Application of ergothioneine in preparation of composition for improving symptoms of age-related intestinal flora disorder accelerated aging of mammals

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses application of ergothioneine in preparation of a composition for improving age-related intestinal flora disorder accelerated aging symptoms of mammals. Modeling administration is carried out on aged mice, and research finds that the ergothioneine can significantly improve the diversity of intestinal flora species of the aged mice, change the composition of intestinal microflora, improve the relative abundance of beneficial bacteria, improve the expression of intestinal metabolism and synthesis related pathways, and show good activity of regulating in-vivo intestinal flora; meanwhile, the ergothioneine can effectively improve symptoms and expressions related to accelerated aging of intestinal flora disorder, for example, inflammation is relieved, hair growth is promoted, and the limb movement coordination ability and the anti-fatigue endurance of the body are improved. Therefore, the ergothioneine can be used as a beneficial supplement of a current diet treatment means, provides a potential medicine for researching and developing diseases caused by intestinal flora disorder in the future, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of ergothioneine in the preparation of a composition for improving age-related intestinal flora disorders and accelerating the symptoms of aging in mammals. Background Art

[0002] The intestinal flora refers to the microbial community residing in the intestinal tract of the body. These microorganisms include bacteria, fungi, viruses, and other microorganisms, which together constitute a complex ecosystem. According to research, the number of bacteria in the human intestine is as many as 1000 species, about 100 trillion, which is 10 times the total number of human cells. The intestinal flora is of great significance to human health. They participate in digestion, absorption, synthesis of nutrients, maintain the intestinal barrier function, and regulate the immune system and metabolic function.

[0003] At present, the imbalance of intestinal flora balance is related to many diseases, and the composition and function of the intestinal microbial community will change under different physiological and pathological conditions. For example, Heravi et al. revealed the connection between intestinal microbiota dysbiosis and Alzheimer's disease (AD) through a systematic review, and found that Ruminococcaceae, Bacteroidaceae, and Actinomycetaceae are related to AD. Li Na et al. used transgenic mice as animal models and found that the abundance of Firmicutes in the intestines of mice in the AD group increased, the abundance of Desulfobacterota decreased, at the genus level, the relative abundance of Ligilactobacillus salivarius increased, and the relative abundances of Alistipes and Eubacterium inertum decreased. Yaping Liu et al. found through radiation enteritis that radiation can cause intestinal flora dysbiosis, radiation caused an increase in the abundance of Proteobacteria and a decrease in the abundance of Bacteroidetes, and at the genus level, the relative abundances of Escherichia-Shigella, Helicobacter pylori, and Enterococcus increased. It can be seen that the intestinal flora changes significantly from the phylum level to the genus level under different physiological or pathological incentives, the dominant flora compositions are different and the relative abundances are also different. At the same time, the metabolites in the serum will change differently, and the related metabolic pathways are completely different, and these pathways may have changed under different pathological conditions. Therefore, how to regulate host health by affecting these microorganisms is the key point of research.

[0004] Aging is a natural phenomenon in the process of life. As time goes by, the functions of various organs in the human body gradually decline, including the imbalance of the intestinal flora. There is a close connection between aging and intestinal flora disorders, and the two interact with each other to form a vicious cycle. With the increase of age, the diversity of the intestinal flora gradually decreases, the number of beneficial bacteria decreases, while the number of harmful bacteria and conditional pathogenic bacteria increases. This intestinal flora disorder phenomenon will accelerate the aging process, specifically manifested as white and dull hair, hair loss, decreased digestive and absorption function, decreased immune function, etc.

[0005] Ergothioneine (EGT) is a sulfur-containing small molecule histidine derivative (trimethylbetaine of L-histidine), which was first isolated and named from rye infected with Claviceps purpurea in 1909. EGT is synthesized by microorganisms such as actinomycetes, cyanobacteria, some fungi (such as Streptomyces, Mycobacterium, etc.) and mushrooms. Mammals cannot synthesize ergothioneine, but can obtain it by absorbing from the diet. As is well known, EGT is considered a physiological protective agent against ultraviolet (UV)-induced ROS production and damage, because it can directly absorb light in the UV range and repair the DNA of cells irradiated with UV. EGT is not only a direct antioxidant, but also can regulate the entire antioxidant system (increase the expression of antioxidant genes). In senescent cells, free radicals such as ROS and RNS increase, leading to the loss of the activity of the longevity protein Sirtuin. And Ergothioneine is a scavenger of ROS and RNS, and it can also enhance the activity of Sirtuins by preventing the oxidative modification of Sirtuin enzymes. However, its role in regulating age-related intestinal microbiota disorders and improving the related symptoms of accelerated aging caused by intestinal microbiota disorders has not been studied and reported. Summary of the Invention

