Abstract
Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by Paracoccidioides brasiliensis Almeida (Onygenales) that requires 1–2 years of treatment. In the absence of drug therapy, the disease is usually fatal, highlighting the need for the identification of safer, novel, and more effective antifungal compounds. With this need in mind, several plants employed in Brazilian traditional medicine were assayed on P. brasiliensis and murine macrophages. Extracts were prepared from 10 plant species: Inga spp. Mill. (Leguminosae), Schinus terebinthifolius Raddi (Anacardiaceae), Punica granatum L. (Punicaceae), Alternanthera brasiliana Kuntze (Amaranthaceae), Piper regnellii CDC. (Piperaceae), P. abutiloides Kunth (Piperaceae), Herissantia crispa L. Briz. (Malvaceae), Rubus urticaefolius Poir (Rosaceae), Rumex acetosa L. (Polygonaceae), and Baccharis dracunculifolia DC. (Asteraceae). Hexane fractions from hydroalcoholic extracts of Piper regnellii and Baccharis dracunculifolia were the most active against the fungus, displaying minimum inhibitory concentration (MIC) values of 7.8 μg/mL and 7.8–30 μg/mL, respectively. Additionally, neither of the extracts exhibited any apparent cytotoxic effects on murine macrophages at 20 μg/mL. Analyses of these fractions using gas chromatography-mass spectrometry (GC-MS) showed that the major components of B. dracunculifolia were ethyl hydrocinnamate (14.35%) and spathulenol (16.02%), while the major components of the hexane fraction of Piper regnellii were 1-methoxy-4-(1-propenyl) benzene (21.94%) and apiol (21.29%). The activities of these fractions against P. brasiliensis without evidence of cytotoxicity to macrophages justify their investigation as a potential source of new chemical agents for the treatment of PCM.
Introduction
Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America (CitationCoutinho et al., 2002). The etiologic agent of this mycosis is the thermodimorphic fungus Paracoccidioides brasiliensis Almeida (Onygenales). The annual incidence rate of PCM in Brazil, the country with the highest disease endemicity, is 10–30 per million inhabitants, and the mean mortality rate estimated for the period from 1980 to 1995 was 1.4 deaths per million inhabitants per year (CitationCoutinho et al., 2002).
In the absence of drug therapy, the disease is usually fatal. The treatment of PCM is usually long, with many patients receiving therapy for 1–2 years or even more (CitationShikanai-Yasuda et al., 2006). Although azoles and other drugs can arrest the progression of PCM, the fibrosis sequelae persist, probably constituting a source of P. brasiliensis that could lead to a relapse in the disease following termination of treatment (CitationOnishi et al., 2000; CitationBorges-Walmsley et al., 2002). The strong toxicity of amphotericin B makes the effective management of severe disease difficult (CitationLorthay et al., 1999). This situation highlights the need for the advent of safe, novel, and effective antifungal compounds. In this regard, plants provide abundant resources of antimicrobial compounds and have been used for centuries to inhibit microbial growth (CitationNicoletti, 2002).
There are some literature reports on the antimicrobial activity of Punica granatum L. (Punicaceae), Schinus terebinthifolius Raddi (Anacardiaceae), Herissantia crispa L. Briz. (Malvaceae), Rubus urticaefolius Poir (Rosaceae), Baccharis dracunculifolia DC. (Asteraceae), and Piper regnellii CDC. (Piperaceae) (CitationMartinez et al., 1996; CitationPrashanth et al., 2001; CitationHoletz et al., 2002; CitationVoravuthikunchai et al., 2004; CitationGuerra et al., 2005; CitationSchomourlo et al., 2006; Johann et al., Citation2007b). CitationSilva and Siqueira (2000) observed that the extracts of R. urticaefolius were capable of inhibiting the growth of Gram-positive and Gram-negative bacteria. Johann et al. (Citation2007b) showed that P. granatum, S. terebinthifolius, R. urticaefolius, B. dracunculifolia, and P. regnellii display activity against Candida spp., Cryptococcos neoformans, and Sporothrix schenkii. CitationSouza et al. (2004) and Johann et al. (Citation2007b) found that Staphylococcus aureus, S. epidermidis, Escherichia coli, Bacillus subtilis, and Candida spp. were resistant to the extracts of Alternanthera brasiliana. However, other authors reported that extracts of this plant act as inhibitors of lymphocyte proliferation (CitationMoraes et al., 1994) and as an antiviral agent against virus herpes simplex 1 (CitationLagrota et al., 1994). CitationJohann et al. (2008) reported that extracts of S. terebinthifolius and P. granatum were able to inhibit the growth of three isolates of Candida albicans. These plant species also showed the best results as inhibitors of adhesion of C. albicans to buccal epithelial cells. Otherwise, Rumex acetosa was active against C. tropicalis whereas Inga spp. were inactive against all the fungal species tested (Johann et al., Citation2007b). The aims of this work were to evaluate extracts of plants used in Brazilian popular medicine against P. brasiliensis and to verify the toxicity of these extracts on murine macrophages.
Materials and methods
Plant materials
Different parts of 10 plant species were sampled for use in this study. The samples included leaves (312 g) of Inga spp. Mill. (Leguminosae), leaves (400 g) and stem (250 g) of Schinus terebinthifolius Raddi (594 g), and fruit peel (546 g) and stems (1520 g) of Punica granatum. The aerial parts (a mix of leaves, stems, and flowers) of the following plants were collected: Alternanthera brasiliana Kuntze (Amaranthaceae), 149 g; Piper regnellii, 191 g; P. abutiloides Kunth (Piperaceae), 301 g; Herissantia crispa, 95 g; Rubus urticaefolius, 141 g; Rumex acetosa L. (Polygonaceae), 45 g; and Baccharis dracunculifolia, 483 g. The plant materials were collected in different regions of Santa Catarina State, Brazil from December 2003 to January 2004. Inga spp. and P. granatum were identified by Professor Barcellos Falkenbergat from the Department of Botany, Federal University of Santa Catarina (UFSC), and voucher specimens were deposited in the FLORA-UFSC herbarium. The remaining plant materials were furnished by EPAGRI (Empresa Agropecuária e Extensão Rural de Santa Catarina) germoplasm bank, Itajaí, SC, Brazil. Botanical names and voucher specimens are listed in .
