Methods of cell regeneration

Abstract

Method of Cell Regeneration: The present invention relates to a composition for inducing regeneration of at least one cell, the composition comprising: The present invention comprises at least one probiotic strain of Bacillus subtilis and at least one amino acid or peptide, wherein the amino acid is selected from glutamine, glutamic acid or a salt thereof, and conjugated glutamine, and the peptide is an oligopeptide having a length of 2 to 10 amino acid units, the amino acid units being naturally occurring amino acids, and at least one amino acid unit being a glutamine or glutamic acid unit.

Description

[Technical field]

[0001] The present invention relates to a composition for inducing cell regeneration and / or reducing the biological age of at least one cell or subject. The present invention also relates to a method of inducing cell regeneration and / or reducing the biological age of a subject by administering the composition to said subject. In particular, the composition is a synbiotic composition comprising a probiotic Bacillus subtilis strain and at least one amino acid. [Background technology]

[0002] Aging is known to involve a gradual loss of function at the molecular, cellular, tissue and organismal levels. Aging of individuals and tissues leads to the development of various diseases. In particular, senescent cells are frequently observed in inflammatory tissues such as chronic rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin injury, and arteriosclerosis, and the accumulation of senescent cells leads to poor division of senescent cells, which makes it impossible to properly repair damaged tissues. At the chromatin level, aging is associated with the progressive accumulation of epigenetic errors that ultimately result in aberrant gene regulation, stem cell exhaustion, senescence, and deregulated cell / tissue homeostasis. In particular, Non-Patent Document 1 found that DNA methylation correlates with chronological age, supporting its epigenetic function in aging. Epigenetic age is highly correlated with biological age and responds to environmental factors that accelerate or slow down the aging process, causing it to deviate substantially from the "true" biological age. Thus, environmental confounders or cues contribute to either accelerated or decelerated aging, while accelerated aging is consistent with negative confounders / factors such as disease, stress, malnutrition, and negative environmental conditions. In contrast, both health and good nutrition are associated with decelerated aging. Acquired aging acceleration (acquired aging age > chronological age) suggests that the underlying tissues age faster than expected based on chronological age, whereas negative values ​​(acquired aging age < chronological age, aging deceleration) suggest that the tissues age slower than expected. Acquired aging acceleration is associated with a significant number of age-related conditions and diseases, such as inflammatory processes. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Horvath S. (2013) Genome Biol.; 14(10): R115 Summary of the Invention [Problem to be solved by the invention]

[0004] As mentioned above, healthy nutrition is known to reduce acquired aging.Therefore, there is a need in the industry for a simple and reliable dietary supplement that can regulate cellular aging and can be used for long-term disease prevention.In particular, there is a need in the industry for a dietary supplement that can be used to rejuvenate senescent cells into young and healthy cells. [Means for solving the problem]

[0005] The present invention aims to solve the above problems by providing a dietary supplement that can delay cellular aging or restore cells to their original / earlier state, thereby reducing the biological age of cells or subjects. As it is a dietary supplement, it is not an essential component of the diet of a subject. However, it has the advantage of reducing the biological age of cells or subjects and / or inducing cell regeneration in a subject. Moreover, being a dietary supplement, it is also a very convenient method / tool ​​that can be used to affect the biological age of cells or subjects, compared to diet and / or physical activity. In particular, the dietary supplement is a synbiotic. More specifically, the dietary supplement includes synbiotics (i.e., including prebiotics and probiotics) and other ingredients that further include the benefits of the dietary supplement according to any aspect of the present invention.

