Mixture of Purified SODs of Plant Origin

Abstract

The present invention relates to an original and specific mixture of purified superoxide dismutases (SODs) of plant origin, characterised in that said mixture is essentially made up of three superoxide dismutases: a manganese superoxide dismutase, a copper and zinc superoxide dismutase and an iron superoxide dismutase provided in two isoforms, which can be obtained from an extract of the hybrid variety F1 of Cucumis melo MA 7950 or the cells thereof cultured in vitro or by transfer and expression of the genes of said SODs in prokaryotic or eukaryotic cells. The specific mixture according to the invention imparts to the compositions containing same a greater effectiveness in the treatment or prevention of diseases linked to inflammatory and/or oxidative stress, such as radiation-induced fibroses, cardiovascular diseases, obesity, atherosclerosis, labial herpes and myopathies, as well as in nutritional, pharmaceutical, veterinary or cosmetic uses.

Claims

1. A mixture of superoxide dismutases of plant origin, characterized in that it is essentially consisting of 3 superoxide dismutases: a manganese superoxide dismutase, a copper and zinc superoxide dismutase and an iron superoxide dismutase in at least two isoforms, the first isoform of iron superoxide dismutase having a molecular weight of between 28 000 and 36 000 Da, the second isoform of iron superoxide dismutase having a molecular weight of between 75 000 and 85 000 Da, said mixture able to be obtained from an extract of the F1 hybrid variety of Cucumis Melo MA 7950 or the cells thereof cultured in vitro or via transfer and expression of the genes of these SODs in prokaryote or eukaryote cells.

2. The mixture of superoxide dismutases according to claim 1, characterized in that said mixture has a total SOD activity equal to or higher than 130 U/mg of said mixture.

3. The mixture of superoxide dismutases according to claim 1 or 2, characterized in that the first isoform of iron superoxide dismutase has a molecular weight of about 32 200 Da.

4. The mixture of superoxide dismutases according to any one of claims 1 to 3, characterized in that the second isoform of iron superoxide dismutase has a molecular weight of about 79 800 Da.

5. The mixture of superoxide dismutases according to any one of claims 1 to 4, characterized in that the accumulated SOD activity of the two isoforms of iron superoxide dismutase is between 20% and 26%, advantageously between 22% and 26% of the total SOD activity of the mixture.

6. The mixture of superoxide dismutases according to any one of claims 1 to 5, characterized in that the accumulated SOD activity of the two isoforms of iron superoxide dismutase is between 20% and 26% of the total SOD activity of the mixture, the activity of the copper and zinc superoxide dismutase is between 60% and 70% of total SOD activity of the mixture, and the activity of the manganese superoxide dismutase is between 7 and 12% of total SOD activity of the mixture.

7. The mixture of superoxide dismutases according to any one of claims 1 to 6, characterized in that the manganese superoxide dismutase has a molecular weight of between 70 000 and 90 000 Da, and the copper and zinc superoxide dismutase has a molecular weight of between 27 000 and 33 000 Da.

8. The mixture of superoxide dismutases according to any one of claims 1 to 7, characterized in that it is able to be obtained by grinding or pressing in an aqueous medium, preferably at a pH of 5 to 9, the F1 hybrid variety of Cucumis Melo MA 7950 or the cells thereof cultured in vitro or via transfer and expression of the genes of these SODs in prokaryote or eukaryote cells, followed by recovery of the supernatant and purification by chromatography, IMAC chromatography in particular.

9. A nutritional, cosmetic or pharmaceutical composition containing as active ingredient a mixture of superoxide dismutases according to any one of claims 1 to 7 and at least one pharmaceutically or cosmetically acceptable food-grade excipient.

10. The cosmetic composition according to claim 9, characterized in that it is intended for external topical use such as care products, shampoos, lotions, gels.

11. The pharmaceutical composition according to claim 9, characterized in that it is intended for administration via topical, oral, nasal or parenteral route, and for example is in the form of a tablet, hard capsule, soft capsule, effervescent tablet, sachet or stick to be diluted, syrup, elixir, herbal tea, chewing gum, spray, aerosol or solution for injection.

