μg/mL (). To determine which plants can be used for further testing and isolation, not only the MIC value is important, but also the total activity (CitationEloff, 1999).
Total activity () revealed that leaf extract of Cajanus cajan has the higher magnitude of antibacterial activity, as the antibacterial component(s) from this plant can be diluted in 30000 mL of solvent and still inhibits growth of Shigella sonnei (total activity = 30000 mL/g), followed by Eugenia uniflora (leaves) against Pseudomonas aeruginosa (26000 mL/g), Baccharis dracunculifolia (aerial parts) against Bacillus cereus and Pseudomonas aeruginosa (22000 mL/g), Solanum palinacanthum (seeds) against Pseudomonas aeruginosa (22000 mL/g) and Solanum concinnum (leaves) against Staphylococcus aureus (14000 mL/g).
It has frequently been reported that Gram (+) bacteria are more susceptible to plant antibiotic substances than Gram (2) bacteria (CitationBurn, 1988). However, our results did not show any selectivity towards Gram (+) bacteria. Among the most active species, Baccharis dracunculifolia, Cajanus cajan and Eugenia uniflora have already been studied for their antimicrobial effects elsewhere. Baccharis dracunculifolia, a native plant from Brazil, is the most important botanical origin for the production of the Brazilian green propolis by honeybees. It produced a bacteriostatic effect on Streptococcus mutans cultures at a concentration of 0.4 mg/mL, suggesting an anticariogenic effect (CitationLeitão et al., 2004). A diversity of extracts from Eugenia uniflora showed positive results against Aspergillus flavus, Bacillus subtilis, Escherichia coli, Klebsiella aerogenes, Mycobacterium phlei, Proteus vulgaris, Pseudomonas aeruginosa, Sarcina lutea, Serratia marcescens, Shigella dysenteriae, Staphylococcus aureus, Trichophyton mentagrophytes (CitationFadeyi & Akpan, 1989; CitationAdebajo et al., 1989), Micrococcus luteus (CitationCoelho de Souza et al., 2004), Candida albicans and Cryptococcus neoformans (CitationBraga et al., 2007). Cajanus cajan showed relatively large inhibition zone when tested against Pseudomonas cichorii, Bacillus subtilis, Salmonella typhimurium and Escherichia coli (CitationKumar et al., 1997), Candida albicans and Cryptococcus neoformans (CitationBraga et al., 2007). Antibacterial activity for Solanum palinacanthum and Solanum concinnum was reported here for the first time.
The brine shrimp lethality assay consists of exposing larvae to plant extract in saline solution and lethality is evaluated after a day. The commercial availability of inexpensive brine shrimp eggs, the low cost, safety, and a feasible protocol, as well as the lack of a need for special equipment, make this method a very helpful bench-top tool for a phytochemistry laboratory (CitationMcLaughlin, 1991). First developed by Meyer (1992), this assay has wide application in the discovery of cytotoxic and other active principles present in plant extracts (CitationMongelli et al., 1996). The results of the brine shrimp toxicity assay can be analyzed in two different ways. Firstly, toxic plant extracts often contain physiologically active constituents and are therefore interesting for further investigation. Secondly, if the test is employed in combination with other mechanism-based test systems, there should be little or no toxicity with regard to a specific mechanism of action in the specific way (CitationNick et al., 1995).
For example, a very positive correlation between the lethality to brine shrimp and antitumoral activity has been established by researchers working on the development of new anticancer drugs from plants at the National Cancer Institute (NCI) in the United States (CitationAnderson et al., 1991). This correlation is considered so good that lethality to brine shrimp is recommended by these authors as an effective pre-screen to existing cytotoxicity and antitumor assays. More recently, it has been shown that there is a very good correlation between the median lethal concentrations (LC50) of plants extracts to brine shrimp larvae and the median lethal doses (LD50) of these same extracts, administered orally in mice (CitationParra et al., 2001). A number of studies have demonstrated the use of the brine shrimp assay to screen plants popularly used as pesticides (CitationMcLaughin & Rogers, 1998), plants having ethnomedical uses related to cancer (CitationMongelli et al., 1996), and trypanocidal activity (CitationZani et al., 1995). In this assay, six out of the 44 extracts screened showed LC50 values smaller than 250 μg/mL, being the extracts of Mikania glomerata and Leonurus sibiricus, the most cytotoxic with LC50 lower than 100 μg/mL (). It would seem that many of the extracts tested in this study for antibacterial activity do not possess toxic effects. It was observed that there were many cases where plant extracts had antibacterial activity but no brine shrimp lethality ( and ).
