U.S. patent application number 10/504124 was filed with the patent office on 2005-06-02 for n-acyl-phosphatidyl-ethanolamines and/or mixtures of n-acyl-ethanolamines with phosphatidic acids or lysophosphatidic acids.
Invention is credited to Cestaro, Benvenuto, Pistolesi, Elvira.
Application Number | 20050118232 10/504124 |
Document ID | / |
Family ID | 27736279 |
Filed Date | 2005-06-02 |
United States Patent
Application |
20050118232 |
Kind Code |
A1 |
Pistolesi, Elvira ; et
al. |
June 2, 2005 |
N-acyl-phosphatidyl-ethanolamines and/or mixtures of
n-acyl-ethanolamines with phosphatidic acids or lysophosphatidic
acids
Abstract
Pharmaceutical, cosmetic and dietetic compositions and
functional foods, constituted by: A) phospholipid mixtures
containing N-acyl-phosphatidyl-ethanolamines (NAPEs) and/or B)
phospholipid mixtures containing N-acyl-ethanol amines (NAEs)
together with phosphatidic acids (PAs) and/or lysophosphatidic
acids (LPAs) with the proviso that said
N-acyl-phosphatidyl-ethanolamines (NAPEs) do not include
N-oleoyl-phosphatidyl-ethanolamine. as well as new
phosphobioflavonic complexes of NAPE or NAE with one or more
bioflavonoids, are disclosed.
Inventors: |
Pistolesi, Elvira; (Milano,
IT) ; Cestaro, Benvenuto; (Milano, IT) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W.
SUITE 800
WASHINGTON
DC
20005
US
|
Family ID: |
27736279 |
Appl. No.: |
10/504124 |
Filed: |
February 10, 2005 |
PCT Filed: |
February 7, 2003 |
PCT NO: |
PCT/EP03/01233 |
Current U.S.
Class: |
424/439 ;
514/78 |
Current CPC
Class: |
A61K 31/164 20130101;
A61P 15/12 20180101; A61P 17/14 20180101; A61K 31/366 20130101;
A61P 17/16 20180101; A23V 2002/00 20130101; A61P 3/04 20180101;
A61P 25/28 20180101; A61K 8/553 20130101; A23V 2250/712 20130101;
A23V 2250/1846 20130101; A23V 2250/026 20130101; A23V 2250/1844
20130101; A23V 2250/1848 20130101; A61P 3/10 20180101; A23J 7/00
20130101; A61P 25/16 20180101; A61P 25/24 20180101; A23V 2002/00
20130101; A61Q 19/06 20130101; A61Q 19/08 20130101; A61P 15/00
20180101; A61P 19/10 20180101; A61P 25/14 20180101; A61P 13/08
20180101; A23L 33/10 20160801; A61K 31/661 20130101; A61P 35/00
20180101 |
Class at
Publication: |
424/439 ;
514/078 |
International
Class: |
A61K 047/00; A61K
031/685 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 13, 2002 |
IT |
MI2002A00270 |
Nov 26, 2002 |
IT |
MI2002A002512 |
Claims
1. Pharmaceutical, cosmetic and dietetic compositions and
functional foods, constituted by: A) phospholipid mixtures
containing N-acyl-phosphatidyl-ethanolamines (NAPEs); and/or B)
phospholipids mixtures containing N-acyl-ethanolamines (NAEs)
together with phosphatidic acids (PAs) and/or lysophosphatidic
acids (LPAs), with the proviso that said
N-acyl-phosphatidyl-ethanolamines (NAPEs) do not include
N-oleoyl-phosphatidyl-ethanolamine.
2. Compositions as claimed in claim 1 wherein the phospholipid
mixtures are constituted by complexes of one or more bioflavones
with NAPE or NAE plus PA and/or LPA.
3. Compositions as claimed in claim 1, also containing amphiphilic
surfactants.
4. Functional foodstuffs containing the compositions claimed in
claim 1.
