EP3240565A1 - Method for obtaining a superoxide dismutase (sod)-concentrated protein extract - Google Patents

Abstract

The invention relates to a method for obtaining a superoxide dismutase (SOD)-concentrated protein extract having a specific activity of at least 700 UI/mg protein, said method comprising a single step of extraction from a plant selected from any species of the Plumbaginaceae family.

Description

 PROCESS FOR OBTAINING A CONCENTRATED EXTRACT P ROTE I SUPEROXIDE

 DISMUTASE (SOD)

The present invention relates to a process for obtaining a concentrated protein extract superoxide dismutase (SOD) and the extract obtained by this method and its use.

 The present invention also relates to a composition comprising the extract obtained by the method of the invention.

 Superoxide dismutases (SOD) are enzymatic proteins that have a powerful antioxidant action against reactive oxygen species (ROS) generated in living organisms (animals and plants) by any type of environmental stress or during natural aging. Catalyzing the elimination of the first formed oxygen species (superoxides) following the reduction of oxygen, SOD is therefore the first line of defense against these EORs, which are strongly implicated in various neurodegenerative diseases (Alzheimer's disease, Parkinson), atherosclerosis, rheumatoid arthritis, Crohn's disease and some cancers (Campana et al., 2004; Valdivia et al., 2009).

SODs are metalloproteins that contain in their active site Cu / Zn, Mn or Fe atoms. These metals play an important role in the mechanism of action of SOD. For example, for Cu / Zn SOD, Cu ²⁺ is first reduced by the superoxide radical (0 ₂ ^" ), which is thus detoxified into oxygen.The reduced copper then yields an electron to a new superoxide molecule which in the presence of two protons, generates H ₂ 0 ₂ , much less toxic than 0 ₂ '^" .

 Due to their antioxidant capacity, SODs are widely used in different fields:

 In cosmetology, SODs protect the skin and hair by maintaining the integrity of the natural keratin structure (FR 7331354).

Experiments conducted at the University Hospital of Besançon (France) have shown that SOD supplementation prepares human skin for sun exposure (Annual Congress for Dermatological Research, Brest, France, May 2005).

In the pharmaceutical industry, drug formulations based on extracts rich in SOD (eg, orgotine) have been proposed to lower the level of EOR strongly implicated in various neurodegenerative or inflammatory diseases. In addition, an oral bioactive plant formula consisting of a SOD-rich melon (Cucumis melo) extract (Vouldoukis et al., 2004) coupled with a molecule of gliadin, a protein extracted from wheat (Menvielle-Bourg, 2005). ), has been proposed: Superoxide dismutase Gliadine (GliSODine) (FR 2729296). This dietary supplement is particularly appropriate to fight against the overproduction of free radicals, especially when the natural defenses of the body are weakened: elderly subjects, sun exposure, smoking, stress, intense physical exercise, etc. It can prevent certain chronic conditions involving oxidative stress or slow their evolution, thus improving the patient's living conditions.

In the food industry, plant extracts rich in SOD (eg PROMUTASE ™ 200, based on melon) are incorporated in foods for sport animals such as racehorses or racing pigeons to increase their resistance to intense efforts. The same extracts are incorporated into feed before and during the transport of farmed fish and crustaceans (shrimp, fry, trout, salmon, etc.) to reduce the mortality rate during transport.

To improve the quality of the milk produced, extracts rich in SOD (example PROMUTASE ™ R) are introduced into the diet of females of dairy farms (cows, goats, etc.).

Extracts rich in SOD are used in some food formulations to improve the growth performance of chickens and turkeys, animals often faced with poorly controlled digestive imbalances. SOD can be of very different origins: animal (for example bovine or porcine), human (for example placental or sanguine), microbial (for example bacteria or yeasts), etc.

 They can also be derived from genetic engineering or chemical synthesis. Several SODs are of plant origin (fungi, seaweeds, spinach, cereals, fruits and vegetables, etc.).

Among the various plants, a variety of melon (Cucumis melo) is rich in SOD (Vouldoukis et al., 2004). This variety of melon is characterized by resistant seeds that have a significant germination capacity, as well as fruits with slow aging (protected from the effects of free radicals). From the foregoing, it is clear that the role of SOD in reducing oxidative stress is very important.

