Compositions of at least 3 organic salts of magnesium to treat disorders of magnesium deficiency such as muscle cramps

Abstract

Described is a composition comprising at least three organic salts of magnesium, preferably magnesium pidolate, magnesium gluconate and magnesium citrate. The invention also relates to the medical use of the composition and the use of the composition as a food supplement, for improving, increasing and / or aiding the absorption and / or bioavailability of magnesium in an individual, preferably an individual of paediatric age.

Description

[0001] DESCRIPTION

[0002] TITLE COMPOSITIONS OF AT LEAST 3 ORGANIC SALTS OF MAGNESIUM TO TREAT DISORDERS OF MAGNESIUM DEFICIENCY SUCH AS MUSCLE CRAMPS

[0003] Technical field

[0004] The present invention relates to a composition comprising at least three organic salts of magnesium.

[0005] Background art

[0006] Magnesium is an essential mineral involved in over 300 biochemical reactions in the human body. It plays a crucial role in energy production, protein synthesis, blood pressure regulation, and muscle and nerve function. When magnesium levels in the blood drop below normal, it is called hypomagnesemia. This condition can have a significant impact on overall health, affecting numerous physiological processes and potentially leading to a range of symptoms and complications.

[0007] Magnesium deficiency in the blood can be attributed to a variety of factors, which are often interconnected. One of the most common causes is insufficient intake through diet, particularly in societies where the consumption of processed foods is high at the expense of magnesium-rich foods such as leafy greens, nuts, seeds and whole grains.

[0008] Children are often prone to dehydration resulting in loss of mineral salts, including magnesium.

[0009] Lifestyle, stress and continuous movement result in children perspiring more than an adult. In order to prevent a child from becoming dehydrated, it is essential to pay attention to everything that causes a loss of body water and to provide an adequate intake of both water and mineral salts, including magnesium, which are important substances for maintaining a well hydrated body. The correct amount of magnesium helps children in their development thanks to its various benefits including: support of the psychological function with help in the quality of sleep, improvement of mood, concentration,correct development of muscle function, as an aid to strengthening bones and teeth and especially at the energy level.

[0010] However, it is not always possible for foods rich in magnesium to be included in the diet of children in sufficient quantities, and sometimes the amounts of magnesium in foods are also low, due to food processing.

[0011] Summary of the invention

[0012] The invention relates to a composition comprising at least three organic salts of magnesium, where said three organic salts of magnesium are selected between: magnesium pidolate, magnesium gluconate, magnesium citrate, magnesium carbonate, magnesium chloride, magnesium fumarate, magnesium glycinate, magnesium L-lactate, magnesium DL-aspartate, magnesium L-aspartate, magnesium salicylate, magnesium sulphate, magnesium aminoate, magnesium phosphate, magnesium acetate, magnesium malate or magnesium picolinate.

[0013] Preferably, the at least three organic magnesium salts are magnesium pidolate, magnesium gluconate, magnesium citrate.

[0014] In one embodiment, the pidolate magnesium is present in a concentration of between 35% and 75% weight / weight (w / w), the magnesium gluconate is present in a concentration of between 20% and 50% w / w, and the magnesium citrate is present in a concentration of between 0.1% and 10% w / w.

[0015] In another embodiment, the pidolate magnesium is present in a concentration of between 40% and 70% weight / weight (w / w), the magnesium gluconate is present in a concentration of between 30% and 45% w / w, and the magnesium citrate is present in a concentration of between 0.5% and 5% w / w.

[0016] In a further embodiment, the pidolate magnesium is present in a concentration of between 50% and 65% w / w (w / w), the magnesium gluconate is present in a concentration of between 30% and 40% w / w, andthe magnesium citrate is present in a concentration of between 1% and 4% w / w.

[0017] Preferably, the composition is formulated for an oral administration, preferably as a pill, tablet, granules, powder in sachet, lozenge, capsule, operculum, syrup, drinkable vial.

[0018] Furthermore, the present invention relates to the above-described composition for use as a medication, preferably for use in the treatment of a pathology or disorder caused by an insufficiency of magnesium levels in an individual.

[0019] Preferably, the individual is of paediatric age, that is, the individual is less than eighteen years of age.

[0020] According to an embodiment, the pathology or disorder caused by an insufficiency of magnesium is selected between: migraine, muscle cramps, sleep disturbances, anxiety, depression, mood variability, osteo-tendon hyperreflexia, muscle incoordination, tremors, paraesthesia, cardiac arrhythmia and arterial hypertension.

[0021] Lastly, the invention relates to the use of the above-described composition as a food supplement, for improving, increasing and / or aiding the absorption and / or bioavailability of magnesium in an individual, preferably in an individual of paediatric age.

[0022] Brief description of the drawings

[0023] Figure 1 shows the bioaccessibility (A) and excreted fraction (B) of magnesium expressed as a percentage (%) of the total amount of magnesium measured at the end of the (complete) digestive process. The results are expressed as mean ± standard deviation.

