Alpha-ketoglutarate of nicotinamide ribose as well as preparation method and application thereof

Abstract

The invention provides alpha-ketoglutarate of nicotinamide ribose as well as a preparation method and application of the alpha-ketoglutarate, and belongs to the technical field of medicines. In the alpha-ketoglutaric acid salt of nicotinamide ribose, the molar ratio of nicotinamide ribose to alpha-ketoglutaric acid is 1: 1. Through efficient combination of the nicotinamide ribose and the alpha-ketoglutaric acid, the synergistic bioavailability of the nicotinamide ribose and the alpha-ketoglutaric acid is optimized, the stability of the nicotinamide ribose is remarkably improved, and the anti-aging effect of the nicotinamide ribose composition is remarkably higher than that of single nicotinamide ribose and alpha-ketoglutaric acid and physical mixing of the nicotinamide ribose and the alpha-ketoglutaric acid.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to α-ketoglutarate of nicotinamide riboside, its preparation method and application. Background Art

[0002] As an important derivative of vitamin B3, nicotinamide riboside (NR) has attracted much attention in recent years due to its significant role in regulating cell metabolism and anti-aging fields. NR participates in various physiological processes by being converted into nicotinamide adenine dinucleotide (NAD + ), including energy metabolism, DNA repair and antioxidant stress, etc. Research shows that supplementing NR can effectively increase the level of NAD + in the body, thereby improving metabolism-related dysfunction, neurodegenerative diseases and cardiovascular diseases associated with aging. However, the chemical stability of NR is poor, it is easy to absorb moisture and degrade, and this defect severely limits its long-term storage and practical application as a drug or nutritional supplement.

[0003] In addition, in the field of preparation and purification of NR, existing processes mostly adopt conventional crystallization or chromatographic separation techniques, but there are problems such as insufficient purity, complex operation or high cost. Strong acidic cation exchange resin has been widely used in the purification of bioactive substances due to its high-efficiency selective adsorption ability, however, its specific application in the purification of NR derivatives still lacks systematic research. Existing purification processes of NR salts are difficult to meet the dual requirements of high purity (>99%) and high stability at the same time, resulting in uneven product quality.

[0004] Existing patent CN11454311A provides a class of organic salts of nicotinamide riboside and their compositions, which are formed by salifying nicotinamide riboside with citric acid or malic acid. The decomposition rate of the organic salt of nicotinamide riboside at 2-8°C for 6 months is 0.99% - 1.44%, and there is still room for further improvement. Secondly, the organic salt of nicotinamide riboside prepared in this patent has not been purified and cannot be directly applied to production. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides α-ketoglutarate of nicotinamide riboside, its preparation method and application. In the α-ketoglutarate of nicotinamide riboside, nicotinamide riboside forms a salt with α-ketoglutaric acid, optimizing their synergistic bioavailability.

[0006] The technical solution of the present invention is as follows:

[0007] The first object of the present invention is to provide that the molar ratio of nicotinamide riboside to α-ketoglutaric acid is 1:1.

[0008] The present invention combines nicotinamide riboside and α-ketoglutaric acid in a molar ratio of 1:1, achieving efficient binding and optimizing their synergistic bioavailability.

[0009] The second object of the present invention is to provide a composition of the α-ketoglutarate of nicotinamide riboside, including the α-ketoglutarate of nicotinamide riboside as described in claim 1.

[0010] The third object of the present invention is to provide a method for preparing the α-ketoglutarate of nicotinamide riboside, characterized by including the following steps:

[0011] (1) Under a protective atmosphere, after reducing nicotinamide riboside chloride, an active reduction intermediate is obtained;

[0012] (2) Under a protective atmosphere, after mixing the active reduction intermediate with α-ketoglutaric acid, an oxidant is added for oxidation, and after filtration, washing, and drying in sequence, a crude product of the α-ketoglutarate of nicotinamide riboside is obtained;

[0013] (3) The crude product of the α-ketoglutarate of nicotinamide riboside is purified through a strongly acidic cation exchange resin to obtain the α-ketoglutarate of nicotinamide riboside.

