Searching for health beneficial n-3 and n-6 fatty acids in plant seeds

Various plant seeds have received little attention in fatty acid research. Seeds from 30 species mainly of Boraginaceae and Primulaceae were analysed in order to identify potential new sources of the n-3 PUFA α-linolenic acid (ALA) and stearidonic acid (SDA) and of the n-6 PUFA γ-linolenic acid (GLA). The fatty acid distribution differed enormously between genera of the same family. Echium species (Boraginaceae) contained the highest amount of total n-3 PUFA (47.1%), predominantly ALA (36.6%) and SDA (10.5%) combined with high GLA (10.2%). Further species of Boraginaceae rich in both SDA and GLA were Omphalodes linifolia (8.4, 17.2%, resp.), Cerinthe minor (7.5, 9.9%, resp.) and Buglossoides purpureocaerulea (6.1, 16.6%, resp.). Alkanna species belonging to Boraginaceae had comparable amounts of ALA (37.3%) and GLA (11.4%) like Echium but lower SDA contents (3.7%). Different genera of Primulaceae (Dodecatheon and Primula) had varying ALA (14.8, 28.8%, resp.) and GLA portions (4.1, 1.5%, resp.), but similar amounts of SDA (4.9, 4.5%, resp.). Cannabis sativa cultivars (Cannabaceae) were rich in linoleic acid (57.1%), but poor in SDA and GLA (0.8, 2.7%, resp.). In conclusion, several of the presented plant seeds contain considerable amounts of n-3 PUFA and GLA, which could be relevant for nutritional purposes due to their biological function as precursors for eicosanoid synthesis. Practical applications N-3 PUFA are important for human health and nutrition. Unfortunately, due to the increasing world population, overfishing of the seas and generally low amounts of n-3 PUFA in major oil crops, there is a demand for new sources of n-3 PUFA. One approach involves searching for potential vegetable sources of n-3 PUFA; especially those rich in ALA and SDA. The conversion of ALA to SDA in humans is dependent on the rate-limiting Δ6-desaturation. Plant-derived SDA is therefore a promising precursor regarding the endogenous synthesis of n-3 long-chain PUFA in humans. The present study shows that, in addition to seed oil of Echium, other species of Boraginaceae (Cerinthe, Omphalodes, Lithospermum, Buglossoides) and Primulaceae (Dodecatheon, Primula), generally high in n-3 PUFA (30–50%), contain considerable amounts of SDA (5–10%). Therefore, these seed oils could be important for nutrition.


Introduction
The n-3 and n-6 polyunsaturated fatty acids (PUFA) are essential for normal human growth and development and further appear to be important for the prevention and treatment of several diseases (coronary heart disease, arthritis, inflammatory disorders). They are involved in the eicosanoid synthesis and are components of cellular membranes [1][2][3].
Modern Western diets are not only low in n-3 PUFA but also too high in n-6 PUFA, deriving mainly from meat and vegetable oils rich in LNA (corn, sunflower and safflower) [1]. Thus, nutritional pro-and anti-inflammatory eicosanoid precursors become imbalanced. The main dietary source of EPA and docosahexaenoic acid (DHA; C22:6c4,7,10, 13,16,19) is marine fish fed on marine microbes (micro algae, diatoms, bacteria). By themselves, mammals and marine fish have a very limited capacity to produce the important n-3 LC-PUFA.
Due to the increasing world population and the overfishing of the seas the supply of n-3 LC-PUFA is limited. Several work groups pursue strategies to develop alternative sources of n-3 PUFA such as transgenic plants, aquaculture of marine microbes and fish, and searching for plant oils rich in n-3 PUFA [4,5]. There are only low amounts of n-3 PUFA in major oil crops such as soybean, sunflower and palm oil [6,7]. The PUFA synthesis occurs in all plant cells, and therefore PUFA are present in the leaves and roots, but mainly in seeds in varying amounts [8]. Several studies examined Boraginaceae species as relevant sources of health beneficial n-3 and n-6 fatty acids [9][10][11][12].
ALA is the most abundant n-3 PUFA in plant oils, such as linseed oil [7,13]. In the human body, the endogenous synthesis of n-3 LC-PUFA by elongation and desaturation of ALA to EPA and particularly to DHA is limited [14]. A further interesting n-3 PUFA is stearidonic acid (SDA; C18:4c6, 9,12,15). SDA is present only in species of few plant families, for example Boraginaceae [15]. Plant-derived SDA is a promising precursor regarding endogenous synthesis of n-3 LC-PUFA in humans, because the ratelimiting D6-desaturation of ALA to SDA already occurred effectively in the plant.
The g-linolenic acid (GLA; C18:3c6, 9,12) is also a D6-desaturation product but of LNA and is found primarily in vegetable oils. High levels of GLA are found mainly in Boraginaceae and in other families such as Ranunculaceae and Primulaceae [12,16]. GLA is described as a fatty acid with anti-inflammatory properties [17]. However, its therapeutic value is controversially discussed (atopic dermatitis, rheumatoid arthritis) [17].
To improve the supply with n-3 LC-PUFA for human nutrition new vegetable oil sources containing precursors, such as SDA, have to be identified. In this work, the fatty acid composition of seeds from various species of plant families potentially high in ALA, SDA and GLA, such as Primulaceae and Boraginaceae, were investigated.

