Hemp (Cannabis sativa L.) Varieties: Fatty Acid Profiles and Upgrading of γ‐Linolenic Acid–Containing Hemp Seed Oils

Hemp seeds (HS) constitute a rich nutrient source and contain γ‐linolenic acid (GLA, 18:3, n‐6), which is a healthy fatty acid (FA). The objectives of this research are i) to look for GLA‐rich varieties of unhusked hemp seeds (UHS) and commercial hulled hemp seeds (HHS); ii) to check the influence of different extracting systems on both oil yield and FA profiles; iii) to test a simultaneous oil extraction/GLA‐enrichment process looking to improve GLA content. Hop and European hackberry seeds (both from Cannabaceae family) are also analyzed for comparative purposes. GLA is the most discriminant FA among UHS varieties, ranging in both UHS and HHS seeds from 0.5% to 4.5% of total FA, while hop seeds are the richest GLA source from Cannabaceae (7.2% of total FA). The extraction system selected for hemp seeds processing has a clear influence on oil yields, although, the FA profiles are slightly modified. The use of n‐hexane and n‐hexane:acetone in extractions allows an improvement in oil yields at the same GLA percentage. A process comprising saponification and subsequent cooling allows the improvement of GLA percentage in both hemp and hop seeds extracts at values higher than 10% of total FA, at high yields (>70%). Practical Applications: The global HS market increases significantly year after year and the demand of hemp products is increasing rapidly. The natural GLA sources in nature are limited, and although hemp contains GLA, this reaches low percentages in the oil. Hemp is a well‐established crop with highly standardized agricultural technologies, thus, the development of any well‐designed processes feasible for application in oil extraction industries, would allow the development of new GLA‐based functional seed oils. This would boost the development of the agricultural and food industries dedicated to revaluing hemp products.     

γ-Linolenic acid concentrates from borage and evening primrose oil fatty acidsvia lipase-catalyzed esterification

γ-Linolenic acid (GLA, all-cis 6,9,12-octadecatrienoic acid) has been enriched from fatty acids of borage (Borago officinalis L.) seed oil to 93% from the initial concentration of 20% by lipase-catalyzed selective esterification of the fatty acids withn-butanol in the presence ofn-hexane as solvent. The immobilized fungal lipase preparation, Lipozyme, used as biocatalyst, preferentially esterified palmitic, stearic, oleic and linoleic acids and discriminated against GLA, which was thus concentrated in the unesterified fatty acids fraction. In the absence of hexane, concentrate containing about 70% GLA was obtained. When the reaction conditions, optimized for borage oil fatty acids, were applied to fatty acids of evening primrose (Oenothera biennis L.) oil, concentrates containing 75% GLA were obtained. From both oils, GLA concentrates were prepared efficiently in short reaction times (1–3 h) at 30–60°C. The process can be applied for the production of GLA concentrates for dietetic purposes.     

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.     

Calendula and Coriandrum – New Potential Oilcrops for Industrial Uses

In a two years' field test Calendula officinalis and Coriandrum sativum were shown to be promising candidates for new crops for industrial oil productions. In Calendula seed oil the main fatty acid is calendic acid containing three conjugated double bonds. The mean oil content is 19.4%, and 62.8% of all fatty acids are calendic acid. Seed yield potential of C. officinalis proved to be excellent, but seed shedding is a major problem. Within the species a wide variation was established for genetic differences in, e.g. date of flowering, flower heads per plant, ray and disk flowers and different achene types. For sowing and harvest common farm machinery can be used. In Coriandrum sativum the main fatty acid is petroselinic acid, an isomer of the oleic acid. The agronomic potential of C. sativum appeared to be very good and no special breeding efforts are necessary to initiate first productions. The oil content was 17.1%, and 82% of all fatty acids consisted of oleic and petroselinic acid. In a 2 ha field trial with C. sativum the seed yield was as high as 25 dt/ha. Further breeding work should be directed to increase the oil content of the fruit and to possibly improve disease resistance.     

