Bioactivity of crude extracts and some constituents of Blutaparon portulacoides (Amaranthaceae)

Phytomedicine 9: 566–571, 2002 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/phytomed

Phytomedicine

Bioactivity of crude extracts and some constituents of Blutaparon portulacoides (Amaranthaceae) M. J. Salvador1, E. O. Ferreira1, E. M. F. Pral2, S. C. Alfieri2, S. Albuquerque1, I. Y. Ito1, and D. A. Dias1 1 2

Faculdade de Ciˆencias Farmacêuticas de Ribeir˜ao Preto – Universidade de S˜ao Paulo, Avenida do café, Ribeir a˜o Preto (SP), Brasil Instituto de Ciências Biomédicas – Universidade de S˜ao Paulo, Avenida Prof. Lineu Prestes, S˜ao Paulo (SP), Brasil

Summary Crude extracts (aerial parts and roots, both dried), methylenedioxyflavonol, and a mixture of acyl steryl glycosides isolated from Blutaparon portulacoides, were assayed for their toxicity against Trypanosoma cruzi trypomastigotes and Leishmania amazonensis amastigotes from axenic cultures. The antimicrobial activity was also investigated, in a screening conducted using fifteen strains of Gram-positive and Gram-negative bacteria, along with the yeasts, Candida albicans and Candida tropicalis. To assess the antibacterial activity of the isolated compounds, the minimum inhibitory concentrations (MICs) were determined. There are no reports of acyl steryl glycosides in the genus Blutaparon and their biological activities are being evaluated for the first time. Key words: Blutaparon portulacoides, Amaranthaceae, methylenedioxyflavonol, acyl steryl glycoside, Trypanosoma cruzi, Leishmania amazonensis, antimicrobial activity

j Introduction The Amaranthaceae family comprises approximately 65 genera and 1000 species of annual and perennial herbaceous plants, shrubs and some trees occurring in tropical, subtropical and temperate regions (Siqueira, 1994/1995). Many plants of this family have been employed in folk medicine in the treatment of several diseases and for their nutritive value (Macedo et al., 1999; De Souza et al., 1998; Gorinstein et al., 1991; Patterson et al., 1991; Si-Man et al., 1988). Blutaparon portulacoides, a well-known species belonging to the Gomphreneae tribe, is commonly found on Brazil’s eastern beaches (Farias and Flores, 1989), where it is known popularly as “capotiraguá”. In folk medicine, B. portulacoides has been employed for the treatment of leukorrhea (Siqueira, 1987). This work reports the isolation and identification of bioactive compounds from ethanolic crude extracts prepared from dried aerial parts and roots of B. portulacoides. Crude extracts, methylenedioxyflavonol (1)

(Ferreira and Dias, 2000), and a mixture of acyl steryl glycosides (1a and 2b) were screened for trypanocidal and leishmanicidal activity in vitro, and also for antimicrobial activity. This is the first report documenting acyl steryl glycosides and their biological activities in the genus Blutaparon.

j Materials and Methods Plant Material

Blutaparon portulacoides (St. Hil) Mears (Amaranthaceae), aerial parts and roots, was collected at Restinga de Maricá, Rio de Janeiro, RJ, Brazil, in January 1995, and identified by Prof. J. C. de Siqueira (Pontificia Universidade Católica, Rio de Janeirao). An authenticated youcher specimen was deposited at the Herbarium of the Department of Biology of the School of Sciences of FFCLRP-USP (SPSFR 02961). 0944-7113/02/09/06-566 $ 15.00/0

Bioactivity of crude extracts of Blutaparon portulacoides General experimental procedures

NMR Analyses were performed on a Bruker DPX-300 spectrometer operating at 300 MHz for 1H and 75 MHz for 13C. Gas chromatograph (GC) analyses were carried out on a Hewlett-Packard 5890 Series II gas chromatograph equipped with a capillary inlet system. The capillary column (30 m × 0.25 mm × 0.25 µm) consisted of fused silica coated with cross-linked methylsilicone (HP1). The carrier gas was hydrogen with a linear velocity of 40 cm/s. Operating conditions wereas follows: the column was equilibrated initially at 100 °C for 1 min and the temperature was then raised at a rate of 5 °C/min up to 250 °C for 20 min. Sample introduction was performed in the split mode at an injection temperature of 260 °C and a Flame Ionization Detector (FID) at 300 °C was used. Extraction and isolation

