A study of the fatty acid and tocochromanol patterns of some Fabaceae (Leguminosae) plants from Turkey I

En este estudio, los contenidos en ácidos grasos, tocoferoles, tocotrienoles y plastocromanol-8 de algunas especies seleccionadas de Fabaceae (Leguminosae), per tenecientes a diferentes géneros (Colutea, Vicia, Lathyrus, Gonocytisus, Lupinus, Hedysarum, Onobrychis, Trigonella) de Turquía, fueron determinadas usando técnicas de GLC y HPLC. Algunas de las especies estudiadas son endémicas de Turquía. Los aceites de semillas de los diferentes taxones de leguminosas contenían los ácidos linoleico, oleico y linolénico como principales componentes. Las proporciones de estos ácidos grasos fueron muy variables entre los géneros de leguminosas. Los ácidos palmítico y esteárico son los principales ácidos grasos saturados en los aceites de semillas. Los patrones de Vicia y Onobrychis mostraron una alta similitud en las medias de las concentraciones de ácidos grasos cualitativos. Los patrones de tocoferoles y tococromanoles en los aceites de semillas resultaron también ser altamente variables entre los géneros investigados aquí. El total de tocoferoles fue superior al de tocotrienoles. Los tocoferoles alfa y gamma fueron los principales tocoferoles encontrados en el conjunto de especies. Los tocotrienoles beta, gamma y delta no se encontraron en la mayoría de los patrones estudiados de leguminosas. Los resultados son discutidos con vistas a los recursos renovables y a la quimiotaxonomía..


INTRODUCTION
The Fabaceae (Leguminosae) is a family of flowering plants comprising about 269 genera and 5100 species (Mabberley, 1997) and it is one of the largest plant families in Turkey and in the world .It has 68 genera and more than 900 species in Flora of Turkey (Davis, 1970(Davis, , 1988;;Seçmen et al., 1989).The family is important as food plants, especially leguminate (beans, gram, peas), oil (soybean, ground nut), but also for tanbarks, timber, copal, gums, insecticides and cultivated ornamentals, as well as medicinal plants (Seçmen et al., 1989;Tsevegsuren et al., 1998).
Leguminosae is well suited with respect to chemical components.Lipids from some more common Leguminosae have been investigated to some extent, other legume lipids have not been studied in any great detail because of their low lipid content and limited or negligible use for oil purposes (Gunstone et al., 1972;Kleiman, 1988;Chowdurry, 1984Chowdurry, , 1986Chowdurry, , 1995;;Ucciani, 1995;Grela and Gunter, 1995).Some species of the family Fabaceae (Leguminosae) are also sources of cheap protein for both humans and animals (Tewatia and Wirk, 1996).
Omega 3-fatty acids are polyunsaturated fatty acids which have been associated with many health benefits (Freeman, 2000).Linoleic acid is needed for a normal immune response and an essential fatty acid deficiency impairs B and T cell-mediated responses (Meydani et al., 1991).Tocopherols naturally present in seeds have been strongly , Grasas y Aceites correlated with the polyunsaturated fatty acids since they counteract the potential oxidative stress caused by fats in the diet (Anttolainen et al., 1995).
Seed oils with a substantial amount of very long chain FAs have attracted attention because of their value for industrial purposes.Furthermore these compounds can be of chemotaxonomic significance (Bagci et al., 2003).The fatty acid and tocopherol composition of plant seed oils can provide characteristic information in order to confirm taxonomical and phylogenetic relationships in the plant kingdom (Goffman et al., 1999;Bagci et al., 2004).Relatively complete data on the fatty acid composition of seed oils from Leguminosae were first used for chemotaxonomic consideration by Wolff and Kwolek, (1971).

Plant samples
Some seed specimens were collected in natural habitats from different regions of Turkey and some of the samples were obtained from the seed bank in Aegean Agricultural Research Institute, Izmir.Studied plant samples from different genera are listed in Table I.

Chemical Analysis
2.2.1.Oil Extraction and preparation of fatty acid methyl esters (FAME) Impurities were removed from the seeds and the cleaned seeds were ground into powder using a ball mill.Lipids were extracted with heptane in a straight through extractor.The triglycerides were transesterified to methyl esters with potassium hydroxide in methanol according to ISO method 5509 (1989).

