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The focus of work presented here was to study and describe the following: (1) The effect of proline series molecules (L-Proline, N‐acetyl‐L‐proline, L‐prolinamide, N‐acetyl‐L‐prolinamide) on the stability of piperacillin and tazobactam considering experimental results and molecular modelling data. This is a very interesting family of molecules, since all of them are based on L- proline, yet significantly different when it comes to their overall charges and protonation states. L‐Proline is neutral, but zwitterionic, N‐acetyl‐ L‐proline is anionic, Lprolinamide is cationic, and N‐acetyl‐L‐prolinamide is neutral without any charged fragments. (2) Utilization of deep eutectic solvent systems (DES) for stabilization of piperacillin and tazobactam, where one of the components is N-acetyl-L-prolinamide. Furthermore, preparation and stability testing of formulations in the form of a deep eutectic solvent that comprise piperacillin and/or tazobactam and at least one N-acetyl amino acid amide was conducted. Some of the evaluated excipients were N-acetyl-D- alaninamide, and as mentioned above, N-acetyl-L- prolinamide. It was discovered that the stability of the formulation is more enhanced in the specific DES systems. More precisely, DES compositions provided improved stability over time (evaluated through specific impurities increase and purity drop) when stored refrigerated (2-8 °C), and at room temperature (25 °C) in comparison to liquid (aqueous) formulations that only contain piperacillin and/or tazobactam. Computational analysis utilized a combination of quantumchemical calculations and molecular dynamics simulations to characterize relevant interactions in solutions in relation with the studied degradation mechanisms, and to help in interpreting the observed experimental trends.

Blackberry wine, a popular fruit wine, is a product of yeast fermentation of natural sugars present in blackberry juice, well known natural source of vitamins. It was shown that blackberry variety, technology of manufactoring and duration of wine storage have influence on amounts of vitamins present in wine. Today, HPLC methods are the most often used for the determination of water-soluble vitamins for evaluation and quality control of wines. Major advantage of these chromatographic methods is their ability to separate different forms of vitamins from very complicated matrix, such as blackberry wine. The contents of ascorbic acid, thiamine, riboflavin, nicotinic acid, pyridoxine and folic acid were determined in fifteen samples of blackberry wine from different regions of Croatia. All samples were filtered through Minisart RC4, 0.45 µ m filters (Sartorius), which did not retain any of the analytes. The samples were injected after filtration or after dilution with 1 M phosphate buffer, pH 5.5. The chromatographic system consisted of a Dionex HPLC Pump P680, Automated Sample Injector ASI-100, Termostatted Column Compartment TCC-100 and UVD340U PDA Detector. Vitamins were separated on a Zorbax SB-C18 (250 x 4.6 mm, 5 µ m ; Agilent Technologies) with suitable guard column. The following solvents were used: solvent A (50 mM sodium phosphate buffer (pH 2.5):MeOH, 90:10, v/v) and solvent B (50 mM sodium phosphate buffer (pH 2.5):MeOH, 10:90, v/v). The mobile phase was filtered and degassed ultrasonically prior to use. The gradient was linearly changed from 0 to 70% B in 22 min, with a post-run time of 3 min (100% B) and a constant flow rate of 1 mL min-1. The column temperature was maintained at 20 °C, the injection volume was 10 µ ; l and the detector was set at 223 nm for folic acid and riboflavin, 245 nm for thiamine and ascorbic acid, 268 nm for nicotinic acid and 292 nm for pyridoxine. Vitamin standards were used to generate characteristic UV-VIS spectra and calibration curves. The analyses were performed in triplicate. Optimized HPLC method with UV detection was validated through linearity, sensitivity, selectivity and repeatability, and it was shown that this method can be used for routine analysis of six vitamins in blackberry wine.

The aim of our study was to investigate the effect of 35 structurally diverse flavonoids on production of reactive oxygen species (ROS) using appropriate cell culture model.

Propolis is one of the richest sources of plant phenolics (flavonoids and phenolic acids), which are widely recognized as rather strong antioxidants. The aim of our work was to use colored stable free radical (DPPH· and ABTS· +) spectrophotometric and thin-layer chromatographic (TLC) assays to study the antioxidative behavior of the phenolics (caffeic acid, galangin and pinocembrin) most commonly present in Croatian propolis samples obtained from different Croatian regions. We propose a mathematical model providing a more sophisticated interpretation of the obtained results and a new parameter named antioxidative efficiency (AOE) is introduced. The kinetic behaviour of chosen standards determined by spectrophotometric assays follows the exponential decrease of the absorption curve. Explained numerically, AOE represents the absolute value of the first derivative of an absorbance curve in the point A0/e (where A0 is the absorbance measured at t = 0 and e is the natural logarithm base). The advantage of this newly introduced parameter is that it provides an easy and accurate mutual comparison between the rates of antioxidative efficiency of different propolis samples. A TLC assay was only applicable in the case of the DPPH· radical. Dose-response curves were described using a linear function with AOE expressed as a coefficient of the slope. The chromatographic method was shown to be very rapid, reliable and easy-to-perform.

Three spectrophotometric methods for the quantitative determination of different flavonoid groups and total phenolics in Croatian propolis samples were optimized and validated. The assay based on the formation of aluminium chloride complex (with galangin as a standard) was applied for the quantification of flavones and flavonols, while 2, 4-dinitrophenylhidrazine method (with pinocembrine as a reference) was used for the quantification of flavanones. Total phenolics content was measured by the Folin-Ciocalteau method using reference solution of caffeic acid : galangin : pinocembrine (1:1:1). Through analytical validation, the most suitable extraction conditions (regarding the time, temperature, and concentration of extraction solvent) were determined, and final conditions for the extraction were established (80% ethanol, 1 hour at the room temperature). Appropriate ratio between the mass of raw propolis and extraction solvent volume was established as well. Applying developed method of extraction, ten propolis tinctures were prepared and subjected to the analysis of general pharmacopoeial parameters which are fundamental for the creation of quality specification (relative density, dry residue of extract, content of ethanol, methanol and 2-propanol). Additionally, we determined the content of waxes as the main inactive constituents in order to observe the level of their migration from crude propolis to the prepared tinctures.

