Use of n-acetyl cysteine in the preparation of weight loss products

Abstract

This invention belongs to the field of pharmaceutical and health food technology, specifically relating to the application of N-acetylcysteine ​​(NAC) in the preparation of weight-loss products. The application of NAC in the preparation of weight-loss products in this invention reduces animal weight gain by promoting the metabolic rate of colonic epithelial cells. This invention achieves targeted delivery of nutrients to the colon by preparing a controlled-release formulation containing NAC that targets the colon, accelerating epithelial cell oxidative metabolism and energy supply, thereby achieving the goal of weight loss while maintaining intestinal health.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and health food technology, specifically relating to the application of N-acetylcysteine ​​in the preparation of weight loss products. Background Technology

[0002] Maintaining a healthy weight is crucial for the health of both humans and pets. Obesity significantly increases the risk of cardiovascular disease and various cancers in humans. Meanwhile, overweight dogs and cats are more prone to arthritis, respiratory problems, diabetes, and other diseases, leading to decreased mobility and a loss of their role as companion animals.

[0003] Body weight is a dynamic balance between nutrient intake and metabolic expenditure. Administering appetite-regulating hormones such as glucagon-like peptide can reduce appetite to some extent, thus achieving weight loss. However, this can only be done via injection and can lead to depression and various behavioral abnormalities in animals. Increasing exercise is also an important weight loss strategy. However, increased exercise can actually increase appetite, and weight rebound is faster after exercise stops. Therefore, identifying the optimal regulatory target and employing a suitable nutritional program are crucial for maintaining a healthy body weight.

[0004] Aside from muscle and adipose tissue, the animal intestine is the primary site of nutrient metabolism. Intestinal epithelial cells serve as a crucial barrier between intestinal microbes and the body's cells. Significant differences in nutrient concentration, oxygen content, and microbiota exist among different intestinal segments, leading to marked variations in oxidative metabolic rates. The hindgut, due to its highly anaerobic environment and low nutrient concentration, has a much lower metabolic rate than the foregut. Numerous studies have found that gut microbiota dysbiosis can lead to pathological inflammation and increased metabolism in the intestine, resulting in ileal or colitis and rapid weight loss. These findings suggest that targeting specific intestinal segments to promote oxidative metabolism is an effective measure for weight loss.

[0005] N-acetylcysteine ​​(NAC), a stable derivative of cysteine, is considered a highly efficient precursor nutrient in nutrition and antioxidant fields. Its core value lies in its ability to directly provide the rate-limiting raw material for the cellular synthesis of reduced glutathione (GSH). Since the absorption rate of glutathione from food is extremely low, oral NAC can rapidly increase intracellular GSH levels, making it a key nutritional strategy for maintaining the body's antioxidant defense system. As a potent antioxidant, NAC can not only directly scavenge various free radicals but also regenerate other antioxidants such as vitamin C and vitamin E, synergistically resisting oxidative stress damage to mitochondrial DNA, proteins, and lipid membranes. However, directly orally administered NAC is rapidly absorbed in the small intestine, making it difficult to reach the slower-evolving hindgut region to exert its effects. Furthermore, there are no reports on the role of NAC in regulating colonic cell metabolism and weight loss. Summary of the Invention

[0006] Addressing at least one deficiency of existing technologies, this invention aims to provide the application of N-acetylcysteine ​​in the preparation of weight-loss products. This invention achieves targeted delivery of nutrients to the colon by preparing a controlled-release formulation containing NAC that targets the colon, accelerating epithelial cell oxidative metabolism and energy supply, thereby achieving weight loss while maintaining intestinal health.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: the application of N-acetylcysteine ​​in the preparation of weight loss products, which reduces the weight gain of animals by promoting the metabolic rate of colonic epithelial cells.

[0008] As one embodiment of the application of N-acetylcysteine ​​in the preparation of weight loss products according to the present invention, the concentration of N-acetylcysteine ​​is 10~100mg / kg.

[0009] As one embodiment of the application of N-acetylcysteine ​​described in this invention in the preparation of weight loss products, the weight loss products are oral controlled-release formulations.

[0010] As one embodiment of the application of N-acetylcysteine ​​in the preparation of weight loss products according to the present invention, the oral controlled-release formulation includes controlled-release pills, controlled-release tablets, and controlled-release capsules.

