Nadh-containing sustained-release pellets and a method for preparing the same

Abstract

The present application relates to the technical field of health products, in particular to a NADH-containing sustained-release pellet and a preparation method thereof.The NADH-containing sustained-release pellet comprises NADH liposomes, an isolation layer and an enteric outer layer.The isolation layer takes calcium-type octenyl succinate starch as the main raw material, and the enteric outer layer is formed by cross-linking of chitosan-sodium tripolyphosphate.The isolation layer in the present application can not only effectively protect the NADH liposomes from the external environment, but also improve the embedding rate by electrostatic force and combination with the enteric outer layer.The preparation process of the present application is simple, and the preparation conditions are mild.The NADH-containing sustained-release pellet prepared has high storage stability, and can realize almost no release of the NADH component in the stomach and precise release in the intestine.

Description

Technical Field

[0001] This invention relates to the field of health product technology, specifically to a sustained-release microsphere containing NADH and its preparation method. Background Technology

[0002] Reduced coenzyme I (NADH, nicotinamide adenine dinucleotide) is a core high-energy coenzyme and electron transporter in living organisms, widely involved in key physiological processes such as cellular respiration, energy metabolism, redox regulation, DNA repair, and antioxidation. In the fields of biocatalysis, biomedicine, synthetic biology, and the health industry, the NAD⁺ / NADH redox pair is a core cofactor in the vast majority of redox enzymatic reactions. Most core metabolic pathways in living organisms rely on NADH's electron transport and proton transfer functions, making it a crucial substance for maintaining efficient biological reactions. Compared to precursors such as NMN, NADH can directly increase the level of effective coenzymes in the body without complex in vivo conversion, resulting in higher bioavailability and significant advantages in anti-aging repair, metabolic regulation, liver protection, and antioxidation. Although NADH has been extensively studied in the field of anti-aging, its extremely unstable chemical properties—on the one hand, exposure to sunlight, oxygen, and moisture in the air can damage its molecular structure, increasing storage difficulties; on the other hand, it is immediately decomposed upon contact with stomach acid—greatly limit its application in the health supplement field, especially in the preparation of oral formulations. Summary of the Invention

[0003] The purpose of this invention is to provide a sustained-release microsphere containing NADH and a method for preparing the same, so as to solve the problems mentioned in the background art.

[0004] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0005] A method for preparing NADH-containing sustained-release microspheres includes the following steps:

[0006] Step 1: Add lecithin, cholesterol, and Tween 80 to anhydrous ethanol and stir at 50°C to dissolve and obtain a liposome solution; disperse NADH in phosphate buffer and stir to obtain an NADH solution; keep the temperature of the liposome solution constant, add the NADH solution, stir for 30-40 min, remove ethanol by rotary evaporation, and filter through a 0.45 μm microporous membrane to obtain NADH liposomes;

[0007] Step 2: Take calcium-type octenyl succinate starch ester, tea polyphenols, and glycerol and disperse them in deionized water. Gelatinize them at 90~95℃ to obtain an isolation layer emulsion. Mix NADH liposomes with the isolation layer emulsion and spray dry to obtain NADH liposome microspheres embedded in the isolation layer.

[0008] Step 3: The NADH liposome microspheres embedded in the isolation layer were added to a sodium tripolyphosphate solution and stirred for 30 min. Then, chitosan solution was added and stirring was continued for 30 min. After centrifugation, separation, washing and drying, NADH sustained-release microspheres were obtained.

[0009] Further, in step 1, the mass ratio of lecithin, cholesterol, vitamin E, Tween 80 and NADH in the NADH liposome is (15~25):(5~10):(3~5):(12~15):(10~15).

[0010] Furthermore, in step 1, the phosphate buffer solution has a pH of 7.4 and a concentration of 0.01 mol / L.

[0011] Further, in step 2, the preparation method of calcium-type octenyl succinate starch ester is as follows: sodium-type octenyl succinate starch ester is mixed with calcium chloride solution and subjected to ion exchange reaction for 3-4 hours. The product is washed with deionized water, filtered until no chloride ions are present in the solution, vacuum dried and pulverized to obtain calcium-type octenyl succinate starch ester.

