High-stability NMNH sustained-release pellet and preparation method thereof

Abstract

The invention relates to the field of health food, in particular to a high-stability NMNH sustained-release pellet and a preparation method thereof. The invention discloses a preparation method of a high-stability NMNH sustained-release pellet, and the stability and the sustained-release performance of NMNH are remarkably improved through the synergistic effect of a multi-layer structure design and a functional material. According to the invention, centrifugal granulation is combined with a double-layer coating technology, sodium carboxymethyl cellulose is used as a binder to prepare a mother pill, and povidone, magnesium stearate first coating slurry and second coating slurry containing amino starch, stearic acid grafted chitosan and stearic acid are sequentially sprayed; and finally, constructing a stable pH-responsive three-dimensional network structure through ionic crosslinking of sodium tripolyphosphate and covalent crosslinking of glutaraldehyde. According to the preparation process, nitrogen protection is adopted in the whole process, the activity of NMNH is guaranteed through a freeze-drying process, and the obtained pellet has excellent mechanical strength, slow-release effect and long-term stability.

Description

Technical Field

[0001] This invention relates to the field of health food technology, specifically to a highly stable NMNH sustained-release microsphere and its preparation method. Background Technology

[0002] Coenzyme I (NAD) + Nicotinamide mononucleotide (NMNH) is an important coenzyme in the human body, mainly involved in metabolic reactions such as the tricarboxylic acid cycle and anaerobic glycolysis, playing a crucial role in human health. NMNH is an effective NAD+... + The enhancer, as the reduced form of β-nicotinamide mononucleotide (NMN), can rapidly and efficiently increase NAD in tissues. + level.

[0003] Currently, NMNH is mainly synthesized through chemical methods. Chinese patent CN202310875029.3 discloses a process for preparing reduced nicotinamide mononucleotide under high temperature and high pH conditions, using reduced nicotinamide adenine dinucleotide (NADH) as a raw material and synthesizing NMNH through enzymatic catalysis at pH 7.5–10.0 and 30–60°C. Chinese patent CN202410452498.9 discloses a high-catalytic-performance glucose dehydrogenase mutant and its application in NMNH synthesis, with an optimal reaction temperature of 30°C and a pH of 9.0 ± 0.5, achieving a 100% conversion rate of β-nicotinamide mononucleotide within 2 hours. However, as a reduced form of NMN, NMNH contains reducing functional groups in its molecular structure, resulting in higher chemical activity and susceptibility to oxidation in air; this also makes it difficult to store stably. Therefore, preparing it as microparticles is beneficial for improving its stability. Summary of the Invention

[0004] The purpose of this invention is to provide a highly stable NMNH sustained-release microsphere and its preparation method, so as to solve the problems mentioned in the background art.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a highly stable NMNH sustained-release microsphere and its preparation method, comprising the following steps: Step 1: Sodium carboxymethyl cellulose was dispersed in water to obtain an adhesive slurry; magnesium stearate, povidone K30, and ethanol were mixed to obtain a first coating slurry; amino starch, stearic acid-grafted chitosan, and stearic acid were added to water and ultrasonically dispersed to obtain a second coating slurry; NMNH and sodium bicarbonate were mixed to obtain a premix. Step 2: Under nitrogen protection at 15-25℃, the premixed material is fed into a centrifugal granulator, and simultaneously sprayed with binder slurry to wet the material before centrifugation and pelletizing. The pellets are then freeze-dried until the water content is 1-1.5 wt.% to obtain master pellets. The master pellets are then added to a centrifugal granulator, and simultaneously sprayed with the first coating slurry for the first coating. After the first coating is completed, the temperature is raised to 30-35℃, and the second coating slurry is sprayed for the second coating. After the coating is completed, heating is stopped, and the pellets are dried with cold air until the water content is 1-1.5 wt.% to obtain coated microparticles. Step 3: The coated microparticles were added to an aqueous solution of sodium tripolyphosphate and stirred for 1-2 hours. Then, glutaraldehyde was added and the reaction was carried out for 10-12 hours. After the reaction was completed, the microparticles were centrifuged and washed with anhydrous ethanol and deionized water. The microparticles were then freeze-dried to obtain highly stable NMNH sustained-release microparticles.

