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Page 1: Poster Presentations - Hacettepe · POSTER PRESENTATIONS ... is one of the most important commercial boron ... bismuth subnitrate (BSN) (as astringents, antacides, antiulcers and

PosterPresentations

Page 2: Poster Presentations - Hacettepe · POSTER PRESENTATIONS ... is one of the most important commercial boron ... bismuth subnitrate (BSN) (as astringents, antacides, antiulcers and

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“From Chemistry to Medicine” DRD 2007

P 01

PRICEITE DOES NOT INDUCE GENOTOXICITY IN HUMAN

LYMPHOCYTES IN VITRO

Hasan TÜRKEZ, Fatime GEYİKOĞLUAtatürk University, Faculty of Arts and Sciences, Department of Biology, 25240 Erzurum, Turkey

Boron does not exist by itself in nature. This element combines with oxygen and other elements to form boric acid, or inorganic salts called borates. People need borates, too, as an important part of a healthy diet and an essential ingredient in many products necessary for an acceptable standard of living. And borates frequently used in indus-trial, cosmetic, and medical settings. Priceite also (or pandermite = Ca4B10O19.7H2O) is one of the most important commercial boron compounds produced in large. The aim of the present study was to investigate the ability of priceite to induce sister-chromatid exchange (SCE) and micronucleus (MN) in cultured human lymphocytes.

With this aim, whole heparinized blood samples were taken from eight healthy non-smoking donors. Thirteen experimental concen-trations of priceite were used, ranging from 5 to 500 mg/L.

The peripheral blood cultures which were applied 400 and 500 mg/L of priceite was found to be sterile. Treatment with priceite did not cause an increase in the frequency of SCE per cell at all other concentrations. Moreover, there were no significant aneugenic and/or clastogenic effects observed in the micronucleus assay.

Our results firstly indicated that priceite is not a genotoxic agent in human blood cultures and safe for use in medical and cosmetic applications.

P 02

THE EFFECTS OF SOME LICHEN SPECIES AGAINST

SISTER-CHROMATID EXCHANGE FREQUENCY INDUCED

BY TITANIUM DIOXIDE IN HUMAN LYMPHOCYTES

Fatime GEYİKOĞLU, Hasan TÜRKEZAtatürk University, Faculty of Arts and Sciences, Department of Biology, 25240 Erzurum, Turkey

Despite the increasing use of factory-made synthetic drugs, natu-ral healing materials have persisted as the treatment of choice for a multitude of health problems in populations throughout the world. Investigations of genotoxicity and anti-genotoxicity can help evalu-ate the safety and effectiveness of herbal health products. Titanium dioxide (TiO2) is widely used in the pharmaceutical and cosmetic industries. It is also used for sterilization of waste water. The pur-pose of this work was to evaluate the effects of four lichen species (Dermotocopon intestiniformis, Pseudevernia furfuracea, Rhizoplaca melanophthalma and Xanthoparmelia pulla) against the genotoxicity induced by titanium dioxide (TiO2) for the first time.

With this aim, whole heparinized blood samples were taken from three healthy non-smoking donors. TiO2 was added to the cultures in concentrations of 5 and 10 μM. After the application of TiO2 and lichen extracts, seperate and together, the sister-chromatid exchange (SCE) test was used to assess DNA damage in lymphocytes.

None of the lichen extracts showed a significant genotoxicity alone. The extract of X. pulla did not show anti-genotoxic activity against the genotoxic effects induced by TiO2. However, D. intestiniformis, P. furfuracea, R. melanophthalma extracts caused significant decreases in titanium-induced SCE frequencies as dose dependent manner. The potency of anti-genotoxic activity was also in the following or-der: P. furfuracea > R. melanophthalma > D. intestiniformis.

Our findings indicated that lichens can be a new resource of therapeu-tic potential as recognized here against to adverse effects of drugs used.

P 03

IN VITRO EFFECTS OF ALUMINUM SULPHATE ON SISTER-

CHROMATID EXCHANGES AND OXIDATIVE STRESS

IN HUMAN BLOOD: PROTECTIVE ROLE OF BISMUTH

SUBNITRATE

Fatime GEYİKOĞLU, Hasan TÜRKEZAtatürk University, Faculty of Arts and Sciences, Department of Biology, 25240 Erzurum, Turkey

Aluminum, which is found in several different forms and oxida-tion states, causes acute and chronic adverse health effects. Medici-nally, the treatment methods with bismuth compounds especially bismuth subnitrate (BSN) (as astringents, antacides, antiulcers and antidiarrheals) have been increased. The goal of the present study was to evaluate the genetic and oxidative effects of aluminum sul-phate (Al2(SO4)3) and BSN in human blood in vitro.

The various concentrations of Al2(SO4)3 (10 and 20 μg/mL) and BSN (0.75, 1.5, 3 and 5 μg/mL) were used. Evaluation of DNA dam-ages was carried out by using Sister-Chromatid Exchange (SCE) method in blood lymphocytes. Oxidative status of erythrocytes was assessed by measuring following oxidative stress markers: reduced glutathione (GSH), superoxide dismutase (SOD), glucose-6- phos-phate dehydrogenase (G6PDH) and catalase (CAT).

The SOD activity increased by Al2(SO4)3 (10 μg/mL) exposure but the ratio of SCEs didn’t change compared to the controls. On the other hand, the high dose of Al2(SO4)3 (20 μg/mL) caused oxidative stress and increased SCE frequency. When the concomitant treat-ment with Al2(SO4)3 of BSN were investigated, BSN exerted an anti-oxidant action at low doses (0.75 and 1.5 μg/mL). It also reduced the formation of SCEs.

This study suggests for the first time that BSN may be admin-istered as a potential protective against the effects of Al2(SO4)3 in which oxidative and genetic damages are clearly involved.

P 04

GST-CDNB ACTIVITIES IN GILTHEAD SEABREAM (SPARUS

AURATA) LIVER CYTOSOL OF DIFFERENT AGES

1Hatice ARDAĞ-AKDOĞAN, 2Alaattin ŞEN1Pamukkale University, Science and Arts Faculty, Department of Chemistry, 20017 Kınıklı, Denizli, Turkey2Pamukkale University, Science and Arts Faculty, Department of Biology, 20017 Kınıklı, Denizli, Turkey

Lipophilic compounds such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls, nitroaromatics, dioxins, drugs, various pesticides and natural residual are readily taken up into the tissues of aquatic organisms where biotransformation via Phase I and Phase II metabolism can in part, determine the fate and toxicity of the xenobiotics. The glutathione S-transferases (GST) represent a ma-jor group of detoxification enzymes. GSTs are a family of phase II detoxification enzymes. It is known that the important changes in drug metabolism occur with ageing because the various factors that have significant influences on xenobiotic metabolizing enzymes are altered by ageing and season.

In this study, gilthead seabream (Sparus aurata) fish liver cytosol of different ages were used as sample. The fish used in this study, Gilthead Seabream (Sparus aurata), were bought from Pinar Fish in İzmir, Aegean coast of Turkey. Glutathione S-transferase (GST) were determined from this Gilthead Seabream (Sparus aurata) fish livers cytosoles.

Though various factors that may have a significant impact on drug metabolism are affected by ageing, our results suggest that some im-

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portant age-related differences in xenobiotic metabolism do occur in Gilthead Seabream liver and are substrate specific, which might af-fect obtaining desired actions and/or responses to drugs, hormones and dietary supplements used during breeding.

GST-CDNB activity increased in gilthead seabream (Sparus au-rata) liver cytosol of different ages (ranging from 1.5 to 20 months). 1.5 months of the fish activity is 17,7 ± 0,5 pmol/dakika/mg protein, 20 months of the fish is 690,9 ± 32,0 pmol/dakika/mg protein.

P 05

THE PROTECTIVE ROLE OF PSEUDOVERNIA FURFURACEA

AGAINST COLLOIDAL BISMUTH SUBCITRATE – INDUCED

GENOTOXICITY IN HUMAN LYMPHOCYTE CULTURES

Hasan TÜRKEZ, Fatime GEYİKOĞLUAtatürk University, Faculty of Arts and Sciences, Department of Biology, 25240 Erzurum, Turkey

Bismuth compounds, especially, colloidal bismuth subcitrate (CBS) have been actively promoted for the treatment of diarrhoea, peptic and duedonal ulcer diseases. And the therapeutic bismuth compounds are now being marred by episodes of serious adverse reactions. On the other hand, the potential of lichens in cellular ac-tivities remains largely unexplored. The aim of this study was to as-sess the efficacy of the lichen Pseudovernia furfuracea (2.5, 5, 10 and 20 μg/mL) on the genotoxicity induced by CBS in the human blood cultures.

With this aim, whole heparinized blood samples were taken from three healthy non-smoking donors. Sister-chromatid exchanges (SCE) and Micronuclei (MN) tests were used for evaluating the ge-notoxicity.

SCEs and MN formations significantly increased by effect of 5 μg/mL CBS when compared with the the control group. However, the decreased rates of such formations indicated that P. furfuracea was anti-genotoxic agent. Our results also showed that the protective role of P. furfuracea was dose-related.

On the basis of data, it is thought that this lichen species can pro-vide anti-genotoxic effects as due to their antioxidant defenses al-though there is no evidence for the content of the lichen species in the present investigation.

P 06

QSAR MODELING ON SIGMA RECEPTOR LIGANDS

Mine YARIM, Ece GÜRDAL, Dilek EROLYeditepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 34755 İstanbul, Türkiye

Sigma (σ) receptors are functional, membrane-bound proteins distributed throughout the brain and peripheral organs. σ1 and σ2 receptor types are clearly established, and further pharmacologi-cal differentiation may be possible. σ1 receptors are implicated in central nervous system (CNS) disorders such as depression, schizo-phrenia and dementia. Further, σ1 receptor agonists have value as neuroactive agents, while antagonists may help alleviate cocaine ad-diction. The significance of the σ receptor system to human health is augmented through the overexpression of σ sites by a number of cancers. Thus, σ receptor ligands might be used for the detection and treatment of malignant neoplastic diseases. σ2 receptors may be of particular prognostic relevance, as the extent of their expression seems indicative of the proliferative status of tumors [1].

Structurally, σ ligands are represented by wide variety of chemical scaffolds, for example (+)-benzomorphans, phenylpiperidines, (+)-pentazocine and haloperidol [2].

Corbera et al. synthesized a series of novel tetrahydroindazole de-rivatives and tested for σ1 and σ2 receptor binding [3].

Since sigma receptors are membrane-bound proteins, isolating and resolving their three-dimensional structure has proven to be dif-ficult. QSAR (quantitative structure-activity relationship) methods assume that biological activity is correlated with chemical structure or properties and that as a consequence activity can be modeled as a function of computable physicochemical attributes. QSAR tech-niques are able to raise a predictive description of global structural requirements for interactions between substrates and receptor by us-ing binding data.

We have described a detailed QSAR study on tetrahydroindazole series, in order to give better picture of action and to rationalize se-lection of substituents.

The QSAR functions in the Molecular Operating Environment (MOE) and SYBYL were used to compute theoretical molecular de-scriptors related to physicochemical properties.

1. Lever JR, Gustafson JL, Xu R, Allmon RL, Lever SZ. Synapse, 59, 350-358, 2006.

2. Vangveravong S, Xu J, Zeng C, Mach RH. Bioorg. Med. Chem., 14, 6988-6997, 2006.

3. Corbera, J. et al. Chem. Med.Chem., 1, 140-154, 2006.

P 07

SYNTHESIS AND DNA-CLEAVING ACTIVITY OF A SERIES

OF SUBSTITUTED ARENEDIAZONIUM IONS

Murat KIZIL, Bircan ÇEKENDicle University, Faculty of Science and Art, Department of Chemistry, 21280 Diyarbakır, Turkey

Aryl radicals and biradicals are generated in biological systems as intermediates of some drugs targeted DNA. Cleavage of DNA via radical attack plays an important role in various biological processes, including chemoteraphy and carcinogenesis. Some antitumor drugs generate aromatic biradicals that are belived to cleave double-strand-ed DNA via hydrogen atom abstraction from the sugar moiety. How-ever, the chemical behavior of substituted phenyl radicals toward DNA has not been extensively investigated. We have investigated the ability of causing the supercoiled DNA cleavage and the free radical formation of the substituted aryl radicals and aryl cations derived from arenediazonium ions.

We prepared the substituted arenediazonium tetrafluoroborates in 22-74 % yields by reaction of the corresponding amine with isoamyl nitrite and aqueous fluoroboric acid in ethanol. The plasmid pBlue-script M13+ DNA was prepared and isolated according to stand-ard protocols using Qiagene plasmid mini preparation kit. Gel was scanned on Gel documentation system (Gel-Doc-XR, BioRad, Her-cules, CA, USA). Bands on the gels were quantified using discovery series Quantity One programme (version 4.5.2, BioRad Co.).

Systematic studies indicate that both aryl radicals as well as aryl cation participates in the DNA cleavage pathway.

It has been found that the substituted arenediazonium salts have capacity to cleave supercoiled DNA to form the open circular Form II DNA and linear Form III DNA.

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“From Chemistry to Medicine” DRD 2007

P 08

ANTIDEPRESSANT-LIKE EFFECT OF SOME NEW ARYL

PROPANE DERIVATIVE COMPOUNDS

1Fulya MORAL, 1Meriç KÖKSAL, 1Mine YARIM, 2S. Sırrı BİLGE, 2S. Elif AKSÖZ 1Yeditepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 34755 İstanbul, Turkey2Ondokuz Mayıs University, Ondokuz Mayıs University, Faculty of Medicine, Department of Pharmacology, 55139, Kurupelit, Samsun, Turkey

In the early 1970s, evidence of the role of serotonin (5-hydroxytry-tamine or 5-HT) in depression began to emerge and the hypothesis that enhancing 5-HT neurotransmission would be available mecha-nism to mediate antidepressant response was put forward. On the basis of this hypothesis, efforts to develop agents that inhibit the up-take of 5-HT from the synaptic cleft were initiated [1].

In recent years, selective serotonin reuptake inhibitors (SSRIs) have become a standard treatment because of their safety profile and few-er side effects than the other classes of antidepressants. Fluoxetine, (±)-N-methy-3-phenyl-3(α,α,α-trifluoro-p-tolyoxy)propylamine hydrochloride, is a potent selective serotonin reuptake inhibitor used for the treatment of major depression which marketed under the well-known trade name Prozac® [2]. Furthermore, some of the undesired effects such as sexual dysfunction, gastrointestinal intol-erance and activating effects such as nervousness, anxiety and im-sonia are showed with all available SSRIs [3, 4]. Therefore, one of the still therapeutic needs is the availability of antidepressants with a more rapid onset of action and less side effects.

In view of these literature results, we aimed modifications on Fluoxetine structure by changing the amine group with benzylpipe-ridine group to reach new antidepressant compounds with a faster onset of action, a better efficacy and less side-effects. The antide-pressant-like effect of this synthesized compounds was studied in comparison with other antidepressants (Fluoxetine, Sertraline, Imi-pramine) in forced swimming test (FST), a validated experimental model of depression in mice. According to antidepressant profile evaluation, three of six showed antidepressant-like effect some of which better than the standarts (fluoxetine, sertraline, imipramine) without changing any locomotor activity.

1. Wong DT, Perry KW, Bymaster FP. Nature Reviews Drug Discovery, 4, 764-774, 2005.

2. Takeuchi, K et al. Bioorg. Med. Chem. Lett., 13, 1903-1905, 2003. 3. Labbate A, Grimes JB, Arana GW. Biol. Psychiatry, 43, 904-907, 1998. 4. Takeuchi K, Kohn TJ, Honigschmidt NA, Rocco VP, Spinazze PG, At-

kinson ST, Hertel W, Nelson DL, Wainscott DB, Ahmad LJ, Shaw J, Threlkeld PG, Wong DT. Bioorg. Med. Chem. Lett., 13, 3939-3942, 2003.

P 09

COMPARATIVE ANALYSIS OF THE COFACTOR

BINDING SITE OF PLASMODIUM VIVAX LACTATE

DEHYDROGENASE WITH SOME OTHER KNOWN

APICOMPLEXAN COUNTERPARTS

1Venhar ÇELİK, 2Dilek TURGUT-BALIK1University of Fırat, Faculty of Arts And Sciences, Department of Biology, 23169 Elazığ, Turkey2Yıldız Technical University, Faculty of Chemical and Metallurgical Engineering, Department of Bioengineering, Davutpaşa Campus, 34210 Esenler-İstanbul, Turkey

Malaria is caused by the protozoan parasite Plasmodium from the phylum Apicomplexa that also includes the important pathogens Toxoplasma, Eimeria, Theileria, Cryptosporidium and Babesia. Ma-laria is one of the greatest causes of human misery and death. Despite continued efforts to control the disease, it remains a major health

problem in many regions of the world. The need for new antimalar-ials comes from the widespread resistance to those in current use. In this study, the use of parasite’s lactate dehydrogenase as an antima-larial drug target is evaluated. The cofactor binding site of Plasmo-dium vivax lactate dehydrogenase was compared to some other api-complexan counterparts by making a single residue change. Residue 163 is a leucine in Plasmodium, a serine in human and a methionine in other apicomplexans. Leucine 163 was replaced by methionine by site directed mutagenesis. It was observed that enzyme was tolerant when leucine 163 residue in its cofactor binding site was replaced with methionine like in its apicomplexan counterpart. This is a fea-ture that distinguishes Plasmodium and other apicomplexan lactate dehydrogenase enzymes from their human lactate dehydrogenase, supporting their suitability as targets in common drug design stud-ies.

P 10

MUTAGENIC ANALYSIS OF ACTIVE SITE LOOP OF

LACTATE DEHYDROGENASE BY MIMICKING EIMERIA

TENELLA IN PLASMODIUM VIVAX

1Venhar ÇELİK, 2Dilek TURGUT-BALIK1University of Fırat, Faculty of Arts and Sciences, Department of Biology, 23169 Elazığ, Turkey2Yıldız Technical University, Faculty of Chemical and Metallurgical Engineering, Department of Bioengineering, Davutpaşa Campus, 34210 Esenler-İstanbul, Turkey

Malaria, the most prevalent and most pernicious parasitic diseases of humans, is estimated to kill approximately three million people each year. The increasing occurence of drug resistance in many en-demic areas emphasizes the need for antimalarial drugs. The gene encoding Plasmodium vivax lactate dehydrogenase is a functinal validation. We have isolated and cloned the lactate dehydrogenase, and overexpressed the protein from Plasmodium vivax. The en-zyme’s structure was also solved from P. vivax recently. Active site loop of the enzyme has been determined by crystal structure studies and shown to be an ideal site for the drug design studies. The active site loop of this enzyme was found to have a pentapeptide insertion and this insertion differentiates this enzyme from its human coun-terpart. Similar insertion sequence was also found in Eimeria tenella lactate dehydrogenase. It was observed in this study that replacing the pentapeptide insertion sequence in Plasmodium vivax with the insertion sequence from Eimeria tenella has unaffected the enzyme activity.

P 11

SYNTHESIS OF AMINO ALCOHOL BASED

CHIRAL LIGANDS AND THEIR APPLICATIONS IN

ENANTIOSELECTIVE DIETHYLZINC ADDITION TO

BENZALDEHYDES

Dilek Işık TAŞGIN, Canan ÜNALEROĞLUHacettepe University, Faculty of Science, Department of Chemistry, 06800 Beytepe-Ankara, Turkey

Among asymmetric catalysis of C-C bond-formation, the enanti-oselective addition of diorganozinc reagents to aldehydes in the pres-ence of a catalytic amount of a chiral ligand is a convenient method for the preparation of optically active secondary alcohols [1]. These structural features are important building blocks for the synthesis of many natural and biologically active compounds, and materials such as liquid crystals [2]. For this purpose, a wide variety of chiral cata-lysts, i.e. amino alcohols, amino thiols, diamines, disulfonamides,

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and diols have been synthesized [3]. Among the chiral ligands re-ported, β-amino alcohols are the most often used chiral ligands [4].

In this study, norephedrine based chiral ligands were synthesized starting from pyrrole carbaldehydes and norephedrine. The synthe-sized (1S,2R)-2-((1H-pyrrol-2-yl)methylamino)-1-phenylpropan-1-ol (1) and (1R, 2S)-2-((1H-pyrrol-2-yl)methyl amino)-1-phenyl-propan-1-ol (2) were used as chiral ligands in the enantioselective addition of diethylzinc to benzaldehyde.

Synthesis of Amino Alcohols: Synthesized imines (1.22 mmol) from pyrrolecarbaldehydes and norephedrine, and NaBH4 (1.35 mmol) in methanol was refluxed for 8 hours. When the reaction was completed, the mixture was cooled to room temperature and quenched with 15 mL water. It was extracted with ether (3x10 mL) and dried over MgSO4. The mixture was filtered and the solvent was evaporated. Crude products 1 and 2 were dissolved in benzene and filtered. The product was obtained after evaporation.

General Procedure for the Enantioselective Diethylzinc Addi-tion to Benzaldehyde: System was dried under vacuum by applying the Schlenk technique for five times. 0.05 mmol chiral ligand was dissolved in dry solvent (5 mL) under the argon atmosphere. Af-ter cooling to 0 oC, 2.2 mmol diethylzinc (1M in hexane) was added with a syringe over a period of 5 min. The mixture was stirred for 5 hours 0 oC and 1 mmol aldehyde was added. The reaction mixture was stirred for 16 hours at room temperature and monitored by TLC. The reaction was quenched with 5 mL of 1 N HCl solution and ex-tracted with ether and dried over anhydrous MgSO4 and the solvent was evaporated under reduced pressure. The crude product was pu-rified by flash column chromatography ( EtOAc:hexane, 1:6).

Amino alcohols were obtained by the reduction of imines with NaBH4 in methanol. Chiral ligands 1 and 2 were obtained in 65 % and 70 % yields, respectively. Characterization of these compounds was carried out by 1H-NMR and 13C-NMR techniques.

The enantioselective addition of diethylzinc to benzaldehyde was carried out in different solvents in the presence of 5 mol % of ligands under argon atmosphere at 0oC to room temperature. Catalytic ac-tivity of 1 and 2 were examined in toluene, hexane, toluene-hexane mixture and dichloromethane (Table 1). The best result was obtained with ligand 2 (70 % yield and 78 % ee) in toluene-hexane solvent mixture.

Table 1. The addition of diethylzinc to benzaldehyde with ligands 1 and 2.

entry ligand solvent yield (%) ee (%) conf.

1 1 Toluene 36 71 S

2 1 Hexane 85 58 S

3 1 Tol.+Hex. 67 66 S

4 2 Toluene 35 74 R

5 2 Hexane 77 64 R

6 2 Tol.+Hex. 70 78 R

7 2 Dichloromethane 17 27 R

1. Roudeau R, Pardo DG, Cossy J. Tetrahedron, 62, 2388, 2006. 2. Melo RPA, Vale JA, Zeni G, Menezes PH. Tetrahedron Lett., 47, 1829,

2006. 3. Xu Q, Zhu G, Pan X, Chan ASC. Chirality, 14, 716, 2002. 4. Cicchi S, Crea S, Goti A, Brandi A. Tetrahedron:Asymmetry, 8, 293,

1997.

P 12

ELECTROCHEMICAL INVESTIGATION OF INTERACTIONS

BETWEEN DNA AND SOME CHEMICALS

1Görkem YALÇIN, 2Murat ÇİZMECİOĞLU, 1Özlem SÖĞÜT, 1Mehmet ÖZSÖZ1Ege University, Faculty of Pharmacy, Department of Analytical Chemistry, İzmir, Turkey2Ege University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, İzmir, Turkey

Determinations of interactions between DNA and drugs are im-portant aspects of biological studies in drug discovery and pharma-ceutical development processes.

The interactions of some molecules with DNA have been inves-tigated by a variety of techniques. There has been growing interest to determine interacts between DNA and some molecules by using electrochemical methods.

In this study, interactions of some chalcone derivatives with DNA were investigated by using electrochemical methods. In recent years chalcone (1,3-diphenyl-2-propen-1-one) derivatives have been synthesized in order to develop active compounds against cancer, malaria, leishmaniase, tuberculosis and cardiovascular diseases. Therefore, determination of interactions between some chalcone derivatives and DNA will give some help to chalcone based drug de-velopment studies.

P 13

GLYCOSYLATION IN ROOM TEMPERATURE IONIC LIQUID

(RTIL) USING UNPROTECTED AND UNACTIVATED

DONORS

1Sultan N. BAYTAŞ, 2Tae-joon PARK, 1Robert J. LINHARDT1Rensselaer Polytechnic Institute, Department of Chemistry and Chemical Biology, Troy, NY, USA2Rensselaer Polytechnic Institute, Department of Chemical and Biological Engineering, Troy, NY, USA

Glycosylation occurs between a donor and an acceptor in the pres-ence of a promoter, which activates the donor. There are various fac-tors that need to be considered when carrying out the synthesis of glycosides including the manipulation of protecting groups in both

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donor and acceptor, the architecture of donor and acceptor and the solvent system [1]. These strategies involve often a large number of synthetic steps. The chemical synthesis of unprotected carbohydrates poses a number of challenges, including poor solubility in most con-ventional organic solvents. Only very polar organic solvents, such as formamide, dimethylformamide, dimethylsulfoxide, and pyridine, easily dissolve significant amounts of sugars. It is important to in-vestigate new solvent systems that dissolve carbohydrates, support glycosylation reactions of unprotected sugars, and facilitate product recovery. Room temperature ionic liquids (RTILs) are becoming increasingly used as solvents for a wide variety of chemical reac-tions [2]. RTILs also display desirable solvent properties and have the potential of replacing conventional volatile organic solvents in carbohydrate chemistry. In this work, we report the glycosylation of various simple, unprotected monosaccharides in RTILs to give ben-zyl glycosides, disaccharides and oligosaccharides. RTILs facilitate the use of unactivated and unprotected donors in these reactions, re-sulting in a simple synthetic strategy involving a single glycosylation step. The synthesis of galactose oligomers was also possible using these novel solvents.

1. Sasaki K, Nagai H, Matsumura S, Toshima K. A novel greener glycosida-tion using an acid-ionic liquid containing a protic acid, Tetrahedron Lett., 44, 5605-5608, 2003.

2. Murugesan S, Linhardt RJ. Ionic liquids in carbohydrate chemistry - cur-rent trends and future directions, Curr. Org. Synth., 2, 437-451, 2005.

P 14

DEVELOPMENT OF VALIDATED METHOD FOR

RISEDRONATE BY HPLC-MS MS FROM BIOLOGICAL

MATERIAL

2Zeynep İrem DİLER, 2Gülay ŞAHİN-KOÇ, 2Hüseyin YALÇINKAYA, 1Durişehvar ÖZER-ÜNAL, 1Dilek EROL1Yeditepe University, Faculty of Pharmacy, Department of Analytical Chemistry, İstanbul, Turkey2Yeditepe University, Yeditepe Sağlık Hizmetleri A.Ş., GLP Laboratory, Acıbadem-İstanbul, Turkey

Risedronate (RS) is a third generation of bisphosphonate class of drugs. It is widely used for the treatment of bone disorders such as osteopotrosis. Determination of RS from biological fluids have dif-ficulties because of its low level in urine and blood. A sentitive and reliable HPLC-MS MS method was developed and validated from human urine.

The extraction method was developed to analyse RS from bio-logical material. In this method TMS-diazomethan derivatization was used followed by solid phase extraction.The mobile phase was MeOH:H20 (80:20; v/v) containing 0.1 % formic acid. The best reso-lution was obtained by using Agilent Zorbax Eclipse XDB reversed phase C18 (150x4.6mm, 5μm) and alendronate was used as an inter-nal standart.

The developed method was applied succesfully to biological ma-terial. The limit of quantitation of RS from biological material was 5 ng/ml. The calibration curve was linear in between 5-400 ng/ml. Precision, recovery, accuracy and stability results were satisfactory for the method developed. The method is suitable for routine analy-sis of bioequivalence studies.

1. Zhu LS., Lapko VN., Lee JW et.al. Rapid Commun. Mass Spectrom., 20(22), 3421-6, 2006.

2. Vallano PT., Shugards SB., Kline WF. et. al. J. Chromatogr. B, 794, 23-33, 2003.

P 15

AMINO TERMINAL ANALYSIS OF THE ACTIVE SITE LOOP

OF LACTATE DEHYDROGENASE FROM THE MALARIA

PARASITE PLASMODIUM VIVAX

1Bünyamin ATMIŞ, 1Dilek SADAK, 1Venhar ÇELİK, 2Dilek TURGUT-BALIK1University of Fırat, Faculty of Arts And Sciences, Department of Biology, 23169 Elazığ-Turkey2Yıldız Technical University, Faculty of Chemical and Metallurgical Engineering, Department of Bioengineering, Davutpaşa Campus, 34210 Esenler-İstanbul,Turkey

Malaria is parasitic disease that threatens nearly half the global population. It is caused by protozoan apicomplexan parasites of the genus Plasmodium; four species cause malaria in man. In this study, amino acid exchanges were made in the amino terminal end of the active site loop of Plasmodium vivax lactate dehydrogenase (LDH) by mimicking Toxoplasma gondii I ve II, Eimeria acervulina, Eimeria tenella ve Theileria parva LDH’s using the site directed mutagenesis method. Although enzymatic activity was decreased compared to that of the wild type protein, some enzymatic activity was present meaning that enzyme was still in contact with its substrate. Decrease in the enzymatic activity indicates that this region is sensitive to changes and this supports the idea that this site could be evaluated as an ideal target for the drug design studies for both Plasmodium and some other apicomplexans.

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AN ANALYSIS TO BE USED IN SCTRUCTURE-BASED DRUG

DESIGN STUDIES: COMPARISON OF ACTIVE SITE LOOPS

OF ENZYMES, PLASMODIUM VIVAX AND TOXOPLASMA

GONDII LACTATE DEHYDROGENASES

1Venhar ÇELİK, 2Dilek TURGUT-BALIK1University of Fırat, Faculty of Arts and Sciences, Department of Biology, 23169 Elazığ, Turkey2Yıldız Technical University, Faculty of Chemical and Metallurgical Engineering, Department of Bioengineering , Davutpaşa Campus, 34210 Esenler-İstanbul, Turkey

Increasing resistance of Plasmodium to the antimalarial agents necessitates the development of new drugs which have different modes of action from the currently existing ones. Present study is targeted to lactate dehydrogenase (LDH) which is a key anaerobic metabolic pathway enzyme. LDH of Plasmodium has strikingly dif-ferent properties compared to its human counterparts. Inhibition of the activity of this enzyme has been shown to kill the parasites in the erythrocytes. Thus, we have isolated and cloned the LDH gene, and overexpressed the protein. The structure of the enzyme from P. vivax (PvLDH) recently reported. A five amino acids insertion in the ac-tive site formed a distinctive cleft on the surface of the PvLDH as did in P. falciparum LDH. This site has been identified as a potential tar-get for the structure based drug design studies. The site is not unique to Plasmodium. The five amino acid insertion was also observed in some other apicomplexan parasites. This site was analysed by mak-ing residue exchanges in the P. vivax LDH active site loop to mimick the same region of Toxoplasma gondii LDH1. It was observed that making residue exchanges in the active site loop of PvLDH was pos-sible without losing enzymatic activity. This observation emphasizes that the active site loop is a crucial region accross the apicomplexan LDHs.

