Food composition for suppressing glycation stress and cosmetic composition for suppressing aging symptoms

Abstract

To provide a food composition for suppressing glycation stress, a food composition for suppressing the formation and / or accumulation of endogenous AGEs (Advanced Glycation End Products), a food composition for suppressing aging caused by the accumulation of endogenous AGEs, and a cosmetic composition for suppressing aging caused by the accumulation of endogenous AGEs. [Solution] A food composition containing Gly-Pro-Hyp for suppressing glycation stress. A food composition containing Gly-Pro-Hyp for suppressing the formation and / or accumulation of endogenous AGEs (Advanced Glycation End Products) is provided. A food composition containing Gly-Pro-Hyp for suppressing aging caused by the accumulation of endogenous AGEs in the dermis. A cosmetic composition containing Gly-Pro-Hyp for suppressing aging caused by the accumulation of endogenous AGEs in the dermis is provided.

Description

[Technical Field]

[0001] The present invention relates to a food composition for inhibiting glycation stress containing Gly-Pro-Hyp, a food composition for inhibiting the decline of biological functions caused by glycation stress containing Gly-Pro-Hyp, a food composition for inhibiting biological inflammation caused by glycation stress containing Gly-Pro-Hyp, a food composition for inhibiting cell death caused by glycation stress containing Gly-Pro-Hyp, an anti-fatigue food composition containing Gly-Pro-Hyp, a food composition for inhibiting the production and / or accumulation of endogenous AGEs (advanced glycation end products) containing Gly-Pro-Hyp, a food composition for inhibiting aging signs caused by the accumulation of endogenous AGEs in the dermis containing Gly-Pro-Hyp, a food composition for inhibiting the production of RAGE (AGE-specific receptor) binding AGEs containing Gly-Pro-Hyp, a food composition for inhibiting the activity (reactivity) of aldehyde compounds in vivo containing Gly-Pro-Hyp, and a food composition for inhibiting pentosidine formation containing Gly-Pro-Hyp. The present invention also relates to a cosmetic composition for inhibiting aging signs caused by the accumulation of endogenous AGEs in the dermis containing Gly-Pro-Hyp, a cosmetic composition for inhibiting glycation stress, a cosmetic composition for inhibiting the decline of biological functions caused by glycation stress, a cosmetic composition for inhibiting biological inflammation caused by glycation stress, a cosmetic composition for inhibiting cell death caused by glycation stress, a cosmetic composition for inhibiting the production and / or accumulation of endogenous AGEs (advanced glycation end products), a cosmetic composition for inhibiting the production of RAGE (AGE-specific receptor) binding AGEs, a cosmetic composition for inhibiting the activity (reactivity) of aldehyde compounds in vivo, and a cosmetic composition for inhibiting pentosidine formation. [Background Art]

[0002] The decline in physical function, i.e., aging, the visible effects of aging (senile signs), and the onset and progression of age-related diseases do not progress uniformly for everyone, but vary from person to person. One of the factors contributing to this is said to be diet. In particular, advanced glycation end products (AGEs), which are formed by the non-enzymatic reaction of sugars and proteins, are said to have strong organ-damaging properties and are associated with many diseases and signs of aging.

[0003] AGEs are broadly classified into two types: exogenous and endogenous. Exogenous AGEs are those found in heat-processed foods, while endogenous AGEs are those produced by the reaction of sugars or carbonyl compounds (sugar metabolites) with proteins in the body. In both cases, proteins and sugars or sugar metabolites bind non-enzymatically to form Schiff bases, followed by the formation of Amadori compounds. These then undergo various structural changes, such as the addition, oxidation, and decomposition of amino acids, to become AGEs. AGEs are not a single structure, but rather a group of structures produced by various reaction pathways and carbonyl compounds, and the structures of all AGEs have not been elucidated. However, in the initial stage of Schiff base formation, the amino group of amino acids is likely to be involved in the reaction. In particular, because this reaction is non-enzymatic, it is known that the amino groups of the side chains of highly reactive arginine and lysine are likely to be involved.

[0004] Both exogenous and endogenous AGEs are harmful to the body, but their mechanisms of action differ. Exogenous AGEs are those found in food, primarily produced by the heating reaction of sugars and proteins, and are absorbed into the body upon ingestion. They then change form through digestion and other processes before reaching the bloodstream. Upon reaching cells via blood circulation, they bind to AGEs receptors, including RAGE (Receptor of AGEs), which is a specific receptor for AGEs. The intracellular signals triggered by this process are involved in inducing oxidative stress and inflammatory stress, thereby influencing the progression of various diseases and signs of aging.

[0005] In contrast, endogenous AGEs are AGEs that are produced when dietary components migrate into the body and react with proteins in the body. Representative endogenous AGEs include pentosidine, crothrin, and carboxymethyllysine (CML). Pentosidine is produced when the pentose ribose binds to arginine and lysine to form a crosslink. Crothrin is a crosslinked product of two lysine residues and glucose, and carboxymethyllysine is produced by the oxidative cleavage of an Amadori compound from the reaction product of glucose and lysine.

[0006] Diabetes mellitus is a representative disease caused by glycation in the body. The accumulation of endogenous AGEs in various parts of the body, including tissues and lesions in diabetic patients, arteriosclerotic lesions, the liver, brain, and lens, and their impact on health have been clarified. Carboxymethyllysine accumulates in the skin and is associated with aging. Pentosidine, which accumulates in tissues throughout the body with age, is a marker of nephropathy and is also involved in fractures and osteoporosis due to bone deterioration, decreased joint flexibility, osteoarthritis of the knee, arteriosclerosis, and aging of the skin, and is being studied as a biomarker (Non-patent Literature 1-3). In addition, AGEs and reaction intermediates in their formation process are sometimes used as biomarkers. HbA1c is an Amadori product produced when hemoglobin A is glycated by glucose and is widely used as an indicator of hyperglycemia. Carboxymethyllysine and fluorescent AGEs are known as evaluation indicators for renal function, anemia, and the degree of arteriosclerosis.

[0007] Furthermore, as blood sugar levels rise, blood aldehyde levels also increase. Sugars such as glucose and fructose that we ingest usually have a cyclic structure, but during sugar metabolism in the body, the ring opens, producing linear aldehydes. These aldehyde compounds significantly influence the induction of biological damage due to the formation of endogenous AGEs (Non-Patent Literature 4). Because aldehyde groups are highly reactive, they can react with substrates without relying on enzymes, and the glycation reaction proceeds at a much faster rate compared to cyclic sugars. This glycation reaction causes intracellular proteins to cross-link and become AGEs, leading to decreased function or dysfunction. This is the primary harm caused by endogenous AGEs. For example, it has been reported that when cell death occurs due to glyceraldehyde-derived AGEs, cross-linking of proteins essential for maintaining the body, such as Heatshock cognate 70 (Non-Patent Literature 5), Caspase-3, and heterogeneous nuclear ribonucleoprotein M (Non-Patent Literature 6), is observed. Furthermore, when the arginine or lysine side chains of insulin precursors are glycated, the amount of insulin produced by insulin decreases. When insulin stored in cells is also glycated, its insulin activity decreases. It is also said that glycation is involved in the aggregation of amyloid-beta in senile plaques (Non-Patent Literature 7), and when cultured nerve cells are treated with glyceraldehyde, changes very similar to the pathological changes in Alzheimer's disease occur, suggesting that the accumulation of endogenous AGEs in the brain may contribute to the onset and progression of Alzheimer's disease (Non-Patent Literature 8).

[0008] As long as we ingest sugar in our life activities, we are susceptible to glycation by aldehyde compounds produced in the body. Furthermore, reaction products between aldehyde compounds produced during alcohol metabolism and lipid metabolism and endogenous proteins are also classified as endogenous AGEs. Many aldehyde compounds are known to be produced during metabolic processes, including glyceraldehyde, 3-deoxyglucosone, glyoxal, methylglyoxal, acetaldehyde, malondialdehyde, and acrolein (Non-Patent Literature 7). In addition to metabolic pathways, it is known that skin lipids can also be peroxidized by ultraviolet light, leading to the formation of AGEs in the skin.

[0009] The second harm caused by endogenous AGEs lies in the generation of reactive oxygen species (ROS). Reactive oxygen species (ROS) are generated during the formation of AGEs within cells (Non-Patent Literature 9), and these damage cells. This triggers inflammation and causes significant damage to genes and other tissues. Normally, reactive oxygen species are removed by mechanisms present in cells, but when glycation impairs this function, the body cannot keep up, resulting in increased susceptibility to cell death. For example, the accumulation of AGEs derived from glyceraldehyde, known as one of the highly reactive aldehyde compounds, in hepatocytes increases the expression of inflammation-related genes such as interleukin-8 (IL-8) (Non-Patent Literature 10) and C-reactive protein (CRP) (Non-Patent Literature 5 and 6), and induces cell death. It has become clear that dysfunction caused by aldehyde-derived AGEs and the generation of ROS are involved in both alcoholic hepatitis and cirrhosis caused by non-alcoholic hepatitis (Non-Patent Literature 11). Furthermore, a constant increase in ROS due to glycation stress stimulates the production of cytokines and chemokines, which causes chronic inflammation. This chronic inflammation is known to be associated with the progression of aging and various diseases. In diabetes, IL-8, one of the cytokines, is involved in the pathogenesis of diabetic vascular complications (neuropathy, retinopathy, nephropathy, arteriosclerosis, etc.) (Non-patent Literature 12), suggesting a link between chronic inflammation due to glycation stress and disease onset. In addition, IL-8 has aspects of being a marker of fatigue in healthy individuals and fatigue (malaise) in various diseases. In diabetes, it has been found that there is a correlation between increases and decreases in IL-8 and scores such as physical fatigue, decreased activity, mental fatigue, and decreased motivation (Non-patent Literature 13), suggesting a link between chronic inflammation due to glycation stress and fatigue.

[0010] Since sugars and sugar metabolites reach the entire body via the bloodstream, all tissues and functional proteins in the body are directly affected by the production of endogenous AGEs. However, it is important to note that the effects of endogenous AGEs are not limited to the dysfunction of the glycated tissue itself or individual cells. Endogenous AGEs leaked through cell death reach other organs via the bloodstream. Alternatively, glycated collagen in the extracellular matrix comes into contact with nearby cells. When these AGEs bind to RAGE, they activate cellular signals, similar to exogenous AGEs, causing oxidative stress and inflammation. This is the third harmful effect of endogenous AGEs. For example, carboxymethyllysine itself contributes to decreased elasticity, wrinkles, sagging, and yellowing of the skin by cross-linking the extracellular matrix of the skin, but when it accumulates, it binds to RAGE in nearby skin fibroblasts, driving signaling and inducing cell death. Fibroblast cell death disrupts skin homeostasis and further accelerates skin aging (Non-Patent Literature 14). Furthermore, RAGE expression itself is enhanced in cells stimulated by AGEs (Non-Patent Literature 15). This means that the RAGE signal can be activated even with small amounts of AGEs, as sensitivity to AGEs increases. RAGE is expressed in a wide range of tissues besides the skin, including blood vessels and vascular pericytes, lungs, kidneys, and immune cells (Non-Patent Literature 16), so it is thought that the AGE-RAGE signaling pathway is activated in a wide range of tissues.

[0011] As described above, the concept of glycation stress encompasses the excessive production of various reducing sugars and aldehydes derived from sugars, lipids, and alcohols in the metabolic process within the body, as well as the generation of endogenous AGEs resulting from these substances, and the subsequent reactions and stress on the body (Non-Patent Documents 17 and 18).

[0012] Glycation stress can occur anywhere in the body—in the blood, extracellular matrix, or within cells. Proteins with longer lifespans are particularly susceptible to its cumulative effects. For example, collagen is a prime example of a protein with a slow metabolic turnover and long lifespan. Not only pentosidine and carboxymethyllysine, but also a wide variety of AGEs (Advanced Glycation End Products) cross-link and denature collagen over long periods. This alters the physical properties of collagen tissue, increasing its stiffness. This, in turn, affects tissue function, degrading health and triggering various symptoms.

[0013] Non-patent document 19 reports that the intake of collagen peptides containing Hyp-Gly and Pro-Hyp reduces AGEs levels in subjects. While the authors consider this effect to be related to the inhibition of the activity of DDP-4, an enzyme that breaks down in lectins, they have not shown any possibility of directly inhibiting the formation of endogenous AGEs.