12. The pharmaceutical composition according to claim 9 or 11, characterized in that it contains another active ingredient, advantageously another antioxidant.

13. The pharmaceutical composition according to any one of claim 9, 11 or 12 for use thereof as medicinal product.

14. Pharmaceutical composition for use according to claim 13, characterized in that the medicinal product is intended to treat or prevent diseases related to oxidative stress and/or inflammation and/or to reinforce the action of other pharmaceutical molecules, and in particular other employed antioxidants, or to treat orphan diseases in animals.

15. Pharmaceutical composition for use according to claim 14, characterized in that the medicinal product is intended to stimulate cell vitality in animals.

16. Pharmaceutical composition for use according to claim 14 or 15, characterized in that the animal is human being.

17. Pharmaceutical composition for use according to claim 13, characterized in that the medicinal product is intended for the treatment of cardiovascular diseases, obesity, arteriosclerosis and labial herpes, in particular in humans.

18. Nutritional composition according to claim 9, characterized in that it is intended for administration via oral route and for example is in the form of a tablet, hard capsule, soft capsule, effervescent tablet, sachet or stick to be diluted, chewing gum, beverages, juices, yoghurt, confectionery, biscuit or bars.

Description

FIGURES

[0127] FIG. 1: Determination of different SODs derived from the F1 hybrid variety of the MA 7950 melon (schematic illustration of the result obtained after detection on acrylamide gel). Total SOD activity (well 1), with 2 mM KCN (well 2), with H.sub.2O.sub.2 (well 3).

[0128] FIG. 2: electrophoretic profile of the different SODs derived from the F1 hybrid variety of the MA 7950 melon (schematic illustration of the result obtained after detection on IEF gel); from left to right: MA 7950 melon (wells 1 and 2), water melon (wells 3 and 4), peach (wells 5 and 6) and nectarine (wells 7 and 8).

[0129] FIG. 3: electrophoretic profile of the different SODs derived from the F1 hybrid variety of the MA 7950 melon (schematic illustration of the result obtained after detection on IEF gel); from left to right: MA 7950 melon (wells 1 and 2), Canari melon (wells 3 and 4), apricot (wells 5 and 6) and cherry (wells 7 and 8).

[0130] FIG. 4: Photograph of two melons just after picking (t0). On the left, a common F1 hybrid melon, on the right a melon derived from the F1 hybrid melon called MA 7950.

[0131] FIG. 5: Photograph of the two same melons taken 7 days later. On the left the F1 hybrid melon of common type, on the right the melon derived from the F1 hybrid variety of the melon known as MA 7950.

EXAMPLES

[0132] The following examples are given for illustration purposes and do not in any manner limit the invention.

Example 1. Specificity of the F1 Hybrid Variety of Melon called MA 7950

[0133] A simple experiment was conducted to demonstrate the particular properties of the melons derived from the F1 hybrid variety called MA 7950, and its antioxidant activity in particular.

[0134] Two melons, one of common type and the other derived from the F1 hybrid variety of melon called MA 7950, were placed in a room at ambient temperature and under atmospheric pressure at D1 (FIG. 4). The melons were kept in this room under the same conditions (ambient temperature, atmospheric pressure) for 7 days (FIG. 5: photograph of the same melons taken at D8). After 7 days, it can be clearly seen that the F1 hybrid melon of common type is in an advanced state of degradation, while the melon derived from the F1 hybrid variety of the melon called MA 7950 still has a good outer appearance. The twofold higher content and composition of the SOD mixture of the F1 hybrid variety of the MA 7950 melon provide the cells of the melon with more efficient resistance against the natural degradation process.

Example 2. Preparation of the Extract and Purification of the Different SODs, Assay of SOD Activity

[0135] 5 g of pulp of Cucumis melo of the F1 hybrid variety called MA 7950 or the cells thereof were cold crushed in a mortar. A volume of 50 mM phosphate buffer (pH: 7.8; EDTA 1 mM; 5% glycerol) equivalent to 3 times the plant mass was added. After homogenisation, the suspension was centrifuged at 5000 g at 4 C. for 30 minutes. The supernatant was recovered and filtered. This crude extract was used for purification of the different SOD forms. The different SODs were purified applying a technique well known in the art: IMAC (Immobilised Metal Affinity Chromatography). For this method, copper was used as metal ion immobilised on the column; attaching of the proteins is dependent on their charge and therefore correlates with the presence of amino acids such as tryptophan, histidine, cysteine. SODs are known to have a high histidine content, justifying the use of IMAC.

