FR2643637A1 - CALCIUM ION COMPLEX WITH AT LEAST ONE UNSATURATED FATTY ACID, BIOMEDIATING AGENT COMPRISING SUCH A COMPLEX, PHARMACEUTICAL COMPOSITION INCORPORATING SAME AND PLANT OR ORGAN EXTRACTS CONTAINING THE SAME - Google Patents

Abstract

The invention concerns a calcium complex. Said complex is characterized in that it comprises the calcium ion combined with at least one unsaturated fatty acid, preferably polyunsaturated, and without addition of ethylene binders. Examples of such polyunsatured fatty acids are 9,12-linoleic acid, 9,12,15-linolenic acid and 5,8,11,14-arachidonic acid. The complex forms a biomediator agent particularly effective for delivering calcium in situ on account of a high bioavailability resulting in the preparation of particularly interesting pharmaceutical compositions, notably for the treatment of pain, fever, inflammation and muscular atony.

Description

Calcium ion complex with at least one unsaturated fatty acid, biomediating agent comprising such a complex, pharmaceutical composition incorporating it and extracts of plants or animal organs containing it.

 The present invention essentially relates to a complex of the calcium ion with at least one unsaturated fatty acid, a biomedical agent comprising such a complex, a pharmaceutical composition incorporating it and extracts from plants and animal organs.

 We know that calcium is today considered a major bioelement.

Only a generation ago, the Ca ion was, more or less, known physiologically as a mineral constituent of bone and an element of the blood plasma necessary for the functioning of the heart and the coagulation of blood. Then, we discovered the role of the calcium ion in the contraction-relaxation mechanism of striated musk, followed by the discovery of calcium pumps whose effectiveness is directly dependent on the concentration of Ca2 + ion ( see The Role of Calcium in Biological
Systems LJ ANGHILERI et al. flight. 1, 2, 3 CRC Press Boca
Raton / Florida 1982).

Today, we know that the calcium ion participates in many biological functions. The most well-known calcium-dependent processes are the transmission of nerve impulses and
The release of neuromediators, The mechanisms of contraction of the striated, cardiac and smooth muscles, and the secretory mechanisms of the exocrine and endocrine glands (see Endocrinology of Calcium Metabolism JA PARSONS Editor Raven Press New York 1982).

 It is thus known that calcium interacts with hormone receptors and regulates the effects of hormones either by acting directly on enzymes, or indirectly by modifying the intracellular pH, The concentration of phosphate and sodium and the availability of magnesium. Calcium is also an essential constituent of plasma membranes which influences their structures, their defarmability and their permeability. Its ability to bind to mucroproteins affects their surface properties, and its effects on microtubules extend to a wide range of functions.

 In vertebrates, calcium homeostasis is regulated by calcium migrations between five compartments: extracellular fluids, the intracellular complex itself subdivided into several sectors, bones and related fluids, the intestinal lumen, and the fluid of renal tubules. The transport between these various compartments is controlled by three hormones: cholecalciferol, parathyroid hormone (PTH) and calcitonin.

 At least three different membrane systems of the cell are involved in the various regulatory processes by changes in concentrations of cytosolic or extracellular Ca 2+. This is how cytoplasmic Ca 2+ concentrations are capable of simultaneously regulating several different activities within the same cell, which gives an idea of the subtlety of the mechanisms involved. The biochemical processes which allow excess calcium to be removed from the cell, to convey it into the extracetlular fluids and then to mineralize it in the urine before excretion are also quite remarkable.

 However, faced with the finesse of these biochemical processes, the therapeutic means for correcting calcium concentrations or inflection of their derivatives remain quite crude.

 The intakes of intracellular calcium, intended for example for the constitution or reconstitution of bone tissues, are relatively controlled and the practitioner has two axes of prescription: supplementation of food intake by taking assimilable salts such as gluconate or calcium glycerophosphate and stimulation of calcium fixation by polypeptide complexes like calcitonin associated with vitamin D factors.

Calcium biomediator, the one whose local variation of concentration is active on biological processes, is on the other hand modulated, in influx or in reflux, by indirect actions; the best known biological intermediates being calmodulins (see 1 - Calcium Transport in Contraction and
Secretion Editors: E. CARAFOLI et al. North Holland Amsterdam 1975 2 - Calcium Regulation and Bone Metabolism vol. 9 Editors: DU

 COllN et al. Excerpta Medica Amsterdam 1986).

 The present invention therefore aims to solve the new technical problem consisting in the supply of a new calcium ion complex capable of constituting a biomediator agent of remarkable quality by allowing a calcium supply in situ thanks to a significant bioavailability.

The present invention also aims to solve the new technical problem consisting in the supply of a new biomediating agent having a high bioavailability, achieving a calcium intake in situ, thanks to the combination of the effects of at least the following two factors, to know :
a) molecular ampiphilia conducive to the passage of cell membranes;
b) chain unsaturation conducive to mobility in biological fluids.

