Application of 5 '-monophosphate nucleotide composition in preparation of anti-aging and life-prolonging medicine and / or functional food

Abstract

The invention discloses application of a 5 '-monophosphate nucleotide composition in preparation of anti-aging and life-prolonging medicines and / or functional foods, and belongs to the technical field of medicines. The composition consists of 5 '-adenosine monophosphate, 5'-cytidine monophosphate, disodium 5 '-guanylate, disodium 5'-uridinemonophosphate and hypoxanthine nucleotide, and is converted into CMP, AMP, UMP, GMP and IMP according to the following acid types in percentage by mass: 15 to 78 percent of CMP, 6 to 44 percent of AMP, 7 to 40 percent of UMP, 7 to 51 percent of GMP and 0 or greater than 0 and not greater than 2.5 percent. According to the composition, through the synergistic interaction of various mechanisms of remarkably reducing the insulin resistance index, enhancing the anticoagulation function of a body, improving blood circulation, adjusting metabolism of necessary mineral substances such as lithium and copper, adjusting intestinal flora ecology and the like, aging is delayed, and the healthy life is prolonged; animal experiment data shows that compared with a control group, the mouse median survival time is prolonged by 9.21-12.6%, and the calculated human life is equivalent to 8.76-12.01 years; human body test data shows that compared with a control group, the composition intervention has the advantage that the DNA methylation median is reduced by 3.08 years, and a new thought is provided for developing the anti-aging and life-prolonging medicine and / or functional food.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to the use of a 5'-monophosphate nucleotide composition in the preparation of anti-aging and life-extending drugs and / or functional foods. Background Art

[0002] Human aging is a complex, multifactorial biological process involving multiple levels, including cells, molecules, tissues, and organs. Mechanisms of aging include decreased cellular antioxidant capacity and disrupted cellular metabolism. Extending lifespan cannot be achieved through a single mechanism; instead, significant results require the synergistic effects of multiple pathways, such as enhancing cellular antioxidant capacity, improving cellular metabolism, and activating cellular regeneration.

[0003] Currently, a wide variety of products are available on the market for 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 scavenging free radicals in the body; nutritional supplements, such as coenzyme Q10 and fish oil, maintain cellular function by providing nutrients necessary for metabolism; health products, such as ginseng and ganoderma, enhance physical fitness by regulating the immune and endocrine systems; and some medications, such as metformin and rapamycin, delay aging by targeting specific biological targets. These products can improve certain aging-related indicators to a certain extent, but their effectiveness is limited.

[0004] However, existing products present numerous challenges. First, most have a single mechanism of action, targeting only a specific aging mechanism and failing to comprehensively improve aging. For example, antioxidants only scavenge free radicals. Furthermore, the mechanisms of action of some existing products are unclear and lack systematic research, making their practical effects difficult to predict. Therefore, developing a product with safe and effective ingredients, no toxic side effects, and the ability to effectively delay aging and extend lifespan has become a crucial and pressing issue.

[0005] Nucleic acids, composed of multiple nucleotides, are essential components of cells. They are divided into deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), playing a key role in the storage and transmission of genetic information and protein synthesis. Nucleotides, as the basic building blocks of nucleic acids, are widely distributed throughout the organs, tissues, and cells of organisms, participating in fundamental life processes such as heredity, development, and growth. Nucleotide compounds have important biological functions and participate in nearly all biochemical reactions in organisms, such as cellular energy metabolism, coenzyme synthesis, and cell signaling. Therefore, nucleotides and their derivatives have great potential in delaying aging. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a use of a 5'-monophosphate nucleotide composition in the preparation of anti-aging and life-extending drugs and / or functional foods.

[0007] The object of the present invention is to achieve the following goals:

[0008] The present invention provides the use of a composition of 5'-monophosphate nucleotides or sodium salts thereof in the preparation of anti-aging and life-extending drugs and / or functional foods.

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

[0010] Based on the above technical solution, further, the mass ratios of each nucleotide in the composition converted into CMP, AMP, UMP, GMP, and IMP acid forms are: 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%.

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

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

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

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

[0015] Based on the above technical solution, further, the functional food and / or medicine contains 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 reduces the insulin resistance index, improves the body's glucose and lipid metabolism ability, and reduces the body's diabetes risk.

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

[0018] Based on the above technical solution, further, 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).

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

[0020] Based on the above technical solution, further animal experimental studies have shown that the composition prolongs the median survival time of mice by 9.21 to 12.6% compared with the control group, which is equivalent to a human lifespan of 8.76 to 12.01 years; human experimental studies have shown that the composition reduces the median age of DNA methylation by 3.08 years compared with the control group by regulating the DNA methylation clock.

[0021] The present invention has the following beneficial effects compared to the prior art:

[0022] (1) The present invention uses a specific ratio of adenosine 5'-monophosphate, cytidine 5'-monophosphate, disodium 5'-guanylate, disodium 5'-uridylate and inosine nucleotide composition, combined with pyrroloquinoline quinone (PQQ), curcumin, resveratrol, 6-gingerol and other nutritional supplements 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, thereby playing an anti-aging and life-prolonging 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 ability 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 fluidity by reducing platelet volume and increasing the average platelet volume; at the same time, it can regulate the stability of the blood system by controlling the proportion of monocytes, red blood cells, 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 metabolic capacity of the human body 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 by reducing the abundance of intestinal Klebsiella pneumoniae, reducing the proportion of potential pathogenic bacteria, and maintaining the functional stability of normal flora.

[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 of the human body from a holistic perspective, alleviating damage to the body caused by the external environment, and achieving the purpose of delaying aging and prolonging healthy lifespan. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0029] Figure 1 This is a graph analyzing the cumulative survival rates of mice in each group in Example 10, where 1 represents the purified feed-fed group (NTs-Free); 2 represents the normal control group (Normal control); 3, 4, and 5 represent the NTs low-, medium-, and high-dose intervention groups (NTs-L, NTs-M, and NTs-H), respectively; 6 represents the nicotinamide mononucleotide-fed 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 intestinal flora in Example 11.

