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XXVIII Savetovanje o lekovitim i aromatičnim biljkama

Biljne lekovite sirovine i biljni proizvodi:

savremeni pristup karakterizaciji, proizvodnji i klasifikaciji

Zbornik apstrakata

Kongresno-muzička dvorana Centar “Millennium”

Vršac, 08-11.10.2008.

XXVIII Savetovanje o lekovitim i aromatičnim biljkama

Biljne lekovite sirovine i biljni proizvodi:

savremeni pristup karakterizaciji, proizvodnji i klasifikaciji

Zbornik apstrakata

Kongresno-muzička dvorana Centar “Millennium”

Vršac, 08-11.10.2008.

XXVIII Savetovanje o lekovitim i aromatičnim biljkama: Biljne lekovite sirovine i biljni proizvodi: savremeni pristup karakterizaciji, proizvodnji i klasifikaciji Zbornik apstrakata

Naučni odbor:

• Nada Kovačević—predsednik

• Silvana Petrović

• Katarina Šavikin

• Vanja Tadić

• Vele Tešević

• Neda Mimica-Dukić

• Silva Dobrić

• Gordana Vuleta

• Branislava Lakušić

• Slađana Šobajić

• Dragoja Radanović

Organizacioni odbor:

• Zoran Maksimović—predsednik

• Tatjana Kundaković

• Đorđe Malenčić

• Danilo Stojanović

• Sofija Đorđević

Sekretarijat:

• Mirjana Anđić

• Dragica Stupar

Farmaceutsko društvo Srbije Bulevar Vojvode Mišića 25 11000 Beograd Telefon: +381 11 264 83 85 Faks: +381 11 264 83 86

PPLLEENNAARRNNAA PPRREEDDAAVVAANNJJAA PPLLEENNAARRYY LLEECCTTUURREESS

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PL1 Diverzitet flore i vegetacije Vršačkih planina Boža Pal Univerzitet u Novom Sadu Prirodno-matematički fakultet, Departman za biologiju i ekologiju, Trg D. Obradovića 2, 21000 Novi Sad

UVOD Vršačke planine se nalaze u jugoistočnom delu Banata i pripadaju Panonskom delu Srbije. Tačna lokacija je između 18 0 i 45 ’ i 19 0 i 15 ’ istočne geografske dužine, odnosno 45 0 i 45 0 i 15’ severne geografske širine u odnosu na Greenwich (Maličević, 2008). Ukupna površina Vršačkih planina je 172 km2, od čega 122 km2 je na teritoriji Srbije (Bugarski, Tomić, 1988). Vršačke planine u geološkom pogledu predstavljaju ostatak nekadašnje Panonske mase, koja se spustila, odnosno potonula posle kretacejske periode u Mezozoiku, tačnije u oligomiocenu, na prelazu paleogena i neogena u tercijeru pre oko 40 - 50 miliona godina (Bukurov, 1950). Posle ovih tektonskih aktivnosti potonuli delovi Panonske mase su poplavljeni vodama Prasredozemnog mora, dok planine na južnom obodu današnje Panonske nizije, kao što su Fruška Gora, Vršačke planine, Meček u Mađarskoj i slavonske planine u novonastalom Panonskom moru bila ostrva, tj. njihovi delovi iznad 250 metara nikada nisu bili potopljeni. Ova činjenica jasno ukazuje na to da je flora i vegetacija Vršačkih planina veoma stara. U orogenetskom i horološkom pogledu Vršačke planine pripadaju tektonskom kompleksu Moravidima, koji se nalaze između tektonskih celina Karpato-Balkanoida na istoku, Šumadida na zapadu, a na jugu se nadovezuju na planine u sklopu srpsko-makedonske mase (Anđelković, 1982; Zeremski, 1985). U geološkom pogledu Vršačke planine čine kristalasti škriljci poreklom iz paleozoika, ponegde prošaran granitom. Na ovoj geološkoj osnovi kasnije su nastali neogeni sedimenti, terigeno-jezerski sedimenti. Iz vremenskog intervala Panonskog mora na Vršačkim planinama su nastali i badenski, sarmatski, panonski i pontijski sedimenti (Kukin et al., 1987) koji ujedno predstavljaju i faze u evoluciji Panonskog mora. Na geološkoj podlozi sadejstvom klime i biljnog pokrivača nastaje pedološki substrat u obliku zemljišta različitog tipa. Na Vršačkim planinama registrovani su sledeći tipovi zemljišta: černozemi, smonice na tercijernim glinama, zatim gajnjače i aluvijalna zemljišta kao što su ritske crnice. (Miljković, 1986). Ovi tipovi zemljišta su stvorili životne uslove za veoma različit biljni svet. Klima Vršačkih planina je umereno kontinentalna sa izraženim godišnjim dobima, međutim spada u toplije regione Vojvodine. Zimski meseci su za oko 0.5 stepeni topliji od vojvođanskog proseka. Interesantna je činjenica da apsolutni maksimumi temperatura su za oko 2 stepena manji u odnosu na vojvođanski prosek. Padavina za oko 50 mm ima više u odnosu na druge delove Vojvodine. Od karakteristika klime važno je još napomenuti vetrove, od kojih je najznačajnija Košava, koja duva čak 171 dan u godini, što ovo područje čini najvetrovitijim u Vojvodini (Bugarski, Tomić, 1987). Međutim, zahvaljujući biljnom pokrivaču, pre svega šumama, negativno delovanje jakih vetrova je delimično ublaženo na Vršačkoj planini, smanjeno je isušivanje i erozivno delovanje na zemljište, što u ekološkom pogledu predstavlja veoma važan faktor ne samo za razvoj nego i za opstanak biljnog sveta.

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VEGETACIJSKI DIVERZITET Dominantni tip vegetacije na Vršačkim planinama je šumska vegetacija, koja ujedno zauzima i najveće površine. Livadska vegetacija je sekundarna, nastaje na mestima gde je šuma iskrčena i ako se ne održava (ispaša, košenje) ponovo će je osvojiti šume. Ranije ne Vršačkim planinama je bilo mnogo više šuma i verovatno sasvim drugačiji diverzitet. Prirodna sukcesija različitih tipova vegetacije, kao i seča šuma formirali su današanju, momentalnu sliku diverziteta šumske vegetacije, koji će se , pre svega u negativnom pravcu i dalje menjati. Danas, na osnovu sintetskih istraživanja, na Vršačkim planinama konstatovano je ukupno 36 šumskih sintaksonomskih jedinica, koje pripadaju klasi eurosibirskih listopadnih šuma klase Querco-Fagetea Br. – Bl. et Wilieger. Iz ove klase konstatovane su tri vegetacijska reda. Red medunčevih šuma Quercetalia pubescentis Br. – Bl. obuhvata kserotermne šumske i žbunaste zajednice, gde ulaze razne vrste hrastova: Quercus pubescens Willd, Q. virgiliana Ten., Q. frainetto Ten., Q. polycarpa , Q. cerris L. i dr. Ovom vegetacijskom redu na Vršačkim planinama pripadaju 4 sveze, 9 asocijacija i 6 subasocijacija. Od sveza bi trebalo istaći Aceri tatarico – Quercion Zólyomi et Jakucs, koja je šumostepska sveza, predstavlja poslednju prirodnu sliku vegetacije Panonske nizije, gde je samo fragmentarno opstala. Njeno prisustvo na Vršačkim planinama jasno ukazuje na tesnu vezu u razvoju flore i vegetacije u Panonskoj niziji i na Vršakim planinama, iako je „ostraovsku floru i vegetaciju“ ovog prostora možemo smatrati reliktnom i veoma starom. Drugi red šumske vegetacije obuhvata bukove šume Fagetalia sylvaticae Pawl. i raščlanjuje se na 3 sveze, jednu podsvezu, 6 asocijacija i 4 subasicijacija. Bukove šume nisu zonalnog tipa, nego su raspoređene mozaično između hrastovih šuma, zbog specifične kontinentalne klime. U ovim šumama dominiraju Fagus moesiaca Maly in A. et G., Carpinus betulus L., Tilia tomentosa Moench, Fraxinus excelsior L. i dr. Treći red šumske vegetacije na Vršačkim planinama je red bele topole Populetalia albae Br. – Bl. koja se javlja u nižim delovima, duž donjih i srednjih tokova potoka, na muljevitom zemljištu. Na Vršačkim planinama obuhvata samo po jednu svezu i asocijaciju. Dominantne vrste u ovim zajednicama su Pupulus alba L., Salix alba L. , S. fragilis L. i dr. (Sučević, 1962; Pekanović, 1991; Kojić et al., 1998). Livadska i livado-stepska vegetacija na Vršačkim planinama rasprostranjena je u podnožju i brdskom pojasu. Zahvaljujući različitim orografskim, pedološkim i klimatskim faktorima livadska vegetacija pripada dvema klasama Molinio-Arrhenatheretea R. Tx. i Festuco-Brometea Br. – Bl. et Tx. Livadske zajednice se dele na močvarne livade Deschampsietalia H-ić, dolinske livade Trifolio-Hordeetalia H-ić i Arrhenathretalia Pawl., kao i na brdske livade Festucetalia valesiacae Br. – Bl. et R. Tx. Ovi tipovi vegetacije obuhvataju ukupno 31 sintaksonomsku jedinicu, tj. 2 klase, 4 reda, 4 sveze, 8 asocijacija i 14 subasocijacija. Livadske i livadostepske zajednice su veoma značajne sa aspekte ispaše i kao senokosi. Močvarnih livada je malo na Vršačkim planinama uglavnom se javljaju u vidu oaza ili traka u dolinama potoka. Dolinske livade su na nadmorskoj visini između 110 – 150 m u dolinama potoka i na okolnom području, nadovezuju se na močvarne livade i nalaze se u zoni hrastovo – grabovih šuma, ali samo na severoistoku Vršačkih planina. Njačešći tip livadske vegetacije su brdske livade, kje se nalaze na nižem pobrđu u visinskom pojasu između 160 – 450 m, mozaično su raspoređene u šumskoj zoni sveze Quercion frainetto B. Jov. na umereno vlažnim i umereno kserotermnim staništima (Včković, 1991; Kojić et al. 1998). Uvidom u sintetski pregled ukupne vegetacije Vršačkih planina može se konstatovati da je vegetacijski diverzitet veoma bogat, jer na oko 120 km2 ima 67 sintaksonomskih jedinica (Tab. 1.).

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Ova činjenica je veoma značajna sa više aspekata: sa aspekta vegetacijskog diverziteta, ukupnog biodiverziteta Vršačkih planina i Srbije, sa aspekta zaštite a i sa aspekta ekoturizma.

Tabela 1. Vegetacijski diverzitet Vršakih planina

Šumski ekosistemi Livadski ekosistemi Klasa 1 2 Red 3 3 Sveza 7 4 Podsveza 1 - Asocijacija 16 8 Subasocijacija 8 14 Ukupno 36 31

FLORISTIČKI DIVERZITET

Flora Vršačkih planina je istraživana već od sredine 19. veka (Heuffel, 1850). Do danas je konstatovano oko 1100 vrsta i podvrsta (Panjković Matanović, 1989) i oko 320 varijeteta i formi (Maličević, 2008). Imajući u vidu, da u flori Srbije danas ima oko 3600 vrsta i podrvrsta (Stevanović et al. 1995) floristički diverzitet Vršačkih planina možemo smatrati izuzetno bogatim. S obzirom da je površina Vršačkih planina planina u Srbiji oko 120 km2 a cele Srbije oko 88 500 km2 lako je izračunati da na 0.011% teritorije Srbije raste 26% ukupne flore Srbije. Najznačajnije biljke Vršačkih planina su endemi i relikti, kojih iako ima malo veoma su važne. Barbarea lepuznica Nyár. je rumunski endem, u celoj Srbiji raste samo na Vršačkim planinamam gde se ujedno nalazi i na severnoj i zapadnoj granici areala (Obradović, 1988). Ova vrsta se nalazi i u prvom tomu Crvene knjige flore Srbije kao krajnje ugrožen takson (Diklić, Lakušić, 1999). Campanula grossekii Heuff. je endem centralnog dela Balkanskog poluostrva, na Vršačkim planinama se nalazi na severozapadnoj granici rasprostranjenja (Obradović, 1988). Cirsium bracycephalum Jur. je panonski endem, veoma retka na vlažnim staništima a u celoj Srbijiraste samo u Vojvodini. Ovi endemi, koji pripadaju različitim fitogeografskim provincijama i okruzima, ujedno ukazuju na uticaj i povezanost flore Vršačkih planina sa dacijskim, balkanskim i panonskim područjima. Od ostatka veoma stare, arhaične flore značajno je spomenuti Ostrya carpinifolia Scop. koja je na Vršačkim planinama ima karakter tercijernog relikta (Obradović, 1988). Ova vrsta je izuzetno retka i neophodno je i dalje proveravati njeno prisutvo kao i stanje populacije. Odsustvo većeg broja relikata je posledica nedostatka specifičnih mikrostaništa tipa refugijum. Pored endemičnih, reliktnih biljaka i biljaka na granicama areala značajne su i one vrste, koje imaju povezujući florni element i ukazuju na florogenetske veze Vršačkih planina sa okolnim područjima Balkana, Ponta i Panonske nizije. Od biljaka sa povezaujućim elementima flore interesantne su Euphorbia pannonica Host i Allium atropurpureum W. et K. jer pripadaju panonsko-balkanskim vrstama (Soó, 1966; 1973) i povezuju floru Balkanskog poluostrva sa florom Vršačkih planina, tj. Panonske nizije. Iris pumila L. i Taraxacum serotinum (W. et K.) Poir. pripadaju pontsko-panonskom flornom elementu (Soó, 1970; 1973) takođe povezujući floru Panonske nizije i okoline Ponta, odnosno Crnog mora.

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Nestale biljke sa Vršačkih planina govore o osiromašenju kako diverziteta flore, tako i ukupnog biljnog genofonda. Među nestalim biljkama su Astragalus varius S. G. Gmelin, koji pripada pontsko-južnosibirsko-turanskom geoelementu i ukazivao je na uticaj suvog i toplog boreala na fromiranje flore na Vršačkim planinama (Boža, 1999). Oxytropis pilosa L. pripada (evroazijskom) kontinentalnom flornom elementu (Soó, 1966) i povezivala je floru livadospeskog karaktera na Vršačkim planinama sa florom istočnih stepa. U biljnogeografskom pogledu Vršačke planine pripadaju srednjeevropskom regionu, srednjeevropsko ilirsko-balkanskom podregionu i panonskoj provinciji (Stevanović, 1995), unutar holarktičke oblasti i eurosibirske podoblasti. Međutim, zbog njenog prelaznog karaktera i veoma specifičnog florističkog i vegetacijskog sastava ubraja se u poseban floristički podokrug Vrsackibrgense okruga Pannonicum (Panjković, 1989). Zbog veoma bogatog vegetacijskog i florističkog diverziteta i uopšte biodiverziteta Vršačkih planina ovaj pre svega šumski ekosistem zaslužuje posebnu pažnju i posebnu zaštitu. Iako je relativno dobro istražen, istraživanja su i dalje neophodna, pre svega zbog veoma izraženih negativnih antropogenih uticaja. Takođe smatram da neki predstavnici flore, koji su autohtone lekovite biljke, posle sveobuhvatnog sagledavanja stanja njhovih populacija mogu postati značajne sirovine za fitofarmaciju i ftoterapiju.

LITERATURA Anđelković, M. (1982): Geologija Jugoslavije. Univerzitet u Beogradu, Beograd.

Boža, P. (1999): Astragalus varius S. G. Gmelin in Crvena Knjiga flore Srbije I ed. Stevanović, V. 102 - 104. Ministarstvo za životnu sredinu Republike Srbije, Biološki fakultet Univerziteta u Beogradu, Zavod za zaštitu prirode Srbije, Beograd

Bugarski, D., Tomić, P. (1987): Klimatske prilike Vršca i okoline in Monografije Vršačkih planina ed. Mihaljev, I. 1 - 63, Matica Srpska, Novi Sad.

Bugarski, D., Tomić, P. (1988): Hidrogtafske karakteristike Vršačkih planina in Monografije Vršačkih planina ed. Mihaljev, I. 1 - 44, Matica Srpska, Novi Sad.

Bukurov, B. (1950): Vršačke planine. Posebna izdanja Matice srpske. Novi Sad.

Diklić, N., Lakušić, D. (1999): Barbarea vulgaris R. Br. subsp. lepuznica (Nyár.) Soó in Crvena Knjiga flore Srbije I ed. Stevanović, V. 221 – 222. Ministarstvo za životnu sredinu Republike Srbije, Biološki fakultet Univerziteta u Beogradu, Zavod za zaštitu prirode Srbije, Beograd

Heuffel, J. (1850): Enumeratio Plantarum in Banatu Temesiensii spontae crescentium et fraquentius cultarum. Windobonae.

Kojić, M., Popović, R., Karadžić, B. (1998): Sintaksonomski pregled vegetacije Srbije. Beograd

Kukin, A., Kemenci, R., Janković, P. (1987): Geologija Vršačkih planina in Monografije Vršačkih planina ed. Mihaljev, I. 1 - 64, Matica Srpska, Novi Sad.

Maličević, M. (2008): Floristički i vegetacijski diverzitet Vršačkih planina. Završni rad. Univerzitet u Novom Sadu, Prirodno-matematički fakultet, Departman za biologiju i ekologiju.

Miljković, S. N. (1986): Zemljišta Vršačkih planina in Monografije Vršačkih planina ed. Mihaljev, I. 1 - 67, Matica Srpska, Novi Sad.

Obradović, M. (1988): Retke i reliktne biljke Vršačkih planina. Matica Srpska, Novi Sad.

Panjković Matanović, V. (1989): Biljnogeografska analiza flore Vršakih planina. Matica srpska, Novi Sad.

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Pekanović, V., (1991): Šumska vegetacija Vršačkih planina. Matica srpska, Novi Sad.

Soó, R. (1966): A magyar flóra és vegetáció renszertani, növényföldrajzi kézikönyve, II. Akadémiai kiadó, Budapest.

Soó, R. (1968): A magyar flóra és vegetáció rendszertani, növényföldrajzi kézikönyve, III. Akadémiai kiadó, Budapest

Soó, R. (1970): A magyar flóra és vegetáció rendszertani, növényföldrajzi kézikönyve, IV. Akadémiai kiadó, Budapest

Soó, R. (1973): A magyar flóra és vegetáció rendszertani, növényföldrajzi kézikönyve, V. Akadémiai kiadó, Budapest

Stevanović, V. (1995): Biogeografska podela teritorije Jugoslavije in Biodiverzitet Jugoslavije ed. Stevanović, V, Vasić, V. 117 – 127. Ekolibri, Beograd, Biološki fakultet, Beograd, Beograd.

Stevanović, V., Jovanović, S., Lakušić, D., Niketić, M (1995): Diverzitet vaskularne flore Jugoslavije sa pregledom vrsta od međunerodnog značaja in Biodiverzitet Jugoslavije ed. Stevanović, V, Vasić, V. 183 – 217. Ekolibri, Beograd, Biološki fakultet, Beograd, Beograd.

Sučević, P. (1962): Šumske fitocenoze Vršačkih planina. Rad Vojvođanskih muzeja br. II, Novi Sad.

Vučković, M. (1991): Livadska i livadostepska vegetacija Vršačkih planina. Matica srpske, Novi Sad.

Zeremski, M. (1985): Geomorfologija Vršačkih planina in Monografije Vršačkih planina ed. Mihaljev, I. 1 - 59, Matica Srpska, Novi Sad.

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PL1 Diversity of flora and vegetation of the Vršac mountains

Boža Pal University of Novi Sad, Faculty of natural sciences, Department of biology and ecology 2 Dositej Obradović square, 21000 Novi Sad, Republic of Serbia

ABSTRACT

The Vršac mountains represent the unique geomorphologic unit in Southeast Banat. They are placed between Banat mountains in the South and East and Pannonian plain in the West (Panjković-Matanović, 1989). The Vršac mountains represent the remainings of old Pannonian mass from post-Cretaceous, Oligo-Miocene period and belong to tectonic row of Moravides, i.e. the Rodop mass (Anđelković, 1982). On very diverse geological basis, diverse pedological layer is developed: cambisols, vertisols (Miljković, 1986). Since their specific position, the Vršac mountains are influenced by various phytogeographic horions: Pannonian, Dacian, Balkan and Pontian province. The Vršac mountains are distinguished by very specific flora and vegetation because of the specific soils and climates. Vegetation diversity of the Vršac mountains is very distinct, with 67 syntaxonomic units, from classes to subassociations. It is important to mention that the diversity of forest ecosystems is richer than the one of meadow ecosystems (37:31 syntaxonomic units). The forest associations belong to class Querco-Fagetea Br.-Bl. & Vilinger, while the meadow associations belong to classes Molinio-Arrhenatheretea R. Tx, and Festuco-Brometea Br.-Bl. & R. Tx. (Maličević, 2008). Floristic diversity of the Vršac mountains is also very diverse. From the beginning of research of flora (Heuffel, 1858) till today, 1100 species are detected in this area in comparison to 3600 species detected on the territory of the whole Serbia. The ratio of area size is 177km2: 88 361km2 (Panjković, Matanović, 1989; Maličević 2008). Flora of the Vršac mountains contains rare, endangered, protected and endemic species. In this work, the representatives of plants from these groups will be presented and their importance will be emphasized. Some of the analyzed plants are Campanula lingulata W. & K., Balkan species; Iris pumila L., Pontian-Pannonian species; Nepeta nuda L., continental species; Barbarea lepuznica Nyarady, Romanian endemic species; Allium atropurpureum W. & K. and Euphorbia pannonica Host are Pannonian-Balkan species, etc. The mentioned taxa are, besides their phytogeographic diversity, important from other aspects as well. Besides the fact that they have limited areal, some of them represent taxonomic problems, they are placed on areal borders and form the vicarian chain. Campanula lingulata is, on Vršac mountains, placed on the northern border of its distribution. Within species Iris pumila there are taxonomic problems that need to be solved. For the populations of this species in Serbia, the place in vicarian chain from Romania to Adriatic sea should be determined. According to some researchers, Nepeta nuda includes also the taxa Nepeta pannonica although samples of this complex from Pannonian plain and mountain region of Serbia clearly differ. Barbarea lepuznica is a Romanian endemic plant and Vršac mountains are the Western border of its distribution. Allium atropurpureum and Euphorbia pannonica belong to connecting elements of flora and imply floro-genetic connections of Pannonian plain and Balkan peninsula.

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REFERENCES Anđelković, M. (1982) Geologija Jugoslavije. Univerzitet u Beogradu. Beograd

Heuffel, J. (1858) Enumeratio Plantarum in Banatu Temesiensi sponte crescentium et frequentius cultarum. Wienna

Maličević M. (2008) Floristički, vegetacijski diverzitet Vršačkih planina Master work, PMF Novi Sad.

Miljković, N. (1986) Zemljišta Vršačkih planina. Matica Srpska. Novi Sad.

Panjković-Matanović, V. (1989) Biljnogeografska analiza flore vršačkih planina. Matica Srpska. Novi Sad.

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PL2 Neke samonikle biljne vrste Srbije i Crne Gore kao potencijalni izvori lekovitih sirovina Slobodan Milosavljević Hemijski fakultet Univerziteta u Beogradu Fitohemijska ispitivanja na Hemijskom fakultetu u Beogradu i Centru za Hemiju (IHTM), od sredine devedesetih godina prošlog veka do danas, obuhvatila su oko 60 samoniklih biljnih vrsta iz Srbije i Crne Gore. Ispitivani su predstavnici sledećih familija: Asteraceae, Apiaceae, Clusiaceae, Gentianaceae, Boraginaceae i Dipsacaceae. U ovom predavanju su prikazani sekundarni metaboliti i njihove biološke aktivnosti sledećih biljnih vrsta: 1. Familija ASTERACEAE: 1.1. Achillea atrata ssp. multifida (DC) HEIM. [1] 1.2. A. clavennae L. [2] 1.3. Amphoricarpos neumayeri Vis. [3]1.4. A. autariatus ssp. autariatus Blečić et Mayer i A. autariatus ssp. bertisceus Blečić et Mayer [4,5]1.5. Centaurea derventana Vis. & Pančić [6] 1.6. C. nicolai Bald. [7] 1.7. Tanacetum larvatum (Gris.) Kanitz. [8] 2. Familija GENTIANACEAE: 2.1. Swertia punctata Baumg. [9] 2.2. Gentiana dinarica Beck. [10] 2.3. G. kochiana Perr. et Song.[11, 12] 2.4. Gentianella austriaca (A&J Kerner) Holub [13, 14] 2.5. G. bulgarica (Velen.) Holub [13] 2.6. G. albanica (Jav.) Holub [13] 2.7. G. crispata (Vis.) Holub [13] Sve navedene vrste sadržavale su biološki aktivna jedinjenja sledećeg tipa: seskviterpenske laktone i flavonoide (Asteraceae) i ksantone, seko-iridoide i C-glukoflavone (Gentianaceae). Biološki testovi su obuhvatili sledeće aktivnosti: antimikrobnu, citototoksičnu, redioprotektivnu i antioksidativnu. Literatura: 1. Aljančić I, Vajs V, Menković N, Karadžic I, Juranić N, Milosavljević S, Macura S. (1999), J. Nat. Prod. 62: 909-911. 2. Trifunović S, Vajs V, Juranić Z, Žižak Ž, Tešević V, Macura S, Milosavljević S. (2006), Phytochemistry 67: 887-893. 3. Djordjević I, Vajs V, Bulatović V, Menković N, Tešević V, Macura S, Janaćković P, Milosavljević S. (2004)Phytochemistry 65: 2337–2345. 4. Djordjević I, Jadranin M, Vajs V, Menković N, Tešević V, Macura S, Milosavljević S. (2006), Z. Naturforsh. B 61: 1437-1442. 5. Atrrog AAB, Natić M, Tosti T, Milojković-Opsenica D, Djordjević I, Tešević V, Jadranin M, Milosavljević S, Lazić M, Radulović S, Tešić Ž. (2008), Biomed. Chromatography, in press. 6. Tešević V, Vajs V, Janaćković P, Todorović N, Djoković D, Marin P, Milosavljević S. (1998), Planta Med. 64: 488. 7. Vajs V, Todorović N, Ristić M, Tešević V, Todorović B, Janaćković P, Marin P, Milosavljević S. (1999), Phytochemistry 52: 383-386. 8. Aljančić I, Vajs V, Bulatović V, Menković N, Milosavljević S. (2001) Biochem. Syst. Ecol. 29: 655-657. 9. Menković N, Šavikin-Fodulović K, Bulatović V, Aljančić I, Juranić N, Macura S, Vajs V, Milosavljević S. (2002), Phytochemistry 61: 415–420. 10. Krstić D, Janković T, Aljančić I, Šavikin-Fodulović K, Menković N, Milosavljević S. (2004) Biochem. System. Ecol. 32: 937–941. 11. Tomić M, Tovilović G, Butorović B, Krstić D, Janković T, Aljančić I, Menković N. (2005) Pharmacol Biochem Behavior 81: 535-542. 12. Isaković A, Janković T, Harhaji Lj, Kostić-Rajačić S, Nikolić Z, Vajs V, Trajković V. (2008) Bioorg. Med. Chem. 16: 5683-5694. 13. Janković T, Krstić D, Aljančić I, Šavikin-Fodulović K, Menković N, Vajs V, Milosavljević S. (2005), Biochem. Syst. Ecol. 33: 729-735. 14. Janković T, Šavikin K, Menković N, Aljančić I, Leskovac A, Petrović S, Joksić G. (2008) Planta Med. 74: 736-740.

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PL2 Some wild growing plants from Serbia and Montenegro as a potential source of herbal drugs Slobodan Milosavljević Faculty of Chemistry, University of Belgrade Phytochemical examinations carried at the Faculty of Chemistry, Belgrade, and the Centre for Chemistry (Institute for Chemistry, Technology and Metallurgy, Belgrade), since the mid-nineties, involved ca. 60 wild-growing plant species from Serbia and Montenegro belonging to the following families: Asteraceae, Apiaceae, Clusiaceae, Gentianaceae, Boraginaceae i Dipsacaceae. In this lecture the secondary metabolites of the species listed above (see 1 and 2) together with their biological activities are are presented. All quoted species contained the following types of biologically active compounds: sesquiterpene lactones and flavonoids (Asteraceae), xanthones, secoiridoids and C-glucoflavones (Gentianaceae). References: 1. Aljančić I, Vajs V, Menković N, Karadžic I, Juranić N, Milosavljević S, Macura S. (1999), J. Nat. Prod. 62: 909-911. 2. Trifunović S, Vajs V, Juranić Z, Žižak Ž, Tešević V, Macura S, Milosavljević S. (2006), Phytochemistry 67: 887-893. 3. Djordjević I, Vajs V, Bulatović V, Menković N, Tešević V, Macura S, Janaćković P, Milosavljević S. (2004)Phytochemistry 65: 2337–2345. 4. Djordjević I, Jadranin M, Vajs V, Menković N, Tešević V, Macura S, Milosavljević S. (2006), Z. Naturforsh. B 61: 1437-1442. 5. Atrrog AAB, Natić M, Tosti T, Milojković-Opsenica D, Djordjević I, Tešević V, Jadranin M, Milosavljević S, Lazić M, Radulović S, Tešić Ž. (2008), Biomed. Chromatography, in press. 6. Tešević V, Vajs V, Janaćković P, Todorović N, Djoković D, Marin P, Milosavljević S. (1998), Planta Med. 64: 488. 7. Vajs V, Todorović N, Ristić M, Tešević V, Todorović B, Janaćković P, Marin P, Milosavljević S. (1999), Phytochemistry 52: 383-386. 8. Aljančić I, Vajs V, Bulatović V, Menković N, Milosavljević S. (2001) Biochem. Syst. Ecol. 29: 655-657. 9. Menković N, Šavikin-Fodulović K, Bulatović V, Aljančić I, Juranić N, Macura S, Vajs V, Milosavljević S. (2002), Phytochemistry 61: 415–420. 10. Krstić D, Janković T, Aljančić I, Šavikin-Fodulović K, Menković N, Milosavljević S. (2004) Biochem. System. Ecol. 32: 937–941. 11. Tomić M, Tovilović G, Butorović B, Krstić D, Janković T, Aljančić I, Menković N. (2005) Pharmacol Biochem Behavior 81: 535-542. 12. Isaković A, Janković T, Harhaji Lj, Kostić-Rajačić S, Nikolić Z, Vajs V, Trajković V. (2008) Bioorg. Med. Chem. 16: 5683-5694. 13. Janković T, Krstić D, Aljančić I, Šavikin-Fodulović K, Menković N, Vajs V, Milosavljević S. (2005), Biochem. Syst. Ecol. 33: 729-735. 14. Janković T, Šavikin K, Menković N, Aljančić I, Leskovac A, Petrović S, Joksić G. (2008) Planta Med. 74: 736-740.

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PL3 Nova dostignuća u gajenju lincure (Gentiana lutea) i brđanke (Arnica montana) u Srbiji Dragoja Radanović Institut za proučavanje lekovitog bilja „Dr Josif Pančić“, Beograd, Tadeuša Košćuška 1 E-mail: dradanovic@mocbilja.rs

Uvod Koren žute lincure (Gentianae radix) i cvet brđanke (Arnicae flos) su na tržištu lekovitog bilja dve izuzetno tražene sirovine. Njihova cena na legalnom tržištu ima stalnu tendenciju rasta a potražnja permannentno nadmašuje ponudu. Obe vrste su slične i po tome što je njihov spontani resurs dramatično smanjen i ugrožen tokom poslednjih decenija a ponuda iz samoniklog izvora je najčešće limitirana vrlo oštrom zakonskom regulativom u gotovo svim zemljama. Tako je brđanka (Arnica montana) uvrštena u nekoliko nacionalnih Crvenih Knjiga, kao što je slučaj u Ukrajini, Češkoj, Litvaniji, Holandiji, Portugalu, Rumuniji, Nemačkoj i Mađarskoj (Lange, 1998). Ekspoatacija žute lincure iz prirode zabranjena je u gotovo svim zemljama Evrope tako da se tržište jednim delom zadovoljava iz „sivog tržišta“ pa se sirovina Gentianae radix ponekad u prometu pojavljuje pod drugim imenima. U takvim okolnostima logično se nameće zaključak da je održivo gajenje ovih dveju vrsta najbolji način za prevazilaženje ovog problema.

U celini gledano iskustva u gajenju lincure i brđanke su vrlo skromna. Tokom nekoliko poslednjih decenija u mnogim zemljama Evrope sprovedena su istraživanja sa ciljem upoznavanja i unapređenja tehnologije gajenja lincure (Barralis et al., 1978; Franz and Fritz 1978; Bezzi et al., 1986; 1996; Galambosi, 1996, 2004). Slično lincuri i o gajenju vrste Arnica montana u pojedinim zemljama Evrope može se naći samo manji broj radova koji se odnose na definisanje prihvatljive tehnologije njenog gajenja (Heger, 1956; Munoz, 1987; Bomme, 1995, 1999; Dachler und Pelzmann, 1999, Galambosi, 2004). Na prostoru Balkana istraživanja u oblasti gajenja lincure i brđanke su novijeg datuma. Pored radova u Srbiji, kojih je u poslednje vreme najviše, publikovani su početni rezultati ispitivanja gajenja nekoliko autohtonih ekotipova lincure na različitim lokalitetima u Sloveniji (Kušar and Baričevič, 2006).

U Srbiji su prvi pokušaji gajenja na otvorenom polju sprovedeni na Šar Planini sa vrstom Arnica chamissonis koja u izvesnim namenama može da se koristi slično kao i A. montana (Roki et al., 2001). Prva eksperimentalna plantaža žute lincure koja je krajem devedesetih zasnovana na planini Suvobor, proizašla je iz istraživačkih projekata Ministarstva nauke koje je sprovodio Institut „Dr Josif Pančić“ i druge naučne institucije iz Srbije ali publikovani rezultati o agrotehničkom aspektu ogleda nisu objavljeni.

Eksperimenti gajenja koji su sprovedeni u okviru više projekata Ministarstva nauke, (iz okvira: Tehnološki razvoj, i Nacionalnog program), u periodu od 2002. do 2008. godine rezultirali su većim brojem radova u koji imaju za predmet agronomski aspekt gajenja lincure (Radanović et al., 2005a i b; 2006; 2007a i b; Nastovska et al., 2007) i brđanke (Radanović et al., 2006; 2007c, d i e, Ristić et al., 2007). Rezultati ovih istraživanja biće sažeti u ovom radu.

Rezultati istraživanja tehnologije gajenja lincure u Srbiji Uslovi uspevanja Kroz eksperimente i putem eksperimentalnih plantaža utvrđeno je da lincura u Srbiji dobro uspeva na planinskim lokalitetima koji se nalaze na nadmorskim visinama iznad 1000. m. Lincura može da raste i na nižim pozicijama ali u tom slučaju postoji veća mogućnost da kultura strada od visokih temperatura. U takvim slučajevima bolje je da lokalitet ne bude direktno izložen suncu u dužem periodu tokom dana.

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Zemljlište za lincuru treba da bude dubine bar 40 - 50 cm a poželjno i dublje. Zemljišta na prirodnim staništima lincure na tri planine (Suvobor, Maljen i Povlen) u Srbiji su u tipu distričnog smeđeg, formirana na eruptivnim stenama sa litičnim kontaktom na 40 – 70 cm. Zemljišta su lakšeg mehaničkog sastava manje ili više skeletoidna, dobre vodopropusnosti. Reakcija zemljišta je srednje i jako kisela, uglavnom uvek ispod 6,4 (pH/H2O) odnosno ispod 5,6 (pH/KCl) (Radanović i sar., 2005a). Ovaj rezultat u izvesnoj meri demantuje do sada preovlađujuće mišljenje da samonikla lincura u Srbiji raste isključivo na krečnim zemljištima. U svakom slučaju kroz sopstveno iskustvo (Radanović i sar., - nepublikovano) u podizanju i praćenju rasta lincure na više eksperimentalnih plantaža u Srbiji i ogleda sprovedenih sa primenom krečnjaka, potvrđeno je da lincura dobro uspeva na srednje do slabo kiselom zemljištu.

Navodnjavanje lincure u početnim godinama gajenja, posebno u prvoj, pokazalo se kao veoma poželjna mera koja doprinosi stabilnosti proizvodnje. Zbog toga, prilikom izbora mesta gajenja treba da se u blizini obezbedi stabilan izvor kvalitetne vode za navodnjavanje.

Zasnivanje plantaža – proizvodnja sadnica Zasnivanje plantaža lincure odvija se putem sadnica. Priozvodnja sadnica lincure za masovnu poljoprivrednu proizvodnju koje po kvalitetu i ceni zadovoljavaju uzgajivače dugo je bila glavni ograničavajući faktor koji je sprečavao bilo kakve ozbiljnije pookušaje da se lincura gaji na ekonomski prihvatljliv način u Srbiji. Beshormonskim postupkom proizvodnje sadnica žute lincure (Gentiana lutea L.) u otvorenim zimskim lejama, koji je proizašao kao rezultat jednog od projekata, se na pouzdan i potpuno prirodan način prekida dormantnost semena žute lincure (Gentiana lutea L.) i pobuđuje njegova klijavost, čime se postiže zadovoljenje agrotehnoloških i ekonomskih zahteva za uspešnu proizvodnju kvalitetnih sadnica. Ovom procedurom je na racionalan način ukomponovan i iskorišćen prirodni uticaj faktora spoljne sredine na podsticanje klijanja semena lincure a definisanim agrotehničkim postupcima eliminišu se ili svode na minimum mogući negativni uticaji edafskih faktora, čime se postiže efikasnost u više od 95 % slučajeva. Sadnice koje se proizvedu ovim postupkom poseduju neuporedivo bolju vitalnost i kvalitet za upotrebu u polju nego što je imaju sadnice proizvedene u stakleniku tokom zime ili uz primenu giberelinske kiseline. Ove sadnice kvalitetom i ekonomskom cenom koštanja zadovoljavaju zahteve koje treba da ima početni sadni materijal za masovnu rentabilnu plantažnu proizvodnju žute lincure.

Ispitivanjima je bilo obuhvaćeno više modela proizvodnje sadnica prema ovom postupku. Sadnice bez zemljišta kao i one proizvedene u kontejnerima na otvorenom su prihvatljive za sociološko - ekonomske uslove proizvodnje u planinskim krajevima Srbije (Nastovska i sar. 2007).

Prinos i kvalitet korena gajene lincure u Srbiji U istraživanju sprovednom sukcesivno u dve vegetacije (2006 i 2007. godine) na oglednim parcelama lincure različite starosti na planini Tari ostvareni su prinosi korena koji po masi i kvalitetu, u određenim okolnostima već u četvrtoj, a izvesnije, u petoj godini gajenja u polju obezbeđuju rentabilnu proizvodnju i dobru zaradu proizvođačima (Radanović i sar., 2008).

Najmanju težinu koren lincure u plantaži ima u II vegetaciji, prosečno oko 11 g suve mase po biljci, koji u slučaju pune gustine po ha (0,7 x 0,25 m) obezbeđuje prinos od 600 kg ha-1. Sadržaj genciopikrina bio je u intervalu 70 – 110 mg g-1 d. w a stepen gorčine iznad 15000. Biljke na kraju III vegetacije postižu masu suvog korena od 25 g po biljci što pri punom sklopu daje prinos od oko 1500 kg ha-1. Sadržaj genciopikrina u korenu bio je na nivou 65 – 110 mg g-1 d. w. a stepen gorčine iznad 10000.

U IV vegetaciji suv koren postiže težinu od oko 70 g po biljci koje mogu obezbediti prinos od oko 4 t korena po hektaru. Sadržaj genciopikrina je u intervalu 44 – 77 mg g-1 d. w. a stepen gorčine iznad 15000. Najviši prinos suvog korena se postiže u V vegetaciji, oko 120 g po biljci, što pruža osnov za prinos od preko 6000 kg ha-1. Sadržaj genciopikrina u korenu je u intervalu 60 – 90 mg g-1 d. w. a stepen gorčine iznad 10000. Odnos težine svežeg i suvog korena varira u intervalu 4 : 1 do 3 : 1 (Grafikon 1.).

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Grafikon 1. Prinos korena gajene lincure (Gentiana lutea) različite starosti u grama po biljci sveže i suve mase (The yeald of the gentiam root obtained in different years of culivation / dry and fresh weight in g per plant)

96,7

202,8

270,9

410,5

67,343,6

12,8

381,5

72,2

3,1 11,0 19,8 25,859,3

108,3 118,6

0

100

200

300

400

500

600

II year /July

II year /October

III year /July

III year /October

IV year /July

IV year /October

V year /July

V year /October

age / season

gram

s

root fesh 06/07 root dry 06/07 Rezultati istraživanja tehnologije gajenja brđanke u Srbiji Uslovi uspevanja Brđanka najbolje uspeva u planinskim predelima sa hladnijom i vlažnom klimom a prema našim iskustvima iz prethodnih godina može se reći da joj u Srbiji najviše odgovarjau nadmorske visine od 900 do 1.500 m.

Poznato je da je brđanka osetljiva na Fe hlorozu pa za uspešan rast i razvoj zahteva kisela zemljišta (pH = 5 – 6). Vrlo je osetljiva na prisustvo karbonata odnosno fiziološki aktivnog kreča. Osetljivost ispoljava čim je sadržaj CaCO3 u zemljištu >1,5 %. U našim planinskim krajevima preovlađuju kisela zemljišta. Prena našim istraživanjima (Radanović i sar., 2004) pH reakcija planinskih zemljišta je bila najčešće u intervalu 4,5 – 6,5 (pH/H2O) pa se može smatrati da su sa toga aspekta zemljišta naših planinskih krajeva pogodna za gajenje brđanke. Pored toga brđanka preferira rastresita, humozna zemljišta sa dobrim vodno – vazdušnim režimom (Dachler und Pelzman, 1999). Većina naših planinskih zemljišta ispunjava i ove uslove pošto su najčešće dobrih fizičkih karakteristika.

Obezbeđenost naših planinskih zemljišta biljnim hranivima je na vrlo niskom nivou, a naročito je izražen nedostatk fosfora (Radanović i sar., 2004). Obzirom da se brđanka gaji zbog cveta, prilikom sprovođenja mera fertilizacije, posebna pažnja treba da se posveti ovom biogenom elementu. Prema našoj analizi rezultata proizvodnje cveta A. chamissonis kod proizvođača u Srbiji, plantaža gajena na zemljištu koje je bilo dobro obezbeđeno hranivima dala je značajno više prinose cveta koji je bio dobrog kvaliteta (Radanović i sar., 2007d).

Razmnožavanje i zasnivanje plantaža brđanke Razmnožavanje – Arnika se razmnožava generativno i vegetativno. Razmnožavanje semenom se češće primenjuje u savremenoj proizvodnji, pogotovu u početnim godinama gajenja.

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Obzirom da je seme veoma sitno (težina 1000 semenki je u intervalu od 1,17 – 1,42 g) direktna setva u polje ne daje zadovoljavajuće rezultate. U eksperimentima direktne setve sprovedenim u Nemačkoj (Bomme, 1999) arnika je sejana u periodu juli – avgust, u redove na rastojanju 30 i 40 cm između redova, pri čemu seme nije pokrivano zemljom a primenjivano je pokrivanje PE folijama. I pored velike pažnje i truda nisu postignuti prinosi koji bi opravdali primenu direktne setve u širokoj praksi pa se proizvodnja sadnica nametnula kao, za sada, sigurniji vid zasnivanja arnike. Kod ovog modela zasnivanja izvode se dve varijante. Zimska proizvodnja sadnica u grejanim staklenicima, započinje setvom u februaru a rasad se u aprilu iznosi napolje na adaptaciju dok se sadnja u tom slučaju najčešće izvodi u prvoj polovini maja. Za klijanje semena i nicanje optimalna temperatura supstrata je od 15 – 20 °C a u ovakvoj proizvodnji danas se često primenjuju kontejneri što značajno povećava kvalitet sadnica i procenat njnihovog prijema u polju. Zbog prolećnih suša potrebno je da se za sadnju obezbedi navodnjavanje, u suprotnom je proizvodnja izložena velikom riziku i cela plantaža može da dođe u pitanje. Letnja proizvodnja sadnica u otvorenim lejama izvodi se na mestu proizvodnje. Leje se zasejavaju krajem maja – početkom juna. Seme se pokriva veoma tankim slojem treseta (0,5 cm) a leje se obavezno zasenjuju i redovno navodnjavaju, prihranjuju i pleve tokom celog leta. Ovako proizvedene sadnice su spremene za rasađivanje na stalno mesto u septembru. Proizvodnja rasada u hladnim lejama je jeftiniji i proizvođačima pristupačniji način.

Vegetativno razmnožavanje brđanke sprovodi se delenjem biljaka (busenova) posle druge i treće godine gajenja. Starije biljke vrste A. montana su pogodne za delenje obzirom da formiraju veliki busen sa 15 – 20 rozeta koji se lako može podeliti, pri čemu se dobijaju sadnice koje imaju rozetu sa pripadajućim delom korena. Sadnja ovakvih sadnica odvija se u septembru ili još bolje krajem avgusta. U uslovima planina Srbije u to vreme je vegetacija još aktivna a ukoliko se obezbedi navodnjavanje posle sadnje postiže se vrlo dobar procenat prijema pre kraja vegetacije. Vegetativni način razmnožavanja teoretski je moguće izvesti i u proleće ali je efikasnost daleko manja nego kod jesenje sadnje. Vrsta A. chamissonis uspešno se razmnožava stolonima, slično pitomoj nani, samo su stoloni sitniji. Pravila u pogledu sadnnje stolona su slična kao i kod pitome nane. I kod A. chamissonis u našim krajevima bolji rezultati su postignuti kad se sadnja stolona obavi u jesen.

Sadnja – Proizvedeni rasad u letnjim lejama kao i sadnice dobijene delenjem bokora rasađuju se na stalno mesto u septembru. Sadnice iz zimske proizvodnje rasađuju se u polje u prvoj polovini maja. Sadi na rastojanju redova, 60 – 70 cm a između biljaka u redu razmak treba da bude 23 – 25 cm. Kod ovakvog načina sadnje, postiže se sklop od oko 60.000 – 72.500 biljaka. U pojedinim evropskim zemljama (Španija, Nemačka) preporučuje se veća gustina biljaka, 100.000 i više po ha ((Munoz, 1987; Bomme, 1999) ali to iziskuje manje rastojanje između redova što nije prilagođeno mehanizaciji koja se uobičajeno upotrebljava za međurednu obradu u našim krajevima. Razmak između redova u praksi je uslovljen raspoloživim sredstvima za međurednu obradu.

Međuredno kultiviranje i okopavanje su najčešće mere nege kojima se sprovodi kontrola korova u kod proizvođača u Srbiji. Herbicid za primenu u arnici nije registovan. Naša istraživanja primene polietilenskih (PE) folija u proizvodnji vrste A. montana (Radanović i sar., 2007e) dala su određene pozitivne rezultate ali ostaje da se reši problem visokih temperatura koje se javljaju pod ovakvim folijama obzirom da ispitivane folije ne propuštaju vazduh i atmosferske padavine.

Prinos i kvalitet cveta gajene brđanke u Srbiji Prinos cveta gajene brđanke se dosta razlikuje u zavisnosti od starosti zasada, uslova gajenja i primenjenih agrotehničkih mera. U eksperimentima i proizvodnji u Nemačkoj, Finskoj i drugim zemljama prinos cveta je varirao u intevalu od 150 – preko 1000 kg/ha suvog cveta (Bomme, 1999; Galambosi, 2004). Najviši prinosi se u principu postižu od II – IV godine.

U tabelama 1, 2 i 3 prikazani su prinosi i kvalitet cveta dve vrste roda Arnica koje su tokom prethodnih nekoliko godina testirane i eksperimentalno gajene u Srbiji (Radanović i sar., 2007d)

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Tabela 1. Kumulativni prinosi cveta A. montana tokom perioda žetve u pojedinim godinama u eksperimentima sprovedenim na Tari (2004 – 2007) (The yields of Arnica montana and harvesting periods in perennial experiments conducted on Tara locality) (Radanović i sar., 2007d)

Flower yield (kg ha-1) (Harvesting period, dd. mm.) Model of crop

establishment Locality

Transplantation date I

vegetation I vegetation

I vegetation

I vegetation

2004 year 2005 year 2006 year 2007 year Nursery plants* Tara

3rd decade of May 2003

30 (06.06.-26.07.)

340 (14.06.-29.06.)

200 (31.05.-26.06.)

100 (18.05.-30.05.)

2006 2007 Nursery plants** Tara (+ PE mulch)

September 2005

90 – 110 (26.05.-22.06.)

2007 year Nursery plants* Tara

3rd decade of May 2006

25 (18.05.- 07.06.)

*- kontejnerske sadnice (60 ml) proizvedene u zatvorenom prostoru u periodu II – IV mesec nursery plants production in plastic house during II – IV month, in containers of 60 ml volume **- sadnice proizvedene u stakleniku tokom perioda II – IV a posle toga držane na otvorenom od V – IX meseca u kontejnerima 500 ml zapremine (nursery plants production in green house during II – IV month and left outdoors during period V – IX month, in containers of 500 ml volume). Tabela 2. Prinosi cveta A. chamissonis var. foliosa i trajanje perioda žetve tokom godine u višegodišnjim eksperimentima i eksperimentalnoj proizvodnji u Srbiji (2004–2007). (The flower yields of Arnica chamissonis var. foliosa and harvesting periods in perennial experiments conducted on Tara and Sevojno) (Radanović i sar., 2007d)

Flower yield (kg ha-1) (Harvesting period, dd. mm.) Model of crop

establishment Locality

Transplantation date I

vegetation II vegetation

III vegetation IV vegetation

2004 year 2005 year 2006 year 2007 year Nursery plants* Tara

October 2003

186 (06.07.-20.08.)

343 (14.06.-29.06.)

426 (22.06.-04.07.)

45 (10.06.-05.07.)

2006 year 2007 year Stolones Tara

October 2005

90 (11.07.-01.08.)

200 (10.06.-12.07.)

Stolones Sevojno

October 2005

159 (10.07.-07.08.)

370 (15.05.-12.07.)

* sadnice proizvedene u otvorenim lejama (VII – IX) (produced in open beds (VII – IX month) at experimental locality)

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Tabela 3. Sadržaj ukupnih seskviiterpenskih laktona u suvom cvetu dve vrrste roda Arnica gajene u periodu 2005 – 2007. u Srbiji (Content of total sesquiterpene lactones in dry flowers of two cultivated Arnica species, during several years of exploitation 2005 - 2007). (Radanović i sar., 2007d)

Year of analysis

Species Season (month / year)

Harvest / date (dd.mm.)

Total sesquiterpene lactones (expressed as helenalin tiglate) in dry Arnica flowers (%)

I harvest / 14. 06. 1,25 III harvest / 20. 06. 1,21 2005. A. montana

established V / 2003. V harvest / 29. 06 1,26 I harvest / 31. 05. 1,49 III harvest / 13. 06. 1,35 A. montana

established V / 2003. IV harvest / 26.06. 1,54 I harvest / 26. 05. 1,55 – 2,49 II harvest / 31. 05. 1,36 – 1,71 III harvest / 06. 06. 1,29 – 2,31 IV harvest / 13. 06. 1,88 – 2,30

2006. A. montana established IX / 2005. on PE mulch

V harvest / 22. 06 2,25 – 2,70 I harvest / 18. 05. 1,45 A. montana

established V / 2003. III harvest / 30. 05. 1,33 I harvest / 20. 05. 1,49 – 3,00 II harvest / 28. 05. 1,37 – 3,12

2007. A. montana established IX / 2005. on PE mulch III harvest / 08. 06 2,10 – 2,49

I harvest / 14. 06. 0,76 III harvest / 20. 06. 0,39 – 0,48 2005. A. chamissonis

Tara - established X / 2003. V harvest / 29. 06 0,24 – 0,40 I harvest / 22. 06. 0,65 III harvest / 30. 06. 0,60 2006. A. chamissonis

Tara - established X / 2003 IV harvest / 04. 07. 0,70 I harvest / 15. 05. 0,64 III harvest / 08. 06. 0,98 A. chamissonis

Sevojno established X / 2005. VI harvest / 12. 07 1,52 I harvest / 10. 06. 0,54 III harvest / 21. 06. 0.64

2007. A. chamissonis Tara - established X / 2005. V harvest / 12. 07 0,65

Na osnovu rezultata naših istraživanja može se zaključiti da u planinskim krajevima Srbije može da se proizvede cvet brđanke dobrog kvaliteta sa aspekta sadržaja ukupnih seskviterpenskih laktona izraženih kroz sadržaj dihelenalin tiglata (Tabela 3). Relativno niski prinosi cveta (Tabela 1 i 2) koji su postignuti u eksperimentima i oglednoj proizvodnji treba da se unaprede kroz dalje usavršavanje agrotehničkih mera među koje po našem mišljenju treba da u prvom redu treba da se uvrste fertilizacioni ogledi kao i definisanje optimalne gustine biljaka prilagopđene za sociološko - ekonomske uslove i nivo agrotehnikčke opremljenosti naših planinskih krajeva. Literatura 1. Barralis G., Chadoeuf R., Desmarest P., 1978. New trends of Gentiana lutea cultivation, Acta Horticulturae, 73, 303

– 306. 2. Bezzi A., Aiello N., Tartarotti M., 1986. La coltivazione di Gentiana lutea L. in ambienti montani, Estrato da

Agricoultura Ricerca n. 62/1986, Instituto di Tecnica e Propaganda Agraria, Roma, Italy.

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3. Bezzi A., Vender C., Scartezzini F., 1996. Dati morfologici e produttivi rilevati su coltivazioni di Gentiana lutea L. (1981 – 1994), Atti del Convegno „Genziana e specie amaro – aromatiche Ricerche ed Applicazioni“, Camerino Universita degli Studi, Camerino (Macerata) Italia, 35 – 56.

4. Dachler M. und Pelzman H. 1999. Arznei- und Gewürzpflanzen, Anbau, Ernte und Aufbereitung, Zweite überarbeitete Auflage, Österreichisher Agrarferlag, 134 – 136.

5. Franz CH., Fritz D., 1978. Cultivation aspects of Gentiana lutea L. Acta Horticulturae., 73: 307 – 314. 6. Franz CH., Bezzi A., Belliardo F., 1996. Variability and breeding of Gentiana lutea L. with respect to bitter

substances, Atti del Convegno „Genziana e specie amaro – aromatiche Ricerche ed Applicazioni“, Camerino Universita degli Studi, Camerino (Macerata) Italia, 29 – 34.

7. Galambosi B., 1996. Experiences of cultivation Gentiana lutea L. in Finland, Atti del Convegno „Genziana e specie amaro – aromatiche Ricerche ed Applicazioni“, Camerino Universita degli Studi, Camerino (Macerata) Italia, 139 – 142.

8. Galambosi B. 2004: Introduction of Arnica montana L. in Finland, Z. Arzn. Gew. Pfl., 9, (4), 174 - 180 9. Heger E. F., 1956. Handbuch des arznei- und gewürzpflanzenbaues, Deutscher Bauernferlag, Leipzig, 261 – 267. 10. Kušar A., Baričevič D., 2006. Cultivation trials of Yelow Gentian (Gentiana lutea L., subsp. symphyandra Murb.) in

west part of Slovenia (in slovenian), Acta agriculturae Slovenica, 87 – 2, september 2006, 213 – 224. 11. Lange, D., 1998. Europe’s medicinal and aromatic plants: their use, trade and conservation. Traffic Europe /

International, Cambridge UK. 12. Nastovski T., Radanović, D., Antić Mladenović, S, Jevđović, R. (2007): Bezhormonska proizvodnja sadnica lincure

(Gentiana lutea) u autentičnim planinskim uslovima Srbije kao preduslov njene uspešne proizvodnje, III simpozijum sa međunarodnim učešćem „Inovacije u ratarskoj i povrtarskoj proizvodnji“, Poljoprivredni fakultet Beograd, 19 – 20 oktobar 2007. g. Zbornik izvoda, 192 – 193.

13. Radanović D., Nešić Lj., Sekulić P., Belić M., Maksimović S., (2005a): Physical and chemical characteristics of the soils of some natural sites of yellow gentian in west Serbia, Zemljište i biljka, 54, 3, 183 – 192

14. Radanović, D., Stepanović, B., Nastovski, T. (2005b): “Some experiences in nursery plants production of Yellow Gentian (Gentiana lutea L.) with an accent on its seed dormancy”, Savremena poljoprivreda, vol. 54, br 3 - 4, str. 474 - 481

15. Radanović, D., Nastovski, T., Pljevljakušić D., Jevđović R. (2006) “Growing results of some MAP species at mountaneous region of Serbia ”, III Conference on MAP of Southeast European Countries, Nitra, Slovak Republic, Proceedings, 84 - 93.

16. Radanović D., Nastovski T, Janković T, Šavikin K, Menković N, Zdunić G (2007a): The amount of secondary metabolites in cultivated Gentiana lutea L., 55th International Congress and Annual Meeting of Society for Medicinal Plant Research, September 2 – 6 2007, Graz, Austria, Abstracts, pp. 916,

17. Radanović D, Marković T., Janković T. (2007b); Morphological and chemical parameters of importance for cultivation of Gentiana lutea L. in mountain region of Serbia, 1st International Scientific Conference: on Medicinal, Aromatic and Spice Plants, Nitra, Slovakia, December 5 – 6, 2007, Book of Scientific Papers, 28 – 32.

18. Radanović D., Nastovski, T., Pljevljakušić, D. (2007c): Ispitivanje mogućnosti gajenja brđanke (Arnica montana, A. chamissonis) u planinskom regionu Srbije, III simpozijum sa međunarodnim učešćem „Inovacije u ratarskoj i povrtarskoj proizvodnji“, Poljoprivredni fakultet Beograd, 19 – 20 oktobar 2007. g. Zbornik izvoda, 202 – 203.

19. Radanović D, Marković T., Antić – Mladenović S. Pljevljakušić D., Ristić M. Krivokuća Đokić D. (2007d); Yield and quality of Arnica (Arnica montana and Arnica chamissonis var. foliosa) cultivated in Serbia, 1st International Scientific Conference: on Medicinal, Aromatic and Spice Plants, Nitra, Slovakia, December 5 – 6, 2007, Book of Scientific Papers, 157 – 161.

20. Radanović D, Pljevljakušić D., Nastovski T., Ristić M. (2007e); Influence of fertilization model and PE mulch on yield and quality of Arnica (A. montana) at dystric cambisol, Zemljište i Biljka, vol. 56. (3) 85 – 95.

21. Radanović D, Marković T., Janković T. Pljevljakušić D. (2008): Yield and quality of yellow gentian (Gentiana lutea) root grown in mountaneous region of Serbia. V Conference on MAP of Southeast European Countries, Brno, 2 – 5 Sept. 2008., Czech Republic, Book of Abstracts, Pg. 119.

22. Ristić M., Krivokuća-Đokić D., Radanović D., Nastovski T.: Essential oil of Arnica montana and Arnica chamissonis, Hemijska industrija, 61(5), 272-277, 2007.

23. Roki, Đ., Menković, N., Šavikin Fodulović, K., Krivokuća Đokić, D., Ristić, M., Grubišić, D., 2001. Flavonoids and Essential Oil in Flower Heads of introduced Arnica chamissonis, Journal of Herbs, Spices & Medicinal Plants, Vol. 8, (4), 19 – 27.

24. EHGA INVENTORY, 2004. PRODUCTION OF HERBS in existing and incoming member - states of the European Union (2004), European Herb Growers Association (Europam). http://www.europam.net/inventory.htm

25. EUROPEAN PHARMACOPOEIA 5.0, Arnica Flower, 01/2005:1391.

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PL3 Achievements in Gentiana lutea and Arnica montana cultivation in Serbia Dragoja Radanović Institute for medicinal plant research „Dr Josif Pančić“, Belgrade, Tadeuša Košćuška 1 E-mail: dradanovic@mocbilja.rs During the period 2002 – 2007. year several plantations of Gentiana lutea, Arnica montana and Arnica chamissonis were established in conventional mode of cultivation, on locality Kaludjerske Bare, at mountain Tara, Serbia (1004 m a. s. l., g. latitude 43°N, 53', 41'' and g. longitude 19°E, 33', 41'').

In two succeeding vegetations, the root yields of different age Yellow Gentian plantations were measured (the II, III, IV and V year old plantations).The lowest dry root weight had plants in their II vegetation – ca. 11 g plant-1, what would, in case of full crop density (0,7 x 0,25 m), give an ideal yield of ca. 600 kg ha-1. The content of gentiopicrin ranged 70 – 110 mg g-1 d. w., and the bitterness value was above 15000. The plants in their III vegetation yielded ca. 25 g plant-1, and accordingly, the ideal yield they could reach in full crop density is ca. 1500 kg ha-1. The content of gentiopicrin ranged 65 – 110 mg g-1 d. w., and the bitterness value was above 10000.

In the IV vegetation, the root reached dry weight of ca. 70 g plant-1 and the yield in ideal full crop density would have been ca. 4000 kg ha-1. The content of gentiopicrin ranged 44 – 77 mg g-1 d. w., and the bitterness value was above 15000. The highest dry root weight had plants in their V vegetation – ca. 120 g per plant, what would, in case of crop density – 50000 plants ha-1, give the dry root yield of ca. 6000 kg ha-1. The content of gentiopicrin ranged 60 – 90 mg g-1 d. w., and the bitterness value was above 10000. The ratio dry : fresh root weight was averagely 1 : 3,5, ranging in interval from 1 : 3 to 1 : 4.

Perennial investigations in cultivation of two Arnica species showed that in mountainous region of Serbia good quality Arnica flower (herbal drug Arnicae flos that fits demanded requirements) may be produced. In further investigation, the low yields should be improved by optimizing agro-technique measures in order to provide economic profitability of production. Investigation should be directed towards determination of optimal crop nutrition and optimal crop density in perennial establishments.

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PL4 How well the chemical diversity of the flora of Hungary is known? Imre Máthé1,2, Judit Hohmann1, Ákos Máthé3

1Institute of Pharmacognosy, Szeged University, 2Institute of Ecology and Botany of the Hungarian Academy of Sciences 3Department of Botany, University, of West Hungary, Mosonmagyaróvár, Hungary Three different types of information on our chemical knowledge of the flowering plants in Hungray is given. Firstly, our Carpathian floristic region is put on the world scale. Some figures are provided on the world’s living entities among them the flowering plants, their distribution in the main floristic regions and their chemistry how well is known. These figures illustrate that the European flora is a small proportion of the plant kingdom. The great number of endangered plants in our regions, too, makes the evaluation of our flora more and more urgent. Secondly, figures based upon database surveys illustrate the past and present tendencies in plant researches. The methods of comparison is characterized by pointing out to the limits, the credibility of database information sources, predominantly Scifinder (Chemical Abstract and Medline Abstracts were used together with patent journals being referred by Scifinder from the beginning of existing these data, very often from the beginning of the last century.) In all, data on some 2200 species present in the Carpathian basin were checked using their valid Latin species names. It has turned out that many species of the Middle European Flora have not been investigated at all. On the contrary, there are many species, predominantly economic plants which are thoroughly investigated but still can not be regarded as satisfactorily investigated ones. This is well illustrated by the increasing number of new publications on these species. This holds true of such ‘classical’ medicinal plants like Digitalis, Salvia, Lavandula, Hypericum, etc. species. Time-dependent variation of the number of publications shows the changes of interest towards certain plants. One is however common in all cases that even the most thoroughly investigated species are the target of even more and more publications. The numbers of patents on plants has increased in the last two decades. An increasing proportion of both scientific publications and patents in Chinese language can be observed in the last decades. Thirdly, some figures introduce the Hungarian medicinal plant research places, their main research profiles and some more characteristic plant genera, the species of which they studied in the last decades. Finally it could be concluded that, despite of the information boom in plant researches, still there are a lot deficiencies in our knowledge even on the plants in the Middle Europe. Nobody else can investigate this flora but the scientist working in this region. In order to get even better knowledge on our flora a stronger cooperation (joint researches) between the institutions of this region should be necessary.