 In addition, given its wide use in various fields of industry, there is a great interest to search for new natural sources hyperproductive of this protein with a high specific activity.

 Since SOD is a fragile protein, it is necessary to have processes that make it possible to obtain it in large quantities or better still, in a smaller quantity but with a high specific activity, without increasing the use of chemical compounds capable of alter the functionality of this protein and decrease its specific activity.

 SOD can be obtained simply by protein extraction from plants comprising it in large quantities and having a high specific activity. It is preferable that such SOD extraction processes do not involve the use of a multitude of solvents, especially organic solvents, which require the implementation of additional purification steps and degrade part of the SOD. .

 In addition, it should be noted that, in the majority of cases, the extraction of SOD from the plants is done by extraction processes which include a SOD enrichment step. The SOD extraction processes currently known require the realization of several steps before obtaining a highly concentrated SOD extract with a high specific activity. Such a process is described, for example, in patent application FR 2,747,044, said process comprising, in addition to the extraction step, a step of purifying the filtrate obtained by extraction with a precipitation agent making it possible to increase the specific activity. of SOD in the final extract. The enzymatic activity of SOD in the extract after the extraction step is 1482 IU / g. This extract is obtained from cereal plants, leguminous plants and oleaginous plants.

 Thus, the main disadvantage of these multistep processes is their high cost and their often long production time. In particular, to obtain a SOD having a high specific activity, an additional purification step typically including a precipitation step is systematically performed. Thus, these multistage processes are difficult to transpose on a large scale and often require the use of several chemical reagents, including solvents, which must be removed from the extract, which is not always easily achievable and increases the cost of the process.

There is therefore a need to have new processes for obtaining SOD with a high specific activity, the implementation of which is simple and fast and the cost is low. In addition to the high specific activity of SOD, the extracts obtained must be clean, that is to say devoid of the chemical reagents that were necessarily used during the extraction process, which will optimize the use of these products. extracts especially in food and feed. It is also desirable that the extract obtained by these methods contain other beneficial active substances, naturally present in plants, such as polyphenols which have antioxidant properties.

 The present invention therefore aims to implement a SOD extraction process having a very high specific activity. This process is simple, fast, inexpensive; it makes it possible to obtain an extract devoid of chemical reagents, it contains other natural substances having a beneficial effect and thus overcomes the disadvantages of the SOD extraction methods known in the prior art.

 The inventors have surprisingly discovered that the protein extracts obtained from the species of a family of littoral plants are so rich in SOD activity that a simple aqueous extraction from the crushed plant, without the addition of other solvents (for example for example, alcoholic solvents) and without additional purification steps, is sufficient to obtain extracts where the specific activity of SOD is at least 700 IU / mg of protein. This level greatly exceeds the highest level (126 IU / mg) reported so far in the literature for a variety of melon (Vouldoukis et al., 2004) considered as a reference among natural sources of SOD.

 Thus, the inventors have implemented a simple process for extracting SOD, the cost of which is low and which makes it possible to obtain very active protein extracts in SOD devoid of chemical reagents frequently used in conventional extraction processes and which contains other natural substances having a beneficial effect, thus overcoming the disadvantages of SOD plant extraction processes of the prior art.

 A first subject of the present invention therefore relates to a process for obtaining a concentrated superoxide dismutase (SOD) protein extract having a specific activity of at least 700 IU / mg of protein comprising a single protein extraction step from of a plant selected from any of the species of the Plombaginaceae family.

For the purposes of the present invention, the term "extraction process" or "extraction" means the implementation of a set of steps for obtaining a protein extract from a source, preferably a plant source. said steps comprising at least one step of recovering a liquid phase after contacting (under appropriate conditions) the solvent with said source, and then a step of purification comprising a precipitation step, in order to obtain the desired protein extract.

 Thus, within the meaning of the present invention, the expression "in a single extraction step" means that a single step is carried out among the steps mentioned above, namely the recovery of the liquid phase after contacting. solvent with the source, preferably the vegetable source. Preferably, the protein extraction method according to the present invention therefore does not include SOD protein purification steps and / or SOD protein enrichment steps.

