U.S. patent application number 10/994175 was filed with the patent office on 2005-06-16 for lipids containing omega-3 and omega-6 fatty acids.
This patent application is currently assigned to ENZYMOTEC LTD.. Invention is credited to Bar-On, Zohar, Ben Dror, Gai, Farkash, Orly, Pelled, Dori, Platt, Dorit, Shulman, Avidor, Zuabi, Rassan.
Application Number | 20050130937 10/994175 |
Document ID | / |
Family ID | 34655264 |
Filed Date | 2005-06-16 |
United States Patent
Application |
20050130937 |
Kind Code |
A1 |
Ben Dror, Gai ; et
al. |
June 16, 2005 |
Lipids containing omega-3 and omega-6 fatty acids
Abstract
A lipid preparation including a glycerophospholipid or salt,
conjugate and derivatives thereof, particularly phosphatidylserine
(PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE),
phosphatidyl-inositol (PI), phosphatidylglycerol (PG) and
phosphatidic acid (PA), and poly-unsaturated fatty acid (PUFA) acyl
groups, particularly long-chain poly-unsaturated fatty acid
(LC-PUFA) acyl groups such as omega-3 and/or omega-6 acyl groups,
wherein said PUFA is covalently bound to said glycerophospholipid.
The preparation possesses an improved bioactivity, and is useful in
the treatment of various cognitive and mental conditions and
disorders and for maintenance of normal functions of brain-related
systems and processes.
Inventors: |
Ben Dror, Gai; (Moshav Ofer,
IL) ; Platt, Dorit; (Shimshit, IL) ; Farkash,
Orly; (Shimshit, IL) ; Zuabi, Rassan; (Afula,
IL) ; Bar-On, Zohar; (Karmiel, IL) ; Shulman,
Avidor; (Kiryat Tivon, IL) ; Pelled, Dori;
(Hod Hasharon, IL) |
Correspondence
Address: |
FLEIT KAIN GIBBONS GUTMAN & BONGINI
COURVOISIER CENTRE II, SUITE 404
601 BRICKELL KEY DRIVE
MIAMI
FL
33131
US
|
Assignee: |
ENZYMOTEC LTD.
MIGDAL HAEMEK
IL
|
Family ID: |
34655264 |
Appl. No.: |
10/994175 |
Filed: |
November 19, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10994175 |
Nov 19, 2004 |
|
|
|
PCT/IL04/00957 |
Oct 21, 2004 |
|
|
|
Current U.S.
Class: |
514/78 ;
426/601 |
Current CPC
Class: |
A61K 31/685 20130101;
A23V 2002/00 20130101; A61P 43/00 20180101; A23L 33/12 20160801;
A21D 2/32 20130101; A23V 2250/1842 20130101; A23V 2250/1942
20130101; A23V 2250/1882 20130101; A23V 2200/322 20130101; A23D
9/013 20130101; A23V 2002/00 20130101; A23D 7/013 20130101; A61P
25/00 20180101 |
Class at
Publication: |
514/078 ;
426/601 |
International
Class: |
A61K 031/685; A23D
009/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 22, 2003 |
IL |
158552 |
Claims
What is claimed is:
1. A lipid preparation, wherein said lipid is selected from the
group consisting of a glycerophospholid and salts, conjugates and
derivatives and thereof and any mixture thereof, and
poly-unsaturated fatty acid (PUFA) acyl groups, particularly
long-chain poly-unsaturated fatty acid (LC-PUFA) acyl groups,
preferably omega-3 and/or omega-6 acyl groups, at a concentration
of least 5% (w/w) of total fatty acids content of said preparation,
preferably more than 10% (w/w), more preferably 20-50% (w/w),
wherein said PUFA is covalently bonded to said lipid.
2. A lipid preparation of claim 1 wherein said lipid is a naturally
occurring lipid, or a synthetic lipid.
3. A lipid preparation of claim 2, wherein said lipid is a
glycerophospholipid in which at least some of the sn-1 or sn-2
groups of the glycerol backbone are substituted with said
poly-unsaturated fatty acid (PUFA) acyl groups.
4. A lipid preparation of claim 1, wherein said lipid is a
glycerophosphlipid of formula I: 4wherein R" represents a moiety
selected from serine (PS), choline (PC), ethanolamine (PE),
inositol (PI), glycerol (PG) and hydrogen (phosphatidic acid--PA),
and R and R', which may be identical or different, independently
represent hydrogen or an acyl group, wherein said acyl group is
selected from saturated, mono-unsaturated or poly-unsaturated acyl
groups (PUFA), particularly long-chain poly-unsaturated fatty acids
(LC-PUFA), more preferably omega-3 and/or omega-6 acyl groups, and
salts thereof, with the proviso that R and R' cannot simultaneously
represent hydrogen, and wherein said polyunsaturated acyl groups
comprise at least 5% (w/w) of total lipid fatty acids, preferably
more than 10% (w/w), and particularly 20-50% (w/w).
5. A preparation of claim 4, wherein R represents hydrogen and R'
represents an acyl group.
6. A preparation of claim 4, wherein R' represents hydrogen and R
represents an acyl group.
7. A preparation of claim 4, wherein said acyl group is an omega-3
acyl group, preferably an eicosapentaenoyl (EPA), a docosahexaenoyl
(DHA) group, or linolenic omega-3 group.
8. A preparation of claim 4, wherein said acyl group is an omega-6
acyl group, preferably an arachidonoyl (ARA) group, or a linoleic
omega-6 group.
9. A preparation of claim 4, wherein said acyl group is a
linolenoyl (18:3) group.
10. A preparation of claim 4, wherein R" represents serine,
choline, ethanolamine, inositol, glycerol, and H.
11. A preparation according to claim 4, wherein the identity and
content of R and R' are predetermined.
12. A preparation of claim 10, wherein R" is serine, characterized
in that it mimics the composition of human brain PS.
13. A preparation of claim 10, wherein R" is serine, characterized
in that it is different from human brain PS and has improved
bioactivity compared to soybean-PS.
14. A PS preparation, wherein said PS is derived from any one of
plant, animal or microorganism source, said preparation being
enriched with PS of formula I, wherein R" represents a serine
moiety.
15. A preparation of claim 12, characterized in that it is
effective at a lower dosage compared to soybean-PS, while having
similar and/or improved bioactivity compared to soybean-PS.
16. A preparation of claim 1, wherein said omega-3 or omega-6 is
more stable than a omega-3 or omega-6 in the free fatty acid form,
bonded to a triglyceride or as an ethyl ester.
17. A preparation of claim 1, characterized in having a reduced or
absent of fish-related organoleptic effects.
18. A preparation of claim 1, said preparation being enriched with
PS of formula I, characterized in having a reduced or absent of
fish-related organoleptic effects.
19. A preparation of claim 1, for use in the reduction and/or
prevention of serum oxidative stress leading to atherosclerosis,
cardiovascular disorders and/or coronary heart disease.
20. A preparation of claim 1, for use in the improvement and
treatment of cognitive and mental conditions and disorders as well
as the maintenance of normal functions of brain-related systems and
processes, preferably ADHD, aging, Alzheimer's disease, Parkinson's
disease, multiple sclerosis (MS), dyslexia, depression, learning
capabilities, intensity of brain waves, stress, anxiety, mental and
psychiatric disorders, concentration and attention, mood, brain
glucose utilization, general cognitive and mental well being,
neurological disorders and hormonal disorders.
21. A preparation of claim 1, said preparation being enriched with
PS of formula I, for use in any one of the improvement and
treatment of ADHD, and reducing ADHD symptoms in children.
22. A preparation of claim 1, for enhancing the bioavailability of
polyunsaturated fatty acids, particularly omega-3 and/or omega-6
fatty acids.
23. A preparation of claim 1, for use in combined improvement of
cognitive and mental functions together with improvement of
additional health disorders or conditions.
24. The preparation of claim 23, wherein said additional health
disorders or conditions are at least one of high blood cholesterol
levels, high triglycerides levels, high blood fibrinogen levels,
HDL/LDL ratio, diabetes, metabolic syndrome, menopausal or
post-menopausal conditions, hormone related disorders, vision
disorders, inflammatory disorders, immune disorders, liver
diseases, chronic hepatitis, steatosis, phospholipid deficiency,
lipid peroxidation, dysrhythmia of cell regeneration,
destabilization of cell membranes, coronary artery disease, high
blood pressure, cancer, hypertension, aging, kidney disease, skin
diseases, edema, gastrointestinal diseases, peripheral vascular
system diseases, allergies, neurodegenerative and psychiatric
diseases.
25. A nutraceutical composition comprising a phospholipid
preparation as claimed in claim 1.
26. A nutraceutical composition of claim 25, in the form of softgel
capsules, tablets, syrups, or any other common dietary supplement
delivery system.
27. A functional food article comprising the phospholipid
preparation of claim 1.
28. The functional food article of claim 27, selected from dairy
products, ice-creams, biscuits, soy products, bakery, pastry and
bread, sauces, soups, prepared foods, frozen foods, condiments,
confectionary, oils and fats, margarines, spreads, fillings,
cereals, instant products, drinks and shakes, infant formulas,
infant foods (biscuits, mashed vegetables and fruits, cereals),
bars, snacks, candies and chocolate products.
29. A pharmaceutical composition comprising the phospholipids
preparation of claim 1, and optionally at least one
pharmaceutically acceptable additive, diluent or excipient.
30. A pharmaceutical composition of claim 29, further optionally
comprising at least one pharmaceutically active agent.
31. The preparation of claim 1, wherein said PUFA has increased
bioavailability to the organism when comparing to a composition
comprising PUFA alone.
Description
RELATED APPLICATION
[0001] The present application is a continuation of International
Patent Application No. PCT/IL2004/000957, filed Oct. 21, 2004, the
contents of which are here incorporated by reference in their
entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to phospholipids and polar
lipids preparations which are enriched with omega-3 and/or omega-6
fatty acids covalently attached to the lipid backbone. The
phospholipid preparations of the invention are particularly useful
as nutraceuticals, food additives and/or pharmaceutical agents for
the treatment of various conditions, in particular related to
cognitive functions.
[0004] 2. Prior Art
[0005] Lipids, and especially polar lipids, nitrogen containing
lipids, and carbohydrate containing lipids (phospholipids,
sphingosines, glycolipids, ceramides, sphingomyelins) are the major
building blocks of cell membranes, tissues, etc. Additionally they
play important roles in signal transduction processes and in a
variety of biochemical and biosynthetic pathways.
[0006] Glycerophospholipids, lipids based on a glycerol backbone
and containing a phosphate head group, are the main building blocks
of cell membranes. Since most, if not all, biochemical processes
involve cell membranes, the structural and physical properties of
membranes in different tissues is crucial to the normal and
efficient functioning of membranes in all biochemical
processes.
[0007] In light of the emerging functional foods category in the
area of dietary lipids many health benefits have been attributed to
the consumption of certain fatty acids. For example, it has been
reported in many research studies that polyunsaturated fatty acids
(PUFA) of the type omega-3 and omega-6, have several health
benefits on cardiovascular disease, immune disorders and
inflammation, renal disorders, allergies, diabetes, and cancer.
