Abstracts

ARTIGO

Antimicrobial activity of Trembleya laniflora , Xyris platystachia and Xyris pterygoblephara

Atividade antimicrobina de Trembleya laniflora , Xyris platystachia e Xyris pterygoblephara

Claiton Pires Ventura; Alaíde Braga de Oliveira; Fernão Castro Braga^* * E-mail: fernao@netuno.lcc.ufmg.br, Tel. +55-31-34996951, Fax +55-31-34996935

Faculdade de Farmácia, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Campus Pampulha, 31270-901, Belo Horizonte, MG, Brazil

ABSTRACT

Trembleya laniflora (D. Don) Cogn. (Melastomataceae), Xyris platystachia Alb. Nilss. (Xyridaceae) and Xyris pterygoblephara Kunth., Brazilian species collected from a biodiversity hotspot for conservation priority, had their antimicrobial activity evaluated against standardized strains of Staphylococcus aureus and Micrococcus luteus, by the agar diffusion assay. All extracts, assayed in the concentration of 2000 µg/disc, were active against M. luteus, whereas S. aureus growth was inhibited only by T. laniflora leaves and X. platystachia aerial parts. Fractionation of the extracts by partition between immiscible solvents resulted in active fractions from extracts originally inactive against S. aureus. Activity was mainly found in low and medium polar fractions. The extract of T. laniflora leaves was also fractionated by silica gel column chromatography and both the HPLC fingerprint and antimicrobial activity of the obtained fractions were distinct of those originated from the partition process.

Keywords:Trembleya laniflora , Xyris platystachia , Xyris pterygoblephara, antimicrobial activity.

RESUMO

As espécies Trembleya laniflora (Melastomataceae), Xyris platystachia (Xyridaceae) e Xyris pterygoblephara foram coletadas na Serra do Cipó, região considerada hotspot para conservação de biodiversidade. A atividade antimicrobiana dessas espécies foi avaliada em ensaios in vitro de difusão em ágar frente a linhagens padronizadas de Staphylococcus aureus e Micrococcus luteus. Todos os extratos, avaliados na concentração de 2000 µg/disco, foram ativos contra M. luteus, enquanto a inibição de crescimento de S. aureus somente foi observada para os extratos de T. laniflora (folhas) e X. platystachia (partes aéreas). A partição dos extratos brutos entre solventes imiscíveis resultou na obtenção de frações ativas, oriundas de extratos originalmente inativos frente a S. aureus, observando-se atividade principalmente para as frações de baixa e média polaridade. O extrato de folhas de T. laniflora foi adicionalmente fracionado por cromatografia em coluna de sílica gel e as frações resultantes apresentaram atividade antimicrobiana e perfis por CLAE distintos daquelas obtidas pela partição entre solventes imiscíveis.

Unitermos:Trembleya laniflora , Xyris platystachia; Xyris pterygoblephara, atividade antimicrobiana.

INTRODUCTION

Brazil is recognized as one of the megadivesity countries, concentrating around 10 to 20% of all plant species in the world (Mittermeier et al., 1997). A total of 10,000 plant species are estimated to occur in Minas Gerais (Mendonça; Lins, 2000). Serra do Cipó is a national park located in this state, in a region classified as a biodiversity hotspot for conservation priority (Myers et al., 2000). The area presents exceptional concentrations of endemic species and is experiencing loss of habitats, contributing to drive many species into extinction. We have previously investigated the antifungal and antibacterial activity of 20 plant species from Serra do Cipó (Cota et al., 2002). Among the active species, three were selected for study in the present work.

Trembleya laniflora (Melastomataceae) is a shrub popularly named "flor-de-lã" (wool flower), used as ornamental species (Pio Corrêa, 1969). T. laniflora grows mainly in rocky soils from campos rupestres, an altitudinal ecosystem covered by open vegetation, being the genus endemic in Brazil. A chemotaxonomic study carried out for the leaves of Melastomataceae species, belonging to the closely related genera Lavoisiera , Microlicia and Trembleya, resulted in the identification of 116 flavonoids, comprising 69 flavonol and 47 flavone glycosides, including kaempferol 3-O-glycosides and quercetin 3-O-glycosides in T. laniflora (Bomfim-Patrício et al., 2001). A literature search indicated no ethnomedical use or biological activity other than antimicrobial described for T. laniflora (Cota et al., 2002).

Xyris species are small shrubs, popularly known as "sempre-vivas" (everlasting plants). Some are collected for ornamental purposes or for medicinal uses, to treat eczemas and dermatitis (Pio Corrêa, 1969). The identification of Xyris species based solely on morphological characters is rather limited (Varanda et al., 2002). Around 90% of the Xyris found in Brazil are endemic (Sajo et al., 1997) and over harvesting has put several species in risk of extinction.

