Screening of Central and South American plant extracts for antimycobacterial activity by the Alamar Blue test

Triagem de extratos de plantas das Américas Central e do Sul para atividade antimicobacteriana pelo teste do Alamar Blue

ARTIGO

Screening of Central and South American plant extracts for antimycobacterial activity by the Alamar Blue test

Triagem de extratos de plantas das Américas Central e do Sul para atividade antimicobacteriana pelo teste do Alamar Blue

Suzana G. LeitãoI, ^*; Oscar Castro^II; Eduardo N. Fonseca^I; Lisieux S. Julião^III; Eliana S. Tavares^III; Rodrigo R.T. Leo^III; Ricardo C. Vieira^III; Danilo R. Oliveira^IV; Gilda G. Leitão^IV; Virginia Martino^V; Valeria Sulsen^V; Ymira A.G. Barbosa^I; Diva P.G. Pinheiro^I; Pedro E. Almeida da Silva^VI; Dulcineia F. Teixeira^VII; Ivan N. Junior^VIII; Maria Cristina S. Lourenço^VIII

^IDepartamento de Produtos Naturais e Alimentos, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Ilha do Fundão, 21941-590, Rio de Janeiro, RJ, Brazil

^IIDepartamento de Química, Universidad Nacional, Heredia, Costa Rica

^IIIDepartamento de Botânica, Instituto de Biologia, Universidade Federal do Rio de Janeiro, Ilha do Fundão, 21951-590, Rio de Janeiro, RJ, Brazil

^IVNúcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, Ilha do Fundão, 21941-590, Rio de Janeiro, RJ, Brazil

^VCátedra de Farmacognosia, Instituto de Química y Metabolismo del Fármaco (UBA – CONICET), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina

^VILaboratório de Micobacterias, Departamento de Patologia, Fundação Universidade Federal do do Rio Grande, Rio Grande, 96200-190, Rio Grande do Sul, Brazil

^VIIFarmanguinhos, Fiocruz, Rua Sizenando Nabuco 100, Manguinhos, 21041-250, Rio de Janeiro, RJ, Brasil

^VIIIInstituto de Pesquisa Clínica Evandro Chagas, Serviço de Bacteriologia, Setor de Testagem de Drogas, Fiocruz, 21045-900, Rio de Janeiro, RJ, Brazil

ABSTRACT

Forty eight ethanolic crude extracts and fractions (hexane, dichloromethane, ethyl acetate and n-butanol) from ten Brazilian plants (Leguminosae, Monimiaceae and Verbenaceae), 1 from Costa Rica (Verbenaceae) and 1 from Argentina (Verbenaceae) were screened for anti-mycobacterium activity against Mycobacterium tuberculosis (ATCC-27294H₃₇Rv), by the Alamar Blue test, at a fixed concentration of 100 µg/mL. Out of the forty eight, seven were active at this concentration, corresponding to Lantana trifolia (hexane and dichloromethane extracts from leaves), Vitex cooperi (methanol:water, 1:1 extract from barks), Lippia lacunosa (hexane and dichloromethane extracts from leaves) and Lippia rotundifolia (hexane and dichloromethane extracts from leaves), all from the Verbenaceae family.

Keywords:Mycobacterium tuberculosis, Lantana trifolia, Vitex cooperi, Lippia lacunosa, Lippia rotundifolia, antimycobacterial activity, 20-hydroxyecdysone.

RESUMO

Quarenta e oito extratos brutos etanólicos e frações (em hexano, diclorometano, acetato de etila e n-butanol) de dez plantas brasileiras pertencentes às famílias Leguminosae, Monimiaceae e Verbenaceae; uma da Costa Rica (Verbenaceae) e uma da Argentina (Verbenaceae) foram ensaiados para verificação da atividade anti-micobacteriana contra Mycobacterium tuberculosis (ATCC-27294H₃₇Rv), pelo teste do Alamar Blue, a uma concentração fixa de 100 µg/mL. Dentre os quarenta e oito extratos e frações estudados, sete mostraram-se ativos na concentração ensaiada - frações em hexano e diclorometano de folhas de Lantana trifolia, extrato em metanol:água, 1:1 de cascas de Vitex cooperi, frações em hexano e diclorometano de folhas de Lippia lacunosa e de Lippia rotundifolia, sendoque todas essas plantas pertencem à família Verbenaceae.