[0012] Furthermore, in step 2, the components in the isolation layer emulsion are present in the following proportions by weight: 4-5 parts calcium-type octenyl succinate starch ester, 1-1.5 parts tea polyphenols, 0.5-1 part glycerol, and 1000 parts deionized water.

[0013] Further, in step 2, NADH liposomes and the isolation layer emulsion are mixed at a mass ratio of 1:(3~4).

[0014] Further, in step 3, the chitosan solution is prepared by dispersing chitosan in a 1 wt.% acetic acid solution, stirring to dissolve, and then adjusting the pH to 4 with NaOH to obtain a 1 wt.% chitosan solution.

[0015] Furthermore, in step 3, the concentration of the sodium tripolyphosphate solution is 1 g / L.

[0016] Furthermore, in step 3, the mass ratio of chitosan, NADH liposome microspheres encapsulated in the isolation layer, and sodium tripolyphosphate is 1:1:(2~3).

[0017] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0018] This invention uses chitosan and sodium tripolyphosphate to encapsulate NADH liposomes to prepare microparticles. Sodium tripolyphosphate molecules contain multiple phosphate anions, which can combine with the cationic amino groups in chitosan molecules through electrostatic interactions. Chitosan-sodium tripolyphosphate forms a cross-linked network structure in the enteric outer layer, thereby preventing NADH from dissolving prematurely in the stomach and ensuring that NADH is released to the maximum extent in the intestine.

[0019] In addition to the outer enteric coating, this invention also prepares an isolation layer to embed NADH liposomes. The components of the isolation layer include calcium-type octenyl succinate starch ester, tea polyphenols, and glycerol. Glycerol, as a plasticizer, forms hydrogen bonds with starch molecules through hydroxyl groups, inserting into the starch polysaccharide chains, weakening the hydrogen bond network of the starch molecules themselves, increasing the free volume of molecules, and improving the toughness of the isolation layer. Tea polyphenols, as natural antioxidants, effectively improve the antioxidant function of the isolation layer, thereby protecting the internal NADH liposomes and improving processing and storage stability.

[0020] In this invention, calcium-form octenyl succinate starch ester is used as the matrix material for the isolation layer. This calcium-form octenyl succinate starch ester is prepared from sodium-form octenyl succinate starch ester via an ion exchange reaction. Compared to sodium-form octenyl succinate starch ester, calcium-form octenyl succinate starch ester has lower viscosity, superior processing performance, and higher encapsulation efficiency. Furthermore, the calcium-form octenyl succinate starch ester carries positively charged calcium ions on its surface. During the preparation of the outer enteric coating, NADH liposome microspheres embedded in the isolation layer are added to a sodium tripolyphosphate solution. The sodium tripolyphosphate is adsorbed onto the surface of the embedding layer through electrostatic attraction. Then, a chitosan solution is added, further enhancing the binding force between the intermediate layer and the enteric outer layer through electrostatic interaction, achieving a better encapsulation effect. During the preparation process, if the NADH liposome microspheres encapsulated in the isolation layer are first added to the chitosan solution, since both the isolation layer and chitosan are positively charged, there is a repulsive force between them. On the one hand, this leads to poor encapsulation effect of the enteric outer shell. On the other hand, in the acidic solution, the starch in the isolation layer absorbs water and swells, causing the internal liposomes to dissolve and NADH to denature in acid, thus affecting the processing effect.

[0021] In summary, the present invention has a simple process, mild preparation conditions, and high production efficiency, making it easy to achieve industrial-scale mass production; the product has high storage stability, and NADH can be precisely controlled to be released almost non-existently in the stomach and slowly released in the intestines, resulting in outstanding effects. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] All raw materials used in this invention are commercially available food-grade raw materials; among them, sodium octenyl succinate starch ester (E1450) was purchased from British Industrial Technology (Nanjing) Co., Ltd., and chitosan was purchased from Qingdao Bozhi Huili Biotechnology Co., Ltd.

