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Revista Brasileira de Plantas Medicinais

Print version ISSN 1516-0572On-line version ISSN 1983-084X

Rev. bras. plantas med. vol.17 no.4 supl.2 Botucatu  2015

http://dx.doi.org/10.1590/1983-084X/14_148 

Articles

Screening antimycobacterial activity of Baccharis dracunculifolia, Centella asiatica, Lantana camara and Pterodon emarginatus

Atividade anti-micobacteriana de Baccharis dracunculifolia, Centella asiatica, Lantana camara e Pterodon emarginatus

R.R.P. MACHADO1  2  * 

R.C. DUTRA3 

F. PITTELLA4 

N.R.B. RAPOSO5 

B. LESCHE6 

R.S. DUARTE7 

G.L.G. SOARES8 

M.A.C. KAPLAN9 

1Universidade Federal do Rio de Janeiro, Programa de Pós-graduação em Biotecnologia Vegetal, Cidade Universitária, Ilha do Fundão, Avenida Carlos Chagas Filho, Rio de Janeiro, RJ, CEP 21941-902, Brasil;

2Faculdade de Ciências Médicas e da Saúde de Juiz de Fora - SUPREMA. Rua Doutor Dirceu de Andrade, 33, Bairro São Mateus, Juiz de Fora, MG, CEP 36025-330, Brasil;

3Universidade Federal de Santa Catarina, Campus Araranguá, Rodovia Jorge Lacerda, Km 35,4, SC 449, Bloco C1, Jardim das Avenidas, Araranguá, SC, CEP 88900-000, Brasil;

4Faculdade de Farmácia e Bioquímica, Universidade Federal de Juiz de Fora, Cidade Universitária, Bairro Martelos, Juiz de Fora, MG, CEP 36036-900, Brasil;

5Universidade Federal de Juiz de Fora, Cidade Universitária, Bairro Martelos, Juiz de Fora, MG, CEP 36036-900, Brasil;

6Universidade Federal de Juiz de Fora, Instituto de Ciências Exatas, Departamento de Física, Campus Universitário, Bairro Martelos, Juiz de Fora, MG, CEP 360036-900, Brasil;

7Universidade Federal do Rio de Janeiro Laboratório de Micobactérias, Instituto de Microbiologia Paulo Góes, Bloco I, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, RJ, CEP 21941-902, Brasil;

8Universidade Federal do Rio Grande do Sul, , Instituto de Biociências, Av. Bento Gonçalves 9500, Campus do Vale, 91501-970 Porto Alegre, RS, Brasil;

9Universidade Federal do Rio de Janeiro, Instituto de Pesquisa de Produtos Naturais, Bloco H, Ilha do Fundão, Rio de Janeiro, RJ, CEP-21941-902, Brasil.

ABSTRACT

The permanent investigation of new antimycobacterial drugs is necessary for the eradication programs of tuberculosis and other mycobacterium-related diseases. The aim of the present study is to search for new sources of antimycobacterial drugs using plant materials. In this study, 11 plant materials (extracts, essential oils and some fractions) obtained from 4 species of medicinal plants traditionally used as general therapeutics for different illnesses and specifically as treatment of tuberculosis, were evaluated using the microplate resazurin assay against 2 species of the Mycobacterium tuberculosis Complex and 3 nontuberculous mycobacteria. The results showed the hexane extract and the essential oil from fruits of Pterodonemarginatus (Vogel) as potential sources of antimycobacterial drugs against 4 species of tested mycobacteria. The hexane fraction of methanol extract from leaves of Centella asiatica also presented significant mycobacterial growth inhibition, but against M. chelonae only. In conclusion, it was possible to contribute to the antimycobacterial investigations by presenting three new samples of plants with significant antimicrobial activity against four Mycobacteriumspp and suggest future studies about the antimycobacterial properties of fruits from P. emarginatus.

Keywords Antimicrobial; Mycobacterium tuberculosis Complex; Microplate Resazurin Assay; Nontuberculous mycobacteria; Plants

RESUMO

A investigação permanente de novas drogas antimicobacterianas é necessária no programa de erradicação da tuberculose e de outras doenças relacionadas com micobactérias. O objetivo deste estudo foi buscar novas fontes de drogas antimicobacterianas usando material vegetal. Neste estudo, 11 materiais de base vegetal (extratos, óleos essenciais e algumas frações) foram avaliados contra 5 espécies de micobactérias. Estes materiais foram obtidos a partir de 4 espécies de plantas medicinais tradicionalmente utilizadas como terapêutica geral para diferentes doenças e, especificamente, no tratamento de tuberculose (Baccharis dracunculifolia, Centella asiatica, Lantana camara, Pterodon emarginatus). Os ensaios foram realizados em microplacas com resazurina contra duas espécies do Complexo Mycobacteriumtuberculosis e 3 espécies de micobactérias não tuberculosas. Os resultados mostraram o extrato hexânico e o óleo essencial de frutos de P.emarginatus como potenciais fontes para drogas antimicobacterianas contra quatro espécies de micobactérias testadas. A fração hexânica do extrato metanólico das folhas de C. asiatica também apresentou significativa inibição do crescimento de micobactérias apenas contra M.chelonae. Em conclusão, foi possível contribuir para as investigações de antimicobacterianos por apresentar três novas amostras de plantas com atividade antimicrobiana significativa contra quatro Mycobacterium spp e sugerir a realização de estudos futuros sobre as propriedades antimicobacterianas de frutos de P. emarginatus.