 The present invention also aims to solve the new technical problem consisting in the supply of a calcium ion biomediating agent of very high bioavailability, combining the two aforementioned factors, as well as a third factor consisting in obtaining of an additional chelating effect of the calcium ion by the combination with functional peptide groups.

The present invention also aims to solve the new technical problem consisting in the supply of a pharmaceutical composition carrying out a calcium supply in situ, making it possible to treat all the pathologies due to the biomediation of calcium, preferably making it possible to carry out therapeutic treatments. following
- analgesic treatment, for example dental neuralgia or headache,
- anti-inflammatory treatment, for example erythema, or rheumatoid arthritis,
- anti-pyretic treatment,
- cardiotonic treatment,
- myotonic treatment, for example rheumatoid arthritis.

 Finally, the present invention also aims to solve the new technical problem consisting in the supply of plant extracts or parts of plants, and extracts of animal organs comprising new calcium ion complexes with effect remarkable biomediator.

 The present invention makes it possible to solve the aforementioned new technical problems for the first time, in a particularly simple manner, usable on an industrial scale.

 Thus, according to a first aspect, the present invention provides a calcium ion complex, characterized in that it comprises the calcium ion combined with at least one unsaturated fatty acid, preferably having from 14 to 26 carbon atoms, more preferably from 16 to 24 carbon atoms. This unsaturated fatty acid can be linear or branched.

 According to an advantageous embodiment of the invention, this complex is characterized in that the unsaturated fatty acid is a polyunsaturated fatty acid, preferably without conjugation of the ethylenic bonds. Preferably, this polyunsaturated fatty acid without conjugation of the ethylenic bonds is chosen from 9,12-linoteic acid, 9,12,15-Linolenic acid, 5,8,11,14-arachidonic acid.

 According to a particularly advantageous embodiment of the invention, the aforementioned calcium ion complex further comprises at least one calcium chelating agent with peptide or pseudopeptide structure, one of the amino acids of which is proline, preferably of molecular weight less than about 1000. Preferably, it is a di- or tripeptide.

 According to an even more preferred characteristic, the di- or tripeptide is chosen from the group consisting of cysteyl-proline, p-glutamyl-proline, glycyl-p-glutamylproline or diketopiperazine of proline.

 According to a second aspect, the present invention also provides a biomediating agent, characterized in that it comprises a calcium ion complex in which the calcium ion is combined with at least one unsaturated fatty acid, preferably having from 14 to 26 carbon atoms, more preferably from t6 to 24 carbon atoms. This unsaturated fatty acid can be linear or branched.

 According to an advantageous embodiment, the abovementioned unsaturated fatty acid is a polyunsaturated fatty acid, preferably without conjugation of the ethylenic bonds.

 According to another preferred embodiment of the invention, the aforementioned polyunsaturated fatty acid without conjugation of the ethylenic bonds is chosen from the group consisting of 9,12-linoleic acid, 9,12,15-linolenic acid, 5,8,11,14-arachidonic acid.

 According to yet another particularly advantageous embodiment of the invention, the abovementioned biomediating agent is further characterized in that it comprises at least one calcium chelating agent with peptide or pseudopeptide structure, one of the amino acids of which is proline. Preferably, this chelating agent is a di- or tripeptide.

 According to an even more preferred characteristic, this chelating agent is chosen from the group consisting of cysteylproline, p-glutamyl-proline, glycyl-p-glutamyl-proline or diketopiperazine of proline.

 According to yet another alternative embodiment of the invention, this complex calcium ion is contained in phospholipid or glycophospholipid form in an extract of plants or parts of plants, preferably belonging to the families of crucifers, Lineas or Resedaceae.

According to yet another alternative embodiment of the invention, the aforementioned complex calcium ion is contained in phospholipid or glycophospholipid form in an extract from animal organs, these animals preferably being fish belonging to the families of Clupeidae, Gadidae,
Salmonids or Scombridae.

 According to a third aspect, the present invention also relates to a pharmaceutical composition, characterized in that it comprises, as active principle, a complex of calcium ion as previously defined or a biomedicating agent as previously defined.

 According to another advantageous characteristic of the pharmaceutical composition according to the invention, it is characterized in that it comprises a concentration of the abovementioned calcium ion complex of between 0.1 and 5%, in percentage by weight relative to the total weight of the pharmaceutical composition.

 According to a particular embodiment of the pharmaceutical composition according to the invention, the latter is characterized in that it is a pharmaceutical composition with analgesic activity preferably formulated for pernasal administration.

 According to another particular embodiment of the pharmaceutical composition according to the invention, it is characterized in that it is a pharmaceutical composition with antipyretic activity, preferably formulated for oral administration.