[0014] Non-patent document 20 reports that the intake of collagen tripeptide (CTP), a material containing 15% Gly-XY (where X and Y are any amino acids) tripeptides, improves vascular elasticity in healthy individuals and simultaneously lowers the concentration of glyceraldehyde-derived AGEs in their serum. However, it was unclear which specific CTP component was responsible for the decrease in serum AGEs concentration, and it was also unclear whether the effect was specific to CTP. [Prior art documents] [Non-patent literature]

[0015] [Non-Patent Document 1] Yagi et al. Glycative Stress Research 2018; 5.2: 119-128. [Non-Patent Document 2] Ohno et al., Journal of clinical biochemistry and nutrition 2015; 57.1: 27-32. [Non-Patent Document 3] Mitsuru Saito. Journal of the Japanese Geriatrics Society 2013; 50.2: 213-217.

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Non-licensed literature 9

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[0016] An object of the present invention is to verify the endogenous advanced glycation end products (AGEs) production inhibitory effect of collagen-derived peptides, and provide a food composition for inhibiting glycation stress, a food composition for inhibiting endogenous AGEs production and / or accumulation, a food composition for inhibiting aging signs caused by endogenous AGEs accumulation, and a cosmetic composition for inhibiting aging signs caused by endogenous AGEs accumulation. [Means for Solving the Problem]

[0017] The present inventors found that Gly-Pro-Hyp inhibits the production of endogenous AGEs, that is, has a glycation stress inhibitory effect, and completed the present invention through further research.

[0018] The present invention provides the following inventions. [1] A food composition containing Gly-Pro-Hyp for suppressing glycation stress. [2] A food composition containing Gly-Pro-Hyp for suppressing the decline of biological functions caused by glycation stress. [3] A food composition containing Gly-Pro-Hyp for suppressing inflammation in the body caused by glycation stress. [4] A food composition containing Gly-Pro-Hyp for suppressing cell death caused by glycation stress. [5] An anti-fatigue food composition containing Gly-Pro-Hyp. [6] A food composition containing Gly-Pro-Hyp for inhibiting the formation and / or accumulation of endogenous AGEs (Advanced Glycation End Products). [7] A food composition containing Gly-Pro-Hyp for suppressing aging caused by the accumulation of endogenous AGEs in the dermis. [8] The food composition described in [7], wherein the early signs of aging are yellowish discoloration. [9] A food composition containing Gly-Pro-Hyp that inhibits the formation of AGEs that bind to RAGE (AGE-specific receptor).

[10] A food composition containing Gly-Pro-Hyp for inhibiting the activity (reactivity) of aldehyde compounds in the body.

[11] The food composition according to

[10] , wherein the preceding aldehyde is glyceraldehyde.

[12] A food composition containing Gly-Pro-Hyp for inhibiting pentosidine formation.

[13] A cosmetic composition containing Gly-Pro-Hyp for suppressing aging caused by the accumulation of endogenous AGEs in the dermis.

[14] The cosmetic composition according to

[13] , wherein the sign of aging is yellowish discoloration.

[15] A cosmetic composition for suppressing glycation stress containing Gly-Pro-Hyp.

[16] A cosmetic composition containing Gly-Pro-Hyp for suppressing the decline in biological function caused by glycation stress.

[17] A cosmetic composition containing Gly-Pro-Hyp for suppressing inflammation in the body caused by glycation stress.

[18] A cosmetic composition containing Gly-Pro-Hyp for suppressing cell death caused by glycation stress.

[19] A cosmetic composition containing Gly-Pro-Hyp for inhibiting the formation and / or accumulation of endogenous AGEs (Advanced Glycation End Products).

[20] A cosmetic composition containing Gly-Pro-Hyp that inhibits the formation of AGEs that bind to RAGE (AGE-specific receptor).

[21] A cosmetic composition containing Gly-Pro-Hyp for inhibiting the activity (reactivity) of aldehyde compounds in the body.

[22] The cosmetic composition according to

[21] , wherein the aldehyde is glyceraldehyde.

[23] A cosmetic composition for inhibiting pentosidine formation containing Gly-Pro-Hyp. [A1] A method for suppressing glycation stress, comprising ingesting or administering a composition containing Gly-Pro-Hyp to a subject. [A2] A method for suppressing a decline in biological function caused by glycation stress, comprising ingesting or administering a composition containing Gly-Pro-Hyp to a subject. [A3] A method for suppressing inflammation in a living organism caused by glycation stress, comprising ingesting or administering a composition containing Gly-Pro-Hyp to a subject. [A4] A method for suppressing cell death caused by glycation stress, comprising ingesting or administering a composition containing Gly-Pro-Hyp to a subject. [A5] A method for suppressing fatigue, comprising ingesting or administering a composition containing Gly-Pro-Hyp to a subject. [A6] A method for inhibiting the formation and / or accumulation of endogenous AGEs (Advanced Glycation End Products), comprising ingesting or administering a composition containing Gly-Pro-Hyp to a subject. [A7] A method for suppressing aging symptoms caused by the accumulation of endogenous AGEs in the dermis, comprising ingesting or administering a composition containing Gly-Pro-Hyp to a subject. [A8] The method according to A7, wherein the aging symptom is yellowish discoloration. [A9] A method for inhibiting the formation of RAGE (AGE-specific receptor)-binding AGEs, comprising ingesting or administering a composition containing Gly-Pro-Hyp to a subject. [A10] A method for suppressing the activity (reactivity) of aldehyde compounds in a living body, comprising ingesting or administering a composition containing Gly-Pro-Hyp to a subject. [A11] The method according to A10, wherein the aldehyde is glyceraldehyde. [A12] A method for inhibiting pentosidine formation, comprising ingesting or administering a composition containing Gly-Pro-Hyp to a subject. [A13] A method for suppressing aging symptoms caused by the accumulation of endogenous AGEs in the dermis, comprising applying a cosmetic composition containing Gly-Pro-Hyp to the skin. [A14] The method according to A13, wherein the aging symptom is yellowish discoloration. [A15] A method for suppressing glycation stress, comprising applying a cosmetic composition containing Gly-Pro-Hyp to the skin. [A16] A method for suppressing a decline in biological function caused by glycation stress, comprising applying a cosmetic composition containing Gly-Pro-Hyp to the skin. [A17] A method for suppressing inflammation in a living organism caused by glycation stress, comprising applying a cosmetic composition containing Gly-Pro-Hyp to the skin. [A18] A method for suppressing cell death caused by glycation stress, comprising applying a cosmetic composition containing Gly-Pro-Hyp to the skin. [A19] A method for inhibiting the generation and / or accumulation of endogenous AGEs (Advanced Glycation End Products), comprising applying a cosmetic composition containing Gly-Pro-Hyp to the skin. [A20] A method for inhibiting the formation of RAGE (AGE-specific receptor)-binding AGEs, comprising applying a cosmetic composition containing Gly-Pro-Hyp to the skin. [A21] A method for suppressing the activity (reactivity) of aldehyde compounds in a living organism, comprising applying a cosmetic composition containing Gly-Pro-Hyp to the skin. [A22] The method according to [A21], wherein the aldehyde is glyceraldehyde. [A23] A method for inhibiting pentosidine formation, comprising applying a cosmetic composition containing Gly-Pro-Hyp to the skin. [Brief explanation of the drawing]

[0019] [Figure 1] Figure 1 is a schematic diagram illustrating the action of AGEs and their inhibition by Gly-Pro-Hyp, as revealed in this application. [Figure 2] Figure 2 is an SDS-PAGE gel image. Lane x shows the marker, and the arrows and numbers on the left indicate the molecular weight (kDa) of each band in the marker. Arrow M indicates native (undenatured) LZ, arrow D indicates LZ dimer, and arrow T indicates LZ trimer. Explanation of each lane: a; LZ (unheated), b and d; LZ + GA, c and e; LZ + GA + Gly-Pro-Hyp, f; LZ + Gly-Pro-Hyp, g; LZ + Gly-Pro-Hyp. [Figure 3] Figure 3 is a graph showing the change in crosslinking degree over 7 days. (Left) Dimer, (Right) Trimer. In both graphs, the solid line (1) represents the control, and the dashed line (2) represents the solution with Gly-Pro-Hyp added. [Figure 4] Figure 4 shows the results of the crosslinking degree analysis for each solution. Dimer (top), trimer (bottom). 1: Control, 2: Gly-Pro-Hyp, 3: Glycine, 4: L-Proline, 5: L-Hydroxyproline, 6: L-Lysine, 7: L-Arginine. [Figure 5] Figure 5 shows the results of the crosslinking degree analysis for each solution. Dimer (top), trimer (bottom). 1: Control (GA only), 2: Gly-Pro-Hyp, 3: Gly-Pro-Pro, 4: Gly-Pro, 5: Pro-Hyp, 6: Hyp-Gly, 7: Gly-Pro-Arg, 8: Gly-Pro-Lys. [Figure 6]Figure 6 shows the results of the crosslinking degree analysis for each solution. Dimer (left), trimer (right). 1: Control (GA only), 2: Gly-Pro-Hyp, 3: Gly-Pro-Ala, 4: Gly-Leu-Hyp, 5: Gly-Pro-Lys, 6: Gly-Pro, 7: Pro-Gly. [Figure 7] Figure 7 shows the results of the crosslinking degree analysis for each solution. Dimer (left), trimer (right). 1: Control (GA only), 2: Gly-Pro-Hyp, 3: Ala-Hyp-Gly. [Figure 8] Figure 8 shows the retention rate of GA. 1h and 24h are reaction times. 1: Control (GA only), 2: Gly-Pro-Hyp, 3: Gly-Pro, 4: Pro-Hyp, 5: Glycine. [Figure 9] Figure 9 shows the retention rates of GA. 1: Control (GA only), 2: 3 mM Gly-Pro-Hyp, 3: 2 mM Gly-Pro-Hyp, 4: 1 mM Gly-Pro-Hyp, 5: 3 mM Pro-Hyp, 6: 3 mM Gly-Pro, 7: 3 mM Glycine. [Figure 10] Figure 10 shows the change in cell viability in GA-treated HepG2 cells. The numbers represent the concentration (mM) of Gly-Pro-Hyp, and +GA indicates 3mM GA treatment. [Figure 11] Figure 11 shows the changes in cell viability in GA-treated HepG2 cells. The numbers represent the concentration (mM) of each peptide, and +GA indicates 3mM GA treatment. (1) Gly-Pro-Hyp, (2) Gly-Pro, (3) Gly-Gly, (4) Glycine, (5) Pro-Hyp. [Figure 12] Figure 12 shows IL-8 gene expression in GA-treated HepG2 cells. +GA indicates 3 mM GA treatment. GPHyp: Gly-Pro-Hyp. [Figure 13] Figure 13 shows the absorption (left) and fluorescence intensity (Ex: 370 nm, Em: 440 nm) (right) of the BSA-containing solution after the glycation reaction. GPHyp: Gly-Pro-Hyp. 1 and 3: BSA only, 2 and 4: BSA + Gly-Pro-Hyp. [Figure 14]Figure 14 shows the absorbance (450 nm) of the solutions after the saccharification reaction. 1: BSA only, 2: BSA + aminoguanidine, 3: BSA + Gly-Pro-Hyp, 4: BSA + Gly-Pro-Lys. [Figure 15] Figure 15 shows the amount of pentosidine produced. 1: Control, 2: Gly-Pro, 3: Gly-Pro-Hyp, 4: Gly-Pro-Ala. [Figure 16] Figure 16 shows the gene expression of Nrf2, HO-1, IL-8, and MMP-1 in GA-treated HDF (human dermal fibroblast) cells. The numbers represent the concentration (mM) of Gly-Pro-Hyp, and +GA indicates 1.5 mM GA treatment. [Figure 17] Figure 17 shows ROS generation in GA-treated HDF cells. The vertical axis represents fluorescence intensity, with intensity increasing as ROS generation increases. GA was added at concentrations ranging from 0 to 2.0 mM. Gly-Pro-Hyp was added at 0 or 8 mM. [Figure 18] Figure 18 shows the expression level of COL1A1 protein in GA-treated HDF cells. The amount of Gly-Pro-Hyp added was 8 mM. [Figure 19] Figure 19 shows the gene expression of IL-8 after 24 hours of exposure of HDF cells to AGEs prepared by reacting BSA and GA. The numbers indicate the concentration (mM) of Gly-Pro-Hyp added, and +AGEs means that the cells were treated with 50 μg / mL of AGEs. [Modes for carrying out the invention]

[0020] The following description of the present invention may be based on representative embodiments and specific examples, but the present invention is not limited to such embodiments. In this specification, numerical ranges represented by "~" mean a range that includes the numbers written before and after "~" as the lower and upper limits.

