Marica Medić-Šarić

Chemical composition of the ethanolic propolis extracts and its effect on HeLa cells.

ETHNOPHARMACOLOGICAL RELEVANCE Propolis is a resinous hive product collected by honeybees from various plant sources. It is widely used in traditional medicine and is reported to have a broad spectrum of pharmacological effects (antibacterial, antihepatoxic, antioxidative, anti-inflammatory, etc.). Thus the aim of this study was to assess cytotoxic effect of various ethanol propolis extractions on the cervical tumor cell line (HeLa) and compare it with their phenolic acids and flavonoids composition. MATERIALS AND METHODS Twenty samples of raw propolis were collected from 17 localities of Croatia (I-XVII), 2 of Bosnia and Hercegovina (XVIII, XIX) and 1 of Macedonia (XX). Reverse phase HPLC was used to determine the chemical composition of polyphenols. Biological experiments were carried out in vitro on cervix adenocarcinoma cell line (HeLa). RESULTS Phenolic acids (ferulic acid, p-coumaric acid, caffeic acid) and flavonoids (tectochrysin, galangin, pinocembrin, pinocembrin-7-methylether, chrysin, apigenin, kaempferol, quercetin) have been determined using HPLC analysis in 20 ethanolic propolis extracts. All samples contain tectochrysin in ranges of 0.1988 mg/g (XVIII) to 1.2004 mg/g (III), while caffeic acid and quercetin have not been found. Flavonoid that is most abundant is galangin in ranges from 0.3706 mg/g (XVII) to 47.4879 mg/g (IX). The samples of propolis numbers I, VI and X applied in the investigated concentration range manifested significant reduction of cell growth. GI(50) value as indicator of cytotoxicity among propolis samples showed that propolis number VII is the most effective (GI(50) =76 μg/ml) followed by propolis nos. XV, XVIII and I. CONCLUSION Antiproliferative and cytotoxic effect of propolis on the HeLa cells is not correlating with the concentration of particular components but on establishing the possible synergistic antiproliferative activity of individual phenolic acid and flavonoids. Differences in the chemical composition lead to diversity in biological activity of propolis samples.

Optimization of TLC analysis of flavonoids and phenolic acids of Helleborus atrorubens Waldst. et Kit.

Numerical methods for the evaluation of the separation power of thirteen thin-layer chromatographic systems for splitting a methanolic extract of leaves of Helleborus atrorubens Waldst. et Kit. into 15 compounds (flavonoids and phenolic acids) have been investigated. For this purpose, the following mathematical approaches have been applied: calculation of the information content (I), determination of discriminating power (DP) and formation of clusters and dendrogram. The most suitable chromatographic system for the separation of investigated compounds is ethyl acetate-formic acid-water (65:15:20, v/v/v).

QSAR study of antioxidant activity of wine polyphenols

Quantitative structure activity relationships (QSAR) were obtained describing the antioxidant activity of the main pharmacologically active polyphenols of wine, using molecular properties and descriptors derived from the 2D and 3D representations of molecules. The best models for the prediction of the ability to scavenge the ABTS radical cation were obtained by polynomial regression analysis using zero-order connectivity index and molar refractivity. Statistically, significant models for lipid peroxidation inhibiting effects of flavonoids were obtained by polynomial and multiple regression using lipophilicity, Balaban index, Balaban-type index and 3D GETAWAY descriptor. The 3D descriptors possess the ability for discrimination of stereoisomers, like catechin and epicatechin. We demonstrated that a size and shape of molecules, as well as steric properties, play an important role in the antioxidant activity of polyphenols.

