Evaluation of in vitro antileishmanial and antimycobacterial activities of Stifftia chrysantha J.C. Mikan extracts

antimicrobiano de diferentes extratos de S. chrysantha contra algumas espécies de micobactérias e formas promastigotas de duas espécies de Leishmania in vitro métodos colorimétricos, de acordo com o microrganismo avaliado. Os resultados mostraram baixa atividade dos extratos contra as culturas de micobactérias. Por outro lado, um efeito inibidor do crescimento foi observado no L. amazonensis (CI50 = 55,16 L. chagasi (CI50 = 72,05 do crescimento das formas promastigotas.


Introduction
Stifftia chrysantha J.C. Mikan (Asteraceae) is popularly known as diadem, fox tail or gold rain due to the lar names of the plant are: rabo-de-cotia, diadema, jambeiro do-mato, pincel.Its main use by the population is ornamental and it is moderately endangered to die out nowadays, according to list annexed to the Decree 19.149 published by the Rio de Janeiro Environmental Protection Bureau (2000).It can be found in Protected Areas such as the National Park of Tijuca, Rio de Janeiro Botanical Garden and Grajaú- Jacareal., 2010).Few studies on the chemical composition of this plant have been reported.Oliveira (1999) descri-licylate in the volatile fractions from fruits ranging from 85% to 95% in the volatile mixture as well as such presence in all aerial parts of the plant during all seasons of the year.
The organic ester methyl salicylate seems to take part in the attraction process of pollinators and in the defense of the plant.Besides, the metabolic conversion of methyl salicylate into salicylic acid and subsequently into acetyl salicylic could justify the ancient use of the plant by quilombo communities for treatment of 2005).This metabolic conversion from methyl saattacks.Salicylic acid is known as an important phytoalexin present in responses to physical and biological stresses suffered by the plant (Durrant et al., 2004).Employing a rapid radiometric method, Lall et al.
Mycobacterium tuberculosis H37Rv exposing the bacteria to the extracts of Polygala myrtifolia (Polygalaceae), a plant that shows high concentration of methyl salicylate.The reported activity found in P. myrtifolia and the similar major compound content present in the aerial parts of Stifftia chrysantha motivated the investigation about red risk species S. chrysantha extracts and also to the pure methyl salicylate against mycobacteria.We also tested the activity of the extracts against promastigote forms of leishmania.The diseases caused by mycobacteria and Leishmania sp.represent important disorders for Public Health, since long times are needed for effective treatment by using drugs that may cause potential side effects (Almeida et al., 2005;Medeiros et al., 2005;Coll et al., 2009;Sundar et al., 2007).Many research works have been performed with the attempt to identify new therapeutically potential drugs against tuberculosis, mycobacteriosis and leishmaniasis.Such nary approaches with in vitro experiments, before in vivo ones and clinical trials (Lahlou et al., 2004).

Collection of aerial parts and roots of S. chrysantha:
Aerial parts and roots of S. chrysantha wers, barks, branches and fruits) were collected under supervision of botanist Roberto L. Esteves in the garden of National Museum of Rio de Janeiro in December 2004.The voucher number is R208153.The plant material was collected early in the morning from the same chosen specimen and it was taken immediately to the laboratory and separately reduced into small pieces.The powdered materials were air-dried.

Preparation of S. chrysantha extracts:
Air-dried and powdered plant materials (50g of leaf; 30g of bark and maceration using hexane as solvent, and followed by methanol.Removal of residual solvent under reduced pressure was performed using a Büchi rotatory evaporator, equipped with warm bath under controlled temperature (40oC).The same procedure was carried out with methanol as solvent extractor.
Extracts used: hexane extract from leaves and bark of S. chrysantha; and methanol extract from leaves and fruits of S. chrysantha.
Methyl salicylate: methyl salicylate was used in the experiments on mycobacteria at the following concentrations: 250 16 Leishmania sp.assays: promastigote forms of Leishmania amazonensis ted from a patient who had diffuse cutaneous form of leishmaniasis) were cultured in Warren medium (BHI, plus hemin and folic acid) and promastigote forms of L. chagasi who had visceral form of leishmaniasis) were cultured in 199 medium, both supplemented with fetal bovine serum and maintained at 24ºC during one week.Fetal bovine serum (FBS) was purchased from Cultilab (BHI) from Himédia (Mumbai, Indian).Hemin, folic acid, and 199 medium were purchased from Sigma ty against promastigote forms of L. amazonensis and L. chagasi was determined using the MTT colorimetric method based on reduction of the salt by mitochondrial dehydrogenases (Mossman et al., 1983).Promastigoste forms in logarithmic growth stage of in vitro growth of both species were used in the explastic tissue-culture plates at the concentrations 2.0 6 L. amazonensis and L. chagasi, respectively.After 1h and at 24ºC, the parasites were exposed to different concentrations of extracts of S. chrysantha previously soluwere 250.0 mL, 15.6 were performed in triplicate and in two independent assays.The promastigote forms were exposed to the extracts, to Amphotericin B (standard drug used as the positive control) and to the DMSO solution at 0.01% for 72h 24ºC.The colorimetry was assessed by absorbance using SPECTRAMAX 190, Molecular analysis, GraFit software version 5 was used.
Experiments using rapidly and slow growing mycobacteria: the susceptibility tests for mycobacteria using extracts were performed in 96-well plates tion following the procedure used by Palomino et al. (2002).All experiments were performed in triplicates and at least three repetitions.Concentrated solutions in sterile water.The total content of DMSO in each well reached 10%, which did not inhibit mycobacterial plates, dilutions of the extract were prepared in Mi- serial dilution (1:2) for both drugs.The plates were sealed and kept at 37ºC for 7 days.On the seventh (diluted in etylenglicol and sterile distilled water) was nic N.V., Geel, Belgium).Extracts were considered as active against mycobacteria when exhibited MIC