[0014] The active reduction intermediate in step (1) is: 1-((3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxin-4-yl)-1,4-dihydropyridine-4-carboxamide.

[0015] In the present invention, the protective atmosphere refers to an atmosphere that does not participate in the reaction, specifically referring to a nitrogen atmosphere, a helium atmosphere, an argon atmosphere, etc.

[0016] In a specific embodiment, step (1) is specifically: under a protective atmosphere, controlling the temperature to be -5 to 0 °C, adding concentrated sulfuric acid to an organic solvent, adding 2,2-dimethoxypropane 5 to 30 minutes after the addition is complete, adding nicotinamide riboside chloride 3 to 10 minutes later, stirring until room temperature, after the reaction is complete, adding a saturated salt solution at -20 to 0 °C, continuing to stir for 1 to 12 hours, filtering, collecting the filtrate and adjusting the pH to 6, a white solid precipitates, washing the white precipitate and drying it, then dissolving it in water, adding sodium bicarbonate and sodium dithionite, continuing to stir for 0.5 to 2 hours, adding ethyl acetate to extract the aqueous phase, collecting the organic phase, and drying to obtain the active reduction intermediate.

[0017] In a specific embodiment, step (2) is specifically as follows: Under a protective atmosphere, control the temperature to -10 to -5 °C, dissolve the active reducing intermediate of step (1) in an organic solvent, add α-ketoglutaric acid, stir for 1 to 12 h until clear, add cobalt acetate tetrahydrate and / or cobalt acetate, the solution turns pink, continue to stir for 5 to 60 min, then add an oxidant, the solution becomes clear, continue to stir for 0.5 to 2 hours, add an organic solvent, precipitates will form, and under a protective atmosphere, filter and wash in sequence, and then dry below -5 °C to obtain the crude α-ketoglutarate salt of nicotinamide ribose.

[0018] In a specific embodiment, in step (2), the oxidant includes at least one of hydrogen peroxide, tert-butyl hydroperoxide, m-chloroperbenzoic acid, peroxyacetone, manganese dioxide, Dess-Martin periodinane, 2-iodoxybenzoic acid, pyridinium chlorochromate, pyridinium dichromate, Jones reagent, and 2,2,6,6-tetramethylpiperidine 1-oxyl.

[0019] In a specific embodiment, the oxidant is at least one of hydrogen peroxide, pyridinium chlorochromate, and tert-butyl hydroperoxide.

[0020] In a specific embodiment, in step (3), the strongly acidic cation exchange resin is Dowex 50×4, Amberlite IR-120, Amberlite IR-124, Purolite C100, Lewatit S108, D001 macroporous strongly acidic styrene-based cation exchange resin, 001×8 type strongly acidic cation exchange resin, 001×13 type strongly acidic cation exchange resin, Dow resin IR100NA, Rohm and Haas AMBERLITE IR120Na, Purolite C100E, Bayer NM60, Lanxess SP112 of Germany, Rohm and Haas AMBERLITE TM IRN97H, Mitsubishi SK1B of Japan, GC104, GC107, GC107B, GC108, GC109, GC110, and one or several of them in series.

[0021] The fourth object of the present invention is to provide the application of the α-ketoglutarate salt of nicotinamide ribose in the field of anti-aging.

[0022] The fourth object of the present invention is to provide the application of the α-ketoglutarate salt of nicotinamide ribose in nutritional supplements.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] 1. The invention uses strongly acidic cation exchange resin for the purification of crude products. Cation exchange resin can selectively adsorb and purify target molecules according to the charge characteristics of molecules, thereby improving the selectivity and efficiency of purification. Through the purification process of cation exchange resin, impurities can be effectively removed, and high-purity products can be obtained. The operation of cation exchange resin is simple and easy to control, and the purification process can be optimized by adjusting the elution conditions. Cation exchange resin has the characteristics of stable chemical properties and strong rigidity, and can be operated under high pH conditions. Cation exchange resin can be reused, and its exchange capacity can be restored through appropriate cleaning and regeneration processes, reducing production costs. Compared with some traditional purification methods, the purification process using cation exchange resin produces less waste and has less impact on the environment. The quality and performance of the products purified by cation exchange resin are usually more stable.