[0006] Aiming at the deficiencies of the existing technologies at present, the present invention aims to construct the application of ergothioneine in the preparation of a composition for improving the symptoms of accelerated aging caused by age-related intestinal microbiota disorders in mammals.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The application of ergothioneine in the preparation of a composition for improving the symptoms of accelerated aging caused by age-related intestinal microbiota disorders, wherein the host of the intestinal microbiota is a mammal, and the composition has one or more of the following effects:

[0009] (1) Alleviating inflammation;

[0010] (2) Promoting hair growth;

[0011] (3) Improving motor coordination ability;

[0012] (4) Improving anti-fatigue ability.

[0013] Since there is a close connection between aging and intestinal microbiota disorders, and the two interact with each other to form a vicious cycle. The manifestations of age-related intestinal microbiota disorders include a decrease in the species diversity of the intestinal microbiota, a change in the composition of the intestinal microbial community, a decrease in the abundance of beneficial bacteria, and an increase in the abundance of harmful bacteria. The symptoms of accelerated aging caused by the intestinal microbiota disorders are manifested as gray and dull hair, hair loss; decreased physical strength and endurance, easy fatigue; weakened motor ability, reduced flexibility, and slow movement; decreased digestion and absorption function; immune dysfunction; chronic low-grade inflammation, etc.

[0014] Furthermore, the composition improves the symptomatic manifestations of aging by regulating the intestinal flora. The effects of regulating the intestinal flora include increasing the species diversity of the intestinal flora, changing the composition of the intestinal microbial community, increasing the abundance of beneficial bacteria, and increasing the expression of intestinal metabolism and synthesis-related pathways.

[0015] Furthermore, the increase in the abundance of beneficial bacteria refers to an increase in the abundance of Firmicutes and Patescibacteria at the phylum level and an increase in the abundance of Mucispirillum and Eubacterium at the genus level.

[0016] Furthermore, the increase in the expression of intestinal metabolism and synthesis-related pathways is manifested as an increase in metabolic pathways such as cysteine and methionine metabolism, amino sugar and nucleotide sugar metabolism, alanine, aspartic acid and glutamic acid metabolism, D-glutamine and D-glutamic acid metabolism, and D-alanine metabolism, and synthetic pathways such as glycolysis / gluconeogenesis and fatty acid biosynthesis and lysine biosynthesis.

[0017] Furthermore, the reduction of inflammation refers to the down-regulation of the expression of IL-1β, IL-17a, Ccl3 and Ccl5 in tissues.

[0018] Furthermore, the mammal is a wild-type mammal.

[0019] Furthermore, the mammal is an aged mammal.

[0020] Furthermore, the mammal includes, but is not limited to, humans, horses, cows, mice, pigs, cats, dogs, rabbits, sheep, monkeys, etc.

[0021] Furthermore, the ergothioneine is administered at a daily dose of 10 - 40 mg / kg. The daily dose can be administered in divided doses or a single dose.

[0022] Furthermore, the ergothioneine in the composition is ergothioneine or one or more nutritionally or pharmaceutically acceptable salts, acids, esters, polymers, analogs or derivatives thereof as the active ingredient.

[0023] Furthermore, the composition may also contain other ingredients, such as other active agents, preservatives, buffers, salts, pharmaceutically acceptable carriers or other pharmaceutically acceptable ingredients. In some embodiments, the composition can be prepared in the form of a food, beverage, nutritional composition or pharmaceutical composition. Depending on the needs of human and / or veterinary applications, the composition is administered by a specifically suitable route.

[0024] Furthermore, the composition can be in any dosage form, including compositions for tablets, pills, injections, granules, capsules, suspensions, suppositories, oral liquids, aerosols, syrups, powders, external reagents, beverages, dietary supplements, nutritional products, health products or foods.

