WO2012143860A1

WO2012143860A1 - Ameliorating cognitive decline

Info

Publication number: WO2012143860A1
Application number: PCT/IB2012/051929
Authority: WO
Inventors: Jian Guan
Original assignee: Jian Guan
Priority date: 2011-04-18
Filing date: 2012-04-18
Publication date: 2012-10-26

Abstract

A method of ameliorating cognitive decline or a related condition, comprising oral administration of an effective amount of one or more compositions of mixed phospholipids and use of a mixed phospholipid composition in the manufacture of an oral formulation for ameliorating cognitive decline or a related condition.

Description

AMELIORATING COGNITIVE DECLINE

FIELD OF THE INVENTION

[0001] The present invention relates to use of one or more phospholipid compositions to ameliorate cognitive decline.

BACKGROUND

[0002] Cognitive decline is associated with disease and aging. Mild cognitive impairment (MCI), or memory decline, is a premonitory symptom associated with many neurodegenerative diseases, as well as a natural phenomenon that occurs during the aging process and after acute brain injury such as stroke and brain trauma. The most common symptom of MCI is loss of short term memory, while other cognitive functions such as the ability to learn, think, speak and write might all be retained. MCI is believed to exist as a transitional disease, and represents a stage of cognitive impairment between normal cognition and dementia. A consequence of continued cognitive decline due to old age is dementia where the sufferer's memory, attention, language skills and problem solving skills are gready reduced or lost.

[0003] Pathologically, cognitive decline due to old age may be characterised by depletion in dopamine, and neuronal and glial cell degeneration. Glial cells, including microglia and astrocytes are thought to provide support to neurons, and are essential for neuronal survival and function. They are also involved in maintaining vascular integrity, and provide both an immune and metabolic function in the brain. The neuro-vascular-glial cell network has been recognised as being critical in maintaining brain function and repair and recovery following injury or as a consequence of various neurodegenerative conditions.

[0004] Phospholipids are amphiphilic lipids that play an essential role in the formation of the lipid bilayer of cell membranes. Sources high in phospholipids include eggs, soy and dairy, and their consumption has been reported to have a number of beneficial health effects in humans (Wehrmuller, 2008).

[0005] The effects of phospholipids on cognition and brain function appear varied, with several trials producing inconsistent results. The lack of sufficient data to support the hypothesis that soy phospholipids reduce cognitive impairment associated with either aging or onset of neurodegenerative diseases, suggests the clinical effectiveness of phospholipids in this area is unclear (Ajmone-Cat et al, 2003; Hoshioka et al, 2007; Tweedy and Garcia, 1982; Well et al, 1995). [0006] Interventions that either ameliorate or prevent cognitive decline, including memory decline, are highly desirable.

[0007] It is an object of the invention to provide an improved or alternative means for ameliorating cognitive decline or to at least provide the public with a useful choice.

SUMMARY OF THE INVENTION

[0008] Accordingly, in a first aspect the present invention relates to a method of ameliorating cognitive decline or a related condition, the method comprising oral administration to a subject in need thereof of an effective amount of one or more mixed phospholipid compositions.

[0009] In various embodiments the mixed phospholipid compositions are selected from

(1) a combination of phosphatidylserine and one or more or two or more of

phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and sphingomyelin,

(2) a combination of phosphatidylserine, phosphatidylcholine and one or more or two or more of phosphatidylethanolamine, phosphatidylinositol and sphingomyelin,

(3) a combination of phosphatidylethanolamine and one or more or two or more of

phosphatidylserine, phosphatidylcholine, phosphatidylinositol and sphingomyelin,

(4) a combination of phosphatidylinositol and one or more or two or more of

phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine and sphingomyelin,

(5) a combination of sphingomyelin and one or more or two or more of phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol,

(6) one or more milk phospholipid extracts,

(7) a hydrolysate of any one or more of (1) to (6), and

(8) any combination of any two or more of (1) to (7),

[0010] In various embodiments the subject has

(1) no symptoms of cognitive decline, or

(2) no clinical symptoms of cognitive decline, or

(3) an age of 25 to 55 years, including 30 to 55, 35 to 55, 40 to 55, 45 to 55, 50 to 55, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 25 to 45, 30 to 45, 35 to 45 or 40 to 45 years, or

(4) a combination of (1) to (3). [0011] In another aspect the present invention relates to use of one or more phospholipid compositions described above or below in the manufacture of an oral formulation for ameliorating cognitive decline in a subject.

[0012] In yet another aspect the present invention relates to one or more phospholipid compositions described above or below for ameliorating cognitive decline in a subject.

[0013] It should be understood that amelioration of cognitive decline may include one or more of delaying onset of cognitive decline, preventing the onset of cognitive decline, reducing the severity of cognitive decline, slowing the progression of cognitive decline and preventing cognitive decline.

[0014] The following embodiments may relate to any of the above aspects.

[0015] In one embodiment the milk is bovine, camelid, caprine, cervine, equine, ovine or porcine milk, or any combination of any two or more thereof.

[0016] In some embodiments the cognitive decline is age-related cognitive decline, age- related memory loss or age-related learning impairment.

[0017] In some embodiments ameliorating cognitive decline includes maintaining or improving cognition, maintaining or improving memory, supporting cognitive function, reducing age-related memory impairment, reducing the risk of memory loss as a result of ageing, maintaining or increasing short term memory, maintaining or increasing memory, maintaining or increasing long term memory, maintaining or increasing ability to learn, maintaining or increasing ability to think, maintaining or increasing ability to speak, maintaining or increasing ability to write, maintaining or increasing attention, maintaining or increasing language skills, maintaining or increasing problem solving skills, reducing or preventing anxiety, reducing or preventing mood swings, reducing or preventing depression, reducing or preventing mental disorders, reducing or preventing age-related mental disorders, or maintaining or increasing social activity, or any combination of any two or more thereof.

[0018] In one embodiment the phospholipid composition comprises one or more phosphatidylethanolamines, one or more phosphatidylinositols, one or more

phosphatidylserines, one or more phosphatidylcholines, one or more sphingolipids (including one or more sphingomyelins, one or more dihydrosphingomyelins, one or more ceramides, one or more cerebrosides, or one or more gangliosides, or any combination of any two or more thereof), one or more lysophospholipids (phospholipids with one fatty acid lost), or any combination of any two or more thereof.

[0019] In one embodiment the phospholipid composition is administered as a component of a lipid composition. Preferred lipid compositions include animal fats and oils, plant oils (including for example soy bean oil or olive oil or a combination thereof), marine oils and fats, algal oils, fish oils, microbial oils and lipids produced by fermentation with microorganisms, or any combination of any two or more thereof. Preferred animal fats include but are not limited to dairy fats, particularly bovine milk fat, including cream.

[0020] In one embodiment the milk phospholipid extract is selected from buttermilk, one or more buttermilk extracts, butter serum, one or more butter serum extracts, beta serum, low- lactose beta serum, one or more beta serum extracts, one or more sphingolipid extracts, one or more milk fat globule membrane lipid extracts, one or more phospholipid extracts, one or more complex lipid extracts and any combination of any two or more thereof.

[0021] In preferred embodiments the milk phospholipid extract is selected from beta serum, low-lactose beta serum, one or more beta serum extracts and any combination of any two or more thereof.

[0022] In some embodiments the phospholipid composition or milk phospholipid extract comprises

(1) about 1 to about 100% w/w lipid, or

(2) about 5 to about 100% w/w lipid, or

(3) about 10 to about 100% w/w lipid, or

(4) about 1 to about 85% w/w phospholipid, or

(5) about 1 to about 70% w/w phospholipid, or

(6) about 5 to about 95% w/w lipid and about 0 to about 75% w/w protein, or

(7) about 15 to about 95% w/w lipid and about 0 to about 75% w/w protein, or

(8) about 5 to about 95% w/w lipid, about 0 to about 75% w/w protein and about 5 to about 85% w/w phospholipid, or

(9) about 15 to about 95% w/w lipid, about 0 to about 65% w/w protein and about 5 to about 70% w/w phospholipid, or

(10) about 5 to about 95% w/w lipid, about 0 to about 75% w/w protein, about 5 to about 85% w/w phospholipid and about 0 to about 5% w/w ganglioside, or (11) about 15 to about 95% w/w lipid, about 0 to about 65% w/w protein, about 5 to about 70% w/w phospholipid and about 0 to about 2.5% w/w ganglioside.

[0023] In some embodiments the phospholipid composition or milk phospholipid extract comprises about 0, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 % w/w protein, and useful ranges may be selected between any of these values (for example, about 0 to about 80%, about 5 to about 80%, about 10 to about 80%, about 15 to about 80%, about 20 to about 80%, about 25 to about 80%, about 30 to about 80%, about 35 to about 80%, about 40 to about 80%, about 45 to about 80%, about 50 to about 80%, about 0 to about 70%, about 10 to about 70%, about 15 to about 70%, about 20 to about 70%, about 25 to about 70%, about 30 to about 70%, about 35 to about 70%, about 40 to about 70%, about 45 to about 70%, about 50 to about 70%, about 0 to about 60%, about 10 to about 60%, about 15 to about 60%, about 20 to about 60%, about 25 to about 60%, about 30 to about 60%, about 35 to about 60%, about 40 to about 60%, about 45 to about 60%, about 0 to about 10%, and about 0 to about 15% w/w protein).

[0024] In some embodiments the phospholipid composition or milk phospholipid extract comprises at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% w/w lipid, and useful ranges may be selected between any of these values (for example, about 5 to about 95%, about 10 to about 95%, about 15 to about 95%, about 20 to about 95%, about 25 to about 95%, about 30 to about 95%, about 35 to about 95%, about 40 to about 95%, about 45 to about 95%, about 50 to about 95%, about 55 to about 95%, about 60 to about 95%, about 65 to about 95%, about 70 to about 95%, about 75 to about 95%, about 80 to about 95%, about 85 to about 95%, about 10 to about 70%, about 15 to about 70%, about 20 to about 70%, about 25 to about 70%, about 30 to about 70%, about 35 to about 70%, about 40 to about 70%, about 45 to about 70%, about 50 to about 70%, about 10 to about 60%, about 15 to about 60%, about 20 to about 60%, about 25 to about 60%, about 30 to about 60%, about 35 to about 60%, about 40 to about 60% w/w lipid).

[0025] In some embodiments the phospholipid composition or milk phospholipid extract comprises less than about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 % w/w carbohydrate, and useful ranges may be selected between any of these values (for example, about 5 to about 80%, about 5 to about 70%, about 10 to about 70%, about 15 to about 70%, about 20 to about 70%, about 25 to about 70%, about 5 to about 60%, about 10 to about 60%, about 15 to about 60%, about 20 to about 60%, about 25 to about 60%, about 5 to about 50%, and about 5 to about 15% w/w carbohydrate). In one embodiment the carbohydrate is lactose, galactose or glucose. In another embodiment the carbohydrate is lactose. [0026] In some embodiments the phospholipid composition or milk phospholipid extract comprises at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% w/w phospholipid, and useful ranges may be selected between any of these values (for example, about 5 to about 95%, about 10 to about 95%, about 5 to about 95%, about 20 to about 95%, about 25 to about 95%, about 30 to about 95%, about 35 to about 95%, about 40 to about 95%, about 45 to about 95%, about 50 to about 95%, about 10 to about 70%, about 5 to about 70%, about 20 to about 70%, about 25 to about 70%, about 30 to about 70%, about 35 to about 70%, about 40 to about 70%, about 45 to about 70%, and about 50 to about 70% w/w phospholipid).

