Improved pharmaceutical compositions comprising omega-3 containing excipients

Abstract

A composition comprising a total lipid concentrate, wherein at least 20% by weight of the total lipid concentrate comprises a polar lipid fraction, and the polar lipid fraction comprises more than about 30% by weight of glycolipids, and the composition comprises 4% or less by weight of the composition as chlorophyll concentrate, as well as formulations and pharmaceutical compositions comprising the lipid-containing composition.

Description

[Technical field]

[0001] This application claims priority to U.S. Patent Application No. 17 / 718,045, filed April 11, 2022, which is a continuation-in-part of U.S. Patent Application No. 16 / 953,978, filed November 20, 2020, both of which are incorporated herein by reference.

[0002] [STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT] Not applicable. [Background technology]

[0003] Safe, readily available, and synergistic excipients are in high demand today in both the pharmaceutical and nutraceutical industries, where the performance of pharmaceutical compositions is held to high standards. Excipients include all ingredients in a pharmaceutical composition that are not considered to be "active pharmaceutical ingredients" (APIs) or other active ingredients. Despite being traditionally considered "non-active" ingredients, excipients are now understood to be potential determinants of the performance of a dosage form, affecting factors including, for example, the color, flavor, and vehicle form of a parenteral pharmaceutical composition, as well as the stability, bioavailability, or patient acceptability of the active ingredients with which they are formulated. Moreover, excipients have been found to account for up to 90% of a drug, and their implementation is monitored by the US Pharmacopeia (USP) and regulated by international and national agencies such as the Food and Drug Administration (FDA). Thus, there is an increasing need for pharmaceutical dosage forms containing safe and beneficial excipients that are simply and inexpensively produced from readily available sources.

[0004] One such excipient component is a fatty acid, such as omega-3 fatty acid. Omega-3 fatty acids can be employed in enteric coatings, which are coatings for capsules, tablets, and other pharmaceutical compositions taken orally. Control of the enteric coating composition can affect both the location and rate of release of the active ingredient in the digestive tract. These compounds are also found in the formulation of topical creams, suppositories, and soaps, for example.

[0005] Omega-3 fatty acids, also called long-chain omega-3 oils, are essential for human health, and their deficiency can have serious adverse health effects. These range from impaired development in the brain and neural networks to our cardiovascular health, and more. Even when employed as excipients, studies have found that omega-3 oils can act as cancer inhibitors. For example, oleic acid, when employed with Herceptin for the treatment of breast cancer, not only functions as a drug delivery vehicle but actually inhibits the breast cancer gene. Other omega-3 acids have been recognized to have antitumor activity and cardiovascular effects, respectively. Today, omega-3 oils are in high demand because only about 800,000 tons of omega-3 fatty acids can be supplied annually for human consumption in aquaculture, fishing, and other marine sources. This is far below the human nutritional requirement of 1.4 million tons currently required to supply the world population with 500 mg of omega-3 fatty acids daily, and will be further exacerbated by population growth. Deficiency of omega-3 fatty acids has been observed worldwide, particularly affecting people living in North America, Central Europe, the Middle East, India, Brazil, and the United Kingdom, with regional and socio-economic variations within these countries.

[0006] Three main omega-3 fatty acids are found in nature: alpha linoleic acid (ALA), with 18 carbons and 3 double bonds, is found, for example, in flaxseed, soybean oil, and olives; docosahexaenoic acid (DHA), with 22 carbons and 6 double bonds, and eicosapentaenoic acid (EPA), with 20 carbons and 5 double bonds, are only produced in aquatic species (algae) and can be extracted from krill or fish that eat the algae, or from the algae themselves. Given the thousands of naturally occurring and easily culturable strains of microalgae, it is possible to select specific fatty acids by choosing the corresponding microalgae strain(s).

[0007] "Working algae", i.e., algae that grow using photosynthesis using sunlight or artificial light, often contain polar lipids that fall into the categories of glycolipids and phospholipids. By their nature, these species have hydrophobic tails and polar (hydrophilic) heads, i.e., they are essentially foreign natural detergents. These substances have a very high bioavailability and therefore easily enter the human bloodstream to achieve the health benefits mentioned above. It has been proven that these diverse and valuable substances are not naturally produced in other sources, which may be of questionable sustainability or have proven difficult to feasibly access. Furthermore, their extraction from microalgae efficiently and in a pure, natural, bioavailable form has not been effectively achieved commercially with success, as unattractive appearance, odor, and texture have hindered existing commercial products. While algae extracts have a higher bioavailability, they have traditionally contained components that cause the oil extracted from algae to appear very dark, almost blackish overall, and to feel highly viscous, causing the extracted oil to resemble a tarry black solid in which the concentration of the omega-3 oils themselves remains relatively low.

[0008] Moreover, to date, existing crude products have not been fully or adequately analyzed and characterized. Indeed, despite decades of research, fully characterized mass balances have not been achieved or published without reporting the inclusion of high levels (e.g., up to 10%) of either "unknown" or "unidentified" substances, or by reporting results only "by difference," placing essentially uncharacterized substances in a bucket simply labeled, for example, "carbohydrates." This is particularly undesirable in the case of dietary supplement and pharmaceutical products.