Zbog sve veće popularnosti istočnjačke tradicionalne medicine, razvoja farmakognozije i potrage za novim ljekovitim supstancijama različite su se skupine biljnih spojeva, posebice polifenolnih molekula, našle u središtu znanstvenih istraživanja. Flavonodi i fenolne kiseline čine neizostavan dio svakodnevne ljudske prehrane, a pokazuju i niz farmakoloških djelovanja. Zajedno s još dvjestotinjak drugih supstancija nalaze se i u propolisu, čiji sastav ovisi o zemljopisnom porijeklu. U ovom radu koristeći in vitro stanične modele (stanice Jurkat i diferencirane stanice THP– 1) ispitano je i međusobno uspoređeno imunomodulacijsko djelovanje 35 strukturno različitih flavonoida, 9 fenolnih kiselina i 12 uzoraka propolisa, primijenjenih u tri različite koncentracije (standardi kao 30, 10 i 3 μ M i ekstrakti kao 30, 10 i 3 μ g/mL standardizirai na suhu tvar). Nakon analize sastava ekstrakata propolisa metodom tekućinske kromatografije visoke djelotvornosti uspoređeno je njihovo djelovanje s djelovanjem standardnih supstancija. Imunomodulacija je praćena kroz promjenu funkcije makrofaga (stvaranje reaktivnih kisikovih vrsta – RKV i indukcija/inhibicija antioksidacijskih enzima superoksid dismutaze – SOD i glutation peroksidaze) te kroz promjene produkcije citokina i kemokina od strane makrofaga i limfocita T. Kvantitativnim istraživanje odnosa strukture i djelovanja ispitani su odnosi elektronskih svojstava pojedinih flavonoida i njihove imunomodulacijske aktivnosti te je na taj način definiran dio strukture odgovoran za djelovanje. Utjecaj na produkciju RKV diklorflurescin metodom. Aktivnost antioksidacijskih enzima određena je spektrofotometrijski, koristeći komercijalno dostupne smjese reagencija. Produkcija citokina i kemokina određena je metodom sandwich ELISA. Hibridna metoda koju tvore genetski algoritmi spregnuti sa strojevima potpornih vektora i metoda slučajne šume korištene su za razvoj QSAR modela, dok je pristup izbaci– jedan– van korišten za internu i eksternu validaciju modela. Flavonoidi, fenolne kiseline i ekstrakti propolisa, ovisno o primijenjenoj koncentraciji, većinom suprimiraju produkciju interleukina (IL)– 2 i proliferaciju Jurkat stanica. Stvaranje proupalnih citokina faktora nekroze tumora (TNF)– α i IL– 1β potaknuto je najvišim koncentracijama flavonoida, a suprimirano najnižim. Produkciju IL– 8 induciraju samo flavoni akacetin i diosmetin i flavonol ramnetin u najvišoj koncentraciji te ekstrakti propolisa u najnižoj koncentraciji. Stvaranje reaktivnih kisikovih vrsta u stanicama izloženim oksidacijskom stresu značajno se smanjuje (za > 50%) zbog djelovanja nekih flavonoida (flavona: krizina, apigenina, luteolina i diosmetina, flavonola: fisetina, kempferola, kvercetina, ramnetina i izoramnetina te kavene kiseline), dok prooksidacijski djeluju samo dva izoflavonoida – formononetin i genistein. Ispitane standardne supstancije i ekstrakti nemaju učinak na aktivnost glutation peroksidaze, dok jue aktivnost SOD moguće stimulirati flavonoidima (posebice flavonima). Atomi u prstenima C i B i na njih vezani supstituenti određuju stabilnost O1– C2 veze te orijentaciju prstena B u prostoru i strukturne su odrednice stimulativnog učinka flavonoida na produkciju TNF– α i IL– 1β . Imunomodulacijski učinci uzoraka propolisa razlikuju se od učinaka standardnih supstancija (flavonoida i fenolnih kiselina) čija je prisutnost u ekstraktima kvalitativno i kvantitativno utvrđena. Različitost u djelovanju samih standardnih supstancija te prisutnost velikog broja nepoznatih komponenti u uzorcima propolisa onemogućavaju predviđanje biološke aktivnosti tako složene smjese.

Flavonoids are a group of phytochemicals ubiquitous in photosynthesizing organisms that display a remarkable array of biochemical and pharmacological activities. As human immune system can be modified by diet, pharmacologic agents, environmental pollutants, it is assumed that certain members of this group of compounds significantly affect the function of the immune system. Using appropriate cell culture model (human macrophage-like cells - differentiated THP-1 cells), the effects of 31 flavonoid representative on the production of selected cytokines that play crucial roles in innate and adaptive immune responses was determined (TNFα , IL-1β ) [1]. TNFα and IL-1β production was measured using ELISA 24 hrs after the addition of chosen flavonoids in 30 μ M concentration. E. coli lipopolysacharide was used as a positive control (TNFα and IL-1β production was considered as maximal, 100%). Un-stimulated cells did not produce cytokines and were used as a negative control. Some of tested flavonoids exhibited immunomodulatory effects and quantitative structure-activity relationships could be established. 26 Mulliken charges and sums there of corresponding to selected substituents were calculated based on B3LYP DFT single point approach. PM3 optimized geometries were used for these calculations. Random forests (RF) and genetic algorithms coupled with support vector machines (GA/SVM) were used for the development of quantitative relationships between percentages of activation of TNFα and IL-1β production and selected partial atomic charges. RF parameters were kept at their default values while GA/SVM parameters were fine-tuned by internal leave-one-out validation loop. Models obtained this way were characterized by average R2 greater than 0.65 for calculated and experimental values while corresponding CV was less than 5%. These values were calculated based on external leave-one-out validation approach repeated ten times for different random seeds. Descriptors highly ranked by both, RF and GA/SVM approaches were extracted and their influence, as well as the influence of different substituents on the TNFα and IL-1β production was evaluated.

The aim of this work was to determine log P values of 30 polyphenols (22 flavonoids and 8 phenolic acids) by shake-flask method. Concentration was determined by HPLC method. Correlations between experimentally obtained log P values and ADME parameters predicted by different computer programs were established.

Analize sadržaja aktivne komponente, onečišćenja u sirovini ili gotovom obliku i druge metode instrumentalne kemijske analize lijekova, za koje ne postoje ili se ne primjenjuju standardizirani analitički postupci, nužno podliježu zahtjevu za ispitivanjem prikladnosti analitičkih svojstava metode za danu primjenu. Nasuprot tome, postupci analize određenih svojstava lijekova koji se provode prema standardiziranim farmakopejskim protokolima poput mikrobioloških analiza, bioloških pokusa i slično, rjeđe se podvrgavaju takvom postupku kojeg nazivamo validacijom metode. Validacija metoda analize lijekova prema europskoj, japanskoj i američkoj farmakopeji sastavni je dio procesa registracije ljekovitih oblika ili farmakološki aktivnih tvari. Osnovni je element sustava osiguranja kvalitete i nužan preduvjet distribucije i terapijske primjene lijeka u Hrvatskoj i inozemstvu.

Propolis (bee glue) is the generic name for the resinous substance collected by honeybees (Apis mellifera, L.) from various plant sources. History of using propolis in folk medicine dates back to the ancient Egyptians, Greeks, and Romans. Nowadays, many in vitro studies confirm its antibacterial, antifungal, antiviral, hepatoprotective, chemopreventive, anti-inflammatory, and antioxidative activities. Its chemical composition is very complex and varies with geographic origin. Although propolis is considered to be a complex mixture of around 200 compounds, all listed activities and potential toxic effects are assumed to originate from various plant phenolics. Quantitative Structure-Activity Relationship (QSAR) and Quantitative Structure-Property Relationship (QSPR) modelling are used increasingly in rational design of bioactive molecules. The most frequently employed approach to obtain QSAR/QSPR models is to perform regression analysis using molecular descriptors. Propolis extracts are currently used for the treatment of oronasal infection. In our work, we analysed 38 propolis extracts from different Croatian regions using liquid chromatography, and investigated their ability to inhibit the growth of Staphylococcus aureus and Streptococcus pyogenes. These activities were correlated with their total phenolic and flavonoid content. In our previous study, we postulated two QSPR models based on topological indices, describing molecular weight and van der Waals volume for group of flavonoids and phenolic acids usually present in Croatian propolis. Topological indices used for molecular modelling are Wiener index and valence connectivity index. The aim of our further study was to check the validity of postulated models. We investigated linear relationship between molecular weight and van der Waals volume data and values obtained using QSPR models. Statistical analysis was performed using STATISTICA v7.0. In our study, we used propolis extracts prepared in 80% ethanol. The analysis of composition was performed using liquid chromatiography, while susceptibility of the gram-positive bacteria to the propolis samples was determined using the disc diffusion method (results are expressed as zone of inhibition – Z.I.). Linear regression analysis was found suitable to describe the predictive power of postulated QSPR models. From 38 samples, we chose 12 which expressed the best antimicrobial activities and correlated the content of phenolic acids and flavonoids with the obtained zones of inhibition. Only in the case of inhibition of Staphylococcus aureus growth, we could observe satisfactory correlation between the content of flavonoids and antimicrobial activity of tested propolis samples. In all other cases, we could not observe any significant relationship. According to our findings, for the antimicrobial activity of propolis samples (considering the growth inhibition of Staphylococcus aureus), more important is the content of flavonoids than phenolic acids.

