A compound liquor based on N-acetyl-D-glucosamine and its preparation method and application

Abstract

The application discloses a kind of compound wine based on N-acetyl-D-glucosamine and preparation method and application thereof, belong to the technical field of biological medicine and the technical field of health food.The active ingredient of the composition is composed of N-acetyl-D-glucosamine 150-250 parts, taurine 20-40 parts, spironolactone extract 5-15 parts and L-arginine 5-15 parts.The application combines the antioxidant synergistic effect of N-acetyl-D-glucosamine and taurine, the free radical scavenging and immunomodulatory effect of spironolactone extract, the immune enhancement and absorption promotion effect of L-arginine, and the four components produce significant synergistic effect in a specific ratio range.Animal experiments show that the composition of the application can significantly improve the SOD, GSH-Px activity of aging model mice, reduce the MDA content, enhance the lymphocyte proliferation capacity, macrophage phagocytosis function and NK cell activity.In addition, the application also provides the application of the above composition in preparing health products for assisting in protecting chemical liver damage.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to a compound medicated wine based on N-acetyl-D-glucosamine, its preparation method, and its application. Background Technology

[0002] Oxidative stress is a process in which the body produces excessive reactive oxygen species (ROS) when exposed to various harmful stimuli, exceeding the body's ability to scavenge oxidants. This leads to an imbalance between the oxidative and antioxidant systems, resulting in tissue damage. Oxidative stress is closely related to the development and progression of various diseases, including aging, tumors, cardiovascular diseases, diabetes, and neurodegenerative diseases. Therefore, developing safe and effective antioxidant functional foods or health products is of great significance.

[0003] N-Acetyl-D-Glucosamine (NAG) is the smallest unit that makes up the chitin component of the exoskeleton of insects and crustaceans, and it is also the basic building block of many important polysaccharides in biological cells. N-Acetyl-D-Glucosamine has anti-inflammatory, anti-tumor, and antioxidant effects, and is widely used in bone and joint health foods, beauty beverages, and dietary supplements.

[0004] The safety of N-acetyl-D-glucosamine has been fully confirmed by toxicological studies. The toxicological test report issued by the Hunan Provincial Center for Disease Control and Prevention (acceptance number: J20090248) shows that: (1) in the acute oral toxicity test, the maximum tolerated dose (MTD) for both male and female Kunming mice was greater than 20.0 g / kg·bw, which is non-toxic; (2) the results of the three genotoxicity tests (Ames test, mouse bone marrow polychromatic erythrocyte micronucleus test, and mouse sperm abnormality test) were all negative, indicating no genotoxicity; (3) at doses of 1.67 g / kg·bw, 3.33 g / kg·bw, and 5.00 g / kg·bw (equivalent to human... SD rats were administered N-acetyl-D-glucosamine via gavage for 90 days at doses of 100, 200, and 300 times the recommended dosage. No significant differences were observed between the male and female rat groups in terms of body weight gain, food intake, food utilization, complete blood count (hemoglobin, hematocrit, erythrocyte count, white blood cell count, platelet count, and white blood cell differential), blood biochemical indicators (total protein, albumin, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, creatinine, blood glucose, cholesterol, and triglycerides), organ weight, and organ / body weight ratio compared to the control group (P>0.05). Gross anatomical and histopathological examinations revealed no abnormalities related to the samples. These data sufficiently demonstrate that N-acetyl-D-glucosamine has extremely high food safety.

[0005] Taurine is a sulfur-containing amino acid found in animals, widely distributed throughout various tissues and organs, primarily existing in free form in interstitial fluid and intracellular fluid. Studies have found that taurine has a wide range of biological functions, including maintaining normal visual function, maintaining osmotic pressure balance, regulating cellular calcium balance, lowering blood sugar, regulating nerve conduction, increasing cardiac contractility, enhancing immunity, strengthening cell membrane antioxidant capacity, and protecting cardiomyocytes. The safety of taurine is widely recognized, and it has been approved by various countries for use as a food additive and dietary supplement.

[0006] Purslane (Portulaca oleracea L.) is an annual herb belonging to the genus Portulaca in the family Portulacaceae. It is a traditional Chinese medicine with both food and medicinal uses, listed by the National Health Commission of China as an item that is both food and medicine. Modern pharmacological studies have shown that purslane extract contains abundant flavonoids, alkaloids, polysaccharides, and other active ingredients, exhibiting significant antioxidant, anti-inflammatory, antibacterial, hypoglycemic, and hypolipidemic pharmacological activities. Purslane polysaccharides can significantly improve the body's immune function, enhance the phagocytic capacity of macrophages, and promote lymphocyte proliferation.

