Kniga na apstrakti book of abstrcts

Cytoprotective effects of a new flavonoid from Astragalus hamosus
Ilina Krasteva
1
, Georgi Momekov
2
, Spiro Konstantinov
2
,
Stefan Nikolov
1
1
Department of Pharmacognosy, 
2
Lab. of Molecular Pharmacology and Experimental Chemotherapy, 
Department of Pharmacology and Toxicology; Faculty of Pharmacy, 
2
Dunav st., 1000 Sofia, Bulgaria; e-mail: [e-mail]
Astragalus hamosus L. (Fabaceae) is annual herb, distributed in Europe - Mediterranean to Armenia, Ukraine
and the Caucasus East; Northern Africa; central and South-western Asia. The plant is used in herbal medicine as
emollient, demulcent, aphrodisiac, diuretic, laxative and good for inflammation, ulcers and leucoderma. It is useful
in treating irritation of the mucous membranes, nervous affections and catarrh.
The phytochemical study of the ethyl acetate extract from the aerial parts of A. hamosus led to the isolation
and structural elucidation of a new flavonol glycoside: 7-O-methyl-kaempferol 4´-
β-D-galactopyranoside (rhamnoc-
itrin 4´-
β-D-galactopyranoside). The cytoprotective effects of this flavonoid were tested in models of gentamicin
and cisplatin-induced cytotoxicity in human renal cells 293T, using the MTT-dye reduction assay. The results obtained
indicate that the compound significantly ameliorated the detrimental effects of the two established nephrotoxins upon
the renal cell line 293T after 72 h incubation.
Macedonian pharmaceutical bulletin 53 (1,2) 288 (2007)
PP - 139
288
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Phytochemical investigation of Astragalus cicer L. (Fabaceae)
Petranka Zdraveva
1
, Ilina Krasteva
1
, Ivanka Pencheva
2
, Stefan Nikolov
1
1
Department of Pharmacognosy, 
2
Department of Pharmaceutical Chemistry, 
2
Faculty of Pharmacy, 
Dunav St., 1000 Sofia, Bulgaria; e-mail: [e-mail]
Many Astragalus species are used in folk medicine for their hepatoprotective, antioxidative, immunostimulant
and antiviral properties. Three groups of chemicals have been described as their pharmacologically active principles: poly-
saccharides, saponins and phenolics. The aim of this study is to determine the composition of phenolic compounds in the
aerial parts of Astragalus cicer L. – a perennial herbaceous plant, widely distributed in central and Eastern Europe and
South-western Asia. The aqueous/alcoholic extracts from the overground parts of the plant were treated with different sol-
vents and purified by column chromatography with various eluents. The ethyl acetate extract were purified over Sephadex
LH-20 and selected fractions were further analysed by HPLC. The optimum separation was achieved using a reversed
phase Tracel Excel RP-C
18
ODS-2, 5
µm (250 x 4.6 mm) column by linear gradient elution mode on a Shimadzu LC–10
Advp (Japan) chromatographic system included UV-VIS detector SPD with fixed analytical wavelengths set at 254 nm.
The mobile phase was composed of methanol-water and acetonitrile-water mixtures in diferent proportions. The obtained
results reveal the presence of two groups compouds - flavonoids (flavones and flavonols) and coumaines.
Macedonian pharmaceutical bulletin 53 (1,2) 289 (2007)
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Prophilactic effect of Urtica dioica seed extrats against acute 
experimental hepatotoxicity in vivo
Tatjana Kadifkova Panovska
1
, Svetlana Kulevanova
2
, Icko Gjorgoski
3
, 
Mirjana Bogdanova
4
, Gordana Petrushevska
5
1
Institute of Toxicology, Faculty of Pharmacy, University "Ss. Cyril and Methodius" Vodnjanska 17, 1000 Skopje, Republic of Macedonia
2
Institute of Pharmacognosy, Faculty of Pharmacy, University "Ss. Cyril and Methodius" Vodnjanska 17, 1000 Skopje, Republic of Macedonia
3
Institute of Biology, Faculty of Natural Sciences and Mathematics, 
University "Ss. Cyril and Methodius" P.B. 126, 1000 Skopje, Republic of Macedonia
4I
nstitute of Clinical Biochemistry, Clinical Center, Vodnjanska 17, 1000 Skopje, Republic of Macedonia
5
Institute of Pathology, Faculty of Medicine, University "Ss. Cyril and Methodius" Vodnjanska 17, 1000 Skopje, Republic of Macedonia
Lipid peroxidation is an important deteriorates reactions which have influence not only in food quality but
also is believed to be associated with some diseases such as carcinogenesis, mutagenesis, ageing, and arteriosclero-
sis. The role of active oxygen and free radicals in tissue damage in such diseases, are becoming increasingly recog-
nized. Cancer, emphysema, cirrhosis, arteriosclerosis, and arthritis have all been correlated with oxidative damage.
