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

Flavonoids are polyphenolic compounds isolated from a wide variety of plants. They are benzo-γ -pyrone derivatives, which resemble coumarin. Flavonoids (together with other polyphenols, e.g. phenolic acids) are attributed to have numerous physiological activities. One of the most concentrated sources of those compounds is propolis. It is resinous substance, collected by the honeybees from various plant sources. Its composition and amount of pharmacologically active compounds depends on the geographic origin. In our work we have chosen to develop a new TLC method, which will be suitable for the analysis of complex mixtures such as propolis. Two-dimensional TLC together with densitometric evaluation seemed to be 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 Croatian geographic regions. To establish the RF values, standard solutions of 9 flavonoids and 6 phenolic acids were first applied as 10 mm bands to the chromatographic plate. Plates were developed in vertical separating glass chambers, previously saturated with 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 wave UV light and under long wave UV light after spraying with 1% AlCl3 ethanol solution and RF values were calculated. All standards were chromatographed again in groups of 3 or 4 together with propolis sample. First, plates were developed in mobile phase 1 (or 2), evaporated, standard solutions were applied again, rotated for 90° and chromatographed again in mobile phase 2 (or 1). The presence (or absence) of all standards was determined according to their RF values and fluorescence colors. Better combination of mobile phases was chosen, mass of present standards was adjusted and those standards were again individually chromatographed together with propolis sample. Quantitative evaluation was carried out using Camag Reprostar 3 densitometer (CAMAG, Switzerland). Two-dimensional TLC was shown to be highly suitable method for the given task. We proved that method could be used in analysis of different propolis samples in order to identify and quantify main pharmacologically active compounds.

propolis ; flavonoids ; phenolic acids ; 2D TLC ; densitometry

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