[0006] Furthermore, in step 1, the preparation method of the amino starch is as follows: S1: Add sodium chloride to corn starch suspension until the sodium chloride content is 1~1.5 wt.%, add sodium hydroxide solution to adjust the pH value of the system to 8~8.5, add epichlorohydrin at a mass ratio of corn starch to epichlorohydrin of 10:(1~1.5), react at 25~30℃ for 18~20h, wash with water until neutral, and vacuum dry to obtain cross-linked starch; S2: Take 3-5 parts of cross-linked starch and add it to 100 parts of water. Add 10-15 parts of sodium periodate solution with a pH of 4-4.5. React at 35-40℃ for 3-5 hours, wash with water until neutral, and vacuum dry to obtain cross-linked oxidized starch. S3: Dissolve spermidine in an aqueous solution, add hydrochloric acid solution to adjust the pH value to 6~6.5, add cross-linked oxidized starch and ultrasonically disperse evenly, react at 35~40℃ for 2~3h, wash with water until neutral, and vacuum dry to obtain amino starch.

[0007] Furthermore, the amino content in amino starch is 1~2 wt.%; Further, in step 1, the method for preparing the stearic acid-grafted chitosan is as follows: Stearic acid was dispersed in ethanol and stirred to obtain a stearic acid-ethanol solution. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added sequentially to the stearic acid-ethanol solution. After stirring, the solution was added to an O-carboxymethyl chitosan-water solution and reacted at 70-80℃ for 6-9 hours. After precipitation with anhydrous ethanol, the crude product was separated and washed to obtain a crude product. The crude product was then dialyzed with water, freeze-dried, and pulverized to obtain stearic acid-grafted chitosan.

[0008] Furthermore, the degree of substitution of stearic acid-grafted chitosan is 3-5%.

[0009] Furthermore, in step 3, the content of each component in the highly stable NMNH sustained-release microspheres, by weight, is as follows: 200-250 parts NMNH, 25-30 parts sodium bicarbonate, 80-90 parts sodium carboxymethyl cellulose, 135-140 parts povidone K30, 8-12 parts magnesium stearate, 5-10 parts amino starch, 45-50 parts stearic acid grafted chitosan, and 6-8 parts stearic acid.

[0010] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: The present invention uses a mixture of NMNH and sodium bicarbonate as a premix, sodium carboxymethyl cellulose solution as a binder slurry, and uses a centrifugal granulator to granulate NMNH. Then, a double-layer coating technology is used to sequentially spray the first coating slurry and the second coating slurry. Finally, the mixture is cross-linked twice in a sodium tripolyphosphate and glutaraldehyde solution to produce NMNH sustained-release microspheres with high stability.

[0011] In this invention, sodium bicarbonate is added to the premix. Sodium bicarbonate provides a favorable alkaline environment during both the preparation process and the storage of the finished product, accelerating the decomposition of NMNH upon contact with water. The first coating slurry is a mixed solution of magnesium stearate, povidone K30, and ethanol, which can rapidly form a film, creating an initial coating layer for preliminary isolation. The second coating slurry consists of stearic acid-grafted chitosan, amino starch, and stearic acid. The amino starch is obtained through cross-linking, oxidation, and spermidine grafting processes, exhibiting excellent antioxidant capacity and serving as one of the sources of stability for the active substances in the sustained-release microcapsules. The stearic acid-grafted chitosan successfully introduces a long-chain fatty acid (stearic acid) onto the amino group of O-carboxymethyl chitosan, retaining the good biocompatibility and film-forming properties of chitosan while enhancing the hydrophobic barrier ability of the coating material through hydrophobic alkyl chains.

[0012] Finally, the coated microspheres were first ionically crosslinked in sodium tripolyphosphate solution, and then covalently crosslinked with glutaraldehyde. This significantly improved the crosslinking degree of stearic acid-grafted chitosan and amino starch, thus forming a stable three-dimensional network structure. Furthermore, the amphiphilic structure of stearic acid-grafted chitosan promoted interfacial compatibility between amino starch and stearic acid, enhancing the synergistic stability of the multi-component coating system. A dense and complete coating layer was formed on the surface of the microspheres, effectively blocking the damage of NMNH to external moisture and oxygen. This dual crosslinking not only improved the chemical and mechanical stability of the coating membrane but also created a precise pH-responsive intestinal sustained-release effect. The release of NMNH was less than 5% in gastric juice (pH=1.2) and gradually and slowly released in intestinal juice (pH=7.4), promoting stable intestinal absorption.

