WO2001091589A1

WO2001091589A1 - Dietary supplement with antioxidant activity comprising an alkanoyl carnitine and a combination of polyphenols extracted from trees or shrubs

Info

Publication number: WO2001091589A1
Application number: PCT/IT2001/000261
Authority: WO
Inventors: Franco Gaetani
Original assignee: Sigma Tau HealthScience SpA
Current assignee: Sigma Tau HealthScience SpA
Priority date: 2000-05-30
Filing date: 2001-05-23
Publication date: 2001-12-06
Anticipated expiration: 2002-11-30
Also published as: AU2001274468A1; ITRM20000298A1; IT1317036B1; ITRM20000298A0

Abstract

A health food/dietary supplement with antioxidant activity is disclosed, comprising as its characterising components an alkanoyl carnitine and a combination of polyphenols extracted from trees or shrubs.

Description

Dietary supplement with antioxidant activity comprising an alkanoyl carnitine and a combination of polyphenols extracted from trees or shrubs

The present invention relates to a dietary supplement containing as its characterising components isovaleryl L-carnitine and a combination of polyphenols extracted from trees such as the Pinaceae (e.g. the maritime or cluster pine) and the Fagaceae (e.g. the chestnut and oak) or from shrubs such as Forsythia.

It has been found that the aforesaid composition is extremely effective in exerting potent antioxidant, vasculo-protective and anti-inflammatory activity on account of the unexpected synergistic effect exerted by its components. Particularly because of its protective effect against free radicals, the composition according to the invention can be used, in both human subjects and animals, for the prevention and treatment of many vascular and peripheral dysfunctions and diseases of an inflammatory and metabolic type, immune deficiencies, learning disorders and disorders related to ageing, as well as in periods of intense muscular activity which make an increased energy supply advisable.

Isovaleryl L-carnitine, a natural component of the carnitine "pool", presents specific activity at the lysosomal level and on cytosolic calcium movements. In addition to sharing the metabolic activities of the other carnitines, it is capable of intervening in proteolytic processes and of protecting a number of organs, such as the liver, against the action of toxic substances.

It is also known that numerous polyphenols can be extracted particularly from the bark and wood of the Pinaceae, Fagaceae and Oleaceae. The Pinaceae above all are trees rich in various classes of polyphenols, mainly including those belonging to the proanthocyanidin class which possess interesting biological properties. Among the Pinaceae, we should mention particularly the red fir (Picea abies), the Finnish pine, the Eastern hemlock (Tsuga canadensis), the Pinus massoniana and the Douglas fir (Pseudotsuga menziesii).

It is above all the polyphenols extracted from the bark of the maritime or cluster pine (Pinus pinaster) which have been most studied from the chemical and biological points of view. The polyphenols extracted from the bark of the maritime pine consist mainly in water-soluble procyanidines, but also in catechins, taxifolins, gallic and vanillic acid, hydrocinnamic, caffeic and ferulic acid, catechin and epicatechin.

Among the Fagaceae, particularly worthy of mention are the Fagus grandifolia, common beech (Fagus sylυatica), sweet chestnut (Castanea satiυa) and English oak (Quercus robur).

Among the Oleaceae, we should mention the shrubs of the genus Forsythia, particularly Forsythia intermedia and Forsythia suspensa.

Various methods of extraction of the above-mentioned polyphenol combinations are known. One very simple, yet effective method is based on the extraction of pine bark powder in water at high temperature, the extract of which, after filtration, is treated with NaCl and ammonium sulphate. After removal of the precipitate by filtration, the extract is treated with ethyl acetate and then with Na2SO₄ and then distilled at low pressure and purified with chloroform.

The proanthocyanidins, which constitute most of the polyphenols collected, are purified by dissolving them again in ethyl acetate and then precipitating them in chloroform and drying. The same method can be used for the extraction of polyphenols from dried Forsythia wood, but more appropriately the method described by Kitagawa can be used (Kitagawa, S., Photochemistry. 23:1636, 1989), this latter publication being incorporated in this description for reference purposes.

The extraction can, however, also be accomplished with other methods, both laboratory and industrial. The quantitative determination of proanthocyanidins is based on the characteristic affinity of collagen for these substances. The most frequently used quantitative determination methods are those described by Roux (Roux, P. G., Nature. 179, 1957), Bate-Smith (Phytochemistrv. 14:1107, 1975) or Lewis using HPLC (Lewis, N. G., J Chromatography. 479, 345, 1987), all these publications being incorporated in this description for reference purposes.