[0025] In a preferred embodiment of the present invention, a senescence mouse model was established for drug administration, and EGT was administered. The experimental results showed that EGT could significantly reduce the expression of IL-1β, IL-17a, Ccl3 and Ccl5 in the serum of mice; after EGT administration, the hair of senescence mice became thicker and smoother, and the whiskers grew normally; the duration, dropping speed and distance of mice in the rotarod test in the EGT group were significantly increased.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] The present invention provides a new use of ergothioneine, which can safely and effectively improve the intestinal flora disorder related to aging in mammals. Through in-vivo experiments on a senescence mouse model, it was found that ergothioneine can significantly increase the species diversity of the intestinal flora and the relative abundance of beneficial bacteria in senescence mice, and increase the expression of metabolic and synthetic related pathways in the intestines of senescence mice, showing good activity in regulating the intestinal flora in vivo and improving the health of the host organism. Ergothioneine can also effectively improve the symptoms related to aging, including reducing inflammation, promoting hair growth, improving limb coordination ability and fatigue endurance. Ergothioneine can be a beneficial supplement to current dietary treatment methods, providing potential drugs for the research and development of diseases caused by intestinal flora disorders in the future, and has good application prospects. Description of the Drawings

[0028] Figure 1 It is a picture of the effect of intragastric administration of EGT to senescence mice for 6 months on the hair growth of senescence mice.

[0029] Figure 2 It is a diagram showing the results of the rotarod fatigue experiment learning and test analysis of mice after intragastric administration of EGT to senescence mice for 6 months.

[0030] Figure 3 It is a diagram showing the results of the content analysis of inflammation-related proteins detected by multi-factor assay of the serum of mice after intragastric administration of EGT to senescence mice for 6 months.

[0031] Figure 4 It is a diagram showing the results of the species diversity analysis of the intestinal flora of mice after intragastric administration of EGT to senescence mice for 6 months.

[0032] Figure 5 It is a diagram showing the results of the analysis of the intestinal microbial community composition of mice after intragastric administration of EGT to senescence mice for 6 months.

[0033] Figure 6 The results of analyzing the composition of the intestinal microbial community in the intestines of mice after intragastric administration of EGT to senescent mice for 6 months are shown in the figure.

[0034] Figure 7 The results of analyzing the effects of EGT on Firmicutes, Patescibacteria, Mucispirillum, and Eubacterium bacteria in the intestinal flora of senescent mice after intragastric administration of EGT for 6 months are shown in the figure.

[0035] Figure 8 The results of analyzing the effects of EGT on differential microbial species and their evolutionary relationships in senescent mice after intragastric administration of EGT for 6 months are shown in the figure.

[0036] Figure 9 The results of analyzing the effects of EGT on the expression of metabolic and synthetic related pathways in the intestines of senescent mice after intragastric administration of EGT for 6 months are shown in the figure. Detailed implementation mode

[0037] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0038] Unless otherwise specified, the test methods used in the embodiments of the present invention are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0039] The experimental animals and the process of model establishment and drug administration adopted in the embodiments of the present invention: 15-month-old SPF-grade male C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (certificate number: SYXK(Yue)2022-0284). The purchased C57BL / 6 mice were housed in the animal center of the Precision Medicine Research Institute of the Guangdong-Hong Kong-Macao Greater Bay Area at 5 mice per cage. After 10 days of adaptation, the mice were randomly divided into a control group and an EGT group, with 10 mice in each group. The mice in the control group were intragastrically administered normal saline every day, and the mice in the EGT group were intragastrically administered 20 mg / kg EGT every day. During the drug administration period, the mice were weighed at fixed times every week and their hair was observed. The experiment ended after 6 months of intragastric administration. Samples were taken for relevant experiments.

[0040] Example 1 Effect of EGT on hair growth in senescent mice

[0041] Experimental method: During the intragastric administration period, the mice were weighed at fixed times every week and the original data was recorded, and the hair sparsity of the mice was observed. After 6 months of intragastric administration, the two groups of mice were photographed.

[0042] Result analysis: As Figure 1 shown, compared with the control group, the hair of the mice in the EGT group was thicker and smoother. The mice in the control group had no whiskers, while the whiskers of the EGT mice grew normally, which had the effect of improving / promoting hair growth in aging mice.

[0043] Example 2 Effect of EGT on the coordination ability and fatigue endurance of aging mice

[0044] Experimental method: After 6 months of gavage in mice, the rotarod fatigue test learning and testing were carried out on the two groups of mice.

[0045] Result analysis: The rotarod fatigue test in mice is to evaluate the effect of drugs on the coordination ability and fatigue endurance of experimental animals. As Figure 2 shown, compared with the control group, the duration, dropping speed and distance of the mice in the EGT group during the rotarod were significantly increased, improving the coordination ability and fatigue endurance of aging mice.

[0046] Example 3 Effect of EGT on the expression of serum inflammatory factors in aging mice

[0047] Experimental method: After 6 months of gavage in mice, the serum of the mice was collected to detect the content of inflammation-related proteins by multi-factor detection.