[0024] Figure 2 shows the viability of intestinal epithelia exposed to digestive fluids in the absence of formulation (0%) and to increasing amounts of FORMULA 1 (A), FORMULA 2 (B), FORMULA 3 (C) and FORMULA 4 (D) for 3 hoursexpressed as a percentage (%) relative to the negative control. The results are expressed as mean ± standard deviation.

[0025] Figure 3 shows TEER of intestinal epithelia exposed to digestive fluids in the absence of formulation (0%) and to increasing amounts of FORMULA 1 (A), FORMULA 2 (B), FORMULA 3 (C) and FORMULA 4 (D) for 3 hours expressed as a percentage (%) relative to the negative control. The results are expressed as mean ± standard deviation.

[0026] Figure 4 shows the bioavailability of magnesium following exposure of the intestinal epithelium to the formulations for 3 hours expressed as a percentage (%) of the amount of magnesium applied to the intestinal epithelium (A) and as apparent permeability (B). The results are expressed as mean ± standard deviation.

[0027] Figure 5 shows the viability (A), apparent permeability (Papp) (B) and TEER % (C) of the intestinal epithelia exposed for 3 hours to the bioaccessible fraction of the formulations. The results are expressed as mean ± standard deviation.

[0028] Detailed description of preferred embodiments of the invention

[0029] A first aspect of the present invention relates to a composition comprising organic salts of magnesium.

[0030] In one embodiment the composition comprises at least three organic magnesium salts. Preferably, said at least three organic salts of magnesium are selected between: magnesium pidolate, magnesium gluconate, magnesium citrate, magnesium carbonate, magnesium chloride, magnesium fumarate, magnesium glycinate, magnesium L-lactate, magnesium DL-aspartate, magnesium L-aspartate, magnesium salicylate, magnesium sulphate, magnesium aminoate, magnesium phosphate, magnesium acetate, magnesium malate or magnesium picolinate.

[0031] In one embodiment the at least three organic magnesium salts are magnesium pidolate, magnesium gluconate and magnesium citrate. Preferably, the composition comprises magnesium pidolate, magnesiumgluconate and magnesium citrate.

[0032] The composition described above preferably comprises the at least three organic magnesium salts in ratios and / or percentages that allow an increased intestinal absorption of magnesium.

[0033] In one embodiment, the composition comprises pidolate magnesium in a concentration ranging from 35% to 75% weight / weight (w / w) relative to the weight of the composition, preferably in a concentration ranging from 40% to 70% w / w, more preferably ranging from 50% to 65% w / w.

[0034] In one embodiment, the composition comprises magnesium gluconate in a concentration ranging from 20% to 50% weight / weight (w / w) relative to the weight of the composition, preferably in a concentration ranging from 30% to 45% w / w, more preferably ranging from 30% to 40% w / w.

[0035] In one embodiment, the composition comprises magnesium citrate in a concentration ranging from 0.1% to 10% weight / weight (w / w) relative to the weight of the composition, preferably in a concentration ranging from 0.5% to 5% w / w, more preferably ranging from 1% to 4% w / w.

[0036] In one embodiment, the composition comprises:

[0037] pidolate magnesium in a concentration of between 35% and 75% weight / weight (w / w) relative to the weight of the composition, magnesium gluconate in a concentration of between 20% and 50% w / w, and magnesium citrate in a concentration of between 0.1% and 10% w / w. Preferably, the composition comprises

[0038] pidolate magnesium in a concentration of between 40% and 70% w / w, magnesium gluconate in a concentration of between 30% and 45% w / w, and magnesium citrate in a concentration of between 0.5% and 5% w / w. In a preferred embodiment of the invention, the composition comprises pidolate magnesium in a concentration of between 50% and 65% w / w, magnesium gluconate in a concentration of between 30% and 40% w / w, and magnesium citrate in a concentration between 1% and 4% w / w.

[0039] The composition according to the present invention may further comprise at least one excipient acceptable for pharmaceutical use, food use(including food supplements) or cosmetic use, which is useful in the preparation of the composition and which is generally biologically safe and non-toxic.

[0040] Preferably, the composition comprises at least one solvent, preferably at least one aqueous solvent, more preferably water.

[0041] Furthermore, the composition preferably comprises at least one sweetening agent, preferably selected from: glucose, fructose, mannose, maltodextrins and stevia. Preferably, the sweetening agent is fructose. Furthermore, the composition preferably comprises at least one sweetener, preferably selected from: erythritol, aspartame, sucralose.

[0042] In a preferred form of the invention, the sweetener is sucralose.

[0043] Preferably, the composition comprises at least one acidifying agent, more preferably selected from citric acid, acetic acid, hydrochloric acid and phosphoric acid. In a preferred form of the invention, the at least one acidifying agent is citric acid.

[0044] In one embodiment, the composition is formulated for oral administration. Preferably, the composition is formulated as a tablet, lozenge, granules, sachet powder, tablet, capsule, operculum, syrup, drinkable vial.

[0045] Alternatively, the composition is formulated as a beverage. The beverage may be water-based and / or fruit-based and / or milk-based. In the particularly preferred embodiment of the invention the beverage is fruit-based, preferably orange-based. If necessary, the drink can be freeze-dried.

