Attorney Docket No. AKBM-14409/US-19/CON
`
`BIOEFFECTIVE KRILL OIL COMPOSITIONS
`
`CROSS-REFERENCE TO RELATED APPLICATIONS
`
`This application is a continuation of US. Patent Application No. 15/915,439, filed March
`
`8, 2018, which is a continuation of US. Patent Application No. 15/589,605, filed May 8, 2017,
`
`now US. Patent No. 10,010,567, which is a continuation of US. Patent Application No.
`
`15/180,439, filed June 13, 2016, now US. Patent No. 9,644,170, which is a continuation of US
`
`Patent Application No. 14/020,162, filed September 6, 2013, now US. Patent No. 9,375,453,
`
`which is a continuation of US. Patent Application No. 12/057,775, filed March 28, 2008, now
`
`10
`
`US Patent No. 9,034,388, which claims the benefit of expired US Provisional Patent
`
`Application No. 60/920,483, filed March 28, 2007, expired US. Provisional Patent Application
`
`No. 60/975,058, filed September 25, 2007, expired US Provisional Patent Application No.
`
`60/983,446, filed October 29, 2007, and expired US Provisional Patent Application No.
`
`61/024,072, filed January 28, 2008, all of which are incorporated by reference herein in their
`
`15
`
`entirety.
`
`FIELD OF THE INVENTION
`
`This invention relates to extracts from Antarctic krill that comprise bioactive fatty acids.
`
`20
`
`BACKGROUND OF THE INVENTION
`
`In the Southern Ocean, off the coast of Antarctica, Antarctic krill (Euphausia superba)
`
`can be found in large quantities, ranging from 300-500 million metric tons of biomass. It feeds on
`
`phytoplankton during the short Antarctic summer. During winter, however, its food supply is
`
`limited to ice algae, bacteria, marine detritus as well as depleting body protein for energy.
`
`In order to isolate the krill oil from the krill, solvent extraction methods have been used.
`
`See, e.g., WO 00/23 546. Krill lipids have been extracted by placing the material in a ketone
`
`solvent (6. g. acetone) in order to extract the lipid soluble fraction. This method involves
`
`separating the liquid and solid contents and recovering a lipid rich fraction from the liquid
`
`fraction by evaporation. Further processing steps include extracting and recovering by
`
`evaporation the remaining soluble lipid fraction from the solid contents by using a solvent such as
`
`ethanol. See, e.g., WO 00/23 546. The compositions produced by these methods are characterized
`
`25
`
`30
`
`1
`
`

`

`Attorney Docket No. AKBM-14409/US-19/CON
`
`by containing at least 75 ug/g astaxanthin, preferably 90 ug/g astaxanthin. Another krill lipid
`
`extract disclosed contained at least 250 ug/g canastaxanthin, preferably 270 ug/g canastaxanthin.
`
`Krill oil compositions have been described as being effective for decreasing cholesterol,
`
`inhibiting platelet adhesion, inhibiting artery plaque formation, preventing hypertension,
`
`controlling arthritis symptoms, preventing skin cancer, enhancing transdermal transport, reducing
`
`the symptoms of premenstrual symptoms or controlling blood glucose levels in a patient. See,
`
`e. g., WO 02/102394. In yet another application, a krill oil composition has been disclosed
`
`comprising a phospholipid and/or a flavonoid. The phospholipid content in the krill lipid extract
`
`could be as high as 60% w/w and the EPA/DHA content as high as 35% (w/w). See, e.g., WO
`
`10
`
`03/01 1873.
`
`Furthermore, nutraceuticals, pharmaceuticals and cosmetics comprising the phospholipid
`
`extract were disclosed. Previously, it was also shown that supercritical fluid extraction using neat
`
`C02 could be used to prevent the extraction of phospholipids in order to extract the neutral lipid
`
`fraction from krill, which comprised of esterifled and free astaxanthin. See, e.g., Yamaguchi et
`
`al., J. Agric. Food Chem. (1986), 34(5), 904-7. Supercritical fluid extraction with solvent
`
`modifier has previously been used to extract marine phospholipids from salmon roe, but has not
`
`been previously used to extract phospholipids from krill meal. See, e.g., Tanaka et al., J. Oleo
`
`Sci. (2004), 53(9), 417—424.
`
`The methods described above rely on the processing of frozen krill that are transported
`
`from the Southern Ocean to the processing site. This transportation is both expensive and can
`
`result in degradation of the krill starting material. Data in the literature showing a rapid
`
`decomposition of the oil in krill explains why some krill oil currently offered as an omega-3
`
`supplement in the marketplace contains very high amounts of partly decomposed
`
`phosphatidylcholine and also partly decomposed glycerides. Saether et al., Comp. Biochem
`
`Phys. B 83B(1): 51-55 (1986). The products offered also contain high levels of free fatty acids.
`
`What is needed in the art are methods for processing krill that do not require transport of
`
`frozen krill material over long distances and the products produced by those methods.
`
`SUMMARY OF THE INVENTION
`
`In a first aspect of the invention is a composition characterized by comprising at least 65%
`
`(w/w) phospholipids.
`
`15
`
`20
`
`25
`
`30
`
`