[0025] 2. The invention forms a salt of α-ketoglutaric acid and nicotinamide riboside at a molar ratio of 1:1, which not only significantly improves the stability of nicotinamide riboside, but also its anti-aging effect is significantly higher than that of single nicotinamide riboside and α-ketoglutaric acid, as well as the physical mixture of nicotinamide riboside and α-ketoglutaric acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a line graph of the survival period data of four groups of mice. DETAILED DESCRIPTION OF THE INVENTION

[0027] The following specific examples are used to further illustrate the present invention, but the implementation manners of the invention are not limited thereto.

[0028] Example 1

[0029] Preparation of nicotinamide riboside α-ketoglutarate:

[0030] Under nitrogen protection, at a temperature of -5 to 0 °C, in anhydrous acetonitrile (60 ml), concentrated sulfuric acid (0.43 ml, 8.00 mmol) was slowly added dropwise. After 5 minutes of addition, 2,2-dimethoxypropane (10.0 eq, 117.53 mmol) was added and stirred. After 5 minutes, nicotinamide riboside chloride (1.00 eq, 11.75 mmol) was added and stirred until room temperature. After the reaction was monitored by TLC and completed, saturated sodium carbonate solution (0.55 eq, 6.00 mmol) was added at 0 °C, and stirring was continued for 1 hour. The system was filtered, and the pH of the filtrate was adjusted to 6. White solid precipitated out. The white precipitate was washed with 60 ml each of methyl tert-butyl ether / methanol and dried. The white solid was dissolved in a small amount of water, sodium bicarbonate (3.00 eq, 33.00 mmol) and sodium dithionite (1.15 eq, 13.00 mmol) were added, and stirring was continued for 1 hour. 50 ml × 2 of ethyl acetate was added to extract the aqueous phase. The organic phase was collected and dried over anhydrous sodium sulfate to obtain a yellow solid, namely the reducing intermediate (1-((3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxin-4-yl)-1,4-dihydropyridine-4-carboxamide), with a yield of 55%.

[0031] Characterization data: (400 MHz, MeOD) 7.01 (s, 1H), 5.95 (d, J = 6.56 Hz, 1H), 4.97 (d, J = 4.08 Hz, 1H), 4.92 - 4.88 (m, 1H), 4.77 - 4.75 (m, 1H), 4.71 - 4.66 (m, 1H,), 4.01 - 3.98 (m, 1H), 3.70 - 3.61 (m, 2H), 2.96 (s, 2H), 1.49 (s, 3H), 1.30 (s, 3H);

[0032] Under nitrogen protection, at a temperature of -10 to -5 °C, the reducing intermediate (1.00 eq, 22.00 mmol) was dissolved in 60 mL of methanol, α-ketoglutaric acid (1.24 eq, 0.0264 mol) was added, and stirring was carried out for 2 h until it became clear. Cobalt(II) acetate tetrahydrate (1.1 eq, 23.2 mmol) was added, and the solution turned pink. After continuing to stir for 30 min, 190 μl of 30% hydrogen peroxide solution was added, and the solution became clear immediately. After continuing to stir for 1 hour, 100 mL of anhydrous methyl tert-butyl ether was added, and precipitation occurred. It was filtered under nitrogen protection, rinsed with anhydrous methyl tert-butyl ether and anhydrous acetonitrile, and dried below -5 °C to obtain the product (0.0123 mol). The molar ratio of nicotinamide ribose (NR) to α-ketoglutaric acid in the product was 1:1, with a yield of 31% and a purity of 94.13%.