The dried and powdered material (aerial parts and roots) of B. portulacoides was extracted exhaustively using hexane, ethyl acetate and ethanol successively to yield crude extracts. The aqueous lyophilized extracts (aerial parts and roots) were prepared in hot (FAHL) and cold (FACL) water, as described by Zucchi et al. (2000). The mixture of acyl steryl glycosides was isolated from the ethanolic extract of roots (3.9 kg). The crude extract (177 g) was submitted to LVC chromatography over silica gel 60H (5–40 µm) using various ratios of hexane, ethyl acetate and methanol as solvents. After TLC, fraction 15 was purified by washes with acetone, yielding 0.100 g of mixture. The methylenedioxyflavonol (0.022 g) was obtained from the crude ethanolic extract (600 g) of the aerial parts (6.2 kg) of the plant. The purification process has been reported previously (Ferreira and Dias, 2000). Bioactivity screening

In vitro trypanocidal activity, using trypomastigote forms of Trypanosoma cruzi (Y strain), was performed according to protocols established previously (Bastos et al., 1999). Blood of infected Swiss albino mice at the peak of parasitemia (7th day post-infection) was collected by cardiac puncture and was mixed with blood of healthy mice to adjust parasite suspension to 106 trypomastigotes forms/ml. Crude extracts (at 4000 µg/ml) and isolated compounds (at 100, 250 and 500 µg/ml) were prepared in dimethyl sulfoxide (DMSO) and were added to the trypomastigote-containing blood samples to provide the final concentrations given above. After 24 h incubation at 4 °C, trypanocidal activity was evaluated by counting the remaining trypomastigotes (Brener, 1962). Negative and positive controls containing, respectively, DMSO and gentian violet were run in parallel. The bioassays were performed in triplicate. To assess the effect of crude extracts and isolated compounds on viability of Leishmania amazonensis

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(strain designation MPRO/BR/72/M 1841) amastigotes, parasites cultivated serially at 33 °C in modified UM-54 medium (Pral et al., 1993; Balanco et al., 1998), pH 5.0, were resuspended in RPMI medium supplemented with 4% fetal calf serum, pH 5.0, and incubated at 33 °C for 24 h with crude extracts (1000 µg/ml) or the isolated compounds (14, 84, and 500 µg/ml) dissolved previously in RPMI or DMSO. Control parasite suspensions were incubated in medium alone or in medium containing DMSO (negative controls). Amastigote viability was assessed colorimetrically by the reduction of a tetrazolium salt (MTT) as described by Mosmann (1983). Absorbances were expressed as percentages relative to untreated controls. Antimicrobial activity was measured by the welldiffusion method (well technique in double layer) (Cole, 1994; Grove and Randall, 1955). Twenty µL of each test-drug solution were applied to 5.0 mm diameter wells. Solutions were prepared in dimethylformamide (DMF) at 5000 µg/ml (for the crude extracts) and 2500 µg/ml for pure compounds. After incubation at 37 °C for 24 h, the inhibition zone, corresponding to the halo (H) formed from well edge to the beginning of the region of microbial growth was measured in millimeters (mm). The minimum inhibitory concentration (MIC) was determined in µg/ml (Tereschuk et al., 1997; Giesbrecht et al., 1987; Washington and Sutter, 1980). In these tests, gentamicin discs (CECON = 10 µg) were used as positive controls. In MIC determination, media containing sterile distilled water and DMF (1:1) were used as negative control for which no inhibitory effect could be observed. The following microorganisms were used: standard strains of Escherichia coli – ATCC 10538; Pseudomonas aeruginosa – ATCC 27853; Micrococcus luteus – ATCC 9341; Staphylococcus aureus – ATCC 25923 and 6548; Staphylococcus aureus 7+ penicilinase producer; Staphylococcus aureus 8– penicilinase nonproducer; Staphylococcus epidermidis 6ep – field strains, Candida albicans – ATCC 1023; Candida albicans-cas and Candida tropicalis-ct – field strains cultivated for 24 hours at 37 °C in Mueller Hinton broth (Difco)-MHb; Enterococcus faecalis – ATCC 10541; Streptococcus mutans – ATCC 25175; Streptococcus mutans (11.1; 9.1; 9.31; 11.22.1) and Streptococcus sobrinus 180.3 – field strains, incubated for 24 hours at 37 °C in Brain Heart Infusion (Difco) – BHI.

