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

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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 13URJUDPDGH3yVJUDGXDomRHP%LRWHFQRORJLD9HJHWDO±&HQWURGH&LrQFLDV0pGLFDVHGD6D~GH &&6 8QLYHUVLGDGH)HGHUDOGR5LRGH-DQHLUR 8)5- &(35LRGH-DQHLUR5LRGH-DQHLUR%UDVLO 2)DFXOGDGHGH&LrQFLDV0pGLFDVHGD6D~GH6835(0$%5.P6DOYDWHUUD&(3-XL] de Fora, Minas Gerais, Brasil. 31~FOHRGH3HVTXLVDGH3URGXWRV1DWXUDLV 1331 8QLYHUVLGDGH)HGHUDOGR5LRGH-DQHLUR 8)5- &&6 %ORFR+&LGDGH8QLYHUVLWiULD&(35LRGH-DQHLUR5LRGH-DQHLUR%UDVLO 4/DERUDWyULR GH 3DUDVLWRORJLD 'HSDUWDPHQWR GH 3DUDVLWRORJLD 0LFURELRORJLD H ,PXQRORJLD 8QLYHUVLGDGH )HGHUDOGH-XL]GH)RUD 8)-) &(3-XL]GH)RUD0LQDV*HUDLV%UDVLO 5/DERUDWyULR GH 0LFREDFWpULDV ,QVWLWXWR GH 0LFURELRORJLD &&6 8QLYHUVLGDGH )HGHUDO GR 5LR GH -DQHLUR 8)5- &(35LRGH-DQHLUR%UDVLO 6'HSDUWDPHQWRGH%RWkQLFD,QVWLWXWRGH%LRFLrQFLDV8QLYHUVLGDGH)HGHUDOGR5LR*UDQGHGR6XO 8)5*6 $Y %HQWR*RQoDOYHV%ORFR,9±3UpGLR&(33RUWR$OHJUH5LR*UDQGHGR6XO%UDVLO *Correspondência: HPDLODQGUHIDUPDFLDUM#\DKRRFRPEU 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 pro- mastigotas 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. amazo- nensis (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. &RUUHVSRQGHQWDXWKRUDQGUHIDUPDFLDUM#\DKRRFRPEU 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 gar- den 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
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