[0007] L-Arginine is a conditionally essential amino acid that is converted into nitric oxide (NO) in the body by nitric oxide synthase (NOS), participating in various physiological processes. L-Arginine can promote growth hormone secretion, enhance immune function, promote wound healing, and protect the cardiovascular system. Studies have shown that L-Arginine deficiency can lead to decreased immune function, while supplementation can significantly enhance cellular and humoral immunity. L-Arginine has been recognized as "Generally Recognized As Safe" (GRAS) by the US FDA.

[0008] Chinese patents CN102199513A and CN103695273A disclose a medicinal wine prepared from N-acetyl-D-glucosamine and taurine, which exhibits synergistic effects of anti-free radical and antioxidant activity. Chinese patent CN108272808B discloses a pharmaceutical composition containing N-acetyl-D-glucosamine and taurine with anti-tumor activity, and its medicinal wine formulation, showing a synergistic anti-tumor effect between the two within a specific ratio range.

[0009] However, there are no existing research reports on combining N-acetyl-D-glucosamine, taurine, purslane extract, and L-arginine to exert a multi-component synergistic antioxidant and immune-enhancing effect. Through inventive research, the applicant has discovered that combining these four components within a specific ratio not only fully leverages the individual antioxidant and immune-enhancing effects of each component but also exhibits a significant synergistic effect among them. The antioxidant and immune-enhancing effects are significantly superior to using them alone or in combination. Furthermore, since all raw materials have sufficient safety data to support their safety, this invention has reliable safety assurance. Summary of the Invention

[0010] The purpose of this invention is to provide a compound wine preparation based on N-acetyl-D-glucosamine. This compound wine preparation uses N-acetyl-D-glucosamine and taurine as the core active ingredients, supplemented with purslane extract and L-arginine. The four ingredients synergistically enhance each other within a specific ratio range, exhibiting excellent antioxidant and immune-enhancing functions, and with high safety.

[0011] Another object of the present invention is to provide a method for preparing the above-mentioned compound wine preparation.

[0012] Another object of the present invention is to provide the application of the above-mentioned compound wine in the preparation of health products for anti-oxidation and immune enhancement.

[0013] To achieve the above objectives, the present invention adopts the following technical solution:

[0014] A compound medicinal wine based on N-acetyl-D-glucosamine, wherein the active ingredients of the compound medicinal wine are composed of the following raw materials in parts by weight: 150-250 parts N-acetyl-D-glucosamine, 20-40 parts taurine, 5-15 parts purslane extract, and 5-15 parts L-arginine.

[0015] Preferably, the active ingredients of the compound medicated wine are composed of the following raw materials in parts by weight: 190-210 parts N-acetyl-D-glucosamine, 28-32 parts taurine, 8-12 parts purslane extract, and 8-12 parts L-arginine.

[0016] More preferably, the active ingredients of the compound medicated wine are composed of the following raw materials in parts by weight: 200 parts N-acetyl-D-glucosamine, 30 parts taurine, 10 parts purslane extract, and 10 parts L-arginine.

[0017] The preparation method of the purslane extract of this invention is as follows: Take the dried whole purslane herb, pulverize it to 20-40 mesh, add 8-12 times its weight (based on the weight of the dried whole purslane herb) of ethanol solution with a mass fraction of 60%-80%, heat and reflux to extract 2-3 times, 1-2 hours each time, combine the extracts, concentrate under reduced pressure to an extract with a relative density of 1.10-1.20 (measured at 60℃), dry under reduced pressure at 60-80℃ until the moisture content is ≤5%, pulverize and pass through an 80-mesh sieve to obtain the extract. The total flavonoid content in the obtained purslane extract should not be less than 5% (calculated as rutin, determined by ultraviolet-visible spectrophotometry).

[0018] The preparation method of the compound medicinal wine of the present invention includes the following steps: (1) Take the liquor as the base, add two-thirds of the weight of N-acetyl-D-glucosamine taurine, and stir until completely dissolved; (2) Cool down to 5-10℃, add N-acetyl-D-glucosamine, stir and mix evenly, and let stand at 5-10℃ for 1-3 hours; (3) Add the remaining taurine, stir and mix evenly, and let stand at 5-10℃; (4) Add purslane extract and L-arginine, and stir to mix evenly; (5) Filter, fill, seal, and store at 5-10℃ for 10-20 days to obtain the product.

[0019] The liquor mentioned in step (1) is an edible liquor with an alcohol content of 30-42 degrees (preferably light-aroma or strong-aroma type), and the ratio of N-acetyl-D-glucosamine to liquor is 200g:8000-12000mL. The purity of the N-acetyl-D-glucosamine is ≥98% (HPLC method), and the purity of the taurine is ≥98% (compliant with the Chinese Pharmacopoeia or food additive standards).

[0020] The mixing method of purslane extract and L-arginine in step (4) is as follows: first, L-arginine is dissolved in anhydrous ethanol, then purslane extract is added, mixed evenly, dried, and then added to the mixture obtained in step (3).

