Kniga na apstrakti book of abstrcts

Localization and variability of flavon aglycons in Teucrium polium
from different localities of the Republic of Macedonia
Gjoshe Stefkov
1
, Marina Stefova
2
, Svetlana Kulevanova
1
1
Faculty of Pharmacy, Vodnjanska 17, Skopje, Macedonia
2
Faculty of Natural Science and Mathematics, Alexandar the Great bb,  Skopje, Macedon
ia
For localization of flavon aglycons in the plant, HPLC analysis were done on 3 different, successive extracts
obtained with rinsing of the whole plant (extract 1), followed by etheric extraction (extract 2) and ethyl acetate extrac-
tion after hydrolysis of the same plant material (extract 3). The examined plant materials were collected from 6 dif-
ferent localities with divergent ecological characteristics and vertical distribution from 0 m above sea level up to
1200 m a.s.l. Luteolin, apigenin, cirsiliol, cirsimaritin and cirsilineol were identified as superficial flavon aglycons.
Luteolin, apigenin, cirsiliol, cirsimaritin, cirsilineol and diosmetin were identified as free tissue-localized flavonoids
and total tissue flavon aglycons. Cirsiliol is quantitatively dominant among the free and superficially localized
flavonoids, while luteolin and apigenin are dominant among the tissue-localized flavonoids, both, as free aglycons
as well as in glycozid form. There is no qualitative difference, nor significant quantitative alterations in the flavonoid
content among the examined plants collected from different localities, which eliminate the ecological conditions as
factor for variability of flavonoid content in this plant. 
Macedonian pharmaceutical bulletin 53 (1,2) 300-301 (2007)
PP - 146
300
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Lokalizacija i varijabilnost na flavonskite aglikoni vo
Teucrium polium od razli~ni regioni na Republika Makedonija
\o{e Stefkov
1
, Marina Stefova
2
, Svetlana Kulevanova
1
1
Farmacevtski fakultet, ul.Vodwanska 17, Skopje, Makedonija
2
Prirodomatemati~ki fakultet, bul.Aleksandar Mkedonski bb., Skopje, Makedonija
Za utvrduvawe na lokalizacijata na flavonskite aglikoni vo rastenieto, napravena e HPLC analiza
na 3 razli~ni, sukcesivno dobieni, ekstrakti i toa so povr{insko plaknewe na celo rastenie (ekstrakt 1),
prosledeno so eterska ekstrakcija (ekstrakt 2) i etilacetatna ekstrakcija po hidroliza na istiot mate-
rijal (ekstrakt 3). Ispituvanite rastitelni primeroci se od 6 razli~ni lokaliteti so divergentni eko-
lo{ki karakteristiki i viso~inska distribucija od 0 m.n.v. do 1200 m.n.v. Identifikuvani se: luteolin,
apigenin, cirsiliol, cirsimaritin, cirsilineol kako povr{inski slobodni flavonski aglikoni. Kako
slobodni tkivni flavonoidi i vkupni tkivni flavonski aglikoni se identifikuvani luteolin, apigenin,
cirsiliol, cirsimaritin, cirsilineol i diosmetin. Cirsiliol e kvantitavno dominaniten me|u slobod-
nite i povr{inski lokaliziranite flavonoidi a luteolinot i apigeninot pome|u tkivnite flavonoidi,
i kako slobodni aglikoni i vo glikozidna forma. Nema kvalitativna razlika,  nitu zna~ajni kvantitativ-
ni razliki vo flavonoidniot sostav pome|u ispituvanite primeroci sobrani od razli~ni lokaliteti {to
gi isklu~uva ekolo{kite uslovi kako faktor na varijabilnost na flavonoidniot sostav kaj ova rastenie.