[0027] In some embodiments the phospholipid composition or milk phospholipid extract comprises at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% w/w of one or more, two or more, three or more, or four or more phospholipids selected independently from phosphatidylcholine,

phosphatidylethanolamine, sphingomyelin, phosphatidylserine, and phosphatidylinositol, and useful ranges may be selected between any of these values (for example, about 0.1 to about 30%, about 0.5 to about 30%, about 1 to about 30%, about 2 to about 30%, about 3 to about 30%, about 4 to about 30%, about 5 to about 30%, about 10 to about 30%, about 15 to about 30%, about 20 to about 30%, about 0.1 to about 5%, about 0.5 to about 5%, about 1 to about 5%, about 2 to about 5%, about 3 to about 5%, about 0.1 to about 10%, about 0.5 to about 10%, about 1 to about 10%, about 2 to about 10%, about 3 to about 10%, about 4 to about 10%, about 5 to about 10%, about 6 to about 10%, about 0.1 to about 20%, about 0.5 to about 20%, about 1 to about 20%, about 2 to about 20%, about 3 to about 20%, about 4 to about 20%, about 5 to about 20%, about 10 to about 20%, about 15 to about 20% w/w of one or more, two or more, three or more, or four or more phospholipids selected independendy from

phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phosphatidylserine, and phosphatidylinositol).

[0028] In some embodiments the phospholipid composition or milk phospholipid extract comprises at least about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5% w/w ganglioside, and useful ranges may be selected between any of these values (for example, about 0.1 to about 1, about 0.1 to about 2, about 0.1 to about 3, about 0.1 to about 4, and about 0.1 to about 5% w/w ganglioside). In various embodiments the ganglioside is independently selected from GM3 and GD3 and combinations thereof. [0029] In some embodiments the phospholipid composition or milk phospholipid extract comprises about 25 to about 35% w/w protein, about 15 to about 25% w/w lipid, and about 5 to about 12% w/w phospholipid, or

about 25 to about 35% w/w protein, about 15 to about 25% w/w lipid, about 5 to about 12% w/w phospholipid, about 5 to about 10% w/w MFGM (milk fat globule membrane) protein, and about 0.2 to about 0.9% w/w ganglioside, or

about 25 to about 35% w/w protein, about 15 to about 25% w/w lipid, about 5 to about 12% w/w phospholipid, about 1 to about 5% w/w phosphatidylcholine, about 1.5 to about 6% w/w phosphatidylethanolamine, about 1 to about 5% w/w sphingomyelin, about 0.5 to about 2% w/w phosphatidylserine, about 0.1 to 2% w/w

phosphatidylinositol, about 5 to about 10% w/w MFGM protein, and about 0.2 to about 0.9% w/w ganglioside, or

about 20 to about 50% w/w protein, about 5 to about 25% w/w lipid, and about 1 to about 15% w/w phospholipid, or

about 20 to about 50% w/w protein, about 5 to about 25% w/w lipid, and about 1 to about 15% w/w phospholipid, and about 1 to about 10% w/w MFGM protein, or about 25 to about 35% w/w protein, about 10 to about 20% w/w lipid, about 4 to about 12% w/w phospholipid, about 1 to about 3% w/w phosphatidylcholine, about 1 to about 4% w/w phosphatidylethanolamine, about 0.5 to about 3% w/w sphingomyelin, about 0.5 to about 1.5% w/w phosphatidylserine, about 0.1 to 1% w/w

phosphatidylinositol, about 1 to about 10% w/w MFGM protein, or

about 40 to about 60% w/w protein, about 25 to about 45% w/w lipid, and about 10 to about 25% w/w phospholipid, or

about 40 to about 60% w/w protein, about 25 to about 45% w/w lipid, about 10 to about 25% w/w phospholipid, about 5 to about 20% w/w MFGM protein, and about 0.5 to about 2.0% w/w ganglioside, or

about 46 to about 52% w/w protein, about 28 to about 40% w/w lipid, about 11 to about 16% w/w phospholipid, about 2 to about 6% w/w phosphatidylcholine, about 3 to about 8% w/w phosphatidylethanolamine, about 2.5 to about 7% w/w

sphingomyelin, about 0.5 to about 3% w/w phosphatidylserine, about 0.5 to 2% w/w phosphatidylinositol, about 5 to about 15% w/w MFGM protein, and about 0.5 to about 0.9% w/w ganglioside, or (10) about 50 to about 70% w/w protein, about 12 to about 32% w/w lipid, and about 5 to about 25% w/w phospholipid, or

(11) about 50 to about 70% w/w protein, about 12 to about 32% w/w lipid, about 5 to about 25% w/w phospholipid, about 2 to about 8 % w/w phosphatidylcholine, about 2 to about 10% w/w phosphatidylethanolamine, about 2 to about 8% w/w sphingomyelin, and about 1 to about 3% w/w phosphatidylserine, about 10 to about 20% w/w MFGM protein, and about 0.5 to about 2.5% w/w ganglioside, or

(12) about 56 to about 65% w/w protein, about 18 to about 28 % w/w lipid, about 8 to

about 20% w/w phospholipid, about 2 to about 8 % w/w phosphatidylcholine, about 2 to about 10% w/w phosphatidylethanolamine, about 2 to about 8% w/w sphingomyelin, and about 1 to about 3% w/w phosphatidylserine, and about 0.5 to 3% w/w

phosphatidylinositol, about 10 to about 20% w/w MFGM protein, and about 0.5 to about 2.5% w/w ganglioside, or

(13) about 0 to about 10% w/w protein, about 85 to about 97% w/w lipid, and about 25 to about 35% w/w phospholipid, or

(14) about 0 to about 10% w/w protein, about 85 to about 97% w/w lipid, about 25 to about 35% w/w phospholipid, about 5 to about 10 % w/w phosphatidylcholine, about 7 to about 13% w/w phosphatidylethanolamine, about 4 to about 9% w/w sphingomyelin, about 2 to about 5% w/w phosphatidylserine, about 1 to about 3% w/w

phosphatidylinositol, about 0 to about 5 % w/w MFGM protein, and about 1 to about 3% w/ w gangliosides, or

(15) less than about 1% w/w protein, about 80 to about 95% w/w lipid, about 60 to about 80% w/ w phospholipid, or

(16) less than about 1% w/w protein, about 80 to about 95% w/w lipid, about 60 to about 80% w/w phospholipid and about 1 to about 5% w/w gangliosides, or

(17) less than about 1% w/w protein, about 80 to about 95% w/w lipid, about 60 to about 80% w/w phospholipid, about 10 to about 20 % w/w phosphatidylcholine, about 18 to about 28% w/w phosphatidylethanolamine, about 10 to about 20% w/w sphingomyelin, about 4 to about 12% w/w phosphatidylserine, about 2 to about 10% w/w

phosphatidylinositol, and about 1 to about 5% w/w gangliosides, or

(18) about 75 to about 99% w/w lipid and about 15 to 35% w/w phospholipid, or

(19) about 80 to about 90% w/w lipid, about 5 to about 15% w/w phosphatidylcholine, about 5 to about 15% w/w phosphatidylethanolamine, about 4 to about 10% w/w sphingomyelin, and about 0.1 to about 2% w/w phosphatidylserine, or (20) about 80 to about 90% w/w lipid, about 20 to 30% w/w phospholipid, about 5 to about 15% w/w phosphatidylcholine, about 5 to about 15% w/w phosphatidylethanolamine, about 5 to about 10% w/w sphingomyelin, about 0.5 to about 1.5% w/w

phosphatidylserine, and about 0.1 to about 1.2% w/w phosphatidylinositol, or

(21) about 75 to about 95% w/w lipid and about 50 to about 90% w/w phospholipid, or

(22) about 80 to about 90% w/w lipid, about 10 to about 30% w/w phosphatidylcholine, about 12 to about 22% w/w phosphatidylethanolamine, about 12 to about 22% w/w sphingomyelin, and about 1 to about 3% w/w phosphatidylserine, or

(23) about 75 to about 95% w/w lipid, about 50 to about 90% w/w phospholipid, about 10 to about 45% w/w phosphatidylcholine, about 12 to about 25% w/w

phosphatidylethanolamine, about 12 to about 25% w/w sphingomyelin, about 1 to about 6% w/w phosphatidylserine, and about 0.5 to 4% w/w phosphatidylinositol, or

(24) about 80 to about 90% w/w lipid, about 65 to about 75% w/w phospholipid, about 10 to about 30% w/w phosphatidylcholine, about 12 to about 22% w/w

phosphatidylethanolamine, about 12 to about 22% w/ w sphingomyelin, about 1 to about 3% w/w phosphatidylserine, and about 0.5 to 3% w/w phosphatidylinositol, or

(25) about 25 to about 45% w/w lipid and about 0.2 to about 1% w/w ganglioside GD3, or

(26) about 25 to about 45% w/w lipid, about 10 to about 30% w/w phospholipids, and about 0.2 to about 1% w/w ganglioside, or

(27) about 25 to about 45% w/w lipid, about 10 to about 30% w/w phospholipids, about 2 to about 5% w/w phosphatidylcholine, about 3 to about 7% w/w

phosphatidylethanolamine, about 2 to about 5% w/w sphingomyelin, about 2 to about 12% w/w phosphatidylserine, about 1 to about 5% w/w phosphatidylinositol, and about 0.2 to about 1% w/w ganglioside, or

(28) about 20 to about 40% w/w lipid and about 0.8 to about 2% w/w ganglioside GD3, or

(29) about 20 to about 40% w/w lipid, about 5 to about 30% w/w phospholipids, and about 0.8 to about 3.5% w/w ganglioside, or

(30) about 20 to about 40% w/w lipid, about 5 to about 30% w/w phospholipids, about 1 to about 5% w/w phosphatidylcholine, about 2 to about 8% w/w

phosphatidylethanolamine, about 0.5 to about 5% w/w sphingomyelin, about 1 to about 10% w/w phosphatidylserine, about 1 to about 6% w/w phosphatidylinositol, and about 0.8 to about 3.5% w/w ganglioside.

[0030] In one embodiment a composition useful herein comprises, consists essentially of, or consists of at least about 0.1 , 0.2, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 99.5, 99.8 or 99.9% by weight of one or more compositions or extracts described above and useful ranges may be selected between any of these foregoing values (for example, from about 0.1 to about 50%, from about 0.2 to about 50%, from about 0.5 to about 50%, from about 1 to about 50%, from about 5 to about 50%, from about 10 to about 50%, from about 15 to about 50%, from about 20 to about 50%_^ from about 25 to about 50%, from about 30 to about 50%, from about 35 to about 50%, from about 40 to about 50%, from about 45 to about 50%, from about 0.1 to about 60%, from about 0.2 to about 60%, from about 0.5 to about 60%, from about 1 to about 60%, from about 5 to about 60%, from about 10 to about 60%, from about 15 to about 60%, from about 20 to about 60%_^ from about 25 to about 60%, from about 30 to about 60%, from about 35 to about 60%, from about 40 to about 60%, from about 45 to about 60%, from about 0.1 to about 70%, from about 0.2 to about 70%, from about 0.5 to about 70%, from about 1 to about 70%, from about 5 to about 70%, from about 10 to about 70%, from about 15 to about 70%, from about 20 to about 70%_^ from about 25 to about 70%, from about 30 to about 70%, from about 35 to about 70%, from about 40 to about 70%, from about 45 to about 70%, from about 0.1 to about 80%, from about 0.2 to about 80%, from about 0.5 to about 80%, from about 1 to about 80%, from about 5 to about 80%, from about 10 to about 80%, from about 15 to about 80%, from about 20 to about 80%_^ from about 25 to about 80%, from about 30 to about 80%, from about 35 to about 80%, from about 40 to about 80%, from about 45 to about 80%, from about 0.1 to about 90%, from about 0.2 to about 90%, from about 0.5 to about 90%, from about 1 to about 90%, from about 5 to about 90%, from about 10 to about 90%, from about 15 to about 90%, from about 20 to about 90%_^ from about 25 to about 90%, from about 30 to about 90%, from about 35 to about 90%, from about 40 to about 90%, from about 45 to about 90%, from about 0.1 to about 99%, from about 0.2 to about 99%, from about 0.5 to about 99%, from about 1 to about 99%, from about 5 to about 99%, from about 10 to about 99%, from about 15 to about 99%, from about 20 to about 99%_^ from about 25 to about 99%, from about 30 to about 99%, from about 35 to about 99%, from about 40 to about 99%, and from about 45 to about 99%).