[0021] The mechanisms of action of each component in the compound medicinal wine of this invention are as follows: N-acetyl-D-glucosamine, as the core active ingredient, possesses direct antioxidant activity, capable of scavenging free radicals in the body and reducing oxidative stress damage. Simultaneously, N-acetyl-D-glucosamine is an important precursor for glycoproteins and glycosaminoglycans, participating in the synthesis of immunoglobulins and playing a crucial role in maintaining normal immune function.

[0022] Taurine, an important sulfur-containing amino acid, possesses significant antioxidant activity. It can directly scavenge reactive oxygen species such as hypochlorous acid and peroxynitrite, protecting cell membranes from oxidative damage. Taurine can also regulate intracellular calcium ion concentration, stabilize cell membrane structure, and enhance the activity of the body's immune cells.

[0023] Purslane extract is rich in active ingredients such as flavonoids and polysaccharides. Among them, purslane flavonoids have significant free radical scavenging ability and lipid peroxidation inhibition activity, while purslane polysaccharides can activate macrophages, promote lymphocyte proliferation, and enhance natural killer cell activity, thereby enhancing the body's immune function through multiple pathways.

[0024] In vivo, L-arginine is catalyzed by nitric oxide synthase (NOS) to produce nitric oxide (NO). NO, as an important signaling and effector molecule, participates in various physiological processes such as immune regulation and vasodilation. L-arginine can also promote T lymphocyte proliferation and enhance the killing activity of macrophages, thus promoting both cellular and humoral immunity.

[0025] Through extensive experimental research, the applicant discovered that when the four components are used in combination within the specific proportions of this invention, not only are the antioxidant and immune-enhancing effects of each component fully realized, but there is also a significant synergistic effect among the four components. Specifically, the synergistic effect of N-acetyl-D-glucosamine and taurine has been confirmed in the prior art; based on this, the addition of purslane extract can further enhance the antioxidant capacity of the system through its rich flavonoid components, while its polysaccharide components and the immunomodulatory effect of L-arginine work together to form a multi-target, multi-pathway immune-enhancing mechanism; and the addition of L-arginine not only enhances immune function, but the NO generated by it can also promote blood circulation, which is beneficial to the absorption and distribution of other active ingredients. Detailed Implementation

[0026] 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.

[0027] Experiment 1: Raw Material Safety Evaluation Test All raw materials used in this invention have undergone rigorous toxicological safety evaluations, and the results are as follows: 1.1 Toxicological tests of N-acetyl-D-glucosamine Test source: Hunan Provincial Center for Disease Control and Prevention (Test Report Acceptance Number: J20090248), and the testing basis is the "Technical Specifications for Inspection and Evaluation of Health Food" (2003 edition).

[0028] (1) Acute oral toxicity test The maximum tolerated dose (MTD) method was used. Twenty Kunming mice (half male and half female) weighing 18-22g were selected. 25.00g of N-acetyl-D-glucosamine was diluted with distilled water to a final volume of 50mL and administered orally to the mice twice via gavage at 4-hour intervals, with each gavage volume being 0.2mL / 10g·bw. The mice were observed for two weeks after gavage, and the signs of poisoning and mortality were recorded.

[0029] The results showed that no obvious signs of poisoning were observed after gavage administration of the sample at a dose of 20.0 g / kg·bw to both sexes of Kunming mice, and no deaths were observed after 14 days. At the end of the experiment, the test animals were euthanized for autopsy, and no obvious abnormalities were found in the major organs, including the liver, spleen, kidneys, stomach, intestines, heart, and lungs. The maximum tolerated dose (MTD) of the sample to both male and female Kunming mice was greater than 20.0 g / kg·bw, classifying it as non-toxic.

[0030] Table 1 Results of acute toxicity test of N-acetyl-D-glucosamine in mice

[0031] (2) Ames test Four Salmonella Typhimurium strains (TA97, TA98, TA100, and TA102) that met the requirements were used in the experiment. Rat liver microsomal enzyme (S-9) induced by polychlorinated biphenyls (PCBs) was used as the in vitro metabolic activation system. Five dosages were set up: 5000 μg / plate, 1000 μg / plate, 200 μg / plate, 40 μg / plate, and 8 μg / plate. Spontaneous reversion, solvent control, and positive mutagen control were also included.

[0032] The results showed that, under both S-9-added and S-9-free conditions, the number of revertant colonies in all four strains did not exceed twice the number of spontaneously revertant colonies, and no dose-response relationship was observed. In contrast, the number of revertant colonies in the positive control group was significantly increased, indicating the effectiveness of the experimental system. The Ames test result was negative, indicating that N-acetyl-D-glucosamine has no mutagenic effect.

