(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2008/0274203 A1
`Bruheim et a].
`(43) Pub. Date:
`NOV. 6, 2008
`
`US 20080274203Al
`
`(54) BIOEFFECTIVE KRILL OIL COMPOSITIONS
`
`Publication Classi?cation
`
`75
`(
`)
`
`1
`1
`1
`nven 0“
`
`I
`
`B h' Vld O'M'kk
`Eeslllalg’o (on)? gNno?e 1
`°
`Tilseth; 13ergen (NO); Sebastiano
`
`Banni; Cagliari (IT); Jeffrey Stuart Cohn; CamperdoWn (AU); Daniele
`Mancinelli Orsta (NO)
`’
`
`(51) Int-Cl
`A61K 35/56
`A61K 31/661
`33/10/585
`‘423D 9/00
`A61K 31/66
`
`(2006.01)
`(200601)
`(
`'
`)
`(200601)
`(2006.01)
`
`Correspondence Addreesi
`Casimir Jones, S.C.
`440 Science Drive, Suite 203
`Madison, WI 53711 (US)
`
`(73) Assignee:
`
`AKER BIOMARINE ASA; Oslo
`(NO)
`
`(21) Appl. No.:
`
`12/057,775
`
`(22) Filed:
`
`Mar. 28, 2008
`
`Related US. Application Data
`
`(60) Provisional application No. 60/ 920,483; ?led on Mar.
`28; 2007; provisional application No. 60/975,058;
`?led on Sep. 25; 2007; provisional application No.
`60/983,446; ?led on Oct. 29; 2007; provisional appli
`cation No. 61/024,072; ?led on Jan. 28; 2008.
`
`(52) us. Cl. ......... .. 424/522; 514/121; 514/78; 514/114;
`426/601
`
`(57)
`
`ABSTRACT
`
`This invention discloses neW krill oil compositions charac
`terized by having high amounts of phospholipids; astaxanthin
`esters and/or omega-3 contents. The krill oils are obtained
`from krill meal using supercritical ?uid extraction in a tWo
`stage process. Stage 1 removes the neutral lipid by extracting
`With neat supercritical CO2 or CO2 plus approximately 5% of
`a co-solvent. Stage 2 extracts the actual krill oils by using
`supercritical CO2 in combination With approximately 20%
`ethanol. The krill oil materials obtained are compared With
`commercially available krill oil and found to be more bioef
`fective in a number of areas such as anti-in?ammation; anti
`oxidant effects; improving insulin resistances and improving
`blood lipid pro?le.
`
`RIMFROST EXHIBIT 1043 page 0001
`
`

`

`Patent Application Publication
`
`Nov. 6, 2008 Sheet 1 0f 19
`
`US 2008/0274203 A1
`
`821'0"
`IOO'O ====3d 9
`vao'om
`
`l
`
`7
`E
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`
`RIMFROST EXHIBIT 1043 page 0002
`
`
`
`0 Sta ‘nda rd" method
`
`
`
`
`
`
`
`

`

`Patent Application Publication
`
`Nov. 6, 2008 Sheet 2 0f 19
`
`US 2008/0274203 A1
`
`FIGURE 2
`
`ctrl
`
`TAG
`
`PLl
`
`Chol
`
`HDL
`
`LDL
`
`RIMFROST EXHIBIT 1043 page 0003
`
`

`

`Patent Application Publication
`
`Nov. 6, 2008 Sheet 3 0f 19
`
`US 2008/0274203 A1
`
`FIGURE 3
`
`350 —
`
`300 —
`
`250 —
`
`200 -
`
`150 -
`
`100 -
`
`50
`
`
`
`658 .3820
`
`CTRL
`
`FO
`
`NKO
`
`Superba
`
`RIMFROST EXHIBIT 1043 page 0004
`
`