[0011] As one embodiment of the application of N-acetylcysteine ​​described in this invention in the preparation of weight loss products, the weight loss products include weight loss drugs, weight loss health foods, and weight loss foods.

[0012] The present invention also claims protection for the use of N-acetylcysteine ​​in the preparation of products that promote the metabolic rate of colonic epithelial cells, wherein the concentration of N-acetylcysteine ​​is 10-100 mg / kg.

[0013] As one embodiment of the application of the N-acetylcysteine ​​described in this invention in the preparation of products that promote the metabolic rate of colonic epithelial cells, the products include pharmaceuticals, health foods, and ordinary foods.

[0014] The common food mentioned in this invention refers to finished products and raw materials intended for human or animal consumption or drinking, including processed food, semi-finished products and unprocessed food.

[0015] This invention also claims protection for the use of an N-acetylcysteine ​​controlled-release formulation in the preparation of weight-loss products, which reduces animal weight gain by promoting the metabolic rate of colonic epithelial cells.

[0016] As one embodiment of the application of the N-acetylcysteine ​​controlled-release formulation of the present invention in the preparation of weight loss products, the amount of N-acetylcysteine ​​added in the weight loss products is 10~100ppm.

[0017] As one embodiment of the application of the N-acetylcysteine ​​controlled-release formulation of the present invention in the preparation of weight loss products, the amount of N-acetylcysteine ​​added in the weight loss products is 10~80ppm.

[0018] As one embodiment of the application of the N-acetylcysteine ​​controlled-release formulation of the present invention in the preparation of weight loss products, the N-acetylcysteine ​​controlled-release formulation is a colon-targeted controlled-release formulation.

[0019] This invention also claims protection for the use of an N-acetylcysteine ​​controlled-release formulation in the preparation of products that promote the metabolic rate of colonic epithelial cells.

[0020] As one embodiment of the application of the N-acetylcysteine ​​controlled-release formulation of the present invention in the preparation of a product that promotes the metabolic rate of colonic epithelial cells, the amount of N-acetylcysteine ​​added in the product that promotes the metabolic rate of colonic epithelial cells is 10~100ppm.

[0021] As one embodiment of the application of the N-acetylcysteine ​​controlled-release formulation of the present invention in the preparation of a product that promotes the metabolic rate of colonic epithelial cells, the amount of N-acetylcysteine ​​added in the product that promotes the metabolic rate of colonic epithelial cells is 10~80 ppm.

[0022] As one embodiment of the application of the N-acetylcysteine ​​controlled-release formulation of the present invention in the preparation of products that promote the metabolic rate of colonic epithelial cells, the N-acetylcysteine ​​controlled-release formulation is a colon-targeted controlled-release formulation.

[0023] Compared with the prior art, the present invention has the following beneficial effects: The results of this study show that colonic instillation of NAC significantly reduces body weight gain and increases blood ammonia levels in C57BL / 6 mice, but does not affect serum MDA levels. Furthermore, NAC was prepared as a colon-targeted controlled-release formulation. In a weaned piglet experiment, hindgut-targeted controlled-release NAC also reduced average daily weight gain and increased feed conversion ratio. This indicates that NAC has the function of weight reduction without affecting intestinal health. Attached Figure Description

[0024] Figure 1 This figure shows the effect of colonic perfusion of NAC on serum ammonia (Figure A) and MDA levels (Figure B) in mice, as described in this embodiment of the invention. * indicates a p-value ≤ 0.05, meaning the difference is statistically significant.

[0025] Figure 2This diagram illustrates the effect of colonic perfusion of NAC on the metabolism of mouse colonic epithelial cells in an embodiment of the present invention. A shows representative images of p-mTOR, mTOR, PCNA, and Tubulin proteins in a colonic Western blot; B shows the grayscale values ​​of p-mTOR / mTOR proteins in a colonic Western blot; C shows the grayscale values ​​of PCNA proteins in a colonic Western blot; D shows representative images of Ubiquitin and Tubulin proteins in a colonic Western blot; E shows the grayscale values ​​of Ubiquitin proteins in a colonic Western blot; F shows the expression of colonic amino acid transporters and metabolism-related mRNAs; G shows the expression of colonic fatty acid metabolism-related mRNAs; and H shows the expression of colonic carbohydrate metabolism-related mRNAs. * indicates p-value ≤ 0.05, indicating statistical significance; ** indicates p-value ≤ 0.01, indicating highly statistical significance; and *** indicates p-value ≤ 0.001, indicating extremely strong statistical significance.

