WO2023005454A1 - 一种延缓衰老的含核苷酸的组合物及其制备方法和应用 - Google Patents

一种延缓衰老的含核苷酸的组合物及其制备方法和应用 Download PDF

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WO2023005454A1
WO2023005454A1 PCT/CN2022/098180 CN2022098180W WO2023005454A1 WO 2023005454 A1 WO2023005454 A1 WO 2023005454A1 CN 2022098180 W CN2022098180 W CN 2022098180W WO 2023005454 A1 WO2023005454 A1 WO 2023005454A1
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parts
nucleotide
nucleotides
compound
containing composition
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French (fr)
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陈玉松
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陈玉松
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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
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    • 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
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    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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    • 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
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • 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
    • A23L33/13—Nucleic acids or derivatives thereof
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    • A23L33/15—Vitamins
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    • 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
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    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
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    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
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    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A61K31/708—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid having oxo groups directly attached to the purine ring system, e.g. guanosine, guanylic acid
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Definitions

  • the invention belongs to the technical field of medicine and health care, and in particular relates to a nucleotide-containing composition with the function of delaying aging and its preparation method and application.
  • the aging of the human body is actually the aging of human cells. After about 40-60 divisions, the cells will die of aging and cannot continue to divide. The reduction of cell viability and ability to divide is the real cause of body aging. In order to delay aging, it is necessary to improve the vigor and vitality of body cells.
  • Cells are composed of cell membrane, cytosol, and nucleus. The basic code of life - gene is hidden in the nucleus. A gene is the entire nucleotide sequence required to produce a polypeptide chain or functional RNA.
  • Nucleic acid is a biological macromolecular compound composed of a variety of nucleotides, and is one of the most basic substances of life. Nucleic acid is widely present in all animals, plant cells, and microorganisms. According to different chemical composition, nucleic acid can be divided into deoxyribose nucleic acid (DNA) and ribonucleic acid (RNA). DNA is the main material basis for storing, replicating and transmitting genetic information, and RNA plays an important role in the process of protein synthesis. There are three types of RNA involved in protein synthesis, transfer RNA (tRNA), messenger RNA (mRNA) and ribosomal RNA (rRNA).
  • tRNA transfer RNA
  • mRNA messenger RNA
  • rRNA ribosomal RNA
  • tRNA plays the role of carrying and transferring activated amino acids; mRNA is the template for protein synthesis; rRNA is the main place for cell protein synthesis.
  • Nucleic acid is not only the basic genetic material, but also occupies an important position in the biosynthesis of proteins, so it plays a decisive role in a series of major life phenomena such as growth, heredity, and variation.
  • a nucleic acid is a polynucleotide whose basic structural unit is a nucleotide.
  • Nucleotide is the basic unit of ribonucleic acid and deoxyribonucleic acid. As nucleic acid is distributed in the nucleus and cytoplasm of various organs, tissues, and cells in the organism, it participates in the genetics, development, and growth of organisms as a component of nucleic acid. basic life activities.
  • Nucleotide compounds have important biological functions, and they participate in almost all biochemical reaction processes in organisms: nucleotides are the precursors of synthetic biological macromolecules ribonucleic acid and deoxyribonucleic acid; adenosine triphosphate (ATP) plays a role in cellular energy Metabolism plays an extremely important role; ATP can also transfer high-energy phosphate bonds to UDP, CDP and GDP to generate UTP, CTP and GTP; adenosine is also several important coenzymes, such as coenzyme I (nicotinamide adenine dinucleoside acid, NAD+), coenzyme II (nicotinamide adenine dinucleotide phosphate, NADP+), flavin adenine dinucleotide (FAD) and coenzyme A (CoA); nucleotides are essential for many basic biological The learning process has a certain regulating effect.
  • ATP adenosine triphosphate
  • ATP
  • the patent discloses a composition containing nicotinamide mononucleotide for anti-aging beauty and skin care products.
  • Niacinamide mononucleotide is used as an active ingredient in the composition of anti-aging beauty and skin care products to prevent rough skin from aging , reduce the appearance of skin wrinkles, keep the skin bright and elastic.
  • the object of the present invention is to provide a nucleotide-containing composition for delaying aging, which is composed of four kinds of mononucleotides of AMP, CMP, GMP, UMP and tea polyphenols, astragalus polysaccharide, curcumin in a certain proportion , resveratrol, marine fish oligopeptide, coenzyme Q10, nicotinamide and other functional active ingredients.
  • the finished product has the effect of delaying the aging of the body and enhancing the vitality of cells.
  • a nucleotide-containing composition for delaying aging which is prepared by compounding nucleotides and functional active ingredients according to a certain ratio, and can delay, improve and prevent aging after use.
  • the compound nucleotides are composed of AMP, CMP, GMP, and UMP; the functional active ingredients are tea polyphenols, astragalus polysaccharides, curcumin, resveratrol, marine fish oligomeric One or more of peptides, coenzyme Q10, and nicotinamide.
  • the weight ratio of the compound nucleotides is: AMP 15.0-25.0%, CMP 15.0-45.0%, GMP 15.0-35.0%, UMP 15.0-30.0%.
  • the weight ratio of nucleotides and functional active ingredients is: 0.5-1.5 parts of compound nucleotides, 0.5-2 parts of tea polyphenols, 0.02-0.1 parts of resveratrol, 0.02-0.08 parts of coenzyme Q10, 0.01-0.01 parts of nicotinamide 0.04 parts; or 0.5-1.5 parts of compound nucleotides, 0.1-0.4 parts of astragalus polysaccharide, 0.1-0.4 parts of curcumin, 0.02-0.1 parts of resveratrol, 0.01-0.04 parts of nicotinamide; or compound nucleotides 0.5-1.5 parts, marine fish oligopeptide 0.2-0.8 parts.
  • the weight ratio of the compound nucleotides is: the weight ratio of the compound nucleotides is: AMP 16.9%, CMP 43.2%, GMP 17.8%, UMP 22.1%
  • the weight ratio of the compound nucleotides and functional active ingredients is: 1.2 parts of compound nucleotides, 1 part of tea polyphenols, 0.05 parts of resveratrol, 0.04 parts of coenzyme Q10, and 0.02 parts of nicotinamide.
  • the weight ratio of the compound nucleotides is: AMP 16.9%, CMP 43.2%, GMP 17.8%, UMP 22.1%, the weight of the compound nucleotides and functional active ingredients The ratio is: 1.2 parts of compound nucleotides, 0.2 parts of astragalus polysaccharide, 0.19 parts of curcumin, 0.05 parts of resveratrol, and 0.02 parts of nicotinamide.
