Use of a composition containing 5'-monophosphate nucleotides for the preparation of a medicament and / or a functional food against aging

Abstract

The application discloses application of a 5'-monophosphate nucleotide composition in preparation of anti-aging and longevity drugs and / or functional foods, and belongs to the technical field of medicines.The composition is composed of 5'-monophosphate adenosine, 5'-monophosphate cytidine, 5'-guanylic acid disodium, 5'-uridylic acid disodium and hypoxanthine nucleotide, and the mass ratio of the acid types of CMP, AMP, UMP, GMP and IMP is respectively as follows: CMP: 15-78%, AMP: 6-44%, UMP: 7-40%, GMP: 7-51%, and IMP is 0 or greater than 0 and not higher than 2.5%. The composition can synergistically enhance various mechanisms such as significantly reducing an insulin resistance index, enhancing body anti-coagulation function, improving blood circulation, regulating metabolism of essential minerals such as lithium and copper and regulating intestinal flora ecology, delays aging and prolongs healthy life span. Animal experiment data show that, compared with a control group, the composition prolongs the median survival time of mice by 9.21-12.6%, and the equivalent human life span is 8.76-12.01 years. Human experiment data show that, compared with the control group, the composition intervention reduces the median age of DNA methylation by 3.08 years, and provides a new idea for developing anti-aging and longevity drugs and / or functional foods.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of compositions containing 5'-monophosphate nucleotides in the preparation of anti-aging drugs and / or functional foods. Background Technology

[0002] As people's living standards improve, China is about to enter an aging society, and delaying aging and achieving healthy longevity are goals for most people. Preventing and delaying aging has become a hot research topic. Human aging is a complex biological process involving multiple factors, including cellular, molecular, tissue, and organ levels. Mechanisms of aging include decreased intracellular antioxidant capacity and cellular metabolic disorders. Extending lifespan cannot be achieved through a single mechanism but requires the synergistic effect of multiple pathways, such as enhancing cellular antioxidant capacity, improving cellular metabolism, and activating cellular regeneration capacity, to achieve significant results.

[0003] Currently, the market offers a wide variety of products aimed at delaying aging or extending lifespan, including antioxidants, nutritional supplements, health products, and some medications. Antioxidants, such as vitamin C and vitamin E, slow aging by eliminating free radicals in the body; nutritional supplements, such as coenzyme Q10 and fish oil, maintain cellular function by providing the nutrients needed for cell metabolism; health products, such as ginseng and reishi mushrooms, enhance physical health by regulating the body's immune and endocrine systems; and some medications, such as metformin and rapamycin, delay aging through specific biological targets. While these products can improve certain aging-related indicators to some extent, their effects are limited.

[0004] However, existing products have many problems. First, most products have a single mechanism of action, targeting only a specific aging mechanism and failing to comprehensively improve the aging process. For example, antioxidants can only eliminate free radicals. Furthermore, the mechanisms of action of some existing products are not clearly defined, and systematic research is lacking, making their practical application effects difficult to predict. Therefore, developing a product with safe, effective, and non-toxic ingredients that can efficiently delay aging and extend lifespan has become a crucial issue that urgently needs to be addressed.

[0005] Nucleic acids are essential components of cells, composed of various nucleotides, and are divided into deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). They play a crucial role in the storage and transmission of genetic information and protein synthesis. As the basic building blocks of nucleic acids, nucleotides are widely distributed in various organs, tissues, and cells of organisms, participating in fundamental life activities such as heredity, development, and growth. Nucleotide compounds have important biological functions, participating in almost all biochemical processes in organisms, such as cellular energy metabolism, coenzyme synthesis, and cell signal transduction. Therefore, nucleotides and their derivatives have enormous potential in delaying aging. Summary of the Invention

[0006] Therefore, the object of the present invention is to provide the use of 5'-monophosphate nucleotide compositions in the preparation of anti-aging and longevity-promoting pharmaceuticals and / or functional foods.

[0007] The objective of this invention is achieved through the following means:

[0008] This invention provides the use of compositions of 5'-monophosphate nucleotides or their sodium salts in the preparation of anti-aging and longevity-promoting pharmaceuticals and / or functional foods.

[0009] Based on the above technical solution, the composition further comprises 5'-adenosine monophosphate, 5'-cytidine monophosphate, disodium 5'-guanylate, disodium 5'-uridine monophosphate, and hypoxanthine nucleotide.

[0010] Based on the above technical solution, further, the mass ratios of each nucleotide in the composition converted to CMP, AMP, UMP, GMP, and IMP acid forms are as follows: CMP: 15-78%, AMP: 6-44%, UMP: 7-40%, GMP: 7-51%, and IMP is 0, or greater than 0 but not higher than 2.5%.

[0011] Based on the above technical solution, the mass ratios of the various nucleotides in the composition converted to CMP, AMP, UMP, GMP, and IMP acid forms are as follows: CMP: 15-45%, AMP: 15-25%, UMP: 15-30%, GMP: 15-35%, and IMP is 0, or greater than 0 but not higher than 2.5%.

[0012] Based on the above technical solution, the dosage form of the drug further includes powder, tablet, soft / hard capsule and oral liquid preparation.

[0013] Based on the above technical solution, the functional food further includes powders, tablets, soft / hard capsules, dairy products, baked goods, and liquid beverages.

[0014] Based on the above technical solution, the functional food and / or medicine further includes one or more nutritional supplements and food or medicine-acceptable excipients; the nutritional supplements include pyrrolizinone (PQQ), curcumin, resveratrol, and 6-gingerol.

[0015] Based on the above technical solution, the functional food and / or medicine further comprises 600-2400 parts of a composition in the form of 5'-monophosphate nucleotide or its sodium salt, 10-40 parts of PQQ, and 100-400 parts of curcumin; or 600-2400 parts of a composition in the form of 5'-monophosphate nucleotide or its sodium salt, 10-40 parts of PQQ, and 5-40 parts of resveratrol; or 600-2400 parts of a composition in the form of 5'-monophosphate nucleotide or its sodium salt, 10-40 parts of PQQ, and 5-40 parts of 6-gingerol.

[0016] Based on the above technical solution, the composition further improves the body's glucose and lipid metabolism by significantly reducing the insulin resistance index, thereby reducing the risk of diabetes.

[0017] Based on the above technical solution, the composition further improves the stability of the body's blood system by regulating the production or metabolism of blood cells such as platelets.

[0018] Based on the above technical solution, the composition further protects the stability of the nervous system and regulates the body's metabolic capacity by regulating the metabolism of essential minerals such as lithium (Li) and copper (Cu).

[0019] Based on the above technical solution, the composition further regulates the balance of intestinal flora by reducing the abundance of Klebsiella pneumoniae, thereby reducing the proportion of potentially pathogenic bacteria and maintaining the functional stability of normal flora.

[0020] Based on the above technical solution, further animal experiments showed that the composition extended the median survival time of mice by 9.21-12.6% compared with the control group, which is equivalent to 8.76-12.01 years in human life. Human trials showed that the composition reduced the median DNA methylation age by 3.08 years compared with the control group by regulating the DNA methylation clock.