[0046] According to a further embodiment of the invention, said composition further comprises a flavouring agent preferably selected from: essential oils, flavours or synthetic mixtures, including but not limited to flavours derived from plants and fruits such as citrus fruits, fruit essences, peppermint oil, mint oil, other mint oils, clove oils, sugar oil, anise and the like, aromatic oils with germ-killing properties such as menthol, eucalyptus, thymol, spices such as cocoa powder and cinnamon powder, as flavouring agents and combinations thereof.

[0047] A second aspect of the present invention relates to the compositiondescribed in detail above for use as a medication.

[0048] A third aspect of the present invention relates to the composition described in detail above for use in the treatment of a pathology or disorder caused by an insufficiency of magnesium levels in an individual. Preferably, the composition is administered to or taken by an individual of paediatric age, preferably by an individual of less than eighteen years of age.

[0049] In one embodiment, the individual has normal blood levels of magnesium, preferably between 1.5 and 2.55 mg / dL.

[0050] In another embodiment, the individual has blood levels of magnesium slightly below normal, that is, is suffering from mild hypomagnesemia, preferably with blood values of magnesium of between 1.2 and 1.7 mg / dL. In a further embodiment, the individual has blood levels of magnesium below normal, that is, is suffering from moderate hypomagnesemia, preferably with blood values of magnesium of between 0.8 and 1.2 mg / dL. In a further embodiment, the individual has blood magnesium levels below normal, that is, is suffering from severe hypomagnesemia, preferably with blood magnesium values of less than 0.8 mg / dL.

[0051] According to an embodiment, the pathology or disorder caused by an insufficiency of magnesium is selected between: migraine, muscle cramps, sleep disturbances, anxiety, depression, mood variability, osteo-tendon hyperreflexia, muscle incoordination, tremors, paraesthesia, cardiac arrhythmia and arterial hypertension.

[0052] Preferably, the composition is taken at least once a day by the individual, more preferably at least twice a day or as needed.

[0053] Practically, the composition may be taken as a dietary supplement, in particular for the purpose of increasing the absorption and / or bioavailability of magnesium in an individual.

[0054] In particular, the invention relates to the use of the composition described in detail above as a dietary supplement, preferably by individuals of a paediatric age, to improve, increase and / or aid the absorption and / or bioavailability of magnesium.Preferably, the invention relates to a non-medical use of the composition described in detail above, more preferably as a food supplement.

[0055] In one embodiment, the composition, for use as a dietary supplement, is taken in combination or in combination with a balanced diet, preferably with a magnesium-rich diet.

[0056] The invention also relates to a method for the treatment of a pathology or disorder caused by magnesium insufficiency. Preferably, said method comprises at least one step of administering the composition described in detail above to an individual in need thereof.

[0057] Preferably, the pathology or disorder caused by an insufficiency of magnesium is selected between: migraine, muscle cramps, sleep disturbances, anxiety, depression, mood variability, osteo-tendon hyperreflexia, muscle incoordination, tremors, paraesthesia, cardiac arrhythmia and arterial hypertension.

[0058] In one embodiment, the individual has normal blood levels of magnesium, preferably between 1.5 and 2.55 mg / dL.

[0059] In another embodiment, the individual has blood levels of magnesium slightly below normal, that is, is suffering from mild hypomagnesemia, preferably with blood values of magnesium of between 1.2 and 1.7 mg / dL. In a further embodiment, the individual has blood levels of magnesium below normal, that is, is suffering from moderate hypomagnesemia, preferably with blood values of magnesium of between 0.8 and 1.2 mg / dL. In a further embodiment, the individual has blood magnesium levels below normal, that is, is suffering from severe hypomagnesemia, preferably with blood magnesium values of less than 0.8 mg / dL.

[0060] EXAMPLE

[0061] In order to evaluate the bioaccessibility and intestinal absorption of magnesium, 4 formulations have been tested:

[0062] Formulation 1 : 80 mg of Magnesium from an innovative mix of Organic Magnesium Salts (75% Pidolate, 2.5% Citrate, 22.5% Gluconate)in a 10 ml vial.

[0063] Formulation 2: 40 mg of Magnesium Citrate (100%).

[0064] Formulation 3: 80 mg Magnesium Pidolate (100%).

[0065] Formulation 4: 40 mg Magnesium Gluconate (100%).

[0066] Determination of the magnesium content in the formulations

[0067] The magnesium content of the formulations was determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis (Nexlon 300D, PerkinElmer). Briefly, 0.5 mL of each formulation was transferred to centrifuge tubes and 1 mL of nitric acid (HNO3, Romil) was added. The samples were then kept at 80°C for 8 hours and, at the end of the digestion, the digest was transferred into a 50 mL vial and brought to a final volume of 50 mL using ultrapure water. The amount of magnesium measured was compared with that declared and the magnesium recovery was calculated as a ratio between these two amounts.

