Purification of proanthocyanidins from lingonberry leaves extracts by gel filtration chromatography

Abstract

Introduction: Proanthocyanidins, which are the most abundant polyphenol class in the human diet, have positive health effects on a variety of pathologies such as inflammation, diabetes, cardiovascular, neurodegenerative diseases, and cancer. Moreover, proanthocyanidins prevent Escherichia coli, which is the main cause of urinary tract infections, from adhering to host urinary tract epithelial cells [1,2]. The present study aimed to establish an efficient fractionation method to obtain a proanthocyanidins fraction from lingonberry leaves extracts, which are generally accepted as a good source of these compounds. Materials and methods: The extraction of proanthocyanidins was conducted using 80% acetone (the sample/solvent ratio of 1:25) until exhaustion. The extract was concentrated by vacuum evaporation and freeze-dried to yield crude aqueous soluble fraction as a light brown powder. For fractionation by column chromatography, the powder was dissolved in 50% methanol (1:40), applied to a Sephadex LH-20 column and eluted successively with water, different ethanol or methanol and acetone aqueous solutions as mobile phase. Each fraction was concentrated by vacuum, freeze-dried and analyzed by reversed-phase HPLC-PDA. Results: The purest proanthocyanidins fraction was achieved when washing applied extract with two columns of water to remove non-phenolic lingonberry constituents, phenolic acids, arbutin, and four columns of 50% ethanol to elute flavonols and monomeric flavan-3-ols. The bound fraction, which was enriched in proanthocyanidins, was eluted with two volumes of 70% acetone. HPLC-PDA results showed that the sum of identified proanthocyanidins (procyanidins A1, A2, B1, B2, B3, C1) was 100.46±3.2 mg/g DW, and according to strong absorbance intensity at the specific to proanthocyanidins detection wavelength of 280 nm, at least 13 unidentified proanthocyanidins were detected in the final fraction. Conclusion.[...]