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Xanthine oxidase (XO) is an important enzyme catalyzing the hydroxylation of hypoxanthine to xanthine and xanthine to uric acid which is excreted by kidneys. Excessive production and/or inadequate excretion of uric acid results in hyperuricemia. This paper presents a detailed review of methods of isolation, determination of xanthine oxidase activity, and the effect of plant extracts and their constituents on it. Determining the content and activities of XO can be used for diagnostic purposes. Testing inhibition of XO is important for detection of potentially effective compounds or extracts that can be used to treat diseases that are caused by increased activity of XO. In vitro bioassays are used to examine test material for XO inhibition, as inhibitors of XO may be potentially useful for the treatment of gout or other XO induced diseases. Several authors reported on the XO inhibitory potential of traditionally used medicinal plants.

Ova doktorska disertacija je koncipirana sa ciljem da ispita polifenolni profil kao i potencijalna biohemijska dejstva ekstakata dobijenih različitim tehnikama ekstrakcije (konvencionalnim i nekonvencionalnim) odabranih biljnih vrsta Lavatera thuringiaca L., Erica carnea L. i Satureja hortensis L. Od konvencionalnih ekstrakcija su primenjene Soksletova (Soxlet) ekstrakcija i maceracija, a od savremenih ultrazvučna ekstrakcija, mikrotalasna ekstrakcija i subkritična ekstrakcija vodom. Takođe, ova istraživanja su usmerena u pravcu pružanja mogućnosti navedenih biljnih ekstrakata kao izvora potencijalnih prirodnih nutraceutika. U tom smislu, u ovoj doktorskoj disertaciji su prezentirani rezultati sledećih ispitivanja: • kvalitativna identifikacija komponenata ekstrakata biljnih vrsti L. thuringiaca L., E. carnea L. i S. hortensis L • kvantitativna analiza komponenata ekstrakata biljnih vrsti L. thuringiaca L., E. carnea L. i S.hortensis L • biohemijska aktivnost ekstrakata biljnih vrsta L. thuringiaca L., E. carnea L. i S. hortensis L • određivanje opšte toksičnosti i genotoksičnosti ispitivanih ekstrakata biljnih vrsta L. thuringiaca L., E. carnea L. i S. hortensis L., kako bi utvrdili mogućnost primene istih kao potencijalnih prirodnih nutraceutika. Na osnovu svih sprovedenih istraživanja,ekstrakti dobijeni iz biljnih vrsta L. thuringiaca L., E.carnea L. i S.hortensis L. su od neprocenjivog naučnog značaja. Oni predstavljaju smernice za dalje proučavanje i primenu ovih biljnih vrsta kao izvora prirodnih nutraceutika u preventivne i terapijske svrhe. Ujedno doprinos naučnom napretku na polju biohemije, medicine, farmacije, farmakoterapije u vidu proširenja saznanja o aktivnim sastojcima biljaka i njihovog pozitivnog delovanja u tretmanu različitih bolesti, ali i u profilaksi radi očuvanja zdravlja kao najvažnijeg resursa sa kojim čovek raspolaže. This doctoral dissertation is designed to examine the polyphenolic profile as well as the potential biochemical effects of extracts obtained by different extraction techniques (conventional and unconventional) of protected plants of the species Lavatera thuringiaca L. Erica carnea L. and Satureja hortensis L. Also, this research is aimed at providing the possibility of these plant extracts as a source of potential natural nutraceuticals. In that sense, in this doctoral dissertation the results of the following examinations are presented:In this sense, the paper presents the results of the following tests: • qualitative identification of components of extracts of plant species L. thuringiaca L., E. carnea L. and S. hortensis L, • • quantitative analysis of components of extracts of plant species L. thuringiaca L., E. carnea L. and S. hortensis L, • • biochemical activity of extracts of plant species L. thuringiaca L., E. carnea L. and S. hortensis L, • • determination of general toxicity and genotoxicity of tested extracts of plant species L. thuringiaca L., E. carnea L. and S. hortensis L., in order to determine the possibility of using them as potential natural nutraceuticals. • Based on all conducted research, extracts were obtained from plant species L. thuringiaca L., E. carnea L. and S. hortensis L. are of inestimable scientific importance. They represent guidelines for further study and application of these plant species as sources of natural nutraceuticals in preventive and therapeutic species. One contribution to scientific progress in biochemistry, medicine, pharmacy, pharmacotherapy in the form of expanding knowledge about the active ingredients of bioca and their positive effects is the beginning of the treatment of a large number of diagnostic tests.

