WO2002058682A2

WO2002058682A2 - An anti-atherosclerosis composition containing carotenoids and method for inhibiting ldl oxidation

Info

Publication number: WO2002058682A2
Application number: PCT/IL2002/000053
Authority: WO
Inventors: Morris Zelkha; Tanya Sedlov; Zohar Nir
Original assignee: Lycored Natural Products Industries Ltd.
Priority date: 2001-01-23
Filing date: 2002-01-21
Publication date: 2002-08-01
Also published as: US20040110849A1; KR20030091972A; AU2002226652B2; NO20033232L; BR0206942A; CA2435613A1; ES2346047T3; WO2002058682A3; NO20033232D0; KR100841238B1; RU2288707C2; PL208263B1; EP1363609B1; JP2004520373A; DE60236787D1; PL361734A1; EP1363609A2; CN1498102A; JP4405151B2; US20080114074A1

Abstract

An anti-atherosclerosis composition comprising a LDL-oxidation inhibiting effective amount of phytoene, phytofluene or mixtures thereof and a method for inhibiting the progression of atherosclerosis.

Description

An Anti-Atherosclerosis Composition Containing Carotenoids and Method for

Inhibiting LDL Oxidation

Field of the Invention

The present invention relates to the field of natural products and uses thereof for inhibiting the progression of atherosclerosis. More particularly, the present invention

relates to compositions comprising phytoene, phytofluene or mixtures thereof and their

use for inhibiting the progression of atherosclerosis.

Background of the Invention

The prevention of LDL oxidation has been acknowledged as an important factor in the

inhibition of the progression of atherosclerosis. There is a variety of anti-atheosclerosis drugs such as the drugs of the vastatin group, hereinafter conventional anti-atherosclerosis

agents. Furthermore, certain carotenoids which are known to be anti-oxidants have been

reported to inhibit LDL oxidation and thus arrest the progression of atherosclerosis (see,

e.g. Krinsky, N., Eree Radical Biology & Medicine, (1989) vol. 7, pp. 617-635 ).

PCT/IL98/00286 discloses the effectiveness of a synergistic mixture of lycopene and

vitamin E in inhibiting LDL-oxidatisn, and hence arresting the progression of

atherosclerosis. The effectiveness of the carotenoids phytoene and phytofluene in inhibiting LDL-oxidation has not been evaluated.

The contribution of atherosclerosis to heart disease is well known. Therefore there is a constant need for novel compositions which effectively inhibit the progression of

•atherosclerosis. Accordingly, it is an objective of the present invention to provide a novel composition

which demonstrates high effectiveness in inhibiting LDL oxidation, thus effectively arresting the progression of atherosclerosis.

A further objective of the present invention is to provide a method for inhibiting LDL

oxidation and hence inhibit the progression of atherosclerosis.

Other objects of the invention will become apparent as the description proceeds.

Summary of the Invention

The present invention provides a method for inhibiting the progression of atherosclerosis,

comprising the administration of an effective anti-LDL-oxidation amount of phytoene, phytofluene or mixtures thereof, to a subject in need thereof.

Further provided by the present invention is the use of a composition comprising

phytoene, phytofluene or mixtures thereof, for inhibiting the oxidation of LDL.

A further aspect of the present invention is the use of phytoene, phytofluene or mixtures

thereof, in the preparation of a medicament for inhibiting the oxidation of LDL and the

progression of atherosclerosis.

The present invention further provides an anti-atherosclerosis composition comprising a

•LDL-oxidation inhibiting effective amount of phytoene, phytofluene or mixtures thereof. Further provided by the present invention is the use of a LDL-oxidation inhibiting

effective amount of phytoene, phytofluene or mixtures thereof, in functional foods,

dietary supplements, food stuff and drinks.

Description of the Drawings

Fig. 1(A),(B) - Effect of crystalline natural lycopene and of tomato oleoresin on copper

ion-induced LDL oxidation.

Fig. 2 (A),(B)- Effect of phytofluene/phytoene on copper ion-induced LDL oxidation.

Fig. 3 - Effect of lycopene, vitamin E, β-carotene, and phytofluene/phytoene on LDL

oxidation.

Fig. 4 - Effect of lycoepene in combination with antioxidants on LDL oxidation.

Fig. 5 - Effect of β-Carotene in combination with antioxidants on LDL oxidation.