Propolis (bee glue) is the generic name for the resinous substance collected by honeybees (Apis mellifera, L.) from various plant sources. Beneficial effects of propolis on human health have been recognized thousands years ago and nowadays many in vitro studies confirm its numerous biological activities. In our in vitro study, we investigated immunomodulatory effects of 10 propolis extracts from different Croatian regions (Vis, Pelješac, Omiš, Gorski Kotar, Veliki Zdenci, Metković, Kutina, Sinj, Beli Manastir, Hum na Sutli) using appropriate cell culture models, and measuring the production of cytokines (TNFα , IL-1β , IL-2) and chemokine (IL-8) that play crucial roles in innate and adaptive immune responses. THP-1 cells were differentiated into macrophage-like cells, and induction of cytokine and chemokine production was measured using sandwich ELISA 24 hrs after the addition of propolis extracts (concentrations of 3, 10, 30 μ g/mL). Inhibition was measured in the same manner using LPS-stimulated cells (lipopolysacharide from E. coli was also used as a positive control). Jurkat cells were stimulated to produce IL-2, and inhibition of cytokine production was determined 24 hrs after the addition of propolis extracts. Cell proliferation was determined by measuring the incorporation of [6-H3] thymidine, while cell viability was determined using bioluminiscent cytotoxicity assay (measuring the release of adenylate kinase from damaged cells). None of the used propolis extracts exhibited cytotoxic effects, but significantly (>50%) suppressed the proliferation of both tested cell lines. Regarding the TNFα production, the propolis extracts from Vis, Pelješac, Omiš and Metković provoked the induction higher than 30%, while none of the used extracts induced the IL-1β production. Intrestingly, all propolis extracts induced IL-8 production. None of the tested extracts exerted the inhibitory effects on TNFα , IL-1β and IL-8 production in LPS-stimulated cells. As all propolis extracts suppressed the proliferation of Jurkat cells, consequently, also suppressed the IL-2 production.

Propolis je smolasti produkt pčelinje zajednice koji se već stotinama godina koristi kao gotovo univerzalni lijek u narodnoj medicini. Različita farmakološka djelovanja (poput antimikrobnog, imunomodulatornog, protuupalnog, citoprotektivnog) pripisuju se propolisu, odnosno njegovim najvažnijim farmakološki aktivnim sastavnicama – polifenolima: flavonidima i fenolnim kiselinama. U novije vrijeme antioksidacijski učinak propolisa zaokuplja sve veću pažnju. Kemijski sastav propolisa ovisi o biljnom pokrovu područja s kojeg dolazi, a time je i njegova biološka aktivnost različita. Cilj ovog rada bio je optimirati uvjete spektrofotometrijske analize antioksidacijske učinkovitosti hrvatskih propolisa koristeći radikal 2, 2'-azino-bis(3-etilbenztiazolin-6-sulfonske kiseline) (ABTS• +), te postaviti odgovarajući matematički model koji omogućuje preciznu usporedbu uzoraka propolisa različitog zemljopisnog porijekla. Matematički model gašenja slobodnog radikala ABTS• + u ovisnosti o koncentraciji antioksidansa, konstruiran je koristeći otopinu standarda kavene kiseline, pinocembrina i galangina. Rezultat je iskazan veličinom antioksidacijska učinkovitost (nagib tangente na krivulju). Nakon optimiranja metode, analizirali smo antioksidacijsku učinkovitost osam sirovih uzoraka propolisa iz različitih dijelova Republike Hrvatske (područje Metkovića, Čisle, Visa, Lonjskog polja, Pelješca, Južnodalmatinskih otoka, Kutine i Gorskog Kotara). Usporedbom antioksidacijske učinkovitosti zaključeno je kako propolisi s područja otoka i priobalja sadrže u svom sastavu najviše biljnih antioksidansa.

The number of research papers, reviews and commercially available software packages relating to the prediction of absorption, distribution, metabolism and elimination (ADME) of pharmaceutical compounds has increased dramatically in recent years. Molecular lipophilicity is a major physico-chemical property, which has impact on oral absorption, as well as permeability, cell uptake, protein binding, blood-brain penetration and metabolism of bioactive substances. The ability to predict drug absorption through the gastro-intestinal barrier is a key issue in the selection of new drug candidates for oral delivery. Passive diffusion is the major mechanism for drug uptake through the intestinal epithelium, driven by a concentration gradient. Lipophilic compounds cross the plasma membrane easily and therefore are mainly transported transcellularly. Cell membrane is relatively impermeable for hydrophilic compounds, and they are transported predominantly via the paracellular route. Besides, excessive lipophilicity is common cause of poor solubility of substances and can lead to incomplete absorption after oral administration. Futhermore, it is generally belived that very lipophilic compounds have greater affinity for plasma-protein binding and are easily transported across blood-brain barrier. Widespread applications of lipophilicity to model biological processes explains the need for rapid and valid procedures to quantify that physico-chemical property. The aim of this work was to investigate the chromatographic behavior of polyphenols (flavonoids and phenolic acids) by reversed-phase thin-layer chromatography (RP-TLC), in order to establish the relationship between chromatographic indices and selected ADME parameters. RP-TLC is rapid, easy to perform technique, which requires small quantities of the sample and allows simultaneous analysis of several compounds. Moreover, RP-TLC allows an easy measurement of a variety of indices (RMw and φ 0), which provide information on the lipophilic behavior of compounds. Chromatography was performed on 10 x 20 cm glass TLC plates precoated with RP-18F254s stationary phase (Merck, Germany). Binary solvent system, methanol – phosphate buffer, was used as a mobile phase. pH of the mobile phase was at least 2 log units lower than pKa values of all investigated compounds (in order to avoid pKa correction, phosphate buffer pH = 2.5 with ionic strength 36 mM was used). Content of organic modifier was varied from 30 – 80% (v/v) in 5% increment. 5 μ L of standard solutions were applied as 8 mm bands by means of 100 μ L microsyringe (Hamilton, USA), mounted on a Linomat 5 applicator (Camag, Switzerland). Ascending development was performed at the room temperature in the saturated, glass chamber (Desaga, Germany). Plates were developed to the distance of 80 mm and after development were dried at the room temperature. Detection was performed at λ = 254 nm using TLC Scanner 3 (Camag, Switzerland). Data acquisition and processing were performed with Wincats software v1.4 (Camag, Switzerland). ADME parameters of selected polyphenols were calculated using several computer programs that cover the main currently used theoretical approaches: CLOGP, KowWin, molinspiration, MolSoft, XLOGP, OSIRIS, DRAGON, ALOGPS v2.1, ChemSilico, Interactive Analysis, VEGA ZZ and SPARC v3. Correlations between RMw and φ 0 values, obtained by RP-TLC, and calculated ADME parameters were analyzed. The solubility parameters calculated with the ALOGPS (r = 0.934), ChemSilico (r = 0.936), Osiris (r = 0.964) and MolSoft (r = 0.965) programs correlate very well with RMw values of the investigated polyphenols. In general, aqueous solubility decreases with increasing RMw values. According to correlation between RMw values and plasma-protein binding data predicted by ChemSilico program, RMw is a useful parameter for evaluation of polyphenols´ plasma-protein binding properties (r = 0.937). Correlations between RMw and calculated log P values are surprisingly poor. The best correlation is found for OSIRIS program (r = 0.890). Comparing the calculated log P values with φ 0 values, obtained by chromatographic analysis, better correlations are found for molinspiration (r = 0.925) and ChemSilico (r = 0.907). Apparently, the φ 0 parameter is more suitable for determination of the polyphenols´ lipophilicity than RMw. Moderate correlations are found between chromatographic indices (RMw and φ 0) and other predicted ADME parameters (polar surface area, blood-brain barrier penetration and human intestinal absorption).