Active oxygen, either in the form of superoxide (O
2
o-
), hydrogen peroxide (H
2
O
2
), hydroxyl radical (OH
o
), or sin-
gled oxygen (
1
O
2
), is a product of normal metabolism and attacks biological molecules, leading to cell or tissue
injury. Antioxidant supplements or foods rich in antioxidants may be used to help the human body in reducing oxida-
tive damage by free radicals and active oxygen. Recently, various phytochemicals and their effects on health, espe-
cially the suppression of active oxygen species by natural antioxidants from teas, spices and herbs, have been inten-
sively studied. The most commonly used antioxidants at the present time are butylated hydroxyanisole (BHA),
butylated hydroxytoluene (BHT), propyl gallate (PG), and tert-butylhydroquinone (TBHQ). However, they are sus-
pected of being responsible for liver damage and carcinogenesis in laboratory animals. Therefore, the development
and utilization of more effective antioxidants of natural origin are desired.
Urtica dioica is an annual herb, belonging to the family Urticaceae. It is known in traditional therapy such
as acute diuretic, natriuretic and hypotensive. Different parts of plant have also been used in the treatment of hyper-
tension, stimulation of proliferation of human lymphocytes, immunostimulation on neutrophilis, beneficial effect on
the prostate tissue and antirheumatic effects.
The aim of the present study is to investigate a possible prophylactic effect of Urtica dioica seed extracts
pre-treatment on prevention of oxidative stress generated by CCl
4
in rats. 
Dose of 25 mg/kg of Urtica dioica extracts was administered i.p. the test groups of female Wistar rats. The
positive control group received CCl
4
and the negative control group received normal saline. In this investigation the
level of lipid peroxidation and serum liver enzyme activities were measured. Histopatological examinations were
also studied.
The CCl
4
treatment of rats increased the lipid peroxidation and liver enzymes, and also decreased the antiox-
idant enzyme levels (superoxide dismutase, catalase, glutathione peroxidase and the levels of glutathione). Pre-treat-
ment the experimental animals with Urtica dioica extracts for 7 days decreased the elevated lipid peroxidation and
liver enzyme levels and also increased the reduced antioxidant enzyme levels. Histopatological studies provided sup-
portive evidence for the biochemical analysis.
The present study has shown that Urtica dioica pre-treatment diminishes oxidative stress in the liver gener-
ated by applying CCl
4
. This finding suggests that Urtica dioica has a cell protection effect against oxidative stress
in rats. Nevertheless, further studies should be carried out for structural, biochemical, and comparative investiga-
tions with other antioxidants in higher animal models for prevention of oxidative stress, before clinical applications.
Macedonian pharmaceutical bulletin 53 (1,2) 290-291 (2007)
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Za{titen efekt na ekstrakti od seme na Urtica dioica nasproti akutna
eksperimentalna hepatotoksi~nost in vivo
Tatjana Kadifkova Panovska
1
, Svetlana Kulevanova
2
, Icko Gjorgoski
3
, 
Mirjana Bogdanova
4
,Gordana Petru{evska
5
1
Institut za Primeneta biohemija, Farmacevtski fakultet, 
Univerzitet “Sv. Kiril i Metodij”, Vodwanska 17, 1000 Skopje, R. Makedonija 
2
Institut za Farmakognozija, Farmacevtski fakultet, 
Univerzitet “Sv. Kiril i Metodij”, Vodwanska 17, 1000 Skopje, R. Makedonija 
3
Institut za Biologija, Prirodno matemati~ki fakultet, 
Univerzitet “Sv. Kiril i Metodij”, P.B. 126, 1000 Skopje, R. Makedonija
4
Institut za Klini~ka biohemija, Klini~ki centar, Vodwanska 17, 1000 Skopje, R. Makedonija
5
Institut za Patologija, Medicinski fakultet, Univerzitet “Sv. Kiril i Metodij”, Vodwanska 17,
1000 Skopje, R. Makedonija 
Lipidnata peroksidacija e va`na reakcija koja ima vlijanie ne samo vrz kvalitetot na hranata tuku
se povrzuva i so odredeni zaboluvawa kako karcinogenezata, mutagenezata, stareeweto i arteriosklero-
zata. Ulogata na aktivniot kislorod i slobodnite radikali vo o{tetuvaweto na tkivata pri ovie zabolu-
vawa stanuva se poprepoznatliva. Kancer, emfizem, ciroza, arterioskleroza i artritis se doveduvaat vo
vrska so oksidativnoto o{tetuvawe. Aktivniot kislorod, nezavisno od formata, superoksid (O
2
•-
), vodoro-
den peroksid (H
2
O
2
), hidroksil radikal (OH•), ili singlet kislorod (
1
O
2
), e produkt na normalniot metabo-
lizam i gi napa|a biolo{kite molekuli, predizvikuvaj}i o{tetuvawe na klekite ili tkivata. Upotrebata
