WO2022161485A1 - 一种包含n-乙酰氨基葡萄糖的组合物及其制备方法和应用 - Google Patents

一种包含n-乙酰氨基葡萄糖的组合物及其制备方法和应用 Download PDF

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WO2022161485A1
WO2022161485A1 PCT/CN2022/074897 CN2022074897W WO2022161485A1 WO 2022161485 A1 WO2022161485 A1 WO 2022161485A1 CN 2022074897 W CN2022074897 W CN 2022074897W WO 2022161485 A1 WO2022161485 A1 WO 2022161485A1
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glucosamine
granules
composition
group
bird
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PCT/CN2022/074897
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French (fr)
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顾利民
王朝东
解胜厚
喻林
冯伟
周锋
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张曼
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/175—Amino acids

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  • the invention belongs to the field of health food, and in particular relates to a composition comprising N-acetylglucosamine and ornithine aspartate, a preparation method thereof, and a preparation method for relieving fatigue, improving immunity and assisting liver protection. Application of health food.
  • Ornithine aspartate is a salt formed by aspartic acid and ornithine, which was initially used clinically to treat acute and chronic liver diseases such as various types of hepatitis, cirrhosis, fatty liver and post-hepatitis syndrome. Elevated blood ammonia and hepatic encephalopathy.
  • Ornithine aspartate is decomposed into aspartic acid and ornithine in the body, among which, ornithine is the initial substrate in the urea cycle, which is the synthesis of ornithine carbamoyltransferase and carbamoyl phosphate
  • the activator of enzymes combined with toxic metabolites such as ammonia in the blood circulation, promotes the synthesis of urea, and accelerates the metabolism of toxic substances in the body, especially ammonia
  • aspartic acid participates in the synthesis of glutamine and nucleic acid in liver cells, which can not only promote the liver
  • the synthesis of glutamine is indirectly involved in the triphosphate cycle and the synthesis of nucleic acid, and provides intermediate products of energy metabolism, promotes the repair and regeneration of liver cells, restores liver function, and enhances liver detoxification.
  • N-acetylglucosamine is a derivative of glucosamine, which is the basic unit of various polysaccharides in living organisms. It also exists widely in nature and is the main component of chitin and chitosan. N-acetylglucosamine has the effect of protecting cartilage tissue and bone joints, and is often used as a dietary supplement.
  • the present invention provides a composition comprising N-acetylglucosamine and ornithine aspartate, and the weight ratio of the two can be (1 ⁇ 10): (10 ⁇ 1), for example (1 ⁇ 8):(8 ⁇ 1), (1 ⁇ 5):(5 ⁇ 1), (1 ⁇ 3):(3 ⁇ 1), exemplarily 1:1.
  • the composition may further comprise fruit extracts, including but not limited to blueberry extract, strawberry extract, cranberry extract, citrus extract, grape skin extract, grape seed extract, mulberry extract and the like, preferably blueberry extract.
  • the fruit extract may be an aqueous extract, an alcohol extract and/or an aqueous alcohol extract; the alcohol may be ethanol, and the aqueous alcohol solution may be a 50-95% (v/v) aqueous ethanol solution.
  • Commercially available fruit extracts can be selected, or extracts obtained by extracting fruits by the following methods: leaching method, enzymatic hydrolysis method, ultrasonic extraction method, microwave-assisted extraction method, or a combination thereof.
  • the weight ratio of N-acetylglucosamine, ornithine aspartate and fruit extract can be (1 ⁇ 10):(10 ⁇ 1):(0.1 ⁇ 5) , for example (1 ⁇ 8):(8 ⁇ 1):(0.1 ⁇ 3), (1 ⁇ 5):(5 ⁇ 1):(0.1 ⁇ 2), (1 ⁇ 3):(1 ⁇ 3): (0.2-1), exemplarily about 5:5:1, 3:3:1, 1:1:1, 5.71:2.85:1, 8.33:1.67:1, 1:9:1.
  • the composition may also optionally contain excipients such as flavoring agents, fillers, binders, wetting agents, lubricants, disintegrants, antioxidants, effervescent agents, etc. one, two or more.
  • excipients such as flavoring agents, fillers, binders, wetting agents, lubricants, disintegrants, antioxidants, effervescent agents, etc. one, two or more.
  • the flavoring agents include, but are not limited to, sucrose, stevioside or aspartame, or a combination of two or more thereof.
  • Such fillers include, but are 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 an aqueous ethanol solution.
  • the lubricant includes, but is not limited to, magnesium stearate, talc, or micronized silica gel, or a combination of two or more thereof.
  • the disintegrants include but are 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 antioxidants include, but are not limited to, vitamin C, sodium sulfite, sodium bisulfite, or sodium metabisulfite, or a combination of two or more thereof.
  • the effervescent agents include, but are not limited to, citric acid and sodium bicarbonate.
  • the composition can be in solid form, such as granules, capsules, tablets, pills, etc.; and can also be in liquid form, such as oral liquid, beverage, milk-containing beverage, and the like.
  • the tablet may be a buccal, chewable or effervescent tablet, and the tablet may be a coated tablet.
  • the capsules can be hard capsules or soft capsules.
  • the pills can be pellets or drop pills.
  • the present invention also provides a preparation method of the composition, comprising mixing the formula amounts of ornithine aspartate, N-acetylglucosamine and optional components, and then preparing granules, capsules, tablets or pill.
  • the composition is a granule, and its preparation method comprises the following steps:
  • Preparation of granulation solution Dissolve the binder of the formula amount and part of the fruit extract in deionized water or purified water to obtain a granulation solution;
  • the present invention also provides the application of the composition for preparing the health food for relieving fatigue.
  • the present invention also provides the application of the composition for preparing the immunity-enhancing health food.
  • the present invention also provides the application of the composition for preparing the health food for auxiliary liver protection.
  • the present invention also provides a method of alleviating fatigue comprising the step of administering a composition of the present invention to an individual in need thereof.
  • the present invention also provides a method of enhancing immunity comprising the step of administering to an individual in need a composition of the present invention.
  • the present invention also provides a method for assisting liver protection, comprising the step of administering the composition of the present invention to an individual in need.
  • the auxiliary liver protection includes, for example, enhancing the antioxidant capacity of the liver, helping to reduce the fat content in hepatocytes, helping to reduce the levels of transaminases such as ALT and AST, and the like.
  • the beneficial effects of the present invention include: the composition comprising N-acetyl glucosamine and ornithine aspartate of the present invention, the two functional components have synergistic effects, and the composition of the present invention is synergistic with ornithine aspartate alone or alone Compared with N-acetylglucosamine, it can significantly and quickly relieve fatigue, enhance the body's immunity, and at the same time have the function of assisting liver protection.
  • FIG. 1 shows the comparison of the swimming time length of mice in Effect Test Example 4.
  • Figure 2 shows the reduced glutathione (GSH) in the liver tissue of mice in the blank group, the alcohol group, the different dose groups of Glucosamine Menbird Granules 1 and the different dose groups of Menbird Granules 1 in the effect test example 4. ) content histogram.
  • Figure 3 shows the content of malondialdehyde (MDA) in the liver tissue of mice in the blank group, the alcohol group, the different dose groups of Glucosamine Menbird Granules 1 and the different dose groups of Menbird Granules 1 in the effect test example 5. Histogram.
  • Figure 4 shows the triglyceride (TG) content in the liver tissue of mice in the blank group, the alcohol group, the different dose groups of Glucosamine Menbird Granules 1 and the different dose groups of Menbird Granules 1 in the effect test example 5. Histogram.
  • Fig. 5 shows the effect of elimination of liver tissue in mice in the blank group, the alcohol group, and the high-concentration Glucosamine Gate Bird Granule 1 group compared with the low-concentration Glucosamine Gate Bird Granule 1 group and the Gate Bird Granule 1 group in the effect test example 5 Efficacy comparison in lipidation.
  • N-acetylglucosamine is abbreviated as "glucosamine”
  • ornithine aspartate is abbreviated as "men bird”.
  • the used ornithine aspartate was purchased from Wuhan Qirui Pharmaceutical Co., Ltd.
  • N-acetylglucosamine was purchased from Maanshan Tiantai Biotechnology Co., Ltd.
