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. New phosphobioflavonic complexes of NAPE or NAE with one or more bioflavonoids are also disclosed.

Claims

1. A composition consisting of N-oleoyl-phosphatidyl-ethanolamine (NOPE) and green tea bioflavonoids wherein said composition is an aggregation of said N-oleoyl-phosphatidyl-ethanolamine (NOPE) and said green tea bioflavonoids, and wherein said composition is present in a hydroalcoholic solution.

2. The composition according to claim 1, wherein the aggregation is present as a dry granular residue.

3. The composition according to claim 2, wherein the bioflavonoid is a catechin selected from epicatechin, epicatechin gallate, epigallocatechin, and gallocatechin.

4. A pharmaceutical composition comprising a composition consisting of N-oleoyl-phosphatidyl-ethanolamine (NOPE) and green tea flavonoids, wherein said composition is an aggregation of said N-oleoyl-phosphatidyl-ethanolamine (NOPE) and said green tea flavonoids, wherein said composition is present in a hydroalcoholic solution.

5. A method of treating excess weight through control of the body weight which comprises administering the composition according to claim 2.

6. A method to treat excess weight through control of body weight which comprises administering the pharmaceutical composition according to claim 4.

7. The method according to claim 6, wherein the administration is oral administration.

8. A method of treating obesity through the control of excess weight which comprises administering the composition according to claim 2.

9. A method of treating obesity through control of excess weight which comprises administering the pharmaceutical composition according to claim 4.

10. The method according to claim 9, wherein the administration is oral administration.

11. A process to produce the composition according to claim 1, comprising the step of suspending N-oleyl-phosphatidyl-ethanolamine under stirring in a hydroalcoholic solution buffered at an acid pH containing green tea bioflavonoids.

12. The process according to claim 11, wherein a resulting emulsion is then dehydrated by spray drying.

13. A pharmaceutical composition comprising the composition according to claim 2.

14. A method to treat excess weight through control of body weight which comprises administering a pharmaceutical composition according to claim 13.

15. The method according to claim 14, wherein the administration is oral administration.

Description

EXAMPLE 1

(1) $98g\mathrm{of}N\text{-}\mathrm{linoleoyl}\text{-}\mathrm{phosphatidylethanolamine}+1g\mathrm{of}d\text{-}\alpha \text{-}\mathrm{tocopherol}+1g\mathrm{of}\mathrm{lipoic}\mathrm{acid}.$

(2) 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

(3) $20g\mathrm{of}N\text{-}\mathrm{eicosapentaenoyl}\text{-}\mathrm{ethanolamine}+60g\mathrm{of}\mathrm{phosphatidic}\mathrm{acid}+80g\mathrm{of}\mathrm{soy}\mathrm{phosphatidylcholine}+1g\mathrm{of}d\text{-}\alpha \text{-}\mathrm{tocopherol}+1g\mathrm{of}\mathrm{lipoic}\mathrm{acid}.$

(4) 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

(5) $20g\mathrm{of}N\text{-}\mathrm{linolenoyl}\text{-}\mathrm{ethanolamine}+40g\mathrm{of}\mathrm{lysophosphatidic}\mathrm{acid}+1g\mathrm{of}d\text{-}\alpha \text{-}\mathrm{tocopherol}+1g\mathrm{of}\mathrm{lipoic}\mathrm{acid}.$

(6) 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

(7) $20g\mathrm{of}N\text{-}\mathrm{gamma}\text{-}\mathrm{linolenoyl}\text{-}\mathrm{phosphatidylethanolamine}+80g\mathrm{of}a\mathrm{mixture}\mathrm{of}\mathrm{lysophospholipids}\left(45\%\mathrm{lysophosphatidylcholine}+35\%\mathrm{lysophosphatidylethanolamine}+20\%\mathrm{lysophosphatidylinositol}\right)1g\mathrm{of}d\text{-}\alpha \text{-}\mathrm{tocopherol}+1g\mathrm{of}\mathrm{lipoic}\mathrm{acid}.$

(8) 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 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

