Abstract
The antimicrobial activity of the crude ethanol extract from Pimenta pseudocaryophyllus (Gomes) L. R. Landrum (Myrtaceae), collected at two locations in Brazil, was investigated. Leaf samples were collected in April 2005 and in September 2005, both in Brasília, DF, Brazil, and in July 2000 in São Gonçalo do Abaeté, MG, Brazil. They were dried, crushed and used to obtain three crude ethanol extracts. The agar diffusion test was used for antimicrobial activity screening and the agar dilution method for determining the minimal inhibitory concentration (MIC). In assay conditions, extracts I, II and III demonstrated antimicrobial activity against Gram-positive Staphylococcus aureus, Micrococcus luteus, M. roseus, Bacillus cereus, B. atrophaeus, and B. stearothermophilus (MIC 0.39062 to 12.5 mg/mL, MIC 0.78125 to 1.5625 mg/mL and MIC 0.39062 to 1.5625 mg/mL, respectively), against Gram-negative Pseudomonas aeruginosa (MIC 0.39062 to 3.125 mg/mL, MIC 1.5625 mg/mL and MIC 0.78125 to 1.5625 mg/mL, respectively), against clinical isolates of Pseudomonas stutzeri (MIC 0.39062 to 0.78125 mg/mL, MIC 0.78125 to 1.5625 mg/mL, MIC 0.78125 to 1.5625 mg/mL, respectively) and also against Candida albicans fungi (MIC 0.19531 mg/mL for the extracts I, II and III). This study showed that the antimicrobial activity of P. pseudocaryophyllus might be considered sufficient to encourage further studies to isolate and identify its active principles. Pharmacological and toxicological studies are also necessary, followed by studies regarding the culturing and managing processes of this vegetable.
Introduction
For the past five decades, the use of antibiotic or antimicrobial substances has represented one of the greatest advances in pharmacotherapy. These substances are among the most common pharmaceutical drugs used in hospital settings and have drastically reduced the risk of infection in the human body. However, microbial resistance is a major problem in the hospital environment, where the use of antimicrobials is imperative (CitationGales et al., 2005; CitationHsueh et al., 2005; CitationMeyer et al., 2005; CitationZhanel et al., 2005). Many infections caused by emerging or multiresistant microorganisms remain without effective therapeutic options, not to mention the serious side effects of some of the drugs available (CitationLima, 2001; CitationTavares, 2001; CitationHardman et al., 2003).
Microorganisms acquire resistance to antimicrobial substances faster than new antimicrobial agents can be developed. Therapeutics have been strongly affected by knowledge concerning new standards of microbial resistance altering choice, dosage and substance combination. The discovery of new, safer and more specific drugs is, therefore, urgent (CitationHelfand & Bonomo, 2005; CitationMeyer, 2005; CitationThomson & Bonomo, 2005).
The development of new antimicrobial agents can be greatly aided by research involving natural products (CitationLima, 2001). The diversity of compounds found in plant species make these organisms promising sources of new antimicrobial agents, with general or specific effects. These findings have motivated several studies in different parts of the world (CitationSouza et al., 2002; CitationCunico et al., 2004; CitationZuque et al., 2004; CitationHernández et al., 2005; CitationMichelin et al., 2005; CitationYasunaka et al., 2005; CitationBugno et al., 2007; CitationOliveira et al., 2007; CitationNair & Chanda, 2007).
Brazil is recognized as a mega-diversity country (CitationMittermeier et al., 1997) and Brazilian folk medicine is substantial. Therefore, the plant Pimenta pseudocaryophyllus (Gomes) L.R. Landrum (Myrtaceae) was selected for this study using an ethno-pharmacological approach. This species is known as “pau-cravo” or “craveiro” and occurs in the Brazilian Atlantic Forest and also rarely in regions of the Brazilian savannah. It is a 4-10 m tall tree. Its leaves are elliptical, obovate or elliptic-oblanceolate, densely covered with long unicellular hairs below and glabrous above. Its inflorescence is a dichasium or a panicle (CitationLandrum, 1986; CitationLorenzi, 2002).
