kitaibelii ( winter savory ) were collected in the period may - june , 2005 , in the region of mountain zlatibor , serbia , as labelled .
the voucher specimen of the collected plant material was confirmed by biljana boin and deposited at the herbarium of the department of pharmacognosy , faculty of medicine , university of novi sad .
all chemicals and reagents were of analytical reagent grade and were purchased from sigma chemical co. ( st louis , mq , usa ) , aldrich chemical co. ( steineheim , germany ) and " zorka " ( abac , serbia ) .
. " vital " , oil and vegetable fat factory ( vrbas , serbia ) .
commercial preparation of liposomes ( pro - lipo s , with 30% soybean phosphatidylcholine ) was obtained from lucas - meyer , hamburg , germany . the dried aerial parts ( moisture content 3.72% ) of winter savory were ground in a grinder ( dlfu bhler miag laboratory disc mill , germany ) with a 2 mm in diameter mesh .
this material ( 100 g ) was macerated with 70 % methanol in water ( 2  2500 ml ) at room temperature for 2  24 h. the obtained extract was concentrated under reduced pressure to remove methanol , and then extracted sequentially with petroleum ether ( 2  200 ml ) , chloroform ( 2  200 ml ) , ethyl acetate ( 2  200 ml ) and n - butanol ( 2  200 ml ) .
the petroleum ether , chloroform , ethyl acetate , n - butanol and remaining water extract were evaporated to dryness under reduced pressure at 40c with a water bath .
the yields , average of triplicate analysis , of the extracts were : petroleum ether , m = 0.580.029 g ; chloroform , m=0.490.024 g ; ethyl acetate , m=0.550.028 g ; n - butanol , m=2.130.106 g and water , m=6.030.30 g. hydroxyl radicals were obtained during a fenton reaction by mixing 200 l 0.3 m 5,5-dimethyl-1- pyroline - n - oxide ( dmpo ) , 200 l 10 mm h2o2 , 200 l 10 mm fe and 10 l of methanol ( blank ) .
the influence of different types of extract on the formation and transformation of hydroxyl radicals was investigated by adding the petroleum ether , chloroform , ethyl acetate , n - butanol and water extracts in the fenton reaction system at the range of concentrations 0.10.5 mg / ml ( probe ) .
the esr spectra were recorded on a bruker 300e , after 5 min , with the following spectrometer settings : field modulation 100 khz , modulation amplitude 0.512 g , receiver gain 210 , time constant 81.92 ms , conversion time 163.84 ms , centre field 3440.00 g , sweep width 100.00 g , x - band frequency 9.64 ghz , power 20 mw , temperature 23c .
the antioxidant activity ( aaoh ) of the extracts was defined as : aaoh = 100  ( ho - hx)/ho [ % ] where ho and hx are the height of the second peak in the esr spectrum of the dmpo - oh spin adduct of the blank and probe , respectively .
oxidation was investigated in a solution containing 1 ml of sunflower oil , 0.0018 g acva , 0.0274 g of -phenyl - n - tert - butylnitrone ( pbn ) and 380 l of various concentrations of methanolic solutions of the investigated extracts ( final concentrations were in range 15 mg / ml ) .
liposome suspension was prepared by adding 1 g of commercial liposomes to 10 ml water and incubated with stirring at 37c for 1h .
oxidation was investigated in the solution containing 1 ml of liposome suspension , 0.0019 g aaph , 0.0274 g of pbn and 380 l of various concentrations of methanolic solutions of the investigated extracts ( final concentrations were in the range 15 mg / ml ) .
lipid oxidation in both systems was measured by following the formation of peroxyl radicals with esr spin trapping method .
the esr spectra of the formed pbn - peroxyl radical spin adducts were recorded with the following spectrometer settings : field modulation 100 khz , modulation amplitude 1.021 g , receiver gain 510 , time constant 20.48 ms , conversion time 327.68 ms , centre field 3440.00 g , sweep width 100.00 g , x - band frequency 9.64 ghz , power 20 mw , temperature 23c .
the antioxidant activity value ( aaloo ) of the extract was defined as : aaloo=100(ho - hx)/ho [ % ] , where ho and hx are the height of the second peak in the esr spectrum of the pbn - ool spin adduct obtained in the absence and in presence of the investigated extracts , respectively .
splitting constants were calculated from computer - generated second derivatives of the spectra , after optimizing signal - to - noise ratios , and were verified by computer simulations . for these investigations
the disc diffusion method and microbroth dilution methods were applied . from the primary isolation medium 23 colonies of investigated bacteria
were taken by flamed loop , suspended in mueller - hinton or sabouraud broth , and they were incubated at 37c . the suspension for inoculation
the number of cells in 1 ml of suspension for inoculation measured by the mcfarland nephelometer was 110 cfu / ml .
a volume of 1 ml of this suspension was homogenized with 9 ml of melted ( 45c ) mueller - hinton or sabouraud dextrose agar and poured into petri dishes . for screening ,
sterile 6 mm discs ( himedia , mumbai , india ) were impregnated with 10 l of 100 mg / ml of satureja montana l. subsp .