[0033] (3) Mouse bone marrow polychromatic erythrocyte micronucleus test The study employed a two-time oral gavage administration method with a 24-hour interval. Fifty Kunming mice weighing 25-30g were randomly divided into 5 groups of 10 mice each, with half males and half females. Cyclophosphamide at a dose of 40 mg / kg body weight (bw) served as a positive control, and distilled water as a negative control. The high, medium, and low doses in the experimental groups were 10.00 g / kg bw, 5.00 g / kg bw, and 2.50 g / kg bw, respectively. Six hours after the last administration, the animals were euthanized by cervical dislocation. Bone marrow smears from the sternum were collected, stained with Giemsa, and 1000 polychromatic erythrocytes (PCEs) were counted from each animal to calculate the micronucleus incidence.

[0034] The results showed that the micronucleus incidence rate in each dose group was not significantly different from that in the negative control group (P>0.05), while the micronucleus incidence rate in the cyclophosphamide positive control group was significantly higher than that in the negative control group (P<0.01). The test result was negative, indicating that N-acetyl-D-glucosamine does not cause chromosome breakage or spindle fiber toxicity.

[0035] (4) Mouse sperm abnormality test Twenty-five male Kunming mice weighing 25-35g were randomly divided into 5 groups of 5 mice each. Cyclophosphamide at a dose of 40mg / kg bw was used as a positive control, and distilled water as a negative control. The experimental groups were given high, medium, and low doses of 10.00g / kg bw, 5.00g / kg bw, and 2.50g / kg bw, respectively. Samples of 50.00g, 25.00g, and 12.50g were diluted with distilled water to 100mL to prepare the corresponding test solutions, which were then administered to the mice via gavage (0.2mL / 10g bw). This was repeated once daily for 5 consecutive days. On day 30 after the last gavage, the animals were sacrificed, and sperm smears were collected from both epididymis, stained with eosin, and 1000 structurally intact sperm were counted from each animal. The incidence of abnormal sperm was calculated.

[0036] The results showed that there was no significant difference in the incidence of sperm abnormalities between the different dosage groups and the negative control group (P>0.05), while the incidence of sperm abnormalities in the cyclophosphamide positive control group was significantly higher than that in the negative control group (P<0.01). The test results were negative, indicating that N-acetyl-D-glucosamine has no mutagenic effect on germ cells.

[0037] (5) 90-day feeding trial Eighty SD rats, half male and half female, were randomly divided into four groups: a control group and three test substance groups (low, medium, and high doses), with 20 rats in each group. The low, medium, and high doses of the sample were set at 1.67 g / kg·bw, 3.33 g / kg·bw, and 5.00 g / kg·bw, respectively (approximately 100, 200, and 300 times the recommended human dose, respectively). The control group was given an equal volume of distilled water, administered by gavage once daily for 90 consecutive days.

[0038] During the experiment, there were no significant differences in body weight gain, food intake, and food utilization rate between the male and female rats in each dose group and the control group (P>0.05). Mid- and late-stage hematological examinations showed no significant differences in hemoglobin, total red blood cell count, hematocrit, platelet count, total white blood cell count, and differential count between the dose groups and the control group (P>0.05). Mid- and late-stage blood biochemical examinations showed no significant differences in total protein, albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen, creatinine, blood glucose, cholesterol, and triglycerides between the dose groups and the control group (P>0.05). There were no significant differences in organ weight and organ / body weight ratio between the dose groups and the control group. Gross anatomical and histopathological examinations revealed no abnormalities related to the samples.

[0039] Table 2. Body weight of rats in the 90-day feeding experiment with N-acetyl-D-glucosamine ($\bar{x}±s$, n=10)

[0040] Note: Compared with the control group, P>0.05 for each dosage group.

[0041] Toxicological studies concluded that the maximum tolerated dose (MTD) of N-acetyl-D-glucosamine in both male and female Kunming mice was greater than 20.0 g / kg·bw, classifying it as non-toxic. All three genotoxicity tests (Ames test, mouse bone marrow polychromatic erythrocyte micronucleus test, and mouse sperm abnormality test) were negative, indicating no genotoxicity. Oral administration of 1.67-5.00 g / kg·bw to SD rats for 90 days showed no significant toxic side effects. These data sufficiently demonstrate that N-acetyl-D-glucosamine has extremely high food safety.

[0042] 1.2 Toxicological tests on N-acetyl-D-glucosamine tinctures To further evaluate the safety of alcoholic beverages with N-acetyl-D-glucosamine as the core ingredient, the Hunan Provincial Center for Disease Control and Prevention was commissioned to conduct a systematic toxicological safety evaluation of alcoholic beverages containing N-acetyl-D-glucosamine (application number: J20100225).