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PL5 Značaj etarskih ulja u determinaciji vrsta rodova Acinos Miller, Calamintha Miller i Micromeria Bentham (Lamiaceae) Slavkovska, V.1, Jančić, R1., Couladis, M.2, Bojović, S.3 1 Institut za botaniku,Farmaceutski fakultet, Vojvode Stepe 450, 11000, Beograd, Srbija; 2 Department of Pharmacognosy and Chemistry of Natural Products, Panepistmiopolis Zografou, 157 71, Athens, Greece 3 Institut za biološka istraživanja „Siniša Stanković“, Bulevar despota Stefana 142, 11000, Beograd, Srbija Vrste rodova Acinos, Calamintha i Micromeria su poznate kao značajne aromatične biljke zbog prisustva znatne količine etarskog ulja. U sistemima klasifikacije vrste ovih rodova su tretirane na različite načine. Često je jedna ista vrsta svrstavana u različite rodove, što dovodi do konfuzije čak i u pogledu granica izmedju samih rodova. U dosadašnjim taksonomskim ispitivanjima rodova Acinos, Calamintha i Micromeria kao osnova za diferencijaciju vrsta korišćene su morfološke, horološke, citološke i neke hemijske karakteristike (Šilić, 1979; Doroszenko, 1986; Marin, 1989, 1996; Marin et al., 1991, 2001). Osobine etarskih ulja su retko korišćene za definisanje ovih taksona i odredjivanje njihovog položaja u sistemu klasifikacije, prvenstveno zbog toga što je poznato da su etarska ulja veoma varijabilna i zavisna od fenoloških faza kao i ekoloških promena, što otežava njihovu primenu kao taksonomskih markera (Vernet, 1976; Kokkini et al., 1991; Slavkovska, 1996; Slavkovska et al., 2001). Da bi se sagledala ukupna varijabilnost i stepen diferenciranosti odabranih taksona, u okviru svake vrste uzet je uzorak iz različitih ekoloških situacija. Analiza etarskih ulja je obuhvatila vrste roda Acinos: A. arvensis (Lam.) Dandy, A. alpinus (L.) Moench, A. hungaricus (Simonkai) Šilić, A. majoranifolius (Miller) Šilić (ukupno 13 populacija); Calamintha: C. grandiflora (L.) Moench, C. sylvatica Bromf., C. vardarensis Šilić, C. glandulosa (Req.) Bentham (ukupno 13 populacija); Micromeria: Micromeria croatica (Pers.) Schott., M. parviflora (Vis.) Reichenb., M. cristata (Hampe) Griseb., M. juliana (L.) Benth. ex Reichenb., iz sekcije Micromeria i M. albanica (Grisebach ex K. Malý) Šilić, M. dalmatica Benth., M. pulegium (Rochel) Benth., M. thymifolia (Scop.) Fritsch iz sekcije Pseudomelissa (ukupno 16 populacija). Varijabilnost etarskih ulja istraživana je u odnosu na 126 kvantitativnih karaktera koji su statistički obrađeni korišćenjem programa Statgraphics Plus i programa FLORA (Karadžić et al., 1998). Rezultati su pokazali da je među vrstama roda Acinos jedino A. majoranifolius vrsta bogata uljem (0.57%) sa dominantnim pulegonom (71.97%), C-3 oksidovanim monoterpenom mentanske klase, dok su ostale vrste siromašne uljem (0.05% - 0.08%), a količina C-3 oksidovanih monoterpena mentanske klase neznatna (0.6% - 9.03%). Dominantne komponente u etarskim uljima ovih vrsta su: monoterpenski ugljovodonici, α-pinen / limonen / ρ-cimen (A. arvensis); α-pinen / limonen (A. alpinus), kao i oksidovani monoterpeni i seskviterpeni i neterpenska jedinjenja, timol / α-terpinil acetat / kariofilen oksid / alkani (A. hungaricus). Među vrstama roda Calamintha samo je C. grandiflora sa malom količinom ulja (0.22%) i niskom koncentracijom C-3 oksidovanih monoterpena mentanske klase (25.04%). Dominantne komponente u etarskom ulju ove vrste su oksidovani monoterpeni, α-terpinil acetat i pulegon i seskviterpen kariofilen oksid. Ostale vrste roda Calamintha su bogate uljem (0.74% - 0.98%) sa dominantnim C-3 oksidovanim monoterpenima mentanske klase (60.87% - 84.28%). Dominantne

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komponenete u etarskim uljima su: pulegon / piperitenon oksid / menton / piperitenon (C. vardarensis i C. glandulosa) i cis-piperiton epoksid (C. sylvatica). Vrste roda Micromeria sekcije Micromeria su siromašne uljem (0.05 – 0.13%) i C-3 oksidovanim monoterpenima mentanske klase (0.01%-7.3%). Dominantne komponente u etarskim uljima vrsta ove sekcije su seskviterpeni, kariofilen oksid (M. croatica, M. juliana), spatulenol (M. parviflora), neidentifikovano jedinjenje RI 1554 /spatulenol (M. cristata). Vrster sekcije Pseudomelissa su bogate uljem (0.76% - 1.70%) i C-3 oksidovanim monoterpenima mentanske klase (51.80% - 82.07%). Dominantne komponenete u etarskim uljima su:pulegon / cis-piperiton epoksid / piperitenon (M. thymifolia), cis-piperiton epoksid / piperitenon oksid (M. albanica), piperitenon / pulegon (M. dalmatica) i izomenton (M. pulegium) Analiza kvalitativnih i kvantitativnih osobina etarskih ulja je pokazala da je varijabilnost količine i kvaliteta ulja najmanja između populacija iste vrste i da se povećava sa rangom taksona. Veća je između vrsta iste sekcije, a još veća između sekcija ili vrsta istog roda. Varijabilnost je posebno izražena unutar roda Micromeria. Vrste sekcije Pseudomelissa su po količini i sastavu etarskog ulja mnogo bliže vrsti A. majoranifolius i čitavom rodu Calamintha, nego sekciji Micromeria istoimenog roda. Količina ulja je manje promenljiva karakteristika u odnosu na sastav. Sastav ulja je pokazivao veću promenljivost u odnosu na količinu, s tim da je ta promenjivost manje izražena unutar roda Calamintha i sekcije Pseudomelissa roda Micromeria. U uljima ovih predstavnika dominirali su različiti C-3 oksidovani monoterpeni, ali su svi bili mentanskog tipa. Unutar roda Acinos i u sekciji Micromeria roda Micromeria heterogenost je bila jače izrazena, tako da je unutar ovih taksona bilo moguće uočiti nekoliko različitih hemijskih tipova. Relativna stabilnost količine i kvaliteta etarskih ulja roda Calamintha i sekcije Pseudomelissa roda Micromeria znači da si ove osobine, verovatno, pod relativno strogom kontrolom prirodne selekcije i da su od adaptivnog značaja. Shodno tome, možemo reći da se osobine etarskog ulja mogu koristiti kao dopunski karakteri za klasifikaciju vrsta, naročito u pogledu sekcije Pseudomelissa roda Micromeria. Predstavnici roda Acinos i sekcije Micromeria roda Micromeria sadrže etarsko ulje u znatno manjoj količini. Sastav etarskog ulja je varijabilan, tako da je adaptivni značaj ovog karaktera očigledno mali. Usled toga, u slučaju ovih vrsta, a naročito vrsta sekcije Micromeria, kao taksonomski karakter, može se tretirati jedino mala količina etarskog ulja. Ovakvi rezultati ukazuju da bi trebalo razmotriti taksonomski status sekcija u okviru roda Micromeria. Imajući u vidu veću sličnost vrsta sekcije Pseudomelissa sa vrstama roda Calamintha nego sa vrstama tipske sekcije, postoji dosta razloga da se ona pregrupiše i priključi rodu Calamintha. U tom slučaju, vrste sekcije Micromeria bi zadržale svoju generičku pripadnost. Naravno, pri tome treba uzeti u obzir i ostale osobine ovih taksona kako bi se realno i bez sumnje sagledala njihova taksonomska diferencijacija. Zahvalnica: Rezultati su deo projekta koji finansira Ministarstvo nauke Republike Srbije.

LITERATURA

1. Doroszenko, M. A. (1986): Taxonomic studies of Satureja complex (Labiatae). Thesis (Ph.D.). University of Edinburgh. Edinburgh.

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2. Karadžić, B., Saco-Jovanović, V., Jovanović, Z., Popović, R. (1998): "FLORA" a database and software for floristic and vegetation analyzes. In: I. Tsekos, M. Moustakas (eds.) Progress in Botanical Research., Kluwer Academic Press, Dodrecht, 69-72.

3. Kokkini, S., Vokou, D. & Karousou, R. (1991): Morphological and Chemical variation of Origanum vulgare L. in Greece. Botanika Cronica, 10, 337-346.

4. Marin, P. (1989): Hemotaksonomska i mikromorfološka studija roda Micromeria Benth. i srodnih rodova iz tribusa Satureineae (Lamiaceae). Doktorska disertacija, PMF-Biološki fakultet, Beograd.

5. Marin, D. P. (1996): A chemotaxonomic study of vacuolar flavonoids frome some Balkan Micromeria Benth. species (Lamiaceae). Archives of biological sciences, 48, 1-2, 49-54.

6. Marin, D.P., Sajdl, V., Kapor, S., Tatić, B. & Petković, B. (1991): Fatty acids of the Saturejoideae, Ajugoideae and Scutellarioideae (Lamiaceae). Phytochemistry, 30, 2979-1982.

7. Marin, D.P, Grayer, J.R., Veitch, C.N., Kite, C.G., Harborne, B.J. (2001): Acacetin glycosides as taxonomic markers in Calamintha and Micromeria. Phytochemistry, 58, 943-947.

8. Slavkovska, V. (1996): Sastav etarskih ulja agregata Satureja montana L. (Lamiaceae) u različitim područjima centralnog dela Balkanskog poluostrva. Magistarska teza, Biološki Fakultet, Univerzitet u Beogradu.

9. Slavkovska,V., Jančić, R., Bojović, S., Milosavljević, S., Đoković, D. (2001): Variability of essential oils of Satureja montana L. and Satureja kitaibelii Wierzb. ex Heuff. From the central part of the Balkan peninsula. Phytochemistry, 57, 71-76

10. Šilić, Č. (1979): Monografija rodova Satureja L., Calamintha Miller, Micromeria Bentham, Acinos Miller i Clinopodium L. u flori Jugoslavije, 172-262. Zemaljski muzej BiH, Sarajevo.

11. Vernet, P. (1976): Analyse genetique et ecologique de la variabilite de l’essence de Thymus vulgaris L. (Labiee). PhD Thesis. Montpellier, France.

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PL5 Significance of essential oils in the determination of the species of genera Acinos Miller, Calamintha Miller and Micromeria Bentham (Lamiaceae) Slavkovska, V.1, Jančić, R1., Couladis, M.2, Bojović, S.3 1 Institute of Botany, Faculty of Pharmacy, Vojvode Stepe 450, 11000, Belgrade, Serbia; 2 Department of Pharmacognosy and Chemistry of Natural Products, Panepistmiopolis Zografou, 157 71, Athens, Greece 3 Institute for Biological Research „Siniša Stanković“, Bulevar despota Stefana 142, 11000, Belgrade, Serbia

Due to the presence of considerable quantity of essential oil, the species of Acinos, Calamintha and Micromeria genera represent important aromatic plants. In the systems of classification the species of Acinos, Calamintha and Micromeria genera are treated in a very different way. Very often one and the same species is put in a different genus, which usually leads to certain confusion even when it comes to setting borders between different genera themselves. In the taxonomic researches of Acinos, Calamintha and Micromeria genera that have been done so far, morphological, chronological, cytological and some chemical characteristics have been used as the basis for the differentiation of the species (Šilić, 1979; Doroszenko, 1986; Marin, 1986, 1996, Marin et al., 1991, 2001). Characteristics of essential oils have rarely been used for the defining of taxons and for the determination of their position in the classification system, because It is a well-known that essential oils are very variable, and dependant on fenological and ecological changes, which complicates their use as taxonomic markers (Vernet, 1976; Kokkini et al., 1991; Slavkovska, 1996; Slavkovska et al., 2001). Within each species, a sample was taken from different ecological situations in order to grasp the total variability and degree of differentiation of the chosen taxons. The following species were included in the essential oil analysis genus Acinos: A. arvensis (Lam.) Dandy, A. alpinus (L.) Moench, A. hungaricus (Simonkai) Šilić, A. majoranifolius (Miller) Šilić (13 populations total); Calamintha: C. grandiflora (L.) Moench, C. sylvatica Bromf., C. vardarensis Šilić, C. glandulosa (Req.) Bentham (13 populations total); Micromeria: Micromeria croatica (Pers.) Schott., M. parviflora (Vis.) Reichenb., M. cristata (Hampe) Griseb., M. juliana (L.) Benth. ex Reichenb., of section Micromeria and M. albanica (Grisebach ex K. Malý) Šilić, M. dalmatica Benth., M. pulegium (Rochel) Benth., M. thymifolia (Scop.) Fritsch of section Pseudomelissa (16 populations total). Essential oil variability was researched according to 126 quantitative characters which were statistically processed, and based on it the taxons were clustered by FLORA programme (Karadžić et al., 1998). Results indicated that among the species of genus Acinos only A. majoranifolius is a species rich in oil (0.57%) with dominant pulegone (71.97%), C-3 oxygenated monoterpene of the menthane class, while the other species have little oil (0.05% - 0.08%), and the quantity of C-3 oxygenated monoterpenes of the menthane class is insignificant (0.6% - 9.03%). Dominant components in the essential oils are: monoterpene hydrocarbons, α-pinene / limonene / ρ-cymene (A. arvensis), α-pinene / limonene (A. alpinus), oxygenated monoterpenes and sesquiterpenes and non-terpene compounds, thymol / α-terpinyl acetate / caryophyllene oxide / alcanes (A. hungaricus). Among the species of genus Calamintha only C. grandiflora has a low oil content (0.22%) and a low concentration of C-3 oxygenated monoterpenes of the menthane class (25.04%). Dominant

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components in the essential oil of these species are oxygenated monoterpenes α-terpinyl acetate and pulegone and sesquiterpene caryophyllene oxide. All the other species are rich in oil (0.74% - 0.98%) with dominant C-3 oxygenated monoterpenes of the menthane class (60.87% - 84.28%). Dominant components in the essential oils are: pulegone / piperitenone oxide / mentone / piperitenone (C. vardarensis and C. glandulosa) and cis-piperitone epoxide (C. sylvatica). Species of genus Micromeria section Micromeria have little oil (0.05 – 0.13%) and C-3 oxygenated monoterpenes of the menthane class (0.01%-7.3%). Dominant components in the essential oils are sesquiterpenes: caryophyllene oxide (M. croatica, M. juliana), spathulenol (M. parviflora), unidentified compound RI 1554 / spathulenol (M. cristata). Species of section Pseudomelissa is rich in oil (0.76% - 1.70%) and C-3 oxygenated monoterpenes of the menthane class (51.80% - 82.07%). Dominant components in the essential oils are: pulegone / cis-piperitone epoxide / piperitenone (M. thymifolia), cis-piperitone epoxide / piperitenone oxide (M. albanica), piperitenone / pulegone (M. dalmatica), and isomentone (M. pulegium). Taking the results of our research in consideration, it is clear that the variability of quantity and quality of oil is the smallest among the populations of the same species and that it increases with the rank of the taxon. It is greater among the species of the same section, and even greater among sections or species of the same genus. Variability is particularly pronounced within genus Micromeria. According to their quantity and composition of essential oil, the species of section Pseudomelissa are much closer to species A. majoranifolius and the whole genus Calamintha than to section Micromeria of the genus Micromeria. The quantity of oil is a less variable characteristic than its composition. Oil composition exhibited a greater variability than its quantity, keeping in mind that the variability was less pronounced within genus Calamintha and section Pseudomelissa genus Micromeria. Different C-3 oxygenated monoterpenes, all of them of the menthane type, dominated in the oils of these representatives. Heterogeneity was more pronounced within genus Acinos and section Micromeria, so that several different chemical type could be noted within these taxons. The relative stability of quantity and quality of essential oils of genus Calamintha and section Pseudomelissa genus Micromeria means that these characteristics are, probably, under relatively strict control of natural selection and are of adaptive significance. Thus, we can say that essential oil characteristics may be used as additional characters for species classification, especially in the case of section Pseudomelissa genus Micromeria. Representatives of genus Acinos and section Micromeria genus Micromeria contain essential oil in a significantly lesser quantity. The essential oil composition is variable, meaning that the adaptive significance of this character is obviously small. Due to that, in the case of these species, and particularly the species of section Micromeria, only the small quantity of essential oil can be treated as a taxonomic character. Such results indicate that the taxonomic status of sections within genus Micromeria should be examined. Keeping in mind the bigger similarity of section Pseudomelissa species with genus Calamintha species than with the species of the typical section, there are a lot of reasons to have it regrouped and added to genus Calamintha. In that case, section Micromeria species would keep their generic affiliation. Of course, the rest of the characteristics of these taxons should also be taken into consideration so that their taxonomic differentiation could be observed precisely and without a doubt.

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Acknowledgements: The results are the part of project of the Ministry of Science of the Republic of Serbia.

REFERENCES

1. Doroszenko, M. A. (1986): Taxonomic studies of Satureja complex (Labiatae). Thesis (Ph.D.). University of Edinburgh. Edinburgh.

2. Karadžić, B., Saco-Jovanović, V., Jovanović, Z., Popović, R. (1998): "FLORA" a database and software for floristic and vegetation analyzes. In: I. Tsekos, M. Moustakas (eds.) Progress in Botanical Research., Kluwer Academic Press, Dodrecht, 69-72.

3. Kokkini, S., Vokou, D. & Karousou, R. (1991): Morphological and Chemical variation of Origanum vulgare L. in Greece. Botanika Cronica, 10, 337-346.

4. Marin, P. (1989): Hemotaksonomska i mikromorfološka studija roda Micromeria Benth. i srodnih rodova iz tribusa Satureineae (Lamiaceae). Doktorska disertacija, PMF-Biološki fakultet, Beograd.

5. Marin, D. P. (1996): A chemotaxonomic study of vacuolar flavonoids frome some Balkan Micromeria Benth. species (Lamiaceae). Archives of biological sciences, 48, 1-2, 49-54.

6. Marin, D.P., Sajdl, V., Kapor, S., Tatić, B. & Petković, B. (1991): Fatty acids of the Saturejoideae, Ajugoideae and Scutellarioideae (Lamiaceae). Phytochemistry, 30, 2979-1982.

7. Marin, D.P, Grayer, J.R., Veitch, C.N., Kite, C.G., Harborne, B.J. (2001): Acacetin glycosides as taxonomic markers in Calamintha and Micromeria. Phytochemistry, 58, 943-947.

8. Slavkovska, V. (1996): Sastav etarskih ulja agregata Satureja montana L. (Lamiaceae) u različitim područjima centralnog dela Balkanskog poluostrva. Magistarska teza, Biološki Fakultet, Univerzitet u Beogradu.

9. Slavkovska,V., Jančić, R., Bojović, S., Milosavljević, S., Đoković, D. (2001): Variability of essential oils of Satureja montana L. and Satureja kitaibelii Wierzb. ex Heuff. From the central part of the Balkan peninsula. Phytochemistry, 57, 71-76

10. Šilić, Č. (1979): Monografija rodova Satureja L., Calamintha Miller, Micromeria Bentham, Acinos Miller i Clinopodium L. u flori Jugoslavije, 172-262. Zemaljski muzej BiH, Sarajevo.

11. Vernet, P. (1976): Analyse genetique et ecologique de la variabilite de l’essence de Thymus vulgaris L. (Labiee). PhD Thesis. Montpellier, France.

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PL6 Novi rezultati hemijskih i farmakoloških ispitivanja cimicifuge (Cimicifuga racemosa)

Dejan Nikolić, Tanja Gödecke, David Lankin, Shao Nong-Chen, Guido F. Pauli, Sharla Powell, Judy L. Bolton i Richard B. van Breemen University of Illinois College of Pharmacy, Department of Medicinal Chemistry and Pharmacognosy, UIC/NIH Center for Botanical Dietary Supplements Research, Chicago, IL 60612, USA

UVOD

Cimicifuga racemosa (L.) Nutt. (syn. Actaea racemosa L. (Ranunculaceae), black cohosh) je lekovita biljka poreklom iz istočnih delova Severne Amerike. Ekstrakti korena cimicifuge se aktivno istražuju kao alternativa hormonskoj terapiji za ublažavanje simptoma menopauze kao sto su napadi vrućine. Proizvodi na bazi ove biljke su ternutno jedni od najpopularnijih i najprodavanijih dijetetskih suplemenata na američkom tržištu. Međutim, tačan mehanizam delovanja još uvek nije u potpunosti razjašnjen. Naš Centar za proučavanje lekovitog bilja ispituje hipotezu da ekstrakti cimicifuge deluju putem stimulacije serotoninskih 5-HT7 receptora [1]. U naporima da se identifikuje aktivna supstanca, razvijena je nova strategija koja se zasniva na primeni modernih hromatografskih tehnika kao stu tečno-masena hromatografija, masena spektrometrija visoke rezolucije i tačnosti kombinovane sa korišćenjem baza podataka o prirodnim proizvodima. Ova strategija omogućava da se identifikuju pojedinačni sastojci ekstrakta bez napornog i dugotrajnog proces izolacije. U toku istraživanja, naš tim je oktrio da cimicifuga sadrži alkaloide različitih strukturnih klasa, što ranije nije bilo poznato. U ovom predavanju biće izložena identifikacija nekoliko novih jedinjenja, uključujući i N-metil serotonin, koji je pokazao visoku aktivnost prema serotoninskim receptorima.

METODE

Metanolni ekstrakt korena cimicifuge razdvojen je na vodenu, hloroformsku, etilacetatnu i butanolnu frakciju. Butanolna frakcija je pokazala najveću aktivnost i stoga je dalje ispitivana. Hromatografska razdvajanja vršena su na Tosoh Amide-80 normalno faznoj koloni koristeći gradijent od acetonitrila do 0,1% mravlje kiseline. Maseni spektri visoke rezolucije i tačnosti snimani su na Micromass Q-TOF-2 hibridnom quadrupole/time-of-flight masenom spectrometru koristeći pozitivni jon elekstrosprej. Za određivanje tačne mase fragmenata, prekurzorni jon je korišćen kao referenti jon. Molekulske formule utvrđene iz ovih merenja su pretraživane u MDL CrossFire Commander bazi podataka. Fragmentacioni spektri su snimani koristeći kolizionu energiju od 25 eV i argon kao kolizioni gas.

REZULTATI I DISKUSIJA LC-MS analiza butanolne frakcije metanolnog ekstrakta cimicifuge pokazala je prisustvo velikog broja azotnih jedinjenja. Jedinjenja sa neparnim brojem atoma azota mogu se prepoznati na osnovu neparne molekulske mase, dok se jedinjenja sa parnim brojem atoma azota mogu identifikovati pomoću masenih spektara viskoke rezolucije i tačnosti. Nakon određivanja elementalne kompozicije, molekulske formule su pretraživane u bazama podataka prirodnih proizvoda. Rezultati

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pretraživanja pružaju korisne informacije u vezi mogućeg identiteta ispitivanog jedinjenja, što s druge strane pomaže da se odredi da li je ispitivano jedinjenje vredno daljeg istraživanja. Identifikacija jedinjenja se dalje nastavlja putem eliminacije, gde se mogući „hitovi” iz baze podataka eliminišu na osnovu daljih informacija koje se dobijaju iz masenih spektara kao sto su broj izmenljivih vodonika ili na osnovu fragmentacije ispitivanog jedinjenja. Nakon nekolliko ciklusa eliminacije, konačna identifikacija se može izrvšiti nakon sinteze pretpostavljenog jedinjenja i upoređivanjem retenticionog vremena i fragmentacije sintetskog standarda sa istim podacima dobijenim za nepoznatu supstancu iz ekstrakta. Na taj način se mnoga jedinjenja iz ekstrakta mogu identifikovati bez dugotrajnog i mukotrpnog procesa izolacije. Koristeći ovaj pristup, utvrđeno je prisustvo nekoliko grupa alkaloida. U prvu grupu spadaju ciklični gvanidinski alkaloidi koji se mogu smatrati derivatima cimipronidina kojeg je naša grupa identifikovala pre neliko godina [2]. U drugu grupu spadaju alkaloidi iz tetrahidroizohinolinske i dihidroizohinolinske grupe, kao sto su salsolinol i norsalsolinol. U treću grupu spadaju beta-karbolinski alkaloidi kao što su 6-hidroksi-N(2)-metil-1,2,3,4-tetrahidro-β-karbolin i 3,4,5,6,-tetrahidro-6-hidroksi-5-metil-1H-azepino[5,4,3-cd]indol, nazvan cimitripazepin (Slika 1). Cimiptripazepin je novi prirodni proizvod. Konačno, N-metil serotonin je identifikovan kao predstavnik indolnih alkaloida. Ovo jedinjenje je potentan i selektivni aktivator serotoninskih 5-HT7 receptora. Ovaj sastojak je

Slika 1. Novi alkaloidi iz cimicifuge

NN

OH

HCH3

N

OH

NCH3

H

najverovatnije odgorovan za uočenu in vitro aktivnost ekstrakta cimicifuge. Međutim, u ovom trenutku nije izvesno da li je isto jedinjenje odgovorno i za in vivo aktivnost ove lekovite biljke. Pored alkaloida, utvrđeno je prisustvo i drugih azotnih jedinjenja. Većina tih jedinjenja su proizvodi normalnog ćelijskog metabolizma kao sto su aminokiseline i nukleobaze.

U zaključku, koristeći moderne analitičke tehnike u ekstraktu korena cimicifuge dokazano je pristutvo alkaloida koji pripadaju različitim strukturnim grupama. Znajući dokazanu biološku aktivnost mnogih alkaloida, ovi novi rezultati će doprineti boljem razumevanju biološke aktivnosti ove biljke i pomoći u razvijanju novih metoda za standardizaciju proizvoda na bazi cimicifuge.

LITERATURA 1. Burdette, J. et al. (2003) J. Agric. Food Chem. 51, 5661-5670. 2. Fabricant, D et al. (2005) J. Nat. Prod. 68, 1266-1270.

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PL6 Recent advances in chemistry and pharmacology of black cohosh (Cimicifuga racemosa)

Dejan Nikolić, Tanja Gödecke, David Lankin, Shao Nong-Chen, Guido F. Pauli, Sharla Powell, Judy L. Bolton and Richard B. van Breemen University of Illinois College of Pharmacy, Department of Medicinal Chemistry and Pharmacognosy, UIC/NIH Center for Botanical Dietary Supplements Research, Chicago, IL 60612, USA

INTRODUCTION Cimicifuga racemosa (L.) Nutt. (syn. Actaea racemosa L. (Ranunculaceae), black cohosh) is a medicinal plant, native to eastern North America. Extracts of black cohosh are under investigation as alternatives to hormone replacement therapy for the relief of menopausal symptoms such as hot flashes. The plant remains one of the most popular and best selling supplements in the United States. However, the exact mechanism(s) of action still remains uncertain. Our Center has focused on the hypothesis that black cohosh might act via modulation of serotonin 5-HT7 receptors [1]. In our efforts to identify possible active constituents, we have implemented a mass spectrometric dereplication strategy that relies on accurate mass measurements combined with database searching to rapidly identify known compounds present in the extracts without the need for time-consuming isolation. During this search, we discovered that black cohosh contains several classes of alkaloids that were previously unknown to exist in this plant. In this presentation we will discuss the identification of several new compounds including N-methyl serotonin which has shown strong activity in 5-HT7- binding assay.

METHODS

A methanolic extract of roots of black cohosh was partitioned between water, chloroform, ethyl acetate and butanol. The biological activity was concentrated in the butanolic fraction. Chromatographic separations were carried out using Tosoh Amide-80 normal phase column using various gradients consisting of acetonitrile to 0.1% formic acid. Accurate mass measurements were obtained using a Micromass Q-TOF-2 hybrid quadrupole/time-of-flight mass spectrometer operated in positive ion electrospray mode. For accurate mass measurements of the product ions, the precursor ion was used as a lock mass. Elemental compositions obtained from these measurements were searched using MDL CrossFire Commander database. Product ion tandem mass spectra were recorded at a collision energy of 25 eV using argon as collision gas.

RESULTS AND DISCUSSION LC-MS analysis of the butanol soluble fraction of a methanolic extract of black cohosh revealed the presence of numerous nitrogen-containing compounds. Compounds with odd numbers of nitrogen may be easily recognized by their odd masses while those with even number of nitrogens were identified by accurate mass measurement. After elemental compositions were determined, the molecular formulas were search against the database of natural products. This search provides important clues into the identity of the compounds which in turn, provides important data as to

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whether the compounds were worth pursuing further. The identification of a compound then follows an elimination scheme whereby database hits are eliminated based on additional mass spectrometric information such as the number of exchangeable hydrogens or fragmentation pattern. After several rounds of elimination, the remaining possibilities could be tested by synthesis of authentic standards and comparison of their retention times and fragmentation patterns with those obtained for compounds present in the extract. In this manner, compounds can be identified without laborious isolation process. Using this approach, we discovered that black cohosh contains several classes of alkaloids. One group was cyclic guanidine alkaloids which can be considered derivatives of cimipronidine [2]. Another group of alkaloids was tetrahydro and dihydroisoquinoline-type alkaloids with salsollinol and norsalsolinol as notable representatives. The third class was β-carbolines with 6-hydroxy-N(2)-methyl-1,2,3,4-tetrahydro-β-carboline and 3,4,5,6,-tetrahydro-6-hydroxy-5-methyl-1H-azepino[5,4,3-cd]indole as representatives (Figure 1). The latter was a new natural product. Finally, among indole alkaloids, Nω-methyl-serotonin was identified as a potent and selective 5-HT7 agonist. This compound is likely responsible for the observed in vitro activity of black cohosh extract. At this point, it is unclear whether this compound can contribute to the in vivo activity of this plant.

NN

OH

HCH3

N

OH

NCH3

H

Figure 1. New alkaloids from black cohosh

Besides alkaloids, other classes of nitrogenous compounds were also identified. Most of them were products of normal cellular metabolism such as amino acids and nucleobases. In conclusion, we have recently discovered that black cohosh contains numerous classes of nitrogenous compounds. Given the proven biological activity of alkaloids it is likely that observed clinical effects of this plant are derived from this group of compounds. These discoveries will necessitate new approaches toward standardization of this popular herb.

REFERENCES

3. Burdette, J. et al. (2003) J. Agric. Food Chem. 51, 5661-5670. 4. Fabricant, D et al. (2005) J. Nat. Prod. 68, 1266-1270.

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PL7 Culex pipiens an old problem revisited: Larvicidal activity of Greek aromatic essential oils Olga Tzakou Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, University of Athens, Panepistimiopolis Zografou, 15771 Athens, Greece Insect transmitted diseases remain a major source of illness and death worldwide. Mosquitoes alone transmit disease to more than 700 million persons annually, and they act as vector for many diseases such as dengue fever, yellow fever, malaria, filariasis and encephalitis of different types such as the West Nile Fever. Although insect-borne diseases currently represent a greater health problem in tropical and subtropical climates, no part of the world is immune to their risks. In the fall of 1999, the West Nile virus, transmitted by mosquitoes, was detected for the first time in the Western Hemisphere. Members of Culex pipiens complex are responsible for the transmission of this vector-borne disease as well as many other dreadful diseases. Since 1947, the year when the first incidence of DDT resistance was encountered, more than 100 mosquito species have been reported to be resistant to one or more insecticides. Experimental data have indicated that such use of aerial toxicants to control the population of adult mosquitoes is not effective, since they are highly domesticated and spraying does not affect many mosquitoes that rest indoors in hidden places. On the contrary, the systematic application of larvicides onto their breeding places constitutes a very effective method. Repeated use of synthetic insecticides for mosquito control has disrupted natural biological control systems and led to resurgences in mosquito populations. It has also resulted in the development of resistance, undesirable effects on non-target organisms, and fostered environmental and human health concern. There is a need to find alternatives to these synthetic pesticides, to find newer insecticides which will be effective, ecofriendly and biodegradable. Plants are regarded as a rich source of bioactive chemicals and they may be an alternative source of mosquito control agents. Natural products are generally preferred because of their less harmful nature to non-target organisms and due to their innate biodegradability. Especially essential oils that have been used for centuries as insecticides and insect repellents hold significant promise in insect vector management. Their lipophilic nature enables their interference with basic metabolic, biochemical, physiological and behavioral functions of insects. The Greek flora with some 5,700 species of Higher Plants, is comparable in size to that of some tropical countries and is extraordinarily rich in endemics (13% of the flora), with many other species endemic to the Balkan region. Many of the Greek taxa are common sources of drugs and insecticides used in traditional medicine. Moreover, Greece is regarded as an important gene depository center for Labiatae and Umbelliferae families. In the current study, the larvicidal properties of the essential oils from 10 species of the Labiatae family (Salvia pomifera subsp. pomifera, S. pomifera subsp. calycina, S. fruticosa, Thymus leucospermus, T. teucrioides, Melissa officinalis, Mentha suaveolens, M. spicata, M. longifolia, Nepeta argolica) and 1 species of the Umbelliferae family (Foeniculum vulgare) were evaluated against the larvae of the mosquito species Culex pipiens biotype molestus. Results indicated a wide range of mortality effect among the studied species, except that of Nepeta argolica which showed interesting repellent activity.

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PL8 Proučavanje sastojaka vrsta roda Anthemis Milica Pavlović Institut za farmakognoziju, Farmaceutski fakultet, V. Stepe 450, 11221 Beograd, Srbija Uvod Rod Anthemis L. pripada familiji Asteraceae (Compositae), tribusu Anthemideae i obuhvata preko 210 vrsta koje su rasprostranjene u Evropi, regionu Mediterana, jugozapadnoj Aziji i manjem delu istočne Afrike [1]. U Flori Evrope opisane su 62 vrste [2], a u Flori Srbije 9 vrsta ovog roda [3]. Dosadašnjim ispitivanjima u vrstama roda Anthemis utvrđeno je prisustvo farmakološki aktivnih sekundarnih metabolita: seskviterpenskih laktona, flavonoida, poliacetilena, etarskog ulja i kumarina [4]. Na osnovu morfoloških i anatomskih karakteristika ploda, rod Anthemis je podeljen na dva podroda, Anthemis i Cota, dok je kao treći podrod dodat Ammanthus. Podrodovi su dalje podeljeni na sekcije na osnovu životnih formi, izgleda palja i karakteristika ploda. U podrodu Cota razlikuju se dve sekcije: Anthemaria i Cota, a podrod Anthemis obuhvata sekcije Hiorthia, Anthemis, Maruta i Chia. Taksonomski položaj roda Anthemis u okviru tribusa još uvek je nerazjašnjen, kao i odnos pojedinih vrsta u okviru sekcija [1]. Prema najnovijim istraživanjima, u okviru projekta Euro+Med Plantbase, rod Anthemis obuhvata 111 vrsta a podrod Cota odvojen je u poseban rod Cota J. Gay, koji obuhvata 24 vrste [5]. U ovom radu ispitivano je šest taksona roda Anthemis, koji su zastupljeni u flori Srbije i Crne Gore, i to tri vrste podroda Cota: A. triumfetti L. (DC.) i A. tinctoria L. iz sekcije Anthemaria i A. austriaca Jacq. iz sekcije Cota, kao i 3 taksona podroda Anthemis: A. ruthenica Bieb. iz sekcije Anthemis, A. cotula L. iz sekcije Maruta i A. cretica subsp. carpatica (Willd.) Grierson (sin. A. carpatica Willd.) iz sekcije Hiorthia.

HEMIJSKA ANALIZA HERBE A. TRIUMFETTI Vrsta A. triumfetti pripada sekciji Anthemaria, podrodu Cota i zastupljena je u flori Srbije i Crne Gore. Do sada nije ispitivana sa aspekta hemijskih sastojaka. Iz herbe u cvetu A. triumfetti izolovano je i identifikovano 19 jedinjenja: iz cikoheksanskog ekstrakta skvalen, kamfor, β-karoten i trans-fitol, a iz metanolno-vodenog ekstrakta kvercetin, hispidulin (6-hidroksiapigenin-6-metiletar), rutin, luteolin-7-O-β-D-glukozid, kvercetin-3-O-β-D-glukozid, kvercetin-7-O-β-D-glukozid, kvercetin-4'-O-β-D-glukozid (spireozid), patuletin-7-O-β-D-glukozid, apigenin-7-O-β-D-glukuronid, hlorogenska kiselina, 3,4-di-O-kafeoilhina kiselina, 3,5-di-O-kafeoilhina kiselina, umbeliferon, skopoletin i skopolin. Među izolovanim flavonoidima dominiraju flavonoli i to glikozidi kvercetina. Većina flavonoida (8 od 9) prethodno je izolovana iz drugih Anthemis vrsta [4]. Interesantno je prisustvo kvercetin-4'-O-glukozida, koji je detektovan u tribusu Anthemideae u vrsti Cotula coronopifolia [6], ali u literaturi nema podataka o njegovom prisustvu u rodu Anthemis. Iako su fenolkarbonske kiseline vrlo često prisutne u pripadnicima familije Compositae [4], 3,4-di-O-kafeoilhina i 3,5-di-O-kafeoilhina kiselina do sada nisu izolovane iz vrsta roda Anthemis. Kumarini su takođe uobičajeni sastojci tribusa

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Anthemideae, ali su do sada iz biljaka roda Anthemis izolovani samo herniarin i skopoletin [7]. U literaturi nema podataka o prisustvu umbeliferona i skopolina u biljkama roda Anthemis. U herbi A. triumfetti utvrđeno je i prisustvo stigmasterola, holesterola, kampesterola, β-sitosterola i 24-metilen-cikolartanola. GC/MS analizom identifikovano je i određeno 16 masnih kiselina u obliku metil estara, među kojima je dominantna heksadekanska (palmitinska) kiselina (28,8%). Pored palmitinske u značajnom procentu su zastupljene i 9,12,15-oktadekatrienska (linolenska) kiselina (16,3%) i (Z,Z)-9,12-oktadekadienska (linolna) kiselina (13,9%).

ANALIZA ETARSKIH ULJA ISPITIVANIH TAKSONA RODA ANTHEMIS U nadzemnim delovima u cvetu A. triumfetti, A. tinctoria, A. austriaca, A. cotula, A. ruthenica i A. cretica ssp. carpatica, prikupljenih sa različitih lokaliteta u Srbiji i Crnoj Gori, ispitivan je sadržaj i sastav etarskih ulja. Količina etarskog ulja u herbi A. triumfetti iznosi 0,22-0,40%, u A. cretica ssp. carpatica 0,34%, A. austriaca 0,25-0,30%, A. ruthenica 0,20-0,32%, A. tinctoria 0,10-0,14% i u A. cotula 0,13-0,17%. Međusobnim poređenjem ispitivanih taksona u pogledu sastava etarskih ulja uočavaju se razlike između njih. Etarska ulja A. triumfetti karakteriše visok sadržaj kamfora (13,8-15,4%), α-pinena (7,8-14,4%) i β-pinena (6,5-16,9%), sa izuzetkom ulja biljaka populacije sa Sinjajevine gde dominira cis-hrizantenol (27,0%). U etarskim uljima A. tinctoria u pet ispitivanih populacija uočava se relativno visok sadržaj 1,8-cineola (9,0-25,8%), dok se od njih razlikuje ulje biljaka populacije sa Kaluđerskih bara gde su najzastupljenije komponente borneol (16,0%) i spatulenol (16,0%). cis-Hrizantenilacetat (17,5-22,0%) je glavni sastojak etarskih ulja A. austriaca a u manjem procentu je zastupljen i u uljima ostalih ispitivanih vrsta podroda Cota (A. triumfetti i A. tinctoria). Dominantna jedinjenja u etarskim uljima A. ruthenica su germakren D (8,3-11,3%) i terpinen-4-ol (6,3-7,3%). Etarska ulja A. cotula karakteriše prisustvo β-kedrena (10,3-19,0%), koji nije detektovan u uljima biljaka ostalih ispitivanih taksona. Etarsko ulje A. cretica ssp. carpatica se jasno odvaja od ulja ostalih vrsta prisustvom cis- i trans-tujona (39,0% i 13,5%). Kod taksona kod kojih su ispitivana etarska ulja sa različitih lokaliteta (A. triumfetti, A. tinctoria, A. cotula i A. ruthenica), uglavnom se uočavaju kvalitativna sličnost u pogledu glavnih komponenata i izvesne kvantitativne razlike između različitih populacija istih vrsta. Izuzetke predstavljaju populacija A. triumfetti poreklom sa Sinjajevine i populacija A. tinctoria sa lokaliteta Kaluđerske bare, koje se znatno razlikuju od ostalih populacija ovih vrsta, što ukazuje na mogućnost postojanja hemotipova. Kod A. austriaca gde su ispitivana dva uzorka sa istog lokaliteta prikupljena u različitim godinama, uočava se sličnost između uzoraka u sastavu i vrlo sličan odnos glavnih komponenata etarskih ulja.

ANALIZA FENOLSKIH SASTOJAKA ISPITIVANIH TAKSONA RODA ANTHEMIS HPLC analizom ispitivani su acetonski i metanolni ekstrakti listova, listića involukruma, jezičastih i cevastih cvetova A. triumfetti, A. tinctoria, A. austriaca, A. ruthenica, A. cotula i A. cretica ssp. carpatica poreklom sa različitih lokaliteta u Srbiji i Crnoj Gori, odnosno površinski (lipofilni, eksterni) flavonoidi i unutrašnji (vakuolarni, polarni) flavonoidi, kumarini i fenolkarbonske kiseline. Na površini listića involukruma, listova, jezičastih i cevastih cvetova ispitivanih taksona roda Anthemis utvrđeno je prisusto flavonoidnih aglikona. Pored apigenina, luteolina i kvercetina, prisutni su i njihovi derivati među kojima su najbrojniji metilovani 6-hidroksi derivati. Derivati kemferola su zastupljeni samo u ekstraktima A. tinctoria.

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Među unutrašnjim flavonoidima detektovani su glukozidi i glukuronidi apigenina, luteolina, kvercetina i patuletina, kao i aglikoni apigenin, luteolin i kvercetin. U vakuolama biljaka svih ispitivanih taksona prisutne su i fenolkarbonske kiseline: hlorogenska, 3,4-, 3,5- i 4,5-dikafeoilhina kiseline. Kumarini, skopoletin, umbeliferon i skopolin uočeni su samo u ekstraktima A. triumfetti.

U pogledu flavonoda prisutnih na površini različitih organa, kao i u raspodeli unutrašnjih flavonoida, ispitivani taksoni se međusobno razlikuju tj. svaki takson ima sopstveni flavonoidni kompleks koji je najsloženiji kod A. triumfetti i A. tinctoria. Flavonoidni kompleksi različitih populacija u okviru jedne vrste uglavnom su kvalitativno slični, sa izvesnim, manjim ili većim, kvantitativnim razlikama. Raspodela aglikona kao i raspodela vakuolarnih flavonoida zavisi kako od ispitivanog taksona, tako i od ispitivanog organa. Dominantne fenolkarbonske kiseline: hlorogenska, 3,5- i 4,5-dikafeoilhina kiseline, zastupljene su u svim taksonima i nisu specifične za određeni organ. Pojedini površinski aglikoni karakteristični su samo za određene taksone: jaceidin (6-hidroksikvercetin-3,6,3'-trimetiletar) za A. triumfetti (nema ga jedino na površini jezičastih cvetova), derivati kemferola i kvercetin-3-metiletar za A. tinctoria i spinacetin (6-hidroksikvercetin-6,3'-dimetiletar) za A. cotula. A. ruthenica razlikuje se od ostalih ispitivanih taksona po odsustvu luteolina i njegovih derivata sa površine ispitivanih organa. U pogledu zastupljenosti vakuolarnih flavonoida, rutin, patuletin-3-O-glukozid i kvercetin-3-O-glukuronid prisutni su samo u ispitivanim vrstama podroda Cota (A. triumfetti, A. austriaca i A. tinctoria). Među unutrašnjim flavonoidima A. tinctoria detektovani su samo flavonoli, dok flavoni izostaju. Određeni flavonoidi karakteristični su samo za pojedine organe analiziranih taksona. Apigenin je površinski aglikon jezičastih cvetova svih ispitivanih taksona. Apigenin i apigenin-7-O-glukozid prisutni su u vakuolama svih belih jezičastih cvetova; izostaju jedino u žutim jezičastim cvetovima A. tinctoria, koji su istovremeno specifični po prisustvu patuletin-7-O-glukozida (karakteristika žutih cevastih cvetova). Kvercetin je prisutan na površini i u vakuolama cevastih cvetova dok u ostalim organima izostaje. Za cevaste cvetove nekih taksona (A. tinctoria, A. austriaca, A. ruthenica i A. cretica ssp. carpatica) specifično je i prisustvo površinskog aglikona izoramnetina (kvercetin-3'-metiletar). Cirsimaritin (6-hidroksiapigenin-6,7-dimetiletar) je detektovan samo na površini involukruma i lista nekih ispitivanih vrsta (A. triumfetti, A. tinctoria i A. ruthenica). Pored toga, uočeno je da su u vakuolama cevastih cvetova među flavonoidima prisutni samo flavonolni heterozidi, tj. heterozidi kvercetina i patuletina. Kvercetin-3-O-glukuronid, prisutan je samo u listovima pojedinih taksona (A. triumfetti, A. tinctoria i A. austriaca). U literaturi postoje podaci o analizi površinskih i vakuolarnih flavonoida listova, jezičastih i cevastih cvetova Anthemis vrsta koje nisu ispitivane u ovom radu [6], gde su na površini ispitivanih organa takođe zastupljeni metilovani derivati 6-hidroksiapigenina, -luteolina, -kvercetina i -kemferola, od kojih je većina detektovana i u ekstraktima naših Anthemis vrsta. U vakuolama je utvrđeno prisustvo flavonolnih i flavonskih heterozida koji su detektovani i u našim vrstama: 7-glukozida i 7-glukuronida apigenina i luteolina, glukozida patuletina i rutina. U poređenju sa literaturom, u Anthemis vrstama ispitivanim u ovom radu detektovan je veći broj površinskih i vakuolarnih flavonoida kao i složeniji flavonoidni kompleks karakterističan za svaku vrstu. Vučković i saradnici [8] iz nadzemnih delova A. cotula izolovali su apigenin i hispidulin koji su takođe prisutni i u ekstraktima biljaka sve tri populacije A. cotula ispitivane u ovom radu.

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STATISTIČKA ANALIZA Klaster analiza, zasnovana na rezultatima analize fenolnih jedinjenja, ukazala je na potpunu hemijsku odvojenost svih populacija vrste A. tinctoria u odnosu na ostale ispitivane taksone roda Anthemis. S obzirom da se u pogledu hemijskog sastava A. tinctoria bitno razlikuje od A. austriaca iz istog podroda Cota, kao i od A. triumfetti iz iste sekcije Anthemaria, može se zaključiti da rezultati hemijske analize nisu u skladu sa postojećom taksonomskom klasifikacijom vrste A. tinctoria. Pored toga uočena je i hemijska bliskost različitih populacija iste vrste, hemijska sličnost lista i involukruma u okviru jedne vrste i sličnost sastojaka jezičastih cvetova svih taksona (osim A. tinctoria). Taksoni A. cretica ssp. carpatica i A. ruthenica su hemijski najsrodniji i za njih se sa određenom distancom vezuje A. cotula, što je u skladu sa njihovom pripadnošću istom podrodu Anthemis. Uočena hemijska bliskost vrsta A. austriaca i A. triumfetti potvrđuje njihovu taksonomsku klasifikaciju u isti podrod Cota.

LITERATURA 1. Oberprieler C. (2001) Taxon 50: 745-762. 2. Gajić M. (1975) Fam. Asteraceae Dumortier.In Josifović M. et al. (Eds) Flora SR Srbije, vol. VII, SANU,

Odeljenje prirodno matematičkih nauka, Beograd. 3. Fernandes R. (1976) Anthemis L. In: Tutin, T.G. et al. (Eds) Flora Europaea, Vol. 4. Cambridge

University Press, Cambridge. 4. Pavlović M. et al. (2006) 34: 449-452. 5. Greuter (2005-2007) Euro+Med PlantBase. http://www.emplantbase.org/home.html 6. Williams C.A. et al. (2001) Biochem Syst Ecol 29: 929-945. 7. Saleh M.M., Rizk A.M., (1974) Planta Med. 25: 60-62. 8. Vučković I. et al. (2006) J Serb Chem Soc 71: 127-133.

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PL8 The study on the constituents of some Anthemis species Milica Pavlović Department of Pharmacognosy, Faculty of Pharmacy, V. Stepe 450, 11221 Belgrade, Serbia

This paper analyses six taxa of genus Anthemis (Asteraceae), present in flora of Serbia and Montenegro, for their chemical constituents. Three species belonging to the subgenus Cota, A. triumfetti and A. tinctoria from section Anthemaria and A. austriaca from section Cota as well as three taxa from subgenus Anthemis, A. ruthenica from section Anthemis, A. cotula from section Maruta and A. cretica ssp. carpatica from section Hiorthia were studied. The chemical analysis of the flowering aerial parts of A. triumfetti led to the isolation and identification of 19 compounds: squalene, camphor, β-carotene, trans-phytol, scopoletin, umbelliferone, hispidulin, quercetin, scopolin, luteolin 7-O-β-D-glucoside, quercetin 3-O-β-D-glucoside, quercetin 7-O-β-D-glucoside, quercetin 4'-O-β-D-glucoside, patuletin 7-O-β-D-glucoside, rutin, apigenin 7-O-β-D-glucuronide, chlorogenic acid, 3,5-di-O-caffeoylquinic acid and 3,4-di-O-caffeoylquinic acid. Also, in the aerial parts of A. triumfetti stigmasterol, cholesterol, campesterol, β-sitosterol and 24-methylene cycloartanol were identified. GC/MS analysis revealed the presence of sixteen fatty acids and among them the main constituents were hexadecanoic acid (palmitic acid); 9,12,15-octadecatrienoic acid (linolenic acid) and (Z,Z)-9,12-octadenoic acid (linoleic acid). Comparison of essential oils of Anthemis species investigated revealed differences in the oil compostion between species. Essential oils of A. triumfetti were characterized by high content of camphor, α-pinene and β-pinene with exception of one population in which oil cis-chrysanthenol was the most dominant. In the essential oils of A. tinctoria the major constituent was 1,8-cineole, except in the oil of one population where borneol and spatulenol were the main constituents. In the essential oils of A. austriaca cis-chrysanthenyl acetate was present in high amounts. The most dominant constituents in the oils of A. ruthenica were germacrene D and terpinene-4-ol. The essential oils of A. cotula differed from others in the presence of β-cedrene, whereas A. cretica ssp. carpatica was distinguished by occurence of high amounts of cis- and trans-thujone. HPLC analysis of the external flavonoids revealed the presence of the free flavonoid aglycones on the surface of all analysed plant parts (involucral bracts, leaves, ray and disc florets) of all taxa. Among them methylated derivatives of 6-hydroxy -apigenin, -luteolin and -quercetin were the most frequent. The flavonoid glycoside pattern of Anthemis species investigated was based on glucosides and glucuronides of apigenin, luteolin, quercetin and patuletin. In the methanolic extacts of Anthemis species analysed chlorogenic acid, 3,4-dicaffeoylquinic, 3,5-dicaffeoylquinic and 4,5-dicaffeoylquinic acid were identified. The coumarins, scopoletin, umbelliferone and scopolin occured only in the methanolic extracts of A. triumfetti. The statistical cluster analyses showed that according to the chemical characteristics A. tinctoria was clearly separated from other Anthemis taxa investigated. In addition, similarities between different populations of one species or between samples of one species from different years of collection were observed. Also, chemical similarities between ray florets of all taxa (except A. tinctoria) and some chemical similarities between involucral bracts and leaves within one species were noticed. A. cretica ssp. carpatica and A. ruthenica were chemically the most similar, whereas A. cotula was closely related to them. A. austriaca was the most similar to the A. triumfetti, with whom is placed in the same subgenus Cota.

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REFERENCES

1. Oberprieler C. (2001) Taxon 50: 745-762. 2. Gajić M. (1975) Fam. Asteraceae Dumortier.In Josifović M. et al. (Eds) Flora SR Srbije, vol. VII, SANU,

Odeljenje prirodno matematičkih nauka, Beograd. 3. Fernandes R. (1976) Anthemis L. In: Tutin, T.G. et al. (Eds) Flora Europaea, Vol. 4. Cambridge

University Press, Cambridge. 4. Pavlović M. et al. (2006) 34: 449-452. 5. Greuter (2005-2007) Euro+Med PlantBase. http://www.emplantbase.org/home.html 6. Williams C.A. et al. (2001) Biochem Syst Ecol 29: 929-945. 7. Saleh M.M., Rizk A.M., (1974) Planta Med. 25: 60-62. 8. Vučković I. et al. (2006) J Serb Chem Soc 71: 127-133.

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UUSSMMEENNAA IIZZLLAAGGAANNJJAA OORRAALL LLEECCTTUURREESS

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U1 Morfološka varijabilnost populacija vrste Leucojum aestivum L. (Amaryllidaceae) u Srbiji Anačkov, G.1, Boža, P.1, Igić, R.1, Vukov, D.1, Dević, B.1, Božin, B.2 1 Prirodno-matematički fakultet, Departman za biologiju i ekologiju, Trg Dositeja Obradovića 2, Novi Sad, Srbija; 2 Medicinski fakultet, Zavod za farmaciju, Hajduk Veljkova 3, Novi Sad, Srbija Galantamin je alkaloid koji se već duže vreme koristi u lečenju poliomijelitisa, a od nedavno i u terapiji alchajmerove bolesti. Ovo jedinjenje se u farmaceutskoj industriji dobija izolacijom iz lukovice pojedinih rodova porodice Amaryllidaceae (Leucojum spp., Galanthus spp. i Narcissus spp.), odnosno sintetskim putem [1]. S obzirom na mogućnost varijabilnosti u kvantitativnom hemijskom sastavu koja se često javlja kao posledica morfološke varijabilnosti biljaka istraživanja su obuhvatila analizu variranja morfoloških parametara vrste Leucojum aestivum duž toka Dunava u Srbiji. Obrađeno je ukupno 28 karaktera i to 21 kvantitativna (morfometrijski i meristički) i 7 kvalitativnih karaktera. Analiza glavnih komponenti (PCA) pokazala je izuzetno mali procenat diferencijacije analiziranih jedinki prema veličini i prema obliku. Posmatrajući karaktere koji doprinose diferencijaciji u odnosu na prvu osu izdvojili su se isključivo visoko varijabilni karakteri vegetativnog regiona, dužina stabla i lista. U nižem stepenu diferencijacije izdvojile su se dve grupe karaktera sa po dve podgrupe, međutim u nedovoljnom procentu koji bi ukazao na jasnu diferenciranost. Izdvojen je samo meristički karakter pojave dva stabla iz jedne lukovice. Diskriminantna analiza na nivou populacija pokazala je da je diferencijacija moguća i na osnovu kvantitativnih karaktera te istraživanja u tom pravcu treba proiširiti. Kvalitativni karakteri pokazali su veći stepen diferenciranosti ovih jedinki. Kao veoma precizna stanja pojedinih karaktera koja determinišu pojedine populacije, pokazal su se i oblik lukovice i margina stabla. Na osnovu ove analize definisali smo dva seta kvalitativnih karaktera koji bi mogli da determinišu posebne intraspecijske taksone. Literatura: 1. Heinrich, M., Teoh, H.L. (2004) J. Ethnopharnacol., 92: 147-162.

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U1 Morphological variability of Leucojum aestivum L. (Amaryllidaceae) populations in Serbia Anačkov, G.1, Boža, P.1, Igić, R.1, Vukov, D.1, Dević, B.1, Božin, B.21 Faculty of Sciences, Department of Biology and Ecology, Trg Dositeja Obradovića 2, Novi Sad, Serbia; 2 Faculty of Medicine, Department of Pharmacy, Hajduk Veljkova 3, Novi Sad, Serbia Galanthamine is an alkaloid used in the treatment of poliomyelitis, and recently, in the treatment of Alzheimer’s disease. In the pharmaceutical industry it is naturally isolated from bulbs of several members of the Amaryllidaceae family (Leucojum spp., Galanthus spp. and Narcissus spp.) as well as synthetically [1]. Regarding to the possible changes in the quantitative chemical composition of the biological source (bulb) related to morphological varibailities, the aim of this study was to investigate morphological characters of several populations of Leucojum aestivum species along the banks of Danube. In the examination 28 characters (21 morphometric and meristic, and 7 qualitative) were evaluated. Principle component analysis (PCA) exhibited low percentage of differentiation of samples analysed regarding the size and shape. Characters related to the differentiation connected to the first principle axes were height of the stem and the leaves longiness. In the lower level of differentiation two groups of characters, with two subgroups were distinguished. However, these characters pointed sufficient to the clear separation. Only the meristic character related to the appearance of two stems from one bulb could be usefull as an parameter for further differentiation. Discriminant analysis at the level of populations showed that differentiation is possible using quantitative feature. On the other hand, qualitative characters, especially the shape of the bulbs and margin of the stem, exhibited vary high level of separation. Regarding to this investigation, two groups of qualitative characters, useful for separation of particular infraspecific taxa could be defined. References: 1. Heinrich, M., Teoh, H.L. (2004) J. Ethnopharnacol., 92: 147-162.

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U2 Plantažno gajenje divlje ruže (Rosa canina L.) i prerada šipurka (Rosae pseudofructus) Brkić Sreta, Pljevljakušić Dejan, Menković Nebojša 1Institut za proučavanje lekovitog bilja „Dr Josif Pančić“, Tadeuša Košćuška 1, 11000 Beograd, Srbija Divlja ruža (Rosa canina L.) je veoma rasprostranjena biljna vrsta koja kao samonikla uspeva u skoro svim krajevima naše zemlje. To je listopadni žbun visok 2-3 m sa dugim, debelim i razgranatim granama koje rastu uspravno. Zbog široke farmakološke primene i visokog nivoa vitamina C u šipurku (Rosae pseudofructus), ova biljka je uvrštena u red lekovitih biljaka za kojom postoji velika tržišna tražnja. U otkupu ove biljne droge tržištu fitopreparata u velikoj meri konkuriše tržište prehrambene industrije, koje perikarp šipurka koriste za izradu marmelade. Ova biljna vrsta se sve više se uvodi u plantažnu proizvodnju koja kao izlaznu sirovinu daje veći plod sa debljim perikarpom u odnosu na polodove sakupljene iz prirode. Postoji nekoliko vrsta divlje ruže, koje se međusobno razlikuju po bujnosti, krupnoći ploda i vremenu zrenja. Divlja ruža ima skromne zahteve u pogledu plodnosti i kvaliteta zemljišta, pa se za njieno gajenje mogu kultivirati i zemljišta slabijeg kvaliteta na različitim nadmorskim visinama. Sa gajenjem šipurka u Srbiji otpočelo se pre 15 godina na dva lokaliteta u Južnom Banatu. Rasad divlje ruže se proizvodi iz semena, a proizvodnjom ove vrste rasada uglavnom se bave proizvođači podloga za kalemljenje ruža. Dozrelo seme ima izuzetno trvtdu semenjaču, pa je, za seme, potrebno prikupljati nedozrele plodove. Za površinu od 1 ha potrebno je obezbediti 1300 – 1500 sadnica. Zbog bujnosti nadzemnog dela sadi se na rastojanju 3 m između redova i 2,5 m u redu. Sadnju je najbolje obaviti u jesen, ali i prolećna sadnja daje dobre rezultate. Ako su sadnice dobro razvijene prvi rod se dobija već u drugoj godini gajenja. Branje se obavlja ručno u periodu avgust – septembar, u zavisnosti od sorte. Kampanja branja traje oko 10 dana. Posle branja, plod šipurka se suši. Sušenje je najbolje obaviti u sušarama, ali je moguće i prirodno sušenje. Osušen plod se prerađuje pomoću odgovarajuće opreme. Prerada se sastoji u dezintegraciji ploda i kasnijem odvajanju perikarpa, semena i dlačica, vazdušnom selekcijom. Prinos se u nakon druge godine povećava, a od četvrte godine dostiže svoj permanentni nivo u visini od oko 12 t/ha sirovog ploda odnosno oko 5 t/ha suvog ploda. Maseni udeo perikarpa je oko 50 %, a semena 48%. U okviru istraživanja, razrađena je i HPLC metoda za analizu sadržaja vitamina C u plodu šipurka. Šipurak je veoma tražena biljna droga, koja ima relativno niske agrotehničke zahteve i podizanje ovakvih plantaža, uz dobru organizaciju rada prilikom berbe, može biti unosan sporedni posao za poljoprivrednike.

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U2 Cultivation of dog rose (Rosa canina L.) and rosehips (Rosae pseudofructus) processing Brkić Sreta, Pljevljakušić Dejan, Menković Nebojša 1Institute for Medicinal Plants Research „Dr Josif Pančić“, Tadeuša Košćuška 1, 11000 Belgrade, Serbia Dog rose (Rosa canina L.) is widespread plant specie which, as wild growing, thrives in almost every part of our country. It is deciduous shrub with long, thick; expand branches which are rising upright. Due to wide pharmacological appliance and high level of vitamin C, in dog rose fruit – rosehip (Rosae pseudofructus), this plant has been ranked as a medicinal plant for which market demand is ample. In ransom of this plant drug, phytotherapy market is in a large part competing by food industry market, which are using Rosehip pericarp for marmalade production. This plant has been, recently, frequently included in plantation production, which gives, as output raw material, bigger fruit with thicker pericarp in comparison with wild crafted fruits. There are several species of dog rose, which differ in growth habit, fruit dimensions and time of maturity. Dog rose has modest requirements regard soil quality and fertility; therefore its cultivation could be established on some low quality soils at different altitudes. Cultivation of this plant, at two selected localities, in South Banat, began 15 years ago. Seedlings production is from seeds, and producers of these seedlings are mainly people who already cultivate grafting rose planting material. Mature seed has very hard lignified seed coat, thus immature fruits collection for seed production is needed. For cultivation area of 1 ha, 1300-1500 seedlings has to be ensured. Due to large growth habit planting density is 3 m between rows and 2.5 m in the row. It is better to perform planting in autumn, but spring planting also gives satisfactory results. If seedlings are well developed, first harvest could be expected already in second year. Picking of rosehips is manual in period of August – September, depending on species and altitude. Picking campaign is about 10 days long. After picking, rosehips has to dry. Drying should be preformed in dryers, but open-air drying is also possible. Dry hips are processed through specialized equipment. Processing consists of hip disintegration, and later separation of pericarp, seeds and inner fruit fur, by air selection. Yield increases after second year and in fourth year reaches its permanent level of about 12 t/ha fresh hips, apropos 5 t/ha of dry hips. Mass portion of pericarp is about 50% and seeds about 48%. In this research, HPLC method for analysis of content of vitamin C, in rosehips, has been developed. Rosehips are much demanded plant drug, which has relatively low agrotechnical requirements and establishment of these plantations, with good labour management during picking campaign, could be very profitable side business for farmers.

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U3 Sistematski skrining na biljne fenole QQQ LC/MS/MS tehnikom Dejan Orčić1, Neda Mimica-Dukić1, Emilija Jovin1, Ivana Beara1, Kristina Balog1

1 Departman za hemiju, Prirodno-matematički fakultet, Trg Dositeja Obradovića 3, 21000 Novi Sad, Srbija LC/DAD/MSn predstavlja standardnu tehniku za analizu biljih fenola, koja istovremeno omogućava razdvajanje kompleksnih smeša hemijski sličnih jedinjenja i pruža strukturne informacije o nepoznatim jedinjenjima, uz minimalnu pripremu uzorka. Dok se MSn rutinski koristi za rasvetljavanje strukture pojedinačnih komponenti, bilo je malo pokušaja da se različiti MS1, MS2 i pseudo-MS3 eksperimenti dostupni kod QQQ primene za sistematski skrining biljnih ekstrakata na fenolna jedinjenja. U ovom radu, različiti biljni ekstrakti (uključujući Eucalyptus gunni, Eucalyptus camaldulensis, Myrtus communis, Galium verum) analizirani su na biljne fenole. Rapid resolution RP HPLC sistem obezbedio je zadovoljavajuće razdvajanje komponenata kompleksne smeše za manje od 15 min, dok je QQQ maseni spektrometar sa elektrosprej jonizacijom (ESI-QQQ MS/MS) omogućio rasvetljavanje njihove strukture i identifikaciju. Demonstrirana je primena različitih MS1, MS2 i pseudo-MS3 modova akvizicije i tehnika kao što je katjonizacija u sistematskoj analizi biljnih fenola. Pokazano je da QQQ, uprkos činjenici da je slabijih mogućnosti po pitanju snimanja spektara (budući da je prilagođen za kvanfitaciju) i ne može potpuno zameniti instrumente sa jonskom zamkom i Q-TOF, kao ni NMR tehniku, može biti od izuzetnog značaja u skriningu na biljne fenole. Zahvalnice: Ministarstvo nauke Republike Srbije (Projekat br. 142036) Literatura: 1. De Rijke E. et al. (2006) J. Chromatogr. A 1112:31-63, 2. Tian Q. et al. (2005) J. Chromatogr. A 1091:72-82, 3. Farag M.A. et al. (2007) Phytochemistry 68:342-254, 4. Lhuillier A. et al. (2007) J. Chromatogr. A 1160:13-20, 5. Cuyckens F. et al. (2004) J. Mass Spectrom. 39:1-15.

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U3 Systematic plant phenolics screening by QQQ LC/MS/MS Dejan Orčić1, Neda Mimica-Dukić1, Emilija Jovin1, Ivana Beara1, Kristina Balog1

1 Department of Chemistry, Faculty of Sciences, Trg Dositeja Obradovića 3, 21000 Novi Sad, Serbia LC/DAD/MSn becomes a standard technique in plant phenolics analysis, providing both means to quickly separate complex mixture of chemically similar compounds and to obtain structural data for unknowns, with minimal sample preparation. While MSn is routinely used for structural elucidation of individual peaks, a few attempts have been made to use QQQ’s various available MS1, MS2 and pseudo-MS3 experiments for systematic screening of plant extracts for phenolic compounds. In this work, various plant extracts (including Eucalyptus gunni, Eucalyptus camaldulensis, Myrtus communis, Galium verum) were analyzed for plant phenolics. Rapid resolution reversed phase LC system provided sufficient separation of complex mixture components within less than 15 min, while electrospray ionization triple-quad mass spectrometer (ESI-QQQ MS/MS) enabled their structural elucidation and identification. The application of different MS1, MS2 and pseudo-MS3 acquisition modes and techniques as cationization is demonstrated in light of systematic screening for plant phenolics. It is shown that, although QQQ is less powerful regarding spectra (being primarily optimized for quantitation) and cannot fully replace ion trap and Q-TOF instruments, as well as NMR technique, it can still be invaluable tool in tentative screening for plant phenolics. Acknowledgements: Ministry of Science, Republic of Serbia (Grant No. 142036) References: 1. De Rijke E. et al. (2006) J. Chromatogr. A 1112:31-63, 2. Tian Q. et al. (2005) J. Chromatogr. A 1091:72-82, 3. Farag M.A. et al. (2007) Phytochemistry 68:342-254, 4. Lhuillier A. et al. (2007) J. Chromatogr. A 1160:13-20, 5. Cuyckens F. et al. (2004) J. Mass Spectrom. 39:1-15.

