propolis ( bee glue ) has a long history of being used as a remedy , dating back to the times of ancient greece and rome .
nowadays , it is still used for the treatment of various diseases , and in products like 
health foods ,  biocosmetics , etc . , because of its versatile biological activities ( 1 ) .
tropical propolis samples , and especially brazilian ones , have shown significant differences in their chemical composition to propolis from temperate zone ( 2,3 ) .
for this reason , brazilian bee glue has recently become a subject of increasing interest for scientists ( 47 ) .
it was found that propolis from different regions of brazil display different chemical composition , depending on the local flora at the site of collection .
( 8) have specified 12 types of brazilian propolis according to its geographical origin , chemical composition and source plant .
the most popular and well studied brazilian propolis is the so - called green or alecrim propolis , which originates from baccharis dracunculifolia ( asteraceae ) ( 912 ) .
till now , no chemical data have been published on red propolis from brazil . in brazil ,
red colored propolis is reported to be typical for cuba , where its plant source was identified as clusia nemorosa ( clusiaceae ) ( 13 ) , and for venezuela , where bees collect it from clusia scrobiculata ( 14 ) . in this study
, we report our results on antibacterial and antioxidant activity of chemical constituents of red brazilian propolis .
nuclear magnetic resonance ( nmr ) spectra were measured on a bruker avance 250 mnr spectrometer ; mass - spectra were measured on a hewlett packard 5972 mass spectrometer system .
propolis ( 61 g ) was cut into small pieces and extracted with 70% ethanol ( 1 : 10 , w / v ) at room temperature for 24 h. the ethanol extract was concentrated in vacuo and extracted successively with petrol ether ( 4060c ) three times .
the petrol ether extract was subjected to column chromatography on silica gel ( 300 g ) with an n - hexane / acetone gradient ( 1 : 0.05/1 : 0.4 ) to produce 20 fractions ( i  xx ) .
fraction i ( 300 mg ) was rechromatographed on a silica gel column eluted with n - hexane / diethyl ether gradient ( 1 : 0.01/1 : 1 ) . the first fraction of this column , i.1 ( 12 mg ) ,
was subjected to gas chromatography - mass spectrometry ( gc - ms ) analysis with a hewlett packard gas chromatograph 5890 series ii plus linked to hewlett packard 5972 mass spectrometer system equipped with a 23 m long , 0.25 mm i d , 0.5 m film thickness hp5-ms capillary column .
helium was used as a carrier gas , flow rate 0.7 ml min , split ratio 1 : 80 , injector temperature 280c , ionization voltage 70 ev . using computer searches on a nist98 ms data library ,
the following compounds were identified in the mixture : trans - anethol 1 ( 13% ) , methyl eugenol 2 ( 14% ) , trans - methyl isoeugenol 3 ( 18% ) , elemicin 4 ( 26% ) and trans - isoelemicin 5 ( 11% ) . the second fraction i.2 ( 37 mg ) , after additional separation by preparative thin layer chromatography ( tlc ) ( silica gel , n - hexane / ethyl methyl ketone 1 : 0.06 ) yielded 20(29)-lupen-3-one 6 ( 6 mg ) and 2,3-epoxy-2-(3-methyl-2-butenyl)-1,4-naphthalenedione 7 ( 8.6 mg ) .
fraction ii ( 300 mg ) was rechromatographed on a silica gel column eluted with n - hexane / diethyl ether gradient ( 1 : 0.01/1 : 1 ) . after further purification by preparative tlc ( silica gel , n - hexane / ethyl methyl ketone 1 : 0.06 ) , a mixture of triterpenic alcohols ( 36 mg ) ( h - nmr ) was obtained .
this mixture was analyzed after silylation , using the above mentioned gc - ms apparatus and the same analysis conditions as with fraction i.1 . using computer searches on a nist98 ms data library , -amyrin 8 ,
-amyrin 9 ( identity also confirmed by comparison with an authentic sample ) , cycloartenol 10 and lupeol 11 were identified .
fraction viii ( 241 mg ) was rechromatographed on a silica gel column , mobile phase n - hexane / acetone gradient ( 1 : 0.05/1 : 0.8 ) .