5. The use of phospholipid mixtures containing A)
N-acyl-phosphatidyl-etha- nolamines (NAPEs); and/or B) phospholipid
mixtures containing N-acyl-ethanolamines (NAEs) together with
phosphatidic acids (PAs) and/or lysophosphatidic acids (LPAs). for
the preparation of medicaments having anorexic activity or of
medicaments or foodstuffs for the treatment of aging, obesity and
excess weight; diabetes; cerebro-degenerative disorders such as
Alzheimer's disease, Parkinson's disease and senile dementia;
stress, depression; tumours; menopaulsal syndromes; osteoporosis;
prostate hypertrophy; skin aging; panniculopathy (cellulitis) and
alopecia.
6. Phosphobioflavonic complexes of bioflavonoids with phospholipids
active components selected from N-acyl-phosphatidyl-ethanolamines
(NAPE) and/or N-acyl-ethanolamines (NAE) plus Phosphatidic Acid
(PA) and/or N-acyl-ethanolamines plus Lysophosphatidic acid (LPA).
Description
[0001] The present invention relates to pharmaceutical, cosmetic
and dietetic compositions and functional foods, constituted by:
[0002] A) phospholipid mixtures containing
N-acyl-phosphatidyl-ethanolamin- es (NAPEs);
[0003] and/or
[0004] B) phospholipid mixtures containing N-acyl-ethanolamines
(NAEs) together with phosphatidic acids (PAs) and/or
lysophosphatidic acids (LPAs), with the proviso that said
N-acyl-phosphatidyl-ethanolamines (NAPEs) do not include
N-oleoyl-phosphatidyl-ethanolamine.
[0005] Also disclosed are new phosphobioflavonic complexes of NAPE
or NAE plus PA and/or LPA, with one or more bioflavonoids.
[0006] N-Acyl-ethanolamines (NAEs) and
N-acyl-phosphatidyl-ethanolamines (NAPEs) are known to be present
in many foods of animal and vegetable origin (H. H. Schmid et al.,
1990, Prog. Lipid Res., 29, 1-43), and are particularly abundant in
foods such as soy, eggs and chocolate (K. D. Chapman et al., 1993,
Arch. Biochem. Biophys, 301, 21-23; E. Di Tomaso et al., 1996,
Nature, 382, 677-678).
[0007] The NAEs are formed in vivo by hydrolysis of a NAPE molecule
that gives rise to a mixture of NAE and a molecule of phosphatidic
acid (PA) which, in turn, can be hydrolysed to lysophosphatidic
acid (LPA) in accordance with the following scheme 1. 1
[0008] GB 2051069 discloses the anti-lipemic and
anti-atherosclerotic activity of N-Oleoyl-phosphatidylethanolamine
(NOPE) and excludes any significant activity of other
N-acyl-derivatives.
[0009] NAEs have also been known for some time for their
interesting pharmacological properties: N-arachidonoyl-ethanolamine
has been demonstrated in vitro to be a cannabinoid receptor agonist
(L. Hanus, 1993, J. Med. Chem., 36, 3032-3034);
N-palmitoyl-ethanolamine, when administered intraperitoneally to
rats, possesses anti-inflammatory and anti-anaphylactic activity
(L. Facci et al., 1995, Proc. Natl. Acad. Sci. USA, 92, 3376-3380);
N-palmitoyl-ethanolamine and N-stearoyl-ethanolamine have proved
useful in the pharmacological treatment of inflammatory disorders
resulting from degranulation of the mast cells (EP-A-0550006); they
also inhibit peroxidation of the mitochondrial membranes in vitro
(N. M. Gulaya et al., 1998, Chem. Phys. Lipids, 97, 49-54);
N-oleoyl-ethanolamine (NOE) has a significant anorexic effect in
the rat, when administered by the intraperitoneal route (F.
Rodriguez de Fonseca et al., 2001, Nature, 414, 209-212). Since it
is well known that NAEs are easily hydrolised to free fatty acids
and ethanolamine in the gastrointestinal tract, its activity by the
oral route is not expected.