[0031] In one embodiment a composition useful herein comprises, consists essentially of, or consists of at least about 0.001, 0.01, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 grams of one or more extracts described above and useful ranges may be selected between any of these foregoing values (for example, from about 0.01 to about 1 grams, about 0.01 to about 10 grams, about 0.01 to about 19 grams, from about 0.1 to about 1 grams, about 0.1 to about 10 grams, about 0.1 to about 19 grams, from about 1 to about 5 grams, about 1 to about 10 grams, about 1 to about 19 grams, about 5 to about 10 grams, and about 5 to about 19 grams).

[0032] In one embodiment a composition useful herein comprises, consists essentially of, or consists of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99, or 99.9 % by weight of recombined, powdered or fresh whole milk or a milk product and useful ranges may be selected between any of these foregoing values (for example, from about 0.1 to about 50%, from about 0.2 to about 50%, from about 0.5 to about 50%, from about 1 to about 50%, from about 5 to about 50%, from about 10 to about 50%, from about 15 to about 50%, from about 20 to about 50%_^ from about 25 to about 50%, from about 30 to about 50%, from about 35 to about 50%, from about 40 to about 50%, and from about 45 to about 50%). The milk product is preferably selected from recombined, powdered or fresh skim milk, recombined or reconstituted whole or skim milk powder, skim milk concentrate, skim milk retentate, concentrated milk, ultrafiltered milk retentate, milk protein concentrate (MPC), milk protein isolate (MPI), calcium depleted milk protein concentrate (MPC), low fat milk, low fat milk protein concentrate (MPC), casein, caseinate, milk fat, cream, butter, ghee, anhydrous milk fat (AMF), buttermilk, butter serum, beta serum, hard milk fat extracts, soft milk fat extracts, sphingolipid extracts, milk fat globular membrane extracts, milk fat globular membrane lipid extracts, phospholipid extracts, complex lipid extracts, colostrum, a colostrum extract, colostrum protein concentrate (CPC), colostrum whey, an immunoglobulin extract from colostrum, whey (including sweet whey, lactic acid whey, mineral acid whey, or reconstituted whey powder), whey protein isolate (WPI), whey protein concentrate (WPC), a composition derived from any milk or colostrum processing stream, a composition derived from the retentate or permeate obtained by ultrafiltration or microfiltration of any milk or colostrum processing stream, a composition derived from the breakthrough or adsorbed extract obtained by chromatographic (including but not limited to ion and gel permeation chromatography) separation of any milk or colostrum processing stream, extracts of any of these milk derivatives including extracts prepared by multistage fractionation, differential crystallisation, solvent fractionation, supercritical fractionation, near critical fractionation, distillation, centrifugal fractionation, or fractionation with a modifier (e.g. soaps or emulsifiers), hydrolysates of any of these derivatives, extracts of the hydrolysates, and any combination of any two or more of these derivatives, including

combinations of hydrolysed and/ or non-hydrolysed extracts. It should be understood that the source of these derivatives may be milk or colostrum or a combination thereof. [0033] In one embodiment a composition useful herein further comprises a pharmaceutically acceptable carrier. In another embodiment the composition is or is formulated as a food, drink, food additive, drink additive, dietary supplement, nutritional product, medical food, enteral feeding product, meal replacement, cosmeceutical, nutraceutical, medicament, or pharmaceutical. In one embodiment the composition is in the form of a tablet, a caplet, a pill, a hard or soft capsule or a lozenge. In one embodiment the composition is in the form of a cachet, a powder, a dispensable powder, granules, a suspension, an elixir, a liquid, or any other form that can be added to food or drink, including for example water, milk or fruit juice. In one embodiment the composition further comprises one or more constituents (such as antioxidants) which prevent or reduce degradation of the composition during storage or after administration. These compositions may include any edible consumer product which is able to carry lipid.

Examples of suitable edible consumer products include aqueous products, baked goods, confectionary products including chocolate, gels, ice creams, reconstituted fruit products, snack bars, food bars, muesli bars, spreads, sauces, dips, dairy products including yoghurts and cheeses, drinks including dairy and non-dairy based drinks, milk, milk powders, sports supplements including dairy and non-dairy based sports supplements, fruit juice, food additives such as protein sprinkles, and dietary supplement products including daily supplement tablets. Suitable nutraceutical compositions useful herein may be provided in similar forms.

[0034] In some embodiments a method of the invention further comprises separate, simultaneous or sequential administration of, or a composition useful herein further comprises, one or more additional agents for ameliorating cognitive decline selected from one or more fatty acids (including but not limited to arachidonic acid), one or more polyunsaturated fatty acids, one or more omega-3 fatty acids (such as one or more of oc-linolenic acid (ALA), stearidonic acid (SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), tetracosapentaenoic acid, and tetracosahexaenoic acid (nisinic acid)), one or more plant extracts (including, for example, St John's Wart, curcuma, curcumin, ginseng, Gotu kola, Ginkgo biloba, and vanilla bean extract - vanillin), one or more vitamins (including but not limited to cyanocobalamin, thiamine, niacin, pyridoxine, pantothenate, folic acid, alpha-tocopherol, ascorbic acid, vitamin C, vitamin A, a vitamin B, vitamin B12, and vitamin E), one or more minerals (including but not limited to calcium, magnesium, selenium and zinc), alpha-lipoic acid, N-acetyl cysteine, L-carnosine, acetyl carnitine, one or more amino acids, one or more essential amino acids (including but not limited to isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, histidine, and tyrosine), co-enzyme Q10, resveratrol, indole-3-propionic acid, dehydroepiandrosterone, melatonin, white willow bark, salicin, one or more anthocyanins (such as from apples, red cabbage or grapes), lycopene, tomato, a tomato extract, ginger, cloves, cinnamon, oregano, turmeric, beans, legumes, soy, soy protein, soy bean oil, thiamine, berries, polyphenols, flavonoids, walnuts, uridine, egg, egg yolks, choline, rosemary, carnosic acid, avocados, artichokes, methylating agents, quercetin, vinpocetine, a catechin, epigallocatechin-3-gallate, alcohol, glutamate, and red wine, and any combination of any two or more thereof.

[0035] In some embodiments a method of the invention further comprises separate, simultaneous or sequential administration of, or a composition useful herein further comprises, one or more additional agents for ameliorating cognitive decline selected from the list including but not limited to one or more nootropic agents, apolipoprotein, one or more anti-inflammatory agents, one or more oestrogen replacements, one or more growth hormones, one or more growth factors, one or more non-steroidal anti-inflammatory drugs (NSAIDs), aspirin, ibuprofen, naproxen, one or more agents for maintaining or improving microcirculation, one or more angiogenic agents, one or more acetylcholinesterase inhibitors (including but not limited to donepezil, galantamine and rivastigmine), one or more NMDA receptor antagonists (for example, memantine), and glutamate, and any combination of any two or more thereof.

[0036] In various embodiments the related condition is selected from the list including but not limited to dementia, young-onset or early-onset dementia, degenerative dementia,

Alzheimer's disease, vascular disease, frontotemporal lobar degeneration (FTLD), dementia with Lewy bodies, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, cognitive impairment associated with schizophrenia, chemotherapy-induced neuropathy, Down's syndrome, Korsakoff s disease, cerebral palsy, epilepsy, neuronal ischemia, neuronal reperfusion injury, neuronal trauma, neuronal haemorrhage, neuronal infection, stroke, neuronal exposure to a toxic substance, age-related mental disorders, anxiety disorders, age-related depression, dementia associated with microvascular disorders (such as diabetes, hypotension, stroke induced vascular dementia, and obesity), dementia associated with a disorder of the immune system, dementia associated with a central nervous system (CNS) disorder, dementia associated with hypotension, dementia associated with obesity, vascular dementia (including post stroke).

[0037] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0038] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

[0039] The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is a bar graph showing the effects of phospholipid compositions on striatal dopamine output where BG is the blank gel control, S-PS is the soy phosphatidylserine treatment diet, M-PS is the milk phosphatidylserine treatment diet and M-PL is the milk phospholipid treatment diet. Compared to the young + BG group, the aged + BG control group showed a significant reduction in TH density in the striatum (*** p < 0.001). A significant effect was also seen in the aged group treated with M-PL (# p < 0.05) compared to the aged + BG control group.

[0041] Figure 2 is two bar graphs showing (A) the effects of age and phospholipid compositions on microglia in the CA4 of the hippocampus, and (B) the effects of age and phospholipid compositions on microglia in the frontal cortex; where in each graph BG is the blank gel control, S-PS is the soy phosphatidylserine treatment diet, M-PS is the milk

phosphatidylserine treatment diet and M-PL is the milk phospholipid treatment diet. The average density of the microglia was significandy reduced in both the hippocampus and the frontal cortex of the aged + BG control group compared to the young + BG control group (*** p < 0.0001) (A and B). The aged rats treated with M-PL showed an increase in reactive microglia in the hippocampus (A) but not in the frontal cortex (B) compared to the aged + BG control group (### p < 0.0001). [0042] Figure 3 is a bar graph showing the number of entries to the platform zone in the Morris Water Maze test of Example 2, examining spatial memory 24h after the acquisition. The memory was significantly reduced in the aged control rats (BG) compared to the young controls (* p<0.05). There was a strong trend towards improved memory in the aged rats fed with DPL compared with the aged controls (# p=0.06).

[0043] Figure 4 is a bar graph showing the number of entries to the platform zone in the Morris Water Maze test of Example 2, examining spatial memory 72h after acquisition trials. The memory decline in aged control rats was not significant compared to the young controls. There was trend towards improved memory in the aged rats fed with DPL compared with the aged controls. While the young adult rats and the aged rats fed with DPL made > 1 entries to the platform zone the aged rats fed with SPS or control gel made < 1 entry to the platform zone, as indicated by the line across the graph.

[0044] Figure 5 is a bar graph showing the initial heading error to the platform zone in the Morris Water Maze test of Example 2, examining spatial memory 72h after the acquisition trials. The aged controls rats made significant more errors compared to the young controls (** p<0.001). The aged rats fed with DPL made less errors compared the aged controls (* p=0.05).

[0045] Figure 6 is a bar graph showing dopamine expression. Compared to the aged rats fed with BG, the dopamine expression in the striatum was higher in the young adult rats (p<0.05). The aged rats with DPL supplementation had a significant increase in dopamine expression compared to the rats fed with BG (p<0.05).

[0046] Figure 7 is a bar graph showing the effect of age and the supplementation of PS/PL on neuronal plasticity in the CA3 sub-regions of the hippocampus. Compared to the aged rats control rats (Aged+BG) the aged rats with DPL supplementation significantly elevated the synaptic density in the CA3 sub-region (* p<0.05).

DETAILED DESCRIPTION OF THE INVENTION

[0047] Milk phospholipids were able to maintain microglia cell populations in aged rats and partially restore the loss of dopamine seen in these rats whereas soy phosphatidylserine was not. The inventors believe that administration of an effective amount of milk phospholipid to a subject not yet suffering from cognitive decline will ameliorate, delay the onset of, prevent the onset of, reduce the severity of, slow the progression of or even prevent cognitive decline. Preferably the subject is at an early stage of brain aging, such as at an age of 22 to 55, or 45 to 55 years.

