NAD liposome, preparation method and application thereof

Abstract

The application discloses NAD liposomes, a preparation method and application thereof, and belongs to the technical field of biology.The NAD liposomes are prepared from NAD, agaric and black bean extract prepared by a special fermentation process, cholesterol, water-soluble chitosan, phosphatidylethanolamine and vitamin E through low-pressure homogenization and high-pressure homogenization.It is verified by experiments that the NAD liposomes have high encapsulation efficiency, drug loading and stability, and the effect of improving sleep is improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to an NAD liposome, its preparation method and application. Background Technology

[0002] Nicotinamide adenine dinucleotide (NAD) is a core redox coenzyme in organisms, widely involved in key physiological processes such as cellular energy metabolism, DNA repair, aging regulation, immune regulation, and oxidative stress response. Current NAD formulations suffer from numerous shortcomings, severely limiting their practical application. Free NAD molecules are extremely unstable, highly sensitive to acidic and alkaline environments, temperature, and light, and have weak permeability. After oral administration, they are easily destroyed by the acidic and alkaline environment of the gastrointestinal tract and encounter the first-pass effect in the liver, making it difficult to penetrate cell membranes and exert their effects. Furthermore, existing conventional NAD formulations generally suffer from poor targeting, short circulating half-life, and inability to continuously and stably release the active ingredient. They also exhibit low encapsulation efficiency and are prone to aggregation and leakage during storage. Therefore, there is an urgent need for a stable NAD delivery formulation with high encapsulation efficiency and high bioavailability, along with a suitable preparation process. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide an NAD liposome, its preparation method and application. The prepared NAD liposome has high encapsulation efficiency, drug loading capacity and stability, and has a significant effect on improving sleep.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an NAD liposome comprising the following raw materials in parts by weight: 10-20 parts NAD, 2-8 parts black fungus and black bean extract, 5-15 parts cholesterol, 5-15 parts water-soluble chitosan, 20-30 parts phosphatidylethanolamine, and 0.5-1.5 parts vitamin E.

[0005] Preferably, the extracts of black fungus and black beans are obtained by fermentation of black fungus and black beans with Leuconostoc mesenteroides.

[0006] The present invention provides a method for preparing the NAD liposomes, comprising the following steps: mixing water-soluble chitosan, black fungus and black bean extracts and NAD to obtain an aqueous phase; adding cholesterol, phosphatidylethanolamine and vitamin E to the aqueous phase, homogenizing under low pressure, then homogenizing under high pressure, and drying to obtain NAD liposomes.

[0007] Preferably, the preparation process of the black fungus and black bean extract includes: mixing the total weight of black fungus and black beans with water at a mass ratio of 1:8-20, inoculating with Leuconostoc mesenteroides for fermentation, centrifuging to collect the supernatant, and drying to obtain the black fungus and black bean extract.

[0008] Preferably, the inoculum amount of Leuconostoc mesenteroides is 0.2-0.8%; the fermentation temperature is 28-32℃ and the fermentation time is 40-54h.

[0009] Preferably, in the preparation of the aqueous phase, the weight ratio of the total weight of water-soluble chitosan, black fungus and black bean extract, and NAD to water is 1:1-5; the mixing temperature is 32-38℃ and the stirring speed is 80-120 rpm.

[0010] Preferably, the conditions for low-pressure homogenization include: a nitrogen atmosphere, a pressure of 30-40 MPa, a temperature of 50-60 °C, and a time of 8-12 min.

[0011] Preferably, the conditions for high-pressure homogenization include: nitrogen atmosphere, pressure of 150-250 MPa, and 3-5 cycles.

[0012] Preferably, the drying conditions include: pre-freezing at -20~-30℃ for 20-28 hours, and then freeze-drying at -55~-65℃ for 40-56 hours.

[0013] This invention provides the application of the NAD liposomes or the NAD liposomes obtained by the preparation method in the preparation of sleep-improving products.

[0014] The beneficial effects of this invention are: The raw materials described in this invention are scientifically and synergistically formulated. Utilizing *Leuconostoc mesenteroides* to ferment black fungus and black beans enhances the content of active ingredients. Combined with vitamin E, this forms a dual antioxidant system, effectively addressing the problem of NAD's easy oxidation and inactivation, significantly improving product stability and the retention rate of active ingredients. Simultaneously, by compounding cholesterol, phosphatidylethanolamine, and water-soluble chitosan into a film, a dense liposome carrier is constructed, effectively improving the NAD encapsulation rate and reducing defects such as liposome leakage, aggregation, and demulsification. Furthermore, animal experiments have verified that the raw materials used in this NAD liposome synergistic effect enhances the sleep-improving effect. Detailed Implementation

[0015] This invention provides an NAD liposome comprising the following raw materials in parts by weight: 10-20 parts NAD, 2-8 parts extracts of black fungus and black beans, 5-15 parts cholesterol, 5-15 parts water-soluble chitosan, 20-30 parts phosphatidylethanolamine, and 0.5-1.5 parts vitamin E. The black fungus and black bean extracts of this invention are obtained by fermentation of black fungus and black beans with Leuconostoc mesenteriae. Unless otherwise specified, the raw materials of this invention can be obtained through commercially available channels well known in the art.