Results and Discussion
The experiments performed in order to evaluate potential antimycobacterial activity of extracts from different parts of S. chrysantha did not show biological activity against any of the mycobacteria tested.This judgment is based on a criterion established by Tosun et al. ( 2004), which considers a substance as active if its MIC < 200 sial.Lima ( 2006) evaluated the antimicrobial activity -nol extracts from leaves of Lafoensia pacari against M. smegmatis sidering hence, the activity limits determined by Tosun et al. (2004).
In our study, two MIC values were determined: one against M. smegmatis which resulted from the methanol extract from leaves (1125.1 ± 0.13 another against M. chelonae (312.5 ± 0.16 -Table 1.These found MIC values may indicate the presence of substances capable of interfering with the metabolism of M. chelonae and M. smegmatis.The other evaluated plant products did not show antimycobacterial activity even at higher concentration.It still has to be discovered the active compounds in the S. chrysantha extracts.It is known that the S. chrysantha extracts contain considerable amounts of quercetin such as protection of the plants against insects, fungal attraction of pollinators due to the remarkable colors of these compounds (Salvador et al., 2008).Boligon (2012) studied antimycobacterial activity of quercetin against slowly and rapidly growing mycobacteria and they encountered a MIC > 200 cate that quercetin is one of the active compounds of the S. chrysantha extract.
Seasonal evaluation of S. chrysantha volatile fractions revealed methyl salicylate as the major consti-of this compound was observed mailing in the fruits portance in the attraction process of pollinators, deattacks (Marques et al., 2012).Methyl salicylate has already been related to antimycobacterial activity by effect of Polygala myrtifolia (Polygalaceae) extract, which contains great quantities of methyl salicylate.In addition, methyl salicylate (83.8%, 89.1% and 97.8%) was found as the main volatile constituent in roots of the P. sabulosa, P. paniculata and P. cyparissia, ressurmise that this compound could be responsible for antimycobacterial properties.
In our study, antimycobacterial activity of methyl saresults revealed no inhibitory activity on the mycobacterial growth (Table 1).The results suggests that the M. tuberculosis H37Rv found by Lall (1999), is not due to the exclusive action of methyl salicylate, the major compounds of that extract.This preliminary screening against Mycobacterium tuberculosis, H37Rv was performed using acetone and water plant extracts.The minimal inhibitory concentration of Polygala myrtifolia te of the good activity displayed by P. myrtifolia extract, no phytochemical separation was performed in the related study.A chemical investigation of the genus Polygala showed the occurrence of a variety of secondary metabolites, such as xanthones, saponins, oligo- (Johannl et al. 2011).Some of these compounds are preparations and could be acting against the M. tuberculosis studied strain.Guided by the considerations concerning about antimycobacterial activity one may guess that the leishmanicidal activity might also be related to the quercetin.re.For example, Sarkar and colleagues (2002) detected reduction of leishmanias in the spleen of hamsters of this phytocompound.This reduction is probably due metabolism (Sen et al., 2008).extracts from different parts of S. chrysantha aggretime that the antimycobacterial and antileishmanial potential activity has been evaluated.Despite the low activity found against mycobacteria culture, the progrowth.However, new studies are necessary in order to elucidate which phytocompounds in the plant material are responsible for the biological activity and how they act against the Leishmania species exposed to the products used in this study.
medium enriched with OADC (Becton-Dikinson) at the following concentrations: 2500 mL; 625 L in each well.Next, 100 L of slow-growing mycobacteria (M.tuberculosis (Monroe) or rapidly growing mycobacteria (M.smegmatis M. abscessus M. chelonae trations of the extracts into the wells were: 1250 mL; 625 39 lebrook 7H9 (Difco) culture medium enriched with OADC (Becton-Dikinson) at 1:25 concentration from the initial suspension and turbidity equivalent to 1.0 in McFarland scale.Standard drugs were used as positive control for inhibition of mycobacteria growth, ---growing mycobacteria (M.tuberculosis and M. bovis) against rapidly growing mycobacteria (M.smegmatis, M. abscessus and M. chelonae) The hexane extract from the bark was active against both Leishmania species (L.amazonensis: IC50 = 38.61± 0.48 L. chagasi: IC50 = 72.05± 4.28 mL), while the methanol extract from the leaves was active only against L. amazonensis (IC50 = 55.16 ± 5.08 tory activity of promastigote forms growth, at least in the range of experiments performed.

Table 1 : Inhibitory effect of Stifftia chrysantha extracts and methyl salicylate against Mycobacterium and Leishmania species of medical interest.
In vitro Antimycobaterial and antileishmanial experiments using extracts of S. chrysantha, methyl salicylate and standard antimicrobials.N.I.: No inhibition; N.E.:Not evaluated.