[0021] There is much knowledge about the adverse effects of endogenous AGEs on the body, including decreased skeletal muscle mass (Yosuke Sugioka et al. Comprehensive Health Checkup 2016; 43(5): 537-542.), onset of mild cognitive impairment (MCI) (Igase M and Igase K, Glycative Stress Research 2018; 5 (1): 045-049), lung function (Kato et al., Tokoha University Faculty of Health Sciences Research Reports 2017, Vol. 4, No. 1), osteogenesis disorders and associated osteoporosis (Sakasai-Sakai et al., (2022). Nutrients, 14(5), 990.), periodontal pockets and severe periodontitis (Shungo Watanabe et al., 2020, Journal of Oral Hygiene Society, 70(3), 129-135.), and pancreatic Langerhans β-cell function (Suh, KS et al., Int. J. Mol. Med. 2017, 40, 539-548.; Borg et al., Islets 2018; 10: 10-24.; Takata et al., Nutrients, 14(2), 332.), decreased vascular function (Ueda S et al., Cardiovasc Ther 2011; 30: 249-54.), arteriosclerotic symptoms including vascular calcification (Yagi and Yonei, Glycative Stress Research 2018; 5(2): 82-85.), increased risk of atherosclerosis and myocardial ischemia (Takata et al., Metabolites 2022; 12(7); 615.), age-related lens opacity and age-related cataracts (Kubo, E. Glycative Stress Research 2020; 7 (4): 283-28), disruption of homeostasis of intervertebral discs (Kimiaki Yokosuka. Journal of the Japanese Society for Low Back Pain 2018; 14(1): 58-62.) and articular cartilage (Kimiaki Yokosuka. Journal of the Japanese Society for Low Back Pain 14.1 (2008): 58-62.), alcoholic hepatitis (Yagi and Yonei. Glycative Stress Research 2018; 5 (4): 177-180 previously published), and the onset and progression of non-alcoholic hepatitis (NASH) (Takeuchi et al., Medical Hypotheses 2015: 84(5); 490-493.), infertility (Yagi and Yonei. Glycative Stress Research 2019; 6 (1): 64-67), decreased renal function (Igase M and Igase K, Glycative Stress Research 2018; 5 (1): 045-049; Kumar Pasupulati A, et al., Biomol Concepts. 2016; 7: 293-309), age-related hearing loss (Tsuda, J., Sugahara, K., & Yamashita, H. Glycative Stress Research 2020; 7 (3): 240-247), decreased intestinal barrier (Matsunaga et al., The 144th Annual Meeting of the Pharmaceutical Society of Japan, March 28-31, 2024) and the resulting inflammation due to the invasion of infectious agents into the body (Miyamoto Junki et al., (2017) Chemistry and Biology, 55(4)), Glycative stress is known to be associated with many diseases and the aging of biological functions, including hair aging (Shimode A, Yagi M, Naito J, et al. Glycative Stress Res. 2014; 1: 37-45). Furthermore, the combination of a weakened skin barrier, reduced blood flow and impaired microcirculation, and a weakened immune system increases the risk of infection with infectious diseases, including COVID-19 (Haasbroek et al., Glycative Stress Research 2020; 7 (3): 232-239). It is no exaggeration to say that without countermeasures against glycation stress, maintaining our quality of life, extending healthy lifespan, and preventing disease will be impossible.

[0022] It is difficult for us to recognize the accumulation of endogenous AGEs in our bodies early on in our normal daily lives. One of the tissues where the effects of glycation are most easily recognized is the skin. The accumulation of AGEs in the skin affects the signs of aging. These signs of aging include wrinkles, age spots, loss of elasticity, sagging, dull skin (dryness), yellowing of the skin (yellowish dullness), and lack of transparency. It is generally well known that dry skin and exposure to ultraviolet rays in daily life are involved in these signs of aging. For example, dryness causes inflammation of the skin, which leads to inflammation, a decrease in collagen production, and the breakdown of collagen fibers, resulting in wrinkles, age spots, loss of elasticity, sagging, and dull skin. Ultraviolet rays also cause DNA damage and inflammation, and are particularly involved in the breakdown of collagen, elastin, and hyaluronic acid in the dermis, their decreased secretion, and the resulting development of age spots and wrinkles. To address signs of aging caused by dryness and UV radiation, various measures are being taken, including UV protection, moisturizing, applying or ingesting ingredients that promote the secretion of collagen, elastin, and hyaluronic acid, or applying or ingesting ingredients that supplement these components.

[0023] However, the onset and progression of aging signs due to the accumulation of AGEs in the skin can sometimes be difficult to prevent with the measures mentioned above. The formation of AGEs in the skin can be classified into several categories, and the countermeasures need to be changed accordingly. The process of AGEs formation in the skin can be broadly divided into three categories: (i) when secreted sebum in the superficial layer of the skin is peroxidized by ultraviolet rays, etc., and reacts with skin components to produce AGEs; (ii) when intercellular lipids in the stratum corneum are peroxidized by ultraviolet rays, etc., and react with skin components such as keratin to produce AGEs; and (iii) when sugars and aldehyde compounds produced in the body reach the dermis and glycate dermal components, including the extracellular matrix, to produce endogenous AGEs (Yagi and Yonei., Glycative Stress Research 2018; 5.1: 50-54.). Of these, (i) and (ii) are often recognized as yellowing (yellowing) of the skin surface, but because it is the surface of the body, countermeasures are easy, such as applying an effective agent directly to the glycated area. Moreover, the turnover of the stratum corneum is 14 days, and it peels off the skin in 28 days (Takahashi, "Basic Cosmetics and Skin (I)." Journal of the Color Materials Association 1989; 62.7: 430-438), so improvement is quick if countermeasures are taken. Improvement can be expected not only by using anti-glycation agents, but also by using UV protection and turnover-promoting agents for about 2 to 6 weeks.

[0024] In contrast, the accumulation of endogenous AGEs in the dermis (iii) takes a very long time to accumulate. Moreover, glycated dermal components become significantly less likely to be turned over, making improvement difficult with short-term measures. Furthermore, it has the characteristic of affecting a wide range of aging signs. The process of endogenous AGEs formation in the dermis begins with sugars and aldehyde compounds that have migrated from the blood to the skin forming disordered crosslinks between collagen fibers and elastin tissues via lysine and hydroxylysine, leading to the accumulation of endogenous AGEs, represented by pentosidine. These endogenous AGEs are brown or fluorescent, and as their accumulation progresses, they directly cause yellowing of the skin (Oguchi. Sugiyama Jogakuen University Practical Application Research Center Research Report 2015; 18.). Then, the loss of elasticity and flexibility of the extracellular matrix due to crosslinking causes the skin to harden and lose its flexibility, which leads to wrinkles. Furthermore, glycation stress causes fibroblast dysfunction, leading to a decrease in collagen and hyaluronic acid secretion, and inflammation similar to that caused by dryness and UV damage (Tsuneki et al., Int. J. Med. Sci. 2021; 18.2: 474.). Moreover, these AGEs stimulate increased melanin production, leading to the formation of age spots (Yonei and Yagi. Glycative Stress Research 2023; 10 (1): 27-42.). It is known that people with high fasting blood glucose levels, high accumulation of AGEs in the skin, and other conditions of severe glycation stress appear older than their actual age, even if they are healthy individuals of the same chronological age (Yonei et al. Journal of the Japan Society of Cosmetic Chemists 2019; 53.2: 83-90., previously published). The progression of aging caused by the accumulation of these endogenous AGEs in the dermis is difficult to fully prevent or improve by simply taking measures against UV radiation, using moisturizing cosmetics, and promoting the production of collagen, elastin, and hyaluronic acid or supplementing these components. It is necessary to implement measures specifically tailored to glycation stress over the long term.

[0025] To reduce glycation stress, the first step is to adopt a diet that suppresses blood sugar spikes, and to avoid excessive alcohol consumption and excessive fat intake, which also produce aldehyde compounds during metabolism. In addition to dietary and exercise guidance, medications to control blood sugar levels may be prescribed, such as alpha-glucosidase inhibitors, which inhibit the action of enzymes that break down ingested carbohydrates into monosaccharides, and dipeptidyl peptidase-4 (DPP-4) inhibitors, which promote incretin secretion.

[0026] However, the aforementioned treatments also have challenges. For example, it has been pointed out that glycation can counteract the effects of drugs, such as AGEs increasing DPP-4 expression in tissues (Tahara et al., Clin. Biochem. 2013; 46: 300-3). Also, while DPP-4 is involved in insulin regulation, fructose and other sugars are not subject to this regulation, so aldehyde sparks derived from fructose and other sugars cannot be prevented. It is important to note that blood glucose levels only indicate glucose concentration and do not indicate the blood concentration of other sugars. For example, sucrose, which we commonly consume in our daily diet, is composed of two sugars, and in its metabolic process, fructose is produced in addition to glucose. It has been pointed out that the ring-opening rate of fructose during metabolism is 300 times higher than that of glucose, and therefore it is more likely to produce aldehydes, which are highly harmful. In fact, studies have shown that when rats are continuously fed glucose, sucrose, or fructose daily, cross-linking of skin collagen progresses with any of these sugars, but it progresses most significantly when fructose is administered (Takao et al., J. of Kyushu Univ. of Health and Welfare. 2021; 22: 83-87). Although fructose is a sugar found in foods we commonly consume, such as fruits and soft drinks, measuring blood fructose levels after ingestion is not common. Furthermore, while pentoses are known to be more reactive with proteins than glucose, blood pentose levels are also not generally measured. In addition, blood concentrations of aldehyde compounds, which are sugar metabolites, are generally unknown. In other words, while blood glucose levels are one indicator, it is important to note that glycation is progressing in ways that cannot be determined from blood glucose levels alone.

[0027] As the harmful effects of glycation stress have become widely recognized, research has also been conducted on countermeasures against monosaccharides and aldehydes that have been produced in the body. For example, aminoguanidine is known as a potent anti-glycation agent that reduces the reactivity of sugars and aldehydes and directly inhibits the AGEs formation reaction (Kumar Pasupulati A et al., Biomol. Concepts 2016; 7: 293-309. Previously cited; Thornalley et al., Biochemical pharmacology 2000; 60.1: 55-65.). However, although the inhibitory effect of aminoguanidine on the formation of endogenous AGEs was demonstrated in animal studies, clinical research was discontinued due to multiple adverse effects, including liver damage. Furthermore, various approaches are being studied, including promoting the breakdown of AGEs, eliminating ROS, and inhibiting AGE-RAGE signaling (Kumar Pasupulati A, et al., Biomol. Concepts 2016; 7: 293-309, previously published). Research continues on ingredients that have anti-glycation effects while also ensuring safety.

[0028] Blood glycation markers correlate with the future onset of glycation-related diseases and aging / senile signs, regardless of whether or not a person currently suffers from a disease. Therefore, taking measures against glycation stress from a young age, or even before drug treatment becomes necessary, is crucial for maintaining good health. One measure that can be incorporated into lifestyle habits is the use of natural products and foods.

[0029] For example, in controlling blood sugar levels, it is recommended to replace carbohydrates with those that have a low glycemic index (GI), to actively consume dietary fiber which has the effect of suppressing sugar absorption, to actively consume foods containing lycopene and catechins which are effective in lowering blood sugar levels, and to consume whey protein which is related to insulin regulation.

[0030] Furthermore, proteins, peptides, and amino acids containing amino groups can exert effects such as preventing the formation of endogenous AGEs by acting as substitutes for biological proteins, as in the case of aminoguanidine. For this reason, the intake of proteins with such effects is also recommended. For example, Japanese Patent Publication No. 2024-083631, "Amino Acid-Containing Protein Glycation Inhibitor," and Japanese Patent Publication No. 2020-169154, "Amino Acid-Containing Protein Glycation Inhibitor," provide protein glycation inhibitors containing multiple amino acids. Japanese Patent Publication No. 2001-39816, "Collagen Crosslinking Inhibitor," provides a collagen crosslinking inhibitor containing hydrolyzed casein, L-lysine, or L-arginine. Japanese Patent Publication No. 2005 / 092363, "Preventive and Therapeutic Agent for Diabetic Complications Using Oligopeptides," provides an agent containing an oligopeptide as an active ingredient, which contains at least two amino acid residues having an amino group or guazinino group in its side chain.

[0031] On the other hand, it has been shown that the intake of collagen tripeptide (CTP), a material containing 15% Gly-XY (X and Y are any amino acids) tripeptides, improves vascular elasticity in healthy individuals and simultaneously lowers the concentration of glyceraldehyde-derived AGEs in their serum (Non-patent Literature 20: Tomosugi et al., J. Atheroscler. Thromb. 2017;24:530-538). However, the CTP used as a test food contained a total of 15% Gly-XY tripeptides with diverse sequences, and also contained 85% high-molecular-weight peptides. Upon ingestion, not only do the tripeptides directly enter the bloodstream, but metabolites of the tripeptides, such as Gly-Pro and Pro-Hyp, which are Gly-X or XY sequence components, are also produced (Yamamoto et al., Biol. Pharm. Bull. 2016; 39.3: 428-434; Yamamoto et al., Biosci. Biotechnol. Biochem. 2015; 79.12: 2026-2033.). High molecular weight peptide metabolites such as Pro-Hyp, Pro-Hyp-Gly, and Hyp-Gly, which are XY, XY-Gly, and Z-Gly sequence components, can also be produced (Ichikawa et al. Int. J. Food Sci. Nutri. 2010; 61.1: 52-60.). And, as with other protein foods, free amino acids also enter the bloodstream. Therefore, it was unclear which specific component was responsible for this effect caused by CTP intake, and it was also unclear whether the effect was specific to CTP in the first place.