 For the purposes of the present application, the term "specific activity" refers to the SOD activity related to the amount of protein, that is to say the activity of the SOD in the protein extract.

 The inventors have discovered that, on average, the extracts obtained directly (without purification and / or enrichment steps) by a simple step of contacting the water with crushed plants of the family Plombaginaceae, family of plants commonly represented in the littoral environment, have a specific activity of at least 700 IU / mg of protein. In particular, this specific activity may be at least 1000 IU / mg, more particularly at least 1200 IU / mg and even more particularly at least 1400 IU / mg.

 For the purposes of the present invention, the abbreviation "IU" designates the enzyme unit of SOD in the international system. The enzymatic unit of SOD is defined by Me Cord and Fridovitch (1969).

 In particular, the extracts obtained by the method of the present invention are concentrated in SOD with a specific activity of between 700 and 2000 IU / mg of proteins, more particularly between 1000 and 1800 IU / mg of proteins, more particularly between 1200 and 1600. Ul / mg protein and even more particularly between 1300 and 1500 IU / mg protein.

 The inventors have carried out comparative tests (see comparative examples) which demonstrate that other littoral species belonging to the Apiaceae, Brassicaceae, Aizoaceae and Chenopodiaceae families exhibit a specific SOD activity of between 20 and 400 IU / mg, that is very high. lower than that of the family Plombaginaceae.

 As used herein, the term "catalytic activity" refers to the SOD activity reported to the source plant biomass.

This catalytic activity is, in the species of the family Plombaginaceae, in a range between 3000 and 14000 IU / g MS (activity units / g of material dry), particularly between 5000 and 13000 IU / g MS and even more particularly between 7000 and 12000 IU / g MS.

 The inventors have also carried out comparative tests (see comparative examples) which demonstrate that the species of the Apiaceae, Aizoaceae, Brassicaceae and Chenopodiaceae families exhibit a SOD catalytic activity of between 120 and 1250 IU / g MS, which is much lower than that of species of the family Plombaginaceae.

 The Plombaginaceae family is a cosmopolitan family of dicotyledonous plants. The species of this family are present everywhere, particularly in saline and cold environments where their adaptability gives them a selective advantage.

 In France, the Plombaginaceae family is represented by the three genera Plumbago, Limonium and Armeria. Among them, the genera Limonium and Armeria are the most frequently encountered of this family. They are found on the entire coastline, from the Channel to the Mediterranean, via the Atlantic coasts.

 Thus, according to a preferred embodiment of the method of the invention, the plant from which the SOD is extracted is selected from the group consisting of the genera Limonium and Armeria.

 Advantageously, the process of the invention is carried out from species of the genera Limonium and Armeria found throughout the French coast, preferably on the French Atlantic coast.

 The genus Armeria contains only one species which is named Armeria maritima.

The genus Limonium includes perennial herbaceous plants with purple flowers that grow on sandy or poor soils of the Atlantic coast and in salt marshes.

 The species of the genus Armeria are perennial plants of the dunes or littoral rocks, whose flowering lasts from July to September.

 According to an even more preferred embodiment of the process of the present invention, the extraction of SOD is carried out from the plants selected from the group consisting of the species Limonium latifolium, Limonium normannicum, Limonium vulgare, Limonium tunetanum, Limonium densiflorum, Limonium Pruinosum, Limonium delicatulum, Limonium spathulatum, Limonium boitardii, Limonium wrightii and Armeria maritima.

All of these species are very rich in SOD, having a very high specific activity that is higher than that of SOD extracted from the organisms used as a source of SOD so far. In addition, these species have the advantage of being easily cultivable in the field, which is not usual for species whose natural environment is characterized by high salinity. In addition, Plombaginaceae species are easily cultivated under controlled conditions (greenhouse), making their use on an industrial scale possible, or even easy, in any season.

 Advantageously, the SOD extraction process is carried out from the species Limonium latifolium which is easily cultivable or accessible in the region where the present invention has been implemented.

 Indeed, Limonium latifolium or sea lavender (also called statice) is a perennial herb that grows on sandy or poor soils of the Atlantic coast and in salt marshes. This sea lavender has antioxidant properties due to its phenolic compounds and contains coloring agents (flowers and roots).