[0026] Figure 3 This is a diagram illustrating the in vitro release pattern of NAC microparticles in an embodiment of the present invention. Detailed Implementation

[0027] 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.

[0028] Unless otherwise specified, the experimental methods used in the examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0029] Example 1: Effects of different doses of NAC administered via colonic perfusion on growth performance in mice 1. To investigate the effects of colonic perfusion with NAC on body weight gain and food intake in mice, 18 eight-week-old C57BL / 6J mice of uniform weight were selected and randomly divided into three groups as follows: Control group: Colonic instillation with normal saline; Treatment group 1: Intracolonic instillation of N-acetylcysteine ​​(NAC) solution at a concentration of 10 mg / kg mouse body weight; Treatment group 2: Intracolonic instillation of N-acetylcysteine ​​(NAC) solution at a dose of 30 mg / kg mouse body weight; The pH of the reagents in each group was adjusted to 7.0. Mice were allowed free access to food and water, 12-hour circulating light, environmental humidity of 70%±10%, and constant temperature of 23℃±1℃ for 4 weeks.

[0030] 2. Determining the effect of colonic perfusion of NAC on growth performance in mice. (1) The specific measurement method is as follows: Mice were fed for 4 weeks, during which they were colonically infused with 10 mg / kg and 30 mg / kg of NAC every 3 days. Body weight and food intake were recorded every 3 days for the purpose of calculating body weight gain and cumulative food intake.

[0031] (2) The results are shown in Table 1. Table 1 shows the effect of NAC perfusion in the colon on the growth performance of mice.

[0032] Table 1. Effects of different doses of NAC administered via colonic perfusion on growth performance in mice.

[0033] Note: Different shoulder marks in the same row (a, b) indicate significant differences (P<0.05).

[0034] The results showed that colonic instillation of 10 mg / kg NAC significantly reduced body weight gain in mice, but had no significant effect on their cumulative food intake.

[0035] Example 2: Effects of colonic perfusion of NAC on serum ammonia and MDA levels in mice 1. To investigate the effects of colonic perfusion of NAC on serum ammonia and MDA levels in mice, twelve 8-week-old C57BL / 6J mice were selected and randomly divided into two groups as follows: Control group: Colonic instillation with normal saline; Treatment group 1: Intracolonic instillation of N-acetylcysteine ​​(NAC) solution at a concentration of 10 mg / kg mouse body weight; The pH of the reagents in each group was adjusted to 7.0. Mice were allowed free access to food and water, 12-hour circulating light, environmental humidity of 70%±10%, and constant temperature of 23℃±1℃ for 4 weeks.

[0036] 2. Determine the effect of colonic perfusion of NAC on serum ammonia and MDA levels in mice.

[0037] (1) The specific determination method is as follows: Mice were colonically instilled with 10 mg / kg NAC every 3 days during the feeding period. After 3 weeks, blood was collected from mice via ocular veins, and serum was separated. Serum ammonia (Megazyme) and MDA (Beyotime Biotechnology Co., Ltd.) were used to detect serum ammonia and MDA levels, respectively.

[0038] (2) The results are as follows Figure 1 As shown in the figure. The experimental results indicate that colonic instillation of 10 mg / kg N-acetylcysteine ​​(NAC) significantly increased the ammonia content in mouse serum, but did not affect the serum MDA content, suggesting that N-acetylcysteine ​​(NAC) may promote intestinal epithelial cell metabolism without causing oxidative stress in epithelial cells and thus inducing enteritis.

[0039] Example 3: Effects of colonic NAC perfusion on colonic epithelial cell metabolism 1. The experiment used mice as the research subjects. Twelve 8-week-old C57BL / 6J mice were selected and randomly divided into two groups as follows: Control group: Colonic instillation with normal saline; Treatment group 1: Intracolonic instillation of N-acetylcysteine ​​(NAC) solution at a concentration of 10 mg / kg mouse body weight; The pH of the reagents in each group was adjusted to 7.0. Mice were allowed free access to food and water, 12-hour circulating light, environmental humidity of 70%±10%, and constant temperature of 23℃±1℃ for 4 weeks.

