Evaluation of in Vitro Antileishmanial and Antimycobacterial Activities of Stifftia Chrysantha J.C

Evaluation of in vitro antileishmanial and antimycobacterial FITOQUÍMICA / PHYTOCHEMISTRYFitoquímica / Phytochemistry activities of Stifftia chrysantha J.C. Mikan extracts

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

Rachel R. P. Machado1,2, André M. Marques 3*; Wilson Valente Júnior4; Elaine S. Coimbra4; Rafael S. Duarte5; Geraldo Luiz G. Soares6; Maria Auxiliadora C. Kaplan3

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Palavras chave: Stifftia chrysantha; diadem; methyl salicilate; Mycobacterium bovis; Mycobacterium smegmatis; leishmaniasis.

Keywords: Stifftia chrysantha; diadem; methyl salicilate; Mycobacterium bovis; Mycobacterium smegmatis; leishmaniasis.

Resumo

Stifftia chrysantha J.C. Mikan é uma planta pertencente à família Asteraceae cujo principal uso pela população pRRUQDPHQWDOHDWXDOPHQWHVHHQFRQWUDVREULVFRPRGHUDGRGHH[WLQomReVDELGRTXHDSODQWDIRLXWLOL]DGDQR WUDWDPHQWRGHDIHFo}HVUHVSLUDWyULDVSRUTXLORPERODV2REMHWLYRGHVWHHVWXGRIRLLQYHVWLJDURSRWHQFLDOHIHLWR antimicrobiano de diferentes extratos de S. chrysantha contra algumas espécies de micobactérias e formas promastigotas de duas espécies de Leishmania2VWHVWHVIRUDPUHDOL]DGRVin vitroXWLOL]DQGR077RX5HVD]XULQDHP 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 H[WUDWRPHWDQyOLFRGDVIROKDVHQRH[WUDWRKH[kQLFRGDFDVFDFRQWUDDVFXOWXUDVGHSURPDVWLJRWDVGHL. amazonensis (CI50 = 55,16 PJP/H[WUDWRPHWDQyOLFRDQGPJP/H[WUDWRKH[kQLFR DQGL. chagasi (CI50 = 72,05 PJ P/H[WUDWRKH[kQLFR 1RYRVHVWXGRVVmRQHFHVViULRVSDUDGHVFREULUDVVXEVWkQFLDVUHVSRQViYHLVSHODLQLELomR do crescimento das formas promastigotas.

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252 Revista Fitos Vol. 7 - nº 04 - outubro / dezembro 2012 Evaluation of in vitro antileishmanial and antimycobacterial Fitoquímica / Phytochemistry activities of Stifftia chrysantha J.C. Mikan extracts

Abstract

Stifftia chrysantha J.C. Mikan is a plant that belongs to Asteraceae family, mainly used for ornamental purposes and it is moderately endangered to die out nowadays. It is known this plant has been used on the treatment of respiratory DIIHFWLRQVE\TXLORPERFRPPXQLWLHV %UD]LOLDQKLQWHUODQGVHWWOHPHQWIRXQGHGE\SHRSOHRI$IULFDQRULJLQ 7KHDLPRI this study was to investigate the potential antimicrobial effect of different extracts from S. chrysantha against some species of mycobacterias and promastigote forms of two Leishmania sp. In vitro assays were performed using FRORULPHWULFPHWKRGVZLWK GLPHWK\OWKLD]RO\O GLSKHQ\OWHWUD]ROLXPEURPLGHRU5HVD]XULQDFFRUGLQJWR the microorganism evaluated. The results showed low activity of extracts against mycobacterial cultures. On the other hand, a growth inhibitory effect was observed in the methanol extract from leaves and on the hexane extract from bark against promastigote culture of L. amazonensis (IC50 = 55.16 PJP/PHWKDQROH[WUDFWDQGPJP/ hexane extract) and L. chagasi (IC50 = 72.05 PJP/KH[DQHH[WUDFW 1HZVWXGLHVDUHQHFHVVDU\WRGLVFRYHUWKH substances that were responsible for the growth inhibition of promastigote forms.