j Results Chemical structures of the methylenedioxyflavonol (1) and acyl steryl glycosides (2a and 2b) isolated from B. portulacoides and showing biological activity are presented in Fig. 1. The methylenedioxyflavonol, 3.5.3′-

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Fig. 1. Chemical structures of the compounds with biological activity, isolated from Blutaparon portulacoides.

trihydroxy-4′-methoxy-6,7-methylenedioxyflavone (1), isolated from the ethanolic extract (aerial parts) of B. portulacoides, was characterized by spectral data and literature comparison (Ferreira and Dias, 2000). The acyl steryl glycosides (2a and 2b) were isolated from the ethanolic extracts of roots and were identified by spectroscopic and GC analysis as well as by comparison with published data. The analysis of the IR spectrum (1730 and 1250 cm–1 of carbonyl ester and 3390 and 1080 cm–1 of hydroxyl groups), along with the observation of several signals attributed to methyl, methylene, carbonilic and olefinic groups in the 1H and 13 C NMR spectra, suggested the presence of a mixture of acyl steryl glycosides. The peaks at δ4.37 (J = 7.5 Hz, confirming the β-position of the sugar) and δ101.76, respectively, in the 1H- and 13C-NMR spectra, indicated glucose as the sugar moiety. The sitosteryl and stigmasteryl moiety was determined by the shifts of the olefinic carbons (Kojima et al., 1990). A sample containing 10 mg of the mixture was submitted to trans-esterification reaction (Luddy et al., 1968) to determine the derived fatty acid attached to glucosil moiety. Comparison with authentic sample by gas chromatography as well as 13C-NMR analysis by allowed the conclusion that palmitate is the fatty acid portion of the molecule. Comparison with published data led us to identify the compounds as sitosteryl and stigmasteryl 3-β-O-glucoside 6′-O-palmitate, isolated previously from Urtica dioica (Chaurasia and Wichtl, 1987) and Myrsine pellucida (Lavaud et al., 1994), respectively. The bioassay results for activity against T. cruzi trypomastigotes and L. amazonensis amastigote-like stages are summarized in Table 1. Aqueous lyophilized extracts from aerial parts and roots (FACL and FAHL), all assayed at 4000 µg/ml, did not interfere appreciably with the viability of T. cruzi trypomastigotes, excepting cold water, FACL extracts prepared from roots which reduced parasite viability to 34.4%. On the other hand, while viability of L. amazonensis amastigotes was not affected significantly by extracts of aerial parts, extracts prepared from roots were clearly leishmanicidal, and

reduced viability to 46.9 (FAHL) and 5.3% (FACL). Ethanolic extracts of aerial parts were non-toxic against T. cruzi trypomastigotes (at 4000 µg/ml), but reduced markedly the viability of L. amazonensis amastigotes at 1000 µg/ml. Ethanolic extracts of roots displayed a moderate effect when assayed with both T. cruzi trypomastigotes and L. amazonensis amastigotes. While the methylenedioxyflavonol, at 500 µg/ml, did not interfere appreciably with viability of both T. cruzi trypomastigotes and L. amazonensis amastigotes the mixtures of acyl steryl glycosides, at 100, 250 and 500 µg/ml were clearly trypanocidal and reduced parasite viability to 27.2, 26.4 and 24.9%, respectively. Toxicity of acyl steryl glycosides was also demonstrated in the bioassay with axenic L. amazonensis amastigotes. In the latter case, parasite viability was reduced to 36.0, 18.2 and 2.9%, when the compounds were assayed at 14, 84, and 500 µg/ml, respectively. That L. amazonensis amastigotes remained viable during the 24 h incubation period was indicated by the results of control parasite suspensions that were incubated in medium alone or in medium containing 0.2% DMSO (% amastigote viability = 98.6%). Aqueous lyophilized extracts (aerial parts and roots) were also screened for antimicrobial activity and were shown to be inactive (at 5000 µg/ml) against all microorganisms tested. As shown in Table 2, ethanolic extracts of aerial parts inhibited the growth of S. aureus (strain 7+ penicilinase producer), four strains of S. mutans, and S. sobrinus. Ethanolic extracts of roots were effective only against S. aureus (strain 7+ penicilinase producer), S. mutans (strain 9.1) and S. sobrinus. The methylenedioxyflavonol was active against S. aureus (ATCC 6538 and strain 7+ penicillinase producer), S. mutans (ATCC 25175, and strains 11.1, 9.1 and 11.22.1) and S. sobrinus. In these experiments, minimal inhibitory concentrations (MIC) varied from 20.0 µg/ml (against S. mutans 11.22.1) to 1250 µg/ml (S. aureus ATCC 6548). The mixture of acyl steryl glycosides was active against S. aureus (ATCC 25923 and strain 7+ penicilinase producer), S. epidermidis,

