Calcium salt of reduced Β-nicotinamide mononucleotide and method for producing same

Abstract

The purpose of the present disclosure is to provide, with regard to reduced β-nicotinamide mononucleotide (NMNH), which is expected to exhibit an excellent anti-aging effect, a calcium salt of NMNH as a novel NMNH salt that is easy to distribute and handle and provide a method for producing the calcium salt of NMNH.　One aspect of the present embodiment is a calcium salt of reduced β-nicotinamide mononucleotide.

Description

Calcium salt of reduced β-nicotinamide mononucleotide and method for producing the same

[0001] The present disclosure relates to calcium salts of reduced β-nicotinamide mononucleotide and methods for producing the same.

[0002] Nicotine adenine dinucleotide (NAD) is an essential compound for energy production in mitochondria in eukaryotes. β-nicotinamide mononucleotide (NMN) is a precursor of NAD. It has been reported that NMN supplementation can effectively increase NAD levels in vivo (see Non-Patent Document 1).

[0003] It has also been reported that an increase in NAD levels in the body activates the sirtuin gene, also known as the longevity gene (see Non-Patent Document 2), and NMN, a precursor of NAD, is attracting attention as a pharmaceutical / functional food ingredient from the perspectives of improving vitality and anti-aging.

[0004] Furthermore, it has been reported that reduced β-nicotinamide mononucleotide (NMNH), a reduced form of NMN, increases NAD more effectively than NMN (see, for example, Non-Patent Document 3), and NMNH is expected to be a more effective anti-aging material than NMN.

[0005] However, NMNH is unstable and presents challenges in storage and distribution. Currently, amorphous disodium salt of NMNH is commercially available on a small scale, but this disodium salt is deliquescent (see Patent Document 1), and therefore, careful management of temperature and humidity is still required.

[0006] Chinese Patent Application Publication No. 115368423

[0007] REVOLLO, Javier R.; GRIMM, Andrew A.; IMAI, Shin-ichiro. “The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells”. Journal of Biological Chemistry, 2004, 279.49: 50754-50763. North BJ, Rosenberg MA, et al. “SIRT2 induces the checkpoint kinase BubR1 to increase lifespan”. EMBO J. 2014 Jul 1;33(13):1438-53.ZAPATA‐PEREZ, Ruben, et al. “Reduced nicotinamide mononucleotide is a new and potent NAD+ precursor in mammalian cells and mice”. The FASEB Journal, 2021, 35.4: e21456.

[0008] The present disclosure aims to provide a calcium salt of NMNH, which is a novel salt of NMNH that is easy to distribute and handle, and to provide a method for producing the calcium salt of NMNH, for which excellent anti-aging effects are expected.

[0009] An example aspect of this embodiment is described as follows.

[0010] [1] A calcium salt of reduced β-nicotinamide mononucleotide. [2] A crystalline calcium salt of reduced β-nicotinamide mononucleotide according to [1]. [3] A calcium salt of reduced β-nicotinamide mononucleotide according to [2], which exhibits characteristic peaks at diffraction angles (2θ±0.2°) of 6.8°, 8.3°, 13.4°, 17.8°, 23.9°, and 27.1° in powder X-ray (Cu-Kα) diffraction. [4] A calcium salt of reduced β-nicotinamide mononucleotide according to [2] or [3], which exhibits the powder X-ray (Cu-Kα) diffraction pattern shown in FIG. 1. [5] The calcium salt of reduced β-nicotinamide mononucleotide according to [2], which exhibits characteristic peaks at diffraction angles (2θ±0.2°) of 7.2°, 12.9°, 15.9°, 18.2°, 22.0°, and 25.6° in powder X-ray (Cu-Kα) diffraction. [6] The calcium salt of reduced β-nicotinamide mononucleotide according to [2] or [5], which exhibits the powder X-ray (Cu-Kα) diffraction pattern shown in Figure 2. [7] A method for producing the calcium salt of reduced β-nicotinamide mononucleotide, which comprises mixing reduced β-nicotinamide mononucleotide and a calcium compound in water to precipitate the calcium salt of reduced β-nicotinamide mononucleotide. [8] A method for producing the calcium salt of reduced β-nicotinamide mononucleotide according to [7], which comprises mixing a calcium compound with an aqueous solution of reduced β-nicotinamide mononucleotide to precipitate the calcium salt of reduced β-nicotinamide mononucleotide. [9] A method for producing a calcium salt of reduced β-nicotinamide mononucleotide according to [8], which comprises mixing a calcium compound with an aqueous solution of reduced β-nicotinamide mononucleotide, and then adding a poor solvent.

[10] A method for stabilizing reduced β-nicotinamide mononucleotide, which comprises mixing reduced β-nicotinamide mononucleotide with a calcium compound in water.

[11] A method for stabilizing reduced β-nicotinamide mononucleotide, which comprises mixing a calcium compound with an aqueous solution of reduced β-nicotinamide mononucleotide.

[12] The method for stabilizing reduced β-nicotinamide mononucleotide according to

[10] or

[11] , further comprising precipitating a calcium salt of reduced β-nicotinamide mononucleotide in an aqueous solution containing reduced β-nicotinamide mononucleotide and a calcium compound.

