Preparation of High-Purity Docosahexaenoic Acid Ethyl Ester from Algal Oil through Enzymatic Ethanolysis.

Docosahexaenoic acid plays a crucial role in infant brain function, and the market demand of high-purity docosahexaenoic acid is continuously increasing. The availability of docosahexaenoic acid in natural fish oil is limited, prompting the exploration of alternative sources like microalgae. For algal oil, enzymatic ethanolysis is preferred to chemical methods because the former is milder and can avoid docosahexaenoic acid oxidation. However, enzymatic methods have generally low yield due to the poor substrate-specificity of lipase to long-chain polyunsaturated fatty acids, affecting the yield and purity of docosahexaenoic acid. Therefore, we developed an efficient process to produce high-purity docosahexaenoic acid ethyl ester from algal oil, by screening lipases, optimizing enzymatic ethanolysis and applying molecular distillation. Lipase UM1 was the best lipase to produce ethyl ester from algal oil with the highest ethyl ester yield (95.41%). Meanwhile, it was a catalyst for the reaction of long-chain polyunsaturated fatty acids with ethanol. The fatty acid docosahexaenoic acid conversion rates exceeded 90%. After molecular distillation, a final product containing 96.52% ethyl ester was obtained with a docosahexaenoic acid content up to 80.11%. Our findings provide an highly effective enzymatic method for the production of high-purity docosahexaenoic acid ethyl esters, with potential commercial applications.

Simultaneous Treatment of Long-chain Monounsaturated Fatty Acid and n-3 Polyunsaturated Fatty Acid Decreases Lipid and Cholesterol Levels in HepG2 Cell.

The n-3 type polyunsaturated fatty acids (n-3PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), from fish oil exhibit health benefits such as triacylglycerol- and cholesterol-lowering effects. Some pelagic fishes contain long-chain monounsaturated fatty acids (LC-MUFAs) such as eicosenoic acid (C20:1), which exert health-promoting effects. However, no study has evaluated beneficial effects of n-3PUFA and LC-MUFA combination. Here, we investigated effects of simultaneous treatment with n-3PUFA (EPA and DHA) and LC-MUFA (cis-5-C20:1 and cis-7-C20:1) and found that n-3PUFA and LC-MUFA combination significantly decreased lipid accumulation and reduced total cholesterol in HepG2 cells. Cholesterol level was significantly lower in DHA + cis-7-C20:1 group than in DHA + EPA group. These results suggest the importance of LC-MUFA as a functional molecule in fish oil.

Separation of high-purity eicosapentaenoic acid and docosahexaenoic acid from fish oil by pH-zone-refining countercurrent chromatography.

The evidence for unique effects of eicosapentaenoic acid and docosahexaenoic acid is growing. Further understanding and exploration of their independent effects in the nutraceutical and pharmaceutical industry is calling for the more efficient separation techniques to overcome the equivalent chain length rule of fatty acids. In this study, free eicosapentaenoic and docosahexaenoic acid were successfully separated by pH-zone-refining countercurrent chromatography for the first time. The different solvent systems and the influence of retainer and eluter concentration on the separation efficiency were investigated. A two-phase solvent system composed of n-heptane/methanol/water (100:55:45, v/v) was selected with 50 mM of trifluoroacetic acid as retainer in the organic phase and 40 mM of ammonium hydroxide as an eluter in the aqueous phase for the separation of 500 mg of free fatty acids from a refined fish oil sample. 79.6 mg of eicosapentaenoic acid and 328.3 mg docosahexaenoic acid were obtained with the purities of 95.5 and 96.9% respectively determined by gas chromatography with mass spectrometry after methyl esterification. The scale-up separation of 1 g of samples from both refined and crude fish oil after urea complexation were also achieved successfully with a markedly increased concentration 150 mM of retainer, producing satisfactory yields and purities of targets.

Effect of spray-drying with organic solvents on the encapsulation, release and stability of fish oil.

