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Knowledge concerning the availability of n-3 fatty acids for humans in prehistoric times is highly relevant in order to draw useful conclusions on the healthy dietary habits for present-day humans. To this end, we have analysed fat from several frozen bison found in the permafrost of Siberia (Russia). A total of 3 bison were included in this study, all them very close to the early Holocene (8,000; 8,200; and 9,300 years BP). All samples were analysed by gas-liquid chromatography-mass spectrometry (GLC-MS) and GLC flame-ionization detection (GLC-FID). Fat samples from two bison showed two well-differenced areas, i.e. brown and white, the latter being saturated fatty acid enriched, corresponding to an intermediate stage of adipocere formation, while the brown ones yielded α-linolenic acid in higher percentages than found in present-day bison. As demonstrated in this work, the subcutaneous fat of bison consumed by Mesolithic hunters contained amounts of n-3 fatty acids in higher quantities than those found in current bison; thus, the subcutaneous fat of bison could have contributed to meet today's recommended daily intake of essential fatty acids for good health in the Mesolithic to a greater extent than previously thought.

Analyses of the nutrients are given for the leaves of wild Plantago major L., P. lanceolata L. and P. media L., from different locations of the southeast of Spain. The proximate composition, mineral elements (Na, K, Ca, Mg, Fe, Cu, Zn, Mn and P), fatty acids, vitamins (vitamin C and carotenes), nitrate and oxalic acid were determined. The results show the low proportion of available carbohydrates, ranging from 1.99 g (P. major) to 2.81 g (P. lanceolata) (per 100 g fresh weight). Oxalic acid was found in low amounts, between 33.5 mg (P. media) and 88.2 mg (P. lanceolata) (100 g f wt). P. major had the highest amounts of vitamin C (45.1 mg/100g f. wt.) and calcium (108mg/100g f. wt.). Polyunsaturated fatty acids were high in all species, ranging from 38.97% (P. media) to 46.07% (P. lanceolata). Nutrient ratios (K/Na; Ca/P and oxalic acid/Ca) were found to be favourable in most cases.

The polyunsaturated fatty acids (PUFA) eicosapentaenoic acid (EPA, 20∶5n−3) and docosahexaenoic acid (DHA, 22∶6n−3), which have several pharmaceutical properties, have been purified from cod liver oil. The process consisted of four main steps: (i) saponification of the oil, (ii) use of urea inclusion adducts method to obtain PUFA, (iii) PUFA methylation, and (iv) argentation silica gel column chromatography of the methylated PUFA. Argentation silica gel chromatography yielded highly pure DHA in the process (100% purity, 64% yiild). For EPA, the recovery in the combined process was 29.6%, and the final purity was 90.6%, owing to the simultaneous elution of other polyunsaturated fatty esters. The recovery in the urea inclusion method was strongly enhanced by application of orbital agitation during the crystallization process, in which EPA yield increased from 60–70% without agitation to 90–97% at 800 rpm; stearidonic acid (18∶4n−3) yield ranged from 60–75% without agitation to 87–95% at 800 rpm, and DHA yield varied from 53–73% without agitation to 85–99% at 800 rpm

The free fatty acids eicosapentaenoic acid (C20:5ω3, EPA) and docosahexaenoic acid (C22:6ω3), obtained from the microalgae Phaeodactylum tricornutum, and the EPA methyl ester were compared with regard to their extent of peroxidation using different storage conditions. Several series were stored according to selected variables: hexane addition versus no addition, 4 °C versus 25 °C, and antioxidant addition (octyl gallate) versus no antioxidant addition, always in the dark. Previously, the EPA and methyl EPA structures were confirmed by NMR spectra to discard the formation of conjugated dienes after the downstream process. The results showed that the stability was higher for methyl EPA than for the free fatty acid, and that peroxidation can be retarded by low temperature storage and mainly by hexane addition. The peroxidation process was evaluated by the peroxide value (spectrophotometric method by iodine absorption), although the conjugated diene absorbance and the loss in percentage of the fatty acid have been tested as good indicators of the peroxidation process. A simple kinetic model that explains the peroxidation process during the initiation and propagation steps is given.

