METHOD FOR EVALUATING THE ABILITY OF A COMPOSITION TO PREVENT MUSCLE DAMAGE AND FATIGUE; FOOD SUPPLEMENT AND DRUG

Abstract

Disclosed is a method for evaluating the ability of a chemical substance or a chemical composition to prevent muscle fatigue and damage induced by physical exertion in humans by comparing the effect the chemical substance or a chemical composition has on a combination of three biological markers in cultured human primary skeletal muscle cells.

Claims

1. A method for evaluating the ability of a chemical substance (5) or of a chemical composition (C) to prevent muscle fatigue and muscle damage induced by physical exertion in human beings, said method comprising: a step a) of culturing human primary skeletal muscle cells; a step b) of differentiating the cells obtained in step a) in order to obtain myotubes; a step c) of bringing a chemical substance (5) or a chemical composition (C) into contact with the cell medium obtained in step b); a step d) of bringing at least one calcium ionophore agent (AI) into contact with the cell medium obtained in step c); a step e) of measuring the level of expression of a combination of biological markers comprising: at least one biological marker (M.sub.1) of glycemic homeostasis, chosen from the elements of the group consisting of the myokines or cytokines produced by muscles, at least one biological marker (M.sub.2) of muscle lesions, chosen from creatine kinase and lactate dehydrogenase, at least one biological marker (M.sub.3) of ATP metabolism, chosen from the elements of the group consisting of lactate, aqueous ammonia and oxypurines, in the culture medium obtained in step d); and a step f) of comparing the levels of expression of each of said three biological markers (M.sub.1), (M.sub.2) and (M.sub.3) measured in step e) with a reference expression level for each of these three biological markers.

2. The method of claim 1 wherein, in step d), the calcium ionophore agent (AI) is selected from the elements of the group consisting of beauvericin, ionomycin and calcimycin (or A23187).

3. The method of claim 2 wherein the calcium ionophore agent (AI) is calcimycin.

4. The method of claim 1 wherein the biological marker (M.sub.1) is interleukin-6, the biological marker (M.sub.2) is creatine kinase, and the biological marker (M.sub.3) is lactate.

5. The method of claim 2 wherein the biological marker (M.sub.1) is interleukin-6, the biological marker (M.sub.2) is creatine kinase, and the biological marker (M.sub.3) is lactate.

6. The method of claim 3 wherein the biological marker (M.sub.1) is interleukin-6, the biological marker (M.sub.2) is creatine kinase, and the biological marker (M.sub.3) is lactate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0203] The following experimental summary illustrates the invention without however limiting it.

A-1)—Choice of Cells and Time Course

[0204] A-1-1)—Culture Step

[0205] The cells retained for carrying out the method which is the subject of the present invention are human primary skeletal muscle cells from the gluteus maximus muscle (in the case of muscle exertion in human beings), and because of their ability to differentiate into myotubes (or muscle fibers).

[0206] In order to evaluate their differentiation capacity, the cells were cultured in 48-well plates at 10 000 cells/well, in a specific culture medium, at 37° C. in a humid atmosphere containing 5% of CO.sub.2. Each condition is carried out in quadruplicate.

[0207] A-1-2)—Differentiation Step

[0208] When the cells reached 70% to 80% confluence, their culture medium was replaced with differentiation medium, so as to obtain myotubes. The differentiation of the myoblasts into myotubes was evaluated by measuring the intracellular creatine kinase activity at the end of several durations, so as to determine the optimal duration of the differentiation step. The intracellular creatine kinase activity is measured with an assay kit by spectrophotometric measurement at 340 nm at different times. The results obtained are expressed in mU/mg of proteins.

[0209] The results obtained showed an optimal creatine kinase activity after a period of 10 days of differentiation, and showed that this creatine kinase activity is greater for a cell passage R3 than for a cell passage R4.

[0210] The differentiation step of the method according to the invention is therefore carried out for a period of 10 days for a cell passage R3.

[0211] A-1-3)—Differentiated-Cell Stress Step

[0212] Several concentrations of the ionophore agent calcimycin (or A23187) were tested on the differentiated cells obtained in the previous step.

[0213] For each of these concentrations, the amount of lactate, the amount of interleukin IL-6 and the level of activity of the creatine kinase (CK) were evaluated.

[0214] The measurement of the amount of lactate and of the amount of interleukin IL-6 was carried out by recovering the supernatants from each culture medium.

[0215] The lactate was quantified with an assay kit by spectrophotometric measurement at 575 nm, compared with a standard range of lactate. The results were expressed in pg/μg of DNA.