[0013] This invention employs freeze-drying throughout the process to avoid thermal decomposition of NMNH, ensuring preservation of its activity. The final product achieves advantages such as long-lasting sustained release, minimal gastrointestinal irritation, and high storage stability due to the synergistic effect of its components. This technical solution is simple, highly controllable, and suitable for industrial production, providing an efficient and safe formulation solution for the oral delivery of NMNH. Detailed Implementation

[0014] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] The raw materials used in this invention and their sources are as follows: NMNH is from Manton Biotechnology (Shenzhen) Co., Ltd., food grade; corn starch is from Shandong Shouguang Juneng Jin Corn Development Co., Ltd., food grade; O-carboxymethyl chitosan is from Beijing Solarbio Technology Co., Ltd., food grade; the remaining raw materials have no additional restrictions and are all commercially available products.

[0016] Example 1: A method for preparing highly stable NMNH sustained-release microspheres, comprising the following steps: Step 1: Sodium carboxymethyl cellulose was dispersed in water to obtain an adhesive slurry; magnesium stearate, povidone K30, and ethanol were mixed to obtain a first coating slurry; amino starch, stearic acid-grafted chitosan, and stearic acid were added to water and ultrasonically dispersed to obtain a second coating slurry; NMNH and sodium bicarbonate were mixed to obtain a premix. The preparation method of the amino starch is as follows: S1: Add sodium chloride to corn starch suspension until the sodium chloride content is 1 wt.%, add sodium hydroxide solution to adjust the pH of the system to 8, add epichlorohydrin at a mass ratio of corn starch to epichlorohydrin of 10:1, react at 25°C for 18 h, wash with water until neutral, and vacuum dry to obtain cross-linked starch; S2: Take 3g of cross-linked starch and add it to 100g of water. Add 10g of sodium periodate solution with pH 4. React at 35℃ for 3h and wash with water until neutral. Vacuum dry to obtain cross-linked oxidized starch. S3: Spermine was dissolved in an aqueous solution, and the pH was adjusted to 6-6.5 with hydrochloric acid solution. Cross-linked oxidized starch was added and ultrasonically dispersed evenly. After reacting at 35°C for 2 hours, the mixture was washed with water until neutral and then vacuum dried to obtain amino starch; wherein the amino content was 1 wt.%. The method for preparing the stearic acid-grafted chitosan is as follows: Stearic acid was dispersed in ethanol and stirred to obtain a stearic acid-ethanol solution. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added to the stearic acid-ethanol solution in sequence, stirred, and then added to an O-carboxymethyl chitosan-water solution. The mixture was reacted at 70°C for 6 hours. After precipitation with anhydrous ethanol, the mixture was separated and washed to obtain a crude product. The crude product was dialyzed with water, freeze-dried, and pulverized to obtain stearic acid-grafted chitosan with a degree of substitution of 3%. Step 2: Under nitrogen protection at 15°C, the premixed material was fed into a centrifugal granulator, and simultaneously sprayed with binder slurry to wet the material before centrifugation and pelletizing. The pellets were then freeze-dried until the water content was 1 wt.% to obtain master pellets. The master pellets were then added to the centrifugal granulator, and simultaneously sprayed with the first coating slurry for the first coating. After the first coating was completed, the temperature was raised to 30°C, and the second coating slurry was sprayed for the second coating. After the coating was completed, the heating was stopped, and the pellets were dried with cold air until the water content was 1 wt.% to obtain coated microparticles. Step 3: The coated microspheres were added to an aqueous solution of sodium tripolyphosphate and stirred for 1 hour. Then, glutaraldehyde was added and the reaction was carried out for 10 hours. After the reaction was completed, the microspheres were centrifuged and washed with anhydrous ethanol and deionized water. The microspheres were then freeze-dried to obtain highly stable NMNH sustained-release microspheres. The components in the highly stable NMNH sustained-release microspheres were, by weight, 200 parts NMNH, 25 parts sodium bicarbonate, 80 parts sodium carboxymethyl cellulose, 135 parts povidone K30, 8 parts magnesium stearate, 5 parts amino starch, 45 parts stearic acid grafted chitosan, and 6 parts stearic acid.