[0043] (1) Acute oral toxicity test The maximum tolerated dose method was used. Twenty Kunming mice (half male, half female) weighing 18-22g were selected. A 5-fold concentrated solution of NAG-containing ethanol (containing 15% ethanol, with approximately 10g NAG / 100mL and 1.5g taurine / 100mL in the concentrate) was administered orally once by gavage at a volume of 0.2mL / 10g·bw. The specific gravity was measured to be 1.008g / mL, equivalent to a dose of 20.16g / kg·bw. Mice were fasted for 16 hours before gavage and observed for two weeks afterward, recording any signs of poisoning and mortality.

[0044] The results showed that no obvious signs of poisoning were observed after gavage administration of 20.16 g / kg bw to both sexes of Kunming mice, and no deaths were observed after 14 days. On day 15, the animals were sacrificed and autopsied; no significant abnormalities were found in the major organs, including the liver, spleen, kidneys, stomach, intestines, heart, and lungs. The maximum tolerated dose (MTD) of the sample to both male and female Kunming mice was greater than 20.16 g / kg bw, classifying it as non-toxic.

[0045] Table 3 Results of acute toxicity test in mice containing NAG-containing alcohol

[0046] (2) Ames test Four strains, TA97, TA98, TA100, and TA102, were used, with five dosages: 5000 μg / plate, 1000 μg / plate, 200 μg / plate, 40 μg / plate, and 8 μg / plate. Spontaneous reversion, solvent control, and positive mutagen control were also included.

[0047] The results showed that, under both S-9-added and S-9-free conditions, the number of revertant colonies in all four strains did not exceed twice the number of spontaneously revertant colonies, and no dose-response relationship was observed. The Ames test results were negative.

[0048] (3) Mouse bone marrow polychromatic erythrocyte micronucleus test The method involved oral gavage twice at 24-hour intervals. Sixty Kunming mice were randomly divided into 6 groups of 10 each, with half males and half females. The high, medium, and low doses in the experimental groups were 10.0 g / kg·bw, 5.0 g / kg·bw, and 2.5 g / kg·bw, respectively. A base alcohol control, a distilled water negative control, and a cyclophosphamide positive control were also included.

[0049] The results showed that the micronucleus incidence rate in each dose group was not significantly different from that in the negative control group and the alcohol-based control group (P>0.05), while the micronucleus incidence rate in the cyclophosphamide positive control group was significantly higher than that in the negative control group (P<0.01). The test result was negative.

[0050] (4) Mouse sperm abnormality test Thirty male Kunming mice were randomly divided into 6 groups of 5 each. The experimental groups were administered three doses: high, medium, and low, at 10.0 g / kg body weight (bw), 5.0 g / kg body weight (bw), and 2.5 g / kg body weight (bw), respectively. A base alcohol control, a distilled water negative control, and a cyclophosphamide positive control were also included. The mice were administered the medication by gavage once daily for 5 consecutive days. On day 30 after the last gavage, the animals were sacrificed, and the epididymis was collected for examination.

[0051] Table 4 Results of sperm abnormality test in mice containing NAG alcohol

[0052] Note: Compared with the negative control group, P<0.01.

[0053] The results showed that there was no significant difference in the incidence of sperm abnormalities between the dosage groups and the solvent control group (alcohol base control) and the negative control group (distilled water control) (P>0.05). The incidence of sperm abnormalities in the cyclophosphamide positive control group was significantly higher than that in the negative control group (P<0.01). The experimental results were negative, indicating that the NAG-containing alcoholic beverage did not have a germ cell mutation-inducing effect.

[0054] (5) 30-day feeding trial One hundred SD rats, half male and half female, were randomly divided into five groups: a blank control group, a base alcohol control group, a low-dose group, a medium-dose group, and a high-dose group, with 20 rats in each group. The low, medium, and high doses were equivalent to 25, 50, and 100 times the recommended human dose, respectively (converted based on NAG content). The test substance was administered by gavage as a 5-fold concentrated solution of NAG-containing alcohol with 15% ethanol (the NAG content in the concentrated solution was approximately 10 g / 100 mL). The blank control group was given distilled water, and the base alcohol control group was given a base alcohol solution containing 15% ethanol. The gavage volume was 1.0 mL / 100 g·bw, administered once daily for 30 consecutive days.

[0055] Table 5. Body weight of rats in a 30-day feeding experiment containing NAG alcohol ($\bar{x}±s$, n=10)

[0056] Note: Compared with the blank control group and the base wine control group, P>0.05.

[0057] During the 30-day feeding period, all groups of animals showed good growth and development, with no abnormal behavior or poisoning symptoms, and no deaths. There were no significant differences in body weight, final weight, weight gain, average food intake, weekly food utilization, and total food utilization rate between the male and female rats in each dosage group and the control group at any time point (P>0.05). Complete blood count and blood biochemical indicators were all within the normal range, with no significant differences between the dosage groups and the control group. Gross anatomical and histopathological examinations revealed no abnormalities related to the samples.