  • the weight ratio of the compound nucleotides is: AMP 24.3%, CMP 28.3%, GMP 19.3%, UMP 28.1%, the weight of the compound nucleotides and functional active ingredients The ratio is: 0.6 parts of compound nucleotides, 0.43 parts of marine fish oligopeptides.
  • the finished state of the composition includes powder, granule, tablet, pill, oral liquid and capsule.
  • Another aspect of the present invention provides a method for preparing the above-mentioned nucleotide-containing composition.
  • the above-mentioned compound nucleotides are weighed according to the proportion, mixed uniformly to obtain a mixed powder, and the mixed powder and the functional active ingredients of the formula are mixed uniformly to obtain Nucleotide-containing compositions.
  • Another aspect of the present invention provides the application of the above-mentioned nucleotide-containing composition in the preparation of medicines, health products or functional foods.
  • the nucleotide-containing composition is added to medicines, health products or functional foods directly or after conventional steps.
  • the medicine, health product or functional food has the functions of eliminating free radicals in the body, inhibiting the oxidative damage caused by free radicals to the body, and delaying the aging of the human body.
  • the anti-aging nucleotide-containing composition provided by the present invention combines four kinds of mononucleotides, AMP, CMP, GMP, and UMP, with various functional substances and vitamins to form a composition to play a synergistic role, and can remove free radicals in the body, inhibit The oxidative damage of free radicals to the body can play a role in delaying aging and make the body full of youth.
  • the compound nucleotides are weighed according to the weight ratio of AMP 16.9%, CMP 43.2%, GMP 17.8%, and UMP 22.1%. Weigh the raw materials in the clean area, mix them evenly with a mixer, and obtain the powder. Sampling according to the quality standards for various indicators After passing the inspection, it is ready for use; then in the clean area, prepare 1.2 parts of compound nucleotides, 1 part of tea polyphenols, 0.05 parts of resveratrol, 0.04 parts of coenzyme Q10, and 0.02 parts of nicotinamide in the clean area. Take the raw materials, mix them evenly with a mixer, and press them into tablets with a tablet weight of 0.3g. The finished product shall be sampled according to the quality standard for inspection of various indicators, and shall be preserved for future use after passing the inspection.
  • the compound nucleotides are weighed according to the weight ratio of AMP 16.9%, CMP 43.2%, GMP 17.8%, and UMP 22.1%. Weigh the raw materials in the clean area, mix them evenly with a mixer, and obtain the powder. Sampling according to the quality standards for various indicators After passing the inspection, it is ready for use; then in the clean area, according to the above-mentioned preparation of 1.2 parts of compound nucleotides, 0.2 parts of astragalus polysaccharide, 0.19 parts of curcumin, 0.05 parts of resveratrol, and 0.02 parts of nicotinamide, weigh The raw materials are uniformly mixed with a mixer, and pressed into tablets with a tablet weight of 0.3g. The finished product shall be sampled according to the quality standard for inspection of various indicators, and shall be preserved for future use after passing the inspection.
  • the compound nucleotides are weighed according to the weight ratio of AMP 24.3%, CMP 28.3%, GMP 19.3%, and UMP 28.1%. Weigh the raw materials in the clean area, mix them evenly with a mixer, and obtain the powder. Sampling according to the quality standards for various indicators After passing the inspection, it is ready for use; then in the clean area, according to the above-mentioned ratio of 0.6 parts of compound nucleotides and 0.43 parts of marine fish oligopeptides, the raw materials are weighed, mixed evenly with a mixer, and pressed into tablets. Weighs 0.3g. The finished product shall be sampled according to the quality standard for inspection of various indicators, and shall be preserved for future use after passing the inspection.
  • the compound nucleotides are weighed according to the weight ratio of AMP 24.3%, CMP 28.3%, GMP 19.3%, and UMP 28.1%. Weigh the raw materials in the clean area, mix them evenly with a mixer, and obtain the powder. Sampling according to the quality standards for various indicators After passing the inspection, it is ready for use; then in the clean area, prepare 1.2 parts of compound nucleotides, 1 part of tea polyphenols, 0.05 parts of resveratrol, 0.04 parts of coenzyme Q10, and 0.02 parts of nicotinamide in the clean area. Take the raw materials, mix them evenly with a mixer, and press them into tablets with a tablet weight of 0.3g. The finished product shall be sampled according to the quality standard for inspection of various indicators, and shall be preserved for future use after passing the inspection.
  • the compound nucleotides are weighed according to the weight ratio of AMP 24.3%, CMP 28.3%, GMP 19.3%, and UMP 28.1%. Weigh the raw materials in the clean area, mix them evenly with a mixer, and obtain the powder. Sampling according to the quality standards for various indicators After passing the inspection, it is ready for use; then in the clean area, according to the above-mentioned preparation of 1.2 parts of compound nucleotides, 0.2 parts of astragalus polysaccharide, 0.19 parts of curcumin, 0.05 parts of resveratrol, and 0.02 parts of nicotinamide, weigh The raw materials are uniformly mixed with a mixer, and pressed into tablets with a tablet weight of 0.3g. The finished product shall be sampled according to the quality standard for inspection of various indicators, and shall be preserved for future use after passing the inspection.
  • this experiment firstly investigated the anti-oxidation performance of the nucleotide-containing composition.
  • Get 1 tablet prepared by Example 1 prepare to be dissolved in the mixed solution of deionized water and ethanol (volume ratio is 1:1) after preparing to weigh, make the mother liquor that concentration is 1mg/mL, carry out DPPH free radical scavenging Determination of rate and determination of ABTS free radical scavenging rate.
  • the mother liquor was diluted 100, 50, 25, 10, and 1 times respectively to prepare gradient dilutions with concentrations of 10 ⁇ g/mL, 20 ⁇ g/mL, 40 ⁇ g/mL, 100 ⁇ g/mL, and 500 ⁇ g/mL.
  • a blank is the absorbance of 150 ⁇ L deionized water ethanol solution and 150 ⁇ L DPPH solution
  • a sample is the absorbance of 150 ⁇ L sample and 150 ⁇ L DPPH solution
  • IC50 is the concentration of the sample when the DPPH free radical scavenging rate reaches 50%.
  • a blank is the absorbance of 0.15mL deionized water ethanol solution and 2.85mL ABTS working solution
  • a sample is the absorbance of 0.15mL sample and 2.85mL ABTS working solution.
  • IC 50 is the concentration of the sample when the ABTS free radical scavenging rate reaches 50%.