Introduction extracts of Polygala myrtifolia (Polygalaceae), a plant that shows high concentration of methyl salicylate. Stifftia chrysantha J.C. Mikan (Asteraceae) is popu- The reported activity found in P. myrtifolia and the simi- larly known as diadem, fox tail or gold rain due to the lar major compound content present in the aerial parts FRORUDQGVKDSHRILWVLQÀRUHVFHQFHV%UD]LOLDQSRSX- of Stifftia chrysantha motivated the investigation about lar names of the plant are: rabo-de-cotia, diadema, WKHELRORJLFDOSRWHQWLDOWKLVQDWLYH%UD]LOLDQHQGDQJH- SRPSRPÀRUGDDPL]DGHHVSRQMDHVSRQMDGHRXUR red risk species S. chrysantha extracts and also to the jambeiro do-mato, pincel. Its main use by the popu- pure methyl salicylate against mycobacteria. We also lation is ornamental and it is moderately endangered tested the activity of the extracts against promastigote to die out nowadays, according to list annexed to the forms of leishmania. The diseases caused by myco- Decree 19.149 published by the Rio de Janeiro Envi- bacteria and Leishmania sp. represent important di- ronmental Protection Bureau (2000). It can be found sorders for Public Health, since long times are needed in Protected Areas such as the National Park of Tijuca, for effective treatment by using drugs that may cause Rio de Janeiro Botanical Garden and Grajaú-Jacare- potential side effects (Almeida et al., 2005; Medeiros et SDJXi5RDGLQ5LRGH-DQHLUR5-%UD]LO &UHVSRHW al., 2005; Coll et al., 2009; Sundar et al., 2007). Many al., 2010). Few studies on the chemical composition of research works have been performed with the attempt this plant have been reported. Oliveira (1999) descri- to identify new therapeutically potential drugs against EHGÀDYRQRLGVLVRODWHGIURPLWVÀRZHUVZKLOH0DUTXHV tuberculosis, mycobacteriosis and leishmaniasis. Such IRXQGDVLJQL¿FDQWSUHGRPLQDQFHRIPHWK\OVD- VWXGLHVKDYHEHHQFKDUDFWHUL]HGE\WKHXVHRISUHOLPL- licylate in the volatile fractions from fruits ranging from nary approaches with in vitro experiments, before in 85% to 95% in the volatile mixture as well as such vivo ones and clinical trials (Lahlou et al., 2004). presence in all aerial parts of the plant during all sea- sons of the year. Material and Methods

The organic ester methyl salicylate seems to take part Reagents: Dimethyl-sulfoxide (DMSO); 3-(4,5-di- in the attraction process of pollinators and in the de- PHWK\OWKLD]RO\O GLSKHQ\OWHWUD]ROLXP EURPLGH fense of the plant. Besides, the metabolic conversion (MTT) and Methyl salicylate were purchased from Sig- of methyl salicylate into salicylic acid and subsequen- PD&KHPLFDO&R6W/RXLV0286$ 5HVD]XULQVR- tly into acetyl salicylic could justify the ancient use of dium salt powder was purchased from Acros Organic the plant by quilombo communities for treatment of N.V., Geel, Belgium). ÀX FROGV DQG UHVSLUDWRU\ DIIHFWLRQV 0DUTXHV HW DO 2005). This metabolic conversion from methyl sa- Collection of aerial parts and roots of S. chrysantha: OLF\ODWHWRVDOLF\OLFDFLGLVDOVRLPSRUWDQWIRUWKHSODQW¶V Aerial parts and roots of S. chrysantha OHDYHV ÀR- GHIHQVH V\VWHP DQG IRU VLJQDOLQJ DJDLQVW SUHGDWRUV¶ wers, barks, branches and fruits) were collected under attacks. Salicylic acid is known as an important phyto- supervision of botanist Roberto L. Esteves in the garden of National Museum of Rio de Janeiro in Decem- alexin present in responses to physical and biological ber 2004. The voucher number is R208153. The plant stresses suffered by the plant (Durrant et al., 2004). material was collected early in the morning from the Employing a rapid radiometric method, Lall et al. same chosen specimen and it was taken immediate- GHWHFWHGDVLJQL¿FDQWLQKLELWLRQRIMycobacte- ly to the laboratory and separately reduced into small rium tuberculosis H37Rv exposing the bacteria to the pieces. The powdered materials were air-dried.