[0009] In summary, highly bioavailable, highly concentrated omega-3, EPA, polar lipid-rich compositions, particularly glycolipid compositions, presented as low viscosity, low chlorophyll content, light to dark amber oils for use as excipients in dietary supplement and pharmaceutical products, particularly dosage forms of such products, whose contents have been completely or nearly completely characterized, are currently unavailable but are highly desirable. Moreover, a method for obtaining such highly desirable compositions directly from an abundant and highly sustainable algae source would be ideal. Summary of the Invention

[0010] The present disclosure describes a solution to the above problem by providing nutritionally and medically useful, preventive and / or therapeutic dosage forms that include low viscosity, low chlorophyll content, omega-3-containing excipients and EPA-containing excipients. All dosage forms of the present invention exist as a combination of active ingredient and excipients and can be produced from specific materials by conventional processing and manufacturing methods known in the art. The dosage form materials utilizing such excipients in the present invention are provided using a liquid-liquid separation process to extract an oil extract from a microalgal biomass. The crude extract is then fractionated into clean and well-characterized fractions, such as polar lipids, polysaccharides, carotenoids, etc., with high efficiency and very high recovery. Thanks to the innovative fractionation process of the present disclosure, a perfect mass balance between the oil extract and the whole algal biomass is possible. Careful partitioning of the desired components into the excipient formulation allows for incorporation into pharmaceutical compositions. Thus, in another aspect, pharmaceutical compositions rich in omega oils and processes for producing them are provided.

[0011] In one embodiment, the disclosed process includes a method of producing an oil composition with low chlorophyll content, comprising obtaining an algae paste, extracting the algae paste with a polar solvent, such as an alcohol, like ethanol, to form an extract of algae lipids, extracting the resulting extract (e.g., with an organic solvent, such as the hydrocarbon hexane or heptane) to separate the non-polar lipid fraction, e.g., transferring the alcohol layer containing pigments and polar lipids to additional processing steps, and adding water to the extracted (e.g.) alcohol layer (e.g., with heptane) followed by sequential extractions to extract the pigment fraction (e.g., with heptane) and separate the polar lipid fraction. Polar lipids can then be obtained from the polar lipid-containing fraction by evaporation, and pigments can also be obtained by evaporation of the pigment-containing fraction.

[0012] In an alternative embodiment, the disclosed process includes a method of producing a low chlorophyll oil composition comprising obtaining an algal biomass containing both polar and non-polar lipid fractions and having a chlorophyll concentrate. The method includes substantially separating the chlorophyll concentrate along with the non-polar lipid fraction, and further includes using the polar properties of the polar and non-polar lipid fractions to separate polar components from non-polar components in the algal biomass. Additional steps include bleaching substantially all of the chlorophyll concentrate from the non-polar containing fraction, and recombining the polar and non-polar lipid fractions to produce a low chlorophyll LC-PUFA oil composition.

[0013] Embodiments of the above process produce attractive compositions for use in both the nutraceutical and pharmaceutical fields, particularly in terms of reduced opacity and viscosity.

[0014] In a related embodiment, a method is provided for fractionating and purifying algal paste into clean, accurately characterized Liquid Extracted Biomass, i.e., residual biomass after extraction ("LEA"), polar lipids, pure neutral lipids, chlorophyll, polysaccharides, and carotenoids components with high overall recovery and reporting complete algal biomass balance. The method includes obtaining an algal paste, extracting the algal paste with a polar solvent, such as an alcohol, like ethanol, to form an alcoholic extract (for example) of algal lipids, extracting the resulting alcoholic extract (for example) with an organic solvent, such as the hydrocarbons hexane or heptane, to separate the non-polar lipid fraction, e.g., transferring the alcoholic layer containing pigments and polar lipids to additional processing steps, and adding water to the extracted alcoholic layer (for example, with heptane) followed by sequential extractions to extract the pigment fraction and separate the polar lipid fraction (for example, with heptane).

[0015] The present disclosure further relates to bioavailable compositions, LC-PUFA compositions, omega-3 compositions, EPA compositions, polar lipid compositions, and glycolipid / phospholipid rich compositions, particularly suitable for use in dietary supplement and pharmaceutical compositions, which can be considered as health or pharmaceutical compositions and have other valuable end products. The starting material of the composition can be derived from the algae described herein. In one embodiment, a composition is disclosed that includes a polar lipid fraction of a total lipid concentration of at least 20% by weight of total lipid, wherein the polar lipid fraction includes at least 40% by weight of glycolipid, and the composition includes 4% or less by weight of the composition as chlorophyll concentrate.

[0016] Compositions produced as described above, in which at least 20% by weight of the total lipids is the polar lipid fraction and at least 40% by weight of said polar lipids is the glycolipid fraction, but the chlorophyll fraction is less than 4% by weight in the composition, can further include formulations containing additive non-polar lipids and / or nutraceutical oils such as DHA, or other beneficial additives as described in more detail below, which also help to provide a particular beneficial combination of oils that are more bioavailable, more nutritious, lighter in color, and less viscous.