[13] The method for stabilizing reduced β-nicotinamide mononucleotide according to any one of

[10] to

[12] , further comprising adding a poor solvent to an aqueous solution containing reduced β-nicotinamide mononucleotide and a calcium compound.

[14] A composition or kit for stabilizing reduced β-nicotinamide mononucleotide, comprising a calcium compound. This specification incorporates the disclosures of Japanese Patent Application No. 2023-218526, from which the present application claims priority.

[0011] The present disclosure provides a novel substance, calcium salt of NMNH, and a method for producing the same. The calcium salt of NMNH is non-deliquescent, highly stable, and easier to distribute and handle than the conventional amorphous disodium salt of NMNH.

[0012] 1 is a diffraction pattern obtained by powder XRD analysis of a crystal of NMNH calcium salt (crystal A). 2 is a diffraction pattern obtained by powder XRD analysis of a crystal of NMNH calcium salt (crystal B). 3 is a diffraction pattern obtained by powder XRD analysis of an amorphous NMNH calcium salt. 4 is a diagram showing the change in weight over time when each NMNH salt tested in Example 8 was stored at 25°C and 60% humidity. In the diagram, "weight (relative value)" indicates a relative value when the weight at the start of storage (storage day 0) is set to 100%. 5 is a diagram showing the change in weight over time when each NMNH salt tested in Example 8 was stored at 40°C and 75% humidity. In the diagram, "weight (relative value)" indicates a relative value when the weight at the start of storage (storage day 0) is set to 100%. 1 is a diagram showing the change over time in NMNH purity when each NMNH salt tested in Example 8 was stored at 25°C and 60% humidity. In the diagram, "NMNH content (relative value)" indicates a relative value when the ratio of NMNH in the powder at the start of storage (storage day 0) is set to 100%. FIG. 2 is a diagram showing the change over time in NMNH purity when each NMNH salt tested in Example 8 was stored at 40°C and 75% humidity. In the diagram, "NMNH content (relative value)" indicates a relative value when the ratio of NMNH in the powder at the start of storage (storage day 0) is set to 100%. FIG. 3 is a diagram showing the change over time in NMNH purity when each NMNH salt tested in Example 9 was stored at 25°C. In the figure, "NMNH content (relative value)" indicates a relative value when the ratio of NMNH in the powder at the start of storage (storage day 0) is set to 100%. This is a diagram showing the change in NMNH purity over time when each NMNH salt tested in Example 9 was stored at 40°C. In the figure, "NMNH content (relative value)" indicates a relative value when the ratio of NMNH in the powder at the start of storage (storage day 0) is set to 100%. This is a diagram showing the change in NMNH purity over time when each NMNH salt tested in Example 9 was stored at 60°C. In the figure, "NMNH content (relative value)" indicates a relative value when the ratio of NMNH in the powder at the start of storage (storage day 0) is set to 100%.

[0013] The present invention will be described in detail below. One aspect of this embodiment is a calcium salt of reduced β-nicotinamide mononucleotide. Another aspect of this embodiment is a method for producing a calcium salt of reduced β-nicotinamide mononucleotide, which comprises mixing reduced β-nicotinamide mononucleotide and a calcium compound in water to precipitate the calcium salt of reduced β-nicotinamide mononucleotide. This embodiment will be described in detail below.

[0014] (Calcium Salt of Reduced β-Nicotinamide Mononucleotide and Method for Producing the Same) The calcium salt of reduced β-nicotinamide mononucleotide has improved storage stability compared to the known disodium salt of amorphous NMNH. According to studies by the present inventors, as the calcium salt of reduced β-nicotinamide mononucleotide, an amorphous calcium salt of reduced β-nicotinamide mononucleotide (amorphous NMNH calcium salt) and a crystal of the calcium salt of reduced β-nicotinamide mononucleotide (crystal of NMNH calcium salt) were obtained. Furthermore, crystal polymorphism was observed for the crystal of NMNH calcium salt, and specifically, two types of crystal were obtained. When analyzed by powder XRD, the NMNH calcium salt that gives a diffraction pattern similar to that of FIG. 1 is also referred to as Crystal A, and the NMNH calcium salt that gives a diffraction pattern similar to that of FIG. 2 is also referred to as Crystal B.

[0015] Reduced β-nicotinamide mononucleotide is a compound represented by the following formula (1):

[0016]

[0017] The calcium salt of reduced β-nicotinamide mononucleotide is usually prepared by converting the phosphate group (—OP(O)(OH)) of the reduced β-nicotinamide mononucleotide into a calcium salt of reduced β-nicotinamide mononucleotide. ２ ) two H ＋ is replaced by one calcium cation, and is usually a compound represented by the following formula (2).

[0018]

[0019] The calcium salt of reduced β-nicotinamide mononucleotide may be amorphous or crystalline, with a preferred embodiment being crystalline. The crystalline form of NMNH calcium salt may be either crystalline A or crystalline B described above.

