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Antioxidant potential of Achillea fraasii and 
A. ageratifolia ssp. serbica (Asteraceae)
Nada Spajic
1
, Jelena Kukic
1
, Silvana Petrovic
1
, Marjan Niketic
2
, Mira Stojanovic
3
1
Institute of Pharmacognosy, Faculty of Pharmacy, Vojvode Stepe 450, 11221 Belgrade, Serbia
2
Natural History Museum, Njegoševa 51, 11000 Belgrade, Serbia
3
Pharmacy Belgrade, Bojanska 16/IV, 10000 Belgrade, Serbia
Achillea fraasii Schultz Bip. and A. ageratifolia (Sm.) Benth & Hook. fil. ssp. serbica. (Nyman) Heimerl.
(Asteraceae) are endemics which grow in the regions of Albania, Greece, Serbia and Montenegro. They are sericeous
tomentose aromatic perennial plants up to 30(40) cm height, with conspicuous white ligulas. A. ageratifolia ssp. ser-
bica have spathulate-lanceolate leaves with entire to crenulate midrib. A. fraasii have 2-pinnatifid leaves with lin-
ear-lanceolate lobes.
Data on chemical composition of these taxa are scarce. Essential oils isolated from aerial parts of these plants
are characterised with high amount of monoterpenes, with 1,8-cineole and camphor dominating. A. fraasii essential
oil exhibited substantial antimicrobial activity against Gram negative bacteria and pathogenic fungi. Surface flavonoid
aglicones were studied in both taxa. In A. fraasii main were methyl derivatives of 6-hydroxy kaempferol and quercetin,
while in A. ageratifolia ssp. serbica methyl derivatives of 6-hydroxy scutellarein and luteolin prevailed.
In this work antioxidant activity of these plants was investigated. Aerial parts of A. fraasii were collected on
mountain Jablanica (Macedonia) in July 2006 and of A. ageratifolia ssp. serbica on mountain Suva planina (Serbia)
in July 2006. Air dried and grounded plant material was than successively extracted with CHCl
3
and then with MeOH.
Antioxidant potential of dry MeOH extracts was determined using several assays: FRAP test was used to determine
total antioxidant activity (TAA) of samples; free radical scavenging activity was estimated trough neutralisation of
stable DPPH radical and inhibition of 2-deoxyribose degradation (scavenging of OH radical). In addition, total and
non-tannin phenolics content in both samples were determined using Folin-Cocalteu reagent and expressed as 
µg of
gallic acid (GA)/mg of dry extract.
Results showed that MeOH extracts of A. fraasii and A. ageratifolia ssp. serbica possess substantial antiox-
idant activity. Both extracts strongly and concentration-dependently scavenged DPPH radical (SC50 43.70 and 50.14
µg/ml, respectively). Inhibition of 2-deoxyribose degradation (scavenging of OH radical) was much more pronounced
with IC
50
values at 12.50 and 40.50 
µg/ml, respectively. Total antioxidant activity was 1.21 and 1.12 µmol Fe
2+
/mg,
respectively. Total and non-tannin phenolics content were 93.50 and 43.92 
µg GA/mg of dry A. faasii extract, respec-
tively, and 87.41 and 33.47 
µg GA/mg of dry A. ageratifolia ssp. serbica extract, respectively.
In all experiments MeOH extract of A. fraasii showed higher antioxidant potential than the appropriate extract
of A. ageratifolia ssp. serbica. This could be explained by higher total and especially non-tannin phenolics content
in the A. fraasii extract.
Macedonian pharmaceutical bulletin 53 (1,2) 309 (2007)
PP - 151
309
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Antimicrobial and bacterial antiadhesive activity 
of Vaccinium vitis-idaea
Simic M.
1
, Vucicevic D.
2
, Milenkovic M.
2
, Miajlovic N.
3
, Savic M.
3
, Kovacevic N.
1
1
Institute of Pharmacognosy, Faculty of Pharmacy, University of Belgrade, V. Stepe 450, 11221 Belgrade, Serbia 
2
Institute of Microbiology and Immunology, Faculty of Pharmacy, V. Stepe 450, 11221 Belgrade, Serbia
3
Institute of Agricultural Economics, Volgina 15, 11000 Belgrade, Serbia
The aim of this work was to investigate antimicrobial activity and antiadhesive properties of juice and extracts
of alpine cranberry, Vaccinium vitis-idaea L. (Vacciniaceae) on the binding of bacteria Escherichia coli to epithelial cells.