[0032] If it remains unclear which components of CTP contribute to the glycation inhibitory effect, the processing of foods containing CTP may unintentionally cause the glycation inhibitory effect to be lost or reduced. Furthermore, if CTP contains both substances that promote and inhibit glycation, it may not function properly. Identifying these components was essential for developing more reliable and effective glycation inhibitors.

[0033] (Food composition) This invention relates to a variety of applications based on the glycation stress-inhibiting effect of food compositions containing Gly-Pro-Hyp.

[0034] The inventors investigated the inhibitory effect of collagen-derived peptides on endogenous AGEs formation using a glyceraldehyde-mediated glycation system and found that many tripeptides with Gly-XY sequences possessed an inhibitory effect on endogenous AGEs formation. From this, it was hypothesized that the Gly-XY tripeptides exert their effect by the reaction of the amino group of glycine at the N-terminus with the aldehyde group of glyceraldehyde, as is known from the action of aminoguanidine, to form a Schiff base. However, after further investigation using various evaluation systems, it was discovered that among the various Gly-XY sequences, Gly-Pro-Hyp exhibited particularly strong activity. Gly-Pro-Hyp has high bioavailability, rapidly entering the bloodstream after ingestion and subsequently reaching tissues (Yamamoto et al., Biol. Pharm. Bull. 2016; 39.3: 428-434; Yamamoto et al., Biosci. Biotechnol. Biochem. 2015; 79.12: 2026-2033). Based on this, we hypothesized that Gly-Pro-Hyp plays a particularly significant role in suppressing endogenous AGEs in CTP, and that this effect can be obtained even with Gly-Pro-Hyp alone, leading us to further our research.

[0035] Since Pro-Hyp, L-proline, and L-hydroxyproline, which constitute part of Gly-Pro-Hyp, did not have an inhibitory effect on endogenous AGEs formation, it was considered almost certain that the performance of Gly-Pro-Hyp was carried out by the reactivity of the N-terminal glycine with the aldehyde. However, the effect of Gly-Pro-Hyp was not only stronger than that of structurally similar Gly-Pro-Pro and Gly-Pro-Ala, but also stronger than that of Gly-Pro and glycine, which have a short molecular structure and a high degree of structural freedom, and even stronger than that of Ala-Hyp-Gly, which also has an amino group at the N-terminus, which was unimaginable from the laws of general chemical reactions.

[0036] Gly-Pro-Hyp is a tripeptide consisting of three amino acid residues, glycine, proline, and hydroxyproline, linked from the N-terminus to the C-terminus. Hydroxyproline is produced as a post-translational modification after protein synthesis, by the introduction of a hydroxyl group to proline by prolyl hydroxylase. Hydroxyproline is specifically present in collagen.

[0037] The tripeptide Gly-Pro-Hyp can be produced by specifically degrading the peptide chain represented by (Gly-XY)n of collagen or gelatin using a collagenase derived from Clostridium or the like, as described in Japanese Patent No. 3146251. The tripeptide Gly-Pro-Hyp can be produced by purifying these collagenase degradation products using liquid chromatography or other methods. The tripeptide Gly-Pro-Hyp can also be produced by known peptide synthesis methods such as "solid-phase synthesis" or "liquid-phase synthesis." For solid-phase synthesis, either the Fmoc method or the Boc method may be used.

[0038] In relation to the present invention, "endogenous AGEs (endogenous advanced glycation end products)" refers to irreversible glycation end products produced in living organisms through multiple chemical reaction steps by the non-enzymatic reaction of reactive carbonyl compounds such as reducing sugars and aldehydes, which are generated during the metabolic processes of sugars, lipids, or alcohols, with proteins. Endogenous AGEs mainly refer to glycation end products that occur on amino acid residues of proteins, but in a broader sense, glycation or glycation-related modifications of lipids or nucleic acids may also be included. Endogenous AGEs include non-crosslinking glycation modifications that occur on amino acid residues of proteins and crosslinking glycation modifications that form crosslinks between or within proteins.

[0039] In relation to the present invention, "glycation stress" refers to a state in which endogenous AGEs are generated and accumulated due to the action of reducing sugars and aldehydes derived from sugars, lipids, and alcohols produced in the metabolic process within the body, resulting in harmful effects, burdens, or impairments on biological functions.

[0040] The present invention relates to a food composition for suppressing glycation stress containing Gly-Pro-Hyp. As shown in the examples described below, Gly-Pro-Hyp has the effect of suppressing the cross-linking of proteins due to glycation. Furthermore, as shown in the examples described below, Gly-Pro-Hyp suppresses the generation of reactive oxygen species due to glycation stress in cells. By ingesting an effective amount of Gly-Pro-Hyp to obtain the effect as a food, for example as a nutritional supplement, the formation of AGEs is suppressed by inhibiting the cross-linking of proteins due to glycation, thereby suppressing glycation stress.

[0041] This invention relates to a food composition containing Gly-Pro-Hyp for suppressing the decline in biological functions caused by glycation stress. As shown in the examples described below, Gly-Pro-Hyp has the effect of suppressing protein cross-linking due to glycation for at least 7 days. The increase in AGE-crosslinked dimers of lysozyme observed as a control in the examples may be accompanied by a decrease in lysozyme activity and can therefore serve as an indicator of decline in biological functions. It is known that protein cross-linking is one of the causes of decline in biological functions through tissue hardening. Because Gly-Pro-Hyp has the effect of suppressing protein cross-linking not transiently but sustainably, it suppresses the decline in biological functions caused by glycation stress. By ingesting an effective amount of Gly-Pro-Hyp to obtain the effect as food, for example as a nutritional supplement, the formation of AGEs is suppressed by inhibiting protein cross-linking due to glycation, and the decline in biological functions caused by glycation stress is suppressed.

[0042] This invention relates to a food composition containing Gly-Pro-Hyp for suppressing inflammation in living organisms caused by glycation stress. As shown in the examples described below, Gly-Pro-Hyp can sustainably suppress the cross-linking of proteins due to glycation, and is therefore thought to be able to suppress endogenous AGEs that cause inflammation. Furthermore, the examples described below show that Gly-Pro-Hyp suppresses the expression of IL-8, one of the inflammatory cytokines. The examples described below also show that Gly-Pro-Hyp suppresses the onset of inflammation caused by glycation stress induced by endogenous AGEs that have been produced in the body. Therefore, Gly-Pro-Hyp has the effect of suppressing inflammation in living organisms caused by glycation stress. By ingesting an effective amount of Gly-Pro-Hyp to obtain the effect as food, for example as a nutritional supplement, the formation of cross-links of proteins due to glycation is suppressed, thereby suppressing the production of AGEs and suppressing inflammation in living organisms caused by glycation stress.

[0043] This invention relates to a food composition containing Gly-Pro-Hyp for suppressing cell death caused by glycation stress. The examples described below demonstrate that Gly-Pro-Hyp can sustainably suppress protein cross-linking due to glycation and also suppress the expression of the inflammatory cytokine IL-8. Furthermore, the examples described below demonstrate that Gly-Pro-Hyp suppresses HepG2 cell death induced by GA (glyceraldehyde) in a concentration-dependent manner. Therefore, Gly-Pro-Hyp has the effect of suppressing cell death caused by glycation stress. By ingesting an effective amount of Gly-Pro-Hyp as a food, such as a nutritional supplement, cell death caused by glycation stress is suppressed.

[0044] This invention relates to a food composition containing Gly-Pro-Hyp for anti-fatigue in living organisms caused by glycation stress. The examples described below show that Gly-Pro-Hyp suppresses the expression of IL-8, one of the inflammatory cytokines. Gly-Pro-Hyp exerts its anti-fatigue effect by suppressing the production of IL-8 caused by glycation stress. By ingesting an effective amount of Gly-Pro-Hyp as food, for example as a nutritional supplement, the production of IL-8 due to glycation stress is suppressed, thereby suppressing fatigue and malaise in living organisms caused by IL-8 production due to glycation stress.

[0045] The inventors investigated the effects of Gly-Pro-Hyp on the biological response to glycation stress using cultured cells. As a result, in glyceraldehyde-exposed HepG2 cells, Gly-Pro-Hyp showed concentration-dependent suppression of inflammatory cytokine IL-8 gene expression and concentration-dependent suppression of cell death. Since the IL-8 gene has the property of increasing due to the accumulation of endogenous AGEs and ROS generation (Kikuchi et al., Biol. Pharm. Bull. 2021; 44: 1399-1402. previously reported), the inventors' experimental results indicate that Gly-Pro-Hyp, by exhibiting the aforementioned aldehyde scavenging ability, suppressed a series of subsequent glycation stresses, and as a result prevented cell dysfunction. In particular, the suppression of cell death leads to the suppression of leakage of endogenous AGEs into the bloodstream. Furthermore, in studies using cultured cells, Gly-Pro, L-glycine, Gly-Gly, and Pro-Hyp were subjected to cell tests under the same conditions, but none of them showed any cell death inhibitory effect, indicating that this structure possesses its own inherent activity.

[0046] This invention relates to a food composition containing Gly-Pro-Hyp for inhibiting the formation and / or accumulation of endogenous AGEs (Advanced Glycation End Products). In this invention, endogenous AGEs refer to AGEs that are produced when dietary components are transferred into the body and react with proteins in the body. For example, pentosidine is an AGE that has a structure that cross-links lysine residues and arginine residues in proteins. As shown in the examples described below, Gly-Pro-Hyp has the effect of inhibiting the formation of pentosidine. Furthermore, Gly-Pro-Hyp can sustainably inhibit the formation of AGEs. Therefore, Gly-Pro-Hyp has the effect of inhibiting the formation of endogenous AGEs and further inhibiting their accumulation. By ingesting an effective amount of Gly-Pro-Hyp to obtain the effect as a food, for example as a nutritional supplement, the formation and / or accumulation of endogenous AGEs (Advanced Glycation End Products) can be suppressed.

[0047] This invention relates to a food composition containing Gly-Pro-Hyp for suppressing aging symptoms caused by the accumulation of endogenous AGEs in the dermis. The aging symptoms may include yellowing of the skin. In this invention, "yellowing of the skin" refers to the yellowing of the skin surface. As shown in the examples described below, the inventors have discovered that Gly-Pro-Hyp suppresses the yellowing of proteins and the generation of fluorescent AGEs. Pentosidine, mentioned above, is also one of the fluorescent AGEs and is a component known to accumulate in the dermis. As described above, brown or fluorescent AGEs accumulated in the skin directly cause yellowing of the skin, and ROS generated by the accumulation cause age spots. The inventors' experimental results show that Gly-Pro-Hyp suppresses the occurrence of yellowing of the skin and age spots caused by the accumulation of AGEs, and effectively works to suppress the progression of aging symptoms caused by glycation stress. Furthermore, as shown in the examples described below, Gly-Pro-Hyp has been shown to suppress the expression of oxidative stress response transcription factors and oxidative stress response genes, as well as the inflammatory mediators IL-8 and MMP-1, in dermal fibroblasts exposed to glycation stress. Therefore, Gly-Pro-Hyp is expected to exert an inhibitory effect on dermal aging by suppressing inflammatory responses caused by glycation stress. By ingesting an effective amount of Gly-Pro-Hyp as a food, such as a nutritional supplement, it is possible to suppress aging caused by the accumulation of endogenous AGEs in the dermis.

[0048] The present invention relates to a food composition containing Gly-Pro-Hyp that binds to RAGE (AGE-specific receptor) and inhibits the formation of AGEs. As shown in the examples described below, it is possible to inhibit the formation of highly toxic AGEs that drive the RAGE signal.

[0049] The inventors investigated the effect of Gly-Pro-Hyp on the formation of RAGE-binding glycated compounds. This is because even if the total amount of endogenous AGEs decreases due to the production of Gly-Pro-Hyp, if RAGE-binding glycated compounds increase, the activation of the RAGE signal, as described above, could occur as a third harmful effect of endogenous AGEs. The inventors' verification using a bovine serum albumin (BSA) glycation model showed that the production of RAGE-binding AGEs decreased when Gly-Pro-Hyp was added to BSA. This data indicates that Gly-Pro-Hyp is not involved in the production of highly toxic endogenous AGEs that drive the RAGE signal and cause a third harmful effect, but rather inhibits their production. Even considering this effect alone, it can be said that Gly-Pro-Hyp may contribute to the suppression of aging, signs of aging, and the onset and progression of disease. By ingesting an effective amount of Gly-Pro-Hyp to obtain the effect, such as in food or nutritional supplements, the production of RAGE (AGE-specific receptor)-binding AGEs can be inhibited.