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INTERACTION OF SOME 3,4-DIHYDROQUINOLIN-(1H)-

2-ONE DERIVATIVES WITH RAT LUNG SEMICARBAZIDE-

SENSITIVE AMINE OXIDASE (SSAO)

1Samiye YABANOĞLU, 2Sevil Görkem SUNAL, 2Akgül YEŞİLADA, 1Gülberk UÇAR1Hacettepe University, Faculty of Pharmacy, Department of Biochemistry, 16100 Ankara, Turkey2Hacettepe University, Faculty of Pharmacy, Department of Basic Pharmaceutical Sciences, 06100 Ankara, Turkey

Since semicarbazide-sensitive amine oxidase (SSAO) is shown to be involved in diabetes, Alzheimer’s and Parkinson diseases, heart and vascular diseases, the synthesis of new compounds as specific SSAO inhibitors suggested to be useful developing novel therapeutic agents. In view of the previous studies indicating that diazoheterocy-clic compounds have been introduced as promising class of revers-ible amine oxidase inhibitors, three compounds with 3,4-dihydro-quinoline-(1H)-2-one structure and their open ring derivatives were synthesized and the interaction of these compounds with SSAO pu-rified from rat lung were evaluated.

The compounds of Q (N-amino-3,4-dihydroquinoline-(1H)-2-one) and QB (1-(Benzyliden-amino)-3,4-dihiydroquinoline-(1H)-2-one) were synthesized by the reduction and the ring closure reaction of o-nitro-cinnamic acid whereas MBK (Tert-butyl-N-[cyclohexyl-carbamoyl-(3-hydroxyphenyl)methyl]-N-phenyl-carbamate), MG (Tert-butyl-N-[cyclohexylcarbamoyl-(3-hidroxyphenyl)methyl]-N-(2-benzoylphenyl)-carbamate) and PCN (2-(3-cyano-2-oxo-4-phe-nyl-2H-quinolin-1-yl-N-cyclohexyl-2-(4’-chlorophenyl)acetamide) were synthesized by one pot Ugi-Knovenagel reaction. The struc-tures of the novel compounds were evaluated using 1H NMR, 13C NMR and MS techniques.

Compound Q, which carries a free amine group appeared as a good substrate for rat liver SSAO suggesting that this relatively small compound may interact with the active site channel of the enzyme through its free amine group. QB, PCN and MG inhibited the en-zyme non-competitively and irreversibly suggesting that these com-pounds may interact with an another hydrophobic binding region outside of the active site of the enzyme. It is concluded that these compounds may have promising features as novel anti-parkinson/anti-Alzheimer agents in case their SSAO inhibitory effects can be supported by in vivo studies.

Keywords: 3,4-Dihydroquinoline-(1H)-2-ones, tissue-bound semicar-bazide-sensitive amine oxidase (SSAO), Ugi-Knovenagel condensation, substrate, inhibition

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SYNTHESIS AND ANALGESIC ACTIVITY OF SOME

BENZIMIDAZOLE DERIVATIVES

1İlhan IŞIKDAĞ, 2Ümide DEMİR, 2Özgür Devrim CAN, 1Yusuf ÖZKAY, 2Yusuf ÖZTÜRK1Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 26470 Eskişehir, Turkey2Anadolu University, Faculty of Pharmacy, Department of Pharmacology, 26470 Eskişehir, Turkey

Heterocyclic compounds having two nitrogen atoms oriented in 1,3-positions in ring are endowed with broad spectrum of phar-maceutical properties. Imidazole and benzimidazole, shown as the instances for these heterocyclics, drugs have broaden scope in rem-edying various dispositions in clinical medicine [1]. Pharmaceutical properties concern antifungal and antimycotic, antiprotozoal, anti-

neoplastic, antiulcer, antihistaminic and antiallergic, antihyperten-sive, anthelmintic, antioxidant, antiviral, antibacterial and antipara-sitic activities, all of which are unique characteristics known from imidazole and benzimidazole derivatives [2]. It is known well that synthetic chemical compounds especially lipophylic ones have vari-ous different effects on central nervous system. Benzimidazoles are examples of these derivatives with their reported pharmacological effects such as anesthetic and hypnotic[3], neuroleptic and antipsy-chotic [4], analgesic [5] and sedative [6] etc. These large research areas of benzimidazoles promted us to study with them.

We synthesized five 2-aryl-4,5-dichloro-(1H)-benzimidazoles via the condensation of 4,5-dichloro-o-phenylenediamine and cor-responding aldehyde derivatives in ethanol with the presence of sodium bisulfite. Their structures were elucidated with 1H-NMR, IR and MASS(Apci) spectral analysis. Tail-clip and tail-immersion tests, were applied in order to investigate probable analgesic effects. Synthesized compounds were applied to mices at a dose of 100 mg/kg (i.p). Morphine (1 mg/kg) was used as positive standart.

All of the synthesized compounds showed analgesic activities in tail-clip and tail-immersion tests. Compound 2 was found as the most active derivative in the series. 1H-NMR and IR spectral datas were obtained as expected. Mass (Apci) spectras of the compounds were agreed well with their molecular weight. The compounds (compounds 1, 2 and 4) including metoxy groups at metha posi-tions and/or hydroxyl group at para position of phenyl ring which is substituted at second position of benzimidazole ring, have higher analgesic activity. On the other hand, addition of nitro group at or-tho position of phenyl ring (compound 3) causes decrease in the an-algesic activity. These findings indicate the importance of chemical sturucture and pharmacological activity relationships of the synthe-sized compounds. In conclusion, it may be suggested that to obtain more active derivatives, containing the same structure with the title compounds, number of synthesis including substituted-p-hydroxy-benzaldehyde and substituted-m-methoxybenzaldehyde derivatives should be increased.

1. Kleeman A, Engel J, Kutscher B, Reichert D, Pharmaceutical Substances, 3rd ed.; Stuttgart: New York, 1999.

2. Nezhad AK, Rad MNS, Mohabatkar H, Asraria Z, Hemmateenejada B, Bioorg. Med. Chem., 13, 1931, 2005.

3. Janssen PAJ, Niemegeers CJE, Schellekens KHL, Lenaerts FM, Arzneim. Forsch., 21, 1234, 1971.

4. Sato M, Arimoto M, Ueno K, Kojima H, Yamasaki T, Sakurai T, Kasahara A, J. Med. Chem. 21, 1116, 1978.

5. Sondhi SM, Singh N, Kumar A, Lozach O, Meijer L, Bioorg. Med. Chem., 14, 3758, 2006.

6. Seredenim SB, Eur. Neuropsychopharm., 6, 111, 1996.

P 19

STUDIES ON THE SYNTHESIS AND ANTIPROLIFERATIVE

ACTIVITY INVESTIGATION OF 2,2’-(3-METHOXYPHENYL)

AND 2,2’-(3-HYDROXYPHENYL)-1H,1H’-[5.5’]-BIS-

BENZIMIDAZOLES

1İlhan IŞIKDAĞ, 1Yusuf ÖZKAY, 2Zerrin İNCESU1Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 26470 Eskişehir, Turkey2Anadolu University, Faculty of Pharmacy, Department of Biochemistry, 26470 Eskişehir, Turkey

The incorporation of the imidazole and benzimidazole nuclei is an important synthetic strategy in drug discovery [1]. Previous observations suggest that substituted benzimidazoles and related heterocycles, which are the structural isosters of nucleotides ow-ing fused heterocyclic nuclei in their structures that allow them

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to interact easily with the biopolymers, possess potential activity with lower toxicities in the chemotherapeutic approach in man [2, 3]. The high therapeutic properties of the related drugs have en-couraged the medicinal chemists to synthesize the large number of novel chemotherapeutic agents. Antitumoral activities of benzimi-dazole and bisbenzimidazole compounds were reported in several studies. Furthermore, there are clinical anticancer drugs, known as Hoechst-33258 and Hoechst-33342 dyes including bisbenzimida-zole structure [4, 5]. Prompted above observations we synthesized the title compounds to investigate their possible antiproliferative effects.

We synthesized two bis-benzimidazoles via the condensation of 3,3’-diaminobenzidine and corresponding aldehyde derivatives in ethanol with the presence of sodium bisulfite. Their structures were elucidated with 1H-NMR, IR and MASS(Apci) spectral analysis. Their antiproliferative effects of the compounds will be determined on HDQ-P1 and HT-29 cancer cell lines by using MTT and BrdU assays.

Both of the bisbenzimidazole derivatives were synthesized and their spectral datas were recorded. 1H-NMR and IR spectral datas were obtained as expected. Mass (Apci) spectras of the compounds were agreed well with their molecular weight. Our synthesis with bisbenzimidazole compounds have recently begun. Further studies are in progress to increase the number of compounds with smilar structures. After reaching adequate number of the compounds, anti-proliferative activity scaning will be started.

1. Townsend LB, Chem. Rev., 67, 533, 1976. 2. Haugwitz RD, Angel RG, Jacobs GA, J. Med. Chem., 25, 969, 1982. 3. Hisano T, Ichıkawa M, Tsumoto K, Tasaki M, Chem. Pharm. Bull., 30,

2996, 1982. 4. Kamal A, Ramul P, Srinivas O, Ramesh G, Kumar PP, Bioorg. Med. Chem.

Lett., 14, 4791, 2004. 5. Alper S, Arpacı, ÖT, Akı, EŞ, Yalçın İ, Farmaco, 58, 497, 2003.

P 20

SYNTHESIS OF 3-PHENETHYLAMINO-1-

PHENYL/SUBSTITUTED PHENYL-1-PROPANONE

HYDROCHLORIDES

1Ebru METE, 2H. İnci GÜL, 1Cavit KAZAZ1Atatürk University, Faculty of Arts and Sciences, Department of Chemistry, 25240 Erzurum, Turkey2Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey

It has been reported that acetophenone derived several mono Mannich bases have cytotoxic, antifungal and anticonvulsant activi-ties [1-5]. In this study, synthesis of 1-aryl-3-phenethylamino-1-pro-panone hydrochlorides has been realised to investigate their biologi-cal activities in future.

Chemical structure of the compounds were confirmed by 1H NMR, 13C NMR, UV, IR and MS spectroscopies. Purity level of them was determined by elemental analyses.

1. Gül Hİ, Vepsalainen J, Gül M, Erciyas E, Hanninen O. Cytotoxic activi-ties of mono and bis Mannich bases derived from acetophenone against Renca and Jurkat cells, Pharm. Acta Helv., 74, 393-8, 2000.

2. Gül Hİ, Gül M, Erciyas E. Syntheses and stability studies of some Man-nich bases of acetophenones and evaluation of their cytotoxicity against Jurkat cells, Arzneimittel Forschung, 52, 628-35, 2002.

3. Gül Hİ, Çalış Ü, Vepsalainen J. Synthesis of some mono-Mannich bases and corresponding azine derivatives and evaluation of their anticonvul-sant activity, Arzneimittel Forschung, 54, 359-64, 2004.

4. Gül Hİ, Şahin F, Gül M, Öztürk S, Yerdelen KO. Evaluation of antimi-crobial activities of several Mannich bases and their derivatives, Arch. Pharm., 338, 335-8, 2005.

5. Gül Hİ, Das U, Pandit B, Li PK. Evaluation of the cytotoxicity of some mono-mannich bases and their corresponding azine derivatives against androgen-independent prostate cancer cells, Arzneimittel Forschung, 56, 850-4, 2006.

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SYNTHESIS OF 1-PHENETHYL-3-AROYL-4-ARYL-4-

PIPERIDINOLS

1Ebru METE, 2H. İnci GÜL, 1Cavit KAZAZ1Atatürk University, Faculty of Arts and Sciences, Department of Chemistry, 25240 Erzurum, Turkey2Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey

It has been reported that piperidinol type of compounds have cy-totoxic, antifungal and anticonvulsant activities [1-4]. In this study, synthesis of 1-phenethyl-3-aroyl-4-aryl-4-piperidinols has been re-alised to investigate their biological activities in future.

Chemical structure of the compounds were confirmed by 1H NMR, 13C NMR, UV, IR and MS spectroscopies. Purity level of them was determined by elemental analyses.

1. Gül Hİ, Çalış Ü, Vepsalainen J. Synthesis and evaluation of anticonvul-sant activities of some bis Mannich bases and corresponding piperidi-nols, Arzneimittel Forschung, 52, 863-9, 2002.

2. Gül M, Gül Hİ, Das U, Hanninen O. Biological evaluation and structure-activity relationships of bis-(3-aryl-3-oxo-propyl)-methylamine hydro-chlorides and 4-aryl-3-arylcarbonyl-1-methyl-4-piperidinol hydrochlo-rides as potential cytotoxic agents and their alkylating ability towards cellular glutathione in human leukemic T cells, Arzneimittel Forschung, 55, 332-7, 2005.

3. Gül Hİ, Şahin F, Gül M, Öztürk S, Yerdelen KO. Evaluation of antimi-crobial activities of several mannich bases and their derivatives, Arch. Pharm., 338, 335-8, 2005.

4. Gül M, Atalay M, Gül Hİ, Nakao C, Lappalainen J, Hanninen O. The ef-fects of some Mannich bases on heat shock proteins HSC70 and GRP75, and thioredoxin and glutaredoxin levels in Jurkat cells, Toxicol In Vitro, 19, 573-80, 2005.

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SYNTHESIS AND ANTITUMOR ACTIVITY OF POLY(2,3-

DIHYDROPYRAN-CO-MALEIC ANHYDRIDE-CO-VINYL

ACETATE)

1Hatice KAPLAN-CAN, 2A. Lale DOĞAN, 3Zakir M. O. RZAEV, 2Ayşegül HASEGELİ-ÜNER, 1Ali GÜNER1Hacettepe University, Faculty of Science, Department of Chemistry, Ankara, Turkey2Hacettepe University, Institute of Oncology, Department of Basic Oncology, Ankara, Turkey3Hacettepe University, Faculty of Engineering, Department of Chemical Engineering, Ankara, Turkey

It is known that the water soluble anhydride-containing copoly-mers as polyanions and their functional derivatives have high biologi-cal and physiological activity, specially antimicrobial and antitumor properties. Polyanions are also known for their potential to stimulate the immune system and invoke activities against tumors, viruses and bacteria. Copolymers of dihydropyran and its derivatives with acrylic acid, which contain tetrahydropyran rings and free carboxylic groups on the polymer backbone, as well as an alternating cyclocopolymer of divinyl ether (acyclic analogy of dihydropyran) with maleic anhydride have exhibited antitumor activities in vitro [1, 2].

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Radical-initiated terpolymerization of 3,4-dihydro-2H-pyran (DHP), maleic anhydride (MA) and vinyl acetate (VA) as a donor-acceptor-donor systems was carried out MEK in the presence of AIBN as initiator at 65oC in nitrogen atmosphere. Determination of some kinetic parameters, constants of thermal−copolymerization of complexed comonomers, terpolymer composition behavior re-lationships of synthesized polymers with alternating structure are described and discussed. Synthesized polymers were characterized by analytical methods (acid number), viscometer, FTIR and thermal (DSC and TGA) methods [3].

In vitro cytotoxicities of synthesized poly(DHP-alt-MA) and poly(DHP-co-MA-co-VA) polymers were evaluated using Raji cells (human Burkitt lymphoma cell line). Antitumor activity of prepared anion-active poly(DHP-alt-MA) and poly(DHP-co-MA-co-VA) polymers were studied by methyl-thiazol-tetrazolium (MTT) test-ing method using calorimetric measurements of chemotherapic ef-fect and quantitative evaluation of LD50 dose for the total number of tumor cells. Hydrolyzed copolymer has sufficiently high antitumor activity, which depends on the amount of hydrogen bonded car-boxylic groups and on their regular distribution in side chain of the functional macromolecules [3].

1. Stolfi RL, Martin DS. Therapeutic activity of maleic anhydride-vinyl ether copolymers against spontaneous, autochthonous murine mam-mary tumors, Cancer Treat Rep., 62(11),1791-6,1978.

2. Ottenbrite, RM. The antitumor and antiviral effects of polycarboxylic acid polymers in biological activities of polymers, ACS Symposium Se-ries 186, Washington DC, 1982.

3. Kaplan Can, H., Doğan, AL., Rzaev, ZMO, Üner, AH, Güner, A. Syn-thesis and Antitumor Activity of Poly(3,4-dihydro-2Hpyran-co-maleic anhydride-co-vinyl acetate), Journal Applied Polymer Science, 96, 2352-2359, 2005.

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ANTIFUNGAL ACTIVITIES OF 3-PHENETHYLAMINO-1-

ARYL-1-PROPANONE HYDROCHLORIDES AND 3-AROYL-

4-ARYL-1-PHENETHYL-4-PIPERIDINOLS AGAINST SOME

PLANT AND HUMAN PATHOGENIC FUNGI

1H. İnci GÜL, 1Canan ÖZELGÜL, 2Ebru METE, 3Fikrettin ŞAHİN, 3Dilşat YURDAKUL1Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey2Atatürk University, Faculty of Arts and Sciences, Department of Chemistry, 25240 Erzurum, Turkey3Yeditepe University, Faculty of Engineering and Architecture, Department of Genetics and Bioengineering, İstanbul, Turkey

1-Aryl-3-phenethylamino-1-propanone hydrochlorides (1, 3, 5, 7, 9, 11, 13, 15, 17, 19) and 3-aroyl-4-aryl-1-phenethyl-4-piperidinols (2, 4, 6, 8, 10, 12, 14, 16, 20) were synthesized. Aryl part was C6H5 for 1, 2; p-CH3C6H4 for 3, 4; p-CH3OC6H4 for 5, 6; p-ClC6H4 for 7, 8; p-FC6H4 for 9, 10; p-BrC6H4 for 11, 12; o, p-(Cl)2C6H3 for 13, 14; p-NO2C6H4 for 15,16; p-HOC6H4 for 17; C4H3S(2-yl) i.e. 2-Thienyl for 19, 20.

The compounds synthesized were tested against 8 plant patho-genic fungi (Rhizoctonia soloni-EB-ML, Fusarium oxysporium CE1, Sclerotinia sclerotiorum FD3, Aspergillus niger FS2, Aspergillus flavus Hak23, Alternaria alternata FS2002, Macrophamina phaseoli CE4, Botrytis cinerea MFD3) and 3 human pathogenic fungi (Microsporum canis-AO5, Candida albicans EA-07, Candida parapsilosis EA-08) us-ing agar dilution assay in the concentration range of 6,25 to 200 μg/ml [1]. Minimal Inhibition Concetrations (MIC) of the compounds were determined. Amphotericin-B was used as a reference compounds. Amphotericin-B was only effective against Macrophamina phaseoli CE4, Aspergillus niger FS2 and Aspergillus flavus Hak23 at 100 mg/ml.

None of the compounds showed antifungal activity at the concen-tration range studied against Fusarium oxysporium-CE1, Aspergillus niger-FS7, Botrytis cinerea-MFD, Candida albicans-EA-07, which are plant pathogenic fungi. MIC values of the compounds (μg/ml) were as follows:

Compound (μg/ml): 13(25), 14(50) against Rhizoctonia soloni-2001; 10, 13, 15 (12.5) , 2, 3, 8, 9, 11, 19, 20 (25), 1 (50), 7 (200) against Microsporum canis-AÖ5, 11 (100) against Sclerotinia sclero-tiorum-FD3; 13(100), 15, 16 (200) against Aspergillus flavus Hak23; 8 (200), 14 (100) against Alternaria alternata-FS2002; 5, 7 (200) against Macrophamina phaseoli-CE4; 9, 13, 15, 17, 19 (100) against Candida parapisilosis EA-08.

1. Gül Hİ, Şahin F, Gül M, Öztürk S, Yerdelen KO. Evaluation of antimi-crobial activities of several mannich bases and their derivatives, Arch. Pharm., 338, 335-8, 2005.

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EVALUATION OF CYTOTOXICITY OF 1-ARYL-3-

PHENETHYLAMINO-1-PROPANONE HYDROCHLORIDES

AND 1-PHENETHYL-3-BENZOYL-4-ARYL-4-PIPERIDINOLS

BY BRINE SHRIMP BIOASSAY

1Murat ÇİZMECİOĞLU, 2H. İnci GÜL, 3Ebru METE1Ege University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 35100 İzmir, Turkey2Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey3Atatürk University, Faculty of Arts and Sciences, Department of Chemistry, 25240 Erzurum, Turkey

Synthesis of the novel 1-aryl-3-phenethylamino-1-propanone hy-drochlorides (series 1) and 1-phenethyl-3-benzoyl-4-aryl-4-piperidi-nols (series 2) were carried out. Evaluation of the cytotoxicity of the compounds has been realised by brine shrimp bioassay as previously described [1]. Chemical structure of the compounds were confirmed by 1H-NMR, 13C-NMR, UV, IR and MS spectroscopic methods. Puririties of the synthesized compounds were determined by elemental analyses.

Of the compounds tested, compounds 13, 15, 17 (from the series 1), 4, 6, 8 (from the series 2) were found effective in brine shrimp bi-oassay (Table 1). Aryl part was (2,4-(Cl)2C6H3) for 13, (4-NO2C6H4) for 15, (4-HOC6H4) for 17, (4-CH3C6H4) for 4, (4-CH3OC6H4) for 6, (4-ClC6H4) for 8. The most effective compound was detected as compound 6 while the least effective one was found as compound 15. Cytotoxicities of these compounds were determined in the range of 13.85 - 3467.52 μg/ml while reference compound 5-fluorouracil exhibited 2.99 μg/ml of cytotoxicity.

Table 1. Cytotoxicity of the compounds in brine shrimp bioassay.

Code Aryl Cytotoxicity (μg/ml)

Series 113 2,4-(Cl)2C6H3 65.79 ± 0.08315 4-NO2C6H4 3467.52 ± 0.46917 4-HOC6H4 316.82 ± 0.122Series 24 4-CH3C6H4 165.70 ± 0.1866 4-CH3OC6H4 13.85 ± 0.1918 4-ClC6H4 573.33 ± 0.156

1. Gül Hİ, Gül M, Hanninen O. Cytotoxic activities of some mono and bis Mannich bases derived from acetophenone in brine shrimp bioassay, Arzneimittel Forschung, 52, 840-3, 2002.

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P 25

CRYSTAL STRUCTURE OF 3-(p-CHLOROBENZOYL)-4-(p-

CHLOROPHENYL)-1-PHENETHYL-4-PIPERIDINOL

1Ertan ŞAHİN, 1Cavit KAZAZ, 1Ebru METE, 2H. İnci GÜL1Atatürk University, Faculty of Science & Arts, Department of Chemistry, 25240 Erzurum, Turkey2Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey

NMR Study: The characterisation of compound 1, 3-(p-chlo-robenzoyl)-4-(p-chlorophenyl)-1-phenethyl-4-piperidinol, have been elucidated on the basis of 1H and 13C NMR spectral data (DEPT, COSY, HMQC, HMBC and NOE). 1H NMR spectra of the com-pound was well resolved and the unambiguous proton chemical-shift assignments were based on the multiplicity pattern of proton reso-nances and also on the use of homonuclear 1H – 1H COSY spectra. The assignments of all carbon resonances of compound were based on the analysis of the HMQC and HMBC spectra.

X-Ray Study: For the crystal structure determination, the single–crystal of the molecule 3-(p-chlorobenzoyl)-4-(p-chlorophenyl)-1-phenethyl-4-piperidinol was used for data collection on a four–circle Rigaku R–AXIS RAPID–S diffractometer equipped with a two–di-mensional area IP detector. The graphite–monochromatized Mo Kα radiation (λ=0.71073 Å) and oscillation scans technique with Δω=5° for one image were used for data collection. The lattice parameters were determined by the least–squares methods on the basis of all reflections with F2>2σ(F2). Integration of the intensities, correction for Lorentz and polarization effects and cell refinement was per-formed using CrystalClear software [1]. The structures were solved by direct methods (SHELXS–97) [2] and non–H atoms were refined by full–matrix least–squares method with anisotropic temperature factors (SHELXL-97) [2].

Compound 1, 3-(p-chlorobenzoyl)-4-(p-chlorophenyl)-1-phene-thyl-4-piperidinol, crystallizes in the monoclinic crystal system with space group P21/a and the following unit cell parameters: a= 10.792.(4) Å, b= 12.863 (5)Å, c= 16.950 (6) Å; α =90°, β= 97.77(4)°, γ = 90°; V= 2331.3(16) Å3 , and Z=4. The compound 1 has intermo-lecular O1-H···N1 [O1-H; 0.820, H···N1; 2.11, O1···N1; 2.878(5) Å, O1···H-N1 156˚], C13···H-O2 [C13-H; 0.970, H···O2; 2.54, C13···O2; 3.373(6) Å, C13···H-O2 144˚] hydrogen bonds.

1. Rigaku (2005), CrystalClear, Version 1.3.6. Rigaku American Corpora-tion, 9009 New Trails Drive, The woodlands, TX 77381–5209, USA.

2. Sheldrick GM. SHELXS97 and SHELXL97, University of Göttingen, Germany, 1997.

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STRUCTURE ELUCIDATION OF MONO MANNICH BASE

1-(p-METHOXYPHENYL)-3-PHENETHYLAMINO-1-

PROPANONE HYDROCHLORIDE AND SEMICYCLIC

MONO MANNICH BASE 3-(p-METHOXYBENZOYL)-4-(p-

METHOXYPHENYL)-1-PHENETHYL-4-PIPERIDINOL USING

1D AND 2D NMR SPECTROSCOPY

1Cavit KAZAZ, 1Ebru METE, 2H. İnci GÜL1Atatürk University, Faculty of Science & Arts, Department of Chemistry, 25240 Erzurum, Turkey2Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey

Mono Mannich base 1-(p-methoxyphenyl)-3-phenethylamino-1-propanone hydrochloride and semicyclic mono Mannich base 3-(p-methoxybenzoyl)-4-(p-methoxyphenyl)-1-phenethyl-4-piperidinol have been synthesized according to the literature process [1].

The structural elucidations of the compound was accomplished using extensive 1D-NMR (1H, 13C, NOE-diff, DEPT) and 2D-NMR (COSY, NOESY, HMQC and HMBC) spectroscopic techniques. Pro-ton and carbon-13 spectra were recorded at 27 °C in DMSO on a Varian mercury-plus (Palo Alto, USA) instrument at a frequency of 400 and 100 MHz, respectively, using a 5 mm ASW PFG probe.

1. Gül M, Gül Hİ, Das U, Hanninen O, Arzneimittel Forschung, 55(6), 332-7, 2005.

P 27

INVESTIGATION OF CASEIN KINASE 2 (CK2) ENZYME

AND ITS BINDING PROPERTIES WITH P53 PROTEIN,

POLYLYSINE AND HEPARIN BY MALDI-MS

1Ömür ÇELİKBIÇAK, 1Cansel TAŞAĞIR, 2Wayne E. CRISS, 1Bekir SALİH1Hacettepe University, Faculty of Science, Department of Chemistry, 06532 Beytepe-Ankara, Turkey2Hacettepe Unıversity, Faculty of Medicine, Oncology Institute, Department of Biochemıstry, 06100 Sıhhiye-Ankara, Turkey

Matrix-assisted Laser Desorption/Ionization, along with elec-trospray ionization, is now among the most important ionization methods for nonvolatile, high molecular weight compounds, in particular peptides, proteins, oligonucleotides, oligosaccharides and synthetic polymers. MALDI has also been successfully used for the investigation of fullerenes, fullerene derivatives, and syntheti-cally prepared dendrimers. Under certain conditions, it has even been shown that MALDI can be used for the study of weakly bound noncovalent complexes [1]. Protein kinase CK2 (Casein Kinase 2) is a ubiquitous serine/threonine protein kinase which is composed of two regulatory β- and two catalytic α- or α’- subunits. Although its precise function in the cell is still unclear there is ample evi-dence that CK2 plays an important role in the regulation of cell proliferation [2]. The activity of CK2 is elevated in tissues with a high proliferation rate, such as tumors and embryonic tissue [2]. The p53 tumor suppressor protein is a potent transcription fac-tor which is activated in response to a variety of DNA-damaging agents. Activation of p53 leads to cell growth arrest or the induc-tion of apoptosis, thereby blocking the survival of genetically dam-aged cells [2]. In the absence of functional p53 genes, the cycle is not arrested and the apoptosis signal is not delivered, so a cell with abnormal DNA is allowed to replicate, thus increasing the chance of cancer developing [3]. P53 is phosphorylated at multiple sites in vivo and by several different protein kinases in vitro including the protein kinase CK2. In addition, it was shown previously by some researchers that mutation of p53 at the CK2 phosphoryla-tion site abolishes the growth suppressor activity of p53 [2]. Be-cause of the importance of the binding properties of p53 to the CK2 enzyme, MALDI-MS analysis of CK2 and p53 proteins were performed and interactions of p53 protein with α and β subunits of CK2 enzyme were investigated. Since CK2 was first discovered, many studies have showed us that the enzymatic activity of CK2 was stimulated by polyamines and inhibited by heparin in vitro [2]. According to these data, existence of interactions can be inferred between these molecules and CK2 and/or between these molecules and CK2 substrates, which probably improve the susceptibility of substrates to be phosphorylated or not. In order to get information about the mechanism of noncovalent interactions between these biomolecules, MALDI-MS was used by selecting suitable matrix, sample preparation and clean up procedures at appropriate pH and solvent conditions.

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1. Zenobi R, Knochenmuss R. Ion Formation in MALDI Mass Spectrom-etry, Mass Spectrometry Reviews, 17, 337–366, 1998.

2. Schuster N, Götz C, Faust M, Schneider E, Prowald A, Jungbluth A, Mon-tenarh M. Wild-Type p53 Inhibits Protein Kinase CK2 Activity, Journal of Cellular Biochemistry, 81, 72-183, 2001.

3. Elliott, WH, Elliott, DC. Biochemistry and molecular biology, 3rd ed., Oxford University Press Inc., New York, 2001, p. 533-534.