These types of fatty acids are naturally occurring mainly in fish
and algae, where they are randomly distributed on the sn-1, sn-2,
and sn-3 positions of the glycerol backbone of triglycerides.
[0008] The professional literature emphasizes the importance of an
adequate diet containing omega-3 fatty acids. Extensive clinical
studies investigating the importance of Docosahexaenoic acid (DHA),
one of the most important omega-3 fatty acids, in the brain, found
that low levels of DHA are associated with depression, memory loss,
dementia, and visual problems. All studies showed a dramatic
improvement in the elderly brain function as blood levels of DHA
increased.
[0009] Other known benefits of DHA include: lower risk of
arrhythmias, reduction in the risk of sudden cardiac death, lower
plasma triglyceride levels and reduced blood clotting tendency.
Furthermore, DHA may have importance in the field of brain
functioning enhancement, baby formula fortification, diabetics and
cancer. Nutritional studies, investigating the importance of DHA in
the brain, found that low levels of DHA are associated with
depression, memory loss, cognitive impairment, dementia and visual
problems.
[0010] The human body does not adequately synthesize DHA. Therefore
it is necessary to obtain it from the diet. Humans obtain DHA from
their diets, initially through the placenta, then from breast milk,
and later through dietary sources, such as fish, red meats, animal
organ meats and eggs. Popular fish like tuna, salmon and sardines
are rich sources. Until recently, the primary source of DHA dietary
supplements has been fish oils. The ability of enzymes to produce
the omega-6 and omega-3 family of products of linoleic and
alpha-linolenic acid declines with age. Because DHA synthesis
declines with age, as we get older our need to acquire DHA directly
from diet or supplements increases. In fact, several recent
publications suggested DHA to be considered as essential fatty acid
[for example, Muskiet, F. et al. (2004) J Nutr. 134(1):183-6].
[0011] Because DHA is important for signal transmission in the
brain, eye and nervous system, many consumers concerned with
maintaining mental acuity are searching for a pure, safe way to
supplement their DHA levels.
[0012] Polyunsaturated acids, in particular long chain, such as
omega-3 and 6, have been shown to confer many valuable health
benefits on the population. The global market for long-chain PUFAs,
including the food segment, is rapidly growing.
[0013] The majority of efforts in the industry are however invested
in the improvement of PUFA processing techniques and in the
creation of higher concentrated grades of PUFA derivatives to
accommodate dietary supplements and functional foods needs.
[0014] The academic and industrial communities are less concerned
regarding the evaluation of different delivery approaches of PUFA
in order to enhance their bio-availability and their efficacy in
term of their known variety of health benefits. These benefits
range from prevention and treatment of CVD, diabetes, cognitive
disorders and/or decline, visual disorders, skin conditions,
learning disorders, etc. Additionally, PUFAs have been shown to
assist in the cognitive and visual development of infants.
SUMMARY OF THE INVENTION
[0015] PUFA-Lipids
[0016] PS-PUFA
[0017] Phosphatidylserine, also known as PS, is a natural
phospholipid with bio-functionality that has made it one of the
most promising dietary supplements in the field of brain nutrition.
PS and its health benefits have been known to the scientific and
nutrition communities since the 1970's. Numerous studies have been
conducted in order to establish this efficacy in a variety of
cognitive and mental functions. Those studies have shown that PS
can improve memory, fight dementia, fight early stages of
Alzheimer's disease, reduce stress and tension, improve attention
span, enhance mood and fight depression, to name but few.
[0018] PS is one of the most important building blocks of cell
membranes in the brain. Hence, the level of PS in brain cell
membranes ensures the fluidity and structure of these membranes.
The normal level ensures normal and efficient signal transduction
processes, efficient glucose consumption, and other biological
pathways that result in normal cognitive and mental functions.
[0019] Since PS is not abundant in human nutrition and since in
many people, especially the elderly, the biosynthetic pathways
responsible for the production of PS are malfunctioning, the levels
of PS in the body and brain are low. This results in a variety of
cognitive and mental disorders, such as depression, memory loss,
short attention span, learning difficulties, etc.
[0020] The supplementation of PS in the diets of elderly people
with such disorders has resulted, in many cases, in dramatic
improvements of these disorders. Over the recent years, studies
have shown that even younger people can benefit from dietary
supplementation of PS. PS has been shown to improve the learning
capabilities of students, improve memory and attention span,
etc.
[0021] It is therefore an object of the present invention to
provide special preparations of PS, for use mainly as
nutraceuticals and as functional food additives.
[0022] PC-PUFA
[0023] As mentioned before, phospholipids are essential components
of all cellular and sub-cellular membranes. Phosphatidylcholine and
phosphatidylethanolamine predominate quantitatively, substantially
constituting the typical bilayer configuration. Phospholipids
belong to the amphipathic molecules with a water-soluble and a
fat-soluble component. In the bilayer configuration the hydrophilic
groups are arranged at the outer and inner side of the membrane
toward the surrounding medium; the lipophilic groups, in contrast,
face each other at the inner side of the bilayer configuration.
[0024] Other important constituents of biological membranes are
cholesterol, glycolipids, and peripheral and integral proteins. The
basic structure of biological membranes is thus a series of
recurrent unities of lipid-protein complexes. The membrane is
asymmetric. The function of the external (cellular) and internal
(sub cellular) membrane systems depends on their composition and on
the integrity of their phospholipid structure. In addition to their
presence in cell membranes, phospholipids constitute structural and
functional elements of the surface mono-layers of lipoproteins and
of surfactants.
[0025] Of utmost importance for the function of biological
membranes is their fluidity, which is decisively influenced by
phospholipids. Besides the content in cholesterol and proteins and
the nature and charge of the polar head groups of phospholipids in
the system, membrane fluidity depends on the length of the chains
of fatty acid residues in the phospholipid molecule, as well as on
the number and type of pairing of their double bonds.
[0026] Phospholipids containing poly-unsaturated fatty acids supply
the organism with important building blocks which improves membrane
fluidity.
[0027] Studies conducted with PUFA-containing phospholipids have
shown the following:
[0028] 1. They are high-energy, basic, structural, and functional
elements of all biological membranes, such as cells, blood
corpuscles, lipoproteins, and the surfactant.
[0029] 2. They are indispensable for cellular differentiation,
proliferation, and regeneration.
[0030] 3. They maintain and promote the biological activity of many
membrane-bound proteins and receptors.
[0031] 4. They play a decisive role for the activity and activation
of numerous membrane-located enzymes, such as
sodium-potassium-ATPase, adenylate cyclase and lipoprotein
lipase.
[0032] 5. They are important for the transport of molecules through
membranes.
[0033] 6. They control membrane-dependent metabolic processes
between the intracellular and intercellular space.
[0034] 7. The polyunsaturated fatty acids contained in them, such
as linoleic acid, are precursors of the cytoprotective
prostaglandins and other eicosanoids.
[0035] 8. As choline and fatty acid donors they have an influence
in certain neurological processes.
[0036] 9. They emulsify fat in the gastrointestinal tract.
[0037] 10. They are important emulsifiers in the bile.
[0038] 11. They codetermine erythrocyte and platelet
aggregation.
[0039] 12. They influence immunological reactions on the cellular
level.
[0040] Phospholipids containing PUFA are theoretically of
importance in all those diseases in which damaged membrane
structures, reduced phospholipid levels, and/or decreased membrane
fluidity are present. This hypothesis is supported by experimental
and clinical investigations of various membrane-associated
disorders and illnesses.
[0041] Studies on the active principle as well as pharmacological
and clinical trials are available on a variety of disturbances. and
diseases related to membrane damages. For example, in liver
diseases the hepatocyte structures are damaged by, for example,
viruses, organic solvents, alcohol, medicaments, drugs, or fatty
food. As a consequence, membrane fluidity and permeability may be
disturbed, and membrane-dependent metabolic processes as well as
membrane-associated enzyme activities may be impaired. This
considerably inhibits the metabolism of the liver.
[0042] Other examples include hyperlipoproteinemia with or without
atherosclerosis, hemorrheological disturbances with an elevated
cholesterol/phospholipid ratio in the membranes of platelets and
red blood cells, neurological diseases, gastro intestinal
inflammations, kidney diseases, and in a variety of aging
symptoms.
[0043] All these very different diseases have in common comparable
membrane disorders. With polyunsaturated phosphatidylcholine
molecules such disorders may be positively influenced, eliminated,
or even improved beyond normal due to the high content in
polyunsaturated fatty acids. Following are some examples of the
mechanisms that mediate this phenomenon:
[0044] 1. HDL particles enriched with
PUFA-containing-phosphatidylcholine are able to take up more
cholesterol from low-density lipoprotein (LDL) and tissues. More
cholesterol can be transported back to the liver. This action on
the cholesterol reverse transport is unique. All other
lipid-lowering agents reduce either the cholesterol absorption in
the body or the cholesterol synthesis in the liver and its
distribution to the periphery. These substances, however, do not
physiologically mobilize the cholesterol already present in the
periphery.
[0045] 2. The cholesterol/phospholipid ratio in membranes,
platelets, and red blood cells decreases and membrane function is
improved up to normalization.
[0046] 3. Peroxidative reactions are reduced, damaged hepatocyte
membrane structures restored, membrane fluidity and function
stabilized, immuno-modulation and cell protection improved, and
membrane-associated liver functions enhanced.
[0047] 4. With the normalization of the cholesterol/phospholipid
ratio, the bile is also stabilized.
[0048] 5. Due to its specific property as a surface-active
emulsifier, PUFA-containing-phosphatidylcholine solubilize fat and
is used in reducing the risk and treatment of fat embolism.
[0049] 6. The substitution with poly-unsaturated-fatty-acids and
choline may have a cytoprotective effect in the brain and activate
neuronal processes.
[0050] 7. Liposomes with polyunsaturated phosphatidylcholine
molecules may act as drug carriers, such as of vitamin E.
[0051] Liver Disease
[0052] Experimental and clinical results support the assumption
that the therapeutic application of
PUFA-containing-phosphatidylcholine has protective and even
curative and regenerative effects on biological membranes of sinus
endothelial cells and hepatocytes. The cytoprotective effect of
PUFA-containing-phosphatidylcholine has been corroborated in 7 in
vitro and in 55 in vivo experiments, in which 20 different models
with five different animal species were used. Types of intoxication
that are known to play a role in the etiology of liver disease have
mostly been applied: chemical substances, medicaments, alcohol,
cholestasis, immunological phenomena, exposure to radiation, and so
on.
[0053] The hepato-protective effects of
PUFA-containing-phosphatidylcholin- e have been confirmed and were
the more pronounced the earlier PUFA-containing-phosphatidylcholine
was administered:
[0054] 1. Structures of membranes were normal or largely
normalized.
[0055] 2. Fatty infiltrations and hepatocyte necrosis could be
diminished or even eliminated.
[0056] 3. Corresponding data were found for lipid peroxidation,
transaminase and cholinesterase activity, and for serum lipids;
liver cell metabolism increased.