[0068] Digestive process

[0069] An quantity of formulation corresponding to 80 mg of magnesium was subjected to an in vitro digestive process. The latter is designed to simulate the human digestive process (divided into the oral, gastric and intestinal compartments) and is based on the use of fluids simulating the physiological secretions typical of the digestive process (saliva, gastric juices, intestinal juices and bile). As the formulations were in liquid form, the oral phase was removed. In order to avoid any possible contamination and interference, the Mg contributions deriving from the simulating digestive fluids and the digestive process were carefully removed. The formulation was weighed and to this was added the fluid simulating the gastric juices (pH 1.3) and the pH was adjusted to 2.5. The resulting mixture was kept under constant stirring at 37°C for 2 hours. Subsequently, the fluid simulating the intestinal juices (pH 8.1) and the sodium bicarbonate were added and the pH was adjusted to a value of 6.5. The resulting mixture was kept under constantstirring at 37°C for another 2 hours. At the end of the digestive process, the magnesium concentration was measured in the complete digests and compared with the applied dose to estimate the overall recovery of the process. After a centrifugation phase at 2750 g for 5 min1, magnesium was quantified in the pellet, equivalent to the excreted fraction, and in the supernatant, corresponding to the bioaccessible fraction. A quantity of 0.5 mL of each sample was transferred into a vial, and 1 mL of HNO3 was added thereto. The samples were then kept at 80°C for 8 hours and, at the end of the digestion process, they were transferred into a vial and brought to a final volume of 50 mL using ultrapure water. Three independent replicates were prepared and analysed by ICP-MS for each sample. The method has an estimated Limit of Determination (LOD) and Limit of Quantitation (LOQ) of 0.07 pg / g and 0.12 pg / g, respectively.

[0070] In vitro model of the intestinal epithelium

[0071] The intestinal absorption of magnesium present in the formulations was determined using an in vitro human intestinal epithelium model, based on human Caco-2 intestinal adenocarcinoma cells (ATCC, HTB-37TM) organised as afunctional monolayer on Transwell® inserts. The Transwell® inserts are characterised by two compartments separated by a microporous membrane: apical (or luminal) and basolateral (or serosal). The Caco-2 cell monolayers grown on microporous membranes consist of polarised cells with morpho-functional characteristics typical of enterocytes, such as the presence of microvilli, tight junctions and p-glycoprotein (P-gp).

[0072] Evaluation of the impact of formulations on the viability of the intestinal

[0073] The impact of the digested formulations on the viability of the intestinal epithelium was evaluated by dose-response curve. Following the digestive process, the supernatant (bioaccessible fraction) resulting from the digestion of the formulations was diluted in digestive fluids. The resultingbioaccessible fraction concentrations (0 to 100%) were added to the apical compartment of the intestinal epithelium in vitro, while the magnesium-free Hank's Balanced Salt Solution (HBSS) was added to the basolateral compartment. As a negative control, digestive fluids were used in the absence of the active ingredient. After 3 hours of incubation, the viability of the intestinal epithelia was assessed by MTS assay. The MTS assay is based on the reduction of the MTS tetrazolium compound by the viable cells to generate the coloured formazan product, quantifiable by measuring its absorbance at 490 nm. In parallel, the impact of the bioaccessible fraction of the formulations analysed on the barrier integrity of the intestinal epithelium was evaluated by measuring the transepithelial electrical resistance (TEER).

[0074] Assessment of intestinal

[0075]

[0076] At the end of the digestive process, the maximum non-toxic concentration of the bioaccessible fraction of the formulations was added to the apical compartment of the intestinal epithelium in vitro, while magnesium-free HBSS was added to the basolateral compartment. Digestive fluids without the active ingredient were used as a negative control. After 3 hours of exposure, the apical and basolateral fractions were recovered and their magnesium content was determined by ICP-MS analysis. The magnesium content was also measured inside the cells. The bioavailability is reported as a percentage (%) of magnesium absorbed with respect to the amount applied to the apical compartment and as apparent permeability (Papp) and calculated with the following formulas:

[0077] Bioavailability (%) = (amount of magnesium (mg) in the basolateral fraction) I (amount of magnesium (mg) applied to the apical compartment) x 100

[0078] Apparent Permeability (Papp) = ( C V) / ( tA Co)where AC / At is the flow of magnesium transported across the monolayer during the incubation time (mM / s), V is the volume of the basolateral compartment (cm3), A is the membrane area (cm2), Co is the initial magnesium concentration applied to the apical compartment.

[0079] and integrity of the barrier of the intestinal epithelium model

[0080] At the end of absorption, the cellular viability and barrier integrity of the intestinal epithelium model were assessed. The cell viability was assessed using the MTS assay, while the barrier integrity was assessed by measuring the transepithelial electrical resistance (TEER) of the cell monolayer and assessing its permeability to Lucifer Yellow, a polar tracer which is unable to permeate through intact tight junctions. The apparent permeability coefficient (Papp, cm / min) was calculated with the following formula:

[0081] Papp = (AC 7) / (A CO)

[0082] where AC / At is the flow of the molecule transported across the monolayer during the incubation time (mM / min), V is the volume of the basolateral compartment (cm3), A is the membrane area (cm2), Co is the initial concentration of the Lucifer Yellow molecule in the apical compartment.