[0136] The SODs derived from the extract of the MA 7950 melon were purified on FPLC apparatus (Pharmacia Amersham) on a Superose HR 10/2 column or Hitrap chelating column (Pharmacia-Biotech). 3 ml of sample were injected via a superloop into the column previously treated with a solution of CuSO4 (600 ml, 23 mmol/ml) and equilibrated with 10 ml of 0.05 M potassium phosphate buffer, pH 7.8. Elution of the SODs was conducted at a constant rate of 1 ml/min with a 10% linear gradient (reached in 10 min) of 0.75 M NH.sub.4Cl solution. The remaining Cu-protein complexes were removed with 5 ml of aqueous 1M EDTA solution. 2 ml fractions containing the different SODs were collected, assayed and immediately desalted on an Amicon PM 10 chamber with 4 volumes of 0.05 M phosphate buffer, pH 7.8, then freeze-dried.

Example 3. Determination of the Different SOD Forms and Molecular Weights

[0137] SOD activity was detected on acrylamide gel under native conditions (conditions under which the protein remains in the native state i.e. such as it is in the cell, as opposed to denaturing conditions in which the protein is linearized) following the method of Beauchamp and Fridovich Anal. Biochem. 44:276-82 (1971). It is based on SOD-inhibited reducing of Nitroblue Tetrazolium (N 55 14 Sigma-Aldrich, France). After the reaction, the different bands corresponding to SOD activity appear in white against a background of dark blue gel. Samples of the mixture of purified SODs (45 l of solution A+15 l migration buffer) were deposited in different wells of an electrophoresis gel (4%/10% acrylamide). Migration time was 75 min at 70 mA and 300V. At the gel resolving step, which migrated under non-denaturing conditions, the gel was cut into 3 portions. The activity of the first was determined following the protocol below, the second was preincubated with 2 mM KCN and the activity determined, finally the third was preincubated with 5 mM H.sub.2O.sub.2 and the activity determined.

SOD Activity Identification Conditions:

[0138] The activity of the purified SODs was measured with a SOD assay kit (19160, Sigma-Aldrich, France). This total SOD activity was higher than 500 IU SOD/mg of proteins and the protein content of the purified extract was higher than 70% (Bradford method, Anal. Biochem. 72: 248-54, 1976). After migration, the gels were immersed: [0139] for 150 min in K+ phosphate buffer (50 mM; pH 7.8) containing 2 mM NBT; [0140] then for 15 min in K+ phosphate buffer (50 mM; pH 7.8) containing 28 mM TEMED and 0.0028 mM riboflavin (a 50 mg solution of riboflavin was prepared in 1 ml of phosphate buffer).

[0141] The gels were then rinsed in K phosphate buffer solution (50 mM; pH 7.8). The electrophoresis gels were subsequently digitized using the Perfect Image photographic capture module and analysed on a Gel Analyst module (Claravision, 2000, France).

[0142] As can be seen in FIG. 1, in the absence of inhibitors, the SOD mixture exhibits two thick bands of SOD activity (well 1), one located towards the top part of the gel indicating proteins of high molecular weight (which migrate less quickly than the proteins of low molecular weight) and the other towards the bottom part of the gel indicating activity due to one or more SODs of low molecular weight.

[0143] In the presence of 2 mM KCN (well 2), only Cu/Zn-SOD is inhibited, this corresponding to the band located at the bottom part of the gel and representing the largest portion of this activity band.

[0144] In the presence of 5 mM H.sub.2O.sub.2 (well 3), Fe-SOD and Cu/Zn-SOD are inhibited. The only remaining band on the gel (top part) corresponds to Mn-SOD (insensitive to KCN and H.sub.2O.sub.2). Mn-SOD represents part of the SOD activity band located at the top of the gel, the other part is Fe-SOD inhibited by H.sub.2O.sub.2. The other form of Fe-SOD is located at the bottom part of the gel close to Cu/Zn-SOD.