[0017] These and other features of the present disclosure are explained in greater detail in the detailed description that follows. [Brief description of the drawings]

[0018] [Figure 1] 1 discloses a schematic diagram of the extraction process steps for an exemplary method of producing oil according to embodiments of the present disclosure. [Diagram 2]2 is a photographic depiction of an embodiment of a composition of the present disclosure produced by the exemplary method described in FIG. 1 having a desired target LC-PUFA concentrate, polar lipid concentrate, omega-3 concentrate, EPA concentrate, glycolipid-rich concentrate, and chlorophyll-depleted concentrate with the desired oil composition bioavailability, color, and viscosity, the embodiment of which is disclosed herein. [Diagram 3] FIG. 1 is a photographic representation of a powder obtained by an embodiment of the process of the present disclosure consisting essentially of non-lipid components, including (for example) polysaccharides, contained in a crude ethanolic algal extract produced by an embodiment of the process described herein. [Figure 4] FIG. 1 is a graph showing the spectral characterization of embodiments of an ethanol extract of Nannochloropsis and products after pigment removal as discussed in Example 1, where Y-axis=AU (Absorption Unit), X-axis=nm (nanometers, wavelength). [Diagram 5] FIG. 1 is a graph showing UV-Visible spectral characterization for an embodiment of an algae extract as discussed in Example 2, Y-axis=AU (Absorption Unit), X-axis=nm (nanometers, wavelength). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The following detailed description is illustrative of the claimed disclosure and is not intended to be limiting. The description clearly enables one skilled in the art to make and use the claimed disclosure, and describes several embodiments, adaptations, variations, alternatives, and applications of the claimed disclosure. It is further understood that the claimed disclosure is not limited to its application only to the details and compositions specifically described in the following description or illustrated by the drawings. The claimed disclosure is capable of other embodiments and can be practiced or carried out in various ways. It is also understood that the words and terms used herein are for the purpose of description and should not be regarded as limiting.

[0020] As used herein, the term "polar lipid" refers to an amphipathic lipid having a hydrophilic head and a hydrophobic tail. Examples of polar lipids include phospholipids and glycolipids.

[0021] The term "non-polar lipid" as used herein refers to a fatty molecule in which the charge distribution is approximately uniform and the molecule does not have positive and negative ends. Examples of non-polar lipids include triglycerides of various fatty acids in oils, such as EPA and palmitoleic acid (including mixtures thereof, such as triglycerides (TAG) containing variations or combinations of such fatty acids).

[0022] The acronyms "EPA" and "DHA" used herein refer to eicosapentaenoic acid and docosahexaenoic acid, respectively, as well as the salt and ethyl ester forms of each compound. In its naturally occurring acid form, EPA is an unsaturated chain with 20 carbons, resulting in a carboxylic acid functional group. However, those skilled in the art will appreciate that natural variants of this acid form include alkali salts, in which the deprotonated carboxylic acid is stabilized by a counter anion, and ethyl esters, in which two more carbons are covalently bonded singly to the SP3 hybridized oxygen to produce an ester. In the ethyl ester form, EPA has 22 carbons. Similarly, the acid form of DHA is an unsaturated chain with 22 carbons, resulting in a carboxylic acid functional group, and is understood to have natural variants, including alkali salts and ethyl ester forms. In the ethyl ester form, DHA has 24 carbons, as a result. The alkali salt forms of eicosapentaenoic acid and docosahexaenoic acid may appear naturally as a result of the particular chemical environment in which they exist. Conversion to the ethyl ester variants is similarly easy, and these variants are also used as drugs, for example, to treat high blood triglyceride levels. Any use of the acronyms "EPA" and "DHA" in this application should not be construed as excluding alkali salt or ethyl ester variants of either compound, unless expressly excluded.

[0023] The term "medical composition" as used herein refers to a composition containing active and non-active ingredients intended to be taken by a patient for therapeutic, curative or preventative purposes. A pharmaceutical composition in which the active ingredient is primarily intended for nutritional support is called a "nutraceutical." A pharmaceutical composition in which the active ingredient is primarily a pharmacological drug product is called a "medicinal product."

[0024] The term "dosage form" as used herein refers to the form of a pharmaceutical composition in which its ingredients are administered. Dosage forms include active and non-active ingredients. Dosage forms may be liquid, solid, or gaseous, including, but not limited to, tablets, pills, capsules, wafers, lozenges, liquid solutions, and the like. Dosage forms may be "swallowable" (referring to parenteral use) and, in addition, "chewable" in that their ingestion is substantially assisted by chewing. Dosage forms may also be "dissolvable", with the preferred route of ingestion being by first dissolving in an aqueous medium such as water.

[0025] The terms "inactive" and "excipient" as used herein refer to any and all compounds and elements that are not the active ingredients of the composition. The definitions herein follow those adopted by the Food and Drug Administration (FDA) as set forth in the Code of Federal Regulations (CFR). Excipient regulations must meet strict requirements for purity and toxicity. Excipients should not necessarily be construed as having no therapeutic benefit. Excipients such as those formed from the omega-3 oil compositions of the present invention may have synergistic and / or individual therapeutic effects as a result of their inclusion in the dosage form. The use of one or more excipients belonging to a particular recognized class (e.g., fillers, softeners, dyes, coatings) does not exclude the use of other recognized classes of excipients within the same embodiment of the present invention.

[0026] Thus, the term "active ingredient" as used herein also refers to the definition adopted by the FDA and set forth in the CFR. By providing a descriptive and non-limiting summary of the term, an active ingredient is meant to refer to any substance intended to directly provide a curative, palliative, therapeutic, or any other pharmaceutical or nutraceutical effect.

[0027] The term "antioxidant" as used herein refers to any substance that stops, inhibits, or otherwise interferes with, and thus adversely affects, chemical oxidation processes. Antioxidants include a wide variety of compounds whose identification and common uses are known to those of skill in the art.