Revista Fitos Vol. 7 - nº 04 - outubro / dezembro 2012 253 Evaluation of in vitro antileishmanial and antimycobacterial Fitoquímica / Phytochemistry activities of Stifftia chrysantha J.C. Mikan extracts

Preparation of S. chrysantha extracts: Air-dried and as the positive control) and to the DMSO solution at powdered plant materials (50g of leaf; 30g of bark and 0.01% for 72h 24ºC. The colorimetry was assessed JRIÀRZHU ZHUHVHSDUDWHO\H[WUDFWHGXQGHUVWDWLF by absorbance using SPECTRAMAX 190, Molecular maceration using hexane as solvent, and followed by 'HYLFHVVSHFWURPHWHUDQGQP¿OWHU)RUWKHUHVXOWV methanol. Removal of residual solvent under reduced analysis, GraFit (ULWKDFXV6RIWZDUH/WG+RUOH\8. pressure was performed using a Büchi rotatory evapo- software version 5 was used. rator, equipped with warm bath under controlled tem- perature (40oC). The same procedure was carried out Experiments using rapidly and slow growing myco- with methanol as solvent extractor. bacteria: the susceptibility tests for mycobacteria using extracts were performed in 96-well plates Extracts used: hexane extract from leaves and bark XVLQJWKHFRORULPHWULFWHVWEDVHGRQUHVD]XULQUHGXF- of S. chrysantha; and methanol extract from leaves tion following the procedure used by Palomino et al. and fruits of S. chrysantha. (2002). All experiments were performed in triplicates and at least three repetitions. Concentrated solutions Methyl salicylate: methyl salicylate was used in the PJP/ RI WKH H[WUDFWV ZHUH SUHSDUHG E\ LQLWLDOO\ experiments on mycobacteria at the following concen- VROXELOL]LQJWKHH[WUDFWVLQ'062DQGVXEVHTXHQWO\ trations: 250PJP/ PJP/ PJP/ PJP/ in sterile water. The total content of DMSO in each 16PJP/DQGPJP/DQGXVLQJ'062DVGLOXHQW well reached 10%, which did not inhibit mycobacterial JURZWK,QÀDWERWWRPHGZHOOSODVWLFWLVVXHFXOWXUH Leishmania sp. assays: promastigote forms of plates, dilutions of the extract were prepared in Mi- Leishmania amazonensis 0+20%U-RVHID LVROD- GGOHEURRN+EURWK %'ORWH86$ FXOWXUH ted from a patient who had diffuse cutaneous form of medium enriched with OADC (Becton-Dikinson) at leishmaniasis) were cultured in Warren medium (BHI, the following concentrations: 2500PJP/ PJ plus hemin and folic acid) and promastigote forms of mL; 625PJP/ PJP/ PJP/ DQG PJP/ L. chagasi 0+20%U33LVRODWHGIURPSDWLHQWV UHVXOWLQJ LQ D ¿QDO YROXPH RI PL in each well. who had visceral form of leishmaniasis) were cultured Next, 100PL of slow-growing mycobacteria (M. tu- in 199 medium, both supplemented with fetal bovine berculosis +5Y ±$7&& 0 ERYLV ± %&* serum and maintained at 24ºC during one week. Fe- (Monroe) or rapidly growing mycobacteria (M. smeg- tal bovine serum (FBS) was purchased from Cultilab matis±$7&&M. abscessus±$7&& &DPSLQDV 6mR 3DXOR %UD]LO %UDLQ KHDUW LQIXVLRQ M. chelonae±$7&& VXVSHQVLRQZDVDGGHG (BHI) from Himédia (Mumbai, Indian). Hemin, folic WRWKHH[WUDFWVROXWLRQVVHSDUDWHO\7KH¿QDOFRQFHQ- acid, and 199 medium were purchased from Sigma trations of the extracts into the wells were: 1250PJ &KHPLFDO&R 6W/RXLV0286$ %LRORJLFDODFWLYL- mL; 625PJP/PJP/PJP/PJP/DQG ty against promastigote forms of L. amazonensis and 39PJP/7KHVXVSHQVLRQVZHUHSUHSDUHGLQ0LGG- L. chagasi was determined using the MTT colorime- lebrook 7H9 (Difco) culture medium enriched with tric method based on reduction of the salt by mito- OADC (Becton-Dikinson) at 1:25 concentration from chondrial dehydrogenases (Mossman et al., 1983). the initial suspension and turbidity equivalent to 1.0 Promastigoste forms in logarithmic growth stage of in McFarland scale. Standard drugs were used as in vitro growth of both species were used in the ex- positive control for inhibition of mycobacteria growth, SHULPHQWV7KH\ZHUHDGGHGWRÀDWERWWRPHGZHOO VXFK DV ULIDPSLFLQ PHWK\OSLSHUD]LQ\OLPLQRPH- plastic tissue-culture plates at the concentrations 2.0 WK\O ULIDP\FLQ±/RW±6,*0$ DJDLQVWVORZ- [ FHOOVP/ DQG [ 6FHOOVP/ RI L. amazo- -growing mycobacteria (M. tuberculosis and M. bovis) nensis and L. chagasi, respectively. After 1h and at DQGFLSURÀR[DFLQ $/'5,&+&KHPLVWU\±/RW 24ºC, the parasites were exposed to different concen- against rapidly growing mycobacteria (M. smegma- trations of extracts of S. chrysantha previously solu- tis, M. abscessus and M. chelonae) at the following ELOL]HG LQ '062 &RQFHQWUDWLRQV & RI WKH H[WUDFWV FRQFHQWUDWLRQVULIDPSLFLQ±±;-6PJP/DQG were 250.0PJP/ PJP/ PJP/ PJ FLSURÀR[DFLQ±±[-7PJP/FRQVLGHULQJD mL, 15.6PJP/DQGPJP/DQGHDFKFRQFHQWUDWLRQ serial dilution (1:2) for both drugs. The plates were were performed in triplicate and in two independent sealed and kept at 37ºC for 7 days. On the seventh assays. The promastigote forms were exposed to GD\LQVWHULOHHQYLURQPHQW/RIUHVD]XULQ the extracts, to Amphotericin B (standard drug used (diluted in etylenglicol and sterile distilled water) was