In the folk medicine of São Goncalo do Abaeté, MG, Brazil, the leaves are used to make tea for influenza. In the county of Campos do Jordão, SP, Brazil, the leaves are infused to make a soothing tea as well as to regulate digestion and menstruation (CitationNakaoka-Sakita et al., 1994). According to CitationLima et al. (2006), the tea from the leaf is also used as refreshment and as a diuretic. The literature data supports that there is antimicrobial activity in the essential oils of two specimens of P. pseudocaryophyllus, collected at two locations in São Paulo state, Brazil, against Candida albicans (ATCC 10231), Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027) and Staphylococcus aureus (ATCC 6538) (CitationLima et al., 2006). The main purpose of this study is to assay the in vitro antimicrobial activity of the crude ethanol extract obtained from the leaves of three samples of P. pseudocaryophyllus collected in two locations in Brazil.
Material and methods
Plant material
P. pseudocaryophyllus leaves were collected in two counties: In São Gonçalo do Abaeté, MG, Brazil at 18°20’58.4” South; 45°55’23.4” West, 864 m altitude, in June 2000, in the fructification period (post-anthesis). The plant material was identified by Carolyn Elinore Barnes Proença, a specialist in Myrtaceae from the University of Brasilia and a voucher specimen was placed in the Herbarium of the Federal University of Goiás, number UFG-27.159.
In Brasília, DF, Brazil at 15°51′51.6″ South; 47°49′43″ West and at 767 m altitude, one sampling was taken in April 2005, during fructification (post-anthesis) and another in September 2005, during flowering (anthesis). These samplings were collected at about 10:00 am in sunny weather. A voucher specimen of the plant material was placed in the Ezechias Paulo Heringer Herbarium of Brasilla Botanical Garden, DF, Brazil, number 21, 745-0, and identified by specialists from this institution.
To obtain the pulverized plant material the leaves were oven dried with forced ventilation at 40°C and then crushed to a fine powder. The pulverized material was used to obtain the crude ethanol extract.
Crude ethanol extract preparation
The crude ethanol extract was obtained by cold maceration according to methodology adapted by CitationFerri (1996). Five parts of ethanol 95% (V/V) were added to one part pulverized plant material and submitted to mechanical agitation for 4 h. It was then filtered in filter paper and concentrated in a Rotary evaporator. The procedure was repeated twice more. The extracts obtained as previously described for each sample of both specimens were identified and stored at room temperature.
Antimicrobial activity screening by the agar diffusion method
The antimicrobial activity screening of the extracts was performed as recommended by the National Committee for Clinical Laboratory Standard (CitationNCCLS, 2003), currently known as Clinical and Laboratory Standards Institute (CLSI). Standard American Type Culture Collection (ATCC) strains were used ().
Table 1. Microorganism strains used in triage.
The microorganisms stocked in simple inclined agar (SIA) at 4°C were subcultured in a thioglycollate broth and were incubated at 37°C for 24 to 48 h for reactivation of the cultures. Following this, they were subcultured in SIA plates and incubated at 37°C for 24 h. Microbial suspensions were then prepared, adding an inoculum to 2 mL of sterile saline solution 0.85% (W/V) until turbidity 1 on the MacFarland scale was obtained. Next, 100 μL of the microbial suspension obtained were added to 10 mL of Mueller Hinton liquefied agar, homogenized and transferred rapidly onto the plates that contained a base layer of 20 mL of Mueller Hinton agar. Orifices of 5-mm diameter were distributed in a circle at equidistant points on the plate leaving the center without orifice.
The crude ethanol extract of each sample was solubilized in ethanol 95% (V/V) at a 1:1 ratio and 10 μL was inoculated into each orifice. This step was performed in triplicate. A control was made with the solvent used in the dilution of the extract. The center of the Gram-positive bacteria plates received a disk of penicillin (Oxoid® 10μg) as positive control, the Gram-negative plates received a disk of erythromycin (Oxoid® 15μg) as positive control. The plates were incubated at 37°C for 24 h. The diameter of the inhibition halos was then measured.
Minimal inhibitory concentration (MIC) bioassay
The agar dilution method was used according to the NCCLS recommendation (2003). Double dilution series were made in 9 test tubes for each sample: 1 g of each crude ethanol extract was added to a first sterile test tube and the volume was completed to 2 mL with ethanol 95% (V/V), then 1 mL of sterile distilled water was added to a sequence of 8 sterile test tubes, an aliquot of 1 mL was transferred from the first test tube to the second and so on, successively, until the ninth tube, and at the end an aliquot of 1 mL was discarded.
