CN115397263B - Composition containing N-acetylglucosamine and preparation method and application thereof - Google Patents
Composition containing N-acetylglucosamine and preparation method and application thereof Download PDFInfo
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Abstract
The composition containing N-acetylglucosamine and ornithine aspartate has a synergistic effect, and compared with the compositions respectively containing ornithine aspartate or N-acetylglucosamine, the composition can remarkably and rapidly relieve fatigue, enhance the immunity of an organism and simultaneously has the function of assisting to protect the liver.
Description
Priority and related applications
The present invention claims priority from a prior application entitled "a composition comprising N-acetylglucosamine and a process for its preparation and use" filed on 29/1/2021 with the intellectual property office of the chinese country under the patent application number 202110128604.4. The entire contents of the prior application are incorporated by reference into this application.
Technical Field
The invention belongs to the field of health-care food, and particularly relates to a composition containing N-acetylglucosamine and ornithine aspartate, a preparation method thereof and application thereof in preparing health-care food for relieving fatigue, improving immunity and assisting in protecting liver.
Background
In modern society, people have fast pace of life, high working pressure, much social reward and lack of movement, and are easy to cause chronic fatigue, so that people feel tired in physical, mental and psychological aspects. Chronic fatigue for a long time can cause harm to physiological functions and psychological aspects of human bodies, such as physical strength deficiency, physical performance reduction, immunity reduction, memory reduction, attention deficit and reaction retardation, and can even cause mental symptoms such as melancholy, anxiety, dysphoria and the like, thereby seriously affecting the quality of life. Chronic fatigue often does not attract attention when organic damage or severe mental symptoms are not caused, and doctors do not have a proper medication method. In addition, social reward, such as drinking, high-fat and high-sugar diet, aggravates the metabolic burden of the liver, easily causes obesity fatty liver, alcoholic fatty liver, and the like, and causes the liver to be in an injured state for a long time. This condition is also often overlooked by people and progresses to severe liver disease. Therefore, there is a general need for a health food that can alleviate fatigue, improve immunity, and assist in protecting the liver.
Ornithine aspartate is a salt formed by aspartate and ornithine, and is clinically used for treating blood ammonia increase and hepatic encephalopathy caused by acute and chronic liver diseases such as various hepatitis, liver cirrhosis, fatty liver, posthepatitis syndrome and the like. Ornithine aspartate is decomposed into aspartic acid and ornithine in vivo, wherein the ornithine is an initial substrate in a urea cycle, is an activator of ornithine carbamyl transferase and carbamyl phosphate synthase, is combined with toxic metabolites such as ammonia in blood circulation, promotes urea synthesis, and accelerates metabolism of toxic substances in vivo, particularly ammonia; aspartic acid participates in synthesis of glutamine and nucleic acid in liver cells, can promote synthesis of glutamine in liver, indirectly participate in triphosphate circulation and nucleic acid synthesis, provide an intermediate product of energy metabolism, promote repair and regeneration of liver cells, recover liver functions and enhance liver detoxification functions.
N-acetylglucosamine (GlcNAc) is a glucosamine derivative, is an essential constituent unit of various polysaccharides in the living body, is widely present in nature, and is a main component of chitin and chitosan. N-acetylglucosamine has the effect of protecting cartilage tissue and bone joints, and is commonly used as a dietary supplement.
However, there is no report on the combined use of ornithine aspartate and N-acetylglucosamine, nor on the preparation of a health food from the two.
Disclosure of Invention
In order to overcome the problems of the prior art, the present invention provides a composition comprising N-acetylglucosamine and ornithine aspartate in a weight ratio of (1-10) to (10-1), such as (1-8) to (8-1), (1-5) to (5-1), (1-3) to (3-1), and illustratively 1:1.
According to the present invention, the composition may further comprise fruit extracts including, but not limited to, blueberry extract, strawberry extract, cranberry extract, orange extract, grape skin extract, grape seed extract, mulberry extract, etc., preferably blueberry extract. The fruit extract can be water extract, alcohol extract and/or alcohol water solution extract; the alcohol may be ethanol, and the aqueous alcohol solution may be 50-95% (v/v) aqueous ethanol solution. The extract can be obtained by extracting fruits with the following method: leaching, enzymatic hydrolysis, ultrasonic extraction, microwave-assisted extraction, or a combination thereof.
According to the invention, the weight ratio of N-acetylglucosamine, ornithine aspartate and fruit extract in the composition may be (1-10) to (10-1) to (0.1-5), for example (1-8) to (8-1) to (0.1-3), (1-5) to (5-1) to (0.1-2), (1-3) to (0.2-1), and may illustratively be about 5:1, 3:1, 1:1, 5.71: 2.85: 1, 8.33: 1.67: 1, 1: 9: 1.
According to the present invention, the composition may also optionally comprise excipients, such as one, two or more of flavoring agents, fillers, binders, wetting agents, lubricants, disintegrants, antioxidants, effervescent agents, and the like.
According to the present invention, the flavoring agent includes, but is not limited to, sucrose, stevioside, or aspartame, or a combination of two or more thereof. The filler includes, but is not limited to, microcrystalline cellulose, lactose, mannitol, starch, or dextrin, or a combination of two or more thereof. The binder includes, but is not limited to, hypromellose, povidone, methylcellulose, or sodium carboxymethylcellulose, or a combination of two or more thereof. The wetting agent includes, but is not limited to, water or aqueous ethanol. Such lubricants include, but are not limited to, magnesium stearate, talc, or aerosil, or a combination of two or more thereof. The disintegrant includes, but is not limited to, sodium carboxymethyl starch, croscarmellose sodium, sodium carboxymethyl starch, hydroxypropyl starch, crospovidone, low substituted hydroxypropyl cellulose, or corn starch, or a combination of two or more thereof. The antioxidant includes, but is not limited to, vitamin C, sodium sulfite, sodium bisulfite or sodium metabisulfite, or a combination of two or more thereof. Such effervescent agents include, but are not limited to, citric acid and sodium bicarbonate.