Fig. 6 - Effect of vitamin E in combination with antioxidants on LDL oxidation.

Fig. 7 - Effect of phytofluene/phytoene in combination with antioxidants on LDL oxidation.

Fig. 8 - Effect of combination of antioxidants on LDL oxidation.

Fig. 9 - Effect of carotenoid mixture on LDL oxidation.

Detailed Description of a Preferred Embodiment of the Invention

The following description is illustrative of preferred embodiments of the invention. The

following description is not to be construed as limiting, it being understood that the

skilled person may carry out many obvious variations to the invention. The description applies to mixtures of phytoene and phytofluene as well as to each one of them separately. Therefore, wherever reference is made to mixtures thereof it is also intended for each of the carotenoids separately as well.

While studying the effect of various carotenoids on LDL oxidation it has been surprisingly found that phytoene, phytofluene or mixtures thereof, are substantially more effective than other carotenoids in inhibiting LDL oxidation.

According to a particular embodiment of the method of the present invention, at least 0.2 mg/day of phytoene, phytofluene or mixtures thereof, are administered to a subject in order to inhibit the oxidation of LDL in the blood and hence arrest the progression of atherosclerosis. Wherein a mixture of said carotenoids is administered, the ratio between phytoene and phytofluene in the mixture is from about 1 :20 to 20:1. Administration may be in daily multiple or single dosages. According to the present method, administration is adopted to achieve an anti-LDL-oxidation effective concentration of phytoene,

phytofluene or mixtures thereof, in the blood, said concentration being at least 0.01 μM.

According to an optional embodiment of the present method, phytoene, phytofluene or mixtures thereof, may be administered in conjunction with conventional anti-atherosclerosis agents.

According to a further aspect of the present invention there is provided a composition comprising an anti-LDL-oxidation effective amount of phytoene, phytofluene or mixtures 'thereof. Said composition comprising at least 0.1 mg of phytoene, phytofluene or mixtures thereof. The composition may further comprise pharmaceutically acceptable

adjuvant, excipients and additives.

A further embodiment of the present invention relates to a solid dosage form for oral

administration, in the form of a tablet, capsule, hard-shell capsule, soft gel or gelcap,

which contains at least 0.1 mg of phytoene, phytofluene or mixtures thereof.

According to yet a further embodiment of the present invention, the use of phytoene,

phytofluene or mixtures thereof, is provided wherein they are added to functional foods

such as bars, dietary supplements, drinks or other food stuff so that said functional foods,

dietary supplements, food stuff and drink provide an inhibitory effect on LDL-oxidation.

According to the present embodiment the functional food, food stuff, dietary supplement

or drink comprise at least 0.1 mg of of phytoene, phytofluene or mixtures thereof.

The following examples illustrate the substantially improved effectiveness of phytoene

and phytofluene over other carotenoids, in the inhibition of LDL oxidation.

Examples

General Procedures All studied compounds were dissolved in THF to a stock solution of lmg/ml. LDL was

dialyzed for 24 hours against phosphate buffered- saline (PBS) prior to its oxidation to

remove any EDTA that can interfere with the oxidation. LDL (lOOμg protein/ml), was

incubated in the absence (Control) or presence of increasing concentrations of each one of

'the studied compounds, as well as, in various combinations including 2, 3 or the 4 constituents at the desired natural distribution. LDL oxidation was induced by the

addition of 5μM of CuSO , for 2 hours at 37°C. LDL oxidation was terminated by

adding ImM Na₂ EDTA and immediate refrigeration at 4°C. The extent of LDL oxidation

was then determined by measuring the amount of thiobarbituric acid reactive substances

(TBARS) and of lipid peroxides (PD) formed. Control LDL was supplemented with only THF.

Example 1; Effect of crystalline natural lycopene and of tomato oleoresin on copper

ion-induced LDL oxidation: Concentration study.

LDL (100 μg of protein/mL) was pre-incubated for 30 minutes at 37°C with increasing

concentrations of crystalline pure lycopene, or with tomato oleoresin (at equivalent

lycopene concentrations), followed by a further incubation for 2 hours at 37°C in the

presence of 5 μmol/L of CuSO₄. LDL oxidation was measured by the TBARS (A) or by

the lipid peroxides (B) assays respectively. Results are given as mean ± SD (n=3).