 According to another particular embodiment of the pharmaceutical composition according to the invention, it is characterized in that it is a pharmaceutical composition with anti-inflammatory activity, preferably formulated for oral administration.

 According to another particular embodiment of the pharmaceutical composition according to the invention, the latter is characterized in that it is a composition with myotonic activity, preferably formulated for topical administration, in particular in the form of 'a gel.

 According to yet another particular embodiment of the pharmaceutical composition according to the invention, it is characterized in that it is a pharmaceutical composition with cardiotonic activity, having a positive inotropic activity without associated positive chronotropic effect, preferably formulated for percutaneous administration.

 It can thus be seen that the invention makes it possible to prepare particularly advantageous pharmaceutical compositions in particular in the treatment of pain, fever, inflammation and muscular atony, which represents a decisive, unexpected technical advantage for L skilled in the art.

 According to an alternative embodiment, the pharmaceutical composition is in the form of capsules proportioned from 20 to 100 mg, in particular 50 mg of extract, or of active principle, these capsules being preferably made gastro-resistant.

 The pharmaceutical compositions according to the invention can be presented in various galenical forms, the route of administration and the dosage being adapted to the desired therapeutic activity.

 For example, for analgesic treatment, the route of administration will be pernasal and the dosage will be 5 mg per spray.

 In addition, in the case of an anti-inflammatory treatment, the route of administration will be oral and the dosage will be 2 capsules dosed at 50 mg per day.

 In the case of antipyretic treatment, the route of administration will be oral and the dosage will be 3 capsules at 50 mg per day.

 In the case of cardiotonic treatment, the route of administration will be percutaneous and the dosage will be 5 mg per day.

 In the case of myotonic treatment, the route of administration will be topical and your dosage will be 4 applications per day of a small amount of gel corresponding to 5 mg.

The present invention also covers a pharmaceutical composition, characterized in that it comprises, as active principle, a natural extract derived from animal or plant organisms, preferably respectively chosen from extracts of fish belonging to the families of Clupeidae, Gadidae, of
Salmonidae or Scombridae, or extracts of plants belonging to the Cruciferous, Lineae or Resedaceae families.

 The present invention also relates to a method for the therapeutic treatment of a mammal, including a human being, characterized in that this mammal is administered a therapeutically effective amount of a calcium ion complex in which the calcium ion is combined with at least one unsaturated fatty acid, linear or branched, preferably having from 14 to 26 carbon atoms, more preferably from 16 to 24 carbon atoms. According to particular variant embodiments of this therapeutic treatment process, analgesic treatment, preferably by pernasal administration; according to another variant, an antipyretic treatment is carried out, preferably by oral administration; according to yet another variant, an anti-inflammatory treatment is carried out, preferably by oral administration; still according to another variant, a myotonic treatment is carried out, preferably by topical administration, in particular in the form of a gel; still according to another variant, cardiotonic treatment is carried out, preferably by percutaneous administration.

 It can generally be said that the calcium ion complex according to the invention, the biomedical agent according to the invention containing it as well as the pharmaceutical compositions containing it make it possible to treat all the pathological conditions which regress or yield by an activation of calcium bioregulation. The mode of action of this calcium ion complex has not yet been elucidated. It is believed that it could fall under the kinin system and involve in particular bradykinin, this being given simply as an indication as being a possible scientific explanation of the mechanism of action.

 According to yet another aspect of the present invention, the plant and fish extracts are produced according to general methods allowing preferably simultaneous extraction of lipids and peptides of low molecular weights. The conditions are optimized to favor the extraction of the unsaturated fatty acids according to the invention either in esterified form, or in free or salified form, as well as your very small peptides, that is to say those whose molecular weight is less than 1000. .

Among the plants, all Cruciferae, Lineas and Resedaceae, can be used, in particular: - Reseda Alba L., R. Lutea L., R. Luteola L., R. Odorata L. and
R. Phyteuma L.

- Oligomeris Subulata (Del) Boiss.

- Ochrademus Baccatus Del.

- Asterocarpus Sesamoides Gay., Clusii Gay.

- Sinapis Galba L. and S. Arvensis L.

- Linum glandulosum Moench, L. Narbonense L., L. Carthaticum L. and
L. Usitatisimum L.

 We can use whole plants harvested in flowers or fruits or seeds. They are then dried out of the light under a stream of air.

 Among fish, all your species rich in unsaturated lipids can be used, in particular those of the families of Cupléidae, Galidae, Salmonidae and Scombridae. The raw material can be the whole fish or the only lipid storage organs. Freezing prior to extraction is advantageous.

 It may be an aqueous or organic extract which results from the action of an aqueous or organic solvent or a mixture of such solvents on the comminuted plants or flesh. It is therefore a question of extracting products which are soluble or miscible in one or the other of these solvents or in both.

