Three spectrophotometric methods for the quantitative determination of different flavonoid groups and total phenolics in Croatian propolis samples were optimised and validated. The assay based on the formation of aluminium chloride complex (with galangin as a standard) was applied to the quantification of flavones and flavonols, while the 2,4-dinitrophenylhydrazine method (with pinocembrine as a reference) was used for the quantification of flavanones. Total phenolic 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 (with respect to time, temperature and concentration of extraction solvent) were determined, and final conditions for the extraction were established (80% ethanol, 1 h at the room temperature). The appropriate ratio between the mass of raw propolis and the extraction solvent volume was also established. By the application of the optimised method of extraction, 10 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, the content of waxes as the main inactive constituents was determined in order to observe the level of their migration from crude propolis to the prepared tinctures.
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 A 0 /e (where A 0 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.
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.
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