Table 1. Minimum inhibitory concentrations (µg/mL) of extracts of selected Brazilian medicinal plants against Paraccocidioides brasiliensis and cytotoxic effects on murine macrophages.
Preparation of the extracts
Plant materials were extracted by maceration with 80% ethanol during 10 days at room temperature. After filtration, the hydroalcoholic extract (EtOH) was concentrated under reduced pressure to afford the crude extracts. These extracts were suspended in distilled water and extracted successively with hexane (HEX), dichloromethane (DCM), and ethyl acetate (AcOEt) (CitationJohann et al., 2008). After separation of the phases, the solvents were removed in a rotary evaporator at 45°C under vacuum. All extracts and fractions were submitted to biological assays.
Analysis by gas chromatography-mass spectrometry
Hexane extracts of B. dracunculifolia and P regnellii were analyzed by gas chromatography coupled to nass spectrometry (GC-MS) using the solid phase micro-extraction (SPME) analysis mode.
Collection of volatiles by SPME
The volatiles of the hexane fractions of B. dracunculifolia and P regnellii were adsorbed in an SPME sampling device and analyzed by GC-MS. Thus, 1 mg of each hexane fraction was transferred to a 2 mL glass vial, which was closed with a cap sealed with a Teflon coated septum (Supelco, USA) and placed in a heat block adjusted to 90°C. An SPME fiber (PDMS/DVB™ 65 µm; Supelco, USA) was inserted through the septum and left in the headspace for 5 min. Before use, the fiber was preconditioned at 230°C for 30 min in the GC injector port (CitationSiqueira et al., 2007).
GC-MS analysis
Gas chromatography-mass spectrometry (GC-MS) analyses were performed on a Shimadzu QP-5050A (Shimadzu, Japan) instrument, equipped with a PTE™-5 column (30 mm, 0.25 mm, 0.25 µm; Supelco, USA), using helium as the carrier gas. The following conditions were employed for all analysis: carrier gas, helium at 22.3 mL/min; injector temperature, maintained at 230°C; column temperature, 3 min at 80°C, 80–300°C at 7°C/min, maintained at 300°C for 5 min, and kept at this temperature for 5 min. The split valve was closed during the first minute of injection and then opened, with a 1:10 ratio. The mass detector was set to scan from 50 to 500 atomic mass units, at a rate of two scans per second. Data acquisition and handling were done via CLASS 5000 Shimadzu software. Raw data files were analyzed by Automated Mass Deconvolution and Identification System software (AMDIS), version 2.1, supplied by the National Institute of Standards and Technology (NIST, USA), and compound identification was performed by comparison of the experimental spectra with those stored in the NIST/EPA/NIH library version 2.0 using the NIST Mass Spectral Search Program.
Paracoccidioides brasiliensis strain maintenance
Three clinical P. brasiliensis strains, Pb01 (ATCC MYA-826), Pb339 (ATCC 32069), and Pb18 (from the fungal collection of the Faculty of Medicine of the University of São Paulo, São Paulo, SP, Brazil), were used in the biological assays.
Inoculum preparation
The strains were maintained at the Laboratory of Mycology, ICB, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil, by weekly passage in solid Fava-Netto medium (CitationLacaz et al., 2002) at 37°C and were used after 7–10 days of fungus growth. Yeast cells in the exponential phase were collected aseptically with a platinum loop and resuspended in a tube containing 5 mL of sterile saline. If large aggregates existed, they were allowed to settle for several minutes, and the supernatants were collected. The suspensions were then diluted in synthetic RPMI medium (Sigma, St. Louis, MO, USA) with l-glutamine buffered to pH 7.0 with 0.165 morpholine propanesulfonic acid (MOPS; Sigma), and prepared according to Clinical and Laboratory Standards Institute (CLSI) document M27-A2 (CitationNCCLS, 2008) to obtain a final inoculum size suitable for the strains (CitationNakai, 2003). After homogenization by vortexing, transmittance was measured at 520 nm and adjusted to 69–70% (CitationHahn & Hamdan, 2000).
Susceptibility test of Paracoccidioides brasiliensis
The plant extracts were dissolved in dimethylsulfoxide (DMSO). Serial dilutions were then performed, using RPMI as a diluent, maintaining a constant volume of 1 mL per tube. The extracts were tested at eight concentrations that ranged from 1000 to 7.8 µg/mL. Volumes of 100 µL of each dilution were distributed in sterile flat-bottom 96-well microplates (Difco Laboratories, Detroit, MI, USA).
Susceptibility was determined by the broth microdilution method. Broth microdilution testing was performed in accordance with the guidelines in the CLSI M27-A2 document (CitationNCCLS, 2008) and CitationNakai (2003). RPMI medium was used without compounds or solvents as a control for growth and sterility. Solvent DMSO at the same volume as used in the assay was used as a control for toxicity. Amphotericin B (Sigma) was included as a positive antifungal control, as stock solutions prepared in DMSO and water, respectively. Two-fold serial dilutions were prepared exactly as outlined in CLSI document M27-A2 (CitationNCCLS, 2008). After inoculation of fungal strains the plates were incubated at 37°C for 72 h. The tests were performed in triplicate in at least two independent experiments. The endpoints were determined visually by comparison with the drug-free growth control well. MIC is expressed in μg/mL and defined as the lowest compound concentration for which the well was optically clear.