`

`Patent Application Publication
`
`Nov. 6, 2008 Sheet 4 0f 19
`
`US 2008/0274203 A1
`
`FIGURE 4
`
`3.50
`
`3.00
`
`2.50
`
`2.00
`
`1.50
`
`1.00
`
`0.50
`
`0.00
`
`
`
`Insulin, microU/L
`
`CTRL
`
`FO
`
`NKO
`
`Superba
`
`RIMFROST EXHIBIT 1043 page 0005
`
`

`

`Patent Application Publication
`
`Nov. 6, 2008 Sheet 5 0f 19
`
`US 2008/0274203 A1
`
`FIGURE 5
`
`1.20
`
`1.00 —
`
`0.80
`
`0.60
`
`0.40
`
`0.20 —
`
`0.00
`
`CTRL
`
`FO
`
`NKC)
`
`Superba
`
`RIMFROST EXHIBIT 1043 page 0006
`
`

`

`Patent Application Publication
`
`Nov. 6, 2008 Sheet 6 0f 19
`
`US 2008/0274203 A1
`
`FIGURE 6
`
`80
`
`7O -
`
`60
`
`pg/ mg of protein
`
`5O -
`
`40
`
`3O -
`
`20
`
`Control
`
`FO
`
`NKO
`
`Superba
`
`RIMFROST EXHIBIT 1043 page 0007
`
`

`

`Patent Application Publication
`
`Nov. 6, 2008 Sheet 7 0f 19
`
`US 2008/0274203 A1
`
`FIGURE 7
`
`300 ‘
`
`250 -~
`
`200 \
`
`100 *
`
`50 A
`
`ms/g
`
`\\\\\\\\ \\\\\\\\\\\\
`\
`
`FO
`
`NKO
`
`Superba.
`
`RIMFROST EXHIBIT 1043 page 0008
`
`

`

`Patent Application Publication
`
`Nov. 6, 2008 Sheet 8 0f 19
`
`US 2008/0274203 A1
`
`FIGURE 8
`
`7O
`6O
`5O
`4O
`3O
`2O
`1O
`
`Control
`
`FO
`
`N KO
`
`Superba.
`
`RIMFROST EXHIBIT 1043 page 0009
`
`

`

`Patent Application Publication
`
`Nov. 6, 2008 Sheet 9 0f 19
`
`US 2008/0274203 A1
`
`FIGURE9
`
`
`
`1800 1600
`
`.‘2 1400 D.
`
`E 1200
`O
`m 1000
`
`g 800
`|<_:
`600
`B 400
`E 200
`
`CTRL
`
`F0
`
`NKO
`
`Superba.
`
`RIMFROST EXHIBIT 1043 page 0010
`
`

`

`Patent Application Publication
`
`Nov. 6, 2008 Sheet 10 0f 19
`
`US 2008/0274203 A1
`
`FIGURE 10
`
`140 60 5
`
`4O
`
`2O
`
`Swim!
`
`FG
`
`NKQ
`
`gamma
`
`RIMFROST EXHIBIT 1043 page 0011
`
`

`

`Patent Application Publication
`
`Nov. 6, 2008 Sheet 11 0f 19
`
`US 2008/0274203 A1
`
`.ohéll
`
`?méli
`
`
`
`3806+ . .‘
`
`
`
`\1 I. i . -
`
`
`
`
`
`1"“ \l‘ i i 7 u - 0-8
`
`U . .| 1 u * I 0 ON
`
`I 0.8
`
`: 550E
`
`RIMFROST EXHIBIT 1043 page 0012
`
`

`

`Patent Application Publication
`
`Nov. 6, 2008 Sheet 12 0f 19
`
`US 2008/0274203 A1
`
`FIGURE 12
`
`Body Weight
`(grams)
`
`40
`
`30
`
`1O
`
`N
`
`HF
`
`HFFO ' HFKO
`
`RIMFROST EXHIBIT 1043 page 0013
`
`

`

`Patent Application Publication
`
`Nov. 6, 2008 Sheet 13 0f 19
`
`US 2008/0274203 A1
`
`FIGURE 13
`
`0.3
`
`Muscle Weight 0'2
`(grams)
`
`0.1
`
`0.0
`
`ac
`
`N
`
`HF
`
`HFFO
`
`HFKO
`
`I
`
`RIMFROST EXHIBIT 1043 page 0014
`
`