  • the nucleotide-containing compositions of Examples 1-3 all have higher antioxidant properties, and the antioxidant properties of the compound nucleotides and functional active ingredients are significant after compounding Enhanced effect, the nucleotide-containing compositions of Examples 1-3 can more effectively scavenge free radicals in the body.
  • nucleotide-containing composition of the present invention has the effect of delaying aging.
  • H 2 O 2 was used to model, and the nucleotide-containing compositions of Examples 1-3 were evaluated. Only the compound nucleotide with the same amount and the same ratio as the tablet of Example 1 was used as the control experiment 1, and the compound nucleotide with the same amount and the same ratio as the tablet of Example 3 was used as the control experiment 1.
  • Control experiment 2 only the composition of tea polyphenols, resveratrol, coenzyme Q10, and nicotinamide in the same amount and the same proportion as the tablet of Example 1 was used as control experiment 3, only the tablet of Example 2 The composition of astragalus polysaccharides, curcumin, resveratrol, and nicotinamide in the same amount and the same ratio was used as the control experiment 4, only the marine fish oligopeptide in the same amount and the same ratio as the tablet in Example 3 As the control experiment 5, and the same dose of ⁇ -nicotinamide mononucleotide (NMN) as the control experiment 6, the cytotoxicity test and the evaluation test of the intracellular ROS level were carried out.
  • NPN ⁇ -nicotinamide mononucleotide
  • control group 1-6 can increase the survival rate of cells after H 2 O 2 treatment, and the nucleotide-containing composition of Examples 1-3 can significantly increase the cell survival rate after H 2 O 2 treatment.
  • the survival rate of the cells was almost indistinguishable from the test results of the blank control group, further illustrating that the nucleotide-containing compositions of Examples 1-3 have excellent cell repairing effects.
  • the nucleotide-containing composition of Example 1-3 can significantly reduce the active oxygen in the model cell, and the nucleoside-containing composition of Example 1-3
  • the acid composition has good protective and repairing effects on oxidatively damaged cells.
  • the mechanism of aging involves many aspects.
  • rapid aging-prone mice SAMP 8, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. company
  • mice long-term feeding with the nucleotide-containing composition of the present invention as an intervention to carry out in vivo research
  • mice's autonomous activity, learning and memory ability mice's autonomous activity, learning and memory ability
  • Brain damage and the improvement of smell and the improvement of muscle attenuation Take 110 mice with rapid aging tendency, with a body weight of (20 ⁇ 5) g, and randomly divide them into 11 groups.
  • the positive control group is fed with basal diet, the negative control group is 3-month-old SAMP 8, and the active control group is fed only with
  • the compound nucleotide with the same amount and the same ratio of the tablet of Example 1 is used as a control experiment 1, and the compound nucleotide with the same amount and the same ratio as the tablet of Example 3 is fed as a control.
  • mice were 9 months old, that is, after 6 months of intervention with the nucleotide-containing composition (one tablet each time, once a day), an open-field experiment was carried out.
  • the open-field behavior analysis mainly reflects the animal's autonomous activity ability and exploration of new environments, habits and accompanying emotional changes, and is an experiment used to evaluate the spontaneous activity ability of mice. Its principle is that mice are naturally afraid of open and bright places, and at the same time, they will have curiosity to explore new things. When mice are anxious, they will reduce the exploration of the central area and tend to stay in the edge area; the number of times of crossing and standing The number of times is a reflection of the animal's exploration behavior and excitability.
  • the time in the central grid reflects the animal's cognitive ability to the environment. Normal animals will avoid the open environment, leave the central grid quickly, and move around.
  • the experimental data are expressed as mean ⁇ standard deviation, and the experimental results are shown in Table 4.
  • nucleotide-containing composition of the example 1-3 group significantly improved the spontaneous activity and exploration ability of the mice prone to aging rapidly.
  • the new object recognition experiment is a learning and memory test method established by using the principle that animals have a tendency to explore new objects. It is used to detect the non-spatial learning and memory ability of mice. It is based on the spontaneous exploration behavior of mice to novel objects. Based on the test of memory retention ability. This method has the characteristic of allowing mice to perform learning and memory tests in a free-moving state, and can more closely simulate human learning and memory behaviors.
  • the experiment also allows testing the formation of long-term or short-term memory mechanisms in animals and the impact evaluation of memory formation at specific stages.
  • This experiment can reflect the spontaneous memory ability of mice, and is currently a new and important behavioral method for evaluating brain function.
  • the olfactory memory experiment was carried out.
  • the learning and memory ability and foraging ability of olfactory animals have a high correlation with the sense of smell, and the change of their olfactory function can be judged by observing and recording the changes in their behavior.
  • the principle of the olfactory memory experiment is that as the number of exposures to an object with a special smell increases, the animal smell adapts. According to this characteristic, the animal's memory ability for the smell is reflected. The experiment is divided into the adaptation period (the first day) and the detection period. period (the next day).
  • the two odors selected in the experiment mint essence and lemon essence odor.
  • the mice have no preference for these two odors.
  • Each odor allows the mice to explore freely for 30s. Repeat four times, and then record the mice sniffing the centrifuge tube each time. time.
  • the sniffing time is defined as: the time spent by the mouse facing the centrifuge tube and the nose is within 1cm of the centrifuge tube, excluding the time spent by the mouse biting or climbing the centrifuge tube.
  • the experimental results are shown in Table 7.
  • mice in the embodiment 1-3 group only need a shorter time to adapt to the above-mentioned gas.
  • the nucleotide-containing composition can improve the odor adaptation behavior of mice, and has a protective effect on the olfactory memory of aged mice.