Unitermos:Mycobacterium tuberculosis, Lantana trifolia, Vitex cooperi, Lippia lacunosa, Lippia rotundifolia, atividade antimicobacteriana, 20-hidroxi-ecdisona.

INTRODUCTION

Tuberculosis (TB) is a systemic disease caused by Mycobacterium tuberculosis, the alcohol-acid resistant bacillus, discovered by Robert Koch more than 120 years ago. Despite the discovery of very effective drugs against it more then 40 years ago, it still remains a serious health problem in many regions of the world, specially in developing nations (Lall; Meyer, 1999). Currently, almost one third of the world population is infected with M. tuberculosis, and each year 2-3 million people die from it (Newton et al., 2002). It is estimated that between the years 2000 and 2020 nearly one billion people will be newly infected, 20 million will develop TB and 35 million will die from the disease (WHO, 2000). It is estimated that in Latin America approximately 600.000 new cases of tuberculosis occur per year. Brazil, with c.a. 129.000 new cases per year, ranks the 13º position within other 22 countries with high-burden of TB incidence. The estimated incidence of TB in Costa Rica and Brazil is 15 and 62/100.000 respectively (WHO, 2005). The emergence of multidrug resistant strains (MDR) of M. tuberculosis as well as human immunodeficiency virus (HIV) infection have greatly amplified the incidence of TB, reaffirming tuberculosis as a primary public health threat. In the past 5 years several reports and review articles appeared in the literature about medicinal plants and natural products with anti-mycobacterium activity (Okunade et al., 2004; Copp, 2003; Newton et al., 2002; Cantrell et al., 2001). Over 350 natural products, mainly from plant species have been assessed for their antimycobacterial activities (Newton et al., 2002). A number have demonstrated significant in vitro antimycobacterial activity and active plant-derived compounds belonging to various chemical classes have been isolated (Newton et al., 2002). In recent years, a notable number of natural product-derived agents has been discovered by employing screening approaches involving cellular or biochemical targets in their assay design (Shu, 1998). In this way, we initiated a screening for anti-mycobacterium activity of some plant extracts against Mycobacterium tuberculosis (ATCC-27294H₃₇Rv), by the Alamar Blue test.

MATERIAL AND METHODS

Plant material

Plant species, collection sites as well as voucher specimen numbers are described in Table 1.

Plant extracts

Plants were extracted as follows, or otherwise, as described in Exceptions. Dried and pulverized plant materials were exhaustively extracted with ethanol and the extract was concentrated under reduced pressure to afford a brown syrup. This residue was suspended in water and extracted successively with organic solvents: hexane, dichloromethane, ethyl acetate and n-butanol, in this order.

Exceptions

Lippia integrifolia - Organic extracts - Air dried plant material (10 g) was ground and extracted by soaking in dichloromethane (100 ml) at room temperature for 24 h and then filtered. The process was repeated 3 times. The filtrates were combined and taken to dryness under vacuum. The plant material was then extracted with methanol under the same conditions. Aqueous extract - Dried ground plant material (50 g) was extracted with hot water (500 ml) by maceration at room temperature for 20 minutes. The extract was filtered and freeze-dried. Vitex cooperi – 90 g of bark and wood were separately extracted twice with ethanol-water (7:3) for 24h. The filtrates were combined and taken to dryness under vacuum.

Fractionation of Vitex cooperi bark methanol-water extract

The methanol-water extract (700 mg) from the bark of V. cooperi was chromatographed on silica gel eluted with mixtures of hexane, dichloromethane, ethyl acetate and methanol, to afford 21 fractions. Fractions 7 (24 mg) and 8 (17 mg) eluted with dichloromethane; 10 (13 mg) and 12 (10 mg) eluted with dichloromethane - ethyl acetate (1:1); and 14 (15 mg) and 15 (10 mg), eluted with ethyl acetate, were active by the Alamar Blue test. 20-Hydroxyecdysone was detected on fraction 17 by TCL and co-TLC with an authentic sample isolated from V. cymosa bark (Santos et al., 2001). Due to the limited amount of the remaining active fractions (c.a. 5 mg were used for the Alamar Blue test), GC-MS analysis was performed in order to identify possible active substances. In fractions 7 and 8 the phytosterols sitosterol and stigmasterol were identified.