`

`Patent Application Publication
`
`Nov. 6, 2008 Sheet 14 0f 19
`
`US 2008/0274203 A1
`
`FIGURE 14
`
`Muscle/Body
`Wt. Ratio
`
`1.0
`
`0.8
`
`05
`
`0.4
`
`0.2
`
`0.0
`
`N
`
`HF
`
`HFFO
`
`HFKO
`
`RIMFROST EXHIBIT 1043 page 0015
`
`

`

`Patent Application Publication
`
`Nov. 6, 2008 Sheet 15 0f 19
`
`US 2008/0274203 A1
`
`FIGURE 15
`
`8000
`
`6000
`
`4000
`
`2000
`
`N
`
`HF
`
`HFFO
`
`HFKO
`
`RIMFROST EXHIBIT 1043 page 0016
`
`

`

`Patent Application Publication
`
`Nov. 6, 2008 Sheet 16 0f 19
`
`US 2008/0274203 A1
`
`FIGURE 16
`
`3.0
`
`2.5
`
`2.0
`
`1.5
`
`1.0
`
`0.5
`
`0.0
`
`N
`
`HF
`
`HFFO
`
`HFKO
`
`RIMFROST EXHIBIT 1043 page 0017
`
`

`

`Patent Application Publication
`
`Nov. 6, 2008 Sheet 17 0f 19
`
`US 2008/0274203 A1
`
`FIGURE 17
`
`15
`
`1O
`
`N
`
`HF
`
`HFFO
`
`HFKO
`
`RIMFROST EXHIBIT 1043 page 0018
`
`

`

`Patent Application Publication
`
`Nov. 6, 2008 Sheet 18 0f 19
`
`US 2008/0274203 A1
`
`FIGURE 18
`
`6O
`
`50
`
`40
`
`30
`
`20
`
`1O
`
`N
`
`HF
`
`HFFO
`
`HFKO
`
`RIMFROST EXHIBIT 1043 page 0019
`
`

`

`Patent Application Publication
`
`Nov. 6, 2008 Sheet 19 0f 19
`
`US 2008/0274203 A1
`
`b
`
`FIGURE 19
`
`140
`
`120
`
`100
`
`80
`
`4O
`
`0
`
`N
`
`HF
`
`HFFO
`
`HFKO
`
`RIMFROST EXHIBIT 1043 page 0020
`
`