 The aqueous extract advantageously results from the exhaustion of a comminuted tissue with water, then from the recovery by an appropriate means of the dry solute, such as lyophilization or nebulization to which a preconcentration such as may be associated. than reverse osmosis. In the case of lyophilization or nebulization, it may be advantageous to use a physiologically inert support agent, for example having a viscosifying or absorbing or dehydrating effect, such as hydroxypropylmethylcellulose or HPMC, silicic acid, a maltodextrin or a dextran.

 An extraction can also be carried out with an organic solvent and the extract obtained can then be concentrated to remove all or part of the organic solvent used. We can possibly end with a chromatographic fractionation of this extract.

 The organic extract advantageously results from the exhaustion by an organic solvent, such as ether, hexane, methanol, ethanol or liquid CO 2 in the supercritical phase, of a ground material, then of recovery by evaporation of the dry solute. As solvent, however, ether, hexane or petroleum ether and more preferably methyltertiobutylether, or liquid CO2 in the supercritical phase, in the pure state or optionally with traces of water or ethanol or the like.

 It is also possible to carry out an extraction by a simultaneous action of the aqueous solvent and the organic solvent; in this case, the latter is preferably alcoholic, in particular ethanol.

 The extracts resulting from the extractive phase are subsequently subjected to calcification. This treatment of the extracts is carried out in two stages, a hydrolysis of their fatty acid esters according to your general saponification processes, this being preferably carried out with a sodium hydroxide solution in an alcoholic medium, then a lime milk calcification in the same medium. Direct calcification is also possible.

 The extracts, before saponification or after calcification, can be discolored and / or deodorized by adsorbents such as active carbon used in continuous columns, or batchwise by addition to the medium and then separation on inert filter media.

Finally, the invention also covers extracts of plants or parts of plants characterized in that they are obtained from plants belonging to the families of
Crucifers, Lineas or Resedaceae; as well as extracts from animal organs, characterized in that they are obtained from fish belonging to the families of Clupeidae, Gadidae, Salmonidae or Scombridae. Preferably these extracts are obtained by the general methods previously described.

 The present invention will now be illustrated from several examples of the invention given simply by way of illustration and which therefore cannot in any way limit the scope of the invention. In the examples, as in the text and in the claims, all the percentages are given by weight, unless otherwise indicated.

Example 1 according to the invention
Example of synthesis of the calcium complex
For example, the methyl ester of α-linolenic acid is dissolved in 100 times its weight of methanol and then it is treated with 3 times its weight of potassium hydroxide solution at 10 X with stirring at reflux for 1 h; the whole is then poured into an aqueous solution of the same volume of calcium chloride at 5 X and the whole is brought to reflux for 5 min before being concentrated to a residual volume of less than one tenth of the initial methanolic solution.

 The precipitate is separated on sintered glass, wrung, washed with 2 times its volume of water and then dried.

This calcium complex of α-linolenic acid according to the invention is thus obtained, which is in the form of a powder of broken white color; its melting point is not clear, there is a loss of water and then decomposition; its low solubility in water and the usual solvents have led to the separate identification of three constituents: water, calcium and alphalinolenic acid corresponding to a polyhydrate of calcium alpha linoleate, the spectrum UV has two absorption maxima at 252 and 320 nm and, in the infrared spectrum, characteristic bands of pure acid, at 1681 and 1651 cm-l are associated with a strong diffuse absorption in the region of 3000 to 3500 cm
Example 2 according to the invention
By proceeding in a similar manner to Example 1, - one can prepare:
The calcium complex of 9,12-linoleic acid, in the form of a whitish-colored powder having the physical and spectral characteristics of a calcium linoleate hydrate, the three components being identified as in example 1.

Example 3 according to the invention
By also proceeding in a similar manner to Example 1, the calcium complex of 5,8,11,14-arachido acid is presented in the form of a whiteish powder having the characteristics physical and spectral of a calcium arachidonate hydrate, the three components being identified as in Example 1.

Example 4
Preparation of an extract according to the invention of plant origin
The plant belonging to the Resedaceae family, namely the Reseda alba L plant, picked whole at the maturity of the seeds and then dried in the rules of the art is coarsely ground and then mechanically agitated for 48 h in a mixture at 600C of 10 times its weight of water and once its weight of alcohol at 95% by volume.

 After cooling to room temperature, the liquid phase is separated from the marc, calcified with lime milk to pH 8, filtered at 0.40 dm, then concentrated 5 times under vacuum below 500C. The concentrate is stirred gently for 15 h, left to stand for 48 h and then filtered under vacuum. The recovered precipitate taken up in twice its weight of cold water then re-filtered, dried, homogenized and conditioned.