[0031] Figure 3 This is the result of the effect of the nucleotide composition in Example 11 on the median DNA methylation age of the elderly. DETAILED DESCRIPTION

[0032] The present invention is described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Obviously, the examples described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative work all fall within the scope of protection of the present invention. The test methods in the following examples, unless otherwise specified, are conventional methods and are carried out according to the test methods or conditions described in the literature in this field or according to the kit instructions. The materials, reagents, instruments, etc. used in the following examples can all be obtained from commercial channels.

[0033] Example 1

[0034] The 5'-monophosphate nucleotide composition used in this example includes adenosine 5'-monophosphate, cytidine 5'-monophosphate, disodium 5'-guanylate, and disodium 5'-uridine. The effective percentages of various nucleotides, 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 are tested separately, and those that meet the requirements are sieved through a 60-mesh sieve for later use; the required amount of each monophosphate nucleotide sample is weighed according to the above ratio, and the total amount is mixed for a mixing time of not less than 40 minutes. The resulting mixed sample is stored at room temperature.

[0036] Example 2

[0037] The 5'-monophosphate nucleotide composition used in this example includes adenosine 5'-monophosphate, cytidine 5'-monophosphate, disodium 5'-guanylate, and disodium 5'-uridine. The effective percentages of various nucleotides, calculated as CMP, AMP, UMP, and GMP, are: CMP 18%, AMP 25%, UMP 24%, and GMP 33%, respectively.

[0038] The above four 5'-monophosphate nucleotides or their sodium salts are tested separately, and those that meet the requirements are sieved through a 60-mesh sieve for later use; the required amount of each monophosphate nucleotide sample is weighed according to the above ratio, and the total amount is mixed for a mixing time of not less than 40 minutes. The resulting mixed sample is stored at room temperature.

[0039] Example 3

[0040] The 5'-monophosphate nucleotide composition used in this example includes adenosine 5'-monophosphate, cytidine 5'-monophosphate, disodium 5'-guanylate, disodium 5'-uridine, and inosine monophosphate. The effective percentages of various nucleotides, calculated as CMP, AMP, UMP, GMP, and IMP, are: CMP 28%, AMP 24%, UMP 28%, GMP 19%, and IMP 1%, respectively.

[0041] The above five 5'-monophosphate nucleotides or their sodium salts are tested separately, and those that meet the requirements are sieved through a 60-mesh sieve for later use; the required amount of each monophosphate nucleotide sample is weighed according to the above ratio, and the total amount is mixed for a mixing time of not less than 40 minutes. The resulting mixed sample is stored at room temperature.

[0042] Example 4

[0043] Compared with Example 1, this embodiment further includes PQQ, curcumin and a filler, specifically as follows:

[0044] The composition used in this example has the following proportions: 5'-monophosphate nucleotide composition prepared in Example 1: 50%, PQQ: 0.7%, curcumin: 8%, and the rest is supplemented with microcrystalline cellulose, corn starch, magnesium stearate, etc.

[0045] After the raw materials are tested and qualified, they are mixed in a HZD-1000 mixer for 30 minutes, and the content of the active ingredients in the fine powder after mixing is determined; the fine powder is granulated and tableted to ensure that the weight of each tablet is between 0.56g±8%.

[0046] Example 5

[0047] Compared with Example 2, this embodiment further includes PQQ, resveratrol and a filler, specifically as follows:

[0048] The composition used in this example has the following proportions: 55% of the 5'-monophosphate nucleotide composition prepared in Example 2, 0.7% of PQQ, and 0.7% of resveratrol, with the remainder being supplemented with microcrystalline cellulose, corn starch, and magnesium stearate.

[0049] After the raw materials are tested and qualified, they are mixed in a HZD-1000 mixer for 30 minutes, and the content of the active ingredients in the fine powder after mixing is determined; the fine powder is granulated and tableted to ensure that the weight of each tablet is between 0.55g±8%.

[0050] Example 6

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

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

[0053] After the raw materials are tested and qualified, they are mixed in a HZD-1000 mixer for 30 minutes, and the content of the active ingredients in the fine powder after mixing is determined; the fine powder is granulated and tableted to ensure that the weight of each tablet is between 0.28g±8%.

[0054] Example 7

[0055] This example examines the antioxidant properties of the compositions prepared in Examples 1-6, and the 5'-monophosphate nucleotide composition prepared in Example 1 is used as an example for illustration.

[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 of 1:1) to prepare a stock solution with a concentration of 1 mg / mL. The DPPH free radical scavenging rate and the ABTS free radical scavenging rate were measured.

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

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

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

[0060]

[0061] Where A blank is the absorbance of 150 μL deionized water ethanol solution and 150 μL DPPH solution, A sample is the absorbance of 150 μL sample and 150 μL DPPH solution; IC 50 It is 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 solution was diluted 100, 50, 25, 10, and 1 times, respectively, to prepare gradient dilution solutions with concentrations of 10 μg / mL, 20 μg / mL, 40 μg / mL, 100 μg / mL, and 500 μg / mL.

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

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

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

[0067]

[0068] Where 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. 50 is the sample concentration when the ABTS free radical scavenging rate reaches 50%.

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

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

[0071]

[0072] The above results show 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, and the antioxidant properties of the 5'-monophosphate nucleotide compositions and the compositions after 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 example investigates the improving effects of the compositions prepared in Examples 1-6 on mitochondrial function, and the following description is made using the 5'-monophosphate nucleotide composition prepared in Example 1 as an example.

