γ-Linolenic and Stearidonic Acids from Boraginaceae of Diverse Mediterranean Origin.

Thirty Boraginaceae species from different tribes were evaluated in a search of γ-linolenic (GLA, 18:3n-6) and stearidonic acid (SDA, 18:4n-3)-rich oils. The high GLA percentages were found in the seed oils of Symphytum bulbosum and S. tuberosum subsp. tuberosum (27.6 and 27.2 % of total fatty acids (FA)), which are unusually high values for GLA-oils in the current literature. On the whole seed, noticeable GLA percentages were found in S. grandiflorum, S. tuberosum subsp. tuberosum and Borago officinalis (7.43, 4.90, and 4.51 g/100 g, respectively). The main SDA-taxa detected in this study were Buglossoides arvensis, B. incrassata and Glandora oleifolia (21.3, 18.9, and 16.3 % of total fatty acids). On total seed weight, Glandora rosmarinifolia showed the highest SDA content (3.57 g/100 g). Finally, the higher FA contents were found in S. grandiflorum and Paramoltkia doerfleri seeds (35.2 and 37.0 g/100 g, respectively). Principal component analysis showed that similarities in FA profiles allow grouping species as botanical criteria for Boraginaceae tribes do, while the FA groupings confirm the metabolic activities of desaturase and elongase enzymes. Data on the FA composition of the seed oils analyzed here suggest their potential use as functional foods and can be considered as novel sources of SDA and GLA.

Liquid chromatography separation of α- and γ-linolenic acid positional isomers with a stationary phase based on covalently immobilized cellulose tris(3,5-dichlorophenylcarbamate).

α-Linolenic acid (ALA) and its most important positional isomer γ-linolenic acid (GLA), are essential fatty acids (vitamin F). Therefore, ALA- and GLA-rich edible oils hold great potential in human and animal nutrition, as well as in nutraceutics and cosmetics. Quality control and nutritional validation of oil products is thus of increasing importance. In the present study, the cellulose tris(3,5-dichlorophenylcarbamate)-based chiral stationary phase was successfully used for separation of ALA and GLA, a major challenge in the liquid chromatography of these isomers. The chromatographic conditions were firstly optimized on a HPLC system with UV detection, and the use of a reversed-phase eluent system made up of aqueous 10 mM ammonium acetate/acetonitrile (40/60, v/v; wspH6.0) with a 25 °C column temperature resulted optimal for the simultaneous discrimination of the two isomers at a 0.5 mL/min flow rate (α = 1.10; RS = 1.21). The method was then optimized for LC-MS/MS implementation. The proposed innovative separation method holds a great potential for the quantification of ALA and GLA in food and biological matrices, thus opening the way to further investigations involving the two positional isomers.

Solid Matrix-Supported Supercritical CO2 Enhances Extraction of γ-Linolenic Acid from the Cyanobacterium Arthrospira (Spirulina) platensis and Bioactivity Evaluation of the Molecule in Zebrafish.

Marine cyanobacteria represent a large untapped source of functional glycolipids enriched with polyunsaturated fatty acids (PUFAs) for human health. However, advanced methods for scalable isolation of diverse species containing high-purity PUFA-rich glycolipids will have to be developed and their possible pharmaceutical and nutraceutical functions identified. This paper introduces a novel solid matrix-supported supercritical CO2 extraction method for scalable isolation of the PUFA γ-linolenic acid (GLA)-enriched glycolipids from the cyanobacterium Arthrospira (Spirulina) platensis, which has been the most widely used among microalgae in the nutraceutical and pharmaceutical industries. Of various porous materials studied, diatomite was the best to facilitate extraction of GLA-rich glycolipids, resulting in an extraction efficiency of 98%. Gamma-linolenic acid made up 35% of total fatty acids (TFAs) in the extracts, which was considerably greater than that obtained with ethanol (26%), Bligh and Dyer (24%), and in situ transesterification (24%) methods, respectively. Lipidomics analysis revealed that GLA was exclusively associated with galactolipids. Pharmaceutical functions of GLA-rich galactolipids were investigated on a zebrafish caudal fin regeneration model. The results suggested that GLA extracted from A. platensis possessed anti-oxidative, anti-inflammatory, and anti-allergic activities, which acted in a concerted manner to promote post-injury regeneration of zebrafish.