Addition of increasing concentrations of lycopene inhibited copper ion-induced LDL

oxidation moderately, in a dose-dependent manner. A maximal 22% and 27% inhibition

in TBARS (Fig 1 A) and in lipid peroxides (Fig IB) formation, respectively, was obtained

on using 50μmol/L of lycopene. However, addition of tomato oleoresin to the lipoprotein,

inhibited LDL oxidation to a much greater extent (about 90% inhibition), with an IC₅₀

(the concentration needed to inhibit LDL oxidation by 50%) of 8.0 μmol/L and 8.4

μmol L of lycopene equivalents for the inhibition of TBARS (Fig 1A) and of lipid

peroxides. (Fig IB) formation, respectively. Example 2; Effect of phytofluene/phytoene on copper ion-induced LDL oxidation:

Concentration study.

Phytofluene/phytoene dose-dependently inhibited CuSO -induced LDL oxidation, as

measured by the TBARS 2(A) and by the lipid peroxides assays 2(B), respectively. The

IC₅₀ of Phytofluene/phytoene in the copper ions-induced LDL oxidation is 0.25 μg/ml for

inhibition of TBARS formation and 0.35 μg/ml for inhibition of lipid peroxides

formation, respectively.

Example 3: Effect of lycopene, vitamin E, β-carotene,, and phytofluene/phytoene on

LDL oxidation.

At the low concentration, which mimics the composition of Lyc-O-Mato^®, Lycopene

(lOμg/ml), vitamin E (2.5μg/ml) and β-carotene (0.4μg/ml) did not significantly affect

LDL oxidation. In contrast, phytofluene/phytoene (1.2μg/ml) substantially inhibited LDL

oxidation by 85-90% as analyzed by TBARS and lipid peroxides assays.

Example 4: Effect of lycoepene in combination with antioxidants on LDL oxidation.

On using Lycopene (10 μg/ml) in combination with each one of the other antioxidants

separately (2 compound in each study), the addition to lycopene to β-carotene (0.4μg/ml)

or vitamin E (2.5μg/ml) inhibited LDL oxidation (TBARS assay) by 22% and 11%

respectively, in comparison to the effect of lycopene alone, whereas the addition of

phytofluene/phytoene (1.2μg/ml) inhibited LDL oxidation by as much as 95%. These

results, presented in Fig. 4 show that the addition to lycopene of β-carotene or vitamin E

contribute, though minimally, to the inhibition of LDL oxidation, whereas

phytofluen/phytoen posses the highest capacity to act as a most potent inhibitor of LDL -8-

oxidation (alone, and in combination with lycopene). Similar results were obtained for

LDL oxidation analysis by the lipid peroxides assay.

Example 5: Effect of β-Carotene in combination with antioxidants on LDL

oxidation.

With reference to Fig. 5, on using β-carotene as the major antioxidant alone or in

combination with the other antioxidants, unlike lycopene, it did not affect at all LDL

oxidation alone or in combination with vitamin E. An inhibitory effect (22%) on LDL

oxidation (TBARS) was observed when β-carotene was added in combination with

lycopene and a most potent inhibition of LDL oxidation (98% inhibition, in the TBARS

assay) was obtained on adding β-carotene together with phytofluene/phytoene. Similar

results were obtained for LDL oxidation analyses by the lipid peroxides assay.

Example 6: Effect of vitamin E in combination with antioxidants on LDL oxidation. On using vitamin E as the major antioxidant alone or in combination with the other

antioxidants, the addition of lycopene but not β-carotene, inhibited LDL oxidation by

12% (TBARS assay). The addition of phytofluene/phytoene to vitamin E resulted in a

98% inhibition in LDL oxidation. Similar results were obtained for the lipid peroxides assay as can be seen in Fig. 6.

Example 7: Effect of phytofluene/phytoene in combination with antioxidants on LDL oxidation.

Referring to Fig. 7, phytofluene/phytoene was found to be the most potent antioxidant

among the carotenoids examined, resulting in up to 90% inhibition of LDL oxidation (TBARS assay). The addition of each of the other antioxidant further reduced LDL

oxidation with the highest contribution observed for lycopene.

Example 8: Effect of combination of antioxidants on LDL oxidation. Analysis of the effect of various combinations of 3 antioxidants together were evaluated.