[0057] 4. The increase of RNA and protein synthesis and of the
liver cell glycogen content indicated a stimulation of the liver
cells.
[0058] 5. Reduced collagen production, collagen/DNA ratio, and
liver hydroxyproline content indicated a reduced formation of
connective tissue.
[0059] The dosage of PUFA-containing-phosphatidylcholine ranged
from 525 to 2,700 mg/day when administered orally, and from 500 to
3,000 mg/day in intravenous application. The duration of treatment
lasted from a few weeks to up to 30 months. The main liver
indications were acute hepatitis, chronic hepatitis, fatty liver,
toxic liver damage, cirrhosis of the liver, and hepatic coma.
[0060] The clinical findings, showing the effectiveness of
PUFA-containing-phosphatidylcholine, can be summarized generally as
follows:
[0061] 1. Accelerated improvement or normalization of subjective
complaints, of clinical findings, and of several biochemical
values
[0062] 2. Better histological results as compared with the control
groups
[0063] 3. A shortened duration of hospitalization
[0064] Promising results were obtained also in renal disorders,
chronic ambulatory peritoneal dialysis,
hyperlipoproteinemia/atherosclerosis, gastrointestinal
inflammation, psoriasis, and more.
[0065] Recent research studies have shown that PUFA-enriched
phospholipids, isolated from rainbow trout embryos, have novel
health benefits. Some of these benefits include the treatment of
tumor cells, inhibition of 5-lipoxygenase activity, reduction of
neutral fat levels (such as cholesterol).
[0066] There is proof that a person who receives enriched
phospholipids nutritionally, these phospholipids cross the
intestinal barrier and the blood-brain barrier, thus reaching the
brain. Recently, investigators from Ponroy Laboratories had
described an experiment in which mice lacking essential fatty
acids, i.e. linoleic acid (18:2 n-6) and .alpha.-linolenic acid
(18:3 n-3), which serve as the sole sources for LC-PUFA, were fed
cerebral phospholipids and the quantity of phospholipids in each
part of the brain measured. These phospholipids were found in the
cytoplasm, in the synapses, and in other parts of the brain [Carrie
et al., (2000) J. Lipid Res. 41, 465-472].
[0067] The utilization of phospholipids enriched with PUFA holds
many potential advantages from a clinical point of view. The
phospholipid may deliver the essential fatty acid to specific
organs or body parts, such as the brain, and assist in the
incorporation of these fatty acids in membranes. Other advantages
may arise from the fact that phospholipids enriched with PUFA will
not have odor problems such as found in the major current
nutraceutical source, the fish oils. Furthermore, some preliminary
clinical studies have shown that PUFA incorporated in phospholipids
possess superior efficacy than PUFA carried by triglycerides. [Song
et al. (2001) Atherosclerosis, 155, 9-18].
[0068] Further studies have shown that the activity of DHA-rich
phospholipid was different from that of DHA-rich triacylglycerol in
spontaneously hypertensive rats [Irukayama-Tomobe et al. (2001)
Journal of Oleo Science, 50(12), 945-950]. Spontaneously
hypersensitive rats (SHR) were fed test lipid diets for six weeks,
which contained 30%-docosahexaenoic acid (DHA) phospholipid
(DHA-PL) extracted from fish roe or 30%-DHA fish oil (DHA-TG). The
control diet contained corn oil in the presence of test lipids.
After feeding, blood pressure in the DHA-TG and DHA-PL diet groups
was found significantly lower compared to the control. Serum fatty
acid content of dihomo-linoleic acid (DHLnA) and Arachidonic acid
(AA) of the DHA-PL diet group was significantly less than the
control or DHA-TG diet group. Serum triacylglycerol, phospholipid
and total cholesterol in the DHA-TG and DHA-PL diet groups were
significantly less than in the control. Liver total cholesterol in
DHA-PL was twice that in the DHA-TG diet group and control. The
mechanism for cholesterol removal from blood by DH-PL would thus
appear to differ from that by DHA-TG. Serum lipid peroxide (LPO) in
the DHA-TG and DHA-PL diet groups was essentially the same as in
the control.
[0069] Many PUFA-containing agents suffer from stability and
quality problems due to the high degree of oxidation of the
polyunsaturated fatty acids. These problems require the
incorporation of antioxidants as well as the utilization of special
measures which attempts to reduce this oxidation. The utilization
of phospholipids as carriers of PUFA may result in enhanced
stability of such products due to the anti-oxidative properties of
phospholipids.
[0070] It seems that one of the most effective transport mechanism
for such essential fatty acids is the attachment of these groups to
phospholipid molecules. The phospholipids have been shown to pass
through the blood-brain barrier and transport the DHA where it is
needed.
[0071] Organoleptic Concerns
[0072] PUFAs are traditionally extracted from coldwater fish.
Despite the healthy image, one of the problems of consumer
acceptance has been the resulting strong, fishy taste. To address
this, microencapsulated forms of omega-3 have been pioneered in the
last 15 years. A further step was the development of egg-containing
products such as DHA-enriched mayonnaise and pasta. DHA-enriched
yogurts, baked goods and broilers were also envisaged.
[0073] There is no other nutritional product or ingredient that is
considered to be an agent of PUFA delivery. All current commercial
products are based on the fatty acids themselves in an encapsulated
form or on foods enriched with PUFA through special animal/crop
feed.
[0074] It is therefore an object of the present invention to
provide lipid preparations enriched with omega-3 or omega-6 fatty
acids, for use mainly as nutraceuticals and as functional food
additives. The composition of said preparation is such that it
provides the preparation with the property of enhancing the
bioavailability of PUFAs. Thus upon its consumption, preferably in
the form of nutraceuticals, food additives or pharmaceutical
compositions, the organism may, in the most efficient way, enjoy
the benefits provided by said preparation, as will be described in
detail below.
[0075] This and other objects of the invention will become apparent
as the description proceeds.
[0076] In a first aspect the present invention provides a lipid
preparation, wherein said lipid is selected from
glycerophospholipids and their salts, conjugates, and derivatives
and any mixture thereof, and poly-unsaturated fatty acid (PUFA)
acyl groups, particularly long-chain poly-unsaturated fatty acid
(LC-PUFA) acyl groups, preferably omega-3 and/or omega-6 acyl
groups, at a concentration of least 5% (w/w) of total fatty acids
content of said preparation, preferably more than 10% (w/w), more
preferably 20-50% (w/w), wherein said PUFA is covalently bound to
said lipid.
[0077] Said lipid may be a naturally occurring lipid, or a
synthetic lipid. Preferably, said lipid is a glycerophospholipid in
which at least some of the sn-1 or sn-2 groups of the glycerol
backbone are substituted with said poly-unsaturated fatty acid
(PUFA) acyl groups.
[0078] In one particular embodiment, said lipid is a
glycerophosphlipid of formula I: 1
[0079] wherein R" represents a moiety selected from serine (PS),
choline (PC), ethanolamine (PE), inositol (PI), glycerol (PG) and
hydrogen (phosphatidic acid--PA), and R and R', which may be
identical or different, independently represent hydrogen or an acyl
group, wherein said acyl group is selected from saturated,
mono-unsaturated or poly-unsaturated acyl groups (PUFA),
particularly long-chain poly-unsaturated fatty acids (LC-PUFA),
more preferably omega-3 and/or omega-6 acyl groups, and salts
thereof, with the proviso that R and R' cannot simultaneously
represent hydrogen, and wherein said polyunsaturated acyl groups
comprise at least 5% (w/w) of total lipid fatty acids, preferably
more than 10% (w/w), and particularly 20-50% (w/w).
[0080] In one more particular embodiment of said preparation, R
represents hydrogen and R' represents an acyl group. Alternatively,
R' represents hydrogen and R represents an acyl group.
[0081] Considering these latter embodiments, when said acyl group
is preferably an omega-3 acyl group, it may be an eicosapentaenoyl
(EPA), a docosahexaenoyl (DHA) group, or linolenic omega-3 group.
And, when said acyl group is preferably an omega-6 acyl group, it
may be an arachidonoyl (ARA) group, or a linoleic omega-6 group. A
further possibility is that said acyl group may be a linolenoyl
(18:3) group.
[0082] In a yet further embodiment of the preparation of the
invention, R" may be any one of serine, choline, ethanolamine,
inositol or glycerol.
[0083] In a further particular embodiment, the identity and content
of R and R' are predetermined.
[0084] The preparation of the invention which comprises the
compound of formula I in which R" is serine, mimics the composition
of human brain PS.
[0085] Nonetheless, the invention also refers to preparations
comprising the compound of formula I in which R" is serine, which
are different from human brain PS, but still have an improved
bioactivity, particularly as compared to soybean-PS. This improved
bioactivity results in beneficial effects on both the learning and
working memory in elderly population, in particularly in
cholinergic impaired conditions like Alzheimer's disease.
[0086] The invention also relates to preparation PS preparation
which mimics the human brain PS, is effective at lower dosage (2-3
fold) compared to soybean-PS, while having similar or improved
bioactivity compared to soybean-PS.
[0087] The PS may be of plant, animal or microorganism source, and
is. enriched with PS of formula I, wherein R" represents a serine
moiety.
[0088] The preparation of the invention may be further enriched
with PS of formula I, characterized in having reduced or absent of
fish-related organoleptic effects. Such preparation may be
particularly suitable for incorporation into chocolate-containing
or dairy-based food articles (including concentrated milk).
[0089] The preparation of the invention may be used in the
improvement and treatment of cognitive and mental conditions and
disorders as well as the maintenance of normal functions of
brain-related systems and processes, preferably ADHD, aging,
Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS),
dyslexia, depression, learning capabilities, intensity of brain
waves, stress, anxiety, mental and psychiatric disorders,
concentration and attention, mood, brain glucose utilization,
general cognitive and mental well being, neurological disorders and
hormonal disorders.
[0090] The preparation of the invention is particularly useful in
enhancing the bioavailability of omega-3 and omega-6 fatty
acids.
[0091] The preparation of the invention may be used in combined
improvement of cognitive and mental functions together with
improvement of additional health disorders or conditions. Such
additional health disorders or conditions may be at least high
blood cholesterol levels, high triglycerides levels, high blood
fibrinogen levels, HDL/LDL ratio, diabetes, metabolic syndrome,
menopausal or post-menopausal conditions, hormone related
disorders, vision disorders, inflammatory disorders, immune
disorders, liver diseases, chronic hepatitis, steatosis,
phospholipid deficiency, lipid peroxidation, dysrhythmia of cell
regeneration, destabilization of cell membranes, coronary artery
disease, high blood pressure, cancer, hypertension, aging, kidney
disease, skin diseases, edema, gastrointestinal diseases,
peripheral vascular system diseases, allergies, neurodegenerative
and psychiatric diseases.
[0092] The preparation of the invention may also be used in the
reduction and/or prevention of serum oxidative stress leading to
atherosclerosis, cardiovascular disorders and/or coronary heart
disease.
[0093] The invention further relates to nutraceutical compositions
comprising a lipid preparation in accordance with the invention.