Palavras-chave antimicrobiano; Complexo Mycobacterium tuberculosis; ensaio em microplacas com resazurina; micobactérias não-tuberculosas; vegetais

INTRODUCTION

The Mycobacterium sp is a genus of the Mycobacteriaceae family. They are aerobic microorganisms found in different habitats and they are able to promote relevant diseases such as tuberculosis, mycobacteriosis and leprosy. These diseases are promoted by different species of Mycobacterium spp such as: Mycobacterium tuberculosis and M. bovis (some etiologic agents of tuberculosis) and the species M. abscessus, M. chelonae or M. smegmatis (some Mycobacterium spp responsible for mycobacteriosis) and M. leprae, the etiologic agent of leprosy. Tuberculosis and leprosy are contagious, and the treatment of these cited diseases takes much longer than treating other types of bacterial infections (6-12 months). Thus the possibility of failure is high and it contributes to the appearance of mutant drug-resistant strains. (Brown-Elliot et al., 2012; Delogu et al., 2013).

The World Health Organization recently published good news about the profile of tuberculosis and leprosy around the world, presenting a reduction of their incidence, but also the requirement of continuous financial support to pursue the eradication of these diseases (WHO 2014a, 2014b). On the other hand, relative to mycobacteriosis cases, the number of mycobacteriosis cases is still growing and most cases are related to immunosuppressive causes and surgical interventions using catheters (Brown-Elliot et al., 2012; Pitombo et al., 2009).

Many studies aiming at new alternatives for treatment for mycobacteriosis and tuberculosis are being performed using plant samples which are evaluated by in vitro tests of susceptibility to possible antimicrobial compounds (Bueno et al., 2011).

According to the literature, numerous investigations using plant samples have been undertaken with respect to their antimycobacterial activities, as can be observed for Baccharis dracunculifolia D.C. (Asteraceae); Centella asiatica (L.) Urb. (Apiaceae), Lantana camara L. (Verbenaceae) and Pterodon emarginatus V. (Fabaceae) (Da Silva Filho et al., 2008; Feresin et al., 2003; Ghisalberti, 2000; Herbert et al., 1994; Kirimuhuzya et al., 2009; Machado et al., 2012a; Suresh et al., 2010; Verdi et al., 2005).

Baccharis dracunculifolia is a native plant of South America and is found in Argentina, Bolivia, Brazil and Uruguay. It is used by the bees to produce the green propolis, which has recognized antimicrobial and anti-inflammatory activities (Feresin et al., 2003; Verdi et al., 2005). The antimicrobial activity of B. dracunculifolia has been studied (Ferronatto, 2007; Da Silva Filho et al., 2008), but the effect of its essential oil against Mycobacterium spp has not been investigated to our knowledge.

Lantana camara is a plant frequently used as an ornament in gardens and is also commonly known for its potentially high toxicity due to the presence of hepatotoxic triterpenes (Ghisalberti, 2000). In addition, this plant is widely used in Africa for the treatment of diseases such as tuberculosis. Previous studies revealed a relevant antimycobacterial activity of this plant against M. tuberculosis using the methanol extract from its leaves (Kirimuhuzya et al., 2009), and the absence of toxicity of its essential oil when it was administered to mice (Machado et al., 2012b).

Centella asiatica is a traditional plant used in Ayurvedic medicine. Numerous investigations have shown its biological properties such as its cytotoxic action against cancer cells, its anti-inflammatory activity, its potential use in treatment of neurological disorders, all found in the water extract from its leaves (Barbosa et al., 2008; Pittella et al., 2009). Currently, it is often related to cosmetics for weight reduction according to scientific evaluations presented by Primastuti et al. (2013). However, its previous medicinal folk use was associated with treatment of leprosy lesions in India (Herbert et al., 1994) and a recent study detected antimycobacterial activity in the ethanol extract of its leaves against M. tuberculosis through microplate assay (Suresh et al., 2010).

Pterodon emarginatus is a native Brazilian plant that has mainly been studied in relation to the treatment of inflammatory processes caused by arthritis and other diseases (Dutra et al., 2009; Hoscheid et al., 2013). Regarding the antimycobacterial activity of this plant, few investigations have been carried out (Lima et al., 2006; Machado et al. 2012a), and due to the high variability of its secondary metabolites (Alves et al., 2013), new explorations must be realized.

This study aims to present novel potential sources of drugs for treating mycobacteriosis and tuberculosis. Therefore, the susceptibility of five mycobacteria species (M. abscessus, M. chelonae, M. smegmatis, M. tuberculosis, M. bovis) to 11 plant samples obtained from four plant species (B. dracunculifolia, C. asiatica, L. camara, P. emarginatus) was evaluated.