Fenolne kiseline su, uz flavonoide, jedna od najpoznatijih i najviše istraživanih skupina biljnih polifenola. Njima je pripisan niz farmakoloških djelovanja – antimikrobno, antitumorsko i antioksidativno. Sastavni su dio niza biljnih preparata (npr. propolisa) te se u sklopu istih u vrijeme prehlade i gripe često koriste u kombinaciji s vitaminima i nesteroidnim protuupalnim lijekovima. U ovom radu korištena je metoda tankoslojne kromatografije (TLC) za ispitivanje kemijskih interakcija između fenolnih kiselina (kavena, ferulična, o-hidroksikumarinska, m-hidroksikumarinska, p-hidroksikumarinska, cimetna kiselina) i nesteroidnih protuupalnih lijekova (acetilsalicilna kiselina, paracetamol, ibuprofen, ketoprofen, diklofenak) te vitamina C i E. Za ispitivanje interakcija korištene su TLC silikagel 60 ploče 20 cm x 10 cm s fluorescentnim indikatorom (Merck) koje su nakon nanošenja uzoraka razvijane u staklenim kromatografskim komorama prethodno zasićenim pokretnom fazom kloroform:metanol:mravlja kiselina, 44:3, 5:2, 5 v/v. Sve standardne otopine pripremljene su u 96 % etanolu (Kemika), osim acetilsalicilne kiseline (vodeni standard). Ovisno o prethodno određenim RF vrijednostima ispitivanih supstancija, izabrano je mjesto nanošenja pojedine fenolne kiseline i lijeka na ploču. Nakon kromatografiranja i sprejanja s 1 % etanolnom otopinom AlCl3 sve supstancije su identificirane pod kratkovalnim (254 nm) i dugovalnim (366 nm) UV svjetlom. Izrazita vizualizacija nastalih kemijskih interakcija postiže se nanošenjem fenolnih kiselina u obliku linije, a otopina ispitivanih lijekova i vitamina u obliku točke (za nanošenje uzoraka korištena je Hamilton igla od 10 μ l). U slučaju pojave kemijske interakcije, linija ispitivane fenolne kiseline se deformira pod utjecajem određenog lijeka. Metoda tankoslojne kromatografije prikladna je metoda za utvrđivanje ovakve vrste interakcija, čime se djelomično može predvidjeti i ponašanje ispitivanih supstancija u ljudskom organizmu.

U posljednje vrijeme antioksidativna aktivnost biljnih preparata zaokuplja sve više pažnje. Nizom in vitro pokusa dokazana je antioksidativna učinkovitost različitih polifenola biljnog porijekla i predloženi su njihovi mehanizmi djelovanja. Posebna pažnja u istraživanjima posvećena je skupini flavonoida i fenolnih kiselina. Propolis (smolasti produkt pčelinje zajednice) jedan je od najkoncentriranijih izvora te skupine biljnih antioksidansa. Sastav propolisa (a time i intenzitet antioksidativnog učinka) ovisi o području njegovog sakupljanja, tj. o sastavu biljnog pokrova pojedine regije. U našem radu koristili smo spektrofotometrijsku metodu s DPPH˙ radikalom kako bismo usporedili antioksidativnu aktivnost devet propolisa iz različitih regija Republike Hrvatske. DPPH˙ je stabilan radikal koji daje ljubičasto obojenje otopinama. Ukoliko je u otopini prisutan antioksidans, primjećuje se pad apsorbancije pri 518 nm. Etanolni ekstrakti sirovih uzoraka propolisa razrijeđeni su u odgovarajućim omjerima i takva razrjeđenja korištena su za ispitivanje antioksidativne učinkovitosti. Razrjeđeni ekstrakti i 0, 3 mM etanolna otopina DPPH˙ pomiješani su u optimalnom omjeru te je mjeren pad apsorbancije pri 518 nm tijekom 2 minute. Na temelju baždarne krivulje dobivene reakcijom odabranih flavonoida različitih koncentracija i otopine DPPH˙, procijenjena je koncentracija antioksidansa u pojedinom uzorku. Za svaki uzorak izračunata je i jednadžba krivulje ovisnosti pada apsorbancije o vremenu. Analizom antioksidativnog kapaciteta ekstrakata propolisa različitog zemljopisnog porijekla te usporedbom brzine reakcije s DPPH˙ radikalom, primjetili smo kako uzorci priobalnog i otočnog područja Republike Hrvatske u svom sastavu posjeduju najviše biljnih antioksidansa.

The increasing use of propolis preparations now days requires adequate analytical procedures for quantitative determination of main active components (different polyphenols, including flavonoids and phenolic acids). Rapid spectrophotometric methods have been used for years and are useful methods for routine control of propolis samples. The aim of this work was to improve and validate two spectrophotometric methods for determination of different groups of flavonoids in propolis. Flavones and flavonols form stable complexes with AlCl3, while flavanones and flavanonols react with 2, 4-dinitrophenylhidrazine (2, 4-DNP). Both given methods were optimized using a mixture of flavonoids. Galangin was later used to make the calibration curve for the method with AlCl3 (absorbance was measured at 415 nm), while pinocembrine was used as a standard for 2, 4-dinitrophenylhidrazine method (λ = 495 nm). After the conditions were optimized, we have investigated the following validation characteristics: linearity, accuracy, repeatability, intermediate precision, stability, and selectivity. To prove that improved and validated methods are suitable for intended purpose, we have analyzed nine ethanolic propolis extracts from different regions of Croatia to establish the differences in their composition. Investigated validation parameters for both methods satisfied the acceptance criteria for natural drug preparations (correlation coefficient ł 0.999 ; recovery – AlCl3 method 99.17 – 101.25 % and 2, 4-DNP method 98.67 – 101.46% ; repeatability (RSD(n = 6)) 1.61% and 4.77% ; intermediate precision (RSD(n = 18)) 3.21% and 4.03%). Tested colorimetric methods showed to be appropriate for establishing the differences in the composition of propolis samples (e.g. composition of propolis sample from peninsula Pelješac: mass concentration g(flavones, flavonols) = 320.95 mg/ml / RSD = 3.84%, g(flavanones, flavanonols) = 546.49 mg/ml / RSD = 3.28% ; composition of propolis sample from central Croatia: g(flavones, flavonols) = 54.87 mg/ml / RSD = 1.93%, g(flavanones, flavanonols) = 254.06 mg/ml / RSD = 3.24%).