[0028] The term "vitamin" as used herein refers to any organic molecule or set of chemically related molecules that are considered by those skilled in the art to be essential micronutrients required for the proper metabolic function of a mammalian organism. Such nutrients generally cannot be synthesized in sufficient quantities in vivo and must be obtained through the diet. For purposes of the claimed invention, many major health organizations consider the following list to constitute known vitamins essential to human health: Vitamin A, Vitamin B1, Vitamin B2, Vitamin B3, Vitamin B5, Vitamin B6, Vitamin B7, Vitamin B9, Vitamin B12, Vitamin C, Vitamin D, Vitamin E, Vitamin K, and in some cases, Choline. It should be noted that the above list is intended to be illustrative rather than limiting. It should be noted that certain vitamins refer to "vitamers," or groups of related chemical compounds organized under a single name. For example, the term "vitamin A" refers to a vitamer that includes trans-retinol and trans-retinyl esters. As used herein, the terms for the vitamins above include all known compounds within the associated vitamers.

[0029] As used herein, the term "mineral" refers to an essential nutrient that is also a chemical element other than oxygen, carbon, hydrogen, and nitrogen. The major mineral nutrients are understood to be calcium, phosphorus, potassium, sodium, and magnesium, but may further include elements present in trace amounts in the human diet, including sulfur, iron, chlorine, cobalt, copper, zinc, manganese, molybdenum, iodine, and selenium.

[0030] To produce the disclosed embodiments of the clarified compositions of algal biomass, an algal paste provided as a dark green or even black highly viscous oil can be obtained using standard steps known to those skilled in the art. See, for example, the production of algal paste and various useful algae that can be employed as described in U.S. Patent No. 8,591,912 B1 (hereinafter "Kadam and Goodall"), which is incorporated herein by reference and further discussed herein.

[0031] The procedure for obtaining an algal biomass extract, the algae initiation and extraction procedure for producing an algal biomass, can include the following steps as part of the liquid-liquid extraction.

[0032] An algal biomass, such as an algal paste, is obtained or produced from a suitable algal species suitable for producing such a target algal biomass. Generally, the microalgae can be harvested by conventional means, including but not limited to filtration, air flotation, and centrifugation, and the algal paste is produced by concentrating the harvested microalgae to a desired weight percent solids. In certain embodiments, the microalgae used with the method of the present invention belong to one of the phyla Chlorophyta, Cyanophyta (Cyanobacteria), and Heterokontophyta. In certain embodiments, the microalgae used with the method of the present invention belong to one of the classes Bacillariophyceae, Eustigmatophyceae, and Chrysophyceae. In certain embodiments, the microalgae used in the methods of the present invention belong to one of the genera Nannochloropsis, Chiarella, Dunaliella, Scenedesmus, Selenastrum, Oscillatoria, Phormidium, Spirulina, Amphora, Trachydiscus, and Ochromonas. Non-limiting examples of microalgae species that can be used with the methods of the present invention include Achnanthes orientalis, Agmenellum spp., Amphiprora hyaline, Amphora coffeiformis, Amphora coffeiformis var. linea, Amphora coffeiformis var. punctata, and the like.punctata, Amphora coffeiformis var. taylori, Amphora coffeiformis var. tenuis, Amphora delicatissima, Amphora delicatissima var. capitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmus falcatus, Boekelovia hooglandii, Borodinella sp. sp., Botryococcus braunii, Botryococcus sudeticus, Bracteococcus minor, Bracteococcus medionucleatus, Carteria, Chaetoceros gracilis, Chaetoceros muelleri, Chaetoceros muelleri var. subsalsum, Chaetoceros sp.), Chlamydomus perigranulata, Chlorella anitrata, Chlorella Antarctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha glucotropha, Chlorella infusionum, Chlorella infusionum var. actophila, Chlorella infusionum var. auxenophila, Chlorella kessleri, Chlorella lobophora, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. lutescenslutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella ovalis, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella protothecoides var. acidicola, Chlorella regularis, Chlorella regularis var. minima regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vannierii vanniellii), Chlorella vulgaris, Chlorella vulgaris forma tertiatertia, Chlorella vulgaris var. autotrophica, Chlorella vulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris fo. tertia, Chlorella vulgaris var. vulgaris fo. viridis, Chlorella xanthella, Chlorella zofingiensis zofingiensis, Chlorella trebouxioides, Chlorella vulgaris, Chlorococcum infusionum, Chlorococcum sp., Chlorogonium, Chroomonas sp., Chrysosphaera sp., Cricosphaera sp., Crypthecodinium cohnii, Cryptomonas sp., Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp.), Dunaliella sp.), Dunaliella bardawil, Dunaliella bioculata, Dunaliella granulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella primolecta, Dunaliella salina, Dunaliella terricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta, Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp., Euglena spp., Franceia sp., Fragilaria crotonensis, Fragilaria sp., Gleocapsa. sp., Gloeothamnion sp., Haematococcus pluvialis, Hymenomonas sp., Isochrysis aff. galbana, Isochrysis galbana, Lepocinclis, Micractinium, Micractinium, Monoraphidium minutum, Monoraphidium sp., Nannochloris sp., Nannochloropsis salina, Nannochloropsis sp., Navicula acceptata, Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa, Navicula saprophila, Navicula sp., Nephrochloris sp., Nephroselmis sp.), Nitschia communis, Nitschia alexandria, Nitschia closterium, Nitschia communis, Nitschia dissipata, Nitschia frustulum, Nitschia hantzschiana, Nitschia inconspicua, Nitschia intermedia, Nitschia microcephala, Nitschia pusilla, Nitschia pusilla elliptica, Nitschia pusilla monoensis monoensis, Nitzschia quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva, Oocystis pusilla, Oocystis sp., Oscillatoria limnetica, Oscillatoria sp., Oscillatoria subbrevis, Parachlorella kessleri, Pascheria acidophila, Pavlova sp., Phaeodactylum trichotomum tricomutum, Phagus, Phormidium, Platymonas sp.), Pleurochrysis carterae, Pleurochrysis dentate, Pleurochrysis sp., Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, Prototheca zopfii, Pseudochlorella aquatica, Pyramimonas sp., Pyrobotrys, Rhodococcus opacus, Sarcinoid chrysophyte, Scenedesmus armatus, Schizochytrium, Spirogyra, Spirulina platensis, Stichococcus sp., Synechococcus sp., Synechocystisf, Tagetes erecta, Tagetes patula, Tetraedron, Tetraselmis sp., Tetraselmis suecica, Thalassiosira weissfrogii weissflogii, Trachydiscus, and Viridiella fridericiana.