Kinetics of supercritical carbon dioxide extraction of borage and evening primrose seed oil

In the present work, high‐pressure extraction of borage (Borago officinalis L.) and evening primrose (Oenothera biennis L.) seed oil, containing the valuable γ‐linolenic acid (GLA), has been investigated. Extraction was performed with supercritical carbon dioxide on a semi‐continuous flow apparatus at pressures of 200 and 300 bar, and at temperatures of 40 and 60 °C. A constant flow rate of carbon dioxide in the range from 0.17 to 0.20 kg/h was maintained during extraction. The extraction yields obtained using dense CO₂ were similar to those obtained with conventional extraction using hexane as solvent. The composition of extracted crude oil was determined by GC analysis. The best results were obtained at 300 bar and 40 °C for both seed types extracted, where the quality of oil was highest with regard to GLA content. The evening primrose seed oil extracted with supercritical fluid extraction was particularly rich in unsaturated fatty acids: up to 89.7 wt‐% of total free fatty acids in the oil. The dynamic behavior of the extraction runs was analyzed using two mathematical models for describing the constant rate period and the subsequent falling rate period. Based on the experimental data, external mass transfer coefficients, diffusion coefficients and diffusivity in solid phase were estimated. Results showed good agreement between calculated and experimental data.     

Gamma‐linolenic and stearidonic acids from Moroccan Boraginaceae

Seeds from 20 species belonging to Boraginaceae, subfamilies Boraginoideae and Heliotropioideae, were surveyed in a search for new sources of γ‐linolenic acid (GLA) and stearidonic acid (SDA). Seed oil content ranged from 7.5% in Echium humile ssp. pycnanthum to 28.8% in Anchusa undulata. GLA ranged from 0.2% of total fatty acids in Heliotropium undulatum to 20.2% in Lithodora maroccana. This last species may be considered as new source of GLA. GLA content was also tested in other Lithodora species from the south east of Spain, to compare GLA percentages among related taxa. GLA amounts in all Echium species reached approximately 12%, in good agreement with previous findings in other European Echium species. SDA ranged from an absence in several Cynoglossum species to 16.2% in Echium humile ssp. pycnanthum, which may be considered as a new source of this fatty acid.     

Preparation of stearidonic acid‐enriched triacylglycerols from Echium plantagineum seed oil

HPLC analysis of Echium plantagineum seed oil shows a complex triacylglycerol (TAG) profile. TAG species were separated on an analytical scale by HPLC and their fatty acid (FA) composition is reported. GLC analyses showed that some TAG fractions reached a stearidonic acid (SDA, 18:4n‐3) percentage significantly higher than that in the original oil. TAG separation on a bigger scale was also essayed, by means of a gravimetric normal‐phase chromatographic column, using silver ion‐silica gel as stationary phase. Gradient elution with solvents of increasing polarity was applied, allowing the separation of valuable TAG species containing γ‐linolenic acid (GLA, 18:3n‐6), α‐linolenic acid (ALA, 18:3n‐3) and SDA as the main constituents (more than 85% of the total FA). An enzymatic hydrolysis reaction showed the distribution of FA in the isolated species of TAG. SDA was the major FA in the sn‐2 position (more than 50% of total FA), followed by ALA (19%) and GLA (18.5%).     

Characterisation of Moringa stenopetala seed oil variety “Marigat” from island Kokwa

The oil from Moringa stenopetala seeds variety Marigat from the island Kokwa was extracted using 3 different procedures including cold press (CP), extraction with n‐hexane and extraction with a mixture of chloroform:methanol (1:1) (CM). The yield of oil was 35.7% (CP) to 44.9% (CM). The density, refractive index, colour, smoke point, viscosity, acidity, saponification value, iodine value, fatty acid methyl esters, sterols, tocopherols (by high‐performance liquid chromatography), peroxide value, Eequation/tex2gif-stack-1.gif at 232 nm and the susceptibility to oxidation measured by the Rancimat method were determined. The oil was found to contain high levels of unsaturated fatty acids, especially oleic (up to 76.40%). The dominant saturated acids were behenic (up to 6.01%) and palmitic (up to 6.21%). The oil was also found to contain high levels of β‐sitosterol (up to 52.19%%of total sterols), stigmasterol (up to 16.53% of total sterols) and campesterol (up to 14.26% of total sterols). α‐, β‐ and δ‐tocopherols were detected up to levels of 98.00, 44.50 and 82.41 mg/kg of oil, respectively. The reduction of the induction period (at 120 °C) of M. stenopetala seed oil ranged from 29.4% to 54.7% after degumming. The M. stenopetala seed oil showed high stability to oxidative rancidity. The results of all the above determinations were compared with those of a commercial virgin olive oil and Moringa oleifera seed oil.     