Cranberry ingestion has long been associated with prevention of urinary tract infection. Cranberries (Vaccinium
macrocarpon) shows antiadherence properties that prevent bacteria Escherichia coli from adhering to uroepithelial
cells in the urinary tract. Adherence of uropathogens is the initial step in the development of urinary tract infections.
Serbian native species, Vaccinium vitis-idaea is also used for the treatment of urinary disorders in the traditional
medicine and the interest of our work was to evaluate its activity.
Fruits of alpine cranberry were collected on Stara planina, Serbia in August 2006. The content of sugar before
and after hydrolysis and the content of free and total acids were determinated titrimetrically. Quantity of total antho-
cyanins was measured spectrophotometrically. All measurements were done in the fruit and juice of alpine cranberry.
Investigation of antimicrobial activity was performed on expressed juice, diluted juice (1:3), fruit chloro-
form extract and fruit amyl alcohol extract. Antibacterial activities of juice and extracts were tested using agar-dif-
fusion method. Tested microorganism strains were Staphylococcus aureus (ATCC6538), Streptococcus pyogenes,
Bacillus subtilis (ATCC6633BB), Pseudomonas aeruginosa (ATCC27853), Escherichia coli (ATCC25922) and
Micrococcus luteus (ATCC10240). Results were expressed as diameters of inhibition zones (mm). Antiadhesive
activity of juice and extracts on the binding of bacteria Escherichia coli, was tested on human epithelial cells of buc-
cal mucosa. After incubation microscopic preparations were stained with gentiana violet and results are given as
number of adherent bacteria E. coli on epithelial cell. 
The juice contained 8.45% of sugar before hydrolysis, 11.76% of total sugar, 26.77 mmol/100 g of free acids,
33.29 mmol/ 100 g of total acids and 0.44 mg/g of anthocyanins. The content of sugar before hydrolysis and sugar
after hydrolysis was not remarkably different in the juice and fruit, while the fruit contained slightly smaller amount
of free and total acids and the higher content of anthocyanins (0.83 mg/g). 
Juice and extracts of Vaccinium vitis-idaea showed antimicrobial activity on the tested strains, except dilut-
ed juice, which showed no effect on the growth of bacteria B. subtillis and P. aeruginosa. The best antimicrobial
activity was obtained with amyl alcohol extract. Inhibition of bacterial growth of alpine cranberry juice and extracts
was in the following order: amyl alcohol extract > chloroform extract > juice > diluted juice.
Antiadhesive properties of the binding of bacteria Escherichia coli to epithelial cells were observed with
juice, diluted juice, chloroform and amyl alcohol extract in the comparison with the control. Undiluted juice and
chloroform extract showed the best prevention of adhering of E. coli to epithelial cells. 
Macedonian pharmaceutical bulletin 53 (1,2) 310 (2007)
PP - 152
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Evaluation of the antifungal and antioxidant activities of essential oils
of Pistacia lentiscus and Pistacia atlantica 
Nabila Benhammou
1
, Fawzia Atik Bekkara
1
,
Tatjana Kadifkova Panovska
2
1
Department of Biology, University Abou Bekr Belkaid  LP 119, Imama Tlemcen, Algeria. 
2
Department of Toxicology, Faculty of Pharmacy, Ss “Cyril and Methodius University” 1000 Skopje, R. Macedonia.
Had with the micro-organisms resistant to antibiotics on the one hand, and continuation of the increase in
the complications of certain cardiovascular, diabetic and inflammatory diseases of other hand, the search for a new
prototype drug to fight these infections is absolutely necessary. Several compounds of secondary metabolites (essen-
tial oils) offer an unlimited hope and a great potential with regard to this problem. For that, we were interested to
analyze and evaluate the antifungal and antioxidant activities of essential oils of Pistacia lentiscus and Pistacia
atlantica of both station of Ain Fezza and Oum El Alou of the area of Tlemcen.
These two plants occupy an appreciable place in traditional and pharmaceutical medicine because of its ther-
apeutic activities: antioxidant, anti-inflammatory drug, antidiabetic and antimicrobial.