This precipitate is in the form of a beige powder, with no clear melting point; the characterization of its constituents is difficult as it stands, due to its low solubility in common solvents. Nevertheless, the solubilized fractions indicate in
UV, IR, NMR and mass spectrometry the presence of water, calcium, fatty acids and peptide derivatives. The fatty acids observed are in particular those of Examples 1 to 3 but, by gas chromatography of their decalcified and then esterified derivatives, it appears in addition for approximately 30% of saturated fatty acids of the palmitic type. Peptide derivatives give by hydrolysis essentially of the proline.

Example 5
Animal extract according to the invention
The thawed flesh of a fresh fish from the family of
Salmonidae, namely Salmo rubor salmon, are mechanically shredded in 5 times its fresh weight in cold water, the liquid phase is separated and the pulp exhausted by 3 times its weight in cold water and then re-separated. The liquid phases are joined, filtered at 0.40 μm and then concentrated 3 times under vacuum. The concentrate is possibly enriched at this stage with fish oil obtained elsewhere if no fatty substance has been released to the surface.

 The concentrate is then treated with stirring with 0.1 times its volume of 95% alcohol by volume and then with sodium hydroxide until pH 9 and it is brought to reflux for 1 h.

 After adding lime milk to raise the pH above 11, an additional reflux of 5 min, the whole is concentrated 3 times then cooled, the precipitate is isolated by centrifugation, dried, homogenized then stored.

 This precipitate is in the form of a beige-colored powder having physical and spectral characteristics similar to those of Example 4.

Example 6
Example of formulation of biomediating agent according to the invention
According to a first alternative embodiment, a biomediation agent according to the invention is formed by the product obtained directly in the raw state in any one of the above examples 1 to 5.

 According to another advantageous variant, the crude extract obtained in Example 1, 2 or 3, in the form of a dry product, is mixed with 5 X of its weight of glycyl-p-glutamyl-proline until perfect homogeneity , then stored. This mixture constitutes the active principle of a pharmaceutical composition according to the invention as formulated for example in the following examples.

Example 7
Pharmaceutical composition according to the invention
Example of geLuS specialty
The product obtained according to any one of the abovementioned examples 1 to 6 is added with its weight of magnesium stearate and 10 times its weight with microcrystalline cellulose then intimately homogenized, the mixture obtained is distributed by means of suitable distribution equipment. in gelatin capsules module 2 at a rate of 240 mg / unit.

 The capsules thus dosed with 20 mg of extract are coated using an apparatus of gum arabic, cellulose ester and titanium oxide. After drying, the capsules obtained are gastro-resistant and packaged in a blister in blister packs of 15 grouped in cases of 2.

Example 8
Pharmaceutical composition according to the invention
Specialty dermal gel
The product obtained according to any one of examples 1 to 6 is dissolved in a mixture of its weight of alcohol at 95% by volume, of its weight of glycerol, of its weight of diethylene glycol monoethyl ether and of 7 times its weight of 'purified water ; carbopol at the rate of 1% is dispersed in the cold solution by non-turbulent mechanical stirring then triethanolamine is added in successive fractions so as to adjust the pH to 6.

 The gel obtained is then packaged in a 30 g polyethylene tube.

Example 9
Pharmaceutical composition according to the invention
Specialty nasal solution
The product obtained in any one of Examples 1 to 6 is dissolved in an amount of 5 mg / ml in an excipient for use as a nasal nebulization based on physiological saline, glycerin and monoethyl ether of diethylene glycol. The preparation is packaged in a 30 ml polyethylene bottle fitted with a nasal spray cannula delivering 0.1 ml by pressure.

Example 10
PHARMACOLOGICAL TESTS
A - Search for an analgesic activity
PRINCIPLE
The intraperitoneal injection of acetic acid in mice causes a pain syndrome, which can be reduced or eliminated by prior injection of an analgesic substance.

 The pain syndrome results in a twisting of the dorso-abdominal musculature and Stretching of the hind legs of the mouse.

MATERIAL AND METHOD
1 - ANIMALS
Male EOPS strain mice (10 per batch) from the Montpellier breeding center were used in this study.

The animals are kept under observation for 4 days before the start of the tests. At the start of the test, the animals weigh an average of 20 g. During the observation period, the animals, divided into cages of 10, receive food and drinking water ad libitum and are subjected to a temperature between 21 and 23 C and a day / dark cycle of 12 h.

2 - PRODUCTS
- Product of Example 4
- Lysine acetylsalicylate (ASPEGI p injectable 0.5 g).

- Paracetamol (DAFALGA
- 1 X solution of acetic acid.

3 - PROTOCOL
The mice are fasted for food 18 h before the test. The acetic acid is injected 30 min after the administration of the test substance (time to). A batch of control animals receives the vehicle.

 The counting of contractions begins at t 5 min and continues for 15 min.

 The effective dose 50 (ED50) is considered to be the dose of analgesic which reduces the number of contractions by 50 X.