[0017] Example 2: A method for preparing highly stable NMNH sustained-release microspheres, comprising the following steps: Step 1: Sodium carboxymethyl cellulose was dispersed in water to obtain an adhesive slurry; magnesium stearate, povidone K30, and ethanol were mixed to obtain a first coating slurry; amino starch, stearic acid-grafted chitosan, and stearic acid were added to water and ultrasonically dispersed to obtain a second coating slurry; NMNH and sodium bicarbonate were mixed to obtain a premix. The preparation method of the amino starch is as follows: S1: Sodium chloride was added to the corn starch suspension until the sodium chloride content was 1.2 wt.%, sodium hydroxide solution was added to adjust the pH of the system to 8.3, epichlorohydrin was added at a mass ratio of corn starch to epichlorohydrin of 10:1.3, the reaction was carried out at 28°C for 19 h, and then washed with water until neutral. The cross-linked starch was obtained by vacuum drying. S2: Take 4g of cross-linked starch and add it to 100g of water. Add 12g of sodium periodate solution with a pH of 4.3. React at 38℃ for 4h and wash with water until neutral. Dry under vacuum to obtain cross-linked oxidized starch. S3: Spermine was dissolved in an aqueous solution, and the pH was adjusted to 6.3 with hydrochloric acid solution. Cross-linked oxidized starch was added and ultrasonically dispersed evenly. After reacting at 37°C for 2.5 h, the mixture was washed with water until neutral and then vacuum dried to obtain amino starch; wherein the amino content was 1.5 wt.%. The method for preparing the stearic acid-grafted chitosan is as follows: Stearic acid was dispersed in ethanol and stirred to obtain a stearic acid-ethanol solution. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added sequentially to the stearic acid-ethanol solution, stirred, and then added to an O-carboxymethyl chitosan-water solution. The mixture was reacted at 75°C for 7 hours. After precipitation with anhydrous ethanol, the mixture was separated and washed to obtain a crude product. The crude product was then dialyzed with water, freeze-dried, and pulverized to obtain stearic acid-grafted chitosan with a degree of substitution of 4.5%. Step 2: Under nitrogen protection at 20°C, the premixed material was fed into a centrifugal granulator, and simultaneously sprayed with binder slurry to wet the material before centrifugation and pelletizing. The pellets were then freeze-dried until the water content was 1.2 wt.% to obtain master pellets. The master pellets were then added to a centrifugal granulator, and simultaneously sprayed with the first coating slurry for the first coating. After the first coating was completed, the temperature was raised to 33°C, and the second coating slurry was sprayed for the second coating. After the coating was completed, heating was stopped, and the pellets were dried with cold air until the water content was 1.2 wt.% to obtain coated microparticles. Step 3: The coated microspheres were added to an aqueous solution of sodium tripolyphosphate and stirred for 1.5 h. Then, glutaraldehyde was added and the reaction was carried out for 11 h. After the reaction was completed, the microspheres were centrifuged and washed with anhydrous ethanol and deionized water. The microspheres were then freeze-dried to obtain highly stable NMNH sustained-release microspheres. The components in the highly stable NMNH sustained-release microspheres were, by weight, 230 parts NMNH, 28 parts sodium bicarbonate, 85 parts sodium carboxymethyl cellulose, 138 parts povidone K30, 10 parts magnesium stearate, 8 parts amino starch, 48 parts stearic acid grafted chitosan, and 7.5 parts stearic acid.

[0018] Example 3: A method for preparing highly stable NMNH sustained-release microspheres, comprising the following steps: Step 1: Sodium carboxymethyl cellulose was dispersed in water to obtain an adhesive slurry; magnesium stearate, povidone K30, and ethanol were mixed to obtain a first coating slurry; amino starch, stearic acid-grafted chitosan, and stearic acid were added to water and ultrasonically dispersed to obtain a second coating slurry; NMNH and sodium bicarbonate were mixed to obtain a premix. The preparation method of the amino starch is as follows: S1: Add sodium chloride to corn starch suspension until the sodium chloride content is 1.5 wt.%, add sodium hydroxide solution to adjust the pH value of the system to 8.5, add epichlorohydrin at a mass ratio of corn starch to epichlorohydrin of 10:1.5, react at 30℃ for 20 h, wash with water until neutral, and vacuum dry to obtain cross-linked starch; S2: Take 5g of cross-linked starch and add it to 100g of water. Add 15g of sodium periodate solution with a pH of 4.5. React at 40℃ for 5h and wash with water until neutral. Dry under vacuum to obtain cross-linked oxidized starch. S3: Spermine was dissolved in an aqueous solution, and the pH was adjusted to 6.5 with hydrochloric acid solution. Cross-linked oxidized starch was added and ultrasonically dispersed evenly. The mixture was reacted at 40°C for 3 hours, washed with water until neutral, and then vacuum dried to obtain amino starch; wherein the amino content was 2 wt.%. The method for preparing the stearic acid-grafted chitosan is as follows: Stearic acid was dispersed in ethanol and stirred to obtain a stearic acid-ethanol solution. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added to the stearic acid-ethanol solution in sequence, stirred, and then added to an O-carboxymethyl chitosan-water solution. The mixture was reacted at 80°C for 9 hours. After precipitation with anhydrous ethanol, the mixture was separated and washed to obtain a crude product. The crude product was then dialyzed with water, freeze-dried, and pulverized to obtain stearic acid-grafted chitosan with a degree of substitution of 5%. Step 2: Under nitrogen protection at 25°C, the premixed material was fed into a centrifugal granulator, and simultaneously sprayed with binder slurry to wet the material before centrifugation and pelletizing. The pellets were then freeze-dried until the water content was 1.5 wt.% to obtain master pellets. The master pellets were then added to the centrifugal granulator, and simultaneously sprayed with the first coating slurry for the first coating. After the first coating was completed, the temperature was raised to 35°C, and the second coating slurry was sprayed for the second coating. After the coating was completed, the heating was stopped, and the pellets were dried with cold air until the water content was 1.5 wt.% to obtain coated microparticles. Step 3: The coated microspheres were added to an aqueous solution of sodium tripolyphosphate and stirred for 2 hours. Then, glutaraldehyde was added and the reaction was carried out for 12 hours. After the reaction was completed, the microspheres were centrifuged and washed with anhydrous ethanol and deionized water. The microspheres were then freeze-dried to obtain highly stable NMNH sustained-release microspheres. The components in the highly stable NMNH sustained-release microspheres were, by weight, 250 parts NMNH, 30 parts sodium bicarbonate, 90 parts sodium carboxymethyl cellulose, 140 parts povidone K30, 12 parts magnesium stearate, 10 parts amino starch, 50 parts stearic acid grafted chitosan, and 8 parts stearic acid.

