Concentrated polyunsaturated fatty acid composition

Abstract

To provide a concentrated lipid composition having improved stability and providing a sustainable source of long chain omega-3 fatty acids.SOLUTION: Provided is a lipid composition comprising DPAn-3 derived from a concentrated seed oil, wherein the composition comprises from 20% to 50% DPAn-3, from 10% to 30% OA, and from 2% to 20% ETA. Preferably, the lipid composition comprises about 36% DPAn-3, about 22% OA, and about 6% ETA, and the seed is Brassicaceae.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 926,239, filed October 25, 2019.

[0003] This embodiment relates to lipid compositions that are enriched with one or more polyunsaturated fatty acids, such as omega-3 DPA, DTA, ETA, or their combinations.These polyunsaturated fatty acid compositions have many health benefits and are improved in stability.These polyunsaturated fatty acid compositions are scalable and can be obtained from a single sustainable source.In some embodiments, omega-3 DPA, DTA, or ETA are combined with oleic acid, which synergizes the beneficial activity of these fatty acids. [Background technology]

[0004] Long-chain omega-3 polyunsaturated fatty acids (LC-omega-3) are widely recognized as important compounds for human and animal health. These fatty acids can be obtained from dietary sources or, to a lesser extent, by conversion of linoleic acid (LA, 18:2 ω-6) or α-linolenic acid (ALA, 18:3 ω-3), all of which are considered essential fatty acids in the human diet. From a nutritional perspective, the most important omega-3 fatty acids are probably α-linolenic acid, eicosapentaenoic acid (EPA, 20:5 ω-3 or 20:5n-3), and docosahexaenoic acid (DHA, 22:6 ω-6 or 22:6n-3). For example, DHA is important for brain and eye development, and EPA has been linked to cardiovascular health.

[0005] Docosapentaenoic acid n-3 (DPAn-3, 22:5 ω-3) is an LC-omega-3 with known health benefits, including reducing inflammation and supporting cardiovascular health. DPAn-3 is also a component of adipose, cardiac, and muscle tissue. Additionally, DPAn-3 is a substrate for conversion to DHA. Therefore, sustainable sources of DPAn-3 are needed.

[0006] Docosatetraenoic acid (DTAn-3, 22:4 ω-3) is a lesser-known LC-omega-3 with relatively undocumented benefits. Therefore, there remains a need for sustainable sources of DTAn-3, at least to provide further characterization of this fatty acid.

[0007] Eicosatetraenoic acid (ETA, 20:4 ω-3) is another LC-omega-3 that may have anti-inflammatory properties. Furthermore, ETA is an intermediate in the biosynthesis of EPA. As with DPA and DTA, sustainable sources of ETA remain needed.

[0008] Eicosatrienoic acid (ETrA) (C20:3 ω-3) may also serve as a substrate for conversion to ETrA and as an intermediate in the biosynthesis of EPA. ETrA may also be related to cognitive health. Therefore, sustainable sources of ETrA are needed.

[0009] In general, the oxidative stability of fatty acids decreases significantly as the number of carbon-carbon double bonds (i.e., degree of unsaturation) increases. As a result, products with increased omega-3 content tend to have a shorter shelf life. DPAn-3, DTAn-3, and ETA are all polyunsaturated fats that are prone to oxidation. Therefore, there is a need for DPAn-3, DTAn-3, or ETA that is stable during and after processing. Summary of the Invention

[0010] The present embodiments provide lipid compositions rich in LC-omega-3 content, such as DPAn-3 (22:5n-3), DTAn-3 (22:4n-3), ETA (20:4n-3), or ETrA (20:3n-3), and methods for obtaining these compositions. In at least one embodiment, the DPAn-3, DTAn-3, ETA, or ETrA fatty acids are sourced from a plant, such as a plant seed oil from the plant family Brassicaceae. In a specific embodiment, the Brassicaceae plant is Brassica juncea. In some embodiments, the composition comprises at least one enriched LC-omega-3 (e.g., DPAn-3, DTAn-3, ETA, or ETrA) obtained from a plant source and at least one other LC-omega-3 obtained from another source. The LC-omega-3 of the present embodiments can be in the form of a free fatty acid, a salt, an ester, a salt of an ester, or a combination thereof. In at least one embodiment, the LC-omega-3 is in the form of an ethyl ester. In at least one embodiment, the LC-moega-3 is in the form of a triglyceride.

[0011] In one aspect, the present embodiments provide compositions enriched with DPAn-3, DTAn-3, ETA, or ETrA, or a combination thereof. For example, at least one embodiment provides a composition comprising about 90% to 99% DPAn-3 (inclusive), e.g., about 95% DPAn-3, about 97% DPAn-3, about 98% DPAn-3, or about 99% DPAn-3. These compositions, i.e., compositions containing very high amounts of DPAn-3, may also contain small amounts (e.g., at least about 0.1% and up to 5%, up to 2%, or up to 1% oleic acid (OA18:1n-9)). For example, these compositions may contain about 96% DPAn-3 and about 1% OA. Another embodiment provides a composition comprising about 80% to 99% DTAn-3 (inclusive) (optionally with up to about 15% OA) or about 90% to 99% DTAn-3 (inclusive). For example, the composition may contain about 80% DTAn-3, about 87% DTAn-3, about 90% DTAn-3, or about 95% DTAn-3. Another embodiment provides a composition containing about 90%-99% ETA (inclusive), e.g., about 93% ETA, about 95% ETA, about 98% ETA, or about 99% ETA. Another embodiment provides a composition containing about 60%-70% DPAn-3 (inclusive) and about 0%-20% ETA (inclusive) (e.g., about 5%-15% ETA (inclusive)) (particularly about 64% DPAn-3 and about 12% ETA). Yet another embodiment provides a composition containing about 40%-95% DTAn-3 (inclusive) and 5%-60% ETA (inclusive).

[0012] In another aspect, the present embodiment provides a composition comprising DPAn-3, ETA, or DTAn-3, and oleic acid (OA, 18:1n-9). In at least one embodiment, for example, the composition comprises about 30-60% DTAn-3 (inclusive) and about 30-60% OA (inclusive) (particularly, about 49% DTAn-3 and about 43.3% OA). In another embodiment, the composition comprises about 60-80% DTAn-3 (inclusive), about 10-20% OA (inclusive), and about 1-10% ETA (inclusive) (particularly, about 74% DTAn-3, about 14% OA, and about 4% ETA). In yet another embodiment, the composition comprises about 80-95% DTAn-3 (inclusive) and about 1-15% OA (inclusive) (particularly, about 87% DTAn-3 and about 6.3% OA). In yet another embodiment, the composition comprises about 40% to 60% DPAn-3 (inclusive), 20% to 40% OA (inclusive), and 2% to 20% ETA (inclusive). In yet another embodiment, the composition comprises 20% to 50% DPAn-3 (inclusive), 10% to 30% OA (inclusive), and 2% to 20% ETA (inclusive). For example, the composition may comprise 30% to 50% DPAn-3 (inclusive), 10% to 30% OA (inclusive), and 2% to 20% ETA (inclusive) (particularly, the composition may comprise 36% DPAn-3, 22% OA, and 6% ETA). In another embodiment, the composition comprises about 35.8% DPAn-3, about 22.0% OA, and about 6.1% ETA. In an alternative example, the composition may contain about 5-20% DPA (inclusive), about 30-60% OA (inclusive), and about 1-10% ETA (inclusive) (particularly, the composition may contain about 10.5% DPA, about 44% OA, and about 4% ETA). In another alternative example, the composition may contain about 20-40% DPAn-3 (inclusive), about 1-10% DTAn-3 (inclusive), about 1-10% ETA (inclusive), about 10-20% ALA (inclusive), about 1-10% LA (inclusive), and about 20-40% OA (inclusive) (particularly, the composition may contain about 28% DPAn-3, about 5% DTAn-3, about 5% ETA, about 14% ALA, about 6% LA, and about 29% OA).In yet another alternative, the composition may comprise about 10%-40% DPAn-3 (inclusive), about 20%-60% ETrA (inclusive), and about 0%-30% OA (inclusive) (particularly, the composition may comprise about 37% ETrA and about 16% DPAn-3, or about 54% ETrA and about 35% DPAn-3). In related aspects of these embodiments, the composition (comprising OA and at least one of DPAn-3, DTAn-3, or ETA) has a synergistic anti-inflammatory effect.

[0013] In another aspect, the present embodiment provides a composition comprising an enriched fraction of DPAn-3, ETA, ETrA, or DTAn-3, optionally together with OA, which is anti-inflammatory. In at least one embodiment, the composition alters cytokine activity. In at least one embodiment, the composition increases cytokine activity associated with decreased inflammation. In at least one embodiment, the composition suppresses cytokine activity associated with increased inflammation. For example, a composition with synergistic anti-inflammatory activity may comprise DPAn-3 and ETA, such as about 64% DPAn-3 and about 12% ETA. In another example, a composition with synergistic anti-inflammatory activity may comprise DTAn-3 and OA, such as about 3%-95% DTAn-3 (inclusive) and OA, about 49% DTAn-3 and about 43.3% OA, or about 87% DTAn-3 and 6.3% OA. In additional embodiments, a composition enriched for DPAn-3, DTAn-3, or ETA is also enriched for ALA. For example, a composition enriched in DPAn-3 (eg, containing at least about 28% DPAn-3) may also contain at least about 14% ALA.

[0014] In a further aspect, the present embodiments provide compositions enriched with DPAn-3, DTA, ETA or ETrA from plant (i.e., botanical) sources, which compositions are more stable and exhibit less degradation during storage than similar compositions in which the DPA, DTA, ETA or ETrA is sourced from fish oil or synthetic products.