[0075] Mitochondria are vital to life and participate in important physiological processes such as ATP production, cell apoptosis, and β-oxidation of fatty acids. Mitochondrial ATP production capacity and mitochondrial membrane potential can reflect the functional status of mitochondria. Mitochondrial ATP production capacity was tested using the 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 supplemented with 1% penicillin / streptomycin and 10% fetal bovine serum in a 5% CO2 incubator at 37°C with saturated humidity. The senescent cell model was prepared by treating with hydrogen peroxide at a concentration of 200 μmol / L for 4 h.

[0077] The specific test process of mitochondrial ATP production capacity is as follows: 2×10 5NIH / 3T3 cells were cultured overnight. After the cells adhered, 200 μmol / L hydrogen peroxide was added for 4 hours. The medium containing hydrogen peroxide was then discarded and a 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 positive control group was treated with normal complete medium without 200 μmol / L hydrogen peroxide, and the model control group was treated with 200 μmol / L hydrogen peroxide and then treated with normal complete medium. The probe plate (XFe96FluxPak, Agilent, 102353-100) was hydrated with hydration solution (XF Calibrant Solution, Agilent, 100840-000) one day in advance and incubated in a 37°C CO2-free incubator for more than 12 h. On the day of the experiment, the experimental culture medium was prepared by adding 1 ml of 2.5 mM glucose (Glucose, Sigma, G7528), 2 mM glutamine (L-glutamine, Sigma, G8540), and 1 mM sodium pyruvate (Sodium pyruvate, Sigma, S8636) to 100 ml of basal culture medium (SeahorseXF Base Medium, Agilent, 102353-100), and adjusting the pH of the solution to 7.4±0.05 with 1N NaOH. The cell culture plate was changed with 40 μL / well of the original culture medium discarded, and 160 μL / well of the test culture medium was added for dilution. 160 μL / well of the test culture medium was then aspirated, and the procedure was repeated 2-3 times, with a final volume of 175 μL per well. The cells were incubated at 37°C for 1 h. The probe plate was dosed with 25 μL / well of drugs, with 2 μM oligomycin (abcam, ab141829) added to well A, 1 μM carbonyl-cyanide-p-trifluoromethoxyphenylhydrazine (FCCP, Sigma, C2920) added to well B, and 1 μM antimycin A (abcam, ab141904) / rotenone (Sigma, R8875) added to well C. The probe plate was calibrated on-board, and the cell culture plate was tested on-board.

[0078] The specific test process of mitochondrial membrane potential is as follows: 2×10 5NIH / 3T3 cells were cultured overnight. After cell attachment, 200 μmol / L hydrogen peroxide was added for 4 hours. The hydrogen peroxide-containing medium was then discarded and replaced with complete medium (DMEM high-glucose medium with 1% penicillin / streptomycin and 10% fetal bovine serum) containing 100 μg / mL of a 5'-monophosphate nucleotide combination. The positive control group was treated with normal complete medium without 200 μmol / L hydrogen peroxide, while the model control group was treated with 200 μmol / L hydrogen peroxide followed by normal complete medium. The cells were then trypsinized, harvested by centrifugation, and incubated with mitochondrial membrane potential assay solution (JC-1) for 2 hours. The cells were then washed with pre-chilled buffer, the supernatant discarded, and the cells were transferred to a new EP tube and fresh pre-chilled buffer added. 100 μL of the cell suspension was transferred to a 96-well plate and the fluorescence intensity of the JC-1 polymer was measured. JC-1 is an ideal fluorescent probe widely used to detect mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix to form polymers, which can produce red fluorescence. The higher the red fluorescence intensity, the higher the mitochondrial membrane potential and the higher the mitochondrial activity.

[0079] The improvement effects of the 5'-monophosphate nucleotide compositions in Examples 2-3 and the compositions in Examples 4-6 on mitochondrial function were detected by referring to the same detection method as above. As control experiment 1, an amount of adenosine 5'-monophosphate equivalent to that of the 5'-monophosphate nucleotide composition of Example 1 was used; as control experiment 2, an amount of cytidine 5'-monophosphate equivalent to that of the 5'-monophosphate nucleotide composition of Example 1 was used; as control experiment 3, an amount of disodium 5'-guanylate equivalent to that of the 5'-monophosphate nucleotide composition of Example 1 was used; as control experiment 4, an amount of disodium 5'-uridylate equivalent to that of the 5'-monophosphate nucleotide composition of Example 1 was used; as control experiment 5, an amount of inosine monophosphate equivalent to that of the 5'-monophosphate nucleotide composition of Example 3 was used; as control experiment 6, a composition containing only PQQ and curcumin in the same amounts and proportions as in the tablet of Example 4 was used; as control experiment 7, a composition containing only PQQ and resveratrol in the same amounts and proportions as in the tablet of Example 5 was used; and as control experiment 8, a composition containing only PQQ and 6-gingerol in the same amounts and proportions as in the tablet of Example 6 was used. The experimental data are expressed as mean ± standard deviation.

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

[0081]