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Abstract

公开一种延缓衰老的含核苷酸组合物，属于医药保健技术领域，含核苷酸组合物包括AMP、CMP、CMP、UMP按照一定配比的复配核苷酸和茶多酚、黄芪多糖、姜黄素、白藜芦醇、海洋鱼低聚肽、辅酶Q10、烟酰胺等功能活性成分，所述的延缓衰老的含核苷酸组合物能够清除体内自由基进而抗氧化，对细胞损伤具有保护和修复作用，延缓机体衰老。

Description

一种延缓衰老的含核苷酸的组合物及其制备方法和应用 技术领域
本发明属于医药保健技术领域，具体涉及具有延缓衰老功能的含核苷酸的组合物及其制备方法和应用。
背景技术
人体的衰老实际上就是人体细胞的衰老，细胞大约在40-60次***后，即老化死亡，不能再继续***。细胞活性和***能力的降低是机体衰老的真正原因。要想延缓衰老就必须提高身体细胞的活力和生命力。细胞是由细胞膜、细胞液、细胞核组成的。细胞核中藏有基本生命密码—基因。基因是产生一条多肽链或功能RNA所需的全部核苷酸序列。
核酸是由多种核苷酸聚合而成的生物大分子化合物，是生命的最基本物质之一。核酸广泛存在于所有动物、植物细胞、微生物体内。根据化学组成不同，核酸可分为脱氧核糖核酸(DNA)和核糖核酸(RNA)。DNA是储存、复制和传递遗传信息的主要物质基础，RNA在蛋白质合成过程中起着重要作用。参与蛋白质合成的RNA有三种，转移RNA(tRNA)、信使RNA(mRNA)和核糖体RNA(rRNA)。tRNA起着携带和转移活化氨基酸的作用；mRNA是合成蛋白质的模板；rRNA是细胞合成蛋白质的主要场所。核酸不仅是基本的遗传物质，而且在蛋白质的生物合成上也占据着重要位置，因而在生长、遗传、变异等一系列重大生命现象中起决定性的作用。核酸是一种多聚核苷酸，它的基本结构单位是核苷酸。
核苷酸是核糖核酸及脱氧核糖核酸的基本组成单位，随着核酸分布于生物体内各器官、组织、细胞的核及胞质中，并作为核酸的组成成分参与生物的遗传、发育、生长等基本生命活动。核苷酸类化合物具有重要的生物学功能，它们参与了生物体内几乎所有的生物化学反应过程：核苷酸是合成生物大分子核糖核酸及脱氧核糖核酸的前身物；三磷酸腺苷(ATP)在细胞能量代谢上起着极其重要的作用；ATP还可将高能磷酸键转移给UDP、CDP及GDP生成UTP、CTP及GTP；腺苷酸还是几种重要辅酶，如辅酶Ⅰ(烟酰胺腺嘌呤二核苷酸，NAD+)、辅酶Ⅱ(磷酸烟酰胺腺嘌呤二核苷酸，NADP+)、黄素腺嘌呤二核苷酸(FAD)及辅酶A(CoA)的组成成分；核苷酸对于许多基本的生物学过程有一定的调节作用。近二十年来，外源核苷酸已广泛应用于婴幼儿配方乳粉以及保健品中，其在人体中使用的安全性已得到科学试验的充分证明。专利(CN104771330A)公开了一种含烟酰胺单 核苷酸抗衰老美容护肤品的组合物，将烟酰胺单核苷酸作为活性成分应用在抗衰老美容护肤品组合物中，能够防止皮肤粗糙老化，减少皮肤皱纹的出现，保持皮肤光鲜有弹性。然而，将核苷酸与功能活性成分组合获得一种安全有效，无毒副作用、协同延缓机体老化的组合物的相关研究还未见报道。
发明内容
鉴于此，本发明的目的在于提供一种延缓衰老的含核苷酸的组合物，是由AMP、CMP、GMP、UMP四种单核苷酸按一定比例与茶多酚、黄芪多糖、姜黄素、白藜芦醇、海洋鱼低聚肽、辅酶Q10、烟酰胺等功能活性成分组成的组合物，所得的成品具有延缓机体老化、增强细胞生命力的功效。
本发明为实现上述目的，通过以下技术方案实现：
一种延缓衰老的含核苷酸组合物，由复配核苷酸和功能活性成分按照一定比例调配制成，使用后可起到延缓、改善、防止衰老的功效。
进一步地，基于以上技术方案，所述复配核苷酸由AMP、CMP、GMP、UMP组成；所述功能活性成分为茶多酚、黄芪多糖、姜黄素、白藜芦醇、海洋鱼低聚肽、辅酶Q10、烟酰胺中的一种或二种以上。
进一步地，基于以上技术方案，所述复配核苷酸的重量配比为：AMP 15.0～25.0％、CMP 15.0～45.0％、GMP 15.0～35.0％、UMP 15.0～30.0％，所述复配核苷酸和功能活性成分的重量配比为：复配核苷酸0.5～1.5份、茶多酚0.5～2份、白藜芦醇0.02～0.1份、辅酶Q10 0.02～0.08份、烟酰胺0.01～0.04份；或者复配核苷酸0.5～1.5份、黄芪多糖0.1～0.4份、姜黄素0.1～0.4份、白藜芦醇0.02～0.1份、烟酰胺0.01～0.04份；或者复配核苷酸0.5～1.5份、海洋鱼低聚肽0.2～0.8份。
进一步地，基于以上技术方案，所述复配核苷酸的重量配比为：所述复配核苷酸的重量配比为：AMP 16.9％、CMP 43.2％、GMP 17.8％、UMP 22.1％，所述复配核苷酸和功能活性成分的重量配比为：复配核苷酸1.2份、茶多酚1份、白藜芦醇0.05份、辅酶Q10 0.04份、烟酰胺0.02份。
进一步地，基于以上技术方案，所述复配核苷酸的重量配比为：AMP 16.9％、CMP 43.2％、GMP 17.8％、UMP 22.1％，所述复配核苷酸和功能活性成分的重量配比为：复配核苷酸1.2份、黄芪多糖0.2份、姜黄素0.19份、白藜芦醇0.05份、烟酰胺0.02份。
进一步地，基于以上技术方案，所述复配核苷酸的重量配比为：AMP 24.3％、CMP  28.3％、GMP 19.3％、UMP 28.1％，所述复配核苷酸和功能活性成分的重量配比为：复配核苷酸0.6份、海洋鱼低聚肽0.43份。
进一步地，基于以上技术方案，所述组合物的成品状态包括粉剂、颗粒剂、片剂、丸剂、口服液、胶囊。
本发明另一方面提供上述含核苷酸组合物的制备方法，按配比称量上述复配核苷酸，混合均匀，获得混合粉，将混合粉和配方量的功能活性成分混合均匀，即得含核苷酸组合物。
本发明另一方面提供上述含核苷酸组合物在制备药品、保健品或功能性食品中的应用。
进一步地，基于以上技术方案，所述含核苷酸组合物直接或经常规步骤处理后添加在药品、保健品或功能性食品中。
进一步地，基于以上技术方案，所述药品、保健品或功能性食品具有清除体内自由基、抑制自由基对机体的氧化损伤、延缓人体衰老的作用。
本发明相对于现有技术具有的有益效果如下：