[0015] The compositions of the present embodiments can be used in feeds, dietary supplements, cosmetics, and other chemical compositions, where they may be useful as intermediates or active pharmaceutical ingredients (APIs). [Brief explanation of the drawings]

[0016] [Figure 1] 1 is a graph showing the better stability of a plant-derived composition enriched in DPAn-3 by double distillation compared to an enriched reference blend. Y-axis: ppm propanal; X-axis: days (0, 3, 5); ○: transesterified, double-distilled retentate obtained from B. juncea; □: transesterified, double-distilled reference blend. [Figure 2] 1 is a graph showing the better stability of a plant-derived composition enriched in DPAn-3 via double distillation and chromatography (approximately 98% DPAn-3) compared to a similarly enriched reference blend. Y-axis: ppm propanal; X-axis: days (0, 3, 5); ○: transesterified, double distilled, chromatographic fraction from B. juncea (approximately 98% DPAn-3); □: transesterified, double distilled, chromatographic reference blend (approximately 90% EPA). [Figure 3] 1 is a graph showing the better stability of a plant-derived composition enriched in DPAn-3 via double distillation and chromatography (approximately 64% DPAn-3) compared to an enriched reference blend. Y-axis: ppm propanal; X-axis: days (0, 3, 5); ○: transesterified, double distilled, chromatographic fraction obtained from B. juncea (approximately 64% DPAn-3); □: transesterified, double distilled, chromatographic reference blend (approximately 58% EPA). DETAILED DESCRIPTION OF THE INVENTION

[0017] It is to be understood that the present invention is not limited to the particular methodology, protocols, and reagents, etc., described herein, as such may vary. Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0018] All patents and other publications cited herein are incorporated by reference for the purpose of describing and disclosing the methodologies described therein, for example, as may be used in connection with the present embodiments, but without providing definitions of terms that conflict therewith. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements regarding the date or contents of these documents are based on information available to the applicant and do not constitute an admission as to the accuracy of the dates or contents of these documents.

[0019] As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Throughout this specification, unless otherwise specified, "comprise," "comprises," and "comprising" are used inclusively rather than exclusively, so that a recited integer or group of integers may include one or more other unrecited integers or groups of integers. The term "or" is inclusive unless modified, for example, by "either." Thus, unless the context dictates otherwise, the word "or" means any one member of a particular list and also includes any combination of members of that list.

[0020] All values ​​are approximate because environmental conditions may cause some variation in fatty acid composition. Values ​​are usually expressed as weight percent of total fatty acids or weight percent of total seeds. Therefore, except for the operating examples or where otherwise indicated, all numbers used herein expressing amounts or reaction conditions should be understood to be modified in all cases by the term "about" unless otherwise stated. "About" generally refers to ±1% of the specified value, but may allow ±5% or ±10% of the specified value as accepted by one of ordinary skill in the art in the relevant context.

[0021] The fatty acid level in the composition of the present invention can be determined using conventional methods known to those skilled in the art. Such methods include, for example, gas chromatography (GC) combined with reference standards, according to the method disclosed in the Examples. In certain methods, fatty acids are converted to methyl esters or ethyl esters before GC analysis. The peak position of the chromatogram can be used to identify each specific fatty acid, and the amount can be determined by integrating the area under each peak. As used herein, unless otherwise stated, the percentage of a specific fatty acid in a sample is determined by calculating the area under the curve of the chromatogram of that fatty acid as a percentage of the total area of ​​the fatty acids in the chromatogram. Generally, this corresponds to weight percent (w / w or wt%). The identity of fatty acids can be confirmed by gas chromatography-mass spectrometry (GC-MS).

[0022] LC-omega-3 fatty acids have health benefits including neurological function, diabetes, cardiovascular health, and lipid regulation, and are also known as anti-inflammatory agents. Docosapentaenoic acid (22:5n-3, or DPAn-3) is valued as an intermediate between EPA and DHA, but it offers numerous benefits in its own right and is, in fact, converted back from DHA. DPA is found in high concentrations in breast milk. Mammalian cells, including human cells, metabolize DPAn-3 into a series of products that are members of a specialized pro-degradative mediator class of PUFA metabolites that promotes the restoration of normal cellular function after inflammation that occurs following tissue injury. Studies examining the antitumorigenic effects of n-3 fatty acids on colorectal cancer have found antiproliferative and pro-apoptotic effects of EPA, DPA, and DHA, with DPA demonstrating the strongest effects in both in vitro and in vivo models. DPA has also been associated with a reduced risk of heart disease. See Yazdi, "Review of the biologic and pharmacological role of docosapentaenoic acid n-3,2" F1000 Research 256 (2014). Thus, DPA is becoming increasingly important as a nutritional and therapeutic supplement. References to DPA or DPA3 herein refer to DPAn-3 unless otherwise specified.

[0023] Compared with the growing wealth of information regarding DHA and EPA, docosatetraenoic acid (22:4n-3, DTAn-3) is a relatively little-known LC-omega-3. DTAn-3 is an elongation of ETA. See, for example, Gregory et al., "Cloning and functional characterization of a fatty acyl elongase from southern bluefin tuna (Thunnus maccoyii)," 155 Comp. Biochem. Physiol B Biochem Mol. Biol. 178 (2010)." Perhaps because the tertiary structure of its acyl chain is similar to that of DPA and DHAn-3, DTAn-3 has been implicated in beneficial mediation of Aβ metabolism in the brain. Amtul et al., "Structural insight into the different effects of omega-3 and omega-6 fatty acids on the production of Aβ peptides and amyloid plaques," 286 J. Biol. Chem. 6100 (2011). Therefore, DTAn-3 may have potential as a nutritional or therapeutic agent. References herein to DTA or DTA3 refer to DTAn-3 unless otherwise specified.

[0024] Eicosatetraenoic acid (ETA, 20:4n-3) is known to be an omega-3 intermediate in the biosynthesis of EPA, DPA, and DHA. See, for example, U.S. Patent No. 7,807,849. The potential anti-inflammatory effects of ETA have been confirmed in relation to arthritis. Bierer & Bui, Improvement of arthritic signs in dogs fed green-lipped mussels (Perna canaliculus), 132 J. Nutr. 1623S (2002). ETA, like other LC-omega-3s, may have potential as a nutritional or therapeutic agent.

[0025] Eicosatrienoic acid (ETrA) (C20:3n-3) is produced by elongation of ALA or omega-3 desaturation of eicosadienoic acid (EDA, 20:2n-6), which can be further desaturated to form ETA. See, e.g., U.S. Patent No. 7,807,849. ETrA is not only a key intermediate in the omega-3 pathway, but has also been identified as one of the LC-omega-3s important for cognitive function in at least honeybees. Arien et al., Omega-3 deficiency impairs honeybee learning, 112 PNAS 15761 (2015).

[0026] Lipid compositions containing LC-omega-3s are typically obtained from marine sources (fish, crustaceans, etc.) or algae. Recently, plants have been genetically engineered to produce commercially suitable amounts of LC-omega-3s, particularly DHA. See, for example, WO 2017 / 219006; WO 2017 / 218969. When using these sources, the starting organic matter is first processed to extract the oil contained therein (commonly referred to as "crude" oil). For example, in the case of plant seeds such as DHA canola or DPA juncea, the seeds are crushed to release the oil, which is then separated from the solids by filtration and / or decantation. If a higher concentration of LC-omega-3s than that contained in crude oil is required, concentration is required. This can be achieved by processing the crude oil to remove unwanted components (e.g., components that adversely affect the color, odor, or stability of the product, or unwanted fatty acids) while maximizing the level of the desired fatty acid components. Additionally, when crude oil is deficient in one or more essential components, it is often blended with concentrated oils from other sources (e.g., fish or algae) to achieve the desired composition.

[0027] The composition of this embodiment can be obtained from a single source. Specific compositions that may be mentioned in this regard are the products listed in Tables 4 and 5 in the Examples, as well as the corresponding embodiments of the present invention discussed elsewhere herein. Furthermore, the composition of this embodiment obtained from a single source can be obtained by providing a lipid mixture obtained from a single source, separating the mixture into multiple portions (e.g., via chromatographic separation), and then combining (blending) a subset of those portions. For example, compositions resulting from combining two or more fractions obtained by chromatographic separation (or other separation methods) are contemplated, and these compositions can also be characterized as being obtained from a single source. For example, a composition containing primarily DPA and ETA (e.g., DPA and ETA together constitute approximately 80% by weight of the total fatty acids present in the composition) can be obtained from a single source. A combination containing primarily DPA and ETA can be obtained by blending the fractions CXZ and CR in Table 5 in appropriate ratios. Other combinations shown to have good in vitro activity can similarly be obtained by blending fractions enriched in desired components (e.g., containing at least 80% of one of the components). The use of a single source facilitates efficient and economical processing of crude oil and production of the lipid compositions of the present invention. The phrase "obtainable from a single source" means that the lipid composition is obtainable from one or more organisms of a single taxonomic class. In certain embodiments, the lipid composition is not derived from multiple organisms of different taxonomic classes, e.g., it is not a mixture of oils obtained from a combination of fish and algae or a combination of fish and plants. In this embodiment, the lipid composition (or "crude" oil from which the composition can be obtained by concentration techniques such as interesterification, distillation, and chromatography) is obtainable from a single population of organisms, e.g., a single plant or herbaceous source. In the context of this embodiment, "vegetable" refers to plant or plant life, as opposed to animal or mineral matter. However, in other embodiments, the composition is obtained from one plant source (e.g., DPA Juncea) and another source (e.g., fish, algae, or a synthetic source).

[0028] In at least one embodiment, the lipid composition has a high level of DPAn-3 compared to the amount of other lipids in the composition. In at least one embodiment, the lipid composition has a high level of DTAn-3 compared to the amount of other lipids in the composition. In at least one embodiment, the lipid composition has a high level of ETA compared to the amount of other lipids in the composition. DPAn-3, DTAn-3, or ETA can be independently provided in the form of a free fatty acid, a salt, an ester, or a salt of an ester, or a combination thereof. For example, the composition can include DPAn-3 in the form of an ester together with ETA in the form of a free fatty acid. In at least one embodiment, DPAn-3, DTAn-3, or ETA is a fatty acid ester, such as an ethyl ester. These embodiments can include additional lipid components, such as other omega-3s, saturated fatty acids, monounsaturated fatty acids, or polyunsaturated fatty acids, in the form of a free fatty acid, a salt, an ester, or a salt of an ester, or a combination thereof.

[0029] Suitable fatty acid ester forms are known to those skilled in the art. For example, nutritionally acceptable or pharmaceutically acceptable fatty acid ester forms include ethyl esters, methyl esters, phospholipids, monoglycerides, diglycerides, and triglycerides (triacylglycerides). Depending on the intended use of the lipid composition, different ester forms may be required. For example, triglycerides are esters derived from glycerol and three fatty acids. Triglycerides are particularly suitable for use in foods intended for human consumption, especially for infants, due in part to the stability of these ester forms against taste and heat treatment (which may be required for such foods). Thus, in one embodiment, a food for human or animal consumption is provided, comprising the lipid composition of the present invention, in which DPAn-3, DTAn-3, or ETA polyunsaturated fatty acids are provided in the form of triglyceride esters. Ethyl esters are particularly suitable for use in dietary supplements, as these ester forms can be efficiently and easily produced. Thus, in at least one embodiment, DPAn-3, DTAn-3, or ETA are independently provided in the form of a fatty acid ethyl ester.