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

[0083] Example 9

[0084] Metabolism generates a large number of oxidative free radicals, which disrupt cellular function, leading to organ aging and, consequently, the aging of the body. Superoxide dismutase (SOD) scavenges harmful superoxide anion free radicals. Glutathione peroxidase (GSH-Px) is an important peroxide-degrading enzyme widely present in the body. It reduces toxic peroxides to non-toxic hydroxyl compounds, thereby protecting the structure and function of cell membranes from peroxide interference and damage. Therefore, SOD and GSH-Px activity can reflect the antioxidant capacity of tissues and are important indicators for assessing aging. MDA is a lipid peroxidation product that is closely related to the generation of free radicals in the body and the degree of lipid peroxidation in tissue cells. It can reflect the oxidative stress damage of tissues and is also an important indicator for assessing aging. This example investigates the effect of the 5'-monophosphate nucleotide compositions of Examples 1-3 and the compositions of Examples 4-6 on delaying aging in SAMP-8 mice (3 months old) prone to rapid aging. The mice were housed in an SPF animal room at an ambient temperature of 22±2°C, a relative humidity of 50%-60%, and a 12h / 12h (8:00-20:00) light pattern to simulate a normal circadian rhythm.The American Institute of Nutrition (AIN-93G) animal feed was used as the basal feed, and the above-mentioned composition was used as the intervention agent. 220 mg / kg BW was administered orally daily for 60 days. The specific process was as follows: 75 aging-accelerated mice (SAMP-8 mice, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), weighing (20±5) g, were randomly divided into 15 groups, with 5 mice in each group, namely, a negative control group (fed only with the basal feed), an experimental group (a group composed of Examples 1-6), a control group 1 (adenosine monophosphate equivalent to the 5'-monophosphate nucleotide composition of Example 1), a control group 2 (cytidine monophosphate equivalent to the 5'-monophosphate nucleotide composition of Example 1), a control group 3 (5'-guanylate disodium equivalent to the 5'-monophosphate nucleotide composition of Example 1), a control group 4 (5'-monophosphate nucleotide equivalent to the 5'-monophosphate nucleotide composition of Example 1), and a control group 5 (5'-monophosphate nucleotide equivalent to the 5'-monophosphate nucleotide composition of Example 1). The control group 7 was composed of the following: control group 7 (composition of PQQ and resveratrol in the same amount and the same ratio as the tablets in Example 5); control group 8 (composition of PQQ and 6-gingerol in the same amount and the same ratio as the tablets in Example 6); and control group 5 (inosine nucleotide in the same amount as the 5'-monophosphate nucleotide composition in Example 3). The control group 6 (composition of PQQ and curcumin in the same amount and the same ratio as the tablets in Example 4) was used as control experiment 7. The control group 8 (composition of PQQ and 6-gingerol in the same amount and the same ratio as the tablets in Example 6) was used as control experiment 7. On the second day after the last feeding, blood was collected from the fundus of each group of mice. The blood was allowed to stand at room temperature until it was completely coagulated, and then centrifuged to separate the serum for later use. The mice were then killed by dislocation, and the livers were quickly separated. After rinsing with sodium chloride solution, a 10% tissue homogenate was prepared, which was centrifuged and the separated tissue homogenate supernatant was set aside. The SOD and GSH-Px activities in mouse serum and liver tissue were detected according to the instructions of the superoxide dismutase (SOD) detection kit and the glutathione peroxidase (GSH-Px) detection kit, respectively. The malondialdehyde (MDA) content of lipid peroxide in mouse serum and liver tissue was determined by thiobarbituric acid spectrometry. The experimental data are expressed as mean ± standard deviation.

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

[0086]

[0087]

[0088] From the above results, it can be seen that compared with the negative control group, the SOD activity and GSH-Px activity in the serum and liver tissue of the mice in the control groups 1-8 were increased, and the MDA content in the serum and liver tissue of the mice was reduced; the various indicators of the composition test groups of Examples 1-6 were significantly better than those of the control groups 1-8. The results show that the 5'-monophosphate nucleotide composition of the present invention and the composition obtained by compounding the 5'-monophosphate nucleotide composition with functional active ingredients can synergistically enhance the body's ability to scavenge free radicals, reduce the degree of tissue cell damage, and delay the body's aging.

[0089] Example 10

[0090] This example investigates the effect of lifelong intervention with the 5'-monophosphate nucleotide composition prepared in Example 1 on the survival time of SAMP8 mice.

[0091] 1. Sample: the 5'-monophosphate nucleotide composition obtained in Example 1 above.

[0092] 2. Experimental Animals: 350 healthy, three-month-old, specific pathogen-free (SPF) male senescence-accelerated model mice (SAMP-8) and model control mice (SAMR1) 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, animals were housed in individual cages. The temperature in the animal room was maintained at 22 ± 2°C, the relative humidity was maintained at 50%–60%, and the light-dark cycle was 12 h:12 h.

[0093] 3. Experimental Grouping and Dosage: After one week of adaptive feeding, SAMP8 mice were randomly divided into 6 groups (n=50) according to body weight: a nucleotide-free group (purified feed, NTs-Free), a normal control group (basal feed, Normalcontrol), low-, medium-, and high-dose intervention groups of the composition described in Example 1 (0.3g / kg, 0.6g / kg, 1.2g / kg, corresponding to NTs-L, NTs-M, and NTs-H, respectively), and a nicotinamide mononucleotide feeding group (NMN, 0.3g / kg). A SAMR1 model control group (basal feed, n=50) was also established. Nucleotide mixtures and nicotinamide mononucleotide were incorporated into the basal feed at different doses for intervention. Starting from the age of three months, each group was given the corresponding feed for lifelong feeding intervention. During the experiment, the animals had free access to water and food. The intervention and grouping of the experimental animals are shown in Table 4.

[0094] Table 4. Experimental animal groups and interventions

[0095]

[0096] 4. Experimental methods:

[0097] During the experiment, mice were housed in single cages, and no other factors interfered with the experiment except for routine operations performed by researchers (cleaning the cages and observing the general situation). The time when the mice were found to have died naturally or were euthanized (that is, the mice were observed to be in extremely poor condition and it was judged that the probability of survival within 48 hours was very small) was recorded in detail for lifespan analysis. Euthanasia was performed for humane reasons, and the euthanized mice were considered to have completed their life cycle and could be used for lifespan analysis. The implementation standards for euthanasia were only based on the consideration of the animal's condition, reference to AAALAC guidelines, relevant literature reading and the team's previous experience in lifelong feeding experiments. The main judgment indicators include: severe lethargy, rapid weight loss (>20% for more than two weeks), severe abdominal distension and body condition score accompanied by signs of pain (judged by combining common mouse pain symptoms such as facial expressions, appearance, abnormal behavior, etc.), no response to stimulation or external behavior, severe ulcers or bleeding tumors, severe hypothermia with abnormal respiratory rate, etc. After the experiment, the survival time of each group of mice was summarized and analyzed. Kaplan-Meier survival analysis was performed using SPSS24.0 software, and Log Rank (Mantel-Cox) was used for inter-group comparison. P < 0.05 was the statistical test cutoff value.