The best known pine bark extract is the extract from the bark of the maritime pine or pinaster from the South-East coast of France commercially obtainable under the Pycnogenol trademark. Though referring hereinafter to this extract, because it is easy to come by commercially, it must be clearly understood that, for the purposes of the present invention, any other extract of pine bark or other trees or shrubs whose characteristics are comparable to those of Pycnogenol can be used, and among these particularly the extract from red fir (Picea abies) and Finnish pine which, like Pycnogenol, present a very high mean concentration of proanthocyanidins (OPC 85-90%). Pycnogenol, like the red fir extract, is characterised by the presence of numerous polyphenol components, the most prominent of which are the proanthocyanidins, natural polyphenols that can be extracted and characterised according to the method described by Bate-Smith (Bate- Smith, E.G., Chemistry and Industry. 377, 1953), this latter publication being incorporated in the present description by reference.

As far as the pine bark extract is concerned, this mainly contains procyanidines which are bipolymers formed by the union of catechin and epicatechin and, considering that these two monomers are capable of combining through two different types of chemical bonds, they may form numerous isomers even to the extent of producing polymers of substantial molecular weight.

The complex biological activity of pine bark extracts and of those from Forsythia, chestnut or oak is due above all to the presence of proanthocyanidins which are water-soluble and of other polyphenols which are characterised by their antioxidant activity and its beneficial effects on vascular and cardiac disorders as well as in terms of enhancing the immune defences and preventing tumour formation or even favouring mitochondrial energy activity.

By means of tests conducted by combining the polyphenols extracted from pine bark with isovaleryl L-carnitine an unexpected synergistic action has been found between these substances, the reciprocal enhancement of whose effects can be usefully exploited for the prevention and treatment of many pathological forms related to the damaging action of free radicals, in which pine bark extracts particularly have proved effective.

In the tests reported here below a commercially available pine bark extract (Pinus pinaster) was used (Pycnogenol) and a red fir bark extract (Picea abies) with a proanthocyanidin content similar to that present in the pinaster bark extract (approximately 80-90%).

The total polyphenol content was determined using the Folin-Ciocolteu and Singleton method (Am. J. Enol. Vitic, lfi:144, 1965), whereas the proanthocyanidin content was measured using the modified Bate- Smith method (Phvtocheirπs ry. 24:1107, 1975), these latter publications being incorporated in this description for reference purposes.

Both isovaleryl L-carnitine and the proanthocyanidins extracted from pine bark, whose pharmacokinetics are well known, are water-soluble and well absorbed.

Anti-h d-peroxidation activity tests

On measuring the anti-lipid-peroxidation activity of isovaleryl L- carnitine and pine and red fir bark extract and of the composition in which the two components were combined, an intense synergistic effect exerted by the composition was detected. In these tests, the protective effect of the composition according to the invention described herein on peroxidation induced by hydroperoxides in the membranes of red blood cells was estimated by measuring the formation of malonaldehyde (MDA) as an indicator of peroxidation.

Venous blood samples were taken from healthy volunteers and collected in heparinated test tubes. After centrifuging and washing three times in phosphate-buffered saline solution (PBS, 0.015 M, pH 7.4), the red blood cells were diluted in 10 mL of a solution containing 10^-3 M of PBS-azide. The haemoglobin concentration was then measured using Drabkin reagent. Lipid peroxidation was induced by exposing a cell suspension contained in 5 ml of PBS-azide with a final haemoglobin concentration of 3.75 mg/ml to hydrogen peroxide (5 and 20 mm of hydrogen peroxide per ampoule containing 5 ml of cell suspension) which was then incubated at 37°C for 1 hour. At the end of the incubation period, lipid preoccupation was determined using the Stocks and Dormancy method (Stocks, J., Dormancy, T. L., Brit. J. Haematology. 20:95, 1971) which measures the formation of malonaldehyde (MDA) which in combination with thiobarbituric acid (TBA) forms a coloured chromogen with absorbance at 532 nm (Bird, R. P., Methods Enzymol.. 105:299, 1984). The above-mentioned publications are incorporated in this description for reference purposes.

For the measurement of the anti-lipid-peroxidation activity exerted by isovaleryl L-carnitine, by pine bark extracts and by the composition in which the two components are combined, samples of these substances were added to the ampoules containing the erythrocyte suspensions at doses of 100 μg/mL of isovaleryl L-carnitine or 100 μg/mL of pinaster bark extract or the same amount of red fir bark extract, or a combination of these products, respectively, at the doses indicated above.