According to the present invention, the composition may be in solid form, such as granules, capsules, tablets, pills, and the like; it may also be in liquid form, such as oral liquid, beverage, milk-containing drink, etc. The tablets may be buccal, chewable or effervescent tablets and the tablets may be coated tablets. The capsule can be a hard capsule or a soft capsule. The pill can be pellet or dripping pill.
The invention also provides a preparation method of the composition, which comprises the steps of mixing the ornithine aspartate, the N-acetylglucosamine and optional components according to the formula amount, and then preparing granules, capsules, tablets or pills.
According to the invention, the composition is a granulate and the preparation method thereof comprises the following steps:
(1) Preparation of granulation solution: dissolving the adhesive and part of the fruit extract in the formula amount in deionized water or purified water to obtain a granulation solution;
(2) Preparation of the particles: mixing the N-acetylglucosamine, ornithine aspartate, the rest fructus fici extract and optional excipient, spraying the granulating solution to granulate, drying, and grading.
The invention also provides application of the composition in preparing health-care food for relieving fatigue.
The invention also provides application of the composition in preparing health-care food for improving immunity.
The invention also provides application of the composition in preparing health-care food for assisting liver protection.
The present invention also provides a method of alleviating fatigue comprising the step of administering to an individual in need thereof a composition of the present invention.
The present invention also provides a method of enhancing immunity comprising the step of administering to an individual in need thereof a composition of the present invention.
The present invention also provides a method for aiding in the protection of the liver, comprising the step of administering to an individual in need thereof a composition of the present invention.
According to the present invention, the liver protection assistance is for example enhancing the antioxidant capacity of the liver, decreasing the fat content in liver cells, decreasing the level of transaminases such as ALT and AST, etc.
The beneficial effects of the invention include: the composition containing N-acetylglucosamine and ornithine aspartate has a synergistic effect of the two functional components, and compared with the ornithine aspartate or the N-acetylglucosamine alone, the composition can remarkably and quickly relieve fatigue, enhance the immunity of an organism and simultaneously has the function of assisting and protecting liver.
Brief Description of Drawings
FIG. 1 shows a comparison of the swimming time lengths of mice in effect test example 4.
Fig. 2 is a bar graph showing the content of reduced Glutathione (GSH) in liver tissues of mice in the blank group, the alcohol group, the different dose groups of amcanda particles 1, and the different dose groups of single amcanda particles 1 in effect test example 4.
Fig. 3 shows a bar graph of Malondialdehyde (MDA) content in liver tissue of mice in the blank group, the alcohol group, the different dose groups of amgoda particles 1, and the different dose groups of single door particles 1 in effect test example 5.
Fig. 4 shows a histogram of Triglyceride (TG) content in liver tissue of mice in the blank group, the alcohol group, the different dose groups of the glucosamine goalpost granule 1, and the different dose groups of the individual goalpost granule 1 in the effect test example 5.
Fig. 5 shows a comparison of the effects of the blank group, the alcohol group, the high concentration glucosamine goalpost granule 1 group in the effect test example 5 in eliminating lipidation of liver tissue in mice compared with the low concentration glucosamine goalpost granule 1 group and the goalpost granule 1 group.
Detailed Description
The present invention will be described in further detail with reference to specific examples. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods. In the following examples, N-acetylglucosamine is referred to as "glucosamine" and ornithine aspartate is referred to as "Ornithogmine". The ornithine aspartate is purchased from Wuhanqirui pharmaceutical industry Co., ltd, the N-acetylglucosamine is purchased from Maanshan Tiantai Biotech Co., ltd, and the blueberry extract is purchased from Nanjing Chuan Biotech Co., ltd.
Preparation example 1 preparation of glucosamine Ornital granule 1
The formulation of amikayao bird granule 1 is as follows:
the preparation process comprises the following steps:
(1) Preparation of granulation solution
(1) Weighing the purified water in the group (1);
(2) adding the blueberry extract in the group (1) into purified water, and stirring for dissolving;
(3) adding the polyvidone K30 in the group (1), and stirring for dissolving for later use;
(2) Preparation of the granules
(1) Adding ornithine aspartate, N-acetylglucosamine and blueberry extract in the group (2) into a wet granulator, setting the rotation speed of a stirring paddle to be 180rpm and the rotation speed of a cutting knife to be 1200rpm, and mixing for 5min;
(2) add granulation solution to the above granulation mass: the rotating speed of the stirring paddle is 180rpm, the rotating speed of the cutting knife is 1200rpm, the rotating speed of the peristaltic pump is 30rpm, and the atomizing pressure is 0.025Mpa;
(3) granulating: the rotating speed of a stirring paddle is 180rpm, the rotating speed of a cutting knife is 1200rpm, and the mixture is granulated for 120s;
(4) and (3) wet granulation: sieving with a sieve mesh with the aperture of 2mm and the rotor rotating speed of 1000rpm;
(5) and (3) drying: drying by a fluidized bed at the air inlet temperature of 70 ℃ until the drying weight loss of the material is less than 3 percent;
(6) dry granulation: sieving with sieve mesh of 0.5mm and rotor speed of 1000rpm to obtain purple to light purple granules, and recording as glucosamine bird granule 1.
Preparation example 2 preparation of amikame particles 2
The formula of the glucosamine bird granules 2 is as follows:
the amikame bird granules 2 were prepared by the method of reference preparation example 1.
Preparation example 3 preparation of amikame particles 3
The formulation of amikayao bird granule 3 is as follows:
referring to the method of preparation example 1, amikame granules 3, which were white to off-white granules, were prepared without addition of blueberry extract.
Preparation example 4 preparation of amikame particles 4
The formulation of amikao bird granules 4 is as follows:
referring to the method of preparation example 1, amifoster bird granules 4 were prepared.
Preparation example 5 preparation of glucosamine Ornital granule 5
The formulation of amikame bird granules 5 is as follows:
referring to the method of preparation example 1, particles 5 were prepared.
Comparative example 1 preparation of Ornital particles 1
The formulation of the Ornital granule 1 is as follows:
referring to the method of preparation example 1, pellets 1 of Ornital were prepared.