[0040] 2. Effects of colonic perfusion of NAC on the metabolism of mouse colonic epithelial cells (1) The specific determination method is as follows: Mice were colonically infused with 10 mg / kg NAC every 3 days during the feeding period. After 4 weeks, colonic tissue was collected, contents were removed, and the remaining tissue was homogenized to extract proteins and RNA. Western blot was used to detect the expression of proteins involved in colonic protein turnover (p-mTOR / mTOR), PCNA proliferation, and ubiquitin degradation of Ubiquitin. Real-time quantitative PCR was used to detect the expression of mRNAs related to carbohydrate, lipid, and amino acid metabolism in the colon. The extracted total RNA was reverse transcribed into cDNA, and then the reaction was performed using an AppliedBiosystems QuantStudio 3 real-time PCR system with the following reaction program: 95℃ preheating for 5 min; 95℃ for 10 s, 60℃ for 30 s, 4 cycles. β-Actin was used as an internal reference gene. -ΔΔCt The primer sequences used for real-time quantitative PCR to determine the relative gene expression level of mRNA are shown in Table 2.

[0041] Table 2 Primers used for real-time quantitative PCR

[0042] (2) The results are as follows Figure 2 As shown.

[0043] The experimental results showed that colonic instillation of 10 mg / kg N-acetylcysteine ​​(NAC) significantly reduced the expression of p-mTOR / mTOR, a key protein turnover signal, and proliferating PCNA protein, while increasing the expression of ubiquitin-degrading Ubiquitin protein. Simultaneously, qPCR results indicated that colonic instillation of 10 mg / kg N-acetylcysteine ​​(NAC) significantly increased the expression of amino acid metabolism mRNA and fatty acid metabolism mRNA, but inhibited the expression of glucose metabolism mRNA. These results suggest that NAC primarily leads to energy deficiency in epithelial cells, resulting in decreased protein synthesis and increased degradation, subsequently prompting epithelial cells to promote amino acid and fatty acid metabolism for energy.

[0044] Example 4: Preparation of colon-controlled release NAC microspheres and verification of their release mechanism 1. Preparation of colon-controlled release NAC microspheres, the specific method is as follows: (1) Weigh out 500 g of NAC powder and 500 g of microcrystalline cellulose and mix them evenly to prepare a powder for later use; (2) Granulation using centrifugal granulation: Weigh 600 g of the prepared powder and put it into a centrifugal granulator. Set the parameters as follows: rotation speed, liquid spray rate, and air inlet rate to 300 rpm, 30 mL / min, and 30 m / s, respectively. After it rolls into master pellets, continue to add small amounts of powder to maintain the streamlined rotation of the microspheres until most of the microspheres have a mesh size of approximately 20-40 mesh. Then place the prepared wet microspheres in a 50℃ oven and dry for 12 h. After that, sieve them through a 16-60 mesh sieve to obtain the final product.

[0045] (3) Preparation of colon coating material: Acrylic resin coating was used, and the coating weight gain was 20%. The coating parameters were: top spray coating, constant flow pump flow rate 0.8-1.2 mL / min, material temperature 32-37℃, air volume 30 m / s, spray pressure 1.0-1.5 bar, and inlet temperature 40-45℃.

[0046] 2. Verification of the in vitro release pattern of coated microcapsules (1) The specific method is as follows: NAC microspheres were placed in simulated gastric fluid (pH=2) and incubated at 37°C for 2 hours in a shaker at 150 rpm. The simulated gastric fluid was then aspirated, and an equal volume of simulated small intestinal fluid (pH=5.5) was added, followed by incubation for 4 hours. The simulated small intestinal fluid was then aspirated, and an equal volume of simulated large intestinal fluid (pH=6.5) was added. Samples were taken every 30 minutes, and the same volume of the same type of fluid was added as needed. The samples were diluted tenfold and added to an ELISA plate, and the OD600 was measured using an ELISA reader. The release rate of the microspheres was calculated based on the standard curve. The simulated gastric fluid, simulated small intestinal fluid, and simulated large intestinal fluid were prepared according to the methods described in Part IV of the Chinese Pharmacopoeia. The method for determining the release rate of the microspheres was based on Method II of the Determination of Dissolution and Release Rate of Sustained-Release Preparations in the 2020 Edition of the Chinese Pharmacopoeia.

[0047] (2) The results are as follows Figure 3 As shown, Figure 3 This describes the in vitro release pattern of NAC microspheres.