Toxicity to murine macrophages
Murine macrophages were obtained as previously described by CitationPaulnock (2000), and the cytotoxicity assay was performed according to CitationSoto et al. (2007).
Results
The three P. brasiliensis strains presented similar susceptibility profiles in relation to the plant extracts tested (). Hexane fractions of the extracts of S. terebinthifolius, R. urticaefolia, B. dracunculifolia, P. abutiloides, P. regnellii, and H. crispa were most active against the fungus when compared with the other fractions of these plants. All P. brasiliensis isolates tested were resistant to the EtOH extract of A. brasiliana at a concentration of 1000 µg/mL.
The hexane fractions of B. dracunculifolia and P. regnellii showed MIC values of between 7.8 and 30 µg/mL and 7.8 µg/mL, respectively, against the three strains of P. brasiliensis (). The chromatogram of the hexane fraction of B. dracunculifolia under the conditions described in the “Materials and methods” section showed 87 peaks. The major components were: ethyl hydrocinnamate (14.35%), caryophyllene (14.45%), δ-cadinene (4.83%), nerolidol (5.95%), spathulenol (16.02%), and viridiflorol (4.37%) (, Appendix). Analysis of the hexane fraction of P. regnellii showed 24 peaks, and seven of them were identified by comparison with the NIST library (v. 2.0) (, Appendix). The major components were 1-methoxy-4-(1-propenyl) benzene (21.94%), α-copaene (8.91%), aromadendrene (12.92%), δ-cadinene (9.82%), dillapiole (11.31%), apiol (23.22%), and β-eudesmol (3.48%). Extracts and fractions of P. granatum, S. terebinthifolius, R. urticaefolius, B. dracunculifolia, P. abutiloides, and Inga spp. did not show cytotoxic activity against murine macrophages ().
Figure 1. Total ion chromatogram of the hexane fraction of Baccharis dracunculifolia. a, ethyl hydrocinnamate, m/z = 178; b, caryophyllene, m/z = 204; c, δ-cadinene, m/z = 204; d, nerolidol, m/z = 222; e, spathulenol, m/z = 220; f, viridiflorol, m/z = 222.
Figure 2. Total ion chromatogram of the hexane fraction of Piper regnellii. a, 1-Methoxy-4-(1-propenyl) benzene, m/z = 148; b, α-copaene, m/z = 204; c, aromadendrene, m/z = 204; d, δ-cadinene, m/z = 204; e, dillapiole, m/z = 222; f, β-eudesmol, m/z = 222, g, apiol, m/z = 222.
Discussion
There are a few reports describing the search for new compounds with the ability to inhibit P. brasiliensis. CitationSan-Blas et al. (1989) demonstrated that ajoene, an alicin derivative isolated from garlic (Allium sativum L., (Liliaceae)), was active against the yeast and filamentous forms of P. brasiliensis. These authors observed a lytic effect when the fungal cells were treated with 200 μM of ajoene.
In our work, we observed that extracts from six out of 10 plants examined exhibited antifungal activity against P. brasiliensis. For P. granatum specifically, the hexane fraction from stems (MIC: 40–71.6 µg/mL) exhibited better antifungal activity against the three clinical isolates of P. brasiliensis than extracts from leaves and fruits, indicating that the plant is a potential source of new antimicrobial compounds (). The presence of flavonoids and tannins in the Punicaceae species is responsible for several biological activities (CitationHussein et al., 1997). However, these substances were in more polar, ethanol or aqueous fractions. On the other hand, CitationPurwantini and Wahyuono (2003) isolated terpenoids, steroids with a stigmastane skeleton esterified by a long-chain fatty acid, from the hexane fraction of P. granatum, which showed antifungal activity against Candida albicans. This finding is in agreement with our observations that the hexane fraction is the most active.
The hexane fraction of the extract from the aerial parts of R. urticaefolius inhibited P. brasiliensis growth at a concentration of 60-500 μg/mL. The fractions of the hydroalcoholic extracts of A. brasiliana did not present any activity against the three clinical isolates of P. brasiliensis. This result contrasts with that of CitationBiavatti et al. (2003), which showed inhibitory activity of the extracts of this plant against oocysts of the protozoa Eimeira acervulina.
We could not find data on the biological activity and phytochemical investigation of the species Inga spp. and H. crispa. Extracts of these traditional medicinal plants were active on P. brasiliensis, and could represent new sources of antifungal compounds.
As the hexane fractions of B. dracunculifolia and P. regnellii showed the best MIC values (7.8–30 and 7.8 µg/mL, respectively) against the three strains of P. brasiliensis (), these extracts were chosen for chromatographic analysis. CitationPessini et al. (2005) showed that the EtOAc extract of P. regnellii presented significant activity against C. albicans and moderate activity against both C. krusei and C. parapsilosis. The compounds eupomatenoid-6, eupomatenoid-5, eupomatenoid-3, and conocarpan were isolated, but only the latter was active against the yeasts (CitationPessini et al., 2005). CitationConstantin et al. (2001) analyzed the essential oil obtained by hydrodistillation of leaves of P. regnellii by GC-MS and found that it was active against Staphylococcus aureus and C. albicans. The analysis showed the presence of mircene (52.6%), linalol (15.9%), β-caryophyllene (8.5%), (E)-nerolidol (4.2%), and limonene (4.1%). In our studies, the chromatographic profile of the volatiles obtained in the GC-MS analysis was different because the samples were obtained using different methodologies (fresh leaves vs. hexane fraction of the hydroalcoholic extract), and because we used solid phase extraction instead of hydrodistillation to capture the volatiles. Further studies will be needed to isolate and characterize the antifungal compounds from extracts of P. regnellii and P. abutiloides. The present study confirms the antifungal properties of the P. regnellii extract and its potential as a source of useful bioactive compounds.
