Use of nucleotide mixtures in the preparation of formulations for the prevention or remission of age-related sarcopenia

Abstract

The present invention discloses the application of a nucleotide mixture in the preparation of a preparation for preventing or alleviating age-related sarcopenia, which belongs to the technical field of medicine and health. The nucleotide mixture is composed of four or five kinds of 5'-mononucleotides or their sodium salts, among which the mass ratios of CMP, AMP, UMP, GMP and IMP after converting each nucleotide into a molecular weight are CMP 23-78%, AMP 6-44%, UMP 7-40%, GMP 7-51%, and IMP 0 or 0 to 2.5%. Nucleotides have small molecular weights, are absorbed relatively quickly by the human body, and have high bioavailability. The present invention has found that the intake of nucleotides can reduce the level of oxidative stress and improve mitochondrial dysfunction, thereby improving muscle function and increasing muscle mass in the elderly human body, and playing a role in the prevention and treatment of sarcopenia. The present invention provides a new means for the preparation of drugs or functional foods for the prevention and treatment of sarcopenia.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical and health care, relates to nucleotide products and their new uses, and particularly relates to the use of a nucleotide mixture generated by enzymatic degradation of ribonucleic acid as a raw material in the preparation of a formulation for the prevention or alleviation of age-related sarcopenia.

Background Art

[0002] In modern society, with the significant decline in the birth rate, the extension of the average life expectancy leads to the rapid aging of the world's population. According to the data released by the National Bureau of Statistics of China, at the end of 2019, the number of elderly people aged 60 or above in China reached 254 million, accounting for 18.1% of the total population, and the number of elderly people aged 65 or above reached 176 million, accounting for 12.6% of the total population. According to the prediction of a certain report, China will shift from an aging society to an aged society around 2022, and the population aged 65 or above will account for more than 14% of the total population.

[0003] The aging of the population poses a major challenge to public health in China and even the world. As China's aging progresses, sarcopenia has attracted more attention as a syndrome closely related to the elderly. Sarcopenia is a clinical syndrome that shows a decrease in skeletal muscle mass and muscle strength related to aging, and / or the loss of physical function associated with them. Sarcopenia has become a global health problem. The muscle mass decreases by 1-2% per year in people aged 50 or above, the muscle strength decreases by 1.5% per year from 50 to 60 years old, and decreases by 3% in the elderly aged 60 or above. With the increase in age, the incidence of sarcopenia increases rapidly, and the morbidity rate of sarcopenia in the elderly population in China is relatively high. According to previous studies, the morbidity rate of sarcopenia in the population aged 60 or above in China reaches 18.6%, and the morbidity rate of sarcopenia in the population aged 80 or above reaches 52%.

[0004] Sarcopenia increases the risk of falls and fractures and reduces the ability to perform daily living activities in older adults, and is associated with heart disease, respiratory disease, and cognitive impairment in older adults. Sarcopenia can lead to motor dysfunction, a decline in quality of life, loss of independence or the need for long-term care, and an increased risk of death. Sarcopenia increases the risk of hospitalization and increases the cost of care and hospitalization during hospitalization. One study found that older adults hospitalized with sarcopenia cost five times more than older adults hospitalized without sarcopenia. In 2000, the direct medical cost of sarcopenia in the United States was estimated at approximately $18.5 billion annually, accounting for 1.5% of total direct medical costs. One study found that a 10% reduction in the prevalence of sarcopenia in the United States could save $1.1 billion in medical costs annually. In China, with the arrival of an aging society, sarcopenia will become a significant economic and social burden on individuals and society if left untreated.

[0005] Sarcopenia has a high prevalence, poses significant health risks, and imposes a substantial economic burden on individuals and society, yet there is no specific cure for it. Nutritional interventions and physical exercise are currently recommended as means of preventing and treating sarcopenia. Exercise interventions are effective therapeutic measures that can improve muscle mass and strength, increase muscle protein synthesis rates, strengthen mitochondrial function, and increase the content of skeletal muscle satellite cells. However, resistance to strength training can occur, potentially weakening its effect on promoting skeletal protein synthesis. Moreover, exercise can sometimes be difficult to achieve for older adults with reduced physical function. Some studies suggest that sarcopenia is closely linked to nutritional deficiencies, making nutritional interventions an important means of treating sarcopenia. The components of nutritional compositions are more natural than drugs and have fewer side effects. Therefore, it is clearly important to explore nutritional compositions that can effectively prevent and alleviate sarcopenia based on a better understanding of the underlying mechanisms of this disease.