[0048] Therapeutic interventions that either ameliorate or prevent cognitive impairment, including memory decline, are highly desirable. In particular, supplementation with dairy phospholipids prior to onset of memory decline, may offer new methods for managing, preventing or reversing the effects of mild cognitive impairment, dementia, Alzheimer's and other neurodegenerative diseases.

1. Definitions

[0049] The term "beta-serum" means an aqueous dairy ingredient separated from dairy streams containing greater than 60% fat which have been through phase inversion from an oil- in-water to a water-in-oil emulsion, as described below. Cream is the preferred starting material for the production of beta-serum. For example beta-serum is produced during the production of butter-oil (also known as anhydrous milk fat or AMF) from cream as shown in Figure 2 of WO 2006/ 041316. Preferably the beta serum is dried; preferably dried beta-serum is a powder.

[0050] The term "comprising" as used in this specification means "consisting at least in part of. When interpreting statements in this specification which include that term, the features, prefaced by that term in each statement or claim, all need to be present but other features can also be present. Related terms such as "comprise" and "comprised" are to be interpreted in the same manner.

[0051] An "effective amount" is the amount required to confer therapeutic effect. The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described by Freireich, et al. (1966). Body surface area can be approximately determined from height and weight of the subject. See, e.g., Scientific Tables, Geigy

Pharmaceuticals, Ardley, New York, 970, 537. Effective doses also vary, as recognized by those skilled in the art, dependent on route of administration, carrier usage, and the like.

[0052] The terms "enrich" and "enriched" mean that the extract or composition has a higher concentration of the named component than is present in whole milk, cream, butter, anhydrous milk fat, buttermilk, butter serum, or beta serum, or the parent extract from which the extract or composition is derived. For example, a phospholipid-enriched extract is an extract that has a higher phospholipid concentration than the whole milk, cream, butter, anhydrous milk fat, buttermilk, butter serum, or beta serum from which it is derived. [0053] The term "extract" means a composition that has been isolated from a source material and that is compositionally different to the source material that the extract was isolated from. For example, a milk fat extract, preferably a bovine milk fat extract, differs

compositionally from the naturally occurring milk fat in whole milk. In alternative embodiments the concentration in the extract is higher than the concentration in whole milk, or in whole colostrum, or in cream from milk, or in cream from colostrum, or in anhydrous milk fat (AMF) from milk, or AMF from colostrum. Preferred source material useful herein includes whole milk, cream, anhydrous milk fat, buttermilk, butter serum, or beta serum, or whey cream from bovine milk.

[0054] Accordingly, the term "milk phospholipid extract" means an isolated extract of non- human mammalian milk fat where the phospholipid concentration of the extract is higher than the phospholipid concentration of naturally occurring non-human mammalian milk fat.

Preferably the concentration of at least one phospholipid or at least one phospholipid and at least one ganglioside in an extract useful herein is at least about 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% higher than the concentration in naturally occurring non-human mammalian milk, and useful ranges may be selected between these values. In alternative embodiments the concentration in the extract is higher than the concentration in whole milk, or in whole colostrum, or in cream from milk, or in cream from colostrum, or in AMF from milk, or AMF from colostrum. A preferred milk phospholipid extract is a phospholipid-enriched milk extract.

[0055] The term "oral administration" includes oral, buccal, enteral and intra-gastric administration.

[0056] The term "pharmaceutically acceptable carrier" is intended to refer to a carrier including but not limited to an excipient, diluent or auxiliary, or combination thereof, that can be administered to a subject as a component of a composition described herein that does not reduce the activity of the composition and is not toxic when administered in doses sufficient to deliver an effective amount of a compound or composition useful herein. The formulations useful herein are administered orally.

[0057] A "subject" is an adult mammal, more preferably an adult mammalian companion animal or adult human. Preferred companion animals include cats, dogs and horses. [0058] The term "prevent" and its derivatives should be interpreted in their broadest possible context. The term should not be taken to imply that a condition is completely prevented. Accordingly, "prevent" broadly includes amelioration and/ or prevention of the onset of the symptoms or severity of a particular condition.

2. Amelioration of cognitive decline

[0059] The compositions described herein may be used to ameliorate cognitive decline by oral administration to a subject. In the context of the invention and use of the compositions described herein to treat cognitive decline, the term "cognition" may separately refer to short term memory, memory, long term memory, ability to learn, ability to think, ability to speak, ability to write, attention, language skills or problem solving skills, or any combination of any two or more thereof. Equally, symptoms of cognitive decline and dementia may include one or more of anxiety, mood swings, depression, mental disorders, age-related mental disorders (e.g., bipolar disorder), and reduced social activity including reduced grooming. The compositions described herein may be used to ameliorate one or more of these symptoms of cognitive decline or to maintain one or more of these facets of cognition or both. It should be understood that amelioration of cognitive decline may include one or more of delaying onset of cognitive decline, preventing the onset of cognitive decline, reducing the severity of cognitive decline and slowing the progression of cognitive decline. In some embodiments the cognitive decline is age-related cognitive decline, age-related memory loss or age-related learning impairment.

[0060] The abbreviated mental test score (AMTS), the mini mental state examination (MMSE), Modified Mini-Mental State Examination (3MS), the Cognitive Abilities Screening Instrument (CASI), and the clock drawing test may all be used to determine and track a subject's cognitive status. MMSE is the preferred test for determining and tracking a subject's cognitive status within the context of this invention. Additionally or alternatively, a general assessment to determine presence or absence of indicators of cognitive decline including assessment based on questions that enquire after the subject's mood or state of mind may be used.

[0061] It is anticipated that the compositions described herein will be administered at least once daily to at least once weekly for at least 1 to 10 years or more. A suitable regime of supplementation or low dose supplementation may be determined by the subject's symptoms and risk factors, for example diabetes.

[0062] In one embodiment a composition useful herein is administered daily for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months, or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 years, and useful ranges may be selected between any of these values (for example, about 1 to about 15, about 2 to about 15, about 3 to about 15, about 4 to about 15, about 5 to about 15, about 6 to about 15, about 7 to about 15, about 8 to about 15, about 9 to about 15 and about 10 to about 15 years).

3. Milk fat and milk fat fractionation

[0063] Milk fat is discussed comprehensively by Fox and McSweeney (2006), hereby incorporated by reference. In addition to lipids, milk fat includes vitamins, sterols, and minor components. See Chapter 1, Composition and Structure of Bovine Milk Lipids, Fox and McSweeney, for a description of naturally occurring bovine milk fat. Fractionation of milk fat is discussed in the Dairy Processing Handbook, 1995, and by Illingworth, 2002, and by Rombaut et al, 2006(b), all hereby incorporated by reference. Seasonal variation of milk fat is discussed by Fox and McSweeney (2006).

[0064] Examples of milk fat extracts useful according to the invention include cream (typically about 20 to about 40% fat by weight, preferably about 40% fat by weight), butter, ghee, anhydrous milk fat (AMF) (typically produced by phase inversion of cream or dehydration of butter), buttermilk, butter serum, beta serum, hard milk fat extracts, soft milk fat extracts, sphingolipid extracts, milk fat globular membrane extracts, milk fat globular membrane lipid extracts, phospholipid extracts, and complex lipid (lipids that yield 3 or more types of hydrolysis product per molecule) extracts, and combinations thereof, and hydrolysates thereof.

[0065] Buttermilk, butter serum, and beta serum are discussed by Bylund, 1995, Rombaut et al, 2005, Rombaut et al, 2006(a), Rombaut et al, 2006(b), and published international application WO 2006/ 041316, for example, all incorporated herein by reference. Buttermilk is a term used to describe the aqueous liquid phase obtained from traditional butter production using a butter making process which may be a batch (churn) process or a continuous (Fritz) process.

Buttermilk is also a term used to describe the aqueous by-product produced by the cream concentration step of the traditional method of producing AMF from cream. This traditional method involves concentration then phase inversion of cream to produce oil that is further concentrated and polished to produce AMF. Finally, buttermilk is also a term used to describe a combination of the secondary skim and beta serum by-products of a two-serum process for AMF production - see for example, Bylund (Ed., 1995) and published international application WO 2006/041316 (see Figure 2) that describe this process in detail. In that two-serum process, the by-product from the cream concentration step is further separated to produce secondary skim and the by-product from the oil concentration step is further separated to produce beta- serum. In the first two instances, the buttermilk is produced before any phase inversion has occurred. In the third instance, the buttermilk is a combination of secondary skim produced before phase inversion and beta serum produced after phase inversion. Concentration and polishing in these processes is typically achieved by centrifugation. Phase inversion is typically achieved by homogenisation. It should be understood that the source of these dairy lipid extracts may be milk or colostrum or a combination thereof.

[0066] Useful starting materials for fractionation include cream, AMF, butter milk, butter serum, or beta serum, from milk or colostrum or a combination thereof.

[0067] Multistage fractionation of milk fat may be carried out by differential crystallisation. Milk fat fractions are heated to a set temperature and the crystallised or solid ("stearin"— hard fraction) and liquid ("olein" - soft fraction) fractions are separated. Multi-step fractionation refers to re-fractionation in a subsequent step of a product of a previous fractionation step. Successive soft fractions may be produced by fractionating parent soft fractions into soft and hard sub -fractions.

[0068] Other fractionation methods include phase inversion, interesterification, glycerolysis, solvent fractionation (such as with ethanol, water, or acetone, used alone or sequentially), supercritical fractionation (see Astaire, et al, 2003, for example), near critical fractionation (see WO 2004/066744, for example), distillation, centrifugal fractionation, suspension crystallisation, dry crystallisation, fractionation with a modifier (e.g. soaps or emulsifiers), ultra-filtration, micro- filtration, and any process for fractionation of lipid known in the art, and combinations of these methods, all as known in the art.

[0069] In one embodiment, the fractionation method is selected from solvent fractionation of cream, AMF, butter milk, butter serum, or beta serum, using ethanol, water, or acetone, alone or sequentially.

[0070] Lipids present in the compositions of the invention may be fully or partially modified, whether naturally, chemically, enzymatically, or by any other methods known in the art, including, for example, glycosylated, sialylated, esterified, phosphorylated or hydrolysed. Lipid hydrolysates may be prepared using known techniques, including but not limited to acid hydrolysis, base hydrolysis, enzymatic hydrolysis using a lipase, for example as described in Fox and McSweeney ((2006), Chapter 15 by HC Deeth and CH Fitz-Gerald), and microbial fermentation. One method of base hydrolysis includes adding 1 % KOH (in ethanol) and heating for 10 minutes. Hydrolysed material may be neutralised with acetic acid or hydrochloric acid.

[0071] Milk fat globule membrane material may be isolated according to the acidification method of Kanno & Dong-Hyun, 1990, and further fractionated into lipid and protein fractions by the addition of methanol, as described by Kanno et al, 1975. A phospholipid extract may be isolated by extracting the lipid mixture with acetone according to the procedure of Pruthi et al, 1970. Lipid residue may be further enriched in milk fat globule membrane lipids by the selective extraction of non-polar lipids with pentane.

[0072] Fractionation methods useful to produce milk fat extracts useful herein are also described in published international patent applications WO 2006/041316, WO 2007/123424, and WO 2007/ 123425 that are each hereby incorporated herein by reference in their entirety.

[0073] Particularly preferred milk fat extracts useful herein include those described in the examples below and those summarised in Table 1. These extracts may be emulsions or dried, and may be powders, optionally with components including flow aids such as lactose added to improve flowability.