Macedonian pharmaceutical bulletin 53 (1,2) 300-301 (2007)
PP - 146
301
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Histomorphological assessment of the toxicity 
to liver, myocardial and renal tissue 
of the Teucrium polium extracts in the treatment of diabetic rats
Gjoshe Stefkov
1
, Gordana Petrusevska
2
, Svetlana Kulevanova
1
1
Faculty of Pharmacy, Vodnjanska 17, Skopje, Macedonia
2
Faculty of Medicine, Vodnjanska 17, Skopje, Macedonia
After ten days of intragastral treatment of streptozotocin-diabetic rats with Teucrium polium extracts, we
have made histomorphological analysis and evaluate the toxicity to the liver, myocardial and renal tissue. Histological
specimens, gained from the two groups of streptozotocin-diabetic rats treated with Teucrium polium (T1 and T2)
were compared with the tissue sections of the control group of health rats (K), group of streptozotocin-diabetic rats
treated with oral antidiabetic glibenclamide (GLC) and group of streptozotocin-diabetic untreated rats (D). 
Analysis of the liver sections in the groups T1, T2 and GLC, showed regular portal spaces and liver lobule,
reduction to normalization of the thickness of the blood vessels media, comparing with untreated diabetic rats.
Significant finding was found in the myocardial sections, where we revealed significant decrease of the vas-
cular wall thickness in the T1, T2 and GLC groups comparing to the findings in the myocardial sections of the untreat-
ed diabetic animals D. 
The renal tissue of the untreated diabetic animals showed alteration of the normal histoanatomical structures
(glycogen accumulation, presence of protein droplets in the tubular epithelial cells, thickened media of the blood
vessels in the renal interstitium, diabetic artheriolopathy). In T1 and T2 groups was found regular structure of the
glomeruli, conserved brush border and significant decrease of the arterial media thickness, comparing to the find-
ings in the untreated diabetic animals.
According to these findings, histomorphological analysis of the liver, myocardial and renal sections from
the treated experimental animals with the extracts with Teucrium polium, did not show significant alterations of the
regular morphology. There were no degenerative changes and signs of toxic effect on the structure of the analyzed
tissues, improvements comparing to the diabetic untreated rats can be observed.
Macedonian pharmaceutical bulletin 53 (1,2) 302-303 (2007)
PP - 147
302
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Histomorfolo{ka procenka na toksi~nosta vrz crniot drob, 
srceto i bubrezite na ekstrakti od Teucrium polium
pri tretman na dijabeti~ni staorci
\o{e Stefkov
1
, Gordana Petru{evska
2
, Svetlana Kulevanova
1
1
Farmacevtski fakultet, Ul. Vodwanska 17, Skopje, Makedonija
2
Medicinski fakultet, Ul. Vodwanska 17, Skopje, Makedonija
Po desetdneven intragastralen tretman na streptozotocin-dijabeti~ni staorci so ekstrakti od
Teucrium polium izvr{ena e histomorfolo{ka analiza i procenka na nivnata toksi~nost vrz crniot drob,
srceto i bubrezite. Procenkata e izvr{ena vrz osnova na sporeduvawe na tkivni ise~ocite dobieni od
dvete grupi streptozotocin-dijabeti~ni staorci tretirani so ekstrakt od Teucrium polium (T1 i T2) spore-
deni so tkivnite ise~oci od grupata na zdravi staorci (K), streptozotocin-dijabeti~ni  netretirani staor-
ci (D) i streptozotocin-dijabeti~ni staorci tretirani so  oralen antidijabetik glibenklamid (GLC).
Pri analiza na crnodrobni ise~oci, kaj grupite T1, T2 i GLC, portnite prostori i crnodrobnite
lobulusi se so regularna gradba, ima redukcija, kon normalizacija na debelinata na yidot (medija) na
krvnite sadovi vo odnos na netriranite dijabeti~ni staorci.
Analizata na srcevi ise~oci kaj netretiranite dijabeti~ni staorci uka`uva na zadebeluvawe na
medijata na yidot na krvnite sadovi dodeka kaj grupite T1, T2 i GLC ima zabele`itelno namaluvawe i po-
dobruvawe na ovaa  patolo{ka promena.