P 28

CYTOTOXICITIES OF N,N’-BIS(3-DIMETILAMINO-1-ARYL-

PROPYLIDENE)HYDRAZINE DIHYDROCHLORIDES

1Kaan KÜÇÜKOĞLU, 2Mustafa GÜL, 1H. İnci GÜL, 3Osmo HANNINEN, 3Mustafa ATALAY1Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Erzurum, Turkey2Atatürk University, Faculty of Medicine, Department of Physiology, Erzurum, Turkey3University of Kuopio, Department of Physiology, Finland

Synthesis of N,N’-bis (3-dimethylamino-1-aryl-propylidene) hy-drazine dihydrochlorides were carried out according to the literature procedure [1] by using acetophenone or substituted acetophenones as the ketone component of the reactions while dimethylamine hydro-chloride was the amine component of the reactions. Substituents were methyl for 2, methoxy for 3, hydroxy for 4 and chloro for 5 at the para position of the phenyl ring while compound 1 was nonsubstituted.

Chemical structures of the compounds were confirmed by 1H NMR. Cytotoxicity of the compounds was determined against Jur-kat cells which is human T lymphocytes cells. Reference compounds were 5-fluorouracil and melphalane.

All compounds have shown more powerful activity than both references, cytotoxicity values of the compounds were in the range of 9.75-13.27μM. The most effective compound was 4 in five com-pounds studied.

1. Gül Hİ, Das U, Pandit B, Li PK, Evaluation of the cytotoxicity of some mono-mannich bases and their corresponding azine derivatives against androgen-independent prostate cancer cells, Arzneimittel Forschung, 56(12), 850-4, 2006.

P 29

SYNTHESIS OF 1-[3-(PIPERIDINOMETHYL)-4-

HYDROXYPHENYL]-3-ARYL-2-PROPEN-1-ONES AND

EVALUATION OF THEIR ANTICONVULSANT ACTIVITIES

1H. İnci GÜL, 1K. Özden YERDELEN, 2Ünsal ÇALIŞ1Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey2Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Sıhhiye-Ankara, Turkey

In this study, Mannich bases with piperidine, 1-[3-(piperidinome-thyl)-4-hydroxyphenyl]-3-aryl-2-propen-1-one, B1-B5, were synthe-sized starting from the chalcones, 1,3-diaryl-2-propen-1-one, A1-A5.

Chemical structures of the compounds have been confirmed by 1H-NMR, 13C-NMR, IR, and UV spectra and elemental analysis. Anticonvulsant activities of the compounds were evaluated by MES, scMet tests. Neurotoxicities of the compounds were also evaluated by rotorod test [1, 2].

None of the compounds showed neurotoxicity at the screening of anticonvulsant activity. Compounds B1-B5 at MES test, compounds B1-B3 at scMet test have shown anticonvulsant activity at different dose levels (30-300 mg/kg) and time periods (1/2 h, 4 h).

To conclude, of the compounds B4, B5 against grand-mal epilep-sia, B1, B2 and B3 against both types of epilepsia, can be choosen

as candidate compounds to develop new anticonvulsant compounds for further studies.

1. Gül Hİ, Çalış Ü, Vepsalainen J. Synthesis of some mono-Mannich bases and corresponding azine derivatives and evaluation of their anticonvul-sant activity, Arzneimittel Forschung, 54, 359-64 2004.

2. Gül Hİ, Çalış Ü, Vepsalainen J. Synthesis and evaluation of anticonvul-sant activities of some bis Mannich bases and corresponding piperidi-nols, Arzneimittel Forschung, 52, 863-9 2002.

P 30

DEVELOPMENT AND OPTIMIZATION OF A SURFACE

AIDED ENZYMATIC DIGESTION SYSTEM TO USE IN

PROTEOMICS STUDIES

1Basri GÜLBAKAN, 1Aslı ÖZTÜRK-ÇAL, 2Talat YALÇIN, 1Bekir SALİH1Hacettepe Unıversity, Faculty of Science, Department of Chemistry, 06532 Beytepe-Ankara, Turkey2İzmir Institute of Technology, Faculty of Science, Department of Chemistry, 35430 İzmir, Turkey

Proteomics is the study of all methods covering isolation, sepa-ration, identification, characterization of all proteins in living or-ganisms and finding their functional roles and expression of the sequences of proteins in all tissues. The anomalies in the synthe-sis and function of the proteins are interrelated with several lethal diseases. Understanding the cause and the therapy of a disease is related to determining and understanding the proteins that are in-volved. After completion of the Human Genome Project, proteom-ics gained more importance. However, since proteins are more complex than that of genes, secondary cleavage reactions namely, enzymatic trypsin reactions are employed to obtain smaller peptide fragments and peptides are then analyzed to obtain structural in-formation about proteins. Thus the success of proteomics is closely related with the success of enzymatic trypsin cleavage. Isolation of proteins from their natural environment, which is a very complex matrix, leads to protein loss and in many cases necessitates the ad-dition of contaminating agents like SDS, CHAPS and sucrose. This also limits the information that is obtained from proteomics studies. The presented study was carried out in order to eliminate the intrin-sic difficulties of conventional in-solution and in-gel trypsin diges-tion protocols and to develop a new and effective digestion method. Proteins samples having different molecular weights and different structural complexity was preconcentrated and purified by using conventional reversed phase column packing materials that are C18 bonded silica, polystyrene-divinylbenzene and modified derivatives thereof and digested while bound to surface. The resulting peptides were then analyzed by MALDI mass spectrometry. The effect of dif-ferent MALDI matrices, sample preparation methods, effect of func-tional group loading onto poly (styrene-divinylbenzene) microbeads was studied. The results were compared by conventional methods using trypsin digestion to test the efficiency of the method.

1. Gevaert K, Vandekerckhove J. Protein identification methods in pro-teomics, Electrophoresis, 21, 1145-1154, 2001.

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P 31

SYNTHESIS OF 1-[3-(DIBENZYLAMINOMETHYL)-4-

HYDROXYPHENYL]-3-ARYL-2-PROPEN-1-ONES AND

EVALUATION OF THEIR ANTIFUNGAL ACTIVITIES

1K. Özden YERDELEN, 1H. İnci GÜL, 2Fikrettin ŞAHİN1Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey2Yeditepe University, Faculty of Engineering and Architecture, Department of Genetics and Bioengineering, 34755-Kayışdağı, İstanbul, Turkey

In this study, Mannich bases with dibenzylamine 1-[3-(diben-zylaminomethyl)-4-hydroxyphenyl]-3-aryl-2-propen-1-one, (C1-C5) were synthesized and the chemical structures of the compounds have been confirmed by 1H-NMR, 13C-NMR, IR, and UV spectra and elemental analysis.

Antifungal activities of the compounds have been tested and com-pared with their precursor chalcones (A1-A5) against 3 fungi species pathogenic in humans [Trichophyton rubrum (Hak-8), Trichophyton mentagrophytes (Hak-9), Microsporum canis (Hak-4)] and 13 fungi species pathogenic in plants [Sclerotinia sclerotiorum, Sclerotinia minor, Alternaria alternate (AA-1121), Aspergillus flavus (Hak-23), Aspergil-lus variecolor (IO-Balik), Fusarium acuminatum, Fusarium oxysporum (ED-10), Fusarium solani (ED-1S), Fusarium tabacinum (ED-1T), Moniliania fructicola (FS-M), Penicillium spp. (P-TY), Rhizopus spp. (R-27), Rhizoctonia solani (EB-ML)] at the concetration range of 2-64 μg/ml using amphotericin–B as the reference compound (1).

Of the compounds, A1 against plant pathogens Sclerotinia scle-rotioruma and Rhizoctonia solani, while C5 against plant pathogen Fusarium oxysporum, have shown 2-4 times more powerful antifun-gal activity compared with amphotericin-B. Of the compounds syn-thesized, A1 and C5, against plant pathogenic fungi can be choosen as candidate compounds for further studies to develop new antifun-gal compounds.

1. Gül Hİ, Şahin F, Gül M, Öztürk S, Yerdelen KO. Evaluation of antimi-crobial activities of several mannich bases and their derivatives, 1, Arch. Pharm., 338(7), 335-8, 2005.

P 32

SYNTHESIS OF 1-[3-(PIPERIDINOMETHYL)-4-

HYDROXYPHENYL]-3-ARYL-2-PROPEN-1-ONES AND

EVALUATION OF THEIR CYTOTOXIC ACTIVITIES AGAINST

HUMAN T LYMPHOCYTES (JURKAT) CELLS AND RAT

SKELETAL MUSCLE DERİVED MYOBLAST CELLS (L6)

1H. İnci GÜL, 2Mustafa GÜL, 1K. Özden YERDELEN, 3Osmo HANNINEN, 3Mustafa ATALAY1Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey2Atatürk University, Faculty of Medicine, Department of Physiology, 25240 Erzurum, Turkey3University of Kuopio, Faculty of Medicine, Department of Physiology, 1627 Kuopio, Finland

In this study, Mannich bases with piperidine, 1-[3-(piperidinome-thyl)-4-hydroxyphenyl]-3-aryl-2-propen-1-one, B1-B5 were synthe-sized starting from the chalcones, 1,3-diaryl-2-propen-1-one, A1-A5. Chemical structures of the compounds have been confirmed by 1H-NMR, 13C-NMR, IR, and UV spectra and elemental analyses.

Cytotoxic activities of the compounds have been tested against rat skeletal muscle derived myoblast cells (L6) and transformed hu-man T lymphocytes (Jurkat). Melphalan and 5-fluorouracil were also tested as reference drugs [1].

All compounds have shown 1.28-5.40 times more powerful cyto-toxicity than 5-FU, and the compounds A3, B1, B2, B3 have shown

1.03-2.76 times more powerful cytotoxicity than melphalan against L6 cells, respectively. Preparation of Mannich bases with piperidine from the chalcones increased the cytotoxicity 1.55, 1.54, 1.33, 1.38 times at the compounds B1, B2, B4, B5 respectively, compared with their corre-sponding chalcones. While all compounds synthesized had 3.20-9.43 times more powerful cytotoxicity than 5-FU against Jurkat cells, ex-cept B1, all other compounds showed 1.11-2.38 times more powerful cytotoxicity than melphalan. Preparation of Mannich bases from the chalcones increased the cytotoxicity 1.42, 1.70, 1.43 times respectively at the compounds B2, B4 and B5 compared with the corresponding chalcones against Jurkat cells.The compounds synthesized have been found more selective against Jurkat cells compared with L6 cells.

Of the compounds synthesized, B2, B4 and B5 can be choosen as candidate compounds for further cytotoxicity studies to develop new cytotoxic compounds.

1. Gül M, Gül Hİ, Das U, Hanninen O, Arzneimittel Forschung, 55(6), 332-7, 2005.

P 33

DETERMINATION OF PHYSICOCHEMICAL

PROPERTIES OF SEVERAL SULFONAMIDES BY LIQUID

CHROMATOGRAPHY IN ACETONITRILE-WATER BINARY

MIXTURES

1Nurullah ŞANLI, 1Güleren ALSANCAK, 2Adil DENİZLİ1Süleyman Demirel University, Faculty of Science and Literature, Department of Chemistry, Isparta, Turkey2Hacettepe University, Faculty of Science, Department of Chemistry, Ankara, Turkey

Sulfonamides are antibacterial compounds commonly used to prevent and to treat diseases in medical and veterinary practice. A sulfonamide contains one basic amino group and one acidic amide group which correspond to pKa1 and pKa2 respectively. The degree of ionization of sulfonamides was strongly related with the in vitro bacteriostatic activity [1].

The use of HPLC retention parameters to determine pKa values has been widely applied [1-2]. Literature studies of the chromato-graphic determination of pKa values of sulfonamides and the effect of the organic modifier content on the pKa values of these com-pounds are scarce, but the investigation of Mengelers et al. should be mentioned [1].

In this study, the dissociation constants of related series of sulfon-amides (sulfodiazine, sulfothiazole, sulfomerazine, sulfomethazine, sulfomonomethoxine, sulfodoxine, sulfomethoxazole) were deter-mined by LC methodology and the effects of the ACN percentage on the pKa values were investigated. The prodigy C-18 was used as stationary phase. On changing the mobile phase of the system, the column was thoroughly equilibrated with studied new mobile phase. The electrode system was calibrated with potassium acid phthalate in organic mixture of the same composition as the mobile phase ac-cording to IUPAC rules. The pH of the mobile phase was measured after the addition of the ACN to properly investigate the effect of pH on the retention of ionizable compounds. The effect of pH on sulfonamides retention was investigated in the range of pH 1.7-9.7. The retention time was plotted against pH value of the mobile phase to calculate pKa2 values. The sigmoidal behaviors of studied sulfona-mides are shown in Figure 1.

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Figure 1. Plots of retention times of sulphonamides against mobile phase pH (4.5-9.0) at 30% (v/v) ACN. Symbols indicate: ■ sulfodiazine, ♦ sulfothiazole, ▲ +sulfomerazine, х sulfomethazine, ж sulfomonomethoxine, • sulfodoxine, sulfomethoxazole).

Their pKa values in ACN – water mixture (50% v/v) were also potentiometricaly determined [3] and compared with the results ob-tained by LC methodology.

The retention data of sulfonamides obtained by Reversed Phase Liquid Chromatography (RPLC) were correlated with the molecu-lar partition coefficient, Po, obtained in octanol-water systems by Countercurrent Chromatography (CCC) [4], and the correlated data fits perfectly.

1. Mengelers MJB, Hougee PE, Janssen LHM, Van Miert AS. Structure-ac-tivity relationships between antibacterial activities and physicochemi-cal properties of sulfonamides, J. Vet. Pharmacol. Therap., 20, 276-283, 1997.

2. Botsoglou NA, Fletouris DJ, Simeonidou EJ, Psomas IE. Retention be-havior of multiple sulfonamides in various liquid chromatographic sys-tems, Chromatographia, 46, 9/10, 477-782, 1997.

3. Qiang Z, Adams C. Potentiometric determination of acid dissociation constants (pKa) for human and veterinary antibiotics, Water Research, 38, 2874-2890, 2004.

4. Carda-Broch S, Berthord A. Countercurrent chromatography for the measurement of the hydrophobicity of sulphonamide amphoteric com-pounds, Chromatographia, 59, 79-87, 2004.

P 34

CONTROLLED RELEASE OF NAPROXEN FROM POLY

(VINYL ALCOHOL) / SODIUM ALGINATE MICROSPHERES

1Oya ŞANLI, 2Ebru KONDOLOT-SOLAK1Gazi University, Arts and Sciences Faculty, Department of Chemistry, 06500 Ankara, Turkey2Gazi University, Atatürk Vocational College, Department of Chemistry, Ankara, Turkey

In this study microspheres of poly(vinyl alcohol)/sodium algi-nate (PVA/Na-Alg) and sodium alginate (Na-Alg) were prepared to encapsulate naproxen sodium drug. Microspheres were prepared by liquid curing method by crosslinking with glutaraldehyde, then characterized by fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC) and scanning electron mi-croscopy (SEM).

Microspheres were also characterized by measuring the particle diameter, equilibrium swelling values and determining release pro-files. Equilibrium swelling experiments indicated that the swelling of the spheres decreased with an increase in crosslinking time and concentration however diameter of the spheres was not affected con-siderably.

The release studies were carried out at three pH values 1.2, 6.8 and 7.4 respectively. The release of diclofenac from the micropheres increased as the drug/polymer ratio decreased. Optimum condition for the preparation and release of the spheres were determined as PVA/Na-Alg: 1/2, drug/polymer: 1/4 and pH: 7.8. The release of these conditions was found as 80.3 % at the end of 6th hour.

P 35

SPECTROPHOTOMETRIC ANALYSIS OF CABERGOLINE IN

PHARMACEUTICAL PREPARATIONS

1Demet SALMAN, 2Nursabah E. BAŞÇI, 1Turkish Republic Instutition of Social Security, Ankara, Turkey2Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, Ankara, Turkey

Cabergoline is a synthetic dopamine agonist having high affinity to D2 receptors and used for early and advanced Parkinson’s patients and hyperprolactinemy disorders [1]. Chromatographic and radio-immunoassay methods have been reported for quantification of ca-bergoline in body fluids [2-5], but there was not any spectrophoto-metric analysis of cabergoline in pharmaceutical preparations in the literature. In this study, simple, fast, reliable and validated UV-VIS and 2nd derivative spectroscopy methods were developed for deter-mination of cabergoline in pharmaceutical preparations.

Determination of cabergoline was performed by UV-VIS spectro-photometry at 280 nm wavelength and by 2nd derivative spectropho-tometry at a range of 227-232 nm. HPLC (Shimadzu SCL-10AVP) connected to an electrochemical detector (Decade) was used in com-parison analysis. Cabergoline substance was donated by Pharmacia. Dostinex® (0.5 mg Cabergoline, Pfizer) and Cabaser® (1, 2 and 4 mg Cabergoline, Pfizer) tablets were used for pharmaceutical applica-tions. Developed methods were validated according to the regula-tions and guidelines of ICH, EMEA and FDA. Validation results were statistically evaluated using related methods at a significant level of 95%.

Developed UV-VIS and 2nd derivative spectrophotometry meth-ods were linear over the range of 1-125 μg mL-1. The limit of detec-tion for the methods was 0.5 μg mL-1 (RSD = 0.75%, Bias = 0.30) and the limit of quantification was 1.0 μg mL-1 (RSD = 0.67%, Bias = 0.33). The highest relative error and relative standard deviation in inter-day and intra-day study for UV-VIS spectrophotometry were 1.10% and 0.63%, respectively. The highest relative error and relative standard deviation in inter-day and intra-day study for 2nd derivative spectrophotometry were 1.10% and 0.70%, respectively. The methods were applied to the analysis of cabergoline in pharma-ceutical preparations and there was no statistically significant differ-ence when the results were compared with the results of comparison method (HPLC/ECD). In conclusion, the developed UV-VIS and 2nd derivative spectrophotometry methods were accurate, sensitive, pre-cise and repeatable and can be applied to the analysis of cabergoline in pharmaceutical preparations as direct, fast and simple methods.

1. Gottwald MD, Bainbridge JL, Dowling GA, Aminoff MJ, Alldredge BK. New pharmacotherapy for parkinson’s disease, Annals of Pharmacothera-phy, 31(10), 1205-17, 1997.

2. Persiani S, Pianezzola E., Broutin F, Fonte G, Benedetti MS. Radioim-munoassay for the synthetic ergoline derivative cabergoline in biological fluids, Journal of Immunoassay, 13(3), 457-76, 1992.

3. Pianezzola E, Bellotti V, Croix RL, Benedetti MS. Determination of ca-bergoline in plasma and urine by high-performance liquid chromatog-raphy with electrochemical detection, Journal of Chromatography, 574, 170-174, 1992.

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4. Allievi C, Dostert P. Quantitative determination of cabergoline in human plasma using liquid chromatography combined with tandem mass spec-trometry, Rapid Communications in Mass Spectrometry, 12, 33-39, 1998.

5. Igarashi K, Hotta K, Kasuya F, Abe K, Sakoda S. Determination of caber-goline and L-dopa in human plasma using liquid chromatography-tan-dem mass spectrometry, Journal of Chromatography B, 792, 55-61, 2003.

P 36

INVESTIGATION OF ANTIMICROBIAL ACTIVITES OF

NEW SULFONYLHYDRAZONE DERİVATİVES ON SOME

MICROORGANISMS

Ümmühan ÖZDEMİR-ÖZMEN, Fatma HAMURCUGazi University, Arts and Sciences Faculty, Department of Chemistry, 06500 Ankara, Turkey

The chemistry of hydrazones has been intensively investigated in recent years, owing to their coordinating capability, pharmacological activity, antibacterial and antifungal properties, Sulfonamide drugs are widely used chemotherapeutic agents with large spectrum of ac-tivity [1]. Methane sulfon amide residue has appeared as a suitable pharmacophoric equivalent to replace functional groups in drug de-sign . Having hydrophilic character, like the sulfonyl group is con-sidered as a suitable pharmacophoric equivalent for replacing func-tional groups in drug design [2]. In previous paper, we reported the antibacterial and cytotoxic effect of methanesulfonic acid hydrazide (msh) [3].

Ethanesulfonic acid hydrazide, C2H5SO2NHNH2, (esh) ; 5-methyl-2-hydroxyacetophenoneethanesulfonylhydrazone (5mafesh) and its Ni(II) ,Co(II) complexes containing sulfonamide and hydrazine fragments were synthesized and their structures were determined by using elemental analysis, NMR, FT-IR, LC-MS, magnetic and conductivity studies. Their antimicrobial activities were investigated against to Escherichia coli ATCC 11230, Bacillus subtilis RSKK 244, Bacillus cereus RSKK 863, Bacillus magaterium RSKK 5117, Salmo-nella enteritidis ATCC 13076, Staphylococcus aureus ATCC 25923 by using MIC’s method. MIC’s was defined as the lowest concentrations of compounds which inhibit the growth of microorganisms.

The antimicrobial results evidently showed that the sulfonamide derivatives possessed a broad spectrum of activity against the tested bacteria (MIC’s values g/mL). All compounds exhibited the most ac-tivity against to Staphylococcus aureus between 60-672μg. The pres-ence of the NH group in the sulfonamides contributes positively to the increase of the activity of compounds against bacteria. In addi-tion the negative charges on the other donor atoms show a tendency to increase the activity [4].

1. Albert A. Selective Toxicity, Chapman and Hall, London, New York, 1985.

2. Topiol S, Sabio M, Erhardt PW, J. Chem. Soc.Perkin Trans., II, 437, 1988. 3. Dodoff NI, Özdemir Ü, Karacan N et al., Z. Naturforsch., 54 b , 1553,

1999. 4. Zanatta N, Alues SH, Coelho HS et al., Bioorganic & Medicinal Chemis-

try, 15, 1947, 2007.

P 37

DETERMINATION OF DISSOCIATION CONSTANTS OF

SEVERAL SULFONAMIDES BY POTENTIOMETRY IN

METHANOL, 2-PROPANOL, TETRAHYDROFURAN-WATER

BINARY MIXTURES

1Senem ŞANLI, 1Ebru ÇUBUK-DEMİRALAY, 2Hale SEÇİLMİŞ, 3Jose Luis BELTRAN1Süleyman Demirel University, Science & Literature Faculty, Department of Chemistry, 32260 Isparta, Turkey2Süleyman Demirel University, Research Centre, 32260 Isparta, Turkey3Barcelona University, Department of Analytical Chemistry, 08028 Barcelona, Spain.

The pKa value is a main item in the biophysical characterization of a drug and may be helpful in predicting the behavior of a drug under in vivo conditions. Sulfonamides (SAs) are typical amphoteric compounds and dissociation pathways of sulfonamides are given in Figure 1. Ka1 and Ka2 are the dissociation constants of the aromatic amine and sulfonic groups respectively.

Fig 1. Scheme of dissociation equilibrium of sulfonamides.

Literature studies of the potentiometric determination of pKa val-ues of sulfonamides are scarce, but the investigation of Qiang and Adams should be mentioned [1]. In addition only a few pKa values of sulfonamides in organic solvent-water binary mixtures can be ob-tained from the literature [2].

Among the pKa determination techniques, the potentiometric titration is most economical of time and if due care is taken; this technique is accurate and has good reproducibility. This paper inves-tigated the potential of potentiometric method to determine the pKa values of sulfonamides that show poor solubility. In this study the effect of the organic modifier type and content on the pKa values of these compounds were also investigated. These solvent mixtures can dissolve drugs more effectively than water and in many cases they are more suitable solvent for the determination of the dissociation constants. Methanol and 2-propanol are closest to water in structure and properties. THF is one of the most widely used dipolar aprotic solvent and is much better differentiating solvent than water.

The pKa1 and pKa2 values of sulfonamide were determined by up-ward titration using 0,025 M KOH. The typical titration curve for sulfametaxazole is shown in Fig 2. In this study PKPOT program was used to correct the effect of ionic strength on pKa determina-tion [3].

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Fig 2. Potentiometric titration curve of sulfomethaxazole in MeOH-H2O, 30% (v/v).

1. Qiang Z, Adams C. Potentiometric determination of acid dissociation constants (pKa) for human and veterinary antibiotics, Water Research, 38, 2874-2890, 2004.

2. Mengelers MJB, Hougee PE. Janssen LHM, Van Miert AS. Structure-ac-tivity relationships between antibacterial activities and physicochemi-cal properties of sulfonamides, J. Vet. Pharmacol. Therap., 20, 276-283, 1997.

3. Barbosa J, Barron D, Beltran JL, Sanz-Nebot V. PKPOT, a program for the potentiometric study of ionic equilibria in aqueous and non-aqueous media, Anal. Chim. Acta, 317, 75-81, 1995.

P 38

IMPROVED ULTRA-PERFORMANCE LIQUID

CHROMATOGRAPHIC DETERMINATION OF INDAPAMIDE

IN HUMAN PLASMA

Zeliha ATEŞ, Sami EREN, Selma ÖZİLHAN, Tuncel ÖZDENNovagenix Bioanalytical R&D Centre, Ankara, Turkey

Indapamide, 3-(aminosulfonyl)-4-chloro-N-(2,3-dihydro-2-me-thyl-1H-indol-1-yl)-benzamide, is an oral antihypertensive and diuretic agent [1]. Indapamide inhibits carbonic anhydrase en-zyme, which reduces the vascular response to noradrenalin and an-giotensin II by inhibiting the transportation of Ca2+ into vascular smooth muscle. It shows diuretic effect by acting at the first parts of distal tubules [2].

A simple, rapid, sensitive and selective method for the analysis of indapamide in human plasma, utilizing ultra performance liquid chromatography (UPLC), has been developed and validated to fulfill FDA guidelines for bioanalytical methods [3]. The analyte and the internal standard, sulfamethazine, were isolated from plasma sam-ples by liquid-liquid extraction with diethyl ether. The assay exhib-ited a linear dynamic range of 1 to 100 ngmL-1 for indapamide in human plasma.

The limit of quantification (LOQ) was 1 ngmL-1 with a relative standard deviation of less than 12.2 %. Inter and intra-day preci-sion (CV %) and accuracy (%) for quality control samples (3, 50, 80 ngmL-1) ranged from 0.55 % to 8.48 % and from 94.62 % to 107.56 % respectively. Furthermore, this method was successfully applied to the pharmacokinetic and bioequivalence study of indapamide tab-lets in healthy male volunteers within 96 h period.

1. Caruso FS, Szabadi RR, Vukovich RA. Am. Heart, 106, 212-220, 1983. 2. Johnston MM, Rosenberg MJ, Yeung AK, Grebow PE. J. Pharm. Sci., 69,

1158-1160, 1980. 3. FDA Guidance for Industry, Bioanalytical Method Validation, May

2001.

P 39

THE EFFECT OF TAUROLIDINE ON THICKNESS OF SCAR

TISSUE IN RABBIT MODEL

1Ali HAYAT, 2Füsun TEMAMOĞULLARI, 3Füsun BABA1University of Harran, Faculty of Veterinary Medicine, Department of Surgery, Şanlıurfa, Turkey2University of Harran, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, Şanlıurfa, Turkey3University of Harran, Faculty of Medicine, Department of Pathology, Şanlıurfa, Turkey

Clinicans have used numerous strategies to combat wound infec-tions, including topical and systemic administration of antibiotics and various antiseptic agents [1]. The antimicrobial properties of taurolidine have been ascribed to the biological active methylol tau-rinamide which reacts with cell wall constituents of microbial patho-gens via methylene iminium ions preventing bacterial adhesion to biological surfaces. Taurolidine has a short half-life and is metabo-lised to taurine, carbon dioxide and water. It has been shown to be non-toxic to human and animals [4]. Povidone iodine is commonly used in clinical practice but dermal hypersensitivity is associated with the use of povidone iodine in humans and small animals [2]. In this study, we have investigated efficacy of 2% taurolidine solu-tion on healing wound standing on the clinical and histopathologic parameters comparing with 10% povidine iodine solution and 0.9% sodium chloride.

Six male and six female rabbits (mean weight: 2500±200) were taken for the study. All rabbits were anesthetized with intramuscu-lar administration of xylazine hydrochloride 10 mg/kg (Rompun, Bayer) and ketamin hydrochloride 50 mg/kg (Ketanes, Alke) Right and left costal regions of rabbits were clipped and the skin was pre-pared for aseptic surgery. Then full-thickness skin wounds (3 cm in diameter) as two cranial and one caudally located were created on each animal using a template prepared from x-ray film. Daily, 2% taurolidine, 10% povidine iodine solution and 0.9% sodium chloride were applied on wounds. Macroscopically wounds were examined from point of the exudation during the postoperative days. Biopsy specimens which were collected on the 4th, 8th, 12th and 16th PODs. Specimens were evaluated according to several histopathologic pa-rameters, such as the thickness of scar tissue.

Macroscopically, the wounds treated with 10% povidine iodine so-lution and 0.9% sodium chloride appeared to have marked fibrinous exudate and crust formation which caused adherence to the gauze. On the other hand. 2% taurolidine gauze applied wounds showed lesser degree of exudate and lesser adherence. Daily applied 2% tau-rolidine reduced the thickness of scar tissue when compared to other two solution. The degree of attachment between dressing material and the wound surface is important [3]. The subjective examination showed that the 2% taurolidine conformed well to the wound surface and it was readily separeted from underlying wound. As a result, 2% taurolidine application to full thickness skin wounds in rabbits posi-tively effected wound healing but the mechanism underlies this fact still needs furter investigations.

1. Burks, RI. Povidone-iodine solution in wound treatment, Phys. Ther., 78, 212-218, 1998.

2. Farstvedt, E, Stashak, TS., Othic, A. Update on Topical Wound Medica-tions, Clin. Tech. Equine Pract., 3, 164-172, 2004.

3. Kılıç, S, Timurkan, N, Ünsaldı, S, Günay, C, İstek, Ö, Yılmaz, B. Compari-son of the Effects of Some Wound Healing Materials on Full Thickness Skin Wounds in Rabbits, Turk. J. Vet. Anim. Sci., 26, 263-272, 2002.

4. Koldehoff, M, Zakrzewski, JL. Taurolidine is effective in the treatment of central venous catheter-related bloodstream infections in cancer pa-tients, International Journal of Antimicrobial Agents, 24, 491-495, 2004.