Comparative example 2 preparation of glucosamine granule 1
The formula of glucosamine granules 1 is as follows:
referring to the method of preparation example 1, glucosamine granules 1 were prepared.
The effects of resisting fatigue, improving immunity and assisting in protecting liver of the product of the invention under a single-dose administration scheme are examined through the following effect test examples 1-3.
Effect test example 1 anti-fatigue test
1.1 test materials: the product of the invention, amminotus particle 1 and amminotus particle 2, is prepared from ornithine aspartate, N-acetylglucosamine and blueberry extract, while amminotus particle 3 does not contain blueberry extract. The test animal is KM mouse, male, with a weight of 18-22g, and provided by research center for experimental animals in Hubei province.
1.2 sample preparation: respectively weighing about 1g of glucosamine bird particles 1, 2, 3, 1 and 1, adding a certain amount of normal saline, and making into 10mg/ml test sample. The negative control gastric perfusion group is given with normal saline, and each experimental group is subjected to gastric perfusion according to the volume of 10 ml/kg. The three glucosamine granule groups, glucosamine granule 1 group and Ornital granule 1 group were all administered at a dose of 100mg/kg.
1.3 Experimental methods: (1) weight swimming test: 10 mice in each group are continuously gavaged for 30 days, 30min after the test object is given for the last time, the tail root of the mouse bears the weight of 5 percent, and the mouse is placed in a swimming box for swimming to observe the time from swimming to death of the mouse. (2) determination of blood lactic acid content: the mice in each group were continuously gavaged for 30 days, 30min after the last administration of the test substance, and then placed in water at 30 deg.C for swimming for 10min, and blood was taken twice (inner canthus blood was taken) immediately before and after swimming for measuring the blood lactic acid value. And (3) determination of serum urea nitrogen content: continuously intragastrically irrigating the mouse for 30 days, 30min after the last administration of the test substance, placing the mouse in water of 30 ℃ for swimming for 90min, after water discharge, resting for 60min, and measuring the content of serum urea nitrogen by taking blood from eyeballs.
1.4 test results
1.4.1 Effect of Nitinol particles of the present invention on the time to weight bearing swimming of mice
The glucosamine bird particles 1, 2 and 3 and the single-component N-acetylglucosamine (glucosamine particles 1) can prolong the weight swimming time of mice, and have significant differences (P is less than 0.01, P is less than 0.05, P is less than 0.01 and P is less than 0.05) compared with a negative control group; wherein the effect of the glucosamine paradoxer granules 1 is optimal, and the effect of the glucosamine paradoxer granules is 3 times and 2 times better than that of the single glucosamine paradoxer granules 1 and the single paradoxer granules 1. The results suggest that amikayamamoto compound granules 1, 2 and 3 all have the anti-fatigue effect. See table 1 for details.
TABLE 1 Effect of the products of the invention on weight bearing swimming time in mice
| Group of | Animal number (only) | Swimming time(s) |
| Negative control group | 10 | 265.66±45.23 |
| Glucosamine Ornital granule 1 | 10 | 412.25±76.87 **# |
| |
10 | 364.25±78.18 * |
| |
10 | 389.25±42.76 **# |
| Glucosamine granule 1 | 10 | 342.23±67.43 * |
| Ornit 1 | 10 | 278.43±37.34 |
Remarking: comparison with negative control group: * P < 0.05, P < 0.01; compared with the N-acetylglucosamine group, # P < 0.05.
1.4.2 Effect of Amycotina according to the invention on the number of blood lactic acids swimming in mice
Compared with a negative control group, the glucosamine anoploid particles 1, 2 and 3 and the single N-acetylglucosamine (glucosamine particles 1) can reduce the blood lactate value of mice after swimming, and have significant differences (P < 0.01, P < 0.05, P < 0.01 and P < 0.05), wherein the effect of the glucosamine anoploid particle 1 is optimal, and the glucosamine anoploid particles 3 and 2 are inferior and superior to the single glucosamine particle 1 and the individual glucosamine anoploid particle 1. See table 2 for details.
TABLE 2 Effect of the products of the invention on the swimming serum lactic acid number of mice
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Remarking: comparison with negative control group: * P < 0.05, P < 0.01; compared with the N-acetylglucosamine group, # P < 0.05.
1.4.3 Effect of Amycotina according to the invention on mouse serum Urea Nitrogen
Compared with a negative control group, the glucosamine paragonium granules 1, 2 and 3 and the single N-acetylglucosamine (glucosamine granules 1) can reduce the blood serum urea nitrogen value of mice, and have significant difference (P is less than 0.01, P is less than 0.05, P is less than 0.01 and P is less than 0.05), wherein the effect of the glucosamine paragonium granules 1 is optimal, and the glucosamine paragonium granules 3 and 2 are superior to that of the single glucosamine paragonium granules 1 and the paragonium granules 1. See table 3 for details.
TABLE 3 Effect of the products of the invention on mouse serum uremic toxins
| Group of | Animal number (only) | Serum urea nitrogen (mmol/L) |
| Negative control group | 10 | 8.65±1.65 |
| Glucosamine nital particles 1 | 10 | 5.71±1.05 **# |
| |
10 | 7.33±1.59 * |
| |
10 | 6.68±1.29 *# |
| Glucosamine granule 1 | 10 | 7.18±1.19 * |
| Ornital grain 1 | 10 | 8.04±1.23 |
Remarking: comparison with negative control group: * P < 0.05, P < 0.01; compared with the N-acetylglucosamine group, # P < 0.05.
Effect test example 2 Immunity improvement test
2.1 test materials: the products of the invention, namely the amikay bird particles 1, the amikay bird particles 2 and the amikay bird particles 3, are tested by KM mice, male animals with the weight of 18-22g, and are provided by the research center of experimental animals in Hubei province.