[0216] The IL-6 was quantified with an assay kit by spectrophotometric measurement at 450 nm, compared with a standard range of IL-6. The results were expressed in pg/μg of DNA.

[0217] The cell layers were lyzed in PBS buffer using a sonication probe and two evaluations were carried out on said cells layers: [0218] An evaluation of the creatine kinase (CK) activity with an assay kit by spectrophotometric measurement at 340 nm at different times. The results were expressed in mU/μg of DNA; and [0219] An evaluation of the amount of DNA by fluorimetric assay using a DNA intercalating agent, the Hoescht reagent, compared with a standard range of DNA.

[0220] The results of the amount of lactate and of IL-6 and the results of the creatine kinase (CK) activity were expressed relative to the amount of DNA present in the layers.

[0221] A statistical analysis by means of the Student's test was carried out in order to compare the amounts of lactate and of IL-6 measured, and the creatine kinase (CK) activity measured for each calcimycin (or A23187) concentration tested and for differentiated cells obtained in the previous step, not combined with calcimycin (or A23187).

[0222] The results obtained made it possible to show that the optimal calcimycin (or A23187) concentration is 1 μmol/liter (or 1 μM).

A-2)—Definition of the Optimal Method.

[0223] The method therefore comprises the following steps: [0224] Step a) of culturing human primary skeletal muscle cells from the gluteus maximus muscle according to the operating conditions described in section A-1-1) above, [0225] Step b) of cell differentiation of the medium obtained in step a) according to the operating conditions described in section A-1-2) above, and in particular for a period of 10 days for a cell passage R3, [0226] Step c) of bringing the culture medium obtained in step b) into contact with the substance or the composition to be tested, [0227] Step d) of bringing the medium obtained in step c) into contact with calcimycin (or A23187) at a concentration of 1 μmol/liter (or 1 μM), [0228] Step e) of measuring the amount of lactate and the amount of interleukin IL-6, on the culture medium supernatant, and of measuring the creatine kinase (CK) activity according to the methods described in section A-1-3) above, [0229] Step f) of comparing the levels of expression of creatine kinase (CK), of IL-6 and of lactate. More specifically, in the context of this step f) the following are calculated:
The ratio R.sub.1=[(N.sup.1.sub.0−N.sup.1.sub.i)×100]/[(N.sup.1.sub.0−N.sup.1)] for the extracellular interleukin IL-6 with:

[0230] N.sup.1 corresponding to the amount of IL-6 measured in the culture medium obtained at the end of the method as described above, without carrying out either step c) or step d) of the method (non-treated and non-stressed differentiated cells),

[0231] N.sup.1.sub.0 corresponding to the amount of IL-6 measured in the culture medium obtained at the end of the method as described above, without carrying out step c) of the method (non-treated but stressed differentiated cells),

[0232] N.sup.1.sub.i corresponding to the amount of IL-6 measured in the culture medium obtained at the end of the method as described above, when a substance or a composition (i) is used in step c) of the method (treated and stressed differentiated cells);

The ratio R.sub.2=[(N.sup.2.sub.0−N.sup.2.sub.i)×100]/[(N.sup.2.sub.0−N.sup.2)] for the intracellular creatine kinase with:

[0233] N.sup.2 corresponding to the creatine kinase activity measured in the culture medium obtained at the end of the method as described above, without carrying out either step c) or step d) of the method (non-treated and non-stressed differentiated cells),

[0234] N.sup.2.sub.0 corresponding to the creatine kinase activity measured in the culture medium obtained at the end of the method as described above, without carrying out step c) of the method (non-treated but stressed differentiated cells),

[0235] N.sup.2.sub.i corresponding to the creatine kinase activity measured in the culture medium obtained at the end of the method as described above, when a substance or a composition (i) is used in step c) of the method (treated and stressed differentiated cells);

[0236] The ratio R.sub.3=[(N.sup.3.sub.0−N.sup.3.sub.i)×100]/[(N.sup.3.sub.0−N.sup.3)] for the extracellular lactate with:

[0237] N.sup.3 corresponding to the amount of lactate measured in the culture medium obtained at the end of the method as described above, without carrying out either step c) or step d) of the method (non-treated and non-stressed differentiated cells),

[0238] N.sup.3.sub.0 corresponding to the amount of lactate measured in the culture medium obtained at the end of the method as described above, without carrying out step c) of the method (non-treated but stressed differentiated cells),

[0239] N.sup.3.sub.i corresponding to the amount of lactate measured in the culture medium obtained at the end of the method as described above, when a substance or a composition (i) is used in step c) of the method (treated and stressed differentiated cells).