`

`US 2008/0274203 Al
`
`Nov. 6, 2008
`
`BIOEFFECTIVE KRILL OIL COMPOSITIONS
`
`[0001] This application claims the benefit of U.S. Prov.
`Appl. 60/920,483, filed Mar. 28, 2007, U.S. Prov. Appl.
`60/975,058, filed Sep. 25, 2007, 60/983,446, filed Oct. 29,
`2007, and U.S. Prov. Appl. No. 61/024,072, filed Jan. 28,
`2008, all of which are incorporated by reference herein in
`their entirety.
`
`FIELD OF THE INVENTION
`
`[0002] This inventionrelates to extracts from Antarctic krill
`that comprise bioactive fatty acids.
`
`BACKGROUND OF THE INVENTION
`
`In the Southern Ocean, off the coast of Antarctica,
`[0003]
`Antarctic krill (Euphausia superba) can be found in large
`quantities, ranging from 300-500 million metric tons of bio-
`mass. It feeds on phytoplankton during the short Antarctic
`summer. During winter, however, its food supplyis limited to
`ice algae, bacteria, marine detritus as well as depleting body
`protein for energy.
`[0004]
`Inorderto isolate the krill oil from the krill, solvent
`extraction methods have been used. See, e.g., WO 00/23546.
`Krill lipids have been extracted by placing the material in a
`ketone solvent (e.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 remain-
`ing soluble lipid fraction from the solid contents by using a
`solvent such as ethanol. See, e.g., WO 00/23546. The com-
`positions produced by these methods are characterized by
`containing at least 75 ug/g astaxanthin, preferably 90 ug/g
`astaxanthin. Anotherkrill lipid extract disclosed containedat
`least 250 g/g canastaxanthin, preferably 270 ug/g canastax-
`anthin.
`
`[0005] Krill oil compositions have been described as being
`effective for decreasing cholesterol, inhibiting platelet adhe-
`sion, inhibiting artery plaque formation, preventing hyperten-
`sion, controlling arthritis symptoms, preventing skin cancer,
`enhancing transdermaltransport, reducing the symptomsof
`premenstrual symptomsor controlling blood glucose levels
`in a patient. See, e.g., WO 02/102394. In yet another appli-
`cation, 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
`03/011873.
`
`Furthermore, nutraceuticals, pharmaceuticals and
`[0006]
`cosmetics comprising the phospholipid extract were dis-
`closed. Previously, it was also shownthat supercritical fluid
`extraction using neat CO, could be usedto prevent the extrac-
`tion of phospholipids in order to extract the neutral lipid
`fraction from krill, which comprised of esterified and free
`astaxanthin. See, e.g., Yamaguchi et al., J. Agric. Food Chem.
`(1986), 34(5), 904-7. Supercritical fluid extraction with sol-
`vent modifier has previously been used to extract marine
`phospholipids from salmonroe, but has not been previously
`used to extract phospholipids from krill meal. See, e.g.,
`Tanakaet al., J. Oleo Sci. (2004), 53(9), 417-424.
`[0007] The methods described aboverely on the processing
`offrozen krill that are transported from the Southern Ocean to
`
`the processing site. This transportation is both expensive and
`can result in degradation ofthekrill starting material. Data in
`the literature showing a rapid decomposition ofthe oil in krill
`explains why somekrill oil currently offered as an omega-3
`supplementin the marketplace contains very high amounts of
`partly decomposed phosphatidylcholine and also partly
`decomposedglycerides. Saether et al., Comp. Biochem Phys.
`B 83B(1): 51-55 (1986). The products offered also contain
`high levels of free fatty acids.
`[0008] Whatis neededin the art are methodsfor processing
`krill that do not require transport offrozen krill material over
`long distances and the products produced by those methods.
`
`SUMMARY OF THE INVENTION
`
`Ina first aspect of the invention is a composition
`[0009]
`characterized by comprising at least 65% (w/w) phospholip-
`ids.
`
`In another aspect of the invention is a composition
`[0010]
`obtained from aquatic or marine sources, characterized by
`comprising 65% (w/w) phospholipids.
`[0011]
`In yet another aspect of the invention is a composi-
`tion obtained from krill, characterized by comprising at least
`65% (w/w) phospholipids.
`[0012]
`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 composi-
`[0013]
`tion 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.
`[0014]
`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 composi-
`[0015]
`tion obtained from krill, characterized by comprising at least
`65% (w/w) phospholipids andat least 580 mg/kg astaxanthin
`esters.
`
`Inyet another aspect, the present invention provides
`[0016]
`akrill oil effective for reducing insulin resistance, improving
`blood lipid profile, reducing inflammationor reducing oxida-
`tive stress.
`
`In some embodiments, the present invention pro-
`[0017]
`vides 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 com-
`position is from about 48% to 60% ona 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-alkylacylphosphatidyl-
`choline, alkylacylphosphatidylethanolamine, and combina-
`tions 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, phosphati-
`dylserine, phosphatidylethanolamine
`and combinations
`thereof. In some embodiments, krill oil composition com-
`prises a blend of lipid fractions obtained from krill. In some
`preferred embodiments, krill is Euphausia superba, although
`otherkrill species also find use in the present invention. Other
`krill species include, but are not limited to E. pacifica, E.
`RIMFROST EXHIBIT 1043
`page 0021
`
`RIMFROST EXHIBIT 1043 page 0021
`
`

