Purification and characterization of lingonberry fruits phenolics

Abstract

Introduction. Phenolic compounds are the main class of secondary metabolites in plants and are highly associated with health-protective properties [1]. However, crude plant phenolic extracts always contain sugars, pectins, proteins, and other impurities, which may limit the application and activity of the phenolics, also due to the presence of thousands of compounds, large phenolic structures, characterization of phenolic profile remains challenging [2,3]. The present study aimed at developing an efficient fractionation method to obtain purified fractions of lingonberry fruits, which are generally accepted as a good source of phenolic compounds. Materials and methods. The extraction of phenolic compounds was conducted by using 70% acetone (the sample/solvent ratio of 1:15) until exhaustion. The obtained extract was evaporated and subjected to freeze-drying. For purification by column chromatography, the powder was dissolved in 50% methanol (1:40), applied to a glass column, already packed with Sephadex LH-20, and eluted successively with water, different concentrations and amounts of ethanol, methanol, and acetone as mobile phase. Obtained fractions were concentrated by vacuum evaporator, freeze-dried, and analyzed by HPLC-PDA, using ACE C18 column (250 mm × 4.6 mm, particle size 3 μm; ACT, UK) in the solvent system with 0.1% trifluoroacetic acid and acetonitrile under gradient elution. Results. The purest, highest yield phenolic fractions were achieved when washing crude extract of lingonberry fruits (marked F1) with one column volume of water to remove non-phenolic lingonberry constituents, as sugars (waste), four volumes of 20% methanol containing 2% formic acid (F2), four volumes of 50% ethanol (F3) and two volumes of 70% acetone (F4). After HPLC-PDA analysis, even 37 phenolic compounds, belonging to subgroups of monophenols, anthocyanins, phenolic acids, flavonols, catechins, and proanthocyanidins were [...].