The nutraceutical composition may be in the form of softgel
capsules, tablets, syrups, or any other common dietary supplement
delivery system.
[0094] Still further, the invention relates to functional food
article comprising the lipid preparation of the invention. Such
functional food article may be selected from dairy products, dairy
drinks, ice-creams, bakery products, confectionary products,
biscuits, soy products, pastry and bread, sauces, condiments, oils
and fats, margarines, spreads, cereals, drinks and shakes, oils and
fats, infant formulas, infant foods (biscuits, mashed vegetables
and fruits, cereals), bars, snacks, candies and chocolate
products.
[0095] In yet a further aspect, the invention relates to
pharmaceutical compositions comprising the lipid preparation of the
invention, and optionally further comprising at least one
pharmaceutically acceptable additive, diluent or excipient. The
pharmaceutical composition of the invention may further optionally
comprise at least one pharmaceutically active agent.
BRIEF DESCRIPTION OF THE FIGURES OF THE DRAWING
[0096] FIGS. 1A-D: Performance of Rats in Acquisition of the
Spatial Morris Maze Task.
[0097] Latency time to platform in the three days of acquisition (2
sessions per day) of aged rats supplemented for three months with
various supplements as detailed below was analyzed using video
camera, with (open squares) or without (closed circuits)
pretreatment of 1 mg/kg of scopolamine.
[0098] FIG. 1A: Rats supplemented with MCT, P<0.007.
[0099] FIG. 1B: Rats supplemented with PS-{overscore (.omega.)}3
P<0.07.
[0100] FIG. 1C: Rats supplemented with SB-PS, P<0.02.
[0101] FIG. 1D: Rats supplemented with LC-PUFA, P<0.03.
[0102] Values represent mean .+-.S.E.M of four to five rats per
supplement.
[0103] Abbreviations: Lat. T., latency time; sec., seconds.
[0104] FIG. 2. Performance of Scopolamine-Treated Rats in the
Morris Water Maze Task in the Spatial Probe Test.
[0105] This graph represents percentage. of time (T.) that aged
rats, supplemented for three months with MCT (open bars),
PS-{overscore (.omega.)}3 (solid bars), SB-PS (dotted bars) or
LC-PUFA (striped bars), spent in different areas after the platform
being removed, was analyzed using video camera, following
pre-treatment of 1 mg/kg of scopolamine. Values represent
mean.+-.S.E.M of four to five rats per supplement. Significance
compared to control group (MCT)*P<0.02 and **P<0.08
[0106] FIGS. 3A-D: Performance of Scopolamine-Induced Rats in
Locating the Platform after its Reposition.
[0107] Latency time to platform on the fifth day of the water maze
test, in which the platform was repositioned between the sessions,
in aged rats supplemented for three months with different
supplements as specified below, was analyzed using video camera,
with (open squares) or without (closed circuits) pretreatment of 1
mg/kg of scopolamine.
[0108] FIG. 3A: Rats supplemented with MCT.
[0109] FIG. 3B: Rats supplemented withPS-{overscore
(.omega.)}3.
[0110] FIG. 3C: Rats supplemented with SB-PS.
[0111] FIG. 3D: Rats supplemented with LC-PUFA.
[0112] Values represent mean .+-.S.E.M of four to five rats per
supplement.
[0113] Abbreviations: Lat. T., latency time; sec., seconds; tr.,
trials.
[0114] FIGS. 4A-B: Phospholipid Levels in Rat Tissues as Measured
using .sup.31P-NMR.
[0115] Lipids were extracted from tissues of aged rats that were
supplemented for three months with MCT (open bars), PS-{overscore
(.omega.)}3 (solid bars), SB-PS (dotted bars) or LC-PUFA (striped
bars). Phospholipids levels were analyzed using a .sup.31P-NMR
machine and the relative levels of phosphatidylcholine of the
different treatments are presented.
[0116] FIG. 4A: Analysis of lipids extracted from the liver.
[0117] FIG. 4B: Analysis of lipids extracted from the brain (cortex
region).
[0118] Values represent mean .+-.S.D. of four to five rat tissues
per supplement. Significance compared to control group
(MCT)*P<0.05 and **P<0.1.
[0119] Abbreviations: Tot. PI., total phospholipids.
[0120] FIG. 5: Parental Scores of ADHD Children According to
Behavioral Rating Scales.
[0121] The graph represents percentage of ADHD children that
demonstrated improvement or lack of improvement in a parental view
following two months of supplementation with canola oil (open
bars), DHA (solid bars) or PS-{overscore (.omega.)}3 (hatched
bars). Rating includes remarks regarding behavioral tendencies at
home, at school, with siblings or peers and teachers feedback.
Values represent percentage of twenty to twenty-five ADHD children
scores per supplement. Note that twelve parents decline to respond
to the questioner and six children did not complete the
supplementation period due to poor taste or severe discipline
problems (mostly the control group).
[0122] Abbreviations: Improv., improvement; Marg. Improve.,
marginal improvement; n.c., no change; Deter., deterioration.
[0123] FIG. 6: Effect of PC-DHA on the Serum Oxidative Stress.
[0124] Apo E.degree. mice were fed for 10 weeks with placebo (open
bars) or PC-DHA (solid bars). Serum lipid peroxide (Ser. per.)
levels were measured using a spectrophotometric assay. Values
represent mean.+-.S.D. of 5 mice per treatment.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
[0125] In a first aspect the present invention provides a lipid
preparation, wherein said lipid is a glycerophospholipid, a salt,
conjugate, and derivative thereof, and any mixture thereof, and
poly-unsaturated fatty acid (PUFA) acyl groups, particularly
long-chain poly-unsaturated fatty acid (LC-PUFA) acyl groups,
preferably omega-3 and/or omega-6 acyl groups, at a concentration
of least 5% (w/w) of total fatty acids content of said preparation,
preferably more than 10% (w/w), more preferably 20-50% (w/w),
wherein said PUFA is covalently bound to said
glycerophospholipid.
[0126] Said lipid may be a naturally occurring lipid, or a
synthetic lipid.
[0127] Preferably, said lipid is a glycerophospholipid in which at
least some of the sn-1 or sn-2 groups of the glycerol backbone are
substituted with said poly-unsaturated fatty acid (PUFA) acyl
groups.
[0128] In one particular embodiment, said lipid is a
glycerophosphlipid of formula I: 2
[0129] wherein R" represents a moiety selected from serine (PS),
choline (PC), ethanolamine (PE), inositol (PI), glycerol (PG) and
hydrogen (phosphatidic acid--PA), and R and R', which may be
identical or different, independently represent hydrogen or an acyl
group, wherein said acyl group is selected from saturated,
mono-unsaturated or poly-unsaturated acyl groups (PUFA),
particularly long-chain poly-unsaturated fatty acids (LC-PUFA),
more preferably omega-3 and/or omega-6 acyl groups, and salts
thereof, with the proviso that R and R' cannot simultaneously
represent hydrogen, and wherein said polyunsaturated acyl groups
comprise at least 5% (w/w) of total lipid fatty acids, preferably
more than 10% (w/w), and particularly 20-50% (w/w).
[0130] In one more particular embodiment of said preparation, R
represents hydrogen and R' represents an acyl group. Alternatively,
R' represents hydrogen and R represents an acyl group.
[0131] Considering these latter embodiments, when said acyl group
is preferably an omega-3 acyl group, it may be an eicosapentaenoyl
(EPA), a docosahexaenoyl (DHA) group, or linolenic omega-3 group.
And, when said acyl group is preferably an omega-6 acyl group, it
may be an arachidonoyl (ARA) group, or a linoleic omega-6 group. A
further possibility is that said acyl group may be a linolenoyl
(18:3) group.
[0132] In a yet further embodiment of the preparation of the
invention, R" may be any one of serine, choline, ethanolamine,
inositol or glycerol.
[0133] In a further particular embodiment, the identity and content
of R and R' are predetermined.
[0134] The preparation of the invention which comprises the
compound of formula I in which R" is serine, mimics the composition
of human brain PS.
[0135] Nonetheless, the invention also refers to preparations
comprising the compound of formula I in which R" is serine, which
are different from human brain PS, but still have an improved
bioactivity, particularly as compared to soybean-PS.
[0136] Traditionally, PS active ingredients used as dietary
supplements were produced by the extraction of animal brains,
particularly bovine brains. The PS extracted from animal brain
tissues, similarly to human brain PS, has a fatty acid composition
which is characterized by relatively higher levels of omega-3
moieties, compared to the levels of omega-3 found in plant
phospholipids. PS has the following structure: 3
[0137] Human brain PS is characterized by over 20-30% PS containing
omega-3 fatty acyls, preferably at the sn-2 position of the
glycerol moiety, and mainly DHA or EPA. As mentioned above,
phospholipids, and PS in particular, are responsible for membrane
structure and physical properties. One of the major physical
properties governed by phospholipids is the fluidity of these
membranes. Omega-3 fatty acids, DHA and EPA in particular, also
have a crucial role in membrane fluidity in light of their unique
3D structure. Therefore, PS with omega-3 fatty acyl moieties, DHA
and EPA in particular, has unique bio-functionality which cannot
stem from just the basic phospholipid skeleton of this
phospholipid.
[0138] Considering the risks involved with prion diseases,
particularly bovine spongiform encephalopathy (BSE), as well as
other disadvantages associated with ingredients obtained from
animal sources, PS supplements are usually prepared using PS
originating from soybean lecithin. This lecithin is enriched,
usually enzymatically, with PS. This method of production results
in PS with a fatty acid profile of soybean phospholipids, which is
characterized by low level of omega-3 fatty acids, and almost no
DHA and EPA. This PS active ingredient is also known as
soybean-PS.
[0139] Although the bio-functionality of soybean-PS in the
improvement of cognitive function has been shown to be similar to
that of bovine-PS, it is still different from human brain PS. It is
a purpose of the present invention to provide a PS ingredient with
a predetermined fatty acid composition that mimics the fatty acid
composition of the human brain PS.
[0140] It is a further object of the present invention to provide a
PS ingredient which, while not identical to naturally occurring
brain PS, is characterized by improved functionality, particularly
in comparison with soybean-PS. This improved PS ingredient has a
predetermined fatty acid composition.
[0141] The PS ingredient of the present invention is enriched with
omega-3 fatty acyls, preferably DHA, EPA or linolenic omega-3.
Furthermore, the PS of this invention is enriched with omega-3
fatty acyls covalently bonded to either or both of the sn-1 or sn-2
positions of the glycerol moiety in the PS backbone.
[0142] The present invention is also related and describes other
phospholipids, such as phosphatidylcholine (PC),
phosphatidylethanolamine (PE), phosphatidyl-inositol (PI),
phosphatidylglycerol (PG) and phosphatidic acid (PA), enriched with
omega-3 fatty acids, preferably DHA, EPA, or linolenic acid which
are covalently bonded at either or both of the sn-1 or sn-2
positions of the glycerol moiety of the phospholipid.
Alternatively, the phospholipids of the invention are enriched with
omega-6 fatty acids.
[0143] When referring to PS in the present description, it should
be taken to mean also any other lipid, such as, but not limited to,
the polar lipids listed above.