[0083] Statistical

[0084]

[0085] All the data are presented as mean ± standard deviation (SD) of three independent experiments. In order to determine if there were statistically significant differences between the various conditions, a T-test analysis was performed. The T-test is a statistical method used to test the differences between the means of a series. The differences between groups were considered significant at p < 0.05. All the statistical analyses were performed with OriginLab software.RESULTS

[0086] Determination of the magnesium content in the formulations

[0087] The magnesium content was measured in the formulations and the values obtained are comparable with those declared. The recovery percentage was calculated, for each formulation, as the ratio of the measured magnesium value to the nominal magnesium value. As shown in Table a, the measured magnesium concentration is compatible with that shown with a recovery of 85.80%, 99.96%, 86.10% and 91.56% for FORMULA 1 , FORMULA 2, FORMULA 3 and FORMULA 4, respectively.

[0088] Table 1 Comparison between measured titer and declared titer of the magnesium-based formulations.

[0089] Declared Titer Measured Titer Recovery (pg / g) (pg / g) (%) FORMULA 1 8000 6864.09 ± 205.74 85.80 ± 0.02% FORMULA 2 4000 3998.52 ± 38.82% 99.96 ± 0.01 FORMULA 3 8000 6887.81 ± 86.10 ± 0.02

[0090] 203.91%

[0091] FORMULA 4 4000 3662.52 ± 57.83 91 .56 ± 0.01

[0092]

[0093] Release of magnesium from the formulations - bioaccessible fraction The bioaccessibility refers to the quantity of active ingredient released from magnesium-based formulations in the gastrointestinal tract and available for intestinal absorption. In order to determine the bioaccessibility of the magnesium, an amount of each formulation equal to 80 mg of magnesium was subjected to the in vitro digestive process. After digestion, three fractions were collected: (i) the complete in order to evaluate the overall recovery of the process; (ii) the supernatant corresponding to the bioaccessible fraction; and (iii) the pellet corresponding to the excreted fraction. The digestion has no impact on the magnesium contained in the formulations as the overall recovery of the process is 101.56 ± 1.27%, 99.97 ± 1.67%, 114.36 ± 2.21 % and 102.64 ± 1.85% for FORMULA 1 , FORMULA 2, FORMULA 3 and FORMULA 4, respectively. As shown in Figure 1 andindicated in Table 2, magnesium is fully released from the formulations with a bioaccessibility value of 100.61 ± 1.68%, 99.40 ±3.92%, 97.62 ± 2.24% and 97.96 ± 3.29% for FORMULA 1 , FORMULA 2, FORMULA 3 and FORMULA 4. The magnesium measured in the excreted fraction represents only 0.020 ± 0.001 % (FORMULA 1 ), 0.008 ± 0.005% (FORMULA 2), 0.032 ± 0.005% (FORMULA 3), and 0.036 ± 0.003% (FORMULA 4) relative to the total magnesium measured at the end of the (complete) digestive process.

[0094] Table 2. Bioaccessibility and excreted fraction of the magnesium expressed as a percentage (%) of the total amount of magnesium measured at the end of the (complete) digestive process. The results are expressed as mean ± standard deviation.

[0095] Formulation Bioaccessibility (%) Excreted fraction (%) FORMULA 1 100.61 ± 1.68 0.020 ± 0.001 FORMULA 2 99.40 ± 3.92 0.008 ± 0.005% FORMULA S 97.62 ± 2.24 0.032 ± 0.005 FORMULA 4 97.96 ± 3.29% 0.036 ± 0.003

[0096]

[0097] Evaluation of the impact of formulations on the viability of the intestinal epithelium

[0098] Before evaluating the bioavailability of magnesium, the impact of the bioaccessible fraction of the formulation on the viability of the intestinal mucosa was evaluated. The Caco-2 monolayers were exposed for 3 hours to increasing amounts of the formulations and the results are shown in Figure 2 and Table 3. The dose response shows that the undiluted bioaccessible fraction (100%) of FORMULA 1 and FORMULA 3 causes a significant reduction in the viability of the intestinal epithelium (Figure 2A, C). Following treatment with FORMULA 2 and FORMULA 4, an impact on cell viability up to the 50% concentration of the bioaccessible fraction is observed (Figure 2B, D). In addition, the evaluation of integrity of the epithelial membrane was also performed by measurement of the TEER andthe results are shown in Figure 3 and Tables 4, 5, 6, 7.

[0099] Combining the vitality and membrane integrity data, for the evaluation of the magnesium absorption in the intestine, the Caco-2 monolayer was exposed to 50% of the bioaccessible fraction of FORMULA 1 and 25% of the bioaccessible fraction for the other 3 formulations.

[0100] Table 3. Vitality of intestinal epithelia exposed to digestive fluids in the absence of formulation (0%) and to increasing amounts of the formulations for 3 hours expressed as a percentage (%) relative to the negative control. The results are expressed as mean ± standard deviation.