[0020] Crystal A is an NMNH calcium salt that, when analyzed by powder XRD, gives a diffraction pattern similar to that shown in Figure 1, and more specifically, is an NMNH calcium salt that shows the powder X-ray (Cu-Kα) diffraction pattern shown in Figure 1. When Crystal A is specified by a more specific diffraction angle (2θ±0.2°), Crystal A is an NMNH calcium salt that shows characteristic peaks at diffraction angles (2θ±0.2°) of 6.8°, 8.3°, 13.4°, 17.8°, 23.9°, and 27.1° in powder X-ray (Cu-Kα) diffraction. Furthermore, in powder X-ray (Cu—Kα) diffraction, Crystal A preferably exhibits characteristic peaks at diffraction angles (2θ±0.2°) of 13.7°, 14.7°, 19.1°, 21.6°, 24.4°, 27.7°, 28.9°, 30.9°, 32.5°, 35.5°, 38.1°, and 41.5°.

[0021] Crystal B is an NMNH calcium salt that, when analyzed by powder XRD, gives a diffraction pattern similar to that shown in FIG. 2 , more specifically, an NMNH calcium salt that shows the powder X-ray (Cu-Kα) diffraction pattern shown in FIG. 2 . Crystal B is specified by a more specific diffraction angle (2θ±0.2°), and Crystal B is an NMNH calcium salt that shows characteristic peaks at diffraction angles (2θ±0.2°) of 7.2°, 12.9°, 15.9°, 18.2°, 22.0°, and 25.6° in powder X-ray (Cu-Kα) diffraction. Furthermore, Crystal B preferably shows characteristic peaks at diffraction angles (2θ±0.2°) of 9.7°, 14.6°, 16.9°, 19.2°, 21.2°, 23.3°, 26.7°, 28.4°, and 31.5° in powder X-ray (Cu-Kα) diffraction.

[0022] Depending on the production conditions for the calcium salt of reduced β-nicotinamide mononucleotide, amorphous NMNH calcium salt and NMNH calcium salt crystals (e.g., crystal A and crystal B) can be produced. There are no particular limitations on the production conditions for obtaining each NMNH calcium salt, and they can be produced, for example, by the methods described in the Examples.

[0023] The method for producing NMNH calcium salt is not particularly limited, and examples thereof include a method in which reduced β-nicotinamide mononucleotide and a calcium compound are mixed in water to precipitate the calcium salt of reduced β-nicotinamide mononucleotide. When mixing NMNH and a calcium compound in water, NMNH and the calcium compound may be added to water, an aqueous solution of NMNH may be prepared in advance and the calcium compound may be added thereto, or an aqueous solution of the calcium compound may be prepared in advance and NMNH may be added thereto; there are no particular limitations. One embodiment of the method for producing NMNH calcium salt includes a method in which a calcium compound is mixed with an aqueous solution of reduced β-nicotinamide mononucleotide (an aqueous solution of NMNH) to precipitate the calcium salt of reduced β-nicotinamide mononucleotide. Hereinafter, the method for producing NMNH calcium salt will be mainly described, which involves mixing a calcium compound with an aqueous solution of NMNH to precipitate the calcium salt of NMNH.

[0024] The aqueous solution of NMNH may be obtained by dissolving reduced β-nicotinamide mononucleotide in water or by dissolving the disodium salt of reduced β-nicotinamide mononucleotide in water, and is not particularly limited. Furthermore, the aqueous solution of NMNH may contain counter ions such as sodium ions and potassium ions, and is not particularly limited.

[0025] The temperature at which the calcium salt of NMNH is precipitated is not particularly limited as long as it is a temperature at which NMNH does not decompose, but is usually 0 to 40°C, preferably 0 to 30°C.

[0026] The pH of the aqueous solution of NMNH and the reaction solution for precipitating the calcium salt of NMNH (a solution obtained by mixing an aqueous solution of NMNH with a calcium compound) is not particularly limited as long as it is within a range in which NMNH does not decompose, but is usually pH 8 to 11, preferably pH 9 to 10. When adjusting the pH, a salt such as sodium hydroxide or an acid such as an organic acid can be used as appropriate.

[0027] The concentrations of NMNH and calcium contained in the reaction solution (a solution obtained by mixing a calcium compound with an aqueous solution of NMNH) decrease over time due to precipitation of the calcium salt of NMNH. The concentrations of NMNH and calcium immediately after mixing with the calcium compound are each independently preferably 20 mM or more, more preferably 80 mM or more. Furthermore, the concentrations of NMNH and calcium may each independently be equal to or lower than the concentration at which they can be dissolved in the solvent, and are preferably 1.5 M or less, more preferably 450 mM or less.

[0028] The aqueous solution of reduced β-nicotinamide mononucleotide may contain components other than reduced β-nicotinamide mononucleotide and water, and the reaction solution (a solution obtained by mixing an aqueous solution of NMNH with a calcium compound) may contain components other than reduced β-nicotinamide mononucleotide, a calcium compound, and water. The components other than reduced β-nicotinamide mononucleotide, a calcium compound, and water (other components) are not particularly limited as long as they do not inhibit the reaction between NMNH and calcium ions and do not decompose NMNH. The other components are not limited to compounds and may also be ions. Examples of the other components include sodium ions when a disodium salt of reduced β-nicotinamide mononucleotide is used. Examples of the other components include alkali metal ions, alkaline earth metal ions, and ammonium ions as cations, and halide ions as anions. The other components may also be organic acids. The other components may be used alone or in combination of two or more.