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P 40

THE QUANTITATIVE DETERMINATION OF FEXOFENADINE

IN HUMAN PLASMA BY LC/MSD SYSTEM

Müberra ŞEN, Evren İŞLEYEN, Selma ÖZİLHAN, Suna TOPTAN, Tuncel ÖZDENNovagenix Bioanalytical R&D Centre, Ankara, Turkey

Fexofenadine hydrochloride, (±)-4-[1-hydroxy-4-[4-(hydroxy-diphenylmethyl)-1-piperidinyl]-butyl]-α, α-dimethyl benzeneacetic acid hydrochloride has an empirical formula, C32H39NO4.HCl with a molecular weight of 538.1 which is the active metabolite of terfena-dine and is a second-generation histamine H1-receptor antagonist in piperidine-class drugs. Fexofenadine is a H1-receptor antagonist that blocks peripheral histamine H1-receptors selectively [1, 2].

A simple, rapid, sensitive and selective LC-MS method was de-veloped and validated for quantification of fexofenadine in human plasma. The LC-MS system was operated under the positive elec-trospray ionisation mode (ESI). After liquid-liquid extraction, fex-ofenadine analysis was performed through a C18 column with a mo-bile phase of acetonitrile: 10 mM ammonium acetate: formic acid, 70:30:0.1 (v/v/v) at a flow rate of 1 mLmin-1 by using loratadine as an internal standard. The lower limit of quantitation was 3 ngmL-1 for fexofenadine.

Results of analysis showed after five days validation process, co-efficient correlation was 0.9993 – 0.9999. In quality control sam-ples, with-in-batch and batch-to batch accuracy ranges were 86.51 – 113.50% and 97.92 – 106.06% respectively; precision ranges were 3.89 – 13.62% and 8.40 – 11.81% respectively. In calibration stand-ard samples, batch-to batch accuracy ranges were 96.40 – 104.17%; batch-to batch precision ranges were 2.44 – 5.80%. Our whole study was conducted according to FDA regulations about bioanalytical method validation process [3]. The presented analytical method that is developed originally and validated in our laboratory was used to evaluate the bioequivalency of two different brand name fexofena-dine products.

1. Simpson K, Jarvis B. Drugs, 59, 301-321, 2000. 2. Dollery C. Therapeutic Drugs 2nd edition Churchill Livingstone, United

Kingdom, A151-A154. 1999. 3. FDA, Bioanalytical Method Validation, Guidance for Industry, May

2001.

P 41

COMPARISON OF THE ROYAL JELLY AND POVIDONE

IODINE ON WOUND HEALING IN RABBITS

1Füsun TEMAMOĞULLARI, 2Ali HAYAT, 3Füsun BABA1University of Harran, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, Şanlıurfa, Turkey2University of Harran, Faculty of Veterinary Medicine, Department of Surgery, Şanlıurfa, Turkey3University of Harran, Faculty of Medicine, Department of Pathology, Şanlıurfa, Turkey

Royal jelly (RJ) has been used worldwide for many years as medi-cal products, health foods and cosmetics [1]. A number of biological and immuno-regulatory actions attributed to RJ have been report-ed. In this study, we have investigated the efficacy of RJ on healing wound standing on the clinically and histopathologically comparing with 10 % povidone iodine and 0,9% sodium chloride.

Six male and six female rabbits weighing about 2500 ± 200 g were anesthetized with i.m. administration of 10mg/kg xylazine hydro-chloride (Rompun, Bayer) and 50mg/kg ketamine hydrochloride (Ketanes, Albe). On dorsal aspect of each animal, two cranially and one caudally located full-thickness skin wounds in 3,14 cm diam-

eter were created using a template prepared from an X-ray film. Fol-lowing incision different wounds of each animal were treated with RJ (83 mg/ml Royal Jelly-Arıjel Co.,Ltd. ), 10% povidon iodine and 0,9% sodium chloride as the control respectively. Then the wounds were closed with sterile gauze and fixed with circular adhesive bands. Wounds were examinated macroscopically and by exudation. The beginning of the wound contraction as the indicator of the begin-ning of healing, granulation of the tissue and the first day of epithe-lization were noted regularly. SPSS 11.0 for Windows was used for statistical analyses.

Whole control wound surface were covered by a thin gelatinous exudate (POD 4). After this exudate was removed, an ongoing healthy granulation and epithelization tissues were determined. RJ gauze-applied wounds showed strong adherence and a lesser degree exudate. Therefore, they required greater tearing force for removal of the dressing. According to other groups, the acceleration of epi-thelization in the RJ treated group appeared to occur between 7 and 9 days clinically as well as histologically. However, adhered strongly and the frequent dressing may delay the healing. The epithelization was completed on POD 16 on RJ and 10 % povidone iodine gauze-applied wounds, whereas it wasn’t completed on 0.9 % sodium chlo-ride gauze-applied wounds and ulceration in central wounds was seen. The granulation tissues on all wounds were noted on PODs 3-5, while epithelization was seen on PODs 6-8. The expansion proc-ess (to POD 4) was followed by the contraction process (to PODs 6-8). The contraction in RJ gauze-applied wounds (to PODs 16) be-came more than other wounds. The thickness of scar tissue was sig-nificantly different on PODs 4 and 12 between RJ groups (P< 0.05). In our study, we did not observe any adverse effects of antiseptics on the thickness of scar tissue, the density of vascular proliferation and the degree of inflammatory cell infiltration in full thickness skin wounds. According to current study, RJ gauze-applied wounds was showed strong adherence, the dressing every day and required an extra force to separate it from them. This force could cause epithelial damage and thus may increase the thickness of scar tissue. In conclu-sion, RJ application in full-thickness skin defects in rabbits acceler-ated wound healing.

1. Hidaka S, Okamoto Y, Uchiyama S, Nakatsuma A, Hashimoto K, Ohni-shi ST, Yamaguchi M. Royal jelly prevents osteoporosis in rats: Beneficial effects in ovariectomy model and in bone tissue culture model, Evid. Based Complement. Alternat. Med., Sep 3(3), 339-48, 2006.

P 42

INVESTIGATION OF CYTOTOXIC EFFECTS OF ANHYDRIDE

CONTAINING WATER-SOLUBLE COPOLYMERS ON L929

MOUSE FIBROBLASTS

1Esin AKBAY, 1Handan SEVİM, 1Özer Aylin GÜRPINAR, 2Hatice KAPLAN-CAN, 1Mehmet Ali ONUR, 3Zakİr M. O. RZAEV, 2Ali GÜNER1Hacettepe University, Faculty of Science, Department of Biology, 06800 Beytepe-Ankara, Turkey2Hacettepe University, Faculty of Science, Department of Chemistry, 06800 Beytepe-Ankara, Turkey3Hacettepe University, Department of Chemical Engineering, Faculty of Engineering, 06800 Beytepe-Ankara, Turkey

Synthetic polymers, water-soluble or in the form of hydrogels, na-noparticles, dendrimers or microspheres, are materials with which we are in daily contact or which are under development as materi-als for medical applications. At the end of the last century, synthetic polymers successfully replaced a number of natural materials, either because the latter were in short supply or because the physicochemi-

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cal characteristics of synthetic polymers exceeded those of materials available from natural sources [1].

In this study, complex-radical copolymerization of maleic anhy-dride (MA), and acrylic acid (AA) and ternary polymerization of maleic anhydride (MA), vinyl acceptor−donor−acetate (VA) and acrylic acid (AA) and, considered as acceptor systems, were carried out in 1,4-dioxane with benzoyl peroxide (BPO) as an initiator at 70 oC under a nitrogen atmosphere. The co- and terpolymer synthe-sized by the use of 1: 1 and 1: 2: 1 molar ratio of initial monomers, re-spectively. Polymer samples were purified by several reprecipitating from anhydrous acetone, n-hexane, diethyl ether and were dried in vacuo at 60 oC to a constant weight with quantitative yields [2]. The cytotoxic effects of poly(MA-co-AA) and poly(MA-co-VA-co-AA) polymers samples were investigated in cell culture. The cytotoxicity was observed on L929 mouse fibroblasts. In the first step, fibroblasts were cultured in DMEM at initial density of 50.000 cells/ml. Follow-ing a 24 hour of incubation, the cell culture medium was removed and fresh medium containing poly(MA-co-AA) and poly(MA-co-VA-co-AA) was added. Poly(MA-co-AA) and poly(MA-co-VA-co-AA) were prepared in five different dilutions (Dilution 1: 0.00114 g/mL; Dilution 2: 0.00057 g/mL; Dilution 3: 0.00028 g/mL; Dilution 4: 0.00014 g/mL; Dilution 5: 0.00007 g/mL). Untreated cells served as controls. The cells were incubated during 5 days. Cell number and cell morphology were investigated at the 1st and 5th days. Propidium iodide/acridine orange (PI/AO) staining was used to assess apopto-sis of treated cells and of the control group.

The results showed that there were differences between poly(MA-co-AA) and poly(MA-co-VA-co-AA) in view of cell proliferation. In poly(MA-co-VA-co-AA) group cell number was higher than poly(MA-co-AA). A relatively few number of apoptotic cells were observed in the Poly(MA-co-VA-co-AA) on day 5. Therefore it can be said that toxicity of Poly(MA-co-AA) was related with the pro-liferation characteristics of cells. Cytotoxicity of results can be ex-plained polyanionic character of the co- and ternary polymers and also poly(MA-co-VA-co-AA) depicts the low cytotoxicity behavior. Vinyl acetate fragments in the terpolymer gives the immobility to the polymer chains and lower polyanionic character [2].

1. Ottenbrite RM, Kaplan AM. Some biologically active copolymers of maleic anhydride, macromolecules as drugs and as carriers for biologi-cally active materials, Annals of the New York Academy of Sciences, 446 (1), 160–168, 1985.

2. Kaplan CH, Doğan AL, Rzaev ZMO, Uner AH, Güner A. Synthesis, characterization and antitumor activity of poly(maleic anhydride-co-vi-nyl acetate-co-acrylic acid), Journal Applied Polymer Science, 100, 3425-3432, 2006.

P 43

TOPIC ADMINISTRATION OF YARROW EXTRACT ON

WOUND IN RABBITS

1Füsun TEMAMOĞULLARI, 2Ali HAYAT, 3Füsun BABA1University of Harran, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, Şanlıurfa, Turkey2University of Harran, Faculty of Veterinary Medicine, Department of Surgery, Şanlıurfa, Turkey3University of Harran, Faculty of Medicine, Department of Pathology, Şanlıurfa, Turkey

Many infectious diseases are known to be treated with herbal remedies throughout the history of mankind. Yarrow belongs to the Asteraceae family and contains aquileic acid, essential oils, tannins, flavonoids and acids. The application of infusions showed positive ef-fects on wound healing and hemorrhages [1]. The aim of the present study was to investigate efficacy of yarrow extract on healing wound

standing on the clinical comparison with 10 % povidone iodine and 0.9 % sodium chloride.

Six male and six female rabbits (2250 ± 100g) were taken for the study. All rabbits were anesthetized with i.m. administration of 10 mg/kg xylazine hydrochloride (Rompun, Bayer) and 50 mg/kg ketamine hydrochloride (Ketanes, Albe). Then full-thickness skin wounds were created (n: 3) on costal sides. Yarrow methanolic ex-tract was prepared by infusion of the aerial parts of the plant (10 days) in methanol at 1:5, w/v. The infusion filtered [2] with gauze was applied to the defect on the right cranial side (yarrow ektract group), and 10 % povidone iodine was applied to the defect on the left cranial side, as for control 0.9 % sodium chloride was applied to the defect on the left caudal side of the same animal. Wound surfaces were examined macroscopically and microscopically from the points of exudation, bleeding, thickness of scar, contraction and epitheliza-tion during the postoperative days (PODs).

Macroscopically, there was much less bleeding, and thicker scar and more contraction was observed in yarrow extract group compared to others. The epithelization in this group was completed on PODs 12. But the wounds treated with 0.9 % sodium chloride and 10 % povidon iodine was not completed on PODs 12. The density of vascular prolif-eration progression was significantly different on PODs 4 and 16 with-in yarrow extract group. Such a relation was not found on PODs 8-12 ((P> 0.05). The degree of inflammatory cell infiltration was signifi-cantly different on PODs 8-12 within the yarrow extract group. Such a relation was not found on PODs 4,12 and 16 (P> 0.05). 10% povidone-iodine is a microbicidal, antiseptic agent. However, it is inactivated by organic material and blood [3]. Candan et.al [4], observed that yarrow possess strong antioxidative activity but low antimicrobial activitiy in vitro. In this study, the degree of inflammatory cell infiltration was more decreased in yarrow extract than 10 % povidone iodine applied wounds on PODs 8-12. This might be a result of reduced bleeding due to yarrow extract Conclusively, we suggested that yarrow extract led limited bleeding, better contraction and decrease of inflammatory cell infiltration in wound treatment process.

1. Teixeira RO, Camparoto ML, Mantoovani MS, Vicentini, VEP. Asses-ment of two medicinal plants Psidium guajava L. and Achillea millefo-lium L. in vitro and in vivo assays, Genetics and Moleculer Biology, 26(4), 551-555, 2003.

2. Baytop, T. Türkiye’de Bitkiler ile Tedavi (Geçmişte ve Bugün), İstanbul Üniversitesi Eczacılık Fakültesi, İstanbul, s.166-167, 1999.

3. Frastvedt E., Stashak TD, Othic A. Update on Topical wound medica-tions, Clinical Techinique Practice, 3, 164-172, 2004.

4. Candan F, Ünlü M, Tepe B, Daferera D, Polissiu M, Sökmen A, Akpulat HA. Antioxidant and antimicrobial activity of the essential oil and meth-anol extracts of Achillea millefolium Subs. Millefolium Afan, Journal of Ethnopharmacology, 87, 215-220, 2003.

P 44

INVESTIGATION OF CELL PROLIFERATION OF

DEXAMETHASONE ON HUMAN PULP AND GINGIVAL

FIBROBLASTS

1Handan SEVİM, 1Esin AKBAY, 1Özer Aylin GÜRPINAR, 2Zafer C. ÇEHRELİ, 1Mehmet Ali ONUR, 1Aşkın TÜMER1Hacettepe University, Faculty of Science, Department of Biology, 06800 Beytepe-Ankara, Turkey2Hacettepe University, Faculty of Dentistry, Department of Pediatric Dentistry, 06100 Sıhhiye-Ankara, Turkey

Although previous studies have suggested that topical use of dex-amethasone in replanted animal teeth enhances healing and results in fewer resorption complications, the effect of dexamethasone on

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the types of human cells involved in the periodontal healing process remains unknown [1, 2].

This study investigated the effects of dexamethasone on cultured human pulp fibroblasts (HPF) and gingival fibroblasts (HGF). HPF and HGF were cultured in DMEM at initial density of 20.000 cells/ml and 30.000 cells/ml, respectively. Following 24h incubation, the cell culture medium was removed and fresh medium containing three different dilutions of dexamethasone (Dilution 1: 0.00001 mM ; Dilution 2: 0.0001mM; Dilution 3: 0.05 mM) were added separate-ly. Untreated cells served as controls. The cells were incubated for 5 days. Cell number and cell morphology were investigated at the 1st, 2nd, 3rd, 4th and 5th days. Propidium iodide/acridine orange (PI/AO) staining was used to assess apoptosis of treated cells and of the control group at 1st and 5th days. In Dilution 1, cell number was significantly higher than those of Dilutions 2 and 3. Compared to other dilutions, the number of apoptotic cells observed in the 3rd dilution was relatively higher than those of other dilutions.

The proliferation of HPF was significantly lower than HGF. The results of this study indicate that the effect of topical administered dexamethasone in an avulsion-type dental trauma varies for both cell-type and concentration.

1. Cabral MC, Costa MA, Fernandes MH. In vitro models of periodontal cells: a comparative study of long-term gingival, periodontal ligament and alveolar bone cell cultures in the presence of beta-glycerophosphate and dexamethasone, Journal of Materials Science, 2007 Feb 1, in press.

2. Soury B, Hentzen D, Vignal M, Christeff N, Doly J. Induction of inter-feron-beta gene expression by dexamethasone in murine L929 cells, Mo-lecular Endocrinology, 9, 199-207, 1995.

P 45

INVESTIGATION OF INTERACTIONS IN POLY(MA-ALT-AA)/

PVP BLENDS

1Hatice KAPLAN-CAN, 1Serap KAVLAK, 1Ali GÜNER, 2Zakir M. O. RZAEV1Hacettepe University, Faculty of Science, Department of Chemistry, Ankara, Turkey2Hacettepe University, Faculty of Engineering, Department of Chemical Engineering, Ankara, Turkey

In the last few decades the development of various hydrophilic materials based on blends and interpolymer complexes of poly(carboxylic acid)s and non-ionic water-soluble polymers is of great importance because of their unique properties and possible applications in medicine [1]. Hydrophilic synthetic polymers have been widely investigated as carrier substances. PVP is an amorphous and biocompatible polymer with a high affinity for water and its in-teraction with water has became the topic of a large body of research [2]. It has been known that some maleic anhydride based polymers with high carboxylic acid content exhibit high biological activities such as inhibitory effect on viruses, bacteria and tumors [3].

In this present study, poly(maleic anhydride-alt-acrylic acid) co-polymer, poly(MA-alt-AA), and poly(N-vinyl-2-pyrrolidone), PVP, are used in the preparation of blends. Poly(MA-alt-AA)/PVP blends, covering a full range of compositions, were prepared by dissolution of both of the polymers in common solvent followed by the solvent removal by drying at ambient temperature. Characterization of blends was carried out by FTIR and Raman spectroscopy.

The FTIR and Raman measurements prooved the establishment of the interactions between copolymer and PVP. The significant chemical shifts in the characteristic frequencies in FTIR and Raman spectra support the strong hydrogen bond formation. Thus compat-ible blends were obtained due to this strong hydrogen bond forma-tion between copolymer and PVP.

1. Khutoryanskiy VV, Cascone MG, Lazzeri L, Nurkeeva ZS, Grigory MA, Mangazbaeva RA. Phase behaviour of methylcellulose-poly(acrylic acid) blends and preparation of related films, Polym. Int., 52, 62-67, 2003.

2. Feldstein MM, Kuptsov SA, Shandryuk GA, Plate NA, Chalykh AE. Coherence of thermal transitions in poly(N-vinyl pyrrolidone)-poly(ethylene glycol) compatible blends 3. Impact of sorbed water upon phase behavior, Polymer, 41, 5349-5359, 2000.

3. Abd El-Rehim HA, El-Hag Ali A, Mostafa TB, Farrag HA. Anti-micro-bial activity of anhydride copolymers and their derivatives prepared by ionizing radiation, Eur. Polym. J., 40, 2203-2210, 2004.

P 46

KINETIC EVALUATION OF THE SUBSTITUTIONS ON

BISBENZIMIDAZOL DERIVATIVES ON THEIR INHIBITORY

ACTIVITIES OF MAMMALIAN DNA TOPOISOMERASE I

1Sevil ZENCİR, 2A. Selcen ALPAN, 2Pınar ALCIL, 2Güneş ÇOBAN, 2H. Semih GÜNEŞ, 1Zeki TOPÇU1Ege University, Faculty of Pharmacy, Department of Pharmaceutical Biotechnology, 35100 İzmir, Turkey2Ege University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 35100 İzmir, Turkey

1H-Benzimidazole derivatives are known to have antibacterial, an-tifungal, antimicrobial, antiprotozoal and antihelmintic activities [1]. We have previously identified a considertable inhibition exerted by a number of 1H-benzimidazole derivatives on the mammalian type I DNA topoisomerases [2]. Topoisomerases are ubiquitous enzymes that regulate the conformational changes in DNA topology by cata-lyzing the concerted breakage and rejoining of DNA strands during many genetic processes including DNA replication, transcription, re-combination and transposition [3]. Because of the increased aware-ness on the targetting of these enzymes as an effective approach for the development of chemotherapeutics, we extended our analysis on the 1H-benzimidazole-generated topoisomerase inhibition. In this study, we synthesized 1,2-bis(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)ethane and showed a significant interference of this compound on the mammalian type I topoisomerase using in vitro plasmid super-coil relaxation assays [4-6]. A known topoisomerase I poision, Camp-tothecin, was used as the reference compound in the evaluation of the inhibition. Our report also includes the effects of the substitutions of the hydrogen atom at the 5- and/or 6- position on 1H-benzimidazol ring with chloro, nitro and methyl hydrogen acceptor or donor atoms/groups on the inhibitory activity of the compound.

1. Güneş HS, Coşar G. Arzneimittel-Forschung/ Drug Res., 42, 1045-1048, 1992. 2. Alpan AS, Güneş HS, Topçu Z. 1H-Benzimidazole Derivatives As Mam-

malian DNA Topoisomerase I Inhibitors, Amnusc., submitted 2007. 3. Wang JC. Ann Rev Biochem, 65, 635-692, 1996. 4. Shriner RL. Upson RW. J Am Chem Soc, 63, 2277-2278, 1941. 5. Topçu Z, Castora FJ. Biochim. Biophys. Acta, 1264, 377-387, 1995. 6. Topçu Z. J. Clin. Pharm. Ther., 26, 405-416, 2001.

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P 47

LC-DAD METHOD FOR THE DETERMINATION OF

pKA VALUES OF SULFONAMIDES AND OPTIMIZING

CHROMATOGRAPHIC SEPARATION BY VARYING SOLVENT

COMPOSITION

1Nurullah ŞANLI, 1Güleren ALSANCAK, 2Zerrin ERDEMGİL, 3Jose Luis BELTRAN, 3Jose BARBOSA1Süleyman Demirel University, Science & Literature Faculty, Departament of Chemistry, 32260 Isparta, Turkey2Anadolu Unıversity, BİBAM, 32260 Eskişehir, Turkey3Barcelona University, Department of Analytical Chemistry, 08028 Barcelona, Spain

Systematic optimization of liquid chromatographic (LC) meth-ods requires an accurate knowledgement of the parameters that influence the separation of the compounds. The linear correlation between the logarithm of retention factor and Reichardt’s polarity parameter have been used to predict the chromatographic behavior of the compounds. Nowadays, acetonitrile-water mixtures are widely used in high performance liquid chromatography (HPLC) and LC retention parameters of the compounds strongly depend on the dis-sociation behavior of the compounds and the pH of the acetonitrile-water mobile phases [1].

The aim of this work is to analyze sulfodiazine, sulfothiazole, sul-fomerazine, sulfomethazine, sulfodoxine, sulfomonometoxine, sulfo-methoxazole like sulfonamides efficiently using HPLC method. The separation conditions have been optimized using the relationships between Reichardt’s polarity parameter and the capacity factors of the sulfonamides. The experimental region was selected in a such way that the capacity factors of the sulfonamides would stay within the limits 1< k < 10 [2]. These limits have been provided when the organic modi-fier content of the mobile phase was in the range of 25 -16% (v/v).

The sulfonamides investigated are ampholytes with weakly basic and acidic characteristic. Because of this molecular structure the re-tention of sulfonamides is expected to pass through a maximum at an intermediate mobile phase pH. The pH dependent retention pro-files have been investigated and the results obtained demonstrated that the changes in retention are consistent with this expectation.

Before the optimization of chromatographic separation dissociation constants of these compounds have been determined by LC-DAD methodology [3]. The results obtained show that pKa1 and pKa2 val-ues are between approximately 2 and 7. The pH of the mobile phase was kept constant at pH 4.50 where the neutral form is predominant. Thus they will present a maximum hydrophobicity around this pH [4]. The greatest retention was obtained for sulfonamides bearing additional methyl or methoxy groups on the R side chain. This sug-gests that the R side chain plays an important role in the hydropho-bic interaction of the compounds with the reversed phase column.

1. Botsoglou NA, Fletouris DJ, Psomas IE. Retention behavior of multiple sulfonamides in various liquid chromatographic systems, Chromato-graphia, 46, 477-481, 1997.

2. Augiar de PF, Bourguignon B, Massart DL. Comparison of models and designs for optimization of the pH and solvent strength in HPLC, Anal. Chim. Acta, 356, 7-18, 1997.

3. Jiménez-Lozano E, Marqués I, Barrón D, Beltrán JL, Barbosa J. Determi-nation of pKa values of quinolones from mobility and spectroscopic data obtained by capillary electrophoresis and diode array dedector, Anal. Chim. Acta, 464, 37-45, 2002.

4. Carda-Boch S, Berthod A. Countercurrent chromatography for the measurement of hydrophobicity of sulfonamide amphoteric compounds, Chromatographia, 59, 79-87, 2004.

P 48

SYNTHESIS AND CHARACTERZATION OF NOVEL

DENDRITIC POLYMERS FOR ANTI-CANCER DRUG

ENCAPSULATION AND RELEASE

1Esra GÜÇ, 2Güngör GÜNDÜZ, 1Ufuk GÜNDÜZ1Middle East Technical University, Department of Biological Sciences, Ankara, Turkey2Middle East Technical University, Department of Chemical Engineering, Ankara, Turkey

Controlled drug delivery by biocompatible artificial systems has become one of the most interested research areas because of the ability to reduce the problems of conventional chemotherapy. By dendritic polymer architectures, controlled drug delivery systems gain a new strategy. Particularly dendritic polyesters are highly investigated due to their unique properties including high degree of branching, nontoxicity and water solubility [1]. Fatty acids are good candidates for polymeric systems with their encapsulation stability for hydrophobic drugs and with their biosafety properties [2]. The aim of this study is to design hyperbranched polyesters with fatty acids, to encapsulate anti-cancer drugs and to study their release for chemotherapeutic purposes.

Dendritic polyesters were based on dipentaerythritol (used as core molecule) and dimethylolpropionic acid (used as repeated end groups) and they were used in stoichiometrical ratios [3]. Ricino-leic acid, a C18 fatty acid was hydrolyzed from castor oil and was conjugated to the end groups of polyesters for effective encapsu-lation of hydrophobic drug. Dendritic polyesters were character-ized by fourier transform infrared spectroscopy (FTIR) and size exclusion chromatography (SEC) analysis. For encapsulation of hydrophobic anti-cancer drug, idarubicin was used. Encapsulation of the drug was followed with spectrophotometric measurements in the wavelenght regions at 540 nm and 578 nm. Interactions of idarubicin and dendritic polyester were shown by FTIR analysis. For drug release profiles samples are placed into dialysis bags and drug delivery were applied against phosphate buffer saline (PBS). Releasing medium were analyzed spectrophotometrically to meas-ure the released drug. For the next study, toxicity effect of unloaded and drug loaded dendritic polyesters on cell culture (MCF-7 breast cancer cell lines) will be investigated by XTT tests and IC 50 values will be determined.

The characterization by FTIR of the obtained hyperbranched polymer have indicated the expected hydroxyl end groups. SEC studies have shown the molecular weight distribution of the dendritic polymer. According to the molecular characterization results, various polymer to drug ratios were tested for encapsula-tion studies.The encapsulation efficieny variations with respect to hyperbranched polymer (HBR) to drug ratio is shown in the Figure 1. The results showed that encapsulation efficiency of the drug was increased by increasing the polymer amount. Con-versely efficiency of encapsulation was decreased with increasing drug ratio. Sustained release profiles of dendritic nanoparticles were obtained and analyzed successfully. The next step in this study will be the determination of cytotoxicity of empty and drug loaded dendritic nanoparticles on MCF-7 cell line. These studies will bring new insights to successful cancer therapy by controlled delivery of anti-cancer drugs.

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Figure 1. Encapsulation efficiency of idarubicin at 25mg, 50mg, 75mg of hyperbranched polymer (HBR). Encapsulation efficiency = Encapsulated drug weight(mg)/Initial drug weight(mg)*100a. aMean ± SEM (n=2)

1. Dhanikula RS, Hildgen P. Synthesis and evaluation of novel dendrimers with a hydrophilic interior as nanocarriers for drug delivery, Bioconju-gate Chem., 17 (1), 29, 2006.

2. Slivniak R, Ezra A, Domb AJ. Hydrolytic degredation and drug release of ricinoleic acid-lactic acid copolyesters, Pharmaceutical Research, 23, 6, 2006.

3. Bat E, Gündüz G, Kısakürek D, Akhmedov İ. M. Synthesis and charac-terization of hyperbranched and air drying fatty acid based resins, Prog. Org. Coat., 55, 330-336, 2006.

P 49

DETERMINATION OF CATECHOL USING

MODIFIED ELECTRODE WITH A COMPOSITE OF

POLY(VINYLFERROCENE) AND POLYANILINE

Muammer KAVANOZ, Nuran ÖZÇİÇEK-PEKMEZ, Kadir PEKMEZ, Attila YILDIZHacettepe University, Faculty of Science, Department of Chemistry, Beytepe-Ankara, Turkey

The determination of phenolic compounds has a great interest in many fields, such as neurochemistry, pharmaceutical and clini-cal chemistry. Some phenols as catechol, resorcinol and chlorogenic acid are found in plants, fruits and herbs. Among them, the catechol has acquired an increasing interest, not only to be a model molecule for bi-phenolic compounds such as dopamine, adrenaline, isopre-nalin, dobutamin and phenylethylamine but also have important pharmacological activities. Therefore, it is very important to develop a sensitive analytical method for the determination of catechol in biological studies. The interest in the determination of this phenolic compound has proportionated the development of several methods for their quantification, such as the chromatographic methods with different detection systems. These methods are very important, but time and reagent consuming are, generally, high. Thus, the develop-ment of new methods, that makes possible the minimal use of rea-gent and a lower time of analysis are very important. In this sense, electrochemical methods involving the development of chemical sensors have been utilized [1, 2].

Polyaniline (PANI) is one of the most promising conducting poly-mers due to its high conductivity, good redox reversibility and good stability in aqueous solutions and air. These properties provide favo-rable conditions for its potential applications in super capacitor and electrocatalysis. Poly(vinylferrocene) (PVF) is also an electroactive polymer. In this work, PVF in deposited composite film was used as an electron transfer mediator in the electrochemical oxidation of catechol due to its reversible redox.

In this study, electropolymerization of aniline in the presence of PVF was carried out by cyclic voltammetry in non-aqueous methyl-ene chloride medium. Thin and more adhesive films were obtained

when PANI and PVF+ClO4- were codeposited. The peaks belonging to both PVF and PANI were observed clearly from their cyclic vol-tammograms. It was also proven that film contains both PANI and PVF using FT-IR spectra. This PANI/PVF+ codeposited film on Pt electrode were used for determination of catechol without using en-zyme. The experimental results indicate that the anodic peak potential of catechol at the PANI/PVF+ modified electrode is lower than that at the Pt electrode in a solution consisting of catechol. The OH group on the PANI chain in the composite film plays an important role in the electron transfer between PANI and catechol in the solution. Opti-mum conditions for the determination of the relationship between the response current and the concentration of catechol are that the poten-tial was set at 0.55 V and the pH of the solution controlled at 4.0. Fig. 1 shows the change in the response current with the concentration of catechol from 3,91 to 500 μM and from 3.91 to 64 000 μM, respective-ly. This modified electrode has a lower working potential and a good operational stability due to reducing the electrode fouling, compared with the direct oxidation of catechol at the bare Pt electrode.