(9) $20g\mathrm{of}a\mathrm{dry}\mathrm{phospholipid}\mathrm{residue}\mathrm{obtained}\mathrm{as}\mathrm{described}\mathrm{in}\mathrm{examples}1\text{-}4\mathrm{above}+200g\mathrm{of}\mathrm{an}\mathrm{oily}\mathrm{solution}\left(\mathrm{olive}\mathrm{oil},\mathrm{soy},\mathrm{corn},\mathrm{sunflower},\mathrm{borage},\mathrm{blackcurrant},\mathrm{fish}\mathrm{or}\mathrm{seaweed}\mathrm{oils},\mathrm{or}\mathrm{mixtures}\mathrm{thereof}\right).$

(10) 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

(11) 100 g of a dry phospholipid residue of N-docosahexanoyl-phosphatidylethanolamine, obtained as described in example 1, is re-suspended under strong stirring for 5 minutes at 45° C. in 900 ml of a hydroalcoholic solution (75% alcohol), buffered to pH 4.5, containing 5% by weight of green tea catechins. 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

(12) 50 g of N-linolenoyl-ethanolamine and 50 g of lysophosphatidic acid (CLPA) are slowly added under strong stirring at 60° 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.

(13) Pharmacological and/or Dietetic Tests

(14) 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.

(15) 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: 1) effect of the compositions on the lipoperoxide levels in the plasma, liver, brain and heart; 2) effect of the compositions on variations in body weight; 3) effect of the compositions on variations in membrane fluidity of ghost erythrocytes and plasma platelets; 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; 5) effect of the compositions on plasma levels of total cholesterol and HDL-cholesterol; 6) effect of the compositions on plasma levels of total triglycerides.

(16) 80 Male rats weighing 150-200 g each were used. The animals were divided into 8 groups of 10 animals: 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: 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%. 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%). 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%). 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 ˜50 mg of NOE and 150 mg of PA) replaced the same quantity of olive oil (olive oil used: 4.50%). 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 ˜50 mg of NOE and 100 mg of LPA) replaced the same quantity of olive oil (olive oil used: 4.75%). 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 ˜75 mg of the preparation described in example 6) replaced the same quantity of olive oil (olive oil used: 4.825%). 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%).

(17) TABLE-US-00001 TABLE 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 + PA) 86% 81% 5) Treated rats (NOE + LPA) 83% 80% 6) Treated rats (NOPE + B.F.) 91% 92% 7) Treated rats (B.F.) 73% 70%

(18) TABLE-US-00002 TABLE 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 + PA) 3.7 ± 0.4 31.8 ± 8.2 88 ± 7 34 ± 9 5) Treated rats 3.0 ± 0.3 33.5 ± 7.8 77 ± 5 34 ± 7 (NOE + LPA) 6) Treated rats 2.8 ± 0.3 29.7 ± 6.8 75 ± 4 30 ± 6 (NOPE + B.F.) 7) Treated rats (B.F.) 5.0 ± 0.5 44.0 ± 7.1 105 ± 7  44 ± 7

(19) TABLE-US-00003 TABLE 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 cholesterol cholesterol triglycerides Body 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.)

(20) TABLE-US-00004 TABLE 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 consumption Reduced (umoles O.sub.2/ glutathione Mitochondrial min per (nmoles × 10.sup.6 membrane 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 408 ± 62 41 ± 5 81% (NOPE) 4) Treated rats 412 ± 58 43 ± 6 82% (NOE + PA) 5) Treated rats 409 ± 63 43 ± 8 83% (NOE + LPA) 6) Treated rats 421 ± 51 45 ± 5 88% (NOPE + B.F.) 7) Treated rats (B.F.) 366 ± 41 38 ± 5 71%

(21) 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.

(22) 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.

(23) 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.

(24) 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.): 1) restores the membrane fluidity of ghost and platelets; 2) improves the antioxidant defences of plasma, liver, brain and heart; 3) limits excessive increases in body weight; 4) limits excessive increases in plasma cholesterol and triglyceride levels; 5) improves the functionality of the mitochondria.

(25) 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.

(26) 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.

(27) 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.