  • blueberry extract was purchased from Nanjing Chuanyuan Biotechnology Co., Ltd.
  • the preparation process is as follows:
  • the rotating speed of the stirring paddle is 180 rpm
  • the rotating speed of the cutting knife is 1200 rpm
  • the rotating speed of the peristaltic pump is 30 rpm
  • the atomization pressure is 0.025Mpa
  • 6Dry whole granulation sieve, the mesh aperture is 0.5mm, and the rotor speed is 1000rpm to obtain purple to light purple granules, which are recorded as Glucosamine Menbird Granules 1.
  • Glucosamine Granules 2 were prepared.
  • Glucosamine Gate Bird Granules 3 were prepared, which were white to off-white granules.
  • Glucosamine Granules 4 were prepared.
  • the recipe for Door Bird Granule 1 is as follows:
  • Glucosamine Granules 1 The formula for Glucosamine Granules 1 is as follows:
  • Glucosamine Granules 1 were prepared.
  • Glucosamine Granules 1 and Glucosamine Granules 2 are prepared from ornithine aspartate, N-acetylglucosamine and blueberry extract, while Glucosamine granules Granule 3 does not contain blueberry extract.
  • the experimental animals were KM mice, male, weighing 18-22 g, provided by Hubei Laboratory Animal Research Center.
  • mice in each group were given continuous gavage for 30 days, and 30 minutes after the last administration of the test substance, the tail root of the mice was loaded with 5% of their body weight, and placed in a swimming box to observe the mice swimming. Swim to death.
  • (2) Determination of blood lactic acid content 10 mice in each group, the mice were continuously fed for 30 days, and 30 minutes after the last administration of the test substance, they were placed in water at 30°C for 10 minutes of swimming, before swimming and immediately after swimming twice. Blood (inner canthus blood) was taken to determine the blood lactate value.
  • mice were continuously given intragastric administration for 30 days, and 30 minutes after the last administration of the test substance, they were placed in water at 30°C for 90 minutes of swimming, and after the water came out, rested for 60 minutes, and blood was taken from the eyeballs to measure the serum urea nitrogen content.
  • the Glucosamine Gate Bird Granules 1, 2 and 3 of the present invention can prolong the swimming time of mice with weight, and compared with the negative control group, there is a significant difference (P ⁇ 0.01) , P ⁇ 0.05, P ⁇ 0.01 and P ⁇ 0.05); among them, Glucosamine Gate 1 had the best effect, followed by Glucosamine 3 and 2, and both were better than the unilateral Glucosamine 1 and Gate Bird granules 1.
  • the results suggest that Glucosamine Menniao Compound Granules 1, 2 and 3 all have anti-fatigue effects. See Table 1 for details.
  • the Glucosamine Gate Bird Granules 1, 2 and 3 of the present invention can reduce the blood lactate value of the mice after swimming, and there are significant differences ( P ⁇ 0.01, P ⁇ 0.05, P ⁇ 0.01 and P ⁇ 0.05), among which Glucosamine Granule 1 had the best effect, followed by Glucomen Bird Granules 3 and 2, and both were better than unilateral Glucosamine Granules 1 and 2.
  • the Glucosamine Gate Bird Granules 1, 2 and 3 of the present invention can reduce the serum urea nitrogen value of mice, and there are significant differences (P ⁇ 0.01, P ⁇ 0.05, P ⁇ 0.01 and P ⁇ 0.05), among which Glucosamine Gate 1 had the best effect, followed by Glucosamine Gate 3 and 2, and both were better than unilateral Glucosamine 1 and Gate Bird Particle 1. See Table 3 for details.
  • Test materials the products of the present invention - Glucosamine Bird Granules 1, Glucosamine Gate Bird Granules 2 and Glucosamine Gate Bird Granules 3, the test animals are KM mice, male, weighing 18-22g, from the experimental animals of Hubei City. provided by the research center.
  • mice were injected with Indian ink (4 times diluted with normal saline) according to 0.1ml/10g tail vein, and the timing was immediately after the ink injection, and 2 and 10 minutes after the ink injection, respectively, from the medial canthal vein Collect 20 ⁇ L of blood, add it to 2 mL of 0.1% Na 2 CO 3 solution, and shake well. With Na 2 CO 3 solution as blank control, the absorbance value (OD) of each tube was measured at a wavelength of 600 nm.
  • each mouse was intraperitoneally injected with 0.2 mL of 2% SRBC suspension. After 5 days of immunization, the eyeballs were removed and blood was collected, centrifuged at 2000 rpm for 10 min to separate the serum, and the serum was diluted 200 times with SA buffer, and 1 mL of the diluted serum was taken. Put it in a test tube, take 1 mL of SA buffer from the control tube, and add 0.5 mL of 10% (v/v) SRBC and 1 mL of guinea pig serum diluted 1:8 SA in turn. After incubating in a 37°C water bath for 20 min, the reaction was terminated in an ice bath.
  • HC 50 sample OD/OD at half hemolysis of SRBC ⁇ dilution factor.
  • DTH Delayed type hypersensitivity
  • mice 2.3.5ConA-induced spleen lymphocyte transformation experiment in mice (MTT method): Mice were killed after the last administration, the spleen was aseptically removed, and placed in a small plate containing an appropriate amount of sterile Hank's solution to prepare a cell suspension. Filter through a 200-mesh sieve. Wash twice with Hank's solution and centrifuge for 10 min each time (1000 r/min). Then, the cells were suspended in 1 mL of complete medium, the number of viable cells was counted, and the cell concentration was adjusted to 3 ⁇ 10 6 cells/mL with RPMI1640 medium.
  • the Glucosamine Gate Bird Granules 1, 2 and 3 of the present invention can significantly increase the phagocytosis index in the carbon end clearance experiment in mice (P ⁇ 0.01, P ⁇ 0.05, P ⁇ 0.01, P ⁇ 0.01); among them, Glucosamine Menbird Granule 1 had the best effect, followed by Glucosamine Menbird Granules 2 and 3, and both were better than the unilateral Menbird Granules 1 and Glucosamine Particle 1. It is suggested that the glucosamine particles 1, 2 and 3 of the present invention can improve the phagocytic function of mouse monocytes-macrophages.
  • the Glucosamine Gate Bird Granules 1, 2 and 3 of the present invention and the unilateral Gate Bird Granule 1 can significantly increase the HC 50 in the mouse serum hemolysin experiment (P ⁇ 0.01, P ⁇ 0.01, P ⁇ 0.05, P ⁇ 0.05), among which Glucosamine Gate 1 had the best effect, followed by Gluco Gate 2, and both were better than the unilateral Gate Bird 1 and Glucosamine 1 .
  • the Glucosamine Menbird Granules 1, 2 and 3 of the present invention can significantly increase the weight difference of the right ear piece of mice (P ⁇ 0.01, P ⁇ 0.01, P ⁇ 0.05, P ⁇ 0.05); among them, Glucosamine Gate 2 had the best effect, followed by Glucosamine 1, and both were better than unilateral Gate Bird 1 and Glucocet 1. It is suggested that Glucosamine Gate Bird Granules 1, 2 and 3 have ameliorating effects on delayed-type allergy in mice.
  • the Glucosamine Gate Bird Granules 1, 2 and 3 of the present invention can significantly increase the proliferation ability of spleen lymphocytes in mice (P ⁇ 0.01, P ⁇ 0.01). 0.01, P ⁇ 0.05, P ⁇ 0.05); among them, Glucosamine Gate 1 had the best effect, followed by Glucosamine 2, and both were better than unilateral Gate Bird 1 and Glucocet 1. It is suggested that glucosamine granules 1, 2 and 3 can enhance the transformation ability of mouse lymphocytes.
  • Test materials the products of the present invention—Glucomen Bird Granule 1, Glucomen Bird Granule 2 and Glucomen Bird Granule 3, the test animals are KM mice, male, weighing 18-22g, from the experimental animals of Hubei City. provided by the research center.
  • mice in each group were given continuous gavage for 30 days.
  • the model control group Glucosamine Menniao Granules group, Glucosamine Granules 1 group and Menniao Granules 1 group were given the test substance for the last time 20 minutes after the last administration.