na antioksidativni preparati ili hrana bogata so antioksidansi mo`e da pomogne na organizmot vo redu-
ciraweto na oksidativnoto o{tetuvawe od slobodnite radikali i aktivniot kislorod. Vo posledno vreme,
intenzivno se prou~uva efektot na fitohemiskite preparati vrz zdravjeto, osobeno supresijata na aktiv-
nite kislorodni vidovi so prirodni antioksidansi od ~aj, za~ini i lekoviti rastenija. Naj~esto upotre-
buvani antioksidansi vo dene{no vreme se butil hidroksi anizol (BHA),  butil hidroksi toluol (BHT),
propil galat (PG), i tri-butilhidrokinon (TBHQ). No se smeta deka ovie supstancii se odgovorni za o{te-
tuvawa na crniot drob i karcinogeneza kaj eksperimentalni `ivotni. Spored ova, neophodno e razvivawe
i iskoristuvawe na poefikasni antioksidansi od prirodno poteklo.
Urtica dioica e ednogodi{no rastenie, {to pripa|a na familijata Urticaceae. Poznato e vo tradicio-
nalnata terapija kako diuretik, natriuretik i hipotenziv. Isto taka, razli~ni delovi od rastenieto se
upotrebuvale vo tretman na hipertenzija, stimulacija na proliferacija na limfociti, imunostimulacija
na neutrofili, efekti vrz prostata i antireumatski efekti.  
Celta na trudot e da se ispita mo`niot za{titen efekt na ekstraktite od seme na Urtica dioica pri
pred-tretman na staorci vo prevencijata na oksidativen stres predizvikan od CCl
4
.
Dozi od 25 mg/kg na Urtica dioica ekstrakti se davani i.p. na test grupi od `enski Wistar  staorci.
Pozitivnata kontrola e tretirana samo so CCl
4
, a negativnata kontrola so fiziolo{ki rastvor. Odredu-
vano e nivoto na lipidnata peroksidacija i aktivnosta na enzimite od crniot drob vo serum. Vr{eni se i
histopatolo{ki ispituvawa. 
Kaj staorcite tretirani so CCl
4
se zgolemuva obemot na lipidna peroksidacija i aktivnosta na
enzimite od crniot drob, a istovremeno se namaluva nivoto na antioksidativnite enzimi (superoksid dis-
mutaza, katalaza, glutation peroksidaza i nivoto na glutation). Pred-tretmanot na eksperimentalnite
`ivotni so ekstrakti na Urtica dioica vo tek na 7 dena ja namaluva lipidnata peroksidacija i nivoto na
enzimite, a go zgolemuva reduciranoto nivo na antioksidativnite enzimi. Histopatolo{kite ispituvawa
se vo prilog na biohemiskite analizi. 
Vo trudot se utvrduva deka pred-tretmanot so Urtica dioica go namaluva oksidativniot stres predi-
zvikan od CCl
4
.  Ovie soznanija uka`uvaat deka Urtica dioica poka`uva za{titen efekt kon kletkite na
staorec pri oksidativen stres. Idnite ispituvawa se usmereni kon prou~uvawe na strukturata, biohemi-
jata kako i sporedbeni studii so drugi antioksidansi vrz poslo`eni `ivotinski modeli za prevenirawe
na oksidativniot 
Macedonian pharmaceutical bulletin 53 (1,2) 290-291 (2007)
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Evaluation of the antioxidant and hepatoprotective effect 
of Helychrisum plicatum extracts
Tatjana Kadifkova Panovska
1
, Svetlana Kulevanova
2
, Icko Gjorgoski
3
, 
Mirjana Bogdanova
4
, Gordana Petrushevska
5
1
Institute of Applied Biochemistry, Faculty of Pharmacy, 
University "Ss. Cyril and Methodius" Vodnjanska 17, 1000 Skopje, Republic of Macedonia
2
Institute of Pharmacognosy, Faculty of Pharmacy, University "Ss. Cyril and Methodius" Vodnjanska 17, 1000 Skopje, Republic of Macedonia
3
Institute of Biology, Faculty of Natural Sciences and Mathematics, 
University "Ss. Cyril and Methodius" P.B. 126, 1000 Skopje, Republic of Macedonia
4
Institute of Clinical Biochemistry, Clinical Center, Vodnjanska 17, 1000 Skopje, Republic of Macedonia
5
Institute of Pathology, Faculty of Medicine, 
University "Ss. Cyril and Methodius" Vodnjanska 17, 1000 Skopje, Republic of Macedonia
Helichrysum plicatum DC (Asteraceae) has been extensively used in folk medicine for the management of
gastric and hepatic disorders, usually in combination with other plants with similar effects. Phytochemical screen-
ing (HPLC) of the H. plicatum from Macedonia proved the presence of apigenin and naringenin as free aglycones
and glycosides of apigenin, naringenin, quercetin and kaempferol in the flowers as well as quercetin and luteolin
glycosides and free luteolin in stems and leaves. Its antioxidant and hepatoprotective properties have not been stud-
ied previously. Among phytochemicals, flavonoids deserve a special mention due to their free radical scavenging
activities and in vivo biological activities that are being investigated by many researchers. The goal of research on
antioxidative characteristics of plant extracts is to discover a potential replacement for synthetic antioxidants (BHT,
BHA), which cause unwanted processes after prolonged used.