[0029] The calcium compound may be any compound that dissolves in the aqueous solution of reduced β-nicotinamide mononucleotide, and examples thereof include calcium halide, calcium acetate, calcium bicarbonate, etc. One type of calcium compound may be used alone, or two or more types may be used. When mixing the calcium compound with the aqueous solution of NMNH, the calcium compound may be in the form of a solid, or may be an aqueous solution of the calcium compound.

[0030] As a method for producing NMNH calcium salt, there may be mentioned, as described above, a method in which a calcium compound is mixed with an aqueous solution of reduced β-nicotinamide mononucleotide to precipitate the calcium salt of reduced β-nicotinamide mononucleotide. However, after mixing the calcium compound with the aqueous solution of reduced β-nicotinamide mononucleotide, a step of adding a poor solvent may also be included. Addition of a poor solvent tends to rapidly precipitate the calcium salt of reduced β-nicotinamide mononucleotide, and therefore addition of a poor solvent can shorten the time required to obtain the calcium salt of reduced β-nicotinamide mononucleotide. Furthermore, according to the studies of the present inventors, when a poor solvent is added to rapidly obtain the calcium salt of reduced β-nicotinamide mononucleotide, the calcium salt of reduced β-nicotinamide mononucleotide tends to become an amorphous calcium salt of reduced β-nicotinamide mononucleotide.

[0031] As the poor solvent, at least one solvent selected from methanol, ethanol, propanol, 2-propanol, acetonitrile, hexane, acetone, and DMSO can be used.

[0032] When a poor solvent is added, the volume of the poor solvent is preferably 10 vol% or more and 100 vol% or less, assuming that the volume of the reaction solution obtained by mixing a calcium compound with an aqueous solution of reduced β-nicotinamide mononucleotide is 100 vol%.

[0033] When producing the calcium salt of reduced β-nicotinamide mononucleotide as crystals, the above-mentioned crystal A and crystal B can be separately produced by, for example, the method described in the Examples.

[0034] Crystal A can be obtained by mixing a calcium compound with an aqueous solution of NMNH to precipitate a calcium salt of NMNH, followed by drying under conditions that do not result in transition to Crystal B. Specific drying conditions include, for example, drying at normal pressure or short-term vacuum drying at a temperature below 40°C. When drying at normal pressure, drying is carried out until the solvent evaporates, for example, for 8 to 48 hours. When vacuum drying is carried out for a short time, the time varies depending on the state of precipitation of the calcium salt of NMNH and its volume, but is, for example, less than 5 hours. On the other hand, Crystal A may be transitioned to Crystal B by carrying out vacuum drying for a long period of time, for example, for 5 to 48 hours.

[0035] To obtain crystalline B by drying at normal pressure, it is preferable to dry under conditions that result in a transition from crystalline A. For example, crystalline B can be obtained by mixing a calcium compound with an aqueous solution of NMNH to precipitate a calcium salt of NMNH, and then drying at normal pressure, usually at a temperature of 40°C or higher and lower than 120°C, preferably at a temperature of 40°C or higher and 90°C or lower. Alternatively, crystalline A may be maintained at a temperature within the above range to transition to crystalline B. Alternatively, amorphous calcium salt of NMNH can be obtained by drying crystalline A at a high temperature, for example, at a temperature of 120°C or higher and 200°C or lower.

[0036] Alternatively, crystalline B may be transformed into crystalline A by suspending it in water. After suspending crystalline B in water, it is preferable to dry it at normal pressure or vacuum dry it for a short time at a temperature below 40°C to obtain crystalline A. From these characteristics, it can be assumed that the calcium salt crystal of NMNH is a hydrate containing water of crystallization. Furthermore, it can be assumed that the calcium salt of NMNH is anhydrous or a mono- to hepta-hydrate depending on its drying state.

[0037] (Uses and Compositions) The calcium salt of reduced β-nicotinamide mononucleotide described above is non-deliquescent and stable, and therefore easier to distribute and handle than the conventional disodium salt of amorphous NMNH, and can be used in various applications where NMNH is used. For example, the calcium salt of reduced β-nicotinamide mononucleotide, or a composition containing the calcium salt of reduced β-nicotinamide mononucleotide, can be used as a food, a nutrient functional food, a food for specified health uses, a nutritional supplement, a beverage, a cosmetic, a pet food, a veterinary drug, a pharmaceutical, a therapeutic drug, or a preventative drug. When used in these applications, the calcium salt of reduced β-nicotinamide mononucleotide is typically used as a composition containing at least one component selected from the group consisting of an excipient, a disintegrant, a lubricant, a binder, an antioxidant, a colorant, an anti-aggregating agent, an absorption enhancer, a solubilizer, a stabilizer, a viscosity modifier, an oil or fat, a surfactant, and an active ingredient other than the calcium salt of reduced β-nicotinamide mononucleotide.

[0038] Other Embodiments The present invention further relates to a method for stabilizing reduced β-nicotinamide mononucleotide, which method comprises mixing reduced β-nicotinamide mononucleotide with a calcium compound in water, or mixing a calcium compound with an aqueous solution of reduced β-nicotinamide mononucleotide.

[0039] The details of the reduced β-nicotinamide mononucleotide, the calcium compound, and the mixture are the same as those described above for the calcium salt of reduced β-nicotinamide mononucleotide and the method for producing the same.