additional purification by preparative tlc ( silica gel , toluene / acetone 1 : 0.2 ) yielded isosativan 12 ( 40.8 mg ) .
fraction x ( 454 mg ) was rechromatographed on a silica gel column with mobile phase n - hexane / acetone gradient ( 1 : 0.05/1 : 1 ) .
further purification by preparative tlc ( n - hexane / ethyl methyl ketone 1 : 0.1 ) led to the isolation of 11.8 mg medicarpin 13 .
fraction xiii ( 620 mg ) was rechromatographed on a silica gel column with mobile phase n - hexane / acetone gradient ( n - hexane / acetone 1 : 0.01/1 : 1 ) to yield 20.5 mg of an inseparable mixture of guttiferone e 14 and xanthochymol 15 .
20(29)-lupen-3-one 6 was identified based on comparison of its eims , h- and c - nmr spectra and optical rotation with literature data ( 15 ) .
2,3-epoxy-2-(3-methyl-2-butenyl)-1,4-naphthalenedione 7 , colorless oil , [ ]d 0 ( c 0.2 , acetone ) .
ms ( ei , 70 ev ) , m / z ( rel . int .
m. ( 28 ) , 227 ( m-15 ) ( 85 ) , 213 ( m-29)(100 ) , 196 ( 64 ) , 171 ( 81 ) , 105 ( 35% ) , 89 ( 36 ) , 69 ( 30 ) . hrms ( ei ) m / z : 242.09553 ( calc . for c15h14o3 :
uv , eims , h- and c - nmr spectra identical with literature data ( 16 ) , [ ]d 0 ( c 0.27 , chloroform ) .
uv , eims , h- and c - nmr spectra identical with literature data ( 17 ) , [ ]d + 184 ( c 0.51 , acetone ) .
the components of this inseparable mixture were identified by comparison of the spectral data for the same mixture published by gustafson et al . ( 18 ) h- and c - nmr , ms ( fig .
1 ) . antimicrobial tests . for the investigation of the antibacterial and antifungal activity , the agar cup method ( 19 ) was used with test strains staphylococcus aureus 209 ( obtained from the bulgarian type culture collection , institute for state control of drugs , sofia ) , escherichia coli wf+ ( obtained from the collection of zimet , central institute of microbiology and experimental therapy , jena , germany ) and candida albicans 562 ( obtained from the bulgarian type culture collection , institute for state control of drugs , sofia ) .
an inhibitory zone with a diameter < 10 mm corresponds to lack of activity ( 10 mm is the diameter of the cup ) .
the test solution ( 0.1 ml ) containing 0.4 mg of each substance in ethanol was applied to every cup ( concentration of the test solution 4 mg ml ) .
dpph free radical scavenging activity was measured according to the procedure described in the literature ( 20 ) .
the decrease of the absorption at 516 nm of the dpph solution after addition of the tested solution was measured .
an aliquot ( 2960 l ) of 0.1 mm ethanolic dpph solution was mixed with 40 l of a 3.6 mm solution of the tested substance .
the petrol ether fraction of the ethanol extract of the investigated propolis sample was subjected to column chromatography on silica gel and several fractions were produced . after further purification by repeated column chromatography and preparative tlc , two complex mixtures , one inseparable mixture of two isomers and four pure compounds were obtained .
the most unpolar fraction , isolated by repeated column chromatography , was of complex composition and was analyzed by gc - ms .
it turned out to be composed of following phenylpropene derivatives : trans - anethol 1 , methyl eugenol 2 , trans - methyl isoeugenol 3 , elemicin 4 and trans - isoelemicin 5 .
of these compounds , methyl eugenol , methyl isoeugenol , elemicin and isoelemicin were found for the first time in propolis .
the composition of this fraction also explains the very unusual anis - like odor of this brazilian red propolis sample .
the second complex mixture ( see materials and methods ) was comprised of triterpenic alcohols , which were identified by means of gc - ms as -amyrin 8 , -amyrin 9 , cycloartenol 10 and lupeol 11 .