[0006] The term "dietary supplement" as used herein is defined as a concentrated source of nutrients or other substances having a nutritional or physiological effect, the purpose of which is to supplement the normal diet (www.efsa.europa.eu / en / topics / topic / food-supplements). The term "synbiotic" as used herein refers to a food ingredient or dietary supplement that combines probiotics and prebiotics in a synergistic form. Thus, a synbiotic composition according to the invention is a composition comprising a probiotic strain and an amino acid, dipeptide or oligopeptide as a prebiotic. In particular, prebiotics and probiotics can be used in combination to support the survival and metabolic activity of the latter, the resulting product belonging to the class of synbiotics. The most recent definition of the term "prebiotic" is "a substrate selectively utilized by a host microorganism to confer a health benefit" (Gibson GR, Nat Rev Gastroenterol Heptanol 2017, 14(8):491-502.), and in addition to specific carbohydrates, also refers to, for example, amino acids and peptides, e.g., dipeptides or oligopeptides. Prebiotics may also include other meaningful bioactive ingredients, e.g., plant extracts, vitamins, etc. In one aspect of the invention, there is provided a use of a composition for inducing regeneration of at least one cell, said composition comprising: at least one probiotic Bacillus subtilis strain, and At least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid and acetylated forms thereof, Includes. As used herein, the term "regeneration of at least one cell" may refer to restoring an aged cell to a young, healthy cell. In particular, a "regenerated (rejuvenated) cell" refers to a aged cell that has been treated with one or more cell reprogramming factors or transiently reprogrammed such that the cell has the transcriptome profile of a younger cell while still retaining one or more cell identity markers. The term "cell" as used herein refers to a whole living cell, naturally occurring or modified. The cell may be isolated from other cells, mixed with other cells in culture, or may be within a tissue (partial or whole) or organism. In particular, the term "mammalian cell" refers to any cell derived from a mammalian subject. The cell may also be a cell derived from the culture and propagation of cells obtained from a subject. The cell may also be genetically modified to express recombinant proteins and / or nucleic acids. In particular, the mammalian cell may be an intestinal cell.

[0007] In a further aspect of the invention there is provided a use of a composition for reducing epigenetic age in at least one subject, said composition comprising: at least one probiotic strain of Bacillus subtilis, and At least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid and acetylated forms thereof, Includes. The composition according to any aspect of the present invention may also include other ingredients. The other ingredients may include one or more plant extracts. In particular, the plant extracts may be selected from turmeric extract, green tea extract, and combinations thereof. More specifically, the composition may also include other ingredients such as zinc, vitamin B6, D-biotin, vitamin B12, vitamin D, pantothenic acid, etc. Even more specifically, the composition according to any aspect of the present invention may include, in addition to components (a) and (b), turmeric extract, green tea extract, zinc, vitamin B6, D-biotin, vitamin B12, vitamin D, and pantothenic acid.

[0008] Biological age is determined by the biological state or condition of an individual or population, taking into account the circumstances of life (stress, nutrition, etc.). The terms "acquired aging (age)", "methylation age" and "biological age" have the same meaning and are used interchangeably in the context of this application. The term "chronological age" refers to the calendar days elapsed since birth / hatching. The term "epigenetic clock" as used herein refers to a biochemical test that can estimate DNA methylation age / epigenetic aging in a specific or tissue-independent manner and predict mortality and remaining time to death (Non-Patent Document 1).

[0009] The compositions according to any aspect of the invention can result in post-translational modifications (PTMs) of histones, which are important steps in the epigenetic regulation of genes. PTMs can then result in changes in gene expression. The term "post-translational modification of histones", which is used interchangeably with the term "histone modification", as used herein, refers to a covalent modification of the N-terminal tail of a histone protein. Examples of histone modifications include, for example, methylation, acetylation, phosphorylation, ubiquitination, and ADP-ribosylation. Histone modifications and other acquired mechanisms are important in regulating gene activity and cellular processes. Different histone modifications regulate different processes, such as transcription, DNA replication, and DNA repair. Deregulation of any of the modifications can shift the balance of gene expression, resulting in abnormal acquired patterns and cellular abnormalities. In one example, one or more of the histone modifications can be methylation. In particular, methylation can occur, for example, at H3-K4, H3-K9, H4-K20, H3-K27, H3-K36, and / or H3-K79. The compositions according to any aspect of the invention may be used to alter gene expression by altering histone PTMs, and may be used to treat diseases associated with histone PTMs.

[0010] The terms "subject" and "individual" as used herein are used interchangeably and may be any living organism. For example, a subject according to any embodiment of the present invention may be any kind of plant or animal, preferably a domesticated animal (or domestic livestock) or livestock, which may be a vertebrate or invertebrate. A representative example of an invertebrate animal that may be useful as a subject according to any embodiment of the present invention may be a shrimp or crab, such as marbled crayfish. A representative example of a vertebrate animal that may be useful as a subject according to any embodiment of the present invention may be a fish or a terrestrial animal, such as a chicken or other livestock that may be cultivated. A subject as used herein may also refer to any vertebrate subject, including, but not limited to, humans and other primates, including non-human primates, such as chimpanzees and other ape and monkey species; livestock, such as cows, sheep, pigs, goats and horses; domestic mammals, such as dogs and cats; rodents, such as mice, rats, rabbits, hamsters and guinea pigs; livestock, such as chickens, turkeys and other galliformes, ducks, geese, and birds, including wild and game birds. In particular, the subject is a mammal. More specifically, the mammal is selected from the group consisting of mouse, rat, guinea pig, dog, mini pig, human, cow, sheep, pig, goat, horse, donkey, and mule.