  • the stomach was given 13ml/kg of 50% ethanol, and the blank control group was given double distilled water. After 16 hours of fasting, the animals were sacrificed for the determination of various biochemical indexes and liver histopathological examination.
  • the liver cell lipid droplet content score of the mice in the model control group was increased, and there was a very significant difference (p ⁇ 0.01) compared with the blank control group, indicating that the modeling was successful.
  • the Glucosamine Gate granules 1, 2 and 3 of the present invention can significantly reduce the content of lipid droplets in mouse liver cells (P ⁇ 0.01, P ⁇ 0.01, P ⁇ 0.01, P ⁇ 0.01, P ⁇ 0.01). ⁇ 0.05, P ⁇ 0.05), among which the effect of Glucosamine Menbird Granule 1 was the best, followed by Glucosamine Menbird Granule 2, and both were better than unilateral Menbird Granule 1 and Glucosamine Granule 1.
  • mice male, weighing 18-22 g, were purchased from Hubei Provincial Center for Disease Control and Prevention. Mice were randomly divided into blank group, Glucosamine Granules 1 group, Glucosamine Gate Bird Granules 1 group, and Gate Bird Granules 1 group according to body weight, with 6 animals in each group.
  • Glucosamine Granules 1, Glucosamine Menniao Granules 1 and Menniao Granules 1 were weighed in a certain amount, added with the corresponding volume of distilled water, vortexed and ultrasonically mixed, and then administered to mice at a volume of 10ml/kg, blank control group Distilled water was given once a day for 15 consecutive days, and then they were subjected to a weight-bearing swimming experiment.
  • test mice were subjected to the above-mentioned weight-bearing swimming experiment.
  • the following Effect Test Example 5 is used to study the effect of the Glucosamine Gate Bird Granule 1 and Gate Bird Granule 1 of the present invention in alleviating the alcoholic liver injury of individuals with continuous alcohol load at different doses.
  • mice Male KM mice (body weight 18-22g) purchased from Hubei Provincial Center for Disease Control and Prevention were randomly divided into blank control group, alcohol model group, Glucosamine Menniao Granules 1 dose group A, Menniao Granules 1 dose group A, ammonia Tangmen Bird Granules 1 dose group B, Men Bird Granules 1 dose group B, Glucosamine Men Bird Granules 1 dose group C, Men Bird Granules 1 dose group C, Glucosamine Men Bird Granules 1 dose group D, Men Bird Granules 1 dose group Group D; 6 animals per group.
  • Glucosamine Menniao Granules 1 and Menniao Granules 1 were respectively weighed to a certain amount, added with corresponding volume of distilled water, vortexed and ultrasonically mixed, and then administered to mice at a volume of 10 ml/kg.
  • the blank control group and the alcohol group were given distilled water.
  • the alcohol group and each sample group were given 52-degree liquor (10ml/kg) by gavage, and the blank control group was given distilled water for 5 days, and the animals were killed after fasting for 16 hours.
  • MDA malondialdehyde
  • GSH reduced glutathione
  • TG triglyceride
  • Glucosamine Gate Bird Granules 1 Dosage Group A 2600mg/kg/d Glucosamine Gate Bird Granules 1 Dosage Group B 1800mg/kg/d Glucosamine Gate Bird Granule 1 dose group C 900mg/kg/d Glucosamine Gate Bird Granules 1 Dosage Group D 450mg/kg/d Gate Bird Granules 1 Dosage Group A 1200mg/kg/d Gate Bird Granules 1 dose group B 800mg/kg/d Gate Bird Granules 1 dose group C 400mg/kg/d Gate Bird Granules 1 dose group D 200mg/kg/d