[0048] Result analysis: As Figure 3 shown, compared with the control group, EGT reduced the expression of serum inflammatory factors in aging mice, and the contents of IL-1β, IL-17a, Ccl3 and Ccl5 in the serum of the mice in the EGT group were significantly reduced.

[0049] Example 4 Effect of EGT on the species diversity of intestinal flora in aging mice

[0050] Experimental method: After 6 months of gavage in mice, at least 3 feces of each mouse were collected into sterilized EP tubes, quickly frozen in liquid nitrogen, and then transferred to a -80°C refrigerator for storage. Later, Megagene Company was entrusted to perform 16srDNA detection, and then species diversity analysis was carried out.

[0051] Result analysis: Points of different colors or shapes represent sample groups under different environments or conditions. Principal component 1 (PCoA1) and principal component 2 (PCoA2) are the two largest differential characteristics for partitioning samples, and the principal component interpretation degree is reflected in the form of percentages. The closer the spatial distance of the samples, the more similar the species composition structure of the samples. As Figure 4 shown, the similarity between the samples in the control group was relatively high, and the similarity between the samples in the EGT group was also relatively high, indicating that the repeatability within the group was relatively good, the sample data was very similar, and the two groups of samples were not in the same area, so they had good discrimination. As Figure 5As shown, compared with the control group, the species diversity of the intestinal flora of mice gavaged with EGT was significantly increased.

[0052] Example 5 Effect of EGT on the Intestinal Microbial Community Composition of Aged Mice

[0053] Experimental method: After 6 months of gavage in mice, at least 3 feces of each mouse were collected into sterilized EP tubes, quickly frozen in liquid nitrogen, and then transferred to a -80°C refrigerator. Later, Megagene Company was entrusted to perform 16srDNA detection, and then species difference analysis was carried out.

[0054] Result analysis: The Venn diagram can analyze the abundances of the common and unique flora at the genus level of the flora of each group of mice, and more intuitively display the similarity and overlap of the species composition in different groups. As Figure 6 shown in A, there were 1907 species in the control group and 2033 species in the group of mice gavaged with EGT, and 213 species were common to the two groups. As Figure 6 shown in B, EGT changed the intestinal microbial community composition of aged mice.

[0055] Example 6 Effect of EGT on the Relative Abundance of Beneficial Bacteria in the Intestines of Aged Mice

[0056] Experimental method: After 6 months of gavage in mice, at least 3 feces of each mouse were collected into sterilized EP tubes, quickly frozen in liquid nitrogen, and then transferred to a -80°C refrigerator. Later, Megagene Company was entrusted to perform 16srDNA detection, and then the relative abundance analysis of bacterial species was carried out.

[0057] Result analysis: Most Firmicutes bacteria are Gram-positive bacteria and play a key role in the nutrition and metabolism of the host through short-chain fatty acid synthesis. As Figure 7 shown in A, after gavage with EGT, the relative abundance of Firmicutes in the intestinal flora of aged mice was significantly increased. Recent studies have found that Patescibacteria has become one of the most important microorganisms in plants, animals and humans. In the body, Patescibacteria and other microorganisms form an organic conjugate to constitute the flora inside the body, thus participating in many life-related processes, such as immunity, nutrient metabolism, antibacterial and antiviral. In addition, Patescibacteria can also provide nutrients for the body, such as amino acids, vitamins, etc., and can also decompose harmful substances, such as drugs, chemicals, etc., to help human health. As Figure 7 shown in B, after gavage with EGT, the relative abundance of Patescibacteria in the intestinal flora of aged mice was significantly increased.

[0058] The intestinal bacterium Mucispirillum competes with Salmonella for anaerobic respiration substrates in the intestine, inhibits the expression of Salmonella virulence factors, and thus defends against colitis. As Figure 7 shown in C, after intragastric administration of EGT, the relative abundance of Mucispirillum in the intestinal flora of aging mice increased significantly. In addition, Eubacterium is an important intestinal bacterium found in the colon of healthy humans and is a core genus of the human gut microbiota. Members of this family are important because many species produce short-chain fatty acids, especially butyric acid. Short-chain fatty acids are generally considered to have multiple important roles in maintaining human health, such as acting as special nutrients and energy components for the intestinal epithelium, protecting the intestinal mucosal barrier, reducing the body's inflammatory level, and enhancing gastrointestinal motility. In more and more studies on the intestinal flora, it has been found that the reduction or lack of Eubacterium is associated with many diseases, such as depression and / or fatigue, obesity, inflammatory bowel disease, type II diabetes, cardiovascular and cerebrovascular diseases, colorectal cancer, autism, sarcopenia in the elderly, intestinal health, and good tumor prognosis and intestinal homeostasis. Therefore, an increase in the relative abundance of Eubacterium is beneficial to the health of the body. As Figure 7 shown in D, the relative abundance of Eubacterium in the intestinal flora of mice in the EGT group was significantly higher than that in the control group.