The study of the antifungal activity reveals a considerable sensitivity according to strains tested. Concerning
the antioxidant capacity, it is expressed by the concentration corresponding to 50% of inhibition (IC
50
), it is equal
to 329.95 mg ml 
-1
. For P. lentiscus, it is impossible to measure the effect of essential oil and to calculate a concen-
tration IC
50
by the system used. 
Macedonian pharmaceutical bulletin 53 (1,2) 311 (2007)
PP - 153
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Anticoagulant effects of rutin and hesperidin chelates
Vesna Kuntic, Ivana Filipovic
Faculty of Pharmacy, University of Belgrade,Vojvode Stepe 450, Serbia
Institute of Oncology and Radiology of Serbia, Belgrade, Serbia
Flavonoids, a ubiquitous group of polyphenolic substances, are present in most plants, seeds, fruit skin, or
peel. They exhibit antibacterial, anti-inflammatory, antiallergic, antimutagenic, antiviral, antineoplastic, anti-throm-
botic, and vasodilatory actions. These pharmacological effects are related to the antioxidant activity of flavonoids,
arising from their ability to scavenge free radicals. Flavonoids also exert antioxidant activity through their interac-
tions with the metal’s ions, primarily Fe (II), Fe (III) and Cu (I), which participate in free radical generating reactions.
Moreover, some experimental data have shown that these chelates are considerably more effective free rad-
ical scavengers than the free flavonoids
1
. By using the 1,1-diphenyl-2-picrylhydrazyl radical scavenging method, de
Souza and de Giovani
2
found antioxidant activities of the quercetin, rutin, galangin, and catechin complexes more
effective then mature flavonoids. Afanas’ev et al 
3
found that Fe(II)- and Cu(II)- rutin complexes were more efficient
free radical scavengers in vitro and ex vivo.
In our previous study
4
, the anticoagulation effect on in vitro plasma coagulation of some flavonoids were
investigated. We also investigated the structure and stability of almost 40 flavonoids chelates with different metal
ions
5-6
. The aim of this work was to compare the effects of flavonoid-metal chelates with the effects of already inves-
tigated effects of mature flavonoids and correlate these effect with chelate structure. We investigated two chelates
of rutin and two chelates of hesperidin: Al (III)-rutin, Al (III)-hesperidin, Cu (II)-rutin and Cu (II)-hesperidin, which
were synthesized and characterized in a way described earlier. Measurements were performed on a coagulometer
ACL 7000 (Instrumental Laboratory). Activated partial thromboplastin time (aPTT), thrombin time (TT), and pro-
thrombin time were measured using APTT-SP (liquid), Thrombin Time (3IU), and PT-Recombinant reagents, which
were all made by Instrumental Laboratory. 
Our results demonstrate that effect of flavonoid-metal chelates strongly depend of chelate stability. Stable
chelates Hesp-Cu (log 
β=11.5) and Rut-Al (log β=10.9) significantly prolong aPTT compared to mature flavonoids,
what supports the hypothesis that chelates are more potent antioxidants then free flavonoids. But unstable chelates
Rut-Cu (log
β=4.7) and Hesp-Al (logβ=3.7) have no significant effects on in vitro plasma coagulation.
Macedonian pharmaceutical bulletin 53 (1,2) 312 (2007)
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Antimicrobial activity of Arnica montana L.
Stevic T., Bigovic D., Samardzic Z., Radanovic D.
Institute for Medicinal Plant Research “Dr Josif Pancic”, Tadeusa Koscuska 1, 11000 Belgrade, Serbia
Arnica montana flowers processed into tinctures, ointments, creams or gels are most commonly used topi-
cally to relieve the pain and inflammation of soft-tissue injuries as boils, bruises, hemorrhoids and sprains (1,2). Due
to possible antiseptic and antibacterial properties, topical arnica has also been used to treat acne, insect bites and
minor skin wounds. It may be effective, as well, in mouth rinses to control some bacteria commonly found in the
mouth. Folk remedies using arnica as a tea or tincture for wounds, bruises, reducing high cholesterol, rheumatic
pains, heart weakness and even asthma.