The phytochemical profile of the extracts is summarized in . Biological activity of the extracts appears to be due to the presence of secondary metabolites such as flavonoids identified in 86% of the plant species, alkaloids (75%), triterpenoids (68%) tannins (59%), antraquinones (39%), steroids (34%), coumarins (32%) and saponins (23%). The most active extracts accumulate flavonoids and alkaloids.
Conclusion
The antibacterial study of 37 plants has identified various extracts with activity against several pathogenic bacteria which might explain their ethnomedical uses for the treatment of various infectious diseases. The plants with the greatest potential as targets for bioassay-guided fractionation are Baccharis dracunculifolia, Cajanus cajan, Eugenia uniflora, Solanum palinacanthum and S. concinnum.
Acknowledgements
The authors are grateful to Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Universidade Federal de Juiz de Fora (UFJF)/Brazil for financial support and to Dr. Fatima Regina Salimena for the botanical identification of some species.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References
- Adebajo AC, Oloki KJ, Aladesanmi AJ (1989): Antimicrobial activity of the leaf extract of Eugenia uniflora. Phytother Res 36: 258–259.
- Al-Fatimi M, Wurster M, Schroder G, Lindequist U (2008): Antioxidant, antimicrobial and cytotoxic activities of selected medicinal plants from Yemen. J Ethnopharmacol 111: 657–666.
- Anderson E, McLaughlin JL, Suffness M (1991): A blind comparison of simple bench-top bioassay and human tumor cell cytotoxities as antitumor prescreens. Phytochemistry 2: 107–111.
- Arora DS, Kaur GJ (2007): Antibacterial activity of some Indian medicinal plants. J Nat Med 61: 313–317.
- Braga FG, Bouzada MLM, Fabri RL, Matos MO, Moreira FO, Scio E, Coimbra ES (2007): Antileishmanial and antifungal activity of plants used in traditional medicine in Brazil. J Ethnopharmacol 111: 396–402.
- Burn P (1988): Amphitropic proteins: A new class of membrane proteins. Trends Biochem Sci 13: 79–83.
- Cáceres A, López B, González S, Berger I, Tada I, Maki J (1998): Plants used in Guatemala for the treatment of protozoal infections. I. Screening of activity to bacteria, fungi and American trypanosomes of 13 native plants. J Ethnopharmacol 62: 195–202.
- Chabot S, Bel-Rhlid R, Chenevert R, Piche Y (1992): Hyphal growth promotion in vitro of the VA mycorrhizal fungus, Gigaspora margarita Becker and Hall, by the activity of structurally specific flavonoid compounds under CO2-enriched conditions. New Phytol 122: 461–467.
- Coelho de Souza G, Haas APS, von Poser GL, Schapoval EES, Elisabetsky E (2004): Ethnopharmacological studies of antimicrobial remedies in the south of Brazil. J Ethnopharmacol 90: 135–143.
- Cordell GA (2000): Biodiversity and drug discovery - a symbiotic relationship. Phytochemistry 55: 463–480.
- Corrêa MP (1984): Dicionário das plantas úteis do Brasil e das exóticas cultivadas. Rio de Janeiro, Instituto Brasileiro de Desenvolvimento Florestal, Vol. 1–6.
- Cos P, Vlietinck AJ, Berghe DV, Maes L(2006): Anti-infective potential of natural products: How to develop a stronger in vitro ‘proof-of-concept’. J Ethnopharmacol106: 290–302.
