plant material - c. brasiliense leaves were collected in cardoso island , december 2010 , 
 in the state of so paulo ( sp ) , brazil . the voucher specimen ( sp 363818 )
was identified 
 by prof dr maria claudia m young and was deposited and authenticated at the herbarium of 
 the botany institute of so paulo , sp . the leaves were dried at 35c in a circulating 
 air oven and triturated in a knife mill ( usiram , so bernardo do campo , sp ) .
plant extraction and purification of compounds - the powdered leaves ( 800 g ) were 
 extracted by exhaustive maceration in ethanol / water ( 9:1 ) at room temperature ( rt ) , 
 filtered and concentrated under vacuum at 40c to obtain an aqueous extract and a 
 dark - green residue .
the dark - green 
 residue from the extract , which had adhered to the glass bottles , was dissolved with 
 dichloromethane and the solvent was completely evaporated at rt to yield a 
 dichloromethane extract ( 25.0 g ) ( brenzan et al . 

the dichloromethane extract ( 25.0 g ) was initially fractionated using chromatography in 
 a vacuum silica - gel column ( 8.0  40 cm ) with hexane , hexane / dichloromethane ( 1:1 ) , 
 dichloromethane , dichloromethane / ethyl acetate ( 9:1 ) , ethyl acetate , methanol and 
 methanol / water ( 9:1 ) . next , the hexane fraction ( 4.0 g ) was further chromatographed on a 
 vacuum silica - gel column and eluted with hexane , hexane / dichloromethane ( 98:2 , 95:5 , 
 90:10 , 80:20 and 50:50 ) , dichloromethane , dichloromethane / ethyl acetate ( 98:2 , 95:5 , 
 90:10 , 80:20 and 50:50 ) , ethyl acetate and methanol . the hexane fraction was evaporated 
 to a residue , which was crystallised using dichloromethane ( 9:1 ) , yielding compound ( 1 ) 
 ( 52.0 mg ) , as described in previous studies ( gasparotto - jnior et al .
fractions 6 - 8 ( 667.0 mg ) , 
 which were eluted in hexane , were chromatographed using thin layer chromatography and 
 combined and then rechromatographed on a silica - gel column chromatograph ( 40  2.0 cm ) 
 eluted with hexane , hexane / dichloromethane ( 98:2 - 50:50 ) , dichloromethane , 
 dichloromethane/ ethyl acetate ( 95:5 - 50:50 ) , ethyl acetate and finally methanol . next
, 
 fractions 1 - 12 , which were eluted in hexane , were combined and evaporated to dryness , 
 obtaining ( 2 ) ( 10.5 mg ) , as described in a previous study ( brenzan et al .
the ethyl acetate fraction ( 1.83 g ) of the 
 dichloromethane extract was rechromatographed through a sephadex lh-20 column and eluted 
 with chloroform / methanol ( 1:1 ) .
fractions 48 - 53 ( 223.8 mg ) were identified as ( 3 ) , as 
 described in a previous study ( brenzan et al . 

structure elucidation - the compounds were identified by chromatography - mass 
 spectrometry in a shimadzu gc / ms 17 a qp 5000 mass spectrometer , db5 column ( 30 m ; 0.32 
 m ) and a micromass quattro - lc instrument equipped with an esi / apci z - spray ion source 
 and by nuclear magnetic resonance ( nmr ) obtained in a bruker drx-400 ( 8.4 t ) and gemini 
 300 ( 7.05 t ) , with tms as the internal standard and a constant temperature of 298 k. 1h 
 nmr ( 300 mhz ) ,
13c nmr ( 75.5 mhz ) , dept , cosy ( 300 mhz ) , hmbc and hsqc analysis was 
 conducted using deuterated solvent ( cdcl3 ) .
the d was obtained using a perkin - elmer 
 model 241 polarimeter ( 20c ; 589 nm ) with dichloromethane and was compared with data 
 from the literature ( gasparotto - jnior et al .
spectroscopic and spectrometry data - the spectroscopic and spectrometry data of 
 compounds ( 1 ) , ( 2 ) and ( 3 ) ( fig .
2005 , brenzan et al . 2008a , b , 2010 , respectively ) . 