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U4 Specijaciona analiza hroma u zemljištu i uzorcima pitome nane, Mentha piperita Đogo S.1,Manojlović D. 2, Ražić S.1 1 Farmaceutski fakultet u Beogradu, Vojvode Stepe 450, 11000, Beograd, Srbija; 2 Hemijski fakultet u Beogradu, Akademski trg 12-16, 11000, Beograd, Srbija Koncentracija u zemljištu je jedan od najznačajnijih ekoloških činilaca koji utiče na usvajanje metala od strane biljaka (1). Usvajanje, odnosno dostupnost metala ne zavisi samo od koncentracije, nego i od sposobnosti zemljišta da održava tu koncentraciju u rastvoru, te sposobnosti biljke da ih selektivno akumulira (2,3). Mehanizam usvajanja nije sasvim selektivan i biljke mogu usvojiti znatno veću količinu jona nego sto je potrebno za optimalno odvijanje životnih procesa (4). Mobilnost hroma u zemljištu, njegova dostupnost biljkama, kao i toksični efekti zavise od oksidacionog stanja i forme u kojoj se element nalazi u zemljištu. Naime, Cr(III) je esencijalna jonska vrsta, dok je Cr(VI) toksičan jer deluje kao jako oksidaciono sredstvo i toksični efekti se ispoljavaju već pri koncentraciji u zemljištu jednakoj ili većoj od 5 mg/kg. Cilj ovog rada je bio ispitivanje kapaciteta zemljišta za vezivanje hroma, uslovi pod kojima bi moglo doći do njegovog povećanog rastvaranja, kao i sposobnost biljke da kontroliše njegovo usvajanje. U tu svrhu je izveden eksperiment u kome je nana, Menthae piperita, kontrolisano gajena, u laboratorijskim uslovima, na zemljištu koje je tretirano sa 40, 80, 120 i 200 mg/kg rastvora Cr(III). Za kontaminaciju zemljišta korišćeni su rastvori Cr(III)-nitrata. Nakon završenog perioda vegetacije, izvršena je mikrotalasna digestija korena i nadzemnog dela biljaka (CEM, MDS-2000) po zvaničnoj proceduri US-EPA 3052: Microwave Assisted Acid digestion of Silicons and organically Based Matrices 3052 (A20) SW 846 Ch 3.2, Metod Code 10136206. Sem toga, radi procene dostupnosti hroma u zemljištu, formi u kojima se nalazi, kao i sposobnosti zemljišta da imobiliše dodati hrom i time spreči ulazak u lanac ishrane, na sve uzorke je primenjena sekvencijalna ekstrakcija u tri faze: 1M rastvor amonijum – acetata, 0,1 M rastvor hidroksilamin – hlorhidrata i smeša 0,2M rastvora oksalne kiseline i amonijum – oksalata. Sadržaj hroma je analiziram primenom metode atomske apsorpcione spektrometrije sa grafitnom kivetom (ETAAS), a kompletna procedura validirana u skladu sa važećim principima analitičke hemije. Zahvalnica:Rad je urađen u okviru Projekta br 142039, koji finansira Ministarstvo za nauku i zaštitu životne sredine. Zahvaljujemo se Dr Radosavu Jevđovicu iz Instituta za ispitivanje lekovitog bilja Dr Josif Pančić u Pančevu na stručnim savetima za gajenje biljaka u laboratoriji i na plantaži. Literatura: 1. Kastori R., (1998), Teški metali u životnoj sredini, Naučni institut za ratarstvo i povrtarstvo, Novi sad 2. Miroslawski J., et al. (1995) Bromat. Chem. Toksykol., 28, 363-372, 3. Lozak A., et al., (2002), Sci. Tot. Environ, 289, 33-40, Mengel K., Kirkby A. E., (1999), Principles of Plant Nutrition, International Potash Institute, Switzerland.

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U4 Speciation analysis of chromium in soil and plant material of Mentha piperita Đogo S.1,Manojlović D. 2, Ražić S.1 1 Farmaceutski fakultet U Beogradu, Vojvode Stepe 450, 11000, Beograd, Srbija; 2 Hemijski fakultet u Beogradu, Akademski trg 12-16, 11000, Beograd, Srbija The most important ecological factor for metal uptake by plants is its concentration in soil (1). Metal uptake does not depend only on concentration, but also on the ability of the soils to keep the metal ions in soil solution and plants ability for making selection as well (2, 3). The uptake mechanism is not completely selective and plant takes more ions that are necessary for optimal life process (4). The mobility of chromium in soil, its accessibility and toxic effects depend on its form and oxidation state. Chromium (III) is an essential ionic species while Cr (VI), being a very strong oxidizing agents, is very toxic even with its concentration in soil equal or higher than 5 mg/kg. The objective of this work was to investigate the soil capacity for chromium binding and conditions that could increase its solubility and capability of plants to control chromium uptake. In order to investigate the plants control of chromium uptake the following experiment was designed: Mentha piperita was cultivated under controlled laboratory conditions on the soils spiked with standard solutions of Cr(III) (40, 80, 120 and 200 ppm). For that purpose Cr(III)-nitrate was used. After the vegetation period was finished, all the plants samples (roots and herbs) were subjected to standard procedure of microwave digestion (US-EPA 3052: Microwave Assisted Acid digestion of Silicons and organically Based Matrices 3052 (A20) SW 846 Ch 3.2, Metod Code 10136206). For the evaluation of chromium bioavailability in soils, its forms and ability of soil to immobilize spiked chromium and prevent its entering the food chain the next experiment was conducted: sequential extraction in three stages was performed, with different chemical treatment (1M solution of amonium – acetate, 0.1 M hidroxilamine– chlorohydrate solution and the mixture of 0.2M oxalic acid and amonium – oxalate). Chromium concentrations were determined by applying atomic absorption spectrometry with graphite furnace (GF-AAS) and overall procedure was validated according to the most important principles of analytical chemistry. Acknowledgement:This work was supported by the Ministry of Science and Environmental Protection under Project No. 142039. The authors wish to thank Dr Radosav Jevđović from the Institute for Medical Plant Research Dr Josif Pančić, Pancevo, for advices in cultivating Mentha piperiata in laboratory and field conditions References: 1. Kastori R., (1998), Teški metali u životnoj sredini, Naučni institut za ratarstvo i povrtarstvo, Novi Sad. 2. Miroslawski J., et al. (1995) Bromat. Chem. Toksykol., 28, 363-372. 3. Lozak A., et al., (2002), Sci. Tot. Environ, 289, 33-40, Mengel K., Kirkby A. E., (1999), Principles of Plant Nutrition, International Potash Institute, Switzerland.).

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U5 Sadržaj vitamina E u masnom ulju pasjeg trna Z. Basić1, S. Cupara2, M. Maksimović1, D. Krstić1

1 Institut za higijenu, Vojnomedicinska akademija, Crnotravska 17, 11000, Beograd, Srbija; 2 Medicinski fakultet, Odsek za farmaciju, Kragujevac, Srbija Biljna ulja karakteriše visok sadržaj vitamina E. Zavisno od tehnološkog postupka dobijanja, ta količina može biti očuvana ili umanjena. Hippophae rhamnoides (pasji trn) je trnovit grm koji divlje raste u Aziji i Evropi, uglavnom uz reke, jezera i morsku obalu. Medicinska upotreba ove biljke je zabeležena još u medicinskim knjigama stare Kine, Grčke i Tibeta [1]. Plodovi su bogati uljem, a sadržaj vitamina E u pulpi i semenu nije jednak [2]. Naš cilj je bio da razvijemo HPLC metodu za određivanje sadržaja tokoferola u ulju i odredimo njihov sadržaj u masnom ulju pasjeg trna komercijalnog porekla i ručno ceđenom. Za uspešno određivanje sadržaja vitamina E u namirnicama neophodno je višestruko prečišćavanje uzorka. Najčešće korišćena metoda pripreme za određivanje vitamina E u ulju je tečno/tečna ekstrakcija. Mi smo primenili ekstrakciju n-heksanom, uparavanje ekstrakta i rekonstituisanje u metanolu uz membran filtraciju [3]. Razdvajanje je vršeno RP-HPLC metodom uz fluorescentni detektor sa promenljivim talasnim dužinama. Za detekciju tokoferola primenjen je 95% metanol kao mobilna faza, sa protokom od 1,5 mL/min i ekscitacionom i emisionom energijom 295/330 nm. Selektivnost i osetljivost fluorescentnog detektora je omogućila detekciju tokoferola u ispitivanim uljima. Određen je sadržaj alfa, beta sa gama i delta tokoferola u komercijalnom i ručno ceđenom ulju u količini od 77,9, 9,9, 0,75 mg/100 g, odnosno 115,6, 5,0 i 0,42 mg/100 g ulja. Možemo zaključiti da je masno ulje pasjeg trna dobar izvor vitamina E, ali da postoji razlika u koncentraciji zavisno od načina dobijanja ulja. Literatura: 1. J.Xing, B.Yang, Y.Dong, B.Wang and others: Effects of sea buckthorn (Hippophae rhamnoides L..) seed and pulp oils on experimental models of gastric ulcer in rats. Fitoterapia. 2002, 73: 644-650. 2. H.Kallio, B.Yang, P.Peippo, R.Tahvonen, R.Pan: Triacyglycerols, Glycerophospholipids, Tocopherols and Tocotrienols in Berrie and Seeds of Two Subspecies (ssp. sinensis and mongolica) of Sea Buckthorn (Hippophea rhamnoides). J. Agric. Food Chem. 2002, 50:3004-9. 3. G.F.M.Ball: Vitamins in foods, Analysis, Bioavailability and Stability, Taylor & Francis 2006, USA, pp 423-426.

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U5 Vitamin E content in Hippophae rhamnoides oil Z. Basić1, S. Cupara2, M. Maksimović1, D. Krstić1

1Institute of Hygiene, Military Medical Academy Belgrade, Crnotravska 17, 11000, Belgrade

2 Faculty of Medicine Kragujevac, Department of Pharmacy, Kragujevac Vegetable oils contain high levels of vitamin E. Depending on the technological method applied, the amount of tocopheroles in oil varies. Hippophae rhamnoides is a small branched tree, grows widely in Asia and Europe, its habitat are river, lake and see shores mostly. Medical use of this plant was noted even in old China, Tibet and Greek medical books [1]. Fruits are rich in oil, but their vitamin E content is depending on whether the oils are obtained from pulp, seed or both pulp and seed of the plant [2]. The aim was to evaluate HPLC method for determination of tocopherols content in pulp and in fatty oil of buckthorn berries seed. For successful vitamin E determination in food formulas sample clean-up steps are necessary. The most common used methodology for vitamin E determination in oils is still solvent extraction. We have applied n-hexane extraction, evaporation and reconstitution in methanol, followed by membrane filtration [3]. Separation was performed with RP-HPLC system including fluorescence detector with changeable wavelength. For vitamin E detection a mobile phase containing 95% MeOH, during flow-rate of 1.5 mL/min, excitation and emission energies were 295/330 nm. The selectivity of the fluorescence detector allowed accurate tocopheroles determination in examined oils. The content of alpha, beta with gamma and delta was 77.9, 9.9, 0.75 mg/100g, i.e. 115.6, 5.0, 0.42 mg/100g. We may conclude that the fatty oil of Hippophae rhamnoides represents a good source of vitamin E, while its amount may vary depending on the applied production method. References 1. J.Xing, B.Yang, Y.Dong, B.Wang and others: Effects of sea buckthorn (Hippophae rhamnoides L.) seed and pulp oils on experimental models of gastric ulcer in rats. Fitoterapia. 2002, 73: 644-650. 2. H.Kallio, B.Yang, P.Peippo, R.Tahvonen, R.Pan: Triacyglycerols, Glycerophospholipids, Tocopherols and Tocotrienols in Berrie and Seeds of Two Subspecies (ssp. sinensis and mongolica) of Sea Buckthorn (Hippophea rhamnoides). J. Agric. Food Chem. 2002, 50:3004-9. 3. G.F.M.Ball: Vitamins in foods, Analysis, Bioavailability and Stability, Taylor & Francis 2006, USA, pp 423-426.

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U6 Flavonoidi, sekoiridoid oleuropein i gastroprotektivna aktivnost suvog ekstrakta listova masline Marković G1, Tadić VM1, Dekanski D2, Arsić I1, Đorđević S1, Runjaić-Antić D1

1 Institut za pručavanje lekovitog bilja „Dr Josif Pančić“, Tadeuša Košćuška 1, 11000 Beograd, Srbija; 2 Služba za biomedicinska ispitivanja, Institut za istraživanje i razvoj, Galenika a.d., 11000 Beograd, Srbija Maslina (Olea europaea L., Oleaceae) raste i gaji se u mediteranskom regionu. Proučavana je iscrpno zbog raširene upotrebe plodova i ulja, koji u nekim područjima sveta predstavljaju važne komponente svakodnevne ishrane. Smatra se da je to razlog smanjenom broju onih koji obolevaju od kardiovaskularnih bolesti u Mediteranu. List masline se uglavnom upotrebljava kao antihipertenziv i hipoglikemik. Navedena upotreba je povezana sa prisustvom sekoiridoida oleaceina i oleuropeina [1,2]. Cilj ove studije bio je da se utvrdi korelacije između pokazanog značajnog gastroprotektivnog efekta ekstrakta lista masline i prisutnih flavonoida i sekoiridoida oleuropeina. Za analizu je upotrebljen suvi ekstrakt masline (OLE, FLA® 943, Flachsmann). Sadržaj ukupnih fenola je određen primenom Folin-Ciocalteu metode [3]. Sadržaj je bio ekstrmno visok – 197,8 mg GA/g ispitivanog ekstrakta. Za identifikaciju i kvantitativnu analizu prisutnih fenolskih jedinjenja primenjena je HPLC metoda. U ispitivanom ekstraktu identifikovana su i kvantifikovana sledeća jedinjenja (Tabela 1): kafena kiselina (1), luteolin-7-O-glukozid (4), apigenin-7-O-glukozid (5), oleuropein (6), kvercetin (7), luteolin (8) i hrizoeriol (10). Iako je u literaturi navedeno prisustvo i vanilina (2), rutina (3) i apigenina (9), u ispitivanom ekstraktu nisu detektovani [4]. Ispitano je i protektivno dejstvo ekstrakta lista masline kod oštećenja želudačne mukoze indukovanog apsolutnim etanolom, na Wistar albino pacovima. Intragastrična primena 1 ml apsolutnog etanola uzrokovala je obimna submukozna krvarenja. Pretretman sa 80 mg/kg ekstrakta lista masline je značajno smanjio nastanak gastričnih lezija. Procenat inhibicije ulkusnog indeksa u odnosu na kontrolnu grupu bio je 82%. Ovaj rezultat ukazuje na snažnu gastroprotektivnu aktivnost ekstrakta lista masline. S obzirom na dokazanu biološku aktvnost fenolkarbonskih kiselina, flavonoida [5] i sekoiridoida oleuropeina [1], sve identifikovane komponente u ekstraktu masline mogu biti odgovorne za pokazanu gastroprotektivnu aktivnost. Table 1. Sadržaj flavonoida, fenolkarbonskih kiselina i oleuropeina u suvom ekstraktu listova masline (EFLA® 943, Flachsmann, *brojevi u tabeli se odnose na jedinjenja ranije pomenuta u tekstu)

Jedinjenje * 1 2 3 4 5 6 7 8 9 10 Sadržaj (%) 0,02 nf nf 0,04 0,07 19,7 0,04 t nf t

nd=nije detektovan; t=trag Zahvalnica: Autori zahvaljuju Ministarstvu za nauku i tehnološki razvoj za finansijsku podršku, projekat br. 20137 Literatura: 1. Hansen K. et al. (1996) Phytomed. 2:319-324. 2. Gonzalez M. et al. (1992) Planta Med. 58: 513-515. 3. Velioglu Y. et al. (1998), J. Agric. Food Chem. 46:4113-4117. 4. Bouaziz M. et al. (2004) J. Agric. Food Chem. 52:5476-5481. 5. Havsteen B.H. (2002) Pharmacol. & Therap. 96:67– 202.

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U6 Flavonoids, secoiridoid oleuropein and gastroprotective activity of olive leaves dry extract Marković G1, Dekanski D2, Tadić VM1, Arsić I1, Đorđević S1, Runjaić-Antić D1

1 Institute for Medicinal Plant Research “Dr Josif Pančić”, Tadeuša Košćuška 1,11000 Belgrade,Serbia; 2Biomedical Research, R&D Institute, Galenika a.d., 11000 Belgrade, Serbia Olive tree (Olea europaea L., Oleaceae) is one of the most important fruit trees in Mediterranean countries, where they cover eight million ha, accounting for almost 98% of the world crop. Olive is widely studied for its alimentary use (the fruits and the oil are important components in the daily diet of a large part of the world’s population), whereas the leaves are important for their secondary metabolites such as secoiridoid compounds oleacein and oleuropein. These compounds are responsible for hypoglycemic and hypotensive activities of olive [1,2]. The present study was carried out to correlate observed gastroprotective effect of investigated olive leaf extract (OLE, EFLA® 943, Flachsmann) and phenolics content. The total phenolics content was determined by the Folin-Ciocalteu method [3] and revealed high amount – 197.8 mg GA/g in investigated extract. The HPLC-DAD analysis of OLE allowed identification and quantification of seven phenolic compounds (Table 1): caffeic acid (1), luteolin-7-O-glucoside (4), apigenin-7-O-glucoside (5), oleuropein (6), quercetin (7), luteolin (8) and chrysoeriol (10). Although literature suggested [4], vanillin (2), rutin (3) and apigenin (9), were not identified in the extract. The protective effect of olive leaf extract (OLE) on gastric mucosal damage induced by absolute ethanol in Wistar albino rats was investigated. Intragastrically (i.g.) applied absolute ethanol (1 ml) caused severe gastric submucosal hemorrhage. Pretreatment (i.g.) with 80 mg/kg of OLE significantly (p<0,001) attenuated gastric lesions. Percent of inhibition in ulcer index in relation to the control group, was 82%. This result suggests that OLE exerts potent gastroprotective activity. Taking into account the potent biological effects of flavonoids [5] and secoiridoids [1] all active components identified in the extract might be responsible for activity observed. Table 1. Determination of flavonoids, phenolcarbonic acids and oleuropein in olive leaves dry extract, (EFLA® 943, Flachsmann, *the numbers refer to compounds mentioned in the text)

Compound * 1 2 3 4 5 6 7 8 9 10 Content (%) 0.02 nf nf 0.04 0.07 19.7 0.04 t nf t

nf = not found; t=trace Acknowledgement: The authors thank Serbian Ministry of Science and Technological Development for financial support, project N020137 References: 1. Hansen K. et al. (1996) Phytomed. 2:319-324. 2. Gonzalez M. et al. (1992) Planta Med. 58: 513-515. 3. Velioglu Y. et al. (1998), J. Agric. Food Chem. 46:4113-4117. 4. Bouaziz M. et al. (2004) J. Agric. Food Chem. 52:5476-5481. 5. Havsteen B.H. (2002) Pharmacol&Therap. 96:67– 202.

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U7 Plantago media L. kao potencijalni prirodni antioksidativni i anttiinflamatorni agens I. Beara1, E. Jovin1, M. Lesjak1 , S. Ljubojević1,D. Orčić1, N. Mimica-Dukic1

1 Departman za hemiju, Prirodno-matematički fakultet, Univerzitet u Novom Sadu, Trg D. Obradovića 3, 21000 Novi Sad, Srbija Rod Plantago L. (Plantaginaceae) obuhvata oko 275 široko rasprostranjenih vrsta. Vekovna upotreba bokvica kao biljnog leka je posledica njihovog astringentnog, anti-toksičnog, antimikrobnog, ekspektorantnog i diuretskog dejstva. Cenjena u tradicionalnoj medicini, Plantago media L. (srednja bokvica) korišćena je kao astringent za lečenje rana, protiv zubobolje i infekcije oka, a njeno seme kao laksativ. U nameri da valorizujemo upotrebu ove biljke u medicini, podvrgli smo je određenim biološkim i farmakološkim testovima. Ovde objavljujemo antioksidantnu i antiinflamatornu aktivnost metanolnih ekstrakta P. media, sakupljene na dva lokaliteta u Srbiji (planine Kopaonik i Zlatar). Sastav ekstrakata je ispitan određivanjem sadržaja ukupnih fenola Folin-Ciocalteu metodom [1] i sadržaja flavonoida pomoću AlCl3 reagensa [2], primenom kolorimetrijske tehnike. Kapacitet hvatanja slobodnih radikala (RSC) ispitan je spektrofotometrijski, merenjem sposobnosti neutralizacije DPPH* (difenilpikrilhidrazil), OH* (hidroksil), O2*– (superoksid anjon) i NO* (azot oksid) radikala [3]. Antiinflamatorna aktivnost ispitana je merenjem inhibicije 12-lipoksigenaze (12-LO), kvantifikacijom 12-LO proizvoda 12-HETE (12-hidroksi-5,8,10,14-eikosatetraenoat) pomoću RP-HPLC/MS/MS. Sadržaj ukupnih fenolnih jedinjenja (izražen kao g ekvivalenata hlorogenske kiseline / g suvog ekstrakta) iznosi 0,18 (lok. Kopaonik) i 0,20 (lok. Zlatar). Sadržaj flavonoida iznosi 9.0 i 9.5 mg ekvivalenata kvercetina / g suvog ekstrakta. Slična RSC aktivnost je utvrđena za oba ekstrakta: DPPH* (IC50 5,8 i 4,4 µg/ml), OH* (IC50 372 i 352 µg/ml), O2*– (IC50 43 i 32 µg/ml) i NO* (IC50 1,27 i 1,38 mg/ml), što upućuje na sličnu ili veću aktivnost od sintetičkih antioksidanata poput BHT (butilovani hidroksitoluen) ili PG (propil galat). Pri koncentraciji od 2 mg/mL oba ekstrakta su pokazala 12-LO inhibitornu aktivnost (73 i 66%). Ovim istraživanjem utvrdili smo i antioksidantnu i antiinflamatornu aktivnost metanolnih ekstrakata P. media i predstavili je kao mogući, novi izvor prirodnih antioksidanta i antiinflamatornih agenasa. Takođe, razvili smo novi in vitro test za određivanje antiinflamatorne aktivnosti. References: 1. Singleton, V.L. et al. (1999) Methods in enzymology, Acad. Press, New York. 2. Chang, C.C et al. (2002) J Food Drug Anal. 10: 178-182. 3. Sanchez-Moreno, C. (2002) Food Sci Tech Int 8: 121-137.

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U7 Plantago media L. as a potential natural antioxidant and antiinflammatory agent I. Beara1, E. Jovin1, M. Lesjak1 , S. Ljubojević1, D. Orčić1, N. Mimica-Dukic1

1 Department of Chemistry, Faculty of Sciences, University of Novi Sad, Trg D. Obradovica 3, 21000 Novi Sad, Serbia The genus Plantago L. (Plantaginaceae) comprises about 275 species found all over the world. Ancient use of plantains as herbal remedies is a consequence of their astringent, anti-toxic, antimicrobial, expectorant and diuretic properties. Respected in traditional medicine, Plantago media L. (hoary plantain) has been mostly used as an astringent for treating wounds, as remedy against toothache and eye infections, and the seeds as a laxative. In order to valorize medicinal use of this plant some more biological and pharmacological tests have been undertaken. Here we report on antioxidative and antiinflammatory activities of methanolic extracts of P. media, collected from two different locations in Serbia (Mountains Kopaonik and Zlatar). Examined extracts have been characterized regarding to their composition by different colorimetric techniques, for instance the content of total phenolic compounds by the Folin-Ciocalteu assay [1] and flavonoids by AlCl3 reagent [2]. Their radical scavenger capacity (RSC) was evaluated towards DPPH* (diphenylpicrylhydrazyl), OH* (hydroxyl), O2*– (superoxide anion) and NO* (nitric oxide) radicals using spectrophotometry [3]. Antiinflammatory activity was examined by means of 12-lipooxygenase (12-LO) inhibition, quantifying the 12-LO product 12-HETE (12-hydroxy-5,8,10,14-eicosatetraenoate) by RP-HPLC/MS/MS. The content of total phenolic compounds (expressed as g hlorogenic acid equivalents / g of dry extract) were 0.18 (loc. Mt. Kopaonik) and 0.20 (loc. Mt. Zlatar). Flavonoid content was 9.0 and 9.5 mg quercetine equiv / g of dry extract. Similar RSC was found for both extracts: DPPH* (IC50 5.8 and 4.4 µg/ml), OH* (IC50 372 and 352 µg/ml), O2*– (IC50 43 and 32 µg/ml) and NO* (IC50 1.27 and 1.38 mg/ml), indicating comparable or higher activity than synthetic antioxidants as BHT (butylated hydroxytoluene) or PG (propyl gallate). At concentration of 2 mg/ml both extracts showed 12-LO inhibitory activity, 73 and 66%, respectively. In this study we approved both antioxidant and antiinflammatory activity of P. media methanolic extracts and introduced P. media as a possible new source of natural antioxidant and antiinflammatory agents. Furthermore, the new in vitro test for antiinflamatory activity was introduced and developed. References: 1. Singleton, V.L. et al. (1999) Methods in enzymology, Acad. Press, New York. 2. Chang, C.C et al. (2002) J Food Drug Anal. 10: 178-182. 3. Sanchez-Moreno, C. (2002) Food Sci Tech Int 8: 121-137.

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U8 Antioksidantna aktivnost ekstrakta ploda štavelja (Rumex crispus L., Polygonaceae) Maksimović Z1,Ćebović T2

1 Institut za farmakognoziju, Farmaceutski fakultet, Univerzitet u Beogradu, Vojvode Stepe 450, 11221 Beograd, Srbija; 2 Institut za biohemiju, Medicinski fakultet, Univerzitet u Novom Sadu, Hajduk Veljkova 3, 21000 Novi Sad, Srbija Rumex crispus L. (štavelj) se u mnogim krajevima smatra invazivnom korovskom vrstom. Rasprostire se rasejavanjem semena, koje kontaminira seme useva, ili se kači za odevne predmete i runo ovaca. Međutim, postoje literaturni podaci da se koren ove biljne vrste koristi u tradicionalnoj medicini u prolećnim kurama “čišćenja krvi”, kao tonik, laksativ, kod reumatizma, oboljenja žuči, i kao adstringent kod hemoroida, krvavljenja itd. Takođe, preporučuje se i protiv čitavog niza kožnih oboljenja (od crvenila, do skorbuta i tuberkuloze limfnih žlezda), protiv žutice i funkcionalnih poremećaja želuca i gastrointestinalnog trakta u celini. U narodnoj medicini Srba je opisana i upotreba plodova ove biljke kod dizenterije, zbog adstringentnog delovanja [1,2]. S druge strane, mladi listovi štavelja su jestivi i ponekad se koriste kao povrće [3]. U ovom radu, antioksidantni potencijal metanolnog ekstrakta zrelih plodova R. crispus ocenjen je određivanjem sposobnosti redukcije Fe(III) jona (FRAP-test), određivanjem sposobnosti neutralizacije slobodnih DPPH radikala (DPPH-test) i određivanjem uticaja na lipidnu peroksidaciju u lipozomima (LP-test). U svim test-sistemima uočena je izrazita in vitro aktivnost ispitivanog ekstrakta (FRAP: 9,9 mmol Fe2+/g; DPPH IC50: 3,7 µg/ml; LP IC50: 4,9 µg/ml), uporediva sa aktivnošću BHT (FRAP: 8,0 µg/ml; DPPH IC50: 19,4 µg/ml; LP IC50: 3,5 µg/ml), ali niža od aktivnosti askorbinske kiseline, rutina i kvercetina koji su korišćeni kao pozitivne kontrolne supstancije. In vivo efekti su praćeni na nekoliko hepatičkih antioksidantnih sistema (intenzitet lipidne peroksidacije (LPx), aktivnost glutation preoksidaze (GSH-Px), peroksidaze (Px), katalaze (CAT) i ksantin oksidaze (XOD), kao i sadržaj redukovanog glutationa (GSH)), nakon intraperitonealne primene ekstrakta u različitim dozama kod pacova, kao i u kombinaciji sa ugljen-tetrahloridom (CCl4). Tretman metanolnim ekstraktom plodova R. crispus dozno zavisno je inhibirao oštećenje jetre indukovano CCl4 snižavanjem aktivnosti LPx i povećanjem sadržaja GSH, dovodeći nivo aktivnosti antioksidantnih enzima do vrednosti bliskih kontrolnim. Naposletku, dobijeni rezultati pokazuju da metanolni ekstrakt ploda R. crispus poseduje specifični antioksidantni potencijal, ukazujući na to da se ekstrakt ploda ove biljke može koristiti ne samo kao hepatoprotektivni agens, nego i u ublažavanju posledica drugih poremećaja izazvanih oksidativnim stresom. Zahvalnica: Ostvareno uz podršku projekta br. 143012 Ministarstva nauke i zaštite životne sredine, Republika Srbija. References: 1. Jančić R et al. (1989). Lekovite biljke SR Srbije, Srpska akademija nauka i umetnosti, Beograd. 2. Tyler VE (1993). The Honest herbal, Pharmaceutical Product Press, New York, London, Norwood (Australia). 3. Vračarić B et al. (1977). Ishrana u prirodi. Vojnoizdavački zavod, Narodna knjiga, Beograd.

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U8 Antioxidant activity of yellow dock (Rumex crispus L., Polygonaceae) fruit extract Maksimović Z1,Ćebović T2

1 Institute of Pharmacognosy, School of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11221 Belgrade, Serbia; 2 Institute of Biochemistry, School of Medicine, University of Novi Sad, Hajduk Veljkova 3, 21000 Novi Sad, Serbia Rumex crispus L. (yellow dock) is considered as a serious invasive weed in many areas. It spreads through the fruits contaminating crop seeds, and sticking to clothing or fleece. However, the roots of this plant have a record of regular use in traditional medicine as a helpful alternative (a term that refers to a drug intended for the treatment of venereal diseases and used synonymously with “blood purifier”), a tonic and laxative, in rheumatism, bilious complaints and as an astringent in hemorrhoids, bleedings, etc. Its roots are largely recommended by herbalists for a range of skin diseases, from a spring eruption, to scurvy and scrofula. The usefulness of this herbal drug has also been declared in icterus and as a tonic to the stomach and gastrointestinal system in general. In Serbian traditional medicine, use of the fruits (“seeds”) of yellow dock also has been described [1]. The fruits have been given with advantage in dysentery, for their astringent action [2]. On the other hand, young leaves of yellow dock are edible and often used as vegetables [3]. Methanol extract of ripe R. crispus fruits was evaluated for its antioxidant potential by assays for ferric-reducing antioxidant power (FRAP), DPPH-free radical scavenging activity (DPPH) and the influence to lipid peroxidation in liposomes (LP). Considerable in vitro activity was observed in all test-systems (FRAP: 9.9 mmol Fe2+/g; DPPH IC50: 3.7 µg/ml; LP IC50: 4.9 µg/ml), comparable to that of BHT (FRAP: 8.0 µg/ml; DPPH IC50: 19.4 µg/ml; LP IC50: 3.5 µg/ml), but lower than the activity of ascorbic acid, rutin and quercetin, used as positive control substances. The in vivo effects were evaluated in several hepatic antioxidant systems (intensity of lipid peroxidation (LPx), activities of glutathione peroxidase (GSH-Px), peroxidase (Px), catalase (CAT) and xanthine oxidase (XOD), as well as reduces glutathione (GSH) content), after the treatment with studied yellow dock extract in different doses, or in combination with carbon tetrachloride (CCl4). Pretreatment with the R. crispus extract inhibited CCl4-induced liver injury by decreasing LPx and increasing GSH content in a dosage dependent manner, bringing the levels of antioxidant enzymes to control values. In summary, the obtained results demonstrated that R. crispus extract might possess a specific antioxidant potential, suggesting that it might be used not only as a hepatoprotective agent, but as an active agent in treatment of other disorders caused by oxidative stress, emphasizing the need of further elucidation of mechanisms of its action. Acknowledgement: Supported by the Grant No. 143012 of the Ministry of Science and Environmental Protection, Republic of Serbia. References: 1. Jančić R et al. (1989). Medicinal plants of SR Serbia, Serbian Academy of Sciences and Arts, Belgrade. 2. Tyler VE (1993). The Honest herbal, Pharmaceutical Product Press, New York, London, Norwood (Australia). 3. Vračarić B et al. (1977). Ishrana u prirodi. Vojnoizdavački zavod, Narodna knjiga, Belgrade.

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U9 Antimutageni potencijal monoterpena u bakterijskom test sistemu

Nikolić B, Berić T, Janković I, Kolarević S, Jovanović P, Jagodić A, Vuković-Gačić B i Knežević-Vukčević J Katedra za mikrobiologiju, Biološki fakultet, Univerzitet u Beogradu, Studentski trg 3/II, 11000 Beograd, Srbija Mutacije su uključene u etiologiju mnogih humanih oboljenja uključujući i kancer. Poznato je da aktivne supstance iz lekovitog i aromatičnog bilja poseduju protektivni efekat u odnosu na mutagene iz životne sredine. U cilju njihove upotrebe u zaštiti zdravlja, inhibitori i modulatori mutagenih agenasa se intenzivno proučavaju. Bioantimutageni potencijali etarskih ulja žalfije (Salvia officinalis L.) i bosiljka (Ocimum basilicum L) i njihovih komponenti, kao i molekularni mehanizmi kojim se to dejstvo ostvaruje, praćeni su u bateriji testova na E. coli K12, a UV-C zračenje je korišćeno kao mutageni agens. Dobijeni rezultati pokazuju uključenost višestrukih mehanizama, ali je primarno pojačanje efekta nukleotidne ekscizione reparacije (NER). U ovom radu ispitivan je bioantimutageni potencijal monoterpena iz etarskih ulja žalfije i bosiljka: 1,8-cineola, kamfora i tujona, prema mutagenezi indukovanoj sa hemijskim agensom 4-nitrokinolin-N-oksidom (4NQO) koji spada u grupu “UV-mimetic”. Inhibitorni potencijal je ispitivan u sojevima E. coli K12: reparativno sposobnom kao i soju defeknom u NER. Sve testirane supstance su redukovale indukovanu mutagenezu samo u reparativno sposobnom soju, dok je bilo kakav inhibitorni efekat izostao u soju defeknom u NER. Ovakav rezultat potvrđuje značajnu ulogu nukleotidne ekscizione reparacije u bioantimutagenom potencijalu testiranih supstanci. Istraživanje uključenosti ostalih bioantimutagenih mehanizama je u toku. Iako postoje ograničenja u ekstrapolaciji rezultata dobijenih na bakterijskim test sistemima na sisare, na osnovu dobijenih rezultata može se steći uvid u molekularne mehanizme antimutageneze testiranih supstanci, kao i njihov potencijalni antimutageni efekat na sisarskim ćelijama.

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U9 Antimutagenic potential of monoterpenes in the bacterial assay system

Nikolić B, Berić T, Janković I, Kolarević S, Jovanović P, Jagodić A, Vuković-Gačić B and Knežević-Vukčević J Laboratory for microbiology, Faculty of Biology, University of Belgrade, Studentski trg 3/II, 11000 Belgrade, Serbia Mutations are implicated in many important human diseases, including cancer. It has been shown that active substances from medicinal and aromatic plants possess protective effects against environmental mutagenic agents. In order to apply them in protection of human health, the inhibitors and modulators of mutagens have been intensively explored. We have tested the bioantimutagenic potential of essential oils of sage (Salvia officinalis L.) and basil (Ocimum basilicum L) and their pure components, as well as elucidated the molecular mechanisms involved. The research was conducted using the battery of tests on E. coli K12 with UV-C irradiation as mutagenic agent. Obtained results indicated that bioantimutagenic potential of all tested substances involves multiple mechanisms, but is primarily based on enhancement of nucleotide excision repair (NER). In this work we elucidated bioantimutagenic potential of monoterpenes from essential oils of sage and basil: 1,8-cineole, camphor and α+β thujone against “UV-mimetic” 4-nitroquinoline-N-oxide (4NQO)-induced mutagenesis. The inhibitory potential was tested in repair proficient and NER deficient strains of E. coli K12. All tested substances significantly reduced induced mutagenesis only in repair proficient strain, while no effect was detected in NER deficient strain. This result confirmed that enhancement of nucleotide excision repair is important component of their bioantimutagenic potential. Further work would quantify the involvement of other bioantimutagenic mechanisms. Although there are limitations in extrapolation bacterial antimutagenicity data on mammalian cells, this study provides the possible range of antimutagenic mechanisms of tested natural products.

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U10 Uticaj ekstrakata lista lovora na biohemijske parametre oksidativnog stresa kod miševa tretiranih sa CCl4 Popović M, Kaurinović B 1Departman za hemiju, Prirodno-matematički fakultet, Univerzitet u Novom Sadu, Trg Dositeja Obradovića 3, 21000 Novi Sad, Srbija

U poslednje dve decenije mnogo pažnje je posvećeno prirodnim antioksidansima zbog uočene veze antioksidanasa i slobodnoradikalskih mehanizama u ljudskom organizmu. Kao rezultat, prirodni antioksidansi u hrani i drugim materijalima postali su najvažnije istraživačko naučno polje. Promene životnih navika u modernom industrijskom društvu, s druge strane, izazvale su rastuću svest da neki sastojci hrane mogu povoljno da utiču na probleme u ishrani. U narodnoj medicini, etarsko ulje lišća lovora koristi se kao karminativ, ekscitoaromatik i u industriji parfema. Biljka lovora sadrži nekoliko klasa sekundarnih biljnih proizvoda. Plod sadrži 30% masti i 1% etarskog ulja. Etarsko ulje uglavnom se sastoji od cineola i α-pinena [1,2]. U ovom radu je ispitivano delovanje ekstrakata lista lovora (etarski, hloroformski, etilacetatni, n-butanolni i vodeni) i u kombinaciji ekstrakata sa ugljentetrahloridom na sledeće biohemijske parametre: aktivnost katalaze (CAT), peroksidaze (Px), glutation peroksidaze (GSHPx), glutation reduktaze (GSHR), ksantin oksidaze (XOD), sadržaj lipidne peroksidacije (LPx) i glutationa (GSH)[3,4]. Navedeni parametri su određivani u homogenatu jetre miševa BALB/C. Sadržaj GSH je statistički značajno smanjio samo n-butanolni ekstrakt lista lovora. Svi ostali ekstrakti lovora povećavaju sadržaj ovog enzima. Aktivnost GSHPx, CAT i XOD su smanjili svi ekstrakti lista lovora. Etarski i vodeni ekstrakt lista lovora, povećavaju aktivnost GSHR, dok je aktivnost GSHR statistički značajno smanjena pri tretiranju sa etilacetatnim i n-butanolnim ekstraktom lista lovora. Intenzitet LPx je statistički značajno smanjen pri tretmanu sa etilacetatnim i vodenim ekstraktom lista lovora, Ostali ekstrakti nisu uticali na intenzitet LPx. Peroksidaznu aktivnost su smanjili etilacetatni i n-butanolni ekstrakt lista lovora. Tretman sa ugljentetrahloridom u odnosu na netretirane eksperimentalne životinje je smanjio intenzitet LPx i aktivnosti CAT i XOD, a povećao aktivnosti GSHPx i Px, dok na sadržaj GSH i aktivnost GSHR nije imao uticaja. Kombinacija ekstrakata sa CCl4 nije bitnije uticala na aktivnost ovih enzima. Na osnovu dobijenih rezultata možemo zaključiti das u ispitivani ekstrakti pokazali određeni protektivni efekat. Razlike koje se javljaju pri dejstvu određenih ekstrakata su verovatno posledica različitog sadržaja flavonoida i drugih antioksidantnih komponenata. 1. Conforti F et al. (2006) Biol Pharm Bull 29(10):2056-2064. 2. Dall′Acqua S et al. (2006) Chem Pharm Bull 54(8):1187-1189. 3. Afanas′ev I et al. (1989) Biochem Pharmacol 38:1763-1769 4. Buege AJ, Aust DS (1988) Methods in enzimology. Academic press. New York.

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U10 Effect od laurel leaves extracts on the biochemical parameters of oxidative stress in rats treated with CCl4 Popović M, Kaurinović B 1Department of chemistry, Faculty of Natural Sciences, University of Novi Sad, Trg Dositeja Obradovića 3, 21000 Novi Sad, Serbia

In the last two decades much emphasis has been given to natural antioxidants, due to the relation of antioxidants to free radical mechanisms in the human body. As a result, naturally occurring nutritive and non-nutritive antioxidants have become a major area of scientific research. Changes of lifestyle in the modern industrialized world, on the other hand, have triggered a growing awareness that particular ingredients of foods may favourably modify diet related problems. In traditional medicine, ether oil from laurel leaves is used as carminative, excito-aromatic and in perfumes industry. The laurel plant contains several classes of secondary plant products. The fruit consists of 30% fat and 1% ether oil. Ether oil mostly contains cineol and α-pinen [1,2]. In this paper the effects of laurel leavesa (etheric, chloroformic, ethyl acetate, n-butanolic and water) on the some biochemical parameters was determined, as well as effects of their combination with CCl4. The investigation biochemical parameters were: activities of peroxidise (Px), catalase (CAT), glutathione peroxidase (GSHPx), xanthine oxidase (XOD), extent of lipid peroxidation (LPx), and the content of reduced glutathione (GSH)[3,4]. This parameters were determined in homogenate of mice liver. Content of GSH was statistically significantly reduced only by n-butanolic extract of laurel leaves. All other extracts from laurel increased the content of this enzyme. Activity of GSHPx , CAT and XOD was reduced by all extract of laurel leaves. Etheric and water extract of laurel leaves increase activity of GSHR, while GSHR activity was statistically significantly reduced by ethylacetate and n-butanolic extracts of laurel leaves. LPx intensity was statistically significantly reduced in treatment with ethylacetate and water extracts of laurel leaves. Other extracts did not affect LPx intensity. Peroxidase activity was reduced by ethylacetate and n-butanolic extract of laurel leaves Treatment with carbon tetrachloride reduced LPx intensity and activities of CAT and XOD in relation to non-treated animals; activity of GSHPx and Px was increased, and they had no effect on GSH and activity of GSHR. Combination of extracts with CCl4 had no significant effects on activity of these enzymes. On the basis of the obtained results it can be concluded that the examined extracts showed a certain protective effect. This in vivo study is of essential importance because it encompassed a number of biochemical parameters related to protection against the oxidative stress. Having in mind that laurel leaves contain flavonoids, carotenes, and polyacetylenes, the protective effect of laurel leaves extracts is due to these groups of compounds, whereas the observed differences are a consequence of different contents of these compounds in particular extracts. 1. Conforti F et al. (2006) Biol Pharm Bull 29(10):2056-2064. 2. Dall′Acqua S et al. (2006) Chem Pharm Bull 54(8):1187-1189. 3. Afanas′ev I et al. (1989) Biochem Pharmacol 38:1763-1769 4. Buege AJ, Aust DS (1988) Methods in enzimology. Academic press. New York.

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U11 Hemijska karakterizacija i antiradikalska aktivnost plodova i džema od borovnice Janković T, Šavikin K, Menković N, Zdunić G Institut za proučavanje lekovitog bilja „Dr Josif Pančić“, Tadeuša Košćuška 1, 11000 Beograd, Srbija

Poznato je da neodgovarajuća ishrana doprinosi smanjenju otpornosti čoveka i dovodi do pojave mnogih oboljenja. Jedan od bitnijih činilaca u ishrani je voće. Značaj voća u ishrani potiče od hranljivih i biološki aktivnih, tzv. sekundarnih materija. Poseban značaj imaju jedinjenja koja smanjuju rizik od malignih oboljenja, oboljenja krvnih sudova, srca i dr. Među najznačajnijim od tih jedinjenja su flavonoidi, antocijani, tanini, i dr. Danas su poznate alternativne terapije (ishrana svežim povrćem i voćem) u lečenju nekih malignih oboljenja. Različite analitičke metode omogućavaju utvrđivanje prisustva i sadržaja aktivnih komponenti u voću i proizvodima od voća. Naš cilj je bio da se jedan proizvod od voća, džem od plodova borovnice, ispita na prisustvo onih supstanci koji imaju pozitivni učinak na ljudsko zdravlje. U uzorcima ploda borovnice, svežim (I) i nakon 9 meseci čuvanja na -180C (II), i u uzorcima džema odmah nakon izrade (I) i nakon 9 meseci čuvanja (II), praćeni su: organoleptička svojstva, sadržaj ukupnih fenola, sadržaj ukupnih antocijana, antiradikalska aktivnost u odnosu na stabilni 2,2-difenil-1-pikrilhidrazil (DPPH) radikal, i mikrobiološka ispravnost. Razrađena je i kvalitativna fingerprintna HPLC metoda za aglikone antocijana u plodu borovnice i džemu od plodova borovnice.

1 - delfinidin 2 - cijanidin 3 - petunidin 4 - peonidin 5 - malvidin

Ukupni antocijani (%)

Ukupni fenoli (mg GAE/g)

Antiradikalska aktivnost (mg/ml)

Uzorak I II I II I II

Plod borovnice 0.61±0.03 0.43±0.04 8.9±0.3 5.2±0.3 0.82±0.06 1.77±0.2 Džem od borovnica 0.09±0.01 0.07±0.02 4.5±0.1 4.0±0.2 1.20±0.07 1.01±0.2 U uzorcima plodova i džemova detektovano je 5 aglikona antocijana: delfinidin i cijanidin (kao dominantni), petunidin, peonidin i malvidin. Sadržaj ukupnih antocijana je u zamrznutim plodovima nakon devet meseci čuvanja opao za 29.5% dok je proces izrade džema, pre svega visoka temperatura, uzrokovao pad u sadržaju ukupnih antocijana za oko 85%. Uslovi čuvanja i procesuiranja su manje uticali na sadržaj ukupnih fenola i antiradikalsku aktivnost. Nakon 9 meseci čuvanja, uzorci džemova su zadržali prvobitna organoleptička svojstva i odgovarali zahtevima Ph. Eur. 6.0 za mikrobiološku ispravnost. Period u toku godine kada je sveži plod borovnice dostupan je relativno kratak. Rezultati našeg istraživanja ukazuju da zamrznuti plodovi borovnice kao i džem od borovnica, sadrže značajnu količinu aktivnih sastojaka, a posebno aktivnost u odnosu na slobodne radikale, te predstavljaju vredan sastojak u ishrani u delu godine kada sveže voće nije dostupno.

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U11 Chemical characterization and radical scavenging activity of bilberry and bilberry jam Janković T, Šavikin K, Menković N, Zdunić G Institute for Medicinal Plants Research „Dr Josif Pančić“, Tadeuša Košćuška 1, 11000 Belgrade, Serbia

Epidemiological studies suggests that diets rich in fruits have been associated with reduced risk of chronic diseases, such as cancer and cardiovascular disease. Benefitial effects have been attributed to the high concentration of phytochemicals, especially polyphenols, compounds with antioxidant potential as well as other health-promoting properties. Significant sources of polyphenolics include highly pigmented berries. Berries are available as fresh and frozen, and also as thermally processed products like jams, jellies, juices. The purpose of the present work was to investigate the effects of domestic processing and storage on organoleptic characteristics, total phenolics and anthocyaninns content, DPPH radical scavenging activity and microbiological quality of bilberry. Part of fresh berries were frozen and stored in a domestic freezer up to 9 months while part were cooked to make jam. Contents of active compounds and radical scavenging activity were studied in fresh and frozen berries and jams (fresh and after 9 months of storage). Fingerprint HPLC method was developed for qualitative analyse of anthocyanin aglycones in bilberry fruits and jam.

1 - delphinidin 2 - cyanidin 3 - petunidin 4 - peonidin 5 - malvidin

Total anthocyanin content (%)

Total phenolics (mg GAE/g)

Radical scavenging activity (IC50 mg/ml)

Sample I II I II I II

Bilberry 0.61±0.03 0.43±0.04 8.9±0.3 5.2±0.3 0.82±0.06 1.77±0.2 Bilberry jam 0.09±0.01 0.07±0.02 4.5±0.1 4.0±0.2 1.20±0.07 1.01±0.2 In all samples five anthocyanins delphinidin, cyanidin, petunidin, peonidin and malvidin, were detected, delphinidin and cyanidin being dominant. The content of total anthocyanins in frozen fruits decreased 29% compared with fresh fruits. Processing of berries to jam affected anthocyanins content (85% loss), but did not significantly influence phenol content and radical scavenging activity. Bilberry jam retained organoleptic characteristics and microbiological quality during storage. The season during which fresh bilberry fruits are available is short. Our results indicate that phenols and antiradical activity are well preserved in frozen bilberry fruits as well as in bilberry jam.

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U12 Antioksidantna i antimikrobna aktivnost ekstrakata i etarskog ulja Eryngium palmatum Vis & Panč., Apiaceae Simić M1, Petrović S1, Perović B1, Milenković M2, Vujisić Lj3, Tešević V3, Niketić M4

1 Institut za farmakognoziju, Farmaceutski fakultet Univerziteta u Beogradu, Vojvode Stepe 450, 11221 Beograd; 2 Institut za mikrobiologiju i imunologiju, Farmaceutski fakultet Univerziteta u Beogradu, V. Stepe 450, 11221 Beograd; 3 Hemijski fakultet, Studentski trg 12-16, 11000 Beograd, Srbija; 4 Prirodnjački muzej, Njegoševa 51, 11000 Beograd, Srbija Vrste roda Eryngium L. (Apiaceae) tradicionalno se koriste kod različitih inflamatornih oboljenja, urinarnih infekcija, kao dijaforetici i za zarašćivanje rana. Eryngium palmatum Vis & Panč., je višegodišnja biljna vrsta rasprostranjena u Srbiji, Bugarskoj, Makedoniji i Albaniji [1]. Rizom (uzorak 1) i listovi (uzorak 2) ove biljke sakupljeni su u Sićevačkoj klisuri aprila 2007. god, osušeni na vazduhu i samleveni. Uzorci su macerovani hloroformom (CHCl3), a zatim metanolom (MeOH) i rastvarači uklonjeni pod sniženim pritiskom. Etarsko ulje izolovano je iz rizoma destilacijom vodenom parom (sadržaj etarskog ulja 0,06 %, m/m). U suvim MeOH ekstraktima listova i rizoma, pomoću Folin-Ciocalteu reagensa [2], određen je sadržaj ukupnih fenola (2,90 i 1,39%) i tanina (0,45 i 0,47%). HPLC analizom u ekstraktu lista detektovani su heterozidi kvercetina i kemferola, a u ekstraktu rizoma galna i rozmarinska kiselina. GC i GC-MS metodama utvrđeno je da su glavne komponente etarskog ulja rizoma falkarinol i oktanal (32,8 i 29,8%). Struktura diacetilena falkarinola potvrđena je i na osnovu IR i 1H NMR spektara. Ukupna redukciona sposobnost MeOH ekstrakata, utvrđena FRAP testom [2], bila je relativno niska (0,15 i 0,14 µmol Fe2+/mg ekstrakta). Neutralizacija DPPH radikala MeOH ekstrakata [3] ostvarena je tek pri višim koncentracijama (SC50= 575,2 i 651,9 µg/ml). Sposobnost neutralizacije OH· radikala MeOH ekstrakata [3] ispitivana je u opsegu koncentracija od 12,5–200 µg/ml. Ekstrakt lista ispoljio je najveću aktivnost pri koncentraciji 25 µg/ml (39,3%), dok je ekstrakt rizoma bio najaktivniji pri najvišoj primenjenoj koncentraciji (33,7%). Anti-DPPH aktivnost etarskog ulja preliminarno je potvrđena TLC-DPPH testom [3]. Antimikrobna aktivnost MeOH i CHCl3 ekstrakata ispitivana je agar difuzionom [4] i bujon mikrodilucionom metodom [5] prema 8 standardnih sojeva bakterija i 2 standardna soja gljivice Candida albicans. Svi ekstrakti ispoljili su antifungalnu aktivnost. Najbolju antibakterijsku aktivnost ispoljili su MeOH ekstrakti rizoma i lista prema standardnim sojevima Staphylococcus epidermidis, Enterococcus faecalis i Escherichia coli. Bujon dilucionom metodom aktivnost ekstrakata ispitivana je u opsegu koncentracija 62,5-500 µg/ml. Analizirani ekstrakti inhibirali su rast mikrooorganizama u najnižoj primenjenoj koncentraciji (62,5 µg/ml), osim CHCl3 ekstrakta rizoma i MeOH ekstrakta lista na Klebsiella pneumoniae i MeOH ekstrakta lista na Pseudomonas aeruginosa (MIC =125 µg/ml). Antimikrobna aktivnost etarskog ulja rizoma, analizirana bujon mikrodilucionom metodom [7] u odnosu na 4 standardna soja bakterija i 2 standardna soja gljivice C. albicans, bila je veoma izražena. Najbolju inhibiciju rasta etarsko ulje je ispoljilo prema S. epidermidis (MIC =7,8 µg/ml), zatim prema S. aureus, Micrococcus luteus, E. faecalis i C. albicans ATCC 10259 (MIC 15,6 µg/ml) i C. albicans ATCC 24433 (MIC =31,2 µg/ml). Literatura: 1. Nikolić V. (1973) Eryngium L., u Josifović M. (ed.). Flora SR Srbije, Vol. V Srpska akademija nauka i umetnosti, Beograd. 2. Rakić S. et al. (2007) Food Chem 104: 830-834. 3. Đorđević S. et al. (2007) J Ethnopharmacol 109: 458-463. 4. Acar JF, Goldstein FW. (1996) Disc susceptibility test. In: Lorian V, editor. Antibiotics in Laboratory Medicine, 4th edition. Williams & Wilkins. Baltimore: 1-51. 5. Candan, F. et al. (2003) J Ethnopharmacol 87:215-220.

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U12 Antioxidant and antimicrobial activity of extracts and essential oil of Eryngium palmatum Vis & Panč., Apiaceae Simić M1, Petrović S1, Perović1 B, Milenković M2, Vujisić Lj3, Tešević V3, Niketić M4

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 University of Belgrade, V. Stepe 450, 11221 Belgrade; 3 Faculty of Chemistry, Studentski trg 12-16, 11000 Belgrade, Serbia; 4 Natural History Museum, Njegoševa 51, 11000 Belgrade, Serbia Eryngium L. species (Apiaceae) are traditionally used against various inflammatory disorders, urinary infections, as a diaphoretic and for wound healing. Eryngium palmatum Vis & Panč is a perennial species widespread in Serbia, Bulgaria, FYR Macedonia and Albania [1]. Rhizome (sample 1) and leaves (sample 2) of this plant were collected in Sićevo gorge in April 2007, air-dried and grinded. The samples were macerated with chloroform (CHCl3), then with methanol (MeOH) and solvents evaporated under reduced pressure. Essential oil was isolated by hydrodistillation from rhizome (essential oil content 0.06 %, w/w). In dried MeOH extracts, with Folin-Ciocalteu reagent [2], the content of total phenols (2.90 and 1.39%) and tannins (0.45 and 0.47%) was determined. By HPLC analysis glycosides of quercetin and kaempferol were detected in leaf extracts, and gallic and rosmarinic acid in rhizome extracts. GC and GC-MS analysis revealed that the main constituents of rhizome essential oil were falcarinol and octanal (32.8 and 29.8%). The structure of diacetylene falcarinol was confirmed on the basis of IR and 1H NMR data. Ferric reducing activity of MeOH extracts, assayed by FRAP test [2], was relatively low (0.15 and 0.14 µmol Fe2+/mg extract). DPPH radical scavenging activity [3] of MeOH extracts was realized only at higher concentrations (SC50= 575.2 and 651.9 µg/ml). OH· radical scavenging ability of MeOH extracts [3] was investigated in the range from 12.5–200 µg/ml. Leaf extract showed the highest activity at a concentration 25 µg/ml (39.3%), while rhizome extract exhibited effect at the highest applied amount (33.7%). Anti-DPPH activity of essential oil was preliminary confirmed by TLC-DPPH test [3]. Antimicrobial activity of MeOH and CHCl3 extracts was investigated by agar diffusion [4] and broth microdilution method [5] against 8 laboratory control strains of bacteria and 2 laboratory control strains of yeast Candida albicans. All extracts showed antifungal activity. The best activity exerted MeOH extracts of rhizome and leaf towards laboratory control strains Staphylococcus epidermidis, Enterococcus faecalis and Escherichia coli. Activity of extracts by broth microdilution method was investigated in the range of concentrations 62.5-500 µg/ml. Examined extracts inhibited the growth of microorganisms at the lowest applied concentration (62,5 µg/ml), except CHCl3 extract of rhizome and MeOH extract of leaf on Klebsiella pneumoniae, and MeOH extract of leaf on Pseudomonas aeruginosa (MIC =125 µg/ml). Antimicrobial activity of rhizome essential oil, analyzed by broth microdilution method [7] against 4 laboratory control strains of bacteria and 2 laboratory control strains of yeast C. albicans, was very pronounced. Essential oil exhibited the best growth inhibition on S. epidermidis (MIC =7.8 µg/ml), then on S. aureus, Micrococcus luteus, E. faecalis, C. albicans ATCC 10259 (MIC =15.6 µg/ml) and C. albicans ATCC 24433 (MIC =31.2 µg/ml). References: 1. Nikolić V. (1973) Eryngium L., u Josifović M. (ed.). Flora SR Srbije, Vol. V Srpska akademija nauka i umetnosti, Beograd. 2. Rakić S. et al. (2007) Food Chem 104: 830-834. 3. Đorđević S. et al. (2007) J Ethnopharmacol 109: 458-463. 4. Acar JF, Goldstein FW. (1996) Disc susceptibility test. In: Lorian V, editor. Antibiotics in Laboratory Medicine, 4th edition. Williams & Wilkins. Baltimore: 1-51. 5. Candan, F. et al. (2003) J Ethnopharmacol 87:215-220.

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U13 Uticaj etarskih ulja na ponašanje i dobro opšte stanje pasa Vučinić Marijana Fakultet veterinarske medicine, Bulevar oslobodjenja 18, 11000 Beograd, Srbija U radu su izneti literaturni podaci koji se odnose na aromatologiju kao meru u sprečavanju nastanka poremećaja ponašanja pasa. Aromaterapija i aromatologija (koja podrazumeva isto što i aromaterapija ali bez masaže) su grane fitoterapije, koje se zasnivaju samo na upotrebi etarskih ulja dobijenih destilacijom i ulja limuna dobijenih hladnim ceđenjem (1). Prema Ceccarelli et al. (2) aromaterapija je naziv za terapeutski pristup čiji su glavni činioci mirisi i olfaktorni sistem. Danas se smatra da olfaktorna stimulacija etarskim uljima predstavlja koristan način obogaćivanja smeštajnih uslova za mnoge životinje koje se drže u zatorenim prostorijama kao što su životinje u zoološkim vrtovima i kućni ljubimci, a radi sprečavanja poremećaja ponašanja prouzrokovanih stresorima u životnom okruženju. Kod životinja u zatvorenim uslovima smeštaja etarska ulja deluju ili umirujuće ili stimulativno i poseduju potencijal smanjivanja stresogenih uticaja. Psi mogu da se drže u različitim uslovima smeštaja kao što su odgajivačnice, prihvatilišta i laboratorije (3). U prvim istraživanjima, publikovanim 2005. godine, ispitivan je učinak četiri etarska ulja (ulje lavande, kamilice, ruzmarina i pitome nane) na ponašanje 55 pasa u prihvatilištu. Psi izloženi uticaju mirisa ulja lavande i kamilice mnogo manje vremena su proveli u lajanju i u kretanju. Ulja pitome nane i ruzmarina su ispoljila suprotan uticaj. Difuzija etarskih ulja sa umirujućim uticajem u smeštajne prostorije prihvatilišta smanjuje stres kod pasa usled buke prouzrokovane lavežom i dovodi do njihove sedacije (4). Sve ovo može da poveća procenat udomljenih pasa jer ispoljavaju oblike ponašanja prihvatljive za posetioce prihvatilišta. Pored toga i posetioci se osećaju prijatnije u zatvorenim prostorijama u kojima udišu mirise umirujućeg dejstva. Drugo istraživanje, čiji su rezultati publikovani 2006. godine, odnosi se na umirujući uticaj ulja lavande na pse koji se uznemire u toku transporta. Psi koji su tokom transporta inhalirali miris lavandinog ulja više vremena su proveli u ležećem ili sedećem položaju, a manje su se kretali i lajali. Aromaterapija u vidu difundovanih mirisa lavande znatno smanjuje uznemirenost pasa u toku transporta. Ovo pozitivno utiče i na koncentraciju i smirenost vozača (5). 1. Steflitsch W. and Steflitsch M. (2008) JHM 5: 74–85. 2. Ceccarelli I. et al. (2003). Brain Res. 1001: 78-86. 3. Wells D.L. (2003) Appl Anim Behav Sci. 85: 307–317. 4. Graham L. et al. (2005) Appl Anim Behav Sci. 91:143-153. 5. Wells D.L. (2006). JAVMA 229: 964-967.

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U13 Effects of essential oils on behavior and welfare of dogs Vučinić Marijana Faculty of Veterinary Medicine, Bulevar oslobodjenja 18, 11000 Belgrade, Serbia The paper deals with literature data on aromathology as a preventive measure of behavioral disorders in dogs. Aromatherapy and aromatology (which is similar to aromatherapy but without massage) are branches of phytotherapy, utilizing only the essential oils produced by distillation and the citrus oils produced by cold pressing (1). According to Ceccarelli et al. (2) aromatherapy is the name given to therapeutic approaches having odors and the olfactory system as main actors. Olfactory stimulation with essential oils is now considered a useful form of environmental enrichment for many captive animals, such as zoo animals and pets in order to prevent behavioral disorders caused by environmental stressors. Essential oils have calming or stimulating effects and the potential to reduce stress in confined animals (1). Domestic dogs can be housed in a variety of confined conditions, including kennels, shelters and laboratories (3). The first study, published in 2005, evaluated the effects of four essential oils (lavender, chamomile, rosemary, and peppermint) on the behavior of fifty-five dogs housed in a rescue shelter. Dogs exposed to lavender or chamomile oils spent less time barking and moving. Peppermint or rosemary oils created the opposite effect. Diffusing essential oils with calming effects into the atmosphere of sheltered dogs may help the inhabitants by reducing noise stress and providing overall sedation (4). Adoption rates may rise, because the animals are exhibiting behaviors that the public views more favorably. Visiting a shelter wherein the humans, too, inhale calming aromas may make for a more pleasant experience for all. The second study, published in 2006, reported that ambient odor of lavender may be beneficial in calming dogs susceptible to travel-induced excitement. Dogs inhaling the ambient lavender spent more time resting and sitting and less time moving and vocalizing. Aromatherapy in the form of diffused lavender odor may make traveling dogs less excited. It may affect the driver’s concentration and alertness (5). 1. Steflitsch W. and Steflitsch M. (2008) JHM 5: 74–85. 2. Ceccarelli I. et al. (2003). Brain Res. 1001: 78-86. 3. Wells D.L. (2003) Appl Anim Behav Sci. 85: 307–317. 4. Graham L. et al. (2005) Appl Anim Behav Sci. 91:143-153. 5. Wells D.L. (2006). JAVMA 229: 964-967.