This study was performed to evaluate the antimicrobial activity of two Arnica Montana extracts, water-
ethanol and propilenglicol (PG). They were tested against Gram-negative (Escherichia coli, Salmonella typhimuri-
um, S.enteritidis, Enterobacter cloacae, Pseudomonas aeruginosa, P.tolaasii, Proteus mirabilis) and Gram-positive
(Staphylococcus aureus, S.epidermidis, Bacillus subtilis, Micrococcus luteus, M.flavus, Sarcina lutea, Listeria mono-
cytogenes) bacteria and human pathogen fungi Candida albicans by disk diffusion and micro-dilution methods.
Mesuring the inhibition zones of microbial growth we obtained greater inhibitig activity of water-alcoholic extract
than PG extract against all tested microorganisms, particulary agaist Gram-negative bacteria. MIC was between 117
and 357 µl/ml depending of taxonomic characteristic of bacteria. According to results from both applied methods,
Arnica montana extracts expressed significant antibacterial effect and may used as natural antibiotic agents.
Macedonian pharmaceutical bulletin 53 (1,2) 313 (2007)
PP - 155
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The regulatory status of medicinal and aromatic plants 
and their products in Federation Bosnia and Herzegovina (FB&H)
Tamara Bosnic, Dzenita Softic, Maida Brackovic, Samra Suvalija, Sasa Pilipovic
Institute for Quality Control of Medicines, Sarajevo, Titova 9, Bosnia & Herzegovina
The use of medicinal and aromatic plant in Bosnia & Herzegovina has long tradition. Many of them have a
traditional reputation for their uses. Herbal products are available through pharmacies, herbal shops and supermar-
kets. Medicinal herbal remedies should meet acceptable standards of quality, safety and efficacy. The safe use of
herbal medicines requires adequate regulation. Regulatory aspect of herbal medicines in different parts of the world
is different. The legal status of herbal products is unclear. The law for herbal medicines in FB&H is partly the same
as for conventional pharmaceuticals and partly is regulated as dietary supplements. There are difficulties in defin-
ing the status of products occupying the borderline between medicines and dietary supplements. European pharma-
copoeia is in use, followed by WHO monographs and the European Scientific Cooperative on phytotherapy mono-
graphs (ESCOP monographs). FB&H lack national monographs on herbal medicines and post-marketing surveillance
of herbal medicines.
Macedonian pharmaceutical bulletin 53 (1,2) 314 (2007)
PP - 156
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Quality control of the herbal drug Equiseti herba (Horsetail) 
from the market
Tamara Bosnic, Dzenita Softic, Diana Jerg-Simanovic, Sasa Pilipovic
Institute for Quality Control of Medicines, Sarajevo, Titova 9, Bosnia & Herzegovina
The drug consists of the dried, sterile green stems of the field horsetail (Equisetum arvense L.). Horsetail has
traditionally been used in Europe as an oral diuretic. The German Commission E expert panel has approved horsetail
for this indication. Horsetail is also occasionally used for osteoporosis, nephrolithiasis, urinary tract inflammation,
and wound healing. Equisetum arvense is mainly collected from the wild. It has the same distribution range as other
Equisetum species. Other related species are Equisetum hiemale L., Equisetum limosum Roth., Equisetum maximum L.
and Equisetum palustre L. Adulterations with Equisetum palustre L. and  Equisetum sylvaticum L. occur frequently.
Samples were picked up from retail pharmacies. The samples were tested according to the Ph.Eur. (1). Identity,
purity and assay were tested. Authentication were performed macro and microscopically and TLC identification test
on flavonoid profile.
Macedonian pharmaceutical bulletin 53 (1,2) 315 (2007)
PP - 157
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Soybean extract as antioxidant active and dietary supplement ingredient
Vesna Ceran, Jovan Popovic, Jelena Cvejic, Milica Atanackovic
Laboratory of Analysis of Natural and Pharmaceutical Products, Department of Pharmacy, 
Faculty of Medicine, University of Novi Sad, Serbia
Among natural products for dietary supplementation there is an increasing interest in the beneficial health
effects of soy derived isoflavones. With regard to the health promoting effects of these phytochemicals, the content
of polyphenols as antioxidant active natural substances may be important in supporting the overall physiological
health effects of isoflavone-rich plants. We screened twenty different soybean genotypes in order to determine their
antioxidant activity.