`

`Attorney Docket No. AKBM-14409/US-19/CON
`
`In another aspect of the invention is a composition obtained from aquatic or marine
`
`sources, characterized by comprising 65% (w/w) phospholipids.
`
`In yet another aspect of the invention is a composition obtained from krill, characterized
`
`by comprising at least 65% (w/w) phospholipids.
`
`In another aspect of the invention is a composition obtained from krill, characterized by
`
`comprising at least 65% (w/w) phospholipids and at least 39% omega-3 fatty acids (w/w).
`
`In yet another aspect of the invention is a composition obtained from krill, characterized
`
`by comprising at least 65% (w/w) phospholipids, at least 39% omega-3 fatty acids (w/w) and at
`
`least 580 mg/kg astaxanthin esters.
`
`In another aspect of the invention is a composition obtained from krill, characterized by
`
`comprising at least 39% omega-3 fatty acids (w/w) and at least 580 mg/kg astaxanthin esters.
`
`In yet another aspect of the invention is a composition obtained from krill, characterized
`
`by comprising at least 65% (w/w) phospholipids and at least 580mg/kg astaxanthin esters.
`
`In yet another aspect, the present invention provides a krill oil effective for reducing
`
`insulin resistance, improving blood lipid profile, reducing inflammation or reducing oxidative
`
`stress.
`
`In some embodiments, the present invention provides compositions comprising: from
`
`about 3% to 10% ether phospholipids on a w/w basis, from about 35% to 50% non-ether
`
`phospholipids on w/w basis, so that the total amount of ether phospholipids and non-ether
`
`phospholipids in the composition is from about 48% to 60% on a w/w basis,
`
`from about 20% to 45% triglycerides on a w/w basis, and from about 400 to about 2500 mg/kg
`
`astaxanthin. In some embodiments, the ether phospholipids are selected from the group
`
`consisting of alkylacylphosphatidylcholine, lyso-alkylacylphosphatidylcholine,
`
`alkylacylphosphatidylethanolamine, and combinations thereof. In some embodiments, the ether
`
`lipids are greater than 90% alkylacylphosphatidylcholine. In some embodiments, the non-ether
`
`phospholipids are selected from the group consisting of phosphatidylcholine, phosphatidylserine,
`
`phosphatidylethanolamine and combinations thereof. In some embodiments, krill oil composition
`
`comprises a blend of lipid fractions obtained from krill. In some preferred embodiments, krill is
`
`Euphausia superba, although other krill species also find use in the present invention. Other krill
`
`species include, but are not limited to E. pacifzca, E. frigida, E. longiroslris, E. lriaccmlha, E.
`
`vallenlini, Meganyctiphanes norvegica, Thysanoessa raschil' and Thysanoessa inermis. In some
`
`10
`
`15
`
`20
`
`25
`
`30
`
`