GC-MS analyses

The GC/MS system used was an Agilent 5973 MSD coupled to an Agilent 6890N gas chromatograph equipped with an automatic injector (5683). Conditions: helium as carrier gas (1 ml.min^-1); fused capillary column: HP-5 (5% phenylmethyl silicone); injector temperature: 270°C, and the column oven program was 70° to 290°C at 4°C min^-1. Detector was operated at 280°C. Injection volume, 1 µl (split 1:10). Data acquisition was obtained with a HP CHEMISTATION Acquisition Software. Substances were identified by comparison of their mass spectra with those in a spectral database (Wiley 7N) and with literature (Budzikiewicz, 1974).

Alamar Blue Test

MABA (Microplate Alamar Blue Assay) susceptibility testing was performed according to the method described by Franzblau et al. (1998). Final concentration of plant extract was 100 µg/ml. Media plus bacteria with and without rifampicin were used as controls. The strain H₃₇Rv (ATCC - 27294) was used for all methodologies. The BACTEC Ò (Becton & Dickinson) radiometric test was used as reference to confirm antimycobacterium activity of plants extracts. Samples were simultaneously screened by both microbiology laboratories.

RESULTS

A total of 48 ethanolic crude extracts and fractions (hexane, dichloromethane, ethyl acetate and n-butanol) from 10 Brazilian plants (Leguminosae, Monimiaceae and Verbenaceae), 1 from Costa Rica (Verbenaceae) and 1 from Argentina (Verbenaceae) were assayed at a fixed concentration of 100 µg/mL - Table 1. Out of the assayed extracts, seven, belonging to the Verbenaceae family presented antimycobacterial activity at this concentration. All extracts were simultaneously assayed by the Alamar Blue Test at the two microbiology laboratories (Fiocruz, Rio de Janeiro and FURG, Rio Grande do Sul), in order to assure an interlaboratorial control.

DISCUSSION

Analysis of the results reported in Table 1 drew our attention to the fact that Lantana trifolia, Lippia lacunosa and Lippia rotundifolia crude ethanolic extracts were inactive, whereas the hexane and dichloromethane fractions obtained from them (except for those from L. trifolia stems) were active at the same final concentration of the original extracts. When the search for biological activity is not based on ethno-pharmacological information, a question often arises which is what kind of extract is the best one to be prepared. In the specific case ofthese three plant extracts we could observe that the pre-purification step of the crude ethanolic extract by liquid-liquid partition played a crucial role in the assessment of the antimycobacterial activity. Literature search (Van Puyvelde et al., 1994; Boily; Van Puyvelde, 1986) showed that a crude ethanolic extract of Lantana trifolia collected in Rwanda, and prepared in the same way as ours had been previously assayed for antimycobacterial activity - M. smegmatis(Boily; Van Puyvelde, 1986) and clinical strains of M. tuberculosis (Van Puyvelde et al., 1994), and similarly, no positive activity was found, corroborating with our results. However, the report on the antimycobacterial activity of another species from the genus Lantana - L. hispida (Jimenez-Arellanes et al. 2003), prompted us to investigate this activity on the Lantana trifolia fractions. The active hexane and dichloromethane fractions from the leaves of Lantana trifolia, Lippia lacunosa and Lippia rotundifolia are now being submitted to bioassay-guided fractionation to locate the active(s) substance(s). Here, we report the bioassay-guided fractionation of Vitex cooperi’s methanol-water extract, in order to isolate their antimycobacterial substances. Fractionation by silica gel column chromatography of this extract led to the separation of 21 fractions, one of which containing ecdysteroids, which are common constituents of the barks of Vitex species (Santos et al., 2001; Suksamram; Sommechai, 1993). Comparative TLC and also co-TLC of this fraction with an authentic sample of 20-hydroxyecdysone, previously isolated in our laboratory from the barks of Vitex cymosa (Santos et al., 2001), showed the presence of this substance in Vitex cooperi’s bark extract. Owing to the fact that the two extracts from V. cooperi (bark and wood) have very similar chromatographic profiles, with the exception of 20-hydroxyecdysone which is present only in the active one (bark), we decided to evaluate the susceptibility of M. tuberculosis to this substance. 20-hydroecdysone was assayed at 100 µg/mL but failed to demonstrate any activity against M. tuberculosis at this concentration. In this way, we searched for the antimycobacterium activity on the other fractions by the Alamar Blue test. Six fractions turned out to be active. Unfortunately, due to the limited amount of them (10 to 15 mg), further purification steps by traditional open column chromatography were unfeasible. In this way, GC-MS analysis was performed in order to tentatively identify possible active substances. In fractions 7 and 8 the phytosterols sitosterol and stigmasterol were identified by comparison of their mass spectra with those in a spectral database (Wiley 7N) and with those in the literature (Budzikiewicz, 1974). These sterols have already been identified as the antimycobacterial principles of the hexane fraction of Morinda citrifolia (Saludes et al., 2002), with MICs of 128 µg/mL (sitosterol) (Okunade et al., 2004) and 32 µg/mL (stigmasterol) (Okunade et al., 2004). Although the MIC reported in literature for sitosterol lies above 100 µg/mL, the fact that fractions 7 and 8 are active at this concentration may be explained by the presence of stigmasterol (MIC 32 µg/mL) in the mixture. Sitosterol has also been found in many active plant extracts against Mycobacterium tuberculosis such as Chamaedora tepejilote(Jimenez et al., 2005), Junellia tridens(Caldwell et al., 2000) and Euphorbia ebracteolata(Zhang et al., 1987), together with other antimycobacterium compounds. Also, in a clinical study (randomised placebo-controlled trial) of the efficacy of beta-sitosterol and its glucoside as adjuvants in the treatment of pulmonary tuberculosis significantly improved weight gain and higher lymphocyte and eosinophil counts in positive TB patients receiving sitosterols in addition to an efficacious antituberculosis regimen has been shown (Donald et al., 1998). Authors propose that sitosterols and their possible mode of action should be evaluated in larger numbers of tuberculosis patients and in diseases with a similar immunopathogenesis.