1 : pure compounds isolated from leaves of calophyllum brasiliense : ( - ) 
 mammea a / bb ( 1 ) , ( - ) mammea b / bb ( 2 ) and amentoflavone ( 3 ) . 
 
high performance liquid chromatography coupled to a ultraviolet detector ( hplc - uv ) 
 analysis - the dichloromethane extract and purified compounds were analysed by hplc - uv 
 using hplc - uv grade solvents and ultrapure water ( milli - q system , millipore , bedford , 
 ma , usa ) and a waters 1525 liquid chromatograph equipped with a binary pump ( lc-10 ad ) , 
 automatic injection valve 135 ( rheodyne ) with a 20 l loop , cto-10avp 
 thermostat - controlled oven compartment ( shimadzu ) and a 2489 uv / visible detector 
 ( waters ) controlled by breeze 2 software ( omnisolv em science , gibbstown , nj , usa ) . a 
 metasil ods column ( 5 m ; 150  4.6 mm ) maintained at 25c was used in the 
 chromatographic analysis .
the separation was conducted in a gradient system , using a 
 mixture of acetonitrile and ultrapure water 55:45 v / v to 80:20 ( 0 - 10 min ) , 80:20 v / v to 
 100% ( 10 - 20 min ) , 100% acetonitrile ( 20 - 25 min ) and 55:45 v / v ( 26 - 30 min ) as the mobile 
 phase , at a flow rate of 0.6 ml / min .
the dichloromethane extract ( 3 
 mg / ml ) and purified compounds ( 1 ) , ( 2 ) and ( 3 ) ( 1 mg / ml ) were dissolved in methanol , 
 filtered through a membrane filter ( millipore , brazil ) and injected ( 20 l ) into the 
 hplc - uv system . compounds ( 1 ) and ( 2 ) were quantified in the dichloromethane and aqueous 
 extracts by hplc - uv using compound ( 1 ) isolated from the c. brasiliense leaves as the 
 standard .
the content of ( 2 ) was calculated using the calibration curves of compound 
 ( 1 ) ; these compounds belong to the same coumarin class and produced uv - spectra with 
 almost the same molar absorptivity at 254 and 366 nm ( crombie et al .
2005 ) . for the calibration 
 curves of compound ( 1 ) , solutions in methanol were used at concentrations ranging from 
 15.63 - 250 g / ml . the extracts were prepared in methanol at 3 mg / ml and filtered through 
 a 0.45-m membrane filter ( millipore , brazil ) .
the statistical analyses of the data were performed using statistica 8.0 
 software ( statsoft inc , tulsa , ok , usa ) .
determination of antimycobacterial activity - to determine the mic against m. 
 tuberculosis h37rv ( atcc 27294 ) , the dichloromethane and aqueous extracts from c. 
 brasiliense leaves and purified compounds ( 1 ) , ( 2 ) and ( 3 ) were subjected to in vitro 
 assays in triplicate .
additionally , the pure compounds were tested against 13 clinical 
 isolates ( 6 susceptible and 7 resistant ) .
the clinical isolates were obtained from the 
 mycobacteria collection of the laboratory of medical bacteriology of the state 
 university of maring , state of paran , brazil .
the antimycobacterial activity was 
 evaluated using rema , according to the procedure by palomino et al .
briefly , 200 l of sterile distilled water was distributed into the 
 outer wells of a microplate ( falcon 3072 , becton - dickinson , lincoln park , nj , usa ) .
the 
 extracts and pure compounds were diluted in dimethyl sulphoxide ( dmso ) ( amresco ) and 
 serial two - fold dilutions from 1,000 - 7.8 g / ml and 250 - 1.9 g / ml , respectively , were 
 made in middlebrook 7h9 ( difco laboratories , detroit , mi , usa ) supplemented with oleic 
 acid , bovine albumin , dextrose and catalase ( oadc ) enrichment ( bbl / becton - dickinson , 
 sparks , md , usa ) .
isoniazid ( difco ) was used as the reference drug at concentrations 
 from 1.0 - 0.007 g / ml .
one hundred microlitres of each bacterial inoculum , standardised 
 according to 1 mcfarland turbidity and diluted 1:20 in middlebrook 7h9 medium 
 supplemented with oadc , was inoculated into the wells .
a growth control containing no 
 drug and a sterile control without bacteria were prepared for each assay .
a control 
 containing 2.5 % ( v / v ) dmso was included to detect inhibitory effects on m. tuberculosis 
 growth .
the plates were covered with lids and the edges were sealed with polyethylene 
 tape .
the plates were placed in a plastic box and incubated at 35c at normal atmosphere 
 for seven days .
next , 30 l of freshly prepared 0.01% resazurin solution ( acros , morris 
 plains , nj , usa ) was added to each well and the plates were reincubated for 24 - 48 h at 
 35c for visual readings . a colour change from blue to pink indicated mycobacterial 
 growth and the mic was interpreted as the lowest concentration that prevented the colour 
 change .
cytotoxicity assay - in vitro cytotoxicity assays were performed on j774g.8 macrophages 
 ( papazisis et al .
the macrophages were 
 maintained in tissue flasks containing rpmi-1640 medium ( gibco , invitrogen co , grand 
 island , ny , usa ) with l - glutamine and supplemented with 10% inactivated foetal bovine 
 serum ( fbs ) ( gibco brl , life technologies ) at 37c in a 5% co2 atmosphere .
the 
 macrophages were plated at 5.0  104 cells per well in a 96-well microplate with 
 rpmi-1640 medium supplemented with 10% inactivated fbs and incubated for 24 h at 37c in 
 5 % co2 .
next , the medium was removed and the cell monolayers were treated with 
 different concentrations of the extracts and pure compounds ( 500 - 3.12 g / ml ) . positive 
 and negative controls ( without drugs ) were included .
the microplate was incubated under 
 the same conditions for 48 h. subsequently , 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl 
 tetrazolium bromide dissolved in phosphate buffered saline at a concentration of 2 g / ml 
 was added to all of the wells except the reaction blank ( mosmann 1983 ) .
the microplate was incubated in the absence of light under 5% 
 co2 at 37c for 4 h. next , 150 l of dmso was added and stirred and the optical density 
 was determined using an automated spectrophotometer ( asys expert plus ) at 492 nm .
the 
 50% cytotoxicity concentration ( ic50 ) was determined by logarithm regression analysis . 