Next, 19 mL of liquefied Mueller Hinton agar were added, then homogenized and quickly transferred onto the plate. In this manner the plates obtained contained the crude ethanol extract of the specimens under study in concentrations ranging from 25 to 0.0976 mg/mL. Control plates were prepared containing the solvent used in the extraction along with Mueller Hinton agar and also containing only Mueller Hinton agar.
The strains used were the standard American Type Culture Collection (ATCC) shown in (except for Serratia marcescens ATCC 14756) and Micrococcus luteus ATCC 9341. Clinical isolates of Staphylococcus aureus and Pseudomonas stutzeri kept in the Bacterial Culture Collection in the Bacteriology Laboratory in the Tropical Pathology and Public Health Institute of the Federal University of Goiás, Brazil, were also used ().
Table 2. Microorganisms obtained from clinical isolates used in MIC determination.
The microorganisms were subcultured in thioglycollate broth and incubated at 37°C for 24 to 48 h for reactivation. Next, they were subcultured in SIA plates and once again incubated at 37°C for 24 h. After the microbial growth, inocula of each microorganism were prepared in 2 mL of sterile saline solution 0.85% (W/V) standardized to an opacity equivalent to 1 on the MacFarland scale. 100 μL of each inoculum was transferred to the Steers inoculator (CitationSteers et al., 1959), applied on the surface of the plates containing the extract dilutions and their respective controls and subsequently incubated at 37°C for 24 h. The smallest concentration capable of inhibiting microbial growth was considered to be the MIC.
Results and discussion
Antimicrobial activity screening by the agar diffusion method
The crude ethanol extracts of the samples of P. pseudocaryophyllus analyzed by agar diffusion test presented antimicrobial activity against all Gram-positive bacteria tested. They presented traces of growth inhibition in C. albicans ATCC 1023. Regarding the Gram-negative bacteria, they presented antimicrobial activity against both strains of P. aeruginosa studied and against S. marcescens ATCC 14756, but did not inhibit the growth of Enterobacter aerogenes ATCC 13048, nor E. cloacae ATCC HMA/FT502 or both E. coli strains analyzed ().
Table 3. Halo averages (mm) of microbial growth inhibition obtained in the agar diffusion test using crude ethanol extracts of P. pseudocaryophyllus specimens and controls.
Several authors have studied the antimicrobial activity of essential oils and plant extracts and have found activity against Gram-positive bacteria (CitationCunico et al., 2004; CitationZuque et al., 2004; CitationFernandes et al., 2005; CitationHernández et al., 2005; CitationMichelin et al., 2005; CitationYasunaka et al., 2005; CitationBugno et al., 2007; CitationOliveira et al., 2007; CitationNair & Chanda, 2007). The antimicrobial activity against Gram-positive bacteria B. cereus, B. atrophaeus and B. stearothermophilus observed in this study was relevant, since these are spore-forming microorganisms, and can survive under adverse conditions for several years. S. aureus is the most common cause of pyogenic infections on the skin or in deeper regions and commonly inhabits the nasal cavity of 40-50% of all human beings (CitationBrooks et al., 2004). Considering the clinical importance of this microorganism, the selection of a few clinical isolates, beside the standard ATCC 29213 strains, was performed in order to determine the MIC of the crude ethanol extracts of P. pseudocaryophyllus.
None of the three extracts inhibited E. coli ATCC 8739 or ATCC 25922 strains. Analogous results were also observed in other plant species (CitationZuque et al., 2004; CitationFernandes et al., 2005; CitationYasunaka et al., 2005).
The P. pseudocaryophyllus extracts did not inhibit E. aerogenes ATCC 13048 and E. cloacae ATCC HMA/TF502, enterobacteria responsible for extra intestinal infections and the cause of a great number of hospital infections. CitationNair and Chanda (2007) found similar results for E. aerogenes ATCC 13048 when researching Psidium guajava L. leaf extracts. On the other hand, the enterobacteria S. marcescens ATCC 14756, which belongs to the same group, was inhibited by the tested extracts.