The extract of B. dracunculifolia and its hexane fraction were active at low concentrations against the three isolates of P. brasiliensis. This fraction was also analyzed by GC-MS and showed the presence of diterpenes and sesquiterpenes (δ-cadinene, spathulenol, caryophyllene, viridiflorol, and nerolidol). These compounds could be responsible for the observed activity against P. brasiliensis, since terpenoids are known for their antimicrobial activities (CitationNicoletti, 2002). Terpenoids as both optical isomers of carvone were found to be active toward many kinds of human pathogenic fungi. The development of C. albicans, C. krusei, and C. tropicalis was also inhibited by a combination of monoterpenes, including terpenin-4-ol, α-pinene, 1,8-cineole, linalool, and α-terpineol. These monoterpenes also inhibited the development of dermatophytes such as Trichophyton mentagrophytes, T. rubrum, and Microsporum gypseum. α-Terpinene also exhibited antifungal activity similar to that of commonly used antifungal drugs (CitationPaduch et al., 2007).
The mechanism of action of terpenoids has not been explained completely, but is speculated to involve the rupture of the cellular membrane and modification of the structure of enzymes (CitationLima et al., 1992). Additionally, it may involve inhibition of the synthesis of 1,3-β-d-glucan, which participates in the synthesis of the cellular wall of fungi (CitationOnishi et al., 2000). According to CitationSantos et al. (1966), nerolidol is the major constituent of the essential oil of B. dracunculifolia. However, in our work, this compound represented only 6% of the total area in the hexane fraction of B. dracunculifolia. This discrepancy could be due to many factors, including differences in chemotype, seasonality, and methods of collection and extraction. CitationLoayza et al. (1995) described the presence of alloaromadendrene and nerolidol in the essential oil of B. dracunculifolia, which is in accordance with our findings. However, bioassay-guided isolation using an assay with P. brasiliensis is necessary to verify the compounds responsible for the antifungal activity.
Macrophages are professional phagocytes that act as the first line of defense, provided by the innate immune system. Resident macrophages are widely distributed in tissues and are one of the primary cell types to sense and respond to microbial invaders. The antifungal activity of macrophages is an interesting and important effector mechanism that involves intercellular and/or intracellular killing of parasites (CitationPaulnock, 2000). In the search for plant extracts with antimicrobial activities, it is very important to show that the extracts are not toxic to these cells, which should be included as a control in the screening procedure (CitationJohann et al., 2008; CitationSantos et al., 2008). Taking this into consideration, our extracts were tested in a bioassay with murine macrophages. Our results show that in spite of their ability to inhibit the isolates of the pathogenic fungus P. brasiliensis, the fractions of extracts of P. granatum, S. terebinthifolius, R. urticaefolius, B. dracunculifolia, P. abutiloides, and Inga spp. are not toxic to these cells. Thus, the results obtained in the present work will encourage future studies characterizing the impact of these extracts or compounds isolated from them on human cells in the quest for new antifungal drugs. This is especially true for the hexane fractions of P. regnellii and B. dracunculifolia, which were the most active against this pathogenic fungus and without apparent cytotoxicity to murine macrophages. Further investigations aimed at identifying their bioactive constituents are under way.
Declaration of interest
We thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG) from Brazil for financial support.
References
- Biavatti MWO, Bellaver MH, Volpato LO, Costa C, Bellaver C (2003): Preliminary studies of alternative feed additives for broilers: Alternanthera brasiliana extract, propolis extract and linseed oil. Rev Bras Cienc Avic 5: 147–151.
- Borges-Walmsley MI, Chen D, Shu X, Walmsley AR (2002): The pathobiology of Paracoccidioides brasiliensis. Trends Microbiol 10: 80–87.
- Constantin MB, Sartorelli P, Limberger R, Henriques AT, Steppe M, Ferreira MJP, Ohara MT, Emerenciano VP, Kato MJ (2001): Essential oils from Piper cernuum and Piper regnellii: Antimicrobial activities analysis by GC/MS and 13C-NMR. Planta Med 67: 771–773.
- Coutinho ZF, da Silva D, Lazéra M, Petri V, Oliveira RM, Sabrosa PC, Wanke B (2002): Paracoccidioidomycosis mortality in Brazil (1980-1995). Cad Saúde Pública 18: 1441–1454.
- Guerra MJM, Barreiro ML, Rodríguez ZM, Rubalcaba Y (2005): Actividad antimicrobiana de um extracto fluido al 80% de Schinus terebinthifolius Raddi (Copal). Rev Cubana Med Trop 5: 23–25.
- Hahn RC, Hamdan JS (2000): Effects of amphotericin B and three azole derivatives on the lipids of yeast cells of Paracoccidioides brasiliensis. Antimicrob Agents Chemother 44: 1997–2000.
- Holetz FB, Pessini GL, Sanches NG, Cortez DAG, Nakamura CV, Dias Filho BP (2002): Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases. Mem Inst Oswaldo Cruz 97: 1027–1031.
- Hussein SAM, Barakat HH, Merfort I, Nawwar MAM (1997): Tannins from the leaves of Punica granatum. Phytochemistry 45: 819–823.
- Johann S, Pizzolatti MG, Donnici CL, Resende MA (2007b): Antifungal properties of plants used in Brazilian traditional medicine against clinically revelant fungal pathogens. Braz J Microbiol 38: 632–637.
- Johann S, Silva DL, Martins CVB, Zani CL, Pizzolatti MG, Resende MA (2008): Inhibitory effect of extracts from Brazilian medicinal plants on the adhesion of Candida albicans to buccal epithelial cells. World J Microbiol Biotechnol 24: 2459–2464.