[0144] In a preferred embodiment, the amount of omega-3
(particularly EPA, DHA or linolenic acid) or omega-6 (particularly
ARA and linoleic acid) fatty acids in the PS ingredient of the
invention is greater than 10% at either or both of the sn-1 or sn-2
positions, preferably at the sn-2 position, preferably over 20% and
most preferably above 40%.
[0145] As mentioned, the desired omega-3/omega-6 fatty acyls can be
bonded at both or only one of the sn-1 and sn-2 positions.
[0146] The fatty acid composition of the PS preparation of this
invention can have a predetermined fatty acid composition similar
to or different from the fatty acid composition found in normal
healthy human brain, provided it has enhanced activity,
particularly compared to the activity of plant PS, for example
soybean-PS.
[0147] The preparation of the omega-3/omega-6-enriched PS
preparation of this invention can be enzymatic, chemical or by
molecular biology methods. Briefly, the PS can be enriched with
omega-3 or omega-6 moieties by enzymatic processes, e.g. enrichment
of a natural phospholipid/lecithin with omega-3 fatty acids by
enzymatic transesterification/esterification followed by
transformation of the head group to serine (using PLD enzymes) to
obtain a PS-omega-3/omega-6 conjugate. Another enzymatic pathway is
to obtain a lecithin or phospholipid source which is naturally rich
in omega-3 acids, such as krill phospholipids, and transform their
head groups to serine. It is to be noted that the fatty acid
composition of the PS obtained by this method has an omega-3
composition which is predetermined by the source of choice (fish,
krill, algae, etc.). Such methods have been thoroughly described in
Applicant's co-pending PCT Application claiming priority from IL
158553.
[0148] The PS-omega-3/omega-6 ingredient of the present invention
can also be prepared by chemical transesterification/esterification
methods that will enrich the sn-1 and 2 positions with omega-3 or
omega-6 acyl residues. Such methods of preparation of PS-6mega-3
and PS-omega-6 have been described in Applicant's co-pending PCT
Application claiming priority from IL 158553.
[0149] Alternatively, the PS ingredient of the present invention
can be prepared by GMO (genetically modified
organisms)/biotechnology methods, for example, providing
phospholipids-producing organisms with omega-3 or omega-6 fatty
acids to obtain phospholipids enriched with omega-3 or omega-6 PS.
It may be preferred to use genetically engineered plants or
microorganisms, to avoid use of animal sources.
[0150] The PS of this invention can have the omega-3 or omega-6
fatty acid composition of a specific lecithin raw material,
relatively rich with omega-3 or omega-6 fatty acids, enriched with
PS to yield a PS ingredient with elevated omega-3 or omega-6 fatty
acids levels, compared to soybean-PS. Such is the case, for
example, when phospholipids from krill are used as the starting
material, as described above.
[0151] In a preferred embodiment the PS enriched with omega-3 or
omega-6 can be soybean-PS or any other PS, from plant, animal, for
example krill, or microorganism source. In a further preferred
embodiment the omega-3 or omega-6 enrichment can be performed on a
lecithin, which in turn is enriched with PS by
transphosphatidylation.
[0152] It is the purpose of this invention to provide a novel PS
ingredient, enriched with omega-3 fatty acids, resulting in an
ingredient with improved efficacy compared to ingredients
containing natural or simply enriched PS.
[0153] The improved PS preparation of this invention exhibits
enhanced activity in the improvement and treatment of cognitive and
mental conditions and disorders as well as the maintenance of
normal functions of brain related systems and processes. These
include, but are not limited to ADHD, multiple sclerosis (MS),
dyslexia, depression, learning capabilities, intensity of brain
waves, stress, mental and psychiatric disorders, neurological
disorders, hormonal disorders, concentration and attention, mood,
brain glucose utilization, and general cognitive and mental well
being.
[0154] The novel lipid preparation of this invention exhibits
enhanced activity in the improvement of cognitive functions, as
detailed hereunder, over omega-3 or omega-6 lipids per se or
soybean-PS. Furthermore, under certain conditions or for all or
specific disorders, the lipid preparation of the invention is
effective at a dosage of less than 100 mg/day. This is lower that
the current recommended daily dosage of soybean-PS (100-300 mg/day)
or omega-3 lipids (approx. 1-2 g/day or more) currently available
in the market. Nonetheless, dosages of 100-600 mg/day are preferred
for enhanced efficacy of the lipid preparation of the
invention.
[0155] An important advantage of the PS preparation of the
invention is that it exhibits multifunctional activity. This
multi-functionality is exhibited by improvement in cognitive and
mental functions, together with improvement of other health
disorders or conditions.
[0156] The enhanced activity of this PS ingredient, as well as its
multi-functionality, may arise from the unique structure of this
ingredient and its influence on the physical and chemical
properties of cell membranes in brain tissues as well as other
organs and tissues.
[0157] The enhanced activity of this PS ingredient, as well as its
multi-functionality, may also be attributed to the enhanced
bioavailability of the omega-3 fatty acids, due to their
incorporation in the PS skeleton. Thus, the omega-3 fatty acids can
be delivered to the brain across the blood-brain barrier, being a
part of the PS molecule, which readily passes this barrier. The PS
functions as a delivery platform for the fatty acids bound thereto,
to various organs and tissues, thereby enhancing their
bioavailability.
[0158] The additional health disorders or conditions which are
affected by the multifunctional PS preparation of the invention
include, but are not limited to high blood cholesterol levels, high
triglycerides levels, high blood fibrinogen levels, HDL/LDL ratio,
diabetes, metabolic syndrome, menopausal or post-menopausal
conditions, hormone related disorders, vision disorders,
inflammatory disorders, immune disorders, liver diseases, chronic
hepatitis, steatosis, phospholipid deficiency, lipid peroxidation,
dysrhythmia of cell regeneration, destabilization of cell
membranes, coronary artery disease, high blood pressure, cancer,
hypertension, aging, kidney disease, skin diseases, edema,
gastrointestinal diseases, peripheral vascular system diseases,
allergies, airways diseases, neurodegenerative and psychiatric
diseases.
[0159] The new ingredients of the invention can be delivered and
utilized in a variety of products. Such products include dietary
supplements, functional foods, pharmaceutical delivery systems,
etc.
[0160] The preparation of pharmaceutical compositions is well known
in the art and has been described in many articles and textbooks,
see e.g., Gennaro A. R. ed. (1990) Remington's Pharmaceutical
Sciences, Mack Publishing Company, Easton, Pa., and especially
pages 1521-1712 therein.
[0161] As dietary supplements, the preparations of the invention
may be used in the form of soft gel capsules, tablets, syrups, and
other common dietary supplements delivery systems.
[0162] As functional foods, the preparations of the invention can
be incorporated and used in a variety of foods, such as dairy
products, ice-creams, biscuits, soy products, pastry and bread,
sauces, condiments, oils and fats, margarines, spreads, cereals,
drinks and shakes, infant formulas, infant foods (biscuits, mashed
vegetables and fruits, cereals), bars, snacks, candies, chocolate
products.
[0163] As pharmaceutical products, the preparations of the
invention can be delivered orally, intravenously, or by any other
conventional or special route of administration.
[0164] The new preparations of the invention may be in the form of
fluid oil, powder, granules, wax, paste, oil or aqueous emulsion,
and any other form that will enable its use in the target
applications.
[0165] Pharmaceutical or nutraceutical formulations comprising the
PS preparation of the invention may include physiologically
acceptable free flowing agents, other additives, excipients,
dessicants and diluents, colorants, aroma and taste ingredients,
and any ingredients that control physical, organoleptic, and other
properties, as well as additional active ingredients, for example
minerals, vitamins, other nutritional additives.
[0166] The utilization of omega-3 lipids in a variety of
applications, and especially as ingredient of functional foods, is
hindered due to their distinct fish odor. Thus, another advantage
of the omega-3 enriched phospholipids ingredients of the invention
is that they have reduced odor or taste of omega-3 acyl moieties,
due to the covalent binding of these groups to the PS backbone.
This increases the vapor pressure of these materials, hence
reducing their distinct aroma. Thus, the covalent binding of the
omega-3 fatty acids to the phospholipid backbone, especially PS,
alters and improves their taste properties. Moreover, the PS
ingredient of the invention also offers enhanced stability to the
oxidation sensitive omega-3 fatty acids. Phospholipids in general,
and PS in particular, are known to act as anti-oxidants and
stabilizers.
[0167] These benefits make the lipid preparation of the invention
highly beneficial and important in a variety of applications and
especially in functional foods, where stability, aroma and taste
are fundamental requirements.
[0168] Furthermore, these novel ingredients can be formulated with
additional lipids for an even enhanced bio-functionality and
efficacy.
[0169] The polar lipids derivatives of PUFA, such as the PS-PUFA
derivatives have exhibited high stability as a preparation and
additionally in several food applications, used in the clinical
trials of the present invention. The stability of these sensitive
compounds is emerging from the covalent combination of
phospholipids, known in the past to be used as preservatives and of
the un-stable PUFA moieties.
[0170] The new ingredients of the invention can be delivered and
utilized in a variety of products. Such products include dietary
supplements, functional foods, pharmaceutical delivery systems,
etc.
[0171] Disclosed and described, it is to be understood that this
invention is not limited to the particular examples, process steps,
and materials disclosed herein as such process steps and materials
may vary somewhat. It is also to be understood that the terminology
used herein is used for the purpose of describing particular
embodiments only and not intended to be limiting since the scope of
the present invention will be limited only by the appended claims
and equivalents thereof.
[0172] It must be noted that, as used in this specification and the
appended claims, the singular forms "a", "an" and "the" include
plural referents unless the content clearly dictates otherwise.
[0173] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated integer or step or group of
integers or steps but not the exclusion of any other integer or
step or group of integers or steps.
[0174] The following Examples are representative of techniques
employed by the inventors in carrying out aspects of the present
invention. It should be appreciated that while these techniques are
exemplary of preferred embodiments for the practice of the
invention, those of skill in the art, in light of the present
disclosure, will recognize that numerous modifications can be made
without departing from the spirit and intended scope of the
invention.
EXAMPLES
Example 1
[0175] Methods:
[0176] Animals and Diet
[0177] Male Wistar rats originated from the same colonies were
obtained from Harlen. Fifty rats were randomly divided into five
dietary supplemented groups, in addition to their normal diet: (i)
a group fed 0.1 g medium-chain triglycerides (MCT)/1 ml supplement
matrix (MCT group); (ii) a group fed 0.1 g DHA/EPA (20/30% of total
fatty acids composition, diluted with MCT to generate 30% (w/w)
LC-PUFA compound) triglycerides/1 ml supplement matrix (LC-PUFA
group); (iii) a group fed 0.1 g soybean lecithin-derived PS (20%
SB-PS w/w)/1 ml supplement matrix (SB-PS group); and (iv) a group
fed 0.1 g PS-{overscore (.omega.)}3 (20% PS w/w, and total LC-PUFA
composition of 30%)/1 ml supplement matrix (PS group). The
supplement matrices were stored at -20.degree. C., and fresh
portions were fed to the rats every day. All supplements were
handled so as to minimize oxidation of the fatty acids. Rats
consumed the diet and water ad libitum. All rats were housed in a
standard environment, in which temperature was maintained at
24.+-.0.5.degree. C., and the relative humidity was kept at
65.+-.5% with 12-h periods of light and dark. Body weight was
measured at the beginning and the end of the treatment period.
