Structural characterization of triacylglycerol in several oils containing gamma-linolenic acid

The differences are reported in the triacylglycerol (TG) structures of oils containing gamma-linolenic acid (GLA) from Oenothera biennis Linn seed oil (OBLO) from the wild plant, evening primrose seed oil (EPO) from a cultured plant, and bio-GLA oil (BIO) from a mold, the physiological functions of which were ascertained by animal testing. Reverse-phase high-performance liquid chromatographic separation detected 12 TG peaks each for OBLO and EPO, and 28 TG peaks for BIO. TG-containing GLA were composed of five molecular species each in OBLO and EPO, and ten molecular species in BIO. The totals of the molecular species containing GLA were 29.8% in OBLO, 23.8% in EPO, and 56.6% in BIO. In OBLO, the GLA level at the sn-2 position of the major TG species was higher than that in EPO. In BIO, the GLA level at the sn-2 position of the major TG species was lower than those in OBLO and EPO.     

Effect of Temperature on the Fatty Acid Composition and Yield of Evening Primrose (Oenothera lamarckiana) Seeds

The oil content and yield of evening primrose (Oenothera lamarckianaL.) seeds decreased significantly at high temperatures (32 ?C)compared with lower ones (25 ?C and below), under controlledgrowing conditions. Moreover, the fatty acid composition ofthe seed oils was also affected by the temperature regime. Higheroleic acid levels and lower linoleic and -linolenic acid (GLA)levels were characteristic of the seeds developed at highertemperatures. The opposite trend was observed at lower temperatures.In field experiments, seeds of O. lamarckiana plants sown inautumn contained lower oleic acid levels, and higher linoleicacid and GLA levels than seeds of spring plantings. This effectwas also demonstrated for seeds developed on the same stem inflorescenceunder increasing temperatures during the season in the field.Changes in fatty acid composition during various stages of seeddevelopment were observed. Key words: Evening primrose, oil, fatty acids, Oenothera spp., gamma linolenic acid     

PERMANENT GENETIC RESOURCES: Isolation and characterization of polymorphic microsatellite loci in common evening primrose (Oenothera biennis)

We developed nine polymorphic microsatellite loci for evening primrose (Oenothera biennis). These loci have two to 18 alleles per locus and observed heterozygosities ranging from 0 to 0.879 in a sample of 34 individuals. In a pattern consistent with the functionally asexual reproductive system of this species, 17/36 pairs of loci revealed significant linkage disequilibrium and three loci showed significant deviations from Hardy-Weinberg equilibrium. The loci will be informative in identifying genotypes in multigenerational field studies to assess changes in genotype frequencies.     

Role of intracellular reactive oxygen species and mitochondrial dysfunction in evening primrose extract-induced apoptosis in Ehrlich ascites tumor cells

Herbal medicines are increasingly being utilized to treat a wide variety of disease processes. Evening primrose extract (EPE) is extracted from Oenothera biennis L., one species of evening primroses, which has been shown to have several pharmacological effects. However, anti-tumor activity in the extract of defatted seeds of O. biennis L. has not been defined thus far. In this study, we identified the major biochemical changes upon EPE treatment and investigated the functional relationship between these changes. We found that EPE-induced apoptosis in Ehrlich ascites tumor cells as evidenced by morphological changes. Furthermore, our results demonstrated rapid increase of intracellular peroxides levels, loss of mitochondrial membrane potential and the release of cytochrome c from mitochondria to cytosol. These results suggest that the rapid increase of intracellular peroxides levels after addition of EPE triggers off induction of apoptosis.     

High frequency of adventitious shoot regeneration from commercial cultivars of evening primrose (Oenothera spp.) using thidiazuron

Evening primrose (Oenothera spp.) is grown commercially for its seed oil that contains gamma linolenic acid (GLA), a valuable food supplement and pharmaceutical. There is considerable interest in the potential of genetic engineering to improve yields of GLA in evening primrose, and attention has focused on the current state of tissue‐culture knowledge in this species which is a prerequisite for genetic transformation. Published protocols for the regeneration of plants from leaf or cotyledon material of Oenothera spp. are available, but these prove unsatisfactory when applied to commercial cultivars used in this study. An efficient method for regenerating three commercial cultivars of evening primrose Rigel, Merlin and Vulcan was developed using thidiazuron (TDZ) as a growth regulator. Explants from one month old seedlings were cultivated in vitro; a large number of buds were induced directly from strips of leaves, cotyledons and stems when cultured on Murashige and Skoog (MS) basal medium containing TDZ and indole‐butyric acid (IBA). Shoots that were excised and placed onto MS basal medium, supplemented with IBA, rooted with 85–90% efficiency. Plantlets were transferred to soil after 6–8 wk. TDZ stimulated the regeneration process, and its effects were enhanced when combined with IBA or indole‐3‐acetic acid (IAA). The methods developed may be a useful advance toward improvement of this oil seed crop through genetic modification.     