[0021] The advantages of this invention over the prior art are as follows:

[0022] (1) The present invention uses a specific ratio of 5'-adenosine monophosphate, 5'-cytidine monophosphate, disodium 5'-guanylate, disodium 5'-uridine monophosphate and hypoxanthine nucleotide, combined with nutritional supplements such as pyrroloquinoline quinone (PQQ), curcumin, resveratrol, and 6-gingerol to synergistically eliminate free radicals in the body, inhibit the oxidative damage of free radicals to the body, and enhance the mitochondrial ATP production capacity and mitochondrial membrane potential, thereby enhancing mitochondrial activity and thus playing an anti-aging and longevity-promoting role.

[0023] (2) The 5'-monophosphate nucleotide composition provided by the present invention can significantly reduce the insulin resistance index, significantly improve the glucose and lipid metabolism of the elderly, and reduce the risk of diabetes in the elderly.

[0024] (3) The 5'-monophosphate nucleotide composition provided by the present invention can enhance blood flow by reducing platelet hematocrit and increasing platelet average volume; at the same time, it can regulate the stability of the blood system by regulating the proportion of monocytes, erythrocytes, basophils and eosinophils in the blood.

[0025] (4) The 5'-monophosphate nucleotide composition provided by the present invention can protect the stability of the nervous system and regulate the human body's metabolic capacity by regulating the metabolism of essential minerals such as lithium (Li) and copper (Cu).

[0026] (5) The 5'-monophosphate nucleotide composition provided by the present invention can regulate the balance of intestinal flora, reduce the proportion of potential pathogens, and maintain the functional stability of normal flora by reducing the abundance of Klebsiella pneumoniae in the intestine.

[0027] (6) The 5'-monophosphate nucleotide composition provided by the present invention exerts a synergistic effect by acting on different mechanisms of the body, maintaining the health status of the human body from the overall level, alleviating the damage caused by the external environment, and achieving the purpose of delaying aging and prolonging healthy life. Attached Figure Description

[0028] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0029] Figure 1 The graph shows the cumulative survival rate of mice in each group in Example 10. In this graph, 1 represents the purified feed group (NTs-Free); 2 represents the normal control group; 3, 4, and 5 are the low, medium, and high dose NTs intervention groups (NTs-L, NTs-M, and NTs-H), respectively; 6 represents the nicotinamide mononucleotide feeding group (NMN); and 7 represents the SAMR1 model control group.

[0030] Figure 2 This is a PCA diagram comparing the overall structural changes of the gut microbiota in Example 11.

[0031] Figure 3 The results show the effect of the nucleotide composition in Example 11 on the median DNA methylation age of older adults. Detailed Implementation

[0032] The present invention will be described in detail below with reference to embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the experimental methods or conditions described in the literature in the art or according to the kit instructions. The materials, reagents, instruments, etc. used in the following embodiments can all be obtained commercially.

[0033] Example 1

[0034] The 5'-monophosphate nucleotide composition used in this embodiment includes 5'-adenosine monophosphate, 5'-cytidine monophosphate, disodium 5'-guanylate, and disodium 5'-uridine monophosphate. The effective percentage contents of each nucleotide, calculated as CMP, AMP, UMP, and GMP, are as follows: CMP 43%, AMP 17%, UMP 22%, and GMP 18%.

[0035] The above four 5'-monophosphate nucleotides or their sodium salts were tested separately, and after passing the tests, they were passed through a 60-mesh sieve for later use. The required amount of each monophosphate nucleotide sample was weighed according to the above proportions, and the mixture was mixed for a time of not less than 40 minutes. The resulting mixed sample was stored at room temperature.

[0036] Example 2

[0037] The 5'-monophosphate nucleotide composition used in this embodiment includes 5'-adenosine monophosphate, 5'-cytidine monophosphate, disodium 5'-guanylate, and disodium 5'-uridine monophosphate. The effective percentage contents of each nucleotide, calculated as CMP, AMP, UMP, and GMP, are as follows: CMP 18%, AMP 25%, UMP 24%, and GMP 33%.

[0038] The above four 5'-monophosphate nucleotides or their sodium salts were tested separately, and after passing the tests, they were passed through a 60-mesh sieve for later use. The required amount of each monophosphate nucleotide sample was weighed according to the above proportions, and the mixture was mixed for a time of not less than 40 minutes. The resulting mixed sample was stored at room temperature.

[0039] Example 3

[0040] The 5'-monophosphate nucleotide composition used in this embodiment includes 5'-adenosine monophosphate, 5'-cytidine monophosphate, disodium 5'-guanylate, disodium 5'-uridine monophosphate, and hypoxanthine nucleotide. The effective percentage content of each nucleotide, calculated as CMP, AMP, UMP, GMP, and IMP, is as follows: CMP 28%, AMP 24%, UMP 28%, GMP 19%, and IMP 1%.

[0041] The above five 5'-monophosphate nucleotides or their sodium salts were tested separately, and after passing the tests, they were passed through a 60-mesh sieve for later use. The required amount of each monophosphate nucleotide sample was weighed according to the above proportions, and the mixture was mixed for a time of not less than 40 minutes. The resulting mixed sample was stored at room temperature.

[0042] Example 4

[0043] Compared with Example 1, this embodiment also includes PQQ, curcumin, and a filler, as detailed below:

[0044] The composition used in this embodiment is formulated as follows: 5'-monophosphate nucleotide composition obtained in Example 1: 50%, PQQ: 0.7%, curcumin: 8%, with the remainder supplemented by microcrystalline cellulose, corn starch, magnesium stearate, etc.

[0045] After the raw materials pass the inspection, they are mixed for 30 minutes using an HZD-1000 mixer. The content of active ingredients in the finely mixed powder is then determined. The finely mixed powder is granulated and compressed into tablets, ensuring that each tablet weighs between 0.56g and 8%.

[0046] Example 5

[0047] Compared with Example 2, this embodiment also includes PQQ, resveratrol, and a filler, as detailed below:

[0048] The composition used in this embodiment is formulated as follows: 5'-monophosphate nucleotide composition obtained in Example 2: 55%, PQQ: 0.7%, resveratrol: 0.7%, with the remainder supplemented by microcrystalline cellulose, corn starch, magnesium stearate, etc.

[0049] After the raw materials pass the inspection, they are mixed for 30 minutes using an HZD-1000 mixer. The content of active ingredients in the finely mixed powder is then determined. The finely mixed powder is granulated and compressed into tablets, ensuring that each tablet weighs between 0.55g and 8%.

[0050] Example 6

[0051] Compared with Example 3, this embodiment also includes PQQ, 6-gingerol, and a filler. Details are as follows:

[0052] The composition used in this embodiment is formulated in the following proportions: 70% of the 5'-monophosphate nucleotide composition obtained in Example 3, 1% of PQQ, 0.8% of 6-gingerol, with the remainder supplemented by microcrystalline cellulose, corn starch, magnesium stearate, etc.

[0053] After the raw materials pass the inspection, they are mixed for 30 minutes using an HZD-1000 mixer. The content of active ingredients in the finely mixed powder is then determined. The finely mixed powder is granulated and compressed into tablets, ensuring that each tablet weighs between 0.28g and 8%.