 The species Limonium latifolium is much richer in SOD specific activity than the plant species previously exploited in the agri-food industry as sources of SOD. This means that it is not necessary to have a large quantity of the plant to obtain a very enzymatically active SOD, hence a lower cost of producing a finished product enriched with SOD.

 Indeed, the specific activity of SOD in the extracts obtained from this species exceeds 1200 units / mg proteins, a much higher level than the specific activity of SOD in the extract obtained from the melon, a species considered so far as the richest in SOD (Vouldoukis et al., 2004).

 In addition, the species L. latifolium is an easily cultivable species, with a faster development cycle than melon, without known predator, hence the possibility of producing this plant under conditions corresponding to the regulations on organic crops.

 Finally, since Limonium latifolium is a halophytic salt-resistant species, salt stress is known to stimulate the synthesis of antioxidant compounds including enzymes (Jithesh et al., 2006, Ben Hamed et al. Li et al., 2008, Hameed et al., 2014), it is conceivable to further amplify the SOD activity in the organs of this plant by a culture under controlled conditions in the presence of salt. Thus, it will be possible to produce it under inexpensive conditions by preventing any development of harmful organisms (weeds, pathogens, etc.) sensitive to salt.

The inventors have thus carried out additional tests which demonstrate that the SOD activity can be increased by 10 to 20% in plants of the species L. latifolium grown in the presence of salt at a concentration of 20 g / L NaCl (ie 2/3 of the salt concentration in seawater).

 Among the species studied from the Plombaginaceae family, the inventors have demonstrated that the species Limonium latifolium has particularly advantageous properties and advantages as described above. The inventors have also demonstrated that other species of the genus Limonium have these same properties and advantages and can thus be easily cultivated under controlled conditions in the presence of salt in order to increase the specific activity of SOD.

 According to a preferred embodiment of the process of the present invention, the extraction is carried out from the aerial parts of the plant, preferably from the leaves which are the fleshier organs and the richest in SOD.

 According to a preferred embodiment, the method of the invention comprises a single extraction step consisting in the recovery of an aqueous phase containing the plant extract after contacting the crushed plant of the family Plombaginaceae with water only. In particular, the inventors have demonstrated that plants of the family Plombaginaceae are so rich in SOD that the simple aqueous extraction without addition of other solvents or chemical substances makes it possible to obtain an extract whose specific SOD activity is from minus 700 IU / mg of protein.

 According to a particularly preferred embodiment, in the process of the present invention, the extraction is carried out in the presence of water or an aqueous buffer of the phosphate buffer type, therefore in the absence of toxic organic solvents of the alcohol type ( eg: ethanol, methanol, propanol, isopropanol, butanol), aromatic solvents (eg pyridine, toluene, chlorobenzene or xylene), carbonyl solvents (eg acetone, dimethyl formamide or methyl pyrrolidone), esters (eg: ethyl acetate), sulfoxides (eg dimethyl sulfoxide), nitriles (eg acetonitrile), halogenated solvents (eg dichloromethane, dichloroethane, chloroform, carbon tetrachloride, chlorobenzene), ether-oxides (eg diethylene glycol, tetrahydrofuran, ethyl ether).

 In some cases, during extraction, glycerin is added, which acts as a stabilizer.

 According to one embodiment, prior to the extraction step, said plant can be washed, cut, crushed, micronized and sieved.

According to another embodiment of the invention, the extraction can be carried out from the plant material previously frozen or dried so as to allow its storage and / or storage. Preferably, the drying of the plant is carried out by lyophilization in order to avoid degradation of the enzyme. A second subject of the present invention relates to an SOD concentrated plant protein extract having a specific activity of at least 700 IU / mg proteins, the extract being obtained by the method of the invention as defined above.

 The extract obtained according to the method of the invention has all or part of the characteristics of said process as described below.

 In particular, the extract obtained by the process of the invention is an extract which does not contain chemical reagents usually used in conventional extraction processes and the elimination of which is frequently linked to the reduction in the quality of the extract since it causes the decline of the specific activity of SOD and the destruction of certain substances naturally present in the plant and having a beneficial effect.