Only when the phytofluen/phytoene fraction was present, in any of the 3 studied

combinations, a substantial, over 90% inhibition in LDL oxidation was observed. When

lycopene, β-carotene and vitamin E were used in combination, in the absence of

phylofluene/phytoene, only minimal inhibition of LDL oxidation was obtained as shown

for both the TBARS and the lipid peroxide assays in Fig. 8.

Example 9: Effect of a Mixture of Carotenoids on LDL oxidation.

The effect of Lyc-O-M ato was compared to a mixture of carotenoids prepared from the

combination of 5 antioxidant compounds in the following concentrations; lycopene

(lOμg/ml), β-carotene (0.4μg/ml), Vitamin E (2.5μg/ml) and phytoene/phytofluene

(1.2μg/ml). Referring to Fig. 9, Lyc-O-Mato^® caused about 70% (TBARS assay)

inhibition in LDL oxidation. The carotenoid mixture inhibitory effect on LDL oxidation

was 96%) (TBARS assay). Similar results were obtained for the lipid peroxides assay.

While embodiments of the invention have been described by way of illustration, it will be

apparent that the invention may be carried out with many modifications, variations and

adaptations, without departing from its spirit or exceeding the scope of the claims.

Claims

1. A method for inhibiting the progression of atherosclerosis, comprising the

administration of an effective anti-LDL-oxidation amount of phytoene, phytofluene or mixtures thereof, to a subject in need thereof.

2. A method according to claim 1 wherein a least 0.2 mg/day are administered to a

subject in need thereof.

3. A method according to claim 1 wherein a mixture of phytoene and phytofluene is

administered.

4. A method according to claim 3 wherein the ratio of phytoene and phytofluene in the

mixture is from about 1:20 to about 20:1.

5. A method according to claim 1 wherein administration is in a daily dose.

6. A method according to claim 1 wherein the phytoene, phytofluene or mixtures thereof, are administered in conjunction with a conventional anti-atherosclerosis

agents.

7. A method according to claim 1 wherein administration is adopted to achieve an anti-LDL-oxidation effective concentration of phytoene, phytofluene or mixtures

thereof, in the blood, said concentration being at least 0.01 μM.

8. An anti-atherosclerosis composition comprising a LDL-oxidation inhibiting effective amount of phytoene, phytofluene or mixtures thereof.

9. A composition according to claim 8 comprising at least 0.1 mg of phytoene.

10. A composition according to claim 8 comprising 0.1 mg of phytofluene.

11. A composition according to claim 8 comprising at least 0.1 mg of a mixture of phytoene and phytofluene.

12. A composition according to claim 11 wherein the ratio of phytoene and phytofluene in the mixture is from about 1 :20 to about 20: 1.

13. A composition according to claim 8 in the form of a solid dosage tablet, capsule, hard-shell capsule, soft gel or gelcap.

14. A composition according to claim 8 comprising a pharmaceutically acceptable adjuvant, excipient or additive.

15. Use of a composition comprising phytoene, phytofluene or mixtures thereof, for inhibiting the oxidation of LDL.

16. Use of a composition according to claim 15 in conjunction with a conventional

anti-atherosclerosis agent.

17. Use of phytoene, phytofluene or mixtures thereof, in the preparation of a medicament for inhibiting the oxidation of LDL and the progression of atherosclerosis.

18. Use according to claim 17 wherein the medicament comprises a LDL-oxidation

inhibiting effective amount of phytoene, phytofluene or mixtures thereof.

19. Use according to claim 18 wherein the medicament comprises at least 0.1 mg of

phytoene.

20. Use according to claim 18 wherein the medicament comprises at least 0.1 mg of

phytofluene.

21. Use according to claim 18 wherein the medicament comprises at least 0.1 mg of a

mixture of phytoene and phytofluene.

22. Use according to claim 21 wherein the ratio of phytoene and phytofluene in the

mixture is from about 1 :20 to about 20: 1.

23. Use of an anti-LDL-oxidation effective amount of phytoene, phytofluene or mixtures

thereof in the preparation of functional foods, dietary supplements, food stuff and

drinks.

24. Use according to claim 23 wherein about 0.1 mg of phytoene, phytofluene or mixtures thereof is added to the functional foods, dietary supplements, food stuff and drinks.

25. A method substantially as described and exemplified.

26. A use substantially as described and exemplified.

27. A composition substantially as described and exemplified.

28. A pharmaceutical composition substantially as described and exemplified.