Figure 1. a, b) Current time for different catechol concentrations. The relationship between the response current and the concentration of catechol, c) from 3.91 μM to 500 μM, d) from 3.91 μM to 64000 μM.

1. Rita de Cassia Silva Luz, Flavio Santos Damos, Adriano Bof de Oliveira, Jo-hannes Beck, Lauro Tatsuo Kubota. Development of a voltammetric sensor for catechol in nanomolar levels using a modified electrode with Cu(phen)2 (TCNQ)2 and PLL, Sensors and Actuators, B 117, 274-281, 2006.

2. Shaolin M. Catechol sensor poly (aniline-co-o- aminophenol) as an electron transfer mediator, Biosensors and Bioelectronics, 21, 1237-1243, 2006.

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P 50

THE UV-SPECTROSCOPIC METHOD FOR DETERMINATION

OF DISSOCIATION CONSTANTS OF SEVERAL

SULFONAMIDES IN WATER AND ACETONITRILE-WATER

BINARY MIXTURES

1Senem ŞANLI, 1Güleren ALSANCAK, 2Jose Luis BELTRAN, 2Jose BARBOSA1Süleyman Demirel University, Science and Literature Faculty, Department of Chemistry, 32260 Isparta, Turkey2Barcelona University, Department of Analytical Chemistry, 08028 Barcelona, Spaın.

Sulfonamides are anti-bacterial and anti-infective drugs com-monly used to treat in medicine and veterinary practice. The physi-cochemical profiling of drugs includes the determination of their dissociation constants. Data on proton dissociation of pharmacolog-ically active substances are of utmost interest for the understanding of stability and solubility of drugs. A major factor of drug permea-tion is their aqueous pKa.

Very often, the main difficulty in the determination of dissociation constants of drugs is their aqueous insolubility that forces the use of spectroscopic method. UV spectroscopy is an excellent method for pKa determination. This method requires very low analyte con-centration and allows suitable absorbance measurement in aqueous solution even for products with low aqueous solubility. Acetonitrile (ACN)-water mixtures are usually employed for pKa determina-tion of water insoluble drugs. Literature shows several extrapolation equations to estimate aqueous pKa from the pKa values determined in acetonitrile-water mixtures [1].

In this study, approximately 1.0 x 10-5 M solution of sulfonamides (sulfadiazine, sulfamethazine, sulfatiazole, sulfamethoxazole, sul-famonomethoxine, sulfamerazine, sulfadimethoxine) in water and ACN – water mixtures were titrated with NaOH in the range of pH 2-11 for determination of dissociation constants [2].

The data evaluation was performed by using STAR program [3]. The spectrum recorded for sulfadiazine at various pH values in ACN-water, 30 % (v/v) is shown in Fig 1. The aqueous pKa obtained from UV data and extrapolations of the data in ACN-water medium is consistent between them and agree with those from literature [4].

Fig 1. UV-spectrum of sulfadiazine obtained at various pH in ACN-water mixture (30 % v/v).

1. Ruiz R, Roses M, Rafols C, Bosch. Critical validation of a new simpler approach to estimate aqueous pKa of drugs sparingly soluble in water, Anal. Chim. Acta, 550, 210-221, 2005.

2. Polster J, Lachmann H. Spectrometric Titrations, VCH, 1989. 3. Jiménez-Lozano E, Marqués I, Barrón D, Beltrán JL, Barbosa J. Determi-

nation of pKa values of quinolones from mobility and spectroscopic data obtained by capillary electrophoresis and a diode array detector, Anal. Chim. Acta, 464, 37-45, 2002.

4. Lin CE, Lin WC, Chen YC, Wang SW. Migration behavior of sulfona-mides in capillary electrophoresis, J. Chromatogr. A, 792, 37-47, 1997.

P 51

DETERMINATION OF PHYSICOCHEMICAL PARAMETERS

AND SAR STUDY ON THE SOME BENZIMIDAZO[1,2-

a]PYRIMIDINE DERIVATIVES

Asiye MERİÇAnadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 26470 Eskişehir, Turkey

Condensed compounds bearing bridgehead nitrogen atom have various activity, ranging from antihelmintic and anticonvulsant to antitumor and antiviral. Last study in our research group was re-vealed the cytotoxicities of some benzimidazo[1,2-a]pyrimidine compounds on non-cancer and cancer cell lines [1]. It is necessary to evaluate their SAR in order to understand the activity mechanism of this type fused azole compounds, properly.

Initially, physicochemical parameters of synthetic compounds were calculated theoretically. Steric {molecular weight (MW), mo-lecular refraction (MR) [2], molecular volume (MV) [3], molecular connectivity index (MCI) [4], paracor (Par) [5-7]}, hydrophobic {partition coefficient [8], hydrophobic substituent constant (π) [9]} and electronic{electronic substituent constant [10]} parameters of 2,4-di- and 2,3,4-trisubstituted benzimidazo[1,2-a]pyrimidine de-rivatives were determined firstly. Then, correlations were constituted between cytotoxicities (IC50) and structural properties of compounds. Some computerized programs were also used for further evaluation.

Table . Training Set of Compounds

Compound R1 R2 R3 Compound R1 R2 R3

1 OH H Me 6 Ph H Me

2 OH H Ph 7 Ph H Ph

3 OH H n-Pr 8 Me H Me

4 OH Ph Me 9 Me Me Me

5 OH Me Me 10 Me Et Me

The SAR results of compounds can be summarized as follows:At positions R1, R2 and R3; hydrophobic, electronic and steric

properties were found important. The existence and abundance of methyl substituent on the structure increase the cytotoxic activity. It can be concluded that the substances abundantly bearing methyl subtituent may evaluate as promising compounds for potential anti-neoplastic activity. 1. Meriç A, İncesu Z, Karayel A, Özbey S. Synthesis of some 2,4-di- and

2,3,4-trisubstituted benzimidazo[1,2-a]pyrimidines and evaluation of their cytotoxicities toward F2408 and 5RP7 cells, Revista de Chimie, 57(11), 1090-1097, 2006.

2. Dunn III WJ. Molar refractivity as an independent variable in quantita-tive structure-activity studies, Eur. J. Med. Chem.-Chim. Ther., 12, 109-112, 1977.

3. Berkem AR, Baykut S., Fizikokimya Kitabı, İstanbul Üniversitesi Yayınları, Sayı: 2735, Kimya Fakültesi, No. 42, 1980.

4. Kier LB, Hall LH. Derivation and significance of valence molecular con-nectivity, J. Pharm. Sci. 70, 583-9, 1981.

5. Quayle QR. The parachors of organic compounds, Chem. Rev., 53, 439-89, 1953.

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6. Sugden S. A relation between surface tension, density, and chemical composition, J. Chem. Soc., 125, 1177, 1924.

7. Vogel AI. Physical properties and chemical constitution. Part IX. Aliphat-ic hydrocarbons, J. Chem. Soc., 133, 1946.

8. Rekker RF, De Kort HM. The hydrophobic fragmental constant, an ex-tension to a 1000 data point set, Eur. J. Med.-Chim. Ther., 14(6), 479-488, 1979.

9. Hansch C, Leo A, Unger SH, Kim KH, Nikaitanı D, Lien EJ. Aromatic substituent constant for structure activity correlations, J. Med. Chem., 16, 1207-26, 1973.

10. Akı-Şener E, Yalçın İ. Kantitatif Yapı-Etki İlişkileri Analizleri (QSAR), Ankara Üniversitesi Eczacılık Fakültesi Yayınları No: 86, 2003. (ISBN 975-482-585-8)

P 52

ENHANCED ENZYME ACTIVITY OF IMMOBILIZED LIPASE

AS BIOCATALYST FOR SYNTHESIS OF ESTERS IN ORGANIC

MEDIA

Taylan K. ÖZTÜRK, Funda KARTAL, Ali KILINÇEge University, Faculty of Science, Department of Biochemistry, İzmir, Turkey

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) catalyze the hydrolysis and the synthesis of esters of glycerol and long-chain fatty acids. The many applications of lipases include special organic syntheses, hydrolysis of fats and oils, modification of fats, flavor en-hancement in food processing, resolution of racemic mixtures and chemical analyses [1].

Research on lipase catalyzed production of various kinds of ester has increased tremendously in the recent past. Esters are present in fats and oils and in natural and synthetic polymers. They are useful intermediates or end products in the chemical industry. Enzymatic production of esters can be achieved either by reaction between free acid and hydroxyl groups of alcohol or by ester exchange or transes-terification (include alcoholysis, acidolysis and interesterification).

Lipase catalized esterification reactions have been actively pur-sued to produce various kinds of commercially important esters. Esters of short chain fatty acids are extremely important aromatic compounds. Esters of short chain alcohols and long chain fatty acids are valuable oleochemicals that may be used as lubricants, diesel fuel and antistatic reagents. Esters of long chain fatty acids and polyhy-dric alcohols like glycerol, sorbitol and other carbohydrates (called-emulsifiers/surfactants) find immense application in food and phar-maceutical industries [2].

In this work the immobilized form of lipase was prepared by covalent attachment of enzyme on crosslinked polyvinyl alcohol [3]. Optimization of reaction conditions for ester synthesis was made by experimenting with different chain length acids and alcohols and ef-fects of organic solvent type on esterification activity were studied. The effects of reaction time and reaction temperature were also stud-ied. Esterification activity of immobilized lipase in organic solvents was measured by GC-FID.

1. Hari Krishna S, Karanth N.G. Production, purification, characterization and applications of lipases. Catalysis Reviews, 44, 499-591, 2002.

2. Rohit S, Yusuf C, Uttam B. Lipases and lipase-catalyzed esterification reactions in nonaqueous media, Biotechnology Advances, 19,627-662, 2001.

3. Kılınç A, Önal S, Telefoncu A. Chemical attachment of porcine pancre-atic lipase to crosslinked poly(vinyl alcohol) by means of adipoyldichlo-ride, Process Biochemistry, 38, 641-647, 2002.

P 53

EFFECT OF USNIC ACID ON TISSUE NITRIC OXIDE

SYNTHASE ACTIVITY AND GLUTATHIONE LEVEL IN

TITANIUM-IMPLANTED SUBJECTS

1Fehmi ODABAŞOĞLU, 2Hayati AYGÜN, 2Ömer Selim YILDIRIM, 3Zekai HALICI, 1Mesut HALICI, 4Zafer OKUMUŞ, 5Ali ASLAN, 6Ahmet ÇAKIR, 7Cavit KAZAZ1Atatürk University, Faculty of Pharmacy, Department of Biochemistry, 25240 Erzurum, Turkey2Atatürk University, Faculty of Medicine, Department of Orthopedics and Traumatology, 25240 Erzurum, Turkey3Atatürk University, Faculty of Medicine, Department of Pharmacology, 25240 Erzurum, Turkey4Atatürk University, Faculty of Veterinary Medicine, Department of Surgery, 25240 Erzurum, Turkey5Atatürk University, Kazım Karabekir Education Faculty, Department of Biology, 25240 Erzurum, Turkey6Atatürk University, Kazım Karabekir Education Faculty, Department of Chemistry, 25240 Erzurum, Turkey7Atatürk University, Faculty of Science, Department of Chemistry, 25240 Erzurum, Turkey

Debris due to the frictions as well as biochemical and magnetic re-actions following orthopedic implantations may play a role in aseptic loosening through initiating a series of complex cellular reactions between bone and implant. Loosening may be related to cytotoxic-ity [1, 2]. Usnic acid (UA) (Fig. 1) is a dibenzofuran derivative bio-synthesised by lichens. Previously, it has been shown that UA has various biological activities [3]. The present study was conducted to evaluate the effect of UA on nitric oxide synthase (NOS) activity and glutathione level (GSH) in Ti-implanted tissues of rabbits.

Fig. 1. Molecular structure of usnic acid (UA)

UA was isolated from a lichen species, Usnea longissima [3]. Eight-een New Zealand rabbits divided into 6 groups, of which femurs of rabbits in 5 groups were subperiostally implanted with Ti. Then, they received UA (30 mg/kg) and olive oil (OO) orally or locally every 3 d for 21 d or received none. Rabbits from the other group served as control. Following euthanasia, tissues around the implant were scrapped and then ground within liquid nitrogen for NOS activity and GSH level.

In the present study, 2.17, 1.6, and 1.7-fold increases were deter-mined in the activities of iNOS (inducible), cNOS (constitutive), and tNOS (total) respectively in Ti-implanted rabbits compared to con-trol rabbits (Table 1). However, both local and oral administration of OO and oral administration of UA decreased NOS activities. Olive oil was more effective than UA when administered locally, whereas UA was more effective than OO when administered orally. Surgi-cal intervention was associated with a 31% reduction in GSH level. Local and oral administration of UA and only local administration of OO alleviated this reduction. In conclusion, UA and OO admin-istration may affect cytotoxicity via suppressing iNOS activity and increasing GSH level.

Keywords: Titanium implant, usnic acid, nitric oxide synthase, glutath-ione

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Table 1. Effects of local and oral-administrated usnic acid (UA) and olive oil (OO) on the activities of nitric oxide synthase enzymes (tNOS, cNOS and iNOS) and amount of glutathione (GSH) in titanium-implanted subperiostal tissues of rabbits. Titanium group (TIT) was compared with healthy group. The 30 mg/kg dose of UA and OO treated groups were compared with TIT group.

Trea

tmen

ts

N

NITRIC OXIDE SYNTHASE (NOS) ACTIVITY

(μmol/min/mg tissue)a

Amount of GSH

(nmol/mg tissue)a

tNOS cNOS iNOS

TIT+UA (local) 3 6.84±0.27** 3.07±0.17** 3.77±0.19** 3.44±0.02*

TIT+UA (oral) 3 2.58±0.19** 2.47±0.18** 0.12±0.07** 3.96±0.04**

TIT+OO (local) 3 3.65±0.27** 3.52±0.25* 0.13±0.02** 3.52±0.12*

TIT+OO (oral) 3 3.40±0.37** 2.85±0.40** 0.55±0.05** 2.51±0.13*

TIT (control) 3 5.43±0.21** 4.39±0.22** 1.04±0.03** 2.91±0.08**

Healthy tissue 3 3.19±0.08 2.72±0.06 0.48±0.02 4.21±0.01

a Means±SEM of tissues of six legs in each group. N: The number of rabbits. *Significant at p<0.05; **Significant at p<0.01.

1. Stea S, Visentin M, Granchi D, Cenni E, Ciapetti G, Sudanese A, Toni A. Apoptosis in peri-implant, Biomaterials, 21, 1393-1398, 2000.

2. Raha S, Robinson BH. Mitochondria, Oxygen free radicals, and apopto-sis, Am. J. Med. Gen., 106, 62-70, 2001.

3. Odabaşoğlu F, Çakır A, Süleyman H, Aslan A, Bayır Y, Halıcı M, Kazaz, C. Gastroprotective and antioxidant effects of usnic acid on indometha-cine-induced gastric ulcer in rats, J. Ethnopharmacology, 103 (1), 59-65, 2006.

P 54

SYNTHESIS AND CHARACTERIZATION OF SOME NOVEL

ISATIN DERIVATIVES IN WHICH ANTICONVULSANT

ACTIVITY IS PREDICTED

Ebubekir SEPTİOĞLU, Mutlu DİLSİZ-AYTEMİR, Ünsal ÇALIŞHacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100-Sıhhiye, Ankara, Turkey

The restrictive treatment of epileptic seizures of the patients lets the researches to find the new agents with more efficient activity and less toxicity [1]. Recently, the anticonvulsant activities of isatin derivatives were reported by various studies [2-4]. In this study, we have synthesized some new isatin derivatives as shown below and will evaluated the anticonvulsant activities of all compounds in fu-ture work.

The synthesized compounds were prepared by Tacconi and col-leagues’ method [5]. Treatment of isatin with sodium hydride in N,N-dimethyl formamide (DMF) at room temperature gave isatin sodium salt. This isatin salt was alkylated with 1,2-dibromoethane in DMF. Reaction of the alkylated isatin as named 1-(2-bromoe-thyl)-1H-indole-2,3-dione with benzoxazol-2-one derivatives gave compounds 1 and 2. Final compounds 3 and 4 which have phenyl-hydrazone group were prepared by heating compounds 1 or 2 with phenylhydrazine in ethanol.

The basic structures of these compounds were confirmed by IR, 1H-NMR, mass spectral and elemental analyses data. The reaction products were assigned structure that were in accordance with their spectroscopic and chemical behaviors. Thus, compounds 1 and 2 showed two strong band at 1771-1766 and 1745-1746 cm–1 in their IR spectra assignable to C=O groups. Also, compounds 3 and 4 have a one stretching band at 1783-1779 cm–1. The ethylene group protons in the 1H-NMR spectra of all compounds appeared as a triplet at 3.31-4.07 ppm for -N-CH2-, a triplet signal at 4.23-4.14 ppm for -N-CH2-CH2-. Characteristic singlet peak of –NH for compounds 3 and 4 were observed at 12.4 ppm.

1. Malawska B. New Anticonvulsant Agents, Curr Top Med Chem 5(1), 69-85, 2005.

2. Popp FD. Potential anticonvulsants. IX. Some isatin hydrazones and re-lated compounds, J. Heteroc. Chem. 21, 1641-1645, 1984.

3. Pandeya SN, Sriram D, Yogeeswari P, Stables JP. Anticonvulsant and neurotoxicity evaluation of 5-(un)-substituted isatin-imino derivatives, Pharmazie, 56, 875-876, 2001.

4. Pandeya SN, Smitha S, Stables JP. Anticonvulsant and sedative-hypnotic activities of N-substituted isatin semicarbazones, Arch Pharm (Weinhe-im), 335(4), 129-134, 2002.

5. Tacconi G, Righetti PP, Desimoni G. Einfache Darstellung von N-substi-tuierten Isatinen, J. Prakt. Chem., 315(2), 339-344, 1973.

P 55

PHENOLIC COMPOUNDS OF SIDERITIS OZTURKII

AND THEIR IN VIVO ANTI-INFLAMMATORY AND

ANTINOCICEPTIVE ACTIVITIES

1Pınar ŞAHİN, 2Esra KÜPELİ, 1İhsan ÇALIŞ, 1Nurten EZER, 3Erdem YEŞİLADA1Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Botany, Ankara, Turkey2Gazi University, Faculty of Pharmacy, Department of Pharmacognosy, Ankara, Turkey3Yeditepe University, Faculty of Pharmacy, Department of Pharmacognosy, İstanbul, Turkey

In Turkey, the genus Sideritis L. (Lamiaceae) is represented by 46 species [1] and some of which are used in traditional medicine for their beneficial and curative effects [2]. Sideritis species growing in Turkey are known to be rich in essential oils, diterpenes, flavonoids and phenylethanoid glycosides [3-5]. In a continuation of our phyto-chemical studies on Turkish Sideritis species, we now report the iso-lation of phenolic compounds from S. ozturkii Aytaç & Aksoy, which is endemic to Turkey, through in vivo bioassay-guided fractionation procedures.

Acetone extract from aerial parts of S. ozturkii and its fractions were investigated for its in vivo anti-inflammatory and antino-ciceptive activities. For the anti-inflammatory activity assessment, carrageenan-induced hind paw edema and for the antinociceptive activity, p-benzoquinone-induced abdominal constriction tests were used [6].

Acetone extract of the plant and its phenolic fraction were found to possess significant inhibitory activity on these models in mice. Ozturkosides A-C were isolated from the active phenolic fraction.

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The structures of isolated compounds were elucidated by spectro-scopic techniques (UV, IR, 1D- and 2D-NMR, MS). Ozturkoside C showed notable antinociceptive and anti-inflammatory activities without inducing any apparent acute toxicity or gastric damage. Al-though the activity of ozturkosides A and B were found insignifi-cant in statistical analysis, some inhibitory effects were observed. Accordingly, it is suggested that these components in phenolic fraction might possibly share the antinociceptive and anti-inflam-matory activities together.

1. Aytaç Z, Aksoy A. A new Sideritis species (Labiatae) from Turkey, Flora Mediterranea, 10, 181-184, 2000.

2. Baytop T. Therapy with Medicinal Plants in Turkey (Past and Present), Nobel Tıp Publications, İstanbul, p.375, 1999.

3. Ezer N, Vila R, Cañigueral S, Adzet T. Essential oil composition of four Turkish species of Sideritis, Phytochem., 41, 203-205, 1996.

4. Akcoş Y, Ezer N, Özçelik B, Abbasoğlu U. Iridoid glycosides from Sideri-tis lycia Boiss & Heldr. and its antimicrobial activities, FABAD J. Pharm. Sci., 23, 99-103, 1998.

5. Şahin FP, Taşdemir D, Rüedi P, Ezer N, Çalış İ. Three new acylated flavon glycosides from Sideritis ozturkii Aytaç & Aksoy, Phytochem., 65, 2095-2099, 2004; 66, 125, 2005.

6. Küpeli E, Harput UŞ, Varel M, Yeşilada E, Saracoğlu İ. Bioassay-guided isolation of iridoid glucosides with antinociceptive and anti-inflamma-tory activities from Veronica anagallis-aquatica L., J. Ethnopharmacol., 102, 170–176, 2005.

P 56

ANTI-INFLAMMATORY EFFECTS OF THE ALPHA-LIPOIC

ACID ON CARRAGEENAN-INDUCED ACUTE AND COTTON

PELLET-INDUCED CHRONIC INFLAMMATION MODELS IN

RATS AND ITS RELATION WITH NITRIC OXIDE SYNTHASE

ACTIVITY

1Fehmi ODABAŞOĞLU, 2Zekai HALICI, 3Hayati AYGÜN, 1Mesut HALICI, 1Yasin BAYIR, 4Ahmet ÇAKIR, 5Elif ÇADIRCI, 1Fadime ATALAY1Atatürk University, Faculty of Pharmacy, Department of Biochemistry, Erzurum, Turkey2Atatürk University, Faculty of Medicine, Department of Pharmacology, Erzurum, Turkey3Atatürk University, Faculty of Medicine, Department of Orthopedics and Traumatology, Erzurum, Turkey4Atatürk University, Kazım Karabekir Education Faculty, Department of Biology, Erzurum, Turkey5Atatürk University, Faculty of Pharmacy, Department of Pharmacology, Erzurum, Turkey

Alpha-lipoic acid (ALA) is a dithiol that is found naturally in mitochondria as the coenzyme for pyruvate dehydrogenase and al-pha-ketoglutarate dehydrogenase. ALA has been shown to combat oxidative stress by quenching a variety of intracellular reactive oxy-gen species (ROS) [1]. ALA has been demonstrated to be effective in preventing pathology in various experimental models in which ROS have been implicated [2]. Mediators such as free radicals, ni-tric oxide, prostaglandins, and cytokines play important roles in the development of acut or chronic inflammation [3]. The present study was conducted to evaluate the effect of ALA on acute and chronic inflammation models in Dawley rats. Additionally, we have investi-gated the alterations in the activity of nitric oxide synthase following oral administration of ALA, indomethacine (IND) and diclofenac (DIC) in CAR-induced paw edema tissues of rats.

The present study was carried out in a total of 72 male Sprague-Dawley rats, weighing 180-190 g. The animals were grouped before the experiments and kept under standard conditions [4]. Animals were assigned randomly for acute inflammation to the control groups receiving either CAR (0.1 ml of 1% per animal) or water and expamintal groups receiving CAR plus ALA (50, 100 and 200 mg/

kg), indomethacine (IND, 25 mg/kg) or diclofenac (DIC, 25 mg/kg). In other series of experiments, the effect of ALA on the proliferative phase of inflammation was investigated using cotton pellet test [5].

In the present study, we found that 1) All doses of ALA, IND and DIC have significantly decreasing effect on the mean weight of the cotton pellets. The anti-proliferative effect of ALA was found as 67.7, 68.9 and 69.9 %, of IND was 83.8 % and of DIC was 76.1 %; 2) ALA reduced the development of CAR-induced paw edema, at a smaller magnitude for ALA than for IND and DIC; and 3) There were 2.57-fold increase in activity of inducible nitric oxide synthase (iNOS) in CAR-induced rats compared to control rats. However, oral adminis-tration of ALA, IND and DIC significantly decreased iNOS activity. All doses of ALA were more effective than IND and DIC for de-creasing iNOS activity. These results suggest that the anti-inflamma-tory effect of ALA on CAR-induced acute and cotton pellet-induced chronic inflammations can be attributed to its decreasing effect on activities of inducible nitric oxide synthase.

Table 1. Effects of ALA, indomethacine (IND) and diclofenac (DIC) on carrageenan (CAR)-induced paw edema and cotton pellet granuloma test in rats.

Treatment Num

ber o

f ani

mal

s

Dos

e m

g/kg

bod

y w

t

Acu

te

antii

nfla

mm

ator

y ef

fect

(Inh

ibiti

on %

)

Chr

onic

an

tiinf

lam

mat

ory

effe

ct (I

nhib

ition

%)

ALA 2x6 50 18.5 67.7

- 2x6 100 29.6 68.9

- 2x6 200 40.7 69.9

DIC 2x6 25 44.4 76.1

IND 2x6 25 55.5 83.8

CONTROLS 2x6 - - -

Table 2. Effects of ALA, IND and DIC on changes in activities of cNOS, iNOS and tNOS in CAR-induced paws (5th hour) of rats.

Treatment Num

ber o

f an

imal

s

Dos

e m

g/kg

bo

dy w

t.

cNO

S

iNO

S

tNO

S

ALA 6 50 2.70±0.53* 0.17±0.02** 2.86±0.55

- 6 100 3.10±0.25* 0.13±0.05** 3.23±0.29*

- 6 200 3.23±0.37* 0.13±0.03** 3.37±0.37*

CAR+DIC 6 25 2.28±0.07 0.18±0.03** 2.46±0.07

CAR+IND 6 25 2.04±0.05 0.22±0.01** 2.25±0.05

CAR 6 - 2.22±0.25 0.36±0.06** 2.58±0.24

HEALTHY 6 - 2.51±0.37 0.14±0.03 2.65±0.37

1. Bilska A, Wlodex L. Lipoic acid- the drug of future?, Pharmacol Report, 57, 570-577, 2005.

2. Odabaşoğlu F. Alpha lipoic acid, Pharma Şark, 1 (4), 12-15, 2006.

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3. Gualillo O, Eiras S, Lago F, Dieguez C, Casanueva FF. Evaluated serum leptin concentrations induced by experimental acute inflammation, J. Ethnopharmacol., 75, 213-218, 2001.

4. CCAC. 1993. Guide to the care and use of experimental animals, Vol I, 2nd Ed. Canadian Council on Animal Care. Bradda Printing Services Inc., Ottawa, ON, Canada.

5. Süleyman H, Odabaşoğlu F, Aslan A, Çakır A, Karagöz Y, Göçer F, Halıcı M, Bayır Y. Antiinflammatory and antiulcer effects of aqueous extract of Lobaria pulmonaria, Phytomedicine, 10 (6-7), 552-557, 2003.

P 57

GASTROPROTECTIVE EFFECT OF MONTELUKAST

(SINGULAIRE) ON INDOMETHACINE-INDUCED GASTRIC

ULCER IN RATS AND ITS RELATION WITH SOME

GLUTATHIONE METABOLISM PARAMETERS

1Günnur ÖZBAKIŞ-DENGİZ, 2Zekai HALICI, 3Fehmi ODABAŞOĞLU, 4Elif ÇADIRCI, 2Halis SÜLEYMAN1Karaelmas University, Faculty of Medicine, Department of Pharmacology, Zonguldak, Turkey2Atatürk University, Faculty of Medicine, Department of Pharmacology, Erzurum, Turkey3Atatürk University, Faculty of Pharmacy, Department of Biochemistry, Erzurum, Turkey4Atatürk University, Faculty of Pharmacy, Department of Pharmacology, Erzurum, Turkey

Non-steroidal anti-inflammatory drugs (NSAID) are widely used in the treatment of pain, fever and inflammation. However, these drugs have some side effects, especially on the gastrointestinal tract. Recently, reactive oxygen species (ROS) have also been shown to play a critical role in gastric ulceration process. The role of ROS in the development of pathogenesis in acute experimental gastric le-sions induced by stress, ethanol and NSAID’s is well known [1]. The glutathione (GSH), glutathione S-transferase (GST) and glutathione reductase (GR) play an important role in the prevention of the gas-tric damages [2]. On the other hand, montelukast (MLK), a selec-tive reversible cysteinyl leukotriene-1 receptor (LTD4 receptor) an-tagonist is used in the treatment of asthma [3] and presently nothing known about its effects on the gastro-intestinal system. We have in-vestigated alterations in the GSH level and the activities of antioxida-tive enzymes (GST and GR), following oral administration of MLK, lansoprazole (LAN), famotidine (FAM) and ranitidine (RAN) in rats with indomethacin (IND)-induced ulcer.

48 male Sprague-Dawley rats, weighing 180–190 g were used in the present study. The animals were grouped before the experiments and kept under standard conditions [4]. MLK (5, 10 and 20 mg/kg), LAN (30 mg/kg), FAM (25 mg/kg) and RAN (25 mg/kg) were administrated orally. 5 min after MLK, LAN, FAM and RAN administrations, IND (25 mg/kg) was administrated to all animals orally. Control group re-ceived only water. After 6 h of all treatments, animals were sacrificed using sodium thiopental (50 mg/kg). The stomachs were removed and opened along the greater curvature and washed with saline [2]. The wideness of ulcer areas were determined using a magnifier and a mil-limeter paper. Tissues were grounded under liquid nitrogen for the determinations of GSH level and GST and GR activities.

In the present study, we found that 1) MLK, LAN, FAM and RAN reduced the development of IND-induced gastric damages, at a greater magnitude for MLK, FAM and LAN than for RAN; 2) MLK and RAN caused an increase in the activity of GST possibly resulted from gastric injury; and 3) MLK and RAN ameliorated depressions in the GSH levels and the GR activity possibly caused by IND ad-ministration. These results suggest that the gastroprotective effect of MLK on IND-induced ulceration can be attributed to its ameliorat-ing effect on the oxidative damage.

Table 1. Effects of the MLK, FAM, RAN and LAN on IND-induced gastric damage in rats. IND was compared with healthy and treated groups with IND group.

Treatment N Dose mg/kg body wt.