[0011] The probiotic strain in the composition according to any aspect of the invention may be selected from Bacillus subtilis DSM 32315, Bacillus subtilis DSM 32540, Bacillus subtilis DSM 32592, and mixtures thereof. In particular, the probiotic strain may be Bacillus subtilis DSM 32315.

[0012] The term "natural amino acid" in the context of the present invention is understood to include the L- and D-forms of the 20 known amino acids. In particular, the L-forms of the amino acids may be used in the compositions according to any aspect of the present invention. In one example, the term "amino acid" also includes analogs or derivatives of those amino acids. The term "free amino acid" as used herein is understood to be an amino acid with its amino functional group and its (α-) carboxyl functional group in free form, i.e., an amino acid that is not covalently attached to another molecule, e.g., not forming a peptide bond. Free amino acids may also be present in the form of salts or hydrates. Reference to an amino acid as part of or in an oligopeptide should be understood to refer to that portion of the respective oligopeptide structure that is derived from the respective amino acid by known mechanisms of biochemistry and peptide biosynthesis. A "peptide" is understood to be a molecule comprising at least two amino acids covalently linked to each other by a peptide bond (R1-CO-NH-R2). The amino acid or peptide in the composition according to any aspect of the present invention may be an oligopeptide having at least two amino acids. In particular, the oligopeptide may contain alanine or glycine. More specifically, the oligopeptide may be selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid, and acetylated forms thereof.

[0013] Compositions according to any aspect of the present invention may be in the form of a pill, capsule, tablet, powder blend, granules or liquid. In one example, the composition according to any aspect of the present invention may be in capsule form, and the capsule may contain 1×10 8 ~2×10 10 Contains CFU of probiotic strain and 50mg-800mg of dipeptides. In particular, the total amount of probiotic strains and amino acids or peptides is at least 10% by weight, in particular at least 50% by weight, more in particular at least 60% by weight, even more in particular at least 70% by weight of the total weight of the composition. In particular, the total amount of plant extracts in a composition according to any aspect of the invention is at least 10% by weight, in particular at least 20% by weight, more in particular 20-40% by weight of the total weight of the composition. In one example, a composition according to any aspect of the invention includes an enteric coating comprising at least one of the following: methyl acrylate-methacrylic acid copolymer, cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymer, shellac, cellulose acetate trimellitate, sodium alginate, and zein.

[0014] In another aspect of the invention, there is provided a composition for use in inducing regeneration of at least one cell, comprising: at least one probiotic Bacillus subtilis strain, and At least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid and acetylated forms thereof, Includes. The use according to any aspect of the invention may be for non-therapeutic use, in particular for non-therapeutic food use. In particular, the composition according to any aspect of the invention may be a dietary supplement and may not be medicated. In another aspect of the invention, there is provided a composition for use in reducing epigenetic age in at least one subject, comprising: at least one probiotic strain of Bacillus subtilis, and At least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid and acetylated forms thereof, Includes. In one example, the composition may also be for "combination therapy" or "adjuvant therapy", in which case the composition according to any aspect of the invention may be used for prophylactic, preventative, and / or therapeutic purposes. The composition according to any aspect of the present invention further comprises one or more plant extracts selected from turmeric extract and green tea extract.