[0010] The present invention relates to pharmaceutical and dietetic
compositions and functional foods, constituted by:
[0011] A) phospholipid mixtures containing
N-acyl-phosphatidyl-ethanolamin- es (NAPEs);
[0012] and/or
[0013] B) phospholipid mixtures containing N-acyl-ethanolamines
(NAEs) together with phosphatidic acids (PAs) and/or
lysophosphatidic acids (LPAs),
[0014] with the proviso that said N-acyl-phosphatidyl-ethanolamines
(NAPEs) do not include N-oleoyl-phosphathidyl-ethanolamine.
[0015] The structural formulas of NAE, PA and LPA are shown in
scheme 2, wherein R.sub.1, R.sub.2 and R.sub.4 are acyl residues of
long-chain fatty acids, in particular residues of palmitic,
stearic, oleic, linoleic, conjugated linoleic, linolenic,
gamma-linolenic, eicosapentaenoic and docosahexanoic acids,
etc.
[0016] The phospholipid mixtures may be present in the compositions
of the invention in the form of their complexes with bioflavonoids.
Said complexes, hereinafter called "phosphobioflavonic complexes",
are a further object of the invention.
[0017] Complexes of phospholipids ouch as lecithins,
phosphatidylcholine, phosphatidylethanolamine and
phosphatidylserine with a number of plant extracts have been
disclosed (U.S. Pat. No. 4,963,527, U.S. Pat. No. 4,895,839, EP
283713). Said complexes are reported to increase the
bioavailability of the plant extract. In the phosphobioflavonic
complexes of the invention, NAPE or NAE plus PA and/or LPA provide
an unexpected synergism for the considered applications and do not
act merely as carriers of bioflavones.
[0018] Said complexes, constituted by aggregation of the
phospholipid active components (NAPE and/or NAE plus PA and/or NAE
plus LPA) with one or more types of bioflavonoids, can be obtained
by suspending a dry phospholipid residue under strong stirring for
a few minutes at a temperature preferably between 40.degree. C. and
65.degree. C. in a hydroalcoholic solution (alcohol preferably
between 70 and 90%), buffered to an acid pH (pH preferably between
3 and 5), containing a fraction of one or more types of
bioflavonoids, preferably in a percentage of between 0.5 and 15% by
weight of the hydroalcoholic solution. When stirring is
interrupted, ethanol is partially evaporated from the resulting
emulsion under vacuum and then dehydrated by spray drying, to
produce a dry granular residue of phosphobioflavonic complexes.
[0019] Examples of bioflavonoids which can be used to produce these
phosphobioflavonic complexes include:
[0020] a) simple polyphenols such as cinnamic, cumaric, caffeic and
ferulic acids;
[0021] b) flavones such as hesperidin, naringenin and
taxifolin;
[0022] c) flavonols such as kaempferol glycoside, quercetin,
quercetin glycoside, myricetin and myricetin glycoside;
[0023] d) isoflavones such as genistein and daidzein;
[0024] e) proanthocyanidins such as procyanidin B1, procyanidin B2,
procyanidin B3 and procyanidin C-1;
[0025] f) anthocyanidins such as pelargonidin, delphinidin,
malvidin and petunidin;
[0026] g) catechins such as epicatechin, epicatechin gallate,
epigallocatechin, catechins and gallocatechins;
[0027] h) tannins.
[0028] As mentioned, molecules of NAPE, NAE, PA and LPA are
naturally present in the lipid fractions of many foodstuffs
normally used in the human diet (soy lecithins, eggs, cocoa, meat,
oily extracts of various seeds, etc.), and can easily be extracted
and isolated to various degrees of purity in accordance with
conventional methods. Alternatively, the NAPE and NAE molecules can
be obtained by synthesis according to chemical processes which have
been known for some time.
[0029] NAE can prepared from ethanolamine and the corresponding
fatty acid, for example in accordance with the methods described
in:
[0030] Roc E. T. et al. (1952), J. Am. Chem. Soc., 74, 3442
[0031] Chandrakumar N. S. et al. (1982), Biochim. Biophys Acta,
711, 357.
[0032] NAPE can prepared from phosphatidylethanolamine and the
corresponding fatty acid chloride or anhydride, in accordance with
the methods described in:
[0033] Schmid P. C. et al. (1988), J. Biol. Chem., 288 (6),
9802
[0034] Epps D. E. et al. (1980), Biochim. Biophys Acta, 618,
420
[0035] GB 2051069 B.