Pharmacological activities of the organic extracts and fatty acid composition of the petroleum ether extract from Haplophyllum tuberculatum leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: Haplophyllum tuberculatum is used in traditional medicine to treat many disorders including inflammation and pain. The aim of this study is to investigate the organic extracts from H. tuberculatum leaves against inflammation, gastric ulcer and pain. MATERIALS AND METHODS: Acute toxicity was studied in vivo to determine the toxic doses of the organic extracts. Anti-inflammatory activity was also evaluated in vivo using carrageenan-induced paw edema in Wistar rats. Gastroprotective activity was tested using the HCl/ethanol-induced gastric ulcer test in rats. Peripheral and central analgesic activities were assessed using the acetic acid-induced writhing test and the hot-plate method, respectively. The chemical composition of the fatty acids in the petroleum ether (PE) extract was determined with GC-MS. RESULTS: At 25, 50 and 100mg/kg PE extract was the most active against inflammation. Percentages inhibition 5h after carrageenan-injection were 51.12; 86.71% and 96.92%, respectively. The same extract at 100mg/kg showed good analgesic activities using the acetic acid-induced writhing test and the hot-plate method. The chloroform, ethyl acetate (EtOAc) and butanolic (n-BuOH) extracts exhibited strong anti-inflammatory, gastroprotective and analgesic activities at 100mg/kg. The GC-FID analysis revealed that the PE extract was rich in γ-linolenic acid (45.50%) followed by palmitic acid (18.48%), linoleic acid (10.73%), erucic acid (4.72), stearic acid (3.96%) and oleic acid (2.57%). CONCLUSION: The results of the present study support the traditional use of the leaves of H. tuberculatum and may possibly serve as prospective material for further development of safe new phytochemical anti-inflammatory, gastroprotective and/or analgesic agents.

Improved γ-linolenic acid production in Mucor circinelloides by homologous overexpressing of delta-12 and delta-6 desaturases.

BACKGROUND: γ-Linolenic acid (GLA) is important because of its nutritional value and medicinal applications. Although the biosynthetic pathways of some plant and microbial GLA have been deciphered, current understanding of the correlation between desaturases and GLA synthesis in oleaginous fungi is incomplete. In previous work, we found that a large amount of oleic acid (OA) had not been converted to linoleic acid (LA) or GLA in Mucor circinelloides CBS 277.49, which may be due to inadequate activities of the delta-12 or delta-6 desaturases, and thus leading to the accumulation of OA and LA. Thus, it is necessary to explore the main contributing factor during the process of GLA biosynthesis in M. circinelloides. RESULTS: To enhance GLA production in M. circinelloides, homologous overexpression of delta-12 and two delta-6 desaturases (named delta-6-1 and delta-6-2, respectively) were analyzed. When delta-6 desaturase were overexpressed in M. circinelloides, up to 43% GLA was produced in the total fatty acids, and the yield of GLA reached 180 mg/l, which were, respectively, 38 and 33% higher than the control strain. CONCLUSION: These findings revealed that delta-6 desaturase (especially for delta-6-1 desaturase) plays an important role in GLA synthesis by M. circinelloides. The strain overexpressing delta-6-1 desaturase may have potential application in microbial GLA production.

Impact of botanical oils on polyunsaturated fatty acid metabolism and leukotriene generation in mild asthmatics.

BACKGROUND: Dietary supplementation with botanical oils that contain n-6 and n-3 eighteen carbon chain (18C)-PUFA such as γ linolenic acid (GLA, 18:3n-6), stearidonic acid (SDA, 18:4n-3) and α linolenic acid (ALA, 18:3n-3) have been shown to impact PUFA metabolism, alter inflammatory processes including arachidonic acid (AA) metabolism and improve inflammatory disorders. METHODS: The diet of mild asthmatics patients was supplemented for three weeks with varying doses of two botanical seed oils (borage oil [Borago officinalis, BO] and echium seed oil [Echium plantagineum; EO]) that contain SDA, ALA and GLA. A three week wash out period followed. The impact of these dietary manipulations was evaluated for several biochemical endpoints, including in vivo PUFA metabolism and ex vivo leukotriene generation from stimulated leukocytes. RESULTS: Supplementation with several EO/BO combinations increased circulating 20-22 carbon (20-22C) PUFAs, including eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and dihommo-gammalinolenic acid (DGLA), which have been shown to inhibit AA metabolism and inflammation without impacting circulating AA levels. BO/EO combinations also inhibited ex vivo leukotriene generation with some combinations attenuating cysteinyl leukotriene generation in stimulated basophils by >50% and in stimulated neutrophils by >35%. CONCLUSIONS: This study shows that dietary supplementation with BO/EO alters 20-22C PUFA levels and attenuates leukotriene production in a manner consistent with a reduction in inflammation.