[0019] Comparative Example 1: Corn starch was used instead of amino starch, and the other parameters were the same as in Example 1.

[0020] Step 1: Sodium carboxymethyl cellulose was dispersed in water to obtain an adhesive slurry; magnesium stearate, povidone K30, and ethanol were mixed to obtain a first coating slurry; corn starch, stearic acid-grafted chitosan, and stearic acid were added to water and ultrasonically dispersed to obtain a second coating slurry; NMNH and sodium bicarbonate were mixed to obtain a premix. The method for preparing the stearic acid-grafted chitosan is as follows: Stearic acid was dispersed in ethanol and stirred to obtain a stearic acid-ethanol solution. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added to the stearic acid-ethanol solution in sequence, stirred, and then added to an O-carboxymethyl chitosan-water solution. The mixture was reacted at 70°C for 6 hours. After precipitation with anhydrous ethanol, the mixture was separated and washed to obtain a crude product. The crude product was dialyzed with water, freeze-dried, and pulverized to obtain stearic acid-grafted chitosan with a degree of substitution of 3%. Step 2: Under nitrogen protection at 15°C, the premixed material was fed into a centrifugal granulator, and simultaneously sprayed with binder slurry to wet the material before centrifugation and pelletizing. The pellets were then freeze-dried until the water content was 1 wt.% to obtain master pellets. The master pellets were then added to the centrifugal granulator, and simultaneously sprayed with the first coating slurry for the first coating. After the first coating was completed, the temperature was raised to 30°C, and the second coating slurry was sprayed for the second coating. After the coating was completed, the heating was stopped, and the pellets were dried with cold air until the water content was 1 wt.% to obtain coated microparticles. Step 3: The coated microspheres were added to an aqueous solution of sodium tripolyphosphate and stirred for 1 hour. Then, glutaraldehyde was added and the reaction was carried out for 10 hours. After the reaction was completed, the microspheres were centrifuged and washed with anhydrous ethanol and deionized water. The microspheres were then freeze-dried to obtain highly stable NMNH sustained-release microspheres. The components in the highly stable NMNH sustained-release microspheres were, by weight, 200 parts NMNH, 25 parts sodium bicarbonate, 80 parts sodium carboxymethyl cellulose, 135 parts povidone K30, 8 parts magnesium stearate, 5 parts amino starch, 45 parts stearic acid grafted chitosan, and 6 parts stearic acid.

[0021] Comparative Example 2: Chitosan was used instead of stearic acid grafted chitosan, and the other parameters were the same as in Example 2.