Antioxidant potential of evening primrose oil administration in hyperlipemic rabbits.

The dietary intake of saturated fatty acids affects arteriosclerosis. We studied the effect of supplementation (15% wt/wt) of a hyperlipemic diet (1.33% cholesterol) with evening primrose oil (EPO) (Oenothera biennis) for 6 weeks in four groups of 10 rabbits each. At the end of this period we determined lipid peroxidation, glutathione content, and glutathione peroxidase, reductase and transferase activities in liver, brain, heart, aorta and platelets. The atherogenic diet increased tissue lipid peroxidation and decreased the protective antioxidant effect of glutathione. Dietary supplementation with EPO reduced tissue lipid peroxidation (61% in liver, 57% in brain, 42% in heart, 24% in aorta, 33% in platelets). Total glutathione was increased, especially in the aorta (90%) and platelets (200%); however, in all tissues the percentage of oxidised glutathione decreased. Evening primrose oil reduced glutathione peroxidase activity and increased the activities of glutathione reductase and transferase. We conclude that in rabbits made hyperlipemic with a diet rich in saturated fatty acids, EPO decreased tissue oxidative stress.     

THE EIGHTY-YEAR PERIOD FOR DR. BEAL'S SEED VIABILITY EXPERIMENT

Darlington , H. T., and G. P. Steinbauer . (Michigan State U., East Lansing.) The eighty-year period for Dr. Beal's seed viability experiment. Amer. Jour. Bot. 48 (4): 321–325. 1961.—Seeds of 20 species of plants were buried 80 years ago on the Michigan State University campus. Bottles have been dug up periodically in the intervening years and the seeds tested for viability. A bottle representing the 80-year period of burial was dug up this spring and the contents distributed uniformly on the surface of sterilized soil in the greenhouse. Of the 20 species whose seeds were buried in 1879, only 3 have survivors as viable seeds. These are the same 3 that had survived the 70-year burial period, namely: curled dock, Rumex crispus; evening primrose, Oenothera biennis; and moth mullein, Verbascum blattaria. The low percentage survival of curled dock (2%) indicates that the longevity of the buried seeds of this species is nearing the end. The situation is more favorable for evening primrose, with 10% survival. The third, moth mullein, has about as many survivors as 10 years ago, a remarkably high figure of 70% viability. Since there has been no significant drop in viability of the buried seeds over the last 30 years, it seems highly probable that longevity of buried seeds of this species may extend over a century. A brief summary is given of the current status of the behavior of buried seeds, based on this and other buried seed experiments.     

Enzymatic fractionation of evening primrose oil by rape lipase: Enrichment of gamma-linolenic acid

Summary Rape lipase discriminates strongly against -linolenic acid (allcis-6, 9, 12–183; GLA). GLA in the fatty acids from evening primrose oil was concentrated from 9.5% to 62% with a 95% yield during esterification of these fatty acids with butanol catalyzed by rape lipase. Hydrolysis of evening primrose oil catalyzed by the rape lipase raised the GLA content in unhydrolyzed acylglycerols to 28%.     

Gamma-linolenic acid provides additional protection against ventricular fibrillation in aged rats fed linoleic acid rich diets

Ligation of the coronary artery in rats produces severe ventricular fibrillation (VF) and malignant cardiac arrhythmia. Mortality increases with the age of the animal. Diets rich in saturated fatty acids (SF) but low in linoleic acid (LA) increase, but diets high in LA and low in SF decrease the severity of VF and mortality in older animals. The effects of an LA enriched diet can be blocked by inhibition of cyclooxygenase suggesting that conversion of LA to eicosanoids is central to the development of VF. Conversion of LA to gamma-linolenic acid (GLA) via delta-6 desaturase is the first step in the process. The activity of delta-6 desaturase declines with age. Thus inclusion of GLA in the diet of older animals may provide an additional benefit over LA alone. Dietary supplements of evening primrose oil (EPO) to one year old rats reduced ischaemic VF more than a supplement of sunflower seed oil (SSO) without GLA. Substitution of borage oil (more GLA than EPO but less LA than either EPO or SSO) was without additional benefit.     