 In order to preserve the enzymatic activity of SOD in the extract of the invention, according to a particular embodiment, the extract of the invention can be kept cold, that is to say, at a temperature of between 4 ° C and -30 ° C, preferably between -2 ° C and -30 ° C, more particularly between -18 ° C and -27 ° C.

 According to another embodiment, the preservation of the extract can be carried out by drying it, preferably drying by freeze-drying.

 Thus, the extract of the present invention may comprise other substances naturally present in the family of Plombaginaceae species having a beneficial effect, such as polyphenols which are known for their antioxidant properties.

 According to a particular embodiment, the extract of the invention mainly contains the SOD Cu / Zn isoform.

 The extract of the present invention has the advantage of being very rich in SOD activity as well as in other antioxidant substances, such as polyphenols, and because of its process for obtaining, not to contain or to contain very few chemical reagents usually used during extraction.

 The extract of the invention is therefore perfectly suitable for use in medicine and / or in human and / or animal cosmetology.

 A third subject of the present invention thus relates to a cosmetic or pharmaceutical composition comprising the extract obtained according to the process of the present invention and a cosmetically or pharmaceutically acceptable vehicle, respectively.

For the purposes of the present invention, the term "pharmaceutically acceptable carrier" means any material suitable for use in a pharmaceutical product. By way of example of a pharmaceutically acceptable vehicle, mention may be made of lactose, optionally modified starch, cellulose, hydroxypropylmethylcellulose, mannitol, sorbitol, xylitol, dextrose, calcium sulphate, calcium phosphate, calcium lactate, dextrates, inositol, calcium carbonate, glycine, bentonite and mixtures thereof.

 Preferably, the pharmaceutically acceptable vehicles are chosen from the group consisting of prolamines, that is to say reserve proteins accumulated by the plants of the Poaceae family such as cereals.

 The pharmaceutical composition and the medicaments according to the invention may be in various forms, in particular in a form chosen from the group consisting of injectable solutions, tablets, capsules, dragees, syrups, suspensions, solutions, powders, granules, emulsions, microspheres. In addition, sustained release dosage forms such as capsules, or optionally coated tablets for sustained release will be preferred.

 The pharmaceutical composition and the medicaments according to the invention are administrable by different routes. By way of examples of administration routes that can be used for the pharmaceutical composition and the medicinal products according to the invention, mention may be made of the oral route, the rectal route, the cutaneous route, the nasal route, the sublingual route, the parenteral route, especially intradermal route. subcutaneous, intramuscular, intravenous, intraarterial, intra-articular, intra-pleural and intraperitoneal.

 The pharmaceutical composition and the medicaments according to the invention can be administered in one or more times or in continuous release. Preferably, the pharmaceutical composition or the medicaments according to the invention are administered in continuous release, such as in the form of an infusion or by means of a pharmaceutical form, such as an optionally coated capsule or tablet, sustained and / or delayed release.

 For the purposes of the present invention, the term "cosmetically acceptable vehicle" means a vehicle adapted for use in contact with human and animal cells, in particular the cells of the epidermis, without toxicity, irritation, allergic response and proportionate to a reasonable benefit / risk ratio.

The cosmetic composition according to the invention may comprise one or more formulation agents or additives of known and conventional use in cosmetic and dermatological compositions such as, by way of examples and in a nonlimiting manner, softeners, colorants, film-forming active agents, surfactants, perfumes, preservatives, emulsifiers, oils, glycols, vitamins such as vitamin E, UV filters, etc. With his knowledge of cosmetics and / or dermatology, the skilled person will know which formulating agents add to the compositions of the invention and in what quantities according to the desired properties. The cosmetic composition according to the invention may be in any form known to those skilled in the field of cosmetology and dermatology with no other pharmaceutical restriction than the application on the face and body. Advantageously, the compositions according to the invention are in the form of a gel, a cream, a lotion, an oil, a milk, a spray, etc.

 Whatever the objective, we can propose a daily application, once or twice a day, for a duration of a few days to several months.

 Since the extract of the invention can be obtained by aqueous extraction without the use of other chemical reagents (alcohols, organic solvents, buffers), it is particularly suitable for use in food and feed.