254 Revista Fitos Vol. 7 - nº 04 - outubro / dezembro 2012 Evaluation of in vitro antileishmanial and antimycobacterial Fitoquímica / Phytochemistry activities of Stifftia chrysantha J.C. Mikan extracts

DGGHG 5HVD]XULQ6RGLXP6DOW3RZGHU$FURV2UJD- of this compound was observed mailing in the fruits nic N.V., Geel, Belgium). Extracts were considered DQGÀRZHUVGXULQJWKHZKROH\HDUVXJJHVWLQJDQLP- as active against mycobacteria when exhibited MIC portance in the attraction process of pollinators, de- JP/ 7RVXQHWDO IHQVH V\VWHP DQGRU IRU VLJQDOLQJ DJDLQVW SUHGDWRUV¶ attacks (Marques et al., 2012). Methyl salicylate has Results and Discussion already been related to antimycobacterial activity by /DOO DQG FRZRUNHUV WKDW YHUL¿HG D VLJQL¿FDQW The experiments performed in order to evaluate po- effect of Polygala myrtifolia (Polygalaceae) extract, tential antimycobacterial activity of extracts from di- which contains great quantities of methyl salicylate. In fferent parts of S. chrysantha did not show biological addition, methyl salicylate (83.8%, 89.1% and 97.8%) activity against any of the mycobacteria tested. This was found as the main volatile constituent in roots of judgment is based on a criterion established by Tosun the P. sabulosa, P. paniculata and P. cyparissia, res- et al. (2004), which considers a substance as active if SHFWLYHO\ 3L]]RODWLHWDO 7KHUHIRUHRQHPLJKW its MIC < 200PJP/+RZHYHUWKLVOLPLWLVFRQWURYHU- surmise that this compound could be responsible for sial. Lima (2006) evaluated the antimicrobial activity antimycobacterial properties. RIH[WUDFWVREWDLQHGIURPGLIIHUHQW%UD]LOLDQSODQWVSH- FLHVDQGYHUL¿HGDQWLP\FREDFWHULDODFWLYLW\RIPHWKD- In our study, antimycobacterial activity of methyl sa- nol extracts from leaves of Lafoensia pacari against OLF\ODWHKDVEHHQHYDOXDWHGIRUWKH¿UVWWLPHDQGWKH M. smegmatis 0,& JP/ 0IRUWXLWXP 0,& results revealed no inhibitory activity on the mycobac- JP/ DQG0SKOHL 0,& JP/ QRWFRQ- terial growth (Table 1). The results suggests that the sidering hence, the activity limits determined by Tosun VLJQL¿FDQW LQKLELWLRQ RI M. tuberculosis H37Rv found et al. (2004). by Lall (1999), is not due to the exclusive action of methyl salicylate, the major compounds of that extract. In our study, two MIC values were determined: one This preliminary screening against Mycobacterium tu- against M. smegmatis which resulted from the me- berculosis, H37Rv was performed using acetone and thanol extract from leaves (1125.1 ± 0.13PJP/ DQG water plant extracts. The minimal inhibitory concentra- another against M. chelonae (312.5 ± 0.16PJP/ UH- tion of Polygala myrtifoliaZDVPJPOEHLQJDOVR ODWHG WR PHWKDQRO H[WUDFW IURP ÀRZHUV DV VKRZQ LQ DFWLYHDJDLQVWWKHUHVLVWDQWVWUDLQDWPJPO'HVSL- Table 1. These found MIC values may indicate the te of the good activity displayed by P. myrtifolia extract, presence of substances capable of interfering with the no phytochemical separation was performed in the metabolism of M. chelonae and M. smegmatis. The related study. A chemical investigation of the genus other evaluated plant products did not show antimyco- Polygala showed the occurrence of a variety of secon- bacterial activity even at higher concentration. It still dary metabolites, such as xanthones, saponins, oligo- has to be discovered the active compounds in the S. VDFFKDULGHVÀDYRQRLGVFRXPDULQVDQGVW\U\OS\URQHV chrysantha extracts. It is known that the S. chrysantha (Johannl et al. 2011). Some of these compounds are extracts contain considerable amounts of quercetin HQDEOH WR EH H[WUDFWHG E\ WKH DFHWRQH DQGRU ZDWHU DQLPSRUWDQWÀDYRQRLG 2OLYHLUD $VLWLVNQRZQ preparations and could be acting against the M. tuber- VHYHUDOIXQFWLRQVFDQEHDWWULEXWHGWRWKHÀDYRQRLGV culosis studied strain. such as protection of the plants against insects, fungal DQG EDFWHULDO FRORQL]DWLRQ YLUDO LQIHFWLRQV DV ZHOO DV The hexane extract from the bark was active against attraction of pollinators due to the remarkable colors both Leishmania species (L. amazonensis: IC50 = of these compounds (Salvador et al., 2008). Boligon 38.61 ± 0.48PJP/L. chagasi: IC50 = 72.05 ± 4.28PJ (2012) studied antimycobacterial activity of quercetin mL), while the methanol extract from the leaves was against slowly and rapidly growing mycobacteria and active only against L. amazonensis (IC50 = 55.16 ± they encountered a MIC > 200PJP/WKDWPLJKWLQGL- 5.08PJP/ 7KHRWKHUH[WUDFWVGLGQRWUHYHDOLQKLEL- cate that quercetin is one of the active compounds of tory activity of promastigote forms growth, at least in the S. chrysantha extract. the range of experiments performed.