[0050] This invention relates to a food composition containing Gly-Pro-Hyp for inhibiting the activity (reactivity) of aldehyde compounds in living organisms. The aldehyde may be glyceraldehyde. As shown in the examples described below, Gly-Pro-Hyp contributes to the suppression of AGEs formation by reducing the activity (reactivity) of aldehyde compounds in living organisms.

[0051] The inventors investigated the reactivity of Gly-Pro-Hyp with aldehydes in more detail. Tests were conducted reacting Gly-Pro-Hyp with glyceraldehyde at near body temperature. The results showed that when Gly-Pro-Hyp was added, the concentration of glyceraldehyde in the solution decreased more rapidly than when other peptides were added. This indicates that Gly-Pro-Hyp has superior aldehyde compound scavenging ability and reacts more preferentially with aldehydes in vivo. While the exact reason why Gly-Pro-Hyp is superior to structurally similar components in scavenging glyceraldehyde is unknown, it is speculated that glyceraldehyde may also be involved in reactions after Schiff base formation. In the process of AGEs formation, Schiff base formation is merely the initial trigger; the AGEs mature through a long process. It has been found that in this process, peptides and glyceraldehyde may be further involved, complicating the crosslinking (Takeuchi et al., Medical Hypotheses 2024; 183: 111248). From this, it is possible that Gly-Pro-Hyp may have a stronger ability to reduce aldehyde activity by capturing glyceraldehyde even after the formation of Schiff bases. By ingesting an effective amount of Gly-Pro-Hyp as food, for example as a nutritional supplement, the activity (reactivity) of aldehyde compounds in the body can be suppressed.

[0052] The present invention relates to a food composition for inhibiting pentosidine formation containing Gly-Pro-Hyp. As shown in the examples described below, Gly-Pro-Hyp has the effect of inhibiting the formation of pentosidine.

[0053] The inventors compared the effects of Gly-Pro-Hyp with those of L-lysine and L-arginine. Lysine and arginine are known to be highly reactive with sugars and aldehydes, and the amino groups of lysine and arginine side chains in proteins are often involved in the glycation crosslinking of proteins in vivo. The results showed that the effect of Gly-Pro-Hyp was slightly weaker than that of lysine but similar to that of arginine, and stronger than that of Gly-Pro-Lys and Gly-Pro-Arg, which contain lysine or arginine in their structure. From these findings, it can be expected that Gly-Pro-Hyp can prevent the formation of endogenous AGEs in vivo by competing with sugars and aldehyde compounds when crosslinking the lysine and arginine side chains of proteins. In fact, when Gly-Pro-Hyp was added to a solution containing three components: ribose (a pentose), L-lysine, and L-arginine, the formation of pentosidine was inhibited in a Gly-Pro-Hyp concentration-dependent manner. Therefore, it was confirmed that Gly-Pro-Hyp has strong reactivity that can compete with lysine and arginine in the glycation reaction and suppress cross-linking. Pentosidine formation can be suppressed by ingesting an effective amount of Gly-Pro-Hyp as food, such as a nutritional supplement.

[0054] In summary, our findings indicate that Gly-Pro-Hyp inactivates aldehydes, which are the starting point of glycation stress, prevents protein cross-linking and accumulation of cross-linked products due to glycation, prevents inflammation associated with ROS generation, prevents cellular dysfunction, and prevents the generation of AGEs that drive RAGE signaling. In other words, the action of Gly-Pro-Hyp has been demonstrated at each point from the starting point to the final stage of glycation stress.

[0055] The inhibitory effect of Gly-Pro-Hyp on glycation stress is expected to reduce the damage to the body associated with glycation, and to slow down the progression of aging and senescence, as well as the onset and progression of diseases. Specifically, it is expected to contribute to the suppression of many diseases, the maintenance of aging, and overall health, including the suppression of yellowing of the skin, decreased skin elasticity, sagging, wrinkles, and age spots; the suppression of split ends and breakage of hair, and the suppression of the progression of aging caused by these; the suppression of fatigue and malaise; the maintenance of skeletal muscle mass and muscle performance and the suppression of decline; the suppression of the onset and progression of MCI and dementia; the maintenance of lung function; the suppression of the onset and progression of osteoporosis; the maintenance of gum health; the suppression of the decline in insulin production function of pancreatic β-cells; the suppression of decline in vascular function; the maintenance of elasticity of blood vessels and vascular endothelial cells and the reduction of the risk of developing arteriosclerosis and myocardial ischemia; the suppression of age-related lens opacity and age-related cataracts; the maintenance of articular cartilage performance; the suppression of the onset and progression of alcoholic hepatitis and NASH; the suppression of infertility; the maintenance of renal function; the improvement of age-related hearing loss; the maintenance of the intestinal barrier; and resistance to infection.

[0056] As mentioned above, endogenous AGEs accumulate over many years and gradually decline physical function as one ages. As evidenced by the fact that type II diabetes most commonly develops after the age of 40, it takes a very long time for glycation stress to cause disease, and it is difficult to recognize its effects in one's youth unless there are special circumstances. On the other hand, slight accumulation and changes in function begin as early as one's 20s, and this accumulation of glycation stress from one's 20s certainly influences the degree to which it manifests in later age groups. For this reason, ideally, it is thought that starting to use Gly-Pro-Hyp at a young age, such as in one's 20s, and continuing to use it as part of one's lifestyle for as long as possible will more effectively suppress physical aging and signs of aging, and lead to an extension of healthy life. The effects of Gly-Pro-Hyp mentioned above can be expected to be measured or felt after continuous use for, for example, six months or more, or one year or more.

[0057] The food composition of the present invention contains Gly-Pro-Hyp as an active ingredient. The food composition of the present invention can be combined with collagen hydrolysate. The food composition of the present invention may be, for example, a collagen hydrolysate in which the content of Gly-Pro-Hyp, the active ingredient of the present invention, is increased. To increase the content of Gly-Pro-Hyp in the collagen hydrolysate, for example, Gly-Pro-Hyp obtained by the above synthesis or purification method can be added to the collagen hydrolysate. Examples of collagen hydrolysates include, but are not limited to, HACP-50, HACP-01, HACP-TF, HACP-CF (Zelice Co., Ltd.), and are not limited by the type of raw animal such as pig, fish, or other. By administering or ingesting the food composition of the present invention in addition to these collagen hydrolysates, the above effects, such as the suppression of glycation stress, can be expected.

[0058] Gly-Pro-Hyp is present in the amino acid sequence of collagen in living organisms at a concentration of about 10%, so collagen hydrolysates obtained by digesting and breaking down collagen or gelatin may contain this tripeptide. Collagen hydrolysates containing Gly-Pro-Hyp can be used as a powder, granules, or liquid, or they can be used in combination with other components. Because the amino acid sequence of collagen has high homology in many organisms, including animals, fish, and mollusks, Gly-Pro-Hyp can be obtained using any type of collagen as a raw material. It can also be obtained by chemical synthesis or by secretion production using microorganisms. In both cases, it can be used in powder or liquid form.

[0059] The food composition of the present invention can be processed into any orally ingestible form of food (e.g., solution, suspension, powder, solid molded product, etc.). The processed food can be consumed as is, or mixed with beverages such as coffee, dairy products such as yogurt, etc.

[0060] Examples of foods, though not limited to the present invention, include supplements such as tablets, granules, capsules, soft capsules, and drinks; carbohydrates (bread, noodles, rice, mochi, etc.); confectionery (cookies, jelly, chocolate, chewing gum, candy, etc.); dairy products (cheese, yogurt, butter, etc.); powdered foods (powdered soup, powdered mousse, powdered jelly, powdered sweeteners); nutritional foods; diet foods; sports nutrition foods; and beverages (fruit juice-containing beverages, fruit juices, vegetable juices, cider, ginger ale, isotonic beverages, amino acid beverages, jelly beverages, coffee beverages, green tea, black tea, oolong tea, barley tea, milk beverages, lactic acid bacteria beverages, cocoa, beer, sparkling wine, third-category beer, non-alcoholic beverages, beer-flavored beverages, liqueurs, chuhai, sake, fruit wine, distilled spirits). In one embodiment of the present invention, the food is a functional food or a food for specified health uses.

[0061] The food composition of the present invention can be taken at a dose of 0.01 to 200 mg per kg of body weight per day, preferably 0.02 to 100 mg, once or several times a day. The dose can be adjusted as appropriate depending on age, body weight, etc. There are no particular restrictions on the duration of intake, but it is preferably two weeks or more, more preferably one month or more, or more than two months.

[0062] The food composition of the present invention can be ingested by mammals and humans. The food composition of the present invention can be administered to subjects, which include mammals and humans.

[0063] Regarding the dosage of Gly-Pro-Hyp, since the glyceraldehyde concentration in the body is said to be between 1 and 100 μM (Ukeda et al., Biosci. Biotechnol. Biochem. 1997; 61: 2039-2042), if one were to try to capture all of it with Gly-Pro-Hyp, it would seem desirable to take a large amount of Gly-Pro-Hyp daily. For example, since Gly-Pro-Hyp reaches the skin and takes effect even with the intake of 1 g of CTP containing 3% Gly-Pro-Hyp, it can be expected that continuous intake of 0.03 g of Gly-Pro-Hyp would also be sufficient to produce an effect. Furthermore, it is thought that taking it before or with meals can effectively counteract aldehyde sparks after meals. Gly-Pro-Hyp is rapidly absorbed via specific transporters in the small intestine, so even when ingested mixed with other components such as amino acids, lipids, and minerals, it is absorbed without being affected and can reach tissues to exert its effects. It can be used in combination with other anti-glycation agents, antioxidants, and other beauty ingredients, which is expected to allow for the creation of foods that can address a wider range of anti-aging needs. It can be used in any form, including solid, tablet, powder, and juice. It can also be used in combination with or added to enteral nutrition, high-nutrient intravenous drips, and peritoneal dialysis, which are known to cause glycation in the body. However, when processing Gly-Pro-Hyp mixed with sugar, heating during the processing process can lead to deactivation of the active ingredient, so it is preferable to keep the heat treatment short or use filter sterilization. Although Gly-Pro-Hyp can be stored for a long time in liquid form, prolonged coexistence with sugar in water may accelerate deactivation, so it is also effective to store it as a powder and mix it immediately before use.

[0064] (Cosmetic composition) This invention relates to a cosmetic composition containing Gly-Pro-Hyp for suppressing aging symptoms caused by the accumulation of endogenous AGEs in the dermis. The aging symptoms may include yellowing of the skin. By applying an effective amount of Gly-Pro-Hyp to the skin, it is expected that aging symptoms caused by the accumulation of endogenous AGEs in the dermis will be suppressed.

[0065] As shown in the examples described below, the inventors discovered that Gly-Pro-Hyp suppresses the yellowing of proteins and the generation of fluorescent AGEs. Pentosidine, mentioned above, is also one of the fluorescent AGEs and is a component known to accumulate in the dermis. As stated above, AGEs that accumulate in the skin and exhibit browning or fluorescence directly cause yellowing of the skin, and the ROS produced by this accumulation cause age spots. The inventors' experimental results show that Gly-Pro-Hyp suppresses the yellowing of the skin and the occurrence of age spots caused by the accumulation of AGEs, and effectively works to suppress the progression of aging caused by glycation stress.

[0066] Furthermore, as shown in the examples described below, Gly-Pro-Hyp has been shown to suppress the expression of oxidative stress response transcription factors and oxidative stress response genes, as well as the inflammatory mediators IL-8 and MMP-1, in dermal fibroblasts exposed to GA. Based on the above, Gly-Pro-Hyp is expected to exert an inhibitory effect on dermal aging by suppressing the inflammatory response caused by glycation stress.

[0067] As shown in the examples described below, Gly-Pro-Hyp has been shown to suppress the generation of reactive oxygen species caused by glycation stress in the dermis. Therefore, it can be expected to contribute to the suppression of inflammation and aging by suppressing the generation of reactive oxygen species.

[0068] As shown in the examples described below, Gly-Pro-Hyp has been shown to inhibit the formation of AGEs (Advanced Glycation End Products) of collagen in skin fibroblasts, or to suppress the decrease in collagen expression caused by glycation stress. Therefore, it is expected that Gly-Pro-Hyp can suppress the decline in collagen function in the skin through this action, and contribute to suppressing the appearance and progression of aging signs.

[0069] This invention relates to a cosmetic composition for suppressing glycation stress containing Gly-Pro-Hyp. It is expected that glycation stress will be suppressed by applying an effective amount of Gly-Pro-Hyp to the skin.