[0098] 5. Experimental Results

[0099] 5.1. Effect of lifelong intervention with the composition described in Example 1 on the survival of SAMP8 mice

[0100] As shown in Table 5, mice in each group began receiving intervention at 3 months of age. Deaths began to occur after 6 months of age. The NMN group first experienced two deaths at 7 months of age, with all other groups experiencing deaths by 8 months. After 12 months of age, the mortality rate in all groups accelerated significantly, 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. By 20 months of age, no mice were left in the NTs-Free, Normal, and NTs-H groups. By 23 months of age, only the NTs-L and NTs-M groups (two each) remained alive. The NTs-L group had the longest survival, at 24.93 months, approaching 25 months of age; the NTs-M group was second, at 23.83 months.

[0101] Table 5. Survival of mice in each group from 3 to 25 months of age

[0102]

[0103]

[0104] 5.2. Effect of lifelong intervention with the composition described in Example 1 on the survival time of SAMP8 mice

[0105] As shown in Table 6, the mean survival time (14.98m) and median survival time (14.30m) of the NTs-L group were higher than those of the other groups, followed by the NTs-M group (mean survival time 14.65m, median survival time 13.87m). Analysis of median survival time showed that the median survival time of the NTs-L group (14.30m) was extended by 12.6%, 5.93%, and 5.38% compared with the NTs-Free group (12.70m), the Normal control group (13.50m), and the NMN group (13.57m), respectively. The median survival time of the NTs-M group (13.87m) was extended by 9.21%, 2.74%, and 2.21% compared with the NTs-Free group (12.70m), the Normal control group (13.50m), and the NMN group (13.57m), respectively.

[0106] Table 6. Effect of lifelong NTs intervention on the survival time of mice in each group

[0107]

[0108] Depend on Figure 1 The cumulative survival rates of the mice were found to be the first to die (corresponding to an age of 6.37 months), and their cumulative survival rates were lower than those of the other groups until 10 months of age. At a cumulative survival rate of 50%, the NMN group corresponded to 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, and 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 those of the other groups. At subsequent ages, the cumulative survival rates of the NTs-L and NTs-M groups were consistently higher than those of the other groups, with the NTs-L group surviving the longest (24.93 months), followed by the NTs-M group (23.83 months). The inter-group comparison using Log Rank (Mantel-Cox) showed that the NTs-L group was better than the NTs-Free group: 2 =5.26, P=0.022; NTs-M and NTs-Free group comparison: χ 2 =5.40, P=0.020, both were statistically significant (P<0.05). The test results of NTs-L group were compared with Normal control group and MNM group (χ 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 (χ 2 =3.07, P = 0.536), and NTs-M group (χ 2 =2.62, P=0.105) showed no statistical difference (P>0.05), the cumulative survival rates of the two groups were significantly higher than those of the other groups (P<0.01).

[0109] The above research results show that long-term intervention with the composition described in Example 1 can effectively reduce the mortality rate of mice, prolong their survival time, and increase their lifespan. The NTs-L group had the longest average lifespan (14.98m), median survival time (14.3m), and maximum survival time (24.93m). Their median survival time was extended by 12.6%, 5.93%, and 5.38% compared to the NTs-Free, Normal control, and NMN groups in this study, respectively.

[0110] In summary, the experimental results demonstrate that long-term intervention with the composition described in Example 1 effectively reduces the mortality rate, prolongs survival, and improves lifespan in mice, significantly enhancing their quality of life. Lifelong intervention with the composition in SAMP8 mice extended median survival by 9.21-12.6% compared to the control group, equivalent to an estimated human lifespan of 8.76-12.01 years.

[0111] Example 11

[0112] This example adopts a randomized, double-blind trial design to evaluate the effect of the 5'-monophosphate nucleotide composition prepared in Example 1 on the elderly population.

[0113] 1. Test methods

[0114] 1.1. Trial Design and Participants

[0115] This trial was an exploratory, 19-week, single-center, double-blind, randomized, placebo-controlled study. Community-dwelling individuals were recruited through advertisements and underwent a comprehensive health assessment, including a clinical health examination, questionnaires, physical function assessment, and anthropometrics. Eligibility was determined according to the following criteria. Inclusion criteria: (1) aged 60-70 years; (2) no serious physical or mental illness; (3) no previous use of nucleotide-related supplements or health foods; (4) able to follow the study protocol and provide informed consent. Exclusion criteria: (1) confirmed diseases such as autoimmune diseases, serious cardiovascular and cerebrovascular diseases, complications of important organs such as liver and kidney, or other serious diseases such as malignant tumors, pancreatic diseases, and mental illness; (2) abnormal screening laboratory test values or other laboratory test results that excluded the researcher from participating in the study at the researcher's discretion; (3) severe visual or hearing impairment that affected 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 before recruitment into the study.

[0116] 1.2. Sample size and randomization

[0117] The sample size for this trial was determined based on the "Technical Specifications for Inspection and Evaluation of Health Foods" issued by the National Health Commission of China, which recommends a minimum of 50 participants per group for human intervention trials. Participants were randomly assigned to the intervention and control groups in a 1:1 ratio. To estimate a 20% loss to follow-up rate, a total of 120 participants were included in the study.

[0118] Participants were randomly assigned to the groups using computer-generated random numbers, and the protocol was known only to the trial designers, who had no involvement in data collection or analysis. The randomization code was sealed and was only unblinded in the event of a serious adverse event.