Capillary GLC
The fatty acid methyl ester composition was determined on three different gas chromatographs, Hewlett-Packard HP5890 (A), HP6890 (B) and Unicam -610 (C) each equipped with a fused silica WCOT capillary and FID: A) Silar 5 CP, 50 m.x 0.25 mm ID, 0.24 m film thickness, nitrogen as carrier gas, 1:50 split ratio, pressure 160 kPa, oven temp.Data analysis was done with a chromatointegrator D 2500 (Merck-Hitachi) and a Chemstation integration software, respectively.Peak identification was achieved by comparison of relative retention times with those obtained from test mixtures of known composition on three different columns.

Tocopherol analysis
Tocochromanols were determined by highperformance liquid chromatography (HPLC) according to the procedure of Balz et al. (1992).An aliquot of a solution of 50 mg oil in 1 ml heptane was injected into an HPLC system via a Rheodyne valve with a sample loop volume of 20 µl.Tocopherols were separated on a LiChrospher 100 Diol phase, 5 m particle size (Merck, Darmstadt, Germany).HPLC column 25 cm x 4.6 mm ID with an additional guard column 4mm long and 4 mm ID, filled with LiChrospher Si 60, 5 µm particle size.The system was operated with an eluent of heptane/tert.-butylmethyl ether (96+4v/v) and detection by a fluorescence detector F-1000 (Merck, Darmstadt) at 295 nanometer excitation wavelength and 330 nm emission wavelength.A D-2500 Chromato Integrator (Merck, Darmstadt) was used for data acquisition and processing.Calibration was done by external standards with α-, β-, γ-and δ-tocopherol (Calbiochem, Bad Soden, Germany).Tocotrienols are calculated with the same response factors as the corresponding tocopherols and plastochromanol-8 was calculated with the same response factor as gamma-tocopherol (Balz et al., 1992).

