Salt of reduced β-nicotinamide mononucleotide, and method for producing same

Abstract

The purposes of the present disclosure are: to provide a novel salt of reduced β-nicotinamide mononucleotide (NMNH), the NMNH being expected to have an excellent anti-aging effect and the salt being easy to distribute and handle; and to provide a method for producing the novel salt of NMNH.　One embodiment of the present invention is a salt of reduced β-nicotinamide mononucleotide that is at least one salt selected from among potassium salts, ammonium salts, and divalent metal salts excluding calcium salts.

Description

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

[0001] The present disclosure relates to 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, disodium salt of amorphous NMNH is commercially available on a small scale, but this disodium salt is deliquescent (see Patent Document 1), and 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 novel salt of NMNH, which is expected to have excellent anti-aging effects, that is easy to distribute and handle, and to provide a method for producing the novel salt of NMNH.

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

[0010] [1] A salt of reduced β-nicotinamide mononucleotide, which is at least one salt selected from divalent metal salts excluding calcium salts, potassium salts, and ammonium salts. [2] The salt of reduced β-nicotinamide mononucleotide according to [1], wherein the divalent metal salt is an iron salt or a magnesium salt. [3] A method for producing a salt of reduced β-nicotinamide mononucleotide, which is at least one salt selected from divalent metal salts excluding calcium salts, potassium salts, and ammonium salts, comprising: a step (I) of preparing a solution containing reduced β-nicotinamide mononucleotide and at least one ion selected from divalent metal ions excluding calcium ions, potassium ions, and ammonium ions in an aqueous solvent; a step (II) of mixing the solution obtained in step (I) with a water-miscible organic solvent to precipitate a salt of reduced β-nicotinamide mononucleotide; and a step (III) of separating the salt precipitated in step (II). [4] The production method according to [3], wherein step (I) is carried out by a method including the following steps (A) and (B): Step (A) of subjecting reduced β-nicotinamide mononucleotide to an anion exchange resin to obtain an anion exchange resin having reduced β-nicotinamide mononucleotide adsorbed thereon; and Step (B) of extracting the reduced β-nicotinamide mononucleotide in the anion exchange resin obtained in step (A) with an aqueous solution containing at least one ion selected from divalent metal ions excluding calcium ions, potassium ions, and ammonium ions. This specification includes the disclosure of Japanese Patent Application No. 2023-218492, from which the present application claims priority.

[0011] The present disclosure provides a novel NMNH salt and a method for producing the same. The resulting NMNH salt does not exhibit deliquescent properties and is easier to distribute and handle than the conventional amorphous disodium salt of NMNH.

[0012] The present invention will be described in detail below. One aspect of this embodiment is a salt of reduced β-nicotinamide mononucleotide, which is at least one salt selected from divalent metal salts excluding calcium salts, potassium salts, and ammonium salts. Another aspect of this embodiment is a method for producing a salt of reduced β-nicotinamide mononucleotide, which is at least one salt selected from divalent metal salts excluding calcium salts, potassium salts, and ammonium salts. This production method includes the steps of: (I) preparing a solution containing reduced β-nicotinamide mononucleotide and at least one ion selected from divalent metal ions excluding calcium ions, potassium ions, and ammonium ions in an aqueous solvent; (II) mixing the solution obtained in step (I) with a water-miscible organic solvent to precipitate a salt of reduced β-nicotinamide mononucleotide; and (III) separating the salt precipitated in step (II). This embodiment will be described in detail below.

[0013] (Salt of reduced β-nicotinamide mononucleotide and method for producing same) The salt of reduced β-nicotinamide mononucleotide is at least one salt selected from divalent metal salts excluding calcium salts, potassium salts, and ammonium salts. Specific examples include divalent metal salts excluding calcium salts of reduced β-nicotinamide mononucleotide, potassium salts of reduced β-nicotinamide mononucleotide, more specifically dipotassium salts of reduced β-nicotinamide mononucleotide, and ammonium salts of reduced β-nicotinamide mononucleotide, more specifically diammonium salts of reduced β-nicotinamide mononucleotide. The salt of reduced β-nicotinamide mononucleotide has a specific cation and has improved storage stability compared to the known disodium salt of amorphous NMNH.