MATERIAL AND METHOD

The plant samples and Mycobacterium spp used are listed in Tables 1 and 2 respectively. All vouchers were deposited in the Herbarium Leopoldo Krieger from the Botany Department of the Federal University of Juiz de Fora (UFJF) – Minas Gerais – Brazil and they were identified by Dr. Fátima Regina Gonçalves Salimena (Herbarium Leopoldo Krieger from Botany Department of the Federal University of Juiz de Fora (UFJF) – Minas Gerais – Brazil) (Tables 1 and 2).

TABLE 1 Overview of evaluated plants. 

Scientific Name (Family) Part Used Extract Voucher Brazilian Folk Name
Hexane Extract
(HEPE)
Ethyl acetate Extract
(EAEPE)
Pterodon emarginatus Vogel (Fabaceae) Fruits Butanol Extract
(BEPE)
Methanol Extract
(MEPE)
Essential Oil
(EOPE)
CESJ 48077 Sucupira, sucupira branca, faveiro
Aqueous Extract
(AECA)
Methanol Extract
Centella asiatica (L.) Urb. (Apiaceae) Leaves (MECA)
Hexane Fraction
(HFCA)
Chloroform-Hexane Fraction
(CHFCA)
CESJ 24610 Centelha, centelha asiática
Lantana camara L. (Verbenaceae) Leaves Essential Oil
(EOLC)
CESJ 46090 Cambará, chumbinho
Baccharis dracunculifolia D.C. (Asteraceae) Aerial Parts Essential Oil
(EOBD)
CESJ 46087 Alecrim-do-campo

TABLE 2 Overview of evaluated mycobacteria. 

Group Species
Mycobacterium tuberculosis Complex Mycobacterium bovis - BCG Moreau
Mycobacterium tuberculosis - H37Rv - ATCC 27294
Non-tuberculous mycobacteria Mycobacterium smegmatis - ATCC 14468
Mycobacterium abscessus - ATCC 5752
Mycobacterium chelonae - ATCC 199777

Pterodon emarginatus

Dry fruits of P. emarginatus were collected in September 2006 in the district of Três Marias (Minas Gerais – Brazil) which belongs to the Cerrado biome (tropical Brazilian savanna).

The extracts were obtained according to the method described by Costa (2000). Fruits (30 g) were cut and subjected to hexane extraction during 24 hours in a Soxhlet extractor. The still pot was replaced in order to exchange solvents. The following solvents were used: ethyl acetate, butanol, and methanol. These solvents were removed by means of a rotary evaporator (R-114, Buchi–Swiss) to obtain respectively hexane (HEPE), ethyl acetate (EAEPE), butanol (BEPE) and methanol (MEPE) extracts.

Fruits were cut and essential oil was extracted in a Clevenger type apparatus during 4 hours. The essential oil (essential oil from P. emarginatus - EOPE) and MEPE were kept at -20°C in an amber flask.

Centella asiatica

Adult leaves of C. asiatica, grown under controlled farming on the campus of Federal University of Juiz de Fora - Brazil (S21º46’46.4” W43º22’13.3” elevation 865 m), were collected and dried at room temperature. The aqueous extract of C. asiatica (AECA) was prepared adding 200 g of chopped dried material in 4 L of water during 24 h without heating, as described in previous studies (Pittella et al., 2009; Primastuti et al., 2013). The final solution was filtered and freeze-dried, a process also known as lyophilization, (Thermo – Thermo Electron, USA) and kept at -80 °C in an amber flask. The methanol extract of C. asiatica (MECA) was obtained using 200 g of dried material exposed to methanol (1900 mL) during 2 h in a Soxhlet extractor. The extract obtained was filtered, concentrated and kept at -80 °C in an amber flask. The fractions were obtained using 10 g of MECA first diluted with 100mL of ethanol (10%). This solution was mixed with 100 mL of hexane in a separatory funnel. The aqueous phase and the hexane phase were then collected after three additional times. Then 100 mL of the aqueous phase was mixed with chloroform-hexane (2:8), so the chloroform-hexane phase was obtained. In this way, the hexane fraction (HFCA) obtained from MECA and the chloroform-hexane fraction (CHFCA), also obtained from MECA, were extracted respectively from the concentration of the hexane phase and the chloroform-hexane phase and afterwards kept at -80 °C in an amber flask.

Lantana camara and Baccharis dracunculifolia

Leaves of L. camara (900 g) and aerial parts of B. dracunculifolia (900 g) were collected in Juiz de Fora - Minas Gerais – Brazil (S21º45’38.8’’, W43º21’59.3’’ elevation 860 m) in February 2007 and dried at room temperature, arranged in thin layers, on a rack with wire-nettings that allow free aeration of plant material. The essential oils obtained from those plants were extracted by hydro-distillation using a Clevenger apparatus during 2 h. The extracted essential oils (Essential Oil of L. camara – EOLC and Essential Oil of B. dracunculifolia -EOBD) were kept at -20 °C in an amber flask.