[0024] Example 1: A method for preparing NADH-containing sustained-release microspheres, comprising the following steps:

[0025] Step 1: Lecithin, cholesterol, and Tween 80 were added to anhydrous ethanol and stirred at 50°C to dissolve and obtain a liposome solution. NADH was dispersed in phosphate buffer and stirred to obtain an NADH solution. While maintaining the temperature of the liposome solution, the NADH solution was added and stirred for 30 min. Ethanol was removed by rotary evaporation. After homogenization for 10 min and ultrasonic dispersion for 5 min, the mixture was filtered through a 0.45 μm microporous membrane to obtain NADH liposomes. The mass ratio of lecithin, cholesterol, vitamin E, Tween 80, and NADH in the NADH liposomes was 15:5:3:12:10. The pH of the phosphate buffer was 7.4, and the concentration was 0.01 mol / L.

[0026] Step 2: Preparation of NADH liposome microspheres embedded in the isolation layer:

[0027] S1: Take 10g of sodium octenyl succinate starch ester and mix it with 1L of calcium chloride solution with a concentration of 0.03mol / L. Carry out the ion exchange reaction at 25℃ for 3h. Wash the product with deionized water and filter until there are no chloride ions in the solution. Dry it in a vacuum drying oven at 1MPa and 40℃ for 3h. Pulverize it to obtain calcium octenyl succinate starch ester.

[0028] S2: Take 4g of calcium-type octenyl succinate starch ester, 1g of tea polyphenols, and 0.5g of glycerol and disperse them in 1000g of deionized water. Gelatinize them at 90℃ for 30min to obtain the isolation layer emulsion.

[0029] S3: Mix NADH liposomes and the isolation layer emulsion at a mass ratio of 1:3, stir for 5 minutes, and then spray dry to obtain NADH liposome microspheres embedded in the isolation layer; the inlet air temperature of the spray dryer is 180℃ and the outlet air temperature is 100℃.

[0030] Step 3: Disperse chitosan in a 1 wt.% acetic acid solution, stir to dissolve, and adjust the pH to 4 with NaOH to obtain a 1 wt.% chitosan solution; disperse sodium tripolyphosphate in deionized water to obtain a sodium tripolyphosphate solution with a concentration of 0.8 g / L; add the NADH liposome microspheres with an isolation layer to the sodium tripolyphosphate solution and stir for 30 min, then add them to the chitosan solution, adjust the pH of the system to 4, and continue stirring for 30 min. After centrifugation, separation, washing, and drying, obtain NADH sustained-release microspheres; wherein, the mass ratio of chitosan, NADH liposome microspheres with an isolation layer, and sodium tripolyphosphate is 1:1:2.

[0031] Example 2: A method for preparing NADH-containing sustained-release microspheres, comprising the following steps:

[0032] Step 1: Add lecithin, cholesterol, and Tween 80 to anhydrous ethanol and stir at 50°C to dissolve and obtain a liposome solution; disperse NADH in phosphate buffer and stir to obtain an NADH solution; keep the temperature of the liposome solution constant, add the NADH solution, stir for 35 min, remove ethanol by rotary evaporation, homogenize for 12 min, sonicate for 8 min, and then filter through a 0.45 μm microporous membrane to obtain NADH liposomes;

[0033] The NADH liposomes contain lecithin, cholesterol, vitamin E, Tween 80, and NADH in a mass ratio of 20:8:4:14:12; the phosphate buffer solution has a pH of 7.4 and a concentration of 0.01 mol / L.

[0034] Step 2: Preparation of NADH liposome microspheres embedded in the isolation layer:

[0035] S1: Take 10g of sodium octenyl succinate starch ester and mix it with 1L of calcium chloride solution with a concentration of 0.03mol / L. Carry out the ion exchange reaction at 25℃ for 3.5h. Wash the product with deionized water and filter until there are no chloride ions in the solution. Dry it in a vacuum drying oven at 1MPa and 45℃ for 3h. Pulverize it to obtain calcium octenyl succinate starch ester.

[0036] S2: Take 4.5g of calcium-type octenyl succinate starch ester, 1.2g of tea polyphenols, and 0.8g of glycerol and disperse them in 1000g of deionized water. Gelatinize them at 92℃ for 30min to obtain the isolation layer emulsion.