Lipid extraction
The dried seeds were finely ground and homogenised with a laboratory mill for small amounts. Total lipids were extracted from 1.0 g samples (or lower) with methanol/chloroform/ water (2:1:1 by volume). The lipid extracts were transesterified with trimethylsulphonium hydroxide (TMSH; 5 mg lipid extract; 200 mL methanol, SeccoSolv 1 MERCK; 100 mL TMSH solution, MACHEREY-NAGEL; Germany, incubation with TMSH at 808C for 15 min) to produce FAME directly within the injector of the GC at 2608C.

Fatty acid analysis
The analysis of FAME extracts was performed by GC (GC-17 V3, Shimadzu, Japan) equipped with a cooled autosampler and FID. A fused-silica capillary column of medium polarity was used (DB-225MS: 60 m Â 0.25 mm id with a 0.25 mm film thickness; Agilent Technologies, US). The initial oven temperature was maintained at 708C for 2 min, then increased by 108C/min to 1808C, then increased by 28C/min to 2208C and held for 5 min. Finally, it was increased by 28C/min to 2308C and held for 15 min. The injector and detector temperatures were maintained at 2608C. Hydrogen was used as carrier gas [18].

Identification and calculation of FAME
The total lipid content of the seeds was expressed as % of the air-dried seeds. The amount of the separated FAME was expressed as % of the total FAME. Various reference standards were used as FAME mix to identify fatty acid peaks: No. 463, 674, (NU-CHEK PREP; INC., US), BR2, BR4, ME 93 (Larodan; Sweden), Supelco 1 37 Component FAME Mix, PUFA No. 3. LabSolutions software for GC (GCsolution; Shimadzu, Japan) was used for peak integration.

Statistics
All statistical analyses were performed using SPSS statistics, version 17.0 (ß2009 SPSS Statistics 17 Inc, Illinois, US) with P 0.05 to indicate significant differences. The means of each species resulted from quadruplicates. The SD is given only for the comparison between the families. The difference of means between families, genera and species was tested with the multivariate post-hoc Scheffé-test.

Results
The total oil content of the 30 analysed species varied from 1.7% in Hippophae rhamnoides (Table 1) to 24.3% in Cynoglossum germanicum ( Table 2). The mean oil content was 15.0 AE 5.4%.