Separation of γ-linolenic and other polyunsaturated fatty acids from Boraginaceae via supercritical CO2.

Essential fatty acids were extracted from Echium amoenum (Boraginaceae) seed oil via supercritical carbon dioxide and the results were compared with conventional Soxhlet method as the base case of 100% recovery. The response surface methodology was used to optimize the effective extraction parameters. The chemical composition of recovered oil was analyzed by polar and non-polar gas chromatograph-flame ionization detector columns. The experimental results indicated that echium seed contained 25 wt% oil and the maximum extraction oil recovery of 92% was obtained via supercritical CO(2) at optimal operating conditions (43°C, 280 bar, 1.5 mL/min, 25 min static time and 130 min dynamic time). At similar operating conditions, applying 2.5 and 5 mol% ethanol as a modifier enhanced the recovery to 96 and 112%, respectively. The results showed that supercritical fluid extraction is a viable technique for separation of constituents such as γ-linolenic acid (7-8%), palmitic acid (6-7%), stearic acid (3-4%), oleic acid (12-13%), linoleic acid (19-20%), α-linolenic acid (40-41%) and stearidonic acid (8-9%) from Boraginaceae.

Feeding stimulant in Cinnamomum camphora for the common bluebottle, Graphium sarpedon nipponum (Lepidoptera: Papilionidae).

The acceptance of camphor tree (Cinnamomum camphora) as a host plant for the larvae of common bluebottle (Graphium sarpedon nipponum) was explained by the presence of feeding stimulants in the leaves. When the active methanol extract of C. camphora leaves was separated into hexane and water layers, both layers showed high feeding activities for the larvae of G. sarpedon nipponum. Bioassay-guided fractionation of the hexane layer resulted in the isolation of a highly active compound, which was identified as a-linolenic acid by nuclear magnetic resonance spectrometry and gas chromatography-mass spectrometry.

[Separation of gamma linolenic acid from evening primrose oil with urea inclusion--orthogonal experiment of optimizing technological parameters and observation of urea inclusion compound I].

OBJECTIVE: The influence on the urea inclusion compound under different conditions (allocated proportion, time of inclusion, temperature of inclusion) were studied through the orthogonal test, and theoretical reference of urea inclusion process for further optimization wound be offered. METHOD: The orthogonal experiment was adopted, and microscope was used to observe the shape, aperture size of the urea inclusion compound under different technological parameters, the GC was employed to inspect the purity of GLA. RESULT: The results indicated that the ratio of fatty acids and urea, inclusion of temperature, time of inclusion had great effect on urea inclusion compound. The three factors and its interactions significantly affected the purity of GLA. The results also showed that the best process was that the ratio of fatty acids and urea was 1 : 3, temperature of inclusion was--15 degrees C, time of inclusion was 24 h. CONCLUSION: Under the best condition, the purity of GLA reach up to 95.575 9%; and it is feasible to observe the shape and the amount of the urea inclusion compound to reflect and guide the urea inclusion technology.