[0037] S3: Mix NADH liposomes and the isolation layer emulsion at a mass ratio of 1:3.5, stir for 8 minutes, and then spray dry to obtain NADH liposome microspheres embedded in the isolation layer; the inlet air temperature of the spray dryer is 185℃ and the outlet air temperature is 105℃.

[0038] Step 3: Disperse chitosan in a 1 wt.% acetic acid solution, stir to dissolve, and adjust the pH to 4 with NaOH to obtain a 1 wt.% chitosan solution; disperse sodium tripolyphosphate in deionized water to obtain a sodium tripolyphosphate solution with a concentration of 1 g / L; add the NADH liposome microspheres with an isolation layer to the sodium tripolyphosphate solution and stir for 30 min, then add them to the chitosan solution, adjust the pH of the system to 4, and continue stirring for 30 min. After centrifugation, separation, washing, and drying, obtain NADH sustained-release microspheres; wherein, the mass ratio of chitosan, NADH liposome microspheres with an isolation layer, and sodium tripolyphosphate is 1:1:2.5.

[0039] Example 3: A method for preparing NADH-containing sustained-release microspheres, comprising the following steps:

[0040] Step 1: Add lecithin, cholesterol, and Tween 80 to anhydrous ethanol and stir at 50°C to dissolve and obtain a liposome solution; disperse NADH in phosphate buffer and stir to obtain an NADH solution; keep the temperature of the liposome solution constant, add the NADH solution, stir for 40 min, remove ethanol by rotary evaporation, homogenize for 15 min, sonicate for 10 min, and then filter through a 0.45 μm microporous membrane to obtain NADH liposomes;

[0041] The NADH liposomes contain lecithin, cholesterol, vitamin E, Tween 80, and NADH in a mass ratio of 25:10:5:15:15; the phosphate buffer solution has a pH of 7.4 and a concentration of 0.01 mol / L.

[0042] Step 2: Preparation of NADH liposome microspheres embedded in the isolation layer:

[0043] S1: Take 10g of sodium octenyl succinate starch ester and mix it with 1L of calcium chloride solution with a concentration of 0.03mol / L. Carry out the ion exchange reaction at 25℃ for 4h. Wash the product with deionized water and filter until there are no chloride ions in the solution. Dry it in a vacuum drying oven at 1MPa and 50℃ for 3h. Pulverize it to obtain calcium octenyl succinate starch ester.

[0044] S2: Take 5g of calcium-type octenyl succinate starch ester, 1.5g of tea polyphenols and 1g of glycerol and disperse them in 1000g of deionized water. Gelatinize them at 95℃ for 30min to obtain the isolation layer emulsion.

[0045] S3: Mix NADH liposomes and the isolation layer emulsion at a mass ratio of 1:4, stir for 10 min and then spray dry to obtain NADH liposome microspheres embedded in the isolation layer; the inlet air temperature of the spray dryer is 190℃ and the outlet air temperature is 110℃.

[0046] Step 3: Disperse chitosan in a 1 wt.% acetic acid solution, stir to dissolve, and adjust the pH to 4 with NaOH to obtain a 1 wt.% chitosan solution; disperse sodium tripolyphosphate in deionized water to obtain a sodium tripolyphosphate solution with a concentration of 1 g / L; add the NADH liposome microspheres with an isolation layer to the sodium tripolyphosphate solution and stir for 30 min, then add them to the chitosan solution, adjust the pH of the system to 4, and continue stirring for 30 min. After centrifugation, separation, washing, and drying, obtain NADH sustained-release microspheres; wherein, the mass ratio of chitosan, NADH liposome microspheres with an isolation layer, and sodium tripolyphosphate is 1:1:3.

[0047] Comparative Example 1: In this comparative example, no isolation layer was used to embed NADH liposome microparticles, and the other parameters were the same as in Example 1.

[0048] Step 1: Lecithin, cholesterol, and Tween 80 were added to anhydrous ethanol and stirred at 50°C to dissolve and obtain a liposome solution. NADH was dispersed in phosphate buffer and stirred to obtain an NADH solution. While maintaining the temperature of the liposome solution, the NADH solution was added and stirred for 30 min. Ethanol was removed by rotary evaporation. After homogenization for 10 min and ultrasonic dispersion for 5 min, the mixture was filtered through a 0.45 μm microporous membrane to obtain NADH liposomes. The mass ratio of lecithin, cholesterol, vitamin E, Tween 80, and NADH in the NADH liposomes was 15:5:3:12:10. The pH of the phosphate buffer was 7.4, and the concentration was 0.01 mol / L.