[0030] Alternatively, fatty acid components can be in the form of "free" fatty acid, i.e., the -COOH form of fatty acid.In certain compositions of the present invention, the composition contains a relatively low level of this form of fatty acid, because it is accompanied by an unpleasant (often "soapy") taste and is less stable than the fatty acid in esterified form.Free fatty acid is usually removed from lipid compositions by alkali or physical refining, as is well known in the art.Therefore, in one embodiment, the total free fatty acid content in lipid composition is less than 5 wt% (for example, less than 3 wt%, particularly less than 2 wt%) of the total fatty acid content of the composition.

[0031] The lipid compositions of the present invention may also contain other components (e.g., other than fatty acids) that originate from the raw material and are not completely removed during the extraction and concentration process. While the exact nature of these other components can vary greatly depending on the raw material, examples of such other components include plant sterols (i.e., plant sterols and plant stanols) present either as free sterols or as sterol esters (such as β-sitosterol, β-sitostanol, Δ5-avenasterol, campesterol, Δ5-stigmasterol, Δ7-stigmasterol, and Δ7-avenasterol, cholesterol, brassicasterol, carinasterol, campesterol, campestanol, or ebricol). Examples of other components include antioxidants such as tocopherols and tocotrienols. Thus, certain lipid compositions of this embodiment may include those containing detectable amounts of one or more phytosterols (e.g., β-sitosterol). Such sterols may be present in amounts of at least about 0.01% by weight or more of the lipid composition, but typically not more than about 1% by weight.

[0032] The composition of the present embodiment is advantageously obtainable from a plant source (a "vegetable" source). "Vegetable-based" refers to at least 70% by weight of the lipids present in the lipid composition being obtained from a vegetable source. Vegetable sources include plant sources, particularly crops such as oilseeds. In at least one embodiment, the lipids are obtained from seed oil crops such as Brassica, e.g., B. juncea or B. napus. For the avoidance of doubt, however, it is not necessary for the composition to be obtained exclusively from such sources, and a proportion (e.g., up to about 30% by weight) of the lipids in the composition of the present embodiment may be obtained from other sources, including marine oils (e.g., fish or shellfish), algal oils, or combinations thereof. In one example, at least 80% by weight, e.g., at least 90% by weight, of the lipids present are obtained from a plant source. In certain embodiments, essentially all (i.e., at least 95%, at least 99%, or about 100%) of the lipids are obtained from a plant source.

[0033] There are many advantages to using plants as a lipid or fatty acid source. For example, marine oil sources are known to contain relatively high levels of contaminants not found in plant materials, such as mercury, PCBs, or fish allergens (e.g., parvalbumin). Furthermore, historical overfishing has depleted fish and crustacean (e.g., krill) resources, which are no longer sustainable. Therefore, the present invention provides a sustainably sourced polyunsaturated fatty acid oil composition containing relatively low levels of undesirable contaminants.

[0034] Thus, the lipid compositions (and feed and pharmaceutical compositions comprising these compositions) are not derived from animals (e.g., marine animals). That is, in such embodiments, the lipid compositions do not contain any components derived from animals such as fish and crustaceans. Lipid compositions that do not contain animal-derived components are believed to be advantageous in terms of lipid content and stability profile that can be achieved using standard purification or concentration procedures.

[0035] In one embodiment, the lipid composition is derived from plants.The plants from which oil is obtained are typically oilseed crops such as mustard, canola, copra, cottonseed, flax, palm kernel, peanut, rapeseed, soybean, and sunflower seed.Compositions obtained solely from plants may be referred to as "vegetable" oil or "plant lipid composition".Suitable plants from which the lipid composition of the embodiment can be obtained (whether on a commercial scale or not) are known to those skilled in the art, and include Brassica (oilseeds such as B.juncea, B.napus, or B.carinata), Arabidopsis thaliana (cress), Linumusitatissimum (flax), Camelina sativa (false flax), Gossypiumhirsutum (cotton), Helianthus sp. (sunflower), Carthamus tinctorius (safflower), Glycinemax (soybean), Zeamays (corn), Sorghum sp., Avena sativa (oats), Trifolium sp. (clover), Nicotiana sp. (e.g., Nicotiana benthamiana or Nicotiana tabacum), Hordeum vulgare (barley), Lupinus angustifolius (lupine), Oryza sp. (rice, such as O. sativa or O. glaberrima), Elaesis guineenis (palm), or Crambea byssinica (crambe, an oilseed from the Brassicaceae family). In at least one embodiment, the plant source is Brassica.

[0036] Suitable sources (including marine and algae sources as well as plant sources) may be naturally occurring or may be genetically modified for the ability to produce omega-3.Examples of genetically modified plant sources for this purpose are known to those skilled in the art.For example, see WO2013 / 185184, WO2015 / 089587, WO2015 / 196250.For example, the genetically engineered rapeseed oil NSB500274 that produces DHA in seed oil is described in WO2017 / 218969 and WO2017 / 219006.The process for obtaining oil from suitable sources is well known in the art.The enrichment of the described omega-3 from these oils will be discussed herein.

[0037] The oil produced by plants and seeds can be extracted, processed and analyzed using techniques routinely practiced in the art.In brief, plant seeds are typically cooked and pressed, and oil is extracted to produce crude oil.The oil can then be degummed, refined, bleached or deodorized.The combination of degumming, refining, bleaching and deodorization has been found to be particularly effective for preparing LC-omega-3-enriched lipid mixtures.Therefore, in one embodiment, lipid composition is obtained from degummed, refined, bleached and / or deodorized seed oil.However, it is not always necessary to process oil, and suitable purification and concentration can be achieved without using these methods.

[0038] Generally, techniques for crushing seeds are known in the art. For example, oilseeds can be tempered by spraying them with water to raise the moisture content, e.g., to 8.5%, and then flaked using smooth rollers with a gap setting of 0.23 mm to 0.27 mm. Depending on the type of seed, water may not be added before crushing. Extraction can also be achieved using an extrusion process, which may or may not be used in place of flaking, or as an add-on process either before or after the screw press.

[0039] In one embodiment, the majority of the seed oil is released by crushing using a screw press. The solids discharged from the screw press are then extracted with a solvent, such as hexane, using a heated column, after which the solvent is removed from the extracted oil. Alternatively, the crude oil produced by the pressing operation can be passed through a settling tank equipped with a slotted wire drain top to remove solids that have appeared in the oil during the pressing operation. This clarified oil can be passed through a plate and frame filter to remove any remaining fine solid particles. Optionally, the oil recovered in the extraction process can be combined with the clarified oil to produce a blended crude oil. Once the solvent has been removed from the crude oil, the pressed and extracted portions are combined and subjected to conventional petroleum processing procedures.

[0040] As used herein, "purified" when used in connection with the lipids or oils described herein typically means that the extracted lipid or oil has been subjected to one or more processing steps to increase the purity of the lipid / oil component. For example, the purification step may include one or more of degumming, deodorizing, bleaching, or drying the extracted oil. However, the term "purified" does not include a transesterification process or another process that alters the fatty acid composition of the lipid or oil of the present invention to increase the LC-omega-3 content as a percentage of the total fatty acid content. In other words, the fatty acid composition of the purified lipid or oil is essentially the same as the fatty acid composition of the unpurified lipid or oil.

[0041] Once extracted from the plant source, the vegetable oil can be refined (purified) using one or more of the following processes, particularly a combination of degumming, alkaline refining, bleaching, and deodorization. Suitable methods are known to those skilled in the art. See, for example, WO2013 / 185184.

[0042] Briefly, degumming is the initial stage of oil refining, and its primary purpose is to remove most phospholipids from the oil. Typically, approximately 2% water containing phosphoric acid is added to crude oil at 70-80°C, resulting in the separation of most phospholipids along with traces of metals and pigments. The insoluble material removed is primarily a mixture of phospholipids. Degumming can be accomplished by adding concentrated phosphoric acid to crude seed oil to convert non-hydratable phospholipids to a hydratable form and chelate any rare metals present. Typically, gums are separated from the seed oil by centrifugation.

[0043] Alkali refining, sometimes called neutralization, is a refining process for treating crude oil. It typically follows degumming and precedes bleaching. Following degumming, the seed oil can be treated by adding an amount of alkaline solution sufficient to titrate out all free fatty acids and phosphates and remove the soaps thus formed. Suitable alkaline materials include sodium hydroxide, potassium hydroxide, sodium carbonate, lithium hydroxide, calcium hydroxide, calcium carbonate, and ammonium hydroxide. Alkali refining is typically performed at room temperature to remove the free fatty acid fraction. The soaps are removed by centrifugation or extraction into a solvent, and the neutralized oil is washed with water. If necessary, any excess alkali in the oil can be neutralized with a suitable acid, such as hydrochloric acid or sulfuric acid.

[0044] Bleaching is a refining process in which oil is heated to 90-120°C for 10-30 minutes in the presence of bleaching earth (0.2%-2.0%) and in the absence of oxygen, operating with nitrogen or steam, or under vacuum. Bleaching is designed to remove unwanted pigments (carotenoids, chlorophyll, etc.); the process also removes oxidation products, trace metals, sulfur compounds, and traces of soaps.

[0045] Deodorization is the treatment of oils and fats at high temperatures (e.g., about 180°C) and low pressures (0.1 to 1 mmHg). This is typically accomplished by introducing steam into the seed oil at a rate of about 0.1 ml / min / 100 ml of seed oil. After about 30 minutes of application, the seed oil is allowed to cool under vacuum. This treatment improves the color of the seed oil and removes most of the volatile or odorous compounds, including any remaining free fatty acids, monoacylglycerols, and oxidation products.

[0046] Winterization is a process sometimes used in the commercial production of oils to separate fats and oils into solid (stearin) and liquid (olein) fractions by crystallization at subambient temperatures. It is typically used to reduce the saturated fatty acid content of the oil.

[0047] The present embodiments relate, in part, to lipid compositions obtained using interesterification techniques. As described herein, crude oil typically contains target fatty acids in the form of triacylglycerols (TAGs). Interesterification is a process that can be used to exchange fatty acids within and between TAGs, or to transfer fatty acids to another alcohol to form esters (e.g., ethyl esters or methyl esters). In the embodiments described herein, interesterification is achieved using enzymatic or chemical means.