[0101] Bioaccessible FORMULA 1 FORMULA 2 FORMULA 3 FORMULA 4 fraction Vitality Vitality Vitality Vitality (%) (%) (%) (%) (%)

[0102] NT (0) 100.00 ± 100.00 ± 100.00 ± 100.00 ±

[0103] 2.46 0.13 2.72 0.72

[0104] 1 95.18 ± 0.76 101.72 ± 98.99 ± 1.68 95.52 ± 0.67

[0105] 0.29

[0106] 2 96.66 ± 1.36 100.17 ± 97.76 ± 1.89 101.70 ±

[0107] 0.34 0.84

[0108] 3.3 95.06 ± 1.83 95.57 ± 3.17 94.57 ± 0.57 98.26 ± 3.98 5 96.16 ± 1.24 98.23 ± 0.58 98.25 ± 1.79 96.85 ± 1.01 6,7 91.86 ± 0.67 93.52 ± 0.45 92.03 ± 1.20 93.92 ± 4.74 10 89.41 ± 3.30 95.16 ± 2.33 89.78 ± 2.16 92.10 ± 1.86 12.5 94.91 ± 1.49 97.73 ± 2.78 90.27 ± 2.78 100.43 ±1.07 16.7 101.93 ± 100.12 ± 100.93 ± 94.12 ± 1.25

[0109] 2.58 1.26 0.63

[0110] 25 99.56 ± 1.29 89.13 ± 0.82 95.99 ± 0.55 90.52 ± 0.48 50 83.52 ± 0.66 24.28 ± 0.79 76.48 ± 1.41 29.07 ± 0.39 100 40.15 ± 0.22 0.00 ± 0.12 22.00 ± 0.29 0.00 ± 0.52

[0111]

[0112] Table 4. TEER % of intestinal epithelia exposed to digestive fluids in the absence of formulation (0%) and to increasing amounts of FORMULA 1 for3 hours expressed as a percentage (%) relative to the negative control. The results are expressed as mean ± standard deviation.

[0113] Bioaccessible TEER (%) TEER (%) TEER (%)

[0114] fraction PrePost24h recovery

[0115] (%) treatment treatment

[0116] NT (0) 100.00 ± 2.09 100.00 ± 2.09 100.00 ± 2.09

[0117] 1 100.00 ± 2.09 57.6 ± 0.00 130.40 ± 1.13

[0118] 2 100.00 ±1.56 51.29 ± 0.52 118.45 ± 1.56

[0119] 3.3 100.00 ± 0.00 51.16 ± 0.00 124.42 ± 1.64

[0120] 5 100.00 ± 0.98 38.54 ± 1.47 117.71 ± 0.49

[0121] 6,7 100.00 ± 1.00 35.81 ± 0.50 130.14 ± 0.50

[0122] 10 100.00 ± 0.49 28.57 ± 0.98 128.57 ± 1.48

[0123] 12.5 100.00 ± 1.01 29.50 ± 1.02 141.73 ± 0.00

[0124] 16.7 100.00 ± 0.48 25.76 ± 1.92 136.27 ± 0.00

[0125] 25 100.00 ±0.46 20.06 ± 0.91 162.13 ± 0.46

[0126] 50 100.00 ± 0.96 18.71 ± 1.44 116.67 ± 1.44

[0127] 100 100.00 ± 1.34 18.30 ± 0.89 17.03 ± 0.89

[0128]

[0129] Table 5. TEER % of intestinal epithelia exposed to digestive fluids in the absence of formulation (0%) and to increasing amounts of FORMULA 2 for 3 hours expressed as a percentage (%) relative to the negative control. The results are expressed as mean ± standard deviation.

[0130] Bioaccessible TEER (%) TEER (%) TEER (%)

[0131] fraction PrePost24h recovery

[0132] (%) treatment treatment

[0133] NT (0) 100.00 ± 1.56 63.84 ± 0.52 101 .48 ± 0.52

[0134] 1 100.00 ± 1.49 49.12 ± 1.98 103.51 ± 0.50

[0135] 2 100.00 ± 0.00 46.53 ± 0.98 104.51 ± 1.47

[0136] 3.3 100.00 ± 0.00 48.90 ± 1.03 108.76 ± 1.03

[0137] 5 100.00 ± 0.49 32.18 ± 1.47 114.88 ± 0.00

[0138] 6,7 100.00 ± 1.49 28.07 ± 0.99 128.07 ± 0.47

[0139]

[0140] 10 100.00 ± 0.48 23.39 ± 0.48 145.08 ± 0.96

[0141] 12.5 100.00 ± 0.97 23.10 ± 0.49 151.72 ± 0.97

[0142] 16.7 100.00 ± 1.00 24.11 ± 2.00 87.23 ± 2.00

[0143] 25 100.00 ± 0.45 20.50 ± 0.45 160.57 ± 1.34

[0144] 50 100.00 ± 1.99 17.60 ± 2.99 19.01 ± 0.99

[0145] 100 100.00 ± 0.45 100.00 ± 0.45 100.00 ± 0.45

[0146]

[0147] Table 6. TEER % of intestinal epithelia exposed to digestive fluids in the absence of formulation (0%) and to increasing amounts of FORMULA 3 for 3 hours expressed as a percentage (%) relative to the negative control. The results are expressed as mean ± standard deviation.