[0058] The toxicological test results of the NAG-containing alcoholic beverage are as follows: The beverage with N-acetyl-D-glucosamine as its core ingredient has a maximum tolerated dose (MTD) greater than 20.16 g / kg·bw in both male and female Kunming mice, classifying it as non-toxic. All three genotoxicity tests (Ames test, mouse bone marrow polychromatic erythrocyte micronucleus test, and mouse sperm abnormality test) were negative. No significant toxic side effects were observed in a 30-day feeding trial. These data demonstrate that the alcoholic beverage with N-acetyl-D-glucosamine as its core ingredient has reliable safety.

[0059] Experiment 2: Study on the synergistic antioxidant effect of quaternary compositions Experimental Groups: Experimental Group 1: N-acetyl-D-glucosamine 200mg (alone) Experimental Group 2: Taurine 30mg (used alone) Experimental Group 3: Purslane extract 10mg (used alone) Experimental Group 4: L-arginine 10mg (alone) Experimental Group 5: N-acetyl-D-glucosamine 200mg + taurine 30mg Experimental Group 6: N-acetyl-D-glucosamine 200mg + Portulaca oleracea extract 10mg Experimental Group 7: N-acetyl-D-glucosamine 200mg + L-arginine 10mg Experimental Group 8: N-acetyl-D-glucosamine 200mg + taurine 30mg + purslane extract 10mg + L-arginine 10mg (the composition of this invention) Experimental Methods: A D-galactose-induced aging mouse model was used. Ninety Kunming mice, weighing 18-22g, half male and half female, were randomly divided into 10 groups (including a blank control group and a model control group), with 10 mice in each group. Except for the blank control group, the mice in the other groups were subcutaneously injected with D-galactose 150mg / kg·bw in the neck and back daily for 42 consecutive days to establish an oxidative stress model. The mice in each test substance group (experiments 1-8) were administered the corresponding test substance by gavage daily at a volume of 0.2mL / 10g·bw, with the test substance prepared as a suspension in distilled water. The blank control group and the model control group were given an equal volume of distilled water. After the last administration on day 42, blood was collected by enucleation, and the mice were euthanized by cervical dislocation. Liver tissue was collected, and the activities of superoxide dismutase (SOD) (xanthine oxidase method), glutathione peroxidase (GSH-Px) (DTNB direct method), and malondialdehyde (MDA) content (thiobarbituric acid method) in serum and liver tissue were measured.

[0060] Experimental results: see Table 6.

[0061] Table 6 Comparison of antioxidant indices among experimental groups ($\bar{x}±s$, n=10)

[0062] Note: Blank control group: No D-galactose injection, distilled water administered by gavage; Model control group: D-galactose 150 mg / kg·bw injection, distilled water administered by gavage; Experimental groups 1-8: D-galactose 150 mg / kg·bw injection, corresponding test substance administered by gavage (prepared as a suspension in distilled water, gavage volume 0.2 mL / 10 g·bw). Compared with the blank control group, P<0.01; compared with the model control group, #P<0.05, ##P<0.01.

[0063] Experimental conclusions: The antioxidant effect of the quaternary composition of this invention (8 experimental groups) is significantly better than that of each single-use group and the binary combination group. Its serum SOD activity recovered to 96.3% of the blank control group, and its serum MDA content decreased to 107.4% of the blank control group, close to the normal level. The antioxidant effect of the quaternary composition is significantly better than the theoretical superposition value, indicating that there is a synergistic effect among the four components.

[0064] Experiment 3: Study on the immune-enhancing effect of the quaternary composition Experimental Groups: Experimental Group 1: N-acetyl-D-glucosamine 200mg + taurine 30mg (binary combination) Experimental Group 2: N-acetyl-D-glucosamine 200mg + taurine 30mg + purslane extract 10mg (ternary combination) Experimental Group 3: N-acetyl-D-glucosamine 200mg + taurine 30mg + L-arginine 10mg (ternary combination) Experimental Group 4: 200 mg N-acetyl-D-glucosamine + 30 mg taurine + 10 mg purslane extract + 10 mg L-arginine (quaternary composition of this invention) Experimental Methods: Fifty Kunming mice, weighing 18-22g, half male and half female, were randomly divided into 5 groups (including a blank control group), with 10 mice in each group. Mice in each experimental group were administered the corresponding test substance (prepared as a suspension in distilled water, 0.2 mL / 10g bw) by gavage daily, while the blank control group received an equal volume of distilled water. The administration was continued for 30 days. The following immune indicators were measured on day 30: (1) Spleen index and thymus index: After weighing, the mice were euthanized by cervical dislocation, and the spleen and thymus were weighed and the organ index (mg / g body weight) was calculated.