[0048] Regarding enzymatic interesterification, in this approach, interesterification is achieved using one or more enzymes, particularly lipases known to be useful for hydrolyzing ester bonds, for example, in glycerides. The enzymes can be position-specific (sn-1 / 3 or sn-2 specific) for fatty acids on triacylglycerides (triglycerides or TAGs), or lipases that prefer some fatty acids over others. Specific enzymes that may be mentioned include Lipozyme 435 (available from Novozymes). This process is typically carried out at ambient temperature. This process is typically carried out in the presence of an excess of alcohol corresponding to the desired ester form (e.g., by using ethanol to form ethyl esters of fatty acids).

[0049] Chemical transesterification uses a strong acid or base as a catalyst. Sodium ethoxide (in ethanol) is an example of a strong base used to form fatty acid ethyl esters by transesterification. The process can be carried out at ambient or elevated temperatures (e.g., up to about 80°C).

[0050] In at least one embodiment, the concentrated lipid composition of this embodiment is obtained using distillation. Molecular distillation is an effective method for removing large amounts of highly volatile components, such as short-chain saturated fatty acids, from crude oil. Distillation is typically carried out under reduced pressure, for example, at less than about 1 mbar. The temperature and time of the process can then be selected to achieve an approximately 50:50 split between the distillate and the residue after several hours (e.g., 1 to 10 hours) of distillation time. Typical distillation temperatures used to produce the lipid composition of the present invention are in the range of 120°C to 180°C, for example, 140°C to 160°C, particularly 145°C to 160°C.

[0051] Multiple distillations can be performed, with each distillation considered complete when an approximately 50:50 split between the distillate and the residue is achieved. Using sequential distillations reduces the overall yield, but optimal results may be obtained by distilling twice (i.e., the product is referred to as "double distillation").

[0052] In addition to distillation, chromatography is an effective method for separating the various components of a fatty acid mixture. Chromatography can be used to increase the concentration of one or more preferred LC-omega-3s in the mixture. Chromatographic separation can be achieved under a variety of conditions, but typically involves the use of fixed-bed chromatography systems or simulated moving-bed systems. These are described below.

[0053] Fixed-bed chromatography systems are based on the concept of permeating a mixture containing components to be separated (usually together with an eluent) through a column (stationary phase) containing a packing of porous material that is highly permeable to fluids. The permeation rate of each component of the mixture depends on the physical properties of that component so that the component is continuously and selectively expelled from the column. Thus, some components tend to be strongly fixed to the stationary phase and will be slower, while other components tend to be weakly fixed and expel from the column after a while.

[0054] The simulated moving bed system is composed of several individual columns containing adsorbent, connected together in series, and operated by shifting the injection points of the mixture and eluent within the system, and also periodically shifting the separated component collection points, so that the overall effect simulates the operation of a single column containing a moving bed of solid adsorbent. Thus, although the simulated moving bed system is composed of a column containing a fixed bed of solid adsorbent through which an eluent passes, similar to a conventional fixed bed system, in the simulated moving bed system, operation is such that it simulates a continuous countercurrent moving bed.

[0055] The columns used in these processes typically contain silica (or modified silica) as the base of the stationary phase. The mobile phase (eluent) is typically a highly polar solvent mixture, often containing one or more protic solvents, such as water, methanol, ethanol, and mixtures thereof. The flow rate of the eluent can be adjusted by those skilled in the art to optimize the efficiency of the separation process. For example, the claimed product can be obtained using a relatively fast eluent flow rate. Using a slower flow rate improves the degree of separation of the FAs contained in the initial mixture, thus allowing for the production of DHA at a higher concentration or purity. Detection methods for LC-PUFAs are known to those skilled in the art and include UV-vis absorption and refractive index detection methods.

[0056] Thus, another aspect of the present embodiment provides a process for producing a lipid composition, the process comprising providing a mixture of fatty acid ethyl esters and then subjecting the mixture to a chromatographic separation process. The present embodiment also provides a lipid composition obtained by such a process. Suitable chromatographic separation conditions include those described herein.

[0057] For example, preparative high performance liquid chromatography (HPLC) technology can be used to obtain concentrated lipid fraction.The specific mobile phase that can be used in chromatographic separation is a mixture of methanol and water (for example, 88% methanol), but this can be changed during the separation process to improve efficiency (for example, to increase the methanol content).The specific stationary phase that can be used is a silica-based stationary phase.Analytical HPLC or other suitable techniques known to those skilled in the art can be carried out on the obtained fraction to identify the fraction that contains a sufficiently high concentration of desired fatty acid, and therefore contains the lipid composition of the present invention.

[0058] Therefore, in at least one embodiment, concentrated fatty acid ethyl esters are obtained by transesterification and distillation of plant-based lipid oils, for example, through any one of the above-mentioned processes.Plant-based lipid oils can be obtained from any plant, particularly oilseed, disclosed herein or known in the art.Before transesterification and distillation, vegetable-based lipid oils can be optionally refined by degumming, alkali refining, bleaching or deodorization.

[0059] The lipid composition of the present invention is useful as an active pharmaceutical ingredient (API) or as the precursor (or "intermediate") of the API that can be obtained from it by further concentration.Such composition will be further enriched with useful LC-omega-3 levels, such as DPAn-3, DTAn-3, ETA, or a combination thereof, or the mixture of the preceding with OA or ALA.The form of these LC-omega-3s can be any pharmaceutically acceptable form, such as free fatty acid, ethyl ester, triglyceride, or a combination thereof.

[0060] The concentration of fatty acids in oils can be further increased by various methods known in the art, such as freeze crystallization, complexation with urea, supercritical fluid extraction, and silver ion complexation. Complexation with urea is a simple and efficient method for reducing the level of saturated and monounsaturated fatty acids in oils. First, the TAGs in the oil are split into their constituent fatty acids, often in the form of fatty acid esters. These free fatty acids or fatty acid esters can then be mixed with an ethanolic solution of urea for complexation, as the fatty acid composition is typically not altered by processing. Saturated and monounsaturated fatty acids readily complex with urea and crystallize upon cooling, which can then be removed by filtration. This enriches the non-urea complexed fraction in LC-omega-3 fatty acids (although short-chain polyunsaturated omega-3 or omega-6 fatty acids may also be enriched using this technique).

[0061] The lipid composition of this embodiment may be bulk oil separated from the source (e.g., plant seed) from which some or all of the lipids are obtained.

[0062] The lipid composition of this embodiment can be used in or as feed. That is, these compositions can be provided in an orally available form. For the purpose of this embodiment, "feed" includes any food or preparation for human consumption that, when taken into the body, helps to nourish or build tissues, or provide energy, and / or maintain, restore, or support proper nutritional status or metabolic function. Feed includes, for example, nutritional compositions for infants or young children, such as infant formula. In the case of feed, fatty acids can also be provided in the form of triglycerides to minimize unpleasant taste and maximize stability.

[0063] The feed comprises the lipid composition described herein, optionally together with a suitable carrier. The term "carrier" is used in its broadest sense and includes any component that may or may not have nutritional value. As those skilled in the art will understand, the carrier must be suitable for use in feed (or be used at a sufficiently low concentration) so as not to have a harmful effect on the organism that consumes the feed. The feed composition can be in the form of a solid or liquid.

[0064] Additionally, the composition may contain edible macronutrients, proteins, carbohydrates, vitamins, or minerals in amounts desired for a particular application, as is well known in the art. The amounts of these ingredients will vary depending on whether the composition is intended for use in a normal individual or an individual with special needs, such as an individual suffering from a metabolic disorder, etc.

[0065] Examples of suitable nutritious carriers include macronutrients such as edible fats (e.g., coconut oil, borage oil, fungal oil, Kuroshio oil, soybean oil, and mono- and diglycerides), carbohydrates (e.g., glucose, edible lactose, and hydrolyzed starch), and proteins (e.g., soybean protein, electrodialyzed whey, electrodialyzed skim milk, milk whey, or hydrolysates of these proteins).

[0066] Vitamins and minerals that may be added to the feeds disclosed herein include, for example, calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and vitamins A, E, D, C, and B complex.

[0067] In another aspect of this embodiment, the lipid composition can be used in a pharmaceutical composition. Such a pharmaceutical composition optionally contains the lipid composition of this embodiment together with one or more pharmaceutically acceptable excipients, diluents, or carriers known to those skilled in the art. Suitable excipients, diluents, or carriers include phosphate-buffered saline, water, ethanol, polyols, wetting agents, or emulsions such as water / oil emulsions. The composition can be in either liquid or solid form, including solutions, suspensions, emulsions, oils, or powders. For example, the composition can be in the form of a capsule tablet, an encapsulated gel, an ingestible liquid (including oil or solution) or powder, an emulsion, or a topical ointment or cream. The pharmaceutical composition can also be provided as an intravenous formulation.

[0068] Specific forms suitable for feed and pharmaceutical compositions include liquid-containing capsules and encapsulated gels. The lipid composition of the present invention can be mixed with other lipids or lipid mixtures (especially plant-based fatty acid esters and fatty acid ester mixtures) before use. The lipid composition of the present invention can be provided with one or more additional components selected from the group consisting of antioxidants (e.g., tocopherols (such as α-tocopherol or γ-tocopherol) or tocotrienols), stabilizers, and surfactants). Tocopherols and tocotrienols are naturally occurring components in various plant seed oils, including canola oil.

[0069] For example, it may be desirable to include isotonicity agents such as sugars, sodium chloride, etc. In addition to such inert diluents, composition can also include adjuvants such as wetting agents, emulsifying agents and suspending agents, sweeteners, flavoring agents and fragrances.In addition to the lipid composition of the present invention, suspension can include suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan ester, microcrystalline cellulose, metahydroxyaluminum, bentonite, agar and tragacanth or the mixture of these substances.

[0070] Solid dosage forms such as tablets and capsules can be prepared using techniques well known in the art.For example, the fatty acid produced according to the method disclosed herein can be tableted with conventional tablet bases such as lactose, sucrose and cornstarch, combined with binders such as acacia, cornstarch or gelatin, disintegrants such as potato starch or alginic acid, and lubricants such as stearic acid or magnesium stearate.Capsules can be prepared by incorporating these excipients into gelatin capsules together with related lipid compositions and optionally one or more antioxidants.

[0071] Possible administration routes of the pharmaceutical composition of this embodiment include, for example, enteral (e.g., oral and rectal) and parenteral. For example, liquid preparations can be administered orally or rectally. Additionally, the homogeneous mixture can be completely dispersed in water and mixed with physiologically acceptable diluents, preservatives, buffers, or propellants under sterile conditions to form a spray or inhalant.