RESULTS AND DISCUSSION
In this study, the fatty acid and tocochromanol compositions of the seeds of some selected Fabaceae species from Turkey were determined.The results of the fatty acid analysis and the oil yield of the taxa belonging to Gonocytisus, Lupinus, Colutea, Vicia, Lathyrus, Hedysarum, Onobrychis and Trigonella genera (totally 17 taxa) are shown in Table I; the tocopherol and tocotrienol contents of the studied taxa are shown in Table II Vicia, Lathyrus, Trigonella and Onobrychis genera have the highest taxa number in Flora of Turkey in family Fabaceae (Davis, 1970;1988) The total lipid of the studied leguminous species were found between 18.7% and 32.3% (Vicia canescens subsp.latistipatula and Trigonella cretica, respectively) (Table I).The oil contents of the studied legumes belonging to different genera showed significant quantitative differences.Although qualitative differences were also found, the fatty acid patterns of the studied leguminous taxa belonging to different genera showed uniform fatty acid (FA) composition.
Analysis showed that low molecular acids (lauric, myristic, pentadecanoic acids) from the saturated fatty acid (SFA) were absent or present at trace levels in the Leguminosae seed oils.Palmitic acid (16:0) was the highest SFA in Trigonella cretica (12.9%),Lupinus varius (12.8%) and Colutea melanocalyx (10.7%) respectively.This is also a very constant lipid constituent in most of the Leguminous genera seed oils.It is possible to say that this fatty acid is not a highly variable component in the leguminous genera pattern.16:1, 16:2, Margaric (17:0) and margaroleic (17:1) acids were not detected or in very small amounts (Table I).
The other SFA of the legume seed oils (20:0 and 24:0, arachidic and lignoceric acid) in the studied species, were shown to be lower than 1% except V. cappadocica and Trigonella cretica.But 22:0 (Behenic acid) was different from these FA in the studied genera patterns.Trigonella, Lathyrus laxiflorus, Onobrychis major, two subspecies of V. canescens and Hedysarum species contained this FA in more than 1%.Some researchers indicated that oils with high levels of long chain SFA such as behenic acid may be difficult for the digesting enzymes in humans and animals (Hilditch et al., 1964;Balogun and Fetuga, 1985;Akpinar et al., 2001).
Among the unsaturated fatty acids (USFA), oleic and linoleic acid were the major constituents of the studied legume seed oil.The highest percentages of oleic acid was determined in Trigonella cretica (46.9%),Onobrychis hypargyrea (34.4%) and Lathyrus laxiflorus subsp.laxiflorus (30.4%) respectively (Table I).On the other hand, Vicia michauxii var.stenophylla (12.3%),Colutea melanocalyx (12.7%),Gonocytisus dirmilensis (13.19%) and Onobrychis huetiana (13.3%) showed the lowest oleic acid composition in the seed oils.Linoleic acid was determined as the major constituent of all the seed oils except Trigonella cretica.This fatty acid comprized more than half of the seed oil in Colutea melanocalyx (62.8%),Gonocytisus dirmilensis (67.4%),Lupinus varius (57.8%),Vicia cappadocica (50.9%) and Onobrychis major (51.7%).A high content of this component was found to be characteristic for the legume seed oil.Vicia patterns show a wide variation in this component and it was also found in small amounts in Trigonella, Vicia michauxii var.stenophylla (Table I).The seed oils of all the investigated species were richer in oleic and linoleic acid than in linolenic acid.In the Akpinar et al. (2001) study, while most of the studied Vicia samples showed this result, Vicia hybrida contrasted from the other Vicia taxa.At the same time , oleic and linoleic acids were determined to be the major unsaturated fatty acid in Psophocarpus tetragonolobus (L.) (Fabaceae) DC. (winged bean) oil which is used as a food in the nutrition of some countries (Higuchi et al., 1982).
Linolenic acid (18:3) in the legume seed oils was generally found to be lower than 10%, except in a few taxa like Hedysarum cappadocicum (21.1%),Vicia michauxii var.michauxii (39.1), two Lathyrus taxa (16.5-17.9%)and Onobrychis huetiana (18.3%) and O. major (11.2%).In the others, it was generally lower than ten percent of the oil.This fatty acid was the most variable component among the leguminous genera when compared with the other constituents.
Total saturated fatty acid (TSFA) levels of the studied legume seed oils did not show variation between genera except in the Lupinus (17.36) and Trigonella (21.5%) patterns.These two samples had high saturated FA compositions when compared with the others.On the other hand Lathyrus laxiflorus (6.2%) and Onobrychis huetiana (7.9%) samples were proven to have a very low concentration of TSFA in the oils.It is possible to say that the total saturated fatty acid content of the legume seed oils is between 10-20%.TUSFA composition of the seed oils is generally between 75-90% in the oil for most of the studied specimens.But Vicia patterns and Onobrychis huetiana containied lower than 70% TUFA (Table I).
Erucic acid (22:1) has undesirable effects on the metabolisms of animals and humans (Baudet, 1976;Feil and Stamp, 1993;James, 1994).But it is not detected in most of the legume seed oils except Gonocytisus dirmilensis (0.2%) and Onobrychis huetiana (1.6%).Garcia-Lopez et al. (2001) reported that some Lupinus sp.oils contained high concentrations of palmitic and linoleic acid and that erucic acid was not reported in any of the lupin oils analyzed while eicosanoic acid (22:0) was found only in Lupinus mexicanus.Some Lupinus seed oils (Lupinus albus L., L. angustifolius L., L. luteus L., and L. mutabilis Sweet.(lupin oil, Tarwi seed oil) are reported as oil plants in the world (Aitzetmuller, 1997).