`

`Attorney Docket No. AKBM-14409/US-19/CON
`
`embodiments, the compositions comprise from about 25% to 30% omega-3 fatty acids as a
`
`percentage of total fatty acids and wherein from about 80% to 90% of said omega-3 fatty acids
`
`are attached to said phospholipids. In some embodiments, the present invention provides a
`
`capsule containing the foregoing compositions.
`
`In further embodiments, the present inventions provide compositions comprising: from
`
`about 3% to 10% ether phospholipids on a w/w basis, and from about 400 to about 2500 mg/kg
`
`astaxanthin. In some embodiments, the compositions further comprise from about 35% to 50%
`
`non-ether phospholipids on w/w basis, so that the total amount of ether phospholipids and non-
`
`ether phospholipids in the composition is from about 38% to 60% on a w/w basis. In some
`
`embodiments, the compositions further comprise from about 20% to 45% triglycerides on a w/w
`
`basis. In some embodiments, the ether phospholipids are selected from the group consisting of
`
`alkylacylphosphatidylcholine, lyso-alkylacylphosphatidylcholine,
`
`alkylacylphosphatidylethanolamine, and combinations thereof. In some embodiments, the ether
`
`lipids are greater than 90% alkylacylphosphatidylcholine. In some embodiments, the non-ether
`
`phospholipids are selected from the group consisting of phosphatidylcholine, phosphatidylserine,
`
`phosphatidylethanolamine and combinations thereof. In some embodiments, krill oil composition
`
`comprises a blend of lipid fractions obtained from krill. In some preferred embodiments, krill is
`
`Euphausia superba, although other krill species also find use in the present invention. Other krill
`
`species include, but are not limited to E. pacifzca, E. frigida, E. longiroslris, E. lriaccmlha, E.
`
`vallenlini, Meganyctiphanes norvegica, Thysanoessa raschil' and Thysanoessa inermis. In some
`
`embodiments, the compositions comprise about 25% to 30% omega-3 fatty acids as a percentage
`
`of total fatty acids and wherein from about 80% to 90% of said omega-3 fatty acids are attached
`
`to said phospholipids. In some embodiments, the present invention provides a capsule containing
`
`the foregoing compositions.
`
`In some embodiments, the present invention provides a composition comprising at least
`
`65% (w/w) of phospholipids, said phospholipids characterized in containing at least 35% omega-
`
`3 fatty acid residues. In some preferred embodiments, the composition is derived from a marine
`
`or aquatic biomass. In some further preferred embodiments, the composition is derived from
`
`krill. In some embodiments, the composition comprises less than 2% free fatty acids. In some
`
`embodiments, composition comprises less than 10% triglycerides. In some preferred
`
`embodiments, the phospholipids comprise greater than 50% phosphatidylcholine. In some
`
`10
`
`15
`
`20
`
`25
`
`30
`
`

`

`Attorney Docket No. AKBM-14409/US-19/CON
`
`embodiments, the composition comprises at least 500 mg/kg astaxanthin esters. In some
`
`embodiments, the composition comprises at least 500 mg/kg astaxanthin esters and at least 36%
`
`(w/w) omega-3 fatty acids. In some embodiments, the composition comprises less than about
`
`0.5g/ 100g total cholesterol. In some embodiments, the composition comprises less than about
`
`0.45% arachidonic acid (w/w).
`
`10
`
`15
`
`In some embodiments, the present invention provides a krill lipid extract comprising at
`
`least 500, 100, 1500, 2000, 2100, or 2200 mg/kg astaxanthin esters and at least 36% (w/w)
`
`omega-3 fatty acids. In further embodiments, the present invention provides a krill lipid extract
`
`comprising at least 100 mg/kg astaxanthin esters, at least 20% (w/w) omega-3 fatty acids, and
`
`less than about 0.45% arachidonic acid (w/w).
`
`In some embodiments, the present invention provides methods comprising administering
`
`the foregoing compositions to a subject in an amount effective for reducing insulin resistance,
`
`reducing inflammation, improving blood lipid profile and reducing oxidative stress.
`
`In some embodiments, the present invention provides a krill lipid extract comprising
`
`greater than about 80% triglycerides and greater than about 90, 100, 500, 1000, 1500, 200, 2100
`
`or 2200 mg/kg astaxanthin esters. In some embodiments, the krill lipid extract is characterized in
`
`containing from about 5% to about 15% omega-3 fatty acid residues. In some embodiments, the
`
`krill lipid extract is characterized in containing less than about 5% phospholipids. In some
`
`embodiments, the krill lipid extract is characterized in comprising from about 5% to about 10%
`
`20
`
`cholesterol.
`
`In some embodiments, the present invention provides a krill meal composition comprising
`
`less than about 50g/kg total fat. In some embodiments, the krill meal composition comprises
`
`from about 5 to about 20 mg/kg astaxanthin esters. In some embodiments, the krill meal
`
`composition comprises greater than about 65% protein. In some embodiments, the krill meal
`
`composition of comprises greater than about 70% protein. In some further embodiments, the
`
`present invention provides an animal feed comprising the krill meal composition.
`
`In some embodiments, the present invention provides methods of increasing flesh
`
`coloration in an aquatic species comprising feeding said aquatic species a composition
`
`comprising the krill meal described above. In some embodiments, the present invention provides
`
`methods of increasing growth and overall survival rate of aquatic species by feeding the krill
`
`meal described above.
`
`25
`
`30
`
`