Although the group of Gram-negative bacteria mentioned above did not present inhibition in the agar diffusion test, all three extracts presented antimicrobial activity against P. aeruginosa ATCC 9027 and ATCC 27253 strains. This microorganism normally inhabits vegetables, water and soil and may be found on the skin, excrement and throat of normal individuals. It is opportunist and can be responsible for several infectious diseases such as localized and urinary infections, serious pneumonia and around 15% of bacteremia caused by Gram-negative germs. It is among the most frequent pathogens in hospital infection cases due to its various virulence factors and to its multi-resistance to several antimicrobial drugs (CitationThomson & Bonomo, 2005). Such findings justified the use of clinical isolates of this microorganism as well as standard strains for MIC determination.
Minimal inhibitory concentration (MIC) bioassay
Considering the results obtained from the screening tests performed previously, the determination of the minimal inhibitory concentration (MIC) for all three crude ethanol extracts was considered necessary and the results are shown in .
Table 4. Minimal inhibitory concentration (mg/mL) of the crude ethanol extracts of P. pseudocaryophyllus leaves.
The results observed in the agar diffusion test are qualitative and present limitations for substances with low diffusion ability in the culture medium. The quantitative step occurred in the agar dilution test.
The antimicrobial activity evidenced by the agar diffusion test for Gram-positive bacteria was confirmed by the agar dilution test. Of all the Gram-positive bacteria tested, 60% (6 bacteria) were inhibited by the sample collected in April 2005 (extract I) at a MIC of 0.78125 mg/ mL and 70% (7 bacteria) by the sample collected in September 2005 (extract II) at a MIC of 1.5625 mg/mL. The specimen from Minas Gerais (extract III) presented an inhibition pattern practically identical to the one collected in Brasilia in April 2005.
Similar observations can be made regarding the MIC of the three extracts against P. aeruginosa and P. stutzeri. Extract I of the Brasilia sample inhibited 50% of the Pseudomonas evaluated at a MIC of 0.78125 mg/mL and 37.5% at an even lower concentration (0.39062 mg/mL). Extract II of the Brasilia sample, on the other hand, inhibited 87.5% of the Pseudomonas at a MIC of 1.5625 mg/mL. Extract III of the Minas Gerais sample presented an inhibition pattern very similar to the one presented by extract I from Brasilia, inhibiting 87.5% of the Pseudomonas at a minimal concentration of 0.78125 mg/mL.
Such results may be explained by differences in extract compositions, since the plant materials were collected in different locations and times of the year. Plant material of the specimens analyzed is rich in phenolic compounds, such as tannins and flavonoids, and in essential oils (CitationPaula et al., 2008). It is probable that these compounds are involved in the antimicrobial activity assessed in this study.
Concerning the essential oils, CitationPaula (2006) verified a substantial variation in chemical yields and compositions, as much in terms of geographical location as in terms of stage of development for all three P. pseudocaryophyllus specimens studied. The fact that extract I presented an inferior MIC than extract II may be related to the higher yield of essential oil (0.8%) and to the higher content of methyl eugenol verified in the first sample. The Minas Gerais specimen (extract III), in spite of presenting an antimicrobial inhibition pattern similar to the Brasilia sample (extract I), had a completely different essential oil composition. Its antimicrobial action can be justified by the predominance of citral (neral and geranial) in its essential oil. It is important to highlight that citral has an intense antiseptic effect, even more potent than phenol (CitationSantos & Mello, 2004; CitationSimões & Spitzer, 2004; CitationZuanazzi & Montanha, 2004) and that eugenol is a well-known antimicrobial agent (CitationDorman & Deans, 2000). Recently, CitationLima et al. (2006) related the differences in the chemical composition of the essential oils of two specimens of P. pseudocaryophyllus to the different MIC observed against E. coli and C. albicans.
Data concerning the antimicrobial activity of extracts obtained from P. pseudocaryophyllus leaves were not found in the literature. However, other species of the Myrtaceae family have been studied regarding antimicrobial activity attributed to essential oils or tannins (CitationBara & Vanetti, 1998; CitationDjipa et al., 2000; CitationEstanislau et al., 2001; CitationCimanga et al., 2002; CitationSaenz et al., 2004; CitationLi et al., 2005; CitationOliveira et al., 2007).