[0145] The intensities of the different bands of SOD activity were integrated using the Gel Analyst module (Claravision, 2000, France), and the ratio of each SOD isoform thus calculated:

TABLE-US-00001 Intensity of SOD without each band inhibitors +KCN +H.sub.2O.sub.2 1 177 160 173 721 62 124 2 442 524 43 594 / Total 619 684 217 315 62 124 Results SOD without as % inhibitors +KCN +H.sub.2O.sub.2 1 28.6 28, 10 2 71.4 7 / Total 100 35 10

[0146] For Mn-SOD (band 3), the intensity of the band is 62 124 for a total intensity of 619 684 (SOD without inhibitors), Mn-SOD therefore represents 10% of total SOD activity. For Cu/Zn-SOD, the intensity of the band is (442 524-43 594)/619 684 and corresponds to 65% of total activity. Finally, Fe-SOD corresponds to 25%, one isoform represents 7% (close to Mn-SOD) and the other 18% (close to Cu/Zn-SOD).

[0147] The above analyses performed on a SOD mixture extracted from the F1 hybrid variety of the Cucumis Melo MA 7950 melon, were carried out under the same conditions on other SODs derived from different fruit: Anasta melon, Spanish melon (Canari), Clipper melon (descendant of the F1 hybrid variety of Cucumis Melo 95LS444), water melon, peach, nectarine, cherry (FIGS. 2 and 3).

[0148] As can be seen in FIGS. 2 and 3, the electrophoresis profiles of the SOD activities derived from different types of fruit differ largely from those of a mixture of purified SODs extracted from the F1 hybrid variety of the Cucumis Melo melon MA 7950 (the furthest to the left).

[0149] The percentages of each form after use of inhibitors (2 mM KCN and 5 mM H202) are given in Table 1.

TABLE-US-00002 TABLE 1 Proportion of the different SOD forms. Cu/Zn-SOD Mn-SOD Fe-SOD Melon MA 7950 65% 10% 25% Water melon 66% 18% 16% Canari melon 83% 9% 8% Anasta melon 84% 13.5% 2.5% Clipper melon 25% 60% 15% Nectarine 81.5% 0% 18.5% Peach 88% 0% 12% Cherry 100% 0% 0% Apricot 0% 0% 0%

[0150] On these same electrophoresis gels, molecular weight markers (proteins of known molecular weights) were placed in the first well of the gel using a marker kit (M 3913, Sigma-Aldrich, France) to calibrate the gel for molecular weight and to determine the molecular weight of the different SODs by comparison.

[0151] The same analyses were performed on mixtures of purified SODs from other fruit, only on fruit containing 3 forms of SOD: water melon, Spanish melon (Canari), other melon variety (Anasta).

[0152] The molecular weights of the different SODs are given in Table 2.

TABLE-US-00003 TABLE 2 Molecular weights of different SOD forms extracted from the F1 hybrid variety of Cucumis Melo MA 7950, from a water melon, Canari melon, Anasta melon and Clipper melon (descendant of the F1 hybrid variety of the Cucumis Melo 95LS444 melon). Cu/Zn SOD Mn SOD Fe SOD Melon MA 7950 31 800 80 600 32 200 79 800 Water melon 41 600 72 200 56 400 Canari melon 44 700 65 900 43 700 Anasta melon 42 600 62 700 44 700 Clipper melon 40 000 95 000 30 000

[0153] It can easily be seen that the mixture of purified SODs extracted from the F1 hybrid variety of Cucumis Melo MB17415 is unique (compared with the other SODs derived from different fruit), in particular on account of the presence of the second isoform of Fe-SOD. It contains 3 classes of SOD (differentiated through their metal group) and 4 isoforms: [0154] Cu/Zn-SOD represents 65% of total SOD activity and has a molecular weight of 31 800 Da. [0155] Mn-SOD represents 10% of total SOD activity and has a molecular weight of 80 600 Da. [0156] 2 different forms of Fe-SOD represent 25% of total activity: One Fe-SOD of 32 200 Da represents 18% of total SOD activity. The other Fe-SOD of 79 800 Da represents 7% of total SOD activity.