[0119] Intervention and Blinding

[0120] 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 a starch excipient) to avoid any suspicion among participants. The design of the placebo capsules was similar to that of 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. Subjects were instructed to take four capsules daily while maintaining a regular lifestyle and dietary habits. The double-blind design ensured that participants and research staff—including investigators, clinicians, data collectors, and follow-up coordinators—were unaware of group assignment. The capsules were packaged in opaque bottles labeled only with the participant identifier. The blinding procedures were explained in detail to the subjects during the informed consent process.

[0121] 1.4. Participation in the trial

[0122] After screening, 122 subjects were finally identified as eligible for participation. The intervention lasted for 19 weeks. During the follow-up period, except for one participant in the nucleotide group who withdrew due to a traffic accident one week after the intervention, the remaining 121 participants completed the trial. A total of 121 participants who completed the intervention were finally included in the statistical analysis. There was no significant difference between the two groups in any baseline characteristics. The average age of the participants was 65.65 (standard deviation 2.59) years, of which 82 (67.21%) were female.

[0123] Data Collection

[0124] In the trial, data collection at baseline (T0), mid-term (T1) and final (T2) was completed by clinical examination and biological sample (blood, urine and feces) collection in the physical examination hospital.

[0125] DNA methylation data were obtained by whole-genome bisulfite sequencing (WGBS), and the sequencing work was outsourced to BGI (Shenzhen, China). SOAPnuke and Bismark software were used for data filtering, alignment, and methylation site mapping.

[0126] Insulin resistance was calculated with the homeostasis model assessment-IR (HOMA-IR), a widely used surrogate marker, using the formula: (fasting plasma glucose [mmol / L]*fasting insulin [mIU / L]) / 22.5.

[0127] Statistical analysis

[0128] Data analysis in this trial was based on the intention-to-treat (ITT) principle, and no interpolation was performed for missing data in the baseline data. Data normality was assessed using the Shapiro-Wilk test, and homogeneity of variance was assessed using the Levene test. Pre-intervention, post-intervention, and between-group comparisons of change were performed using the independent-samples T test (for normally distributed data) or the Mann-Whitney U test (for nonnormally distributed data), depending on the distribution of the variables. Effect sizes were calculated using the Cohen's d value, which is the difference between the two group means. d = 0.2 is considered a small effect size, d = 0.5 is considered a medium effect size, and d = 0.8 is considered a large effect size.

[0129] We analyzed all outcome measures using generalized estimating equations (GEEs), specifying an exchangeable working correlation structure. All baseline and endpoint data were used. Model 1 included independent variables including group, time, the interaction term between group and time, the baseline value of the dependent variable (a continuous variable), age, and sex. We also constructed Model 2, which additionally adjusted for dietary nucleotide intake. Similarly, Model 3 further adjusted for dietary purine intake. The intervention effect was determined using the coefficient of the interaction term in the model and its 95% confidence interval (CI).

[0130] 2. Test results

[0131] 2.1 Effects of nucleotide composition on minerals in the elderly

[0132] This study used t-tests to analyze the effects of exogenous nucleotide intervention on various mineral levels in the elderly. The results are shown in Table 7, which showed that at the intervention endpoint (T2), there were significant differences between the intervention group and the control group.

[0133] Table 7. Effects of nucleotide composition on body minerals

[0134]

[0135] Independent sample t-test results showed that lithium (Li) concentrations were significantly lower in the nucleotide intervention group (1.57±0.37 ng / ml) than in the control group (1.74±0.52 ng / ml) at the intervention endpoint (T2), P=0.033, Cohen's d=-0.39. Furthermore, copper (Cu) concentrations at the intervention endpoint (1012.67±114.74 ng / ml) were significantly higher than in the control group (969.44±109.94 ng / ml), P=0.039, Cohen's d=0.38.

[0136] The results of this study showed that exogenous nucleotide intervention significantly affected the levels of some minerals, particularly at the intervention endpoint (T2), where the difference between the intervention and control groups was more pronounced, suggesting that nucleotides may play a role in mineral metabolism. Regarding lithium (Li), the nucleotide intervention group had significantly lower Li concentrations at the intervention endpoint (T2) than the control group. Lithium, a trace element involved in neuromodulation, plays an important role in mood stability, neuronal signaling, and neuroprotection. Decreased Li concentrations may indicate that nucleotide intervention affected Li absorption, distribution, or excretion. Given that lithium is primarily excreted through the kidneys, nucleotides may affect its renal tubular reabsorption or excretion mechanisms. Regarding copper (Cu), the nucleotide intervention group had significantly higher Cu concentrations at the intervention endpoint (T2) than the control group. Copper is an essential trace element that plays a role in iron metabolism, antioxidant defense, immune function, and neurotransmission. Increased Cu concentrations may indicate that nucleotide intervention promoted Cu absorption or affected Cu regulation in the body. Previous studies have shown that nucleotides may indirectly promote Cu metabolism and utilization by affecting the expression of metal transporters or modulating oxidative stress.

[0137] 2.2 Effects of nucleotide composition on glucose and lipid metabolism in the elderly

[0138] 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).

[0139] Table 8. Test results of glucose and lipid metabolism indicators of subjects

[0140]

[0141] The results showed 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), with a statistically significant difference (P=0.02), and the effect size between the two groups was medium (d=-0.43). From baseline to the end of the intervention, 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), with a statistically significant difference (P=0.03), and the effect size between the two groups was medium (d=-0.41).

[0142] These results suggest that exogenous nucleotide supplementation can significantly improve glucose and lipid metabolism in the elderly, particularly in terms of the insulin resistance index. Exogenous nucleotide supplementation is associated with a significant reduction in the insulin resistance index, a change that may help maintain blood glucose homeostasis and reduce the risk of diabetes in the elderly.