`

`Attorney Docket No. AKBM-l4409/US-l9/CON
`
`In some embodiments, the present invention provides methods of producing krill oil
`
`comprising: a) providing krill meal, and b) extracting oil from said krill meal. In some
`
`embodiments, the krill meal is produced by heat-treating krill. In some embodiments, the krill
`
`meal is stored prior to the extraction step. In some embodiments, the extracting step comprises
`
`extraction by supercritical fluid extraction. In some embodiments, the supercritical fluid
`
`extraction is a two step process comprising a first extraction step with carbon dioxide and a low
`
`concentration of a co-solvent (e.g., from about l-10% co-solvent) and a second extraction step
`
`with carbon dioxide and a high concentration of a co-solvent (e.g., from about 10-3 0% co-
`
`solvent). In preferred embodiments, the co-solvent is a C1-C3 monohydric alcohol, preferably
`
`ethanol. In some embodiments, the present invention provides oil produced by the foregoing
`
`method.
`
`In some embodiments, the present invention provides methods of production of krill oil
`
`comprising: a) providing fresh krill, b) treating said fresh krill to denature lipases and
`
`phospholipases in said fresh krill to provide a denatured krill product, and c) extracting oil from
`
`said denatured krill product. In some embodiments, the denaturation step comprises heating of
`
`said fresh krill. In some embodiments, the denaturation step comprises heating said fresh krill
`
`after grinding. In some embodiments, the methods further comprise storing said denatured krill
`
`product at room temperature or below between the denaturation step and the extraction step. In
`
`some embodiments, the enzyme denaturation step is achieved by application of heat. In some
`
`embodiments, the extraction step comprises use of supercritical carbon dioxide, with or without
`
`use of a polar modifier. In some embodiments, the extraction step comprises use of ethanol. In
`
`some embodiments, the extraction step is comprises ethanol extraction followed by acetone to
`
`precipitation of phospholipids. In some embodiments, the denatured krill product is a meal. In
`
`some embodiments, the present invention provides oil produced by the foregoing method.
`
`In some embodiments, the present invention provides a composition comprising oil
`
`extracted from krill having a phosphatidylcholine content of greater then about 50% (w/w). In
`
`some embodiments, the oil has a phosphatidylcholine content of greater then about 70% (w/w).
`
`In some embodiments, the oil has a phosphatidylcholine content of greater then about 80% (w/w).
`
`In some embodiments, the composition comprises less than 2% free fatty acids. In some
`
`embodiments, the composition comprises less than 10% triglycerides. In some embodiments, the
`
`10
`
`15
`
`20
`
`25
`
`30
`
`