Screening of microbial lipases and evalutaion of their potential to produce glycerides with high gamma linolenic acid concentration

Gamma-linolenic acid (GLA, 18:3, cis- 6,9,12-octadecatrienoic acid), an important compound in n-6 eicosanoid family biosynthesis, occurs in the lipids of a few plant and microbial sources. This study focused on the screening of microbial strains with suitable lipase activity for enrichment of GLA by selective hydrolysis of the borage oil (21.6 percent of GLA/total fatty acids). Firstly, 352 microrganisms were tested for their lipolytic capacity using screening techniques on agar plates containing borage oil, strains were then selected and screened for their activity (U/mg) using both submerged fermentation (SmF) and solid state fermentation (SSF). The rate of hydrolysis and the selective preference of these hydrolytic enzymes towards fatty acids, with a special focus on enrichment of GLA were studied and compared with those obtained by two commercially-available lipases. Only one of the lipases tested during this study displayed selectivity, discriminating the GLA during the hydrolysis reaction. Using the enzymatic extract from Geotrichum candidum as a biocatalyst of the reaction, it was possible to obtain a percentage of 41.7 percent of GLA in acylglycerols fraction when the borage oil was treated in a fixed-bed reactor for 24 hours at 30ºC.

Purification and identification of bovine cheese whey fatty acids exhibiting in vitro antifungal activity.

Milk lipids contain several bioactive factors exhibiting antimicrobial activity against bacteria, viruses, and fungi. In the present study, we demonstrate that free fatty acids (FFA) derived from the saponification of bovine whey cream lipids are active in vitro at inhibiting the germination of Candida albicans, a morphological transition associated with pathogenicity. This activity was found to be significantly increased when bovine FFA were enriched in non-straight-chain FFA. At low cell density, this non-straight-chain FFA-enriched fraction was also found to inhibit in a dose-dependant manner the growth of both developmental forms of C. albicans as well as the growth of Aspergillus fumigatus. Using an assay-guided fractionation, the main components responsible for these activities were isolated. On the basis of mass spectroscopic and gas chromatographic analysis, antifungal compounds were identified as capric acid (C10:0), lauroleic acid (C12:1), 11-methyldodecanoic acid (iso-C13:0), myristoleic acid (C14:1n-5), and gamma-linolenic acid (C18:3n-6). The most potent compound was gamma-linolenic acid, with minimal inhibitory concentration values of 5.4 mg/L for C. albicans and 1.3 mg/L for A. fumigatus, in standardized conditions. The results of this study indicate that bovine whey contains bioactive fatty acids exhibiting antifungal activity in vitro against 2 important human fungal pathogens.

Gamma-linolenic acid from Cape Verdian Boraginaceae.

The distribution of gamma-linolenic acid (GLA) was studied in the seed oil of six Cape Verdian Boraginaceae. The GLA ranges from trace levels in the three Heliotropum surveyed to 22.2% on saponifiable oil in Echium stenosiphon. All Echium species had GLA percentages in their seed oil around 20%. Echium seeds yield saponifiable oil from 15.8% (E. stenosiphon) to 17.3% (E. hypertropicum).

[Study on the encapsulation technique of high purity gamma-linolenic acid, part 1--Single factor, and two-factor tests for encapsulation process].

OBJECTIVE: To study the effects of all factors during the process of urea encapsulation of gamma-linolenic acid on the purity and yield. METHOD: To observe the material proportions, time, temperature and purity using single-factor and two-factor tests. RESULT: Single-factor test showed that the optimal ratio of all materials (oil, urea and 95% ethanol) was 1:3:8. A 30% purity after single encapsulation process was obtained, at the best temperature range was - 15 degrees C-20 degrees C, for 24 hours. Two-factor test showed that the optimal ratio of oil, urea and ethanol was 1:3:8, where the concentration of ethanol was 90%-95%. The purity reached 90% or higher, with three-time encapsulation process. CONCLUSION: An optimized process was identified where material ratios, encapsulation time, temperature, and ethanol concentrations were determined using single-factor and two-factor tests.

Production of gamma-linolenic acid by Cunninghamella echinulata cultivated on glucose and orange peel.

A newly isolated strain of Cunninghamella echinulata grown on glucose produced significant quantities of biomass and cellular lipids in media with high C/N ratio. The oil yield from glucose consumed increased after nitrogen exhaustion in the growth medium, but gamma-linolenic acid (GLA) content in cellular oil systematically decreased during the lipid accumulation process. When lipid accumulation was completed, GLA concentration in the cellular lipids progressively increased. The highest GLA production (720 mg/l) was achieved in medium with a C/N ratio equal to 163. C. echinulata was also able to grow on orange peel. The C/N ratio in the orange peel decreased from 50 to 26 during solid-state fermentation. Maximum oxygen uptake was observed during assimilation of reducing sugars, whereas a polygalacturonase activity was detected after reducing sugars had been exhausted. The maximum GLA production was 1.2-1.5 mg/g of fermented peel, calculated on a dry weight basis. After enrichment of the pulp with inorganic nitrogen and glucose, an increase in the production of oil and GLA was observed.