[0014] As the divalent metal salt, an iron salt or a magnesium salt is one of the preferred embodiments. As the ammonium cation constituting the ammonium salt, the most common NH ４ ＋The ammonium cation may be, but is not limited to, a primary ammonium cation, a secondary ammonium cation, a tertiary ammonium cation, or a quaternary ammonium cation. The ammonium cation may be, for example, NR ４ ＋ (wherein each R is independently a hydrogen atom or an organic group). Examples of the organic group include an alkyl group and an aryl group, and an alkyl group having 1 to 10 carbon atoms and an aryl group having 1 to 10 carbon atoms are preferred. The ammonium cation is NH ４ ＋ is one of the preferred embodiments.

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

[0016]

[0017] The salt of reduced β-nicotinamide mononucleotide can be prepared by, for example, converting the phosphate group (—OP(O)(OH)) of the reduced β-nicotinamide mononucleotide into a salt of the phosphate group (—OP(O)(OH) ２ ) two H ＋ is replaced by one divalent metal ion, by two potassium ions, or by two ammonium ions, and is, for example, a compound represented by the following formula (2), (3), or (4).

[0018]

[0019] In the formula (2), M ２＋ is a divalent metal ion (divalent cation) excluding calcium ion, and iron ion (Fe ２＋ ) or magnesium ions (Mg ２＋ In formula (4), each R is independently a hydrogen atom or an organic group. Examples of the organic group include an alkyl group and an aryl group, and an alkyl group having 1 to 10 carbon atoms and an aryl group having 1 to 10 carbon atoms are preferred. In one preferred embodiment, all of the Rs are hydrogen atoms.

[0020] The production conditions for obtaining each NMNH salt are not particularly limited, and they can be produced, for example, by the method described in the Examples. Examples of methods for producing NMNH salts include a method comprising the steps of: (I) preparing a solution containing reduced β-nicotinamide mononucleotide and at least one ion selected from divalent metal ions other than calcium ions, potassium ions, and ammonium ions in an aqueous solvent; (II) mixing the solution obtained in step (I) with a water-miscible organic solvent to precipitate a salt of reduced β-nicotinamide mononucleotide; and (III) separating the salt precipitated in step (II).

[0021] In a preferred embodiment, the aqueous solvent is water. In step (I), reduced β-nicotinamide mononucleotide or disodium salt of reduced β-nicotinamide mononucleotide may be added to the aqueous solvent.

[0022] Furthermore, in order to supply at least one ion selected from divalent metal ions (excluding calcium ions), potassium ions, and ammonium ions, at least one compound or salt selected from compounds or salts of divalent metals (excluding calcium), compounds or salts of potassium, and compounds or salts of ammonium can be added. These compounds or salts may be used alone or in combination of two or more. When these compounds or salts are added to an aqueous solvent, they may be solid or may be aqueous solutions of these compounds or salts. The compounds or salts of divalent metals (excluding calcium), compounds or salts of potassium, and compounds or salts of ammonium are not particularly limited, and examples thereof include iron(II) chloride, magnesium chloride, potassium chloride, and ammonium chloride. Hydrates of these compounds may also be used.

[0023] Step (I) may be carried out by a method comprising the following steps (A) and (B): step (A) of subjecting reduced β-nicotinamide mononucleotide to an anion exchange resin to obtain an anion exchange resin having reduced β-nicotinamide mononucleotide adsorbed thereon, and step (B) of extracting the reduced β-nicotinamide mononucleotide in the anion exchange resin obtained in step (A) with an aqueous solution containing at least one ion selected from divalent metal ions excluding calcium ions, potassium ions, and ammonium ions.