Diabetic retinopathy is the commonest cause of blindness in the population aged 20 to 79 years. The molecular basis of the disease is metabolic dysfunction caused by hyperglycemia, which leads to oxidative damage of biomolecules and inflammation in the retina. Oxidative stress and inflammation cause damage to the bloodretinal barrier, impaired blood flow and ischemia in the retina, while vascular endothelial growth factor (VEGF) is considered to be the most important factor of disease progression. Melatonin is a hormone that is synthesized in the pineal gland and retina. It is considered a circadian rhythm regulator, but it also exerts significant antioxidant and anti-inflammatory effects and is easily transported through the blood-retinal barrier due to its physiochemical properties. The aim of this study is to determine the effects of melatonin supplementation on the parameters of serum glucose and lipid metabolism, as well as on the parameters of oxidative damage, inflammation, and neoangiogenesis in retina of rats with streptozotocin-nicotinamide induced type 2 diabetes mellitus. Besides from hyperglycemia and dyslipidemia, a reduced serum melatonin concentration was found in experimental animals with type 2 diabetes mellitus. Oral supplementation with melaton at a dose of 0.2 mg/kg of body weight led to a significant increase in serum melatonin concentration. Animals treated with melatonin had reduced serum tumor necrosis factor α (TNF-α) levels, which is important due to the impact of this proinflammatory cytokine on the blood-retinal barrier permeability and the leukostasis. Melatonin reduced the concentration of oxidative damage markers, in particular the lipid peroxidation (thiobarbituric acid reactive substances, TBARS) and protein oxidation (advanced oxidation protein products, AOPP) markers in the retina, ,as well as, the concentration of an enzyme inducible nitric oxide synthase (iNOS ), which contributes to the oxidative damage through the production of reactive nitrogen species. Melatonin, in addition, leads to a decrease in the retinal concentration of VEGF and the matrix of metalloproteinase 9 (MMP-9), which prevents the damage to the blood-retinal barrier and the neoangiogenesis. The use of melatonin can, therefore, be considered in the prevention or in the therapy of changes in the retina associated with type 2 diabetes mellitus.

In this dissertation, the influence of diamagnetic metal ions Mg (II) and Ca (II) on the autoxidation of polyphenol compounds, containing one or two ortho-dihydroxyl groups, in aqueous solutions at different pH values, was studied. The autoxidation of polyphenols distributed in nature, such as: catechol, caffeic acid, rosmarinic acid, chlorogenic acid, rutin and hematoxylin was investigated. For this purpose, the following instrumental methods were applied: UV/Vis spectrophotometry with subsequent hemometric treatment of UV/Vis spectra, ESR spectroscopy, high performance liquid chromatography (HPLC) and mass spectrometry (MS). It has been shown that the pH value of the solution and/or the presence and type of metal ions affect the speed and manner of the autoxidation processes of the investigated compounds. Applied techniques have enabled the determination of the number and structure of intermediate products as well as stable autoxidation products. The obtained results allow a more detailed insight into mechanisms of antioxidative activities of studied polyphenols and a more comprehensive view of their biochemical behavior with the implications for further human use.

Priloženi rukopis sumira sintezu šest novih derivata 3-(1-aminoetiliden) hroman-2,4-diona, označenih kao 2a-f, i dva nova derivata 4-hidroksi-3-(1-imino- etil)-2H-hromen-2-ona označenih kao 2g-h, odnosno evaluaciju njihove in vivo anti- koagulativne aktivnosti nakon intraperitonealne i oralne aplikacije na odraslim pacovima Wistar soja, sa ciljem procene njihovog kliničkog antikoagulativnog potencijala, farmakologije i toksičnosti. The enclosed manuscript summarizes the efforts of synthesis and evaluation of six novel 3-(1-aminoethylidene)chromane-2,4-diones, namely 2a-f, as well as of two novel 4- hydroxy-3-(1-iminoethyl)-2H-chromene-2-one derivatives, namely 2g-h, as in vivo anticoagulants by means of intraperitoneal and per os administration to adult male Wistar rats. Condensates were screened to expose their anticoagulant potential, pharmacology and toxicity.

Plants accumulate metals from the soil during growth. Environmental polution can lead to increased metal uptake into the body through food. Therefore, plants have medicinal properties but also can have toxic effects on humans. It is necessary to determine the metal content in the soil, plants, their parts and extracts prepared from them. Based on the obtained results the content of metals and systematic analysis will be determined plant uptake factor (PUF), daily intake of metal (DIM) and the index of health risks (HQ). All this should point to the possibility of safe use of plants from Serbia in nutrition and treatment (in the form of teas and extracts).

