MXPA05010674A - Use of red algae subjected to extreme temperature and light conditions - Google Patents

Abstract

The present invention refers to nutritional, dietetic or nutraceutical compositions from red macroalgae subjected to extreme conditions of temperature and luminosity, or extracts obtained from such algae. The invention further refers to the use of said red microalgae as an antioxidant agent.

Description

USE OF RED ALGAE SUBMITTED TO EXTREME TEMPERATURE AND LUMINOSITY CONDITIONS The present invention deals with the use of red macroalgae subjected to extreme conditions of temperature and luminosity, or extracts obtained from such algae, as antioxidants. The invention finds application especially in the nutritional, dietary, and nutraceutical domains. The nutritional value of algae has been known for a long time, for centuries, the populations of the countries of Southeast Asia consume some (Nisizawa K. et al., Hydrobiol. (1987), vol.151 / 1542, pp.5-29), particularly for its flavors and frequently as a "sea vegetable" ("sea vegetable") or a seasoning vegetable from the sea. In western countries, algae are used essentially as a source of phyco-colloids, mainly to obtain alginates, carrageenans or agars, to widely recognized thickeners, gelatinizing or stabilizing properties (Me Huhh D., Hydrobiol. (1991), vol. .221, pp.19-29). The nutritional value of algae is, however, very well established, with a wealth of minerals, fibers, proteins, essential amino acids and vitamins (Burtin P., Electron, J. Environ, Agrie, Food Che. (2003), vol.2 (4) ISSN: 1579-4377; Jimenez-Escrig et al., Arch. Latinoam. de Nutr. (1999), vol.49, pp.114-120; Carbini L. et al., Riv. Sci. Aliment. (1998), vol.27, pp.169-173). In addition, nowadays, it is admitted that it is necessary to fight better, in our daily life, against the environmental factors that induce an oxidative stress (ÜV, contamination, etc) with a deleterious effect on our organism, such as a very large production of free radicals or oxygenated reactive species. An antioxidant supplement is actually one of the most vivid nutritional recommendations ("Des antioxydants pour nous aider a vieillir en bonne santé" (Antioxidants to help us age healthy), Nutranews, Décembre 2001). In addition, it is one of the main demands of the manufacturers of food supplements, or of alicaments in the nutraceutical industry, with the launch in the market of products with multiple virtues, particularly antioxidants, the "multi-nutrients" ("Les multi-nutriments" , des suppléments nutritionnels essentiels "(Multi-nutrients, essential nutritional supplements), Nutranews, Avril 2003). The antioxidant potential of algae is widely documented in the state of the art. The "antioxidant activity of macroalgae, fresh or dried, makes the object of a recent study (Jimenez-Escrig A. et al., J. Sci. Food Agrie. (2001), vol.81, pp.530-534) where it is concluded, on the one hand, to a correlation between the antioxidant power and the richness of the algae in polyphenolic substances that are the phloroglucinols, and, on the other hand, to the antioxidant potential of the brown or brown algae superior to that of the red algae. should note in this article, for the species "rhodophyte" Chondrus crispus of usual quality (or standard), the non detection of an antioxidant activity The patent application FR-A-2 655 268 describes the use of extracts of brown algae or brown, red or green for the preparation of compositions, particularly foodstuffs with an anti-radical activity.It is mentioned that brown algae have the most important antiradical activity.No information was disclosed regarding the nature of the substances responsible for this activity of algal origin. On the other hand, it should be emphasized that red algae of usual quality, generally exploited for their richness in phyco-colloids, are harvested ordinarily and classically in the proliferation phase. The metabolism that they present is then completely different from that of the algae still in a vegetative state. For example, in the particular case of Chondrus crispus algae, an examination of its composition demonstrates a very rapid consumption of the dipeptide accumulated in the vegetative phase, the dipeptide citrulinil-arginine, at the time of the return of spring and conditions favorable to the growth of the plant (Laycock M.V. et al., Can J. Biochem. (1981), vol.59, pp.522-527). In a general way, it is observed during the growth of the algae, at the same time an increase of the content in phyco-colloids and a decrease of those compounds with energetic value (proteins, amino acids, etc), because the latter 'are actively consumed. In the case of Chondrus crispus algae, this could explain the low nutritional value granted by the authors in conclusion of a study on the effect of algal diets on the growth of marine molluscs (Mai K. et al., Aquaculture (1994), vol. .128, pp.115-130). The literature reports that some red macroalgae can, during winter conditions with low water temperatures and a minimum luminous intensity, capture nitrogen (nitrate ions, nitrite, ammonium) from coastal waters and store it in the form of small peptides and / or amino acids. free (gigartinina, citrulina, arginina, ornítina, etc) variables according to the species. The said red algae naturally identified with such a profile, at the end of the winter were successively Grateloupia turuturu (Miyazawa K. et al., Bulletin of the Japanese Society of Scientific Fisheries (1974), vol.40, pp.815-818) , Ahnfel tia plicata, Gracilaria sp. , Petrocelis middendorfii, Polyides rotundus, Polysiphonia lanosa, Rhodomela confervoides (Laycock MV et al., Can J. Biochem. (1977), vol.55, pp.27-30) and Chondrus crispus (Laycock MV et al., Can J Biochem. (1981), vol.59, pp.522-527).