[0074] Values in Table 1 are typical values and were determined as follows. Proteins levels were determined by total nitrogen multiplied by 6.38. Phospholipid levels were determined by 3¹P NMR. Ganglioside levels were determined as described in published international application WO2009/ 020405 incorporated herein by reference in its entirety.

Table 1 - Examples of phospholipid extracts of bovine milk

ND— not determined; <0.01— trace amounts

4. Compositions for ameliorating cognitive decline

[0075] A composition useful herein may be formulated as a food, drink, food additive, drink additive, dietary supplement, nutritional product, medical food, enteral feeding product, meal replacement, cosmeceutical, or pharmaceutical. Appropriate formulations may be prepared by an art skilled worker with regard to that skill and the teaching of this specification.

[0076] In one embodiment, compositions useful herein include any edible consumer product which is able to carry lipid. Examples of suitable edible consumer products include powders, liquids, confectionary products including chocolate, gels, ice creams, reconstituted fruit products, snack bars, food bars, muesli bars, spreads, sauces, dips, dairy products including yoghurts and cheeses, drinks including dairy and non-dairy based drinks (such as milk drinks and yogurt drinks), milk powders, sports supplements including dairy and non-dairy based sports supplements, food additives such as protein sprinkles, and dietary supplement products including daily supplement tablets. Suitable nutraceutical compositions useful herein may be provided in similar forms.

[0077] A pharmaceutical composition useful according to the invention may be formulated with an appropriate pharmaceutically acceptable carrier (including excipients, diluents, auxiliaries, and combinations thereof) selected for oral administration according to standard pharmaceutical practice. For example, a composition useful according to the invention can be administered orally as a powder, liquid, tablet or capsule. Suitable formulations may contain additional agents as required, including emulsifying, antioxidant, flavouring or colouring agents, and may be adapted for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release.

[0078] Capsules can contain any standard pharmaceutically acceptable materials such as gelatin or cellulose. Tablets can be formulated in accordance with conventional procedures by compressing mixtures of the active ingredients with a solid carrier and a lubricant. Examples of solid carriers include starch, sugar and bentonite. Active ingredients can also be administered in a form of a hard shell tablet or a capsule containing a binder, e.g., lactose or mannitol, a conventional filler, and a tabletting agent.

[0079] The compositions useful herein may be used alone or in combination with one or more other agents. The agent may be a food, drink, food additive, drink additive, food component, drink component, dietary supplement, nutritional product, medical food, nutraceutical, medicament or pharmaceutical.

[0080] When used in combination with another agent, the administration of a composition useful herein and the other agent may be simultaneous or sequential. Simultaneous

administration includes the administration of a single dosage form that comprises all components or the administration of separate dosage forms at substantially the same time. Sequential administration includes administration according to different schedules, preferably so that there is an overlap in the periods during which the composition useful herein and other agent are provided.

[0081] It should be understood that additional agents (whether food based or

pharmaceutical agents) may also be employed in a method according to the invention where they are administered separately, simultaneously or sequentially with a composition useful herein.

[0082] As will be appreciated, the dose of the composition administered, the period of administration, and the general administration regime may differ between subjects depending on such variables as the age, sex and/ or general health of a subject. However, by way of general example, the inventors contemplate administration of from about 1 mg to about 1000 mg per kg body weight of a mixed phospholipid composition useful herein is administered per day, preferably about 50 to about 500 mg per kg per day, alternatively about 150 to about 410 mg/kg/ day or about 110 to about 310 mg/kg/ day. In one embodiment, the inventors contemplate administration of from about 1 mg to about 250 mg per kg body weight of a composition useful herein. [0083] It should be appreciated that administration may include a single daily dose or administration of a number of discrete divided doses as may be appropriate.

[0084] Various aspects of the invention will now be illustrated in non-limiting ways by reference to the following examples.

EXAMPLES

EXAMPLE 1 - The effect of milk phospholipids on age-related memory decline and changes to neurons, glial and cerebral capillaries in aged rats

[0085] This example describes the effect of two different milk phospholipid compositions on age-related memory decline and associated changes to neurons, glia and cerebral capillaries in the brains of aged rats.

Material and Methods

[0086] All animal experiments were approved by the University of Auckland Animal Ethics Committee. Milk phospholipids were obtained from Fonterra Co-operative Group Limited, New Zealand. The mixed milk phospholipid referred to below was Extract 6 from Table 1 above. Soybean phosphatidylserine (PS) was SharpPS™ 60p phosphatidylserine from

Persapiens Ltd, Israel.

1. Animal model

[0087] Forty one aged (27-33 months old) and 20 young (2-3 months old) male rats were divided into six treatment groups as follows: a) Aged + blank gel (BG) (n= 11)

b) Aged + S-PS - soy phosphatidylserine (34mg PS/rat/ day, n= 10)

c) Aged + M-PS - milk phosphatidylserine (34mg PS/ rat/ day, n=9)

d) Aged + M-PL - mixed milk phospholipid (31mg PS/rat/ day, n=l l)

e) Young + blank gel (n= 13)

f) Young + no gel feeding (n=7)

2. Feeding regime

[0088] The phospholipid treatments were dissolved in 50mL of 70% ethanol and mixed with 1L of dissolved gelatine/ sucrose mixture at 50°C. The mixture was transferred into 12.5mL ice cube trays and refrigerated at 4 °C. Rats were fed one cube per day over the 8 week course of treatment. The phospholipid profiles of each of the compositions are detailed in Table 2. Table 2: Phospholipid profiles of each treatment composition as a proportion of total phospholipids (% w/w)

Phospholipid S-PS M-PS M-PL

Phosphatidylserine (PS) 83.3 90.9 12.1

Phosphatidylcholine (PC) 0 0 23.2

Phosphatidylethanolamine (PE) 0 2.5 34.8

Phosphatidylinositol (PI) 0 5.4 8.8

Sphingomyelin (SM) 0 0 20.8

3. Behavioural testing

3.1 Novel Object Recognition Test (NORT)

[0089] A NORT test was carried out both before and after the 8 week feeding regime. Rats were allowed to individually spend 10 minutes in the arena prior to testing. Five acquisition trials and 1 testing trial were carried out over 3 days.

[0090] Two trials (3 hours apart) were conducted each day. For the acquisition trials, two identical objects were placed in the middle of the arena ( 5cm apart) and each rat was given 5 minutes to explore each object. For the testing trial, one of the objects (now familiar to the rat) was replaced with a novel object 3 hours after the fifth acquisition trial. The time spent exploring the two objects was recorded using a stopwatch and the discrimination ratio (novel/ familiar object) was analysed to determine familiarity and novelty recognition.

[0091] The difference between the discrimination ratio in pre- and post-supplemented rats was used to quantify the treatment effects of the phospholipid compositions on the progression of memory decline.

3.2 Morris Water Maze Test

[0092] The Morris Water Maze tests were carried out in a quiet room with multiple distal cues surrounding the apparatus (e.g. a white door, a bright yellow bucket and a colourful picture on the wall). A white circular pool (0.6m x 1.3m) was filled with 20-22°C water and a submerged white platform ( 0cm x 0cm) was located in the north-east quadrant of the pool (20cm from the sidewall).

[0093] Four acquisition and one probe trial were carried out over 4 days. For the acquisition trials, rats were placed in the pool at a random starting position (consistent for each trial) with their head pointed towards the sidewall. Each rat was given 90 seconds to locate and mount the hidden platform. Once located, each rat remained on the platform for 5 seconds before being removed from the pool. The latency, swimming speed and total distance travelled to locate the platform was recorded and analysed with automated tracking software (ANY-maze, v4.2, Stoelting, USA). Rats that were unable to locate the platform within 90 seconds were guided to the platform. Acquisition trials were carried out at 6 minute intervals.

[0094] For the probe trial, the platform was removed from the pool and the rat was allowed to spend 30 seconds in the pool searching for the absent platform before being removed from the pool. The latency to first entry, the time spent in, and the number of entries in the northeastern quadrant of the pool were recorded and analysed to measure retention of spatial memory. Rats with improved spatial memory should make earlier and more entries into the NE quadrant and spend more time in the NE quadrant during the 30 second trial. The probe trial was carried out 2h after the last acquisition trial on day 4.

3.3 Elevated Plus Maze Test

[0095] An elevated maze apparatus consisting of two open arms (flat platform, 50cm x 10cm) and two enclosed arms (flat platform enclosed by walls, 50cm x 0cm x 40cm) perpendicularly intersecting to form a central platform, was set-up. The maze was elevated 50cm above the floor. The rat was placed on the central platform, facing an open arm, and allowed to explore the maze freely for 10 minutes.

[0096] Anxiety-like behaviour was measured by the number of entries to and the time spent in the enclosed arms, and the average time spent in the open arms. Arm entry was defined as all four paws being inside the arm.

4. Tissue collection

[0097] Rats were deeply anaesthetised with pentobarbital (125mg/kg intraperitoneally) and blood samples collected via cardiac puncture. The rats were transcardiacally perfused with normal saline until the outflow from the heart ran clear.

[0098] The brains were excised and separated into the two hemispheres. The left hemisphere was frozen with dry ice and stored at -80°C before being analysed for

phosphatidylserine content. The right hemisphere was fixed in situ with 4% paraformaldehyde for subsequent histological analysis. 4.1 Brain tissue preparation

[0099] The right hemispheres were fixed for at least 48 hours, then transferred into 25% sucrose and stored at 4°C until they sank in solution. Sequential coronal sections (25μτη) were collected with every twelfth section pooled together. The sections from each sample pool were used for one parameter of staining, which was performed simultaneously across all age and treatment groups.

4.2 Histological and Immunohistological staining

[00100] Primary antibodies against Glial Fibrillary Acidic Protein (GFAP), Tyrosine Hydroxylase (TH) and Rat Endothelial Cell Antigen (RECA)-1 were used to mark astrocytes, dopamine terminals and cerebral capillaries, respectively.

[00101] The sections were pretreated with 1% H₂0₂ in 50% methanol for 30 minutes to quench any endogenous peroxidase activity, and then blocked with 1.5% horse serum/PBS at room temperature. The sections were then incubated at 4°C for 48 hours with monoclonal antibodies against each of the proteins as detailed in Table 3.

Table 3: Primary antibodies used for immunohistochemical staining

Primary Ab Details and concentration

Anti-GFAP Mouse (Sigma) - 1 :10000

Anti-TH Rabbit (Protos Biotech Corporation) - 1: 000

Anti-RECA- Mouse (AbCam) - 1 :10000

[00102] Sections were then incubated with either biotinylated horse anti-mouse or goat anti- rabbit secondary antibodies (Sigma) at a 1 :500 concentration at 4°C overnight. ExtrAvidin (Sigma, 1:500) was applied for 3 hours at room temperature, followed was 0.05% DAB to produce a brown reaction product.

[00103] For specific visualisation of reactive microglia, one set of sections was incubated overnight at 4°C with isolectin B-4 (Sigma) at a 1:5000 concentration with Tris-buffered saline before developing with DAB. The stained sections were then mounted on gelatine-coated microscope slides, dehydrated in alcohol and xylene and coverslipped. [00104] For histological assessment, the sections were mounted on gelatine-coated slides, stained with Thionin for 2 minutes, dehydrated through graded alcohol and xylene and coverslipped.

[00105] The average density of the TH terminal staining in the striatum and the isolectin B-4 in the hippocampus was observed under light microscope (Nikon 800) and images captured using image analysis software (SigmaScan Pro 5.0, SPSS, USA) at 4x magnification. The total number of neurons with nuclei was counted in the CA-3 sub-regions of the hippocampus at lOx magnification. The volume of the cerebral cortex was also measured.