[0006] Nucleotides, composed of a pentose sugar, a base, and a phosphate group, are the basic building blocks of nucleic acids. Nucleotides originate from endogenous synthesis in the human body and exogenous synthesis through enzymatic degradation techniques. Recent research suggests that exogenous nucleotides are essential nutrients under certain physiological conditions. These nucleotides may be absorbed and utilized by tissues in actively metabolizing tissues and organs, or under conditions of rapid growth, such as stress, immune antigen administration, liver damage, and starvation. This can optimize tissue function by reducing the depletion of de novo or salvage synthesis within the body. Furthermore, enzymatic degradation of nucleic acids into nucleotides outside the body eliminates the need for in-vivo degradation, facilitating digestion and absorption. Currently, there are no reported roles for the prevention or remission of sarcopenia in exogenous nucleotides obtained through enzymatic degradation techniques. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] This invention, through research, has discovered that ingesting nucleotides improves mitochondrial dysfunction and reduces oxidative stress, thereby improving skeletal muscle function and increasing muscle mass in aged SAMP8 mice. Currently, non-protein nutritional supplements such as nucleotides are not available on the market for the treatment of sarcopenia. Innovative research and development for these products could provide new products for the prevention and mitigation of sarcopenia in middle-aged and elderly individuals. If this technology is applied to the medical industry, it can fully meet the health and medical needs of sarcopenia patients.

[0008] The present invention aims to provide a novel method for the prevention and treatment of sarcopenia via nutritional supplements by offering the use of a low-molecular-weight, rapidly absorbable nucleotide mixture in the preparation of formulations for the prevention and remission of sarcopenia, and to establish a stable, effective, and reproducible experimental animal modeling method. [Means for solving the problem]

[0009] To achieve the above objective, the present invention employs the following technical means. One aspect of the present invention relates to the use of a nucleotide mixture in the preparation of a formulation for the prevention or remission of age-related sarcopenia, wherein the nucleotide mixture is a 5'-mononucleotide mixture.

[0010] The nucleotide mixture consists of four or five types of 5'-mononucleotides or their sodium salts, and the mass ratios of CMP, AMP, UMP, GMP, and IMP after molecular weighting of each nucleotide are 23-78% for CMP, 6-44% for AMP, 7-40% for UMP, 7-51% for GMP, and 0 or 2.5% or more for IMP.

[0011] Furthermore, the mass ratios of CMP, AMP, UMP, GMP, and IMP in the nucleotide mixture after converting each nucleotide to a molecular weight are 43% for CMP, 17% for AMP, 21% for UMP, 18% for GMP, and 1% for IMP.

[0012] Furthermore, the formulation may be a powder, a tablet, a soft sac, a hard sac, a beverage, or an oral liquid.

[0013] Furthermore, the 5'-mononucleotide mixture can improve skeletal muscle function, increase muscle mass, and slow the onset and progression of sarcopenia in older adults by improving mitochondrial dysfunction and reducing oxidative stress. [Effects of the Invention]

[0014] In this invention, we have discovered that an existing substance, namely a mixture of 5'-mononucleotides, has the function of preventing and delaying sarcopenia in aged SAMP8 mice, and further discovered that it has new applications in the preparation of drugs or functional foods for the prevention and treatment of sarcopenia and other diseases. Animal experiments have verified that nucleotide intake can lower the level of oxidative stress in the mouse body, improve mitochondrial dysfunction, enhance muscle function in aged mice, and increase muscle mass, and we declare that the nucleotide mixture has remarkable efficacy in the prevention and treatment of sarcopenia. [Brief explanation of the drawing]

[0015] [Figure 1] This figure shows the effect of a nucleotide mixture on SOD in the muscle tissue of aged SAMP8 mice. [Figure 2] This figure shows the effect of nucleotide mixtures on SDH enzyme activity in muscle tissue of aged SAMP8 mice. [Figure 3] This figure shows the effect of nucleotide mixtures on ATP content in muscle tissue of aged SAMP8 mice. [Figure 4] This figure shows the effect of a nucleotide mixture on mitochondria in the soleus muscle of aged SAMP8 mice. [Figure 5] This figure shows the effect of nucleotide mixtures and single nucleotides on ATP content in muscle tissue of aged SAMP8 mice. [Modes for carrying out the invention]

[0016] The following unrestricted examples are provided solely to give those skilled in the art a more general understanding of the present invention and do not limit the invention in any way.