Ulcer index (UI)

% Inhibition

IND+MLK 6 5 2.98±0.88** 59.1

IND+MLK 6 10 2.21±0.91** 69.7

IND+MLK 6 20 2.03±0.49** 72.2

IND+FAM 6 25 0.16±0.08*** 97.8

IND+RAN 6 25 2.54±0.76** 65.2

IND+LAN 6 30 0.0±0*** 100

IND 6 25 7.29±0.43*** -

Healthy 6 - - -

Table 2. Effects of the MLK, RAN and LAN on GSH, GST and GR in rat’s IND-induced tissues. IND was compared with healthy and treated groups with IND.

Treatment N Dose (mg/kg)

GST ACTIVITY

(EU)

GR ACTIVITY

(EU)

GSH LEVEL

IND+MLK 6 5 26,29±0,5* 34.5±0.6* 3.31±0.06*

IND+MLK 6 10 28,22±0,7* 29.6±0.8* 3.76±0.04*

IND+MLK 6 20 33,08±0,8** 27.6±0.7** 4.24±0.05**

IND+LAN 6 30 33,65±0,4** 43.1±0.6* 4.85±0.03**

IND+RAN 6 25 17,33±0,8* 25.2±0.2** 4.17±0.10**

IND 6 25 23,53±0,3* 37.7±0.9** 3.05±0.04**

HEALTHY 6 - 28,60±0,4 25.6±0.5 4.12±0.04

1. Das, D, Bandyopadhyay, D, Bhattacharjee, M, Banerjee, RK. Hydroxyl radical is the major cousative factor in stress-induced gastric ulceration, Free Radical Biol. Med., 23, 8-18, 1997.

2. Odabaşoğlu F, Çakır A, Süleyman H, Aslan A, Bayır Y, Halıcı M, Kazaz, C. Gastroprotective and antioxidant effects of usnic acid on indometha-cine-induced gastric ulcer in rats, J. Ethnopharmacology, 103 (1), 59-65, 2006.

3. Wenzel, SE. Leukotriene receptor antagonists and related compounds, Can. Respir. J., 6, 189-193, 1999.

4. CCAC. Guide to the care and use of experimental animals, Vol I, 2nd Ed. Canadian Council on Animal Care. Bradda Printing Services Inc., Ottawa, ON, Canada, 1993.

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P 58

GASTROPROTECTIVE EFFECT OF AMIODARONE ON

INDOMETHACIN-INDUCED GASTRIC ULCER IN RATS AND

ITS RELATION WITH MYELOPEROXIDASE AND SOME

ANTIOXIDANT ENZYMES

1Günnur ÖZBAKIŞ-DENGİZ, 2Fehmi ODABAŞOĞLU, 3Zekai HALICI, 4Elif ÇADIRCI, 2Mesut HALICI, 3Halis SÜLEYMAN1Karaelmas University., Faculty of Medicine, Department of Pharmacology, Zonguldak, Turkey2Atatürk University, Faculty of Pharmacy, Department of Biochemistry, Erzurum, Turkey3Atatürk University, Faculty of Medicine, Department of Pharmacology, Erzurum, Turkey4Atatürk University, Faculty of Pharmacy, Department of Pharmacology, Erzurum, Turkey

Amiodarone (AMD){2-butyl-3-(3’;5’diiodo-4’α-diethyl-amino-ethoxy-benzoyl) -benzofuran} is a multiple ion (Ca++, Na+, K+) chan-nel blocker drug and is also a non competitive α- and β-blocker in cardiac cell. It is an effective anti-arrhythmic and is used to treat a wide variety of ventricular and supraventicular tachyarrhytmias [1]. Clinical use of AMD is limited because of its potential for develop-ing numerous adverse side effects. Of greatest concern is AMD-in-duced pulmonary toxicity (AIPT), due to the potential for mortality. However, hepatotoxicity and other adverse effects are also of clinical importance. This drug can also modulate thyroid function and phos-pholipid metabolism [1-3]. Non-steroidal anti-inflammatory drugs (NSAID) are widely used in the treatment of pain, fever and inflam-mation. However, these drugs have some side effects, especially on the gastrointestinal tract. Recently, reactive oxygen species (ROS) have also been shown to play a critical role in gastric ulceration proc-ess. The role of ROS in the development of pathogenesis in acute experimental gastric lesions induced by stress, ethanol and NSAID’s is well known [4]. ROS damage membrane proteins via causing lipid peroxidation in membranes by attacking to unsaturated fatty acids [5]. Oxygen-handling cells have antioxidant enzymes that are able to protect them against. The enzymatic antioxidant defenses include superoxide dismutase (SOD), catalase (CAT) and myeloperoxidase (MPx), as marker of ulceration process [6, 7]. These antioxidants also play an important role in the prevention of the gastric damages. We have investigated alterations in the activities of SOD, CAT and MPx, following oral administration of AMD, lansoprazole (LAN) and ranitidine (RAN) in rats with indomethacin (IND)-induced ul-cer.

42 male Sprague-Dawley rats, weighing 180–190 g were used in this study. The animals were grouped before the experiments and kept under standard conditions [8]. AMD (25, 50 and 100 mg/kg body weight doses prepared as suspension in water) and positive controls [LAN (30 mg/kg body weight) and RAN (25 mg/kg body weight)] were administrated orally to the assigned groups of rats. 5 min after AMD, LAN and RAN administrations, IND (25 mg/kg body weight) was administrated to all animals orally. One group was assigned as control group, which received only water. After 6 h of all treatments, animals were sacrificed using sodium thiopental (50 mg/kg). The rats’ stomachs were removed and opened along the greater curvature and then washed with serum physiological solution. The wideness of ulcer areas was determined using a magnifier and a mil-limeter paper. Then tissues grounded within liquid nitrogen for as-says of SOD, CAT and MPx activities.

In the present study, we found that 1) AMD, LAN and RAN re-duced the development of IND-induced gastric damages, at a greater magnitude for AMD and LAN than for RAN; 2) AMD and RAN al-leviated increase in the activity of CAT enzyme resulting from ulcer; 3) AMD and RAN ameliorated depression in the activities of SOD enzyme caused by IND administration; and 4) All doses of AMD caused an amplification in MPx activity resulting from induced gas-

tric ulcers. These results suggest that the gastroprotective effect of AMD on IND-induced ulceration can be attributed to its ameliorat-ing effect on the oxidative damage, but can not be attributed to its effect on MPx activity, because of high iodine content, amiodarone can activate MPx activity in rat stomach [9].

Keywords: Amiodarone; Indomethacin; Gastroprotective effect; Myeloperoxidase; Antioxidant enzymes

Table 1. Effects of different doses of the amiodarone and single dose of ranitidine and lansoprazole on indomethacin-induced gastric damage in rats. Three doses of amiodarone, lansoprazole and ranitidine treated groups were compared with indomethacin group.

Treatment N Dose mg/kg body wt.

Ulcer index (UI)a

[Ulcerated area / Total stomach area] x 100

% Inhibitionb

6 25 2.75 ±0.70 * 54.5

Amiodarone 6 50 2.28 ±0.83 ** 62.3

6 100 0.89 ±0.46 *** 85.3

Ranitidine 6 25 1.64 ±0.85 ** 72.8

Lansoprazole 6 30 0 ±0 *** 100

Indomethacin 6 25 6.04 ±1.27 *** -

Healthy group 6 - 0 ±0 -

aMean damage index ±SEM of six animals in each group. N: The number of rats.

b% Inhibition in ulcer index in relation to indomethacin group. *Significant at p<0.05; **Significant at p<0.01; *** Significant at p<0.005. as compared with indomethacin group.

Table 2. Effects of different doses of the amiodarone and single dose of ranitidine on the activity of superoxide dismutase (SOD), catalase (CAT) and myeloperoxidase (MPx) enzymes in rat’s indomethacin (IND)-induced gastric tissue.

Treatment N Dosemg/kg body

wt

CAT(mmol/min/

mg tissue)

SOD(mmol/min/mg

tissue)

MPx Activity(μmol/min/mg

tissue)

6 25 115,99±1,20 94,20±0.98* 10,22±0.08

IND+AMD 6 50 90,17±1,20* 107,45±0.81** 11,47±0.26*

6 100 74,05±1,10* 125,92±0.28** 13,51±0.14*

IND+RAN 6 25 50,68±0,84* 116,08±0.06** 5,73±0.11*

IND 6 25 117,89±0,36* 74,57±0.52** 9,75±0.07*

Healthy rats 6 - 74,93±0,95 122,35±1.13 7,65±0.61

N: The number of rats. Results are means ± SE of three measurements. IND group was compared with healthy group. Treated groups were compared with IND group. *Significant at p<0.05; **Significant at p<0.01 as compared with IND group.

1. Mason JW. Amiodarone, New. Eng.. J. Med. 316, 455-466. 1987. 2. Vrobel TR, Miller PE, Mostow ND, Rakita, L. A general overview of

amiodarone toxicity: its prevention, detection, and management, Prog. Cardiovasc. Dis. 31, 393-426, 1989.

3. Figge HL, Figge J. The effects of amiodarone on thyroid hormone func-tion: A review of the physiology and clinical manifestations, J. Clin. Phar-macol., 30, 588-595, 1990.

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4. Das D, Bandyopadhyay D, Bhattacharjee M, Banerjee RK. Hydroxyl radi-cal is the major cousative factor in stress-induced gastric ulceration, Free Radical Biol. Med. 23, 8-18, 1997.

5. Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the degenerative diseases of aging, Proc. Natl. Acad. Sci., 90, 7915-7922, 1993.

6. Mates JM, Perez-Gomez C, Nudez de Castro I. Antioxidant enzymes and human diseases, Clinical Biochemistry, 32, 595-603, 1999.

7. Elliot SN, Wallace JL.. Neutrophil-mediated gastrointestinal injury, Ca-nadian Journal of Gastroenterology, 12, 559-568, 1998.

8. CCAC. 1993. Guide to the care and use of experimental animals, Vol I, 2nd Ed. Canadian Council on Animal Care. Bradda Printing Services Inc., Ottawa, ON, Canada.

9. Magnusson RP, Taurog A, Dorris ML. Mechanisms of thyroid peroxi-dase- and lactoperoxidase catalyzed reactions involving iodide, J. Biol. Chem., 259, 13783-13790, 1984.

P 59

SYNTHESIS OF SOME 5-CHLORO-6-(THIAZOL–4-YL)-2-

OXO-3H-BENZOTHIAZOLE DERIVATIVES AS POTENTIAL

COX-2 INHIBITORS

Selcen ADAK, Deniz S. DOĞRUER, Serdar ÜNLÜGazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Ankara, Turkey

It is known that inhibition of the enzyme cyclooxygenase (COX) is the principal mechanism for the efficacy of NSAIDs. Two distinct and independently COX isoforms have been identified COX-1 and COX-2 [1]. Interruption of COX-1 activity can lead to life-threating gatro-intestinal (GI) toxicity of perforation, ulceration and bleeding. In the meantime, COX-2 is induced upon inflammatory stimuli and is responsible for progression of inflammation [2]. Therefore, many studies have been focused on COX-2 inhibitors to reduce the risk of GI complications. The greatest research activity in the field of COX-2 inhibitors has been made in the synthesis and pharmacological testing of the class of diaryl heterocyclics [3].

Some compounds bearing 5-chloro-2-oxo-3H-benzothiazole ring have been reported to have anti-inflammatory activity [4]. In addi-tion, potent selective COX-2 inhibitors which bear thiazole struc-ture as a central ring have been reported in the literature [5, 6]. In the design of new compounds, development of the hybrid molecules through the combination of different pharmacophores in one struc-ture may lead to compounds with increased analgesic and anti-in-flammatory activities.

Therefore, these observations prompted us to synthesize six new compounds which bear a central thiazole ring whose 2,4-positions are substituted with phenyl and 5-chlorobenzothiazolone moieties, respectively. Structures of the synthesized compounds have been confirmed by IR, 1H-NMR and elemental analysis. Their COX-2 in-hibitory potency will be evaluated by using in vitro human whole blood assay.

1. Menozzi G, Merello L, Fossa P, Mosti L, Piana A, Mattioli F. 4-Substitut-ed 1,5-diarylpyrazole, analogues of celecoxib: synthesis and preliminary evaluation of biological properties, IL Farmaco, 58, 795-808, 2003.

2. Shin SS, Noh MS, Byun YJ, Choi JK, Kim JY, Lim KM, Ha JY, Kim JK, Lee CH, Chung S. 2,2-Dimethyl-4,5-diaryl-3(2H)furanone derivatives as selective cyclooxygenase-2 inhibitors, Bioorg. Med. Chem., 11, 165-168, 2001.

3. Dannhardt G, Laufer S. Structural approaches to explain the selectivity of COX-2 inhibitors: Is there a common pharmacophore, Curr. Med. Chem., 7, 1101-1112, 2000.

4. Önkol T, Doğruer DS, Ito S, Şahin MF. Synthesis and antinociceptive activity of (5-chloro-2-benzothiazolinon-3-yl)acetamide derivatives, Ar-chiv der Pharmazie, 333(10), 337-340, 2000.

5. Kontogiorgis CA, Hadjipavlou-Litina DJ. Non steroidal anti-inflamma-tory and anti-allergy agents, Curr. Med. Chem., 9, 89-98, 2002.

6. Kalgutkar AS, Zhao Z. Discovery and design of selective clooxygenase-2 inhibitors as non-ulcerogenic, anti-inflammatory drugs with potential utility as anti-cancer agents. Curr. Drug Targets, 2, 79-106, 2001.

P 60

SYNTHESIS AND ANTIMICROBIAL EVALUATION

OF 6-SUBSTITUTED-3(2H)-PYRIDAZINONE-2-YL

ACETOHYDRAZİDE DERIVATIVES

1Mehtap GÖKÇE, 1Gülay YELKEN, 2Serpil POLAT, 3Seda TEZCAN, 2Mehmet SERİN1Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06330 Ankara, Turkey2Mersin University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Mersin, Turkey3Mersin University, Faculty of Medicine, Department of Medicinal Microbiology, Mersin, Turkey

Several antibiotics have been prescribed and found to be effective on various infectious disorders. However, the appearance of multi-drug resistant Gram-positive bacteria, in particular, methicillin-re-sistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) is causing a serious menace. Moreover, the emer-gence of vancomycin resistant MRSA can be anticipated in foresee-able future. For the treatment of these intractable infections, a new antibacterial agent is needed. Furthermore Synthetic antibiotics are widely prescribed drugs because of their safety, good tolerance, broad antibacterial spectrum and less resistance.

Due to favorable presence a pyridazinone moiety in known active structures, pyridazinone derivatives provoked a special interest in the search for new antibacterial agents [1-4]. Meanwhile hyrazide-hydrazones have been claimed to exhibit appreciable antimicrobial activity [5-9]. On the basis of these observations a new series of 6-substituted-3(2H)-pyridazinone-2-yl acetohydrazide (I) was syn-thesized using an appropriate synthetic route. The structure eluci-dation of new compounds was based on the relevant 1H-NMR and IR spectral characteristics and purity of the products was confirmed both by TLC and elemental analyses. All the target compounds were evaluated for their in vitro antimicrobial activity against Sta-phylococcus aureus, Bacillus subtilis as examples for Gram positive bacteria, Escherichia coli, Pseudomonas aeruginosa as examples for gram negative bacteria and Candida albicans, Candida parapsilosis as representative of fungi. The minimum inhibitory concentration (MIC) was determined for test compounds as well as for reference standards.

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1. Sönmez M., Berber I., Akbaş E. Eur. J. Med. Chem., 41(1), 101-5, 2006. 2. Fuks B, Talaga P, Huart C, Henichart JP, Bertrand K, Grimee R, Lorent G.

Eur. J. Pharmacol., 519(1-2), 24-30, 2005. 3. Akbaş E, Berber I, Şener A, Hasanov B. Farmaco, 60(1), 23-6, 2005. 4. Takaya M. Yakugaku Zasshi., 113(9), 676-81, 1993. 5. Koçyiğit-Kaymakçıoğlu B, Orçun E, Ünsalan S, Kandermirli F, Shvets N,

Rollas S, Antony D., Eur. J. Med. Chem., 41(11), 1253-61, 2006. 6. Bijev A, Arzneimittel Forschung, 56(2), 96-103, 2006. 7. Küçükgüzel G, Kocatepe A, De Clercq E, Şahin F, Gulluce M. Eur. J. Med.

Chem., 41(3), 353-9, 2006. 8. Sriram D, Yogeeswari P, Madhu K. Bioorg. Med. Chem. Lett., 16(4), 876-

8, 2006. 9. Grover G, Kini SG. Eur. J. Med. Chem., 41(2), 256-62, 2006.

P 61

SYNTHESIS AND ANTIMICROBIAL EVALUATION OF

5-CHLORO-2(3H)-BENZOXAZOLINONE-3-ACETYL-2-(p--

SUBSTITUTED BENZAL)HYDRAZONE DERIVATIVES

1Tijen ÖNKOL, 1Mehtap GÖKÇE, 1Ali Ulvi TOSUN, 2Serpil POLAT, 2Mehmet SERİN, 3Seda TEZCAN1Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06330 Ankara, Turkey2Mersin University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Mersin, Turkey.3Mersin University, Faculty of Medicine, Department of Clinical Microbiology, Mersin, Turkey

Since it is a common agreement of multidrug-resistant bacteria are major cause of failure in the treatment of infectious diseases, the need for the synthesis novel antibiotics is a reality.

The structural and therapeutic diversity coupled with commercial viability of small molecules has fascinated organic and medicinal chemists. It has been reported in the literature that hydrazide-hydra-zones have been demonstrated to possess antibacterial, and antifun-gal activities [1-4].

Furthermore, it is reported in the literature that 2(3H)-benzoxa-zolinone derivatives can exhibit diverse activities. Particularly antibac-terial [3] and antifungal [4] activities have been scrutinized intensively. In addition, it has been published that chlorinated 2(3H)-benzoxa-zolinone compounds have valuable fungicidal properties [5].

These observations led us to synthesize a series of Schiff bases combining 5-chloro-2(3H)-benzoxazolinone, hydrazide and benzal-dehyde moieties in the same molecule. Synthesized 5-chloro-2(3H)-benzoxazolinone-3-acetyl-2-(p-substituted benzal)hydrazone I de-rivatives were evaluated for screening antibacterial and antifungal activities on microorganisms respectively on four bacteria and two Candida species.

R1= H, F, Cl, Br, CH

3, OCH

3, OH

1. Metwally KA, Abdel-Aziz LM, Lashine el-SM, Husseiny MI, Badawy RH., Bioorg. Med. Chem., 14(24), 8675-8682, 2006.

2. Koçyiğit-Kaymakçıoğlu B, Orçun E, Ünsalan S, Kandemirli F, Shvets N, Rollas S, Antony D., Eur. J. Med. Chem., 41(11), 1253-1261, 2006.

3. Mincheva ZP, Kalcheva VB, Golovinsky EG., Pharmazie, 48(11), 859-860, 1993.

4. Wang HX, Liu F, Ng TB., Comp. Biochem. Physiol., 30(3), 379-388, 2001. 5. Basel EM, Riehen JB., US Patent 2,922,794 (Cl. 260-304), January 26,

1960.

P 62

SYNTHESIS OF 6-(SUBSTITUE THIAZOL–4-YL)-2-OXO-

3H-BENZOXAZOLE DERIVATIVES AS POTENTIAL COX-2

INHIBITORS

Şeyma CANKARA, Serdar ÜNLÜGazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Ankara, Turkey

Selective inhibition of cyclooxygenase-2 (COX-2) which is being induced during inflammation is off interest since the resulting drugs lack the gastric ulceration side effects associated to classical NSAIDs. Since 2-oxo-3H-benzoxazole [1] and thiazole [2]ring system bear good anti-inflammatory property and the 2,4-diaryl/heteroaryl structures might also be important for selective COX-2 inhibitory activity, we hereby describe the biological consequences of incorpo-ration of a 2-oxo-3H-benzoxazole ring as one of the aryl substituents and the effect of a 2,4-diarylsubstitution pattern around the thiazole ring on the in vitro activity of the resulting derivatives towards COX-2 inhibition.

Thus, we have designated a series of 6-(substitute thiazole–4-yl)-2-oxo-3H-benzoxazole derivatives and these compounds were pre-pared by the reaction of 6-bromoacetyl-2-oxo-3H-benzoxazole and substituted thiobenzamides. The result of inhibitory potency against COX was evaluated using human whole blood assay [3].

1. Ünlü S, Önkol T, Dündar Y, Ökcelik B, Küpeli E, Yeşilada E, Noyanalpan N, Şahin MF. Synthesis and Analgesic and Anti-inflammatory Activity of Some New (6-Acyl-2-benzoxazolinone and 6-Acyl-2-benzothiazolinone Derivatives with Acetic Acid and Propanoic Acid Residues, Arch. Pharm. Pharm. Med. Chem., 336, 353–361, 2003.

2. Narender M, Reddy MS, Sridhar R, Nageswar YVD, Rama Rao K. Aque-ous phase synthesis of thiazoles and aminothiazoles in the presence of β-cyclodetrin, Tetrahedron Letters, 46, 5953-5955, 2005.

3. Patrignani P, Panara Mr, Greco A, Fusco O, Natoli C, Iacobelli S, Cipol-lone F, Ganci A, Creminon C, Maclouf J, Patrono C. Biochemical and Pharmacological Characterization of the Cyclooxygenase Activity of Hu-man Blood Prostaglandin Endoperoxide Synthases, The Journal of Phar-macology and Experimental Therapeutics, 271, 1705-1712, 1994.

P 63

INHIBITION EFFECT OF NEW SULFONAMIDE DERIVATIVES

ON CARBONIC ANHYDRASE

Ümmühan ÖZDEMİR-ÖZMEN, Fatma ARSLAN, Fatma HAMURCUGazi University, Arts and Sciences Faculty, Department of Chemistry, 06500 Ankara, Turkey

Sulfonamides have been known as powerful inhibitors of carbonic anhydrase (CA) [1]. Since sulfonamides interact with the active site of CA, the active site charge requirements and the orientations of sulfonamide groups are essential for the inhibitory powers of these drugs. Understanding the mechanism of the inhibition of CA isoen-

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zymes with sulfonamide derivatives at the molecular level have been reported to be helpful for the rational design of novel derivatives with minimum side effects [2].

In the present study, inhibitory effects of ethanesulfonic acid hy-drazide (esh), 5-methyl-2-hydroxyacetophenone ethanesulfonylhy-drazone (5mafesh) and its Ni(II) complex on CAII have been inves-tigated. Enzyme activity was determined using p-nitrophenylacetate as substrate. According to the corresponding IC50 and Ki values, 5mafesh was found as the most potent inhibitor of CAII.

1. Arslan O, Küfrevioğlu Öİ, Nalbantoğlu B., Bioorg. & Med. Chem., 5(3), 515, 1997.

2. Chakravarty S , Kannan KK., J. of Mol. Biol., 243, 298, 1994.

P 64

DESIGN AND SYNTHESIS OF NOVEL TIAZOLE DERIVATIVES

AS POTENTIAL CYCLOOXYGENASE- 2 (COX-2) INHIBITORS

Leyla UZUN, Tijen ÖNKOL, Serdar ÜNLÜGazi University, Faculty of Pharmacy Department of Pharmaceutical Chemistry, 06330 Ankara, Turkey

The major side effects associated with the currently available NSAIDs are gastrointestinal (GI) hemorrhagia and ulceration which result from the nonselective inhibition of COX enzymes, namely COX-1 and COX-2. After the discovery of the second isoform (COX-2) that is expressed in inflammatory cells, but not in gastric mucosa, novel NSAIDs with selective COX-2 inhibitory activity resulted to be more useful for the treatment of inflamatory diseases [1] without gastric side effects.

For this reason, the series of 2,4-diarylthiazole derivatives were designed and synthesized for their evaluation as selective COX-2 in-hibitors in an enzymatic assay using human whole blood. We selected thiazole ring to synthesize the title compounds since thiazole ring have been reported to have analgesic and anti-inflammatory activities [2]. The synthesis of 6-(substituted thiazol-4-yl)-2- oxo-3H-benzo-thiazoles were accomplished by the reaction of 6- bromoacetyl-2-oxo-3H-benzothiazole with o/p-substituted thiobenzamides using microwave-assisted synthesis. Physical and chemical properties of synthesized compounds have been confirmed by using their melting points, IR, 1H-NMR and elemental analysis.

1. Ulbrich H, Fiebich B, Dannhardt G. European Journal of Medicinal Chemistry, 37, 953-959, 2002.

2. Norender M, Reddy MS, Sridhar R, Nageswar YVD, Rao KR. Aqueous phase Ssynthesis of thiazoles and aminothiazoles in the presence of β-cyclodextrin, Tetrahedron Letters, 46, 5953-55, 2005.

P 65

SYNTHESIS OF 5-CHLORO-2-OXO-6-(2-SUBSTITUTED

PHENYL-1,3-THIAZOL-4-YL)-3H-BENZOXAZOLE

DERIVATIVES AND EVALUATION OF THEIR COX-1/COX-2

INHIBITORY ACTIVITIES

Şölen URLU, Serdar ÜNLÜGazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06330 Ankara, Turkey

The therapeutic efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) results from the inhibition of cyclooxygenases (COX) which were shown to exist as two distinct isoforms, namely, COX-1 and COX-2. COX-1 is constitutively expressed as a house keeping enzyme in nearly all tissues and mediates physiological responses. On the other hand, COX-2 is expressed by cells involved in inflam-mation and has emerged as the isoform that is primarily responsi-ble for the synthesis of prostanoids involved in acute and chronic inflammatory states. It was hypotesised that selective inhibition of COX-2 might have therapeutic actions similar to those of NSAIDs, but without causing gastrointestinal side effects [1].

Our ongoing studies towards the derivatives of chlorzoxazone

[2]and thiazole with anti-inflamatory activities [3] prompted us to design new compounds which could be selective COX-2 inhibitors. For this purpose some 5-chloro-2-oxo-6-(2-substituted phenyl-1,3-thiazol-4-yl)-3H-benzoxazole derivatives were synthesized by con-densation of 6-bromoacetyl-5-chloro-2-oxo-3H-benzoxazole and appropriate thiobenzamides derivatives in diethylenglicoldimethyl-ether. The activities of the compounds for possible COX inhibitory activity will be performed by enzyme immunoassay method.

1. Brune K, Hinz B. Selective cylooxygenase-2 inhibitors: similarities and differences, Scand. J. Rheumatol., 33, 1-6, 2004.

2. Park, JY, Kim KA, Park PW, Ha JM. Effect of high-dose aspirin on CYP2E1 activity in healty subjects measured using chlorzoxazone as a probe, Journal of Clinical Pharmacology, 46, 109-114, 2006.

3. Kazzouli Sl, Berteina-Raboin S, Mouaddib A, Guillaumet G. Solid support synthesis of 2,4-disubstituted thiazoles and aminothiazoles, Tet-rahedron Letters, 43, 3193-3196, 2002.

P 66

PHYSICOCHEMICAL CHARACTERIZATION OF

BIODEGRADABLE CARDIOVASCULAR STENTS

Can SARISÖZEN, Betül ARICA, Sema ÇALIŞ, A. Atilla HINCALHacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 06100 Ankara, Turkey

The aim of our study was to realize physicochemical characteriza-tion and in vitro evaluation of prepared biodegradable cardiovascular stents for preventing restenosis of blood vessels. Stents were prepared from solution-cast biodegradable formed polymeric films and a model drug prednisolone acetate (PA) was incorporated into the stents.

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For this purpose, two types of synthetic biopolymer (PLGA 75:25 and PLGA 50:50) were dissolved in dichloromethane separately to have a final concentration of 25% (w/v) in which PEG 4000 was add-ed as a plasticizer. The polymer solutions cast onto the aluminum pans (in 3x2 cm area). After evaporation of dichloromethane, the polymer films were cut into strips and coiled on the cylindrical rods to form a helical shaped stents.

Two types of stents were used in our study: stents formed by a polymer film including PA and stents formed by a polymer film in-cluding PA containing spray-dried chitosan microspheres which has been developed by our group previously. The characteristics of these prepared stents such as wall thickness, diameter, length, morphology were measured and evaluated.

Differential Scanning Calorimetry (DSC) and Attenuated Total Reflectance (ATR)-FTIR studies were also performed. Samples were heated under nitrogen atmosphere in aluminum hermetic pans and thermograms recorded a range of (20°C to 300°C at a heating rate of 10°C/min). ATR-FTIR studies were performed to determine if there were drug present on the surface of the stents or not. Stents which were prepared by PLGA 75:25 polymer were analyzed for specific surface area analyzes. In vitro drug release testing was performed to evaluate the drug release form the stents. The test was also performed in PBS solution containing 0.5%(w/v) sodium lauryl sulphate and 0.05%(w/v) sodium azide at pH 7.4.

The prepared stents had outer diameters approximately 3mm and their length were 1.5cm. The polymer wall thicknesses of empty stents were measured using a micrometer and determined as 136μm±0.05. Based on ease of handling for perivascular placement, this film thick-ness was assessed to be optimum. Thin films (100μm) tended to fold and 200μm films were not elastic and easy to crack. Incorporation of drug or drug loaded microspheres did not interfere during the forma-tion of homogenous films and helical stent structure. Prepared stents seemed to have smooth surface without any visible defects and cracks. Incorporated drug or microspheres were homogenously distributed in the polymer matrix. The ATR-FTIR spectrums showed that biode-gradable stent surfaces were free of drug and microspheres.

Thermograms of the formulations are given in Figure 1. As it can be seen from the graphics, melting peak of PA disappeared after it was incorporated into the stents. The results for the specific surface area analyzes are given in Table 1. Although the release of PA from the stents seemed to be very slow following the initial release pe-riod which was about 1-5 days in PBS buffer, a significant amount of the drug was determined to be released at the second release period which started on the 5th day and on the 63th day. This might be ex-plained by the slow degradation of the PLGA polymer in the release medium. Nevertheless, approximately 5-62% of the PA from the PA-loaded stents and 10-13% of the PA from the chitosan microspheres containing stents were released in 63 days.

Table 1. Characterization of biodegradable stent formulations.