[0022] Step 1: Sodium carboxymethyl cellulose was dispersed in water to obtain an adhesive slurry; magnesium stearate, povidone K30, and ethanol were mixed to obtain a first coating slurry; amino starch, chitosan, and stearic acid were added to water and ultrasonically dispersed to obtain a second coating slurry; NMNH and sodium bicarbonate were mixed to obtain a premix. The preparation method of the amino starch is as follows: S1: Sodium chloride was added to the corn starch suspension until the sodium chloride content was 1.2 wt.%, sodium hydroxide solution was added to adjust the pH of the system to 8.3, epichlorohydrin was added at a mass ratio of corn starch to epichlorohydrin of 10:1.3, the reaction was carried out at 28°C for 19 h, and then washed with water until neutral. The cross-linked starch was obtained by vacuum drying. S2: Take 4g of cross-linked starch and add it to 100g of water. Add 12g of sodium periodate solution with a pH of 4.3. React at 38℃ for 4h and wash with water until neutral. Dry under vacuum to obtain cross-linked oxidized starch. S3: Spermine was dissolved in an aqueous solution, and the pH was adjusted to 6.3 with hydrochloric acid solution. Cross-linked oxidized starch was added and ultrasonically dispersed evenly. After reacting at 37°C for 2.5 h, the mixture was washed with water until neutral and then vacuum dried to obtain amino starch; wherein the amino content was 1.5 wt.%. Step 2: Under nitrogen protection at 20°C, the premixed material was fed into a centrifugal granulator, and simultaneously sprayed with binder slurry to wet the material before centrifugation and pelletizing. The pellets were then freeze-dried until the water content was 1.2 wt.% to obtain master pellets. The master pellets were then added to a centrifugal granulator, and simultaneously sprayed with the first coating slurry for the first coating. After the first coating was completed, the temperature was raised to 33°C, and the second coating slurry was sprayed for the second coating. After the coating was completed, heating was stopped, and the pellets were dried with cold air until the water content was 1.2 wt.% to obtain coated microparticles. Step 3: The coated microspheres were added to an aqueous solution of sodium tripolyphosphate and stirred for 1.5 h. Then glutaraldehyde was added and the reaction was carried out for 11 h. After the reaction was completed, the microspheres were centrifuged and washed with anhydrous ethanol and deionized water. The microspheres were then freeze-dried to obtain highly stable NMNH sustained-release microspheres. The components in the highly stable NMNH sustained-release microspheres were, by weight, 230 parts NMNH, 28 parts sodium bicarbonate, 85 parts sodium carboxymethyl cellulose, 138 parts povidone K30, 10 parts magnesium stearate, 8 parts amino starch, 48 parts chitosan, and 7.5 parts stearic acid.

[0023] Comparative Example 3: In step 3, only glutaraldehyde was used to crosslink the surface of the coated microspheres, and the other parameters were the same as in Example 3.

[0024] Step 1: Sodium carboxymethyl cellulose was dispersed in water to obtain an adhesive slurry; magnesium stearate, povidone K30, and ethanol were mixed to obtain a first coating slurry; amino starch, stearic acid-grafted chitosan, and stearic acid were added to water and ultrasonically dispersed to obtain a second coating slurry; NMNH and sodium bicarbonate were mixed to obtain a premix. The preparation method of the amino starch is as follows: S1: Add sodium chloride to corn starch suspension until the sodium chloride content is 1.5 wt.%, add sodium hydroxide solution to adjust the pH value of the system to 8.5, add epichlorohydrin at a mass ratio of corn starch to epichlorohydrin of 10:1.5, react at 30℃ for 20 h, wash with water until neutral, and vacuum dry to obtain cross-linked starch; S2: Take 5g of cross-linked starch and add it to 100g of water. Add 15g of sodium periodate solution with a pH of 4.5. React at 40℃ for 5h and wash with water until neutral. Dry under vacuum to obtain cross-linked oxidized starch. S3: Spermine was dissolved in an aqueous solution, and the pH was adjusted to 6.5 with hydrochloric acid solution. Cross-linked oxidized starch was added and ultrasonically dispersed evenly. The mixture was reacted at 40°C for 3 hours, washed with water until neutral, and then vacuum dried to obtain amino starch; wherein the amino content was 2 wt.%. The method for preparing the stearic acid-grafted chitosan is as follows: Stearic acid was dispersed in ethanol and stirred to obtain a stearic acid-ethanol solution. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added to the stearic acid-ethanol solution in sequence, stirred, and then added to an O-carboxymethyl chitosan-water solution. The mixture was reacted at 80°C for 9 hours. After precipitation with anhydrous ethanol, the mixture was separated and washed to obtain a crude product. The crude product was then dialyzed with water, freeze-dried, and pulverized to obtain stearic acid-grafted chitosan with a degree of substitution of 5%. Step 2: Under nitrogen protection at 25°C, the premixed material was fed into a centrifugal granulator, and simultaneously sprayed with binder slurry to wet the material before centrifugation and pelletizing. The pellets were then freeze-dried until the water content was 1.5 wt.% to obtain master pellets. The master pellets were then added to the centrifugal granulator, and simultaneously sprayed with the first coating slurry for the first coating. After the first coating was completed, the temperature was raised to 35°C, and the second coating slurry was sprayed for the second coating. After the coating was completed, the heating was stopped, and the pellets were dried with cold air until the water content was 1.5 wt.% to obtain coated microparticles. Step 3: The coated microspheres were added to an aqueous solution of glutaraldehyde and stirred for 12 hours. After the reaction was completed, the mixture was centrifuged and washed with anhydrous ethanol and deionized water. The microspheres were then freeze-dried to obtain highly stable NMNH sustained-release microspheres. The components in the highly stable NMNH sustained-release microspheres were, by weight, 250 parts NMNH, 30 parts sodium bicarbonate, 90 parts sodium carboxymethyl cellulose, 140 parts povidone K30, 12 parts magnesium stearate, 10 parts amino starch, 50 parts stearic acid grafted chitosan, and 8 parts stearic acid.