[0017] (Examples) (Example 1) 1. The nucleotide mixture in this example is obtained by mixing five kinds of 5'-mononucleotides or their sodium salts according to the proportion that CMP accounts for 43 wt.%, AMP accounts for 17 wt.%, UMP accounts for 21 wt.%, GMP accounts for 18 wt.%, and IMP accounts for 1 wt.%. 2. The specific preparation method is as follows. (1) Inspect the five kinds of 5'-mononucleotides or their sodium salts separately, and use them after passing the quality inspection. (2) Screen the five kinds of 5'-mononucleotides or their sodium salts that have passed the quality inspection through a 60-mesh screen. (3) Calculate and weigh the amount of each required nucleotide sample according to the proportion, add them all, and mix for more than 40 minutes. Store the obtained sample at room temperature.

[0018] (Example 2) The nucleotide mixture in this example is obtained by mixing five kinds of 5'-mononucleotides or their sodium salts according to the proportion that CMP accounts for 78 wt.%, AMP accounts for 6 wt.%, UMP accounts for 8 wt.%, GMP accounts for 7 wt.%, and IMP accounts for 1 wt.%. The preparation method is the same as that of Example 1.

[0019] (Example 3) The nucleotide mixture in this example is obtained by mixing five kinds of 5'-mononucleotides or their sodium salts according to the proportion that CMP accounts for 23 wt.%, AMP accounts for 44 wt.%, UMP accounts for 25 wt.%, GMP accounts for 7 wt.%, and IMP accounts for 1 wt.%. The preparation method is the same as that of Example 1.

[0020] (Example 4) The nucleotide mixture in this example is obtained by mixing five kinds of 5'-mononucleotides or their sodium salts according to the proportion that CMP accounts for 23 wt.%, AMP accounts for 17 wt.%, UMP accounts for 40 wt.%, GMP accounts for 19 wt.%, and IMP accounts for 1 wt.%. The preparation method is the same as that of Example 1.

[0021] (Example 5) The nucleotide mixture in this example is obtained by mixing five types of 5'-mononucleotides or their sodium salts in proportions of CMP (24 wt.%), AMP (17 wt.%), UMP (7 wt.%), GMP (51 wt.%), and IMP (1 wt.%). The preparation method is the same as in Example 1.

[0022] (Example 6) I. Materials and Methods 1. Sample: The nucleotide mixture sample obtained in Examples 1 to 5 above.

[0023] 2. Experimental Animals: Rapid aging model mice, namely SAMP8 mice, and control group SAMR1 mice were selected as experimental animals. A total of 78 SF-grade male mice, aged 10-12 weeks, were purchased from the Department of Experimental Animal Science, Peking University. These mice were placed individually in cages and allowed to eat and drink freely. The animal room temperature was kept within a range of 25°C ± 1°C, relative humidity between 50% and 60%, and indoor lighting was controlled with a 12h / 12h light-dark alternation cycle.

[0024] 3. Experimental grouping and dosage: Mice are fed adaptive diets for one week, then divided into six groups (n=12): a group without nucleotides, a normal control group (basal diet, SAMP8-age control), a SAMR1 model control group (basal diet), a low-dose nucleotide intervention group (0.3g / kg + basal diet), a medium-dose nucleotide intervention group (0.6g / kg + basal diet), and a high-dose nucleotide intervention group (1.2g / kg + basal diet). Nucleotides are mixed into the basal diet according to the different dosages used for the intervention. At the experimental node, six more 7-month-old SAMP8 mice are designated as the adolescent control group (SAMP8-yong control).

[0025] 4. Experimental method: 4.1 Grip Strength Measurement According to the study, SAMP8 mice have the highest muscle mass at 7 months of age, enter the pre-sarcopenia stage from 8 months of age, and enter the sarcopenia stage at 10 months of age. Therefore, grip strength tests were performed on the mice three times at 7, 9, and 11 months of age, with five measurements taken each time and the maximum value recorded. 4.2 Measurement of body composition of mice We will use an MRI imaging system to measure the body composition of mice (lean muscle and fat content). 4.3 Detection of antioxidant enzyme activity The enzyme activity of SOD and succinate dehydrogenase (SDH) in the tibialis anterior muscle is detected using a reagent kit. 4.4 Measurement of ATP content in muscle tissue The ATP content in muscle tissue is measured using an ATP content testing kit. 4.5 Observation of Mitochondrial Morphology and Structure A transmission electron microscope is used to observe indicators such as the number, morphology, and membrane structure integrity of mitochondria.

[0026] 5. Statistical Methods: All results are expressed as mean ± standard deviation. One-way ANOVA is performed using SPSS 18.0 software for statistical testing, and p < 0.05 is considered statistically significant.