[00106] Images of GFAP -positive astrocytes and REC A- 1 -positive capillary vessels were acquired from the hippocampus, thalamus and frontal cortex at 1 Ox magnification using NIS elements F, Nikon imaging software. The average number of astrocytes and the total length of cerebral capillaries in these brain regions were measured using Metamorph v 6.2.6 image analysis software (Molecular Devices) and a high throughput image analysis assay. The

AddFIattenBgdAngiogenesis assay opens each captured image, performs a Flatten Background transformation to even the intensity of immuno staining across the image, and performs

Metamorph Angiogenesis Tube Formation application to each image. The number of astrocytes and total tubule length is measured. At least 8 sequential sections were used for measurements and the averages of two images used for data analysis.

5. Data analysis

[00107] Data collected from the acquisition trials of the Morris Water Maze and NORT tests and body weights were analysed using two-way ANOVA. The data collected from the probe trials, the Elevated Plus Maze test and the histological and immunohistochemical staining were analysed using one-way ANOVA. The aged— blank gel group was used as the control for both the age and treatment effects of the phospholipid compositions tested. The data were presented as mean ± SEM.

Results

1. Body weight and mortality

[00108] Body weight varied significandy between the groups, but not across the time points. Compared the aged + blank gel group, a significant loss of body weight in the aged groups treated with the two dairy-based phospholipid compositions was observed. [00109] The body weight of the young + blank gel control group increased, and was well maintained in the aged + blank gel control group.

[00110] No significant difference in age related mortality was observed between the different aged groups. No mortality was observed in the young control groups, while 5 rats died or were euthanized prior to initiation of the feeding regime.

2. Novel Object Recognition Test

[00111] A significant difference in novelty recognition was observed between pre- and post- treatment (p < 0.0001). Compared to pre-treatment, novelty recognition was significantly reduced in groups fed with either S-PS or M-PL after 8 weeks of treatment, indicating a decline in novelty recognition with age.

3. Mortis Water Maze Test

[00112] The latency to the hidden platform was significantly different between each of the treatment groups and across the training days. Post-hoc analysis shows that the latency to the platform was significandy reduced in young control rats compared to aged control rats, indicating a clear age-related deficit in spatial learning.

[00113] The latency to the hidden platform was significantly increased in aged + M-PS (p < 0.05) and aged + M-PL (p < 0.01) groups. There was no significant difference between then aged + S-PS and the aged + blank gel groups.

[00114] The total distance travelled to the platform was significandy different between each of the test groups and the training days. The distance travelled to the platform was significantly reduced in the young + blank gel group compared to the aged + blank gel group on days 2, 3 and 4. In contrast, the distance travelled to locate the platform on day 3 of the acquisition trial was significandy longer in the aged + M-PS (p < 0.01) and aged + M-PL (p < 0.01) groups compared to the aged + blank gel control group.

[00115] A significant difference in swimming speeds was observed between the aged and young groups. The young + blank gel control group swam faster compared to the aged + blank gel group (p < 0.01). There was no significant difference in swimming speed between the aged groups.

[00116] Compared to the aged + blank gel group, the young + blank gel group made more entries to ( p < 0.05) and spent more time swimming in the NE quadrant (p < 0.05) with a reduced latency to the NE quadrant (p < 0.05). No significant difference was observed between the aged groups.

4. Elevated Plus Maze Test

[00117] A modest treatment effect was observed in aged rats treated with M-PL, with this group spending more time in the open arms/per entry compared to the aged + blank gel.

Neither age nor treatment altered the time spent in or the number of entries to the open arms.

5. Histological and immunohistochemical staining

[00118] No age- or treatment-related alterations in cortical volume and number of CA3 neurons were observed.

[00119] Compared to the young + blank gel (BG) group, the aged + blank gel control group showed a significant reduction in TH density in the striatum (p < 0.001), indicating age-related dopamine depletion in the nigro-striatal pathway. A modest treatment effect was seen in the aged group treated with M-PL (p < 0.05) compared to the aged + blank gel control group (Figure 1), suggesting a positive treatment effect of dairy-based phospholipid compositions on preventing dopamine depletion and/ or restoring dopamine neurotransmission.

[00120] The average density of the microglia was significantly reduced in both the hippocampus and the frontal cortex of the aged + blank gel control group compared to the young + blank gel control group (p < 0.0001). The aged rats treated with M-PL showed an increase in reactive microglia in the hippocampus but not in the frontal cortex compared to the aged + blank gel control group (p < 0.0001), as shown in Figure 2.

[00121] An age, but not treatment effect was observed on GFAP-positive astrocyte measurements (p < 0.001). Compared to young + blank gel control rats, GFAP-positive astrocytes were significandy reduced in all the sub-regions of the hippocampus of the aged + blank gel control group. No significant difference was observed between the aged rats either treated or not treated with phospholipid compositions.

[00122] Capillary density was reduced in aged rats compared to young rats, with clear morphological damage observed in the aged rats. A significant reduction in the total length of the capillaries in the aged + blank gel control rats examined in the CAl-2, CA3 and CA4 sub- regions of the hippocampus, the cerebral cortex and the thalamus was observed. No difference was observed between the aged groups treated or untreated with phospholipid compositions. Discussion

[00123] The results of this study suggest an age-related deficit in both learning and memory. Neither dairy nor soy-based phosphatidylserine improved the age-related deficits in learning and memory compared to the aged controls.

[00124] The immunoreactivity of both astrocytes and microglia were significantly reduced with age in the brain regions associated with memory. Treatment with M-PL was shown to increase glial cell density, indicating a glial cell protective.

EXAMPLE 2 - The effect of dairy phospholipid on cognitive decline in aged tats.

Materials and methods

1. Animal model

[00125] All animal experiments were approved by the Animal Ethics Committee at the University of Auckland. Aged (22-23 months old) and young (3 months old) male rats

(Fisher/Norway Brown hybrid) were purchased from Harlan, USA. The aged rats were randomly divided into treatment groups (Table 4). Experiments were initiated at 23-24 months of age for aged rats and at 4-5 months of age for young adult rats immediately. The duration of the experiments was 27 weeks and the rats were 30-31 months of age for aged groups and 11-12 months of age for young adult group at the end of the experiment.

Table 4: Experimental groups

2. Diets

[00126] Diets were formulated using flavoured gelatine ( 0%) containing sucrose ( 0%). The phospholipid was Soybean PS (SPS, 34mg/ rat/ day, SharpPS™ 60p, Persapiens Ltd, Israel) or DPL, a mixture of phospholipid similar (total 255mg/rat/day; Fraction 6 from Table 1, Fonterra Co-operative Group Limited, New Zealand). Briefly, the phospholipid was dissolved in water using a food processor and mixed with 1L dissolved gelatine/ sucrose mixture at 50°C. The mixture was then transferred into ice cube trays and firmed at 4°C. Each cube contained 12.5ml gelatine with or without phospholipid and each rat was fed with one cube per day. The diet phospholipid profiles are shown in Table 5.

Table 5 Phospholipid diet profiles (mg/ rat/ day)

Soy-PS I Mixed Dairy PL

Sphingomyelin (SM) 32

3. Experimental Procedure

[00127] Open field test (OFT) and Novel Object Recognition Test (NORT) were carried out in all rats prior to supplementation. Rats in each of the supplementing groups were fed with either gelatine-formulated phospholipid or blank gelatin (BG) as the control, daily for 4 months. A group of young adult rats were also fed with BG (Young+BG) and used to indicate any age- related difference by comparing to the group of aged+BG. OFT, NORT, dark-light boxes test, Elevated Zero Maze (EZM) and Morris Water Maze (MWM) tests were then performed after the supplementation trials completed. The body weights of the rats were measured weekly throughout the experiments. Rats were killed for tissue collection immediately after the MWM tests.

4. Behavioural tests

(a) Open field tests (OFT)

[00128] The tests were carried out before and after the 4 months supplementation trials. This one day test was carried out in a wooden box with an open top (30 x 60 x 60cm). Rat was allowed to move within the arena freely for 30 min. The distance travelled, the resting time and the time spent in grooming were recorded and analyzed for locomotor function.

(b) Novel Object Recognition Tests (NORT)

[00129] Two NORT tests were carried out both before and after the 4 months of daily supplementation. The testing arena is a wooden box with an open top (30 x 60 x 60cm), the same arena used for OFT. Three acquisition trials and 2 testing trial were carried out in the next 4 days of testing, with 2 trials, 3 hours apart on each testing day. For the acquisition trials, two identical objects were placed in the middle of the arena, side by side 5 cm apart and each rat was allowed to spend 5 min exploring both objects. For testing trial, one of the familiar objects was replaced with a novel object either 4 hours or 24 hours after the last acquisition trial. The time spent on exploring the familiar and novel objects was recorded using a stopwatch and the discrimination ratio novel/ familiar + novel objects was analyzed to determine familiarity and novelty recognition. The difference of the discrimination ratio between the pre- and post- supplementation was used to quantify the treatment effects on the progression of memory decline.

(c) Morris Water Maze test

[00130] Apparatus: The MWM tests were carried out in a quiet room with multiple distal cues around the testing apparatus (e.g. a white door, a bright yellow triangle, a squire red sign and a colourful picture on the wall). A white circular pool (0.6 x 2.2m in diameter) was filled with water (maintained at 20-22oC) and a submerged transparent platform ( 0cm in diameter and 2cm below water) was located in the middle of north-east (NE) quadrant. The 1 cup of milk powder was dissolved in the water to make pool background colour evenly white. The water was refilled every second day.

[00131] Acquisition testing: Rats were given 4 trials on each testing day for 4 days, with 0 min intervals between trials. The starting position of test was randomized over each testing day and each testing trials, which was remained the same for all rats. The platform was remained in the northeast (NE) quadrant throughout the tests. Each rat was put on the platform for 0 second to learn the presence of the platform prior to the first acquisition trials. During each trial, the rat was placed in the water with its head pointed towards the sidewall and allowed to swim for 20 sec to locate and mount the hidden platform. The rat was remained on the platform for 10 sec before being removed. The latency, total distance travelled and swimming speed taken to reach the platform was recorded and analysed with automated tracking software (ANY-maze, v4.2, Stoelting, USA). Rats that were unable to locate the platform within 90 sec were guided to the platform.

[00132] Probe trial: Two probe trials were used to examine spatial memory at 24 hours and 72 hours after the last acquisition trial on day 5 and 7. In the probe trial, the platform was removed from the pool and the rat was allowed to spend 30 sec in the pool searching for the absent platform before being removed from the pool. The number of entries to the platform zone and the initial heading errors from the platform zoon were recorded and analyzed for the retention of spatial memory. Rats with improved spatial memory should make more entries to the platform zoon with smaller heading errors to the target during 30 sec probe trial.

(d) Elevated Maze

[00133] The procedure for measuring basal anxiety-like behaviour with the Elevated Maze has been described previously (Jacobson, J., et al., Co-relation of cellular changes and spatial memory during aging in rats Experimental Gerontology 2008. 43: p. 928-938.). The zero shaped apparatus ( m in diameter) divided into 4 equal proportion, two open platforms ( 0cm wide) are between two enclosed (by walls 30cm high) tunnels. The zero maze was elevated 50 cm above the floor. The rat was placed on one central platform and allowed to explore the maze freely for 5 minutes. The definition of an arm entry was when all four paws were inside the arm. As more anxious rats are predicted to spend more time in the enclosed arms, the number of entries to and the time spent in the open arms were recorded and used as a measure of anxiety behaviour.