one of the pure compounds isolated was also of triterpenic nature : the ketone 20(29)-lupen-3-one 6 [ identified by comparison of spectral information with literature data ( 13 ) ] , found for the first time in propolis .
this compound has recently been found to possess antibiotic activity against bacteria and fungi , and antioxidant activity similar to that of tocopherol ( 15 ) .
its structure was determined as 2,3-epoxy-2-(3-methyl-2-butenyl)-1,4-naphthalenedione on the basis of its ms , infrared , h- and c - nmr spectra .
the mass- and h - nmr spectra of our compound were identical with the literature data ( no c - nmr data have been published ) .
compound 7 , obtained synthetically from a natural product , demonstrated antibacterial , antifungal and cytotoxic properties ( 22 ) .
two isoflavonoids were isolated and identified : the isoflavan isosativan 12 and the pterocarpan medicarpin 13 , based on comparison of their spectral properties with literature data ( including absolute stereochemistry of 13 , confirmed by optical rotation measurements ; 12 was racemic ) .
compounds 12 and 13 were till now found only in cuban propolis ( 16,17 ) .
this fact suggests that cuban red propolis and brazilian red propolis might have a common plant source , but a plant that produces isoflavonoids as components of its exudates is not yet known .
the presence of isoflavonoids suggests some plant of the leguminosae family but further studies are needed for confirmation .
especially 13 is of particular interest : it is an important plant phytoalexin well known for its antimicrobial and especially antifungal activity ( 23 ) .
compounds 14 and 15 are double bond isomers which occur as an inseparable mixture , but the structures were deduced by comparison of the spectral data of the mixture with the values for 14 and 15 from the literature ( 2225 ) .
h- and c - nmr spectra of the mixture were virtually identical to those of 14 and 15 and all hmbc correlations were fully consistent with these structures . moreover , gustafson et al . ( 18 ) reported the isolation of the same inseparable mixture from clusia rosea leaves as the active anti - hiv principle .
these compounds have been detected as traces in red cuban propolis ( 13 ) originating from c. rosea floral resins . in cuban propolis nemorosone , another polyisoprenylated benzophenone ,
is the most important constituent ( 13 ) . in our case , however , the mixture of 14 and 15 is among the major components of the extract .
moreover , the presence of isoflavonoids in our sample is an indication that another plant source could be involved , as isoflavonoids have never been found in the resins of clusiaceae plants .
three of the isolated compounds were tested for their antibacterial and radical scavenging activity against dpph radicals .
the results indicated that the isoflavonoids 12 and 13 are important antimicrobial components of red propolis , especially concerning the activity against c. albicans .
this is not surprising , taking into consideration that pterocarpans are known for their antifungal activity and play a defensive role in many plants due to this activity ( 26 ) .
the mixture showed also significant radical scavenging activity against dpph , obviously it is one of the most important antioxidant components of the extract .
the identification of new propolis constituents in red brazilian propolis , most of them having antibacterial , antimycotic and antiradical activities , is a further confirmation of the fact that propolis , independently of its plant source and chemical composition , always possesses antimicrobial and antioxidant activity .
this is due to the role that propolis plays in the hive : it is the  chemical weapon of bees against pathogen microorganisms and the elements of weather .
however , in different propolis types , different chemical constituents are responsible for the valuable activities ( 27 ) .
the results obtained demonstrate once again that propolis remains a fascinating subject for further studies and application to cam .
nmr data of 7 in cdcl3 ,  in ppm ( j in hz ) hmbc , heteronuclear multiple bond correlation . antimicrobial and antiradical activity of isolated compounds mean of three measurements .
 in a new propolis type , red brazilian propolis , 14 compounds were identified ( six of them new for propolis ) , among them simple phenolics , triterepenoids , isoflavonoids , prenylated benzophenones and a naphthoquinone epoxide ( isolated for the first time from a natural source ) . 
 three of the major components demonstrated significant antimicrobial activity , and two ( obtained as inseparable mixture ) possessed radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl ( dpph ) . 
Introduction
Materials and Methods
Results and Discussion
Figures and Tables