[0072] The lipid compositions described herein can provide many advantages typically associated with long-chain polyunsaturated fatty acids.For example, the lipid compositions described herein as described above and the pharmaceutical compositions described above can be used to treat or prevent cardiovascular disease, protect against death in cardiovascular disease patients, reduce overall serum cholesterol levels, reduce hypertension, increase HDL:LDL ratio, reduce triglycerides, or reduce apolipoprotein B levels, as can be determined using tests well known to those skilled in the art.Therefore, one aspect of this embodiment provides a method for treating (or preventing) diseases and conditions using lipid compositions described herein.

[0073] As used herein, the terms "treatment," "treat," and "treating" refer to reversing, alleviating, or inhibiting the progression of a disease or disorder described herein, or delaying, eliminating, or reducing the incidence or onset of a disorder or disease as described herein compared to what would occur in the absence of measures taken. As used herein, the terms "prevent," "prevention," and "preventing" refer to reducing the risk of acquiring or developing a given condition, or reducing or inhibiting the recurrence or occurrence of the condition in a subject who is not ill.

[0074] Typical dosages of specific fatty acids range from 0.1 mg to 20 g, taken 0.1 to 5 times daily (maximum 100 g daily), particularly from about 10 mg to about 1 g, 2 g, 5 g, or 10 g daily (taken in one or more doses). As known in the art, a minimum of about 300 mg / day of fatty acids, particularly LC-omega-3, is desirable. However, it will be understood that any amount of fatty acid can be beneficial to the subject. To enhance absorption of omega-3 fatty acids, oral dosage forms can be taken with meals. When used as a pharmaceutical composition, the dosage of the lipid composition administered to a patient is determined by one of ordinary skill in the art and depends on various factors, such as the patient's weight, age, overall health, past medical history, and immune status.

[0075] The composition of this embodiment is a readily available composition that may have an improved stability profile and may contain a mixture of fatty acids in which the relative ratio of omega-3 and omega-6 fatty acids is particularly beneficial to human health. Stability can be assessed using a variety of methods known to those skilled in the art. Such methods include the Rancimat method, assessment of propanal formation (particularly suitable for omega-3 fatty acids), assessment of hexanal formation (particularly suitable for omega-6 fatty acids), the "peroxide value" method (e.g., using AOCS official method Cd8-53), and the "p-anisidine value" method (e.g., using AOCS official method Cd18-90). In the examples, the composition of this embodiment has been shown to have a superior stability profile compared to a reference blend (a reference blend with a similar composition of major LC-PUFAs but containing a significant amount of animal (fish) or synthetically derived lipids).

[0076] The compositions of the present embodiments also have advantages in efficacy, less toxicity, half-life, potency, fewer sequelae, metabolism, or pharmacokinetic profile (e.g., higher oral bioavailability and / or lower clearance) or other useful pharmacological, physical, or chemical properties compared to prior art lipid compositions. [Example]

[0077] Example 1 - Extraction of DPA Juncea Oil from Seeds Brassica juncea NUBJ1207 (ATCC accession number PTA-125954) was grown in a tent in California, USA. Seeds were harvested and stored at room temperature until crushed. NUBJ1207 produces large amounts of DPAn-3 (over 10%) in its seed oil.

[0078] Seeds (4.92 kg) were crushed to produce DPA oil using a Kern Kraft KK80 screw press. The temperature of the expeller collar heater was set to the maximum setting on the thermostat. The initial ambient and choke temperatures were 20°C, and the choke distance was set at 73.92 mm. Seeds were fed without stopping the expeller, with oil and meal continuously collected, until all the seeds were crushed.

[0079] The auger rotation speed, the temperature of the meal, and the exiting oil were monitored throughout the press. A crude oil yield of 1.02 kg (20.7%) was obtained. After filtration to remove fines, the yield was 0.96 kg (19.4%). The oil profile (fatty acid content) of this preparation (designated BrJ) is shown in Table 1.

[0080] Example 2 - Reference Blend Oil Pure fish oil contains low levels of ALA fatty acid and significantly high levels of EPA and DHA. The reference oil blend was designed to be similar in composition to the filtered DPA Juncea oil obtained in Example 1. Because DPA was not available in comparable amounts from other sources, EPA was selected as a comparator for inclusion in the reference oil because it has five double bonds. This was achieved by blending EPA-rich fish oil, flaxseed oil, and high-OA sunflower oil. The resulting reference blend oil also had a similar total omega-3 content to DPA Juncea oil.

[0081] More specifically, semi-refined sardine oil (19.40 kg, 48.5%), crude high oleic sunflower oil (9.52 kg, 23.8%), and crude linseed oil (11.08 kg, 27.7%) were added to a dry, nitrogen-flushed reactor equipped with a mechanical stirrer, and the mixture was stirred for 2 hours at ambient temperature in an inert atmosphere. The reference oil (designated Rf) was discharged from the reactor and stored under nitrogen until use.

[0082] Table 1 compares the example DPA-Juncea with the reference blend oil. [Table 1]

[0083] Example 3 - Enzymatic Interesterification of Crude DPA Juncea Oil The following enzymatic interesterification procedure was carried out on approximately 5 kg of crude triglyceride oil obtained in Example 1 to produce fatty acid ethyl esters (FAEEs).

[0084] 100% undenatured ethanol (2.0 kg) and the crude triglyceride oil (0.95 kg) obtained in Example 1 were added to a dry nitrogen-flushed reactor equipped with a mechanical stirrer, and the mixture was stirred. 100 g of Lipozyme 435 (Novozymes A / S) was added to the mixture, and the mixture was heated at 40°C for 21 hours. 1 1 H NMR spectrum showed the reaction was complete.

[0085] The mixture was cooled to 20°C. The mixture was discharged from the reactor and filtered through a 4 μm polypropylene filter cloth on a 20 L Neutcher filter. The reactor was rinsed with ethanol (2 × 1.25 L) and petroleum spirits (2.5 L), which were used to sequentially wash the filter cake. Petroleum spirits (2.5 L) and water (2 L) were added to the resulting crude reaction mixture, and the mixture was thoroughly mixed in the reactor and then allowed to settle, after which two phases were formed.

[0086] The petroleum spirit layer was removed, and the aqueous layer was further extracted with petroleum spirit (1 x 5 L and 1 x 2.5 L). The combined petroleum spirit layers were dried over anhydrous magnesium sulfate (approximately 1 kg), filtered, and concentrated in vacuo to give crude DPA FAEE as a yellow oil (yield: 99%). Yield: 99.0%.

[0087] Enzymatic interesterification of the crude triglyceride reference blend oil (5.0 kg) obtained according to Example 2 was completed using the process described in the previous example. The product was obtained as a yellow oil.

[0088] Example 4 - Concentration of interesterified oil by vacuum distillation A standard procedure for removing the more volatile components of a fatty acid ethyl ester (FAEE) mixture by vacuum distillation is described below. The FAEE from Example 3 was subjected to distillation to produce two fractions: a distillate fraction containing a small amount of DPAEE, and a less volatile residue fraction containing most of the DPAEE. Distillative separation was achieved by passing the transesterified crude oil through a Pope 2-inch (50 mm) thin film still under vacuum, equipped with two 1000 ml collection flasks to collect the distillate and residue. The fatty acid composition of each was analyzed. Vacuum was supplied by an Edwards 3 rotary pump and measured by an Ebro VM2000 vacuum gauge.

[0089] The oil was fed to the still at 4 mL / min using a Cole-Palmer Instrument Company easy-load II peristaltic pump. The still motor was set at 325 rpm, and a water condenser was used to condense the distillate. The feed was continued until one receiver flask was full (relatively equal amounts of distillate and residual oil were observed). Crude DPAFAEE was distilled under these conditions, with the heater bands initially set at 153 °C. The objective was to obtain a 50:50 split of distillate:residue. During the first 45 minutes of the experiment, the heater band temperature was lowered to 143 °C to reduce the percentage of distilled oil, and then the still was allowed to equilibrate. After several minutes, the heater band temperature was adjusted lower to 141 °C. The remainder of the distillation was carried out at 141 °C. The total distillation time was 145 minutes. Yield: 52.1% distillate, 47.1% residual. Thus, the volume was reduced by 50%, while maintaining 80% DPAEE in the residual oil. This produced an oil containing approximately 19% DPAEE from an oil that originally contained approximately 10% DPAEE.

[0090] A portion of the residue from the above distillation was again subjected to removal of more volatile components by distillation under standard conditions, with the heater band temperature set at 145°C, again aiming for a 50:50 split. The heater band temperature was increased to 153°C and held at that temperature over 20 minutes to increase the fraction of distilled oil. However, after 50 minutes at 153°C, the distillate flow rate was found to be too high, and the heater band temperature was reduced to 151°C for the remainder of the distillation. The total distillation time was 95 minutes. Yield: 53.0% distillate, 46.4% residue. This produced an oil with approximately 35% DPAEE. Although the amount of oil obtained from the double distillation was reduced by a factor of four, the oil retained approximately 65% ​​of the original DPAEE.

[0091] A reference oil was distilled under similar conditions.

[0092] Example 5 - Chromatographic separation of FAEEs from DPA-Juncea The FAEEs obtained in Example 4 (i.e., FAEEs obtained from crude DPA-Juncea oil by transesterification and double distillation) were subjected to chromatographic separation by preparative HPLC. Preparative HPLC on a 1 g scale followed by vacuum concentration yielded fractions containing >85% DPAEE (and >85% EPAEE from the reference blend oil). A second preparative HPLC experiment was performed to obtain single fractions of either 50–85% DPA-enriched oil or 40–60% EPA-enriched oil. Additional preparative HPLC experiments were performed using a different column. All other FAEE fractions were collected and analyzed for purity by HPLC, and the desired pure fractions were concentrated under vacuum. In this manner, fractions enriched in OAEE, LAEE, ALAEE, ETAEE, EPAEE, and DPAEE were also obtained from one or more oils.

[0093] Preparative HPLC Method A: This method used an HPLC system equipped with a Waters Prep 4000 system, a 10 ml loop Rheodyne injector, a 300 x 40 mm Deltarep C18 column, a Waters 2487 dual wavelength detector, and a chart recorder. The system was equilibrated with an 88% methanol / water mobile phase at 70 mL / min. The detector was set at 215 nm and a full scale of 2.0 absorbance units, and the chart was run at 6 cm / hr. 1.0 g of FAEE oil was dissolved in a minimal amount of 88% methanol / water and injected onto the column via the Rheodyne injector. Approximately 250 mL fractions were collected when the solvent front appeared after approximately 7 minutes. 47 fractions were collected over 150 minutes. After 106 minutes, the mobile phase was changed to 90% methanol / water. After 116 minutes, the mobile phase was changed to 94% methanol / water. After 134 minutes, the mobile phase was changed to 100% methanol. After the final fraction was collected, the column was washed with 100% methanol at 70 mL / min for an additional hour.