Fish-oil (FO) was encapsulated with hydroxypropylcelullose (HPC) by conventional spray-drying with water (FO-water) and solvent spray-drying with ethanol (FO-EtOH), methanol (FO-MeOH) and acetone (FO-Acet) in order to study the effect of the solvent on the encapsulation efficiency (EE), microparticle properties and stability of FO during storage at 40 °C. Results showed that FO-Acet presented the highest EE of FO (92.0%), followed by FO-EtOH (80.4%), FO-MeOH (75.0%) and FO-water (71.1%). A decrease of the dielectric constant increased the EE of FO, promoting triglyceride-polymer interactions instead of oil-in-water emulsion retention. FO release profile in aqueous model was similar for all FO-microparticles, releasing only the surface FO, according to Higuchi model. Oxidative stability of FO significantly improved by spray-drying with MeOH, both in surface and encapsulated oil fractions. In conclusion, encapsulation of FO by solvent spray-drying can be proposed as an alternative technology for encapsulation of hydrophobic molecules.

The Association of Fatty Acid Levels and Gleason Grade among Men Undergoing Radical Prostatectomy.

BACKGROUND: Epidemiological data suggest that omega-6 (ω-6) fatty acids (FAs) may be associated with cancer incidence and/or cancer mortality, whereas ω-3 FAs are potentially protective. We examined the association of the ratio of ω-6 to ω-3 FA (ω-6:ω-3) and individual FA components with pathological results among men with prostate cancer (PCa) undergoing radical prostatectomy. METHODS: Sixty-nine men were included in the study. Components of ω-6 (linoleic acid (LA), arachidonic acid (AA), and dihomo-γ-linolenic acid (DGLA)) and ω-3 (docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)) were analyzed by liquid chromatography/mass selective detector separation. Logistic regression analysis was performed to determine association of FA with pathological high grade (Gleason ≥4+3) disease. RESULTS: The were 35 men with low grade disease (Gleason ≤3+4) and 34 men with high grade disease. Men with low grade disease were significantly younger (58y vs 61y, p = 0.012) and had lower D'Amico clinical classification (p = 0.001) compared to men with high grade disease. There was no significant association of ω-6:ω-3 with high grade disease (OR 0.93, p = 0.78), however overall ω-6, ω-3, and individual components of ω-6 and ω-3 FAs except EPA were significantly associated with high grade disease (ω-6: OR 3.37, 95% CI: 1.27,8.98; LA: OR 3.33, 95% CI:1.24,8.94; AA: OR 2.93, 95% CI:1.24,6.94; DGLA: OR 3.21, 95% CI:1.28,8.04; ω-3: OR 3.47, 95% CI:1.22,9.83; DHA: OR 3.13, 95% CI:1.26,7.74). ω-6 and ω-3 FA components were highly correlated (Spearman ρ = 0.77). CONCLUSION: Higher levels of individual components of ω-6 and ω-3FAs may be associated with higher-grade PCa. IMPACT: Studies into the causative factors/pathways regarding FAs and prostate carcinogenesis may prove a potential association with PCa aggressiveness.

Canola engineered with a microalgal polyketide synthase-like system produces oil enriched in docosahexaenoic acid.

Dietary omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5) are usually derived from marine fish. Although production of both EPA and DHA has been engineered into land plants, including Arabidopsis, Camelina sativa and Brassica juncea, neither has been produced in commercially relevant amounts in a widely grown crop. We report expression of a microalgal polyketide synthase-like PUFA synthase system, comprising three multidomain polypeptides and an accessory enzyme, in canola (Brassica napus) seeds. This transgenic enzyme system is expressed in the cytoplasm, and synthesizes DHA and EPA de novo from malonyl-CoA without substantially altering plastidial fatty acid production. Furthermore, there is no significant impact of DHA and EPA production on seed yield in either the greenhouse or the field. Canola oil processed from field-grown grain contains 3.7% DHA and 0.7% EPA, and can provide more than 600 mg of omega-3 LC-PUFAs in a 14 g serving.