[0178] The PS-{overscore (.omega.)}3 compound used in this study
mimics the fatty acids composition of the mammalian brain PS, with
respect to its DHA content (20%). Generally, in animal cells, the
fatty acid composition of PS varies from tissue to tissue, but does
not appear to resemble the precursor phospholipids, either because
of selective utilization of specific molecular species for
biosynthesis or because or re-modeling of the lipid via
deacylation-reacylation reactions. In human plasma,
1-stearoyl-2-oleoyl and 1-stearoyl-2-arachidonoyl species
predominate, but in brain and many others related tissues
1-stearoyl-2-docosahexaenoyl species are very abundant [O'Brien et
al. (1964) J Lipid Res. 5(3):329-38]. An early work by Yabuuchi et
al. [Yabuuchi et al. (1968) J Lipid Res. 9(1):65-7] established
that the DHA content in bovine gray matter is up to 30% of the
total fatty acids composition; most of the total amount of DHA was
located at the sn-2 position (60%). It was the bovine brain PS that
Toffano and Bruni reported in the early 1980's to be a
pharmacologically active compound, which counteracts age-related
changes in the central nervous system [Toffano et al. (1980)
Pharmacol. Res. Commun. 12:829-845].
[0179] Behavioral Testing
[0180] Water maze test, which was developed by Morris [Stewart, C
A. and Morris, R G. (1993) The water maze. In: Behavioural
Neuroscience: A Practical Approach. Vol. 1 (Saghal, A., ed.), pp.
107-122. Oxford University Press, New York, N.Y.], uses a circular
tank (137 cm diameter, 35 cm deep) constructed of opaque white
plastic. It is filled with water (21-22.degree. C.) to a depth of
28 cm, and the water is rendered opaque by the addition of soluble,
nontoxic white latex paint. In the place version of the maze, the
rat develops a spatial map of the extra-maze cues, which it then
uses to locate the platform. Thus the distance swum to the platform
and the time taken in doing so should decrease over testing
sessions (days) as the rat learns the location of the platform.
Moreover, it is expected that if the rat has learned the location
of the platform in relation to the extra-maze cues, its initial
response on the probe trial will be to swim directly to the
quadrant in which it expects to find the platform. Thus the
distance swum (and time spent) in the target quadrant should be
greater than that in the other two quadrants (excluding the start
quadrant). The distance swum to the platform as well as the latency
to reach the platform were monitored using the video-based tracking
system. The behavioral testing was conducted during the dark cycle,
when rats are normally most active.
[0181] The pool was located in a test room in which there were many
extra-maze spatial cues. On the first three days, the rats were
required to locate the hidden platform (15.5 cm.times.15.5 cm)
situated 1 cm below the surface of the water. There were two
acquisition testing sessions per day, with four trials per session.
On each trial, the rat was placed, facing the wall, in one of the
four quadrants in the tank, and allowed to swim for a maximum of 60
seconds. Once the rat found the platform, it remained there for 5
seconds before being returned to the holding cage, which was kept
warm on a heating pad. If the rat failed to find the platform in
that time, it was placed on it for 5 seconds before being returned
to the holding cage. Each of the eight trials conducted each day
was started from a different quadrant, with the order determined
pseudorandomly (not twice from the same quadrant) and varying from
day to day. The intertrial interval (ITI) was 120 seconds, counted
from the end of one trial to the beginning of the next. On fourth
day, followed by a session as abovementioned, the platform was
removed from the tank, and a probe trial was conducted by placing
the rat in the quadrant opposite to that of the platform and then
allowing it to swim for 60 seconds. The day following the probe
trial, the rats were tested with a session in which the maze was
set up as previously described, followed by a session in which the
platform was repositioned to the center of the opposite quadrant.
The latency to find the platform on each trial was recorded.
Scopolamine (1 mg/Kg) was intraperitoneally (i.p.) administered
30-minutes before the indicated trials.
[0182] Lipid Extraction and NMR Analyses
[0183] At the end of the behavioral testing, the rats were
anesthetized with Halothane and then decapitated. Liver and brain
tissues were quickly removed and stored (at -80.degree. C.). The
lipid fraction of the rat tissues were extracted using a modified
version of the technique described by Bligh and Dyer 1959 [Bligh
and Dyer, (1959) Can. J. Biochem. Physiol. 37, 911-917]. Briefly,
500-700 mg and 300-1200 mg of liver and brain tissues,
respectively, were homogenized in a solution of CDCl3, methanol and
CS-EDTA (1:2:2 v:v:v). The homogenates were further agitated using
ultrasonic bath (10 min, 80.degree. C.), followed by additional
vigorous shaking (20 min). The relative ratio of the phospholipids
in the homogenates was measured using high-resolution .sup.31P-NMR
at 121. MHZ using a 7.06 Tesla General Electric spectrometer.
[0184] These homogenates were further analyzed for their fatty
acids distribution. First, the lipids extracts were desalted by
reverse-phase chromatography using an RP-18 column [Williams et al.
(1980) J. Neurochem.; 35, 266-269]; diheptadecanoyl
phosphatidylcholine was added as internal standard before the
loading on the column. Phospholipids were separated from neutral
lipids, such as cholesterol, on silica gel plates (Merck 60)
developed in isohexane: ether: formic acid 80:20:2 (v:v:v). The
phospholipids spot was visualized by spraying primulin solution and
compared with authentic phospholipids standards. Henicasonoic
methyl ester (C21:0) was added as a 2nd internal standard and the
phospholipids were converted to methyl esters by mild acid
hydrolysis with 1% methanolic H2SO4 overnight at 50.degree. C. The
fatty acids profile of the different samples was determined by
gas-liquid chromatography.
[0185] Results
[0186] Anti-dementia effects of bovine brain cortex-derived PS
(BC-PS) has been demonstrated by several double-blind,
placebo-controlled studies, see review by [Kidd P. (1996) Alt Med
Rev. 1(2):70-84]. In the past decade both BC-PS and soybean
lecithin transphosphatidylated PS (SB-PS) were shown to recover the
scopolamine-induced amnesia in rodent, although the fatty acids
composition is considerably different between these compounds
[Zanotti A et al. (1986) Psychopharmacology (Berl). 90(2):274-5.;
Claro F. et al. (1999) Physiol Behav. 67(4):551-4; Sakai M. (1996)
Nutr Sci Vitaminol. (Tokyo) 42(1):47-54; Furushiro M et al. (1997)
Jpn J Pharmacol. 75(4):447-50]. The means of PS administration in
these studies was predominantly intravenous or intraperitoneal;
although Furushiro et al. described also oral administration of
SB-PS that antagonized amnesic effects of scopolamine. However, in
the latter study the investigator used a considerable high dose of
SB-PS, ranging between 60 to 240 mg/Kg.
[0187] In the presented study, rat diet was supplemented with the
above-mentioned treatments (diets i, ii, iii, iv and v) for three
months before the maze test was performed. In the acquisition stage
(FIG. 1A-1D) there is an expected and marked increase in the
latency time to find the platform after the administration of
scopolamine (1 mg/Kg) of all groups. Although the latency curves of
MCT and PS-.quadrature.3 groups are similar, there is a
statistically smaller difference in the latency change, induced by
scopolamine, in the PS-{overscore (.omega.)}3 group with respect to
the latency presented by the MCT group (P-value<0.07 Vs.
P-value<0.0007, respectively). Similarly, the groups treated
with SB-PS or LC-PUFA, demonstrated a reduced effect of scopolamine
on their learning curves, with respect to the MCT group (see FIG.
1A-1D). Having all groups learn the task at a similar rate,
resembles data presented by Blokland et al. [Blokland et al. (1999)
Nutrition 15(10): 778-83], which showed no difference between PS
obtained from different sources and the empty vehicle, in a water
maze test.
[0188] What is particular to the present trial is the accelerated
rate in learning the task under the scopolamine sedation. This was
not demonstrated previously [Furushiro et al. (1997) id ibid.;
Suzuki et al., (2000) Jpn. J. Pharmacol. 84, 86-8]. Note that in
these studies the rodent faced a different task (passive
avoidance). In Suzuki et al. 2001 (J. Nutr. 131: 2951-6) the
investigators utilized considerably older rats (24-25 months old)
than the ones tested in the present trial. The latency time in the
acquisition step was considerably longer for the aged rats compared
to the young ones that were tested (eight weeks). Interestingly,
although the latency time in the present trial of non-sedated rats
is somewhat comparable to the younger rats tested by Suzuki et al.
[Suzuki et al. (2001) id ibid.], the scopolamine-induced amnesia
latency time in the MCT group resembles the one obtained at the
described study for elderly rats. In conclusion, scopolamine
induced a comparable long latency time in the control group (MCT).
This effect was augmented to a different extent by long-term
treatment of rats with either PS or LC-PUFA.
[0189] In the probe trial, the rats treated with PS-{overscore
(.omega.)}3 showed a distinctively higher tendency than MCT-treated
ones (P<0.085) to be present at the zone in which the platform
was located during the acquisition of the task.(FIG. 2), indicating
that the rats had learned the spatial location of the platform.
Moreover, PS-{overscore (.omega.)}3 treated rats presented a
reduced tendency (P<0.08) to swim in the periphery zone, but
rather spent in the central zone. These latter indications,
presented by the PS-{overscore (.omega.)}3 group are related to a
higher adventurous characteristic and could be somewhat correlated
with the open field behavior trial. Interestingly, in Blokland et
al. [Blokland et al. (1999) id ibid.] BC-PS treated mice
demonstrated a non-significant but clear tendency to be less
adventurous in the open field behavior trial, by spending less time
in the center area. With respect to the remarkable learning
abilities demonstrated by the rats that were treated with
PS-{overscore (.omega.)}3, it is interesting to compare their
performance in the Morris water maze task in the spatial probe test
to the one obtained by the SB-PS treated animals by Suzuki et al
[Suzuki et al (2001) id ibid.]. Though the percent of time spent in
the quadrant where the platform was located is similar
(.about.45%), it is remarkable that the dosage in the current study
was merely one third of the administration levels in Suzuki et al
2001 (20 mg/kg vs. 60 mg/kg, respectively). Indeed, in the present
study there was no significant change in the time that the SB-PS
(20 mg/kg) treated rats spent in this quadrant when compared with
the values obtained by the MCT-treated group [FIG. 1C and FIG. 1A,
respectively]. In summary, the PS-{overscore (.omega.)}3 treated
group learning abilities were markedly higher than the control, in
a considerably low level of PS administration. In addition, the
rats treated with PS-{overscore (.omega.)}3 were less conservative
and more adventurous in studying the maze in the absence of the
platform.