[0024] The method including steps (A) and (B) is preferred because it allows cations other than those constituting the desired salt of reduced β-nicotinamide mononucleotide (cations other than the target) to be excluded.

[0025] The temperature at which step (I) is carried out is not particularly limited as long as NMNH does not decompose, but is usually 0 to 40°C, preferably 0 to 30°C.

[0026] The pH of the solution obtained in step (I) 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 the ions contained in the solution decrease over time due to precipitation of NMNH salts. Immediately after preparation of the solution, the concentrations of NMNH and the ions are each independently preferably 20 mM or more, more preferably 80 mM or more. Furthermore, the concentrations of NMNH and the ions 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] In step (II), the solution obtained in step (I) is mixed with a water-miscible organic solvent to precipitate a salt of reduced β-nicotinamide mononucleotide. The water-miscible organic solvent is preferably an organic solvent that is miscible with water but acts as a poor solvent for the salt of reduced β-nicotinamide mononucleotide, and for example, at least one solvent selected from methanol, ethanol, propanol, 2-propanol, acetonitrile, hexane, acetone, and DMSO can be used.

[0029] Step (II) is usually carried out under stirring. The temperature when step (II) is carried out is not particularly limited, but is usually 0 to 40° C., preferably 0 to 30° C. The time when step (II) is carried out is not particularly limited, but is usually more than 0 hour and 10 hours or less, preferably 1 to 5 hours.

[0030] Step (III) is a step of separating the salt precipitated in step (II), and can be performed by, for example, filtration, removal of the supernatant, etc. The separated salt (salt of NMNH) may be washed with alcohol or the like.

[0031] The separated salt (salt of NMNH) may be subjected to a drying step such as drying under normal pressure or drying under reduced pressure.

[0032] (Uses, Compositions) The salts of reduced β-nicotinamide mononucleotide described above do not exhibit deliquescent properties, and are therefore easier to distribute and handle than conventional amorphous disodium salts of NMNH, and can be used in a variety of applications where NMNH is used. For example, salts of reduced β-nicotinamide mononucleotide or compositions containing salts of reduced β-nicotinamide mononucleotide can be used as foods, nutrient functional foods, foods for specified health uses, nutritional supplements, beverages, cosmetics, pet foods, veterinary drugs, pharmaceuticals, therapeutic drugs, and prophylactic drugs. When used in these applications, they are typically used as compositions containing a salt of reduced β-nicotinamide mononucleotide and 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, a surfactant, and an active ingredient other than a salt of reduced β-nicotinamide mononucleotide.

[0033] The present embodiment will be described below with reference to examples, but the present disclosure is not limited to these examples.

[0034] The conditions for high performance liquid chromatography (HPLC), ion chromatography, and measurement of iron ion concentration in the examples are shown below.

[0035] (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 or 260 nm

[0036] (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

[0037] (Conditions for measuring iron ion concentration) Detection method: Colorimetric determination using IRON BIO manufactured by Roche (This detection method consists of three steps: (1) liberation of iron ions in the sample under acidic conditions, (2) reduction of iron (III) to iron (II) by ascorbic acid, and (3) formation of a complex between FerroZine and iron (II), and is detected by absorbance at 552 nm). Detection wavelength: 552 nm

[0038] Example 1 (Preparation of iron salt of reduced β-nicotinamide mononucleotide) 1.0043 g (2.55 mmol) of disodium salt of reduced β-nicotinamide mononucleotide (NMNH) and 0.3803 g (3.00 mmol) of iron (II) chloride were dissolved in 10 mL of water under a nitrogen atmosphere. A precipitate was deposited when the resulting solution was stirred at room temperature, and a larger amount of precipitate was deposited by adding 10 mL of ethanol. The precipitate was filtered, washed with 20 mL of 50% ethanol and 20 mL of 100% ethanol, and dried under reduced pressure to obtain the iron salt of reduced β-nicotinamide mononucleotide.