the selectivity index ( si ) was determined using the ic50/mic ratio for each compound 
 tested .
the dichloromethane extract showed activity against m. tuberculosis h37rv with a mic of 
 62.5 g / ml . the aqueous extract showed lower activity ( mic 125 g / ml ) ( table i ) . 

table i anti - mycobacterium tuberculosis h37rv activity and cytotoxicity of 
 extracts and pure compounds obtained from leaves of calophyllum 
 brasilienseextracts / compoundsmic g / ml ( m)ic50 g / ml  sd ( m  sd)siextracts  dichloromethane62.536.67  4.710.59  aqueous125.0ntntpure compounds  ( - ) mammea a / bb31.2 ( 76.85)33.30  3.30 ( 82.51  8.0)1.06  ( - ) mammea b / bb31.2 ( 83.87)27.50  2.50 ( 73.92  6.7)0.88  amentoflavone >
ic50 : 
 50% cytotoxic concentration ; mic : minimal inhibitory concentration ; nt : not 
 tested ; sd : standard deviation ; si : ic50/mic . 
 
ic50 : 
 50% cytotoxic concentration ; mic : minimal inhibitory concentration ; nt : not 
 tested ; sd : standard deviation ; si : ic50/mic .
the fractionation of the dichloromethane extract led to the isolation of the coumarins 
 ( 1 ) and ( 2 ) and the biflavonoid ( 3 ) , with the last one as the major constituent of the 
 extract ( fig .
1 ) . the spectral data of the 
 compounds were in agreement with the literature ( gasparotto - jnior et al .
, we identified 
 compounds ( 1 ) ( peak 1 ) , ( 2 ) ( peak 2 ) and ( 3 ) ( peak 3 ) with retention times of 21.0 , 15.5 
 and 5.5 min , respectively ( fig .
2 ) . for the 
 determination of the contents of compounds ( 1 ) and ( 2 ) in the extracts from c. 
 brasiliense ,
the regression equation y = 61589x + 52298 was used , which showed different 
 concentrations of the compounds .
the dichloromethane extract showed a significantly 
 higher concentration of compound ( 1 ) ( 31.86  2.6 g / mg ) than the aqueous extract ( 16.67 
  1.4 g / mg ) ( p < 0.001 ) . compound ( 2 ) was detected at a concentration of 8.24  1.1 
 g / mg in the dichloromethane extract , but it was not possible to quantify ( 2 ) in the 
 aqueous extract .
compound ( 3 ) was not quantified because it did not demonstrate 
 activity . 