`

`Attorney Docket No. AKBM-14409/US-19/CON
`
`composition comprises at least 500 mg/kg astaxanthin esters. In some embodiments, the
`
`composition comprises less than about 0.45% arachidonic acid (w/w).
`
`In some embodiments, the present invention provides composition comprising odorless
`
`krill oil. In some embodiments, the odorless krill oil comprises less than about 10 mg/kg (w/w)
`
`trimethylamine. In some further embodiments, the present invention provides an odorless krill
`
`oil produced by the method comprising: extracting a neutral krill oil from a krill oil containing
`
`material by supercritical fluid extraction to provide a deodorized krill material, wherein said
`
`neutral krill oil contains odor causing compounds and extracting a polar krill oil from said
`
`deodorized krill material by supercritical fluid extraction with a polar entrainer to provide an
`
`10
`
`essentially odorless krill oil.
`
`In some embodiments, the present invention provides a composition comprising krill oil
`
`containing less than about 70 micrograms/kilogram (w/w) astaxanthin esters. In some
`
`embodiments, the compositions comprise less than about 50 micrograms/kilogram (w/w)
`
`astaxanthin esters. In some embodiments, the compositions comprise less than about 20
`
`micrograms/kilogram (w/w) astaxanthin esters. In some embodiments, the compositions
`
`comprise less than about 5 micrograms/kilogram (w/w) astaxanthin esters.
`
`In some embodiments, the present invention provides a krill oil produced by the process
`
`comprising: pumping fresh krill from a trawl onto a ship, heating the krill to provide a krill
`
`material, and extracting oil from the krill material.
`
`In further embodiments, the present invention provides a blended krill oil composition
`
`comprising: from about 45% to 55% w/w phospholipids, from about 20% to 45% w/w
`
`triglycerides, and from about 400 to about 2500 mg/kg astaxanthin. In some embodiments, the
`
`blended krill oil product comprises a blend of lipid fractions obtained from Euphausia superba.
`
`In some embodiments, the composition comprises from about 25% to 30% omega-3 fatty acids as
`
`a percentage of total fatty acids and wherein from about 80% to 90% of said omega-3 fatty acids
`
`are attached to said phospholipids.
`
`In still other embodiments, the present invention provides a Euphausia superba krill oil
`
`composition comprising: from about 30% to 60% w/w phospholipids, from about 20% to 50%
`
`triglycerides, from about 400 to about 2500 mg/kg astaxanthin, and from about 20% to 35%
`
`omega-3 fatty acids as a percentage of total fatty acids in said composition, wherein from about
`
`70% to 95% of said omega-3 fatty acids are attached to said phospholipids.
`
`15
`
`20
`
`25
`
`30
`
`

`

`Attorney Docket No. AKBM-l4409/US-l9/CON
`
`In still further embodiments, the present invention provides a dietary supplement
`
`comprising encapsulated Euphausia superba krill oil comprising from about 30% to 60% w/w
`
`phospholipids, from about 20% to 50% triglycerides; from about 400 to about 2500 mg/kg
`
`astaxanthin, and from about 20% to 35% omega-3 fatty acids as a percentage of total fatty acids
`
`in said composition, wherein from about 70% to 95% of said omega-3 fatty acids are attached to
`
`said phospholipids.
`
`In some embodiments, the present invention provides methods of making a Euphausia
`
`superba krill oil composition comprising: contacting Euphausia superba with a polar solvent to
`
`provide a polar extract comprising phospholipids, contacting Euphausia superba with a neutral
`
`solvent to provide a neutral extract comprising triglycerides and astaxanthin, combining said
`
`polar extract and said neutral extract to provide Euphausia superba krill oil comprising from
`
`about 30% to 60% w/w phospholipids, from about 20% to 50% triglycerides, from about 400 to
`
`about 2500 mg/kg astaxanthin, and from about 20% to 35% omega-3 fatty acids as a percentage
`
`of total fatty acids in said composition, wherein from about 70% to 95% of said omega-3 fatty
`
`acids are attached to said phospholipids. In some embodiments, the methods further comprise the
`
`step of encapsulating the Euphausia superba krill oil. In some embodiments, the present
`
`invention provides a Euphausia superba krill oil produced by the methods described above.
`
`In some embodiments, the present invention provides methods of producing a dietary
`
`supplement comprising, contacting Euphausia superba with a polar solvent to provide an polar
`
`extract comprising phospholipids, contacting Euphausia superba with a neutral solvent to provide
`
`a neutral extract comprising triglycerides and astaxanthin, combining said polar extract and said
`
`neutral extract to provide Euphausia superba krill oil comprising from about 30% to 60% w/w
`
`phospholipids, from about 20% to 50% triglycerides, from about 400 to about 2500 mg/kg
`
`astaxanthin, and from about 20% to 35% omega-3 fatty acids as a percentage of total fatty acids
`
`in said composition, wherein from about 70% to 95% of said omega-3 fatty acids are attached to
`
`said phospholipids, and encapsulating said Euphausia superba krill oil.
`
`In some embodiments, the present invention provides methods of reducing diet-induced
`
`hyperinsulinemia, insulin insensitivity, muscle mass hypertrophy, serum adiponectin reduction or
`
`hepatic steatosis comprising in a subject exposed to a high fat diet: administering to said subject
`
`exposed to a high fat diet an effective amount of a krill oil composition under conditions such that
`
`a condition selected from the group consisting of diet-induced hyperinsulinemia, insulin
`
`10
`
`15
`
`20
`
`25
`
`30
`
`