This metabolic modification within the alga was exploited for applications in dermatology. Thus, the European patent application EP 1 350 517 A1 (submitted by the applicant) describes its dermatological use, as care and treatment agents for skin and fañaras, of chemical molecules analogous to citrulinil-arginine and of an extract enriched in polypeptides containing this dipeptide. Following these investigations, the applicant noted that red macroalgae subjected to extreme conditions of temperature and luminosity, _ so that certain fractions obtained from the extraction of such algae, have a strong antioxidant character, and this, unexpectedly with respect to a comparison with the same red algae, but collected in full growth (called "standard algae") or for the fractions obtained from said algae. The applicant has also noticed, during comparative studies with reference antioxidants and a chemical replication of the dipeptide citrulinil-arginine, that this antioxidant character can not be explained only by the accumulation of the dipeptide in the alga subjected to extreme conditions of temperature and luminosity. He concluded that other constituents accumulated in this algae quality during the winter period also contribute to antioxidant power, and identified some of these constituents.

Thus, according to a first aspect, the invention relates to the nutritional, dietetic or nutraceutical use of red macroalgae subjected to extreme conditions of temperature and luminosity, or of extracts obtained from such algae, as an antioxidant agent. Preferably, red macroalgae are selected from Grateloupia turuturu, Ahnfeltia plicata, Gracilaria sp. , Petrocelis middendorfii, Polyides rotundus, Polysiphonia lanosa, Rhodomela confervoides, Gymnogongrus devoniensis, Callophyllis crassifolia, Callophyllis crenulata, Callophyllis megalocarpa, Callophyllis pínnata and Chondrus crispus, the latter species being preferred. For "red macroalgae subjected to extreme conditions of temperature and luminosity", it is necessary to understand algae in vegetative phase that were exposed to a cold water with a reduced luminosity, allowing the formation of antioxidant constituents, but also energy. These particular conditions can be either natural winter conditions, or artificial conditions with a process of cultivation in ponds that reproduce the natural conditions indicated above. The "sensitive" parameters for such artificial conditions are particularly described for the species Chondrus crispus (Laycock M.V. et al., Can J. Biochem. (1981), vol.59, pp.522-527).