Determination of the Isoelectric Points of the Different SODs

[0157] This analysis technique is of interest to obtain additional information on the pHi of the isoforms of the SODs purified from melon. It is therefore an activity technique. The separation of the SODs as a function of their charge is obtained by isoelectric focusing (IEF). The SODs migrate in an electric field and become immobilised when the pH environment causes them to lose their net charge. The isoelectric points of the different SOD forms can be determined on IEF gels by comparison with markers of known isoelectric point (IEF markers 3.6-9.3, Reference 56733, Sigma-Aldrich, France)

[0158] The samples deposited on gels (30% acrylamide/acrylamide bis, 50% glycerol, ampholines pH 3.5-10 Amersham, ammonium persulfate and Temed) migrate at 300 V and 20 mA in a sample buffer (glycerol: 75% v/v, ampholines: 2% v/v) and a migration buffer (25 mM NaOH for the cathode and 25 mM CH3COOH for the anode). Isoelectric point markers are also deposited on a gel well. Detection of the bands of SOD activity is obtained as described in paragraph 2. By comparison with the bands of the pHi markers, the pHi values of the different isoforms are determined. For the Anasta melon, the low proportion of Fe-SOD (2.5%) does not allow identification of the different pHi values. The isoelectric points of the different SOD forms of the 3 varieties (MA 7950; water melon, Canari melon) are given in Table 3.

TABLE-US-00004 TABLE 3 Isoelectric points (pHi) of the different SOD forms extracted from the F1 hybrid variety of Cucumis Melo MA 7950, water melon and Canari melon. Cu/Zn SOD Mn SOD Fe SOD Melon MA 7950 4.3 4.1 4.4 4.4 4.7 5.3 5.5 5.7 5.85 6.1 Water melon 4.6 4.25 4 4.7 4.7 5.2 5.8 6 6.1 Canari melon 4.4 4.4 4 5.7 5.8 5.9 6

[0159] It is noted that the mixture of purified SODs, extracted from the F1 hybrid variety of Cucumis Melo MA 7950 and characterized by their metal group, molecular weight and pHi, is unique; it contains: [0160] Cu/Zn-SOD represents 65% of total SOD activity, has a molecular weight of 31 800 Da and pHi of 4.3; [0161] Mn-SOD represents 10% of total SOD activity, has a molecular weight of 80 600 Da and has 7 isoforms of different pHi values between 4 and 6; [0162] 2 different forms of Fe-SOD represent 25% of total activity: One Fe-SOD of 32 200 Da represents 18% of total SOD activity. The other Fe-SOD of 79 800 Da represents 7% of total SOD activity. They have a pHi of 4.4 and 4.7.

[0163] In 1 mg of the mixture of the invention, there are about 0.71 mg of Cu/Zn SOD, 0.06 mg of Mn-SOD and 0.23 mg of Fe-SOD.

Example 4. Efficacy of the Mixture of Purified SODS of the Invention on a Mmarker of Oxidative Stress: the Superoxide Anion (O.SUB.2.) Produced by NADPH Oxidase, and on an Inflammation Marker: TNF

[0164] In macrophages, the use of molecules such as interferon gamma or LPS induce an oxidative burst (over-production of the superoxide radical due to stimulation of NADPH-oxidase) and an inflammatory response (increased production of TNF and of interleukins).

[0165] The effect was evaluated of the different doses of a mixture of purified SODs of the invention on the production of the superoxide radical (O.sub.2.) and on the production of an inflammatory cytokine (TNF ), on a cell line of murine RAW 264.7 macrophages.