after incubation for 2448 hours in the thermostat , inhibition zone diameters , zi , ( including disc ) were measured and expressed in mm .
the presence of the inhibition zone indicates the activity of tested extracts against bacteria or yeasts .
the minimum inhibitory concentration ( mic ) was reported as the lowest concentration of the extracts capable of inhibiting the growth of the bacterium tested .
the mic was determined by the broth macrodilution test   using an inoculum of 110 cfu / ml .
final concentrations of the investigated extracts were 6.25 , 12.5 , 25 , 50 , 75 and 100 mg / ml . both tests were done in three replications .
pseudomonas aeruginosa ( atcc 27853 , gram negative ) , escherichia coli ( atcc 25922 , gram negative ) , staphylococcus aureus ( atcc 25923 , gram positive ) , sarcina lutea ( atcc 9341 , gram positive ) , bacillus cereus ( atcc 10707 , gram positive ) , saccharomyces cerevisiae ( 112 , hefebank weihenstephan ) and candida pseudotropicalis ( clinical isolate ) microorganism strains were employed for the determination of antimicrobial activity . penicillin ( 10 iu / disc ) and amoxicillin ( 25 g / disc ) were used as reference standards as obtained from bioanalyse co. , ltd . ,
ankara , turkey . in parallel with the antibacterial investigation of satureja montana l. subsp .
kitaibelii extracts , pure solvent was tested too , and it did not exhibit any antibacterial activity ( data are not shown ) .
bacteria were obtained from the stock cultures of the microbiology laboratory , faculty of technology , university of novi sad .
all measurements were carried out in triplicate , and presented as mean  sme or  sd .
regression analyses and significance of differences were carried out using a spss statistical software package ( spss for windows , 8.0 , 1997 , spss inc . , chicago , il , usa ) .
kitaibelii ( winter savory ) were collected in the period may - june , 2005 , in the region of mountain zlatibor , serbia , as labelled .
the voucher specimen of the collected plant material was confirmed by biljana boin and deposited at the herbarium of the department of pharmacognosy , faculty of medicine , university of novi sad .
all chemicals and reagents were of analytical reagent grade and were purchased from sigma chemical co. ( st louis , mq , usa ) , aldrich chemical co. ( steineheim , germany ) and " zorka " ( abac , serbia ) .
. " vital " , oil and vegetable fat factory ( vrbas , serbia ) .
commercial preparation of liposomes ( pro - lipo s , with 30% soybean phosphatidylcholine ) was obtained from lucas - meyer , hamburg , germany .
the dried aerial parts ( moisture content 3.72% ) of winter savory were ground in a grinder ( dlfu bhler miag laboratory disc mill , germany ) with a 2 mm in diameter mesh .
this material ( 100 g ) was macerated with 70 % methanol in water ( 2  2500 ml ) at room temperature for 2  24 h. the obtained extract was concentrated under reduced pressure to remove methanol , and then extracted sequentially with petroleum ether ( 2  200 ml ) , chloroform ( 2  200 ml ) , ethyl acetate ( 2  200 ml ) and n - butanol ( 2  200 ml ) . the solvent extractions were performed in separation funnels , shaking for 10 min .
the petroleum ether , chloroform , ethyl acetate , n - butanol and remaining water extract were evaporated to dryness under reduced pressure at 40c with a water bath .
the yields , average of triplicate analysis , of the extracts were : petroleum ether , m = 0.580.029 g ; chloroform , m=0.490.024 g ; ethyl acetate , m=0.550.028 g ; n - butanol , m=2.130.106 g and water , m=6.030.30 g.
hydroxyl radicals were obtained during a fenton reaction by mixing 200 l 0.3 m 5,5-dimethyl-1- pyroline - n - oxide ( dmpo ) , 200 l 10 mm h2o2 , 200 l 10 mm fe and 10 l of methanol ( blank ) .
the influence of different types of extract on the formation and transformation of hydroxyl radicals was investigated by adding the petroleum ether , chloroform , ethyl acetate , n - butanol and water extracts in the fenton reaction system at the range of concentrations 0.10.5 mg / ml ( probe ) .
the esr spectra were recorded on a bruker 300e , after 5 min , with the following spectrometer settings : field modulation 100 khz , modulation amplitude 0.512 g , receiver gain 210 , time constant 81.92 ms , conversion time 163.84 ms , centre field 3440.00 g , sweep width 100.00 g , x - band frequency 9.64 ghz , power 20 mw , temperature 23c .
the antioxidant activity ( aaoh ) of the extracts was defined as : aaoh = 100  ( ho - hx)/ho [ % ] where ho and hx are the height of the second peak in the esr spectrum of the dmpo - oh spin adduct of the blank and probe , respectively .
oxidation was investigated in a solution containing 1 ml of sunflower oil , 0.0018 g acva , 0.0274 g of -phenyl - n - tert - butylnitrone ( pbn ) and 380 l of various concentrations of methanolic solutions of the investigated extracts ( final concentrations were in range 15 mg / ml ) .