B]—Evaluation of Substances and of Compositions According to the Method Which is the Subject of the Present Invention.

[0240] The composition (C.sub.1) was prepared by mixing its various constituent ingredients by successively introducing its constituent ingredients into a mixer equipped with a mechanical stirrer system equipped with flat stirrer blades or impeller-type blades, at a temperature of 25° C.

The composition C.sub.1 consists, for 100% of its weight, of:

[0241] 38.2% of zinc gluconate,

[0242] 31.0% by weight of D-αtocopheryl acetate,

[0243] 23.18% by weight of corn starch,

[0244] 6.02% by weight of silica dioxide,

[0245] 1.6% by weight of a dry residue of red wine, sold under the name Provinol™, and comprising a polyphenolic acid content of 90% by weight.

[0246] The results obtained by carrying out the method of evaluation which is the subject of the present patent application are set out in Table 1 below:

TABLE-US-00001 TABLE 1 Intracellular creatine kinase Extracellular Product tested Extracellular IL- activity (pg/μg of lactate (pg/μg of (% by weight) 6 (pg/μg of DNA) DNA) DNA) Non-treated and N.sup.1 = 3.95 ± 1.56 N.sup.2 = 38.07 ± 5.36 N.sup.3 = 14.62 ± 1.87 non-stressed differentiated cells Non-treated and N.sup.1.sub.0 = 12.29 ± 1.43 N.sup.2.sub.0 = 16.86 ± 8.04 N.sup.3.sub.0 = 24.92 ± 4.77 stressed differentiated cells Differentiated cells N.sup.1.sub.i = 11.47 ± 1.42 N.sup.2.sub.i = 12.66 ± 2.16 N.sup.3.sub.i = 25.13 ± 1.74 treated with R.sub.1 = 10 R.sub.2 = −20 R.sub.3 = −2 Provinol ™.sup.(*.sup.) (0.00005%) and stressed Differentiated cells N.sup.1.sub.i = 12.44 ± 1.80 N.sup.2.sub.i = 10.73 ± 2.28 N.sup.3.sub.i = 28.33 ± 6.43 treated with R.sub.1 = −2 R.sub.2 = −29 R.sub.3 = −33 GlnZn.sup.(**.sup.) (0.00015% of Zn) and stressed Differentiated cells N.sup.1.sub.i = 16.55 ± 5.99 N.sup.2.sub.i = 26.88 ± 2.82 N.sup.3.sub.i = 27.34 ± 3.43 treated with R.sub.1 = −51 R.sub.2 = 47 R.sub.3 = −24 vitamin E.sup.(***.sup.) (0.001%) and stressed Differentiated cells N.sup.1.sub.i = 7.00 ± 0.72 N.sup.2.sub.i = 33.24 ± 9.35 N.sup.3.sub.i = 16.12 ± 4.59 treated with (C.sub.1) R.sub.1 = 63 R.sub.2 = 77 R.sub.3 = 85 (0.001%) and stressed .sup.(*.sup.)Provinol ™ is a dry residue of red wine, comprising a polyphenolic compound content of 90% by weight for 100% of its weight, i.e. a weight proportion of polyphenolic compounds of 1.44%. .sup.(**.sup.)the zinc gluconate comprises a zinc divalent cation content of 12.2% .sup.(***.sup.)the vitamin E is D-α tocopheryl acetate

Analysis of the Results

[0247] The ratios R.sub.1, R.sub.2 and R.sub.3 are below 40, for the cells treated with Provinol™ (dry residue of red wine based on polyphenolic compounds). The same is true for the cells treated with zinc gluconate. It is deduced from this that neither Porvinol™ nor zinc gluconate and, consequently, the zinc divalent cation, can be selected alone for preventing muscle fatigue and muscle damage induced by physical exertion in human beings.

[0248] The ratios R.sub.1 and R.sub.3 are below 40, and the ratio R.sub.2 is above 40 (R.sub.2=47), for the cells treated with D-α-tocopheryl acetate. This product therefore cannot also be selected for preventing muscle fatigue and muscle damage induced by physical exertion in human beings.

[0249] Conversely, the ratios R.sub.1, R.sub.2 and R.sub.3 are all above 40 for cells treated with the composition (C.sub.1).

[0250] This composition (C.sub.1) can be selected for preventing muscle fatigue and muscle damage induced by physical exertion in human beings.