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 densitometric 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% AlCl 3 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 fluorescence colors. A better 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.