INVESTIGACIÓN Fatty acid composition of leaf lipids of some Alchemilla L ( Rosaceae ) species from Northeast Anatolia ( Turkey )

Se han estudiado mediante cromatografía gaseosa la imposición en ácidos grasos de los lípidos totales de hojas de 23 especies correspondientes a cinco series del género Alchemilla L. Los ácidos grasos identificados y cuantificados son C 12:0, C 14:0, C 16:0, C 18:0, C 20:0, C 22:0, O 24:0, C 26:0 y C 28:0 como saturados, y C 16:1, C 18:1, C 18:2 y C 18:3 como insaturados, siendo el O 18:2 y C 18:3 los mayoritarios. El contenido más elevado de ácidos grasos se observó en A. hirtipedicellata (78,34%) de la ser. Elatae, A.erytropoda (68,74%) de la ser. Pubescentes, A. rizensis (76,47%) de la ser. Sericeae, A. dura (73,94%) de la ser Calycinae y A. psedocartalinica (74,58 %) de la ser. Vulgares. El ácido graso C 18:1 se determinó en cantidades traza, mientras que los ácidos C 20:0, C 22:0, C 24:0 y C 28:0 no se determinaron en la mayoría de estas especies. Se discuten el valor taxonómico y las implicaciones filogenéticas de los resultados.


INTRODUCTION
The value of fatty acid patterns to establish systematic relationships among plants is becoming increasingly important.There are many studies in the literature dealing with phylogenetic and taxonomical aspects in relation to fatty acid composition in higher plants (Shorland, 1963;Harborne and Turner, 1984;Hegnauer, 1989).
The genus, Alchemilla L. (Rosaceae) includes more than 1000 species with a holarctic distribution.The genus also occurs on mountains of eastern Africa, from Abyssinia to the Cape of Good Hope, in Madagascar, South India, Sri Lanka and Java (Izmailow, 1981).
Alchemilla L. is made of perennial herbs with woody rhizome (Pawlowski and Walters, 1972).
According to Pawlowski and Walters (1972) all Alchemilla species in Turkey belong to section Alchemilla including 3 subsections and 6 series with 50 species.With more recent studies the number of Alchemilla species in Turkey reached to 66 (Kalheber, 1994;Hayirlioglu and Beyazoglu, 1997;Hayirlioglu-Ayaz and Beyazoglu, 1997;Hatyirlioglu-Ayaz, 1997).The leaves and roots of some species of Alchemilla are well-known folk medicinal plants in Turkey and used as diuretic, reinforce and against constipation (A.pseudocartalinica ÔUZ. and A. arvensis (L.) Scop) (Baytop, 1984).However, in spite of extensive taxonomical and cytological studies, chemotaxonomical data concerning Alchemilla are very scarce.This study is the first report on fatty acid composition in leaves of some Alchemilla species selected from 5 series, namely, ser.Elatae, Pubescentes, Sericeae, Calycinae and Vulgares.
In the present paper, we report a comparative study on the fatty acids composition in leaves of 23 species of Alchemilla L. distributed in Northeast Anatolia (Turkey).Leaves were ground into a fine powder with a Waring blender and lipids were extracted with chloroform:methanol (2:1, v:v) according to Bligh and Dyer (1959).The extract was filtered through a sintered funnel, followed by 4 washes with the solvent mixture.The pooled filtrate was subjected to Folch washing (Folch et al., 1957) using 0.9% sodium chloride one fourth of the total filtrate to remove water-soluble impurities.The chloroform: methanol phase was subsequently evaporated to near dryness in a rotary vacuum evaporator, and the residue was again dissolved in 10 ml of chloroform.A suitable aliquot was evaporated to dryness to determine the oil content by weighting.The lipids were saponified and fatty acids methylated according to Folch et al. (1957).Fatty acid methyl esters were prepared from three independent lipid extraction of leaf samples.

Gas Chromatography Analysis of Fatty Acid Methyl Esters
The methylated sample solutions were analyzed with a Varian 3300 gas chromatograph (GC) equipped with a flame ionization detector (FID) and a HP-1 silica capillary column (crosslinked methyl silicone gum, 0.17 inm film thickness, 25 m, 0.32 mm i.d.).Hydrogen was used as the carrier gas at a flow rate of ca.40 cm/s, and the column oven temperature was programmed from 100 °C to 290 °C at 6 °C/min heating rate.The injector and detector temperatures were held at 260 °C and 290 °C, respectively.Peak areas were measured with a Merck-Hitachi D-2000 integrator.Peaks were identified by the comparison with standard fatty acid methyl esters.Methyl heptadecanoate (Me-17:0) was used as internal standard.The C 18:2 and C 18:3 acids are overlapped with the HP-1 column and not properly separated, so their amounts were calculated as their sum.
The chemistry confirms the morphological differences.Thus, for the unsaturated C 18:2 and C 18:3 acids, the values varied between 50.82 and 78.34% for ser.Eiatae, between 49.01 and 68.74% for ser.Pubescentes, between 51.34 and 73.94% for ser.Calycinae, and between 65.44 and 74.88% for ser.
(c) Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://grasasyaceites.revistas.csic.esReher (1991) investigated the seed fatty acid composition of 7 species of the Sanguisorbeae and 7 species of the Potentilleae subfamilies of Rosaceae.The content of C 18:3 and C 18:2 amounted together to more than 70% in 11 species.The ratio of linolenic acid to linoleic acid was shown to be significantly higher in Potentilleae (1.4 to 2.4) than in Sanguisorbeae (0.4 to 10).The C 18:1, C 18:2 and C 16:0 acids were also identified as the major fatty acids in the seeds of cherry laurels (Ayaz et al., 1995) and almonds (García-López et al., 1996).
Consequently, the G 18:2, G 18:3 and G 16:0 acids were identified as the main fatty acids in Alchemilla leaves as well as the seeds of most species of Rosaceae family.From a chemotaxonomic point of view, the prese'nt results suggest that fatty acid composition explains remarkable differences between species and series in the genus Alchemilla.
The highest content of G 18:2 and G 18:3 acids was found for ser.Elatae, Sericeae and Galycinae, respectively.The content of G 16:0 acid was generally found higher in species of ser.Pubescentes than in other series.Our results suggest that fatty acid composition G 16:0, G 18:2 and G 18:3 acids can be used as taxonomic markers in the genus Alchemilla.