 Thus, the present invention further relates to a food or nutraceutical composition comprising a protein extract obtained according to the method of the invention and a food additive or a nutraceutically acceptable vehicle, respectively.

 The food additives may be chosen from all the food additives well known to those skilled in the art, for example, in the group comprising preservatives, food colorants, antioxidants, lactates, citrates, orthophosphates, malates, adipates, alginates, gums, di- and triphosphates, etc.

 For the purpose of the present invention, the term "nutraceutical composition" is intended to mean a composition relating to a product made from food and marketed in the form of a tablet, powder or potion having a physiological beneficial effect against chronic diseases.

 The term "nutraceutically acceptable carrier" in the sense of the present invention, any material that is suitable for use in a nutraceutical product. As a nutraceutically acceptable carrier, mention may be made of dicalcium phosphate, calcium alginate, magnesium carbonate, magnesium stearate, silicon dioxide, calcium chloride, and the like.

 The nutraceutical composition according to the invention is administrable especially orally, for example, in the form of breakable tablets or not, film-coated or not, granules, capsules, capsules, or in the form of free powders preferably packaged in single sachets. , or compressed powder.

When the extract obtained by the process of the invention is intended for use in a food and / or nutraceutical composition, it is preferable to carry out an aqueous extraction by implementing the method of the invention and not an extraction using reagents that may cause discomfort and / or disorder digestive agents such as, for example, protease inhibitors and / or reducing or fixing agents phenols.

 According to one embodiment, said extract comprises of the order of 50000 to 200000 IU / g of solids, preferably of the order of 100000 to 150000 IU / g, or, by weight, approximately 1 to 4% preferably 2 to 3% of superoxide dismutase in the dry extract.

 For the purposes of the present invention, the term "dry extract" is intended to mean the dry residue which remains after desiccation of an extract and, for example, by vacuum evaporation at 40 ° C. or by freeze-drying.

 In addition, the composition according to the invention may comprise from 0.1% to 20%, more particularly from 0.1% to 10% or from 0.5% to 5%, in particular from 1 to 3% of superoxide dismutase. by weight relative to the total weight of the composition.

 According to one embodiment, the pharmaceutical composition according to the invention comprises from 5% to 20%, particularly from 5% to 15% and more particularly from 5% to 10% of superoxide dismutase by weight relative to the total weight of the composition. .

 According to a particular embodiment, the food composition and / or the nutraceutical composition according to the invention may comprise from 0.1% to 5%, particularly from 0.5% to 3% and more particularly from 1 to 2% of superoxide. dismutase by weight relative to the total weight of the composition.

 A fourth aspect of the present invention relates to a cosmetic use of a protein extract as defined according to the invention as an antioxidant cosmetic agent, in particular for combating aging of the skin and / or integuments, against the rays. UV, etc.

 A fifth aspect of the invention relates to the use of a SOD protein extract as defined above for the preparation of cheeses and / or the preservation of dairy products.

 In cheese production, SOD can couple to catalase and in this way effectively inhibit the anarchic oxidation of lipids in dairy products, allowing prolonged storage of milk.

 A sixth aspect of the present invention relates to the SOD protein extract as defined above as a medicament.

 In particular, the invention relates to a SOD protein extract according to the invention for its use in the prevention and / or treatment of diseases selected from the group comprising:

 psychiatric diseases, preferably schizophrenia;

- neurodegenerative diseases, preferably Alzheimer 's disease or Parkinson' s disease; cardiovascular diseases, preferably myocardial infarction or atherosclerosis;

 inflammatory diseases, preferably Crohn's disease or rheumatoid arthritis;

 - chronic diseases, preferably diabetes;

 cancerous diseases, preferably selected from the group consisting of skin cancer, breast cancer, esophageal cancer, stomach cancer, liver cancer, colon cancer and lung cancer .

 The present invention is further illustrated by the examples described below.

EXAMPLES

1. Material and methods

1.1 Plant material

Species tested are usually found on sand dunes, rocks or salt marshes along the Breton coastline.

 The species tested are Limonium normannicum, L. vulgare, L. latifolium, L. tunetanum, L. densiflorum, L. pruinosum, L. delicatulum, L. spathulatum and Armeria maritima.