[0016] The present invention also provides a method for preparing the NAD liposomes, comprising the following steps: mixing water-soluble chitosan, black fungus and black bean extracts and NAD to obtain an aqueous phase; adding cholesterol, phosphatidylethanolamine and vitamin E to the aqueous phase, homogenizing under low pressure, then homogenizing under high pressure, and drying to obtain NAD liposomes.

[0017] In this invention, the preparation process of the black fungus and black bean extract includes: mixing black fungus and black beans with water at a mass ratio of 1:8-20, inoculating with Leuconostoc mesenteroides bacterial solution for fermentation, centrifuging to collect the supernatant, and drying to obtain the black fungus and black bean extract. This invention uses specific strains to ferment and decompose macromolecular substances in black fungus and black beans into small molecule active ingredients, making sleep-aiding substances more easily absorbed by the human body and resulting in a more significant sleep-aiding effect.

[0018] In this invention, the inoculum amount of *Leuconostoc mesenteroides* culture is 0.2-0.8% (volume percentage), preferably 0.5%; the fermentation temperature is 28-32℃ and the time is 40-54 hours, preferably 30℃ and 48 hours. The suitable inoculum amount, fermentation temperature, and time of this invention are beneficial for the microbial fermentation of *Auricularia auricula-judae* and black beans, obtaining active ingredients effective in promoting sleep.

[0019] In this invention, during the preparation of the aqueous phase, the weight ratio of water-soluble chitosan, black fungus and black bean extracts, NAD to water is 1:1-5, preferably 1:3; the mixing temperature is 32-38℃, and the stirring speed is 80-120 rpm, preferably 35℃, and the stirring speed is 100 rpm. This invention selects suitable material-to-water ratios, temperatures, and stirring speeds to ensure the water-soluble components are fully dissolved and dispersed, forming a homogeneous and stable aqueous phase system. This protects the heat-sensitive active ingredients, reduces bubble and aggregation problems, facilitates subsequent liposome formation, and improves encapsulation efficiency and product stability.

[0020] In this invention, the low-pressure homogenization conditions include: a nitrogen atmosphere, a pressure of 30-40 MPa, a temperature of 50-60°C, and a time of 8-12 min; the high-pressure homogenization conditions include: a nitrogen atmosphere, a pressure of 150-250 MPa, and 3-5 cycles. In this invention, low-pressure homogenization is responsible for pre-emulsification, system stabilization, and activity protection, while high-pressure homogenization is responsible for refinement, high encapsulation, and strong stability. Together, they achieve the preparation of NAD liposomes with high encapsulation efficiency and high stability.

[0021] This invention provides the application of the NAD liposomes or the NAD liposomes obtained by the preparation method in the preparation of sleep-improving products.

[0022] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0023] Unless otherwise specified, the following embodiments are all conventional methods.

[0024] Example 1 A type of NAD liposome is made from the following raw materials in parts by weight: 15 parts NAD, 5 parts black fungus and black bean extract, 10 parts cholesterol, 10 parts water-soluble chitosan, 25 parts phosphatidylethanolamine, and 1 part vitamin E.

[0025] The preparation steps of the NAD liposomes are as follows: (1) The preparation of Leuconostoc mesenteroides bacterial culture was as follows: Leuconostoc mesenteroides (trade code BMZ133569, purchased from Ningbo Mingzhou Biotechnology Co., Ltd.) was inoculated into MRS solid medium and cultured at 37°C for 24 h. The strain after two generations of activation was then inoculated into MRS liquid medium and cultured at 36°C with shaking until the bacterial concentration reached 5 × 10⁻⁶. 9 cfu / mL, to obtain Leuconostoc mesentery bacterial suspension; The wood ear fungus and black beans were dried and pulverized separately until they passed through a 10-mesh sieve. The above weight parts were then weighed and mixed evenly. Water was added in 15 times the weight of the total materials and mixed evenly. The mixture was soaked for 12 hours to obtain a material mixture. Enterococcus membranaceus bacterial solution was added to the material mixture at an inoculation rate of 0.5% (volume percentage). The mixture was fermented at 30°C for 48 hours, sterilized, centrifuged, and the supernatant was collected and dried to obtain wood ear fungus and black bean extracts.