Effects of Triterpenes on the Mutagenicities of Various Mutagens toward Salmonella

The effects of different dietary levels of gamma-linolenic acid (GLA) on lipid metabolism was studied in rats using a combination of evening primrose oil (EPO) and palm oil (PLO). EPO compared to PLO significantly reduced liver cholesterol and triglyceride after 4 weeks of feeding, and the effect remained even when EPO was mixed with PLO at the same weight basis. The serum triglyceride level also tended to be low on feeding EPO. Neither liver Δ6-desaturase and phospholipase A₂ activities nor aortic production of prostacyclin and thromboxane A₂ production by platelets were influenced significantly by the fat type, suggesting a peculiar effect of PLO. The percentage of arachidonic acid in liver, serum, and aortic phosphatidylcholine depended on the dietary level of GLA. A more distinct increase in arachidonic acid was observed in tissue triglycerides of rats fed EPO. GLA appears to exert favorable effects on lipid metabolism even when the P/S ratio was lowered from 13.7 of EPO to 1.8 of the 1:1 mixture of EPO and PLO.     

Physiological effects of γ-linolenic acid and sesamin on hepatic fatty acid synthesis and oxidation

Interrelated effects of γ-linolenic acid (GLA) and sesamin, a sesame lignan, on hepatic fatty acid synthesis and oxidation were examined. Rats were fed experimental diets supplemented with 0 or 2 g/kg sesamin (1:1 mixture of sesamin and episesamin) and containing 100 g/kg of palm oil (saturated fat), safflower oil rich in linoleic acid, or oil of evening primrose origin containing 43% GLA (GLA oil) for 18 days. In rats fed sesamin-free diets, GLA oil, compared with other oils, increased the activity and mRNA levels of various enzymes involved in fatty acid oxidation, except for some instances. Sesamin greatly increased these parameters, and the enhancing effects of sesamin on peroxisomal fatty acid oxidation rate and acyl-CoA oxidase, enoyl-CoA hydratase and acyl-CoA thioesterase activities were more exaggerated in rats fed GLA oil than in the animals fed other oils. The combination of sesamin and GLA oil also synergistically increased the mRNA levels of some peroxisomal fatty acid oxidation enzymes and of several enzymes involved in fatty acid metabolism located in other cell organelles. In the groups fed sesamin-free diets, GLA oil, compared with other oils, markedly reduced the activity and mRNA levels of various lipogenic enzymes. Sesamin reduced all these parameters, except for malic enzyme, in rats fed palm and safflower oils, but the effects were attenuated in the animals fed GLA oil. These changes by sesamin and fat type accompanied profound alterations in serum lipid levels. This may be ascribable to the changes in apolipoprotein-B-containing lipoproteins.     

The safety of evening primrose oil in epilepsy

The concern that evening primrose oil might cause epilepsy or seizures, or reduce the threshold for seizures, originated from two papers published in the early 1980s. These original reports are re-examined, and the association of evening primrose oil with seizures is shown to be spurious. Not only are linoleic acid and gamma-linolenic acid safe in epilepsy, with prolonged oral administration of linoleic acid and alpha-linolenic acid (in a 4:1 mixture) protecting rats from having seizures in four different epilepsy models, but the evening primrose oil-derived omega-6 fatty acid arachidonic acid inhibits sodium ion currents and synaptic transmission, while the evening primrose oil-derived eicosanoid prostaglandin E(1) appears to have anticonvulsant activity. In light of these findings, it is suggested that formularies should now remove seizures or epilepsy as a side-effect of evening primrose oil, and should remove a history of seizures or epilepsy as a contraindication to taking evening primrose oil.     

Possible involvement of delta-6-desaturase in control of melanoma growth by gamma-linolenic acid.