[0166] The cells were cultured in an RPMI medium (Roswell Park Memorial Institute Medium) to which were added: fcetal calf serum (10%, Life Technologies), a penicillin-streptomycin antibiotic solution (1%, Life Technologies) and fungizone (0.25%, Life Technologies) in T 175 cm.sup.2 culture dishes (Dutscher, Brumath, France) in a humid atmosphere enriched with 5% CO.sub.2 at 37 C. The cells were counted and passaged every three days using a Thoma coverslip and then distributed in culture dishes at a concentration of 250 000 cells/ml. The cells were subsequently incubated with a mixture of purified SODs of the invention (0 to 100 IU SOD/ml ) for 12 hours. Thereafter, they were detached from the support and suspended at a concentration of 10.sup.6 cells/ml in RPMI. 890 l of this suspension were placed in the presence of 100 l of 1 mM lucigenin and incubated at 37 C. for 30 minutes. After these 30 minutes, and at the time of measurement, 10 l of 10.sup.7 M PMA were added (Phorbol-12-Myristyl-13-Acetate, Sigma Chemical; St louis Mo.; USA) to measure the production of the superoxide radical. To stimulate the production of TNF-, lipopolysaccharide (LPS) was added to the wells 12 h before this measurement.

[0167] The production of O.sub.2was measured using a chemiluminescence technique (Chen et al., Am. J. Renal. Physiol. 289 F 749-53 2005) in the presence of lucigenin (10 M), a bioluminescence probe specific to the superoxide anion. In brief, in the presence of O.sub.2, lucigenin changes over from a fundamental state to an excited state. On its return to the fundamental state, lucigenin emits photons. The number of photons emitted is proportional to the formation of O.sub.2. The intensity of luminescence is recorded by a luminescence microplate reader (Synergy 2 Biotek, USA). The results are expressed in hits/mg of proteins.

[0168] The production of TNF in the macrophages is measured by ELISA with a DY 510 analysis kit (R&D system, Mineapolis, USA). The results are a mean of 3 independent repeats.

[0169] As can be seen in Tables 4 and 5, the mixture of SODs derived from the MA 7950 line modulate the oxidative and inflammatory response of the line of murine macrophages by inhibiting the production of the superoxide anion by NADPH oxidase, and the production of TNF.

TABLE-US-00005 TABLE 4 Effect of different doses of a mixture of purified SODs extracted from the F1 hybrid variety of Cucumis Melo MA 7950 on the production of the superoxide anion. (SOD (UI/ml) 0 5 10 50 100 % activation 100 5 79.5 5.5 66.4 3.2 30.5 2.5 19.6 2.4

TABLE-US-00006 TABLE 5 Effect of different doses of the mixture of MA 7950 SODs on the production of TNF (SOD (UI/ml) 0 5 10 50 100 TNF (pg/ml) 721 12 454 17 166 16 69 7 59 5

Example 5: Efficacy of the Mixture of Purified SODs of the Invention on the Endogenous Synthesis of Antioxidant Enzymes

[0170] A mixture of purified SODs of the invention was encapsulated following the method described in international application WO2006030111, to allow use via oral route. 10 male Wistar rats (bred by Janvier, Le Genest-St-Isle, France) aged 3 weeks were used.

[0171] On arrival, they were randomly divided into groups and housed in plastic cages in a controlled environment at a temperature of 231 C., hygrometry of 70% and 12-hour photoperiod (12 h light/12 h darkness). The rats were handled in accordance with NIH guidelines (National Research Council).

[0172] The animals had free access to food and water. Food consumption was measured daily and the body weight of the animals was recorded every other day.

[0173] The animals were separated into two groups of 5.

[0174] One group received a standard diet (EF R/M control E 15000-00, SSNIFF, Germany)

[0175] The other group was given the standard diet (EF R/M control E 15000-00, SSNIFF, Germany) as well as a dose of 4 IU SOD (or U/mg SOD) of the mixture of the invention per day for 28 days.

[0176] At the end of the experimental period of 28 days, the animals were anesthetized with an intraperitoneal injection of pentobarbital. The liver was infused with 0.15M NaCl, then sampled and stored at 80 C.

[0177] The expression of the SOD and GPx antioxidant enzymes was determined by Western-blot.

[0178] The results are given in Table 4. As can be seen, the ingestion of the mixture of SODs derived from the MA 7950 melon induces the expression of the antioxidant enzymes SOD and GPx in Wistar rats in the absence of oxidative stress.

