ABSTRACT
Plants accumulate different types of phenolic material in their tissue as a response to biotic as well as abiotic stress. Monomeric polyphenols and smaller oligomers can serve as protection against ultraviolet radiation or prevent oxidative tissue damage, while larger molecules such as tannins can be the plant's reaction to an infection or physical damage. Therefore, characterization, profiling, and quantification of diverse phenolics can provide valuable information about the plant and the stress status at any given time. A method was developed that allows the extraction of polyphenols and tannins from leaf tissue, followed by fractionation and quantification. Extraction was performed with liquid nitrogen and 30% acetate-buffered ethanol. The method was tested with four cultivars under varying extraction conditions (solvent strength and temperature) and showed great improvements of the chromatography that would otherwise be impacted by tannins. The separation of tannins from smaller polyphenols was achieved by bovine serum albumin precipitation and resuspension in a urea-triethanolamine buffer. Tannins were reacted with ferric chloride and analyzed spectrophotometrically. Monomeric non-protein-precipitable polyphenols were then analyzed via HPLC-DAD from the supernatant of the precipitation sample. This way, a more complete spectrum of compounds can be analyzed from the same plant tissue extract. With the fractionation suggested here, hydroxycinnamic acids and flavan-3-ols can be separated and quantified with good accuracy and precision. Possible applications include the assessment of plant stress and response monitoring using the total concentrations of polyphenols and tannins, as well as the ratios between those compound classes.