- Cowan MM (1999): Plant products as antimicrobial agents. Clin Microbiol Rev 12: 564–582.
- Dixon RA, Dey PM, Lamb CJ (1983): Phytoalexins: Enzymology and molecular biology. Adv Enzimol 55: 1–69.
- Duarte MCT, Figueira GM, Sartoratto A, Rehder VLG, Delarmelina C (2005): Anti-Candida activity of Brazilian medicinal plants. J Ethnopharmacol 97: 305–311.
- Dunstan C A, Noreen Y, Serrano G, Cox P A, Perera P, Bohlin L (1997): Evaluation of some Samoan and Peruvian medicinal plants by prostaglandin biosynthesis and rat ear edema assays. J Ethnopharmacol 57: 35–56.
- Eloff J N (1999): Is it possible to use herbarium specimens to screen for antibacterial components in some plants. J Ethnopharmacol 67: 355–360.
- Eloff JN (2004): Quantifying the bioactivity of plant extracts during screening and bioassay guided fractionation. Phytomedicine 11: 370–371.
- Fadeyi MO, Akpan UE (1989): Antibacterial activities of the leaf extracts of Eugenia uniflora Linn. (synonym Stenocalix michelli Linn.) Myrtaceae. Phytother Res 3: 154–155.
- Finney DJ (1971): Probit Analysis. Cambridge, Cambridge University Press, p. 31.
- Geyid A, Abebe D, Debella A, Makonnen Z, Aberra F, Teka F, Kebede T, Urga K, Yersaw K, Biza T, Mariam BH, Guta M (2005): Screening of some medicinal plants of Ethiopia for their antimicrobial properties and chemical profiles. J Ethnopharmacol 97: 421–427.
- Ghazal SA, Abuzarqa M, Mahasneh AM (1992): Antimicrobial activity of Polygonum equiseriforme extracts and flavonoids. Phytother Res 6: 265–269.
- Heinrich M, Gibbons S (2001): Ethnopharmacology in drug discovery: An analysis of its role and potential contribution. J Pharm Pharmacol 53: 425–432.
- Howard MD, Chittawong V, Payne AM (1990): Cotton boll weevil antifeedant activity and antifungal activity (Rhizoctonia solani and Pythium ultimum) of extracts of the stems of Wedelia biflora. J Agr Food Chem 38: 1591–1594.
- Iwu MW, Duncan AR, Okunji CO (1999): New antimicrobials of plant origin, in: Janick, J.(Ed.), Perspectives on New Crops and New Uses. ASHS Press, pp. 457–462.
- Kumar S, Bagchi GD, Darokar MP (1997): Antibacterial activity observed in the seeds of some coprophilous plants. Int J Pharmacog 35: 179–184.
- Leitão DPD, Silva da AA, Polizello ACM, Bastos JK, Spadaro ACC (2004): Comparative evaluation of in vitro effects of Brazilian green propolis and Baccharis dracunculifolia extracts on cariogenic factors of Streptococcus mutans. Biol Pharm Bull 27: 1834–1839.
- Lima LA, Siani AC, Brito FA, Sampaio ALF, Henriques MGMO, Riehl CAS (2007): Correlation of anti-inflammatory activity with phenolic content in the leaves of Syzygium cumini (l.) skeels (Myrtaceae). Quím Nova 30: 860–864.
- Lorenzi H, Matos FJA (2002): Plantas medicinais do Brasil. São Paulo, Nova Odessa-Instituto Plantarum, p. 512.
- Machado TB, Pinto AV, Pinto MCFR, Leal ICR, Silva MG, Amaral ACF, Kuster RM, Netto dos Santos KR (2003): In vitro activity of Brazilian medicinal plants, naturally occurring naphthoquinones and their analogues, against methicillin-resistant Staphylococcus aureus. Int J Antimicrob Ag 21: 279–284.
- Mahasneh AM, Abbas JA, Eloqlah AA (1996): Antimicrobial activity of extracts of herbal plants used in the traditional medicine of Bahrain. Phytother Res 10: 251–253.