(e) Dark-Light box tests:

[00134] This is other form test for anxiety-like behaviour. The testing arena is made of transparent plastic box (60x60x60cm) with open top and its half was occupied by an enclosed black box (30x60x40cm) with a small door ( 0x8cm) open to the light area of the arena. During the test the rat was put into the light-open area facing away from the dark box and allowed to explore both areas freely for 5 min. The time spent in and the number of entry to the light area were recorded and analysed for anxiety like behaviour

5. Tissue collection

[00135] Rats were deeply anesthetized with pentobarbital (125mg/kg, i.p.). The rats were transcardially perfused with normal saline till the outflow from heart ran clear. The brains were separated into two hemispheres, the left hemisphere was frozen on dry ice immediately and stored in— 80°C before being used for analyzing protein and RNA profiles. The right hemisphere was fixed in situ with 4% paraformaldehyde for histological analysis. Bone, liver, small intestine and spleen samples were collected.

[00136] Brain tissue preparation: The right hemispheres were fixed for at least 48 hours, then transferred into 25% sucrose and kept at 4°C till they sank. Sequential coronal sections (25um) were collected with every 12 sections pooled together (e.g. 1, 13, 25, 37 and so on). The sections from each sample pool were used for one parameter of staining, which was performed simultaneously across all age/ treatment groups.

Results

1. General wellbeing

[00137] During 22 weeks of experiments the body weight of the young control rats gradually increased from 400g to 490g and the body weight of the aged rats were stable and maintained between 500-550g with a slight increase in body weight during the supplementation period and a slight weight loss during the period of behavioural tests. 2. Novel object recognition tests (NORT):

The novel recognition tests were carried out both before and after the 4 month supplementation. The ability for novelty recognition was examined both 4h and 24h after the acquisition phase. There was no difference between groups.

3. Open field tests

[00138] The young adult rats travelled significantly more compared to the aged rats fed with blank gel (p<0.05, n=10-12) suggesting the aged rats were less active. The general activity was similar in all aged groups before supplementation. Age did not alter grooming activity and it was similar in all aged groups examined before supplementation. The same test was repeated after 4months period of supplementation. In general both total distance travelled and grooming activity were reduced after supplementation in all groups, particularly in the young rats. The general activity was similar between the aged groups fed with different supplementation regimes. The grooming activity after supplementation was similar between the young and aged groups fed with BG. However, a strong trend of increase in grooming activity was found in the aged rats fed with DPL compared to the aged rats fed with BG (p=0.07, n=9-10).

4. Elevated maze test

[00139] The data showed a significant age effect in both parameters measured, but there was no difference between the aged groups fed with phospholipid and blank-gel.

5. Dark/light boxes test

[00140] The data also showed an increase in anxiety in the aged rats and there was no difference between the aged groups.

6. MWM test

[00141] As shown in Figure 3, the number of entries to the platform zoon was examined for spatial memory 24h after the acquisition. The memory was significantly reduced in the aged control rats (BG) compared to the young controls (p<0.05). There was a strong trend towards improved memory in the aged rats fed with DPL compared with the aged controls (#p=0.06).

[00142] As shown in Figure 4, the number of entries to the platform zone was examined for spatial memory 72h after acquisition trials. The memory decline in aged control rats was not significant compared to the young controls. There was trend towards improved memory in the aged rats fed with DPL compared with the aged controls. While the young adult rats and the aged rats fed with DPL made > 1 entries to the platform zone the aged rats fed with SPS or control gel made < 1 entry to the platform zone.

[00143] As shown in Figure 5, the initial heading error to the platform zone was also examined for spatial memory 72h after the acquisition trials. The aged controls rats made significant more errors compared to the young controls (**p<0.001). The aged rats fed with DPL made less errors compared the aged controls (*p=0.05).

7. Histology

[00144] The right hemispheres were used for histological analysis.

(a) Microglial cells

[00145] Microglial cells were visualised using IB4 staining and evaluated by measuring the density of IB4 staining in the CA4 sub-regions of the hippocampus. The density of IB4 staining was similar between the aged and young rats fed with BG (data not shown). The density of microglial cells was also similar between the aged rats with and without supplementation (data not shown).

(b) Capillaries

[00146] The vascular density was also examined using immunohistochemical staining of RECA- and evaluated using the same image analyser. The total length of capillaries was measured for demonstrating the changes in vascular degeneration and remodelling. Vascular density in the hippocampus was similar between two aged control groups, as well as the aged rats with and without supplementation (data not shown).

8. Changes in dopamine

[00147] As shown in Figure 6, compared to the aged rats fed with BG, the dopamine expression in the striatum was higher in the young adult rats (p<0.05). The aged rats with DPL supplementation has a small, but significant increase in dopamine expression compared to the rats fed with BG (p<0.05).

9. Plasticity in the hippocampus

[00148] As shown in Figure 7, compared to the aged rats control rats (Aged+BG) the aged rats with DPL supplementation significantly elevated the synaptic density in the CA3 sub-region (*p<0.05), indicating increased neuronal plasticity in the CA3 sub-regions of the hippocampus. INDUSTRIAL APPLICATION

[00149] The present invention has utility in ameliorating cognitive decline. The described compositions may be employed as foods, drinks, food additives, drink additives, dietary supplements, nutritional products, medical foods, nutraceuticals, medicaments or

pharmaceuticals .

[00150] Those persons skilled in the art will understand that the above description is provided by way of illustration only and that the invention is not limited thereto.

REFERENCES

Ajmone-Cat, M.A. et al "Effects of phosphatidylserine on p38 mitogen activated protein kinase, cyclic AMP responding element binding protein and nuclear factor kappaB activation in resting and activated microglial cells" J Neurochem (2003) 84: 413-416

Astaire J. C, Ward R., German J. B., and Jimenez -Flores R. (2003) Concentration of Polar MFGM Lipids from Buttermilk by Microfiltration and Supercritical Fluid Extraction J. Dairy Sci. 86, 2297-2307

Bylund, G. (Ed.) Dairy processing handbook. 1995 Tetra Pak Processing Systems AB, S-221 86 Lund, Sweden.

Fox PF, McSweeney PLH (eds), Advanced Dairy Chemistry, Volume 2 - Lipids, 3rd Ed, Springer Science + Business Media, Inc., 2006.

Freireich EJ, Gehan EA, Rail DP, Schmidt LH, Skipper HE (1966) Quantitative comparison of toxicity to anticancer agents in mouse, rat, hamster, dog, monkey and man. Cancer Chemother Rep 50: 219—244.

Hashioka, S. et al "Phosphatidlyserine and phosphatidylcholine-containing liposomes inhibit amyloid beta and interferon-gamma-induced microglial activation" Free Radic Biol Med (2007) 42: 945-954.

Illingworth, D., Fractionation of fats. In Physical Properties of Lipids (Marangoni A G & Narine S S, Eds), pp. 411- 448. Marcel Dekker, New York (2002).

Kanno C & Dong-Hyun K (1990). A simple procedure for the preparation of bovine milk fat globule membrane and a comparison of its composition, enzymatic activity, and electrophoretic properties with these prepared by other methods. Agric. Biol. Chem., 54(l l):2845-2854.

Kanno C, Shimizu M & Yamachi K (1975). Isolation and physiochemical properties of a soluble glycoprotein fraction of milk fat globule membrane. Agric. Biol. Chem., 39(9):1835-1842.

Pruthi T D, Narayanan K M & Bhaleerao V R (1970). The role of milk phospholipids in the autoxidation of butterfat— I. Indian Journal of Dairy Science, 23:248-251.

Rombaut R, Camp JV, Dewettinck K., Analysis of phospho- and sphingolipids in dairy products by a new HPLC method. J Dairy Sci. (2005) 88(2):482-8.

Rombaut R, Dejonckheere V, Dewettinck K., Microfiltration of butter serum upon casein micelle destabilization. J Dairy Sci. (2006) (a) 89(6):1915-25.

Rombaut R., Van Camp J. & Dewettinck K., Phospho- and sphingolipid distribution during processing of milk, butter and whey, International Journal of Food Science & Technology, (2006)(b) 41 (4):435-443.

Tweedy, J. R. and Garcia, C.A. "Lecithin treatment of cognitively impaired Parkinson's patients" Eur J Clin Invest. (1982) 12: 87-90.

Wehrmuller, K. "Impact of dietary phospholipids on human health" Agroscope Liebefekd-Posieux Science Summary (2008) No. 524

Wells, K. et al "Neural membrane phospholipids in Alzheimer disease" Neurochem Res. (1995) 20: 1329-1333.

Claims

WHAT I CLAIM IS . A method of ameliorating cognitive decline or a related condition, the method comprising oral administration to a subject in need thereof of an effective amount of one or more mixed phospholipid compositions. 2. A method of claim 1 wherein the mixed phospholipid compositions are selected from (1) a combination of phosphatidylserine and one or more or two or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and sphingomyelin, (2) a combination of phosphatidylserine, phosphatidylcholine and one or more or two or more of phosphatidylethanolamine, phosphatidylinositol and sphingomyelin, (3) a combination of phosphatidylethanolamine and one or more or two or more of phosphatidylserine, phosphatidylcholine, phosphatidylinositol and sphingomyelin, (4) a combination of phosphatidylinositol and one or more or two or more of phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, (5) a combination of sphingomyelin and one or more or two or more of phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, (6) a hydrolysate of any one or more of (1) to (5), and (7) any combination of any two or more of (1) to (6). 3. A method of claim 1 wherein the mixed phospholipid compositions is one or more milk phospholipid extracts or a hydrolysate thereof. 4. A method of claim 1, 2 or 3 wherein the subject has (1) no symptoms of cognitive decline, or (2) no clinical symptoms of cognitive decline, or (3) an age of 25 to 55 years. 5. A method of any one of claims 1 to 4 wherein ameliorating cognitive decline includes maintaining or increasing maintaining or improving cognition, maintaining or improving memory, supporting cognitive function, reducing age-related memory impairment, reducing the risk of memory loss as a result of ageing, short term memory, maintaining or increasing memory, maintaining or increasing long term memory, maintaining or increasing ability to learn, maintaining or increasing ability to think, maintaining or increasing ability to speak, maintaining or increasing ability to write, maintaining or increasing attention, maintaining or increasing language skills, maintaining or increasing problem solving skills, reducing or preventing anxiety, reducing or preventing mood swings, reducing or preventing depression, reducing or preventing mental disorders, reducing or preventing age-related mental disorders, or maintaining or increasing social activity, or any combination of any two or more thereof. 6. A method of any one of claims 1 to 5 wherein the phospholipid composition is selected from buttermilk, one or more buttermilk extracts, butter serum, one or more butter serum extracts, beta serum, low-lactose beta serum, one or more beta serum extracts, one or more sphingolipid extracts, one or more milk fat globule membrane lipid extracts, one or more phospholipid extracts, one or more complex lipid extracts and any combination of any two or more thereof. 7. A method of any one of claims 1 to 6 wherein the phospholipid composition comprises about 5 to about 95%w/w phospholipid). 8. A method of any one of claims 1 to 7 wherein the phospholipid composition comprises at least about 0.1% w/w of two or more, three or more, or four or more phospholipids selected independently from phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phosphatidylserine, and phosphatidylinositol. 9. A method of any one of claims 1 to 8 wherein the phospholipid composition comprises

(1) about 25 to about 35% w/w protein, about 5 to about 25% w/w lipid, and about 5 to about 12% w/w phospholipid, or

(2) about 25 to about 35% w/w protein, about 5 to about 25% w/w lipid, about 5 to about 12% w/w phospholipid, about 5 to about 10% w/w MFGM (milk fat globule membrane) protein, and about 0.2 to about 0.9% w/w ganglioside, or