Lipase‐mediated hydrolysis of flax seed oil for selective enrichment of α‐linolenic acid

Polyunsaturated fatty acids (PUFA) are important ingredients of human diet because of their prominent role in the function of human brain, eye and kidney. α‐Linolenic acid (ALA), a C18, n‐3 PUFA is a precursor of long chain PUFA in humans. Commercial lipases of Candida rugosa, Pseudomonas cepacea, Pseudomonas fluorescens, and Rhizomucor miehei were used for hydrolysis of flax seed oil. Reversed phase high performance liquid chromatography followed by gas chromatography showed that the purified oil contained 12 triacylglycerols (TAGs) with differences in fatty acid compositions. Flax seed oil TAGs contained α‐linolenic acid (50%) as a major fatty acid while palmitic, oleic, linoleic made up rest of the portion. Among the four commercial lipases C. rugosa has preference for ALA, and that ALA was enriched in free fatty acids. C. rugosa lipase mediated hydrolysis of the TAGs resulted in a fatty acid mixture that was enriched in α‐linolenic to about 72% yield that could be further enriched to 80% yield by selective removal of saturated fatty acids by urea complexation. Such purified ALA can be used for preparation of ALA‐enriched glycerides. Practical applications : This methodology allows purifying ALA from fatty acid mixture obtained from flax seed oil by urea complexation.     

Enrichment of γ-linolenic acid from evening primrose oil and borage oilvia lipase-catalyzed hydrolysis

Lipase-catalyzed selective partial hydrolysis of evening primrose (Oenothera biennis L.) seed oil and borage (Borago officinalis L.) seed oil led to an increase in the level of γ-linolenic acid (GLA; 18∶3n−6) in the unhydrolyzed acylglycerols. Thus, in evening primrose oil, the GLA level could be raised from 9.4% in the starting material to 46.5% in the unhydrolyzed acylglycerols by means of a lipase fromCandida cylindracea. Selective hydrolysis of borage oil with Pancreatin led to an increase in the GLA content from 20.4% in the oil to 33.5% in the unhydrolyzed acylglycerols. Partial hydrolysis of borage oil with lipase fromC. cylindracea raised the GLA content of the acylglycerols to 47.8%.     

Mass Spectrometric Confirmation of γ-Linolenic Acid Ester-Linked Ceramide 1 in the Epidermis of Borage Oil Fed Guinea Pigs

Ceramide 1 (Cer1), a Cer species with eicosasphingenine (d20:1) amide‐linked to two different ω‐hydroxy fatty acids (C30wh:0:C32wh:1), which are, in turn, ester‐linked to linoleic acid (LNA; 18:2n‐6), plays a critical role in maintaining the structural integrity of the epidermal barrier. Prompted by the recovery of a disrupted epidermal barrier with dietary borage oil [BO: 36.5 % LNA and 23.5 % γ‐linolenic acid (GLA; 18:3n‐6)], in essential fatty acid (EFA)‐deficient guinea pigs, we further investigated the effects of BO on the substitution of ester‐linked GLA for LNA in these two epidermal Cer1 species by LC–MS in positive and negative modes. Dietary supplementation of BO for 2 weeks in EFA‐deficient guinea pigs increased LNA ester‐linked to C32wh:1/d20:1 and C30wh:0/d20:1 of Cer1. Moreover, GLA ester‐linked to C32wh:1/d20:1, but not to C30wh:0/d20:1, of Cer1 was detected, which was further confirmed by the product ions of m/z 277.2 for ester‐linked GLA and m/z 802.3 for the deprotonated C32wh:1/d20:1. C20‐Metabolized fatty acids of LNA or GLA were not ester‐linked to these Cer1 species. Dietary BO induced GLA ester‐linked to C32wh:1/d20:1 of epidermal Cer1.     