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U14 Terapijski potencijal karvakrola i timola u lečenju salmoneloznih infekcija kod brojlera Ratajac Radomir1, Jezdimirović Milanka2, Lako Branislav3, Orlić Dušan1, Stojanović Dragica1, Stojanov Igor1, Potkonjak Aleksandar3

1Naučni institut za veterinarstvo Novi Sad, Rumenački put 20, 21000 Novi Sad, Srbija; 2Fakultet veterinarske medicine, Bulevar VJ 18, 11000, Beograd, Srbija; 3Poljoprivredni fakultet, Departman za veterinu, Trg Dositeja Obradovića 8, 21 000, Novi Sad, Srbija

Kontrola (preventiva i terapija) infektivnih bolesti životinja, naročito zoonoza, je neophodna jer uprkos savremenom unapređenju higijene klanja i tehnike proizvodnje, bezbednost hrane je sve važnije pitanje kada se radi o zdravlju stanovništva (1). Upotreba antibiotika u borbi sa patogenim mikroorganizmima nosi sa sobom i opasnosti, kao što su razvoj rezistentnih sojeva bakterija na pojedine antibiotike, kao i postojanje rezidua antibiotika u namirnicama životinjskog porekla. Kao jedna od mogućih alternativa sintetskim lekovima, jeste upotreba eteričnih ulja (EU), koja u manjem ili većem stepenu imaju jedno ili više farmakoloških dejstava od kojih je za veterinarsku medicinu posebno značajno antibakterijsko dejstvo. Cilj ovog istraživanja je bio da se ispita antimikrobna aktivnost karvakrola i timola, kako pojedinačno tako i u kombinaciji protiv Salmonella Enteritidis u uslovima in vitro, kao i mogućnost njihovog korišćenja u terapijske svrhe kod brojlera, veštački inficiranih sa Salmonella Enteritidis. Antimikrobna osetljivost izolata Salmonella Enteritidis(SE) poreklom od živine ispitana je Agar dilucionim testom, određivanjem minimalne inhibitorne koncentracije (MIK). Ispitivanje terapijskog efekta karvakrola, timola i njihove kombinacije je izvršeno na brojlerima, oba pola linijskog hibrida provenijencije Hybro G. U starosti od 10 dana pilići su veštački inficirani per os sa 0,1 ml suspenzije SE (inokulum je sadržao 1-2 108 CFU/ml). Ogled je izveden na ukupno 90 jedinki podeljenih u 5 grupa: jedinkama u grupi Ia je aplikovan karvakrol, u grupi Ib je aplikovan timol, u grupi Ic je aplikovana kombinacija karvakrola i timola (u odnosu 1:1), u grupi IIa je aplikovan amoksicilin i grupa IIb nije bila lečena. Ukupna doza sastojaka EU i njihove kombinacije je bila 60 mg/kg telesne mase, a amoksicilin je aplikovan u dozi od 16 mg/kg telesne mase. Antibakterijska sredstva su davana kontinuirano tokom 7 dana u vodi za piće, pet dana posle veštačke infekcije. Terapijski efekat je praćen izolacijom SE iz jetre i cekuma, kod žrtvovanih jedinki 3, 7 i 14 dana od početka terapije, odnosno u starosti od 17, 21 i 28 dana.U spomenutim intervalima je iz svake grupe žrtvovano po 6 jedinki. Karvakrol i timol su pokazali dobru antibakterijsku aktivnost, a njihov MIK je bio 0,156 mgml-1, odnosno µlml-1. MIK smeše timola i karvakrola, koja je dovela do inhibicije porasta S. Enteritidis, iznosilo je 0,312 µlml-1 podloge. Do sličnih rezultata su došli i drugi istraživači koji su određivali MIK eteričnih ulja prema Salmonella spp. (S. Typhimurium); karvakrol i timol su se pokazli kao najaktivniji, jer su imali najniže vrednosti MIK-a ( za karvakrol 1,0mmol l-1= 0,137mgml-1, za timol 1,0 mmol l-1, odnosno 0,1502 mgml-1) (4), takođe i druga istraživanja ukazuju da su vrednosti MIK-a za karvakrol iznosile 0,225-0,25 µlml-1 podloge, a za timol 0,056 µlml-1 podloge (2, 3). U ogledu in vivo karvakrol i timol su doveli do izlečenja 72,22% jedinki, dok je kombincija karvakrola i timola pokazala nešto slabiji terapijski efekat, u 44,44% slučajeva. Značajna razlika u broju jedinki, kod kojih nije izolovana salmonela koristeći mikrobiološke metode, zapažena je između oglednih grupa koje su tretirane EU i kontrolne nelečene grupe, što može da se pripiše antibakterijskoj efikasnosti eteričnih ulja. Utvrđena antibakterijska aktivnost i terapijski efekat ispitivane kombinacije aktivnih sastojaka eteričnih ulja (timola i karvakrola) ne ukazuje na postojanje sinergizma između ovih supstanci. Imajući na umu ukupni broj izlečenih životinja, u odnosu na broj žrtvovanih jedinki u eksperimentalnim grupama, može se zaključiti da procenat izlečenih životinja tretiranih karvakrolom i timolom je bio 72,22%, što je za 5% manje u odnosu na kontrolnu grupu lečenu sa amoksicilinom (77,78%). Literatura: 1. Burt Sara(2004): Essential oils: their antibacterial properties and potential applications in foods – a review, 2004.,International Journal of Food Microbiology 94, p. 223-253. 2. Cosentino, S. et al. (1999) In vitro antimicrobial activity and chemical composition of Sardinian Thymus essential oils., Letters in Applied Microbiology 29, 130– 135. 3. Kim, J. et al. (1995) Antibacterial activity of some essential oil components against five foodborne pathogens. Journal of Agricultural and Food Chemistry 43, 2839-2845. 4. Olasupo N.A. et al. (2003) Activity of natural antimicrobial compounds against Escherichia coli and Salmonella enterica serovar Typhimurium, Letters in Applied Microbiology, 36, p. 448-451.

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U14 The therapeutic potentials of carvacrol and thymol in treating broiler infection caused by Salmonella enteridis Ratajac Radomir1, Jezdimirović Milanka2, Lako Branislav3, Orlić Dušan1, Stojanović Dragica1, Stojanov Igor1, Potkonjak Aleksandar3

1Naučni institut za veterinarstvo Novi Sad, Rumenački put 20, 21000 Novi Sad, Srbija; 2Fakultet veterinarske medicine, Bulevar VJ 18, 11000, Beograd, Srbija; 3Poljoprivredni fakultet, Departman za veterinu, Trg Dositeja Obradovića 8, 21 000, Novi Sad, Srbija

The control (prevention and therapy) of infective animal diseases, especially zoonosis, is necessary because, in spite of the modern improvements in slaughter hygiene and food production techniques, food safety has been an increasingly important public health issue (1). In the struggle against pathogen microorganisms using antibiotics has its disadvantages, like developing resistance of bacteria strains to certain antibiotics, presence of antibiotic residua in the food of animal origin etc. The use of essential oils (EOs) is a possible alternative to synthetic drugs. EOs have one or more pharmacological effects, but for veterinary medicine the antibacterial effect is of great importance. The aim of this study was to investigate the antimicrobial activity of essential oil constituents – carvacrol and thymol, and their combination against filed strain Salmonella Enteritidis in the laboratory (determination of MIC) and in vivo studies on broilers. Antimicrobial sensitivity of Salmonella Enteritidis (SE) isolate (field strain originating from poultry) was tested in vitro conditions determining MIC (minimal inhibitory concentration) and was done by Agar Dilution Test. For examining therapeutic effect in vivo condition the chickens of both sex were used, linear hybrid proficiency Hybro G. At the age of 10 days the chicken were artificially infected per os with 0.1 ml suspension of SE (inoculum contained 1-2 108 CFU/ml). The trial was carried out on total number of 90 broilers divided in five experimental groups (Ia – carvacrol; Ib – thymol; Ic – combination of carvacrol and thymol (1:1) administered in doses of 60 mg/kg of body weight and IIa – amoxicillin administered in doses of 16 mg/kg of body weight) and one control group (IIb) without therapy, each consisting of 18 animals. Therapy was administered in drinking water continually over seven days, five days after the infection of broilers. The therapeutic effect in treating SE infection was examined by isolating the causative agent from the liver and caecum of sacrificed broilers on the days 3, 7 and 14 after the beginning of the therapy, i.e. age of the birds 17, 21 and 28 day of live. In the mentioned intervals, six birds were sacrificed from every experimental group. Minimal inhibitory concentration of carvacrol and thymol that prevented growth of Salmonella was 0.156 µl (mg) ml-1 media. The combination of active substance showed lower activity and their MIC was 0.312 µlml-1 media. The sensitivity of SE in in vitro conditions on carvacrol and thymol was expected, because the previous research pointed that carvacrol’s MIC on Salmonella spp (S. Typhimurium) was 1.0 mmol l-1= 0.137mgml-1, for thymol 1.0 mmol l-1 = 0.1502 mgml-1 (4). Also some other trials showed that MIC for carvacrol was 0.225 – 0.25 µlml-1 media and for thymol MIC was 0.056 µlml-1 media (2, 3). In vivo trials, carvacrol and thymol reduced the number of infected birds in 72.22% of the cases, and the efficiency of combinations of carvacrol and thymol were 44.44%. Significant differences were observed between broilers in group Ia, Ib, Ic and control group IIb. This means that the number of animals in the experimental groups (treated with EOs), where Salmonella was not isolated using microbiological methods, was significantly higher than the number of animals in the control group IIb, what can be attributed to antibacterial effect of EOs. Antibacterial activity and therapeutic effect of the examined combination of active EOs components (thymol and carvacrol) do not show existence of synergism between these substances. Having in mind the total number of cured animals, comparing to the number of sacrified animals in the experimental group, it can be concluded that the percentage of cured animals treated with carvacrol and thymol was 72.22%; 5% lesser comparing to the control group treated with amoxicilline (77.78%). References: 1. Burt Sara(2004): Essential oils: their antibacterial properties and potential applications in foods – a review, 2004.,International Journal of Food Microbiology 94, p. 223-253. 2. Cosentino, S. et al. (1999) In vitro antimicrobial activity and chemical composition of Sardinian Thymus essential oils., Letters in Applied Microbiology 29, 130– 135. 3. Kim, J. et al. (1995) Antibacterial activity of some essential oil components against five foodborne pathogens. Journal of Agricultural and Food Chemistry 43, 2839-2845. 4. Olasupo N.A. et al. (2003) Activity of natural antimicrobial compounds against Escherichia coli and Salmonella enterica serovar Typhimurium,Letters in Applied Microbiology, 36, p. 448-451.

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U15 Antimikrobna aktivnost etarskih ulja i potencijalna primena u proizvodnji sireva Dragoslava Radin1, Branislava Arsenović2 1 Poljoprivredni fakultet, Nemanjina 6, 11080 Beograd, Srbija; 2 Instiut za proučavanje lekovitog bilja «Dr Josif Pančić», Tadeuša Košćuška 1, 11000 Beograd,Srbija

Etarska ulja mnogih lekovitih i aromatičnih biljaka imaju antimikrobnu aktivnost prema velikom broju bakterija, virusa, kvasaca i plesni, i predstavljaju potencijalno bogat izvor novih biocida i konzervanasa. Među mnogim testiranim, etarska ulja timijana (Thymus vulgaris L.), origana (Origanum vulgare L.), nane (Mentha piperita L.), cimeta (Cinnamomum ceylanicum Blume), žalfije (Salvia officinalis L.), karanfilića (Eugenia caryophyllata Thunb.), ruzmarina (Rosmarinus officinalis L.), i lovora (Laurus nobilis L.) poseduju najjača antimikrobna svojstva (1). Najvažnije aktivne komponente etarskih ulja su fenoli, terpeni i aldehidi i osnovni mehanizam delovanja je usmeren ka fosfolipidnoj dvoslojnoj citoplazmatičnoj membrani mikroorganizama. Po svojoj prirodi, fenolna jedinjenja su hidrofobna što im omogućava prodiranje u lipide membrane čime narušavaju njenu strukturu, membrana postaje više permeabilna, dolazi do poremećaja hemiosmoze, toka elektrona, aktivnog transporta, koagulacije ćelijskog sadržaja, inaktivacije metabolitičkih enzima i oštećenja DNK. Generalno, antimikrobna aktivnost je više izražena prema Gram-pozitivnim nego Gram-negativnim bakterijama, zbog, kako se smatra, lipopolisaharida ćelijskog zida Gram-negativnih bakterija, koji sprečavaju penetraciju antimikrobnih komponenti i njihovo pripajanje za DNK, što vodi ka smrti ćelije. Ovo je tačno za mnoga ispitivana etarska ulja, ali su na primer origano, karanfilić i cimet efikasni protiv obe grupe bakterija. Etarska ulja koja najjače deluju na patogene koji se prenose hranom, kao što su Listeria monocytogenes, Escherichia coli O157:H7, Bacillus cereus, Staphylococcus aureus, Clostridium botulinum, Salmonella enteritidis, sadrže visoki procenat fenolnih jedinjenja kao što su karvakrol, eugenol i timol. Karvakrol i timol imaju sposobnost da dezintegrišu spoljašnju membranu Gram-negativnih bakterija, pri čemu dolazi do oslobađanja lipopolisaharida i povećanja permeabilnost citoplazmatične membrane prema ATP-u. Proizvodnja sireva sa dodatkom etarskih ulja aromatičnih i lekovitih biljaka ima veliki marketinški potencijal. U novije vreme opaža se trend proizvodnje nisko slanih sireva, što je u skladu sa apelom Svetske zdravstvene organizacije koji se odnosi na smanjenje konzumiranja soli u cilju redukcije incidence kardiovaskularnih oboljenja. Da bi se očuvala zdravstvena bezbednost proizvoda ukazuje se potreba da se smanjeni nivo soli nadomesti drugim bioaktivnim agensima. Etarska ulja, zahvaljujući svojim antimikrobnim aktivnostima, mogu imati veoma značajnu ulogu u sprečavanju rasta patogenih bakterija (na pr. Listeria monocytogenes, Salmonella enteritidis) u sirevima (2). Ne treba izgubiti iz vida činjenicu da hrana, kao vrlo kompleksni sistem, za postizanje antimikrobnih efekata zahteva primenu veće koncentracije etarskih ulja ili njihovih komponenti nego što je potrebno primeniti u in vitro uslovima. Ustanovljeno je da je za postizanje efekta u mekom siru potrebna 25-100 puta veća koncentracija (3). Iako je većina etarskih ulja klasifikovana kao Generally Recognized As Safe (GRAS), njihova je upotreba kao konzervanasa često limitirana usled uticaja koji mogu imati na ukus i miris proizvoda, jer antimikrobne doze mogu da pređu organoleptički prihvatljiv nivo. Jedna od opcija kojom se može prevazići ovaj problem je vrlo pažljiv odabir većeg broja etarskih ulja sa sinergističkim dejstvom, u koncentracijama koje neće stvarati neželjene promene. Kao poseban aspekt primene etarskih ulja treba istaći veliki značaj koji imaju u promociji zdravlja ljudi svojim antikancerogenim, antioksidativnim i antiinflamatornim efektima. Literatura: 1. Bakkali F. et al. (2008) Food and Chemical Toxicology 46:446-475. 2. Smith-Palmer A. et al. (2001) Food Microbiology, 18: 463-470. 3. Mendoza-Yepes M.J. et al. (1997) Journal of Food Safety 17:47-55.

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U15 Antimicrobial properties of essential oils and potential application in cheese production Dragoslava Radin1, Branislava Arsenović2

1 Faculty of Agriculture, Nemanjina 6, 11080 Belgrade, Serbia; 2 Institute for Medicinal Plants Research “Dr Josif Pančić”, Tadeuša Košćuška 1, 11000 Belgrade, Serbia The antimicrobial properties of aromatic and medicinal plants’ essential oils (EO) are well established against wide spectra of microbes, including bacteria, viruses, yeasts and fungi. EOs represents potentially good resources of new biocides and food preservatives. Among many tested, essential oils of thyme (Thymus vulgaris L.), oregano (Origanum vulgare L.), mint (Mentha piperita L.), cinnamon (Cinnamomum ceylanicum Blume), salvia (Salvia officinalis L.), clove (Eugenia caryophyllata Thunb.), rosemary (Rosmarinus officinalis L.), and bay (Laurus nobilis L.) were found to possess the strongest antimicrobial properties (1). The major active EOs components are phenols, terpens, and aldehydes, and the action of these substances is principally performed against the cell cytoplasmic membrane of microorganisms. An important characteristic of EOs and their components is their hydrophobicity, which enables them to partition in the lipids of bacterial cell membrane, disturbing the structures and rendering them more permeable, disrupting the proton motive force, electron flow, active transport, coagulation of cell contents, inactivation of metabolic enzymes, and damage to DNA. They are generally more inhibitory against Gram-positive than against Gram-negative bacteria. Cell wall lipopolysaccharide of Gram-negative bacteria inhibits the penetration of antimicrobial agents, and their attachments to DNA that causes the death of cells. While this is true of many essential oils, there are some which are effective against both bacteria groups (oregano, clove, cinnamon). Generally, the EOs possessing the strongest antibacterial properties against food borne pathogens such as Listeria monocytogenes, Escherichia coli O157:H7, Bacillus cereus, Staphylococcus aureus, Clostridium botulinum, Salmonella enteritidis, contain a high percentage of phenol compounds such as carvacrol, eugenol and thymol. Carvacol and thymol are able to disintegrate the outer membrane of Gram-negative bacteria, releasing lipopolysaccharides and increasing the permeability of the cytoplasmic membrane to ATP. Cheese production with application of aromatic and medicinal plants’ essential oils has very good marketing potential. Nowadays, trend of low salt cheese production could be recognized, that is in accordance with recent call of the World Health Organization for a world-wide reduction in the consumption of salt in order to reduce the incidence of cardio-vascular disease. If the level of salt in foods is reduced, it is possible that other bioactive compounds will be needed to maintain the safety of foods. One such possibility is the use of EOs as antibacterial additives and natural food preservatives. EOs could be used as antimicrobial agents in dairy product, since they prevented growth and reduce viability of L. monocytogenes and S. enteritids in cheese (2). Food systems due to their complex structures require higher amounts of essential oils or their components than for in vitro activity. It has been recorded that the ratio for soft cheese should be 25- to 100-fold (3). Although the majority of EOs are classified as Generally Recognized As Safe (GRAS), their application as food preservatives could be limited due to the fact that the concentrations required for efficacy in cheese could raise concerns regarding changes in the organoleptic properties. The possible solution could be exploitation of synergistic effects of EOs and minimizing the concentrations required. As a special advantage of EOs application in food systems, their important role in promoting human health by anticancer, antioxidative and anti-inflammatory properties, should be acknowledged.

References: 1. Bakkali F. et al. (2008) Food and Chemical Toxicology 46:446-475. 2. Smith-Palmer A. et al. (2001) Food Microbiology, 18: 463-470. 3. Mendoza-Yepes M.J. et al. (1997) Journal of Food Safety 17:47-55.

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PPOOSSTTEERR PPRREEZZEENNTTAACCIIJJEE PPOOSSTTEERR PPRREESSEENNTTAATTIIOONNSS

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P1 Submerzne livade u okolini Sombora Evica Markoš1, Stevan Markoš2 1 Apoteka "Salvia", Venac R. Putnika 32, 25000, Sombor, Srbija 2 ZC "Dr R.Simonović" KV Dispanzer, Mirna 3, 25000, Sombor, Srbija U sastavu submerznih livada u "Specijalnom rezervatu prirode Gornje Podunavlje", našli smo Ceratophyllum submersum L. (u celosti potopljen u vodi) i Myriophyllum spicatum L. Potamogeton crispus L. i Vallisneria spiralis L. (koje su potopljene, ali su im cvetne stabljike iznad vode). Želeli smo da, koliko nam je to moguće, utvrdimo hemijski sastav ovih biljaka. Poznato je da submerzne biljke, kao i submerzni delovi drugih akvatičnih biljaka imaju veći sadržaj mikroelemenata od flotantnih delova tih biljaka (1). Cilj istraživanja je da se utvrdi mogućnost izolovanja skroba i proteina iz Myriophyllum spicatum (2). Ispitivane su i antioksidantne osobine ove biljke (2). Na ispitivanim submerznim livadama najveću biljnu masu imaju vrste M. spicatum i Potamogeton crispus. Tokom maja 2007. god. sakupili smo cele ove biljke, osušili ih u hladu i ispitali hemijski sastav po propisima Ph.Jug.Ed.IV i V. Dobijeni rezultati su prikazani na tabeli:

H e m i j s k e r e a k c i j e Droga Ph.Jug.IV Ph.Jug.V

Alkaloidi belančevine triptofan.

mono- saharidi antrahinoni skrob tanini

katehol ksantini

M.spicati herba – + + – + – –

P.crispi herba – + + – + + +

Ugljeni hidrati i proteini nemaju veliku primenu u terapiji, ali se koriste u farmaceutskoj i prehrambenoj industriji (3). U letnjim mesecima, često već u maju, P. crispus, a naročito M. spicatum, se javljaju u velikim količinama. Izvađene iz vode ove dve biljke se brzo suše zadržavajući izgled i boju. Sve to govori da bi se mogle koristiti kao izvor proteina i skroba. Treba međutim misliti i na mogućnost narušavanja ekološke ravnoteže u tako osetIjivim životnim staništima, kao što su vodeni biotopi. Literatura 1. Stojanović S.et alter.(I994), Biljni svet kanala Vrbas-Bezdan, Univerzitet u N.Sadu, 51 2. Štajner D.et alter.(1997), Arh.farm.5, 612, 613 3. Kovačević N.(2000), Osnovi farmakognozije, Beograd, 31O, 332

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P1 Submerged meadows around Sombor Evica Markoš1 , Stevan Markoš2

1 Pharmacy „Salvia”, Venac R. Putnika 32, 25000, Sombor, Srbija; 2 HC „Dr. R. Simonović” CV Dispansery, Mirna 3, 25000, Sombor,Srbija In the „submerged meadows” of „Special nature reservation Gornje Podunavlje” we found Ceratophyllum submersum L. (this plant is wholly submerged), Myriophyllum spicatum L., Potamogeton crispus L. and Vallisneria spiralis L. (this plants are also submerged, but their flowers are emerging from water on stout stalk). We wanted to know something about the chemical ingradients of this plants. It is known that submerged plants and submerged parts of other aquatic plants have higher level of mikroelements than the flotant parts.(1) There are papers representing the posibility for separate starch and proteins from M. spicatum.(2) The antioxidant characteristic of this plant was also investigated.(2) The greatest biomass at the investigated,submerged meadows have M. spicatum and P. crispus. ln May 2007. we’ve collected and dry them in shade. Chemical analyses according to Ph.Jug.Ed.IV and V procedurs was done. Positive reactions of M. spicatum herba were on: proteins (triptofanic), monosaharids, starch. Herba P. crispus has positiv reactions on: proteins (triptofanic), monosaharids starch, catehinic, tannins, xanthins. Carbonichydrats and proteins are not of big importance for therapy, their use in the pharmaceutical and nutritive industry is meaningful.(3) In summer, often in May, P. crispus and specially M. spicatum are growing in large quantitis. Taking this plants out of water they dry quickly and retain form and colour. Consequently, this plants may be raw material for proteins and starch. But it is necessary to consider the possibility of probable disturbances of the sensitive water habitat.

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P2 Mikoze značajnih lekovitih biljaka u Srbiji Pavlović Snežana1, Stojšin Vera2, Stojanović Saša3, Starović Mira3

1 Institut za proučavanje lekovitog bilja »Dr J. Pančić«, T. Košćuška, 11000 Beograd, Srbija 2 Poljoprivredni fakultet, Departman za zaštitu bilja i životne sredine, Trg Dositeja Obradovića 8, 21000 Novi Sad, Srbija 3 Institut za zaštitu bilja i životnu sredinu, Teodora Drajzera 9, 11000 Beograd, Srbija

Tokom višegodišnjeg istraživanja praćena je patogena mikopopulacija najvažnijih vrsta lekovitog i aromatičnog bilja koje se plantažno gaji na imanju Instituta za proučavanje lekovitog bilja u Pančevu i kod kooperanata. Ispitivanjem su obuhvaćene sledeće vrste: Altheae officinalis, Echinacea purpurea i E. angustifolia, Hypericum perforatum i Salvia officinalis. Sa biljaka koje su ispoljavale različite simptome oboljenja dobijeno je preko 200 izolata gljiva. Mikroskopskim pregledom ustanovljeno je da je najveći broj izolata pripada vrstama gljiva iz roda Fusarium i Sclerotinia, po nekoliko izolata pripadalo je gljivama iz rodova Aspergillus, Penicillium, Botrytis, Cladosporium, Chetomium, Doratomices, Erysphae, Myrothecium, Phoma, Puccinia, Epicoccum i dr. Ukupno je determinisano 31 vrsta gljiva iz 20 rodova i to: 15 vrsta iz 11 rodova na Athaea officinalis, 11 vrsta iz 9 rodova na Echinacea angustifolia, 14 vrsta iz 10 rodova na Echinacea purpurea, 13 vrsta iz 8 rodova na Hypericum perforatum i 16 vrsta iz 6 rodova na Salvia officinalis. Po mestu na kome su gljive izolovane, dominiraju gljive koje naseljavaju seme ispitivanih lekovitih biljaka, a u manjoj meri koren i nadzemne organe. Većina izolovanih vrsta gljiva (35%) na ispitivanim lekovitim biljkama pripadala je rodu Fusarium. Od 11 determinisanih vrsta roda Fusarium, F. oxysporum, F. proliferatum, F. semitectum i F. verticillioides su izolvani sa četiri domaćina, F. equiseti sa dva, a ostale vrste F. arthrosporoides, F. avenaceum, F. graminearum, F. solani, F. sporotrichioides i F. subglutinans sa po jednog domaćina. Na Altheae officinalis identifikovano je šest vrsta roda Fusarium (F. verticillioides, F. proliferatum, F. oxysporum, F. semitectum i F. solan), na E. angustifolia tri (F. oxysporum, F. proliferatum i F. semitectum), na E. purpurea pet (F. equiseti, F. oxysporum, F. proliferatum, F. semitectum i F. verticillioides), na Hypericum perforatum 6 (F. oxysporum, F. subglutinans, F. gramineaeum. F. sporotrichioides, F. verticillioides i F. proliferatum) i na žalfiji pet vrsta (F. semitectum, F. avenaceum, F. verticillioides, F. equiseti i F. arthrosporoides). Testovi za proveru patogenosti pokazali su da su svi izolati Fusarium vrsta patogeni prema svojim domaćnima, u kontrolisanim uslovima. Izolati F. oxysporum i F. verticillioides sa belog sleza ehinacea i kantariona začajno su umanjili energiju klijanja i klijavost vrsta semena sa kojih su i izolovani. Klijanci belog sleza reagovali su jakom nekrozom inokulisanih korenčića svim vrstama roda Fusarium, a značajno različit stepen nekroze nađen je kod korenčića žalfije. Iz nekrotičnih delova belog sleza i ehinacea izolovana je gljiva Sclerotinia sclerotiorum. Svi izolati S. sclerotiorum ispoljili su značajnu patogenost prema sadnicama belog sleza, žalfije i ehinacea. Po prvi put su registrovane na lekovitim biljkama prouzrokovači pepelnice (Erysiphe cichoracearum) na kantarionu, Oidium spp. na žalfiji i E. purpurea, kao i Phoma sp. na belom slezu i ehinaceama. Sclerotinia sclerotiorum nađena je na E. purpurea i E. angustifolia. Vrste iz roda Fusarium su takođe po prvi put determinisene do vrste i ispitana je njihova patogenost.

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P2 Mycoses of more important medicinal plants in Serbia Pavlović Snežana1, Stojšin Vera2, Stojanović Saša3, Starović Mira3

1 Institute for Medicinal Plant Research “Dr Josif Pančić”, T. Košćuška, 11000 Belgrade, Serbia 2 Faculty of Agriculture, Trg Dositeja Obradovića 8, 21000 Novi Sad, Serbia 3 Institute for Plant Protection and Environment, Teodora Drajzera 9, Belgrade, Serbia During the several year lasting investigation of pathogenic mycopopulation of the most important medicinal and aromatic plant species cultivated at the experimental field of the Institute for Medicinal Plant Research “Dr Josif« Pančić” in Pančevo and in cooperative. Investigation covered following species: marshmallow (Althea officinalis), coneflower (Echinacea purpurea and Echinacea angustifolia), St. John's wort (Hypericum perforatum) and garden sage (Salvia officinalis). From plants with different symptoms of diseases over 200 isolates of phytopathogens was obtained. Microscopic analysis reviled that the biggest number of isolates belonged to the species of taxa Fusarium and Sclerotinia, whilst few further isolates belonged to the taxa Aspergillus, Penicillium, Botrytis, Cladosporium, Chetomium, Doratomices, Erysphae, Myrothecium, Phoma, Puccinia, Epicoccum, etc. In whole, 31 phytopathogenic species, belonging 20 Taxa, were determined, as follows: 15 species from 11 Taxa in Althaea officinalis, 11 species from 9 Taxa in Echinacea angustifolia, 14 species from 10 Taxa in Echinacea purpurea, 13 species from 8 Taxa in Hypericum perforatum, and 16 species from 6 Taxa in Salvia officinalis. According to the point of isolation, pathogenic species were dominant in seeds of medicinal and aromatic plants and in lesser extent in roots and over-grounds organs. Tests for the evaluation of pathogenicity reviled that all isolates of Fusarium species and Sclerotinia sclerotiorum are pathogenic toward their host plants in controlled conditions. For the first time Erysiphe cichoracearum was recorded in St. John's wort, Oidium spp. in garden sage, and E. purpurea and Phoma sp. in marshmallow and coneflower, as well as Sclerotinia sclerotiorum in E. purpurea and E. angustifolia. Fungi belonging to the genus Fusarium also were for the first time recorded and determined to the species level. Pathogenicity of these important causal agents of different diseases in medicinal and aromatic plants was evaluated too.

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P3 Pregled dendroflore Vršačkih planina koja se koristi u narodnoj medicini Štrbac Orhideja JP “Varoš”, 26300, Vršac, Srbija Razvojem urbanog načina života umeće sakupljanja i upotrebe lekovitog bilja je donekle potisnuto. Čovek sve manje vremena provodi u prirodi i sve je manje upoznat sa lekovitim i hranjivim svojstvima samoniklih biljka, posebno drvenastih. Na osnovu prikaza najznačajnijih vrsta dendroflore na Vršačkim planinama sa kratkim sadržajem njihovih lekovitih supstanci i hranjivih materija, može se zaključiti da su postojeće fitocenoze dobar izvor farmaceutski značajnih sirovina. U ovom radu prikazane su najvažnije drvenaste biljke na Vršačkim planinama koje se tradicionalno koriste u lekovite svrhe. Mnoge od njih imaju jestive plodove bogate vitaminima, taninima, flavonoidima, šećerima ili mastima, kao na primer kupine, trnjine, lešnici, drenjine, divlje jabuke, kruške, šljive, trešnje... Neki od ovih plodova koriste se za pripremanje ukusnih džemova, želea, sokova i alkoholnih i drugih napitaka. Osušeni ili sveži plodovi, kora i cvetovi koriste se za spravljanje čajeva, sirupa, marmelada - glog, lipa, šipak, zova… Na Vršačkim planinama je identifikovano oko 30 vrsta koje imaju značajniju primenu u tradicionalnoj medicini. Terenskim istraživanjima, identifikovani su: Acer campestre L. (klen), A. tataricum L. (žestika), Cornus mas L. (dren), Corylus avellana L. (leska), Crataegus monogyna Jacq. (glog), Euonymus europaeus Scop. (kurika), Fagus moesiaca (Domin, Maly) Czeczoti (bukva), Fraxinus ornus L. (crni jasen), Gleditsia triacanthos L. (gledičija), Hedera helix L. (bršljan), Ligustrum vulgare L. (kalian), Malus sylvestris Mill. (divlja jabuka), Pinus nigra Arn. (crni bor), Pinus silvestris L. (beli bor), Prunus avium L. (divlja trešnja), P. cerasifera L., Pyrus pyraster Burgst. (divlja kruška), Quercus cerris L. (cer), Q. robur L. (lužnjak), Q. petraea Liebl. (kitnjak), Robinia pseudoacacia L. (bagrem), Rosa canina L. (pasja ruža), R. gallica L. (galska ruža), Rubus fruticosus L. (kupina), R. spinosa L. (ostruga), Salix alba L. (bela vrba), Salix fragilis L. (krta vrba), Sambucus nigra L. (zova), Sorbus domestica L. (oskoruša), S. torminalis (L.) Crantz. (brekinja), Tilia cordata Mill. (pozna lipa), T. platyphyllos Scop. (rana lipa), Ulmus minor Mill. (poljski brest), Vitis vinifera L. (vinova loza). Literatura: 1. Vukićević E. (1987) Dekorativna dendrologija. Naučna knjiga, Beograd. 2. Tucakov J. (1971) Lečenje biljem. Kultura, Beograd. 3. Lukić P. (1988) Farmakognozija. Farmaceutski fakultet, Beograd.

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P3 Summary on woody plants from the Vršac Mountains used in traditional medicine Štrbac Orhideja JP “Varoš”, 26300, Vršac, Srbija Nowadays urban way of living dominates, besides a small number of people who knows medical uses of woody plants; there is nothing to remind us of their great importance. Trees and shrubs are a vital element in our surroundings. But all too often, trees and shrubs are taken for granted. Based upon the field investigations on the Vršac Mountains, a conclusion that existing phytocenoses are a rich source of raw materials with pharmaceutical importance could be drawn. This paper presents the most important woody plants from the Vršac Mountains and their traditional medical use. Most of these species have edible fruits rich in vitamins, tannins, flavonoids, sugars or essential oils, such as blackberries, dewberries, nuts, apples, pears, plums, cherries etc. Some of these fruits are also used to produce delicious jams, marmalades, jellies, juices, syrups, alcoholic beverages and other drinks. The dried or fresh fruits, flowers or leaves are used to make medicinal herbal teas (hawthorn, linden, dog rose, elder…) In this paper, a list of the most traditionally used woody plants from Vršac Mountains is given. By field investigations, some 30 species are identified and shortly described, that tells us about significance of many woody plants of Vršac Mountains with medicinal properties. These are: Acer campestre L. (Field Maple), A. tataricum L. (Tatar Maple), Cornus mas L. (European Cornel), Corylus avellana L. (Common Hazel), Crataegus monogyna Jacq. (Common Hawthorn), Euonymus europaeus Scop. (European Spindle), Fagus moesiaca (Domin, Maly) Czeczoti (Beech), Fraxinus ornus L. (Manna Ash), Gleditsia triacanthos L. (Honey Locust), Hedera helix L. (Common Ivy), Ligustrum vulgare L. (Wild Privet), Malus sylvestris Mill. (Forest Apple), Pinus nigra Arn. (Black Pine), Pinus silvestris L. (Scots Pine), Prunus avium L. (Wild Cherry), P. cerasifera L. (Cherry Plum), Pyrus pyraster Burgst. (Pear), Quercus cerris L. (Turkey Oak), Q. robur L. (Pedunculate Oak), Q. petraea Liebl. (Sessile Oak), Robinia pseudoacacia L. (Black Locust), Rosa canina L. (Dog Rose), R. gallica L. (Gallic Rose), Rubus fruticosus L. (Blackberry), R. spinosa L., Salix alba L. (White Willow), Salix fragilis L. (Crack Willow), Sambucus nigra L. (Elder), Sorbus domestica L. (Service Tree), S. torminalis (L.) Crantz. (Wild Service Tree), Tilia cordata Mill. (Small-leaved Linden), T. platyphyllos Scop. (Large-leaved Linden, Ulmus minor Mill. (Field Elm), Vitis vinifera L. (Grape Vine). References: 1. Vukićević E. (1987) Dekorativna dendrologija. Naučna knjiga, Beograd. 2. Tucakov J. (1971) Lečenje biljem. Kultura, Beograd. 3. Lukić P. (1988) Farmakognozija. Farmaceutski fakultet, Beograd.

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P4 Kultivacija uskolisne bokvice (Plantago lanceolata L.) za potrebe farmaceutske industrije Pljevljakušić Dejan, Brkić Sreta, Radanović Dragoja, Zdunić Gordana, Janković Teodora, Krivokuća-Đokić Dragana 1Institut za proučavanje lekovitog bilja „Dr Josif Pančić“, Tadeuša Košćuška 1, 11000 Beograd, Srbija Uskolisna bokvica (Plantago lanceolata L.) je biljka koja ima široku farmakološku primenu, ali se najčešće potenciraju njene spazmolitičke osobine u sirupima za kašalj. Kvalitet sirovine dobijen sakupljanjem u prirodi je veoma nizak, a do sada postignuti rezultati kultivacije bokvice pokazuju ogroman skok u kvalitetu. Iz tog razloga se u Evropi gaji na preko 100 ha. Uskolisna bokvica je višegodišnja biljka, ali se kultivacija planira na period od dve godine. Godišnje potrebe industrije fitopreparata, u Srbiji, za drogom Plantaginis folium prevazilaze desetine tona, što uskolisnu bokvicu svrstava u red lekovitih biljaka za kojom postoji velika i stalna tržišna tražnja. Prethodna istraživanja u evropskim zemljama unapredila su tehnologiju gajenja bokvice u intenzivnu ratarsku kulturu. U sprovedenom dvogodišnjem eksperimentalnom gajenjenju plantaže su zasnivane iz direktne setve sa setvenom normom 15 kg/ha. U 2006. god. zasnovano je 10 ari, a u 2007. god. 24 ara. Dobijeni rezultati pokazuju da se u prvoj godini, u našim agroekološkim uslovima, mogu očekivati 2-3 otkosa sa ukupnim prinosom oko 2 t/ha suvog lista, a u drugoj godini 4-5 otkosa sa ukupnim prinosom oko 4 t/ha. Odnos sveže prema suvoj masi kretao se oko 1: 8. Uzorci su ispitivani prema zahtevima Evropske farmakopeje 6.0. U svim uzorcima, sadržaj ukupnih derivata orto-dihidroksicinamične kiseline izražen kao akteozid je bio veći od 1.5% što je zahtev Ph. Eur. 6.0. Započeta istraživanja na gajenju bokvice, u predelu Južnog Banata, pokazala su da je ovo potencijalno veoma prinosna i ekonomski isplativa kultura. Na osnovu preliminarnih rezultata je uočeno da prilikom kultivacije posebnu pažnju treba obratiti na pravovremenu obradu zemljišta, na postžetvenu doradu biljne sirovine i na pojavu imaga buvača.

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P4 Cultivation of ribwort plantain (Plantago lanceolata L.) for pharmaceutical industry demand Pljevljakušić Dejan, Brkić Sreta, Radanović Dragoja, Zdunić Gordana, Janković Teodora, Krivokuća-Đokić Dragana 1Institute for Medicinal Plants Research „Dr Josif Pančić“, Tadeuša Košćuška 1, 11000 Belgrade, Serbia Ribwort plantain (Plantago lanceolata L.) is plant which has wide pharmacological appliance, but the most frequently it has been emphasized by its spasmolytic properties utilized in syrups for cough. Quality of wildcrafted raw material is low and achieved results in plantain cultivation shows huge step forward regard quality. Therefore, cultivation of plantain in EU has been established at more than 100 ha. Ribwort plantain is perennial plant, but duration of cultivation has been projected on two years period. Demand per year for plant drug Plantaginis folium, for Serbian pharmaceutical industry, overcome tenths tons, what rank this plant in order of medicinal plants for which exist big and permanent market demand. Prior research in EU countries has been promoted technology of plantain to intensive farming culture. In conducted two years experimental cultivation, plantain plantations were established through direct sowing, with sowing rate of 15 kg/ha. In 2006 and 2007, 0.1 ha and 0.24 ha plantain plantations has been established, respectively. Attained data shows that in the first year of cultivation, in our agroecological climate, 2-3 leaf harvesting can be expected, with overall yield of about 2 t/ha of dry leaves, and, in the second year, 4-5 harvestings, with overall yield of about 4 t/ha of dry leaves. Drying ratio was about 1:8. Samples were investigated according to European Pharmacpoeia 6.0. In all samples content of total ortho-dihydroxycinnamic acid derivates expressed as acetoside were higher than 1.5 per cent, which is the requirement of Ph. Eur. 6.0. Conducted research, in the field of plantain cultivation, at locality of South Banat, shows that this plant has very high yield and economic potential. During cultivation special attention has to be heeded on forehand soil preparation, postharvest processing of raw material and on appearance of Halticinae imago.

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P5 Uticaj pojedinih agrotehničkih mera na prinos droge belog sleza Babić Milica1, Marinković Branko 1, Jaćimović Goran 1, Crnobarac Jovan 11 Poljoprivredni fakultet, Trg D. Obradovića 8., 21000 Novi Sad, Srbija. Obzirom da količine samoniklog sleza ne mogu da zadovolje narasle potrebe, beli slez se sve više gaji. Istraživanja tehnologije proizvodnje ove biljne vrste u našoj zemlji su vrlo skromna. Iz tih razloga, sproveden je eksperiment koji može da doprinese boljem poznavanju tehnologije gajenja ove vrste. U ogledu sa dve domaće sorte belog sleza, izvedenom u Mošorinu, ispitivan je efekat različitih doza azotnog đubriva (Ø, 50, 80, 120 kg ha-1), četiri veličine vegetacionog prostora i dva načina razmnožavanja na prinos suvog korena sleza. Najveći prinos suvog korena (4.264 kg ha-1) postignut je na varijanti bez azota, što je bilo za očekivati obzirom da je ogled postavljen na baštenskom zemljištu obilno đubrenom stajnjakom. Najveći prinos dobijen je pri najmanjem razmaku između biljaka u redu (20 cm). Pri direktnoj setvi semena u polje prinos korena bio je za 1.664 kg ha-1 veći nego pri zasnivanju kulture putem sadnica.

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P5 Influence of certain agro-technical practices on yield of marshmallow drug Babić Milica1, Marinković Branko 1, Jaćimović Goran 1, Crnobarac Jovan 11 Faculty of Agriculture, D. Obradovića Square 8., 21000 Novi Sad, Serbia. Since present quantities of wild marshmallow do not satisfy the growing demand, marshmallow is cultivated more and more. In our country, researches concerning the cultivation technology of this plant species are poor. Goal of this research was to improve the cultivation methods by application of some agricultural measures. In experiment with two domestic marshmallow varieties, carried out in Mošorin, we explored the effects of different N-rate (Ø, 50, 80, 120 kg ha-1), 4 growing areas and 2 planting methods on the dry root yield of marshmallow. The highest dry root yield (4.264 kg ha-1) was achieved without use of nitrogen, which was expected since the trial was abundantly fertilized with farmyard manure. The highest root yield was on lowest spacing between plants into row (20 cm). The analysis of reproduction method showed that using the direct sowing method the yield was 1.664 kg ha-1 higher than using the plant transplantation.

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P6 Kontejnerska proizvodnja rasada lekovitog, aromatičnog i začinskog bilja Damir Beatović, Slavica Jelačić, Đorđe Moravčević Univerzitet u Beogradu, Poljoprivredni fakultet Nemanjina 6, 11081 Zemun-Beograd Proizvodnja rasada lekovitog, aromatičnog i začinskog bilja kod nas se još uvek odvija na klasičan način u lejama (po sistemu »golih žila«). U savremenoj povrtarskoj i cvećarskoj proizvodnji široko je prihvaćena kontejnerska proizvodnja rasada, po sistemu sadnica sa zaštićenim korenovim sistemom (2). Od svih sistema kontejnerske proizvodnje rasada najracionalnijim i najpraktičnijim se pokazao »speedling system«, koji predstavlja sistem polistirenskih ili polipropilenskih kontejnera (tacni) sa ćelijama različite zapremine u koji se stavlja supstrat i vrši setva (3). Tokom višegodišnjih istraživanja u stakleniku Poljoprivrednog fakulteta u Beogradu u proizvodnji rasada lekovitog, aromatičnog i začinskog bilja korišćeni su različiti kontejnerski sistemi (1). Istraživanjem je obuhvaćen veći broj lekovitih, aromatičnih i začinskih vrsta: bosiljak (Ocimum basilicum L.), matičnjak (Melissa officinalis L.), žalfija (Salvia officinalis L.), ehinaceja (Echinacea angustifolia D.C.), lavanda (Lavandula angustifolia Mill), majoran (Majorana hortensis Moench), ruzmarin (Rosmarinus officinalis L.) i miloduh (Hyssopus officinalis L.). Istraživanja sa kontejnerskom proizvodnjom rasada lekovitog, aromatičnog i začinskog bilja ukazuju na značajne prednosti u odnosu na klasičnu proizvodnju rasada. Rezultati ovih istraživanja su deo projekta Ministarstva nauke Republike Srbije TR 20108: Samoniklo i gajeno lekovito bilje biozona Srbije u funkciji održivog razvoja brdsko-planinskih regija – prvi deo. Literatura: 1. Beatović, D. i sar., (2006): Modeliranje proizvodnje rasada bosiljka – izbor kontejnera. Arhiv za polj. nauke. Vol.67, No 238. str. 103-109. 2. Hanić, E. (2000): Značaj supstrata, kontejnera i hormona u rasadničarskoj proizvodnji, Univerzitet »Džemal Bijedić« Mostar, Studij za mediteranske kulture, str.1-260. 3. Marković, V. i sar., (1992): Kontejnerska proizvodnja rasada, Savr. poljoprivreda, Vol 40, broj 1-2, str. 11-14.

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P6 Container production of medicinal, aromatic and seasoning herbs seedlings Damir Beatović, Slavica Jelačić, Đorđe Moravčević University of Belgrade, Faculty of Agriculture Nemanjina 6, 11081 Zemun-Belgrade Medicinal, aromatic and seasoning herb seedlings in our country is still produced in a classical way in leje (according to the „bare thread“ system). In the modern vegetable and horticultural production containter production of seedlings has been accepted widely, according to the system of seedlings with protected root systems (2). Of all container production systems, the most rational and the most practical one proved to be the so-called »speedling system«, which represents the system of polystyrene and polypropylene containers (plates) with cells of different volumes to which substrate is added and seedlings are set out (3). In the course of several years of research in the glasshouse of the Faculty of Agriculture of Belgrade different container systems were used in the production of medicinal, aromatic and seasoning herb seedlings (1). The research included a large number of medicinal, aromatic and seasoning species: basil (Ocimum basilicum L.), lemon balm (Melissa officinalis L.), sage (Salvia officinalis L.), echinacea (Echinacea angustifolia D.C.), lavender (Lavandula angustifolia Mill), marjoram (Majorana hortensis Moench), rosemary (Rosmarinus officinalis L.) and hyssop (Hyssopus officinalis L.). The reasearch conducted with the container production of medicinal, aromatic and seasoning herb seedlings point out to significant advantages as compared to classical production of seedlings. The results of this research are a part of the project Ministry of Science Republic of Serbia TR 20108: Wild and grown medicinal herbs in Serbian biozones, in the function of sustainable development of hilly and mountainous regions .

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P7 Kvalitativna i kvantitativna svojstva bele (Sinapis alba) i crne slačice (Brassica nigra) Marjanović – Jeromela Ana1, Marinković Radovan1, Jaćimović Goran2, Mrđa Jelena1

1 Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30., 21000 Novi Sad, Srbija. 2 Poljoprivredni fakultet, Trg D. Obradovića 8., 21000 Novi Sad, Srbija. Bela (Sinapis alba) i crna slačica (Brassica nigra) su visoko vredne sirovine u proizvodnji senfa. Obe vrste pripadaju porodici Brassicaceae i u Srbiji se gaje na malim površinama. Potrebe za ovim aromatičnim biljkama daleko su veće od dosadašnje proizvodnje u našoj zemlji. U cilju boljeg razumevanja zahteva ovih vrsta prema uslovima spoljašnje sredine u našem regionu, u toku dve vegetacione sezone ispitivana su najznačajnija kvantitativna i kvalitativna svojstava. U radu su navedene razlike u brzini proticanja fenofaza, mase 1000 semena, hektolitarske mase, prinosu semena kao i sadržaja ulja i proteina. Datum cvetanja, u proseku, kod crne slačice je bio 16. 05, a kod bele 14. 05. Vreme tehnološke zrelosti za obe vrste nastupilo je 04. 07. Masa 1000 semena (g), prosečno, kod crne slačice je iznosila 2,22, a kod bele 6,95 g. Sadržaj ulja (%) je kod crne slačice iznosio 26,20, a kod bele 20,42 %. Hektolitarska masa (kg/hl) kod crne slačice bila je 71,2 a kod bele 73. Uticaj spoljašnje sredine bio je statistički značajan izvor variranja za navedena svojstva.

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P7 Qualitative and quantitative traits of white (Sinapis alba) and black mustard (Brassica nigra) Marjanović – Jeromela Ana1, Marinković Radovan1, Jaćimović Goran2, Mrđa Jelena1

1 Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia. 2 Faculty of Agriculture, D. Obradovića Square 8., 21000 Novi Sad, Serbia White mustard (Sinapis alba) and black mustard (Brassica nigra) are very approve raw materials in mustard production. Both species belongs to Brassicaceae family and in Serbia are growing in small areas. Requirements in these aromatic plants are higher than previous production in Serbia. Because of better understanding requirements of these species to environmental conditions in our region, during two growing seasons were studying the most significant qualitative and quantitative properties. In this paper indicated differences in tempo of flow through growth stages, the mass of 1000 seeds, hectolitre mass, grain yield as well as oil and protein content. Flowering date in average for black mustard was May 16, and may 14 for white mustard, respectively. Date of technological maturity for both species was July 4. The mass of 1000 seeds (g), averaged 22.2 at black and 6.95 at white mustard. Oil content at black mustard was 26.20 % and hectolitre mass was 178.0. White mustard had oil content of 20.42 %, while hectolitre mass was 182.5. Environmental impacts were statistically significant source for mentioned traits fluctuation.

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P8 Kvantitativna HPLC analiza flavonoida i fenolkarbonskih kiselina u etanolnom ekstraktu plodova gloga metodom eksternog standarda Tadić VM1, Marković G1, Dobrić S2, Đorđević S1, Arsić I1, Runjaić-Antić D1

1 Institut za pručavanje lekovitog bilja „Dr Josif Pančić“, Tadeuša Košćuška 1, 11000 Beograd, Srbija; 2 Vojnomedicinska akademija, Crnotravska 17, 11000 Beograd, Srbija Crataegus monogyna Jacq. i C. oxyacantha L. [sin. C. laevigata (Poiret) DC.], Rosaceae, predstavljaju lekovite biljke koje se od davnina primenjuju u evropskoj herbalnoj i kineskoj tradicionalnoj medicini [1]. U Evropi, cvetovi, listovi i plodovi gloga, ili njihova kombinacija, tradicionalno se upotrebljavaju kao adstringenti, antispazmodici, kardiotonici, diuretici, kao sredstva sa hipotenzivnim i antiaterosklerotičnim efektom. Posebno je opravdana upotreba proizvoda koji sadrže glog u tretmanu različitih problema koji se javljaju kod oslabljenog srca, kada terapija digitalisovim kardiotoničnim heterozidima još nije potrebna, naročito kod hipertenzije sa insuficijencijom miokarda, kod blagih oblika srčane aritmije i ateroskleroze [2]. Iako se u praksi više koriste proizvodi koji sadrže ekstrakte listova i cvetova gloga zbog dobro izbalansiranog sadržaja antocijana i drugih flavonoidnih jedinjenja, i proizvodi sa ekstraktima plodova gloga se često javljaju. Cilj ovog rada je bio određivanje flavonoida i fenolkarbonskih kiselina u etanolnom ekstraktu plodova gloga HPLC metodom. Kvantitativnom HPLC analizom razvijenom za određivanje sadržaja flavonoida, metodom eksternog standarda, u etanolnom ekstraktu ploda gloga, utvrđeno je da su u hiperozid i izokvercitrin glavne komponente. Hiperozid (7) je u najvećem procentu zastupljen u ispitivanom ekstraktu (Tabela 1). U ekstraktu su identifikovani i određeni i hlorogenska kiselina (1), viteksin (6), rutin (8), izokvercitrin (9), i kvercetin (10) (Tabela 1). Ukupni sadržaj flavonoida, određen i metodom opisanoj u DAB X [3], iznosio je 0,14%. Primenom naše HPLC metode, ukupni sadržaj aglikona flavonoida, izražen kao kvercetin, iznosio je 0,18%. Zadovoljavajući nivo hidrolize podrazumevao je odsustvo pikova u HPLC hromatogramu koji su odgovarali hiperozidu i izikvercitrinu, koji su u najvećem procentu zastupljeni u ispitivanom ekstraktu, i postojanje samo pika koji je odgovarao kvercetinu. Pik koji je odgovarao viteksinu, i u HPLC hromatogramu hidrolizata je identifikovan, što je i očekivano, s obzirom da je reč o C-glikozidu. Kafena kiselina (2), viteksin-2”-O-ramnozid (3), cijanidin-hlorid (4) i (-)-epikatehin (5) u ispitivanom ekstraktu nisu detektovani, iako literaturni podaci navode prisustvo ovih jedinjenja u plodovima gloga. Tabela 1. Kvantitativna anliza flavonoida i fenolkarbonskih kiselina u etanolnom ekstraktu plodova gloga (*brojevi se odnose na jedinjenja pomenuta u tekstu) Jedinjenje * 1 2 3 4 5 6 7 8 9 10 10a

Sadržaj (%) 0,02 ndb nd nd nd t 0,14 t 0,03 0,01 0,18 aSadržaj posle kisele hidrolize; bnije detektovan; t=trag Zahvalnica: Autori zahvaljuju Ministarstvu za nauku i tehnološki razvoj za finansijsku podršku, projekat br. 20137 Literatura: 1. Commission E, monograph: Crataegi folium cum flore. Bundesanzeiger, 133, 7.19.1994. 2. Weihmayr, T et al. (1996) Fortschr. Med. 114:27-29. 3. Weißdornblätter mit Blüten (1991) Deutsches Arzneibuch, Band 2, Monographien R-Z. Amlitche Ausgabe Deutscher Apotheker Verlag, Govi-Verlag GmbH, Stuttgart, Frankfurt. 4. Bisset NG, Wichtl M. (2001) Herbal Drugs and Phytopharmaceuticals. Medpharm GmbH Scientific Publishers, Stuttgart.

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P8 Quantitative HPLC analysis of flavonoids and phenolcarbonic acids in hawthorn berries ethanol extract, using the external standard method Tadić VM1, Marković G1, Dobrić S2, Đorđević S1, Arsić I1, Runjaić-Antić D1

1 Institute for Medicinal Plant Research “Dr Josif Pančić”, Tadeuša Košćuška 1,11000 Belgrade,Serbia; 2

Institute for Scientific Information, Military Medical Academy, Crnotravska 17, 11000 Belgrade, Serbia

Crataegus monogyna Jacq. and C. oxyacantha L. [sin. C. laevigata (Poiret) DC.], Rosaceae, are medicinal plants with a long history of use in European herbal and traditional Chinese medicine [1]. In Europe, Crataegus (hawthorn) leaves, flowers, and both green (unripe) and red (ripe) berries or a combination thereof have traditionally been used as astringent, antispasmodic, cardiotonic, diuretic, hypotensive, and antiatherosclerotic agents. Hawthorn is particularly used in the treatment of various heart problems, angina pectoris, hypertension with myocardial insufficiency, mild alterations of cardiac rhythm, and atherosclerosis [2]. According to European Pharmacopoeia 6.0, hawthorn berries consist of the dried false fruits of these two species or their mixture. Over the years the preferred used of leaf and flowers drug presumably originated from its traditional use whereby it is assumed to exhibit a balanced level of procyanidins and other subgroups of flavonoids. Nevertheless, the dried berries are frequently used for herbal preparations. The present study was carried out to develop HPLC method for flavonoids and phenolcarbonic acids quantification in ethanol extract of hawthorn berries. By quantitative HPLC analysis, using the external standard method, hyperoside (7) was determined as the major flavonol component, and composed 0.14% of the extract. Other identified components were chlorogenic acid (1), vitexin (6), rutin (8), isoquercitrin (9), and quercetin (10) (Table 1). Total flavonoids content, applying method described in DAB X [3] revealed the percentage of 0.14%. Determined by our HPLC procedure, total flavonoid aglycones represented 0.18%, expressed as quercetin. The successful hydrolysis procedure comprehended the absence of the peaks corresponding to hyperoside and isoquercitrin, as the main components in the investigated extract, and the presence of only one peak corresponding to quercetin in the HPLC chromatogram. Vitexin, whose presence was confirmed in the hydrolisate, as C-glycoside was not hydrolyzed, what was expected. As determined only in the traces, rutin and vitexin, in original extract and vitexin in the extract after hydrolysis did not significantly contribute to the total flavonoid and total flavonoid aglycones content. Caffeic acid (2), vitexin-2”-O-rhamnoside (3), cyanidin-chloride (4) and (-)-epicatechine (5), although literature data suggested [1,4] were not identified in the extract. Based on obtained HPLC fingerprint of investigated extract, hyperoside and isoquercitrine were detected as main constituents in flavonol group of compounds.

Table 1. Quantitative determination of flavonoids and phenolcarbonic acids in ethanol hawthorn berries extract (*the numbers refer to compounds mentioned in the text)

Compound * 1 2 3 4 5 6 7 8 9 10 10a

Content (%) 0.02 nfb nf nf nf t 0.14 t 0.03 0.01 0.18 aContent determined after acid hydrolysis; bnot found; t=trace

Acknowledgement: The authors thank Serbian Ministry of Science and Technological Development for financial support, project N020137 References: 1. Commission E, monograph: Crataegi folium cum flore. Bundesanzeiger, 133, 7.19.1994. 2. Weihmayr, T et al. (1996) Fortschr. Med. 114:27-29. 3. Weißdornblätter mit Blüten (1991) Deutsches Arzneibuch, Band 2, Monographien R-Z. Amlitche Ausgabe Deutscher Apotheker Verlag, Govi-Verlag GmbH, Stuttgart, Frankfurt. 4. Bisset NG, Wichtl M. (2001) Herbal Drugs and Phytopharmaceuticals. Medpharm GmbH Scientific Publishers, Stuttgart.

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P9 Ispitivanje limunskog bosiljka (Ocimum basilicum var. citriodorum)

Slavica Jelačić, Damir Beatović Univerzitet u Beogradu, Poljoprivredni fakultet, Nemanjina 6, 11081 Zemun-Beograd

U okviru vrste Ocimum basilicum L. postoji veći broj varijeteta koji se razlikuju po opštoj morfološkoj strukturi i građi, a zatim i po sadržaju i kompoziciji etarskog ulja (1,2). Bosiljak je lekovita, aromatična i začinska biljka sa dugogodišnjim tradicijom gajenja u našoj zemlji. U našoj zemlji dominiraju dve forme bosiljka krupnolisni i sitnolisni zeleni. Dominantne komponente etarskog ulja u našim populacijama bosiljka su: linalol, estragol, geraniol i 1,8 cineol. Tokom višegodišnjih istraživanja prikupljen je veći broj uzoraka bosiljka iz različitih populacija na teritoriji Srbije (3). Takođe, na osnovu višegodišnjeg kolekcionisanja i introdukcije genotipova iz različitih delova Evrope, nabavljan je limunski bosiljak (Ocimum basilicum L. var. citriodorum). Na osnovu preliminarnih istraživanja hemijskih osobina limunskog bosiljka dobijeni su sledeći rezultati. Prosečan sadržaj etarskog ulja u suvoj herbi je 0,8%; radi se o geranialnom hemotipu etarskog ulja bosiljka u čijem hemijskom sastavu su zastupljeni geranial (32,00%), neral (24,94%), linalol (8,28%), β-kariofilen (6,82%), cis α-bisabolen (6,37%), kariofilen oksid (4,25%), α-bergamoten (2,68%), germakren D (1,55%), α-humulen (1,05%). Rezultati ovih istraživanja su deo projekta Ministarstva nauke Republike Srbije TR 20108: Samoniklo i gajeno lekovito bilje biozona Srbije u funkciji održivog razvoja brdsko-planinskih regija – prvi deo. Literatura: 1. Grayer, R.J. et. al. (1996): Intraspecific taxonomy and essential oil chemotypes in sweet basil, Ocimum basilicum. Phytochem., 43:1033-1039. 2. Иванова, K. В., 1990. Внутривидовая классификация базилика огородного (Ocimum basilicum L.) Труди по прикл. ботанике, генет. и селекции, 133, 41-49. 3. Jelačić, S. et, al. (2004): Genotypes of Basil (Ocimum basilicum L.) in Serbia and Montenegro. 3rd CMAPSEC. 5-8 September 2004, Nitra. Slovakia. p. 42-43.

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P9 Research of citric basil (Ocimum basilicum var. citriodorum)

Slavica Jelačić, Damir Beatović University of Belgrade, Faculty of Agriculture, Nemanjina 6, 11081 Zemun-Belgrade

Within the species Ocimum basilicum L. there a great number of varieties which differ from one another by to their overall morphological structure and substance and ultimately according to their content and composition of essential oils (1,2). Basil is a medicinal, aromatic and seasoning herb with long-lasting growing tradition in our country. In our country two forms of basil are predominant – large-leaved and small-leaved green basil. The predominant components of ethereal oils in our populations of basil are: linalol, estragol, geraniol and 1,8 cineol. During the many years of research, a large number of basil populations was collected on the territory of Serbia (3). Also, based on many years of collection and introduction of genotypes from different parts of Europe, citric basil (Ocimum basilicum L. var. citriodorum) was obtained. Based on the preliminary research of the chemical characteristics of this kind of basil the following results were obtained: average contents of ethereal oil in dry herb was 0,8%; this was a geranial chemotype of basil ethereal oil, the chemical composition of which includes gearnial (32,00%), neral (24,94%), linalol (8,28%), β-caryophyllene (6,82%), cis α-bisabolene (6,37%), caryophyllene oxiode (4,25%), α-bergamotene (2,68%), germacrene D (1,55%), α-humulene (1,05%). The results of this research are a part of the project Ministry of Science Republic of Serbia TR 20108: Wild and grown medicinal herbs in Serbian biozones, in the function of sustainable development of hilly and mountainous regions –the first part

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P10 Resurs i kvalitet aromatičnog lekovitog bilja submediteranskog i mediteranskog pojasa Crne Gore Menković N, Tasić S, Zdunić G, Šavikin K Institut za proučavanje lekovitog bilja „Dr Josif Pančić“, Tadeuša Košćuška 1, 11000 Beograd, Srbija

Biološka aktivnost aromatičnih biljaka delimično je zasnovana na aktivnosti etarskog ulja, pored drugih prisutnih biološki aktivnih sastojaka. Etarska ulja pokazuju niz efekata među kojima: antimikrobni, spazmolitični, iritirajući. Na kvalitet etarskog ulja mogu uticati različiti faktori kao što su genotip, fenofaza (važno zbog definisanja optimalnog vremena prikupljanja biljnog materijala), ekološki faktori (faktori sredine), način obrade biljne sirovine, način izolovanja etarskog ulja. U našem radu smo istraživali planinu Orjen i oblast koja obuhvata obodne delove masiva, okrenute jugu i jugozapadu. Uzorci biljaka su sakupljeni u fazi cvetanja u periodu juni-juli 2007. god. Nakon izolovanja etarskih ulja, urađena su njihova fizičko-hemijska ispitivanja i poređenje rezultata sa referentnim standardima. Na osnovu rezultata ispitivanja sadržaja etarskog ulja, gasne i gasno-masene analize, kao i resursa aromatičnog bilja na istraživanim lokalitetima, odabrane su aromatične biljne vrste koje se mogu racionalno sakupljati. Ispitivane su vrste Salvia officinalis, Helichrysum italicum, Satureja montana, Myrtus communis, Achillea millefolium, Laurus nobilis, Origanum heracleoticum, Thymus serpyllum i Pinus heldreichii. Zaključci: Sadržaj etarskog ulja A. millefolium je osrednji, kvalitet etarskog ulja solidan, prisustvo hamazulena u etarskom ulju ukazuje da je reč o hemotipu koji se na tržištu traži. Zbog značajne rasprostranjenosti smilja na pojedinim lokacijama u okviru ispitivane oblasti urađeno je nekoliko analiza na sadržaj etarskog ulja. Ispitivani uzorci su davali veoma različite rezultate. Najniži rezultat za ispitivane uzorke je bio 0,16% i može se smatrati da je sasvim zadovoljavajući za industrijsku proizvodnju etarskog ulja. Ispitivani uzorci su imali i do 0,45% etarskog ulja, što se smatra veoma visokom vrednošću. Pored sadržaja etarskog ulja, značajan je i njegov hemijski sastav sa dominantnim komponentama α-pinenom i γ-kurkumenom u visokoj koncentraciji. Rezultati su u granicama karakterističnim i standardnim za jadransko priobalje. Vrste L. nobilis, O. heracleoticum i M. communis daju droge veoma dobrog kvaliteta sa visokim sadržajem etarskog ulja. Sadržaj etarskog ulja grančica P. heldreichii je ispitivan u većem broju uzoraka. Rezultati su varirali u granicama očekivanih vrednosti. Svi ispitivani uzorci lista žalfije su imali preko 2% etarskog ulja (prema zahtevu Ph.Eur.6., minimum iznosi 1,5%), što ukazuje na izuzetno dobar kvalitet droge Salviae folium. Pored sadržaja etarskog ulja značajan je i njegov sastav. U tom smislu je rađena i gasnohromatografska analiza uzoraka sa različitih lokaliteta. Rezultati su u granicama karakterističnim i standardnim za jadransko priobalje. Za vrstu S. montana važe slične napomene kao i za vrste S. officinalis i H. italicum. Sadržaj etarskog ulja je zadovoljavajući (1,30-1,45%), sastav takođe (dominantan timol), a gasnohromatografska analiza ukazuje da je etarsko ulje po kvalitetu u granicama karakterističnim i standardnim za jadransko priobalje. Sadržaj etarskog ulja kod vrste T. serpyllum je bio neuobičajeno visok. Naime, pod nazivom T. serpyllum prihvataju se različite vrste roda Thymus. Samim tim i sadržaj i sastav etarskog ulja ne mogu biti precizno definisani. Nekoliko ispitivanih uzoraka je bilo izuzetno dobrog kvaliteta. U pogledu resursa, značajne za sakupljanje su vrste: H. italicum (sa ocenom zastupljenosti 4), M. communis (3), S. officinalis (5) i S. montana (4). Literatura: 1. Ph. Yug. IV. 2. Braun-Blanquet J. 1965. Plant sociology - the study of plant communities. Hafner, London. 3. Wilmanns, O.(1989): Vergesellschaftung und Strategie-Typen von Pflanzen mitteleuropäischer Rebkulturen, Phytocoenologia, 18: 83-128

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P10 Resource and quality of aromatic medicinal plants from submediterranean and mediterranean regions of Montenegro Menković N, Tasić S, Zdunić G, Šavikin K Institute for Medicinal Plants Research „Dr Josif Pančić“, Tadeuša Košćuška 1, 11000 Belgrade, Serbia Biological activity of aromatic plants is partially due to the activity of essential oil present in the plant. Essential oils possess a lot of pharmacological activities such as antimicrobial, antispasmodic, irritating. Quality of the essential oil depends of different factors such as genotype, fenophase, ecological factors, preparation of plant material, and methods of oil extraction. In our study, we examined mountain Orien and regions on the south and south-west shoulder of the massive. Samples were collected in the phase of flowering in June-July 2007. The oils were obtained by separate hydrodistillation using a Clevenger-type apparatus. The percentage yield (%) of the oils was calculated on moisture-free basis. Qualitative and quantitative analysis was carried out using GC/FID and GC/MS. The identification of constituents was performed by matching their mass spectra and retention indices with those obtained from authentic samples and/or NIST/Wiley spectra libraries, using different types of search (PBM/NIST/AMDIS) and available literature data. The percentage compositions were obtained from electronic integration measurements using flame ionization detection (FID; 2500C). Investigated species were Salvia officinalis, Helichrysum italicum, Satureja montana, Myrtus communis, Achillea millefolium, Laurus nobilis, Origanum heracleoticum, Thymus serpyllum and Pinus heldreichii. According to the resource results and chemical analysis, suggestions for rational exploitations are presented. Essential oil of A. millefolium possessed good quality with expected amount of chamazulene. Helichrysum italicum was found in great quantity on different localities and samples were collected from all of them. The amount of essential oil varied in the samples from 0.16% - 0.45%. Domimant compounds, α-pinene and γ-curcumene, was found in great amounts. Results were typical for plants growing on Mediteranian areas. Also, high essential oil content was found in leaves of L. nobilis and M.communis and aerial parts of O. heracleoticum. All samples of S. officinalis leaves possessed more then 2% of essential oil which pointed out a good qualitu of the drug Salviae folium. GC and GC/MS chromatograms were typical for plants growing at Adriatic coast. The amount of the essential oil in the aerial parts of S. montana varied from 1.3%-1.45% with thymol as dominant compound. High amount of essential oil was found in some samples of T. serpyllum aerial parts. Taking in account the resource of medicinal plants growing at the investigated area, plants significant for rational exploitation are H. italicum (assesment of quantity 4), M. communis (3), S. officinalis (5) i S. montana (4). References: 1. Ph. Yug. IV. 2. Braun-Blanquet J. 1965. Plant sociology - the study of plant communities. Hafner, London. 3. Wilmanns, O.(1989): Vergesellschaftung und Strategie-Typen von Pflanzen mitteleuropäischer Rebkulturen, Phytocoenologia, 18: 83-128

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P11 Hemijska karakterizacija uljanih ekstrakata herbi nekih Hypericum vrsta Zdunić G1, Šavikin K1, Petrović S2

1 Institut za proučavanje lekovitog bilja “Dr Josif Pančić”, T. Košćuška 1, 11000 Beograd, Srbija; 2 Institut za farmakognoziju, Farmaceutski fakultet, V. Stepe 450, 11221 Beograd, Srbija Uljani ekstrakt herbe (vršnog dela u cvetu) kantariona, Hypericum perforatum L. (Oleum Hyperici), je poznato lekovito sredstvo koje se koristi u tradicionalnoj medicini mnogih zemalja. Nalazi primenu u tretmanu rana, opekotina prvog stepena, akutnih povreda, kontuzija i mijalgija, a interno kod poremećaja organa za varenje [1]. Cilj ovog istraživanja je bio hemijska karakterizacija uljanih ekstrakata drugih Hypericum vrsta: H. androsaemum L., H. hirsutum L., H. richeri Vill., H. maculatum Crantz, H. acutum Mnch. i H. barbatum Jacq. Uljani ekstrakti su pripremljeni na tri različita načina: maceracijom sveže herbe u suncokretovom ulju na direktnoj sunčevoj svetlosti tokom 40 dana [2], prethodnom maceracijom suve herbe 96% EtOH, koja se nastavlja ekstrakcijom suncokretovim uljem uz zagrevanje na vodenom kupatilu i istovremeno uklanjanje etanola [3], i direktnom ekstrakcijom suve herbe suncokretovim uljem tokom 3 h uz zagrevanje na vodenom kupatilu [4]. Ispitivan je uticaj različitih faktora u postupku izrade ekstrakata (ekstrakcija svežeg ili osušenog biljnog metarijala, vrsta ekstragensa, sunčeva svetlost i temperatura), na sadržaj flavonoida u dobijenim ekstraktima. Kvalitativna i kvantitativna HPLC analiza uljanih ekstrakata izvršena je na aparatu Hewlett Packard HPLC model 1090; DAD detektor (HP 1040); kolona Lichrospher RP-18 (5 µm, 250×4 mm I.D). Za kvantitativnu analizu kvercetina i I3,II8-biapigenina, kao standardne supstance korišćena su jedinjenja prethodno izolovana iz vrste H. richeri [5], a njihov sadržaj izražen u mg/ml uljanog ekstrakta. Sve analize su urađene u tri ponavljanja. U analiziranim uljanim ekstraktima hipericin nije identifikovan, ali su (osim u ekstraktu H. androsaemum) detektovane komponente koje najverovatnije predstavljaju proizvode raspadanja hipericina. Hiperforin i njegovi derivati takođe nisu identifikovani. U uljanim ekstraktima ispitivanih Hypericum vrsta identifikovani su kvercetin i I3,II8-biapigenin (u ekstraktu H. androsaemum samo kvercetin). Sadržaj flavonoida zavisio je kako od biljne vrste, tako i od primenjenog postupka ekstrakcije. Najveća količina ovih jedinjenja utvrđena je u ekstraktima dobijenim prethodnom maceracijom suve herbe 96% EtOH, koja se nastavlja ekstrakcijom suncokretovim uljem uz zagrevanje i istovremeno uklanjanje etanola. Među njima, uljani ekstrakt vrste H. maculatum (Slika 1) je imao najveći sadržaj kvercetina (0,1906 mg/ml) i I3,II8-biapigenina

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Literatura: 1. Blumenthal, M. et al. (eds) (1998) The Complete German Commission E Monographs. American Botanical Council. Austin. 2. Tucakov, J. (1996) Lečenje biljem. Rad. Beograd. 3. Shikov A. et al. (2004) Rastitel’nye masla i maslyanye ekstrakty: tehnologiya, standardizatsiya, svoistva. Ryskii vratch. Moskva. 4. Shass Е. Yu. (1952) Fittoterapiya, An SSSR. Zdunić, G. et al. (2006) International symposium on organic chemistry, Book of abstracts 65, Sofija.