  • W/V tissue homogenate
  • liver Take 0.5 g of liver, add 5 mL of normal saline and grind it into a fine slurry (10% liver homogenate), centrifuge at 3000 rpm for 10 minutes at room temperature, and take the supernatant as the sample. Tested with triglyceride kit.
  • the cross section was taken from the middle of the left lobe of the liver, and the frozen section was stained with Sudan III or Oil Red O.
  • Microscopic examination The pathological changes of cells were recorded from one end of the field of view of the liver, and the entire tissue section was continuously observed with a 40x objective lens. Observe the distribution, extent and area of lipid droplets in the liver. The scoring criteria are shown in Table 14 below:
  • test results show that each dose group of Menniao Granule 1 and each formula dose group of Glucosamine Menniao Granule 1 can promote the expression of GSH to a certain extent (see Figure 2).
  • the liver GSH level of dose B was significantly increased, and the effect of Glucosamine Men Bird Granule 1 dose group A was more significant than that of Men Bird Granule 1 dose group A. Oxidative liver protection has a certain effect.
  • test results show that each dose group of Menniao Granule 1 and each formula dose group of Glucosamine Menbird Granule 1 can inhibit the production of MDA to a certain extent (see Figure 3), and in dose group B, the effect of Glucosamine Menbird Granule 1 Compared with the door bird granule 1, it is more significant, showing that both the glucosamine door bird granule 1 and the door bird granule 1 have the effect of anti-oxidation and liver protection.
  • the lipid droplet scores are shown in Table 15 below. See also the lipid droplet photograph shown in FIG. 5 .

Abstract

包含N-乙酰氨基葡萄糖和门冬氨酸鸟氨酸的组合物，两种功能性组分具有协同作用，与分别包含单独的门冬氨酸鸟氨酸或者单独的N-乙酰氨基葡萄糖的组合物相比，该组合物能够显著快速地缓解疲劳、增强机体的免疫力，同时具有辅助保肝护肝的功能。

Description

一种包含N-乙酰氨基葡萄糖的组合物及其制备方法和应用
优先权与相关申请
本发明要求享有于2021年1月29日向中国国家知识产权局提交的，专利申请号为202110128604.4，名称为“一种包含N-乙酰氨基葡萄糖的组合物及其制备方法和应用”的在先申请的优先权。在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明属于保健食品领域，具体涉及一种包含N-乙酰氨基葡萄糖和门冬氨酸鸟氨酸的组合物、其制备方法及其用于制备缓解疲劳、提高免疫力、辅助保肝护肝的保健食品的应用。
背景技术
现代社会中，人们的生活节奏快，工作压力大，社交应酬多，加之缺乏运动，容易造成慢性疲劳，使人们在体力、脑力和心理上都会感到疲劳。长期的慢性疲劳会对人体生理功能和心理方面造成危害，例如体力不足、体能下降、免疫力下降、记忆力下降、注意力不集中、反应迟钝，甚至会导致忧郁、焦虑、烦躁等精神症状，严重影响生活质量。在尚未造成器质性损伤或者引起严重的精神症状时，慢性疲劳往往不会引起人们的注意，医生也并没有合适的药物治疗方法。此外，社交应酬，例如饮酒、高脂高糖饮食，加重肝脏代谢负担，容易造成肥胖性脂肪肝、酒精性脂肪肝等，使肝脏长期处于损伤状态。这种状态也往往被人们忽视，进而发展成严重的肝脏疾病。因此，存在对于能够缓解疲劳、提高免疫力、辅助保肝护肝的保健食品的普遍需求。
门冬氨酸鸟氨酸为门冬氨酸和鸟氨酸形成的盐，最初被临床上用于治疗因急、慢性肝病如各型肝炎、肝硬化、脂肪肝和肝炎后综合症等引发的血氨升高及肝性脑病。门冬氨酸鸟氨酸在体内分解为门冬氨酸和鸟氨酸，其中，鸟氨酸是尿素循环中的起始底物，是鸟氨酸氨基甲酰转移酶和氨基甲酰磷酸合成酶的活化剂，与血液循环中氨等有毒代谢物结合，促进尿素合成，加速体内有毒物质特别是氨的代谢；天门冬氨酸则参与肝细胞中谷氨酰胺和核酸的合成，既可促进肝脏内谷氨酰胺的合成，又间接参与三磷酸循环和核酸的合成，并提供能量代谢的中间产物，促进肝细胞自身的修护和再生，恢复肝脏功能，增强肝脏解毒功能。
N-乙酰氨基葡萄糖(GlcNAc)是氨基葡萄糖的衍生物，是生物体内多种多糖的基本组成单位，还广泛存在于自然界中，是几丁质和壳聚糖的主要成分。N-乙酰氨基葡萄糖具有保护软骨组织和骨关节的作用，常用作膳食补充剂。
然而，尚且没有关于门冬氨酸鸟氨酸和N-乙酰氨基葡萄糖两者联合使用的有关报道，也 没有将两者制成保健食品的有关报道。
发明内容
为克服现有技术中的问题，本发明提供了一种组合物，其包含N-乙酰氨基葡萄糖和门冬氨酸鸟氨酸，二者的重量配比可以为(1～10):(10～1)，例如(1～8):(8～1)，(1～5):(5～1)，(1～3):(3～1)，示例性地可为1:1。
根据本发明，所述组合物还可以包含水果提取物，包括但不限于蓝莓提取物、草莓提取物、蔓越莓提取物、香橙提取物、葡萄皮提取物、葡萄籽提取物、桑葚提取物等，优选蓝莓提取物。所述水果提取物可以为水提取物、醇提取物和/或醇水溶液提取物；所述醇可以为乙醇，所述醇水溶液可以为50-95％(v/v)的乙醇水溶液。可以选用市售的水果提取物，也可以通过以下方法对水果进行提取获得的提取物：浸提法、酶解法、超声波提取法、微波辅助萃取法，或其组合。
根据本发明，所述组合物中，N-乙酰氨基葡萄糖、门冬氨酸鸟氨酸和水果提取物的重量配比可以为(1～10):(10～1):(0.1～5)，例如(1～8):(8～1):(0.1～3)，(1～5):(5～1):(0.1～2)，(1～3):(1～3):(0.2～1)，示例性地可为约5:5:1、3:3:1、1:1:1、5.71:2.85:1、8.33:1.67:1、1:9:1。