All cultivars were grown and harvested on experimental fields at the Institute of Field and Vegetable Crops,
Novi Sad. After extraction with methanol/water (8:2, v/v) the content of total polyphenols in soybeans was quanti-
fied and their radical scavenging ability were evaluated. The total polyphenols content was determined by the Folin-
Ciocalteu spectrofotometric method [1]. Radical scavenging activity was quantified by the DPPH (2,2-diphenyl-1-
picryl-hidrazyl) method and expressed in EC50 values, which represents the amount of antioxidant necessary to
decrease the initial DPPH concentration by 50 percent [2].
The highest total polyphenols content was 2.13 mg/g soy and the lowest one was 3.45 mg/g, while EC50
values of soybean extracts ranged from 3.44 to 1.40 mg/ml. There was a significant negative correlation between the
content of total polyphenols and EC50 values (P value < 0.05). The results obtained by t-test showed that there was
significant difference between different genotypes of soybean (P value < 0.05). 
Appropriate extracts and preparations of soybeans may be regarded as effective natural and functional dietary
supplements due to their content of polyphenolic substances and to their radical scavenging activity. Considering
significant differences among different soy genotypes, on the basis of the results of this work, some cultivars could
be recommended as potential food and supplement ingredients.
Macedonian pharmaceutical bulletin 53 (1,2) 316 (2007)
PP - 158
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Investigation on antioxidant activity of extracts 
of Alchemilla velebitica Borbas. (Rosaceae)
Rusica Kolakovic
1
, Bosko Bondzulic
1
, Jelena Kukic
1
, Silvana Petrovic
1
, Milka Jadranin
2
, 
Miroslav Novakovic
2
, Dejan Gocevac
2
1
Institute of Pharmacognosy, Faculty of Pharmacy, Vojvode Stepe 450, 11221 Belgrade, Serbia
2
Institute of Chemistry, Technology and Metallurgy, Njegoseva 12, 11000 Belgrade, Serbia
In this work was investigated antioxidant activity of Alchemilla velebitica Borbas. (Rosaceae), species that
grows in the region of Dinaric Mountains, in Balkan. 
Roots and aerial parts of this plant were collected on mountain Durmitor (Montenegro), in July 2005, dried
and than successively extracted with CHCl
3
and with MeOH. 
Antioxidant potential of dry MeOH extracts of A. velebitica root (AVK) and herb (AVH) was estimated by
their ability to inhibit lipid peroxidation (LP) and to scavenge OH and DPPH radicals, along with determination of
total phenolics content. 
Inhibition of lipid peroxidation was investigated using TBA test, where LP was induced by Fe
2+
/ascorbate sys-
tem in liposomes. Malondialdehyde (MDA), main product of LP, reacts with thiobarbituric acid (TBA) forming purple
coloured complex, which absorbs at 532 nm. Results of this test indicated strong activity of the investigated extracts:
AVK and AVH exhibited 50% of activity in concentrations below 70 
µg/ml (62.5 and 65.0 µg/ml, respectively).
Effect of the extracts on generation of OH radical was examined in the Fe
3+
-EDTA-H
2
O
2
-deoxyribose sys-
tem, following degradation of 2-deoxyribose into TBA-reactive substances and measuring pink coloured complex
at 532 nm. Inhibition of OH radical was moderate, but neither of extracts reached 50% of activity.
In the presence of molecules having ability to donate a hydrogen atom, deep violet colour of DPPH radical
rapidly alters to light yellow. DPPH scavenging activity of AVK and AVH was initially evaluated spectrophotomet-
rically, with results demonstrating strong and concentration-dependent activity (AVK and AVH had SC
50
values at
6.30 and 6.48 
µg/ml, respectively). Post-column derivatization with DPPH radical was then used for rapid detection
of radical scavenging components.The analytes separated by HPLC react post-column with the DPPH and in the
presence of an antioxidant negative peak was detected, due to decreasing of absorbance of DPPH.
Total phenolics content in AVK and AVH, determined using Folin-Cocalteu reagent, was 0.36 and 0.30 mg
of gallic acid (GA)/mg of dry extract, respectively. Both extracts contained high tannin amount: 0.34 and 0.26 mg
GA/mg, respectively. 
Differences in phenolics content, especially in tannin content, are in correlation with expressed antioxidant
activity of the investigated MeOH extracts of A. velebitica root and herb.
Macedonian pharmaceutical bulletin 53 (1,2) 317 (2007)
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