[0033] Characterization data: (400 MHz, MeOD): δ 9.72 (s, 1H), 9.42 - 9.43 (d, 1H), 9.01 - 9.03 (d, 1H), 8.25 - 8.30 (m, 1H), 6.18 - 6.19 (d, 1H), 4.42 - 4.46 (m, 2H), 4.30 - 4.32 (t, 1H), 4.01 - 4.05 (dd, 1H), 3.76 - 3.89 (m, 3H), 2.59 (t, 2H);

[0034] MS(ESI+): 256.96 [M+1], MS(ESI-): 145.12 [M-1]

[0035] Take 200 g of Dowex 50W×4 and 100 g of Amberlite IR-120 to pack the column. Dissolve nicotinamide riboside α-ketoglutarate in a small amount of ultrapure water. Use an aqueous solution of 0.01% (v / v) α-ketoglutarate / 0.05% (v / v) trifluoroacetate as the mobile phase, with a flow rate of 5 ml / min. Purify through a flush chromatographic system, monitor the absorption at 254 nm, discard the first and last volumes, collect the middle fraction, and freeze-dry to obtain the pure product with a yield of 83% and a purity of 99.47%.

[0036] Example 2

[0037] Preparation of nicotinamide riboside α-ketoglutarate:

[0038] Under nitrogen protection, at a temperature of -5 to 0 °C, slowly add concentrated sulfuric acid (0.43 ml, 8.00 mmol) to anhydrous acetonitrile (30 ml). After adding, add 2,2-dimethoxypropane (10.0 eq, 117.53 mmol) and stir. After 5 minutes, add nicotinamide riboside chloride (1.00 eq, 11.75 mmol) and stir until room temperature. After monitoring the reaction by TLC and completion, add saturated sodium carbonate solution (0.55 eq, 6.00 mmol) at -10 °C and continue stirring for 2 hours. Filter the system, adjust the pH of the filtrate to 6, and white solid will precipitate. Wash the white precipitate with 30 ml each of methyl tert-butyl ether / methanol and dry. Dissolve the white solid in a small amount of water, add sodium bicarbonate (3.00 eq, 33.00 mmol) and sodium dithionite (1.15 eq, 13.00 mmol), continue stirring for 1 hour, add 100 ml × 2 of ethyl acetate to extract the aqueous phase, collect the organic phase, and dry over anhydrous sodium sulfate to obtain a yellow solid with a yield of 74%.

[0039] Characterization data: (400 MHz, MeOD) 7.01 (s, 1H), 5.95 (d, J = 6.56 Hz, 1H), 4.97 (d, J = 4.08 Hz, 1H), 4.92 - 4.88 (m, 1H), 4.77 - 4.75 (m, 1H), 4.71 - 4.66 (m, 1H,), 4.01 - 3.98 (m, 1H), 3.70 - 3.61 (m, 2H), 2.96 (s, 2H), 1.49 (s, 3H), 1.30 (s, 3H);

[0040] Under nitrogen protection, at a temperature of -10 to -5 °C, dissolve the reducing intermediate (1.00 eq, 22.00 mmol) in 120 mL of methanol, add α-ketoglutaric acid (1.24 eq, 0.0264 mol), stir overnight until clear, add cobalt(II) acetate tetrahydrate (1.1 eq, 23.2 mmol), the solution turns pink. After continuing to stir for 5 min, add 380 μL of 30% hydrogen peroxide solution, the solution immediately becomes clear. After continuing to stir for half an hour, add 200 mL of anhydrous methyl tert-butyl ether, precipitate forms. Filter under nitrogen protection, rinse with anhydrous methyl tert-butyl ether and anhydrous acetonitrile, dry below -5 °C to obtain the product (0.0145 mol). The molar ratio of nicotinamide ribose (NR) to α-ketoglutaric acid in the product is 1:1, yield: 42%, purity 95.96%.