[0025] Experiment: Microspheres with a specification of 0.2 g / sphere were prepared according to the preparation methods in Examples 1-3 and Comparative Examples 1-3 and tested.

[0026] Microsphere storage stability test: Six samples were taken from each group and numbered 1 to 6. After being placed in an environment of 25℃ and 60% relative humidity for 1h, 2h, 3h, 4h, 5h, and 6h (corresponding to samples numbered 1 to 6), the capsules were broken in 100mL of alkaline aqueous solution (the alkaline aqueous solution was a mixture of sodium carbonate and sodium bicarbonate, containing 1g of sodium carbonate and 1g of sodium bicarbonate per 100mL). The purity of NMNH was analyzed by HPLC, and the detection method is shown in Table 1 below.

[0027] Table 1. The formula for calculating the retention rate of NMNH is: Retention rate = (NMNH content after rupture × 100 / initial NMNH content) × 100% The experimental results are shown in Table 2.

[0028] Table 2. Sustained-release effect test: The release of the samples in gastric juice and intestinal juice was tested respectively; 10g of the sample was weighed and added to an intelligent dissolution apparatus containing artificial simulated gastric juice (pH=1.2), and kept at a constant temperature of 37℃ and a rotation speed of 250rpm; every 1 hour, 2.00mL of supernatant was quickly taken out and 2.00mL of fresh mother liquor was added at the same time, and sampling was continued for 2 hours; using the same method, the drug release of each sample pellet in artificial simulated intestinal juice (pH=7.4) at 37℃ was determined, and every 2 hours, 2.00mL of supernatant was quickly taken out and 2.00mL of fresh mother liquor was added at the same time, and sampling was continued for 6 hours. Because NMNH exhibits poor stability in acidic and slightly alkaline environments, making effective monitoring difficult, the release of its oxidation product, NMN, was tested to aid in elucidating the release rate of NMNH. Ultraviolet-visible spectrophotometry (referencing Chinese Pharmacopoeia 2015, Part IV, General Chapter 0401) was used to measure absorbance at a wavelength of 265 nm. A control group was also established, prepared with water to a solution containing approximately 0.04 mg of NMN per mL, and the same method was used to determine the cumulative release rate per tablet. The experimental results are shown in Table 3.

[0029] Table 3.

[0030] Conclusion: The data from Example 1 and Comparative Example 1 show that amino starch has better antioxidant properties and can improve the stability of microspheres compared with ordinary food-grade corn starch. In addition, in gastric juice and intestinal juice, the cross-linked network formed by amino starch and stearic acid grafted chitosan through covalent and hydrogen bonding disintegrates slowly and has a good sustained-release effect.

[0031] Data from Example 2 and Comparative Example 2 show that, compared with chitosan, stearic acid-grafted chitosan has an amphiphilic molecular structure, acting as a molecular bridge and interfacial compatibility between amino starch and stearic acid, thereby enhancing the strong interaction between amino starch and stearic acid and forming a coating with a complete structure and uniform composition. During room temperature storage, it exhibits good hydrophobic properties, preventing the microspheres from absorbing moisture and accelerating NMNH decomposition, thus improving storage stability. Simultaneously, the complete and uniform coating structure demonstrates excellent pH response, resulting in slower release of the microspheres into gastric and intestinal fluids, which is beneficial for stable intestinal absorption.