[0040] The stabilization method may further include precipitating the calcium salt of reduced β-nicotinamide mononucleotide in an aqueous solution containing reduced β-nicotinamide mononucleotide and a calcium compound. The aqueous solution, the precipitation method, and the type of calcium salt of reduced β-nicotinamide mononucleotide obtained thereby (amorphous, crystalline, crystal A, crystal B, etc.) are not particularly limited. For example, the calcium salt of reduced β-nicotinamide mononucleotide and the method for producing it may be similar to those described above.

[0041] The stabilization method may include further adding a poor solvent to an aqueous solution containing reduced β-nicotinamide mononucleotide and a calcium compound. The aqueous solution and the poor solvent are not particularly limited. For example, the poor solvent may be the same as those described above for the calcium salt of reduced β-nicotinamide mononucleotide and the method for producing the same.

[0042] Thus, calcium compounds can be used to stabilize reduced β-nicotinamide mononucleotide. Accordingly, the present invention also relates to the use of calcium compounds in stabilizing reduced β-nicotinamide mononucleotide, and to compositions or kits for stabilizing reduced β-nicotinamide mononucleotide, which contain calcium compounds. The state of the calcium compound used in this case is not particularly limited, and it may be solid or liquid. Furthermore, the compositions and kits may contain at least one component selected from, for example, excipients, disintegrants, lubricants, binders, antioxidants, colorants, anti-aggregating agents, absorption enhancers, solubilizers, stabilizers, viscosity modifiers, oils and fats, and surfactants. The calcium compound may be provided as a kit, which may include, in addition to the calcium compound, a dilution solution, additives, mixing means, antisolvents, instructions, and the like. Multiple types of calcium compounds may be included, and each reagent may be provided in the same container or in different containers.

[0043] Typically, the stabilization method of the present invention improves one or more of the following: deliquescence, morphological stability, chemical stability, environmental stability to humidity, thermal stability, or oxidative stability.

[0044] The present embodiment will be described below with reference to examples, but the present disclosure is not limited to these examples.

[0045] The measurement conditions for powder X-ray diffraction (XRD), thermogravimetric analysis (TGA), high performance liquid chromatography (HPLC), and ion chromatography in the examples are shown below.

[0046] (Powder XRD measurement conditions) Apparatus: Rigaku MiniFlex II X-ray: Cu-Kα Angle: 2θ=2 to 60° Voltage: 30 kV Current: 15 mA Scan speed: 2° / min Divergence slit: 1.25° Scattering slit: 1.25° Receiving slit: 0.3 mm

[0047] (TGA measurement conditions) Apparatus: STA200 manufactured by Hitachi Sample container: Aluminum pan (GAA-0068) Heating rate: 5°C / min Gas flow rate: Nitrogen 50 mL / min

[0048] (HPLC measurement conditions) Apparatus: Shimadzu SIL20 series Column: COSMOSIL 3PBr Packed Column, 3.0 mm I.D. (inner diameter) x 150 mm Mobile phase: 20 mM ammonium formate / methanol = 95 / 5 (v / v) Flow rate: 0.4 mL / min Detection wavelength: 340 nm

[0049] (Ion Chromatography Measurement Conditions) Apparatus: Dionex Integration HPIC manufactured by Thermo Scientific Column: Dionex IonPac CS16 Mobile phase: 30 mM methanesulfonic acid Flow rate: 1 mL / min

[0050] Example 1 Preparation of Amorphous NMNH Calcium Salt 1.25 g of amorphous NMNH disodium salt was dissolved in 20 mL of MilliQ water, and then 0.33 g of calcium chloride was added and dissolved while stirring at 22° C. After confirming the dissolution of calcium chloride, 20 mL of ethanol as a poor solvent was rapidly added, and the mixture was stirred at 22° C. for 3 hours to obtain a suspension.

[0051] The precipitate was collected from the suspension by filtration and dried in vacuo at 25°C to obtain 1.07 g of powder. When the obtained powder was analyzed by powder XRD, the diffraction pattern shown in Figure 3 was obtained, and no characteristic peaks were detected. This result confirmed that amorphous NMNH calcium salt was obtained.

[0052] Example 2 Preparation of NMNH calcium salt crystals (crystal A) 2.52 g of amorphous NMNH disodium salt was dissolved in MilliQ water, adjusted to pH 10.0 with 30% sodium hydroxide, and made up to 20 mL. 5 mL of 1.2 M calcium chloride aqueous solution was added to this solution, and the mixture was stirred at 22°C for 3 hours to obtain a suspension.

[0053] The precipitate was filtered from the suspension, washed with ethanol, and then dried in a room at 24° C. for 16 hours to obtain 2.18 g of powder. When the obtained powder was analyzed by powder XRD, it showed the diffraction pattern shown in FIG. 1 (diffraction pattern of Crystal A).

[0054] Furthermore, 67.4 mg of the powder was dissolved in 50 mL of MilliQ water, and the NMNH content was measured using HPLC with a detection wavelength of 340 nm. The result was 71.2% (w / w). Also, 62.7 mg of the powder was dissolved in 100 mL of MilliQ water, and the calcium content was measured using ion chromatography. The result was 8.8% (w / w). The NMNH to calcium content ratio was calculated from these contents, and it was found that the NMNH and calcium were contained in a molar ratio of 1:1.