[0041] Characterization data:

[0042] (400 MHz, MeOD): δ 9.72 (s, 1H), 9.42 - 9.43 (d, 1H), 9.01 - 9.03 (d, 1H), 8.25 - 8.30 (m, 1H), 6.18 - 6.19 (d, 1H), 4.42 - 4.46 (m, 2H), 4.30 - 4.32 (t, 1H), 4.01 - 4.05 (dd, 1H), 3.76 - 3.89 (m, 3H), 2.59 (t, 2H)

[0043] MS(ESI+): 256.96 [M+1], MS(ESI-): 145.12 [M-1];

[0044] Take 200 g of Dowex 50×4 and pack the column. Dissolve nicotinamide ribose α-ketoglutarate in a small amount of ultrapure water. Use an aqueous solution of 0.01% (v / v) α-ketoglutarate as the mobile phase, with a flow rate of 5 ml / min. Purify through a flush chromatographic system, monitor the absorption at 254 nm, discard the first and last volumes, collect the middle fraction, and freeze-dry to obtain the pure product, with a yield of 88% and a purity of 99.64%.

[0045] Example 3

[0046] Preparation of nicotinamide riboside α-ketoglutarate:

[0047] Under nitrogen protection, at a temperature of -5 to 0 °C, in anhydrous acetonitrile (60 ml), concentrated sulfuric acid (0.23 ml, 4.00 mmol) was slowly added dropwise. After 30 minutes of addition, 2,2-dimethoxypropane (4.0 eq, 57.23 mmol) was added and stirred. After 5 minutes, nicotinamide riboside chloride (1.00 eq, 4.85 mmol) was added and stirred until room temperature. After the reaction was monitored by TLC and completed, saturated sodium carbonate solution (0.55 eq, 2.56 mmol) was added at -20 °C, and stirring was continued overnight. The system was filtered, and the filtrate was adjusted to pH 6, and white solid precipitated. The white precipitate was washed with 60 ml each of anhydrous acetonitrile / methanol and dried. The white solid was dissolved in a small amount of water, sodium bicarbonate (3.00 eq, 16.30 mmol) and sodium dithionite (1.15 eq, 5.60 mmol) were added, and stirring was continued for 1 hour. Ethyl acetate (70 ml × 2) was added to extract the aqueous phase, and the organic phase was collected and dried over anhydrous sodium sulfate to obtain a yellow solid with a yield of 82%.

[0048] Characterization data: (400 MHz, MeOD) 7.01 (s, 1H), 5.95 (d, J = 6.56 Hz, 1H), 4.97 (d, J = 4.08 Hz, 1H), 4.92 - 4.88 (m, 1H), 4.77 - 4.75 (m, 1H), 4.71 - 4.66 (m, 1H,), 4.01 - 3.98 (m, 1H), 3.70 - 3.61 (m, 2H), 2.96 (s, 2H), 1.49 (s, 3H), 1.30 (s, 3H);

[0049] Under nitrogen protection, at a temperature of -10 to -5 °C, the reducing intermediate (1.00 eq, 22.00 mmol) was dissolved in 120 mL of methanol, α-ketoglutaric acid (1.24 eq, 0.0264 mol) was added, and stirring was continued overnight until clear. Cobalt acetate tetrahydrate (1.1 eq, 23.2 mmol) was added, and the solution turned pink. After continuing to stir for 5 min, 600 μl of 30% TBHP solution was added, and the solution became clear. After continuing to stir for half an hour, 100 mL of anhydrous methyl tert-butyl ether was added, and precipitation occurred. It was filtered under nitrogen protection, washed with anhydrous acetonitrile, and dried below -5 °C to obtain the product (0.0145 mol). The molar ratio of nicotinamide riboside (NR) to α-ketoglutaric acid in the product was 1:1, the yield was 32%, and the purity was 97.86%.

[0050] Characterization data:

[0051] (400 MHz, MeOD): δ 9.72 (s, 1H), 9.42 - 9.43 (d, 1H), 9.01 - 9.03 (d, 1H), 8.25 - 8.30 (m, 1H), 6.18 - 6.19 (d, 1H), 4.42 - 4.46 (m, 2H), 4.30 - 4.32 (t, 1H), 4.01 - 4.05 (dd, 1H), 3.76 - 3.89 (m, 3H), 2.59 (t, 2H)

[0052] MS(ESI+): 256.96 [M + 1], MS(ESI-): 145.12 [M - 1];

[0053] Load 50 g of Amberlite IR-120 onto a column. Dissolve nicotinamide riboside α-ketoglutarate in a small amount of ultrapure water. Use an aqueous solution of 0.01% (v / v) TFA as the mobile phase with a flow rate of 10 ml / min. Purify through a flush chromatography system, monitor the absorption at 254 nm, discard the initial and final volumes, collect the middle fraction, and freeze-dry to obtain the pure product with a yield of 58% and a purity of 99.09%.