[0027] II. Experimental Results of Example 1 1. Effects of nucleotide mixtures on mouse grip strength According to the results in Table 1, grip strength tests of 7-month-old mice showed that the grip strength of mice in the high-dose nucleotide intervention group and the SAMR1 group was significantly stronger than that of the normal control group (P<0.05). Grip strength tests of 9-month-old mice showed that the grip strength of mice in the low-dose nucleotide intervention group, the medium-dose nucleotide intervention group, the high-dose nucleotide intervention group, and the SAMR1 group was significantly stronger than that of the group without nucleotides and the normal control group. Grip strength tests of 11-month-old mice also showed that the grip strength of mice in the low-dose nucleotide intervention group, the medium-dose nucleotide intervention group, the high-dose nucleotide intervention group, the adolescent control group, and the SAMR1 group was significantly stronger than that of the group without nucleotides and the normal control group. These results verify that nucleotide intervention can improve muscle function and enhance grip strength in aged SAMP8 mice.

[0028] [Table 1]

[0029] III. Experimental Results of Example 1 Table 2 shows that the results for body composition indicate that muscle mass in the medium-dose nucleotide intervention group, high-dose nucleotide intervention group, adolescent control group, and SAMR1 group is significantly greater than that in the no-nucleotide group and normal control group. The difference in the adolescent control group is statistically significant compared to the no-nucleotide group, normal control group, and low-dose nucleotide intervention group, but not statistically significant compared to the medium-dose nucleotide intervention group and high-dose nucleotide intervention group. This indicates that the nucleotide mixture can significantly increase muscle mass in aged mice. Furthermore, the results for the ratio of muscle to body weight show that the ratio of muscle in the high-dose nucleotide intervention group is significantly higher than that of the normal control group and adolescent control group (P<0.05). The fat content and ratio of fat to body weight in the high-dose nucleotide intervention group are all significantly lower than those of the adolescent control group, but the differences among the other groups are not statistically significant. The above results indicate that the nucleotide mixture has a good effect on the treatment of age-related sarcopenia.

[0030] [Table 2]

[0031] As can be seen in Figure 1, the SOD enzyme activity in the high-dose nucleotide intervention group, the adolescent control group, and the SAMR1 group is significantly higher than in the group without nucleotides and the normal control group. This indicates that the nucleotide mixture has a good antioxidant effect. In Figure 1, # This indicates that the difference is statistically significant compared to the group without nucleotides. * This indicates that the difference is statistically significant compared to the normal control group.

[0032] As can be seen in Figure 2, SDH enzyme activity in the medium-dose and high-dose nucleotide intervention groups is significantly higher than in the group without nucleotides and the normal control group. SDH is one of the marker enzymes that reflect mitochondrial function. The fact that intervention with a nucleotide mixture increases SDH enzyme activity in the muscles of aged mice indicates that the nucleotide mixture is useful in improving mitochondrial dysfunction in the muscles of aged mice. In Figure 2, # This indicates that the difference is statistically significant compared to the group without nucleotides. * This indicates that the difference is statistically significant compared to the normal control group.

[0033] As can be seen in Figure 3, the ATP content in the other groups is significantly higher compared to the group without nucleotides. The ATP content in the high-dose nucleotide intervention group is significantly higher than that of the normal control group. This indicates that nucleotide intervention has a positive effect on increasing the ATP content of mouse muscle. In Figure 3, # This indicates that the difference is statistically significant compared to the group without nucleotides. * This indicates that the difference is statistically significant compared to the normal control group.

[0034] As can be seen in Figure 4, transmission electron microscopy images of mouse soleus muscle show that the mitochondrial membrane structure of mice without nucleotides is severely damaged, and the mitochondrial ridges are absent. Mitochondria in the muscle of normal control mice are also damaged to varying degrees, with vacuoles appearing in the mitochondria and incomplete membrane structures. Mitochondrial damage in the muscle of mice in the low-dose, medium-dose, and high-dose nucleotide intervention groups is less than in the nucleotide-free group and the normal control group, and the mitochondrial membrane has a relatively intact structure with visible mitochondrial ridges. This indicates that intervention with a nucleotide mixture can improve mitochondrial dysfunction in muscle.

[0035] IV. Effects of the nucleotide mixture in Examples 2-5 on the body components of mice. Referencing the experiment in Example 1 on the effects of the nucleotide mixture on the body components of mice, the effects of the nucleotide mixtures in Examples 2 to 5 on the body components of mice are tested. The results are shown in Table 3.

[0036] [Table 3]

[0037] 5. Conclusion of the Experiment This study investigates the efficacy of a nucleotide mixture in the prevention and treatment of age-related sarcopenia by establishing control groups without nucleotides and a normal control group. Animal studies showed that nucleotides improved skeletal muscle function and increased muscle mass in aged SAMP8 mice by improving mitochondrial dysfunction and reducing oxidative stress, thus playing a role in the prevention and treatment of sarcopenia. These experimental results suggest that the nucleotide mixture has remarkable efficacy in treating sarcopenia and possesses the potential to be used as a novel drug for sarcopenia treatment.