[0036] Another method for the preparation of NAPE by means of the
enzyme phospholipase D, disclosed in U.S. Pat. No. 4,783,402, is
illustrated in the scheme below: 2
[0037] wherein
[0038] R.sub.1, R.sub.2 and R.sub.4 represent the alkyl chain of
saturated, mono- or polyunsaturated fatty acids with 12-22 C
atoms;
[0039] R.sub.3 represents a residue of choline, ethanolamine,
inositol, glycerol, serine.
[0040] The therapeutically effective doses of preparations based on
NAPE and/or NAE plus PA and/or LPA vary:
[0041] a) in the case of NAPE from 0.5 to 50 mg, and preferably
from 1 to 10 mg/day per kg of body weight;
[0042] b) in the case of NAE plus PA and/or LPA from 0.5 to 100 mg,
and preferably from 2 to 20 mg/day/kg of body weight. In this
mixture of NAE+PA and/or LPA, the percentage of NAE can vary
between 1 and 70%, and preferably between 25 and 50% of the total
co-mixed lipids.
[0043] The compositions of the invention may also contain other
nutritional components which further implement the therapeutic
properties and benefits of NAPE and/or the mixtures of NAE+PA
and/or LPA. Examples of these components are:
[0044] a) vitamins and vitamin-like factors such as vitamin E,
vitamin C, .beta.-carotenes, vitamin A, vitamin D, lipoic acid and
CoQ;
[0045] b) extracts of vegetables and/or medicinal plants based on
mono- and diterpenes, saponins, and phytosterols;
[0046] c) proteins, peptides or aminoacids and their derivatives
such as glutathione, carnosine, arginine, glutamine, carnitine,
creatine and taurine;
[0047] d) trace elements and mineral salts such as Ca, Mg, Cr, Se,
Va, Zn and Cu;
[0048] e) mixtures of natural amphiphilic detergents such as
phospholipids and lysophospholipids; glycolipids; amphiphilic
proteins; mono- and diglycerides; bile acids or salts able to
incorporate NAPE and/or mixtures of NAE+PA and/or LPA in lipid
emulsions of various types which help to increase their absorption
and bioavailability in vivo.
[0049] The active components, stored as dehydrated granulates or
powders, can be used as such or in the form of aqueous or oily
solutions to make various galenical preparations such as gelatin
capsules, tablets, dragees, sachets, effervescent and
non-effervescent cachets, chewing gum, etc.
[0050] Said active components in the form of dehydrated granulates
or powders can also be used to make various functional foods:
[0051] a) mixed with oils to make sundry dressings, sauces, creams,
mayonnaise, etc;
[0052] b) mixed with flour to make bread, pasta, crackers, biscuits
and other baked products;
[0053] c) added to fruit juices and squashes, mineral waters, soft
drinks and other drinks;
[0054] d) added to milk and derivatives thereof (yoghurt, flans,
ricotta and cheese).
[0055] The pharmaceutical or dietetic compositions of the invention
have proved surprisingly active in:
[0056] a) controlling excess weight and consequently reducing the
risks connected with excess weight and obesity;
[0057] b) improving the functionality of the mitochondria and the
production of cell energy;
[0058] c) increasing the antioxidant defences in the various
tissues;
[0059] d) improving the "fluidity" of the cell membranes and
consequently the functionality of the membrane proteins (enzymes,
receptors, carriers of essential nutrients and trace elements,
etc.).
[0060] The preparations of the invention can therefore be used as
adjuvants in the treatment of aging and many metabolic disorders
connected with it (obesity and excess weight; diabetes;
cerebro-degenerative disorders such as Alzheimer's disease,
Parkinson's disease and senile dementia; stress, depression;
tumours; menopausal syndromes; osteoporosis; prostate hypertrophy;
skin aging; panniculopathy (cellulitis); and alopecia), possibly in
combination with known drugs or diet supplements.