[0026] (2) Mix the water-soluble chitosan, black fungus and black bean extract, NAD and water in a water bath at 35°C and 100 rpm until homogeneous to obtain the aqueous phase. Cholesterol, phosphatidylethanolamine, and vitamin E were added sequentially to the aqueous phase according to the above weight proportions. The mixture was then subjected to low-pressure homogenization for 10 minutes under a nitrogen atmosphere at a pressure of 35 MPa and a temperature of 55 °C to obtain a homogenate. The homogenate was then placed in a high-pressure microfluidic device and subjected to high-pressure microfluidic treatment at a pressure of 200 MPa under a nitrogen atmosphere, and the mixture was circulated 4 times to obtain a liposome solution.

[0027] (3) The liposome solution was pre-ly lyophilized at -25℃ for 24h and then lyophilized at -60℃ for 48h to obtain NAD liposomes.

[0028] Example 2 A type of NAD liposome is made from the following raw materials in parts by weight: 10 parts NAD, 2 parts black fungus and black bean extract, 5 parts cholesterol, 5 parts water-soluble chitosan, 20 parts phosphatidylethanolamine, and 0.5 parts vitamin E.

[0029] The preparation method is the same as in Example 1.

[0030] Example 3 A type of NAD liposome is made from the following raw materials in parts by weight: 20 parts NAD, 8 parts black fungus and black bean extract, 15 parts cholesterol, 15 parts water-soluble chitosan, 30 parts phosphatidylethanolamine, and 1.5 parts vitamin E.

[0031] The preparation method is the same as in Example 1.

[0032] Comparative Example 1 The difference from Example 1 is that the extracts of black fungus and black beans and their related preparation steps are omitted, and 15 parts of NAD are replaced with 20 parts. All other steps are the same as in Example 1.

[0033] Comparative Example 2 The difference from Example 1 is that Leuconostoc mesenteroides was replaced with Lactobacillus plantarum (trade code BMZ134607, purchased from Ningbo Mingzhou Biotechnology Co., Ltd.), while the rest of the steps are the same as in Example 1.

[0034] Comparative Example 3 The difference from Example 1 is that chitosan and phosphatidylethanolamine are replaced with medium-chain triglycerides and soybean lecithin, while the rest of the steps are the same as in Example 1.

[0035] Comparative Example 4 The difference from Example 1 is that the low-pressure homogenization operation in step (2) is replaced with ultrasound (frequency: (35kHz, power 250W, processing time 20min), the remaining steps are the same as in Example 1.

[0036] Experiment 1: Determination of Encapsulation Efficiency, Drug Loading, and Stability Prepare a 100 μg / ml solution of NAD by dissolving it in pure water; then dilute each solution to 10 ml with 0.5, 1, 2, 3, 4, 6, 7, and 8 ml of the solution. Measure the absorbance and perform a linear regression analysis on the absorbance against the mass concentration to plot a standard curve.

[0037] Free NAD content was determined by dialysis: Equal amounts of liposome samples from each group were placed in dialysis bags and dialyzed at room temperature in the dark for 12 hours. The free NAD content in the dialysate was measured using a UV spectrophotometer. Drug loading and encapsulation efficiency were then determined. Encapsulation efficiency (%) = (Total NAD mass - Free NAD mass) / Total NAD mass × 100%. Drug loading is the percentage of effectively encapsulated NAD in the liposomes relative to the total mass of the liposomes. The formula for calculating drug loading is: Drug loading (%) = Encapsulated NAD mass / Total dry weight of liposomes × 100%.

[0038] Each group of liposome samples was stored at 4℃ in a sealed container away from light for 30 days. The encapsulation efficiency of the liposomes before and after storage was measured, and the encapsulation retention rate was calculated. Simultaneously, the presence of stratification, precipitation, or flocculation was observed. Encapsulation retention rate (%) = Encapsulation efficiency after 30 days of storage / Initial encapsulation efficiency × 100%. All experimental data are the average of 10 parallel experiments.

[0039] Table 1. Results of liposome encapsulation efficiency, drug loading, and stability assays for each group.

[0040] As shown in Table 1, the encapsulation efficiency, drug loading, and stability of the liposomes prepared in Examples 1-2 were significantly higher than those in Comparative Examples 1-4, indicating that there was a significant synergistic effect among the components of the formulation. The dedicated fermentation system can optimize the activity of raw materials and improve the liposome membrane structure. The stepwise compounding process can effectively eliminate raw material agglomeration, regulate vesicle morphology, reduce leakage of active ingredients, and greatly improve the uniformity of liposome dispersion and storage stability.

[0041] Experiment Example 2 1. Laboratory Animals: All animal experiments in this study were conducted in accordance with the "Technical Specifications for Inspection and Evaluation of Health Foods" (2003 edition), and the implementation plan was reviewed and approved by the ethics committee. SPF mice were fed and acclimatized under laboratory conditions for 3 days, with free access to food and water.