Whatever the "wintry" environment retained, natural or reproduced, an important feature of the invention is that the quality of the algae sought is obtained only when the algae is exposed to the following conditions: - a harvest phase of the algal species at end of the winter season (northern winter), preferably between March and early April; - an "exposure" of the algal species to a cold water at a temperature between 0 and 8 ° C, preferably between 0 and 4 ° C; - an "exposure" of the algal species to a reduced luminosity preferably with a luminous intensity comprised between 40 and 120 langley.day-1 PAR; - an "exposure" of the algal species to a water containing nitrate and ammonium ions, preferably a water containing nitrate ions between 1 to 6 μmol; an "exposure" of the algal species to a water of low salinity, preferably a water of salinity comprised between 10 and 30 per thousand. These conditions are those that one finds in subarctic zones (north of 45 ° parallel N). The coasts of Nova Scotia in Canada is, for example, a particularly favorable biotope to obtain the quality of the required algae. According to a preferred embodiment of the invention, a "uni-algal" biomass of red algae, for example of Chondrus crispus, is first obtained after a rigorous selection of an algae quality devoid of any other "parasitic" algae . Then it is placed in a pond (at the end of autumn / beginning of winter) then it is kept in filtered seawater throughout the winter, without agitation or oxygenation, under the conditions described above. Later it is harvested at the end of the vegetative period (March / April), before the algae pass in the proliferative phase. This seaweed is finally carefully washed, and then dried. The "exposed" algae is characterized by a high content of antioxidant and energetic constituents, and includes particularly: - the dipeptide citrulinil-arginine, - free amino acids taurine and citrulline and / or its derivatives, - amino acids type "mycosporin", - the ions zinc and / or selenium, - the floridóside. The derivatives of taurine can be N-dimethyl-taurine and N-trimethyl-taurine, and those of citrulline the amino acids lividin, grateloupina, gigartinina et gongrina. Preferably, the "exposed" algae includes: - from 2 to 10% citrulinyl-arginine by weight of dry matter, - from 6 to 30 mg of taurine in 1 g of dried seaweed, - from 6 to 30 mg of citrulline in 1 g of dried seaweed, - of 0.7 to 1.5 mg of amino acid type "mycosporin" in 1 g of dry seaweed, - from 20 to 80 ppm of zinc and from 5 to 40 ppm of selenium, - from 3 to 6% of floridose by weight of dry matter, its nitrogen content, around 4%, is indicative of its content in proteins / peptides / free amino acids all confused. Red macroalgae subjected to extreme conditions of temperature and luminosity are therefore characterized by a higher protein or peptide content than standard red macroalgae. In algae subjected to extreme conditions of temperature and luminosity: - the dipeptide citrulinil-arginine is particularly interesting because it constitutes a source of arginine, a semi-essential amino acid described by participating in the energy metabolism of tissues, under the form of phospho-arginine and constitute a support for glycolysis (Ga.de G. et al., Comp.biochem.physiol. (1986), vol.83B, pp.255-272); - citrulline, from the dipeptide or the free accumulated in the algae, is also capable of representing an interesting energy value because it is a precursor of ornithine, glutamic acid and proline; - Taurine has interesting antioxidant properties against the phenomenon of lipid peroxidation via an affinity for membrane phospholipids and a stabilization of cell membranes (Birdsall TC, Alternative Med. Rev. (1998), vol.3, pp.128- 136); Taurine is commonly used in nutrition because a supplement in this amino acid constitutes an energy contribution whose origin would also be a capacity to modulate glycolysis (Redmond HP et al., Nutrition, (1998), vol 14, pp. 599-604; JB Lombardini et al., Ed Plenum Press, NY (1992)); - "mycosporin" type amino acids, such as palitin, shinorin and asterin, have protective properties demonstrated against ultraviolet irradiation (Bandaranayake WM, Nat. Prod. Rep. (1998), vol.15, pp.159- 172); Zinc ions are known for their antioxidant properties (Zago MP, Free Radie, Biol. Med. (2001), vol.31, pp.266-274), and selenium is a necessary cofactor for the activity of certain antioxidant enzymes (Tappel AL, Curr Top, Cell, Regul. (1984), vol.24, pp.87); 2-glycerol-galactopyranoside or floridoside is an anti-stress onosaccharide (Simon-Colin C, J. Appl. Phycol. (2002), vol.14, pp.123-127). On the other hand, it is obvious that other constituents usually identified in red macroalgae (mineral salts, fibers, etc.) also contribute to the nutritional interest of algae subjected to extreme conditions of temperature and luminosity.