By applying more instrumental methods (UV-Vis spectrophotometry, HPLC chromatography, ICP emission spectrophotometry), the antioxidant activity, phenolic and mineral composition of the pulp and peel extracts of selected types of fruit (Cydonia oblonga, Prunus persica, Malus domestica) were determined and the effect of methanol and acetone solvent of determined composition was investigated. The obtained results are discussed, compared with the literature and analyzed statistically.

The relationships between structure, spectral characteristics, and colour of the aglycone moiety of cyanidin and its 3-glucosidic substitution in cyanidin 3-O- β-glucopyranoside were studied as a function of pH, using a combination of techniques (UV-Vis spectrophotometry, colorimetry, and HPLC-ESI/MS). The absorption and fluorescence emission spectra of cyanidin and Cy3Glc at pH 5.5 in aqueous solution have been studied. The colour variation, colour intensity and stability at various pH values (2.0, 4.0, 7.0 and 9.0) of cyanidin and Cy3Glc were investigated during a period of 8 hours storage at 25 ± 0.1ºC, as well as, in the aqueous solution at pH 7.0 and under the conditions of the elevated temperature (55.0 ± 0.1ºC) which simulate the rapid food ageing. The interactions of cyanidin and Cy3Glc with model lipid membranes differing in surface charge and phase state were investigated using DSC and fluorescence emission polarization spectrometry.

In this dissertation the influence of divalent diamagnetic metal ions (Mg(II), Ca(II), Sr(II), Ba(II), Zn(II), Cd(II) and Pb(II)) on vicinal trihydroxy phenolic compounds autoxidation in aqueous solutions was examined. Autoxidation processes of pyrogallol (the simplest compound with appropriate structure), gallic acid and ethyl gallate were studied with the application of UV/Vis spectrophotometry with chemometric data processing, EPR spectroscopy, High-performance liquid chromatography (HPLC) and Mass spectrometry (ESI-MS). It was determined that pH value of solution and presence and type of diamagentic metal ions have great influence on investigated compounds autoxidation processes rates and mechanism. With the aid of applied techniques the structures of transition free radical species and stable autoxidation processes products were determined. Obtained results enabled better understanding of investigated compounds autoxidation processes which is of great importance since gallic acid is found in many plants, while ethyl gallate is used as an antioxidant in food.

Colorectal cancer is one of the most frequent neoplastic diseases in human population and one of the most frequent causes of death. There are a lot of pathological factors, involved in the process of colon cancer initiation, propagation and progression, among other ROS, who can activate Nuclear transcriptional factor kappa B (NF-B), who controls genes involved in tumor progression.Apoptosis is a process involved in the development of tumors, where blocking apoptosis avoids removal of malignant cells.The matrix metalloproteinase’s generally function to degrade proteoglycans and matrix glycoprotein’s and facilitate the development and metastasis of tumors. Enzymes of purine nucleotide metabolism may be very important in tumor proliferation. The aim of the present study wasthat in 50 patients with colorectal cancer to assess the levels of oxidative stress , quantitative expression of NF-B, level of apoptosis expressed in endonuclease activity and Bcl2 / Bax ratio, the activity of matrix metalloproteinase 9, and the activity of the enzyme of purine nucleotides in homogenates of tumor tissue, the tissue immediately surrounding the tumor and healthy colon tissue. The concentrations of TBARS and AOPP, and enzyme activities were determined by appropriate spectrophotometric methods, quantitative expression of NF-B, Bcl2 and Bax, and the activity of MMP-9 were determined by appropriate immunofluorescence methods. The concentrations of TBARS, AOPP, MMP-9 activity, xanthine oxidase, adenosine dezaminaze and quantitative expression of NF-B and Bcl2 in the tumor tissue increased compared to healthy colon tissue. Catalase activity and quantitative expression of Bax was decreased in tumor tissue compared to healthy colon tissue. The tissue surrounding the tumor also had significantly higher TBARS, AOPP, NF-B, Bcl2, and increased activity of MMP-9, ADA, XO compared to healthy tissue. Our results show that the tumor has an increased level of oxidative stress, increased levels of markers of proliferation, but decreased levels of apoptosis compared to healthy colon tissue. The surrounding tissue is also proliferativeactive with reduced levels of apoptosis. These facts may be relevant in determining the operating margin removal of the tumor, followed by the creation of individual therapeutic strategies in inoperable condition or in patients with bone metastases. Correlation with histological findings, tumor stage and other clinical characteristics of each patient would enable a more accurate assessment of invasiveness and aggressiveness of the tumor and the selection of appropriate therapy.