- Lacaz CS, Porto E, Martins JEC, Heins-Vaccari EM, Melo NT (2002): Tratado de Micologia Médica Lacaz. São Paulo, Sarvier, pp. 639–729.
- Lagrota MHC, Wigg MD, Santos MMG, Miranda MMFS, Camara FP, Couceiro JNSS, Costa SS (1994): Inhibitory activity of extracts of Alternanthera brasiliana (Amaranthaceae) against the herpes simplex virus. Phytother Res 8: 358–361.
- Lima EOG, Paulo MQ, Giesbrecht AM (1992): In vitro antifungal activity of essential oils against clinical isolates of dermatophytes. Rev Microbiol 23: 235–238.
- Loayza I, Abujder D, Aranda R, Jakupovic J, Collin G, Deslauriers H, Jeand FI (1995): Essential oils of Baccharis salicifolia, B. latifolia and B. dracunculifolia. Phytochemistry 38: 381–389.
- Lorthay O, Denning DW, Dupont B (1999): Endemic mycoses: A treatment update. J Antimicrob Chemother 43: 321–331.
- Martinez MJ, Betancourt J, Alonso-Gonzfilez N, Jauregui A (1996): Screening of some Cuban medicinal plants activity for antimicrobial. J Ethnopharmacol 52: 171–174.
- Moraes VL, Santos LF, Castro SB, Loureiro LH, Lima AO, Souza ML, Yien LM, Rossi-Bergmann B, Costa SS (1994): Inhibition of lymphocyte activation by extracts and fractions of Kalanchoe, Alternanthera, Paullinia and Mikania species. Phytomedicine 1: 199–204.
- Nakai T, Uno J, Ikeda F, Jauregui A (2003): In vitro antifungal activity of micafungin (FK463) against dimorphic fungi: Comparison of yeast-like and mycelial forms. Antimicrob Agents Chemother 47: 1376–1381.
- NCCLS (2008): Performance Standards for Antimicrobial Susceptibility Testing; Ninth Informational Supplement. NCCLS document M100-S9. Wayne, PA, National Committee for Clinical Laboratory Standards, pp. 120–126.
- Nicoletti M (2002): Fitoterapia: Curcuma zedoaria (Christm.) var. roscoe, uma possibilidade terapêutica como antifúngica de uso tópico. Infarma 14: 60–65.
- Onishi MM, Thompson J, Curotto S, Dreikorn M, Rosenbach C, Douglas G, Abruzzo A, Flattery L, Kong A, Cabello F, Vicente F, Pelaez MT, Diez I, Martin G, Bills R, Giacobbe A, Dombrowski R, Schwartz S, Morris G, Harris A, Tsipouras K, Wilson M, Kurtz. B (2000): Discovery of novel antifungal (1,3)-β-d-glucan synthase inhibitors. Antimicrob Agents Chemother 44: 368–377.
- Paduch R, Kandefer-Szerszen M, Trytek M, Fiedurek J (2007): Terpenes: Substances useful in human healthcare. Arch Immunol Ther Exp 55: 315–327.
- Paulnock DM (2000): Macrophages: A Practical Approach. New York, Oxford University Press, pp. 211.
- Pessini GL, Dias BP, Nakamura CV, Cortez DAG (2005): Antifungal activity of the extracts and neolignans from Piper regnellii (Miq.) C.DC. var. pallescens (C.DC.) Yunck. J Braz Chem Soc 16: 1130–1133.
- Prashanth D, Asha MK, Amit A (2001): Antibacterial activity of Punica granatum. Fitoterapia 72: 171–173.
- Purwantini I, Wahyuono S (2003): Isolation and identification of antifungal (Candida albicans) compound from the hull of delima fruits (Punica granatum L.). Baglan Biologi Farmasi 14: 150–159.
- San-Blas G, San-Blas F, Gil F, Mariño L, Apitiz-Castro R (1989): Inhibition of growth of the dimorphic fungus Paracoccidioides brasiliensis by ajoene. Antimicrob Agents Chemother 33: 1641–1644.
- Santos AO, Ueda-Nakamura T, Dias Filho BP, Veiga Junior VF, Pinto AC, Nakamura CV (2008): Effect of Brazilian copaiba oils on Leishmania amazonensis. J Ethnopharmacol 120: 204–208.
- Santos SR, Terence RMM, Pinto AJDA, Brilho CC, Donalísio MR, Souza CJ (1966): A new Brazilian essential oil from Baccharis dracunculifolia. Rivista Ital Essenze 98: 428–429.
- Schomourlo G, Mendonça-Filho RR, Alviano CS, Costa SS (2006): Screening of antifungal agents using ethanol precipitation and bioautography of medicinal and food plants. J Ethnopharmacol 96: 563–568.
- Shikanai-Yasuda MA, Telles-Filho FQ, Mendes RP, Colombo AL, Morreti ML (2006): Guideliness in paracoccidioidomycosis. Rev Soc Bras Med Trop 39: 279–310.
- Silva JP, Siqueira AM (2000): Accíon antibacteriana de extratos hidroalcohólicos de Rubus urticaefolius. Rev Cubana Plant Med 5: 26–29.
- Siqueira EP, Alvez TMA, Zani CL (2007): Fingerprint of volatiles from plant extract based on SPME-GC-MS. Rev Bras Farmacogn 17: 565–571.
- Soto K, Garza KM, Murr LE (2007): Cytotoxicity effects of aggregated nanomaterials. Acta Biomater 3: 351–358.
- Souza GC, Haas APSG, Poser L, Schapoval ES, Elisabetsky E (2004): Ethnopharmacological studies of antimicrobial remedies in the south of Brazil. J Ethnopharmacol 90: 135–143.