CitationDjipa et al. (2000) points out that tannins present in different plant extracts do not present a common pattern of microbial inhibition, since not all tannins have antimicrobial action and each extract can have different concentrations of antimicrobial tannins. This observation is important for the present study, since it is noteworthy that among the group of Gram-negative bacteria evaluated, the extracts proved to be more active against Pseudomonas ssp. Considering tannins are partly responsible for the antimicrobial activity of the extracts analyzed in this work, this fact may be related to the content of antimicrobial tannins in the extracts with specificities against P. aeruginosa and P. stutzeri. Therefore, more precise studies to clarify the chemical composition not only of tannins but also of the essential oil components responsible for the antimicrobial activity of P. pseudocaryophyllus leaves will soon be vital. According to CitationBarreiro (2001), such compounds could serve as raw material for the production of pharmaceutical drugs or also serve as prototypes for the synthesis of new pharmaceutical drugs. Hence, the production of antimicrobial substances active against P. aeruginosa, a microorganism that presents multi-resistance to the most modern antimicrobial substances (CitationMakedou et al., 2005) will be invaluable.
The activity of the three extracts against the Gram-positive bacteria studied and, especially against the Gram-negative bacteria Pseudomonas ssp. has significant clinical meaning. Such results indicate promising propositions for controlling microorganisms and for the production of new antimicrobial drugs as well as positively influencing the ethno-medicinal use of this plant. Nevertheless, in order for this plant to become raw material for the production of herbal medicines, phytochemical, pharmacological and toxicological studies are necessary, with a view to guaranteeing the effective and safe application of this plant. Our research group is now focused on these subjects. Besides, studies on the possibility of cultivation and domestication to enable the use of this plant without causing negative environmental, economic and social impacts are also recommended.
Acknowledgements
The authors thank the Fundação de Apoio à Pesquisa (FUNAPE/UFG), the Faculdade de Farmácia /UFG, the Instituto de Ciências Biológicas/UFG and the Universidade Estadual de Goiás (UEG), all in Brazil. The authors thank Sharon Lois Vinaud for language assistance.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References
- Bara MTF, Vanetti MCD (1997/1998): Estudo da atividade antibacteriana de plantas medicinais, aromáticas e corantes naturais. Rev Bras Farmacogn 7/8:23–34.
- Barreiro EJ (2001): Desenho de fármacos a partir de produtos naturais, in: Yunes RA, Calixto JB, eds., Plantas Medicinais sob a ótica da Química Medicinal Moderna. Chapecó, Argos, pp. 237–296.
- Brooks GF, Butel JS, Morse SA (2004): Jawetz, Melnick & Adelberg’s Medical Microbiology. 23rd edition, New York, Lange/McGraw-Hill Publishing, pp. 202–229.
- Bugno A, Nicoletti MA, Almodóvar AAB, Pereira TC, Auricchio MT (2007): Antimicrobial efficacy of Curcuma zedoaria extract as assessed by linear regression compared with commercial mouth rinses. Braz J Microbiol 38: 440–445.
- Cimanga K, Kambu K, Tona L, Apers S, De Bruyne T, Hermans N, Totté J, Pieters L, Vlietinck AJ (2002): Correlation between chemical composition and antibacterial activity of essential oils of some aromatic medicinal plants growing in the Democratic Republic of Congo. J Ethnopharmacol 79: 213–220.
- Cunico MM, Carvalho JLS, Kerber VA, Higakino CEK, Cruz Almeida SC, Miguel MD, Miguel OG (2004): Atividade antimicrobiana do extrato bruto etanólico de raízes e partes aéreas de Ottonia martiana Miq. (Piperaceae). Rev Bras Farmacogn 14: 97–103.
- Djipa CD, Delmée M, Quetin-Leclercq J (2000): Antimicrobial activity of bark extracts of Syzygium jambos (L.) Alston (Myrtaceae). J Ethnopharmacol 71: 307–313.
- Dorman HJD, Deans SG (2000): Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J Appl Microbiol 88: 308–316.
- Estanislau AA, Barros FAS, Pena AP, Santos SC, Ferri PH, Paula JR (2001): Composição química e atividade antibacteriana dos óleos essenciais de cinco espécies de Eucalyptus cultivadas em Goiás. Rev Bras Farmacogn 11: 95–100.
- Fernandes TT, Santos ATF, Pimenta FC (2005): Atividade antimicrobiana das plantas Plathymenia reticulata, Hymenaea courbaril e Guazuma ulmifolia. Revista de Patologia Tropical 34: 113–122.
