Method for Obtaining a Superoxide Dismutase (SOD)-Concentrated Protein Extract

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.

Claims

1. A method for obtaining a superoxide dismutase (SOD)-concentrated protein extract having a specific activity of at least 700 IU/mg protein, characterized in that it comprises a single step of protein extraction from a plant selected from any one of species of the family Plumbaginaceae.

2. The method according to claim 1, wherein the plant is selected from the group consisting of the genus Limonium and Armeria.

3. The method according to claim 1 or 2, wherein the plant is 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 and Armeria maritima.

4. The method 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. The method according to any one of claims 1 to 4, wherein the single extraction step comprises collecting a liquid phase after contacting a solvent with said plant, said solvent preferably being an aqueous solvent.

6. The method according to any one of claims 1 to 5, wherein the extraction is carried out in the absence of organic solvents selected from the group comprising alcohols, aromatic solvents, carbonyl-containing solvents, esters, nitriles, halogenated solvents and ether-oxide solvents.

7. An SOD-concentrated plant protein extract having 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. The protein extract according to claim 7 comprising about 50,000 to 200,000 IU/g, preferably about 100,000 to 150,000 IU/g dry extract, or, by weight, around 1% to 4%, preferably 2% to 3% superoxide dismutase in the dry extract.

9. A cosmetic or pharmaceutical composition characterized in that it comprises an extract according to claim 7 or 8 and a cosmetically or pharmaceutically acceptable carrier, respectively.

10. A dietary 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 carrier, respectively.

11. The composition according to claim 9 or 10 comprising from 0.1% to 20%, more particularly from 0.1% to 10%, even more particularly from 0.5% to 5%, in particular from 1% to 3% superoxide dismutase by weight relative to the total weight of the composition.

12. A cosmetic use of the protein extract according to claim 7 or 8 or of a composition according to claim 9 or 11 as an antioxidant cosmetic agent, in particular for combating aging of the skin and/or skin appendages and/or for UV protection.

13. A use of the protein extract according to claim 7 or 8 or of a composition according to claim 10 or 11 for preparing cheeses and/or preserving dairy products.

14. The protein extract according to claim 7 or 8 or the composition according to claim 9 or 11 as a drug.

15. The protein extract or the composition according to claim 14 for use in the prevention and/or in the treatment of diseases selected from the group comprising: psychiatric illnesses, 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 cancer diseases, preferably selected from the group comprising skin cancer, breast cancer, esophageal cancer, stomach cancer, liver cancer, colon cancer and lung cancer.

Description

EXAMPLES

1. Materials and Methods

[0105] 1.1 Plant Material

[0106] The species tested are typically found among the sand dunes, salt-water rocks and marshes along the Brittany coast.

[0107] The species tested are Limonium normannicum, L. vulgare, L. latifolium, L. tunetanum, L. densiflorum, L. pruinosum, L. delicatulum, L. spathulatum and Armeria maritima.

[0108] 1.2 Cultivation

[0109] Samples of the aerial parts of all these species were taken at the end of spring in their natural habitat.

[0110] Moreover, plants of the species L. latifolium were cultivated under the following controlled conditions: Photoperiod: 8 h/16 h (night/day); Temperature: 14/23 C. (night/day); Relative humidity: 50-70%; Salinity: 0, 5, 10, 20 g/L NaCl.

[0111] The SOD protein extraction is carried out immediately after the leaves are taken from the mother plants or after they have been preserved at 80 C. To that end, the leaves are ground in water or phosphate buffer (100 mM, pH 7.8) for 15 min at 4 C. After filtration or centrifugation at 14,000 g for 30 min at 4 C., the supernatant is collected (liquid phase). The soluble protein content and the enzymatic activities are determined from the supernatant.

[0112] 1.3 Assay of Soluble Proteins

[0113] The soluble protein content is determined in the supernatant of each enzyme extract according to the Bradford (1976) method.