[0190] Finally, the most prominent and outstanding data obtained in
the present study was the response to the repositioning of the
platform. All groups presented a shorter latency in finding the
platform at the first session, when compared to the one obtained by
the MCT-treated group, under scopolamine sedation (FIG. 3A-3D).
These data suggest that LC-PUFA, and more potently PS, can
attenuate scopolamine-induced amnesia, as previously presented by
other studies (see selected references above).
[0191] Surprisingly, in the second session, there were no
differences between the latency in finding the platform after its
repositioning in all groups but the PS-{overscore (.omega.)}3
treated group. In fact, it seemed that in all treatments but the
PS-{overscore (.omega.)}3 there was no learning process of the
position of the platform. The PS-{overscore (.omega.)}3 group
presented a remarkably different behavior; it seemed that there was
no lag in the learning of the repositioned platform in the rat
treated with this anti-muscarinic drug. The ability of the
PS-{overscore (.omega.)}3 treated group to locate the platform
after it had been repositioned seemed to be contradictory with the
result obtained earlier in the spatial probe test (FIG. 2), where
these rats showed preference for the third quadrant. Pearce and
colleagues [Pearce et al. (1998) Nature 396: 75-77] attempted to
resolve this discrepancy, by describing two means for memorizing a
specific spatial location. One is to use a cognitive map that
encodes information about the geometric relationship between the
object and several land marks (the cognitive map method) and the
other is the use of heading vectors that specify the direction and
distance from a single landmark to the object (the heading vector
method). In the present test, the rats could locate the platform
from the above-mentioned cues and/or from the distance and
direction with respect to the walls. In the acquisition and the
spatial probe test, both methods contributed to the score of
finding the platform. However, in the repositioning test, the
cognitive abilities which are related to the heading vector method
and the short-term memory (working memory), made the difference.
The heading vector method, because the distance from the wall was
not effected by the repositioning (just the quadrant), and the
working memory due to the benefits in memorizing the areas already
explored that enable an effective search in the pool.
[0192] It has been previously reported that the mechanism by which
PS attenuates the scopolamine effect could be attributed not only
to a beneficial effect on the cholinergic circuitry, but PS could
also have an effect on the serotonergic neuronal system [Furushiro
et al. (1997) id ibid.]. It appears that the presented data could
be the result of more than one neuronal system alteration, possibly
the dopaminergic. In an earlier study [Drago et al. (1981)
Neurobiol Aging, 2(3):209-13], it was suggested that the alteration
in the obtained behavioral changes between BC-PS treated aged rats
to their control could be attributed not only to the modifications
in cholinergic and serotonergic transmission, as described above,
but also through affecting the catecholaminergic (like dopamine)
system. In this study the facilitated acquisition of active
avoidance behavior as studied in shuttle-box and pole jumping test
situations, and the retention of active and passive avoidance
responses were improved in the PS-treated rats. Tsakiris [Tsakiris,
S. (1984) Z Naturforsch [C], 39(11-12):1196-8] reported on an
indirect effect of PS on the dopamine related adenylyl cyclase,
through membrane fluidity mechanism. Interestingly, it has also
been reported [Chalon, et al. (1998) J Nutr.; 128(12):2512-9] that
enriched diet with high level of (n-3) PUFA could result in an
effect on the cortical dopaminergic function. It is conceivable
that the existence of LC-PUFA on the backbone of the phospholipids
was highly beneficial in terms of such a multi-neurotransmitter
mechanism.
[0193] The biochemical analyses of the present results in liver
tissues (FIG. 4A) shows that in rats supplemented with PS for three
months (SB-PS and PS-{overscore (.omega.)}3) there was a notable
increase in the levels of the primer phospholipids, i.e.
phosphatidylcholine (PC). These data is consistent with early
observations regarding the liver and its major role in the
phospholipids uptake and the primary metabolism of most fatty
acids. Wijendran and colleagues [Wijendran et al. (2002) Pediatr.
Res. 51:265-272] described a study in which baboons were fed
labeled LC-PUFA on the backbone of PC and triglycerides, and
demonstrated that the levels of incorporation of LC-PUFA on a
phospholipid backbone to the liver was higher than the extent of
incorporation of LC-PUFA on the triglycerides backbone. In
addition, PS levels of rats fed with PS-{overscore (.omega.)}3 were
elevated in cortex tissues analyses of phospholipids distribution
(FIG. 4B), comparing with MCT. Interestingly, the phospholipids
fatty acids profile of these cortices (Table 1) demonstrate a
marked elevation in the DHA content of the rats fed with
PS-{overscore (.omega.)}3 (P=0.007). Similar elevation was noted
for LC-PUFA fed rats, however to a reduced extent compared with
PS-{overscore (.omega.)}3 treatment (14.6 versus 17.5, respectively
PS-{overscore (.omega.)}3) and MCT (14.6 versus 12.3, respectively
P=0.02). This difference in the DHA levels between the two omega-3
groups might suggest enhanced bioavailability of DHA when it is
esterified to the backbone of phospholipids rather than to
triglycerides. Similar conclusions were drawn by Lemaitre-Delaunay
and colleagues [Lemaitre-Delaunay et al. (1999) J. Lipid Res.;
40:1867-1874], when they had study the kinetics and metabolic fate
of labeled DHA on triglycerides versus its enrichment in
lysophsphaytidylcholine, and by Wijendran et al. [Wijendran et al.
(2002) id ibid.] in the above-mentioned baboons study.
[0194] Interestingly, this increase in DHA content in the cortices
of both PS-{overscore (.omega.)}3and LC-PUFA fed rats is
accompanied with a statistically significant decrease in the levels
of oleic acids and to somewhat lower extent of linoleic acid (Table
1) in the phospholipids fraction. Similar changes in the ratios of
the fatty acids profile was demonstrated by others, by feeding
rodents with dietary fats enriched with LC-PUFA [for example:
Yamamoto et al. (1987) J. Lipid Res. 28: 144-151]. The SB-PS group
showed a very similar profile to the MCT group.
[0195] In sum, the improved performance in the Morris water maze
test of the PS-{overscore (.omega.)}3 treated rats under
scopolamine sedation strongly supports the potency of PS-{overscore
(.omega.)}3 as an anti-dementia and age-associated memory
impairment effects. This cognitive enhancement is further supported
by the biochemical evidence of the elevated phospholipids levels in
the liver and brain tissues (FIG. 4A-4B), and with elevated levels
of DHA attached to the phospholipids from the cortex of the
PS-{overscore (.omega.)}3 fed rats.
[0196] Table 1 summarizes the effect of dietary LC-PUFA from
different sources on the fatty acids profile in cerebral
phospholipids from elderly Wistar rats. Fatty acids from the
purified phospholipids fraction were analyzed by gas-liquid
chromatography. The major fatty acids are expressed as % of total
fatty acids in the phospholipids. Values represent mean .+-.S.D. of
four different rats per treatment. Statistical significant between
different supplements and MCT group is presented as followed:
*P<0.05;**P<0.01.
1TABLE 1 Fatty acids MCT LC-PUFA SB-PS PS-{overscore (.omega.)}3
C16:0 12.9 .+-. 1.4 14.6 .+-. 4.7 13.7 .+-. 4.7 13.6 .+-. 4.4 C16:1
1.0 .+-. 0.7 1.0 .+-. 0.3 1.5 .+-. 0.4 1.5 .+-. 0.8 C18:0 17.9 .+-.
1.0 20.1 .+-. 1.3* 17.2 .+-. 2.8 18.0 .+-. 5.5 C18:1 36.5 .+-. 1.8
32.0 .+-. 2.8* 37.0 .+-. 6.8 30.7 .+-. 4.1* (n - 9) C18:1 3.7 .+-.
0.5 4.3 .+-. 0.2* 4.0 .+-. 0.3 4.8 .+-. 1.5 (n - 7) C18:2 7.2 .+-.
0.7 4.5 .+-. 0.6** 7.1 .+-. 2.6 5.1 .+-. 2.7 C20:1 2.5 .+-. 0.5 2.9
.+-. 0.8 2.1 .+-. 0.4 2.3 .+-. 0.3 C22:6 12.3 .+-. 1.7 14.6 .+-.
0.6* 12.4 .+-. 3.2 17.5 .+-. 2.4** C24:1 3.4 .+-. 1.0 3.3 .+-. 1.3
2.8 .+-. 0.9 2.0 .+-. 1.2* rest 2.7 .+-. 0.1 2.8 .+-. 0.4 2.1 .+-.
0.9 4.5 .+-. 3.0
Example 2
PS-omega-3 in the Treatment of ADHD Children
[0197] Attention-deficit/hyperactivity disorder (ADHD) encompasses
a broad constellation of behavioural and learning problems and its
definition and diagnosis remain controversial [Kamper (2001) J.
Pediatr. 139:173-4; Richardson et al. (2000) Prostaglandins Leukot.
Essent. Fatty Acids, 63(1-2):79-87]. The etiology of ADHD is
acknowledged to be both complex and multi-factorial. Traditionally,
ADHD is the diagnosis used to describe children who are
inattentive, impulsive, and/or hyperactive. Roughly 20-25% of
children with ADHD show one or more specific learning disabilities
in math, reading, or spelling [Barkley, R. A. (1990)
Attention-deficit hyperactivity disorder: a handbook for diagnosis
and treatment. New York: Guilford Press]. Children with ADHD often
have trouble performing academically and paying attention, and may
be disorganized, have poor self-discipline, and have low
self-esteem. A conservative estimate is that 3-5% of the school-age
population has ADHD [American Psychiatric Association. Diagnostic
and statistical manual of mental disorders. 4th ed. (DSM-IV)
Washington, DC: American Psychiatric Association, 1994]. Treatments
for ADHD include behavior therapy and medications, mainly
methylphenidate (Ritalin.TM.). Psychostimulant drugs and
antidepressants are often used to calm children with ADHD, with an
effectiveness rate of .about.75% (Swanson et al. Except Child 1993;
60:154-61). The advantages of using these medications include rapid
response, ease of use, effectiveness, and relative safety.
Disadvantages include possible side effects, including decreased
appetite and growth, insomnia, increased irritability, and rebound
hyperactivity when the drug wears off [Ahmann et al. (1993)
Pediatrics; 91:1101-6]. Moreover, these medications do not address
the underlying causes of ADHD. Thus, studies to elucidate the
potential contributors to the behavior problems in ADHD may lead to
more effective treatment strategies for some children.
[0198] Omega-3 fatty acids are specifically implicated in
maintaining central nervous system function. Deficiency of n-3
fatty acids in rats and monkeys has been associated with
behavioral, sensory, and neurological dysfunction [Yehuda et al.
(1993) Proc. Natl. Acad. Sci. USA; 90:10345-9; Reisbick et al.