Bubre`noto tkivo  na netretiranite dijabeti~ni staorci poka`aa otstapuvawa od normalnata
gradba na histoanatomskite strukturi (glikogena akumulacija, proteinurija, zadebelena medija na sadovi
vo intersticiumot, dijabeti~na arteriolopatija). Kaj T1 i T2 grupite se voo~uva regularna gradba na
glomerulite, za~uvana ~etkasta granica i zna~ajno namaluvawe na medijata na arteriskite krvni sadovi,
sporedeno so onie od netretiranite dijabeti~ni `ivotni.
Spored ova, histomorfolo{kiot staorci pregled na ise~oci od crn drob, srce i bubrezi na grupite
eksperimentalni, tretirani so ekstrakti od Teucrium polium ne uka`uva na pozna~itelni otstapuvawa od
regularnata morfologija, ne se zabele`uvaat degeneraciski promeni i znaci na toksi~en efekt vrz struk-
turata na ispituvanite organi a mo`at da se zabele`at podobruvawa vo odnos na dijabeti~nite netreti-
rani staorci.
Macedonian pharmaceutical bulletin 53 (1,2) 302-303 (2007)
PP - 147
303
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In vitro investigation of the insulinotropic effect of several flavonoids
in INS-1E insulinoma cells
Gjoshe Stefkov
1
, Anna Jager
2
, Per Molgaard
2
, Knud Josefsen
3
, Svetlana Kulevanova
1
1
Faculty of Pharmacy, Vodnjanska 17, Skopje, Macedonia
2
Faculty of Pharmaceutical Sciences, Jagtvej 162, 2200 Copenhagen, Denmark
3
Bartholin Institute, Panum, 2200 Copenhagen, Denmark
The insulinotropic effect of pure flavon and flavanon, some flavonoid glycosides (apigenin-7-glycoside, luteolin-
7-glycoside and rutin) and some flavonoid aglycones (kaempferol and quercetin), all in the concentration of 500 mg/mL,
was studied. The insulinotropic effect was studied in clonal INS-1E insulinoma cells, isolated from radiation-induced
rat insulinoma. Glucose-induced insulin secretion was dose-related and similar to rat islet response. The target flavonoids
were added in INS-1E cells cultured in medium both with 3 mmol/L glucose (basal condition) and in medium with 20
mmol/L glucose (high glucose condition) .The results of the insulin secretion, measured with ELISA, have revealed
significant increase of the quantities of secreted insulin in the stimulated INS-1E cells with flavonoids (figure 1)
Figure 1. quantities of secreted insulin in INS-1E cells stimulated with certain flavonoids.
It is obvious that the most potent, in the concentration of 500 mg/mL, are the pure flavon and flavanon (330 %-
380 % increase of insulin secretion), but there is different figure, with quite equal bars, if we calculate the insulin
secretion in INS-1E cells towards molar concentrations of the same flavonoids. In the both cases we can conclude
that the examined flavonoids pose positive insulinotropic effect.
Acknowledgment: All the research were done at the Faculty of Pharmaceutical Sciences and Bartholin
Institute, Panum, Copenhagen, Denmark as a part of the TEMPUS project for reconstruction of the pharmaceutical
education in Macedonia.