Preparation of Mycobacterial Suspension

The species of Mycobacterium tuberculosis Complex (Table 2) were seeded on Lowenstein-Jensen (LJ) culture medium, kept at 37oC, and 21 days afterwards colonies were harvested to form a suspension of turbidity equal to 1.0 McFarland Standard. From this suspension a second one was prepared diluting the first one 1:20 (v/v) in Middlebrook 7H9 broth (BD-lot.2112134-USA) enriched with Middlebrook OADC (Oleic Albumin Dextrose Catalase - SIGMA-ALDRICH). This second suspension was used for assays evaluating antimycobacterial activities.

The species of Non-tuberculous mycobacteria (Table 2) were seeded on LJ and after seven days the colonies were harvested to form a suspension of turbidity equal to 1.0 McFarland Standard. From this suspension a second one was prepared diluting the first one 1:25 (v/v) in Muller Hinton Broth (Becton Dickinson and Company).

Evaluation of antimicrobial activity

The tests of the potential antimycobacterial activity of the plant materials (extracts, essential oils and some fractions) and the standard antibiotics [rifampicin (Lot 780773 – SIGMA) and ciprofloxacin (Lot 17850 – ALDRICH Chemistry)] were performed in plates with 96 wells using redox reaction of sodium resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide - Resazurin Sodium Salt Powder Acros Organic N.V., Geel, Belgium) according to Machado et al. (2012a; 2012c).

Each sample of 5000 mg of prepared plant materials (Table 1) was diluted with 100 µL of DMSO (dimethyl sulfoxid - SIGMA) and 900 µL of supplemented 7H9 (liquid culture medium added with OADC) for the assays with mycobacteria of the Mycobacterium tuberculosis Complex (Table 2), or 900 µL of Mueller Hinton Broth (Becton Dickinson and Company) for the assays with Non-tuberculous mycobacteria (Table 2) (Machado et al., 2012c) The solution of rifampicin was prepared using ethylene glycol (Difco) as diluent. The rifampicin concentration of the solution was 256μg/ml and the final percentage of ethylene glycol in the solution was 0.1%. The solution of ciprofloxacin was prepared using 7H9 as diluent. The solutions of plant materials, the DMSO solution (10%), the ethylene glycol and the antibiotics solutions were tested simultaneously in triplicates in different wells of microplates and the DMSO solution and ethylene glycol solution were not able to inhibit the mycobacterial growth. The final concentrations of plant material solutions evaluated in microplates were: 1250 µg/mL; 625 µg/mL; 312.5 µg/mL and so on until 4.88 µg/mL. The final concentrations of rifampicin solutions tested in microplates were: 4.00 µg/mL; 2.00 µg/mL; 1.00 µg/mL and so on until 0.03 µg/mL. The final concentrations of ciprofloxacin solution tested in microplates were 0.50 µg/mL; 0.25 µg/mL; 0.125 µg/mL and so on until 0.0039 µg/mL. The procedures of dilution of the antibiotic followed the description in Machado et al. (2012c).

The wells on the border of each microplate were filled with water in order to reduce the evaporation of the samples which occupied the inner part of the microplates.

To obtain the cited concentrations, all wells of the remaining inner part of the microplates were firstly filled with 100 µL of culture medium. Then all wells of the first line of the inner part (second line of the total plate) received 100 µL of the prepared solution of the plant material or antibiotic solutions. Next a serial dilution was created by passing 100 µL from top to bottom along the rows thus reducing the concentrations always by a factor 1:2 from one line to the following. Finally all wells of the inner part received 100 µL of the mycobacterial suspension. The filled microplate were kept at 37 ºC. The readout was performed after 7 days, dropping 10 µL of resazurin ( 0.01% - diluted in ethylene glycol and sterile distilled water) into the wells. The change of color from blue to pink indicated the mycobacterial growth.

The minimal inhibitory concentration (MIC) less than 200 µg/mL were considered potential antimycobacterial agents (Tosun et al., 2004).

The MIC values were determined based on the means of triplicates. In addition, all procedures were repeated at least three times with periodicity of 2 weeks, performing each one in triplicate. When a repetition of an experiment gave a deviating result, the number of repetitions was increased in order to guarantee the reliability of the results.

RESULTS AND DISCUSSION

The MIC values of ciprofloxacin against tested non-tuberculous mycobacteria (Mycobacterium smegmatis: 0.014 ± 0.005 µg/mL, M. chelonae: 0.015 ± 0.010 µg/mL, M. abscessus: 0.2 ± 0.2 µg/mL) and the MIC values of rifampicin against evaluated Mycobacterium tuberculosis Complex (M. tuberculosis: 0.051 ± 0.031 µg/mL and M. bovis: 0.030 ± 0.003 µg/mL) demonstrated the occurrence of susceptibility to all tested mycobacteria, as was expected. These findings are in accordance with results in the literature (Cavusoglu et al., 2012; Ribeiro et al., 2004).

The evaluation of antimycobacterial activity of plant materials showed promising results, since four of the investigated bacterial species were susceptible to some of the plant samples tested. The data are demonstrated in (Tables 3 and 4).