2 : chromatograms of the standard ( - ) mammea a / bb ( rt = 21.3 min ) ( a ) and 
 dichloromethane ( b ) and aqueous ( c ) extracts from leaves of calophyllum 
 brasiliense .
chromatographic conditions : metasil ods column ; mobile phase : 
 acetonitrile - water 55:45 v / v to 80:20 ( 0 - 10 min ) , 80:20 v / v to 100% ( 10 - 20 
 min ) , 100% acetonitrile ( 20 - 25 min ) and 55:45 v / v ( 26 - 30 min ) ; flow rate 0.6 
 ml / min ; temperature 25c ; detection : 336 nm ; ( 1 ) : ( - ) mammea a / bb ; ( 2 ) : ( - ) 
 mammea b / bb ; ( 3 ) amentoflavone . 
 
both coumarins ( 1 ) and ( 2 ) isolated from the dichloromethane extract showed anti - m . 

tuberculosis h37rv activity , with a mic of 31.2 g / ml ( 76.85 m ) and 31.2 g / ml ( 83.87 
 m ) , respectively ( table i ) .
compound ( 3 ) showed 
 low activity against m. tuberculosis h37rv ( mic > 500 g / ml ) ( table i ) .
we also evaluated the antimycobacterial activity of compounds ( 1 ) and ( 2 ) against six 
 susceptible and seven resistant clinical m. tuberculosis isolates .
the mic values ranged 
 between ( 1 ) 31.2 g / ml ( 76.85 m ) and 62.5 g / ml ( 153.94 m ) and ( 2 ) 31.2 g / ml ( 83.87 
 m ) and 62.5 g / ml ( 168.01 m ) ( table ii ) . 

table ii anti - mycobacterium tuberculosis clinical isolates activity of isoniazid 
 and pure compounds obtained from leaves of calophyllum brasiliensem .
tuberculosis clinical 
 isolatessusceptibility profilemics g / ml ( m ) 
 h(1)(2)1susceptible0.03 ( 0.22)31.2 ( 76.85)31.2 ( 83.87)13638susceptible0.03 ( 0.22)62.5 ( 153.94)62.5 ( 168.01)3susceptible0.03 ( 0.22)31.2 ( 76.85)31.2 ( 83.87)11susceptible0.03 ( 0.22)31.2 ( 76.85)31.2 ( 83.87)16susceptible0.03 ( 0.22)31.2 ( 76.85)31.2 ( 83.87)4851susceptible0.03 ( 0.22)62.5 ( 153.94)31.2 ( 83.87)1193h , r , e , s1 ( 7.29)62.5 ( 153.94)62.5 ( 168.01)91h , s2 ( 14.58)62.5 ( 153.94)31.2 ( 83.87)3614h , r , e , s , et1 ( 7.29)31.2 ( 76.85)31.2 ( 83.87)4250h , r2 ( 14.58)31.2 ( 76.85)31.2 ( 83.87)73ah , r , z4 ( 29.17)31.2 ( 76.85)31.2 ( 83.87)17h2 ( 14.58)62.5 ( 153.94)31.2 ( 83.87)40h , r2 ( 14.58)62.5 ( 153.94)31.2 ( 83.87)results are expressed as mean of experiments performed in triplicate .
e : 
 ethambutol ; et : ethionamide ; h : isoniazid ; mic : minimal inhibitory 
 concentration ; r : rifampicin ; s : streptomycin ; z : pyrazinamide ; ( 1 ) : ( - ) 
 mammea a / bb ; ( 2 ) : ( - ) mammea b / bb . 
 