2.2 sample preparation and grouping: respectively weighing about 1g of glucosamine bird particles 1, 2, 3, 1 and 1, adding a certain amount of normal saline, and making into 10mg/ml test sample. The negative control was gavaged with saline and each experimental group was gavaged at a volume of 10 ml/kg. The dosage of the three glucosamine paradox granule groups, the glucosamine paradox granule 1 group and the paradox granule 1 group is 100mg/kg. The experimental animals were randomly grouped into groups of 10 animals each, and each group of animals was gavaged 1 time per day for 30 consecutive days.
2.3 Experimental methods:
2.3.1 measurement of body weight, organ/body weight ratio: the body weight of the mice was monitored and recorded weekly, the mice were sacrificed after the last administration, the thymus and spleen were taken, weighed on an electronic analytical balance, and the organ/body weight ratio was calculated.
2.3.2 mouse carbon end clearance experiment: injecting India ink (diluted 4 times physiological saline) into tail vein of 0.1ml/10g, timing immediately after injection, collecting blood 20 μ L from inner canthus venous plexus at 2 and 10min, respectively, adding into 2mL0.1% Na 2 CO 3 Shaking up in the solution. With Na 2 CO 3 The solution was used as a blank and the absorbance (OD) of each tube was measured at a wavelength of 600 nm. The mice are sacrificed, the liver and spleen are taken out, weighed and phagocytic index a =is calculatedK1/3 × body weight/(liver weight + spleen weight), K = (lgOD 1-lgOD 2)/(t 2-t 1).
2.3.3 serum hemolysin experiment: intraperitoneal injection of 0.2mL of SRBC suspension per mouse, taking blood from eyeball after 5 days of immunization, centrifuging at 2000rpm for 10min to separate serum, taking serum to dilute 200 times with SA buffer solution, taking 1mL of diluted serum to put in a test tube, taking 1mLSA buffer solution from a control tube, and sequentially adding 0.5mL of 10% (v/v) SRBC and 1:8SA diluted guinea pig serum 1mL. After incubation in a 37 ℃ water bath for 20min, the reaction was stopped with an ice bath. Centrifuge at 2000rpm for 10min. Taking 1mL of supernatant, adding 3mL of Dow's reagent, taking 0.25mL of 10% (v/v) SRBC and adding the Dow's reagent to 4mL in another test tube, and fully mixing to obtain a half hemolysis value. Standing for 10min, measuring absorbance at 540nm wavelength, and calculating HC 50 。HC 50 = OD/OD at half hemolysis of SRBC of sample x dilution factor.
2.3.4 Delayed Type Hypersensitivity (DTH) assay: depilating abdominal skin of mice within a range of about 3cm × 3cm by barium sulfide, applying 1% DNFB solution (acetone: sesame oil = 1: 1) in an amount of 50. Mu.L for sensitization, uniformly applying 10. Mu.L of DNFB to both sides of the right ear of the mice after 5 days for attack, dislocating the cervical vertebrae after 24 hours to kill the mice, cutting off the left and right ear shells, removing 8 mm-diameter ear pieces by a puncher, weighing, and expressing the degree of DTH by the difference between the left and right ear weights
2.3.5 ConA-induced mouse splenic lymphocyte transformation assay (MTT method): mice were sacrificed after the last administration, spleens were aseptically taken, placed in small dishes containing appropriate amounts of sterile Hank's solution, made into cell suspensions, and filtered through a 200 mesh screen. Wash 2 times with Hank's solution, and centrifuge each time for 10min (1000 r/min). Then, the cells were suspended in 1mL of complete culture medium, the number of viable cells was counted, and the cell concentration was adjusted to 3X 10 by RPMI1640 culture medium 6 one/mL. The cell suspension was further added to a 24-well plate in two wells, 1mL per well, 75. Mu.L of ConA solution (equivalent to 7.5. Mu.g/mL) was added to one well, and the other well was used as a control, and the percent by weight of CO was 5% 2 And culturing for 72 hours in a carbon dioxide incubator at 37 ℃.4 hours before the end of the culture, 0.7mL of the supernatant was gently aspirated from each well, and 0.7mL of calf serum-free RPMI1640 culture medium was added thereto together with 50. Mu.L of MTT (5 mg/mL) per well, and the culture was continued for 4 hours. After the culture is finished, 1mL of acidic isopropanol is added into each hole, and the mixture is blown and beatenAnd (4) homogenizing to completely dissolve the purple crystals. Then, the cells were divided into 96-well culture plates, each of which was prepared as 3 parallel wells, and the optical density was measured at a wavelength of 570nm using a microplate reader. The proliferation capacity of lymphocytes was expressed as the optical density value of the ConA plus wells minus the optical density value of the ConA not plus wells.
2.4 test results
2.4.1 Effect of Aminosugar Ornital particles of the invention on mouse body weight, organ/body weight ratio
As can be seen from Table 4, there was no significant difference in body weight and thymic body ratio (P > 0.05) among mice in the glucosamine granules 1, 2 and 3, the glucosamine granule 1 and the granule 1 groups of the present invention compared to the negative control group, suggesting that glucosamine granules 1, 2 and 3 had no significant effect on the weight gain and thymic gland of the mice.
TABLE 4 Effect of the products of the invention on mouse body weight and thymic body ratio
| Group of | Animal number (only) | Initial weight (g) | Terminal weight (g) | Thymus/body weight (mg/g) |
| Negative control group | 10 | 20.14±1.65 | 29.24±3.05 | 1.14±0.15 |
| Glucosamine Ornital granule 1 | 10 | 20.51±1.98 | 28.91±2.29 | 1.21±0.22 |
| |
10 | 20.38±1.18 | 29.31±4.10 | 1.16±0.17 |
| |
10 | 20.47±2.05 | 29.27±3.18 | 1.20±0.08 |
| Glucosamine granules 1 | 10 | 20.28±1.23 | 29.39±4.17 | 1.14±0.27 |
| Ornital grain 1 | 10 | 20.23±2.12 | 29.21±3.88 | 1.23±0.11 |
2.4.2 Effect of Amycotina bird particles of the invention on mouse carbon dust clearance
As can be seen from Table 5, compared with the negative control group, the glucosamine Ornital particles 1, 2 and 3, and the single Ornital particle 1 of the present invention can significantly increase the phagocytic index in the mouse carbon clearance test (P < 0.01, P < 0.05, P < 0.01); wherein the effect of the glucosamine paragonite particle 1 is optimal, and the effect of the glucosamine paragonite particle is 2 times and 3 times, and both the effect is superior to that of the single paragonite particle 1 and the glucosamine particle 1. The suggestion shows that the glucosamine nital particles 1, 2 and 3 can improve the phagocytic function of mouse mononuclear-macrophages.