CYTOTOXIC EFFECTS OF THE RED SEA SOFT CORAL SARCOPHYTON TROCHELIOPHORUM.

The present study describes the in vitro cytotoxic effects of soft coral (Sarcophyton tiocheliophorum). Soft corals of genus Sarcophyton were reported to contain compounds that are active against brine shrimp and promote paclitaxel cytotoxicity in the human colon cancer Caco-2 cell line. The n-hexane extract of the soft coral Sarcophyton tiocheliophorum induced significant dose-dependent toxicity (LC50 96.7 ppm) compared with ethyl acetate (LC50. 120 ppm). We reported the most active cytotoxic level to be correspondence to LC50 values of 20.2, 59.2 ppm and 18.9 and 26 ppm. Accordingly, bio-assay guided fractionation was conducted to identi- fy the bioactive compounds. Arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid were characterized based on GC-MS analyses. Our results demonstrate the value of marine products as a natural source of medicinally interesting cytotoxic compounds.

Development of botanical and fish oil standard reference materials for fatty acids.

As part of a collaboration with the National Institutes of Health's Office of Dietary Supplements and the Food and Drug Administration's Center for Drug Evaluation and Research, the National Institute of Standards and Technology has developed Standard Reference Material (SRM) 3274 Botanical Oils Containing Omega-3 and Omega-6 Fatty Acids and SRM 3275 Omega-3 and Omega-6 Fatty Acids in Fish Oil. SRM 3274 consists of one ampoule of each of four seed oils (3274-1 Borage (Borago officinalis), 3274-2 Evening Primrose (Oenothera biennis), 3274-3 Flax (Linium usitatissimum), and 3274-4 Perilla (Perilla frutescens)), and SRM 3275 consists of two ampoules of each of three fish oils (3275-1 a concentrate high in docosahexaenoic acid, 3275-2 an anchovy oil high in docosahexaenoic acid and eicosapentaenoic acid, and 3275-3 a concentrate containing 60% long-chain omega-3 fatty acids). Each oil has certified and reference mass fraction values for up to 20 fatty acids. The fatty acid mass fraction values are based on results from analyses using gas chromatography with flame ionization detection (GC-FID) and mass spectrometry (GC/MS). These SRMs will complement other reference materials currently available with mass fractions for similar analytes and are part of a series of SRMs being developed for dietary supplements.

A meta-analysis shows that docosahexaenoic acid from algal oil reduces serum triglycerides and increases HDL-cholesterol and LDL-cholesterol in persons without coronary heart disease.

Certain algae contain the (n-3) fatty acid DHA, yet the relation between algal oil supplementation and cardiovascular disease risk factors has not been systematically examined. Our objective was to examine the relation between algal oil supplementation and cardiovascular disease risk factors. We conducted a systematic review of randomized controlled trials published between 1996 and 2011 examining the relation between algal oil supplementation and cardiovascular disease risk factors and performed a meta-analysis of the association between algal oil DHA supplementation and changes in the concentrations of TG, LDL-cholesterol (LDL-C), and HDL-cholesterol (HDL-C). We identified 11 randomized controlled trials with 485 healthy participants that evaluated the relation between algal oil DHA supplementation and TG, LDL-C, and HDL-C. The median dose of algal DHA was 1.68 g/d. The pooled estimate for the change in TG concentration was -0.20 mmol/L (95% CI: -0.27 to -0.14), 0.23 mmol/L (95% CI: 0.16-0.30) for LDL-C, and 0.07 mmol/L (95% CI: 0.05-0.10) for HDL-C. DHA supplementation from algal oil, a marine source of (n-3) fatty acids not extracted from fish, may reduce serum TG and increase HDL-C and LDL-C in persons without coronary heart disease.

Improving stability and activity of cross-linked enzyme aggregates based on polyethylenimine in hydrolysis of fish oil for enrichment of polyunsaturated fatty acids.