本发明提供的延缓衰老的含核苷酸组合物通过AMP、CMP、GMP、UMP四种单核苷酸搭配多种功能性物质、维生素形成组合物，协同发挥作用，能够清除体内自由基，抑制自由基对机体的氧化损伤，可以起到延缓衰老的作用，使机体充满年轻态。
具体实施方式
下面结合具体实施例对本发明的作进一步解释说明，这些实施例应被理解为仅是举例说明，而非以任何方式限制本发明的范围。下述实施例中，如无特殊说明，所使用的实验方法均为常规方法，所用材料、试剂等均可从生物或化学公司购买。
实施例1
复配核苷酸按照重量配比AMP 16.9％、CMP 43.2％、GMP 17.8％、UMP 22.1％在洁净区称取原料，用混合机混合均匀，既得粉剂，按质量标准的规定取样进行各项指标的检验，检验合格后备用；然后在洁净区按照上述制备的复配核苷酸1.2份、茶多酚1份、白藜芦醇0.05份、辅酶Q10 0.04份、烟酰胺0.02份配比，称取原料，用混合机混合均匀，压成片剂，片重为0.3g。成品按质量标准的规定取样进行各项指标的检验，检验合格后保存备用。
实施例2
复配核苷酸按照重量配比AMP 16.9％、CMP 43.2％、GMP 17.8％、UMP 22.1％在洁净 区称取原料，用混合机混合均匀，既得粉剂，按质量标准的规定取样进行各项指标的检验，检验合格后备用；然后在洁净区按照上述制备的复配核苷酸1.2份、黄芪多糖0.2份、姜黄素0.19份、白藜芦醇0.05份、烟酰胺0.02份配比，称取原料，用混合机混合均匀，压成片剂，片重为0.3g。成品按质量标准的规定取样进行各项指标的检验，检验合格后保存备用。
实施例3
复配核苷酸按照重量配比AMP 24.3％、CMP 28.3％、GMP 19.3％、UMP 28.1％在洁净区称取原料，用混合机混合均匀，既得粉剂，按质量标准的规定取样进行各项指标的检验，检验合格后备用；然后在洁净区按照上述制备的复配核苷酸0.6份、海洋鱼低聚肽0.43份配比，称取原料，用混合机混合均匀，压成片剂，片重为0.3g。成品按质量标准的规定取样进行各项指标的检验，检验合格后保存备用。
实施例4
复配核苷酸按照重量配比AMP 24.3％、CMP 28.3％、GMP 19.3％、UMP 28.1％在洁净区称取原料，用混合机混合均匀，既得粉剂，按质量标准的规定取样进行各项指标的检验，检验合格后备用；然后在洁净区按照上述制备的复配核苷酸1.2份、茶多酚1份、白藜芦醇0.05份、辅酶Q10 0.04份、烟酰胺0.02份配比，称取原料，用混合机混合均匀，压成片剂，片重为0.3g。成品按质量标准的规定取样进行各项指标的检验，检验合格后保存备用。
实施例5
复配核苷酸按照重量配比AMP 24.3％、CMP 28.3％、GMP 19.3％、UMP 28.1％在洁净区称取原料，用混合机混合均匀，既得粉剂，按质量标准的规定取样进行各项指标的检验，检验合格后备用；然后在洁净区按照上述制备的复配核苷酸1.2份、黄芪多糖0.2份、姜黄素0.19份、白藜芦醇0.05份、烟酰胺0.02份配比，称取原料，用混合机混合均匀，压成片剂，片重为0.3g。成品按质量标准的规定取样进行各项指标的检验，检验合格后保存备用。
实施例6
为了验证本发明的含核苷酸组合物延缓衰老的作用，本实验首先通过考察含核苷酸组合物的抗氧化性能。
取1片实施例1制备的片剂，准备称重后溶解于去离子水和乙醇的混合溶液中(体积比为1:1)，制得浓度为1mg/mL的母液，进行DPPH自由基清除率的测定和ABTS自由基 清除率的测定。
1.DPPH自由基清除率的测定：
将母液分别稀释100，50，25，10，1倍制备成浓度为10μg/mL，20μg/mL，40μg/mL，100μg/mL，500μg/mL的梯度稀释液。
分别取上述浓度150μL样品和150μL浓度为60mg/L的DPPH溶液(无水乙醇稀释)置于96孔板中，混合均匀，30℃避光保存，30min后使用酶标仪测量519nm下吸光度值。DPPH自由基清除率公式如下：
Figure PCTCN2022098180-appb-000001
式中，A空白为150μL去离子水乙醇溶液和150μL DPPH溶液的吸光度，A样品为150μL样品和150μL DPPH溶液的吸光度；IC 50为DPPH自由基清除率达到50％时样品的浓度。
2.ABTS自由基清除率的测定：
(1)将母液分别稀释100，50，25，10，1倍制备成浓度为10μg/mL，20μg/mL，40μg/mL，100μg/mL，500μg/mL的梯度稀释液。
(2)将7mmol/L的ABTS溶液和140mmol/L的过硫酸钾溶液以62.5：1的比例混合，室温避光静置过夜，形成ABTS储备液，使用前用去离子水稀释成5mmol/L的ABTS工作液。
(3)分别取0.15mL上述稀释液样品与2.85mL ABTS工作液混合，30℃下静置8min，以去离子水为空白在734nm下测定吸光度。
ABTS自由基清除率公式如下：
Figure PCTCN2022098180-appb-000002
式中，A空白为0.15mL去离子水乙醇溶液和2.85mL ABTS工作液的吸光度，A样品为0.15mL样品和2.85mL ABTS工作液的吸光度。IC 50为ABTS自由基清除率达到50％时样品的浓度。
参考上述同样的检测方法检测实施例2中含核苷酸组合物片剂以及实施例3中含核苷酸组合物片剂的抗氧化性能，以仅有与实施例1的片剂相同量及相同配比的复配核苷酸作为对照实验1，以仅有与实施例3的片剂相同量及相同配比的复配核苷酸作为对照实验2，仅有与实施例1的片剂相同量及相同配比的茶多酚、白藜芦醇、辅酶Q10、烟酰胺的组合 物作为对照实验3，仅有与实施例2的片剂相同量及相同配比的黄芪多糖、姜黄素、白藜芦醇、烟酰胺的组合物作为对照实验4，仅有与实施例3的片剂相同量及相同配比的海洋鱼低聚肽作为对照实验5，实验数据以均值±标准偏差表示，实验结果如表1所示。
表1.含核苷酸组合物的抗氧化性能
Figure PCTCN2022098180-appb-000003
结果表明，与对照组1-5相比，实施例1-3的含核苷酸组合物均具有较高的抗氧化性能，复配核苷酸和功能活性成分复配后抗氧化性能呈现显著增强的效果，实施例1-3的含核苷酸组合物能够更有效的清除机体的自由基。
实验例7