`

`US 2008/0274203 Al
`
`Nov. 6, 2008
`
`frigida, E. longirostris, E. triacantha, E. vallentini, Meganyc-
`tiphanes norvegica, Thysanoessa raschii and Thysanoessa
`inermis. In some 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 attachedto said phospholipids. In
`some embodiments, the present invention provides a capsule
`containing the foregoing compositions.
`[0018]
`In further embodiments, the present inventions pro-
`vide 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 compo-
`sitions further comprise from about 35% to 50% non-ether
`phospholipids on w/w basis, so that the total amountof ether
`phospholipids and non-ether phospholipids in the composi-
`tion 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 embodi-
`ments, the ether phospholipids are selected from the group
`consisting of alkylacylphosphatidylcholine,
`lyso-alkyla-
`cylphosphatidylcholine,
`alkylacylphosphatidylethano-
`lamine, and combinationsthereof. In some embodiments, the
`ether lipids are greater than 90% alkylacylphosphatidylcho-
`line. In some embodiments, the non-ether phospholipids are
`selected from the group consisting of phosphatidylcholine,
`phosphatidylserine, phosphatidylethanolamine and combi-
`nations thereof. In some embodiments, krill 011 composition
`comprises a blend oflipid fractions obtained from krill. In
`some preferred embodiments, krill is Huphausia superba,
`although other krill species also find use in the present inven-
`tion. Other krill species include, but are not limited to E.
`pacifica, E. frigida, E. longirostris, E.. triacantha, EF. vallen-
`tini, Meganyctiphanes norvegica, Thysanoessa raschii and
`Thysanoessa inermis. In some embodiments, the composi-
`tions comprise about 25% to 30% omega-3 fatty acids as a
`percentageoftotal fatty acids and wherein from about 80% to
`90% of said omega-3 fatty acids are attached to said phos-
`pholipids. In some embodiments, the present invention pro-
`vides a capsule containing the foregoing compositions.
`[0019]
`In some embodiments, the present invention pro-
`vides a composition comprising at least 65% (w/w) of phos-
`pholipids, 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 somefurther 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% triglyc-
`erides. In some preferred embodiments, the phospholipids
`comprise greater than 50% phosphatidylcholine. In some
`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 0.5 g/100 g total cho-
`lesterol. In some embodiments, the composition comprises
`less than about 0.45% arachidonic acid (w/w).
`[0020]
`In some embodiments, the present invention pro-
`vides 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 providesa krill lipid extract comprising at
`
`least 20% (w/w)
`least 100 mg/kg astaxanthin esters, at
`omega-3 fatty acids, and less than about 0.45% arachidonic
`acid (w/w).
`[0021]
`In some embodiments, the present invention pro-
`vides methods comprising administering the foregoing com-
`positions to a subject in an amount effective for reducing
`insulin resistance, reducing inflammation, improving blood
`lipid profile and reducing oxidative stress.
`[0022]
`In some embodiments, the present invention pro-
`vides 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 astaxanthinesters. In some embodi-
`ments, 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 com-
`prising from about 5% to about 10% cholesterol.
`[0023]
`In some embodiments, the present invention pro-
`vides a krill meal composition comprising less than about 50
`g/kg total fat. In some embodiments, the krill meal composi-
`tion 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 embodi-
`ments, 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.
`[0024]
`In some embodiments, the present invention pro-
`vides methods of increasing flesh coloration in an aquatic
`species comprising feeding said aquatic species a composi-
`tion 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.
`[0025]
`In some embodiments, the present invention pro-
`vides methods ofproducing krill 011 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 1-10% co-solvent) and a second extraction step with
`carbon dioxide and a high concentration of a co-solvent(e.g.,
`from about 10-30% co-solvent). In preferred embodiments,
`the co-solvent is a C,-C, monohydric alcohol, preferably
`ethanol. In some embodiments, the present invention pro-
`vides oil produced by the foregoing method.
`[0026]
`In some embodiments, the present invention pro-
`vides methods of production of krill oil comprising: a) pro-
`viding 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 dena-
`tured krill product. In some embodiments, the denaturation
`step comprises heating of said fresh krill. In some embodi-
`ments, the denaturation step comprises heating said fresh krill
`after grinding. In some embodiments, the methods further
`comprise storing said denatured krill product at room tem-
`perature or below between the denaturation step and the
`extraction step. In some embodiments, the enzyme denatur-
`ation step is achieved by application ofheat. In some embodi-
`RIMFROST EXHIBIT 1043
`page 0022
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`RIMFROST EXHIBIT 1043 page 0022
`
`