[0061] The invention therefore also concerns the use of
phospholipid mixtures containing
[0062] A) N-acyl-phosphatidyl-ethanolamines (NAPEs);
[0063] and/or
[0064] B) phospholipid mixtures containing N-acyl-ethanolamines
(NAEs) together with phosphatidic acids (PAs) and/or
lysophosphatidic acids (LPAs)
[0065] for the preparation of medicaments having anorexic activity
or of medicaments or foodstuffs for the treatment of aging, obesity
and excess weight; diabetes; cerebro-degenerative disorders such as
Alzheimer's disease, Parkinson's disease and senile dementia;
stress, depression; tumours; menopausal syndromes; osteoporosis;
prostate hypertrophy; skin aging; panniculopathy (cellulitis) and
alopecia.
[0066] The invention is illustrated in greater detail in the
following examples.
EXAMPLE 1
[0067] 98 g of N-linoleoyl-phosphatidylethanolamine+1 g of
d-.alpha.-tocopherol+1 g of lipoic acid.
[0068] The various compounds are dissolved and mixed in 10 volumes
of chloroform: methanol (2:1, vol/vol). The solvent is evaporated
under vacuum, and the resulting dry residue is re-suspended in an
aqueous solution buffered to physiological pH to form an aqueous
mixture of a phospholipid emulsion containing the active component
(N-linoleoyl-phosphatidylethanolamine). The aqueous mixture can be
frozen and dehydrated to obtain a dry residue of the phospholipid
active component.
EXAMPLE 2
[0069] 20 g of N-eicosapentaenoyl-ethanolamine+60 g of phosphatidic
acid+80 g of soy phosphatidylcholine+1 g of d-.alpha.-tocopherol+1
g of lipoic acid.
[0070] The various compounds are dissolved in chloroform-methanol
and treated as described in example 1 to obtain an aqueous mixture
of a phospholipid emulsion containing the active components
(N-eicosapentaenoyl-ethanolamine and phosphatidic acid). The
aqueous mixture can be frozen and dehydrated to obtain a dry
phospholipid residue of the active components as described in
example 1.
EXAMPLE 3
[0071] 20 g of N-linolenoyl-ethanolamine+40 g of lysophosphatidic
acid+1 g of d-.alpha.-tocopherol+1 g of lipoic acid.
[0072] The various compounds are dissolved in chloroform-methanol
and treated as described in example 1 to obtain an aqueous mixture
of a phospholipid emulsion containing the active components
(N-linolenoyl-ethanolamine and phosphatidic acid). The aqueous
mixture can be frozen and dehydrated to obtain a dry phospholipid
residue of the active components as described in example 1.
EXAMPLE 4
[0073] 20 g of N-gamma-linolenoyl-phosphatidylethanolamine+80 g of
a mixture of lysophospholipids (45% lysophosphatidylcholine+35%
lysophosphatidylethanolaniine+20% lysophosphatidylinositol)+1 g of
d-.alpha.-tocopherol+1 g of lipoic acid.
[0074] The various compounds are dissolved in chlotoform-methanol
and treated as described in example 1 to obtain an aqueous mixture
of a phospholipid emulsion containing the active constituent
(N-gamma-linolenoyl-phosphatidylethanolamine). The aqueous mixture
can be frozen and dehydrated to obtain a dry phospholipid residue
of the active constituent as described in example 1.
EXAMPLE 5
[0075] 20 g of a dry phospholipid residue obtained as described in
examples 1-4 above+200 g of an oily solution (olive oil, soy, corn,
sunflower, borage, blackcurrant, fish or seaweed oils, or mixtures
thereof).
[0076] 20 g of dry phospholipid residues is slowly dissolved in 200
g of oily solution under slow, continuous stirring. The
phospholipids of the dry residues are restructured in the oily
solutions to form an oil-dispersed micellar organisation containing
the active components.
EXAMPLE 6
[0077] 100 g of a dry phospholipid residue of
N-docosahexanoyl-phosphatidy- lethanolamine, obtained as described
in example 1, is re-suspended under strong stirring for 5 minutes
at 45.degree. C. in 900 ml of a hydroalcoholic solution (75%
alcohol), buffered to pH 4.5, containing 5% by weight of green tea
catechins and epicatechins. The resulting emulsion is then cooled
to room temperature and dehydrated by spray drying to form a dry
granular residue of phosphobioflavonic complexes of
N-docosahexanoyl-phosphatidylethanolamine and green tea
catechins.
