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

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 10.1590/1983-084X/ 14_148


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(WHO , 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).
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 antiinflammatory 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 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 Pterodon emarginatus (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 Mycobacterium spp and suggest future studies about the antimycobacterial properties of fruits from P. emarginatus.Keywords: Antimicrobial, Mycobacterium tuberculosis Complex, Microplate Resazurin Assay, Nontuberculous mycobacteria, Plants.
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).

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 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 chloroformhexane (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 37 o C, 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.
T h e s p e c i e s o f N o n -t u b e r c u l o u s 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).
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 mg/mL; 625 mg/mL; 312.5 mg/mL and so on until 4.88 mg/mL.The final concentrations of rifampicin solutions tested in microplates were: 4.00 mg/mL; 2.00 mg/mL; 1.00 mg/mL and so on until 0.03 mg/mL.The final concentrations of ciprofloxacin solution tested in microplates were 0.50 mg/mL; 0.25 mg/mL; 0.125 mg/mL and so on until 0.0039 mg/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 mL of culture medium.Then all wells of the first line of the inner part (second line of the total plate) received 100 mL of the prepared solution of the plant material or antibiotic solutions.Next a serial dilution was created by passing 100 mL 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 mL of the mycobacterial suspension.The filled microplate were kept at 37 o C. The readout was performed after 7 days, dropping 10 mL 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 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.
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).
Pterodon emarginatus was the most promising species evaluated, showing significant toxic action by the HEPE against M. smegmatis (78.125 mg/mL) and M. chelonae (39.1 mg/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 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 mg/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 mg/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 mg/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.
According to Kiliç (2006), the transcaryophyllene was able to inhibit the growth of M. tuberculosis, but the MIC value was 514 mg/ mL, a MIC value higher than the value founded for EOPE (46 mg/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 mg/ 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)   essential oil does not represent a promising source for antimycobacterial drugs, since its MIC value was 729 mg/mL against M. tuberculosis, exceeding the limit of MIC value determined as 200 mg/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 mg/mL and 78.125 mg/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.

TABLE 1 .
Overview of evaluated plants.

TABLE 2 .
Overview of evaluated mycobacteria.

TABLE 3 .
Minimal Inhibitory Concentrations of mycobacterial growth determined from in vitro tests with fruits of P. emarginatus 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.
(Altaf et al., 2010) bovis and M. tuberculosis(Altaf et al., 2010).However, the results obtained (78.125 mg/mL, 312.5 mg/mL and 625 mg/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.
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

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 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.