[0049] Step 2: Chitosan was dispersed in a 1 wt.% acetic acid solution, stirred until dissolved, and then the pH was adjusted to 4 with NaOH to obtain a 1 wt.% chitosan solution. Sodium tripolyphosphate was dispersed in deionized water to obtain a sodium tripolyphosphate solution with a concentration of 0.8 g / L. NADH liposome microparticles were added to the sodium tripolyphosphate solution and stirred for 30 min, then added to the chitosan solution. The pH of the system was adjusted to 4, and stirring was continued for 30 min. After centrifugation, separation, washing, and drying, NADH sustained-release microparticles were obtained. The mass ratio of chitosan, NADH liposomes, and sodium tripolyphosphate was 1:1:2.

[0050] Comparative Example 2: The preparation order in step 3 was adjusted in this comparative example, and the remaining parameters were the same as in Example 2.

[0051] Step 1: Add lecithin, cholesterol, and Tween 80 to anhydrous ethanol and stir at 50°C to dissolve and obtain a liposome solution; disperse NADH in phosphate buffer and stir to obtain an NADH solution; keep the temperature of the liposome solution constant, add the NADH solution, stir for 35 min, remove ethanol by rotary evaporation, homogenize for 12 min, sonicate for 8 min, and then filter through a 0.45 μm microporous membrane to obtain NADH liposomes;

[0052] The NADH liposomes contain lecithin, cholesterol, vitamin E, Tween 80, and NADH in a mass ratio of 20:8:4:14:12; the phosphate buffer solution has a pH of 7.4 and a concentration of 0.01 mol / L.

[0053] Step 2: Preparation of NADH liposome microspheres embedded in the isolation layer:

[0054] S1: Take 10g of sodium octenyl succinate starch ester and mix it with 1L of calcium chloride solution with a concentration of 0.03mol / L. Carry out the ion exchange reaction at 25℃ for 3.5h. Wash the product with deionized water and filter until there are no chloride ions in the solution. Dry it in a vacuum drying oven at 1MPa and 45℃ for 3h. Pulverize it to obtain calcium octenyl succinate starch ester.

[0055] S2: Take 4.5g of calcium-type octenyl succinate starch ester, 1.2g of tea polyphenols, and 0.8g of glycerol and disperse them in 1000g of deionized water. Gelatinize them at 92℃ for 30min to obtain the isolation layer emulsion.

[0056] S3: Mix NADH liposomes and the isolation layer emulsion at a mass ratio of 1:3.5, stir for 8 minutes, and then spray dry to obtain NADH liposome microspheres embedded in the isolation layer; the inlet air temperature of the spray dryer is 185℃ and the outlet air temperature is 105℃.

[0057] Step 3: Disperse chitosan in a 1 wt.% acetic acid solution, stir to dissolve, and adjust the pH to 4 with NaOH to obtain a 1 wt.% chitosan solution; disperse sodium tripolyphosphate in deionized water to obtain a sodium tripolyphosphate solution with a concentration of 1 g / L; add NADH liposome microspheres with an isolation layer to the chitosan solution, stir for 30 min, then add sodium tripolyphosphate solution, adjust the pH of the system to 4, and continue stirring for 30 min. After centrifugation, separation, washing, and drying, obtain NADH sustained-release microspheres; wherein, the mass ratio of chitosan, NADH liposome microspheres with an isolation layer, and sodium tripolyphosphate is 1:1:2.5.

[0058] Comparative Example 3: In this comparative example, starch was used instead of calcium-type cross-linked octenyl succinate starch ester, and the other parameters were the same as in Example 3.