[0042] Grouping, gavage administration, and mouse condition observation: Mice were randomly divided into 7 groups: normal control group, model group, Example 1 group, and Comparative Examples 1-4 groups, with 10 mice in each group. Except for the normal control group, which was fed normally, the other groups underwent conventional methods to establish a sleep disorder model, creating mouse models of insomnia and sleep disturbance. After successful modeling, the treatment groups were administered 0.03 g / kg of medication by gavage daily, while the normal control and model groups received an equal volume of physiological saline, for 28 consecutive days. After the last administration, mice were fasted but allowed free access to water for 12 hours, during which sleep behavior experiments and brain tissue index detection were performed. The mice's condition was observed throughout the experiment. All groups maintained uniform dosage and feeding conditions to eliminate external interference.

[0043] Sleep behavior experiment: On day 28, 30 minutes after gavage, except for the normal control group, the other groups were given a single intraperitoneal injection of sodium pentobarbital (30 mg / kg). The time of administration, the time of disappearance of the righting reflex and the time of recovery of the righting reflex were recorded. The sleep latency and sleep duration of the mice were calculated (the sleep latency was defined as the time from the injection of sodium pentobarbital to the disappearance of the righting reflex, and the sleep duration was defined as the time from the disappearance of the righting reflex to the reappearance of the righting reflex). The results are shown in Table 2.

[0044] Brain tissue marker detection: Brain tissue homogenate was collected after mouse sacrifice, centrifuged, and the supernatant was collected. The contents of 5-HT, GABA, and NE in the brain tissue were detected according to the instructions of a commercially available ELISA kit. The results are shown in Table 2.

[0045] After observing the condition of mice in each group for 28 days by gavage, all mice in each group were in good condition. No adverse reactions were observed when fed the liposomes prepared in Example 1 and Comparative Examples 1-4 of this invention within 28 days, and the mice were in good mental condition.

[0046] Table 2. Results of liposome-mediated sleep improvement in each group.

[0047] Table 2 shows that the sleep-improving effect of the NAD liposomes prepared in Example 1 is significantly higher than that of Comparative Examples 1-4. The synergistic effect of each component in this formula is prominent, maximizing the activation of the calming and sleep-aiding activities of NAD and plant fermentation extracts. The product has significant efficacy advantages, good reproducibility, and good application and promotion value.

[0048] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An NAD liposome, characterized in that, It includes the following ingredients by weight: 10-20 parts NAD, 2-8 parts black fungus and black bean extract, 5-15 parts cholesterol, 5-15 parts water-soluble chitosan, 20-30 parts phosphatidylethanolamine, and 0.5-1.5 parts vitamin E.

2. The NAD liposomes as described in claim 1, characterized in that, The extracts of black fungus and black beans were obtained by fermentation of black fungus and black beans with Leuconostoc mesenteroides.

3. The method for preparing NAD liposomes as described in claim 1 or 2, characterized in that, The process includes the following steps: water-soluble chitosan, black fungus and black bean extracts, and NAD are mixed to obtain an aqueous phase; cholesterol, phosphatidylethanolamine and vitamin E are added to the aqueous phase, homogenized under low pressure, then homogenized under high pressure, and dried to obtain NAD liposomes.

4. The preparation method according to claim 3, characterized in that, The preparation process of the black fungus and black bean extract includes: mixing the total weight of black fungus and black beans with water at a mass ratio of 1:8-20, inoculating with Leuconostoc mesenteroides for fermentation, centrifuging to collect the supernatant, and drying to obtain the black fungus and black bean extract.

5. The preparation method according to claim 4, characterized in that, The inoculum size of Leuconostoc mesenteroides was 0.2-0.8%; the fermentation temperature was 28-32℃ and the fermentation time was 40-54h.

6. The preparation method according to claim 3, characterized in that, In the preparation of the aqueous phase, the weight ratio of water-soluble chitosan, black fungus and black bean extract, NAD to water is 1:1-5; the mixing temperature is 32-38℃ and the stirring speed is 80-120rpm.

7. The preparation method according to claim 3, characterized in that, The conditions for low-pressure homogenization include: nitrogen atmosphere, pressure of 30-40 MPa, temperature of 50-60 °C, and time of 8-12 min.

8. The preparation method according to claim 3, characterized in that, The conditions for high-pressure homogenization include: nitrogen atmosphere, pressure of 150-250 MPa, and 3-5 cycles.

9. The preparation method according to claim 3, characterized in that, The drying conditions include: pre-freezing at -20~-30℃ for 20-28 hours, followed by freeze-drying at -55~-65℃ for 40-56 hours.

10. The use of NAD liposomes as described in any one of claims 1-2 or NAD liposomes obtained by the preparation method as described in any one of claims 3-9 in the preparation of sleep-improving products.

Images