[0054] Example 4

[0055] Preparation of nicotinamide riboside α-ketoglutarate:

[0056] Under nitrogen protection, at a temperature of -5 to 0 °C, slowly add concentrated sulfuric acid (0.43 ml, 8.00 mmol) dropwise to anhydrous acetonitrile (30 ml). After 5 minutes of addition, add 2,2-dimethoxypropane (10.0 eq, 117.53 mmol) and stir. After 5 minutes, add nicotinamide riboside chloride (1.00 eq, 11.75 mmol) and stir until room temperature. After monitoring the reaction to completion by TLC, add saturated sodium carbonate solution (0.55 eq, 6.00 mmol) at -10 °C and continue stirring for 2 hours. Filter the system, adjust the pH of the filtrate to 6, and a white solid will precipitate. Wash the white precipitate with 30 ml each of methyl tert-butyl ether / methanol and dry. Dissolve the white solid in a small amount of water, add sodium bicarbonate (3.00 eq, 33.00 mmol) and sodium dithionite (1.15 eq, 13.00 mmol), continue stirring for 1 hour, add ethyl acetate 100 ml × 2, extract the aqueous phase, collect the organic phase, and dry over anhydrous sodium sulfate to obtain a yellow solid with a yield of 74%.

[0057] Characterization data: (400 MHz, MeOD) 7.01 (s, 1H), 5.95 (d, J = 6.56 Hz, 1H), 4.97 (d, J = 4.08 Hz, 1H), 4.92 - 4.88 (m, 1H), 4.77 - 4.75 (m, 1H), 4.71 - 4.66 (m, 1H,), 4.01 - 3.98 (m, 1H), 3.70 - 3.61 (m, 2H), 2.96 (s, 2H), 1.49 (s, 3H), 1.30 (s, 3H);

[0058] Under nitrogen protection, at a temperature of -10 to -5 °C, dissolve the reducing intermediate (1.00 eq, 22.00 mmol) in 120 mL of methanol, add α-ketoglutaric acid (1.24 eq, 0.0264 mol), stir overnight until clear, add cobalt(II) acetate tetrahydrate (1.1 eq, 23.2 mmol), the solution turns pink, continue to stir for 5 min, then add 400 μL of saturated aqueous PCC solution, the solution becomes clear immediately, continue to stir for half an hour, add 200 mL of anhydrous methyl tert-butyl ether, precipitate appears, filter under nitrogen protection, wash with anhydrous methyl tert-butyl ether and anhydrous acetonitrile, dry below -5 °C to obtain the product (0.0145 mol). The molar ratio of nicotinamide ribose (NR) to α-ketoglutaric acid in the product is 1:1, yield: 36%, purity 95.30%.

[0059] Characterization data:

[0060] (400 MHz, MeOD): δ 9.72 (s, 1H), 9.42 - 9.43 (d, 1H), 9.01 - 9.03 (d, 1H), 8.25 - 8.30 (m, 1H), 6.18 - 6.19 (d, 1H), 4.42 - 4.46 (m, 2H), 4.30 - 4.32 (t, 1H), 4.01 - 4.05 (dd, 1H), 3.76 - 3.89 (m, 3H), 2.59 (t, 2H)

[0061] MS(ESI+): 256.96 [M + 1], MS(ESI-): 145.12 [M - 1];

[0062] Take 40 g each of Amberlite IR-120 and Amberlite IR-124, a total of 120 g, to pack the column. Dissolve nicotinamide ribose α-ketoglutarate in a small amount of ultrapure water, use 0.01% (v / v) formic acid aqueous solution as the mobile phase, with a flow rate of 1 ml / min, purify through a flush chromatographic system, monitor the absorption at 254 nm, discard the first and last volumes, collect the middle fraction, and freeze-dry to obtain the pure product, with a yield of 76% and a purity of 99.55%.