[0065] (2) ConA-induced T lymphocyte proliferation and LPS-induced B lymphocyte proliferation (MTT assay): Spleen was aseptically harvested, and spleen cell suspension was prepared (concentration 1×10⁻⁶). 7Add 100 μL of MTT solution (5 mg / mL, 20 μL per well) to a 96-well plate, and add ConA (final concentration 5 μg / mL) or LPS (final concentration 10 μg / mL) to the plate. Incubate at 37°C and 5% CO2 for 48 hours. Add MTT solution (5 mg / mL, 20 μL per well) 4 hours before the end of the incubation. After the incubation is complete, add DMSO (150 μL per well), vortex to mix, and measure the OD value at 570 nm using a microplate reader.

[0066] (3) Phagocytic function of peritoneal macrophages (chicken red blood cell phagocytosis method): 1 mL of 2% starch solution was injected into the peritoneum of mice. 24 hours later, 1 mL of 1% chicken red blood cell suspension was injected into the peritoneum. The mice were sacrificed 30 minutes later. The peritoneal fluid was smeared, incubated at 37°C for 30 minutes, stained with Giemsa, and 200 macrophages were counted under a microscope. The phagocytic percentage and phagocytic index were calculated.

[0067] (4) NK cell activity (lactate dehydrogenase release method): Spleen cells were aseptically removed and a suspension of spleen cells was prepared as effector cells. YAC-1 cells were used as target cells with an effector-to-target ratio of 50:1. The cells were cultured at 37°C and 5% CO2 for 4 hours. The OD value of the supernatant was measured using an LDH kit, and the NK cell activity was calculated.

[0068] Experimental results: see Table 7.

[0069] Table 7 Comparison of immune indicators among experimental groups ($\bar{x}±s$, n=10)

[0070] Note: Compared with the blank control group, P<0.01; there were significant differences between experimental group 4 and other experimental groups (P<0.05).

[0071] Experimental conclusion: The quaternary composition of the present invention is significantly superior to the binary and ternary compositions in all immune indicators, indicating that the addition of purslane extract and L-arginine can significantly enhance the immune-enhancing function of the N-acetyl-D-glucosamine and taurine composition, and there is a synergistic effect among the four components.

[0072] Experiment 4: Optimization Study of Medicated Wine Preparation Process Experimental Group 1 (Process of this Invention): Take 10,000 mL of 36% ABV liquor, add two-thirds of its weight of taurine (20 g), dissolve completely, cool to 8°C, add N-acetyl-D-glucosamine (200 g), mix well, let stand at 8°C for 2 hours, add the remaining taurine (10 g), mix well, and let stand at 8°C. Separately, take 10 g of L-arginine, dissolve in 500 g of anhydrous ethanol, add 10 g of purslane extract, mix well, dry, and add to the aforementioned mixture. Filter, fill into 10 bottles, seal the bottle caps tightly, and store at 8°C for 15 days. After outer packaging, the medicinal liquor is obtained.

[0073] Experimental Group 2 (Control Process): Take 10,000 mL of baijiu with an alcohol content of 36 degrees, and add 30 g of taurine, 200 g of N-acetyl-D-glucosamine, 10 g of purslane extract and 10 g of L-arginine. Mix well and fill into 10 bottles to obtain the medicinal wine.

[0074] Experimental methods: Take the medicinal wines from the different experimental groups mentioned above and detect the content of N-acetyl-D-glucosamine (HPLC method, referring to the method in the Chinese Pharmacopoeia), taurine content (amino acid analyzer method, referring to GB / T 5009.169 "Determination of Taurine in Food"), and total flavonoid content in purslane extract (ultraviolet-visible spectrophotometry, with rutin as the reference standard). Place them at 40℃ for 24 hours, at room temperature for 24 hours, and then at 0℃ for 24 hours, repeating the cycle 3 times, and then detect the content of each component again.

[0075] Experimental results: see Table 8.

[0076] Table 8. Comparison of the stability of medicinal wines made using different processes

[0077] Experimental conclusion: The medicinal wine prepared by the stepwise low-temperature preparation process of the present invention exhibits excellent stability after thermal cycling tests, with the content change rate of each active ingredient being less than 3%. In contrast, the content of N-acetyl-D-glucosamine decreased by more than 10% and the total flavonoid content decreased by more than 18% in the control process, indicating poor stability. This fully demonstrates the scientific significance and practical value of the preparation process of the present invention.

[0078] Preparation Examples Example 1 The active ingredients of the medicinal wine are: 1950g N-acetyl-D-glucosamine, 290g taurine, 100g purslane extract, and 100g L-arginine.

[0079] Preparation method: Take 100L of 36% ABV liquor, add two-thirds of its weight of taurine (193.3g), dissolve completely, cool to 8℃, add 1950g of N-acetyl-D-glucosamine, mix well, let stand at 8℃ for 2 hours, add the remaining taurine (96.7g), mix well, and let stand at 8℃. Separately, take 100g of L-arginine, dissolve in 5000g of anhydrous ethanol, add 100g of purslane extract, mix well, dry, and add to the above mixture. Filter, fill into 100 bottles, seal the bottle caps tightly, and store at 8℃ for 15 days. After packaging, the medicinal liquor is obtained.