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P11 Chemical characterization of the oil extracts of some Hypericum species Zdunić G1, Šavikin K1, Petrović S2

1 Institute for Medicinal Plant Research “Dr. Josif Pančić”, T. Košćuška 1, 11000 Belgrade, Serbia; 2 Institute of Pharmacognosy, Faculty of Pharmacy, V. Stepe 450, 11221 Belgrade, Serbia Oil extract of flowering tops of Hypericum perforatum L. (Oleum Hyperici) is popular remedy in traditional medicine of many countries. Externally it is traditionally used for the treatment of wounds, first-degree burns, acute and contused injuries, myalgia and internally for dyspeptic complaints [1]. The aim of this study was to investigate chemical composition of the oil extracts of other Hypericum species: H. androsaemum L., H. hirsutum L., H. richeri Vill., H. maculatum Crantz, H. acutum Mnch. and H. barbatum Jacq. The oil extracts were prepared on three different ways: by maceration of the fresh flowering tops in sunflower oil exposed to sunlight for 40 days [2], by previously maceration of dried flowering tops in 96% EtOH, followed by extraction with sunflower oil by heating on water bath [3], and by digestion of dried flowering tops in sunflower oil for 3 h on water bath [4]. Influence of following factors: fresh or dried plant material, solvent system, use of sunlight or heating systems during extraction on variability of constituents was evaluated. HPLC method for qualitative and quantitative analyses of the oil extracts was developed. Analyses were carried out on a Hewlett Packard HPLC model 1090; DAD detector (HP 1040); column Lichrospher RP-18 (5 µ, 250×4 mm I.D). Quantification of quercetin and I3,II8-biapigenin was done using the standard compounds previosly isolated from H. richeri [5]. Their content is presented as mg/ml of the oil extract. All experiments were done in triplicate. According to the HPLC profiles, hypericin could not be found, but other compounds with the same fluorescence were identified, probably originated from brakedown products of hypericin (with exception of H. androsaemum). Hyperforin and its derivatives were not detected in tested oil extracts. In the investigated oil extracts quercetin and I3,II8-biapigenin were identified (in the oil extract of H. androsaemum quercetin only). Variation of different factors during the preparation of the oil extracts affected the amount of flavonoids, where type of solvent was the most noticable. Oil extracts prepared by maceration in 96% EtOH, followed by extraction with sunflower oil by heating on water bath had the highest content of these flavonoids. Among them, oil extract of H. maculatum (Figure 1) had the highest content of quercetin (0.1906 mg/ml) and I3,II8-biapigenin (0.1587 mg/ml).

References: 1. Blumenthal, M. et al. (eds) (1998) The Complete German Commission E Monographs. ABotanical Council. Austin. 2. Tucakov, J. (1996) Lečenjbiljem. Rad. Beograd. 3. Shikov A. et al. (2004) Rastitel’nye masla i maslyanye ekstrakty: tehnologstandardizatsiya, svoistva. Ryskii vratch. Moskva. 4. Shass Е. Yu. (1952) Fittoterapiya, An SSSR. 5. Zdunić, G. et al. (2006) International symposium on organic chemistry, Book of abstracts 65, Sofija.

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P12 Sadržaj elagne kiseline u plodu, semenu i soku od maline Slavoljub Tasić1, Zorica Juranić2, Biljana Veljković3, Željko Žižak2

1 Institut za proučavanje lekovitog bilja, „Dr.Josif Pančić“,Tadeuša Košćuška 1, 11000 Beograd, Srbija 2 Institut za onkologiju i radiologiju Srbije, Pasterova 14, 11000 Beograd, Srbija 3 Agronomski fakultet Čačak, Cara Dušana 34, 32000 Čačak, Srbija Elagna kiselina je polifenol prisutan u različitom biljnom materijalu, a posebno u plodovima jagodičastog voća (malina, kupina, jagoda). U poslednjih desetak godina vršena su intenzivna istraživanja koja su imala za cilj da pokažu i dokažu povezanost prisustva elagne kiseline u ovim plodovima sa njihovim delovanjem u prevenciji nekih vrsta kancera. Cilj ovog rada je bio da razvijemo metodu za kvalitativno i kvantitativno odredjivanje elagne kiseline u prirodnim preparatima/proizvodima. U tom smislu od 2003 godine započinju šira istraživanja, ali i saradnja izmedju Instituta za proučavanje lekovitog bilja, Instituta za onkologiju i radiologiju, Instituta za voćarstvo i Udruženja proizvodjača maline, pri čemu je centar okupljanja upravo malina. Identifikacija i odredjivanje sadržaja elagne kiseline uradjeno je postupkom tečne hromatografije. Korišćen je detektor sa diodnim nizom, kolona RP-18 (LiChrospher 250mm x 4mm x 5µm). Za hromatografsko razdvajanje je korišćen gradijentni sistem sa mobilnim fazama (A) acetonitril i (B) voda (0-5 min. 5% A, 5-20 min. 100% A), i protok od 1ml/min. Snimanje podataka je vršeno na 255 nm. Sadržaj elagne kiseline u uzorcima je odredjivan metodom eksterne kalibracije. Upotrebljen je standard elagne kiseline (Sigma Chemicals). Korelacioni faktor je 0,999. Pre injektovanja svi uzorci su filtrirani kroz 0,45 µm celulozni filter. U ovom radu prikazani su rezultati odredjivanja sadržaja elagne kiseline u plodu različitih sorti maline, ali i sokovima maline. Ispitivani su sokovi dostupni u prodavnicama, kao i ručno cedjenim sokovima od gajenog ploda maline. Pored toga ispitivan je sadržaj elagne kiseline u ostatku nakon cedjenja soka. Dobijeni su sledeći rezultati: Malina plod, različiti uzorci gajenih sorti - od 0,2 do 4,8 mg/100g „Next“ sok malina, 1 litar, SB=043082 - 1,4 mg/100g „Next“ raspberry juice, 200ml, SB=052249 - 1,7 mg/100g Malina sok, cedjena malina - od 0,7 do 2,7 mg/100g Malina, ostatak nakon cedjenja soka - od 3,9 do 5,8 mg/100g Sumiranjem i analizom svih rezultata može se zaključiti da je sadržaj elagne kiseline najviši u pokožici i semenkama tj. u ostatku nakon cedjenja soka. Stoga preporučujemo da se u ishrani, a takodje i radi prevencije razvoja malignih oboljenja u zdravih osoba, maksimalno koristi čitav plod maline ili nativni sok dobijen od celog ploda bez odvajanja semenke. Zahvalnica: 1. Institut za voćarstvo Čačak, prof dr. Svetislav Petrović, na ustupljenim uzorcima maline Literatura: 1. Zorica Juranić et al.(2005); Food Chemistry, vol 93, Issue 1, 39-45.

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P12 Ellagic acid content in raspberry berries, seeds and juice Slavoljub Tasić1, Zorica Juranić2, Biljana Veljković3, Željko Žižak2

1 Institute for Medicinal Plant Research, „Dr.Josif Pančić“, Tadeuša Košćuška 1, 11000 Belgrade, Serbia 2 Institute for Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia 3 Faculty of Agronomy Čačak, Cara Dušana 34, 32000 Čačak, Serbia Ellagic acid is a naturally occurring plant polyphenol present in some fruits (especially raspberries, blackberries, and strawberries). Many recent investigations are dealing with connection of ellagic acid in these fruits and protecting effect from some forms of cancer. The goal of this work was to develop a method for determination of ellagic acid in natural products. For that purpose in 2003 year cooperation among Institute for Medicinal Plants Research, Institute for Oncology and Radiology, Fruit Research Institute, and Raspberry Manufacturer Society started, having investigation of raspberry as motive grouping. Identification, and determination of ellagic acid contents were done by HPLC. Liquid chromatograph with Diode-Array detector, and RP-18 column (LiChrospher 250mm x 4mm x 5µm) were used. The flow rate of mobile phase was 1 ml/min, A: ACN; B:H20, and an elution gradient (0-5 min. 5% A, 5-20 min. 100% A). Aquisition was carried out at 255nm. An external calibration method, using ellagic acid (Sigma Chemicals) as standard was used. Correlation was 0,999. Before injection, all samples were filtered through cellulose 0,45 µm filters. In this paper we present results of the ellagic acid content in different samples of raspberry, raspberry juice, and residuals (seeds, epidermis, and cuticle) after expression of juice. The results are as follow: Raspberry, different samples/cultivars - from 0,2 to 4,8 mg/100g „Next“ raspberry juice, 1 liter, SN=043082 - 1,4 mg/100g „Next“ raspberry juice, 200ml, SN=052249 - 1,7 mg/100g Raspberry juice /home made/ - from 0,7 to 2,7 mg/100g Residuals /after expression of juice/ - from 3,9 to 5,8 mg/100g Summing the results of analyses we can conclude that ellagic acid content is the highest in residuals after expressing of juice (probably in seeds). Thus we suggest the use of whole raspberries and/or juice obtained from whole berries without removing seeds, both for nutrition and for prevention of cancer diseases in healthy people. Acknowledgements: 1. Fruit Research Institute Čačak, prof dr. Svetislav Petrović, for specimens of raspberry fruit References: 1. Zorica Juranić et al.(2005); Food Chemistry, vol 93, Issue 1, 39-45.

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P13 Fitohemijska karakterizacija nepolarnog ekstrakta Viscum album L. Ćebović T1, Popović M2, Lepojević Ž3, Mimica-Dukić N2 1 Katedra za biohemiju, Medicinski fakultet, Hajduk Veljkova 3, 21000, Novi Sad, Srbija; 2Departman za hemiju, Prirodno-matematički fakultet, Trg D. Obradovića 5, 21000, Novi Sad, Srbija; 3 Tehnološki fakultet, Bul. cara Lazara 1, 21000, Novi Sad, Srbija S obzirom da se većina studija o imeli (Viscum album L.) bavi njenim polarnim ekstraktima, bogatim lektinima i viskotoksinima [1], odlučili smo da pažnju usmerimo na ispitivanje nepolarnih konstituenata ove biljke. Određeni broj gasova se može prevesti u tečno stanje (samo pri temperaturama nižim od superkritične temperature) tako da se ponašaju kao rastvarači. Ugljen dioksid, CO2, se smatra najpodesnijim restvaračem za ekstrakciju bioaktivnih molekula iz biljnog materijala. Tečni CO2 ohlađen ispod kritične temperature (31,3ºC) pokazuje visoku solvatacionu sposobnost koja ne zavisi mnogo od primenjenog pritiska. Na temperaturama višim od 31,3ºC i pritiscima ispod 72 bara (superkritično stanje CO2), solvataciona sposobnost CO2 široko varira u zavisnosti od pritiska i temperaturnog opsega. Adekvatan izbor pritiska i temperature omogućava selektivnu ekstrakciju prirodnih proizvoda, kao i frakcionisanje jedinjenja. U ovom radu, pokušali smo da postignemo selektivnu ekstrakciju nekoliko klasa jedinjenja prisutnih u listovima imele primenjujući superkritičnu CO2 ekstrakciju, i da ih identifikujemo kuplovanjem ekstrakcije sa GC/MS analizom. Uočili smo da je najbolja ekstrakcija postignuta pri pritisku od 350 bara i temperaturi od 34-35ºC. Nekoliko nepolarnih klasa jedinjenja (C23-C30 ugljovodonici, slobodne masne kiseline, slobodni masni alkoholi, aldehidi i alkil estri), kao i dva vitamina su identifikovani u CO2 ekstraktu lista V. album. Takođe, analizirana je i isparljiva frakcija, koja je sadržala nekoliko novih terpenskih molekula: seskviterpen trans-α-bergamoten, trans-β-farnezen, lakton loliolid i vomifoliol. Nije pronađena literatura koja potvrđuje prisustvo pomenutih jedinjenja u ispitivanom biljnom materijalu. Literatura: 1. Bussing A. 2000. Biological and pharmacological properties of Viscum album L. In Mistletoe – the genus Viscum, Bussing A (ed). Harwood Academic Publishers: Amsterdam, 123-182.

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P13 Phytochemical analysis of the non-polar Viscum album L. extract Ćebović T1, Popović M2, Lepojevic Z3, Mimica-Dukić N2 1 Biochemistry Department, School of Medicine, Hajduk Veljkova 3, 21000, Novi Sad, Serbia; 2Chemistry Department, Faculty of Sciences, Trg D. Obradovića 5, 21000, Novi Sad, Serbia; 3Faculty of Technology, Bul. cara Lazara 1, 21000, Novi Sad, Serbia As most of the studies on mistletoe (Viscum album L.) concern its polar extracts, rich in lectins and viscotoxins, it was decided to focus on the investigation of nonpolar constituents of this plant. Certain number of gases can be transferred to liquid state (only at temperatures below supercritical temperature) and act as solvents. Carbon dioxide, CO2, is considered the most advantageous solvent for extraction of bioactive molecules from the plant material. Liquid CO2 cooled below critical temperature (31.3ºC) exhibits high solvatation ability that does not much depend on the applied pressure. At temperatures higher than 31.3ºC and at pressures above 72 bars (supercritical state of CO2), solvatation ability of CO2 varies widely depending on the pressure and temperature ranges. Appropriate choice of pressure and temperature enables selective extraction of natural products, as well as fractionation of compounds. In this paper, we have tried to achieve selective extraction of several classes of compounds present in mistletoe leaves using supercritical CO2 extraction, and to identify them by coupling extraction with GC/MS analysis. We have observed that the best extraction was achieved at 350 bars and 34-35ºC. Several non-polar classes of compounds (C23-C30 hydrocarbons, free fatty acids, free fatty alcoholes, aldehydes and alkyl esters), as well as two vitamins were identified in the CO2 extract of V. album. Also, volatile fraction was analyzed, containing several new terpene molecules: sesquiterpene trans-α-bergamotene, trans-β-farnesene, lactone loliolide and vomifoliol. In the available literature, there are no data about presence of those compunds in the V. album.

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P14 Fenolna jedinjenja dve vrste Marrubium (M. peregrinum L. and M. vulgare) iz Vojvodine E. Jovin1, N. Mimica-Dukić1, A. Toth1, I. Beara1, K. Balog1, D. Orčić1

1 Departman za hemiju, Prirodno-matematički fakultet, Univerzitet u Novom Sadu, Trg D. Obradovića 3, 21000 Novi Sad, Srbija Među vrstama Marrubium samo je M. vulgare (očajnica) detaljno ispitana. Preparati očajnice koriste se kao ekspektoranti i tonici posebno protiv hroničnog kašlja i astme [1]. Četiri vrste Marubium su prisutne u flori Srbije [2], ali ne postoje podaci o njihovom hemijskom sastavu i biološkoj aktivnosti. Namera ovog istraživanja je bila da se uporede sastavi dve samonikle vrste Marrubium u Vojvodini - M. peregrinum, sakupljene sa četiri lokaliteta i M. vulgare sa Velebita, ispitujući hemijski sastav (pomoću HPLC-DAD and HPLC/MS/MS tehnike) njihovih metanolnih ekstrakata. U nameri da pojednostavimo tumačenje rezultata, razdvojili smo, na osnovu polarnosti, jedinjenja metanolnog ekstrakta u četiri grupe (tečno-tečna ekstrakcija hloroformom, etil-acetatom, 1-butanolom i vodom) [3]. Na osnovu HPLC-hromatograma, uočava se da etil acetatna frakcija M. peregrinum sadrži najviše aglikona, a da su flavoni/flavonolni glikozidi i fenilpropanoidi prisutni i u etil acetatnoj i u 1-butanolnoj frakciji (ali u različitim odnosima). Najzastuplenija jedinjenja su: kvercetin-3-O-β-D-glukozid, apigenin-7-O-glukozid, p-metoksi-cimetna kiselina, kempferol-3-O-β-D-glukozid, apigenin-7-O-(3``-p-kumaril)-glukozid, izoramnetin-3-O-β-D-rutinosid, kempferol, apigenin, kvercetin. Kod vrste M. vulgare 1-butanolna frakcija je najbogatija fenolnim jedinjenjima. Najzastupljenije komponente su: apigenin-7-O- glukozid, p-metoksi-cimetna kiselina, apigenin-7-O-(3``-p-kumaril)-glukozid, apigenin, kempferol-3-O-β-D-glukozid. Hloroformska i vodena frakcija imaju manji sadržaj fenolnih jedinjenja. Dve ispitane vrste razlikuju se značajno u kvalitativnom sastavu, pri čemu M. peregrinum ima veći sadržaj fenolnih jedinjenja, a time i potencijal za dalja biološka i farmakološka ispitivanja. References: 1. Tyler, Varro E. (1993) The Honest Herbal. Pharmaceutical Product Press, NY. 2. Diklić, N. (1974) Marrubium in Josifović (ed.) Flora SR Srbije, tom VI, SANU, Beograd. 3. Ribereau-Gayon, P. (1972), Plant Phenolics. Dunod Editeur, Paris.

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P14 Phenolic compounds in two Marrubium species (M. peregrinum L. and M. vulgare) in Vojvodina E. Jovin1, N. Mimica-Dukić1, A. Toth1, I. Beara1, K. Balog1, D. Orčić1

1Department of Chemistry, University of Novi Sad, Trg Dositeja Obradovica 3, 21000 Novi Sad, Serbia Among Marrubium species only M. vulgare (white horehound) has been extensively investigated. Preparations of horehound are mainly used as expectorants and tonics with considerable benefit for chronic cough and asthma [1]. Four Marubium species had been recognized in Serbian flora [2], but there are no available data on their chemistry and biological activity. The aim of this study was to compare two Marrubium species indigenously growing in Vojvodina - M. peregrinum, collected on four localities and M. vulgare from Velebit, by examining the chemical composition (HPLC-DAD and HPLC/MS/MS) of their methanol extracts. In order to simplify forthcoming results, we separated compounds maintained in methanol extracts into four groups on the basis of their polarity (by liquid-liquid extraction with chloroform, ethyl acetate, 1-butanol and water) [3]. Based on the HPLC-profiles, it is evident that the ethyl acetate fractions of M. peregrinum contain the majority of aglycones, but some flavone/flavonol glycosides and phenylpropanoids are present in both ethyl acetate and 1-butanol fractions (although in different ratios). The dominant compounds are: quercetin-3-O-β-D-glucoside, apigenin-7-O-glucoside, p-methoxy-cinnamic acid, kaempferol-3-O-β-D-glucoside, apigenin-7-O-(3``-p-coumaryl)-glucoside, isoramnetin-3-O-β-D-rutinoside, kaempferol, apigenin, quercetin. In M. vulgare species 1-butanol fraction is the most abundant in phenolic content. The dominant compounds are: apigenin-7-O-glucoside, p-methoxy-cinnamic acid, apigenin-7-O-(3``-p-coumaryl)-glucoside, apigenin, kaempferol-3-O-β-D-glucoside. Chloroform and water fractions contain less phenolic compounds. Between two examined species there is significant difference only in qualitative composition, representing M. peregrinum as more abundant in phenolic compounds, and thus more promising for the further bioclogcal and pharmacological screening. References: 1. Tyler, Varro E. (1993) The Honest Herbal. Pharmaceutical Product Press, NY. 2. Diklić, N. (1974) Marrubium in Josifović (ed.) Flora SR Srbije, tom VI, SANU, Beograd. 3. Ribereau-Gayon, P. (1972), Plant Phenolics. Dunod Editeur, Paris.

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P15 Antioksidantna svojstva lista soje Popović B1, Štajner D.1, Čanadanović-Brunet,J.21 Poljoprivredni fakultet, Trg Dositeja Obradovića 8, 21000 Novi Sad, Srbija 2 Tehnološki fakultet, Bulevar Cara Lazara 1, 21000 Novi Sad, Srbija

Poslednjih godina u svetu se intenzivno ispituju različti proizvodi od soje i njihov značaj u ishrani čoveka i u medicini. Upotreba soje u ishrani smanjuje rizik od pojave koronarnih bolesti, osteporoze i različitih vrsta karcinoma [1]. Pretpostavlja se da su nosioci biološke aktivnosti soje izoflavoni , genistein, daidzein i njihovi glikozidi [2]. Pored izoflavona, soja takodje sadrži mnoge lipo- i hidrosolubilne antioksidante [3]. Za razliku od zrna soje, antioksidantne aktivnosti lista soje još uvek nisu dobro ispitane. Stoga je cilj ovog rada bio da se ispita antioksidantna aktivnost različitih ekstrakata lista soje (H2O, n-BuOH, EtOAc, CHCl3 i Et2O ekstrakt) zbog njihove moguće primene u medicini i farmaciji. Antioksidantna aktivnost je u in vitro testovima odredjena pomoću DPPH i NO skevindžer metoda. Za odredjivanje ukupne antioksidantne aktivnosti primenjena je FRAP metoda. Takođe je ispitivana antioksidantna aktivnost (DPPH i NO skevindžer aktivnosti i ESR metoda) fosfatno puferskih (pH 7) ekstrakata lista pojedinih sorti soje. Ispitivana su četiri genotipa: Venera, Meli, Ana i Novosađanka. Svi spitivani ekstrakti lista soje su pokazali značajnu, koncentraciono zavisnu antioksidativnu aktivnost sa IC50=320-823 µg/ml u DPPH testu i IC50=210-508 µg/ml) u NO testu. Ukupna antioksidantna aktivnost određena FRAP metodom je takođe bila značajna kod svih uzoraka. Naša ispitivanja soje ukazuju na veliki antioksidantni potencijal lista, odnosno prisustvo komponenti sa visokim antiradikalskim potencijalom u listu ove biljke. Kod svih ispitivanih genotipova soje pomoću ESR metode utvrđeni su skevindžer efekti veći od 70%. Ipak, ustanovljene su i intraspecijske razlike između pojedinih genotipova. Najbolje antioksidantne karakteristike utvrđene su kod genotipa Ana sa najvećom DPPH skevindžer aktivnošću (59,7%), FRAP vrednošću (4,80 FRAP jedinica) i skevindžer efektom (95,97%) što preporučuje ovaj genotip kao prirodni izvor moćnih antioksidanata. Najveću NO skevindžer aktivnost pokazao je genotip Meli (66,3%). Mogućnost primene sojinog lista u proizvodnji hrane i za dobijanje farmaceutskih preparata trebala bi u budućnosti detaljnije da se ispita. Literatura: 1. Ho et al. (2002) Pharmacoter 56:289. 2. Lucas et al. (2001) J.Nutr 131:211. 3. Prakash et al. (2006) Food Chem 104:783.

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P15 Antioxidant properties of soy leaf Popović B1, Štajner D.1, Čanadanović-Brunet,J.21 Faculty of Agriculture, Trg Dositeja Obradovića 8, 21000 Novi Sad, Serbia 2 Faculty of Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia The health benefits of soybean products have been extensively investigated. Consumption of soybean reduces the risk of certain types of cancers and osteoporosis, exhibit antiatherosclerotic activity and decrease the risk of coronary diseases [1]. It is believed that major isoflavons in soybean, genistein, daidzein and their glycosides may be responsible for these medicinal benefits [2]. Beside isoflavones, soybean also contains many structurally different lipo- and hidrosoluble antioxidants [3]. In contrast to soybean, antioxidant and free radical scavenger investigation of soy leaf are very rare. Therefore, the present study was conducted to evaluate antioxidant potential and free radical-scavenging activity of soy leaf extracts (H2O, n-BuOH, EtOAc, CHCl3 and Et2O) for their possible utilization in foods or pharmaceutical supplements. The in vitro antioxidant activity of the plant extract was determined by DPPH and NO scavenging activity methods. For total antioxidant capacity determination, FRAP method was used. Antioxidant potential and free radical-scavenging activity (by DPPH assay, NO radical inhibition and ESR) of phosphate buffer (pH 7) leaf extracts in some varieties of soy were also investigated. Four genotypes of soy were investigated: Venera, Meli, Ana and Novosadjanka. In the present study, all investigated soy leaf extracts showed remarkable and concentration-dependent free radical scavenging activities. According to the results obtained by DPPH method, the extracts showed high scavenger abilities (IC50=320-823 µg/ml). NO scavenger abilities of the extracts were also high (IC50=210-508 µg/ml). Total antioxidant capacity determined by FRAP method approved that all soy leaf extracts possessed significant antioxidant potential. Our investigations showed that leaf extracts of different soy genotypes possesses antioxidant and free radical scavenging activity. All genotypes showed scavenger effects, higher than 70%. This report also suggests intraspecific antioxidant differences between investigated soy genotypes. The best antioxidant characteristics expressed genotype Ana with the highest DPPH radical scavenger capacity (59.7%), ferric reducing antioxidant power (4.80 FRAP units) and scavenger effect (95.97%) what recommended this genotype as a source of potent natural antioxidants. The highest NO radical inhibition showed genotype Meli (66.3%). The possibility to use soy leaf in food and pharmaceuticals preparation should be further explored. References: 1. Ho et al. (2002) Pharmacoter 56:289. 2. Lucas et al. (2001) J.Nutr 131:211. 3. Prakash et al. (2006) Food Chem 104:783.

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P16 Sadržaj polifenola i antioksidativna aktivnost u različitim genotipovima sojinog semena Jelena Cvejić1, Vesna Tepavčević1, Đorđe Malenčić2, Jegor Miladinović3 1 Odsek za farmaciju, Medicinski fakultet, Univerzitet u Novom Sadu, Hajduk Veljkova 3, Novi Sad, 2100 , Srbija; 2 Poljoprivredni fakultet, Univerzitet u Novom Sadu, Trg Dositeja Obradovića 8, Novi Sad,21000, Srbija 3 Institut za ratarstvo i povrtarstvo, Maksima Gorkog 30, Novi Sad, 21000, Srbija Produkti na bazi ekstrakta sojinog semena imaju povoljne efekte na zdravlje, što se objašnjava prisustvom izoflavona i drugih polifenolnih komponeneti. Cilj ovog rada je kvantifikacija sadržaja izoflavona, flavonoida i ukupnih polifenola i ispitivanje antioksidativne aktivnosti sojinog semena u cilju uspostavljenja kriterijuma za selekciju genotipova sa najpovoljnijim hemijskim sastavom. Davadeset različitih genotipova soje upotrebljenih u ovom radu su uzgajani na eksperimentalnim poljima Instituta za ratarstvo i povrtarstvo u Novom Sadu. Nakon ekstrakcije smešom metanol-voda (8/2, v/v), sadržaj izoflavona je određen pomoću C18 reverzno fazne hromatografije [1]. Sadržaj ukupnih polifenola i flavonoida je određen spektrofotometrijski [2]. Antioksidativna aktivnost sojinih ekstrakata je testirana u odnosu na stabilni 2,2-diphenil-1-picril-hidrazil (DPPH) radikal [3]. Rezultati su pokazali statistički značajnu korelaciju između sadržaja ukupnih polifenola i antioksidativne aktivnosti sojinih ekstrakata (P<0.05). Genotipovi soje sa visokim sadržajem ukupnih polifenola su imali visoku antioksidativnu aktivnost, i obrnuto. Korelacija između sadržaja ukupnih izoflavona i antioksidativne aktivnosti nije ustanovljena, kao ni između sadržaja flavonoida i antioksidativne aktivnosti. Potrebno je ispitati antioksidativni potencijal individualnih izoflavona i flavonoidnih komponenti da bi se ustanovilo tačno poreklo antioksidativne aktivnosti u soji. Dobijeni rezultati pokazuju da je pored sadržaja izoflavona, za selekciju genotipova potrebno uzeti u obzir i sadržaj ukupnih polifenola, kao markera antioksidativne aktivnosti u soji. Zahvalnost: Ministarstvo nauke Republike Srbije Reference: 1. Lee, J., Renita, M. et al. (2004) J Agric Food Chem 52(9):2647-2651. 2. Marckam, K. R. (1989) Methods in Plant Biochemistry. Academic Press. London. 3. Abe, N., Murata, T. et al. (1998) Biosci Biotech Biochem 62:661-662.

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P16 Polyphenolic content and antioxidant activity in different genotypes of soybean Jelena Cvejić1, Vesna Tepavčević1, Đorđe Malenčić2, Jegor Miladinović3 1 Department of Pharmacy, Faculty of Medicine, University of Novi Sad, Hajduk Veljkova 3, Novi Sad, 2100 , Serbia; 2 Faculty of Agriculture, University of Novi Sad, Trg Dositeja Obradovića 8, Novi Sad,21000, Serbia 3 Institute of Field and Vegetable Crops, Maksima Gorkog 30, Novi Sad, 21000, Serbia The beneficial health effects of products based on soybean extracts may be due to the presence of isoflavones and other polyphenolic compounds. The aim of this assay was to quantify the content of isoflavones, flavonoids and total polyphenolics and to investigate antioxidant activity of soybean seeds in order to establish a selection criterion for seeds with favorable chemical composition. Twenty different soybean genotypes used in this assay were cultivated on experimental fields at the Institute of Field and Vegetable Crops, Novi Sad. After extraction with methanol-water (8/2, v/v), isoflavone content was determined by C18 reversed phase high-performance liquid chromatography coupled with photodiode array detector [1]. Polyphenolic and flavonoid contents were determined spectrophotometrically [2]. Antioxidant activities of soybean extracts were tested in reaction with stable 2,2-diphenyl-1-pycril-hydrazil (DPPH) radical [3]. Correlation between total polyphenolic content and antioxidant activity of soybean extracts was found to be statistically significant (P<0.05). The highest contents of total polyphenols were found in cultivars which also displayed the highest total antioxidant activity. Conversely, genotypes poor in polyphenol compounds showed low levels of DPPH-radical scavenging activity. No such correlation was found between content of isoflavones and antioxidant activity or content of flavonoids and antioxidant activity. It would be necessary to analyze antioxidant potential of individual isoflavone and flavonoid compounds in order to investigate the origin of antioxidant activity in soybeans. The results suggested that beside isoflavone content, total polyphenol content should be considered an important characteristic of soybean seeds and a useful marker of antioxidant activity in soybeans. Acknowledgements (italic): Ministry of Science, Republic of Serbia References: 1. Lee, J., Renita, M. et al. (2004) J Agric Food Chem 52(9):2647-2651. 2. Marckam, K. R. (1989) Methods in Plant Biochemistry. Academic Press. London. 3. Abe, N., Murata, T. et al. (1998) Biosci Biotech Biochem 62:661-662.

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P17 Određivanje izoflavona u fitopreparatima i semenu soje

Mira Stojanović1, Jelena Kukić2, Mirjana Simić2, Ljubinka Lemajić1, Nada Kovačević2

1Apoteka „Beograd“, Bojanska 16/IV,11000 Beograd, Srbija; 2Institut za farmakognoziju, Farmaceutski fakultet Univerziteta u Beogradu, Vojvode Stepe 450, 11221 Beograd

Seme soje [Glycine max (L.) Merr., Fabaceae] sadrži fosfolipide, proteine, fitosterole i izoflavone. Glavni izoflavonski heterozidi soje su genistin i daidzin. Dokazani su različiti mehanizmi delovanja izoflavona: vezivanje za estrogene receptore, inhibicija fosforilacije tirozina, indukcija diferencijacije i inhibicije DNK topoizomeraze, inhibicija pojedinih faza ćelijskog ciklusa, indukcija apoptoze, inhibicija angiogeneze i antioksidatna aktivnost [1]. Ispitivani fitopreparat se zahvaljujući estrogenom delovanju izoflavona primenjuje za ublažavanje menopauzalnih tegoba. Izvršena je identifikacija izoflavona i određen je njihov sadržaj u fitopreparatu i semenu soje, pomoću HPLC metode. Korišćeni su komercijalni uzorci: kapsule koje sadrže 133 mg ekstrakta semena soje standardizovanog na 40 mg sojinih izoflavona i sirovo seme biološki uzgajane soje. Za ekstrakciju izoflavona iz uzoraka primenjena je modifikovana AOAC metoda za određivanje izoflavona u proizvodima od soje [2, 3]. HPLC analiza uzoraka je urađena na aparatu Agilent 1100, korišćenjem kolone Zorbax Eclipse XDB-C18 (4,6×250 mm; 5 µm), sa DAD detektorom. Mobilna faza se sastojala iz faze: A) H2O/H3PO4, pH=2,75 i B) 10% A u acetonitrilu. Razdvajanje komponenata u uzorcima vršeno je pri protoku mobilne faze od 0,8 ml/min, uz korišćenje gradijenta, pri temperaturi 25ºC. Izoflavoni su identifikovani poređenjem UV spektara i retencionih vremena njihovih pikova sa pikovima standardnih supstanci: daidzina, daidzeina, genistina i genisteina (λ=254 nm) [4]. Pripremljeni su rastvori standardnih supstanci daidzeina i genisteina (0,02-0,2 mg/ml), konstriusane odgovarajuće kalibracione krive, pomoću kojih je izračunata količina izoflavonskih aglikona i preračunat sadržaj odgovarajučih izoflavona u ispitivanim uzorcima.U ekstraktima fitopreparata i semena soje dokazano je prisustvo daidzina, genistina i njihovih aglikona. U cilju daljeg ispitivanja, izvršena je kisela hidroliza dobijenih ekstrakata fitopreparata i semena soje korišćenjem 6 M HCl tokom različitih vremenskih intervala zagrevanja (3, 6, 9, 12 i 15 sati). Hidrolizom dolazi do razgradnje izoflavonskih heterozida daidzina i genistina (Rt=9,475 i Rt=12,649 min) na odgovarajuće aglikone daidzein i genistein i povećanja njihovih pikova (Rt=25,515 i Rt=29,216 min). U hidrolizatu semena soje posle 15 sati nema značajnijeg porasta koncentracije aglikona u odnosu na njihove koncentracije u hidrolizatu posle 12 sati. Nasuprot tome, u hidrolizatu fitopreparata posle 15 sati uočeno je smanjenje njihove koncentracije u odnosu na hidrolizat posle 12 sati. Period od 12 sati zagrevanja je optimalan period za hidrolizu izoflavonskih heterozida soje (daidzina i genistina). Rezultati HPLC analize fitopreparata pokazuju da je ukupan sadržaj izoflavona 20,481 mg u 1 kapsuli, što ne odgovara deklarisanom sastavu. Razlog ovog odstupanja može biti nedovoljna količina ekstrakta semena soje u preparatu ili nezadovoljavajući kvalitet ekstrakta semena soje. Ispitivani uzorak semena soje sadrži 45,307 mg daidzeina i 49,709 mg genisteina (računato na 100 g semena soje), tj. ispitivani uzorak sadrži zadovoljavajuću količinu daidzeina, dok je količina genisteina značajno manja [5].

Literatura: 1. PDR for Herbal Medicines (2004) Thompson Medical Economics, Montvale. 2. Delmonte P. et al. (2006) Journal of Chromatography A 1107(1-2):59-69, 3. AOAC International (2001) Official Methods of Analysis of AOAC International. 17th ed., Method 2001.10. The Association, Gaithersburg, MD. 4. Mabry T. J. et al. (1971) The Systematic Identification of Flavonoids. Thomas M. B. Springer-Verlag. 5. http://www.isoflavones.info/isoflavones-content.php 19.12.2007.

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P17 Determination of isoflavones in herbal products and soybean Mira Stojanović1, Jelena Kukić2, Mirjana Simić2, Ljubinka Lemajić1, Nada Kovačević2

1Belgrade Pharmacy, Bojanska 16/IV, 11000 Belgrade, Serbia; 2Institute of Pharmacognosy, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11221 Belgrade, Serbia Soybean [Glycine max (L.) Merr., Fabaceae] contains phospholipids, proteins, phytosterols and isoflavones. Main soybean isoflavones are genistein and daidzein. Isoflavones exhibit various mechanisms of activity: binding to estrogen receptors, inhibition of protein tyrosine phosphorylation, induction of differentiation and inhibition of DNA topoisomerases, inhibition of specific cell cycle events, induction of apoptosis, inhibition of angiogenesis and antioxidant activity [1]. Investigated herbal product is used for treatment of menopausal complaints, due to its estrogenic activity. HPLC method was used for identification and quantification of isoflavones in herbal product and soybean. Commercial samples were: capsules, containing 133 mg of soybean extract standardised on 40 mg of soy isoflavones, and seed of organic Soya. Modified AOAC method was applied for extraction of isoflavones from samples [2, 3]. HPLC analysis was performed on Agilent 1100, using Zorbax Eclipse XDB-C18 column (4.6×250 mm; 5 µm), with DAD detector. Samples were gradiently eluted with two phase system (A=H2O/H3PO4, pH=2.75; B=10% A in acetonitrile), flow rate of 0.8 ml/min, at 25ºC. Identification of isoflavones was performed by comparison of their UV spectra and peak retention times with standards: daidzin, daidzein, genistin and genistein (λ=254 nm) [4]. Calibration curves were obtained using standard solutions of daidzein and genistein (0.02-0.2 mg/ml), quantity of isoflavone aglycones was determined and amount of isoflavones in samples was calculated. In herbal product and soybean extracts the presence of daidzin, genistin and their aglycones were confirmed. For further analysis, acid hydrolysis was performed using 6 M HCl during different periods of heating (3, 6, 9, 12, 15 h). Isoflavon glycosides daidzin and genistin (Rt=9.475 and Rt=12.649 min) were hydrolysed to corresponding aglycones, which resulted in their peak enlargement (Rt=25.515 and Rt=29.216 min). In soy seed hydrolisate there was no significant increase of aglycone concentration after 15 hours of heating, compared to aglycone concentration after 12 h of heating. On the contrary, in herbal product hidrolisate, after 15 h of heating, decrease in aglycone concentration was detected, compared to that after 12 h. Period of 12 h of heating is optimal for soy isoflavones hydrolysis (daidzin and genistin). HPLC analysis of herbal product showed that total isoflavones content was 20.481 mg per capsule, which was not in correlation with declared content. The reason for this may be lower amount of soy seed extract in the product, or inappropriate quality of extract. Analysed sample of soy seed contained 45.307 mg of daidzein and 49.709 mg of genistein (per 100 g of soy seed), which means that investigated sample contained appropriate amount of daidzein, while amount of genistein is significantly lower [5]. References: 1. PDR for Herbal Medicines (2004) Thompson Medical Economics, Montvale. 2. Delmonte P. et al. (2006) Journal of Chromatography A 1107(1-2):59-69, 3. AOAC International (2001) Official Methods of Analysis of AOAC International. 17th ed., Method 2001.10. The Association, Gaithersburg, MD. 4. Mabry T. J. et al. (1971) The Systematic Identification of Flavonoids. Thomas M. B. Springer-Verlag. 5. http://www.isoflavones.info/isoflavones-content.php 19.12.2007.

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P18 Antimikrobna aktivnost ekstrakata Geranium robertianum i G. phaeum Mirjana Simić1, Dragana Vučićević2, Marina Milenković2, Nada Kovačević1

1 Institut za farmakognoziju, Farmaceutski fakultet, V. Stepe 450, 11221 Beograd, 2 Institut za mikrobiologiju i imunologiju, Farmaceutski fakultet, V. Stepe 450, 11221 Beograd Ispitano je antimikrobno delovanja nadzemnih i podzemnih delova vrsta Geranium robertianum L. i Geranium phaeum L. (Geraniaceae). Osušen i usitnjen biljni materijal ekstrahovan je metanolom (70%), postupkom maceracije i ekstrakti upareni pod sniženim pritiskom. Sadržaj ukupnih polifenola i tanina određen je Folin-Cicalteu metodom [1]. Preliminarno ispitivanje antioksidantnog delovanja izvršeno je određivanjem sposobnosti neutralizacije DPPH radikala [2]. Antimikrobna aktivnost je ispitana agar difuzionim metodom [3] i bujon dilucionim metodom [4] od 1,56-50 µg/ml na MH agaru/bujonu za bakterije, ili Sabouraud agaru/bujonu za gljivice. Testirano je sedam standardnih sojeva bakterija i dva standardna soja gljivice. Antimikrobni efekat ispitivanih ekstrakta je upoređen sa aktivnošću antibiotika, koji su korišćeni kao pozitivne kontrole. U metanolnom ekstraktu herbe i metanolnom ekstraktu rizoma G. robertianum sadržaj polifenola nije se značajno razlikovao (18,3-18,7% ukupnih polifenola i 12,4-12,8% tanina), dok je u metanolnom ekstraktu rizoma G. phaeum određeno više ukupnih polifenola (23,9%) i tanina (10,1%) nego u ekstraktu herbe (10,5% ukupnih polifenola, 4,8% tanina). Ekstrakti herbe i rizoma G. robertianum pokazali su sličnu sposobnost neutralizacije DPPH radikala (IC50= 7,4 µg/ml). Antioksidantna aktivnost metanolnog ekstrakta rizoma G. phaeum je veća (IC50= 6,7 µg/ml) od delovanja metanolnog ekstrakta herbe (IC50= 17,5 µg/ml). Antimikrobna aktivnost ekstrakata herbe i rizoma G. robertianum i G. phaeum obuhvatala je Gram pozitivne bakterije: S. aureus, B. subtilis, M. luteus (prečnik zone inhibicije rasta 9-20,5 mm, MIC 1,56-12,5 µg/ml, MBC 6,25-25 µg/ml), Gram negativnu K. pneumoniae (prečnik zone inhibicije rasta 8-10 mm, MIC 12,5 µg/ml, MBC 25 µg/ml) i gljivicu C. albicans (prečnik zone inhibicije rasta 10-18 mm, MIC 12,5 µg/ml, MBC 12,5-25 µg/ml). Ekstrakt herbe G. phaeum je izraženo inhibirao S. aureus (MIC 1,56 µg/ml, MBC 12,5 µg/ml). Uočena je aktivnost ekstrakta rizoma G. phaeum na E. faecalis, Gram pozitivnu bakteriju, na koju ostali ekstrakti nisu delovali (prečnik zone inhibicije rasta 14,5 mm, MIC 12,5 µg/ml, MBC 12,5 µg/ml). Antimikrobna aktivnost, sadržaj polifenola, sposobnost neutralizacije DPPH radikala se kod ekstrakata herbe i rizoma jednogodišnje vrste G. robertianum nisu značajno razlikovale. U poređenju sa ekstraktom herbe, ekstrakt rizoma, višegodišnje vrste G. phaeum sadrži više ukupnih polifenola i tanina, izraženija je antioksidantna aktivnost, kao i inhibicija većeg broja mikroorganizama. Literatura: 1. Makkar HPS et al. (2000) Quantification of Tannins in Tree Foliage – A Laboratory Manual. FAO/IAEA. Vienna. 2. Bondet V. et al. (1997) Lebensm.-Wiss. u.-Technol. 30: 609-15. 3. Acar JF, Goldstein FW. (1996) Disc susceptibility test. In: Lorian V, editor. Antibiotics in Laboratory Medicine, 4th edition. Williams & Wilkins. Baltimore: 1-51. 4. Candan, F. et al. (2003) J Ethnopharmacol 87: 215.

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P18 Antimicrobial activity of Geranium robertianum and G. phaeum extracts Mirjana Simić1, Dragana Vučićević2, Marina Milenković2, Nada Kovačević1

1 Institute of Pharmacognosy, Faculty of Pharmacy, V. Stepe 450, 11221 Belgrade, Serbia; 2 Institute of Microbiology and Immunology, Faculty of Pharmacy, V. Stepe 450, 11221 Belgrade, Serbia Antimicrobial activity of aerial and subterranean parts of Geranium robertianum L. and Geranium phaeum L. (Geraniaceae) species was investigated. Dried and grounded plant material was extracted with methanol (70%), by a maceration process and extracts were dried under reduced pressure. Total phenolics and tannins contents were determined using Folin-Ciocalteu method [1]. Preliminary antioxidant activity was evaluated by DPPH radical scavenging activity [2]. Antimicrobial activity was assessed by agar diffusion method [3] and broth microdilution method [4] from 1.56-50 µg/ml on MH agar/broth (for bacteria) and Sabouraud agar/broth (for yeasts). Antibacterial and antifungal activity was tested against seven laboratory control strains of bacteria and two strains of yeast. Activity of extracts was compared to the effect of antibiotics, which were used as positive controls. Contents of phenolics in methanol extract of aerial parts and in extract of rhizome of G. robertianum were not significantly different (18.3-18.7% total phenolics and 12.4-12.8% tannins). In methanol extract of rhizome of G. phaeum higher contents of total phenolics (23.9%) and tannins (10.1%) than in aerial parts extract (10.5% total phenolics, 4.8% tannins) were determined. Both extracts of G. robertianum showed similar DPPH radical scavenging activity (IC50= 7.4 µg/ml). Antioxidant activity of G. phaeum rhizome extract (IC50= 6.7 µg/ml) was higher than G. phaeum aerial parts extract (IC50= 17.5 µg/ml). G. robertianum and G. phaeum extracts exerted antimicrobial activity against Gram positive bacteria: S. aureus, B. subtilis, M. luteus (diameters of zones of inhibition 9-20.5 mm, MIC 1.56-12.5 µg/ml, MBC 6.25-25 µg/ml), Gram negative K. pneumoniae (diameters of zones of inhibition 8-10 mm, MIC 12.5 µg/ml, MBC 25 µg/ml) and yeast C. albicans (diameters of zones of inhibition 10-18 mm, MIC 12.5 µg/ml, MBC 12.5-25 µg/ml). G. phaeum aerial parts extract pronouncedly inhibited bacteria S. aureus (MIC 1.56 µg/ml, MBC 12.5 µg/ml). Rhizome extract of G. phaeum inhibited the growth of E. faecalis, Gram positive bacteria; which was not affected by the other extracts (diameter of zone of inhibition 14.5 mm, MIC 12.5 µg/ml, MBC 12.5 µg/ml. Antimicrobial activity, phenolics content, DPPH radical scavenging activity were not significantly different for aerial parts or rhizome extracts of annual species G. robertianum. In the comparison with aerial parts extract, rhizome extract of perennial species G. phaeum contained higher content of total phenolics and tannins, antioxidant activity was more pronounced and wider range of microorganisms was inhibited. References: 1. Makkar HPS et al. (2000) Quantification of Tannins in Tree Foliage – A Laboratory Manual. FAO/IAEA. Vienna. 2. Bondet V. et al. (1997) Lebensm.-Wiss. u.-Technol. 30: 609-15. 3. Acar JF, Goldstein FW. (1996) Disc susceptibility test. In: Lorian V, editor. Antibiotics in Laboratory Medicine, 4th edition. Williams & Wilkins. Baltimore: 1-51. 4. Candan, F. et al. (2003) J Ethnopharmacol 87: 215.

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P19 Fenolski sastojci i antimikrobna aktivnost četiri endemične balkanske biljne vrste Tomić A1, Petrović S1, Kukić J1, Milenković M2, Niketić M3

1 Institut za farmakognoziju, Farmaceutski fakultet, V. Stepe 450, 11221 Beograd, Srbija; 2 Institut za mikrobiologiju i imunologiju, Farmaceutski fakultet, V. Stepe 450, 11221 Beograd, Srbija; 3 Prirodnjački muzej, Njegoševa 51, 11000 Beograd, Srbija U radu su ispitani fenolski sastojci i antimikrobna aktivnost ekstrakata četiri endemične balkanske biljne vrste: Cicerbita pancicii (Vis.) Beauverd (balkanska planinska endemična vrsta) i Leucanthemum illyricum Vogt & Greuter (dinarska endemična vrsta) iz familije Asteraceae i Grafia golaka (Hacq.) Reichenb. (apeninsko balkanska planinska vrsta) i Pancicia serbica Vis. (endemična planinska vrsta jugoistočnih Dinarida i Šar-planine) iz familije Apiaceae. Nadzemni delovi u cvetu C. pancicii i G. golaka sakupljeni su na planini Maglić, nadzemni delovi u cvetu P. serbica na Jahorini (Bosna) i cvasti L. illyricum na Durmitoru u Crnoj Gori, tokom jula 2004. god. Na vazduhu osušen i fino usitnjen biljni materijal ekstrahovan je hloroformom (CHCl3), a potom metanolom (MeOH). Za analizu su korišćeni suvi MeOH ekstrakti. Ukupni antioksidantni potencijal i sposobnost inhibicije lipidne peroksidacije MeOH ekstrakata C. pancicii i L. illyricum [1] i sposobnosti neutralizacije DPPH radikala MeOH ekstrakata G. golaca i P. serbica [2] ispitani su ranije. U ovom radu, HPLC analizom uz upotrebu eksternih standarda i akviziciju spektara na 350 nm, u ekstraktima C. pancicii i L. illyricum identifikovani su hlorogenska kiselina, luteolin, luteolin 7-O-glukozid i detektovane dikafeoilhina kiseline. U ekstraktu L. illyricum utvrđeno je i prisustvo apigenina, njegovih heterozida i derivata. U ekstraktu G. golaka detektovani su hlorogenska kiselina i heterozidi kvercetina, a u ekstraktu P. serbica hlorogenska kiselina, izokvercitrin i heterozidi derivata kvercetina [3]. Antimikrobna aktivnost analiziranih ekstrakata ispitana je agar-difuzionom [4] i bujon-mikrodilucionom metodom (u opsegu koncentracija od 15-250 µg/ml) [5], prema Gram(+) bakterijama Staphylococcus aureus ATCC 25923, S. epidermidis ATCC 12228, Micrococcus luteus ATCC 9341, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633, Gram(–) bakterijama Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae NCIMB 9111, Escherichia coli ATCC 25922, i gljivici Candida albicans (ATCC 10259 i ATCC 24433). U bujon-mikrodilucionom testu ekstrakti L. illyricum i G. golaca pokazali su identičnu antimikrobnu aktivnost: minimalna inhibitorna koncentracija (MIC) za P. aeruginosa iznosila je 30 µg/ml, dok je rast ostalih mikroorganizama inhibiran i u najnižoj primenjenoj koncentraciji (15 µg/ml). Ekstrakt C. pancicii razlikovao se od prethodnih samo po tome što je MIC vrednost za E. coli iznosila 30 µg/ml. Ekstrakt P. serbica pokazao je nešto slabiju antimikrobnu aktivnost i prema E. faecalis, B. subtilis., K. pneumoniae i C. albicans (MIC = 30 µg/ml). Generalno se može zaključiti da su ispitivani ekstrakti pokazali međusobno sličnu i solidnu značajnu antimikrobnu aktivnost. Identifikovani fenolski sastojci mogu se smatrati odgovornim za pokazano antimikrobno delovanje, kao i za ranije utvrđenu antioksidantnu aktivnost. Literatura: 1. Tomić A. et al. (2005) 8. Simpozijum o flori jugoistočne Srbije i susednih regiona. Niš. Knjiga apstrakata, str. 87-88. 2. Tomić A. et al. (2005) 8. Simpozijum o flori jugoistočne Srbije i susednih regiona. Niš. Knjiga apstrakata, str. 79. 3. Mabry T.J. et al. (1970) The Systematic Identification of Flavonoids. Springer-Verlag Berlin, Heidelberg. 4. Acar J.F., Goldstein F.W. (1996) Disc Susceptibility Test. In: Lorian V., ed. Antibiotics in Laboratory Medicine, 4th edition. Williams & Wilkins. Baltimore. 5. National Committee for Clinical Laboratory Standards - NCCLS (2001). In: Pa Wayne, editor. Document M100-S11: Performance Standards for Antimicrobial Susceptibility Testing, 11th Information Supplement

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P19 Phenolic compounds and antimicrobial activity of four Balkan endemic plant species Tomić A1, Petrović S1, Kukić J1, Milenković M2, Niketić M3

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 Natural History Museum, Njegoševa 51, 11000 Belgrade, Serbia In this study phenolic compounds and antimicrobial activity of extracts of four endemic Balkan plant species were examined: Cicerbita pancicii (Vis.) Beauverd (Balkan endemic plant) and Leucanthemum illyricum Vogt & Greuter (Dinaric endemic plant) from Asteraceae family, and Grafia golaka (Hacq.) Reichenb. (Apennine-Balkan mountain species) and Pancicia serbica Vis. (endemic mountain species of south-eastern Dinarides and Mt. Šara) from Apiaceae family. Aerial blooming parts of C. pancicii and G. golaka were collected on Mt. Maglić, aerial blooming parts of P. serbica on Mt. Jahorina (Bosnia), umbel of L. illyricum on Mt. Durmitor (Montenegro), in july 2004. Air-dried and well powdered plant material was extracted with chloroform (CHCl3), and then with methanol (MeOH). Dry MeOH extracts were used for analyses. Total antioxidant capacity and inhibition of lipid peroxidation of C. pancicii and L. illyricum MeOH extracts [1], and scavenging activity against DPPH radical of G. golaca and P. serbica MeOH extracts [2] were previously examined. In this study, using HPLC analyses with external standards and activation of spectra at 350 nm, in C. pancicii and L. illyricum extracts chlorogenic acid, luteolin, luteolin-7-O-glucoside were identified, and dicaffeoilquinic acids were detected. In L. illyricum extract presence of apigenin, its glycosides and derivates was estimated as well. In G. golaka extract chlorogenic acid and quercetin glycosides were detected, while in P. serbica extract chlorogenic acid, isoquercitrin and glycosides of quercetin derivates were detected [3]. Antimicrobial activity of analyzed extracts was investigated using agar-diffusion [4] and broth-microdilution method (in concentration range from 15-250 µg/ml) [5], against Gram(+) bacteria Staphylococcus aureus ATCC 25923, S. epidermidis ATCC 12228, Micrococcus luteus ATCC 9341, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633, Gram(–) bacteria Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae NCIMB 9111, Escherichia coli ATCC 25922, and yeast Candida albicans (ATCC 10259 and ATCC 24433). In broth-microdilution assay L. illyricum and G. golaca extracts exhibited identical antimicrobial activity: minimal inhibitory concentration (MIC) for P. aeruginosa was 30 µg/ml, while growth of other microorganisms was inhibited in the lowest concentration applied (15 µg/ml). C. pancicii extract differed from previous only in MIC value for E. coli which was 30 µg/ml. P. serbica extract showed slightly weaker antimicrobial activity against E. faecalis, B. subtilis., K. pneumoniae and C. albicans as well (MIC = 30 µg/ml). Generally it could be concluded that examined extracts exhibited similar and solid antimicrobial activity. Identified phenolic compounds could be regarded as responsible for exhibited antimicrobial activity, as well as for previously estimated antioxidant effects. References: 1. Tomić A. et al. (2005) 8th Symposium on the Flora of Southeastern Serbia and Neighbouring Regions. Niš. Book of Abstracts. pp. 87-88. 2. Tomić A. et al. (2005) 8th Symposium on the Flora of Southeastern Serbia and Neighbouring Regions. Niš. Book of Abstracts, p. 79. 3. Mabry T.J. et al. (1970) The Systematic Identification of Flavonoids. Springer-Verlag Berlin, Heidelberg. 4. Acar J.F., Goldstein F.W. (1996) Disc Susceptibility Test. In: Lorian V., ed. Antibiotics in Laboratory Medicine, 4th edition. Williams & Wilkins. Baltimore. 5. National Committee for Clinical Laboratory Standards - NCCLS (2001). In: Pa Wayne, editor. Document M100-S11: Performance Standards for Antimicrobial Susceptibility Testing, 11th Information Supplement.

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P20 Hemijski sastav i antimikrobna aktivnost etarskog ulja Stachys atherocalyx K. Koch Pavlović M1, Petrović S1 , Milenković M2, Kukić J1, Couladis M3, Tzakou O3, Niketić M4 1 Institut za farmakognoziju, Farmaceutski fakultet, V. Stepe 450, 11221 Beograd, Srbija 2 Institut za mikrobiologiju i imunologiju, Farmaceutski fakultet, V. Stepe 450, 11221 Beograd, Srbija 3 Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, Panepistimiopolis, 15771 Athens, Greece 4 Prirodnjački muzej, Njegoševa 51, 11000 Beograd, Srbija Rod Stachys L. (Labiatae) obuhvata oko 300 vrsta od kojih je 17 zastupljeno u flori Srbije i Crne Gore [1]. U tradicionalnoj medicini vrste roda Stachys se koriste za lečenje glavobolje, neuralgija, kao tonici, antidijaroici, kod dispepsije i za lečenje rana i upala kože [2]. S. atherocalyx K. Koch je višegodišnja, zeljasta biljka rasprostranjena u centralnom i istočnom delu Balkanskog poluostrva (Albanija, Grčka, Makedonija, Srbija, Bugarska i Rumunija), a ima je i u Anatoliji i kavkaskom regionu. Pripada taksonomski komplikovanoj grupi S. recta i veoma je slična vrsti S. recta L. [3]. U ovom radu predstavljeni su rezultati kvalitativne i kvantitativne analize i antimikrobne aktivnosti etarskog ulja herbe S. atherocalyx, koje do sada nije proučavano. Nadzemni delovi u cvetu prikupljeni su u okolini Prizrena, jula 2007. god. Etarsko ulje je izolovano destilacijom vodenom parom i analizirano GC i GC/MS tehnikama. Sadržaj etarskog ulja iznosi 0,04%, m/m, računato na osušeni materijal. Najveći deo ulja čine seskviterpenski ugljovodonici (36,1%) i neterpenska jedinjenja (35,4%). Monoterpenska jedinjenja zastupljena su u maloj količini (5,5%). Glavne komponente ulja su: (E)-kariofilen (19,9%), 1-okten-3-ol (19,5%), fitol (9,2%) i germakren D (8,2%). Antimikrobna aktivnost etarskog ulja ispitivana je mikrodilucionom metodom [4] prema Gram(+) bakterijama: Staphylococcus aureus ATCC 25923, S. epidermidis ATCC 12228, Micrococcus luteus ATCC 9341, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633, Gram(–) bakterijama: Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae NCIMB 9111, Escherichia coli ATCC 25922 i gljivici Candida albicans (ATCC 10259 i ATCC 24433). Najveću bakteriostatsku i baktericidnu aktivnost etarsko ulje herbe S. atherocalyx ispoljilo je prema B. subtillis (MIC 12, 5 µg/ml i MBC 100 µg/ml), a zatim prema E. faecalis (MIC 50 µg/ml i MBC 100 µg/ml). Analizirano ulje pokazalo je i značajnu antifungalnu aktivnost: minimalna inhibitorna koncentracija (MIC) za oba soja C. albicans iznosila je 50 µg/ml, dok je minimalna baktericidna koncentracija (MBC) za oba soja bila 100 µg/ml. Literatura: 1. Petrović S. et al. (2006) Flavour Fragr J 21: 250-252. 2. Kukić J. et al. (2007) Pharm Biol 45: 560-563. 3. Batisberger M. et al. (2006) Willdenowia 36: 205-216. 4. Candan, F. et al. (2003) J Ethnopharmacol 87:215-220.