[0055] These results confirmed that crystals of NMNH calcium salt were obtained.

[0056] Example 3 Preparation of NMNH calcium salt crystals (crystal B) 2.52 g of amorphous NMNH disodium salt was dissolved in MilliQ water, adjusted to pH 10.0 with 30% sodium hydroxide, and made up to 20 mL. 5 mL of 1.2 M calcium chloride aqueous solution was added to this solution, and the mixture was stirred at 22°C for 3 hours to obtain a suspension.

[0057] The precipitate was filtered from the suspension, washed with ethanol, and then vacuum dried at 25° C. for 19 hours to obtain 1.67 g of powder. When the obtained powder was analyzed by powder XRD, it showed the diffraction pattern shown in FIG. 2 (diffraction pattern of Crystal B).

[0058] Furthermore, 70.6 mg of the powder was dissolved in 50 mL of MilliQ water, and the NMNH content was measured using HPLC with a detection wavelength of 340 nm. The result was 82.6% (w / w). Furthermore, 81.6 mg of the powder was dissolved in 100 mL of MilliQ water, and the calcium content was measured using ion chromatography. The result was 9.8% (w / w). The NMNH to calcium content ratio was calculated from these contents, and it was found that the NMNH and calcium were contained in a molar ratio of 1:1.

[0059] These results confirmed that crystals of NMNH calcium salt were obtained.

[0060] Example 4 (Preparation of NMNH calcium salt crystals (crystal A)) 18.07 g of amorphous NMNH disodium salt was dissolved in 180 mL of MilliQ water. In order to separate sodium ions and NMNH from this aqueous solution, 56.08 g of anion exchange resin was added and mixed for 2 hours, and NMNH was adsorbed onto the resin. After removing the supernatant, the resin was washed by mixing with 180 mL of MilliQ water. After performing this washing operation twice, the resin and 180 mL of 1 M potassium chloride aqueous solution were mixed for 2 hours to obtain 193.0 g of NMNH eluate. Furthermore, this eluate was diafiltered with a total of 292.1 g of MilliQ water and then concentrated to obtain 133.0 g of NMNH aqueous solution. The results of analyzing each component contained in the NMNH aqueous solution by HPLC and ion chromatography are shown below. NMNH: 0.24M Na: 0.04M K: 0.54M Cl: 0.05M

[0061] 10 mL of the NMNH aqueous solution was dispensed into a test tube, and 2.4 mL of a 1 M calcium acetate aqueous solution was added thereto, followed by stirring at 22° C. for 16 hours to obtain a suspension.

[0062] The precipitate was filtered from the suspension, washed with ethanol, and then vacuum dried at 25° C. for 3 hours to obtain 0.74 mg of powder. When the obtained powder was analyzed by powder XRD, it showed a diffraction pattern similar to that of Crystal A in FIG.

[0063] Furthermore, 54.7 mg of the powder was dissolved in 50 mL of MilliQ water, and the NMNH content was measured using HPLC with a detection wavelength of 340 nm. The result was 72.9% (w / w). Furthermore, 51.0 mg of the powder was dissolved in 100 mL of MilliQ water, and the calcium content was measured using ion chromatography. The result was 8.8% (w / w). The NMNH to calcium content ratio was calculated from these contents, and it was found that the NMNH and calcium were contained in a molar ratio of 1:1.

[0064] These results confirmed that crystals of NMNH calcium salt were obtained.

[0065] Example 5 (Transition from Crystal B to Crystal A) Crystal B of NMNH calcium was stored indoors for 12 days. After storage, the diffraction angle of Crystal B was confirmed by powder XRD, and no change in the diffraction pattern was observed since the crystal was obtained.

[0066] 0.39 g of crystalline B was suspended in 3.78 mL of MilliQ water and stirred overnight at 22 °C. The slurry was collected on filter paper, washed with ethanol, and then dried overnight indoors to obtain a powder. The diffraction angle of the obtained powder was confirmed by powder XRD, and a transformation to crystalline A was confirmed.

[0067] Furthermore, 9.587 mg of Crystal A and 9.594 mg of Crystal B were heated at 25 to 200°C in a thermogravimetric analyzer (TGA) and the weight change was measured. As a result, the weight change (weight loss) was 22.2% for Crystal A and 12.0% for Crystal B. From these results, it was speculated that Crystal A and Crystal B were hydrates with different hydration numbers.

[0068] Example 6 (Transition from Crystalline A to Crystalline B and Amorphous Salt) Crystalline A was placed on an aluminum dish and dried in a dry heat dryer at 40°C, 60°C, 90°C, 120°C, or 150°C for 3 hours. The diffraction angle of the dried powder was confirmed by powder XRD. The diffraction patterns of Crystalline B were detected at 40°C, 60°C, and 90°C, but no characteristic peaks were observed at 120°C and 150°C. Furthermore, 0.14 g of the amorphous powder dried at 120°C was suspended in 1 mL of MilliQ water, stirred for 2 hours, and then collected on filter paper and dried overnight at room temperature to obtain a powder. The diffraction angle of the resulting powder was confirmed by powder XRD, and the diffraction pattern of Crystalline A was detected.