[0054] Example 7

[0055] This embodiment examines the antioxidant properties of the compositions prepared in Examples 1-6. The following description uses the 5'-monophosphate nucleotide composition prepared in Example 1 as an example.

[0056] The 5'-monophosphate nucleotide composition prepared in Example 1 was weighed and dissolved in a mixed solution of deionized water and ethanol (volume ratio 1:1) to obtain a mother liquor with a concentration of 1 mg / mL. The DPPH free radical scavenging rate and ABTS free radical scavenging rate were then determined.

[0057] 1. Determination of DPPH free radical scavenging rate:

[0058] The mother liquor was diluted 100, 50, 25, 10, and 1 times to prepare gradient dilutions with concentrations of 10 μg / mL, 20 μg / mL, 40 μg / mL, 100 μg / mL, and 500 μg / mL, respectively.

[0059] Take 150 μL of the sample at the above concentration and 150 μL of 60 mg / L DPPH solution (diluted with anhydrous ethanol) and place them in a 96-well plate, mix well, and store at 30°C in the dark. After 30 min, measure the absorbance at 519 nm using a microplate reader. The formula for DPPH free radical scavenging rate is as follows:

[0060] DPPH free radical scavenging rate (%) =

[0061] In the formula, Ablank represents the absorbance of 150 μL of deionized water ethanol solution and 150 μL of DPPH solution, and Asample represents the absorbance of 150 μL of sample and 150 μL of DPPH solution; IC0 50 The concentration of the sample when the DPPH free radical scavenging rate reaches 50%.

[0062] 2. Determination of ABTS free radical scavenging rate:

[0063] (1) The mother liquor was diluted by 100, 50, 25, 10 and 1 times to prepare gradient dilutions with concentrations of 10 μg / mL, 20 μg / mL, 40 μg / mL, 100 μg / mL and 500 μg / mL respectively.

[0064] (2) Mix 7 mmol / L ABTS solution and 140 mmol / L potassium persulfate solution in a ratio of 62.5:1, let stand overnight at room temperature in the dark to form ABTS stock solution, and dilute with deionized water to 5 mmol / L ABTS working solution before use.

[0065] (3) Take 0.15 mL of the above diluted sample and mix it with 2.85 mL of ABTS working solution. Let it stand at 30°C for 8 min. Use deionized water as a blank and measure the absorbance at 734 nm.

[0066] The formula for ABTS free radical scavenging rate is as follows:

[0067] ABTS Free radical scavenging rate (%) =

[0068] In the formula, A blank is the absorbance of 0.15 mL of deionized water ethanol solution and 2.85 mL of ABTS working solution, and A sample is the absorbance of 0.15 mL of sample and 2.85 mL of ABTS working solution. IC 50 The concentration of the sample when the ABTS radical scavenging rate reaches 50%.

[0069] The antioxidant properties of the 5'-monophosphate nucleotide compositions in Examples 2-3 and the compositions in Examples 4-6 were tested using the same detection method described above. Control Test 1 used an amount of 5'-adenosine monophosphate equal to that of the 5'-monophosphate nucleotide composition in Example 1; Control Test 2 used an amount of 5'-cytidine monophosphate equal to that of the 5'-monophosphate nucleotide composition in Example 1; Control Test 3 used an amount of disodium 5'-guanylate equal to that of the 5'-monophosphate nucleotide composition in Example 1; and Control Test 4 used an amount of 5'-uridine equal to that of the 5'-monophosphate nucleotide composition in Example 1. Disodium phosphate was used as control test 4; hypoxanthine nucleotide in the same amount as the 5'-monophosphate nucleotide composition of Example 3 was used as control test 5; a composition containing only the same amount and ratio of PQQ and curcumin as the tablet of Example 4 was used as control test 6; a composition containing only the same amount and ratio of PQQ and resveratrol as the tablet of Example 5 was used as control test 7; and a composition containing only the same amount and ratio of PQQ and 6-gingerol as the tablet of Example 6 was used as control test 8. The test data are expressed as mean ± standard deviation, and the test results are shown in Table 1.

[0070] Table 1. Antioxidant properties of nucleotide-containing compositions

[0071]

[0072] The above results indicate that, compared with the control groups 1-8, the 5'-monophosphate nucleotide compositions of Examples 1-3 and the compositions of Examples 4-6 all have higher antioxidant properties. The antioxidant properties of the 5'-monophosphate nucleotide compositions and the compositions of the 5'-monophosphate nucleotide compositions and the functional active ingredients are significantly enhanced, indicating that the compositions of Examples 1-6 can effectively scavenge free radicals in the body.

[0073] Example 8

[0074] This embodiment examines the effect of the compositions prepared in Examples 1-6 on improving mitochondrial function. The following description uses the 5'-monophosphate nucleotide composition prepared in Example 1 as an example.

[0075] Mitochondria are essential for life, participating in important physiological processes such as ATP production, apoptosis, and fatty acid β-oxidation. Mitochondrial ATP production capacity and mitochondrial membrane potential can reflect the functional state of mitochondria. Mitochondrial ATP production capacity was detected using a Seahorse XFe96 (Agilent) analyzer, and mitochondrial membrane potential was detected using JC-1 fluorescent dye.

[0076] Cell culture conditions: Mouse embryonic fibroblasts NIH / 3T3 (purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured in DMEM high glucose medium containing 1% penicillin / streptomycin and 10% fetal bovine serum, and cultured in a 5% CO2 incubator at 37°C under saturated humidity; the senescent cell model was obtained by intervention with 200 μmol / L hydrogen peroxide for 4 h.

[0077] The specific experimental procedure for assessing mitochondrial ATP production capacity is as follows: 2 × 10⁶ cells were seeded in each well of a 96-well cell culture plate (excluding the four corner background wells). 5NIH / 3T3 cells were cultured overnight. After cell adhesion, 200 μmol / L hydrogen peroxide was added for 4 h. The hydrogen peroxide-containing medium was then discarded, and complete medium containing 100 μg / mL of 5'-monophosphate nucleotides (DMEM high-glucose medium containing 1% penicillin / streptomycin and 10% fetal bovine serum) was added. The experimental group that was not treated with 200 μmol / L hydrogen peroxide and then added normal complete medium served as the positive control group, while the experimental group that was treated with 200 μmol / L hydrogen peroxide and then added normal complete medium served as the model control group. The cells were cultured for 24 h. One day in advance, hydrate the probe plate (XFe96FluxPak, Agilent, 100840-000) with hydration solution (XF Calibrant Solution, Agilent, 102353-100) and incubate it in a CO2-free incubator at 37°C for at least 12 hours. On the day of the experiment, prepare the experimental culture medium by adding 1 ml of 2.5 mM glucose (Sigma, G7528), 2 mM glutamine (Sigma, G8540), and 1 mM sodium pyruvate (Sigma, S8636) to 100 ml of basal medium (SeahorseXF BaseMedium, Agilent, 102353-100). Adjust the pH of the solution to 7.4 ± 0.05 with 1 N NaOH. Change the medium in the cell culture plate, discarding 40 μL / well of the original medium, and dilute with 160 μL / well of the test culture medium. Then, aspirate 160 μL / well and repeat 2-3 times, with a final volume of 175 μL per well. Incubate at 37°C for 1 h. Add 25 μL / well of the drug to the probe plate. Add 2 μM oligomycin (Oligomycin, abcam, ab141829) to well A, 1 μM carbonyl-cyano-p-trifluoromethoxyphenylhydrazine (FCCP, Sigma, C2920) to well B, and 1 μM Antimycin A (abcam, ab141904) / Rotenone (Sigma, R8875) to well C. Calibrate the probe plate on the instrument. Analyze the cell culture plate on the instrument.