[0059] Step 1: Add lecithin, cholesterol, and Tween 80 to anhydrous ethanol and stir at 50°C to dissolve and obtain a liposome solution; disperse NADH in phosphate buffer and stir to obtain an NADH solution; keep the temperature of the liposome solution constant, add the NADH solution, stir for 40 min, remove ethanol by rotary evaporation, homogenize for 15 min, sonicate for 10 min, and then filter through a 0.45 μm microporous membrane to obtain NADH liposomes;

[0060] The NADH liposomes contain lecithin, cholesterol, vitamin E, Tween 80, and NADH in a mass ratio of 25:10:5:15:15; the phosphate buffer solution has a pH of 7.4 and a concentration of 0.01 mol / L.

[0061] Step 2: Preparation of NADH liposome microspheres embedded in the isolation layer:

[0062] S1: Take 5g starch, 1.5g tea polyphenols and 1g glycerin and disperse them in 1000g deionized water. Gelatinize them at 95℃ for 30min to obtain the isolation layer emulsion.

[0063] S2: Mix NADH liposomes and the isolation layer emulsion at a mass ratio of 1:4, stir for 10 min and then spray dry to obtain NADH liposome microspheres embedded in the isolation layer; the inlet air temperature of spray drying is 190℃ and the outlet air temperature is 110℃.

[0064] Step 3: Disperse chitosan in a 1 wt.% acetic acid solution, stir to dissolve, and adjust the pH to 4 with NaOH to obtain a 1 wt.% chitosan solution; disperse sodium tripolyphosphate in deionized water to obtain a sodium tripolyphosphate solution with a concentration of 1 g / L; add the NADH liposome microspheres with an isolation layer to the sodium tripolyphosphate solution and stir for 30 min, then add them to the chitosan solution, adjust the pH of the system to 4, and continue stirring for 30 min. After centrifugation, separation, washing, and drying, obtain NADH sustained-release microspheres; wherein, the mass ratio of chitosan, NADH liposome microspheres with an isolation layer, and sodium tripolyphosphate is 1:1:3.

[0065] The sustained-release microspheres prepared in Examples 1-3 and Comparative Examples 1-3 were immersed in simulated colonic solution SCF (37°C, pH=6.8) for 12 h. The total NADH release over 12 h was used as the actual encapsulation amount, and the encapsulation efficiency was calculated. The results are shown in Table 1. The formula for calculating the encapsulation efficiency is as follows:

[0066] Encapsulation rate = (Total NADH release over 12 hours / Theoretical total NADH content) × 100%;

[0067] Table 1.

[0068] project Encapsulation rate / % Example 1 93.87% Example 2 91.56% Example 3 92.39% Comparative Example 1 52.62% Comparative Example 2 79.54% Comparative Example 3 81.63%

[0069] The sustained-release microspheres prepared in Examples 1-3 and Comparative Examples 2-3 were soaked in simulated gastric acid solution SGF (37°C, pH=2) for 2 h, and then soaked in simulated colonic solution SCF (37°C, pH=6.8) for 10 h. The cumulative release rate of NADH was tested at 2, 4, 6, and 12 h, and the results are shown in Table 2. The formula for calculating the cumulative release rate is:

[0070] Cumulative release rate = (Cumulative release amount of NADH / Actual total content of NADH) × 100%;

[0071] The total release of NADH from the microcapsules over 12 hours is considered the actual total content of NADH.

[0072] Table 2.

[0073] 2-hour cumulative release rate 4-hour cumulative release rate 6-hour cumulative release rate 12h cumulative release rate Example 1 0.85% 18.57% 48.63% 98.96% Example 2 0.92% 20.61% 50.77% 98.48% Example 3 1.04% 22.32% 52.39% 99.74% Comparative Example 2 13.68% 67.46% 99.67% / Comparative Example 3 3.27% 35.39% 63.95% 99.62%