Dijetarnim polifenolima (posebice flavonoidima i fenolnim kiselinama) pripisuje se širok spektar bioloških učinaka. Studije kojima su dokazana njihova brojna djelovanja uglavnom zanemaruju pitanje njihove bioraspoloživosti te stvarne koncentracije u plazmi. In vitro i in vivo istraživanja bioraspoloživosti potencijalnih lijekova su dugotrajna i zahtjevna, stoga je korištenje računalnih programa za predviđanje bioraspoloživosti u ranim fazama razvoja novih lijekova vrlo značajno. Bioraspoloživost lijekova odraz je prvenstveno fizikalno-kemijskih parametara uključenih u procese apsorpcije i eliminacije. Lipofilnost je svojstvo koje utječe na gotovo sve navedene procese te se može koristiti kao parametar za predviđanje bioraspoloživosti ovih spojeva. Primjenom najnovijih računalnih programa i baza podataka proučeni su neki od najvažnijih parametara koji određuju bioraspoloživost polifenola: lipofilnost (log P i log D), HIA (engl. human intestinal absorption), PSA (engl. polar surface area), prolazak kroz krvno-moždanu barijeru (BBB, engl. blood-brain barier) i vezanje za proteine plazme (PB, engl. protein binding). Za predviđanje lipofilnosti korišteno je 15 računalnih programa koji se zasnivaju na različitim teorijskim pristupima. Korištenjem programa ChemSilico i ProLogP, također je ispitan i lipofilni profil polifenola (promjena lipofilnosti ovisno o promjeni pH medija, tj. log D). Promjena lipofilnosti ovisno o temperaturi (23 i 37°C) ispitana je korištenjem programa SPARC. Dobiveni rezultati obrađeni su primjenom statističkog programa StatSoft v.6.0. Interkorelacija log P vrijednosti dobivenih različitim programima pokazala je značajnu korelaciju cLogP i miLogP programa. Također značajna korelacija zapažena je između CSLogP, odnosno cLogP programa i vezanja za proteine plazme. S porastom temperature uočen je linearan pad lipofilnosti.

In this work by using molecular modeling procedures, we investigated the relationships between structure and properties of flavonoids and phenolic acids, usually present in Croatian propolis. Topological indices used for correlation analysis include Wiener index, W, valence connectivity index,  v, Balaban index, J, information-theoretic index, I, and Schultz Index, MTI. All indices were calculated using TAM software. Investigated physicochemical properties were molecular weight (M.w.), van der Waals volume (Vw) and lipophilicity (log P). Van der Waals volume was calculated according to the method described by Moriguchi and log P values were calculated using different computer program. The best QSPR models were obtained using Wiener index and valence connectivity index.

Slobodni radikali su vrlo reaktivni kemijski spojevi s nesparenim elektronom u vanjskoj ljusci. Sudjeluju u razvoju niza bolesti, a jedan od najčešće istraživanih štetnih učinaka je oštećenje biomembrana. Danas je poznato niz antioksidansa koji se upotrebljavaju u prevenciji bolesti, no podaci o učinkovitosti su još uvijek kontradiktorni. Flavonoidi pripadaju porodici biljnih antioksidansa, čija je popularnost porasla unazad posljednjih par desetljeća. Ubikvitarni su u biljnom kraljevstvu i ne mogu se sintetizirati u ljudskom organizmu. Antioksidativni potencijal ove skupine spojeva čini ih zanimljivima za in vivo istraživanja. Jedan od poznatih izvora flavonoida je propolis – produkt pčelinje zajednice kojem se uz antioksidativno, pripisuju i drugi fiziološki učinci. Cilj našeg istraživanja bio je ispitati da li svakodnevno uzimanje propolisa tijekom 30 dana ima utjecaja na lipidnu peroksidaciju u ljudskom organizmu. U studiji je dobrovoljno sudjelovalo 47 ispitanika (muškaraca i žena) u dobi od 20 do 30 godina, podijeljenih u kontrolnu i test skupinu. Odabranim kriterijima uključivanja i isključivanja iz studije oponašala se prosječna populacija. Tijekom studije bilo je zabranjeno uzimanje dijetetskih suplemenata i lijekova, te je ograničeno uzimanje hrane bogate flavonoidima. Analiza lipidnih peroksida provedena je TBARS metodom. Uzorci krvi uzimani su u svakih 14 dana tijekom trajanja studije (ukupno 60 dana). U prvih 30 dana uzorci su uzeti 2 puta kako bi se odredila bazna linija lipidnih peroksida dobrovoljaca i zatim 2 puta tijekom slijedećih 30 dana (period primjene propolisa). Za ispitivanje je korišten preparat propolisa deklariranog sastava EPID BUSTINE (ekstrakt propolisa u prahu, proizvođač Specchiasol, Busolengo – Verona, Italija). Dobrovoljci u test skupini uzimali su 1 dozu propolisa tri puta dnevno (jedna doza sadrži 16, 25 mg flavonoida, što dnevno iznosi 48, 75 mg). Rezultati ANOVA-e združenih skupina pokazuju statistički značajan pad (p = 0, 05) koncentracije lipidnih peroksida nakon četverotjedne primjene preparata propolisa.

Propolis je pčelinji proizvod koji se upotrebljava kao pomoćno sredstvo za liječenje bakterijskih upala gornjih dišnih puteva, tretmana Herpes Labialis-a, za jačanje imuniteta te u obliku krema i masti za liječenje kožnih oboljenja. Danas je niz različitih preparata dostupno u ljekarnama. Kako ti preparati podliježu regulativi o dodacima prehrani, točna deklaracija sastava nije nužna. Povećana upotreba preparata propolisa zahtijeva i više znanja o njihovom sastavu. Metoda tankoslojne kromatografije jednostavna je kromatografska metoda prikladna za brzo kvalitativno ispitivanje sastava kompleksnih smjesa. U našem radu ispitali smo sastav jedanaest komercijalno dostupnih etanolnih ekstrakata. Ispitivane su glavne farmakološki aktivne sastavnice propolisa - flavonoidi i fenolne kiseline. Kao standardi odabrani su flavonidi pinocembrin, galangin, kvercetin, akacetin, apigenin, kemferol, krizin i fenolne kiseline kavena i ferulična kiselina, koji se najčešće javljaju u sastavu sirovih uzoraka propolisa. Određeni volumeni etanolnih otopina standarda (c = 0.2 mg/mL) i ekstrakata propolisa naneseni su u obliku 10 milimetarskih vrpca na HPTLC silikagel 60 ploče s fluorescentnim indikatorom F254s. Svaki uzorak propolisa kromatografiran je sa standardima na sobnoj temperaturi u staklenim komorama prethodno zasićenim pokretnom fazom n-heksan:etil acetat:octena kiselina, 29:5:6 (v/v). Prisutnost navedenih spojeva utvrđena je vizualizacijom kromatograma pod kratkovalnim (λ = 254 nm) i dugovalnim (λ = 366 nm) UV svijetlom nakon prskanja s 1 % etanolnom otopinom aluminij-triklorida. Kromatogrami su dokumentirani CAMAG Reprostar 3 sustavom. Metoda tankoslojne kromatografije pokazala se prikladnom za brzu kvalitativnu analizu sastava komercijalno dostupnih preparata. Kvantifikacija rezultata je slijedeći korak potreban kako bi se uistinu utvrdila vrijednost ekstrakata dostupnih u ljekarnama.