TABLE 5 Effect of the products of the invention on the phagocytic index of mice
| Group of | Animal number (only) | Phagocytic index |
| Negative control group | 10 | 3.39±0.34 |
| Glucosamine nital particles 1 | 10 | 5.01±0.62 **# |
| |
10 | 4.88±0.41 *# |
| |
10 | 4.18±0.49 * |
| Glucosamine granules 1 | 10 | 3.42±0.38 |
| Ornit 1 | 10 | 4.60±0.70 ** |
Remarking: comparison with negative control group: * P < 0.05, P < 0.01; compared to the ornithine aspartate group, # P < 0.05.
2.4.3 Effect of Aminosugar bird particles of the present invention on humoral immunity of mouse serum hemolysin experiment
As can be seen from Table 6, compared with the negative control group, the amgodan bird granules 1, 2 and 3, and the single-component amgodan bird granule 1 of the present invention all significantly increased HC in the mouse serum hemolysin experiment 50 (P < 0.01, P < 0.05), wherein the effect of the glucosamine paragonium granules 1 is optimal, and the effect of the glucosamine paragonium granules is 2 times better than that of the single paragonium granules 1 and the single paragonium granules 1.
Table 6 product of the invention versus mouse HC 50 Influence of (2)
| Group of | Animal number (only) | HC 50 |
| Negative control group | 10 | 85.29±10.01 |
| Glucosamine Ornital granule 1 | 10 | 102.28±8.40 **# |
| |
10 | 98.08±9.21 **# |
| |
10 | 92.08±7.11 *# |
| Glucosamine granules 1 | 10 | 86.12±6.43 |
| Ornital grain 1 | 10 | 93.11±10.10 * |
Remarking: comparison with negative control group: * P < 0.05, P < 0.01; compared to the ornithine aspartate group, # P < 0.05.
2.4.4 Effect of the invention on mouse cellular Immunity (delayed type allergy test)
As can be seen from Table 7, compared with the negative control group, the glucosamine paragonite granules 1, 2 and 3 of the present invention and the single paragonite granule 1 can significantly increase the weight difference of the right ear of the mouse (P < 0.01, P < 0.05); wherein the effect of the glucosamine paradoxus particle 2 is optimal, and the effect of the glucosamine paradoxus particle is 1 times lower than that of the single-side paradoxus particle 1 and the glucosamine particle 1. It was suggested that pellets 1, 2 and 3 of Amycota birds had an ameliorating effect on delayed-type allergy in mice.
TABLE 7 Effect of the products of the invention on the difference in weight of the left and right ear pieces in mice
| Group of | Animal number (only) | Difference in weight of left and right ear (mg) |
| Negative control group | 10 | 1.09±0.12 |
| Glucosamine nital particles 1 | 10 | 1.98±0.23 **# |
| |
10 | 2.15±0.27 **## |
| |
10 | 1.47±0.38 * |
| Glucosamine granule 1 | 10 | 1.20±0.17 |
| Ornit 1 | 10 | 1.67±0.20 * |
Remarking: comparison with negative control group: * P < 0.05, P < 0.01; compared with ornithine aspartate group, # P < 0.05, # P < 0.01.
2.4.5 Effect of Amycotina paradisi particles of the invention on ConA-induced splenic lymphocyte transformation Capacity in mice
As can be seen from Table 8, compared with the negative control group, the glucosamine Ornital granules 1, 2 and 3, and the single Ornital granule 1 of the invention can significantly increase the mouse spleen lymphocyte proliferation capacity (P < 0.01, P < 0.05); wherein the effect of the glucosamine paragonite particle 1 is optimal, and the effect of the glucosamine paragonite particle is 2 times of that of the glucosamine paragonite particle 1 and the glucosamine particle 1, and both are superior to that of the single species of paragonite particles 1 and glucosamine particles 1. It was suggested that the glucosamine Ornital particles 1, 2 and 3 could enhance the transforming ability of mouse lymphocytes.
TABLE 8 Effect of the products of the invention on the transforming ability of mouse lymphocytes
| Group of | Animal number (only) | Lymphocyte proliferative Capacity (OD value) |
| Negative control group | 10 | 0.20±0.08 |
| Glucosamine nital particles 1 | 10 | 0.35±0.07 **# |
| |
10 | 0.31±0.13 **# |
| |
10 | 0.25±0.11 * |
| Glucosamine granule 1 | 10 | 0.19±0.05 |
| Ornital grain 1 | 10 | 0.27±0.08 * |
Remarking: comparison with negative control group: * P < 0.05, P < 0.01; compared with ornithine aspartate group, # P < 0.05, # P < 0.01.
Effect test example 3 auxiliary liver protection test
3.1 test materials: the products of the invention, namely the amikay bird particles 1, the amikay bird particles 2 and the amikay bird particles 3, are tested by KM mice, male animals with the weight of 18-22g, and are provided by the research center of experimental animals in Hubei province.
3.2 sample preparation: respectively weighing about 1g of glucosamine bird particles 1, 2, 3, 1 and 1, adding a certain amount of normal saline, and making into 10mg/ml test sample. The negative control was gavaged with saline and each experimental group was gavaged at a volume of 10 ml/kg. The dosage of the amikao granule group, the amikao granule 1 group and the amikao granule 1 group is 100mg/kg.
3.3 Experimental methods: 10 mice in each group were continuously subjected to intragastric administration for 30 days, and 20min after the test substances were administered to the model control group, the amikame granule group 1 and the amikame granule group 1 for the last time, 13ml/kg of 50% ethanol was administered to the intragastric administration for one time, double distilled water was administered to the blank control group, and after fasting for 16 hours, the animals were killed to perform various biochemical index determinations and liver disease histological examination.