[0143] 2.3 Effects of nucleotide composition on blood routine in the elderly

[0144] This part of the study aimed to comprehensively evaluate the effects of exogenous nucleotides on routine blood counts in the elderly, focusing on analyzing multiple hematological parameters to explore their potential impact on the immune system and overall health of the elderly. The study included routine hematological parameters such as white blood cell count, platelet count, and hemoglobin, as well as immune cell-related parameters such as the percentage and absolute values of lymphocytes, neutrophils, monocytes, eosinophils, and basophils.

[0145] Table 9. Test results of glucose and lipid metabolism indicators of subjects

[0146]

[0147]

[0148] The above results show that at baseline, there were no significant differences in the various blood routine indicators between the two groups of elderly people (P>0.05), indicating that the baseline blood routine conditions of the two groups were comparable. The inter-group difference analysis of the changes before and after the intervention (T2-T0) showed that the platelet counts of the two groups changed in opposite directions. The platelet counts of the NTs group decreased while those of the C group increased (NTs: -11.60±30.76, C: 6.87±51.56), and the difference was statistically significant (P=0.019). The effect size between the groups was medium (d=0.43); the platelet count of both groups increased, and the change in the NTs group was less than that of the C group (NTs: 0.01±0.03, C: 0.02±0.03), and the difference was statistically significant (P=0.037). The effect size between the groups was medium (d=0.39); the percentage of basophils in both groups increased. The basophil percentage decreased in the NTs group (-0.01±0.12, -0.05±0.11), with a near-significant difference (P=0.069), and a small effect size between the groups (d=0.34). The absolute basophil count decreased in both groups, with a greater change in the NTs group (0.00±0.01, -0.00±0.01, -0.00±0.01), with a statistically significant difference (P=0.037), and a small effect size between the groups (d=0.39). Overall, nucleotide intervention induced some changes in routine blood tests, particularly in platelet-related parameters. Specifically, platelet count and platelet hematocrit decreased in the NTs group, while mean platelet volume increased. The decrease in platelet count may reflect the potential effects of exogenous nucleotides on platelet production or function, suggesting that they may affect platelet number by regulating cell production or metabolic pathways in the blood. At the same time, the increase in platelet volume indicates an increase in platelet volume, which may mean that individual platelets have become larger or more active. The increase in platelet volume is generally closely related to the functional activity of platelets and may have a certain impact on the blood coagulation process. In summary, the changes in platelet-related indicators in the NTs group may indicate that nucleotides have a certain regulatory effect on the blood system, especially in physiological processes such as immune response and blood coagulation, and may play a certain biological role.

[0149] 2.4 Effects of nucleotide composition on the metagenome of elderly people

[0150] This study used high-throughput sequencing technology to analyze the metagenomics of an elderly population to explore the effects of exogenous nucleotides on the gut microbiome. First, intestinal samples were collected from participants, and total DNA was extracted using standardized DNA extraction methods. Subsequently, the extracted DNA samples were subjected to metagenomic sequencing using the DNBSEQ platform to obtain raw sequencing data. All raw data underwent rigorous quality control, and software tools were used to remove host genome sequences to ensure the accuracy and reliability of the final data. The processed sequence data were assembled, and k-mer assembly and contig generation were performed using MEGAHIT. Gene prediction was then performed using MetaGeneMark to obtain information on relevant genes. On this basis, species classification was completed using the Kraken2 tool, and sequences were aligned to further calculate the abundance of each microorganism in the sample.

[0151] To fully understand the effects of exogenous nucleotides on the microbial community, we performed diversity analysis on the measured gene abundance tables, species abundance tables, and functional abundance tables. We assessed the differences in microbial communities between different groups by calculating α diversity (species richness and evenness) and β diversity (differences in community structure). PCA dimensionality reduction was used to further visualize the differences between samples, helping to reveal the potential effects of nucleotide intervention on microbial community structure. GEE was used to assess the effects of exogenous nucleotide intervention on α diversity and differential microbial communities. Differences were considered statistically significant when P < 0.05 in GEE analysis.

[0152] Table 10. Effects of nucleotide composition on the diversity of intestinal α-microorganisms in the elderly

[0153]

[0154] *T0 represents the baseline time point, and T2 represents the end point of the intervention (19 weeks).

[0155] *P values are based on generalized estimating equations that account for group-by-time interactions and covariates, with assessments conducted at baseline and the end point of the intervention. Adjusted covariates included baseline value, age, and sex.

[0156] *Only alpha diversity indicators with P values < 0.05 are listed in the table.

[0157] Table 11. Nucleotide composition significantly affects the intestinal flora of the elderly

[0158]

[0159] *T0 represents the baseline time point, and T2 represents the end point of the intervention (19 weeks).

[0160] *P values are based on generalized estimating equations that account for group-by-time interactions and covariates, with assessments conducted at baseline and the end point of the intervention. Adjusted covariates included baseline value, age, and sex.

[0161] *Only bacterial species with P values < 0.05 are listed in the table.

[0162] Regarding alpha diversity, at the family level, significant differences were observed in the Shannon and Simpson indices before and after intervention (T2 vs. T0). The mean difference between the groups for the Shannon index was -0.25 (P = 0.026), and the mean difference for the Simpson index was -0.059 (P = 0.019). Alpha diversity indices at other levels did not show statistical significance. Nucleotide intervention reduced both the Shannon and Simpson indices, indicating changes in the richness and uniformity of the microbiome. A decrease in the Shannon index may indicate a decrease in the abundance of certain bacterial groups, leading to some adjustments in microbial structure, while a change in the Simpson index may indicate a shift in the proportion of dominant species within the microbiome. These changes may be related to nucleotide regulation of host metabolism, the immune system, and competition among intestinal microbiota. Microbial diversity is crucial for maintaining intestinal homeostasis. Moderate changes in diversity may reflect optimized microbial function rather than simply imbalance, suggesting that nucleotide intervention may promote structural adaptation of the microbiome, thereby optimizing its ecological stability.