TABLE 3 Minimal Inhibitory Concentrations of mycobacterial growth determined from in vitro tests with fruits of P. emarginatus 

Plant materials M. smegmatis M. chelonae M. abscessus M. tuberculosis M. bovis
(MIC-µg/mL)
HEPE 78.125 39.1 > 1250 312.5 625
EAEPE 625 > 1250 > 1250 > 1250 625
BEPE > 1250 > 1250 > 1250 > 1250 > 1250
MEPE > 1250 > 1250 > 1250 > 1250 625
729 46
EOPE > 1250 I II III > 1250 I I I I 78.125
II 39.1
312.5 625 1250 19.55 78.125

Notes: HEPE - Hexane Extract of P. emarginatus; EAEPE - Ethyl-Acetate Extract of P. emarginatus; BEPE - Butanol Extract of P. emarginatus; MEPE - Methanol Extract of P. emarginatus; EOPE - Essential Oil of P. emarginatus. MIC values were obtained as mean values of three assays. As the results of all triplicates were uniform, the table contains no standard deviation. In two cases the three assays I, II and III gave different values and they were listed separately. Promising results with MIC values smaller than 200µg/mL are shown in boldface.

TABLE 4 Minimal Inhibitory concentrations of mycobacterial growth determined from in vitro tests with C. asiatica leaves, L. camara leaves and B. dracunculifolia aerial parts 

Plant materials M. smegmatis M. chelonae M. abscessus M. tuberculosis M. bovis
(MIC-µg/mL)
AECA > 1250 > 1250 > 1250 > 1250 > 1250
MECA > 1250 > 1250 > 1250 > 1250 > 1250
HFCA > 1250 78.125 > 1250 > 1250 > 1250
CHFCA > 1250 > 1250 > 1250 > 1250 > 1250
EOLC > 1250 > 1250 > 1250 > 1250 > 1250
729
EOBD > 1250 > 1250 > 1250 I II III > 1250
312.5 625 1250

Notes: AECA - Aqueous Extract of C. asiatica; MECA - Methanol Extract of C. asiatica; HFCA - Hexane Fraction of C. asiatica; CHFCA - Chloroform-Hexane Fraction of C. asiatica; EOLC - Essential Oil of L. camara; EOBD - Essential Oil of B. dracunculifolia. MIC values were obtained as mean values of three assays. As the results of all triplicates were uniform, the table contains no standard deviation. In one case the three assays I, II and III gave different values and they were listed separately. Promising result with MIC value smaller than 200µg/mL is shown in boldface.

Pterodon emarginatus was the most promising species evaluated, showing significant toxic action by the HEPE against M. smegmatis(78.125 µg/mL) and M. chelonae (39.1 µg/mL). According to Dutra et al. (2012b) the previous phytochemical analysis of HEPE revealed occurrence of tannins, flavonoids and leucoanthocyanidins. These chemical classes of substances were absent in MEPE, which was not able to inhibit the mycobacterial growth (Table 3) suggesting that the actives belong to one or more of the cited phenolic chemical classes.

M. bovis is often used as a model to investigate M. tuberculosis. M. smegmatis has been used as model for M. bovis and M. tuberculosis (Altaf et al., 2010). However, the results obtained (78.125 µg/mL, 312.5 µg/mL and 625 µg/mL, the MIC values of HEPE against M. smegmatis, M. tuberculosis and M. bovis, respectively) indicate that such use is of limited value, since the active substances in the extract HEPE that inhibit the growth of M. smegmatis do not affect M. bovis and M. tuberculosis.

Despite the low antimycobacterial activity of HEPE against M. tuberculosis and M. bovis this extract deserves attention, since previous study of the biological properties of HEPE revealed it to be toxic against Artemia salina (LD50 = 8.06 µg/mL), but not against mice, when it was used topically as ointment (Dutra, 2008).

The inhibitory action of the EOPE on the M. tuberculosis development in vitro was significant according to Tosun et al., (2004), presenting a mean MIC value of 46 µg/mL. As can be seen in Table 3, this mean value is composed of MIC values that increase with time, reaching a saturation value of 78.125 µg/mL. This suggests that EOPE contains at least two active substances, one of them is unstable on a scale of time of a few weeks and the other one seems to be stable. The stable compound seems to affect M. bovis in the same way, but the inhibitory action of the unstable ones are limited to M. tuberculosis.

The results of Table 3 were experimentally verified at least three times (each one in triplicates) with periodicity of two weeks. In the cases where a variable behavior was found, such as EOPE against M. chelonae and EOPE against M. tuberculosis, 6 and 7 assays were performed respectively. This precaution was used to test the reliability, while revealing unstable substances that may be involved in some activities.

Previous investigation of the currently used EOPE revealed its chemical composition by GC/MS, showing the following major compounds: trans-caryophyllene (35.9%), β-elemene (15.3%) and germacrene D (9.8%) (Dutra et al., 2012a).