[0114] 1.4 Measurement of SOD Activities

[0115] Superoxide dismutases catalyze the reaction:

2 O.sub.2.sup..(superoxide anion)+2 H+.fwdarw.O.sub.2+H.sub.2O.sub.2

[0116] Since the substrate for SODs is highly unstable and has a very short lifespan (10.sup.9 s), the assay methods are very indirect. The method proposed by Beauchamp and Fridovich (1969), modified by Scebba et al. (1999), evaluates SOD by its capacity to inhibit a flux of superoxide anion generated by illuminated riboflavin. The superoxide radicals produced by this system reduce nitro blue tetrazolium (NBT) to blue formazan.

[0117] Two series of tubes were prepared. The first are controls, kept in the dark and containing a mixture in 50 mM pH 7.8 phosphate buffer consisting of EDTA (0.1 mM), methionine (13 mM), NBT (75 M), riboflavin (2 M) and the enzyme extract. The second series of tubes is for determining SOD activity. These tubes contain the same reaction mixture but they are kept for 15 min under 15 W lighting. The absorbance measurement is carried out at 560 nm. One SOD enzyme unit corresponds to the amount of plant extract capable of inducing 50% inhibition of the nitro blue tetrazolium reduction reaction.

[0118] The catalytic activity of the enzyme is thus related to the tissue mass having been the object of the extraction whereas the specific activity, for its part, is related to the amount of proteins in the extract.

2. Results

[0119] The results obtained by measurements of SOD catalytic activity and specific activity in extracts obtained from plants of the family Plumbaginaceae by the method of the invention are shown in table 1 below:

TABLE-US-00001 TABLE 1 Results of measurements of SOD catalytic activity and specific activity in species of Plumbaginaceae. Catalytic activity Specific activity Species (units/g DM) (units/mg protein). Family Plumbaginaceae Limonium normannicum 12709.40 1588.68 L. vulgare 10945.94 1368.24 L. latifolium 7812.50 1228.38 L. tunetanum 8051.61 1006.45 L. densiflorum 8836.72 1104.59 L. pruinosum 8027.38 1003.42 L. delicatulum 3703.70 752.78 L. spathulatum 4743.72 705.91 Armeria maritima 9280.74 1784.76

[0120] These results clearly show that the method for extracting SOD protein from species of Plumbaginaceae, according to the present invention, makes it possible to obtain, simply and rapidly, SOD-concentrated protein extracts having a specific activity of at least 700 IU/mg protein and a catalytic activity of at least 3000 IU/g dry matter.

COMPARATIVE EXAMPLES

[0121] Under the same protein extraction conditions as those described above (aqueous extraction or extraction in phosphate buffer), comparative examples were prepared from halophytic plants of the families Apiaceae, Aizoaceae, Brassicaceae and Chenopodiaceae.

[0122] The results obtained are presented in table 2 below.

TABLE-US-00002 TABLE 2 Results of comparative tests of the catalytic activity and the specific activity of of the SODs in species of various families halophytic plants. Catalytic activity Specific activity Species (units/g DM) (units/mg protein) Family Plumbaginaceae Limonium normannicum 12709.40 1588.68 L. vulgare 10945.94 1368.24 L. latifolium 7812.50 1228.38 L. tunetanum 8051.61 1006.45 L. densiflorum 8836.72 1104.59 L. pruinosum 8027.38 1003.42 L. delicatulum 3703.70 752.78 L. spathulatum 4743.72 705.91 Armeria maritima 9280.74 1784.76 Family Apiaceae Crithmum maritimum 1216.55 405.52 Eryngium maritimum 687.62 172.56 Family Brassicaceae Cakile maritima 879.31 314.04 Crambe maritima 1250 155 Family Aizoaceae Mesembryanthemum crystallinum 160 20 Family Chenopodiaceae Suaeda fruticosa 144 18 Arthrocnemum indicum 176 22 Halocnemum strobilaceum 160 20

[0123] These results clearly show that the SOD catalytic activity and specific activity of species of the family Plumbaginaceae are much higher than those of the species studied of other families.

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