[0070] This invention relates to a cosmetic composition for suppressing the decline in biological functions caused by glycation stress. By applying an effective amount of Gly-Pro-Hyp to the skin, it is expected that the decline in biological functions caused by glycation stress will be suppressed.

[0071] This invention relates to a cosmetic composition for suppressing inflammation in living organisms caused by glycation stress. By applying an effective amount of Gly-Pro-Hyp to the skin, it is expected that inflammation in living organisms caused by glycation stress will be suppressed.

[0072] This invention relates to a cosmetic composition for suppressing cell death caused by glycation stress. By applying an effective amount of Gly-Pro-Hyp to the skin, it is expected that cell death caused by glycation stress will be suppressed.

[0073] This invention relates to a cosmetic composition for inhibiting the formation and / or accumulation of endogenous AGEs (Advanced Glycation End Products). By applying an effective amount of Gly-Pro-Hyp to the skin, it is expected that the formation and / or accumulation of endogenous AGEs will be suppressed.

[0074] This invention relates to a cosmetic composition for inhibiting the formation of RAGE (age-specific receptor)-binding AGEs. By applying an effective amount of Gly-Pro-Hyp to the skin, it is expected that the formation of RAGE (age-specific receptor)-binding AGEs will be inhibited.

[0075] This invention relates to a cosmetic composition for inhibiting the activity (reactivity) of aldehyde compounds in living organisms. By applying an effective amount of Gly-Pro-Hyp to the skin, it is expected that the activity (reactivity) of aldehyde compounds in living organisms will be suppressed. The aldehyde may also be glyceraldehyde.

[0076] This invention relates to a cosmetic composition for inhibiting pentosidine formation. By applying an effective amount of Gly-Pro-Hyp to the skin, it is expected that pentosidine formation will be inhibited.

[0077] Gly-Pro-Hyp has a molecular weight of less than 500 daltons and can penetrate the skin, so it can also be effective when applied topically. However, it is important to note that the site and manifestation of its effects may differ depending on whether it is used for a short period or a long period. For short-term use, it can be expected to suppress glycation in the epidermis, where cell turnover is rapid. Specifically, it can be expected to suppress yellowing of the skin caused by the peroxidation of secreted sebum and intercellular lipids in the stratum corneum by ultraviolet rays, which then reacts with skin components. As mentioned above, since the turnover of the skin epithelium is about 28 days, it is thought that improvement can be felt or observed even with use for 2 to 6 weeks, or at most less than 6 months. On the other hand, symptoms caused by the accumulation of endogenous AGEs in the dermis take a very long time to manifest, so it is difficult to feel its effects with short-term use, and long-term use is required. By using it for at least six months, ideally for one to five years, or even throughout one's life, it is believed that the yellowing of the deep layers of the skin caused by the accumulation of endogenous AGEs, wrinkles and decreased skin elasticity caused by the transformation of the extracellular matrix into AGEs, and age spots caused by ROS associated with AGEs accumulation will be suppressed, and an improvement in signs of aging can be felt or observed.

[0078] Gly-Pro-Hyp can be applied to the skin as a solution mixed with a liquid medium such as water. When applying Gly-Pro-Hyp, it penetrates the applied area, so the Gly-Pro-Hyp solution does not necessarily need to be highly concentrated; a Gly-Pro-Hyp concentration of 0.001(w / v)% or higher is desirable, ideally 0.01(w / v)% or higher. The form of application is not limited, but it can be in the form of lotions, emulsions, gels, creams, shampoos, conditioners, hair tonics, bath additives, sheet masks, skin patches, and iontophoresis cosmetics. It is also effective to store it as a powder and mix it with lotions, creams, or iontophoresis cosmetics just before use. [Examples]

[0079] The present invention will be described more specifically based on the following examples, but the present invention is not limited to these examples.

[0080] Example 1: Lysozyme Crosslinking Inhibition Test Part 1 <Method> The AGE formation inhibitory effect of Gly-Pro-Hyp was investigated using a glyceraldehyde glycation model of lysozyme (LZ). LZ is a naturally occurring antimicrobial substance with a molecular weight of 14,000 Da and consisting of approximately 130 residues, and is found in egg white, saliva, tears, and nasal mucus. It has 6 lysine residues and 11 arginine residues, and is susceptible to glycation, making it suitable as a glycation model. 349.5 μM LZ was added to phosphate buffer (pH 7.4), 10 mM glyceraldehyde (GA), and 4 mM Gly-Pro-Hyp, and the mixture was reacted at 37°C for 7 days. The reaction solution was mixed with an equal amount of Ezapply (ATTO) and reacted in an aluminum block at 100°C for 5 minutes to prepare a sample for SDS-PAGE. This sample was subjected to SDS-PAGE using a 15% acrylamide gel, and then stained with CBB stain to detect bands. The reaction with GA cross-links LZ molecules, producing dimers and trimers. The bands representing LZ dimers and trimers were identified from the positions of markers run simultaneously, and the intensity of the bands was compared with and without the addition of Gly-Pro-Hyp.

[0081] <Result> Figure 2 shows images of the stained gels. When LZ and GA reacted, dimers and trimers clearly appeared. On the other hand, when Gly-Pro-Hyp was added in addition to LZ and GA, the bands of dimers and trimers became fainter. This indicates that Gly-Pro-Hyp suppressed glycation crosslink formation. This data suggests that Gly-Pro-Hyp contributes to suppressing the decline in biological function caused by glycation stress by suppressing the generation and accumulation of endogenous AGEs.

[0082] Example 2: Lysozyme Crosslinking Inhibition Test Part 2 <Method> Next, the time course of LZ crosslinking was tracked. 300 μM LZ was added to phosphate buffer (pH 7.4), 4 mM GA and 4 mM Gly-Pro-Hyp were added, and the mixture was reacted at 37°C for 3, 5, and 7 days. A control was prepared by adding only 4 mM GA to LZ and treating it similarly. After the reaction, each solution was mixed with an equal amount of Ezapply (ATTO) and reacted in an aluminum block at 100°C for 5 minutes to prepare samples. These samples were subjected to SDS-PAGE using a 15% acrylamide gel and then stained with CBB stain. After imaging the gels, the bands of LZ crosslinking-induced dimers and trimers were analyzed using ImageJ. For both dimers and trimers, the degree of crosslinking was calculated using the band intensity of the control solution on day 7 as 100%.

[0083] <Result> Figure 3 shows the change in the degree of crosslinking over time. For both the dimer and trimer, the degree of crosslinking increased with longer reaction times. On the other hand, when Gly-Pro-Hyp was added, the degree of crosslinking was persistently kept low. This indicates that the inhibitory effect of Gly-Pro-Hyp on LZ glycation is not transient.

[0084] Example 3: Lysozyme Crosslinking Inhibition Test Part 3 <Method> Next, the crosslinking inhibitory effect of Gly-Pro-Hyp was compared with that of other components. 4 mM GA and 4 mM of various compounds (Gly-Pro-Hyp, glycine, L-proline, L-hydroxyproline, L-lysine, or L-arginine) were added to a phosphate buffer (pH 7.4) containing 300 μM lysozyme (LZ), and the mixture was reacted at 40°C for 72 hours. Control solutions containing only LZ and GA were also prepared and operated simultaneously. After the reaction, each solution was mixed with an equal amount of Ezapply (ATTO) and reacted in an aluminum block at 100°C for 5 minutes to prepare samples. These samples were subjected to SDS-PAGE using a 15% acrylamide gel and then stained with CBB stain. After capturing images of the gels, the intensity of the dimer and trimer bands was analyzed using ImageJ, and the ratio of the band intensity of each solution to the control solution was defined as the degree of crosslinking. Hereafter, all values ​​are shown as mean ± standard deviation.

[0085] <Result> Figure 4 shows the analysis results for dimers and trimers. Of the six components examined, L-proline and L-hydroxyproline produced cross-linking substances equivalent to those of the control, and no glycation inhibitory effect was observed. The other components showed a lower degree of cross-linking than the control, and an inhibitory effect on cross-linking formation was observed. The inhibitory effect on trimer formation was, in order from strongest to weakest, arginine > lysine > Gly-Pro-Hyp > glycine. On the other hand, the degree of dimer formation inhibition was, in order from strongest to weakest, lysine > arginine = Gly-Pro-Hyp > glycine. This data indicates that Gly-Pro-Hyp has a higher glycation inhibitory effect than glycine, and that it may show a glycation inhibitory effect equivalent to that of arginine.

[0086] Example 4: Lysozyme Crosslinking Inhibition Test Part 4 <Method> The action of Gly-Pro-Hyp was compared with structurally similar components. 4 mM GA and 4 mM of various compounds (Gly-Pro-Hyp, Gly-Pro-Pro, Gly-Pro, Pro-Hyp, Gly-Pro-Arg, or Gly-Pro-Lys) were added to phosphate buffer (pH 7.4) containing 300 μM lysozyme (LZ), and the mixture was reacted at 40°C for 72 hours. Control solutions containing only LZ and GA were also prepared and operated simultaneously. After the reaction, each solution was mixed with an equal amount of Ezapply (ATTO) and reacted in an aluminum block at 100°C for 5 minutes to prepare samples. These samples were subjected to SDS-PAGE using a 15% acrylamide gel and then stained with CBB stain. After capturing images of the gels, the intensity of the dimer and trimer bands was analyzed using ImageJ, and the ratio of the band intensity of each solution to the control solution was defined as the degree of crosslinking.

[0087] <Result> Figure 5 shows the analysis results. Of the six components examined, no effect was observed with Pro-Hyp, but the other peptides showed a lower degree of cross-linking than the control, indicating an inhibitory effect on cross-linking formation. The inhibitory effect on dimer formation was in the order of Gly-Pro-Hyp > Gly-Pro = Gly-Pro-Arg > Gly-Pro-Pro > Gly-Pro-Lys. The inhibitory effect on trimer formation was in the order of Gly-Pro-Hyp > Gly-Pro > Gly-Pro-Arg > Gly-Pro-Pro > Gly-Pro-Lys. Generally, peptides containing lysine and arginine are said to be easily associated with glycation (cross-linking) reactions, but the effect of Gly-Pro-Hyp was stronger than those, and peptides with a similar structure to Gly-Pro-Hyp showed at least a stronger inhibitory effect than Gly-Pro-Lys.

[0088] Example 5: Lysozyme Crosslinking Inhibition Test, Part 5 <Method> The action of Gly-Pro-Hyp was compared with structurally similar components. 4 mM GA and 4 mM of various compounds (Gly-Pro-Hyp, Gly-Pro-Ala, Gly-Leu-Hyp, or Gly-Pro-Lys) were added to phosphate buffer (pH 7.4) containing 300 μM lysozyme (LZ), and the mixture was reacted at 40°C for 24 hours. Control solutions containing only LZ and GA were also prepared and operated simultaneously. After the reaction, each solution was mixed with an equal amount of Ezapply (ATTO) and reacted in an aluminum block at 100°C for 5 minutes to prepare samples. These samples were subjected to SDS-PAGE using a 15% acrylamide gel and then stained with CBB stain. After capturing images of the gels, the intensity of the dimer and trimer bands was analyzed using ImageJ, and the ratio of the band intensity of each solution to the control solution was defined as the degree of crosslinking.

[0089] <Result> Figure 6 shows the analysis results. All six components examined showed an inhibitory effect on cross-linking formation. For both dimers and trimers, the inhibitory effect on cross-linking formation was strongest in the order of Gly-Pro-Hyp > Gly-Pro-Ala > Gly-Leu-Hyp > Gly-Pro-Lys. It was found that peptides other than lysine had a stronger effect than peptides containing lysine, and in particular, Gly-Pro-Hyp had a strong effect.

[0090] Example 6: Lysozyme Crosslinking Inhibition Test, Part 6 <Method> The action of Gly-Pro-Hyp was compared with that of XY-Gly sequence peptides. 4 mM GA and 4 mM of various compounds (Gly-Pro-Hyp or Ala-Hyp-Gly) were added to phosphate buffer (pH 7.4) containing 300 μM lysozyme (LZ), and the mixture was reacted at 40°C for 72 hours. Control solutions containing only LZ and GA were also prepared and operated simultaneously. After the reaction, each solution was mixed with an equal amount of Ezapply (ATTO) and reacted in an aluminum block at 100°C for 5 minutes to prepare samples. These samples were subjected to SDS-PAGE using a 15% acrylamide gel and then stained with CBB stain. After capturing images of the gels, the intensity of the dimer and trimer bands was analyzed using ImageJ, and the ratio of the band intensity of each solution to the control solution was defined as the degree of crosslinking.

[0091] <Result> Figure 7 shows the analysis results. Both components showed glycation inhibitory effects. Although glycine in Gly-Pro-Hyp and alanine in Ala-Hyp-Gly both share an amino group, Gly-Pro-Hyp showed a stronger glycation inhibitory effect.