(3) about 25 to about 35% w/w protein, about 5 to about 25% w/w lipid, about 5 to about 12% w/w phospholipid, about 1 to about 5% w/w phosphatidylcholine, about 1.5 to about 6% w/w phosphatidylethanolamine, about 1 to about 5% w/w sphingomyelin, about 0.5 to about 2% w/w phosphatidylserine, about 0.1 to 2% w/w phosphatidylinositol, about 5 to about 10% w/w MFGM protein, and about 0.2 to about 0.9% w/w ganglioside, or

(4) about 20 to about 50% w/w protein, about 5 to about 25% w/w lipid, and about 1 to about 5% w/w phospholipid, or

(5) about 20 to about 50% w/w protein, about 5 to about 25% w/w lipid, and about 1 to about 5% w/w phospholipid, and about 1 to about 0% w/w MFGM protein, or

(6) about 25 to about 35% w/w protein, about 10 to about 20% w/w lipid, about 4 to about 12% w/w phospholipid, about 1 to about 3% w/w phosphatidylcholine, about 1 to about 4% w/w phosphatidylethanolamine, about 0.5 to about 3% w/w sphingomyelin, about 0.5 to about

1.5% w/w phosphatidylserine, about 0.1 to 1% w/w phosphatidylinositol, about 1 to about 10% w/w MFGM protein, or

(7) about 40 to about 60% w/w protein, about 25 to about 45% w/w lipid, and about 10 to about 25% w/w phospholipid, or

(8) about 40 to about 60% w/w protein, about 25 to about 45% w/w lipid, about 10 to about 25% w/w phospholipid, about 5 to about 20% w/w MFGM protein, and about 0.5 to about 2.0% w/w ganglioside, or

(9) about 46 to about 52% w/ w protein, about 28 to about 40% w/ w lipid, about 11 to about 16% w/w phospholipid, about 2 to about 6% w/w phosphatidylcholine, about 3 to about 8% w/w phosphatidylethanolamine, about 2.5 to about 7% w/w sphingomyelin, about 0.5 to about 3% w/w phosphatidylserine, about 0.5 to 2% w/w phosphatidylinositol, about 5 to about 15% w/w MFGM protein, and about 0.5 to about 0.9% w/w ganglioside, or

(10) about 50 to about 70% w/w protein, about 12 to about 32% w/w lipid, and about 5 to about 25% w/w phospholipid, or

(12) about 56 to about 65% w/w protein, about 18 to about 28 % w/w lipid, about 8 to about 20% w/w phospholipid, about 2 to about 8 % w/w phosphatidylcholine, about 2 to about 10% w/w phosphatidylemanolamine, about 2 to about 8% w/w sphingomyelin, and about 1 to about 3% w/w phosphatidylserine, and about 0.5 to 3% w/w phosphatidylinositol, about 10 to about 20% w/w MFGM protein, and about 0.5 to about 2.5% w/w ganglioside, or

(14) about 0 to about 10% w/w protein, about 85 to about 97% w/w lipid, about 25 to about 35% w/w phospholipid, about 5 to about 10 % w/w phosphatidylcholine, about 7 to about 13% w/w phosphatidylethanolamine, about 4 to about 9% w/w sphingomyelin, about 2 to about 5% w/w phosphatidylserine, about 1 to about 3% w/w phosphatidylinositol, about 0 to about 5 % w/w MFGM protein, and about 1 to about 3% w/w gangliosides, or

(15) less than about 1% w/w protein, about 80 to about 95% w/w lipid, and about 60 to about 80% w/w phospholipid, or

(16) less than about 1% w/w protein, about 80 to about 95% w/w lipid, and about 60 to about 80% w/w phospholipid and about 1 to about 5% w/w gangliosides, or

(17) less than about 1% w/w protein, about 80 to about 95% w/w lipid, about 60 to about 80% w/w phospholipid, about 10 to about 20 % w/w phosphatidylcholine, about 18 to about 28% w/w phosphatidylethanolamine, about 10 to about 20% w/w sphingomyelin, about 4 to about 12% w/w phosphatidylserine, about 2 to about 10% w/w phosphatidylinositol, and about

1 to about 5% w/w gangliosides, or

(18) about 75 to about 99% w/w lipid and about 15 to 35% w/w phospholipid, or

(19) about 80 to about 90% w/w lipid, about 5 to about 15% w/w phosphatidylcholine, about 5 to about 15% w/w phosphatidylethanolamine, about 4 to about 10% w/w

sphingomyelin, and about 0.1 to about 2% w/w phosphatidylserine, or

(20) about 80 to about 90% w/w lipid, about 20 to 30% w/w phospholipid, about 5 to about 15% w/w phosphatidylcholine, about 5 to about 15% w/w phosphatidylethanolamine, about 5 to about 10% w/w sphingomyelin, about 0.5 to about 1.5% w/w phosphatidylserine, and about 0.1 to about 1.2% w/w phosphatidylinositol, or

(22) about 80 to about 90% w/w lipid, about 10 to about 30% w/w phosphatidylcholine, about 12 to about 22% w/w phosphatidylethanolamine, about 12 to about 22% w/w

sphingomyelin, and about 1 to about 3% w/w phosphatidylserine, or

(23) about 75 to about 95% w/w lipid, about 50 to about 90% w/w phospholipid, about 10 to about 45% w/w phosphatidylcholine, about 12 to about 25% w/w phosphatidylethanolamine, about 12 to about 25% w/w sphingomyelin, about 1 to about 6% w/w phosphatidylserine, and about 0.5 to 4% w/w phosphatidylinositol, or

(24) about 80 to about 90% w/w lipid, about 65 to about 75% w/w phospholipid, about 10 to about 30% w/w phosphatidylcholine, about 12 to about 22% w/w phosphatidylethanolamine, about 12 to about 22% w/w sphingomyelin, about 1 to about 3% w/w phosphatidylserine, and about 0.5 to 3% w/w phosphatidylinositol, or

(27) about 25 to about 45% w/w lipid, about 10 to about 30% w/w phospholipids, about 2 to about 5% w/w phosphatidylcholine, about 3 to about 7% w/w phosphatidylethanolamine, about

2 to about 5% w/w sphingomyelin, about 2 to about 12% w/w phosphatidylserine, about 1 to about 5% w/w phosphatidylinositol, and about 0.2 to about 1% w/w ganglioside, or

(30) about 20 to about 40% w/w lipid, about 5 to about 30% w/w phospholipids, about 1 to about 5% w/w phosphatidylcholine, about 2 to about 8% w/w phosphatidylethanolamine, about 0.5 to about 5% w/w sphingomyelin, about 1 to about 10% w/w phosphatidylserine, about 1 to about 6% w/w phosphatidylinositol, and about 0.8 to about 3.5% w/w ganglioside. 0. A method of any one of claims 1 to 9 wherein the method further comprises separate, simultaneous or sequential administration of one or more additional agents for ameliorating cognitive decline.

11. A method of any one of claims 1 to 0 wherein the related condition is selected from dementia, young-onset dementia, degenerative dementia, Alzheimer's disease, vascular disease, fronto temp oral lobar degeneration (FTLD), dementia with Lewy bodies, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, cognitive impairment associated with schizophrenia, chemotherapy-induced neuropathy, Down's syndrome, Korsakoff s disease, cerebral palsy, epilepsy, neuronal ischemia, neuronal reperfusion injury, neuronal trauma, neuronal haemorrhage, neuronal infection, stroke, neuronal exposure to a toxic substance, age- related mental disorders, anxiety disorders, age-related depression, dementia associated with microvascular disorders (such as diabetes, hypotension, stroke induced vascular dementia, and obesity), dementia associated with a disorder of the immune system, dementia associated with a central nervous system (CNS) disorder, dementia associated with hypotension, dementia associated with obesity, vascular dementia (including post stroke).

12. Use of one or more mixed phospholipid compositions in the manufacture of an oral formulation for ameliorating cognitive decline or a related condition.

13. A use of claim 12 wherein the mixed phospholipid composition is selected from

(1) a combination of phosphatidylserine and one or more or two or more of

(3) a combination of phosphatidylethanolamine and one or more or two or more of phosphatidylserine, phosphatidylcholine, phosphatidylinositol and sphingomyelin, (4) a combination of phosphatidylinositol and one or more or two or more of phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine and sphingomyelin,

(6) a hydrolysate of any one or more of (1) to (5), and

(7) any combination of any two or more of (1) to (6).

14. A use of claim 12 wherein the mixed phospholipid compositions is comprises one or more milk phospholipid extracts or a hydrolysate thereof.

15. A use of claim 12, 13, or 14 wherein the subject has

(1) no symptoms of cognitive decline, or

(2) no clinical symptoms of cognitive decline, or

(3) an age of 25 to 55 years.

16. A use of any one of claims 12 to 15 wherein ameliorating cognitive decline includes maintaining or increasing maintaining or improving cognition, maintaining or improving memory, supporting cognitive function, reducing age-related memory impairment, reducing the risk of memory loss as a result of ageing, short term memory, maintaining or increasing memory, maintaining or increasing long term memory, maintaining or increasing ability to learn, maintaining or increasing ability to think, maintaining or increasing ability to speak, maintaining or increasing ability to write, maintaining or increasing attention, maintaining or increasing language skills, maintaining or increasing problem solving skills, reducing or preventing anxiety, reducing or preventing mood swings, reducing or preventing depression, reducing or preventing mental disorders, reducing or preventing age-related mental disorders, or maintaining or increasing social activity, or any combination of any two or more thereof.

17. A use of any one of claims 12 to 16 wherein the phospholipid composition comprises one or more phosphatidylethanolamines, one or more phosphatidylinositols, one or more phosphatidylserines, one or more phosphatidylcholines, one or more sphingolipids (including one or more sphingomyelins, one or more dihydrosphingomyelins, one or more ceramides, one or more cerebrosides, or one or more gangliosides, or any combination of any two or more thereof), one or more lysophospholipids (phospholipids with one fatty acid lost), or any combination of any two or more thereof.

8. A use of any one of claims 12 to 7 wherein the phospholipid composition is selected from buttermilk, one or more buttermilk extracts, butter serum, one or more butter serum extracts, beta serum, low-lactose beta serum, one or more beta serum extracts, one or more sphingolipid extracts, one or more milk fat globule membrane lipid extracts, one or more phospholipid extracts, one or more complex lipid extracts and any combination of any two or more thereof.

19. A use of any one of claims 12 to 8 wherein the phospholipid composition comprises about 5 to about 95% w/w phospholipid).

20. A use of any one of claims 12 to 9 wherein the phospholipid composition comprises at least about 0.1% w/w of two or more, three or more, or four or more phospholipids selected independently from phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phosphatidylserine, and phosphatidylinositol.

21. A use of any one of claims 12 to 20 wherein the phospholipid composition comprises a composition as defined in claim 9.

22. A use of any one of claims 12 to 21 wherein the use further comprises separate, simultaneous or sequential administration of one or more additional agents for ameliorating cognitive decline.

23. A use of any one of claims 2 to 22 wherein the related condition is selected from dementia, young-onset dementia, degenerative dementia, Alzheimer's disease, vascular disease, fronto temp oral lobar degeneration (FTLD), dementia with Lewy bodies, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, cognitive impairment associated with schizophrenia, chemotherapy-induced neuropathy, Down's syndrome, Korsakoff s disease, cerebral palsy, epilepsy, neuronal ischemia, neuronal reperfusion injury, neuronal trauma, neuronal haemorrhage, neuronal infection, stroke, neuronal exposure to a toxic substance, age- related mental disorders, anxiety disorders, age-related depression, dementia associated with microvascular disorders (such as diabetes, hypotension, stroke induced vascular dementia, and obesity), dementia associated with a disorder of the immune system, dementia associated with a central nervous system (CNS) disorder, dementia associated with hypotension, dementia associated with obesity, vascular dementia (including post stroke).