As can be seen from the results presented in Table 1, which shows MDA formation after a 1-hour incubation of red blood cells with hydrogen peroxide, both isovaleryl L-carnitine and pine bark extracts are capable of reducing the peroxidation induced by hydrogen peroxide, but the greatest protective effect is that produced by the combination of isovaleryl L-carnitine and pine bark extract, thus demonstrating the unexpected synergistic action of the composition.

Table 1

Treatment MDA production (n ol MDA g Hb)

Controls 748.5±49.5

Isovaleryl L-carnitine 535.5±38.8

P.B.E. (*) 485.5±39.8

R.F.B.E. (**) 475.8±29.8

Isovaleryl L-carnitine + P.B.E. 180.8±31.1

Isovaleryl L-carnitine + R.F.B.E. 177.2±30.4

(*) P.B.E. = pinaster bark extract (**) R.F.B.E. = red fir bark extract

Experimental atherosclerosis tests

To evaluate the vascular protective and anti-atherosclerotic activity of the composition according to the invention, experimental atherosclerotic lesions were produced in Wistar rats using the modified Malinow method (Malinow, M.R., Atherosclerosis. 48-: 105, 1983), this latter publication being incorporated in this description for reference purposes. This method can be used to induce pronounced atherosclerotic lesions by administering the animals an atherogenic diet containing 24% casein, 10% cotton oil, 2% cholesterol, 61% sugar, and 200 mUST of Vit. D₂/g diet and 3% sodium chloride.

The diet with this composition was administered for 6 consecutive weeks both to control rats and to different groups of rats treated with isovaleryl L-carnitine (200 mg/kg) or with 150 mg/kg of pinaster or red fir bark extract or with the composition in which the two components were combined at the doses indicated above.

After 6 weeks of treatment, both the control animals and the treated animals were sacrificed, and the atherosclerotic lesions induced were evaluated by using a morphometric method to measure the thickness of the abdominal aorta and by assessing the intensity of the Sudan IV staining induced. The severity of the lesions was assessed using a scoring system from 1 to 5.

On the basis of the examinations performed, it was found that the animals treated with isovaleryl L-carnitine and with pine bark extract presented an approximately 30% reduction in atherosclerotic damage as compared to the control animals, as determined both morphometrically and using Sudan IV staining.

The reduction of the damage is practically identical in the animals treated with isovaleryl L-carnitine and in those treated with both pine bark and red fir bark extracts.

When, however, isovaleryl L-carnitine was combined with pine bark extract,, the almost complete disappearance of the atherosclerotic lesions induced was detected, and thus the results of these tests also demonstrated a potent, unexpected synergistic protective effect.

Some non-limiting examples of compositions according to the present invention are given hereinbelow.

1) Isovaleryl L-carnitine 500 mg

Maritime pine bark exctract 100 mg

(proanthocyanidine titre: 85-90%)

2) Isovaleryl L-carnitine 500 mg

Maritime pine bark exctract 50 mg

(proanthocyanidine titre: 75-85%)

Red fir bark exctract 50 mg

(proanthocyanidine titre: 75-85%) 3) Isovaleryl L-carnitine 400 mg

Maritime pine bark exctract 50 mg (proanthocyanidine titre: 85-90%) β-carotene 5 mg

Vit. E 5 mg

Vit. Be 5 mg

Vit. PP 10 mg

Vit. C 50 mg

Selenomethionine 50 μg

Magnesium stearate 5 mg

Zinc glycinate 5 mg

4) Isovaleryl L-carnitine 400 mg

Red fir bark exctract 50 mg (proanthocyanidine titre: 85-90%)

Grape seed extract 50 mg

(polyphenols titre: 90%)

Forsythia extract 50 mg

(polyphenols titre: 70%)

Citroflavonoids 50 mg

Vit. E 5 mg β-carotene 5 mg

Vit. C 50 mg

Coenzyme io 20 mg

Selenomethionine 50 μg

The composition of the invention may also comprise both L-carnitine and further alkanoyl L-carnitines (such as acetyl, propionyl, butyryl and valeryl L-carnitine). It has been found that supplementation of the characterizing composition (i.e. isovaleryl L-carnitine + poliphenol extract from the bark and wood of the aforesaid plants) with the aforesaid alkanoyl L-carnitines further enhances the synergistic effect of the composition. What is meant by a pharmacologically acceptable salt of isovaleryl L- carnitine, L-carnitine or any other alkanoyl L-carnitine is any salt of these with an acid which does not give rise to unwanted toxic or side effects. These acids are well known to pharmacologists and to experts in pharmaceutical technology.