Samples
Oil content (%) Oil Means with different superscript numbers indicate differences between species ( 1 highest value of a species is underlined, post-hoc Scheffé-test P<0.05; for a better overview the comparison between different species is given only up to the third [3] highest value).
From a nutritional point of view, Echium oil could increase the n-3 PUFA intake in humans. In addition, it has been shown that highly concentrated SDA was converted to EPA more efficiently than ALA in humans [20]. Application of natural Echium oil effectively results in an enrichment of EPA in human tissues ( [21,22]; unpublished results [23]). E. plantagineum was established as a SDArich Echium species [12]. In 2007, the US Food & Drug Administration declared refined Echium oil as novel food for the market and the oil from E. plantagineum is available as food ingredient (e.g., INCROMEGA V3 with 12.5% SDA; Croda GmbH, Germany). Furthermore, a new biotechnologically-derived soybean oil with 20% SDA was created (SDA-20; Monsanto Company, MO, US) [24].
To our knowledge, the only report on the fatty acid profile of Omphalodes linifolia [11] to date shows a much lower percentage of SDA (3.5%) as well as of ALA (8.8%) than presented herein (Table 2). However, in Cerinthe minor we detected similar SDA and ALA portions (7.3, 29.0%, resp.; [11]) ( Table 2). Although the closely related C. major was not subject of our analyses, it is worth mentioning that despite high ALA, it contains only marginal amounts of SDA [11]. Well corresponding to our data, Buglossoides purpureocaerulea (syn. Lithospermum purpurocaeruleum) was previously been found to be a relevant source of SDA with 7% [9]. Among Lithospermum species, L. arvense has been found to be relatively rich in SDA with a portion up to 17% [9,11]. For seeds of Lithospermum officinale, varying levels of SDA ranging from 7.0 to 13.3% have been reported [11] that were generally higher than the present finding (5.4%; Table 2). In general, Echium species were formerly and herein confirmed to be good sources of SDA ( [12], Table 2).
In addition to high SDA portions, we detected comparably high GLA accounting for 17% in both Omphalodes linifolia and Buglossoides purpureocaerulea (Table 2). Also, the analysed Echium species as well as Alkanna graeca and orientalis, Anchusa officinalis and Lithospermum officinale were relatively rich in GLA with about 10% [11] (Table 2).
SDA and GLA are D6-desaturation products derived from ALA and LNA, respectively. Thus, SDA and GLA appear jointly in few seed oils such as of Echium species with a unique SDA to GLA ratio of 1:1 (Table 2), most likely due to high activity of the D6-desaturase [19]. The D6-desaturase is classified as an acyl-lipid desaturase that inserts a further double bond between the pre-existing double bond and the carboxyl end of the fatty acid. Its expression strongly depends on tissue and plant species [13]. New data show that D6-desaturases of different species, even of the same genus (e.g., Primula), have different substrate preferences, for example, the D6-desaturase with n-3 selectivity [27,28]. Consequently, in the analysed Primula species the SDA portion was considerably higher than the GLA portion (5:2; Table 2), despite similar LNA and ALA portions.
The branched-chain fatty acid C17:0iso found in P. denticulata could have been originated from surface waxes. However, no respective data were available.
The oils from Moltkia suffructicosa and Cynoglossum species were found to be relatively rich in n-9 VLC-MUFA, such as erucic acid with 7.5 and 9.1%, respectively ( Table 2). Erucic acid is a predominant component of rapeseed oils (e.g., Brassica napus up to 45%).
For reasons of health, the level of erucic acid in oils and fats intended for human consumption is limited by government regulation to a maximum of 2 and 5% of total fatty acids in the USA and EU, respectively. However, since erucic acid is the precursor of nervonic acid, higher erucic acid contents like in Cynoglossum species correlate with higher nervonic acid contents (up to 3.6%; Table 2). Nervonic acid, for instance, is considered crucial for biosynthesis of nerve cell myelin. VLC-MUFA-rich oils might therefore be relevant as nutraceuticals for the treatment of disorders involving demyelination, such as adrenoleukodystrophy and multiple sclerosis [29]. Comparable amounts of these n-9 VLC-MUFA to the herein presented Cynoglossum species have previously been found in C. divaricatum [10]. Interestingly, neither erucic acid nor nervonic acid were found in C. officinale and C. nebrodense [16], despite comparable amounts of eicosenoic acid (5.1% vs. 5.5%; Table 2).
From the economic point of view, various parameters have to be taken into account (e.g., oil content and oil yield of seeds, growth period, cultivation conditions). In addition, the availability and sustainability of the new oil sources and for the use in human nutrition the existence of toxic compounds, for example pyrrolizidine alkaloids in Boraginaceae, should be investigated.
In general, the oil content and the fatty acid profile can differ considerably between and even within one species, reflecting the influence of factors like vegetation, climate, degree of ripeness, management regimes, soil cultivations, testing site. In addition, minor genetic variations [12] and not least different methodical parameters (fatty acid transmethylation and GC conditions) could influence the fatty acid profile, too.
In conclusion, an important strategy to reduce the n-6/n-3 ratio in the diet is to increase its n-3 PUFA content. Considering the overfishing of the seas, the integration of n-3 PUFA-rich plant oils, particularly SDA-containing oils, into the diet could be a promising step forward towards ensuring the n-3 PUFA supply of an increasing world population. It is therefore assumed that screening of the fatty acid composition of less-researched plant seed oils may provide a nutritional benefit.