This study examined the effects of linoleic acid (LA) and gamma-linolenic acid (GLA) on BL6 melanoma growth in cell culture and of safflower oil (SFO) which contains LA and evening primrose oil (EPO) which contains GLA, on melanoma growth when grown in mice. The delta-6-desaturase activity of the melanoma cells in the two systems was also examined and an attempt made to relate the activity of the enzyme to the effects of GLA on cell and tumour growth. LA and GLA were found to be equipotent in inhibiting growth of the in vitro cultured BL6 cells which were found to contain an appreciable level of delta-6-desaturase activity. EPO was however found to be a more potent promoter of in vivo melanoma growth in mice than SFO. Melanomas grown in mice were found to lack delta-6-desaturase activity suggesting that the EPO diet, by providing GLA, was able to compensate for the loss of enzyme activity in the melanomas. The possibility that melanomas in mice have a requirement for GLA for growth while in in vitro cultured cells excess GLA inhibits the growth of the cells through an increase in lipid peroxidation is discussed.     

Protective Effect of Borage Seed Oil and Gamma Linolenic Acid on DNA: In Vivo and In Vitro Studies

Borage (Borago officinalis L.) seed oil has been used as a treatment for various degenerative diseases. Many useful properties of this oil are attributed to its high gamma linolenic acid content (GLA, 18:3 ω-6). The purpose of this study was to demonstrate the safety and suitability of the use of borage seed oil, along with one of its active components, GLA, with respect to DNA integrity, and to establish possible in vivo toxic and in vitro cytotoxic effects. In order to measure these properties, five types of assays were carried out: toxicity, genotoxicity, antigenotoxicity, cytotoxicity (using the promyelocytic leukaemia HL60 cell line), and life span (in vivo analysis using the Drosophila model). Results showed that i) Borage seed oil is not toxic to D. melanogaster at physiological concentrations below 125 µl/ml and the studies on GLA indicated non-toxicity at the lowest concentration analyzed ii) Borage seed oil and GLA are DNA safe (non-genotoxic) and antimutagenic compared to hydrogen peroxide, thereby confirming its antioxidant capacity; iii) Borage seed oil and GLA exhibited cytotoxic activity in low doses (IC₅₀ of 1 µl/ml and 0.087 mM, respectively) iv) Low doses of borage seed oil (0.19%) increased the health span of D. melanogaster; and v) GLA significantly decreased the life span of D. melanogaster.Based on the antimutagenic and cytotoxic effects along with the ability to increase the health span, we propose supplementation with borage seed oil rather than GLA, because it protects DNA by modulating oxidative genetic damage in D. melanogaster, increases the health span and exerts cytotoxic activity towards promyelocytic HL60 cells.     

Antioxidant factors in plant foods and selected oilseeds

The effect of dietary factors on health promotion and disease prevention has been an issue of interest since antiquity and has become a subject of renewed research activity in recent years. Many of the components involved are antioxidative in nature and include phenolic compounds. These phenolics exist in the free, esterified, glycosylated and polymeric forms. Scrutiny of the source materials, their subsequent extraction under optimized conditions and evaluation of activity, followed by fractionation and structure elucidation of active components is generally necessary. Meals obtained from oilseeds, such as canola, mustard, flax, borage, and evening primrose, after oil extraction, contained a variety of antioxidative factors. The antioxidative effect of source materials, their extracts or fractions thereof, depended on the seed type, the content and chemical nature of their active components.     

Fatty acid and prostaglandin metabolism in children with diabetes mellitus. II. The effect of evening primrose oil supplementation on serum fatty acid and plasma prostaglandin levels

Our previous study demonstrated that levels of dihomo-gamma-linolenic acid (DGLA) and arachidonic acid in serum total lipids decreased in association with increased plasma levels of prostaglandins E2 (PGE2) and F2 alpha (PGF2 alpha) in patients with insulin-dependent diabetes mellitus. In this study, 11 children with insulin-dependent diabetes mellitus completed a double-blind, placebo-controlled study to assess the effect of dietary supplementation with gamma-linolenic acid (GLA) on serum essential fatty acid and plasma PGE2 and PGF2 alpha levels. GLA was given as the seed oil from the evening primrose (EPO) and all patients received either EPO capsules (containing 45 mg of GLA and 360 mg of linoleic acid) or indistinguishable placebo capsules for 8 months. Initially patients took 2 capsules daily for 4 months then 4 capsules daily for a further 4 months. All patients were assessed at the start of the study, after 4 months and at the end of the study, by measuring serum essential fatty acid and plasma PGE2 and PGF2 alpha levels. After administration of 4 capsules daily the DGLA levels increased and PGE2 levels decreased significantly (p less than 0.01) in the EPO compared with the placebo group. Neither fatty acid nor PGE2 and PGF2 alpha levels were altered by administration of 2 EPO capsules daily. This suggests that the altered essential fatty acid and PG metabolism in diabetes may be reversed by direct GLA supplementation.     