liposome suspension was prepared by adding 1 g of commercial liposomes to 10 ml water and incubated with stirring at 37c for 1h .
oxidation was investigated in the solution containing 1 ml of liposome suspension , 0.0019 g aaph , 0.0274 g of pbn and 380 l of various concentrations of methanolic solutions of the investigated extracts ( final concentrations were in the range 15 mg / ml ) .
lipid oxidation in both systems was measured by following the formation of peroxyl radicals with esr spin trapping method .
the esr spectra of the formed pbn - peroxyl radical spin adducts were recorded with the following spectrometer settings : field modulation 100 khz , modulation amplitude 1.021 g , receiver gain 510 , time constant 20.48 ms , conversion time 327.68 ms , centre field 3440.00 g , sweep width 100.00 g , x - band frequency 9.64 ghz , power 20 mw , temperature 23c .
the antioxidant activity value ( aaloo ) of the extract was defined as : aaloo=100(ho - hx)/ho [ % ] , where ho and hx are the height of the second peak in the esr spectrum of the pbn - ool spin adduct obtained in the absence and in presence of the investigated extracts , respectively .
splitting constants were calculated from computer - generated second derivatives of the spectra , after optimizing signal - to - noise ratios , and were verified by computer simulations .
for these investigations the disc diffusion method and microbroth dilution methods were applied . from the primary isolation medium 23 colonies of investigated bacteria
were taken by flamed loop , suspended in mueller - hinton or sabouraud broth , and they were incubated at 37c . the suspension for inoculation
the number of cells in 1 ml of suspension for inoculation measured by the mcfarland nephelometer was 110 cfu / ml .
a volume of 1 ml of this suspension was homogenized with 9 ml of melted ( 45c ) mueller - hinton or sabouraud dextrose agar and poured into petri dishes . for screening ,
sterile 6 mm discs ( himedia , mumbai , india ) were impregnated with 10 l of 100 mg / ml of satureja montana l. subsp .
after incubation for 2448 hours in the thermostat , inhibition zone diameters , zi , ( including disc ) were measured and expressed in mm .
the presence of the inhibition zone indicates the activity of tested extracts against bacteria or yeasts .
the minimum inhibitory concentration ( mic ) was reported as the lowest concentration of the extracts capable of inhibiting the growth of the bacterium tested .
the mic was determined by the broth macrodilution test   using an inoculum of 110 cfu / ml .
final concentrations of the investigated extracts were 6.25 , 12.5 , 25 , 50 , 75 and 100 mg / ml .
pseudomonas aeruginosa ( atcc 27853 , gram negative ) , escherichia coli ( atcc 25922 , gram negative ) , staphylococcus aureus ( atcc 25923 , gram positive ) , sarcina lutea ( atcc 9341 , gram positive ) , bacillus cereus ( atcc 10707 , gram positive ) , saccharomyces cerevisiae ( 112 , hefebank weihenstephan ) and candida pseudotropicalis ( clinical isolate ) microorganism strains were employed for the determination of antimicrobial activity . penicillin ( 10 iu / disc ) and amoxicillin ( 25 g / disc ) were used as reference standards as obtained from bioanalyse co. , ltd . , ankara , turkey . in parallel with the antibacterial investigation of satureja montana l. subsp .
kitaibelii extracts , pure solvent was tested too , and it did not exhibit any antibacterial activity ( data are not shown ) .
bacteria were obtained from the stock cultures of the microbiology laboratory , faculty of technology , university of novi sad .
all measurements were carried out in triplicate , and presented as mean  sme or  sd . regression analyses and significance of differences
were carried out using a spss statistical software package ( spss for windows , 8.0 , 1997 , spss inc . ,
 the antioxidant activity of different satureja montana l. subsp . 
 kitaibelii extracts was tested by measuring their ability to scavenge reactive hydroxyl radical during the fenton reaction , using esr spectroscopy . also , the influence of these extracts on lipid peroxyl radicals obtained during lipid peroxidation of : ( i ) sunflower oil ( 37 c , 3h ) induced by 4,4-azobis(4-cyanovaleric acid ) ( acva ) and ( ii ) liposomes induced by 2,2-azobis(2- amidino - propane)dihydrochloride ( aaph ) was studied . 
 n - butanol extract had the best antioxidant activity ( 100% at 0.5 mg / ml in fenton reaction system ; 89.21% at 5 mg / ml in system i ; 83.38% at 5 mg / ml in system ii ) . 
 the antioxidant activities of the extracts significantly correlated with total phenolic content . 
 the antimicrobial activity of satureja montana l. subsp . 
 kitaibelii extracts was investigated . 
 petroleum ether , chloroform and ethyl acetate extracts expressed a wide range of inhibiting activity against both grampositive and gram - negative bacteria . 
Experimental Section
Plant material and chemicals
Preparation of Extracts
Hydroxyl radical assay
Oxidation of Sunflower Oil Induced by Azo-initiator ACVA
Oxidation of the PC Liposomes Induced by Azo-initiator AAPH
Antimicrobial activity test
Statistical analysis