Bioactivity of crude extracts of Blutaparon portulacoides E. coli, S. mutans (strain 9.1) and S. sobrinus; MIC values were in the range of 50 and 500 µg/ml. As indicated in Table 2, the extracts and the compounds assayed were ineffective against M. luteus (ATCC 9341), S. aureus (strain 8-penicilinase non-producer),

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P. aeruginosa (ATCC 27853), E. faecalis (ATCC 10541) and the Candida strains tested. The gentamicin disc (CECON = 10 µg), here used as a positive control against all bacteria assayed, produced a halo of inhibition (H) of 18 mm.

Table 1. Activities of crude extracts and compounds isolated from Blutaparon portulacoides. aThe activity against Trypanosoma cruzi is expressed as percentage (%) of the trypanomastigotes viable. bEffect of extracts and compounds on viability of Leishmania amazonensis amastigotes is expressed as percentage (%) relative to untreated control. The experiments were run in triplicate. Extract or compound

% viability of T. cruzi trypomastigotes (µg/ml) a

% viability of L. amazonensis amastigotes (µg/ml) b

FACL – aerial parts FAHL – aerial parts FACL – roots FAHL – roots Ethanolic extract – aerial parts Ethanolic extract – roots Methylenedioxyflavonol (1) Acyl steryl glycosides (2a and 2b) Acyl steryl glycosides Acyl steryl glycosides

63.2 (4000) 78.4 (4000) 34.4 (4000) 78.4 (4000) 99.2 (4000) 47.2 (4000) 70.0 (500) 24.9 (500) 26.4 (500) 27.2 (100)

68.9 (1000) 96.1 (1000) 5.3 (1000) 2.8 (1000) 44.3 (1000) 44.3 (1000) 83.8 (500) 2.9 (500) 18.2 (84) 36.0 (14)

Table 2. Antimicrobial activity of crude extracts and isolated compounds (methylenedioxyflavonol and mixture acyl steryl glycosides) with MIC values (µg/ml) from Blutaparon portulacoides. Microorganisms

M. luteus (ATCC 9341)a S. aureus (ATCC 6538)a S. aureus (ATCC 25923)a S. aureus (7+)b S. aureus (8–)b S. epidermis (6ep)b E. coli (ATCC 10538)a P. aeruginosa (ATCC 27853)a E. faecalis (ATCC 10541)a S. mutans (ATCC 25175)a S. mutans (11.1)b S. mutans (9.1)b S. mutans (9.3)b S. mutans (11.22.1)b S. sobrinus (180.3)b C. albicans (ATCC 1023)a C. albicans (cas)b C. tropicalis (ct)b

Tested material A

B

C

D

H

H

H

MIC

H

MIC

– – – 15 – – – – – – 8 9 9 11 7 – – –

– – – 8 – – – – – – – 8 – – 7 – – –

– 6 – 12 – – – – – 9 10 8 – 7 6 – – –

– 1250 – 160 – – – – – 80 80 40 – 20 160 – – –

– – 7 7 – 6 9 – – – – 6 – – 6 – – –

– – 50 50 – 500 50 – – – – 500 – – 500 – – –

A – ethanolic extract – aerial parts; B – ethanolic extract – roots; C – methylenedioxyflavonol (1); D – mixture of acyl steryl glycosides (2a and 2b); H – halo of inhibition; – without inhibition of the development; MIC – minimum inhibitory concentration; astandard strain; bfield strain (oral cavity). The values indicate diameter of inhibition in mm and MIC (µg/ml).