The microalga Phaeodactylum tricornutum was cultured under various conditions in an external tubular photobioreactor and in a bubble column. The proximate composition (moisture, ash, crude protein, available carbohydrates, fiber, lipids and energy), nitrate, nucleic acids, mineral elements (Na, K, Ca, Mg, Fe, Cu, Zn, Mn, P, S, As, Pb, Cd), C/N ratio, fatty acids and pigments (total carotenoids, carotenoid class and chlorophylls) were analyzed. On average, the biomass contained 36.4% crude protein, 26.1% available carbohydrates, 18.0% lipids, 15.9% ash and 0.25% neutral detergent fiber, on a dry wt basis. The mineral element contents in 100 g dry biomass were: Ca (1910 mg), K (1720 mg), Na (1430 mg), S (1050 mg), Mg (555 mg), Zn (373 mg), (Mn 31.4 mg), Cu (8.4 mg), and P (269 mg). Toxic heavy metal contents were negligible. The fatty acid content was (on percent dry wt): 0.65% in 14:0;1.28% in 16:0; 1.48% in 16:lω7; 0.34% in 16:2ω4; 0.75% in 16:3ω4 and 2.50% in 20:5ω3. Nutrient composition of biomass was highly influenced by means of the residence time in the culture bioreactor and the external irradiance. The biomass produced at low external irradiance was richer in protein and eicosapentaenoic acid.

Nineteen species of the genus Echium (Fam. Boraginaceae) collected in Macaronesia were surveyed in a search for new sources of γ-linolenic acid (GLA, 18:3ω6). High amounts of this acid were found in all of them, ranging from 9.15% (E. plantagineum) to 26.31% (E. callithyrsum) of total seed fatty acids. The amounts of GLA related to total seed weight were also significant, ranging from 1.77% (E. sventenii) to 5.02% (E. nervosum). In addition, considerable amounts of stearidonic acid (SA, 18:4ω3) were detected, ranging from 3.03% (E. auberianum) to 12.94% (E. plantagineum) of total fatty acids. These data allow us to consider the members of the genus Echium from Macaronesia as one of the richest sources of γ-linolenic acid found so far in nature. The results obtained from multivariable data analysis and the taxonomic relationships among the species is discussed.

The polyunsaturated fatty acids (PUFA) eicosapentaenoic and arachidonic acids (EPA and AA), which have several pharmaceutical properties, have been purified from the red microalga Porphyridium cruentum. The process consists of only four main steps: (i) simultaneous extraction and saponification of the microalgal biomass; (ii) urea inclusion method (iii) PUFA esterification (iv) argentated silica gel column chromatography of the urea concentrate. Total AA and EPA recoveries reached 39.5% and 50.8% respectively for a purity ∼97% for both fatty acids. Therefore, recovery of highly pure PUFA could be improved in organisms that are rich in two or more fatty acids of interest. The results of several procedures for AA and EPA recovery from several authors by using this microalga were compared.