`

`US 2008/0274203 Al
`
`Nov. 6, 2008
`
`ments, the extraction step comprises use of supercritical car-
`bon dioxide, with or without use of a polar modifier. In some
`embodiments, the extraction step comprises use ofethanol. In
`some embodiments, the extraction step is comprises ethanol
`extraction followed by acetoneto precipitation of phospho-
`lipids. In some embodiments, the denatured krill productis a
`meal. In some embodiments, the present invention provides
`oil produced by the foregoing method.
`[0027]
`In some embodiments, the present invention pro-
`vides a composition comprising oil extracted from krill hav-
`ing a phosphatidylcholine content of greater then about 50%
`(w/w). In some embodiments, the oil has a phosphatidylcho-
`line 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% trig-
`lycerides. In some embodiments, the composition comprises
`at least 500 mg/kg astaxanthinesters. In some embodiments,
`the composition comprises less than about 0.45% arachidonic
`acid (w/w).
`[0028]
`In some embodiments, the present invention pro-
`vides 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 embodi-
`ments, the present invention provides an odorless krill oil
`producedby 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 polarkrill oil from said deodorized krill material
`by supercritical fluid extraction with a polar entrainer to pro-
`vide an essentially odorless krill oil.
`[0029]
`In some embodiments, the present invention pro-
`vides 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)astaxanthinesters.
`[0030]
`In some embodiments, the present invention pro-
`vides 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.
`[0031]
`In further embodiments, the present invention pro-
`vides a blendedkrill 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 astax-
`anthin. 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.
`[0032]
`In still other embodiments, the present invention
`provides a Euphausia superba krill oil composition compris-
`ing: 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 percentageoftotal fatty acids in said compo-
`sition, wherein from about 70% to 95% of said omega-3 fatty
`acids are attached to said phospholipids.
`
`In still further embodiments, the present invention
`[0033]
`provides a dietary supplement comprising encapsulated
`Euphausia superba krill oil comprising from about 30% to
`60% w/w phospholipids; from about 20% to 50% triglycer-
`ides; 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 attachedto said
`phospholipids.
`[0034]
`In some embodiments, the present invention pro-
`vides methods of making a Euphausia superba krill oil com-
`position comprising: contacting Euphausia superba with a
`polar solventto provide a polar extract comprising phospho-
`lipids; contacting Fuphausia 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 attachedto 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 Fuphausia superba krill oil pro-
`duced by the methods described above.
`[0035]
`In some embodiments, the present invention pro-
`vides methods of producing a dietary supplement compris-
`ing; contacting Euphausia superba with a polar solvent to
`provide an polar extract comprising phospholipids; contact-
`ing Euphausia superba with a neutral solvent to provide a
`neutral extract comprising triglycerides and astaxanthin;
`combining said polar extract and said neutral extract to pro-
`vide Euphausia superba krill o11 comprising from about 30%
`to 60% w/w phospholipids; from about 20% to 50% triglyc-
`erides; 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 attachedto said
`phospholipids; and encapsulating said Euphausia superba
`krill oil.
`
`In some embodiments, the present invention pro-
`[0036]
`vides methods of reducing diet-induced hyperinsulinemia,
`insulin insensitivity, muscle mass hypertrophy, serum adi-
`ponectin reduction or hepatic steatosis comprising in a sub-
`ject exposedto 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 insensitivity, muscle mass hypertrophy, serum adi-
`ponectin 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 encap-
`sulated.
`In some preferred embodiments,
`the effective
`amount of a krill oil composition is from 0.2 grams to 10
`gramsof said krill oil composition. In some embodiments, the
`krill oi1 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 011 composition comprises a blend of
`RIMFROST EXHIBIT 1043
`page 0023
`
`RIMFROST EXHIBIT 1043 page 0023
`
`