Cross-linking of enzyme aggregates from recombinant Geotrichum sp. lipase based on polyethylenimine (PEI) was applied to hydrolyze fish oil for enrichment of polyunsaturated fatty acids successfully. Through acetone precipitation and cross-linking of physical aggregates using glutaraldehyde in the presence of PEI, firmly cross-linked enzyme aggregates (PEI-CLEAs) were prepared. They could maintain more than 65% of relative hydrolysis degree after incubation in the range of 50-55 °C for 4 h and maintain more than 85% of relative hydrolysis degree after being treated by acetone, tert-butyl alcohol and octane for 4 h. PEI-CLEAs increased hydrolysis degree to 42% from 12% by free lipase. After five batch reactions, PEI-CLEAs still maintained 72% of relative hydrolysis degree. Hydrolysis of fish oil by PEI-CLEAs produced glycerides containing concentrated EPA and DHA in good yield. PEI-CLEAs had advantages over general CLEAs and free lipase in initial reaction rate, hydrolysis degree, thermostability, organic solvent tolerance and reusability.

Eicosapentaenoic and docosahexaenoic acids enriched polyunsaturated fatty acids from the coastal marine fish of Bay of Bengal and their therapeutic value.

Eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) enriched polyunsaturated fatty acids (PUFA) significantly present in marine fish oil emerge as preventive agents for combating many health problems specially in chronic or metabolic disorders. The fish in the coastal area of Bay of Bengal has remained unexplored with respect to EPA/DHA enriched PUFA content in its oils, although it may be a potential source in harnessing the health benefit. In this study, seven varieties of the coastal fish were analysed for the content of EPA/DHA. The one locally known as lotte, (Harpadon nehereus) though has low content of total lipids, was found to have high EPA/DHA in its oil. The phospholipids rich fraction was extracted from the total fish oil. The EPA/DHA enriched PUFA was isolated to investigate the potential use for health benefits. EPA/DHA is found to act as protective agent against mercury poisoning studied in cell culture as well as in animal mode. It is found to be highly preventive in diabetes. The lotte is available in the coastal area of Bay of Bengal adjoining West Bengal, India in large scale and it is the first report showing EPA/DHA enriched PUFA in these fish oil that can be availed to harness in important health benefits.

Protective effect of Mediterranean fish oil extracts on heat-induced denaturation of albumin.

Three oily extracts, obtained by acetone extraction from the entrails of different varieties of Mediterranean fishes, such as mackerel (Scomber scombrus), sardine (Sardina pilchardus) and horse mackerel (Trachurus mediterraneus), were characterized to determine their unsaturated fatty acid content. In an in-vitro model, their inhibitory effect was then evaluated against protein aggregation and their protective efficacy against heat-induced albumin denaturation assessed. The fish oil extracts tested in this study presented a significant amount of unsaturated fatty acids; in particular the extract obtained from the entrails of horse mackerel proved to have higher concentrations of DHA (docosahexaenoic acid) and oleic acid compared with the other two oils. The in-vitro study revealed an interesting protective effect of the oil extracts (particularly the horse mackerel extract) against heat-induced denaturation of albumin.

In vitro percutaneous absorption studies and in vivo evaluation of anti-inflammatory activity of essential fatty acids (EFA) from fish oil extracts.

The aim of the present study was to evaluate the in vitro percutaneous absorption and the in vivo anti-inflammatory activity of EPA and DHA fatty acids from three oily extracts, obtained by acetonic extractions from the entrails of different varieties of Mediterranean fishes such as mackerel (Scomber scombrus), sardine (Sardina pilchardus) and horse mackerel (Trachurus mediterraneus). In the first part of our research, we focused our attention on the characterization of the oily extracts to determine their omega-3 polyunsaturated fatty acid content, then, we evaluated the in vitro percutaneous absorption through excised human skin (stratum corneum/epidermis membranes; SCE) of EPA and DHA contained in the extracts. In the second part, the fish oil which guaranteed the best in vitro permeation profile of these omega-3 fatty acids was studied in order to evaluate its inhibiting ability towards the in vivo UVB-induced skin erythema. From the results obtained, all the fish oils tested in this study presented significant amounts of omega-3 fatty acids EPA and DHA, and particularly sardine oil extract showed higher concentrations of these substances compared to the other two fish oils. The in vitro experiments revealed interesting fluxes of these compounds from sardine extract through the stratum corneum/epidermis membranes and an appreciable anti-inflammatory activity against UVB-induced erythema in human volunteers was also observed.