Propolis je proizvod pčelinje zajednice - nastaje miješanjem različitih biljnih smola i salivarnih enzima pčela. U košnici se koristi kao zaštita od atmosferilija i različitih nametnika. Glavnim farmakološki aktivnim sastavnicama propolisa smatraju se polifenoli – flavonoidi i fenolne kiseline. Ti spojevi posjeduju niz djelovanja – antibakterijsko, antiviralno, protuupalno, antioksidativno te mnoga druga. Propolis je u narodnoj medicini prisutan već od 300-te godine prije Krista, a danas njegova povećana upotreba zahtijeva razvoj i validaciju analitičkih metoda kojima je moguće utvrditi njegov sastav. Postoji nekoliko jednostavnih spektrofotometrijskih metoda (metoda s aluminij-trikloridom, 2, 4-dinitrofenilhidrazinom i Folin-Ciocalteu reagensom) kojima je moguće utvrditi postotak farmakološki aktivnih sastavnica u etanolnom ekstraktu propolisa. U našem radu izabrali smo metodu s Folin-Ciocalteu reagensom, kao prikladnu metodu određivanja sadržaja ukupnih polifenola. Optimirani su uvjeti provođenja analize te je sama metoda validirana. Za izradu baždarne krivulje pri određivanju ukupnih polifenola (flavonoida i fenolnih kiselina) korištena je otopina galangina, pinocembrina i kavene kiseline kao standardna otopina. Uzorak je pripremljen za mjerenje prema propisu u postojećoj literaturi te je mjerena apsorbancija pri 760 nm. Postupak je ponovljen s ekstraktom propolisa. Ukupna koncentracija polifenola u ekstraktu izražena je kao masena koncentracija galangina, pinocembrina i kavene kiseline prema izrazu: γ (mg/mL) = γ standard ekvivalent (mg/mL) x faktor razrijeđenja. Validacijom spektrofotometrijske metode s Folin-Ciocalteu reagensom dokazali smo kako je analitička metoda prikladna za predviđenu namjenu, tj. za određivanje ukupnog sadržaja polifenola u etanolnom ekstraktu propolisa.

Molecular lipophilicity was studied using salicylamide as a model drug. Log P value for the target compound was experimentally determined by "shake-flask" method and calculated using nine different computer programs: based on atom contributions, based on atom/fragment contributions, based on 13 structural parameters, based on fragmental contributions, based on atom-type electrotopological-state indices and neural network modeling, and based on topological structure descriptors. Our analysis demostrates perfect agreement between experimentally observed log P value of salicylamide and calculated value by CSLogP program, based on topological structure descriptors and electrotopological indices.

Flavonoids are one of the largest groups of natural compounds known. They are reputated to have numerous physiological activities. There are many food supplies which contain flavonoids, but one of the most important source is propolis. In propolis, main active substances except flavonoids are phenolic acids. Nowdays, the use and list of preparations of propolis became nearly endless, and demands an accurate analytical method to define the substances in this natural product. It is easily analysed by chromatographic methods, but before testing some new type of propolis it is better to optimize chromatographic conditions. The aim of this study is to optimize chromatographic conditions in TLC of flavonoids and phenolic acids as standard compounds that may be present in Croatian propolis. We used 9 different mobile phases and using information theory and numerical taxonomy methods compared by application of computer search program KT1, we found the most appropriate mobile phase, the optimal combination of two and three mobile phases for separation of standards.

Phenolic acids and flavonoids, with various biological activities, are considered to be main compounds in Croatian propolis - a natural product produced by the honeybee whose composition depends on its geographic origin. Flavonoids are a group of plant phytochemicals that cannot be synthesized by humans. They are benzo-γ -pyrone derivatives which resemble coumarine and are divided in six groups (flavanols, flavones, flavonols, flavanons, isoflavons and anthocyanidins) according to the substituents on the main ring. Many studies have been made confirming their antiseptic, antifungal, antibacterial, antiviral, anti-inflamatory and antioxydant properties. The QSPR models are simply approximations of real systems primarily developed and used in scientific work to represent, predict or estimate phenomena of interest. In QSPR approach, molecular structure is described by a number which can be calculated from molecular topology, e. g. topological indices. We investigated correlation between some topological indices (Wiener index, Connectivity index, Balaban index, Shultz index and Shannon index) and properties such as molecular weight, Van der Waals volume, melting point and log P. List and structures of investigated compounds are given in Figure 1. All indices used in the modeling procedure were calculated by the TAM program for personal computer. We have investigated topological indices which are applicable to QSPR studies of phenolic acids' and flavonoids' properties. Log P values, which were not available from literature, were calculated using ALOGPs program (for phenolic acids) and KowWin program (for flavonoids). We have investigated linear, nonlinear and multivariate relationships between topological indices and properties of flavonoids. Different properties were best modeld with different regressions and different indices. The best models of examined physicochemical properties are based on linear regression with the Wiener index (plot of Wiener index vs. molecular weight is given in Figure 2), Connectivity index (0) and Connectivity index (1) (plots of Connectivity indices vs. Van der Waals volume are given in Figures 3 and 4), and polynomial regression with the Shultz index (plot of Shultz index vs. Van der Waals volume is given in Figure 5). In conclusion, molecular weight and van der Waals volume are shown to be useful parameters for structure-property analysis and could be used in further estimation of structure-activity relationships because these parameters can be easily calculated for wide variety of moleculs (e. g. phenolic acids and flavonoids).