根据本发明，所述组合物还可以任选地包含赋形剂，例如矫味剂、填充剂、粘合剂、润湿剂、润滑剂、崩解剂、抗氧化剂、泡腾剂等中的一种、两种或更多种。
根据本发明，所述矫味剂包括但不限于蔗糖、甜菊苷或阿司巴甜，或其中两种或更多种的组合。所述填充剂包括但不限于微晶纤维素、乳糖、甘露醇、淀粉或糊精，或其中两种或更多种的组合。所述粘合剂包括但不限于羟丙甲纤维素、聚维酮、甲基纤维素或羧甲基纤维素钠，或其中两种或更多种的组合。所述润湿剂包括但不限于水或乙醇水溶液。所述润滑剂包括但不限于硬脂酸镁、滑石粉或微粉硅胶，或其中两种或更多种的组合。所述崩解剂包括但不限于羧甲基淀粉钠、交联羧甲基纤维素钠、羧甲基淀粉钠、羟丙基淀粉、交联聚维酮、低取代羟丙基纤维素或玉米淀粉，或其中两种或更多种的组合。所述抗氧化剂包括但不限于维生素C、亚硫酸钠、亚硫酸氢钠或焦亚硫酸钠，或其中两种或更多种的组合。所述泡腾剂包括但不限于柠檬酸和碳酸氢钠。
根据本发明，所述组合物可以为固体形式，例如颗粒剂、胶囊剂、片剂、丸剂等；也可以为液体形式，例如口服液，饮料，含乳饮品等。所述片剂可以是口含片、咀嚼片或者泡腾片，所述片剂可以是包衣片。所述胶囊剂可以是硬胶囊或软胶囊。所述丸剂可以是微丸或滴丸。
本发明还提供所述组合物的制备方法，包括将配方量的门冬氨酸鸟氨酸、N-乙酰氨基葡萄糖以及可选的组分混合，然后制成颗粒剂、胶囊剂、片剂或丸剂。
根据本发明，所述组合物为颗粒，其制备方法包括以下步骤：
(1)制粒溶液的制备：将配方量的粘合剂以及部分水果提取物溶于去离子水或纯化水中，得制粒溶液；
(2)颗粒的制备：将配方量的N-乙酰氨基葡萄糖、门冬氨酸鸟氨酸、剩余的水果提取物以及任选的赋形剂混合，喷洒制粒溶液制粒，再经干燥、整粒即得。
本发明还提供所述组合物用于制备缓解疲劳的保健食品中的应用。
本发明还提供所述组合物用于制备提高免疫力的保健食品中的应用。
本发明还提供所述组合物用于制备辅助保肝护肝的保健食品中的应用。
本发明还提供一种缓解疲劳的方法，其包括将本发明的组合物给予有需要的个体的步骤。
本发明还提供一种提高免疫力的方法，其包括将本发明的组合物给予有需要的个体的步骤。
本发明还提供一种辅助保肝护肝的方法，其包括将本发明的组合物给予有需要的个体的步骤。
根据本发明，所述辅助保肝护肝例如增强肝脏的抗氧化能力、辅助减轻肝细胞中的脂肪含量、辅助降低转氨酶如ALT和AST水平等。
本发明的有益效果包括：本发明的包含N-乙酰氨基葡萄糖和门冬氨酸鸟氨酸的组合物，两种功能性组分具有协同作用，与单独的门冬氨酸鸟氨酸或者单独的N-乙酰氨基葡萄糖相比，能够显著快速地缓解疲劳、增强机体的免疫力，同时具有辅助保肝护肝的功能。
附图简述
图1示出了效果试验例4中小鼠游泳时间长度的对比。
图2示出了效果试验例4中空白组、酒精组、氨糖门鸟颗粒1的不同剂量组与门鸟颗粒1单方的不同剂量组的小鼠肝组织中还原型谷胱甘肽(GSH)含量的柱状图。
图3示出了效果试验例5中空白组、酒精组、氨糖门鸟颗粒1的不同剂量组与门鸟颗粒1单方的不同剂量组的小鼠肝组织中丙二醛(MDA)含量的柱状图。
图4示出了效果试验例5中空白组、酒精组、氨糖门鸟颗粒1的不同剂量组与门鸟颗粒1单方的不同剂量组的小鼠肝组织中甘油三酯(TG)含量的柱状图。
图5示出了效果试验例5中空白组、酒精组、高浓度氨糖门鸟颗粒1组与低浓度氨糖门鸟颗粒1组和门鸟颗粒1组相比在小鼠中消除肝组织脂质化方面的功效对比。
具体实施方式
下文将结合具体实施例对本发明做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。在下面的实施例中，N-乙酰氨基葡萄糖简称为“氨糖”，门冬氨酸鸟氨酸简称为“门鸟”。所用的门冬氨酸鸟氨酸购自武汉启瑞药业有限公司，N-乙酰氨基葡萄糖购自马鞍山天泰生物科技有限公司，蓝莓提取物购自南京川源生物科技有限公司。
制备实施例1氨糖门鸟颗粒1的制备
氨糖门鸟颗粒1的配方如下：
Figure PCTCN2022074897-appb-000001
制备工艺如下：
(1)制粒溶液的配制
①称取(1)组的纯化水；
②将(1)组的蓝莓提取物加入纯化水中，搅拌溶解；
③再加入(1)组的聚维酮K30，搅拌溶解，备用；
(2)颗粒的制备
①将(2)组的门冬氨酸鸟氨酸、N-乙酰氨基葡萄糖和蓝莓提取物加入湿法制粒机，设置搅拌桨转速180rpm，切割刀转速1200rpm，混合5min；
②将制粒溶液加入上述制粒物料：搅拌桨转速180rpm，切割刀转速1200rpm，蠕动泵转速30rpm，雾化压力0.025Mpa；
③制粒：搅拌桨转速180rpm，切割刀转速1200rpm，制粒120s；
④湿整粒：过筛，筛网孔径2mm，转子转速1000rpm；
⑤干燥：流化床干燥，进风温度70℃，干燥至物料干燥失重小于3％；
⑥干整粒：过筛，筛网孔径0.5mm，转子转速1000rpm，得紫色至浅紫色颗粒，记为氨糖门鸟颗粒1。
制备实施例2氨糖门鸟颗粒2的制备
氨糖门鸟颗粒2的配方如下：
Figure PCTCN2022074897-appb-000002
Figure PCTCN2022074897-appb-000003
参考制备实施例1的方法制成氨糖门鸟颗粒2。
制备实施例3氨糖门鸟颗粒3的制备
氨糖门鸟颗粒3的配方如下：
Figure PCTCN2022074897-appb-000004
参考制备实施例1的方法，不添加蓝莓提取物，制成氨糖门鸟颗粒3，其为白色至类白色颗粒。
制备实施例4氨糖门鸟颗粒4的制备
氨糖门鸟颗粒4的配方如下：
Figure PCTCN2022074897-appb-000005
参考制备实施例1的方法，制备氨糖门鸟颗粒4。
制备实施例5氨糖门鸟颗粒5的制备
氨糖门鸟颗粒5的配方如下：
Figure PCTCN2022074897-appb-000006
参考制备实施例1的方法，制备颗粒5。
对比实施例1门鸟颗粒1的制备
门鸟颗粒1的配方如下：
Figure PCTCN2022074897-appb-000007
参考制备实施例1的方法，制成门鸟颗粒1。
对比实施例2氨糖颗粒1的制备
氨糖颗粒1的配方如下：
Figure PCTCN2022074897-appb-000008
参考制备实施例1的方法，制成氨糖颗粒1。
通过以下效果试验例1～3考察本发明的产品在单剂量给药方案下的抗疲劳、提高免疫力和辅助保肝护肝效果。
效果试验例1抗疲劳试验
1.1试验材料：本发明的产品——氨糖门鸟颗粒1和氨糖门鸟颗粒2由门冬氨酸鸟氨酸、N-乙酰氨基葡萄糖和蓝莓提取物制备而成，而氨糖门鸟颗粒3不含有蓝莓提取物。试验动物为KM小鼠，雄性，体重18-22g，由湖北省实验动物研究中心提供。
1.2样品制备：分别称取氨糖门鸟颗粒1、氨糖门鸟颗粒2、氨糖门鸟颗粒3、氨糖颗粒1和门鸟颗粒1约1g，加入一定量生理盐水，配制成10mg/ml的供试品。阴性对照灌胃组给予生理盐水，各实验组均按照10ml/kg体积灌胃。三个氨糖门鸟颗粒组、氨糖颗粒1组和门鸟颗粒1组的给药剂量均为100mg/kg。
1.3实验方法：(1)负重游泳试验：每组10只小鼠，连续灌胃30天，末次给受试物后30min，小鼠尾根部负重5％体重，置游泳箱中游泳观察小鼠自游泳至死亡时间。(2)血乳酸含量的测定：每组10只小鼠，小鼠连续灌胃30天，末次给受试物后30min，放入30℃的水中游泳10min，游泳前、游泳后即刻分别二次取血(内眦取血)测定血乳酸值。(3)血清尿素氮含量的测定：小鼠连续灌胃30天，末次给受试物后30min，放入30℃的水中游泳90min，出水后，休息60min，眼球取血测定血清尿素氮含量。
1.4试验结果
1.4.1本发明的氨糖门鸟颗粒对小鼠负重游泳时间的影响
本发明的氨糖门鸟颗粒1、2和3，以及单方N-乙酰氨基葡萄糖(氨糖颗粒1)均能延长小鼠负重游泳时间，与阴性对照组比较，有显著性差异(P<0.01，P＜0.05，P＜0.01和P＜0.05)；其中氨糖门鸟颗粒1效果最优，氨糖门鸟颗粒3和2次之，并且均优于单方的氨糖颗粒1和门鸟颗粒1。结果提示，氨糖门鸟复方颗粒1、2和3均具有抗疲劳作用。详见表1。
表1本发明产品对小鼠负重游泳时间的影响
Figure PCTCN2022074897-appb-000009
组别 动物数(只) 游泳时间(s)