The seed oils of the leguminous species contained very low levels of monoenoic fatty acids which were lower than polyenoic FA. (Table I).This is important for the quality of oils consumed as a food resource.
The results of the present study, as far as unsaturated fatty acid content is concerned, are supported by previous Leguminous studies (Sengupta and Basu, 1978;Daulatab et al., 1987;Tharib and Veitch, 1983;Hamberg and Fahlstadius, 1992;Liu et al., 1995).All these studies showed that the saturated and particularly unsaturated FA contents of Fabaceae seed oils are closely allied to each other and that the main components in the oils are linoleic-oleic type fatty acids.
Tocopherols, together with tocotrienols and plastoquinones, are known as tocochromanols (Seher and Ivanov, 1973;Velasco et al., 2000) and some of them exhibit vitamin E activity.With this study some Turkish leguminous seed oils were examined as potential sources of natural tocopherols and tocotrienols.The tocochromanol (tocopherol and tocotrienol) derivatives α, β, γ and δ-tocopherols and tocotrienols and 8-plastochromanol were detected in some of the studied Fabaceae seed oils (Table II).α and γ-tocopherol were detected as the most abundant tocopherol components in all of the studied taxa except Lathyrus inconspicuus, Onobrychis huetiana, and O. hypargyrea.This is also reported for some Fabaceae taxa from Bulgarian Flora (Arachis hypogaea, Gleditsia triacanthos, and Robinia pseudoacacia) (Ivanov and Aitzetmuller, 1998).Colutea and Trigonella species had the highesttocopherol level (85.7 and 89.4% respectively) in all of the studied taxa.On the other hand, Hedysarum cappadocicum (69.9%),Vicia michauxii var.stenophylla (80.2%),V.cappadocica (93.4%), and in general Onobrychis species (except O. huetiana and O. hypargyrea) were characterized by the high content of gamma tocopherol in each oil (Table II).tocopherol was determined in the Lathyrus inconspicuus(51.7%),L. laxiflorus subsp.laxiflorus (46.7%) and O. huetiana (34.3%) at a high level, but it was not detected in most of the others (Table II).Onobrychis genera patterns showed the highest fatty acid and tocochromanol variability in the studied Leguminosae.This requires further investigation in view of both fatty acid and vitamin resources as well as chemotaxonomic relationships.
The tocochromanol analysis of the Fabaceae seed oils from Turkey showed that they contained low percentages of tocotrienols.The highest -tocotrienol was found in Onobrychis hypargyrea (65.6%),Lupinus varius (45.9%) and Gonocytisus dirmilensis (23.3%) and the other taxa had lower than 10%.Other tocotrienols were either not found or determined in very small amounts .Plastochromanol -8-was found in trace amounts in most of the studied species, except two Lathyrus taxa (Table II).The analysis results showed that the total tocopherol content of the studied Fabaceae species was higher than the total tocotrienol level (Table II).On the other hand, there are some clues on the variation of the tocochromanols in family patterns.Lupinus, Gonocytisus and Onobrychis genera were demonstrated to have different tocopherol and tocotrienol patterns in means of individual and total amounts compared to other genera.It can be possible to differentiate the taxa in the following groups: 1) Having high -tocopherol, like Colutea and Trigonella patterns.2) Having high γ -tocopherol, as in Hedysarum, Vicia and some Onobrychis patterns.
It is determined that linoleic and oleic acid are abundant components in most of the leguminous genera and this may be a characteristic of the family or of some genera.But some results showed that the linoleic -palmitic type FA pattern is typical for some genera like Cassia nodosa, Berlinia auriculata, Bauhinia monandra, Parkia clappertoniana (Balogun and Fetuga, 1985), some Astragalus sp (Bagci and Vural, 2001), some Ebenus species reported from Turkey (Azcan et al., 2001) or linoleic -oleic -palmitic type, like in some Lathyrus species (Bagci et al., 2001;Bagci and Sahin, 2004) and some Crolataria species (Fabaceae) (Chowdury and Banerjii, 1995).The seed oils of all leguminous members contained very low levels of linolenic acid, both in this study and in reports from the literature, cited above (Hemavathy and Prabhakar, 1989).
Such a favourable composition of USFA in most of the legume seed oils suggests that some of these species may have potential as renewable sources in oilseed crop for the food and oil industry, if growth and different regional yields can be improved.The present study shows the infrafamilial variability for fatty acid and tocochromanols in Fabaceae.With this study, it was confirmed that patterns of fatty acids 382 Grasas y Aceites and tocochromanols may be a good chemotaxonomic criterion for this family, particularly with regard to the studied taxa.However, further studies are required to confirm the results obtained so far.The evaluation of fatty acids and tocochromanols in a wider range of species of the Fabaceae is suggested as a powerful tool that might contribute to characterize the chemotaxonomic and evolutionary relationships among the tribes and genera of Fabaceae.