[0078] The specific experimental procedure for mitochondrial membrane potential is as follows: Seed 2 × 10⁶ cells per well of a 96-well cell culture plate (excluding the four background wells). 5NIH / 3T3 cells were cultured overnight. After cell adhesion, 200 μmol / L hydrogen peroxide was added for 4 h. The hydrogen peroxide-containing medium was then discarded, and complete medium containing 100 μg / mL of a 5'-monophosphate nucleotide combination (DMEM high-glucose medium containing 1% penicillin / streptomycin and 10% fetal bovine serum was added; the group without 200 μmol / L hydrogen peroxide treatment and then added to normal complete medium served as the positive control, while the group treated with 200 μmol / L hydrogen peroxide treatment and then added to normal complete medium served as the model control) was cultured for 24 h. Cells were digested with trypsin, centrifuged to collect the cells, and incubated with mitochondrial membrane potential detection solution (JC-1) for 2 h. Cells were then washed with pre-cooled buffer, the supernatant was discarded, and the cells were collected into new EP tubes with fresh pre-cooled buffer added. 100 μL of cell culture was transferred to a 96-well plate, and the fluorescence intensity of the JC-1 polymer was detected. JC-1 is an ideal fluorescent probe widely used for detecting mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix to form a polymer, which can produce red fluorescence. The higher the intensity of the red fluorescence, the higher the mitochondrial membrane potential and the higher the mitochondrial activity.

[0079] The effects of the 5'-monophosphate nucleotide compositions in Examples 2-3 and the compositions in Examples 4-6 on improving mitochondrial function were detected using the same detection method described above. Control experiment 1 used an amount of 5'-adenosine monophosphate equal to that of the 5'-monophosphate nucleotide composition of Example 1; control experiment 2 used an amount of 5'-cytidine monophosphate equal to that of the 5'-monophosphate nucleotide composition of Example 1; control experiment 3 used an amount of 5'-guanylate disodium equal to that of the 5'-monophosphate nucleotide composition of Example 1; control experiment 4 used an amount of 5'-uridine disodium equal to that of the 5'-monophosphate nucleotide composition of Example 1; control experiment 5 used an amount of hypoxanthine nucleotide equal to that of the 5'-monophosphate nucleotide composition of Example 3; control experiment 6 used a composition containing only the same amount and ratio of PQQ and curcumin as the tablets of Example 4; control experiment 7 used a composition containing only the same amount and ratio of PQQ and resveratrol as the tablets of Example 5; and control experiment 8 used a composition containing only the same amount and ratio of PQQ and 6-gingerol as the tablets of Example 6. The experimental data are expressed as mean ± standard deviation.

[0080] Table 2. Antioxidant properties of nucleotide-containing compositions

[0081]

[0082] The results above show that, compared with the model control group, the mitochondrial ATP production capacity and mitochondrial membrane potential of mouse embryonic fibroblasts in control groups 1-8 were improved. The improvement effect of the composition test group of Examples 4-6 on the mitochondrial ATP production capacity and mitochondrial membrane potential of mouse embryonic fibroblasts was significantly better than that of control groups 1-8. The results indicate that the composition of the 5'-monophosphate nucleotide composition and the functional active ingredients of the present invention can synergistically improve the mitochondrial ATP production capacity and mitochondrial membrane potential of mouse embryonic fibroblasts, significantly improve mitochondrial function, increase mitochondrial activity, and keep the body cells in a highly viable state.

[0083] Example 9

[0084] Metabolism produces a large number of free radicals, which damage cell function, lead to organ aging, and thus contribute to overall aging. Superoxide dismutase (SOD) can scavenge harmful superoxide anion free radicals. Glutathione peroxidase (GSH-Px) is an important peroxide-degrading enzyme widely present in the body. It can reduce toxic peroxides to non-toxic hydroxyl compounds, thereby protecting cell membrane structure and function from peroxide interference and damage. Therefore, SOD and GSH-Px activities can reflect the antioxidant capacity of tissues and are important indicators for evaluating aging. MDA, a lipid peroxidation product, is closely related to the production of free radicals and the degree of lipid peroxidation in tissue cells. It can reflect the oxidative stress damage state of tissues and is also an important indicator for evaluating aging. This embodiment investigates the effects of the 5'-monophosphate nucleotide compositions in Examples 1-3 and the compositions in Examples 4-6 on delaying aging in rapidly aging-prone SAMP-8 mice (3 months old). The mice were housed in an SPF-grade animal facility at an ambient temperature of 22±2℃ and a relative humidity of 50%-60%, with a 12h / 12h (8:00-20:00) light pattern to simulate normal diurnal rhythms.Using the American Institute of Nutrition (AIN-93G) animal feed as the basal diet, and the above-mentioned composition as the intervention, the mice were fed 220 mg / kg BW by gavage daily for 60 days. The specific process is as follows: 75 rapidly aging mice (SAMP-8 mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), weighing (20±5) g, were randomly divided into 15 groups of 5 mice each. The groups were: negative control group (fed only the basal diet), experimental group (composition groups of Examples 1-6), control group 1 (equivalent to 5'-adenosine monophosphate of the 5'-monophosphate nucleotide composition of Example 1), control group 2 (equivalent to 5'-cytidine monophosphate of the 5'-monophosphate nucleotide composition of Example 1), control group 3 (equivalent to 5'-guanylate disodium of the 5'-monophosphate nucleotide composition of Example 1), and control group 4 (equivalent to 5'-guanylate disodium of the 5'-monophosphate nucleotide composition of Example 1). The following mice were used as control experiments: 1) Disodium uridine monophosphate; 2) Control group 5 (equivalent to the 5'-monophosphate nucleotide composition of Example 3); 3) Control group 6 (containing only the same amount and ratio of PQQ and curcumin as the tablets of Example 4); 4) Control group 7 (containing only the same amount and ratio of PQQ and resveratrol as the tablets of Example 5); 5) Control group 8 (containing only the same amount and ratio of PQQ and 6-gingerol as the tablets of Example 6). On the second day after the last feeding, blood was collected from the fundus of each group of mice. After the blood was allowed to coagulate completely at room temperature, the serum was separated by centrifugation for later use. The mice were then euthanized by dislocation, and the liver was quickly separated. After rinsing with sodium chloride solution, a 10% tissue homogenate was prepared. The homogenate was centrifuged, and the supernatant of the separated tissue homogenate was used for later use. The activities of SOD and GSH-Px in mouse serum and liver tissue were detected according to the instructions of the superoxide dismutase (SOD) assay kit and the glutathione peroxidase (GSH-Px) assay kit, respectively. The content of malondialdehyde (MDA) peroxide in mouse serum and liver tissue was determined by the thiobarbituric acid spectrophotometric method. The experimental data are expressed as mean ± standard deviation.