Oral supplementation with gamma-linolenic acid extracted from Mucor circinelloides improves the deformability of red blood cells in hemodialysis patients.

BACKGROUND: The development of abnormalities in red blood cell (RBC) deformability in patients undergoing hemodialysis remains a major problem, because it is related to peripheral microcirculation, oxygen supply, and various complications in such patients. gamma-Linolenic acid (GLA; 18:3n-6), one of the polyunsaturated fatty acids and a precursor of prostaglandin E(1), is reported to have a favorable effect on the deformability of circulating blood cells in diabetic patients. METHODS: In order to clarify the efficacy of GLA on RBC deformability in 7 patients undergoing maintenance hemodialysis, we examined in a pilot study the changes in the deformability of RBC and the changes in the phospholipid fatty acid composition in both plasma and RBC membrane before and after high-dose oral supplementation with GLA derived from Mucor circinelloides for 12 weeks. RESULTS: Before supplementation, the micropore passage time of RBC suspension, which is an indicator of RBC deformability, in these patients was markedly longer than that in healthy control subjects. After administering GLA, the prolonged passage time of the patients both rapidly and steadily decreased and nearly reached control levels. Light microscopic observations of RBCs using Giemsa stain revealed a decreased number of poikilocytes after supplementation. An analysis of the fatty acid composition before treatment and 8 weeks after starting the treatment showed the dihomo-gamma-linolenic acid (DGLA; 20:3n-6) level in the plasma to have increased (p < 0.05), while the arachidonic acid (AA; 20:4n-6) concentration in the RBC membrane decreased (p < 0.05). The level of DGLA in the RBC membrane, the level of GLA, and the ratio of GLA + DGLA/AA in plasma and RBC membrane did not change significantly; however, these all tended to increase. CONCLUSION: The results of this pilot study indicate that the oral supplementation of GLA extracted from M. circinelloides improves the poor RBC deformability in hemodialysis patients, partly by inducing changes in the composition of fatty acids in plasma and RBC membrane.

Safflower petals: a source of gamma linolenic acid.

Safflower petals have been shown to have a lot of medicinal and therapeutic values. Indian safflower petal samples were analyzed for the red pigment carthamin, protein and oil contents. The petal oil (4.0-5.8%) was further analyzed for its fatty acids followed by alpha linolenic acid (15-19%) and palmitic acids (14-16%). Gamma linolenic acid, which has a lot of therapeutic value was present to an extent of 2-3%; decanoic and dodecanoic acids (2-5%) were also present.

Separation of representative lipid compounds of biological membranes and lipid derivatives from peroxidized polyunsaturated fatty acids by reversed phase high-performance liquid chromatography.

A complex mixture of different lipid compounds, including phosphatidylcholine, phosphatidylserine, all trans-retinol, 15(S)-hydroperoxyeicosatetraenoic acid, D-alpha-tocopherol, saturated and unsaturated fatty acids can be separated by reversed phase HPLC by using a C-18, 120 mm x 4 mm, 3 microns particle size column and a step gradient from acetonitrile/water (1:1; v:v) to 100% acetonitrile at a flow rate of 0.8 ml/min. By applying this elution condition, separation of various groups of lipid hydroperoxides and lipid derivatives, each one originating from a different in vitro peroxidized polyunsaturated fatty acid, can be obtained. Simultaneous detection is carried out by a diode array detector at a wavelength accumulation range set up between 195 and 400 nm. The possibility of simultaneously having such a large number of measurements renders this chromatographic method particularly suitable in studies concerning lipid peroxidation where, in addition to the detection of free radical-induced lipid hydroperoxides, data on some key antioxidant molecules, i.e. vitamin A and E, as well as that of structural compounds of biological membranes, i.e. phosphatidylcholine and phosphatidylserine, can be achieved.