[0148] Bioaccessible TEER (%) TEER (%) TEER (%)

[0149] fraction PrePost24h recovery

[0150] (%) treatment treatment

[0151] NT (0) 100.00 ± 2.65 55.81 ± 1.59 106.74 ± 0.53

[0152] 1 100.00 ± 0.50 53.68 ± 1.49 112.63 ± 0.50

[0153] 2 100.00 ±0.96 51.69 ± 0.48 108.11 ± 1.91

[0154] 3.3 100.00 ± 1.50 59.01 ± 0.50 107.77 ± 0.50

[0155] 5 100.00 ± 1.91 43.24 ± 0.96 116.89 ± 0.96

[0156] 6,7 100.00 ± 0.49 37.98 ± 1.48 133.24 ± 0.00

[0157] 10 100.00 ± 0.47 29.24 ± 0.00 132.89 ± 0.00

[0158] 12.5 100.00 ± 0.45 25.71 ± 0.45 129.21 ± 0.45

[0159] 16.7 100.00 ± 1.40 27.72 ± 1.87 146.53 ± 0.93

[0160] 25 100.00 ± 0.90 23.25 ± 0.45 165.61 ± 2.70

[0161] 50 100.00 ± 0.92 24.51 ± 0.46 29.08 ± 1.39

[0162] 100 100.00 ± 0.94 24.50 ± 0.94 17.88 ± 0.94

[0163]

[0164] Table 7. TEER % of intestinal epithelia exposed to digestive fluids in the absence of formulation (0%) and to increasing amounts of FORMULA 4 for 3 hours expressed as a percentage (%) relative to the negative control. The results are expressed as mean ± standard deviation.Bioaccessible TEER (%) TEER (%) TEER (%)

[0165] fraction PrePost24h recovery

[0166] (%) treatment treatment

[0167] NT (0) 100.00 ± 2.21 66.41 ± 1.10 107.42 ± 0.55

[0168] 1 100.00 ± 1.00 49.65 ± 2.01 108.87 ± 0.50

[0169] 2 100.00 ±0.00 48.57 ± 1.01 105.36 ± 0.51

[0170] 3.3 100.00 ± 0.00 70.66 ± 2.92 105.37 ± 0.58

[0171] 5 100.00 ± 0.52 35.16 ± 0.00 35.16 ± 0.00

[0172] 6,7 35.16 ± 0.00 35.16 ± 0.00 35.16 ± 0.00

[0173] 10 100.00 ±0.52 24.72 ± 2.61 167.16 ± 0.52

[0174] 12.5 100.00 ± 0.49 23.37 ± 0.97 158.76 ± 0.00

[0175] 16.7 100.00 ± 0.92 21.24 ± 1.39 168.95 ± 1.39

[0176] 25 100.00 ±0.00 22.15 ± 0.95 128.52 ± 1.42

[0177] 50 100.00 ± 0.00 21.43 ± 1.01 19.29 ± 1.01

[0178] 100 19.29 ± 1.01 19.29 ± 1.01 19.29 ± 1.01

[0179]

[0180] Assessment of intestinal magnesium absorption

[0181] In order to assess the magnesium absorption, the intestinal epithelia were exposed for 3 hours to 50% concentration of the bioaccessible fraction of FORMULA 1 and 25% concentration of the bioaccessible fraction of FORMULA 2, FORMULA 3, and FORMULA 4. At the end of the incubation, the magnesium was quantified in the apical fraction (fraction applied on the epithelium), in the basolateral fraction (fraction absorbed) and in the intracellular fraction in order to evaluate the amount of magnesium retained by the intestinal epithelium cells. The percentage absorption of magnesium after 3 hours of exposure is shown in Figure 4A and Table 8. FORMULA 4 has the highest percentage bioavailability value compared to the other formulations and this difference is statistically significant (3.74 ± 0.33% for FORMULA 4 vs 2.78 ± 0.31%, 2.07 ± 0.02% and 2.18 ± 0.02% for FORMULA 1 , FORMULA 2 and FORMULA 3, respectively). The statistical analysis also showed a significant difference between FORMULA 2 and FORMULA 3 (2.07 ± 0.02% vs 2.18 ± 0.02%, respectively). The same trendis observed for the apparent permeability (Figure 4B and Table 8). Table 9 shows the p-value values derived from the T-test for comparison between the different formulations. A p-value of less than 0.05 (represented in the table by a *) indicates a statistically significant difference between the formulations. In addition to the fraction of active ingredient absorbed by the intestinal epithelium, the magnesium was also quantified within the cells. The amount of active ingredient measured in the intracellular compartment after 3 hours of exposure is less than the LOQ (0.07 pg / g), suggesting that the magnesium is not retained by the cells but passes directly into the basolateral compartment.

[0182] Table 8. Bioavailability of the magnesium following exposure of the intestinal epithelium to the formulation for 3 hours expressed as a percentage (%) of the amount of magnesium applied to the intestinal epithelium and as apparent permeability. The results are expressed as mean ± standard deviation.