3.4 test results
3.4.1 Effect of Amycotina according to the invention on the content of Malondialdehyde (MDA) in homogenates of mouse liver
As can be seen from Table 9, the MDA content in the liver homogenate of the mouse in the model control group is increased, and the MDA content is very different from that in the blank control group (p is less than 0.01), which indicates that the modeling is successful. Compared with a model control group, the glucosamine anoplophora granules 1, 2 and 3 and the single Ornithogalus granules 1 can obviously reduce the MDA content (P is less than 0.01, P is less than 0.05) in liver homogenate of an alcoholic liver injury mouse; wherein the effect of the glucosamine paradoxus particle 2 is optimal, and the effect of the glucosamine paradoxus particle is 1 times lower than that of the single-side paradoxus particle 1 and the glucosamine particle 1.
TABLE 9 Effect of the products of the invention on the Malondialdehyde (MDA) content of liver homogenates in mice
/>
| Group of | Animal number (only) | MDA(nmol/mg prot) |
| Blank control group | 10 | 3.09±0.25 |
| Model control group | 10 | 7.34±1.03 && |
| Glucosamine Ornital granule 1 | 10 | 4.78±0.89 **# |
| |
10 | 3.78±0.75 **## |
| |
10 | 5.13±1.18 * |
| Glucosamine granules 1 | 10 | 7.21±0.87 |
| Ornital grain 1 | 10 | 5.24±0.75 * |
Remarking: comparison with blank control: and & P is less than 0.01; p < 0.05, P < 0.01 compared to model control; compared with ornithine aspartate group, # P < 0.05, # P < 0.01.
3.4.2 Effect of Amycotina anglica particles of the invention on the content of reduced Glutathione (GSH) in liver homogenates of mice
As can be seen from Table 10, the liver homogenate of the mouse in the model control group has a reduced GSH content, and has a very significant difference (p is less than 0.01) compared with that of the blank control group, which indicates that the modeling is successful. Compared with a model control group, the glucosamine anoplophora granules 1, 2 and 3 and the single Ornithogalus granules 1 can obviously increase the content of GSH (P is less than 0.01, P is less than 0.05) in liver homogenate of mice with alcoholic liver injury; wherein the effect of the glucosamine paragonite particle 1 is optimal, and the effect of the glucosamine paragonite particle is 2 times of that of the glucosamine paragonite particle 1 and the glucosamine particle 1, and both are superior to that of the single species of paragonite particles 1 and glucosamine particles 1.
TABLE 10 Effect of the products of the invention on reduced Glutathione (GSH) content in liver homogenates of mice
| Group of | Animal number (only) | GSH (mg/g liver) |
| Blank control group | 10 | 9.76±0.45 |
| Model control group | 10 | 7.14±0.63 && |
| Glucosamine nital particles 1 | 10 | 9.57±0.77 **# |
| |
10 | 9.38±0.69 **# |
| |
10 | 8.88±0.65 * |
| Glucosamine granule 1 | 10 | 7.19±0.56 |
| Ornital grain 1 | 10 | 8.91±0.34 * |
Remarking: comparison with blank control: and & P is less than 0.01; p < 0.05, P < 0.01 compared to model control; compared with ornithine aspartate group, # P < 0.05, # P < 0.01.
3.4.3 Effect of Aminocotimen particles of the present invention on the content of Triglyceride (TG) in liver homogenate in mice
As can be seen from Table 11, the TG content in the liver homogenate of the mouse in the model control group is increased, and the TG content is very different from that in the blank control group (p is less than 0.01), which indicates that the modeling is successful. Compared with a model control group, the glucosamine anoplophora particles 1, 2 and 3 and the single-prescription anoplophora particle 1 can obviously reduce the TG content (P is less than 0.01, P is less than 0.05, and P is less than 0.05) in liver homogenate of a mouse with alcoholic liver injury; wherein the effect of the glucosamine paragonite particle 1 is optimal, and the effect of the glucosamine paragonite particle is 2 times of that of the glucosamine paragonite particle 1 and the glucosamine particle 1, and both are superior to that of the single species of paragonite particles 1 and glucosamine particles 1.
TABLE 11 Effect of the products of the invention on the Triglyceride (TG) content of liver homogenates of mice
| Group of | Animal number (only) | TG (mu mol/g liver) |
| Blank control group | 10 | 7.16±0.39 |
| Model control group | 10 | 15.87±2.19 && |
| Glucosamine Ornital granule 1 | 10 | 10.36±1.79 **# |
| |
10 | 11.78±2.63 **# |
| |
10 | 13.47±2.49 * |
| Glucosamine granules 1 | 10 | 15.58±2.77 |
| Ornital grain 1 | 10 | 12.18±2.39 * |
Remarking: comparison with blank control: and & P is less than 0.01; p < 0.05, P < 0.01 compared to model control; compared with ornithine aspartate group, # P < 0.05, # # P < 0.01.
3.4.4 Effect of Amycotina avian particles of the invention on histopathological Scoring of mouse liver
As can be seen from Table 12, the hepatic cell lipid drop content score of the mice in the model control group is increased, and is very different from that in the blank control group (p is less than 0.01), which indicates that the modeling is successful. Compared with a model control group, the glucosamine Ornithogalus granules 1, 2 and 3 and the single aspartic acid ornithine can obviously reduce the fat drop content scores of mouse liver cells (P is less than 0.01, P is less than 0.05), wherein the effect of the glucosamine Ornithogalus granule 1 is optimal, and the effect of the glucosamine Ornithogalus granule is 2 times better than that of the single glucosamine Ornithogalus granule 1 and the single glucosamine Ornithogalus granule 1.