Evaluation of antiaggregatory activity of flavonoid aglycone series

BackgroundAmong natural compounds, present in every day diet, flavonoids have shown beneficial effect in prevention of cardiovascular diseases that can be attributed, at least partially to the described antiaggregatory activity i.e. antiplatelet effects of flavonoids. Due to the ever increasing pharmacological interest in antiplatelet agents a systematic experimental evaluation of large flavonoid series is needed.MethodsA set of thirty flavonoid aglycones has been selected for the evaluation. All measurements of aggregation were done under standardized and firmly controlled in vitro conditions. The whole blood samples, multiple platelet functional analyzer and adenosine diphosphate (ADP) as a weak agonist of aggregation were selected for this purpose.ResultsThe results were expressed as minimal concentration of flavonoid that can significantly lower the platelet aggregation compared to the corresponding untreated sample (minimal antiaggregatory concentration - MINaAC). All analyzed flavonoids exhibited antiaggregatory activity MINaAC ranging from 0.119 μM to 122 μM, while the most potent representatives were 3,6-dihydroxyflavone (0.119 μM) and syringetin (0.119 μM).ConclusionsMeasurable antiplatelet activity established at submicromolar flavonoid concentrations suggests that even a dietary consumption of some flavonoids can make an impact on in vivo aggregation of platelets. These findings also point out a therapeutical potential of some flavonoids.

QSAR Study of Antimicrobial Activity of Some 3-Nitrocoumarins and Related Compounds†

A new class of antimicrobial agents, 3-nitrocoumarins and related compounds, has been chosen as a subject of the present study. In order to explore their activity and molecular properties that determine their antimicrobial effects, QSAR models have been proposed. Most of the 64 descriptors used for the development were extracted from semiempirical and density functional theory (DFT) founded calculations. For this study literature data containing results of microbiological activity screening of 33 coumarin derivatives against selected clinical isolates of C. albicans (CA) and S. aureus (SA) have been selected. Multivariate predictive models based on random forests (RF) and two hybrid classification approaches, genetic algorithms (GA) associated with either support vector machines (SVM) or k nearest neighbor (kNN), have been used for establishment of QSARs. An applied feature selection approach enabled two-dimensional linear separation of active and inactive compounds, which was a necessary tool for rational candidate design and descriptor relevance interpretation. Candidate molecules were checked by cross-validated models, and selected derivatives have been synthesized. Their antimicrobial activities were compared to antimicrobial activities of the representative derivatives from the original set in terms of minimal inhibitory concentration (MIC) against chosen SA and CA ATCC strains. High ranking of descriptors consistent with the degree of hydrolytic instability of selected compounds is common to models of antimicrobial activity against both microorganisms. However, descriptor ranking indicates different antimicrobial mechanisms of action of chosen coumarin derivatives against selected microbial species.

A comparison of the nutritional value and food safety of organically and conventionally produced wheat flours

Growing interest in organic agriculture has prompted this study aiming to evaluate nutritional content of wheat flours originating from organic and conventional production systems. Obtained results showed that organic samples had significantly lower protein content and lower levels of Ca, Mn and Fe compared to conventional samples. Protein digestibility and levels of K, Zn and Mo were significantly higher in organic than in conventional wheat flours. Regarding undesirable metals, significantly higher levels of As and Cd were found in conventional compared to organic wheat flours. Although the mean concentrations of zearalenone and ochratoxin A were higher in conventional than in organic flours, this difference was not significant. This study revealed that organic agriculture has the potential to yield products with some relevant improvements in terms of high quality proteins and microelements contents, while the reduction in contamination with toxic elements and mycotoxins may be accomplished.

Evaluation of Antioxidative Activity of Croatian Propolis Samples Using DPPH· and ABTS·+ Stable Free Radical Assays

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.

Quantitative Analysis of Flavonoids and Phenolic Acids in Propolis by Two-Dimensional Thin Layer Chromatography