[0033] Preferred algae include those that are rich sources of LC-PUFA oils, for example. Exemplary of such algae strains are algae varieties of Nannochloropsis or Chlorella.

[0034] Algal biomass in algal paste or other suitable form can be processed as follows: extracted (e.g., with a polar solution (such as an alcohol, including ethyl alcohol)) to form an alcohol extract of algal lipids with low water content (e.g., to form an Ethanol Extract of Nannochloropsis Lipids (hereafter referred to as "EEN" for short)), and the resulting EEN is extracted (e.g., with an organic solvent, such as the hydrocarbons hexane or heptane) to separate the non-polar lipid (e.g., triglycerides, waxes, carotenes) fraction, thereby forming a "Non-polar Lipid Fraction (F#1 in FIG. 1)" in the heptane layer. The alcohol layer contains the pigments and polar lipids and can be moved to additional processing steps. See FIG. 1. Other polar solvents, such as carbon dioxide or a mixture of carbon dioxide and ethanol, are also contemplated.

[0035] Subsequent processing steps may include adding water to the extracted polar solvent layer (e.g., with heptane) and then sequentially extracting (e.g., with heptane) to extract the pigment fraction (see FIG. 1, heptane layer, F#3) and to separate the polar lipid fraction (see FIG. 1, water-alcohol layer, F#2). The polar lipids can be obtained by evaporation from the F#2 fraction, and the pigments can be obtained by evaporation from the F#3 fraction. The F#1 layer may contain a certain amount of chlorophyll and carotenoids, which can be removed by methods known to those skilled in the art, for example, methods known for the production of edible oils. Examples of these methods include adsorption-filtration using silica gel, bleaching clays such as B80, T41, and activated carbon. The selective removal of chlorophyll from the F#1 fraction results in a more or less liquid to semi-solid oil that is almost clear to dark amber in color, including red, in this embodiment.

[0036] In an alternative embodiment, a method for producing a low chlorophyll composition is provided, where the algal biomass or algal paste so obtained contains both polar and non-polar lipid fractions and also has a chlorophyll concentrate. The polar properties of the polar and non-polar lipid fractions of the biomass are used to separate the polar components from the non-polar components in the algal biomass, including substantially separating the chlorophyll concentrate along with the non-polar lipid fraction. As shown in Example 2, additional steps include bleaching substantially all of the chlorophyll concentrate from the non-polar containing fraction and recombining the polar and non-polar lipid fractions to produce a low chlorophyll oil composition.

[0037] In addition to the low chlorophyll content aspect of the composition, the present invention also provides isolated oils derived at least in part from algae rich in LC-PUFAs, including at least one omega-3 fatty acid, such as, but not limited to, EPA or DHA, in the form of total and hydrolyzable phospholipids and total and hydrolyzable glycolipids extracted by the above process. The oils produced by such processes are unexpectedly high in polar lipids, such as phospholipids and glycolipids. Krill krill oil is known to have a higher bioavailability in mammals than fish oils, which contain almost exclusively neutral lipids (triglycerides), due to its phospholipids (polar lipids). See Jan Philipp Schuchardt et al., Lipids in Health and Disease, 2011, 10:145. Surprisingly, oils extracted from algae such as Nannochloropsis using the methods of the present invention have shown bioavailability to mammals that is even higher than krill oil.

[0038] After isolating the algal oil, it can be incorporated into pharmaceutical formulations. The active ingredients of pharmaceutical formulations can include, but are not limited to, pharmaceuticals, vitamins or dietary supplements, other omega-3 containing oils, or antioxidants. Incorporating the isolated algal oil can include, but is not limited to, direct mixing with the active ingredient, by incorporating into other excipients understood to provide an optimal dosage form, and by coating the surface of the ingredient and dosage form with the algal oil or a mixture of algal oil and other excipient species. In one preferred embodiment, the isolated algal oil can be incorporated by applying the algal oil to the outside of a gel capsule, pill, or other consumable dosage form to increase the lipophilicity of the dosage form. The following examples illustrate the invention for purifying and determining certain components of the oil composition, and for producing LC-PUFA-rich, low chlorophyll, low polysaccharide oil as further described herein. [Example]