The invention also relates to extracts, particularly alcoholic, hydro-alcoholic or hydro-glycolic extracts, of these red macroalgae exposed to extreme conditions of temperature and luminosity. These extracts are obtained by techniques well known to the specialist, and include the same antioxidant and energy constituents as those of the "exposed" red macroalgae, preferably in the amounts mentioned below; - at most 15% citrulinyl-arginine by weight of extract, - at most 45 mg of taurine in 1 g of extract, - at most 45 mg of citrulline in 1 g of extract, - at most 2.25 mg of "icosporin" type amino acids in 1 g of extract, - at most 120 ppm of zinc and at most 60 ppm of selenium, - at most 9% of floridoside by weight of extract. The red "exposed" macroalgae according to the invention, or the extracts of such algae, are advantageously used as an active ingredient for the preparation of compositions with nutritional and antioxidant function, such as nutritional, dietetic or nutraceutical compositions. Thus, according to another aspect, the invention relates to a nutritional, dietetic or nutraceutical composition containing one or several species of red macroalgae subjected to extreme conditions of temperature and luminosity (or one or several extracts of these algae). Generally, red macroalgae or their extracts are transformed (a) (as) into a micronized powder or paste, and then mixed (a) with the excipients commonly used in nutrition, dietetics or nutraceuticals to form the said composition. The composition of the invention may also contain other active ingredients according to the needs of the domain under consideration. It can be packaged in capsules, tablets, paper, etc ... The invention is illustrated by the following examples and compositions, given only as an indication. Example 1: Determination of the antioxidant activity of an ethanolic fraction from Chondrus crispus "exposed" Algae of the species Chondrus crispus subjected to extreme conditions of temperature and luminosity according to the operative method indicated above, were then washed and dried. An ethanolic fraction is obtained according to the following extraction protocol: the ground alga is first humidified with water at room temperature. Then it is subjected to a direct extraction with ammonia ethanol under stirring at room temperature. After filtration, the extract is successively concentrated, adjusted with water, decolorized with active carbon, filtered, and again concentrated. The extract thus obtained, contains particularly: citrulinil-arginine, taurine and methyl derivatives, saturated fatty acids, monosaccharides, floridósido, amino acids type "mycosporin", citrulina, mineral salts such as zinc, and selenium. i) measurement of the "sequestering" activity of the hydroxyl radical (OH °) The method, described by Reliman, A. et al. (British J. Pharmacol. (1997), vol.122, pp.1702-1706), is used to measure the rate constant of sequestration of the hydroxyl radical Ks (OH °) The extract according to the invention is compared to mannitol (reference sequestrant), to the dipeptide citrulinil-arginine (product of synthesis) and ascorbic acid (reference sequestrant), whose Ks (OH °) is measured according to Cabelli D.E., J. Phys.

Chem. (1983), vol.87, pp.1809-1812). Table 1 shows the average values obtained from four independent experiments.

Table 1 ii) Measure of antioxidant power "global 1 The production system of reactive species derived from oxygen, xanthine oxidase / hypoxanthine + EDTA, described by Nowak D. et al. (Biomed, Biochem. Acta (1991), vol.50, pp.265-272), was used to determine the antioxidant power of an ethanolic fraction from Chondrus crispus "exposed", This antioxidant power, characterized by the sum of the effects against superoxide anion (02 ° ~), to hydrogen peroxide (H2O2), and OH °, is expressed by a percentage of protection of a "detector" molecule, deoxyriboose (Table 2).

Table 2 Example 2: Determination of the antioxidant activity of a hydro-alcoholic fraction from Chondrus crispus "exposed" Algae of the Chondrus crispus species have been collected subjected to extreme conditions of temperature and luminosity according to the operating procedure indicated above, then washed and dried. A hydroethanolic fraction (80 EtOH / 20 H2O) is obtained according to the following extraction protocol: the ground alga is first humidified by water at room temperature. It is then subjected to a direct extraction with an ammoniacal hydro-ethanolic solution, under stirring at room temperature. After the filtration, the. The extract is successively concentrated, adjusted with water, discolored to active carbon, filtered and again concentrated.

The extract thus obtained, contains particularly: citrulinil-arginine, taurine, amino acids type "mycosporin", L-citruline, mineral salts such as zinc, floridoside, and selenium. i) measurement of the "sequestering" activity of the hydroxyl radical (OH °) The protocol used is that described in example 1. The results are shown in table 3.

Table 3 ii) Measurement of the "global" antioxidant power The protocol used is that described in example 1. The extract according to the invention is compared to the dipeptide citrulinil-arginine and to mannitol. The results are shown in table 4.

Table 4 Example 3: Determination and comparison of the antioxidant activities of hydro-alcoholic extracts of Chondrus crispus "exposed" and "standard" Hydro-alcoholic algae extracts of "exposed" and "standard" quality are obtained according to the same extraction protocol. The determination of its antioxidant power was then carried out with a general oxidation model by Nowack et al., Using the production system of reactive species, xanthine oxidase / hypoxanthine (Biomed, Biochem. Acta (1991), vol.50, pp. .265-272). As indicated in the preceding examples 1 and 2, the antioxidant power is expressed as a percentage of protection from deoxyribose (or percentage of oxidation inhibition of deoxyribose). A variant (table 6) consisting of not adding EDTA, was introduced to better distinguish the effect of chelating compounds contained in the extracts, and its contribution to the overall antioxidant effect.