[0039] 10.3 mg of the iron salt of reduced β-nicotinamide mononucleotide was dissolved in 10 mL of 20 mM EDTA aqueous solution to prepare an aqueous solution of NMNH salt. The NMNH content of the aqueous solution of NMNH salt was measured using HPLC with a detection wavelength of 340 nm, resulting in a content of 26.0% (w / w). The NMN content was measured using HPLC with a detection wavelength of 260 nm, resulting in a content of 12.6% (w / w). The aqueous solution of the NMNH salt was also analyzed using IRON BIO, resulting in a content of 8.0% (w / w). From these contents, the content ratio of NMNH, NMN, and iron was calculated, resulting in a molar ratio of NMNH, NMN, and iron of 1.00:0.48:1.92. The high ratio of iron to NMNH is due to the oxidation of a portion of NMNH during operation in air.

[0040] Example 2 (Preparation of magnesium salt of reduced β-nicotinamide mononucleotide) 0.2003 g (0.50 mmol) of disodium salt of reduced β-nicotinamide mononucleotide (NMNH) and 0.1546 g (0.76 mmol) of magnesium chloride hexahydrate were dissolved in 10 mL of water. 15 mL of ethanol was added to the resulting solution, and the mixture was stirred at room temperature overnight to form a precipitate. The precipitate was filtered, washed with 20 mL of 75% ethanol and 20 mL of 100% ethanol, and dried under reduced pressure to obtain the magnesium salt of reduced β-nicotinamide mononucleotide.

[0041] 8.0 mg of the magnesium salt of reduced β-nicotinamide mononucleotide was dissolved in 20 mL of water to prepare an aqueous solution of NMNH salt. The NMNH content of the aqueous solution of NMNH salt was measured using HPLC with a detection wavelength set to 340 nm, and the result was 79.0% (w / w). Furthermore, the aqueous solution of NMNH salt was analyzed by ion chromatography to measure the magnesium content, which was 4.3% (w / w). From these contents, the content ratio of NMNH to magnesium was calculated, and the NMNH to magnesium content was found to be in a molar ratio of 1.00:0.76. A small amount of sodium was detected in the obtained salt, and considering the molar ratio of NMNH to magnesium, it was thought that some sodium salt was present.

[0042] Example 3 (Preparation of magnesium salt of reduced β-nicotinamide mononucleotide) 5.0043 g of disodium salt of reduced β-nicotinamide mononucleotide (NMNH) was weighed out and dissolved in 50 mL of water, and 18 mL of the solution was applied to 5.9393 g of anion exchange resin. After stirring for 1 hour, the resin was washed three times with water, and 20 mL of 1 M magnesium chloride was added and stirred for 1 hour. 80 mL of ethanol was added to the resulting supernatant and stirred overnight. The resulting precipitate was washed with 20 mL of 80% ethanol and 20 mL of 100% ethanol and dried under reduced pressure to obtain the magnesium salt of reduced β-nicotinamide mononucleotide.

[0043] 10.1 mg of the magnesium salt of reduced β-nicotinamide mononucleotide was dissolved in 20 mL of water to prepare an aqueous solution of NMNH salt. The NMNH content of the aqueous solution of NMNH salt was measured by HPLC with a detection wavelength set to 340 nm, and the result was 69.9% (w / w). Furthermore, the aqueous solution of NMNH salt was analyzed by ion chromatography to measure the magnesium content, which was 4.8% (w / w). From these contents, the content ratio of NMNH to magnesium was calculated, and it was found that NMNH and magnesium were contained in a molar ratio of 1.00:0.95.

[0044] Example 4 (Preparation of dipotassium salt of reduced β-nicotinamide mononucleotide) 5.0043 g of disodium salt of reduced β-nicotinamide mononucleotide (NMNH) was weighed and dissolved in 50 mL of water, and then 20 mL of the solution was taken, to which 0.2361 g of potassium chloride was added. After stirring, 80 mL of methanol was added and the mixture was stirred overnight. The resulting precipitate was washed with 80% methanol and 100% methanol and dried under reduced pressure to obtain dipotassium salt of reduced β-nicotinamide mononucleotide.