- Ferri PH (1996): Química de produtos naturais: métodos gerais, in: Distasi LC, ed., Plantas Medicinais: Arte e Ciência. São Paulo, Editora da Universidade Estadual Paulista, pp. 129–156.
- Gales AC, Jones RN, Andrade SS, Sader HS (2005): Antimicrobial susceptibility patterns of unusual non fermentative Gram-negative bacilli isolated from Latin America: Report from the SENTRY Antimicrobial Surveillance Program (1997-2002). Mem Inst Oswaldo Cruz 100: 671–677.
- Hardman JG, Limbird LE, Gilman AG (2003). As Bases Farmacológicas da Terapêutica, tenth edition, Rio de Janeiro, McGraw-Hill, pp. 859–1033.
- Helfand MS, Bonomo RA (2005): Current challenges in antimicrobial chemotherapy: the impact of extended-spectrum β-lactamases and metallo-β-lactamases on the treatment of resistant Gram-negative pathogens. Curr Opin Pharmacol 5: 452–458.
- Hernández T, Canales M, Avila JG, García AM, Martínez A, Caballero J, Romo de Vivar A, Lira R (2005): Composition and antibacterial activity of essential oil of Lantana achyranthifolia Desf. (Verbenaceae). J Ethnopharmacol 96: 551–554.
- Hsueh P, Chen W, Luh K (2005): Relationships between antimicrobial use and antimicrobial resistance in Gram-negative bacteria causing nosocomial infections from 1991–2003 at a university hospital in Taiwan. Int J Antimicrob Agents 26: 463–472.
- Landrum LR (1986): Monography 45: Campomanesia, Pimenta, Blepharocalyx, Legrandia, Acca, Myrrhinium, and Luma (Myrtaceae). Flora Neotropica 4: 72–115.
- Li Y, Xu C, Zhang Q, Liu JY, Tan RX (2005): In vitro anti-Helicobacter pylori action of 30 Chinese herbal medicines used to treat ulcer diseases. J Ethnopharmacol 98: 329–333.
- Lima EO (2001): Plantas e suas propriedades antimicrobianas: Uma breve análise histórica, in: Yunes RA, Calixto JB, eds, Plantas Medicinais sob a ótica da Química Medicinal Moderna. Chapecó, Argos, pp. 481–501.
- Lima MEL, Cordeiro I, Young MCM, Sobra MEG, Moreno PRH (2006): Antimicrobial activity of the Pimenta pseudocaryophyllus (Gomes) L. R. Landrum (Myrtaceae) native from São Paulo State – Brazil. Pharmacologyonline 3: 589–593.
- Lorenzi H (2002): Árvores Brasileiras. Manual de Identificação e Cultivo de Plantas Arbóreas do Brasil, second edition, Nova Odessa, Instituto Plantarum de Estudos da Flora, pp. 262–262.
- Makedou KG, Tsiakiri EP, Bisiklis AG, Chatzidimitriou M, Halvantzis AA, Ntoutsou K, Alexiou-Daniel S (2005): Changes in antibiotic resistance of most common Gram-negative bacteria isolated in intensive care units. J Hosp Infect 60: 245–248.
- Meyer AL (2005): Prospects and challenges of developing new agents for tough Gram-negatives. Curr Opin Pharmacol 5: 490–494.
- Meyer E, Schwab F, Jonas D, Ruden H, Gastmeier P, Daschner FD (2005): Temporal changes in bacterial resistance in German intensive care units, 2001-2003: data from the SARI (surveillance of antimicrobial use and antimicrobial resistance in intensive care units) project. J Hosp Infect 60: 348–352.
- Michelin DC, Moreschi PE, Lima AC, Nascimento GGF, Paganelli MO, Chaud MV (2005): Avaliação da atividade antimicrobiana de extratos vegetais. Rev Bras Farmacogn 15: 316–320.
- Mittermeier RA, Gil PR, Mittermeier CG (1997): Megadiversity: Earth’s Biologically Wealthiest Nations. Agrupacíon Sierra Madre, Mexico City, CEMEX, pp. 39–49.
- Nair R, Chanda S (2007): In-vitro antimicrobial activity of Psidium guajava L. leaf extracts against clinically important pathogenic microbial strains. Braz J Microbiol 38: 452–458.