`

`Attorney Docket No. AKBM-14409/US-19/CON
`
`insensitivity, muscle mass hypertrophy, serum adiponectin reduction and hepatic steatosis is
`
`reduced. The present invention is not limited to any particular krill oil composition. In some
`
`embodiments, the krill oil composition is a Euphausia superba krill oil composition. The present
`
`invention is not limited to any particular formulation of krill oil. In some embodiments, the krill
`
`oil composition is encapsulated. In some preferred embodiments, the effective amount of a krill
`
`oil composition is from 0.2 grams to 10 grams of said krill oil composition. In some
`
`embodiments, the krill oil composition comprises: from about 45% to 55% w/w phospholipids,
`
`from about 20% to 45% w/w triglycerides, and from about 400 to about 2500 mg/kg astaxanthin.
`
`In some embodiments, the krill oil composition comprises a blend of lipid fractions obtained
`
`from Euphausia superba. In some embodiments, the krill oil composition comprises from about
`
`25% to 30% omega-3 fatty acids as a percentage of total fatty acids and wherein from about 80%
`
`to 90% of said omega-3 fatty acids are attached to said phospholipids. In some embodiments, the
`
`krill oil composition comprises from about 30% to 60% w/w phospholipids, from about 20% to
`
`50% triglycerides, from about 400 to about 2500 mg/kg astaxanthin, and from about 20% to 35%
`
`omega-3 fatty acids as a percentage of total fatty acids in said composition, and wherein from
`
`about 70% to 95% of said omega-3 fatty acids are attached to said phospholipids.
`
`In some embodiments, the present invention provides methods of reducing diet-induced
`
`hyperinsulinemia, insulin insensitivity, muscle mass hypertrophy, serum adiponectin reduction or
`
`hepatic steatosis comprising in a subject consuming a high fat diet or a normal fat diet:
`
`administering to said subject consuming a high fat diet or a normal fat diet an effective amount
`
`of a krill oil composition under conditions such that a condition selected from the group
`
`consisting of diet-induced hyperinsulinemia, insulin insensitivity, muscle mass hypertrophy,
`
`serum adiponectin reduction and hepatic steatosis is reduced. The present invention is not limited
`
`to any particular krill oil composition. In some embodiments, the krill oil composition is a
`
`Euphausia superba krill oil composition. The present invention is not limited to any particular
`
`formulation of krill oil. In some embodiments, the krill oil composition is encapsulated. In some
`
`preferred embodiments, the effective amount of a krill oil composition is from 0.2 grams to 10
`
`grams of said krill oil composition. In some embodiments, the krill oil composition comprises:
`
`from about 45% to 55% w/w phospholipids, from about 20% to 45% w/w triglycerides, and from
`
`about 400 to about 2500 mg/kg astaxanthin. In some embodiments, the krill oil composition
`
`comprises a blend of lipid fractions obtained from Euphausia superba. In some embodiments,
`
`10
`
`15
`
`20
`
`25
`
`30
`
`