EXAMPLE 7
[0078] 50 g of N-linolenoyl-ethanolamine and 50 g of
lysophosphatidic acid (CLPA) are slowly added under strong stirring
at 60.degree. C. and emulsified for 10 minutes in 900 ml of a
hydroalcoholic solution (85% alcohol) buffered to pH 4.0,
containing 10% by weight of a mixture of catechins, epicatechins
and proanthocyanidins extracted from grape seeds. When stirring is
arrested, the resulting emulsion is cooled to room temperature and
dehydrated by spray drying to form a dry granular residue of
phosphobioflavonic complexes of N-linolenoyl-ethanolamine and
grape-seed bioflavonoids.
[0079] Pharmacological and/or Dietetic Tests
[0080] A series of experimental tests on rats and clinical tests on
man have been carried out to study the pharmacological and/or
dietetic characteristics of the composition of the invention.
[0081] In the experimental tests, the rats were given a
high-calorie, high-triglyceride, high-cholesterol diet. The
following parameters were evaluated after twenty days
treatment:
[0082] 1) effect of the compositions on the lipoperoxide levels in
the plasma, liver, brain and heart;
[0083] 2) effect of the compositions on variations in body
weight;
[0084] 3) effect of the compositions on variations in membrane
fluidity of ghost erythrocytes and plasma platelets;
[0085] 4) effect of the compositions on the functionality of the
hepatic mitochondria, evaluated by measuring: a) O.sub.2
consumption; b) reduced glutathione; c) the potential of the
mitochondrial membranes;
[0086] 5) effect of the compositions on plasma levels of total
cholesterol and HDL-cholesterol;
[0087] 6) effect of the compositions on plasma levels of total
triglycerides.
[0088] 80 Male rats weighing 150-200 g each were used. The animals
were divided into 8 groups of 10 animals:
[0089] 1.sup.st group: control (C); 10 animals (control at time 0)
were used as is, and 10 were given a standard high-calorie,
high-fat, high-cholesterol diet for 20 days, consisting of:
[0090] casein: 20%; mixture of trace elements and mineral salts:
3.5%; mixture of vitamins: 0.1%; choline bitartrate: 0.2%;
cellulose: 2%; cholesterol: 0.5%; sodium cholate: 0.25%;
saccharose: 58.44%, lard: 10.0% and olive oil: 4.9%.
[0091] 2.sup.nd group: treated with N-oleoyl-ethanolamine as such
(NOE); the animals were given the same diet as the controls for 20
days, except that 50 mg of NOE replaced the same quantity of olive
oil (olive oil used: 4.85%).
[0092] 3.sup.rd group: treated with
N-oleoyl-phosphatidylethanolamine prepared as described in example
1 (NOPE); the animals were given the same diet as the controls for
20 days, except that 50 mg of NOPE (prepared as described in
example 1) replaced the same quantity of olive oil (olive oil used:
4.85%).
[0093] 4.sup.th group: treated with
N-oleoyl-ethanolamine+phosphatidic acid prepared as described in
example 2: (NOE+PA); the animals were given the same diet as the
controls for 20 days, except that 400 mg of the preparation
described in example 2 (containing .about.50 mg of NOE and 150 mg
of PA) replaced the same quantity of olive oil (olive oil used:
4.50%).
[0094] 5.sup.th group: treated with
N-oleoyl-ethanolamine+lysophosphatidic acid prepared as described
in example 3: (NOE+LPA); the animals were given the same diet as
the controls for 20 days, except that 150 mg of the preparation
described in example 3 (containing .about.50 mg of NOE and 100 mg
of LPA) replaced the same quantity of olive oil (olive oil used:
4.75%).