[0094] Analytical HPLC was performed on all fractions (as well as HPLC methods A and B above and below), and the "control" fractions, which contained primarily DPA, were combined (yield: 22%). Sample analysis was performed using an HPLC system equipped with a Waters 600E pump controller, a 717 autosampler, a 2996 photodiode array detector, and a 2414 refractive index detector. Analysis was performed on a 150 x 4.6 mm Alltima C18 column using either an isocratic 90% methanol / water or 95% methanol / water mobile phase at 1.0 mL / min. Data collection and processing were performed with Waters Empower3 software.

[0095] This approach yielded the fraction designated "A fr" in Table 2. [Table 2]

[0096] In a variation of this method to isolate high-purity DPAEE, Method A was modified as follows: After 96 minutes, the mobile phase was changed to 90% methanol / water. After 109 minutes, the mobile phase was changed to 94% methanol / water. After 120 minutes, the mobile phase was changed to 100% methanol. After collecting the final fraction, the column was washed with 100% methanol at 70 mL / min for an additional hour. Thirty-nine fractions were collected over 120 minutes. Analytical HPLC was performed on all fractions to determine their purity and FAEE profile, which closely matched that of Fraction A. Based on this, the following fractions were combined: A1 fractions 25-30 and A1 fractions 36-37.

[0097] Another variation of the AHPLC approach was performed with the following changes: After 107 minutes, the mobile phase was changed to 90% methanol / water. After 119 minutes, the mobile phase was changed to 94% methanol / water. After 130 minutes, the mobile phase was changed to 100% methanol. After the final fraction was collected, the column was washed with 100% methanol at 70 mL / min for an additional hour. 40 fractions were collected over 129 minutes. This isolated highly pure DPAEE, and the fractions were designated A2 fr25-30 and A2 fr38-39.

[0098] The fractions from Method A were then subjected to further purification steps and analysis. To obtain moderately pure DTA3EE, fractions A fr39-40, A1 fr36-37, and A2 fr38-39 were combined and extracted with petroleum spirits (3 x 300 mL). The combined petroleum spirit layers were dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. This preparation was designated AP (yield: 260 mg).

[0099] Preparative HPLC Method B: For the isolation of medium-purity DPAEE by preparative HPLC, double-distilled FAEE oil was chromatographed on a modified Deltarep C18 column under standard conditions. The objective was to collect a single DPA fraction containing 50–85% DPA. Starting with 1.07 g of double-distilled DPAEE, single fractions were collected from 72 min (15 min before the DPA peak) to 120 min (15 min after the end of the DPA peak) under the following conditions: After 105 min, the mobile phase was changed to 90% methanol / water; after 120 min, the mobile phase was changed to 94% methanol / water; and after 124 min, the mobile phase was changed to 100% methanol. After collecting the final fraction, the column was washed with 100% methanol at 70 mL / min for an additional hour. A single fraction (designated Bfr1) was evaporated for GC analysis (yield: 338 mg).

[0100] In a related approach, HPLC method B was modified as follows: after 96 minutes, the mobile phase was changed to 90% methanol / water; after 111 minutes, the mobile phase was changed to 94% methanol / water; and after 116 minutes, the mobile phase was changed to 100% methanol. After the final peak eluted from the column, the column was washed with 100% methanol at 70 mL / min for an additional hour. Analytical HPLC was performed on the fraction designated B1 fr1, which determined a purity and FAEE profile closely matching that of B fr1.

[0101] In another related approach, HPLC method B was modified as follows: After 104 minutes, the mobile phase was changed to 90% methanol / water. After 119 minutes, the mobile phase was changed to 94% methanol / water. After 126 minutes, the mobile phase was changed to 100% methanol. After the final peak eluted from the column, the column was washed with 100% methanol at 70 mL / min for an additional hour. Single fractions were collected from 15 minutes before the DPA peak to 15 minutes after the end of the DPA peak. Analytical HPLC was performed on the fraction designated B2 fr1, and the purity and FAEE profile were determined to be nearly identical to those of fraction B fr1. This procedure was repeated to prepare fractions B1 fr1 and B2 fr1.

[0102] Bfr1, B1fr1, and B2fr1 were then combined and extracted with petroleum spirits (3 x 3 L). The combined petroleum spirit layers were dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. This preparation was designated BL (yield: 1.1 g).

[0103] The concentrated DPAEE extract BL was chromatographed under standard conditions using a 94% methanol / water mobile phase. A total of eight fractions were collected over 24 minutes. Analytical HPLC was performed on all fractions to determine their purity and FAEE profile, which closely matched that of BL. Based on this, the following fractions were combined and designated BM fr1-8:

[0104] Subsequently, BMfr1-8 were combined and extracted with petroleum spirits (3 x 300 mL). The combined petroleum spirits layers were dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. This DPAEE extract was designated BN (yield: 809 mg).

[0105] Preparative HPLC Method C: An alternative separation of high-purity DPAEE was performed as follows. Crude DPA-juncea-derived EPAEE double-distilled oil (1.57 g) was chromatographed using a 250 × 50 mm Gemini-NXC18 column under standard conditions with the following modifications: fractions were collected from the beginning of the EPAEE peak at approximately 58 min. A total of 21 fractions were collected over 73 min. After 111 min, the mobile phase was changed to 90% methanol / water. After 127 min, the mobile phase was changed to 94% methanol / water. After 137 min, the mobile phase was changed to 100% methanol. After collecting the final fraction, the column was washed with 100% methanol at 70 mL / min for an additional hour. Analytical HPLC was performed on all fractions, and based on this, the following fractions were combined and concentrated in vacuo for GC analysis. Yields are shown in Table 3. [Table 3]

[0106] Fraction Cfr10-13 was extracted with petroleum spirits (3 x 300 mL). The combined petroleum spirit layers were dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The remaining aqueous layer was then also concentrated in vacuo to determine the completeness of the extraction procedure. This process yielded the Cfr10-13 and Cfr10-13aq in the yields shown in Table 3 above.

[0107] A variation of Method C was performed with the following changes: After 93 minutes, the mobile phase was changed to 90% methanol / water. After 116 minutes, the mobile phase was changed to 94% methanol / water. After 124 minutes, the mobile phase was changed to 100% methanol. After collecting the final fraction, the column was washed with 100% methanol at 70 mL / min for an additional hour. A total of 23 fractions were collected over 68 minutes. The fractions were combined as follows: C1 fr5-6, C1 fr10-13, and C1 fr20-21.

[0108] Another variation of Method C was performed with the following changes: After 96 minutes, the mobile phase was changed to 90% methanol / water. After 111 minutes, the mobile phase was changed to 94% methanol / water. After 117 minutes, the mobile phase was changed to 100% methanol. After collecting the final fraction, the column was washed with 100% methanol at 70 ml / min for an additional hour. A total of 23 fractions were collected over 70 minutes. The fractions were combined as follows: C2 fr5-6, C2 fr10-13, and C2 fr20-21.

[0109] Another variation of Method C was performed with the following changes: After 99 minutes, the mobile phase was changed to 90% methanol / water. After 115 minutes, the mobile phase was changed to 94% methanol / water. After 126 minutes, the mobile phase was changed to 100% methanol. After collecting the final fraction, the column was washed with 100% methanol at 70 mL / min for an additional hour. A total of 24 fractions were collected over 75 minutes. Analytical HPLC was performed on all fractions to determine purity and FAEE profiles that closely matched those of the C fractions, and the following fractions were combined accordingly. The fractions were combined as follows: C3 fractions 5-6, C3 fractions 10-13, and C3 fractions 20-22.

[0110] An additional variation of Method C was performed with the following changes: After 92 minutes, the mobile phase was changed to 90% methanol / water. After 109 minutes, the mobile phase was changed to 94% methanol / water. After 116 minutes, the mobile phase was changed to 100% methanol. After collecting the final fraction, the column was washed with 100% methanol at 70 mL / min for an additional hour. A total of 24 fractions were collected over 72 minutes. Analytical HPLC was performed on all fractions to determine purity and FAEE profiles that closely matched those of the C fractions, and the following fractions were combined accordingly. Fractions were combined as follows: C4 fractions 5-6, C4 fractions 10-13, and C4 fractions 20-21.

[0111] Following these procedures, fractions C, C1, C2, C3, and C4 were combined and extracted with petroleum spirits (3 x 300 mL). The combined petroleum spirit layers were dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. This preparation contained high ETAn-3EE and was designated CR (yield: 217 mg).

[0112] Further, following the initial HPLC procedure, fractions C19-20, C120-21, C220-21, C320-22, and C420-21 were combined and extracted with petroleum spirits (3 x 300 mL). The combined petroleum spirit layers were dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. This preparation contained high DTAn-3EE and was designated CS (yield: 254 mg).

[0113] Following the initial preparative HPLC procedure, fractions C, C1, C2, C3, and C4 were combined and extracted with petroleum spirits (3 x 3 L). The combined petroleum spirit layers were dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. These preparations provided highly pure DPAEEs designated CT1 (yield: 1.088 g) and CT2 (yield: 417 mg).

[0114] The concentrated DPAEE extract (417 mg, CT2) was chromatographed under standard conditions using a 94% methanol / water mobile phase, and four fractions were collected over 12 min. Analytical HPLC was performed on all fractions, determining a purity and FAEE profile nearly identical to that of CT2. Based on this, the following fractions were combined and designated CXfr2-4. Furthermore, the concentrated DPAEE extract (1.088 mg, CT1) was chromatographed under standard conditions using a 94% methanol / water mobile phase, and six fractions were collected over 16 min. Analytical HPLC was performed on all fractions, determining a purity and FAEE profile nearly identical to that of CT1. Based on this, the following fractions were combined and designated CZfr3-6.

[0115] The concentrated DPAEE preparations CX fr2-4 and CZ fr3-6 were then combined and extracted with petroleum spirits (3 x 300 mL). The combined petroleum spirit layers were dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. This preparation was designated CXZ (yield: 1.0 g).

[0116] Preparative HPLC Method D: An alternative separation of intermediate-purity DPAEE was also used to collect single DPA fractions containing 50–85% DPA. Crude DPA-Juncea-derived FAEE double-distilled oil (1.57 g) was chromatographed using a 250 x 50 mm Gemini-NXC18 column under standard conditions with the following modifications: Single fractions were collected from 15 min before the DPA peak to 15 min after the end of the DPA peak. After 90 min, the mobile phase was changed to 90% methanol / water. After 100 min, the mobile phase was changed to 94% methanol / water. After 113 min, the mobile phase was changed to 100% methanol. After the final peak eluted from the column, the column was washed with 100% methanol at 70 mL / min for an additional 1 h. Single fractions were extracted with petroleum spirits (3 x 300 mL). The combined petroleum spirits layers were dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo for GC analysis. This fraction was identified as D fr1 (yield: 587 mg).