[0080] Example 2 The active ingredients of the medicinal wine are: 2000g N-acetyl-D-glucosamine, 300g taurine, 100g purslane extract, and 100g L-arginine.

[0081] The preparation method is the same as in Example 1.

[0082] Example 3 The active ingredients of the medicinal wine are: 2050g N-acetyl-D-glucosamine, 310g taurine, 120g purslane extract, and 100g L-arginine.

[0083] The preparation method is the same as in Example 1.

[0084] Example 4 The active ingredients of the medicinal wine are: 2000g N-acetyl-D-glucosamine, 300g taurine, 80g purslane extract, and 80g L-arginine.

[0085] The preparation method is the same as in Example 1.

[0086] Example 5 Preparation of capsules: Take 200g of N-acetyl-D-glucosamine, 30g of taurine, 10g of purslane extract, and 10g of L-arginine, mix them evenly, pulverize them through a 100-mesh sieve, add 250g of starch as a filler, mix evenly, and fill into No. 0 capsule shells. Each capsule contains 0.3g (containing about 0.17g of active ingredients), resulting in about 1600 capsules.

[0087] Example 6 Preparation of granules: Take 200g of N-acetyl-D-glucosamine, 30g of taurine, 10g of purslane extract, and 10g of L-arginine, mix them evenly, add 150g of dextrin and 100g of sucrose, mix well, use a 70% ethanol solution as a wetting agent to make a soft mass, granulate it through a 14-mesh sieve, dry it at 60℃ until the moisture content is ≤5%, granulate it, and package it into 3g bags (containing about 0.17g of active ingredients), thus obtaining about 160 bags of granules.

[0088] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the compositions and methods disclosed in the embodiments, those skilled in the art can understand them by referring to the above description, and further details will not be repeated here.

[0089] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A compound medicinal wine based on N-acetyl-D-glucosamine, characterized in that, The active ingredients of the compound medicated wine are composed of the following raw materials in parts by weight: 150-250 parts N-acetyl-D-glucosamine, 20-40 parts taurine, 5-15 parts purslane extract, and 5-15 parts L-arginine.

2. The compound medicinal wine according to claim 1, characterized in that, The active ingredients of the compound medicated wine are composed of the following raw materials in parts by weight: 190-210 parts N-acetyl-D-glucosamine, 28-32 parts taurine, 8-12 parts purslane extract, and 8-12 parts L-arginine.

3. The compound medicinal wine according to claim 2, characterized in that, The active ingredients of the compound medicated wine are composed of the following raw materials in parts by weight: 200 parts N-acetyl-D-glucosamine, 30 parts taurine, 10 parts purslane extract, and 10 parts L-arginine.

4. The compound medicinal wine according to any one of claims 1-3, characterized in that, The preparation method of the purslane extract is as follows: Take the dried whole purslane herb, pulverize it to 20-40 mesh, add 8-12 times its weight of 60%-80% ethanol solution, heat and reflux to extract 2-3 times, 1-2 hours each time, combine the extracts, concentrate under reduced pressure to an extract with a relative density of 1.10-1.20 (measured at 60℃), dry under reduced pressure at 60-80℃ until the water content is ≤5%, pulverize and pass through an 80-mesh sieve to obtain the extract.

5. A method for preparing the compound medicinal wine according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Take the liquor as the base, add two-thirds of the weight of N-acetyl-D-glucosamine taurine, and stir until completely dissolved; (2) Cool down to 5-10℃, add N-acetyl-D-glucosamine, stir and mix evenly, and let stand at 5-10℃ for 1-3 hours; (3) Add the remaining taurine, stir and mix evenly, and let stand at 5-10℃; (4) Add purslane extract and L-arginine, and stir to mix evenly; (5) Filter, fill, seal, and store at 5-10℃ for 10-20 days to obtain the product.

6. The preparation method according to claim 5, characterized in that, The matrix in step (1) is a type of liquor with an alcohol content of 30-42 degrees, and the ratio of N-acetyl-D-glucosamine to liquor is 200g:8000-12000mL.

7. The preparation method according to claim 5, characterized in that, The mixing method of purslane extract and L-arginine in step (4) is as follows: first, L-arginine is dissolved in anhydrous ethanol, then purslane extract is added, mixed evenly, dried, and then added to the mixture obtained in step (3).

8. The use of the compound wine preparation according to any one of claims 1-4 in the preparation of antioxidant health products.

9. The use of the compound wine preparation according to any one of claims 1-4 in the preparation of health products that enhance immunity.

10. The use of the compound wine preparation according to any one of claims 1-4 in the preparation of a health product for the auxiliary protection against chemically induced liver damage.

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