[0063] Stability test:

[0064] The product of Example 1 was divided and filled into 14 bottles, 50 mg per bottle. Nitrogen was filled in, and the bottles were sealed. First, one bottle (initial sample) was tested for each, and the other 12 bottles were stored in two groups, 6 bottles in each group. Then they were stored at 2 - 8 °C and at room temperature of 25 °C respectively. One sample was regularly tested every month, and nicotinamide riboside chloride and the physical mixture of nicotinamide riboside / α-ketoglutaric acid (molar ratio 1:1) were used as controls.

[0065] The purity was detected by HPLC, and the decomposition rate was calculated:

[0066] Mobile phase for HPLC test: Isocratic elution with 5% water (0.1% formic acid) + 95% methanol (0.1% formic acid) (both v / v)

[0067] Wavelength: 254 nm

[0068] Temperature and humidity: 23.0 °C, 54% RH

[0069] Sample dissolution: Dissolved in methanol

[0070] Chromatographic column: ODS-2, 4.6 * 250 mm, 5 μm, pressure constant at 12 - 13 Mpa

[0071] Flow rate: 1.0 mL / min Injection volume: 5 μL

[0072] Running time: ≥15 min

[0073] Table 1. Purity and decomposition rate at 25 °C are as follows:

[0074]

[0075]

[0076] Note: Decomposition rate = (Initial sample purity - Purity of the sample after 6 months) / Initial sample concentration × 100%

[0077] Table 2. Purity and decomposition rate at 2 - 8 °C are as follows:

[0078] Time (month) Example 1 Nicotinamide riboside chloride Physical mixture group (1:1) 0 99.47％ 99.90％ 99.90％ 1 99.46％ 99.52％ 96.52％ 2 99.38％ 99.26％ 94.23％ 3 99.30％ 98.74％ 88.29％ 4 99.25％ 98.30％ 85.98％ 5 99.19％ 98.18％ 80.31％ 6 99.11％ 98.10％ 80.00％ Decomposition rate 0.36％ 1.80％ 19.92％

[0079] Mouse survival period test (as shown in Figure 1 shown):

[0080] To evaluate the anti-aging effect of nicotinamide riboside α-ketoglutarate, C57 / BL male aged mice, 18 months old, were purchased from the Animal Center of West China Hospital, Sichuan University. There were 15 mice in each of the four groups (nicotinamide riboside chloride, α-ketoglutaric acid, Example 1, physical mixture group (1:1)). They were raised under standard experimental conditions, with free access to food and water, and a light cycle of 12 / 12 hours. Intragastric administration was carried out at 10 am every day at a dose of 300 mg / kg / d (calculated based on the final product), lasting for 180 days from the start of the experiment or until the natural death of the mice, and the number of surviving individuals was recorded. This experiment has obtained the approval of the Animal Ethics Committee of Sichuan University, and the relevant regulations on the welfare of laboratory animals in the country were followed during the experimental process. The results showed that Example 1 could help extend the lifespan of mice compared with nicotinamide riboside chloride, α-ketoglutaric acid, and the physical mixture group.

[0081] The above-disclosed are only the preferred embodiments of the present invention. Of course, the scope of the rights of the present invention cannot be limited thereby. Therefore, equivalent changes made according to the claims of the present invention still fall within the scope covered by the present invention.

Claims

1. α-Ketoglutarate of nicotinamide riboside, characterized in that, The molar ratio of nicotinamide riboside to α-ketoglutaric acid is 1:

1.

2. A composition of nicotinamide riboside α-ketoglutarate, characterized in that, It includes the α-ketoglutarate of nicotinamide riboside described in claim 1.