Macedonian pharmaceutical bulletin 53 (1,2) 304-305 (2007)
PP - 148
304
^ETVRTI KONGRES NA FARMACIJATA NA MAKEDONIJA SO ME\UNARODNO U^ESTVO
FOURTH CONGRESS OF PHARMACY OF MACEDONIA WITH INTERNATIONAL PARTICIPATION
0
0,5
1
1,5
2
2,5
3
3,5
3mMG 20
mM
G
20
mM
G-
DM
SO
Ap
i-7
-G
Lu
t-7
-G
Kaem
p
Que
r
Ru
tin
Fl
av
on
Fl
av
anon

In vitro ispituvawe na insulinotropniot efekt na opredeleni
flavonoidi kaj INS-1E kletki
\o{e Stefkov
1
, Knul Jozefsen
3
, Ana Jager
2
, Pjer Molgard
2
, Svetlana Kulevanova
1
1
Farmacevtski fakultet, ul. Vodwanska 17, Skopje, Makedonija
2
Fakultet za farmacevtski nauki, ul. Jagtvej 162,2200 Kopenhagen, Danska
3
Bertolin institut, Panum, 2200 Kopenhagen, Danska
Izvr{eno e istra`uvawe na insulinotropniot efekt na opredeleni flavonoidi. Ispituvano e
dejstvoto na ~ist flavon i flavonon, flavonoidni glikozidi (apigenin-7-glikozid, luteoin-7-glikozid,
rutin) nekoi flavonoidni aglikoni (kemferol, kvercetin). Raboteno e so koncentracija od 500 µg/ml. Vo
ovie istra`uvawa se koristeni monoklonalni INS-1E kletki, homogena kleto~na linija, izolirani od
roditelskite  INS-1, insulinomski kletki (predizvikani so radiacija) dobieni od staorec. Kaj ovoj model
glukoza-induciranata sekrecija na insulin e dozno-zavisna i sli~na na onaa od izolirani pankreati~ni
ostrovca od staorec.
Ispituvaweto e izvedeno vrz INS-1E kletki koi se nao|aat vo medium so 3 mmol/L glukoza (normal-
na fiziolo{ka sostojba) kako i vo medium so visoka koncentracija na glukoza (20 mmol/L) glukoza (model
za 
β-kletki vo hiperglikemi~na sostojba). Izvr{enoto merewe na koli~inite na izla~en insulin so elisa
metod poka`uva 
Rezultatite od izvr{enoto merewe na koli~inite na izla~en insulin so elisa metod poka`uva
zna~ajno poka~uvawe na koli~estvoto izla~en insulin kaj stimuliranite  INS-1E kletki (slika 1). 
Slika 1. Koli~estvo izla~en insulin od INS-1E kletki vo medium so 20 mmol/L glukoza pri stimu-
lacija so ispituvanite flavonoidi (500 µg/ml)
Od ispituvanite flavonoidi, vo koncentracija od 500 µg/ml kaj INS-1E kletki vo medium so 20 mmol/L
glukoza, najgolema potencija (330-380 %) poka`aa ~istitiot falvon i flavanon. Pri procenka na
koli~estvoto izla~en insulin vo zavisnost od molarnite koncentracii na flavonoidite mo`e da se
zaklu~i deka site ispituvani flavonoidi davaat pozitiven insulinotropen efekt.
Blagodarnost: Site istra`uvawaa bea napraveni za vreme na studiskiot prestoj na Fakultetot
za farmacevtski nauki i Panum institutot vo Kopenhagen, Danska, vo sklop na TEMPUS proektot
za rekonstrukcija na farmacevtskata edukacija vo Makedonija.
Macedonian pharmaceutical bulletin 53 (1,2) 304-305 (2007)
PP - 148
305
^ETVRTI KONGRES NA FARMACIJATA NA MAKEDONIJA SO ME\UNARODNO U^ESTVO
FOURTH CONGRESS OF PHARMACY OF MACEDONIA WITH INTERNATIONAL PARTICIPATION
0
0,5
1
1,5
2
2,5
3
3,5
3mMG 20
mM
G
20
mM
G-
DM
SO
Ap
i-7
-G
Lu
t-7
-G
Kaem
p
Que
r
Ru
tin
Fl
av
on
Fl
av
anon

In vitro investigation of the insulinotropic effect of the 
Teucrium polium extracts in INS-1E insulinoma cells
Gjoshe Stefkov
1
, Anna Jager
2
, Per Molgaard
2
, Knud Josefsen
3
, Svetlana Kulevanova
1
1
Faculty of Pharmacy, Vodnjanska 17, Skopje, Macedonia
2
Faculty of Pharmaceutical Sciences, Jagtvej 162, 2200 Copenhagen, Denmark
3
Bartholin Institute, Panum, 2200 Copenhagen, Denmark
The insulinotropic effect of different concentrations of Teucrium polium extract and its fractions were stud-
ied. The insulinotropic effect was studied in clonal INS-1E insulinoma cells, isolated from radiation-induced rat
insulinoma. Glucose-induced insulin secretion was dose-related and similar to rat islet response. The extract and its
fractions were added in INS-1E cells cultured in medium both with 3 mmol/L glucose (basal condition) and in medi-
um with 20 mmol/L glucose (high glucose condition). The results of the insulin secretion, measured with ELISA,
have revealed significant increase of the quantities of secreted insulin in the stimulated INS-1E cells with flavonoids.