阴性对照组 10 265.66±45.23
氨糖门鸟颗粒1 10 412.25±76.87 **#
氨糖门鸟颗粒2 10 364.25±78.18 *
氨糖门鸟颗粒3 10 389.25±42.76 **#
氨糖颗粒1 10 342.23±67.43 *
门鸟颗粒1 10 278.43±37.34
备注：与阴性对照组比较：*P＜0.05，**P＜0.01；与N-乙酰氨基葡萄糖组比较#P＜0.05。
1.4.2本发明的氨糖门鸟颗粒对小鼠游泳血乳酸值的影响
相比阴性对照组，本发明的氨糖门鸟颗粒1、2和3，以及单方N-乙酰氨基葡萄糖(氨糖颗粒1)均能降低小鼠游泳后的血乳酸值，有显著性差异(P<0.01，P＜0.05，P＜0.01和P＜0.05)，其中氨糖门鸟颗粒1效果最优，氨糖门鸟颗粒3和2次之，并且均优于单方的氨糖颗粒1和门鸟颗粒1。详见表2。
表2本发明产品对小鼠游泳血乳酸值的影响
Figure PCTCN2022074897-appb-000010
Figure PCTCN2022074897-appb-000011
备注：与阴性对照组比较：*P＜0.05，**P＜0.01；与N-乙酰氨基葡萄糖组比较#P＜0.05。
1.4.3本发明的氨糖门鸟颗粒对小鼠血清尿素氮的影响
相比阴性对照组，本发明的氨糖门鸟颗粒1、2和3，以及单方N-乙酰氨基葡萄糖(氨糖颗粒1)均能降低小鼠血清尿素氮值，有显著性差异(P<0.01，P＜0.05，P＜0.01和P＜0.05)，其中氨糖门鸟颗粒1效果最优，氨糖门鸟颗粒3和2次之，并且均优于单方的氨糖颗粒1和门鸟颗粒1。详见表3。
表3本发明产品对小鼠血清尿毒素的影响
Figure PCTCN2022074897-appb-000012
组别 动物数(只) 血清尿素氮(mmol/L)
阴性对照组 10 8.65±1.65
氨糖门鸟颗粒1 10 5.71±1.05 **#
氨糖门鸟颗粒2 10 7.33±1.59 *
氨糖门鸟颗粒3 10 6.68±1.29 *#
氨糖颗粒1 10 7.18±1.19 *
门鸟颗粒1 10 8.04±1.23
备注：与阴性对照组比较：*P＜0.05，**P＜0.01；与N-乙酰氨基葡萄糖组比较#P＜0.05。
效果试验例2提高免疫力试验
2.1试验材料：本发明的产品——氨糖门鸟颗粒1、氨糖门鸟颗粒2和氨糖门鸟颗粒3，试验动物为KM小鼠，雄性，体重18-22g，由湖北省实验动物研究中心提供。
2.2样品制备和分组：分别称取氨糖门鸟颗粒1、氨糖门鸟颗粒2、氨糖门鸟颗粒3、氨糖颗粒1和门鸟颗粒1约1g，加入一定量生理盐水，配制成10mg/ml的供试品。阴性对照灌胃给予生理盐水，各实验组均按照10ml/kg体积灌胃。三个氨糖门鸟颗粒组、氨糖颗粒1组和门鸟颗粒1组给药剂量均为100mg/kg。试验动物随机分组，每组10只，各组动物每天灌胃1次，连续灌胃30天。
2.3实验方法：
2.3.1体重、脏器/体重比值的测定：每周监测、记录小鼠体重，末次给药后处死小鼠，取胸腺和脾脏，电子分析天平称重，计算脏器/体重比值。
2.3.2小鼠碳末廓清实验：小鼠按0.1ml/10g尾静脉注射印度墨汁(生理盐水4倍稀释)，墨汁注入后立即计时，于注入墨汁后第2、10min，分别从内眦静脉丛取血20μL，加入到2mL0.1％Na 2CO 3溶液中，摇匀。以Na 2CO 3溶液作空白对照，于600nm波长测定各管吸光值(OD)。处死小鼠，取肝、脾脏，称重，计算吞噬指数a＝K1/3×体重/(肝重+脾重)，K＝(lgOD1－lgOD2)/(t2-t1)。
2.3.3血清溶血素实验：每鼠腹腔注射2％SRBC悬液0.2mL，免疫5天后摘眼球取血，2000rpm离心10min分离血清，取血清用SA缓冲液稀释200倍，取稀释后的血清1mL置试管内，对照管取1mLSA缓冲液，依次加入10％(v/v)SRBC 0.5mL、1:8SA稀释的豚鼠血清1mL。置37℃水浴中孵育20min后，冰浴终止反应。2000rpm离心10min。取上清液1mL，加都氏试剂3mL，另取10％(v/v)SRBC 0.25mL加都氏试剂至4mL于另一支试管内，充分混匀，作为半数溶血值。放置10min后，于540nm波长测定各管吸光度值，计算HC 50。HC 50＝样品OD/SRBC半数溶血时的OD×稀释倍数。
2.3.4迟发型超敏反应(DTH)实验：用硫化钡将小鼠腹部皮肤脱毛约3cm×3cm范围，涂以1％DNFB溶液(丙酮：麻油＝1:1)50μL致敏，5天后将10μL DNFB均匀涂抹于小鼠右耳两面进行攻击，24小时后颈椎脱臼处死小鼠，剪下左右耳壳，用打孔器取下8mm直径的耳片、称重，以左右耳重量之差值表示DTH的程度
2.3.5ConA诱导的小鼠脾淋巴细胞转化实验(MTT法)：末次给药后处死小鼠，无菌取脾，置于盛有适量无菌Hank’s液的小平皿中，制成细胞悬液，经200目筛网过滤。用Hank’s液洗2次，每次离心10min(1000r/min)。然后将细胞悬浮于1mL完全培养液中，计数活细胞数，用RPMI1640培养液调整细胞浓度为3×10 6个/mL。再将细胞悬液分两孔加入24孔培养板中，每孔1mL，在其中一孔加75μL ConA液(相当于7.5μg/mL)，另一孔作为对照，置5％CO 2，37℃二氧化碳孵箱中培养72h。培养结束前4h，每孔轻轻吸去上清液0.7mL，加入0.7mL不含小牛血清的RPMI1640培养液，同时加入MTT(5mg/mL)50μL/孔，继续培养4h。培养结束后，每孔加入1mL酸性异丙醇，吹打混匀，使紫色结晶完全溶解。然后分装到96孔培养 板中，每个孔作3个平行孔，用酶标仪，以570nm波长测定光密度值。淋巴细胞的增殖能力用加ConA孔的光密度值减去不加ConA孔的光密度值表示。
2.4试验结果
2.4.1本发明氨糖门鸟颗粒对小鼠体重、脏器/体重比值的影响
由表4可见，相比阴性对照组，本发明的氨糖门鸟颗粒1、2和3组、氨糖颗粒1组和门鸟颗粒1组小鼠的体重以及胸腺脏体比均无明显差别(P＞0.05)，提示氨糖门鸟颗粒1、2和3对小鼠的体重增长以及胸腺无明显影响。
表4本发明产品对小鼠体重和胸腺脏体比的影响
Figure PCTCN2022074897-appb-000013
组别 动物数(只) 初始重量(g) 末期重量(g) 胸腺/体重(mg/g)
阴性对照组 10 20.14±1.65 29.24±3.05 1.14±0.15
氨糖门鸟颗粒1 10 20.51±1.98 28.91±2.29 1.21±0.22
氨糖门鸟颗粒2 10 20.38±1.18 29.31±4.10 1.16±0.17
氨糖门鸟颗粒3 10 20.47±2.05 29.27±3.18 1.20±0.08
氨糖颗粒1 10 20.28±1.23 29.39±4.17 1.14±0.27
门鸟颗粒1 10 20.23±2.12 29.21±3.88 1.23±0.11
2.4.2本发明的氨糖门鸟颗粒对小鼠碳末廓清的影响
由表5可见，相比阴性对照组，本发明的氨糖门鸟颗粒1、2和3，以及单方的门鸟颗粒1均能显著升高小鼠碳末廓清实验中的吞噬指数(P＜0.01，P＜0.05，P＜0.01，P＜0.01)；其中氨糖门鸟颗粒1效果最优，氨糖门鸟颗粒2和3次之，并且均优于单方的门鸟颗粒1和氨糖颗粒1。提示本发明的氨糖门鸟颗粒1、2和3均可提高小鼠单核-巨噬细胞的吞噬功能。
表5本发明产品对小鼠吞噬指数的影响
Figure PCTCN2022074897-appb-000014
组别 动物数(只) 吞噬指数
阴性对照组 10 3.39±0.34
氨糖门鸟颗粒1 10 5.01±0.62 **#
氨糖门鸟颗粒2 10 4.88±0.41 *#
氨糖门鸟颗粒3 10 4.18±0.49 *
氨糖颗粒1 10 3.42±0.38
门鸟颗粒1 10 4.60±0.70 **
备注：与阴性对照组比较：*P＜0.05，**P＜0.01；与门冬氨酸鸟氨酸组比较#P＜0.05。
2.4.3本发明的氨糖门鸟颗粒对小鼠血清溶血素实验体液免疫的影响
由表6可见，相比阴性对照组，本发明的氨糖门鸟颗粒1、2和3，以及单方的门鸟颗粒1均能显著升高小鼠血清溶血素实验中的HC 50(P＜0.01，P＜0.01，P＜0.05，P＜0.05)，其中氨糖门鸟颗粒1效果最优，氨糖门鸟颗粒2次之，并且均优于单方的门鸟颗粒1和氨糖颗粒1。