Non-limiting examples of such salts are the following: chloride; bromide; iodide; aspartate, acid aspartate; citrate, acid citrate; tartrate; phosphate, acid phosphate; fumarate, acid fumarate; glycerophosphate; glucose phosphate; lactate; maleate, acid maleate; mucate; orotate; oxalate, acid oxalate; sulphate, acid sulphate; trichloroacetate; trifluoroacetate and methane sulphonate.

Among these salts, isovaleryl L-carnitine acid fumarate (US 5,227,518) is particularly preferred.

A list of FDA-approved pharmacologically acceptable acids is given in Int. J. Pharm.. 33, 1986, 201-217, the latter publication being incorporated in the present specification by reference.

The supplement of the invention may further comprise vitamins, coenzymes, mineral substances, aminoacids and antioxidants. The supplement may be manufactured in the form of tablets, lozenges, capsules, pills, granulates, syrups, vials or drops.

Claims

1. A food/dietary supplement which comprises the following characterizing ingredients:

(a) isovaleryl L-carnitine or a pharmacologically acceptable salt thereof; and

(b) a poliphenol combination extracted from trees or shrubs.

2. The supplement of claim 1, wherein the trees belong to the Pinaceae family.

3. The supplement of claim 2, wherein the Pinaceae are selected from the group comprising the maritime pine (Pinus pinaster), the red fir (Picea abies), the Finnish pine, the Eastern hemlock (Tsuga canadensis), the Pinus massoniana and the Douglas fir (Pseudotsuga menziesiϊ).

4. The supplement of claim 1, wherein the trees belong to the Fagaceae family.

5. The supplement of claim 4, wherein the Fagaceae are selected from the group comprising the Fagus grandifolia, the common beech (Fagus sylυatica), the sweet chestnut (Castanea satiυa) and the English oak (Quercus robur).

6. The supplement of claim 1, wherein the shrubs belong to the Oleaceae family, Forsythia genus.

7. The supplement of claim 6, wherein the Forsythia species are Forsythia intermedia and Forsythia suspensa.

8. The supplement of claim 3, wherein the weight ratio between isovaleryl L-carnitine or an equimolar amount of a pharmacologically acceptable salt thereof and the maritime pine or red fir extract ranges from 10:1 to 2:1.

9. The supplement of claim 8, in unit dosage form, comprising:

Isovaleryl L-carnitine 500 mg

Maritime pine bark exctract 100 mg

(proanthocyanidine titre: 85-90%)

10. The supplement of claim 8, in unit dosage form, comprising:

Isovaleryl L-carnitine 500 mg

Maritime pine bark exctract 50 mg

(proanthocyanidine titre: 75-85%)

Red fir bark exctract 50 mg

(proanthocyanidine titre: 75-85%)

11. The supplement of claim 8, in unit dosage form, comprising:

Isovaleryl L-carnitine 400 mg

Vit. E 5 mg

Vit. Bi 5 mg

Vit. Be 5 mg

Vit. PP 10 mg

Vit. C 50 mg

Selenomethionine 50 μg

Magnesium stearate 5 mg

Zinc glycinate 5 mg

12. The supplement of any of the preceding claims, for the prevention of vascular, cardiac, central or peripheral nervous system alterations, immune deficiencies, learning and ageing-related disorders as well as for meeting increased muscular energy requirements.

13. The supplement of any of the preceding claims, further comprising a "carnitine" selected from the group consisting of L- carnitine, acetyl L-carnitine, propionyl L-carnitine, butyryl L-carnitine and valeryl L-carnitine or the pharmacologically acceptable salts or mixtures thereof.

14. The supplement of anyone of the preceding claims which further comprises vitamins, coenzymes, mineral substances, aminoacids, and antioxidants.

15. The supplement of any of the preceding claims wherein the pharmacologically acceptable salt is selected from the group comprising: chloride; bromide; iodide; aspartate, acid aspartate; citrate, acid citrate; tartrate; phosphate, acid phosphate; fumarate, acid fumarate; glycerophosphate; glucose phosphate; lactate; maleate, acid maleate; mucate; orotate; oxalate; acid oxalate; sulphate, acid sulphate; trichloroacetate; trifluoroacetate and methane sulphonate.

16. A method for the prevention and/or treatment of vascular, cardiac, central or peripheral nervous system alterations and for the prevention of learning and ageing-related disorders and immunodeficiencies as well as for meeting increased muscular energy requirements, which comprises administering to an individual in need thereof a combination composition comprising the following ingredients:

(a) isovaleryl L-carnitine or a pharmacologically acceptable salt thereof; and

(b) a poliphenol combination extracted from Pinaceae, Fagaceae and Oleaceae.