[0039] [Example 1] 2.1 Process Protocol for this Example (see Figure 1) 1) Weigh out 100g of Nannochloropsis algae paste (22-27% solids in water). (Step 1, Figure 1) 2) The algae paste was placed in a 2L flask and 850ml of alcohol was added. (Step 1, Figure 1) 3) The algae was extracted at a temperature of 70° C. for 45 minutes with vigorous stirring (Step 1, FIG. 1). 4) The solid algae residue was filtered from the ethanol extract (vacuum filtration). (Step 2, FIG. 1) 5) The ethanol extract from the previous step was placed in a separatory funnel (2 L), 300 mL of heptane was added to the resulting extract, stirred vigorously for 2 minutes, the layers were separated, and the top layer was carefully screened and placed in a separate flask to obtain about 120 mL of green heptane layer. (Step 3, Figure 1) 6) An additional 100 mL of heptane was added to the ethanol layer, stirred vigorously for 2 minutes, the layers were separated (ethanol layer-bottom, and heptane layer-top), and the top layer was carefully selected and combined with the heptane layer from the previous step (yielding ~200-220 mL of combined green heptane layer, fraction F#1) (Step 4, Figure 1). 7) 1g of silica gel was added to the obtained total heptane layer, and after vigorously stirring for 5 minutes, the slurry was filtered through a layer of 1g of silica gel (instead of silica gel, activated carbon or T41 bleaching earth may be used). Due to the property differences of various silica gels, activated carbons, and bleaching earths, the actual amount of materials should be adjusted in the factory. (Step 8, Figure 1) 8) The bottom layer (ethanol layer) from step 6 of this protocol was removed, 350 mL of water and 200 mL of heptane were added, and the mixture was shaken vigorously for 2 min. The separated phases were allowed to settle for about 5 min, after which the top layer was carefully screened and placed into a separate flask (~400 mL of green heptane layer was obtained from this step). (Step 5, Figure 1) 9) Repeatedly extract the lower layer in the above step using 200 mL of heptane (3 x 200 mL). Carefully select the top layer and combine it with the heptane layer from the previous step (~1000-1020 mL of combined green heptane layer was obtained, fraction F#3). (Step 5, Figure 1) 10) The extracted bottom layer contained the purified polar lipids (F#2). This fraction can be purified, for example, by 3 g of Amaze-N bleach adsorbent from Helix Chromatography (15 E. Palantine Rd. #118, Prospect Heights, IL 60070; helixchrom.com) (or a similar adsorbent can be used, if necessary). 11) The resulting fraction was evaporated in vacuum with heating below 45°C.

[0040] The above process steps and test results demonstrate a highly efficient and exemplary liquid-liquid extraction method for removing chlorophyll and carotenoid fractions from phototrophic (autotrophic) algae extracts such as Nannochloropsis or Chlorella lipids. By using the above embodiment of the disclosed method, not only was over 99% of chlorophyll a and b and pheophytin removed from the ethanol extract of Nannochloropsis (EEN), but also about 2 / 3 of the carotenoids (medium polar carotenoids) were removed. In so doing, at least 90% by weight, preferably more than 95% by weight, more preferably more than 97.5% by weight, even more preferably more than 98.0% by weight, and most preferably more than 99% by weight of the original mass balance was fully preserved and can be characterized for its major components without leaving the majority of the mass balance intact, whether characterized simply "by subtraction" or "by difference". See Table 1: Extraction Mass Balance (Composition) and Table 2: Major Components of Ethanol Extract of Nannochloropsis for Mass and Weight % Analysis of Composition. (Representing 99.86% of the total algal biomass by weight.) See Figure 4 for spectral characterization of Nannochloropsis ethanol extract after pigment removal.

[0041] [Table 1]

[0042] [Table 2]

[0043] While viscosity measurements may vary to some extent depending on factors such as temperature, compositional concentrations of various components in the formulation, as described herein, after combining the polar and neutral lipid fractions at 25°C, the viscosity reading measured for an embodiment of the Nannochloropsis extract produced as described was shown to be 165,000 mPa.'s.

[0044] In summary, the product shown in Example 1 can be decomposed into three main fractions of the ethanol extract of Nannochloropsis edulis (EEN): 1) fraction (F#1), non-polar lipids, mainly triglycerides; 2) fraction (F#2), polar lipids including glycolipids and phospholipids; and 3) fraction (F#3), a medium polar fraction including diglycerides and monoglycerides, free fatty acids (FFA's), carotenoids and chlorophyll. The purified F#1 and F#2 fractions can be used as valuable lipid sources rich in palmitoleic acid and eicosapentaenoic acid (EPA). F#3 is a concentrate of natural pigments including chlorophyll, astaxanthin, and zeaxanthin, etc., which are also valuable as food additives. Fractions F#1 to F#3 can be used as food additives, respectively, and are valuable raw materials with high biological potential.

[0045] [Example 2] A dark green paste sample of algal biomass was produced by the method generally described in U.S. Patent No. 8,591,912 B1 (see generally, column 6, line 62 to column 9, line 3) and discussed herein (see, e.g., paragraph 0021). The algal biomass paste was extracted with hot absolute ethanol. Specifically, 66 g algal paste, 3×250 mL ethanol, 75° C., 30′, each stirred at 500 rpm, and centrifuged at 4450 rpm for 10 min to obtain an algal extract sample.

[0046] Analysis of the oil extracts established that the predominant polar lipids in the algal samples were 1) glycolipids (monogalactosyldiglyceride (MGDG) and digalactosyldiglyceride (DGDG) and 2) phospholipids (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol) (see Table 3 herein).

