Amino-Acid Composition of Asperula odorata Herb

Abstract

The genus Asperula L. (Rubiaceae Juss.) numbers over 200 species in the world flora that are distributed in Europe, North America, Ukraine, Russia (Urals, Siberia), and the Caucasus. The genus is represented by 40 species in Ukraine, the most common of which is A. odorata L. [1]. The plant is not official but is widely used in folk medicine as a diuretic, sudorific, sedative, and wound-healing agent. The herb of A. odorata has previously yielded phenolcarboxylic acids, coumarins, flavonoids, tanning agents, iridoids, and steroidal saponins; the subterranean organs, anthracene-derivatives of the alizarin group [2, 3]. We studied the component composition of essential oil from the herb. Antimicrobial and antifungal activity was found for the CHCl 3 extract from the herb of this plant [4]. The goal of the present research was to study the amino-acid composition of A. odorata herb as the air-dried sample collected during flowering in May-June 2012 in Ivano-Frankovsk Region of Ukraine (herbarium specimen No. 14/12, preserved in the herbarium of the Pharmacognosy Dept., NPhU). The content of total N was determined on an EA-3000 elemental analyzer (EuroVector, Italy) using a thermalconductivity detector (TCD). The analyzed raw material contained 3.1% N. The protein content calculated using the common factor of 6.25 was 19.38%. Free amino acids were determined by adding to the raw material an aqueous solution (0.1 N) of HCl containing -mercaptoethanol (0.2%) and irradiating in an ultrasonic bath for 2 h at 50°C. Total free and bound amino acids in a weighed portion of raw material (10 mg) were determined by adding aqueous HCl (6 N) containing -mercaptoethanol (0.4%) and irradiating in an ultrasonic bath for 24 h at 110°C [5]. Quantitative analysis of free and bound amino acids in the studied samples was performed on an Agilent Technologies Model 1100 liquid chromatograph connected to a G1379A flow vacuum de-gasser, a G13111A 4-channel low-pressure gradient pump, a G1313A automated injector, a G13116A column thermostat, and a G1316A diode-matrix detector. Standard solutions of amino acids (TU 6-09-3147-83) were used for the quantitative determination. Chromatography used a column (4.6 50 mm) packed with octadecylsilyl sorbent (1.8 m, Zorbax-XDB-C18) and a protective guard-column. The mobile phase was solution A (0.05 M aqueous NaOAc adjusted to pH 6.5 with 1–2% AcOH with added 0.3% THF) and solution B (0.10 M aqueous NaOAc and CH 3 CN in a 23:22 ratio adjusted to pH 6.5 with 1–2% AcOH). The mobile phase flow rate was 1.5–2 mL/min. The eluent operating pressure was 220–275 kPa, column thermostat temperature 50°C, sample volume 2 L. Detection was made using a UV detector with measurement scale 1.0, scan time 0.5 s, detection wavelength 265 nm. Amino acids were identified by retention time and comparison with standards. Table 1 presents results for the quantitative contents of free and bound amino acids in A. odorata herb. The study identified in the herb 21 free and 19 bound amino acids, 9 of which were essential (valine, isoleucine, leucine, lysine, arginine, histidine, methionine, threonine, phenylalanine). Table 1 shows that aspartic and glutamic acids, proline, leucine, alanine, serine, glycine, and arginine dominated in A. odorata herb. The content of free amino acids was 0.07%; of bound, 0.96%. A calculation of the protein content from the amino-acid content according to the RF State Pharmacopoeia [6] found that A. odorata herb contained 7.71% protein. Thus, the difference in protein content determined by the different methods was apparently explained by several factors, e.g., the hypothetical value of the factor for calculating the N content of the raw material protein and the presence in the raw material of other classes of N-containing compounds.