- Voravuthikunchai S, Lortheeranuwat A, Jeeju W, Sririrak T, Phongpaichit S, Supawita T (2004): Effective medicinal plants against enterohaemorrhagica Escherichia coli O157:H7. J Ethnopharmacol 94: 49–54.
Appendix
List of peaks with percentage relative abundance
B. dracunculifolia
Ethyl hydrocinnamate (m/z; % relative abundance): (41; 1.1), (42; 2.4), (43; 3.0), (44; 0.2), (45; 1.3), (50; 4.4), (51; 14.0), (52; 3.2), (53; 1.5), (54; 0.1), (55; 1.2), (61; 0.2), (62; 1.0), (63; 3.9), (64; 1.2), (65; 9.8), (66; 1.7), (74; 1.1), (75; 1.2), (76; 2.2), (77; 20.6), (78; 14.0), (79; 18.6), (80; 1.3), (87; 0.2), (89; 2.4), (91; 56.0), (92; 5.6), (102; 2.3), (103; 15.2), (104; 99.9), (105; 40.4), (107; 40.8), (108; 3.0), (131; 1.8), (132; 1.9), (133; 10.6), (134; 1.1), (149; 1.2), (178; 26.8), (179; 3.0), (180; 0.2).
Caryophyllene (m/z; % relative abundance): (40; 7.1), (41; 76.9), (42; 4.6), (43; 8.5), (44; 3), (50; 1.7), (51; 8.2), (52; 6.3), (53; 29.9), (54; 4.0), (55; 35.6), (56; 7.6), (57; 5.8), (63; 2.5), (64; 1.0), (65; 18.3), (66; 5.7), (67; 38.7), (68; 9.5), (69; 75.4), (70; 4.7), (71; 3.1), (77; 43.9), (78; 12.0), (79; 76.3), (80; 19.2), (81; 38.6), (82; 11.4), (83; 3.5), (89; 1.0), (91; 85.8), (92; 25.9), (93; 99.9), (94; 20.6), (95; 21.6), (96; 3.0), (97; 1.2), (103; 5.1), (104; 2.5), (105; 62.3), (106; 34.2), (107; 48.3), (108; 11.8), (109; 20.5), (110; 3.2), (111; 3.4), (112; 5), (113; 1), (115; 5.0), (116; 1.9), (117; 6.9), (118; 1.9), (119; 40.6), (120; 44.7), (121; 30.5), (122; 8.7), (123; 6.0), (128; 1.6), (129; 1.5), (130; 6), (131; 4.2), (133; 92.1), (134; 24.4), (135; 13.2), (136; 6.6), (137; 1.8), (145; 1.5), (146; 1.1), (147; 28.9), (148; 26.7), (149; 7.4), (150; 1.0), (151; 1), (159; 4), (160; 1.0), (161; 33.0), (162; 9.4), (163; 2.8), (175; 9.9), (176; 6.1), (177; 8); (189; 16.6), (190; 2.5), (204; 6.5), (205; 1.1).
δ-Cadinene (m/z; % relative abundance): (41; 20.8), (42; 1.5), (43; 6.5), (51; 2.0), (52; 1.1), (53; 6.5), (55; 12.2), (56; 1.1), (57; 1.5), (65; 4.3), (66; 1.4), (67; 10.0), (68; 1.8), (69; 12.2), (70; 1.2), (77; 13.0), (78; 3.4), (79; 22.2), (80; 4.3), (81; 19.6), (82; 2.2), (83; 1.4), (91; 24.4), (92; 12.2), (93; 33.5, (94; 12.2), (95; 7.4), (96; 1.0), (103; 1.9), (104; 1.7), (105; 38.2), (106; 7.8), (107; 9.6), (108; 2.5), (109; 3.1), (115; 3.1), (116; 1.3), (117; 4.1), (118; 2.2), (119; 34.8), (120; 10.0), (121; 10.0), (122; 2.7), (123; 1.5), (127; 1.3), (128; 1.6), (129; 1.6), (131; 3.1), (132; 1.4), (133; 18.3,), (134; 10.0), (135; 7.4), (136; 7.0), (145; 2.7), (147; 6.1), (148; 6.1), (149; 2.1), (150; 1.4), (159; 2.0), (160; 2.7), (161; 99.9), (162; 14.3), (163; 1.4), (175; 2.1), (176; 1.6), (189; 11.3), (190; 1.6), (204; 33.0), (205; 5.1).
Nerolidol (m/z; % relative abundance): (40; 2.7), (41; 58.6), (42; 4.1), (43; 41.0), (44; 1.3), (45; 1.1), (51; 1.3), (53; 9.6), (54; 1.5), (55; 27.2), (56; 2.1), (57; 4.6), (58; 1.1), (65; 3.6), (66; 1.3), (67; 24.9), (68; 9.1), (69; 99.9), (70; 8.1), (71; 37.0), (72; 2.3), (77; 7.2), (78; 1.2), (79; 19.2), (80; 9.7), (81; 24.1), (82; 7.6), (83; 5.4), (84; 1.1), (91; 11.9), (92; 3.4), (93; 56.0), (94; 7.2), (95; 10.9), (96; 1.8), (97; 3.7), (105; 6.5), (106; 1.5), (107; 27.5), (108; 3.9), (109; 11.8), (110; 1.3), (111; 2.2), (119; 9.4), (120; 3.2), (121; 13.1), (122; 4.2), (123; 13.9), (124; 1.6), (133; 4.5), (134; 3.2), (135; 5.4), (136; 18.3), (137; 3.0147; 1.8), (148; 2.6), (149; 1.1), (161; 12.7), (162; 2.2).