According to Kiliç (2006), the trans-caryophyllene was able to inhibit the growth of M. tuberculosis, but the MIC value was 514 µg/mL, a MIC value higher than the value founded for EOPE (46 µg/ml) in the present work. This finding suggests that the potential activity of the EOPE may not be exclusively due to the presence of trans-caryophyllene, but due to a minor constituent present in EOPE.

According to the PubMed and Scielo databases, the potential of essential oil from leaves of L. camara (EOLC) against mycobacteria was evaluated for the first time. The tests revealed that this essential oil was not able to inhibit the growth of evaluated Mycobacterium species until 1250 µg/mL of EOLC (Table 4).

The composition of the currently used EOLC was described in a previous study that showed its leishmanicidal potential (Machado et al., 2012b). This previous study presented germacrene D, caryophyllene and farnesene as the major compounds. Thus, despite the absence of antimycobacterial activity of EOLC, the described composition of EOLC and EOPE (Dutra et al., 2012a; Machado et al., 2012b) suggests that the germacrene D is not related to the antimycobacterial activity revealed by EOPE in this work, since both essential oils presented germacrene D as one of the major compounds.

The essential oil of B. dracunculifolia (EOBD) was investigated against Mycobacterium species. According to the results obtained this essential oil does not represent a promising source for antimycobacterial drugs, since its MIC value was 729 µg/mL against M. tuberculosis, exceeding the limit of MIC value determined as 200 µg/mL by Tosun et al., (2004).

On the other hand, the low toxicity of this plant material has been described in other works revealing no toxic alteration in mice that received approximately 11mg/mL of different extracts of B. dracunculifolia from different parts of the plant (Fabri et al., 2011; Lemos et al., 2007; Menezes, 2005). Furthermore, the authors did not find any register in the literature of intoxication with this plant by the population.

The M. chelonae was susceptible to HEPE and HFCA presenting the following MIC values, 39.1 µg/mL and 78.125 µg/mL, respectively. In view of the activity of HEPE against M. chelonae and M. smegmatis and of the toxic action of HFCA only against M. chelonae, it is possible to conclude that there are at least two active substances in HEPE able to inhibit the growth of these mycobacteria. These active substances present in the HEPE, according to Dutra et al. (2012b), belong to the following chemical classes of secondary metabolism: tannins, flavonoids and leucoantocyanidins.

Considering the phytochemical analysis of MECA performed by Silva (2008) and the fact that HFCA is derived from MECA, it can be concluded that their active compounds are included in one of the following chemical classes: flavonoids, triterpenic glycosides, coumarins and tannins. Additionally, despite the verified antimycobacterial activity of HFCA, the MECA was not able to inhibit the growth of evaluated mycobacteria. This fact can be explained considering the preparation performed to obtain MECA. During the hot extraction with methanol, both polar and apolar components were extracted. Thus MECA is composed of a mix of components of different polarities. When the preparation of a fraction from MECA was performed using the hexane, apolar compounds were concentrated. Thus, the absence of antimycobacterial activity of MECA and the presence of antimycobacterial activity of HFCA can be related to a greater amount of the active substance in the tested volume of the latter plant material than in the evaluated volume of MECA.

According to Andrade et al. (2008), the high presence of lipids in the cell wall of mycobacteria leads apolar substances to present a higher probability of acting against these microorganisms. Thus, the presence of antimycobacterial activity in EOPE, HEPE and HFCA, which are composed of apolar substances, are in accordance with the literature.

The M. chelonae is classified as a component of the M. fortuitum complex, that is closely related to M. abscessus and M. immunogenum, which is sometimes known as the M. chelonae/abscessus group (Brown-Elliott & Wallace, 2005).

Despite the proximity of M. abscessus to M. chelonae, the results show differences between them, since M. abscessus was not susceptible to the active substances against M. chelonae present in HEPE and HFCA.

The results showed that none of the tested plant materials were able to inhibit the growth of M. abscessus. These data support the continuous need for research about new antimicrobial drugs against M. abscessus, which is often known as the most pathogenic and resistant non-tuberculosis Mycobacterium associated with pulmonary and soft tissue injuries (Griffith et al., 2007; Thomson et al., 2013).

CONCLUSIONS

The present work showed new potential sources of antimycobacterial drugs and pointed to Pterodon emarginatus as the most promising plant species. Comparing the results obtained for EOLC and EOPE to the literature records, we suggest that some compounds found in the essential oil from P. emarginatus fruits, such as the trans-caryophyllene and the germacrene D, are not responsible for the inhibitory action of the mycobacterial growth. Additionally, the present work presented one more biological property for Centella asiatica, since the hexane fraction of methanol extract from its leaves was active against Mycobacterium chelonae, a relevant non-tuberculosis Mycobacterium.

ACKNOWLEDGMENTS

The authors thank the Fundação de Amparo a Pesquisa e Extensão de Minas Gerais (FAPEMIG) for the financial support (Process no. CEX–APQ-00246-10) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the scholarship furnished to RRRP (number of process: 158347/2013-7) and to MACK. We thank Marlei da Silva Gomes (Laboratory of Mycobacterium, Center of Health Sciences - Federal University of Rio de Janeiro - Brazil) for invaluable help with handling mycobacteria.