The efficacy of the product of Example 4 was evaluated with respect to paracetamol and aspegic. The product of Example 4 and the paracetamol were the subject of an ED50 determination;
4 - DOSES USED
- Product of Example 4 - 10, 20, 50 and 100 mg / kg.

 - ASPEGI at 30Q mg / kg.

 - PARACETAMOL 100, 160, 200, 320.

 The dilutions are adjusted to administer 10 ml / kg.

5 - EXPRESSION OF RESULTS
The number of contractions is noted for each animal and is compared between the different lots by the F tests of SNEDECOR and Student t.

 The percentage of protection of the animals treated is calculated relative to the number of colics obtained in the controls.

 The results are expressed in Figures 1, 2 and 3.

B - Search ~ for anti-inflammatory activity
(Product of Example 4)
CARRAGENIN OEDEMA - WINTER TECHNIQUE (1)
INTRODUCTION
The injection of carrageenan under the plantar fascia of the rat's hind leg causes an acute inflammatory reaction which can be reduced by anti-inflammatory substances.

^ TRIEL AND ZETHODES
1 - ANIMALS
Wistar mat rats (12 per lot) from the Montpellier breeding center were used in this study.

The animals are kept under observation for 4 days before the start of the tests. At the start of the test, the animals weigh an average of 90 g. During the observation period, the animals distributed in cages of 5 receive food and drinking water ad libitum and are subjected to a temperature between 21 and 23 C and a day / dark cycle of 12 h.

2 - PRODUCTS
- Product of Example 4
- SATIA lambda type carrageenan. CECA SA

 - Excipients.

- physiological serum
- 3 X gummy water.

3 - PROTOCOL
The rats are fasted on food the day before the test.

Carrageenan (0.05 ml - 1 gX solution in physiological saline) is injected into the plant cushion of the right hind paw 1/2 h after intraperitoneal administration of the study substance. A batch of control animals serves as a control and receives the excipient. For each dose of substance, the concentrations of the suspensions used are adjusted so as to administer exactly 1 ml per 100 g of body weight.

 The plethysmographic measurements (2) are made before and 1 h, 2 h, 3 h and 4 h after the administration of the carrageenan.

4 - DOSES USED
- Product of Example 4
- 100, 200, 400 mg / kg intraperitoneally.

5 - EXPRESSION OF RESULTS
The percentage increase in paw volume is obtained for each animal according to the following formula
Measured volume - Initial volume Z = Initial volume - x 100
The swelling percentages are analyzed by the F tests of SNEDECOR and t of STUDENT (3).

The anti-inflammatory activity is expressed by the percentage reduction in edema in the treated rats compared to the controls according to the following formula
Z Control swelling - X Treatment swelling
X = X Control swelling x 100
RESULTS
The results are recorded in Tables I, II and graphs of Figures 4 and 5.

TABLE I
Z increased leg volume
TREATMENTS 1 h 2 h 3 h 4 h
WITNESSES 25 + 8 50 + 16 52 + 17 46 + 4
Product of Example 4
100 mg / kg 17 + 8 -16 + 12 12 + 11 * 13 + 12 *
200 mg / kg 17 + 4 21 + 13 * 26 + 13 * 19 + 10 *
400 mg / kg 12 + 7 13 + 9 * 19 + 14 + 15 + 10
* Significantly different from witnesses
TABLE II
X of inhibition of edema
TREATMENTS 1 h 2 h 3 h 4 h
Product of Example 4
100 mg / kg 29 67 76 70
200 mg / kg 31 57 50 57
400 mg / kg 49 73 62 66
C - Search for antipyretic activity
INTRODUCTION
Administration of brewer's yeast in rats causes hyperthermia which can be reduced by antipyretic substances.

MATERIALS AND METHODS
1 - ANIMALS
Male rats of the Wistar strain (12 per batch) from the Montpellier breeding center were used in this study.

The animals are kept under observation for 4 days before the start of the tests. At the start of the test, the animals weigh an average of 200 g. During the observation period, the animals, divided into cages of 5, receive food and drinking water ad libitum and are subjected to a temperature between 21 and 23 C and a day / dark cycle of 12 h.

2 - PRODUCTS
- Product of Example 4
- Paracetamol (DAFALGA)
- Excipients
- physiological serum
- yeast
3 - PROTOCOL
The animals (10 per batch) are fasted 16 h before the test. On the day of the test, the rats are weighed and receive an injection of brewer's yeast (2 g / kg) subcutaneously. 3 hours later, the test substances are administered intraperitoneally. The temperature of each animal is recorded before and after the induction of hypertermia by brewer's yeast as well as after treatment-every hour for 4 h using a thermocouple consisting of a rectal probe and d a reading device.

4 - DOSES USED
- Products of Example 4
- 50 and 100 mg / kg intraperitoneally in suspension
in water for injection.

- Paracetamol
- 200 and 400 mg / kg intraperitoneally in suspension
in gummy water at 3 X.