Therefore, we can suggest that the presence of these phytosterols in the bark extract of Vitex cooperi may account for the antimycobacterial activity of this plant.

CONCLUSION

The screening of 48 south and central America plant extracts for antimycobacterium activity led to the discovery of 7 active ones, all from the Verbenaceae family. With the exception of Vitex cooperi ‘s methanol:water extract, all the others were obtained by liquid-liquid partition of their crude ethanolic extracts, showing that pre-purification steps played a crucial role in the assessment of the antimycobacterial activity. The results obtained also pointed out a trend of activity concentration towards the more lipophilic extracts (hexane and dichloromethane). In the case of the active polar methanol-water extract from V. cooperi, bioassay-guided fractionation by column chromatography led to the identification of two phytosterols in the less polar (lipophilic) fractions of this extract, which may account for the antimycobacterium activity of this plant. These results are in agreement with recent reports that the cell wall of Mycobacterium tuberculosis has high permeability to hydrophobic compounds (Korycka-Machala et al., 2005).

The therapeutic arsenal to the treatment of tuberculosis is reduced; furthermore the treatment is long. This is a serious problem that has worsened in the last times because of the increase of TB cases with resistant Mycobacterium tuberculosis strains. Therefore, the search for new drugs from natural origin is urgent and extremely important to developing countries like Brazil and Costa Rica, which are the richest countries in terms of biodiversity.

AKNOWLEDGEMENTS

Collaborative work was performed under the auspices of the Iberoamerican Program for Science and Technology (CYTED), Project X.11:PIBATUB. S. G. Leitão is indebted to CNPq (Edital Universal n. 477060/2004-8) for financial support. Some of us are indebted to CAPES and CNPq for fellowships, and for UNICEF/UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases (TDR).

Received 11/16/05

Accepted 02/16/06

S. G. Leitão wishes to dedicate this work in memory of Prof. Nikolai Sharapin who kindly introduced the author to the CYTED Program

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Publication Dates

• Publication in this collection
  26 Feb 2008
• Date of issue
  Mar 2006

History

• Accepted
  16 Feb 2006
• Received
  16 Nov 2005