α-Linolenic acid and unusual fatty acids of the ω3 and ω6 series play an important role in the modulation of human metabolism. The presence of these acids in the leaves of several edible wild plants has recently been reported. In this study, six edible wild species were selected in order to establish the fatty acid compositions in their leaf lipids. Thus, young leaves from Amaranthusviridis L.(blet), Chenopodiumalbum L. (goosefoot), Crithmummaritimum L. (rock samphire), Plantagomajor L. (plantain), Portulacaoleracea L. (purslane) and Verbenaofficinalis L. (vervain) yielded 1.50, 2.20, 3.02, 1.46, 3.81, and 2.28 g of lipids per 100 g dry plant material. Silica gel chromatography yielded 0.64 g (Plantagomajor) to 2.19 g (Crithmummaritimum) neutral lipids, 0.37 g (Plantagomajor) to 1.60 g (Portulacaoleracea) glycolipids, and 0.26 g (Crithmummaritimum) to 0.57 g (Verbenaofficinalis) phospholipids per 100 g (dry weight). Gas chromatography (GC) showed the major fatty acids to be 18 : 3ω3, 18 : 2ω6 and 16 : 0 in all fractions, with high concentrations of 18 : 3ω3 in the glycolipid fraction. GC-mass spectrometric analyses did not reveal the presence of unusual fatty acids. Carotenes were found in high concentrations, ranging from 30.5 mg/100 g (Chenopodiumalbum) to 89.2 mg/100 g (Portulacaoleracea). The analyzed plants are rich sources of essential fatty acids (18 : 2ω6 and 18 : 3ω3) and also of carotenes.

The composition of tender leaves from five species of the family Cruciferae (Alliaria petiolata, Cardaria draba, Cakile maritima, Capsella bursa-pastoris and Sisymbrium irio) from different locations of the southeast of Spain (Almeria) was investigated. The proximate composition, mineral elements (Na, K. Ca, Mg, P, Fe, Cu, Zn and Mn), fatty acids, vitamin C, carotenoids and oxalic acid contents were determined. The available carbohydrate contents ranged from 1.4% 8.2 %. Vitamin C contents were high, specially in Alliaria petiolata (261 mg/lOO g). Carotenoid content was relatively high in Alliaria petiolata (13.3 mg). Mineral elements were present at higher concentrations than is common for other green leafy vegetables. Fiber amounts were also remarkable (1.96-5.38 g/IOO g). Il.le w3 series of fatty acids were predominant in the three species (Capsella bursa-pastoris 54.02 76). Oxalic acid values and ratios of oxalic acid/Ca were moderate, being higher in C. maritima. We believe that these

RAPD analysis has been performed for 15 plant populations, covering eight different infraspecific taxa ofSideritis pusilla(Lange) Pau, member of the Lamiaceae endemic to southeastern Spain, and their putative parental speciesSideritis hirsutaL. andSideritis leucanthaCav. Genetic distances, together with the presence of numerous fixed molecular markers differentiatingS. pusillafrom its putative ancestors, indicate that it should be considered as a true species. Cladistic and populational analysis led to the allocation ofS. pusillapopulations into three major groups—osteoxylla, flavovirensandpusilla/almeriensis—which include taxa previously described as ranging from the varietal to the specific level. Low genetic differentiation among groups revealed by a reduced number of specific molecular markers justify their assignment under the infraspecific range. Moreover, the existence of both morphological and biogeographical differences, supports a status for these groups as subspecies ofS. pusilla.Highly significant (P

Analyses of the nutritional composition of tender leaves of three species of Sonchus (S. asper L, S oleraceus L and S tenerrimus L) from different locations in the south east of Spain were carried out. The proximate composition, as well as the content of mineral elements (Na, K, Ca, Mg, P, Fe, Cu, Zn and Mn), fatty acids, vitamin C, carotenoids and oxalic acid were determined. The results, which referred to fresh weight, emphasised the low proportion of available carbohydrates. Vitamin C contents were high, and ranged from 457 mg kg−1 (S tenerrimus) to 779 mg kg−1 (S oleraceus). Carotenoids were found in a high proportion (158 mg kg−1) in S oleraceus. Mineral element contents were similar to other green leafy vegetables. Fibre was present in amount above 30 g kg−1 in the three species. Essential fatty acids of the ω3 series were highest in S oleraceus (44·97%). It is believed that these species of Sonchus could be used for nutritional purposes, due to the high concentrations of nutrients that they contain. © 1998 SCI.

