Amino Acids from Lichens of the Genera Cladina and Cladonia

Abstract

The protein content in samples of the studied species were determined by the Lowry method [4], which is based on formation of a colored complex from the reaction of the protein with an alkaline solution of copper sulfate and sodium tungstate and molybdate. The protein content in C. stellaris was 0.48%; C. mitis, 0.65; and C. uncialis, 0.62. Amino acids in the aqueous extract that was passed beforehand over a column of Al 2 O 3 (h = 4 cm, d = 2 cm) in order to remove phenolic compounds were detected using TLC. The eluate was chromatographed in ascending mode on Silufol plates using PrOH–H 2 O (70:30, 1), BuOH–HOAc–H 2 O (8:2:2, 2), and EtOH (96%)–NH 4 OH (conc.) (16:4.5, 3). Plates were preheated at 110°C for 30 min. Extract (0.005 mL) was placed at the origin of the chromatography plate using a micropipette. The plate with sample was dried in air for 15 min and placed into a previously saturated chamber. When the solvent front reached the end of the plate, the plate was removed, dried in air to remove completely the solvent, treated with alcoholic ninhydrin (0.5%), and heated in a drying cabinet at 60°C for 10 min [5, 6]. Spots of amino acids were separated best using solvent system 1. Lichen C. stellaris showed five spots with R f1 0.024 (violet) and R f2 0.36, R f3 0.41, R f4 0.51, and R f5 0.63 (orange); C. mitis, two spots with R f1 0.65 (violet) and R f2 0.72 (pink); C. uncialis, two spots with R f1 0.64 and R f2 0.73 (pink). The color intensities of the spots were different. Thus, lichen C. stellaris contained at least five free amino acids; C. mitis and C. uncialis, at least two. The amino-acid composition was studied further using an AAA 339 amino-acid analyzer (Czech Rep.). Total amino acids were extracted from ground lichens (~10.0 g, accurate weight) using refluxing EtOH (80%, 150 mL) in a 250-mL roundbottomed flask on a boiling-water bath for 1 h. Pulp was separated by centrifugation and rinsed with EtOH (80%, 20 mL). The combined extracts were purified of phenolic substances by passage over a column of Al 2 O 3 . The resulting extract was evaporated to a small volume (10 mL), and treated with EtOH (50 mL) to precipitate polymeric compounds. The precipitate was separated by centrifugation. The filtrate was evaporated to a dry solid that was dissolved in citrate buffer (5 mL), a part (100 L) of which was passed over the column (0.6 22 cm, DF-6 ion-exchange resin, 50–65°C, flow rate 25 mL/h) of the amino-acid analyzer. The eluent was sodium citrate buffer of sequential pH values 5.2, 4.0, and 2.0. The qualitative composition of the amino acids in the samples was determined from retention times. The internal standard was a standard mixture of 17 amino acids. The quantitative contents of the detected amino acids were calculated in mg% of the absolute dry raw material. Peak areas were used as the quantitative parameter. The amino-acid analyzer was calibrated using nanomolar standard amino-acid samples. Table 1 presents the analytical results.