There were no significant changes in the insulin secretion in the INS-1E cells cultured in 3 mmol/L glucose medi-
um. There is a dose-effect related secretion in the concentration interval between 5-500 mg/mL of the extract and
slight down alteration at concentration of 1000 mg/mL. Most potent of the tested fractions, in the concentration of
500 mg/mL, was the water fraction after liqid/liqid extraction with ethyl acetate of the hydrolyzed extract (acid
hydrolysis). This fraction increased the insulin secretion in the INS-1E cells in 20 mmol/L glucose medium up to
350 %, although the ethyl acetate fraction have shown smaller but also significant effect. These results show that
examined extract and its fraction have pose positive insulinotropic effect.
Acknowledgment: All the research were done at the Faculty of Pharmaceutical Sciences and Bartholin
Institute, Panum, Copenhagen, Denmark as a part of the TEMPUS project for reconstruction of the pharmaceutical
education in Macedonia.
Macedonian pharmaceutical bulletin 53 (1,2) 306-307 (2007)
PP - 149
306
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In vitro ispituvawe na insulinotropniot efekt na  ekstrakt od
Teucrium polium kaj INS-1E kletki
\o{e Stefkov
1
, Knul Jozefsen
3
, Ana Jager
2
, Pjer Molgard
2
, Svetlana Kulevanova
1
1
Farmacevtski fakultet, ul.Vodwanska 17, Skopje, Makedonija
2
Fakultet za farmacevtski nauki, ul. Jagtvej 162, 2200 Kopenhagen, Danska
3
Bertolin institut, Panum, 2200 Kopenhagen, Danska
Ispituvan e insulinotropniot efekt na razli~ni koncentracii na ekstrakt od Teucrium polium od
Makedonija, kao i na nekolku negovi frakcii. Vo ovie istra`uvawa se koristeni monoklonalni INS-1E
kletki, homogena kleto~na linija, izolirani od roditelskite  INS-1, insulinomski kletki (predizvikani
so radiacija) dobieni od staorec. Kaj ovoj model glukoza-induciranata sekrecija na insulin e dozno-zav-
isna i sli~na na onaa od izolirani pankreati~ni ostrovca od staorec. 
Ispituvaweto e izvedeno vrz INS-1E kletki koi se nao|aat vo medium so 3 mmol/L glukoza (normal-
na fiziolo{ka sostojba) kako i vo medium so visoka koncentracija na glukoza (20 mmol/L) glukoza (model
za b-kletki vo hiperglikemi~na sostojba). Izvr{enoto merewe na koli~inite na izla~en insulin so elisa
metod poka`uva poka~uvawe na koli~estvoto izla~en insulin kaj stimuliranite  INS-1E kletki so 500 µg/ml
ekstrakt od Teucrium polium od 250 % vo odnos na kontrolnata grupa kletki vo medium so 20 mmol/L glukoza.