Identification of n‐6 Monounsaturated Fatty Acids in Acer Seed Oils

Seed oils from Acer species are a potential source of the nutraceutical fatty acids, nervonic acid (cis‐15‐tetracosenoic acid, NA), and γ‐linolenic acid (cis‐6,9,12‐octadecatrienoic acid, GLA). To further characterize the genus, seed fatty acid content and composition were determined for 20 species of Acer. Fatty acid content ranged from 8.2% for Acer macrophyllum to over 36% for A. mono and A. negundo. The presence of very‐long‐chain fatty acids (VLCFA), with chain length of 20‐carbons or greater, and GLA were characteristic features of the seed oils. In all species, erucic acid (cis‐13‐docosenoic acid, EA) was the predominant VLCFA with the highest level of NA being only 8.6% in A. olivianum. Regioselective lipase digestion demonstrated that VLCFA are largely absent from the sn‐2 position of seed triacylglycerol, whereas GLA is primarily located at this position. Five Acer species contained low levels (<2%) of cis‐12‐octadecenoic acid and cis‐14‐eicosenoic acid, uncommon n‐6 fatty acids not previously reported from Acer.     

Lipophilic components in black currant seed and pomace extracts

The nature of the fatty acids and other lipophilic components in extracts from black currant seed and pomace (containing seed) were investigated, with a view to highlighting any potential uses. The same non‐hydroxylated fatty acids were the major components in both types of extract, but total levels were less in pomace (75 582 mg 100 g^?1 oil) than in seed alone (90 972 mg 100 g^?1 oil) and there were less unsaturated fatty acids, including GLA (8653 and 12 625 mg 100 g^?1 oil, respectively), but long chain n‐20:0 – n‐30:0 fatty acids (4080 and 437 mg 100 g^?1 oil, respectively) were greatly increased in pomace. Phytosterols (mainly β‐sitosterol), saturated n‐20:0 – n‐30:0 policosanols, ω‐hydroxy fatty acids (mainly 16‐hydroxy 16:0) and 2‐hydroxy fatty acids (mainly 2‐hydroxy 24:0) were present at much greater levels in pomace (2496, 2097, 958 and 46 mg 100 g^?1 oil, respectively) than in seed (553, 108, 161, and 1 mg 100 g^?1 oil, respectively). The pomace extract is a useful source of fatty acids, phytosterols and policosanols with potential functional properties. Practical applications: The study investigated the lipophilic components in isohexane extracts from black currant seed and pomace (containing seed). Only pomace extracts had substantial amounts of phytosterols and policosanols that have potential as cholesterol‐lowering agents, whereas fatty acids such as GLA, that has anti‐inflammatory properties, are mainly in the seed.     

Kigelia pinnata Seed Oil – A Moderate Source of Vernolic Acid

Vernolic acid represents 22.3% of the constituent fatty acids of the speed oil of an additional hitherto unexamined species of Bignoniaceae Kigelia pinnata. Its identification is based on comparative informations from thin-layer chromatography, infrared analysis, gas liquid chromatography and nuclear magnetic resonance spectroscopy with that of reference sample of Vernonia anthelmintica seed oil. The other fatty acids in this oil are: 14:0 (0.4), 16:0 (25.4), 18:0(0.9), 18:1 (8.9) and 18:2 (42.0%). K. pinnata is the first species of Bignoniaceae to be reported to contain vernolic acid in moderate amount.     

Restricted-Range Boraginaceae Species Constitute Potential Sources of Valuable Fatty Acids

The aim of this work was to establish the richness in γ-linolenic acid (GLA, 18:3n-6) and stearidonic acid (SDA, 18:4n-3) of the seed oil of several restricted-range Boraginaceae species, in a search for new valuable oils as advantageous alternatives to the commercially available sources of both polyunsaturated fatty acids. To this end, seeds of selected Boraginaceae species were collected and analyzed. The highest GLA contents (% total fatty acids) were found in the seed oils of Symphytum caucasicum M.Bieb. (22.9 %), Anchusa undulata subsp. undulata (Ten.) Cout. (22.0 %), Anchusa puechii Valdés (20.0 %), Glandora nitida Thomas (19.2 %), Echium pininana Webb & Berth. (17.1 %) and Pentaglottis sempervirens (L.) L. H. Bailey (17.0 %). With regard to SDA, the highest percentage was found in the seed oil of Echium cantabricum (M. Laínz) Fdez. Casas & M. Laínz (14.7 %), followed by Lappula patula (Lehm.) Asch ex Gürke (13.6 %). It is noticeable that several GLA-enriched species stand under a great threat of extinction, thus revealing the importance of the preservation of the natural ecosystems for endangered species.     

