Concentrates of polyunsaturated fatty acids from Lappula squarosa seed oil

Abstract

Polyunsaturated fatty acids (PUFA), gamma-linolenic (GLA), alpha-linolenic (ALA), and stearidonic (SA), exhibit high biological activity [1]. Various attempts have been made to prepare PUFA fractions enriched in one PUFA or another for use in drugs and dietary products. A method based on the different solubilities of sodium salts of FA in EtOH that was used for this enabled PUFA concentrates to be obtained from seed oil of Echium fastuosum, Borago officinalis, and Anchusa azurea (Boraginaceae) [2]. We obtained good results for the isolation of FA fractions enriched in ALA and GLA from linseed oil and seed oil of B. officinalis by using low-temperature crystallization at –80°C from CH3CN:Me2CO (7:3) [3]. The resulting fraction (mother liquor) from linseed oil, which contains 55% ALA, consisted of 88.5% ALA; from total FA of B. officinalis with 23.4% GLA, 88.3% of this FA. Crystallization of FA from hexane solution (10%) at –70°C enabled GLA and SA concentrates to be obtained from E. fastuosum seed oil. This method could be used on an industrial scale [4]. Fractions with high GLA and SA contents could be obtained by chromatographic methods such as HPLC, CC over silica gel and over silica gel impregnated with AgNO3, and centrifugal preparative chromatography (CPC). However, they are suitable only for laboratory studies [5]. Extraction by supercritical CO2 in order to isolate and enrich oils in GLA and SA did not give the desired result [6–8]. Fractionation of urea complexes of FA was used to isolate epoxytetraenoic acid from total FA of fish and microalgae [9]; a mixture of GLA with ALA, from FA of Ribes nigrum seed oil [10] and from FA of seed oil of A. azurea, E. fastuosum, and S. scophila [11]. As a rule, a urea:FA ratio of 4:1 (by mass) is used [9]. Production of FA fractions enriched with PUFA using urea complexes (clathrates) depends primarily on the initial set of PUFA and their content in the oil. The main difficulty in producing FA fractions with a high SA content is the presence in oils of SA and its precursor ALA. Until now, plants with oil that contains only SA have not been found [1]. Studies of seed lipids from several species of the family Boraginaceae indigenous to the Republic of Bashkortostan found that Lappula squarosa seed lipids contained >70% PUFA including >16% SA [12]. Separation of the FA from Lappula seed oil as the urea clathrates afforded fractions enriched with PUFA. For this, mild alkaline hydrolysis (10% KOH solution in MeOH, 70–80°C, 30 min) isolated from the oil the total FA. EtOH was used as the solvent to prepare the urea complexes because it was shown earlier that enrichment of PUFA in it occurred better by using FA from B. officinalis seed oil as an example [13]. FA were separated in two steps [13]. In the first step, a mixture of FA (0.8 g) was treated with a saturated solution of urea in EtOH (FA:urea ratio 1:3 by mass), stirred on a magnetic stirrer with heating (60°C) until totally dissolved, and left at room temperature for 3 h and then at 10°C for 24 h. The resulting clathrate crystals were separated on a Schott filter and washed with cold saturated urea solution in EtOH to produce mother liquor 1 and a precipitate of crystalline FA urea complexes, clathrates 1. The yield of FA fractions (mass% of starting FA) was 36.25 and 57.5, respectively (Table 1). Next, the FA compositions of the mother liquor and clathrates were analyzed. The mother liquor was evaporated to half the volume in a rotary evaporator, diluted with H2O, and extracted with Et2O in order to isolate the FA from it. The Et2O extracts were washed with H2O and dried over anhydrous Na2SO4. The solvent was evaporated. The resulting FA (0.29 g, mother liquor 1) were methylated by diazomethane and studied by GC (Table 1).