- Nakaoka-Sakita M, Aguiar OT, Yatagai M, Igarashi T (1994): óleo essencial de Pimenta pseudocaryophyllus var. pseudocaryophyllus (Gomes) Landrum (Myrtaceae) I: Cromatografia a gás/espectrometria de massa (CG/EM). Rev Inst Flor 6: 53–61.
- NCCLS (National Committee for Clinical Laboratory Standards) (2003): Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-A6, sixth edition. Wayne, PA, NCCLS. pp. 1–47.
- Oliveira GF, Furtado NAJC, Silva Filho AA, Martins CHG, Bastos JK, Cunha WR, Silva MLA (2007): Antimicrobial activity of Syzygium cumini (Myrtaceae) leaves extract. Braz J Microbiol 38: 381–384.
- Paula JAM (2006): Estudo Farmacognóstico e Avaliação da Atividade Antimicrobiana das Folhas de Pimenta pseudocaryophyllus (Gomes) L. R. Landrum – Myrtaceae. Goiânia, Universidade Federal de Goiás, pp. 91–110.
- Paula JAM, Paula JR, Bara MTF, Rezende MH, Ferreira HD (2008): Estudo farmacognóstico das folhas de Pimenta pseudocaryophyllus (Gomes) L.R. Landrum – Myrtaceae. Rev Bras Farmacogn 18: 265–278.
- Saenz MT, Tornos MP, Alvarez A, Fernadez MA, García MD (2004): Antibacterial activity of essential oils of Pimenta racemosa var. terebinthina and Pimenta racemosa var. grisea. Fitoterapia 75: 599–602.
- Santos SC, Mello JCP (2004): Taninos, in: Simões CMO, Schenkel EP, Gosmann G, Mello JCP, Mentz LA, Petrovick PR, eds, Farmacognosia: da Planta ao Medicamento. Florianópolis, Ed. da UFSC, pp. 615–656.
- Simões CMO, Spitzer V (2004): Óleos voláteis, in: Simões CMO, Schenkel EP, Gosmann G, Mello JCP, Mentz LA, Petrovick PR, eds., Farmacognosia: da Planta ao Medicamento. Florianópolis, Ed. da UFSC, pp. 467–495.
- Souza LKH, Oliveira CMA, Ferri PH, Santos SC, Oliveira Júnior JG, Miranda ATB, Lião LM, Silva MRR (2002): Antifungal properties of Brazilian cerrado plants. Braz J Microbiol 33: 247–249.
- Steers E, Foltz EL, Graaves VS (1959): An inocula replicating apparatus for continued testing of bacterial susceptibility to antibiotics. Antibiot Chemother 9: 307–311.
- Tavares W (2001): Manual de Antibióticos e Quimioterápicos Antiinfecciosos, third edition. São Paulo, Atheneu, pp. 40–520.
- Thomson JM, Bonomo RA (2005): The treatment of antibiotic resistance in Gram-negative pathogenic bacteria: β-lactams in peril! Curr Opin Microbiol 8: 518–524.
- Yasunaka K, Abe F, Nagayama A, Okabe H, Lozada-Pérez L, López-Villafranco E, Muñiz EE, Aguilar A, Reyes-Chilpa R (2005): Antibacterial activity of crude extracts from Mexican medicinal plants and purified cumarins and xanthones. J Ethnopharmacol 97: 293–299.
- Zhanel GG, Hisanaga TL, Laing NM, DeCorby MR, Nichol KA, Palatnick LP, Johnson J, Noreddin A, Harding GKM, Nicolle LE, Hoban DJ (2005): Antibiotic resistance in outpatient urinary isolates: final results from the North American Urinary Tract Infection Collaborative Alliance (NAUTICA). Int J Antimicrob Agents 26: 380–388.
- Zuanazzi JAS, Montanha JA (2004): Flavonóides, in: Simões CMO, Schenkel EP, Gosmann G, Mello JCP, Mentz LA, Petrovick PR, eds., Farmacognosia: da Planta ao Medicamento. Florianópolis, Ed. da UFSC, pp. 577–614.
- Zuque ALF, Watanabe ES, Ferreira AMT, Arruda ALA, Resende UM, Bueno NR, Castillo RO (2004): Avaliação das atividades antioxidante, antimicrobiana e citotóxica de Couepia grandiflora Benth. (Chrysobalanaceae). Rev Bras Farmacogn 14: 129–136.