[0047] [Table 3]

[0048] The bioavailable low chlorophyll content oils, polar lipid rich oils, LC-PUFA rich oils, and omega-3 rich oils embodiments of the present disclosure quantified herein were produced from the starting materials using the following additional steps. 1) Polar lipids were separated from a mixture of nonpolar lipids, chlorophyll, and other components based on differences in polarity. 2) Chlorophyll was bleached from the remaining non-polar lipid components using a well-developed protocol for bleaching vegetable oils in general, as described in Example 1 above, e.g., 1 g of silica gel was added to the total heptane layer obtained, and after vigorously stirring for 5 minutes, the slurry was filtered through a layer of 1 g of silica gel (activated carbon or T41 bleaching earth may be used instead of silica gel). Due to the property differences of various silica gels, activated carbons, and bleaching earths, the actual amounts of materials should usually be adjusted in the factory. 3) The polar lipid fraction from step (1) above was combined with the bleached non-polar lipids from step (2) above.

[0049] Generally, a low viscous, nearly clear to light brown oil composition was obtained that was rich in bioavailable polar lipids and had a low chlorophyll content. See FIG. 5 for spectral analysis. The composition was a waxy solid at ambient temperatures of about 70° F. The composition melts when warmed and exhibits low viscosity when mixed with other oils such as triglycerides.

[0050] Analysis of the oil composition of the bioavailable oil embodiments of the present disclosure produced using the above processes described herein demonstrated that an oil was obtained from the algal biomass having the following components and characteristics (column 1), and component ranges (column 2), as shown in Table 4.

[0051] [Table 4]

[0052] Minor variations in the weight percentages of the components and other properties of the oil disclosed in this application can be obtained by modifying the process used as known to those skilled in the art. However, preferably, the weight percentage of the polar lipid fraction in the total lipid concentrate of the oil produced is greater than 20% by weight, preferably greater than 30% by weight, more preferably greater than 40% by weight, even more preferably greater than 50% by weight, and even more preferably greater than about 70% by weight. Also, preferably, the weight percentage of chlorophyll concentrate in said oil product is less than 4% by weight of the total oil product, more preferably less than 3.0%, even more preferably less than 2.0%, even more preferably less than 1.0%, less than 0.75%, less than 0.50%, less than 0.2%, and even more preferably less than 0.1%. Also, preferably, the weight percentage of polysaccharide concentrate in said oil product is less than about 4% by weight of the total oil product, more preferably less than 3.0%, even more preferably less than 2.5%, less than 2.0%, even more preferably less than 1.0%, less than 0.5%, or lower. Furthermore, the weight percent of glycolipids as a weight percent of total polar lipids is greater than 20%, preferably greater than 30%, more preferably greater than about 40%, even more preferably greater than 50%, 60%, 70%, and even more preferably greater than 80%, and the weight percent of glycolipids of the total oil composition is greater than 10%, more preferably greater than 20%, and even more preferably greater than about 25%. Furthermore, the weight percent of phospholipids as a weight percent of polar lipids is greater than 20%, more preferably greater than 30%, and even more preferably greater than 35%, and the weight percent of phospholipids of the total oil composition is greater than 20%, more preferably greater than 30%, and even more preferably greater than 40%. Without being bound to any particular theory, applicants believe that the combination of these properties, including the very low chlorophyll concentration in the oil product, produces an attractive, light color, almost clear to amber, or even darker amber color, in a preferred embodiment, almost clear to amber. Additionally, the concentration of EPA in the total oil product content is preferably at least 20% by weight, more preferably at least 25% by weight, and even more preferably at least about 30% by weight or more.Similarly, the omega-3 content in the oil product is at least 20% by weight, more preferably at least 25% by weight, and even more preferably at least 30%, 40% by weight or more are produced. Taken as a whole, the weight percent LC-PUFA content of the oil product is at least 20% by weight, at least 25% by weight, 30% by weight, 40% by weight, and 50% by weight.

[0053] Enhanced bioavailability of the oil produced was also achieved. Additionally, unlike some prior art methods of extracting and fractionating microalgae, the disclosed methods do not involve harsh chemical reactions, such as the use of strong mineral acids, which often destroy polar lipids and can significantly degrade other valuable fractions of the algal biomass.

[0054] Additionally, embodiments of the disclosed process result in a powder of non-lipid components, such as polysaccharides, in the crude ethanolic algal extract. See FIG.

[0055] It should also be noted that the LC-PUFA-containing high polar content oil, omega-3-containing high polar content oil, and EPA-containing high polar content oil of the present disclosure have a significantly reduced weight percentage of chlorophyll, including: 1) chlorophyll a, 2) protochlorophyll a, and 3) methyl chlorophyllide. Preferably, the weight percentage of chlorophyll in the composition is less than 4% of the total weight percentage of the composition, preferably less than 3%, more preferably less than 2%, even more preferably less than 1%, even more preferably less than 0.5%, and even more preferably less than about 0.1%.

[0056] In another embodiment of the composition of the present disclosure, the composition has an increased weight percentage of carotenoids, e.g., carotene (alpha and beta), and zeaxanthin (yellow pigments), and several other components including canthaxanthin and zeaxanthin (red pigments), with the total carotenoids comprising greater than 0.5% of the total weight of the extract, preferably 1% or more.