Spathulenol (m/z; % relative abundance): (40; 3.5), (41; 68.8), (42; 3.2), (43; 99.9), (44; 1.7), (45; 3.7), (51; 5.5), (52; 2.8), (53; 15.3), (54; 1.8), (55; 27.4), (56; 1.4), (57; 5.2), (58; 3.8), (59; 3.1), (63; 1.4), (64; 1.0), (65; 9.6), (66; 3.1), (67; 24.4), (68; 2.7), (69; 34.9), (70; 2.4), (71; 20.1), (72; 2.4), (77; 19.5), (78; 6.2), (79; 31.6), (80; 7.1), (81; 22.8), (82; 15.9), (83; 8.3), (84; 1.2), (85; 3.9), (91; 42.2), (92; 10.9), (93; 39.7), (94; 10.6), (95; 19.6), (96; 3.7), (97; 5.0), (99; 2.2), (103; 2.9), (104; 4.7), (105; 34.1), (106; 20.0), (107; 29.1), (108; 6.2), (109; 10.6), (110; 2.7), (111; 1.5), (115; 3.4), (116; 1.5), (117; 14.0), (118; 4.8), (119; 42;1), (120; 14.2), (121; 17.5), (122; 4.7), (123; 6.3), (124; 1.1), (125; 2.4), (128; 2.3), (129; 2.7), (130; 1.8), (131; 20.8), (132; 5.9), (133; 16.7), (134; 10.3), (135; 11.2), (136; 2.7), (137; 2.4), (143; 1.7), (144; 1.8), (145; 17.2), (146; 15.2), (147; 21.7), (148; 3.5), (149; 12.7), (150; 4.5), (151; 1.6), (159; 33.5), (160; 9.4), (161; 3.8), (162; 23.5), (163; 5.0), (164; 1.4), (173; 2.9), (174; 2.4), (177; 8.6), (178; 1.1), (187; 17.1), (188; 2.5), (202; 16.1), (203; 3.1), (205; 33.2), (206; 5.5), (220; 5.6).
Viridiflorol (m/z; % relative abundance): (41; 60.9), (43; 99.9), (53; 13.0), (55; 33.7), (57; 5.3), (59; 7.3), (65; 5.8), (67; 3.07), (68; 3.9), (69; 53.8), (70; 4.3), (71; 19.6), (77; 12.9), (79; 21.0), (80; 5.0), (81; 33.8), (82; 18.5), (83; 9.5), (91; 17.2), (93; 25.6), (94; 7.2), (95; 25.7), (96; 10.3), (97; 4.7), (105; 24.6), (106; 6.5), (107; 25.9), (108; 15.4), (109; 49.8), (111; 4.7), (119; 14.0), (120; 4.3), (121; 21.4), (122; 21.7), (123; 8.2), (133; 10.0), (135; 11.3), (136; 5.7), (139; 7.8), (147; 11.3), (148; 8.7), (149; 9.3), (161; 30.6), (162; 7.1), (164; 5.2), (189; 14.5), (204; 17.0).
P. regnellii
1-Methoxy-4-(1-propenyl) benzene (m/z; % relative abundance): (41; 2.2), (50; 6.4), (51; 11.6), (52; 4.1), (53; 4.0), (55; 5.1), (62; 2.9), (63; 8.2), (64; 2.4), (65; 6.4), (74; 3.7), (75; 2.8), (77; 24.6), (78; 11.2), (79; 15.9), (80; 1.1), (89; 5.1), (90; 1.8), (91; 16.0), (92; 2.3), (93; 1.6), (102; 4.0), (103; 15.9), (104; 5.1), (105; 22.3), (106; 2.4), (107; 1.9), (115;18.1), (116; 7.2), (117; 28.7), (118; 3.7), (119; 2.5), (121; 17.1), (122; 1.7), (131; 4.9), (132; 4.4), (133; 24.0), (134; 2.2), (147; 55.2), (148; 99.9), (149; 10.1).
α-Copaene (m/z; % relative abundance): (41; 33.6), (42; 2.8), (43; 1.9), (44; 3.8), (51; 3.2), (52; 1.3), (53; 8.0), (55; 23.4), (56; 6.4), (57; 2.6), (63; 1.5), (65; 6.3), (66; 1.3), (67; 7.0), (69; 14.5), (70; 3.0), (73; 1.0), (77; 13.1), (78; 4.2), (79; 11.2), (80; 4.2), (81; 32.0), (82; 2.7), (83; 1.2), (91; 29.2), (92; 18.8), (93; 25.9), (94; 3.9), (95; 4.5), (103; 3.4), (104; 2.5), (105; 97.6), (106; 11.3), (107; 14.4), (108; 5.2), (109; 1.6), (115; 4.8), (116; 2.0), (117; 6.4), (118; 4.2), (119; 93.3), (120; 28.6), (121; 4.9), (122; 1.5), (127; 1.3), (128; 2.7), (129; 2.6), (130; 1.2), (131; 3.9), (132; 1.5), (133; 8.3), (134; 2.9), (145; 2.2), (146; 1.1), (147; 4.2), (148; 1.1), (159; 3.2), (161; 99.9), (162; 13.6), (189; 1.7), (204; 20.4), (205; 3.5).