5 - EXPRESSION OF RESULTS (see figure 6)
The homogeneity of the temperatures before and after induction of the hyperthermic was verified by an analysis of variance.

The significance of hyperthermia after injection of brewer's yeast was confirmed for each batch by a t-test of
STUDENT.

 The difference measured from baseline, measured after 1 h, 2 h and 4 h of treatment (differential temperature) was calculated for each animal and then compared between the control batch and the batches treated by an analysis of variance.

RESULTS
Average deviations from Base temperature
Witnesses 1.08 +09 1.18 + 09 1.24 + 04 1.07 + 13
Product of Example 4
50 mg / kg 0.43 + 12 0.57 + 13 0.54 + 14 0.59 + 09
Product of Example 4
100 mg / kg -0.80 + 12 -0.70 + 15f -0.36 + 38 0.21 + 16
ParacetamoL 200 mg / kg -1.02 + 23 * -1.60 + 26 .1.63 + 38 * -1.13 + 44 *
ParacetamoL 400 mg / kg. 1.52 + 25 *. 1.75 + 27 *. 1.87 + 35 * 1.43 + 46
* Significant difference compared to controls
CONCLUSION
Examination of the results shows that, under our experimental conditions, The product of Example 4 reduces the hyperthermia induced by Brewer's yeast significantly compared.

to witnesses.

 Although its effectiveness is less than that of paracetamol, the product of Example 4, because of its low toxicity, has a significant therapeutic benefit.

D - Search for cardiac activity (cardiotonic)
The cardiac activity of the product sample
Example 4 was sought by the method of isolated rat atria.

A - TRIAL AND METHODS
1 - ASSEMBLY OF THE PREPARATION
Rats of both sexes with an average weight of 300 g are knocked out and the heart is quickly removed and placed in a beaker containing oxygenated solution of CHENOWETH-KOELLE (SCK) of the following composition (mM): NaCl, 1.20 ; KCl, 5.63; CaCl2, 2.0; dextrose, 9.7; MgCl2, 2.0; NaHC03, 26.0.

 The right and left atria are detached from the heart and placed in an insulated organ tank containing SCK permanently oxygenated and maintained at a temperature of 370C using a circulation pump. The Free portion of the headset is attached to a Plexiglas electrode, while the other end (detached from the heart) is pinched by a clip which is then connected to an isometric recorder.

 The right atrium beats spontaneously and allows you to follow the chronotropic effect by recording the rhythm. The left atrium, electrically driven at the frequency of 1.6 Hz, with a pulse duration of 5 ms and a voltage double the threshold using a Grass stimulator, makes it possible to measure the inotropic effect by evaluation of voltage variations.

Chronotropic (right atrium) inotropic (left atrium) responses are recorded on Physiograph (Narco-bio
Systems). The basal values are 195 beats / minute (right atrium) and 0.5 g (left atrium), respectively.

 The earpieces thus arranged are only used after 45 minutes of waiting. During this period, the bath liquid is replaced every 15 min.

2 - STUDY PROTOCOL
The product of Example 4 was added directly to the tank with an isolated organ in the form of a concentrated solution 100 times.

 After obtaining the induced maximum tension, by the administration of a first dose of product of example 4, the following dose is added without washing and so on.

 The concentrations indicated correspond to the cumulative concentrations present in the tank.

 The results are expressed as a percentage change compared to the basal values measured before any administration.

B - RESULTS
The results of a typical experiment are illustrated in Figure 7.

 These results indicate that the product of Example 4 has positive inotropic activity without positive chronotropic activity.

 The average value, over three experiments, of the increase in voltage induced by 0.30 mg of product of Example 4 per mt is 90 + 5 Z. It is 50 + 5 Z for 0.15 mg / ml .

 Under similar conditions, isoprenaline (1Q-6M) induces an increase in blood pressure of 200 Z, but also an increase in rhythm of approximately 100 Z.

C - CONCLUSION
The product of Example 4 has a positive inotropic activity without positive chronotropic activity.

 These pharmacological properties appear potentially very interesting in the context of the treatment of heart failure.

Claims (27)