(1994) Physiol. Behav. 55:231-9; Enslen et al. (1991) Lipids;
26:203-8]. Several studies have focused on essential fatty acid
metabolism in children with ADHD [Colquhoun et al. (1981) Med
Hypotheses; 7:673-679]. Children with hyperactivity have been
reported to be more thirsty than normal children and have symptoms
of eczema, asthma, and other allergies [Mitchell et al. (1987)
Clin. Pediatr.; 26:406-11]. For example, in a cross-sectional study
in 6-12-y-old boys recruited from central Indiana, it was showed
that 53 subjects with ADHD had significantly lower proportions of
key fatty acids in the plasma polar lipids [arachidonic acid (AA;
20:4n-6), eicosapentaenoic acid (EPA; 20:5n-3), and docosahexaenoic
acid (DHA; 22:6n-3)] and in red blood cell total lipids (20:4n-6
and 22:4n-6) than did 43 control subjects [Stevens et al. (1995)
Am. J. Clin. Nutr.; 62:761-8]. However, recent publications
[Hirayama et al. (2004) Eur. J. Clin. Nutr.; 58(3):467-73; Voigt et
a. (2001) J Pediatr.; 139(2):189-96] that investigated whether DHA
supplementation would result with ameliorate the symptoms in ADHD
children, suggested that careful attention should be paid as to
which fatty acid(s) is used. In these studies DHA supplementation
had demonstrated only marginal if any beneficial effects.
[0199] Recently, it has been suggested that one of the possible
solutions to the nutrient deficiencies which are common in ADHD,
could be PS supplementation [Kidd (2000) Altern Med Rev.;
5(5):402-28].
[0200] Method
[0201] Subjects and diet
[0202] Ninety 8-to-13-year old children diagnosed according to the
DSM-IV as ADHD, were assigned randomly, in a double-blind fashion
to receive PS-{overscore (.omega.)}3 (300 mg/d; containing total
450 mg/d DHA/EPA), 450 mg/d DHA/EPA or canola oil (30 per group)
for two months, while not taking stimulant medication or other
supplements. Characterizing the subject as ADHD included a score
lower than -1.8 in the Test of Variables of Attention.
[0203] Data Analysis
[0204] At the conclusion of the trial, ADHD children were scored
according to parental behavioural rating scales (Connors' Rating
scale).
[0205] Results and Discussion
[0206] Use of complementary therapies is particularly common among
patients with chronic, incurable, or frequently relapsing
conditions. For example, use of complementary and alternative
medical therapies (CAM) is common in children with cancer, asthma,
and cystic fibrosis. Parents or subjects who seek CAM typically do
so because such therapies are more consistent with their values,
are more empowering, and are perceived as more natural and less
risky than conventional treatments. The majority of these patients
do not abandon mainstream therapies but use herbs and other forms
of CAM as adjunctive treatments. Only a minority (<40%) talk
with their pediatricians about their use of CAM. Because of the
stigma and side effects that accompany use of stimulant
medications, many families turn to CAM to treat ADHD. Typically,
only 70% of children respond to stimulants such as Ritalin.TM., and
of those who do, approximately half report side effects from their
medications. In an Australian survey of 290 families seen at a
multidisciplinary referral center for ADHD, 64% had tried at least
one "other therapy," most commonly dietary restriction,
multivitamin supplementation, and occupational therapy
[Stubberfield et al. (1999) J Paediatr Child Health; 35:450-3].
[0207] In the presented study the different supplementation was
formulated into a popular chocolate paste (see below). Using this
matrix enable the parents to administer the treatments in a
non-conventional form to their children and provided a reduced
organoleptic effect characteristic of the marine-derived compounds
(see below).
[0208] The parental rating survey, at the end of the treatment
period, measured the attention deficit, hyperactivity and
impulsivity of the children, as well as the aggression as assessed
by parents, teachers, siblings and peers. The results indicate a
distinctively large placebo effect. This effect is somewhat reduced
if the placebo-treated ADHD children that failed to complete the
study due to severe behavioral deterioration are taken into
consideration. It seemed that most of these children insisted on
reassigning for Ritalin.TM. administration. However, the present
data also clearly demonstrate PS-{overscore (.omega.)}3 as a potent
agent. All in all, .about.70% of the parents of the PS-{overscore
(.omega.)}3 treated ADHD children indicated some improvement in the
behavioural score of their children, whereas 50% of these parents
provided clear indications for multiple beneficial effect of the
supplement on their children behavior. This prominent effect is
2.2-fold higher than the improvement obtained by placebo
(.about.30%). Comparison of the parental scoring of LC-PUFA on ADHD
children behavior with the parallel rating that followed three
months of PS-{overscore (.omega.)}3 administration, point at the
latter to have a higher score. While both compounds demonstrated
similar extent of marginal improvement, PS-{overscore (.omega.)}3
had a marked higher rate of substantial improvement (47% versus.
35%, respectively) with the lowest rats of lack or deteribrating
effects (21% & 11% versus 26% and 17%, respectively). These
effects of PS-{overscore (.omega.)}3 supplementation could be
attributed to both enhanced bioavailability of omega-3 fatty acids
and through PS well documented effects on mood, stress and
anxiety.
Example 3
Effect of PC-DHA Consumption in ApoE.degree. Mice
[0209] Methods
[0210] Animal Diet
[0211] Apolipoprotein E deficient (ApoE.degree.) mice [Hayek T. et
al. (1994) Biochem. Biophys. Res. Commun. 201:1567-1574] at 8 weeks
of age, were assigned randomly (5 mice each) to LC-PUFA enriched
lecithin (30% omega-3 of total fatty acids composition; PC-DHA
group) or placebo. The mice were fed, besides the regular chow
diet, once every three days with either 25 .mu.l PC-DHA or PBS, via
oral gavage, during 10 weeks.
[0212] Each mouse consumed approximately 5 mL of water/day, and 5 g
of chow/day.
[0213] Serum Lipids Peroxidation
[0214] Serum was diluted 1:4 in PBS. Serum susceptibility to
oxidation was determined by incubating serum sample with 100 mM of
the free radical generating compound, 2'-2'-azobis
2'-amidinopropane hydrochloride (AAPH), which is an aqueous soluble
azo compound that thermally decomposes to produce peroxyl radicals
at a constant rate. The formation of thiobarbituric reactive
substances (TBARS) and of lipid peroxides was measured and compared
to serum that was incubated under similar conditions, but without
AAPH.
[0215] Results and Discussion:
[0216] ApoE.degree. mice are widely used as an animal model for
atherosclerosis as they develop severe hypercholesterolemia and
atherosclerotic lesions on a chow diet. Moreover, accelerated
atherosclerosis is associated with increased lipid peroxidation of
plasma lipoproteins and arterial cells in these mice [Hayek T. et
al. (1994) id ibid.; Keidar S. (1998) Life Sci. 63:1-11].
[0217] FIG. 6 shows how prolonged PC-DHA consumption by
ApoE.degree. mice resulted in a clear tendency (P<0.10) to
reduce the serum susceptibility to MPH-induced oxidation by 16% (in
comparison to placebo).
[0218] Organoleptic Issues
[0219] The utilization of omega-3 lipids in a variety of
applications, and especially as ingredient of functional foods, is
hindered due to their distinct fish odor. Thus, another advantage
of the omega-3 enriched phospholipids ingredients of the invention
is that they have reduced odor or taste of omega-3 acyl moieties,
due to the covalent binding of these groups to the PS backbone.
This increases the vapor pressure of these materials, hence
reducing their distinct aroma. Thus, the covalent binding of the
omega-3 fatty acids to the phospholipid backbone, especially PS,
alters and. improves their taste properties. Moreover, the PS
ingredient of the invention also offers enhanced stability to the
oxidation sensitive omega-3 fatty acids. Phospholipids in general,
and PS in particular, are known to act as anti-oxidants and
stabilizers.
[0220] These benefits make this novel phospholipids' preparation of
the invention highly beneficial and important in a variety of
applications and especially in functional foods, where stability,
aroma and taste are fundamental requirements.
[0221] Furthermore, these novel ingredients can be formulated with
additional lipids for an even enhanced bio-functionality and
efficacy.
[0222] The starting compound used for the above-mentioned clinical
trial in ADHD patients, was LC-PUFA enriched PS mixed with fish
oil. Originally, this product and the control fish oil were
formulated in food products like energy bars; however the responses
from expert panels were categorically devastating, pointing at
severe organoleptic problems. In order to overcome this taste
barrier the PS-{overscore (.omega.)}3 product of the invention was
de-oiled. The end-product of this process was a paste that when
reformulated with either inert or dominant--organoleptic saturated
fats could be easily formulated in chocolate bars, chocolate
spread, chocolate coated cornflakes, low-fat dairy products or
concentrated milk. Each one of these formulations had an evidently
reduced organoleptic objection from both the expert panels and the
trial volunteers.
[0223] The polar lipids derivatives of PUFA, such as the PS-PUFA
derivatives have exhibited high stability as a preparation and
additionally in several food applications, used in the clinical
trials of this invention. This stability, of these sensitive
compounds is emerging from the covalent combination of
phospholipids, known in the past to be used as preservatives and of
the un-stable PUFA moieties.
[0224] The stability of a commercially prepared fish oil (omega-3
fatty acid) for laboratory rodent diet [Lytle et al. (1992) Nutr
Cancer, 17(2):187-94] or as an enrichment in spreadable fats
[Kolanowski et al. (2001) Int J Food Sci Nutr.; 52(6):469-76] was
addressed by several studies as the public awareness towards the
beneficial effects of LC-PUFA increased. A major effort was
directed at maintaining the oxidative stability of the fish oil, as
these fatty acids are subject to rapid and/or extensive oxidation
and other chemical changes by exposure to air, light, or heat
during processing or when stored for various lengths of time. The
common solution presented in these studies was supplementation the
fish oil matrix with antioxidants like butylated hydroxytoluene,
butylated hydroxyquinone and alpha-tocopherol, or alternatively,
dilution of concentrated fish oil to a limit of 1% in a saturated
fats matrix. However, Song and colleagues [Song et al. (1997)
Biosci Biotechnol Biochem.; 61(12):2085-8] had already evaluated
the peroxidative stability of DHA-containing oils the form of
phospholipids, triglycerides, and ethyl esters in the dark at
25.degree. C. in a bulk phase during 10 weeks storage. They had
shown that DHA-containing oil in the form of phospholipids was more
resistant to the oxidative degradation of DHA than that in the form
of triglycerides and ethyl esters in a bulk phase.
[0225] The abovementioned PS-{overscore (.omega.)}3 containing
products utilized for the clinical studies were tested for their
shelf-life and stability in room temperature. The enriched
PS-{overscore (.omega.)}3 formulated in condensed milk (1 g product
per 10 ml milk) was analyzed by .sup.31P-NMR for stability in
cycles of freeze-thawing for a week and was found to be stable. In
the second phase PS-{overscore (.omega.)}3 in a chocolate paste
matrix (0.75 g product per 20 g chocolate spread) was tested for
stability after two weeks storage in room temperature. This
formulation also presented a stable percentage of PS, in
.sup.31P-NMR analysis. In conclusion, we had been able to establish
that {overscore (.omega.)}-3 containing phospholipids are highly
stable in room temperature, as well as in freezing-thawing cycles,
as oppose to {overscore (.omega.)}-3 containing triglycerides known
to rapidly decay after antioxidant consumption.
* * * * *