[0059] In the phylogenetic tree, species with no significant differences are uniformly colored yellow, and differential species Biomarkers are colored according to the group. Red nodes represent microbial taxa that play important roles in the EGT group, green nodes represent microbial taxa that play important roles in the control group, and the colors of other circles have similar meanings. As Figure 8 can be seen, compared with the control group, the differential species and their phylogenetic relationships in mice given intragastric administration of EGT were significantly improved.

[0060] Example 7 Effects of EGT on the expression of metabolic and synthetic related pathways in aging mice

[0061] Experimental method: After 6 months of intragastric administration of mice, at least 3 feces of each mouse were collected into a sterilized EP tube, quickly frozen in liquid nitrogen, and then transferred to a -80°C refrigerator. Later, Megagene Company was commissioned to perform 16srDNA detection, and then functional prediction analysis was carried out.

[0062] KO, Pathway, and EC information were obtained through PICRUSt2, and KEGG pathway clustering analysis was performed on the above common species, and the top 30 species with relative abundance were displayed and clustered. As Figure 9It can be seen that intragastric administration of EGT increases the expression of metabolism- and synthesis-related pathways in the intestines of aging mice, such as metabolic pathways including Cysteine and methionine metabolism, Amino sugar and nucleotide sugar metabolism, Alanine aspartate and glutamate metabolism, D-Glutamine and D-glutamate metabolism, D-Alanine metabolism, etc., and synthesis pathways including Glycolysis / Gluconeogenesis, Fatty acid biosynthesis, Lysine biosynthesis, etc.

[0063] Obviously, the specific implementation manners described above only further elaborate on the purpose, technical solutions, and beneficial effects of the present invention. It should be understood that the above is only a specific example of the present invention and is not used to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. The application of ergothioneine in preparing a composition for improving the symptoms of accelerated aging caused by age-related intestinal flora disturbance in mammals, characterized in that, The composition has one or more of the following effects: (1) Reduce inflammation; (2) Promote hair growth; (3) Improve motor coordination ability; (4) Improve fatigue resistance.

2. The use according to claim 1, characterized in that: The composition improves the symptoms of aging by regulating the intestinal flora. The effects of regulating the intestinal flora include increasing the species diversity of the intestinal flora, changing the composition of the intestinal microbial community, increasing the relative abundance of beneficial bacteria, and increasing the expression of intestinal metabolism and synthesis-related pathways.

3. The use according to claim 2, characterized in that: The increasing the abundance of beneficial bacteria refers to increasing the abundance of Firmicutes and Patescibacteria at the phylum level, and increasing the abundance of Mucispirillum and Eubacterium at the genus level.

4. The use according to claim 2, characterized in that: The expression of pathways related to intestinal metabolism and synthesis is increased, including cysteine ​​and methionine metabolism, amino sugar and nucleotide sugar metabolism, alanine, aspartic acid and glutamate metabolism, D-glutamine and D-glutamate metabolism, d-alanine metabolic pathway; glycolysis / gluconeogenesis, fatty acid biosynthesis, and lysine biosynthesis pathway.

5. The use according to claim 1, characterized in that: The reduction of inflammation refers to down-regulation of IL-1β, IL-17a, Ccl3 and Ccl5 in tissues.

6. The use according to claim 1, characterized in that: The mammal is a wild-type elderly mammal.

7. The use according to claim 6, characterized in that: The mammals include humans, horses, cows, mice, pigs, cats, dogs, rabbits, sheep, and monkeys.

8. The use according to any one of claims 1 to 7, characterized in that: The ergothioneine is administered in a daily dose of 10 to 40 mg / kg.

9. The use according to any one of claims 1 to 7, characterized in that: The ergothioneine in the composition is ergothioneine or its nutritionally and pharmaceutically acceptable single or multiple salts, acids, esters, polymers, analogs or derivatives as an active ingredient.

10. The use according to any one of claims 1 to 7, characterized in that: The composition is in any dosage form, including tablets, pills, injections, granules, capsules, suspensions, suppositories, oral solutions, aerosols, syrups, powders, external agents, beverages, dietary supplements, nutritional products, health products or food compositions.

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