REFERENCES

ALTAF, M. et al. Evaluation of the Mycobacterium smegmatis and BCG models for the discovery of Mycobacterium tuberculosis inhibitors. Tuberculosis, v.90, n.6, p.333-337, 2010. [ Links ]

ANDRADE, C.H. et al. Abordagem racional no planejamento de novos tuberculostáticos inibidores da InhA, enoil-ACP redutase do M. tuberculosis. Brazilian Journal of Pharmaceutical Sciences, v. 44, n.2, p.167-179, 2008. [ Links ]

ALVES, S.F. et al. Chemical variability of the essential oils from fruits of Pterodon emarginatus in the Brazilian Cerrado. Revista Brasileira de Farmacognosia, v.23, n.2, p.224-229, 2013. [ Links ]

BARBOSA, N.R. et al. Centella asiatica water extract inhibits iPLA2 and cPLA2 activities in rat cerebellum. Phytomedicine: International Journal of Phytotherapy and Phytopharmacology, v.15, n.10, p.896-900, 2008. [ Links ]

BROWN-ELLIOTT, B.A.; WALLACE, R.J. Infections caused by nontuberculous mycobacteria. In: MANDELL, G.L. et al. (Eds.). Principles and practice of infectious diseases. 6. ed. Philadelphia: Elsevier Churchill Livingstone, 2005. p.2090-2916. [ Links ]

BROWN-ELLIOTT, B.A. et al. Antimicrobial susceptibility testing, drug resistance mechanisms, and therapy of infections with nontuberculous mycobacteria. Clinical Microbiology Reviews, v.25, n.3, p.545-582, 2012. [ Links ]

BUENO, J. et al. Antimycobacterial natural products – an opportunity for the Colombian biodiversity. Revista Española de Quimioterapia, v.24, n.4, p.175-183, 2011. [ Links ]

CAVUSOGLU, C. et al. Evaluation of antimicrobial susceptibilities of rapidly growing mycobacteria by sensitive RAPMYCO panel. New Microbiologica, v.35, n.1, p.73-76, 2012. [ Links ]

COSTA, A.F. Farmacognosia experimental. 3. ed. Lisboa: Fundação Calouste. Gulbenkian, 2000. 993p. [ Links ]

DA SILVA FILHO A.A. et al. Antimicrobial activity of the extract and isolated compounds from Baccharis >dracunculifolia D. C. (Asteraceae). Journal of Biosciences, v.63, n.1-2, p.40-6, 2008. [ Links ]

DELOGU, G. et al. The biology of Mycobacteriumtuberculosis infection. Mediterranean Journal of Hematology and Infectious Diseases, v.5, p.e2013070, 2013. [ Links ]

DUTRA, R.C. Avaliações fitoquímicas e farmacológicas das sementes de Pterodon emarginatus Vogel. 2008. 488f. Dissertação (Mestrado - Área de concentração em Genética/Biotecnologia) - Instituto de Ciências Biológicas, Universidade Federal de Juiz de Fora, Juiz de Fora. [ Links ]

DUTRA, R.C. et al. Antiulcerogenic and anti-inflammatory activities of the essential oil from Pterodon emarginatus seeds. Journal of Pharmacy and Pharmacology, v.61, n.2, p.243-250, 2009. [ Links ]

DUTRA, R.C. et al. Chemical composition and cytotoxicity activity of the essential oil of Pterodon emarginatus. Revista Brasileira de Farmacognosia, v.22, n.5, p.971-978, 2012a. [ Links ]

DUTRA, R.C. et al. Caracterização fitoquímica e citogenética das sementes de Pterodon emarginatus Vogel. Revista Técnico Científica do IFSC, v.3, n.1, p.99-109, 2012b. [ Links ]

FABRI, R.L. et al. Potencial antioxidante e antimicrobiano de espécies da família Asteraceae. Revista Brasileira de Plantas Medicinais, v.13, n.2, p.183-189, 2011. [ Links ]

FERESIN, G.E. et al. Constituents of the Argentinian medicinal plant Baccharis grisebachii and their antimicrobial activity. Journal of Ethnopharmacology, v.89, n.1, p.73-80, 2003. [ Links ]

FERRONATTO, R. et al . Atividade antimicrobiana de óleos essenciais produzidos por Baccharis dracunculifolia D.C. e Baccharis uncinella D.C. (Asteraceae). Revista Brasileira de Farmacognosia, v. 17, n. 2, 2007. [ Links ]

GHISALBERTI, E.L. Lantana camara L. (Verbenaceae). Fitoterapia, v.71, n.2000, p.467-486, 2000. [ Links ]

GRIFFITH, D.E. et al. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. American Journal of Respiratory and Critical Care Medicine, v.175, n.4, p.367-416, 2007. [ Links ]

HERBERT, D. et al. In vitro experiments with Centellaasiatica: investigation to elucidate the effect of an indigenously prepared powder of this plant on the acid-fastness and viability of M. tuberculosis. Indian Journal of Leprosy, v.66, n.1, p.65-68, 1994. [ Links ]