The evaluation of plant extracts antioxidant and hepatoprotective capacity is not easy task, as many meth-
ods can be used to determine this activity, and substrates, conditions, analytical methods, and concentrations can
affect the estimated activity. This paper reports a study in which antioxidative and hepatoprotective activity of
methanolic, ethyl acetate, and after hydrolysis H. plicatum extracts in in vitro and in vivo conditions are tested. 
The antioxidative effect was followed by three in vitro methods: evaluation the free radical scavenging capac-
ity (DPPH method), inhibition of hydroxyl radical production and protection of 
β-carotene-linoleic acid system. The
hepatoprotective activity was investigated using rats with CCl
4
-induced liver damage. Specific biochemical param-
eters (glutathione peroxidase, superoxide dismutase, reduced glutathione and total antioxidative status) were esti-
mated in blood and in liver homogenate. Lipid peroxidation in CCl
4
-intoxicated rats was evidenced by a marked
increment in the levels of thiobarbituric acid reactive substances. Histopatological examinations of the liver were
undertaken to monitor the status of the liver. Silymarin was used as a standard to compare the hepatoprotective activ-
ity of the extracts.
Investigate extracts showed radical scavenging activity with IC
50
from 6 to 11 mg/ml. The extracts are capa-
ble to reacting with OH• radical with inhibition of its production ranged between 33-58%. The high preventive activ-
ity against the bleaching of beta-carotene (15-49% of initial value after 120 minutes) was also observed. The antiox-
idative activity of the extracts in the experimental systems was compared with that of reference substances: luteolin,
quercetin, BHA, BHT and sylimarin (the main agent of the well-known milk thistle - Silybum marianum L.). In in
vivo studies, the biochemical parameters in groups treated with Helichrysum plicatum extracts at a dose of 25 mg
kg
-1
, have shown significantly different value than that of the CCl
4
-treated group. The liver biopsy of all experimen-
tal rat groups treated with Helichrysum plicatum extracts showed significant restoration of normal histomorfologi-
cal pattern of liver cells. 
Results of this study suggest that Helichrysum plicatum represent a natural source with antioxidant and
hepatoprotective potential.
Macedonian pharmaceutical bulletin 53 (1,2) 292-293 (2007)
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Procenuvawe na antioksidativniot i hepatoprotektivniot
efekt na ekstraktite od Helychrisum plicatum
Tatjana Kadifkova Panovska
1
, Svetlana Kulevanova
2
, Icko Gjorgoski
3
, 
Mirjana Bogdanova
4
, Gordana Petru{evska
5
1
Institut za Primeneta biohemija, Farmacevtski fakultet, 
Univerzitet 
“
Sv. Kiril i Metodij
”
, Vodwanska 17, 1000 Skopje, R. Makedonija 
2
Institut za Farmakognozija, Farmacevtski fakultet, 
Univerzitet 
“
Sv. Kiril i Metodij
”
, Vodwanska 17, 1000 Skopje, R. Makedonija 
3
Institut za Biologija, Prirodno matemati~ki fakultet, Univerzitet “Sv. Kiril i Metodij”,
P.B. 126, 1000 Skopje, R. Makedonija
4
Institut za Klini~ka biohemija, Klini~ki centar, Vodwanska 17, 1000 Skopje, R. Makedonija
5
Institut za Patologija, Medicinski fakultet, Univerzitet “Sv. Kiril i Metodij”,
Vodwanska 17, 1000 Skopje, R. Makedonija 
Helichrysum plicatum DC (Asteraceae) intenzivno se koristi vo narodnata medicina pri gastri~ni i
hepati~ni zaboluvawa, voobi~aeno vo kombinacija so drugi rastenija so sli~en efekt. Fitohemiskata analiza
(HPLC) na H. plicatum od Makedonija uka`uva na prisustvo na apigenin i naringenin kako slobodni aglikoni
i glikozidi na apigenin, naringenin, kvercetin i kemferol vo cvetovite kako i kvercetin i luteolin glikozi-
di i sloboden luteolin vo steblata i listovite. Negovite antioksidativni i hepatoprotektivni karakteris-
tiki dosega ne se prou~uvani. Od fitohemiskite supstancii, flavonoidite pretstavuvaat poseben interes za
istra`uva~ite, so ogled na nivnata aktivnost za fa}awe na slobodnite radikali i in vivo biolo{kata aktivnost.