e : 
 ethambutol ; et : ethionamide ; h : isoniazid ; mic : minimal inhibitory 
 concentration ; r : rifampicin ; s : streptomycin ; z : pyrazinamide ; ( 1 ) : ( - ) 
 mammea a / bb ; ( 2 ) : ( - ) mammea b / bb .
the cytotoxicity assays , which were used to evaluate the safety of these compounds for 
 mammalian cells , were performed only with the dichloromethane extract and pure compounds 
 ( 1 ) and ( 2 ) , which showed antimycobacterial activity .
the dichloromethane extract and 
 the pure compounds showed si ranges from 0.59 - 1.06 ( table i ) .
in the present study , we evaluated the antimycobacterial activity of dichloromethane and 
 aqueous extracts from the leaves of c. brasiliense and pure compounds ( 1 ) , ( 2 ) and ( 3 ) 
 isolated from the dichloromethane extract . no relevant difference in mic values between the dichloromethane and aqueous extracts 

the similarity in the mic 
 values for the nonpolar and polar extracts may be attributed to the coumarin contents in 
 the aqueous extract , which was demonstrated by hplc - uv , albeit at lower concentrations 
 than that in dichloromethane extracts .
the mic values of coumarins ( 1 ) and ( 2 ) isolated from the dichloromethane extract 
 encourage us to consider these compounds as promising candidates for anti - tb drugs . 

compound ( 3 ) showed a low activity against m. tuberculosis , which is in agreement with a 
 previous study .
( 2001 ) , 
 working with natural biflavonoids isolated from the seed kernels of rhus succedanea and 
 garcinia multiflora , which included compound ( 3 ) , observed no activity against m. 
 tuberculosis h37rv .
although compounds ( 1 ) and ( 2 ) showed mics higher than those of the reference drug 
 isoniazid ( mic 0.01 - 0.2 g / ml ) , these compounds are comparable to the mics of other 
 first - line anti - tb drugs , such as pyrazinamide ( mic 20 - 100 g / ml at ph = 5.5 or 6.0 ) 
 ( inderlied & salfinger 1999 ) . as observed in this study , the mic values for the resistant isolates , of which five were 
 mdr , were equivalent to those of the sensitive isolates .
this encourages us to consider 
 these compounds as potential candidates for the development of new anti - tb drugs that 
 act against resistant bacilli .
a drug that can target these bacilli is undoubtedly 
 desirable because this severe form of tb has been increasing worldwide .
the si is used to estimate the therapeutic window of a drug and to identify drug 
 candidates for further studies . according to orme 
 ( 2001 ) ,
( 2012 ) , a candidate as a new 
 drug must have an si  10 .
the extract and pure compounds studied here exhibited lower 
 sis , indicating low therapeutic selectivity .
however , these compounds deserve attention 
 and , in the future , a microparticles study of these compounds , as well as the antitumour 
 activity will be conducted . here
we report the activity of extracts and coumarins from the leaves of c. brasiliense 
 against m. tuberculosis .
the results of this study provide a strong basis for the 
 selection of new anti - tb drugs .
additional studies are required to increase the 
 effectiveness of coumarins against m. tuberculosis and to decrease their toxicity to a 
 host .
 we evaluated the in vitro anti - mycobacterium tuberculosis activity and the 
 cytotoxicity of dichloromethane extract and pure compounds from the leaves of 
 calophyllum brasiliense . 
 purification of the dichloromethane extract yielded the pure 
 compounds ( - ) mammea a / bb ( 1 ) , ( - ) mammea b / bb ( 2 ) and amentoflavone ( 3 ) . 
 the 
 compound structures were elucidated on the basis of spectroscopic and spectrometric 
 data . 
 the contents of bioactive compounds in the extracts were quantified using high 
 performance liquid chromatography coupled to an ultraviolet detector . 
 the anti - m . 
 
 tuberculosis activity of the extracts and the pure compounds was evaluated using a 
 resazurin microtitre assay plate . 
 the cytotoxicity assay was performed in j774g.8 
 macrophages using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide 
 colourimetric method . 
 the quantification of the dichloromethane extract showed ( 1 ) 
 and ( 2 ) at concentrations of 31.86  2.6 and 8.24  1.1 g / mg of extract , 
 respectively . 
 the dichloromethane and aqueous extracts showed anti - m . 
 tuberculosis 
 h37rv activity of 62.5 and 125 g / ml , respectively . 
 coumarins ( 1 ) and ( 2 ) showed 
 minimal inhibitory concentration ranges of 31.2 and 62.5 g / ml against m. 
 tuberculosis h37rv and clinical isolates . 
 compound ( 3 ) showed no activity against m. 
 tuberculosis h37rv . 
 the selectivity index ranged from 0.59 - 1.06 . 
 we report the 
 activity of the extracts and coumarins from the leaves of c. brasiliense against m. 
 tuberculosis . 
MATERIALS AND METHODS
RESULTS
DISCUSSION