[0069] [Example 7] (Hygroscopicity test) 0.9988 g of amorphous NMNH calcium salt obtained in Example 1, 0.9229 g of crystal A obtained in Example 2, and 1.0051 g of crystal B obtained in Example 3 were weighed out. In addition, 1.3229 g of amorphous NMNH disodium salt was weighed out after vacuum drying overnight at 25°C.

[0070] Each powder was allowed to stand in a thermo-hygrostat at 25°C and 60% humidity for 24 hours, and then its weight was reconfirmed. As a result, weight gains of 0.2% for Crystal A, 1.1% for Crystal B, 3.9% for amorphous NMNH calcium salt, and 15.5% for amorphous NMNH disodium salt were confirmed. Furthermore, particles of NMNH disodium salt had adhered to each other, causing a loss of powder fluidity, but Crystal A, Crystal B, and amorphous NMNH calcium salt all maintained their powder fluidity.

[0071] After that, they were left to stand for 7 days in a room at a temperature of 24°C and a humidity of approximately 40%. As a result, the amorphous NMNH disodium salt completely lost its powder form and solidified, while the NMNH calcium salt, both amorphous and crystalline, maintained its powder state.

[0072] Example 8 Hygroscopicity and Stability Test in an Open System 17.49 g of amorphous NMNH disodium salt was dissolved in 150 mL of MilliQ water, and then 34.7 mL of a 1.9 M aqueous calcium chloride solution was added while stirring at 22°C to obtain a suspension. The precipitate was filtered from the suspension, washed with ethanol, and then vacuum-dried at 25°C for 30 hours to obtain an NMNH calcium salt powder. When the peaks were confirmed by powder XRD, the diffraction pattern shown in Figure 2 (diffraction pattern of crystal B) was observed.

[0073] Separately, 15.63 g of amorphous NMNH disodium salt was dissolved in 250 mL of MilliQ water, and 4.13 g of calcium chloride powder was added. Then, 250 mL of ethanol was rapidly added and the mixture was stirred for 3 hours to obtain a suspension. The precipitate was filtered from the suspension, washed with ethanol, and then vacuum-dried at 25°C for 48 hours to obtain NMNH calcium salt powder. Powder XRD peak analysis revealed the diffraction pattern shown in Figure 3, confirming that the product was amorphous.

[0074] Open containers containing approximately 1.0 g of each of the resulting crystalline and amorphous NMNH calcium salts and commercially available amorphous and crystalline NMNH disodium salts were prepared for NMNH content measurement and weight measurement, respectively. These containers were left standing in an open system for two weeks in a thermo-hygrostat at 25°C and 60% humidity, or at 40°C and 75% humidity. The NMNH content was measured by recovering a small amount of each salt. Weight changes were calculated by weighing the weighing containers without recovering any salt. Measurements were performed at the start of storage (storage day 0) and at appropriate times after 1, 4, 7, and 14 days. Appearance was also inspected.

[0075] The NMNH content was measured by HPLC in the same manner as in Example 2. The NMNH content was calculated as a percentage of the weight of the salt recovered and subjected to measurement. The relative NMNH content was calculated by setting the NMNH content at the start of storage (crystalline NMNH calcium salt: 82% (w / w); amorphous NMNH calcium salt: 73% (w / w); amorphous NMNH disodium salt: 81% (w / w); crystalline NMNH disodium salt: 72% (w / w)) as 100%.

[0076] The relative weight values ​​were calculated with the weight at the start of storage (storage day 0) set as 100%.

[0077] The results of the weight change under each condition are shown in FIGS. 4 and 5, and the results of the NMNH content are shown in FIGS. 6 and 7.

[0078] As shown in Figures 4 and 5, both amorphous ("Na salt (amorphous)") and crystalline ("Na salt (crystalline)") NMNH disodium salt showed a significant weight gain due to moisture absorption, resulting in a loss of powder fluidity and adhesion of particles to each other. The weight gain of crystalline NMNH disodium salt was lower (approximately 18% increase after 14 days of storage) than amorphous NMNH disodium salt (approximately 26% increase after 14 days of storage) at 25°C and 60% humidity. However, at 40°C and 75% humidity, the mass gains of crystalline and amorphous NMNH disodium salt were comparable (approximately 35% to 40% increase after 14 days of storage).

[0079] On the other hand, both amorphous ("Ca salt (amorphous)") and crystalline ("Ca salt (crystalline)") NMNH calcium salt maintained the fluidity of a powder. Regarding weight change, as shown in Figures 4 and 5, amorphous NMNH calcium salt ("Ca salt (amorphous)") showed a smaller weight increase than NMNH disodium salt (an increase of about 6 to 9%), and crystalline NMNH calcium salt ("Ca salt (crystalline)") showed almost no weight increase during 14 days of storage (an increase of about 3 to 4%). This confirmed that NMNH calcium salt has excellent storage stability as a powder, even when stored under high-temperature and high-humidity conditions for a longer period than in Example 7.