TABLE-US-00007 TABLE 6 Quantification of the expression of SOD and glutathione peroxidase (GPX) in the liver of male Wistar rats SOD GPX control animals 100 100 animals treated with a mixture of 235 8 161 5 purified SODs of the invention

[0179] In a situation of oxidative stress, this stock of antioxidant proteins can be activated and used, allowing a better cell response to oxidative stress.

Example 6: Effect of the SOD Mixture of the Invention on the Expression of the NPPA and RXFP1 Genes

[0180] The effect of the mixture of purified SODs of the invention on the genes involved in cardiac hypertrophy was evaluated on a model of spontaneously hypertensive rats (SHR). These spontaneously hypertensive rats have cardiac hypertrophy originating from genetic cross breeding, and can be used on reception therefore having the advantage of being ready-for-use.

[0181] The different mixtures of purified SODs of the invention were encapsulated following the method described in international application WO2006/030111 to allow use via oral route.

[0182] 40 male SHR rats (bred by Janvier, Le Genest-St-Isle, France) aged 3 weeks were used. On arrival, they were randomly divided into groups and housed in plastic cages in a controlled environment at a temperature of 231 C., hygrometry of 70% and photoperiod of 12 hours (12 h light/12 h darkness). The rats were handled in accordance with NIH guidelines (National Research Council).

[0183] The animals had free access to food and water. Food consumption was measured daily and the body weight of the animals was recorded every other day.

[0184] The animals were separated into 4 groups of 10 (groups 1, 2, 3 and 4).

[0185] Group 1 received a standard diet (EF R/M control E 15000-00, SSNIFF, Germany)

[0186] Group 2 received the standard diet (EF R/M control E 15000-00, SSNIFF, Germany) for 4 days as well as a dose of 4 IU SOD (or U/mg SOD) per day of the SOD mixture derived from the MA7950 melon of the invention.

[0187] Groups 3 and 4 received the standard diet (EF R/M control E 15000-00, SSNIFF, Germany) for 4 days and a dose of 4 IU SOD (or U/mg SOD) per day of the SOD mixture derived from the Canari melon (group 3) or Anasta melon (group 4)

[0188] After the 4 experimental days, the animals were anesthetized with an intraperitoneal injection of pentobarbital. The heart was taken, and assay of ANP and RXFP1 performed by Western blot on the left ventricle (LV).

[0189] Protein extraction from the LV was conducted on ice with 20 mM Tris buffer (pH 6.8) containing 150 mM NaCl, 1mM 1% EDTA, 20% Triton, 0.1% SDS, and 1% cocktail of protease inhibitors. After centrifuging (1500 rpm, 15 min at 4 C.), the supernatant was collected and the tissue proteins separated by SDS PAGE. An equivalent amount of proteins was deposited on 10 or 15% acrylamide gels with a concentration gel of 4% acrylamide. Migration took place in Tris-glycine-SDS buffer from Sigma-Aldrich (Saint Quentin Fallavier, France). After separation, the proteins were transferred onto nitrocellulose membranes (Whatman, Dassel, Germany).

[0190] Quantification was conducted after standardisation in the membranes by expressing the density of each band of interest relative to GAPDH in one same line. The results are expressed as changes relative to the placebo group. ** p<0.01 compared with the placebo group.

[0191] The results are given in Table 7 below.

TABLE-US-00008 TABLE 7 Quantification of ANP and RXFP1 expression in the heart of SHR rats. ANP RXFP1 Control animals 100 100 Animals treated with mixture of SODs 54 147 derived from MA7950 melon ** ** Animals treated with mixture of SODs 98 103 derived from Canari melon (NS) (NS) Animals treated with mixture of SODs 97 96 derived from Anasta melon (NS) (NS) (NS): non-significant; **: significant

[0192] As can be seen in Table 7 above, solely the ingestion of the mixture of SODs derived from the MA 7950 melon (SOD mixture of the invention) leads to modulation of the expression of the ANP and RXFP1 proteins, of which the NPPA and RXFP1 genes are involved in cardiovascular pathologies, in particular cardiac hypertrophy and obesity. This modulation, as explained above, is beneficial in the event of cardiovascular disease (reduction of ANP concentration and increase in RXFP1 concentration).