Seasonal evaluation of S. chrysantha volatile fractions revealed methyl salicylate as the major consti- WXHQW RI ÀRZHUV IUXLWV DQG OHDYHV7KH KLJK FRQWHQW

Revista Fitos Vol. 7 - nº 04 - outubro / dezembro 2012 255 Evaluation of in vitro antileishmanial and antimycobacterial Fitoquímica / Phytochemistry activities of Stifftia chrysantha J.C. Mikan extracts N.I. ) L. chagasi JP/ P ( 50 IC L. species of medical interest. amazonensis g/mL) P M. bovis M. Mycobacterium and Leishmania g/mL) ( P tuberculosis M. N.I. N.I. N.I. N.I. N.I. g/mL) ( P , methyl salicylate and standard antimicrobials. abscessus S. chrysantha M. N.I. N.I. N.I. N.I. g/mL) ( P chelonae extracts and methyl salicylate against 312.5 ± 0.16 M. g/mL) ( P Stifftia chrysantha Stifftia ( smegmatis 1125.1 ± 0.13 1125.1 0.018 ± 0.012 ± 0.015 0.014 ± 0.26 0.21 ------Antimycobaterial and antileishmanial experiments using extracts of Rifampicin ------0.818 ± 1.280 ± 0.001 0.004 ------Bark (Hex) N.I. N.I. N.I. N.I. N.I. PRODUCTMICMIC MIC MIC &LSURÀR[DFLQ Fruit (MeOH) N.I N.I. N.I. N.I. N.I. N.I. N.I. Leaves (Hex) N.I. N.I. N.I. N.I. N.I. N.I. N.I. EVALUATED Amphotericin B ------0.9 ± 0.0001 1.9 ± 0.0001 Leaves (MeOH) Flower (MeOH) N.I. Methyl salicylate N.I. N.I. N.I. N.I. N.I. N. E. N. E. N.I.: No inhibition; N.E.: Not evaluated. Table 1: Inhibitory effect of Table In vitro

256 Revista Fitos Vol. 7 - nº 04 - outubro / dezembro 2012 Evaluation of in vitro antileishmanial and antimycobacterial Fitoquímica / Phytochemistry activities of Stifftia chrysantha J.C. Mikan extracts