Analyses are given of the nutritional composition of tender leaves of three species of Chenopodium (C. album L., C. murale L. and C. opulifolium Shraeder) from different locations of southeast Spain (Almeria). The proximate composition, mineral elements (Na, K, Ca, Mg, P, Fe, Cu, Zn and Mn), fatty acids, vitamin C, carotenoids and oxalic acid contents were determined. Low proportions of available carbohydrates were found. Vitamin C contents were high, especially in C. album (155mg/100g). Carotenoids were found in high proportion (12.5mg/100g) in C. album. Mineral element contents were higher than in other green leafy vegetables. Fiber amounts were also remarkable (4–6g/100g). The ω3 series of essential fatty acids were major in the three species (C. album, 45.33%). Oxalic acid values and ratios oxalic acid/(Ca + Mg) were moderate, being higher in C. murale. We believe that these species of Chenopodium could be used for nutritious purposes, due to the amount and diversity of nutrients they contain.

Seeds from 50 species belonging to the family Caryophyllaceae, subfamilies Alsinoideae, Silenoideae, and Spergulaceae, were surveyed in a search for new sources of γ-linolenic acid (GLA) having low levels of other interfering PUFA and therefore appropriate for GLA purification. It was detected mainly in Alsinoideae species, with a maximum of 15.6% of total FA in Minuartia laricifolia subsp. ophiolitica. Different amounts of stearidonic acid (18∶4n−3) were present in the Alsinoideae species, ranging from undetectable levels in seven species to 3.30% of total FA in Stellaria media. An ANOVA showed a taxonomical correlation between GLA percentages and the genus/subfamily within this family.

Production of 2-monoacylglycerols (2-MAGs) by selective hydrolysis of the triacylglycerols (TAGs) of Echium plantagineum seed oil and Marinol and further purification was carried out. Three purification methods, including silica gel column chromatography, liquid–liquid extraction and low-temperature crystallization were assayed. Partial acyl migration during the purification step is always observed. Acyl migration rates were similar both for the column chromatography and for the liquid–liquid extraction methods, and resulted in 1-MAG/2-MAG ratios higher than 1.0. Fatty acid (FA) profiles of 2-MAGs after enzyme hydrolysis showed that the major FAs were stearidonic acid (56.9% of total FA in 2-position) and docosahexaenoic acid (63.6% of total FA in 2-position) for E. plantagineum seed oil and Marinol, respectively.

The cytotoxic effects of extracts of the tomato variety "Racimo" have been evaluated through the use of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at several concentrations. Three extracts-ethanol-water, petroleum ether, and in vitro digested tomato-exhibited in vitro cytotoxicity against the proliferation of the cultured cancer cell line HT-29. The concentration that caused 50% inhibition of cancer cell growth occurred (GI(50)) of the different extracts for HT-29 cells was 62.5 μg/mL for the petroleum ether extract and 87.0 μg/mL for the digested tomato extract. For the ethanol-water extract, it was not possible to determine this parameter at the assayed extract concentrations. These results clearly indicate that after the digestion process, the less polar substances, such as carotenoids and sterols, are bioavailable as active species against cancer cells. The GI(50) levels for tomato extracts are similar to those values reported for medicinal plants. The results of the MTT assay on nonmutagenic CCD-18 cells showed a lack of negative effect on cell growth, which indicates that tomato extracts act selectively on HT-29 tumor cells. (1)H-Nuclear magnetic resonance spectra confirmed the presence of known compounds with accepted cytotoxic activity against tumor lines (lycopene and β-carotene). The high cytotoxicity for HT-29 cells showed by the petroleum ether extract might be due to the simultaneous presence in the extract of both carotenoids and glyceryl esters of fatty acids. The results of this work clearly indicate the importance of carotenoid consumption on colon tumor proliferation and prevention, and also the importance of the dietary fats in carotenoid bioavailability.