3. The preparation method of α-ketoglutarate of nicotinamide riboside according to claim 1, characterized in that, It includes the following steps: (1) Under a protective atmosphere, after reducing nicotinamide riboside chloride, an active reduction intermediate is obtained; (2) Under a protective atmosphere, after mixing the active reduction intermediate with α-ketoglutaric acid, an oxidant is added for oxidation, and after filtration, washing, and drying in sequence, a crude product of the α-ketoglutarate of nicotinamide riboside is obtained; (3) The crude product of the α-ketoglutarate of nicotinamide riboside is purified by a strongly acidic cation exchange resin to obtain the α-ketoglutarate of nicotinamide riboside.

4. The preparation method of α-ketoglutarate of nicotinamide riboside according to claim 3, characterized in that, Step (1) is specifically as follows: Under a protective atmosphere, the temperature is controlled at -5 to 0 °C. Concentrated sulfuric acid is added to an organic solvent. After 5 to 30 minutes of adding, 2,2-dimethoxypropane is added. After 3 to 10 minutes, nicotinamide riboside chloride is added, and it is stirred until room temperature. After the reaction is completed, a saturated salt solution is added at -20 to 0 °C, and stirring continues for 1 to 12 hours. After filtration, the pH of the filtrate is adjusted to 6, and a white solid precipitates. The white precipitate is washed and dried, then dissolved in water, sodium bicarbonate and sodium dithionite are added, and stirring continues for 0.5 to 2 hours. Then, ethyl acetate is added to extract the aqueous phase, and the organic phase is collected and dried to obtain the active reduction intermediate.

5. The preparation method of α-ketoglutarate of nicotinamide riboside according to claim 3, characterized in that, Step (2) is specifically as follows: Under a protective atmosphere, the temperature is controlled at -10 to -5 °C. The active reduction intermediate obtained in step (1) is dissolved in an organic solvent, α-ketoglutaric acid is added, and stirring is carried out for 1 to 12 h until it becomes clear. Cobalt acetate tetrahydrate and / or cobalt acetate are added, and the solution turns pink. After stirring continues for 5 to 60 min, an oxidant is added, and the solution immediately becomes clear. After stirring continues for 0.5 to 2 hours, an organic solvent is added, and a precipitate precipitates. Under a protective atmosphere, after filtration and washing in sequence, it is dried below -5 °C to obtain the crude product of the α-ketoglutarate of nicotinamide riboside.

6. The preparation method of α-ketoglutarate of nicotinamide riboside according to claim 3, wherein In step (2), the oxidant includes at least one of hydrogen peroxide, tert-butyl hydroperoxide, m-chloroperbenzoic acid, peroxyacetone, manganese dioxide, Dess-Martin periodinane, 2-iodoxybenzoic acid, pyridinium chlorochromate, pyridinium dichromate, Jones reagent, and tetramethylpiperidine oxide.

7. The preparation method of α-ketoglutarate of nicotinamide riboside according to claim 6, characterized in that, The oxidant is at least one of hydrogen peroxide, pyridinium chlorochromate, and tert-butyl hydroperoxide.

8. The preparation method of α-ketoglutarate of nicotinamide riboside according to claim 3, characterized in that, In step (3), the strongly acidic cation exchange resin is one or more of Dowex 50×4, Amberlite IR-120, Amberlite IR-124, Purolite C100, Lewatit S108, D001 macroporous strongly acidic styrene-based cation exchange resin, 001×8 type strongly acidic cation exchange resin, 001×13 type strongly acidic cation exchange resin, Dow resin IR100NA, Rohm and Haas AMBERLITE IR120 Na, Purolite C100E, Bayer NM60, Lanxess SP112 of Germany, Rohm and Haas AMBERLITE TM IRN97H, Mitsubishi SK1B of Japan, GC104, GC107, GC107B, GC108, GC109, GC110 in series connection.

9. The application of the α-ketoglutarate of nicotinamide riboside described in claim 1 in the field of anti-aging.

10. The application of the α-ketoglutarate of nicotinamide riboside described in claim 1 in nutritional supplements.

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