Guided by the considerations concerning about anti- species of Polygala. Brazilian Journal of Microbiology, mycobacterial activity one may guess that the leishma- v. 42, p. 1065-1075. nicidal activity might also be related to the quercetin. 7KLVK\SRWKHVLVLVVXSSRUWHGE\¿QGLQJVLQWKHOLWHUDWX- Lall, N.; Meyer, J.J.M. 1999 - In vitro inhibition of drug- re. For example, Sarkar and colleagues (2002) detec- resistant and drug-sensitive strains of Mycobacterium ted reduction of leishmanias in the spleen of hamsters tuberculosis by ethnobotanically selected South of this phytocompound. This reduction is probably due African plants. Journal of Ethnopharmacology, v. 66, WRDQLQWHUIHUHQFHRITXHUFHWLQLQWKHOHLVKPDQLD¶VLURQ p. 347-354. metabolism (Sen et al., 2008). Lahlou, M. 2004 - Methods to study the phytochemis- ,QFRQFOXVLRQWKHELRORJLFDODFWLYLWLHVYHUL¿HGIURPWKH try and activity of essential oils. Phytotherapy Resear- extracts from different parts of S. chrysantha aggre- ch, v. 18, p. 435-448. JDWHDUHOHYDQWYDOXHWRWKHSODQWRQFHLWZDVWKH¿UVW time that the antimycobacterial and antileishmanial /LPD 05) $]HYHGR;LPHQHV (&3 /XQD - potential activity has been evaluated. Despite the low Goulart-Sant`Ana, A.E. 2006 - The antibiotic activity of activity found against mycobacteria culture, the pro- VRPH%UD]LOLDQPHGLFLQDOSODQWVRevista Brasileira de PDVWLJRWHFXOWXUHVXIIHUHGDVLJQL¿FDQWLQKLELWLRQRILWV Farmacognosia, v. 16, p. 300-306. growth. However, new studies are necessary in order to elucidate which phytocompounds in the plant mate- Marques, A.M.; Garcia, A.I.C.; Esteves, R.; Lima, rial are responsible for the biological activity and how M.C.H.P.; Araújo-Filho, H.C.; Kaplan, M.A.C. 2005 - they act against the Leishmania species exposed to Potencialidades da fração volátil de Stifftia chrysantha the products used in this study. Mikan. In: XXIX Jornada Giulio Massarani de Iniciação &LHQWt¿FD$UWtVWLFDH&XOWXUDOGD8)5-5LRGH-DQHL- Acknowledgment: ro, Livro de Resumos, Rio de Janeiro, p. 38-39.

We thank Marlei da Silva Gomes (Microbiology Institu- Marques, A.M.; Lima, C.H.P.; Esteves, R.; Araújo- WH±&HQWURGH&LrQFLDVGD6D~GH±8)5- IRUSUHSD- -Filho, H.C.; Kaplan, M.A.C. 2012 - Evaluation of the ration of materials for mycobacterial assays. volatile components and the seasonal variation of the methyl salicylate from Stifftia chrysantha Mikan E\+6630(*&06Boletín Latinoamericano y del References Caribe de Plantas Medicinales y Aromáticas, v. 11, p. 413-419. Almeida, P.; Oliveira, M..M.; Hinrichsen, S.L.; Kawas- saki, A.M.; Lima, E.H.M. 2005 - Tuberculose. In: Doen- Medeiros, I.M.; Nascimento, E.L.T.; Hinrichsen, S.L. ças Infecciosas e Parasitárias. 1ª edição, ed.: Guana- 2005 - Leishmanioses (Visceral e Tegumentar). In: bara Koogan, Rio de Janeiro. p. 281-296. Doenças Infecciosas e Parasitárias. 1ª edição, ed.: Guanabara Koogan, Rio de Janeiro. p. 398-409. %ROLJRQ$$JHUWW9-DQRYLN9&UX]5&&DP- pos, M.M.A.; Guillaume, D.; Athayde, M.L.; Santos, Mossman, T. 1983 - Rapid colorimetric assay for cellu- A.R.S. 2012 - Antimycobacterial activity of the frac- lar growth and survival: applications to proliferation tions and compounds from Scutia buxifolia. Revista and cytotoxicity assay. Journal of Immunology Metho- Brasileira de Farmacognosia, v. 22, n.1, p. 45-52. ds, v. 16, p.55-63.

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Received in October 2012. Accepted in January 2013.

258 Revista Fitos Vol. 7 - nº 04 - outubro / dezembro 2012