[0038] (Example 7) The nucleotide mixture is prepared according to the method described in Example 1. Experimental animals: Rapid aging model mice, namely SAMP8 mice, and control group SAMR1 mice were selected as experimental animals. SF-grade male mice aged 10-12 weeks were placed one per cage, allowing them to eat and drink freely. The animal room temperature was kept within a range of 25°C ± 1°C, relative humidity between 50% and 60%, and indoor lighting was controlled with a 12h / 12h light-dark alternation cycle.

[0039] Experimental grouping: Mice were fed adaptive diets for one week, then divided into five groups (n=10): a nucleotide-free group, a SAMP8 normal control group (basal diet), a SAMR1 model control group (basal diet), a nucleotide mixture group (1.2g / kg nucleotide mixture + basal diet), and a nucleotide mixture + amino acid group (1.2g / kg nucleotide mixture + amino acids + basal diet). Interventions were performed by feeding the mice from the different groups.

[0040] Measurement of ATP content in muscle tissue: The ATP content in muscle tissue is measured using an ATP content test kit.

[0041] As can be seen in Figure 5, the ATP content in the other groups is significantly higher compared to the group without nucleotides. The addition of amino acids to the nucleotide mixture has little effect on increasing the ATP content in mouse muscles. This indicates that amino acids play a very small or almost no role in this process. In Figure 5, # This indicates that the difference is statistically significant compared to the group without nucleotides. * This indicates that the difference is statistically significant compared to the normal control group. Anyone skilled in the art can make many possible changes and modifications to the technical means of the present invention, or modify them into equivalent embodiments, without departing from the scope of the technical solutions of the present invention, based on the technical content set forth above. Therefore, simple changes, equivalent changes and modifications made to the above embodiments in accordance with the technical interior of the present invention without departing from the scope of the technical means of the present invention should still be included within the scope of the protection of the present invention.

[0042] (Note) (Note 1) Use of nucleotide mixtures in the preparation of formulations for the prevention or remission of age-related sarcopenia. (Note 2) The use described in Appendix 1, characterized in that the nucleotide mixture consists of four or five types of 5'-mononucleotides or their sodium salts, and each nucleotide is converted to molecular weights of CMP, AMP, UMP, GMP, and IMP, with the mass ratio being 23-78% for CMP, 6-44% for AMP, 7-40% for UMP, 7-51% for GMP, and 0 or 2.5% or less for IMP. (Note 3) The use described in Appendix 1 or 2, characterized in that each nucleotide in the nucleotide mixture is converted to molecular weights of CMP, AMP, UMP, GMP, and IMP, with the mass ratio being 43% CMP, 17% AMP, 21% UMP, 18% GMP, and 1% IMP. (Note 4) The use described in Appendix 1, characterized in that the preparation is a powder, a tablet, a soft sac, a hard sac, a beverage, or an oral liquid. (Note 5) The use described in Appendix 1 or 2, characterized in that the 5'-mononucleotide mixture improves mitochondrial dysfunction and reduces oxidative stress, thereby improving skeletal muscle function in the elderly, increasing muscle mass, and delaying the onset and progression of sarcopenia.

Claims

1. The use of nucleotide mixtures in the preparation of formulations for the prevention or remission of age-related sarcopenia, The use of the nucleotide mixture is characterized in that the nucleotide mixture consists only of five types of 5'-mononucleotides or their sodium salts, and each nucleotide is converted to molecular weights of CMP, AMP, UMP, GMP, and IMP, with the mass ratio being 23-78% for CMP, 6-44% for AMP, 7-40% for UMP, 7-51% for GMP, and 1% for IMP.

2. The use according to claim 1, characterized in that each nucleotide in the nucleotide mixture is converted to molecular weights of CMP, AMP, UMP, GMP, and IMP, with the mass ratio being 43% CMP, 17% AMP, 21% UMP, 18% GMP, and 1% IMP.

3. The use according to claim 1, characterized in that the preparation is a powder, a tablet, a soft sac, a hard sac, a beverage, or an oral liquid.

4. The use according to claim 1, characterized in that the nucleotide mixture is a 5'-mononucleotide mixture, and the 5'-mononucleotide mixture improves mitochondrial dysfunction and reduces oxidative stress, thereby improving skeletal muscle function in elderly people, increasing muscle mass, and delaying the onset and progression of sarcopenia.

Images