[0085] Table 3. Results of SOD, GSH-Px, and MDA levels in mice fed the compositions of Examples 1-6

[0086]

[0087] As can be seen from the above results, compared with the negative control group, the SOD activity and GSH-Px activity in the sera and liver tissues of the mice in Control Groups 1-8 were increased, and the MDA content in the sera and liver tissues of the mice was decreased; the indexes of the composition test groups in Examples 1-6 were significantly better than those in Control Groups 1-8, indicating that the 5'-mononucleotide composition of the present invention and the composition after the compounding of the 5'-mononucleotide composition and functional active ingredients can synergistically enhance the ability of the body to scavenge free radicals, reduce the degree of tissue cell damage, and delay the senescence of the body.

[0088] Example 10

[0089] This example examines the effect of lifelong intervention with the 5'-mononucleotide composition prepared in Example 1 on the survival time of SAMP8 mice.

[0090] 1. Sample: The 5'-mononucleotide composition obtained in Example 1 above.

[0091] 2. Experimental animals: Three-month-old healthy specific pathogen free (SPF)-grade male senescence-accelerated mouse prone 8 (SAMP-8) and model control mouse SAMR1 mice, a total of 350, were purchased from the Laboratory Animal Center of Peking University Health Science Center (Laboratory Animal Production License No.: SCXK (Beijing) 2016-0010; Laboratory Animal Use License No.: SYXK (Beijing) 2016-0041). During the experiment, the animals were housed individually in cages, the temperature of the animal room was controlled at 22±2°C, the relative humidity was maintained at 50% - 60%, and the light-dark cycle was 12h:12h.

[0092] 3. Experimental grouping and dosage: After one week of adaptive feeding, the SAMP8 mice were randomly divided into 6 groups (n = 50) according to body weight: nucleotide-free group (purified feed, NTs-Free), normal control group (basic feed, Normal control), low, medium, and high-dose intervention groups of the composition described in Example 1 (0.3 g / kg, 0.6 g / kg, 1.2 g / kg, corresponding to NTs-L, NTs-M, NTs-H respectively) and nicotinamide mononucleotide feeding group (NMN, 0.3 g / kg), and a SAMR1 model control group (basic feed, n = 50) was established. The nucleotide mixture and nicotinamide mononucleotide were incorporated into the basic feed at different dosages for intervention. Since the mice were three months old, each group was given the corresponding feed for lifelong feeding intervention, and the animals had free access to water and food during the experiment; the intervention and grouping of the experimental animals are shown in Table 4.

[0093] Table 4. Grouping and intervention of experimental animals

[0094]

[0095] 4. Experimental Methods:

[0096] During the experiment, mice were housed individually in cages, and no other factors interfered except for routine procedures performed by researchers (cleaning the cages and general observation). The times when mice were found to have died naturally or were euthanized (i.e., observed to be in extremely poor condition and judged to have a very low probability of survival within 48 hours) were recorded in detail for lifespan analysis. Euthanasia was performed for humane reasons, and euthanized mice were considered to have a complete lifespan and could be used for lifespan analysis. The criteria for euthanasia were based solely on the animal's condition, referring to AAALAC guidelines, relevant literature review, and the team's previous experience in lifelong feeding experiments. Key indicators included: severe lethargy, rapid weight loss (>20% over two weeks), severe abdominal distension and physical condition score accompanied by signs of pain (assessed by facial expressions, appearance, behavioral abnormalities, and other common mouse pain symptoms), unresponsiveness to stimuli or external behaviors, severe ulcers or hemorrhagic tumors, and severe hypothermia accompanied by abnormal respiratory rate. After the experiment, the survival time of mice in each group was summarized and analyzed using SPSS 24.0 software. Kaplan-Meier survival analysis was performed, and Log Rank (Mantel-Cox) was used for intergroup comparisons. P<0.05 was used as the statistical threshold.

[0097] 5. Experimental Results

[0098] 5.1. Effect of lifelong intervention with the composition described in Example 1 on the survival of SAMP8 mice.

[0099] As shown in Table 5, intervention began in all groups of mice at 3 months of age. Mortality began gradually after 6 months of age, with the NMN group experiencing the first two deaths at 7 months, followed by deaths in all other groups at 8 months. After 12 months of age, the mortality rate accelerated significantly in all groups, with the NTs-L and NTs-M groups showing lower mortality rates than the other groups. At 18 months of age, the NTs-L and NTs-M groups had the highest number of surviving mice, with survival rates of 26% and 20%, respectively. At 20 months of age, no mice survived in the NTs-Free, Normal, and NTs-H groups. By 23 months of age, only the NTs-L and NTs-M groups had surviving mice (2 mice each). The NTs-L group had the longest survival time at 24.93 months, close to 25 months, followed by the NTs-M group at 23.83 months.

[0100] Table 5. Survival status of mice in each group from 3 to 25 months of age.

[0101]

[0102] 5.2. Effect of lifelong intervention with the composition described in Example 1 on the survival time of SAMP8 mice.

[0103] As shown in Table 6, the mean survival time (14.98 m) and median survival time (14.30 m) of the NTs-L group were both higher than those of the other groups, followed by the NTs-M group (mean survival time 14.65 m, median survival time 13.87 m). Analyzing the median survival time, the median survival time of the NTs-L group (14.30 m) was 12.6%, 5.93%, and 5.38% longer than that of the NTs-Free (12.70 m), Normal control (13.50 m), and NMN group (13.57 m), respectively. The median survival time of the NTs-M group (13.87 m) was 9.21%, 2.74%, and 2.21% longer than that of the NTs-Free (12.70 m), Normal control (13.50 m), and NMN group (13.57 m), respectively.

[0104] Table 6. Effects of lifelong intervention with NTs on survival time of mice in each group

[0105]

[0106] Depend on Figure 1 The results showed that the NMN group mice died first (corresponding to age 6.37 months), and their cumulative survival rate was lower than that of other groups before 10 months of age. At a cumulative survival rate of 50%, the NMN group mice were at an age of 13.57 months, higher than the NTs-Free (12.70 months), Normal control (13.50 months), and NTs-H (12.77 months) groups, but lower than the NTs-L (14.30 months) and NTs-M (13.87 months) groups. At 14 months of age, the cumulative survival rates of the NTs-L and NTs-M groups were 52% and 46%, respectively, higher than other groups. In subsequent months, the cumulative survival rates of the NTs-L and NTs-M groups were consistently higher than other groups, with the NTs-L group having the longest survival time (24.93 months), followed by the NTs-M group (23.83 months). Intergroup comparisons using Log Rank (Mantel-Cox) revealed that, compared to the NTs-Free group, the NTs-L group showed: χ² 2 =5.26, P =0.022; Ratio of NTs-M to NTs-Free groups: χ² 2 =5.40, P =0.020, all of which showed statistical significance. P <0.05). The test results for NTs-L compared to the Normal control and MNM groups were (χ²) 2 =5.40, P=0.071), (χ 2 =3.07, P =0.08). SAMR1 was the model control group, except for the NTs-L group (χ² = 0.08). 2 =3.07, P =0.536), and NTs-M group (χ²) 2 =2.62, P =0.105) compared to no statistically significant difference ( P >0.05, and the cumulative survival rate was significantly higher than that of the other groups ( P <0.01).