Nema signifikantni promeni vo koli~estvata izla~en insulin kaj kontrolnata i stimuliranite grupi
na kletki vo normalen medium so 3 mmol/L glukoza. Vo koncentraciskiot interval od 5 µg/ml do 500 µg/ml
ima doza-efetkt zavisnost dodeka so koncentracija od 1000 µg/ml, efektot e zna~aen no pomal od onoj na
predizvikan so 500 µg/ml. Od ispituvanite frakcii, vo koncentracija od 500 µg/ml kaj INS-1E kletki vo
medium so 20 mmol/L glukoza, najgolema potencija  poka`a vodenata frakcija posle izvr{ena hidroliza
na ispituvaniot ekstrakt (354% zgolemuvawe na koli~estvoto izla~en insulin), iako i drugata, etilac-
etatna frakcija poka`a zna~ajno poka~uvawe na koli~estvoto izla~en insulin.
Ovie rezultati uka`uvaat na zna~aen pozitiven insulinotropen efekt na ekstrati od Teucrium polium.
Blagodarnost: Site istra`uvawaa bea napraveni za vreme na studiskiot prestoj na Fakultetot
za farmacevtski nauki i Panum institutot vo Kopenhagen, Danska, vo sklop na TEMPUS proektot
za rekonstrukcija na farmacevtskata edukacija vo Makedonija.
Macedonian pharmaceutical bulletin 53 (1,2) 306-307 (2007)
PP - 149
307
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Antioxidant potential of Achillea millefolium from Vrsa
c
ki breg
Jelena Gavrilovic
1
, Jelena Kukic
2
, Zvezdana Doslov-Kokorus
2
, Zoran Maksimovic
2
1
FID “Dr Jovan Tucakov”, Vojvode Stepe 450, 11221 Belgrade, Serbia
2
Institute for pharmacognosy, Faculty of Pharmacy, Vojvode Stepe 450, 11221 Belgrade, Serbia
The aim of this study was to investigate and compare antioxidant activity of two samples of Achillea mille-
folium L. (Asteraceae). 
Plant material was collected from two localities at Vršacki breg (Southern Banat region, in the vicinity of
the town of Vršac) during July 2006 (sample I and II, respectively). Aerial parts of investigated plants were dried in
a shaded and well-ventilated place and coarsely grounded. In both samples tannin and flavonoid contents were deter-
mined, according to the appropriate Pharmacopoeial procedures. Each sample was then extracted with 70 % acetone
and with water (drug:extract ratio = 1:50, w/v). Antioxidant potential of obtained extracts was determined using two
assays: FRAP test (to determine total antioxidant activity, TAA) and DPPH assay (to study free radical scavenging
capacity). In addition, total and non-tannin phenols in all extracts were determined, using Folin-Ciocalteu procedure.
Tannin content, determined according to Rhataniae radix monograph (Yugoslav Pharmacopoeia V), was
4.45 and 3.85 % in samples I and II, respectively. Flavonoids in samples I and II were determined according to pro-
cedure described in monograph Crataegi folium cum flores (DAB 10) and represent 0.90 and 0.16%, respectively. 
Investigated acetone and aqueous extracts of A. millefolium samples possess substantial antioxidant activi-
ty, with the acetone extract of sample II being the most active one. Results are presented in Table 1.
Extracts of sample II showed higher antioxidant activity than the appropriate extracts of sample I. It is also
evident that acetone extracts of both samples possess higher activity than the corresponding aqueous extracts. This
could be explained by differences in total and non-tannin phenolics content in the investigated extracts.
Macedonian pharmaceutical bulletin 53 (1,2) 308 (2007)
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a
ml of extract/ml; 
b
ìg Fe
2+
/ml extract;  
c
mg of gallic acid/ml extract  
Phenolics
c 
Extract 
SC
50 
DPPH
a 
FRAP value
b
 
total 
non-tannin 
tannins 
Achillea I  - acetone 
77.21 
1.190 
0.208 
0.206 
0.003 
Achillea I  - water 
303.75 
0.804 
0.134 
0.112 
0.022 
Achillea II  - acetone 
14.62 
1.284 
0.458 
0.458 
0.000 
Achillea II  - water 
90.89 
0.481 
0.156 
0.117 
0.039