Polymer PA or Microspheres

Length (cm)

Diameter (mm)

Thickness (μm)

Specific Surface

Area

PLGA 75:25

None (Empty Stent)

1.5 3 136.5±5 μm 1,32 m2/g

PLGA 75:25

PA Incorporated

1.5 3 140±3.6 μm 2,14 m2/g

PLGA 75:25

Microsphere Incorporated

1.5 3 145±4 μm 0,44 m2/g

PLGA 50:50

None (Empty Stent)

1.5 3 109±8 μm -

PLGA 50:50

PA Incorporated

1.5 3 119.3±4.2 μm -

PLGA 50:50

Microsphere Incorporated

1.5 3 118.7±3.2 μm -

P 67

CO-ADMINISTRATION OF A NITRIC OXIDE SYNTHASE

INHIBITOR AND MELATONIN EXERTS AN ADDITIVE

ANTIDEPRESSANT-LIKE EFFECT IN THE MOUSE FORCED

SWIM TEST

1Yusuf ERGÜN, 2Ufuk Güney ERGÜN, 3Özlem ORHAN, 4Ekrem KÜÇÜK1Kahramanmaraş Sütçü İmam University, School of Medicine, Department of Pharmacology, 46100 Kahramanmaraş, Turkey2Kahramanmaraş Sütçü İmam University, School of Medicine, Department of Family Medicine, 46100 Kahramanmaraş, Turkey3Kahramanmaraş Sütçü İmam University, School of Medicine, Department of Psychiatry, 46100 Kahramanmaraş, Turkey4Osmaniye High School of Science, Osmaniye, Turkey

Previous studies have shown that nitric oxide synthase inhibi-tors and melatonin are as efficacious as conventional antidepres-sant drugs in pre-clinical antidepressant screening procedures such as Porsolt swim test. The aim of the present study was to assess the combination therapy of these two distinct arrays of drugs.

Porsolt swim test was conducted to resemble the symptomatology of major depressive disorder, and an open filed locomotor activity was also used.

NG-nitro-L-arginine (L-NNA) at doses 3 to 30 mg/kg and me-latonin at 3 and 10 mg/kg were examined in the forced swim test in mice. 3 mg/kg L-NNA slightly but not significantly reduced the duration of immobility, and increasing the dose to 10 mg/kg was suf-ficient to attain a significant reduction (Fig. 1). On the other hand, the maximal dose of L-NNA (30 mg/kg) had no effect although a non-significant small increase was observed (Fig. 1). The results obtained with L-NNA were in accordance with a U-shape effect. 3 mg/kg melatonin was found to be ineffective while a statistically sig-nificant decrease in the duration of immobility was found at the dose of 10 mg/kg (Fig. 1). The combination of ineffective doses (3 mg/kg, each) of L-NNA and melatonin revealed no further inhibition in the duration of immobility and the most effective doses (10 mg/kg, each) caused a more pronounced reduction when compared to those of each drug alone (Fig. 1). None of the drugs effected locomotor activity over the dose range applied (Fig. 2).

In conclusion, the combined therapy with L-NNA and melatonin seems to have an additive effect and may be considered as a feasible candidate in attenuating the symptoms of major depressive disorder.

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Figure 1. The effects of saline, 1% alcohol, NG-nitro-L-arginine (L-NNA: 3, 10 and 30 mg/kg) and melatonin (3 and 10 mg/kg) on the duration of immobility (sec) in forced swim test. Values presented as mean ± SEM, n=10. * P<0.05 (versus control, saline or 1% alcohol), ** P<o.o5 (versus L-NNA or melatonine alone) analysis of variance (ANOVA) with Bonferroni correction.

Figure 2. The effects of saline, 1% alcohol, NG-nitro-L-arginine (L-NNA: 3, 10 and 30 mg/kg) and melatonin (3 and 10 mg/kg) on the squares entered in locomotor activity test. Values presented as mean ± SEM, n=10.

1. Trullas R, Skolnick P. Functional antagonists at the NMDA receptor complex exhibit antidepressant actions, Eur. J. Pharmacol., 185, 1-10, 1990.

2. Harkin AJ, Bruce KH, Craft B, Paul IA. Nitric oxide synthase inhibitors have antidepressant-like properties in mice. 1. Acute treatments are ac-tive in the forced swim test, Eur. J. Pharmacol., 372, 207-213, 1999.

3. Karolewicz B, Paul IA, Antkiewicz-Michaluk L. Effect of NOS inhibitor on forced swim test and neurotransmitters turnover in the mouse brain, Pol. J. Pharmacol., 53, 587-596, 2001.

4. Overstreet DH, Pucilowski O, Retton MC, Delangrange P, Guardiola-Le-maitre B. Effects of melatonin receptor ligands on swim test immobility, Neuroreport, 9, 249-253, 1998.

5. Mantovani M, Pértile R, Calixto JB, Santos ARS., Rodrigues ALS. Me-latonin exerts an antidepressant-like effect in the tail suspension test in mice: evidence for involvement of N-methyl-D-aspartatereceptors and the L-arginine-nitric oxide pathway, Neurosci. Lett., 343, 1-4,2003.

6. Heiberg IL, Wegener G, Rosenberg R. Reduction of cGMP and nitric oxide has antidepressant-like effects in the forced wimming test in rats, Behav. Brain Res., 134, 479-484, 2002.

P 68

A UTILITY OF FURAN CORED COMPOUNDS; SYNTHESIS

OF NITROGEN TETRACYCLES, ISOBENZOFURANOL AND

ISOBENZOTHIOPHENOL

Muhsin KARAARSLAN, Ersen GÖKTÜRK, Aydın DEMİRCANUniversity of Niğde, Faculty of Sciences & Letters, Department of Chemistry, Kampus, 51100 Niğde, Turkey

Intramolecular Diels–Alder (IMDA) reactions involving furane as a diene form oxanorbornenes that have been used in the synthesis of numerous complex targets and as an intermediate in the synthesis

of natural products such as carbohydrates and prostaglandins [1]. A series of key precursors to the IMDA reaction of furane diene (2a–c) have been prepared via facile alkylation and protection. While the cycloaddition process for (3a–c) was afforded in hot toluene, a com-mercial microwave (2450 MHz) was used for the synthesis of (5a–b) (Figure 1).

Figure 1. i. Br2, Et3N, DCM, 0°C, 98%; NaBH4, CeCl3.7H2O, MeOH, 0°C, 97%; PBr3, Pyridine, PhH, 0°C� reflux, 68%; KOtBu, CHBr3, Pentane, 0°C; Heat 155°C, 1h, 78%; 2,3-dibromocyclopentene or 2,3-dibromocyclohexene, K2CO3, THF, reflux, 3d; (Boc)2O, DMAP, DCM, 0°C, 2h.

Treatment of fused oxy- and thio-heterotricycles (5a–b) with bo-rontriluoride-etherate in dichloromethane at –78°C cleaved Epoxy-Bridge and concomitant aromatisation gave the isobenzo-furanol and thiophenol (6a–b) in 72–76% yields respectively (Figure 2) [2].

Figure 2.

1. Lipshutz BH. Chem. Rev., 86, 795, 1986. Wang Q, Padwa A, Rh(I)-Cata-lyzed ring opening of an IMDAF-derived oxabicyclo cycloadduct as the key step in the synthesis of ±-epi-Zephyranthine, Org. Lett., 6, 2189, 2004.

2. Demircan A, Karaarslan M, Turac E.. A facile synthesis of heterotricycles from furfurylbromoalkenes using thermal IMDA cycloaddition. Hetero-cyclic Commun., 3&4, 233, 2006. Karaarslan M, Göktürk E, Demircan A. Thermal intramolecular Diels–Alder reaction of furan; Synthesis of ni-trogen tetracycles, isobenzofuran and isobenzothiophene, J. Chem. Res., 2, 117, 2007.

P 69

THE DETERMINATION OF ANTHOCYANINS

DELPHINIDIN AND PEONIDIN IN FRESH FRUITS BY HIGH

PERFORMANCE LIQUID CHROMATOGRAPHY

Nafiz Öncü CAN, Göksel ALTIOKKAAnadolu University, Faculty of Pharmacy, Department of Analytical Chemistry, Eskişehir, Turkey

Anthocyanins constitute one of the major groups of natural pig-ments and are responsible for many of the colours of fruits and vegetables [1]. It has been demonstrated that anthocyanins possess some positive therapeutic effects [2, 3] and are in use as nutritional supplements in many countries [4]. The aim of this study was to de-velop a simple and precise procedure for the determination of two anthocyanins, delphinidin and peonidin, and indicate their levels in some fresh fruits consumed in Turkey.

11 fruit samples, including pomegranate, cherry and grape, were examined using high performance liquid chromatography coupled

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to photodiode array detection. Two anthocyanins and cyanidin (in-ternal standard) were separated using a gradient elution technique by a reversed phase column. Pumping the mobile phase at a flow rate of 0.8 mL.min-1, analytes were detected at 520 nm wavelength within an average time of 18 min. Samples were prepared from fresh fruits by taking their juices with direct pressing which was followed by filtration through 0.2 μm PVDF filters, and injected into the sys-tem with no further extraction or concentrating steps.

Figure 1. A typical chromatogram of pomegranate fruit sample (DP: Delphinidin, PN: Peonidin, IS: Internal standard)

The method was found to be linear between the ranges about 80 – 400 ngmL-1, giving symmetrically sharp and repeatable analyte peaks. The overall limits of quantitation (LOQ) were 58.4 and 50.1 ngmL-1 for delphinidin (DP) and peonidin (PN), respectively. It was observed that the amount of anthocyanins detected in fruits reach to the maximum due to freshness and exist especially in dark coloured fruits.

1. Kong JM, Chia LS, Goh NK, Chia TF, Brouillard R. Analysis and biologi-cal activities of anthocyanins, Phytochemistry, 64, 923-933, 2003.

2. Dell’Agli M, Busciala A, Bosisio E. Vascular effects of wine polyphenols, Cardiovasc. Res., 63, 593-602, 2004.

3. Kowalczyk E, Krzesinski P, Kura M, Szmigiel B, Blaszczyk J. Anthocy-anins in medicine, Pol. J. Pharmacol., 55, 699-702, 2003.

4. Bravo L. Polyphenols: chemistry, dietary sources, metabolism, and nutri-tional significance, Nutr. Rev., 56, 317-333, 1998.

P 70

SCREENING OF ANTHOCYANINS PELARGONIDIN AND

MALVIDIN IN COMMERCIAL FRUIT JUICES USING LIQUID

CHROMATOGRAPHY COUPLED TO DIODE ARRAY

DETECTION

Göksel ALTIOKKA, Nafiz Öncü CANAnadolu University, Faculty of Pharmacy, Department of Analytical Chemistry, Eskişehir, Turkey

Commercial fruit juices are one of the most common consumed food products that contain many naturally occurring compounds, such as anthocyanins, which have therapeutic effects against cancer and cardiovascular diseases [1, 2]. Development of a cheap and re-producible method for the determination of two anthocyanins, pel-argonidin and malvidin, in fruit juices was introduced in this study.

8 samples of different fruit juices were examined using liquid chromatography coupled to photodiode array detection. Utilizing the sugar-free aglycone cyanidin as an internal standard, two an-

thocyanins were resolved by reversed phase technique within about 16 minutes. Analytes were detected at 520 nm wavelength and mo-bile phase was pumped through the column at a flow rate of 0.8 mL.min-1. Samples were injected to the system directly, following a simple filtration through 0.2 μm PVDF filters, without any extrac-tion or concentration steps.

Figure 1. A typical chromatogram of grape juice sample (PG: Pelargonidin, MV: Malvidin, IS: Internal standard).

Anthocyanins were detected in ppb levels with adequate chroma-tographic resolution. Linearities were obtained between the ranges of 78.1 – 390.7 ppb for pelargonidin and 88.15 – 440.7 ppb for mal-vidin. On the whole, malvidin and pelargonidin levels were found to be relatively high in grape products, such as wine and grape juice, in accordance with the previous studies on these compounds.

Keywords: Pelargonidin, malvidin, food analysis, liquid chromatography

1. Bravo L. Polyphenols: chemistry, dietary sources, metabolism, and nutri-tional significance. Nutr. Rev., 56, 317-333, 1998.

2. Dell’Agli M, Busciala A, Bosisio E. Vascular effects of wine polyphenols, Cardiovasc. Res., 63, 593-602, 2004.

P 71

RAPID DETERMINATION OF ACRYLAMIDE IN

POTATO CHIPS BY HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY WITH DIODE ARRAY DETECTION

Nafiz Öncü CAN, Göksel ALTIOKKAAnadolu University, Faculty of Pharmacy, Department of Analytical Chemistry, Eskişehir, Turkey

Acrylamide is one of the heat-generated food toxicants, which was frequently found in various foodstuffs such as potato chips, biscuits and coffee [1, 2]. It has been demonstrated that acrylamide possesses some neurotoxic and carcinogenic effects [3, 4] and is classified in Group 2A as a possible carcinogen to humans by International Agency for Research on Cancer [5]. The aim of this study was to develop an inexpensive, simple and sensitive method for determi-nation of acrylamide and indicate acrylamide levels of some potato products produced in Turkey.

Acrylamide concentration of the samples was examined using high performance liquid chromatography combined to diode ar-ray detection. Acrylamide and methacrylamide (internal standard) were separated using isocratic elution technique by a reversed phase column (GL Sciences, Inertsil® ODS-3). Mobile phase (acetonitrile:water, 2:98, v/v) was pumped at 0.5 mL.min-1 flow rate and analytes

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were detected at 200 nm wavelength within an average retention time of 15 min. Samples were prepared by solid phase extraction and injected into the system with no further extraction or pre-concentra-tion steps.

Figure 1. A typical chromatogram of homemade potato chip sample (AA: Acrylamide, MAA: Methacrylamide)

Acrylamide was detected in ppb levels with adequate chromato-graphic resolution. The developed method was validated according to the recommendations of International Conference of Harmoni-zation – Q2(R1) and system suitability parameters were tested ac-cording to United States Pharmacopeia. The method was found to be linear in the ranges of 2.9-14.2 ppb, giving LOD and LOQ con-centrations of 0.93 and 2.82 ppb, respectively. As a result, it was ob-served that the amount of acrylamide in potato chips varies due to cooking conditions and was found to be relatively high in many of the samples.

Keywords: acrylamide, food analysis, liquid chromatography, Turkish foods

1. Tareke E, Rydberg P, Karlsson P, Eriksson S, Tornqvist M. Acrylamide: a cooking carcinogen? Chem. Res. Toxicol., 13, 517-522, 2000.

2. Tareke E, Rydberg P, Karlsson P, Eriksson S, Tornqvist M. Analysis of acr-ylamide, a carcinogen formed in heated foodstuffs, J. Agric. Food Chem., 50, 4998-5006, 2002.

3. JECFA - Joint FAO/WHO Expert Committee on Food Additives, Sum-mary and conclusions of the sixty-fourth meeting, JECFA/64/SC, p. 7-17, World Health Organization WHO, Rome, Italy, 2005.

4. Shipp A, Lawrence G, Gentry R, McDonald T, Bartow H, Bounds J, Mac-donald N, Clewell H, Allen B, van Landingham C. Acrylamide: Review of toxicity data and dose-response analyses for cancer and noncancer ef-fects, Crit. Rev. Toxicol., 36, 481-608, 2006.

5. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Volume 60: Some Industrial Chemicals, p. 389-433, International Agen-cy for Research on Cancer (IARC),Geneva, Switzerland, 1997.

P 72

IMPROVED HIGH PERFORMANCE LIQUID

CHROMATOGRAPHIC SEPARATION OF CYANIDIN AND

PETUNIDIN IN FRESH VEGETABLES USING AN ODS-3

REVERSED-PHASE COLUMN

Göksel ALTIOKKA, Nafiz Öncü CAN, Zeki ATKOŞARAnadolu University, Faculty of Pharmacy, Department of Analytical Chemistry, Eskişehir, Turkey

As a subgroup of the polyphenols family, anthocyanins are known as one of the best natural antioxidant compounds that can be found

in many of the vegetables such as red cabbage and turnip [1]. An-thocyanins, which act as colouring pigments in plants, have been shown to be strong antioxidants with potential health benefits [2]. These properties make these compounds preferable as nutritional supplements and possible therapeutic agents against cancer and car-diovascular diseases [3, 4]. The aim of this study was to investigate the levels of two major anthocyanins, cyanidin and petunidin, in vegetables using an improved liquid chromatographic method.

Anthocyanin content of some commonly consumed vegetables was determined using an ODS-3 reversed phase column (GL Sci-ences, Inertsil ODS-3, 3 μm, 150 x 4.6 mm) with excellent accuracy (recovery more than 99%) and precision (RSD% < 0.9). Both com-pounds were separated with adequate resolution factor which was higher than 3. Cyanidin and petunidin signals were found to be line-ar between the ranges of 80.8 – 404.0 ng/mL and 85.8 – 429.0 ng/mL, giving limit of detection levels of 7.9 and 9.1 ng/mL for cyanidin and petunidin, respectively. No intensive extraction steps were applied in order to keep the methodology as simple as possible; direct pressing was applied to the homogenized samples, and extracted juice was injected to the system after filtration.

Figure 1. A typical chromatogram of turnip sample (CY: Cyanidin, PT: Petunidin, IS: Internal standard)

Developed method was validated according to the recommenda-tions of International Conference of Harmonization. As a result, it can be concluded that both anthocyanins exist at high levels espe-cially in red cabbage and turnip, while there is no evidence was ob-served in tomato and red pepper. Further investigations are advised to construct a general profile of anthocyanins in vegetables.

1. Bravo L. Polyphenols: chemistry, dietary sources, metabolism, and nutri-tional significance, Nutr. Rev., 56, 317-333, 1998.

2. Kong JM, Chia LS, Goh NK, Chia TF, Brouillard R. Analysis and biologi-cal activities of anthocyanins, Phytochemistry, 64, 923-933, 2003.

3. Kowalczyk E, Krzesinski P, Kura M, Szmigiel B, Blaszczyk J. Anthocy-anins in medicine, Pol. J. Pharmacol., 55, 699-702, 2003.

4. Dell’Agli M, Busciala A, Bosisio E. Vascular effects of wine polyphenols, Cardiovasc. Res., 63, 593-602, 2004.

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P 73

MOLECULAR MODELLING STUDIES OF

BENZO[α]CARBAZOLE DERIVATIVES AS ESTROGEN

RECEPTOR INHIBITORS

Tuğba TAŞKIN, Fatma SEVİNHacettepe University, Faculty of Science, Department of Cemistry, Ankara, Turkey

There have been some researches about a number of 5,6-dihydro-11-alkylbenzo[α]carbazoles derivatives with one or two hydroxy groups in the aromatic rings for their binding affinities for the estro-gen receptor [1, 2]. According to analysis, the best conditions for the receptor binding were provided by one hydroxy group at C-3 and a second one at position 8 or 9. These results prompted us to elucidate and visualize this experimental researches by using computational and molecular docking methods.

Our conformatinal analysis consist of first generating conformers by using Gaussian 03 software and then optimizing with AM1, PM3, DFT-B3LYP/6-31G* methods. QSAR [3] is used to determine which molecular descriptors preferences that can be rapidly computed and describe the structure, size, topology and other properties of a mol-ecule. The most statistically significant descriptors like substituent hydrophobicity constant (π), electrophilicity index (ω) and heat of formation energy (HF) are selected using stepwise multiregression analysis (SMLR) to validate biochemical activity. According to the results of analysis, the most fitting equation is determined to describe the relationship between Log1/C and three independent variables for each method. In addition, predicted biological activities have been compared with observed activities.

Furthermore, to visualize the interactions with 5,6-dihydro-11-alkylbenzo[α]carbazole derivatives-binding sites on selected a-chain of estrogen receptor, ICM Pro.3.4. was performed. Results are used to compare physical and chemical properties in determining the conformational preference. Then the most stable conformers, 2 and 3 compounds were brought onto the ER and than the system are fully optimized allowing both systems interactions by using the potential virtual screening of this method. The results are discussed in terms of the nature of the these compounds, ligands recognition on the estrogen receptor.

1. Angerer E, Prekajac J. Journal of Medicinal Chemistry, 29, 3, 1986. 2. Katzenellenbogen JA, Katzenellenbogen BS. Chem. Biol., 3, 529, 1996. 3. Pahsa FA, Srıvastava HK, Singh PP. International Journal of Quantum

Chemistry, 104, 87–100, 2005.

P 74

RAPID AND SENSITIVE VOLTAMMETRIC DETERMINATION

OF ASCORBIC ACID IN TABLET DOSAGE FORMS AND

SOME FRUIT JUICES

1Selehattin YILMAZ, 1Gökçe AŞKIN, 1Sultan YAĞMUR, 1Gülen TÜRKER, 1Gülşen SAĞLIKOĞLU, 1Hüseyin ERDUĞAN1Çanakkale Onsekiz Mart University, Faculty of Arts and Sciences, Department of Chemistry, 17020 Çanakkale, Turkey2Çanakkale Onsekiz Mart University, Faculty of Arts and Sciences, Department of Biology, 17020 Çanakkale, Turkey

Ascorbic acid, a water-soluble vitamin, is important in forming collagen, a protein that gives structure to bones, muscles, and blood vessels. Also it helps to take of ferrum into body. Ascorbic acid is one of the most ubiquitous vitamins ever discovered. Besides play-ing a paramount role as an antioxidant and free radical scavenger, it has been suggested to be an effective antiviral agent. Ascorbic acid is usually administered orally. Because of the notable loss of the in-fused ascorbic acid in the urine, daily doses of 200 mg are needed to

maintain normal concentrations of about 60 M in plasma It is the most common electroactive biological compound [1-4]. So, the elec-trochemical oxidation of ascorbic acid has been carried out at glassy carbon electrode in various aqueous solution in the pH range of 0.64-10.15 (Britton-Robinson, acetat, phosphate buffers and 0.5 M sulfu-ric acide solution) by cyclic (CV) and differential pulse voltammetry (DPV). The best results were obtained for the quantitative determi-nation of ascorbic acid by DPV technique in 0.2 M acetate buffer ( pH 3.49) at 0.342 V. The diffusion controlled nature of the peak was established. This electroanalytical procedure enabled to determine ascorbic acid in the concentration range 8x10-6-8x10-4 M. The regres-sion equation was obtained as Ip (μA) = 5.29 x10-3 C (M) + 0.035 (r = 0.998). Limit of detection (LOD) and limit of quantitation (LOQ) were obtained as 5.16x10-7 M and 1.72x10-6 M, respectively. Based on this study, simple, rapid, selective, and sensitive DPV technique was developed for the quantitative determination of ascorbic acid in pharmaceutical dosage forms and some fruit juices. The proposed voltammetric technique was validated and good recoveries were ob-tained in tablet dosage forms and some fruit juices.

1. Erdurak-Kılıç CS, Uslu B, Doğan B, Özgen U, Özkan SA, Coşkun M. Anodic voltammetric behaviour of ascorbic acid and its selective deter-mination in pharmaceutical dosage forms and some rosa species of Tur-key, J. Analytical Chemistry, 61, 1113-1120, 2006.

2. Sweetman SC. in Martindale: The Complete Drug Reference, 33rd ed., London: Pharmaceutical, 2002, p. 1389.

3. Hardman JG, Limbird LE. Goodmann and Gilmann’s the pharmacologi-cal basis of therapeutics, 9th ed. [CD-ROM], New York: McGraw-Hill, 1996.

4. Pamuk F. Biochemistry (in Turkish), 1st ed. Ankara, Gazi press, 2000, p.138-139.

P 75

ONE-POT SYNTHESIS AND STRUCTURAL ELUCIDATION

OF SOME NEW BIGINELLI COMPOUNDS

İnci Selin DOĞAN, Selma SARAÇHacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Ankara, Turkey

3,4-Dihydropyrimidin-2(1H)-ones(thiones) have been reported to possess diverse pharmacological properties, such as antibacte-rial, antiviral, antitumor, antiinflammatory, antihypertensive, cal-cium channel blocker, α-1a-antagonist and neuropeptide Y (NPY) antagonist effects. The biological activity of some alkaloids isolated recently has also been attributed to the dihydropyrimidine moiety [1, 2]. Therefore, synthesis of these compounds is still of great in-terest. Earlier, we have described the synthesis and calcium antago-nistic activity of some 5-acetyl-3,4-dihydro-6-methyl-4-(substituted phenyl)-2(1H)-pyrimidinone derivatives [3]. In continuation of our study, we aimed to report the synthesis of some new 5-acetyl–6-me-thyl-4-(substituted phenyl)-3,4-dihydropyrimidin-2(1H)-thiones which are expected to have calcium channel blocker and antibacte-rial activities.

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The title compounds were prepared by using the Biginelli three-component cyclocondensation reaction of acetylacetone, substituted benzaldehyde and thiourea under strongly acidic conditions [4]. The reaction was carried out simply by stirring the mixture of the three components dissolved in absolute ethanol with a catalytic amount of hydrochloric acid at room temperature. The solid products were isolated after cooling the reaction mixture.

The reactions were completed within 15-20 hours in 30%-40% yields. The structures of the compounds were confirmed by IR, 1H-NMR, 13C-NMR, mass spectra and elemental analysis. The calcium channel blocker and antibacterial activities of the com-pounds are in progress.

1. Kappe CO. Biologically active dihydropyrimidones of the Biginelli-type – A literature survey, Eur. J. Med. Chem., 35, 1043-1052, 2000.

2. Zorkun İS, Saraç S, Çelebi S, Erol K. Synthesis of 4-aryl-3,4-dihydropyri-midine-2(1H)-thione derivatives as potential calcium channel blockers, Bioorg. Med.Chem., 14, 8582-8589, 2006.

3. Yarım M, Saraç S, Ertan M, Batu ÖS, Erol K. Synthesis, structural elu-cidation and pharmacological properties of some 5-acetyl-3,4-dihydro-6-methyl-4-(substituted phenyl)-2(1H)-pyrimidinones, Il Farmaco, 54, 359-363, 1999.

4. Biginelli P.Aldehyde-urea derivatives of aceto- and oxaloacetic acids. Gazz. Chim. Ital., 23, 360-416, 1893.

P 76

AN EXPERIMENTAL DESIGN APPROACH TO SELECTING

THE OPTIMUM LIQUID CHROMATOGRAPHIC

CONDITIONS FOR THE DETERMINATION OF LOCAL

ANESTHETICS

1Aysun DİNÇEL, 2Nursabah E. BAŞÇI1Hacettepe University, Faculty of Medicine, Department of Pharmacology, Ankara, Turkey2Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, Ankara, Turkey

A sensitive high performance liquid chromatographic (HPLC) method has been developed and validated for the simultaneous determination of four local anesthetics: lidocaine, proparacaine, bupivacine and oxybuprocaine. A full factorial design was used. The chromatographic separation was achieved using a Bondesil C8 (4.6 x 2.5 mm i.d., particle size 5 μm) analytical column. An optimised mobile phase consisted of acetonitrile and sodium dihydrogen phos-phate (pH=3.0, 20 mM) (30:70, v/v) at a flow rate of 1.2 ml min-1. Local anesthetics detection was performed by UV/Vis detector at 220 nm. The retention times for lidocaine, proparacaine, bupivacine and oxybuprocaine 5.74, 9.28, 16.84 and 26.26 min, respectively. This method was linear in the range of 50-5000 ng ml-1 for lidocaine and proparacaine and 100-5000 ng ml-1 for bupivacine and oxybu-procaine. The limit of detection (LOD) was 25 ng ml-1 for lidocaine, proparacaine and 30 ng ml-1 for bupivacine and oxybuprocaine. The limit of quantification (LOQ) was found to be 50 ng ml-1 for lido-caine, proparacaine and 100 ng ml-1 for bupivacine, oxybuprocaine (RSD ≤ 15 %, n=6).

Keywords: Local anesthetics, validation, experimental design and meth-od optimization

P 77

STUDIES ON NEW OXIME ESTER DERIVATIVES OF

NAFIMIDONE AS POTENTIAL ANTICONVULSANT

COMPOUNDS

1Burcu SELİMOĞLU, 2Arzu KARAKURT, 1Sevim DALKARA1Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Ankara, Turkey2İnönü University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 44280 Malatya, Turkey

Nafimidone [1-(2-naphthyl)-2-(imidazol-1-yl)ethanone] is one of the two representatives of (arylalkyl)imidazole anticonvulsants [1] and nafimidone alcohol is a major and active metabolite of nafimidone. Nafimidone oxime ethers also have anticonvulsant activities [2]. In this project we prepared ten new nafimidone oxime ester derivatives. These compounds (I-X) have been synthesized by esterification of nafimidone alcohol with benzoyl and substituted benzoyl chlorides.

Total synthesis scheme starting from 2-acetylnaphthalene is given below:

Their structural elucidation were realized by IR, 1H-NMR, Mass spectral data and elemental analysis.

1. Walker KAM, Wallach BM, Hirschfeld RD. 1-(Naphthylalkyl)-1H-imi-dazole Derivatives, a New Class of Anticonvulsant Agents. J. Med. Chem., 24, 67-74, 1981.

2. Karakurt A, Dalkara S, Özalp M, Özbey S, Kendi E, Stables JP. Synthesis of Some 1-(2-naphthyl)-2-(imidazole-1-yl)ethanone Oxime and Oxime Ether Derivatives and Their Anticonvulsant and Antimicrobial Activi-ties, Eur. J. Med. Chem., 36, 421-433, 2001.

Acknowledgement This project was supported by Hacettepe University Scientific Research

Fund, Project no: 06 D03 301 001.

P 78

SEPARATION OF SEVEN QUINOLONES USING HPLC:

DETERMINATION OF LEVOFLOXACIN IN PLASMA AND

AMNIOTIC FLUID

1Emirhan NEMUTLU, 1Sedef KIR, 2Sinan BEKSAÇ1Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, 06100 Ankara, Turkey2Hacettepe Unıversity, Faculty of Medicine, Department of Obstetrics and Gynecology, 06100 Ankara, Turkey

A reversed-phase high-performance liquid chromatographic method is described for the separation and determination of seven quinolones (ciprofloxacin, levofloxacin, oxolinic acid, lomefloxacin moxifloxacin, enoxacin, and pefloxacin) in plasma and amniotic fluid. Chromatographic separation of the quinolones was performed on a Zorbax Eclipse XDB-C18 column (150 mm x 4.6 mm i.d.) in connec-tion to a Phenomenex C18 column (4 mm x 3.0 mm i.d.). The op-timum assay conditions were found with experimental designs. Op-timum conditions were 15 mM citrate buffer, pH 3.2, 9 % MeCN, 5

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% MeOH, 5 mM TMAB, 1.5 mL min-1 flow rate and 40 °C column temperature. Photodiode array detector was set to 280 nm. Marbo-floxacin (MAR) was used as internal standard. The retention times of the quinolones in optimum conditions were 6.0 min for MAR, 6.7 min for enoxacin, 7.8 min for levofloxacin, 8.5 min for pefloxacin, 9.3 min for ciprofloxacin, 11.2 min for lomefloxacin, 16.3 min for moxi-floxacin and 17.3 min for oxolonic acid. A simple and efficient solid phase extraction was applied for sample preparation of plasma and amniotic fluid. The validation studies of the method for the analysis of levofloxacin was performed according to FDA guidelines and the linearity range was found as from LOQ to 30 μg mL-1. The LOD and LOQ were 0.010 and 0.035 μg mL-1, respectively. The absolute recover-ies from plasma and amniotic fluid were 98.0 and 95.9, respectively. In intra-day and inter-day studies, the developed method was found to be accurate and precise with a bias value less than 2.6 % and the RSD value less than 5 %. The robustness studies were performed with an experimental design. All validation data were showed that the method was accurate, precise, linear, robust, and specific. Finally, the method has been applied to quantification of levofloxacin in amniotic fluid and plasma for investigation fetal transformation of levofloxacin.

