Ana Mornar

Identification and Quantification of Flavonoids and Phenolic Acids in Burr Parsley (Caucalis platycarpos L.), Using High-Performance Liquid Chromatography with Diode Array Detection and Electrospray Ionization Mass Spectrometry

A sensitive method coupling high-performance liquid chromatography (HPLC) with diode-array detector (DAD) and electrospray ionization mass spectrometry (MS) was optimized for the separation and identification of phenolic acids, flavonoid glycosides and flavonoid aglycones in the extract of burr parsley (Caucalis platycarpos L.). Fragmentation behavior of flavonoid glycosides and phenolic acids were investigated using ion trap mass spectrometry in negative electrospray ionization. The MS, MSn and UV data together with HPLC retention time (TR) of phenolic acids and flavonoids allowed structural characterization of these compounds. Caffeoylquinic acid (CQA) isomers, p-coumaroyl-quinic acids (p-CoQA), feruloylquinic acids (FQA), dicaffeoylquinic acids (diCQA), luteolin-7-O-rutinoside, apigenin-7-O-rutinoside as well as isolated chrysoeriol-7-O-rutinoside have been identified as constituents of C. platycarpos for the first time. An accurate, precise and sensitive LC-DAD method for quantification of four phenolic acids (3-O-caffeoylquinic, caffeic, p-coumaric, o-coumaric acid), four flavonoid glycosides (luteolin-7-O-glucoside, apigenin-7-O-glucoside, quercetin-3-O-galactoside, quercetin-3-O-rhamnoside), and three flavonoid aglycones (luteolin, apigenin, chrysoeriol) in C. platycarpos extract was validated in terms of linearity, limit of detection, limit of quantification, precision and accuracy. 3-O-caffeoylquinic acid was the predominant phenolic acid and luteolin-7-O-glucoside was the predominant flavonoid glycoside.

Simultaneous determination of lovastatin and citrinin in red yeast rice supplements by micellar electrokinetic capillary chromatography

Lovastatin is a main component of Monascus purpureus fermented red rice contributing to the lipid-lowering effect. Citrinin is a toxic fermentation by-product which can be found as a contaminant. An accurate, simple and rapid micellar electrokinetic capillary chromatographic method was developed for the first time for simultaneous determination of lovastatin present in lactone and hydroxy acid forms and citrinin in red rice products provided by different manufacturers and formulated in various dosage forms. Separation was achieved within only 2 min using 20 mM of phosphate buffer at pH 7.0 and 30 mM of sodium dodecyl sulphate at an applied voltage of 25 kV. Sensitivity crucial for detecting citrinin was enhanced by using an extended light path capillary. The results showed that the content of lovastatin and its acid form in dietary supplements were considerably different indicating the need for improved standardization in order to ensure efficiency and safety of these products.

Development of a Rapid LC/DAD/FLD/MSn Method for the Simultaneous Determination of Monacolins and Citrinin in Red Fermented Rice Products

Red fermented rice is used worldwide by many patients as an alternative therapy for hyperlipidemia; however, the discovery of a toxic fermentation byproduct, citrinin, causes much controversy about the safety of red mold rice products. A new and fast high-performance liquid chromatography method was developed and validated for simultaneous determination of cholesterol-lowering compounds monacolin K (lovastatin), monacolin K hydroxy acid, and other monacolins present in red fermented rice as well as nephrotoxic mycotoxin citrinin in a single run using connected diode array and fluorescence and mass spectrometric detectors. The proposed method was successfully applied for the analysis of red fermented rice food samples and various dietary supplements also containing other natural lipid-lowering agents. The deviations between label content and levels of active compounds found in investigated samples as well as high batch-to-batch variation found in one product indicate that the regular quality control of red fermented rice products is of great importance.

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.

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.

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.

Investigation of the Flavonoids in Croatian Propolis by Thin-Layer Chromatography

Flavonoids and phenolic acids with a variety of biological activity are considered to be the main compounds in propolis–a natural product produced by the honey bee. TLC can be used for rapid screening of pharmacologically active components and to establish the difference between different propolis samples. Our goal was to optimize chromatographic conditions for separation of flavonoids and phenolic acids and to apply the optimized method for analysis of propolis samples from different geographic regions of Croatia. For chromatographic analysis we used 20 cm × 20 cm glass-backed TLC plates coated with 0.25 mm layers of silica gel 60 F254. Ethanolic standard solutions (80%) of the flavonoids and phenolic acids (10 μL) were applied to the plates. Chromatograms were developed at room temperature by ascending development in previously saturated vertical, flat-bottomed glass chambers with glass lids. Visualization was performed in short- and long-wavelength UV light and in long-wavelength UV light after spraying with different reagents. After calculation of RF values numerical taxonomy methods were used to test the efficiency of 11 mobile phases and to optimize chromatographic conditions for separation of 19 standard solutions. We established the most appropriate mobile phases (chloroform–methanol–(98–100%) formic acid, 44.1 + 3 + 2.35, and n-hexane–ethyl acetate–glacial acetic acid, 31 + 14 + 5) for separation of standards. The results obtained were used for analysis of propolis samples. TLC was shown to be a highly suitable method for rapid analysis of propolis samples. It can be used to establish differences between the amounts of pharmacologically active compounds in propolis from different geographic regions of Croatia.

ADME Data for Polyphenols Characterized by Reversed-Phase Thin-Layer Chromatography

The chromatographic behavior of polyphenols (flavonoids and phenolic acids) has been investigated by reversed-phase thin-layer chromatography (RP-TLC) to establish relationships between chromatographic data and selected ADME (absorption, distribution, metabolism, and elimination) data. Good correlation between chromatographic RM values (a measure of the interactions of the solute with the lipophilic stationary phase and with the hydrophilic mobile phase) and the concentration of the buffer in the mobile phase (r ≥ 0.994) might be taken as a measure of the suitability of the system for estimation of the lipophilicity of the polyphenols. Experimentally obtained RMw values (a measure of the interactions of the solute with the lipophilic stationary phase and with pure water as mobile phase) and ϕ0 (the volume fraction of organic modifier/buffer in the mobile phase for which retention is zero) were compared with ADME data calculated by software packages based on different theoretical approaches. From our results we could establish correlations between chromatographic behavior and lipophilicity, solubility, plasma-protein binding, and oral absorption of the polyphenols investigated.

Pharmacokinetic Parameters of Statin Drugs Characterized by Reversed Phase High-Performance Liquid Chromatography

The aim of this study was to evaluate the usefulness of reversed-phase high performance liquid chromatography in building models that would allow prediction of ADME (absorption, distribution, metabolism, and elimination) parameters of statin drugs in humans. The retention time of each drug was measured using C-18 and C-8 columns with an eluent acetonitrile-water. Furthermore, chromatographic data were compared with physicochemical properties and ADME data calculated by use of 17 different software programs. According to obtained results the calculated values correlate fairly well with chromatographic data which confirm the utility of liquid chromatographic technique for determination of pharmacokinetic parameters.