`

`Attorney Docket No. AKBM-14409/U S-19/CON
`
`the krill oil composition comprises from about 25% to 30% omega-3 fatty acids as a percentage
`
`of total fatty acids and wherein from about 80% to 90% of said omega-3 fatty acids are attached
`
`to said phospholipids. In some embodiments, the krill oil composition comprises from about
`
`30% to 60% w/w phospholipids, from about 20% to 50% triglycerides; from about 400 to about
`
`2500 mg/kg astaxanthin, and from about 20% to 35% omega-3 fatty acids as a percentage of total
`
`fatty acids in said composition, and wherein from about 70% to 95% of said omega-3 fatty acids
`
`are attached to said phospholipids.
`
`In some embodiments, the present invention provides methods of inducing diuresis in a
`
`subject comprising: administering to said subject an effective amount of a krill oil composition
`
`under conditions such that diuresis is induced. In some embodiments, the present invention
`
`provides methods of increasing muscle mass in a subject, comprising:
`
`administering to said subject an effective amount of a krill oil composition under conditions such
`
`that muscle mass is increased. In some embodiments, the present invention provides methods of
`
`decreasing protein catabolism in a subject, comprising: administering to said subject an effective
`
`amount of a krill oil composition under conditions such that protein catabolism is decreased. In
`
`some embodiments, the present invention provides methods of decreasing lipid content in the
`
`heart of a subject, comprising: administering to said subject an effective amount of a krill oil
`
`composition under conditions such that lipid content in the heart of the subject is decreased. In
`
`some embodiments, the present invention provides methods of decreasing lipid content in the
`
`liver of a subject, comprising: administering to said subject an effective amount of a krill oil
`
`composition under conditions such that lipid content in the liver of the subject is decreased.
`
`DESCRIPTION OF THE FIGURES
`
`Figure l. 3 lP NMR analysis of polar lipids in krill oil.
`
`Figure 2. Blood lipid profiles in Zucker rats fed different forms of omega-3 fatty acids
`
`(TAG = FO, PLl = NKO and PL2 = Superba).
`
`Figure 3. Plasma glucose concentration in Zucker rats fed different forms of omega-3
`
`fatty acids.
`
`10
`
`15
`
`20
`
`25
`
`Figure 4. Plasma insulin concentration in Zucker rats fed different forms of omega-3 fatty
`
`30
`
`acids.
`
`10
`
`

`

`Attorney Docket No. AKBM-14409/US-19/CON
`
`Figure 5. Estimated HOMA-IR values in Zucker rats fed different forms of omega-3 fatty
`
`acids.
`
`Figure 6. The effect of dietary omega-3 fatty acids on TNF
`
`
`
`
`
`
`production by peritoneal
`
`macrophages.
`
`Figure 7. The effect of dietary omega-3 fatty acids on lipid accumulation in the liver.
`
`Figure 8. The effect of dietary omega-3 fatty acids on lipid accumulation in the muscle.
`
`Figure 9. The effect of dietary omega-3 fatty acids on lipid accumulation in the heart.
`
`Figure 10. Relative concentrations of DHA in the brain in Zucker rats supplemented with
`
`omega-3 fatty acids.
`
`Figure 11. Mean group body weights (g) in the collagen-induced male DBA/ 1 arthritic
`
`mice. B - PL2 is the krill oil group. * p<0.05, significantly different from Group A (Positive
`
`Control - Fish Oil) and Group C (Control).
`
`Figure 12.
`
`Body weight for the various treatment groups.
`
`Figure 13.
`
`Muscle weight for the various treatment groups.
`
`Figure 14.
`
`Muscle to body weight ratio for the various treatment groups.
`
`Figure 15.
`
`Serum adiopnectin levels (ng/ml) for the various treatment groups.
`
`Figure 16.
`
`Serum insulin levels for the various treatment groups.
`
`Figure 17.
`
`Blood glucose (mmol/l) levels in the various treatment groups.
`
`Figure 18.
`
`HOMA-IR values for the various treatment groups.
`
`Figure 19.
`
`Liver triglyceride levels (umol/g) for the various treatment groups.
`
`10
`
`15
`
`20
`
`DEFINITIONS
`
`As used herein, "phospholipid" refers to an organic compound having the following
`
`25
`
`general structure:
`
`11
`
`