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j Discussion Here we report for the first time from the genus Blutaparon, the isolation of a mixture of acyl steryl glycosides displaying in vitro activity against T. cruzi trypomastigotes and L. amazonensis amastigotes, as well as antibacterial activities. Chagas’ disease affects more than 18 million people in Latin America, leading to approximately four hundred thousand deaths per year. The two major strategies used for controlling the disease are to keep the insect vector population under control in endemic regions and to prevent infection by blood transfusion, considered an important Source of infection (WHO, 1993). Gentian violet is the only effective compound available which efficiently eliminates the parasite from the blood prior to its transfusion (Bastos et al., 1999). However, despite its efficacy, there are several restrictions for use (Nussenzweig et al., 1953). On the other hand, patients with chronic Chagas’ disease are still waiting for more effective drugs (Dias, 1993). Thus, investigation of new drugs for therapeutic or prophylactic treatment is very welcome. Leishmaniases comprise a group of cutaneous, mucocutaneous and visceral diseases distributed worldwide, with around two million new cases being registered per year. The incidence of the disease has increased in the last several years and chemotherapy, although available, is toxic, difficult to apply in the field and not always effective (Martín et al., 1998). Drugs of first choice include pentavalent antimonials such as Nmethylglucamine antimonate (Glucantime) or sodium stibogluconate (Pentostam). Chemotherapy usually requires multidose schemes and often induces side effects (Croft, 1988). In addition, visceral leishmaniasis clinically resistant to antimony has been reported. Thus, a search for new, more effective drugs against amastigotes, the parasite stages that are ultimately responsible for the clinical manifestations of leishmaniasis, should be encouraged. The results documented here indicate that compounds isolated from B. portulacoides, particularly acyl steryl glycosides, display trypanocidal and leishmanicidal activity in vitro and can thus be considered as potential candidate drugs in the treatment of these two parasitic diseases. However, their toxicological effects, as well as the molecular mechanisms by which these drugs affect cell growth, should be determined prior considering them for systematic use. Considering that leishmaniasis not only manifest as a visceral disease but also as a cutaneous infection, it would be interesting to determine the efficacy of tropical administration (ointments) of the compounds. With microorganisms as etiological agents of infectious diseases, and with the difficulties emerging

today in chemotherapy of bacterial diseases due to resistance and tolerance of microorganisms to drugs used currently, the interest in research of antimicrobial substances and discovery of new chemotherapeutic agents has intensified. This screening conducted with extracts and compounds isolated from B. portulacoides revealed that the antimicrobial activity was associated mainly with Gram-positive bacteria. The methylenedioxyflavonol was active mainly against the strains of Streptococcus analyzed, against five of the six strains tested. The MIC values obtained for the methylenedioxyflavonol (40 µg/ml against S. mutans strain 9.1, and 160 µg/ml against S. sobrinus strain 180.3) were considerably smaller than the MIC value determined for the mixture of acyl steryl glycosides (500 µg/ml for the two respective strains). Except for the mixture of acyl steryl glycoside, all other drugs assayed were inactive against S. epidermidis (strain 6ep) and E. coli (ATCC 10538). Thus, our results explain and justify, at least in part, the popular use of B. portulacoides for the treatment of infections (leukorrhea). Overall, the results obtained in this study indicate the potential of B. portulacoides as a source of new trypanocidal, leishmanicidal and antibacterial compounds.

Acknowledgements

We are grateful to Professor J. C. de Siqueira for identifying the plant material and to FAPESP, CAPES and CNPq for their financial support.

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j Address M. J. Salvador, Departemento de Fisica e Quimica, Faculdade de Ciências Farmacêuticas de Ribeir a˜ o Preto – Universidade de S a˜ o Paulo, Avenida do café, s/n, CEP 14040-903 – Ribeir˜ao Preto (SP), Brasil Tel.: ++55-16-602-4248; fax: ++55-16-502-4161; e-mail: [email protected]