[0048] The experimental results showed that by using artificial gastric fluid (SGF) at pH 2, artificial small intestinal fluid (SIF) at pH 5.5, and artificial large intestinal fluid (LIF) at pH 6.5 to determine the in vitro release pattern of β-Ala microspheres, NAC microspheres were not released in artificial gastric fluid and artificial small intestinal fluid, but were completely released within 1 hour in artificial large intestinal fluid.

[0049] Example 5: Effects of dietary supplementation with different doses of colonically controlled-release NAC pellets on pig growth performance 1. To investigate the effects of dietary supplementation with different doses of colonically controlled-release NAC pellets on the growth performance of pigs, 150 weaned piglets aged 28 days were selected and randomly divided into 5 groups according to their weight, with 5 pens in each group and 6 pigs in each pen. The groups are as follows: Control group: Basic diet supplemented with blank pellets; Treatment 1: The basal diet was supplemented with microspheres containing 20 ppm NAC; Treatment 2: The basal diet was supplemented with microspheres containing 40 ppm NAC; Treatment group 3: The basal diet was supplemented with microspheres containing 80 ppm NAC; Treatment 4: The basal diet was supplemented with 80 ppm of NAC powder.

[0050] The microparticles used in processing groups 1 to 3 were prepared according to the preparation method in Example 4.

[0051] All groups were allowed free access to feed and water during the experiment, with 24-hour light, a constant temperature of 26℃±1℃, and an ambient humidity of 60%±10%, for 28 days. During the experiment, piglets were fed twice daily, at 8:00 AM and 5:00 PM, with free access to feed and water, and good ventilation in the pens was ensured. At the end of the experiment, the piglets were weighed, and the average daily feed intake, average daily weight gain, and feed conversion ratio were calculated.

[0052] 2. Effects of dietary supplementation with different doses of colonically controlled-release NAC pellets on the growth performance of weaned piglets. (1) The specific measurement method is as follows: During the experiment, feed intake was recorded every 7 days. After the experiment, the feed was weighed, and the average daily feed intake, average daily weight gain, and feed conversion ratio were calculated.

[0053] (2) The results are shown in Table 3.

[0054] Table 3 Effects of dietary supplementation with NAC microcapsules on growth performance of weaned piglets

[0055] The experimental results showed that adding 80 ppm of NAC microcapsules to the diet significantly reduced the average daily weight gain of weaned piglets and increased the feed conversion ratio. Meanwhile, the results from treatments 1-3 and treatment 4 indicated that adding N-acetylcysteine ​​powder (not prepared as a controlled-release formulation) to the diet in treatment 4 ultimately failed to reduce the average daily weight gain of weaned piglets, and the feed conversion ratio was similar to that of the control group.

[0056] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. The application of N-acetylcysteine ​​in the preparation of weight loss products, characterized in that, By promoting the metabolic rate of colonic epithelial cells, it reduces the weight gain of animals.

2. The application as described in claim 1, characterized in that, The concentration of N-acetylcysteine ​​is 10~100 mg / kg.

3. The application as described in claim 1, characterized in that, The weight loss product is an oral controlled-release formulation.

4. The application as described in claim 1, characterized in that, The weight loss products include weight loss medicines, weight loss health foods, and weight loss foods.

5. The application of N-acetylcysteine ​​in the preparation of products that promote the metabolic rate of colonic epithelial cells, characterized in that, The concentration of N-acetylcysteine ​​is 10~100 mg / kg.

6. The application as described in claim 5, characterized in that, The products include pharmaceuticals, health foods, and general foods.

7. The application of an N-acetylcysteine ​​controlled-release formulation in the preparation of weight-loss products, characterized in that, By promoting the metabolic rate of colonic epithelial cells, it reduces the weight gain of animals.

8. The application as described in claim 7, characterized in that, The amount of N-acetylcysteine ​​added to the weight loss product is 10~100ppm; The N-acetylcysteine ​​controlled-release formulation is a colon-targeted controlled-release formulation.

9. The application of an N-acetylcysteine ​​controlled-release formulation in the preparation of products that promote the metabolic rate of colonic epithelial cells.

10. The application as described in claim 9, characterized in that, The amount of N-acetylcysteine ​​added to the product that promotes the metabolic rate of colonic epithelial cells is 10~100ppm; The N-acetylcysteine ​​controlled-release formulation is a colon-targeted controlled-release formulation.

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