[0015] In a further aspect of the invention, there is provided a method for reducing epigenetic age in at least one subject, comprising administering to said subject a therapeutically effective amount of: at least one probiotic strain of Bacillus subtilis, and At least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid and acetylated forms thereof, and administering to a subject in need thereof a composition comprising: In another aspect of the invention, there is provided a method for inducing regeneration of at least one cell, comprising: at least one probiotic strain of Bacillus subtilis, and At least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid and acetylated forms thereof, and administering to a subject in need thereof a composition comprising: The probiotic strain is selected from the group consisting of Bacillus subtilis DSM 32315, Bacillus subtilis DSM 32540, Bacillus subtilis DSM 32592 and mixtures thereof, and / or the composition further comprises one or more plant extracts selected from turmeric extract and green tea extract. A therapeutically effective dose or amount of the composition according to any aspect of the present invention can be administered to a subject in need thereof. By "therapeutically effective dose or amount" is intended an amount capable of inducing at least one cell regeneration that can result in a long-term positive therapeutic response in a subject in need of tissue repair or regeneration, such as an amount that restores function and / or generates new tissue at the treatment site. Thus, for example, a "positive therapeutic response" is an improvement in an age-related disease or condition associated with the treatment, and / or an improvement in one or more symptoms of an age-related disease or condition associated with the treatment, such as restoration of tissue functionality, relief of pain, improved stamina, increased muscle strength, increased mobility, and / or improved cognitive function. The exact dose required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the mode of administration, etc. The appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation based on the information provided herein. An embodiment of the present invention will be described with reference to the drawings. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 is a graph showing methylation changes in 18 subjects confirming a decrease in the subjects' biological age after taking a dietary supplement according to any embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The above describes preferred embodiments, and as will be understood by those skilled in the art, may be subject to variations or modifications in design, structure, or operation without departing from the scope of the claims. These variations are intended to be encompassed, for example, by the claims. EXAMPLES

[0018] A proof-of-concept study to evaluate the impact of a dietary supplement (SAMANA® FORCE containing Bacillus subtilis DSM32315, L-alanyl-L-glutamine, turmeric extract, green tea extract, zinc, vitamin B6, D-biotin, vitamin B12, vitamin D, and pantothenic acid) on short-chain fatty acid production and the microbiome in healthy subjects This is a proof-of-concept study to investigate the effect of Samana Force, a dietary supplement consisting of probiotics, dipeptides, plant extracts, and vitamins and minerals, on the alteration of the gut microbiota and the production of short-chain fatty acids. In addition, stool and blood biomarkers of intestinal barrier and GI health were evaluated. In addition, tolerability and bowel function parameters (stool frequency, stool consistency, and GI symptoms) were evaluated during the study intervention. Test product, dose and mode of administration: SAMANA® FORCE contains 2 billion colony forming units of Bacillus subtilis DSM32315, 290 mg L-alanyl-L-glutamine, 90 mg turmeric extract (approximately 70-80% curcumin), 90 mg green tea extract (approximately 50% EGCG), 5 mg zinc, 0.56 mg vitamin B6, 20 μg D-biotin, 0.75 μg vitamin B12, 4 μg vitamin D, and 2.4 mg pantothenic acid. Mode of administration: 1 capsule in the morning (soft capsule with or without breakfast) and 1 capsule in the evening (soft capsule with or without dinner), taken without chewing, with water. Intervention period: 2 weeks run-in period, followed by 4 weeks intervention period and 2 weeks follow-up period Evaluation criteria: The primary focus of this study was to determine the effect of Samana® Force on changes in fecal and blood short-chain fatty acid (SCFA) production over a 4-week feeding period. Additionally, the gut microbiota, as assessed by 16S-rRNA, was analyzed. Statistical methods: This study was performed as an exploratory proof of concept study. Changes over time were examined. All data obtained in this study and documented in eCRFs, questionnaires and diaries were listed and summarized with descriptive statistics or frequency tables as appropriate. All statistical tests were performed two-sided. A p-value of less than 5% was considered statistically significant. Bioinformatics was applied for the microbiome analysis by a sequencing company. Results and conclusions: A significant increase in SCFA butyrate was observed over the study period (baseline vs. 2 weeks p=0.0278, baseline vs. 4 weeks p=0.0728). To what extent this can be mainly attributed to the probiotic Bacillus subtilis and the metabolism of the provided dipeptide, or whether this results from a further shift in the microbiota associated with an increase in amino acid conversion, cannot be derived from the data. However, the data clearly show the effect of the supplement on the microbiome, which also led to changes in gut biomarkers, but not stool frequency and consistency. A significant increase in secretory IgA was identified (baseline vs. 4 weeks p=0.0151 (n=17)), indicating an immune stimulating effect. In this study population, despite the absence of any symptomatic clinical signs or history of gastrointestinal disorders, possibly due to a rather unhealthy dietary habit characterized by low fiber and low amounts of fruits and vegetables, significantly elevated leaky gut and calprotectin biomarkers were identified.Furthermore, significant reductions in total cholesterol (baseline vs. 4 weeks p=0.0037) and LDL cholesterol (baseline vs. 4 weeks p=0.0313) were observed.Highly significant reductions in fasting total GLP-1 and PYY were observed (baseline vs. 4 weeks, p<0.001), possibly due to the influence of the microbiome or other direct modes of action of the ingredients in SAMANA® FORCE. Conclusion: In conclusion, over a four-week intervention with SAMANA® FORCE, the pilot study indicates a significant increase in the SCFA butyrate in fecal samples accompanied by an intriguing shift in the microbiome, enhancing known butyrate producers such as Faecalibacterium prausnitzii, as well as metabolic biomarkers and gut hormones. EXAMPLES