[0107] The above results indicate that long-term intervention with the composition described in Example 1 can effectively reduce the mortality rate, prolong the survival time, and improve the lifespan of mice. The NTs-L group had the longest average lifespan (14.98m), median survival time (14.3m), and longest survival time (24.93m). Compared to the NTs-Free, Normalcontrol, and NMN groups in this study, the median survival time was extended by 12.6%, 5.93%, and 5.38%, respectively.

[0108] In summary, the experimental results show that long-term intervention with the composition described in Example 1 can effectively reduce the mortality rate, prolong the survival time, and improve the lifespan of mice, thus having a highly positive promoting effect on the quality of life. Its lifelong intervention in SAMP8 mice extended the median survival time by 9.21–12.6% compared to the control group, which is equivalent to an estimated human lifespan of 8.76–12.01 years.

[0109] Example 11

[0110] This embodiment employs a randomized, double-blind experimental design to evaluate the application effect of the 5'-monophosphate nucleotide composition prepared in Example 1 in the elderly population.

[0111] 1. Test Methods

[0112] 1.1. Experimental Design and Participants

[0113] This trial is an exploratory, 19-week, single-center, double-blind, randomized, placebo-controlled study that recruits community residents through advertising for a comprehensive health assessment, including clinical health check-ups, questionnaires, physical function assessments, and anthropometry. Eligibility was determined according to the following criteria: Inclusion criteria: (1) Age between 60 and 70 years; (2) No serious physical or mental illness; (3) No prior use of nucleotide-related supplements or health foods; (4) Ability to follow the study protocol and provide informed consent. Exclusion criteria: (1) Diagnosed diseases such as autoimmune diseases, serious cardiovascular and cerebrovascular diseases, complications of major organs such as the liver and kidneys, or other serious diseases such as malignant tumors, pancreatic diseases, and mental illnesses; (2) Abnormal screening laboratory test values ​​or other laboratory test results, excluding the researcher's judgment; (3) Severe visual or hearing impairments that affect communication; (4) Participation in other clinical trials within the past 6 months; (5) Use of foods or drugs related to the function being tested. All participants provided informed written consent in accordance with the Declaration of Helsinki prior to their recruitment into the study.

[0114] 1.2. Sample size and randomization

[0115] The sample size for this trial was determined according to the "Technical Specifications for Inspection and Evaluation of Health Foods" issued by the National Health Commission of China, which recommends at least 50 participants per group in human intervention trials. Participants were randomly assigned to the intervention group and the control group in a 1:1 ratio. To estimate a 20% loss to follow-up rate, a total of 120 participants were included in the study.

[0116] Participants are randomly assigned to groups using computer-generated random numbers. The protocol is known only to the trial designers, who are not involved in data collection or analysis. The randomization code is sealed and is only unblinded in the event of a serious adverse event.

[0117] 1.3. Intervention and blinding

[0118] This trial included a nucleotide intervention group and a placebo control group. The intervention was administered in capsule form to ensure consistency in appearance, taste, and formulation between the placebo (pure starch excipient) and the intervention (the 5'-monophosphate nucleotide composition described in Example 1 plus starch excipient), thus avoiding any suspicion among participants. The placebo capsules were designed similarly to the intervention capsules. Each capsule in the nucleotide intervention group contained 0.1 g of starch excipient and 0.3 g of the 5'-monophosphate nucleotide composition described in Example 1, while each placebo capsule contained 0.4 g of starch excipient. Participants were instructed to take four capsules daily while maintaining a regular lifestyle and diet. The double-blind design ensured that participants and researchers, including investigators, clinicians, data collectors, and follow-up coordinators, were unaware of group assignments. The capsules were packaged in opaque bottles labeled only with the participant identifier. The blinding procedure was explained in detail to participants during the informed consent process.

[0119] 1.4. Participation in the Experiment

[0120] After screening, 122 participants were selected as eligible to participate. The intervention lasted for 19 weeks. During the follow-up period, except for one participant in the nucleotide group who withdrew after one week of intervention due to a traffic accident, the remaining 121 participants completed the trial. A total of 121 participants who completed the intervention were ultimately included in the statistical analysis. There were no significant differences between the two groups in any baseline characteristics. The mean age of the participants was 65.65 years (standard deviation 2.59), of whom 82 (67.21%) were female.

[0121] 1.5. Data Collection

[0122] In the trial, data collection at the baseline (T0), intermediate (T1), and final (T2) periods was conducted by the hospitals that performed clinical examinations and collected biological samples (blood, urine, and stool).

[0123] DNA methylation data were acquired through whole-genome bisulfite sequencing (WGBS), with the sequencing work outsourced to BGI Genomics (Shenzhen, China). SOAPnuke and Bismark software were used for data filtering, alignment, and methylation site localization.

[0124] Insulin resistance was calculated using the Homeostasis Model for Insulin Resistance (HOMA-IR) method, a widely used surrogate indicator. The formula is: (Fasting blood glucose [mmol / L]) Fasting insulin (mIU / L) / 22.5.

[0125] 1.6. Statistical Analysis

[0126] The data analysis in this study followed the intention-to-treat (ITT) principle, and no imputation was performed on missing data in the baseline data. The Shapiro-Wilk test was used to assess data normality, and the Levene test was used to assess homogeneity of variance. Based on the distribution characteristics of the variables, the independent samples t-test (for normally distributed data) or the Mann-Whitney U test (for non-normally distributed data) was used to compare pre-intervention, post-intervention, and changes between groups. The effect size was calculated using Cohen's d-value, derived from the difference between the means of the two groups, where d=0.2 was a small effect size, d=0.5 was a moderate effect size, and d=0.8 was a large effect size.

[0127] We used generalized estimating equations (GEE) to analyze all outcome measures, specifying an exchangeable work-related structure. Data from all baselines and endpoints were used in the trial. Independent variables in Model 1 included group, time, group-time interaction terms, baseline dependent variable (a continuous variable), age, and sex. We also constructed Model 2, further adjusting for dietary nucleotide intake. Similarly, Model 3 further adjusted for dietary purine intake. The intervention effect was determined by the coefficients of the interaction terms in the models and their 95% confidence intervals (CIs).

[0128] 2. Test Results

[0129] 2.1 Effects of nucleotide compositions on mineral content in the elderly

[0130] This study used a t-test to analyze the effects of exogenous nucleotide intervention on the levels of multiple minerals in elderly individuals. The results are shown in Table 7. The results showed that there were significant differences between the intervention group and the control group at the intervention endpoint (T2).