[0092] Example 7: Effect on the remaining rate of GA in solution, Part 1 <Method> It is considered that the background of the saccharification cross-linking production inhibitory effect observed in the above Examples 1 to 6 is that the amino group of the peptide reacts with the aldehyde group of GA involved in cross-linking, thereby reducing its reactivity. Therefore, the GA concentration in the reaction solution was measured using MBTH (3-Methyl-2-benzothiazolinonehydrazone Hydrochloride) reagent, which develops color by binding to aldehydes, and the effect of components such as Gly-Pro-Hyp on the residual rate of GA in the reaction solution was verified. First, 3 mM of Gly-Pro-Hyp (or Gly-Pro, Pro-Hyp, glycine, aminoguanidine) was added to 0.2 M phosphate buffer (pH 7.4) containing 3 mM of GA, mixed, and reacted at 40°C for 1 hour or 24 hours. Separately, a solution containing only GA was prepared as a control and treated in the same manner. Next, after these solutions were appropriately diluted with phosphate buffer (pH 7.4) to 150 µL, 2.85 mL of 0.2 M acetate buffer and 1 mM of 0.01% 3-Methyl-2-benzothiazolinonehydrazone Hydrochloride were added, mixed, and reacted at 50°C for 30 minutes. Separately, a freshly prepared GA solution was prepared as a standard solution and subjected to the MBTH method in the same manner as the above-mentioned samples. The absorbance at 310 nm was measured for all these solutions. The GA concentration in each sample solution and control solution was calculated from the absorbance of the standard solution.

[0093] <Results> The analysis results are shown in Figure 8. After 24 hours of reaction, the GA concentration was equivalent in all solutions, but a difference was observed after 1 hour of reaction, suggesting that components containing glycine in their structure contribute to the consumption (deactivation) of GA. The GA residual rate was in the order of Gly-Pro-Hyp < Gly-Pro < glycine from the lowest, and it was found that among the peptides containing glycine, Gly-Pro-Hyp exhibited the strongest effect. This data shows that Gly-Pro-Hyp rapidly traps GA. From these results, it was demonstrated that Gly-Pro-Hyp contributes to the inhibition of AGEs production by reducing the activity (reactivity) of aldehyde compounds in vivo.

[0094] Example 8: Effect on the remaining rate of GA in solution, Part 2 <Method> The GA concentration in the reaction solution with each component was measured using the MBTH method, and the residual GA rate of each component was measured for verification. First, a sample solution was prepared by adding 1-3 mM Gly-Pro-Hyp, 3 mM Gly-Pro, 3 mM Pro-Hyp, or 3 mM glycine to 0.2 M phosphate buffer (pH 7.4) containing 3 mM GA, mixing, and reacting at 40°C for 3 hours. Separately, a solution containing only GA was used as a control and reacted in the same manner. Next, these solutions were appropriately diluted with phosphate buffer (pH 7.4) to 150 μL, to which 2.85 mL of 0.2 M acetate buffer and 1 mM 0.01% 3-Methyl-2-benzothiazolinonehydrazone Hydrochloride were added and mixed, and reacted at 50°C for 30 minutes. Separately, a GA solution prepared on the spot was used as a standard solution and subjected to the MBTH method in the same manner as the samples described above. The absorbance at 310 nm was measured for all of these solutions. The GA concentration in each sample solution and the control solution was calculated from the absorbance of the standard solution.

[0095] <Result> Figure 9 shows the analysis results. The GA retention rate was lower with 3mM Gly-Pro-Hyp, 3mM Gly-Pro, and 3mM glycine compared to the control (GA only), while the GA retention rate with Pro-Hyp was similar to the control. This suggests that components containing glycine contribute to the consumption (inactivation) of GA. In 1-3mM Gly-Pro-Hyp solutions, the GA retention rate decreased in a concentration-dependent manner with Gly-Pro-Hyp addition. Furthermore, even with 1mM Gly-Pro-Hyp, the GA retention rate was lower than with 3mM Gly-Pro and 3mM glycine, indicating that the effect of Gly-Pro-Hyp was the strongest.

[0096] Example 9: Effect on cell viability of glycation-treated cells, Part 1 <Method> HepG2 cells, dispersed in DMEM medium (Dulbecc's modified Eagle agar) containing 10% fetal bovine serum (FBS) at a concentration of 60,000 cells / mL, were seeded in 96-well plates at a rate of 100 μL / well. These were cultured overnight in a 37°C CO2 incubator. The following day, the culture medium was changed to DMEM medium containing 0.5% FBS, and the cells were cultured for another overnight. The next day, Gly-Pro-Hyp solutions dissolved at concentrations of 0, 2, 4, and 8 mM were added to DMEM medium containing 2% dialysis FBS, and the cells were incubated in a 37°C CO2 incubator for 2 hours. Next, 3 mM GA was added to these cells, and they were cultured for 24 hours. After culturing, cell viability was measured using CellTiter-Glo (Promega).

[0097] <Result> Figure 10 shows the analysis results. Gly-Pro-Hyp suppressed GA-induced cell death in HepG2 cells in a concentration-dependent manner. It is thought that Gly-Pro-Hyp suppressed cell death by inhibiting glycation stress in the body, thereby contributing to the suppression of functional decline and reduction of ROS production.

[0098] Example 10: Effect on cell viability of glycated cells, Part 2 <Method> HepG2 cells, dispersed in DMEM medium containing 10% FBS at a concentration of 60,000 cells / mL, were seeded into 96-well plates at a rate of 100 μL / well. These were cultured overnight in a 37°C CO2 incubator. The following day, the culture medium was changed to DMEM medium containing 2% dialysis FBS, and the cells were cultured overnight. The next day, Gly-Pro-Hyp, Gly-Pro, Gly-Gly, Pro-Hyp, and glycine solutions dissolved at concentrations of 0, 2, 4, and 8 mM were added to the cells in DMEM medium containing 2% dialysis FBS, and the cells were incubated in a 37°C CO2 incubator for 2 hours. Next, 3 mM GA was added to these cells, and they were cultured for 24 hours. After culturing, cell viability was measured using CellTiter-Glo (Promega).

[0099] <Result> Figure 11 shows the analysis results. Gly-Pro-Hyp suppressed cell death in a concentration-dependent manner. On the other hand, no concentration-dependent effect was observed with the other peptides.

[0100] Example 11: Effects on IL-8 gene expression <Method> HepG2 cells, dispersed in DMEM medium containing 10% FBS at a concentration of 57,000 cells / mL, were seeded into 24-well plates at a rate of 500 μL / well. These were cultured overnight in a 37°C CO2 incubator. The following day, the culture medium was changed to DMEM medium containing 2% dialysis FBS, and the cells were cultured overnight. The next day, Gly-Pro-Hyp solutions dissolved at concentrations of 0, 2, 4, and 8 mM were added to the cells in DMEM medium containing 2% dialysis FBS, and the cells were incubated in a 37°C CO2 incubator for 2 hours. Next, 3 mM GA was added to the cells, and they were cultured for 16 hours. After culture, FastGene... TM Total RNA was extracted using an RNA purification kit (Nihon Genetics). IL-8 mRNA expression was measured using quantitative RT-PCR and evaluated using the ΔΔCt method.

[0101] <Result> Figure 12 shows the analysis results. Gly-Pro-Hyp suppressed IL-8 mRNA cell expression induced by GA addition in a concentration-dependent manner. This data indicates that Gly-Pro-Hyp suppressed inflammation by reducing the accumulation of endogenous AGEs and ROS in cells through the suppression of glycation stress.

[0102] Example 12: Effect on coloration / fluorescent AGEs formation related to yellowing <Method> The accumulation of brown or fluorescent AGEs in the skin causes yellowing and blemishes. Therefore, we investigated the effect of Gly-Pro-Hyp on the generation of fluorescent AGEs in a GA-treated model of bovine serum albumin (BSA). First, 4 mM Gly-Pro-Hyp and then 10 mM GA were added to 0.2 M phosphate buffer (pH 7.4) containing 10 mg / mL of BSA, mixed, and incubated at 37°C for 7 days. A fixed volume of this solution was added to 10K nanosep (PALL), and BSA and low molecular weight components were separated by centrifugation at 12,000 × g. After centrifugation, a fixed volume of phosphate buffer was added to the remaining solution on the membrane containing BSA, and the mixture was redispersed. The absorption (420 nm) and fluorescence intensity (Ex: 370 nm, Em: 440 nm) of this solution were measured using a microplate reader to evaluate the degree of coloration and fluorescence generation of BSA derived from AGEs after reaction with GA.

[0103] <Result> Figure 13 shows the analysis results. The absorption at 420 nm of the BSA solution after reaction with GA decreased when Gly-Pro-Hyp was added, suggesting that the formation of colored BSA-AGEs was suppressed. Furthermore, the fluorescence intensity was also significantly suppressed by the addition of Gly-Pro-Hyp, suggesting that the formation of fluorescent BSA-AGEs was suppressed. These results suggest that Gly-Pro-Hyp suppresses the formation of endogenous AGEs that exhibit browning or fluorescence, indicating that it can suppress the yellowing of the skin caused by the accumulation of endogenous AGEs.

[0104] Example 13: Effect on the formation of RAGE-binding AGEs <Method> AGEs generated in vivo subsequently bind to RAGE, a receptor on the cell surface, inducing oxidative stress and inflammatory responses, which are involved in aging and the onset and progression of disease. In other words, if AGEs are generated but do not drive the RAGE signal, their harmful effects will be limited to a narrower range. Therefore, using a GA glycation model of bovine serum albumin (BSA), we investigated how AGEs binding to RAGE change with and without the addition of Gly-Pro-Hyp. First, 0.2M phosphate buffer (pH 7.4) containing 10 mg / mL of BSA was mixed with 0 or 4 mM Gly-Pro-Hyp, Gly-Pro-Lys, or AG. Then, 4 mM GA was added to each solution and mixed, and incubated at 37°C for 72 hours. 5 μL of this solution was placed on a Circulex AGE-RAGE in vitro Binding Assay Kit (MBL), and absorption at 450 nm was measured using a microplate reader. BSA without added GA was similarly tested and used as a baseline. In the Circulex AGE-RAGE in vitro Binding Assay Kit (MBL), soluble RAGE in the reagent binds to either GA-derived AGEs coated on the bottom or AGEs in the sample, and the RAGE that ultimately binds to the GA-derived AGEs coated on the bottom is detected by absorbance. In other words, a higher absorbance at 450 nm indicates that there were fewer RAGE-binding AGEs in the sample.

[0105] <Result> Figure 14 shows the analysis results. The solution obtained by reacting BSA with Gly-Pro-Hyp showed higher absorbance compared to BSA alone, suggesting a reduction in RAGE-binding AGEs similar to that of aminoguanidine (AG), a potent anti-glycation agent. Furthermore, the reduction in RAGE-binding AGEs by Gly-Pro-Hyp was higher than that of Gly-Pro-Lys, which has a similar structure and contains lysine. This suggests that Gly-Pro-Hyp inhibits the formation of highly toxic AGEs that drive the RAGE signal.

[0106] Example 14: Pentosidine formation inhibition test <Method> The action of Gly-Pro-Hyp was compared with that of peptides having Gly at the N-terminus. Substrate solutions were prepared using 200 mM sodium phosphate buffer (pH 7.4) with 40 mM ribose, 40 mM L-lysine hydrochloride, and 40 mM L-arginine. To these equal volumes, Gly-Pro, Gly-Pro-Hyp, and Gly-Pro-Ala, prepared using 200 mM sodium phosphate buffer (pH 7.4), were added to final concentrations ranging from 2.5 mM to 20 mM. The final concentration of each substrate solution was 10 mM. The substrate solutions and mixtures of the various synthetic peptides were reacted at 60°C for 24 hours. For all of these solutions, the fluorescence intensity at 385 nm was measured after irradiation with 335 nm excitation light, and the amount of pentosidine produced was calculated as the fluorescence amount.

[0107] <Result> Figure 15 shows the analysis results. Gly-Pro, Gly-Pro-Hyp, and Gly-Pro-Ala all inhibited pentosidine production in a concentration-dependent manner. Among them, Gly-Pro-Hyp had a higher pentosidine production inhibitory effect than Gly-Pro and Gly-Pro-Ala at all concentrations. This result indicates that Gly-Pro-Hyp contributes to suppressing various aging symptoms and disease onset caused by the accumulation of pentosidine in the body. Lysine and arginine, whose side chains have high reactivity with sugars, are known to be strongly involved in AGEs formation reactions, but Gly-Pro-Hyp was shown to have activity strong enough to compete with lysine and arginine in glycation reactions.