[0019] Differences in biological age (start and end) using blood samples As described in Example 1, DNA was extracted from the entire blood samples obtained from 18 subjects administered SAMANA (registered trademark) FORCE. In particular, 18 male subjects with poor eating habits were included in the study. The methylation patterns of blood cells were analyzed 4 weeks before and after the supplementation of SAMANA FORCE. As a result, 36 samples analyzed in this test were obtained. The DNA methylation analysis was performed at Elysium Health Inc. using the APEX platform that tests blood samples using the Horvath Clock. The results are shown in Figure 1. Additives such as butyrate-forming symbiotics and curcumin appear to show a tendency to slow down aging. For all 18 subjects, the biological age was determined at two time points through the DNA methylation profile. Table 1 shows the biological age difference between V1 (starting point) and V3 (ending point) of the subjects. When the value is positive, the age decelerates as V1 > V3, and when it is negative, the age accelerates, and the subject is older at V3 than at the start of the test (V1 < V3). *= Subject 108 had a fever at time point V3 and was invalidated.

[0020]

Table 1

Claims

1. The use of a composition for inducing the regeneration of at least one cell, wherein the composition is as follows: At least one probiotic strain of Bacillus subtilis, and At least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid, and their acetylated forms, Includes, use.

2. The use of a composition for reducing epigenetic aging in at least one subject, wherein the composition is as follows: At least one probiotic strain of Bacillus subtilis, and At least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid, and their acetylated forms, Includes, use.

3. The use according to claim 2, wherein the subject is a mammal.

4. The use according to claim 1, wherein the cells are mammalian cells.

5. The use according to claim 1, wherein the composition further comprises one or more plant extracts selected from turmeric extract and green tea extract.

6. The use according to claim 1, wherein the probiotic strain is selected from the group consisting of Bacillus subtilis DSM32315, Bacillus subtilis DSM32540, Bacillus subtilis DSM32592, and mixtures thereof.

7. The use according to claim 1, wherein the total amount of the probiotic strain and the dipeptide is at least 40% by mass of the composition, and / or the total amount of the plant extract is at least 10% by mass of the total mass of the composition.

8. The use according to claim 1, wherein the composition comprises an enteric coating, and the enteric coating comprises at least one of the following: methyl acrylate-methacrylic acid copolymer, cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymer, shellac, cellulose acetate trimellitate, sodium alginate, and zein.

9. A composition for use in inducing the regeneration of at least one cell, the following: At least one probiotic strain of Bacillus subtilis, and At least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid, and their acetylated forms, A composition containing the following:

10. A composition for use in reducing epigenetic aging in at least one subject, comprising the following: At least one probiotic strain of Bacillus subtilis, and At least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid, and their acetylated forms, A composition containing the following:

11. Furthermore, the composition according to claim 9 comprises one or more plant extracts selected from turmeric extract and green tea extract.

12. The composition according to claim 9, wherein the probiotic strain is selected from the group consisting of Bacillus subtilis DSM32315, Bacillus subtilis DSM32540, Bacillus subtilis DSM32592, and mixtures thereof.

13. A method for reducing epigenetic aging in at least one subject, the following: At least one probiotic strain of Bacillus subtilis, and At least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid, and their acetylated forms, A method comprising administering a composition containing to a subject for which it is required.

14. A method for inducing the regeneration of at least one cell, the following: At least one probiotic strain of Bacillus subtilis, and At least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid, and their acetylated forms, A method comprising administering a composition containing to a subject for which it is required.

15. The method according to claim 13, wherein the probiotic strain is selected from the group consisting of Bacillus subtilis DSM32315, Bacillus subtilis DSM32540, Bacillus subtilis DSM32592, and mixtures thereof.

16. The composition further comprises one or more plant extracts selected from turmeric extract and green tea extract. The method according to claim 13, further comprising one or more plant extracts selected from turmeric extract and green tea extract.

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