`

`Attorney Docket No. AKBM-14409/US-19/CON
`
`ii
`
`o—c—R1
`
`ii
`
`—o—c—R2
`
`o
`
`o—P—o—R3
`
`1
`
`wherein R1 is a fatty acid residue, R2 is a fatty acid residue or —OH, and R3 is a —H or nitrogen
`
`containing compound choline (HOCHZCH2N+(CH3)3OH'), ethanolamine (HOCHZCHzNHz),
`
`inositol or serine. R1 and R2 cannot simultaneously be OH. When R3 is an —OH, the compound
`
`is a diacylglycerophosphate, while when R3 is a nitrogen-containing compound, the compound is
`
`a phosphatide such as lecithin, cephalin, phosphatidyl serine or plasmalogen.
`
`An “ether phospholipid” as used herein refers to a phospholipid having an ether bond at
`
`position 1 the glycerol backbone. Examples of ether phospholipids include, but are not limited
`
`to, alkylacylphosphatidylcholine (AAPC), lyso—alkylacylphosphatidylcholine (LAAPC), and
`
`alkylacylphosphatidylethanolamine (AAPE). A “non-ether phospholipid” is a phospholipid that
`
`does not have an ether bond at position 1 of the glycerol backbone.
`
`As used herein, the term omega-3 fatty acid refers to polyunsaturated fatty acids that have
`
`the final double bond in the hydrocarbon chain between the third and fourth carbon atoms from
`
`the methyl end of the molecule. Non-limiting examples of omega-3 fatty acids include,
`
`5,8,11,14,17-eicosapentaenoic acid (EPA), 4,7,10,13,16,19-docosahexanoic acid (DHA) and
`
`10
`
`15
`
`7,10,13,16,19-docosapentanoic acid (DPA).
`
`As used herein, astaxanthin refers to the following chemical structure:
`
`12
`
`

`

`Attorney Docket No. AKBM-14409/US-19/CON
`
`O A
`
`s used herein, astaxanthin esters refer to the fatty acids esterifled to OH group in the
`
`astaxanthin molecule.
`
`As used herein, the term w/w (weight/weight) refers to the amount of a given substance in
`
`a composition on weight basis. For example, a composition comprising 50% w/w phospholipids
`
`means that the mass of the phospholipids is 50% of the total mass of the composition (i.e., 50
`
`grams of phospholipids in 100 grams of the composition, such as an oil).
`
`13
`
`

`

`Attorney Docket No. AKBM-14409/US-19/CON
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`This invention discloses novel krill oil compositions characterized by containing high
`
`levels of astaxanthin, phospholipids, included an enriched quantities of ether phospholipids, and
`
`omega-3 fatty acids. The krill oils compositions are extracted from krill meal using supercritical
`
`fluid extraction (SFE) with a co-solvent modifier. The krill meal has been processed on board a
`
`ship in Antarctica using live krill as starting material in order to ensure the highest possible
`
`quality of the krill meal. The krill oils are extracted from the krill meal in two stages, in step 1 the
`
`neutral fraction is extracted using neat supercritical C02 or in combination with 5% ethanol. The
`
`neutral fraction consisted mostly of triglycerides and cholesterol. In stage 2, the polar lipids
`
`(phospholipids) are extracted by adding at least 20% ethanol to the supercritical C02 extraction
`
`medium.
`
`The present invention provides methods to avoid decomposition of glycerides and
`
`phospholipids in krill oil and compositions produced by those methods. The product obtained by
`
`these new methods is virtually free of enzymatically decomposed oil constituents. The solution to
`
`the problem is to incorporate a protein denaturation step on fresh krill prior to use of any
`
`extraction technology. Denaturation can be achieved by thermal stress or by other means. After
`
`denaturation, the oil can be extracted by an optional selection of nonpolar and polar solvents
`
`including use of supercritical carbon dioxide. Krill is adapted to a very efficient nutrient digestion
`
`at very low temperatures. Therefore the enzymes are sensitive to heat and the step of applying
`
`thermal denaturation of lipases and phospholipases does not imply use of very high temperatures.
`
`Surprisingly, it has been found that the use of mild denaturation conditions can greatly enhance
`
`the quality of krill oil.
`
`Additionally, a major obstacle of several processes of extraction is the cost of removing
`
`water. This is particularly true for methods feasible for extraction of highly unsaturated lipids
`
`where freeze drying has been regarded as the method of choice to avoid oxidative breakdown of
`
`lipids. However, the lipids in krill are surprisingly stable against oxidative deterioration.
`
`Therefore, a process including moderate use of heat in the