- Matos FJA (1997): Introdução à Fitoquímica Experimental. Fortaleza, EUFC.
- McLaughin JL, Rogers LL (1998): The use of biological assays to evaluate botanicals. Drug Inf J 32: 513–524.
- McLaughlin JL (1991): Crown gall tumours on potato discs and brine shrimps lethalithy: Two simple bioassays for higher plant screening and fractionation, in: Hostettmann Ked.,pd., Assays for Bioactivity, Academic Press, San Diego, pp. 1–32.
- Meyer BN, Ferrigni NR, Putnam, JE, Jacobsen LB, Nichols DE, McLaughlin JL (1982): Brine shrimp: A convenient general bioassay for active plant constituents. Planta Medica 45: 31–34.
- Mongelli E, Martino V, Coussio J, Ciccia G (1996): Screening of Argentine medicinal plants using the brine shrimp microwell cytotoxicity assay. Int J Pharmacog 34: 249–254.
- NCCLS (National Committee for Clinical Laboratory Standards) (2002): Reference method for broth dilution antifungal susceptibility testing of yeasts, in: Approved standard M27–A2. Wayne, PA, NCCLS.
- Nick A, Rali T, Sticher O (1995): Biological screening of traditional medicinal plants from Papua New Guinea. J Ethnopharmacol 49: 147–156.
- Parra AL, Yhebra RS, Sardinas IG, Buela LI (2001): Comparative study of the assay of Artemia salina L. and the estimate of the medium lethal dose (LD50 value) in mice, to determine oral acute toxicity of plant extracts. Phytomedicine 8: 395–400.
- Paulo W, Ribeiro JEG, Pinto DS (2000): Safety and efficacy of Eupatorium laevigatum paste as therapy for buccal aphthae: Randomized, double-blind comparison with triamcinolone 0.1% oral base. Adv Ther 17: 272–281.
- Perez C, Pauli M, Bazerque P (1990): An antibiotic assay by the well agar method. Acta Biologiae et Medicine Experimentalis 15: 113–115.
- Phillipson JD, O`Neill MJ (1989): New leads to the treatment of protozoal infections based on natural product molecules. Acta Pharmaceutica Nordica 1: 131–144.
- Rieser MJ, Gu ZM, Fang L, Wood KV, McLaughlin JL (1996): Five novel mono-tetrahydrofuran ring acetogenins from the seeds of Annona muricata. J Nat Prod 59: 100–108.
- Rios JL, Recio MC (2005): Medicinal plants and antimicrobial plants from Psydrax and Oxyanthus species. Phytochemistry 31: 567–570.
- Sato M, Fujiwara S, Tsuchiya H, Fujii M, Iinuma M, Tosa H, Ohkawa Y (1996): Flavones with antibacterial activity against cariogenic bacteria. J Ethnopharmacol 54: 171–176.
- Sheldon J, Balick MJ, Laird SA (1997): Medicinal Plants: Can utilization and conservation coexist? New York, The New York Botanical Garden p. 104.
- Tomoko N, Takashi A, Hiromu T, Yuka I, Hiroko M, Munekazu I, Totshiyuki T, Tetsuro I, Fujio A, Iriya I, Tsutomu N, Kazuhito W (2002). Antibacterial activity of extracts prepared from tropical and subtropical plants on methicillin-resistant Staphylococcus aureus. J Health Sci 48: 273–276.
- Tsuchiya H, Sato M, Miyazaki T, Fujiwara S, Tanigaki S, Ohyama M, Tanaka T, Linuma M (1996): Comparative study of the antibacterial activity of phytochemical flavanones against methicillin-resistant Staphylococcus aureus. J Ethnopharmacol 50: 27–34.
- Zani CL, Chaves PPG, Queiroz R, De Oliveira AB, Cardoso JE, Anjos AMG, Grandi TSM (1995): Brine shrimp lethality assay as a prescreening system for anti-Trypanosoma cruzi activity. Phytomedicine 2: 47–50.