Several jujube varieties from the southeast of Spain were analyzed for fatty acid and carotene contents. Triglycerides having medium-chain fatty acids were most abundant in all samples. The main fatty acids were 12:0 (18.3 +/- 9.97), 10:0 (12.5 +/- 19.0), 18:2n6 (9.27 +/- 7.26), 16:1n7 (8.50 +/- 5.77), 16:0 (7.25 +/- 4.35), and 18:1n9 (5.34 +/- 2.52) on total saponifiable oil. The fruits yield 1.33 +/- 0.17 g/100 g saponifiable oil on a dry weight basis. Fatty acid profiles of fruits were found to be influenced by their developmental stage. Multivariable data analyses show that the samples could be grouped on the basis of their fatty acid content. Carotenes were found to be in good agreement with other fruits, varying from 4.12 to 5.98 mg/100 g on a dry weight basis. The contribution to vitamin value reach a mdium of 38 microg RE/100 g on a fresh weight basis.

Four edible oils, linseed, borage and sunflower seed oils, and the liver oil of shortfin mako (Isurus oxyrinchus), have been fractionated by open column liquid chromatography, using partially argentated silica gel as stationary phase, looking for purify polyunsaturated fatty acids (PUFA). By this method, linoleic (LA, 18:2n6),α-linolenic (ALA, 18:3n3), γ-linolenic (GLA, 18:3n6), stearidonic (SDA, 18:4n3), eicosapentaenoic (EPA, 20:5n3) and docosahexaenoic (DHA, 22:6n3) fatty acid esters have been isolated from these oils with high purity percentages and yields.Los aceites comestibles de lino, girasol, borraja y el de hígado de marrajo (Isurus oxyrinchus), se fraccionaron mediante cromatografía líquida en columna abierta, usando como fase estacionaria gel de sílice parcialmente impregnado con nitrato de plata, con el objetivo de purificar varios ácidos grasos poliinsaturados (PUFA). Mediante este método, se aislaron los ésteres metílicos de los ácidos linoleico (LA, 18:2n6), α-linolénico (ALA, 18:3n3), γ- linolénico (GLA, 18:3n6), estearidónico (SDA, 18:4n3), eicosapentaenoico (EPA, 20:5n3) y docosahexaenoico (DHA, 22:6n3) con altas purezas y rendimientos.

Fourteen species of the genus Echium (Fam. Boraginaceae) collected in the Macaronesia were surveyed in a search for high levels of γ-linolenic acid (GLA, 18:3ω6) in the seed oil. High amounts of this fatty acid were found in all of them, ranging from 18.85% (E. pitardii var. pitardii ) to 27.42% ( E. gentianoides ) on total seed fatty acids. The GLA content related to total seed weight was also significant, ranging from 1.26% ( E. handiense ) to 8.22% ( E. gentianoides ). In addition, considerable amounts of stearidonic acid (SA, 18:4ω3) were detected, ranging from 3.78% ( E. bonnetii var. bonnetii ) to 8.81% ( E. pininana ) on total fatty acids. Besides all the perennial species, the four herbaceous Echium taxa endemic to the Macaronesia also showed high GLA percentages. This is in contrast to the low GLA level found in continental Echium species, all of them bearing an herbaceous habit. These results are in good agreement with the available genetic data and show the ability of GLA to discriminate between Macaronesian and continental Echium species. The analysis of five other Macaronesian species belonging to plant families rich in GLA are also reported.

Leaves from 25 Macaronesian Echium (Boraginaceae) species have been surveyed for hydrocarbon compounds. These plants were previously reported as the major source of γ-linolenic acid so far found in nature. In addition, six European Echium species and the common Borago officinalis have been analysed for comparative purposes. High squalene amounts were found in all Echium plants from the Macaronesia, ranging from 3.73% in E. simplex to 20.11% in E. fastuosum . Squalene was almost absent from all European Echium species, and the same is true for B. officinalis . The relatively high oil content (2.27%) in leaves of E. fastuosum raises the total squalene amount to about 0.46% within this tissue. The main fatty acid component in the leaf was α-linolenic acid (ALA, 18:3ω3), ranging in the Macaronesian Echium from 9.32% in E. acanthocarpum to 54.45% in E. simplex . Possible utilisation of these plants as a commercial source of squalene and hypotheses about its physiological role in the plant are discussed.