The results are shown below, they are obtained from 3 independent tests, they allow a comparison of the antioxidant potential between the "exposed" and "standard" extracts of Chondrus crispus, at different concentrations.

Table 5 Table 6 Example 4: Total nitrogen proportion and "amino acid" profile of Chondrus crispus "exposed" and "standard" algae.

The first results of an in vivo study carried out in man can also illustrate the use of said algae according to said invention. Now, we give examples of nutritional, dietetic or nutraceutical compositions with a considerable antioxidant potential and a high energy value according to the invention.

Composition 1: (capsule presentation) • Micronized powder of Chondrus crispus and / or Gymnogongrus devoniensis (200 mg / capsule). • excipients csp 1 capsule: microcrystalline cellulose, magnesium stearate.

Composition 2: (presentation in capsule) • Dry extract of Chondrus crispus and / or Gymnogongrus devoniensis (150 mg / capsule). • excipients csp 1 capsule: microcrystalline cellulose, starch, magnesium stearate.

Composition 3: (tablet presentation) • Micronized powder of Chondrus crispus and / or Gymnogongrus devoniensis (250 mg / tablet). • excipients csp 1 tablet: microcrystalline cellulose, sodium carboxymethylcellulose, colloidal silica, magnesium stearate.

Claims (11)

1. Nutritional, dietetic or nutraceutical use of red macroalgae subjected to extreme conditions of temperature and luminosity, or extracts from such algae, as an antioxidant agent.

2. Use according to claim 1, characterized in that the red acroalgae are chosen within the species Grateloupia turuturu, Ahnfeltia plicata, Gracilaria sp. , Petrocelis middendorfii, Polyides rotundus, Polysiphonia lanosa, Rhodomela confervoides, Gymnogongrus devoniensis, Callophyllis crassi folia, Callophyllis crenulata, Callophyllis megalocarpa, Callophyllis pinnata and Chondrus crispus.

3. Use according to claim 1 or 2, characterized in that the red macroalgae are of the species Chondrus crispus.

4. Use according to one of claims 1 to 3, characterized in that macroalgae subjected to extreme conditions of temperature and luminosity, or their extracts, include the following constituents: - the dipeptide citrulinil-arginine, - free amino acids taurine and citrulline and / or its derivatives, - "mycosporin" type amino acids, - zinc and / or selenium ions, - floridoside.

5. Use according to claim 4, characterized in that said red macroalgae include: - from 2 to 10% of citrulinil-arginine by weight of dry matter, - from 6 to 30 mg of taurine in 1 g of dried seaweed, - from 6 to 30 mg of citrulline in 1 g of dried seaweed, - of 0.7 to 1.5 mg of amino acid type "mycosporin" in 1 g of dry seaweed, - from 20 to 80 ppm of zinc and from 5 to 40 ppm of selenium, - from 3 to 6% of floridose by weight of dry matter.

6. Use according to claim 4, characterized in said extracts include: - at most 15% of citrulinil-arginine by weight of extract, - at most 45 mg of taurine in 1 g of extract, - at most 45 mg of citrulline in 1 g of extract, - at most 2.25 mg of amino acid type "mycosporin" in 1 g of extract, - at most 120 ppm of zinc and at most 60 ppm of selenium, - at most 9% of floridóside in weight of extract.

7. Use according to one of claims 4 to 6 characterized in that the "mycosporin" type amino acids are chosen within a group, such as palitin, shinorin and asterin.

8. Use according to one of claims 1 to 7, characterized in that the extracts are alcoholic, hydro-alcoholic or hydro-glycolic extracts.

9. Use according to one of claims 1 to 8 characterized in that said red macroalgae are presented in the form of a micronized powder or a paste.

10. Nutritional, dietetic or nutraceutical composition of red macroalgae subjected to extreme conditions of temperature and luminosity, or extracts from such algae.

11. Composition according to claim 10, characterized in that said red macroalgae or said extracts are such as defined in one of claims 2 to 9.