为进一步验证本发明的含核苷酸组合物具有延缓衰老的作用，进行了体外实验进行评价。以人脐静脉内皮细胞、PC-12细胞(大鼠肾上腺嗜铬细胞瘤细胞)为模型，采用H 2O 2造模，对实施例1-3的含核苷酸的组合物进行评价实验，以仅有与实施例1的片剂相同量及相同配比的复配核苷酸作为对照实验1，以仅有与实施例3的片剂相同量及相同配比的复配核苷酸作为对照实验2，仅有与实施例1的片剂相同量及相同配比的茶多酚、白藜芦醇、辅酶Q10、烟酰胺的组合物作为对照实验3，仅有与实施例2的片剂相同量及相同配比的黄芪多糖、姜黄素、白藜芦醇、烟酰胺的组合物作为对照实验4，仅有与实施例3的片剂相同量及相同配比的海洋鱼低聚肽作为对照实验5，并以相同剂量的β-烟酰胺单核苷酸(NMN)为对照实验6，进行细胞毒性试验和细胞内ROS水平的评价试验。
1.细胞毒性试验
将对数生长期的细胞消化成单细胞悬液，调节细胞浓度为10 5个/mL，接种于96孔细胞培养板，每孔100μL，每组6个平行孔。接种24小时后，细胞贴壁生长，弃上清，加入终浓度200μmol/L的H 2O 2的培养基培养4h，然后替换为终浓度分别为50μg/mL、10μg/mL的组合物培养液继续培养24小时。实验同时设置不加H 2O 2的空白对照和只加H 2O 2的模型组，用MTT法测定细胞存活状况：每孔加入50μL MTT储备液，终浓度为2mg/mL。继续培养4小时后，弃去上清，每孔加DMSO 100μL，10min后用自动酶标仪于490nm测定各孔吸光度A，计算细胞的存活率。实验数据以均值±标准偏差表示，实验结果如表2所示。
表2.人脐静脉内皮细胞和PC-12细胞的存活率
Figure PCTCN2022098180-appb-000004
上述结果表明，与模型对照组相比，对照组1-6能够提高H 2O 2处理后细胞的存活率，实施例1-3的含核苷酸组合物能够显著提高H 2O 2处理后细胞的存活率，几乎和空白对照组试验结果无明显区别，进一步说明，实施例1-3的含核苷酸组合物具有非常优异的修复细胞效果。
2.细胞内ROS水平的评价试验
过氧化氢作用细胞后产生大量ROS，攻击细胞膜、蛋白质及核酸，导致细胞损伤。将对数生长期的细胞消化成单细胞悬液，调节细胞浓度为10 5个/mL，接种于96孔细胞培养板，每孔100μL，每组6个平行孔。接种24小时后，细胞贴壁生长，弃上清，加入终浓度200μmol/L的H 2O 2的培养基培养4h，然后替换为终浓度分别为50μg/mL的组合物培养液继续培养24小时。实验同时设置不加H 2O 2的空白对照和只加H 2O 2的模型组，用活性氧检测试剂盒进行细胞内活性氧的状况评价：每孔加入10μmol/L的DCFH-DA母液(DMSO溶解)，终浓度为1μmol/L。继续培养2小时后，弃去上清，每孔加PBS 100μL洗涤2次，离心后使用200μL的PBS重悬细胞，使用荧光酶标仪于激发波长485nm，发射波长为525nm条件下测定各孔荧光强度，计算细胞的活性氧含量。实验数据以均值±标准偏差表示，实验结果如表3所示。
表3.人脐静脉内皮细胞和PC-12细胞内的活性氧含量
  人脐静脉内皮细胞荧光强度 PC-12细胞荧光强度
模型对照 19845±214 12538±159
对照实验1 12370±171 8649±139
对照实验2 15955±168 10936±150
对照实验3 9795±196 7242±205
对照实验4 10154±108 7085±160
对照实验5 11985±210 9054±175
对照实验6 13128±175 9752±124
实施例1 5697±99 6048±118
实施例2 5543±135 5847±142
实施例3 6499±160 7016±187
上述结果表明，和模型对照以及对照组1-6的组合物相比，实施例1-3的含核苷酸组合物能够显著降低模型细胞内的活性氧，实施例1-3的含核苷酸组合物对氧化损伤的细胞具有良好的保护和修复作用。
实验例8
衰老的机制涉及多个方面，为了更好的验证本发明的含核苷酸组合物对延缓衰老的作用，我们以快速老化倾向小鼠(SAMP 8，购自北京维通利华实验动物技术有限公司)为模型动物，以本发明含核苷酸组合物为干预物进行长期喂养来开展体内研究，考察实施例1-3中含核苷酸的组合物对小鼠自主活动能力、学习记忆能力、脑损伤及嗅觉的改善作用以及肌肉衰减的改善作用。取110只快速老化倾向小鼠，体重为(20±5)g，随机分成11组，以饲喂基础饲料为阳性对照组，以3月龄的SAMP 8为阴性对照组，以仅饲喂有与实施例1的片剂相同量及相同配比的复配核苷酸作为对照实验1，以仅饲喂有与实施例3的片剂相同量及相同配比的复配核苷酸作为对照实验2，仅饲喂有与实施例1的片剂相同量及相同配比的茶多酚、白藜芦醇、辅酶Q10、烟酰胺的组合物作为对照实验3，仅饲喂有与实施例2的片剂相同量及相同配比的黄芪多糖、姜黄素、白藜芦醇、烟酰胺的组合物作为对照实验4，仅饲喂有与实施例3的片剂相同量及相同配比的海洋鱼低聚肽作为对照实验5，并以相同剂量的β-烟酰胺单核苷酸(NMN)为对照实验6，结合多种行为形态学检测方法验证本发明的含核苷酸组合物对延缓衰老的作用效果。
1.含核苷酸的组合物对小鼠自主活动能力的改善---开阔场实验
本研究在实验动物9月龄时，即含核苷酸的组合物干预6个月后(每次1片，每天1次)，进行开阔场实验。开阔场行为分析主要反映动物自主活动能力和对新环境的探索，习惯以及伴随的情绪变化，是用于评价小鼠自发活动能力的实验。它的原理是小鼠有畏惧空旷明亮场地的天性，同时面对新鲜事物又会产生好奇心去探索，小鼠焦虑时，会减少探索中央区域，倾向于待在边缘区域；跨格次数及站立次数是动物探索行为及兴奋性的反映，中央格时间反映动物对环境的认知能力，正常动物会避开空旷环境，迅速离开中央格，沿周边活动。实验数据以均值±标准偏差表示，实验结果如表4所示。
表4.含核苷酸的组合物对小鼠自主活动能力的影响
组别 站立次数 跨格次数 进入中央格次数 中央格停留时间(s) 粪便个数
阳性对照组 3.0±5.4 60.6±12.2 4.4±2.6 46.4±19.2 4.9±5.3
阴性对照组 15.2±3.8 121.4±20.1 2.6±2.2 16.0±8.2 2.4±1.9
对照实验1 7.6±7.6 78.2±17.8 3.2±2.0 25.2±24.9 3.0±1.7
对照实验2 5.0±3.2 70.8±5.6 4.5±2.2 29.1±14.5 4.3±1.6