[0163] β diversity analysis compared the overall structural changes of intestinal flora through PCA diagram ( Figure 2 ), showing the results of a beta diversity analysis of metagenomic data. Principal component analysis (PCA) visualizes the distribution of different sample groups in 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 of data, reflecting the variability between samples and the distance between groups. This PCA plot allows for intuitive observation of the similarities and differences between different sample groups. The results show that there were no significant differences in microbial composition between the nucleotide intervention group and the control group, indicating that the overall microbial structure did not vary significantly between the groups.

[0164] Analysis of differentially expressed species revealed statistically significant differences in some species before and after intervention at both the genus and species levels. At the genus level, the mean difference between the groups for Klebsiella was -0.035 (P = 0.022); at the species level, the mean difference between the groups for Klebsiella pneumoniae was -0.028 (P = 0.030). This suggests that exogenous nucleotide intervention may affect the abundance of specific intestinal bacterial species. Furthermore, nucleotide intervention led to a significant decrease in the abundance of Klebsiella pneumoniae, a phenomenon of important biological significance. Klebsiella pneumoniae is an opportunistic pathogen that can become a potential pathogen in the context of intestinal microbial imbalance, host immunity, or antibiotic use. It is associated with antibiotic resistance, chronic inflammation, and metabolic disorders. The decrease in its abundance suggests that nucleotides may inhibit the colonization and growth of this bacterium by modulating intestinal barrier function, enhancing host immunity, or affecting metabolic competition among microbiota. It is worth noting that although the abundance of certain bacteria changed, nucleotides did not have a significant effect on the overall bacterial community structure, indicating that their role may be to locally regulate the balance of the bacterial community rather than comprehensively reshape the bacterial community ecology, which may help reduce the proportion of potential pathogens while maintaining the functional stability of the normal bacterial community.

[0165] In summary, this study found that the effects of exogenous nucleotide intervention on the intestinal microbiota were primarily reflected in reduced α-diversity and a decrease in the abundance of specific bacterial species (such as Klebsiella pneumoniae), but did not significantly affect the overall microbial structure. This suggests that nucleotides may play a potential health-promoting role by moderately regulating the intestinal microbial ecology and optimizing microbial composition without disrupting microbial homeostasis.

[0166] 2.5 Effect of nucleotide composition on median DNA methylation age in the elderly

[0167] This trial used whole genome methylation sequencing (WGBS) technology to explore the methylation status of the genome, and used SOAPnuke software (version v1.5.6) independently developed by BGI for data quality control. After comparing the data of 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 biological aging markers. This significant reduction was consistent across all models (P < 0.01 for models 2 and 3). The results are as follows Figure 3 shown.

[0168] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. The technical solutions described in the above embodiments may be modified, or some or all of the technical features therein may be replaced by equivalents. However, these modifications or replacements 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. Use of a composition of 5'-monophosphate nucleotides or their sodium salts in the preparation of anti-aging and life-extending drugs and / or functional foods, characterized in that: The composition consists of 5'-adenosine monophosphate, 5'-cytidine monophosphate, 5'-guanylate disodium, 5'-uridylate disodium and inosine nucleotide; the mass ratios of the nucleotides in the composition converted into acid forms of CMP, AMP, UMP, GMP and IMP are respectively: 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%.

2. The use according to claim 1, characterized in that The mass ratios of various nucleotides in the composition converted into acid forms of CMP, AMP, UMP, GMP and IMP are as follows: CMP: 15-45%, AMP: 15-25%, UMP: 15-30%, GMP: 15-35%, and IMP is 0, or greater than 0 and not higher than 2.5%.

3. The use according to claim 1, characterized in that The dosage forms of the drug include powder, tablet, soft / hard capsule and oral liquid preparation; The functional foods include powders, tablets, soft / hard capsules, dairy products, baked products and liquid beverages.

4. The use according to claim 1, characterized in that The functional food and / or medicine further comprises one or more nutritional supplements and excipients acceptable to food or medicine; the nutritional supplements include pyrroloquinoline quinone (PQQ), curcumin, resveratrol, and 6-gingerol.

5. The use according to claim 4, characterized in that The functional food and / or medicine contains 600-2400 parts of a composition in the form of 5'-monophosphate nucleotides or their sodium salts, 10-40 parts of PQQ, and 100-400 parts of curcumin; or 600-2400 parts of a composition in the form of 5'-monophosphate nucleotides or their sodium salts, 10-40 parts of PQQ, and 5-40 parts of resveratrol; or 600-2400 parts of a composition in the form of 5'-monophosphate nucleotides or their sodium salts, 10-40 parts of PQQ, and 5-40 parts of 6-gingerol.

6. The use according to claim 1, characterized in that The composition significantly reduces the insulin resistance index, improves the body's glucose and lipid metabolism ability, and reduces the body's diabetes risk.

7. The use according to claim 1, characterized in that The composition significantly improves the stability of the body's blood system by regulating the production or metabolic pathways of blood cells such as platelets.

8. The use according to claim 1, characterized in that The composition protects the stability of the nervous system and regulates the metabolic capacity of the body by regulating the metabolism of essential minerals such as lithium (Li) and copper (Cu).

9. The use according to claim 1, characterized in that The composition regulates the balance of intestinal flora by reducing the abundance of intestinal Klebsiella pneumoniae, reduces the proportion of potential pathogenic bacteria, and maintains the functional stability of normal flora.

10. The use according to claim 1, characterized in that Animal experiments showed that the composition prolonged the median survival time of mice by 9.21 to 12.6% compared with the control group, which is equivalent to a human lifespan of 8.76 to 12.01 years. Human experiments showed that the composition reduced the median age of DNA methylation by 3.08 years compared with the control group by regulating the DNA methylation clock.

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