1.2 Crop management Samples of aerial parts of all these species were collected in late spring in their natural habitat.

 In addition, plants of the species L. latifolium were cultivated under the following controlled conditions: Photoperiod: 8h / 16h (night / day); Temperature: 14/23 ° C (night / day); Relative humidity: 50-70%; Salinity: 0, 5, 10, 20 g / L NaCl.

 Extraction of SOD proteins is carried out immediately after the leaves are taken from the mother plants or after preservation at -80 ° C. For this, the leaves are ground in water or a 100 mM pH phosphate buffer. 7.8 for 15 minutes at 4 ° C. After filtration or centrifugation at 14,000 g for 30 minutes at 4 ° C., the supernatant is collected (liquid phase). The soluble protein content as well as the enzymatic activities are determined from the supernatant.

1.3 Determination of soluble proteins

The soluble protein content is determined in the supernatant of each enzymatic extract according to the method of Bradford (1976). 1.4 Measurement of SOD activities

Superoxide dismutases catalyze the reaction: 2 0 ₂ ^"(superoxide anion) + 2 H + - 0 ₂ + H ₂ 0 ₂

The substrate of SOD is very unstable and has a very short life span (10 ^"9 s), so the assay methods are very indirect.The method proposed by Beauchamp and Fridovich (1969), modified by Scebba et al. 1999), evaluated SOD by its ability to inhibit superoxide anion flux generated by illuminated riboflavin.The superoxide radicals produced by this system reduce the nitroblue tetrazolium (NBT) to formazan blue.Two sets of tubes were prepared The former are controls, maintained in the dark and containing a mixture in 50 mM phosphate buffer pH 7.8 composed of 0.1 mM EDTA, methionine (13 mM), NBT (75 μl), riboflavin (2 The second series of tubes is intended for the determination of the SOD activity and these tubes contain the same reaction mixture but they are maintained for 15 minutes under a lighting of 15 W. The measurement of the absorbance is carried out at 560 nm. SOD nzymatic corresponds to the amount of plant extract capable of inducing a 50% inhibition of the nitroblue tetrazolium reduction reaction.

The catalytic activity of the enzyme is then related to the mass of tissues that have been extracted while the specific activity is related to the amount of protein in the extract.

2. Results

The results obtained by the measurements of the catalytic activity and the specific activity of SOD in the extracts obtained from plants of the family Plombaginaceae by the process of the invention are shown in Table 1 below:

Catalytic Activity Specific Activity

Species

 (Unit / g MS) (Unit / mg Prot).

 Plombacinaceae family

Limonium normannicum 12709.40 1588.68 L. seen the station 10945.94 1368.24

L. latifolium 7812.50 1228.38

 L. tunetanum 8051, 61 1006.45

 L. densiflorum 8836.72 1,104.59

 L. pruinosum 8027.38 1003.42

 L. delicatulum 3703.70 752.78

 L. spathulatum 4743.72 705.91

 Armeria maritima 9280.74 1784.76

Table 1: Results of measurements of catalytic activity and specific activity of SOD in Plombaginaceae species.

 These results clearly show that the method of protein extraction of SOD from Plombaginaceae species, according to the present invention, makes it possible to obtain in a simple and fast way concentrated SOD protein extracts having a specific activity of at least 700 IU. / mg protein and catalytic activity of at least 3000 IU / g dry matter.

COMPARATIVE EXAMPLES Under the same conditions of protein extraction (aqueous extraction or extraction in a phosphate buffer) as that described above, comparative examples were made from the halophyte plants of the Apiaceae, Aizoaceae, Brassicaceae and Chenopodiaceae families.

The results obtained are shown in Table 2 below.

 Activity Activity

 Specific Catalytic Species

 (Unit / g MS) (Unit / mg prot.)