[0057] In another alternative embodiment of the present disclosure, the oil composition with high bioavailable polar lipids and low chlorophyll content including EPA, which is an embodiment disclosed herein, can be provided as a formulation to which other useful ingredients are added. These other useful ingredients can be added alone or in combination with one or more other ingredients, such as other essential oils, dietary supplements, and dietary supplements. Specific examples include, but are not limited to: 1) other omega-3-containing oils or ingredients, such as DHA and EPA (e.g., in the form of neutral lipids extracted as a product of the present invention or supplied exogenously), lysolipids from the present invention, or ethyl esters of DHA or EPA supplied exogenously; 2) antioxidants, such as carotenoids (including the carotenoid fractions of the present invention), including astaxanthin, lutein, zeaxanthin, lycopene, carotene (alpha and beta), cryptoxanthin, and mixtures thereof; 3) vitamins, such as vitamins C and D; 4) cannabinoids, such as cannabidiol (CBD); and 5) other combinations. It is understood that such formulations containing some of these species with less colored and / or less viscous compositional profiles can reduce the overall color profile and viscosity of formulations containing fatty acid compositions with high polar lipids, high glycolipids, and low chlorophyll concentrations produced by embodiments of the methods disclosed herein. Thus, this can be accomplished, for example, by including non-polar lipids re-added from the original biomass stock or an external source, or by producing formulations that exhibit such attributes.

[0058] An example of a dietary supplement formulation comprising a blend of the polar EPA fractions described above with DHA (omega-3) may advantageously be in a ratio of 10-90 or 90-10, where the preferred level of the polar EPA formulation component is mixed at 20-50%. Applications of such formulations include use as a delivery system for other neutral lipids and ingredients to be formulated with the primary dietary / health supplement for cardiovascular health, mood, antidepressant, etc. This may be DHA, other neutral forms of EPA, or mixtures thereof. Astaxanthin at levels of 0.04%-10%, preferably 0.1%-2%, more preferably 0.2%-1%, may also be advantageously formulated with the pure polar EPA lipids or blends thereof with neutral EPA and / or EPA. Another ingredient that may be advantageously added to such formulations is coenzyme Q10 at levels of about 1-50%, preferably about 2-20%, relative to the polar EPA, either pure or in said formulations.

[0059] In addition to other attributes, formulation blends with added neutral lipids may be useful for targeting various viscosity levels, such as, for example, less than 50,000 mPa·s, preferably less than 10,000 mPa·s, more preferably less than about 2,000 mPa·s, and most preferably less than about 300 mPa·s.

[0060] In view of the above, it will be seen that the several objects and advantages of the disclosure are achieved and other advantageous results attained.

[0061] Since various modifications may be made to the above configurations without departing from the scope of the present disclosure, all matter contained in the above description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense.

Claims

1. A formulation comprising a composition containing a total lipid concentrate, wherein at least 20% by weight of the total lipid concentrate comprises a polar lipid fraction, more than about 30% by weight of the polar lipid fraction comprises glycolipids, the composition contains chlorophyll, and the composition contains 1.0% or less of its weight as a chlorophyll concentrate, a composition, and One or more additives selected from the group consisting of other omega-3-containing oils, antioxidants, vitamins, and minerals, and A formulation comprising.

2. The formulation according to claim 1, wherein the antioxidant is selected from the group consisting of astaxanthin, lutein, zeaxanthin, lycopene, α-carotene, β-carotene, ubiquinone, and cryptoxanthin.

3. The formulation according to claim 1, wherein astaxanthin is present at 0.04% to 10% of the formulation.

4. The formulation according to claim 1, wherein ubiquinone is present at 1 to 50% of the formulation.

5. The formulation according to claim 1, wherein ubiquinone is present at 2 to 20% of the formulation.

6. The formulation according to claim 1, wherein the one or more additives are DHA.

7. The formulation according to claim 1, wherein the one or more additives are ALA.

8. The formulation according to claim 1, wherein the composition of the formulation contains 0.1% or less of its weight as a chlorophyll concentrate.

9. The formulation according to claim 1, wherein the vitamin is selected from the group consisting of vitamin A, β-carotene, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D2, vitamin D3, vitamin E, vitamin K, and choline.

10. The formulation according to claim 1, wherein the mineral is selected from the group consisting of calcium, phosphorus, potassium, sodium, magnesium, sulfur, iron, chlorine, cobalt, copper, zinc, manganese, molybdenum, iodine, and selenium.

11. The formulation according to claim 1, wherein the composition further comprises at least 25% total EPA by weight.

12. The formulation according to claim 11, wherein the composition contains 0.5% or less of the weight of the composition as a chlorophyll concentrate.

13. The formulation according to claim 1, wherein the composition contains EPA and contains 0.5% or less of the weight of the composition as a chlorophyll concentrate. ​ A pharmaceutical composition comprising an excipient, wherein the excipient is a composition comprising a total lipid concentrate, wherein at least 20% by weight of the total lipid concentrate comprises a polar lipid fraction, more than about 30% by weight of the polar lipid fraction comprises glycolipids, the composition comprises chlorophyll, and the composition comprises 1.0% or less by weight thereof as a chlorophyll concentrate, a composition, and one or more additives selected from the group consisting of other omega-3-containing oils, antioxidants, vitamins, and minerals, and an active ingredient. A pharmaceutical composition.

15. The pharmaceutical composition according to claim 14, wherein the total lipid concentrate is present on the outer surface of the pharmaceutical composition.

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