对照实验3 6.5±3.2 70.6±11.2 2.6±1.8 27.3±19.3 2.7±1.9
对照实验4 5.0±4.9 90.8±10.8 3.0±1.6 22.3±10.8 4.5±2.0
对照实验5 4.1±2.4 85.0±12.5 4.2±2.4 26.5±10.5 3.8±1.7
对照实验6 3.6±5.9 82.3±16.8 4.4±6.1 30.0±16.3 3.2±2.0
实施例1 14.5±4.5 107.9±16.7 2.4±3.2 18.5±16.7 2.3±1.6
实施例2 14.2±4.3 103.6±21.2 1.6±1.8 19.8±19.3 2.7±1.9
实施例3 12.6±2.6 95.1±20.2 2.5±1.7 20.3±20.2 2.2±3.3
综上可知，与对照组1-6相比，实施例1-3组的含核苷酸的组合物明显改善了快速老化倾向小鼠的自发活动性和探索能力。
2.含核苷酸的组合物对小鼠非空间学医记忆能力的改善---新物体识别实验
本研究在实验动物9月龄时，即含核苷酸的组合物干预6个月后(每次1片，每天1次)，进行新物体识别实验。新物体识别实验是利用动物先天对新物体有探索倾向的原理而建立的学习记忆测试方法，用于检测小鼠的非空间学习记忆能力，它是建立在小鼠对新异物体的自发探索行为的基础上的，对记忆保持能力的测试。该方法具有让老鼠在自由活动状态下进行学习记忆测试的特点，能更近似地模拟人类的学习记忆行为。同时，通过新物体(形状、大小等)的灵活变换，该实验还允许测试动物长期或短期记忆机制的形成以及在特定阶段的记忆形成的影响评判。该实验能反映小鼠自发的记忆能力，是目前一种新型的评价脑部功能的重要行为学方法。
表5.含核苷酸的组合物对小鼠新旧物体探索时间(s)的影响
组别 旧物体 新物体 识别指数
阳性对照组 16.2±3.0 23.0±10.7 46.9
阴性对照组 15.2±10.8 35.5±10.5 70.0
对照实验1 16.8±9.2 29.3±9.6 63.5
对照实验2 20.5±6.8 30.0±8.2 59.4
对照实验3 18.1±7.0 29.8±10.5 62.5
对照实验4 20.6±7.5 32.3±11.9 61.0
对照实验5 19.9±5.4 32.0±9.0 61.6
对照实验6 17.4±8.2 23.6±9.4 57.6
实施例1 13.7±9.5 34.7±12.9 71.7
实施例2 16.1±4.7 37.9±5.4 70.2
实施例3 18.1±9.9 35.8±12.6 66.4
由上述实验结果可知，与对照组1-6相比，实施例1-3组小鼠对新物体的探索时间均 明显长于旧物体，说明了实施例1-3的含核苷酸的组合物明显改善了快速老化倾向小鼠的自发的记忆能力。
3.含核苷酸的组合物对小鼠脑损伤的改善---筑巢实验
本研究在实验动物9月龄时，即含核苷酸的组合物干预6个月后(每次1片，每天1次)，进行筑巢实验。筑巢行为是小鼠的一种本能行为，操作简单、无损伤，可以反映小鼠脑损伤情况。有研究显示，小鼠筑巢行为能力受到神经递质的调控以及众多脑区的控制。脑区功能正常完好的小鼠，其筑巢能力优秀。在实验前一天弃去旧垫料，放入定量刨花，每个笼内均匀放入16块软纸片(4.5cm×4.5cm)，作为小鼠作巢材料。分别于实验开始后12h、18h、24h对各组小鼠筑巢效果进行四分制盲法评分，实验得分结果以三位实验人员的平均打分值，记为该时间点小鼠筑巢行为的得分，实验结果如表7所示。
表6.各组小鼠在12h、18h、24h的筑巢评分
组别 12h 18h 24h
阳性对照组 1.6±0.7 2.5±0.9 2.6±0.6
阴性对照组 2.7±0.8 4.9±1.0 6.3±0.5
对照实验1 2.4±0.5 3.2±0.7 3.8±0.7
对照实验2 1.9±0.7 2.7±0.9 3.3±0.8
对照实验3 2.2±0.6 3.2±0.4 3.9±0.4
对照实验4 2.1±0.4 3.3±0.5 3.7±0.5
对照实验5 1.8±0.9 2.9±1.0 3.5±0.7
对照实验6 1.7±1.0 2.7±0.8 3.1±0.5
实施例1 2.3±0.4 4.2±0.4 5.3±0.5
实施例2 2.5±0.8 4.5±1.1 4.9±1.1
实施例3 2.0±0.5 4.0±0.6 4.6±0.7
由上述实验结果可知，在12h(筑巢初期)时，各组小鼠的筑巢质量无明显差异；随着小鼠筑巢时间的延长、每组小鼠撕咬软纸片迹象增多，筑巢质量也均有提高，在筑巢24h时，实施例1-3组小鼠筑巢质量得分较高，几乎和阴性对照组试验结果无明显区别，表明实施例1-3的含核苷酸组合物能够有效提升衰老小鼠筑巢能力以及改善小鼠脑区受损程度。
4.含核苷酸的组合物对小鼠嗅觉记忆的改善---嗅觉记忆实验
本研究在实验动物9月龄时，即含核苷酸的组合物干预6个月后(每次1片，每天1次)，进行嗅觉记忆实验。嗅敏动物的学习记忆能力、寻食能力等与嗅觉有很高的相关性，可以通过观察、记录其行为学的改变，从而判断其嗅觉功能的变化。嗅觉记忆实验原理是随着某种具有特殊气味的物体暴露次数的增加，动物气味产生适应，根据这一特点，反应 动物对该气味的记忆能力，实验分为适应期(第一天)和检测期(第二天)。实验中选择的两种气味：薄荷香精、柠檬香精气味，小鼠对这两种气味没有偏好，每种气味允许小鼠自由探索30s，分别重复四次，然后记录小鼠每次嗅探离心管的时间。嗅探时间定义为：当小鼠朝向离心管且鼻子距离离心管周围1cm范围内所花费的时间，排除小鼠啃咬或攀爬离心管所花费的时间，实验结果如表7所示。
表7.含核苷酸的组合物对小鼠嗅探时间(s)的影响
Figure PCTCN2022098180-appb-000005
由表7结果可知，与阳性对照实验组和对照实验1-6相比，实施例1-3组小鼠仅需较短的时间就能够适应上述气体，由此可知，实施例1-3的含核苷酸组合物能够改善小鼠的气味适应行为，对老年小鼠的嗅觉记忆具有保护作用。
对于任何熟悉本领域的技术人员而言，在不脱离本发明技术方案范围情况下，都可利用上述揭示的技术内容对本发明技术方案做出许多可能的变动和修饰，或修改为等同变化的等效实施例。因此，凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所做的任何简单修改、等同变化及修饰，均应仍属于本发明技术方案保护的范围内。