[0045] 10.9 mg of dipotassium salt of reduced β-nicotinamide mononucleotide was dissolved in 20 mL of water to prepare an aqueous solution of NMNH salt. The NMNH content of the aqueous solution of NMNH salt was measured by HPLC with a detection wavelength set to 340 nm, and the result was 61.9% (w / w). Furthermore, the aqueous solution of NMNH salt was analyzed by ion chromatography to measure the potassium content, which was 18.3% (w / w). From these contents, the content ratio of NMNH to potassium was calculated, and the NMNH to potassium content was found to be 1.00:2.55 molar ratio.

[0046] Example 5 (Preparation of diammonium salt of reduced β-nicotinamide mononucleotide) 3.5130 g of disodium salt of reduced β-nicotinamide mononucleotide (NMNH) was weighed out and dissolved in 35 mL of water, and 10 mL of the solution was applied to 3.0098 g of anion exchange resin. After stirring for 4 hours, the resin was washed three times with water, and 10 mL of a 1 M aqueous ammonium chloride solution was added and stirred for 2 hours. 60 mL of ethanol was added to the resulting supernatant and the mixture was stirred overnight. The supernatant was removed and dried under reduced pressure to obtain the diammonium salt of reduced β-nicotinamide mononucleotide.

[0047] 19.8 mg of the diammonium salt of reduced β-nicotinamide mononucleotide was dissolved in 20 mL of water to prepare an aqueous solution of NMNH salt. The NMNH content of the aqueous solution of NMNH salt was measured by HPLC with a detection wavelength set at 340 nm, and the content was found to be 58.1% (w / w). The aqueous solution of NMNH salt was also analyzed by ion chromatography to determine the ammonium (NH ４ ＋ The content of NMNH and ammonium was measured and found to be 8.1% (w / w). From these contents, the content ratio of NMNH and ammonium was calculated and found to be 1.00:2.59 by molar ratio.

[0048] [Example 6] (Deliquescense Test) Each powder was allowed to stand in a thermo-hygrostat at 25°C and 60% humidity for 24 hours, and then the shape was visually confirmed. It was found that the particles of NMNH disodium salt had adhered to each other and had lost their powder fluidity, but the NMNH salts of Examples 1 to 5 all maintained their powder fluidity.

[0049] 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.

[0050] 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. A salt of reduced β - nicotinamide mononucleotide, which is at least one salt selected from divalent metal salts excluding calcium salts, potassium salts, and ammonium salts.

2. The salt of reduced β - nicotinamide mononucleotide according to claim 1, wherein the divalent metal salt is an iron salt or a magnesium salt.

3. A method for producing a salt of reduced β - nicotinamide mononucleotide, which is at least one salt selected from divalent metal salts excluding calcium salts, potassium salts, and ammonium salts, the method comprising: Step (I) of preparing a solution containing reduced β - nicotinamide mononucleotide and at least one ion selected from divalent metal ions excluding calcium ions, potassium ions, and ammonium ions in an aqueous solvent; Step (II) of mixing the solution obtained in Step (I) with a water - miscible organic solvent to precipitate a salt of reduced β - nicotinamide mononucleotide; and Step (III) of separating the salt precipitated in Step (II).

4. The production method according to claim 3, wherein Step (I) is carried out by a method including the following Steps (A) and (B): Step (A) of subjecting reduced β - nicotinamide mononucleotide to an anion - exchange resin to obtain an anion - exchange resin adsorbed with reduced β - nicotinamide mononucleotide; Step (B) of extracting the reduced β - nicotinamide mononucleotide in the anion - exchange resin obtained in Step (A) with an aqueous solution containing at least one ion selected from divalent metal ions excluding calcium ions, potassium ions, and ammonium ions.

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