`

`US 2008/0274203 Al
`
`Nov. 6, 2008
`
`lipid fractions obtained from Euphausia superba. In some
`embodiments, the krill 011 composition comprises from about
`25% to 30% omega-3 fatty acids as a percentageoftotal 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 011 composition comprises from about
`30% to 60% w/w phospholipids; from about 20% to 50%
`triglycerides; from about 400 to about 2500 mg/kg astaxan-
`thin; 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.
`[0037]
`In some embodiments, the present invention pro-
`vides methods of reducing diet-induced hyperinsulinemia,
`insulin insensitivity, muscle mass hypertrophy, serum adi-
`ponectin reduction or hepatic steatosis comprising in a sub-
`ject consuminga high fat diet or a normalfat diet: adminis-
`teringto said subject consuminga high fat diet or a normalfat
`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 insensi-
`tivity, 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 011 composition is encapsulated. In
`some preferred embodiments, the effective amountofa krill
`oil composition is from 0.2 gramsto 10 gramsofsaid 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 o11 composition comprises from about 25% to 30%
`omega-3 fatty acids as a percentage oftotal fatty acids and
`wherein from about 80% to 90% of said omega-3 fatty acids
`are attachedto 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 percentageoftotal fatty
`acids in said composition, and wherein from about 70% to
`95% of said omega-3 fatty acids are attached to said phos-
`pholipids.
`[0038]
`In some embodiments, the present invention pro-
`vides methods of inducing diuresis in a subject comprising:
`administering to said subject an effective amountof 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: admin-
`istering 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 pro-
`vides 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 condi-
`tions such that lipid content in the heart of the subject is
`
`decreased. In some embodiments, the present invention pro-
`vides methods of decreasing lipid content in the liver of a
`subject, comprising: administering to said subject an effec-
`tive amountofa krill oil composition under conditions such
`that lipid contentin theliver of the subject is decreased.
`
`DESCRIPTION OF THE FIGURES
`
`FIG. 1.31P NMRanalysisof polar lipids in krill oil.
`[0039]
`FIG. 2. Bloodlipid profiles in Zucker rats fed dif-
`[0040]
`ferent forms of omega-3 fatty acids (TAG=FO, PLI=NKO
`and PL2=Superba).
`[0041]
`FIG. 3. Plasma glucose concentration in Zuckerrats
`fed different forms of omega-3 fatty acids.
`[0042]
`FIG. 4. Plasma insulin concentration in Zuckerrats
`fed different forms of omega-3 fatty acids.
`[0043]
`FIG. 5. Estimated HOMA-IR values in Zuckerrats
`fed different forms of omega-3 fatty acids.
`
`[0044]
`FIG. 6. The effect of dietary omega-3 fatty acids on
`
`
`
`TNFU
`production by peritoneal macrophages.
`[0045]
`FIG. 7. The effect of dietary omega-3 fatty acids on
`lipid accumulation in theliver.
`[0046]
`FIG. 8. The effect of dietary omega-3 fatty acids on
`lipid accumulation in the muscle.
`[0047]
`FIG. 9. The effect of dietary omega-3 fatty acids on
`lipid accumulation in the heart.
`[0048]
`FIG. 10. Relative concentrations of DHA in the
`brain in Zucker rats supplemented with omega-3 fatty acids.
`[0049]
`FIG. 11. Mean group body weights (g) in the col-
`lagen-induced male DBA/1 arthritic mice. B-PL2is the krill
`oil group. * p<0.05, significantly different from Group A
`(Positive Control—Fis