Human immunodeficiency virus type 1 (HIV-1) is the agent of the acquired immunodeficiency sydrom (AIDS). HIV-1 integrase is an enzyme that mediates the integration of HIV-1 DNA into a host chromosome and is essential to replication of the virus. HIV-1 integrase is an attractive target for therapeutic development against AIDS since there are no known cellular enzymes that function similary, raising the hope that integrase inhibitors could be relatively non-toxic. Recent reports have appeared on high HIV-1 integrase inhibitory potency of certain biscoumarin analogues. In the previous studies it was shown that the active pharmacophore consisted of a coumarin dimer containing an aryl substituent on the central linker methylene. QSAR studies demostrated that the aromatic hydroxyl group and lipophilicity of the aryl substituent on the central linker methylene were associated with bioactivity of HIV-1 integrase inhibitors. The aim of our work was to prepare 3, 3'-(4-hydroxybenzylidene)bis[4-hydroxycoumarin] and investigate its lipophilicity using potentiometric titration method and theoretical approaches. The potentiometric titration method was chosen as the easiest method of log P measurment, and provides detailed information on the partitioning characteristics of target compound at all pH values. pKa, log P and log D values were determinated using Sirius instrument GLpKa. Experimentally determinated log P value (3.180) was correlated with log P values predicted by different computer programs: Hyperchem 7.0 (2.508), XLOGP 2.0 (4.22), KowWin (3.0772), MLOGP (2.519), CLOGP (4.129), milogP (4.188), ALOGPS 2.1 (3.1), IA logP (3.93) Vega-Crippen (5.0823) and Vega-Broto (6.847). The best correlation between experimentally determinated and calculated values was found for the KowWin and ALOGPS 2.1 programs. The correlation between experimental and calculated values was consider in order to evaluate the predictive power of the approaches applied to compute the lipophilicity of biscoumarins. The e-state indices encode relevant information and their implementation in powerful statistical tools as the neural networks seems to offer a promising alternative for more consistent log P value.

Molecular lipophilicity (log P) is a prime physicochemical descriptor of xenobiotics with relevance to their biological properties. It plays an important role in the pharmacokinetic behavior of the drug molecules, biological activities, toxicological properties and the hydrophobic interactions of drugs with their receptors. Because experimental measurements are time consuming and difficult, computational methods are very valuble tools for prediction lipophilicity of large sets of compounds in QSPR studies, particulary at the screening stage. A number of different computer programs based on different theoretical approaches have been recently developed. The previous studies have shown better predictive power for simple organic molecules than for complex drug molecules. In our work, the predictive power of seven computer programs (HyperChem, miLogP, ALOGPs, IA logP, CLOGP, KowWin and XLOGP) was evaluated comparing obtained calculated with experimental log P values from the literature. 100 chemically and pharmacologicaly heterogeneus drug molecules with complex fuctional groups were used as a test set. Statistical analysis of results was performed using StatSoft 6.0 program. Regression analysis and Chi-Square - test were applied for evaluation of validity of the computer programs. Our analysis demostrates that the highest correlation of log P values predicted by a computer program to experimental data is attributed to KowWin program (r = 0.97 and Chi-Square = 8.19). In our further work an enlargement of the database will be done.

The aim of this study was to establish the difference in composition of pharmacologically active compounds between raw propolis samples from different parts of Croatia. We used two dimensional TLC together with densitometric evaluation to establish the concentration of flavonoid aglicones and phenolic acids present in ethanolic extract of propolis samples.

Flavonoids belong to the recently popular phytochemicals ; they are ubiquitous in plant kingdom and cannot be synthesised by humans. Biochemical effects of flavonoids in living systems can be divided into four categories - (a) binding affinity to biological polymers, (b) binding of heavy metal ions, (c) catalysis of electron transport and (d) ability to scavenge free radicals. Antioxidant potential is one of the earliest functions discussed for this group of compounds and preferred interpretation of that effect lies in the radical-scavenging properties. One of the most concentrated sources of flavonoids available is propolis - resinous material collected by honeybees. The aim of our study was to investigate whether or not daily intake of propolis has influence on antioxidant defence enzymes (such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT)) in human subjects. Analysis of SOD and GPx activity was performed using RANSOD and RANSEL antioxidant kits (Randox Laboratories Ltd., Crumlin, United Kingdom), while the catalase activity was measured according to the method of Aebi (Methods Enzymol. 105, 1984). Fourty healthy male and female volunteers aged between 20 and 30 years were included in our study and were divided into two groups (study and control group). Inclusion and exclusion criteria were developed to mimic the average population ; ingestion of any dietary supplements and medications was forbidden and the intake of flavonoid rich diet was limited. During the study, blood samples were taken four times - two times in the period of 14 days before propolis supplementation (to establish the baseline status of antioxidant enzymes of all volunteers) and two times during propolis therapy. Propolis supplementation for study group began after their baseline blood samples were taken and lasted for 30 days. Propolis product used was EPID BUSTINE (powdered propolis extract soluble in water ; Specchiasol, Bussolengo - Verona, Italy). Volunteers received 1 dose three times per day during the given period (one dose contains 16, 25 mg of flavonoids ; daily intake was 48, 75 mg) and the blood samples were taken 2 times, every 14 days. It is expected that elevation of daily intake of flavonoids is associated with the reduction of the expression of antioxidative defence enzymes. The statistical significance of the results is discussed.

In our work log P value for 3, 3´-(2-metoxybenzilidene)bis[4-hydroxycoumarin] (MBbisHC) was experimentally determinated (˝shake-flask˝ method) and calculated using six computer programs: HyperChem 7.0 (based on atom contributions), XLOGP (based on atom contributions), KowWin (based on atom/fragment contributions), CLOGP (based on fragmental contributions), ALOGPS 2.1 (based on atom-type electrotopological-state indices and neural network modeling), and IA logP (based on atom-type electrotopological-state indices and neural network modeling). The experimental and calculated log P values were correlated. There is nearly perfect agreement between experimentally observed and calculated log P (HyperChem 7.0) value.

Flavonoids constitute one of the largest groups of naturally occuring phenols and they are ubiquitous in green plants. Phenolic acids and flavonoids, with various biological activities, are considered to be main compounds in propolis – a natural product produced by the honeybee whose composition depends on its geographic origin. Our previous work showed that TLC is suitable method for rapid screening of pharmacologically active compounds. In our present study we wanted to develop a method more suitable for quantitative analysis. We used 2D TLC together with multiple develpoment of chromatograms to obtain better separation of the complex mixture. For chromatographic analysis we used 20 x 20 cm glass-backed TLC plates coated with 0.25 mm layers of silica gel 60 F254 (Merck, Darmstadt). First, we developed the chromatogram in mobile phase composed of chloroform : methanol : acetic acid (44.1:3:2.4) and after evaporation and rotation for 90°, we used n-hexane : ethyl acetate : acetic acid (31:14:5) for the second development [1]. Chromatograms were developed at the room temperature by ascendenting multiple development in previously saturated vertical flat-bottom glass chambers with glass lid (CAMAG, Switzerland). We investigated the presence of 17 flavonoids and 4 phenolic acids in our ethanolic propolis extract [2]. First, we established the RF values of all standards in the given mobile phases and divided them into 5 groups. Each group of standards (10 μ l of 80 % ethanolic solution in three different concentrations) together with the propolis sample was chromatographed on the same plate. Visualization was performed under short (λ = 254 nm) and long wave (λ = 366 nm) UV light, in long wave UV light after spraying with 5% ethanolic solution of AlCl3 and exposing to NH3 vapour and after spraying with different reagents (diazotized sulphanilic acid [3], Folin-Ciocalteu reagent, mixture of 1% ferric chloride and 1% potassium ferricyanide [4]). After establishing the presence of certain standard, composition of the mobile phases will be modified if it will be necessary. All present standards will be chromatographed again and densitometric evaluation of their concentration will be performed. [1] Jasprica I., Smolčić-Bubalo A., Mornar A. and Medić-Šarić M.: J. Planar Chromatogr. 2004 (accepted) [2] Park Y. K. and Ikegaki M.: Biosci. Biotechnol. Biochem. 1998, 62, 2230-2232. [3] Markham K. R. :Techniques of Flavonoid Identification. Academic Press, London 1982. [4] Harborne J. B.: Phytochemical Methods. John Wiley & Sons, Inc., New York 1973.