TABLE 12 Effect of the products of the invention on mouse hepatocyte lipid droplet content scores
| Group of | Animal number (only) | Hepatocyte lipid droplet content score |
| Blank control group | 10 | 0.15±0.03 |
| Model control group | 10 | 3.47±0.28 && |
| Glucosamine Ornital granule 1 | 10 | 1.38±0.26 **## |
| |
10 | 1.66±0.36 **# |
| |
10 | 2.37±0.46 * |
| Glucosamine granule 1 | 10 | 3.28±0.41 |
| Ornit 1 | 10 | 2.08±0.19 * |
Remarking: comparison with blank control: and & P is less than 0.01; p < 0.05, P < 0.01 compared to model control; compared with ornithine aspartate group, # P < 0.05, # # P < 0.01.
The results in tables 9 to 12 above all show that the pellets 1, 2 and 3 of the present invention have protective effect on liver injury.
The following effect test example 4 was conducted to investigate the effect of the glucosamine mince granules 1 and the mince granules 1 of the present invention on the function of alleviating physical fatigue.
Effect test example 4 physical fatigue test for alleviating amycotina particles
4.1 animal groups
KM mice, males, weighing 18-22g were purchased from Hubei provincial center. Mice were randomly divided into blank group, glucosamine particles 1 group, glucosamine granules 1 group, and Ornital granules 1 group according to body weight, with 6 animals per group. Respectively weighing a certain amount of glucosamine particles 1, glucosamine Ornital particles 1 and Ornital particles 1, adding distilled water with corresponding volume, mixing uniformly by vortex and ultrasound, intragastrically administering to mice according to the volume of 10ml/kg, administering distilled water to a blank control group, 1 time per day, and performing a load swimming experiment after 15 days.
4.2 weight swimming time test protocol and results
The test mice were subjected to the above-mentioned weight swimming test 15 days after the administration.
30min after the tested mouse is last dosed, a 5 percent weight lead sheet is loaded on the tail root of the mouse, the mouse is placed in a swimming box for swimming to observe the time from swimming to death, the water depth in the swimming box is not less than 30cm, and the water temperature is 25 +/-1.0 ℃. The results show that the swimming time of the amikao bird particle 1 group was significantly increased relative to the control group, both exceeding the amikao bird particle 1 group and the amikao particle 1 group. This result demonstrates that the pellets of the invention have a fatigue-relieving effect and in particular a synergistic effect of two active ingredients glucosamine and muscovy (see figure 1).
The following effect test example 5 was conducted to investigate the effect of the amikae granules 1 and the amikae granules 1 of the present invention on the alleviation of alcoholic liver injury in individuals with continuous alcohol load at different doses.
Effect test example 5 Amycorma particles for alleviating Long-term alcoholic liver injury test
5.1 animal groups
Male KM mice (weight 18-22 g) purchased from the disease control center in northHubei province were randomly divided by weight into a blank control group, an alcohol model group, a glucosamine gate bird granule 1 dose group a, a glucosamine gate bird granule 1 dose group B, a glucosamine gate bird granule 1 dose group C, a glucosamine gate bird granule 1 dose group D, and a gate bird granule 1 dose group D; each group contained 6 animals. Weighing a certain amount of glucosamine Ornithodion 1 and Ornithodion 1 respectively, adding distilled water with corresponding volume, performing vortex ultrasonic mixing, performing intragastric administration on mice according to the volume of 10ml/kg, administering distilled water to a blank control group and an alcohol group for 1 time every day, continuously performing intragastric administration on the alcohol group and each sample group for 52-degree white spirit (10 ml/kg) after 10 days, administering distilled water to the blank control group for continuous administration and 5 days of white spirit, killing animals after fasting for 16 hours, and detecting the content change of Malondialdehyde (MDA), reduced Glutathione (GSH) and Triglyceride (TG) in the liver. And detecting the content changes of GSH, MDA and TG according to the instruction of the commercial kit.
Details of the specific test groupings are shown in table 13 below.
TABLE 13 animal grouping and dosing regimens
| Amikame bird granule 1 dose group a | 2600mg/kg/d |
| Amikame bird granule 1 dose group B | 1800mg/kg/d |
| Glucosamine Ornital granule 1 dose group C | 900mg/kg/d |
| Glucosamine Ornital granule 1 dose group D | 450mg/kg/d |
| Ornital grain 1 dose group A | 1200mg/kg/d |
| Ornital granule 1 dose group B | 800mg/kg/d |
| Dosage group C of Ornithogalus granules 1 | 400mg/kg/d |
| Dosage group D of Ornithogalus granules 1 | 200mg/kg/d |
5.2 detection of hepatic tissue Malondialdehyde (MDA)
Taking about 0.5g of liver, washing with normal saline, wiping, weighing, shearing, placing in a homogenizer, adding 0.2M phosphate buffer solution, homogenizing for 10s at 20000r/min, repeating for 3 times at intervals of 30s to obtain 5% tissue homogenate (W/V), centrifuging for 5-10 min at 3000r/min, and taking supernatant for detection.
5.3 liver homogenate reduced Glutathione (GSH) assay
Taking 0.5g of liver, adding 5mL of physiological saline, fully grinding into fine slurry (10% liver homogenate), uniformly mixing, taking 0.5mL of slurry, adding 0.5mL of 4% sulfosalicylic acid, uniformly mixing, centrifuging at 3000rpm at room temperature for 10 minutes, and taking supernatant as a sample. And (3) detecting by adopting a reduced glutathione kit.
5.4 liver homogenate Triglyceride (TG) assay
0.5g of liver is taken, 5mL of physiological saline is added, the mixture is fully ground into fine slurry (10% liver homogenate), the mixture is centrifuged for 10 minutes at 3000rpm at room temperature, and supernatant is taken as a sample. And (3) detecting by adopting a triglyceride kit.