[0131] Table 7. Effects of nucleotide compositions on the body's mineral content

[0132]

[0133] Independent samples t-test results showed that, in terms of changes in lithium (Li) concentration, at the intervention endpoint (T2), the lithium concentration in the nucleotide intervention group (1.57±0.37 ng / ml) was significantly lower than that in the control group (1.74±0.52 ng / ml). P =0.033, Cohen's d=-0.39. Furthermore, the concentration of copper (Cu) at the intervention endpoint (1012.67±114.74 ng / ml) was significantly higher than that in the control group (969.44±109.94 ng / ml). P =0.039, Cohen's d=0.38.

[0134] The results of this study indicate that exogenous nucleotide intervention significantly affected the levels of some minerals, especially at the intervention endpoint (T2), where the difference between the intervention group and the control group was more pronounced, suggesting that nucleotides may play a role in mineral metabolism. Regarding lithium (Li), the lithium concentration in the nucleotide intervention group was significantly lower than that in the control group at the intervention endpoint (T2). Lithium, as a trace element related to neuroregulation, plays an important role in mood stability, neuronal signaling, and neuroprotection. Its decreased level may suggest that nucleotide intervention affected lithium absorption, distribution, or excretion to some extent. Considering that lithium is mainly excreted through the kidneys, nucleotides may affect its renal tubular reabsorption or excretion mechanism. Regarding copper (Cu), the copper concentration in the nucleotide intervention group was significantly higher than that in the control group at the intervention endpoint (T2). Copper is an essential trace element in the body, playing an important role in iron metabolism, antioxidant defense, immune function, and neurotransmission. Its increased concentration may indicate that nucleotide intervention promoted copper absorption or affected the regulation of copper in the body. Previous studies have shown that nucleotides may indirectly promote copper metabolism and utilization by affecting the expression of metal transport proteins or regulating oxidative stress levels.

[0135] 2.2 Effects of nucleotide compositions on glucose and lipid metabolism in the elderly

[0136] This part of the experiment aims to comprehensively evaluate the effects of exogenous nucleotides on glucose and lipid metabolism in the elderly, mainly through analysis of indicators such as fasting blood glucose (FBG) and insulin resistance index (HOMA-IR).

[0137] Table 8. Results of glucose and lipid metabolism indicators in the subjects

[0138]

[0139] The results above show that from baseline to the midpoint of the intervention, fasting blood glucose decreased in the NTs group and increased in the C group (NTs: -0.13±0.66, C: 0.20±0.84), and the difference was statistically significant. P =0.02, and the effect size between groups was moderate (d=-0.43). From baseline to the intervention endpoint, the insulin resistance index decreased in the NTs group and increased in the C group (NTs: -0.27±1.45, C: 0.19±0.74), and the difference was statistically significant. P =0.03), and the between-group effect size was moderate (d=-0.41).

[0140] These results indicate that exogenous nucleotide supplementation can significantly improve glucose and lipid metabolism in older adults, particularly in terms of insulin resistance index. The supplementation of exogenous nucleotides was associated with a significant reduction in insulin resistance index, a change that may help maintain glycemic homeostasis and reduce the risk of diabetes in older adults.

[0141] 2.3 Effects of nucleotide compositions on blood routine tests in the elderly

[0142] This part of the experiment aims to comprehensively evaluate the effects of exogenous nucleotides on blood routine tests in elderly individuals, focusing on analyzing multiple hematological indicators and exploring their potential effects on the immune system and overall health of the elderly. The experiment includes routine hematological indicators such as white blood cell count, platelet count, and hemoglobin, as well as immune cell-related indicators such as the percentage and absolute values ​​of lymphocytes, neutrophils, monocytes, eosinophils, and basophils.

[0143] Table 9. Results of glucose and lipid metabolism indicators in the subjects

[0144]

[0145] The results above indicate that at baseline, there were no significant differences in any of the blood routine indicators between the two groups of elderly individuals. P The value >0.05 indicates that the baseline blood routine conditions of the two groups are comparable. Analysis of the differences between groups in the changes before and after the intervention (T2-T0) shows that the platelet count changes in the two groups are opposite: the platelet count decreased in the NTs group and increased in the C group (NTs: -11.60±30.76, C: 6.87±51.56), and the difference is statistically significant. P =0.019), the effect size between groups was moderate (d=0.43); platelet-weighted hematocrit increased in both groups, but the change in NTs group was smaller than that in group C (NTs: 0.01±0.03, C: 0.02±0.03), and the difference was statistically significant. P =0.037, the effect size between groups was moderate (d=0.39); the percentage of basophils decreased in both groups, but the change in the percentage of basophils in the NTs group was smaller than that in the C group (NTs: -0.01±0.12, C: -0.05±0.11), and the difference was close to statistical significance. P =0.069), the effect size between groups was small (d=0.34); the absolute value of basophils decreased in both groups, but the change in the absolute value of basophils in the NTs group was greater than that in the C group (NTs: 0.00±0.01, C: -0.00±0.01), and the difference was statistically significant. P=0.037), with a small effect size between groups (d=0.39). Overall, nucleotide intervention caused some changes in blood routine indicators, especially in platelet-related indicators. Specifically, platelet count and hematocrit decreased in the NTs group, while mean platelet volume increased. The decrease in platelet count may reflect the potential influence of exogenous nucleotides on platelet production or function, suggesting that they may affect platelet count by regulating cell production or metabolic pathways in the blood. Meanwhile, the increase in hematocrit indicates an increase in platelet volume, which may mean that individual platelets are larger or more active. Increased platelet volume is usually closely related to platelet functional activity and may have some impact on blood coagulation. In conclusion, the changes in platelet-related indicators in the NTs group may indicate that nucleotides have a certain regulatory role in the blood system, especially in physiological processes such as immune responses and blood coagulation, and may play a certain biological role.

[0146] 2.4 Effects of nucleotide compositions on metagenomics in older adults

[0147] This study employed high-throughput sequencing technology to analyze the metagenomics of an elderly population and explore the impact of exogenous nucleotides on the gut microbiota. First, gut samples were collected from participants, and total DNA was extracted using standardized DNA extraction methods. Subsequently, metagenomic sequencing was performed on the extracted DNA samples using the DNBSEQ platform to obtain raw sequencing data. All raw data underwent rigorous quality control, with host genome sequences removed using software tools to ensure the accuracy and reliability of the final data. Genome assembly was performed on the processed sequence data. MEGAHIT was used for k-mer assembly to generate contigs, followed by gene prediction using MetaGeneMark to obtain relevant gene information. Based on this, species classification was performed using the Kraken2 tool, sequence alignment was performed, and the abundance of each microorganism in the samples was calculated.

[0148] To comprehensively understand the impact of exogenous nucleotides on the microbial community, we performed diversity analysis on the measured gene abundance, species abundance, and functional abundance tables. By calculating α-diversity (species richness and evenness) and β-diversity (differences in community structure), we assessed the differences in microbial communities between different groups. PCA dimensionality reduction was used to further visualize the differences between samples, helping to reveal the potential impact of nucleotide intervention on microbial community structure. GEE was used to assess the impact of exogenous nucleotide intervention on α-diversity and differential microbial communities. P A value <0.05 is considered statistically significant.

[0149] Table 10. Effects of nucleotide compositions on gut α-microbiota diversity in older adults

[0150]

[0151] T0 represents the baseline time point, and T2 represents the intervention endpoint time point (19 weeks).