Enzyme-assisted acidolysis of menhaden and seal blubber oils with γ-linolenic acid

Oils containing both n−3 and n−6 fatty acids have important clinical and nutritional applications. Lipase-catalyzed acidolysis of seal blubber (SBO) and menhaden oils (MO) with γ-linolenic acid (GLA) was carried out in hexane. The process variables studied for lipase-catalyzed reaction were concentration of enzyme (100–700 units/g of oil), reaction temperature (30–60°C), reaction time (0–48 h), and mole ratio of GLA to triacylglycerols (TAG) (1∶1 to 5∶1). Two lipases chosen for acidolysis reaction were from Pseudomonas species (PS-30) and Mucor miehei. Lipase PS-30 was chosen over Mucor (also known as Rhizomucor) miehei to catalyze the acidolysis reaction owing to higher incorporation of GLA. For the acidolysis reaction, optimal conditions were a 3∶1 mole ratio of GLA to TAG, reaction temperature of 40°C, reaction time of 24 h, and an enzyme concentration of 500 units/g of oil. Under these conditions, incorporation of GLA was 37.1% for SBO and 39.6% for MO.     

One-Step Extraction and Concentration of Polyunsaturated Fatty Acids from Fish Liver

The fatty acids (FA) eicosapentaenoic acid (20:5ω-3; EPA) and docosahexaenoic acid (22:6ω-3; DHA), which have several health benefits, have been concentrated from mako shark liver (Isurus oxyrinchus). The process was carried out in one single step, in which fish liver oil was simultaneously extracted, saponified and concentrated. Additionally, the polyunsaturated fatty acids (PUFA) concentrate was winterized to crystallize the remaining saturated FA, resulting in a further increase in the concentration of DHA and EPA. Two variables, temperature and water concentration in the saponification mixture, were optimized to increase the concentration of ω-3 PUFA. Best results were obtained at 12 °C and 0% water content in the mixture, obtaining 17.8% purity and 77.6% yield of EPA; DHA purity and yield were 33.3 and 82.2%, respectively.     

Chemical Properties and Fatty Acid Composition of Thunbergia fragrans Roxb. Seed Oil

The physicochemical and fatty acid compositions of seed oil extracted from Thunbergia fragrans were determined. The oil content, free fatty acids, peroxide value, saponification value and iodine value were 21.70 %, 2.25 % (as oleic acid), 9.6 (mequiv. O₂/kg), 191.71 (mg KOH/g) and 127.84 (g/100 g oil) respectively. The fatty acid profiles of the methyl esters showed the presence of 90.16 % unsaturated fatty acids and 9.84 % saturated fatty acids. Palmitoleic acid, which is usually found in marine foods and is unique in seed oils of botanical origin, was the major component (79.24 %). The oil can also be used in industries for the preparation of liquid soaps, shampoos and alkyd resin.     

Purification of GLA-Triglycerides from Evening Primrose Oil by Gravimetric Column Chromatography

Gravimetric normal-phase silver ion–silica gel column chromatography has been used for the novel application of purification of GLA-containing triglycerides (GLA-TGs) from evening primrose seed oil (EPO). Gradient elution with increasing polarity enabled separation of valuable TG species containing γ-linolenic acid (GLA, 18:3n-6). Enzymatic hydrolysis revealed the distribution of fatty acids (FAs) in the isolated TG species, with GLA in the sn-2 position in different percentages, depending on the degree of unsaturation. A novelty of this work was the successful use of the procedure to improve the purification of raw GLA species from EPO up to preparative scale, thus enabling use of this methodology for industrial purposes.     

Preparation of γ-linolenic acid rich triacylglycerol from borage oil via a two-step lipase-catalyzed reaction

γ-Linolenic acid (GLA) rich triacylglycerol (TAG) was successfully synthesized from glyceride, instead of glycerol, and fatty acid (FA) via Lipozyme TL IM-catalyzed esterification as a novel strategy. In the first step, GLA was enriched into glyceride fraction from borage oil by Candida rugosa lipase-catalyzed hydrolysis. The glyceride was separated from the reaction mixture by molecular distillation. GLA was enriched from 20.64% in borage oil to 45.94% in the glyceride fraction under optimum conditions. In the second step, the Lipozyme TL IM-catalyzed synthesis of TAG was carried out with the glyceride, and the FA obtained by saponification of a portion of the glyceride. The optimum conditions were the temperature of 50°C, the enzyme loading of 10%, and the vacuum level of 20 mmHg, respectively. The maximum TAG content of approximately 92% was achieved after 12 h under the optimum conditions.