Docosahexaenoic acid, a ligand for the retinoid X receptor in mouse brain.

The retinoid X receptor (RXR) is a nuclear receptor that functions as a ligand-activated transcription factor. Little is known about the ligands that activate RXR in vivo. Here, we identified a factor in brain tissue from adult mice that activates RXR in cell-based assays. Purification and analysis of the factor by mass spectrometry revealed that it is docosahexaenoic acid (DHA), a long-chain polyunsaturated fatty acid that is highly enriched in the adult mammalian brain. Previous work has shown that DHA is essential for brain maturation, and deficiency of DHA in both rodents and humans leads to impaired spatial learning and other abnormalities. These data suggest that DHA may influence neural function through activation of an RXR signaling pathway.

Preliminary safety assessment of an arachidonic acid-enriched oil derived from Mortierella alpina: summary of toxicological data.

An arachidonic acid-enriched oil (AA-oil), derived from Mortierella alpina was subjected to a programme of studies to establish its preliminary safety for use in infant nutrition. This was addressed at two levels: (1) HPLC analysis of metabolites produced by the production strains at various conditions, and (2) an evaluation of the toxicity of the final product. The following studies were carried out on the AA-oil: gene mutation assays in bacteria and mammalian cells in vitro; chromosome aberration assays both in vitro and in vivo and acute and subacute (4-wk) oral toxicity in the rat. No known mycotoxins were produced by the production strains under the conditions tested. Further, the oil did not show mutagenic or clastogenic activity and the acute oral toxicity, expressed as the LD50 value, exceeded 20 ml/kg body weight, that is, 18.2 g/kg body weight. In the subacute oral toxicity study the AA-oil was tested as such and in combination with a docosahexaenoic-enriched oil (DHA-oil) derived from fish oil at a ratio of 2:1 (AA:DHA). This was done because high dose levels of AA may result in adverse effects; DHA can compensate for these effects. Furthermore, human milk contains both AA and DHA at a ratio of AA:DHA of 2 to 3:1. No obvious signs of toxicity were observed. Levels of phospholipids and triglycerides tended to be decreased in the highest dose groups. The no-observed-adverse-effect level of the AA-oil in the subacute 4-wk toxicity study was placed at the highest levels tested, namely 3000 mg AA-oil/kg body weight/day as such and in the combination of 3000 mg AA-oil and 1500 mg DHA-oil/kg body weight/day. This corresponds to an intake of 1000 mg AA/kg body weight/day, which represents approximately 37 times the infant intake of AA in human milk.

Inhibitory effects of purified eicosapentaenoic acid and docosahexaenoic acid on growth and metastasis of murine transplantable mammary tumor.

We studied the effects of purified linoleic acid, eicosapentaenoic acid(EPA) and docosahexaenoic acid (DHA) on mammary tumor MM48 the transplanted into C3H/He mice. The growth of the primary tumor was significantly inhibited in EPA- and DHA-treated groups as compared with control and/or linoleic acid-treated groups (p < 0.05). The number of metastatic tumors in the lungs tended to be inhibited in the EPA- and DHA-treated groups and promoted in the linoleic acid-treated group, as compared with the control group, but not to a statistically significant extent. The EPA-treated group survived significantly longer than the control group (p = 0.002), while the DHA-treated group survived longer than the control group although the difference was not significant (p = 0.070). However, there was no significant difference between EPA- and DHA-treated groups on primary tumor growth, number of lung metastases or survival time of animals.