[0117] Distilled fatty acid ethyl esters (FAEEs) of a reference blend were similarly subjected to chromatographic separation under similar conditions. Analytical HPLC was performed on all fractions, and the "control" fractions, which contained primarily EPA, were combined.

[0118] The following table shows examples of fatty acid content of DPA Juncea crude oil and various concentration steps. (Oil was analyzed by GC-FID as is well known in the art. FAEE ID was established using Supelco 37 FAME standard mix transesterified into a FAEE mix.) [Table 4]

[0119] A more detailed presentation of the FA content of the various enriched fractions of this embodiment is shown in Table 5. [Table 5]

[0120] In some embodiments, fractions can be mixed to achieve a desired concentration of a particular FAEE. For example, a concentrated DPA fraction can be mixed with another fraction or an oil from a different source (e.g., DHA canola oil or another canola oil) to provide a lipid composition containing approximately 45% DPAn-3. In at least one embodiment, the composition contains 20%-50% DPAn-3, 10%-30% OA, and 2%-20% ETA (all ranges inclusive).

[0121] Example 6 - Oil Stability Test Selected oils were subjected to a light-induced, time-course, accelerated oxidation study using solid-phase microextraction (SPME) headspace GC / MS to determine whether plant-derived oils exhibited greater stability than the marine-derived reference blend oils observed in practice.

[0122] Headspace GC-MS stability testing was performed as follows: Headspace analysis was performed on the concentrated product described above to assess the amount of propanal released under specific conditions. An increased level of propanal release indicates a decrease in the stability of the test material.

[0123] SPME (Solid Phase Microextraction) method: A 65 μm PDMS / DVB TableFlex fiber (Supelco Fiber Kit 57284-u) was selected. The fiber was conditioned in a Triplus RSH conditioning station at 250 °C for 10 minutes before use. Samples were incubated at 40 °C for 1 minute before extraction.

[0124] GC method: ThermoScientific TRACE 1310 GC ThermoScientific TR-DIOXIN 5 MS column, 0.25 mm id, 30 m film 0.1 μm. Split injection 250 °C Split 83, 1.2 ml He / min. GC Ramp: 40 °C 1 min to 100 °C at 5 °C / min, then 300 °C at 50 °C / min.

[0125] A common MS-specific column was used, which showed excellent synergy for headspace analysis. A slow initial temperature ramp was employed to maximize volatile separation before ramping up to maintain column performance. A split injection was employed to avoid the need for cryogenic cooling of the inlet and improve column resolution.

[0126] The following ions were monitored on a Thermo Scientific DFS high-resolution double-focusing MS, TRACE1310, with a Triplus RSH autosampler using high-resolution multi-ion detection (MID) (LINEARELECTRICSCAN) at 10,000 resolution: m / z 57 propanal-H (-H recorded a higher dynamic range but was not used). Perfluorokerosene (PFK) was used as the calibration and lock mass standard at m / z 51, 69, and 93.

[0127] MS method: ThermoScientific DFS high-resolution GC-MS, low resolution (1000), full scan from 35 to 350 Da at 0.5 s / scan. Standards: Propanol and hexanal standard dilutions were used in supplied commercial canola oil. These standard mixtures were then added in a volume of 100 μl to a 20 ml headspace vial.

[0128] A full scan was employed to monitor all development products rather than specific molecules.

[0129] Stability Results: Table 6 below shows the results obtained from the DPA Juncea oil compared to the reference preparation obtained from the HPLC concentration of Example 5 at T=0, T=3, and T=5 days. The test samples were kept at ambient temp on a light box and under fluorescent tube lighting during this period. The m / z 57 molecular ion was analyzed, and the mass chromatogram clearly shows the appearance of propanal at 1.37 minutes at room temperature. The evolution of propanal is quantified in the table below, and the data are also shown in Figures 1-3. The DPA Juncea oil released substantially less propanal, indicating improved stability of the FAEE fraction compared to the reference composition. [Table 6]

[0130] These data indicate that the FAEEs prepared from DPA Juncea oil have superior stability compared to the FAEEs from the reference blend oil.

[0131] Example 7 - Regulation of inflammatory cytokine production The DPA, DTA, and ETA lipid compositions of this embodiment modulate the immune system. The immune system is an organized, complex network of biological structures and processes that protect against infection. For example, cytokines and chemokines directly mediate cell-cell interactions, modulating the responses of target immune cells and promoting inflammation. These responses result in a coordinated attack by the immune system to eradicate foreign pathogens and initiate the healing process. As a result, the inflammatory process plays an important protective role in immunity. Furthermore, the study of cytokines and chemokines is essential for understanding the immune system and its multifaceted responses to most antigens, as well as pathologies such as autoimmune diseases, allergic reactions, sepsis, and cancer. While immune responses can be helpful in protecting against pathogens, excessive or inappropriate immunity can be harmful. For example, it has been proposed that chronic inflammation may contribute to diverse diseases such as type 2 diabetes, metabolic syndrome, liver disease, arthritis, atherosclerosis, cancer, colitis, and neurodegenerative diseases. Therefore, immune suppression may also be beneficial.

[0132] In this example, lipid compositions derived from DPA-Juncea are investigated to confirm their immunomodulatory activity and compare their activity with that of synthetic counterparts. The comparison demonstrates that, in at least one embodiment, the plant-derived lipid compositions described herein are distinguishable from their synthetic counterparts.

[0133] Preparation of Test Materials: The exemplary lipid composition for comparison in this example comprises a concentrated FAEE composition as described herein mixed with synthetic fatty acids and a reference oil fraction for comparison.

[0134] Free fatty acids were prepared from the FAEEs of the previous examples: BrJDD (double distilled DPA Juncea FAEE), BN, CS, and AP. Petroleum spirits (10 ml) and fatty acid ethyl esters (150 mg) were added to a 100 ml two-necked round-bottom flask to form a clear solution. Lipozyme 435 (150 mg) was added, followed by deionized water (5 ml), and the mixture was vigorously stirred while the flask was immersed in an oil bath maintained at 40 °C. The reaction mixture was stirred until the majority of the FAEE was seen to have been reduced and no further hydrolysis was observed. 1 The reaction mixture was checked daily by HNMR and TLC. Upon completion, the cloudy white reaction mixture was cooled to room temperature. The mixture was diluted with 100 ml of fresh petroleum spirits and transferred to a separatory funnel. The clear aqueous phase was removed, and the organic phase was filtered under vacuum to remove all solids. The clear filtrate was dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo to give a viscous oil. Samples were 1 The product was analyzed by H NMR (CDCl3) and purified by radial chromatography (4 mm silica, eluting with 100% DCM in 100% petroleum spirits, then 95:5 DCM:MeOH). Fractions were analyzed via TLC developed using 75:25 petroleum spirits:EtOAc. The developed plate was sprayed with basic bromocresol spray, and fractions showing bright yellow spots were selected. 1 The fractions containing free fatty acids were combined, concentrated in vacuo, placed in a vial, and sealed under nitrogen.

[0135] Free fatty acids were prepared from flaxseed (Flx) and high oleic sunflower (HOS) TAG oils as follows: Petroleum spirits (60 ml) and triglyceride oil (1000 mg) were added to a 250 ml two-necked round-bottom flask to form a clear solution. Lipozyme 435 (1000 mg) was added, followed by deionized water (40 ml), and the mixture was vigorously stirred while the flask was immersed in an oil bath maintained at 40°C. The reaction mixture was stirred until the majority of the triglyceride signals at 5.2, 4.2, and 4.1 ppm had decreased and no further hydrolysis was observed. 1The reaction mixture was checked daily by HNMR and TLC. Upon completion, the cloudy white reaction mixture was cooled to room temperature. The mixture was diluted with 100 ml of fresh petroleum spirits and transferred to a separatory funnel. The clear aqueous phase was removed, and the organic phase was filtered under vacuum to remove all solids. The clear filtrate was dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo to give a viscous oil. The crude oil was 1 The product was analyzed by H NMR (CDCl) and purified by radial chromatography (4 mm silica, eluting with 100% DCM in 100% petroleum spirits, then 95:5 DCM:MeOH). Fractions were then analyzed via TLC developed using 75:25 petroleum:EtOAc. The developed plate was sprayed with basic bromocresol spray, and fractions showing bright yellow spots were selected. 1 The fractions containing free fatty acids were combined, concentrated in vacuo, placed in a vial, and sealed under nitrogen.

[0136] Additionally, DPA (synthetic DPA) and ETA (synthetic ETA) were purchased for use as comparators in the cell assay.

[0137] Various blends of free fatty acids were prepared as follows. [Table 7]

[0138] The FA content of the FFA components and FFA blends was determined by GC and is shown in Table 8. [Table 8]

[0139] Modulation test: Briefly, spleen cells (spleen cells) are collected from female mice (BALB / cmic) and cultured in 96-well plates. The cells are exposed to each oil preparation (dilution) and lipopolysaccharide (LPS, usually derived from or otherwise mimicking E. coli bacteria) to stimulate a cytokine response in the presence of the test oil preparation. Subsequently (after 24 hours of exposure to oil preparation dilutions and LPS), the medium from the stimulated cells is tested for the presence of cytokines (i.e., cytokines released from the cells into the medium). Standard kits or sets of cytokines and antibodies useful for identifying or quantifying cytokine modulation are commercially available. For example, the Milliplex Map Mouse Cytokine / Chemokine Magnetic Bead Panel (Millipore #MCYTMAG-70K-PX32) contains a premix of antibodies recognizing the following cytokine / chemokine analytes: Eotaxin / CCL11, G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17, IP-10, KC, LIF, LIX, MCP-1, M-CSF, MIG, MIP-1α, MIP-1β, MIP-2, RANTES, TNF-α, and VEGF. Data are normalized for cytokines to unstimulated controls.

[0140] The data may indicate that vegetable-derived DPA-rich lipid compositions have unexpected regulatory activity compared to synthetic lipid compositions.

[0141] Example 8 - Inhibition of inflammatory cytokines A preliminary in vitro study of the effects of various free fatty acid (FFA) preparations on endotoxin (LPS)-activated human blood cells was performed. More specifically, the assay measured the in vitro effects of various FFA preparations on the ability of LPS-stimulated human peripheral blood mononuclear cells (PBMCs) to produce pro- and anti-inflammatory cytokines, chemokines, and growth factors.