 Plombaginaceae family

 Limonium normannicum 12709.40 1588.68

 L. seen the station 10945.94 1368.24

 L. latifolium 7812.50 1228.38

 L. tunetanum 8051, 61 1006.45

 L. densiflorum 8836.72 1,104.59

 L. pruinosum 8027.38 1003.42

 L. delicatulum 3703.70 752.78

 L. spathulatum 4743.72 705.91

Armeria maritima 9280.74 1784.76 Apiaceae family

 Crithmum maritimum 1216.55 405.52

 Eryngium maritimum 687.62 172.56

 Brassicaceae family

 Cakile maritima 879.31 314.04

 Crambe maritima 1250 155

 Aizoaceae family

 Mesembryanthemum crystallinum 160 20

 Family Chenopodiaceae

 Suaeda fruticosa 1 4 18

 Arthrocnemum indicum 176 22

 Halocnemum strobilaceum 160 20

Table 2: Comparative test results of catalytic activity and specific activity of SOD in species of different families of halophyte plants.

These results clearly show that the catalytic activity and the specific activity of SOD of Plombaginaceae family species are clearly superior to those of the studied species of the other families.

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 EP 2 747 044

Claims

1. Process for obtaining a concentrated protein extract of superoxide dismutase (SOD) having a specific activity of at least 700 IU / mg of proteins, characterized in that it comprises a single protein extraction step starting from a plant selected from any of the species of the family Plombaginaceae. The method of claim 1, wherein the plant is selected from the group consisting of Limonium and Armeria genera. The method of claim 1 or 2, wherein the plant is selected from the group consisting of Limonium latifolium, Limonium normannicum, Limonium vulgare, Limonium tunetanum, Limonium densiflorum, Limonium pruinosum, Limonium delicatulum, Limonium spathulatum, Limonium booitardii and Armeria maritima. 4. A process according to any one of claims 1 to 3, wherein the extraction is carried out from the aerial parts of the plant, preferably from the leaves. 5. Process according to any one of claims 1 to 4, wherein the single extraction step comprises the recovery of a liquid phase after contacting a solvent with said plant, said solvent being preferably a solvent. aqueous. 6. Process according to any one of claims 1 to 5, wherein the extraction is carried out in the absence of organic solvents chosen from the group comprising alcohols, aromatic solvents, carbonyl solvents, esters, nitriles, solvents. halogenated and ether-oxide solvents. 7. Plant protein extract concentrated in SOD with a specific activity of at least 700 IU / mg protein, characterized in that it is obtained by the method according to any one of claims 1 to 6. 8. Protein extract according to claim 7 comprising of the order of 50000 to 200000 IU / g, preferably of the order of 100,000 to 150,000 IU / g of solids, or, by weight, about 1 to 4%, preferably 2 to 3% superoxide dismutase in the dry extract. 9. Cosmetic or pharmaceutical composition characterized in that it comprises an extract according to claim 7 or 8 and a cosmetically or pharmaceutically acceptable vehicle, respectively. 10. Food or nutraceutical composition, characterized in that it comprises a protein extract according to claim 7 or 8 and a food additive or a nutraceutically acceptable vehicle, respectively. 11. Composition according to any one of claims 9 or 10 comprising from 0.1% to 20%, more particularly from 0.1% to 10%, more particularly from 0.5% to 5%, in particular from 1% to 10% by weight. at 3% superoxide dismutase by weight relative to the total weight of the composition. 12. Cosmetic use of a protein extract according to claim 7 or 8 or a composition according to claim 9 or 11 as an antioxidant cosmetic agent, in particular for combating the aging of the skin and / or superficial body growths and / or UV protection. 13. Use of a protein extract according to claim 7 or 8 or a composition according to claim 10 or 11 for the preparation of cheeses and / or the preservation of dairy products. Protein extract according to claim 7 or 8 or composition according to claim 9 or 11 as a medicament. The protein extract or composition of claim 14 for use in preventing and / or treating diseases selected from the group consisting of: psychiatric diseases, preferably schizophrenia;  neurodegenerative diseases, preferably Alzheimer's disease or Parkinson's disease;  cardiovascular diseases, preferably myocardial infarction or atherosclerosis;  inflammatory diseases, preferably Crohn's disease or rheumatoid arthritis;  chronic diseases, preferably diabetes, and  cancerous diseases, preferably selected from the group consisting of skin cancer, breast cancer, esophageal cancer, stomach cancer, liver cancer, colon cancer and lung cancer .