[0095] 6.sup.th group: treated with "phosphobioflavonic complexes"
of N-oleoyl-phosphatidylethanolamine and green tea bioflavones
(B.F.) prepared as described in example 6 (NOPE+B.F.). The animals
were given the same diet as the controls for 20 days, except that
50 mg of NOPE and 25 mg of B.F. (corresponding to .about.75 mg of
the preparation described in example 6) replaced the same quantity
of olive oil (olive oil used: 4.825%).
[0096] 7.sup.th group: treated with green tea bioflavones (B.F.).
The animals were given the same diet as the controls for 20 days,
except that 25 mg of B.F. replaced the same quantity of olive oil
(olive oil used: 4.875%).
1TABLE I Percentage variations in membrane fluidity of ghost
erythrocytes and plasma platelets (expressed as a % of the control
values at time 0) of the rats before and after 20 day diet
treatment. Membrane fluidity Membrane fluidity (ghost erythrocytes)
(plasma platelets) 1A) Control rats at time 0 100% 100% 1B) Control
rats after 72% 69% a 20-day diet 2) Treated rats (NOE) 72% 70% 3)
Treated rats (NOPE) 84% 81% 4) Treated rats (NOE + 86% 81% PA) 5)
Treated rats (NOE + 83% 80% LPA) 6) Treated rats (NOPE + 91% 92%
B.F.) 7) Treated rats (B.F.) 73% 70%
[0097]
2TABLE II Lipoperoxide levels [expressed as nmoles of
malonyldialdehyde (MDA) per gram of tissue or per ml of plasma] in
the plasma, livers, brains and hearts of the rats before and after
20 day diet treatment. MDA MDA MDA MDA PLASMA LIVER BRAIN HEART 1A)
Control rats 2.5 .+-. 0.5 25.5 .+-. 5.9 55 .+-. 4 24 .+-. 5 at time
0 1B) Control rats 5.1 .+-. 0.6 44.2 .+-. 8.2 108 .+-. 6 45 .+-. 6
after a 20-day diet 2) Treated rats (NOE) 5.0 .+-. 0.6 44.1 .+-.
8.2 106 .+-. 7 44 .+-. 9 3) Treated rats (NOPE) 3.8 .+-. 0.4 33.1
.+-. 6.5 85 .+-. 9 32 .+-. 7 4) Treated rats (NOE + 3.7 .+-. 0.4
31.8 .+-. 8.2 88 .+-. 7 34 .+-. 9 PA) 5) Treated rats (NOE + 3.0
.+-. 0.3 33.5 .+-. 7.8 77 .+-. 5 34 .+-. 7 LPA) 6) Treated rats
(NOPE + 2.8 .+-. 0.3 29.7 .+-. 6.8 75 .+-. 4 30 .+-. 6 B.F.) 7)
Treated rats (B.F.) 5.0 .+-. 0.5 44.0 .+-. 7.1 105 .+-. 7 44 .+-.
7
[0098]
3TABLE III Variation in body weight and total cholesterol, HDL
cholesterol and triglyceride levels in the plasma of the rats
before and after 20 day diet treatment. Total HDL Total Body
cholesterol cholesterol triglycerides weight (mg dl.sup.-1) (mg
dl.sup.-1) (mg dl.sup.-1) (gm) 1A) Control rats 35.6 .+-. 1.8 26.2
.+-. 1.4 50.2 .+-. 7.7 180 .+-. 12 at time 0 1B) Control rats 126.2
.+-. 13.5 29.4 .+-. 1.6 82.5 .+-. 9.5 224 .+-. 19 after a 20-day
diet 2) Treated rats 120.4 .+-. 12.7 28.9 .+-. 2.8 80.5 .+-. 6.8
221 .+-. 16 (NOE) 3) Treated rats 110.3 .+-. 10.1 31.6 .+-. 3.9
71.4 .+-. 8.7 209 .+-. 18 (NOPE) 4) Treated rats 103.9 .+-. 12.4
29.9 .+-. 2.0 70.4 .+-. 10.5 208 .+-. 14 (NOE + PA) 5) Treated rats
101.7 .+-. 8.9 32.1 .+-. 3.8 68.5 .+-. 7.9 206 .+-. 20 (NOE + LPA)
6) Treated rats 80 .+-. 7.5 31.4 .+-. 3.9 60.2 .+-. 6.4 191 .+-. 14
(NOPE + B.F.) 7) Treated rats 123.5 .+-. 12.4 29.3 .+-. 1.5 80.7
.+-. 7.1 218 .+-. 17 (B.F.)