The elucidation of the sources of n-3 fatty acids available for the humans in the Upper Palaeolithic and Neolithic is highly relevant in order to ascertain the availability of such nutrients in that time frame as well as to draw useful conclusions about healthy dietary habits for present-day humans. To this end, we have analysed fat from several frozen mammals found in the permafrost of Siberia (Russia). A total of 6 specimens were included in this study: 2 mammoths, i.e. baby female calf called "Lyuba" and a juvenile female called "Yuka", both specimens approximately from the same time, i.e. Karginian Interstadial (41,000 and 34,000 years BP); two adult horses from the middle Holocene (4,600 and 4,400 years BP); and two bison very close to the Early Holocene (8,200 and 9,300 years BP). All samples were analysed by gas-liquid chromatography-mass spectrometry (GLC-MS) and GLC-flame ionization detector (GLC-FID). As demonstrated in this work, the fat of single-stomached mammals often consumed by Palaeolithic/Neolithic hunters contained suitable amounts of n-3 and n-6 fatty acids, possibly in quantities sufficient to meet the today's recommended daily intake for good health. Moreover, the results also suggest that mammoths and horses at that time were hibernators.

The nutritional composition of the marine eustigmatophyte Nannochloropsis spp. cultured in an indoor chemostat under continuous illumination was analyzed. Proximate composition, (moisture, ash, crude protein, available carbohydrates, fiber, lipids, and energy), nitrate, nucleic acid, mineral element (Na, K, Ca, Mg, Fe, Cu, Zn, Mn, Pb, Cd, Cr, Ni, Co, and S), fatty acid, and pigment (carotenoids and chlorophyll) concentrations were determined. On average, the biomass contained 37.6% (w/w) available carbohydrates, 28.8% crude protein, and 18.4% total lipids. Mineral in 100 g of dry biomass were as follows: Ca (972 mg), K (533 mg), Na (659 mg), Mg (316 mg), Zn (103 mg), Fe (136 mg), Mn (3.4 mg), Cu (35.0 mg), Ni (0.22 mg), and Co (0.1 mg). Toxic heavy metal contents (Cd and Pb) were negligible. Fatty acid content was as follows (on percent dry weight): 0.6% of 14:0, 5.0% of 16:0; 4.7% of 16:1omega7, 3.8% of 18:1omega9, 0.4% of 18:2omega6; 0.7% of 20:4omega6, and 2.2% of 20:5omega3. Nutrient composition of the biomass was highly influenced by residence time in the photobioreactor. The biomass harvested for short residence times was richer in protein and eicosapentaenoic acid than biomass harvested for high residence time.

Forty-nine plant species from Spain, belonging to the Boraginaceae, Scrophulariaceae, Onagraceae, and Ranunculaceae families, were surveyed in a search of new sources of γ-linolenic acid (18∶3ω6, GLA). Fatty acid profiles from seeds, stems, roots, flowers and leaves were determined. GLA was detected mainly in seed and root tissues. High GLA amounts were found in seeds of Boraginaceae species, with a maximum of 20.25% of total fatty acids in Myosotis nemorosa. Within the Scrophulariaceae the highest GLA content (10.17%) was found in Scrophularia sciophila. Variable amounts of stearidonic acid, (18∶4ω3, SDA) were present in Boraginaceae species, ranging from 0.08% of total seed fatty acids in Anchusa azurea to 21.06% in Echium asperrimum. SDA was also very abundant in all organs of Asperugo procumbens. A multivariate analysis was performed using our results and those reported for other plant species belonging to the same families in order to investigate a possible correlation between the fatty acid profile and the genera within these families.