[0183] Formulation Formulation Papp (x 10'6cm / s) FORMULA 1 2.78 ± 0.31 1.69 ± 0.18 FORMULA 2 2.07 ± 0.02 1.25 ± 0.01 FORMULA S 2.18 ± 0.02 1.32 ± 0.01 FORMULA 4 3.74 ± 0.33 2.31 ± 0.12

[0184]

[0185] Table 9. P-value values calculated by T-test for comparison between the different formulations. *Statistically significant difference, p < 0.05

[0186] p-value

[0187] Formulation vs vs vs vs FROMULA 1 FROMULA 2 FROMULA 3 FROMULA 4 FORMULA 1 - 0.056 0.078 0.021* FORMULA 2 0.056 - 0.010* 0.013* FORMULAS 0.078 0.010* - 0.015* FORMULA 4 0.021* 0.013* 0.015* -

[0188]

[0189] Vitality and integrity of the barrier of the intestinal epithelium model Parallel to the evaluation of the absorption, the impact of the formulations on the viability and integrity of the intestinal epithelium barrier was monitored. No significant alterations in the viability (Figure 5A) and apparent permeability (Figure 5B and 5C) of the intestinal epithelium were observed following treatment with the maximum non-toxic concentration of the bioaccessible fraction of each formulation.

[0190] Conclusions

[0191] Based on the results obtained, the four magnesium-based formulations are characterised by a high bioaccessibility (about 100%) (the highest score was obtained from Formulation 1). The absorption of the magnesium released from FORMULA 4 is significantly higher than the other 3 formulations and has a value of 3.74 ± 0.33% with respect to the amount of magnesium applied to the intestinal epithelium, corresponding to an apparent permeability value of 2.31 ± 0.12 x 10-6 cm / s.

[0192] In addition, the FORMULA 3 exhibits statistically higher bioavailability than FORMULA 2. Under the experimental conditions used, the formulations did not induce adverse effects at the level of the intestinal epithelium.

Claims

CLAIMS1. A composition comprising at least three organic salts of magnesium, where said at least three organic salts of magnesium are selected between: magnesium pidolate, magnesium gluconate, magnesium citrate, magnesium carbonate, magnesium chloride, magnesium fumarate, magnesium glycinate, magnesium L-lactate, magnesium DL-aspartate, magnesium L-aspartate, magnesium salicylate, magnesium sulphate, magnesium aminoate, magnesium phosphate, magnesium acetate, magnesium malate or magnesium picolinate.

2. The composition according to claim 1 , wherein the at least three organic salts of the magnesium are magnesium pidolate, magnesium gluconate and magnesium citrate.

3. The composition according to claim 1 or 2, whereinthe magnesium pidolate is present in a concentration of between 35% and 75% weight / weight (w / w),the magnesium gluconate is present in a concentration of between 20% and 50% w / w, andthe magnesium citrate is present in a concentration of between 0.1% and 10% w / w.

4. The composition according to any one of claims 1 to 3, wherein the magnesium pidolate is present in a concentration of between 40% and 70% weight / weight (w / w),the magnesium gluconate is present in a concentration of between 30% and 45% w / w, andthe magnesium citrate is present in a concentration of between 0.5% and 5% w / w.

5. The composition according to any one of claims 1 to 4, whereinthe magnesium pidolate is present in a concentration of between 50% and 65% weight / weight (w / w),the magnesium gluconate is present in a concentration of between 30% and 40% w / w, andthe magnesium citrate is present in a concentration of between 1% and 4% w / w.

6. The composition according to any one of claims 1 to 5, formulated for an oral administration.

7. The composition according to claim 6, formulated as a pill, tablet, granules, powder in sachet, lozenge, capsule, operculum, syrup, drinkable vial.

8. The composition according to any one of claims 1 to 7, for use in the treatment of a pathology or a disorder caused by an insufficiency of magnesium levels in an individual.

9. The composition for the use according to claim 8, wherein the individual is younger than eighteen years old.

10. The composition for the use according to claim 8 or 9, wherein the pathology or disorder caused by an insufficiency of magnesium is selected between: migraine, muscle cramps, sleep disturbances, anxiety, depression, mood variability, osteo-tendon hyperreflexia, muscle incoordination, tremors, paraesthesia, cardiac arrhythmia and arterial hypertension.

11. The composition for use according to any one of claims 8 to 10, wherein the individual has blood levels of magnesium ranging from 1.5 to 2.55 mg / dL.

12. The composition for use according to any one of claims 8 to10, wherein the individual has blood levels of magnesium ranging from 1.2 to 1.7 mg / dL.

13. The composition for use according to any one of claims 8 to 10, wherein the individual has blood levels of magnesium ranging from 0.8 to 1.2 mg / dL.

14. The composition for use according to any one of claims 8 to10, wherein the individual has blood levels of magnesium of less than 0.8 mg / dL.

15. A food supplement comprising the composition according to any one of claims 1 to 7 and at least one carrier and / or an excipient.

16. A non-therapeutic use of the composition according to any one of claims 1 to 7 or of the supplement according to claim 15, for improving, increasing and / or aiding the absorption and / or bioavailability of magnesium in an individual.

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