Aromadendrene (m/z; % relative abundance): (40; 12.1), (41; 99.9), (42; 8.3), (43; 16.2), (44; 1.4), (50; 2.4), (51; 10.6), (52; 5.9), (53; 25.6), (54; 5.3), (55; 35.4), (56; 3.7), (57; 3.7), (63; 3.7), (64; 2.7), (65; 16.5), (66; 6.4), (67; 35.8), (68; 5.7), (69; 34.8), (70; 2.2), (71; 1.8), (76; 9), (77; 36.5), (78; 12.8), (79; 50.7), (80; 10.4), (81; 32.3), (82; 10.2), (83; 4.3), (89; 1.8), (91; 67.8), (92; 16.5), (93; 45.5), (94; 13.1), (95; 15.9), (96; 3.3), (102; 1.8), (103; 5.4), (105; 50.2), (106; 16.5), (107; 36.5), (108; 9.1), (109; 7.1), (115; 6.0), (116; 3.3), (117; 8.9), (119; 32.3), (120; 13.2), (121; 16.9), (122; 13.3), (123; 2.7), (127; 1.7), (128; 3.3), (129; 2.2), (131; 1.7), (132; 4.5), (133; 31.6), (134; 9.5), (135; 7.4), (145; 2.6), (147; 22.1), (148; 15.9), (149; 4.2), (159; 1.4), (161; 33.3), (162; 5.6), (163; 1.2), (175; 3.2), (176; 2.1), (189; 9.3), (204; 11.6), (205; 2.4).
δ-Cadinene (m/z; % relative abundance): (41; 20.8), (42; 1.5), (43; 6.5), (51; 2.0), (52; 1.1), (53; 6.5), (54; 8), (55; 12.2), (56; 1.1), (57; 1.5), (65; 4.3), (66; 1.4), (67; 10.0), (68; 1.8), (69; 12.2), (70; 1.2), (77; 13.0), (78; 3.4), (79; 22.2), (80; 4.3), (81; 19.6), (82; 2.2), (83; 1.4), (91; 24.4), (92; 12.2), (93; 33.5), (94; 12.2), (95; 7.4), (96; 1.0), (103; 1.9), (104; 1.7), (105; 38.2), (106; 7.8), (107; 9.6), (108; 2.5), (109; 3.1), (110; 5), (115; 3.1), (116; 1.3), (117; 4.1), (118; 2.2), (119; 34.8), (120; 10.0), (121; 10.0), (122; 2.7), (123; 1.5), (124; 5), (127; 1.3), (128; 1.6), (129; 1.6), (130; 9), (131; 3.1), (132; 1.4), (133; 18.3), (134; 10.0), (135; 7.4), (136; 7.0), (145; 2.7), (147; 6.1), (148; 6.1), (149; 2.1), (150; 1.4), (159; 2.0), (160; 2.7), (161; 99.9), (162; 14.3), (163; 1.4), (175; 2.1), (176; 1.6), (189; 11.3), (190; 1.6), (204; 33.0), (205; 5.1).
Dillapiole (m/z; % relative abundance): (40; 1.2), (50; 1.7), (51; 3.0), (52; 2.1), (53; 3.3), (55; 1.2), (59; 1.0), (62; 1.3), (63; 2.7), (64; 1.1), (65; 6.8), (66; 3.8), (67; 1.3), (69; 1.9), (76; 1.1), (77; 7.4), (78; 4.0), (79; 3.7), (81; 1.8), (83; 2.2), (90; 1.9), (91; 6.0), (92; 1.6), (93; 4.2), (94; 1.4), (95; 1.3), (103; 2.2), (105; 1.9), (106; 4.9), (107; 2.0), (117; 1.9), (118; 1.3), (119; 2.2), (121; 6.8), (131; 1.6), (133; 4.5), (134; 3.9), (135; 1.8), (145; 1.3), (147; 1.6), (149; 16.3), (150; 2.3), (151; 1.4), (161; 8.5), (162; 1.9), (163; 2.9), (165; 1.6), (175; 1.7), (176; 1.0), (177; 18.1), (178; 2.4), (179; 1.4), (180; 2.4), (191; 12.1), (192; 2.8), (193; 1.7), (195; 10.5), (196; 1.0), (207; 26.1), (221; 3.0), (222; 99.9), (223; 12.9).
β-Eudesmol (m/z; % relative abundance): (41; 44.4), (43; 25.2), (52; 4.7), (55; 22.3), (59; 99.9), (65; 10.5), (67; 30.5), (69; 10.8), (77; 12.9), (78; 8.5), (79; 20.6), (81; 23.7), (83; 10.6), (88; 2.6), (89; 2.0), (93; 14.3), (102; 1.2), (103; 2.5), (105; 13.7), (106; 5.3), (107; 10.3), (109; 16.5), (121; 13.1), (127; 1.3), (133; 7.6), (135; 6.0), (151; 1.5), (160; 1.6), (161; 12.7), (164; 10.8), (191; 1.4), (202; 1.1), (212; 9), (223; 8).
Apiol (m/z; % relative abundance): (40; 1.2), (50; 1.7), (51; 3.0), (52; 2.1), (53; 3.3), (55; 1.2), (59; 1.0), (62; 1.3), (63; 2.7), (64; 1.1), (65; 6.8), (66; 3.8), (67; 1.3), (69; 1.9), (76; 1.1), (77; 7.4), (78; 4.0), (79; 3.7), (81; 1.8), (82; 5), (83; 2.2), (90; 1.9), (91; 6.0), (92; 1.6), (93; 4.2), (94; 1.4), (95; 1.3), (103; 2.2), (105; 1.9), (106; 4.9), (107; 2.0), (117; 1.9), (118; 1.3), (119; 2.2), (120; 5), (121; 6.8), (131; 1.6), (132; 4), (133; 4.5), (134; 3.9), (135; 1.8), (145; 1.3), (147; 1.6), (149; 16.3), (150; 2.3), (151; 1.4), (161; 8.5), (162; 1.9), (163; 2.9), (165; 1.6), (175; 1.7), (176; 1.0), (177; 18.1), (178; 2.4), (179; 1.4), (180; 2.4), (191; 12.1), (192; 2.8), (193; 1.7), (194; 1), (195; 10.5), (196; 1.0), (207; 26.1), (221; 3.0), (222; 99.9), (223; 12.9).