Flavonoids are polyphenolic compounds present in a wide variety of plants. They are benzo-γ-pyrone derivatives, which resemble coumarin. Flavonoids (with other polyphenols, e. g. phenolic acids) are believed to have a variety of physiological activity. One of the most concentrated sources of those compounds is propolis, a resinous substance collected by the honeybees from various plant sources. Its composition, and thus its content of pharmacologically active compounds, depends on geographic origin. We have developed a new TLC method suitable for analysis of complex mixtures such as propolis. Two-dimensional TLC with densitometry evaluation seemed an appropriate method for rapid screening of active compounds and quantifying the concentration of flavonoid aglycones and phenolic acids present in propolis extracts. After developing the method we tested it by analyzing and comparing the composition of three raw propolis samples from different geographic regions of Croatia. To establish the RF values, standard solutions of nine flavonoids and six phenolic acids were first applied to the chromatographic plate as 10-mm bands. Plates were developed in vertical glass chambers previously saturated with the mobile phases - n -hexane-ethyl acetate-glacial acetic acid, 31 + 14 + 5 (v/v), (mobile phase 1) or chloroform-methanol-formic acid, 44 + 3.5 + 2.5 (v/v), (mobile phase 2). After drying, bands were visualized under short- and long-wavelength UV light; the plates were then sprayed with 1% AlCl3 ethanol solution and viewed again under long-wavelength UV light. RF values were calculated. All standards were chromatographed again in groups of 3 or 4 together with a propolis extract. First, plates were developed with mobile phase 1 (or 2), the mobile phase was evaporated, standard solutions were applied again, and the plate was rotated through 90° and chromatographed again with mobile phase 2 (or 1). The presence (or absence) of all standards was determined according to their RF values and fluoresfluorescence colors. Abetter combination of mobile phases was chosen, the amounts of standards present were adjusted, and the standards were again individually chromatographed with the propolis extract. Quantitative evaluation was performed with Camag Reprostar 3 densitometer. Two-dimensional TLC was shown to be a highly suitable method for the task. We proved the method could be used for analysis of different propolis samples to identify and quantify the main pharmacologically active compounds.

The content of essential and toxic elements in Croatian propolis samples and their tinctures

Summary There is a general lack of scientific literature dealing with the mineral composition of propolis, despite the fact that the presence of minerals in plant tissues can significantly affect the pharmacotherapeutic properties of derived products. Raw propolis samples collected from different Croatian regions and their respective tinctures were therefore analysed for mineral composition by atomic emission/absorption spectrometry. Contents of macro- and trace elements (Ca, K, Mg, Na, Al, B, Ba, Cr, Cu, Fe, Mn, Ni, Sr and Zn) were determined. Levels of toxic elements (As, Cd, Hg, and Pb) were also analysed, since they are possible environmental contaminants that could be transferred into propolis products for human consumption. Following the European Pharmacopoeia requirements for quality control of herbal drugs, crude propolis samples were also analysed for total ash content. Significant correlation between the total mineral content and total ash of raw propolis (r = 0.9273) showed that total ash might serve as the first information that justifies the need for detailed mineral analysis of tested samples. Among analysed minerals, Ca, Mg, K, Al, Fe and Zn were found to be the most abundant in raw propolis, ranging from 12.06 (Mg) to 932.6 mg/100g (Zn). Mineral contents of all investigated tinctures were significantly lower compared to the respective raw propolis samples. Extractability was variable due to influence of the sample matrix and correlated with the mineral content of raw propolis only in the case of Fe, Mg, Sr and Zn. All samples met the World Health Organization (WHO) standards regarding the maximum permitted amounts of Hg, Pb, As and Cd except for two raw propolis samples with increased lead content indicating pollution of the collection areas.

Analysis of phenolic components in Croatian red wines by thin-layer chromatography

A thin-layer chromatographic (TLC) method has been used for determination of phenolic compounds in two samples of wine commercially available in Croatia. Sample preparation was by liquid—liquid extraction with diethyl ether at pH 2.0. Extracts and standard solutions were applied to 20 cm × 20 cm silica gel 60 F254 TLC plates. After treatment of the developed plates with ammonia vapor, phenolic compounds were detected on chromatograms by their colors or by fluorescence in UV light at λ= 244 nm and at λ= 366 nm before and after spraying with 1% ethanolic AlCl3. The efficiency of eleven mobile phases was 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 was shown that the best mobile phase for separation of phenolic compounds in the wine extracts was benzene—ethyl acetate—formic acid, 30 + 15 + 5 (v/v). Six phenolic compounds were identified in the tested samples.