P 79

INVESTIGATION FOR NATURAL QUORUM SENSING

INHIBITORS: ALTERNATIVE TO ANTIBIOTICS?

1Gülgün BOŞGELMEZ-TINAZ, 1Seyhan ULUSOY, 1Fatma Filiz COŞKUN-ARI, 2Aysel UĞUR, 2Özgür CEYLAN1Süleyman Demirel University, Faculty of Arts and Sciences, Department of Biology, Isparta, Turkey2Muğla University, Faculty of Arts and Sciences, Department of Biology, 480000 Muğla, Turkey

As the bacterial resistance to multiple antibiotics increases, it is becoming more difficult to treat infections and, in many cases, the available therapeutic options are severely limited. Hence, there is a growing urgency to search for novel targets and the development of new antimicrobials. Millions of dollars are devoted to antibiotics that almost immediately start to become less therapeutically useful when they finally reach the marketplace. For this reason, pharma-ceutical companies are now focusing on finding novel bacterial tar-gets and new ways to control and eradicate bacterial infections [1]. To infect a host and cause a disease, bacteria produce an array of viru-lence determinants that contribute to pathogenesis. It is now known that many different Gram-negative pathogens communicate via production of small, diffusible N-acyl homoserine lactone (AHL) derivatives as signaling molecules to coordinate virulence determinant production. This event is called “quorum sensing (QS)”. A variety of physiologi-cal process in a range of bacterial species is regulated by QS, including antibiotic biosynthesis, biofilm differentiation and the production of virulence determinants in animal and plant pathogens [2]. Since many pathogens use QS to regulate virulence, strategies intended to interfere with signaling systems will likely have many potential applications. Biotechnological research is now focused on the development of AHL antagonists. In medicine, usage of such molecules represents a novel therapeutic approach offering the opportunity to attenuate virulence, and thus infection, by blocking cell-to-cell communication [3]. This approach is highly attractive because it does not impose harsh selective pressure for the development of resistance as with antibiotics [1].

Various plant samples were screened for anti-QS activities using bioassays with Chromobacterium violaceum (ATCC 12472) and Erwinia carotovora. Plant materials were dried and extracted using chloroform, methanol and water.

In this study, we tested 46 terrestrial plants materials for their ability to inhibit QS-regulated behaviors in different bacterial spe-

cies. The chloroform-soluble compounds extracted from Scor-zonera sandrasica were found to inhibit violacein production, a QS-regulated behavior in C. violaceum which is used as a model system for QS inhibition studies. This extract was also able to in-hibit QS-regulated carbapenem antibiotic production in an im-portant plant pathogen E. carotovora. In addition, some of these plant materials such as Origanum onites L and Nigella sativa have strong anti-bacterial effect against some bacterial species. As the regulation of many bacterial processes is controlled by QS systems, the finding of natural compounds acting as QS inhibitors suggests an attractive tool to control and handle detrimental infec-tions caused by human, animal and plant pathogens.

1. Hentzer M, Giskov M. Pharmacological inhibition of quorum sensing for the treatment of chronical bacterial infections, J. Clin. Invest., 112, 1300-1307, 2003.

2. Fuqua C, Parsek MR, Greenberg EP. Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing, Annu. Rev. Genet., 35, 439-468, 2001.

3. Finch RG, Pritchard DI, Bycroft BW, Stewart GSAB. Quorum sensing-a novel target for anti-infective therapy, J. Antimicrob. Chemother., 42:569-571, 1998.

P 80

DEVELOPMENT OF A CAPILLARY ZONE

ELECTROPHORESIS METHOD FOR THE SIMULTANEOUS

ANALYSIS OF OLMESARTAN MEDOXOMIL AND

HYDROCHLOROTHIAZIDE

Mustafa ÇELEBİER, Sacide ALTINÖZHacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, Ankara, Turkey

Olmesartan medoxomil is a selective AT1 subtype angiotensin II receptor antagonist [1]. Hydrochlorothiazide is a thiazide diuretic that is commonly used in combination with other antihypertensive agents. It has been reported that the combination of olmesartan medoxomil and hydrochlorothiazide is a safe, well tolerated, and ef-fective option for antihypertensive therapy, demonstrating greater blood pressure reduction than monotherapy [2]. In the literature, there hasn’t been any method described for the simultaneous analy-sis of olmesartan medoxomil and hydrochlorothiazide. A capillary zone electrophoresis method with ultraviolet detection for the simul-taneous analysis of olmesartan medoxomil and hydrochlorothiazide in synthetic tablets was developed. A fused silica capillary (50 μm internal diameter, 48.5 cm total length, 40 cm effective length) was used and the separation was obtained by 40 mM pH 9.5 borate buffer followed by detection with an ultraviolet detector at 210 nm. The analysis were performed at 30 °C with the application of 3 seconds hydrodynamic injection at 50 mbar pressure and a applied potential of 30 kV. Diflunisal was used as internal standard. The developed method was validated according to the literature [3].

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1. Mire DE, Silfani TN, Pugsley MK. A review of the structural and func-tional features of olmesartan medoxomil, an angiotensin receptor block-er, Journal of Cardiovascular Pharmacology, 46, 585-593, 2005.

2. Steven GC, Michael AW, Antonia CW, Donald JH. Evaluation of antihy-pertensive therapy with the combination of olmesartan medoxomil and hydrochlorothiazide, AJH, 17, 252–259, 2004.

3. ICH Steering Committee. Text and Methodology Q2 (R1). Harmanized Tripartite Guideline, 2005.

P 81

CYP1A2, CYP2A6, NAT2 AND XANTHINE OXIDASE

ACTIVITIES IN TURKISH VOLUNTEERS

1Aysun DİNÇEL, 2Nursabah E. BAŞÇI, 1Atila BOZKURT1Hacettepe University, Faculty of Medicine, Department of Pharmacology, 06100 Ankara, Turkey2Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, 06100 Ankara, Turkey

A RP-HPLC method has been developed for the determination of caffeine metabolites in urine for the evaluation of the CYP1A2, CYP2A6, xanthine oxidase (XO) and N-acetyl-transferase-2 (NAT-2) activities in 101 Turkish volunteers (47 males and 54 females). Spot urine samples were analyzed 5 h after 100 mg caffeine con-sumption. The urinary caffeine metabolites, 1,7-dimethylxanthine (17X), 1,7-dimethyluric acid (17U), 1,3-dimethyluric acid (13U), 3-methylxanthine (3X), 1-methylxanthine (1X), 1-methyluric acid (1U), theobromine (37X), and 5-acethylamino-6-formylamino-3-methyluracil (AFMU) were analysed. CYP1A2, CYP2A6, XO and NAT-2 activities were estimated from the metabolic ratios (AFMU + 1U + 1X)/17U, 17U/17X, 1U/(1X + 1U) and AFMU/(AFMU + 1U + 1X), respectively. Urine samples were extracted with chloro-form/isopropanol (85:15, v/v) and separated on a reversed phase C18 (4.6 x 2.5 mm i.d., particle size 5 μm) analytical column with acetic acid/tetrahydrofuran/acetonitrile/water (1:2.5:44:925, v/v/v/v). Peaks were monitored with UV detection at 280 nm. The en-zyme activities of CYP1A2, CYP2A6, NAT2 and XO were found as 5.28±6.12, 0.22±0.11, 0.33±0.17, and 0.65±0.16 (mean±SD), respec-tively. Smoking and gender were not affected CYP1A2, and CYP2A6 activities. The developed RP-HPLC method was validated and suc-cessfully applied for the evaluation of CYP1A2, CYP2A6, XO and NAT-2 activities. These results are comparable to those reported for Caucasian populations previously.

Keywords: CYP1A2, CYP2A6, XO, NAT-2, HPLC, caffeine

P 82

DETERMINATION OF PAEONIFLORIN AND

OXYPAEONIFLORIN IN PAEONIA SPECIES USING

VALIDATED HPLC METHOD

1Semra KOYUNOĞLU, 2Sedef KIR, 3Ali A. DÖNMEZ, 4İhsan ÇALIŞ1Hacettepe University, Faculty of Pharmacy, Department of Basic Pharmaceutical Sciences, 06100 Sıhhiye-Ankara, Turkey2Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, 06100 Sıhhiye-Ankara, Turkey3Hacettepe University, Faculty of Science, Department of Biology, 06100 Sıhhiye-Ankara, Turkey4Hacettepe University, Faculty of Pharmacy, Department of Pharmacognosy, 06100 Sıhhiye-Ankara, Turkey

Paeonia L., the largest genus in the family Paeoniacea, is repre-sented by 7 species in the flora of Turkey. Paeonia Radix is one of the most important herbal drugs in traditional Chinese medicine, and as well as in different countries. Significant chemical and pharmaco-

logical studies have been conducted on the roots of Paeonia species. Monoterpen glycosides are the active constituents for this plant spe-cies. Among the terpen glycosides, paeoniflorin (1) and oxypaeoni-florin (2) are the main compounds.

In the present study, reversed-phase high performance liquid chromatographic (HPLC) method with diode array detection for the determination of the 1 and 2 has been developed. Compounds 1 and 2 were isolated from Paeonia species using chromatographic meth-ods. The structure elucidations of 1 and 2 were achieved by combina-tion of 1D and 2D-NMR experiments and mass spectrometry. The HPLC separation was achieved on a Nucleosil 100-5 C18 (5 μm, 250 x 4.6 mm) column using a mobile phase composed of acetonitrile:10 mM pH 3.5 phosphate buffer (20:80 v/v) at a flow rate of 1 mL min-1 . A wavelength of 230 nm for diode array detection was selected. The internal standard was metronidazol.

The retention times were 4.1 min for 1 and 7.8 min for 2. The developed method was optimized by changing the chromatographic parameters such as organic solvent ratio and pH of mobile phase. Optimal conditions were selected by calculating capacity and tail-ing factors of each peaks and by identifying the resolution for the separation. Low retention time, symmetric peak, high peak area and high resolution were performed by optimized method. The validated method was found to be linear, accurate, precise, sensitive, rugged and robust. The linear ranges were 0.25-80 μg/ml for 1 and 0.25-60 μg/ml for 2. The developed method was found to be useful for de-termination of compounds 1 and 2 in Paeonia species. By the help this method, 3.47 %- 5.46 % paeoniflorin (1), and 0.049 % -0.422 % oxypaeoniflorin (2) were found from 2 g of the crude samples of P. mascula, P. daurica, P. peregrina, P. tenuifolia, P. mascula subsp. bod-urii collected from the flora of Turkey.

P 83

STABILITY OF LIQUID AND SOLID FORMS OF

RECOMBINANT HUMAN INTERFERON

Bilgen ÖZBATIR, Filiz ÖNERHacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Biotechnology, 06100 Ankara, Turkey

Due to the patient compliance problems of injectable formulations, alternative routes for peptide-protein drugs are under investigation in recent years. Mucosal route seems advantageous for administra-tion of peptide drugs due to the direct systemic effect and avoidance of first hepatic elimination [1]. The aim of this study was to develop a stable lyophilized solid lozenge form for mocosal application of rHuIFN-α2b which has a molecular mass of 19.269 kDa and has a significant therapeutic potential in treatment of some cancers and infectious diseases.

Studies on the therapeutic effects of low dose oral mucosal for-mulations of interferon present promising results in some references [2]. In this study we prepared a solid lozenge form and liquid form of rHuIFN-α2b (recombinant human interferon alfa 2b) and exa-mined the stability of the active substance in vitro by comparing with the commercial product. Composition of the formulated forms of interferon alpha is shown in Table I.

Experiments were performed in an artificial saliva medium (pH 6.7) in order to simulate the medium in the oral cavity. Liquid and solid forms were kept at 4oC and 25oC for 1 and 4 months and the amount of active substance was determined quantitatively by HPLC and qualitatively by SDS-PAGE. No degradation product was found and rHuIFN-α2b was kept stable for 1 month at both 4oC and 25oC temperatures.

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Table I. Composition of the liquid and solid interferon formulations used in the study

Components

Liquid formulation Solid formulation

rHuIFN-α2b rHuIFN-α2b

Sodium phosphate dibasic anhydrous Gelatin

Sodium phosphate monobasic monohydrate Glycerol

Disodium EDTA Sucrose

Polysorbate 80 (Tween 80) Water for injection

Phenol

Sodium chloride

Water for injection.

1. Shojaei AH. Buccal mucosa as a route for systemic drug delivery: A re-view, J. Pharm. Pharmaceut. Sci.,1(1), 15-30, 1998.

2. Ship JA., Fox PC, Michalek JE, Cummins MJ, Richards AB. Treatment of pimary Sjögren’s syndrome with low-dose natural human interferon- α administered by the oral mucosal route: a phase II clinical trial, Journal of Interferon and Cytokine Research, 19, 943-951, 1999.

P 84

HPLC-ECD DETERMINATION OF EPINEPHRINE PLASMA

CONCENTRATIONS IN VARIOUS DENTAL PATIENTS

1Şeyda DEMİRCAN, 2Ayşe Gül AKALIN, 1Filiz SAYIN, 2Gökçe MERAL, 2Ferda TAŞAR, 1Sedef KIR, 1Nursabah E. BAŞÇI1Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, 06100, Sıhhiye-Ankara, Turkey2Hacettepe University, Faculty of Dentistry, Department of Oral Surgery, 06100-Sıhhiye-Ankara, Turkey

A sensitive and reliable reversed-phase high performance liquid chromatographic (RP-HPLC) method with electrochemical detector (ECD) has been developed for the determination of local anesthetic agent added epinephrine in plasma obtained during dental surgery. For this aim, the changes of plasma levels of epinephrine have been measured in the blood samples taken at five different times (0, 3, 15, 30 and 60 minutes) after administration of the local anesthetic. The method was based on the use of a Nucleosil 100-5 C18 (5 μm, 250 x 4,6 mm i.d.) as analytical column with a mobile phase containing methanol: 0.1 mol L-1 citrate buffer (10:90, v/v, pH 2.5) with a flow rate of 1.2 mL min-1. Electrochemical detector was set to 800 mV. Retention times of epinephrine and isoprotrenole (internal standard, IS) were 8.0 and 19.0 min, respectively. The plasma assay was vali-dated for the parameters such as specificity, accuracy and extraction recovery and was applied to three different patient groups. The re-covery of epinephrine after extraction from spiked plasma was 97 ± 0.03 % (mean±SD). The method was linear over the range of 15-200 ng mL-1 (r2 = 0.9992). The detection limit (LOD) was found to be 5 ng mL-1 (signal-to-noise ratio = 3) and the quantitation limit (LOQ) was 15 ng mL-1 for epinephrine. The described HPLC-ECD method is simple, selective and can successfully be applied with high degrees of precision and accuracy for the quantitative determination of epinephrine in plasma samples. The pharmacokinetic profiles for epinephrine in the healthy young, healthy older and ASA II CVS di-seased older dental patients after the administration of local anesthe-sia with epinephrine were obtained.

Keywords: Epinephrine; HPLC; Electrochemical Detector (ECD); Vali-dation; Plasma; Dental Patients

P 85

ANTIMICROBIAL ACTIVITY OF 3-HYDROXY-6-METHYL-2-

SUBSTITUTED 4H-PYRAN-4-ONE DERIVATIVES

1Ekrem KILIÇ, 2Mutlu DİLSİZ-AYTEMİR1Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, 06100 Sıhhiye-Ankara, Turkey2Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Sıhhiye-Ankara, Turkey

Over the past several years the emergence of organisms resistant to nearly all the class of antimicrobial agents has become a serious public health concern. For these reasons the research of new antimi-crobial agents with novel modes of action represents a main target in chemotherapy. It has been shown that hydroxypyran-4H-one deriva-tives have antimicrobial activity [1, 2]. In our previous studies, we synthesized some Mannich bases of 3-hydroxy-6-methyl-4H-pyran-4-one derivatives and investigated their antimicrobial activity [3].

Antifungal activities of the compounds evaluated in vitro against fungi such as Candida albicans (ATCC 90028), C. krusei (ATCC 6258) and C. parapsilosis (ATCC 90018). They were also tested against Gram-positive bacteria such as Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212) and Gram-negative bac-teria such as Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) by using microdilution broth method recommended by National Committee for Clinical Laboratory Standards (NCCLS)

[4, 5]. The minimum inhibitory concentrations (MIC) were defined as the lowest concentrations of the compounds that prevented visible growth. Fluconazole and Ampicillin were used as the standards for antifungal and antibacterial tests, respectively. The screening data indicates that compound 5 (MIC: 16 μg/ml), which was carrying (4-chlorophenyl)phenylmethyl substituent showed antibacterial activity against S. aureus. The other compounds had no valuable inhibitory activity. Compound 5 (MIC: 16 μg/ml) was also the most effective compound towards C. albicans than the others (MIC: 32-64 μg/ml).

1. Aytemir MD, Erol DD, Hider RC, Özalp M. Synthesis and evaluation of antimicrobial activity of new 3-hydroxy-6-methyl-4-oxo-4H-pyran-2-carboxamide derivatives, Turk J. Chem., 27(6), 757-764, 2003.

2. Kayahara H, Shibata N, Tadasa K, Kotani T. Amino acid and peptide de-rivatives of kojic acid and their antifungal properties Agric. Biol. Chem., 54(9), 2441-2442, 1990.

3. Aytemir MD, Çalış Ü, Özalp M. Synthesis and evaluation of anticonvul-sant and antimicrobial activities of 3-Hydroxy-6-methyl-2-substituted 4H-pyran-4-one derivatives. Arch. Pharm. Pharm. Med. Chem. 337, 281-288, 2004.

4. National Committee for Clinical Laboratory Standards (NCCLS), Meth-ods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, Approved Standard M7-A, 37(2), Villanova, PA. (1997).

5. National Committee for Clinical Laboratory Standards (NCCLS), Refer-ence method for broth dilution susceptibility testing of yeast, Approved Standard M27-A, 17(9) Villanova, PA. (1997).

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May 17-20, 2007

International Symposium on Drug Research and Development

P 86

INHIBITION OF RAT LIVER MONOAMINE OXIDASE (MAO)-

A AND –B BY GENTIANELLA CAUCASEA (Loddiges ex Sms)

HOLUB EXTRACTS

1Tayfun ERSÖZ, 1Zeliha Ş. AKDEMİR, 2Samiye YABANOĞLU, 1, 3Funda Nuray YALÇIN, 1Duygu KAYA, 4İ. İrem ÇANKAYA, 2Gülberk UÇAR1Hacettepe University, Faculty of Pharmacy, Department of Pharmacognosy, 06100 Ankara, Turkey2Hacettepe University, Faculty of Pharmacy, Department of Biochemistry, 06100 Ankara, Turkey3Hacettepe University, Faculty of Pharmacy, Department of Pharmacy Management, 06100 Ankara, Turkey4Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Botany, 06100 Ankara, Turkey

Monoamine oxidase (MAO), which is found in two forms desig-nated as MAO-A and -B, plays an essential role in the metabolism of the biogenic amines [1]. MAO-B inhibitors are shown to be useful in the treatment of Parkinson’s and Alzheimer’s diseases [2] while MAO-A inhibitors are known as antidepressant and antianxiety agents [3]. Since severe side-effects have been observed with some classical MAO inhibitors [4], new inhibitors devoid of these adverse effects are needed. The presence of plant-derived MAO inhibitors suggests that such plant extracts might be useful as potential neu-roprotectans in the treatment or prevention of depression, psychosis, schizophrenia, Alzheimer’s and Parkinson’s diseases [5].

Gentianella caucasea is a biennial stem to 30 cm grown in North-ern and Northeastern Anatolia [6]. Preliminary works on Gen-tianella caucasea showed the presence of secoiridoid, flavonoid and xanthone compounds in the chemical composition. Xanthones are known to possess MAO inhibitor activity [7].The present study was designed to investigate the MAO inhibitory activities of the metha-nol, petrolum ether, chloroform and the remaining water extracts prepared from the aerial parts of Gentianella caucasea.

MAO was purified from the rat liver and MAO-A and –B activities were determined according to a previous method [8]. Kinetic data for interaction of the enzyme with the abstracts were determined us-ing Microsoft Excel package program. IC50 values were determined from plots of inhibitory activity percentage, calculated in relation to a sample of the enzyme treated under the same conditions wit-hout extracts, versus extract concentration. All Gentianella extracts tested inhibited rat liver MAO isoforms in a dose-and time-depen-dent manner. Incubation of the enzyme with the extracts at 37 0C for 60 min. increased the inhibitory action of the extracts. Metha-nol extract inhibited MAO-B isoform selectively with IC50 value of 38.2±2.26 μg dry weight/mL whereas chloroform, petrolum ether, and water extracts inhibited MAO-A selectively with IC50 values of 58.7±4.31, 64.33±5.06 and 102.13±7.55 μg dry weight/mL, respec-tively. Xanthones of Gentianella caucasea may be a possible source of pharmaceuticals for the treatment and prevention of depression, Parkinson’s and Alzheimer’s diseases. Phytochemical study on the title plant is carrying on.

1. Loscher W, Lehman H, Teschendorf H, Traut M, Gross, G. Inhibition of monoamine oxidase type A, but not type B, is an effective means of in-ducing anticonvulsant activity in the kindling model of epilepsy. J Phar-macol Exp Ther 288: 984-992, 1999.

2. Uçar G, Gökhan N, Yeşilada A, Bilgin A. 1-N-substituted thiocarbamoyl-3-phenyl-5-thienyl-2-pyrazolines: A novel cholinesterase and selective monoamine oxidase-B inhibitors for the treatment of Parkinson’s and Alzheimer’s diseases. Neurosci Lett 382: 327-331, 2005.

3. Ertuğrul A, Uçar G, Başar K, Demir B, Yabanoglu S, Uluğ B. Influence of clozapine on platelet serotonin, monoamine oxidase and plasma seroto-nin levels. Pscyhiatr Res 149: 49-57, 2007.

4. Power MB, Hackett LP, Dusci LJ, Ilett KF. Antidepressant toxicity and the need for identification and concentration monitoring in overdose. Clin Pharmacokin 29: 154-171, 1995.

5. Haraguchi H, Tanaka Y, Kabbashi A, Fujioka T, Ishizu T, Yagi A. Mono-amine oxidase inhibitors from Gentiana lutea. Phytochemistry 65: 2255-2260, 2004.

6. Pritchard NM, Gentianella “in Flora of Turkey and the East Aegean Is-lands “(Ed. Davis PH) University Press, Vol 6 Edinburg, 1978.

7. Suzuki O, Katsumata Y, Oya M, Chari VM, Vermes B, Wagner H, Hos-tettmann K. Inhibition of type A and B monoamine oxidases by naturally occuring xanthones Planta Med 42: 17-21, 1981.

8. Holt A, Sharman DF, Baker GB, Pelcic MM. Continuous spectrophoto-metric assay for monoamine oxidase and related enzymes in tissue ho-mogenates. Anal Biochem 244: 384-392, 1997.

P 87

NEW MOLECULARLY IMPRINTED MICROSPHERES FOR

COLONIC DELIVERY OF 5-AMINOSALICYLIC ACID

Yeliz TUNÇ, Ersin BAYKARA, Kezban ULUBAYRAMHacettepe University, Faculty Pharmacy, Department of Basic Pharmaceutical Sciences, Ankara, Turkey

Molecular imprinting has become an increasingly active field of study for the construction of new highly stable molecularly imprin-ted polymers (MIPs) that possess selective molecular recognition properties [1]. MIPs have a large number of potential applications such as seperation process, immunoassays and antibody mimics, biosensor recognition elements, and catalysis and artifical enzymes [2]. Recently, researchers have applied the molecular recognition properties of imprinted polymers for enhanced control in the release of pharmaceutical compounds [3].

In this study, feasibility of molecularly imprinted polymeric mi-crospheres (MIPs) has been investigated for colonic delivery of 5-aminosalicylic acid (5-ASA), an active agent used in treatment of ulcerative colitis and Crohn’s disease. For the first time, 5-ASA im-printed microspheres were prepared by a single step precipitation polymerization of 2-(diethylamino) ethyl methacrylate (DEAEMA; functional monomer) and trimethylolpropane trimethacrylate (TRIM; crosslinker). The microspheres were prepared in the diame-ter range between 1.2 and 2.7 by varying polymerization conditions. The release characteristics of 5-ASA imprinted and non-imprinted microspheres were determined and molecular imprinting effect on release behaviors was evaluated. We present a precipitation po-lymerization method for preparing uniform molecularly imprinted microspheres in micron range, quickly and cleanly. Monodisperse polymer particles with good spherical shapes and smooth surfaces were obtained. Furthermore, the results show that the imprinted mi-crospheres have a slower 5-ASA release in the initial stages than the non-imprinted microspheres, because of the interaction of the drug molecules with the recognition sites in the imprinted microspheres. This result indicates that molecular imprinting may have a potential for controlled delivery of drugs.

1. Tunç Y, Hasırcı N, Yeşilada A, Ulubayram K. Comonomer effects on binding performances and morphology of acrylate-based imprinted polymers, Polymer, 47, 6931-6940, 2006.

2. Zhang H, Ye L, Mosbach K. Non-covalent molecular imprinting with emphasis on its application in separation and drug development, J. Mol. Recognit., 19, 248-259, 2006.

3. Pepppas, N.A, Intelligent therapeutics: Biomimetic systems and nanote-chnology in drug delivery, Adv. Drug Del. Rev., 56, 1529-1531, 2004.

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P 88

BIOLOGICALLY ACTIVE COMPOSITE SCAFFOLD FOR

TISSUE ENGINEERING APPLICATIONS

1Sinan GÜVEN, 2Nesrin HASIRCI, 3Sevda MÜFTÜOĞLU, 1Kezban ULUBAYRAM1Hacettepe University, Faculty of Pharmacy, Department of Basic Pharmaceutical Sciences, Ankara, Turkey2Middle East Technical University, Faculty of Arts and Sciences, Department of Chemistry, Ankara, Turkey3Hacettepe University, Faculty of Medicine, Department of Histology and Embrology, Ankara, Turkey

Tissue engineering plays a vital role in regenerative medicine in order to create new tissues and organs from cultured cells for trans-plantation [1]. In scaffold oriented tissue engineering, the scaffolds should mimic the structure and biological function of native extra-cellular matrix which provide mechanical support and regulate cell activities [2].

In the present study biologically active composite scaffolds were developed from natural polymers by tissue engineering approach and tested in vitro. Freeze-dried scaffolds composed of chitosan, gelatin and dermatan sulfate were treated with different stirring rates, freez-ing temperatures and molding. Among the prepared scaffolds at dif-ferent conditions, scaffolds (SC) prepared at 500 rpm and frozen at -80°C were chosen for further studies. These scaffolds achieved 0.512 MPa tensile strength, 9.165 MPa tension modulus and 3.428 MPa compression modulus. Besides in lysozyme containing degradation medium they conserved their integrity and lost about 30% of their initial weight in 30 days period. Mechanical and enzymatic degrada-tion tests showed that scaffolds have physical integrity for the tissue engineering applications. To mimic the natural tissue and enhance cell growth, biologically active arginine-glycine-aspartic acid-serine (RGDS) peptides and platelet derived growth factor-BB (PDGF-BB) were immobilized on selected scaffolds. Fibroblast cells were seeded on the scaffolds containing RGDS, (SC-RGD), and PDGF-BB, (SC-RGD-GF), and incubated in media either free of serum or contain-ing serum. Scaffolds immobilized with RGDS and PDGF-BB had the highest attached cell number by the day 15. According to scan-ning electron microscopy (SEM) results, cells on modified scaffolds demonstrated better cell morphology and attachment. Based on the obtained results, it can be concluded that RGDS-PDGF immobilized chitosan-gelatin-dermatan sulfate systems have a great potential to be used as a scaffold for soft tissue engineering applications.

1. Langer R, Vacanti JP, Tissue Engineering, Science, 260, 920-926, 1993. 2. Marler JJ, Upton J, Langer R, Vacanti JP. Transplantation of cells in matri-

ces for tissue regeneration, Advanced Drug Delivery Reviews, 33, 65–182, 1998.

P 89

EFFECT OF CORIANDER (Coriander sativum L) AND

PARSLEY (Petroseleinum sativum Hoffm.) EXTRACTS ON

PLATELET AGGREGATION AND BLOOD COAGULATION

Muhammad Jamal SHAMMOUTUniversity of Jordan, Analytical Toxicology, Amman, Jordan

Certain herbs may cause pharmacological effects on some car-diovascular aspects such as platelet aggregation and blood coagula-tion during homeostasis processes. In this study, the volatile oils and crude extracts of Coriander (Coriander sativum L) and Parsley (Pet-roseleinum sativum Hoffm.) were isolated using steam distillation and heating under reflux techniques. The effects of herb’s prepara-

tions on platelets aggregation were investigated in vitro using adeno-sine-5’-diphosphate (ADP) and arachidonic acid (AA) as agonists.

The volatile oil of coriander seeds and the ethanolic extracts of leaves of coriander and parsley exhibited inhibitory effects on plate-let aggregation induced by ADP and AA in dose response manner.

The effects of aspirin and dimethylsulfoxide (DMSO) on platelet aggregation induced by ADP and AA were also studied. It was found that Aspirin exhibited a strong inhibitory effect on platelet aggrega-tion induced by ADP or AA, while DMSO exhibited an inhibitory effect on platelets.

A combination of aspirin with coriander and parsley extracts was studied in terms of its effect on platelet aggregation. İt was found that the combination of aspirin and coriander seed’s volatile oil extract were approximately additive effect on platelet aggregation.

The effects of coriander and parsley extracts on blood coagulation were also investigated. The effects of plant preparations were inves-tigated by determination of their effects on activated partial thromo-plastine time (APTT). None of tested extracts affected prothrombine time (PT) values.

As a result, the coriander’s seeds volatile oil and parsley leaves extracts are of the interest for open new approaches towards expla-nation of the beneficial effects on several manifestations of cardio-vascular diseases like hypertension, arteriosclerosis and ischemic diseases and it could be assessed as useful natural source of pharma-ceutical agents.


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