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P20 Chemical composition and antimicrobial activity of Stachys atherocalyx K. Koch essential oil Pavlović M1, Petrović S1 , Milenković M2, Kukić J1, Couladis M3, Tzakou O3, Niketić M4 1 Institute of Pharmacognosy, Faculty of Pharmacy, V. Stepe 450, 11221 Belgrade, Serbia 2 Institute of Microbiology and Immunology, Faculty of Pharmacy, V. Stepe 450, 11221 Belgrade, Serbia3 Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, Panepistimiopolis, 15771 Athens, Greece 4 Natural History Museum, Njegoševa 51, 11000 Belgrade, Serbia The genus Stachys L. (Labiatae) comprises about 300 species, 17 of which are found in flora of Serbia and Montenegro [1]. Some of Stachys species are used in traditional medicine against headache, neuralgia, dyspepsia, as tonic, antidiarrheal drugs and wound healing agents [2]. S. atherocalyx K. Koch is a perennial plant widespread in the C. & E. Balkans (Albania, Greece, FYROM, Serbia, Bulgaria, Romania) and also occurs in Anatolia and Caucasus region. It belongs to the taxonomically complicated group of S. recta L., and it is very similar to the species S. recta L. [3]. In this paper we report the results of qualitative and quantitative analysis of essential oil as well as the results of antimicrobial activity of S. atherocalyx essential oil, which was not the subject of previous studies. The flowering aerial parts were collected nearby Prizren in July 2007. Essential oil was isolated by hydrodistillation and analyzed by GC and GC/MS. The oil yield was 0.04% (w/w), calculated on dry weight basis. Sesquiterpene hydrocarbons (36.1%) and nonterpenoid compounds (35.4%) were the main classes of constituents. Monoterpenes were present in low amount (5.5%). The main constituents of the oil were: (E)-caryophyllene (19.9%), 1-octen-3-ol (19.5%), phytol (9.2%) and germacrene D (8.2%). Antimicrobial activity of the oil was tested using the microdilution method [4] against Gram (+) bacteria Staphylococcus aureus ATCC 25923, S. epidermidis ATCC 12228, Micrococcus luteus ATCC 9341, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633, Gram(–) bacteria Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae NCIMB 9111, Escherichia coli ATCC 25922 and a yeast Candida albicans (ATCC 10259 and 24433). The best antimicrobial activity was detected against B. subtillis (MIC 12. 5 µg/ml and MBC 100 µg/ml), followed by the effect against E. faecalis (MIC 50 µg/ml and MBC 100 µg/ml). The oil exhibited significant antifungal activity: minimal inhibitory concentration (MIC) against both strains of C. albicans was 50 µg/ml, whereas minimal bactericidal concentration (MBC) was 100 µg/ml. Literatura: 1. Petrović S. et al. (2006) Flavour Fragr J 21: 250-252. 2. Kukić J. et al. (2007) Pharm Biol 45: 560-563. 3. Batisberger M. et al. (2006) Willdenowia 36: 205-216. 4. Candan, F. et al. (2003) J Ethnopharmacol 87:215-220.

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P21 Fitohemijski skrining i antioksidantna svojstva vrste Euphorbia nicaeensis All.

Đ. Malenčić1, P. Boža2, J. Luković2, L. Zorić2 i B. Kiprovski2

1 Poljoprivredni fakultet, Trg Dositeja Obradovića 8, 21000 Novi Sad, Srbija 2 Institut za biologiju, Prirodno – matematički fakultet, Trg Dositeja Obradovića 1, 21000 Novi Sad, Srbija Cilj rada bio je fitohemijski skrining i određivanje antioksidantnih svojstava lista i cvasti samonikle vrste Euphorbia nicaeensis All., sa dva lokaliteta u Vojvodini (Zobnatica i Rimski Šanac). Fitohemijskim skriningom ustanovljeno je dominantno prisustvo fenolnih jedinjenja, pri čemu je pozitivna hemijska reakcija na prisustvo flavonoida u listu biljaka sa prvog lokaliteta bila naročito izražena. Za oba ispitivana organa, sadržaj flavonoida bio je vrlo visok i kretao se od 0,57-0,84 g rutina 100 g-1 s.m. Visok sadržaj superoksid-radikala (O2.-) i hidroksil-radikala (.OH) ukazuju da su biljke ispitivane vrste bile izložene oksidativnom stresu, ali utvrđen sadržaj malonil-dialdehida (91,5-179,2 nmol MDA g-1 sv.m.), visok sadržaj ukupnih fenola (23,1-30,3 mg katehina g-1 s.m.), kao i aktivnost antioksidantnog enzima, superoksid dismutaze (SOD; 1345-1591 U g-1 sv.m.) ukazuje na efikasan antioksidantni sistem zaštite. Aktivnost uklanjanja DPPH-radikala, kao mera ne-enzimske antioksidantne aktivnosti 70% acetonskog ekstrakta ispitivane vrste, bila je iznad 50%, što kategorizuje datu vrstu u srednje aktivne.1 Literatura: 1. Lee S., Mbwambo Z., Chung H., Luyengi L., Gamez E., Mehta R., Kinghorn A., Pezzuto J. (1998): Evaluation of the antioxidant potential of natural products., Combinatorial Chem. & High Throughoutput Screening, 1, 35.

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P21 Phytochemical screening and antioxidant characteristics of Euphorbia nicaeensis All.

Dj. Malenčić1, P. Boža2, J. Luković2, L. Zorić2 i B. Kiprovski2

1 Faculty of Agriculture, Trg Dositeja Obradovića 8, 21000 Novi Sad, Serbia 2 Institute of biology, Faculty of Science, Trg Dositeja Obradovića 1, 21000 Novi Sad, Serbia In this study, phytochemical screening and investigation of antioxidant characteristics of leaves and cyatia of wild growing plant Euphorbia nicaeensis All., from two localites of Vojvodina (Zobnatica and Rimski Sanac), have been conducted. Phytochemical screening showed dominant presence of phenolic compounds with positive chemical reaction on flavonoids presence. Their presence was especially expressed in leaves of plants from the first locality but in both examinated organs the determined amount of total flavonoids was very high (0.57-0.84 g rutin 100 g-1 dry matter). High quantities of superoxide (O2.-) and hydroxyl (.OH) radicals shows that investigated plants were exposed to oxidative stress, but determined quantities of malonyl-dialdehyde (91.5-179.2 nmol MDA g-1 fresh weight), high quantities of total phenols (23.1-30.3 mg catechine g-1 dry matter), as well as activity of antioxidant enzyme, superoxide dismutase (SOD; 1345-1591 U g-1 fresh weight), shows on effective antioxidant ability. DPPH-radical scavenging activity as a measure of non-enzymatic antioxidant activity of 70% acetone extracts was higher than 50%, which categorize this extract as moderately active.1 References: 1. Lee S., Mbwambo Z., Chung H., Luyengi L., Gamez E., Mehta R., Kinghorn A., Pezzuto J. (1998): Evaluation of the antioxidant potential of natural products., Combinatorial Chem. & High Throughoutput Screening, 1, 35.

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P22 Fitohemijski skrining i antioksidantna svojstva vrste Centaurea sadleriana Janka

Đ. Malenčić1, M. Popović1, P. Boža2, J. Luković2 i M. Kodranov2

1 Poljoprivredni fakultet, Trg Dositeja Obradovića 8, 21000 Novi Sad, Srbija 2 Institut za Biologiju, Prirodno – matematički fakultet, Trg Dositeja Obradovića 1, 21000 Novi Sad, Srbija U radu su analizirani list i cvast dve populacije samonikle vrste Centaurea sadleriana Janka, sa dva različita lokaliteta u Vojvodini (Zobnatica i Rimski Šanac). Urađen je fitohemijski skrining radi kvalitativnog određivanja sekundarnih biomolekula u populacijama sa različitih tipova staništa i analizirana su antioksidantna svojstva (sadržaj kiseoničnih radikala i antioksidantni sistem zaštite). Fitohemijska analiza biljnog materijala ukazala je na dominantno prisustvo antocijanina i leukoantocijanina u cvastima, zatim flavonoida i tanina u oba biljna organa, slobodnih hinonskih derivata, alkaloida i saponina u listovima. Ispitivani kiseonični radikali (O2.- i .OH) detektovani su u velikoj količini što ukazuje da su biljke bile izložene oksidativnom stresu. Dobijene visoke vrednosti za aktivnost antioksidantnog enzima SOD, lipidnu peroksidaciju i količinu redukovanog glutationa ukazuju na efikasan antioksidantni zaštitni sistem. Aktivnost uklanjanja DPPH-radikala, kao mera ne-enzimske antioksidantne aktivnosti, bila je iznad 50%, što kategorizuje datu vrstu u srednje aktivne.1

Literatura: 1. Lee i sar. (1998): Evaluation of the antioxidant potential of natural products, Combinatorial Chem. & High Throughoutput Screening, 1, 35.

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P22 Phytochemical screening and antioxidant properties of Centaurea sadleriana Janka

Đ. Malenčić1, M. Popović1, P. Boža2, J. Luković2,i M. Kodranov2

1 Faculty of Agriculture, Trg Dositeja Obradovića 8, 21000 Novi Sad, Serbia 2 Institute for Biology, Faculty of Science, Trg Dositeja Obradovića 1, 21000 Novi Sad, Serbia In this study, leaves and petioles of two population of wild growing species Centaurea sadleriana Janka, from a different localites of Vojvodina (Zobnatica and Rimski Sanac), have been phytochemicaly examined. Phytochemical screening was performed to identify the presence of secondary biomolecules i.e. natural products in plant organs. Also, antioxidant characteristics were analysed (quantity of oxygen radicals and antioxidant ability). Phytochemical screening of plant material showed dominant presence of anthocyanins and leucoanthocyanins in petiole, flavonoids and tannins in both investigated organs, as well as free quinone derivatives, alkaloids and saponins in the leaves. Oxygen free radicals (O2.- i .OH) were detected in greater quantities, which indicate that the plants were exposed to negative environmental stimuli resulting in oxidative stress. Data obtained for antioxidant enzyme SOD activity, lipid peroxidation and quantity of reduced glutation were high, which indicate effective antioxidant protective system. Results for DPPH-radical scavenging activity categorize species Centaurea sadleriana Janka as moderately active 1. References: 1. Lee i sar. (1998): Evaluation of the antioxidant potential of natural products., Combinatorial Chem. & High Throughoutput Screening, 1, 35.

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P23 Ispitivanje redoks-kapaciteta ekstrakata Laurus nobilis L. Kaurinović B1, Popović M1. 1Departman za hemiju, Prirodno-matematički fakultet, Univerzitet u Novom Sadu, Trg Dositeja Obradovića 3, 21000 Novi Sad, Srbija Lovor je karakterističan za Malu Aziju, odakle se proširio na Mediteran i na ostale zemlje sa sličnom klimom. Tokom istorije, ovo moćno drvo je imalo religiozno značenje pobede i igra glavnu ulogu u klasičnim mitologijama. Postoji verovanje da je Hermes izmislio vatru trljajući listove nara i lovora jedan o drugi. U grčkoj mitologiji pripisivao se Apolonu, bogu zdravlja i medicine, koji ga je proglasio svetim drvetom i simbolom časti. Kao osnovne komponente, ulje sadrži laurosterin, estar glicerola sa laurinskom kiselinom, seskviterpenoide (kostunol i dehidrokostuslakton), dok ostatak čine masti: trigliceridi sa laurinskom, miristinskom i elainskom kiselinom. Aroma potiče uglavnom od terpena (cineol, terpineol, α- i β-pinene, citral), ali i cimetne kiseline i njenog metil-estra. Cilj istraživanja je bio da se ispita uticaj ekstrakata lovora na redoks kapacitet. Sirovi metanolni ekstrakt lista i ploda lovora je podvrgnut ekstrakciji sa 70% metanolom. Nakon uparavanja do suva, ostatak je rastvoren u vodi i ekstrahovan sa etrom, hloroformom, etilacetatom i n-butanolom. Pripremljeni su 10%-ni(v/v) rastvori ekstrakata u 50%-nom etanolu. Redoks kapacitet ekstrakata je određivan spektrofotometrijskim metodama koje su zasnovane na reakciji redukcije Fe3+ u Fe2+ (FRAP metoda, metoda po Yen i Chen-u).[1,2] Količine ukupnih flavonoida kretale su se od 5,36 (plod lovora) do 6,88 mg/1g (list lovora). Među ekstraktima lista lovora najbogatiji flavonoidima pokazao se EtOAc ekstrakt, dok se u H2O ekstraktu nalaze najmanje količine flavonoida. Skoro identični rezultati dobijeni su ispitivanjem količine flavonoida u ekstraktima ploda lovora jer se najviše flavonoida nalazi u EtOAc ekstraktu, a najmanje u H2O ekstraktu. FRAP metodom je utvrđeno da najveći redoks kapacitet poseduje etilacetatni ekstrakt, a najmanji etarski ekstrakt lovora. Metodom po Yen i Chen-u su potvrđeni rezultati dobijeni FRAP metodom. I ovom metodom etilacetatni ekstrakt, kao rastvor srednje polarnosti pokazuje najveći redoks kapacitet. Etarski ekstrakt, koji sadrži nepolarne supstance, lovora pokazuje najmanji redoks kapacitet. Ovo ukazuje na to da u EtOAc ekstraktu ima dosta slobodnih flavonoida. Et2O ekstrakt lovora ima najmanje polarnih komponenti, odnosno fenolnih jedinjenja koja su antioksidanti, dok su redoks kapaciteti �polarnih� ekstrakata veoma slični. Literatura: 1. Benzie IFF, Strain JJ (1999) Methods Enzymol 299:15-17. 2. Yen GH, Chen, HY (1997) J Agric Food Chem 43:27-32

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P23 Investigation of redox capacity of Laurus nobilis L. extracts Kaurinović B1, Popović М1 1Department of chemistry, Faculty of Natural Sciences, University of Novi Sad, Trg Dositeja Obradovića 3, 21000 Novi Sad, Serbia

Laurel is native to Near East and Anatolia from where it had spread to Mediterranean and other regions with similar climate. Dirong history this powerfull tree was a religious symbol of victory and it plays a major role in classical mythology. A legend says that the god Hermes invented fire by scrubbing leaves of laurel and granate against each other. In Greek mythology it was the atribute of Apollo, god of health and medicine, who regareded it the holy tree and the symbol of honour. The basic constituents, essential oil of laurel contains laurosterine, ester of glycerol with lauric acid, sesquiterpenoides (costunol and dehydrocostus-lactone), while the residues represent lipids: triglycerides with lauric, myristic and elaic acid. Laurel aroma originates mainly from terpenes (cineol, terpineol, α- and β-pinene, citral), and also cinnamic acid and its methyl-ester. The aim of this research was to investigate the redox capacity of the laurel extract. Crude methanol extracts of macerated laurel leaves and poppies were obtained in extraction with 70% methanol. After evaporation to dryness, dry matter was dissolved in water and extracted with ether, chloroform, ethyl-acetate, and n-butanol, thus leaving water solution as well. 10% (v/v) solutions of extracts in 50% ethanol were prepared. Redox capacity of extracts was investigated spectrometrically, using methods based on reduction of Fe3+ into Fe2+ (FRAP method, method according to Yen and Chen).[1,2] Total amounts of flavonoids ranged from 5.36 (laurel poppy) to 6.88 mg/1g (laurel leaves). Of single laurel leaves extracts, the most abundant in flavonoids was EtOAc extract, while H2O extract contains the smallest amount of flavonoids. Almost identical results were obtained in investigation of laurel poppies – largest amounts were detected in EtOAc extract, and the smallest in H2O extract. FRAP method established that the ethyl-acetate extract exhibits the highest redox capacity, while the lowest capacity was achieved with ether extract. Method according to Yen and Chen confirmed these results – the highest redox capacity of ethyl-acetate extract and the lowest capacity of ether extract, which contains nonpolar substances. This points out that EtOAc extracts contains lots of free flavonoids. Et2O extract of laurel contains the least polar components (phenolic antioxidants) while of ″polar″ extracts exhibit similar redox capacities. References: 1. Benzie IFF, Strain JJ (1999) Methods Enzymol 299:15-17.

2. Yen GH, Chen, HY (1997) J Agric Food Chem 43:27-32.

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P24 Ispitivanje antioksidantnog potencijala ekstrakata Ruscus aculeatus L., R. hypoglossum L. i R. alexandrinus Garsault Hadžifejzović N1, Petrović S2, Kukić J2, Savić A2, Nahrstedt A3 1 MADAUS GmbH, Colonia Allee 15, 51067 Keln, Nemačka; 2 Institut za farmakognoziju, Farmaceutski fakultet, Vojvode Stepe 450, 11221 Beograd, Srbija; 3 Institut za farmaceutsku biologiju i fitohemiju, Hittorfstrasse 56, 48149 Minster, Nemačka Rizom veprine, Rusci rhizoma (Ruscus aculeatus L.) koristi se za dopunsku terapiju simptoma hronične venske insuficijencije i hemoroida. Njegovi karakteristični sastojci su steroidni saponini, ruskozid, ruscin, deglukoruskozid i deglukoruscin [1]. Nadzemni delovi biljke (Rusci herba), koriste se isključivo tradicionalno za stimulaciju diureze, uglavnom u Sredozemlju i na Bliskom istoku. Sadrže različite O- i C-heterozide flavonoida, dok su saponini prisutni u maloj količini [2]. U ovom radu ispitivan je antioksidantni potencijal ekstrakata herbe i rizoma vrste R. aculeatus, herbe R. hypoglossum L. i herbe R. alexandrinus Garsault. Osušen i fino usitnjen biljni materijal ekstrahovan je 70% metanolom (MeOH). Za analizu su korišćeni suvi MeOH ekstrakti dobijeni nakon uparavanja rastvarača pod sniženim pritiskom. Deo suvog MeOH ekstrakta herbe R. aculeatus rastvoren je u destilovanoj vodi i izmućkan metilenhloridom (CH2Cl2) u cilju uklanjanja hlorofila i drugih lipofilnih sastojaka. Za ispitivanje je korišćen suvi ekstrakt dobijen nakon uparavanja vode (označen kao MeOH ekstrakt herbe R. aculeatus bez lipofilnih materija). Ostatak vodenog rastvora suvog MeOH ekstrakta herbe R. aculeatus sukcesivno je reekstrahovan etiacetatom (EtOAc) i butanolom (BuOH). Za analizu su korišćeni suvi EtOAc i BuOH ekstrakt. Iz ispitivanih ekstrakata prethodno su izolovani različiti flavonoidi, fenolske kiseline i amidi hidroksicimetne kiseline [2]. U cilju procene antioksidantnog potencijala ekstrakata, ispitivana je sposobnost neutralizacije 2,2-difenil-1-pikrihidrazil (DPPH) [3] i OH• radikala [4]. Sadržaj ukupnih fenola određen je spektrofotometrijski, korišćenjem Folin-Ciocalteu reagensa [5]. Najbolju anti-DPPH aktivnost ispoljili su EtOAc i BuOH ekstrakt herbe R. aculeatus. U koncentracijama 158 i 173 µg/ml doveli su do neutralizacije 50% DPPH radikala (SC50 vrednosti). MeOH ekstrakti herbi R. hypoglossum i R. alexandrinus bili su najmanje aktivni (SC50 = 518 i 541 µg/ml). Sposobnost neutralizacije DPPH radikala bila je u korelaciji sa sadržajem ukupnih fenola. U EtOAc i BuOH ekstraktu herbe R. aculeatus utvrđen je najveći (79,24 i 46,84 µg galne kiseline/mg suvog ekstrakta), a u MeOH ekstraktima herbi R. hypoglossum i R. alexandrinus najmanji sadržaj ukupnih fenola (22,86 i 34,69 µg galne kiseline/mg suvog ekstrakta). U cilju ispitivanja sposobnosti neutralizacije OH• radikala, ekstrakti su primenjeni u opsegu koncentracija od 12,5-200 µg/ml. MeOH ekstrakt herbe R. aculeatus bio je najaktivniji i doveo do 50% inhibicije degradacije 2-dezoksiriboze u koncentraciji 71,8 µg/ml (IC50 vrednost). U opsegu koncentracija 50-200 µg/ml ostali ispitivani ekstrakti herbe R. aculeatus ostvarili su inhibiciju preko 40% (42,57-49,28%). MeOH ekstrakt rizoma R. aculeatus ostvario je maksimum inhibicije (43.31%) pri koncentraciji od 200 µg/ml. Maksimalni efekti MeOH ekstrakata herbi R. hypoglossum i R. alexandrinus (42,23 i 47,48 µg/ml) ispoljeni su takođe pri najvećoj primenjenoj koncentraciji. Literatura: 1. ESCOP Monographs (2003) ESCOP. Exeter. Thieme. New York. 2. Hadžifejzović N. (2006) Flavonoide und Hydroxyzimtsäureamide in Ruscus aculeatus L. und in anderen verwandten Arten. Strukturaufklärung, Verteilung und physiologische Bedeutung. PhD Thesis. University of Münster, Germany. 3. Cuendet M. et al. (1997) Helv Chim Acta 80:1144-1152. 4. Lee J.-C. and Lim K.-T. (2000) Food Sci Biotechnol 9:83-88. 5. Velioglu Y.S. et al. (1998) J Agric Food Chem 46:4113-4117.

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P24 Investigation on antioxidant capacity of extracts of Ruscus aculeatus L., R. hypoglossum L. and R. alexandrinus Garsault Hadžifejzović N1, Petrović S2, Kukić J2, Savić A2, Nahrstedt A3 1 MADAUS GmbH, Colonia Allee 15, 51067 Köln, Germany; 2 Institute of Pharmacognosy, Faculty of Pharmacy, Vojvode Stepe 450, 11221 Belgrade, Serbia; 3 Institute of Pharmaceutical Biology and Phytochemistry, Hittorfstrasse 56, 48149 Münster, Germany

Butcher's Broom, Rusci rhizoma (Ruscus aculeatus L.) is officially used for supportive therapy for symptoms of chronic venous insufficiency and hemorrhoids. Its characteristic constituents are steroidal saponins, such as ruscoside, ruscin, deglucoruscoside and deglucoruscin [1]. Aerial parts of this plant (Rusci herba) are used only traditionally as diuretic, mostly in the Mediterranean and in the Middle East. They contain various flavonoid O- and C-glycosides, while saponins are present in small amount [2]. In this work we tested antioxidant potency of various extracts of R. aculeatus herb and rhizome, of R. hypoglossum L. herb and R. alexandrinus Garsault herb. Dried and finely grounded plant material was extracted with 70% MeOH, solvent was evaporated under reduced pressure and dry MeOH extracts were used for investigation. One part of dry MeOH extract of R. aculeatus herb was dissolved in distilled water and extracted with CH2Cl2 in order to remove chlorophyll and other lipophyllic substances. Dry extract, obtained after water evaporation, and marked as MeOH extract of R. aculeatus herb without lipophyllic matters, was used. The other part of water solution of dry MeOH extract of R. aculeatus herb was successively reextracted with EtOAc and BuOH, and dry EtOAc and BuOH extracts were obtained. From investigated Ruscus extracts various flavonoids, phenolic acids and hydroxycinnamic acid amides were previously isolated [2]. In order to investigate antioxidant capacity of the extracts, a tests of scavenging on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical [3] and inhibition of generation of OH• radicals were used [4]. Total phenolics were determined spectrophotometrically using Folin-Ciocalteu reagent [5]. The best anti-DPPH activity was exhibited by EtOAc and BuOH extracts of herb of R. aculeatus. Concentrations at which they scavenged DPPH radical by 50% (SC50 values) were 158 and 173 µg/ml, respectively. MeOH extracts of herbs of R. hypoglossum and R. alexandrinus were the least active, with SC50 values 518 and 541 µg/ml, respectively. Results of DPPH radical assay were correlated with total phenolics. EtOAc and BuOH extracts of herb of R. aculeatus had the highest phenolics content (79.24 and 46.84 µg gallic acid/mg dry extract, respectively). MeOH extracts of herbs of R. hypoglossum and R. alexandrinus had the lowest content of total phenolics (22.86 and 34.69 µg gallic acid/mg dry extract, respectively). For determining the inhibition of OH• radicals production, the extracts were applied in concentration range from 12.5-200 µg/ml. MeOH extract of R. aculeatus herb was most active and inhibited 2-deoxyribose degradation by 50% (IC50 value) at 71.8 µg/ml. In concentration range from 50-200 µg/ml other extracts of R. aculeatus herb exhibited activity higher than 40% (42.57-49.28%). MeOH extract of R. aculeatus rhizome showed maximal activity (43.31%) at 200 µg/ml. The uppermost activities against OH• radical of R. hypoglossum and R. alexandrinus herb extracts (42.23 and 47.48 µg/ml, resp.) was also shown at the highest concentration applied. References: 1. ESCOP Monographs (2003) ESCOP. Exeter. Thieme. New York. 2. Hadžifejzović N. (2006) Flavonoide und Hydroxyzimtsäureamide in Ruscus aculeatus L. und in anderen verwandten Arten. Strukturaufklärung, Verteilung und physiologische Bedeutung. PhD Thesis. University of Münster, Germany. 3. Cuendet M. et al. (1997) Helv Chim Acta 80:1144-1152. 4. Lee J.-C. and Lim K.-T. (2000) Food Sci Biotechnol 9:83-88. 5. Velioglu Y.S. et al. (1998) J Agric Food Chem 46:4113-4117.

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P25 Povećanje antimutagene aktivnosti monoterpena na t-BOOH indukovanu mutagenezu u prisustvu enzima jetre pacova Dragana Mitić-Ćulafić, Sanja Ćakić, Milena Janković, Branka Vuković-Gačić and Jelena Knežević-Vukčević Katedra za Mikrobiologiju, Biološki Fakultet, Univerzitet u Beogradu, Studentski trg 3/II, 11000 Beograd, Srbija Reaktivne kiseonične vrste (ROS) mogu nastati u toku normalnog ćelijskog metabolizma ali je njihova produkcija značajno povećana delovanjem jonizujućeg zračenja i različitih hemjskih agenasa. Oksidativna oštećenja DNK su jedana od najčešćih pretnji genomskoj stabilnosti i mogu doprineti pojavi kancerogeneze, dijabetesa i starenja. Ćelije poseduju mehanizme antioksidativne zaštite i DNK reparacione enzime, ali i neka prirodna jedinjenja mogu delovati protektivno na ROS indukovana DNK oštećenja. Cilj ovog rada je bio ispitivanje protektivne uloge linaloola, mircena i eukaliptola na t-butil hidroperoksid (t-BOOH) indukovanu mutagenezu. U tom cilju, koristili smo E. coli WP2 oxyR soj, koji je deficijentan u indukciji enzima antioksidativne zaštite. Eksperimenti su rađeni sa i bez S9 frakcije. S9 frakcija sadrži enzime jetre pacova koji su uključeni u metabiličku transformaciju i detoksifikaciju ksenobiotika. Dobijeni rezultati ukazuju da u prisustvu enzima S9 frakcije dolazi do povećanja antimutagenog potencijala linaloola, mircena i eukaliptola. Jači protektivni efekat linaloola, mircena i eukaliptola u prisustvu S9 može biti rezultat njihovog prevođenja u metaboličku formu koja je aktivnija u zaštiti od ROS. Naši rezultati ukazuju da su prirodni proizvodi efikasni u zaštiti od okidativne mutageneze i predstavljaju podstrek za dalja izučavanja u studijama hemoprevecije.

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P25 Enhanced antimutagenic effect of monoterpenes against t-BOOH induced mutagenesis in the presence of rat liver enzymes Dragana Mitić-Ćulafić, Sanja Ćakić, Milena Janković, Branka Vuković-Gačić and Jelena Knežević-Vukčević Chair of Microbiology, Faculty of Biology, University of Belgrade, Studentski trg 3/II, 11000 Belgrade, Serbia Reactive oxygen species (ROS) are generated by normal metabolic functions or by environmental exposure to ionizing radiation and chemicals. Oxidative DNA damage is one of the most common threats to genome stability and may contribute to carcinogenesis, diabetes and ageing. In addition to cellular antioxidative defense mechanisms and DNA repair enzymes, some natural compounds can act as protective substances against ROS induced DNA damage. The aim of this work is to investigate the protective capacity of monoterpenes: linalool, myrcene and eucalyptol against t-butyl hydroperoxide (t-BOOH) induced oxidative mutagenesis. For this purpose, we used E. coli WP2 oxyR strain, which is deficient in induction of antioxidative enzymes. The experiments were conducted with or without S9 fraction. The S9 fraction contains rat liver enzymes involved in metabolic transformation and detoxification of xenobiotic. The obtained results suggest that in the presence of S9 enzymes antimutagenic potential of linalool, myrcene and eucalyptol was enhanced. Higher antimutagenic effect of linalool, myrcene and eucalyptol might be attributed to their processing by S9 enzymes to more protective metabolic form. Our results indicate that natural products have substantial protective efficiency against oxidative mutagenesis and encourage for further chemoprevention studies.

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P26 Ispitivanje antioksidantnog potencijala voćnih čajeva Maksimović Z1, Gavrilov B2

1 Institut za farmakognoziju, Farmaceutski fakultet, Univerzitet u Beogradu, Vojvode Stepe 450, 11221 Beograd, Srbija; 2 Hemofarm a.d., Beogradski put bb, 26300 Vršac, Srbija Najrazličitije voćne vrste se od davnina koriste u ishrani ljudi i predstavljaju bogat izvor vitamina, minerala i dijetnih vlakana. Intenzivnim istraživanjima u oblasti ispitivanja antioksidativnog potencijala različitih namirnica koje se koriste u ljudskoj ishrani, utvrđeno je da sveže voće predstavlja bogat izvor antioksidanasa. Antioksidansi u voću su: vitamini C, A, E, beta-karoten, flavonoidni polifenoli, fenolne kiseline i njihovi estri, minerali (Se, Mn) i dr. Za razliku od svežeg voća, voćni čajevi se uglavnom konzumiraju kao osvežavajući napici, radi uživanja. Imajući u vidu da su voćni čajevi ukusni i da ih rado uzima veliki broj ljudi, oni predstavljaju značajan sekundarni izvor antioksidanasa u ishrani. U ovom radu, antioksidantni potencijal 5 komercijalnih monokomponentnih voćnih čajeva (borovnica, jabuka, šipurak, šljiva i kruška) ocenjen je određivanjem sposobnosti redukcije Fe(III) jona (FRAP-test) i određivanjem sposobnosti neutralizacije slobodnih DPPH radikala (DPPH-test) i upoređen sa antioksidantnim potencijalom sintetskog sredstva BHT. Pored toga, u svim uzorcima analiziran je sadržaj vitamina C [1], flavonoida [2] i tanina [3]. Utvrđeno je da je sadržaj vitamina C izuzetno nizak u voćnim čajevima u poređenju sa svežim voćem i da stoga voćni čajevi ne predstavljalju značajan izvor vitamina C u ishrani. Međutim i pored niskog sadržaja, vitamin C u značajnoj meri učestvuje u antioksidativnoj aktivnosti voćnih čajeva. Od ispitanih uzoraka ustanovljeno je da šipurak ima najizraženiji antioksidantni potencijal, što je u pozitivnoj korelaciji sa sadržajem vitamina C i tanina. Najniži antioksidantni potencijal imaju šljiva i kruška, dok je potencijal borovnice i jabuke ocenjen kao intermedijeran. U slučaju šipurka, u najvećoj meri odgovorni za antioksidativnu aktivnost su vitamin C i tanini, a u manjoj meri flavonoidi. Kod jabuke, glavni nosioci antioksidativnog potencijala su flavonoidi, a u manjoj meri vitamin C i tanini. Kod kruške, glavni nosioci antioksidativnog potencijala su flavonoidi i tanini, u manjoj meri vitamin C. Kod šljive su prvenstveno flavonoidi i tanini odgovorni za antioksidativnu aktivnost, a vitamin C u manjoj meri. Na kraju, kod borovnice, za antioksidativnu aktivnost u najvećoj meri odgovorni su flavonoidi i vitamin C, a u manjoj meri tanini. Zahvalnica: Ostvareno uz podršku projekta br. 143012 Ministarstva nauke i zaštite životne sredine, Republika Srbija. Literatura: 1. Mirić M, Stanimirović D (2001) Praktikum iz bromatologije, Grafopan, Beograd. 2. Deutsches Arzneibuch (1996) 10. Ausgabe, Band 3. Deutscher Apotheker Verlag Stuttgart, Govi-Verlag GmbH, Frankfurt. 3. Jugoslovenska farmakopeja 2000 (2001) Peto izdanje, Vol. 3, Savremena administracija, Beograd.

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P26 Antioxidant potential of some fruit teas Maksimović Z1, Gavrilov B2

1 Institute of Pharmacognosy, School of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11221 Belgrade, Serbia; 2 Hemofarm a.d., Beogradski put bb, 26300 Vršac, Serbia Since antiquity, various fruits are considered as important constituents of human diet, being major and rich sources of a number of vitamins, minerals and dietary fibers. It is established, by intensive research in the domain of antioxidant potential of different food constituents, that fresh fruit is the essential source of natural antioxidants. The antioxidants present in fresh fruit are vitamins (C, A, E and beta-carotene), flavonoids, phenolic acids with resembling esters, minerals (Se, Mn) etc. Unlike fresh fruit, fruit teas are consumed as refreshing drinks mostly, just for enjoyment. Taking into account that fruit teas are tasty and readily taken by a number of people, they could be considered as important secondary source of natural antioxidants in human common diet. In this research, the aqueous extracts of five commercial fruit teas (bilberry, apple, rose hips, plum and pear) were evaluated for their antioxidant potential by assays for ferric-reducing antioxidant power (FRAP) and DPPH-free radical scavenging activity (DPPH), and compared to the same feature of synthetic antioxidant BHT, which has been used as a positive control substance. In all samples, the vitamin C [1], flavonoid [2] and tanin [3] contents were determined, too. It was established that the vitamin C content is very low in fruit teas, compared to available literature data on fresh fruit, suggesting that fruit teas are of a minor importance as the sources of vitamin C in diet. However, although being low, vitamin C contributes to overall antioxidant potential of fruit teas to a great extent. Among samples investigated, rose hips expressed the most evident antioxidant activity, which correlated well to the vitamin C and tannins content. The lowest antioxidant potential was recorded in plum and pear, while the same feature in bilberry and apple was assessed as intermediate. In rose hips, vitamin C and tannins were identified as constituents responsible for observed effect, while flavonoids were of a minor importance. In apple, the most active constituents were flavonoids, while vitamin C and tannins were in the background. In pear, flavonoids and tannins, but not vitamin C contributed mostly to observed activity. In plum, flavonoids and tannins come to a first place, while in bilberry the antioxidant activity is mainly influenced by flavonoids and vitamin C. Acknowledgement: Supported by the Grant No. 143012 of the Ministry of Science and Environmental Protection, Republic of Serbia. References: 1. Mirić M, Stanimirović D (2001) Praktikum iz bromatologije, Grafopan, Belgrade. 2. Deutsches Arzneibuch (1996) 10. Ausgabe, Band 3. Deutscher Apotheker Verlag Stuttgart, Govi-Verlag GmbH, Frankfurt. 3. Yugoslav Pharmacopoeia 2000 (2001) Fifth issue, Vol. 3, Savremena administracija, Belgrade.

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P27 Antioksidantna, antimikrobna aktivnost i fenolski sastojci metanolnih ekstrakata Laserpitium zernyi Hayek (Apiaceae) Popović V1, Petrović S1, Milenković M2, Tomić A1, Niketić M3 1 Institut za farmakognoziju, Farmaceutski fakultet, V. Stepe 450, 11221 Beograd, Srbija; 2 Institut za mikrobiologiju i imunologiju, Farmaceutski fakultet, V. Stepe 450, 11221 Beograd, Srbija; 3 Prirodnjački muzej, Njegoševa 51, 11000 Beograd, Srbija Laserpitium zernyi Hayek (Umbelliferae) je skardo-pindska endemična vrsta rasprostranjena u planinskim predelima centralnog Balkana (jugozapadna Srbija, zapadna Makedonija, istočna Albanija i severozapadna Grčka). Ranije je smatrana podvrstom vrste L. siler L., subsp. zernyi (Hayek) Tutin [1,2]. Nadzemni delovi biljke u cvetu sakupljeni su na planini Jablanica, u Makedoniji, tokom jula 2006. god. Osušene i usitnjene cvasti, i herba bez cvasti macerovani su odvojeno u hloroformu (CHCl3), a zatim u metanolu (MeOH) i rastvarači upareni pod sniženim pritiskom. Suvi MeOH ekstrakt cvasti (uzorak 1) i suvi MeOH ekstrakt herbe bez cvasti (uzorak 2) korišćeni su za dalja ispitivanja. U ekstraktima, uzorcima 1 i 2, korišćenjem Folin-Ciocalteu reagensa, utvrđeno je da sadržaj ukupnih fenola iznosi 14,92 i 6,83 % (izraženo kao galna kiselina) [3]. HPLC analizom, korišćenjem standardnih supstanci, u oba uzorka identifikovani su hlorogenska kiselina i flavoni (apigenin, luteolin, apigenin 7-O-glukozid i luteolin 7-O-glukozid). Antiradikalska svojstva uzoraka ispitivana su na osnovu sposobnosti neutralizacije 2,2-difenil-1-pikrilhidrazil radikala (DPPH) i OH• radikala. Ukupna antioksidantna aktivnost određena je FRAP testom [3]. Ekstrakt cvasti (uzorak 1) i ekstrakt herbe bez cvasti (uzorak 2) ispoljili su koncentraciono zavisnu sposobost neutralizacije DPPH radikala (SC50 = 38,95 i 109,54 µg/ml). Oba analizirana ekstrakta pokazala su relativno visoku sposobnost inhibije OH• radikala (SC50 vrednost za ekstrakt cvasti iznosila je 10,32 µg/ml, a za ekstrakt herbe bez cvasti 35,97 µg/ml). Ukupna antioksidantna aktivnost ekstrakata (uzorka 1 i 2) iznosila je 1,55 i 0,68 µmol Fe2+/mg suvog ekstrakta. Antimikrobna svojstva ekstrakata ispitivana su agar-difuzionom [4] i mikrodilucionom [5] metodom prema Gram(+) bakterijama: Staphylococcus aureus ATCC 25923, S. epidermidis ATCC 12228, Micrococcus luteus ATCC 9341, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633, Gram(–) bakterijama: Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae NCIMB 9111, Escherichia coli ATCC 25922, i gljivici Candida albicans (ATCC 10259 i ATCC 24433). Oba ekstrakta ispoljila su najveću antimikrobnu aktivnost prema C. albicans, a ekstrakt herbe bez cvasti (uzorak 2) i prema B. subtilis (MIC 30 µg/ml). Aktivnost uzorka 2 prema svim ostalim testiranim sojevima bakterija, kao i uzorka 1 prema većini testiranih sojeva bakterija bila je identična (MIC 62,5 µg/ml). Ekstrakt cvasti pokazao je nešto slabiju aktivnost prema sojevima M. luteus, E. faecalis i P. aeruginosa (MIC 125 µg/ml). Može se zaključiti da je ekstrakt cvasti L. zernyi ispoljio značajno veću antioksidantnu aktivnost i neznatno slabije antimikrobno delovanje u odnosu na ekstrakt herbe bez cvasti. Literatura: 1. Tutin T.G. (1968) Laserpitium L. In: Flora Europea 2, Tutin T.G. et al. (editors), Cambridge University Press, Cambridge. 2. Micevski K. (2005) Flora na Republika Makedonija 1(6), Makedonska Akademija na Naukite i Umetnostite, Skopje. 3. Kukic J. et al. (2006) Biol. Pharm. Bull. 29:725-729. 4. Acar J.F., Goldstein F.W. (1996) Disc Susceptibility Test. In: Lorian V., editor. Antibiotics in Laboratory Medicine, 4th edition. Williams & Wilkins. Baltimore. 5. National Committee for Clinical Laboratory Standards - NCCLS (2001). In: Pa Wayne, editor. Document M100-S11: Performance Standards for Antimicrobial Susceptibility Testing, 11th Informational Supplement.

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P27 Antioxidant, antimicrobial activity and phenolic compounds of methanol extracts of Laserpitium zernyi Hayek (Apiaceae)

Popović V1, Petrović S1, Milenković M2, Tomić A1, Niketić M3 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 Natural History Museum, Njegoševa 51, 11000 Belgrade, Serbia

Laserpitium zernyi Hayek (Umbelliferae) is Scardo-Pindic endemic plant distributed in the mountain regions of C. Balkans (S. W. Serbia, W. FYROM, E. Albania, N. W. Greece). It was treated earlier as a subspecies of closely related L. siler L., subsp. zernyi (Hayek) Tutin [1,2]. The aerial parts of this plant were collected in FYROM at Mt. Jablanica in July 2006, during the period of full flowering. Air-dried and powdered flowers, as well as herb without flowers were macerated in CHCl3 and then in MeOH. Solvents were evaporated under low pressure and dry MeOH extract of flowers (sample 1) and dry MeOH extract of herb without flowers (sample 2) were used for this investigation. In L. zernyi extracts (samples 1 and 2), using Folin-Ciocalteu reagent, 14.92 and 6.83 % of total phenolics (calculated as gallic acid) was determined [3]. Using HPLC in both samples chlorogenic acid and flavones (apigenine, luteoline, apigenine 7-O-glucoside and luteoline 7-O-glucoside) were identified. Radical scavenging activity (RSC) of extracts was estimated by their ability to scavenge 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and OH radical. Total antioxidant activity was determined using FRAP assay [3]. Extract of flowers (sample 1) and extact of herb without flowers (sample 2) showed significant and concentration-dependent DPPH radical scavenging activity (SC50 = 38.95 and 109.54 µg/ml). Both extracts showed high OH scavenging activity, and SC50 for samples 1 and 2 were 10.32 and 35.97 µg/ml. Total antioxidant activity (TAA) of samples 1 and 2 was 1.55 and 0.68 µmol Fe2+/mg of dry extract. The antimicrobial properties of extracts were tested by agar diffusion [4] and microdillution [5] methods against Gram(+) bacteria Staphylococcus aureus ATCC 25923, S. epidermidis ATCC 12228, Micrococcus luteus ATCC 9341, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633, Gram(–) bacteria Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae NCIMB 9111, Escherichia coli ATCC 25922, and yeast Candida albicans (ATCC 10259 and ATCC 24433). Both samples showed highest antimicrobial activity against C. albicans, and extract of herb without flowers (sample 2) also against B. subtilis (MIC 30 µg/ml). Activity of sample 2 against all the other tested bacteria, and the activity of sample 1 against the most of the tested bacterial strains were identical (MIC 62.5 µg/ml). Extract of flowers showed lower antimicrobial activity against M. luteus, E. faecalis and P. aeruginosa (MIC 125 µg/ml). In conclusion, extract of flowers of L. zernyi had significantly higher antioxidant activity, and some lower antimicrobial effect compared to extract of herb without flowers. References: 1. Tutin T.G. (1968) Laserpitium L. In: Flora Europea 2, editor. Tutin T.G. et al., Cambridge University Press, Cambridge. 2. Micevski K. (2005) Flora na Republika Makedonija 1(6), Makedonska Akademija na Naukite i Umetnostite, Skopje. 3. Kukic J. et al. (2006) Biol. Pharm. Bull. 29:725-729. 4. Acar J.F., Goldstein F.W. (1996) Disc Susceptibility Test. In: Lorian V., editor. Antibiotics in Laboratory Medicine, 4th edition. Williams & Wilkins. Baltimore. 5. National Committee for Clinical Laboratory Standards - NCCLS (2001). In: Pa Wayne, editor. Document M100-S11: Performance Standards for Antimicrobial Susceptibility Testing, 11th Informational Supplement.

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P28 Antioksidantna aktivnost metanolnih ekstrakata herbe Epilobium dodonaei Vill. (Oenotheracae) Relja Suručić1, Mirjana Simić1, Branislava Lakušić2, Tatjana Kundaković1, Milica Pavlović1, Nada Kovačević1 1Institut za farmakognoziju i 2Institut za botaniku, Farmaceutski fakultet Univerziteta u Beogradu, Vojvode Stepe 450, 11221 Beograd Ispitivano je antioksidantno delovanja metanolnih ekstrakata herbe Epilobium dodonaei Vill. (Oenotheracae) [1]. Biljni materijal je prikupljen sa tri lokaliteta: u okolini Kolašina (Crna Gora), sa padina Galičice (Makedonija) i u klisuri Belog Rzava (Srbija). Osušen i usitnjen biljni materijal ekstrahovan je metanolom (70%), postupkom maceracije, i ekstrakti upareni do suva. Sadržaj ukupnih polifenola određen je Folin-Ciocalteu metodom [2]. Antioksidantno delovanje procenjeno je na osnovu ukupne redukcione sposobnosti ekstrakata (FRAP metodom) [3], sposobnosti neutralizacije DPPH radikala [4] i sposobnosti inhibicije lipidne peroksidacije [5]. U metanolnim ekstraktima herbe E. dodonaei sa različitih lokaliteta, sadržaj polifenola je bio u rasponu 13,22 - 14,19%. Najviši sadržaj polifenola je određen u ekstraktu E. dodonei iz klisure Belog Rzava. Na osnovu HPLC analize može se pretpostaviti da ispitivani ekstrakti sadrže: elagnu kiselinu, galnu kiselinu, kvercetin, kvercetin-3-O-glukozid, hiperozid, heterozid apigenina, enotein i miricetin. U svim ekstraktima utvrđeno je prisustvo elagne kiseline, galne kiseline i enoteina, dok je jedino u ekstraktu E. dodonaei sa lokaliteta Kolašin prisutan miricetin. Ispitivanjem ukupne redukcione sposobnosti ekstrakata utvrđena je aktivnost u rasponu od 2,64 - 2,73 µmola Fe2+/mg ekstrakta. Metanolni ekstrakti E. dodonei pokazali su izraženu sposobnost neutralizacije DPPH radikala (SC50

= 4,10 - 4,48 µg/ml). Ispitivanjem sposobnosti inhibicije lipidne peroksidacije utvrđena je visoka aktivnost (IC50 = 8,67 - 9,30 µg/ml). Sadržaj polifenola, ukupna redukciona sposobnost, sposobnost neutralizacije DPPH radikala i inhibicije lipidne peroksidacije metanolnih ekstrakata herbe E. dodonaei prikupljene sa različitih lokaliteta, nisu se značajno razlikovali. Utvrđen je nešto veći sadržaj polifenola i izraženija antioksidantna aktivnost ekstrakta herbe E. dodonaei prikupljene u klisuri Belog Rzava. Literatura: 1. Raven, P.H. (1968): Epilobium L., U: Tutin, T.G. et al. (eds.). Flora Europaea, Vol. 2. Cambridge University Press., Cambridge 2. Makkar H.P.S. et al. (2000) Quantification of Tannins in Tree Foliage – A Laboratory Manual. FAO/IAEA. Vienna. 3. Szőllősi, R., Varga Szőllősi, I. (2002) Act Biol Szeg 46:125-7. 4. Bondet, V. et al. (1997) Lebensm.-Wiss. u.- Technol. 30:609-15. 5. Afanas’ev, I. B. et al. (1989) Biochem. Pharmacol. 38:1763-9.

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P28 Antioxidant activity of methanol extracts of Epilobium dodonaei Vill. (Oenotheracae) herb Relja Suručić1, Mirjana Simić1, Branislava Lakušić2, Tatjana Kundaković1, Milica Pavlović1, Nada Kovačević1 1Institute of Pharmacognosy and 2Institute of Botany, Faculty of Pharmacy, V. Stepe 450, 11221 Belgrade, Serbia Antioxidant activity of aerial parts of Epilobium dodonaei Vill. (Oenotheraceae) [1] methanol extracts was investigated. Plant material was collected from three localities: near Kolašin (Montenegro), Galičica slopes (FYR Macedonia) and Beli Rzav canyon (Serbia). Dried and grounded plant material was extracted with methanol (70%), by a maceration process and extracts were dried till dryness. Total phenolics contents were determined using Folin-Ciocalteu method [2]. Antioxidant activity was evaluated by ferric ion reducing power assay (FRAP method) [3], DPPH radical scavenging activity [4] and inhibition of lipid peroxidation assay [5]. Contents of phenols in methanol extracts of E. dodonaei from different localities varied from 13.22% - 14.19%. The highest value of phenolic constituents’ content was determined in the samples collected at Beli Rzav canyon. HPLC analyses indicated possible presence of ellagic acid, gallic acid, quercetin-3-O-glucoside, hyperoside, quercetine, apigenin glycoside, oenothein and myricetin. Ellagic acid, gallic acid and oenothein were present in all extracts, while myricetin was detected only in E. dodonaei extract from Kolašin vicinity. Ferric reducing activity of extracts varied from 2.64 - 2.73 µmol Fe2+/mg extract. Methanol extracts of E. dodonei showed significant DPPH radical scavenging activity (SC50 = 4.10 - 4.48 µg/ml). Inhibition of lipid peroxidation was high for all extracts (IC50 = 8.67 - 9.30 µg/ml). Total phenolics content, ferric ion reducing power, DPPH radical scavenging activity, inhibition of lipid peroxidation were not significantly different for E. dodonaei extracts from different localities. Extract of E. dodonaei collected at Beli Rzav canyon contained slightly higher content of total phenolics and antioxidant activity was more pronounced. References: 1. Raven, P.H. (1968): Epilobium L., U: Tutin, T.G. et all. (eds.). Flora Europaea, Vol.2. Cambridge University Press., Cambridge 2. Makkar HPS et al. (2000) Quantification of Tannins in Tree Foliage – A Laboratory Manual. FAO/IAEA. Vienna. 3. Szőllősi, R., Varga Szőllősi, I. (2002) Act Biol Szeg 46:125-7. 4. Bondet, V. et al. (1997) Lebensm.-Wiss. u.-Technol. 30:609-15. 5. Afanas’ev, I. B. et al. (1989) Biochem. Pharmacol. 38: 1763-9.

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P29 Antimikrobna aktivnost matičnog luga usninske kiseline Goran Nikolić1, Saša Zlatković2, Siniša Đorđević1, Tatjana Kundaković3 1 Tehnološki fakultet, Bulevar oslobođenja 124, 16000 Leskovac, Srbija; 2 Actavis Trading Ltd., Đorđa Stanojevića 12, 11070 Novi Beograd, Srbija; 3 Farmaceutski fakultet, Vojvode Stepe 450, 11000 Beograd, Srbija; Lišajevi su predstavnici simbiotskog društva gljiva i algi. Sastavne komponente mnogih vrsta lišajeva su pokazale određena antibiotička svojstva [1]. Od onih koji su do sada proučavani, ističu se lišajevi čiji je aktivni princip usninska kiselina. Usninska kiselina je žuta kristalna supstanca prirodnog porekla (Sl. 1), izolovana iz lišaja Usnea barbata, sa izraženom antibakterijskom aktivnošću.

Slika 1. Strukturna formula usninske kiseline C18H16O7

Proučavanje usninske kiseline je od naročitog značaja zbog mogućnosti njene praktične primene za izradu farmaceutskih i kozmetičkih preparata. U literaturi je opisan i patentiran veliki broj postupaka izolovanja usninske kiseline iz različitih lišajeva, kao i sinteza njene natrijumove soli [2-5]. Međutim, u literaturi nema podataka o ispitivanju aktivnosti matičnog luga usninske kiseline, koji nastaje kao sporedni proizvod u procesu njenog dobijanja. Poznato je da usninska kiselina posle više godina čuvanja u neadekvatnim uslovima veoma malo izgubi antibiotska svojstva u odnosu na sveže izolovanu supstancu. Ta činjenica je otvorila ideju iskorišćenja matičnog luga sa praktičnog stanovišta. Cilj ovog rada je ispitivanje antimikrobne aktivnosti matičnog luga usninske kiseline sa aspekta njegove primene za izradu fitopreparata i kozmetičkih proizvoda. Matični lug je dobijen po postupku koji je patentiran [3]. Korelacijom dobijenih podataka sa aktivnostima kristalne usninske kiseline izolovane iz Usnea barbata i Na-usninata utvrđeno je da je ispitivani matični lug sačuvao kvalitativno i kvantitativno antibiotska svojstva. Potvrđena je aktivnost matičnog luga usninske kiseline na sledeće test mikroorganizme: Echerichia coli 95, Bacillus subtilis ATCC 6633, Staphylococus aureus ATCC 6538, Micrococcus flavus ATCC 10240, Sarcina lutea ATCC 9341, Micrococcus pyogenes ATCC 6538P, Candida albicans ATCC 10231. Literatura: 1. K.O. Vartia (1973) Antibiotics in lichens. In: V. Ahmadjian, M.E. Hale (Eds.), The Lichens, Academic Press, New York, 547–561. 2. M. Stankovic, S. Stankovic, Z. Djordjevic, S. Djordjevic (1989) Yu Pat 43978. 3. S. Stanković, I. Djordjevic, Z. Kocić (2001) Pat WO 01/95900 and EP 1294373. 4. M. Cocchietto, N. Skert, P. Nimis, G. Sava (2002) Naturwissenschaften 89 : 137. 5. T. Kristmundsdottir, R. Aradottir, K. Ingolfsdottir, R. Ogmundsdottir (2002) J. Pharm. Pharmacol. 54(11) : 1447.

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P29 Antimicrobial activity of usnic acid mother liquor Goran Nikolić1, Saša Zlatković2, Siniša Đorđević1, Tatjana Kundaković3 1 Faculty of technology, Bulevar oslobođenja 124, 16000 Leskovac, Serbia; 2 Actavis Trading Ltd., Đorđa Stanojevića 12, 11070 Novi Beograd, Serbia; 3 Faculty of pharmacy, Vojvode Stepe 450, 11000 Beograd, Serbia; The lichens are the representatives of symbiotic union of fungi and algae. The constitutional components of many lichen species show some antibiotic activities [1]. According the literature, the lichens, containing usnic acid as an active principle have major significance. Usnic acid is a yellow crystal substance of natural origin (Fig. 1), isolated from the lichen Usnea barbata, with significant antibacterial activity.

Figure 1. Structure of usnic acid, C18H16O7

The study of usnic acid is of particular importance because of its practical application in the production of pharmaceutical and cosmetic preparations. Extended number of patented and described procedures for the isolation of usnic acid from various lichens, and synthesis of Na-usninate, is described in the literature [2-5]. However, there is no data in the literature about investigation of usnic acid mother liquor obtained as a by-product in the production process. That was the reason for the investigation of the antimicrobial activity of usnic acid mother liquor with respect to its application for the production of pharmaceutical and cosmetic preparations. The mother liquid was produced according the patented procedure [3]. Correlation of obtained data with the activity of crystal usnic acid isolated from Usnea barbata and Na-usninate have shown that tested mother liquor retained antibiotic properties qualitatively and quantitatively. The high activity of usnic acid and Na-usninate against the following test microorganisms was confirmed: Echerichia coli 95, Bacillus subtilis ATCC 6633, Staphylococus aureus ATCC 6538, Micrococcus flavus ATCC 10240, Sarcina lutea ATCC 9341, Micrococcus pyogenes ATCC 6538P, Candida albicans ATCC 10231. References: 1. K.O.Vartia (1973) Antibiotics in lichens.In:V. Ahmadjian, M.E. Hale (Eds.),The Lichens, Academic Press,New York, 547–561. 2. M. Stankovic, S. Stankovic, Z. Djordjevic, S. Djordjevic (1989) Yu Pat 43978. 3. S. Stanković, I. Djordjevic, Z. Kocić (2001) Pat WO 01/95900 and EP 1294373. 4. M. Cocchietto, N. Skert, P. Nimis, G. Sava (2002) Naturwissenschaften 89 : 137. 5. T. Kristmundsdottir, R. Aradottir, K. Ingolfsdottir, R. Ogmundsdottir (2002) J. Pharm. Pharmacol. 54(11) : 1447.

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P30 Antimikrobna aktivnost vrsta Ampelopsis brevipedunculata i Parthenocissus tricuspidata Kundaković T1, Milenković M2, Grubin J3, Nikolić G4, Kovačević N1, Lakušić B5

1 Institut za farmakognoziju, Farmaceutski fakultet, Vojvode Stepe 450, 11221 Beograd, Srbija

2Institut za mikrobiologiju i imunologiju, Farmaceutski fakultet, Vojvode Stepe 450, 11221 Beograd, Srbija 3Ministarstvo za nauku, Njegoševa 12, 11000 Beograd, Srbija 4Tehnološki fakultet, Bulevar oslobođenja 124, 16000 Leskovac, Srbija 5 Institut za botaniku, Farmaceutski fakultet, Vojvode Stepe 450, 11221 Beograd, Srbija

Listovi i cvasti vrsta Ampelopsis brevipedunculata i Parthenocissus tricuspidata sakupljeni su juna 2007. godine u Botaničkoj bašti u Beogradu (Srbija). Osušeno lišće i cvasti macerovani su cikloheksanom (1:10; 2 puta po dva dana), a zatim metanolom (70%, V/V) istim postupkom ekstrakcije. Antimikrobna aktivnost cikloheksanskih i metanolnih ekstrakata je testirana in vitro. Dve različite metode su korišćene za određivanje antimikrobne aktivnosti: agar-difuziona [1] i mikrodiluciona metoda [2]. Testirane Gram-pozitivne i Gram-negativne bakterije i gljivice su Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Micrococcus luteus (ATCC 10240), Enterococcus faecalis (ATCC 29212), Bacillus subtilis (ATCC 6633BB), Bacillus cereus (ATCC11778), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (NCIMB 9111) i Candida albicans (ATCC 10259 and 24433). MIC predstavlja najnižu koncentraciju ekstrakta koja ne izaziva vidljiv rast mikroorganizama. Ispitivani biljni ekstrakti su pokazali slabu antimikrobnu aktivnost protiv testiranih bakterija i gljivica, osim cikloheksanskog ekstrakta cvasti i metanolnih ekstrakata cvasti i listova P. tricuspidata koji su pokazali umerenu aktivnost protiv C. albicans i M. luteus (MIC 3,25 mg/ml). Literatura: 1. Acar J.F., Goldstein F.W. (1996) Disc susceptibility test. In Antibiotics in laboratory medicine ed. Lorian V, Williams & Wilkins, Baltimore, London, Los Angeles, Sydney, p. 1 – 52; 2. Candan F. et al. (2003) J Ethnopharmacol 87(2-3): 215-220.

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P30 Antimicrobial activity of Ampelopsis brevipedunculata and Parthenocissus tricuspidata Kundaković T1, Milenković M2, Grubin J3, Nikolić G4, Kovačević N1, Lakušić B5

1Department of Pharmacognosy, Faculty of Pharmacy, Vojvode Stepe 450, 11221 Belgrade, Serbia

2Department of Microbiology and Immunology, Faculty of Pharmacy, Vojvode Stepe 450, 11221 Belgrade, Serbia 3Ministry of Science, Njegoševa 12, 11000 Belgrade, Serbia 4Faculty of Technology, Bulevar oslobođenja 124, 16000 Leskovac, Serbia 5 Department of Botany, Faculty of Pharmacy, Vojvode Stepe 450, 11221 Belgrade, Serbia The leaves and inflorescences of Ampelopsis brevipedunculata and Parthenocissus tricuspidata were collected in June 2007 in Botanical Garden in Belgrade (Serbia). The dried leaves and inflorescences were macerated with cyclohexane (1:10; 2 x two days), followed by methanol (70%, V/V) extraction using the same procedure. The antimicrobial activities of cyclohexane and methanol extracts were tested in vitro. Two different methods were used for the determination of antimicrobial activities: agar-diffusion method and broth microdilution assay. The tested Gram-positive and Gram-negative bacteria and fungi were Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Micrococcus luteus (ATCC 10240), Enterococcus faecalis (ATCC 29212), Bacillus subtilis (ATCC 6633BB), Bacillus cereus (ATCC11778), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (NCIMB 9111) and Candida albicans (ATCC 10259 and 24433). The MIC is defined as the lowest concentration of the extract at which the microorganism does not demonstrate visible growth. The tested plant extracts have shown weak antimicrobial activity against tested bacteria and fungi, except cyclohexane extract of the inflorescence and the methanol extract of the inflorescence and leaves of P. tricuspidata which have shown moderate antimicrobial activity against C. albicans and M. luteus with MIC 3.25 mg/ml. References: 1. Acar J.F., Goldstein F.W. (1996) Disc susceptibility test. In Antibiotics in laboratory medicine ed. Lorian V, Williams & Wilkins, Baltimore, London, Los Angeles, Sydney, p. 1 – 52; 2. Candan F. et al. (2003) J Ethnopharmacol 87(2-3): 215-220.

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P31 Efekat ekstrakta herbe Teucrium scordium L. subsp. scordioides (Lamiaceae) na oštećenje jetre pacova izazvano ugljentetrahloridom Kundaković T1, Milenković M2, Topić A3, Kovačević N1, Lakušić B4, Stanojković T5

1 Institut za farmakognoziju, Farmaceutski fakultet, Vojvode Stepe 450, 11000 Beograd, Srbija

2Institut za mikrobiologiju i imunologiju, Farmaceutski fakultet, Vojvode Stepe 450, 11000 Beograd, Srbija 3 Institut za medicinsku biohemiju, , Farmaceutski fakultet, Vojvode Stepe 45011000 Beograd, Srbija 4 Institut za botaniku, Farmaceutski fakultet, Vojvode Stepe 450, 11000 Beograd, Srbija 5 Institut za onkologiju i radiologiju Srbije, Pasterova 4, 11000 Beograd, Srbija Infuz, pripremljen od nadzemnih delova T. scordium koristi se u tradicionalnoj medicini na području Balkana kao gorko aromatično sredstvo i holagog sa sposobnošću da leči rane i ublažava groznicu [1,2]. Pored vrlo detaljnih ispitivanja hepatotoksičnosti nekih Teucrium vrsta, posebno T. chamaedrys [3], nema podataka o uticaju T. scordium L. subsp. scordioides Schreb. na jetru. Nadzemni delovi vrste T. scordium subsp. scordioides sakupljeni su na lokalitetu Oblačina (Prokuplje, Serbia) u septembru 2007, tokom perioda cvetanja. Biljni materijal je ekstrahovan cikloheksanom na sobnoj temperaturi, a zatim dihlormetanom i metanolom. Hepatotoksičnost metanolnog ekstrakta T. scordium subsp. scordioides testirana je in vivo nakon i.p. administracije CCl4 kod pacova. Oštećenje jetre je praćeno na osnovu nivoa specifičnih enzima, AST i ALT, kao i MDA, u serumu i homogenatu jetre. Nije bilo značajnog efekta ispitivanog metanolnog ekstrakta u dozama od 25; 50 i 100 mg/kg i.p. na lipidnu peroksidaciju u homogenatu i serumu, kao i na nivoe AST i ALT u serumu u poređenju sa životinjama tretiranim CCl4-om. Na osnovu ispitivanih parametara, metanolni ekstrakt T. scordium nema protektivno delovanje na jetru, ali takođe ne pokazuje ni hepatotoksičnost. Literatura: 1. Gostuški R. (1979) Lečenje lekovitim biljem, 7th ed. Narodna knjiga, Beograd, pp. 190-191; 2. Tucakov J. (1971) Lečenje biljem, Kultura, Beograd, pp. 518-520; 3. Kouzi S.A. et al. (1994) Chem. Res. Toxicol. 7, 850-856.

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P31 Effects of Teucrium scordium L. subsp. scordioides (Lamiaceae) on carbontetrachloride-induced hepatotoxicity in rats Kundaković T1, Milenković M2, Topić A3, Kovačević N1, Lakušić B4, Stanojković T5. 1Department of Pharmacognosy, Faculty of Pharmacy, Vojvode Stepe 450, 11000 Belgrade, Serbia

2Department of Microbiology and Immunology, Faculty of Pharmacy, Vojvode Stepe 450, 11000 Belgrade, Serbia 3 Institute for medicinal biochemistry, Faculty of Pharmacy, Vojvode Stepe 450, 11000 Belgrade, Serbia 4Department of Botany, Faculty of Pharmacy, Vojvode Stepe 450, 11000 Belgrade, Serbia 5Institute for Oncology and Radiology of Serbia, Pasterova 4, 11000 Belgrade, Serbia. The infusum from aerial parts of T. scordium has been used in traditional medicine on Balkan as as bitter aromatic, cholagogue with wound healing and fever reducing properties [1,2]. Besides very detailed studies about hepatotoxicity of some Teucrium species, especially T. chamaedrys [3], there were not any data about the effects of T. scordium L. subsp. scordioides Schreb. in liver. The aerial parts of T. scordium subsp. scordioides were collected on locality Oblačina (Prokuplje, Serbia) in Septembar 2007, during the period of full flowering. The plant material was extracted with cyclohexane on room temperature, followed by extraction with dichlormethane and methanol. Hepatotoxicity of methanol extract of T. scordium subsp. scordioides was tested in vivo after i.p. administration of CCl4 in rats. The damage of hepatocytes was measured according to the level of specific enzymes, AST and ALT, as well of MDA, in serum and liver homogenate. There were no significant effects of tested methanol extract in doses of 25; 50 and 100 mg/kg i.p. on lipid peroxidation in homogenate and serum, as well on the levels of AST and ALT in serum compared to CCl4-treated animals. Concerning the studied paramethers, the methanol extract of T. scordium had not any protective activity in liver, but also any hepatotoxicity. References: 1. Gostuški R. (1979) Lečenje lekovitim biljem, 7th ed. Narodna knjiga, Beograd, pp. 190-191; 2. Tucakov J. (1971) Lečenje biljem, Kultura, Beograd, pp. 518-520; 3. Kouzi S.A. et al. (1994) Chem. Res. Toxicol. 7, 850-856.