The chemistry and biological activity of Xyris have been poorly investigated. Metabolites obtained from this genus include isocoumarins from X. indica (Ruangrungsi et al., 1995), anthraquinones from X. semifuscata (Fournier et al., 1975) and flavonoids from X. itatiayensis , X. longiscapa and X. obtusiuscula (Varanda et al., 2002). Recently, we reported the isolation of a new anthraquinone from X. pilosa, active against Fusarium oxysporum (Cota et al., 2004). Except the antimicrobial effect (Cota et al., 2002; Cota et al., 2004), no other biological activity or any chemical data has been described for X. platystachia and X. pterygoblephara, species selected for the study.

The diversity of compounds found in plant species make these organisms promising sources of new antimicrobial agents, with general or specific effects (Amaral et al., 2006; Leitão et al., 2006). The interest in plant secondary metabolites with antimicrobial properties has revived as a consequence of microbial resistance development against the antibiotics in clinical use (Rocha et al., 2004; Lima et al., 2006; Oliveira et al., 2006), especially in the case of opportunistic infections affecting immunocompromised patients (Klausmeyer et al., 2004). Considering that the species selected for the study occur in an ecosystem with a high degree of endemism, it is feasible to infer that they are a potential source of bioactive compounds. Hence, the main goal of this work was to assay the antimicrobial activity of extracts and fractions from T. laniflora , X. platystachia and X. pterygoblephara.

MATERIAL AND METHODS

Plant materials

The species Trembleya laniflora (D. Don) Cogn., Xyris pterygoblephara Kunth. and Xyris platystachia Alb. Nilss. were collected in Minas Gerais state, Brazil, at Serra do Cipó National Park and APA Morro da Pedreira. The plants were identified by botanists from the Fundação Zoo-Botânica, Belo Horizonte, Brazil, where voucher specimens are deposited, under numbers BHZB 2340, BHZB 2496 and BHZB 2492, respectively.

Plant material extraction and preliminary fractionation

The plants were dried separately, at 40 ºC, for 72 h. The extracts of T. laniflora (leaves, stems), X. pterygoblephara (aerial parts) and X. platystachia (aerial parts) were prepared by exhaustive percolation with ethanol. The extracts were concentrated to residue by removing the solvents in a rotavapor, at 50 ºC. Data obtained for the dry extracts are shown in Table 1. Portions (2 g) of the dry extracts were suspended in MeOH/H₂O (1:1; 120 mL) and sequentially partitioned with equal volumes (120 mL) of n-hexane, CH₂Cl₂ and EtOAc. MeOH was removed in a rotavapor, before partitioning the extract suspension with CH₂Cl₂ and EtOAc. Solvents were removed in a rotatory evaporator, at maximum temperature of 50 °C, and the obtained residues are displayed in Table 2. Emulsions were generated during the partition of the extract from T. laniflora leaves between n-hexane (Emulsion 1), dichloromethane (Emulsion 2) and ethyl acetate (Emulsions 3 and 4). HPLC analysis carried out for the emulsions indicated distinct profiles from the obtained fractions and for this reason they were concentrated separately and had their residues evaluated in the antimicrobial assays.

Chromatographic fractionation of the extract from T. lanifora leaves

The crude extract of T. laniflora leaves (22.8 g) was chromatographed on a silica gel column (70-230 mesh, Merck), employing a gradient elution of n-hexane (79.6 mg, TL1), n-hexane:CH₂Cl₂ (1:1) (361.7 mg, TL2), CH₂Cl₂ (689.4 mg, TL3), CH₂Cl₂:EtOAc (1:1) (1639.3 mg, TL4; 1239.7 mg, TL5), EtOAc (2205.2 mg, TL6), MeOH (80.9 mg, TL7; 11589.8 mg, TL8) and MeOH:H₂O (1:1) (1201.6 mg, TL9).

Bacterial cultures and growth conditions

Staphylococcus aureus ATCC 25923 and Micrococcus luteus ATCC 9341 were employed as test organisms. The cultures were grown in agar medium, in tubes kept in a slating position, at 36 ºC, for 24 h. Cultures were maintained in plates, at 4 °C, in nº 1 antibiotic agar.

Antimicrobial assay

The antibacterial activity of extracts and fractions was evaluated by the disk diffusion method. For the assays, solutions of the extracts and fractions were prepared in MeOH to concentrations of 100 and 50 mg/mL, respectively. Suspensions of microorganisms were prepared in peptone saline solution. The transmittance of the inoculum suspension was adjusted to 50 ± 1%, at 580 nm. Seeded agar plates were prepared by pouring 20 mL of nº 1 antibiotic agar into each plate. After medium solidification, each plate was overlaid with 5 mL medium containing 0.05% of the inoculum suspension. Sterile paper discs (6 mm diameter) were impregnated with 20 µL of the extracts (2000 µg/disc) or fractions (1000 µg/disc). The discs were placed in duplicate onto the plates and incubated for 24 h, at 37 ºC. The experiments were carried out in six replicates. The results (mean value plus standard deviation) were recorded by measuring the zones of growth inhibition surrounding the discs. Chloramphenicol (3 µg/disc) was included in the assays as positive control, whereas control disks contained solvent only (MeOH) as negative control.