[0142] First, triplicate human PBMC samples were plated in 96-well plates and stimulated with four different LPS doses relative to an LPS-free control (0, 1, 10, 100, and 1000 ng / mL). Each series was also incubated with a single FFA preparation, Rfdd (see Table 8), at three different doses (relative to the FFA-free control) in DMSO vehicle. The cellular distribution of the samples was determined based on flow cytometry analysis of cell surface markers (CD3, CD4, CD8, CD14, CD19, and CD56). Table 9 provides detailed information on the source and cellular content of the human cell samples. [Table 9]

[0143] The production of 38 different cytokines / chemokines / growth factors (MILLIPLEX Map Human Cytokine / Chemokine Magnetic Bead Panel - Premix 38-plex - Immunology Multiplex Assay (Millipore HCYTA-60K-PX38)) was compared between these cell samples. The markers in this assay kit were EGF, eotaxin / CCL11, G-CSF, GM-CSF, IFNα2, IFNγ, IL-1α, IL-1β, IL-RA, IL-2, IL-3, IL-4, and IL-5. The analytes were IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17A, IL-17E / IL-25, IL-17F, IL-18, IL-22, IP-10, MCP-1, M-CSF, MIG, MIP-1α, MIP-1β, PDGF-AA, PDGF-AB / BB, RANTES, TNFα, TNFβ, and VEGF-A. Data showed that 1 ng / mL LPS exhibited a potent stimulatory effect on the levels of multiple analytes.

[0144] In the absence of FFA preparations, LPS generally activated monocytes as expected. Specifically, the following factors were induced by LPS: eotaxin (slightly), G-CSF, GM-CSF, IFNγ, IL-1α and IL-1β, IL-1RA, IL-6, IL-8 (slightly), IL-10, IL-12(p40) (2 / 3 samples), IL-17e, IL-18, IL-22, MIP1α, MIP1β, RANTES (slightly), TNFα, and TNFβ. In contrast, three factors were suppressed by LPS: IL-2, IP10, and MCP-1 (2 / 3 samples). However, the reason for the suppression of these three factors by LPS is unclear. Note that T cell-derived cytokines were generally not activated, suggesting that B cells may also contribute to LPS-induced expression. Upon stimulation with 1 ng / mL LPS, Rfdd exhibited a moderate inhibitory effect on D1 PBMCs, as seen in the reduction of cytokines such as IFNγ, IL-1β, IL-1RA, IL-12, and TNFα. PBMCs from D1 were selected for further study.

[0145] Thaw D1 PBMCs and place 1 x 10 per well in a 96-well plate. 5 Cells were seeded at a density of 1000 x g / ml. Next, the seeded cells were treated in duplicate with a blinded test FFA preparation (see Example 7) serially diluted 1:3, with the highest dose approximately 30 μM. For context, the typical total FFA content in the blood of normal individuals after an overnight fast is approximately 580 μM. The treated cells were then stimulated with 1 ng / mL LPS. Control wells were set up with untreated PBMCs stimulated or unstimulated with 1 ng / mL LPS in the presence of vehicle (0.1 or 0.3% DMSO). Cell-free supernatants were collected 24 hours after treatment and analyzed using the Human 38-plex Cytokine / Chemokine / Growth Factor Panel AMilliplexMapKit (Millipore HCYTA-60KPX38) described above. With regard to inhibition, an inhibitory dose was considered a preparation that produced a signal less than 50% of the control (LPS-stimulated, no added FFA) signal. To analyze the data in the context of FFA content, a simple 0-5 scale of inhibition, primarily based on the 30 μM data, was created based on the following qualitative aspects: 0 was used for no or very little inhibition. IFNγ inhibition, as well as inhibition of the IL-1 series cytokines (IL-1α, IL-1β, IL-1RA) and the chemokines IP-10 and MCP-1, were considered low and ranked from 1 to 2.5. If TNFα was also inhibited, a score of 3 was given. A score greater than 3 indicates inhibition of some T cell cytokines. If all cytokines were inhibited, a maximum of 5 was assigned. The results are shown in Table 10 (FA content is rounded; 0 means <0.5). [Table 10-1] [Table 10-2]

[0146] Generally speaking, several FFA preparations showed potent inhibitory effects at a dose of 30 μM on more than half of the analytes (>50% reduction compared to the LPS-stimulated control): combined high B. juncea DPAn-3 fraction (approximately 95.8% DPAn-3); synthetic ETA; and SynDEHOS2 (synthetic DPA and EPA with oleic acid).

[0147] Some compounds had strong inhibitory effects. IFNγ was universally suppressed, at least weakly, by all FFA preparations. Other types of minimal suppression tended to involve the cytokines IL-1α, IL-1β, and IL-1RA (IL-1 series) or the chemokines IP-10 and MIP-1. The most potent inhibitory preparations also suppressed classical T cell cytokines such as IL-2-7, IL-13, IL-15, and IL-22. The most inhibitory preparations also suppressed TNFα, which can be considered a representative marker of potent suppression. In this preliminary study, at least one DPA preparation derived from DPA Juncea (approximately 96% DPAn-3 from the C series technology described herein) appeared to be more inhibitory than similar synthetic DPA. Overall, preparations containing higher amounts of DPAn-3, DTAn-3, or ETA demonstrated inhibitory activity in this assay. ETA, DTAn-3, and DPAn-3 all exhibited significant inhibitory activity when present as the major components of the preparations. Surprisingly, the combination of DPAn-3 and ETA was an effective inhibitor of inflammatory cytokines in this assay.

[0148] Immunomodulatory activity was also observed in FFA preparations provided at approximately 10 μM. More specifically, stimulatory signals were classified as signals greater than 130% of those stimulated with LPS (no FFA added), and inhibitory signals were classified as signals less than 70% of those stimulated with LPS (no FFA added). At 10 μM, the following preparations showed only stimulatory responses (see Table 8 for FFA content): BrJdd, BN, Bfr1FlxHOS, and Rfdd. At 10 μM, the following preparations showed inhibitory responses: CR (IFNγ, IL-12(p40), and IP-10 only), AP (IL-12(p40), and IL-17F only), DHAfr21-25 (IL12(p40), and IL-17F only), HOS (substantial), and SynDEHOS1 (six cytokines). At 10 μM, the following preparations showed both stimulatory and inhibitory responses: CXZ, CS, BrJddFlxHOS, BrJddFlxHOS1, A fr39-40HOS, A fr19-20HOS, SynDPA, SynDHOS, SynETA, SynDEHOS, and SynDEHOS2.

[0149] Furthermore, with regard to stimulation, GM-CSF was generally the only cytokine consistently increased in the 30 μM preparations (>150% compared to control in 15 / 21 formulations).

[0150] Although the foregoing embodiments have been described in some detail by way of illustration and example, for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications can be practiced within the scope of the invention, which is limited only by the appended claims.

[0151] The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Claims

1. A lipid composition enriched for at least one of DPAn-3, DTAn-3, ETA, or ETrA, as described herein.

2. A lipid composition enriched in at least one of DPAn-3, DTAn-3, ETA, or ETrA, wherein the at least one of DPAn-3, DTAn-3, ETA, or ETrA is enriched from a plant source.

3. The lipid composition of claim 2, wherein the DPAn-3, DTAn-3, ETA, or ETrA is an ethyl ester or triglyceride.

4. 4. The lipid composition of claim 2 or claim 3, wherein the composition exhibits improved stability compared to a similar composition derived from a marine source.

5. A lipid composition comprising DPAn-3 derived from concentrated seed oil, said composition comprising 20% ​​to 50% DPAn-3, 10% to 30% OA, and 2% to 20% ETA.

6. 6. The lipid composition of claim 5, comprising about 36% DPAn-3, about 22% OA, and about 6% ETA.

7. A lipid composition comprising DPAn-3 derived from concentrated seed oil, said composition comprising about 10.5% DPAn-3, about 44% OA, and about 4% ETA.

8. A lipid composition comprising DPAn-3 derived from concentrated seed oil, said composition comprising 60% to 70% DPAn-3 and 0% to 20% ETA.

9. A concentrated lipid composition derived from seed oil containing 90% to 99% DPAn-3.

10. A lipid composition comprising DTAn-3 derived from concentrated seed oil, said composition comprising 40% to 95% DTAn-3 and 5% to 60% ETA.

11. A concentrated lipid composition derived from seed oil containing 90% to 99% DTAn-3.

12. A concentrated lipid composition derived from seed oil containing 90% to 99% ETA.

13. A lipid composition comprising DPAn-3 derived from concentrated seed oil, said composition comprising 10% to 40% DPAn-3 and 20% to 60% ETrA, and 0% to 30% OA.

14. 14. The composition of claim 13, comprising about 37% ETrA and about 16% DPAn-3.

15. 15. The lipid composition according to any one of claims 1 to 14, wherein the plant or seed is of the Brassicaceae family.

16. 16. The lipid composition of claim 15, wherein the Brassicaceae family is B. juncea or B. napus.

17. 17. The lipid composition of claim 16, wherein the B. juncea is NUBJ1207, ATCC Accession No. PTA-125954.

18. A lipid composition that suppresses inflammatory cytokine production, comprising DPAn-3 and ETA.

19. 19. The composition of claim 18, wherein the DPAn-3 and ETA are concentrated from a vegetable oil.

20. 19. The composition of claim 18, comprising about 28% DPAn-3, about 5% DTAn-3, about 5% ETA, about 14% ALA, about 6% LA, and about 29% OA.

21. A lipid composition that suppresses inflammatory cytokine production, the lipid composition comprising about 74% DTAn-3, about 14% OA, and about 4% ETA.

22. A lipid composition that suppresses inflammatory cytokine production, the lipid composition comprising about 96% DPAn-3 and about 1% OA.

23. A lipid composition that suppresses the production of inflammatory cytokines, the lipid composition comprising about 96% DPAn-3 enriched from seed oil of B. juncea, NUBJ1207, ATCC Accession No. PTA-125954.

24. 24. The lipid composition of any one of claims 1 to 23, wherein the composition is provided in the form of a tablet, capsule, encapsulated gel, ingestible liquid or powder, or topical ointment or cream.

25. 25. A lipid composition as defined in any one of claims 1 to 24 for use in treating or preventing cardiovascular disease, protecting against mortality in patients with cardiovascular disease, lowering overall serum cholesterol levels, lowering high blood pressure, increasing the HDL:LDL ratio, lowering triglycerides, or lowering apolipoprotein B levels.

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