[0099]
4TABLE IV Variations in hepatocellular oxygen consumption, membrane
potential of mitochondria and reduced hepatocellular glutathione
content in control rats at time 0 and after 20 day diet treatment.
Hepatocellular O.sub.2 Reduced consumption glutathione
Mitochondrial (umoles O.sub.2/min (nmoles .times. 10.sup.6 membrane
per 10.sup.7 cells) cells) potential 1A) Control rats 480 .+-. 60
48 .+-. 5 100% at time 0 1B) Control rats after 360 .+-. 45 36 .+-.
4 68% a 20-day diet 2) Treated rats (NOE) 368 .+-. 52 36 .+-. 6 70%
3) Treated rats (NOPE) 408 .+-. 62 41 .+-. 5 81% 4) Treated rats
(NOE + PA) 412 .+-. 58 43 .+-. 6 82% 5) Treated rats (NOE + LPA)
409 .+-. 63 43 .+-. 8 83% 6) Treated rats (NOPE + B.F.) 421 .+-. 51
45 .+-. 5 88% 7) Treated rats (B.F.) 366 .+-. 41 38 .+-. 5 71%
[0100] When the membrane fluidity of the ghost erythrocytes and
plasma platelets is measured, TMA-DPH in accordance with the method
described by Caimi F. et al., 1999, Thromb. Hoemost., 82 pp. 149,
is used as the fluorescent probe.
[0101] Malonyldialdehyde is assayed in accordance with the
procedure described by K. Yagi et al., 1982, in "Lipid Peroxides in
Biology and Medicine", Academic Press, New York, 99. 324-340.
[0102] Hepatocellular O.sub.2 consumption, mitochondrial membrane
potential and reduced glutathione content are assayed in accordance
with the methods described by T. M. Hagen et al., 1999, FASEB J.,
13, 99. 411.
[0103] The data set out in Tables I, II, III and IV demonstrate
that administration of compositions containing the active
components (NOPE; NOE+PA; NOE+LPA; NOPE+B.F.):
[0104] 1) restores the membrane fluidity of ghost and
platelets;
[0105] 2) improves the antioxidant defences of plasma, liver, brain
and heart;
[0106] 3) limits excessive increases in body weight;
[0107] 4) limits excessive increases in plasma cholesterol and
triglyceride levels;
[0108] 5) improves the functionality of the mitochondria.
[0109] These effects, obtainable by oral administration of the
formulations prepared in accordance with the invention (NOPE;
NOE+PA; NOE+LPA; NOPE+B.F.), are always statistically significant.
It is important to note that no statistically significant benefit
can be obtained by administering equivalent oral doses of
N-oleoyl-ethanolamines as such.
[0110] The data set out above demonstrate the surprising synergy of
action observed between NAPE and/or NAE+PA and the various
bioflavonoid molecules; the therapeutic results obtainable by
administering the "phosphobioflavonic complexes" of NAPE (see data
set out in Tables I, II, III and IV) and NAE plus PA and/or LPA are
always far higher than the sum of the benefits obtainable with
single separate administrations of equivalent doses of NAPE (or
NAE) and bioflavonoids.
[0111] In all the diet treatment tests carried out on man, the
effects obtainable by orally the formulations claimed by the
invention (NAPE; NAE+PA; NAE+LPA; NAPE+B.F. and NAE plus PA and/or
LPA+B.F.) always provided highly significant results and
advantages, both in preventing biological signs of aging
(improvement in mitochondrial activity, better membrane fluidity,
improvement in plasma antioxidant defences, and limited weight
increase) and improving the clinical parameters tested in relation
to prevention of aging, and many of the metabolic disorders
associated therewith. It is noteworthy that also in humans no
significant benefit can be obtained by administering equivalent
oral doses of N-oleoyl-ethanolamine as such.
* * * * *