[0152] The p-value is based on a generalized estimation equation. The model considers the interaction between group and time, as well as covariates. The assessment time points include baseline and intervention endpoint. Adjusted covariates include baseline value, age, and sex.

[0153] The table only lists α diversity indicators with p-values ​​< 0.05.

[0154] Table 11. Significantly Differential Species Affecting the Gut Microbiota of Elderly Patients with Nucleotide Compositions

[0155]

[0156] T0 represents the baseline time point, and T2 represents the intervention endpoint time point (19 weeks).

[0157] The p-value is based on a generalized estimation equation. The model considers the interaction between group and time, as well as covariates. The assessment time points include baseline and intervention endpoint. Adjusted covariates include baseline value, age, and sex.

[0158] The table only lists bacterial species with a P value < 0.05.

[0159] Regarding alpha diversity, at the scientific level, both the Shannon index and the Simpson index showed significant differences in changes between groups before and after the intervention (T2 vs. T0). Specifically, the mean difference in the Shannon index between groups was -0.25. P = 0.026; the inter-group mean difference of the Simpson index was -0.059. P= 0.019. No statistical significance was observed at other levels of α-diversity indices. Nucleotide intervention reduced the Shannon and Simpson indices, indicating changes in the richness and evenness of the gut microbiota. The decrease in the Shannon index may indicate a reduction in the abundance of certain bacterial groups, leading to some adjustment in the microbiota structure, while the change in the Simpson index may suggest a shift in the proportion of dominant species within the microbiota. This change may be related to nucleotide regulation of host metabolism, the immune system, and competition among gut microbiota. Microbiota diversity is crucial for maintaining gut homeostasis; moderate changes in diversity may reflect optimized microbiota function rather than simple microbiota imbalance, suggesting that nucleotide intervention may promote adaptive structural adjustments in the microbiota, thereby optimizing its ecological stability.

[0160] β-diversity analysis compared changes in the overall structure of the gut microbiota using PCA plots. Figure 2 This figure illustrates the β-diversity analysis results of metagenomic data. Principal component analysis (PCA) was used to visualize the distribution of different sample populations in a two-dimensional space. Each color represents a different sample group (red, green, blue, yellow), and each point represents the position of a sample in the principal component space. The ellipses shown in the figure represent the distribution range of each group, reflecting the variability between samples and the distance between populations. This PCA plot allows for a direct observation of the similarities and differences between different sample groups. The results show no significant difference in microbial composition between the nucleotide intervention group and the control group, indicating that there are no significant changes in the overall microbial community structure between the groups.

[0161] Differential species analysis showed that some species exhibited statistically significant differences before and after the intervention at both the genus and species levels. At the genus level, the mean difference between groups for Klebsiella was -0.035. P = 0.022; at the species level, the mean difference between groups of Klebsiella pneumoniae was -0.028. P= 0.030. This indicates that exogenous nucleotide intervention may affect the abundance of specific gut microbiota species. Furthermore, nucleotide intervention led to a significant decrease in the abundance of Klebsiella pneumoniae, a phenomenon with important biological significance. Klebsiella pneumoniae is an opportunistic pathogen that can become a potential pathogen in cases of gut microbiota imbalance, host immunodeficiency, or antibiotic use, and is associated with antibiotic resistance, chronic inflammation, and metabolic disorders. Its decreased abundance suggests that nucleotides may inhibit the colonization and growth of this bacterium by regulating intestinal barrier function, enhancing host immunity, or affecting metabolic competition among microbiota. Notably, although the abundance of some bacteria changed, nucleotides did not significantly affect the overall microbiota structure, indicating that their role may be local regulation of microbiota balance rather than a comprehensive reshaping of the microbiota ecology. This may help reduce the proportion of potential pathogens while maintaining the functional stability of the normal microbiota.

[0162] In summary, this study found that exogenous nucleotide intervention primarily affected gut microbiota by reducing α-diversity and decreasing the abundance of specific bacterial species (such as Klebsiella pneumoniae), but did not significantly impact the overall microbiota structure. This suggests that nucleotides may exert a potential health-promoting effect by moderately regulating gut microbiota ecology and optimizing microbiota composition without disrupting microbiota homeostasis.

[0163] 2.5 Effect of nucleotide composition on median DNA methylation age in older adults

[0164] This study employed whole-genome methylation sequencing (WGBS) to investigate the methylation status of the genome, and used BGI's proprietary SOAPnuke software (version v1.5.6) for data quality control. Comparing data from the nucleotide intervention group and the placebo control group, the nucleotide group showed a mean difference of β = -3.08 years (95% confidence interval: -5.07 to -1.10, P = 0.0023 for Model 1), indicating a significant reduction in biomarkers of aging. This significant reduction was consistent across all models (P < 0.01 for Model 2 and Model 3). Figure 3 As shown.

[0165] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; modifications or equivalent substitutions may be made to the technical solutions described in the foregoing embodiments, and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of compositions and nutritional supplements containing 5'-monophosphate nucleotides in the preparation of anti-aging pharmaceuticals and / or functional foods, characterized in that, The composition comprises 5'-adenosine monophosphate, 5'-cytidine monophosphate, disodium 5'-guanylate, disodium 5'-uridine monophosphate, and hypoxanthine nucleotide; the mass ratios of the nucleotides in the composition converted to CMP, AMP, UMP, GMP, and IMP acid forms are as follows: CMP: 15-45%, AMP: 15-25%, UMP: 15-30%, GMP: 15-35%, and IMP: 0-2.5%, with the IMP content not being zero; The composition can extend the median survival time of mammals by 9.21-12.6%; the composition can reduce the median DNA methylation age in humans by 3.08 years by regulating the DNA methylation clock. The composition significantly reduces the insulin resistance index, improves the body's ability to metabolize glucose and lipids, and reduces the risk of diabetes. The composition significantly improves the stability of the body's blood system by regulating platelet production or metabolic pathways; The composition protects the stability of the nervous system and regulates the body's metabolic capacity by regulating the metabolism of essential minerals such as lithium (Li) and copper (Cu). The composition regulates the balance of intestinal flora by reducing the abundance of Klebsiella pneumoniae in the intestine, thereby reducing the proportion of potentially pathogenic bacteria and maintaining the functional stability of normal flora. The aforementioned drugs and / or functional foods also contain food or pharmaceutically acceptable excipients; the aforementioned nutritional supplements include one or more of pyrroloquinoline quinone (PQQ), curcumin, resveratrol, and 6-gingerol; The drug and / or functional food contains 600-2400 parts of a composition containing 5'-monophosphate nucleotide, 10-40 parts of PQQ, and 100-400 parts of curcumin; or 600-2400 parts of a composition containing 5'-monophosphate nucleotide, 10-40 parts of PQQ, and 5-40 parts of resveratrol; or 600-2400 parts of a composition containing 5'-monophosphate nucleotide, 10-40 parts of PQQ, and 5-40 parts of 6-gingerol.

2. The application according to claim 1, characterized in that, The dosage forms of the drug include powder, tablets, soft / hard capsules, or oral liquid formulations; The functional foods include powders, tablets, soft / hard capsules, dairy products, baked goods, or liquid beverages.

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