Istra`uvawata na antioksidativnite karakteristiki na rastitelnite ekstrakti se so cel da se otkrie soodvet-
na zamena za sintetskite antioksidansi (BHT, BHA), koi mo`at da predizvikaat nesakani procesi posle prodol-
`ena upotreba. 
Procenuvaweto na antioksidativniot i hepatoprotektivniot kapacitet na rastitelnite ekstrakti e
odgovorna zada~a, so ogled na golemiot broj na metodi {to mo`at da se primenat za opredeluvaweto na ovaa
aktivnost, kako i mo`noto vlijanieto na razli~nite supstrati, uslovi, analiti~ki metodi i koncentracii. Vo
ovoj trud ispituvana e antioksidativnata i hepatoprotektivnata aktivnost na metanolnite, etilacetatnite
ekstrakti kako i na ekstraktite posle hidroliza na H. plicatum vo in vitro i in vivo uslovi. 
Antioksidativniot efekt e sleden so tri in vitro metodi: procenuvawe na kapacitetot za fa}awe na
slobodnite radikali (DPPH test), inhibicija na produkcijata na hidroksil radikal i za{tita na 
β-karoten-
linolenska kiselina sistemot. Hepatoprotektivnata aktivnost e ispituvana vrz staorci so CCl
4
inducirano
o{tetuvawe na crniot drob. Specifi~nite biohemiski parametri (glutation peroksidaza, superoksid dismu-
taza, reduciran glutation i totalen antioksidativen status) se odreduvani vo krv i homogenat od crn drob.
Lipidnata peroksidacija kaj CCl
4
intoksiciranite staorci e opredeluvana preku nivoto na tiobarbiturna
kiselina reaktivnite supstancii. Za sledewe na sostojbata na crniot drob, vr{eni se histopatolo{ki ispitu-
vawa. Silimarinot se upotrebuva kako standard za sporedba na hepatoprotektivnata aktivnost na ekstraktite. 
Ispituvanite ekstrakti poka`uvaat aktivnost za fa}awe na slobodnite radikali so IC
50
od 6 do 11
mg/ml. Ekstraktite interferiraat so OH• radikalot so inhibicija na negovata produkcija pome|u 33 i 58%.
Isto taka, poka`uvaat izrazena preventivna aktivnost kon obezbojuvaweto na 
β-karotenot (15 do 49% od
po~etnata vrednost, posle 120 minuti). Antioksidativnata aktivnost na ekstraktite vo eksperimentalnite
sistemi e sporeduvana so aktivnosta na referentnite supstancii: luteolin, kvercetin, BHA, BHT i silimarin
(aktivniot princip na mle~en trn - Silybum marianum L.). Vo in vivo ispituvawata, biohemiskite parametri
vo grupite tretirani so ekstrakti na  Helichrysum plicatum vo doza od 25 mg kg
-1
poka`uvaat signifikantni raz-
liki vo odnos na grupite tretirani so CCl
4
.  Biopsijata na crniot drob kaj site eksperimentalni grupi na staor-
ci, tretirani so ekstrakti na Helichrysum plicatum poka`uva signifikantno vra}awe na normalnata histopa-
tolo{ka struktura na kletkite na crniot drob.  
Rezultatite od ova ispituvawe uka`uvaat deka Helichrysum plicatum pretstavuva priroden izvor so
antioksidativen i hepatoprotektiven potencijal. 
Macedonian pharmaceutical bulletin 53 (1,2) 292-293 (2007)
PP - 142
293
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FOURTH CONGRESS OF PHARMACY OF MACEDONIA WITH INTERNATIONAL PARTICIPATION