Dose-dependent effects of dietary gamma-linolenic acid on rat spleen lymphocyte functions.

Feeding rodents a diet rich in evening primrose oil (EPO), which contains 5-10 g gamma-linolenic acid (GLA)/100 g total fatty acids, has been shown to decrease lymphocyte proliferation and natural killer cell activity. However, EPO contains a very high level of linoleic acid which itself can affect lymphocyte functions and it is not clear to what extent the effects of EPO can be attributed to GLA. The current study investigated the effect of two levels of GLA in the rat diet upon immune cell functions; the level of linoleic acid was maintained below 30 g/100 g total fatty acids. Weanling rats were fed on high fat (178 g/kg) diets which contained 4.4 g or 10 g GLA/100 g total fatty acids in place of a proportion of linoleic acid. The total polyunsaturated fatty acid content and the n-6 to n-3 polyunsaturated fatty acid ratio of the diet were maintained at 35 g/100 g total fatty acids and 7, respectively. The fatty acid compositions of the serum and of spleen leukocytes were markedly influenced by that of the diet, with an increase in the proportions of GLA and dihomo-gamma-linolenic acid when the diets containing GLA were fed; these diets also increased the proportion of arachidonic acid in spleen leukocytes. Spleen lymphocyte proliferation in response to concanavalin A was significantly reduced (by 60%) by feeding the diet containing the higher level of GLA, but not by the diet containing the lower level of GLA. Spleen natural killer cell activity and prostaglandin E (PGE) production by spleen leukocytes were not significantly affected by inclusion of GLA in the diet, although there was a tendency towards decreased natural killer cell activity by cells from rats fed the high GLA diet. Thus, this study shows that dietary GLA is capable of altering the fatty acid composition of cells of the immune system and of exerting some immunomodulatory effects, but that the level of GLA in the diet must exceed 4.4 g/100 g total fatty acids for these effects to become apparent.     

THE NINETY-YEAR PERIOD FOR DR. BEAL'S SEED VIABILITY EXPERIMENT

In 1879 Dr. W. J. Beal selected seeds of 23 different species of locally common plants, mixed 50 seeds of each species with moist sand in unstoppered one-pint bottles, and buried the bottles in a sandy knoll to be unearthed and the viability of the seeds tested periodically. The year 1970 marked the ninetieth year the seed had been buried, and the thirteenth bottle was recovered to test for seed viability. Of the three species which had germinated in the 1960 test (curly dock, Rumex crispus; evening primrose, Oenothera biennis; and moth mullein, Verbascum blattaria), only V. blattaria had viable seed with 20% germination. No other species germinated. All ten seedlings of V. blattaria were grown to maturity, and seeds were then harvested to study the possible deviations from normality and the requirements for seed germination. All seedlings emerging from the first progeny seed appeared normal. The most prominent requirement for germination was light, and this is a possible explanation of why the seeds had remained viable but dormant for so long a period. One-third of the freshly harvested seed germinated in darkness and, furthermore, redrying of dark-moistened seed in the absence of light induced additional germination. Germination of dark-moistened seed was not completely restored when the still moist seeds were subsequently exposed to light. However, when dark-moistened seeds were dried and then remoistened in the light, germination was about 95 %. About 5 % of the seed did not germinate under the conditions used. We find that 5 % of the population of V. blattaria seeds are dormant for unknown reasons, that 30 % will germinate if supplied only with moisture, and that 65 % are inhibited and require light and moisture simultaneously for germination. Supplying this 65 % of the population with moisture in darkness results in the development of a second type of inhibition which is no longer light reversible. It appears that the simultaneous requirement for light and moisture is an important factor in permitting V. blattaria seeds to remain dormant during prolonged burial.     

Additions to the list of wild edible plants preservable by the deep freeze method

While the author (Gaertner 1962) has previously reported on the freezing, preservation and preparation of native wild plants of Ontario for culinary purposes, this communication concerns itself primarily with three common weeds: sow thistle (Sonchus spp.), evening primrose (Oenothera biennis L.) and sheep sorrel (Rumex acetosella L.), which might add to our winter diet. Two of the three plants are European introductions, usually considered extremely troublesome when present in the field; the third is a native biennial. In addition, the use of the unopened male cones of pine (Pinus spp.) and spruce (Picea spp.) are described.