HPLC characterization of fractions

Analysis were carried out on a Merck-Hitachi apparatus (Germany) composed of pump L-6200A, automatic injector AS-2000A, UV-VIS detector L-4250 and integrator D-2500. An ODS column (150 × 4.0 mm I.D., 5 µM) was employed (Merck, Germany) at a temperature of 40 °C and flow rate of 1.0 mL/min. Analysis were performed at 220 nm. A linear gradient of H₂O (A) and CH₃CN (B) was employed: 0 min 90% A, 10% B; 60 min 10% A, 90% B, followed by 10 min of isocratic elution. Solvents used were of HPLC grade (Merck, Germany) and were degassed by sonication before use. Fractions were dissolved in MeOH to a concentration of 5 mg/mL. After centrifugation at 10,000 r.p.m, the sample solutions (30 µL) were automatically injected.

Phytochemical analysis

The presence of saponins, alkaloids, coumarins, anthraquinones, flavonoids, triterpenes and tannins was evaluated in the ethanol extracts, by TLC analysis, according to Wagner et al. (1984).

RESULTS AND DISCUSSION

The ethanol extracts of Trembleya laniflora , Xyris platystachia and Xyris pterygoblephara have been previously evaluated in antimicrobial assays, against different strains of bacteria and fungi (Cota et al., 2002; Cota et al., 2004). The three species were particularly active against S. aureus and M. luteus, and for this reason both were selected as test organism in the present work. Aliquots from the crude ethanol extracts were initially fractionated by partition between immiscible solvents and the obtained results are displayed in Table 2.

All assayed extracts were active against M. luteus, whereas S. aureus growth was inhibited only by the extracts from T. laniflora leaves and X. platystachia aerial parts. In the previous work (Cota et al., 2002), the extracts from T. laniflora stems and X. pterygoblephara aerial parts were active against S. aureus, while X. platystachia did not show inhibitory effect against this microorganism. Such contradictory results may be explained by differences in extract compositions, since the plant materials were collected in distinct locations.

Phytochemical analysis (Wagner et al., 1984) of the ethanol extracts gave positive results for saponins, triterpenes and tannins. Flavonoids were also detected in the three species, except in the stems of T. laniflora, whereas coumarins were solely present in the aerial parts of X. platystachia and X. pteryglobephara. Several flavonoids (Harborne; Williams, 2002), coumarins (Borges et al., 2005), tannins (Chung et al., 1998), saponins (Wallace, 2004) and triterpnes (Katerere et al., 2003) have been reported to posses antimicrobial activity. Therefore, the presence of metabolites from these classes in the assayed species might explain their antimicrobial activity here reported.

The four extracts were submitted to preliminary fractionation by partition between immiscible solvents and the resulting fractions had their antimicrobial activity evaluated. Active fractions were obtained from extracts originally inactive against S. aureus (Table 3). This result demonstrates the importance of a preliminary fractionation when assaying the antimicrobial activity of plant extracts, once the low concentration of the active compounds may impair their detection in crude extracts.

Partition between immiscible solvents is an adequate approach for the preliminary separation of complex matrices, such as vegetal extracts. However, scaling up this procedure to obtain quantities of material for further studies is frequently time consuming and production of emulsions is almost impossible to avoid. Fractionation by silica gel column chromatography constitutes an alternative to overcome these limitations. It should be reminded, however, that different physical phenomena are involved in these procedures, namely solubility in the first and adsorption in the second. Hence, applying these methods to the same matrix might result in fractions with distinct compositions and activities.

In order to confirm this supposition, fingerprint profiles were registered by HPLC for fractions of the extract from T. laniflora leaves obtained by partition between immiscible solvents and by fractionation on a silica gel column. Besides, the antimicrobial activity of the chromatographic fractions was also assayed.

Dichloromethane fractions originated from both approaches showed distinct HPLC profiles (Figure 1) and antimicrobial effects: while the fraction obtained by partition was significantly active against M. luteus and S. aureus (Table 3), the chromatographic one (TL3) showed no activity against both microorganisms (Table 4). On the other hand, HPLC analysis of the dichloromethane / ethyl acetate (1:1) fraction (TL4), originated from the chromatographic fractionation, showed a more related profile to that of the dichloromethane fraction obtained by partition between immiscible solvents (Figure 1). However, TL4 was active solely against M. luteus. These results clearly confirm our hypothesis that fraction constitution, and therefore biological activity, depends on the procedure adopted for fractionation.

In conclusion, the results here reported corroborate the popular use of the species to treat microbial diseases and also demonstrate the relevance of a preliminary fractionation for detecting active fractions, when assaying the antimicrobial effect of plant extracts.

ACKNOWLEDGEMENTS

This work was financed by funds from Fundo Fundep, UFMG, Brazil. CNPq is also acknowledged for a research fellowship (F.C.B.).

Received 06/18/06. Accepted 01/25/07

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