[0032] Data from Example 3 and Comparative Example 3 show that when glutaraldehyde is used alone for cross-linking to prepare the coating, the release rate of the microparticles in the gastric juice environment is greatly increased. However, the microparticles prepared by the double cross-linking method have a low release rate in acidic gastric juice, effectively preventing NMNH from being oxidized in a strong acid environment, thereby improving the absorption rate of active substances in the intestine.

[0033] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A process for the preparation of high stability NMNH sustained release pellets, characterized by: Comprising the following steps: Step 1: Mixing NMNH powder and sodium bicarbonate powder to obtain a premix under the protection of nitrogen at 15~25℃, and then putting the premix into a centrifugal granulator, and spraying binder slurry to wet the premix for centrifugal pill-making, and then freeze-drying to obtain mother pills; adding the mother pills into the centrifugal granulator, and spraying the first coating slurry for the first coating, and then increasing the temperature to 30~35℃, and continuously spraying the second coating slurry for the second coating, and then stopping heating after the coating is completed, and then blowing cold air to dry to obtain coated pills; Step 2: Adding the coated pills into a sodium tripolyphosphate aqueous solution, stirring for 1~2h, and then adding glutaraldehyde and reacting for 10~12h, and then centrifuging and washing with anhydrous ethanol and deionized water, and then freeze-drying to obtain high-stability NMNH sustained-release pills.

2. The process for the preparation of high stability NMNH sustained release pellets as claimed in claim 1, wherein: In step 1, the binder slurry is a sodium carboxymethyl cellulose aqueous solution.

3. The process for the preparation of high stability NMNH sustained release pellets as claimed in claim 1, wherein: In step 1, the first coating slurry is obtained by mixing magnesium stearate, povidone K30 and ethanol.

4. The process for the preparation of high stability NMNH sustained release pellets as claimed in claim 1, wherein: In step 1, the second coating slurry is obtained by mixing amino starch, stearic acid grafted chitosan, stearic acid and water.

5. The process for the preparation of high stability NMNH sustained release pellets as claimed in claim 4, wherein: The preparation method of the amino starch comprises the following steps: S1: adding sodium chloride to a corn starch suspension until the sodium chloride content is 1~1.5wt.%, adding sodium hydroxide solution to adjust the pH value of the system to 8~8.5, and adding epichlorohydrin, and then reacting at 25~30℃ for 18~20h, and then washing to neutral, and then vacuum drying to obtain cross-linked starch; S2: taking the cross-linked starch and adding it into water, and then adding sodium periodate solution with a pH value of 4~4.5 dropwise, and then reacting at 35~40℃ for 3~5h, and then washing to neutral, and then vacuum drying to obtain cross-linked-oxidized starch; S3: dissolving spermidine in an aqueous solution, adding hydrochloric acid solution to adjust the pH value to 6~6.5, adding cross-linked-oxidized starch and uniformly dispersing by ultrasonic, and then reacting at 35~40℃ for 2~3h, and then washing to neutral, and then vacuum drying to obtain amino starch.

6. The process for the preparation of high stability NMNH sustained release pellets as claimed in claim 5, wherein: In S3, the amino content in the amino starch is 1~2wt.%.

7. The process for the preparation of high stability NMNH sustained release pellets as claimed in claim 4, wherein: In step 1, the preparation method of the stearic acid grafted chitosan is as follows: dispersing stearic acid in ethanol to obtain a stearic acid-ethanol solution; adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide into the stearic acid-ethanol solution in sequence, stirring, and then adding into an O-carboxymethyl chitosan-aqueous solution, and then reacting at 70~80℃ for 6~9h, precipitating with anhydrous ethanol, separating, washing to obtain a crude product, adding water for dialysis, freeze-drying, and crushing to obtain stearic acid grafted chitosan.

8. The process for the preparation of high stability NMNH sustained release pellets as claimed in claim 7, wherein: The degree of substitution of the stearic acid grafted chitosan is 3~5%.

9. The process for the preparation of high stability NMNH sustained release pellets as claimed in claim 1, wherein: In step 2, in the high-stability NMNH sustained-release pills, the content of each component is 200~250 parts of NMNH, 25~30 parts of sodium bicarbonate, 80~90 parts of sodium carboxymethyl cellulose, 135~140 parts of povidone K30, 8~12 parts of magnesium stearate, 5~10 parts of amino starch, 45~50 parts of stearic acid grafted chitosan, and 6~8 parts of stearic acid.

10. NMNH sustained-release pills prepared by the method according to any one of claims 1~9.

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