表6本发明产品对小鼠HC 50的影响
Figure PCTCN2022074897-appb-000015
组别 动物数(只) HC 50
阴性对照组 10 85.29±10.01
氨糖门鸟颗粒1 10 102.28±8.40 **#
氨糖门鸟颗粒2 10 98.08±9.21 **#
氨糖门鸟颗粒3 10 92.08±7.11 *#
氨糖颗粒1 10 86.12±6.43
门鸟颗粒1 10 93.11±10.10 *
备注：与阴性对照组比较：*P＜0.05，**P＜0.01；与门冬氨酸鸟氨酸组比较#P＜0.05。
2.4.4本发明的氨糖门鸟颗粒对小鼠细胞免疫的影响(迟发型变态反应实验)
由表7可见，相比阴性对照组，本发明的氨糖门鸟颗粒1、2和3，以及单方的门鸟颗粒1均能显著增加小鼠右耳片重量差值(P＜0.01，P＜0.01，P＜0.05，P＜0.05)；其中氨糖门鸟颗粒2效果最优，氨糖门鸟颗粒1次之，并且均优于单方的门鸟颗粒1和氨糖颗粒1。提示氨糖门鸟颗粒1、2和3对小鼠迟发型变态反应具有改善作用。
表7本发明产品对小鼠左右耳片重量差值的影响
Figure PCTCN2022074897-appb-000016
组别 动物数(只) 左右耳片重量差值(mg)
阴性对照组 10 1.09±0.12
氨糖门鸟颗粒1 10 1.98±0.23 **#
氨糖门鸟颗粒2 10 2.15±0.27 **##
氨糖门鸟颗粒3 10 1.47±0.38 *
氨糖颗粒1 10 1.20±0.17
门鸟颗粒1 10 1.67±0.20 *
备注：与阴性对照组比较：*P＜0.05，**P＜0.01；与门冬氨酸鸟氨酸组比较#P＜0.05，##P＜0.01。
2.4.5本发明的氨糖门鸟颗粒对ConA诱导小鼠脾淋巴细胞转化能力的影响
由表8可见，相比阴性对照组，本发明的氨糖门鸟颗粒1、2和3，以及单方的门鸟颗粒1均能显著增加小鼠脾淋巴细胞增殖能力(P＜0.01，P＜0.01，P＜0.05，P＜0.05)；其中氨 糖门鸟颗粒1效果最优，氨糖门鸟颗粒2次之，并且均优于单方的门鸟颗粒1和氨糖颗粒1。提示氨糖门鸟颗粒1、2和3可增强小鼠淋巴细胞的转化能力。
表8本发明产品对小鼠淋巴细胞转化能力的影响
Figure PCTCN2022074897-appb-000017
组别 动物数(只) 淋巴细胞增殖能力(OD值)
阴性对照组 10 0.20±0.08
氨糖门鸟颗粒1 10 0.35±0.07 **#
氨糖门鸟颗粒2 10 0.31±0.13 **#
氨糖门鸟颗粒3 10 0.25±0.11 *
氨糖颗粒1 10 0.19±0.05
门鸟颗粒1 10 0.27±0.08 *
备注：与阴性对照组比较：*P＜0.05，**P＜0.01；与门冬氨酸鸟氨酸组比较#P＜0.05，##P＜0.01。
效果试验例3辅助保肝护肝试验
3.1试验材料：本发明的产品——氨糖门鸟颗粒1、氨糖门鸟颗粒2和氨糖门鸟颗粒3，试验动物为KM小鼠，雄性，体重18-22g，由湖北省实验动物研究中心提供。
3.2样品制备：分别称取氨糖门鸟颗粒1、氨糖门鸟颗粒2、氨糖门鸟颗粒3、氨糖颗粒1和门鸟颗粒1约1g，加入一定量生理盐水，配制成10mg/ml的供试品。阴性对照灌胃给予生理盐水，各实验组均按照10ml/kg体积灌胃。氨糖门鸟颗粒组、氨糖颗粒1组和门鸟颗粒1组给药剂量均为100mg/kg。
3.3实验方法：每组10只小鼠，连续灌胃30天，模型对照组、氨糖门鸟颗粒组、氨糖颗粒1组和门鸟颗粒1组最后一次给予受试物后20min，一次灌胃给予50％乙醇13ml/kg，空白对照组给双蒸水，禁食16h后处死动物进行各项生化指标测定和肝脏病理组织学检查。
3.4试验结果
3.4.1本发明的氨糖门鸟颗粒对小鼠肝匀浆中丙二醛(MDA)含量的影响
由表9可见，模型对照组小鼠肝匀浆中MDA含量增高，与空白对照组相比有极显著差异(p＜0.01)，表明建模成功。相比模型对照组，本发明的氨糖门鸟颗粒1、2和3，以及单方的门鸟颗粒1均能显著降低酒精性肝损伤小鼠肝匀浆中MDA含量(P＜0.01，P＜0.01，P＜0.05，P＜0.05)；其中氨糖门鸟颗粒2效果最优，氨糖门鸟颗粒1次之，并且均优于单方的门鸟颗粒1和氨糖颗粒1。
表9本发明产品对小鼠肝匀浆中丙二醛(MDA)含量的影响
Figure PCTCN2022074897-appb-000018
组别 动物数(只) MDA(nmol/mg prot)
空白对照组 10 3.09±0.25
模型对照组 10 7.34±1.03 &&
氨糖门鸟颗粒1 10 4.78±0.89 **#
氨糖门鸟颗粒2 10 3.78±0.75 **##
氨糖门鸟颗粒3 10 5.13±1.18 *
氨糖颗粒1 10 7.21±0.87
门鸟颗粒1 10 5.24±0.75 *
备注：与空白对照组比较：&&P＜0.01；与模型对照组比较*P＜0.05，**P＜0.01；与门冬氨酸鸟氨酸组比较#P＜0.05，##P＜0.01。
3.4.2本发明的氨糖门鸟颗粒对小鼠肝匀浆中还原型谷胱甘肽(GSH)含量的影响
由表10可见，模型对照组小鼠肝匀浆中GSH含量降低，与空白对照组相比有极显著差异(p＜0.01)，表明建模成功。相比模型对照组，本发明的氨糖门鸟颗粒1、2和3，以及单方的门鸟颗粒1均能显著升高酒精性肝损伤小鼠肝匀浆中GSH含量(P＜0.01，P＜0.01，P＜0.05，P＜0.05)；其中氨糖门鸟颗粒1效果最优，氨糖门鸟颗粒2次之，并且均优于单方的门鸟颗粒1和氨糖颗粒1。
表10本发明产品对小鼠肝匀浆中还原型谷胱甘肽(GSH)含量的影响
Figure PCTCN2022074897-appb-000019
组别 动物数(只) GSH(mg/g肝)
空白对照组 10 9.76±0.45
模型对照组 10 7.14±0.63 &&
氨糖门鸟颗粒1 10 9.57±0.77 **#
氨糖门鸟颗粒2 10 9.38±0.69 **#
氨糖门鸟颗粒3 10 8.88±0.65 *
氨糖颗粒1 10 7.19±0.56
门鸟颗粒1 10 8.91±0.34 *
备注：与空白对照组比较：&&P＜0.01；与模型对照组比较*P＜0.05，**P＜0.01；与门冬氨酸鸟氨酸组比较#P＜0.05，##P＜0.01。
3.4.3本发明的氨糖门鸟颗粒对小鼠肝匀浆中甘油三酯(TG)含量的影响
由表11可见，模型对照组小鼠肝匀浆中TG含量升高，与空白对照组相比有极显著差异(p＜0.01)，表明建模成功。相比模型对照组，本发明的氨糖门鸟颗粒1、2和3，以及单方的门鸟颗粒1均能显著降低酒精性肝损伤小鼠肝匀浆中TG含量(P＜0.01，P＜0.01，P＜0.05， P＜0.05)；其中氨糖门鸟颗粒1效果最优，氨糖门鸟颗粒2次之，并且均优于单方的门鸟颗粒1和氨糖颗粒1。
表11本发明产品对小鼠肝匀浆中甘油三酯(TG)含量的影响
Figure PCTCN2022074897-appb-000020
组别 动物数(只) TG(μmol/g肝)
空白对照组 10 7.16±0.39
模型对照组 10 15.87±2.19 &&
氨糖门鸟颗粒1 10 10.36±1.79 **#
氨糖门鸟颗粒2 10 11.78±2.63 **#
氨糖门鸟颗粒3 10 13.47±2.49 *
氨糖颗粒1 10 15.58±2.77
门鸟颗粒1 10 12.18±2.39 *
备注：与空白对照组比较：&&P＜0.01；与模型对照组比较*P＜0.05，**P＜0.01；与门冬氨酸鸟氨酸组比较#P＜0.05，##P＜0.01。
3.4.4本发明氨糖门鸟颗粒对小鼠肝脏组织病理学评分影响
由表12可见，模型对照组小鼠肝细胞脂滴含量评分升高，与空白对照组相比有极显著差异(p＜0.01)，表明建模成功。相比模型对照组，本发明氨糖门鸟颗粒1、2和3，以及单方门冬氨酸鸟氨酸均能显著降低小鼠肝细胞脂滴含量评分(P＜0.01，P＜0.01，P＜0.05，P＜0.05)，其中氨糖门鸟颗粒1效果最优，氨糖门鸟颗粒2次之，并且均优于单方的门鸟颗粒1和氨糖颗粒1。
表12本发明产品对小鼠肝细胞脂滴含量评分影响