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P32 Poređenje spazmolitičkih efekata vrste Arbutus unedo poreklom iz Crne Gore i Grčke Branković S.1, Pavlović D.2, Kitić D.2, Kovačević N., Veljković S.1 1Institut za fiziologiju, Medicinski fakultet, Bul. dr Zorana Đinđića 81, 18000 Niš 2Studijska grupa za Farmaciju, Medicinski fakultet, Bul. dr Zorana Đinđića 81, 18000 Niš 3 Institut za farmakognoziju, Farmaceutski fakultet, Vojvode Stepe 450, 11000 Beograd Arbutus unedo L. koristi se u tradicionalnoj medicini u terapiji gastrointestinalnih poremećaja, bolesti kože, urinarnih infekcija i arterijske hipertenzije [1,2,3]. Etanolni ekstrakti listova vrste Arbutus unedo sakupljenih u Crnoj Gori (AuM) i Grčkoj (AuG) ispitivani su na spazmolitičku aktivnost na izolovanom ileumu pacova [4]. I AuM i AuG inhibiraju spontane kontrakcije ileuma pacova na koncentraciono zavisan način (opseg koncentracija između 0,03 i 10 mg/mL). AuG pokazuje veću aktivnost od Aum (p<0,05). AuM (EC50=1,43±0,51 mg/mL) i AuG (EC50=2,04±0,39 mg/mL) redukuju kontraktilni odgovor na acetilholin (ACh), kada se kumulativno dodaju u vodeno kupatilo (0,01-10 µg/ml). Ovi rezultati pokazuju da etanolni ekstrakt vrste Arbutus unedo deluje kao spazmolitik na izolovani ileum pacova. AuM i AuG verovatno ispoljavaju spazmolitički efekat posredstvom holinergičkih mehanizama. Reference:1. Tucakov, J. (1997): Lečenje biljem, 7 th edition, Rad, Beograd. 2. Ziyyat, A., Boussairi, E.-H.(1998) Phytotherapy research Vol. 12, 110–113 3. Afkir, S.et al. (2008) Journal of Ethnopharmacology 116 288–295 4. Rojas, A. et al. (1996) Planta Medica 62, 154–159.

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P32 Comparative study of intestinal spasmolytic effects of Arbutus unedo from Montenegro and Greece Branković S.1, Pavlović D.2, Kitić D.2, Kovačević N., Veljković S.1 1Institute for Physiology, Faculty of Mwdicine, University of Niš, Niš 2Departmant of Pharmacy, Faculty of Medicine, University of Niš, Niš 3 Institute for farmacognosy, Faculty of Pharmacy, University of Belgrade, Belgrade Arbutus unedo L. is traditionally used to treat gastrointestinal disorders, skin desease, urinary infections and arterial hypertension [1, 2, 3]. The ethanolic extracts from Arbutus unedo leaves collected in Montenegro (AuM) and in Greece (AuG) were studied for spasmolytic effect in the rat ileum [4]. Both AuM and AuG inhibited spontaneous contraction of the rat ileum in a concentration-dependent manner (concentrations span between 0.03 and 10 mg/mL). The AuG extract was more active than AuM one (p<0.05). AuM (EC50=1.43±0.51 mg/ml) and AuG extracts (EC50=2.04±0.39 mg/ml) reduced contractile response to acetylcholine (ACh) cumulatively added to bath (0.01-10 µg/ml). These data indicate that the ethanolic extract of Arbutus unedo acts as a spasmolytic in the rat ileum. The spasmolytic effects of AuM and AuG ethanolic extracts are possibly mediated through the cholinergic mechanism. References:1. Tucakov, J. (1997): Lečenje biljem, 7 th edition, Rad, Beograd. 2. Ziyyat, A., Boussairi, E.-H.(1998) Phytotherapy research Vol. 12, 110–113 3. Afkir, S.et al. (2008) Journal of Ethnopharmacology 116 288–295 4. Rojas, A. et al. (1996) Planta Medica 62, 154–159.

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P33 Kardioprotektivni efekat rezveratrola Novaković A1, Gojković-Bukarica Lj2, Kanjuh V3 1Institut za farmakologiju, Farmaceutski fakultet u Beogradu, Srbija; 2Institut za farmakologiju, Medicinski fakultet u Beogradu, Srbija; 3Srpska akademija nauka i umetnosti, Beograd, Srbija. Trihidroksistilben, rezveratrol, nađen je u oko 72 biljne vrste, kao što su Polygonum cuspidatum (“Kojokon” -Japan) i grožđe. Poslednjih godina, brojne epidemiološke studije pokazale su inverznu korelaciju između upotrebe crnog vina i incidence kardiovaskulrnih bolesti. Smatra se da je rezveratrol, kao jedna polifenolna komponenta prisutna u značajnim količinama u crnom vinu, delom odgovoran za kardioprotektivno delovanje vina. U mehanizam kardioprotektivnog delovanja rezveratrola verovatno su uključeni antioksidativno, antitrombocitno i vazodilatatorno delovanje. Mehanizam vazodilatacije prouzrokovane rezveratrolom još uvek nije poznat. Cilj rada bio je da se ispitaju efekti i mehanizam vazorelaksantnog delovanja rezveratrola na izolovanoj aorti pacova, sa i bez endotela. Eksperimenti su izvedeni na izolovanoj aorti pacova koja je bila prekontrahovana fenilefrinom (1 µM). Rezveratrol je koncentracijski-zavisno relaksirao aortu pacova sa i bez endotela. Visoko selektivni blokator ATP-senzitivnih K+ kanala, glibenklamid, kao i neselektivni blokator velikih kalcijum-senzitivnih K+ kanala, tetraetilamonijum nisu antagonizovali rezveratrolom indukovanu relaksaciju aorte pacova. Karibdotoksin, selektivni blokator kalcijum-senzitivnih K+ kanala nije inhibirao relaksaciju indukovanu rezveratrolom. 4-Aminopiridin, neselektivni blokator volatažno-zavisnih K+ (Kv) kanala, i margatoksin, blokator Kv1 kanala, antagonizovali su relaksaciju aorte pacova, prouzrokovanu rezveratrolom. Rezveratrol je potentno relaksirao aortu pacova sa i bez endotela. Izgleda, da su 4-aminopiridin- i margatoksin-senzitivni K+-kanali smešteni u membrani vaskularnih glatko-mišićnih ćelija aorte pacova, uključeni u mehanizam relaksacije prouzrokovane rezveratrolom. Zahvalnica: Ministarstvu za nauku i tehnologije (Srbije)

Literatura: Novakovic A, et.al. (2006) BCPT. 99(5):360-4.

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P33 Cardioprotective effect of resveratrol Novaković A1, Gojković-Bukarica Lj2, Kanjuh V3 1Department of Pharmacology, Faculty of Pharmacy; Belgrade, Serbia; 2Department of Pharmacology, Medical Faculty, Belgrade, Serbia; 3 Academy of Sciences and Arts, Belgrade, Serbia. The trihydroxystilbene, resveratrol, is a naturally occurring phenolic substance present in a variety of plants such as Polygonum cuspidatum (“Kojokon” in Japanese) roots, and grapes. Interest in this compound has expanded in recent years, when a numerous epidemiological studies showed an inverse correlation between red wine consumption and incidence of cardiovascular diseases. It seems that resveratrol might be partly responsible for cardiovascular benefits associated with wine consumption. The mechanism of cardiovascular benefits probably includes vasorelaxation, antioxidant and anti-platelet effects of resveratrol. The mechanisms by which resveratrol causes vasodilatation are uncertain. The aim of this study was to investigate the mechanism (s) of resveratrol-induced vasorelaxation in rat aorta with endothelium and without endothelium. Rat aorta were precontracted with phenylephrine (1 µM). Resveratrol induced a concentration-dependent relaxation of rings with endothelium and without endothelium. Highly selective blocker of ATP-sensitive K+ channels, glibenclamide as well as nonselective blockers of calcium-sensitive K+ channels, tetraethylammonium did not block resveratrol-induced relaxation of rat aorta rings. Charybdotoxin, a blocker of calcium-sensitive K+ channels did not affect the resveratrol-induced relaxation. 4-Aminopyridine, non selective blocker of voltage-gated K+ (KV) channels, and margatoxin that inhibits KV1 channels abolished relaxation of rat aorta rings induced by resveratrol. We have shown that resveratrol can induce relaxation of rat aorta with endothelium and without endothelium. It seems that 4-aminopyridine– and margatoxin–sensitive K+ channels located in vascular smooth muscle mediated relaxation of rat aorta produced by resveratrol. Acknowledgements: Ministry of Science and Technology (Serbia). References: Novakovic A, et.al. (2006) BCPT. 99(5):360-4.

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P34 Uticaj ekstrakata belog luka (Allium sativum L., Alliaceae) na delovanje nekih lekova Biljana Božin1, Neda Mimica-Dukić2, Isidora Samojlik3, Goran Anačkov4, Ružica Igić4

1Zavod za farmaciju, Medicinski fakultet, Hajduk Veljkova 3, 21000 Novi Sad, Srbija 2Departman za hemiju, PMF, Trg D. Obradovića 3, 21000 Novi Sad, Srbija 3Zavod za farmakologiju i toksikologiju, Medicinski fakultet, Hajduk Veljkova 3, 21000 Novi Sad, Srbija 4Departman za biologiju i ekologiju, PMF, Trg D. Obradovića 2, 21000 Novi Sad, Srbija Beli luk (Allium sativum L., Alliaceae) je od davnina imao važnu ulogu u ljudskom postojanju. Korišten je kao hrana, začin, ali i za prevenciju i lečenje određenih bolesti, zahvaljujući svojim lekovitim svojstvima. Epidemiološka, klinička i predklinička istraživanja pokazala su blisku povezanost između konzumacije belog luka i izlečenja određenih oboljenja. Za ovu biljku vezuje se širok spektar lekovitih dejstava kao što su hipolipemijsko, antiaterosklerotsko, hipoglikemijsko, antikoagulantno, antihipertenzivno, antimikrobno i hepatoprotektivno [1]. Međutim, potvrđene su i određene komplikacije i neželjena delovanja koja se javljaju nakon konzumacije belog luka, a jedna od glavnih je produženo krvarenje, kao i halitoza, mučnina, hipotenzija, glavobolja i nadutost. Pored toga, zabeležene su i interakcije belog luka sa određenim hipoglikemicima, kardiovaskularnim lekovima i MAO inhibitorima [2,3]. Cilj ovog rada bio je da se na miševima ispita uticaj metanolnih ekstrakata belog luka na poremećaj motorne koordinacije indukovan midazolamom i na analgetski efekat kodeina. Ispitivani metanolni ekstrakti dobijeni su iz „mladog“ belog luka, iz samlevene i osušene zrele lukovice belog luka (po tipu bezmirisnih komercijalnih preparata) i iz sveže lukovice belog luka. U ekstraktima je određen sadržaj ukupnih fenola i flavonoida. Životinje su podeljene u 4 grupe, u saglasnosti sa peroralnim tretmanom određenim ekstraktima (tri eksperimentalne i kontrolna grupa) i leka. Aplikacija ekstrakata trajala je 5 dana. Petog dana je, 2h nakon aplikacije ekstrakta primenjen određeni lek, a merenje je izvršeno u saglasnosti sa farmakološkim protokolom. Svi ispitivani ekstrakti ispoljili su značajnu redukciju disbalansa motorne aktivnosti izazvane midazolamom, kao i redukciju analgetskog delovanja kodeina. Sam beli luk nije doveo do motorne diskoordinacije i analgetskog efekta, već je njegova aplikacija izazvala promenu efekta ispitivanih lekova. Dobijeni rezultati ukazuju na interakcije između midazolama i kodeina s jedne, i belog luka s druge strane, pa je preporuka da se dodatno ispitaju moguće interakcije fitopreparata na bazi belog luka, s obzirom na njihovu vrlo široku primenu, kod ljudi. Literatura: 1. Bisset N.G., Wichtl M. (2001) Herbal Drugs and Phytopharmaceuticals. CRC Press, Boca Raton, London, New York, Washington D.C. 2. Blumenthal R. 1999. The Complete German Commission E Monographs. American Botanical Council, Austin. 3. Tessier D.J., Bash D.S. (2003) J Surg Res 114 (1), pp. 30-36.

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P34 Influence of garlic (Allium sativum L., Alliaceae) extracts on pharmacodynamic effects of some drugs Biljana Božin1, Neda Mimica-Dukić2, Isidora Samojlik3, Goran Anačkov4, Ružica Igić4

1Department of Pharmacy, Faculty of Medicine, Hajduk Veljkova 3, 21000 Novi Sad, Serbia 2Department of Chemistry, Faculty of Sciences, Trg D. Obradovića 3, 21000 Novi Sad, Serbia 3Department of Pharmacology and Toxicology, Faculty of Medicine, Hajduk Veljkova 3, 21000 Novi Sad, Serbia 4Department of Biology and Ecology, Faculty of Sciences, Trg D. Obradovića 2, 21000 Novi Sad, Serbia Garlic (Allium sativum L., Alliaceae) has played one of the most important dietary as well as medicinal role in many cultures for centuries. It has been used as a spice and condiment, for flavoring, and due to its potential benefits in preventive and curative treatment. Epidemiological, clinical, and preclinical studies have shown the close relation between dietary habits, including garlic intake, and the occurrence of diseases. A wide array of therapeutic effects such as hypolipidaemic, antiatherosclerotic, hypoglycaemic, anticoagulant, antihypertensive, antimicrobial, and hepatoprotective have been reported [1]. However, the most common complication from garlic use is prolonged bleeding, as well as side effects that include halitosis, nausea, hypotension, headache and bloating. Drug-herb interactions with hypoglycemics, cardiovascular medications and monoamine oxidase inhibitors have been reported, too [2,3]. The aim of the study was to examine the effects of methanol extracts derived from garlic midazolam induced impairment in motor coordination and analgesic effect of codeine, in mice. The examined extracts were obtained from the immature garlic plant, from grounded and air-dried (prepared as an Aged Garlic Extract) and fresh garlic bulbs, with determined content of total phenolics and flavonoids. The animals were divided according to pretreatment peroral regime with particular extract during 5 consecutive days into 4 groups (control and three extracts’ group). Drugs tested were injected 2 h after the particular extract application on 5th day and the measurements were done according to survey protocol. All three extracts reduced motor impairment caused by midazolam and decreased the analgesic effect of codeine. Regarding the fact that garlic alone do not induce sleep, motor discoordiantion and analgesic effect and that its usage produce changes in tested drug effects, it can be recommended that the interaction between drugs and phytopreparations containing garlic should be additionally examined/confirmed in human population. References: 1. Bisset N.G., Wichtl M. (2001) Herbal Drugs and Phytopharmaceuticals. CRC Press, Boca Raton, London, New York, Washington D.C. 2. Blumenthal R. 1999. The Complete German Commission E Monographs. American Botanical Council, Austin. 3. Tessier D.J., Bash D.S. (2003) J Surg Res 114 (1), pp. 30-36.

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P35 In vivo citotoksičnost ekstrakta Viscum album L. za ćelije Ehrlich-ovog tumora Ćebović T1, Popović M2, Spasić S3 1 Katedra za biohemiju, Medicinski fakultet, Hajduk Veljkova 3, 21000, Novi Sad, Srbija; 2Departman za hemiju, Prirodno-matematički fakultet, Trg D. Obradovića 5, 21000, Novi Sad, Srbija; 3Katedra za biohemiju, Farmaceutski fakultet, Bul. Vojvode Stepe 450, 11221, Beograd, Srbija Dosadašnja farmakološka ispitivanja imele (Viscum album L.) odnosila su se uglavnom na terapijske efekte njenih polarnih ekstrakata i komponenata [1]. U ovom radu, ispitivali smo nepolarne aktivne principe V. album i njihovu biološku aktivnost. Za selektivnu ekstrakciju i identifikaciju jedinjenja iz listova V. album koristili smo ekstrakciju sa CO2 pod superkritičnim uslovima kuplovanu sa gasnom hromatografijom/masenom spektrometrjom (GC/MS). U ekstraktu smo identifikovali nekoliko klasa nepolarnih jedinjenja, kao i dva vitamina. Uz to, analizirali smo i isparljivu frakciju koja je sadržala nekoliko novih terpenskih molekula. Dalje smo proverili hipotezu da V. album ekstrakt pokazuje citotoksičnu aktivnost za ćelije Ehrlich-ovog karcinoma (EAC) in vivo zahvljujući indukciji oksidativnog stresa. Miševima su ćelije EAC implanirane pre, za vreme i nakon pretretmana sa ekstraktom. Značajo smanjenje incidence karcinoma je uočno u svim grupama koje su primale ekstrakt V. album u poređenju sa EAC kontrolnom grupom. Najveće smanjenje je uočeno kod miševa koji su pretretirani ekstrakom V. album, iako je značajno smanjen broj ćelija EAC tumora uočen i kod životinja koje su imale već razvijenu formu tumora (uznapredovalu prolferaciju tumorskih ćelija). Aktivnosti ispitivanih antioksidantnih enzima u ćelijama tumora EAC ukazivale su na odsustvo oksidativnog stresa. Međutim, promene u aktivnostima antioksidantnih enzima uočene nakon administracije ekstrakta V. album bi mogle biti posledica indukcije oksidativnog stresa u ćelijama tumora EAC. Literatura: 1. Bussing A. 2000. Biological and pharmacological properties of Viscum album L. In Mistletoe – the genus Viscum, Bussing A (ed). Harwood Academic Publishers: Amsterdam, 123-182.

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P35 Cytotoxic effects of the Viscum album L. extract on Ehrlich tumour cells in vivo Ćebović T1, Popović M2, Spasić S3 1 Biochemistry Department, School of Medicine, Hajduk Veljkova 3, 21000, Novi Sad, Serbia; 2Chemistry Department, Faculty of Sciences, Trg D. Obradovića 5, 21000, Novi Sad, Serbia; 3Biochemistry Department, School of Pharmacy, Bul. Vojvode Stepe 450, 11221, Belgrade, Serbia To date most pharmacological studies on mistletoe (Viscum album L.) have focused on the therapeutic properties of its polar extracts [1]. Here, we examined the non-polar constituents of V. album and their biological activities. We used supercritical CO2 extraction coupled with gas chromatography/mass spectrometry (GC/MS) to selectively extract and identify compounds in V. album leaves. We identified several non-polar classes of compounds, as well as two vitamins in the extract. In addition, we analyzed a volatile fraction that contained several novel terpene molecules. We tested the hypothesis that the V. album extract exhibits cytotoxic properties in Ehrlich carcinoma (EAC) cells in vivo due to the induction of oxidative stress. Mice were implanted with EAC cells before, at the time of or after pretreatment with the extract. A significant reduction in the incidence of cancer was observed in all groups that received the V. album extract compared to the EAC control group. The largest decrease was observed in mice pretreated with the V. album extract, although significantly reduced numbers of EAC cells were also observed in animals with developed carcinoma. The activities of antioxidative enzymes in the EAC cells suggested the absence of oxidative stress. However, changes in the antioxidative enzymes activities observed after administration of the V. album extract might be due to the induction of oxidative stress in the EAC cells. References: 1. Bussing A. 2000. Biological and pharmacological properties of Viscum album L. In Mistletoe – the genus Viscum, Bussing A (ed). Harwood Academic Publishers: Amsterdam, 123-182.

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P36 Biljni ekstrakti – novi stimulatori rasta

Radmila Marković1, Petrujkić B1., Svetlana Grdović1, Milena Krstić2, Šefer D.2

1 Fakultet veterinarske medicine, Katedra za ishranu i botaniku, Bul. oslobođenja 18, 11000 Beograd, Srbija 2 Fakultet veterinarske medicine, Katedra za hemiju Bul. oslobođenja, 11000 Beograd, Srbija

U prirodne stimulatore rasta (NGP's – natural growth promoters) spadaju fitobiotici, probiotici, prebiotici, zakišeljivači i enzimi koji utiču na poboljšanje zdravlja kroz stimulaciju eubioze digestivnog trakta. Fitobiotici su funkcionalni sastojci hrane koji se dobijaju iz aromatičnih biljaka, začina i etarskih ulja (Jovanović i sar, 2004). Etarska ulja predstavljaju najkoncentrovaniji oblik fitogenih aditiva i pojačavaju ukus, podržavaju varenje i modifikuju mikrofloru. To su mešavine lipofilnih, tečnih, isparljivih i aromatskih supstanci. Da bi se pospešilo korišćenje ekstrakata biljaka i etarskih ulja kao AGP (antibiotic growth promoter) alternativa, sprovedeno je istraživanje koje je obuhvatilo 94 proizvodjača hrane iz 13 zemalja, 7 iz EU i 6 van EU. Cilj velikog broja istraživanja je bio da se utvrdi efikasnost biljnih estrakata u ishrani životinja. Pregledom najčešće zajedno korišćenih ekstrakata i organskih kiselina potvrđen je veći prirast, bolja konverzija hrane i niži morbiditet kod prasadi u kombinaciji u poređenju sa upotrebom organskih kiselina. Pri upotrebi ekstrakata u hrani za krmače uočeno je poboljšanje od 100 g na dan kod prasadi i 300 g po zalučenom prasetu (Raper, 2008). Kod živine, sličan napredak u vezi sa ekstraktima uočen je u prirastu i konverziji kako na obrocima baziranim na pšenici tako i na obrocima baziranim na kukuruzu. Iz sprovedenih istraživanja može se zaključiti da se ekstrakti često ubacuju u hranu za životinje (čak iako način delovanja i efikasnost nisu potpuno razjašnjeni kod svih ekstrakata). Zato je urađen pregled naučnih istraživanja sa aktivnim supstancama za hranu za svinje i živinu, uključujući karvakol koji se nalazi u origanu, cimet aldehid koji se prirodno javlja u cimetu i oleorezina paprike (ekstrakt ljute paprike). Iz dobijenih rezultata tri ogleda sa dobijeno je prosečno poboljšanje u dnevnom prirastu prasadi i konverzijom hrane od 14.1% i 6.5% Reference Jovanović N, Marković Radmila, Sinovec Z. (2004): Tradicionalna medicina u savremenom pristupu ishrani, Clinica veterinaria, Budva, p. 90-94. Raper G. (2008): Increasing interest for plant extracts, Feed Tech, volume 12, No 3, p. 12-13.

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P36 Plant extracts – new growth promoters Radmila Marković1, Petrujkić B1., Svetlana Grdović1, Milena Krstić2, Šefer D.2

1 Faculty of veterinary medicine, Department of nutrition and botany, Bul. oslobođenja 18, 11000 Belgrade, Serbia 2 Faculty of veterinary medicine, Department of chemistry Bul. oslobođenja, 11000 Belgrade, Serbia In natural growth promoters (NGP's) phytobiotics, probiotics, prebiotics, acidifying matters and enzymes are classified. They all affect health throughout stimulation of eubisoses in gut. Phytobiotics are functional ingredients of feed which are derived from aromatic plants, spices and ether oils (Jovanović et al., 2004). Essential oils represent most concentrated form of phytogenic additives; they increase palatability, improve digestion, and modify micro organisms in guts. They are mixtures of lipophylic, liquid, volatile and aromatic substances. In order to increase use of plant extracts like alternative to use of antibiotic growth promoters (AGP), trial which includes 94 producers from 13 different countries (7 EU and 6 non EU countries) was undertaken. Main aim of numerous trials was to evaluate efficiency of plant extracts in animal nutrition. By review of combined use of extracts with organic acids higher daily gain better feed conversion and lower morbidity were determined in comparison with use of only organic acids. When extracts were used in sow feed increase of 100 grams a day per piglet and 300 grams per weaner were determined (Raper, 2008). In poultry, similar improvements with use of extracts were determined and especially in daily gain and conversion as well as on diets based on wheat and on corn based diets. From these trials it can be concluded that extracts are often used in feed (even in cases when mode of action and efficiency are not fully understood yet). Because of all that review of scientific trials with active substances in swine and poultry feed was done. From results achieved it can be seen that daily gain was higher for 14.1% and conversion was lower for 6.5% when extract of chilly pepper, oregano and cinnamon were used. References Jovanović N, Marković Radmila, Sinovec Z. (2004) Tradicionalna medicina u savremenom pristupu ishrani, Clinica veterinaria, Budva, p. 90-94. Raper G. (2008) Increasing interest for plant extracts, Feed Tech, volume 12, No 3, p. 12-13.

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P37 Primena etarskih ulja u pčelarstvu Jevrosima Stevanović1, Marijana Stanimirović2, Miloš Stanić1 1 Katedra za biologiju, Fakultet veterinarske medicine, Bul. oslobođenja 18, 11000 Beograd, Srbija 2Jugohemija-Vetagra, Resavska 31, 11000 Beograd, Srbija U radu su date osnovne informacije o primeni etarskih ulja u pčelarstvu. Primena etarskih ulja ili komponenti etarskih ulja predstavlja atraktivnu alternativu za sintetske akaricide koji se u pčelarstvu koriste za suzbijanje ektoparazitskog krpelja pčela Varroa destructor. Laboratorijska ispitivanja velikog broja različitih etarskih ulja i njihovih komponenata pokazala je da većina etarskih ulja ima izuzetno akaricidno dejstvo. Međutim, prilikom primene u košnicama, samo nekoliko etarskih ulja pokazalo je dovoljnu efikasnost: timol, eukaliptol i ulja četinara. Ova ulja mogu se koristiti pojedinačno ili u mešavini sa drugim etarskim uljima ili komponentama etarskih ulja. Mortalitet krpelja nakon primene tih formulacija u košnicama iznosi 45-49%, mada ima podataka o efikasnosti preko 90% u slučaju etarskih ulja nekih endemskih vrsta biljaka dalekog istoka. Osim toga, preparati na bazi etarskih ulja ostavljaju veoma malo rezidua u medu i drugim pčelinjim proizvodima. Ako se primenjuju kao fumiganti, efikasnost timola i drugih etarskih ulja protiv Varroa krpelja zavisi u velikoj meri od temperature, sezone i jačine pčelinjeg društva. U konvencionalnom pčelarenju, etarska ulja se koriste komplementarno sa biotehničkim metodama kao sastavni deo programa suzbijanja krpelja V. destructor, dok su u organskom pčelarenju isključivo etarska ulja dozvoljena za suzbijanje ektoparazita. Međutim, za pouzdano i efikasno suzbijanje krpelja u košnicama, neophodna je optimizacija primene etarskih ulja. Za sada, postoji samo nekoliko komercijalnih formulacija na bazi etarskih ulja ili njihovih komponenti namenjenih za suzbijanje Varroa krpelja. Antimikrobna aktivnost etarskih ulja testirana je i radi procene mogućnosti njihove primene protiv Paenibacillus larvae, uzročnika američke truleži, najopasnije bolesti pčelinjeg legla. Od brojnih etarskih ulja koja su testirana, najbolji efekat ispoljilo je ulje grejpfruta Citrus paradisi, što se može pripisati vrsti i procentu isparljivih komponenti u tom ulju, kao što su limonen (69.9%) i mircen (9.6%). Upotreba etarskih ulja ili njihovih specifičnih isparljivih komponenti u suzbijanju P. larvae može predstavljati alternativni način kontrole ove ozbiljne bolesti, zato što te supstance ne ostavljaju toksične rezidue niti u medu, niti u ostalim pčelinjim proizvodima.

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P37 Appliance of essential oils in beekeeping Jevrosima Stevanović1, Marijana Stanimirović2, Miloš Stanić1 1Department of Biology, Faculty of Veterinary Medicine, Bul. oslobodjenja 18, 11000 Belgrade, Serbia 2Jugohemija-Vetagra, Resavska 31, 11000 Belgrade, Serbia The purpose of this paper is to give basic information about appliance of essential oils in beekeeping. Essential oils and essential oil components offer an attractive alternative to synthetic acaricides for the control of Varroa destructor. Many kinds of essential oils and their components have been evaluated in laboratory screening tests and most of them show excellent acaricidal activities. However, few of them have proven successfully when tested in the field: thymol, eucalyptol and wintergreen oil. These oils can be applied singly or blended with other essential oils or essential oil components. Mite mortality is 45-49% when these formulations are used, although there are data about efficacy over 90% in case of essential oils of some endemic plant species from the fat east. In addition, their residues in honey and other bee products are low, even after long-term treatments. When applied as fumigants, the effectiveness of thymol and other essential oils against Varroa mites depends greatly on temperature, time of the year and colony strength. Along with biotechnical control measures, essential oils can be used in the integrated control of V. destructor in conventional beekeeping and in organic beekeeping, essential oils represent the only permitted substances for the control of external parasites. For reliable effective control of mites in the hives, efforts are necessary to optimize the use of these essential oils. Currently, there are very few commercial formulations based on essential oils or their components to be use to control Varroa mite. Essential oils have been tested in order to reveal their antimicrobial properties against Paenibacillus larvae, the causal agent of American foulbrood, the most serious disease of honey bee brood. Among many evaluated plant essences, best results were achieved with the essential oil of grapefruit Citrus paradisi. This property could be attributed to the kind and percentage of the volatile components of the oil, like limonene (69.9%) and myrcene (9.6%). The use of essential oils or their specific volatile components individually against P. larvae may represent an alternative scope for the control of this serious disease because it does not leave toxic chemical residues in honey nor in other bee products.

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P38 Uticaj eugenola na pasažu crevnog sadržaja kod pacova Milanka Jezdimirović1, Nevenka Aleksić1, Saša Ivanović1, Saša Trailović1, Radomir Ratajac2

1Fakultet veterinarske medicine, Bulevar oslobođenja 18, 11000 Beograd, Srbija 2Naučni institut za veterinarstvo Novi Sad, Rumenački put 6, 21000 Novi Sad, Srbija. Preporuka Evropske agencije za procenu lekova (EMEA), američke Uprave za hranu i lekove (FDA), kao i Svetske zdravstvene organizacije (WHO) je da se sintetski lekovi, kad god je moguće, zamene bezbednim i efikasnim fitofarmacima. Visoko-razvijene zemlje Evrope u poslednjih nekoliko godina koriste fitofarmake za preveniranje i lečenje različitih bakterijskih i protozoarnih infekcija životinja, ali i kao stimulatore rasta. Ovakvim prilazom izbegava se prisustvo rezidua sintetskih lekova i njihovih metabolita u jestivim tkivima lečenih životinja, odnosno obezbeđuje se zdrava hrana za ishranu ljudi. Nekoliko godina unazad u veterinarskoj medicini se sa uspehom koristi fitofarmak na bazi eteričnog ulja origana indikovan za preveniranje kokcidioze i salmoneloze kod živine i svinja. Poznato je da eterična ulja lekovitih i začinskih biljaka ili njihovi sintetski aktivni principi imaju niz farmakoloških dejstava od kojih su za veterinarsku medicinu posebno značajni antimikrobno, antimikotičko, antiinflamatorno i antitoksično dejstvo. Relativno je malo podataka o farmakodinamskim dejstvima široko primenjivanog eugenola na pojedine sisteme organa. Cilj rada bio je da se ispita uticaj različitih doza eugenola na motilitet creva pacova, kao i da se ustanovi jačina njegovog dejstva. Dobijeni efekat primenjenih doza eugenola na motilitet creva upoređivan je sa efektom primenjenih doza atropin-sulfata. Eksperiment je izveden na 56 pacova soja Wistar, podeljenih u 6 eksperimentalnih i jednu kontrolnu grupu. Gastričnom sondom je aplikovana suspenzija barijum-sulfata, a neposredno posle aplikacije na isti način dat je eugenol u dozi od 100, 150 i 300 mg/kg t.m, kod prve tri eksperimentalne grupe i atropin-sulfat u dozi od 0,1, 0,5 i 1 mg/kg t.m. kod preostale tri grupe. Kontrolna grupa pacova dobijala je samo barijum-sulfat. Vreme do pojave barijum-sulfata u fecesu predstavlja vreme pasaže izraženo u minutima. Vreme pasaže crevnog sadržaja kod kontrolne grupe pacova iznosilo je prosečno 197,5 minuta. Prosečno vreme pasaže posle primene eugenola u dozi od 100 mg/kg iznosilo je 294 minuta, posle primene doze od 150 mg/kg 408 minuta, а posle primene najviše testirane doze 357 minuta. Eugenol u svim primenjenim dozama produžava vreme pasaže crevnog sadržaja. Produženje je, u poređenju sa kontrolom, statistički značajno posle primene eugenola u dozi od 100 i visoko značajno posle doze od 150 mg/kg. Eugenol u dozi od 100 i 150 mg/kg pokazao je intenzivniji spazmolitički efekat u odnosu na najvišu testiranu dozu. Intenzitet spazmolitičkog efekta eugenola primenjenog u dozi od 100 i 150 mg/kg odgovara jačini spazmolitičkog efekta atropina datog u dozi od 0,1 i 0,5 mg/kg. Eugenol primenjen p.o. u dozi od 100 i 150 mg/kg može da se koristi kao spazmolitik i ima efikasnost kao i atropin dat u dozi od 0,1 i 0,5 mg/kg. Literatura: 1. Maksimović Z. (2005) Antioksidativni potencijal lekovitog bilja, Prooksidativni efekti ksenobiotika i antioksidativna zaštita, Kurs kontinuirane edukacije, Farmaceutski fakultet, Beograd. 2. Leal-Cardoso J.H. et al. (2002) Inhibitory actions of eugenol on rat isolated ileum, Can. J. Physiol. Pharmacol. 80:901-6. 3. Limma C.C. et al. (2000) Relaxant and antispasmodic actions of methyleugenol on guinea-pig isolated ileum, Planta Med, 66:408-11.

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P38 The influence of eugenol on intestinal passage in rats

Milanka Jezdimirović1, Nevenka Aleksić 1, Saša Ivanović 1, Saša Trailović1, Radomir Ratajac2

1Faculty of veterinary medicine, Bulevar oslobođenja 18, 11000 Belgrade, Serbia 2Veterinary research institute Novi Sad, Rumenački put 6, 21000 Novi Sad, Serbia. There is a recommendation from the European Medicines Agency (EMEA), Food and Drug Administration (FDA) as well as World Health Organisation (WHO) that synthetic drugs should be replaced with safe and efficacious phytopharmaceutics. In recent years highly developed European countries have been using phytopharmaceutics for prevention and treatment of various bacterial and protozoal infections as well as growth stimulators in animals. Thus, the presence of residues of synthetic drugs and their metabolites in animal tissues is avoided, i.e. healthy food for human consumption is provided. The phytopharmaceutic from oregano etheric oil indicated for coccidiosis and salmonellosis prevention has been successfully used in veterinary medicine in the last several years. The fact that etheric oils from medicinal and spice herbs or their synthetic active principles have a wide range of pharmacological effects (antimicrobial, antimycotic, anti-inflammatory and antitoxic) being particularly important in veterinary medicine, is reknown. There is little evidence on the pharmacodynamic effects of wide-used eugenol on certain organ systems. The aim of the present study was both the investigation of the influence of various doses of eugenol on the intestinal motility in rats, as well as the estimation of its intensity. The effect of eugenol was compared with that of atropine sulphate. The experiment was conducted on 56 Wistar rats, divided into 6 experimental plus a control group. Barium sulphate suspension was given by gastric gavage immediately prior to eugenol in doses 100, 150 and 300 mg/kg BW to the first three experimental groups and atropine in doses 0.1, 0.5 and 1 mg/kg BW to the remaining experimental groups. The control group received barium sulphate only. The time until the appearance of barium sulphate in faeces indicated the intestinal passage time (expressed in minutes). In the control group the intestinal passage time was 197.5 minutes in average. The average time of food passage in the group that received eugenol 100 mg/kg was 294 minutes, after 150 mg/kg of eugenol it was 408 minutes аnd in rats with the highest tested dose of eugenol 357 minutes. In all doses applied eugenol prolonged the intestinal passage time. The prolongation was statistically significant after the 100 mg/kg dose and statistically highly significant with the dose of 150 mg/kg, in comparison to the control. The spasmolytic effect of eugenol in doses 100 i 150 mg/kg BW was more intensive than that of the highest dose tested. The intensity of the spasmolytic effect of eugenol applied in doses 100 and 150 mg/kg corresponded to the spasmolytic effect of atropine given in doses of 0.1 and 0.5 mg/kg, respectively. Eugenol in p.o. doses of 100 and 150 mg/kg may be used as spasmolytic and its efficacy is equal to the efficacy of atropin given in doses 0.1 and 0.5 mg/kg. References: 1. Maksimović Z. (2005) Antioksidativni potencijal lekovitog bilja, Prooksidativni efekti ksenobiotika i antioksidativna zaštita, Kurs kontinuirane edukacije, Farmaceutski fakultet, Beograd. 2. Leal-Cardoso J.H. et al. (2002) Inhibitory actions of eugenol on rat isolated ileum, Can. J. Physiol. Pharmacol. 80:901-6. 3. Limma C.C. et al. (2000) Relaxant and antispasmodic actions of methyleugenol on guinea-pig isolated ileum, Planta Med, 66:408-11.

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P39 Antiretroviralno dejstvo propolisa prema HIV-1 in vitro Dušica Stevanović1, Jevrosima Stevanović2 1 Biohemijska laboratorija, Zdravstveni centar Zaječar, Rasadnička bb, 19000 Zaječar, Srbija 2 Katedra za biologiju, Fakultet veterinarske medicine, Bul. oslobodjenja 18, 11000 Beograd, Srbija Ovaj rad daje informacije o dejstvu propolisa prema virusu HIV-1 procenjivanom in vitro na ćelijama koje ovaj virus najčešće inficira in vivo, odnosno na CD4+ limfocitima i mikroglijalnim ćelijama u kulturi. Propolis je jako lepljiv smolasti pčelinji proizvod. Pčele sakupljaju smolu sa drveća i lisnih pupoljaka i mešaju je sa voskom, polenom i sekretima svojih pljuvačnih žlezda. Zbog svojih lekovitih svojstava, propolis se koristi od antičkog doba. Najpoznatije dejstvo propolisa je antimikrobno, pri čemu je efikasan protiv širokog spektra mikroorganizama, uključujući i brojne oportunističke patogene koji se javljaju kod bolesnih od AIDS-a. Pre deset godina, koristeći CEM ćelijsku liniju, Harish i sar. (1997) dokazali su da propolis jako inhibira ekspresiju virusa HIV-1. Nedavno, Gekker i sar. (2005) ispitivali su antiretroviralnu aktivnost propolisa u ćelijama koje HIV-1 primarno napada, odnosno u CD4+ limfocitima i mikroglijalnim ćelijama. U tom ispitivanju, propolis je inhibirao ekspresiju virusa i efekat je direktno zavisio od koncentracije (najveći inhibitoni efekat uočen je pri primeni 66.6 µg/ml propolisa i iznostio je 85% u kulturama CD4+ limfocita, odnosno 98% u kulturama mikroglialnih ćelija). Mehanizam antiviralnog dejstva propolisa u CD4+ limfocitima obuhvata inhibiciju ulaska virusa u ćelije. Obzirom da antiretroviralna terapija podrazumeva korišćenje kombinacije lekova različitog mehanizma dejstva, bilo je neophodno ispitati moguće interakcije između propolisa i standardnih antiretroviralnih agenasa. U tom cilju, propolis je dodat u kulture CD4+ limfocita i mikroglijalnih ćelija koje su simultano tretirane zidovudinom ili indinavirom. Uočen je aditivni antiviralni efekat propolisa na zidovudin koji je inhibitor reversne transkriptaze, dok na indinavir, proteazni inhibitor, propolis nije imao efekta (Gekker i sar., 2005). Rezultati ovog in vitro ispitivanja ukazuju na potrebu kliničkog ispitivanja propolisa u tretmanu HIV-1 infekcija. Literatura: 1. Harish et al. (1997) Drugs Exp Clin Res 23, 89–96. 2. Gekker et al. (2005) J Ethnopharmacol 102, 158–163.

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P39 Antiretroviral activity of propolis against HIV-1 in vitro Dušica Stevanović1, Jevrosima Stevanović2 1 Biochemical Laboratory, Health Centre Zaječar, Rasadnička bb, 19000 Zaječar, Serbia 2 Department of Biology, Faculty of Veterinary Medicine, Bul. oslobodjenja 18, 11000 Belgrade, Serbia The paper gives information about anti-HIV-1 activity of propolis in vitro evaluated in the major cell types that are infected by this virus in vivo, i.e. CD4+ lymphocytes and microglial cell cultures. Propolis is a strongly adhesive resinous bee product. The resin is collected by honey bees from trees and leaf buds and combined with wax, pollen and salivary gland secretion. Used for medicinal purposes since antiquity, propolis possess broad spectrum antimicrobial activity, including activity against many of the opportunistic pathogens associated with the acquired immunodeficiency syndrome (AIDS). A decade ago, using a cell line (CEM cells), Harish et al. (1997) demonstrated that propolis potently inhibited HIV-1 expression. More recently, Gekker et al. (2005) investigated antiretroviral activity of propolis in the primary cell targets of HIV-1, i.e. CD4+ lymphocytes and microglial cell cultures. In that study propolis inhibited viral expression in a concentration-dependent manner (maximal suppression of 85 and 98% was observed at 66.6 µg/ml propolis in CD4+ and microglial cell cultures, respectively). The mechanism of propolis antiviral property in CD4+ lymphocytes appeared to involve, in part, inhibition of viral entry. Since antiretroviral therapy involves the use of drug combinations with different mechanisms of action, it was necessary to investigate potential interactions between propolis and standard antiretroviral agents. Thus, propolis was added to CD4+ lymphocyte and microglial cell cultures that were simultaneously treated with zidovudine or indinavir. While propolis had an additive antiviral effect on the reverse transcriptase inhibitor zidovudine, it had no noticeable effect on the protease inhibitor indinavir (Gekker et al., 2005). The results of this in vitro study support the need for clinical trials of propolis in the treatment of HIV-1 infection. References: 1. Harish et al. (1997) Drugs Exp Clin Res 23, 89–96. 2. Gekker et al. (2005) J Ethnopharmacol 102, 158–163.

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P40 Uporedno ispitivanje efikasnosti eugenola i permetrina u lečenju šuge svinja Nevenka Aleksić1, Milanka Jezdimirović1, Zoran Kulišić1, Radomir Ratajac2

1Fakultet veterinarske medicine, Bulevar oslobođenja 18, 11000 Beograd, Srbija 2Naučni institut za veterinarstvo Novi Sad, Rumenački put 6, 21000 Novi Sad, Srbija. Široka i/ili neadekvatna primena akaricida kod domaćih životinja često dovodi do razvoja rezistencije ektoparazita na primenjivane supstance [1]. Zbog toga postoji potreba za ispitivanjem efikasnosti i bezbednosti primene novih lekova koji bi se koristili u cilju lečenja artropodoza životinja. Pored brojnih novosintetisanih akaricida sve veći značaj imaju fitofarmaci. Za neke biljke je poznato da imaju akaricidno i insekticidno dejstvo (cimet, karanfilić, lavanda, korijander, nana, duvan, timijan, ren). Eterična ulja ovih biljaka sadrže više aktivnih sastojaka (u različitim procentima), od kojih su za akaricidno delovanje značajni fenoli. Eterično ulje karanfilića (Syzygium aromaticum, syn. Eugenia aromaticum ili Eugenia caryophyllata), sadrži 80-90 % aktivnog sastojka eugenola [2, 3, 4]. U radu je ispitivano akaricidno dejstvo eugenola na šugarce kod svinja i upoređivano sa permetrinom. Akaricidna efikasnost eugenola je ispitivana kod 32 svinje (četiri grupe po osam životinja) prirodno inficirane šugarcem Sarcoptes scabiei var. suis. Životinje su tretirane 10%, odnosno 20% rastvorom eugenola u etanolu i propilen-glikolu, koji je utrljavan u kožu. Prva i druga grupa svinja tretirane su 10% rastvorom eugenola, prva jednokratno, a druga trokratno (interval između aplikacije eugenola bio je sedam dana). Treća i četvrta grupa tretirana je 20% rastvorom eugenola, jednokratno (treća grupa), odnosno trokratno (četvrta grupa). Efikasnost permetrina primenjivanog u koncentraciji od 1% testirana je na 16 svinja (dve grupe po osam životinja), prema preporuci proizvođača. U prvoj grupi permetrin je primenjen jednokratnim utrljavanjem u kožu, a u drugoj na isti način, ali trokratno (interval između aplikacija permetrina iznosio je sedam dana). Kontrolna grupa svinja (osam inficiranih životinja) nije tretirana. Uzorci kože za pregled na šugu uzimani su sa promenjenih mesta na telu kod tretiranih i kontrolnih svinja, 7, 14, 21. i 28. dana od prvog tretmana, odnosno od početka ogleda. U lečenju šuge, eugenol u koncentraciji od 10%, primenjen jednokratno ispoljio je efikasnost kod 75,0%, a primenjen trokratno kod 100,0% životinja. U koncentraciji od 20%, primenjen jednokratno pokazao je efikasnost kod 87,5% tretiranih životinja, a posle trokratne primene njegova efikasnost u terapiji šuge iznosila je 100,0%. Maksimalna efikasnost (100,0%) u lečenju sarkoptes-šuge svinja postignuta je već posle druge primene 20 eugenola. Permetrin u koncentraciji 1% bio je efikasan kod 62,5 % svinja inficiranih šugarcima kada je primenjen jednokratno i kod 75,0% životinja posle trokratne primene. Na osnovu dobijenih rezultata akaricidne efkasnosti eugenola u lečenju šuge svinja može da se zaključi da ovaj aktivni sastojak eteričnog ulja karanfilića može da se koristi u ovu svrhu, jer je njegova efikasnost značajno veća od efikasnosti permetrina, a bezbednost zadovoljavajuća. Literatura: 1. Bishop Y. (2001) The veterinary formulary, 5th ed, The British Veterinary Associacion. 2. Gayoso C.W. et al. (2005) Sensitivity of fungi isolated from onychomycosis to Eugenia cariophyllata essential oil and eugenol, Fitotherapy, 76:247-9. 3. Jezdimirović M. i sar, (2006) Efikasnost eugenola u lečenju šuge svinja, Vet Glasnik, 60:33-42. 4. Kim S, Yi J.H, Tak J.H., Ahn Z.J. (2004) Acaricidial activity of plant essential oils against Dermanyssus galinae, Vet Parasitol, 120:297-304. 5. http://oznet.ksu.edu/library/entml2/c676.pdf Lice, mange and other swine insect problems.

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P40 The investigation of comparative efficacy of eugenol and permethrin in pig mange Nevenka Aleksić1, Milanka Jezdimirović1, Zoran Kulišić1, Radomir Ratajac2

1Faculty of veterinary medicine, Bulevar oslobođenja 18, 11000 Belgrade, Serbia 2Veterinary research institute Novi Sad, Rumenački put 6, 21000 Novi Sad, Serbia. Wide and/or inadequate administration of acaricides in domestic animals frequently leads to resistance of ectoparasites to substances applied [1], giving reason to the investigation of efficacy and safety of new medicines which can be used for treatment of arthropodoses of animals. Apart from novel synthetic acaricides, phytopharmaceutics have been of increasing importance. It is known that certain plants have acaricidal and insecticidal effects (cinnamon, clove, lavander, coriander, menthe, tobacco, thyme, horseradish). The essential oils of these herbs contain several active ingredients (in various quantities), the most important with acaricide activity being phenols. The essential oil of clove (Syzygium aromaticum, syn. Eugenia aromaticum or Eugenia caryophyllata) contains 80-90% of the active ingredient eugenol [2, 3, 4]. In this research the acaricide effect of eugenol on swine sarcoptic mange was investigated and compared with permethrine. The acaricide efficacy of eugenol was tested on 32 pigs (four groups, eight animals per group) with naturally acquired mange infection caused by Sarcoptes scabiei var. suis. The animals were treated with 10 % and 20 % eugenol solution in ethanol and propylenglycol, rubbed into skin. The first and the second group were treated with 10 % eugenol solution, the first group once and the second one three times (in seven-day intervals). The third and the fourth group were treated with 20% eugenol solution, once and three times, respectively. The efficacy of 1% permethrin was tested on 16 pigs (two groups, eight animals per group), as recommended. Permethrin was applied by rubbing into skin, once to the first group and three times to the second group of pigs (in seven-day intervals). The control group (eight infected pigs) remained untreated. Skin samples from affected sites of the body were collected from treated and control pigs on days 7, 14, 21 and 28 after the first treatment, i.e. from the beginning of the experiment. The efficacy of eugenol 10% in mange therapy after single application was 75.0% and 100.0% after triple treatment. In 20% concentration, eugenol expressed efficacy in 87.5% and 100.0% of animals when applied once and three times, respectively. Maximal efficacy (100.0%) in the treatment of pig mange was achieved after the second treatment with 20% eugenol. Permethrin 1% was efficacious in 62.5% of pigs infected with mange mites when applied once and in 75.0 % of animals after triple treatment. According to the results of acaricide efficacy of eugenol in the therapy of pig mange it can be concluded that this active ingredient of clove essential oil can be used in this purpose due to its efficacy which is significantly higher in comparison with the efficacy of permethrin, as well as satisfactory safety. References: 1. Bishop Y. (2001) The veterinary formulary, 5th ed, The British Veterinary Associacion. 2. Gayoso C.W. et al. (2005) Sensitivity of fungi isolated from onychomycosis to Eugenia cariophyllata essential oil and eugenol, Fitotherapy, 76:247-9. 3. Jezdimirović M. i sar, (2006) Efikasnost eugenola u lečenju šuge svinja, Vet Glasnik, 60:33-42. 4. Kim S, Yi J.H, Tak J.H., Ahn Z.J. (2004) Acaricidial activity of plant essential oils against Dermanyssus galinae, Vet Parasitol, 120:297-304. 5. http://oznet.ksu.edu/library/entml2/c676.pdf Lice, mange and other swine insect problems.

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P41 Isparljivi sastojci nativnog i termički tretiranog semena hrasta Rakić S1, Tešević V2, Jadranin M3, Petrović S4, Kukić J4, Povrenović D5, Šiler-Marinković S5 1Institut za prehrambenu tehnologiju i biohemiju, Poljoprivredni fakultet, Nemanjina 6, 11080 Zemun, Beograd, Srbija; 2Hemijski fakultet, Studentski trg 12-16, 11000 Beograd, Srbija; 3Institut za hemiju, tehnologiju i metalurgiju, Njegoševa 12, 11000 Beograd, Srbija; 4Institut za farmakognoziju, Farmaceutski fakultet, V. Stepe 450, 11221 Beograd, Srbija; 5Tehnološko-metalurški fakultet, Karnegieva 4, 11000 Beograd, Srbija Upotreba termički obrađenog hrastovog semena u ishrani, naročito u oblasti Mediterana i u Srbiji, ima dugu istoriju. Hrastovo seme se tradicionalno koristi kao adstringent, antidijaroik i antidot. Ono sadrži hranljive sastojke (ugljene hidrate, amino kiseline, proteine i masno ulje), kao i različite polifenole sa antioksidantnim delovanjem [1]. U ovom radu ispitivani su isparljivi sastojci nativnog i termički tretiranog semena dve vrste hrasta: cera, Quercus cerris i lužnjaka, Q. robur (Fagaceae). U literaturi se mogu naći podaci o isparljivim komponentama termički obrađenog hrastovog drveta [2]. Takođe, ima dosta podataka o isparljivim komponentama različitih vrsta semena (nativnih i termički obrađenih) [3,4], ali isparljivi sastojci hrastovog semena do sada nisu proučavani. Zreli hrastovi žirevi su oljušteni, semena osušena na sobnoj temperaturi i samlevena (sirovo seme). Deo uzorka je zagrevan na 200°C tokom 10 min (termički tretirano seme). Izolovanje isparljivih jedinjenja izvršeno je kontinualnom hidrodestilacijom sa CH2Cl2 u aparatu po Likens-Nickerson-u. Uzorak (10 g) je pomešan sa 150 ml destilovane vode i ekstrahovan pomoću 7 ml CH2Cl2 tokom 2 h. U ovako dobijenim metilenhloridnim ekstraktima GC/FID metodom utvrđena je relativna zastupljenost komponenata, a kombinovanom GC/MS metodom izvršena njihova identifikacija. U uzorcima nativnih semena identifikovano je ukupno 28 isparljivih jedinjenja, od čega 20 u semenu cera (92,98% detektovanih sastojaka) i 15 u semenu lužnjaka (91,91% detektovanih sastojaka). Najzastupljenija isparljiva komponenta semena cera je palmitinska kiselina (53,79% detektovanih sastojaka). Glavne isparljive komponente semena lužnjaka su β-eudezmol i palmitinska kiselina (39,90 i 24,87% detektovanih sastojaka). U termički tretiranim uzorcima identifikovano je ukupno 48 isparljivih jedinjenja, od čega 29 u semenu cera (96,33% detektovanih sastojaka) i 31 u semenu lužnjaka (94,99% detektovanih sastojaka). U isparljivim frakcijama, između ostalog, identifikovani su derivati furana, koji nastaju termičkom degradacijom ugljenih hidrata, i alkilpirazini, proizvodi termičke degradacije proteina. Furani i alkilpirazini su odgovorni za aromu koja nastaje tokom termičkog tretmana mnogih semena. U isparljivoj frakciji analiziranih uzoraka najzastupljeniji su bili furani: furfural (u semenu cera 60,58% i lužnjaka 51,73% detektovanih sastojaka) i 5-metil-furfural (9,45 i 8,62% detektovanih sastojaka). U semenu lužnjaka u značajnoj količini prisutan je i 2-hidroksi-1-metoksi etilfuran (9,90% detektovanih sastojaka). Sadržaj palmitinske kiseline nakon termičkog tretmana je značajno niži (u semenu cera 2,21% i lužnjaka 0,59% detektovanih sastojaka). Senzorna svojstva ispitivanih semena hrasta bila su međusobno gotovo identična, uprkos uočenim razlikama u kompoziciji njihovih isparljivih frakcija. Termički tretman značajno poboljšava njihove organoleptičke karakteristike. Literatura: 1. Rakić S. et al. (2007) Food Chem 104:830-834. 2. Nonier M.F. et al. (2006) J Anal Appl Pyrol 75:181-193. 3. Maga J.A. (1991) Nuts. In: Maarse, H. (Ed.). Volatile Compounds in Foods and Beverages. Marcel Dekker Inc. New York. 4. Siegmund B., Murkovic M. (2004) Food Chem 84:367–374.

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P41 Volatile constituents of native and thermally treated Oak acorn kernels Rakić S1, Tešević V2, Jadranin M3, Petrović S4, Kukić J4, Povrenović D5, Šiler-Marinković S5 1Department of Food Technology and Biochemistry, Faculty of Agriculture, Nemanjina 6, 11080 Belgrade-Zemun; 2Faculty of Chemistry, Studentski trg 12-16, 11000 Belgrade; 3Institute for Chemistry, Technology and Metallurgy, Njegoševa 12, 11000 Belgrade; 4Institute of Pharmacognosy, Faculty of Pharmacy, V. Stepe 450, 11221 Belgrade, Serbia; 5 Faculty of Technology and Metallurgy, Karnegieva 4, 11000 Belgrade, Serbia Usage of thermally treated Oak acorn kernels in nutrition, especially in the Mediterranean region and in Serbia, has a long history. Oak kernels are traditionally used as an astringent, antidiarrhoeal and antidote. They contain nutritional components (carbohydrates, amino acids, proteins and fixed oils), as well as various polyphenols with antioxidant properties [1]. In this work volatile constituents of native and thermally treated acorn kernels of Turkish Oak, Quercus cerris and English Oak, Q. robur (Fagaceae) were investigated. Data concerning volatiles from thermally treated Oak wood are available [2], as well as data about volatiles from various seeds (native and thermally treated) [3,4], but none dealing with those from Oak kernels. The mature Oak acorns were shelled out, the kernels dried at room temperature and powdered (native kernels). One part of these was heated at 200°C for 10 min (thermally treated kernels). Isolation of volatiles was performed using continuous hydrodistillation with CH2Cl2 in Likens-Nickerson apparatus. Sample (10 g) was mixed with distilled water (150 ml) and extracted with CH2Cl2 (7 ml) for 2 h. Obtained CH2Cl2 extracts were quantitatively and qualitatively analysed using GC/FID and GC/MS techniques, respectively. In samples of native kernels total of 28 volatile compounds were identified, of which 20 in Turkish Oak kernels (92.98% of detected compounds) and 15 in English Oak kernels (91.91% of detected compounds). The most prominent compound in Oak kernels was palmitic acid (53.79% of detected compounds). The main compounds in English Oak acorns were β-eudesmol and palmitic acid (39.90 and 24.87% of detected compounds, respectively). In thermally treated samples total of 48 volatile constituents were identified, of which 29 in Turkish Oak kernels (96.33% of detected compounds) and 31 in English Oak kernels (94.99% of detected compounds). In volatile fractions, among others, furane derivatives, products of thermal degradation of carbohydrates, and alkylpyrazines, products of protein thermal deprivation were identified. Furanes and alkylpyrazines are volatile flavour compounds which are formed during the thermal treatment of various seeds. The main volatile compounds in roasted Turkish and English Oak kernels were furanes: furfural (60.58 and 51.73% of detected compounds) and 5-methyl-furfural (9.45 and 8.62%). In English Oak kernels 2-hydroxy-1-methoxy ethylfurane (9.90% of detected compounds) was also detected in substantial amount. Palmitic acid content in samples after thermal degradation was significantly lower (2.21 and 0.59% of detected compounds, in Turkish and English Oak kernels, respectively). Flavours of investigated Quercus kernels are almost identical, in spite of differences in composition of their volatile fractions. Thermal treatment of kernels significantly improves their organoleptic characteristics. References: 1. Rakić S. et al. (2007) Food Chem 104:830-834. 2. Nonier M.F. et al. (2006) J Anal Appl Pyrol 75:181-193. 3. Maga J.A. (1991) Nuts. In: Maarse, H. (Ed.). Volatile Compounds in Foods and Beverages. Marcel Dekker Inc. New York. 4. Siegmund B., Murkovic M. (2004) Food Chem 84:367–374.

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P42 Uticaj hibrida na sadržaj karotina i vitamina C u korenu mrkve Đorđe Moravčević1, Vukašin Bjelić1, Slavica Jelačić1, Damir Beatović1 1Univerzitet u Beogradu, Poljoprivredni fakultet, Nemanjina 6, 11080 Zemun – Beograd, Srbija

Ispitivani su sledeći hibridi mrkve: Nanco, Tamino, Puma, Jaguar i Major. Prosečan sadržaj karotina iznosi 14,93 mg%. Najveću količinu karotina stvarao je hibrid Puma. Ta količina iznosi 17,23 mg%. Na drugom mestu je hibrid Jaguar, gde je količina karotina dostigla 15,91 mg%. Sledi hibrid Nanco, koji je stvorio 14,52 mg% karotina. Na kraju se nalaze hibridi Tamino i Major, gde količina karotina iznosi 13,87 mg% i 13,12 mg%. Prosečan sadržaj vitamina C dostigao je 10,62 mg%. Ovde se najbolje pokazao hibrid Nanco, koji je stvarao 11,45 mg% vitamina C. Na drugom mestu je hibrid major, sa 11,06 mg% vitamina C. Hibridi Jaguar i tamino zauzimaju drugo i treće mesto, gde vrednosti za sadržaj vitamina C iznose 10,52 mg% i 10,38 mg%. Poslednji je hibrid Puma, sa 9,74 mg% vitamina C. Mrkva se najviše ceni po visokom sadržaju karotina, koji u organizmu prelazi u vitamin A. Umereno je bogata vitaminom C, kao i drugim vitaminima. Odlikuje se lepim izgledom i prijatnim ukusom. Koristi se kao hrana i lekovito sredstvo.

References: 1. Grassman, J., Schnitzler, H., Habegger, R. (2007): Evaluation of different coloured carrot cultivars on atioxidative capacity based on their carotenoid and phenolic contents. International Journal of Food Sciences and Nutrition, V. 58, No 8, 603-611. 2. Kidmose, U., Hansen, S.I., Christensen, L.P., Edelenbos, M., Larsen, E.I. and Norbek, R. (2004): Effects of genotype, root size, storage, and processing on bioactive compounds in organically grown carrots (Daucus carota L.). Journal of Food Science, V. 69, No 9, 71-78. 3. Kvasnikov, B.V., Židkova, N.I. (1981): Selekcija morkovi na povišenije karotina. Kačestvo ovošćnih i bahčevih kultur. Kolos, Moskva, 60-68. 4. Nicolle, C., Simon, G., Rock, E., Amouroux, P. (2004): Genetic variability influences carotenoid, vitamin, phenolic, and mineral content in whote, yellow, purple, orange, and dark-orange carrot cultivars. Journal of the American Society for Horticultural Science, V. 129, No 4, 523-529. 5. Simon, P.W., Wolff, X.J. (1987): Carotenes in typical and dark orange carrots. Journal of Agricultural and Food Chemistry, 35, 8, 1017-1022.

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P42 The effect of hybrids on the contents of carotene and vitamin C in the carrot root Djordje Moravčević1, Vukašin Bjelić1, Slavica Jelačić1, Damir Beatović1 1University of Belgrade, Faculty of Agriculture, Nemanjina 6, 11080 Zemun – Belgrade, Serbia

The following hybrids of carrot were used: Nanco, Tamino, Puma, Jaguar and Major. Average contents of carotene was 14.93 mg%. The greatest quantity of carotene was produced by the hybrid Puma. This quantity amounted to 17.23 mg%. It was followed by the hybrid Jaguar, where the quantity of carotene was 15.91 mg%, hybrid Nanco, which produced 14.52 mg% of carotene, and finally hybrids Tamino and Major, where the quantity of carotene was 13.87 mg% and 13,12 mg% respectively. The average contents of vitamin C reached 10.62 mg%. Hybrid Nanco rated first according to this criterion and produced 11.45 mg% of vitamin C. It was followed by the hybrid Major, with 11.06 mg% of vitamin C. Hybrids Jaguar and Tamino came second and third, where the contents of vitamin C were 10.52 mg% and 10,38 mg% respectively. The last hybrid was Puma, with 9.74 mg% of vitamin C. Carrot is mostly appreciated for its high contents of carotene which is transformed in the body into vitamin A. It is moderately rich in vitamin C, as well as other vitamins. It is charaterized by nice appearance and taste. It is used both as food and as a medicinal agent. References: 1. Grassman, J., Schnitzler, H., Habegger, R. (2007): Evaluation of different coloured carrot cultivars on atioxidative capacity based on their carotenoid and phenolic contents. International Journal of Food Sciences and Nutrition, V. 58, No 8, 603-611. 2. Kidmose, U., Hansen, S.I., Christensen, L.P., Edelenbos, M., Larsen, E.I. and Norbek, R. (2004): Effects of genotype, root size, storage, and processing on bioactive compounds in organically grown carrots (Daucus carota L.). Journal of Food Science, V. 69, No 9, 71-78. 3. Kvasnikov, B.V., Židkova, N.I. (1981): Selekcija morkovi na povišenije karotina. Kačestvo ovošćnih i bahčevih kultur. Kolos, Moskva, 60-68. 4. Nicolle, C., Simon, G., Rock, E., Amouroux, P. (2004): Genetic variability influences carotenoid, vitamin, phenolic, and mineral content in whote, yellow, purple, orange, and dark-orange carrot cultivars. Journal of the American Society for Horticultural Science, V. 129, No 4, 523-529. 5. Simon, P.W., Wolff, X.J. (1987): Carotenes in typical and dark orange carrots. Journal of Agricultural and Food Chemistry, 35, 8, 1017-1022.

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P43 Antioksidativno delovanje nekih vrsta samoniklih gljiva

Senka S. Vidović1, Zoran P. Zeković1, Ibrahim O. Mujić2, Jelena V. Živković3, Vesna T. Tumbas1 1Tehnološki fakultet, Univerzitet u Novom Sadu, Bulevar Cara Lazara 1, 21000 Novi Sad, Srbija 2 Biotehnički fakultet, Univerzitet u Bihaću, Kulina Bana 2, Bihać, Bosna i Hercegovina 3 Medicinski fakultet, Univerzitet u Nišu, Bulevar Zorana Đinđića 81, 18 000 Niš, Srbija Antioksidanti štite organizam od posledica izazvanih oksidativnim stresom. U poslednjih nekoliko godina oni se iz tog razloga intenzivno proučavaju. Gljive se kao prirodni fitonutritijenti već dugo vremena upotrebljavaju u starim orjentalnim kulturama. Ustanovljeno je da one imaju antitumorno i antivirusno delovanje kao i da imaju osobinu da deluju kao imunomodulatori. Ispitano je antioksidativno delovanje četiri samonikle vrste gljiva sa područja Cazinske Krajine (Hrvatska). Pripremljeni su etanolni ekstrakti ovih gljiva i određen je njihov kapacitet delovanja na DPPH radikale kao i antioksidativno delovanje metodom “reducing power“. Takođe određen je sadržaj ukupnih fenola i ukupnih flavonoida u dobijenim ekstraktima. Ispitivane gljive Polyporus umbellatus, Ganoderma applantum, Clitocybe nebularis, Piptoporus betulinus su ekstrahovane 50% etanolom. Određen je kapacitet delovanja ekstrakata na DPPH radikale, na osnovu čega je dobijena IC50 vredost koja iznosi 0.089, 0.071, 0.075, 0.078 mg/ml, redom. Primenom metode “Reducing power“ i standardnih antioksidanata, kao sto su butilen hidroksianizol (BHA) i vitamin C, utvrđen je sledeći redosled antioksidativnog delovanja: vitamin C > BHA > Ganoderma applantum > Clitocybe nebularis > Piptoporus betulinus > Polyporus umbellatus pri koncentraciji od 1 mg/ml. Antioksidativno delovanje ispitivanih ekstrakata gljiva zavisilo je od sadržaja ukupnih fenola.