5.5 liver Pathology detection
Taking the material from the middle part of the left lobe of the liver as a cross section, freezing the cross section, and staining with Sudan III or oil red O. Microscopic examination: pathological changes of cells were recorded starting from one end of the liver field and the whole tissue section was observed continuously with a 40-fold objective. The distribution, extent and area of lipid droplets in the liver were observed. The scoring criteria are shown in table 14 below:
TABLE 14 liver pathology test Scoring criteria
5.6 test results
Test results show that each dosage group of the Ornithogalum granules 1 and each formula dosage group of the Ornithogalum granules 1 can promote the expression of GSH to a certain extent (see figure 2), the liver GSH level of the Ornithogalum granules 1 and the dosage groups A and B of the Ornithogalum granules 1 is obviously increased, the effect of the dosage group A of the Ornithogalum granules 1 is more obvious compared with the dosage group A of the Ornithogalum granules 1, and the fact that the Ornithogalum granules 1 have certain efficacy on the liver protection function based on GSH antioxidation under the dosage of A is shown.
The test results show that each dosage group of the Ornithogalum granules 1 and each formula dosage group of the Ornithogalum granules 1 can inhibit the production of MDA to a certain extent (see figure 3), and in the dosage group B, the effect of the Ornithogalum granules 1 is more obvious compared with that of the Ornithogalum granules 1, and the Ornithogalum granules 1 both have the effects of resisting oxidation and protecting liver.
The experimental results show that the dosage groups A, B and C of the 1 st glucosamine anorex particles and the dosage groups A, B and D of the 1 st glucosamine anorex particles can effectively reduce the content of TG in the liver, and the glucosamine anorex particles 1 are suggested to have the effects of reducing fat and protecting the liver (see figure 4).
The lipid drop score is given in table 15 below. See also the fat droplet photograph shown in fig. 5.
TABLE 15 fatty drop score results for liver pathology testing
The results show that compared with the alcohol group, the liver fat droplets of the mice in the high concentration amikay bird granule 1 group (2000 mg/kg/d) are obviously reduced and approach to the blank group, while the liver fat droplets of the mice in the low concentration amikay bird granule 1 group (250 mg/kg/d) and the door bird granule 1 group are not obviously changed compared with the alcohol group, which indicates that the amikay bird granule 1 has the effects of reducing fat and protecting liver, and is especially obvious under the high concentration condition.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Embodiments of the present invention are exemplified above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Claims (33)
1. A composition comprises N-acetylglucosamine and ornithine aspartate, and the weight ratio of the N-acetylglucosamine to the ornithine aspartate is (1-10) to (10-1).
2. The composition as in claim 1, wherein the weight ratio of N-acetylglucosamine to ornithine aspartate is (1-8) to (8-1).
3. The composition as in claim 1, wherein the weight ratio of N-acetylglucosamine to ornithine aspartate is (1-5) to (5-1).
4. The composition as in claim 1, wherein the weight ratio of N-acetylglucosamine to ornithine aspartate is (1-3) to (3-1).
5. The composition according to claim 1, wherein the weight ratio of N-acetylglucosamine to ornithine aspartate is 1.
6. The composition of any one of claims 1 to 5, wherein the composition further comprises a fruit extract.
7. The composition of claim 6, wherein the fruit extract comprises blueberry extract, strawberry extract, cranberry extract, orange extract, grape skin extract, grape seed extract, mulberry extract.
8. The composition of claim 6, wherein the fruit extract is a blueberry extract.
9. The composition as in claim 6, wherein the weight ratio of the ornithine aspartate, the N-acetylglucosamine and the fruit extract is (1-10), (10-1) and (0.1-5).
10. The composition as in claim 6, wherein the weight ratio of the ornithine aspartate, the N-acetylglucosamine and the fruit extract is (1-8), (8-1) and (0.1-3).
11. The composition as in claim 6, wherein the weight ratio of ornithine aspartate, N-acetylglucosamine and fruit extract is (1-5), (5-1) and (0.1-2).
12. The composition as in claim 6, wherein the weight ratio of ornithine aspartate, N-acetylglucosamine and fruit extract is (1-3) to (0.2-1).
13. The composition according to claim 6, wherein the weight ratio of ornithine aspartate, N-acetylglucosamine and fruit extract is 5.
14. The composition according to claim 6, wherein the weight ratio of ornithine aspartate, N-acetylglucosamine and fruit extract is 3.
15. The composition according to claim 6, wherein the weight ratio of ornithine aspartate, N-acetylglucosamine and fruit extract is 1.
16. The composition of any one of claims 1 to 5 and 7 to 15, wherein the composition further comprises an excipient.
17. The composition of claim 16, wherein the excipient is one, two or more of a flavoring agent, a coloring agent, a filler, a binder, a wetting agent, a lubricant, a disintegrant, an antioxidant, an effervescent agent, and the like.
18. The composition of claim 6, wherein the composition further comprises an excipient.
19. The composition of claim 18, wherein the excipient is one, two or more of a flavoring agent, a coloring agent, a filler, a binder, a wetting agent, a lubricant, a disintegrant, an antioxidant, an effervescent agent, and the like.
20. The composition of any one of claims 1 to 5, 7-15, and 17 to 19, wherein the composition is in a solid form or a liquid form.
21. The composition of claim 20, wherein the solid form is a granule, capsule, tablet, or pill.
22. The composition of claim 20, wherein the liquid form is an oral liquid or a beverage.
23. The composition of claim 20, wherein the liquid form is a milk-containing drink.
24. The composition of claim 6, wherein the composition is in a solid form or a liquid form.
25. The composition of claim 24, wherein the solid form is a granule, capsule, tablet, or pill.
26. The composition of claim 24, wherein the liquid form is an oral liquid or a beverage.
27. The composition of claim 24, wherein the liquid form is a milk-containing drink.
28. The composition of claim 16, wherein the composition is in a solid form or a liquid form.
29. The composition of claim 28, wherein the solid form is a granule, capsule, tablet, or pill.
30. The composition of claim 28, wherein the liquid form is an oral liquid or a beverage.
31. The composition of claim 28, wherein the liquid form is a milk-containing drink.
32. A method of preparing a composition according to any one of claims 1 to 21, 24, 25, 28 and 29, comprising mixing the formula amounts of ornithine aspartate, N-acetylglucosamine and optionally the components, and then making into granules, capsules, tablets or pills.
33. Use of a composition according to any one of claims 1 to 31 for the preparation of a health food product capable of relieving fatigue, enhancing immunity and/or aiding in the protection of the liver.
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