U protetskoj se terapiji upotrebljava vrlo mnogo plemenitih slitina različita sastava. Plemeniti metali su sinonim za biokompatibilnost. Zlato je jedna od najinertnijih plementih kovina, a srebro jedna od najreaktivnijih. Cilj rada bio je razvijanje i raščlamba kromatografskih mrlja čistoga zlata i srebra (Aurodent, Celje, Slovenija). Kovine su uzorkovane na sobnoj temperaturi otopinom voda-HCl(conc.) (v/v 10:1) tijekom 10 s pri stalnom naponu struje od 4,5 V. Otopine kationa iz čistih kovina i 10 µl standardnih otopina soli nanesene su na HPTLC kromatografske ploče (Merck, Darmstadt, Njemačka) 10 x 10 cm presvučene celulozom. Ploče su razvijene u staklenoj kromatografskoj komori zasićenoj mobilnom fazom izo-amilni alkohol: HCl(conc.) : acetonitril u omjeru 5,4 : 4,3 : 0,3 do visine od približno 8 cm. Kada su razvijene sušene su u struji vrućega zraka, a kationi su vizualizirani prskanjem zasićenim etanolnim otopinama difenilkarbazida, alizarina, kvercetina te 0,1% otopinom ditizona u kloroformu. Nakon prskanja i sušenja ploče su izložene parama amonijaka i učinjena je vizualizacija. Uzorkovano zlato i srebro međusobno se razlikuje bojom kromatografskih mrlja i utvrđenim Rf vrijednostima. Metodom anodnoga uzorkovanja slitina i identifikacijom kationa tankoslojnom kromatogafijom moguće je dokazati postojanje određenih kationa s udjelom mase većim od 1%, ne oštećujući pritom sam uzorak. Metoda se pokazala prikladnom za uzorkovanje kovina zlata i srebra, što je nastavak dosadašnih istraživanja u identifikaciji određenih sastavnica u pojedinim slitinama ili u inkorporiranome protetskom radu., Different precious dental alloys are used in prosthodontic therapy. Precious metals are the synonym for biocompatibility. Gold is one of the most inert and silver the most active presious metals. The purpose of this study was to develop an analyze the chromatographic spots of pure gold and silver (Aurodent, Celje, Slovenia). The sampling was performed in a water-HCl(conc.) solution in a 10 : 1 volume ratio at room temperature using a 4,5 V battery. Dissoluted cations and solutions of standard elements (salts of gold and silver) were applied to HPTLC plates (Mareck, Darmstadt, Germany) 10 x 10 cm precoated with cellulose. Plates were developed in a vertical separating chamber to a height of approximately 8 cm saturated with iso-amyl (3.methyl-1-butanol) - HCl (36,5%) - acetonitrile in a volume ratio 5,4 : 4,3 : 0,3. After development, plates were dried in (a steam of) hot air and the cations were visualized by spraying with the saturated ethanolic solutions of alizarin, diphenylcarbazide, quercetin and 0,1% chlorophorm solution of dithizone. After drying, plates were exposed to NH3 vapor. According to the Rf value and the color of the spots, the difference between gold and silver were obtained. Anodic sampling with thin-layer chromatography is a suitable, nondestructive method for identification of cations presents in different dental alloys. It is possible to identify the cations with their mass ratio W > 1%. The results have shown that the described method is suitable for analysis of gold and silver and it can be used in future work to identify those cations in dental alloys of unknown composition.

Different dental alloys are used for producing prostodontics appliance. The nonprecious alloys are the most commonly used for metal-ceramics bridges and crowns. They are developed by modification of the steel composition. Iron as a minor component segregates on grain boundaries of alloy surface and create chemical bonding between metal and ceramic. Six alloys of different composition were analysed. The analysis was performed by anodic sampling in water-HCl (conc.) solution in a 10:1 volume ratio [1]. Alloy sampling was timing and voltage controlled (20 s, 4.5 V) and transfer of dissoluted cations to the chromatographic plates took 15 s. Metal cations were separated and identified by Thin Layer Cromatography [2]. Standard solutions (FeSO4 0.1 mg/ml and FeCl3 0.1 mg/ml) and alloy samples were applied to TLC plates (Merck, Darmstadt, Germany) 10 x 10 cm, precoated with cellulose. For the cromatography the standard solutions (10 μ l volume) and the alloy samples were applied 1 cm from the lower edge of the plates. Plates were developed in the saturated glass chamber (DESAGA) to the distance of ca 8 cm with iso-amyl alchohol (3-methyl-1-butanol HPLC grade, SIGMA-ALDRICH) - HCl (36.5%, p.a., KEMIKA) - acetonitrile (p.a. Merck, Darmstadt) 8.8 + 0.7 + 0.5. The developing time was ca 2 hours and the temperature 23˚ C. After separation the plates were dried in a steam of warm air for aproximately 1 minute and the spots were visualised by spraying with the saturated ethanolic solutions of alizarin, diphenylcarbazide and quercetin [3]. The plates were dried and exposed to NH3 vapour. Pink (identifacation solution - diphenylcarbazide), purple (identifacation solution – alizarin) and brown (identifacation solution – quercetin) spots of iron were visible at the distance of 6.8 cm from the start (Rf value was 0.85). The difference between Rf value and the colours of the standard solutions (FeSO4 and FeCl3) spots could not be found. The method described is suitable for the separation and identification of iron cations no matter of charge.

Thin-layer chromatography (TLC) method are studied for determination of phenolic compounds in two samples of commercially available wines from the Croatia. Sample preparation method using liquid-liquid extraction with diethyl ether at pH 2.0 was used. Extracts and standard solutions were applied on silica gel 60 F254 TLC plates (20 × 20 cm). The efficiency of eleven mobile phases, used for this purpose, has been tested by three mathematical techniques: calculation of the information content (I) derived from Shannon 's equation ; determination of discriminating power (DP) and formation of clusters and dendrogram. It has been shown that the most favourable mobile phase for separation phenolic compounds in extract of wine “ Frankovka” is benzene-ethyl acetate-formic acid, 30 + 15 + 5 (v/v), while for wine "Merlot" optimal mobile phases are chloroform-methanol-formic acid, 15 + 3 + 2 (v/v) and ethyl acetate-chloroform-formic acid, 24 + 21 + 5 (v/v). Six phenolic compounds have been identified in the tested samples.

Pripravke za koje se zahtijeva da budu sterilni potrebno je prije njihova puštanja na tržište još jednom provjeriti kako bi se izbjegle neugodne posljedice moguće kontaminacije, pogotovo patogenim mikroorganizmima. Validacijom sustava za ispitivanje sterilnosti provjeravaju se tehnološke karakteristike sustava i sukladnost s namjeravanom upotrebom.