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P43 Antioxidant properties of selected wild mushrooms

Senka S. Vidovic1, Zoran P. Zekovic1, Ibrahim O. Mujic2, Jelena V. Zivkovic3, Vesna T. Tumbas1 1Faculty of Technology, University Novi Sad, Bulevar Cara Lazara 1, Novi Sad, Serbia 2 Biotechnical Faculty, University Bihać, Kulina Bana 2, Bihać, Bosnia and Herzegovina 3 Medical Faculty, University Niš, Bulevar Zorana Đinđića 81, Niš, Serbia Natural antioxidants have been extensively investigated in past few years for their capacity to protect organisms from damage caused by oxidative stress. Mushrooms have a history of use in traditional oriental cultures. They were found to have certain antitumor, anitviral and immunomodulating effects. The antioxidant properties of four wild mushrooms, collected from Cazinska Krajina (Croatia), were investigated. Mushroom ethanolic extracts were screened for their free radical-scavenging capacity and reducing power. Total phenolic and total flavonoid content of mushrooms were also determined. For extracts obtained with 50% ethanol from Polyporus umbellatus, Ganoderma applantum, Clitocybe nebularis and Piptoporus betulinus, DPPH free radical-scavenging activity was found to exhibit IC50 value at 0.089, 0.071, 0.075, 0.078 mg/ml extract concentration, respectively. Reducing power, compared with reducing power of standard antioxidants such are ascorbic acid and butylate dhydroxianisol (BHA), decreased in following order: ascorbic acid > BHA > Ganoderma applantum > Clitocybe nebularis > Piptoporus betulinus > Polyporus umbellatus for extract concentration of 1 mg/ml. Positive correlation between total phenolic content and antioxidant capacity of mushrooms has been found in this study.

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P44 Biološke aktivnosti otpadnog čvrstog materijala Salvia spp. nakon procesa hidrodestilacije etarskog ulja Veličković D1, Stanisavljević I2, Stojičević S3, Marinković V1, Lazić M2, Veljković V2 1 Zdravlje-Actavis, Vlajkova 199, 16000 Leskovac, Srbija 2 Tehnološki fakultet, Univerzitet u Nišu, Bulevar oslobodjenja 124, 16000 Leskovac, Srbija 3 Hemijska industrija Nevena A.D., Djordja Stamenkovića St,16000 Leskovac, Srbija Enormne količine aromatičnih biljaka, kao što su i vrste roda Salvia, koriste se širom sveta za produkciju etarskih ulja, gradeći pritom ogroman deo čvrstog otpadnog ostatka. Iz razloga što otpadni biljni materijal može sadržati biološke komponente [1,2], on može biti eksploatisan kao sekundarna sirovina za dobijanje različitih bioaktivnih komponenata putem ekstrakcije [2,3], što je tehnološki, ekonomski i ekološki opravdano. U ovom radu, ispitivana je antioksidantna i antimikrobna aktivnost dve vrste roda Salvia (obična žalfija - Salvia officinalis L. i lepljiva žalfija - Salvia glutinosa L.), kao i količina ukupnih fenola i flavonoida. Za ispitivanje su korišćeni ekstrakti herbe (H) i rezidualnog biljnog materijala (SPM) koji zaostaje nakon hidrodestilacije etarskih ulja. Za ekstrakciju su korišćeni rastavrači različite polarnosti (70 % V/V vodeni rastvor etanola i metanol). Ekstrakti su dobijeni ultrazvučnom ekstrakcijom i maceracijom. Pokazalo se da antioksidantna i antimikrobna aktivnost ekstrakata zavisi od ekstrakcionih uslova i tipa biljnog materijala. SPM ekstrakti S. officinalis sadržali su više flavonoida od ekstrakata S. glutinosa. Takođe, SPM ekstrakti su pokazali, generalno, veću antimikrobnu aktivnost prema svim korišćenim mikroorganizmima u odnosu na H ekstrakte. Na osnovu dobijenih rezultata, može se zaključiti da SPM navedenih vrsta roda Salvia predstavlja sirovinu za pripremu potencijalno korisnih ekstrakata. Zahvalnice: Rad je podržan od strane Ministarstva nauke i zaštite životne sredine Republike Srbije (Projekat 142073). Literatura: 1. Veličković D. et al. (2006) Ultrason. Sonochem. 13:150-156. 2. Veličković D. et al. (2007) J. Serb. Chem. Soc. 72:73-80. 3. Veličković D. et al. (in press) Biochem. Eng. J.

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P44 Biological activities of waste solid residues from the Salvia sp. essential oil hydrodistillation process Veličković D1, Stanisavljević I2, Stojičević S3, Marinković V1, Lazić M2, Veljković V2 1 Zdravlje-Actavis, 199 Vlajkova St, 16000 Leskovac, Serbia 2 Faculty of Technology, University of Niš, 124 Bulevar oslobodjenja St, 16000 Leskovac, Serbia 3 Chemical Industry Nevena A.D., Djordja Stamenkovića St,16000 Leskovac, Serbia Enormous quantities of aromatic plants such as the Salvia sp. are used in the production of essential oils all over the world, thereby creating a huge waste solid residue. Since these residual plant material may contain biological compounds [1,2], they could be exploited as secondary raw materials for obtaining different bioactivities using solvent extraction [2,3], with possible technological, economical and ecological justification. In this work, it was examined antioxidant and antimicrobial activities of two Salvia sp. (garden sage - Salvia officinalis L. and glutinous sage - Salvia glutinosa L.), as well as total phenols and flavonoids amount. For investigation it were used herb (H) and spent plant material (SPM) remaining after the hydrodistillation of essential oils, as and solvents of different polarity (70 % V/V aqueous ethanol and methanol). The extracts were obtained by ultrasound-assisted extraction and maceration. It was shown that antioxidant and antimicrobial activity of the extracts depended on the extraction operating conditions and the type of plant materials. SPM extracts of S. officinalis contained more flavonoids than those of S. glutinosa. The SPM extracts were shown to posses, generally, a higher antimicrobial activity against the all microorganisms than the H extracts. Based on the results obtained, one could conclude that the SPM from the Salvia sp. essential oil hydrodistillation process could be used as possible sources of exploitable natural products. Acknowledgements: The Ministry of Science and Environmental Protection of the Republic of Serbia supported this work (Project 142073). References: 1. Veličković D. et al. (2006) Ultrason. Sonochem. 13:150-156. 2. Veličković D. et al. (2007) J. Serb. Chem. Soc. 72:73-80. 3. Veličković D. et al. (in press) Biochem. Eng. J.

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P45 Solubilizacija resveratrola u žučnim kiselinama M. Atanacković1, M. Poša1, V. Krstonošić1, J. Cvejić1, Lj. Gojković-Bukarica3 1Zavod za Farmaciju, Medicinski fakultet, Hajduk Veljkova 3, 21000, Novi Sad, Srbija. 2Zavod za Farmakologiju, Toksikologiju i Kliničku farmakologiju, Medicinski fakultet, P.O.Box 840, 11129, Beograd, Srbija.

Poznavanje procesa formiranja molekulskih agregata žučnih kiselina u vodenim sredinama je veoma važno za razumevanje solubilizacije liposolubilnih supstanci u biološkim sistemima. Micelarna solubilizacija, koja povećava rastvoljivost slabo rastvornih organskih supstanci u vodi inkorporacijom u micele, igra bitnu ulogu u biološkim procesima kao što su adsorpcija i transfer supstanci u tkiva [1]. Kritična micelarna koncentracija (CMK) je karakteristična vrednost za žučne kiseline koja opisuje tendenciju molekula da se samoasociraju ili formiraju mešane micele. Resveratrol (trans-3,5,4’-trihidroksistilben) je fitoaleksin koji nastaje u vinovoj lozi pod uticajem gljivičnih infekcija i njegova blagotvorna dejstva na zdravlje široko su poznata. Utvrđeno je da poseduje antioksidativna i antikancerogena svojstva, ali mu je oralna bioraspoloživost mala [2]. Solubilizacija resveratrola u žučnim kiselinama ispitivana je na sobnoj temperaturi nakon 18 sati mešanja 1mg/ml resveratrola u rastvorima žučnih kiselina u puferu pH 7. Kontrolni rastvor je bio rastvor 1mg/ml resveratrola u puferu pH 7 pripremljen na isti način. Korišćene su natrijumove soli 12-monoketodeoksiholne (12-MKDH) i 3,7,12-triketoholne kiseline (3,7,12-TKH) u koncentracijama 0.5, 1, 1.5 CMK za svaku kiselinu. Detekcija i kvantifikacija je vršena pomoću HPLC sistema sa UV detekcijom.

0.5 CMK 1 CMK 1.5 CMK Na-12MKDH 2,78 3,96 82,62

Na-TKH 50,05 87,56 93,99 *Kontrolni rastvor 0.006 mg/ml Obe žučne kiseline poboljšale su rastvorljivost resveratrola (Tabela 1). Ali, Na-TKH kiselina pokazala je očigledno veći solubilizacioni efekat na 0,5 i 1 CMK vrednostima. Ova pojava se može objasniti činjenicom postoje dve vrste interakcija: hidrofobne interakcije između svake žučne kiseline i resveratrola i interakcije između OH grupa resveratrola i keto grupa kiselina. Stoga, usled prisustva dodatnih keto grupa, stabilnost 3,7,12-TKH micela se povećava i solubilizacija resveratrola raste. 1. De Castro. et al. (2001) J. Pharm. Biomed. Anal 24: 595–602. 2. Walle et al. (2004) Drug. Metab. Dispos. 32(12): 1377-1382.

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P45 Solubility of resveratrol in bile acids

M. Atanacković1, M. Poša1, V. Krstonošić1, J. Cvejić1, Lj. Gojković-Bukarica3 1Department of Pharmacy, Faculty of Medicine, Hajduk Veljkova 3, 21000, Novi Sad, Serbia. 2Department of Pharmacology and Toxicology and Clinical Pharmacology, Faculty of Medicine, P.O.Box 840, 11129, Belgrade. Serbia.

The knowing of the process of formation of molecular aggregates of bile acids in aqueous media is important in view of the solubilization of liposoluble substances in biological systems. Micellar solubilization, the enhanced aqueous solubility of otherwise poorly soluble organic substances, which is caused by their incorporation into micelles, plays a very important role in biological processes, such as adsorption and transfer of materials in living tissues [1]. Critical micellar concentration (CMC) is an important characteristic of bile acids as its value determines the tendency of bile acid molecules to self-associate or form mixed micelles. Resveratrol (trans-3,5,4’-trihydroxystilbene) is a phytoalexin produced by grapevines in response to fungal infection and benefits of trans-resveratrol and its glucoside to health is widely reported. It has been shown that it possesses antioxidative and anticarcinogenic properties, but its oral bioavailability is poor [2]. Solubilization of resveratrol in bile acids solutions was investigated after 18 hours mixing on room temperature 1mg/ml of resveratrol in bile acids solutions in pH 7 buffer. Control solution was 1mg/ml of resveratrol in buffer solution pH 7 prepared in the same way. Sodium salts of 12-monoketodeoxycholic (12-MDCA) and 3,7,12-triketocholic acids (3,7,12-TCA) were used, in concentrations 0.5, 1, 1.5 CMC for each acid, respectively. Detection and quantification were performed using HPLC system with UV detection.

0.5 CMC 1 CMC 1.5 CMC Na-12MKDC 2,78 3,96 82,62 Na-TKC 50,05 87,56 93,99

* Control solution 0.006 mg/ml Both bile acids improved solubility of resveratrol (Table 1.). But, Na-TKC showed obviously bigger solubilization effect on 0,5 and 1 CMC values. This effect can be explained with the fact that except hydrophobic interactions of both acids with resveratrol, interactions between keto groups of bile acids and OH groups of resveratrol occur. Therefore, due to the presence of additional keto groups, stability of 3,7,12-TKC micelles is improved and solubilization of resveratrol is enhanced. 1. De Castro. et al. (2001) J. Pharm. Biomed. Anal 24: 595–602. 2. Walle et al. (2004) Drug. Metab. Dispos. 32(12): 1377-1382.

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P46 Biljni ekstrakti u kozmetičkim proizvodima za tretman celulita Jakšić I, Savić S, Vuleta G 1 Farmaceutski fakultet, Vojvode Stepe 450, Beograd, Srbija

Celulit predstavlja kozmetički neprihvatljivu pojavu koja se javlja kod oko 85% žena nakon puberteta. Etiologija celulita je i danas nejasna, mada postoje brojne teorije. U tretmanu celulita, biljni ekstrakti se mogu primeniti lokalno, oralno ili injekcionim putem (mezoterapija). Cilj rada bio je da se napravi pregled najčešće korišćenih biljnih ekstrakata i preporučenih koncentracija u kozmetičkim proizvodima za tretman celulita, i iznesu podaci dobijeni istraživanjem mehanizama kojim biljni ekstrakti i njihovi aktivni sastojci deluju protiv celulita. Dugo godina su metilksantini veoma interesantni za tretman celulita. Deluju na nivou adrenergičkih receptora kao β-agonisti i stimulišu proces lipolize preko receptora koji se nalaze na površini adipocita [1]. Najčešće se koristi kofein zbog zadovoljavajuće penetracije kroz stratum corneum. On deluje na nivou adipocita, stimuliše lipolizu, inhibira fosfodiesterazu i tako povećava nivo cAMP koji je neophodan za hidrolizu triglicerida. S druge strane, kofein poboljšava i tkivnu mikrocirkulaciju, a obično se koristi u koncentraciji od 1 do 2% [2]. Danas se u kozmetičkim proizvodima za tretman celulita široko koristi ekstrakt zelenog čaja (Camellia sinensis, Theaceae), zbog sadržaja kofeina (2-4%), i drugih aktivnih principa koji deluju drugačijim mehanizmima. Listovi zelenog čaja bogati su polifenolima (8-12%), kao i katehinima kojima se pripisuje antiinflamatorna aktivnost, naročito na nivou slobodnih radikala. Najjači antioksidans među katehinima je epigalokatehingalat koga sadrže sve vrste čaja (crni, beli, zeleni...), ali se u zelenom čaju nalazi u najvećoj koncentraciji [1, 3]. Ekstrakt bršljana (Hedera helix, Araliaceae), je često korišćeni sastojak ovih kozmetičkih proizvoda zbog antiinflamatornog efekta, kao i povoljnog uticaja na mikrocirkulaciju i prevenciju pojave edema. Bogat je saponozidima, među kojima je najvažniji hederakozid C, i flavonoidima (rutozid, rutinozid). Saponozidi deluju protektivno na krvne sudove i smanjuju im permeabilnost, poboljšavaju limfnu drenažu i redukuju edem, dok flavonoidi imaju antiinflamatorno dejstvo. Zbog moguće pojave alergijskih reakcija, preporučuje se koncentracija do 2% [1, 2]. Ilex paraguariensis (mate) je u poslednjih nekoliko godina dobio značajnu pažnju, između ostalog, zbog antioksidativnog efekta, pošto sadrži nešto veću količinu polifenola, u poređenju sa zelenim čajem. Takođe, mate i zeleni čaj se razlikuju i po vrsti polifenola koje sadrže, s obzirom da je mate bogat hlorogenskom kiselinom, a ne sadrži katehine [4]. Ekstrakt gotu kole (Centella asiatica, Apiaceae), se u tretmanu celulita primenjuje i mezoterapijom, što podrazumeva aplikaciju malih količina proizvoda direktno u dermis pomoću igala dužine samo 4 mm, čime se zaobilazi epidermalna barijera. S obzirom na široku primenu biljnih ekstrakata u tretmanu celulita, javlja se potreba za relevantnim naučnim ispitivanjima njihove efikasnosti i bezbednosti. Različiti faktori utiču na celulit, poput režima ishrane, fizičke aktivnosti, gubitka telesne mase, što sve otežava evaluaciju efikasnosti ovih preparata. Tu su i mnogi predisponirajući faktori poput pola, rase, genetskog nasleđa, telesne težine i starosti. Imajući sve ovo u vidu, kao i činjenicu da formiranje celulita traje godinama, ne može se očekivati brz efekat nakon primene bilo kog kozmetičkog proizvoda sa navedenim biljnim ekstraktima, bez obzira na reklamne poruke koje prate ove proizvode. Literatura: 1. Oborska A. (2007) Personal Care (May):37-40. 2. Hexsel D. et al. (2005) Dermatol Surg 31: 866-872. 3. Thornfeldt C. (2005) Dermatol Surg 31: 873-880. 4. Heck C. I. et al. (2007) J Food Sci 72: 138-151.

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P46 Plant extracts in cosmetic products used in the treatment of cellulite Jakšić I, Savić S, Vuleta G 1 Faculty of Pharmacy, Vojvode Stepe 450, Belgrade, Serbia Cellulite is an aesthetically unacceptable cosmetic problem which appears in approximately 85% of women after puberty. In spite of the numerous theories, its etiology is still uncertain. In the treatment of cellulite, cosmetic products containing plant extracts can be administered topically, orally or by intradermotherapy (mesotherapy). This article reviews plant extracts most commonly used, their active ingredients and recommended concentrations in anti-cellulite products. Methylxanthines are ingredients of considerable interest for the treatment of cellulite. They act as β-adrenergic agonists and stimulate lipolysis through receptors placed on the fat-cell surface [1]. Caffeine is frequently used due to its satisfactory penetration through stratum corneum. It stimulates lipolysis by phosphodiesterase inhibition, which leads to the increase in cAMP concentration necessary for triglyceride hydrolysis. Furthermore, caffeine stimulates microcirculation and is commonly used in concentration 1-2% [2]. Green tea extract (Camellia sinensis, Theaceae), which is widely used in anti-cellulite cosmetic products, contains caffeine (2-4%), as well as other active ingredients affecting cellulite through various mechanisms. Green tea leaves are rich in polyphenols (8-12%), and catechins which are ingredients responsible for antioxydant and anti-inflammatory activity. The most potent antioxydant among catechins is epigallocatechin gallate, present in all teas (black, white, green...), but green tea contains the highest concentration [1, 3]. Common Ivy extract (Hedera helix, Araliaceae), is a common ingredient of these cosmetic products due to its anti-inflammatory activity, as well as its influence on microcirculation and oedema prevention. The extract is rich in saponins, especially hederacoside C, and flavonoids (rutosid, rutinosid). Saponins protect blood vessels and decrease their permeability, influence lymphatic drainage and reduce oedema, while flavonoids have anti-inflammatory activity. Because of the possible allergic reactions, concentration up to 2% is recommended [1, 2]. Ilex paraguariensis (mate tea), has attracted considerable attention recently, mainly for its antioxydant activity, owing to the significant content of polyphenols, in comparison to green tea. Mate and green tea contain different polyphenols, mate is rich in chlorogenic acid and has no catechins [4]. Gotu Kola extract (Centella asiatica, Apiaceae), can be used by mesotherapy in the treatment of cellulite. In mesotherapy, small amounts of the product is administered directly in dermis using 4 mm needles, in order to avoid epidermal barrier. Considering the increasing use of plant extracts in cellulite treatment, relevant efficacy and safety studies are required. Various factors influence cellulite, such as diet, physical exercise, weight loss, making the evaluation of product’s efficacy even more difficult. Many predisposing factors, like gender, race, genetics, body weight and age, should also be taken into consideration. In conclusion, since cellulite takes several years to be formed and visible, fast effect after the use of cosmetic products containing any of the extracts mentioned above should not be expected, regardless of the accompanying commercial messages. References: 1. Oborska A. (2007) Personal Care (May):37-40. 2. Hexsel D. et al. (2005) Dermatol Surg 31: 866-872. 3. Thornfeldt C. (2005) Dermatol Surg 31: 873-880. 4. Heck C. I. et al. (2007) J Food Sci 72: 138-151.

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P47 Biljne sirovine u kozmetičkim proizvodima za decu i bebe Lukić M, Savić S, Vuleta G Farmaceutski fakultet, Vojvode Stepe 450, 11000, Beograd, Srbija; Kozmetički proizvodi za decu formulišu se kao posebna grupa proizvoda za osetljivu kožu, namenjenu najmlađima. Pri tome se uvek razmatraju poslednja naučna saznanja značajna sa aspekta bezbednosti i kompatibilnosti, uz veoma brižljiv odabir sirovina [1]. Trend u svetu, a sve više i kod nas, jeste primena tzv. „prirodne kozmetike“ u kojima su glavni sastojci biljne sirovine, pre svega biljni ekstrakti i masna ulja. Da bi se koristile kao sirovine za izradu kozmetičkih proizvoda za decu i bebe, neophodno je da biljni ekstrakti budu visokog kvaliteta, što je moguće obezbediti primenom smernica za izbor sirovina, validiranih proizvodnih procesa i odgovarajućom analitičkom kontrolom [2]. Biljni ekstrakti ili izolovani aktivni biljni principi koriste se u kozmetičkim proizvodima za decu i bebe kao aktivne supstance antiinflamatornog, antiseptičnog i epitelizantnog dejstva, a biljna ulja često su prisutna kao emolijensi i premašćivači. Biljne sirovine, najčešće prisutne u proizvodima dečije kozmetike su: ekstrakti kamilice, nevena, aloe gel/ekstrakt, ekstrakt korena gaveza, ekstrakt pšenice, bademovo, sojino i maslinovo ulje i ulje žutog noćurka. Kamilica je jedna od najčešće korišćenih biljaka i uglavnom se koriste ekstrakti kamilice. Sa kozmetičkog aspekta, najznačajniji sastojci ekstrakta su: terpenoidi (α-bisabolol, α-bisabolol oksid A, α-bisabolol oksid B), flavonoidi (glikozidi apigenina, luteolina i kvercetina) i kumarini (umbeliferon, metilumbeliferon i herniarin). Antiinflamatorno, umirujuće i antioksidativno delovanje su najznačajniji efekti na koži aktivnih sastojaka kamilice, a pre svega se pripisuju α-bisabololu, azulenu i apigeninu [3]. Na tržištu postoje standardizovani i prečišćeni biljni ekstrakti, kao i izolovani aktivni biljni principi, koji se koriste u proizvodnji kozmetičkih proizvoda za decu kao: Herbalia® Chamomila-Cognis, Nemačka; Camomile Flower CO2-se extract (integr.)-Flavex® Naturextracte, Nemačka; H/E Chamomile-Dow Corning, SAD; Bisabolol-BASF, Nemačka. Ekstrakt cveta nevena nalazi primenu u gotovo svim vrstama preparata dečije i bebi kozmetike za negu i zaštitu. Ovaj ekstrakt sadrži karotenoide (β-karoten, flavoksantin, flavohrom...), flavonoide (glikozidi izoramnetina, kvercetina), triterpenske alkohole i saponine koji deluju antiiritantno, antiseptično i zaceljujuće [4]. U grupu ekstrakata koji zadovoljavaju visoke zahteve za kvalitet i bezbednost spadaju: H/E Marigold-Dow Corning, SAD; Marigold Flower CO2-se extract (integr.)-Flavex® Naturextracte, Nemačka. Aloe gel/ekstrakt se široko koristi u preparatima za decu i bebe. Gel sadrži 0,7-1,5% suvog ostatka, a 25% tog ostatka čine polisaharidi [5]. Primena aloje u kozmetičkim proizvodima zasnovana je na protivupalnom, emolijentnom i vlažećem efektu gela/ekstrakta (Aloveria®-Cognis, Nemačka; Aloe Con UP 1 FG, Aloe Con UP 10 FG, Aloe Oil Extract #101-C, Aloe Con UP 200-Jan Dekker, Velika Britanija, su samo neke od sirovina na tržištu). S obzirom na veoma široku primenu određenih biljnih ekstrakata i ulja u kozmetičkim proizvodima za decu i bebe, godinama se sprovode brojna naučna ispitivanja njihove bezbednosti i efikasnosti, ali je još uvek malo relevantnih podataka o tome. Literatura: 1. IKW Working Group „Baby Cosmetics“ (2004) SÖFW Journal 130:30-6. 2. Martinez S. et al. (2007) Skin Care Forum issue 42 http://www.scf-online.com/. 3. Areus-Corell M. (2001) Scin Care Forum issue 24 http://www.scf-online.com/. 4. Patri F., Silano V. (2002) Plants in cosmetics: Plants and plant preparations used as ingredients for cosmetic products Vol. 1 Councile of Europe. 5. Fox C. (1997) Cosm&Toilet. 112:67-84

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P47 Plant raw materials in cosmetic products for children and babies Lukić M, Savić S, Vuleta G Faculty of Pharmacy, Vojvode Stepe 450, 11000, Belgrade, Serbia Cosmetic products for children are formulated as a special group of products for sensitive skin, intended for the youngest population. Special attention is given to the latest scientific knowledge important in respect to safety and compatibility, with a very careful choice of raw materials [1]. The latest world trend, accepted in our country as well, is the implementation of the so-called “natural cosmetics” which comprises mainly of plant raw materials, primarily of plant extracts and vegetable oils. In order for the plant extracts to be used in the production of cosmetic products for children and babies, it is necessary to be of constantly high quality, which can be acquired through the implementation of guidelines for the selection of the raw materials, validated production processes and appropriate analytic control [2]. Plant extracts or contained actives with anti-inflammatory, antiseptic and anti-irritation properties are the most frequently used active substances in cosmetic products for children and babies, while vegetable oils are often present as emollients and fatting agents. Commonly used plant raw materials are: camomile extracts, marigold extract, aloe gel/extract, extracts of comfrey root, oat extract, almond, soybean and olive oils and evening primrose oil. A usual choice in these products is camomile, in extract form. From the cosmetic aspect, the most important components of the extract are: terpenoids (α-bisabolol, α-bisabolol oxide A and B), flavonoids (glucosides of apigenin, luteolin and quercetin) and coumarins (umbelliferone, methylumbelliferone and herniarin). Anti-inflammatory, soothing and antioxidant effects of camomile active ingredients are the most important ones, and they are attributed to α-bisabolol, azulene and apigenin [3]. There are standardized and refined plant extracts on the market, as well as isolated active plant principles which are used in the production of cosmetic products for children such as: Herbalia® Chamomila-Cognis, Germany; Camomile Flower CO2-se extract (integr.)-Flavex® Naturextracte, Germany; H/E Chamomile-Dow Corning, USA; Bisabolol-BASF, Germany. Marigold flower extract is implemented in almost all types of cosmetics products for care and protection of children and babies skin. This extract contains carotenoids (β-carotene, flavoxanthin, flavochrome), flavonoids (isorhamnetin glycoside, quercetin), triterpen alcohols and saponins which have anti-irritating, antiseptic and healing effect [4]. Some of trade mark group of extracts satisfying high quality and safety demands are: H/E Marigold-Dow Corning, USA; Marigold Flower CO2-se extract (integr.)-Flavex® Naturextracte, Germany. Aloe gel/extract is widely used in the cosmetics for children and babies. The gel contains 0.7-1.5% of dry residue, while 25% of that residue is made up of polysaccharides [5]. The implementation of aloe in cosmetic products is based on its anti-inflammatory, emollient and moisturizing effects. Aloveria®-Cognis, Germany; Aloe Con UP 1 FG, Aloe Con UP 10 FG, Aloe Oil Extract #101-C, Aloe Con UP 200-Jan Dekker, UK, are just some of the raw materials on the market. Considering wide use of certain plant extracts and oils in cosmetic products for children and babies, their safety and efficacy have been the subject of numerous scientific studies over the years; however there is a lack of relevant data on this issue. Literature: 1. IKW Working Group „Baby Cosmetics“(2004) SÖFW Journal 130:30-6. 2. Martinez S. et al. (2007) Skin Care Forum issue 42 http://www.scf-online.com/. 3. Areus-Corell M. (2001) Scin Care Forum issue 24 http://www.scf-online.com/. 4. Patri F., Silano V. (2002) Plants in cosmetics: Plants and plant preparations used as ingredients for cosmetic products Vol. 1 Councile of Europe. 5. Fox C. (1997) Cosm&Toilet. 112: 67-84.

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P48 Etarsko ulje čajevog drveta u farmaceutskim preparatima za lokalnu primenu Vučeljić Ivana1, Milanović Isidora2, Jelić Mladenka3

1 Z.U. apoteka "Divpharm", B.Dimitrijevića 26, 12000 Požarevac, Srbija 2Visoka zdravstvena škola strukovnih studija, Cara Dušana 254,11080 Beograd, Srbija 3OŠ”Svetislav Golubović - Mitraljeta”, Dalmatinske zagore 2, 11273 Beograd, Srbija Etarsko ulje čajevog drveta dobija se destilacijom lista drveta Melaleuca alternifolia, koje raste u uskim suptropskim obalskim regionima Novog Južnog Velsa i Kvinslenda u Australiji. Primarno sadrži monoterpene, a terpinen-4-ol je sastojak koji ispoljava najširi antimikrobni spektar u odnosu na druge komponente (1). Ispitivanja su pokazala da ovo ulje poseduje, širok spektar antimikrobnog dejstva, na Escherichia coli, Streptococcus i Staphylococcus vrste. Takodje, njegove aktivne komponente inhibiraju i rast Candida albicans (2). Rezultati studije izvedene na Univerzitetu u Minesoti, u kojoj je ispitivan uticaj ulja čajevog drveta kod infekcije kože i podkožnog tkiva (uzrokovane Staphylococcus aureus), pokazali su da se, ono kao dopunska terapija, može koristiti zbog antimikrobnih, antiinflamatornih efekata i sposobnosti da aktivira monocite. Uočeni su i neki sporedni efekti, pri lokalnoj primeni etarskog ulja čajevca u niskim koncentracijama, od kojih se kontaktni dermatitis javlja najčešće (3). Studija, izvedena u Nemačkoj i objavljena 2006 godine, o in vitro ispitivanjima antibakterijskog i antigljivičnog dejstva većeg broja etarskih ulja, na populaciji mikroorganizama sa vaginalne sluzokože u toku infekcije, pokazala je da najjače dejstvo su imala etarska ulja limunove trave, lavande i čajnog drveta(4). U ispitivanju sprovedenom na 45 pacijenata sa akutnim suvim i vlažećim kontaktnim dermatitisom, tokom 9 meseci u lečenju je korišćena smeša etarskih ulja, u kojoj je bilo prisutno i etarsko ulje čajevog drveta, uporedo sa određenim kortikosteroidnim preparatima. Postignuto je željeno terapijsko dejstvo, a sporedni efekti nakon primene smeše etarskih ulja bili su znatno slabije izraženi u odnosu na kortikosteroidne preparate (5). Etarsko ulje čajevog drveta ulazi u sastav nekih farmaceutskih preparata za dopunsku terapiju oboljenja kože ili vaginalnih preparata (vaginalni gel, vagitorije). Na našem tržištu, zastupljeni su proizvodi Australijskog proizvođača Thursday Plantation Health: gel za tretman akni koji sadrzi 20% etarskog ulja čajevog drveta i gel za odstranjivanje vašiju i gnjida. Na evropskom tržištu mogu se naći i preparati druge namene: Femisan® vaginalni gel (Altermed Corporation a.s., Olomouc, Česká republika), koji sadrži kombinaciju etarskih ulja karanfilića i čajevca, sa dodatkom mlečne kiseline i vagitorije (Tea Tree Oil® Suppository Pessary, Thursday Plantation, Australija) sa 200mg etarskog ulja čajevca. Oba preparata se koriste kod upalnih procesa praćenih pojačanim vaginalnim sekretom i dovode od smanjenja upale. Literatura: 1.Carson CF et al: Antimicrobial activity of the major components of the essential oil Malaleuca alternifolia,2002,J of Applied Bacteriology,vol 78 (3):264-9; 2.Reichling J et al: Bioactive essential oils used in phytomedicine as anti-infective agents: Australian tea tree oil and manuka oil, 2002, Acta Phytotherapeutica, vol1:26-32; 3.Halcon L. et al: Staphylococcus aureus and wounds: A review of tea tree oil as a promising antimicrobial, 2004, American Journal of Infection Control, vol 32(7):402-408; 4. Schwiertz A. et al: In vitro activity of essential oil on microorganisms isolated from vaginal infections,2006, Int J Aromatherapy, vol 16 (3):169-174; 5.Hadji-Minaglou F. et al.: The potential role of specific essential oils in the replacement of derma corticoid drugs (strong, medium and weak) in the treatment of acute dry and weeping dermatitis, 2005, Int J Aromatherapy, vol 15(2): 66-73;

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P48 Tea tree essential oil in pharmaceutical preparation for external use Vučeljić Ivana1, Milanović Isidora2, Jelić Mladenka3

1 Z.U. apoteka "Divpharm", B.Dimitrijevića 26, 12000 Požarevac, Serbia 2Visoka zdravstvena škola strukovnih studija, Cara Dušana 254,11080 Beograd, Serbia 3OŠ”Svetislav Golubović - Mitraljeta”, Dalmatinske zagore 2, 11273 Beograd, Serbia Tea tree oil is made by distillation of aromatic leaves of Maleleuca alternifolia tree. This type of tree is grown on the narrow north coast of subtropical region of New South Wales and Queensland in Australia. Tea Tree oil is primarily consisted of monoterpens and terpinen-4-ol is an ingredient which indicates the widest antibacterial spectrum in comparison to other ingredients (1). The tea tree oil, as it is shown in the study, has a large spectrum of antibacterial activity on Escherichia coli, Streptococcus and Staphylococcus species. Also, essential oil ingredients inhibit the growth of Candida albicans (2). Results of the research which was carried out at the University of Minnesota show that skin infections as well as infections of underlying skin tissues (caused by Staphylococcus aureus) are successfully treated by Tea tree oil. The oil is used as additional therapy for its antibacterial and anti-inflammatory effects and capability to invoke monocytes. It is noticed that some side effects can appear during local application of tea tree oil in low concentration and that Contact dermatitis is one of the most common(3). The study of in vitro testing of antibacterial and antifungal activity of essential oils was carried out in Germany and than published in 2006. Results of the study show that lemongrass, tea tree and lavender oil have the highest effects on bacteria isolated from existing vaginal infections (4). In the study, which included 45 patients who suffered from acute dry or wet contact dermatitis, was used a mixture of essential oils. The treatment lasted for nine months and the mixture included the usage of tea tree oil together with corticoid preparation. The results show that if we use tea tree oil the side effect of corticoid preparation will be avoided. And the aim of the study was to try to avoid this kind of side effects (5). Tea tree oil is essential part of some pharmaceutical preparations and it is additional therapy for the treatment of skin infections and diseases. It is also an essential component of vaginal preparations (for example, vaginal gel). The products of Thursday Plantation Health (Australia) which are available on our domestic market are: gel for acne treatment which contains 20% of the tea tree oil as well as nit and lice gel. On European market there are some other products of a different kind of usage: Femisan® vaginal gel (Altermed Corporation a.s., Olomouc, Check Republic), which is a combination of Tea Tree Oil and Clove essential oils, lactic acid and vaginal suppositories (Tea Tree Oil® Suppository Pessary, Thursday Plantation, Australia). This combination contains 200 mg of tea tree oil. Both of these preparations are used during inflammatory process which is followed by vaginal secretion in order to lessen symptoms of infection. Literature: 1.Carson CF et al: Antimicrobial activity of the major components of the essential oil Malaleuca alternifolia,2002,J of Applied Bacteriology,vol 78 (3):264-9; 2.Reichling J et al: Bioactive essential oils used in phytomedicine as antiinfective agents: Australian tea tree oil and manuka oil, 2002, Acta Phytotherapeutica, vol1:26-32; 3.Halcon L. et al: Staphylococcus aureus and wounds: A review of tea tree oil as a promising antimicrobial, 2004, American Journal of Infection Control, vol 32(7):402-408; 4. Schwiertz A. et al: In vitro activity of essential oil on microorganisms isolated from vaginal infections,2006, Int J Aromatherapy, vol 16 (3):169-174; 5.Hadji-Minaglou F. et al.: The potential role of specific essential oils in the replacement of derma corticoid drugs (strong, medium and weak) in the treatment of acute dry and weeping dermatitis, 2005, Int J Aromatherapy, vol 15(2): 66-73.

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Radionica II – Workshop II Savremene analitičke tehnike Contemporary analytical techniques R1 Primena Rapid Resolution tečne hromatografije u analizi materijala biljnog porekla Zbog kompleksnosti materijala biljanog porekla, bitno je unaprediti metode za analizu sadržaja i određivanje pojedinačnih komponenti u uzorcima. Poboljšanje se postiže boljim razdvajanjem pojedinačnih komponenti u kraćem vremenu analize što se u gasnoj hromatografiji postiže prelaskom sa pakovanih na kapilarne kolone, a u tečnoj hromatografiji primenom kolona punjenih česticama manjim od 2 µm. Primenom metoda brze hromatografije dobije se poboljšanje kako u brzini tako i u rezoluciji što će se demonstrirati analizom biljnog ekstrakta na Rapid Resolution HPLC sistemu. R1 Application of Rapid Resolution High Performance Liquid Chromatography in analysis of herbal originated samples Because of complicity of herbal-originated materials, it is important to improve methods for analysis of composition and determination of each compound in samples. This improvement can be achieved with better separation of individual components within shorter time frame. In gas chromatography this is achieved by changing packed columns with capillary columns and in liquid chromatography with usage of columns with sub 2 µm particles. Appling fast chromatography methods can improve speed as well as resolution, which will be demonstrated with analysis of herbal extract on Rapid Resolution HPLC system.

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Radionica III – Workshop III Biljni lekoviti proizvodi – regulativa Herbal medicinal products - regulations Moderatori: Silvana Petrović, Dragan Đurović R1 Herbal medicinal products in Europe - viewpoint of the regulatory authorities experience in Greece Ioanna Chinou 1 University of Athens, School of Pharmacy, Div. of Pharmacognosy and Chemistry of Natural Products, University Campus of Zografou,Athens 15771 Athens, Greece All over the world medicinal plants have been used in health care through ancient times. Eventhough selection and documentation of manifold receipts have a long tradition the formal regulation of herbal medicinal products is almost new and National regulation is replaced little by little by implementation of European legislation. The aim of this presentation is mainly to discuss this implementation of the current European regulations at the level of EU authorities in terms of quality, safety and efficacy of herbal products. Within the group of herbal or botanical products there is a large variation with regard to the properties and the legal status worldwide of them. Some herbal products come closer to or are medicines, while others are close or even identical to foods such as dietary supplements, functional foods, novel foods or just foods with health claim. The European Union has recently considered medicinal use of herbal products through the Traditional Herbal Medicinal Products Directive (Directive 2004/27EC amending Directive 2001/83/EC as regards THMPs), while use of herbal preparations under food law is covered in the Food Supplements Directive (FSD) 2002/46/EC. Regulations on nutritional and health claims and the addition of vitamins and minerals and certain other substances to foods were adopted on October 12, 2006. (Directive 2006/C80E/02 and 03). The Herbal Medicinal Products Committee (HMPC) at the European Medicines Agency (EMEA, London) has drafted and adopted a couple of guidelines which are intended to support assessment of (traditional) herbal medicinal products considering their particular characteristics. One of the major tasks of the HMPC is to establish community monographs and list entries of herbal substances or combinations. The development of such documents will lead to a more harmonised view of the member states and at the end it should ideally generate a common market of herbal medicinal products. Currently, about 45 monographs have been finalised and in three years there will be probably a set of more than 100 monographs available. The regulatory framework differentiates between marketing authorisation of herbal medicinal products and registration of traditional herbal medicinal products, for which the efficacy must be plausible based on their long-standing use instead of being proven by clinical trials. In many cases, the distinction between traditional herbal medicinal products and food supplements containing herbal products without nutritional value but having physiological effects remain difficult and controversial. Borderline products, as well as advantages and disadvantages of both uses (as medicines and nutritional with health claims), have to be pointed out, as well as the consumers protection. A viewpoint of the regulatory Authorities Experience in Greece will be discussed in details, through several examples as well as the legal situation in the EU regarding the use of vitamins, minerals and other ingredients with a nutritional or physiological effect (including amino acids, herbals/botanicals and others) in food supplements and foods in general. Except of the food legislation, mostly referred to rules in that area, the issue of the "borderline" products will also be covered.

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R2 Karakterizacija i status biljnih proizvoda Dragan Đurović1 1 Agencija za lekove i medicinska sredstva Srbije, Vojvode Stepe 458, 11152, Beograd, Srbija Biljni proizvodi su jedinstveni. Samo biljni proizvodi se mogu naći u širokom rasponu proizvoda: biljni lekoviti proizvodi (biljni lekovi, tradicionalni biljni lekovi) i biljni suplementi/kozmetika. Neki biljni lekovi se izdaju na recept, a veći deo su OTC lekovi. Cilj rada je da se predstavi karakter biljnih proizvoda, ukaže na značaj klasifikacije i određivanje njihovog statusa. Agencija za lekove i medicinska sredstva Srbije izdaje stručna mišljenja o klasifikaciji proizvoda na osnovu Zakona o lekovima i medicinskim sredstvima (Sl. glasnik RS br. 84/04, 85/05-dr. zakon). Definisani su biljni lekovi, tradicionalni biljni lekovi i dodaci ishrani, utvrđeni zahtevi u pogledu dosijea i regulative. Direktiva o tradicionalnim lekovima pruža: regulatornu mogućnost za registraciju leka bez kliničkih studija, pomaže u održavanju tradicionalnih proizvoda u prometu, bezbedne proizvode potrošačima i pomaže u definisanju granice između lekova i dodataka ishrani. U radu je predstavljena hijerarhija biljnih proizvoda na osnovu vrste zahteva, upotrebe i prikazana je piramida biljnih lekovitih proizvoda prema regulativi EU. Data je veza između nivoa dokaza i karaterizacije biljnih lekovitih proizvoda. Direktivom 2001/03 su dati kriterijumi za opravdanost dobro ustanovljene upotrebe za biljne lekovite proizvode. Kod klasifikacije „graničnih“ proizvoda mogu da nastanu određeni problemi. Utvrđene su granice: prva granica između lekova sa dobro ustanovljenom upotrebom (ustanovljeno delovanje i dobro poznata bezbednost) i tradicionalnom upotrebom (farmakološki efekti se mogu pretpostaviti), gde je granica čista naučna procena, indikacije i prateća dokumentacija. Druga granica je između lekova (izlečiti, unaprediti, dijagnostifikovati bolest) i dodataka ishrani (fiziološki efekat, održavanje zdravlja). Klasifikacija proizvoda podrazumeva jasno rangiranje između medicinske indikacije i zdravstvene izjave za dodatke ishrani/kozmetika. Lek ili nije lek? Podnosilac zahteva za klasifikaciju proizvoda podnosi Agenciji za lekove i medicinska sredstva Srbije odgovarajuću dokumentaciju. Sve karakteristike proizvoda (između ostalog sastav proizvoda/leka, njegove farmakološke karakteristike/osobine, način upotrebe, obim njegove prodaje, koliko ga potrošač/korisnik poznaje, kao i rizici do kojih upotreba može da dovede) moraju se uzeti u obzir kada se vrši klasifikacija tih proizvoda. Na osnovu dostavljene dokumentacije i regulative, Agencija za lekove i medicinska sredstva Srbije izdaje stručno mišljenje: lek ili nije lek zasnovano na naučnim činjenicama i karakteristikama proizvoda. Karakterizacija (klasifikacija) biljnih proizvoda je osnova za primenu odgovarajućeg zakona, određivanja statusa razmatranog proizvoda, kao i stavljanja biljnih lekovitih proizvoda u promet.

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R2 Characterization and status of herbal products Dragan Đurović1 1Medicines and Medical Devices Agency of Serbia, Vojvode Stepe 458, 11152, Belgrade, Serbia Herbal products are unique. Only herbal products may be found in wide range of products: herbal medicinal products (herbal medicines, traditional herbal medicines) and herbal supplements/cosmetics. Some herbal medicines are prescription-only medicines, and most of them are OTC medicines. Aim of this work is to present the character of herbal products, point out the significance of classification and to determine their status. Medicines and Medical Devices Agency of Serbia issues expert opinions on product classification on the basis of Law on Medicines and Medical Devices („Official Gazette of the Republic of Serbia“ No. 84/04 and 85/05, other law). Herbal medicines, traditional herbal medicines and food supplements are defined, and requirements concerning dossiers and legislation are determined. Directive on traditional medicines gives: regulatory possibility for medicine registration without clinical studies, help in keeping traditional medicines on the market, safe products for consumers and help in defining borderline between medicines and food supplements. This work presents herbal product hierarchy on the basis of the application type and product use. Pyramid of herbal medicinal products is shown according to EU legislation. This work also shows connection between evidence level and herbal medicinal product characterization. Directive 2001/03 gives criteria for justifiability of well-established use of herbal medicinal products. Certain problems might occur during classification of „borderline“ products. Following borderlines are determined: first borderline is between medicines with well-established use (established action and well-known safety) and traditional use (pharmacological effects might be presumed), where borderline is clear scientific assessment, indications and submitted documentation; second borderline is between medicines (cure, improve, diagnose illness) and food supplements (physiological effects, health maintenance). Product classification implies clear ranking between medical indication and health statement for food supplements/cosmetics. Is it medicine or not? Product classification applicant submits appropriate documentation to Medicines and Medical Devices Agency of Serbia. All product characteristics (among others: product/medicine composition, its pharmacological characteristics/properties, usage, sales volume, in what extent are consumers familiar with it, as well as the risks resulting from its use) must be considered when classifying these products. On the basis of submitted documentation and legislation, Medicines and Medical Devices Agency of Serbia issues expert opinion: medicine or non-medicine, taking into consideration scientific facts and product characteristics. Herbal product characterization (classification) is the basis for application of appropriate law, determination of the status of discussed product as well as for marketing of herbal medicinal products.

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R3 Potvrda kvaliteta kao uslov za stavljanje biljnih lekovitih proizvoda u promet Svjetlana Mihaljica1, Snježana Zeljković2 1 Agencija za lekove i medicinska sredstva, Vojvode Stepe 458, 11152, Beograd, Srbija; 2 Agencija za lekove i medicinska sredstva, Vojvode Stepe 458, 11152, Beograd, Srbija Cilj ove prezentacije je da se utvrde opšti aspekti kvaliteta biljnih lekovitih proizvoda (za humanu i veterinarsku upotrebu), uključujući tradicionalne biljne lekove za humanu upotrebu. Konzistentan kvalitet proizvoda biljnog porekla može biti obezbeđen ako su polazni materijali definisani na strog i detaljan način, posebno ako se koristi specifična botanička identifikacija biljke. Takođe je, da bi se obezbedio materijal postojanog kvaliteta, važno da se zna geografsko poreklo i uslovi pod kojima je biljna droga (biljna supstanca) dobijena. Sve biljne droge i/ili preparati biljnih droga (biljni preparati) (npr. ekstrakti) koji ulaze u sastav biljnog lekovitog proizvoda su definisani njihovim proizvodnim procesom i specifikacijama. Tako se količina standardizovanih biljnih droga/ preparata biljnih droga izražava kroz sadržaj terapijski aktivnih konstituenata, količina kvantifikovanih biljnih droga/ preparata biljnih droga izražava kroz sadržaj određenih konstituenata (aktivni markeri), a kod ostalih biljnih droga/ preparata biljnih droga nisu poznati ni terapijski aktivni konstituenti ni aktivni markeri. Izrada biljnog lekovitog proizvoda iz biljne droge i/ili preparata biljne droge i/ili vitamina i/ili minerala trebalo bi da bude opisana. Opis treba da uključi sve detalje proizvodnog procesa. Podaci o farmaceutskom razvoju i validacija procesa takođe bi trebalo da budu obezbeđeni. Kontrola polaznog materijala uključuje:

- kontrolu biljnih droga i preparata biljnih droga u skladu sa njihovim specifikacijama: test postupci i prihvatljivi kriteriji;

- kontrolu vitamina i minerala (ako je primenljivo); - kontrolu ekscipijenasa.

Kontrolni testovi na gotovom proizvodu trebalo bi da obezbede kvalitativnu i kvantitativnu analizu aktivnih principa. Specifikacija treba da uključi ispitivanje aktivnih markera gde su konstituenti sa terapijskom aktivnošću nepoznati. U slučaju biljnih droga ili preparata biljnih droga sa konstituentima poznate terapijske aktivnosti, ovi konstituenti trebalo bi da budu specificirani i kvantifikovani. Za tradicionalne biljne lekovite proizvode za humanu upotrebu koji sadrže vitamine i/ili minerale, vitamini i/ili minerali trebalo bi takođe da budu specificirani i kvantifikovani. Testovi stabilnosti se izvode na biljnoj drogi, preparatu biljne droge i gotovom biljnom proizvodu određujući stabilnost svakog aktivnog principa ili pokazujući odgovaraće „fingerprint’’ hromatograme onda kada je to opravdano. Dakle, praćenjem svih navedenih parametara može se proceniti kvalitet biljnog leka koji je jedan od najvažnijih uslova za njegovo stavljanje u promet. Literatura: 1. Direktiva 2004/24/EC., 30. april 2004. 2. CPMP/QWP/2819/00 Rev 1, Smernica za kvalitet biljnih lekovitih proizvoda/tradicionalnih biljnih lekovitih proizvoda, 30. mart 2006. 3. CPMP/QWP/2820/00 Rev 1, Smernica za specifikaciju: Test procedure i prihvatljivi kriteriji za biljne supstance, biljne preparate/tradicionalne biljne lekovite proizvode, 30. mart 2006. 4. EMEA/HMPC/246816/05, Smernica za dobru poljoprivrednu praksu i praksu sakupljanja polaznog materijala biljnog porekla, revidirana januar 2006. 5. Evropska farmakopeja, 6. izdanje, 2008.

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R3 Quality verification as the condition for marketing authorization of herbal medicinal products Svjetlana Mihaljica1, Snježana Zeljković2 1 Medicines and Medical Devices Agency of Serbia, 458, Vojvode Stepe Street, Belgrade 11152, Republic of Serbia; 2 Medicines and Medical Devices Agency of Serbia, 458, Vojvode Stepe Street, Belgrade 11152, Republic of Serbia This presentation scope is to identify general quality aspects of herbal medicinal products (for human and veterinary use) including traditional herbal medicinal products for human use. Consistent quality for product of herbal origin can be assured if starting materials are defined in a rigorous and detailed manner, particulary the specific botanical identification of the plant material use. It is also important to know the geographical source and the conditions under which the herbal drug (herbal substance) is obtained to ensure material of consistent quality. All herbal drugs and/or herbal drug preparations (herbal preparations) (e.g. extracts) in the herbal medical product are defined by their production process and their specifications. So, standardised herbal drugs/herbal drug preparations are adjusted to a given content of constituents with known therapeutic activity, quantified herbal drugs/herbal drug preparations are adjusted to defined range of constituens (active markers), and other herbal drugs/herbal drug preparations are active principles for which neither constituens with known therapeutic activity nor active markers are known. Preparation of herbal medicinal product from herbal drug and/or herbal drug preparation and/or vitamins and/or minerals should be described. Description should include all details of their production process. Information on development pharmaceutics and prosess validation should also be provided. Control of starting materials include:

- control of herbal drugs and herbal drug preparations in accordance with their specifitations: test procedures and acceptance criteria;

- control of vitamins and minerals (if applicable); - control of excipients.

Control tests on the herbal medicinal product should allow the qualitative and quantitative analysis of the active principles. A specification should include markers examination where constituens with known therapeutic activity are unknown. In the case of herbal drugs or herbal drug preparations with constituents of known therapeutic activity, these constituents should be specified and determined. For traditional herbal medicinal products for human use containing vitamins and/or minerals, the vitamins and/or minerals should also be specified and determined. Stability tests should be carried out on the herbal drug, the herbal drug preparation and the finished herbal product determining active principle stability or demonstrating appropriate fingerprint chromatograms if it is justified. Therefore, quality of a herbal medicinal product, as one of the most important condition for its marketing authorization, can be evaluated by observing all mentioned parameters. References: 1. Directive 2004/24/EC., 30. April 2004. 2. CPMP/QWP/2819/00 Rev 1, Guideline on Quality of Herbal Medicinal Products/Tradicional Herbal Medicinal Products, 30 March 2006. 3. CPMP/QWP/2820/00 Rev 1, Guideline on Specifications: Test Procedures and Acceptance Criteria for Herbal Substances, Herbal Preparations and Herbal Medicinal Products/Traditional Herbal Medicinal Products, 30 March 2006. 4. EMEA/HMPC/246816/05, Guideline on Good Agricultural and Collection Practice for starting materials of Herbal Origin, Adopted January 2006. 5. European Pharmacopoeia, 6th Edition 2008.

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R4 Zahtevi za efikasnost biljnih lekovitih proizvoda: kada i kako? Miroslav M. Savić 1 Institut za farmakologiju, Farmaceutski fakultet, Univerzitet u Beogradu, Vojvode Stepe 450, 11221, Beograd, Srbija Da li biljne lekove treba procenjivati i registrovati na osnovu istih kriterijuma kvaliteta, bezbednosti i efikasnosti kao i konvencionalne lekove? Na ovo klasično pitanje davani su različiti odgovori u različitim vremenima i zemljama. Decenijama, američka Uprava za hranu i lekove je biljne proizvode smatrala dijetetskim suplementima, a ne lekovima; u nekim zemljama, poput Nemačke i Francuske, dovoljna je bila pojednostavljena procedura registracije, bez ubedljivih dokaza o efikasnosti, dok su u Velikoj Britaniji kriterijumi bili istovetni za sve lekove, što je često, zbog nedostatka dokaza o efikasnosti, biljne proizvode skretalo na slobodno tržište, koje nema jasne mehanizme regulatorne kontrole [1-3]. Savremena medicina zasnovana na dokazima zahteva potvrdu efikasnosti leka u dvostruko slepim randomizovanim kontrolisanim ispitivanjima. Iako stalno raste, broj takvih ispitivanja sprovedenih sa biljnim lekovima još uvek nije impresivan. Međutim, ne samo da su klinička ispitivanja sa biljnim lekovima malobrojnija u odnosu na ona sa konvencionalnim lekovima, već su ona i češće manjkava i pristrasna (nedostatak statističke snage, pseudo-randomizacija, neadekvatna kontrolna grupa, otvoreni dizajn). Dodatno, sastojci prisutni u biljnim proizvodima koji se pripremaju iz iste biljne droge mogu značajno da variraju u zavisnosti od brojnih faktora, što otežava ekstrapolaciju podataka dobijenih sa jednim proizvodom [2-4]. Aktuelna legislacija u Evropskoj uniji pravi razliku između biljnih proizvoda koji poseduju kompletne podatke potrebne za dobijanje standardne autorizacije za promet (dobro-utemeljeni biljni lekoviti proizvodi), i onih za koje se efikasnost, na osnovu dugotrajne primene i iskustva, može smatrati uverljivom (tradicionalni biljni lekoviti proizvodi) [5]. Drugim rečima, ovde je princip pouzdanosti rezultata zamenjen principom uverljivosti tradicionalne primene. Ovakav pristup bi mogao da bude vrlo ranjiv, imajući u vidu da puna procena bezbednosti jednog leka nije moguća ukoliko nedostaju adekvatna (a ponekad i bilo kakva) klinička ispitivanja. Konačno, na marginama savremene legislacije se nalazi tradicionalni herbalizam, koji čak i ne koristi konvencionalne patofiziološke termine i propisuje individualizovane biljne mešavine [3]. Skorašnje istraživanje je pokazalo da su do sada publikovana ukupno samo dva randomizovana kontrolisana ispitivanja prakse individualizovanog herbalizma; u oba slučaja, potvrdni rezultati su izostali [2]. U zaključku, budućnost ispitivanja efikasnosti biljnih i konvencionalnih lekova je zajednička. Svaki biljni lek za koji je pokazano da je prihvatljivo bezbedan i efikasan treba široko prihvatiti. S druge strane, trenutnu mogućnost da neki (“tradicionalni”) biljni proizvodi mogu da dobiju status leka u odsustvu rezultata iz kliničkih ispitivanja treba posmatrati kao privremeno stanje kompromisa, a ne izbor nauke. Buduća legislacija mora da pruži jasan podsticaj proizvođačima za sprovođenje rigoroznog istraživanja efikasnosti svih biljnih proizvoda za koje se očekuje da budu lekovi. Treba imati u vidu da su, iako neefikasni i nebezbedni, pijavice, purgativi i emetici vekovima bili uverljivi tradicionalni lekovi. Literatura: 1. Ferner RE, Beard K. (2005) Br Med J 331:62-3. 2. Guo R. et al. (2007) Postgrad Med J 83:633-7. 3. Ernst E. (2007) Postgrad Med J 83:615-6. 4. Ernst E. (2003) Br Med J 327:881-2. 5. Anonimous (2004). Official Journal of the European Union 47: L136/85-90.

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R4 Efficacy requirements for herbal medicinal products: when and how? Miroslav M. Savić 1 Department of Pharmacology, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11221, Belgrade, Serbia Should herbal medicines be judged and licensed by the same criteria of quality, safety and efficacy as conventional medicines? This classical question has been answered differently in different times and countries. For decades, the United States Food and Drug Administration regarded herbal products as dietary supplements, not medicines; in some countries, such as Germany and France, a simplified registration procedure, without conclusive evidence of efficacy, was sufficient, while in the United Kingdom the criteria were indistinguishable for all medicines, which, due to the lack of evidence of efficacy, frequently relegated herbal products to the free market without clear regulatory control [1-3]. The contemporary evidence based medicine requires proof of efficacy from double-blind randomized controlled trials. Though steadily increasing, the number of such studies performed with herbal medicines so far is not impressive. Not only that the clinical trials with herbal medicines are fewer than with the conventional drugs, but also these studies are more frequently flawed and biased (lack of statistical power, pseudo-randomization, inadequate control group, open design). Additionally, herbal products prepared from the same herbal drug can vary considerably in their constituents depending on many factors, which hampers extrapolation of the data obtained from a single product [2-4]. The current legislation in the European Union makes a distinction between herbal products possessing complete data needed for granting the standard marketing authorization (well-established herbal medicinal products), and those for which efficacy can be regarded as plausible, based on the long-standing use and experience (traditional herbal medicinal products) [5]. In other words, the principle of reliability of results is substituted with the principle of plausibility of traditional use. Such an approach might be highly vulnerable, because it is impossible to fully judge the safety of a medicine if adequate (or, sometimes, any) clinical studies are lacking. Finally, on the margins of current legislation stays traditional herbalism, which does not even use the conventional pathophysiological terms and prescribes individualized herbal mixtures [3]. A recent survey revealed that only two randomized controlled trials of practise of individualised herbalism have ever been published; both of them failed to give affirmative results [2]. In conclusion, the future of efficacy investigation of herbal medicines is the same as for conventional ones. Any herbal medicine shown to be reasonably safe and effective shall be broadly accepted. On the other hand, the current option that some (“traditional”) herbal products may be granted a medicine status in the absence of data from clinical trials shall be viewed as a temporary state of compromise, rather than state of the art. The future legislation has to give a clear impetus to manufacturers for conducting rigorous research on the efficacy of all herbal products aimed to be medicines. One must have in mind that leeches, purgatives and emetics, though ineffective and unsafe, used to be plausible traditional medicines for centuries. References: 1. Ferner RE, Beard K. (2005) Br Med J 331:62-3. 2. Guo R. et al. (2007) Postgrad Med J 83:633-7. 3. Ernst E. (2007) Postgrad Med J 83:615-6. 4. Ernst E. (2003) Br Med J 327:881-2. 5. Anonimous (2004). Official Journal of the European Union 47: L136/85-90.

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R5 Neželjena dejstva biljnih lekovitih proizvoda – prijavljivanje, informisanje, regulatorne mere Kovačević I. 1, Ilić M. 1, Petronijević M. 1 1 Agencija za lekove i medicinska sredstva Srbije, Vojvode Stepe 458, 11152, Beograd, Srbija I pored činjenice da su uzrok visoke stope morbiditeta i mortaliteta, neželjene reakcije na lekove često ostaju neprepoznate od strane zdravstvenih stručnjaka i neadekvatno tretirane. Neželjena dejstva lekova predstavljaju klinčki problem koji je poseban izazov savremenoj medicini. Rezultati brojnih ispitivanja u kojima su proučavane posledice neželjenih dejstava lekova ukazuju da su neželjene reakcije uzrok oko 5% hospitalizacija, jedan od 10 najčešćih uzroka mortaliteta i da značajno povećavaju ukupne troškove zdravstvenog sistema. Na žalost, u toku razvoja novog leka nije moguće identifikovati sva neželjena dejstva. Razlozi za to uključuju sprovođenje preregistracionih kliničkih ispitivanja u manjoj populaciji ispitanika (do 5000), odsustvo posebnih kategorija pacijenata i ograničeno vreme trajanja (1-3 godine), čime se potvrđuje samo kratkoročna bezbednost u relativno maloj i pažljivo selektovanoj grupi ispitanika. Kontinuirano praćenje karaktera i učestalosti ispoljavanja neželjenih dejstava lekova u prometu omogućava konstantno sagledavanje odnosa koristi i rizika primenjene farmakoterapije, odnosno generisanje signala kao mogućeg novog neželjenog dejstva. Na ovaj način moguće je evidentirati odložene, odnosno kasne neželjene reakcije koje se javljaju nekoliko meseci, pa čak i godina od početka primene leka, kao i one efekte koji se usled male incidencije ispoljavanja mogu uočiti tek u postmarketinškoj fazi, kada lek primenjuje veliki broj pacijenata. Analiza prijavljenih slučajeva neželjenih dejstava lekova omogućava identifikaciju povećanog rizika od primene leka u specifičnim populacijama bolesnika (gerijatrijska, pedijatrijska populacija, period trudnoće i dojenja...), što je osnova za proaktivan pristup u smislu sprovođenja restriktivnih mera i konsekutivne prevencije ispoljavanja neželjenih reakcija u rizičnim grupama bolesnika. Poseban problem u farmakovigilanci, kada je u pitanju primena biljnih lekova, baziran je na stavu da ovi lekovi omogućavaju bezbednu terapiju, pri čemu se često ne poznaju mogući neželjeni efekti, potrebna upozorenja i mere opreza pri primeni, kao ni njihov potencijal da stupaju u klinički značajne interakcije. U radu su saopštena dosadašnja iskustva Agencije za lekove i medicinska sredstva Srbije (ALIMS) u primeni važećeg Zakona o lekovima i medicinskim sredstvima u oblasti farmakovigilance biljnih lekova. Pažnja je usmerena na primer kineskog biljnog leka, za koji je sistemom spontanog prijavljivanja identifikovan potencijalni rizik od nastanka ozbiljnih hepatotoksičnih reakcija. U procesu izdavanja dozvole za stavljanje u promet ovog leka, a na osnovu procene da mogući terapijski efekat ne nadmašuje potencijalne rizike i ozbiljne klinčke manifestacije uzrokovane hepatičkom disfunkcijom, zaključeno je da je odnos koristi i rizika negativan, čime nije ispunjen bezbednosni aspekt kao jedan od tri osnovna kriterijuma koje lek mora da ispuni da bi se našao u prometu – kvalitet, efikasnost i bezbednost. Dosadašnje iskustvo pokazuje da je sistem spontanog prijavljivanja u Srbiji na niskom nivou, tako da se trenutno stanje od oko 300 prijava neželjenih reakcija godišnje, mora unaprediti i to prvenstveno motivacijom zdravstvenih radnika, koji u ovom sistemu imaju najvažniju ulogu. Ispunjavajući dužnost prijavljivanja suspektnih neželjenih reakcija na lekove Nacionalnom centru za farmakovigilancu, zdravstveni radnici omogućavaju adekvatno reagovanje regulatornih autoriteta u skladu sa novim bezbednosnim informacijama, čime suštinski doprinose i omogućavaju sprovođenje racionalne farmakoterapije.

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R5 Adverse reactions to herbal medicinal products – reporting, communication, regulatory measures Kovačević I. 1, Ilić M. 1, Petronijević M. 1 1 Medicines and Medical Devices Agency of Serbia,, Vojvode Stepe 458, 11152, Beograd, Srbija In spite of the high extent of morbidity and mortality caused by adverse drug reactions (ADRs), identification and treatment of ADRs by health care professionals are usually inadequate. Undesirable drug effects indicate clinical problem which is particular challenge for modern medicine. Numerous studies investigating consequences of ADRs have shown that ADRs count for about 5% of all hospital admissions, represent one of ten leading causes of mortality, and significantly increase overall health care cost. Unfortunately, it is not possible to identify all ADRs during drug development. The reasons for this are conducting pre-marketing clinical trials in small population (up to 5000 of subjects), absence of specific populations of patients and limited duration (between 1 and 3 years), providing only evidence for short-term safety in relatively small and carefully selected group of subjects. Continuous monitoring of character and incidence of ADRs during post-marketing phase enables constant reevaluation of benefit/risk ratio of the applied pharmacotherapy, i.e. identification of signals as a potentially new adverse reactions. In this way, it is possible to evident delayed ADRs, i.e. reactions that occur few months, or even years, after starting therapy, as well as those effects that, due to low incidence, cannot be observed earlier than in post-marketing phase when drug is used by larger number of patients. The analysis of reported ADR cases enables identification of raised risk of using drug in specific patients’ populations (geriatrics, pediatrics, during pregnancy and lactation…), providing ground for proactive approach - conducting restrictive measures and consequentially preventing adverse reactions in patients exposed to higher risk for their manifestation. Particular problem in pharmacovigilance referring to the use of herbal medicines, comes from the attitude that these drugs provide safe therapy, while possible adverse reactions, necessary warnings and precautionary, as well as their potential to cause clinically significant interactions, usually are not known. The present article brings out experience of the Agency for Medicines and Medical Devices of Serbia in the application of the current Law on Medicines and Medical Devices in the field of the herbal medicinal products’ pharmacovigilance. Attention is directed to the example of the Chinese herbal medicinal product for which potential risk of serious hepatotoxic reactions has been identified through the system of spontaneous reporting. In the process of marketing authorisation, based on the evaluation that possible therapeutic effect does not exceed potential risks and serious clinical manifestations of hepatic dysfunction, conclusion was made that benefit/risk ratio was negative, meaning that safety aspect was not fulfilled as one of three fundamental criteria required for putting drug on the market – quality, efficacy, and safety. Experience shows that system of spontaneous reporting in Serbia is at low level, and current situation of reporting about 300 cases of adverse drug reactions annually has to be improved, primarily by motivating health care professionals who have essential role in this system. By fulfilling the duty of ADRs reporting to National Pharmacovigilance Centre, health care professionals enable regulatory authorities to draw optimal regulatory measures based on safety reasons, giving in that way essential contribution to conducting rational pharmacotherapy.

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