[0108] Example 15: Manufacture of cosmetics containing Gly-Pro-Hyp Lotions and liquid formulations containing Gly-Pro-Hyp can be manufactured with the following compositions. [Table 1]

[0109] [Table 2] This raw material contains 0.9% Gly-Pro-Hyp. It can be mixed with other cosmetic ingredients as appropriate to produce the final product.

[0110] [Table 3]

[0111] Example 15: Effects on gene expression in GA-treated dermal fibroblasts <Method> HDF cells dispersed in DMEM medium containing 10% FBS were placed in 24-well plates at a rate of 6,000 cells / cm². 2 Seeds were seeded in 500 μL / wells to achieve the desired concentration. These were cultured overnight in a 37°C CO2 incubator. The next day, the culture medium was changed to DMEM medium containing 2% dialysis FBS, and the cells were cultured for another overnight. The following day, Gly-Pro-Hyp solution dissolved at a concentration of 0 or 8 mM was added to the cells in DMEM medium containing 2% dialysis FBS, and the cells were incubated in a 37°C CO2 incubator for 2 hours. Next, 1.5 mM GA was added to these cells, and they were cultured for 16 hours. After culturing, FastGene TM Total RNA was extracted using an RNA purification kit (Nihon Genetics). Using this sample, mRNA expression of Nuclear factor erythroid 2-related factor 2 (Nrf2), Heme Oxygenase-1 (HO-1), IL-8, and Matrix metalloproteinase-1 (MMP-1) was measured by quantitative RT-PCR and evaluated using the ΔΔCt method with β-actin as an internal standard.

[0112] <Result> The results are shown in Figure 16. Gly-Pro-Hyp suppressed the mRNA expression of Nrf2 and HO-1, which had been elevated by GA addition, to levels similar to those of the control without GA addition. Nrf2 is a gene encoding an oxidative stress response transcription factor, and HO-1 is an oxidative stress response gene whose expression is induced downstream of Nrf2. This data indicates that Gly-Pro-Hyp reduced ROS production by suppressing the accumulation of endogenous AGEs in dermal fibroblasts. Furthermore, Gly-Pro-Hyp reduced the mRNA expression of IL-8 and MMP-1, which had been elevated by GA addition, to levels similar to those of the control without GA addition. This data indicates that Gly-Pro-Hyp suppressed the expression of IL-8 and MMP-1, which are inflammatory mediators, as a result of reducing the accumulation of endogenous AGEs and the subsequent ROS production. In other words, Gly-Pro-Hyp is expected to suppress glycation stress in the dermis, as well as inflammation caused by glycation stress, and contribute to the suppression of aging symptoms.

[0113] Example 16: Impact on ROS generation <Method> HDF cells dispersed in DMEM medium containing 10% FBS were placed in 96-well plates at a rate of 6,000 cells / cm². 2 Seeds were seeded at a concentration of 100 μL / well. These were cultured overnight in a 37°C CO2 incubator. The next day, the culture medium was changed to DMEM medium containing 2% dialysis FBS, and cultured overnight. The following day, Gly-Pro-Hyp solution dissolved at a concentration of 0 or 8 mM was added to the cells in DMEM medium containing 2% dialysis FBS, and the cells were incubated in a 37°C CO2 incubator for 2 hours. Next, 0, 1.5, or 2.0 mM GA was added to these cells, and they were cultured for 5 hours. Next, each well was washed twice with HBSS (Hanks equilibrium salt solution, Fujifilm Wako Pure Chemical Industries), and then stained with ROS Assay Kit-Photo-oxidation Resistant DCFH-DA-(DOJINDO) for 30 minutes. After 30 minutes, each well was washed twice with HBSS, HBSS was added again, and then the cells were photographed with a microplate reader at an excitation wavelength of 511 nm and an fluorescence wavelength of 530 nm.

[0114] <Result> Figure 17 shows the analysis results. The fluorescence intensity showed a gradual upward trend in proportion to the concentration of GA added. When Gly-Pro-Hyp was co-added, the increase in fluorescence intensity was suppressed. This result suggests that Gly-Pro-Hyp suppressed the accumulation of endogenous AGEs in skin fibroblasts, thereby suppressing the subsequent generation of ROS. It should be noted that the number of viable cells remained almost unchanged within the reagent concentration range used in this experiment.

[0115] Example 17: Effects on collagen protein expression in GA-treated dermal fibroblasts <Method> HDF cells dispersed in DMEM medium containing 10% FBS were placed in 12-well plates at a rate of 6,000 cells / cm². 2The cells were seeded at a concentration of 1 mL / well. These were incubated overnight in a 37°C CO2 incubator. The next day, the culture medium was changed to DMEM medium containing 2% dialysis FBS, and the cells were incubated overnight. The following day, Gly-Pro-Hyp solution dissolved at a concentration of 0 or 8 mM was added to the cells in DMEM medium containing 2% dialysis FBS, and the cells were incubated in a 37°C CO2 incubator for 2 hours. Next, 0, 1.5, and 2.0 mM GA were added to these cells, and they were incubated for 24 hours. The following day, each well was washed with ice-cold PBS, and 200 μL of RIPA buffer (Nacalai Tesque) containing cOmplete(R) and Mini was added to each well, and the cells were incubated on ice for 15 minutes. After incubation, the cell lysate was scraped off using a scraper and collected in a 1.5 mL microcentrifuge tube. This was centrifuged at 14,000 × g for 10 minutes, and the supernatant was collected. The protein concentration in the supernatant was measured using a BCA PROTEIN ASSAY KIT (BDL). Simultaneously, the supernatant was mixed with the same volume of Ezapply (ATTO) and heated at 98°C for 5 minutes to obtain an SDS sample. Approximately 20 μL of this SDS sample was subjected to electrophoresis on a 7.5% acrylamide gel and then Western blotting. Anti-COL1A1(3G3) antibody (Santa Cruz) was used as the primary antibody for collagen detection at a concentration of 1:1000, and Anti-mouse IgG (Proteintech) was used as the secondary antibody at a concentration of 1:4000. Similarly, β-actin rabbit mAb (Cell signaling technology) was used as the primary antibody for β-actin detection at a concentration of 1:1000, and Anti-rabbit IgG (Cell signaling technology) was used as the secondary antibody at a concentration of 1:2000. Detection was performed using ECL Prime Detection Reagent (Cytiva), and after a 1-minute reaction, detection was performed rapidly with LAS500 (Cytiva). Images of the bands acquired with ECL500 were analyzed using ImageJ, and the expression level of COL1A1 was normalized with β-actin.

[0116] <Result> The results are shown in Figure 18. COL1A1 (type I collagen α1 chain) expression was reduced in proportion to the GA addition concentration. Adding 1.5 mM GA reduced expression to approximately 60% of the control condition (no GA added), and adding 2.0 mM GA reduced it to approximately 30% of the control condition (no GA added). This suggests that GA treatment may have weakened collagen expression itself, or that collagen fibers in the extracellular matrix were glycated, denatured to a state where they were difficult to recognize by primary antibodies. In a similar experiment conducted by Sakai-Sakasai et al. (Nutrients 2022, 14, 990) using osteoblasts, a decrease in collagen band intensity was observed, and the cause was considered to be either decreased collagen expression or reduced antibody recognition due to glycation. On the other hand, adding Gly-Pro-Hyp to 1.5 mM GA restored COL1A1 expression to a level equivalent to the control condition (no GA added). When Gly-Pro-Hyp was added to 2.0 mM GA, recovery was better than when Gly-Pro-Hyp was not added. This data indicates that Gly-Pro-Hyp is influential in suppressing the AGEs formation of collagen or the decrease in collagen expression caused by glycation stress. In other words, Gly-Pro-Hyp may contribute to suppressing the decline in collagen function in the skin and inhibiting the appearance and progression of aging signs through this action.

[0117] Example 18: Effects of BSA-derived AGEs exposure on IL-8 gene expression As mentioned earlier, when collagen and other components in the extracellular matrix of skin cells are glycated, endogenous AGEs are generated. These AGEs activate cellular signals via RAGE in nearby fibroblasts, causing oxidative stress and inflammation. This is known to disrupt skin homeostasis and accelerate skin aging. We investigated whether Gly-Pro-Hyp can suppress inflammation caused by endogenous AGEs already present in the skin.

[0118] <Method> 1. Preparation of BSA-derived AGEs 18 mg of GA and 3.9 mg of diethylenetriamine-N,N,N',N'',N''-pentaacetic acid (DTPA) were dissolved in 5 mL of phosphate buffer (pH 7.4), and then 50 mg of BSA was added and dispersed. This was filtered through a 0.2 μm filter and sterilized. This was added to a tube, sealed, and reacted at 37°C for 7 days. 2.5 mL of the reaction mixture was applied to a PD-10 column (Cytiva) and eluted with 3.5 mL of PBS. The eluate was further filtered several times through a 10K ultrafiltration membrane (PALL) to remove glyceraldehyde. The concentration of BSA-derived AGEs in the purified product was measured using a BCA assay kit (BDL).

[0119] 2. Cell stimulation by BSA-derived AGEs HDF cells dispersed in DMEM medium containing 10% FBS were placed in 24-well plates at a rate of 6,000 cells / cm². 2 Seeds were seeded at a concentration of 500 μL / well. These were cultured in a 37°C CO2 incubator until subconfluent. Next, the culture medium was changed to DMEM medium containing 2% dialysis FBS and cultured overnight. The following day, BSA or BSA-derived AGEs at a concentration of 50 μg / mL and Gly-Pro-Hyp solution at a concentration of 0 or 8 mM were added to the DMEM medium containing 2% dialysis FBS, and after several days of culture, FastGene TM Total RNA was extracted using an RNA purification kit (Nihon Genetics). IL-8 mRNA expression was measured using quantitative RT-PCR with this sample and evaluated using the ΔΔCt method with β-actin as an internal standard.

[0120] <Result> Figure 19 shows the analysis results. Gly-Pro-Hyp reduced IL-8 mRNA cell expression, which had increased due to the addition of BSA-derived AGEs, to below the control level without BSA-derived AGEs. This data indicates that Gly-Pro-Hyp suppresses the development of inflammation caused by oxidative stress induced by endogenous AGEs already produced in the body. From this effect, it can be expected that it can reduce skin aging (appearance and progression of aging signs) caused by inflammation.

Claims

1. A food composition containing Gly-Pro-Hyp for suppressing glycation stress.

2. A food composition containing Gly-Pro-Hyp for suppressing the decline of biological functions caused by glycation stress.

3. A food composition containing Gly-Pro-Hyp for suppressing inflammation in the body caused by glycation stress.

4. A food composition containing Gly-Pro-Hyp for suppressing cell death caused by glycation stress.

5. An anti-fatigue food composition containing Gly-Pro-Hyp.

6. A food composition containing Gly-Pro-Hyp for inhibiting the formation and / or accumulation of endogenous AGEs (Advanced Glycation End Products).

7. A food composition containing Gly-Pro-Hyp for suppressing aging symptoms caused by the accumulation of endogenous AGEs in the dermis.

8. The food composition according to claim 7, wherein the early aging symptom is yellowish discoloration.

9. A food composition containing Gly-Pro-Hyp for inhibiting the formation of AGEs that bind to RAGE (AGE-specific receptor).

10. A food composition containing Gly-Pro-Hyp for inhibiting the activity (reactivity) of aldehyde compounds in living organisms.

11. The food composition according to claim 10, wherein the preceding aldehyde is glyceraldehyde.

12. A food composition containing Gly-Pro-Hyp for inhibiting pentosidine formation.

13. A cosmetic composition containing Gly-Pro-Hyp for suppressing aging caused by the accumulation of endogenous AGEs in the dermis.

14. The cosmetic composition according to claim 13, wherein the sign of aging is yellowish discoloration.

15. A cosmetic composition containing Gly-Pro-Hyp for suppressing glycation stress.

16. A cosmetic composition containing Gly-Pro-Hyp for suppressing the decline in biological functions caused by glycation stress.

17. A cosmetic composition containing Gly-Pro-Hyp for suppressing inflammation in the body caused by glycation stress.

18. A cosmetic composition containing Gly-Pro-Hyp for suppressing cell death caused by glycation stress.

19. A cosmetic composition containing Gly-Pro-Hyp for inhibiting the formation and / or accumulation of endogenous AGEs (Advanced Glycation End Products).

20. A cosmetic composition containing Gly-Pro-Hyp for inhibiting the formation of AGEs that bind to RAGE (AGE-specific receptor).

21. A cosmetic composition containing Gly-Pro-Hyp for inhibiting the activity (reactivity) of aldehyde compounds in living organisms.

22. The cosmetic composition according to claim 21, wherein the aldehyde is glyceraldehyde.

23. A cosmetic composition containing Gly-Pro-Hyp for inhibiting pentosidine formation.

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