Figure PCTCN2022074897-appb-000021
组别 动物数(只) 肝细胞脂滴含量评分
空白对照组 10 0.15±0.03
模型对照组 10 3.47±0.28 &&
氨糖门鸟颗粒1 10 1.38±0.26 **##
氨糖门鸟颗粒2 10 1.66±0.36 **#
氨糖门鸟颗粒3 10 2.37±0.46 *
氨糖颗粒1 10 3.28±0.41
门鸟颗粒1 10 2.08±0.19 *
备注：与空白对照组比较：&&P＜0.01；与模型对照组比较*P＜0.05，**P＜0.01；与门冬氨酸鸟氨酸组比较#P＜0.05，##P＜0.01。
上述表9至表12中的结果均表明，本发明的氨糖门鸟颗粒1、2和3对肝损伤具有保护作用。
以下效果试验例4用于研究本发明的氨糖门鸟颗粒1和门鸟颗粒1在缓解体力疲劳功能的效果。
效果试验例4氨糖门鸟颗粒缓解体力疲劳试验
4.1动物分组
KM小鼠，雄性，体重18-22g购买于湖北省疾控中心。小鼠根据体重随机分成空白组、氨糖颗粒1组、氨糖门鸟颗粒1组、门鸟颗粒1组，每组动物6只。氨糖颗粒1、氨糖门鸟颗粒1和门鸟颗粒1分别称取一定量后，加入相应体积蒸馏水，涡旋超声混匀后，按照10ml/kg体积灌胃给药小鼠，空白对照组给予蒸馏水，每日1次，连续15天后，对其进行负重游泳实验。
4.2负重游泳时间检测方案和结果
在受试小鼠给药15天后，对其进行上述负重游泳实验。
在受试小鼠末次给药后30min，在小鼠尾根部负重5％体重铅片，置游泳箱中游泳观察小鼠自游泳至死亡时间，游泳箱中水深不少于30cm，水温25℃±1.0℃。结果显示，氨糖门鸟颗粒1组游泳时间相对于对照组明显增加，均超过门鸟颗粒1组和氨糖颗粒1组。这一结果说明，本发明的氨糖门鸟颗粒具有缓解疲劳的作用以及特别是氨糖和门鸟两种活性成分的协同作用(参见图1)。
以下效果试验例5用于研究本发明的氨糖门鸟颗粒1和门鸟颗粒1在不同剂量下缓解连续酒精负荷的个体的酒精肝损伤的效果。
效果试验例5氨糖门鸟颗粒对缓解长时间酒精肝损伤试验
5.1动物分组
将购自湖北省疾控中心的雄性KM小鼠(体重18-22g)根据体重随机分成空白对照组、酒精模型组、氨糖门鸟颗粒1剂量组A、门鸟颗粒1剂量组A，氨糖门鸟颗粒1剂量组B、门鸟颗粒1剂量组B、氨糖门鸟颗粒1剂量组C、门鸟颗粒1剂量组C、氨糖门鸟颗粒1剂量组D，门鸟颗粒1剂量组D；每组动物6只。氨糖门鸟颗粒1和门鸟颗粒1分别称取一定量后，加入相应体积蒸馏水，涡旋超声混匀后，按照10ml/kg体积灌胃给药小鼠，空白对照组和酒精组给予蒸馏水，每日1次，连续10天后，将酒精组及各样品组灌胃给予52度白酒(10ml/kg)，空白对照组给蒸馏水，连续给药及白酒5天，禁食16小时后处死动物，检测肝脏中丙二醛(MDA)、还原型谷胱甘肽(GSH)和甘油三酯(TG)的含量变化。根据商品化试剂盒说明书进行检测GSH、MDA和TG含量变化。
具体试验分组详情见下表13。
表13动物分组和给药方案
氨糖门鸟颗粒1剂量组A 2600mg/kg/d
氨糖门鸟颗粒1剂量组B 1800mg/kg/d
氨糖门鸟颗粒1剂量组C 900mg/kg/d
氨糖门鸟颗粒1剂量组D 450mg/kg/d
门鸟颗粒1剂量组A 1200mg/kg/d
门鸟颗粒1剂量组B 800mg/kg/d
门鸟颗粒1剂量组C 400mg/kg/d
门鸟颗粒1剂量组D 200mg/kg/d
5.2肝组织丙二醛(MDA)检测
取肝脏约0.5g，生理盐水冲洗、拭干、称重、剪碎，置匀浆器中，加入0.2M磷酸盐缓冲液，以20000r/min匀浆10s，间歇30s，反复进行3次，制成5％组织匀浆(W/V)，3000r/min离心5～10min，取上清液检测。
5.3肝匀浆还原型谷胱甘肽(GSH)测定
取肝脏0.5g，加生理盐水5mL充分研磨成细浆(10％肝匀浆)，混匀后取浆液0.5mL加4％磺基水杨酸0.5mL混匀，室温下3000rpm离心10分钟，取上清液即为样品。采用还原型谷胱甘肽试剂盒检测。
5.4肝匀浆甘油三酯(TG)测定
取肝脏0.5g，加生理盐水5mL充分研磨成细浆(10％肝匀浆)，室温下3000rpm离心10分钟，取上清液即为样品。采用甘油三酯试剂盒检测。
5.5肝脏病理学检测
从肝左叶中部做横切面取材，冰冻切片，苏丹Ⅲ染色或油红O染色。镜检：从肝脏的一端视野开始记录细胞的病理变化，用40倍物镜连续观察整个组织切片。观察脂滴在肝脏的分布、范围和面积。评分标准见下表14：
表14肝脏病理学检测评分标准
Figure PCTCN2022074897-appb-000022
5.6试验结果
试验结果显示门鸟颗粒1各剂量组及氨糖门鸟颗粒1各配方剂量组均可一定程度上促进GSH的表达(参加图2)，氨糖门鸟颗粒1与门鸟颗粒1剂量组A、剂量B的肝脏GSH水平明显升高，且氨糖门鸟颗粒1剂量组A效果相对于门鸟颗粒1剂量组A更为显著，显示在A剂量下氨糖门鸟颗粒1对基于GSH抗氧化的护肝功能有一定功效。
试验结果显示门鸟颗粒1各剂量组及氨糖门鸟颗粒1各配方剂量组均可一定程度上抑制MDA的产生(参见图3)，且在剂量组B中，氨糖门鸟颗粒1效果相对于门鸟颗粒1更为显著，显示氨糖门鸟颗粒1与门鸟颗粒1均具有抗氧化、护肝的功效。
实验结果显示氨糖门鸟颗粒1剂量组A、B、C与门鸟颗粒1剂量组A、B、D可有效降低肝脏中TG含量，提示氨糖门鸟颗粒1具有降脂护肝作用(参见图4)。
脂滴评分见下表15。另外可参见图5示出的脂滴照片。
表15肝脏病理学检测脂滴评分结果
Figure PCTCN2022074897-appb-000023
结果显示，与酒精组相比，高浓度氨糖门鸟颗粒1组(2000mg/kg/d)小鼠的肝脏脂滴明显减少，趋近于空白组，而低浓度氨糖门鸟颗粒1组(250mg/kg/d)与门鸟颗粒1组小鼠的肝脏脂滴相较于酒精组无明显改变，提示氨糖门鸟颗粒1具有降脂护肝作用，特别是在高浓度条件下尤其明显。
以上对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
在上文对本发明的实施方式进行了示例性说明。然而本发明并不限于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (9)

  1. 一种组合物，其包含N-乙酰氨基葡萄糖和门冬氨酸鸟氨酸，二者的重量配比为(1～10):(10～1)，例如(1～8):(8～1)，(1～5):(5～1)，(1～3):(3～1)，示例性地可为1:1。
  2. 根据权利要求1所述的组合物，其中所述组合物还包含水果提取物，包括但不限于蓝莓提取物、草莓提取物、蔓越莓提取物、香橙提取物、葡萄皮提取物、葡萄籽提取物、桑葚提取物等，优选蓝莓提取物；
    优选地，所述组合物中，门冬氨酸鸟氨酸、N-乙酰氨基葡萄糖和水果提取物的重量配比为(1～10):(10～1):(0.1～5)，例如(1～8):(8～1):(0.1～3)，(1～5):(5～1):(0.1～2)，(1～3):(1～3):(0.2～1)，示例性地为5:1:1、3:3:1或1:1:1。
  3. 根据权利要求1或2的组合物，其中所述组合物还任选地包含赋形剂，例如矫味剂、着色剂、填充剂、粘合剂、润湿剂、润滑剂、崩解剂、抗氧化剂、泡腾剂等中的一种、两种或更多种。
  4. 根据权利要求1-3中任一项所述的组合物，所述组合物可以为固体形式，例如颗粒剂、胶囊剂、片剂、丸剂等；也可以为液体形式，例如口服液，饮料或含乳饮品。
  5. 根据权利要求1-4中任一项所述的组合物的制备方法，包括将配方量的门冬氨酸鸟氨酸、N-乙酰氨基葡萄糖以及可选的组分混合，然后制成颗粒剂、胶囊剂、片剂或丸剂。
  6. 根据权利要求1-4中任一项所述的组合物用于制备保健食品中的应用，所述保健食品可以缓解疲劳、提高免疫力和/或辅助保肝护肝。
  7. 一种缓解疲劳的方法，其包括将权利要求1-4中任一项所述的组合物给予有需要的个体的步骤。
  8. 一种提高免疫力的方法，其包括将权利要求1-4中任一项所述的组合物给予有需要的个体的步骤。
  9. 一种辅助保肝护肝的方法，其包括将权利要求1-4中任一项所述的组合物给予有需要的个体的步骤。
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