[0074] Conclusion: The data in Table 1 show that the NADH-containing sustained-release microspheres prepared in Examples 1-3 of this invention have higher encapsulation rates. In Comparative Example 1, no isolation layer was used to encapsulate the NADH liposome microspheres. When chitosan and sodium tripolyphosphate were used to encapsulate the NADH liposomes, pH changes disrupted the stability of the NADH liposomes, resulting in a low encapsulation rate. In Comparative Example 2, the order of step 3 was adjusted. According to Table 2, the sustained-release microspheres in Comparative Example 2 released more quickly in the stomach. Therefore, the reason can be inferred to be: because the surface of calcium octenyl succinate starch ester is positively charged, it repels the positively charged chitosan, resulting in poor chitosan encapsulation. Subsequent addition of sodium tripolyphosphate makes it difficult to form a complete enteric coating layer on its surface, thus resulting in poor acid resistance and rapid release in the stomach. In Comparative Example 3, ordinary starch was used instead of calcium-type octenyl succinate starch ester. Although ordinary starch can form an isolation layer, its encapsulation effect is worse than that of Example 3 because the surface of ordinary starch does not carry a positive charge and it is prone to swelling after absorbing water. Therefore, the release rate in the stomach is higher and the release speed in the intestine is faster.

[0075] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0076] 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 method for preparing NADH-containing sustained-release microspheres, characterized in that: Includes the following steps: Step 1: Add lecithin, cholesterol, and Tween 80 to anhydrous ethanol and stir at 50°C to dissolve and obtain a liposome solution; disperse NADH in phosphate buffer and stir to obtain an NADH solution; keep the temperature of the liposome solution constant, add the NADH solution, stir for 30-40 min, remove ethanol by rotary evaporation, and filter through a 0.45 μm microporous membrane to obtain NADH liposomes; Step 2: Take calcium-type octenyl succinate starch ester, tea polyphenols, and glycerol and disperse them in deionized water. Gelatinize them at 90~95℃ to obtain an isolation layer emulsion. Mix NADH liposomes with the isolation layer emulsion and spray dry to obtain NADH liposome microspheres embedded in the isolation layer. Step 3: The NADH liposome microspheres embedded in the isolation layer were added to a sodium tripolyphosphate solution and stirred for 30 min. Then, chitosan solution was added and stirring was continued for 30 min. After centrifugation, separation, washing and drying, NADH sustained-release microspheres were obtained.

2. The method for preparing NADH-containing sustained-release microspheres according to claim 1, characterized in that: In step 1, the mass ratio of lecithin, cholesterol, vitamin E, Tween 80 and NADH in the NADH liposome is (15~25):(5~10):(3~5):(12~15):(10~15).

3. The method for preparing NADH-containing sustained-release microspheres according to claim 1, characterized in that: In step 1, the phosphate buffer solution has a pH of 7.4 and a concentration of 0.01 mol / L.

4. The method for preparing NADH-containing sustained-release microspheres according to claim 1, characterized in that: In step 2, the preparation method of calcium-type octenyl succinate starch ester is as follows: sodium-type octenyl succinate starch ester is mixed with calcium chloride solution and subjected to ion exchange reaction for 3-4 hours. The product is washed with deionized water, filtered until no chloride ions are present in the solution, vacuum dried and pulverized to obtain calcium-type octenyl succinate starch ester.

5. The method for preparing NADH-containing sustained-release microspheres according to claim 1, characterized in that: In step 2, the components in the isolation layer emulsion are as follows by weight: 4-5 parts calcium-type octenyl succinate starch ester, 1-1.5 parts tea polyphenols, 0.5-1 part glycerol, and 1000 parts deionized water.

6. The method for preparing NADH-containing sustained-release microspheres according to claim 1, characterized in that: In step 2, NADH liposomes and the isolation layer emulsion are mixed at a mass ratio of 1:(3~4).

7. The method for preparing NADH-containing sustained-release microspheres according to claim 1, characterized in that: In step 3, the chitosan solution is prepared by dispersing chitosan in a 1 wt.% acetic acid solution, stirring to dissolve, and then adjusting the pH to 4 with NaOH to obtain a 1 wt.% chitosan solution.

8. The method for preparing NADH-containing sustained-release microspheres according to claim 1, characterized in that: In step 3, the concentration of the sodium tripolyphosphate solution is 1 g / L.

9. The method for preparing NADH-containing sustained-release microspheres according to claim 1, characterized in that: In step 3, the mass ratio of chitosan, NADH liposome microspheres encapsulated in the isolation layer, and sodium tripolyphosphate is 1:1:(2~3).

10. NADH-containing sustained-release microspheres prepared by the method according to any one of claims 1 to 9.