[0080] As shown in Figures 6 and 7, the NMNH content of both amorphous ("Na salt (amorphous)") and crystalline ("Na salt (crystalline)") NMNH disodium salt decreased linearly with increasing storage days. The results for amorphous and crystalline NMNH disodium salt were similar under all conditions. Under conditions of 25°C and 60% humidity (Figure 6), the NMNH content of NMNH disodium salt was reduced by half after 14 days of storage, whereas the NMNH content of amorphous NMNH calcium salt ("Ca salt (amorphous)") remained at a good level (approximately 85% after 14 days of storage), and the NMNH content of crystalline NMNH calcium salt ("Ca salt (crystalline)") remained almost unchanged (approximately 100% after 14 days of storage).

[0081] Under conditions of 40°C and 75% humidity (Figure 7), the NMNH content of NMNH disodium salt was reduced by half after one day of storage, while the NMNH content of crystalline NMNH calcium salt ("Ca salt (crystalline)") was slightly reduced (approximately 84% after 14 days of storage), but maintained a good level even after 14 days of storage. This confirmed that NMNH calcium salt is excellent not only in stability as a powder but also in chemical stability.

[0082] Example 9 (Stability Test in a Closed System) The crystalline and amorphous NMNH calcium salts and crystalline NMNH disodium salt used in Example 8 were used. Each NMNH salt (approximately 0.3 g) was placed in an aluminum heat-sealed pouch, lightly evacuated, and then sealed. The aluminum heat-sealed pouch was left to stand in a thermo-hygrostat at 25°C or 40°C, or in an incubator at 60°C. A portion of each salt was collected together with the pouch to check the appearance and measure the NMNH content. Measurements were performed at the start of storage (storage day 0) as well as at appropriate times of 1 day, 3 days, 7 days, and 21 days after the start of storage.

[0083] The crystalline NMNH disodium salt melted at 60° C. and completely lost its powder form. On the other hand, the crystalline and amorphous NMNH calcium salts did not change in appearance even at 60° C. This confirmed that NMNH calcium salt is more stable as a salt than NMNH sodium salt.

[0084] The NMNH content remained almost unchanged in either powder at 25°C (Fig. 8) or 40°C (Fig. 9). On the other hand, at 60°C (Fig. 10), the NMNH content of crystalline NMNH sodium salt ("Na salt (crystalline)") rapidly decreased, reaching 3% after 7 days of storage. In contrast, the NMNH calcium salt, both crystalline and amorphous, showed little decrease in NMNH content. The NMNH content of crystalline NMNH calcium salt ("Ca salt (crystalline)") after 7 days of storage was approximately 90%, and that of amorphous NMNH calcium salt ("Ca salt (amorphous)") after 7 days of storage was approximately 78%, maintaining high quality.

[0085] From the above, it was confirmed that NMNH calcium salt has extremely excellent chemical stability.

[0086] The upper and / or lower limits of the numerical ranges described herein can be arbitrarily combined to define a preferred range. For example, the upper and lower limits of the numerical ranges can be arbitrarily combined to define a preferred range, the upper limits of the numerical ranges can be arbitrarily combined to define a preferred range, and the lower limits of the numerical ranges can be arbitrarily combined to define a preferred range. Furthermore, in this application, numerical ranges expressed using the symbol "to" include the numerical values ​​written before and after the symbol "to" as the upper and lower limits, respectively.

[0087] Although the present embodiment has been described in detail above, the specific configuration is not limited to this embodiment, and any design changes that do not deviate from the gist of the present disclosure are also included in the present disclosure. All publications, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims

1. Calcium salt of reduced β - nicotinamide mononucleotide.

2. The calcium salt of reduced β - nicotinamide mononucleotide according to claim 1, which is crystalline.

3. The calcium salt of reduced β - nicotinamide mononucleotide according to claim 2, which shows characteristic peaks at diffraction angles (2θ ± 0.2°) of 6.8°, 8.3°, 13.4°, 17.8°, 23.9°, and 27.1° in powder X - ray (Cu - Kα) diffraction.

4. The calcium salt of reduced β - nicotinamide mononucleotide according to claim 2, which shows the powder X - ray (Cu - Kα) diffraction pattern shown in Figure 1.

5. The calcium salt of reduced β - nicotinamide mononucleotide according to claim 2, which shows characteristic peaks at diffraction angles (2θ ± 0.2°) of 7.2°, 12.9°, 15.9°, 18.2°, 22.0°, and 25.6° in powder X - ray (Cu - Kα) diffraction.

6. The calcium salt of reduced β - nicotinamide mononucleotide according to claim 2, which shows the powder X - ray (Cu - Kα) diffraction pattern shown in Figure 2.

7. A method for producing a calcium salt of reduced β - nicotinamide mononucleotide, which comprises mixing reduced β - nicotinamide mononucleotide and a calcium compound in water to precipitate the calcium salt of reduced β - nicotinamide mononucleotide.

8. The method for producing a calcium salt of reduced β - nicotinamide mononucleotide according to claim 7, which comprises mixing a calcium compound into an aqueous solution of reduced β - nicotinamide mononucleotide to precipitate the calcium salt of reduced β - nicotinamide mononucleotide.

9. The method for producing a calcium salt of reduced β - nicotinamide mononucleotide according to claim 8, which comprises adding a poor solvent after mixing a calcium compound into an aqueous solution of reduced β - nicotinamide mononucleotide.

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