 1. Calcium complex, characterized in that it comprises the calcium ion combined with at least one unsaturated fatty acid, preferably having from 14 to 26 carbon atoms, more preferably from 16 to 24 carbon atoms, linear or branched.  2. Complex according to claim 1, characterized in that the abovementioned unsaturated fatty acid is a polyunsaturated fatty acid, preferably without conjugating its ethylenic sounds.  3. Complex according to claim 2, characterized in that the aforementioned polyunsaturated fatty acid is chosen from the group consisting of 9,12-linoleic acid, 9,12,15-linolenic acid and Acid 5, 8,11,14-arachidonic.  4. Complex according to one of claims 1 to 3, charac terized in that it further comprises at least one calcium chelating agent with peptide or pseudopeptide structure, one of the amino acids of which is proline, preferably of weight molecular less than about 1,000.  5. Complex according to claim 3, characterized in that the chelating agent comprises a di- or a tripeptide.  6. -CompLexe according to claim 5, characterized in that the aforementioned di- or tripeptide is chosen from the group consisting of cysteyl-proline, p-glutamyl-proline, glycylp-glutamyl-proline and diketopiperazine of proline .  7. Biomediator, characterized in that it comprises the calcium ion combined with at least one unsaturated fatty acid, preferably having from 14 to 26 carbon atoms, more preferably from 16 to 24 carbon atoms, linear or branched .  8. Biomediator according to claim 7, characterized in that the aforementioned unsaturated fatty acid is a polyunsaturated fatty acid, preferably without conjugate its ethylenic bonds.  9. Biomediator according to claim 8, characterized in that the aforementioned polyunsaturated fatty acid is chosen from The group consisting of 9/12-linoleic acid, 9,12,15 linolenic acid, 5,8,11,14-arachidonic acid.  10. Biomediator according to one of claims 7 to 9, characterized in that it further comprises at least one calcium chelating agent with peptide or pseudopetide structure, of which One of the amino acids is proline, preferably with a molecular weight of less than about 1,000.  11. Biomediator according to claim 10, characterized in that the chelating agent is a di- or a tripeptide.  12. Biomediator according to claim 11, characterized in that the aforementioned di- or tripeptide is chosen from the group consisting of cysteyl-proline, p-glutamyL-proline, glycyl-p-glutamyl-proline and diketopiperazine Proline.  13. Biomediator according to one of claims 7 to 12, characterized in that the aforementioned calcium complex is contained in phospholipid or glycophospholipid form in a natural extract from animal or plant organisms.  14. Biomediation agent according to claim 13, characterized in that the natural plant extract is an extract of plants belonging to the families of Cruciferae, Lineas or Resedaceae.  15. Biomediating agent as claimed in claim 13, charac terized in that The above-mentioned animal organ extract is an extract of fish belonging to the families of Clupeidae, Gadidae, Salmonidae or Scombridae.  16. Pharmaceutical composition, characterized in that it comprises, as active principle, a calcium complex as defined in any one of claims I to 6, or a biomedical agent as defined in any one of claims 7 at 15.  17. Pharmaceutical composition according to claim 16, characterized in that the calcium complex is present at a concentration of 0.1 to 5 in percentage by weight relative to the total weight of the composition.  18. Pharmaceutical composition, characterized in that it comprises, as active principle, a natural extract derived from animal or plant organisms, preferably respectively chosen from fish extracts belonging to the families of Clupeidae, Gadidae, Salmonidae or Scombridae, or extracts of plants belonging to the families of crucifers, Linear or Resedaceous, said natural extract from animal or plant organisms, containing a calcium complex as defined according to any one of claims 1 to 6.  19. Pharmaceutical composition according to one of claims 16 to 18, characterized in that it is a pharmaceutical composition with analgesic activity, preferably formulated for pernasal administration.  20. Pharmaceutical composition according to one of claims 16 to 18, characterized in that it is a pharmaceutical composition with antipyretic activity, preferably formulated for oral administration.  21. Pharmaceutical composition according to one of claims 16 to 18, characterized in that it is a pharmaceutical composition with anti-inflammatory activity, preferably formulated for oral administration.  22. Pharmaceutical composition according to one of claims 16 to 18, characterized in that it is a composition with myotonic activity, preferably formulated for topical administration, in particular in the form of a gel.  23. Pharmaceutical composition according to one of claims 16 to 18, characterized in that it is a pharmaceutical composition with cardiotonic activity, having a positive inotropic activity without associated positive chronotropic effect, preferably formulated for percutaneous administration. .  24. Pharmaceutical composition according to one of claims 16 to 18, characterized in that it is in the form of capsules dosed with 50 mg of extract or active principle, these capsules being preferably made gastro-resistant.  25. Extract of plants or parts of plants, characterized in that it is obtained from plants belonging to the families of Crciferous, Lineae or Resedaceae and in that it contains a calcium complex as defined according to L any of claims 1 to 6.  26. Extract from animal organs, characterized in that it is obtained from fish belonging to the families of Clupeidae, Gadidae, Salmonidae or Scombridae and in that it contains a calcium complex as defined in any one of claims 1 to 6.  27. Process for preparing extract of plants or parts of plants or organs of animals, as defined in claim 25 or 26, characterized in that it is an aqueous or organic extract which results from the action of an aqueous or organic solvent or a mixture of such solvents on a ground material of plants or flesh, optionally followed by concentration and recovery by an appropriate means of the dry solute, such as by example by lyophilization or nebulization, and in that the extracts are subjected to direct or indirect calcification, the extracts having preferably been discolored and / or deodorized.