HOSCHEID, J. et al. Inhibitory effect of the hexane fraction of the ethanolic extract of the fruits of Pterodon pubescens Benth in acute and chronic inflammation. Evidence-Based Complementary and Alternative Medicine, 7 p., 2013. Available at: http://dx.doi.org/10.1155/2013/272795. Acessed on: 24 Jan. 2015. [ Links ]

KILIÇ, T. Analysis of essential oil composition of Thymbra spicata var. spicata: antifungal, antibacterial and antimycobacterial activities. Zeitschrift für Naturforschung. C, Journal of Biosciences, v.61, n.5-6, p.324-328, 2006. [ Links ]

KIRIMUHUZYA, C. et al. The anti-mycobacterial activity of Lantana camara a plant traditionally used to treat symptoms of tuberculosis in South-western Uganda. African Health Sciences, v.9, n.1, p.40-45, 2009. [ Links ]

LEMOS, M. et al. Baccharis dracunculifolia, the main botanical source of Brazilian green propolis, displays antiulcer activity. The Journal of Pharmacy and Pharmacology, v.59, n.4, p.603-608, 2007. [ Links ]

LIMA, M.R.F. et al. The antibiotic activity of some Brazilian medicinal plants. Journal of Ethnopharmacology, v.105, n.1-2, p.300-306, 2006. [ Links ]

MACHADO, R.R.P. et al. Metabolic activity interferometer: a powerful tool for testing antibiotics. Journal of Sensors, v.2012, 7p, 2012a. Available at: http://dx.doi.org/10.1155/2012/493797. Accessed on: 24 Jan. 2015. [ Links ]

MACHADO, R.R.P. et al. Essential oil from leaves of Lantana camara: a potential source of medicine against leishmaniasis. Revista Brasileira de Farmacognosia, v.22, n.5, p.1011-1017, 2012b. [ Links ]

MACHADO, R.R.P. et al. Evaluation of in vitro antileishmanial and antimycobacterial activities of Stifftia chrysantha J.C. Mikan extracts. Revista Fitos, v. 7, p. 252-258, 2012c. [ Links ]

MENEZES, H. Avaliação da atividade antiinflamatória do extrato aquoso de Baccharis dracunculifolia (Asteraceae). Arquivos do Instituto Biológico, v.72, p.33-33, n. Suplemento2, 2005. [ Links ]

PITOMBO, M.B. et al. Infections by rapidly growing mycobacteria resistant to disinfectants: a national matter? Revista Brasileira de Ginecologia & Obstetrícia, v.31, n.11, p.529-533, 2009. [ Links ]

PITTELLA, F. et al. Antioxidant and cytotoxic activities of Centella asiatica (L) Urb. International Journal of Molecular Sciences, v.10, n.9, p.3713-3721, 2009. [ Links ]

PRIMASTUTI, R.F. et al. Effect of a combination of extract of Centella asiatica L. leaves and extract of green coffee (Coffea canephora robusta P.) Beans in a cream preparation for grade 1-3 cellulite and slimming. Makara Journal of Science, v.17, n.1, p.1-5, 2013. [ Links ]

RIBEIRO, M.O. et al. Evaluation of rapid microplate assays using cellular-viability indicators to determine patterns of susceptibility to isoniazid and rifampicin in Mycobacterium tuberculosis strains. Jornal Brasileiro de Pneumologia, v.30, n.4, p.365-370, 2004. [ Links ]

SILVA, F.P. Caracterização fitoquímica e atividades biológicas de Centella asiatica (L.) Urban. 2008. 300p. Dissertação (Mestrado - Área de concentração em Genética e Biotecnologia) - Instituto de Ciências Biológicas, Universidade Federal de Juiz de Fora, Juiz de Fora. [ Links ]

SURESH, M. et al. Anti-mycobacterial effect of leaf extract of Centella asiatica (Mackinlayaceae). Research Journal of Pharmacy and Technology, v.3, n.3, p.872-876, 2010. [ Links ]

THOMSON, R. et al. Mycobacterium abscessus isolated from municipal water - a potential source of human infection. BMC Infectious Diseases, v.13, p.241, 2013. [ Links ]

TOSUN, F. et al. Antimycobacterial screening of some Turkish plants. Journal of Ethnopharmacology, v.95, n.2-3, p.273-275, 2004. [ Links ]

VERDI, L.G. et al. The Baccharis genus(Asteraceae): chemical, economic and biological aspects. Química Nova, v.28, n.1, p.85-94, 2005. [ Links ]

WHO - World Health Organization. Leprosy elimination. 2014a. Available at: http://www.who.int/lep/en/. Accessed on: 24 Jan. 2015. [ Links ]

WHO - World Health Organization. World TB Day 24 March 2013. 2014b. Available on: http://www.who.int/campaigns/tb-day/2013/en/index.html. Accessed on: 24 Jan. 2015. [ Links ]

Received: November 11, 2014; Accepted: March 03, 2015

*Autor para correspondência: rachel.machado@suprema.edu.br

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