Claims (10)

  1. 一种延缓衰老的含核苷酸组合物，其特征在于，由复配核苷酸和功能活性成分按照一定比例调配制成，所述复配核苷酸由AMP、CMP、GMP、UMP组成；所述功能活性成分为茶多酚、黄芪多糖、姜黄素、白藜芦醇、海洋鱼低聚肽、辅酶Q10、烟酰胺中的一种或两种以上的组合。
  2. 根据权利要求1所述的含核苷酸组合物，其特征在于，所述复配核苷酸的重量配比为：AMP 15.0～25.0％、CMP 15.0～45.0％、GMP 15.0～35.0％、UMP 15.0～30.0％，所述复配核苷酸和功能活性成分的重量配比为：复配核苷酸0.5～1.5份、茶多酚0.5～2份、白藜芦醇0.02～0.1份、辅酶Q10 0.02～0.08份、烟酰胺0.01～0.04份；或者复配核苷酸0.5～1.5份、黄芪多糖0.1～0.4份、姜黄素0.1～0.4份、白藜芦醇0.02～0.1份、烟酰胺0.01～0.04份；或者复配核苷酸0.5～1.5份、海洋鱼低聚肽0.2～0.8份。
  3. 根据权利要求1-2所述的含核苷酸组合物，其特征在于，所述复配核苷酸的重量配比为：AMP 16.9％、CMP 43.2％、GMP 17.8％、UMP 22.1％，所述复配核苷酸和功能活性成分的重量配比为：复配核苷酸1.2份、茶多酚1份、白藜芦醇0.05份、辅酶Q10 0.04份、烟酰胺0.02份。
  4. 根据权利要求1-2所述的含核苷酸组合物，其特征在于，所述复配核苷酸的重量配比为：AMP 16.9％、CMP 43.2％、GMP 17.8％、UMP 22.1％，所述复配核苷酸和功能活性成分的重量配比为：复配核苷酸1.2份、黄芪多糖0.2份、姜黄素0.19份、白藜芦醇0.05份、烟酰胺0.02份。
  5. 据权利要求1-2所述的含核苷酸组合物，其特征在于，所述复配核苷酸的重量配比为：AMP 24.3％、CMP 28.3％、GMP 19.3％、UMP 28.1％，所述复配核苷酸和功能活性成分的重量配比为：复配核苷酸0.6份、海洋鱼低聚肽0.43份。
  6. 根据权利要求3、4或5所述的含核苷酸组合物，其特征在于，所述组合物的成品状态包括粉剂、颗粒剂、片剂、丸剂、口服液、胶囊。
  7. 权利要求1-6任一项所述的含核苷酸组合物的制备方法，其特征在于，按配比称量所述复配核苷酸，混合均匀，获得混合粉，将混合粉和配方量的功能活性成分混合均匀，即得含核苷酸组合物。
  8. 权利要求1-6任一项所述的含核苷酸组合物在制备药品、保健品或功能性食品中的应用。
  9. 根据权利要求8所述的应用，其特征在于，所述含核苷酸组合物直接或经常规步骤处理后添加在药品、保健品或功能性食品中。
  10. 根据权利要求9所述的应用，其特征在于，所述药品、保健品或功能性食品具有清除体内自由基、抑制自由基对机体的氧化损伤、延缓人体衰老的作用。
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CN114939153B (zh) * 2022-05-09 2023-04-28 大连双迪科技股份有限公司 一种抗衰老的含有尿苷酸、腺苷酸和酵母肽的组合物及其应用
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040001817A1 (en) * 2002-05-14 2004-01-01 Giampapa Vincent C. Anti-aging nutritional supplement
CN1559290A (zh) * 2004-02-18 2005-01-05 上海纳贝生物技术有限责任公司 一种保健食品软胶囊及制备方法
CN101401649A (zh) * 2008-11-17 2009-04-08 珍奥集团股份有限公司 可增强机体免疫力的组合物
CN101559214A (zh) * 2009-05-27 2009-10-21 珍奥集团股份有限公司 具有增强免疫功能作用的组合物及其用途
CN111821323A (zh) * 2020-07-30 2020-10-27 李兴春 一种延缓衰老的保健胶囊

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101147779B (zh) * 2006-09-21 2011-04-06 珍奥集团股份有限公司 一种含有核苷酸的组合物、其制备方法及其用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040001817A1 (en) * 2002-05-14 2004-01-01 Giampapa Vincent C. Anti-aging nutritional supplement
CN1559290A (zh) * 2004-02-18 2005-01-05 上海纳贝生物技术有限责任公司 一种保健食品软胶囊及制备方法
CN101401649A (zh) * 2008-11-17 2009-04-08 珍奥集团股份有限公司 可增强机体免疫力的组合物
CN101559214A (zh) * 2009-05-27 2009-10-21 珍奥集团股份有限公司 具有增强免疫功能作用的组合物及其用途
CN111821323A (zh) * 2020-07-30 2020-10-27 李兴春 一种延缓衰老的保健胶囊

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"XIANDAI KANGSHUAILAO FANGLUE", 30 September 2009, PEOPLE'S MILITARY MEDICAL PUBLISHING HOUSE, CN, ISBN: 7509129265, article LI, GUI ET AL.: "Aging and Anti-aging", pages: 65 - 67, XP009543479 *

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